Transcript document

On-resin and in-solution
synthesis of cyclic peptides
Gábor Mező
Research Group of Peptide Chemistry
Hungarian Academy of Sciences
Eötvös L. University
Budapest, Hungary
ERASMUS Teaching Mobility Grant
Konstanz
28th October 2008
Synthesis of cyclopeptides
What is the reason for the synthesis of cyclopeptides?
1. Natural compounds: antibiotics, hormones, toxins, enzymes,
immunoglobulines, depsipeptides, etc.
 gramicidine S (antibiotic):
Val-Orn-Leu-D-Phe-Pro
Pro-D-Phe-Leu-Orn-Val
 somatostatine (hormone):
H-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH
 a-conotoxin GI (toxin):
H-Glu-Cys-Cys-Asn-Pro-Ala-Cys-Gly-Arg-His-Tyr-Ser-Cys-NH2
 phalloidine (toxin in mushrooms):
CR4
CO
CO
NH
O
NH
CR1
NH
CO
NH
CO
CR3
NH
O
N
H
CO
CR2
S
CO
NH
2. Increasing or change the biological activity of the cyclic peptides:
eg. Somatostatine derivative with high antitumour activity;
H-D-Phe-Cys-Tyr-D-Trp-Lys-Cys-Thr-NH2
3. Stabilization of secondary and tertiary structures:
eg. improvement of the hormone-receptor interaction (increased
selectivity);
Leu-enkephaline:
Cyclic derivative:
H-Tyr-Gly-Gly-Phe-Leu-OH
Selective for
m-receptor
H-Tyr-Dab-Pro-Phe-Leu
Dab = a,g-diaminobutiric acid; gNH2-CH2-CH2-CH2-COOH
aNH
2
4. Inreasing the enzyme stability:
GnRH-III (antitumour activity):
Pyr-His-Trp-Ser-His-Asp-Trp-Lys-Pro-Gly-NH2
Pyr-His-Trp-Ser-His-Asp-Trp-Lys-Pro-Gly-NH2
Pyr = pyroglutamic acid
5. Study of the structural elements:
c(b-Ala-Ala-b-Ala-Pro) has g-turn conformation
6. Templates: e.g. synthesis of miniproteins
G
P
K
K
K
C
C
K
S
P
G
The template contains amide bonds in the cycle
and it is stabilized by disulfide cross-linkage.
Selective protection of Lys residues allows
the attachment of 4 different peptide chains.
S
Arrangement of cyclic peptides
homodetic
only amide bonds in the cycle
heterodetic
disulfide bridge, thioether bond
lactone, ether, oxime thiazolidine bond
Homodetic cyclic peptides
(only amide bonds in the cycle)
(head to tail)
Coupling of the N- and C-termini
(side-chain to tail)
Coupling of side chain amino group
(Lys, Orn) to C-terminal carboxyl group
(side-chain to side-chain)
Coupling of side chain amino and carboxyl
groups (Lys, Orn, Asp, Glu)
(side-chain to head)
Coupling of side chain carboxyl group
(Asp, Glu) to N-terminal amino group
Connection of two side chains with a peptide chain
(branched)
Synthesis of cyclic peptides:
 Solid phase synthesis of linear precursor peptide
 Cyclisation in solution or on solid support
Cyclisation in solution:
 cyclisation in diluted solution (0.1-0.2 mg (mmol)/mL to prevent
cyclodimerisation or polymerisation.
 preparation of cyclic dimers: a more concentrated solution is necessary.
e.g. synthesis of Gramicidine S from a pentapeptide precursor peptide:
H-Val-Orn(Z)-Leu-D-Phe-Pro-ONp
concentration (mol/L)
Val
cyclopentapeptide
(cyclo-semigramicidine S)
cyclodecapeptide
(gramicidine S)
3x10-3
1/3
2/3
3x10-4
1/2
1/2
Gly
only cyclopentapeptide has been formed
The efficiency of cyclisation is always highly sequence dependent!
Cyclisation on solid support:
 the peptides are attached to the resin (capacity of 0.2-0.4 mmol/g)
(pseudo dilution); intramolecular cyclisation is prefered over intermolecular reaction.
 reagents can be removed by filtration; easier workup procedure.
Efficiency of cyclisation depends on:
 Sequence (also protecting groups) and the conformation of the
linear precursor peptide;
 Size of the cycle:
6 amino acids in the cycle is an ideal size. Larger cycles may be formed
easily, while smaller cycles are difficult to be formed (it needs cis peptide
bond, Pro, Gly help). Diketopiperazine (2 amino acids-2 cis peptide bonds);
O
C
R”
N
CH
H
CH
trans
O
R’
H
C
R”
O
N
CH
CH
cis
C
R’
NH
R1-CH
NH
CH-R2
C
O
diketopiperazine
 Solvent: the solvent polarity has an influence on the secondary structure;
 Concentration: low concentration helps the intramolecular reaction;
 Temperature: at higher temperature the molecules are more flexible.
Protecting groups:
 lactam ring: orthogonal (selectively removable) protecting groups
on amino and carboxyl groups:
-----Lys(X)--------Glu(Y)---Asp(Y)-------Lys(Y)--------Glu(X)----X removal
-----Lys--------Glu(Y)---Asp(Y)-------Lys(Y)--------Glu----cyclisation
-----Lys--------Glu(Y)---Asp(Y)-------Lys(Y)--------Glu----Y removal
-----Lys--------Glu---Asp-------Lys--------Glu---- disulfide bridge: selective Cys protection in case of 2 disulfide bonds
 other type of cycles: unprotected peptide precursors
Synthesis in solution of ”head to tail”
type cyclic peptides
Experimental aspects:
 synthesis of linear precursor peptide on solid support;
 cleavage of the peptide with free N- and C-terminus;
 fully or semi-protected side chains;
 there is a difference in structure and solubility of fully or semi-protected
peptides (purification is much easier in case of the later one);
 choose an amino acid at the C-terminus which has no or less racemisation;
(Gly, Pro, Ala);
 use excess of coupling agents (no uronium type ones, HBTU, TBTU);
 precursor peptides containing turn structure give better results in
cyclisation when the turn is inside the sequence.
Boc chemistry:
Fully protected peptides can be prepared only on photo or alkaline cleavable
resins (expensive and complicated). Oxime resin (see on resin cyclisation).
