1. Solid phase peptide synthesis Applications of Boc/Bzl strategy
Part I
Applications of Boc/Bzl strategy
Gábor Mező
Research Group of Peptide Chemistry
Hungarian Academy of Sciences
Eötvös L. University
Budapest, Hungary

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. immunostimulator peptides; muramyl dipeptide tuftsin derivatives
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
Transporter peptides:
penetratin
oligoarginine
HIV-Tat protein
Applications of
synthetic peptides
Toxins:
conotoxins
spider toxins
snake toxins
ionchanel blockers
Carriers:
templates
miniproteins
Peptides
for structural studies:
turn mimicking cyclic peptides
Enzymes and
enzyme inhibitors:
Ribonuclease A

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 új vegyület kerül gyógyszerként bevezetésre.
Ezek között a peptidek száma egyre növekszik.

6. Peptidek a piacon piacon pre-regisztrációs fázis klinika-II
Rekombináns fehérjék: > ~ ~60
Monoklonális ellenanyagok: > > >45
Szintetikus peptidek: > > >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
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;
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;
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 R X
anchoring
deprotection
AA2
coupling (-H2O)

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

11. STRATEGIES Boc/Bzl: 2,6-Cl2Bzl Bzl HF Boc TFAN O Cl R 3
TFA HF
Boc-Asp(OBzl)-Gly-Tyr(2,6-Cl2Bzl)-Merrifield resin
Boc
Bzl
2,6-Cl2Bzl

12. Fmoc/tBu: .. tert-butyl TFA Fmoc Wang-resin piperidineH C 2 O 3 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. p-hydroxymethyl-phenyl-
Type of resins for Boc-chemistry C H 2 Cl P
Merrifield (chloromethyl) resin
NH3 N
Aminomethyl resin
Starting resin for the synthesis
of many other resins O
PAM resin (phenyl-acetamidomethyl)
p-hydroxymethyl-phenyl-
acetic acid (handle)
+ DIC

15. Attachment of the first amino acid to Merrifield and PAM resins2 Cl P
Boc-Aaa-O-Cs+
DMF, 50oC, 48h O N
Boc-Aaa-
Boc-Aaa-OH
DIC + 10%DMAP RT
DCM-DMF (1:3) h
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
DIC +
Boc-Aaa-O

16. Benzhydrylamine resin (BHA):C H N 2 P
Boc-Aaa-OH
DCC/HOBt
H(R) O
Boc
4-Methyl-benzhydrylamine resin (MBHA): 3
too stable under acid cleavage conditions
(only; HF!) C H N 2 P
Boc-Aaa-OH
DCC/HOBt C H N P
H(R) O
Boc
The final cleavage results in peptides with carboxamide (CONH2) group at the C-terminus.

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) C H 2
-OH (Ser, Thr, Tyr)
Side chain functional group protecting group name (abbreviation) HF
intramolecular
intermolecular N O OR R + OH
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:

19. without a-NH protection
Side chain functional group protecting group name (abbreviation)
2,6-dichlorobenzyl
(2,6-di-Cl-Bzl) C H 2
-OH (Tyr) Cl O Br
2-bromobenzyl-
oxycarbonyl
(2-Br-Z)
But < 2-Br-Z < cHex < 2,6-di-Cl-Bzl < Bzl
0,05% 0,1% 0,5% ,0% %
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. not for longer peptides!
--C----C---C------C---
Acm S
Side chain functional group protecting group name (abbreviation)
4-methylbenzyl
(Meb)
-SH (Cys) C H 2 3 O
4-methoxylbenzyl
(Mob)
Stability vs TFA is
not good enough;
not for longer peptides! NH
acetamidomethyl
(Acm)
For selective deprotection
Meb HF 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
Bzl
---C-----NH2 SH +
in acidic buffer
(pH 5-6)
at pH > 7
Neutral or basic
condition is not
appropriate for
asymmetrical
disulfide bond
formation!

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

23. Succinimide ring formation (Asp):
-Asp-Gly- N H C O 2
Bzl
-Asu-Gly-
- BzlOH
Asp-X; X = Gly, Arg, Ala, Ser, Asx
~30%
~70%
a-Asp-peptide
b-Asp-peptide
+H2O
Molecular weight is the same in both cases; HPLC separation of isomers in case
of small peptides; enzymatic degradation amino acid analysis.

