1. Peptide conjugation and cyclisation chemistry
for synthetic antigen development
Gábor Mező
Research Group of Peptide Chemistry,
Hungarian Academy of Sciences, Eötvös University
Budapest, Hungary
2005

2. Synthetic antigens Aim: synthetic vaccines – prevention of infections
Increasing of immunogenicity of small epitope peptides
(size, conformation)
Application of multi copy of the epitopes (B- and T-cell epitopes)
Prevention of the fast degradation of epitope peptides
Synthetic antigens
Aim: synthetic vaccines – prevention of infections
diagnostic tools – effective and selective demonstration of
the presence of infections in organism.
How can be realized the point of wievs?
coupling of the epitope to carrier molecules (conjugation)
preparation of cyclic derivatives of epitopes (cyclisation)
synthesis of peptides containing epitope as repeat unit
(oligomerisation, chemical ligation)
Point of wievs:

3. Carrier molecules Natural compounds Synthetic products
BSA, KLH, ovalbumine,
tetanus toxoid, dextrane
Synthetic products
biodegradable
biocompatible, but
non-degradable
Polymers
polylisine
branched chain polypeptide
polytuftsin
N-vinyl-pirrolidone maleic acid copolymer
stirene-maleic acid copolymer
Molecules with defined
structure
lysine dendrimers
sequential oligopeptides

4. Carrier molecules applied in conjugates of
epitope peptides derived from HSV gD-1
Oligotuftsin derivative (T20): H-[Thr-Lys-Pro-Lys-Gly]4-NH2
Sequential oligopeptide (SOC): Ac-[Lys-Aib-Gly]4-OH
Lysine tree (MAP): H-Lys-Lys(H-Lys)-Arg-Arg-b-Ala-NH2
Carriers with well defined composition (four conjugation sites):
Natural compound as carrier molecule (multiple conjugation sites)
Keyhole limpet hemocyanine (KLH)
Polymer carrier molecule (multiple conjugation sites):
Branched chain polpeptide (XAK)
L-Ser or L-Glu
oligo-DL-Ala
polylysine

5. Applied epitope regions of HSV-1 glycoprotein D
9LKMADPNRFRGKD21L22
9-21 of HSV-1 gD is the optimal epitope from the N-terminal (1-23) part
13D, 16R, 17F residues are essential for antibody recognition;
14PN15 b-turn like structure under appropriate conditions;
11M can be replaced by Nle resulting in easier synthesis;
22L prevents the succinimid formation during the synthesis
of 9-21-amide derivative.
Applied epitope regions of HSV-1 glycoprotein D
268LAPEDPEDSALLEDPVGTVA287
281DPVG284 minimal epitope available for antibody production
as a part of conjugate;
DP highly acid sensitive peptide bond.
272DPEDSALL279, 276SALLEDPVG284, 278LLEDPVGTVA287
were used for preparation of cyclic epitope peptides from this region.

6. Bond formation in conjugation reactions
Amide bond: needs COOH group (compound 1) and NH2 (compound 2);
N- or C-terminal or side chain (Glu, Asp, Lys) functional groups;
there are more functional (COOH, NH2)groups in the peptides;
protected or semiprotected peptides for conjugation;
removal of protecting groups may cause side reaction;
in case of protein or polymer conjugates the side reactions
can’t be well detected and the side product can’t be removed.
Disulfide bridge: needs thiol (Cys) group on both compounds;
symmetrical disulfie bridge is more stable than asymmetrical;
unprotected peptide fragments can be used.
Chemoselective ligations: eg. thioether bond, thiazolidine ring formation
unprotected peptide fragments can be used.
Bifunctional coupling agents: homo- and hetero bifunctional reagents