Eg. Synthesis of a semiprotected peptide for cyclisation:
272Asp-Pro-Glu-Asp-Ser-Ala-Leu-Leu279
HSV gD-1 epitope peptide
Boc-Leu-Leu-Asp(OcHex)-Pro-Glu(OcHex)-Asp(OcHex)-Ser(Bzl)-Ala-R
1) 33%TFA/DCM
2) 1M TMSOTf-thioanisole/TFA; 45min, 0oC
H-Leu-Leu-Asp(OcHex)-Pro-Glu(OcHex)-Asp(OcHex)-Ser-Ala-OH
BOP-HOBt-DIEA (3:3:6 equiv) in DMF
RT, 4hrs
cyclo(Leu-Leu-Asp(OcHex)-Pro-Glu(OcHex)-Asp(OcHex)-Ser-Ala)
HF-p-cresol (9:1, V/V)
0oC, 90min
cyclo(Leu-Leu-Asp-Pro-Glu-Asp-Ser-Ala)
cyclo(Asp-Pro-Glu-Asp-Ser-Ala-Leu-Leu)
Fmoc chemistry:
cyclo(Cys-D-Phe-Arg-Gly-Asp)
RGD peptides prevent cell addhesion
Fmoc-Asp(OtBu)-Cys(Trt)-D-Phe-Arg(Pbf)-Gly-ClTrt
1) 2%DBU-2%piperidene/DMF (2+2+5+10 min)
2) AcOH-MeOH-DCM (1:1:8, V/V), RT, 30 min
H-Asp(OtBu)-Cys(Trt)-D-Phe-Arg(Pbf)-Gly-OH
BOP-HOBt-DIEA (3:3:6 equiv) in DMF
RT, 16hrs
cyclo(Asp(OtBu)-Cys(Trt)-D-Phe-Arg(Pbf)-Gly)
TFA-water-EDT-thioanisole-phenol
(10mL:0.5mL:025mL:0.5mL:0.75g)
RT, 2hrs
cyclo(Asp-Cys-D-Phe-Arg-Gly)
cyclo(Cys-D-Phe-Arg-Gly-Asp)
Cys: dimerisation
or conjugation
Synthesis of ”head to tail” type cyclic peptides
on resin
Application of oxime resin:
P
C
NO2
HO
 The peptide-resin bond is stable
in acids, but cleavable by amines.
 Compatible only with Boc chemistry.
 However, in situ neutralisation is
necessary.
N
C
NO2
p-nitrobenzophenone oxime resin
Boc-Aaa(X)-OH
+DCC/DCM
NH2-PEPTIDE-O
P
N
c(PEPTIDE)
NO2
Boc-Aaa-O
C
N
Synthesis of cyclic peptides
and protected peptide fragments
P
C
NO2
Boc-PEPTIDE-O
N
P
NH2-Aaa(X)-OY
Boc-PEPTIDE-Aaa(X)-OY
Synthesis of cyclo(Arg-Gly-Asp-Phg):
Boc-Aaa4-Aaa3-Aaa2-Aaa1-O-N=C(resin) (0.5 mmol/g capacity)
1) 25% TFA/DCM
2) TEA-AcOH (2 equiv each)/DMF, RT, 24h
3) HF
cyclo(Aaa4-Aaa3-Aaa2-Aaa1)
Aaa4
1. Arg(Tos)
2.
Phg
Gly
Aaa3
Aaa2
Aaa1
yield
monomer
dimer
Gly
Asp(OcHex)
Phg
65%
75%
20%
Asp(OcHex) 65%
50%
25%
Arg(Tos)
Gly
Phg
Arg(Tos)
Gly
70%
40%
40%
Asp(OcHex)
Phg
Arg(Tos)
53%
10%
ND
3. Asp(OcHex)
4.
+ oxime-resin
Formation of the cyclic dimer can be decreased by using a resin with lower capacity.
The efficacy of the cyclisation highly depends on the sequence.
H-bonds in linear compounds determine an effective cyclisation
(according to NMR studies).
Attachment of a side chain functional group to the resin:
Applied amino acids: His, Asp, Glu, Lys
NO2
NO2
R-CH2-NH2
+
NO2
F
NO2
R-CH2-NH
(AM-PS-DVB)
F
F
1) Boc-His-OH
2) DCC/HOBt
3) H-Gly-OBzl
R
Boc-His(Dnp)-Gly-His(Dnp)-Gly-His(Dnp)-Gly-OBzl
NO2
1) 40% HBr/AcOH
50% TFA/DCM
(1:1,v/v), 2x0.5h
2) Pyridine
3) EEDQ, 2x24h, RT
in pyridine-toluene
His(Dnp)-Gly
Gly
His(Dnp)-Gly
1)
2)
3)
4)
His(Dnp)
RSH/DMF
Boc-Gly-OH
Boc-His(Dnp)-OH
Boc-Gly-OH
Boc-His(Dnp)-OH
R-CH2-NH
NO2
R
His-Gly
Gly
His-Gly
His
Boc-NH-CH-CO-NH-CH2-COOBzl
Attachement of Asp and Glu to resins are the most common ones:
Coupling to hydroxymethyl type (PAM or Wang) resins results in
Asp or Glu at the end of the synthesis.
Coupling to amine type (MBHA, Rink-Amide MBHA) resins results in
Asn or Gln at the end of the synthesis.
