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Solid phase peptide synthesis
Part I
Applications of Boc/Bzl strategy
Gábor Mező
Research Group of Peptide Chemistry
Hungarian Academy of Sciences
Eötvös L. University
Budapest, Hungary
Outline
 Why peptide synthesis is necessary
Solid phase peptide synthesis
(idea, comparison with the synthesis in solution);
 Resins;
 Protecting groups;
 Synthetic protocol;
 Monitoring;
 Cleavage procedures;
 Side reactions;
Immune peptides:
synthetic antigens;
vaccines
diagnostic tools
immunostimulator peptides;
muramyl dipeptide
tuftsin derivatives
Transporter peptides:
penetratin
oligoarginine
HIV-Tat protein
Peptides
for structural studies:
turn mimicking cyclic peptides
Hormones:
oxytocin
vasopressin
insulin
somatostatin
GnRH
etc.
Applications of
synthetic peptides
Carriers:
templates
miniproteins
Neuropeptides:
substance P
cholecystokinin
neurotensin
Antibiotics:
tachikinin
gramicidine S
Toxins:
conotoxins
spider toxins
snake toxins
ionchanel blockers
Enzymes and
enzyme inhibitors:
Ribonuclease A
Why chemists are needed?
Gene expression is very popular, relatively easy and cheap method:
it is good for long linear peptides or proteins containing L-amino acids.
However:
 no D-amino acids
 no unnatural amino acids
 no post translation (Hyp, Pyr, glyco- and phosphopeptides)
 no branches
 no cyclic peptides
 no fluorescent or isotop labeling
Peptides as drugs: there are not too many, because of the price and their
fast biodegradation.
“Peptides have and will continue to be important sources of lead compounds in many
drug discovery programs. However, due to their generally poor pharmacokinetic
properties and hydrolytic instability, natural peptide structures are usually
substituted with mimics of the actual peptide constuction.”
Peptidek mint gyógyszerek ?
 A peptidekhez, fehérjékhez számos biológiai és élettani funkció kapcsolható.
 Ezért a '60-as évektől a jövő gyógyszereinek gondolták.
 Előnyök: nagy specifitás, magas aktivitás, viszonylag kis dózis, kicsi toxicitás,
kevés mellékhatás.
 Hátrány: gyors lebomlás, magas költségek.
2000-ben a világ gyógyszeriparának kb. 265 milliárd USD bevételéből
28 milliárd USD a peptidek és fehérjék bevételéből származott.
Évente 35-40 új vegyület kerül gyógyszerként bevezetésre.
Ezek között a peptidek száma egyre növekszik.
Peptidek a piacon
piacon
pre-regisztrációs fázis
klinika-III
klinika-II
Rekombináns fehérjék:
>50
~40
~60
Monoklonális ellenanyagok:
>20
>20
>45
Szintetikus peptidek:
>40
>20
>60
 GnRH szuper-agonisták és antagonisták:
tumor terápia
 Szomatosztatin analógok:
tumor terápia
 ACE (angiotenzin konvertáló enzim) inhibitorok:
vérnyomás szabályozás
 HIV proteáz inhibítorok:
AIDS ellen
 Vazopresszin, Oxitocin, ACTH:
hormonok
 Kalcitoninok:
oszteoporózis ellen
 Immunstimuláló peptidek:
szervezet védekező
képességének növelése
A. Loffet J. Peptide Science (2002) 8, 1-7.
PEPTIDE SYNTHESIS
Coupling of amino acids:
NH2-CH(R)-COOH + NH2-CH(R’)-COOH
- H2O
NH2-CH(R)-CO-NH-CH(R’)-COOH; NH2-CH(R’)-CO-NH-CH(R)-COOH;
NH2-CH(R)-CO-NH-CH(R)-COOH; NH2-CH(R’)-CO-NH-CH(R’)-COOH;
+ oligomers and polymers with different composition
Protecting groups: amino-; carboxyl-; side chain protecting groups
X-NH-CH(R)-COOH + NH2-CH(R’)-COOY
- H2O
X-NH-CH(R)-CO-NH-CH(R’)-COOY;
Removal of the protecting groups together or selectively
Synthesis in solution
Synthesis on resin (SPPS)
 time consuming;
 fast;
 manual;
 synthesizer (or manual);
 1.1-1.2 equiv amino acid derivatives  3-10 equiv amino acid derivatives
and coupling reagent for acylation;
and coupling reagents for acylation;
 side chain protecting groups for
Lys, Asp, Glu, (Cys);
 side chain protecting groups for
all functional groups
 coupling: less than 90% conversion;  coupling: over 99.5% conversion;
 purification after each steps;
 purification at the end;
 large scale;
 rather small scale;
 cheap.
 expensive.
