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Chemical Synthesis of Peptides and Peptide Libraries

Prof. Victor J. Hruby

1The screen versions of these slides have full details of copyright and acknowledgements

Chemical Synthesis of Peptidesand Peptide Libraries

Prof Victor J Hruby

1

Prof. Victor J. HrubyRegents Professor of Chemistry

Professor of Biochemistry and Molecular BiophysicsProfessor of Neuroscience

Professor of Medical PharmacologyProfessor of the Arizona Research Laboratories

Professor of BIO5

Introduction

• Peptides and proteins are of central importance to all biological processes

• Methods for their synthesis by chemical means are thus of central importance to understanding biology

f f

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• Excellent methods for the chemical synthesis of peptides and peptidomimetics have been developed in past 100 years (indeed most robust of any class of organic compounds)

• Primary focus of this discussion is, therefore, synthesis of peptides and peptidomimetics with desired chemical and physical properties for biological applications

• Are the catalysts of the chemicals reactions in life processes

• Are the structural scaffolds of most living systems

• Are the mediators of energy transduction

Why peptides and proteins?they do everything!

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• Are the messengers and modulators of information transduction

• Can readily adapt their structures to recognize all other structures

• Can readily change their 3 dimensional structures in response to the environment

• Can incorporate the universe of structures into their structures




Some misconceptions about peptidesas suitable drugs candidates

• Peptides are not stable in biological systems

– Some are, some are not

– All can be made stable with retention of bioactivity

• Peptides have poor biodistribution

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• Peptides have poor biodistribution

– Often not true if stable and not given as bolus

– Can interact with blood proteins

• Peptides do not cross membrane barriers

– Many do already and methods are being developed

– That utilize a variety of mechanisms

Advantages of peptides

• Native ligand often a peptide or peptide fragment of a protein

• Relative stability easily built in

• Potent

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• Selectivity can be built in

• Multiple activities can be built in

• Can be designed to cross BBB

Torsional angles for peptide backbone and side chains

6Parameters often utilized for topographical design of peptide ligand

ωφ ψ

gauche (+), χ1= +60o gauche (-), χ1= -60o trans, χ1= ±180o




The Ramachandran plot

ϕ – ψ map of N-acetyl alanine N’-methyl amide using

ECEPP/2 potentials*

Definition of backbone torsion angles ϕ and ψ

7* Data of Rotherman, I.K.,Lambert, M.H.,

Gibson K.D. and Scheraga, H.A. J. Biomol. Struct. Dyn., 7, 421 (1989)

φ

ψ

What structures do we want to stabilize by design and synthesis – natures’

evolved choices• Backbone

– α-helix

– β-sheets

– β-turns; hairpin turns

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β ; p

– Extended structures

• Side chain group – CHI space – gauche (-); gauche (+); trans

• Important caveat – some conformational flexibility may be critical

• Biological systems are inherently dynamic

Uses of conformational constraint

• Determine bioactive conformation – template

– a. Agonists; b. Antagonists

• Improve receptor selectivity

– a. Agonists; b. Antagonists

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• Stabilize peptide against proteolytic degradation

• Improve bioavailability

– Blood-brain barrier; half-life in vivo

• Minimize structure

– Stable pharmacophore; no toxicity

• Peptide mimetic design




Chi-1/Chi-2 plot for Tyr

L- Tyrosine

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• To more clearly define chi space in peptide, protein and peptidomimetic structure

• For scaffold design in combinatorial chemistry

• To examine significance of chirality in chi space in molecularrecognition in biological systems

• To examine uses of chi space constraints of key

Needs and uses for topographical constraints

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pharmacophore residues:

– To provide selectivity for receptor types and subtypes, for enzymes, etc.

– To provide antagonists

– For providing ability to cross membranes

– To stabilize peptides from protease degradation

• For design of receptors and acceptors

Novel Chi constrained amino acids

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e. (2S,3R)-TMT

Chi-1/Chi-2 plots for L- and D-Tyr and β-Me-3’,-5’-Me2-Tyr

X axes=χ1Y axes=χ2

a. L-Tyrc. (2S,3S)-TMT

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b. D-Tyrd. (2R,3R)-TMT f. (2R,3S)-TMT

Solid phase synthesis considerations• Solid support, polymer

– Stable to organic chemistry used

• Linkers – for appropriate c-terminal

• Synthetic organic chemistry

– C- to N-terminal assembly

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– Minimal racemization

– N⟨ protection

– Orthogonal side chain protection

– Macrocyclic synthesis methods

– Well developed

– Disulfides; lactams; lactones; side chain to backbone; backbone-backbone, etc.

