peptide conjugation and cyclisation chemistry for synthetic antigen development gábor mező...
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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
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:
Carrier molecules
A) Natural compounds
BSA, KLH, ovalbumine,tetanus toxoid, dextrane
B) 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
Carrier molecules applied in conjugates ofepitope 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--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
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 -turn like structure under appropriate conditions;11M can be replaced by Nle resulting in easier synthesis;22L prevents the succinimid formation during the synthesisof 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.
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.
Bond formation in conjugation reactions
Bifunctional coupling agents: homo- and hetero bifunctional reagents
NH2NH2
poly[Lys(DL-Alam)]; AK
H-SALLEDPVG-NH2
H-SALLQDPVG-NH2
H-SALLENPVG-NH2H-SALLED-OHH-DPVG-NH2
NHNHCOCO
NHNH
CO CO
NHNH
COCO
NHNH
CO CO
NHNH
CO 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 sulphonateBOP: benzotriazol-1-yl-oxy-tris-dimethylamino phosphonium hexafluorophosphate
*
*
*
Conjugation with amide bond formationMező, G. et al. J. Peptide Science 8, 107 (2002)
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/DMFAc-(Lys-Aib-Gly)4-PAM
Fmoc
1. 40% piperidine/DMF2. Boc-Leu-OH/DIC/HOBt
Ac-(Lys-Aib-Gly)4-PAMBoc-Leu-
Ac-(Lys-Aib-Gly)4-PAM
-
Boc-LK(ClZ)NleAD(OBzl)PNR(Tos)FR(Tos)GK(ClZ)D(OBzl)L-
1. 50% TFA/DCM
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-OHH-LKNleADPNRFRGKDL- Mező, G. … Tsikaris, V. et al. Bioconjugate Chem. 14, 1260 (2003)
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
NaIO4O CH-CO-R’
NH2-CH-CO-R
HS-CH2
CH-CO-R’CH2-S
R-CO-CH-NH
Application of amino/thiol type heterobifunctionalcompounds
N
-OCO-(CH2)2-S-S-
O
OSPDP
N-succinimidyl-3-(2-pyridyldithio)-propionate
NH2NH2
NH2NH2
poly[Lys(DL-Alam)]; AK
SPDP
NHNH2
NH2NH2
N
-OCO-(CH2)2-S-S-
HS
NHNH2
NH2NH2
-OCO-(CH2)2-S-S
in buffer solution pH= 7.5-8.5
Carlson et al. Biochem. J. 173, 723 (1978)
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
Application of Cys(Npys) derivatives
N
R-CO-CH-CH2-S-S-
R’-HN
-Cys(Npys)-Npys: 3-nitro-2-pyridinesulphenyl
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)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)
Synthesis of branched chain polypeptide-epitope peptide conjugates
N
O-CO-CH-CH2-S-S-
HNF
F
F F
FCO
O
CH3C
CH3
CH3
Boc-Cys(Npys)-OPfp
NH2NH2
NH2NH2
1. Boc-Cys(Npys)-OPfp in DMF-water (9:1)2. 95%TFA-5%water
NHNH2
NH2NH2
N
-OCO-CH-CH2-S-S-
NO2
NO2
NH2
HS
in buffer solution pH= 5.5
NHNH2
NH2NH2
-OCO-CH-CH2 -SS
NH2
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.