Synthesis of ah-ACTH (1-39) in solution took months for several chemists;
A 39-mer peptide by SPPS 2 days, 1 day cleavage, 1-2 days purification,
1 week altogether for 1 chemist.
SOLID PHASE PEPTIDE SYNTHESIS
Bruce Merrifield published in 1963
Nobel Prize in Chemistry in 1984
The idea:
P
T
P
X
AA1
anchoring
R
P
P
T
AA2
P
AA1
T
AA1
R
deprotection
P
R
T
AA2
coupling (-H2O)
AA1
R
T
P
P
AA2
AA1
R
deprotection
coupling
T
repetitive
cycle
P
P
P
AAn
AA2
AA1
cleavage
+
final deprotection
AAn
AA2
AA1
R
STRATEGIES
Boc/Bzl:
CH2
Bzl
Boc
O
C
CH3
C
O
HF
CH2
H3C
2,6-Cl2Bzl
Cl
Cl
O
C
O
O
CH2
NH
CH
CH2
C NH
O
CH3
TFA
CH2
C
O
NH
CH
C
O
CH2
O
HF
Boc-Asp(OBzl)-Gly-Tyr(2,6-Cl2Bzl)-Merrifield resin
R
Fmoc/tBu:
H3C
tert-butyl
Fmoc
C
H3C
H3C
C
H3C
CH3
TFA
O
CH3
R
C
O
C O
O
C
H
..
NH
CH2
O
C
H2C
NH
CH
H2C
C
O
NH
CH
C
O
CH2
O
CH2
O
Wang-resin
piperidine
Fmoc-Asp(OtBu)-Tyr(tBu)-Wang resin
RESINS
 Can be functionalised;
 Chemical stability (it must be inert to all applied chemicals);
 Mechanical stability (it shouldn’t brake under stirring);
 It must swell extensively in the solvents used for the synthesis;
 Peptide-resin bond should be stable during the synthesis;
 Peptide-resin bond can be cleaved effectively at the end of the synthesis;
The basic of the most common used resins:
polystyrene-1,4-divinylbenzene (1-2%) copolymer
+
polymerisation
Type of resins for Boc-chemistry
Merrifield (chloromethyl) resin
Cl CH2
Aminomethyl resin
NH3
P
H2N
CH 2
P
Starting resin for the synthesis
of many other resins
O
HO CH2
O
HO CH2
CH2 C
NH
CH2
PAM resin (phenyl-acetamidomethyl)
P
CH2 C
OH
p-hydroxymethyl-phenylacetic acid (handle)
+ DIC
Attachment of the first amino acid to Merrifield and PAM resins
Cl CH2
Boc-Aaa-O-Cs+
P
DMF, 50oC, 48h
Boc-Aaa-O CH2
O
HO CH2
CH2 C
NH
CH2
P
P
Boc-Aaa-OH
DIC + 10%DMAP
DCM-DMF (1:3)
RT
1h
O
Boc-Aaa-O CH2
O
Boc-Aaa-O
CH2
CH2 C
OH
CH2 C
DIC + H2N CH2
NH
CH2
P
Peptide-PAM resin bond is more TFA stable than Peptide-Merrifield resin bond.
The final cleavage
at the C-terminus.
results in peptides with carboxyl (COOH) group
P
Benzhydrylamine resin (BHA): too stable under acid cleavage conditions
(only; HF!)
Boc-Aaa-OH
H2N
CH
DCC/HOBt
P
O
Boc NH CH(R) C
NH CH
P
4-Methyl-benzhydrylamine resin (MBHA):
CH3
H3C
Boc-Aaa-OH
H2N
CH
The final cleavage
the C-terminus.
