1 / 44

Peptide conjugation and cyclisation chemistry for synthetic antigen development

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

oistin
Download Presentation

Peptide conjugation and cyclisation chemistry for synthetic antigen development

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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 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)

  3. Carrier molecules • Natural compounds • 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 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)

  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. 268LAPEDPEDSALLEDPVGTVA287 281DPVG284minimal 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 * Mező, G. et al. J. Peptide Science8, 107 (2002) H-SALLEDPVG-NH2 NH2 NH NH NH2 NH NH NH NH NH NH NH NH CMC (35.0%) poly[Lys(DL-Alam)]; AK H-SALLQDPVG-NH2 BOP (26.3%) H-DPVG-NH2 H-SALLENPVG-NH2 * H-SALLED-OH BOP (23.8%) BOP (40.4%) CMC (51.6%) * CO CO CO CO CO CO CO CO CO CO CMC: N-cyclohexyl-N’(2-morpholinoethyl)carbodiimide methyl p-toluene sulphonate BOP: benzotriazol-1-yl-oxy-tris-dimethylamino phosphonium hexafluorophosphate

  8. Tandem synthesis of conjugate SOC4([Nle11]-9-22) Boc/Bzl strategy on PAM (phenyl-acetamidomethyl) resin: 1. 50% TFA/DCM 2. Ac2O/DIEA/DMF Ac-(Lys-Aib-Gly)4-PAM Boc-(Lys-Aib-Gly)4-PAM Fmoc Fmoc 1. 40% piperidine/DMF 2. Boc-Leu-OH/DIC/HOBt 1. 50% TFA/DCM 2. 5% DIEA/DCM 3. Boc-Aaa(X)-OH 13x 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)L- - 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 ofhaloacyl group to the carrier B: -HCl R-SH + Cl-CH2-CO-NH-R’ R-S- CH2-CO-NH-R’ Thiazolidine ring: Ser in the carrier is converted to the glyoxyl moiety HS-CH2 CH2-S HO-CH2 NaIO4 NH2-CH-CO-R CH-CO-R’ O CH-CO-R’ NH2-CH-CO-R’ R-CO-CH-NH

  10. NH NH2 NH NH2 NH2 NH2 SPDP NH2 NH2 NH2 NH2 NH2 NH2 -OCO-(CH2)2-S-S O N N -OCO-(CH2)2-S-S- -OCO-(CH2)2-S-S- O HS Application of amino/thiol type heterobifunctional compounds SPDP N-succinimidyl-3-(2-pyridyldithio)-propionate Carlson et al. Biochem. J.173, 723 (1978) poly[Lys(DL-Alam)]; AK in buffer solution pH= 7.5-8.5

  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. N R-CO-CH-CH2-S-S- R’-HN Application of Cys(Npys) derivatives • 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 NH2 NH NH NH2 NH2 NH2 N O-CO-CH-CH2-S-S- HN NO2 CO F F O H3C CH3 F C 1. Boc-Cys(Npys)-OPfp in DMF-water (9:1) 2. 95%TFA-5%water CH3 Boc-Cys(Npys)-OPfp F F NH2 NH2 N NH2 NH2 NH2 NH2 NH2 NH2 -OCO-CH-CH2 -S -OCO-CH-CH2-S-S- S NO2 HS in buffer solution pH= 5.5

  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; NH3 NH2 NH2 NH3 NH2 NH2 • 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. + + - OH COO - OH COO NH3 NH2 NH2 NH2 NH3 NH2 - OH COO - OH 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) 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

  16. 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% 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 h100% 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 2H-LKNleADPNRFRGKDLC-NH2 3 H-CFRHDSGY-NH2 4 H-CGGGGGFRHDSGY-NH2 5H-FRHDSGYC-NH2 6 H-FRHDSGYGGGGGC-NH2 7 GlpHWSHDWK(H-C)PG-NH2 8 GlpHWSHDWK(Ac-C)PG-NH2 Mező, G., Manea, M. et al. J. Peptide Science10, 701 (2004)

  17. Conjugation of [Nle]11-9-22 epitope peptide from HSV gD-1 to SAK carrier molecule L-Ser oligo-DL-Ala polylysine NH-CO-CH2Cl 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 48.5 41.3 30.1 21.7 Subst.pept.(%) 44 nd nd 22 9 7 NH-CO-CH2 S H-Cys-OH H-9LKNleADPNRFRGKDL22C-NH2 Mező et al. Bioconjugate Chemistry14, 1260 (2003)

