Efficient enzyme-catalyzed synthesis of peptide secondary amides for use as serine proteinase inhibitors

Journal of Molecular Catalysis B: Enzymatic 80 (2012) 58–66

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Efficient enzyme-catalyzed synthesis of peptide secondary amides for use as serine proteinase inhibitors Maxim E. Sergeev a,∗ , Tatiana L. Voyushina a , Olga A. Sergeeva b , Galina G. Belozerskaya b a b

Laboratory of Protein Chemistry after V. M. Stepanov, Institute of Microbial Genetics, Moscow, Russia Laboratory for Pathology and Pharmacology of Hemostasis, Hematology Research Center, Russian Academy of Medical Sciences, Moscow, Russia

a r t i c l e

i n f o

Article history: Received 18 October 2011 Received in revised form 19 April 2012 Accepted 20 April 2012 Available online 3 May 2012

a b s t r a c t An efficient and straightforward approach toward enzymatic synthesis of secondary amides of peptides was developed. A number of peptide derivatives containing amide moiety at C-terminus was obtained via subtilisin catalysis in good yields under mild conditions. Biological activity of some peptamides is evaluated. © 2012 Elsevier B.V. All rights reserved.

Keywords: Peptides Peptamides Enzyme Catalysis Medicinal chemistry

1. Introduction 1.1. Statement of significance Cyclic amides of amino acids and peptides, which are inhibitors of serine proteinases such as proteinases of haemostasis system (thrombin, plasmin, etc.), can be used in medicine as anti-bleeding or anti-thrombotic agents [1–3]. There is also a number of reports covering the applicability of peptide amides for the treatment of various conditions such as obesity and obesity-related diseases (in which case they act as the ligands for melanocortin receptors), hypertension, congestive heart failure and other blood pressure-related disorders (renin inhibitors), cancer and immune dysfunctions (tyrosine phosphatase inhibitors) [4–7]. Also, imidazole derivatives of peptides and amino acids can be directly converted into ␣-aminoaldehydes and peptidoaldehydes, which in turn are inhibitors of plasmin and thrombin [8–10]. Affinity chromatography is another important field where cyclic amides of peptides are extensively used often offering many advantages over other ligands. The affinity purification of proteins is based on the functional characteristics of enzymes and their binding affinity to the different ligands making it possible to separate the required

∗ Corresponding author at: Laboratory of Protein Chemistry after V. M. Stepanov, Institute of Microbial Genetics, 1st Dorozhny pr., 1, Moscow, 117545, Russia. Tel.: +7 495 315 37 38; fax: +7 495 315 05 01. E-mail address: [email protected] (M.E. Sergeev). 1381-1177/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.molcatb.2012.04.019

enzyme from an enzymatic mixture or a biological sample [11–16]. The peptidic ligands with better specificity to the enzyme are required, for example peptidoaldehydes and peptide cyclic amides [17–21]. Thus, until the present work there existed no satisfactory general procedure for the synthesis of such compounds, especially cyclic amides, which are more stable than peptidoaldehydes. 1.2. Known synthetic procedures Prior to our studies Kikumoto et al. prepared the N-protected arginyl-piperidines by the mixed anhydrides method starting from N-protected arginine and 4-methylpiperidine with subsequent deprotection of the product [22]. However, this method includes the steps of protection–deprotection of arginine side-group, which procedure is pretty hard. Synthesis of morpholine derivatives of leucine and arginine via coupling of N-protected amino acids and morpholine in the presence of 1-hydroxybenzotriazole (HOBt) and dicyclohexylcarbodiimide (DCC) at room temperature is described earlier. The reaction yields were reasonably high (up to 80%) though it took up to four days for the reaction to be completed [21]. The piperidine derivatives of tryptophan-like compounds were prepared in low yields (40–60%) by Reichert et al. via coupling of the carboxylic acid derivatives and the piperidine with Mukayama’s reagent (2-chloro-1-methylpyridinium iodide (CMPI)) as the coupling agent [23]. Our procedure described earlier allows to obtain secondary amides of amino acids with high yields [24], but in further synthesis the yield of final tripeptide product decreases dramatically when the conventional methods of peptide synthesis

