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PSEUDO-DYNAMIC COMBINATORIAL LIBRARIES:
A RECEPTOR-ASSISTED COMBINATORIAL CHEMISTRY APPROACH TO
DRUG DISCOVERY
Jeremy D. Cheeseman
A thesis submitted to the Faculty of Graduate Studies and Research
of Mc Gill University in partial fulfilment of the requirements of the degree of
Doctor of Philosophy
Department of Chemistry
McGill University
Montréal, Québec, Canada
May 2004
© Jeremy D .. Cheeseman 2004
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"'1-fe who makes a 6east of himseg: 8ets rid of the yain of 6ein8 a man."
-I}-{unter s. rfhomyson
ABSTRACT
Emerging methods of combinatorial chemistry involve receptor assistance to combine
synthesis and screening. Binding to the receptor alters either the thermodynamics or
kinetics of synthesis. Dynamic combinatorial chemistry uses reversible synthesis where
binding to the receptor shifts the equilibrium to make more of the best binders. In target
accelerated synthesis, binding of the starting materials to the receptor speeds up the
synthesis of the best-binding compounds. We report a new receptor-assisted method
-pseudo-dynamic combinatorial chemistry- where binding to a receptor slows the
destruction of the best-binding compounds. In pseudo-dynamic libraries, synthesis and
destruction of library members are separate, irreversible reactions. Extending the
destruction reaction amplifies binding differences similar to a kinetic resolution of
enantiomers. Initial libraries of two to eight dipeptides, sorne containing an aryl
sulfonamide moiety that binds to carbonic anhydrase, showed that a ratio of> 100: 1 of the
best binding dipeptide over the next best was possible. These experiments also suggested
that the selectivity is related to the number of compounds in a library, with more library
members producing higher selectivity (a highly desirable result opposite that seen in
traditional dynamic libraries). Expansion of these libraries to include compounds
containing sulfonamides, aryl sulfonamides, sulfamates and hydroxamic acids further
support postulations as to the origins of the high selectivity of these systems, and take the
number of compounds screened by a pseudo-dynamic library closer to practical levels for
drug discovery.
RÉSUMÉ
Les nouvelles méthodes de la chimie combinatoire impliquent une cible biologique pour
combiner la synthèse et la détection des résultats d'une librairie. La liaison d'un composé
à la cible change les aspects thermodynamiques, ou cinétiques de la synthèse. Les
librairies combinatoires dynamiques utilisent une synthèse réversible pour déplacer
l'équilibre en faveur des composés qui se lient à la cible. Dans les systèmes où la cible
favorise la synthèse, la vitesse de synthèse est améliorée pour les composés qui se lient le
plus fortement à la cible. On a créé un nouveau système combinatoire -les librairies
combinatoires pseudo-dynamiques- dont la liaison d'un composé à la cible ralentisse sa
destruction. Dans les systèmes pseudo dynamiques, la synthèse et la destruction de la
bibliothèque sont des processus séparés et irréversibles. Dans nos premières expériences,
on a créé des inhibiteurs dipeptidiques contre l'anhydrase carbonique. La synthèse
utilisait des esters activés qui se couple avec des aminoacides. La destruction était
l'hydrolyse catalysée par Pronase. En utilisant un processus similaire aux résolutions
cinétiques, le prolongement de la destruction amplifie les différences des affinités des
composés pour la cible. Ces systèmes peuvent distinguer entre les composés ayant une
différence dans les constantes de liaison de moins d'un facteur de deux. Les expériences
récentes ont augmenté le nombre des composés, et démontrent que dans les systèmes
pseudo-dynamiques le fait d'avoir plus des composés dans le système améliore la
sélectivité de la cible pour son inhibiteur le plus puissant.
ii
ACKNOWLEDGEMENTS
1 would like to thank my research supervisor, Prof essor Romas J. Kazlauskas,
whose extensive knowledge and multifaceted approach to chemistry has in great part
shaped my own scientific style. 1 am also grateful (tongue in cheek) for his technological
savvy in the form of video conferencing, database support and email, which more than
compensated for his lack of physical presence during the greater part of my studies. But
in aIl seriousness, 1 am grateful for the opportunity to have worked so independently.
1 also wish to thank Professor James L. Gleason, with whom we collaborated on
this project. He always pushed me to my limits, without which 1 would have foundered
early on in my studies.
1 want to thank Andrew D. Corbett for his often tireless synthetic efforts, for
editing this thesis, and for taking sorne of the nights off my hands when 1 just couldn't
stay awake for another shift on the library. Without his efforts, this project would not
have been possible.
1 would like to thank David Soriano deI Arno, who has taken over the job of
supplying me with molecules to play with, and who l'm sure will take this project to the
nextlevel.
The department and especially my lab have been quite dynamic, with many
changes in personnel and location in a short time and l'm grateful to aIl who came and
went. l'd especially like to thank Seongsoon Park, Chris Savile, Paul Mugford, Krista
Morely and James Ashenhurst along with the rest of the Gleason lab for a great time, at
least while we were aIl in the same countries.
iii
l would like to thank Renée Charron, Chantal Marotte, Fay Nurse, Paulette
Henault, Carol Brown and Sandra Aerssen for great administrative support.
Financial assistance from McGill University and the department of chemistry in
the form of Alma Mater Travel Grants, the Parsini Diwan prize in chemistry, and the
Robert Zamboni Prize in chemistry, and from NSERC and CIHR for post-graduate
fellowships is gratefully acknowledged.
iv
TABLE OF CONTENTS
Abstract
Résumé
Acknowledgements
Table of Contents
Abbreviations
List of Figures
List of Schemes/Tables/Miscellaneous
General Contribution of Authors
Chapter One Introduction to Receptor Assisted Combinatorial
Chemistry in Drug Discovery
1.1 Combinatorial Chemistry
1
11
111
v
X11
XV111
XXIV
xxvii
1
2
v
1.2 Objectives, Methods and Terms in Drug Discovery Applications of Receptor-
Assisted Combinatorial Chemistry
1.3 Receptor Assisted Systems Under Thermodynamic Control:
1.4
Dynamic Combinatorial Libraries
1.3.1 DCLs requiring "lock in" reactions
1.3.2 Systems with in situ detection
1.3.3 Limitations to DCLs
Receptor Assisted Systems That Add Kinetic Control
1.4.1 Receptor-Accelerated Synthesis
1.4.2 Limitations In RAS
1.4.3 Affinity Chromatography
References
5
10
11
15
21
25
26
28
29
32
ChapterTwo Amplification of Screening Sensitivity Through Selective
Destruction: Theory and Screening of a Library of Carbonic
Anhydrase Inhibitors 36
Abstract 37
2.1 Introduction 37
vi
2.2 Theory: Finding the Best Inhibitor by Destruction of Poorer
Inhibitors
2.3 Results
2.3.1 Synthesis of 4'-Sulfonamidophenylalanine Dipeptides
2.3.2 Inhibition of Carbonic Anhydrase
2.3.3 Selective Extraction of Inhibitors By Carbonic Anhydrase
2.3.4 Selective Protection oflnhibitors by Carbonic Anhydrase
from Hydrolysis
2.3.4.1 Screening ofProteases
2.3.4.2 Selective Protection oflnhibitors
2.4 Discussion/Conclusions
Contribution of Authors
References
Chapter Three First Generation Pseudo-Dynamic Libraries
Abstract
40
43
43
44
46
49
49
49
54
58
59
61
62
vii
3.1 Designing the First Pseudo-Dynamic Combinatorial Library
3.1.1 Synthesis of the Library
62
64
3.1.2 Binding to the Receptor and Destruction ofUnbound Library Members
66
3.1.3 Recycling Destruction Products and Iteration of Synthe sis 69
3.2 Results of the Integrated Processes in the First Pseudo-Dynamic Combinatorial
Library 70
3.3 Discussion/Conclusion 75
Contribution of Authors 77
References 77
Chapter Four Pseudo-Dynamic Combinatorial Libraries: A New Receptor-
Assisted Approach For Drug Discovery 78
Abstract 79
4.1 Introduction 79
4.2 Experimental Design 80
viii
4.3 Results 83
4.4 Discussion/Conclusion 86
Contribution of Authors 88
References 88
Chapter Five Amplification and Selectivity in, Expansion and Modeling of
Pseudo-Dynamic Combinatorial Libraries
91
Abstract
5.1 Origins of the Amplification Maxima in the Eight-Memberedp-DCL
5.1.1 Introduction
5.1.2 Testing Enzyme Purity
5.1.3 Lowering the Concentration ofNucleophiles
92
92
92
95
97
5.1.4 Discussion: A Steady State Concentration of the Strongest Binding
Dipeptide 102
ix
5.2 The Effect of the Number of Inhibitors on Amplification and Selectivity in
5.3
5.4
5.5
5.6
Pseudo-Dynamic Combinatorial Libraries
5.2.1 Introduction
104
104
5.2.2 Two- and Three-Inhibitor 16 h Cycle P-DCLs 104
5.2.3 Discussion: Fewer Inhibitors Increase Amplification, but Decrease
Se1ectivity in P-DCLs 106
Expansion ofthe Pseudo-Dynamic Combinatorial Library 107
5.3.1 Introduction: New Library Members and P-DCL Scheme 107
5.3.2 Results
5.3.3 Discussion
Preliminary Modeling of Pseudo-Dynamic Combinatorial Libraries
5.4.1 Introduction
5.4.2 Modeling Synthesis
5.4.3 Mode1ing Receptor-Binding
5.4.4 Modeling Destruction 117
110
111
112
112
113
115
5.4.5 Discussion: The Integrated Model and its Comparison to Experiment
119
Overall Conclusions
Future Endeavours
5.6.1 Improving the p-DCL Model
5.6.2 Fundamental Experimentation inp-DCLs
122
124
124
125
x
5.6.3 Expansions and Miniaturizations 125
Contribution of Authors 126
References 126
Chapter Six Experimental Section 129
6.1 Experimental Section for Chapter Two 130
6.2 Experimental Section for Chapter Three 141
6.3 Experimental Section for Chapter Four 145
6.5 Experimental Section for Chapter Five 149
References 156
Contribution to Knowledge 157
Appendix 1 Amplification of Screening Sensitivity through Selective Destruction:
Theory and Screening of a Library of Carbonic Anhydrase Inhibitors
xi
Journal of the American Chemical Society 2002, 124,5692-5701 © 2002
American Chemical Society
Appendix II Pseudo-Dynamic Combinatorial Libraries: A Receptor-Assisted Approach
for drug Discovery Angewandte Chemie International Edition English
2004,43,2432-2436 © 2004 Wiley InterScience
XII
ABBREVIATIONS
AA amino acid
Ac acetyl
AcCN acetonitrile
AChE acetylcholine esterase
Alasa [3-sulfonamidoalanine
Aq aqueous
Ar aryl
Arg argmme
Asp aspartic acid
Asn asparagme
BICINE N,N-bis(2-hydroxyethyl)glycine
Bn benzyl
BOC tert -butoxycarbony 1
BSA bovine serum albumine
Bu butyl
Bz benzoyl
oC degree Celsius
CA carbonic anhydrase
cat. catalystlcatalytic
Cbz benzyloxycarbonyl
CC combinatorial chemistry
Xlll
Con A concanavalin A
d doublet
DCL dynamic combinatoriallibrary
dd doublet of doublets
ddd doublet of doublet of doublets
de diastereomeric excess
DMAP 4-( dimethy lamino )pyridine
DMF N,N-dimethylformamide
DMSO dimethy lsulfoxide
DNA deoxyribonucleic acid
DTT dithiothreitol
EDC-HCI 1-(3-dimethylamino )propyl-3-~-
ethy lcarbodiimide hydrochloride
Etoc ethoxycarbonyl
ee enantiomeric excess
eq. equivalents
Et ethyl
g gram(s)
Gal galactose
GC gas chromatography
Glc glucose
GluNHoH L-glutamic acid y-monohydroxamate
Gly glycine
xiv
h hour(s) .~-
HMPA hexamethylphosphoramide
HPLC high performance liquidchromatography
HTS high-throughput screening
Hz hertz
i iso
l inhibitor
ICso concentration required for 50% inhibition
J coupling constant
Ka affinity constant
kI destruction rate constant
Kd dissociation constant
Kr inhibition constant
ks synthetic rate constant
Ks equilibrium constant of synthesis
L litre
LCMS high performance liquid
chromatograhy /mass spectrometry
Leu leucine
Lyssa lysine-E-sulfamide
m meta
Il micro
m milli, multiplet
xv
M moles per litre
Man mannose
mCPBA meta-chloroperoxybenzoic acid
Me methyl
MIC minimum inhibitory concentration
mL millilitre
mmol millimole
mol mole
MS mass spectrometry
m/z mass to charge ratio
n normal
NANA N-acetylneuraminic acid ~-
ND not determined
NHSSu N-hydroxysulfosuccinamide
n nano
NMR nuclear magnetic resonance
p para or pseudo
PEG polyethylene glycol
PFP pentafluorophenol
pH -log [H+]
Ph phenyl
PhMe toluene
Phe phenylalanine
xvi
Phesa
pKa
Pr
Pro
R
RACe
RAS
RNA
rt
s
s
SM
STP
t
T
t
tert
tetra-FP
TEA
tri-FP
TFA
THF
Tyr
4' -sulfonamidophenylalanine
-log(Ka)
propyl
proline
receptor
receptor-assisted combinatorial chemistry
receptor-accelerated synthesis
ribonucleic acid
room temperature
secondary
singlet
starting material
4-sulfo-2,3,5,6-tetrafluorophenol
tertiary
biological target
triplet
tertiary
tetrafluorophenol
triethylamine
trifluorophenol
trifluoroacetic acid
tetrahydrofuran
tyrosine
XVll
UV
Val
w/w
WGA
Xyl
ultraviolet
Valine
weight by weight comparison
wheat germ agglutinin
xylose
xviii
LIST OF FIGURES
Figure 1.1: ParaUel Synthesis 3
Figure 1.2: Split-Pool Synthesis 4
Figure 1.3: Three receptor-assisted combinatorial methods 10
Figure 1.4: The first dynamic combinatoriallibrary 12
Figure 1-5: An example of a DCL from O. Ramstrom and l-M. Lehn 13
Figure 1-6: Creation of an imine library against neuraminidase using a diamine and
several ketones 15
Figure 1-7: A DCL made from NANA aldolase catalyzed aldol formation in the
presence of wheat germ agglutinin 16
Figure 1-8: An oxindole library created in the presence of cyclin-dependent kinase-2
(CDK-2) crystals 17
Figure 1-9: An example of "tethering" usmg DCL principles from Sunesis
pharmaceuticals 18
xix
Figure 1-10: Extended tethering with caspase-3, a protease with several binding pockets
(SI-S4) 20
Figure 1-11. The dynamic combinatoriallibrary equilibria yield a higher amount of
good inhibitor 22
Figure 1-12. Theoretical effect of a DCL in which the combination of three different
starting materials (A, B and C) yields a library of trimers 25
Figure 1-13. Nicolaou's vancomycin monomer scaffold 27
Figure 1-14. An example ofreceptor-accelerated synthesis from W. G. Lewis et al.
28
Figure 1-15. The bis(salicylaldimiato)zinc DNA-binding complex affinity column
experiment 30
Figure 1.16. The affinity column-UV generator loop by Nelen and Eliseev 32
Figure 2-1. Predicted ratio of the total (bound and unbound) concentrations of two
hypothetical inhibitors 43
xx
Figure 2-2. Selective concentration of the sulfonamide 4a over non-inhibitor 5 into the
carbonic anhydrase-containing compartment of a two compartment vessel
47
Figure 2-3. Selective concentration of sulfonamides 4a-d over non-inhibitor 5 into the
carbonic anhydrase-containing compartment of a two compartment vessel
48
Figure 2-4. Reaction design for the selective destruction experiments 50
Figure 2-5. Selective protection from destruction of sulfonamide 4a over non-inhibitor
5 by carbonic anhydrase 51
Figure 2-6. Selective protection from destruction of sulfonamide 4a over 4 b by
carbonic anhydrase 52
Figure 2-7. Selective protection from destruction of sulfonamide 4a over 4c by
carbonic anhydrase 53
Figure 2-8. Selective protection from destruction of sulfonamides by carbonic
anhydrase 54
xxi
Figure 2-9. The graph shows theoretical and experimental ratios for the screening
experiments 56
Figure 3-1. Schematic of a hypothetical pseudo-dynamic combinatoriallibrary 64
Figure 3-2. Solid phase, aqueous synthesis of a library of dipeptides 65
Figure 3-3. The dipeptide library can now interact with the receptor, carbonic
anhydrase 67
Figure 3-4. The destruction of unbound library members 68
Figure 3-5. The three-chambered p-DCL experimental set up 69
Figure 3-6. P-DCL oftwo cycles, 24 h each 73
Figure 3-7. Two 24 h cycles, with a delay in the addition of the destruction chamber
74
Figure 3-8. Four 12 h cycles with half the amount of dipeptide formed per cycle
75
xxii
Figure 4-1. Schematic of the pseudo-dynamic combinatoriallibrary experiment
82
Figure 4-2. Structures and competitive inhibition constants for the CA-catalyzed
hydrolysis of p-nitrophenyl actetate 84
Figure 4-3. Pseudo-dynamic library experiments 86
Figure 5-1. Quantification of CA binding sites 96
Figure 5-2. Six 16-h cycles of an eight-membered p-DCL with [nucleophiles] = 4 mM
99
Figure 5-3. Twelve 16-h cycles of an eight-membered p-DCL with [nucleophiles] = 4
mM 100
Figure 5-4. Twelve 16-h cycles of an eight-membered p-DCL with [nucleophiles] = 4
mM 101
Figure 5-5. Results of a four membered p-DCL, with two inhibitors 105
Figure 5-6. Results of a six membered p-DCL over six 16-hour cycles with three
inhibitors 106
xxiii
Figure 5.7. The expanded library members 109
Figure 5.8. Expanded library scheme 110
Figure 5.9. Uncorrected UV absorbance of the final two remaining members of a 30
memberedp-DCL 111
Figure 5-10. General scheme for library synthe sis 114
Figure 5-11. Two inhibitors lA and lB compete for a biological target T 115
Figure 5-12. The destruction of any unbound inhibitor 1 govemed by the inhibition
constant and the kinetic rate constant 118
Figure 5-13. Theoretical curve for the ratio oftwo inhibitors in apDCL 119
LIST OF SCHEMESrrABLES/MISCELLANEOUS
Box 1.1. Terms in receptor-assisted combinatorial chemistry. 6
Table 1.1. Drug discovery approaches using receptor-assisted combinatorial '
chemistry 9
xxiv
Scheme 2-1. Aryl sulfonamide-based dipeptide libraries as inhibitors of carbonic
anhydrase 39
Scheme 2-2. Destruction of inhibitors 40
Scheme 2-3. Preparation of 4'sulfonamidophenylalanine dipeptides 44
Table 2.1.
Table 3.1.
Table 3.2.
Inhibition of carbonic anhydrase by sulfonamides 1 and 2, sulfonamide
dipeptides 4a-d, and dipeptide 5 45
Distribution of products from the library described in Figure 3.1 66
Inhibition constants for the components of the first generation p-DLCs
71
Scheme 4.1. Creation of a pseudo-dynamic library of dipeptides 81
Table 5.1.
Table 5.2.
Effect on synthetic rate and yield due to changing the nucleophile
concentration 98
Effect on yield and selectivity due to changing the nucleophile,
electrophile and number of cycles in the eight-membered library 101
xxv
Table 5.3. Parameters used to generate Figure 5.6 127
Table 6.1. Amounts of (1) used in static library experiments 151
Table 6.2. Variations of the eight-membered, 16 hour library 152
xxvi
General Contribution of Authors
The research project described in this thesis was carried out as a coIlaborative
effort between the research groups of professors James L. Gleason and Romas J.
Kazlauskas of the department of chemistry of Mc Gill University. Ronghua Shu was
responsible for the initial protease screening in section 2.3.4.1. Andrew D. Corbett,
Jonathan Croteau and David Soriano deI Arno, under the supervision of professor J. L.
Gleason, developed aIl che mi cal syntheses described in this thesis. R. J. Kazlauskas
developed the theory in section 2.2. Andrew D. Corbett or David Soriano with the author
performed sorne library experiments together. The author developed and carried out aIl
other work, including library design, described in this thesis. Chapters two and four are
adapted from published manuscripts reprinted in the appendices 1 and II respectively.
xxvii
CHAPTERONE
INTRODUCTION: RECEPTOR-ASSISTED COMBINATORIAL CHEMISTRY
IN DRUG DISCOVERY
1
1.1 Combinatorial Chemistry
For millennia, human beings have used products from nature to alleviate
symptoms from, or to cure disease. Little known to our ancestors, leaves, bark, and other
natural sources were effective because of active ingredients within them. Purifying out
the active ingredients became possible as chemistry developed starting in the
Renaissance. With the onset of globalization in the late 1800s, researchers realized that
there were many natural products in use as pharmaceuticals in various parts of the world,
but attaining these compounds in levels that would meet demand was difficult or
impossible. In the late 1800s, acetylsalicylic acid (Aspirin™) was synthesized in a
chemicallaboratory, and an alternate source to the natural one (willow bark) was finally
available. This advancement began what is now known as drug discovery. Throughout
the 1900s, new synthetic methods have been developed and numerous drugs have been
synthesized.
In the 1980's, with the onset ofmacromolecular modeling and rapid advances in
numbers of known biological structures, rationally designing drugs based on careful
inspection of pharmacophores aspired to create potent pharmaceuticals. However, tailor
making the perfect drug proved far more difficult than initially anticipated. Additionally,
creating new, potent drugs one at a time was not supplying demand. If one could not then
successfully design a tight binder from scratch, could one be created by chance?
Combinatorial chemistry emerged in the early 1990's as a way to create many
compounds in a short period of time. A plethora of potential drugs are made at the same
time (called a library), providing hundreds and even thousands of new molecules in the
2
same time it had taken to make one. Potential drug leads are usually synthesized using
one of two techniques: parallel synthe sis (Figure 1.1) or split pool synthesis (Figure 1.2).
After the library is made it is screened against a biological receptor (a membrane
receptor, enzyme, DNA, RNA etc.) in a separate step. This screening usually involves a
specialized assay that needs to be designed for each new receptor.
0101.1 ... <:::> : ! 1 !
a
C;;i~i+i"'? r
<jiliti"'t Figure 1.1: Parallei synthesis. Starting materials (represented by an open circle, square, black circle and
open oval) are aIl subjected to the same set of reaction conditions in separate vessels, resulting in
modification "a". Performing subsequent reactions and purifications ( ... n) yields products that can then be
screened against a biological receptor, R, and detected using a specific assay (represented by "D"). Because
aIl reactions happened in separate vials, aIl products are separate at the end of the synthetic procedure and
identifying the ones that bind to the receptor is straightforward. Another of this method's strengths is that it
is easy to automate. Drawbacks of parallei synthesis include the difficulty in creating libraries greater than
hundreds of compounds, and the need to have a separate, specific assay to detect binding to the receptor.
3
Figure 1.2: Split-Pool combinatorial synthesis (two steps shown) and library screening. In split pool
synthesis an initial mixture of starting materials (usually on solid support) are reacted with a variety of
compounds under various conditions (represented on the far left by three reaction arrows with a circle,
triangle or square representing three hypothetical sets of conditions). This results (in this illustration) in
three vessels, one containing products from the first reaction, one containing products of the second and so
on (the first "split"). The three products are then mixed together (pooled), split into three portions each of
which are then again subjected to three different sets of reactions. Each new round of splitting and pooling
exponentially increases the number of compounds in the library. In the example shown, three initial
compounds undergo three reactions in two split/pool rounds to give 27 different products. Step "a"
represents addition of the receptor, R. Step "b" represents addition of a detection method (symbolized by
"D"). Step "c" represents deconvolution of library and identification of drug lead. Split/pool techniques can
make libraries of millions of compounds, however deconvoluting which compounds bind to the receptor
can be very difficult. This method also requires a separate, receptor-specific screening step.
This completely random approach has not, in large part, proven successful. The
libraries were simply not large enough, or did not contain high enough diversity to
generate potent receptor-binding compounds. Since increasing the size of the library
renders its screening more and more difficult, a shift back towards a more rational design
is taking place, only now using element of combinatorial synthesis.
4
1.2 Objectives, Methods and Terms in Drug Discovery Applications of Receptor-
Assisted Combinatorial Chemistry
Combinatorial chemistry methods currently favour focused libraries, which use
templates or functional groups known to bind to the desired receptor.[1,2] An emerging
method in combinatorial chemistry, receptor-assisted combinatorial chemistry (RACC),
not only uses focused libraries, but also adds stoichiometric amounts of the receptor
during the synthesis of the library. Addition of the receptor biases the synthesis toward
the best binding compounds, thereby combining the synthesis and screening into one
step. In addition, analysis avoids specific receptor assays, but detects increased amounts
of the best-binding compounds with established chemical methods such as HPLC, MS,
NMR or even X-ray crystallography.
Three main RACC methods have emerged: dynamic combinatorial libraries,
receptor-accelerated synthesis, and a new method, pseudo-dynamic combinatorial
libraries (Table 1.1, Figure 1.3). These methods respectively use thermodynamic control,
kinetic control or both to increase the relative amounts of the best binding compounds
during synthesis. This relative increase can be defined as amplification (see Box 1.1 for
definitions). The amplification should reflect the relative binding constants of the library
members and selectivity is defined so as to measure this relationship. The selectivity for a
pair of library members is their amplification divided by their respective binding
constants. Methods based on thermodynamic control are in equilibrium, so the
amplification does not normally exceed the binding constants and the maximum
selectivity is usually below one. On the other hand, methods based on kinetic control can
5
yield selectivities weIl beyond one. Selectivities beyond one are called enhancements.
Enhancements enable methods to easily distinguish library members with similar binding
constants.
amplification - ratio of the amount of a library member synthesized in the presence of
a receptor as compared to its amount in the absence of the receptor, [IA]receptor/ [IA]no
receptor. In pseudo-dynamic libraries, aIl compounds are destroyed in the absence of the
receptor, so the amplification is infinite. In these cases, it is more useful to compare
yield, which is the concentration of a compound in the binding chamber compared to
the concentration of the receptor, [IA]/[R]. The maximum yield is 100%.
casting vs. molding - casting forms a small molecule using a receptor binding site as a
template. Drug discovery seeks to cast a drug lead using the receptor. Molding forms a
receptor by surrounding a small molecule target. For example, mol ding could form a
crown ether-like macro cycle around an ion.
enhancement - selectivity beyond one; that is, the ability to amplify library members
beyond their relative binding constants. Enhancement allows RACC to distinguish
between library members with similar binding constants.
receptor - entity to which the library members should bind. In drug discovery
applications, receptors can be cell membrane receptors, enzymes, interfaces for
protein-protein interaction, sites on RNA or DNA, etc. In supramolecular receptor
building applications, receptors are the supramolecules that are evolved to bind the
guest molecule.
selectivity - in DCL and RAS selectivity is the amplification of two library members
6
lA and lB, where lA is the stronger binder, compared to their relative binding constants,
[([IA]receptor/[IA]no receptor)/([IB]receptor![IB]no receptor)][(Ka )/(Ka )rl. In p-DCL, since aIl
lA lB
compounds are destroyed in the absence of the receptor, any detectable amount of a
library member would result in an amplification ofinfinity. For p-DCL, the selectivity
is [[IA]receptor![IB]receptor] [(Ka ) /(Ka )rl, which compares the relative concentrations of
lA lB
the inhibitors to their relative binding constants.
tethering - a dynamic combinatorial method involving formation of disulfides where
one component is a cysteine residue on the receptor. Tethering focuses the binding to
the region near the cysteine.
Box 1.1: Terms in Receptor-assisted combinatorial chemistry.
Dynamic combinatorial libraries (DCLs) use reactions in equilibrium to form
mixtures of library members. The receptor binds the tightest-binding library members,
removing them from solution. The synthetic equilibrium shifts to increase the amounts of
these tightest binding library members according to LeChâtelier' s principle. As
mentioned above, the amplification does not normaIly exceed the relative binding
constants of the library members. In other words, the selectivity rarely exceeds one and
there is little or no enhancement in DCLs.
In receptor-accelerated synthesis (RAS) the receptor binds several starting
components and promotes their coupling due to proximity, forming a new, tighter-
binding species. The rate-acceleration of this coupling reaction identifies the best binding
compounds and determines the amplification. Selectivity arises from two factors: binding
of the starting components and the ability of the receptor to catalyze the reaction. The
7
binding of the starting components is presumably an equilibrium, but the binding
constants of the components may not match their contribution to binding in the final
products. If the product exhibits tighter binding than do the composite starting materials,
then this binding increases selectivity, but if the binding decreases in the final product,
then selectivity is decreased. The ability of the receptor to accelerate coupling is essential
for amplification and selectivity because there will be no amplification if the library
components also form products outside the receptor's binding pocket. Since the receptor
is not normally a catalyst for this coupling reaction, this can be a challenging
requirement. This coupling reaction should be irreversible, thus, the synthesis is
kinetically controlled. This kinetic control permits high selectivity and enhancements,
although the level of enhancement is difficult to predict.
A new receptor-assisted method, pseudo-dynamic combinatorial chemistry, uses
an irreversible library synthe sis, combined with an irreversible destruction reaction that
regenerates sorne of the starting materials (Figure 1.3). These starting materials are then
re-used in a new round of synthesis. Thermodynamically controlled binding to a receptor
protects strong binding library members from the kinetically controlled destruction
process. Amplification results from the receptor protecting bound library members from
the destruction because in the absence of receptor, the destruction reaction removes aIl
library members. Iterative synthetic cycles allow the tightest binding library members to
build up in the system, thus increasing the absolute amounts. The selectivity cornes
partially from the initial, reversible binding of the library members to the receptor, but
mainly from the kinetically controlled destruction of the weaker binders. The selectivity
can exceed one and increases as the destruction reaction proceeds.
8
The purpose of this introduction will be to give an overview of first dynamic
combinatorial libraries, and then of receptor-accelerated synthesis, with examples as to
how they have been applied to drug discovery. A detailed description of the development
of the destruction process in pseudo-dynamic combinatorial libraries (P-DCLs) will be
the focus of chapter two. The first generation of p-DCLs will be described in chapter
three. The optimization of amplification and selectivity in a p-DCL will comprise chapter
four. Finally, chapter five will deal with both the expansion of this library and theoretical
characterizations of the processes involved inp-DCL systems.
Table 1.1 Drug Discovery Approaches using Receptor-Assisted Combinatorial Chemistry
Approach Description How ~~Gbinding is How How selectivity arises (is used amplification enhancement possible)
arises Dynamic Reversible reaction creates Thermodynamic Bound Stronger binders are bound
--- combinatorial a library; binding to the approach; shift products are to relatively stronger chemistry receptor shifts the equilibrium synthesized degrees (no enhancement)
equilibrium more to balance eguilibrium
Receptor- Binding of components to Kinetic approach; Receptor Stronger binding starting accelerated receptor accelerates increase rate of accelerates materials are more synthesis synthesis ofbest inhibitors synthesis coupling of available for receptor-
starting induced coupling materials due (enhancement difficult and to their impossible to predict) proximity when bound
Pseudo- Formation and destruction Kinetic 1. Receptor 1. Receptor selects stronger dynamic of library are separate destruction protects binders combinatorial irreversible reactions; enhancesthe binders from 2. Destruction culls weak chemistry binding slows destruction thermodynamic destruction binders
reactions selectivity 2. Iterations of 3. Iterations introduce synthesis library members again, allows strong binders have chance compounds to to take the place ofweaker build up (high levels of
enhancement)
9
DCL: Thermodynamic ,- (receptor binding shifts synthetic equilibrium)
0-0 00 Library Synthesis t>--O Receptor
C> 0-<] Re<,pto--l
RAS: Kinetic (receptor binding accelerates synthesis)
... Recepto~ ~ l ........ Recep~---l~~ Receptor-",,,,,
pDCL: Thermodynamic + Kinetic (receptor b. inding slows destruction) (J ~ 0 LibrarySynthesis 9 r Receptor
~'o r-'''O~ Re<eptofh
D Figure 1.3: Three receptor-assisted combinatorial methods. In dynamic combinatorial libraries (DCLs),
because library synthesis and binding to the receptor are reversible, thermodynamic pressures govern both
amplification and selectivity. In receptor-accelerated synthesis (RAS), starting materials with strong
receptor-affinity come together in the active site and form products due to their proximity, forming a
stronger inhibitor due to kinetic pressures. In pseudo-dynamic combinatoriallibraries (P-DCLs), the term
pseudo-dynamic refers to an irreversible library synthesis, coupled with a complimentary irreversible
destruction that takes the place of the reversible library synthesis in traditional DCLs. Kinetic destruction
enhances the thermodynamic selectivity in these systems.
1.3 Receptor Assisted Systems Under Thermodynamic Control:
Dynamic Combinatorial Libraries
In the early 20th century LeChatelier showed that secondary reactions could shift
an equilibrium. U sing this princip le to discover tight binding molecules is more recent. In
the early 1990's, researchers used it to discover DNA-binding molecules, where binding
10
to the biomolecule shifted a synthetic equilibriumP1 Similarly, equilibrating starting
materials can surround a template and form a receptor.[41
1.3.1 DeLs Requiring "Lock-In" Reactions
Ivan Huc and Jean-Marie Lehn first used LeChâtelier's princip le to identify
enzyme inhibitors and identified the key requirements of a dynamic combinatoriallibrary
(DCL) in 1997.[51 The creation of a library should be reversible, occur in aqueous media,
with a detectable shift in equilibrium. Huc and Lehn chose a well-characterized,
inexpensive receptor, carbonic anhydrase, for this proof of principle study.
Arylsulfonamides are known to inhibit carbonic anhydrase by binding to its active site
zinc ion. Their DCL consisted of 12 imines that were formed from the reversible
combinations of four amines and three aldehydes in water at pH 6. One of the aldehydes
contained an aryl sulfonamide that gave sorne products receptor-affinity. To detect these
imines by HPLC without shifting the equilibrium, they used a "lock-in" reaction -
irreversible reduction of the imines with NaBH3CN to the corresponding amines (Figure
1.4).
11
A
B
R' HPLC NaCNBH3 'NH Detection
carbonic anhydrase carbonic anhydrase -imine-
A -reduction of bound imine? H R'
~NH VH~
~SO,NH, mest ampl~ied
HJl.-O.
tr-S03H
~HN-.,/'N Ph 1 II~
CO,H 0 H 0 1 A S03H
o H~CO,H
U H,N'J('N
°H~ y C02H
HN-.,/' Ph""( Il ~
CO,H 0H~ y CO,H
œ~N H 1 ~ ,,&
CO,H
H~ V--S02NH2
Figure 1.4: The first dynamic combinatoriallibrary.151 A) Imines are formed from the aqueous, reversible
combination of amines and aldehydes in aqueous media, and can subsequently bind to the receptor,
carbonic anhydrase. Reduction of the imines with NaCNBH3 to the corresponding amines is necessary to
detect the amplified library members by HPLC. B) 12 library members are created from the combination of
four amines and three aldehydes (one ofwhich contains a receptor-binding aryl sulfonamide).
The reduction step made the results somewhat ambiguous, as it was not clear whether
compounds that were amplified by the receptor, or compounds more able to undergo
reduction (possibly due to reagent accessibility because they were free in solution) were
being reduced. This "lock-in" reduction added a level of complexity undesirable in a
DCL. AU early DCLs required sorne form oflock-in reaction.
12
Although many linking reactions are possible, dynamic combinatorial libraries
most often use formation of hydrazones, imines or disulfides to create the library, with
disulfide exchange being the most common. Disulfides rapidly equilibrate at pHs greater
than 8, but are "locked in" below pH 5. In one early mode! system a library of disulfide-
linked sugar dimers was equilibrated in the presence of the plant lectin, concanavalin A,
which binds mannose-rich oligosaccharides. Equilibration of the disulfides took place in
the presence of the receptor, after which the results were locked in by lowering the pH. [6]
The receptor was immobilized, allowing any unbound compounds to be filtered off,
simplifying the subsequent HPLC analysis. Mannose-containing dimers were the only
ones detected (Figure 1.5).
OH OH
HO~", OH OH ~
HO~cJ
HO~~f6\ HO-S:-?-f6, HO~ HO~ Con A
+ OH OH 0-... ~== ... OHOH cYS
~.\ ~q 1
H~O - ~ HO~oY OH
o 0
sugar-0-o-~~s-s~~-o-o-sugar
Sugars = D-mannose (Man), D-galactose (Gal), D-glucose, L- arablnase (Ara), D-xylase (XyI)
n = 3 for Man and Gal n = 2 for Gal, Gle, Ara and Xyl
OH OH
HO~ï-'6\ HO~
Con A OH OH '"
HO~O S HO 1
J
Figure 1.5: An example of a DeL from O. Ramstrëim and J.-M. Lehn.[6] Top: Disaccharides are attached to
sulfides using various linker lengths, and are equilibrated in the presence of plant lectin concanavalin A.
Bottom: The structure of the linker.
More recent applications of DCLs have found pharmaceutically promlsmg
inhibitors. In a recent example from Therascope Pharmaceuticals (now Alantos
13
Pharmaceuticals), an imine library was created from the condensation of a diamine and
over 50 ketones (Figure 1.6)pl This library was synthesized in the presence and absence
of neuraminidase, a key enzyme in the propagation of the influenza virus. After reduction
of the imines, Le/MS analysis identified several hits. The researchers performed two
control experiments to ensure that amplification of library members was due only to
binding to the neuramidase active site. In one of these controls, the library was
synthesized in the presence of bovine serum albumin (BSA). If amplification of an
apparent hit took place, it was not due to binding to neuraminidase. In another control
experiment, the library was synthesized in the presence of neuraminidase and a known,
potent inhibitor, zanamavir. If amplification of a hit happened in this case, it was not due
to active site binding, and was therefore deemed to be a false positive. One of the hits
(bottom right of Figure 1.6) was eliminated as a possibility by control experiments
because it was amplified in the presence of BSA, and in the presence of neuramidase and
zanamavir.
The DeL experiment gave several hits, however, the relative amplifications were
puzzling. The compound that was most prevalent in the Le/MS trace, with a high level of
amplification, was not a potent inhibitor at aIl. The strongest inhibitor was amplified
approximately three-fold less (Figure 1.6). Explanations ofhow these results are possible
in sorne DeLs will come in section 1.3.3.
14
HO 0
NaCNBH3 9 lC/MS Detection ..
HN" "NH2
~NHAC HO)()
HO 0
,,,.X,,'" IIY 2 R, ......... R2 NHAc
Neuramidase
H20, pH =6 ,~~~" IIY 2 R, ......... R
2 NHAc
Unknown Amplifications
HO 0
"~,Q,~~ ~NHAC
largest amounts detected by lCIMS Amplification Factor> 30 Amplification Factor> 90 (highest) Amplification Factor = 84 KI = 85 nM (strongest binder) ~ = 92 nM KI = 700 nM
HO 0
"",9"" ,-A NHAc HO-1 -
Amplified in DCl but Eliminated by Control Experiments
Figure 1.6: Creation of an imine library against neuraminidase using a diamine and several ketones. (7) The
library gave several hits, identifying potential drug leads against this enzyme. However, the most prevalent,
and the most amplified species were not the strongest binders.
1.3.2 DeL Systems with in situ Detection
Although detecting bound library members us mg HPLC reqU1res a lock-in
reaction, other methods can detect library members in situ. In a recent example,[8] Kubota
et al. used difference NMR to detect amplified members of a receptor library formed
from (en)Pd(N03)2 and several pyridyl-appended bridging ligands around a small guest
mole cule (e.g. CBr4). Although not a drug discovery application, modifications of this
technique might detect drug-like molecules bound to a receptor.
Turner,[9] in the first example of dynamic library synthesis using an enzyme, made
a library of aldols using an aldolase. N-acetylneuraminic acid aldolase (NANA aldolase)
c1eaves sialic acid to N-acetylmannosamine and pyruvate (Figure 1.7). By adding excess
pyruvate, the synthesis equilibrium was pushed towards formation of aldol products.
Amplification of sialic acid resulted when a small (three-membered) library was
15
synthesized and incubated for 160 h in the presence of the plant lectin, wheat germ
agglutinin (WGA). The researchers were able to take aliquots of the reaction mixtures at
various time points, denature both the receptor and the aldolase (which rendered the
library products stable as the aldolase could no longer catalyze the reverse reaction) and
immediate1y analyze them by HPLC.
o
R2 R, ~oNa ~OHOH OH OHOH OH HO~OH 0 3 R ONa WGA Ho .......... ·) ... J~~--ol--ONa
HO 2 2 R 0 ..-===="'" ~ HN=tfV 11 NANA aldolase ' HO 1 0 Aé HO 0
a) R, = NHAc, R2 = CH20H a) R, = NHAc, R2 = CH20H Sialic acid: mes! ampified b) R, = OH, R2 = CH20H b) R, = OH, R2 = CH20H c) R, = OH, R2 = H c) R, = OH, R2= H
Figure 1.7: A DeL made from NANA aldolase catalyzed aldol formation in the presence ofwheat germ
agglutinin (WGA).[9] Amplification of the mM inhibitor, sialic acid (la) occurs at the expense of non
inhibitors 1 band 1 c when W GA is present during library synthesis.
