RU2717689C2 - Peptide substrates of cysteine peptides of family of papain - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to the field of bioorganic chemistry and relates to new substrates for determination of enzymatic activity of peptidases. Disclosed is a peptide substrate of cysteine peptides of the family of papain, which is a compound of general formula (I) A-Xaa-Gln-B, where A is an N-protective group which is Glp, Xaa is an amino acid Phe, B is a marker fluorogenic group which is 7-amido-4-methylkumaride (AMS) or 4-nitroanilide (pNA).

EFFECT: peptide substrate is used to create test systems for detecting peptidases, which is carried out in solution, in gel after electrophoresis in native conditions, in systems in vivo.

2 cl, 1 dwg, 2 tbl, 9 ex

Description

Technical field

The invention relates to the field of bioorganic and biomedical chemistry, biochemistry, enzymology, cytology, and relates to new substrates for determining the enzymatic activity of peptidases, their preparation, use as highly specific and selective chromogenic and fluorogenic substrates for cysteine peptidases of the papain family (in the presence of enzymes of other clans), applications for specific testing of cathepsin L in natural mixtures of various cathepsins; applications for selective testing of cathepsin L activity using fluorogenic compounds in in vivo systems, use in the development of test systems and testing the activity of cysteine peptidases C1 family directly in the gel, use for the search glutaminspetsifichnyh peptidases having an ability to cleave the gliadins, and their toxic peptide ..

Technical field

Peptidases (proteases) play a key role in all metabolic processes. The enzymatic activity of peptidases is determined using various substrates. The most convenient and effective among them are peptide substrates that are highly specific and selective for a specific group of enzymes.

Ovarian cysteine proteinases in the teleost Fundulus heteroclitus: Molecular cloning and gene expression during vitellogenesis and oocyte maturation, Mol. Reproduct. Development, 67 (2004) 282-294;

C. Delbarre-Ladrat, M. Lamballerie-Anton, V. Verrez-Bagnis, Calpain and cathepsin activities in post mortem fish and meat muscles, Food Chemistry 101 (2007) 1474-1479; S. Subramanian, S. MacKinnona, N. Rossa, A comparative study on innate immune parameters in the epidermal mucus of various fish species, Comp. Biochem. Physiol. 148B (2007) 256-263; O. Vasiljeva, T. Reinheckel, C. Peters, D. Turk, V. Turk, B. Turk. Emerging roles of cysteine cathepsins in disease and their potential as drug targets. Curr. Pharm.Design, 13 (2007) 385-401; V. Turk, V. Stroka, O. Vasiljeva. M. Renko, T. Sun, B. Turk, D. Turk, Cysteine cathepsins: from structure, function and regulation to new frontiers, Biochim. Biophys. Acta (BBA)-Proteins and Proteomics 1824 (2012) 68-88). В настоящее время серьезная роль отводится пищеварительным цистеиновым пептидазам насекомых, в том числе и таких опасных вредителей, как колорадский жук, большой и малый мучные хрущаки, тараканы и т.д. (Р.Т. Cristofoletti, A.F. Ribeiro, W.R. Terra, The cathepsin L-like proteinases from the midgut of Tenebrio molitor larvae: sequence, properties, immunocytochemical localization and function. Insect Biochem. Mol. Biol. 35 (2005) 883-901; K. Gruden, A.G. Kuipers, G. Guncar, N. Slapar, B. Strukelj, M.A. Jongsma, Molecular basis of Colorado potato beetle adaptation to potato plant defence at the level of digestive cysteine proteinases, Insect Biochem. Mol. Biol. 34 (2004) 365-375; K.S. Vinokurov, E.N. Elpidina, B. Oppert, S. Prabhakar, D.P. Zhuzhikov, Y.E. Dunaevsky, M.A. Belozersky, Fractionation of digestive proteinases from Tenebrio molitor (Coleoptera: Tenebrionidae) larvae and role in protein digestion, Comp. Biochem. Physiol. 145B (2006) 138-146; K.S. Vinokurov, E.N. Elpidina, D.P. Zhuzhikov, B. Oppert, D. Kodrik, F. Sehnal, Digestive proteolysis organization in two closely related Tenebrionid beetles: red flour beetle (Tribolium castaneum) and confused flour beetle (Tribolium confusum), Arch. Insect Biochem. Physiol. 70 (2009) 254-279).Cysteine peptidases constitute one of the main classes of proteolytic enzymes and are characterized by the presence of a Cys residue in the active center. Most cysteine peptidases currently known belong to the C1 family, also called the papain family (ND Rawlings, AJ Barrett, Introduction: The Clans and Families of Cysteine Peptidases, in: ND Rawlings, GS Salvesen (Eds.), Handbook of Proteolytic Enzymes, Third edition, Academic Press, London, 2013, v. 2, pp. 1743-1776). Cysteine peptidases of the C1 family were found in viruses (GN Rudenskaya, DV Pupov, Cysteine proteinases of microorganisms and viruses, Biochemistry (Mosc). 73 (2008) 1-13), bacteria (J. Potempa, R. Pike, J. Travis, The multiple forms of trypsin-like activity present in various strains of Porphyromonas gingivalis are due to the presence of either Arg-gingipain or Lys-gingipain, J. Infect. Immunol. 63 (1995) 1176-1182), protozoa (C. Serveau , G. Lalmanach, MA Juliano, J. Scharfstein, L. Juliano, F. Gauthier, Investigation of the substrate specificity of cruzipain, the major cysteine proteinase of Trypanosoma cruzi, through the use of cystatin-derived substrates and inhibitors, Biochem. J 313 (1996) 951-956; J. Cazzulo, V Stoka, V. Turk, The major cysteine proteinase of Trypanosoma cruzi: a valid target for chemotherapy of Chagas disease, Curr Pharm Des. 7 (2001) 1143-1156; DC Greenbaum, A. Baruch, M. Grainger, Z. Bozd ech, KF Medziharadszky, J. Engel, J. DeRisi, AA Holder, M. Bogyo, A role for the protease falcipain 1 in host cell invasion by the human malaria parasite, Science 298 (2002) 2002-2006; M. Sajid, JH McKerrow, Cysteine proteases of parasitic organisms, Mol. Biochem. Parasitol. 120 (2002) 1-21; YA Sabnis, PV Desai, PJ Rosenthal, MA Avery, Probing the structure of falcipain-3, a cysteine protease from Plasmodium falciparum: Comparative protein modeling and docking studies, Protein Science 12 (2003) 501-509; CR Caffrey, AP Lima, D. Steverding, Cysteine peptidases of kinetoplastid parasites, Adv Exp Med Biol. 712 (2011) 84-99; ZZ Dou, VB Carruthers, Cathepsin proteases in Toxoplasma gondii, Adv. Exp.Med. Biol. 712 (2011) 49-61), plants (H H.-H. Otto, T. Schirmeister, Cysteine proteases and their inhibitors, Chem. Rev. 97 (1997) 133-171; DF Gruis, DA Selinger, JM Curran, R. Jung, Redundant proteolytic mechanisms process seed storage proteins in the absence of seed-type members of the vacuolar processing enzyme family of cysteine proteases, Plant Cell. 14 (2002) 2863-2882; VK Dubey, MV Jagannadham, Procerain, a stable cysteine protease from the latex of Calotropis procera, Phytochemistry 62 (2003) 1057-1071) and vertebrates (M. Yamashita & S. Konagaya, High activities of cathepsins B, D, H and L in the white muscle of chum salmon in spawning migration, Comp. Biochem. Physiol. 95B, (1990) 149-152, H.-H. Otto, T. Schirmeister, Cysteine proteases and their inhibitors, Chem. Rev. 97 (1997) 133-171; TS Uinuk-Ool , N. Takezaki, N. Kuroda, F. Figueroa, A. Sato, IE Samonte, WE Mayer, J. Klein, Phylogeny of Antigen-Processing Enzymes: Cathepsins of a Cephalochordat e, an Agnathan and a Bony Fish, Scand. J. Immunol. 58 (2003) 436-448; M. Fabra &

Ovarian cysteine proteinases in the teleost Fundulus heteroclitus: Molecular cloning and gene expression during vitellogenesis and oocyte maturation, Mol. Reproduct. Development, 67 (2004) 282-294;

C. Delbarre-Ladrat, M. Lamballerie-Anton, V. Verrez-Bagnis, Calpain and cathepsin activities in post mortem fish and meat muscles, Food Chemistry 101 (2007) 1474-1479; S. Subramanian, S. MacKinnona, N. Rossa, A comparative study on innate immune parameters in the epidermal mucus of various fish species, Comp. Biochem. Physiol. 148B (2007) 256-263; O. Vasiljeva, T. Reinheckel, C. Peters, D. Turk, V. Turk, B. Turk. Emerging roles of cysteine cathepsins in disease and their potential as drug targets. Curr. Pharm.Design, 13 (2007) 385-401; V. Turk, V. Stroka, O. Vasiljeva. M. Renko, T. Sun, B. Turk, D. Turk, Cysteine cathepsins: from structure, function and regulation to new frontiers, Biochim. Biophys. Acta (BBA) -Proteins and Proteomics 1824 (2012) 68-88). At present, insect digestive cysteine peptidases play a serious role, including such dangerous pests as the Colorado potato beetle, large and small flour flies, cockroaches, etc. (R.T. Cristofoletti, AF Ribeiro, WR Terra, The cathepsin L-like proteinases from the midgut of Tenebrio molitor larvae: sequence, properties, immunocytochemical localization and function. Insect Biochem. Mol. Biol. 35 (2005) 883-901 ; K. Gruden, AG Kuipers, G. Guncar, N. Slapar, B. Strukelj, MA Jongsma, Molecular basis of Colorado potato beetle adaptation to potato plant defense at the level of digestive cysteine proteinases, Insect Biochem. Mol. Biol. 34 (2004) 365-375; KS Vinokurov, EN Elpidina, B. Oppert, S. Prabhakar, DP Zhuzhikov, YE Dunaevsky, MA Belozersky, Fractionation of digestive proteinases from Tenebrio molitor (Coleoptera: Tenebrionidae) larvae and role in protein digestion, Comp Biochem. Physiol. 145B (2006) 138-146; KS Vinokurov, EN Elpidina, DP Zhuzhikov, B. Oppert, D. Kodrik, F. Sehnal, Digestive proteolysis organization in two closely related Tenebrionid beetles: red flour beetle (Tribolium castaneum ) and confused flour beetle (Tribolium confusum), Arch. Insect Bio chem. Physiol. 70 (2009) 254-279).