NH2NH2
NH2NH2
OH OH
OHOH
NH2NH2
NH2NH2
NH3NH3
NH3NH3
COOCOO
COOCOO
AK CAK (100%) SAK CSAK (27%) EAK CEAK (54%)
+ +
+ +
--
--
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 8.0-8.5)
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
Time Peptide (dimer)
1 2 3 4 5 6 7 8
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
1 H-CLKNleADPNRFRGKDL-NH2
2 H-LKNleADPNRFRGKDLC-NH2
3 H-CFRHDSGY-NH2
4 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)
L-Ser
oligo-DL-Ala
polylysineNH-CO-CH2Cl
NH-CO-CH2ClNH-CO-CH2
NH-CO-CH2
SH-Cys-OH
SAK:ClAcOPcp 1:1 1:0.8 1:0.6 1:0.5 1:0.4 1:0.3
Subst. Cl (%) 46.5 45.9 48.5 41.3 30.1 21.7
Subst.pept.(%) 44 nd nd 22 9 7
Conjugation of [Nle]11-9-22 epitope peptide from HSV gD-1 to SAK carrier molecule
H-9LKNleADPNRFRGKDL22C-NH2
Mező et al. Bioconjugate Chemistry 14, 1260 (2003)
N
ON
O
O O
O
OMBS
N-(3-maleimido-benzoyloxy)-succinimide
Kitagawa,T. et al. J. Biochem. 79, 233 (1976)
NH2
NH2
NH2
KLH
NH2
NH
NH2
N
O
O
O
O
+
NH2
NH
NH2
N
O
O
O
O
H-9LKNleADPNRFRGKDL22C-NH2
H-9LKNleADPNRFRGKDL22C-NH2
H
in PBS-DMF, 30 min, RTthen Sephadex G25, 10mM PBS (pH 6)
S
Synthesis of HSV gD1 [Nle]11-9-22Cys-KLHconjugate
PBS solution is adjuted to pH 7.5
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
1. Fmoc cleavage(20% piperidine/DMF)
2. Chloroacetylation(ClAc-OPcp/DMF)
Boc-[Thr(Bzl)-Lys(ClZ)-Pro-Lys(ClAc)-Gly]4-MBHA
1. Boc cleavage(33% TFA/DCM)
2. 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
H-9LKNleADPNRFRGKDL22C-NH2
S
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/100L pmol/100L
T20(9-22C) 4 9202.5/9202.1 3.4 0.72
SOC(9-22C) 4 8281.4/8281.2 2.9 0.70
MAP(9-22C) 4 7919.4/7942.4 7.5 n.i.
SAK(9-22C) 9 3.4 1.50
SAK(9-22C) 22 0.5 0.74
SAK(9-22C) 44 1.3 1.30
KLH(9-22C) 270 13.6 157.0
9-22 1641.9/1642.0 51.2 3.00
Fmoc-Lys(Fmoc)-Lys(Fmoc-Lys(Fmoc))-Ser-Ser--Ala- R =
FmocFmoc
Fmoc
FmocMAP core
Fmoc-Cys(StBu)- Antigen -Lys(Mtt)-Asp(OtBu)-Pro-
Fmoc-Cys(StBu)- Antigen -Lys(Mtt)-Asp-(OtBu)Pro-
Fmoc-Cys(StBu)- Antigen -Lys(Mtt)-Asp(OtBu)-Pro-
Fmoc-Cys(StBu)- Antigen -Lys(Mtt)-Asp(OtBu)-Pro-Synthesis usingFmoc chemistry
1. 95%TFA-5%TIS2. Fmoc-Ser(tBu)-OH DCC/HOBt in DMF
Fmoc-Cys(StBu)- Antigen -Lys(Fmoc-Ser(tBu))-Asp(OtBu)-Pro-
Fmoc-Cys(StBu)- Antigen -Lys(Fmoc-Ser(tBu))-Asp(OtBu)-Pro-
Fmoc-Cys(StBu)- Antigen -Lys(Fmoc-Ser(tBu))-Asp(OtBu)-Pro-
Fmoc-Cys(StBu)- Antigen -Lys(Fmoc-Ser(tBu))-Asp(OtBu)-Pro-
Multiple cyclic antigene peptideSpetzler, J.C., Tam, J.P. Peptide Research 9, 290 (1996)
Fmoc-Cys(StBu)- Antigen -Lys(Fmoc-Ser(tBu))-Asp(OtBu)-Pro-
1. 20% piperidine/DMF2. TFA/TIS/thioanisole/water(92.5:2.5:2.5:2.5, V/V)
NH2-Cys(StBu)- Antigen -Lys(NH2-Ser)-Asp-Pro-
OH
NH2-Cys(StBu)- Antigen -Lys(O=CH-CO)-Asp-Pro-
NaIO4 in 10mM PBS solution (pH 6.8) then HPLC purification
Tris-(2-carboxyethyl)phosphine10mM Na-acetate buffer (pH 4.2), Rt, 48h
OH
Antigen -Lys-Asp-Pro-
OH
COS
NH Antigen = peptide derived from V3 loop of gp120 HIV
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, 12h0.1M Tris-buffer (pH 8)
0.1 mg/mL peptide conc.