Congreve et al. [10] detected an oxindole bound to cyclin-dependent kinase-2
(CDK-2) by X-ray crystallography (Figure 1.8). The oxindole library formed from
hydrazines and isatins in DMS0I20% H20 in the presence of prote in crystals. Crystal
structures identified which oxindole bound to the protein. An inhibitor with an IC50 of 30
nM was detected directly by observation of its electron density, and a non-inhibitor was
not detected. Although a seemingly amazing result, this research is plagued by several
problems. First, it is not clear how the receptor, present in very low amounts compared to
the amount of products formed, influences the synthesis. The researchers state that a
small amount of products could be diffusing into the crystals, where they undergo
another, separate dynamic equilibrium, however this is speculative. In addition, the
results may be skewed towards detection of compounds that more easily diffuse into the
16
crystals. This seems likely as severallibrary members that were equally as potent as the
one detected were not se en in electron density maps. Finally, the general applicability of
this method is highly questionable as the CDK-2 crystals used in this study are unusually
robust. Most macromolecular crystals would have little chance of standing up to the harsh
conditions required for library synthe sis (80% DMSO in this case).
Direct Detection by X-Ray
Figure 1.8: An oxindole library created in the presence of cyclin-dependent kinase-2 (CDK-2) crystals.1101
Detection of bound library members does not require a lock-in reaction as X-ray crystal structures of the
receptor-inhibitor crystal complex can be analyzed directly. This method, however, may not be widely
applicable as many macromolecular crystals are too fragile to be present during a library synthesis.
A recent modification of thermodynamic receptor-assistance is "tethering" whose
key advantages are first, to focus binding to a particular region on a receptor, and second,
to detect the results of the experiment in situ by running a mass spectrograph of the
equilibrated receptor-inhibitor complex. In this method the thiol of a cysteine residue
(either existing near the site of interest or added by protein engineering) reacted with a
library of disulfides. These disulfides can undergo exchange with the cysteine thiol to
form a receptor-disulfide. The most stable receptor-disulfides were formed from original
disulfides containing fragments that bound the receptor the most tightly. MS analysis of
the receptor-disulfides identified the bound species. [11] By screening 7000 disulfides in
batches of 5-20 compounds, tethering identified a potent inhibitor of interleukin 2, a
17
target in immune-disorder therapy (Figure 1.9).[12] The screening required small batches
to distinguish between species with identical molecular weights, and with similar binding
constants.
1. Introduce Cys Mutations Near Active Site M 10 Mutations. Y31C & l72C Found to be Closest to Binding Site
~-~ 2. DCl Using Disulfide Exchange between Cys-SH and 7000 Disulfides (Tethering)
+ ' ~SH ~
Il-2 l. Disulfide Exchange
S_S-R s-S
~ Binding Stabilizes@'1 Disulfide Bond R
Il-2 Il-2
3. Improve Design of a Known Inhibitorwith Tethering "Hif'
o ~~NH2 H NH N :
N~H i\ o
(Y"o
o ~~NH2 H NH N •
N rH k Fur1her "'1 improvements
IC50 = 3 ~M A('A CI
B 0.2 ~M < IC50 < 3.0 ~M IC50 = 0.06 ~M
Figure 1.9: An example of "tethering" using DCL princip les from Sunesis pharmaceuticalsY2) 1. Crystal
structure-guided mutations introduced a cysteine near the binding site of IL-2. 2. Each mutant is
individually screened against 7000 disulfide-containing fragments (in batches of 5-20) using dynamic
combinatorial interactions. MS identified most stable disulfides. 3. A known inhibitor of IL-2 is improved
50-fold by modifying it with the tethering "hit".
Recent expansions of the tethering approach include extended tethering, which
has been used to find a sub-micromolar inhibitor of cysteine aspartyl protease-3 (caspase-
3). Caspase-3 is a receptor involved in apoptosis and a target in cancer therapy.[13] In
extended tethering an "extender" molecule is used to irreversibly alkylate a cysteine
residue near the enzyme's active site. This extender contains a free sulfhydryl that can
then undergo dynamic interchange with a library of disulfides in a tethering process. The
18
advantage of extended tethering cornes from the variety of extender molecules that can be
added to the receptor. Depending on their size and orientation, these extenders can direct
disulfide exchange towards different binding pockets of a receptor.
Caspase-3 is a protease, and normally binds peptides. It has a series of shallow
binding pockets (Sl-S4) in which individual amino acids normally bind. The extender
molecules used in this study included an aspartyl moiety that allows binding to the SI
pocket. A cysteine residue, known by X-ray structure data to be near this pocket, was
modified with two extenders of different lengths (Figure 1.10). Extender A was shorter,
and was expected to direct binding to the S4 pocket, while extender B would direct
binding to the more distal S2 pocket. Separate dynamic interchange experiments between
caspase-3 with either extender A or Band 7000 disulfides (in batches of between 8-20)
revealed two independent hits by MS, one for each extender. The two compounds, made
from either the extender A disulfide, or extender B disulfide were synthesized in
bioactive form (replacement of the labile disulfide with either methylenes or a sulfone).
They proved to be sub-micromolar inhibitors of caspase-3, and X-ray diffraction showed
that they bound to different pockets of the receptor.
19
1. Modify Receptor (with Native Active Site Cys) with 2 Different "Extender" Molecules
54 Caspase-3B
N CI 0 0
1. ~0~~Y'5.Â.. CI 0 HO C) 8
2 (Extender A)
ÇÇCI 0 ~ 0
OR1. 1 A O~~,~.Â.. I~\ s
CI 0 00 H02C (Extender B)
2. NH30H
o H )lyNY'5H
s~\ on () _ C02H, V <-JI 51 54 52
Caspase-3A
o HO~ )lyN,g ~ ~
h\"O"n -A _ \ ~ V <-JI
51 54 52 Caspase-3B
2. DCl Using Disulfide Exchange between Extender A or B, and 7000 Disulfides (Extended Tethering)
4~'Ç;" n _ 1 \ ~ ~ ~ + ,
51 54 52 ~, Caspase-3A
o HO~ )lyN'g ~ /,
s~ \ éi 5H C02H
51 54 Caspase-3B
R 1 5 + ,
~,
Disulfide ExchangEl.
Disulfide ExchangEl.
3. Replace Bio-Inactive Functionality of Extended Tethering Hits
Figure 1.10: Extended tethering with caspase-3, a protease with several binding pockets (SI_S4).[13] 1.
Irreversible alkylation of a Cys residue in the active site of the receptor with either extender molecule A, or
the longer, B. 2. DCL with 7000 disulfides in sm ail batches with either extender A-modified caspase-3
(Caspase-3A) or Caspase-3B gives independent hits for each extender. 3. Synthesis ofbioactive versions of
the hit molecules.
20
1.3.3 Limitations in DeLs
CUITent research is also addressing sorne of the selectivity and amplification
issues associated with systems under thermodynamic control. The level of selectivity
possible can be a problem in traditional DCLs, as aU compounds made are present to
sorne degree in the equilibrated system. Several compromises have been made to
overcome this problem. As previously described, tethering screens compounds in small
batches and then combines the hits from each batch in a new, enriched experiment. In
another example the selectivity problem was overcome by creating sub-libraries in which
each library left out a particular starting compound. Starting materials whose absence
resulted in no significant amplification of any library members were identified as being
crucial for binding, thus "deconvoluting" the resultsY41 However, large libraries remain
difficult to screen because the level of selectivity possible for even the strongest binding
library member over the next strongest cannot be higher than the ratio of their binding
constants, [15, 161limiting the enhancement level of a DCL to one (see Box 1.1).
The analytical problems caused by this lack of enhancement become more
apparent in large libraries. Although the selectivity between two compounds will remain
the same regardless of the library size, the absolute differences in concentration will
become smaller and smaUer as the library size increases. This aspect of DCLs is
illustrated in the following explanation and derivation.
Dynamic combinatoriallibrary experiments contain two equilibria: an equilibrium
for the synthesis of inhibitors and an equilibrium for binding of the inhibitors to the
receptor (Figure 1.11). The binding equilibrium removes the good inhibitors from the
21
synthe sis equilibrium and, to re-establish equilibrium, the synthesis produces more of the
good inhibitors than it would in the absence of receptor. The following derivation shows
that the ratio of good inhibitor to a poor inhibitor depends linearly on the ratio of the
binding constants.
~D comma" atartlng
materlal.
synthesls
new Inhlbltor. targat - Inhlbltor complexee
Figure 1.11. The dynamic combinatorial library equilibria yield a higher total amount of a good inhibitor.
Binding of the inhibitor to a target removes it from the synthesis equilibrium so the synthesis produces
more of the good inhibitor. However, there are limits to the level ofselectivity a DeL can exhibit.
Given a typical DeL in which library members lA - IN are synthesized under
equilibrium conditions, and then can reversibly interact with a receptor (biological
receptor or target T):
K K SM] SIA
lA a lA IA-T A
SM] K K
SI.
lB a 18 -. IB-T •
SM] K K
SIe
lc ale Ic-T c ..
K K SM] SIN. IN a IN .. I~T
N
and where equations 1-3 define respectively SM]x the starting material used for
synthesizing any inhibitor, Ix, KS]x the equilibrium constant for synthe sis of inhibitor, Ix,
KaJ the equilibrium constant for binding ofinhibitor, Ixto the target, and [Ix ], the total x T
of bound and unbound forms of lx:
22
Equations 1-3 define the concentrations of a particular inhibitor lA :
K = [lA] slA [ SM
IA]
K = [TelA ]
alA [T][IA] (1,2,3)
[IAJ=[IA]+[Te lA]
Then combining equations 1-3 and solving for [lAT] gives equation 4.
(4)
The fraction of [lA] relative to the rest of the components in a library of N members is: T
[lAT] KS/JSM/J{KaIJT] + 1) (5)
[IBT
] + [le T] + ... + [IN T] KsI. [SMI• ](KaIJT] + 1)+ KslJ SMIc ](KadT] + 1)+ ... + KslJ SM/J(KadT] + 1)
Under conditions oftight binding of aH components and an excess oftarget, Kalx [T]» 1
for aH components, equation 5 will simplify to:
The highest selectivity due to receptor binding in DeLs arises when the synthetic
equilibria lie far to the reactant side, and the synthesis equilibria are equal for aH library
members. Under these ideal conditions, the concentration of starting materials is
approximately the same for aHlibrary members and equation 5 simplifies further to:
[lAT] _ K alA
[IBT
] + [leT] + .... + [INT
] - K alB + K alc + .... + KaIN
(7)
Equation 7 shows that the relative concentration of aH lA in the system relative to
aIl other library members at equilibrium is proportional to the ratio of its binding constant
23
to the sum of the binding constants of aH other library members. This would mean that in
a library of 1000 compounds (a practical size for drug discovery), if lA bound ten times
more strongly than aIl other compounds (differences in binding are often much smaller
than this) then lA would comprise only ~ 1 % of the total amount of library members
present. This situation would be aIl but impossible to analyze by conventional methods. It
should be noted that equation 7 depicts an ideal case where either the same starting
material forms aIllibrary members (as in the case of geometrical isomerization), or a case
in which the change in concentration of starting materials is negligible with respect to the
concentration of products. More complex cases are currently being modelled.
Amplification of weak or non-binders can occur in certain types of libraries. In
cases where several different starting materials combine to form potential binders,
products that represent a statistical mixture of these starting materials can be amplified
independently of their binding affinity. For example, it has been shown that in a
theoretical system in which equal amounts of components A, B and C couple to make
trimeric library members (A3, A2B, ABC, AB2 etc), and only component A imparts
receptor-affinity to any member forme d, then a library member with an ABC
composition is amplified 500 fold over one composed of three A units (A3), the
theoretically strongest binder (Figure 1.12).[17] This is a highly undesirable result, as the
amplified compounds should only represent tight binding. [18]
24
~.
A A A R Rillillili ,X. pt-À pt~B .. < < X-X
C B A B / C / 'c A- A-
Theoretical Perturbation: Incorporation of component A gives 300-fold stability (receptor-affinity)
Relative Stability
o Library ~ C omposition
A K-À
900
0.1
B B ' 'c / 'c B- A-
1 300
1.6 45.7
Figure 1.12: Theoretical effect of a DCL in which the combination of three different starting materials (A,
Band C) yields a library oftrimers.[17] If component A (and only A) contained a receptor-binding moiety,
and hence imparted stability to any trimeric product, the amplification of the most stable library member is
actually decreased dramatically. Instead, a product that has only moderate affinity, but represents a
statistical mixture of starting materials is the most amplified compound. This undesirable amplification is
due to entropic effects that favour the formation ofthe ABC trimer over the A3 trimer.
1.4 Receptor Assisted Systems That Add Kinetic Control
Receptor-assisted systems under purely thermodynamic control have not yet been
shown to be able to achieve selectivities above one. Systems that use kinetic factors to
induce the formation of tight-binding compounds can overcome this limitation. Two
methods will be described: receptor-accelerated synthesis and affinity chromatography.
In receptor-accelerated synthesis, a rate of product formation is the key to amplification
and selectivity. Although not yet applied to receptor-assisted combinatorial systems,
affinity chromatography uses the rate of elution of compounds to provide a kinetic-based
resolution.
25
1.4.1 Receptor-Accelerated Synthesis
In receptor-accelerated synthesis (RAS), a library of starting materials
competitively binds to a receptor, and undergoes an irreversible cross-coupling to form a
tight binding ligand. The binding of the starting materials to the receptor brings them
close to one another, speeding up a reaction that would not have occurred in solution.
Thus, only precursors that bind the receptor tightly make products, and the amplification
and selectivity of product formation cornes from both receptor-affinity and the ability of
the receptor to accelerate the coupling.
Early, moderately successful, examples of RAS used either an enzyme's active
site as a template in which ligands were synthesized, or used small molecules as
templates to couple larger molecules. An early example coupled alkyl chlorides to 0.
mercaptotosylamide in the presence of carbonic anhydrase. [19] o.-Mercaptotosylamide has
a sulfonamide group that binds to the active site zinc. Comparisons of HPLC traces with
and without the addition of carbonic anhydrase showed a two-fold increase in selectivity
for binders with a nine-fold difference in binding constants. Thus, in this case the
selectivity was less than the difference in binding constants (enhancement of less than
one).
In a second example, Nicolaou and co-workers dimerized vancomycin analogues
by either disulfide formation or olefin metathesis in the presence of a fragment of the
vancomycin receptor (Figure 1.13). In parallel experiments, one dimer formed two times
faster than a control compound with no receptor affinity.[20] Further parallel experiments
26
showed dimerization rate accelerations directly related to receptor affinity. Had the same
results been obtained in a one-pot system, they would have achieved enhancement levels
approaching one.
1: X=SAc n = 2, 3, 4, 7, 8 R= D-LeuNMe
HO~ ~OH ~N~otb
n:o" :_-~IO:I O~~~ OH 3: N OH 0 H
X = HC=CH2 H N ~N N,R NH H ~ H
n = 1, 2, 3, 7 ~ 0 0 R = H, D-LeuNMe HO 1 0 ~-Ala, L-Asn, y-Abu ~ ...., AH NH2 L-Arg, L-Phe - 1
HO OH
Figure 1.13: Nicolaou's vancomycin monomer scaffold.!20] Dimerizations of various analogues of the
thioacetate 1 using NaOH, H20 and air were faster in the presence of a binding motif of the vancomycin
receptor. Dimerizations of analogues of3 by olefin metathesis were less successful.
Mosbach's group enhanced the rate of a nucleophillic aromatic substitution using
an enzyme active site as a template. The receptor kallikrein induced selection of a new
polyheteroaromatic inhibitor from a five-membered library. This system produced
enhancements approaching 1.5, indicating that the relative amount of a binder to its
closest competing compound was 1.5 times higher than their relative binding constants.
This is higher than the thermodynamically govemed maximum of one possible in a DCL.
Further investigations showed that a molecularly imprinted polymer could increase the
coupling rate using the same starting materials and could provide nearly as high
amplification and selectivity as the natural enzyme. [21]
The most successful example of RAS yielded a femtomolar inhibitor of
acetylcholine esterase (AChE) by optimizing the linker length and orientation between a
27
micromolar and a nanomolar inhibitor of AChE. Two poylaromatic AchE inhibitors
attached to linkers of varying lengths with terminal azides or acetylenes coupled to form
64 new potentially stronger inhibitors. Only linkers of the correct length and in the
correct orientation brought the reactive functionalities of two bound starting materials
close enough for a [3+2] dipolar cycloaddition to occur (Figure 1.14).[22]
"HGI (YNyj ~
HN '(CH2)riN3 AChE
TZ2-6 (n = 2-6)
Figure 1.14: An example of receptor-accelerated synthesis from W. G. Lewis et al.122] Eight azides and
eight acetylenes were combined in the presence of acetylcholine esterase (AChE). Optimizing the linker
length and geometry between a micromolar and a nanomolar inhibitor allowed a [3 + 2] dipolar
cycloaddition to form a femtomolar inhibitor. This was possible because both starting materials could bind
to adjacent sites on the receptor.
1.4.2 Limitations in RAS
The two challenges of RAS are binding the starting materials to the receptor and
significantly speeding up their coupling. For amplification to occur, the receptor must
tightly bind the two starting materials simultaneously. This requirement could eliminate
cases where two weakly binding species link to form a strong binding one. Similarly,
optimization of substituents around a tightly binding mole cule many be difficult with
RAS if the substituent fragments do not bind weIl. RAS is probably best suited to
28
optimize linkers between two molecules that bind weIl at adjacent sites. The need to
significantly speed up product formation is also challenging because the receptor is not
necessarily a catalyst for the desired reaction simply by holding the two reactants close to
one another. To detect this rate acceleration and to minimize the spontaneous reaction in
solution, researchers have not carried out RAS with aIl library components present
simultaneously. Rather, they tested each combination of starting materials in a series of
parallel experiments.
1.4.3 Affinity Chromatography
For decades researchers have used affinity chromatography to isolate tight
binding compounds from a static mixture. Recent advances in affinity chromatography
such as affinity capillary electrophoresis[23] and frontal affinity chromatography coupled
with mass spectrometry (F ACMSi24] continue to extend the usefulness of these methods.
Applying affinity chromatography to a dynamic mixture of compounds requires either a
lock-in reaction, or sorne other form of deconvolution to permit identification of the tight
binding compounds. However, using the elution of the mixture as a kinetic step in
removing unbound species can lead to higher levels of selectivity than is possible in
DCLs.
In an early example of using affinity chromatography to select a tight binder from
an equilibrating mixture, Miller and co-workers combined eight salicylaldimines with
zinc dichloride to form a library of 36 bis(salicylaldiminato )zinc complexes on an affinity
column of cellulose resin on which poly (dA-T) DNA was immobilized (Figure 1.15).[25]
29
After equilibration, the mixture was eluted from the column, and was expected to be rich
in non-binding complexes. The complexes were then hydrolyzed with TF A to the
salicylaldimine "monomers" which were then derivitized with 2-naphthoyl chloride (for
UV detection). Subsequent HPLC analysis of the eluate was expected to show the
monomers that did not lead to DNA-binding Zn complexes. Subsequent control
experiments and analysis revealed that one of the most tightly retained monomers formed
a complex that exhibited an inhibition constant of 1 /-lM. Other monomers that were not
detected by HPLC were eliminated as possible hits by the control experiments, which
showed that they either bound to the column as monomers, rather than as complexes, or
that they bound to cellulose rather than to DNA.
Incubation on Affinity Column
1. Elution of Unbound Complexes 2. Lyophilization
Jrtzn~;'N~ (J-(/' ·"o-<=>
1. TFA (hydrolysis of Zn complex) 2. 2-naphlhoyl chloride (derivitizaton for UV deleclion) 3. Separation/Delection (HPLC)
~N·R ~OH
Monomers that do not form DNA-Binding Complexes
R = (CH2l:!CH20H
(CH2)2CH20Me
H2C~ (R&S) N
H2C
UO 1
(R&S)
H2C~ H2C~F
~1?: v.-.o··~:Zn'r:o:Q.
Sirongest DNA-Binding Complex (Kt = 11JM)
Figure 1.15: The bis(salicylaldiminato)zinc DNA-binding complex affinity column experiment.[25] 36
complexes reversibly form on a column ofpoly (dA-T). Elution of the mixture should be rich in complexes
that do not bind to the column. Detection of the salicylaldimine monomers ofthese complexes shows which
monomers do not lead to productive complexes, and allows for identification of strong binding ones.
30
Eliseev and Nelen used iterative affinity chromatography in conjunction with UV
induced isomerization of dienoic acids to enrich the arginine-binding compounds in a
mixture. The cis and trans alkene isomers of dienoic acids were passed through a column
where they could bind to immobilized arginine (Figure 1.16).[26] The eluted dienoic acids,
enriched in the less-tightly binding trans isomers, were photoisomerized and passed
through the column again. After thirty cycles of Arg binding and UV -induced
isomerization of eluted library members, the amount of the tightest binding, cis, cis
compound was 50% greater than after one cycle. The enhancement could be as high as
~2.7 for the tightest binding, cis,cis compound. This enhancement is higher than that
possible in a thermodynamic system because this experiment included a kinetic
component - the elution of more weakly bound compounds, and used iterative cycles to
increase the enhancement achieved through one binding/elution/isomerization cycle.
Iterative chromatography systems require immobilization of a receptor onto a column,
and a process that can randomize the compounds in the column eluate. This method has
not yet been used in a drug discovery application, but needs only the above two
conditions to be fulfilled before it can be.
31
0- -0 cis.cis: Strongest Binder
Kinetic flow rate
Figure 1.16: The affinity column-UV generator loop by Nelen and Eliseev.[26) A mixture of isomers is
passed through the arginine column. UV-induced isomerization to their most thermodynamically stable
state occurs only to compounds that do not bind. Re-introduction of the mixture to the column adds more
tight binders. Combining the thermodynamic binding with the rate of introduction of the new library
compounds (kinetic) results in amplification and high selectivity for the strongest arginine binder.
References
1. M. A. Rouhi, Chem. Eng. News, 2003, 81 (41), 77-78, 82-83, 86, 88-91.
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Lehn, Angew. Chem. Int. Ed. Engl. 2003,240-243; s) V. Patroniak, P. N. W.
Baxter, J.-M. Lehn, M. Kubicki, M. Nissinen, K. Rissanen, Eur. J Inorg. Chem.
2003,22,4001-4009; t) W. G. Skene, E. Couzigne, J.-M. Lehn, Chem. Eur. J
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6. O. Ramstrom, l-M. Lehn, ChemBioChem, 2000, 1,41-48.
7. M. Hochgürtel, R. Biesinger, H. Kroth, D. Piecha, M. W. Hofmann, S. Krause, O.
Schaaf, C. Nicolau, A. V. Eliseev, J Med. Chem. 2003,46, 356-358.
8. Y. Kubota, S. Sakamoto, K. Yamaguchi, M. Fujita, Proc. Nat. Acad. Sci. USA,
2002, 99, 4854-4856.
9. R. J. Lins, S. L. Flitsch, N. J. Turner, E. Irving, S. A. Brown, Angew. Chem. Int.
Ed. Engl. 2002,41,3405-3407.
33
10. M. S. Congreve, D. J. Davis, L. Devine, C. Granata, M. O'Reilly, P. G. Wyatt, H.
Jhoti, Angew. Chem. Int. E.d Engl. 2003,42, 4479-4482; Angew. Chem.2003,
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Il. D. A. Erlanson, A C. Braisted, D. R. Raphael, M. Randal, R M. Stroud, E. M.
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O'Brien, Nature Biotechno/. 2003,21,308-314.
14. T. Bunyapaiboonsri, O. Rarnstrorn, S. Lohrnan, J.-M. Lehn, L. Peng, M.
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18. For other reviews, see: a) A. Ganesan Angew. Chem. Int. Ed Engl. 1998,37,
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Chimique 2000,51-54; c) C. Karan, B.L. Miller Drug Discov. Today 2000,2,67-
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e) J.-M. Lehn, AV. Eliseev Science 2001,291,2331-2332; f) O.Rarnstrorn, J.-M.
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Sanders Curr. Op. Chem. Biol., 2002, 6, 321-327. h) O. Rarnstrorn, T.
34
Bunyapaiboonsri, S. Lohman, J.-M. Lehn Biochim. Biophys. Acta 2002, 1572
178-186.
19. R. Nguyen, I. Huc, Angew. Chem. Int. Ed. Engl. 2001,40, 1774-1776; Angew.
Chem. 2001,113,1824-1826.
20. K. C. Nicolaou, R. Hughes, S. Y. Cho, N. Winssinger, C. Smethurst, H.
Labischinski, R. Endermann, Angew. Chem. Int. Ed. Engl. 2000,39, 3823-3828;
Angew. Chem. 2000,112,3981-3986
21. Y. Yu, L. Ye, K. Haupt, K. Mosbach, Angew. Chem. Int. Ed. Engl. 2002,41,
4459-4463; Angew. Chem. 2002, 114,4639-4643.
22. W. G. Lewis, L. G. Green, F. Grynszpan, Z. Radic, P. R. Carlier, P. Taylor, M. G.
Finn, K. B. Sharpless, Angew. Chem. Int. Ed. Engl. 2002,41, 1053-1057; Angew.
Chem. 2002,114, 1095-1099.
23. Y.-H. Chu, L. Z. Avila, J. M. Gao, G. M. Whitesides, Ace. Chem. Res. 1995,28,
461-468.
24. D. C. Schriemer, D. R. Bundle, L. Li, O. Hindgaul, Angew. Chem. Int. Ed. Engl.
1998,37,3383-3387; Angew. Chem. 1998,110,3625-3628.
25. B. Klekota, M. H. Hammond, B. L. Miller Tet. Lett. 1997,38,8639-8642.
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35
CHAPTERTWO
AMPLIFICATION OF SCREENING SENSITIVITY THROUGH SELECTIVE
DESTRUCTION: THEORY AND SCREENING OF A LIBRARY OF CARDONIC
ANHYDRASE INHIBITORS
36
Abstract
Application of RACC to larger libraries is chaUenging because of the need to
accurately measure the amount of each inhibitor. In this chapter, we dramatically simplify
this analysis by adding a reaction that destroys the unbound inhibitors. This works similar
to a kinetic resolution, with the best inhibitor being the last one remaining. We
demonstrate this method for a static library of several sulfonamide inhibitors of carbonic
anhydrase. Four sulfonamide containing dipeptides, 4a-d, were prepared and their
inhibition constants measured. These inhibitors migrated to the carbonic anhydrase
compartment of a two-compartment vessel. Although higher concentrations of the better
inhibitors were observed in the carbonic anhydrase compartment, the concentration
differences were smaU (1.83 : 1.71 : 1.54 : 1.46 : 1 for 4a : 4b : 4c : 4d : 5, where 5 is a
non-inhibiting dipeptide EtOC-Phe-Phe). Addition of a protease rapidly cleaved the
weaker inhibitors (4d and 5). Intermediate inhibitor 4c was cleaved at a slower rate and
at the end of the reaction, only 4a and 4b remained. In a separate experiment, the ratio of
4a to 4b was found to increase over time to a final ratio of nearly 4: 1. This is greater than
the ratio of their inhibition constants (approx. 2:1). The theoretical model predicts that
these ratios would increase even further as the destruction proceeds. This removal of
poorer inhibitors simplifies identification of the best inhibitor in a complex mixture.
2.1 Introduction
Synthesis using combinatorial chemistry (CC) aUows testing of hundreds of
thousands of drug candidates using high throughput screening (HTS) techniques.
37
Although this rapid pace has changed drug development, the search for faster and more
efficient testing methods continues due to the lack of drugs found using CC coupled with
HTS,. One promising method is in situ screening of mixtures such as in dynamic
combinatoriallibraries. [1] Dynamic combinatorial libraries are equilibrating mixtures of
organic molecules. Equilibration in the presence of a therapeutic target increases the
equilibrium amounts of those library members that bind tightly to that target. The
difference in library composition with and without a stoichiometric amount of target
identifies the best inhibitors.
To render dynamic combinatorial chemistry practical in drug discovery, methods
must be developed to screen dynamic combinatoriallibraries with thousands of members.
This screening is complicated because it is often difficult to measure the concentration of
each library member in the absence and presence of a target. Further, the libraries will
likely contain not one, but many good inhibitors because many library members have
similar structures and thus similar binding constants. In these cases, adding the target
increases the concentration of many library members, rather than a single member, and
makes analysis very difficult or impossible. Eliseev and Nelen[2] estimated that a dynamic
library combined with an affinity column containing the target would yield one major
compound (>50%) only if KstronglKweak was at least n, where n is the number of
members of the library. Thus, for one member to predominate in a library of 1000
members, that member must would have to bind > 1000 times stronger than the others, an
unlikely possibility. This inability to distinguish between inhibitors of similar binding
constants is a major limitation of the current dynamic combinatoriallibraries.
38
This chapter describes a screening method that enhances the ability to detect the
best inhibitor in a mixture of similar inhibitors. The key to the method is an irreversible
destruction reaction that destroys the unbound and weakly bound inhibitors, similar to a
kinetic resolution. The best inhibitor is the one in greatest concentration after a certain
period of time has been allowed for destruction of more weakly bound library members.
The necessary period of time would presumably be determined by the binding strengths
of the inhibitors. We demonstrate that this method works for a static library and discuss
its potential application to a dynamic system.
Our library targets carbonic anhydrase and consists of dipeptides with an N-
terminal 4'-sulfonamidophenylalanine (1, Phesa)Yl These dipeptides can either bind to
carbonic anhydrase or be cleaved by a protease (Scheme 2.1). This cleavage increases the
ratio of the strongest binder relative to weaker binders. Importantly, the ratio may
increase to values significantly greater than the ratio of the binding constants, thus
overcoming the limitation identified by Eliseev and N el en and making it easy to identify
the best inhibitor in the mixture.
~ 0 83
Ji ..;...... selective
R{' N C02H pressure H ~
1-';::'
R ,Q
, , , , , , ,
!lœE) : carbonic i anhydrase , 1 l hydrolysis of
poorer inhibitors
membrane
ffJ O
H 83
2 + H2NÀ
C02H
1'<:::
R1
,Q
1 R1 = S02NH2 R2 = H
Scheme 2.1: Aryl Sulfonamide-Based Dipeptide Libraries as Inhibitors of Carbonic Anhydrase. Strong
binding inhibitors will be bound to carbonic anhydrase and protected. Weaker inhibitors will be hydrolyzed
by a protease.
39
2.2 Theory: Finding the Best Inhibitor by Destruction of Poorer Inhibitors
Section 1.2.3 (Limitations in DCLs) outlined the theoreticallimits in selectivity of
a traditional DCL. One way to enhance the concentration differences between inhibitors
with similar binding constants is to add an irreversible reaction that removes the
unbound, poorer inhibitor (Scheme 2.2). This situation is similar to a kinetic resolution of
enantiomers. As the destruction reaction winnows away the poorer inhibitors, the relative
concentration of the best inhibitor increases exponentially. The analysis below is similar
to that for kinetic resolutions. [4]
tergal· Inhlbltor complexes
dlSloclatlon D+O destruction DO .. [7 <l
Inhlbltora
Scheme 2.2: Destruction of inhibitors. The free concentration of the poorer inhibitor is higher, thus it is
destroyed more readily. This destruction reaction exponentially increases the relative concentration of the
good inhibitor similar to a kinetic resolution.
Consider two inhibitors, lA and lB, that compete for a target, T, and are also
destroyed by an irreversible reaction to yield P and Q with rates of k dl and k dl A B
40
Kdl k -""--- T-/A --!.a.... T / dIA P (1) +A~
K k T / dlB T 1 dIB Q (2)
-B -- +B-
The rate of disappearance of inhibitor lA is
dr/A] = -k [I 1 (3) dt dIA A
if [lA] is the total concentration ofbound and unbound forms ofinhibitor lA, it can be T
shown that
(4)
Upon solving for lA and substituting into equation 3, the rate of disappearance of lA is
given by
d[IAl kdlA Kdl)IA) --=-
KdI + [Tl A
dt (5)
Under our experimental conditions the concentration of the free target, T, will be much
larger than the dissociation constant, K dlA typical of our library members, so [ 11 » K dIA ,
therefore equation 5 simplifies to
dt [Tl (6)
A similar equation is obtained for inhibitor lB. The ratio oftheir partial reaction rates is
(7)
This equation shows that the relative rate of disappearance of the two inhibitors depends
linearly on their total concentration, their relative binding ability and their relative rate of
destruction. For simplicity, we define S as the product of the relative binding abilities and
41
relative rates of destruction of the two inhibitors. If the rates of destruction of the two
inhibitors are equal, then S is the ratio of the inhibition constants and will be greater than
one if lA is a stronger inhibitor of the target than lB.
Upon integration of equation 7, one finds that the ratio of the total amounts of the
two inhibitors varies exponentially with S (equation 8), where [lA ] represents the initial T 0
total concentration of inhibitor lA. This exponential relationship enhances the ability to
detect small differences as the destruction reaction progresses.
In([JBT 1/[JBT ]0) = S
In([JAT 1/[JA
T ]0)
(8)
By measuring the relative concentration of the two inhibitors during the
destruction reaction, the value of Scan be determined using equation 8. Alternatively,
equation 9 below, which expresses [lA ], [ lB ], [ lA ] , and [ lB] in terms of the T T TO TO
conversion, C, and the ratio of the total concentrations of the two inhibitors, can be used
to determine S.
ln[(l- C)(~)] = S
ln[(l- C)(l :RR)] (9)
These predictions are shown graphically for several values of S in Figure 2.1. If
the rates of destruction of the two inhibitors are equal, then S is the ratio of the inhibition
constants. As the destruction reaction proceeds (conversion increases from zero to one),
the ratio of the total amounts of the two inhibitors, [lA ]/[ lB ], varies when S is not equal T T
to one. When S is large (e.g., 40), the relative concentration of the good inhibitor
increases steeply near 50% conversion. When S is small (e.g., 2), the relative
concentration of the good inhibitor increases steeply near 90% conversion. In either case,
42
the ratio of the total amounts of the two inhibitors, [lA ]/[ lB ], can be much larger than T T
the value of S.
o 0.1 02 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Conversion
Figure 2.1: Predicted ratio of the total (bound and unbound) concentrations oftwo hypothetical inhibitors,
lA and lB as a function of the degree of conversion for given values of S. The degree of conversion is the
fraction of the total amount of inhibitors that have been destroyed. The calculated lines follow equations 8
and 9 where the initial total concentration of the inhibitors is one. This graph shows that the ratio of the two
inhibitors can be much larger than the value of S, even for values of Sas low as 2.
2.3 Results
2.3.1 Synthesis of 4'-Sulfonamidophenylalanine Dipeptides
(S)-4'-Sulfonamidophenylalanine (1 or PhesJ was prepared from (S) -N-
acetylphenylalanine by a modification of the procedure described by Escher et al. [5) Thus,
chlorosulfonylation of N-acetylphenylalanine in chlorosulfonic acid at 60 oC followed by
ammonolysis afforded N-acetyl-4'-sulfonamidophenylalanine. Direct purification of this
intermediate proved difficult. Therefore it was deacetylated using hog kidney acylase 1,[6)
43
and the resulting free amino acid 1 was purified by ion exchange chromatography and
recrystallization. Using this procedure, 1 was prepared as an analytically pure solid in
40% yield from N-acetyl-Phe. The a-amino group was selectively blocked using ethyl
chloroformate under standard Schotten-Baumann conditions. The requisite dipeptides
were then prepared by coupling 2 with tert-butyl amino acid esters using EDCIHOBTpl
followed by trifluoroacetic acid mediated deprotection of the ester function to afford
dipeptides 4a-d (Scheme 2.3). No acylation of the sulfonamide nitrogen was observed
under either the Schotten-Baumann or peptide coupling conditions. Dipeptide Etoc-Phe-
Phe (5), which does not contain a sulfonamide group and serves as a control, was
prepared by standard methods.
1:1 0 RI
Et02CJr~~02R2
H2N's~ d''b 2
TFA CH-"I r 3 R = t·Bu • "'" 2 L-. 4 R2 = H
5
a)RI=Bn b) RI = H
c) RI = i-Bu
Scheme 2.3: Preparation of 4'-Sulfonamidophenylalanine dipeptides.
2.3.2 Inhibition of Carbonic Anhydrase
Sulfonamides 1 and 2 as weIl as sulfonamide dipeptides 4a-d aIl inhibited the
carbonic-anhydrase-catalyzed hydrolysis of 4-nitrophenyl acetate (NP A). The inhibition
was competitive and Lineweaver-Burk plots revealed similar inhibition constants, which
44
varied by only a factor of 10 (Table 1). The parent amino acid 1 (Phesa) was the poorest
sulfonamide inhibitor (KI = 13 /lM), while dipeptides 4a (Etoc-Phesa-Phe) and 4b (Etoc-
Phesa-Gly) were the best sulfonamide inhibitors (KI = 1.2 /lM, 2.5 /lM respectively).
Dipeptides 4c (Etoc-Phesa-Leu) and 4d (Etoc-Phesa-Pro) showed slightly higher inhibition
constants (4.4 /lM, 9.4 /lM respectively). Other simple sulfonamides also inhibit carbonic
anhydrase with similar inhibition constants. [8) As expected, the dipeptide lacking a
sulfonamide group, 5, did not inhibit carbonic anhydrase.
Table 2.1: Inhibition of carbonic anhydrase by sulfonamides 1 and 2, sulfonamide dipeptides 4a-d, and
dipeptide 5.
Compound KI (/lM)a
Phesa (1) 13 ± 1.6
Etoc-Phesa (2) 12 ± 1.4
Etoc-Phesa-Phe (4a) 1.2 ± 0.2
Etoc-Phesa-Gly (4b) 2.5 ± 0.5
Etoc-Phesa-Leu (4c) 4.4 ± 0.7
Etoc-Phesa-Pro (4d) 9.4 ± 1.6
Etoc-Phe-Phe (5) »1000b
a Competitive inhibition constants for the carbonic anhydrase-catalyzed hydrolysis of p-nitrophenyl
acetetate (PNPA) at 25°C in phosphate buffer (10 mM pH 7.5). A typical procedure was to add carbonic
anhydrase solution (lOO.O flL, 0.05 mg/mL) containing inhibitor (0.0-100.0 flM in most cases) to an
acetonitrile solution of pNPA (5.0 flL, 2.0-32 mM) and follow the release of p-nitrophenoxide
spectrophotometrically at 404 nM. b No inhibition detected at an inhibitor concentration of 1 mM.
45
2.3.3 Selective Extraction of Inhibitors By Carbonic Anhydrase
First, we demonstrated that a strongly binding inhibitor concentrates into the
carbonic anhydrase-containing compartment ofa two-compartment vessel (c.f., figure 2.4
without Pronase). The two compartments were created by suspending a dialysis bag
containing a solution of bovine carbonic anhydrase[9] (~0.33 mM, 10 mg/mL) in a
solution of phosphate buffer. The dialysis membrane (12 kDa cutoff) separated the two
compartments so that small molecules such as the sulfonamide dipeptides could diffuse
freely across the membrane, while carbonic anhydrase (30 kDa) could not. Both
compartments initially contained a mixture of 0.16 mM sulfonamide dipeptide 4a and
0.19 mM non-inhibitor dipeptide 5. Over several hours the total sulfonamide
concentration increased in the inside compartment containing carbonic anhydrase and
decreased in the outside compartment as analyzed by HPLC (Figure 2.2). On the other
hand, the concentrations of the non-inhibitor 5 remained similar in both compartments.
This result showed that tight binding to a target could concentrate a good inhibitor into
one compartment of a two-compartment reaction vessel.
46
[dipeptide) (mM)
0.3 Outside Chamber
-e~~------____ 5 0.2
0.1
------4a o+-----~----~----~----~
o 3 6 Time (h)
9 12
Figure 2.2:(10) Selective concentration of the sulfonamide 4a over non-inhibitor 5 into the carbonic-
anhydrase-containing compartment of a two-compartment vesse!. One compartment contained carbonic
anhydrase (0.34 mM), while both compartments (20 mL each) initially contained equal concentrations of
sulfonamide 4a (0.16 mM) and non-inhibitor 5 (0.19 mM). The sulfonamide diffused freely across the
dialysis membrane and concentrated in the carbonic-anhydrase-containing compartment as shown. In
contrast, the concentrations of non-inhibitor 5 remained similar in both compartments. After 12 h, the
concentration of sulfonamide 4a in the outside compartment decreased to 0.04 mM and increased in the
inside compartment to 0.28 mM (total of free and carbonic anhydrase-bound). The final ratio of 4a to 5 in
the carbonic anhydrase chamber was 1.75:1.
In a similar experiment using a mixture of inhibitors, we could further detect
differences in relative inhibition constants. A more tightly binding inhibitor concentrated
in the carbonic anhydrase compartment to a greater extent than a less tightly binding
inhibitor. Starting with an equimolar mixture of sulfonamide dipeptides 4a-d and the
non-inhibitor 5 in both compartments, the sulfonamide dipeptides concentrated into the
carbonic anhydrase compartment, Figure 2.3. The fraction of dipeptide in the carbonic
anhydrase compartment varied: 88% for 4a, 82% for 4b, 74% for 4c, 70% for 4d, and
48% for the non-inhibitor 5 (or a ratio of 1.83 : 1.71 : 1.54 : 1.46: 1 for 4a : 4b : 4c : 4d :
47
5). The order of highest to lowest concentration in the carbonic anhydrase chamber
reflects the order of the binding constants of the inhibitors.