O
Tachikinine antagonist:
O CH2
CO
CH2
Boc-NH-CH-CO-OFm
CH2 C
NH CH2
P
PAM
Peptide chain elongation
by Boc/Bzl strategy
(In situ neutralisation is suggested)
Boc-Tyr(BrZ)-D-Trp-Val-D-Trp-D-Trp-Arg(Tos)-Asp(PAM)-OFm
1) 50% TFA/DCM (5+15 min)
2) 20% piperidine/DMF (3+7 min)
3) BOP-DIEA (3:6 equiv)/DMF (2x3h)
cyclo[Tyr(BrZ)-D-Trp-Val-D-Trp-D-Trp-Arg(Tos)-Asp(PAM)]
Yield: 72%
Purity: 65%
HF-anisole-DMS (10:1:0.5, v/v), 1h, 0oC
cyclo[Tyr-D-Trp-Val-D-Trp-D-Trp-Arg-Asp]
Part of pp60c-src retrovirus (autophosphorilation of tyrosine kinase):
Fmoc-Glu-ODmb, DIC, DMAP
(Dmb=2,4-dimethoxybenzyl)
O
HO
CH
R (0.45 mmol/g capacity)
O CH
2
2
CO
Leu R
HMPA-linker
Fmoc-Glu-ODmb
Peptide synthesis; Fmoc strategy
O
R
Fmoc-Ala-Ala-Arg-(Mtr)-D-Phe-Pro-Glu(OtBu)-Asp(OtBu)-Asn-Tyr(tBu)-Glu-ODmb
1) 1% TFA/DCM (6x5 min)
2) 20%piperidine/DMF (3+10 min)
O
R
NH2-Ala-Ala-Arg-(Mtr)-D-Phe-Pro-Glu(OtBu)-Asp(OtBu)-Asn-Tyr(tBu)-Glu-OH
1) BOP-HOBt-NMM (3:3:6 equiv), 5h, RT
2) TFA-phenol(95:5, v/v)
Ala-Ala-Arg-D-Phe-Pro-Glu-Asp-Asn-Tyr-Glu
Yield: 26%
ca. 20% D-Glu
Racemisation is the main side reaction:
His > Asp > Glu
Epimerisation might occur: - attachment of the first amino acid to the resin
- removal of the C-terminal protecting group (with
base)
- cyclisation
Less racemisation by using:
Boc-Asp(O-Cs+)-OFm
+
O
Br CH
2
CH C
2
NH CH
2
Attachment of dipeptide derivatives:
eg. Fmoc-Glu-Gly-OAll, Boc-Glu-Ahx-OFm, etc.
However, Asp derivatives may form succinimide ring
Because of the epimerisation, in many cases the cyclisation in solution using
protected peptides is more effective. It is sequence dependent. The
C-terminal amino acid can be chosen in case of cyclisation in solution.
P
On resin cyclisation using side chain functional group(s)
Head to side chain:
NH2-Aaa1-Aaa2-Aaa3-Aaa4-Asp-Aaa6-......-R
X must be cleaved selectively
Tail to side chain:
OX
Q-Aaa1-Aaa2-Lys-Aaa4-Aaa5-Aaa6-Asp(R)-OX
Y
X and Y must be cleaved together, selectively, under the same conditions
Side chain to side chain:
Q-Aaa1-Aaa2-Lys-Aaa4-Aaa5-Aaa6-Asp-Aaa7-...-R
OX
Y
X and Y must be cleaved together selectively under the same conditions
Strategy
R
Boc/Bzl
PAM, MBHA
Boc/cHex*
BHA
Fmoc/tBu
Wang, Rink, etc
Fmoc/tBu
Wang, Rink, etc
Fmoc/tBu* Wang, Rink,
Fmoc/Bzl
PAM, MBHA
X
Y
Q
OFm
OBzl
OAll
ODmab
ODmb
OtBu
Fmoc
(Cl)Z
Aloc
(iv)Dde
Mtt
Boc
Boc
Choc
Fmoc/Boc
Fmoc/Boc
Fmoc/Boc
Z
Synthesis of an epitope peptide of FMDV (foot-and-mouth disease virus) C (134-155)
cyclic derivative (solution vs. solid phase)
H-TTETASARGDLAHLTTTHAKHL-NH2
Z
E
D
OtBu
H KH
TFA
R
Boc
E
D
E
H KH NH2
Boc
NH2
CO
E
NH
D
D
R
Boc
E
NH2
D
NH2
NH
13%
H
E
CO
H KH
R
NH2
BOP/DIEA/DMF
OtBu Trt Trt
H KH
R
Boc
E
D
H KH
R
NH
CO
HF
H KH
Aloc
COOH
NH
CO
HF
E
H KH
BOP/DIEA/DMF
Boc
R
Pd(PPh3)4, morpholine
in DMF/THF (3:1, V/V)
OcHex
H KH
H KH
OtBu Trt Trt
COOH
NH2
CO
H
D
D
OAll
Fmoc
E
OcHex
D
E
1) PhSH/DIEA/DMF
2) 2%DBU, 20%Pip/DMF
BOP/DIEA/DMF
E
R Boc
H KH
OcHex
COOH
Z
D
OFm
Boc
OcHex
Z
OtBu Trt Trt
OcHex Dnp Dnp
OcHex Trt Trt
TFA
D
H KH
NH
NH2 H
5%
E
CO
D
H KH
NH2
NH <5%
Synthesis of a bicyclic peptide by a
combination of on-resin and in solution cyclisation
Mező, G. et al. Tetrahedron 54, 6757-6766 (1998).
Choc-SALLE(OBzl)D(OcHex)PVGK(ClZ)- BHA
1M TMSOTf-thioanisole/TFA
(30 min, 0oC)
Choc-SALLED(OcHex)PVGK- BHA
PyBOP (6eq), DIEA (12 eq)/DMF
(24h, RT)
Choc-SALLED(OcHex)PVGK- BHA
HF-p-cresol-DTT (10ml:1g:0.1g)
(1h, OoC)
Choc= cyclohexyloxycarbonyl
Cyclohexyl type protecting groups
as well as peptide-BHA resin bond
are stable in TMSOTf/TFA solution
at 0oC.
H-SALLEDPVGK-NH2
PyBOP (6eq), DIEA (12 eq)/DMF
(24h, RT, c=0.1 mg/ml)
-SALLEDPVGK-NH2
Cyclopeptides containing disulfide bridge(s)
Peptides and proteins with different number of disulfide bridges:
eg. oxytocin (1), conotoxins (2-3), defensins (3), ribonuclease A (4), etc.
Synthetic strategy of cyclic peptides depends on the number of disulfide bridges.
The main side reactions in the syntheses:
cyclic dimer formation (intermolecular)
polymerisation (intermolecular)
disulfide isomers (intramolecular)
Disulfide isomers: in case of 2 disulfide bridges
3 disulfide bridges
4 disulfide bridges
XXCXXXCXCXXXC
XXCXXXCXCXXXC
3 isomers
15 isomers
105 isomers
XXCXXXCXCXXXC
 The primary structure (sequence) determines the conformation and in this way
the disulfide isomer composition. We might have a slightly influence on it.
 The proper disulfide isomer formation is much easier in case of natural
compounds than the artificial ones.
 In neutral solution (biological test) the bridges may open and refold in a way
to get the conformation with minimal energy.
 Disulfide bridge is not as stable as amide or thioether bond.
