turner the of - collectionscanada.gc.ca€¦ · and the bromination of pyrimidine derivatives...
TRANSCRIPT
The Study of &Cyciodextrin Interactions Sugars Using "Inhibition Kinetics" And The Brornination of Pyrimidine Derivatives
Isabelle Turner
A Thesis
in
The Department
of
Chemistry and Biochemistry
Presenïed in Parüal Fuifilment of the Requirements for the Degree of Master of Scienœ at
Concordia University Montréal, Québec, Canada
August 1 999
O Isabelle Turner, 1999
National Library BiMiothèque nationale du Canada
Acquisitions and Acquisitims et Bibliog mphic Services services bibliographiques 395 Wellington Street 395. nie Wsll-ngior, OrtawaON K1AûN4 OeEawaON K I A W Canada CaMda
The author has granted a non- L'auteur a accordé une licence non exclusive Licence aliowing the exclusive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distnite or seii reproduire, prêter, distribuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/fiim, de
reproduction sur papier ou sur format électronique.
The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation.
ABSTRACT
The Study of fbCyclodextrin Interactions with Sugars Using Ynhibiüon Kinetics"
And The Bromination of Pyrimidine Derivatives
Isabelle Turner
PART 1: The binding of &cyclodexîrin @CD) with sugars was studied using
"inhibition kinetics". The rates of hydroiysis of trimethyl orthobenzoate and
benzaldehyde dimethyl acetal in acidic solution were monitoreâ using a single
beam stopped-flow spectrophotometer. Both substrates exhiba saturation kinetics
and 1:1 binding with &CD, and the rates of hydrolysis are retarded by the
cyclodextn'n, in agreement with earlier studies. The retarded rates of hydrolysis in
the presence of PCD are modulated by the addition of guests that bind to the CD,
and "in hibition constants" (KI) may be determined. Several aldoses were studied.
Pentoses were found to bind modestly to PCD (K, = 100 - 1 O00 mM) and hexoses
hardly bind at al1 (K, > 500 mM). The results obtained with the two probe reacüons
agree well with each other, and the K, values are compared to the disparate values
in literature.
PART II: The aqueous bromination of 2-aminopyrimidine (2-AP) involves two
major steps. and the first step fons an addition product, an intemediate that was
previously observed by NMR. The second step, which is quite slow. involves
iii
elimination of water from the intermediate to yield the Sbromo derivath The focus
of this investigation was on the initial fast step to try to discover how the
in termediate is formed. The kinetics of the bromination of 2-aminopwmidine and
several related derivathes have been studied using a stopped-flw apparatus. The
mechanisms were investigated by constnicting pH-rate profiles for each mpound.
The pH-rate profiles for 2-amino pyrimidine and 1.2-d ihydro-2-imino-1 -methyl-
pyrimidine (M2A) dernonstrate the complexity of these reactions and the evidenœ
that more than one mechanism is invoived. This statement is especial true for the
latter compound where mixed orders of readions were found. For comparative
purposes the kinetics of bromination of 2-amino4,6dimethyl-pyrimidine (2ADP).
2-aminopyridine (2-APy). 2dimethylaminopyridine (2-DAPy), aniline and N, N-
dimethylaniline were also sstdied.
I would like to thank my supeMsor Professor Oswald S. Tee for his patience
and his support. I am grateful for his teachings of both life and science. Thank you
for being such a good pedagogue and above al1 a patient English teacher.
I am appreciative of Dr. Timothy A Gadosy and Dr. Joanne Tumbull for
readily accepting to be on my cornmittee, even if they knew what they were getting
into.
I am pleased to have met and exchanged with a large number of fellow
students in the Department of Chemistry and Biochernistiy. Several have become
friends and I h o p to keep in touch. Special thanks to Alexei A. Fedortchenko,
Delna Ghadiali, Michael John Boyd, Lee Fadet, Araz Jakalian, Michael Harvey and
Donald Paquette. Good luck to Paul Loncke, Ogaritte Jennifer Yazbeck, and Dons
Badaan in reaching their acadernic goals.
Finally I could not have cornpleted this thesis without the support and love
of rny family and friands.
=Tout ce que l'homme pouvait gagner au jeu de la peste et de la vie, c'&ait la connaissance et la mémoire..
Albert Camus La Peste
vii
Table of Contenîs
................................................... List of Figures xi
................................................ List of Schemes xiv
ListofTables .................................................. xvi
............................................. List of Abbreviations xix
PART I The Stuw of ~Cyclodextn'n lnteracions wrah Sugats Using "hhibifrbn
Nnetrcs"
4-Introduction ................................................... 2
1 . 1 Supramolecular Chemistry and Molecular Recognition ........... 2
........................................... 1.2 Cyclodextrins 4
1.2.1 Stmcture, Chernical and Physical Properties ............ 5
1.2.2 Inclusion Complexes and Kinetics .................... 6
1.2.3 Effect of CDS on reactions ......................... 11
1.3 Probe Reactions, Hydrolysis of TM06 and BDMA ............. 11
1.4 Alcohols as Guests ..................................... 17
1.5Sugarsasguests ....................................... 17
2.Results&Discussion .......................................... 19
2.1 Effects of guests ....................................... 19
........................................ 2.2 Sugars structure 28
3. Conclusion .................................................. 37
................................................. 4.Expenmental 38
4.1Materiats ............................................. 38
viii
4.2 Kinetic Experiments ..................................... 38
4.3 Calculation of Rate Constants ............................. 39
....................... 4.4 Calculation of Dissociation Constants 39
PART N The Bmmination of Pyrimidine Derivatives
5.lntroduction .................................................. 42
5.1 Pyrimidine Structure and Properbies ........................ 42
................ 5.1.1 Bromination of Pyrimidine Derivatives -44
............ 5.1 -1 -1 Brornination of 2-Aminopyrimidine 44
5.f . 1 -2 Bromination of 1.2dihydro-2-imino-1-
methylpyrimidine ................................ 45
5.1 -1 -3 Bromination of 2-Amino4.6-dimethylpfimidine . . 45
5.2 Pyridine Structure and Properties .......................... 46
.................. 5.2.1 Bromination of Pyridine Derivatives 47
.............................. 5.2.1.1 Bromination of 2-Arninopyridine 47
....... 5.2.1.2 Brornination of 2-Dirnethylaminopyridine 48
5.3 Aniline Structure and Properties ........................... 49
5.3. f Bromination of Aniline ............................ 50
.................. 5.3.2 Bromination of N, KDirnethylaniline 51
........................................ 5.4 Simple Kinetics 51
.......................................... 6.Results&Discussion 54
................................... 6.1 Bromination of Aniline 54
6.2 Bromination of Arninopyridine ............................. 57
.......................... 6.3 Bromination of Aminopyridimidine 61
ix
7.Conclusion .................................................. 80
8.mperimental ................................................. 81
8.1 Materials ............................................. 81
8.2 Kinetic Experiments ..................................... 81
............................ 8.3 Calculations of Rate Constants 83
......................................... 8.4p&€stimation 84
8.5 Observation of the Bromination of 2AP Intemediate by 'H NMR . . 84
9. References .................................................. 87
10.AppendixA ................................................. 93
1tAppendixB ................................................. 98
List of Figures
Figure 1.1
Figure 1 -2
Figure 1.3
Figure 2.1
Figure 2.2
Figure 2.3
Figure 2.4
Figure 2.5
Figure 2.6
Figure 2.7
Figure 2.8
Catalysis of the Cleavage of nNitrophenyl Alkanoate by B-CD. 12
.. Retardation of the Hydrolysis of TMOB in 0.1 M HCI by $40. 12
Retardation of the Hydrolysis of TM08 and BDMA in 0.1 M HCI
byf3GD. ........................................... 14
pK, estimated values from this work yg literature pK, values from
~ o y a m a et al.'' ...................................... 25
pK, estimated values from this work literature pKl values from
Hacket et al." ....................................... 25
pK, estimated values from this work for benzaldehyde dimethyl acetal
vs pKl estimated values for Acetophenone dimethyl acetal. .... 26 - pK, estimated values from this work for benzaldehyde dimethyl acetal
vs pK, estimated values from this work for Tnmethyl orthobenzoate. - .................................................. 26
pK, estimated values for Acetophenone dimethyl acetal 1 ~ s pK,
. . estimated values from this work for Trimeth yl orthobenzoate. 27
AG0 values from the literature AG0 averaged values of BDMA and
TMOB from this work. ................................. 27
bb. scaled according to the k, value for the hydrolysis of TM06 in the
presence of 1 mM of B-CD. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
bb scaled according to the value for the hydrolysis of BDMA in the
presence of 5 mM of BGD. ............................. 30
xi
Figure 2.9
Figure 2.1 0
Figure 2.1 1
t
Figure 2.1 2
Figure 6.1
Figure 6.2
Figure 6.3
Figure 6.4
Figure 6.5
Figure 6.6
Figure 6.7
Figure 6.8
scaled according to the k, value for the hydroiysis of TM06 in the
presence of 1 mM of B-CD. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
scaled acwrding to the k,, value for the hydrolysis of BDMA in the
presenceof5 mM of P-CD. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . 31 scaled according to the value for the hydrolysis of TM08 in the
presence of 1 mM of p-CD. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
scaled according to the Id value for the hydrolysis of BDMA in the
presence of 5 mM of PGD. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
pH rate profile for the bromination of N,Mimethylaniline and aniline.
............................. . . . . . W . . . . . . . . . . . . . . . 54
Hammett plot for the bromination of parasubstituted anilines.
Substihients: F, Br, CI. Me, NHAc, NH;. . . . . . . . . . . . . . . . . . . 56
pH rate profile for the bromination of 2-aminopyridine and 2-dimethyf-
aminopyridine.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
Hammett plot for the bromination of rnonosubstituted 2-Arninopyridine
and 2-Dimethylaminopyridine at 20°C. Substituents: Br, CI, NO,. 59
pH rate profile for the bromination of 2-amino4.6-dimethylpynmidine.
.................................................. 61
pH rate profile for the bromination of 2-aminopyrimidine. . . . . . 63
pH rate profile for the bromination of 1,2-dihydro-1 -imino-1 methyl
pyrimidine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
Traces of (a) first-order kinetics, (b) rnixed-order kinetics and
xii
Figure 81.1.1
Figure 81.3.1
Figure B i .3.2
Figure B I .4.1
Figure 81.5.1
Figure 81.6.1
Figure 81.6.2
Figure 81.7.1
(c) zerwrder kinetics ............................ 71
The Dependence of k,* on the aniline concentration at a pH
of 1.14 for experiment IT-196M and a pH of 0.852 for
........... ............... experiment IT-197M.. ; 100
Dependence of kt* on the Paminopyndine concentration at
............. pH4.43 .................... .... 102
Dependence of the amplitude on the brornine concentration at
..... pH4.407 for the bromination of 2-aminopyndine 103
Dependence of k,Oh on the 2-dimethylaminopyridine
concentration al pHd.865 ....................... 1 05
Dependence of klob on the 2-amino4,6-dimethylpyrirnidine
concentration minus the bromine concentration at pH=1.429.