20 a) Outside Chamber
16
12L-.-________ -+ __________ ~
[dipeptide 1 (mM) 8
5
4d 4c
4 4b 4a
o+----.----.---~----~---,
o 10 20 30 40 50
Time(h)
20
16
12 [dipeptide)
(mM) 8
4
0
b) Inside Chamber 4a 4b
~ ___ --::::::64c 4d
r-.----------+-----------s
0 10 20 JO 40 50
Time(h)
Figure 2.3:[101 Selective concentration of the sulfonamides 4a-4d over non-inhibitor 5 into the carbonic-
anhydrase-containing compartment of a two-compartment vessel separated by a dialysis membrane. One
compartment contained carbonic anhydrase (0.485 mM), while both compartments (20 mL each) initially
contained equal concentrations of sulfonamides 4a-4d and non-inhibitor 5 (-0.11 mM each). The
sulfonamides diffused freely across the dialysis membrane and concentrated in the carbonic-anhydrase-
containing compartment. In contrast, the concentration of non-inhibitor 5 increased slightly in the outer
compartment.
These results show that is possible to distinguish between inhibitors, but the
differences in concentration are small, especially among inhibitors of similar strength.
Even comparing the best inhibitor (4a) with a non-inhibitor (5) gives a concentration
differing by less than a factor of two. To enhance this difference in concentration, we
explored the use of proteases to destroy the poorer inhibitors.
48
2.3.4 Selective Protection of Inhibitors by Carbonic Anhydrase from Hydrolysis
2.3.4.1 Screening of Proteases.
We screened twenty-two commercially available proteases to identify those that
could hydrolyze the dipeptide Etoc-Phesa-Phe (4a). All proteases showed sorne activity.
Using 0.1 mg ofprotease and 2 /lmol (2 mM) dipeptide 4a, the five most active proteases
(a-chymotrypsin, protease from Streptomyces casepitosus, proteinase K, Pronase from
Streptomyces grise us, protease from Bacillus thermoproteolyticus rokko) cleaved all of
the dipeptide within twenty four hours, while two moderately active proteases (protease
N "Amano", protease from Bacillus polymyxa) cleaved all of the dipeptide within forty
eight hours. The remaining proteases cleaved less than half of the dipeptide after seventy
two hours. We chose one of the most active yet inexpensive enzymes, Pronase from
Streptomyces griseus, for subsequent experiments. Pronase was found to cleave all five
dipeptides (4a-d and 5), although the glycine and proline dipeptides (4b and 4d) were
cleaved more slowly (80-90% hydrolysis within 24 h). To ensure high cleavage rates,
larger amounts of Pronase were used in the competitive degradation experiments
described below.
2.3.4.2 Selective Protection of Inhibitors
We compared the ability of carbonic anhydrase to prote ct sulfonamide inhibitor
4a from hydrolysis while allowing a non-inhibitor, 5, to be hydrolyzed. An experiment
49
similar to that described above, except with Pro nase added to the outer chamber was set
up (Figure 2.4). In the Pronase containing chamber, both dipeptides were rapidly cleaved
to the constituent pieces within 15 minutes. On the other hand, the inside compartment
showed a steady decrease in the concentration of non-inhibitor 5 over 12 h (Figure 2.5),
while the concentration of sulfonamide 4a remained nearly constant (a decrease of 9%
over 12 h). [11] After even just 6 h, the ratio 4a to 5 in the inside compartment was 3.7 : 1
and continued to increase to greater than 20:1 after 12 h. By comparison, the experiment
that does not contain Pronase had a final ratio of 4a to 5 of 1.75 : 1.
Outer Cham ber Inner Cham ber
l~!!a ~ HO, • H NHEtOC
l 1"'" : R1 R,
jl 1 carbonic lil anhydrase e' .' Il lI:
~l pronase 1
~!!a
Ho, • HEtOC
H • CA 1'" ,
Inhlbltor 1 CA comple.
Figure 2.4: Reaction design for the selective destruction experiments. The dipeptides can diffuse across the
dialysis membrane into either chamber. One chamber contains carbonic anhydrase, the other contains
Pronase. Dipeptides in the Pronase cham ber are rapidly cleaved to their constituent pieces. Carbonic
anhydrase prevents strong binding dipeptides from diffusing across the membrane and thus slows their
hydrolysis.
50
0.2 Inside Chamber
0.16 n-:-----.--.----=----"""4a
0.12 [dipeptide]
(mM) 0.08
0.04
0+-__ .-__ ~ ____ ~== __ ~5
o 3 6
Time(h)
9 12
Figure 2.5:[10] Selective protection from hydrolysis of sulfonamide 4a over non-inhibitor 5 by carbonic
anhydrase. A vessel containing two compartments of equal volume (20 mL each) separated by a dialysis
membrane was filled with a solution of. sulfonamide 4a (0.16 mM) and non-inhibitor 5 (0.19 mM). The
inside compartment contained carbonic anhydrase (0.34 mM), while the outside compartment contained
Pronase. The protease rapidly cleaved the dipeptides in the outside compartment to the corresponding
amino acids (data not shown). The non-inhibitor 5 diffused freely across the dialysis membrane and was
cleaved by the protease. In contrast, the inhibitor 4a bound to carbonic anhydrase in the inside
compartment was not consumed at a significant rate. After 6 h, the concentration of sulfonamide 4a in the
inside compartment decreased by only 6% (0.15 mM), while the concentration ofnon-inhibitor 5 decreased
to 0.041 mM during the same time period (ratio = 3.7:1).
In a similar experiment, dipeptides 4a and 4b, which have very similar binding
constants, were exposed to carbonic anhydrase and Pronase. In this experiment, the
dipeptides were placed only in the carbonic anhydrase chamber and an excess of carbonic
anhydrase was used (1.6:1 ratio of CA to dipeptides) so that the conditions adhered
rigorously to the theory described above. As expected due to the excess of target and tight
binding ofboth dipeptides, the hydrolysis of 4a and 4b was slow. However, as in the first
reaction, the weaker binder, 4b, was consumed at a higher rate (Figure 2.6). After 193 h,
83% of the total dipeptides have been hydrolyzed and the ratio of 4a to 4b was 3.8:1.
51
This final ratio is in excess of the ratio of the independently determined binding constants
of the dipeptides (2.1 :1).
0.3 Inside Cham ber
0.25 • • 0.2 t
• [dipeptide) 0 15
(mM) .
0.1
0.05 4a
4b 0
0 40 80 120 160 200
Time(h)
Figure 2.6:[10] Selective protection from hydrolysis of dipeptide 4a over 4b by carbonic anhydrase. A
reaction vessel was separated into two compartments (20 mL each) by a dialysis membrane. The inside
compartment contained carbonic anhydrase (13.6 f.1mol), dipeptide 4a (4.3 f.1mol) and dipeptide 4b (4.3
f.1mol) in 20 mL ofbuffer. The outer compartment contained Pronase (5 mg) dissolved in 20 mL ofbuffer.
The time dependence of the concentration in the carbonic anhydrase chamber is shown in the figure. At
83% conversion (193 h) the ratio of 4a to 4b was 3.8:1.
In a related experiment, we compared two sulfonamide dipeptides 4a and 4c,
which also have similar inhibition constants (Figure 2.7). In this experiment, both the
inside and outside chambers initially contained equal concentrations of the dipeptides,
and total concentration of dipeptides was in excess (2.1: 1 ratio of dipeptides to CA). The
result was a much faster decrease in concentration of both dipeptides initially present in
the carbonic anhydrase chamber. This faster rate reflects the rapid release of one
equivalent of Phesa (2) from the Pronase chamber. Although 2 is a weaker binder than
52
either 4a or 4c, enough of it was produced such that it could displace a small amount of
4a and 4c from the carbonic anhydrase binding pocket, thus accelerating their hydrolysis
by Pronase. However, the net result was still the same. After 6 h, 93% of 4c was
hydrolyzed after 6 h, but only 58% of 4a was hydrolyzed. Thus, the ratio of
concentrations was 6: 1, which is much larger than the 1.6: 1 ratio observed in a control
experiment which did not contain Pronase and larger than the 3.7: 1 ratio of their binding
constants.
0.2 Inside Chamber
0.16
0.12 [dipeptide]
(mM) 0.08
0.04
4a
0+-______ ~------~----~4c
o 2 4 6
Time (h)
Figure 2.7:[10) Selective protection from hydrolysis of dipeptide 4a over 4c by carbonic anhydrase. A
reaction vessel was separated into two compartments (20 mL each) by a dialysis membrane. The inside
compartment contained carbonic anhydrase (0.34 mM), while the outside compartment contained Pronase
(4 mg). Both compartments initially contained similar concentrations of dipeptide 4a (0.l6 mM) and
dipeptide 4c (0.14 mM). The protease rapidly cleaved the dipeptides in the outside compartment to the
corresponding amino acids (data not shown). The time course of the reaction in the carbonic anhydrase
chamber is shown in the figure. After 6 h, 93% of 4c inside the CA chamber had been hydrolyzed, while
only 58% of 4a had hydrolyzed. A control experiment that did not contain carbonic anhydrase showed an
equal rate ofhydrolysis for the two dipeptides in the chamber not containing Pronase.
53
FinaHy, an experiment containing aH five dipeptides (4a-d and 5) was conducted
using an excess of carbonic anhydrase (ratio of CA to dipeptides is 1.2: 1). The
experiment was consistent both with the theory and with the prior results. Dipeptide 5
was cleaved rapidly while dipeptides 4a-d disappeared at rates that corresponded to their
binding constants (Figure 2.8). Weaker inhibitors rapidly diffuse into the outside
chamber, and may occupy aH the available protease sites, causing the apparent lag-time
for destruction of stronger inhibitors at the beginning of the experiment. More detailed
analysis of this phenomena is required.
0.25 Inside Chamber
0.2
0.15 [di peptide)
(mM) 0.1
40 80 120 160 200
Time (h)
Figure 2.8:[10] Selective protection from hydrolysis of dipeptides by carbonic anhydrase. A reaction vessel
separated into two compartments (20 mL each) by a dialysis membrane was set up. The inside
compartment contained carbonic anhydrase (25.6 I1mol) and dipeptides 4a-d and 5 (4.3 I1mol each) in 20
mL of buffer. The outer compartment contained Pronase (5 mg) dissolved in 20 mL of buffer. The time
dependence of the concentrations in the carbonic anhydrase chamber is shown in the figure.
2.4 Discussion/Conclusions
54
As expected, the four sulfonamide dipeptides 4a-d aIl inhibit carbonic anhydrase
competitively with similar inhibition constants (within a factor of 10 of each other).
Classical kinetics using initial rates easily identified these differences, but these classical
methods are slow and require measuring each inhibitor separately. This becomes
laborious for libraries containing thousands of members.
To rapidly identify the best inhibitor, we used competitive binding to carbonic
anhydrase in one compartment of a two-compartment cell. The inhibitors concentrated
into the carbonic anhydrase compartment of a two-compartment cell. Higher
concentrations of the better inhibitors were observed in the carbonic anhydrase
compartment, but the concentration differences were small (1.83 : 1.71 : 1.54 : 1.46 : 1
for 4a : 4b : 4c : 4d : 5). If the mixture contained a thousand dipeptides, this competitive
experiment would not identify the best inhibitor because it would be hard to separate aU
the dipeptides and the differences in concentration with and without target would be
small.
Although this experiment does not include a dipeptide-synthesis step and thus is
not a dynamic library, the diffusion across the membrane mimics a synthesis step in a
dynamic library in that both are equilibrium processes. For the diffusion process, in the
absence of a target, each compartment should contain equal amounts of each inhibitor. In
the presence of the target, the carbonic anhydrase chamber contains more of the tight
binding inhibitors. Thus, the equilibrium for the diffusion reaction has shifted.
A non-selective destruction of the library members should enhance differences in
the relative concentrations of the members bound to the target. The po or binding
members are destroyed at a higher rate than the strong-binding members and as the
55
degradation progresses, the ratio improves exponentially in favour of the latter. This was
observed in our library, where dipeptide hydrolysis by Pronase was used as the
destruction process. In a competition experiment between a strong and weak binder (4a
vs. 5), the ratio of 4a to 5 in the carbonic anhydrase chamber increased from 1.75:1 in the
absence ofPronase to 3.7:1 in the presence ofPronase (at 45% conversion). Furthermore,
this ratio continued to increase to >20: 1 as the reaction progressed. A second experiment
with two species with very similar KIS (4a vs. 4 b) had a final ratio of 3.8: 1 when the ratio
of the binding constants was 2.1: 1. As shown in Figure 2.9a, these results follow the
theoretical model closely. Similar results were obtained for an experiment containing two
inhibitors (4a and 4c) where an excess of a weaker binder, Phesa (2), was generated in the
reaction mixture. The presence of 2 accelerated the rate of cleavage of 4a and 4c but, as
can be se en in Figure 2.9b, the ratio of dipeptides during the course of the reaction still
followed the theoretical model. At 70% conversion, the ratio of 4a to 4c was 6: 1, which
is much larger than the 1.6: 1 ratio observed in a reaction not containing Pronase. In all
cases, the model indicates that the ratios should continue to increase if the reactions are
carried out for even longer periods. In experiments with a large number of library
members, this increase will be critical in allowing the tightest binding species to be easily
identified. [12]
56
a) 5
4
Ratio 3
4a/4b 2
•
b) 10
8
Ratio 6
4a/4c 4
2
o+---~----~----~--~~--~ o+---~----~----~--~--~
o 0.2 0.4 0.6 0.8 o 0.2 0.4 0.6 0.8
Conversion Conversion
Figure 2.9: The graph shows theoretical and experimental ratios for the screening experiments. Theoretical
lines are shown as smooth lines. The S values correspond to the ratios of the experimentally determined
binding constants. The data points show the experimentally determined ratios at different conversions for a)
4a/4b (c.f. Fig. 6) and b) 4a/4c (c.f. Fig. 7).
One potentiallimitation of this screening method is selectivity in the destruction
reaction. For example, Pronase c1eaves dipeptide 4c at a much slower rate than dipeptide
4a. In such a case, S from equations 10-12 will not be equal to the ratio of the binding
constants and thus the degradation reaction will not follow the theoretical curves of figure
1. To accommodate this situation, we used a large amount of protease and, more
importantly, we employed a dialysis membrane to separate the target-inhibitor complexes
from the protease. In this setup, the rate limiting step in the destruction reaction is not the
protease-catalyzed c1eavage but diffusion across the dialysis membrane. Unlike the
protease-catalyzed c1eavage, the rate of diffusion does not vary significantly with the
structure of the inhibitor and the result is that the destruction reaction follows the
theoretical curve. Although Pronase accepts a wide variety of peptides, substrate
specificity of the enzyme may become problematic if highly diverse libraries are studied.
A dipeptide that is not c1eaved by Pronase would be retained in the reaction mixture, even
57
if it did not bind to carbonic anhydrase. One way to alleviate this problem would be to
use a mixture of enzymes with a wide range of specificities. Altematively, it is important
to note that the de gradation reaction is not limited to enzymatic processes. Other
chemical degradation methods can be envisioned, depending upon the type of library
being studied. For example, a library based on disulfide exchange could be degraded by
adding a reducing agent (e.g. a phosphine) to cleave any unbound disulfides.
Altematively, physical methods for removal of unbound inhibitors (e.g. adsorption to a
solid phase, extraction) should accomplish the same effect as a chemical degradation.
Another potential limitation of this screening method, and indeed for methods
based on the dynamic combinatorial library technique, is the need for stoichiometric
amounts of the target. The initial experiments reported here used large amounts of
carbonic anhydrase (100-500 mg/experiment) as we expect to apply it to a dynamic
library process where the best inhibitor will actually be isolated and characterized.
However, for purely analytical screening purposes, the methods can easily be scaled
down using smaller compartments, assuming that more sensitive analytical tools are used
(e.g., mass spectroscopy). These modifications could reduce the amount oftarget needed
to <0.1 mg/experiment, an amount that is easily accessible for targets that have been
cloned and over-expressed.
In conclusion, we have developed a method for screening mixtures of compounds
against a therapeutic target that readily identifies the best binder in a library. The method
works by selectively degrading the poorer inhibitors with an enzyme. This results in a
significant improvement in the ability to distinguish between inhibitors that have very
close binding constants. We plan to extend this method to dynamic libraries with the goal
58
of improving the enhancement observed in synthesis of good inhibitors in the presence of
a therapeutic target.
Contribution of Authors
Romas J. Kazlauskas developed the theory in section 2.2
Andrew D. Corbett and Jonathan Croteau developed and performed the chemical
synthe sis described in section 2.3.1
Ronghua Shu performed the screening of proteases in section 2.3.4.1
The author (Jeremy D. Cheeseman) developed and performed an other experiments
described in this chapter.
References
1. Reviews: a) A. Ganesan, Angew. Chem. Int. Ed Eng. 1998,37,2828-2831; b) J.
M. Lehn, Chem. Eur. J 1999,5,2455-2463; c) G. R. L. Cousins, S. A. Poulsen, J.
K. M. Sanders, Curr. Opin. Chem. Biol. 2000, 4, 270-279; d) 1. Huc, R. Nguyen,
Comb. Chem. High Throughput Screening 2001, 4, 53-74.
2. a) A. V. Eliseev, M. 1. Nelen, J Am. Chem. Soc. 1997,119, 1147-1148; b) A. V.
Eliseev, M. 1. Nelen, Chem. Eur. J 1998, 4, 825-834.
3. Glaucoma patients often take carbonic anhydrase inhibitors to reduce the pressure
in the eye. An commercial inhibitors contain a sulfonamide moiety. We chose
carbonic anhydrase as a test case for inhibitor design and screening methods.
4. The analysis below follows closely the mathematical treatment for kinetic
resolutions. For examples see: a) V. S. Martin, S. S. Woodard, T. Katsuki, Y.
Yamada, M. Ikeda, K. B. Sharpless, J Am. Chem. Soc. 1981,103,6237-6240; b)
59
C. S. Chen, Y. Fujimoto, G. Girdaukas, C. J. Sih, J. Am. Chem. Soc. 1982, 104,
7294-7299; c) H. B. Kagan, J. C. Fiaud, Top. Stereochem. 1988, 18, 249-330.
5. E. Escher, M. Bernier, P. Parent, Helv. Chim. Acta. 1983,66, 1355-1365.
6. Researchers often use hog kidney acylase to resolve enantiomers of N-acetyl
amino acids. For examples see: a) H. K. Chenault, J. Dahmer, G. M. Whitesides,
J. Am. Chem. Soc. 1989, 111, 6354-6364; b) S. M. Roberts, Ed., Preparative
Biotransformations, Wiley: Chichester 1992-1998, Module 1:14 In our case, this
intermediate was already enantiomericaUy pure. We used hog kidney acylase to
cleave the acetyl group under mil der conditions than those required by chemical
cleavage methods.
7. EDC = 1-(3-dimethylaminopropyl)-3-ethy1carbodiimide; HOBT 1-
hydroxybenzotriazole.
8. For example, Nguyen and Huc investigated a simple sulfonamides with inhibition
constants of ~0.1 to 1 IlM (R. Nguyen, 1. Huc, Angew. Chem. Int. Ed 2001,40,
1774-1776), while Doyon et al. investigated other simple sulfonamides with
inhibition constants of ~0.001 IlM (J. B. Doyon, E. A. M. Hansen, c.-y. Kim, J.
S. Chang, D. W. Christianson, R. D. Madder, J. G. Voet, T. A. Baird Jr., C. A.
Fierke, A. Jain, Org. Lett. 2000,2, 1189-1192).
9. These experiments required stoichiometric amounts of carbonic anhydrase. We
used an inexpensive mixture of isozymes from bovine sources. Although material
was not pure carbonic anhydrase, we calculated the concentrations assuming it
was pure. Thus, the true concentration will be less than the number given.
10. Lines drawn in aU figures (except Figure 2.1 and Figure 2.9) are for illustration
purposes only. They do not represent theoreticallines of any sort.
Il. Both 4a and 5 diffused through the membrane at identical rates with a half-life of
about three hours. (Data not shown.)
12. The reaction mixture will contain the products of the degradation reactions.
However, in most cases, this method will be applied to combinatoriallibraries
and, as such, the degradation products wiU often be the common starting materials
used to make the library members. Thus only a limited number of degradation
products will be produced.
60
CHAPTER THREE
FIRST GENERATION PSEUDO-DYNAMIC COMBINATORIAL LIBRARIES
61
Abstract
This chapter describes the integration of the screening process developed in
chapter two with an in situ library synthesis from solid phase in the first pseudo-dynamic
combinatorial library. The library experiments took place in a three-chambered reaction
vessel that separated the synthesis resin and protease from the receptor, carbonic
anhydrase. Our library consisted of two series of four dipeptides each. Dipeptides 4a-d
carried an inhibitory arylsulfonamide moiety that could bind to the zinc metalloezyme,
carbonic anhydrase. Dipeptides 5a-d served as negative controls. The selectivity for the
strongest binding dipeptide (4a) was greater than 50 : 1 in a library with cycle times of 12
and 24 hours. However, the amplification of 4a was less than 1 %.
3.1: Designing the First Pseudo-Dynamic Combinatorial Library: Description of
the Three Key Processes: Synthesis of the Library, Binding to the Receptor, and
Destruction/Recycling of Unbound library Members
Amplification and selectivity are the two aspects of any receptor-assisted
combinatorial system that need to be optimized. High selectivity for only strong binding
compounds ensures that only compounds that might provide useful drug leads survive
screening. Amplification of the selected compounds is necessary for their detection and
characterization. Although the selective destruction process in chapter two provided
selectivity higher than is normally possible in traditional DeLs, this selectivity was
brought about by a destruction process that left only small amounts of the tight binders
62
left in the system.[I] Ideally, the amount of a tight binder after a highly selective
experiment should be easy to detect and characterize. Pseudo-dynamic libraries were
developed so that not only could the receptor select its tightest binders from a mixture,
but also increase the amounts of these binders over time, facilitating their detection.
Pseudo-dynamic libraries (P-DCLs) combine kinetic and thermodynamic events
to increase amplification and selectivity. They include the irreversible (and hence
kinetically govemed) formation of a library in situ. They then allow members of the
library to interact with a receptor in a thermodynamic process common to DCLs. The
synthesis and binding is coupled to a kinetic destruction process that removes unbound
library members. Finally, re-using some of the species left over from the destruction
process in a new round of library synthesis allows the library to undergo another cycle of
binding and destruction.
This system has several features that can potentially improve its ability to select
and amplify tight binders over that se en in other RACC systems, and over the selective
protection from destruction alone (Figure 3.1). The initial binding to the receptor should
select library members based on their relative binding affinities, similar to a traditional
DCL, but the destruction process should increase the selectivity beyond the levels
possible in a DCL. Recycling starting materials in the re-creation of the library will re
introduce tight binders to the receptor, allowing them to re-compete for binding sites with
other, weaker library members left over from the first cycle of synthesis, binding and
destruction.
63
(,J-(> 0 "'~, s,.~ <>? ~=R=ece=p=tor==", Receptor: 0 ~·O -.. .. ·6~ ~h
D Figure 3.1: Schematic of a hypothetical pseudo-dynamic combinatoriallibrary.
3.1.1 Synthesis of the Library
The tirst process in a p-DCL is an irreversible library synthesis. Based on
previous resultsY] we made a library of dipeptides, sorne with an aryl sulfonamide
moiety able to bind to the receptor, carbonic-anhydrase (CA). Previous studies showed
that TentaGel-supported tetrafluorophenol active esters react cleanly with free amino
acids in water under alkaline (pH 8-10) conditions to form dipeptides. [2] Aqueous peptide
synthesis was necessary to ensure receptor stability, and a solid phase approach would
allow for periodic replenishment of starting materials. The initial library consisted of
eight dipeptides, four ofwhich had a CA-binding aryl sulfonamide moiety (Figure 3.2).
64
x= SOzNHz, H R,=Bn,Rz=H R, = CHzipr, Rz = H R,=H,Rz=H R,RZ = (CHzla
1 ,&: 1 ,&: HZNOZS~ H~
H~ ~",COZH H~ ~",COZH Eloc 0 Eloc 0
Eloc-Phe..G1y (4b) Eloc-PheGly (5b)
Eloc..phePhe (5a)
Figure 3.2: Solid phase, aqueous synthesis of a library of dipeptides. Top: the general scheme of library
synthesis in pseudo-dynamic libraries. Bottom: The solid phase synthesis of the first generation pseudo-
dynamic library. A nucleophile (amino acid) attacks that N-Etoc-protected active ester of an amino acid on
the tetrafluorophenol-TentaGel resin. Sorne dipeptides contain an aryl sulfonamide moiety that can bind to
carbonic anhydrase.
The tirst generation p-DCLs coupled nucleophiles (glycine, proline, leucine and
phenylalanine) to active esters of phenylalanine and 4'-sulfonamidophenylalanine.
Previous studies showed a general trend going from pH 6 to 10 that favoured dipeptide
synthesis over hydrolysis from the resinYl Since 9.0 was the highest pH at which the
receptor was stable for several days, the library synthesis was investigated at pH 9.0
using four different concentrations of nucleophile (10, 5, 4, and 3 mM, Table 3.1). The
65
nuc1eophile was in excess relative to the active ester in every case. The product
distribution did not change significantly with varying nuc1eophile concentration, giving
an average ratio of 4b : 4d : 4a : 4c of 3.0 : 1.5 : 1.2 : 1.0. We now had a versatile,
aqueous, solid phase synthetic strategy for the generation of a dipeptidic library of
potential CA inhibitors.
Table 3.1. Distribution of products from the library described in Figure 3.1.
Concentration 4b 4d 4a 4c
(mM)
10 (16 equiv) 44% 25% 17% 14%
5 (8.0 equiv) 46% 21 % 18% 14%
4 (6.4 equiv) 44% 23% 18% 15%
3 (4.8 equiv) 44% 22% 18% 16%
Average ratio 3.0 1.5 1.2 1.0
3.1.2 Binding to the Receptor and Destruction ofUnbound Library Members
A kinetic synthesis can generate a library of dipeptides. In a p-DCL, the synthesis
is followed by an initial, reversible interaction of library members with the receptor,
providing selectivity based on relative binding affinities (Figure 3.3). At this point, one of
the major drawbacks to DCLs, that being the amplification of non-binding library
members (see section 1.2.3 and Figure 1.10),[3] has already been overcome because the
66
library synthesis is irreversible. This means that the product distribution is based only on
the relative synthetic rates of each library member and their binding affinities, and
entropic factors cannot affect the product distribution, which can be the case in fully
reversible systems.
The selectivity should be similar to that seen in the experiments in which a
receptor selectively extracted inhibitors based on their binding affinities (described in
section 2.3.3, Figures 2.2 and 2.3). However, library members that are synthesized to
greater degrees will be present in larger quantities and will be able to more effectively
compete for binding sites, which would alter the results to favour the most readily formed
dipeptide rather than the tightest binder.
« P Receptor 0 6 t> == Receptor. ...... h ~
Figure 3.3: The dipeptide library can now interact with the receptor, carbonic anhydrase. This is a
thermodynamic process that gives initial selectivity for tight binders, but is not great enough to overcome
synthetic biases without an added destruction reaction.
The kinetic destruction reaction is the same as described in chapter 2, namely a
non-selective protease catalyzed cleavage of the dipeptides to their constituent amino
acids (by Pronase from Streptomyces griseus). This destruction reaction gives greater
67
selectivity than is possible in DCLs by weaning away weakly bound library members.
This process is analogous to the experiments in which a receptor selectively protected
strong binders from a destruction reaction (described in section 2.3.4.2, Figures 2.4-2.8).
The destruction reaction was hoped to be able to overcome any synthetic bias, and
increase the selectivity many fold over that seen from the thermodynamic binding alone.
o <> ? Receptor Recepto9
o r=6~ ~'b o
Figure 3.4: The destruction of unbound library members. Top: Schematic of the process. Bottom: Sorne
aryl-sulfonamide dipeptides bind to CA and are protected from the Pronase catalyzed hydrolysis to their
constituent amino acids. One half of each dipeptide is recycled in a new round of synthesis, while the other
builds up in the system with each successive destruction cycle.
Carbonic anhydrase (as weIl as many other receptors) has surface-exposed amines
that could react with the active esters used to create the library. A dialysis bag that would
allow the dipeptides, but not macromolecules to cross, was therefore employed to
separate the receptor and resin. The isolation of the resin also allowed for the extraction
of used resin from the system and its easy replacement with fresh reagents. We had
previously made a two-chambered system in which the receptor was separated from a
protease. This concept was extended to form a three-chambered vessel consisting of two
68
dialysis bags, one containing the resin (the synthesis chamber, 10 mL), and one
containing the protease (destruction chamber 20 mL), suspended in a solution of carbonic
anhydrase (binding chamber 20 mL) in a 100 mL container.
Chamber 1: Synthesis
Pronase
Figure 3.5: The three-chambered p-DCL experimental set up. A library of dipeptides is synthesized from
active esters on solid support in chamber 1. The dipeptides can then diffuse into the binding chamber
(chamber 2) and interact with the receptor, carbonic anhydrase. If they are not bound to the receptor, they
will diffuse into chamber 3 and be destroyed by Pronase. The non-N-protected amino acids can then diffuse
into the synthesis chamber again, where new active ester is added in a new round of synthesis, binding and
destruction. MWCO = molecular weight cut off.
3.1.3 Recycling Destruction Products and Iteration of Synthesis
Up to this point, that is the end of the first cycle of synthesis, binding and
destruction, the pseudo-dynamic library is very similar to the systems described in
chapter two. In those static libraries, pre-made dipeptides were added to a chamber
containing CA, which kept them from diffusing into a destruction chamber. The main
69
difference in a full p-DCL is the process of iteration, where new active esters are added,
and can react with freed starting materials produced by the protease. By re-synthesizing
the library, aIl the compounds have another chance to bind to the receptor. As in the first
cycle, tighter binders will be able to occupy relatively more binding sites. In this second
round, however, the tight binders will be able to replace weaker binders left over from the
first cycle. This will increase the relative amounts of the best binders in the binding
chamber, effectively increasing the selectivity over that seen in the static library cases.
Rather than continuously being destroyed, re-synthesis also allows compounds to
build up in the system. This gives larger overall quantities of compounds, introduced to
the receptor, and gives more material at the end of an experiment, further facilitating
analysis. The ability to re-use starting materials that are formed is also important, as
without it, aIl the compounds generated in the destruction process would continue to
build up in the system over time. This aspect of the first generation p-DCLs will be
discussed at the end ofthis chapter, and will become important in chapter 4.
3.2 Results of the Integrated Processes in the First Pseudo-Dynamic
Combinatorial Library
The first moderately successfullibraries showed that the selectivity could indeed
be increased not only over that of DCLs, but over that of the static libraries described in
chapter 2. Pseudo-dynamic experiments vary with respect to cycle time and the number
of equivalents (with respect to the receptor) of dipeptides that are synthesized in each
cycle. The first generation experiments coupled amino acids Gly, Pro, Leu and Phe with
70
active esters of phenylalanine and 4'-sulfonamidophenylalanine. The amino acids were
regenerated by hydrolysis catalyzed by Pronase. Control experiments established that the
hydrolysis rate was similar for an compounds, and was at least four times faster than
diffusion across the dialysis membrane separating the binding and destruction chambers.
The competitive inhibition constants of an species that would be present in the library
were measured at pH 9.0 (Table 3.2) so that the results of the library experiments could
be accurately interpreted.
Table 3.2: Inhibition constants for components of the first generation p-DCLs.
Compound KI (J.tM)a
Phesa(l) 8±2
Etoc-Phesa (2) 13±2
Etoc-Phesa-Phe (4a) 0.45±0.09
Etoc-Phesa-Gly (4b) 0.75±0.1
Etoc-Phesa-Leu (4c) 1.26±0.03
Etoc-Phesa-Pro (4d) 6.6±0.2
Etoc-R-Phe (5a-d) »1000b
a Competitive inhibition constants for the carbonic anhydrase catalyzed hydrolysis of p-nitrophenyl acetate
(PNPA) at 25°C in BICINE buffer (lOmM pH 9.0). A typical procedure was to add carbonic anhydrase
solution (100.0 !lL, 0.05 mg/mL) containing inhibitor (0.0-100.0 !lM in most cases) to an acetonitrile
solution of pNPA (5.0 !lL, 2.0-32 mM) and follow the release of p-nitrophenoxide spectrophotometrically
at 404 nM. b No inhibition detected at an inhibitor concentration of 1 mM.
71
The results of a p-DCL using two, 24 h cycles are shown in Figure 3.6. In this
experiment CA (28.0 mmol, 1.0 eq) and Pronase (catalytic) were suspended in 20 mL
each of a 5 mM stock solution of amino acids in BICINE buffer at pH 9.0. The Pronase
solution was placed in a dialysis bag to form the destruction chamber, and was suspended
in the CA solution in a 100 mL plastic cup (the binding chamber). The synthesis chamber
had a 160 mmol (~5. 7 eq) portion of resin composed of equal amounts of the active esters
of phenylalanine and 4'-sulfonamidophenylalanine.
The graph shows only the three strongest dipeptidic inhibitors, 4a (KI = 0.45 ~M),
4b (Kr = 0.75 ~M), and 4c (Kr = 1.3 ~M). AlI eight dipeptides were present at the
beginning of each cycle, but the non-sulfonamides and the weakest sulfonamide, 4d had
all been removed from the binding chamber within four hours. As was observed in the
static library synthesis (section 3.1.1), 4b is synthesized to a greater degree than any other
dipeptide. However, by 10 hours its concentration is lower than that of the strongest
binding dipeptide, 4a. This shows that the destruction process provides at least enough
selective pressure to overcome a two-fold synthetic bias. At the end of two cycles, only
the strongest binding dipeptide can be observed in the binding chamber. This shows that
the iterative process does increase the selectivity over that of the static library
experiments.
However, despite this excellent selectivity, the amount of the best binder at the
end of the experiment represents less than 1 % of the total available receptor binding sites,
and seems to actually decrease over successive cycles. This is the opposite of what was
expected from the initial design of these systems.
72
0.07
0.06
0.05
0.04 mM
0.03
0.02
0.01
0
0 10 20 30 40
Time (h)
Figure 3.6: P-DCL of two cycles, 24 h each. The strongest binder (4a, KI = 0.45 !lM) dominates in each
cycle, giving high selectivity. However, the amounts of the best binder decrease over successive cycles,
rather than increasing as expected. The receptor is present at a concentration of 1.4 mM.
Figure 3.7 shows the results of a very similar experiment to that of Figure 3.6.
The only difference in this case is that the destruction chamber was not added for six
hours after the initiation of a new cycle. The rationale behind this design was to observe
if the destruction process was taking place too rapidly to allow compounds to build up
over time, giving the apparent lack of amplification seen in Figure 3.6. This did not prove
to be the case, as the strongest binding compound, although still present as the only
binder left after two 24 h cycles, seems to go down in concentration with successive
cycles rather than the opposite.
73
0.08
0.07
0.06
0.05
mM 0.04
0.03
0.02
0.01
0
0 10 20 30 40
Time (h)
Figure 3.7: Two 24 h cycles, with a 6 h delay in the addition of the destruction chamber. As in Figure 3.6,
excellent selectivity is observed, but no build up of the best binder occurs over successive cycles. The
receptor is present at a concentration of 1.4 mM.
Finally, in Figure 3.8 the amount of dipeptides formed in each cycle is halved to
80 ~mol (~2.9 eq in each portion of resin), while the cycle time is also halved to 12 h,
and the number of cycles is doubled. The selectivity is not quite as pronounced as in the
24 h cases, but the strongest binder is still the only one observable after two cycles.
Again, the total amount of the strongest binder drops with successive cycles to occupy
less than 1 % of available binding sites at the end of four cycles.
74
0.05
0.04
0.03
mM 0.02
0.01
0
0 10 20 30 40
Time (h)
Figure 3.8: Four 12 h cycles with half the amount of dipeptide formed per cycle as compared to Figures
3.6 and 3.7. The selectivity remains very high, but the amplification decrease over successive cycles is even
more pronounced. The receptor is present at a concentration of 1.4 mM.
3.3 Discussion/Conclusion
Pseudo-dynamic libraries use two coupled, but irreversible processes in place of
the equilibrium reaction used by traditional DeLs. The level of selectivity shown by just
a few cycles of a pseudo-dynamic library is higher than in any receptor-assisted method
to date. The in situ library synthesis was successful in the initial cycle, and in the
subsequent cycles that used material generated from the destruction of unbound
compounds. So long as the equivalents of nucleophiles in the system at aU times are high
with respect to the active esters, there should be no bias from the library as to which
compounds are re-synthesized, hence each cycle should give the strongest binders an
opportunity to replace weaker ones. Additionally, as long as diffusion across the
membrane to the dialysis chamber is the rate-limiting step in destruction, there should be
no bias in the system with respect to differences in the hydrolysis rates of the library
members.
75
The selectivity of these systems can overcome significant (two-fold) synthetic
bias. This is a result that would not be possible in a DCL. In a DCL, aIl the processes are
reversible, which leads to the concentrations of each compound being linearly dependent
on the product of its binding and synthetic constants (see section 1.2.3 for derivations).
The kinetic destruction and iteration of the binding of compounds to the receptor resolve
an important issue in receptor-assisted combinatorial systems in that, as the synthe sis
becomes more diverse and complex, the chance of having identical rates and levels of
synthesis for each library member becomes increasingly difficult. Overcoming synthetic
bias is an important asset of p-DCLs.
Although the selectivity of these initial systems was promising, the lack of any
amplification over successive cycles was puzzling. The cause of this unfavourable result
was quickly identified as being inherent in the synthesis of the library. In each cycle, a
portion of the dipeptide, but not aIl, is recycled in the new round of synthesis. In these
first generation libraries, the amino acid containing the aryl sulfonamide moiety used for
receptor binding was the un-recycled partner (Figure 3.4). Although not a strong
inhibitor, N-EtocPhesa (2) does inhibit CA with a KI of ~ 13 /lM (Table 3.2). Large
amounts of this compound will rapidly build up in the system, first from hydrolysis of the
active ester by water, and then from the destruction of every unbound sulfonamide
containing library member. This compound can easily achieve concentrations at which it
can out-compete even the strongest binding library member. The likelihood of the
recycling partner being of paramount importance in amplification over cycles was the
subject of the next generation of p-DCLs, and is addressed in the libraries created in
Chapter4.
76
Contribution of Authors
Andrew D. Corbett developed and performed the synthe sis described in section 3.1.1
The author (Jeremy D. Cheeseman) developed and performed aIl other experiments
described in this chapter.
References
1. J. D. Cheeseman, A.D. Corbett, R. Shu, J. Croteau, J. L. Gleason, R. J. Kazlauskas J
Am. Chem. Soc. 2002,124,5692-5701.
2. A. D. Corbett, J. L. Gleason Tetrahedron Lett. 2002,43, 1369-1372.
3. Z. Grote, R. Scopelliti, K. Severin, Angew. Chem. Int. Ed. Engl. 2003,42,3821-3825;
Angew. Chem. 2003,115,3951-3955.
77
CHAPTER FOUR
PSEUDO-DYNAMIC COMBINATORIAL LIBRARIES: A NEW RECEPTOR
ASSISTED APPROACH FOR DRUG DISCOVERY
78
Abstract
This chapter contains the results of a pseudo-dynamic library in which the amino
acid containing the inhibitory arylsulfonamide moiety is recycled in each new round of
synthesis. The library was made of two new series of four dipeptides each, 7a-d
(sulfonamides) and 8a-d (negative controls). In these libraries, the importance of cycle
time on selectivity becomes apparent. Using eight-hour cycles proved insufficient to
overcome a synthetic bias that favoured 7b, the third strongest binder. However,
lengthening the cycle time to 16 hours improved the selectivity profile to give the
strongest binding dipeptide, 7d in greater than 100 : 1 selectivity and 29% yield.
4.1 Introduction
Emerging methods of combinatorial chemistry incorporate receptor assistance to
combine synthesis and screening. [1] Binding to stoichiometric amounts of a receptor alters
either the thermodynamics or kinetics of library synthesis. Dynamic combinatorial
libraries[2] use a thermodynamic approach where binding shifts a synthetic equilibrium to
increase the amounts of the best binding compounds, in accordance with 's principle.
These libraries usually identify the tightest binding library members, but sorne
experimental conditions can give small or misleading changes in concentrationY] An
alternative method, receptor-accelerated synthesis, uses a kinetic approach. [4] Starting
components that bind to the receptor can couple to create a new, tight-binding compound.
The receptor accelerates the coupling of the better fitting starting components due to their
79
close proximity, but requires that both components bind tightly to the receptor. Here we
demonstrate a new method, a pseudo-dynamic library, which adds a kinetic contribution
to traditional dynamic libraries to dramatically increase the selectivity.
A pseudo-dynamic combinatorial library combines an irreversible synthesis of
library members with an irreversible destruction step. Those library members that bind to
the receptor are protected from destruction. Subsequent synthesis reuses fragments from
destroyed library members, thus amplifying the amounts of the better binders at the
expense of the lesser ones. In cases where one of the fragments contains a binding motif,
this should be the fragment that is reused. Otherwise it can build up in the system and
decrease any amplification that could arise from the reintroduction of the library. The
separate irreversible synthesis and destruction steps allow adjustment to optimize both
the amplification and selectivity.
4.4 Experimental Design
We developed a pseudo-dynamic library of eight dipeptides to identify the best
inhibitor of carbonic anhydrase (CA). CA, a zinc metalloenzyme, is a therapeutic target
for glaucoma and is inhibited by aromatic sulfonamides, which coordinate to the zinc ion.