Solid supports

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OO

OH [ ]

[ ] [ ]OO

OO

OHOH




Merifield Wang SASRIN HALHF 50% TFA/DCM 2% TFA/DCM 0.2% TFA/DCM

Benzhydryl Rink Sieber TritylHF 20% TFA /DCM 1% TFA /DCM 2 10% TFA /DCM

Acid labile linkers

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HF 20% TFA /DCM 1% TFA /DCM 2-10% TFA /DCM

R1=H, R2=HR1=H, R2=OMeR1=H, R2=MeR1=Cl, R2=H

R=H or R=Me X=O or X=NH

Solid phase peptide synthesis

NH

NH

OH

O

O

O

R3R1

NH2

R2

R3 R3 CouplingAttachment Deprotection Coupling

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CleavageNH

NH

OH

O

O

O

R3R1

NH2

R2

PG

NH

NH

O

O

O R3

R2

NH

X

O

R3

PG

Attachment

NH

O

O

R3

PG

Deprotection

NH2

O

O

R3

NH

O

R2

XPG

Coupling

OH

Attachment Deprotection Coupling

Peptide synthesis: coupling reagents

N NN

NN

OH

N NN

N

OH

NN

NN

O P+NN N

NN

NN

NN

NN NN

N

DIC HOBt HOAt

PF - PF6-

PF -

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O P+N

NO P+

NN

NO P+

NN

O P+N

N

C+ NMe2

Me2N

N+

NN

O

C+ NMe2

Me2N

N N+

NN

O

BOP AOP PyBOP PyAOP

PF6- PF6

6 PF6

HBTU PF6- HATU

TBTU BF4-

PF6-




Peptide synthesis: Boc strategy

O

O

SO

O

NH

O

OOH

O

CO2 NH2O

O

+ + ++NH

O

OO

O CF3CO2H

Boc

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Boc-HNOH

OBoc-HN

OH

O

Boc-HNOH

O

Boc-HNOH

O

NH

O

Boc-HNOH

O

N

NSO

O

Boc-HNOH

O

NH

NHNH

SO

O

Boc-HNOH

O

NH

O

O

Boc-HNOH

O

O

Peptide synthesis: Fmoc strategy

Fmoc

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Strategy for peptide-targeted molecular design

21




• Combinatorial chemistry

– The development of chemical methods that will produce large collections of molecules that are present as a statistically valid mixture or entity

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• Chemical library

– A large mixture of molecules randomly generated that can be evaluated for ‘properties’

Combinatorial chemistry strategy

• Synthesize a large random library of selected concentrations

• Isolate or determine the ligands that bind to a specific acceptor molecule

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p p

• Determine the structure of the compound

• Establish consensus structures if possible

• Note: parallels molecular cloning or molecular antibody technology

Requirements for obtaining statisticallyvalid combinatorial libraries

• Reactions must go to completion or nearly so

• Beads must be of uniform size

• Loading of beads must be uniform at the single

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bead level

• All spliting steps must give uniform and essentiallyequivalent distribution

• If reaction mixtures are used must have detailedknowledge of rates




Widely used methodologies that can usecombinatorial chemistry

• Geysen’s multipin method

• Tea bag method of houghten

• One peptide (ligand) – one bead

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• Proportioning-mixing method

• Deconvolution methods

• Positional scanning method

• Multiple release methods

• Chemical encoding-sequence tagging

• Phage display libraries and other molecular biological methods

Library types• Peptide libraries

– Simple chemistry

– Simple structure determination

– Large number of building blocks

– In some cases expensive building blocks

• Peptide mimetic libraries

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• Peptide mimetic libraries– More complicated chemistry