0.1M Tris-buffer (pH 8)3-4h
1-2mg/ml final concentration
Preparation of cyclic epitope peptides derived from 272-279 sequence of HSV-1 glycoprotein D
CO-NH-DPEDSALLC-CONH2
CH2 S
S S
Jakab, A., Mező, G. et al. (submitted)
NH-DPEDSALL-CO NH-LLDPEDSA-CO=
Boc-Leu-Leu-Asp(OcHex)-Pro-Glu(OcHex)-Asp(OcHex)-Ser(Bzl)-Ala-R
1. 33% TFA/DCM2. 1M TMSOTf-thioanisole/TFA (m-cresol) 45 min, 0oC
H-Leu-Leu-Asp(OcHex)-Pro-Glu(OcHex)-Asp(OcHex)-Ser-Ala-OHBOP-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
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 272-279
region of HSV gD-1
200 220 240 260 280-120000
-100000
-80000
-60000
-40000
-20000
0
20000
40000
60000
80000
100000 water TFE25 TFE50 TFE75 TFE
/d
eg
cm2 d
mo
l -1
/nm200 220 240 260 280
-100000
-80000
-60000
-40000
-20000
0
20000
40000
60000
80000
100000 disulfide thioether linear
/
deg
cm
2 dm
ol-1
/nm
200 220 240 260 280
-30
-20
-10
0
10
20
CD spectra of H-LLDPEDSA-OH
TFE TFE50 water
*10
-3 /d
eg c
m2 dm
ol-1
/nm
180 200 220 240 260 280-150
-100
-50
0
50
100
CD spectra of c(DPEDSALL)
TFE TFE50 water
*1
0-3 /
de
g cm
2 dm
ol-1
/nm
0 10 20 30 40
-0,05
0,00
0,05
0,10
0,15
0,20
Cyclopeptide with disulfide bond - 0 h
A21
4
t /min
10 20 30 40
0,0
0,1
Cyclopeptide with disulfide bond - 24 h
A21
4
t /min
0 10 20 30 40-0,04
0,00
0,04
0,08
0,12
0,16
0,20
Linear peptide - 0 hA
214
t /min
0 10 20 30 40-0,04
0,00
0,04
0,08
0,12
0,16
Linear peptide - 24 h
A21
4
t /min
Enzyme digestion of 272-279 epitope and cyclic
(disulfide) derivative by Aminopeptidase M
0
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 peptide(DL 6: 1:125000 dilution)
OD
495
Binding of monoclonal antibody DL6 to linear and cyclic epitope peptides of HSV gD-1
(Competition ELISA)
0
0,2
0,4
0,6
0,8
1
1,2
1,4
1,6
1,8
2
0
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 peptide(DL 6: 1:125000
OD
495
H-DPEDSALL-NH2
260-284
267-281
Target antigen: 0.5 g 260-284 peptide / well
c(CH2CO-DPEDSALLC)-NH2
c(CDPEDSALLC)-NH2
H-LLDPRDSALL-OH
9-22-Acp-C-272-279
0%
20%
40%
60%
80%
100%
0 24 48 72 96
Time (hours)
Pep
tid
e%
0%
20%
40%
60%
80%
100%
0 24 48 72 96
Time (hours)
Pep
tid
e%
H-LLEDPVGTVA-NH2
c(LLEDPVGTVA)
H-c(CLLEDPVGTVAC)-NH2
c(CH2CO-LLEDPVGTVAC)-NH2
0%
20%
40%
60%
80%
100%
0 60 120 180
Time (min)
Pep
tid
e(%
)
0%
20%
40%
60%
80%
100%
0 60 120 180
Time (min)
Pep
tid
e(%
)
H-LLEDPVGTVA-NH2
c(LLEDPVGTVA)
H-c(CLLEDPVGTVAC)-NH2
c(CH2CO-LLEDPVGTVAC)-NH2
Enzymatic cleavage of linear and cyclic peptides derived from 278-287 region of HSV gD-1
50% human serum
lysosoma
Tugyi, R., Mező, G., et al. J. Peptide Science (in press)
Boc-L-K-X-A-D-P-N-R-F-K-G-K-D-L-MBHA
ClZ OcHex Tos ClZ
Meb Fmoc OcHex
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 ClZ
Meb ClAc OcHex
HF-m-cresol–p-thiocresol
(10mL:0.5mL:0.5g)
90 min, 0oCpurificationRP-HPLC
H-L-K-X-A-D-P-N-R-F-K-G-K-D-L-NH2
SH ClAcCyclisation (adding peptide in small portion
0.1M Tris-buffer to the solution) (pH 8.1)
H-9L-K-X-A-D-P-N-R-F-K-G-K-D-L22-NH2
S CH2-CO
Synthesis of cyclic derivatives of 9-22 sequence from HSV gD-1
Arg was replaced by Lys in position 18