............................................ 106
Dependence of k,& on the 2-aminopyrimidine concentration
at pHd.852 for experiment IT-93M and a pH of 4.34 for
experiment lT-94M ............................. 108
Dependence of the amplitude on the bromine concentration at
pH=0.852 for the bromination of Barninopyrimidine . . . 109
Dependence of klobs on the 1 ,BdihydroQ-imino-l -
methylpyrimidine concentration minus the bromine
concentration at pH4.465 for experiment IT-124M and a pH
of 5.56 for expenment IT-125M ................... 1 1 1
xiii
Ust of Schemes
Scheme 1.1 Schematic Representation of a Molecular Complex . The Host molecule is
................... convergent and the Ouest molecule is divergent 3
Scheme 1.2 The Molecular dimensions of the Three Different Cyclodextrins (a.. p- and
y-CD) .................................................... 5
Scheme 1.3 Structure of an a-CD Molecule and a Schernatic Representation of the
Binding site of a CD Molecule .................................. 7
Scheme 5.1 Structure of Pyrimidine Molecule .............................. 42
Scheme 5.2 Equilibrium between Tautomeric forms of 2-AP i.e . the Amino and the lmino
. . . form and between the Hydroxy and the Ketone form of Pyrimidone 43
Scheme 5.3 Bromination of 2-AP in D,O to observe the intemiediate by NMR
spectroscopy . ............................................ 44
Scheme 5.4 The Bromination of M2A in D, O to observe a similar intermediate by NMR
............................................. spectroscopy 45
Scheme 5.5 Structure of 2.Amin04,6~dimethyIpyrimidine ..................... 46
Scheme 5.6 The structure of Pyridine and the partial and overall charge distribution . 47
Scheme 5.7 The bromination of 2.Aminopyridine ........................... 48
Scheme 5.8 The structura of 2.dimethylaminopyridine . . . . . . . . . . . . . . . . . . . . . . . 48
Scheme 5.9 Resonnance structures of Aniline . . ........................... 49
Scheme 5.10 Resonnace structures of an aryiamrnoniurn ion . . . . . . . . . . . . . . . . . . 50
Scheme 5 . 1 1 The bromination of Aniline ................................... 51
xiv
........................ Scheme 5.1 2 The bromination of N. N4imethyl aniline 51
....... Scheme 6.1 Reacüon of both fom of 2-AP with bromine in acidic solution. 64
........... Scheme 6.2 Formation of the intermediate in the bromination of 2.AP 65
Scheme 6.3 Bromine attack follawed by water attack and addition .............. 65
...... Scheme 6.4 Proposed mechanism for the bromination of 2.aminopyrimidine 67
Scheme 6.5 Suggested pathways for the bromination of 2-AP and M2A ......... 69
Scheme6.6 ........................................................ 72
Scheme6.7 ........................................................ 73
Scheme6.8 ........................................................ 75
Scheme 6.9 Proposed mechanism for the bromination of M2A ................ 79
Scheme B I Lotus 1-2-3 spreadsheet for the brornination of aniline ............ 113
List of Tables
. . ......... Table 1 0 Dissociation Constant (Ks) of PCD-guest Complexes 8
Table 1.1 Constants for the Hydrolysis of Acetals in the presenœ of
Cyclodextn'ns ........................................ 16
............ Table 1 -2 @CD host-guest dissociation constants. K, (mM) 18
Table 2.1 Dissociation constants for the @CD.guest} complexes ....... 19
Table 2.2 Correlation coefficients for the comparison of the pK, values -mated
using dafierant probe reactions .......................... 23
Table 2.3 Correlation coefficients for the comparison of the pK, values estimated
ta the pK, values from the literature ....................... 24
............................... Table 6.1 Bromination Parameters 55
Table 6.2 Hammett Equation Parameten for Aniline ................. 57
............................... Table 6.3 Bromination Parameters 58
. . . . . . . . Table 6.4 Hammett Equation Parameten for 2APy and 2.DAPy 59
Table 6.5 Bromination Parameters ............................... 62
............................... Table 6.6 Bromination Parameters 64
............................ Table 8.1 Parameters for Bromination 82
Table 8.2 H NMR Shifts ....................................... 86
Table 81 . 1 Raw Data for the Bromination ([Br& = 0.0250 mM) of Aniline (1 . 00
........................................ mM) at 25OC 99
Table 81.1 -1 Raw Data for the Dependenœ of k, * on [Aniline] using [Br& =
0.0250 r n ~ at 25OC ................................... 99
xvi
Table 81 .2 Raw Data for the Bromination ([Br& = 0.0250 mM) of N,Ndimethyl-
............................ aniline (0.50 mM) at 25OC 1 O0
Table 81.3 Raw Data for the Bromination of 2-Arninopyridine (1 .O0 mM) at 25OC
................................................. 101
Table 81.3.1 Raw Data for the Dependence of k,Ob on [2-Aminopyndine] using
[Br& = 0.05W mM at 25OC ............................ 102
Table 61 3.2 Raw Data for the dependence of the Amplitude on the [Br& at 25OC,
for the bromination of 2-Aminopyridine ................... 103
Table 81.4 Raw Data for the Bromination ( [ & J O = 0.0250 mM) of 2-dimethyl-
aminopyridine (1 -00 mM) a1 25% ....................... 104
Table 81.4.1 Raw Data for the Dependence of k,* on [2-dimethylaminopyridine]
using [BrJo = 0.0500 mM at 2S°C ....................... 104
Table 81.5 Raw Data for the Bromination of 2-Amino4,6-dimethylpyrimidine
(1.00 mM) at 25OC ................................... 1 05
Table 8 1 5 .1 Raw Data for the Dependence of ktobt on (2-amino-4,6-dimethyl-
pyrimidine] using [BrJ, = 0.0500 mM at 25OC .............. 1 06
Table 81.6 Raw data for the Bromination of 2-Aminopyrirndine (1 -00 mM at 25OC
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 0 7
Table 81.6.1 Raw Data for-the Dependence of k,Ob on [2-aminopyrimidine] using
[Br& = 0.0500 mM at 25OC ............................ 1 08
Table 81.6.2 Raw Data for the Dependence of the Amplitude on the [Br&, for the
Bromination of 2-aminopyrimidine at 25OC ...... .- ......... 109
Table BI .7 Raw Data for the bromination ([Br& = 0.0250 mM) of 1,2-dihydro-2-
xvii
imino-lniethyipyrimidine (0.250 mM) at 2S°C . . . . . . . . . . . . . 1 10
Table B 1.7.1 Raw Data for the Dependenœ of - kob. on [f ,2dihydro-2-irnino-1-
meti~yi~rnidine] using [Br&, = 0.0500 mM at 2S°C . . . . . . . . . 11 1
Table B i .8 Raw Data for the Bromination ([Br$ = 0.0250 mM) of Synthesued
1,2dihydro-2-imino-1-methylpyndmaine (0.250 mM) at 2S°C . 1 12
PART 1
The Stud of
1 In-
1.1 Supramolecukr Chemistry and Molecular Recognition
During the late sixües a new field was developed. an explosion in the
investigations of hghG structured complexes mth the goal of mimicking enzymes.
Supramolecular chemistry is the study of the fundons and the structures of
superniolecules which are formed during the associations of substrates and
recepton.' Many biological processes, such as substrate binding to an enzyme.
and enzymic cataiysis, exploit supramolecular properties.
Following the growing interest in supramolecular chemistry Cam and
coworkem in 1977, defined the ternis to be used in host-guest complexation.
Suprarnolecular complexes were defined as 'cornposed of two or more molecules
or ions held together in unique structural relationships by electrostatic forces other
than those of full covalent bonds."' The forces that are usualiy involved include Van
der Waals, hydrophobic, dipolar and polar interactions. Covalent bonds are
excluded since they lead to a molecule instead of a complex. A molecular wmplex
requires a minimum of one host and one guest component. The host is known as
'an organic molecule or ion whose binding sites converge in the complex."' The
guest is therefore 'any molecule or ion whose binding sites diverge in the
cornplex."' A simple representation of a molecular complex can be seen in Scheme
1.1.
Host Guest Complex
Scheme 1.1 Schematic Represenation of a Molecular Cornplex- The Host molecuîe is convergent and the Guest mofecule is divergent-
For a guest to bind to a potential host severaf requirements must be met:
both the host and the guest must camplement each other i-e., the geometry, size
and shape must allow appropriate interactions to occur; the host should have a
degree of flexibility and of rigidity to allow the access of the guest to the binding
site.' When interaction is made possible. the function of the supiamolecular
complex can be investigated. Funaional properties of a supermolecule include
catalysis, transport and rnolecular recognition.'
The main requirement of molecular recognition is that the receptor and its
substrates be in contact over a large area? In order to satisfy this requirernent
several factors play a key role. The first factor involves diffusion. Encounters
between a host and a guest occur through diffusion. If a complex is fomed upon
contact, the rate of cornplex formation is diffusioncontrolled. On the other hand. if
some adjustments to structure of the host andlor the guest are needed
(preorganisation), the rate of complexation will be ~ lower.~ When the surfaces of
both mofecules corne in contact. multiple weak bonds are fomed and w-ll hold until
random thermal motions lead to the dissociation of the mole~ules.~
3
Therefore the second fadar involves thermal motions. A general correlation
behveen binding Wngth and the rate of dissociation is the stronger the binding the
slower the rate of dissociation- In the case of a fast rate of dissociation, the total
energy of the bonds formed is negligible in contrast with that of thermal motion, the
molecules dissociate very rapidly. If the binding is strong. the bond energy is high
resulting in a slow rate of dissociation.' Preorganisation will play a major role in
detennining the binding powr,' this is because by adjusting their structures the
malecules enhance the fit and the number of noncovalent interactions.
Corn plementa rïty wïil be invohred in structural recognition ,' not only does it increase
the number of noncovalent bonds fomed, it also increases the area of contact,
satisfying the main requirement of molecular recognition.
The third and last factor involves molecular motion. All three kinds of
molecular motions are significant in bnnging the surfaces of interacting molecules
together. These motions are: translational, vibrational and rotationaL6
Many scientists have contributed to the studies of host-guest chemistry and
have elaborated potential applications. some of which are drug design, new
analyücal and diagnostic procedures, enzyme-analogous catalysis in water, etc ... 7
Other researchers focussed on designing and constnicting hosts. adding to the Iist
of existing hosts. Cornmon hosts incfude crown ethers, cryptands and cyclodextrins.
1.2 Cyclodextrins
Cyclodextrins were first diswvered from a culture of Bacillus amylobacter
4
grown on a starch medium by Villiers in 1891 ? Following this discovery, Schardinger
was able to isolate a bacillus (Bacillus macerans) which yields cyclodextrins upon
treatrnent with starch? Through years of extensive work by various researchers
secrets of the cyclodextnns have unfolded revealing their structures and chernical
and physical properties.
The reaction of the enzyme cyclodextrinase on starch is relatively speufic,
fornting only a few byproducts? The main products are three different cyclodex&ins,
a-, p, and y-, varying in the number of glucose units as follow: 6 units, 7 units and
8 units (Scheme 1.2). Several methods can be used to isolate them including: gel
chromatography, column chromatograp hy, HPLC, etc.'#'
Scheme 1.2 The Molecular dimensions of the Three Different Cyclodextrins (a-, f3- and y-CD).
1.2.1 Structure, Chemical and Physical Properties
The doughnut-shape of CDS is one of their interesting features (Scheme
1.3). This parücular shape is allowed because the undistorted D(+)-glucopyranose
units in the C l conformation are attached by a-1.4-linkages?** A wnsequenœ of
the Cl wnformation is that the sewndary hydroxy groups. which are situated on
the C-2 and C-3 positions of the glucose un*. are located on the larger opening of
the CD, and the primary hydroxy groups are located on the smalier side of the CD?
The seaindary hydroxy groups of adjacent glucose un& (G2 and C-3) are
involved in hydrogen bonding, resulting in stabilkation of the shape of the molecuk
and affecüng its solubility in ~ a t e r . ~ Secondary hydroxyl groups are fairly rigid and
do not rotate whereas free rotation of the primaiy hydroxyl groups is allowed and
they can partially block the cavity by rotating.'
The pnmary characteristic of this toms shape is its cavity. The cavity of CDS
are mainly wmposed of C-H groups and glucosidic oxygens which makes ït
relatively apolar? The fact that the electmn pairs of the glycosidic oxygen bridges
are directed towards the inside of the cavity produces a high eledron density and
gives some Lewis-base charader?
1.2.2 Inclusion Complexes and Kinetics
The CD cavity can accommodate a variety of organic guests,* however,
strong interactions are usually associated with hydrophobic molecules. The only
requirement for inclusion of guests appean to be spatial and therefore the dïfFerent
cavity sites allow a-CD to include benzene derivatives, for P-CD to include
naphthalene and for y-CD to include anthracene?
Inclusion complex formation is usually associateci with unfavouraMe entropy
changes and favourable enthalpy changes.' The favourable enthalpy change is
thought to be caused by: the Van der Waals interactions between the CD and its
guest (London dispersion attraction and dipole-dipole intera~tions)~, hydrogen
bonding between the hydroxyl groups of the CD and the guest, high energy water
molecules released upon complex formation, and strain energy being released in O
the macromolecular ring of the CD.'
Secondary Hydroxy \
Hydropho ic Cavity P
Primary Hydroxy
Scheme 1.3 Structure of an a-CD Molecule and a Schematic Representation of the Binding site of a CD Molecule. !
The study of the complexes formed by CDS with their guests can be undertaken by
various methods depending on the type of information sought. When essential
information about factors involved in complexation are needed. dissociation
constants are easily measured. The dissociation constant of a complex is an
equilibnum constant (i$) for the dissociation of two or more molecules that form this
cornplex? Ks relates to the stabilii of the wmplex. " and from equation 1 -0 the
Gibbs fke energy can be obtained.
LW = -RT In K (1 -0)
The stronger a guest binds to a host the lower the Ks value. As previousfy
mentioned, alis is because the binding energy is higher and usually results in a
slower rate of dissociation.' Several factors influence the binding strength and
hence the dissociation constant value. These factors were enumerated and
explained in Section 1.1.
Some exam pies of K, values for binding to PCD are presented in Table 1 -0.
The larger the Ks value is the lesser interaction there is between the host and the
guest Note how the values depend on the guest size and shape.
Table 1.0 Dissociation Constants (KJ of PCD-Guest Complexes
Guest Ks, mM Reference
CH3-CH2-CH2-OH
n-propanol
n-butanol
Cyclo hexanol
C yclohexanone
Acetophenone 8.1 1 I 0.55 14
One of the methods that w e have ernployed to calculate dissociation constant
values invoives the use of 'inhibition kineti~s''.'~-'* Using saturation kinetics several
equations can be defined:
The first equation (1 -1) involves the conversion of the substrate (S) to the produd
(P) without the involvement of a 'catalyst". The rate constant of the reaction is
therefore written as k,, for 'uncataiyzed". Equation (1 -2) describes the conversion of
S to P in the presenœ of CD. The substrate can form a 1 :1 complex with CD (S-CD)
and then be converted to P. The dissociation constant of this complex is expressed
as Ks (equation 1.3). k, represents the rate constant of the readion from the S.CD
wmplex to P (c is used for 'mtalyzed") From equations (1.1, 1.2 and 1.3). equation
(1.4) is derived, where k, is the observed (measurable) rate constant. Experiments
can be camed out where oniy the [CD] is varied and k, is measured. Analysis of
the results using equation (1.4) provides calculated values for the rate constant k,
and the dissociation constant Ks.
