Transcript Document

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
Synthetic antigens
Aim: synthetic vaccines – prevention of infections
diagnostic tools – effective and selective demonstration of
the presence of infections in organism.
Point of wievs:

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
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)
Carrier molecules
A) Natural compounds
BSA, KLH, ovalbumine,
tetanus toxoid, dextrane
Polymers
 polylisine
 branched chain polypeptide
 polytuftsin
 N-vinyl-pirrolidone - maleic acid copolymer
 stirene-maleic acid copolymer
B) Synthetic products
•
•
biodegradable
biocompatible, but
non-degradable
Molecules with defined
structure
 lysine dendrimers
 sequential oligopeptides
Carrier molecules applied in conjugates of
epitope peptides derived from HSV gD-1
Carriers with well defined composition (four conjugation sites):
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
Polymer carrier molecule (multiple conjugation sites):
Branched chain polpeptide (XAK)
L-Ser or L-Glu
oligo-DL-Ala
polylysine
Natural compound as carrier molecule (multiple conjugation sites)
Keyhole limpet hemocyanine (KLH)
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.
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.
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
Conjugation with amide bond formation
*
Mező, G. et al. J. Peptide Science 8, 107 (2002)
NH2
NH2
CO
NH
H-SALLEDPVG-NH2
CMC (35.0%)
poly[Lys(DL-Alam)]; AK
H-DPVG-NH2
BOP (23.8%)
*
CO
NH
CO
NH
CO
NH
H-SALLQDPVG-NH2
BOP (26.3%)
H-SALLED-OH
H-SALLENPVG-NH2
BOP (40.4%)
CMC (51.6%)
CO
NH
CO
NH
CO
NH
CO
NH
CO
NH
CMC: N-cyclohexyl-N’(2-morpholinoethyl)carbodiimide methyl p-toluene sulphonate
BOP: benzotriazol-1-yl-oxy-tris-dimethylamino phosphonium hexafluorophosphate
CO
NH
*
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
Fmoc
1. 40% piperidine/DMF
2. Boc-Leu-OH/DIC/HOBt
1. 50% TFA/DCM
2. 5% DIEA/DCM
13x
3. Boc-Aaa(X)-OH
Ac-(Lys-Aib-Gly)4-PAM
Boc-Leu-
Ac-(Lys-Aib-Gly)4-PAM
Boc-LK(ClZ)NleAD(OBzl)PNR(Tos)FR(Tos)GK(ClZ)D(OBzl)LHF-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)
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’
B:
-HCl
R-S- CH2-CO-NH-R’
Thiazolidine ring: Ser in the carrier is converted to the glyoxyl moiety
HO-CH2
NH2-CH-CO-R’
NaIO4
HS-CH2
O CH-CO-R’
NH2-CH-CO-R
CH2-S
CH-CO-R’
R-CO-CH-NH
Application of amino/thiol type heterobifunctional
compounds
NH2
O
NH2
-OCO-(CH2)2-S-S-
N
O
NH2
SPDP
N-succinimidyl-3-(2-pyridyldithio)-propionate
NH2
Carlson et al. Biochem. J. 173, 723 (1978)
poly[Lys(DL-Alam)]; AK
SPDP
NH2
NH -OCO-(CH2)2-S-S-
N
NH2
NH -OCO-(CH2)2-S-S
HS
NH2
NH2
in buffer solution
pH= 7.5-8.5
NH2
NH2
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
R-CO-CH-CH2-S-S-
N
R’-HN
-Cys(Npys)Npys: 3-nitro-2-pyridinesulphenyl
Matsueda et al. Chem. Lett. (1981) 737
 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)
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.
Drijfhout et al. Int. J. Pept. Prot. Res. 32, 161 (1988)
In case of synthetic carriers: Cys(Npys) is attached to the carrier then
react with Cys containing epitope peptide.
Mező et al. Bioconjugate Chem. 11, 484 (2000)
Free Cys on the carrier may cause cross-linkage during the storage.