X = Hcy (mimicking Met in the cycle) or Cys
Sclosser, G., Mező, G. et al. Biophys. Chem. 106, 155 (2003)
Mimicking of Met in cyclopeptides containing thioether bond
--NH-CH-CO--- ---NH-CH-CO--
CH2
CH2
S
CH2
CH2
CH2
CH2
CH2 CO NH
Hcy ------ ------ Lys
--NH-CH-CO--- ---NH-CH-CO--
CH2
CH2
CH3
S
CH2
CH2
CH2
CH2
NH2
Met Lys Hcy ClAc-Lys
--NH-CH-CO--- ---NH-CH-CO--
CH2
CH2
SH
CH2
CH2
CH2
CH2
Cl-CH2-CO-NH
- HCl
200 220 240 260 280-12000
-10000
-8000
-6000
-4000
-2000
0
2000
4000
[] M
R (
de
g c
m2 /d
mo
l)
wavelength (nm)
200 220 240 260 280-10000
-8000
-6000
-4000
-2000
0
2000
[] M
R (
de
g c
m2 /d
mo
l)
wavelength (nm)
CD-spectra and amide I peaks in FT-IR spectrumof cyclic epitope peptides
(cm-1) [%]
Peptide
High-freqency region
Solvated amides
-turns -turns
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)
H-LK[HcyADPNRFK]GKDL-NH2 H-LK[CADPNRFK]GKDL-NH2
waterwater-TFE (1:1)TFE
waterwater-TFE (1:1)TFE
Mean average NMR stucture of
cyclic epitope peptides
N-terminal
C-terminal
16Arg13Asp
H2C
S
H2C
CO
HN
H2C
CH2
H2C
CH2
H2C
N-terminal
C-terminal
13Asp 16Arg
CH2
S
H2C CO
HN
CH2H2C
H2C
CH2
CH2
H-LK[HcyADPNRFK]GKDL-NH2, conformer „A” H-LK[HcyADPNRFK]GKDL-NH2, conformer „B”
N-teminalC-terminal
13Asp 16Arg
H2CCH2
CO
HN
CH2CH2
H2C
CH2
S
H-LK[CADPNRFK]GKDL-NH2
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
Fmoc
Fmoc
Boc/Fmoc*
Boc/Fmoc
Boc-LK(ClZ)MAD(OcHex)PNR(Tos)FR(Tos)GK(ClZ)D(OcHex)LAhxK(Fmoc)AhxC(Meb)-MBHA
2%DBU + 2% pipridine 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 terminal; Ac2O/DIEA in DMF
Boc-LK(ClZ)MAD(OcHex)PNR(Tos)FR(Tos)GK(ClZ)D(OcHex)LAhxKAhxC(Meb)-MBHA
Ac-C(Acm)GFLG-
1. deBoc; 33% TFA/DCM, 2+20 min2. ClAc2O/DIEA in DMF, 30 min
ClAc-LK(ClZ)MAD(OcHex)PNR(Tos)FR(Tos)GK(ClZ)D(OcHex)LAhxKAhxC(Meb)-MBHA
Ac-C(Acm)GFLG-
HF-p-cresol-DTT (10ml:1g:0.1g), 90min, 0oC
ClAc-LKMADPNRFRGKDLAhxKAhxC-NH2
Ac-C(Acm)GFLG-
Cyclisation in 0.1M Tris buffer (pH 8.0), 3-4h, RT
CH2CO-LKMADPNRFRGKDLAhxKAhxC-NH2
Ac-C(Acm)GFLG-
[CH2CO-LKMADPNRFRGKDLAhxC]-NH2 59.1 28.6
[CH2CO-AhxLKMADPNRFRGKDLAhxC]-NH2 22.7 28.0
[CH2CO-LKMADPNRFRGKDLAhxC]AhxGFLGC(Acm)-NH2 300.7 57.8
[CH2CO-LKMADPNRFRGKDLAhxK{Ac-C(Acm)GFLG}AhxC]-NH2 65.4 88.8
[CH2CO-LKNleADPNRFRGKDLAhxK{Ac-C(Acm)GFLG}AhxC]-NH2 37.9 89.3
H-LKMADPNRFRGKDL-NH2 4.0 2.4
H-LKNleADPNRFRGKDL-NH2 18.9 2.8
H-LKMADPNRFKGKDL-NH2 25.9 8.0
H-LKNleADPNRFKGKDL-NH2 25.4 5.0
H-LK[CADPNRFK(CH2CO)]GKDL-NH2 >6000 7900
H-LK[HcyADPNRFK(CH2CO)]GKDL-NH2 4443 2300
Direct Competition
Reactivity of 9-22 epitope derivatives against A16 mAb
Data are in pmol range
CH2CO-LKMADPNRFRGKDLAhxKAhxC-NH2
Ac-C(Acm)GFLG-
I2 or Tl(tfa)3oxidation
CH2CO-LKMADPNRFRGKDLAhxKAhxC-NH2
Ac-CGFLG-
CH2CO-LKMADPNRFRGKDLAhxKAhxC-NH2
Ac-CGFLG-
DTT
CH2CO-LKMADPNRFRGKDLAhxKAhxC-NH2
Ac-CGFLG-
CH2CO-LKMADPNRFRGKDLAhxKAhxC-NH2
Ac-CGFLG-
Ag-triflate
carrierAc-[TKPKG]4-NH2
CH2CO
CH2CO-LKMADPNRFRGKDLAhxKAhxC-NH2
Ac-CGFLG-
Dimer of cyclic peptide
Conjugate containing 4 cyclicepitope peptide
Synthesis of cyclic dimers and conjugates containing cyclic epitope peptides
O2
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.