Depending on the reaction, CDS cm have one of two effedo. The first case
is catalysis, which implies that with increasing [CD] k, will increase as seen in
Figure 1.1. This is because the 'uncataiyred' rate constant (k,,) in equation (1 -4) is
srnaller than the ucatalyzed" rate constant (k& CDS have been known to catalyre
a large number of react i~ns.~* '~ Catalysis of a reaction by CDS, or a catalyst,
resides in their ability to lower the fke energy of the transition statemS This can be
done via nonavaient or covalent interactions. Nonco~ len t catalysis may involve . one of Wo possible effects, microsoivent effect or conformational effect? Covalent
catalysis involves the formation of covalent inter me dia te^.^^^ The second case is
retardation, where &< k. Therefore k, decreases with increasing [CD] (Figure
1.2)-
1.3 Probe Reactions, Hydrolysis of TM06 and BDMA
Of the reactions that were found to be retarded by CDS, two were of
parD'cular interest, the hydrolysis of trimethyl orthobenzoate and the hydrolysis of
benzaldehyde dimethyl acetal. Both readions were well known and had previously
been ~tudied.""~ In their own nght these reactions were interesting because they
are relatively fast and their products are chromophores which made them suitable
for stopped-flow experiments.
Figure 1.1 Catalysis of the Cleavage of m-Nitrophenyl Alkanoate by a- CD?
Figure 1.2 Retardation of the Hydrolysis of TMOB in 0.1 M HCI by B-CD.
In the case of acid catalyzed hydrolysis of TMOB, two major steps are
involved :
acid PhC(OH)(OMe), - PhC(0)OMe + MeOH (1 -6)
or base
Where the first step (1.5) invohres general-acid cataiysis and is rate-lirniting.qq
Appearanœ of the product, methyl benzoate, can be monitored by meawring the
absorbanœ increase at 228 nm.
The acid cataiyzed hydroiysis of BDMA follows a similar course.
H+ PhCH(OMe), + H,O - PhC(OH)(OMe) + MeOH (1 -7)
acid PhCH(OH)(OMe) - PhCHO + MeOH (1 -8)
or base
Step (1 -7) is the acid catalysed conversion of the acetal to the hemiaœtal, followed
by step (1 -8) where a molecule of methanol is eliminated by acid or base ~atalysis.'~
Benzaldehyde appearance can be monitored by measuring the absorbanœ
increase at 252 nm.
TM06 0 BDMA
Figure 1.3 Retardation of the Hydroiysis of TMOB and BDMA in 0.1 M HCI by 8 CD.
Discovery of the effect that PCD has on these rea~t ions '~ '~ lead to the
conclusion that they could be used as probe reactions. By making use of inhibition
kinetics, and carrying several expenrnents under the same conditions with the
exception of the [CD], which was varied (Figure 1.3). pertinent information (k, and
Ks) could be estimated. Analysis of the results yielded the parameten presented in
Table 1.1
To exploit inhibition kinetics as probes. equations have to be elaborated. By
adding an 'inhibitof or a guest. Le. a molecule that binds to CD. to the reaction
mixture, with increasing concentration, an increase in the observed rate of the
reaction is expected. The guest would compete with the substrate for the CD.
therefore increasing the amount of free substrate available for hydrolysis.
The guest can fom a cornplex with the CD and the dissociation constant can
be expressed as equation (2.0).
An estimation of the [CD] compared to the [CD],, is obtained as follows:
[CD], = [CD] + [CD-Il (2-1 )
[Ilo = [l] + [CD-Il (2-2)
The [CD] is estimated using equation (1 -4) where [CD] may be isolateci ta yield:
AI1 the parameten are known with the exception of K, which can therefore be
calculated.
Table 1.1 Constants for the Hydroiysis of Acetals in the Presenœ of Cyclodextrins-
Substrate k,,,s-' &, mM k, s" uk
a In 0.1 0 M HCI, at 25 O C . Values of &, K, and k, were obtained by non-linear fming of equation (1 -4).
See ref. 12 ' Value indistinguishable from zero. See ref. 48 Acetophenone dimethyl acetal in 0.010 M HCI, at 25 OC. See ref. 46 Benzaldehyde diethyl acetal in 0.1 M Chloroacetate buffer. See ref. 49 See ref. 50
1.4 Alcohols as Guests
The use of inhibition kinetics to calculate the dissociation constants of
different CD cornpiexes was proven to yield satïsfactory r e ~ u l t s . ~ * ~ ~ * * ~ particulaily
when alcohols were used as gue~ts.'*'~ Table 1.2 illustrates the results obtained
with TMOB and BDMA hydrolysis as probe reacbons in determining the dissociation
constant of alcohols to PCD.
Comparing & values with Iiterature values and values obtained with a
different probe madion demonstrated strong agreement.1213 The validity of this
method established, a new goal couiâ be undertaken: the study of the interactions
of &CD with sugars.
1.5 Sugars as guests
In previous yean, several publications presented the interactions of CDS with
s u g a r ~ . ' ~ ' ~ Surpnsingly, the resuits of some groups seem to disagree strongly with
the results of other groups. Due to their size and properties, it was believed that
sugan wouM interact weakly with CDS. Hirsh et al. estimated with a blood glucose
meter a dissociation constant (4) for {$-CD.D-glucose} complex of 2.38 mM.' In
contrast, Hacket et al. determined by Ruorometric cornpetition titrations in water a
K, value of 1667 mM for the same complex.17 From the wnflicting results cornes the
interest to study the interaction of cycloheptaamylose, P-CD, with several sugars
using a difierent method: kinetics measurernents.
Table 1.2 PCD host-guest dissociation constants. K, (mM)
1-Hexanol 4.07kO. 18
1 -Heptano1 1.27I0.01
3-Hexanol 16.2k0.6
tert-Butanol 20.311 .O
Cyclohexanol 1 -51iû.09
' Acetophenone dimethyl acetal, in 0.01 M HCI at 25 OC. Product increase monitored at 244 nrn. See ref- 46
From the effect of guests on the retardation of the hydrolysis of trimethyl orthobenzoate by CDS in 0.10 M aqueous HCI.12
From the effect of guests on the retardation of the hydrolysis of benzaldehyde dimethyl acetal by CDS in 0.1 0 M aqueous HCI.12
From displaœment of a fluorescent probe. at pH 11.6 (0.2 M phosphate buffer)." Mainly ref. 15.
' Other literature values.52
2.4 Effects of guests
As seen in the introduction. W D retards the hydroiysis of TMOB and
BDMA. By adding a guest that binds to N D , and that campetes with the substrate
for the CD, an incmase in the obsenred rate should be observed. An increase in
the k, is expcded because binding of the guest will lower the amount of free CD
available, increase the fkee subotrate, resulting in faster hydroiysis. The
dissociation constant for the @-CD-guest} complex (6) can be estimated by
exploiting the same methodokgy as in 'inhibition kineticsn. In this case, the reaction
is slowed dom instead of cataiysed and the inhibitor (sugar) increases the
observed rate. Equations 1.4 and 2.4 derived in the Introduction and the h, k, and
Ks values from Table 1.1 are needed to estimate the K, values. The estimation is
done by obtaining k, for a reacüon inciuding a guest (sugar) at several known initial
concentrations. The K, values estimated in this work are shown in Table 2.1.
Table 2.1 Dissociation constants for the {&CD.guest) complexes
Guest Lit8 Litb TMOB BDMA ADMA'
Pentoses
D-ara binose 1430 667 1060*80 1500e20 1 07Oî45
L-arabinose 1430 949190 1 05Oi50 1 23e54
D-2deoxyribose 106 63i16 145k3 1 34î1 2
D-l yxose 233. 394S4 456e1 4Mk11
0-ribose 389 159 209I16 252i11 21 018
D-xyiose 1 O00 625 573i20 - 757k13 834î75
L-xylose 480k32 9581124 775î74
DL-xylose 427S6 1020i85 786i30
Hexoses
D-fructose 7 170186 18OOi90 1670*100
Dgalactose 2000 3920M90 2960i300 3500î185
D-gluwse 1670 2630ï130 47201400 5390k730
0-mannitol 3650I21 O 8720i2020 4250Q60
D-mannose 1000 1460*190 2790Q35 2540i180
L-rhamnose 503i76 1640&140 1 1 90î58
D-sorbitol 120011 50 3420k180 41 1 Oi290
L-sorbose 665146 829176 969i27
Others
P-D-iactose 2200182 1 040011 700 5550i1760
sucrose 22001380 31 90k290 6030I2060
D-glucosamine.HCI 81 3148 847I27 1040*110
Reference 18. Reference 17. Hydrolysis of acetophenone dimethyl acetal in 0.01 M HCI (monitored for
acetophenone at 244 nm). Work perfomied by Samer s us sain.^
The "inhibition kinetic" method can oniy be used if the guest binds in the
cavity of the CD. This is because the probe substrate is bound inside the CD cavity.
The interior of the CD is relatively hydrophobic which prevents solvated protons
from reaching the substrate and supresses its hydrolysis. If the gwst interads with
the exterior of the PCD, this will not be monitored. It is possible that the sugars
associate outside of the cavity, a point that Hacket et al. have raised. "and that has
been demonstrated by NMR to occur between protons on the outside the CD host
cavity and those of cyclohexanecar6oxyiate in unpublished work by Gadre et a/.,
cited by Hacket et al. l7 If the sugars interact with the exterior of the &CD cavity,
the actual binding constant of the (sugar.&CD) cornplex may be different than the
estimated K, value. As an example, if a sugar is bound on the exterior of the cavity
is blocking the opening of the CD, then the substrate is "lockedn in the CD cavity
and cannot be hydrolysed. The rate of hydrolysis would be lower than if the sugar
was bound in the CD cavity, leading to a lower estimated concentration of bound
sugar, resulting in a larger dissociation constant for the {sugar.&CD} cornplex.
One cannot condude that the sugan are actually interacting on the exterior of the
CD cavity since there is a lack of prwf, nevertheless. many indications suggest that
they are. In fad this might explain the observed enhancement in solubility of the &
CD in the presence of glucose noticed by Hacket et al."
The resutts obtained by Hirsch et al? may refled a similar ambiguity: their
dissociation constants for {glucose.PCD) are very small which implies strong
binding, and the method they used only monitored the free glucose, it could not
differentiate between glucose interading in the CD cavity or on the exterior of the
CD cavity. If the glucose is binding to the exterior of the CD cavïity. kss fke glucose
will be detected, and a lower K, values will be estimated. This could be s possible
explanation for the estimatecl K, value of 2.38 mM for (glucose.gCD} cornplex. More
over, Hi- et al. found that the dissociation constants for the @lucose.a-CD} and
{gluwse.&CD} complexes were indistinguishable m i n experimental enor, which
is not usually the case for large molecules like glucose. The a-CD cavity is of
smaller diameter (0.6 nm) than &CD (0.8 nm) and the general binding pattern is
that PCD can accommodate branched and cyclic compounds better. as seen in
several publications and s t ~ d i e s . ~ ~ * ~ ~ If the glucose is binding to the exterior of the
CD cavity, 1 is possible that the K, values are relatively the same for a-CD and &
CD, if not, the dissociation constants are unlikely to be so similar.
The titration microcalorimetry used by de Namor et al? is another technique
that cannot distinguish between the sugar interacting with the exterior or the interior
of the CD cavity. And the same thinking as for the Hirsch et al. values may apply
here, since the K, values reported by de Namor et al. are significantly lower than the
K, values by Hacket et al., Aoyama et al. or this work (see Figure 2.6). One has to
keep in mind that at the moment, al1 this thinking is purely speculative and that
further studies and experiments have to be perfomed in order ta confirrn or reject
it .
The TM06 and BOMA values were obtained under the same acidic
conditions and using the same sugar solutions, therefore cornparing them is one
way of detennining if the estimated K, values are reasonable. Figures 2.3 and 2.4
22
presents the correlation of Ge BDMA values with the ADMA (obtained in l o w r
[HCI]) values as well as with the TM06 vakies. Figure 2.5 shows the conelation of
the ADMA values with the TMOB values. The conelation coefficient values are
shown in Table 2.2,
Table 2.2 Correlation coefficients for the cornparison of the pK, values estirnateci osing different probe readions
Correlation Pentoses Hexoses Othe-
- - -
BDMA ADMA 0-977 0.880 0.885
BDMA vs TM06 0.937 0.81 9 0.894
ADMA TMOB 0.949 0.855 0.965
In general. the values seem to agree, which is significant since most of the
K, values obtained were large (explained further). Within experimental error the
results show appreciable constancy, the difference in the K, values are most likely
due ta the distinct probe reactions. A low K, value is estimated more precisely due
to the strong binding: the effed of a guest is greater when it interacts strongly with
its host, the increase in k, is larger therefore the range of the values used in the
calculations is bigger, resulting in less enor. The fact most sugan interact weakly
with P-CD is a major source of enor, as one can notice in Table 2.1, the lower K,
values have usually smaller relative error values. It is also important to note that the
pentoses values agree more closely than the hexoses or the othen sugars values,
23
which is fùrther proof that estimation of a binding constant for weakfy interacüng
host and guest is difficutt. When cornparing the estirnated values with the literature
values the correlation coefficient values obtained are presented in Table 2.3.
Table 2.3 Correlation coefficients for the cornparison of the pK, values estimated to the pK, values from the literature
Correlation TM06 BDMA ADMA
Referenœ 1 8. Referenœ 17.