M.E. Sergeev et al. / Journal of Molecular Catalysis B: Enzymatic 80 (2012) 58–66

are used, such as activated-esters synthesis, or HOBt/DCC-coupling of amino acid derivatives [25–27]. Synthesis of aspartic protease inhibitors using Mukayama aldol condensation led to target compounds in moderate yields of 20–60%. This procedure was also a complicated one requiring multi-step synthesis [28]. Enzyme-catalyzed synthetic procedures spawned great application field since it became now possible to obtain the desired target compounds with excellent yields under mild conditions and in most cases the purification procedures for reaction mixtures are simple and short. Also, in case of enzymatic catalysis, the acylating agents with non-protected side groups can be used and the optical purity of the product can be preserved. Use of enzymes with broad substrate specificity made it possible to carry out the experiments with different nucleophiles, which in turn led to the synthesis of a wide spectrum of potentially bioactive compounds with possible use in various medical areas. A number of reports dealing with the enzyme-catalyzed synthesis of bioactive compounds using different enzymes [29,30]. Maruyama et al. described the synthesis of sugar amino acid esters using proteases (Subtilisin Carlsberg and others) [31]. It was shown that conversion of starting material in organic media such as pyridine at 40 ◦ C did not exceed 50% after 24 h. Application of enzyme–surfactant complex allows to achieve ∼65% conversion at these conditions but the yields of target compounds are still low. The chemoenzymatic synthesis of peptidomimetics based on (R)-3,4-diaminobutanoic acid utilizing combination of solidphase synthesis and enzymatic catalysis with lipase B from Candida antarctica was reported. The yield of title compound was found to be about 76% after 3 days at 30 ◦ C [32]. There are also some reports about subtilisin-catalyzed peptide synthesis under high pressure, but the yields of these reactions are still low (about 30%) [33,34]. Some papers dealt with enzyme-catalyzed synthesis of primary amide derivatives of peptides deals with few primary amines only and the yields of the acylation reaction catalyzed by subtilisin BPN’ were as high as 40–70% [40]. The research of enzyme-catalyzed synthesis of various substrates and enzyme-inhibitors has been the most important purpose of our laboratory for many years. We have reported a number of efficient methods for the synthesis of peptides and different peptide-derivatives which utilized thermolysin, various subtilisins, trypsin, ␣-chymotrypsin and others [35–39]. The most effective among the enzymes used was subtilisin [41]. Highly aqueous media are undesirable in enzymatic reactions due to easy hydrolysis of acylating agent [42]. After having been bound on the solid-phase carrier, the enzyme becomes potentially vulnerable to the carrier hydrate water; but due to the lack of free water molecules in solvent hydrolysis process was strongly reduced [43,44]. Earlier we demonstrated that the usage of solid-phase-bound subtilisin-72 (serine proteinase from Bacillus subtilis st. 72) distributed on porous carrier Silochrom-C80 allowed to carry out the synthesis in organic media, which were generally non-specific for enzymatic synthesis due to enzyme inactivation by organic solvent. We have also proved the feasibility of peptamide synthesis by acylation of amino acid amides with peptide esters. The reaction between For-Ala-Phe-OMe and LysPip afforded the tripeptide ForAlaPhe-LysPip in high yield [45]. By this procedure we synthesized the two novel synthetic peptide amides, namely formyl-l-alanyl-l-phenylalanyll-lysyl-piperidide (For-Ala-Phe-Lys-Pip) and formyl-l-alanyl-lphenylalanyl-l-lysyl-morpholide (For-Ala-Phe-Lys-Mrf) of formulae 1 and 2 (Fig. 1), and tested them for the plasmin-inhibiting activity in vitro and in vivo. In vitro experiments proved that these peptides are good inhibitors of euglobulin factor XIIa-dependent lysis (time of thrombi degradation about 21–23 s while 40–50 s with tranexamic acid or ␧-aminocaproic acid which are standard bleeding-terminating agents) and plasmin inhibition constants Ki are 19.20 ␮M and 19.08 ␮M correspondingly. In vivo experiments

