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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
2
• 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!
3
• 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
Chemical Synthesis of Peptides and Peptide Libraries
Prof. Victor J. Hruby
2The screen versions of these slides have full details of copyright and acknowledgements
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
4
• 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
5
• 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
Chemical Synthesis of Peptides and Peptide Libraries
Prof. Victor J. Hruby
3The screen versions of these slides have full details of copyright and acknowledgements
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
8
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
9
• Stabilize peptide against proteolytic degradation
• Improve bioavailability
– Blood-brain barrier; half-life in vivo
• Minimize structure
– Stable pharmacophore; no toxicity
• Peptide mimetic design
Chemical Synthesis of Peptides and Peptide Libraries
Prof. Victor J. Hruby
4The screen versions of these slides have full details of copyright and acknowledgements
Chi-1/Chi-2 plot for Tyr
L- Tyrosine
10
• 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
11
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
12
Chemical Synthesis of Peptides and Peptide Libraries
Prof. Victor J. Hruby
5The screen versions of these slides have full details of copyright and acknowledgements
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
13
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
14
– 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
15
OO
OH [ ]
[ ] [ ]OO
OO
OHOH
Chemical Synthesis of Peptides and Peptide Libraries
Prof. Victor J. Hruby
6The screen versions of these slides have full details of copyright and acknowledgements
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
16
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
17
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 -
18
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-
Chemical Synthesis of Peptides and Peptide Libraries
Prof. Victor J. Hruby
7The screen versions of these slides have full details of copyright and acknowledgements
Peptide synthesis: Boc strategy
O
O
SO
O
NH
O
OOH
O
CO2 NH2O
O
+ + ++NH
O
OO
O CF3CO2H
Boc
19
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
20
Strategy for peptide-targeted molecular design
21
Chemical Synthesis of Peptides and Peptide Libraries
Prof. Victor J. Hruby
8The screen versions of these slides have full details of copyright and acknowledgements
• Combinatorial chemistry
– The development of chemical methods that will produce large collections of molecules that are present as a statistically valid mixture or entity
22
• 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
23
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
24
bead level
• All spliting steps must give uniform and essentiallyequivalent distribution
• If reaction mixtures are used must have detailedknowledge of rates
Chemical Synthesis of Peptides and Peptide Libraries
Prof. Victor J. Hruby
9The screen versions of these slides have full details of copyright and acknowledgements
Widely used methodologies that can usecombinatorial chemistry
• Geysen’s multipin method
• Tea bag method of houghten
• One peptide (ligand) – one bead
25
• 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
26
• 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
27
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
Chemical Synthesis of Peptides and Peptide Libraries
Prof. Victor J. Hruby
10The screen versions of these slides have full details of copyright and acknowledgements
Some chemical reactions examined in chemical library format
• Acylations
• Alkylations, alkynations, reductive alkylations, etc.
• Carbanion chemistry- grignards, organocadmiums, etc.
C b i ti
28
• 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
29
• 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
30
• 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
Chemical Synthesis of Peptides and Peptide Libraries
Prof. Victor J. Hruby
11The screen versions of these slides have full details of copyright and acknowledgements
Desired factors to control in drug design
• Potency
• Selectivity
• Stability- metabolism
• Penetration through membrane barriers
31
• 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
32
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
Chemical Synthesis of Peptides and Peptide Libraries
Prof. Victor J. Hruby
12The screen versions of these slides have full details of copyright and acknowledgements
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
35
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
36
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
Chemical Synthesis of Peptides and Peptide Libraries
Prof. Victor J. Hruby
13The screen versions of these slides have full details of copyright and acknowledgements
Biologically active peptides derived from pro-opiomelanocortin
γ-MSH α-MSH ACTH β-MSH β-Endorphin
37
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
38
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
Chemical Synthesis of Peptides and Peptide Libraries
Prof. Victor J. Hruby
14The screen versions of these slides have full details of copyright and acknowledgements
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
Chemical Synthesis of Peptides and Peptide Libraries
Prof. Victor J. Hruby
15The screen versions of these slides have full details of copyright and acknowledgements
Frogs treated with MT-II (left) and α-MSH (right) –1 week later
43
Local topographical constraints
44
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
45
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)
Chemical Synthesis of Peptides and Peptide Libraries
Prof. Victor J. Hruby
16The screen versions of these slides have full details of copyright and acknowledgements
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
47
• 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
48
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
Chemical Synthesis of Peptides and Peptide Libraries
Prof. Victor J. Hruby
17The screen versions of these slides have full details of copyright and acknowledgements
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
Chemical Synthesis of Peptides and Peptide Libraries
Prof. Victor J. Hruby
18The screen versions of these slides have full details of copyright and acknowledgements
Conclusions regarding factors that increase peptide permeability through membranes
• Stability against proteolytic degradation
• Appropriate hydrophobic substitutions
• Redistribution of electron density
52
– 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
54
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