Four of the eight dipeptides in our library contain 4'-sulfonamidophenylalanine (Phesa, 1),
and thus should bind to CA, while the remaining four contain only Phe and serve as
negative controls. The irreversible synthesis of dipeptides used a solid-supported
coupling of activated esters with an amino acid in aqueous solution (Scheme 1).
TentaGel-supported tetrafluorophenol active esters react cleanly with free amino acids in
80
water under alkaline (PH 8-10) conditions to form dipeptides. [5] A non-selective protease
from Streptomyces griseus (Pronase) destroyed these dipeptides by catalyzing their
irreversible hydrolysis. [6] This library differs from that described in chapter 3 of this
thesis in that the nucleophillic, recycled starting material contained the sulfonamide
binding motif. This arrangement prevented the build up of 4'-sulfonamidophenylalanine
in the system, hopefully allowing for greater amplification of inhibitory dipeptides.
H2Ny C02H
~ V--x 1 X: S02NH2 6X:H
Pronase
X~
~ = 0 C02Et
HO C)...N)l .NoR 2 H l 2 R,
7X:S02NH2 8X:H
Series: a R, : CH2Ph, R2: H bR,: H, R2 : H cR, : CH2CH(CH3n, R2 : H d R, R2 = (CH2h
Scheme 4.1: Creation of a pseudo-dynamic library of dipeptides.
The pseudo-dynamic library was prepared in a three-chambered reaction vessel
formed by suspending two dialysis bags in a surrounding solution (Figure 4.1). One
dialysis bag (the synthesis chamber) contained the active esters; the other dialysis bag
(hydrolysis chamber) contained the protease, while the surrounding solution (screening
chamber) contained the carbonic anhydrase. Adding nucleophiles 1 and 6 to the synthesis
chamber generated the dipeptide library. These dipeptides diffused into the surrounding
solution where they could bind to carbonic anhydrase, and further diffused into the
hydrolysis chamber where Pronase cleaved them. This cleavage regenerated 1 and 6,
which could diffuse back into the synthesis chamber to repeat the cycle. This
arrangement prevented Pronase-catalyzed destruction and active ester-mediated
81
modification of the receptor, CA, and also permitted periodic replenishment of the
activated ester to regulate the rate of synthesis.
Synthesis Chamber Screening Chamber Hydrolysis Chamber
Figure 4.1: Schematic of the pseudo-dynamic combinatorial library experiment. Reaction of two free
amino acids (Phesa (1) and Phe (6» with four solid-supported active esters (N-Etoc-Phe, N-Etoc-Gly, N-
Etoc-Leu and N-Etoc-Pro) creates an eight-member library.
The experiments used four active ester resins derived from N-Etoc-Phe, N-Etoc-
Gly, N-Etoc-Leu and N-Etoc-Pro (0.8 equiv each), nucleophiles 1 and 6 (6.4 equiv each),
carbonic anhydrase (28 Ilmo1, 1 equiv) and Pro nase (25 mg/mL). The large amount of
Pronase made diffusion across the dialysis membrane the rate-limiting step for
hydrolysis; hence, aIl dipeptides were cleaved at similar rates in spite of the substrate
selectivity of Pronase. Periodic addition of fresh portions of active ester resin (defined as
the cycle time) regulated the overall rate of library synthesis. We conducted three
experiments with this system using cycle times of 8, 12 and 16 h. HPLC analysis of
aliquots from the screening chamber showed the progress ofthe experiments (Figure 4.3).
82
4.5 Results
Two control experiments established, first, that the synthetic process afforded aU
the expected dipeptides and, second, that the sulfonamide-containing dipeptides inhibited
carbonic anhydrase. Combining equal amounts of the four active esters with Phesa (1) as
the nucleophile produced four dipeptides 7a : 7b : 7c : 7d in a ratio of 18 : 44 : 15 : 23.
Not surprisingly, coupling of 1 with the less hindered glycine ester to produce 7b was
more efficient than with the more hindered phenylalanine, leucine, or proline esters. In
spite of these differences aIl four dipeptides formed in significant amounts. Using
phenylalanine as the nucleophile gave similar results. For the second control experiment,
aIl eight dipeptides were prepared individuaIly and their ability to inhibit the CA
catalyzed hydrolysis of p-nitrophenyl acetate (Figure 4.2) was measured. As expected the
sulfonamide-containing dipeptide competitively inhibited this hydrolysis with inhibition
constants of 1.1-8.7 IlM, while the non-sulfonamide dipeptides showed no detectable
inhibition. Dipeptide 7d was the best inhibitor, with an inhibition constant of 1.1 IlM and
dipeptide 7c was the next best inhibitor with an inhibition constant of2.5 IlM. Compound
1 also inhibits CA (KI = 13 IlM), but approximately ten-fold less effectively than the
tightest binding dipeptide (7d).
83
(7a) EtocPhePhesa KI = 8. 7 ~M
~H
o "" 1 H N OH
Et02C" 0~ ~O V
(8a) EtocPhePhe I<j» 1.0 mM
"...1 o ;$S02NH2
~ Il OH EtOC" ~N
2 H o
(7b) EtocGlyPhesa KI = 5.6 ~M
H o;$""IH
N Il OH EtOC" ~N
2 H o
(8b) EtocGlyPhe KI» 1.0 mM
;$H
o "" 1 H N OH
Et02C .... 0~ y 0
(8c) EtocLeuPhe I<j» 1.0 mM
""""J A!," U ~Ày0H o
(7d) EtocProPhesa KI = 1.1 ~M
Figure 4.2: Structures and competitive inhibition constants of the library members for the CA-catalyzed
hydrolysis of p-nitrophenyl acetate. The non-sulfonamide compounds showed no detectable inhibition at 1
mM.
In the first pseudo-dynamic experiment (8-hour cycle, Figure 4.3a), the cycle time
was too short for the destruction reaction to remove the less effective inhibitors. During
the first four hours of each cycle, the screening chamber contained all eight dipeptides,
indicating that all eight had formed as expected. At the end of each 8 h cycle, prior to the
next addition of active ester, the hydrolysis had removed the four non-sulfonamide
dipeptides, leaving only the four sulfonamide dipeptides. At the end of six cycles of
active ester addition, dipeptide 7b was present in the highest amount (58% yield, relative
to CA), followed by 7d (33%), 7e (27%) and 7a (8%), respectively. These relative
amounts differ from their relative binding constants. Rather, the higher yield of 7b
reflects its more favourable rate of synthesis. In addition, the sum of all sulfonamide
dipeptides at 48 h was greater than the amount of target (126% yield). This high yield
shows that unbound dipeptides remained and that the destruction reaction had not had
enough time to distinguish between the different sulfonamide inhibitors.
84
Lengthening the cycle time from 8 h to 12 h yielded the best three inhibitors with
the relative amounts in the order of their inhibition constants (Figure 4.3b). Although
sulfonamides 7b, 7c and 7d were present in high concentrations early in the experiment,
at the end of four cycles, the concentration of these weaker binding dipeptides had
diminished substantially. The tightest binding dipeptide, 7d, was present in the highest
amount (15% yield relative to CA), followed by 7c (5%) and 7b (1.5%). Notably, the
ratio at the end of the experiment (10.1 : 3.5 : 1) exceeded the ratio of their binding
constants (5.1 : 2.2 : 1). None of the weaker binding 7a or of the non-sulfonamide
dipeptides remained at the end of the experiment.
The selectivity of the dynamic process improved even further upon extending the
cycle time to 16 h (Figure 4.3c). The initial synthesis during the first cycle favoured
dipeptide 7b, the most rapidly synthesized dipeptide, but this dipeptide disappeared in
later cycles where the main competition was between 7 d and 7 c, the tightest binding
dipeptides. After four cycles (64 h), only these two remained and the ratio of their
concentrations (13 : 1) was significantly higher than the ratio of their binding constants
(2.3 : 1). After three more cycles the selectivity increased to > 1 00 : 1 in favour of the
strongest binding dipeptide, 7d. The yield was 29% relative to the amount of CA and
corresponded to 4 mg of dipeptide. Thus, adjusting the relative rate of library synthesis
and destruction optimized the selectivity so that only the best binding dipeptide remained
and in a good overall yield.
85
a) 1.20
1.00
0.80
0.60
[7]/mM 0.40
0.20
0.00 Ji~~k::=:=~=:=:::::==::==~
b) 0.30
0.25
0.20
0.15
[7]/mM 0.10
c)
0.05
0.60
0.50
0040
0.30
[7]/mM 0.20
0.10
o 10
10
20 30 40 50
tlh ----
20 30 40 50
tlh----
0.00 __ --~ __ ~--.......... __ ...... _ __.
o 20 40 60 80 100
tlh----
Figure 4.3: Pseudo-dynamic Iibrary experiments. Concentrations of sulfonamide containing dipeptides 7a
(e), 7b C"), 7c (.) and 7d (+) over the course of experiments a) 8 h per cycle, b) 12 h per cycle and c) 16
h per cycle.
4.4 Discussion/Conclusion
The selectivity in the pseudo-dynamic library is significantly greater than that in
many traditional dynamic libraries. The optimum conditions produced only the single,
86
tightest-binding dipeptide (> 1 00: 1 selectivity), while a traditional approach would yield a
mixture because the binding constants for the two tightest-binding dipeptides differed by
only 2.3-fold. This higher selectivity greatly simplifies the analysis, as only one
compound need be identified and characterized. The optimization of a pseudo-dynamic
library arises through control of the relative rates of synthesis and destruction. We
previously showed that a destruction reaction operating on a static library in the presence
of a receptor distinguishes between library members with very similar binding constants,
selectively removing the weaker binding speciesJ6] However, when selectivity arises
from destruction alone, significant amounts of the best-binding library member must be
destroyed to achieve high ratios of good binder to slightly poorer binder. This leaves only
a small amount of the best binder for analysis. In pseudo-dynamic libraries, the high
selectivity also stems from the competition between binding to the receptor and
destruction.
The iterative nature of the experiment also contributes to the high selectivity.
Toward the end of each cycle, cleavage by Pronase has reduced the amounts of weak
binding dipeptides, leaving dipeptide 7 d as the major species bound to CA. The
subsequent burst of synthesis produces a mixture of all dipeptides, which compete for the
smaller amount of free target. Pronase then rapidly cleaves all unbound species, which
would consist of a higher proportion of weak binders. Continued action of Pronase
further increases the ratio in favour of the bound species, following our static model,
resulting ultimately in high selectivity for the tightest-binding species.
Our static model of pseudo-dynamic combinatorial libraries[6] indicates that
selectivity stems from the relative binding constants of the inhibitors, not their absolute
87
affinity for the target. Thus, we expect that pseudo-dynamic combinatoriallibraries will
also work with even tighter binding inhibitors, but would require longer cycle times to
distinguish between these more tightly binding inhibitors. lndeed, we are currently
expanding to larger pseudo-dynamic libraries to disco ver such tighter binding inhibitors.
Contribution of Authors
Andrew D. Corbett developed and performed the solution-phase synthesis to acquire pure
samples of the library members shown in figure 4.2.
The author (Jeremy D. Cheeseman) developed and performed aH other experiments
described in this chapter.
References
1. a) A. Ganesan Angew. Chem. Intl. Ed. Engl. 1998, 37, 2828-2831; Angew. Chem.
1998,110,2989-2992; b) I. Huc, I-M. LehnActua/ité Chimique 2000, 51-54; c)
C. Karan, B.L. Miller Drug Discov. Today 2000,2,67-75; d) R. Nguyen, I. Huc
Comb. Chem. High-Throughput Screen. 2001, 4, 53-74; e) J.-M. Lehn, A.V.
Eliseev Science 2001, 291, 2331-2332; f) O.Ramstrom, J.-M. Lehn Nat. Rev.
Drug Disc. 2002, 1, 26-36; g) S. Otto, R.L.E. Furlan, IK.M. Sanders Curr. Op.
Chem. Biol., 2002,6,321-327. h) O. Ramstrom, T. Bunyapaiboonsri, S. Lohman,
I-M. Lehn Biochim. Biophys. Acta 2002, 1572 178-186.
2. a) I. Huc, J.-M. Lehn Proc. Nat!. Acad. Sci. USA 1997,94, 2106-10; b)
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P.G.Swann, RA. Casanova, A. Desai, M.M. Frauenhoff, M. Urbancic, U.
Slomczynska, A.l Hopfinger, G.C. LeBreton, D.L. Venton Biopolymers 1997,
40,617-625; c) B. Klekota, M.H. Hammond, B.L. Miller Tetrahedron Lett., 1997,
38, 8639-8642; d) B. Klekota, B.L. Miller Tetrahedron, 1999, 55, 11687-11697;
O. Ramstrom, l-M. Lehn ChemBioChem 2000,1,41-48; e) C. Karan, B.L. Miller
J Am. Chem. Soc., 2001, 123, 7455-7456; f) RJ. Lins, S.L. Flitsch, N.J. Turner,
E. Irving, S.A Brown Angew. Chem. Int. Ed Engl. 2002,41,3405-3407; Angew.
Chem. 2002, 114, 3555-3557; g) M. Hochgurtel, H. Kroth, D. Piecha, M.W.
Hofmann, K.C. Nicolaou, S. Krause, O. Schaaf, G. Sonnenmoser, A.V. Eliseev
Proc. Nat!. Acad. Sei. USA, 2002, 99, 3382-3387; h) I. C. Choong, W. Lew, D.
Lee, P. Pham, M.T. Burdett, J.W. Lam, C. Wiesmann, T.N. Luong, B. Fahr, W.L.
DeLano, R.S. McDowell, D.A Allen, D.A Erlanson, E.M. Gordon, T. O'Brien J
Med Chem. 2002, 45, 5005-5022; i) S. Otto, RL.E. Furlan, J.K.M. Sanders,
Science, 2002, 297, 590-593; j) D.A. Erlanson, J.W. Lam, C. Wiesmann, T.N.
Luong, R.L. Simmons, W.L. DeLano, I.C. Choong, M.T. Burdett, W.M.
Flanagan, D. Lee, E.M. Gordon, T. O'Brien Nature Biotechnol. 2003, 21, 308-
314; k) AC. Braisted, J.D. Oslob, W.L. Delano, l Hyde, RS. McDowell, N.
Waal, C. Yu, M.R. Arkin, B.C. Raimundo J Am. Chem. Soc. 2003, 125, 3714-
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3. a) A.V. Eliseev, M.I. Nelen J Am Chem Soc. 1997, 199, 1147-1148; b) J.S.
Moore, N.W. Zimmerman Org. Lett. 2000,2,915-918; c) Z. Grote, R. Scopelliti,
K. Severin Angew. Chem. Int. Ed Engl. 2003,42, 3821-3825; Angew. Chem.
2003,115,3951-3955.
89
4. a) K.C. Nieolaou, RHughes, S.Y. Cho, N. Winssinger, C. Smethurst, H.
Labisehinski, R Endermann Angew. Chem. Int. Ed. Engl. 2000, 39, 3823-3828;
Angew. Chem. 2000,112,3981-3986; b) R Nguyen, I. Huc Ang. Chem. Int. Ed.
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90
CHAPTER FIVE
AMPLIFICATION AND SELECTIVITY IN, EXPANSION AND MODELING OF
PSEUDO-DYNAMIC COMBINATORIAL LIBRARIES
91
Abstract
Pseudo-dynamic combinatoriallibraries are different from other receptor-assisted
methods because they use irreversible synthesis and complementary destruction reactions
bridged by binding to a receptor. This chapter describes experiments that show that
neither an impurity in carbonic anhydrase, nor the concentration of nucleophile 1 affect
the yield of the strongest binder, 7d in the eight-membered, 16 hour cycle experiment
described in chapter four (Figure 4.3c). Further experiments show that fewer compounds
in the library can raise the amplification of 7d, but simultaneously lower the selectivity in
this p-DCL. Expanding the library to 30 compounds results in selection of a single library
member but requires a longer cycle time, probably due to that inhibitor's increased
receptor-affinity. Finally, a preliminary mathematical model of p-DCLs shows that the
relative contribution of the kinetic destruction to selectivity is greater than that of
thermodynamic receptor-binding in these systems.
5.1 Origins of Amplification Maxima in the Eight-Membered p-DCL
5.1.1 Introduction
There are two elements that need to be optimized in any receptor-assisted
combinatoriallibrary: amplification and selectivity. By the definition of amplification
given in chapter one (Box 1.1), any amount of library member left at the end of the
experiment counts as amplification in a p-DCL because without the receptor, the amounts
92
of any compound left at the end of a p-DCL experiment will depend on its level of
synthesis, but more importantly, on the amount of time given for destruction. The
equation for amplification ([IA]receptor![IA]no receptor) becomes artificially large given long
cycle times (in which [IA]no receptor becomes very low). Because of this unique feature of p
DCLs, analyzing the final yield of a library member (from chapters three and four,
"yield" refers to the percentage of receptor binding sites occupied by a library member)
gives a more useful measure of the level of its amplification.
The six, 16-hour cycle experiment (Figure 4.3c) gave a selectivity of 7d : 7c of
>50 : 1. Recycling the amino acid that carries the receptor-recognition moiety of the
dipeptide in each new round of synthesis resulted in a 29% yield of the strongest library
member (7d), much higher than the <1 % yield of the strongest inhibitor when this amino
acid was not recycled. This 29% yield (corresponding to ~4 mg of7d in the system) was
easily detectable by HPLC, and could have been characterized by NMR and MS if
necessary (the extra NMR and MS analysis of the library material was not necessary
because a pure sample of the compound had previously been prepared and analyzed by
these methods). Although this amount of material was easily analyzed, the amount of an
inhibitor present in the screening chamber depends on the amount of receptor available to
bind it. Smaller-scale p-DCLs will be needed in the future because sorne receptors, due to
low bacterial expression levels, rarity in nature, and high costs, are not as readily
available as carbonic anhydrase. In a small-scale p-DCL, analysis of a library hit will
require that the yield of the tight-binding compounds be as high as possible. Since we
hope to exp and the pseudo-dynamic library method to other, less readily available
biological targets, we decided to explore ways of improving the yield of 7d in the eight-
93
membered library (depicted in Figure 4.3c) to determine the features of a p-DCL that
govern amplification.
These explorations took two directions. First, we wanted to be sure that our
enzyme was pure. The carbonic anhydrase used for the experiments described in chapters
three and four was an inexpensive mixture of isozymes from bovine erythrocytes. An
impurity in the measured amount of CA would have made our estimate of its molar
amount in the experiments artificially high. The yield is calculated by dividing the molar
amount of 7d by the molar amount of CA in the screening chamber, hence an impurity in
CA would have made the yield of 7 d appear to be lower than it actually was. Thus, we
needed to quantify the amount of CA active sites present in the commercial sample. The
results described in section 5.1.2 show that an impurity was not significantly affecting the
yield of7d.
The second direction we undertook was to lower the systemic concentration of
nucleophiles in the library experiments themselves. In chapter three we discovered that
the amplification of library members suffered because the replenishable activated amino
acid on solid support carried the main zinc-binding element of the dipeptide and was not
recycled in each successive cycle. It therefore built up successive over cycles, allowing it
to effectively compete for receptor binding sites. However, in the improved libraries of
chapter four in which the recycled nucleophillic amino acid carried the zinc-binding unit,
large amounts of this nucleophile were still being used to ensure efficient dipeptide
synthesis. These high amounts could have allowed the nucleophile to again effectively
compete for binding sites, lowering the yield of the true strongest binder. The results that
94
will be described in section 5.2.3 suggest that high nucleophile concentrations were not
the cause of the low yield either.
5.1.2 Testing Enzyme Purity
To quantitate CA active sites, we performed a series of parallel, two-chamber
experiments in which CA occupied a binding chamber and Pronase occupied a
destruction chamber. Various amounts of a known, sub-IlM inhibitor (4a) were added to
the binding chamber (4.0 eq, 2.5 eq and 1.0 eq with respect to CA) in separate, parallel
experiments. The association constant Ka for 4a was ~lx106 M- I and was therefore
expected to bind to nearly aIl available CA active sites based on its potency. In the
experiments where there was more than one equivalent of 4a, the excess amount should
not have been able to bind, and would therefore rapidly diffuse out of the binding
chamber and be destroyed by Pronase. Once the excess 4a had been removed, the amount
of the remaining, bound dipeptide could be estimated by observing the inflection point in
the rate of its disappearance. The amount of inhibitor left at the point at which its
destruction becomes slow should give an estimate of the amount of receptor active sites
present. The decrease in 4a concentration in each parallel experiment that used an excess
of the inhibitor should aIl exhibit an inflection point at the same concentration: that at
which the remainder is bound to CA. The experiments that did not have an excess of 4a
should not exhibit an inflection point. The results of this experiment are summarized in
Figure 5.1.
95
1.8
1.6
<:S 1.4
] 1.2
5 LO t-;;::::--_"':'''"'''II;::-:;;::-:-~q::::;:::;;:::-i 0.8
$ 0.6
0.4
0.2
O.O+--~~-~-~-~-~-~
o 10 12 14
Time(h) 2.1 1.6 l.l 0.6
Equivalents of CA
Figure 5.1:(1) Quantification of CA binding sites by observing the differences in the rate of destruction of
bound and unbound inhibitor 4a. Varying equivalents of 4a were placed in a chamber containing CA in
each experiment (3.9 eq (+), 2.3 eq (.) hatched lines, and 1.0 eq (». Diffusing into a second chamber
containing Pronase should rapidly destroy excess amounts of 4a. The point at which the rate of
disappearance of 4a becomes much lower is the point at which the rate-determining step in the destruction
changes from diffusion across the membrane (fast) to dissociation from CA (slow). The molar amount of
4a left at this point should correlate with the amount of CA active sites present. a) The concentration profile
over time of 4a. The y-axis shows the concentration of 4a in terms of equivalents of CA. The solid
horizontal line extrapolated to the y-axis corresponds to the point of the greatest rate of change in 4a
concentration in the 3.9 eq experiment and crosses the axis at -0.98 eq of CA (the x-axis value is taken
from the midpoint between the two equivalents of CA exhibiting the highest rate of change of [4a]. The
large hatched horizontal line extrapolated to the y-axis corresponds to the point of the greatest rate of
change in 4a concentration in the 2.3 eq experiment and crosses the axis at -1.1 eq of CA. There was no
significant rate of change in 4a concentration in the 1.0 eq experiment (as expected). A blank experiment in
which there was no CA showed rapid hydrolysis of aIl 4a (data not shown). b) The % rate of change of 4a
concentration versus amounts of 4a (in terms of CA equivalents) in order to better iIIustrate the inflection
point on the concentration versus time graph. Looking from left to right on the graph, one can see the
decreasing amounts of 4a measured along the x-axis. The y-axis shows the % 4a concentration of the
concentration measured in the previous time point (aIl time points are exactly one hour long). The solid
vertical line corresponds to the point of the greatest rate of change in 4a concentration in the 3.9 eq
experiment and extrapolates to -0.98 eq of CA. and the large hatched verticalline corresponds to the point
of the greatest rate of change in 4a concentration in the 2.3 eq experiment and extrapolates to -1.1 eq of
CA.
96
In the experiments using 4.0 and 2.5 eq of 4a, the inflection point in the rate of its
destruction (at 0.98 eq and 1.1 eq of CA respectively) correlated with approximately
100% of the amount of CA that was supposed to be present in the binding chamber.
Further, in the experiment with 1.0 eq of 4a, no inflection point was observed. A control
experiment with no CA showed rapid destruction of aIl 4a added to the binding chamber
as expected. These results showed that the molar amounts of CA as calculated from its
weight were accurate measures of the molar amounts of binding sites. This shows that an
impurity was certainly not accounting for 70% of the calculated mass of CA, and was not
significantly affecting the calculated yield in the library experiments.
5.1.3 Lowering the Concentration of Nucleophiles
In the libraries of chapter four, high concentrations of nucleophiles 1 and 6 were
used to ensure efficient synthesis of the library. The amount of 1 initially added to the
synthe sis chamber was eight times the amount of any one of the eight dipeptides
synthesized in one cycle. Since 1 is a moderate inhibitor of CA, its high concentrations in
the system may have allowed it to effectively compete for binding sites with more potent
library members. Initial calculations showed that the amounts of 1 in the 16 h library
experiment were high enough that if aIl of it was present in the binding chamber at once,
it could occupy approximately 50% of CA binding sites. We tested whether or not
minimizing the amount of 1 would free CA binding sites and allow for higher levels of
dipeptide binding.
97
First, static libraries were synthesized using different concentrations of 1 to
determine the minimum concentration of nucleophile needed for efficient synthesis. The
results of the se experiments are summarized in Table 5.1.
Table 5.1: Effect on synthetic rate and yield due to changing the concentration ofnucleophile 1.
Maximum ~ield {l:!moQ Time to
Concentration of 1 reach
Experiment (mMt
7a 7b 7c 7d maXlmu m yield
~h2 A 10 2.5 6.5 2.1 3.7 1.0 B 5.0 2.7 6.5 2.4 4.9 2.5 C 4.0 3.2 8.0 2.9 5.1 3.0 D 3.0 3.4 7.8 3.2 4.9 4.0 E 1.0 1.6 4.1 1.8 3.8 48
a In ail cases there was an excess of nucleophile with respect to the total amount of activated amino acids.
In the previous experiment (Figure 4.3c), the amount of nucleophile added was
approximately 9 mM (close to experiment A in Table 5.1). With 10 mM ofnucleophile,
the maximum synthetic yield of aIllibrary members was reached in one hour. The yield
of aIl dipeptides in experiments A-D were approximately the same, but took increasingly
longer to maximize as the concentration of nucleophile was lowered. Experiment E, in
which only 1 mM of nucleophile was used, was the only one to show significantly lower
yield, probably because at this low concentration, water is able to out-compete 1 in the
coupling reaction.
These results showed that a 3 mM concentration of nucleophile (Table 5.1,
Experiment D) was the lowest possible that could be used to ensure a comparable
coupling yield to the 10 mM nucleophile case. However, the rate of library synthe sis was
98
much slower, taking four hours to reach the maximum yield. We did not want significant
attenuation of the synthesis, so we performed a new pseudo-dynamic library experiment
identical to the 16-h/cycle experiment in Figure 4.3c, but with a combined nucleophile
concentration of 1 and 6 of 4 mM (Figure 5.2).
004
0.3
0.3
0.2 (mM)
0.2
0.1
0.1
0.0 0 20 40 60 80 100
Time (h)
Figure 5.2: Six 16-h cycles of an eight-membered p-DCL with [nucleophiles] = 4 mM. Concentrations of
sulfonamide containing dipeptides 7a (.), 7b ( .... ), 7c (e) and 7d (+) over the course of experiment show a
similar pattern to a 16 h experiment with [nucleophiles] = 9 mM (Figure 4.3c). However, the yield of 7d
drops from 29% to 23% and the ratio between 7d and 7c drops from > 1 00 : 1 to 4 : 1.
Rather than increasing the yield, this experiment gave a lower yield of dipeptide
7d (23%). The selectivity also dropped significantly. This was not what we had expected.
We had hoped, based on comparisons of diffusion and library synthe sis rates using 4 mM
ofnucleophiles that there would be no appreciable difference from experiment 4.3c in the
amounts or rates of dipeptides that were entering the screening chamber. However, since
the yield of 7d was lower, and since it appeared as though the concentration of 7d was
increasing at the end of the sixth cycle (Figure 5.2), we hypothesized that the 4 mM level
of nucleophiles must have attenuated the introduction of library members to the receptor
99
to such a degree that they had not had enough time to build up in the screening chamber.
Renee, the experiment was repeated but allowed to undergo more than six cycles (Figure
5.3).
0.45
0.40
0.35
0.30
0.25 (mM)
0.20
0.15
0.10
0.05
0.00
0 50 100
Time (h)
150 200
Figure 5.3: Twelve 16-h cycles of an eight-membered p-DCL with [nucleophiles] = 4 mM. Concentrations
of sulfonamide containing dipeptides 7a (.), 7b (.â.), 7c (e) and 7d (+) over the course of experiment
show a similar pattern to a 16 h experiment with [nucleophiles] = 9 mM (Figure 4.3c). The yield and
selectivity return to those of the experiment shown in Figure 4.3c (30% yield of7d and a ratio of7d : 7c of
> 1 00 : 1), but do not get higher.
The extended cycles brought the yield and selectivity back up to Figure 4.3c
levels, but did not increase them. Additional experiments were conducted in which the
amount of electrophiles (and hence the amounts of the eight dipeptides synthesized each
cycle) were increased (Figure 5.4). These experiments did not improve the yield, and
lowered the selectivity, even over 16 cycles. The results of the investigations into
increasing the yield are summarized in Table 5.2.
100
0.50
0.45
0.40
0.35
0.30
mM 0.25
0.20
0.15
0.10
0.05
0.00
0 50 100 150 200 250
Time (h)
Figure 5.4: Sixteen 16-h cycles of an eight-membered p-DCL with [nucleophiles] = 4 mM, and increased
amounts of electrophiles added each cycle. Concentrations of the strongest binder 7d (+) plateaus at 30%
over the course of the experiment, and sulfonamide containing dipeptides 7a (.), 7b ( ... ), 7c (e) are not
completely removed from the screening chamber.
Table 5.2: Effect on yield and selectivity due to changing nucleophile, electrophile and number of cycles
in the eight-membered p-DCL using a 16 h cycles time.
Equivalents of Equivalents of
# Cycles to activated amino Maximum
Number nucleophile 1 acid added each ratio between
reach Yield of cycles with respect
cycle with respect 7d and 7c maximum of7db
to CAa
toCA ratio
6c 9.6 3.2 > 100:1 4 30 6d 4.5 3.2 4:1 6 23 12e 4.5 3.2 > 100:1 8 31 16f 4.5 9.6 3:1 16 30
a In alllibraries Phe (6) was present in the same amounts as 1, but because 6 is a non-inhibitor, its presence
did not affect the yield or selectivity.
b Yield refers to the percentage of CA binding sites occupied by inhibitor 7d.
c Experiment 4.3c.
d Experiment 5.2.
e Experiment 5.3.
101
fExperiment 5.4.
Neither extending the same library to 16 cycles, nor adding more active esters per
cycle improved the yield of 4d. Thus we concluded that in this particular system, the
concentration of nucleophiles in the system was not affecting the yield of the strongest
binder.[2]
5.1.4 Discussion: A Steady State Concentration of the Strongest Binding Dipeptide
Since aspects of the synthesis were not significantly affecting the end yield of the
tightest binder, and the enzyme was pure, we hypothesized that the maximum yield for
dipeptide 7 d in an eight-membered library using a 16 h cycle time must have been
reached. The only two factors that remain that can affect the final amount of the tightest
binder in libraries with equal numbers of members are the absolute binding strength of
the inhibitor, and the time allowed for its destruction (the effect of the number of
inhibitors on the yield and selectivity will be discussed in section 5.3). If an inhibitor has
a high binding strength, it binds tightly to a receptor and spends less time in the unbound
state. This leaves less of the compound available for destruction, and ensures that its
bound levels remain at a certain level. After X number of cycles of synthesis and
destruction, a steady state concentration of the inhibitor will be reached, as each cycle it
is synthesized to the same degree, has to compete for binding sites to the same degree,
and spends the same time equilibrating with the receptor and being destroyed. The
number of cycles (X) needed to reach this steady state will depend on the amounts of
library members that are synthesized each cycle.
102
A second factor contributing to the final yield is the time allowed for destruction
before the next synthetic burst. The longer the time allowed for destruction, the more of
the compound will unbind and be destroyed. In chapter four we saw that the time allowed
for destruction was crucial in determining the selectivity. Shorter destruction times allow
more synthesis relative to destruction, and hence give higher levels of alllibrary members
in the system, and thus higher amplifications. However, if the destruction time is not long
enough to remove all but the most strongly binding compound the selectivity will
necessarily be lower. The selectivity and amplification must be simultaneously optimized
in each new p-DCL.
It is important to note that the conclusion that decreasing the cycle time leads to
increased yield, although seemingly true for the 16 h cycle, is not directly applicable to
all systems. In chapter four, (Figure 4.3b), the cycle time was shorter (12 h), but the yield
of7d was only 15%. This lower yield with a shorter cycle time seemingly contradicts the
conclusion that shorter cycle times lead to higher yields. However, in the 12 h experiment
inhibitor 7b was still present to sorne degree in the system at the end of four cycles.
Dipeptide 7b is synthesized 1.5-fold more rapidly than 7d per cycle. The effect ofthis
higher level of the compound could have been to make it an artificially better binding
competitor. The effect of the higher levels of 7b on the yield of 7d, and other library
members at this shorter cycle time are not yet fully understood. However, sorne
experiments (section 5.2) help explain the relative effects of having greater or fewer
inhibitors in the library with a 16-h cycle time.
103
5.2 The Effect of the Number of Inhibitors on
Amplification and Selectivity in Pseudo-Dynamic Combinatorial Libraries
5.2.1 Introduction
The effect of increasing the number of library members in a DCL is to dilute the
absolute amplification of library members (chapter one). This results in a decrease in the
absolute concentration differences between inhibitors as the library size increases. We
hypothesized that since a p-DCL had added kinetic control that this would not be the case
in our system. As a preliminary investigation into this question, we performed two, six
16-h cycle experiments, one in which only the two strongest inhibitors (7c and 7d) were
made, and another in which the strongest inhibitor was left out of the 1ibrary, creating a
library of 7a-c.
5.2.2 Two- and Three-Inhibitor 16 h Cycle P-DCLs
We performed an analogous experiment to the one described in Figure 4.3c, but
using a four-membered library consisting of two inhibitors 7c and 7d, and negative
controls 8c and 8d (Figure 5.5). After six-16 h cycles the ratio of7d : 7c was only 10 : 1,
much lower than the> 100: 1 in the analogous eight-membered experiment. The yield of
7d was ~40%, and that of 7c was ~4%, both of which were higher than the ~30% and
~O% respectively, se en in the eight-membered system. These results suggest that the
104
yields of aU library members increase when there are fewer library members in the
system and that as a consequence ofthis the selectivity is lower.
0.70
0.60
0.50
0.40 mM
0.30
0.20
0.10
0.00
0 20 40 60 80 100
Time (h)
Figure 5.5: Results of a four memberedp-DCL, with two inhibitors 7c (e) and 7d (+) over six-16 h
cycles. The ratio of7d : 7c is lower than in the analogous eight-membered library at only 10 : 1. The yield
of each dipeptide is higher than in the eight-membered library at -40% for 7 d and -10% for 7 c.
In a similar experiment to that of Figure 5.4, a six-membered library was created,
this time excluding the strongest binder, 7d (and its corresponding negative control, 8d).
The results (Figure 5.6) showed that 7 c (now the strongest inhibitor) was present in
highest concentration, but with the weaker inhibitors 7a and 7b still present (at less than
1 % yield each) after six cycles. The yield of 7 c was ~9%, which is higher than in the
four-membered library containing the potent 7d where the yield of 7c was only ~4%, and
than in the eight-membered library in which it was ~O%.
105
0.50
0.40
0.30
mM 0.20
0.10
0.00
0 20 40 60 80 100
Time (h)
Figure 5.6: Results of a six membered p-DCL over six 16-hour cycles with three inhibitors 7a (.), 7b ( .... )
and 7c (e). 7c, now the strongest inhibitor in the library is selected as the strongest binder by the p-DCL
system. At 9%, the final yield of7c is higher than it was in the four-membered library. The yields of7a and
7b are both ~1%. Note: The sampling frequency in this experiment was higher than in Figure 5.5. Ali
experiments in which the sampling frequency was high showed the same cyclical behaviour in each
inhibitor's concentration as seen in the above graph (for example, the experiments shown in Figure 4.3a and
b). However, for the sake of comparison of Figure 4.3a and b to 4.3c, sorne of the data points were omitted
for clarity,
5.2.3 Discussion: Fewer Inhibitors Increase Amplification, but Decrease Selectivity
inP-DCLs
The above results demonstrate that more inhibitors in the system lead to more
competition for binding sites. Furthermore, a weak binder will be able to compete more
effectively with a library member of medium strength than with a strong one. A
comparison between the results in Figure 5.4 and Figure 4.3c shows that removing
weaker binders 7 a and 7b from the system leads to less overall competition for binding
sites. This in turn increases the yields of both 7d and 7c, and gives higher amplification
106
for both compounds. This consequently results in a lower selectivity between the two.
The yield of the second strongest binder, 7c increases from 0% to 4% and the yie1d of7d
rises from 30% to 40%. Adding weaker inhibitors should decrease the yields ofboth 7d
and 7c, but decrease that of7c to a greater degree.
Comparison of the results shown in Figures 5.4 and 5.5 show that 7d has almost
double the effect on the levels of 7c than do 7a and 7b combined (7c's concentration rose
to 4% due to the lack of 7a and 7b (Figure 5.7) but rose even more (to 9%) due to the
lack of only 7d. Competition for binding sites has a relative effect on each inhibitor that
favours strong binders, depending on the number of other compounds present, and on the
binding strengths of those compounds.
The number of compounds in the library represents a third factor influencing the
amplification of the strongest binder, and interestingly, fewer inhibitors lead to lower
selectivity. Further experiments in the 16 h system will be needed to determine whether
the converse of this result is true, that is, that more inhibitors leading to higher selectivity.
5.3 Expansion of the Pseudo-Dynamic Combinatorial Library
5.3.1 Introduction: New Library Members and P-DCL Scheme
With several successful experiments, and a greater understanding of the nature of
pseudo-dynamic libraries, the next step was to exp and the size of the library by adding
more electrophilic amino acids, and new nuc1eophilic amino acids. In the experiments
described thus far experiments we had chosen an aryl sulfonamide moiety to impart
107
carbonic anhydrase affinity because this unit is known to display affinity for the zinc ion
in the active site of CA. [3] Studies have indicated that hydroxamic acids can impart even
higher affinity for zinc.[4] Our existing library consisted of aryl sulfonamides (series 7)
and negative controls (series 8). We added three new series of amino acids carrying
potential receptor-binding functionalities: alkyl sulfonamides based on sulfonamido
alanine (series 9) to complement the aryl sulfonamides, sulfamic acids based on
sulfonamido lysine (series 10) out of interest, and hydroxamic acids formed from
y-hydroxyglutamine (series 11) which would hopefully generate stronger inhibitors than
the micromolar library hits from series 7 (Figure 5.7). This brought our total number of
nucleophiles to five.
We used six amino acids as our solid-supported electrophiles: Phe (a), Leu (c),
Pro (d), Val (e), Ala (t) and pipecolic acid (g). Ala served as a replacement for Gly (b),
which was not included because its retention time was too low using the HPLC
conditions necessary to separate aIl the compounds in the new library. Val and pipecolic
acid were added to increase diversity. Pipecolic acid was especially interesting because of
its structural similarity to proline, which had formed the dipeptide 7d (EtocProPhesa), the
strongest library member in our previous experiments. This gave six electrophiles that,
when combined with the five nucleophiles, would give 30 library members (Figure 5.7).
108
Recycled Nucleophiles
o H2NyÂ-OH
R3
+
Replenishable Electophiles
Eto.C 0
Rz'N0oH ~1
Series (7): Phe.a Series (8): Phe
H 0
o H2N0oH
" o,:.S;o NH2
Series (9): Ala.a
o H2N0oH
)h HN,~O
~,s .. o NH2
Series (10): Lys ..
o H2N0oH
~O HN
OH Series (11): GIUNHOH
E102C,Nyt-X H 0 0 H 0 H 0
Et02Cfx
E102C,N yÂ-x E102C'NJ E102C,N0x Et02C,N0x
QY --( \.Jx CH.
(a):Phe (c):Leu (d):pro
Representative dieptide •
~ 0 C02EI . ~N HO C ......... N . 2
2 H R1 R '------', , ,
7-11 a,c-g
A.-
(e):Val (f):Ala (g):Pipecolic acid
Figure 5.7: The expanded library members. 30 dipeptides are synthesized in five series based on the
coupling offive nucleophillic amino acid-derivatives (7: Phe.", 8: Phe, 9: Ala.a, 10: Lys.a, and 11: GlUNHOH)
with six N-Etoc protected electrophillic amino acids (a: Phe, c: Leu, d, Pro, e: Val, f: Ala, g: Pipecolic acid.
(X = TentaGel resin).
We synthesized each of the thirty library members separately by adding each of
the nucleophiles to each of the electrophiles on solid support and analyzed them for
purity by HPLC. Control experiments showed that Pronase hydrolyzed an the dipeptides
at similar rates (aIl faster than diffusion across the dialysis membrane). We hoped to
discover a tighter binder than in previous cases in which we used six 16-h cycles to
achieve apparent absolute selectivity for compound 7d, so we performed three paralIel
library experiments with cycle times of 16 h, 24 h, and 32 h. An components were scaled
down two-fold compared to previous libraries, but otherwise we used the same
experimental procedure and three-chambered vessel (Figure 5.8).
109
(Synthesis Cham ber) F
(TentaGel~F 1'" 1 0 C0
2Et + H02C'(NH2 pH 9.0
F.... O~NR2 R3
F R'
(Screening Cham ber)
carbon le anhydrase
replenishable solid-supported •• recylcling of nucleophilic electrophllc amlno acid amlno acid (inhibltory ••••••••••••••••••••••••••••
coupling partner)
(Destruction Chamber)
Pronase from S.griseus
o C02Et . .Â...-N Innocuous by·products
HO l. 'R2
R'
Figure 5.8: Expanded library scheme. The dashed lines represent dialysis membranes separating the three
chambers (synthesis, screening and destruction) similar to ail previous Iibrary set-ups.