Synthesis of branched chain polypeptide-epitope
peptide conjugates
NH2
F
NH2
F
O-CO-CH-CH2-S-S-
F
F
F
NH2
1. Boc-Cys(Npys)-OPfp
in DMF-water (9:1)
2. 95%TFA-5%water
NH2
HN
NO2
CO
O
NH2
NH2
H3C C CH3
CH3
NH -OCO-CH-CH2-S-S-
N
Boc-Cys(Npys)-OPfp
NH2
NH-OCO-CH-CH2 -S
S
NH2
NO2
HS
NH2
NH2
N
in buffer solution
pH= 5.5
NH2
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.
NH3+
NH2
+
NH
NH
3
2
NH2
NH2
COO OH
COO OH
NH2
AK
OH
NH2
CAK (100%)
SAK
NH2
OH
NH2
CSAK (27%)
COO -
+
NH3
EAK
COO -
NH3+
CEAK (54%)
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)
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
Oxidation of Cys containing epitope peptides
Time
Peptide (dimer)
1
2
3
4
5
6
7
8
5 min
nd*
nd
36%+
27%
2%
2%
31%
0%
1h
22%
5%
86%
76%
3%
3%
90%
15%
2h
43%
10%
93%
88%
4%
4%
98%
27%
4h
68%
16%
100%
100%
nd
nd
100%
41%
6h
90%
23%
nd
nd
10%
10%
nd
nd
8h
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
0.5mg/mL peptide concentration in 0.1 M Tris buffer; pH 8.2 (in a closed tube)
1
H-CLKNleADPNRFRGKDL-NH2
5
H-FRHDSGYC-NH2
2
H-LKNleADPNRFRGKDLC-NH2
6
H-FRHDSGYGGGGGC-NH2
3
H-CFRHDSGY-NH2
7
GlpHWSHDWK(H-C)PG-NH2
4
H-CGGGGGFRHDSGY-NH2
8
GlpHWSHDWK(Ac-C)PG-NH2
Mező, G., Manea, M. et al. J. Peptide Science 10, 701 (2004)
Conjugation of [Nle]11-9-22 epitope peptide from
HSV gD-1 to SAK carrier molecule
L-Ser
oligo-DL-Ala
NH-CO-CH2Cl
polylysine
H-9LKNleADPNRFRGKDL22C-NH2
NH-CO-CH2
NH-CO-CH2Cl
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
Subst.pept.(%) 44
nd
48.5 41.3 30.1 21.7
nd
22
9
Mező et al. Bioconjugate Chemistry 14, 1260 (2003)
7
NH-CO-CH2
S
H-Cys-OH
Synthesis of HSV gD1 [Nle]11-9-22Cys-KLH
conjugate
NH2
O
O
+
NH2
NH2
N O
O
KLH
N
MBS
N-(3-maleimido-benzoyloxy)succinimide
O
Kitagawa,T. et al. J. Biochem.
79, 233 (1976)
O
in PBS-DMF, 30 min, RT
then Sephadex G25, 10mM PBS (pH 6)
H-9LKNleADPNRFRGKDL22C-NH2
NH2
H
O
O
NH2
N
O NH
O
NH2
S
O
O
H-9LKNleADPNRFRGKDL22C-NH2
PBS solution is adjuted to pH 7.5
NH2
N
O NH
O
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
S
H-9LKNleADPNRFRGKDL22C-NH2
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.