Protecting groups
Chemistry
Cleavage
HF
CH2
CH3
4-methylbenzyl
(Meb)
Boc
CH2
OCH3
4-methoxylbenzyl
(Mob)
Boc
CH2 NH C CH3
acetamidomethyl
(Acm)
O
CH2 NH C
CH3
C CH3
O CH3
CH2 NH C
O
CH2
trimethylacetamidomethyl
(Tacm)
phenylacetamidomethyl
(Phacm)
Boc, Fmoc
Boc, Fmoc
Boc, Fmoc
HF, TFMSA,
TMSOTf
Hg-, Ag-, Tl-salt
Hg- or Ag-salt
I2, Tl(tfa)3
Hg- or Ag-salt
I2, Tl(tfa)3
Hg- or Ag-salt
I2, Tl(tfa)3
Peniciline
amidohydrolase
Protecting groups
NO2
S
S
N
N
Cleavage
3-nitro-2-pyridinesulphenyl
(Npys)
Boc
RSH (e.g. Cys)
Bu3P, base
2-pyridinesulphenyl
(SPyr)
Boc
RSH (e.g. Cys)
Bu3P, base
trityl (Trt)
OCH3
CH3O
Chemistry
Trimethoxytrityl
(TMTr)
OCH3
Fmoc
TFA, I2
Hg-, Ag-, Tl-salt
Fmoc
1% TFA, I2
Hg-, Ag-, Tl-salt
Protecting groups
CH3
tert-butyl
C CH3
Chemistry
Boc, Fmoc
CH3
CH3
S
RSH, Bu3P,
reducing agents
9-fluorenylmethyl
(Fm)
Boc
piperidine, DBU
NH3/MeOH
2,4,6-trimethoxylbenzyl
(Tmob)
OCH3
Fmoc
7% TFA, I2,
Tl(tfa)3
S-tert-butyl
CH3
H
OCH3
CH2
OCH3
HF (20oC)
TCMS/DPSO
Hg-salt, NpsCl
Fmoc
C CH3
C
Cleavage
C H2
Peptide cleavage from the resin
Boc-strategy:
HF: p-cresol (anisole):DTT (reducing agent) = 10mL:1g:0.1g
1M TMSOTf (TFMSA):thioanisole:EDT:m-cresol:TFA = 1.8:1.2:0.6:0.2:6.8
1-2 h, 0oC
(v/v)
Fmoc-strategy:
Reagent K:
TFA:phenol:water:thioanisole:EDT = 82.5:5:5:5:2.5 (v/v)
Reagent R:
TFA:thioanisole:EDT:anisole = 90:5:3:2 (v/v)
Reagent B:
TFA:phenol:water:TIS = 88:5:5:2 (v/v)
1-4 h, RT
Preparation of peptides containing cysteine residues
for oxidation (cyclisation)
Buffer solution:
0.1 M Tris.HCl or 0.2 M phosphate buffer (pH 8-9)
Reducing agent:
10-100 mM DTT or 1.0-0.3 M b-mercaptoethanole
Denaturation:
6 M guanidium HCl or 8 M urea
Reduction: 2-20 h, RT
Purification: acidify, then gel filtration or HPLC
Basic methods for disulfide bond formation
SX
SX
SX
cleavage
-2X
SH
SX
I2 oxidation
- 2 XI
SH
selective
cleavage
-Y
1.
2.
3.
4.
S
Air oxidation
DTNB (Ellman’s Reagent)
K3Fe(CN)6
DMSO/1M HCl
d+SZ
SH
d-
oxidation
-2H
S
SZ
SY
- HZ
S
S
1. iodine oxidation
2. Tl(tfa)3/TFA
3. TCMS/DPSO
S
S
1. Cys(Npys)
2. Cys(SEt)
3. Cys(SPyr)
Andreu et al.: Methods in molecular Biology Vol. 35: Peptide Synthesis Protocols
(Eds.: M.W. Pennington and B.M. Dunn), Humana Press Inc. Totowa, NJ (1994) pp. 91-170
Disulfide bond formation in case of free cysteines
1. K3Fe(CN)6: duVigneud et al.: J Biol Chem 237, 1563-1566 (1962)
Synthesis of oxitocin and its derivatives:
H-Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2
 Dissolve the peptide at a concentration of 0.1-1 mg/mL in buffer at pH 7-8;
 Add 20% excess of 0.01 M K3Fe(CN)6 solution to the mixture during 30 min;
 Adjust the pH to 5 with AcOH.
The procedure is good for the preparation of cyclic peptides with
one disulfide bond and small size of cycle
The efficacy of the cyclisation (disulfide bond formation) can be checked with
Ellman’s Reagent: 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB)
Ellman: Arch Biochem Biophys 82, 70-77 (1959)
Ellman’s Reagent
COOH
O2N
COOH
S
NO2
S
HS-R
O2N
COOH
S
S-R
+
If there is a free thiol group, the solution
has yellow colour.
NO2
S
COOH
(yellow)
2. Ellman’s Reagent (DTNB) can be used for disulfide bond formation:
COOH
O2N
S
S
NO2
COOH
R-S-S-R
+
2 HS-R
2 S
NO2
COOH
Application of solid phase Ellman’s Reagent for disulfide bond formation
O2N
S
OC
NH
R
HN
NH
OC
NO2
S
CO
(Lys)
R = PEG-PS
JACS 120, 7226 (1998)
• Dissolve the peptide in 0.1 M Na2HPO4
buffer, pH 6.6
• buffer:CH3CN:CH3OH = 2:1:1.3 (v/v/v)
(0.8 mM peptide concentration)
• 50 mg solid phase Ellman’s Reagens
( ~ 15 equiv to SH)/DCM
• washing DMF 2x1min, DCM 1x1 min
• 2-5 h stirring at 25oC
• filtration
• lyophilization
Advantages:
The reaction sites are far from each other, intramolecular reaction is prefered .
The resin can be reactivated.
3. Random air oxidation:
Usually for multiple disulfide bond formation in the same time (slow oxidation);
Peptide concentration: 0.1-0.5 mg/mL, 4-25oC, 24-120 h, pH 7.5-8.5 buffer solution;
Additives: Guanidium.HCl (against aggregation),
Cys, mixture of oxidized-reduced (10:1, mol/mol) glutathione (improving mechanism)
4. DMSO/1M HCl oxidation:
Acidic solution usually prevents the refolding of disulfide isomers;
Especially for the formation of one disulfide bond;
Not for peptides with Cys at the N-terminus (DMSO oxidation in neutral solution);
No extra reduction with DTT after Hg- or Ag-salt cleavage step.