59

Table 1 Gradient elution for HPLC analysis. Time, min

Flow rate, ml/min

%A

%B

0 10 15 20

1 1 1 1

80 40 40 80

20 60 60 20

on thiopental-anaesthetized Shinshilla rabbits showed that bleeding of liver surface wound (with a size approximately 2 cm × 2 cm) was arrested in about 60–70 s vs 205 s with standard anti-bleeding agent (␧-aminocaproic acid) [41]. 2. Materials and methods 2.1. Enzyme and chemicals Subtilisin-72 was isolated and purified by common methods from B. subtilis st. 72. All other chemicals used were of analytical grade and purchased from Sigma–Aldrich and Serva. 2.2. Analytical methods The aminolysis reaction was monitored by RP-HPLC analysis carried out in a system (Beckman System Gold) composed of a column (Beckman C18 5 ␮M, 250 mm × 4.6 mm) and a UV/vis detector (Beckman System Gold, model 167,  = 220 nm). The compounds were eluted by gradient elution with an eluent system of 5% acetonitrile/H2 O (A) and 95% acetonitrile/H2 O (B), both containing 0.05% of trifluoroacetic acid (see Table 1). 2.3. Determination of enzyme activity Z-Ala-Ala-Leu-pNA solution in DMF (50 ml, 5 mg/mL) was added to 2 ml of 0.05 M Tris/HCl buffer (pH 8.3) containing 1.5 mM CaCl2 and the mixture was incubated at 20 ◦ C for 10 min. Then, immobilized enzyme (20 mg subtilisin/250 mg silochrom) was added to initiate the reaction and the suspension was incubated at 20 ◦ C with stirring for 1–4 d. Thereafter, the absorbance of reaction mixture was measured at  410 nm. Qualitative curves are plotted in coordinates A410 – reaction time. 2.4. Enzymatic synthesis of peptide amides General procedure for the synthesis of N␻ -(benzyloxycarbonyl)l-alanyl-l-alanyl-l-(O-methyl)glutamic acid (imidazol-1-yl)amide (Cbz-AlaAlaGlu(OMe)-Im): subtilisin-72 (from B. subtilis, strain 72) (20 mg, 0.69 ␮mol) was dissolved in phosphate buffer (pH 7.5, 600 ␮l). The obtained solution was deposited dropwise on the surface of silochrom-C80 (250 mg), placed in flat-bottom flask as a ‘single-layer’. The flask was placed under high vacuum and dried over phosphorous pentoxide for 3 days. N␻ -(benzyloxycarbonyl)-l-alanyl-l-alanyl-l-(Omethyl)glutamic acid methyl ester (46 mg, 100 ␮mol) was dissolved in 1.5 ml of freshly distilled acetonitrile to prepare solution A. Imidazole (68 mg, 1000 ␮mol) was dissolved in 1 ml of freshly distilled acetonitrile to prepare solution B. The solutions A and B were mixed together and the catalyst was added immediately after being unvacuumed. The flask was sealed and the reaction mixture was stirred by means of orbital shaker for 5 days at room temperature (about 18–22 ◦ C). The catalyst was filtered from acetonitrile and washed thoroughly with methanol (3 × 10 ml) under sonication. The fraction with less product content was discarded. The remaining fraction was filtered and evaporated. The residue was dissolved in methanol (500 ␮l) and precipitated

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M.E. Sergeev et al. / Journal of Molecular Catalysis B: Enzymatic 80 (2012) 58–66