Figure 2.1 shows the values from this work plotted against the Aoyama1'
values and Figure 2.2 shows the estimated K, values compared to the Hacket17
values. The K, values obtained in our work tend to agree well with the literature
values from these two publications, even though the rnethods used were very
ditferent In contrast, when looking at Figure 2.6, one c m obviously see that the AG0
values, and therefore the K, values, from de Namor et al. and Hirsch et al. do not
correlate with our values. This may be due to the fad mentioned previousiy (sugars
interacting with the exterior of the cavity) or the difference in the techniques used.
pKl Estimated YS pKl Aoyama
+ pK (BDMA) pK(ADMA) PK (-1
pK, Aoyama values
Figure 2.1 pK, estimated values from this worù yg literature pK, values from Aoyama et al. .la
pKi Estimated us pKI Hacket
pK ( W B ) pK (BDMA) pK (ADMA)
-1 J O
pK( Hacket values
Figure 2.2 pK, estimateci values from this work literature pK, values from Hacket et al-.''
Figure 2.3 pK, estimated values from this work for benzaldehyde dimethyi acetal pK, estimated values for Acetophenone dirnethyl aœtal.
pK Pentoses pK Hexoses pK Ohers
Figure 2.4 pK, estimated values from this work for benzaldehyde dimethyl acetal .
pK, estimated values from this work for Trimethyl orthobenzoate.
Pentoses Hemses Others
Figure 2.5 pK, esümated values for Acetophenone dimethyl acetal pK, estimated values from this work for Trimethyl orthobenzoate.
Aoyama x de Narnor 0' Hacket
Hirsch
Figure 2.6 AG0 values from the iiterature AG0 averaged values of BDMA and TMOB from this work.
2.2 Sugars structure
The structure of most of the sugars used in this work are shown in Appendix
A. The majonty of hem are found to be in the pyranose form in so~ution."~ In
general, there is an equilibrium between the a and f a n of the sugars. At this
point, the anomer or anomen present in solution and mutarotation is not the main
concem. This is because mutarotation is promoted by acids and bases and
temperature dependent, the rate of the reaction may double or triple every
increment of 10 OC? ln this case. the reactions were carrïed at room temperature
and a low acid concentration. The important factor is which cyclic fonn the sugars
adopt For some sugan, only one cyclic form is found. as an example, a-D-
glucopyranose exhibits a simple mutarotation suggesting that only the a- and
pyranoses exist in ~olution.~ For other sugars, both cyclic forms are present, and
in the D-ribose case. no chair conformation is preferred ('C, or 'C,)? Which makes
it dinicult to determine what type of structure is interacting with PCD.
In general, the pentoses bind more tightly to W D than the hexoses. Figures
2.7,2.8, 2.9 and 2.10 present the k, y the sugar mncentrations for the pentoses
and hexoses. By looking at those graphics one can see the effect that adding
sugan has. The tighter the sugar binds. the more effect can be seen, as an
example in figures 2.7 and 2.8. 2deoxy-D-ribose (2deoxy-D-erythm-pentose)=
demonstrates the larger increase in k, with increasing concentration. Using TMOB
as a probe reaction, the dissociation complex of Z-deoxy-D-ribose was estirnated
to be 63 i 16 mM- In the case of BDMA, it was estimated to be 145 I 3 mM, Which
explains wh y a larger effect for 2deoxy-Dribose is seen in Figure 2.7 than in Figure
28
2-8,
The lowest dissociatÏon constant was found to be for the cornplex (2deoxy-
D-ribose.wD). The reason for thf most Iikeiy stands in thc stnich~re of this sugar.
Pdeoxy-0-ribose is the most important 2deory-aidose sinœ it is a component of
deoxyrïbonucleic acids (DNA). The fom and conformation of this sugar in solution
was not studied and therefore is not knomi with certainty. In DNA, 2 d e o x y - & n i
exists in the furanose f ~ n n , ~ s o this migM be the case in solution. The sugar could
also be found in an acyciic form. Both these forms could explain why this sugar
binds the most tightly, sinœ no omet sugars adopt them ni solution (with the
exception of the alcohols).
0 Larabinose 0-deoxyribose L-xybse DL-)sr bse
[Pentoses],, mM
Figure 2.7 scaled y~ [Pentoses], for the hydrolysis of TtdOB in the presence of 1 mM of PCD.
Figure 2.8 scaled [Pentoses], for the hydrolysis of BDMA in the presenœ of 5 mM of &CD.
t I I I I 1 1
O 100 200 300 400 500 600
[Hexoses],, mM
Figure 2.9 scaled [Hexoses], for the hydrolysis of TM08 in the presence of 1 mM of &CD.
30
Figure 2.10 k, scaled [Hexosesl, for the hydroiysis of BDMA in the presence of 5 mM of N D .
The xyloses al1 show relatively the same effect with both probe readons.
The configuration of these sugars does not seem to affect their binding to W D . In
the case of the arabinoses one can see the same pattern: the configuration does
not seem to affect the binding. By contrast, the conformation seems to be slightly
more important with respect to the binding. D-arabinose adopts the 'C,
conformation in solution and the D-xylose adopts the 'C, in solution. Even though
both those sugan have three hydroxyl groups in the equatorial position and one in
the axial position. their dissociation constants differ only by approximately a factor
of 2. The anomer also seems to be a factor in the case of D-arabinose and 0-
xylose. The different anomers indicate the "stenc arrangement, with respect to the
conformational disposition."' This explaim why both sugars have the same number
of axial and equatorial groups while they have different conformations.
a-D-Lyxose has a %, prefened conformation and two axial hydroxyl groups
31
and two equatorial hydroxy groups. a-û-xylose adopts the same conformation but
diiffer in the number of axial hydroxyi groups shce those hiuo sugars are bpirners.
The effect of adding those hno sugars are similar when using TMOB. but differ
slightly when using BDMA. This may be caused by the difkrent probe reacüons.
a-0-Ribose does not have a preferred conformation in solutionu. and it can
be found in the pyranose form (76-ûû%) and the furanose fom (2420%). Therefore
several structures of ribose wïll be in solution, this rnay explain why it binds the
tightest after 2deory-0-ribose. Dribose is the tnie epimer (2-epimer) of D-
arabinoseY, but those epimen do not have similar binding constants (they differ by
a factor of approximately 5).
The hexoses show les of an effect on the kinetics and this is reflecîed in the
dissociation constants estimated. The hexoses are larger molecules and might not
be as well accommodated by the PCD as the pentoses. D-mannitol is one of the
sixcarbon carbohydrates that did not bind tightly to the cyclodextrin. This compound
is found in an acyclic fom in solution and was thought of having easy access to
P-CD cavity. The other alcohol studied, D-sorbitol (D-glucÏtol), did bind more tightly
to PCD. This sugar is also found in an acydic fomi in solution but with a bentehain
arrangement,- due to an unfavouable 1,3 interaction between two oxygen atoms.
The bent-chain arrangement rnay facilitate the accommodation of D-sorbitol by &
CD. resulting in a lower dissociation constant.
D-glucose and D-galactose are 4-epimers and behave in the same rnanner
for both probe reactions. D-glucose and O-mannose are true epimers and differ in
their effed with TMOB and BDMA. The position of the hydroxyl group on C-2 seems
32
an important one in the binding of the sugars to FCD sinœ all of the true epimers
studied were found to behave difFerently and have large variation in their binding
constants.
The sugan that have the lowest dissociation constant are L-rhamnose and
L-sorbose. L-rhamnose is a deoxy sugar (6-deoxy-L-mannose) and L-sorbose is a
ketose (D-xylo-henlose) wïth a carbonyl group on C-2.2-Deoxy-0-ribose was the
pentose with the lowest dissociation constant and here again we have a deoxy
sugar for the hexoses. More over, both 2deoxy-D-ribose and L-rhamnose contain
carbonyl groups (not on C-2). It seems that the sugars that posses a carbonyi group
are better accomrnodated by &CD.
a-D-Fructose is .an hexose that can be found both in the pyranose form
(68.4-76.0%) and the furanose form (31 -624%). a-D-Ribose is a pentose that is
also found in both forms, and it is interesting to see that they both bind the tightest
after the deoxy sugars (L-rhamnose and 2deoxy-D-ribose). Again. this may be
explained by the different structures found in solution. It is important to note that
fructose (D-arabino-hexulose) is a ketose with a carbonyl group at carbon atom no.
2.
Figures 2.1 1 and 2.1 2 present k, the sugars concentrations for various
monosaccharides, both pentoses and hexoses. When sugars adopt the pyranose
form, some of the pentoses and hexoses have the sarne structure. that differs only
by the R group on carbon 5. It is interesting to compare them and see if they behave
in the same manner.
0-ribose - Pm- D-ybse L-aiabinose L-rhamnose
Figure 2.1 1 k, scaled [sugars], for the hydrolysis of TMOB in the presence of 1 mM of PCD.
Figure 2.12 k, scaled y~ [sugars], for the hydrolysis of BDMA in the presence of 5 mM of PCD.
The firsthno sugars to be compared are Dglucose and 0-xyiose. They adopt
the same confornation. but do not have the same anomer le. that the hydroxyl
group on C-4 is in the a position for O-xylose and in the position for Dglucose. By
looking at Figures 2.1 1 and 2.12 one can see that Dglucose does not bind as tighüy
to W D as 0-xybse. Two hdors could be involved: The R group on G 5 an& the
position of the hydroxyl group on C-1. a-D-Iyxose and PD-mannose are similar to
a-0-xylose and @Dglucose, they both have the same conformation and difFer by
their anomer. Hem again, the a-aidopentose binds more tightly to &CD than the
aldohexose. Now. FD-galactose and a-L-anbinose do not have the same
conformation or anomer. These two aldoses also have different dissociation
constants.
The resuits in Table 2.1 are clear, the pentoses have lower dissociation
constants than the hexoses, therefore the R group on C-5 is a major factor in the
binding of the carbohydrates to W D , which was also observed by Aoyama et al.''
It also becomes evident that the a anomers bind more tightly to PCD than the P
anomers. This leads to the conclusion that both the R group on C-5 and the position
of the hydroxyl group on the Cl are important factors involved in the interaction of
PCD to monosaccharides.
D-glucosamine hydrochloride has the same structure as 0-glucose, with the
exception of having an NH, group on G2. The glucosamine has a lower dissociation
constant than D-glucose. This agrees well with the previous statement that "the
position of the hydroxyl group on C-2 seems an important one in the binding of the
sugars to &CD since al1 of the tnie epimers studied were found to behave
35
differently and have large variation in their binding constants:
Two oligosaccharides were studied, &0-bctose and sucrose. lt is interestng
to note that both these sugars have, within experimental eror, approximately the
same dissociation constants as D-glucose. For sucrose, this might indicate that not
al1 the sugar is accommodated in the &CD cavity and that either the glucose or
fructose part of the moiecule binds to the cyclodextrin. The same couki appîy to
D-lactose, where either the Dgalactose or Oglucose part of the molecule could
interact with the W D - Sinœ the values for those two sugars a n similar, it is most
likely the glucose part of the molecule that binds to the cyclodextrin.
Since the K, values from the (B-CD.oligosaccharides) complexes are so hrge
(over a 1000 mM) they indicate that very little interactions are ocairring betiiineen the
P-CD and the sugars. A value over a 1000 mM is almost meaningless and hardly
reproducible. It is possible that the values for PD-lactose and D-glucose are similar
because of a large deviation rather than an indication of what part of the molecule
is involved in binding .
The rate of hydrdysis of trimethg orthobenroate and benzaldehyde dimethyl acetal
were confirmed to be slowed dom by P-CD; By exploiting the saturation type
kinetics involved and using 'inhibition kinetics', the dissociation constants (K,) for
several (B-CD.sugar} complexes were esh'mated. These K, values were found to
conelate well with the values obtained using the acidic hydrolysis of acetophenone
dirnethyl acetal as a probe reactim." It was al- demonstrateâ that the results
obtained in this work agreed tolerabiy with the results of Hacket et al." and of
Aoyama et al.,'' but aiere are strong disagreements between ouf results and those
of de Namor et al?' and of Hirsch et al.'' These conflicting results do not seem to
indicate flaws in the technique used. It could possibly mean that the sugars are
binding with the exterior of the cyclodextrin, as well as in the cavity. The viability of
the acidic hydrolysis of TM06 and BDMA as probe reactions in estimating
dissociation constants for the binding of guests to CDS had been established
before'l'' and this work provides further evidence to confinn this statement. This
work did not provide astonishing results as it was expected that B-CD would interact
only slightly with sugars. Pentoses were found to bind modestly to PGD (K, = 100 - 1 000 mM) and hexoses hardly bind at al1 (K, w 500 mM).
4.11 Materials
P-Cyclodextrin was purchased from Wacker C hernie (Munich. Gemany).
Trimethyl orthobenzoate (TMOB), benzalâehyde dimethyi acetal (BDMA) and al1 the
sugan were obtained from Aldrich Chernical Co. Standard HCI solutions were
purchased from A & C Chernicals (Montrkal).
4.2 Kinetic Experiments
The kinetic experirnents were camed out by using a single beam stopped-
flow spectrophotometer by Applied Photophysics modal SXI 7MV. In the stopped-
flow apparatus the mixing is 1:1 and so the concentrations of the fwo solutions
before and after mixing dHer by a factor of 2. The observation cell was kept at 25.0
r 0.1 O C . The sugar solutions were prepared in 0.10 M HCI. A concentration after
mixing of 1 mM of FCD was used for the hydrolysis of TMOB. The concentration
of TMOB and BDMA after mixing was 5 mM (50 pl of 0.1 M solution in 50 ml flask
before mixing) The concentration afler mixing of &CD for the hydrolysis of BDMA
was 5 mM.
The hydrolysis of TMOB in 0.10 M HCI was monitored for changes in
absorbance at 228 nm for the appearance of the product, methyl benzoate. The
hydrolysis of BDMA under the same conditions was monitored for changes in
absorbance at 252 nm for the appearance of its product, benzaldehyde.