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

  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 H-9LKNleADPNRFRGKDL22C-NH2

  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) 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

  21. Fmoc Fmoc Fmoc Fmoc MAP core 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- 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- Multiple cyclic antigene peptide Spetzler, J.C., Tam, J.P. Peptide Research9, 290 (1996) Fmoc-Lys(Fmoc)-Lys(Fmoc-Lys(Fmoc))-Ser-Ser-b-Ala- R = Synthesis using Fmoc chemistry 1. 95%TFA-5%TIS 2. Fmoc-Ser(tBu)-OH DCC/HOBt in DMF

  22. Fmoc-Cys(StBu)- Antigen -Lys(Fmoc-Ser(tBu))-Asp(OtBu)-Pro- NH2-Cys(StBu)- Antigen -Lys(NH2-Ser)-Asp-Pro- NH2-Cys(StBu)- Antigen -Lys(O=CH-CO)-Asp-Pro- S CO Antigen NH Antigen -Lys-Asp-Pro- OH 1. 20% piperidine/DMF 2. TFA/TIS/thioanisole/water(92.5:2.5:2.5:2.5, V/V) NaIO4 in 10mM PBS solution (pH 6.8) then HPLC purification OH Tris-(2-carboxyethyl)phosphine 10mM Na-acetate buffer (pH 4.2), Rt, 48h OH = peptide derived from V3 loop of gp120 HIV

  23. 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) Number of atoms in the cycle 24 32 30 Preparation NH-DPEDSALL-CO see next slide air oxidation, 12h 0.1M Tris-buffer (pH 8) 0.1 mg/mL peptide conc. S S NH2-CDPEDSALLC-CONH2 CH2 S 0.1M Tris-buffer (pH 8) 3-4h 1-2mg/ml final concentration CO-NH-DPEDSALLC-CONH2 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%

  25. Solution conformation of linear and cyclic epitope peptidesderived from 272-279 region of HSV gD-1 CD-spectra of H-DPEDSALL-NH2, c(CH2-CO-DPEDSALLC)-NH2andH-c(CDPEDSALLC)-NH2 in TFE CD-spectra of H-DPEDSALL-NH2 linear peptide in water-TFE mixtures disulfide 100000 100000 water thioether 80000 TFE25 80000 linear 60000 TFE50 60000 TFE75 40000 -1 40000 -1 TFE 20000 dmol dmol 20000 0 2 2 0 -20000 / deg cm /deg cm -20000 -40000 -40000 Q Q -60000 -60000 -80000 -80000 -100000 CD spectra of H-LLDPEDSA-OH CD spectra of c(DPEDSALL) -100000 -120000 200 220 240 260 280 200 220 240 260 280 l /nm 20 l /nm 100 10 -1 50 -1 dmol dmol 0 2 2 0 /deg cm /deg cm -10 -50 TFE -3 -3 *10 TFE TFE50 *10 -20 Q Q TFE50 water -100 water -30 -150 180 200 220 240 260 280 200 220 240 260 280 l /nm l /nm

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

  27. BindingofmonoclonalantibodyDL6 to linear and cyclic epitope peptides of HSV gD-1 (Competition ELISA) 2 2 2 1,8 1,8 1,8 1,6 1,6 1,6 H - DPEDSALL - NH 1,4 1,4 1,4 2 c(CH2CO-DPEDSALLC)-NH2 1,2 1,2 1,2 c(CDPEDSALLC)-NH2 495 495 1 1 1 H-LLDPRDSALL-OH OD OD 0,8 0,8 0,8 9-22-Acp-C-272-279 0,6 0,6 0,6 260-284 0,4 0,4 0,4 267-281 0,2 0,2 0,2 0 0 0 1,95 1,95 3,9 3,9 7,8 7,8 15,6 15,6 31,3 31,3 62,5 62,5 125 125 250 250 500 500 1000 1000 2000 2000 n / n / pmol peptid pmol peptid e e ( ( DL 6 DL 6 : : 1:125000 1:125000 dilution) Target antigen: 0.5 mg 260- 284 peptide / well