P
DCC/HOBt
O
Boc NH CH(R) C
NH CH
results in peptides with carboxamide (CONH2) group at
P
Coupling capacity of the resin
 Preloaded resins are commercially available
(coupling capacity, written on the box is expressed in mmol/g);
 BHA and MBHA resin (the NH2 content is given on the box)
Attachment of the first amino acid is usually performed with
100% yield; the resin capacity will be the same;
 Coupling of p-hydroxymethyl-phenoxy acetic acid containing Bocamino acid to aminomethyl-resin represents a similar situation;
 Attachment of Boc-amino acid derivative to Merrifield or PAMresin (Kjeldahl N analysis, elemental analysis, amino acid analysis
or titration by pycric acid after Boc-removal: colour test)
Kjeldahl N analysis:
Amino acid analysis:
 cc. H2SO4 for 24 hrs
 add base
 NH3 destillation into water
 titration with 4mM H2SO4
 calculation of % N to mmol/g
 Lys (2N), His (3N), Arg (4N)
 6M HCl in an evacuated and
stopped tube (hydrolysis)
 heating at 110oC for 24 hrs
 evaporation, neutralisation
 amino acid analysis
(quantitative)
Applied side chain protecting groups in Boc-chemistry
Side chain functional group
protecting group
-OH (Ser, Thr, Tyr)
CH2
However in case of Tyr:
H
OR
name (abbreviation)
benzyl (Bzl)
H
+ R
O
O
R+
HF
CH2
NH
CH
CH2
C
NH
CH
O
CH2
C
O
CH
C
O
intermolecular
intramolecular
20-100% side product!
OH
The side reaction
can not be avoided
by using scavangers.
Mw: +90.05
CH2
R+ can be caught by scavangers
R
NH
NH
CH
C
O
Side chain functional group
protecting group
Cl
2,6-dichlorobenzyl
(2,6-di-Cl-Bzl)
CH2
-OH (Tyr)
name (abbreviation)
Cl
O
N acyl shift;
do not keep the peptide
without a-NH protection
for long time !
O
C O CH2
Br
It is not commercially available
Electrophilicity order of carbocations:
But < 2-Br-Z < cHex < 2,6-di-Cl-Bzl < Bzl
0,05% 0,1% 0,5%
5,0%
20%
(amount of 3-alkyltyrosine in the peptide)
2-bromobenzyloxycarbonyl
(2-Br-Z)
cyclohexyl
(cHex)
Side chain functional group
-SH (Cys)
Stability vs TFA is
not good enough;
not for longer peptides!
For selective deprotection
protecting group
name (abbreviation)
CH2
CH3
4-methylbenzyl
(Meb)
CH2
OCH3
4-methoxylbenzyl
(Mob)
CH2 NH C CH3
acetamidomethyl
(Acm)
O
Eg.
Meb
--C----C---C------C--Acm
HF
Acm
S
--C----C---C------C--S
SH
--C----C---C------C---
Acm
S
S
SH
Meb
I2 or Tl(tfa)3
Hg(II)- or Ag(I)-salt !
Acm
S
air
oxidation
S
--C----C---C------C--Acm
Acm
Side chain functional group protecting group
NO2
-SH (Cys)
S
name (abbreviation)
3-nitro-2-pyridinesulphenyl
(Npys)
N
stable in the presence of acids
cleavable by bases
or thiols
Not for Fmoc-chemistry!
For the synthesis of asymmetrical disulfide dimers
Eg.
Npys
--------C-------R
HF
Npys
--------C-------OH
Bzl
--------C-------OH
S
S
--------C-------OH
---C-----NH2
S
S
---C-----NH2
+
SH
in acidic buffer
---C-----NH2 (pH 5-6)
--------C-------OH
at pH > 7
S
S
---C-----NH2
Neutral or basic
condition is not
appropriate for
asymmetrical
disulfide bond
formation!
Side chain functional group protecting group
eNH (Lys)
2
Z is not stable enough
in TFA;
branches in the peptide !
O
C O CH2
O
C O CH2
Cl
name (abbreviation)
benzyloxycarbonyl
(Z)
2-chlorobenzyloxycarbonyl
(2-Cl-Z)
wCOOH (Asp, Glu)
OBzl is not stable enough
in TFA;
lead to ringclosure
side reaction !
O CH2
benzyl(ester)
(OBzl)
O
cyclohexyl(ester)
(OcHex)
Succinimide ring formation (Asp):
H 2C
NH
CH
Asp-X; X = Gly, Arg, Ala, Ser, Asx
O
O
C
C
OBzl
C
NH CH2
O
C
H2C
- BzlOH
NH
N
CH
CH2 C
O
C
O
O
-Asp-Gly-
-Asu-Gly-
+H2O
+H2O
O
H 2C
NH
CH
C
C
O
OH
NH CH2
O
~30%
a-Asp-peptide
C
O
H2C
NH
C
N
CH2 C
O
CH
C
O
OH
~70%
b-Asp-peptide
Molecular weight is the same in both cases; HPLC separation of isomers in case
of small peptides; enzymatic degradation
amino acid analysis.