24. Pyroglutamic acid formation at the N-terminal of the peptide (Glu):2
Bzl R
-BzlOH
Pyroglutamic acid formation at the N-terminal of the peptide (Glu):
M= Mcalc-18
NH2
-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-N H C N 2 NH R
Side chain functional group protecting group name (abbreviation)
wCONH2 (Asn, Gln)
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. H2
NH2
DCC
Boc

26. Side chain functional group protecting group name (abbreviation)NO 2
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 H
(His) p t
imidazol group SO C 3
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.
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. thiazolidine-4-charboxylic acid
Side chain functional group protecting group name (abbreviation) N H
(His) p t
imidazol group
benzyloxymethyl
(Bom) (p) O C H 2
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
Work up the peptide as soon as possible !
hemiacetale acetale
However, Bom must not be used in case of peptides containing Cys at the N-terminus:
HNH-CH-CO- - -
CH2 SH
H2C=O
-H2O
NH-CH-CO- - - S
H2C
M=Mcalc+12
thiazolidine-4-charboxylic acid
(thioproline)
Use Cys as scavanger under the cleavage
condition to catch the formaldehyde !

28. Racemisation H R N C R’ O ActO C:- O:- DL and LL dipeptide derivatives
Acyl-L-Aaa-OAct B:
-ActOH
5(4H)-oxazolone
-H+
C:-
O:-
Pseudo aromatic
system
+H+ DL
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. Side chain functional group protecting group name (abbreviation)
-NH-C-NH2 NH C H 3 S O
p-toluolsulfonyl
or tosyl (Tos)
4-methoxy-2,3,6-
trimethylbenzene-
sulfonyl (Mtr)
guanidino group
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)
2,3,6-trimethyl-
benzenesulfonyl or
mesitelenesulfonyl
(Mts)

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 C
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
M2 = Mcalc
M3 = Mcalc
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) !
(Trp)

31. Side chain functional group protecting group name (abbreviation)
-CH2-CH2-S-CH3
sulfide (Met) 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-CH2-S-CH2
- CH3OH
M = Mcalc in case of Bzl
M = Mcalc 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
Wash the resin 3x with DCM; min each
Cleavage of Boc protection with 33%TFA/DCM; 2+20min
Wash the resin 5x with DCM; min each
(Shrinking the resin with 25%dioxan/DCM)
Neutralisation 3-4x with 5-10%DIEA/DCM; 1 min each
Wash the resin 4x with DCM; min each
Coupling: Boc-amino acid derivative-DCC-HOBt in DCM-DMF *
(3 equiv each calculated to the resin capacity); 60 min
Wash the resin 2x with DMF; min each
Wash the resin 2x with DCM; min each
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. Ninhydrin monitoring OH H N + NH2-R 2 O In case of Pro: O-
Isatin test:
3% isatin + 5% Boc-Phe-OH
dissolved in benzylalcohol
+ ninhydrin test solution
Colour of resin is red to black
Ninhydrin monitoring O H 2
+ NH2-R OH N
blue l(570nm) O- +
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

35. Monitoring with bromophenol blue
3’,3”,5’,5”-tetrabromophenolsulfonphtalein: Br O H S
NH2-R -
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. 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 C H 2
C O P O
R1-CH NH
CH-R2
NH2 HO +
Pro-Pro
Pro-Gly
Gly-Pro
D-Aaa-Pro
Pro-D-Aaa
cis-peptide bonds
(Preactivation is necessary) CF3COO-+NH3-CHRCO
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
Diketopiperazine formation:

37. Boc cleavage flow chart
Does the peptide
contain His(Dnp)?
yes no
Remove Dnp
contain N-terminal Boc group?
Remove Boc
Is the peptide (protecting groups)
compatible with HF, TMSOTf, TFMSA? HF
contain Trp(For)?
Deformylate
Trp(For) or
”Low-high” HF cleavage
HF cleavage
TMSOTf
TFMSA
contain Trp(For) or Met(O)?
TMSOTf cleavage
”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); C H 3 S N O R HF OH +
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 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.
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:H 3
Cresol is prefered in case of Glu: O
M = Mcalc O N H N H O
Don’t use indole as scavanger for Trp in strong acids N H
R-CH2
M = Mcalc
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 N H R
R=H (Ser), CH3 (Thr) H3 +
This reaction can be reversed by
either neutralizing with NH4OH or
relyophilisation from 5% NH4HCO3

42. ”Pull-push” mechanism in the presence of thioanisole
SiMe3-O3S-CF3
SiMe3 C H 2 C H 2 O C H 2 R O C H 2 R + S C H + 3 S C H 3
CF3SO3-
m-cresol
H2O (NH4F) +
HO-SiMe3 OH C H 2 R C H
CF3SO3H 3 + C H 2
Thioanisole = reversible scavanger
Cresol = irreversible scavanger HO S C H 3
Don’t use reversible scavanger alone !

43. ”Low-high” HF cleavage
Standard HF cleavage (SN1):
10 mL HF
g scavanger (anisole, p-cresol)
0.1 g DTT or 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 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
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: C
NO2 P N HO
Boc-Aaa(X)-OH
+DCC/DCM
p-nitrobenzophenone oxim resin
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.
NH2-PEPTIDE-O
c(PEPTIDE)
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):NH CO
CR2
CR1 O
CR3
CR4
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 = 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 P 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