The most extensive group of the C1 family is cysteine peptidases of mammals and humans, the so-called cathepsins (DP Dickinson, Cysteine peptidases of mammals: their biological roles and potential effects in the oral cavity and other tissues in health and disease, Crit. Rev. Oral Biol. Med . 13 (2002) 238-275). According to bioinformatics analysis of the genome, only 11 different cysteine cathepsins B, C, F, H, K, L, O, S, V (L2), X, W are known in humans today. The main function of cysteine cathepsins is non-specific lysosomal protein degradation (ND Rawlings, AJ Barrett, Introduction: The Clans and Families of Cysteine Peptidases, in: ND Rawlings, GS Salvesen (Eds.), Handbook of Proteolytic Enzymes, Third edition, Academic Press, London, 2013, v. 2, pp. 1743-1776). However, their participation in more specific processes has also been shown: regulation of the cell cycle (V. Turk, V. Stroka, O. Vasiljeva. M. Renko, T. Sun, B. Turk, D. Turk, Cysteine cathepsins: from structure, function and regulation to new frontiers, Biochim. Biophys. Acta (BBA) -Proteins and Proteomics 1824 (2012) 68-88), the processing of certain proteins, enzymes and hormones (Ueno T., Linder S., Rice WR, Johansson J., Weaver T.E. Processing of pulmonary surfactant protein B by napsin and cathepsin H, J. Biol. Chem. 279 (2004) 16178-16184), presentation of antigens (LC Hsing, AY Rudensky. The lysosomal cysteine proteases in MHC class II antigen presentation , Immunol. Rev. 207 (2005) 229-241).

D. Ko, С. Wei, J. Henderson, Е. del Re, L. Hsing, A. Erickson, C. Cohen, M. Kretzler, D. Kerjaschki, A. Rudensky, B. Nikolic, J. Reiser, Proteolytic processing of dynamin by cytoplasmic cathepsin L is a mechanism for proteinuric kidney disease. J. Clin. Invest. (2007) 117, 2095-104). Катепсины участвуют также в таких воспалительных заболеваниях как панкреатит (катепсин В), ревматоидный артрит и остеоартрит (катепсины В, K, L) (О. Vasiljeva, Т. Reinheckel, С. Peters, D. Turk, V. Turk, В. Turk. Emerging roles of cysteine cathepsins in disease and their potential as drug targets. Curr. Pharm.Design, 13 (2007) 385-401). Катепсины В и H являются возможными маркерами внутритканевых болезней легких (М. Kimura, K. Tani, J. Miyata. The significance of cathepsins, thrombin and aminopeptidase in diffuse interstitial lung diseases. J. Med. Invest. 52 (2005) 93-100). Показано, что катепсин L участвует в возникновении диабета (R. Maehr, J.D. Mintern, А.Е. Herman,

D. Mathis, С. Benoist, H.L. Ploegh, Cathepsin L is essential for onset of autoimmune diabetes in NOD mice. J. Clin. Invest. 115 (2005) 2934-2943). Есть данные, что катепсин В участвует в возникновении болезни Альцгеймера, вызывая апоптоз нейронов (Gan L., S. Ye, A. Chu, K. Anton S. Yi, V. Vincent, D. von Schack, D. Chin, J. Murray, S. Lohr, L. Patthy, M. Gonzalez-Zulueta, K. Nikolich, R. Urfer. Identification of cathepsin В as a mediator of neuronal death induced by Abeta-activated microglial cells using a functional genomics approach. J Biol Chem. 279 (2004) 565-572).Cysteine cathepsins have a special role in the development of various pathological processes. Thus, cathepsins B and L are involved in the growth and spread of tumors (ND Rawlings, AJ Barrett, Introduction: The Clans and Families of Cysteine Peptidases, in: ND Rawlings, GS Salvesen (Eds.), Handbook of Proteolytic Enzymes, Third edition, Academic Press, London, 2013, v. 2, pp. 1743-1776). An elevated level of cathepsin B was observed with multiple injuries and septic shock, and secretion of mature active cathepsins B and L by macrophages into the intercellular medium in chronic inflammation was detected (S. Sever, M. Altintas, S. Nankoe,

D. Ko, C. Wei, J. Henderson, E. del Re, L. Hsing, A. Erickson, C. Cohen, M. Kretzler, D. Kerjaschki, A. Rudensky, B. Nikolic, J. Reiser, Proteolytic processing of dynamin by cytoplasmic cathepsin L is a mechanism for proteinuric kidney disease. J. Clin. Invest. (2007) 117, 2095-104). Cathepsins are also involved in inflammatory diseases such as pancreatitis (cathepsin B), rheumatoid arthritis and osteoarthritis (cathepsins B, K, L) (O. Vasiljeva, T. Reinheckel, C. Peters, D. Turk, V. Turk, B. Turk Emerging roles of cysteine cathepsins in disease and their potential as drug targets. Curr. Pharm. Design, 13 (2007) 385-401). Cathepsins B and H are possible markers of interstitial lung diseases (M. Kimura, K. Tani, J. Miyata. The significance of cathepsins, thrombin and aminopeptidase in diffuse interstitial lung diseases. J. Med. Invest. 52 (2005) 93-100 ) Cathepsin L has been shown to be involved in the onset of diabetes (R. Maehr, JD Mintern, A.E. Herman,

D. Mathis, C. Benoist, HL Ploegh, Cathepsin L is essential for onset of autoimmune diabetes in NOD mice. J. Clin. Invest. 115 (2005) 2934-2943). There is evidence that cathepsin B is involved in the onset of Alzheimer's disease, causing apoptosis of neurons (Gan L., S. Ye, A. Chu, K. Anton S. Yi, V. Vincent, D. von Schack, D. Chin, J. Murray, S. Lohr, L. Patthy, M. Gonzalez-Zulueta, K. Nikolich, R. Urfer. Identification of cathepsin In as a mediator of neuronal death induced by Abeta-activated microglial cells using a functional genomics approach. J Biol Chem 279 (2004) 565-572).

Thus, given the diversity and complexity of the tasks “solved” by these enzymes during cellular proteolysis, it is obvious that there is a serious need to create a complex of highly specific and selective peptide substrates for testing this class of enzymes. Meanwhile, the detection of cysteine peptidases is currently difficult due to the fact that the synthetic substrates commonly used to determine the activity of this group of proteinases are not selective.

Currently, almost all the used substrates of cysteine proteinases of the C1 family (peptides containing Arg and Lys in the P1 position) are not selective for this class of enzymes, since they are good substrates of trypsin-like proteinases. The currently known chromogenic substrates of the cysteine proteinases of the C1 family can be divided into several groups. The benzoylarginine esters and amides are low sensitive and poorly soluble in water (for papain K _M = 14 mM and 33 mM, respectively) (LJ Brubacher, MR Zaher, A kinetic study of hydrophobic interactions at the S1 and S2 sites of papain, Can. J Biochem. 57 (1979) 1064-1072). Chromophore n-nitrophenyl substrates (G. Lowe, Y. Yuthavong, Kinetic specificity in papain-catalysed hydrolyses, Biochem. J. 124 (1971) 107-115) are prone to spontaneous hydrolysis, which makes it difficult to correctly determine the activity of cysteine proteinases. The most widely used chromogenic substrates are n-nitroanilides of arginine-containing peptides. These compounds have good solubility and high stability in aqueous mixtures, satisfactory kinetic characteristics (for papain K _M = 3 mM, k _cat = 0.8 s ^-1 ) (JE Mole, HR Horton, Kinetics of papain-catalyzed hydrolysis of -N -benzoyl-L-arginine-p-nitroanilide, Biochemistry 12 (1973) 816-822). The fluorogenic cysteine proteinase substrates used also have dual specificity and limited applicability when working with crude enzyme preparations (Barrett, An improved color reagent for use in Barrett's assay of Cathepsin B, Anal. Biochem. 76 (1976) 374-379; S. Zucker, D. Buttle, J. Nicklin, A. Barrett, The proteolytic activities of chymopapain, papain, and papaya proteinase III, Biochem. Biophys. Res. Commun. 828 (1985) 196-204; J. Tchoupe, T. Moreau, F Gauthier, J. Bieth, Photometric or fluorometric assay of cathepsin B, L and H and papain using substrates with an aminotrifluoromethylcoumarin leaving group, Biochim. Biophys. Acta, 1076 (1991) 149-151; M. Cezari, L. Puzer, M. Juliano, A. Carmona and A. Juliano, Cathepsin In carboxydipeptidase specificity analysis using internally quenched fluorescent peptides. Biochem J. 368 (2002) 365-369). The main disadvantages of all of the mentioned substrates is the need for complex multistage synthesis to be obtained, and, as a result, a low yield of optically pure product. All of these substrates are produced by leading world companies specializing in the production of reagents for research in the field of biochemistry, molecular biology, biotechnology: BACHEM (Germany), SIGMA (USA), FLUKA (Switzerland). This list of influential manufacturers of cysteine proteinase substrates indicates the importance of these products and the market demand for their production.