7. Conjugation with amide bond formation
NH2
poly[Lys(DL-Alam)]; AK
H-SALLEDPVG-NH2
H-SALLQDPVG-NH2
H-SALLENPVG-NH2
H-SALLED-OH
H-DPVG-NH2 NH CO
BOP (23.8%)
CMC (51.6%)
BOP (40.4%)
BOP (26.3%)
CMC (35.0%)
CMC: N-cyclohexyl-N’(2-morpholinoethyl)carbodiimide methyl p-toluene sulphonate
BOP: benzotriazol-1-yl-oxy-tris-dimethylamino phosphonium hexafluorophosphate *
Conjugation with amide bond formation
Mező, G. et al. J. Peptide Science 8, 107 (2002)

8. Tandem synthesis of conjugate SOC4([Nle11]-9-22)
Boc/Bzl strategy on PAM (phenyl-acetamidomethyl) resin:
Boc-(Lys-Aib-Gly)4-PAM
Fmoc
1. 50% TFA/DCM
2. Ac2O/DIEA/DMF
Ac-(Lys-Aib-Gly)4-PAM
1. 40% piperidine/DMF
2. Boc-Leu-OH/DIC/HOBt
Boc-Leu- -
Boc-LK(ClZ)NleAD(OBzl)PNR(Tos)FR(Tos)GK(ClZ)D(OBzl)L-
2. 5% DIEA/DCM
3. Boc-Aaa(X)-OH
13x
HF-p-cresol (95:5, V/V), 1.5h, -8 -0oC
Ac-(Lys-Aib-Gly)4-OH
H-LKNleADPNRFRGKDL-
Mező, G. … Tsikaris, V. et al. Bioconjugate Chem. 14, 1260 (2003)

9. Attachment of epitope peptide containing thiol group
to carrier molecules
Bonds: disulfide bridge, thioether bond, thiazolidine ring
Disulfide bridge: thiol group on the carrier is needed
Cysteine or cystine in the protein
(partially reduction in the second case is necessary)
Incorporation of bifunctional reagents
Attachment of Cys-derivative to the carrier
Thioether bond: coupling of haloacyl group to the carrier
R-SH + Cl-CH2-CO-NH-R’ R-S- CH2-CO-NH-R’ B:
-HCl
NH2-CH-CO-R’
HO-CH2
Thiazolidine ring: Ser in the carrier is converted to the glyoxyl moiety
NaIO4
O CH-CO-R’
NH2-CH-CO-R
HS-CH2
CH-CO-R’
CH2-S
R-CO-CH-NH

10. Application of amino/thiol type heterobifunctional
compounds
NH2 N
-OCO-(CH2)2-S-S- O
SPDP
N-succinimidyl-3-(2-pyridyldithio)-propionate
Carlson et al. Biochem. J. 173, 723 (1978)
poly[Lys(DL-Alam)]; AK NH
NH2
-OCO-(CH2)2-S-S
SPDP NH
NH2 N
-OCO-(CH2)2-S-S- HS
in buffer solution
pH=

11. Disadvantage of heterobifunctional reagents:
decrease the number of amino groups
involve the introduction of a hydrophobic spacer moiety
(both decrease the water solubility of the conjugate compared to the
parent macromolecule)
the disulfide formation between the activated thiol of the carrier and
the Cys containing peptide proceeds at neutral or slightly alkaline pH
dimerisation of Cys containing epitope peptide
unstable carrier-peptide bond
prefered symmetrical disulfide bridges
intra- and/or intermolecular cross-linkage of conjugate
lost solubility
Some of the bifunctional reagents have antigenic property

12. Npys: 3-nitro-2-pyridinesulphenyl
Application of Cys(Npys) derivatives N
R-CO-CH-CH2-S-S-
R’-HN
Stable in acids, however decomposes
in alkaline solution:
It is only compatible with Boc/Bzl
strategy in SPPS
React with thiols (Cys) in slightly
acidic solution (pH 5-6)
-Cys(Npys)-
Npys: 3-nitro-2-pyridinesulphenyl
Matsueda et al. Chem. Lett. (1981) 737
Incorporation of Cys(Npys) to the epitope peptide or to the carrier:
In case of proteins (BSA, KLH): protein is partially reduced and then
react with Cys(Npys) containing peptide.
In case of synthetic carriers: Cys(Npys) is attached to the carrier then
react with Cys containing epitope peptide.
Free Cys on the carrier may cause cross-linkage during the storage.
Drijfhout et al. Int. J. Pept. Prot. Res. 32, 161 (1988)
Mező et al. Bioconjugate Chem. 11, 484 (2000)