  28. H-LLEDPVGTVA-NH2 100% 100% 80% 80% c(LLEDPVGTVA) (%) (%) 60% 60% Peptide Peptide H-c(CLLEDPVGTVAC)-NH2 40% 40% 20% 20% c(CH2CO-LLEDPVGTVAC)-NH2 0% 0% 0 0 60 60 120 120 180 180 Time (min) Time (min) Enzymatic cleavage of linear and cyclic peptides derived from 278-287 region of HSV gD-1 100% 100% H-LLEDPVGTVA-NH2 80% 80% 50% human serum c(LLEDPVGTVA) % % 60% 60% Peptide Peptide H-c(CLLEDPVGTVAC)-NH2 40% 40% 20% 20% c(CH2CO-LLEDPVGTVAC)-NH2 0% 0% 0 0 24 24 48 48 72 72 96 96 Time ( Time ( hours hours ) ) lysosoma Tugyi, R., Mező, G., et al. J. Peptide Science (in press)

  29. Meb Fmoc OcHex Boc-L-K-X-A-D-P-N-R-F-K-G-K-D-L-MBHA ClZ OcHex Tos ClZ Meb ClAc OcHex H-L-K-X-A-D-P-N-R-F-K-G-K-D-L-MBHA ClZ OcHex Tos ClZ SH ClAc H-L-K-X-A-D-P-N-R-F-K-G-K-D-L-NH2 Synthesis of cyclic derivatives of 9-22 sequence from HSV gD-1 1. deFmoc (2%DBU,2%piperidine/DMF) 2. ClAcOPcp(5equiv)/DMF 3. deBoc (33%TFA/DCM) HF-m-cresol–p-thiocresol (10mL:0.5mL:0.5g) 90 min, 0oC purification RP-HPLC 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 CH2 CH2 CH2 CH3 NH2 Cl-CH2-CO-NH CH2 CH2 S CH2 SH CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 --NH-CH-CO--- ---NH-CH-CO-- --NH-CH-CO--- ---NH-CH-CO-- Met Lys Hcy ClAc-Lys CH2 CO NH S - HCl CH2 CH2 --NH-CH-CO--- ---NH-CH-CO-- Hcy ------ ------ Lys

  31. CD-spectra and amide I peaks in FT-IR spectrum of cyclic epitope peptides H-LK[CADPNRFK]GKDL-NH2 H-LK[HcyADPNRFK]GKDL-NH2 water water-TFE (1:1) TFE water water-TFE (1:1) TFE - 1 (cm ) [%] n Peptide High - freqency Solvated b g - turns - turns region amides 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)

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

  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: Fmoc 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 Boc/Fmoc* Boc/Fmoc

  34. Boc-LK(ClZ)MAD(OcHex)PNR(Tos)FR(Tos)GK(ClZ)D(OcHex)LAhxK(Fmoc)AhxC(Meb)-MBHABoc-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 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)GFLG- Cyclisation in 0.1M Tris buffer (pH 8.0), 3-4h, RT CH2CO-LKMADPNRFRGKDLAhxKAhxC-NH2 Ac-C(Acm)GFLG-

  35. Reactivity of 9-22 epitope derivatives against A16 mAb Direct Competition H-LKMADPNRFRGKDL-NH2 4.0 2.4 H-LKNleADPNRFRGKDL-NH218.92.8 H-LKMADPNRFKGKDL-NH225.98.0 H-LKNleADPNRFKGKDL-NH225.4 5.0 H-LK[CADPNRFK(CH2CO)]GKDL-NH2>6000 7900 H-LK[HcyADPNRFK(CH2CO)]GKDL-NH24443 2300 [CH2CO-LKMADPNRFRGKDLAhxC]-NH2 59.1 28.6 [CH2CO-AhxLKMADPNRFRGKDLAhxC]-NH222.7 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 Data are in pmol range

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

  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 281-284 epitope of HSV gD1 (”guest”) Asp-Pro-Val-Gly (DPVG) Mező, G. et al. J. Peptide Research55, 7 (2000) Core epitope of MUC1 (”guest”) Pro-Asp-Thr-Arg (PDTR) Drakopoulou, E., Mező, G. et al. J. Peptide Science6, 175 (2000) Succesfull synthesis if the conformation ”host” and (”guest”) sequence is similar.

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

  39. Synthesis of oligomers of epitope peptides MUC-1: bulid upfrom 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. 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

  41. 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 1. EDC/HOBt in DMF 2. HF-p-cresol (95:5) Choc-VTSAPDTRPAPGSTAPPAHG-OH H-VTSAPDTRPAPGSTAPPAHG-NH2 H-VTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHG-NH2

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

  43. Competition ELISA using C595 mAb H-APDTRPAPG-NH2 56.3 mmol/dm3 H-APDTRPAPGC-NH2 53.2 mmol/dm3 H-APDTRPAPGC-NH2 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

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

More Related