Disulfide bond formation in one step with deprotection of cysteines
A) Fuji et al.: Chem Pharm Bull 35 2339-2347 (1987)
SX
1.2 equiv. Tl(tfa)3/TFA
2 % anisol
0oC, 1-2 h
SX
-tfa-+X
S
SX
Tl
tfa
B) Akaji et al.: J Chem Soc Chem Commun 1991, 167-169
SX
SX
Ph2SO + MeSiCl3
-MeSiCl2O-+X
[MeSiCl2OSPh2]+-Cl
S
SX
S+
tfa
Ph
Ph
-tfa-+X
S
S
-Ph2S
S
S++X
Tl
-X+
tfa
-Tl(tfa)
X(A): Acm, Mob,
tBu, Trt, Tmob
S
S
X(B): Acm, Mob, Meb,
tBu, Tacm
S
S
Synthesis of a-conotoxin-HSV chimeric peptide
Mező et al.: J. Pept. Res. 55, 7-17 (2000)
276SALLEDPVGTVA287
H-ECCNPACGRHYSC-NH2
HSV gD-1 epitope
a-conotoxin GI
H-YCCNPACGDPVGC-NH2
a-conotoxin-HSV chimera
Chimeric peptides are unnatural constructs consisting of bioactive compounds from
at least two different peptide(s) and/or proteins or two sequences from different
parts of the same protein.
Fmoc-Y(tBu)C(Acm)C(Trt)N(Trt)PAC(Acm)GD(OtBu)PVGC(Trt)- R
1. 20% piperidine/DMF
2. 95% TFA-5%EDT
H-YC(Acm)CNPAC(Acm)GDPVGC-NH2
A) air oxidation or B) DTNB at pH 8.3
H-YC(Acm)CNPAC(Acm)GDPVGC-NH2
1 loop (3-13)
C)
D)
E)
I2/MeOH or AcOH
1. AgOTf/TFA; 2. 50%DMSO/1M HCl (V/V)
Tl(tfa)3/TFA
2 loops (2-7, 3-13)
H-YCCNPACGDPVGC-NH2
Synthesis of a-conotoxin MI
with Acm and tBu protecting groups on Cys residues
Akaji et al.: J Chem Soc Chem Commun 1991, 167-169, Tetrahedron Lett. 33, 1073-1076 (1992)
Fmoc-GR(Pbf)C(tBu)C(Acm)H(Trt)PAC(tBu)GK(Boc)N(Trt)Y(tBu)S(tBu)C(Acm)- R
1. 20% piperidine/DMF
2. TFA-scavangers
H-GRC(tBu)C(Acm)HPAC(tBu)GKNYSC(Acm)-NH2 (I)
Oxidation with iodine
20 equiv I2 in CH3OH-water (4:1, v/v), 15 min 25oC
1 mM peptide concentration
H-GRC(tBu)CHPAC(tBu)GKNYSC-NH2
(II)
Oxidation with Ph2SO (10 eq)-CH3SiCl3 (150 eq)
in TFA, 10 min, 25oC
1 mM peptide concentration
H-GRCCHPACGKNYSC-NH2
R = Rink-Amide MBHA resin
(III)
Synthesis of a-conotoxin GI
with Acm and tBu protecting groups on Cys residues
Fmoc-Glu(OtBu)-Cys(Acm)-Cys(tBu)-Asn(Trt)-Pro-Ala-Cys(Acm)-Gly-Arg(Pbf)-His(Trt)-Tyr(tBu)-Ser(tBu)-Cys(tBu)- R
1.
2.
2%piperidine-2%DBU/DMF (2+2+5+10 min)
TFA-phenol-thioanisole-EDT-water
(10ml:0.75g:0.5mL:0.25mL:0.5mL), 90 min, RT
H-Glu-Cys(Acm)-Cys(tBu)-Asn-Pro-Ala-Cys(Acm)-Gly-Arg-His-Tyr-Ser-Cys(tBu)-NH2
Oxidation with iodine
5equiv I2 in AcOH-water (4:1, v/v), 1h RT
1 mM peptide concentration
H-Glu-Cys-Cys(tBu)-Asn-Pro-Ala-Cys-Gly-Arg-His-Tyr-Ser-Cys(tBu)-NH2
Oxidation with Ph2SO (10 equiv)-CH3SiCl3 (150 equiv)
in TFA, 20 min, RT
1mM peptide concentration
H-Glu-Cys-Cys-Asn-Pro-Ala-Cys-Gly-Arg-His-Tyr-Ser-Cys-NH2
a-conotoxin GI/2
a-conotoxin GI/1
18.9 min
3500
17.2 min
1000
3000
intensity, cps
intensity, cps
800
2500
2000
1500
600
LCPackings, C18, 300 mm, 3 m, 15 cm,
A: 2% ACN, 0.1% TFA, B: 95% ACN, 0.1% TFA
0 min: 5%B, 2 min: 5 %B, 30 min: 50%B
5mL/min, 5 pmol peptide, 5mL, partial loop
400
1000
200
500
0
0
0
10
20
30
0
10
time, min
[M+H]+
20000
12000
intensity, cps
intensity, cps
1437.6
[M+H]+
20000
8000
12000
4000
0
1428
8000
1432
1436
1440
1444
1448
m/z
intensity, cps
intensity, cps
30
1437.6
16000
16000
20
time, min
1452
15000
15000
10000
5000
10000
0
1428
1432
1436
1440
1444
1448
m/z
5000
4000
0
0
1000
1500
m/z
2000
2500
1000
1500
2000
m/z
Voyager DE-Pro MALDI, reflectron mode, CCA matrix (10 mg/mL, ACN:H2O=1:1, 0.1% TFA)
2500
1452
Enzymatic degradation using subtilysin
a-conotoxin GI/2
a-conotoxin GI/1
ECCNPACGRHYSC
ECCNPACGRHYSC
ECCNPACGR
SC
3
CGRHYSC
3
4,0x10
4
2,0x10
+H2O
3
2,0x10
0,0
600
900
1200
m/z, Th
1500
intensity, cps
intensity, cps
6,0x10
4
1,5x10
4
1,0x10