13. Synthesis of branched chain polypeptide-epitope
peptide conjugates N
O-CO-CH-CH2-S-S- HN F CO O C
H3C
CH3
Boc-Cys(Npys)-OPfp
NH2
1. Boc-Cys(Npys)-OPfp
in DMF-water (9:1)
2. 95%TFA-5%water NH
-OCO-CH-CH2-S-S-
NO2 HS
in buffer solution
pH= 5.5
-OCO-CH-CH2 -S S

14. Advantage of the use of Cys(Npys):
No change in the number of amino groups (no significant influence on
the solubility);
Reaction with thiol group can be carried out in slightly acidic condition;
less dimer formation of epitope peptide
the formed disulfide bridge between the carrier and epitope
peptide is more stable
However, stability study is necessary under the conditions used for biological
assays. In neutral solutions refolding of disulfide bridges may occur. Artificial
disulfide bridges may not be chemically and/or biologically stable.
NH2 OH
NH3
COO
AK CAK (100%) SAK CSAK (27%) EAK CEAK (54%) + -

15. Conjugation with thioeter bond formation
Advantages of thioether bond:
application of non-protected peptide precursors
(vs. amide bond formation)
chemically and biologically stable bond between the carrier and
epitope peptide (vs. disulfide bridge)
non-immunogenic bond
(vs. some bifunctional coupling agents)
easy coupling (between ClAc and SH groups), good yield
(usually better than in case of amide or disulfide bond formation)
Conjugation with thioeter bond formation
Disadvantages:
coupling is carried out in slightly alkaline solution (pH )
Cys containing peptides can dimerize (especially Cys at N-terminal)
very active BrAc derivatives can be used effectively only when no other
nucleophilles are present except Cys
unreacted haloacetyl group should be blocked with an excess of Cys

16. Oxidation of Cys containing epitope peptides
Time Peptide (dimer)
5 min nd* nd 36%+ 27% 2% 2% 31% 0%
1 h 22% 5% 86% 76% 3% 3% 90% 15%
2 h 43% 10% 93% 88% 4% 4% 98% 27%
4 h 68% 16% 100% 100% nd nd 100% 41%
6 h 90% 23% nd nd 10% 10% nd nd
8 h 100% 30% nd nd nd nd nd nd
24 h nd 62% nd nd 15% 13% nd 82%
* no data; + percentage of dimer present in the reaction mixture according to area under the peak in HPLC chromatogram
H-CLKNleADPNRFRGKDL-NH2
H-LKNleADPNRFRGKDLC-NH2
H-CFRHDSGY-NH2
H-CGGGGGFRHDSGY-NH2
5 H-FRHDSGYC-NH2
6 H-FRHDSGYGGGGGC-NH2
7 GlpHWSHDWK(H-C)PG-NH2
8 GlpHWSHDWK(Ac-C)PG-NH2
Oxidation of Cys containing epitope peptides
0.5mg/mL peptide concentration in 0.1 M Tris buffer; pH 8.2 (in a closed tube)
Mező, G., Manea, M. et al. J. Peptide Science 10, 701 (2004)

17. Conjugation of [Nle]11-9-22 epitope peptide from
L-Ser
oligo-DL-Ala
polylysine
NH-CO-CH2Cl
NH-CO-CH2 S
H-Cys-OH
SAK:ClAcOPcp 1:1 1:0.8 1:0.6 1:0.5 1:0.4 1:0.3
Subst. Cl (%)
Subst.pept.(%) nd nd
Conjugation of [Nle] epitope peptide from
HSV gD-1 to SAK carrier molecule
H-9LKNleADPNRFRGKDL22C-NH2
Mező et al. Bioconjugate Chemistry 14, 1260 (2003)