O N

N O

O HN

HN HN

O O

HCl

HN NH2

HN

O O

HCl

NH2

HN

O

O 1

2

Fig. 1. Structures of synthetic peptide-amides formyl-l-alanyl-l-phenylalanyl-l-lysyl-piperidide hydrochloride (For-Ala-Phe-Lys-Pip*HCl) 1 and formyl-l-alanyl-lphenylalanyl-l-lysyl-morpholide hydrochloride (For-Ala-Phe-Lys-Mrf*HCl) 2.

with diethyl ether (5 ml). The resulted solid was filtered out and dried in vacuum. Title compound was obtained as a cream solid, yield 96%. 1 H NMR (400 MHz, d6-DMSO): ı 1.2–1.3 (6H, 2*CH -Ala), 3 2.1–2.3 (m, 4H, 2*CH2 , Glu), 3.6 (s, 3H, GluOCH3 ), 4.3–4.4 (m, 2H, 2*CH-Ala), 4.7–4.8 (m, 1H, CH-Glu), 5.1 (s, 2H, CH2 -Cbz), 6.9 (d, 1H, CH-Im), 7.3–7.4 (m, 5H, C6 H5 -Cbz), 7.6 (s, 3H, 3*NH), 8.1–8.2 (d, 1H, CH-Im), 8.7 (s, 1H, CH-Im) ppm. MS (MALDI-TOF): 474.5 [M+H]. HRMS (ES+): 473.1915 found, 473.1910 calculated. Scaled-up experiment, utilizing 1.5 g (3.2 mmol) of peptide ester, resulted in the same yield of desired product without significant increase of hydrolysis product formation. In a similar way, using appropriate N-protected peptidic esters and cyclic amines as starting materials the following peptamides were obtained: N␻ -(2,4-dinitrophenyl)-l-alanyl-l-alanyl-l-leucine (piperidin-1-yl)amide (Dnp-AlaAlaLeu-Pip): Orange solid. 1 H NMR (400 MHz, d6-DMSO): ı 0.8–0.9 (d, 6H, 2*CH 3 Leu), 1–1.1 (m, 2H, CH2 -Leu), 1.3–1.35 (m, 1H, CH-Leu), 1.4–1.6 (m, 6H, 2*m-CH2 -Pip + p-CH2 -Pip), 1.23/1.47 (dd, 6H, 2*CH3 -Ala), 3.9/4.2/4.6 (m, 3H, CH-(Ala/Ala/Leu), 3.1–3.3 (dd, 4H, 2*o-CH2 -Pip), 7, 8 (dd, 2H, 5,6-CH-Dnp), 8.55 (s, 3H, 3*NH), 8.8 (s, 1H, 3-CHDnp) ppm. MS (MALDI-TOF): 507.2 [M+H]. HRMS (ES+): 506.5528 found, 506.5521 calculated. N␻ -(2,4-dinitrophenyl)-l-alanyl-l-alanyl-l-leucine (3carboxy-4-hydroxyphenyl)amide (Dnp-AlaAlaLeu-ASA): Yellow solid. 1 H NMR (400 MHz, d6-DMSO): ı 0.8–0.9 (d, 6H, 2*CH -Leu), 3 1–1.1 (m, 2H, CH2 -Leu), 1.3–1.35 (m, 1H, CH-Leu), 1.23/1.47 (dd, 6H, 2*CH3 -Ala), 3.9/4.2/4.6 (m, 3H, CH-(Ala/Ala/Leu), 7, 8 (dd, 2H, 5,6CH-Dnp), 6.9–7.4 (2*d, 2H, 2*5,6-CH-ASA), 8.3 (s, 1H, 3-CH-ASA), 8.8 (s, 1H, 3-CH-Dnp), 9.0 (s, 3H, 3*NH) ppm. 4-OH- and 3-COOHprotons overlapped by NH signals. MS (MALDI-TOF): 575.6 [M+H]. HRMS (ES+): 574.2015 found, 574.2023 calculated. N␻ -(2,4-dinitrophenyl)-l-alanyl-l-alanyl-l-leucine (morpholin-4-yl)amide (Dnp-AlaAlaLeu-Mrf): Orange solid. 1 H NMR (400 MHz, d6-DMSO): ı 0.8–0.9 (d, 6H, 2*CH -Leu), 3 1–1.1 (m, 2H, CH2 -Leu), 1.3–1.35 (m, 1H, CH-Leu), 1.23/1.47 (dd, 6H, 2*CH3 -Ala), 3.9/4.2/4.6 (m, 3H, CH-(Ala/Ala/Leu), 3.1–3.2 (dd, 4H, 2*o-CH2 -Mrf), 3.5–3.6 (m, 4H, 2*m-CH2 -Mrf), 7–8 (dd, 2H, 5,6CH-Dnp), 8.55 (s, 3H, 3*NH), 8.8 (s, 1H, 3-CH-Dnp) ppm. MS (MALDI-TOF): 509.6 [M+H].