Known chromogenic peptide substrate of the formula Glp-Phe-Leu-pNA, selective for determining the activity of cysteine proteinases. The synthesis of the substrate and its use for testing preparations of cysteine proteinases of various degrees of purity was described in the journal Analytical Biochemistry (I.Yu. Filippova, EN Lysogorskaya, ES Oksenoit, GN Rudenskaya, VM Stepanov, L-Pyroglutamyl-L-phenylalanyl-L-leucine- p-nitroanilide - a chromogenic substrate for thiol proteinase assay, Anal Biochem. 143 (1984) 293-297). This substrate is manufactured by BACHEM (Germany) and PEPTAIDS INSTITUTES Inc. (Japan) and is used to study cysteine proteinases from various sources, including cathepsins B from the human brain and bovine (Azaryan A., Agatyan G. and Galoyan A, Cathepsin B from bovine, Biochemistry (Moscow) 52 (1987) 2033 -2037), digestive proteinases of one of the most dangerous agricultural pests - the Colorado potato beetle (CJ Bolter, MA Jongsma, Colorado potato beetles (Leptinotarsa decemlineata) adapt to proteinase inhbitors induced in potato leaves by methyl jasmonate, J. Insect Physiol. 41 (1995) 1071 -1078). However, the use of this drug is limited by its low solubility in water and the need to use high concentrations of organic solvents (20% dimethylformamide or dimethyl sulfoxide).

To avoid these problems, a chromogenic substrate Glp-Phe-Ala-pNA and fluorogenic analogues of this substrate, Glp-Phe-Ala-AMC and Glp-Phe-Ala-AFC (Patent No. 1198082 (USSR), publ. In B. I. 1985, N 46; Semashko TA New selective peptide substrates of cysteine peptidases of the papain M. family, 2011 Abstract of dissertation (http://www.chem.msu.su/rus/theses/avtoreferat/2011/ 2011-06-14-semachko.pdf; Semashko T.A., Vorotnikova E.A., Sharikova VF, Vinokurov KS, Smirnova YA, Dunaevsky YE, Belozersky MA, Oppert B., Elpidina EN, Filippova IY Selective chromogenic and fluorogenic peptide substrates for the assay of cysteine peptidases in complex mixtures, Anal. Biochem. 49 (2014) 179-187). These alanine-containing peptidomimetics are They are selective for cysteine peptidases, but lose to commercial arginine-containing substrates in terms of hydrolysis efficiency.These substrates were successfully used both for testing purified enzymes and for characterizing cysteine peptidases in complex multicomponent natural mixtures (insect digestive peptidase extracts).

Also known is the chromogenic glutamine-containing peptide substrate Z-Ala-Ala-Gln-pNA, disclosed in (IA Goptar, T.A. Semashko, SA Danilenko, EN Lysogorskaya, ES Oksenoit, DP Zhuzhikov, MA Belozersky, YE Dunaevsky, B Oppert, I.Yu. Filippova, EN Elpidina, Cysteine digestive peptidases function as post-glutamine cleaving enzymes in tenebrionid stored product pests, Comparative Biochemistry and Physiology - In Biochemistry and Molecular Biology, 161 (2012) 148-154), however this the substrate has low kinetic characteristics.

The substrate of the formula Glp-Phe-Leu-pNA (I.Yu. Filippova, EN Lysogorskaya, ES Oksenoit, GN Rudenskaya, VM Stepanov, L-Pyroglutamyl-L-phenylalanyl-L-leucine-p-nitroanilide - a chromogenic substrate for thiol proteinase assay, Anal Biochem. 143 (1984) 293-297).

The closest analogue (prototype) is a peptide substrate of the general formula A-XaaYaaB, where A = Glp (pyroglutamyl), Abz (o-aminobenzoyl); B = pNA (p-nitroanilide), AMS (7amido-4-methylcoumarin) and AFC (7-amido-4-trifluoromethylcoumarin); Haa = Phe, Val; Yaa = Ala (Semashko TA, New selective peptide substrates of cysteine peptidases of the papain family, M., 2011 (http://www.chem.msu.su/eng/theses/avtoreferat/2011/2011-06-14-semachko .pdf). The disadvantage of the prototype is the low catalytic (kinetic) parameters, which, despite the selectivity, reduces the efficiency of their use.

Disclosure of Invention

The present invention is the creation of new synthetic substrates for testing cysteine peptidases of the papain family, resistant to trypsin-like peptidase hydrolysis.

The problem is solved by new peptide substrates of cysteine peptidases of the papain family of general formula (1):

A is the N-protective group of the urethane or acyl type;

Haa - a proteinogenic or non-proteinogenic amino acid or dipeptide that meets the requirements of the substrate specificity of the cysteine peptidases of the papain family (C1 family);

B - marker chromogenic or fluorogenic group, for quantitative control of the cleavage of substrates by spectrophotometric or fluorometric methods.

Preferably, the N-protecting groups of the urethane type are selected from the series Z (benzyloxycarbonyl), Boc (tert-butyloxycarbonyl) or acyl type, for example, Glp (pyroglutamyl), Suc (succinyl), Bz (benzoyl), Ac (acetyl), For (formyl )

Preferably, a proteinogenic or non-proteinogenic amino acid or dipeptide meeting the substrate specificity requirements of the cysteine peptidases of the papain family (C1 family) is selected from the group consisting of Phe, Phe-4-Cl (4-chloro-phenylalanine), Phe-3,4-Cl ₂ ( 3,4-dichloro-phenylalanine), homoPhe (homophenylalanine), Phg (phenyl-glycine), Tyr, Leu, Val, Ile, Pro, His, Arg, Lys, Thr (OBzl) (O-benzyltreonine), Thr (tBu ) (O-tert-butyltreonine), Ser (OBzl) (O-benzylserine), Ser (tBu) (O-tert-butylserine), Cys (SBzl) (S-benzylcysteine), Ala-Ala, Ala-Gly, Gly -Ala, Gly-Gly.

Preferably, the chromogenic or fluorogenic groups are selected from the range including AMS (7-amido-4-methylcoumaride), βNA (beta-naphthylamide), AFC (7-amido-4-trifluoromethylcoumaride), pNA (n-nitroanilide) to determine the enzymatic activity peptidases. The place of hydrolysis of the substrates is shown by the arrow.

The problem is also solved by the use of the inventive peptide substrate to create test systems for the detection of peptidases, while the detection can be carried out in solution, in a gel after electrophoresis under native conditions, in vivo systems.

The inventive substrate of the general formula (1) can be used to determine the enzymatic activity of peptidases as highly specific and selective chromogenic and fluorogenic substrates for cysteine peptidases of the papain family (in the presence of enzymes from other clans); to determine the enzymatic activity of peptidases for the selective detection of cathepsin L in natural mixtures of various cathepsins (eg, B and L); to determine the enzymatic activity of peptidases for selective testing of cathepsin L activity using fluorogenic compounds in in vivo systems: cells and their organelles, intercellular space, tissues, as well as to determine the enzymatic activity of peptidases to search for glutamine-specific peptidases with the ability to cleave gliadins and their toxic peptides .

The technical result of the claimed peptide substrate is its high efficiency (kinetic characteristics) and selectivity for cysteine peptidases of the C1 family.

The inventive substrate allows to achieve an efficiency of hydrolysis in comparison with the prototype and analogues by 3-5 times, and in some cases, for example, for cathepsin L - even an order of magnitude. The glutamine-containing substrates we offer have better kinetic characteristics than the alanine-containing substrates and Z-Ala-Ala-Gln-pNA described above. All of the proposed chromogenic and fluorogenic substrates are selective for cysteine peptidases of the C1 family and are not hydrolyzed by peptidases of other clans. This is an important advantage over the widespread commercially available arginine-containing substrates: Z-Phe-Arg-pNA, Z-Phe-Arg-AMC, Z-Arg-Arg-pNA, Z-Arg-Arg-AMC also cleaved with trypsin-like serine peptidases. This allows the use of the proposed glutamine-containing substrates for testing cysteine peptidases in the presence of enzymes of other clans.

Also, the claimed peptide substrate is characterized by high activity of the cysteine peptidases of the papain family, such as plant peptidases papain and ficin, as well as animal peptidases - lysosomal and digestive cathepsins L, according to the proposed selective substrates, which are highly specific. In particular, among cathepsins of animal origin, given the lower activity of cathepsin B in relation to these substrates, their use allows specific testing of cathepsin L in natural mixtures of these two cathepsins (for example, in the digestive system of beetle pests of grain stocks, in lysosomes).