4.3 Calculation of Rate Constants
Using the instrument manufacturer's software, the observed rate constants
(kod were calwlated by fitting the absorbanœ increase with a single exponential
function. The absorbance traces were composeci of 400 points. For the purpose of
this work, the first 15 points were not taken into account when calculating b, to
allow for the small induction p e r i d 2 Each recorded k, value is derived from the
average of 5 - 10 absoibance traces. Absorbanœ traces extended over 7 half-ives
for the hydroiysis of TMOB and 8 haClives for the hydroiysis of BDMA.
4.4 Calculation of Dissociation Constants
The calculation of K, values was carried out following the procedure
explained in the Introduction. In a spread sheet (see Scheme 4.1) the [CD] was
estimated using equation 2.5:
and this was used to estirnate K,. through equation 2.4. A dissociation constant was
calculated for each concentration of guest or "inhibitor" added. The K, values were
then averaged and a graph of k, vs b , was plotted to verify the linear
relationship. A sample calculation is shown in Scheme 4.1, overleaf.
The k, values are entered in the second column of the spread sheet. and
these values are scaled according to the Ig,. The calculated rate constant (163
was detenined using the known parameters presented in Table 1.1. In Scheme
4.1, b, was calculated for the hydrolysis of TMOB in 0.1 0 M HCI in the presence
of 1 rnM of &CD in solution and was found to be 2.188 s-'. Different factors may
influence the k, obtainad for a [CD], = 1 mM and [guestb = O mM: the precïse [CD]
and the [HCI]. By scalirig the obsenred rate constants to a calculated rate constant
cornparison by gnphic representation is facilitateci, sinœ the initial point for al1
inhibition analysis are the same.
Scheme 4.1 Calculation of the dissociation constant of D-glucosamine to N D using a Lotus 1-2-3 spread sheet.
PART Il
The Bromination of Pwimidine Derivatives
5JQQd-
5.1 Pyrimidine Sbutaii. and Properüm
Pyrimidine derivatives are important heterocydes since they are essential
components of nudei aads as well as some coenzymes and d r ~ g s . ~ Pyrimidine,
*rtsetf, is composed of a six-membered anniatic ring containing two ni&ogen atoms,
as seen in Scheme 5.1. The pyrimidine mokcuie is symmetrical with two planes of
symmetry, with one plane and axis of symmetry going through carbons 2 and 5.
which makes positions on carbons 4 and 6 equivaient, as well as positions 1 and
3 equivalent Yïelding an overall C, syrnmetry.
Scheme 5.1 Structure of Pyrimidine Molecule
The nitrogens are powemil electron-withdrawing atoms resulting in electron-
deficient positions 2.4. and 6? Their separate effects are reinforcd because the
nitrogen. atoms are meta to each other? The reaeüvity of substituent g roups found
in positions 2,4 and 6 will reflect the electron deficiency. Therefore, if a halogen
atorn is found in one of these positions, the halogen would be easily attacked by a
nucleophiie.
The Iposition is much less eledrondeficient due to the general inductive
effectn Consequenüy. eiectrophiles would attack at this point Nitration, nitrosation,
halogenation may easily be carried at this position.
When eiectron-releasing substituents are introduced. the pyrimidine reacts
more as an aromatic ring because the rr-electrons are restored. This facilitates
electrophilic reacüons at the 5-position. Some of these substituents are hydroxy and
amino groups. As seen in Scheme 5.2, pyrimidines with these substituents in the
2,4 and 6 positions may be in equilibrium with their tautomeric forms. As an
example, 2-aminopyrimidine will be tautomeric mai its ûimino fom. Of course. the
equilibrium will lie towards the more energetically favoured structure, and in the
case of 2-aminopyrimidine. Brown and coworken. found that in solution the major
tautomeric fom is the amino fomP
Arnino
H ydroxy
Scheme 5.2 Equilibrium between Tautomeric forms of 2-AP 'e. the Arnino and the lmino fom and between the Hydroxy and the Ketone form of . Pyrimidone.
5.1.1 Brominrtion of Pyrimidine Derivatives
5.1 A.1 Bromination of 2-Aminopyrimidine
The brornination of 2-aminopyrimidine (Z-AP) has been largely studied through the
yearqna and it invohres two major steps. First a fast $tep where addition of water
and attack by bromine occur. The second step is slow elimination of water to yield
the 5-bromo produd. Further investigation into this reaction provided key
information about the intemediate. This intermediate was isolated and observed by
NMR by ~anerjee," who also had obsenred similar intermediates for the
bromination of pyn'midone and uracil derivatives. Barbien' et al. had observed
analogous intermediates in the bromination of pyrimidine sulphonamides in
methanol solution?
2-aminopyrimidine intenned iate 5-bromo produd
Scheme 5.3 Bromination of 2-AP in D,0 to observe the intermediate by NMR spectroswpy.
Following the observation of the intermediate in the bromination of 2-AP, an
interesting question anses, how is this intemediate formed? Does it first involve the
attack of bromine then foifowed by addition of M e r or the inverse? This question
was addressed by an undergraduate student in a CHEM 450 research project and
thesis in 1980 but with no conaete conclu~ion.~
5.1 .1.2 Bromination of 1,2-dihydm-2-imino4-rnethylpyrimidine
The addition of bromine to 1,2dihydro-2-imino-1 -methylpyrimidine was
studied in 1980, here again. a similar intermediate was isolated and observed by
NMR? In this case, can the same mechanism as for 2-AP by applied? If it c m . do
al1 the pyrimidine derivatives read with brornine in the same manner? More
derivatives need to be studied.
020 DCI
Scheme 5.4 The Bromination of M2A in D,O to observe a sirniiar intermediate by NMR spectroswpy.
5.1 -1 -3 Bromination of 2-Amino4,6dimethylpyrimidine
The literature contains very little information about the bromination of 2-
amino4,6dimethy(Wnmidine (2ADP). The articles usually involve product studies.
Studying the bromination of 2-ADP could give insghts about the mechanism
involved, furthemore, it could be compareci with other pyrimidine derivatives.
Scherne 5.5 Structure of 2-Amin04.6dimethylpyrïmidine.
5.2 Pyridine Structure and Properties
The broad interest in pyridine and its derivatives has produced a lot of
literature? Pyridine is similar to pyrimidine, but it only contains one nitrogen atom.
The molecufe is relatively flat with bond angles close to 120'. The nitrogen atom is
sp2 hybridized and will contribute one s electron to the aromatic sextet. The other
TI electrOns are provided by the carbon atorns.
Since the nitrogen atom is electron withdrawing it will have some negative
charge. Partial positive charges are found at the 2,4 and 6 positions. creating an
overall charge distribution of negative for the nitrogen atom and posiüve for the ring
(Scheme 5.6). The properties of pyridine are parallel to b e n ~ e n e , ~ but with
differences due to the presence of the nitrogen atom. The electronegative nitrogen
causes the pyridine to be more polar than benzene. Eledrophilic substitution
reacüons can ocair but with more difficulty than with benzene, and primarily at
positions 3 and 5.
Scheme 5.6 The structure of Pyridine and the partial and overall charge distribution.
Electrondonating groups are ortho and para direding groups. Therefore. if
a substituent is at the 2 position it further activates position 3 and 5 for electrophilic
attack.
5.2.1 Bromination of Pyridine Derivatives
5.2.9.1 Bromination of 2-Aminopyridine
Frorn the literature, it is known that the bromination of 2-aminopyridine (2-
APy) yields a mixîure of products. Wth excess brornine, the major products are 33-
dibromo and 5-bromo-2-aminopyridine. To a lesser extent, the 3-bromo derivative
is obtained. Occasionally. one gels Sbromo and 3.5.6-tribromo derivatives. 27-29
CH3,
Scheme 5.7 The bromination of 2-Aminopyridine.
No study on the mechanisms involved have been undertaken. The interest in this
type of study is for comparative reasons.
5.2.1 -2 Bromination of 2-Dimethykminopyridine
In the case of the bromination of 24imethylaminopyridine (2-DAPy), the
literature states that with a limiting amount of bromine a mixîure of the mono (5-
brorno-2-dimethyl aminopyridine and dibrorno (3,5-dibromo-2-
dimethylaminopyridine) derivatives is obtained?
Scheme 5.8 The structure of 2-dimethylaminopyndina.
5.3 Aniline Structure and Propertks
Aniline is an important compound in organic chemistry, not only for its
chemical and physical properties. but for its historical background. In 1856. Henry
Perkin oxidized impure aniline with potassium dichromate, yielding a purple
pigment. which gave hirn the idea of using it as a dye. Following this discovery,
Perkin resigned his post with Hoffinan and created his own chemical Company. To
produce his dye on a brge-scak he had to elaborne a methd for prepanng aniline,
the early beginnings of organic synthesis and of the synthetic dye industry?
Aniline is a benzene molecule with an arnino substituent It is less basic than
simple alkylamines, because the electron lone-pair of the nitrogen atom is
delocalized by orbital overlap with the B electron system of the aromatic ring and
therefore less available for bonding?
Scheme 5.9 Resonanœ structures of Aniline.
The protonation of the amino group of aniline is less favoured, because the
resonance stabilization present in aniline is lost. due to the fact Bat only two
principal resonance structures are possible for the arylammoniurn ion:
Scheme 5.10 Resonanœ structures of an arylammonium ion.
Subslituents which decrease the reactiv'i of aromatics towards eiectrophilic
attack also decrease the basicity of anilines. As an example, the pK,, of aniline is
equal to 4.58 and that of the pbromoaniline is equal to 3.86? The basicity is
affected by deadivating groups (eledron-withdrawing groups) because they make
the arornatic ring electron-poor. If the amino group is protonated. a deactivating
substituent will demase the stability of the posiovely charged ion, yielding a smaller
AGO for protonation and a lower p&.
5.3.1 Bromination of Aniline
It is known that the bromination of aniline takes place rapidly and yields the
2,4,6-tribrorninated product The amino group being so strongly acüvating, and
ortho- and para- directing, it is hard to stop at the monosubstituted producta
Scheme 5.1 1 The bromination of Aniline.
Similarly, the bromination of N,N,-dimethylaniline is rapid and with exœss
bromine the ûibromo aniline is obtained.
Scheme 5.12 The brornination of N,N-dimethyl aniline.
For the purposes of wmparison with that of aminopyrimidines and aminopyridines.
the study of the bromination of aniline and its derivative was considered important.
5.4 Simple Kinetics
To further understand the reaction of bromine with the aminopyrimidines,
aminopyridines and anilines previously mentioned, kinetic equations can be derived.
51
These equations date to the rate of the reaction and express the dependance of
the rate on different factors such as the concentration. The kineüc equations can
help resohre the mechanism puzzle.
Bromination is usualiy camed out under acidic conditions and the substrate
can be found in the protonated or unprotonated form. Assuming that the bromine
only attacks the unprotonated form. equation 2.7 would represent the reaction
equation where S is the substrate. P is the product, K, is the equilibrium constant
between the protonated and unprotonated fonn of the substrate (equation 3.0) and
k, is the second order rate constant for the brominaüon of the substrate. From the
type of traces obtained, it can be established that the rate of the reaction will be
equal to the disappearance of bromine. as shown in equation (2.8a). Sinœ the
disappearance of bromine is what is measured, k2ObS is used for the obsenrable
second order rate constant The rate of the reaction should also be equal to that
required by the scherne (eq. 2.7) of the reaction as seen in equation (2.8b).
rate = -- = k20bl [SIO [BrJ
rate = k, [SI [BrJ
Knowing that both equations (2.8a) and (2.8b) are equal, one may isolate k,& and
obtain equation (2.9). Where [SI and [SI, differ due to the protonation of some
52
substrate. The substrate concentration may be substihited using equation (3.0) to
yield equation (3.1).
A low pH, Le. at a pH lower than the pK, value, equation 3.1 may be simplifieci to
equation (3.2). The logarithmic version of this equatîon is shown in (3.3).
log k2- = log k, Y + pH (3-3)
A plot of log k2ObS ys pH should yield a Iinear graphic where the intercept is equal to
the log k,. This situation is applicable to the following substrates. where the pH of
the experiments was always much lower than the pK, values of the protonated fonn
of each substrate: 24Py, 2-DAPy, 2-ADP, aniline and N,Ndimethylaniline.
6.1 Bromination of Aniline
As seen in the Introduction, equation 3.3 can be useâ to express pH
dependence of the the second order rate constant k2. The requirement of this
equation is that a dope of approximately 1 is obtained when log kZ* is plotted
against pH. The pH rate profiles obtained for aniline and N,Mimethylaniline (F~ure
6.1) meet the requirements of equation 3.3 and the calculated resub are tabulatecl
in Table 6.1.
log k,pb = log k2K, + pH (3-3)
N, Wdimethy bniline
Aniline
Figure 6.1 pH rate profile for the Bromination of N, Ndimethylaniline and aniline. The points with open symbols (o and 0) were excluded from the calculations. This is mostly due to the high ionic strength at low pH.
Table 6.1 Bromination Panmeters
N, Ndimeth ylanilinea Aniline8
log k? 8.54
r 0-999
a For an ionic strength of 0.1 M. Reference 38
The value bapg is the value before any corrections for the tribromide ion has been
made in Table 6.1. The reason for estimating this value is that a value for N,N-
dimethylaniline was available from the Merature. Bell and Ramsden detennined an
apparent secondorder rate constant of 3.16 x 10' M1 se' for an ionic strength of
0 .O5 M ," whereas. the value for this work is 3.46 x 1 OB iW1 S-' for an ionic strength
of 0.10 M. These values are the same within experimental error, given the different
ionic strengths. The simple Iinear relationship between the log of k2- and pH leads
to the conclusion that direct bromine attack on the unprotonated substrates is
involved .