ECCNPA
3
5,0x10
0,0
600
900
1200
m/z, Th
1500
H-Glu-Cys(Acm)-Cys(tBu)-Asn-Pro-Ala-Cys(Acm)-Gly-Arg-His-Tyr-Ser-Cys(tBu)-NH2
Oxidation with Ph2SO (10 equiv)-CH3SiCl3 (150 equiv)
in TFA, 20 min, RT
1mM peptide concentration
H-Glu-Cys-Cys-Asn-Pro-Ala-Cys-Gly-Arg-His-Tyr-Ser-Cys-NH2
Catalytical amount of Cys
in Tris-buffer at pH 8.0
1h, RT
H-Glu-Cys-Cys-Asn-Pro-Ala-Cys-Gly-Arg-His-Tyr-Ser-Cys-NH2
Synthesis of humane insuline
E Q C C T S I C S L Y Q L
V
E N
G I
A-21
Y
S
C N
S
S
G S H L V E
C
S
Q H L
A L Y
B-1
V N
L V
F
C
G
E
R
Biosynthesis in Langerhans-Insel
G
of pancrease. It regulates the level
F
of blood sugar content
F
B-30
T Y
P
K
T
A-1
Insulin is derived from proinsuline (1 peptide chain, 84 amino acid);
Insuline with hormone activity is formed after cleavage of connecting (C)-peptide;
Two intermolecular and one intramolecular disulfide bridges;
Selective disulfide bond formation is necessary in chemical synthesis;
A-chain
OCH 3
tBu
Fmoc-Thr-O
H 3CO
-HN CH
Fmoc-Asp-OtBu
OCH2-R
O CH2
CH2
P
1. SPPS
2. 1M HBF4-thioanisole
1. SPPS
2. TFA-scavangers
tBu
B-chain
SH
Acm
H-FVNQHLCGSHLVEALYLVCGERGFFYTPKT-OH
tBu
H-GIVEQCCTSICSLYQLENYCN-NH2
N
SH
Acm
Acm
S S
N
SPy
H-FVNQHLCGSHLVEALYLVCGERGFFYTPKT-OH
Mix in 8M urea cont. buffer (pH 8.5)
tBu
tBu
H-GIVEQCCTSICSLYQLENYCN-NH2
Acm
Acm
S
S
H-FVNQHLCGSHLVEALYLVCGERGFFYTPKT-OH
Iodine oxidation
tBu
tBu
H-GIVEQCCTSICSLYQLENYCN-NH2
S
Acm
Iodine oxidation
S
Acm
H-FVNQHLCGSHLVEALYLVCGERGFFYTPKT-OH
tBu
tBu
H-GIVEQCCTSICSLYQLENYCN-NH2
CH3SiCl3
S
S
Ph2SO
S
S
H-FVNQHLCGSHLVEALYLVCGERGFFYTPKT-OH
S
S
H-GIVEQCCTSICSLYQLENYCN-NH2
S
S
S
S
H-FVNQHLCGSHLVEALYLVCGERGFFYTPKT-OH
Structural determination by thermolysin degradation
Main cleavage sites of thermolysine are X-Leu and X-Phe
Synthesis of (225-232/225’232’) bis-cystinyl fragment
of hinge region of humane IgG1
E. Wünsch, L. Moroder et al.: Int J Pept Prot Res, 32, 368-383 (1988)
H-Thr-Cys-Pro-Pro-Cys-Pro-Ala-Pro-OH
H-Thr-Cys-Pro-Pro-Cys-Pro-Ala-Pro-OH
• synthesis in solution by fragment condensation using Boc-chemistry;
• air oxidation after removal of protecting groups;
• ca. 90% yield of the parallel cyclic dimer peptide.
Synthesis of antiparallel cyclic dimer peptide with selective protecting groups
resulted in 50% antiparallel and 10% parallel cyclic dimer peptides.
When the antiparallel compound was kept in reductive buffer solution
(peptide:phosphine 10:1 mol/mol; 0.1M NH4OAc; pH 6.8), in 5 days 90% of parallel
derivative was present in the reaction mixture.
The influence of thiols on the folding pattern:
Cys, reduced glutation, extra Cys in the sequence.
Glutation: g-L-glutamyl-L-cistenyl-glycine
Nps-Thr(tBu)-Cys(Acm)-Pro-Pro-Cys(StBu)-Pro-Ala-Pro-OH
Bu3P
H-Thr(
tBu)-Cys(Acm)-Pro-Pro-Cys(SH)-Pro-Ala-Pro-OH
Boc-N=N-Boc (di-tert-butyl azodicarboxilate)
H-Thr(
tBu)-Cys(Acm)-Pro-Pro-Cys(Boc-N-NH-Boc)-Pro-Ala-Pro-OH
+
t
HO-Pro-Ala-Pro-Cys(Acm)-Pro-Pro-Cys(SH)-Thr( Bu)-H
H-Thr(
tBu)-Cys(Acm)-Pro-Pro-Cys-Pro-Ala-Pro-OH
HO-Pro-Ala-Pro-Cys(Acm)-Pro-Pro-Cys-Thr(
tBu)-H
10 equiv I2 in 80% AcOH
H-Thr(
tBu)-Cys-Pro-Pro-Cys-Pro-Ala-Pro-OH
HO-Pro-Ala-Pro-Cys-Pro-Pro-Cys-Thr(
tBu)-H
TFA/anisole-methylindole
H-Thr-Cys-Pro-Pro-Cys-Pro-Ala-Pro-OH
HO-Pro-Ala-Pro-Cys-Pro-Pro-Cys-Thr-H
On-resin disulfide bond formation
(synthesis of oxitocine)
Boc-Cys(Fm)-Tyr(BrZ)-Ile-Gln-Asn-Cys(Fm)-Pro-Leu-Gly- MBHA
piperidine-DMF-ethylmercaptane
(10:10:0.7, v/v), 3h, RT
0.35 mmol/g
Boc-Cys-Tyr(BrZ)-Ile-Gln-Asn-Cys-Pro-Leu-Gly- MBHA
oxidation