Pyroglutamic acid formation at the N-terminal of the peptide (Glu):
O
C
BzlO
CH2
CH2
CH2
NH2
R
CH
C
NH CH
O
C
-BzlOH
O
C
CH2
NH
CH
R
C
O
NH CH
O
C
O
M= Mcalc-18
Don’t prepare peptides containing Gln at the N-terminus
They are not present in the nature!
O
NH2
C
CH2
CH2
CH2
NH2
CH
R
C
O
NH CH
C
O
-NH3
O
C
CH2
NH
CH
R
C
NH CH
O
C
O
M= Mcalc-17
QXNAD: X= K(21%), Arg, His(18%), Ala(11%), Leu(8%), Tyr(7%), Asp(4%), Glu(2%)
After 48h at pH 7, 37oC: His(51%), Arg(32%), Leu(19%), Tyr(22%), Asp(21%)
Acidic pH, elevated temperature, X= D-Aaa; increase the Glp content
Side chain functional group
protecting group
O
wCONH (Asn, Gln)
2
O
O
NH
C
not necessary, but;
CH2
CH
C
NH CH
NH2
deBoc, deXan
CH
O
NH2
Boc
CH2
CH2
NH2
C
O
O
C
increase the solubility of
Boc-Gln-OH and Boc-Asn-OH,
eliminate the nitryl formation.
R
O
33%TFA/DCM
O
NH CH
C
DCC
CH2
CH2
NH
CH
C
OH
N
C
O
R
C
xantyl (Xan)
Why do we use Xan protecting group?
CH2
Boc-NH
name (abbreviation)
C
O
CH2
CH2
Boc
NH
CH
C
O
OH
Side chain functional group
protecting group
(His)
p
N
N
SO 2
H
t
imidazol group
p-toluolsulfonyl
or tosyl (Tos) (t)
It is too sensitive in the presence of weak acids
like HOBt, however, it is too stable in HF.
Protection of tN prevents
the alkylation or acylation
of imidazol ring, but not
the epimerisation of His;
Protection of pN prevents
also the epimerisation.
CH3
name (abbreviation)
NO2
NO2
dinitrophenyl
(Dnp) (t)
Special cleavage procedure:
thiophenol:DIEA:DMF = 3:3:4 (V/V/V)
several times; long reaction time (yellow colour)
Boc-Aaa1-Aaa2(Bzl)-His(Dnp)-....-Resin
thiophenol
Boc-Aaa1-Aaa2(Bzl)-His-....-Resin
TFA
NH2-Aaa1-Aaa2(Bzl)-His-....-Resin
HF
NH2-Aaa1-Aaa2-His-....-OH
Side chain functional group
protecting group
name (abbreviation)
(His)
p
N
N
benzyloxymethyl
(Bom) (p)
CH2 O CH2
H
t
imidazol group
Cleavage of Bom results in Bzl+ and CH2O;
Formaldehyde can react with nucleophiles:
 H2C=O + eNH2-Q
 H2C=O + OH-R
Work up the peptide as soon as possible !
H+
H2C=N-Q (Schiff base)
H2C-OH
H2C-O-R
O-R
O-R
hemiacetale
acetale
However, Bom must not be used in case of peptides containing Cys at the N-terminus:
H2C=O
SH
CH2
-H2O
HNH-CH-CO- - -
Use Cys as scavanger under the cleavage
condition to catch the formaldehyde !