mol/mol
[M+H]+
calc/found
direct ELISA
ng/100mL
competition ELISA
pmol/100mL
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
51.2
3.00
9-22
1641.9/1642.0
Multiple cyclic antigene peptide
Spetzler, J.C., Tam, J.P. Peptide Research 9, 290 (1996)
Fmoc
Fmoc-Lys(Fmoc)-Lys(Fmoc-Lys(Fmoc))-Ser-Ser-b-Ala- R
Fmoc
Fmoc
=
Fmoc
Synthesis using
Fmoc chemistry
MAP core
Fmoc-Cys(StBu)- Antigen -Lys(Mtt)-Asp(OtBu)-ProFmoc-Cys(StBu)- Antigen -Lys(Mtt)-Asp(OtBu)-ProFmoc-Cys(StBu)- Antigen -Lys(Mtt)-Asp-(OtBu)ProFmoc-Cys(StBu)- Antigen -Lys(Mtt)-Asp(OtBu)-Pro-
Fmoc-Cys(StBu)- Antigen -Lys(Fmoc-Ser(tBu))-Asp(OtBu)-ProFmoc-Cys(StBu)- Antigen -Lys(Fmoc-Ser(tBu))-Asp(OtBu)-ProFmoc-Cys(StBu)- Antigen -Lys(Fmoc-Ser(tBu))-Asp(OtBu)-ProFmoc-Cys(StBu)- Antigen -Lys(Fmoc-Ser(tBu))-Asp(OtBu)-Pro-
1. 95%TFA-5%TIS
2. Fmoc-Ser(tBu)-OH
DCC/HOBt in DMF
Fmoc-Cys(StBu)- Antigen -Lys(Fmoc-Ser(tBu))-Asp(OtBu)-Pro1. 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-ProNaIO4 in 10mM PBS solution (pH 6.8)
then HPLC purification
OH
NH2-Cys(StBu)- Antigen -Lys(O=CH-CO)-Asp-ProTris-(2-carboxyethyl)phosphine
10mM Na-acetate buffer (pH 4.2), Rt, 48h
S
NH
CO
OH
Antigen = peptide derived from
V3 loop of gp120 HIV
Antigen -Lys-Asp-ProOH
Preparation of cyclic epitope peptides derived from
272-279 sequence of HSV-1 glycoprotein D
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
S
S
NH2-CDPEDSALLC-CONH2
CH2
S
CO-NH-DPEDSALLC-CONH2
Jakab, A., Mező, G. et al. (submitted)
Number of atoms
in the cycle
Preparation
24
see next slide
32
air oxidation, 12h
0.1M Tris-buffer (pH 8)
0.1 mg/mL peptide conc.
30
0.1M Tris-buffer (pH 8)
3-4h
1-2mg/ml final concentration
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%
Solution conformation of linear and cyclic epitope
peptides derived from 272-279 region of HSV gD-1
CD-spectra of H-DPEDSALL-NH2 linear peptide
in water-TFE mixtures
100000
water
TFE25
TFE50
TFE75
TFE
20000
60000
-1
40000
80000
0
40000
20000
2
2
-1
Q /deg cm dmol
60000
-20000
-40000
-60000
0
-20000
-40000
-80000
-60000
-100000
-80000
-120000
disulfide
thioether
linear
100000
Q / deg cm dmol
80000
CD-spectra of H-DPEDSALL-NH2,
c(CH2-CO-DPEDSALLC)-NH2 and H-c(CDPEDSALLC)-NH2
in TFE
-100000
200
220
240
260
280
200
220
l /nm
CD spectra of H-LLDPEDSA-OH
260
280
CD spectra of c(DPEDSALL)
50
0
Q*10 /deg cm dmol
-1
100
TFE
TFE50
water
2
-1
0
-10
-3
-3
Q*10 /deg cm dmol
2
20
10
240
l /nm
-20
TFE
TFE50
water
-50
-100
-30
200
220
240
l /nm
260