The inventive peptide substrate is selective for cathepsin L, which is a marker of many diseases, allows the use of substrates for selective testing of the enzyme in vivo systems. The presence of marker chromogenic or fluorogenic groups in substrates allows them to be used to create test systems and test activity directly in the gel after electrophoresis under native conditions.

Also, the claimed peptide substrate is characterized by high activity against postglutamine-cleaving peptidases, hydrolyzing gliadins and their toxic peptides. This makes it possible to use substrates for screening peptidases, potential candidates for creating drugs for celiac disease (an autoimmune disease of the gastrointestinal tract caused by undegraded immunogenic Gln-containing peptides), as well as for producing gluten-free products.

Brief Description of the Drawings

In FIG. 1 shows elution profiles of cysteine peptidases from the intestinal extract of T. castaneum larvae with Sephadex G-75 on substrates Glp-Phe-Gln-pNA, Z-Phe-Arg-pNA and Z-Arg-Arg-pNA at pH 5.6 in the presence of 6 mM Cys. Peaks 1 and 4 relate to non-enzymatic protein fractions. Peaks 2 and 3 contain the activity of impurity serine trypsin-like peptidases and cysteine cathepsins B and L, tested on the non-selective substrate Z-Phe-Arg-pNA. Peak 2 predominantly contains cathepsin B activity, selectively tested for Z-Arg-Arg-pNA substrate. In peak 3, predominantly cathepsin L activity elutes selectively from the Glp-Phe-Gln-pNA substrate.

DETAILED DESCRIPTION OF THE INVENTION

The structure of the substrates corresponds to the specificity requirements of cysteine peptidases of the C1 family: key position P1 (Schechter I., Berger A. On the size of the active site in proteases. I. Papain. Biochem Biophys Res Commun., 1967, 27 (2), 157- 162) is a small-sized residue of glutamine (Gln). At position P2, which is crucial for the specificity of these peptidases, are the hydrophobic residues Xaa = Phe, Phe-4-Cl, Phe-3,4-Cl ₂ , homoPhe, Phg, Tyr, Leu, Val, Ile, Pro, His, Thr (OBzl), Thr (tBu), Ser (OBzl), Ser (tBu), Cys (SBzl) or Ala-Ala, Ala-Gly, Gly-Ala, Gly-Gly dipeptides.

All these derivatives satisfy the requirements for substrate specificity of the C1 family cysteine peptidases (Chang A., Scheer M., Grote A., Schomburg I., Schomburg D. BRENDA, AMENDA and FRENDA the enzyme information system: new content and tools in 2009. Nucleic Acids Res., 2009, 37, 588-592.http: //www.brenda-enzymes.org: ND Rawlings, GS Salvesen (Eds.), Handbook of Proteolytic Enzymes, Third edition, Academic Press, London, 2013, v. 2, pp. 1743-1776).

The N-terminal grouping of substrates (A) is represented by standard N-protective groups used in peptide synthesis - benzyloxycarbonyl (Z), tert-butyloxycarbonyl (Boc), benzoyl (Bz), acetyl (Ac), formyl (For), succinyl (Suc ) (Gershkovich A.A., Kibirev V.K. Chemical synthesis of peptides. Naukova Dumka, Kiev, 1992). In addition, it is proposed to use the remainder of pyroglutamic acid (Glp) as the N-terminal group. Glp is a natural amino acid and can serve as a natural defense of the amino groups of peptides and proteins. The introduction of the Glp residue also improves the solubility of substrates in aqueous-organic mixtures, since pyroglutamic acid is more hydrophilic than most standard N-protecting groups. The effectiveness of using the Glp residue as the N-terminal group was demonstrated by us earlier (I.Yu. Filippova, EN Lysogorskaya, ES Oksenoit, GN Rudenskaya, VM Stepanov, L-Pyroglutamyl-L-phenylalanyl-L-leucine-p-nitroanilide - a chromogenic substrate for thiol proteinase assay, Anal Biochem. 143 (1984) 293-297). Known marker groups were used as C-terminal residues (B): chromogenic n-nitroanilide (pNA) and fluorogenic 4-amino-7-methylcoumaryl (AMC), 4-amino-7-trifluoromethylcoumaryl (AFC) and beta-naphthylamide (βNA ) groups, the presence of which ensures the convenience and accuracy of the control of enzymatic activity by changing the spectral and fluorescence characteristics of substrates during hydrolysis (Gershkovich A.A., Kibirev V.K. Chromogenic and fluorogenic peptide substrates of proteolytic enzymes. Bioorg. Chemistry, 1988.14 ( 11), 1461-1488).

As a marker of chromogenic or fluorogenic groups (B), selected from the series of AMS (7-amido-4-methylcoumaride), βNA (beta-naphthylamide), AFC (7-amido-4-trifluoromethylcoumaride), pNA (n-nitroanilide) - to determine the enzymatic activity of peptidases.

Peptide substrates can be obtained by any method known in the art (Gershkovich A.A., Kibirev V.K. Chemical synthesis of peptides. Naukova Dumka, Kiev, 1992; Houben-Weyl Methods of Organic Chemistry: Synthesis of Peptides and Peptidomimetics, 4th edition 2004. Edited by Goodman M., Toniolo C, Moroder L., Felix AV E22a, p. 785. Thieme, Stuttgart, New York). Substrates of the above general formula (1) can be prepared by peptide condensation by dicyclohexylcarbodiimide (DCCA) and / or using activated esters in the presence of, for example, pentafluorophenol. (examples 1-4).

The carboxy components are: (a) pyroglutamyl amino acids (see examples 1 and 2), (b) N-protected amino acids (Z, Suc, Boc, Bz, Ac, For) or dipeptides (see example 3). The amino component is represented by the Gln residue containing a C-terminal fluorogenic or chromogenic group: AMS, βNA, AFC, pNA.

For the synthesis of Glp-containing carboxylic components, an approach based on the preparation of the corresponding methyl esters of Glp-amino acids (according to the method of activated esters) followed by their enzymatic (example 1) or alkaline (example 2) saponification was used. To obtain N-protected amino acids or dipeptides, standard methods of introducing protective groups were used (Example 3) (Gershkovich A.A., Kibirev V.K. Chemical peptide synthesis. Naukova Dumka, Kiev, 1992) or commercially available N-protected derivatives of amino acids were used and dipeptides (e.g., BACHEM, Germany). In all cases, to obtain pyroglutamyl derivatives (amino acids and peptides), an approach based on the use of pentafluorophenyl ester of pyroglutamic acid GlpOPfp was used (Semashko et al., 2008)

Peptide condensation was carried out in an environment of anhydrous organic solvents (usually dimethylformamide (DMF), dichloromethane (DCM), acetonitrile (AN), introducing into the reaction equimolar amounts of carboxylic and amine component, pentafluorophenol and 5-10% excess of DCCA (Gershkovich A.A., Kibi VK Chemical synthesis of peptides. Naukova Dumka, Kiev, 1992).

The homogeneity of substances was determined on Silufol plates (Czech Republic).

Chromatography was performed in the following systems:

chloroform - ethanol (9: 1) (A)

ethyl acetate-pyridine-acetic acid-water (75: 27: 17: 8) (B);

n-butanol-pyridine-water-acetic acid (15: 1012: 3) (C);

n-butanol-acetic acid-water (3: 1: 1) (D);

n-butanol-acetic acid-water (40: 7: 4) (E)

benzene-acetone-acetic acid (100: 25: 4) (F).

Peptide substrates were analyzed on a Milichrom A-02 liquid chromatograph (EkoNova, Russia) and Altex Model 110 A (USA) on a ProntoSil 120-5C18AQ column (2.0 × 75 mm) with elution with a solution of 0.1% CF ₃ COOH in linear acetonitrile gradient (flow rate 1 ml / min) from 0 to 80% in 20 minutes (A) and on a Nucleosil C18 column (4.6 × 250 mm) (Biochemical, Russia) with elution with a 1% CF ₃ COOH solution in a linear gradient acetonitrile (flow rate 1 ml / min) from 30 to 70% in 30 minutes (B). Detection was carried out at 214 nm, 280 nm and 350 nm.

Spectrophotometric measurements were performed on Genesys 10 S UV spectrophotometers (Spectronics, USA), as well as on an ELx808 microplate photometer (BioTek, USA) in 96-well plates.

Fluorescence measurements were performed on Fluoroskan Ascent microplate fluorometers (Thermo Scientific, USA) and Flx808 (BioTek, USA) in 96-well plates, as well as on a Cary Eclipse spectrofluorimeter (Varian, USA).

The structures of the obtained compounds were confirmed using 1H-NMR spectroscopy. NMR spectra were recorded in deuterodimethyl sulfoxide on a Brucker AC-300 spectrometer (Germany) at a frequency of 300 MHz. Chemical shifts are given in parts per million (δ, ppm) relative to the internal standard of tetramethylsilane (δ 0,000 ppm).

Amino acid analysis was performed on a Hitachi 835 automatic amino acid analyzer (Japan) after acid hydrolysis of 6 M HCl samples at 105 ° C in vacuum ampoules for 48 h.

Hydrolysis of the obtained glutamine-containing substrates was carried out with various cysteine peptidases of the C1 family - plant enzymes: papain, bromelain, ficin, as well as lysosomal cathepsins B and L of mammals and humans, respectively, and the digestive cathepsin L of the insect pest Tribolium castaneum. All investigated substrates were digested with cysteine peptidases of the C1 family. The glutamine-containing substrates we offer have better kinetic characteristics than the prototype and its analogues Glp-Phe-Ala-AMC, Glp-Phe-Ala-AFC, Glp-Phe-Ala-pNA and Z-Ala-Ala-Gln-pNA ( example 5).