S
M=Mcalc+12
CH2
H2C
NH-CH-CO- - thiazolidine-4-charboxylic acid
(thioproline)
Racemisation
H
R
N
C
H
C
R’
C
O
O
ActO
B:
R
-ActOH
N
C
O
H
C
C
R’
O
5(4H)-oxazolone
Acyl-L-Aaa-OAct
-H+
R
C
N
O
C
C
H
R’
O
+H+
R
-H+
C
Direct proton withdrawn
or oxazolone formation;
No racemisation in case
of Gly and Pro (through
oxazolone);
No racemisation with
uretane type proecting
groups (Boc, Fmoc);
His: proton transfer
N
O
C:C
R
R’
N
C
O
O
C
O:-
DL
H-L-Aaa-OY
DL and LL dipeptide derivatives
R
Pseudo aromatic
system
C
N
O
R’
C
C
C
R’
O:-
Side chain functional group
-NH-C-NH2
(Arg)
NH
guanidino group
protecting group
O
O
Lability in acids:
O
Mtr > Mts > Tos
S
Cleavage:
Tos only in HF
(TFMSA or TMSOTf at RT;
not recommended)
Mts all of them
Mtr TFA for extended time
(it was used in Fmoc-chem.)
CH3
S
CH3
CH3
O
CH3
O
CH3
OCH3
S
O
CH3
name (abbreviation)
p-toluolsulfonyl
or tosyl (Tos)
2,3,6-trimethylbenzenesulfonyl or
mesitelenesulfonyl
(Mts)
4-methoxy-2,3,6trimethylbenzenesulfonyl (Mtr)
CH3
In the synthesis of oligo-Arg (cellpenetrating peptide) use Mts or Mtr protection
Application of sulfonyl type protecting groups: the protection of Trp is suggested
Side chain functional group
protecting group
(Trp)
N
H
indole
Side reaction under
acidic condition:
H-C
O
name (abbreviation)
formyl (For)
Special cleavage is necessary:
20% piperidine/DMF before HF cleavage
or low-high HF cleavage procedure
 oxidation
 alkylation
O
C O
cyclohexyloxycarbonyl (Hoc)
Oxidation of Trp results in oxyindolyl and kynureninyl derivatives; pink colour
Alkylation by tert-butyl cation resulted under TFA cleavage of Boc-group
* M1 = Mcalc+ 56.06
*
*
N
H
*
M2 = Mcalc+ 112.12
M3 = Mcalc+ 168.18
In case of the application of Trp without
any protection, add anisole and indole as
scavangers to the TFA cleavage mixture
(10mL TFA : 0.3mL anisole : 0.1g indole) !
Side chain functional group
-CH2-CH2-S-CH3
sulfide (Met)
Side reaction under
acidic conditions:
 oxidation
 alkylation
protecting group
-CH2-CH2-S-CH3
name (abbreviation)
sulfoxide (O)
O
Removal: N-methylmercaptoacetamide, low-high HF,
NH4I, TMSOBr+thioanisole
Oxidation of Met results in its sulfoxide form.
Alkylation by benzyl or tert-butyl cation:
+
-CH2-CH2-S-CH3
-CH2-CH2-S-CH3
-CH2-CH2-S-CH2
- CH3OH
CH2
M = Mcalc + 76.03 in case of Bzl
M = Mcalc + 42.05 in case of tBu
In case of the application of Met without any protection, add anisole and
Met as scavangers as well as DTT as reductive agent to the TFA cleavage
mixture (10mL TFA : 0.3mL anisole : 0.1g Met : 0.1g DTT) !
Synthetic protocol of Boc-strategy
1)
2)
3)
4)
5)
6)
7)
(-) yellow
Wash the resin 3x with DCM; 0.5-1.0 min each
Cleavage of Boc protection with 33%TFA/DCM; 2+20min
Wash the resin 5x with DCM; 0.5-1.0 min each
(Shrinking the resin with 25%dioxan/DCM)
(+) blue
Neutralisation 3-4x with 5-10%DIEA/DCM; 1 min each
Wash the resin 4x with DCM; 0.5-1.0 min each
Coupling: Boc-amino acid derivative-DCC-HOBt in DCM-DMF *
(3 equiv each calculated to the resin capacity); 60 min
8) Wash the resin 2x with DMF; 0.5-1.0 min each
9) Wash the resin 2x with DCM; 0.5-1.0 min each
10) Ninhydrin monitoring **
* The ratio of DCM and DMF depends on the solubility of the amino acid
derivatives; DCM-DMF = 4:1 or 2:1 (V/V) in most cases.
However, in case of Arg, Gln, Asn DCM:DMF 1:4 or 1:2 (V/V) is prefered.
**When coupling is carried out to Pro, the ninhydrin assay can’t be used.
Application of isatin test or bromophenol blue test is necessary.