5.3.2 Results
In the first cycle of each of the 16-h, 24-h and 32-h cycle time experiments, the
non-inhibitory series 8 had disappeared after the first 4 h, series 9 had disappeared after 9
h, series 10 had disappeared after 12 h, and after 16 h, even series 7 was gone, leaving
only the six members of series 11 left in the screening chamber. In each successive cycle
(whether 16-h, 24-h or 32-h) series 8-10 were initially observed, but had aH depleted to
unobservable levels by the end of the cycle.
After six, 16-h cycles, aH the members from series 11 were still present in the
screening chamber. It took three 24 h cycles to wean away compounds 11a and 11d (the
Phe- and Ala-based dipeptides). One more 24-h cycle removed 11b (the Val dipeptide),
and a total of six were needed to remove 11f (the Pipecolic acid dipeptide). This left two
compounds present at the end of six 24-h cycles. These two dipeptides, 11e (the Leu
dipeptide) and 11d (the Pro dipeptide) were also the only compounds to survive five
cycles of the 32 h library. After the sixth cycle, 11e was no longer observable, leaving
11d as the sole survivor of a 30-member library after a total of 192 hours (Figure 5.9).
110
6' 2.5E+06
t 2.0E+06
~ of 1.5E+06
Sl J:>.
~ I.OE+06
~ ~ 5.0E+05
8 = ;;J O.OE+OO F---,----,---,-----'lI-r----,
o 50 100 150 200 250
Time (h)
Figure 5.9: Uncorrected UV absorbance of the final two remaining members of a 30 membered p-DCL,
lld (+) and lle (.) over six-32 hour cycles. The final remaining library member is lld, EtocProGluNHOH.
5.3 Discussion
Compound 11d was the sole survivor of six-32 h cycles in the pseudo-dynamic
library. We have not yet determined the inhibition constant ofthis compound, but aH the
evidence to date suggests that its survival is due to its being the strongest binder in the 30
membered library. Resolution of 11d from other series 11 members required a much
longer cycle time than did the members of series 7 in the eight-membered library (Figure
4.3c). Series 7, and most notably dipeptide 7d was able to survive several16 h cycles in
the eight-membered case in which there were no stronger binders in the system.
However, the Phesa series (7) was eliminated from the library in less than 16 h in the first
cycle of each of the 16 h, 24 h and 32 h cycles in the expanded library. This suggests that
the selectivity in p-DCLs does in fact depend on the relative binding strengths of the
library members, and that stronger binders in the system will indeed help eliminate
weaker ones, making the hit compounds easier to identify. Hence, p-DCLs are biased
111
towards the retention of strong binders, which is a fundamental requirement for success
in receptor-assisted combinatorial chemistry. However, the results of this experiment,
including the yield and KI of Bd need to be confirmed with an authentic sample.
5.4 Preliminary Modeling of Pseudo-Dynamic Combinatorial Libraries
5.4.1 Introduction
A fundamental problem in traditional dynamic libraries is that the amplification of
tight binders becomes diluted as the number of binders in the library increases. Alllibrary
members will be present in amounts corresponding to their binding affinities in an
equilibrated system because of the thermodynamic control over both amplification and
selectivity. In large libraries, the relative concentration differences between two binders
will not change as compared to a small library, but the absolute amounts of all library
members will decrease, making the differences in concentrations difficult to observe. We
sought to determine whether the same is the same true for pseudo-dynamic libraries. We
also wanted to know the degree to which kinetics allow for greater selectivity than
thermodynamics alone, what the effects were of having stronger inhibitors in the system,
and how much the cycle time would need to be adjusted to distinguish between inhibitors
of nanomolar strengths. We attempted to develop a mathematical model the pseudo
dynamic library to answer these questions.
In a p-DCL, three distinct processes occur: library synthe sis, binding to the
receptor, and destruction of unbound library members. Once the library is synthesized,
112
the library members must then traverse a dialysis membrane to interact with the receptor
where they can equilibrate based on binding affinity. Another membrane must then be
crossed before the last process, a kinetic destruction of unbound library members, can
occur. Destruction products can then diffuse back through both membranes to initiate
another round of synthesis and begin the cycle again. After the first cycle, aIl these
processes occur simultaneously in a p-DCL experiment. Modeling the entire process
simultaneously with one equation proved difficult, so the mathematical model consists
instead of three functions applied in succession, which are iterated over several cycles.
This is a major assumption of the model that may render it less accurate when analyzing
full p-DCLs. However, this model does allow for comparison of each process (synthesis,
binding and destruction) with experimental results. We found that the three processes in
the model correlate weIl with experiment, and that the integrated model gave similar
results of amplification and selectivity to an analogous experiment (shown in Figure 5.5).
5.4.2 Modeling Synthesis
Library synthe sis consists of coupling between a nucleophile and an electrophile.
The electrophile is an activated amino acid on solid support, so this reaction may exhibit
pseudo-first order kinetics. However, library members must diffuse across a dialysis
membrane before entering the screening chamber. Since diffusion is a first order process,
a first order equation should accurately model the apparent rate of synthesis (due to
synthesis and diffusion) in our p-DCL. Control experiments have shown that alllibrary
members diffuse accross 1 and 12 kDa MWCO membranes at similar rates. Higher (or
113
lower) order rate equations can easily be applied to model synthe sis as required by each
new type of synthesis. [5]
Synthesis of a dipeptide using a solid supported active ester is illustrated in Figure
5.10, where AA is the solid supported electrophile, Nu is the nucleophile, 1 is the
dipeptide inhibitor formed by the coupling reaction, and ks is the first order rate constant.
AA+ Nu ks. 1
Figure 5.10: General Scheme for library synthesis. AA is the solid supported activated amino acid, Nu is
the nucleophile, l is the dipeptide inhibitor formed by the coupling reaction and k. is the tirst order rate
constant for the coupling reaction.
The rate of the reaction of nucleophile to inhibitor, [1] is shown by equation 1.
v = _ d[Nu] = d[I] = k [Nu] dt dt S
The relationship between [1] and [Nu] is shown by equation 2.
[I] = [Nuo ] - [Nu] (2)
The simplest differential equation from Equation 1 is Equation 3.
d[Nu] =-k dt [Nu] S
(3)
The definite integral of 3 is given by Equation 4.
[Nu], In--2 = -k (t - t )
[Nu]'1 S 2 1 (4)
Using [Nu]o as the nucleophile concentration at tl = 0 and rearrangement gives Equation
5.
114
(5)
Substituting 5 into 2 gives Equation 6.
(6)
Equation 6 gives [1] in terrns oftime and initial [Nu]o, with a rate constant (ks) that can be
modified depending on the synthetic rates of individual inhibitors in the library. [2]
5.4.3 Modeling Receptor-Binding
The second event in a p-DCL is binding to the receptor. This is a
thermodynamically governed equilibration that favours tight binders. The relative
amounts of two compounds competing for binding sites were determined in order to
model this event (Figure 5.11).
lA + T KaA
IA-T ,
K'A
IB+ T KaB
IB-T K'B
Figure 5.11: Two inhibitors lA and lB compete for a biological target T (the receptor) with binding affinities
KaA and KaB' and inhibition constants K1A and KIB where Ka = K/l.
The equation for the inhibition constant of any inhibitor 1 is given by:
(7)
115
The total concentration of bound and unbound forms of 1 can be expressed as:
(8)
where [10 ] is the concentration ofinhibitor 1 after synthesis, and [1] is the concentration of
1 that is not bound to the receptor-target T (i.e. excess 1 from the synthesis).
Equation 6 is true for two inhibitors lA and lB if one assumes tight binding, that is,
at equilibrium nearly an the target is bound to an inhibitor (true for sub millimolar
inhibitors under our typical experimental conditions [7]).
(9a)
or equivalently:
(9b)
Taking the ratio of Equation 7 for each inhibitor lA and lB, gives:
(10)
Solving for [lA • T] gives:
(11)
Substituting Equation 9 into 8 in terms of [lB] gives:
(12)
Substituting Equations 12 and 9b into Il gives Equation 13.
(13)
Multiplying through gives the quadratic equation 10:
116
(KIIA - K1IJ[IA e Ty + (KhJ IBo]- K1IJTJ + KIJTJ + KIIJIAo])[IA eT]- K1IJTJ[ lA 0] = 0 (14)
Equation 10 can be solved using the quadratic formula and gives:
Similarly, solving for [lB eT] gives:
Equations 15 and 16 give the concentrations of each of the inhibitors lA and lB
bound to the receptor given that their initial concentration in the binding chamber after
synthesis [lAo] and [IBo], the concentration of the receptor-target T, and their inhibition
constants are known. AU of these parameters are easily accessible in an actual
experiment, and provide wide versatility in the parameters that can be tested in model p-
DCLs. Unfortunately, modeling more than two inhibitors at once becomes very complex.
5.4.4 Modeling Destruction
The last step in ap-DCL is the destruction ofunbound inhibitors (Figure 5.12). To
model this, we assumed that the equilibration process was fast compared to diffusion
(typical kon rates for sulfonamides range between 0.0033-31 x106 M-1s-1, or at least 3
associations of inhibitor to receptor per second at a (typical) 1 mM concentration of
library member, and koffrates range from 0.01-0.05 S-I)JS] After equilibrating, aH inhibitor
117
present in excess of the receptor was assumed to be hydrolyzed before the destruction
equation was applied.
I·T T+I _k_d_ .. ~ Nu + AA
Figure 5.12: The destruction of any unbound inhibitor 1 governed by its inhibition constant KI and its
kinetic rate constant kd. The destruction rate for ail compounds in the library should be the same, so the
removal of compounds should be based only on how far they lie on the unbound side ofthe equilibrium.
From Chapter two the destruction rate of an inhibitor 1 is given by equation 17.
d[IT] kdIKI)IT] --=-
dt [T]
Integrating equation 13 gives:
(18)
where [Ir] is the concentration of free inhibitor, [la] is the concentration of the inhibitor
bound to the receptor-target after the equilibrium of binding, [1'] is the concentration of
free target, t is time, kd is the destruction rate constant and Kd is the inhibition constant 1 1
ofthe inhibitor, I. Solving 18 for [Ir] gives equation 15:
(19)
Equation 19 gives [Ir] in terms of time, with manipulable parameters for the destruction
rate kd, and the inhibition constant KI. The concentration of free receptor-target T is
assumed to be constant. Although strictly not true, at higher conversions of inhibitor to
118
starting materials (at which points, from chapter two the selectivity starts to increase
dramatically), the [11»[Ir], and changes in [1] due to the change in concentration of 1
become negligible.
5.4.5 Discussion: The Integrated Model and its Comparison to Experiment
We applied Equations 6, 15 or 16 (for either lA or lB respectively) and 19 in
succession in a spreadsheet and then iterated the three equations over time using 16 h
cycles. Since the theoretical curves modelled two inhibitors it was directly compared to
the two inhibitor experiment shown in Figure 5.4 to normalize the ks and kd parameters to
give similar amounts of synthesis and destruction each cycle. [10] A curve was generated
showing the ratio of [lA] : [lB] over six, 16 h cycles (Figure 5.13).
6
= ... ~ 5 :S -= 4 0: ... < 3 ... ;§ ..Q
2 :El .s .. 1 '" .. =:
0
0 16 32 48
Time (h)
64 80 96
Figure 5.13:[9) Theoretical curve of the ratio oftwo inhibitors with K1s of 1.0 J.1M (+) and a 2.5J.1M (e)
generated from the above equations. Parameters for the curve were generated to most closely mimic the
amounts of synthesis and destruction observed in a 16 h experiment with two inhibitors (Figure 5.5). The
relative strengths of these inhibitors were chosen to correspond to those of the two strongest library
members, 7d and 7c respectively.
119
In this theoretical experiment, the ratio of the stronger inhibitor to the weaker
starts at one, corresponding to the equal synthesis of each inhibitor. The ratio jumps
during the short equilibration process (short compared to the time needed for synthesis
and diffusion-see reference #8) and then grows quickly over the destruction process. The
ratio drops towards one as a new cycle of synthe sis occurs, and then rises somewhat due
to the thermodynamic equilibration, and even more during the next destruction. Over the
next few cycles, the ratio seems to plateau reaching approximately 5.6 : 1 (lA: lB) after
six cycles.
This result is consistent with the steady state concentrations the inhibitors reach
after six 16-h cycles shown in Figure 5.4. The stronger inhibitor's concentration increases
with each successive cycle, and the weaker inhibitor's concentration decreases, which is
also observed experimentally. The ratio between the two inhibitors is 5.6 : 1 in the model,
compared with a ratio of approximately 10 : 1 seen experimentally.
The ratio of the two inhibitors after the first equilibration process is 1.7 : 1. This
theoretical number models the thermodynamic effect in the first cycle of the p-DCL. It
can be directly compared with an earlier result shown in chapter two, Figure 2.3, in which
inhibitors are allowed to selectively concentrate into a chamber containing carbonic
anhydrase (with no Pronase, therefore only thermodynamic effects could take place). In
that experiment, the ratio between the two strongest inhibitors 4a and 4b (with K1s of 1.2
/lM and 2.5 /lM respectively) was 1.1 : 1. The theoretical model may give a higher ratio
because there is an initial excess of each inhibitor associating with CA, which would
increase the thermodynamic selectivity, and because the modelled inhibitors have a
slightly higher ratio of inhibition constants.
120
After the first round of destruction, the ratio of inhibitors is 4.2 : 1. This higher
ratio is a result of the kinetic destruction influence. This theoretical result can be
compared with those depicted in Figure 2.6 in which 4a is selectively protected from
destruction over 4b. The ratio of 4a : 4b after 183 h in this experiment was 3.8 : 1. These
results cannot be directly compared with the theoretical ones because the theoretical
result from the first cycle did not start with the same concentration of inhibitors (the
equilibration process happened first). However, if one takes the destruction process alone,
and applies it to two inhibitors of the same strengths as 4a and 4b over 183 h, one finds a
final ratio of ~ 10 : 1. In the example shown in Figure 2.6, the starting concentration of the
weaker 4b was slightly higher than that of 4a, which would lower the final ratio.
However, discrepancy between these two numbers cannot as yet be fully explained.
The model seems to correlate with experiment. It suggests that the
thermodynamic contribution common to all p-DCLs plays a smaller role in determining
selectivity than does the kinetic destruction. The model also shows a potential drawback
to p-DCLs. Since the time needed to achieve high selectivity depends only on the relative
binding constants, p-DCLs can distinguish between inhibitors with similar binding
constants. However, the absolute strength ofthe binders will determine the time per cycle
needed by the destruction reaction to resolve them. If the strengths of the inhibitors are
increased ten-fold, the system requires a ten-fold increase in the destruction rate, or a ten
fold increase in the cycle time will be needed to achieve the same selectivity as when the
inhibitors were ten-fold weaker. The instability of many biological targets may render
these lengths of time impractical. However, this model does not take into account the
possible effect of having large library sizes.
121
The theoretical model assumes tight binding. This may not always be true of aIl
libraries and therefore the model will not predict what will happen when there are no
potent inhibitors in the library. This model also treats synthesis, binding and destruction
as completely separate events, which is not the case in an experimental p-DCL in which
diffusion of starting materials causes sorne attenuation of synthesis in aIl cycles but the
first, and thus may alter sorne of the theoretical results from reality. The final, main
limitation is that these theoretical models can only mode! two inhibitors at a time, and
thus cannot address the fundamental issue of increased selectivity in the presence of more
competition.
5.5 Overall Conclusions
The selectivity of a p-DCL is high enough to distinguish between inhibitors with
similar binding constants. Because decreasing the number of library members leads to
lower selectivity, it may follow that increasing the library size can actually improve the
selectivity. However, resolving stronger inhibitors requires longer cycles. The level of
amplification is a result of the strength of the inhibitor and the time given for destruction
per cycle.
In pseudo-dynamic libraries, thermodynamics provide the essential initial
selective binding to the receptor, but, as in dynamic libraries, this selectivity is often low.
The kinetic destruction during the temporary absence of synthesis winnows away non-,
and poor inhibitors and greatly improves the selectivity for the best binders. Iteration of
the synthesis-binding-destruction cycle aIlows betier binders to build up in the system,
122
giving amplification. Iteration also improves selectivity by re-introducing strong binders
to the system, allowing them to replace weaker binders that were not completely removed
in the previous destruction cycle.
The yield of any compound in a p-DCL is dependent on three factors: its absolute
binding strength, the length of time the destruction reaction is allowed to proceed
unabated by new synthesis, and the strength and number of other competitive binders in
the library. It is possible to allow the destruction process to run for too long, which would
decrease the yield of the best binding compound to undetectable levels. Therefore, in
each new p-DCL, because the selectivity increases as the destruction reaction proceeds, it
is necessary to optimize the cycle time to allow the destruction process to run long
enough to achieve good selectivity without decreasing the yield too much.
The selectivity is higher than in DCLs, and the particular receptor type should not
influence the success of these systems as it does in RAS. Further, no lock-in reaction is
needed as the library members are synthesized irreversibly. By combining the inherent
thermodynamic selectivity of a receptor towards a library of inhibitors of various
strengths with a kinetic removal of weak binders, the selectivity of a receptor-assisted
combinatorial system can be vastly increased .. Being able to iterate synthesis and
destruction steps, and to optimize the amount of resolution allowed to occur, gives a level
of control here-to-fore impossible in receptor-assisted methods.
The mathematical model appears to indicate that the destruction process is the
main determinant of selectivity. Since the destruction rate depends on the strength of the
inhibitors in the library (how fast they can unbind the receptor and be destroyed) stronger
inhibitors will require longer cycle times for resolution. The resolution time required by
123
very potent inhibitors (and hence the most interesting ones) might not be compatible with
receptor stability. This raises an important design issue in p-DCLs, in which receptor
stability must be made compatible with the apparatus and length of time needed for
selectivity. This may become a difficult requirement when using unstable receptors such
as membrane-spanning proteins.
Another design issue in p-DCLs is the nature of the kinetic destruction
component. The part of a p-DCL is not limited to peptide hydrolysis, but could include
other chemical reactions that destroy unbound library members, or physical separation
steps that remove them. An optimal destruction method must be designed to complement
each new type of library synthesis.
5.6 Future Endeavours
5.6.1 Improving the p-DCL Model
Currently the model can only handle two inhibitors at a time. This major
limitation must be overcome if the model is to have any predictive value for library
experiments practical size for drug discovery. Existing models in the field of systems
biology may be helpful directions to take in this regard. [11] The model also shows sorne
fundamental weaknesses in its treatment of certain variables, such as the concentration of
free target (assumed to be constant during the destruction process). Other assumptions
(such as the assumption that aH target is bound by inhibitors at the end of the
124
equilibration process) need to be eliminated as well before the model can be trusted to
give accurate predictions of the outcome of unknown p-DCLs.
5.6.2 Fundamental Experimentation in p-DCLs
Sorne future experiments in the eight-membered system will include further
characterization of the origins of amplification and selectivity with specific focus on the
effect of having more inhibitors in the system. A 12 hour cycle p-DCL experiment such
as the one shown in Figure 4.3b but with five or six inhibitors instead of four will directly
determine whether more inhibitors indeed leads to greater selectivity. Other 16 hour cycle
experiments using varying equivalents of each of the weaker inhibitors 7a, 7b, and 7c
will help determine the relative effects of inhibitors of various potency on the
amplification of the strongest inhibitor 7d. A 16-hour cycle experiment with no 7c will
also help determine the relative effects of weaker inhibitors 7a and 7b on 7d, and will be
directly comparable with the knockout experiment described in Figure 5.8.
5.6.3 Expansions and Miniaturizations
Future projects will be focused around the practical application of the p-DCL
method. Synthesis of new potential inhibitors will continue to be needed to create large,
125
diverse libraries suitable for p-DCLs. Since these experiments necessarily use
stoichiometric amounts of the receptor, a practical application would also require that the
experiment take place in a very small reaction vessel, so as not to use copious amounts of
the receptor. To this end, miniaturization of the experimental design is one of the most
important aspects that needs to be developed.
A new reaction vessel has only to prevent synthetic, or proteolytic modification of
the receptor. Dialysis equipment, including Microdialysis Buttons™ that separate JlL
volumes are available from Hampton Research (Hampton Research; Laguna Niguel,
CA)y21 These would be ideal reaction vessels in which to perform small-scale p-DCLs.
However, before this is possible, a new, soluble solid-phase library synthesis needs to be
developed because the small volumes will not be sufficient to swell a resin such as
TentaGel. Derivitized supports based on polyethylene glycol may be suitable for these
purposes.
Contribution of Authors
The author (Jeremy D. Cheeseman) with David Soriano deI Amo developed and
performed the synthesis of the library members shown in Figures 5.9-5.13.
The author (Jeremy D. Cheeseman) deveioped the theoretical model and performed all
other experiments described in this chapter.
References
126
1. Lines drawn in aIl figures (except Figure 5.12) are for illustration purposes only.
They do not represent theoreticallines of any sort.
2. This is not to say that the amount of nucleophile (assuming it carries the
inhibitory moiety) will not affect the yield in other systems. If the relative strength
of the nucleophile to the library members is increased, the nucleophile will
probably have a more notice able effect.
3. C. T. Supuran, A. Scozzafava Expert. Opin. Ther. Patents, 2002,12,217-242 and
references therein.
4. L. R. Scolnick, A. M. Clements, J. Liao, L. Crenshaw, M. HeIlberg, J. May, T. R.
Dean, D. W. ChristiansonJ Am. Chem. Soc., 1997,119,850-851.
5. It is important to note that for the purposes of examining amplification and
selectivity alone, the exact order of the synthesis is not important. For modeling
purposes it is only necessary to have the required manipulable parameters [Nu]
and ks.
6. Adapted from: 1. Tinoco Jr., K. Sauer, J. C. Wang, Physical Chemistry:
Principles and Applications in Biological Sciences 3rd Ed; Prentice Hall: Upper
Saddle River, New Jersey, 1995; pp 331-333.
7. In our experiments a typical receptor concentration [T] is 1.5 mM. Since
K = [1- T] , for Equation 9 to be true, a, [l][T] [I-T]»[l],or Ka [T]~lO.Inour ,
system, this equates to Ka, ~ 1.4xl0-4 M- 1, or a KIt of 0.15 mM or better. AlI our
inhibitors fall well below this limiting KI.
8. B. W. Clare, C. T. Supuran, Eur. J Med Chem. 1997,32,311-319.
127
9. J. D. Cheeseman, A. D. Corbett, R. Shu, J. Croteau, J. L. Gleason, R. J.
Kazlauskas J. Am. Chem. Soc. 2002,124,5692-5701.
10. Table 5.3: Parameters used to generate Figure 5.6
Inhibitor Kj(M) [Nu] (M) ks (sol) [1] (M) kd (soi)
lA 1.0xlO06 4.0xlO03 2.0xlOoS 1.4xlO03 3.0xlO02
lB 2.5xlO06 4.0xlO03 2.0xl00S 1.4xl003 3.0xlO02
Il. Q.-H. Chen, D. B. Bylund, Receptors & Signal Transduction, 1997, 7, 73-84 and
references therein.
12. Hampton Research Tools Catalogue, 2004, 11, 77-80.
128
CHAPTERSIX
EXPERIMENTAL SECTION
129
6.1 Experimental Section for Chapter 2
General Experimental:. p-Nitrophenyl acetate (PNPA), carbonic anhydrase (CA, from
bovine erythrocytes, a mixture of isozymes, C-3934) and proteases were purchased from
Sigma unless otherwise noted and used without further purification. HPLC analyses were
conducted using a Phenomenex-Cs reversed phase HPLC column (10 x 250 mm) with
detection at 220 nm, unless noted. Elemental analyses were obtained from Quantitative
Technologies Inc. Whitehouse, NJ. High resolution mass spectra were obtained from
Université de Sherbrooke, Sherbrooke, QC.
4'-Sulfonamidophenylalanine (1): N-Acetylphenylalanine (37.7 g, 178 mmol, 1 eq) was
added in portions over a 1 h period to neat chlorosulfonic acid (110 mL, 1.65 mol, 9.5 eq)
at -10°C. The resulting yellow solution was stirred at -10°C for 2.5 h, at 25 oC for 2.5
h, and then heated to 60 oC until gas evolution had ceased (approx. 12 h). The resulting
orange solution was cooled to 0 oC and poured carefully onto 750 mL of ice (Caution:
exotherm!). The resulting mixture was extracted with ethyl acetate (3 x 1 L) and the
combined organic layers were dried over Na2S04, filtered and concentrated in vacua to
afford the sulfonyl chloride (45.1 g, 83%) as an orange solid which was used immediately
without further purification .. IH NMR «CD3)2S0) 8.26-8.21 (d, IH, J = 8.5 Hz), 7.55 (d,
2H, J= 6.9 Hz), 7.22 (d, 2H, J= 6.8 Hz), 4.49-4.34 (m, IH), 3.13-3.00 (dd, IH, J= 14.4
and 6.8 Hz), 2.92-2.79 (dd, IH, J= Il.0 and 10.2 Hz), 1.80 (s, 3H).
The sulfonyl chloride was dissolved in 28% N~OH (240 mL) and the resulting
solution was heated at reflux for 3 h. After cooling to 0 oC, the solution was acidified to
130
pH 1 by addition of 3 M H2S04 (ca. 200 mL) and extracted with ethyl acetate (3 x 500
mL). The combined organic extracts were dried over Na2S04, filtered and concentrated in
vacuo to afford the sulfonamide (29.9 g, 71 %) as a white solid. The N-acetyl sulfonamide
could not be purified to homogeneity by either chromatography or recrystallization. IH
NMR «CD3)2S0) Ô 8.29-8.24 (d, IH, J= 8.5 Hz), 7.77 (d, 2H, J= 3.9 Hz), 7.45 (d, 2H, J
= 6.9 Hz), 7.33 (s, 2H), 4.53-4.41 (m, IH), 3.20-3.09 (dd, IH, J= 14.2 and 6.8 Hz), 3.01-
2.87 (dd, IH, J= 11.2 and 10.1 Hz), 1.80 (s, 3H).
A suspension of the sulfonamide (20.0 g, 69.9 mmol, 1 eq) in distilled water (300
mL) was adjusted to pH 5.00 with LiOH (900 mg). A 0.25 M solution of Na2HP04 (85
mL) was used to raise the pH to 7.50. Acylase 1 from hog kidney (200 mg, 17.8 U/mg,
3560 U) was added as an aqueous solution (12 mL) and the resulting solution was stirred
at 21°C for 70 h. The solution was then acidified to pH 1.0 with 3M H2S04 and extracted
with ethyl acetate (3 x 500 mL), the organic layer was then dried with anhydrous sodium
sulphate and concentrated in vacuo to afford 2.28 g (11 %) of the sulfonamide starting
material. The aqueous layer was the neutralized with 2 M NaOH and concentrated. The
solution was then applied to an Amberlite 120(plus) acidic ion exchange column. The
column was rinsed with water until the eluent was at pH 6.0 and the it was rinsed with
0.50M NH40H solution until the eluent became basic. The basic wash was concentrated
in vacuo and recrystallized from water to afford provided 4'-sulfonamidophenylalanine as
a white solid (11.60 g, 68%). IH NMR (D20 / DCI) Ô 7.62 (d, 2H,J= 8.1 Hz), 7.26 (d,
2H, J= 8.1 Hz), 4.14 (t, IH, J= 6.8 Hz), 3.19-3.12 (dd, IH, J= 14.6 and 5.7 Hz),3.08-
3.01 (dd, IH, J = 14.4 and 6.9 Hz). l3e NMR (D20 / DCI)
131
8170.73,140.451,139.49,130.38,126.55,53.49,35.19. FABMS in satNaCI
m/z 267 (M+ Na, C9H12N204SNa requires 267).
N-Ethoxycarbonyl-4'-sulfonamidophenylalanine (2): Ethyl chloroformate (398 ilL,
4.17 mmol, 1.10 eq) was added to a two phase mixture of 4'-sulfonamidophenylalanine
(925 mg, 3.73 mmol, 1 eq) in 1,4-dioxane (25 mL) and sat. NaHC03 solution (25 mL) at
o oC and the resulting solution was stirred for 6 h at 0 oC. The mixture was extracted with
ethyl acetate (100 mL), the aqueous layer was acidified to pH 1 by addition of2 M HCI
(ca. 20 mL) and then extracted with ethyl acetate (3 x 50 mL). Latter organic extracts
were combined, dried over Na2S04, filtered and concentrated in vacuo to afford the ethyl
carbamate (879 mg, 83%) as an analytically pure oil. IH NMR «CD3)2CO) 8 7.85 (d,
2H, J= 7.1 Hz), 7.51 (d, 2H, J= 6.9 Hz), 6.54 (s, 2H), 6.45 (d, 1H, J= 6.7 Hz), 4.62-4.45
(m, 1H), 4.01-3.97 (q, 2H, J= 2.4 Hz), 3.41-3.28 (dd, 1H, J= 11.3 and 4.0 Hz), 3.16-3.05
(dd, 1H, J = 10.4 and 7.8 Hz), 1.13 (t, 3H, J = 6.0 Hz). HR-CIMS (mlz): [MH+]
calculated for C12H17N206S, 317.0807; found, 317.0817.
Et02C-(4'-S02NH2)Phe-Gly-O-t-butyl (3b): EDC-HCI (136 mg, 0.711 mmol, 1.10 eq),
HOBT (87.3 mg, 0.646 mmol, 1.00 eq) and triethylamine (269 mL, 1.94 mmol, 3.00 eq)
were added to a solution of 2 (204 mg, 0.646 mmol, 1 eq) in THF (3 mL) at 0 oC.
Glycine t-butyl ester-HCl (119 mg, 0.711 mmol, 1.10 eq) was added and the resulting
solution was allowed to warm to 21°C while stirred for 13 h, at which point the bulk of
the THF was removed by concentration in vacuo. The residue was dissolved in ethyl
acetate (45 mL) and extracted with 0.1 M HCI (3 x 25 mL) and sat. NaHC03 solution (3
132
x 25 mL). The organic layer was dried over Na2S04, filtered and concentrated in vacuo.
The solid residue was purified by mixed solvent recrystallization (ethyl acetate/hexanes)
to afford 193 mg (70%) of 3b. lH NMR «CD3)2CO) ô 7.82 (d, 2H, J= 7.5 Hz), 7.63 (s,
1H), 7.50 (d, 2H, J= 7.5 Hz), 6.52 (s, 2H), 6.40 (d, 1H, J= 7.5 Hz), 4.50 (m, 1H), 4.00-
3.88 (m, 4H), 3.40 (dd, 1H, J= 14.1 and 4.2 Hz), 3.02 (dd, 1H, J= 13.5 and 9.9 Hz), 1.45
(s, 9H), 1.12 (t, 3H, J= 6.9 Hz). BC NMR «CD3)2CO) ô 171.48, 168.93, 156.33, 142.70,
130.06, 126.18, 81.04, 60.48, 55.92,41.79, 37.83, 27.52, 14.22. Analysis calculated for
ClsH27N307S C, 50.34; H, 6.34; N, 9.78. Found: C, 50.33; H, 6.35; N, 9.73.
Et02C-(4'-S02NH2)Phe-Gly-OH (4b): TFA (7 mL) was added to a solution of3b (175
mg, 0.409 mmol, 1 eq) in CH2Ch (8 mL) and the solution was stirred for 25 min at 21°C
under an atmosphere of argon. The solvents were removed in vacuo, and the residue was
purified by recrystallization from acetone to afford 121 mg (79 %) of 4b. lH NMR
(CD30D) ô 8.55 (s, 1H), 7.83 (d, 2H, J= 7.2 Hz), 7.46 (d, 2H, J= 7.2 Hz), 4.45-4.42 (m,
1H), 4.02-3.98 (q, 2H, J=6.8), 3.95-3.92 (m, 1H), 3.32-3.25 (m, 2H), 2.97-2.89 (dd, 1H,
J= 13.5 and 9.9 Hz), 1.18-1.14 (t, 3H, J= 6.8). l3C NMR (CD3CD) ô 173.0, 171.8,
157.3, 148.7, 142.4, 137.6, 129.8, 126.0,60.9,56.0,37.7, 13.7. HR-CIMS (mlz): [MH+]
calculated for Cl4H20N307S, 374.1022; found, 374.1030.
Et02C-(4'-S02NH2)Phe-Phe-O-t-butyl (3a): Prepared as for 3b to afford to afford 173
mg (72%). lH NMR (CDCh) ô 7.80 (d, 2H, J= 7.8 Hz), 7.33-7.24 (m, 5H), 7.09 (d, 2H, J
= 6.0 Hz), 6.41 (d, 1H, J= 5.7 Hz), 5.19 (d, 1H, J= 6.1 Hz), 4.96 (s, 2H), 4.75-4.61 (m,
1H), 4.52-4.38 (m, 1H), 4.10-4.03 (q, 2H, J= 6.9 Hz), 3.20-2.96 (m, 4H), 1.39 (s, 9H),
133
1.21 (t, 3H, J= 6.8 Hz). l3C NMR (CDCh) Ô 170.34,170.11,142.21,140.84,136.03,
130.38, 129.65, 128.67, 127.31, 126.96, 82.93, 77.44, 61.76, 53.86, 38.40, 38.21,28.16,
14.69. Analysis calculated for C25H33N307S C, 57.79; H, 6.40; N, 8.09. Found: C, 57.71;
H, 6.34; N, 7.96.
Et02C-(4'-S02NH2)Phe-Leu-O-t-butyl (3e): Prepared as for 3 b to afford 227 mg
(78%). IH NMR «CD3)2CO) Ô 7.81 (d, 2H, J= 8.4 Hz), 7.55 (m, 2H), 7.48 (d, 2H, J=
8.1 Hz), 6.51 (m, 1H), 6.31 (m, 1H), 4.50 (m, 1H), 4.39 (m, 1H), 4.00-3.95, (q, 2H, J=
5.7), 3.32-3.26 (dd, 1H, J= 13.8 and 3.9 Hz), 3.05-2.97 (dd, 1H, J= 13.6 and 9.6 Hz),
1.75-1.64 (m, 2H), 1.61-1.56 (m, 2H), 1.45 (s, 9H), 1.12 (t, 3H, J= 7.4 Hz), 0.94-0.90
(m, 7H). l3C NMR «CD3)zCO) Ô 171.8, 171.0, 156.4, 142.5, 130.1, 126.2, 81.0, 60.5,
55.7, 51.6, 41.1, 37.8, 27.5, 24.8, 22.5, 21.3, 19.5, 14.2. Analysis calculated for
C22H35N307S C, 54.42; H, 7.26; N, 8.65. Found: C, 54.28; H, 1.27; N, 8.46.
Et02C-(4'-S02NH2)Phe-Pro-O-t-butyl (3d): Prepared as for 3 b to afford 312 mg
(67%). IH NMR «CD3)2S0) Ô 7.69 (d, 2H, J = 7.6 Hz), 7.48 (d, 2H, J = 7.0 Hz),7.36 (d,
2H, J = 7.2 Hz), 7.28 (s, 2H), 4.36 (m 1H), 4.19 (m, 1H), 3.85 (t, 2H, J = 6.7 Hz), 3,65
(m, 2H), 2.97-2.94 (dd 1H, J= 11.7 and 4.2 Hz), 2.82-2.74 (dd 1H, J= 11.1 and 9.2 Hz),
2.14 (m, IH), 1.90 (m, 2H), 1.77 (m, IH), 1.35 (s, 9H), 1.04 (t, 3H, J= 7.3 Hz). l3C NMR
«CD3)2S0)Ô 171.6, 170.4, 156.8, 143.0, 142.7, 130.5, 126.1,81.0,60.5,60.1,54.7,47.1,
36.4, 29.2, 28.3, 25.3, 15.2. Analysis calculated for ~lh7N307S C, 50.34; H, 6.34; N,
9.78. Found: C, 50.33; H, 6.35; N, 9.73.
134
Et02C-(4'-S02NH2)Phe-Phe-OH (4a): Prepared as for 4b to afford 270 mg (68 %). lH
NMR (CD30D) ô 8.19 (d, 1H, J= 9.3 Hz), 7.80 (d, 2H, J= 8.4 Hz), 7.38 (d, 2H, J= 9.3
Hz), 7.27-7.21 (m, 5H), 7.07 (d, 1H, J= 8.7 Hz), 4.68-4.63 (m, 1H), 4.40-4.35 (q, 2H, J
=6.1 Hz), 3.24-3.18 (dd, 1H, J=13.9 and 5.2 Hz), 3.18-3.11 (dd, 1H, J=14.3 and 5.5 Hz),
3.04-2.97 (dd, 1H, J= 13.9 and 8.2 Hz), 2.88-2.80 (dd, 1H, J= 13.9 and 9.7 Hz), 1.18-
1.14 (t, 3H, J= 6.9 Hz). l3C NMR (CD3CD) ô 173.1, 172.3, 157.2, 142.3, 137.0, 129.8,
129.2, 128.3, 126.6, 126.0, 60.9, 55.9, 53.9, 37.6, 37.2, 13.7. HR-CIMS (m/z): [MH+]
calculated for C2lH26N307S, 464.1491; found, 464.1501.
Et02C-(4'-S02NH2)Phe-Leu-OH (4c): Prepared as for 4b to afford 174 mg (80 %). lH
NMR (CD3CD) ô 7.82 (d, 2H, J= 8.4 Hz), 7.68 (d, 1H, J= 8.1 Hz), 7.45 (d, 2H, J= 8.1
Hz), 4.47-4.42 (m, 2H), 4.01-3.95 (q, 2H, J= 6.4 Hz), 3.31-3.29 (m, 1H), 3.25-3.3.19
(dd, 1H, J = 13.9 and 4.9 Hz), 2.95-2.87 (dd, 1H, J = 13.9 and 9.7 Hz), 1.71-1.62 (m,
2H), 1.18-1.13 (t, 3H, J= 7.1 Hz), 0.97-0.91 (m, 6H). l3C NMR (CD3CD) ô 174.6,
172.7, 157.2, 142.3, 129.8, 126.7, 126.0, 60.9, 55.8, 50.9, 40.4, 37.7, 24.8, 22.2, 20.6,
13.7. HR-CIMS (m/z): [MH+] calculated for ClsH2SN307S, 430.1648; found, 430.1654.
Et02C-(4'-S02NH2)Phe-Pro-OH (4d): Prepared as for 4b except recrystallized from
iso-propanol / hexanes to afford 94.5 mg (55 %). lH NMR (CD30D) ô 7.83 (d, 2H, J =
8.1 Hz), 7.50 (d, 2H, J= 8.4 Hz), 7.25 (d, 1H, J= 3.9 Hz), 4.66-4.61 (dd, 1H, J= 8.8 and
5.5 Hz), 4.47-4.43 (dd, 1H, J= 8.4 and 3.9 Hz), 4.02-3.95 (q, 2H, J= 7.2 Hz), 3.80-3.75
(m, 1H), 3.56-3.51 (m, 1H), 3.32-3.29 (m, 1H), 3.21-3.14 (dd, 1H, J= 13.9 and 5.2 Hz),
135
2.96-2.89 (dd, IH, J= 13.8 and 8.7 Hz), 2.27-2.21 (m, IH), 2.05-1.96 (m, 2H), 1.19-1.16
(t, 3H, J=7.2 Hz. BC NMR (CD3CD) ô 174.0, 171.1, 157.3, 141.9, 130.1, 129.1, 128.2,
126.0, 60.8, 59.4, 53.9, 37.0, 29.0, 24.6, 13.7. HR-CIMS (mlz): [MH+] calculated for
C17H24N307S, 414.1335; found, 414.1325.
Measurement of Inhibition Constants: Kinetic constants for carbonic anhydrase (CA)
were measured according to Pocker and Stone using p-nitrophenyl acetate (PNPA) as the
substrateYl The CA-catalyzed hydrolysis of pNPA was followed spectrophotometrically
at 25 OC in a 96-well microplate spectrophotometer by monitoring the appearance of p
nitrophenolate at 404 nm. The values of Km and V max were determined by measuring
the hydrolysis rate as a function of the pNP A concentration. To determine the inhibition
constants, the values of Km and V max were re-determined in the presence of varying
amounts of inhibitor. Since the values of Km for pNP A increased in the presence of the
inhibitor, but the values of V max remained unchanged, we concluded that the inhibition
is competitive. The concentration of inhibitor that increased the Km for pNP A by a factor
oftwo is the inhibition constant. A typical procedure was to add CA solution (100.0 mL)
with inhibitor to acetonitrile solution of pNPA (5.0 mL). In the assay solution, the
concentration of inhibitor ranges from 0.0 to 6.0 J.lM, while the concentration of pNPA
ranged from 0.2 to 2.5 mM. The microplate was shaken for 5 s before the first reading
and for 3 s between readings.
Selective Concentration of EtOC-Phesa-Phe (4a) Over EtOC-Phe-Phe, (5) Into a
Compartment Containing Carbonic Anhydrase: A solution of 4a (2.9 mg, 6.3 mmol)
136
and 5 (2.9 mg, 7.5 mmol) in 0.01 M aqueous phosphate buffer (pH 7.5, 40 mL) was
divided into two equal portions. Carbonic anhydrase (0.20 g, approx 6.7 mmol) was
dissolved in the first portion and the resulting solution (20.0 mL) was transferred to a
dialysis bag (12,000 MW cutoff, Sigma D-0655). This dialysis bag was suspended in the
second portion and the reaction vessel was shaken gently (200 rpm) at 30 oC. Aliquots
were removed periodically from each compartment, heated to 80 oC until a white
precipitate formed (~5 min), centrifuged and the supematant filtered through a 0.22 /lm
pore filter. The amount of dipeptides was measured by HPLC using a Zorbax C8 column
and 40/60/0.1 water/methanol/trifluoroacetic acid at 0.40 mL/min. After 12 hours 88% of
4a (retention time Il.4 min) had accumulated inside the dialysis bag while only 42% of 5
(retenti on time 25.5 min) was found inside the bag.