Table 1 contains data on the efficiency of hydrolysis with cysteine peptidases of different origin of the chromogenic substrate Glp-Phe-Gln-pNA and fluorogenic substrates Glp-Phe-Gln-AMC and Z-Ala-Ala-Gln-βNA. The proposed glutamine-containing substrates are compared with the prototypes Glp-Phe-Ala-AMC, Glp-Phe-Ala-AFC and Glp-Phe-Ala-pNA. The hydrolysis efficiency was evaluated by the specificity coefficient of the enzymatic reaction k _cat / K _M , which characterizes both the rate of the enzymatic reaction (k _cat value) and the affinity of this enzyme for this substrate (K _M value). The higher the k _cat / K _M value, the better the substrate. Fluorogenic substrates with AMS and AFC groups (columns 3-5) were approximately more efficiently cleaved by all studied enzymes than their chromogenic analogues (columns 1 and 2) and fluorogenic substrates with a β-naphthylamide group. Comparison of the kinetic parameters of the hydrolysis of Glp-Phe-Gln-pNA (column 1) and Glp-Phe-Gln-AMC (column 3) with their alanine-containing chromogenic prototype Glp-Phe-Ala-pNA (column 2) and fluorogenic prototypes Glp- Phe-Ala-AMC and Glp-Phe-Ala-AFC (columns 4, 5) convincingly show the advantage of the glutamine-containing substrates we offer. The most effective in relation to the proposed substrates papain, but in the case of cathepsins animals the values of the coefficient of specificity k _cat / K _{M are} quite high.

For practical purposes, a more convenient assessment of the hydrolytic efficiency of peptidases is a characteristic of their specific activity (examples 6.7). The specific activity value shows how much substrate (in moles) is cleaved by a certain amount of enzyme (in this case, we pray) per unit time (in this case, per minute) (see examples 6 and 7). The higher the specific activity value given, the more efficiently the substrate is hydrolyzed by this enzyme.

Table 2 in the corresponding rows shows the data on the specific activity of a number of studied family C1 cysteine peptides in relation to the synthesized chromogenic substrate Glp-Phe-Gln-pNA (column 2) and fluorogenic substrates Glp-Phe-Gln-AMC, Glp-Val-Gln -AMC, Z-Pro-Gln-βNA, Z-Ala-Ala-Gln-βNA (columns 4-7). Columns 1 and 3 show the values of the specific activity of hydrolysis by the indicated cysteine peptidases of alanine-containing prototypes: the chromogenic substrate Glp-Phe-Ala-pNA (column 1) and the fluorogenic substrate Glp-Phe-Ala-AMC (column 3). Among all the studied peptidases, papain, the ancestor of the C1 family, showed the highest activity on all substrates. The most promising for all enzymes among these substrates are Glp-Phe-Gln-pNA and Glp-Phe-Gln-AMC. A significant advantage of these glutamine-containing substrates (columns 2 and 4) compared to their prototypes (columns 1 and 3, respectively) is that with their use a higher specific activity of all tested peptidases is observed, i.e. hydrolysis is achieved in a shorter time and with less enzyme. Particularly noticeable is the advantage of Glp-Phe-Gln-pNA and Glp-Phe-Gln-AMC in the case of cysteine cathepsins of various origins, with cathepsin L. showing the highest activity on this substrate.

The synthesized chromogenic and fluorogenic substrates are selective for cysteine peptidases of the C1 family and are not hydrolyzed by peptidases of other clans - chymotrypsin, trypsin and subtilisin serine peptidases, thermolysin metallopeptidases and pepsin aspartyl peptidases (table 2). This allows the use of the proposed glutamine-containing substrates for testing cysteine peptidases in the presence of enzymes of other clans, and is an important advantage over the widely used commercially available arginine-containing substrates: Z-Phe-Arg-pNA, Z-Phe-Arg-AMC Z -Arg-Arg-pNA, Z-Arg-Arg-AMC, which are also actively cleaved by trypsin-like serine peptidases.

Given the lower activity of cathepsin B (21 ± 1, 23 ± 9 (Msubstrate / Enzyme * min) for Glp-Phe-Gln-pNA and Glp-Phe-Gln-AMC substrates, respectively) compared to cathepsin L (68 ± 3, 97 ± 9 (Msubstrate / Enzyme * min) for Glp-Phe-Gln-pNA and Glp-Phe-Gln-AMC substrates, respectively) (table 1), their use allows specific and selective testing of cathepsin L in natural mixtures of these two cathepsins ( for example, in the digestive system of grain pest beetles, in lysosomes).

The selectivity of the proposed substrates can be demonstrated in an experiment on fractionation of a complex multicomponent enzyme mixture from the digestive complex of Tribolium castaneum larvae of the flour meal, in which cathepsins L and B dominated among cysteine peptidases (A. Martynov, E. Elpidina, L. Perkin, B. Oppert , Functional analysis of C1 family cysteine peptidases in the larval gut of Tenebrio molitor and Tribolium castaneum. BMC genomics. 16 (2015) 75-89)) (Example 9). In FIG. 1 shows elution profiles of cysteine peptidases from the intestinal extract of T. castaneum larvae with Sephadex G-75 on substrates Glp-Phe-Gln-pNA, Z-Phe-Arg-pNA and Z-Arg-Arg-pNA at pH 5.6 in the presence of 6 mM Cys. Peaks 1 and 4 relate to non-enzymatic protein fractions. Peaks 2 and 3 contain the activity of impurity serine trypsin-like peptidases and cysteine cathepsins B and L, tested on the non-selective substrate Z-Phe-Arg-pNA. Peak 2 predominantly contains cathepsin B activity, selectively tested for Z-Arg-Arg-pNA substrate. In peak 3, predominantly cathepsin L activity elutes selectively from the Glp-Phe-Gln-pNA substrate. It should be noted that only the use of Glp-Phe-Gln-pNA allowed selective testing of cathepsin L and differentially detect cathepsins L and B.

It is also possible selective testing of cathepsin L activity using fluorogenic compounds in in vivo systems: cells and their organelles, intercellular space, and tissues.

To determine the enzymatic activity of the studied enzyme in a homogeneous solution, an aqueous solution of the inventive substrate is used with the addition of a polar organic solvent necessary for complete dissolution of the substrate. Usually this is an aqueous solution with the addition of 2-5% organic solvent, for example, dimethylformamide, dimethyl sulfoxide, ethanol, methanol, propanol, isopropanol, acetonitrile, dioxane can be used. The criterion for choosing a particular solvent is both the solubility of the substrate (the higher the solubility, the better), and the effect of this solvent on the activity of the enzyme to be determined (a solvent is chosen in the presence of which the enzyme activity is lost to the least extent).

In the case of determining activity in a gel, gels from polyacrylamide (PAG), starch, agarose, gelatin, and cellulose can be used as a polymer matrix.

The presence of marker chromogenic or fluorogenic groups in substrates allows their use in the creation of test systems in solution (examples 6 and 7) and the detection of activity directly in the gel after electrophoresis under native conditions (example 8).

The test system for determining the enzymatic activity in a solution includes a cysteine peptidase preparation, a buffer solution with a pH of 5 to 7 containing ethylenediaminetetraacetic acid (EDTA) (final concentration 1 mM), a solution of a reducing SH reagent (for example, cysteine, mercaptoethanol, dithiothreitol) (final concentration from 2 to 6 mM), a solution of the substrate in 2.5% organic solvent (final concentration of chromogenic substrates from 1 to 5 mM, fluorogenic from 10 to 100 μM) (examples 6 and 7).

The test system for post-electrophoretic testing of the activity of cysteine peptidases of the C1 family directly in the gel includes a gel plate (polyacrylamide or similar), in which electrophoresis (EF) of the studied peptidase preparation was carried out under native conditions (in the absence of sodium dodecyl sulfate) at pH 6.8 -7.2 according to the method described in (KS Vinokurov, B. Oppert, EN Elpidina, Anal. Biochem. (2005)) and a developing solution consisting of a buffer with a pH of 5-7 containing EDTA (final concentration 1 mM) reducing SH reagent (e.g. cis thein, mercaptoethanol, dithiothreitol) (final concentration from 2 to 6 mM) with the addition of 50-100 μM substrate solution in a 2.5% organic solvent. After an incubation of 5-15 minutes, the localization sites of proteolytic activity are detected by the formed fluorescence bands at 350-370 nm (Example 8).

The proposed glutamine-containing substrates can be effective in the search for glutamine-specific peptidases with the ability to cleave gliadins and their toxic peptides. This creates the prospect of using substrates for screening peptidases - potential candidates for creating drugs for celiac disease (an autoimmune disease of the gastrointestinal tract caused by undegraded immunogenic Gln-containing peptides), as well as for producing gluten-free products.

