THE SOLID PHASE PEPTIDE SYNTHESIS IS SLIGHTLY DIFFRENT FROM PEPTIDE SYNTHESIS WHICH IS DISCUSSED HERE, ITS SYNTHESIS WITH STRUCTURE ANS BASICS ARE DISCUSSED WHICH WILL BE VERY USEFUL FOR READERS.

1. Solid phase peptide synthesis
Part I
Applications of Boc/Bzl strategy

2. 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;

3. Applications of
synthetic peptides
Immune peptides:
synthetic antigens;
vaccines
diagnostic tools
immunostimulator peptides;
muramyl dipeptide
tuftsin derivatives
Hormones:
oxytocin
vasopressin
insulin
somatostatin
GnRH
etc.
Neuropeptides:
substance P
cholecystokinin
neurotensin
Antibiotics:
tachikinin
gramicidine S
Toxins:
conotoxins
spider toxins
snake toxins
ionchanel blockers
Enzymes and
enzyme inhibitors:
Ribonuclease A
Carriers:
templates
miniproteins
Peptides
for structural studies:
turn mimicking cyclic peptides
Transporter peptides:
penetratin
oligoarginine
HIV-Tat protein

4. 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.”

5. 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.

6. Peptidek a piacon
Rekombináns fehérjék: >50 ~40 ~60
Monoklonális ellenanyagok: >20 >20 >45
Szintetikus peptidek: >40 >20 >60
piacon pre-regisztrációs fázis klinika-II
klinika-III
 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.

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

8. Synthesis in solution Synthesis on resin (SPPS)
 time consuming;
 manual;
 1.1-1.2 equiv amino acid derivatives
and coupling reagent for acylation;
 side chain protecting groups for
Lys, Asp, Glu, (Cys);
 coupling: less than 90% conversion;
 purification after each steps;
 large scale;
 cheap.
 fast;
 synthesizer (or manual);
 3-10 equiv amino acid derivatives
and coupling reagents for acylation;
 side chain protecting groups for
all functional groups
 coupling: over 99.5% conversion;
 purification at the end;
 rather small scale;
 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.

9. SOLID PHASE PEPTIDE SYNTHESIS
Bruce Merrifield published in 1963
Nobel Prize in Chemistry in 1984
The idea:
T
P
AA1 RX
anchoring
T
P
AA1 R
deprotection
P
AA1 R
T
P
AA2
coupling (-H2O)
T
P
AA2
P
AA1 R

10. T
P
AA2
P
AA1 R deprotection
coupling
repetitive
cycle
T
P
AAn
P
AA2
P
AA1 R
AAn
AA2 AA1
cleavage
+
final deprotection

11. STRATEGIES
Boc/Bzl:
CH2
NH CH
CH2
C
O
O
C
O
NH CH2 C
O
NH CH C
O
O
CH2
O
CH2
ClCl
CH2 RCH3 C O
CH3
CH3
C
O
TFA
HF
HF
Boc-Asp(OBzl)-Gly-Tyr(2,6-Cl2Bzl)-Merrifield resin
Boc
Bzl
2,6-Cl2Bzl

12. Fmoc/tBu:
NH
C
CH2 O C NH CH
CH2
C
O
O
C
O
H
C
CH3
CH3CH3
NH CH C
O
O
CH2
CH2 O CH2
O
O
C
CH3
CH3CH3
C
R
Fmoc
tert-butyl
..
piperidine
TFA
Wang-resin
Fmoc-Asp(OtBu)-Tyr(tBu)-Wang resin

13. 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

14. Type of resins for Boc-chemistry
CH2Cl P
Merrifield (chloromethyl) resin
NH3
CH2NH2 P
Aminomethyl resin
Starting resin for the synthesis
of many other resins
CH2OH CH2 C
O
OH
CH2OH CH2 C
O
NH CH2 P
PAM resin (phenyl-acetamidomethyl)
p-hydroxymethyl-phenyl-
acetic acid (handle)
+ DIC