Selective Concentration of Etoc-Phesa-Phe (4a) from a Mixture of Etoc-Phesa-Leu
(4c), Etoc-Phesa-Gly (4b), and Etoc-Phe-Phe (5) by Carbonic Anhydrase: Dipeptides
4a (2.0 mg, 4.3 /lmol), 4b (1.6 mg, 4.3 /lmol), 4c (1.9 mg, 4. /lmol) 4d (1.8 mg, 4.3
/lmol) and 5 (1.7 mg, 4.3 /lmol) were dissolved in 40 mL of 10 mM KH2P04 buffer, pH
7.5 containing 0.1 mg/mL penicillin G (to avoid bacterial growth). Carbonic anhydrase
(CA) (0.29 g, 9.7 /lmol, 0.45 eq) was dissolved in 20 mL ofthis solution and placed in a
dialysis bag (the bag was washed in ddH20 for 1 hr, rinsed in EtOH once and then
washed again with ddH20). The bag was suspended in the remaining 20 mL of inhibitor
solution in a lOO-mL container and shaken at 60 rpm on a 3 dimensional orbital shaker at
room temperature for 49 hrs. Samples (1 mL) were taken periodically from inside and
outside the dialysis bag, heated in an 80°C water bath for 5 min and then centrifuged for
137
10 min. The supernatant was filtered through a 0.22 I-tM sterile filter. The supernatant
(700 I-tL) was added to MeOH (300 I-tL) to form the HPLC sample (30 % MeOH, 70%
aqueous). The sample was run on a Phenomenex C8 reverse phase column under the
following conditions: 0-15 min 30% MeOH, 70% H20, 15-60 min 37% MeOH 63%
H20, 60-90 min 62% MeOH, 38% H20. The peak are as were monitored: PhesaGly: 7.9
min, PhesaPro: 17.6 min, PhesaLeu: 54.5 min, PhesaPhe: 60.0 min, PhePhe: 69.5 min. The
percentages are accurate to +/- 2%. AlI non-sterile apparatus used was autoclaved prior to
use to avoid bacterial growth.
Screening of Proteases for the Hydrolysis of Etoc-Phe-Phe Dipeptide (4a): The
protease to be screened (0.1 mg) was added to a solution of 4a (1.0 mg, 2.2 Ilmol) in 0.01
M aqueous phosphate buffer (pH 7.5). The solution was kept at 30 oC and aliquots were
removed periodically worked up as above, and analyzed by HPLC.
Selective Protection of Inhibitors from Hydrolysis by Carbonic Anhydrase: A
solution of 4a (3.0 mg, 6.5 mmol) and 5 (2.8 mg, 7.3 mmol) in 0.01 M aqueous
phosphate buffer (pH 7.5, 40 mL) was divided into two equal portions. Carbonic
anhydrase (0.20 g, approx 6.7 mmol) was dissolved in the first portion and the resulting
solution (20.0 mL) was transferred to a dialysis bag (12,000 MW cutoff, Sigma D-0655).
Pronase from Streptomyces griseus (Sigma P-5147, 4 mg) was dissolved in the second
portion and the dialysis bag was then suspended in the resulting solution. The reaction
vessel was then shaken gently (200 rpm) at 30 oC and aliquots were removed periodically
from each compartment, worked up as above, and analyzed by HPLC. After 30 min,
138
neither substrate was detectable in the solution outside the dialysis bag. Inside the dialysis
bag, 78% of 5 had hydrolyzed while only 6% of 4a had hydrolyzed after 6 h. In a control
experiment containing no carbonic anhydrase, inside the dialysis bag, 76% of 4a and
80% of 5 had hydrolyzed after 6 h.
Selective Binding of Etoc-Phesa-Phe (4a) over Etoc-Phesa-Leu (4c): A solution of 4a
(3.3 mg 7.1 /lmol) and 4c (3.6 mg, 8.4 /lmol) in 0.01 M aqueous phosphate buffer (pH
7.5,40 mL) was divided into two equal portions. Carbonic anhydrase (0.20 g, approx 6.7
mmol) was dissolved in the first portion and the resulting solution (20.0 mL) was
transferred to a dialysis bag (12,000 MW cutoff, Sigma D-0655). This dialysis bag was
suspended in the second portion and reaction vessel was shaken gently (200 rpm) at
30°C. Aliquots were removed periodically from each compartment, worked up as above,
and analyzed by HPLC using a Zorbax C8 column. After 12 hours 98% of 4a had
accumulated inside the dialysis bag while only 60% of 4c was found inside the bag.
Hydrolysis of Etoc-Phesa-Gly (4b) and Etoc-Phesa-Phe (4a) in the Presence of
Carbonic Anhydrase: PhesaPhe 4a (2.0 mg, 4.3 /lmol) and PhesaGly 4b (1.6 mg, 4.3
/lmol), were dissolved in 20 mL of 10 mM KH2P04 buffer, pH 7.5. Carbonic anhydrase
(CA) (0.4090 g, 13.6 /lmol, 1.60 eq) was dissolved in this solution and placed in a
dialysis bag (the bag was washed in ddH20 for 1 h, rinsed in EtOH once and then washed
again with ddH20). The bag was suspended in 20 mL of the phosphate buffer containing
Pro nase from Streptomycese griseus (5.0 mg, 0.01 eq) in a 150 mL beaker and shaken at
150 rpm at 30 oC for 313 hrs. Samples (1 mL) were taken periodically from inside
139
worked up as above and analyzed by HPLC. After 193 h, only 71% of 4a had hydrolyzed
while 93% of 4b had hydrolyzed.
Hydrolysis of Etoc-Phesa-Leu (4c) and Etoc-Phesa-Phe (4a) in the Absence of
Carbonic Anhydrase: A solution of 4a (2.9 mg 6.3 ~mol) and 4c (2.4 mg, 5.6 ~mol) in
0.01 M aqueous phosphate buffer (pH 7.5, 40 mL) was divided into two equal portions.
The first portion was transferred to a dialysis bag (12,000 MW cutoff, Sigma D-0655).
Pronase from Streptomyces griseus (Sigma P-5147, 4 mg) was dissolved in the second
portion and the dialysis bag was then suspended in the resulting solution. The reaction
vessel was then shaken gently (200 rpm) at 30 oc and aliquots were removed periodically
from each compartment, worked up as above, and analyzed by HPLC using a Zorbax C8
column. After 30 min, neither substrate was detectable in the solution outside the dialysis
bag. After 8 h, 86% of 4a and 88% of 4c inside the dialysis bag had hydrolyzed.
Hydrolysis of Etoc-Phesa-Leu (4c) and Etoc-Phesa-Phe (4a) in the Presence of
Carbonic Anhydrase: A solution of 4a (2.9 mg, 6.3 ~mol) and 4c (2.4 mg, 5.6 ~mol) in
0.01 M aqueous phosphate buffer (pH 7.5, 40 mL) was divided into two equal portions.
Carbonic anhydrase (0.14 g, approx 4.7 mmol) was dissolved in the first portion and the
resulting solution (20.0 mL) was transferred to a dialysis bag (12,000 MW cutoff, Sigma
D-0655). Pronase from Streptomyces griseus (Sigma P-5147, 4 mg) was dissolved in the
second portion and the dialysis bag was then suspended in the resulting solution. The
reaction vessel was then shaken gently (200 rpm) at 30 oC and aliquots were removed
140
periodically from each compartment, worked up as above, and analyzed by HPLC. After
6 h, 93% of 4c had hydrolyzed while only 58% of 4a was hydrolyzed.
Hydrolysis of Etoc-Phesa-Phe (4a), Etoc-Phesa-Gly (4b) Etoc-Phesa-Leu (4c), Etoc
Phesa-Pro (4d) and Etoc-Phe-Phe (5), in the Presence of Carbonic Anhydrase:
PhesaPhe 4a (2.0 mg, 4.3 !-lmol), PhesaGly 4b (1.6 mg, 4.3 !-lmol), PhesaLeu 4c (1.9 mg, 4.
!-lmol), PhesaPro 4d (1.8 mg, 4.3 !-lmol) and PhePhe 5 (1.7 mg, 4. !-lmol) were dissolved in
20 mL of 10 mM KH2P04 buffer, pH 7.5. Carbonic anhydrase (CA) (0.7670 g, 25.6
!-lmol, 1.20 eq) was dissolved in this solution and placed in a dialysis bag (the bag was
washed in ddH20 for 1 h, rinsed in EtOH once and then washed again with ddH20). The
bag was suspended in 20 mL of the phosphate buffer containing Pronase from
Streptomycese griseus (4.9 mg, 0.01 eq) in a 150 mL beaker and shaken at 150 rpm at 30
oC for 193 hrs. Samples (l mL) were taken, worked up as above, and analyzed by HPLC.
6.2 Experimental Section for Chapter 3
Resin Preparation[21: Amino-Tentagel resin (2.0g, 0.88 mmol, 1.0 eq, Novabiochem)
was swelled in distilled THF (30 mL) in a 50-mL coarse-fritted peptide synthesis vessel
(Chemglass). 2,3,5,6-Tetrafluoro-4-hydroxy-benzoic acid (925mg, 4.40 mmol, 5.0 eq,
Aldrich) was added to the resin suspension and the reaction vessel was shaken lightly for
5 min. Pyridine (1.52 mL, 17.6 mmol, 20 eq) was added followed by
diisopropylcarbodiimide, (690 I!L, 4.40 mmol, 5 eq). The resulting mixture containing
the resin suspended in a milky white solution was gently rocked for 16 h. During this
141
time the solution becomes clear. The resin was washed with THF (2x50 mL), DMF (2x50
mL), CH2Ch (50 mL), DMF (50 mL),and then THF (50 mL). The resin was then
suspended in THF «30 mL) and again treated with 2,3,5,6-tetrafluoro-4-hydroxy-benzoic
acid, pyridine and DIC in the manner described above. After the final wash, the resin was
swelled in THF (30 mL), tert-butylamine (1.85 mL, 17.6 mmol, 20.0 eq) was added and
the mixture was gently rocked for 16 h. The resin was subsequently washed with THF
(4x50 mL) and was ready for use.
Resin Activation and Quantification: The batch of resin prepared above was swelled in
distilled THF (20 mL). A 0.50 M solution of EtOC-Phe-OH (10.2 mL, 8.80mmol, 10 eq)
in THF was added, followed by diisopropy1carbodiimide (1.38 mL, 8.80 mmol, 10.0 eq)
and the resulting mixture was gently rocked for 16 h. The resin was washed THF (2x50
mL) and DMF (2x50 mL), and then rocked for 2 h in THF (30 mL). The resin was
washed with THF (2x50 mL), DMF (2x50 mL), CH2Ch (1x50 mL), and then THF (2x50
mL).
To quantify the amount of active ester on the resin, it was suspended in THF (30
mL), tert-butylamine (1.85 mL, 17.6 mmol, 20.0 eq) was added, and the resulting mixture
was gently rocked for 16 h. The resin was then washed with THF (3x50 mL). The filtrate
was concentrated in vacuo and the residue was taken up in ethyl acetate and washed with
O.lM HCI (3x30 mL), water (30 mL) and saturated NaHC03 solution (3x 30ml). The
organic layer was dried over anhydrous Na2S04 and the solvent removed in vacuo to
afford 212mg (0.72 mmol) of EtOC-Phe-N-t-Bu as a foamy off-white solid, indicating an
142
82% loading of active ester relative to the initial number of amine termini on the
T enta Gel resin.
General Procedure for Testing Pronase Activity Towards Dipeptide Hydrolysis: A
series ofPronase solutions containing 0.1 mg (~5 nmol), 10 mg (~0.5 /lmol), 100 mg (~5
/lmol) and 0.5 g (~20 /lmol) are prepared in either 30 mM NaH2P04 buffer containing 0.1
mg/mL PEN Na, pH 7.5, or 30 mM BI CINE buffer containing 0.1 mg/mL PEN Na, pH
9.0 in steri1ized glass vials. Approximately 10 /lmol of the dipeptide is added to each vial
containing Pronase, and to one vial with only buffer (control). The vials are then shaken
gently for several days, with samples taken during the first four hours, and then semi
daily. Each sample is heated to 80°C for ~5 min., centrifuged for 10 min and filtered
through a 0.22 um sterile filter to form the HPLC sample. Samples are then run through a
Phenomenex C8 (2) column monitoring at 220 nm under various separation conditions.
Eight Membered p-DCL: Gly, Pro, Leu, Phe as Nucleophiles, EtocPhe and
EtocPhesa as Electrophiles on Solid Support: A stock amino acid solution was prepared
in 500 mL ddH20 by adding Phe (0.413 g, 2.50 nunol), Leu (0.328 g, 2.50 mmol), Pro
(0.288 g, 2.50 nunol), Gly (0.188 g, 2.50 nunol), BI CINE (2.45 g, 30 mM) and PEN-Na
(0.05 g, 0.1 g/mL). Each amino acid was 5 mM. This stock was adjusted to pH 9.0 and
used to dissolve/suspend aIl components of the system. Tentagel resin with activated
ester, either 0.45 g or 0.9 g, (~0.80 E-4 or 1.6E-4 respectively) mol active ester, (~1.5 or
3 eq EtocPhesa (2), 1.5 or 3 eq EtocPhe wrt CA) was dissolved in 10 mL AA stock (this
gave 1.25 eq of AA's wrt resin in the chamber to begin). This suspension was placed in a
143
washed dialysis bag (MWCO 12 kDa) and agitated manually for ~10 sec to promote resin
swelling. It was then placed in a 100 mL container. Pronase (0.5 g) was dissolved in 20
mL AA stock, injected into a dialysis bag (MWCO 1 kDa) and placed beside the resin
bag in the container. CA (0.84 g, 2.8E-5 mol, 1 eq) was dissolved in 20 mL AA stock and
poured over the two bags in the container forming the immersion solution. The eq of each
AA in the whole system wrt CA was 9.75. The system was set to stirring on a TW3
orbital shaker (Rose Scientific) for 48 hrs. The entire synthe sis chamber was replaced
(new resin) after either 12 or 24 hrs. In sorne cases the entire Pronase chamber was
replaced (new Pronase in new AA stock) after 24 hrs. 200 !AL samples were taken every 2
hrs, heated to 80°C for ~ 5 min., centrifuged for 10 min and filtered through a 0.22 um
sterile filter to form the HPLC sample. Samples were mn through a Phenomenex C8 (2)
column monitoring at 220 nm under the following conditions: 0-30 min gradient 100%
H20/O.l%TFA, 0% MeOH to 70% H20/O.1%TFA, 30% MeOH; 30-70 min 63%
H20/O.l% TFA, 37% MeOH; 70-90 min, gradient 63% H20/0.l%TFA, 37% Me OH to
38% H20/O.1 %TFA, 62% MeOH. The main peaks monitored were EtocPhesaGly (4b) at
25.2 min, EtocPhesaLeu (4c)at 57.4 min and EtocPhesaPhe (4a)at 58.6 min.
Variations:
1) In experiment 3.7 Pronase chamber was not added to the system for 6 hrs after each
synthetic cycle was begun.
2) Synthetic cycle times were either 12 hrs (3.8) or 24 hrs (3.6 and 3.7).
3) In (3.7) the Pronase chamber was removed after 24 hrs and replaced after 30 hrs.
4) In (3.6 and 3.7) 0.9 g resin portions were used. In (3.8) 0.45 g resin portions were
used
144
5) AlI experiments had 4 cycles each.
6.3 Experimental Section for Chapter 4
General Experimental Procedures: AU chemicals and enzymes were purchased from
Sigma-Aldrich Canada with the foUowing exceptions. Acylase 1 from hog kidney was
obtained from Fluka. Amino and bromo NovaSyn Tentagel resins were obtained from
Caledon-NovaBiochem. AU chemicals were used without further purification with the
foUowing exceptions. Tetrahydrofuran was distilled from sodium benzophenone ketyl.
Methylene chloride and triethylamine were distilled from calcium hydride. AU amino
acids used were natural L-enantiomers. AU solid phase syntheses, with the exception of
the library experiments, were carried out in coarse-fritted peptide synthesis vessels
obtained from Chemglass. NMR spectra were recorded at 270, 300, or 400 MHz for IH
and 67.5, 75, and 100 MHz for BC. Elemental analyses were performed by Quantatative
Technologies Inc, Whitehouse, NJ, USA. High-resolution mass spectra were performed
by Université de Sherbrooke, Sherbrooke, QC, Canada.
In aU cases where yields were determined by HPLC, molar absorptivity values
were determined with solutions of known concentration.
Procedures for Preparation of Authentic Standards
Et02C-Gly-(4'-S02NH2)Phe-OH (7h). 2 M NaOH (702 ~L, 1.40 mmol, 4.0 eq) was
added to a solution of Et02C-Gly-(4'-S02NH2)Phe-OMe (136 mg, 0.351 mmol, 1.0 eq)
in methanol (8.0 mL) and aUowed to stir at 21°C for 12 h. 2 M HCI (702 ~L, 1.40 mmol,
145
4.0 eq) was added and the methanol was removed in vacuo. The aqueous solution was
then acidified to pH 1 with 2 M HCI (1.0 mL) and then extracted with ethyl acetate (3 x
30 mL). The organic layer was dried over Na2S04, filtered and concentrated in vacuo to
afford 129 mg of3b (99%) as a white solid. IH NMR «CD3)2S0) Ô 7.93 (d, IH, J= 8.0
Hz), 7.71 (d, 2H, J= 8.4 Hz), 7.36 (d, 2H, J= 8.0 Hz), 7.15 (s, 2H), 7.0 (s (broad), IH),
4.52-4.46 (m, IH), 3.98 (q, 2H, J = 6.4 Hz), 3.63-3.51 (m, 2H), 3.15-3.10 (m, IH), 3.01-
2.96 (dd, IH, J = 13.8 and 8.6 Hz), 1.17-1.13 (t, 3H, J = 7.0). 13C NMR «CD3)2S0) Ô
173.1,169.8,157.2,143.0,142.3,130.3,126.2,60.7, 53.9,43.9,37.2,15.5. HR-CIMS
(m/z): [MH+] calculated for C14H19N307S, 373.0944; found, 373.0949.
Et02C-Phe-(4'-S02NH2)Phe-OH (7a). Prepared using the general procedure to afford
162 mg (84%). IH NMR «CD3)2S0) Ô 8.32 (d, IH,J= 7.6 Hz), 7.70 (d, 2H,J= 8.0 Hz),
7.40 (d, 2H, J = 8.0 Hz), 7.30 (s, 2H), 7.27-7.16 (m, 5H), 7.15-7.14 (m, IH), 4.48-4.45
(m, IH), 4.23-4.19 (m, IH), 3.87-3.84 (m, 2H), 3.16-3.11 (dd, IH, J= 13.6 and 5.2 Hz),
3.03-2.97 (dd, IH, J= 13.4 and 9.0 Hz), 2.94-2.91 (m, IH), 2.69-2.63 (m, IH), 1.08-1.05
(t, 3H, J = 6.8 Hz). 13C NMR «CD3)2S0) Ô 173.1, 172.5, 156.6, 142.9, 142.2, 138.7,
130.4, 129.8, 128.7, 126.9, 126.2, 60.6, 56.6, 53.9, 38.0, 37.1, 15.4. HR-CIMS (mlz):
[MH+] calculated for C21H26N307S, 464.1491; found, 464.1483.
Et02C-Leu-(4'-S02NH2)Phe-OH (7c). Prepared following the general procedure to
afford 727 mg (58%). IH NMR «CD3)2S0) Ô 8.10 (d, IH, J= 8.1 Hz), 7.68 (d, 2H, J=
8.4 Hz), 7.37 (d, 2H, J= 7.8 Hz), 7.29 (s, 2H), 7.16 (d, IH, J= 8.9 Hz), 4.18-4.41 (m,
IH), 3.99-3.92 (m, 3H), 3.14-3.09 (m, IH), 3.01-2.94 (m, IH), 1.62-1.52 (m, IH), 1.40-
146
1.32 (m, 2H), 1.16-1.12 (t, 3H, J= 6.9 Hz), 0.86-0.81 (m, 6H). l3C NMR ((CD3)2S0) Ô
. 173.1, 156.6, 142.9, 142.3, 130.3, 126.1, 60.6, 53.7, 37.0, 24.9, 23.9, 22.3, 15.5. HR
CIMS (m/z): [MH+] calculated for C18H28N307S, 430.1648; found, 430.1654.
Et02C-Pro-(4'-S02NH2)Phe-OH (7d): Prepared following the general procedure to
afford 278 mg (91%). IH NMR ((CD3)2S0) Ô 8.19-8.15 (m, 1H), 7.69 (d, 2H, J = 7.6
Hz), 7.41-7.38 (m, 2H), 7.30 (s, 2H), 4.50-4.40 (m, 1H), 4.18-4.06 (m, 1H), 4.01-3.95 (m,
1H), 3.83-3.79 (q, 2H, J= 6.0 Hz), 3.31-3.24 (m, 2H), 3.17-3.09 (m, 2H), 3.01-2.95 (m,
2H), 2.03-1.98 (m, 1H), 1.71-1.62 (m, 2H), 1.15-1.10 (t, 2H, J = 7.0 Hz). l3C NMR
((CD3)zSO) Ô 173.3, 173.1*, 172.7, 172.6*, 154.9*, 154.6, 142.9, 142.7*, 130.4*, 130.1,
126.0,61.2*,61.1,60.3*,60.0,53.9*,53.6,47.6,47.2*, 36.9, 31.7, 30.5*, 24.5*, 23.7,
15.5*, 15.3 (* indicates minor rotamer).HR-CIMS (m/z): [MH+] calculated for
C17H23N307S, 413.1257; found, 413.1266.
Measurement of Diffusion Rates Out of Dialysis Bags: Resorufin (random amounts
similar molecular weight to the dipeptide library members) was dissolved in X a mL of
ddH20 and placed in a dialysis bag of a chosen MWCO. This dialysis bag was suspended
in a 100 mL container with ya mL of ddH20. Samples were taken initially and after every
30 min from inside and outside the dialysis bag. Samples are observed for absorbance at
574 nm. A graph of increasing absorbance versus time in the outside chamber was then
made to estimate the time needed for diffusion.
a The volumes will vary depending on which system is being mimicked. Usually they are
both 20 mL.
147
Measurement of Inhibition Constants: The CA-catalyzed hydrolysis of pNPA at 25°C
was followed spectrophotometrically on a 96-well microplate spectrophotometer by
monitoring the appearance of p-nitrophenolate at 404 nm. A typical procedure was to add
CA solution (100.0 mL, 3-5 mg/ml) with inhibitor (0.0-6.0 /lM) to acetonitrile solution of
pNPA (5.0 IlL, 0.2-2.5 mM). The microplate was shaken for 5 s before the first reading
and for 3 s between readings.
General Procedure for Pseudo-Dynamic Combinatorial Libraries: A buffer solution
was prepared in 100 mL ddH20 with BICINE (0.490 g, 30 mM) and PEN-Na (10 mg, 0.1
g/mL). The buffer was adjusted to pH 9.0 and used to dissolve/suspend aIl components of
the system. Phesa (1) (66.2 mg, 0.27 mmol, 9.6 eq) and Phe (6) (44.6 mg, 0.27 mmol, 9.6
eq) were dissolved in 10 mL ofbuffer, which was then re-adjusted to pH 9.0. EtocGly,
EtocPro, EtocLeu and EtocPhe Tentagel active esters (each 125 mg, 22.5 /lmol, 0.80 eq)
were suspended in this solution. The resulting suspension was placed in a washed dialysis
bag (MWCO 12 kDa) and agitated manually for ~ 10 sec to promote resin swelling. It was
then placed in a 100 mL container. Pronase (0.5 g) was dissolved in 20 mL buffer, the
resulting solution was added to a dialysis bag (MWCO 1 kDa) to form the destruction
chamber, and was placed beside the other resin bag in the container. CA (0.84 g, 28
/lmol, 1 eq) was dissolved in 20 mL buffer and poured over the two bags in the container
forming the immersion solution (screening chamber). The system was set to stirring on an
orbital shaker. New resin was added directly to the synthe sis compartment after every 8,
12 or 16 h, depending upon the experiment. After two cycles, the solution inside the
148
synthesis chamber was filtered and the filtrate was used to suspend the next portion of
resin. 200 ~L samples were taken from the screening chamber every 4 hrs, heated to
80°C for ~5 min., centrifuged for 10 min and filtered through a 0.22 /lm sterile filter to
form the HPLC sample. Samples were run through a Phenomenex C8 (2) column
monitoring at 220 nm under the following conditions: 0-120 min gradient 80%
H20/O.1%TFA, 20% MeOH to 0% H20/0.1%TFA, 100% Me OH. The peaks monitored
were EtocGly Phesa (7b) at Il.3 min, EtocProPhesa (7 d) at 18.5 min, EtocLeuPhesa (7 c) at
39.8 min, EtocGlyPhe (8b) at 41.0 min, EtocPhePhesa (7a) at 43.5 min, EtocProPhe (8d)
at 52.7 min, EtocLeuPhe (8c) at 69.8 min, and EtocPhePhe (8a) at 70.6 min.
6.4 Experimental Section for Chapter 5
Quantification of CA Active Sites:[4] Four solutions of CA (0.3 g, 10 /lmol in each)
were prepared in 20 mL of 30 mM BICINE buffer containing 0.2 mg/mL PEN Na, pH
9.0.23 mg (50 /lmol) of EtocPhesaPhe (4a) was dissolved in one ofthese solutions for the
5.0 eq experiment. Similarly, 12 mg (30 /lmol) of 4a for the 2.5 eq, and 4.5 mg (10 /lmol)
of 4a for the 1.0 eq and blank experiments were prepared. Four 20 mL solutions of
Pronase (0.01 g in each) were prepared in the BICINE buffer, put inside dialysis bags
(MWCO 12 kDa) and suspended in the Ca/4a solutions inside a 150 mL container. The
solutions were agitated gently on a TW3 orbital shaker (Rose Scientific) for two days.
Samp1es were taken every hour for the first 10 hours, and once the next day. Each sample
was heated to 80°C for ~5 min., centrifuged for 10 min and filtered through a 0.22 /lm
sterile filter to form the HPLC sample. Samples were run through a Phenomenex C8 (2)
149
column monitoring at 220 nm under the following conditions: 0-120 min gradient 80%
H20/O.1 % TF A, 20% MeOH to 0% H20/O.1 % TF A, 100% MeOH. 4d absorbance was
monitored at 47.5 min.
Static Library Syntheses With Varying Concentrations of Nucleophile: A buffer
solution was prepared in 100 mL ddH20 with BICINE (0.490 g, 30 mM) and PEN-Na
(10 mg, 0.1 g/mL). The buffer was adjusted to pH 9.0 and used to dissolve/suspend aIl
components of the system. Phesa (1 t was dissolved in 50 mL of buffer in the 1 mM, 3
mM, 4 mM and 5 mM experiments and in 25 mL of buffer in the 10 mM experiment.
EtocGly, EtocPro, EtocLeu and EtocPhe Tentagel active esters (a total of 250 mg, 0.45
mmol, 1 eq per experiment) were suspended in this solution inside a peptide synthesis
vessel. The reactions were set to stirring on a TW3 orbital shaker (Rose Scientific) at 60
rpm. 200 I-tL samples were taken from the vessels at various intervals (at least every half
hour for the first 8 hours), and were then centrifuged for 10 min and filtered through a
0.22 /lm sterile filter to form the HPLC sample. Samples were run through a Phenomenex
C8 (2) column monitoring at 220 nm under the following conditions: 0-120 min gradient
80% H20/0.1 % TF A, 20% MeOH to 0% H20/0.1 % TF A, 100% MeOH. The peaks
monitored were EtocGlyPhesa (7b) at Il.3 min, EtocProPhesa (7 d) at 18.5 min,
EtocLeuPhesa (7c) at 39.8 min EtocGlyPhe (8b) at 41.0 min, EtocPhePhesa (7a) at 43.5
min, EtocProPhe (8d) at 52.7 min, EtocLeuPhe (8c) at 69.8 min, and EtocPhePhe (8a) at
70.6 min.
150
a Table 6.1: Amounts of(l) used in static library experiments.
Equivalents of(l) with Experiment Mass of(l) (mg) Amount of (1) (f.lmol) respect to total activated
ImM 3mM 4mM 5mM 10mM
12 37 49 61 61
amino acids 50 1.1 150 3.3 200 4.4 250 5.6 250 5.6
Eight Membered 16 h p-DCL Experiments: A buffer solution was prepared in 100 mL
ddH20 with BICINE (0.490 g, 30 mM) and PEN-Na (10 mg, 0.1 g/mL). The buffer was
adjusted to pH 9.0 and used to dissolve/suspend aIl components of the system. Phesa (lt
and Phe (6)b were dissolved in 10 mL ofbuffer. EtocGly, EtocPro, EtocLeu and EtocPhe
Tentagel active estersa were suspended in this solution. The resulting suspension was
placed in a washed dialysis bag (MWCO 12 kDa) and agitated manually for ~10 sec to
promote resin swelling. It was then placed in a 100 mL container. Pronase (0.5 g) was
dissolved in 20 mL buffer, the resulting solution was added to a dialysis bag (MWCO 1
kDa) to form the destruction chamber, and was placed beside the other resin bag in the
container. CA (0.84 g, 28 /lmol, 1 eq) was dissolved in 20 mL buffer and poured over the
two bags in the container forming the immersion solution (screening chamber). The
system was set to stirring on a TW3 orbital shaker (Rose Scientific) at 60 rpm. New resin
was added directly to the synthe sis compartment after every 16 h. After every two cycles,
the solution inside the synthesis chamber was filtered and the filtrate was used to suspend
the next portion of resin. 200 /lL samples were taken from the screening chamber every 4
hrs, heated to 80°C for ~5 min., centrifuged for 10 min and filtered through a 0.22 /lm
151
sterile filter to form the HPLC sample. Samples were run through a Phenomenex C8 (2)
column monitoring at 220 nm under the following conditions: 0-120 min gradient 80%
H20/O.1 % TF A, 20% MeOH to 0% H20/0.1 % TF A, 100% MeOH. The peaks monitored
were EtocGlyPhesa (7b) at 11.3 min, EtocProPhesa (7d) at 18.5 min, EtocLeuPhesa (7c) at
39.8 min EtocGlyPhe (Sb) at 41.0 min, EtocPhePhesa (7a) at 43.5 min, EtocProPhe (Sd)
at 52.7 min, EtocLeuPhe (Sc) at 69.8 min, and EtocPhePhe (Sa) at 70.6 min.
a Table 6.2: Variations of the eight membered, 16 hour library
Equivalents of Equivalents of activated Number of nucleophile 1 amino acid added each
cycles with respect to cycle with respect to CAb CA
6 9.6 3.2 6 4.5 3.2 12 4.5 3.2 16 4.5 9~
b In alllibraries Phe (6) was present in the same amounts as 1, but because 6 is a non-inhibitor, its presence did not affect the yield or selectivity.
Four Membered p-DCL: A buffer solution was prepared in 100 mL ddH20 with
BICINE (0.490 g, 30 mM) and PEN-Na (10 mg, 0.1 g/mL). The buffer was adjusted to
pH 9.0 and used to dissolve/suspend aIl components of the system. Phesa (1) (33.0 mg,
0.135 mmol, 4.8 eq) and Phe (6) (22.3 mg, 0.135 mmol, 4.8 eq) were dissolved in 10 mL
of buffer, which was then re-adjusted to pH 9.0. EtocPro EtocGly and EtocPhe Tentagel
active esters (each 125 mg, 22.5 ~mol, 0.80 eq) were suspended in this solution. The
resulting suspension was placed in a washed dialysis bag (MWCO 12 kDa) and agitated
manually for ~ 10 sec to promote resin swelling. It was then placed in a 100 mL container.
Pronase (0.5 g) was dissolved in 20 mL buffer. The resulting solution was added to a
dialysis bag (MWCO 1 kDa) to form the destruction chamber and was placed beside the
other resin bag in the container. CA (0.84 g, 28 ~mol, 1 eq) was dissolved in 20 mL
152
buffer and poured over the two bags in the container forming the immersion solution
(screening chamber). The system was set to stirring on a TW3 orbital shaker (Rose
Scientific) at 60 rpm. New resin was added directly to the synthesis compartment after
every 16 h. After two cycles, the solution inside the synthesis chamber was filtered and
the filtrate was used to suspend the next portion of resin. 200 !AL samples were taken
from the screening chamber approximately every 8 hrs, heated to 80°C for -5 min.,
centrifuged for 10 min and filtered through a 0.22 /lm sterile filter to form the HPLC
sample. Samples were run through a Phenomenex C8 (2) column monitoring at 220 nm
under the following conditions: 0-120 min gradient 80% H20/0.1%TFA, 20% Me OH to
0% H20/0.1%TFA, 100% MeOH. The peaks monitored were EtocProPhesa (7d) at 18.5
min, EtocLeuPhesa (7c) at 39.8 min, EtocProPhe (8d) at 52.7 min, and EtocLeuPhe (8c)
at 69.8 min.
Six Membered p-DCL: A buffer solution was prepared in 100 mL ddH20 with BICINE
(0.490 g, 30 mM) and PEN-Na (10 mg, 0.1 g/mL). The buffer was adjusted to pH 9.0 and
used to dissolve/suspend all components of the system. Phesa (1) (49.5 mg, 0.202 mmol,
7.9 eq) and Phe (6) (33.4 mg, 0.20 mmol, 7.9 eq) were dissolved in 10 mL of buffer,
which was then re-adjusted to pH 9.0. EtocPro EtocGly and EtocPhe Tentagel active
esters (each 125 mg, 22.5 /lmol, 0.80 eq) were suspended in this solution. The resulting
suspension was placed in a washed dialysis bag (MWCO 12 kDa) and agitated manually
for -10 sec to promote resin swelling. Tt was then placed in a 100 mL container. Pronase
(0.5 g) was dissolved in 20 mL buffer. The resulting solution was added to a dialysis bag
(MWCO 1 kDa) to form the destruction chamber and was placed beside the other resin
153
bag in the container. CA (0.84 g, 28 J.lmol, 1 eq) was dissolved in 20 mL buffer and
poured over the two bags in the container forming the immersion solution (screening
chamber). The system was set to stirring on a TW3 orbital shaker (Rose Scientific) at 60
rpm. New resin was added directly to the synthesis compartment after every 16 h. After
two cycles, the solution inside the synthesis chamber was filtered and the filtrate was
used to suspend the next portion of resin. 200 J.lL samples were taken from the screening
chamber approximately every 8 hrs, heated to 80°C for ~5 min., centrifuged for 10 min
and filtered through a 0.22 J.lm sterile filter to form the HPLC sample. Samples were run
through a Phenomenex C8 (2) column monitoring at 220 nm under the following
conditions: 0-120 min gradient 80% H20/0.1%TFA, 20% MeOH to 0% H20/0.1%TFA,
100% MeOH. The peaks monitored were EtocGlyPhesa (7b) at Il.3 min, EtocProPhesa
(7d) at 18.5 min, EtocGlyPhe (8b) at 41.0 min, EtocPhePhesa (7a) at 43.5 mm,
EtocProPhe (8d) at 52.7 min, and EtocPhePhe (8a) at 70.6 min.
General Procedure for 30 Membered p-DCLs: A buffer solution was prepared in 100
mL ddH20 with BI CINE (0.490 g, 30 mM) and PEN-Na (10 mg, 0.1 g/mL). The buffer
was adjusted to pH 9.0 and used to dissolve/suspend aIl components of the system. Phesa
(1) (38.7 mg, 0.101 mmol, 7.21 eq), Alasa-TFA (30.5 mg, 0.101mmol, 7.21 eq), Lyssa
TFA (36.6 mg, 0.101mmol, 7.21 eq), GlUNHOH (17.5 mg, 0.101 mmol, 7.21 eq) and Phe
(6) (17.5 mg, 0.101 mmol, 7.21 eq) were dissolved in 5 mL ofbuffer. EtocAla, EtocVal,
EtocPro, EtocPipecolic acid, EtocLeu and EtocPhe Tentagel active esters (each 80 mg,
14.4 J.lmol, 1.03 eq) were suspended in this solution. The resulting suspension was placed
in a washed dialysis bag (MWCO 12 kDa) and agitated manually for ~1O sec to promote
154
resin swelling. It was then placed in a 100 mL container. Pronase (0.25 g) was dissolved
in 10 mL buffer, the resulting solution was added to a dialysis bag (MWCO 1 kDa) to
form the destruction chamber, and was placed beside the other resin bag in the container.
CA (0.42 g, 14 Ilmol, 1 eq) was dissolved in 10 mL buffer and poured over the two bags
in the container forming the immersion solution (screening chamber). The system was set
to stirring on a TW3 orbital shaker (Rose Scientific). New resin was added directly to the
synthesis compartment after every 16, 24 or 32 h, depending on the experiment. After
every two cycles, the solution inside the synthesis chamber was filtered and the filtrate
was used to suspend the next portion of resin. 200 ilL samples were taken from the
screening chamber every 4-16 hrs, heated to 80°C for ~5 min., centrifuged for 10 min and
filtered through a 0.22 Ilm sterile filter to form the HPLC sample. Samples were run
through a Phenomenex C8 (2) column monitoring at 220 nm under the following
conditions: 0-180 min gradient 100% H20/0.1 %TFA, 0% MeOH/0.1 %TFA to 0%
H20/O.I%TFA, 100% MeOH/O.1%TFA. The peaks monitored were EtocProPhesa (7d) at
24.5 min, EtocValPhesa (7e) at 25.0 min, EtocAlaPhesa (7t) at 42.5 min, EtocPipPhesa
(7g) at 70.8 min, EtocLeuPhesa (7e) at 78.1 min, EtocPhePhesa (7a) at 82.4 min,
EtocProPhe (8d) at 58.2 min, EtocValPhe (8e) at 61.2 min, EtocAlaPhe (8t) at 82.1 min,
EtocPipPhe (8g) at 90.0 min, EtocLeuPhe (8e) at 100.1 min, EtocPhePhe (8a) at 108.3
min, EtocProAlasa (ge) at 36.4 min, EtocValAlasa (9b) at 47.3 min, EtocAlaAlasa (9d) at
57.5 min, EtocPipAlasa (9t) at 60.1 min, EtocLeuAlasa (ge) at 67.7 min, EtocPheAlasa
(9a) at 70.6 min, EtocProLyssa (lOe) at 43.8 min, EtocValLyssa (lOb) at 55.1 min,
EtocAlaLyssa (lOd) at 57.9 min, EtocPipLyssa (lOt) at 67.6 min, EtocLeuLyssa (lOc) at
71.8 min, EtocPheLyssa (lOa) at 75.4 min, EtocProGluNHoH (Ile) at 35.6 min,
155
EtocValGluNHoH (l1b) at 41.9 min, EtocAlaGluNHoH (l1d) at 57.2 min, EtocPipGluNHoH
(11f) at 59.9 min, EtocLeuGluNHoH (l1c) at 67.3 min, and EtocPheGluNHoH (l1a) at 69.7
min,
References
1. Y. Pocker, J. T. Stone Biochemistry 1968, 7,3021-3031.
2. A. D. Corbett, J. L. Gleason Tetrahedron Lett. 2002,43, 1369-1372
3. J. D. Cheeseman, A. D. Corbett, R. Shu, J. Croteau, J. L. Gleason, R. J.
Kazlauskas J. Am. Chem. Soc. 2002,124,5692-5701.
4. J. B. Chaires, N. Dattagupta, D. M. Crothers Biochemistry, 1982,21, 3933-
3940.
156
CONTRIBUTION TO KNOWLEDGE
The nature of evolution dictates that there will forever be a need for new synthetic
compounds for use as drugs against disease. Combinatorial chemistry has accelerated the
process of discovering drug leads, and receptor assistance in combinatorial systems has
improved upon the se ideas to evolve new, small molecules.
Pseudo-dynamic libraries exhibit selectivity higher than in any other receptor
assisted system, and as such, are promising in their potential application to the drug
discovery process. The first p-DCL system, its optimization, expansion and theoretical
development enable this method to be applied to other biological targets. This is a highly
versatile method in that it does not require any specific type of receptor to be efficient,
and it does not necessarily require high technology in its application.