280
-150
180
200
220
240
l /nm
260
280
Enzyme digestion of 272-279 epitope and cyclic
(disulfide) derivative by Aminopeptidase M
Linear peptide - 24 h
Linear peptide - 0 h
0,20
0,16
0,16
0,12
0,12
214
A
A
214
0,08
0,08
0,04
0,04
0,00
0,00
-0,04
0
-0,04
10
20
30
0
40
10
20
t /min
30
40
t /min
Cyclopeptide with disulfide bond - 0 h
Cyclopeptide with disulfide bond - 24 h
0,20
0,15
0,1
A214
A214
0,10
0,05
0,00
0,0
-0,05
0
10
20
t /min
30
40
10
20
30
t /min
40
Binding of monoclonal antibody DL6 to linear
and cyclic epitope peptides of HSV gD-1
(Competition ELISA)
2
1,8
1,6
H-DPEDSALL-NH2
c(CH2CO-DPEDSALLC)-NH2
OD 495
1,4
1,2
c(CDPEDSALLC)-NH2
1
H-LLDPRDSALL-OH
9-22-Acp-C-272-279
0,8
0,6
260-284
267-281
0,4
0,2
0
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)
Target antigen: 0.5 mg 260-284 peptide / well
Enzymatic cleavage of linear and cyclic peptides
derived from 278-287 region of HSV gD-1
100%
H-LLEDPVGTVA-NH2
50% human serum
c(LLEDPVGTVA)
60%
H-c(CLLEDPVGTVAC)-NH2
40%
20%
c(CH2CO-LLEDPVGTVAC)-NH2
0%
0
24
48
72
96
Time (hours)
H-LLEDPVGTVA-NH2
100%
lysosoma
Peptide (%)
Peptide %
80%
80%
c(LLEDPVGTVA)
60%
H-c(CLLEDPVGTVAC)-NH2
40%
20%
c(CH2CO-LLEDPVGTVAC)-NH2
0%
0
60
120
Time (min)
Tugyi, R., Mező, G., et al. J. Peptide Science (in press)
180
Synthesis of cyclic derivatives of 9-22 sequence
from HSV gD-1
Meb
Fmoc
OcHex
1. deFmoc (2%DBU,2%piperidine/DMF)
Boc-L-K-X-A-D-P-N-R-F-K-G-K-D-L-MBHA
ClZ
OcHex Tos
2. ClAcOPcp(5equiv)/DMF
ClZ
3. deBoc (33%TFA/DCM)
Meb
HF-m-cresol–p-thiocresol
purification
RP-HPLC
SH
(10mL:0.5mL:0.5g)
ClAc
OcHex
H-L-K-X-A-D-P-N-R-F-K-G-K-D-L-MBHA
ClZ
90 min, 0oC
OcHex Tos
ClZ
ClAc
H-L-K-X-A-D-P-N-R-F-K-G-K-D-L-NH2
Cyclisation (adding peptide in small portion
0.1M Tris-buffer
(pH 8.1)
to the solution)
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)
Mimicking of Met in cyclopeptides containing
thioether bond
CH3
NH2 CH2
Cl-CH2-CO-NH
CH2
S
CH2
SH
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
--NH-CH-CO--- ---NH-CH-CO-Met
--NH-CH-CO--- ---NH-CH-CO--
Lys
CH2 CO
Hcy
NH
ClAc-Lys
CH2
S
CH2
CH2
CH2
CH2
CH2
--NH-CH-CO--- ---NH-CH-CO--
Hcy ------ ------ Lys
- HCl
CD-spectra and amide I peaks in FT-IR spectrum
of cyclic epitope peptides
4000
H-LK[HcyADPNRFK]GKDL-NH2
H-LK[CADPNRFK]GKDL-NH2
2000
2
0
[Q]MR (deg cm /dmol)
2
[Q]MR (deg cm /dmol)
2000
-2000
-4000
water
water-TFE (1:1)
TFE
-6000
-8000
0
-2000
-4000
water
water-TFE (1:1)
TFE
-6000
-8000
-10000
-10000
-12000
200
220
