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Microwave-assisted solid-phase peptide synthesis of the 60–110 domain of human pleiotrophin on 2-chlorotrityl resin

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Abstract

A fast and efficient microwave-assisted solid phase peptide synthesis (MW-SPPS) of a 51mer peptide, the main heparin-binding site (60–110) of human pleiotrophin (hPTN), using 2-chlorotrityl chloride resin (CLTR-Cl) following the 9-fluorenylmethyloxycarbonyl/tert-butyl (Fmoc/tBu) methodology and with the standard N,N′-diisopropylcarbodiimide/1-hydroxybenzotriazole (DIC/HOBt) coupling reagents, is described. An MW-SPPS protocol was for the first time successfully applied to the acid labile CLTR-Cl for the faster synthesis of long peptides (51mer peptide) and with an enhanced purity in comparison to conventional SPPS protocols. The synthesis of such long peptides is not trivial and it is generally achieved by recombinant techniques. The desired linear peptide was obtained in only 30 h of total processing time and in 51% crude yield, in which 60% was the purified product obtained with 99.4% purity. The synthesized peptide was purified by reversed phase high performance liquid chromatography (RP-HPLC) and identified by electrospray ionization mass spectrometry (ESI-MS). Then, the regioselective formation of the two disulfide bridges of hPTN 60–110 was successfully achieved by a two-step procedure, involving an oxidative folding step in dimethylsulfoxide (DMSO) to form the Cys77–Cys109 bond, followed by iodine oxidation to form the Cys67–Cys99 bond.

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Abbreviations

Acm:

Acetamidomethyl

ACN:

Acetonitrile

AcOH:

Acetic acid

Boc:

tert-Butyloxybarbonyl

1-BuOH:

N-butanol

Bzl:

Benzyl

CLTR-Cl:

2-Chlorotrityl chloride

DCM:

Dichloromethane

DIC:

N,N′-diisopropylcarbodiimide

DIPEA:

Diisopropylethyamine

DMF:

N,N′-dimethylformamide

DMSO:

Dimethylsulfoxide

EDT:

1,2-Ethanedithiol

ESI-MS:

Electrospray ionization mass spectrometry

Fmoc:

9-Fluorenylmethyloxycarbonyl

HOBt:

1-Hydroxybenzotriazole

hPTN:

Human pleiotrophin

i-PrOH:

Isopropanol

MeOH:

Methanol

MW-SPPS:

Microwave-assisted solid-phase peptide synthesis

PTN:

Pleiotrophin

rhPTN:

Recombinant hPTN

tBu:

tert-Butyl

RP-HPLC:

Reversed phase high performance liquid chromatography

TFA:

Trifluoroacetic acid

TFE:

2,2,2-Trifluoroethanol

Tol:

Toluene

Trt:

Trityl

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Acknowledgments

This work was supported by the E.U.-European Social Fund (75%) and the Greek Ministry of Development-GSRT (25%) (Grant PENED2003, 036Δ560). Special thanks to Eldrug SA for providing access to CEM Liberty automated microwave peptide synthesizer.

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Authors and Affiliations

  1. Department of Chemistry, University of Patras, 265 04, Patras, Greece

    Irene Friligou, Dimitrios Gatos, John Matsoukas & Theodore Tselios

  2. Laboratory of Molecular Pharmacology, Department of Pharmacy, University of Patras, 265 04, Patras, Greece

    Evangelia Papadimitriou

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  1. Irene Friligou
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  2. Evangelia Papadimitriou
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  3. Dimitrios Gatos
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  4. John Matsoukas
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  5. Theodore Tselios
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Correspondence to Theodore Tselios.

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Friligou, I., Papadimitriou, E., Gatos, D. et al. Microwave-assisted solid-phase peptide synthesis of the 60–110 domain of human pleiotrophin on 2-chlorotrityl resin. Amino Acids 40, 1431–1440 (2011). https://doi.org/10.1007/s00726-010-0753-6

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