15. CH2Cl P
Boc-Aaa-O-Cs+
DMF, 50oC, 48h
CH2OH CH2 C
O
NH CH2 P
CH2OBoc-Aaa- CH2 C
O
NH CH2 P
Boc-Aaa-OH
DIC + 10%DMAP RT
DCM-DMF (1:3) 1h
Peptide-PAM resin bond is more TFA stable than Peptide-Merrifield resin bond.
The final cleavage results in peptides with carboxyl (COOH) group
at the C-terminus.
Attachment of the first amino acid to Merrifield and PAM resins
CH2
OBoc-Aaa- CH2
C
O
OH
CH2NH2 PDIC +
CH2Boc-Aaa-O P

16. The final cleavage results in peptides with carboxamide (CONH2) group at
the C-terminus.
CHNH2
P
Boc-Aaa-OH
DCC/HOBt
CHNH PCCH(R)NH
O
Boc
Benzhydrylamine resin (BHA):
CHNH2
P
Boc-Aaa-OH
DCC/HOBt
CHNH PCCH(R)NH
O
Boc
4-Methyl-benzhydrylamine resin (MBHA):
CH3 CH3
too stable under acid cleavage conditions
(only; HF!)

17. 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 Boc-
amino acid to aminomethyl-resin represents a similar situation;
 Attachment of Boc-amino acid derivative to Merrifield or PAM-
resin (Kjeldahl N analysis, elemental analysis, amino acid analysis
or titration by pycric acid after Boc-removal: colour test)
Kjeldahl N 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)
Amino acid analysis:
 6M HCl in an evacuated and
stopped tube (hydrolysis)
 heating at 110oC for 24 hrs
 evaporation, neutralisation
 amino acid analysis
(quantitative)

18. Applied side chain protecting groups in Boc-chemistry
benzyl (Bzl)CH2-OH (Ser, Thr, Tyr)
Side chain functional group protecting group name (abbreviation)
HF
intramolecular intermolecular
NH CH C
O
CH2
OR
NH CH C
O
CH2
O
H R+
NH CH C
O
CH2
O
H
R+
NH CH C
O
CH2
OH
R
R+ can be caught by scavangers
However in case of Tyr:
20-100% side product!
The side reaction
can not be avoided
by using scavangers.
Mw: +90.05

19. Side chain functional group protecting group name (abbreviation)
2,6-dichlorobenzyl
(2,6-di-Cl-Bzl)CH2-OH (Tyr)
Cl
Cl
O CH2
Br
C
O
2-bromobenzyl-
oxycarbonyl
(2-Br-Z)
But < 2-Br-Z < cHex < 2,6-di-Cl-Bzl < Bzl
0,05% 0,1% 0,5% 5,0% 20%
Electrophilicity order of carbocations:
(amount of 3-alkyltyrosine in the peptide)
cyclohexyl
(cHex)
It is not commercially available
O N acyl shift;
do not keep the peptide
without a-NH protection
for long time !

20. --C----C---C------C---
Acm Acm
S S
--C----C---C------C---
S S
S S
Side chain functional group protecting group name (abbreviation)
4-methylbenzyl
(Meb)
-SH (Cys) CH2 CH3
CH2 CH3O
4-methoxylbenzyl
(Mob)
Stability vs TFA is
not good enough;
not for longer peptides!
CH2 NH C
O
CH3 acetamidomethyl
(Acm)
For selective deprotection
--C----C---C------C---
Acm Acm
Meb Meb
HF
--C----C---C------C---
Acm Acm
SH SH
air
oxidation
I2 or Tl(tfa)3
Hg(II)- or Ag(I)-salt !
Eg.