Aniline was expeded to be less reactive towards bromine than N,N-
dimethylaniline. This is because aniline is less basic than N.Ndimethyianiline as
stated in the Introduction. T& effect of two methyl groups on the nitrogen atom is
moderate, and k, was expeded to be slghtly higher for N,Mimethyianiline. In order
to compare the value obtained for aniline with the Iiterature. a Hammett plot. Figure
6.2, was wnstructed using the values for substituted anilines taken from Katritzky
et al?'
Figure 6.2 Hammett plot for the bromination of psubstituted aniline^.^ Subsütuents: F, Br, CI. Me, NHAc, NH,'.
From the Hammett equationYb (3.4), the rate constant for the unsubsMuted
compound can be estimated-
log k = log k, + po (3-4)
In this equation p is the reaction constant and o depends only on the substituent.
From the plot of log k, a,', the intercept is equal to log k, for the unsubstiuted
aniline.
The results from this plot are shown in Table 6.2.
Table 6.2 Hammett Equation Parameten for aniline8
Aniline
r 0.989
S ~ O P ~ (P) 4-40 i 0.33
log k2 8.97 i 0.1 3
k2 9.33 x I O a M" s"
a From the literature datan in Figure 6.1.
There seems to be a discrepancy between the value of 9 x I O 8 M-' S'. estimated
from the Hammett plot, and the value of 5 x I O 8 M-' s-' measured in this work. This
may have arisen because the literature results3' were obtained in fairly strong acid
and had to be extrapolated to aqueous solution.
6.2 Bromination of Arninopyrïdine
In the case of the pyridines, similar pH rate profiles were obtained, as seen
in Figure 6.3. To compare the values of k, obtained in this work for the
aminopyridines (Table 6.3). to eariier results. Two Hammett plots were constructed
from the values for six derivatives studied by Katntzky et aLn, these plots gave the
parameters presented in Table 6.4, according to which the values of k, for 2APy
and 2-DAPy should be 2 x 107 M-' s-' and 2 x 10' M" s-'. whereas our measured
values are 9.4 x 106 M-' s-' and 1.5 x 10' M-' s-' respecüvely.
Figure 6.3 pH rate profile for the bromination of 2-aminopyndine and 2dimethyC aminopyridine. The points with open syrnbols (n and O ) were excluded from the c@culations-
Table 6.3 Bromination Parameters
. -- -~
pK,
Slope
log k2 K,
k2
log k2
r
a For an ionic strength of 0.1 M. Reference 38 This work
Figure 6.4 Hammett plot for the bromination of monosubstituted 2-Arninopyridine and 2-Dimethylaminopyridine at 20°C. Substihients: Br, CI, NO,.
Table 6.4 Hammett Equation Parameters for 2-APy and 2-DAPÿ
S ~ O P ~ (P) -6.78 * 0-43 -5.80 2 0.88
log k2 7.35 i 0.22 8.40 i 0.45
k2 2.24 x 1 O7 M-l S-' 2-51 x 1 0' M-' S-'
' From the data3' in Figure 6.4.
The major source of the discrepancy for 2-APy is probably due to the limited
number of points in the Hammett plot. Only three data points were available, and
so any slight deviation in the slope may result in a large variation in the y intercept
(log ka value. It is interesting to note that the ratios of the second order rate
constants (2-DAPy12-APy) are quite sirnilar. The ratio for our work is 15.8 whereas
the ratio for the values estimated fmm the literahire is 1 1.2.
The object of stud ying aniline, 2-aminopyridine (2-APy), and their N, N-
dirnethyl derivatives, was to observe the effect of an ka-nitrogen (=N-) in a
benzene ring. From our value of k, for aniline and 2-APy, and the p+ of 4.4 obtained
by Katritrky et al?. we estimate a value of
a,' = log (9.35 x IO6) - log (5.11 x 108)14.4 = 0.4
Assuming the effects of two aza-nitrogens in a ring are additive, then for bromine
attack on 2-aminopyrimidine:
log k 2 = l ~ ( 5 . 1 1 ~103-4-4(2~0.4)=5.19
And so an estimate of k2 is 1.5 x I O 5 M-l s-'.
Altematively, we can use a value of a,* - 0.55 for aza-nitrogen, derived
earlier by Kanitzky's group.= Using this value and the same p+ gives an estimated
k, of 7.4 x 1 O3 M-' s-'. So, we expected 2-aminopynmidine (2-AP) to react with
bromine with a rate constant in the vicinity of 7400 to 150000 M-l s-'. As shown in
the next section, we actually found a value of 2.5 x I O 4 M-' se', which is within the
range mentioned above.
In a similar way, we can estimate a value of k, to be expected for bromine
attack on 2-amino4,6-dïmethylpytimïdine (2-ADP). Proceeding as above, and using
O,' = O,' = -0.31",
log k2 = 8.71 - 4.4 (2 x 0.55 + 2(-0.31)) = 6.60
60
and k, 4 x 1 O6 M1 s-l. which is very close to the value achially recorded in the next
section.
6.3 Bromination of 2-A~ninopyridirnidine (2dP)
. Before we discuss the resuîts obtained for the bromination of 2-AP, let us
brïefly look at the bromination of 2-amino-4.6dimethylpyrhidine (2-ADP). No
Iiterature values are available for this particular campound. Sinœ the studies of
aniline derivatives and aminopyndine derivatives yielded satisfactory resuits, the
assumption is that the results obtained for 2-ADP are also satisfactory. The kinetic
parameters are presented in Table 6.5.
Figure 6.5 pH rate profile for the bromination of 2-amino4.6dimethylpyrimidine.
Table 6.5 Brominatian Parameters
-- -
P& 4.8Sb
Slope 1.17 I 0-01
109 kz K, 1.92 i 0-07
k2 5.87 x 1 O6 M-l
log b 6-77
r 0.999
a For an ionic strength of O. 1 M. Reference 38
The value of k, = 5.9 x 106 M' s-' in Table 6.5 is very dose to that estimateci
above (4 x I O 6 M-' s-'). Accordingly, it is concluded that 2-ADP reads simply by
direct attack of bromine on its free base fom-
The pH rate profile of 2-aminopyrimidine is not straightfomard. as seen in
Figure 6.6. In fact, the 's" type curve indicates that more than one mechanism is
involved ,
Figure 6.6 pH rate profile for the bromination of 2-aminopyrimidine.
At low pH (O - 2) one mechanism is involved and at higher pH (2.5 - 6)
another is taking part. Thinking in ternis of kinetics, one can derive an equation,
which would correspond to the curve in Figure 6.6. Using this equation. the pK, of
2-AP could be estimated.
In order to derive a kinetic equation, one must consider which species are
reacting. The reactions were camed out under acidic conditions, therefore both the
protonated and unprotonated foms of the substrate are present in solution. If the
bromine can react with both foms, with k, as the rate constant for the bromine
reacting with the protonated form and k,' for the bromine reacting with the
unprotonated fom (Scheme 6.1). then, equation 3.5 can be derived.
Scheme 6.1 Reacüon of both forms of 2-AP with btomine in acidic solution.
The results obtained by fitüng this equation are shown in Tabie 6.6. Note, in
particular. that a pK, of 3.66 was estimated, whereas the literature pK, for 2AP is
3.54 at 20 OC? The agreement between these two vaiues is obvious and it is an
indication that the equation derived is appropriate. at least in part. Confirmation is
also brought about by the fad that the data points are lying close to the curve.
Table 6.6 Bromination Parameters
P& 3.66 i 0.05
k2 1200 i 60 M-' s-'
k2' 25200 * 1200 M-' s-'
r 0-996
a For an ionic strength of 0.1 M at 25 O C .
Any viable mechanism for the bromination of 2-AP must be in accordanœ
with the observed kinetics, the pH dependenœ of the rate. and the formation of the
observed intemiediate. As mentioned in the Introduction, the reaction of 2-AP with
bromine in water ieads inioally to the fornation of an addition intermediate, 6, rather
than a substitution product (Scheme 6.2). This formation has been confimed by
new NMR experiments describecl in the Experimental section. Conceivably, the
intermediate 6 could arise from bromine attack followed by water attack and
addition, as in Scheme 6.3.
Scheme 6.2 Formation of the intermediate in the bromination of 2-AP.
Scheme 6.3 Bromine attack followed by water attack and addition.
65
Altemativeiy. there could be the addition of water to 2-AP. followed by bromine
attack and water attack (Scheme 6.4). A simibr mechanism has been shami for the
bromination of 2- and ~pyrimidone." Part of the object of this work was to try to
differentiate between the two pathways in Scheme 6.3 and 6.4. - The mechanism in Scheme 6.4 could follow the pathway described below.
Addition of water to the fke base form 1, probabiy via the cation 2, can give 4 or 5.
Of the three hydrated species (3,4, and 5), the cation 3 would be dominant at a low
pH. However, structures 4 and 5 are strong guanidine-type bases and should be
very reactive towards bromine. Structure 5 is in gray to illustrate that the paths
leading from structures 4 and 5 are kinetically equivalent In contrast, the path 6om
the protonated fom 3 would have a different dependence on the pH. Attack of
bromine on 3.4 or 5, fdlowed by addition of a second water molecule. would yield
the observed intemediate 6.
The k', value of 2.5 x I O 4 M" s-' obtained for the bromination of 2-AP at pH
3 - 6 agrees with the range of values predicted for bromine attack on 2-AP in the
previous section. Therefore, the reaction of 2-AP seems to occur, at least in part,
in the same fashion as the bromination of aniline and 2-APy, by direct attack of
brornine, as in Scheme 6.3.
H@ CK~~ - - Ka"
Q Strong base
. . -9 9
Ka' - c~~~ 7 H H
H2
al Dominant fom
at low pH
Scheme 6.4 Proposed mechanism for the bromination of 2-aminopyrimidine.
At lover pH values (pH c 2). another mechanism is taking place and it is
harder to explain what is happening. The mechanism in Scheme 6.4 is complex
because there are sevenl equilibria involved. Of course, there is an equilibrium
between the N o fomis of the substrate. 1 and 2. but there may be smali amounts
of the hydrated cation 3 and its flee base foms 4 and 5. Since the pH rate profile
for the bromination is Rat at pH 2 (Figure 6.6) the reactive species must be the
protonated fom 2 or some other prolonateci fom in equiIibrium wwi fn. most
probably the hydrated fom 3 (Scheme 6.4). Support for the idea that the reactike
fom is the d i o n 3, in equilibrium wiai the cation 2, cornes frorn observations made
for the &on 7 (Scherne 6.5) which reacts with an observed second order rate
constant of 1 O00 M" s-', very close to the value of 1200 M-l s-' for 2. So. t is
proposed that 2 reacts via the hydrated fomi 3, and 7 reacts via its hydrated fom
10 (Scheme 6.5).
Because it is a guanidine, 1.2-dihydro-2-irnino-1 -meth yl pyrimidine (M2A) is
very basic. with a p h of 10.75~, and it exists as its cation 7 at al1 pHs studied. We
chose to study this system because the cation 7 could serve as a mode1 of the
protonated f o m of 2-AP, 2.
Scheme 6.5 Suggested pathways for the bromination of 2-AP and M2A.
The pH rate profile for the bromination of M2A is very interesüng and quite
cornplex, as seen in Figure 6.7. Assuming that this pyrimidine reacts with bromine
in a similar way as 2-AP, at least two mechanisms must be involved. one at low pH
( O to 3.5) and one or more at higher pH (4 to 7). At low pH, the pH rate profile for
7 is almost identical to the pH rate profile for the bromination of 2-AP, therefore we
propose a similar mechanism as for 2AP, as shown in Scheme 6.5. Since M2A is
very basicw, the cation 7 of the substrate is the predominant fom in solution, but
structures 8 and 1 0 could be available due to pre-equilibrium hydration, which
leaves the rate determining step to be the attack by bromine. Bromine attack on the
cation 10 could explain the pH-rate profile (Figure 6.7) at low pH.
- 1st order 0 mi>aed order
fast raie slow rate
Figure 6.7 pH rate profile for the bromination of 1.2dihydro-1 imino-1 -methyl pyrimidine.
Around pH 2. we observe a transioon from first-order kinetics (figure 6.8 (a))
through mixed order (b) to zero-order (c). Again. sirnilar behaviour was observed
previously for the bromination of 2- and 4-pyrimidone~.~ The change in order is
consistent with a change of rate-limiting step from bromine attack to hydrate
formation. To understand where these dïfterent orders arise h m , kinetic equations
(a) pH = 0.465 .
(b) pH = 2.12
Figure 6.8 Traces of (a) first-order kinetics, (b) mixedorder kinetics and (c) zero- order kinetics
We have considered the following posibili.
Scheme 6.6
The total amount of 8 will be found in two forms, namely, 8 and 10. but since
8 is a very strong base, we can make the following assumption.
and
We followed the reaction by monitoring the disappearance of bromine and
according to Scherne 6.6 the rate is given by:
rate = = k, [IO] [Bq dt
As long as 8 and 10 are in rapid equilibrium,
and substituting for [8] from (3.7)
Using the steady state assumption on ( 8 * 10) equation 4.1 = 0, and so we can
isoiate [l O].