-conotoxin GI (”host”)
H-ECCNPACGRHYSC-NH2
281-284 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)
Synthesis of HSV gD1 epitope peptide-conotoxin chimera
Fmoc-YCCNPACGDPVGC-Rink AM
tBu Trt
TrtAcm
Trt
Acm
OtBu1. 20% piperidine/DMF
2. 95%TFA-5% EDT (V/V)
H-YCCNPACGDPVGC-NH2
Acm Acm
DTNB
phosphate buffer(pH8.3), 1h, RT
H-YCCNPACGDPVGC-NH2
Acm Acm
H-YCCNPACGDPVGC-NH2
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
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.
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)- -Thr(tBu)-Arg(Pmc)-Pro-Ala-Pro-Gly-Ser(tBu)-Thr(tBu)-Ala-Pro-ClTrt
TFE-DCM (3:7)
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-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, 10874 (1998)
DIC/HOBt (3-fold excess)
(3-fold excess)
Choc-VTSAPDTRPAPGSTAPPAHG-Merrifield
BzlBzl Bzl
Bzl Bzl BomOcHex
Mts
Boc-VTSAPDTRPAPGSTAPPAHG-MBHA
BzlBzl Bzl
Bzl Bzl BomOcHex
Mts
1M TMSOTf-thioanisole/TFA
Choc-VTSAPDTRPAPGSTAPPAHG-OH
OcHex
H-VTSAPDTRPAPGSTAPPAHG-NH2
OcHex
1. EDC/HOBt in DMF2. HF-p-cresol (95:5)
H-VTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHG-NH2
MUC1 dimer synthesis by fragment condensation using semiprotected peptides
Boc-VTSAPDTRPAPGSTAPPAHGC-MBHA
BzlBzl Bzl
Bzl Bzl BomOcHex
Tos
Meb
ClAc-VTSAPDTRPAPGSTAPPAHG-MBHA
BzlBzl Bzl
Bzl Bzl BomOcHex
Tos1. 33% TFA/DCM (2+20 min)
2. HF- p-cresol/DTT (10ml: 1g :0.1g) 90min, 0oC
HF- p-cresol (10ml: 1g) 90min, 0oC
ClAc-VTSAPDTRPAPGSTAPPAHG-NH2
H-VTSAPDTRPAPGSTAPPAHGC-NH2
Tris buffer (pH 8.2)2h, RT
CH2CO-VTSAPDTRPAPGSTAPPAHG-NH2
H-VTSAPDTRPAPGSTAPPAHGC-NH2
MUC1 dimer synthesis by chemical ligation
H-VTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHG-NH2
CH2CO-VTSAPDTRPAPGSTAPPAHG-NH2
H-VTSAPDTRPAPGSTAPPAHGC-NH2
H-APDTRPAPG-NH2
H-APDTRPAPGC-NH2
H-APDTRPAPGC-NH2
H-VTSAPDTRPAPGSTAPPAHG-NH2
56.3 mol/dm3
53.2 mol/dm3
25.9 mol/dm3
0.62 mol/dm3
0.78 mol/dm3
Competition ELISA using C595 mAb
Conjugation method has no significant influence on binding capacity
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)