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

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

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


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-




Multiple cyclic antigene peptideSpetzler, J.C., Tam, J.P. Peptide Research 9, 290 (1996)


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


CH2

CH2

CH3

S

CH2

CH2

CH2

CH2

NH2

Met Lys Hcy ClAc-Lys


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


Ac-C(Acm)GFLG-

I2 or Tl(tfa)3oxidation


Ac-CGFLG-


Ac-CGFLG-

DTT


Ac-CGFLG-


Ac-CGFLG-

Ag-triflate

carrierAc-[TKPKG]4-NH2

CH2CO


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


MUC1 dimer synthesis by chemical ligation

H-VTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHG-NH2

CH2CO-VTSAPDTRPAPGSTAPPAHG-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)

Peptide conjugation and cyclisation chemistry for synthetic antigen development

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