Letters in Peptide Science

, Volume 10, Issue 5–6, pp 589–596 | Cite as

Chemical synthesis of kurtoxin, a T-type calcium channel blocker

  • Hideki Nishio
  • Yuji Nishiuchi
  • Masanori Ishimaru
  • Terutoshi Kimura
Article

Summary

Kurtoxin isolated from the venom of scorpion,Parabuthus transvaalicus, is a 63-residue peptide with four intramolecular disulfide bonds which inhibits low-threshold T-type Ca2+ channels. Kurtoxin was synthesized by native chemical ligation involving the coupling of (1–26)-thioester peptide and Cys27-(28–63)-peptide. The former was synthesized by standard solid-phase peptide synthesis (SPPS) with Boc chemistry, while the latter was sequentially assembled from three protected segments onto a resin-bound C-terminal segment in a chloroform-phenol mixed solvent followed by deprotection reaction using HF. Each protected segment used for the coupling on a solid support was prepared on anN-[9-(hydroxymethyl)-2-fluorenyl] succinamic acid (HMFS) resin and detached from the resin by treatment with 20% Et3N in DMF to produce it in the form of an α-carboxylic acid. Synthetic kurtoxin obtained after the oxidative folding reaction was found to be identical with the natural product by means of several analytical procedures, and its disulfide structure was determined for the first time to be Cys12-Cys61, Cys16-Cys37, Cys23-Cys44 and Cys27-Cys46 by peptide mapping, sequence analysis and mass measurements.

Key words

a mixture of chloroform and phenol DIC/HOAt method epimerization kurtoxin segment condensation reaction T-type calcium channel 

Abbreviations

Boc

tert. butoxycarbonyl

BrZ

2-bromobenzyloxycarbonyl

Bzl

benzyl

CHCA

α-cyano-4-hydroxy cinaminic acids

cHx

cyclohexyl

ClZ

2-chlorobenzyloxycarbonyl

CSPPS

convergent solid phase peptide synthesis

CZE

capillary zone electrophoresis

DIEA

N, N-diisopropylethylamine

DMF

N, N-dimethylformamide

EDC

1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

Et3N

triethylamine

For

formyl

HATU

N-[(dimethylamino)-1H-1,2,3-trtiazolo[4,5-b]pyridine-1-ylmethylene]-N-methyl-methanaminium hexafluorophosphateN-oxide

HBTU

N-[(1H-benzotriazol-1-yl) (dimethylamino)methylmethanaminium hexafluorophosphateN-oxide

HFIP

1,1,1,3,3,3-hexafluoro-2-propanol

HMFS

N-[9-(hydroxymethyl)-2-fluorenyl] succinamic acid

HOAt

1-hydroxy-7-azabenzotriazole

HOBt

1-hydroxybenzotriazole

HOOBt

3,4-dihydro-3-hydroxy-4-oxo-benzotriazine

4-MeBzl

4-methylbenzyl

NMP

1-methyl-2-pyrrolidinone

SPPS

solid phase peptide synthesis

TFA

trifluoroacetic acid

Tos

p-toluenesulfonyl

Trt

trityl

Xan

xanthenyl

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References

  1. 1.
    Uchitel, O.D., Toxicon, 35 (1997) 1161.PubMedCrossRefGoogle Scholar
  2. 2.
    Meves, H., Simard, J.M. and Watt, D.D., Ann. NY Acad. Sci., 479 (1986) 113.PubMedGoogle Scholar
  3. 3.
    Chuang, R.S., Jaffe, H., Cribbs, L. Perez-Reyes, E. and Swarz, K.J., Nat. Neurosci., 1 (1998) 668.PubMedCrossRefGoogle Scholar
  4. 4.
    Sidach, S.S. and Mintz, I.M., J. Neurosci, 22 (2002) 2023.PubMedGoogle Scholar
  5. 5.
    Dawson, P.E., Muir, T.W., Clark-Lewis, I. and Kent, S.B.H., Seience, 266 (1994) 776.Google Scholar
  6. 6.
    Schnoelzer, M., Alewood, P., Jones, A., Alewood, D. and Kent, S.B.H., Int. J. Pept. Protein Res., 40 (1992) 180.Google Scholar
  7. 7.
    Lyoyd-Williams, P., Albericio, F., Giralt, E., Chemical Approach to the Synthesis of Peptides and Proteins. CRC Press LLC, Boca Raton, FL, 1997.Google Scholar
  8. 8.
    Tomalia, D.A., Naylor, A.M. and Goddard, W.A., Angew. Chem. Int. Ed. Engl., 29 (1990) 138.CrossRefGoogle Scholar
  9. 9.
    Nishiuchi, Y., Nishio, H., Inui, T. and Kimura, T., in E. Benedetti and C. Pedone (Eds.), PEPTIDE 2002. Edizioni Ziino, Napoli, Italy, 2002, pp. 236–237.Google Scholar
  10. 10.
    Inui, T., Bódi, J., Nishio, H., Nishiuchi, Y. and Kimura, T., Lett. in Pept. Sci., 8 (2002) 319.CrossRefGoogle Scholar
  11. 11.
    Hackeng, T., Griffin, J.H. and Dawson, P.E., Proc. Natl. Acad. Sci. USA, 96 (1999) 10068.PubMedCrossRefGoogle Scholar
  12. 12.
    Rabanal, F., Giralt, E., Albericio, F., Tetrahedron, 51 (1995) 1449.CrossRefGoogle Scholar
  13. 13.
    Nishiuchi, Y., Nishio, H., Inui, T., Bódi, J., Kimura, T., J. Pept. Sci., 6 (2000) 84.PubMedCrossRefGoogle Scholar
  14. 14.
    Nishiuchi, Y., Nishio, H., Inui, T. Kimura, T. and Sakakibara, S., Tetrahedron Lett., 37 (1996) 7529.CrossRefGoogle Scholar
  15. 15.
    Bódi, J., Nishiuchi, Y., Nishio, H., Inui, T. and Kimura, T., Tetrahedron Lett., 39 (1998) 7117.CrossRefGoogle Scholar
  16. 16.
    König, W. and Geiger, R., Chem Ber., 103 (1970) 2034.PubMedGoogle Scholar
  17. 17.
    Carpino, L.A., J. Am. Chem. Soc., 115 (1993) 4397.CrossRefGoogle Scholar
  18. 18.
    Bouhauala-Zahar, B., Benkhalifa, R. Srairi, N., Zenouaki, I., Ligny-Lemaire, C., Drenet, P., Sampieri, F., Pelhate, M., Ayeb, M., Ménez, A., Karoui, H. and Ducancel, F., Eur. J. Biochem., 269 (2002) 2831.CrossRefGoogle Scholar

Copyright information

© Kluwer Academic Publishers 2004

Authors and Affiliations

  • Hideki Nishio
    • 1
  • Yuji Nishiuchi
    • 1
  • Masanori Ishimaru
    • 1
  • Terutoshi Kimura
    • 1
  1. 1.Protein Research FoundationPeptide Institute Inc.OsakaMinoh-shiJapan

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