Thus, the advantage of the claimed peptide substrate are:

1. Increased efficiency compared to the prototype and analogues. The hydrolysis efficiency was evaluated by the specificity coefficient of the enzymatic reaction k _cat / K _M , which characterizes both the rate of the enzymatic reaction (k _cat value) and the affinity of this enzyme for this substrate (K _M value). The higher the k _cat / K _M value, the better the substrate. Fluorogenic substrates with AMS and AFC groups (columns 3-5) were approximately more efficiently cleaved by all studied enzymes than their chromogenic analogues (columns 1 and 2) and fluorogenic substrates with a β-naphthylamide group. Comparison of the kinetic parameters of the hydrolysis of Glp-Phe-Gln-pNA (column 1) and Glp-Phe-Gln-AMC (column 3) with their alanine-containing chromogenic prototype Glp-Phe-Ala-pNA (column 2) and fluorogenic prototypes Glp- Phe-Ala-AMC and Glp-Phe-Ala-AFC (columns 4, 5) convincingly show the advantage of the glutamine-containing substrates we offer. (Table 1; Example 5).

2. High activity of cysteine peptidases of the papain family, such as plant peptidases papain and ficin, as well as animal peptidases — lysosomal and digestive cathepsins L, according to the proposed substrates, which are highly selective (table 2, examples 6.7). A significant advantage of glutamine-containing substrates in comparison with their prototype is that with their use there is a higher specific activity of all tested peptidases (table 2, columns 1 and 2, 3 and 4). This can significantly increase the sensitivity of the analysis without loss of selectivity.

3. The selectivity of the vast majority of glutamine-containing substrates in relation to cysteine peptidases of the C1 family. The synthesized substrates are cleaved only by cysteine peptidases of the C1 family and are not hydrolyzed by peptidases of other clans (table 2). This allows the use of the proposed glutamine-containing substrates for testing cysteine peptidases in the presence of enzymes of other clans, and is an important advantage over the widespread commercially available arginine-containing products: Z-Phe-Arg-pNA, Z-Phe-Arg-AMC Z -Arg-Arg-pNA, Z-Arg-Arg-AMC, which are also actively cleaved by trypsin-like serine peptidases.

4. The possibility of selective detection of various cathepsins of animal origin. Given the lower activity of cathepsin B (table 2) with respect to glutamine-containing substrates, their use allows specific testing of cathepsin L in natural mixtures of these two cathepsins (for example, in the digestive system of beetle pests of grain stocks, in lysosomes). The selectivity of the proposed substrates was demonstrated in an experiment on fractionation of a complex multicomponent enzyme mixture from the digestive complex of Tribolium castaneum larvae of the flour meal, in which cathepsins L and B dominated among cysteine peptidases (A. Martynov et al, 2015). Testing the activity of cathepsins L and B after chromatographic separation was performed using the proposed substrate Glp-Phe-Gln-pNA, which has partial selectivity for cathepsin B of a commercial substrate Z-Arg-Arg-pNA and a non-selective commercial substrate Z-Ala-Phe-Arg -pNA. Only the use of Glp-Phe-Gln-pNA allowed selective testing of cathepsin L and differentially detect cathepsins L and B (Fig. 1).

5. Selective testing of cathepsin L activity using fluorogenic compounds in in vivo systems: cells and their organelles, intercellular space, tissues.

6. Possibilities of using the proposed substrates; biomedical aspect.

6.1. The presence of marker chromogenic or fluorogenic groups in substrates allows them to be used to create test systems and test activity directly in the gel after electrophoresis under native conditions.

6.2. The selectivity of the proposed substrates in relation to cathepsin L, which is a marker of many diseases, allows them to be used for selective testing of the enzyme in in vivo systems.

6.3. The proposed glutamine-containing substrates can be effective in the search for glutamine-specific peptidases with the ability to cleave gliadins and their toxic peptides. This creates the prospect of using substrates for screening peptidases, potential candidates for creating drugs for celiac disease (an autoimmune disease of the gastrointestinal tract caused by undegraded immunogenic Gln-containing peptides), as well as for producing gluten-free products.

The present invention is illustrated by specific examples of execution, which are not the only possible, but clearly demonstrates the possibility of achieving the desired technical result.

Example 1

Synthesis of pyroglutamyl-L-phenylalanyl-L-glutaminyl-7-amino-4-methylcoumaride (Glp-Phe-Gln-AMC)

GlogOPfp pyroglutamic acid pentafluorophenyl ester

To a suspension of 2.58 g (20 mmol) of pyroglutamic acid and 3.70 g (20 mmol) of pentafluorophenol in 90 ml of abs. ethyl acetate at 0 ° C was added 4.53 g (22 mmol) of DHA in 10 ml of abs. ethyl acetate. Stirred for 1 h at 0 ° C and 3 h at 20 ° C. The precipitate of dicyclohexylurea (DCM) was filtered off and washed with ethyl acetate. The filtrate and washings were evaporated in vacuo. The dry residue was washed with hexane. Dried over paraffin in a vacuum. Yield 5.3 g (90%). R _ƒ = 0.76 (A); 0.5 (B).

Phenylalanine Methyl PheOMe

To a suspension of 3.9 g of phenylalanine methyl ester hydrochloride in 100 ml of abs. ethyl acetate was added dropwise 2.5 ml of abs. triethylamine and left under stirring for 1 h. The precipitate was filtered off, the solution was evaporated in vacuo. The rest is oil. Yield 3.2 g (98%).

Pyroglutamyl-phenylalanine methyl ester Glp-PheOMe

(с 1; ДМФ). Аминокислотный анализ: Glu 1,06; Phe 1,0.To 3.2 g (18 mmol) of phenylalanine methyl ester was added 5.3 g (18 mmol) of pentafluorophenyl ether of pyroglutamic acid in 20 ml of tetrahydrofuran, stirred for 20 minutes, evaporated in vacuo, the residue was dissolved in 80 ml of ethyl acetate and washed with 5% citric acid acid, water. An additional peptide was extracted from the washings with a mixture of ethyl acetate and n-butanol, 8: 1. The combined extracts were washed with citric acid and water. The ethyl acetate solution and the extracts were combined, dried with anhydrous Na ₂ SO ₄ and evaporated in vacuo. The oil crystallized in the cold under abs. ether. Yield 3.3 g (63%). Mp 28-30 ° C, R _f 0.79 (B), 0.83 (C);

(s 1; DMF). Amino acid analysis: Glu 1.06; Phe 1.0.

Pyroglutamyl-L-Phenylalanine Glp-Phe

(c 0,5; DMF-вода 1:1).To a solution of 1 g (3.38 mmol) of Glp-PheOMe in 27 ml of 0.1 M ammonium bicarbonate (pH = 8), 10 ml of a solution of α-chymotrypsin (1 mg / ml in water) was added. The reaction mixture was stirred for 5 hours at 37 ° C, then evaporated in vacuo. The resulting oil was crystallized by treatment with ether and ethyl alcohol, followed by distillation of the solvents in vacuo. Dried over P ₂ O ₅ . Yield 0.88 g (88%). Mp 159-161 ° C; R _f 0.48 (C),

(c 0.5; DMF-water 1: 1).

Pyroglutamyl-L-phenylalanyl-L-glutaminyl-7-amino-4-methylcoumaride Glp-Phe-Gln-AMC

To a solution of 0.059 g (0.21 mmol) of Glp-Phe and 0.081 g (0.21 mmol) of HBr × Gln-AMC in a mixture of 1.5 ml of acetonitrile and 5 ml of DMF containing 30 μl (0.21 mmol) of triethylamine at while cooling to 0 ° C and stirring, 0.054 g (0.26 mmol) of DHA was added. The reaction mixture was left under stirring at room temperature overnight, the precipitate formed was filtered off, and the filtrate was evaporated. The resulting oily substance was dissolved in ethyl acetate: butanol (80:20). The organic layer was washed with 3% NaHCO ₃ , water, 5% citric acid, dried over anhydrous Na ₂ SO ₄ and evaporated to dryness. The product was crystallized from abs. diethyl ether. Yield 0.060 g (52%). Amino acid analysis: Glu: Phe 2.1: 1; R _f 0.77 (C), 0.62 (D); HPLC 13.5 min (gradient A), 8.9 min (gradient B); Mr (calc / calc.) 561.5 / 561.4; 1 H NMR, DMSO-D 6, δ, ppm. - 1.2 s (3H, C H ₃ in the AMS), 1.5-1.9 m (2H, -C H ₂ -CH ₂ -CO-NH ₂ in Gln and 1H -NH-CO-CH ₂ - With H ₂ -CH- in Glp), 2-2.4 m (4H: 2H, -CH ₂ -C H ₂ -CO-NH ₂ Gln and 2H -NH-CO-C H ₂ -C H ₂ -CH - in Glp), 2.5 s (2Н, C H ₂ -CO-О, AMS), 2.7 m (1Н, α-С Н Phe), 3.35 d (2Н, C H ₂ - in Phe ), 4.5 d (1H, —NH — CO — CH ₂ —CH ₂ —C H — in Glp), 7.2-8.2 m (5H, C ₆ H ₅ in Phe).

Similarly, compounds of the general formula Glp-Xaa-Gln-B were prepared, where Xaa = Phe-4-Cl, Phe-3,4-Cl ₂ , homoPhe, Phg, Leu, Tyr; B = AMC, βNA, AFC.

Example 2

Synthesis of pyroglutamyl-L-valyl-L-glutaminyl-7 amino-4-methylcoumaride (Glp-Val-Gln-AMC)

ValOMe Roller HCl Methyl Ester Hydrochloride

5 g (0.043 mol) of valine were dissolved in 50 ml of abs. methanol. 6 ml (0.043 mol) of thionyl chloride were added to the solution cooled to 0 ° C with stirring. The solution was then stirred for 24 hours, evaporated to dryness in vacuo, and the oil was again treated with methanol and thionyl chloride under the conditions described above. The resulting crystalline material was washed with abs. ether, dried in vacuo over alkali. Yield 6.2 g (86%). R _ƒ = 0.54 (D).