SubstiMing for [fol in equation 3.8 yields
The above rate equation cannot explain a change of rate-lirnibing step near
pH 2 with pH because it has no term in [Hq (or [OH']) in the denominator.
Accordingly, the mechanism in Scheme 6.6 is not viable.
Consider now, another possibility.
Scheme 6.7
In this mechanism the cation 90 is formed in a pre-equilibriurn from 7, and the ftee
base fom 8 is fomied from f O. However, 8 is a strong, guanidine-type base which
would form slowly from 10 and it is an enamine so it should react rapidly with
73
bromine. We will consider 8 as a steady-state intemediate:
and so,
But K,, = [10]Im, and [IO] = K,, m
Thus, according to Scheme 6.7 the rate of bromination is
For the rate equation in (4.7) there are two exheme situations.
(i) k, [Hq >> k, [BrJ, in which case
This situation corresponds to rate-limiting attack of bromine on 8, and it requires
first order kinetics for high m and fixed [H+l. The corresponding pseudo-first
order rate constant (= k, K,, k2 m / k, [Hl) should increase with increasing pH.
(ii) k, [BrJ >> k, [Hl. In this case. the acidity is low enough that reprotonation of
8 to 10 cannot compete with fast bromine attack and
-d[BrJ/dt=k,K,J] (4.9)
In other words, the formation of 8 is the rate-limiting step and the kinetics are
pseudo-zero order.
ûverall, Scheme 6.7 and its rate equation (4.7) require the pseuddirst order
rate constant to Vary wiüi lI[H) at high [Hl whereas at low [H? there should be
pseudo-zerwrder behaviour and no pH dependence. Unfominateiy, such
behaviour was not observeci.
The third and final possbility tha will be considered is the mechanisrn shown
in Scheme 6.8.
Scheme 6.8
For this scheme,
rate = k, [ lO] [BrJ
Using the steady state assumption on 10:
d [IO] 1 dt = k, [7m - k, [IO] [Hq - k, [BrJ [IO] = O (5- 1
However, [ I H ~ = m [WK. (5-3)
and. substituting for [IO] and r H 7
This rate equation, which is appropriate for the mechanisrn in Scheme 6.8, can
explain the pH-rate profile for the bromination of 7 in the pH range O - 3.
Equation (5.6) has no dependence on the [Hl, and it corresponds to rate-limiting
attack of bromine on the hydrated cation 10, formed in a preequilibrium from the
cation 7. This mechanism is exactly that proposed earlier in Scheme 6.5.
At higher pH, if k, [BrJ >> k-, [H7 then
Equation (5.7) has a [H l term in the numerator and therefore the rate would
decrease with increasing pH. Since this equation is zero-order with respect to
bromine, the rate-limiting step involves the formation of 10 by water attack on 7H'.
Thus, the mechanism presented in Scheme 6.8 and the rate equation (5.4)
can explain the observed change in rate-limiting step. At firot glane, the dication
7H' is not easy to conceive. but it is not far-fetched since double protonation has
been observed ni the case of Barnina-2,4dimethyipyrÏmidine (6ADP) (pK, = 6.90;
pK2 = -0.14).*
By obseMng the pH rate profile for the bromination of 2-AP (Figure 6.6). one
can see that two points (O) were not taken into account to estimate the p K value.
These points were not taken into account because mixeâ order kinetics were
observed at a pH of about 2.3 - 2.5. If we consider applying a similar mechanism as
in Scheme 6.8 to 2-AP, we rnust consider the possibility of a dication. A dication
could be viable ifwe think in ternis of pK, and pK2. By camparison with 6-ADP. 2-
AP should have a pK, = 3.50 and pK, - -2 or -3. which is not far from the pHs at
which we performed the experiments. It seems likely that there is also a change of
ratelimiting step in the bromination of the cation of 2-AP, near pH 2.5, but the zero
order process never bewmes truly established because attack of bromine on the
free base form of 2AP takes over at pH > 2.5 (Figure 6.6).
Returning to the subject of the bromination of 7, at higher pH (4 - 7). the pH
rate profile is cornplex. in addition to the various orders obtained two unexpected
results were noticeâ. First, in the pH region of 4.5 to 6 for log k2- vs pH a dope of
- 1.7 was calculated, which suggests that two protons are rernoved in the reacüon.
in the case of the bromination of 2-AP a slope of - 1 had been calculated. The
second surprise was that two difFerent rates could be observed at the highest pHs.
A very rapid rate and a slow rate were seen. For the moment, we cannot explain
these various observations, and further experiments, with the same compound and
with similar derivathes. will have to be camed out to resolve this enigma.
The only problem with performing other experiments is the reproducibilïfy
factor. Whik working with the various substrates (aniline, N,N-dimethylaniline, 2-
APy, PDAPy, 2-ADP and 2-AP), the results obtained were easily reproduced. 1
whereas in the case of M2A reproducibility was very hard to attain. In a science
where precision is a key factor, substrates like M2A can rnake Our work tediow and
interesting at the same tirne.
Scheme 6.9 Proposed mechanism for the bromination of M2A.
79
Direct bromination seems to be involved for the bromination of aniline, N,N-
dimethylaniline, 2-DAPy. 2-APy and 2-ADP. These substrates exhibit a simpk linear
relationship beîween kg be and pH. The pH rate profile of the bromination of 2-AP
is slig htfy more cornplex as it is a sigrnoidal w rve. A mechanism for the bromination
of 2AP was pmposed that agrees with the data coliected. A similar mechanism was
proposed forthe bromination of M2A Further experiments will have to be perfomed
to detemine whether the mechanism is proper. The pH rate profile of the
bromination of M2A was unusual. ieading to mixed observations. New substrates
should be studied to further investigate the mechanism involved. As an example,
2-am in&methylpp*midine is available in the la boratory . It would also be
interesting to perfom substituent effect studies to evaluate the effect of the
presence or absence of a singk nitrogen group or both nitrogen groups in the
aromatic ring.
8.1 Materials
2-Aminopyrimidine, 2-amino4,6dimethylpyrirnidine. 2-aminopyrÏdine, 2-
dimethyl aminopyridine. aniline, N.Ndimethylaniline and the bromine were
purchased from Aldrich Chemical Co. 2-Aminopyrimidine and 2-amino4,6-
dimethylpyrimidine were recrystallïzed previous to their use. Standard HCI solutions
were obtained from A & C Chernicals (Montréal).
1 :2-Oihyd ro-2-imino-1 -methylpyrimidine (M2A) hyd riod ide was synthesized
by methyiation of 2-aminopyrimidine following Brown et al.?A total yield of 64.7 %
was obtained. The conesponding perchlorate salt was synthesized following Tee
et al. procedure? The total yield obtained was 79.3%. The 'H NMR shifts are
shown in Table 8.2. The 'H NMR chernical shifts are almost identical for the
reference compound (M2A) as for the newly synthesized M2A.
8.2 Kinetic Expriment.
The kinetic experiments were carried out by using a single beam stopped-
flow spectrophotometer by Applied Photophysics model SX17MV. Bromination was
followed by monitoring absorbance decrease at 267 nm (tnbromide ion band) for the
disappearanœ of bromine. In the stopped-flow apparatus the mixing is 1:l and so
the concentrations before and after mixing differ by a factor of 2. The observation
cell was kept at 25.0 k 0.1 OC. Bromine solutions were prepared in 0.1 M KBr to
maintain the ionic strength.
Table 8.1 Parameters for Brornination
Compound
-
[Br& u=~M [substrate] used before mixing, M
2-AP 5 x l o 4
1 x 104
2-ADP 5 x lo4
MZA 5 x 104
i x 104
2-APy 1 x l o 4
2-DAPY 5~ 105
aniline 5 x lo4
N, Ndimethylaniline 5 x lo4
For the pH range of 0.1 5 to 2.12, HCI solutions were used. For the pH range
of 2.20 to 3.40 chloroacetic buffers were used. For pH between 4.10 to 5-30,
acetate buffers were used. And finally for pH between 6.40 to 7.70 phosphate
buffen were used. Succinic buffers were used for a pH range of 4-40 to 5-90.'
a Succinic buffer reacts slowly with bromine, and to prevent any problems the bMer was only added to the substmte.
82
With the instrument manufacturer's software, the obsewed first order rate
constants (w were calailated by fitting the absorbance trace with a single
exponential. The absorbanœ traces were composed of 400 points. The trace
analyzed was an average of 5 - 10 individual traces. In some cases the trace
exhibited different or mixed orden, when this occuned the absorbanœ trace was
fitted with two exponentiah or a linear portion followed by a single exponential.
From the observed rate constants, the second order rate constants were
calculated using a spreadsheet (see Appendbc B). A value of kZw is calculateci by
dividing k,* by ([substratel - [Br&) for reasons given earlier.m Then is obtained
by dividing k,* by the fraction of Br, present in solution. The free bromine in
solution is influenced by two equilibria: i) formation of tribromide ion (eq. 8.1); ii)
hyd rolysis to hypobrornous acid (eq. 8.2).
K,
Br, + H20 * H' + B f + HOBr K2
For these equilibria, the constantsaa are:
In fact, under the reaction conditions used, the co~~ection for HOBr is only signifiant
The fraction of Br, is calculated using these dissociation constants, the [Br] and
[Hq and equation (8.3).
In each case where the firotorder disappearance of bromine was observeci.
rate = = kIe [BrJ dt
and it was verified that the reactions were also first order in substrate:
rate = kIe [B rJ = ktobr [SI [B rJ (8-7)
In other words, k,& values Vary with [substrate]. The relevant data are given in the
Appendix B.
8.4 p K Estimation
The pK, of 2-DAPy was estimated by t i i n g a 0.05 M solution of 2-DAPy
with a 0.10 M HCI solution at 22 OC. The experiment was camed out by monitoring
the pH change using an Accumet mode1 25 piilion metre by Fisher Scientific. The
calculations were carried using a Lotus 1-2-3 spreadsheet, based on the method
described by Albert and Serjeant?
8.5 Observation of the Bromination of 2-AP intemediate by 'H NMR
Approximately 0.005 moles of 2-AP (- 0.47 g) was dissoived in 5 ml solution
of 2 M DCI in D,O. The DCI solution was prepared by adding - 1 g of 37 wt% DCI
(F.W. 37.47) in D20 and diluting to the 5 ml mark with D20. The brornine solution
was prepared by adding 0.005 moles (- 0.79 g) of bmmine to 5 ml of D,O.
Approximately 0.5 ml of the 2-AP solution was mixed in a NMR tube with 0.5 ml of
the bromine solution and a 'H NMR spectnim was nn. In addition to the two 2-AP
peaks found at 8.65 and 7.15 ppm. two peaks characteristic of the intemediate
appeared upfieid at 5.33 and 4.49 ppm (TaMe 8.2). By adding more bromine and
therefore saturaüng the mixture. the 'H NMR spedrum showed that the intemiediate
peaks increased and the starting material peaks disappeared.
D. J. Cram, Angew. Chem. Int Ed. Engl., 27,1009.19ûû.
J.-M. Lehn, Scknce, 227,849,1985.
J. Szejüi, Cyclodextnns and theri Inclusion Complexes, Akademiai KÏadO,
Budapest, 1982.
M. L. Bender and M. Komiyama, Cyclodexfn'n Chemism Springer-Veriag,
New York, 1978.
~mceed in~s of the First Intemational Symposium on Cyclodexains, J.
Szejtli (Ed.), O. Reidel Publishing Company, Boston, 1982.
B. Alberts, D. Bray, J. Lewis, M. Raff, K. Roberts. J. D. Watson, M o l d a r
Biology of the Cell, ed., Garland Publishing, New York, 1994, pp. 89-97
H.-J. Schneider, Angew. Chem. /nt. Ed. Engl., 30, 1417, 1991.
A. Lehninger, D. Nelson, M. Cox, Principles of Biihemisfry. P' ed, Woral
Publisher, New York, 1993. p. 64
0. S. Tee, Adv. Phys. Org. Chern.. 29,1,1994.
O. S. Tee, R. A. Donga, J. Chem. Soc., Pedùn Trans. 2,2763, 1996.
E. Cordes, H. Bull, Chem. Rev., 74,581, 1974.
O. S. Tee, A. A. Fedortchenko, P. Lim Sm, J. Chem. Soc., Perkin Thans. 2.
123,1998.
1. E. Turner, The Effect of Cyclodexffris on the Hydrdysis of TMethyl
Orthobenzoate and its use to Probe Guest Binding. CCHEM 450 Research
project & Thesis, Conwrdia University, 1997.
14. a) O. S Tee, T. k Gadosy and J. B. Giorgi, Can. J. Chem., 74. 736. 1996.
b) O. S Tee, A A Fedortchenko. P. G. Loncke, T. A. Gadosy, J. Chem Soc.,
Perkin Trans- 2, 1243,1996.
15. Y. Matsui, T. NWhioka and T. Fujita, Top. CUR Chem., 128,61, 1985.
16. W. Hirsch, T. Muller, R. Pizer, P.J. Ricatto, Can. J. Chem., 73, 12 , 1995.
17. F. Hacket, J.4. Coteron, H .4 Schneider, V.P. Kazachenko, Can. J. Chem.,
75, 52,1997.
18. Y. Aoyama, Y. Nagai, J.4 Otsuki. K. Kobayashi, H. Toi, Angew. Chem. Int.
Ed. Engl., 31, 745, 1992.
19. A.F. Danif de Namor, P.M. Blackett, MC. Cabakiro, J.M. Al Rad, J. Chem.
Soc. Farady Tmns., 90. 845, 1994.