157
JACS ARTICLES
Published on Web 04/2612002
Amplification of Screening Sensitivity through Selective Destruction: Theory and Screening of a Library of Carbonic
Anhydrase Inhibitors
Jeremy D. Cheeseman, Andrew D. Corbett, Ronghua Shu, Jonathan Croteau, James L. Gleason,* and Romas J. Kazlauskas*
Contribution from the Department of Chemistry, McGill University, 801 Sherbrooke St. West, Montreal, Quebec H3A 2K6, Canada
Received September 17, 2001
Abstract: A new method for identifying enzyme inhibitors is to conduct their synthesis in the presence of the targeted enzyme. Good inhibitors form in larger amounts than poorer ones because the binding either speeds up synthesis (target-accelerated synthesis) or shifts the synthesis equilibrium (dynamic combinatorial libraries). Several groups have successfully demonstrated this approach with simple systems, but application to larger libraries is challenging because of the need to accurately measure the amount of each inhibitor. ln this report, we dramatically simplify this analysis by adding a reaction that destroys the unbound inhibitors. This works similar to a kinetic resolution, with the oost inhibitor being the last one remaining. We demonstrate this method for a static library of several sulfonamide inhibitors of carbonic anhydrase. Four sulfonamidecontaining dipeptides, EtOC-Phesa-Phe (4a), EtOC-Phesa-Gly (4b), EtOC-Phesa-Leu (4c) and EtOC-PhesaPro (4d), were prepared and their inhibition constants measured. These inhibitors migrated to the carbonic anhydrase compartment of a two-compartment vessel. Although higher concentrations of the better inhibitors were observed in the carbonic anhydrase compartment, the concentration differences were small (1.83:1.71 :1.54:1.46:1 for 4a:4b:4c:4d:5, where 5 is a noninhibiting dipeptide EtOC-Phe-Phe). Addition of a protease rapidly cleaved the weaker inhibitors (4d and 5). Intermediate inhibitor 4c was cleaved at a slower rate, and at the end of the reaction, only 4a and 4b remained. In a separate experiment, the ratio of 4a to 4b was found to increase over time to a final ratio of nearly 4:1. This is greater than the ratio of their inhibition constants (approximately 2:1). The theoretical model predicts that these ratios would increase even further as the destruction proceeds. This removal of poorer inhibitors simplifies identification of the best inhibitor in a complex mixture.
Introduction
Synthesis using combinatorial chemistry allows testing of hundreds of thousands of drug candidates using high throughput screening techniques. Although this rapid pace has revolutionized drug development, the search for faster and more efficient testing methods continues. One promising method is in situ screening of mixtures such as in dynarnic combinatorial libraries.! Dynamic combinatorial libraries are equilibrating mixtures of organic molecules. Equilibration in the presence of a therapeutic target increases the equilibrium amounts of those library members that bind tightly to that target. The difference in library composition with and without a stoichiometric amount of target identifies the best inhibitors.
Dynarnic libraries are still in the developmental stage, and only a few examples have been reported.2 For example, Ramstrom and Lehn3 created a dynamic library of 10 di-
* To whom correspondence should be addressed. E-mail: jim.gleason@ mcgill.ca and [email protected]. (1) Reviews: Ganesan, A. Angew. Chem., Int. Ed. 1998,37,2828-2831; Lehn,
J.-M. Chem. Eur. J. 1999, 5, 2455-2463; Cousins, G. R. L.; Poulsen, S.A.; Sanders, J. K. M. Curr. Opin. Chem. Biol. 2000,4,270-279; Huc, 1.; Nguyen, R. Comb. Chem. High Throughpul Screening 2001, 4, 53-74.
5692 • J. AM. CHEM. SOC. 2002, 124,5692-5701
saccharides by disulfide exchange starting from a mixture of monosaccharide tItiols. The library was screened against concanavalin A, which binds mannose-rich oligosaccharides. A mannoside homodimer was the strongest binder in the library. When the disulfide exchange was carried out in the presence of concanavaHn A, the amount of mannoside homodimer present increased by 40%. This increase in the amount of mannoside homodimer identifies it as the best-binding disaccharide. Analysis of a larger 21-member library was more difficult because HPLC did not resolve each member. Nevertheless, the mannoside homodimer was clearly favored in tItis library as weIl.
To make a real impact on drug discovery, methods must be developed to screen dynarnic combinatorial libraries with thousands of members. This screening is complicated because
(2) For examples aimed towards biological targets, see: (a) Huc, 1.; Lehn, J.-M. Proe. Nol/. Aead. Sei. U.SA. 1997, 94, 2106-2110. (h) Nicolaou, K. C.; Hughes, R.; Cho, S. Y.; Winssinger, N.; Smethurst, c.; Labiscbinski, H.; Endermann, R. Angew. Chem., Int. Ed. 2000,39,3823-3828. (c) Karan, c.; Miller, B. L. J. Am. Chem. Soc. 2001, 123, 7455-7456. (d) Bunyapaiboonsri, T.; Ramstriim, O.; Lohmanu, S.; Lehn, J.-M.; Peng, L.; Goeldner, M. ChemBioChem 2001, 2, 438-444. (e) Nguyen, R.; Huc, 1. Angew. Chem., Inl. Ed. 2001,40, 1774-1776.
(3) Rarnstrôm, O.; Lehn, J.-M. ChemBioChem 2000,1,41-48.
10.1021118017099+ CCC: $22.00 02002 America" Chemlcal Society
Screening Sensitivity through Selective Destruction
Scheme 1. Aryl Sulfonamide-Based Dipeptide Libraries as Inhibitors of Carbonic Anhydrasea
7 0 ~3
ff .... N NÂCOH 2 H 2
1'" R h-
selective pressure lœt)
carbonic anhydrase , 1 hydrolysis of
poorer inhibitors
mem rane
HI 0 R
ff2"N OH + H2N~C02H 1'"
R, h-
1 R, = S02NH2 R2 = H
a Strong binding inhibitors will be bound to carbonic anhydrase and protected. Weaker inhibitors will be hydrolyzed by a protease.
it is often difficult to measure the concentration of each library member in the absence and presence of a target. Further, the libraries will likely contain not one but many good inhibitors because many library members have similar structures and thus similar binding constants. In these cases, adding the target increases the concentration of many library members, rather than a single member, and makes analysis very diffieult or impossible. Eliseev and Nelen4 estimated that a dynarnic library combined with an affinity column containing the target would yield one major compound (>50%) only if KstronglKweak was at least n, where n is the number of members of the library. Thus, for one member to predominate in a library of 1000 members, that member would have to bind > 1000 times stronger than the others, an unlikely possibility. This inability to distinguish between inhibitors of similar binding constants is a major limitation of the CUITent dynarnic combinatorial libraries.
In this report, we propose a screening method that enhances the ability to detect the best inhibitor in a mixture of similar inhibitors. The key to the method is an irreversible destruction reaction that destroys the unbound and weakly bound inhibitors, similar to a kinetic resolution. The best inhibitor is the last one remaining. We demonstrate that this method works for a statie library and discuss its potential application to a dynarnic system.
Our library targets carbonie anhydrase and consists of dipeptides with an N-terrninal 4' -sulfonarnidophenylalanine (1, Phesa).5 These dipeptides can either bind to carbonic anhydrase or be c1eaved by a protease, Scheme 1. This c1eavage increases the ratio of the strongest binder relative to weaker binders. Importantly, the ratio may increase to values significandy greater than the ratio of the binding constants, thus overcoming the limitation identified by Eliseev and Nelen and making it easy to identify the best inhibitor in the mixture.
Theory
Finding the Best Inhibitor by Shifting the Equilibrium To Make More of the Better Inhibitors. Most dynarnic combinatorial library experiments contain two equilibria: an
(4) (a) Eliseev, A. V.; Nelen, M. J. J. Am. Chem. Soc. 1997, 119, 1147-1148. (b) Eliseev, A. V.; Nelen, M. J. Chem. Eur. J.1998, 4, 825-834.
(5) G1aucorna patients oflen talce carbonic anhydrase inhibitors to reduce the pressure in the eye. Ali commercial inhibitors contain a sulfonamide moiety. We chose carbonic anhydrase as a test case for inhibitor design and screening methods.
ARTICLES
Scheme 2. Dynamic Combinatorial Library Equilibria Yield a Higher Total Amount of a Good Inhibitora
~D~D.O ~6) ~ newtnhlbltors target. Inhlbhor
compte ...
a Binding of the inhibitor to a target removes it from the synthesis equilibrium so that the synthesis produces more of the good inhibitor.
equilibrium for the synthesis of inhibitors and an equilibrium for binding of the inhibitors to the target, Scheme 2. The binding equilibrium removes the good inhibitors from the synthe sis equilibrium, and to reestablish equilibrium, the synthesis produces more of the good inhibitors than it would in the absence of target. First, we show that the ratio of good inhibitor to a poor inhibitor depends linearly on the ratio of the binding constants.
Consider a common starting material, SM, in equilibrium with two inhibitors, A and B, which can each bind reversibly to a target, T, to form complexes T'A and T'B
KsA KaA ====:!:: A + T =====:!:: (1)
SM KsB KaB
==:!::B+T==:!:: (2)
If [AT] is the total of bound and unbound forms of A ([AT] = [A] + [T'A]), it can be shown that under typical conditions (e.g., tight binding and an excess of target at a high concentration), the equilibrium ratio of the total amounts of the two inhibitors, [AT] and [BT], depends linearly on their relative association constants (eq 3, see Supporting Information for a derivation).6
(3)
For the ideal case where there is one good inhibitor in a pool of noninhibitors, these equilibria indeed will yield the good inhibitor. For example, assurning the rates of synthesis are equal, a mixture of two inhibitors differing in their inhibition constants by a factor of 10 will give a 1:1 mixture in the absence of target (50 mol % of better inhibitor) but gives al: 10 mixture in the presence of target (91 mol % of better inhibitor). Similarly, a mixture of 1000 inhibitors would yield 0.1 mol % of each inhibitor in the absence of target, but in the presence of target, the poorer inhibitors would decrease to 0.09 mol % each, while the one good inhibitor would increase to 0.9 mol %. This very simple example is already a difficult analysis problem. The more common situation where many inhibitors with similar binding constants are present may become difficult or impossible to analyze.
Finding the Best Inhibitor by Destruction of Poorer Inhibitors. One way to enhance the concentration differences between inhibitors with similar binding constants is to add an irreversible reaction that removes the unbound poorer inhibitor.
(6) If the binding is not tight or the target is not in excess at high concentration, then the concentrations of the inhibitors will he mOre similar than those discussed in the texl above, and il will he even harder to distingnish which is the better inhibitor.
J. AM. CHEM. SOC •• VOL. 124. NO. 20. 2002 5693
ARTICLES
Scheme 3. Destruction of Inhibitors·
&\I~ ~ I=:>>> q) dl •• oc~tJon
tergat • Inhibltor complexe.
o + 0 d ••• ,uctoo 8 D Inhlbltors <J
a The free concentration of the poorer inhibitor is higher, thus it is destroyed more readily. This destruction reaction exponentially increases the relative concentration of the good inhibitor similar to a kinetic resolution.
This situation is similar to a kinetic resolution of enantiomers.7
As the destruction reaction winnows away the poorer inhibitors, the relative concentration of the best inhibitor increases exponentially (Scheme 3). The analysis below is similar to that for kinetic resolutions.7
Consider two inhibitors, A and B, that compete for a target, T, and are also destroyed by an irreversible reaction to yield P and Q with rates of k2A and k2B.
KdA kzA T'A=T+A-P
The rate of disappearance of inhibitor A is
d[A] -=-k [A] dt 2A
(4)
(5)
(6)
If [AT] is the total concentration of bound and unbound forms of inhibitor A, it can be shown that
(7)
Upon solving for [A] and substituting into eq 6, the rate of disappearance of A is given by
d[A] --=
dt (8)
When the target is in excess of the inhibitor, the concentration of the free target, T, will be much larger than the dissociation constant, KdA, so [T] » KdA' therefore, eq 8 simplifies to
d[A]
dt (9)
A similar equation is obtained for inhibitor B. The ratio of their partial reaction rates is
KdB k2B where S = - x - (10)
K dA k2A
This equation shows that the relative rate of disappearance of the two inhibitors depends linearly on their total concentration, their relative binding ability, and their relative rate of destruction. For simplicity, we define S as the product of the relative binding abilities and relative rates of destruction of the
5694 J. AM. CHEM. SOC •• VOL. 124, NO. 20, 2002
12
10
8
2
o
Cheeseman et al.
o 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
Conversion
Figure 1. Predicted ratio of the total (bound and unbound) concentrations of two hypothetical inhibitors, A and B, as a function of the degree of conversion for given values of S. The degree of conversion is the fraction of the total amount of inhibitors !hat have been destroyed. The calcu1ated Hnes follow eqs 11 and 12 where the initial total concentration of the inhibitors is one. This graph shows that the ratio of the two inhibitors can be much larger !han the value of S, even for values of S as low as 2.
two inhibitors. If the rates of destruction of the two inhibitors are equal, then S is the ratio of the dissociation constants and will be greater than one if A is a stronger inhibitor of the target than B.
Upon integration of eq 10, one finds that the ratio of the total amounts of the two inhibitors varies exponentially with S (eq Il), where [AT]O represents the initial total concentration of inhibitor A. This exponential relationship enhances the ability to detect small differences as the destruction reaction progresses.
(11)
By measuring the relative concentration of the two inhibitors during the destruction reaction, the value of S can be determined using eq 11. Alternatively, eq 12 below, which expresses [AT], [BT] , [AT]O, and [BT]O in terms of the conversion, C, and the ratio of the total concentrations of the two inhibitors, can be used to determine S.
_ln....:;[~(1_-_C).....:....:...(2....;/('-1_+_R..:..:»-=-] = S ln[(l - C)(2R/(1 + R»]
[AT] + [BT] where C = 1 - [AT]o + [BT]o
These predictions are shown graphically for several values of S in Figure 1. If the rates of destruction of the two inhibitors are equal, then S is the ratio of the dissociation constants. As the destruction reaction proceeds (conversion increases from 0 to 1), the ratio of the total amounts of the two inhibitors, [AT]! [BT], varies when S ~ 1. When S is large (e.g., 40), the relative concentration of the good inhibitor increases steeply near 50% conversion. When S is small (e.g., 2), the relative concentration of the good inhibitor increases steeply near 90% conversion.
(7) The analysis below follows closely the mathematical treatment for kinetic resolutions. For examples, see: Martin, V. S.; Woodard, S. S.; Katsuki, T.; Yamada, Y.; Ikeda, M.; Sharpless, K. B. J. Am. Chem. Soc. 1981, 103, 6237-6240; Chen, C. S., Fujimoto, Y., Girdaukas, G., Sih, C. J. J. Am. Chem. Soc. 1982, 104, 7294-7299; Kagan, H. B., Fiaud, J. C. Top. Stereochem. 1988, 18, 249-330.
Screening Sensitivity through Selective Destruction ARTICLES
Scheme 4. Preparation of 4'-Sulfonamidophenylalanine Dipeptides
~ 1. CIS03H, t;, H~02H EtO~CI Et02C,N ........... :. C~H 2 NH OH' NHz-Xaa-OtBu
. 4 ~ --v' NaHC03 --v-3. hog kldney H N 1 H~ 1 dioxane H ~ 1 EDe. HOBT, CH~12 acylase 1 2 ',R~ .& 2 ,R~ .--:;
o 0 0 0 1 2
In either case, the ratio of the total amounts of the two inhibitors, [Ar]/[Br], can be much larger than the value of S.
Results
Preparation of 4'-Sulfonamidophenylalanine Dipeptides. (S)-4'-Sulfonamidophenylalanine (1 or Phe.,.) was prepared from (S)-N-acetylphenylalanine by a modification of the procedure described by Escher et al. 8 Thus, chlorosulfonylation of Nacetylphenylalanine in chlorosulfonic acid at 60 oC followed by ammonolysis afforded N-acetyl-4' -sulfonamidophenylalanine. Direct purification of this intermediate proved difficult. Therefore, it was deacetylated using hog kidney acylase 1,9 and the resulting free amine acid 1 was purified by ion-exchange chromatography and recrystallization. Using this procedure, 1 was prepared as an analytically pure solid in 40% yield from N-acetyl-Phe. The a-amino group was selectively blocked using ethyl chloroformate under standard Schotten-Baumann conditions. The requisite dipeptides were then prepared by coupling 2 with terl-butyl amino acid esters using EDCIHOBT,IO followed by trifluoroacetic acid-mediated deprotection of the ester function to afford dipeptides 4a-d (Scheme 4). No acylation of the sulfonamide nitrogen was observed under either the Schotten-Baumann or peptide-coupling conditions. Dipeptide EtOC-Phe-Phe (5), which does not contain a sulfonamide group and serves as a control, was prepared by standard methods.
Inhibition of Carbonic Anhydrase. Sulfonamides 1 and 2 as weIl as sulfonamide dipeptides 4a-d all inhibited the carbonic anhydrase-catalyzed hydrolysis of 4-nitrophenyl acetate (pNPA). The inhibition was competitive and Lineweaver-Burk plots revealed similar inhibition constants, which varied by only a factor of 10 (Table 1). The parent amino acid 1 (Phes.) was the poorest sulfonamide inhibitor (KI = 13 ,uM), while dipeptides 4a (EtOC-Phesa-Phe) and 4b (EtOC-Phesa-Gly) were the best sulfonamide inhibitors (KI = 1.2 and 2.5 ,uM, respectively). Dipeptides 4c (EtOC-Phesa-Leu) and 4d (EtOC-Phesa-Pro)
(8) Escher, E.; Bernier, M.; Parent, P. Helv. Chim. Acta 1983, 66, 1355-1365.
(9) Researchers oflen use hog kidney acylase to resolve enantiomers of N-acetyl amino acids. Chenault, H. K.; Dahmer, J.; Whitesides, G. M. J. Am. Chem. Soc. 1989, 11 J, 6354-6364; Roberts, S. M., Ed. Prepara/ive Bio/ransformations; Wiley: Chichester 1992-1998; Module 1: 14. In our case, !his intennediate was a1readyenantiomerically pure. We used hog kidney acylase to c1eave the acetyl group under milder conditions than those required by chemical c1eavage methods.
(10) EDC = 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide; HOBT = l-hydroxybenzotriazole.
5
Table 1. Inhibition of Carbonic Anhydrase by Sulfonamides 1 and 2, Sulfonamide Dipeptides 4a-d, and Dipeptide 5
compound K, (uM)'
Phes.(l) EtOC-Phes• (2) EtOC-Phe..-Phe (4a) EtOC-Phe..-Gly (4b) EtOC-Phe..-Leu (4c) EtOC-Phesa-Pro (4d) EtOC-Phe-Phe (5)
13 ± 1.6 12 ± 1.4
1.2 ± 0.2 2.5 ± 0.5 4.4 ± 0.7 9.4 ± 1.6
»100()b
a Competetive inhibition constants for the carbonic anhydrase-catalyzed hydrolysis of p-nitrophenyl actetate (pNPA) at 25 °C.in phosphate bu~fer (l0 mM pH 7.5). A typical procedure was to add carbomc anhydrase solution (100 ilL, 0.05 mg/mL) containing inhibitor (0.0-100 IlM in most cases) to an acetonitrile solution of pNPA (5.0 ilL, 2.0-32 mM) and follow the release of p-nitrophenoxide spectrophotometrically at 404 nM. b No inhibition detected at an inhibitor concentration of 1 mM
showed slightly higher inhibition constants (4.4 and 9.4 ,uM, respectively). Other simple sulfonamides also inhibit carbonic anhydrase with similar inhibition constants. 1 1 As expected, the dipeptide lacking a sulfonamide group, 5, did not inhibit carbonic anhydrase.
Selective Extraction of Inhibitors by Carbonic Anhydrase. First, we demonstrated that a strongly binding inhibitor concentrates into the carbonic anhydrase-containing compartment of a two-compartrnent vessel (cf. Figure 4 without Pronase). The two compartrnents were created by suspending a dialysis bag containing a solution of bovine carbonic anhydrase 12 (0.34 mM) in a solution of phosphate buffer. The dialysis membrane (12-kDa cutoff) separated the two compartments so that small molecules such as the sulfonamide dipeptides could diffuse freely across the membrane, while carbonic anhydrase (30 kDa) could not. Both compartrnents initially contained a mixture of 0.16 mM sulfonamide dipeptide 4a and 0.19 mM noninhibitor dipeptide 5. Over several hours the total sulfonamide concentration increased in the inside compartrnent containing carbonic
(11) For example, N guyen and Huc investigated a simple sulfonarnides with inhibition constants of ~O.I-l ,uM (Nguyen, R.; Huc, I. Angew. Chem., Int. Ed. 2001,40, 1774-1776), while Doyon et al. investigated other simple sulfonarnides with inhibition constants of ~O.OOI ,uM (Doyon, J. B.; Hansen, E. A. M.; Kim, C.-Y.; Chang, J. S.; Christianson, D. W.; Madder, R. D.; Voet, J. G.; Baird, T. A., Jr.; Fierke, C. A.; Jain, A. Org. Leu. 2000, 2, 1189-1192).
(12) These experiments required stoichiometric amounts o~ carbonic anhydrase. We used an inexpensive mixtore of isozymes from bovme sources. Alth~ugh material was not pure carbonic anhydrase, we calcu1ated the concentrations assuming it was pure. Thus, the true concentration will be less than the number given.
J. AM. CHEM. SOC .• VOL. 124, NO. 20, 2002 5695
ARTICLES
[dipeptide) (mM)
0.3 a) Outside Chamber
-...--a-_____ S
0.2
0.1
-------4a
O+-----,------r----~----~
o 3 6 Tlme (h)
9 12
0.3
0.2
[dipeptide) (mM)
0.1
Cheeseman et al.
b) Ioside Chamber _---.--~4a
-!...._e_.....--_---- s
O+-----.------r-----r----~
o 3 6
Time(h)
9 12
Figure 2.13 Selective concentration of the sulfonamide 4a over noninhibitor S into the carbonic-anhydrase-containing compartment of a two-compartment vesse!. One compartment contained carbonic anhydrase (0.34 mM), while both compartments (20 mL each) initially contained equal concentrations of sulfonarnide 4a (0.16 mM) and noninhibitor 5 (0.19 mM). The sulfonamide diffused freely across the dialysis membrane and concentrated in the carbonicanhydrase-containing compartment as shown In contras!, the concentrations of noninhibitor S remained similar in both compartments. After 12 h, the concentration of sulfonarnide 4a in the outside compartment decreased to 0.04 mM and increased in the inside compartment to 0.28 mM (total of free and carbonic anhydrase-bound). The final ratio of 4a to 5 in the carbonic anhydrase chamber was 1.75: 1.
20 a) Outside Chamber
16
12&-.-__________ ~----------__ 5 [dipeptide) I~;::::::-___ ....... __
(mM) 8 1 . 4 _--, ____ -=~ d
4 4c
~::::::=====:::4b 4a O+----,-----r----~--~r---~
o 10 20 30 40 50
TIme(h)
Idipeptidel (mM)
20
16
b) Ioside Chamber 4a 4b
__ ~ __ -----==~4c 4d
12~~~=-------~-~'---------~~---------'S
8
4
O+-----r---~-----r----,---~ o 10 20 30 40 50
Time(h)
Figure 3.13 Selective concentration of the sulfonamides 4a-4d over noninhibitor 5 into the carbonic-anhydrase-containing compartment of a two-compartment vessel separated by a dialysis membrane. One compartment contained carbonic anhydrase (0.485 mM), while both compartments (20 mL each) initially contained equal concentrations of sulfonarnides 4a-4d and noninhibitor 5 (~O.ll mM each). The sulfonamides diffused freely across the dialysis membrane and concentrated in the carbonic-anhydrase-containing compartment. In contrast, the concentration of noninhibitor S increased slightly in the outer compartment.
anhydrase and decreased in the outside compartment (Figure 2). Alternatively, the concentrations of the noninhibitor 5 remained similar in both compartments. This result showed that tight binding to a target could concentrate a good inhibitor into one compartment of a two-compartment reaction vesse!.
In a similar experiment using a mixture of inhibitors, we could further detect differences in relative inhibition constants. A more tightly binding inhibitor concentrated in the carbonic anhydrase compartment to a greater extent than a less tightly binding inhibitor. Starting with an equimolar mixture of sulfonamide dipeptides 4a-d and the noninhibitor 5 in both compartments, the sulfonamide dipeptides concentrated into the carbonic anhydrase compartment, Figure 3. The fraction of dipeptide in the carbonic anhydrase compartment varied: 88, 82, 74, 70, and 48% for 4a, 4b, 4c, 4d, and the noninhibitor 5, respectively (or a ratio of 1.83: 1. 71: 1.54: 1.46: 1 for 4a:4b:4c:4d:5). The order of highest to lowest concentration in the carbonic anhydrase chamber reflects the order of the binding constants of the inhibitors.
These results show that is possible to distinguish between inhibitors, but the differences in concentration are small, especially among inhibitors of similar strength. Even comparing the best inhibitor (4a) with a noninhibitor (5) gives a concentration differing by less than a factor of 2. To enhance this
5696 J. AM. CHEM. SOC •• VOL. 124, NO. 20,2002
difference in concentration, we explored the use of proteases to destroy the poorer inhibitors.
Screening of Proteases. We screened 22 commercially available proteases to identify those that could hydrolyze the dipeptide EtOC-Phesa-Phe (4a). AU proteases showed sorne activity. Using 0.1 mg of protease and 2.umol (2 mM) dipeptide 4a, the five most active proteases (a-chymotypsin, protease from Streptomyces casepitosus, proteinase K, Pronase from Streptomyces grise us, and protease from Bacillus thermoproteolyticus rokko) cleaved all of the dipeptide within 24 h, while two moderately active proteases (protease N "Amano", protease from Bacillus polymyxa) cleaved a11 of the dipeptide within 48 h. The remaining proteases cleaved less than half of the dipeptide after 72 h. We chose one of the most active yet inexpensive enzymes, Pronase from S. grise us, for subsequent experiments. Pronase was found to cleave all five dipeptides (4a-d and 5), although the glycine and proline dipeptides (4b and 4d) were cleaved more slowly (80-90% hydrolysis within 24 hl. To ensure high cleavage rates, larger amounts of Pronase were used in the competitive degradation experiments described below.
Selective Protection of Inhibitors by Carbonic Anhydrase from Hydrolysis. We compared the ability of carbonic anhydrase to protect sulfonamide inhibitor 4a from hydrolysis while allowing a noninhibitor, 5, to be hydrolyzed. An experiment
Screening Sensitivity through Selective Destruction
Outar Chambar Inner Chamber
Ho.z~ H NHEtOC .,~ ::::;:::::b:: HO ~ NHEtOC ; .,~ : 2 H
pronase 1 1" .... ,
H~HEIOC 1"
" "'
! 1 ::
~i "' .si 1 carbonic ml anhydrase
~ ~I
!
~
Ho,c"- NHEtOC R.~ " • CA 1 ... .,
Inhlbltor 1 CA complex
Figure 4. Reaction design for the selective destruction experiments. The dipeptides can diffuse across the dialysis membrane into either chamber. One chamber contains carbonic anhydrase, the other contains Pronase. Dipeptides in the Pronase charnber are rapidly cleaved to their constituent pieces. Carbonic anhydrase prevents strong binding dipeptides from diffusing across the membrane and thus slows their hydrolysis.
0.2 Ioside Chamber
0.16 r-~"'---,--""--=------,, 4a
0.12 [dipeptide)
(mM) 0.08
0.04
+-__ ,-___ -r __ -r~=_~5
3 6 9 12
Time (h)
Figure 5.13 Selective protection from hydrolysis of sulfonamide 4a over noninlùbitor 5 by carbonic anhydrase. A vessel containing two cornpartrnents of equal volume (20 mL each) separated by a dialysis membrane was filled with a solution of. sulfonamide 43 (0.16 mM) and noninlùbitor 5 (0.19 mM). The inside compartment contained carbonic anhydrase (0.34 mM), while the outside compartment contained Pronase. The protease rapidly cleaved the dipeptides in the outside compartment to the corresponding amino acids (data not shown). The noninhibitor 5 diffused freely across the dialysis membrane and was cleaved by the protease. In contrast, the inhibitor 43 bound to carbonic anhydrase in the inside compartrnent and was not consumed at a significant rate. After 6 h, the concentration of sulfonamide 43 in the inside compartment decreased by oruy 6% (0.15 mM), while the concentration of noninhibitor 5 decreased to 0.041 mM during the sarne time period (ratio = 3.7:1).
similar to that described above, except with Pronase added to
the outer chamber was set up (Figure 4). On the one hand, in
the Pronase-containing chamber, both dipeptides were rapidly
cleaved to the constituent pieces within 15 min. On the other
hand, the inside compartment showed a steady decrease in the
concentration of noninhibitor 5 over 12 h (Figure 5), while the
concentration of sulfonarnide 4a remained nearly constant
(a decrease of 9% over 12 h).14 After even just 6 h, the ratio,
4a:5, in the inside compartment was 3.7:1 and continued to
increase to greater than 20:1 after 12 h. By comparison, the
experiment which does not con tain Pronase had a final ratio of
4a to 5 of 1.75:1. In a simlar experiment, dipeptides 4a and 4b, which have
very similar binding constants, were exposed to carbonic
anhydrase and Pronase. In this experiment, the dipeptides were
(13) Lines drawn in ail figures (except Figure 1 and Figure 9) are for illustration purposes only. They do not represent theoretical Iines of any sort.
(14) Both 4a and 5 diffused through the membrane at identical rates with a half-Iife of about 3 h. (Data not shown.)
0.3 Ioside Chamber
0.25 •
0.2.,..,...-__
[dipeptide) 0 15 (mM) •
0.1
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0.05 43
0+-__ ~ ____ '-__ ~r-__ -r __ ~4b
o 40 80 120 160 200
Time(h)
Figure 6.13 Selective protection from hydrolysis of dipeptide 43 over 4b by carbonic anhydrase. A reaction vessel was separated into Iwo compartments (20 mL each) by a dialysis membrane. The inside compartment contained carbonic anhydrase (13.6 mol), dipeptide 43 (4.3 mol) and dipeptide 4b (4.3 mol) in 20 mL ofbuffer. The outer compartrnent contained Pronase (5 mg) dissolved in 20 mL of buffer. The time course of the reaction in the carbonic anhydrase chamber is shown in the figure. At 83% conversion (193 h) the ratio of 4a to 4b was 3.8:1.
0.2
0.16
0.12 [dipeptide)
(mM) 0.08
0.04
Ioside Chamber
4a
o +-----.....,..-----,.----i 4c
o 2 4 6
Time(h)
Figure 7.n Selective protection from hydrolysis of dipeptide 4a over 4c by carbonic anhydrase. A reaction vessel was separated into two compartments (20 mL each) by a dialysis membrane. The inside compartment contained carbonic anhydrase (0.34 mM), while the outside compartment contained Pronase (4 mg). Both compartments initially contained similar concentrations of dipeptide 43 (0.16 mM) and dipeptide 4c (0.14 mM). The protease rapidly cleaved the dipeptides in the outside compartment to the corresponding amino acids (data not shown). The time course of the reaction in the carbonic anhydrase charnber is shown in the figure. After 6 h, 93% of 4c inside the CA chamber had been hydrolyzed, white oruy 58% of 4a had hydrolyzed A control experiment which did not contain carbonic anhydrase showed an equal rate of hydrolysis for the two dipeptides in the chamber not containing Pronase.
placed only in the carbonic anhydrase chamber, and an excess
of carbonic anhydrase was used (1.6: 1 ratio of CA to dipeptides)
so that the conditions adhered rigorously to those of the theory
described above. As expected, due to the excess of target and
tight binding of both dipeptides, the hydrolysis of 4a and 4b
was slow. However, as in the first reaction, the weaker binder,
4b, was consumed at a higher rate (Figure 6). After 193 h, 83% of the total dipeptides had been hydrolyzed, and the ratio of 4a
to 4b was 3.8:1. This final ratio is in excess of the ratio of the
independently determined binding constants of the dipeptides
(2.1:1).
In a related experiment, we compared two sulfonamide
dipeptides 4a and 4c which also have similar inhibition constants
(Figure 7). In this experiment, both the inside and outside
chambers initially contained equal concentrations of the dipep
tides, and the total concentration of dipeptides was in excess
(2.1: 1 ratio of dipeptides to CA). The result was a much faster
hydrolysis of both dipeptides in the carbonic anhydrase chamber.
This faster rate reflects the rapid release of 1 equiv of PheSA
J. AM. CHEM. SOC .• VOL. 124, NO. 20, 2002 5697
ARTICLES
(2) from the Pronase chamber. Although 2 is a weaker binder than either 4a or 4c, enough of it was produced such that it could displace a small amount of 4a and 4c from the carbonic anhydrase binding pocket, thus accelerating their hydrolysis by Pronase. However, the net result was still the same. After 6 h, 93% of 4c was hydrolyzed after 6 h, but only 58% of 4a was hydrolyzed. Thus, the ratio of concentrations was 6:1, which is much larger than the 1.6: 1 ratio observed in a control experirnent which did not contain Pronase and larger than the 3.7:1 ratio of their binding constants.
Fina11y, an experiment containing a11 five dipeptides (4a-d and 5) was conducted using an excess of carbonic anhydrase (ratio of CA to dipeptides is 1.2:1). The experiment was consistent both with the theory and with the prior results. Dipeptide 5 was cleaved rapidly while dipeptides 4a-d disappeared at rates which corresponded to their binding constants (Figure 8).
Discussion
As expected, the four sulfonamide dipeptides 4a-d all inhibit carbonic anhydrase competitively with similar inhibition constants (within a factor of 10 of each other). Classical kinetics using initial rates easily identified these differences, but these classical methods are slow and require the separate measurement of each inhibitor. This becomes laborious for libraries containing thousands of members.
To rapidly identify the best inhibitor, we used competitive binding to carbonic anhydrase in one compartrnent of a twocompartrnent ce11. The inhibitors concentrated into the carbonic anhydrase compartment of a two-compartment ce11. Higher concentrations of the better inhibitors were observed in the carbonic anhydrase compartrnent, but the concentration differences were small (1.83:1.71:1.54:1.46:1 for 4a:4b:4c:4d:5). If the mixture contained 1000 dipeptides, this competitive experiment would not identify the best inhibitor because it would be difficult to separate all the dipeptides, and the differences in concentration with and without target would be small.
Although this experiment does not include a dipeptidesynthesis step and thus is not a dynamic library, the diffusion across the membrane mimics a synthesis step in a dynamic library in that both are equilibrium processes. For the diffusion process, in the absence of a target, each compartment should contain equal amounts of each inhibitor. In the presence of the target, the carbonic anhydrase chamber con tains more of the tight-binding inhibitors. Thus, the equilibrium for the diffusion process has shifted.
A nonselective destruction of the library members should enhance differences in the relative concentrations of the members bound to the target. The poor binding members are destroyed at a rate higher than that for the strong-binding members, and as the degradation progresses, the ratio improves exponentially in favor of the latter. This was observed in our library, where dipeptide hydrolysis by Pronase was used as the destruction process. In a competition experiment between a strong and weak binder (4a vs 5), the ratio of 4a to 5 in the carbonic anhydrase chamber increased from 1.75:1 in the absence of Pronase to 3.7:1 in the presence of Pronase (at 45% conversion). Furthennore, this ratio continued to increase to > 20: 1 as the reaction progressed. A second experiment with two species with very similar Kj's (4a vs 4b) had a final ratio of 3.8: 1 when the ratio of the binding constants was 2.1: 1. As
5698 J. AM. CHEM. SOC .• VOL. 124, NO. 20, 2002
0.2S
0.2
O.lS [dipeptide)
(mM) 0.1
O.OS
o
Inside Chamber
40 80 120 Time (h)
Cheeseman et al.
160
4a 4b
200
Figure 8.13 Selective protection from hydrolysis of dipeptides by carbonic anhydrase. A reaction vessel separated into two compartments (20 mL each) by a dialysis membrane was set up. The inside compartment contained carbonic anhydrase (25.6 mol) and dipeptides 4a-d and 5 (4.3 mol each) in 20 mL of buffer. The outer compartment contained Pronase (5 mg) dissolved in 20 mL of buffer. The time course of the reaction in the carbonic anhydrase charnber is shown in the figure.
shown in Figure 9a, these results fo11ow the theoretical model closely. Similar results were obtained for an experirnent containing two inhibitors (4a and 4c) where an excess of a weaker binder, PheSA (2), was generated in the reaction mixture. The presence of 2 accelerated the rate of cleavage of 4a and 4c, but as can be seen in Figure 9b, the ratio of dipeptides during the course of the reaction still fo11owed the theoretical model. At 70% conversion, the ratio of 4a to 4c was 6: 1, which is much larger than the 1.6:1 ratio observed in a reaction not containing Pro nase. In all cases, the model indicates that the ratios should continue to increase if the reactions are carried out for even longer periods. In experiments with a large number of library members, this increase will be critical in allowing the tightest binding species to be easily identified. 15
One potentiallimitation of this screening method is selectivity in the destruction reaction. For example, dipeptide 4c is cleaved by Pronase at a much slower rate than that for dipeptide 4a. In such a case, S from eqs 10-12 will not be equal to the ratio of the binding constants, and thus the degradation reaction will not fo11ow the theoretical curves of Figure 1. To accommodate this situation, we used a large amount of protease, and more importantly, we employed a dialysis membrane to separate the target-inhibitor complexes from the protease. In this setup, the rate-limiting step in the destruction reaction is not the proteasecatalyzed cleavage but diffusion across the dialysis membrane. Unlike the protease-catalyzed cleavage, the rate of diffusion does not vary significantly with the structure of the inhibitor, and the result is that the destruction reaction fo11ows the theoretical curve. Although Pronase accepts a wide variety of peptides, substrate specificity of the enzyme may become problematic if highly diverse libraries are studied. A dipeptide which is not cleaved by Pronase would be retained in the reaction mixture even if it did not bind to carbonic anhydrase. One way ta alleviate this problem would be to use a mixture of enzymes with a wide range of specificities. Altematively, it is important to note that the degradation reaction is not limited to enzymic processes. Other chernical degradation methods can be envisioned, depending upon the type of library being studied. For
(15) The reaction mixture will contain the products of the degradation reactions. However, in most cases, this method will be applied to combinatorial libraries, and as sucb, the degradation products will often be the common starting materials used to make the library members. Thus, only a limited number of degradation products will be produced.
Screening Sensitivity through Selective Destruction
a) 5
4
Ratio 3
4a/4b 2
• • O+------r------r------r------r-----~
o 0.2 0.4 0.6 0.8
Conversion
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b) 10
8
Ratio 6
4a/4c 4
2
0
0 0.2 0.4 0.6 0.8
Conversion Figure 9. The graph shows theoretical and experimental ratios for the screening experiments. Theoretical lines are shown as smooth \ines. The S values correspond to the ratios of the experimentally determined binding constants. The data points show the experimentally deteITnined ratios at different conversions for a) 4a/4b (cf. Figure 6) and b) 4a/4c (cf. Figure 7).
example, a library based on disulfide exchange could be degraded by adding a reducing agent (e.g., a phosphine) to cleave any unbound disulfides. Altematively, physical methods for removal of unbound inhibitors (e.g., adsorption to a solid phase, extraction) should accomplish the same effect as a chemical degradation.
Another potential limitation of this screening method, and indeed for methods based on the dynamic combinatoriallibrary technique, is the need for stoichiometric amounts of the target. The initial experiments reported here used large amounts of carbonic anhydrase (100-500 mg/experiment) as we expect to apply it to a dynamic library process where the best inhibitor will actually be isolated and characterized. However, for purely analytical screening purposes, the methods can easily be scaled down using smaller compartments, assurning that more sensitive analytical tools are used (e.g., mass spectroscopy). These modifications could reduce the amount of target needed to <0.1 mg/experiment, an amount that is easily accessible for targets that have been cloned and overexpressed.
In conclusion, we have developed a method for screening mixtures of compounds against a therapeutic target which readily identifies the best binder in a library. The method works by selectively degrading the poorer inhibitors with an enzyme. This results in a significant improvement in the ability to distinguish between inhibitors which have very close binding constants. We plan to extend this method to dynamic libraries with the goal of improving the enhancement observed in synthesis of good inhibitors in the presence of a therapeutic target.
Experimental Section
General Experimental p-Nitrophenyl acetate (PNPA), carbonic anhydrase (CA, from bovine erythrocytes, a mixture of isozymes, C-3934) and proteases were purchased from Sigma unless otherwise noted and used without further purification. HPLC analyses were conducted using a Phenomenex-Cs reversed phase HPLC column (10 mm x 250 mm) with detection at 220 mn, unless otherwise noted. Elemental analyses were obtained from Quantitative Technologies Inc., Whitehouse, NJ. High-resolution mass spectra were obtained from Université de Sherbrooke, Sherbrooke, QC.
4'-Sulfonamidophenylalanine (1). N-Acetylphenylalanine (37.7 g, 178 mmol, 1 equiv) was added in portions over a I-h period to neat chlorosulfonic acid (110 mL, 1.65 mol, 9.5 equiv) at -10 OC. The resulting yellow solution was stirred at -10 oC for 2.5 h, at 25 oC for 2.5 h, and then heated to 60 oC until gas evolution had ceased (approximately 12 hl. The resulting orange solution was cooled to 0 oC and poured carefully onto 750 mL of ice (Caution: exotherrn!).
The resulting mixture was extracted with ethyl acetate (3 x 1 L), and the combined organic layers were dried over Na2S04, filtered, and concentrated in vacuo to afford the sulfonyl chloride (45.1 g, 83%) as an orange solid which was used immediately without further purification. IH NMR «CD3hSO) Ô 8.26-8.21 (d, tH, J = 8.5 Hz), 7.55 (d, 2H, J = 6.9 Hz), 7.22 (d, 2H, J = 6.8 Hz), 4.49-4.34 (m, tH),3.13-3.00 (dd, tH, J = 14.4 and 6.8 Hz), 2.92-2.79 (dd, tH, J = 11.0 and 10.2 Hz), 1.80 (s, 3H).