– Difficult structure determination

– Unlimited number of building blocks

– Generally less expensive building blocks

– Often have not been optimized for valid

– Combinatorial science

Synthesis of hetero-bivalent ligands

O

O

O

H2N PS/DVB

HN

NH

O

ONH

HN

NH

HN

H2N

O

O O

OtBuON

O

O

O

O O

CCK-6H

OO

O

N

N

H2N O

CCK-6

OO

O

NH

OOOH2N

PEGOH

CCK-6H CCK-6

CCK-6

Boc

NH23

I II

III IV

Rink resin

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HN

O

PEGO CCK-6

PEGO[PG]n CCK-6PEGO

HN

O

ONH

OHN

ONH

OHN

ONH

OHN

ON BocOtBu

OtBuO

NNTrt

MSH-7

NH

HNHN Pbf

O

N

O

HN

O

NNH

O

O

HN

n

IV

IV

V

Fmoc/tBu SPPS PEGOPGPGPG

Fmoc/tBu SPPS

TFA (85%), H2O (5%)

Thioanisone (5%),Ethanedithiol (5%)

Ht-bivalent ligands




Some chemical reactions examined in chemical library format

• Acylations

• Alkylations, alkynations, reductive alkylations, etc.

• Carbanion chemistry- grignards, organocadmiums, etc.

C b i ti

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• Carbene insertion

• Condensation reactions- Aldol, Claisen, Mannic, Wittig, etc.

• Cycoadditions- Diels-Alder, dipolarophiles, click, etc.

• Cyclization reactions- heterocyclics turn mimetics, aromatics

Some chemical reactions examined in chemical library format (continued)

• Libraries from libraries

• Nucleophilic additions- Michael, etc.

• Nucleophilic substitutions

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• Metal catalyzed- Heck reaction, Suzuki coupling, etc.

• Carbamates, ureas, etc. formation

• Oxidations

• Reductions

• Multicomponent reactions

• Prepare combinatorial library- hexapeptides

– One peptide one bead- 206- 64x106 peptides; 1-4 days- one large library

– Deconvolution- 206; 400 sub-libraries, 2-20 days

• Screen Library assay developed

Combinatorial chemistry drug lead time chart - for peptides

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• Screen Library- assay developed

– One peptide- one bead- 206; 2-3 days to 3-4 weeks

– Deconvolution- 206; 8 to 40 sub-libraries per day, 5-50 days

• Structure determination of hits

– One peptide- one bead- 4-40 per day/instrument

– Deconvolution- chose structures to synthesize

• Resynthesis and test hits; one to three weeks




Desired factors to control in drug design

• Potency

• Selectivity

• Stability- metabolism

• Penetration through membrane barriers

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• Penetration through membrane barriers

– BBB

– Gut

– Cells

• Biodistribution/elimination

Working hypothesis (chemical) derivedfrom biological collaborations regarding ligand

design, biological activity and behavior

Small changes in structure can produce critical changes in biological activity which can lead

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changes in biological activity which can lead to traumatic changes in behavior

How to apply global conformational constraint approach to design highly potent

peptide hormones

33Mosberg, H.I., Hurst R., Hruby V.J., Gee K., Yamamura H.I., Galligan J.J., and Burks T.F., Proc Natl Acad Sci U S A 1983, 80:5871-5874




How to apply topographical constraint approach to design potent

peptide hormone analogues

34Qian, X., Hruby, V. J. et al., J. Am. Chem. Soc. 1996, 118, 7280-7290

Binding affinities and biological potencies of DPDPE and four stereoisomers

of [TMT1]DPDPEBinding data, IC50 ± SE, nM

Peptide [3H]CTOP GPI (μ) MVD (δ) Selectivityμ/δ

DPDPE 380 1780609± 70 1 6± 0 2 7300± 1700 4 1± 4 6

Bioassay, EC50 ± SE, nM

Selectivityμ/δ

[3H][p-CIPhe4]DPDPE

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DPDPE 380 1780[(2S,3S)TMT1]DPDPE 3.4 1.7