240
260
200
280
220
240
260
wavelength (nm)
wavelength (nm)
-1
n (cm ) [%]
Peptide
H-LK[HcyADPNRFK]GKDL-NH2
H-LK[CADPNRFK]GKDL-NH2
High-freqency
region
Solvated
amides
b-turns
g-turns
1676 (15)
1674 (20)
1661 (43)
1660 (44)
1644 (10)
1643 (6)
1629 (14)
1629 (18)
280
Mean average NMR stucture of
cyclic epitope peptides
H-LK[HcyADPNRFK]GKDL-NH2, conformer „A”
H-LK[HcyADPNRFK]GKDL-NH2, conformer „B”
16
13
Asp
13
Arg
16
Asp
Arg
C-terminal
N-terminal
N-terminal
C-terminal
H2C
CH2
CH2
H2C
CH2
HN
H2C
H2C
H2C
CH2
S
H2C
CH2
H2C
C
O
H-LK[CADPNRFK]GKDL-NH2
16
13
Arg
Asp
N-teminal
C-terminal
H2C
S
CH2
C
O
H
N
H2
C
C
H2
CH2
C
H2
H2
C
S
HN
C
O
CH2
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
Fmoc
CH2-CO-AhxLKMADPNRFRGKDLAhxC-NH2
Fmoc
CH2-CO-LKMADPNRFRGKDLAhxCAhxGFLGC(Acm)-NH2
Fmoc
CH2-CO-LKMADPNRFRGKDLAhxK[Ac-C(Acm)GFLG]AhxC-NH2
Boc/Fmoc*
CH2-CO-LKNleADPNRFRGKDLAhxK[Ac-C(Acm)GFLG]AhxC-NH2
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)GFLG1. 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
Ac-C(Acm)GFLG-
HF-p-cresol-DTT (10ml:1g:0.1g), 90min, 0oC
ClAc-LKMADPNRFRGKDLAhxKAhxC-NH2
Ac-C(Acm)GFLGCyclisation in 0.1M Tris buffer (pH 8.0), 3-4h, RT
CH2CO-LKMADPNRFRGKDLAhxKAhxC-NH2
Ac-C(Acm)GFLG-
Reactivity of 9-22 epitope derivatives against
A16 mAb
Direct Competition
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
H-LK[HcyADPNRFK(CH2CO)]GKDL-NH2
[CH2CO-LKMADPNRFRGKDLAhxC]-NH2
>6000
7900
4443
2300
59.1
28.6
22.7
28.0
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
[CH2CO-AhxLKMADPNRFRGKDLAhxC]-NH2
[CH2CO-LKMADPNRFRGKDLAhxC]AhxGFLGC(Acm)-NH2
Data are in pmol range
Synthesis of cyclic dimers and conjugates
containing cyclic epitope peptides
CH2CO-LKMADPNRFRGKDLAhxKAhxC-NH2
Ac-C(Acm)GFLG-
I2 or Tl(tfa)3
oxidation
Ag-triflate
CH2CO-LKMADPNRFRGKDLAhxKAhxC-NH2
DTT
Ac-CGFLGAc-CGFLGCH2CO-LKMADPNRFRGKDLAhxKAhxC-NH2
Dimer of cyclic peptide
O2
CH2CO-LKMADPNRFRGKDLAhxKAhxC-NH2
Ac-CGFLGAc-CGFLGCH2CO-LKMADPNRFRGKDLAhxKAhxC-NH2
Ac-[TKPKG]4-NH2
CH2CO
Ac-CGFLG-
CH2CO-LKMADPNRFRGKDLAhxKAhxC-NH2
carrier
Conjugate containing 4 cyclic
epitope peptide
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
281-284 epitope of HSV gD1 (”guest”)
Asp-Pro-Val-Gly (DPVG)
Mező, G. et al. J. Peptide Research 55, 7 (2000)
Core epitope of MUC1 (”guest”)
Pro-Asp-Thr-Arg (PDTR)
Drakopoulou, E., Mező, G. et al. J. Peptide Science 6, 175 (2000)
Succesfull synthesis if the conformation ”host” and (”guest”) sequence is similar.