HRMS (ES+): 508.2277 found, 508.2282 calculated. N␻ -(2,4-dinitrophenyl)-l-alanyl-l-alanyl-l-leucine (imidazol-1-yl)amide (Dnp-AlaAlaLeu-Im): Yellow solid. 1 H NMR (400 MHz, d6-DMSO): ı 0.87–0.92 (d, 6H, 2*CH -Leu), 3 1.05–1.15 (m, 2H, CH2 -Leu), 1.3–1.35 (m, 1H, CH-Leu), 1.2/1.4 (dd, 6H, 2*CH3 -Ala), 3.9/4.2/4.6 (m, 3H, CH-(Ala/Ala/Leu), 6.9 (1H, d, 4CH-Im), 8.08 (1H, d, 5-CH-Im), 7, 8 (dd, 2H, 5,6-CH-Dnp), 8.55 (s, 3H, 3*NH), 8.7 (1H, s, 3-CH-Im), 8.8 (s, 1H, 3-CH-Dnp) ppm. MS (MALDI-TOF): 490.6 [M+H]. HRMS (ES+): 489.2001 found, 489.1972 calculated. N␻ -(2,4-dinitrophenyl)-l-alanyl-l-alanyl-l-(O-methyl) glutamic acid (piperidin-1-yl)amide (Dnp-AlaAlaGlu(OMe)-Pip): Yellow solid. 1 H NMR (400 MHz, d6-DMSO): ı 1.1–1.25 (6H, 2*CH -Ala), 3 1.4–1.6 (m, 6H, 2*m-CH2 -Pip + p-CH2 -Pip), 2–2.2 (m, 4H, 2*CH2 , Glu), 3.1–3.3 (dd, 4H, 2*o-CH2 -Pip), 3.55 (s, 3H, GluOCH3 ), 4.3–4.4 (m, 2H, 2*CH-Ala), 4.7–4.8 (m, 1H, CH-Glu), 7–8 (dd, 2H, 5,6-CHDnp), 7.6 (s, 3H, 3*NH), 8.8 (s, 1H, 3-CH-Dnp) ppm. MS (MALDI-TOF): 537.2 [M+H]. HRMS (ES+): 536.2236 found, 536.2231 calculated. N␻ -(2,4-dinitrophenyl)-l-alanyl-l-alanyl-l-(O-methyl) glutamic acid (morpholin-1-yl)amide (Dnp-AlaAlaGlu(OMe)Mrf): Yellow solid. 1 H NMR (400 MHz, d6-DMSO): ı 1.1–1.25 (6H, 2*CH -Ala), 2–2.2 3 (m, 4H, 2*CH2 , Glu), 3.1–3.2 (dd, 4H, 2*o-CH2 -Mrf), 3.5–3.6 (m, 4H, 2*m-CH2 -Mrf), 3.55 (s, 3H, GluOCH3 ), 4.3–4.4 (m, 2H, 2*CH-Ala), 4.7–4.8 (m, 1H, CH-Glu), 7–8 (dd, 2H, 5,6-CH-Dnp), 7.6 (s, 3H, 3*NH), 8.8 (s, 1H, 3-CH-Dnp) ppm. MS (MALDI-TOF): 579.3 [M+H]. HRMS (ES+): 578.2020 found, 578.2023 calculated. N␻ -(2,4-dinitrophenyl)-l-alanyl-l-alanyl-l-(O-methyl) glutamic acid diethylamide (Dnp-AlaAlaGlu(OMe)-DEA): Yellow solid. 1 H NMR (400 MHz, d6-DMSO): ı 0.9–1 (m, 6H, 2*CH -Et), 3 3.3–3.4 (m, 4H, 2*CH2 -Et), 1.1–1.25 (6H, 2*CH3 -Ala), 2–2.2 (m, 4H, 2*CH2 , Glu), 3.55 (s, 3H, GluOCH3 ), 4.3–4.4 (m, 2H, 2*CH-Ala), 4.7–4.8 (m, 1H, CH-Glu), 7–8 (dd, 2H, 5,6-CH-Dnp), 7.6 (s, 3H, 3*NH), 8.8 (s, 1H, 3-CH-Dnp) ppm. MS (MALDI-TOF): 525.3 [M+H]. HRMS (ES+): 524.2033 found, 524.2031 calculated. N␻ -(2,4-dinitrophenyl)-l-alanyl-l-alanyl-l-(O-methyl) glutamic acid (pyridin-4-yl)amide (Dnp-AlaAlaGlu(OMe)-APy): Yellow solid. 1 H NMR (400 MHz, d6-DMSO): ı 1.1–1.25 (6H, 2*CH -Ala), 3 2–2.2 (m, 4H, 2*CH2 , Glu), 3.55 (s, 3H, GluOCH3 ), 4.3–4.4 (m, 2H,