Boc-Cys-Tyr(BrZ)-Ile-Gln-Asn-Cys-Pro-Leu-Gly- MBHA
1. 50%TFA/DCM
2. HF-anisole (9:1, v/v)
H-Cys-Tyr(BrZ)-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2
Oxidation: 1. Air oxidation in DMF at pH 7 for 1h
2. Air oxidation in DMF at pH 8 for 1h
3. DTNB (0.5 equiv) in DMF at pH7.5 for 2h
4. diethyl-azodicarboxilate in DMF at pH7.5 for 2h
60% yield
63% yield
65% yield
24% yield
Cyclisation via thioeter bond formation
Advantages of thioether bond:
 application of non-protected peptide precursors
(vs. amide bond formation)
 chemically and biologically stable bond (vs. disulfide bridge)
 easy coupling (between ClAc and SH groups), good yield
(usually better than in case of amide or disulfide bond formation)
Disadvantages:
 coupling is carried out under slightly alkaline conditions (pH 8.0-8.5)
 Cys containing peptides can dimerize (especially Cys at N-terminus)
 very active BrAc derivatives can be used effectively only when no other
nucleophiles are present except Cys
Boc-LK(ClZ)MAD(OcHex)PNR(Tos)FR(Tos)GK(ClZ)D(OcHex)LAhxK(Fmoc)AhxC(Meb)-MBHA
2%DBU + 2% piperidine in DMF, 2+2+5+10 min
Boc-LK(ClZ)MAD(OcHex)PNR(Tos)FR(Tos)GK(ClZ)D(OcHex)LAhxKAhxC(Meb)-MBHA
1. Fmoc synthesis; Fmoc-Aaa-OH/DIC/HOBt (3equiv)
2. Acetylation of the N-terminus; Ac2O/DIEA in DMF
Boc-LK(ClZ)MAD(OcHex)PNR(Tos)FR(Tos)GK(ClZ)D(OcHex)LAhxKAhxC(Meb)-MBHA
Ac-C(Acm)GFLG1. deBoc; 33% TFA/DCM, 2+20 min
2. ClAcOPcp in DMF, 2-3h
ClAc-LK(ClZ)MAD(OcHex)PNR(Tos)FR(Tos)GK(ClZ)D(OcHex)LAhxKAhxC(Meb)-MBHA
Ac-C(Acm)GFLGHF-p-thiocresol-m-cresol (10ml:0.5g:0.5ml), 90min, 0oC
ClAc-LKMADPNRFRGKDLAhxKAhxC-NH2
Ac-C(Acm)GFLGCyclization in 0.1M Tris buffer (pH 8.0), 3-4h, RT
CH2CO-LKMADPNRFRGKDLAhxKAhxC-NH2
Ac-C(Acm)GFLG-
Synthesis of cyclic dimers and conjugates
containing cyclic epitope peptides
CH2CO-LKMADPNRFRGKDLAhxKAhxC-NH2
Ac-C(Acm)GFLG-
I2 or Tl(tfa)3
oxidation
Ag-triflate
CH2CO-LKMADPNRFRGKDLAhxKAhxC-NH2
DTT
Ac-CGFLGAc-CGFLGCH2CO-LKMADPNRFRGKDLAhxKAhxC-NH2
Dimer of cyclic peptide
M: 5316
O2
CH2CO-LKMADPNRFRGKDLAhxKAhxC-NH2
Ac-CGFLGAc-CGFLGCH2CO-LKMADPNRFRGKDLAhxKAhxC-NH2
Ac-[TKPKG]4-NH2
CH2CO
Ac-CGFLG-
CH2CO-LKMADPNRFRGKDLAhxKAhxC-NH2
carrier
Conjugate containing 4 cyclic
epitope peptides
112 amino acid residues
(M: 12902)
Intens.
x106
4
5 min
4+
665.7
3
1.2
2
3+
887.1
28.3
1.0
1
MW calc.: 2659.22
0.8
Abs220 nm
MW exp.: 2658.60
0
19.5
0.6
250
500
750
1000
1250
1500
1750
2000
2250
m/z
0.4
0.2
0.0
4+
603.7
Intens.
x107
10
20
30
40
50
4
Time (min)
3
3+
804.5
2
1
MW calc.: 2410. 48
2+
1206.0
MW exp.: 2410.60
0
250
500
750
1000
1250
1500
1750
2000
2250
m/z
3h
27.5
11+
12+ 935.5
857.6
MW calc.:10279.76
MW exp.:10279.50
0.6
0.75
0.4
10+
1028.9
13+
791.8
0.50
14+
735.3
9+
1143.1
0.0
0.00
400
500
600
700
800
900
1000
1100
25.6
0.2
0.25
29.4
Abs220 nm
1.00
26.9
1.25
0.8
27.8
Intens.
x107
28.4
1.0
1200
1300
1400
16
m/z
18
20
22
24
26
28
30
32
34
36
38
40
Time (min)
„2-copies epitope“ conjugate
Intens.
10+
766.6
Intens.
x107
7+
720.1
x106
3.0
8
2.5
6
6+
839.9
2.0
11+
697.1
9+
851.7
1.5
4
8+
630.2
1.0
2
5+
1007.7
MW calc.: 5034.24
12+
639.1
0.5
8+
958.0
MW exp.: 5033.50
MW calc.: 7657.00
MW exp.: 7656.30
0.0
0
400
600
800
1000
1200
1400
m/z
400
600
800
1000
1200
1400
m/z
12+
1076.2
Intens.
x107
24 h
2.5
MW calc.: 12902.52
MW exp.: 12902.20
2.0
2.0
27.8
11+
1173.9
1.8
13+
993.5
1.5
1.6
1.0
1.4
29.4
14+
922.6
15+
861.2
0.5
1.0
0.8
10+
1291.1
9+
1434.5
0.0
600
800
1000
1200
1400
1600
m/z
0.6
28.4
Abs220 nm
1.2
0.4
0.2
0.0
10
20
30
Time (min)
40
50
Intens.