[(2S,3R)TMT1]DPDPE 850 0% at

60 μM >35000

77000± 6000 22 23

0% at 104 9% at 104 n/a n/a

[(2R,3R)TMT1]DPDPE[(2R,3S)TMT1]DPDPE

4270± 820

722± 126

609± 70 1.6± 0.2

211± 33

5.0± 1.0

3500± 230

7300± 1700

293± 1

(5± 3)x104

75% at 82 μM

4.1± 4.6

168± 37

1.76± 0.32

2190± 780

28% at 10 μM

The binding affinity (ic50, nm) of the first generationof potent and δ opioid receptor selective non-peptide

mimetics designed from our group

OH

Compounds[3H]-DMGO(μ receptor)

850[(2S,3R)TMT1]-DPDPE*

IC50 (nM) Selectivity (μ/δ)[3H]-pCIDPDPE(δ receptor)

4720± 820 5.0± 0.10

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H3CN N

OH

N

(H3C)3C

N

OMe

N

(H3C)3C

N

1.28

2020

>8000 1840 NA

780± 72

17,000± 3000

610± 310

8.4± 1.6

*Xinhua Qian et al., J. Am. Chem. Soc. 1996, 118, 7280-7290




Biologically active peptides derived from pro-opiomelanocortin

γ-MSH α-MSH ACTH β-MSH β-Endorphin

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γ-MSH YVMGHFRWDRF

ACTH SYSMEHFRWGKPVGKKRRPVKVYPNGAEDSAEAFPLEF

α-MSH Ac–SYSMEHFRWGKPV–NH2

β-MSH AEKKDEGPYRMEHFRWGSPPKD–OH

Important structuresAc-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2

α-MSH

H-Tyr-Val-Met-Gly-His-Phe-Arg-Trp-Asp-Arg-Phe-Gly-OH

γ-MSH

Ac-Ser-Tyr-Ser-Nle-Glu-His-DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH2

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NDP-α-MSH (Super-agonist)

Ac-Nle-Asp-His-DPhe-Arg-Trp-Lys-NH2

MT-II (Super-agonist)

Ac-Nle-Asp-His-DNal(2')-Arg-Trp-Lys-NH2

SHU-9119 (Potent antagonist, MC3R/MC4R)

NMR structures of MT-II and SHU-9119

MT ll

39

MT-ll

SHU-9119




Bioactivities of Agouti, SHU-9119 and MT-II

rMC3-R mMC4-R

Antagonist AntagonistAgouti

IC50>100 nM IC50= 3.9 nM

40

50

Antagonist AntagonistSHU-9119

Agonist AgonistMT-II

IC50= 4.5 nM

EC50= 0.27 nM

50

IC50= 0.36 nM

EC50= 0.057 nM

ICV administration of MC4 receptor antagonist SHU-9119 blocks ICV MT-II inhibition of feeding

41

Change in body weight after 10 days on MT-II

42




Frogs treated with MT-II (left) and α-MSH (right) –1 week later

43

Local topographical constraints

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Comparative biological activities of β- MeTrp α- MSH analogues

Peptide activity in the frog skin bioassay

Relativea

potencyEC50

Value (nM)Peptide Structure α-MSH Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2 1.0 0.10

MTlI Ac-Nle-Asp-His-DPhe-Arg-Trp-Lys-NH2 1.0 0.10

I 0 225 0 44

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I Ac-Nle-Asp-His-DPhe-Arg-β-MeTrp-Lys-NH2(3S,2S)

0.225 0.44

II 1.6 0.06

III 0.0035 28.6

IV 0.3 0.30

a- All peptide activities were tested at a range concentrations and compared to the half-maximal effective dose of α-MSH in the frog skin (10-10 M) bioassays

Ac-Nle-Asp-His-DPhe-Arg-β-MeTrp-Lys-NH2(3S,2R)

Ac-Nle-Asp-His-DPhe-Arg-β-MeTrp-Lys-NH2(3R,2S)

Ac-Nle-Asp-His-DPhe-Arg-β-MeTrp-Lys-NH2(3R,2R)




Prolonged bioactivities of MT-II analogues contain all four isomers of β-Me-Trp

46

A systematic approach to peptide and peptidomimetic delivery through the BBB

• Develop potent, receptor selective, efficacious and stable ligands

• Site specific proteolytic cleavage of stable peptides to bioactive ligand in the brain- prodrugs

– Enzyme specific structural vectors

M i i ff t f li hili it hi hili it d d i

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• Maximize effects of lipophilicity, amphiphilicity and dynamics for penetration through the BBB

• Enhancement of peptide half life in circulation

– Binding to blood- born proteins

• Use of carrier-mediated transport at BBB

• Designed glycopeptides, lipopeptides, etc.