M.E. Sergeev et al. / Journal of Molecular Catalysis B: Enzymatic 80 (2012) 58–66

2*CH-Ala), 4.7–4.8 (m, 1H, CH-Glu),7–8 (dd, 2H, 5,6-CH-Dnp), 8.8 (s, 1H, 3-CH-Dnp), 7.5 (s, 3H, 3*NH), 7.6–8.6 (2*dd, 4H, 4*CH-Py) 8.1 (d, 1H, 5-CH-Im) ppm. MS (MALDI-TOF): 546.2 [M+H]. HRMS (ES+): 545.1875 found, 545.1870 calculated. N␻ -(2,4-dinitrophenyl)-l-alanyl-l-alanyl-l-(O-methyl) glutamic acid (imidazol-1-yl)amide (Dnp-AlaAlaGlu(OMe)-Im): Yellow solid. 1 H NMR (400 MHz, d6-DMSO): ı 1.1–1.25 (6H, 2*CH -Ala), 2–2.2 3 (m, 4H, 2*CH2 , Glu), 3.55 (s, 3H, GluOCH3 ), 4.3–4.4 (m, 2H, 2*CHAla), 4.7–4.8 (m, 1H, CH-Glu), 6.9 (d, 1H, CH-Im), 7–8 (dd, 2H, 5,6CH-Dnp), 8.8 (s, 1H, 3-CH-Dnp), 7.6 (s, 3H, 3*NH), 8.1 (d, 1H, 5-CHIm), 8.8 (s, 1H, 2-CH-Im) ppm. MS (MALDI-TOF): 520.1 [M+H]. HRMS (ES+): 519.1717 found, 519.1714 calculated. N␻ -(2,4-dinitrophenyl)-l-alanyl-l-alanyl-l-(O-methyl) glutamic acid (indol-1-yl)amide (Dnp-AlaAlaGlu(OMe)-Ind): Yellow solid. 1 H NMR (400 MHz, d6-DMSO): ı 1.2–1.3 (6H, 2*CH -Ala), 3 2.1–2.3 (m, 4H, 2*CH2 , Glu), 3.6 (s, 3H, GluOCH3 ), 4.3–4.4 (m, 2H, 2*CH-Ala), 4.7–4.8 (m, 1H, CH-Glu), 6.2–8.2 (m, 6H, Ind), 7–8 (dd, 2H, 5,6-CH-Dnp), 7.6 (s, 3H, 3*NH), 8.8 (s, 1H, 3-CH-Dnp) ppm. MS (MALDI-TOF): 569.3 [M+H]. HRMS (ES+): 568.1815 found, 568.1819 calculated. N␻ -(2,4-dinitrophenyl)-l-alanyl-l-alanyl-l-arginine (morpholin-4-yl)amide (Dnp-AlaAlaArg-Mrf): Yellow solid. 