Pyroglutamyl Valine Methyl Ester Glp-ValOMe

To 3.3 g (20 mmol) of HClxValOMe dissolved in 30 ml of DMF was added 3 ml of Et ₃ N, stirred. Then, 5.8 g (20 mmol) of GlpOPfp in 30 ml of DMF was added to the reaction mixture. After completion of the reaction, the precipitate was filtered off, and DMF was evaporated to dryness. The resulting substance was dissolved in 150 ml EtOAc, the precipitate was filtered off and the organic phase was washed with citric acid and water. Dried over anhydrous Na ₂ SO ₄ , filtered and evaporated in vacuo. It was crystallized under petroleum ether, dried over alkali in vacuo. Yield 2.20 g (48%). R _ƒ = 0.12 (F).

Pyroglutamyl-L-Valine Glp-Val

0.59 g (2.54 mmol) of Glp-ValOMe was dissolved in 5 ml of methyl alcohol, 1.5 ml of 2N was added. NaOH and stirred at room temperature for 1.5 hours. The solvent was evaporated, cold water was added and the pH was adjusted to 3. It was extracted from water with ethyl acetate: butanol (60:40, the solution was washed with water, the solvent was evaporated and the residue was precipitated with petroleum ether. Yield 0.360 g (62%). R _f = 0.39 (B) and 0.62 (D).

Pyroglutamyl-L-valyl-L-glutaminyl-7-amino-4-methylcoumaride Glp-Val-Gln-AMS

To a solution of 0.050 g (0.21 mmol) of GlpVal and 0.081 g (0.21 mmol) of HBr × Gln-AMC in a mixture of 1.5 ml of AN and 5 ml of DMF containing 30 μl (0.21 mmol) of TEA while cooling to 0 ° C. While stirring, 0.054 g (0.26 mmol) of DHA was added. The reaction mixture was left under stirring at room temperature overnight, the precipitate formed was filtered off, and the filtrate was evaporated. The resulting oily substance was dissolved in 3.2 ml of a mixture of ethyl acetate: butanol (80:20), the precipitate formed was filtered off and the filtrate volume was adjusted to 10 ml with the same mixture of ethyl acetate and butanol. The organic layer was washed with 3% NaHCO ₃ , water, 5% citric acid, dried over anhydrous Na ₂ SO ₄ and evaporated to dryness. The product was crystallized from abs. diethyl ether. Yield 0.052 g (48%). Amino acid analysis: Glu: Val = 2.2: 1; R _f 0.51 (C), 0.41 (D); HPLC 12.0 min (gradient A), 7.9 min (gradient B); Mr (calc. / Dec.) 513.2 / 513.1; 1H NMR, DMSO-D6, δ, ppm -1.2 d (3H, C H ₃ in Val), 1.65 d (3H, C H ₃ in Val), 2.5 s (3H, C H ₃ in AMC), 2.6-2.75 m (2H, -C H ₂ -CH ₂ -CO-NH ₂ Gln), 3.3 s (2H, C H ₂ AMC) 7.91 s (1H, -C (O) -N H -CH- Val, 8.34 d (1H, -C (O) -N H -CH- in Glp).

Similarly, compounds of the general formula Glp-Xaa-Gln-B were prepared, where Xaa = Val, Pro, Ile, His, Thr (OBzl), Thr (tBu), Ser (OBzl), Ser (tBu), Cys (SBzl); B = AMC, βNA, AFC.

Example 3

Synthesis of carbobenzoxy-L-alanyl-L-alanyl-L-glutaminyl-β-naphthylamide Z-Ala-Ala-Gln-βNA

Benzyloxycarbonyl-L-alanyl-L-alanine Z-Ala-Ala

With vigorous stirring, to a solution of 5 g (0.031 mol) of alanyl-alanine and 7.8 g of sodium bicarbonate (0.09 mol) in 60 ml of water was added dropwise 9 ml (0.062 mol) of carbobenzoxychloride. The reaction mixture was stirred for 1 h at 0 ° C and 2 h at 20 ° C. Unreacted carbobenzoxychloride was extracted with ether. The aqueous fraction was acidified with 5 N HCl (Congo red), the precipitate was filtered, washed with 0.2 N hydrochloric acid and water. Dried in vacuo over alkali. Yield 8 g (80%). Mp 150-151 ° C. R _f = 0.75 (C), 0.72 (E).

Carbobenzoxy-L-alanyl-L-alanyl-L-glutaminyl-β-naphthylamide Z-Ala-Ala-Gln-βNA

To a solution of 0.154 g (0.5 mmol) of HCl × Gln-βNA and 0.147 g (0.5 mmol) of Z-Ala-Ala in 4 ml of DMF containing 70 μl (0.5 mmol) of TEA was added with cooling and stirring 0.134 g (0.65 mmol) of DHA. The reaction mixture was stirred for 1 h while cooling and left overnight at room temperature. The precipitate of DCMG was separated and the filtrate was evaporated to dryness. The resulting solid residue was dissolved in 15 ml of a mixture of ethyl acetate: butanol (80:20). The organic layer was washed with 3% NaHCO ₃ (2 × 1 ml), water (2 × 1 ml), 5% citric acid (2 × 1 ml), dried over anhydrous Na ₂ SO ₄ and evaporated to dryness. The product crystallized under abs. diethyl ether. Yield 0.134 g (49%). Amino acid analysis: Ala: Glu = 1.8: 1; R _f 0.81 (C), 0.79 (D); HPLC 16.8 min (gradient A); Mr (calc / calc.) 547.6 / 547.6; 1H NMR, DMSO-D6, δ, ppm -1.1 d (3H, C H ₃ in Ala), 1.25 d (3H, C H ₃ in Ala), 1.4-1.7 m (2H, -C H ₂ -CH ₂ -CO- NH ₂ Gln), 1.75 m (2H, -CH ₂ -C H ₂ -CO-NH ₂ Gln), 3.35 m (1H, α-C H Gln), 4.1 m (1H, α- С Н Ala), 4.5 m (1Н, α-С Н Н Ala), 5.3 d (2Н, -О-С Н -С ₆ Н ₅ ), 6.75 s (1H, aromatic CH in A β-cycle βNA), 7.2-7.7 m (5H, C ₆ H ₅ in Cbz-), 7.8-8.4 m (4H, aromatic CH in βNA B-cycle).

Compounds of the general formula A-Xaa-Gln-B, where A = Z, Boc, Bz, Ac, For, Suc; Haa = Arg, Lys, Pro, Ala-Ala, Ala-Gly, Gly-Ala, Gly-Gly; B = AMC, βNA, AFC, pNA.

Example 4

Synthesis of pyroglutamyl-L-phenylalanyl-L-glutaminyl-n-nitroanilide (Glp-Phe-Gln-pNA)

Boc-Phe-Gln-pNA tert-butyloxycarbonyl-L-phenylalanyl-L-glutaminyl-n-nitroanilide

A solution of 4 g (9.3 mmol) of Boc-Phe-OPfp and 3.0 g (7.9 mmol) of H-Gln-pNA in DMF was stirred at room temperature for 12 hours. After evaporation on a rotary evaporator, the resulting oily residue was dissolved in a mixture ethyl acetate and isopropanol. The organic layer was washed with 5% H ₂ SO ₄ , 5% NaHCO ₃ , 20% NaCl, dried over anhydrous Na ₂ SO ₄ and evaporated to dryness. Yield: 3.6 g (90%).

Phenylalanyl-L-glutaminyl n-nitroanilide Glp-Phe-Gln-pNA (trifluoroacetate) TFA x H-Phe-Gln-pNA

A mixture of trifluoroacetic acid (30 ml) and DCM (20 ml) was added to Boc-Phe-Gln-pNA (3.60 g; 7.0 mmol) and stirred for 30 minutes. The reaction mixture was evaporated on a rotary evaporator to a viscous oil. Diethyl ether was added to the residue, triturated, the precipitate formed was filtered off and washed with ether and petroleum ether. Dried in vacuo. Yield: 3.60 g (97%).

Pyroglutamyl-L-phenylalanyl-L-glutaminyl-n-nitroanilide (Glp-Phe-Gln-pNA)

To a solution of Glp-OPfp (2.14 g; 7.25 mmol) in DMF was added 3.4 g (6.50 mmol) of TFAxPhe-Gln-pNA, 0.44 ml (6.5 mmol) of triethylamine and left under stirring for the night. The reaction mixture was evaporated in vacuo, the resulting oily residue was dissolved in a mixture of ethyl acetate and propanol and washed with 10% NaHCO ₃ solution and saturated NaCl solution. The solution was dried over Na ₂ SO ₄ and evaporated in vacuo. The solid residue was triturated with dry ethyl acetate and petroleum ether. The crystalline precipitate was filtered off, washed with ethyl acetate and dried in vacuo.

Yield: 1.763 g (52%). Amino acid analysis: Glu: Phe 1.98: 1.1; R _f 0.75 (C), 0.60 (D); HPLC 13.0 min (gradient A), 8.2 min (gradient B).