20. J . McMu rry, Organic Chemistry, 7hid Edition, 6 rooks/Cole, Pacific Grove,
1992.
2 1 . D. J. Brown, The Pyrimidines, Vol. 1 6 of The Chemistry of Hetemcyclic
Compounds. A. Weissberger Ed., Interscience, New York, 1962.
22. D. J. Brown, E. Hoetger, S. F. Mason, J. Chem. Soc., 4035, 1955.
23. A. R. Katritzky, M. Kingsland and 0. S. Tee, J. Chem. Soc. (B), 1484, 1968.
24. S. Banerjee, The Mechanism of Bromination of Mono- and Dioxo-
Pyrimidines, M.Sc. Thesis, Sir George Williams University, 1974.
25. F. Platis, Bromination of Z-Aminopyrimidine and Related Compounds, CHEM
450, Concordia University, 1980.
26. W. Barbien, L. Bernardi, G. Palamidessi, and M. T. Venturi, Tefrahdmn
Lett., 2931,1968.
27. R. A. Bames, F. Brody, and P. R. Ruby, Pynane and Ifs Derivafives, Patt
One, Vol. 14 of The Chemistry of Hete~~~ycl ic Compounds, A* Weissberger
Ed., Interscience, New York, 1960.
28. H. E. Mertel, R. He Minoni, E. N. Shaw, L E. Tenenbaurn, and H. L. Yale,
Pyrïdine and Ifs Denvathes, Patt Two, Vol. 14 of The Chemisûy of
Hefemcyclic Compounds, A- Weissberger Ed . , I nterscience, New York,
1961.
29. J. P. Wibaut, G. M. Kraay, Rec. Tmv. Chim, 1085,1924.
30. A. E. Tschitschibabin, 1- L. Knunianz, Ber., 61,427,1928.
31. R. S. Shallenger and G. G. Birch, Sugar Chemistry, Avi Publishing Company,
Wesport, 1975.
32. R. J. Ferrier and P. M. Collins, Penguin Libraty of Physical Sciences,
Monosaccharide Chemistry, Peng uin Education, Middlesex, 1 972.
33. H. S. El Khadem, CarBohydrate Chemism The Monosacchandes anf Their
Oligomers, Academic Press, San Diego, 1 988.
34. R. D. Guth rie and J. Honeyman, introduction to Cahohydrafe Chemistry.
Fourth Edifion, Cfarendon Press, Oxford, 1974.
35. R. S. Shallenberger, Advanced Sugar Chemistry, Pnnciple of Sugar
Stereochemisfry, Avi Publishing Company, Westport, 1982.
36. R. P. Bell and E. N. Ramsden, J. Chem. Soc., 161,1958.
P. J. Brignell, P. E. Jones and A. R. Katritay, J. Chem. Soc. (B), 117,1970.
Handbook of Bhchemisby and Mokular Biology, Third ed., PhysicaI and
Chemkal Data, Vdume I, G. D-Fasman (Ed.), CRC Press, Cleveland, 1976.
D.J. Brown, E. Hoerger and S.F. Mason, J. Chem. Soc.. 4035,1955.
O.S. Tee and S. Banerjee, J. Org. Chem., 44,18,3256,1979.
D.W. Griffith$ and M.L. bender, A&. Catalyss. 23,209 1973.
J.H. Fend fer and E. J. Fendkr, Catalysis in Uicellar and ~ ~ a ~ m m 0 1 8 ~ ~ 1 a r
Systems. Academic Press, New yorù, 1975.
M. Komiyama and M.L. Bender, The Chemism of Enzyme Action. M.I.Page,
Ed., Elsevier, Amsterdam, 19û4.
O.S. Tee, Carbohydr. Res., 192.181 1989.
A. Albert and €.P. Serjeant, The Detemination of Ionkation Constants, A
Laboratory Manual. b Edioon, Chapman and Hall, New York, 1984.
S. Hussain, Inhibaon and Cataljsis Effects of Cyclodextnns, CO-OP Work
Tem Report, Concordia University, 1997.
X. Du, The Cleavage of Aryl Alkanoates Esters by Cyclodexfrins. MM. Sc.
Thesis, Concordia University. 1989.
A.A. Fedortchenko. Stabilizaüon of Transition States of Organic Reactions
by Cyclodextnns, Micelles, and Dendrimers, MM. Sc. Thesis, Concord ia
University. 1997.
A. A. Fedortchenko, Ph. D., work h progress.
Patrick Lirn Soo, The Retardafion of Acetal Hydmiysis by Cyclodextiins and
its use in Probng Cyclodextnn-guest binding, CHEM 450 Research project
8 Thesis, Concordia University, 1997.
51. K A Connors, Bindjng Constants, m e Measurnent of Molecular Complex
Stability, Wiley-lnte-enœ Publication, New York, 1987.
52. (a) 1. Tabushi, K. Shimokawa, N. Shimizu, H. Shirakata and K. Fujii, J. Am.
Chem. Soc., 98, 7855, 1976; (b) 1. Tabushi. N. Shimizu, T. Sugimoto. M.
Shiozuka and K, Yamamura, J. Am. Chem Soc., 99, 71 00, 1977; O Y.
Matsui, K. Ogawa. S. Mikami, M. Yoshimoto and K. Mochida. Bull. Chem.
Soc. Jpn., 60,1219,1987; (cf) Y. Aoyama, Y. Nagai, J. Otsuki, K. Kobayashi
and H. Toi, Angew. Chem., Int. Ed. Engl.. 31,745,1992; (e) Y. Aoyama, J.
Otsuki, Y. Nagai, K. Kobayashi and H. Toi, Tetrahedmn Lefi., 33, 3775,
1992-
53. (a) O. S. Tee, The Hydrogen-Deutenum Exchange of Some Simple
Pyrimidines, Ph-D. Thesis, University of East Anglia, 1967; (b) A-R. Katntd<y,
M. Kingsland and 0. S. Tee, J. Chem. Soc. (B), 1484,1968.
54. (a) T. H. Lowry and K. S. Richardson, Mechanism and Theory h Organic
Chemistry. Td Edition, Harper & Row, New York, 1981, p. 1 31, Table 2.2; (b)
Ibid, p. 132.
55. (a) O.S. Tee and S. Banerjee, Can. J. Chem., 52.45 1, 1974; (b) O.S. Tee
and S. Banerjee, J. Org. Chem., 44, 18, 3256, 1979; (c) O S Tee and M.
Paventi, J. Og. Chem., 45,2072,1980; (d) O.S. Tee and M. Paventi, J. Org.
Chem., 45,2072,1981.
56. D.D. Pemn and I.H. Pitman, J. Chem. Soc., 7071,i965.
57. K.L Wemhawski and D. Shugar, Photochem. And PhotobM, 2,377,1963.
58. O.S. Tee and C.G. Berks. J. Oq- Chem., 45,830,19ûû.
59. R.P. Bell and EN. Ramsden, J- Chem. Soc., 1294,1958.
60. J.M. Pink, Can. JI chem., 48, 1 169, 1970.
Pentoses:
&DAm binose Pyranose forrn 97 %. Preferred conformation 'C,
a u - L y x o ~ Pyranose forh 99.5 1, Preferred conformation %,
a-0-ri bose Pyranose fom 76-80 %, No preferred conformation
a-D-Xylose Pyranose fom 99.5 %. Preferred conformation 'Cl
Hexoses:
a-ûlfructose Pyranose fomi 68.4 - 76.0 %, Preferred conformation 'C,
B-D-Oalactore Pyranose fom 93 - 100%. Prefened conformation 4G,
~-DGlucose Pyranose fom 100%. Preferred conformation 'C,
B-D-Mannose Pyranose fom ? 00%. Preferred confornation 4C,
Others:
Alcohols:
CYOH I
H-C-OH I
HO-C-H I
H-C-OH I
H-C-OH I
CH20H
CH20H I
HO-C-H I
HO-C-H I
H-C-OH I
H-C-OH I
Sugars with uncertain configuration andlor conformation andlor anomec
L-Rhamnose monohydrate
D-Glucosamine hydrochloride
97
Table 61 -1. Raw Data for the Bromination ([Br& = 0.0250 mM) of Aniline . (1 -00 mM) at 25°C.
Table B1.1.1 Raw Data for the Dependence of k,-on [Aniline] using [Br& = 0.0250 mM at 25OC.
Exp. # [Aniline] - [Br$, mM kto., s-'
a The experiments were performed at a pH of 1.14. The experiments were performed at a pH of 0.852.
Figure B I -1.1 The Dependenœ of kq* on the aniline concentration at a pH of 1 -14 for experiment IT-196M and a pH of 0.852 for experiment IT-197M.
Table 61.2. Raw Data for the Bromination ([Br& =0.0250 mM) of N,K dimethylaniline (0.500 mM) at 25OC.
Table 81.3. Raw Data for the Bromination of 2-Aminopyridine (1 -00 mM) at 25OC. -
lT-1 O3M
IT-96M
IT-113M
IT-111 M
IT-1 i OM
TT-99M
IT-l02M
Il-101 M
IT-1 OOM
IT-1 OSM
IT-1 O4M
IT-l12M
IT-106M
IT-107M
4.60 x I O 4
1-30 x 1 O=
3.00 x 1 O4
3.80 x I O a
8.50 x I O 4
1.52 x IO-'
3.95 x 1 Oe2
9.29 x 10'~
2.86 x 1 O-'
1.04
2.68
9.06
22.5
71 -4
a The [Br& used was 0.0500 mM for al1 the experiments with the exception of IT- 11 0M through IT-113M where0.0250 mM was used instead.
Table B1.3.1 Raw Data for the ûependenœ of k,OD. on [2-aminopyndine] using [Br$ = 0.0500 mM a 25%.
a The expenments were performed at a pH of 4.43.
Figure 81.3.1 Dependenœ of kl* on the 2aminopyridine concentration at pH=4.43.
Table B1.3.2 Raw Data for the Dependence of the Amplitude on the [Br&, at 25OC. for the bromination of Zaminopyridine.
a The experiments were performed at a pH of 4.41.
Figure B I -3.2 Dependence of the amplitude on the brornine concentration at pHa.407 for the bromination of 2-aminopyridine.
Table 61 A. Raw Data for the Bromination ([Br&, = 0.0250 mM) of 24imethyC aminopyridine (1 -00 mM) at 25OC.
Table BI .4.1 Raw Data for the Dependence of k,-on [2- dimethylaminopyridineJ using [Br& = 0.0500 rnM at 2S°C.
a The expenments were performed at a pH of 0.865.
Figure 61 -4.1 Dependence of k1* on the 2dimethylaminopyridine concentration a pH= 0.865.
Table BI .S. Raw Data for the Bromination of 2-Amin~.6dimethylpynynmidine (1 -00 mM) at 25OC.
- --
a The [Br& used was 0.0250 mM for al1 the experiments with the exception of IT- 129M where 0,0126 mM was used instead.
Table B1.5.1 Raw Data for the Dependence of kqm on [2-arnino-4.6- dimethylpyrimidine] using [Br& = 0.0500 mM at 25%.
' The experiments were perforrned at a pH of 1.44.
Figure B I .5.1 Dependenœ of kte on the 2-amin04~6dimethylpyrimidine concentration minus the bromine concentration at pH4.429.
Table B1.6. Raw Data for the Bromination of 2-Aminopyrimidine (1 -00 mM) at 25°C-
a These experiments were not taken into amunt when calculating the pK, value.
107
Table 61.6.1 Raw Data for the ûependence of kfa on [2-aminopyrimidine] using [Br& = 0.0500 mM at 25OC.
a The experiments were peiformed at a pH of 0.852. The experiments were performed at a pH of 4.34.
O 0 0.0 0.5 1 -0 1.5 2.0 2.5
[Barninopyrimidine] - [Br&, mM
Figure B I .6.1 Dependence of kIh on the 2-aminopyrimidine concentration at pH=0.852 for expenment IT-93M and a pH of 4.34 for experiment IT-94M.
Table Bi -6.2 Raw Data for the Dependence of the Amplitude on the [Br$ for the Bromination of 2-aminopyrimidine at 2S°C.
-- -
Amplitude
a The experiments were performed at a pH of 1.44.
Figure 81.6.2 Dependence of the amplitude on the bromine concentration at pH=0.852 for the bromination of 2-aminopyrimidine.
Table 61.7. Raw Osta far the Bromination aBr& =0.0250 mM) of 1.24ihydro-2- imino-lmethyipyrimidine (0.250 rnM) at 25OC.
Table Bi.7.1 Raw Data for the Dependence of k,*on [1,2-dihydro-2-imino- 1-methylpyrimidine] using [Br&, = 0.0500 mM a 25OC.
a The experiments were performed at a pH of 0.465. The experiments were perfomed at a pH of 5.56.
Figure B1.1.1 Dependence of k,* on the 1.2dihydro-2-imino-1- methylpyrïmidine concentration minus the bromine concentration at pHz0.465 for experiment IT-124M and a pH of 5.56 for experiment IT-125M.
Table B I .8. Raw aata for the Bromination (IBrJ,, = 0.0250 mM) of Synthesized 1.2- dihydro-2mino-lmethflpflmidine (0.250 mM) at 25%.
1-42 x 10''
2.06 x 1 O-'
' Two different rates were obsenred, a fast rate and a slow rate.
112
[ m: oz- KI = 0.0562 M
Scheme B1 Lotus 1-2-3 spreadsheet for the bromination of aniline.