18. Synthesis of HSV gD1 [Nle]11-9-22Cys-KLH
MBS
N-(3-maleimido-benzoyloxy)-
succinimide
Kitagawa,T. et al. J. Biochem.
79, 233 (1976)
NH2
KLH NH +
H-9LKNleADPNRFRGKDL22C-NH2 H
in PBS-DMF, 30 min, RT
then Sephadex G25, 10mM PBS (pH 6) S
Synthesis of HSV gD1 [Nle] Cys-KLH
conjugate
PBS solution is adjuted to pH 7.5

19. Conjugation of [Nle]11-9-22 epitope peptide from
HSV gD-1 to T20 carrier
Boc-[Thr(Bzl)-Lys(ClZ)-Pro-Lys(Fmoc)-Gly]4-MBHA
Fmoc cleavage
(20% piperidine/DMF)
Chloroacetylation
(ClAc-OPcp/DMF)
Boc-[Thr(Bzl)-Lys(ClZ)-Pro-Lys(ClAc)-Gly]4-MBHA
Boc cleavage
(33% TFA/DCM)
Cleavage
(HF-p-thiocresol-m-cresol)
(10ml:0.5g:0.5ml)
H-[Thr-Lys-Pro-Lys(ClCH2CO)-Gly]4-NH2
Conjugation
H-9LKNleADPNRFRGKDL22C-NH2
(0.1M Tris buffer, pH 8.0, 72 h)
H-[Thr-Lys-Pro-Lys(CH2CO)-Gly]4-NH2 S

20. Advantage of well-characterised carrier molecules:
conjugation can be followed by HPLC and/or MS
the conjugate can be purified by HPLC
the product can be characterised by MS and amino acid analysis
the conjugate has a defined structure
Conjugate epitope/conj [M+H] direct ELISA competition ELISA
mol/mol calc/found ng/100mL pmol/100mL
T20(9-22C) /
SOC(9-22C) /
MAP(9-22C) / n.i.
SAK(9-22C)
SAK(9-22C)
SAK(9-22C)
KLH(9-22C) /

21. Multiple cyclic antigene peptide
Fmoc
Spetzler, J.C., Tam, J.P. Peptide Research 9, 290 (1996)
Fmoc-Lys(Fmoc)-Lys(Fmoc-Lys(Fmoc))-Ser-Ser-b-Ala- R =
MAP core
Fmoc-Cys(StBu)- Antigen -Lys(Mtt)-Asp(OtBu)-Pro-
Fmoc-Cys(StBu)- Antigen -Lys(Mtt)-Asp-(OtBu)Pro-
Synthesis using
Fmoc chemistry
1. 95%TFA-5%TIS
2. Fmoc-Ser(tBu)-OH
DCC/HOBt in DMF
Fmoc-Cys(StBu)- Antigen -Lys(Fmoc-Ser(tBu))-Asp(OtBu)-Pro-

22. then HPLC purification
Fmoc-Cys(StBu)- Antigen -Lys(Fmoc-Ser(tBu))-Asp(OtBu)-Pro-
1. 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-Pro-
NaIO4 in 10mM PBS solution (pH 6.8)
then HPLC purification OH
NH2-Cys(StBu)- Antigen -Lys(O=CH-CO)-Asp-Pro-
Tris-(2-carboxyethyl)phosphine
10mM Na-acetate buffer (pH 4.2), Rt, 48h OH
Antigen -Lys-Asp-Pro- OH CO S NH
Antigen
= peptide derived from
V3 loop of gp120 HIV

23. Preparation of cyclic epitope peptides derived from
Synthesis of cyclopeptides:
amide bond (protected precursor peptide)
disulfide bridge (stability of the bond may be not appropriate)
thioether bond (stable bond formation from unprotected precursor)
NH-DPEDSALL-CO
NH2-CDPEDSALLC-CONH2
Number of atoms
in the cycle 24 32 30
Preparation
see next slide
air oxidation, 12h
0.1M Tris-buffer (pH 8)
0.1 mg/mL peptide conc.
3-4h
1-2mg/ml final concentration
Preparation of cyclic epitope peptides derived from
sequence of HSV-1 glycoprotein D
CO-NH-DPEDSALLC-CONH2
CH S S
Jakab, A., Mező, G. et al. (submitted)

