Biomedical Chemistry: Research and Methods, 2018, 1(4), e00057

An Improved Procedure for the Preparation of Thrombin Low Molecular Weight Substrates - Peptide p-Nitroanilides

A.A Chistov1,2, A.V. Talanova1, M.V. Melnikova1, S.S. Kuznetsova1, E.F. Kolesanova1*

1Institute of Biomedical Chemistry, 10 Pogodinskaya str., Moscow119121 Russia; *e-mail: EKolesanova@yandex.ru
2Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry RAS, 16/10 Miklukho-Maklaya str.,
Moscow GSP-7, 117997 Russia

Key words: chromogenic substrates; p-nitroanilides; solid-phase peptide synthesis; Bio-Beads SM-2; adsorption; thrombin

DOI:10.18097/BMCRM00057

The whole version of this paper is available in Russian.

Low molecular weight chromogenic thrombin peptide substrates, p-nitroanilides of short peptides protected at their N-terminal amino group, were prepared by solid-phase peptide synthesis on polystyrene-divinylbenzene polymer with trityl groups with preliminary attached p-phenylene diamine moiety. After the cleavage from the resin peptide p-aminoanilides were mildly oxidized to p-nitroanilides with the mixture of potassium sulfate and persulfate. Adsorption onto polymer support Bio-Beads SM-2 with further elution by acetonitrile allowed easy separating peptide p-nitroanilides from the oxidizer and obtaining the thrombin chromogenic substrate preparations with the target substance contents of not less than 95% and yields of 30 - 40%. Thrombin effectively catalyzed hydrolysis of the prepared substrates with KM and Vmax values of 29 - 134 µM and 0.03 - 1/16 µM/s, respectively.

CLOSE
Table 1. Characteristics of synthesized peptide p-nitroanilide thrombin substrates.

CLOSE
Table 2. Kinetic parameters of the synthesized substrate hydrolysis under the thrombin action. .

ACKNOWLEDGEMENTS

Authors are grateful to Ms. Natalia A. Kazmina and Ms. Alena V. Kolesnichenko for excellent technical assistance. Element analysis of the p-phenylenediamine-modified resin was made at the Institute of Organic Chemistry RAS/ Peptide synthesis was performed on the equipment of the IBMC Core Facility @Human proteome@, which was supported by the Russian Ministry of Education and Science in the frame of the Agreement No. 14.621.21.0017 (project unique identifier RFMEFI62117X0017). The work was made in the frame of the Program of Basic Scientific Research for State Academies of Sciences for 2013-2020.

SUPPLEMENTARY

Supplementary materials are available at http://dx.doi.org/10.18097/BMCRM00057

REFERENCES

  1. Semashko T.A., Vorotnikova E.A., Sharikova V.F., Vinokurov K.S., Smirnova Y.A., Dunaevsky Y.E., Belozersky M.A., Oppert B., Elpidina E.N., Filippova I.Y. (2014) Selective chromogenic and fluorogenic peptide substrates for the assay of cysteine peptidases in complex mixtures, Anal. Biochem. 449, 179–187. DOI
  2. Rijkers D.T.S., Adams H.P.H.M., Hemker H.C., Tesser G.I. (1995) A convenient synthesis of amino acid p-nitroanilides; synthons in the synthesis of protease substrates, Tetrahedron, 51(41), 11235–11250. DOI
  3. Loeffen R., Winckers K., Ford I., Jukema J.W., Robertson M., Stott D.J., Lowe G.D. (2014) Associations Between Thrombin Generation and the Risk of Cardiovascular Disease in Elderly Patients: Results From the PROSPER Study, J.Gerontol.A Biol.Sci.Med.Sci. 1758-535X (Electronic), 982–988. DOI : 10.1093/gerona/glu228 DOI
  4. Sheehan J.J., Tsirka S.E. (2005) Fibrin-modifying serine proteases thrombin, tPA, and plasmin in ischemic stroke: A review, Glia 50(4), 340–350. DOI
  5. Walker C.P.R., Royston D. (2002) Thrombin generation and its inhibition: a review of the scientific basis and mechanism of action of anticoagulant therapies, Br. J. Anaesth. 88(6), 848–863. DOI
  6. Backes B.J., Harris J.L., Leonetti F., Craik C.S., Ellman J.A. (2000) Synthesis of positional-scanning libraries of fluorogenic peptide substrates to define the extended substrate specificity of plasmin and thrombin, Nat. Biotechnol. 18(2), 187–193. DOI
  7. Rijkers D.T., Wielders S.J., Tesser G.I., Hemker H.C. (1995) Design and synthesis of thrombin substrates with modified kinetic parameters, Thromb. Res. 79(5-6), 491–499. DOI
  8. Erlanger B.F., Kokowsky N., Cohen W. (1961) The preparation and properties of two new chromogenic substrates of trypsin, Arch. Biochem. Biophys. 95, 271-278. DOI
  9. Kaspari A., Schierhorn A., Schutkowski M. (1996) Solid-phase synthesis of peptide-4-nitroanilides, Int. J. Pept. Protein Res. 48(5), 486-494. DOI
  10. Bernhardt A., Drewello M., Schutkowski M. (1997) The solid-phase synthesis of side-chain-phosphorylated peptide-4-nitroanilides, J. Pept. Res. 50(2), 143-152. DOI
  11. Alsina J., Yokum T.S., Albericio F., Barany G. (1999) Backbone Amide Linker (BAL) Strategy for N(alpha)-9-Fluorenylmethoxycarbonyl (Fmoc) Solid-Phase Synthesis of Unprotected Peptide p-Nitroanilides and Thioesters, J. Org. Chem., 64(24), 8761-8769. DOI
  12. Kwon Y., Welsh K., Mitchel, A.R., Camarero J.A. (2004) Preparation of peptide p-nitroanilides using an aryl hydrazine resin, Org. Lett. 6(21), 3801-3804. DOI
  13. Burdick P.J., Struble M.E., Burnier J. (1993) Solid Phase Synthesis of Peptide para -Nitroanilides, Tetrahedron Lett., 34, 2589–2592. DOI
  14. Mergler M., Dick F., Gosteli J., Nyfeler R. (2000) AProtected peptide p-nitroanilides by solid-phase synthesis, Lett. Pept. Sci. 7, 1. DOI
  15. Abbenante G., Leung D., Bond T., Fairlie D.P. (2001) An efficient Fmoc strategy for the rapid synthesis of peptide para-nitroanilides, Lett. Pept. Sci., 7, 347–351. DOI
  16. Bio-Beads® SM Hydrophobic and Polar Interaction Adsorbents Instruction Manual. Bio-Rad Laboratories.