21. Side chain functional group protecting group name (abbreviation)
3-nitro-2-pyridinesulphenyl
(Npys)
-SH (Cys) S
N
NO2
For the synthesis of asymmetrical disulfide dimers
stable in the presence of acids
cleavable by bases
or thiols
Not for Fmoc-chemistry!
Eg.
--------C-------R
Npys
HF
--------C-------OH
Npys
Bzl
---C-----NH2
SH
+ in acidic buffer
(pH 5-6)
---C-----NH2
S
--------C-------OH
S--------C-------OH
S
--------C-------OH
S
---C-----NH2
S
---C-----NH2
S
at pH > 7
Neutral or basic
condition is not
appropriate for
asymmetrical
disulfide bond
formation!

22. O
O CH2
Cl
C
2-chlorobenzyl-
oxycarbonyl
(2-Cl-Z)
Side chain functional group protecting group name (abbreviation)
O
O CH2C
benzyloxycarbonyl
(Z)
eNH2 (Lys)
Z is not stable enough
in TFA;
branches in the peptide !
wCOOH (Asp, Glu)
O CH2
O
benzyl(ester)
(OBzl)
cyclohexyl(ester)
(OcHex)
OBzl is not stable enough
in TFA;
lead to ringclosure
side reaction !

23. Succinimide ring formation (Asp):
-Asp-Gly-
NH CH C
O
NH CH2 C
O
CH2
C
OBzl
O
-Asu-Gly-
NH CH
N
CH2
C
C
O
CH2
O
C
O
NH CH- BzlOH
Asp-X; X = Gly, Arg, Ala, Ser, Asx
NH CH C
O
NH CH2 C
O
CH2
C
OH
O
~30%
NH CH
CH2
C
C
O
O
NH CH
N CH2 C
O
OH
~70%
a-Asp-peptide b-Asp-peptide
+H2O
+H2O
Molecular weight is the same in both cases; HPLC separation of isomers in case
of small peptides; enzymatic degradation amino acid analysis.

24. NH2 CH C
O
NH CH C
O
CH2
C
OBzl
O
CH2
R
NH CH C
O
NH CH C
O
CH2
CO
CH2
R-BzlOH
Pyroglutamic acid formation at the N-terminal of the peptide (Glu):
M= Mcalc-18
NH2 CH C
O
NH CH C
O
CH2
C
NH2
O
CH2
R
NH CH C
O
NH CH C
O
CH2
CO
CH2
R-NH3
M= Mcalc-17
Don’t prepare peptides containing Gln at the N-terminus
They are not present in the nature!
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

25. O
Boc-NH CH C
O
NH CH C
O
CH2
C
NH
O
CH2
R
Side chain functional group protecting group name (abbreviation)
wCONH2 (Asn, Gln)
O
xantyl (Xan)
33%TFA/DCM deBoc, deXan
Why do we use Xan protecting group?
not necessary, but;
increase the solubility of
Boc-Gln-OH and Boc-Asn-OH,
eliminate the nitryl formation.
NH2 CH C
O
NH CH C
O
CH2
C
NH2
O
CH2
R
NH CH C
O
OH
CH2
C
N CH2
DCC
Boc
O
NH CH C OH
CH2
C
NH2
O
CH2
Boc

26. Side chain functional group protecting group name (abbreviation)
NO2
Protection of tN prevents
the alkylation or acylation
of imidazol ring, but not
the epimerisation of His;
Protection of pN prevents
also the epimerisation.
N N
H
(His)
p
t
imidazol group
SO2 CH3
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.
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
Boc-Aaa1-Aaa2(Bzl)-His-....-Resin
NH2-Aaa1-Aaa2(Bzl)-His-....-Resin NH2-Aaa1-Aaa2-His-....-OH
HF
TFA
thiophenol

27. However, Bom must not be used in case of peptides containing Cys at the N-terminus:
Side chain functional group protecting group name (abbreviation)
O CH2CH2N N
H
(His)
p
t
imidazol group
benzyloxymethyl
(Bom) (p)
Cleavage of Bom results in Bzl+ and CH2O;
Formaldehyde can react with nucleophiles:
 H2C=O + eNH2-Q H2C=N-Q (Schiff base)
H+
 H2C=O + OH-R H2C-OH
O-R
H2C-O-R
O-R
hemiacetale acetaleWork up the peptide as soon as possible !
HNH-CH-CO- - -
CH2
SH
H2C=O
-H2O
NH-CH-CO- - -
CH2
S
H2C
thiazolidine-4-charboxylic acid
(thioproline)
M=Mcalc+12
Use Cys as scavanger under the cleavage
condition to catch the formaldehyde !