Example 5

Determination of kinetic parameters of the hydrolysis of Gln-containing fluorogenic substrates by cysteine peptidases of the C1 family

5 μl of enzyme preparations of known concentration were added to the microplate cell. Added 5 μl of 40 mm EDTA and 10 μl of 120 mm Cys, after which the contents of the cells were brought to a final volume of 195 μl of 0.1 M universal buffer with a pH of 5.6. The resulting reaction mixture was incubated for 20 minutes at room temperature. Next, 5 μl of 10-100 μM substrate solutions in DMF were added and the fluorescence intensity was measured at zero time. The mixture was then incubated at 37 ° C, determining the change in fluorescence intensity at various time intervals. The measurements were carried out at λ _ex = 360 nm and λ _em = 440 nm for the AMS group; λ _ex = 355 nm and λ _em = 460 nm for the βNA fluorophore (k _fl = 0.081 μM) and at λ _ex = 400 nm and λ _em = 505 nm for the AFC fluorophore (k _fl = 0.08 μM).

Kinetic parameters were calculated by nonlinear regression using the OriginPro 7.5 program according to the standard Michaelis-Menten equation:

where y is the initial reaction rate, which was determined as the derivative at time zero of the function of the dependence of the amount of product released on time, M / min; x is the concentration of the substrate in the sample, M; a = k _cat * [E] / K _M , b = k _cat * [E], where [E] is the concentration of the enzyme in the sample.

Example 6

Hydrolysis of Gln-containing fluorogenic substrates with cysteine peptidases of the C1 family and peptidases of other clans

5 μl of a known concentration enzyme preparation was added to the microplate well. The contents of the cells were adjusted to a final volume of 195 μl with 0.1 M universal pH 5.6 buffer containing 0 0002 mmol EDTA and 0.0012 mmol Cys for cysteine peptidases, Tris-HCl buffer, pH 8.0, for chymotrypsin, trypsin, subtilisin and thermolysin and acetate buffer, pH 3.5, for pepsin. The reaction mixture was incubated for 20 min at room temperature, 5 μl of a 1 mM substrate solution in DMF was added, and the fluorescence intensity was measured at zero time. The mixture was then incubated at 37 ° C, determining the change in fluorescence intensity at various time intervals. The measurements were carried out at λ _ex = 360 nm and λ _em = 440 nm. The enzyme activity was calculated by the formula:

where A _beats - the activity of the drug, M _Subst / M _farms min

dI / dt is the initial fluorophore cleavage rate, which was determined as the derivative at time zero of the function of the dependence of the amount of the released fluorophore on time, cu / min;

для АМС-группировки, при которой интенсивность флуоресценции раствора равно 1 условной единице (определяется в специальном опыте путем построения калибровочной прямой);

for the AMS group, in which the fluorescence intensity of the solution is 1 conventional unit (determined in a special experiment by constructing a calibration line);

c _farms - the concentration of the enzyme in the sample, M

The obtained data on the specific activity of a number of the studied C1 family cysteine peptides in relation to fluorogenic Gln-containing substrates Glp-Phe-Gln-AMC, Glp-Val-Gln-AMC, Z-Pro-Gln-βNA, Z-Ala-Ala-Gln ββNA are given in table. 2 (columns 4-7). Column 3 shows the values of the specific activity of hydrolysis by the indicated cysteine peptidases of the alanine-containing prototype Glp-Phe-Ala-AMC.

Example 7

Hydrolysis of Gln-containing chromogenic substrates with cysteine peptidases of the C1 family

5 μl of a known concentration enzyme preparation was added to the microplate well. Added 5 μl of 40 mm EDTA and 10 μl of 120 mm Cys, after which the contents of the cells were brought to a final volume of 195 μl of 0.1 M universal buffer with a pH of 5.6. The reaction mixture was incubated for 15 min at room temperature and 5 min in an incubator at 37 ° C. Next, 5 μl of 10 mM substrate solution in DMF was added and the absorbance of the solution was measured at 405 nm at the initial time. The mixture was then incubated at 37 ° C, determining the change in absorbance at 405 nm every 3 minutes.

To calculate the specific activity A _beats (M _substr / M _farms * min) used the formula:

where dD ₄₀₅ / dt is the initial rate of n-nitroaniline cleavage at time zero (determined by the slope of the linear portion of the kinetic curve, p.u./min;

k _chr = 157 μM for the n-nitroanilide group, in which the absorption of the solution is 1 conventional unit (determined in a special experiment by constructing a calibration line)

c _farms - the final concentration of the enzyme in the reaction mixture, M.

The obtained data on the specific activity of a number of the studied C1 cysteine peptidases with respect to chromogenic substrates: Ala-containing prototype Glp-Phe-Ala-pNA and Gln-containing substrate Glp-Phe-Gln-pNA are given in table. 2 (columns 1 and 2, respectively).

Example 8

Native electrophoresis and post-electrophoretic activity detection

Electrophoresis of the preparations was performed under native conditions in a 12% separating and 4% concentrating polyacrylamide gel (PAGE) in a buffer containing 35 mM HEPES and 43 mM imidazole at pH 7.4. On the gel track, 20 μl of papain was applied in the same buffer with D ₂₈₀ = 0.05, 0.1, 0.25, 0.5, 1.0 p.u. Electrophoresis was usually carried out in the direction from the cathode to the anode at a current of 10 mA for 40 min.

After electrophoresis was completed to visualize the protein bands, the gel block was placed in a 0.1% Coomassie G-250 solution in ethanol / acetic acid / water (30:10:60 volume%), washed with the same solution without dye, and the gels were photographed.

Post-electrophoretic testing of activity on n-nitroanilide substrates was carried out by applying nitrocellulose membranes saturated with specific substrates to the PAAG block, in which electrophoretic separation of proteins was previously performed. For this, the gel was placed for 20 minutes in 10 ml of 0.1 M universal buffer, pH 5.6, containing 1 mm DTT. In parallel, in a solution containing 250 μl of 20 mM substrate in DMF in 10 ml of 0.1 M universal buffer, pH 5.6, containing 1 mM DTT, the nitrocellulose membrane was soaked and incubated in an incubator at 37 ° C for 30 minutes. Then, the nitrocellulose membrane was applied to the gel after electrophoresis and incubated at 37 ° C for 60 minutes in a closed Petri dish. After incubation, the membrane was transferred to a new Petri dish and the diazotization reaction of n-nitroaniline released as a result of hydrolysis was carried out successively with three freshly prepared reagents:

1. 10 ml of an aqueous solution of NaNO ₂ (0.01 g in 10 ml of 1 M HCl), incubated for 5 minutes, then the solution was drained.

2. 10 ml of an aqueous solution of ammonium amidosulfate (0.05 g in 10 ml of 1 M HCl), left for 5 minutes, the solution was selected

3. 10 ml of a solution of N- (1-naphthyl) ethylenediamine dihydrochloride (0.005 g in 10 ml) in 50% ethanol, left for 5 minutes, the solution was selected.

The localization of proteolytic activity was determined by the formed pink bands on a white background of the membrane.

Post-electrophoretic testing of activity on fluorogenic substrates was carried out after incubation of the gel block in a solution of fluorogenic substrate. The gel after electrophoresis was placed for 10 minutes in 10 ml of 0.1 M universal buffer, pH 5.6, containing 1 mM DTT, 2.5% DMF and 125 μM fluorogenic substrate and incubated at 37 ° C. Localization of proteolytic activity was determined by visualization of fluorescent bands under a UV lamp at 366 nm.

The results obtained indicate the possibility of using the proposed substrates for direct detection and error-free localization in the gel of a protein band belonging to the studied enzyme for further analysis.

Example 9

Digestion of digestive peptidases from Tribolium castaneum larvae

2.1 ml of the extract (D ₂₈₀ = 5.9) obtained from the intestine of T. castaneum larvae, after centrifugation at 16,000 rpm for 1 h, was applied at 4 ° C on a Sephadex G-75 column (2.5 × 120 cm) in 0.01 M phosphate buffer, pH 5.6, containing 10 ^-4 M TLCK (N-α-tosyl-L-lysine-chloromethyl ketone) and 10 ^-4 M chymostatin. 9 ml fractions were collected. Protein concentration was evaluated by absorbance at 280 nm. Enzymatic activity in eluate fractions, taking 20-80 μl aliquots, was determined using substrates Glp-Phe-Gln-pNA, Z-Phe-Arg-pNA, Z-Arg-Arg-pNA at pH 5.6 in the presence of 6 mM cysteine and 1 mm EDTA.

Thus, the use of the proposed selective substrates makes it possible to unambiguously identify the activity of cysteine peptidases in such a complex natural mixture as the digestive complex of T. castaneum larvae.

¹ Data from the work [Semashko T.A., Vorotnikova E.A., Sharikova VF, Vinokurov KS, Smirnova YA, Dunaevsky YE, Belozersky MA, Oppert B., Elpidina EN, Filippova IY Selective chromogenic and fluorogenic peptide substrates for the assay of cysteine peptidases in complex mixtures, Anal. Biochem. 49 (2014) 179-187].

Claims (6)

1. The peptide substrate of the cysteine peptidases of the papain family of General formula (I): A-Xaa-Gln-B (I), where A is an N-protecting group, which is Glp, Haa - amino acid Phe, In - marker fluorogenic group, represents 7-amido-4-methylcoumaride (AMS) or 4-nitroanilide (pNA). 2. The use of the peptide substrate according to claim 1 for creating test systems for detecting peptidases, which is carried out in solution, in a gel after electrophoresis under native conditions, in in vivo systems.

Images