24. Synthesis of cyclic epitope peptide with amide bond formation
NH-DPEDSALL-CO
NH-LLDPEDSA-CO =
Boc-Leu-Leu-Asp(OcHex)-Pro-Glu(OcHex)-Asp(OcHex)-Ser(Bzl)-Ala-R
1. 33% TFA/DCM
2. 1M TMSOTf-thioanisole/TFA (m-cresol)
45 min, 0oC
H-Leu-Leu-Asp(OcHex)-Pro-Glu(OcHex)-Asp(OcHex)-Ser-Ala-OH
BOP-HOBt-DIEA (3:3:6 equiv) in DMF
2x12h, RT
0.5mg/mL peptide concentration
Leu-Leu-Asp(OcHex)-Pro-Glu(OcHex)-Asp(OcHex)-Ser-Ala
HF-p-cresol (10mL:1g)
Leu-Leu-Asp-Pro-Glu-Asp-Ser-Ala
R: Merrifield resin; D-Ala content < 1% Yield: 20%
Synthesis of cyclic epitope peptide
with amide bond formation

25. CD-spectra of H-DPEDSALL-NH2 linear peptide
in water-TFE mixtures
CD-spectra of H-DPEDSALL-NH2,
c(CH2-CO-DPEDSALLC)-NH2 and H-c(CDPEDSALLC)-NH2
in TFE
Solution conformation of linear and cyclic epitope peptides derived from region of HSV gD-1
200
220
240
260
280
-80000
-60000
-40000
-20000
20000
40000
60000
80000
100000
water
TFE25
TFE50
TFE75
TFE Q
/deg cm 2
dmol -1 l
/nm
disulfide
thioether
linear
/ deg cm
-30
-20
-10 10 20
CD spectra of H-LLDPEDSA-OH
*10 -3
180
-150
-100
-50 50
100
CD spectra of c(DPEDSALL)

26. Enzyme digestion of 272-279 epitope and cyclic
(disulfide) derivative by Aminopeptidase M 10 20 30 40
-0,05
0,00
0,05
0,10
0,15
0,20
Cyclopeptide with disulfide bond - 0 h A
214
t /min
0,0
0,1
Cyclopeptide with disulfide bond - 24 h
-0,04
0,04
0,08
0,12
0,16
Linear peptide - 0 h
Linear peptide - 24 h

27. Binding of monoclonal antibody DL6 to linear
0,2
0,4
0,6
0,8 1
1,2
1,4
1,6
1,8 2
1,95
3,9
7,8
15,6
31,3
62,5
125
250
500
1000
2000
n /
pmol peptid e (
DL 6 :
1:125000
dilution) OD
495
Binding of monoclonal antibody DL6 to linear
and cyclic epitope peptides of HSV gD-1
(Competition ELISA) H -
DPEDSALL NH
Target antigen: 0.5
mg 260-
284 peptide / well
c(CH2CO-DPEDSALLC)-NH2
c(CDPEDSALLC)-NH2
H-LLDPRDSALL-OH
9-22-Acp-C

28. Enzymatic cleavage of linear and cyclic peptides
derived from region of HSV gD-1
100%
100%
H-LLEDPVGTVA-NH2
80%
80%
c(LLEDPVGTVA)
50% human serum % %
60%
60%
Peptide
Peptide
40%
40%
H-c(CLLEDPVGTVAC)-NH2
20%
20%
c(CH2CO-LLEDPVGTVAC)-NH2 0% 0% 24 24 48 48 72 72 96 96
Time (
Time (
hours
hours ) ) 0%
20%
40%
60%
80%
100% 60
120
180
Time (min)
Peptide
(%)
H-LLEDPVGTVA-NH2
c(LLEDPVGTVA)
H-c(CLLEDPVGTVAC)-NH2
c(CH2CO-LLEDPVGTVAC)-NH2
lysosoma
Tugyi, R., Mező, G., et al. J. Peptide Science (in press)