The sulfonyl chloride was dissolved in 28% NH40H (240 mL), and the resulting solution was heated at reflux for 3 h. After cooling to 0 oC, the solution was acidified to pH 1 by addition of 3 M H2S04 (ca. 200 mL) and extracted with ethyl acetate (3 x 500 mL). The combined organic extracts were dried over Na2S04, filtered, and concentrated in vacuo to afford the sulfonamide (29.9 g, 71 %) as a white solid. The N-acetyl sulfonamide could not be purified to homogeneity by either chromatography or recrystallization. IH NMR «CD3hSO) Ô 8.29-8.24 (d, tH, J = 8.5 Hz), 7.77 (d, 2H, J = 3.9 Hz), 7.45 (d, 2H, J = 6.9 Hz), 7.33 (s, 2H), 4.53-4.41 (m, tH), 3.20-3.09 (dd, IH, J = 14.2 and 6.8 Hz), 3.01-2.87 (dd, tH, J = 11.2 and 10.1 Hz), 1.80 (s, 3H).
A suspension of the sulfonamide (20.0 g, 69.9 mmol, 1 equiv) in distilled water (300 mL) was adjusted to pH 5.00 with LiOH (900 mg). A 0.25 M solution of Na2HP04 (85 mL) was uSed to raise the pH to 7.50. Acylase 1 from hog kidney (200 mg, 17.8 U/mg, 3560 U) was added as an aqueous solution (12 mL), and the resulting solution was stirred at 21°C for 70 h. The solution was then acidified to pH 1.0 with 3 M H2S04 and extracted with ethyl acetate (3 x 500 mL); the organic layer was then dried with anhydrous sodium sulfate and concentrated in vacuo to afford 2.28 g (II %) of the sulfonamide starting rnaterial. The aqueous layer was neutralized with 2 M NaOH and concentrated. The solution was then applied to an Amberlite 120(plus) acidic ion-exchange column. The column was rinsed with water until the eluent was at pH 6.0, and then it was rinsed with 0.50 M ~OH solution until the eluent became basic. The basic wash was concentrated in vacuo and recrystallized from water to afford the provided 4'sulfonamidophenylalanine as a white solid (11.60 g, 68%).IH NMR (D20IDCI) Ô 7.62 (d, 2H, J = 8.1 Hz), 7.26 (d, 2H, J = 8.1 Hz), 4.14 (t, tH, J = 6.8 Hz), 3.19-3.12 (dd, tH, J = 14.6 and 5.7 Hz),3.08-3.01 (dd, tH, J = 14.4 and 6.9 Hz). l3C NMR (D20IDCI) Ô 170.73, 140.451, 139.49, 130.38, 126.55,53.49,35.19. FABMS in saturated NaCI mlz 267 (M + Na, C9H12N20 4SNa requires 267.)
N-Ethoxycarbonyl-4'-sulfonamidophenylalanine (2). Ethyl chloroforrnate (398 JlL, 4.17 mmol, 1.10 equiv) was added to a two-phase mixture of 4'-sulfonamidophenylalanine (925 mg, 3.73 mmol, 1 equiv) in 1,4-dioxane (25 mL) and saturated NaHC03 solution (25 mL) at 0 oC, and the resulting solution was stirred for 6 h at 0 oC. The mixture was extracted with ethyl acetate (100 mL), and the aqueous layer was acidified to pH 1 by addition of 2 M HCI (ca. 20 mL) and then extracted with ethyl acetate (3 x 50 mL). Latter organic extracts were combined, dried over Na2S04, filtered, and concentrated in vacuo to afford the
J. AM. CHEM. SOC •• VOL. 124, NO. 20, 2002 5699
ARTICLES
ethyl carbamate (879 mg, 83%) as an analytically pure oil. IH NMR «C03hCQ) à 7.85 (d, 2H, J = 7.1 Hz), 7.51 (d, 2H, J = 6.9 Hz), 6.54 (s, 2H), 6.45 (d, IH, J = 6.7 Hz), 4.62-4.45 (m, lH), 4.01-3.97 (q, 2H, J = 2.4 Hz), 3.41-3.28 (dd, lH, J = 11.3 and 4.0 Hz), 3.16-3.05 (dd, lH, J = 10.4 and 7.8 Hz), 1.13 (t, 3H, J = 6.0 Hz). HR-CIMS (mlz): [MH+] calculated for CI2H17N206S, 317.0807; found, 317.0817.
Et01C.(4'.SOzNH1)Phe-Gly.O-terl.butyl (3b). EDC'HCl (136 mg, 0.711 mmol, 1.10 equiv), HOBT (87.3 mg, 0.646 mmol, 1.00 equiv), and triethylamine (269 pL, 1.94 mmol, 3.00 equiv) were added to a solution of 2 (204 mg, 0.646 mmol, 1 equiv) in THF (3 mL) at 0 oc. Glycine tert-butyl ester'HCI (119 mg, 0.711 mmol, 1.10 equiv) was added, and the resulting solution was allowed to warm to 21°C while stirring for 13 h, at which point the bulk of the THF was removed by concentration in vacuo. The residue was dissolved in ethyl acetate (45 mL) and extracted with 0.1 M HCI (3 x 25 mL) and saturated NaRC03 solution (3 x 25 mL). The organic layer was dried over Na2S04, tiltered, and concentrated in vacuo. The solid residue was purified by mixed solvent recrystallization (ethyl acetatelhexanes) to afford 193 mg (70%) of 3b. IH NMR «CD3hCO) à 7.82 (d, 2H, J = 7.5 Hz), 7.63 (s, IH), 7.50 (d, 2H, J = 7.5 Hz), 6.52 (s, 2H), 6.40 (d, lH, J = 7.5 Hz), 4.50 (m, lH), 4.00-3.88 (m, 4H), 3.40 (dd, lH, J = 14.1 and 4.2 Hz), 3.02 (dd, lH, J = 13.5 and 9.9 Hz), 1.45 (s,9H), 1.12 (t, 3H, J = 6.9 Hz). 13C NMR «CD3hCO) à 171.48, 168.93, 156.33, 142.70, 130.06,126.18,81.04,60.48,55.92,41.79,37.83,27.52, 14.22. Analysis calculated for CI8H27N307S C, 50.34; H, 6.34; N, 9.78. Found: C, 50.33; H, 6.35; N, 9.73.
Et01C·(4'·SOlNHZ)Phe·Gly·OH (4b). TFA (7 mL) was added to a solution of 3b (175 mg, 0.409 mmol, 1 equiv) in CH2Ch (8 mL), and the solution was stirred for 25 min at 21°C under an atmosphere of argon. The solvents were removed in vacuo, and the residue was purified by recrystallization from acetone to afford 121 mg (79%) of 4b. IH NMR (C030D) à 8.55 (s, lH), 7.83 (d, 2H, J = 7.2 Hz), 7.46 (d, 2H, J = 7.2 Hz), 4.45-4.42 (m, lH), 4.02-3.98 (q, 2H, J = 6.8), 3.95-3.92 (m, IH), 3.32-3.25 (m, 2H), 2.97-2.89 (dd, lH, J = 13.5 and 9.9 Hz), 1.18-1.14 (t, 3H, J = 6.8). 13C NMR (CD3CD) à 173.0, 171.8,157.3,148.7,142.4,137.6,129.8,126.0,60.9, 56.0, 37.7,13.7. HR-ClMS (mlz): [MW] calculated for CI4H2oN3Û7S, 374.1022; found, 374.1030.
Measurement of Inhibition Constants. Kinetic constants for carbonic anhydrase (CA) were measured according to Pocker and Stone usingp-nitrophenyl acetate (PNPA) as the substrate.16 The CA-catalyzed hydrolysis of pNPA was followed spectrophotometrically at 25 oC in a 96-well microplate spectrophotometer by monitoring the appearance of p-nitrophenolate at 404 nm. The values of Km and V max were deterrnined by measuring the hydrolysis rate as a function of the pNPA concentration. To deterrnine the inhibition constants, the values of Km and V max were redetermined in the presence of varying amounts of inhibitor. Since the values of Km for pNPA increased in the presence of the inhibitor, but the values of Vrnax remained unchanged, we concluded that the inhibition is competitive. The concentration of inhibitor that increased the Km for pNPA by a factor of 2 is the inhibition constant. A typical procedure was to add CA solution (100.0 pL) with inhibitor to acetonitrile solution of pNPA (5.0 pL). In the assay solution, the concentration of inhibitor ranges from 0.0 to 6.0 pM, while the concentration of pNPA ranged from 0.2 to 2.5 mM. The microplate was shaken for 5 s before the fust reading and for 3 s between readings.
Selective Concentration of EtOC·Phe..·Phe (4a) over EtOC·PhePhe, (5) into a Compartment Containing Carbonic Anhydrase. A solution of 4a (2.9 mg, 6.3 pmol) and 5 (2.9 mg, 7.5 pmol) in 0.01 M aqueous phosphate buffer (pH 7.5, 40 mL) was divided into two equal portions. Carbonic anhydrase (0.20 g, approx. 6.7 pmol) was dissolved in the first portion, and the resulting solution (20.0 mL) was transferred to a dialysis bag (12 OOO-MW cutoff, Sigma 0-0655). This dialysis bag was suspended in the second portion, and the reaction vessel was shaken gently (200 rpm) at 30 oC. Aliquots were removed periodically
(16) Poeker, Y.; Stone, J. T. Biochemistry 1968, 7, 3021-3031.
5700 J. AM. CHEM. SOC •• VOL. 124, NO. 20, 2002
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from each compartment, heated to 80 oC until a white precipitate forrned (~5 min), and centrifuged, and the supematant was filtered through a 0.22-pm pore tilter. The amount of dipeptides was measured by HPLC using a Zorbax C8 colurun and 40160/0.1 water/methanolltrifluoroacetic acid at 0.40 mL/min. After 12 h 88% of 4a (retention time 11.4 min) had accumulated inside the dialysis bag while only 42% of 5 (retention time 25.5 min) was found inside the bag.
Selective Concentration Of EtOC·Phe...Phe (4a) from a Mixture of EtOC·Phe. •• Leu (4c), EtOC·Phe..·Gly (4b), and EtOC·Phe-Phe (5) by Carbonlc Anhydrase. Dipeptides 4a (2.0 mg, 4.3 pmol), 4b (1.6 mg, 4.3 pmol), 4c (1.9 mg, 4. pmol) 4d (1.8 mg, 4.3 pmol), and 5 (1.7 mg, 4.3 pmol) were dissolved in 40 mL of 10 mM KH2P04 buffer, pH 7.5 containing 0.1 mg/mL penicillin G (to avoid bacterial growth). Carbonic anhydrase (CA) (0.29 g, 9.7 pmol, 0.45 equiv) was dissolved in 20 mL of this solution and placed in a dialysis bag (the bag was washed in ddH20 for 1 h, rinsed in EtOH once, and then washed again with ddH20). The bag was suspended in the remaining 20 mL of inhibitor solution in a lOO-mL container and shaken at 60 rpm on a three-dimensional orbital shaker at room temperature for 49 h. Samples (1 mL) were taken periodically from inside and outside the dialysis bag, heated in an 80 oC water bath for 5 min, and then centrifuged for 10 min. The supernatant was tiltered through a 0.22-pM sterile tilter. The supematant (700 pL) was added to MeOH (300 pL) to forrn the HPLC sample (30% MeOH, 70% aqueous). The sample was run on a Phenomenex C8 reverse phase colurun under the following conditions: 0-15 min 30% MeOH, 70% H20, 15-60 min 37% MeOH 63% H20, 60-90 min 62% MeOH, 38% H20. The peak areas were monitored: Phes.Gly: 7.9 min, PhesaPro: 17.6 min, Phe •• Leu: 54.5 min, Phes.Phe: 60.0 min, PhePhe: 69.5 min. The percentages are accurate to ±2%. Ali nonsterile apparatus used was autoclaved prior to use to avoid bacterial growth.
Screening of Proteases for the Hydrolysis of EtOC·Phe·Phe Dipeptide (4a). The protease to be screened (0.1 mg) was added to a solution of 4a (1.0 mg, 2.2 mol) in 0.01 M aqueous phosphate buffer (pH 7.5). The solution was kept at 30 oC, and aliquots were removed periodically, worked up as above, and analyzed by HPLC.
Selective Protection of Inhibitors from Hydrolysis by Carbonic Anhydrase. A solution of 4a (3.0 mg, 6.5 pmol) and 5 (2.8 mg, 7.3 IImol) in 0.01 M aqueous phosphate buffer (pH 7.5, 40 mL) was divided into two equal portions. Carbonic anhydrase (0.20 g, approx 6.7 IImol) was dissolved in the first portion, and the resulting solution (20.0 mL) was transferred to a dialysis bag (12 OOO-MW cutoff, Sigma 0-0655). Pronase from S. griseus (Sigma P-5147, 4 mg) was dissolved in the second portion, and the dialysis bag was then suspended in the resulting solution. The reaction vessel was then shaken gently (200 rpm) at 30 oC, and aliquots were removed periodically from each compartment, worked up as above, and analyzed by HPLC. After 30 min, neither substrate was detectable in the solution outside the dialysis bag. Inside the dialysis bag, 78% of 5 had hydrolyzed, while only 6% of 4a had hydrolyzed after 6 h. In a control experiment containing no carbonic anhydrase, inside the dialysis bag, 76% of 4a and 80% of 5 had hydrolyzed after 6 h.
Selective Binding of EtOC·Phe..·Phe (4a) over EtOC·Phe..·Leu (4c). A solution of 4a (3.3 mg 7.1 mol) and 4c (3.6 mg, 8.4 mol) in 0.01 M aqueous phosphate buffer (pH 7.5, 40 mL) was divided into two equal portions. Carbonic anhydrase (0.20 g, approx 6.7 IImol) was dissolved in the first portion, and the resulting solution (20.0 mL) was transferred to a dialysis bag (12 OOQ-MW cutoff, Sigma 0-0655). This dialysis bag was suspended in the second portion, and the reaction vessel was shaken gently (200 rpm) at 30 oC. Aliquots were removed periodically from each compartment, worked up as above, and analyzed by HPLC using a Zorbax C8 colurun. After 12 h 98% of 4a had accumulated inside the dialysis bag, while only 60% of 4c was found inside the bag.
Hydrolysis of EtOC·Phe..·Gly (4b) and EtOC·Phe..·Phe (4a) in the Presence of Carbonic Anhydrase. PhesaPhe 4a (2.0 mg, 4.3 pmol)
Screening Sensitivity through Selective Destruction
and Phe",Gly 4b (1.6 mg, 4.3 .umol) were dissolved in 20 mL of 10 mM KH2P04 buffer, pH 7.5. Carbonic anhydrase (CA) (0.4090 g, 13.6 .umol, 1.60 equiv) was dissolved in this solution and placed in a dialysis bag (the bag was washed in ddH20 for 1 h, rinsed in EtOH once, and then washed again with ddH20). The bag was suspended in 20 mL of the phosphate buffer containing Pronase from S. griseus (5.0 mg, 0.01 equiv) in a 150-mL beaker and shaken at 150 rpm at 30 oc for 313 h. Samples (1 mL) were taken periodically from inside, worked up as above, and analyzed by HPLC. After 193 h, only 71% of 4a had hydrolyzed, while 93% of 4b had hydrolyzed.
Hydrolysis of EtOC·Phes.·Leu (4c) and EtOC·Phe,...Phe (4a) in the Absence of Carbonic Anhydrase. A solution of 4a (2.9 mg 6.3 mol) and 4c (2.4 mg, 5.6 mol) in 0.01 M aqueous phosphate buffer (pH 7.5, 40 mL) was divided into two equal portions. The first portion was transferred to a dialysis bag (12 OOO-MW cutoff, Sigma D-0655). Pronase from S. griseus (Sigma P-5147, 4 mg) was dissolved in the second portion, and the dialysis bag was then suspended in the resulting solution. The reaction vessel was then shaken gently (200 rpm) at 30 oC, and aliquots were removed periodically from each compartment, worked up as above, and analyzed by HPLC using a Zorbax C8 column. After 30 min, neither substrate was detectable in the solution outside the dialysis bag. After 8 h, 86% of 4a and 88% of 4c inside the dialysis bag had hydrolyzed.
Hydrolysis of EtOC·Phe,...Leu (4c) and EtOC·Phe, •• Phe (4a) in the Presence of Carbonic Anhydrase. A solution of 4a (2.9 mg, 6.3 mol) and 4c (2.4 mg, 5.6 mol) in 0.01 M aqueous phosphate buffer (pH 7.5, 40 mL) was divided into two equal portions. Carbonic anhydrase (0.14 g, approx 4.7 .umol) was dissolved in the first portion, and the resulting solution (20.0 mL) was transferred to a dialysis bag (12000-MW cutoff, Sigma D-0655). Pronase from S. griseus (Sigma P-5147, 4 mg) was dissolved in the second portion, and the dialysis
ARTICLES
bag was then suspended in the resulting solution. The reaction vessel was then shaken gently (200 rpm) at 30 oC, and aliquots were removed periodically from each compartrnent, worked up as above, and analyzed by HPLC. After 6 h, 93% of 4c had hydrolyzed, while only 58% of 4a was hydrolyzed.
Hydrolysis of EtOC·Phe, •• Phe (4a), EtOC·Phe .. ·Gly (4b) EtOC· Phe,.·Leu (4c), EtOC·Phe,.·Pro (4d) and EtOC·Phe-Phe (5), in the Presence of Carbonic Anhydrase. Phes.Phe 4a (2.0 mg, 4.3 .umol), Phes.Gly 4b (1.6 mg, 4.3 .umol), Phe.,.Leu 4c (1.9 mg, 4.3 .umol), Phes.Pro 4d (1.8 mg, 4.3 .umol), and PhePhe 5 (1.7 mg, 4.3 .umol) were dissolved in 20 mL of 10 mM KH2P04 buffer, pH 7.5. Carbonic anhydrase (CA) (0.7670 g, 25.6 .umol, 1.20 equiv) was dissolved in this solution and placed in a dialysis bag (the bag was washed in ddH20 for 1 h, rinsed in EtOH once, and then washed again with ddH20). The bag was suspended in 20 mL of the phosphate buffer containing Pronase from S. griseus (4.9 mg, 0.01 equiv) in a 150-mL beaker and shaken at 150 rpm at 30 oC for 193 h. Samples (1 mL) were taken, worked up as above, and analyzed by HPLC. Data for this experiment is shown in Figure 8.
Acknowledgment. We thank Merek Frosst Ine., BioChem Pharma Ine., Boehringer Ingelheim Ine., AstraZeneea Ine., and FCAR for generous funding of this researeh. J.D.C. thanks NSERC for a postgraduate fellowship. The reviewers are aeknowledged for helpful eomments and suggestions.
Supporting Information Available: A derivation of eq 3 and eharaeterization data for 3a,c,d and 4a,c,d (PDF). This material is available free of eharge via the Internet at http://pubs.aes.org.
JA017099+
J. AM. CHEM. SOC •• VOL. 124, NO. 20, 2002 5701
Supporting Information
Amplification of Screening Sensitivity Through Selective Destruction. Theory
and Screening of a Library of Carbonic Anhydrase Inhibitors.
Ronghua Shu, Andrew D. Corbett, Jeremy D. Cheeseman, Jonathan Croteau, James L. Gleason*
and Romas J. Kazlauskas*
Department of Chemistry, McGill University, 801 Sherbrooke Street West,
Montréal, QC, Canada, H3A 2K6.
Derivation of the equation for an equilibrium ratio of products in adynamie combinatorial
library.
Consider a common starting material, SM, in equilibrium with two inhibitors, A and B,
which can each bind reversibly to a target, T, to form complexes TVA and TVB
KsA KaA A+T T·A (1 )
SM KsB
B+T KaB
T·B (2)
Equations 3-5 define respectively KSA' the equilibrium constant for synthesis of inhibitor A,
KaA' the equilibrium constant binding of inhibitor A to the target, and [AT]' the total ofbound
and unbound forms of A.
K = [A] sA [SM]
(3)
K = [T-A] aA [T][A]
(4)
[Ar]=[A]+[T-A] (5)
Combining these equations and solving for [AT] yields equation 6.
(6)
Dividing this equation by an analogous equation for [BT] yields equation 7.
[Ar] _ KsAKaA[T] + 1) [BT ] - K sB (KaB [T]+l)
(7)
Under typical conditions (tight binding and an excess of target at a high concentration), the
product of the association constant (KaA or KaB) and free target concentration is much greater
than one so the equation simplifies to equation 8, below. l
[Ar] = KsA x KaA [BT ] K sB X K aB
Characterization Data:
(8)
Et02C-(4'-S02NH2)Phe-Phe-O-t-butyl (3a). Prepared as 3b to afford to afford 173 mg (72%).
IH NMR (CDCh) Ô 7.80 (d, 2H, J= 7.8 Hz), 7.33-7.24 (m, 5H), 7.09 (d, 2H, J= 6.0 Hz), 6.41
(d, 1H,J= 5.7 Hz), 5.19 (d, 1H,J= 6.1 Hz), 4.96 (s, 2H), 4.75-4.61 (m, 1H), 4.52-4.38 (m, 1H),
4.10-4.03 (q, 2H, J = 6.9 Hz), 3.20-2.96 (m, 4H), 1.39 (s, 9H), 1.21 (t, 3H, J = 6.8 Hz). 13C NMR
(CDCh) Ô 170.34, 170.11, 142.21, 140.84, 136.03, 130.38, 129.65, 128.67, 127.31, 126.96,
82.93, 77.44, 61.76, 53.86, 38.40, 38.21, 28.16, 14.69. Ana1ysis calculated for C2sH33N307S C,
57.79; H, 6.40; N, 8.09. Found: C, 57.71; H, 6.34; N, 7.96.
Et02C-(4'-SÛ2NHüPhe-Leu-O-t-butyl (3e). Prepared as 3b to afford 227 mg (78%). IH NMR
«CD3)2CO) Ô 7.81 (d, 2H, J = 8.4 Hz), 7.55 (m, 2H), 7.48 (d, 2H, J= 8.1 Hz), 6.51 (m, 1H),
6.31 (m, 1H),4.50(m, 1H),4.39 (m, 1H),4.00-3.95,(q,2H,J=5.7),3.32-3.26 (dd, 1H,J= 13.8
and 3.9 Hz), 3.05-2.97 (dd, 1H, J= 13.6 and 9.6 Hz), 1.75-1.64 (m, 2H), 1.61-1.56 (m, 2H), 1.45
(s,9H), 1.12 (t, 3H, J= 7.4 Hz), 0.94-0.90 (m, 7H). I3C NMR «CD3)2CO) Ô 171.8, 171.0, 156.4,
1 If the binding is not tight or the target is not in excess at high concentration, then the
concentrations of the inhibitors will be more similar than that discussed in the text above and it
will be even harder to distinguish which is the better inhibitor.
142.5, 130.1, 126.2,81.0,60.5,55.7,51.6,41.1,37.8,27.5,24.8,22.5, 21.3, 19.5, 14.2. Analysis
calculated for C22H3sN307S C, 54.42; H, 7.26; N, 8.65. Found: C, 54.28; H, 1.27; N, 8.46.
Et02C-(4'-SÜzNH2)Phe-Pro-O-t-butyl (3d). Prepared as 3b to afford 312 mg (67%). IH NMR
«CD3)2S0) ù 7.69 (d, 2H, J= 7.6 Hz), 7.48 (d, 2H, J= 7.0 Hz),7.36 (d, 2H, J= 7.2 Hz), 7.28 (s,
2H), 4.36 (m IH), 4.19 (m, IH), 3.85 (t, 2H, J = 6.7 Hz), 3,65 (m, 2H), 2.97-2.94 (dd IH, J =
11.7 and 4.2 Hz), 2.82-2.74 (dd IH, J= 11.1 and 9.2 Hz), 2.14 (m, IH), 1.90 (m, 2H), 1.77 (m,
IH), 1.35 (s, 9H), 1.04 (t, 3H, J = 7.3 Hz). 13C NMR «CD3)2S0)
ù 171.6,170.4,156.8,143.0,142.7,130.5,126.1,81.0,60.5, 60.1, 54.7, 47.1, 36.4, 29.2, 28.3,
25.3, 15.2. Analysis calculated for CI8H27N307S C, 50.34; H, 6.34; N, 9.78. Found: C, 50.33;
H, 6.35; N, 9.73.
Et02C-(4'-S02NH2)Phe-Phe-OH (4a). Prepared as 4b to afford 270 mg (68 %). IH NMR
(CD30D) Ù 8.19 (d, IH,J= 9.3 Hz), 7.80 (d, 2H,J= 8.4 Hz), 7.38 (d, 2H,J= 9.3 Hz), 7.27-7.21
(m, 5H), 7.07 (d, IH, J= 8.7 Hz), 4.68-4.63 (m, IH), 4.40-4.35 (q, 2H, J =6.1 Hz), 3.24-3.18
(dd, IH, J=13.9 and 5.2 Hz), 3.18-3.11 (dd, IH, J=14.3 and 5.5 Hz), 3.04-2.97 (dd, IH, J= 13.9
and 8.2 Hz), 2.88-2.80 (dd, IH, J= 13.9 and 9.7 Hz), 1.18-1.14 (t, 3H, J= 6.9 Hz). 13C NMR
(CD3CD) Ù 173.1, 172.3, 157.2, 142.3, 137.0, 129.8, 129.2, 128.3, 126.6, 126.0,60.9,55.9,53.9,
37.6, 37.2, 13.7. HR-CIMS (mlz): [MH+] calculated for C2IH26N307S, 464.1491; found,
464.1501.
Et02C-(4'-S02NH2)Phe-Leu-OH (4c). Prepared as 4b to afford 174 mg (80 %). IH NMR
(CD3CD) Ù 7.82 (d, 2H, J= 8.4 Hz), 7.68 (d, IH, J= 8.1 Hz), 7.45 (d, 2H, J= 8.1 Hz), 4.47-
4.42 (m, 2H), 4.01-3.95 (q, 2H, J= 6.4 Hz), 3.31-3.29 (m, IH), 3.25-3.3.19 (dd, IH, J= 13.9 and
4.9 Hz), 2.95-2.87 (dd, IH, J= 13.9 and 9.7 Hz), 1.71-1.62 (m, 2H), 1.18-1.13 (t, 3H, J= 7.1
Hz), 0.97-0.91 (m,6H). 13C NMR (CD3CD) Ù 174.6,172.7, 157.2, 142.3, 129.8, 126.7, 126.0,
60.9,55.8,50.9,40.4,37.7,24.8,22.2,20.6, 13.7. HR-CIMS (mlz): [MH+] calculated for
CI8H28N307S, 430.1648; found, 430.1654.
Et02C-(4'-S02NH2)Phe-Pro-OH (4d). Prepared as 4b except recrystallized from iso-propanol /
hexanes to afford 94.5 mg (55 %). IHNMR (CD30D) Ù 7.83 (d, 2H,J= 8.1 Hz), 7.50 (d, 2H, J
= 8.4 Hz), 7.25 (d, IH,J= 3.9 Hz), 4.66-4.61 (dd, IH,J= 8.8 and 5.5 Hz), 4.47-4.43 (dd, IH,J
= 8.4 and 3.9 Hz), 4.02-3.95 (q, 2H, J= 7.2 Hz), 3.80-3.75 (m, IH), 3.56-3.51 (m, IH), 3.32-3.29
(m, IH), 3.21-3.14 (dd, IH, J= 13.9 and 5.2 Hz), 2.96-2.89 (dd, IH, J= 13.8 and 8.7 Hz), 2.27-
2.21 (m, IH), 2.05-1.96 (m, 2H), 1.19-1.16 (t, 3H,J=7.2 Hz. 13CNMR(CD3CD) Ô 174.0,171.1,
157.3, 141.9, 130.1, 129.1, 128.2, 126.0,60.8,59.4,53.9,37.0,29.0,24.6, 13.7. HR-CIMS
(m1z): [MH+] ca1culated for C17H24N307S, 414.1335; found, 414.1325.
?t!smmunications
© 2004 Wiley·VCH Verl.g GmbH & Co. KG.A, Weinheim
Selective Binding '~p
Pseudodynamic Combinatorial Libraries: A Receptor-Assisted Approach for Drug Discovery**
Andrew D. Corbett, Jeremy D. Cheeseman, Romas 1 Kazlauskas, * and James L. Gleason*
Emerging methods of combinatorial chemistry incorporate receptor assistance to combine synthesis and screening.[I] Stoichiometric binding to a receptor alters either the thermo-
[*) A. D. Corbett, J. D. Cheeseman, Prof. R. J. Kazlauskas,+ Prof. J. L Gleason Department of Chemistry, McGili University 801 Sherbrooke St. West, Montréal, QC, H3A 2 K6 (Canada) Fax: (+ 1) 514·398·3797 E·mail: [email protected].
ri Current address: University of Minnesota Department of Biochemistry Molecular Biology and Biophysics and The Biotechnology Institute 1479 Gortner Avenue, Saint Paul, MN 55108 (USA) Fax: (+ 1) 612·625·5780
[**) The authors thank FQRNT for support ofthis research through the Soutien aux Equipes and VRQ programs, Prof. Sidney M. Hecht (University ofVirginia) for the discussion that led to this research, and Prof. Sijbren Otto, Prof. Jeremy K. M. Sanders, and Prof. K. Barry Sharpless for reading the manuscript.
• Supporting information for this article is available on the WWW under http://www.angewandte.org or from the author.
Angew. Chem. Inl. Ed. 2004, 43, 2432 -2436
dynamics or kinetics of library synthesis. Dynamic combinatorial libraries[21 use a thermodynamic approach where binding shifts a synthetic equilibrium to increase the amounts of the best-binding compounds, in accordance with LeChâtelier's principle. These libraries usually identify the library members that bind the tightest, but sorne experimental conditions can give small or misleading changes in concentrationYl An alternative method, receptor-accelerated synthesis, uses a kinetic approach.f41 Starting components that bind to the receptor can couple to create a new, tight-binding compound. The receptor accelerates the coupling of the bcttcr-fitting ~tnrting çomponçnt~ n~ n rc~ult of thcir proximity, but requires that both components bind tightly to the receptor. Here we demonstrate a new method, a pseudodynamic library, which adds a kinetic contribution to traditional dynamic libraries to dramatically increase the selectivity.
A pseudodynamic combinatorial library combines an irreversible synthesis of library members with an irreversible destruction step. Those library members that bind to the receptor are protected from destruction. Subsequent synthesis reuses fragments from destroyed library members, thus amplifying the amounts of the better binders at the expense of the lesser ones. The separate irreversible synthesis and destruction steps allow adjustment to optimize both the amplification and selectivity.
We developed a pseudodynamic library of eight dipeptides to identify the best inhibitor of carbonic anhydrase (CA). Carbonic anhydrase, a zinc metalloenzyme, is a therapeutic target for glaucoma and is inhibited by aromatic sulfonamides, which coordinate to the zinc ion. Four of the eight dipeptides in our library contain 4'-sulfonamidophenylalanine (Phesa, 1), and thus should bind to CA, while the remaining four contain only Phe and serve as negative controls. The irreversible synthesis of dipeptides used a
solid-supported coupling of activated esters with an amino acid in aqueous solution (Scheme 1). TentaGel-supported tetrafluorophenyl active esters react cleanly with free amino
Pronase
,x. = so,NW, 4X-H
Series: a R' " CH"ph. R·" H bR' =H,R'=H ç R' .. CH,CH(CH,h. R' '" H d R' R' '" {CH,l,
Scheme 1. Creation of a pseudodynamic library of dipeptides.
acids in water under alkaline (pH 8-10) conditions to form dipeptides.[51 A nonselective protease from Streptomyces griseus (Pronase) destroyed these dipeptides by catalyzing their irreversible hydrolysis.f61
The pseudodynamic library was prepared in a threechambered reaction vessel formed by suspending two dialysis bags in a surrounding solution (Figure 1). One dialysis bag (the synthesis chamber) contained the active esters and the other dialysis bag (hydrolysis chamber) contained the protease, while the surrounding solution (screening chamber) contained the carbonic anhydrase. Adding nucleophiles 1 and 2 to the synthesis chamber generated the dipeptide library. These dipeptides diffused into the surrounding solution where they could bind to carbonic anhydrase and then diffuse into the hydrolysis chamber where Pronase cleaved them. This
Figure 1. Schematic representation of the pseudodynamic combinatoriallibrary experiment. Reaction of two free amino acids (Phe .. (1) and Phe (2» with four solid-supported active esters (N-EtO,C-Phe, N-EtO,C-Gly, N-EtO,C-Leu, and N-EtO,C-Pro) creates an eight-member library. MWCO = molecular-weight eut off.
Angew. Chem. Inl. Ed. 2004, 43, 2432 -2436 www.angewandte.org © 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 2433
cleavage regenerated 1 and 2, which could diffuse back into the synthesis chamber to repeat the cycle. This arrangement prevented Pronase-catalyzed destruction and active-estermediated modification of the receptor (CA) and also permitted periodic replenishment of the activated ester to regulate the rate of synthesis.
The experiments used four active ester resins derived from N-EtOzC-Phe, N-Et02C-Gly, N-Et02C-Leu, and NEtOzC-Pro (0.8 equiv each), nucleophiles 1 and 2 (6.4 equiv each), carbonic anhydrase (28 J.lffiol, 1 equiv), and Pronase (25 mgmL-1
). The large amount of Pronase made diffusion across the dialysis membrane the rate-limiting step for hydrolysis; hence, ail the dipeptides were cleaved at similar rates in spite of the substrate selectivity of Pro nase. Periodic addition of fresh portions of active ester resin (defined as the cycle time) regulated the overall rate of library synthesis. We conducted three experiments with this system using cycle times of 8, 12, and 16 h. HPLC analysis of aliquots from the screening chamber showed the progress of the experiments (Figure 2).
Tho control experiments established, first, that the synthetic process afforded all the expected dipeptides and, second, that the sulfonamide-containing dipeptides inhibited carbonic anhydrase. Combining equal amounts of the four active esters with Phesa (1) as the nucleophile produced four dipeptides 3a-d in a ratio of 18:44:15 :23. Not surprisingly, the coupling of 1 with the less-hindered glycine ester to produce 3b was more efficient than with the more-hindered phenylalanine, leucine, or proline esters. In spite of these differences, ail four dipeptides formed in significant amounts. The use of phenylalanine as the nucleophile gave similar results. For the second control experiment, ail eight dipeptides were prepared individually and their ability to inhibit the CA-catalyzed hydrolysis of p-nitrophenyl acetate (Scheme 2) was measured. As expected, the sulfonamide-containing dipeptide competitively inhibited this hydrolysis, with inhibition constants of 1.1-8.7 J.IM, while the non-sulfonamide dipeptides showed no detectable inhibition. Dipeptide 3d was the best inhibitor,
with an inhibition constant of 1.1 J.IM, and dipeptide 3e the next best, with an inhibition constant of 2.5 J.IM. Compound 1 also inhibits CA (Ki = 13 J.IM), but approximately tenfold less effectively than the tightest binding dipeptide (3d).
In the first pseudo-dynamic experiment (8-hour cycle, Figure 2a), the cycle time was too short for the destruction reaction to rem ove the less-effective inhibitors. During the first four hours of each cycle, the screening chamber contained ail eight dipeptides, thus indicating that ail eight had formed as expected. At the end of each 8 h cycle, prior to the next addition of active ester, the hydrolysis had removed the four non-sulfonamide dipeptides, thus leaving only the four sulfonamide dipeptides. At the end of six cycles of active ester addition, dipeptide 3b was present in the highest amount (58% yield, relative to CA), followed by 3d (33%), 3 e (27 %), and 3 a (8 %). These relative amounts differ from their relative binding constants. The higher yield of 3 b instead reflects its more favorable rate of synthesis. In addition, the sum of ail the sulfonamide dipeptides at 48 h was greater than the amount of target (126 % yield). This high yield shows that unbound dipeptides remained and that the destruction reaction had not had enough time to distinguish between the different sulfonamide inhibitors.
Lengthening the cycle time from 8 h to 12 h yielded the best three inhibitors, in relative amounts in the order of their inhibition constants (Figure 2b). Although sulfonamides 3bd were present in high concentrations early in the experiment, the concentration of these weaker binding dipeptides had diminished substantially at the end of four cycles. The tightest binding dipeptide (3d) was present in the highest amount (15% yield relative to CA), followed by 3e (5%) and 3b (1.5 %). Notably, the ratio at the end of the experiment (10.1:3.5:1) exceeded the ratio of their binding constants (5.1:2.2:1). None of the weaker binding 3a or of the nonsulfonamide dipeptides remained at the end of the experiment.
The selectivity of the dynamic process improved even further upon extending the cycle time to 16 h (Figure 2c). The
;$:
S02NH
2 SO NH 1 SO NH
;$:
2 2 SO NH
;$:
22 1 ;$:22 o '" l '" 1 H Il 0 '" H 0 0 '"
..... N~ OH H Il N Il OH Et02C Il Et02C i ~ ..... N~ OH Eto,C ..... Y'N N-...../"-- OH
0:= 0 Et02C ~ ---,/ H 0 '-) ~
1 0 1 0 ~ 3a: El02CPhePhe.. 3b: Et02CGlyPhesa 3e: Et02CLeuPhe.. 3d: Et02CProPhesa
Ki = 8.7 IJM Ki = 5.61JM Ki = 2.51JM Ki = 1.1 IJM
;$:
1 H ,;? H ;$:1 H ;$:H ..... ~J '" OH H w;$1 ~ W '" OH Et02C, W : 1 Et02C , N N~ OH EtO C ..... ~N N-...../"-- OH
V= H 0 Et02C'" ~ 2 --/ H 0 \.J ~
1 0 1_ 0 ~ 4a: EtO,CPhePhe 4b: Et02CGlyPhe 4e: EtO,CLeuPhe 4d: Et02CProPhe
Ki» 1.0 mM Ki» 1.0 mM Ki» 1.0 mM Ki» 1.0 mM
Scheme 2. Competitive inhibition constants of the library members for the CA·catalyzed hydrolysis of p·nitrophenyl acetate. The non-sulfonamide compounds showed no detectable inhibition at l mM.
2434 © 2004 Wiley·YCH Yerl.g GmbH & Co. KG.A. Weinheim www.angewandte.org Ange",. Chem. Int Ed. 2004. 43. 2432 -2436
a) 1.20
1.00
0.80
0.60 [3]1mM 0.40
0.20
10 20 30 40 SO tlh
b) 1.20
1.00
0.80
0.60
0.40
[31/mM 0.20
0.00 0 10 20 30 40 SO
tlh
c) 0.60
O.SO
0.40
0.30
0.20 (3)/mM
0.10
20 40 60 80 100
tlh----
Figure 2. Pseudodynamic library experiments. Concentrations of sulfo· namide containing dipeptides 3a (el, 3b ("'l, 3c (_l, and 3d (+l over the course of experiments: a) 8 h per cycle, bl 12 h per cycle, and cl 16 h per cycle.
initial synthesis during the first cycle favored dipeptide 3 b, the most rapidly synthesized dipeptide, but this dipeptide disappeared in later cycles where the main competition was between 3 d and 3 c, the tightest binding dipeptides. After four cycles (64 h), only these two remained and the ratio of their concentrations (13:1) was significantly higher than the ratio of their binding constants (2.3:1). The selectivity increased to > 100:1 in favor of the strongest binding dipeptide 3d after three more cycles. The yield was 29 % relative to the amount of CA and corresponded to 4 mg of dipeptide. Thus, adjusting the relative rate of the library synthesis and destruction optimized the selectivity so that only the best-binding dipeptide remained and was present in a good overall yield.
The selectivity in the pseudodynamic library is significantly greater than that in many traditional dynamic libraries. The optimum conditions produced only the single, tightestbinding dipeptide (> 100:1 selectivity), while a traditional approach would yield a mixture because the binding constants for the two tightest-binding dipeptides differed by only 2.3-fold. This higher selectivity greatly simplifies the analysis, as only one compound need be identified and characterized. The optimization of a pseudodynamic library arises through control of the relative rates of synthesis and destruction. We previously showed that a destruction reaction operating on a
An ewahdte g::'Chemie
static library in the presence of a receptor distinguishes between library members with very similar binding constants, selectively removing the weaker-binding species,!6] However, when selectivity arises from destruction alone, significant amounts of the best-binding library member must be destroyed to achieve high ratios of good binder to slightly poorer binder. This situation leaves only a small amount of the best binder for analysis. The high selectivity in pseudodynamic libraries also stems from the competition between binding to the receptor and destruction.
The iterative nature of the experiment also contributes to the high selectivity. Cleavage by Pronase has reduced the amounts of weak-binding dipeptides toward the end of each cycle, which leaves dipeptide 3 d as the major species bound to CA. The subsequent burst of synthesis produces a mixture of ail dipeptides which compete for the smaller amount of free target. Pronase then rapidly c1eaves al1 unbound species, which would consist of a higher proportion of weak binders. Following our static model, continued action of Pronase further increases the ratio in favor of the bound species, which results ultimately in high selectivity for the tightest-binding speices.
Our static model of pseudodynamic combinatorial libraries16] indicates that selectivity stems from the relative binding constants of the inhibitors, not their absolute affinity for the target. Thus, we expect that pseudodynamic combinatorial libraries will also work with even tighter-binding inhibitors, but would require longer cycle times to distinguish between these more tightly binding inhibitors. Indeed, we are currently expanding our studies to larger pseudodynamic libraries to discover such tighter-binding inhibitors.
Received: January 15, 2004 [Z53769] Published Online: March 31, 2004
eywords: combinatorial chemistry . drug design. enzyme inhibitors . kinetics . receptors
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