28. Racemisation
C
R
O
N
H
C
R’
H
C
ActO O
Acyl-L-Aaa-OAct
B:
-ActOH
5(4H)-oxazolone
C
R
O
N
C
R’
H
C
O
-H+
C
R
O
N
C:-
R’
C
O
C
R
O
N
C R’
C
O:-
C
R
O
N
C R’
C
O:-
Pseudo aromatic
system
+H+
C
R
O
N
C
R’
H
C
O
DL
-H+
H-L-Aaa-OY
DL and LL dipeptide derivatives
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

29. 2,3,6-trimethyl-
benzenesulfonyl or
mesitelenesulfonyl
(Mts)
Side chain functional group protecting group name (abbreviation)
-NH-C-NH2
NH
CH3S
O
O
p-toluolsulfonyl
or tosyl (Tos)
CH3S
O
O
CH3
CH3
CH3
O
4-methoxy-2,3,6-
trimethylbenzene-
sulfonyl (Mtr)
guanidino group
CH3S
O
O
CH3
CH3
Lability in acids:
Mtr > Mts > Tos
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.)
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
(Arg)

30. Side chain functional group protecting group name (abbreviation)
indole
H-C
O
formyl (For)
Special cleavage is necessary:
20% piperidine/DMF before HF cleavage
or low-high HF cleavage procedure
OC
O
cyclohexyloxy-
carbonyl (Hoc)
Side reaction under
acidic condition:
 oxidation
 alkylation
Oxidation of Trp results in oxyindolyl and kynureninyl derivatives; pink colour
Alkylation by tert-butyl cation resulted under TFA cleavage of Boc-group
N
H
*
*
*
*
M1 = Mcalc+ 56.06
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) !
N
H
(Trp)

31. Side chain functional group protecting group name (abbreviation)
-CH2-CH2-S-CH3
sulfide (Met)
-CH2-CH2-S-CH3
O
sulfoxide (O)
Side reaction under
acidic conditions:
 oxidation
 alkylation
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-CH2-S-CH2
- CH3OH
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) !
Removal: N-methylmercaptoacetamide, low-high HF,
NH4I, TMSOBr+thioanisole

32. Synthetic protocol of Boc-strategy
1) Wash the resin 3x with DCM; 0.5-1.0 min each
2) Cleavage of Boc protection with 33%TFA/DCM; 2+20min
3) Wash the resin 5x with DCM; 0.5-1.0 min each
4) (Shrinking the resin with 25%dioxan/DCM)
5) Neutralisation 3-4x with 5-10%DIEA/DCM; 1 min each
6) Wash the resin 4x with DCM; 0.5-1.0 min each
7) 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 **
(-) yellow
(+) blue
* 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.

33. 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).

34. Isatin test:
3% isatin + 5% Boc-Phe-OH
dissolved in benzylalcohol
+ ninhydrin test solution
Colour of resin is red to black
Ninhydrin monitoring
O
OH
OH
O
2 + NH2-R
O
OH
N
O
O
blue l(570nm)
O-
OH
N
+
In case of Pro:
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
NH
O
O

35. Monitoring with bromophenol blue
3’,3”,5’,5”-tetrabromophenolsulfonphtalein:
Br
Br
O
Br
Br
OH
S
O
O
OH
NH2-R
Br
Br
O
Br
Br
OH
S
O
O
O-
HNH2-R
+
lmax = 429 nm 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

