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Chemoenzymatic synthesis of new fluorogenous substrates for cysteine proteases of the papain family

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Abstract

A chemoenzymatic synthesis was developed for new highly specific fluorogenic substrates for cysteine proteases of the papain family, Abz-Phe-Ala-pNA (I) and Glp-Phe-Ala-Amc (II) (Abz, pNA, Glp, and Amc are o-aminobenzoyl, p-nitroanilide, pyroglutamyl, and 4-amino-7-methylcoumaride, respectively). Substrate (I) was obtained in an aqueous-organic medium using native chymotrypsin. Substrate (II) was synthesized in DMF-MeCN by the treatment with chymotrypsin and subtilisin Carlsberg immobilized on polyvinyl alcohol cryogel. Hydrolysis of substrate (I) with papain, ficin, and bromelain was accompanied by a 15-fold increase in fluorescence intensity, and that of substrate (II), by a change in the fluorescence spectrum. Unambiguity of enzymatic hydrolysis of the substrates after the Ala residue was shown. The specific activity of the substrate hydrolysis with papain, bromelain, and ficin and was determined. Papain showed the greatest activity for both substrates. The activity of all proteases under study was essentially higher for substrate (II), than for substrate (I). The lowest detectable papain concentrations were 2.4 × 10−10 M for (I) and 1.2 × 10−11 M for (II). A high selectivity of cysteine proteases for Glp-Phe-Ala-Amc was established.

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Abbreviations

Abz-OH:

o-aminobenzoic acid

Amc:

4-amino-7-methylcoumaride

ChT and iChT:

native and immobilized chymotrypsin

DCC:

N,N′-dicyclohexylcarbodiimide

Glp:

pyroglutamyl

HOBt:

N-hydroxybenzotriazole

iSL:

immobilized subtilisin

pNA:

p-nitroanilide

PVA:

polyvinyl alcohol

TEA:

triethylamine. All amino acids are of L-series

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Authors and Affiliations

  1. Faculty of Bioengineering and Bioinformatics, Moscow State University, Vorob’evy gory, Moscow, 119991, Russia

    T. A. Semashko

  2. Faculty of Chemistry, Moscow State University, Vorob’evy gory, Moscow, 119991, Russia

    E. N. Lysogorskaya, E. S. Oksenoit, A. V. Bacheva & I. Yu. Filippova

Authors
  1. T. A. Semashko
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  2. E. N. Lysogorskaya
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  3. E. S. Oksenoit
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  4. A. V. Bacheva
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  5. I. Yu. Filippova
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Corresponding author

Correspondence to I. Yu. Filippova.

Additional information

Original Russian Text © T.A. Semashko, E.N. Lysogorskaya, E.S. Oksenoit, A.V. Bacheva, I.Yu. Filippova, 2008, published in Bioorganicheskaya Khimiya, 2008, Vol. 34, No. 3, pp. 376–381.

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Semashko, T.A., Lysogorskaya, E.N., Oksenoit, E.S. et al. Chemoenzymatic synthesis of new fluorogenous substrates for cysteine proteases of the papain family. Russ J Bioorg Chem 34, 339–343 (2008). https://doi.org/10.1134/S1068162008030151

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  • DOI: https://doi.org/10.1134/S1068162008030151

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