Chemical ligation procedures

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Chemical ligation procedures in peptide and
protein chemistry
Gábor Mezo Research Group of Peptide
Chemistry Hungarian Academy of Sciences Eötvös L.
University Budapest, Hungary ERASMUS Teaching
Mobility Grant Konstanz 30th October 2008
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Synthesis of peptide dendrimers with oxime,
hydrazone, and thiazolidine linkages
J Shao, JP Tam JACS 117, 3893 (1995)
O
NH2OCH2C- VA20
(OxmVA20)4-TLCP
CHOCO
Lys
CHOCO
O
O
O
Lys-Ala-OH
VA20 -C(CH2)2CNHNC-C)4-TLCP
(HydVA20)4-TLCP
Lys
CHOCO
O
SH
NH2CH-C- VA20
CHOCO
O
O
(HC-C)4-TLCP
(ThzVA20)4-TLCP
VA20 VMEYKARRKRAAIHVMLALA model peptide with
helical structure
1. NaOH 2. HCl
(MeO)2CHCO
Boc
Lys
(MeO)2CHCO
Lys
Boc
1. TFA 2. (MeO)2CH2COONaBOP/DIEA
Lys-Ala-OMe
Lys-Ala-OMe
Lys
(MeO)2CHCO
Boc
Lys
Boc
(MeO)2CHCO
(dimethyl acetal form)
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-NH-CH2-CONH2
NH2-CH2-CO-
CH2
CH2
S
S
T-S-S-B disulfide
T-cell (Cys(Pys) either at N- or C-terminus) and
B-cell (Cys at N-terminus)
O
O
B-S-S-T disulfide
N
H
N
H
2
2
O
H
O
H
NaIO4
N
H
O
N
H
O
N
H
O
2
O
H
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Native chemical ligation with Asp or Glu as
C-terminal residues
M. Villain et al. Eur. J. Org. Chem. 3267 (2003)

• After native chemical ligation 20-30 isomer (b-
  (Asp), g (Glu) peptide bond)
• Migration of thioester moiety
• The side chain of Asp or Glu must be protected
• Boc-chemistry is prefered for the synthesis of
  thioester
• Side chain protecting groups must be stable
  under acidic conditions
• Clevable by base or reduction (hydrogenation)

In case of Glu
Cleavage of BrZ from Tyr OFm alkylation can occur!
O-CO-
9-fluorenylmethyl ester (OFm)
0.1M Na2CO3-10 2-mercaptoethanol (pH 9) for 2h,
37oC
20 piperidine-20 DMF-10 2-mercaptoethanol (pH
13) for 15 min, RT
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Native Chemical Ligation at DBSB
size synthetized failed
41-70 45 1
71-100 37 5
101-130 24 5
131-164 9 1
unpublished data from Hubert Gaertner and Paolo
Botti
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Production of a thioester of the N-terminal
fragment
1. Express in E. coli 2. Affinity
purification
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