Synthesis of HSV gD1 epitope peptide-conotoxin
chimera
tBu Trt
OtBu Trt
Fmoc-YCCNPACGDPVGC-Rink AM
Acm Trt Acm
H-YCCNPACGDPVGC-NH2
Acm
1. 20% piperidine/DMF
2. 95%TFA-5% EDT (V/V)
DTNB
phosphate buffer
(pH8.3), 1h, RT
Acm
Tl(tfa)3/TFA/anisole
H-YCCNPACGDPVGC-NH2
H-YCCNPACGDPVGC-NH2
Acm
Acm
DPVG specific antibody was produced:
Immunogenicity:
bicyclic > monocyclic> linear
IgM antybody binding to chimera:
linear > bicyclic > monocyclic
DTNB (Ellman reagent) = 5,5’-dithio-bis(2-nitrobenzoic acid)
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.
Krambovitis, E. et al. J. Biol. Chem. 273, 10874 (1998)
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
TFE-DCM (3:7)
20% piperidine/DMF
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
+
(3-fold excess)
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
DIC/HOBt (3-fold excess)
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
TFA-water-phenol-EDT-thioanisole (82.5:5:2.5:5)
H-[Pro-Ala-His-Gly-Val-Thr-Ser-Ala-Pro-Asp-Thr-Arg-Pro-Ala-Pro-Gly-Ser-Thr-Ala-Pro]5-OH
MUC1 dimer synthesis by fragment condensation
using semiprotected peptides
OcHex
Bzl
Mts
Bzl
Bom
Choc-VTSAPDTRPAPGSTAPPAHG-Merrifield
Bzl
Bzl
Bzl
OcHex
Bzl
Mts
Bzl
Bom
Boc-VTSAPDTRPAPGSTAPPAHG-MBHA
Bzl
Bzl
Bzl
1M TMSOTf-thioanisole/TFA
OcHex
OcHex
Choc-VTSAPDTRPAPGSTAPPAHG-OH H-VTSAPDTRPAPGSTAPPAHG-NH2
1. EDC/HOBt in DMF
2. HF-p-cresol (95:5)
H-VTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHG-NH2
MUC1 dimer synthesis by chemical ligation
OcHex
Bzl
Tos
Bzl
Bom
Boc-VTSAPDTRPAPGSTAPPAHGC-MBHA
Bzl
Bzl
Bzl
Meb
OcHex
Bzl
Tos
1. 33% TFA/DCM
(2+20 min)
2. HF- p-cresol/DTT
(10ml: 1g :0.1g)
90min, 0oC
Bom
ClAc-VTSAPDTRPAPGSTAPPAHG-MBHA
Bzl
H-VTSAPDTRPAPGSTAPPAHGC-NH2
Tris buffer (pH 8.2)
2h, RT
Bzl
Bzl
Bzl
HF- p-cresol (10ml: 1g)
90min, 0oC
ClAc-VTSAPDTRPAPGSTAPPAHG-NH2
H-VTSAPDTRPAPGSTAPPAHGC-NH2
CH2CO-VTSAPDTRPAPGSTAPPAHG-NH2
Competition ELISA using C595 mAb
H-APDTRPAPG-NH2
H-APDTRPAPGC-NH2
H-APDTRPAPGC-NH2
56.3 mmol/dm3
53.2 mmol/dm3
H-VTSAPDTRPAPGSTAPPAHG-NH2
25.9 mmol/dm3
H-VTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHG-NH2
0.62 mmol/dm3
H-VTSAPDTRPAPGSTAPPAHGC-NH2
CH2CO-VTSAPDTRPAPGSTAPPAHG-NH2
0.78 mmol/dm3
Conjugation method has no significant influence on binding capacity
Zoltán Bánóczi
Marilena Manea
Szilvia Bősze
Michael Przybylski
Ágnes Hilbert
Annamária Jakab
Gitta Schlosser
Zsolt Skribanek
(Konstanz, Germany)
Eliander Oliveria
Mari-Luz Valero
Regina Tugyi
David Andreu
Katalin Uray
(Barcelona, Spain)
Ferenc Hudecz
(Budapest, Hungary)
Eugenia Drakopoulou
Claudio Vita
(Saclay, France)
Sytske Welling Wester
Matty Feilbrief
(Groningen, Netherland)
Vassilios Tsikaris
(Ioannina, Greece)