36. Diketopiperazine formation:
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
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
CH2C O P
O
R1-CH
NH
C
O
CH-R2
NH2
C
O
R1-CH
NH C
O
NH
CH-R2
CH2 PHO
+ Pro-Pro
Pro-Gly
Gly-Pro
D-Aaa-Pro
Pro-D-Aaa
cis-peptide bonds
(Preactivation is necessary) CF3COO-+NH3-CHRCO

37. 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 no
Deformylate
Trp(For) or
”Low-high” HF cleavage
HF cleavage
TMSOTf TFMSA
Does the peptide
contain Trp(For) or Met(O)?
TMSOTf cleavage
no
yes
”Low-high” TFMSA cleavage
Standard TFMSA cleavage

38. 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
3S
NH
O
O
R
HF
CH
3S
NH
OH
O
+
O
NH
O
M = Mcalc- 47.0

39. 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.

40. Cresol is prefered in case of Glu:
O CH3
NH
O
O
NH
O
Don’t use indole as scavanger for Trp in strong acids
M = Mcalc+ 90.05
M = Mcalc+ 117.1
N
H
H
N
H
R-CH2
H
H
Indole dimerisation can occur also
in case of peptides containing Trp
at the N-terminus resulting in dimer
peptide connected through indole rings.
Asp-Pro bond might be cleaved under acidic condition
Use dried materials and equipments !

41.  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
O
NH
R
O
OH
NH
R=H (Ser), CH3 (Thr)
NH
R
O
O
OH
NH
NH3
R
O
O
O
NH+
This reaction can be reversed by
either neutralizing with NH4OH or
relyophilisation from 5% NH4HCO3

42. ”Pull-push” mechanism in the presence of thioanisole
CH2OCH2R
SiMe3-O3S-CF3
SCH3
OCH2R
SiMe3 CH2
S CH3+
+
CF3SO3
-
H2O
(NH4F)
OHCH2R
m-cresol
HO
CH3HO-SiMe3 +
CH2
SCH3
CF3SO3H
+
Thioanisole = reversible scavanger
Cresol = irreversible scavanger
Don’t use reversible scavanger alone !

43. ”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-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):
 standard HF cleavage
new HF and scavangers
 45 min
 0oC
Low HF:
Met(O) Met
Trp(For) 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
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%)

44. Synthesis of ”head to tail” type cyclic peptides
on resin
Application of oxim resin:
CNO2
P
N
HO
Boc-Aaa(X)-OH
+DCC/DCM
p-nitrobenzophenone oxim resin
CNO2
P
N
Boc-Aaa-O
The peptide-resin bond is stable
in acids, but cleavable by amines.
Compatible just with Boc chemistry.
However in situ neutralisation is
necessary.
CNO2
P
N
NH2-PEPTIDE-O
c(PEPTIDE)
CNO2
P
N
Boc-PEPTIDE-O NH2-Aaa(X)-OY
Boc-PEPTIDE-Aaa-OY
Synthesis of cyclic peptides
and protected peptide fragments

45. 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

46.  phalloidine (toxin in mushrooms):
N
H
S
NHCO
CO
CR2
NH
CO
CR1
NHO
CONH
ONH
CR3
CO
NH
CR4
CO
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

47. 3. Structure stabilization:
eg. for improvement of the hormone-receptor interaction (increased
selectivity);
Leu-enkephaline: H-Tyr-Gly-Gly-Phe-Leu-OH
Cyclic derivative: H-Tyr-Dab-Pro-Phe-Leu
Dab = a,g-diaminobutiric acid; gNH2-CH2-CH2-CH2-COOH
aNH2
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
Selective for
m-receptor

48. 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
K
C
K
P
P
K
C
K
G
S
S
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.
Arrangement of cyclic peptides
homodetic heterodetic
only amide bonds in the cycle
disulfide bridge, thioether bond
lacton, ether, oxime thiazolidine bond