When might be double coupling necessary
 Incorporation of the 10-15th amino acids;
 Attachment to Pro;
 Coupling of amino acids containing a branch on b-C atom (Val, Ile, Thr);
 Attachment to these amino acids;
 Coupling of Arg or attachment to Arg;
 Attachment to e-amino group of Lys (synthesis of branched peptides).
Influence on the efficacy of the coupling:
 Solvent: change DCM-DMF to NMP (N-methyl-pyrolidone)
 Coupling reagent: change DCC/HOBt to BOP, HBTU or HATU
Application of these expensive reagents is suggested for the third coupling.
If the nynhidrine test is still blue make acetylation to block the
unreacted amino groups (acetic anhydride and DIEA in DMF).
Ninhydrin monitoring
OH
2
O
OH
O
OH
N
+ NH2-R
O
O
blue l(570nm)
OH
In case of Pro:
O
+
N
O-
There is no difference
between the colour of
ninhydrine and the product
yellow
Test solutions:
 40 g phenol in 10 mL abs. EtOH
 65 mg KCN in 100 mL d.i. water
(take 2 mL and dilute with 98 mL pyridine)
 2.5 g ninhydrin in 50 mL abs. EtOH
O
Isatin test:
NH
O
3% isatin + 5% Boc-Phe-OH
dissolved in benzylalcohol
+ ninhydrin test solution
Colour of resin is red to black
Monitoring with bromophenol blue
3’,3”,5’,5”-tetrabromophenolsulfonphtalein:
HO
Br
HO
Br
Br
S OH
Br
O
lmax = 429 nm







Br
Br
O
O
Br
NH2-R
O
O
S O-
O
+
Br
HNH2-R
lmax = 600 nm
the change of the colour is because of salt formation (non covalent bond)
highly sensitive
the coupling can be followed (blue
green
yellow)
application of amine-free DMF is necessary
1% BB solution in dimethylacetamide; 2-3 drops to the reaction mixture
25 mL 0.04M solution for analysis
available for checking the coupling to Pro
In situ neutralisation
Apply this method when there is a danger of:
diketopiperazine formation; Pro containing dipeptide
pyroglutamic acid formation; Glu(Bzl), Gln on the N-terminus
”difficult” sequence, aggregation; a-helical or b-sheet structure
Synthesis cycle: Deprotection with 100% TFA 2x1 min
Wash with DMF 30 sec flow wash
Coupling: Boc-Aaa-derivative:DIC:HOBt (4 equiv each)
+ 1.5 equiv DIEA (calculated to the resin capacity
Wash with DMF 30 sec flow wash
(Preactivation is necessary) CF3COO-+NH3-CHRCO
Diketopiperazine formation:
O
C O CH2
NH2
R1-CH
NH
C
CH-R2
O
P
HO CH2
O
C
NH
R1-CH
NH
+
CH-R2
C
O
cis-peptide bonds
P
Pro-Pro
Pro-Gly
Gly-Pro
D-Aaa-Pro
Pro-D-Aaa
Boc cleavage flow chart
Does the peptide
contain His(Dnp)?
yes
no
Remove Dnp
Does the peptide
contain N-terminal Boc group?
yes
no
Remove Boc
Is the peptide (protecting groups)
compatible with HF, TMSOTf, TFMSA?
HF
Does the peptide
contain Trp(For)?
yes
TFMSA
TMSOTf
TMSOTf cleavage
no
Deformylate
HF cleavage
Trp(For) or
”Low-high” HF cleavage
Does the peptide
contain Trp(For) or Met(O)?
yes
no
”Low-high” TFMSA cleavage
Standard TFMSA cleavage
Why is it necessary to remove Boc-group before
cleavage with strong acids?
 tert-butyl cation is a very effective alkylating agent;
 long cleavage time, high cation concentration;
 the best scavanger to trap the tert-butyl cation is water;
 however water can’t be used with strong acids because of splitting
of peptide bonds;
 there are some special side reactions, eg. in case of peptides containing
Met at the C-terminal (homoserine lactone formation);
CH
3
S
+ CH
3
S
HF
NH
O
O
R
NH
OH
O
M = Mcalc- 47.0
NH
O
O
Problems with the cleavage procedures
HF : needs a special teflon instrument. However all the applied
protecting groups can be cleaved. Cleavage time is 45-60 min at 0oC,
but in case of Arg(Tos), Cys(Meb), Asp(OcHex), Glu(OcHex) 90 min is
recommended. Anisole, p-cresol and DTT as scavangers are used.