29. Synthesis of cyclic derivatives of 9-22 sequence from HSV gD-1
Boc-L-K-X-A-D-P-N-R-F-K-G-K-D-L-MBHA
ClZ
OcHex
Tos
Meb
Fmoc
1. deFmoc (2%DBU,2%piperidine/DMF)
2. ClAcOPcp(5equiv)/DMF
3. deBoc (33%TFA/DCM)
H-L-K-X-A-D-P-N-R-F-K-G-K-D-L-MBHA
ClZ
OcHex
Tos
Meb
ClAc
HF-m-cresol–p-thiocresol
(10mL:0.5mL:0.5g)
90 min, 0oC
purification
RP-HPLC
H-L-K-X-A-D-P-N-R-F-K-G-K-D-L-NH2 SH
ClAc
Cyclisation (adding peptide in small portion
0.1M Tris-buffer to the solution)
(pH 8.1)
Arg was replaced by Lys in position 18
X = Hcy (mimicking Met in the cycle)
or Cys S
CH2-CO
H-9L-K-X-A-D-P-N-R-F-K-G-K-D-L22-NH2
Sclosser, G., Mező, G. et al. Biophys. Chem. 106, 155 (2003)

30. Mimicking of Met in cyclopeptides containing
thioether bond
--NH-CH-CO NH-CH-CO--
CH2 S
CH2 CO NH
Hcy Lys
CH3
NH2
Met Lys Hcy ClAc-Lys SH
Cl-CH2-CO-NH
- HCl

31. CD-spectra and amide I peaks in FT-IR spectrum
of cyclic epitope peptides n
(cm - 1
) [%]
Peptide
High
freqency
region
Solvated
amides b
turns g
H-LK[HcyADPNRFK]GKDL-NH2
1676 (15)
1661 (43)
1644 (10)
1629 (14)
H-LK[CADPNRFK]GKDL-NH2
1674 (20)
1660 (44)
1643 (6)
1629 (18)
water
water-TFE (1:1)
TFE

32. Mean average NMR stucture of cyclic epitope peptides
N-terminal
C-terminal 16
Arg 13
Asp H 2 C S O N
H-LK[HcyADPNRFK]GKDL-NH2, conformer „A”
H-LK[HcyADPNRFK]GKDL-NH2, conformer „B” N-
teminal
H-LK[CADPNRFK]GKDL-NH2

33. New analogue with increased size of cycle; dimerization, conjugation
H-LK[HcyADPNRFK]GKDL-NH2 and H-LK[CADPNRFK]GKDL-NH2
have very low binding activity on A16 mAb.
New analogues:
CH2-CO-LKMADPNRFRGKDLAhxC-NH2
CH2-CO-AhxLKMADPNRFRGKDLAhxC-NH2
CH2-CO-LKMADPNRFRGKDLAhxCAhxGFLGC(Acm)-NH2
CH2-CO-LKMADPNRFRGKDLAhxK[Ac-C(Acm)GFLG]AhxC-NH2
CH2-CO-LKNleADPNRFRGKDLAhxK[Ac-C(Acm)GFLG]AhxC-NH2
Fmoc
Boc/Fmoc*
Boc/Fmoc

34. Boc-LK(ClZ)MAD(OcHex)PNR(Tos)FR(Tos)GK(ClZ)D(OcHex)LAhxK(Fmoc)AhxC(Meb)-MBHA
2%DBU + 2% pipridine in DMF, 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 terminal; Ac2O/DIEA in DMF
Ac-C(Acm)GFLG-
1. deBoc; 33% TFA/DCM, 2+20 min
2. ClAc2O/DIEA in DMF, 30 min
ClAc-LK(ClZ)MAD(OcHex)PNR(Tos)FR(Tos)GK(ClZ)D(OcHex)LAhxKAhxC(Meb)-MBHA
HF-p-cresol-DTT (10ml:1g:0.1g), 90min, 0oC
ClAc-LKMADPNRFRGKDLAhxKAhxC-NH2
Cyclisation in 0.1M Tris buffer (pH 8.0), 3-4h, RT
CH2CO-LKMADPNRFRGKDLAhxKAhxC-NH2