Designed peptide ligands that are stable

to proteolytic degradationin vitro and in vivo

H-Tyr-DPen-Gly-Phe-D-Pen-OH

H-Tyr-DPen-Ala-Phe-DPen-OH

Ac-Ser-Tyr-Ser-Nle-Glu-His-DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH2

Ac-Nle-Asp-His-DPhe-Arg-Trp-Lys-NH2

Enkephalin analogues

Melanotropin analogues

Deltorphin analogues

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Tyr-DPen-Phe-Asp-Pen-Nle-Gly-NH2

Tyr-DPen-Phe-His-Pen-Met-Asp-NH2

β-Mpa-Tyr-Ile-Glu-Asn-Cys-Pro-Lys-Gly-NH2

Pen-DPhe(p-Me)-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2

p

DPhe-Cys-Tyr-DTrp-Arg-Thr-Pen-Thr-NH2

Oxytocin analogues

Mu opioids derived from somatostatin




Modification at C-terminal

• Modification at C-terminal:

– Critical activity difference at rat NK1 receptor

TY005: Tyr-DAla-Gly-Phe-Met-Pro-Leu-Trp-O-3,5-Bn(CF3)2

TY027: Tyr-DAla-Gly-Phe-Met-Pro-Leu-Trp-NH-3,5-Bn(CF3)2

TY025: Tyr-DAla-Gly-Phe-Met-Pro-Leu-Trp-NH-Bn

49

y p

– Less substance P antagonist activity difference at guinea pig ileum (species difference)

– Act as an “address region” for opioid activities

• Expected activities in human

– Opioid activity: TY025 > TY027 > TY005

– SP antagonist activity: TY025 = TY027 > TY005

TY005

No.

24.7358.822.3O-3,5-Bn(CF3)2

Antagonist(Ke; nM)

Agonist(IC50; nM)

Agonist(IC50; nM)

Substance POpioid (μ)Opioid (δ)

GPI/LMMPMVD

C-terminal

Tyr-DAla-Gly-Phe-Met-Pro-Leu-Trp-R

δ, μ opioid agonist and substance P antagonist functional activities (ex vivo)

50

247.0N.T.N.T.L-732,138N.T.54.7258.1Morphine9.961.14.8NH-BnTY025

10.0487.914.5NH-3,5-Bn(CF3)2TY027

MVD: mouse vas deferensGPI/LMMP: guinea pig isolated ileum/ longitudinal muscle with myenteric plexus• Opioid activity (δ-opioid selective): TY025 > TY027 > TY005

• Guinea pig SP antagonist activity: TY025 = TY027 > TY005

Brain distribution using in situ perfusion technique

• Both TY025 and TY005 can penetrate blood brain barrier very well

10

100

Potentially il bled

via

sing

leys

is

Current study

51

0.1

1

10

Vascularcomponent

availablefor activity

Kin

(µl/m

in/g

), C

alcu

late

time

poin

t ana

ly

The peptides were radiolabeled with I125

and used for the experiment to detect the trace of I125 in brain




Conclusions regarding factors that increase peptide permeability through membranes

• Stability against proteolytic degradation

• Appropriate hydrophobic substitutions

• Redistribution of electron density

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– Especially aromatic rings

• Side chain conformations

• Ability to assume more compact structure in membrane vs. aqueous environment

• Conformational flexibility

• Carrier/saturable transporters at BBB

Key reasons why peptides will be the drugs of the future

• Nature has chosen peptides and proteins to do everything, especially in multicellular life

• Are initiators, modulators, and controlling moieties in most biological functions

• Have low or minimal toxicities especially when compared

53

to current drugs

• Broad spectrum of biological activities in health and disease- hormones, neurotransmitters, growth factors, cell function, metabolism, etc.

• Ability to infinitely modulate properties in chemical and three dimensional conformational space

• Requires better delivery systems than current oral delivery

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