TMSOTf : 1 M TMSOTf-thioanisole/TFA solution in the presence of
m-cresol and EDT at 0oC for 120 min.
Arg(Tos), Cys(Meb), Asp(OcHex), Glu(OcHex) and BHA resin
are not cleavable under these conditions.
TFMSA : 10% TFMSA- 10% thioanisole in TFA at RT for 1.5-2hrs.
EDT and m-cresol are recommended as scavangers.
Arg(Tos), Cys(Meb), Asp(OcHex), Glu(OcHex) and BHA resin
are not compatible with this method.
More side reactions than in case of TMSOTf.
Desalting is necessary at the end.
O
Cresol is prefered in case of Glu:
M = Mcalc+ 90.05
CH3
O
O
NH
NH
O
Don’t use indole as scavanger for Trp in strong acids
H
H
M = Mcalc+ 117.1
H
N
Indole dimerisation can occur also
H
in case of peptides containing Trp
at the N-terminus resulting in dimer
R-CH2
peptide connected through indole rings.
N
H
Asp-Pro bond might be cleaved under acidic condition
Use dried materials and equipments !
 2-mercaptopyridine (10 equiv.) was suggested to prevent Met(O)
formation or Met(O) reduction under HF cleavage. However it
decreases the acidity of HF, so some protecting groups (eg. Tos)
can’t be removed effectively. Add Met and DTT to eliminate
Met(O) formation under HF cleavage.
 N-O acyl shift in case of Ser or Thr
HO
R
HO
O
NH
NH
R
O
NH
NH
O
O
R=H (Ser), CH3 (Thr)
This reaction can be reversed by
either neutralizing with NH4OH or
relyophilisation from 5% NH4HCO3
O
O
+
NH3
R
NH
O
”Pull-push” mechanism in the presence of thioanisole
SiMe3-O3S-CF3
R
SiMe3
CH2 O CH2
R
CH2 O
CH2
+
S CH3
+
CF3SO3-
CH3 S
m-cresol
H2O
(NH4F)
HO-SiMe3 + R CH2 OH
CF3SO3H
Thioanisole = reversible scavanger
Cresol = irreversible scavanger
Don’t use reversible scavanger alone !
CH3 S
CH3
+
HO
CH2
”Low-high” HF cleavage
Standard HF cleavage (SN1):
 10 mL HF
 0.5-1.0 g scavanger (anisole, p-cresol)
 0.1 g DTT or 0.5-1.0 mL EDT or DMS
as reducing agent
 45-90 min depending on the protecting
groups
 from -15oC to 0oC, depending on the
sequence (side reactions)
Low HF:
Met(O)
Trp(For)
”Low-high” HF cleavage (SN2+ SN1):
First step (low);there is no carbocation
 2.5 mL HF
 0.75 g p-cresol + 0.25 g p-thiocresol
 6.5 mL DMS
 2-3 hrs
 0oC
 evaporation of HF and DMS
(it takes quite a long time)
Second step (high):
Met
Trp
100% cleavable:
Arg(Mtr), Arg(Mts), Asp(OBzl)Glu(OBzl),
Lys(Z), Lys(ClZ), Ser(Bzl), Thr(Bzl),
Tyr(BrZ), Tyr(Bzl), Merrifield resin
 standard HF cleavage
new HF and scavangers
 45 min
 0oC
Cys(Mob), Tyr(2,6-Cl2Bzl), PAM resin (<80-85%)
His(Bom) (<60%)
The other protecting groups
can be cleaved just by high
HF procedure.
TFMSA (15%)-DMS(30%)-TFA(55%)
Synthesis of ”head to tail” type cyclic peptides
on resin
Application of oxim resin:
P
C
NO2
HO
The peptide-resin bond is stable
in acids, but cleavable by amines.
Compatible just with Boc chemistry.
However in situ neutralisation is
necessary.
N
C
NO2
p-nitrobenzophenone oxim 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-OY
Synthesis of cyclopeptides
What is the reason of cyclopeptides synthesis?
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. Structure stabilization:
eg. for 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. Increased 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: for eg. 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 fixed by disulfide cross-linkage.
Selective protection of Lys residues allows
attachment of 4 different peptide chains.
S
Arrangement of cyclic peptides
homodetic
only amide bonds in the cycle
heterodetic
disulfide bridge, thioether bond
lacton, ether, oxime thiazolidine bond