35. Reactivity of 9-22 epitope derivatives against
[CH2CO-LKMADPNRFRGKDLAhxC]-NH
[CH2CO-AhxLKMADPNRFRGKDLAhxC]-NH
[CH2CO-LKMADPNRFRGKDLAhxC]AhxGFLGC(Acm)-NH
[CH2CO-LKMADPNRFRGKDLAhxK{Ac-C(Acm)GFLG}AhxC]-NH
[CH2CO-LKNleADPNRFRGKDLAhxK{Ac-C(Acm)GFLG}AhxC]-NH
H-LKMADPNRFRGKDL-NH
H-LKNleADPNRFRGKDL-NH
H-LKMADPNRFKGKDL-NH
H-LKNleADPNRFKGKDL-NH
H-LK[CADPNRFK(CH2CO)]GKDL-NH >
H-LK[HcyADPNRFK(CH2CO)]GKDL-NH
Direct Competition
Reactivity of 9-22 epitope derivatives against
A16 mAb
Data are in pmol range

36. Synthesis of cyclic dimers and conjugates
CH2CO-LKMADPNRFRGKDLAhxKAhxC-NH2
Ac-C(Acm)GFLG-
I2 or Tl(tfa)3
oxidation
Ac-CGFLG-
DTT
Ag-triflate
carrier
Ac-[TKPKG]4-NH2
CH2CO
Dimer of cyclic peptide
Conjugate containing 4 cyclic
epitope peptide
Synthesis of cyclic dimers and conjugates
containing cyclic epitope peptides O2

37. Synthesis of peptide chimeras
Peptide chimera: combination of peptide sequences from different
peptides and/or proteins.
The ”host” peptide serve the basic sequence of the chimeric peptide,
and one of the possible antigen presenting sequence (loop or turn)
is replaced by the ”guest” sequence.
a-conotoxin GI (”host”)
H-ECCNPACGRHYSC-NH2
epitope of HSV gD1 (”guest”)
Asp-Pro-Val-Gly (DPVG)
Core epitope of MUC1 (”guest”)
Pro-Asp-Thr-Arg (PDTR)
Succesfull synthesis if the conformation ”host” and (”guest”) sequence is similar.
Mező, G. et al. J. Peptide Research 55, 7 (2000)
Drakopoulou, E., Mező, G. et al. J. Peptide Science 6, 175 (2000)

38. Synthesis of HSV gD1 epitope peptide-conotoxin
chimera
Fmoc-YCCNPACGDPVGC-Rink AM
tBu
Trt
Acm
OtBu
1. 20% piperidine/DMF
2. 95%TFA-5% EDT (V/V)
H-YCCNPACGDPVGC-NH2
DTNB
phosphate buffer
(pH8.3), 1h, RT
Tl(tfa)3/TFA/anisole
DTNB (Ellman reagent) = 5,5’-dithio-bis(2-nitrobenzoic acid)
DPVG specific antibody was produced:
Immunogenicity:
bicyclic > monocyclic> linear
IgM antybody binding to chimera:
linear > bicyclic > monocyclic

39. Synthesis of oligomers of epitope peptides
MUC-1: bulid up from tandem repeat unit of a 20-mer peptide;
APDTRPAPGSTAPPAHGVTS, APDTR is the main epitope;
Thr are highly glycosilated;
in many human tumours of epithelia origin the produced
mucin is overexpressed and underglycosilated;
the free peptide chain is recognised as an antigen;
effective detection of antibodies may help in early diagnosis.
Epitope peptides may be used as diagnostic tool:
Increasing the number of epitopes results in higher antibody recognition.
Synthesis of oligomers from the repeat unit.