x107
1.0
7+
760.4
6+
887.0
0.8
0.6
8+
665.5
0.4
5+
1064.1
0.2
MW calc.: 5316.42
9+
591.6
MW exp.: 5315.80
0.0
400
600
800
1000
1200
1400
m/z
Enzymatic cleavage of linear and cyclic peptides
derived from 278-287 region of HSV gD-1
100%
H-LLEDPVGTVA-NH2
50% human serum
c(LLEDPVGTVA)
60%
H-c(CLLEDPVGTVAC)-NH2
40%
20%
c(CH2CO-LLEDPVGTVAC)-NH2
0%
0
24
48
72
96
Time (hours)
H-LLEDPVGTVA-NH2
100%
lysosoma
Peptide (%)
Peptide %
80%
80%
c(LLEDPVGTVA)
60%
H-c(CLLEDPVGTVAC)-NH2
40%
20%
c(CH2CO-LLEDPVGTVAC)-NH2
0%
0
60
120
180
Time (min)
Tugyi, R., Mező, G., et al. J. Peptide Science 11, 642-649
Formation of thiazolidine or oxime ring
1. Oxidation of Ser to a-ketoaldehyde:
-Lys-
H2N
NaIO4
pH 6.8, 2min
O
HO
O
-LysO
H
2. Reaction of N-terminal Cys with aldehyde (thiazolidine ring formation):
H2C=O +
SH
-H2O
CH2
HNH-CH-CO- - -
S
CH2
H2C
NH-CH-CO- - -
3. Reaction of hydroxylamine with aldehyde (oxime formation):
R-CH2-O-NH2
+
H2C=O
-H2O
R-CH2-O-N=CH2
Multiple cyclic antigen peptide
Spetzler, J.C., Tam, J.P. Peptide Research 9, 290 (1996)
Fmoc
Fmoc-Lys(Fmoc)-Lys(Fmoc-Lys(Fmoc))-Ser-Ser-b-Ala- R
Fmoc
Fmoc
=
Fmoc
Synthesis by
Fmoc chemistry
MAP core
Fmoc-Cys(StBu)- Antigen -Lys(Mtt)-Asp(OtBu)-ProFmoc-Cys(StBu)- Antigen -Lys(Mtt)-Asp(OtBu)-ProFmoc-Cys(StBu)- Antigen -Lys(Mtt)-Asp-(OtBu)ProFmoc-Cys(StBu)- Antigen -Lys(Mtt)-Asp(OtBu)-Pro-
Fmoc-Cys(StBu)- Antigen -Lys(Fmoc-Ser(tBu))-Asp(OtBu)-ProFmoc-Cys(StBu)- Antigen -Lys(Fmoc-Ser(tBu))-Asp(OtBu)-ProFmoc-Cys(StBu)- Antigen -Lys(Fmoc-Ser(tBu))-Asp(OtBu)-ProFmoc-Cys(StBu)- Antigen -Lys(Fmoc-Ser(tBu))-Asp(OtBu)-Pro-
1. 1%TFA-5%TIS
2. Fmoc-Ser(tBu)-OH
DIC/HOBt in DMF
Fmoc-Cys(StBu)- Antigen -Lys(Fmoc-Ser(tBu))-Asp(OtBu)-Pro1. 20% piperidine/DMF
2. TFA/TIS/thioanisole/water(92.5:2.5:2.5:2.5, V/V)
NH2-Cys(StBu)- Antigen -Lys(NH2-Ser)-Asp-ProNaIO4 in 10mM PBS solution (pH 6.8)
then HPLC purification
OH
NH2-Cys(StBu)- Antigen -Lys(O=CH-CO)-Asp-ProTris-(2-carboxyethyl)phosphine
10mM Na-acetate buffer (pH 4.2), Rt, 48h
S
NH
CO
OH
Antigen = peptide derived from
V3 loop of gp120 HIV
Antigen -Lys-Asp-ProOH
Cyclisation in solution by oxime bond formation
Pallin, TD, Tam, JP. J. Chem Soc. Chem. Commun. 1995, 2021-2022
NH-Gly-Ile-Gly-Pro-Gly-Arg(Pmc)-Ala-Phe-Gly-Lys-bAla-O- Wang
OC-CH2-O-NHBoc
BocHN
HO
O
TFA/scavangers
NH-Gly-Ile-Gly-Pro-Gly-Arg-Ala-Phe-Gly-Lys-bAla-OH
H2N
OC-CH2-O-NH2
O
HO
NaIO4 in buffer solution, pH 6.8; 2 min
NH-Gly-Ile-Gly-Pro-Gly-Arg-Ala-Phe-Gly-Lys-bAla-OH
O
OC-CH2-O-NH2
O
H
NH-Gly-Ile-Gly-Pro-Gly-Arg-Ala-Phe-Gly-Lys-bAla-OH
N
OC-CH2-O
O
H
Backbone cyclisation
Byk, G., Gilon, Ch. J. Org. Chem. 57, 5687 (1992)
Application of side chain amino-, carboxyl- or thiol groups in cyclisation
may inactivate the peptide
Solution: N-backbone cyclisation (N in amide bond)
R1
N
H
N
O
O
R2
R3
N
H
N
O
R1
O
R4
N
H
O
N to N backbone
R1
N
H
O
N
H
O
R2
R3
N
H
N
H
O
R2
R3
N
H
O
N
O
R4
N backbone to N-terminal
N
O
R1
O
R4
N
H
N backbone to side chain
= -R-S-S-R’-; -R-CO-NH-R’-; -R-CH2-S-R’-
N
O
O
R2
R3
N
H
O
N
H
N backbone to C-terminal
O
R4
Building-blocks
NH2-(CH2)n-NH2 + Br- CHR-COOH
Boc-NH-(CH2)n
Fmoc-N-CHR-COOH
Fmoc-chemistry
NH2-(CH2)n-NH-CHR-COOH
Fmoc-NH-(CH2)n
Boc-N-CHR-COOH
Boc-chemistry
On-resin acylation of N-alkylated amino acid derivatives is very difficult.
Dipeptide formation in solution and then it can be attached to the peptide-resin.
Z-NH-(CH2)n
Boc-NH-CHR-CO-N-CHR’-COOH
BzlO-CO-(CH2)n
Boc-NH-CHR-CO-N-CHR’-COOH
Bzl-S-(CH2)n
Boc-NH-CHR-CO-N-CHR’-COOH
Preparation of building blocks
is quite complicated
Reductive alkylation
Kaljuste, K., Undén, A. Int. J. Pept. Prot. Res. 43, 505-511 (1994)
NH2-APK(ClZ)Y(BrZ)- MBHA
Boc-Val-al
NaCNBH3
Boc-VY[CH2NH]APK(ClZ)Y(BrZ)- MBHA
S(Meb)-3-mercaptopropanal,
NaCNBH3
Boc-VY[CH2N]APK(ClZ)Y(BrZ)- MBHA
1. Boc-Aaa-OH derivatives
2. Boc-Leu-al, NaCNBH3
3. S(Meb)-3-mercaptopropanal,
NaCNBH3
CH2CH2CH2-S-Meb
Boc-LY[CH2N]S(Bzl)PGK(ClZ)VY[CH2N]APK(ClZ)Y(BrZ)- MBHA
CH2CH2CH2-S-Meb
CH2CH2CH2-S-Meb
1. TFMSA/DMS, 2. HF
H-LY[CH2N]SPGKVY[CH2N]APKY-NH2
Meb-S-CH2CH2CH2
Reduced peptide bonds;
Missing CO-NH, hydrogen bonds?
Influence on the structure.
CH2CH2CH2-S-Meb
oxidation
H-LY[CH2N]SPGKVY[CH2N]APKY-NH2
CH2CH2CH2-S CH2CH2CH2-S