40. + Fmoc-Pro-Ala-His(Trt)-Gly-Val-Thr(tBu)-Ser(tBu)-Ala-Pro-Asp (OtBu)-
-Thr(tBu)-Arg(Pmc)-Pro-Ala-Pro-Gly-Ser(tBu)-Thr(tBu)-Ala-Pro-ClTrt
20% piperidine/DMF
NH2-Pro-Ala-His(Trt)-Gly-Val-Thr(tBu)-Ser(tBu)-Ala-Pro-Asp (OtBu)-
TFE-DCM (3:7)
-Thr(tBu)-Arg(Pmc)-Pro-Ala-Pro-Gly-Ser(tBu)-Thr(tBu)-Ala-Pro-OH +
Fmoc-[Pro-Ala-His(Trt)-Gly-Val-Thr(tBu)-Ser(tBu)-Ala-Pro-Asp (OtBu)-
-Thr(tBu)-Arg(Pmc)-Pro-Ala-Pro-Gly-Ser(tBu)-Thr(tBu)-Ala-Pro]2-ClTrt
H-[Pro-Ala-His-Gly-Val-Thr-Ser-Ala-Pro-Asp-
-Thr-Arg-Pro-Ala-Pro-Gly-Ser-Thr-Ala-Pro]5-OH
TFA-water-phenol-EDT-thioanisole (82.5:5:2.5:5)
Krambovitis, E. et al. J. Biol. Chem. 273, (1998)
DIC/HOBt (3-fold excess)
(3-fold excess)

41. MUC1 dimer synthesis by fragment condensation
Choc-VTSAPDTRPAPGSTAPPAHG-Merrifield
Bzl
Bom
OcHex
Mts
Boc-VTSAPDTRPAPGSTAPPAHG-MBHA
1M TMSOTf-thioanisole/TFA
Choc-VTSAPDTRPAPGSTAPPAHG-OH
H-VTSAPDTRPAPGSTAPPAHG-NH2
1. EDC/HOBt in DMF
2. HF-p-cresol (95:5)
H-VTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHG-NH2
MUC1 dimer synthesis by fragment condensation
using semiprotected peptides

42. MUC1 dimer synthesis by chemical ligation
Boc-VTSAPDTRPAPGSTAPPAHGC-MBHA
Bzl
Bom
OcHex
Tos
Meb
ClAc-VTSAPDTRPAPGSTAPPAHG-MBHA
1. 33% TFA/DCM
(2+20 min)
2. HF- p-cresol/DTT
(10ml: 1g :0.1g)
90min, 0oC
HF- p-cresol (10ml: 1g)
ClAc-VTSAPDTRPAPGSTAPPAHG-NH2
H-VTSAPDTRPAPGSTAPPAHGC-NH2
Tris buffer (pH 8.2)
2h, RT
CH2CO-VTSAPDTRPAPGSTAPPAHG-NH2
MUC1 dimer synthesis by chemical ligation

43. Competition ELISA using C595 mAb
H-VTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHG-NH2
CH2CO-VTSAPDTRPAPGSTAPPAHG-NH2
H-VTSAPDTRPAPGSTAPPAHGC-NH2
H-APDTRPAPG-NH2
H-APDTRPAPGC-NH2
H-VTSAPDTRPAPGSTAPPAHG-NH2
56.3 mmol/dm3
53.2 mmol/dm3
25.9 mmol/dm3
0.62 mmol/dm3
0.78 mmol/dm3
Competition ELISA using C595 mAb
Conjugation method has no significant influence on binding capacity

44. Zoltán Bánóczi
Szilvia Bősze
Ágnes Hilbert
Annamária Jakab
Gitta Schlosser
Zsolt Skribanek
Regina Tugyi
Katalin Uray
Ferenc Hudecz
(Budapest, Hungary)
Sytske Welling Wester
Matty Feilbrief
(Groningen, Netherland)
Marilena Manea
Michael Przybylski
(Konstanz, Germany)
Eliander Oliveria
Mari-Luz Valero
David Andreu
(Barcelona, Spain)
Eugenia Drakopoulou
Claudio Vita
(Saclay, France)
Vassilios Tsikaris
(Ioannina, Greece)