1 H NMR (400 MHz, d6-DMSO): ı 1.25–1.46 (m, 2H, ␣-CH -Arg), 2 1.2/1.4 (m, 2*CH3 -Ala), 1.7 (m, 1H, ␤-CH2 -Arg), 3.26 (m, 1H, ␥-CH2 Arg), 3.2–3.6 (m, 8H, 4-CH2 -Mrf), 3.9 (m, 2H, 2*CH-Ala), 7.1 (d, 1H, 6-CH-Dnp), 7.7 (s, 7H, 3*NH-Ala-Ala-Arg + NH + NH + NH2 -Arg), 8.5 (d, 1H, 5-CH-Dnp), 8.7 (s, 1H, 3-CH-Dnp) ppm. MS (MALDI-TOF): 553.6 [M+H]. HRMS (ES+): 552.2444 found, 551.2452 calculated. N␻ -(2,4-dinitrophenyl)-l-alanyl-l-alanyl-l-arginine (imidazol-1-yl)amide (Dnp-AlaAlaArg-Im): Yellow solid. 1H NMR (400 MHz, d6-DMSO): ı 1.25–1.46 (m, 2H, ␣-CH2 -Arg), 1.2/1.4 (m, 2*CH3 -Ala), 1.7 (m, 1H, ␤-CH2 -Arg), 3.26 (m, 1H, ␥-CH2 Arg), 3.9 (m, 2H, 2*CH-Ala), 6.9 (d, 1H, 4-CH-Im), 7.1 (d, 1H, 6-CHDnp), 7.7 (s, 7H, 3*NH-Ala-Ala-Arg + NH + NH + NH2 -Arg), 7.9 (d, 1H, 5-CH-Im), 8.5 (d, 1H, 5-CH-Dnp), 8.7 (s, 1H, 3-CH-Dnp), 8.7 (s, 1H, 2-CH-Im) ppm. MS (MALDI-TOF): 533.5 [M+H]. HRMS (ES+): 532.2155 found, 532.2142 calculated. N␻ -(2,4-dinitrophenyl)-l-alanyl-l-alanyl-l-arginine (pyrazol-1-yl)amide (Dnp-AlaAlaArg-Pyr): Yellow solid. 1 H NMR (400 MHz, d6-DMSO): ı 1.25–1.46 (m, 2H, ␣-CH 2 Arg), 1.2/1.4 (m, 2*CH3 -Ala), 1.7 (m, 1H, ␤-CH2 -Arg), 3.26 (m, 1H, ␥-CH2 -Arg), 3.9 (m, 2H, 2*CH-Ala), 6.2 (d, 1H, 4-CH-Pyr), 7.1 (d, 1H, 6-CH-Dnp), 7.4 (d, 1H, 3-CH-Pyr), 7.7 (s, 7H, 3*NH-AlaAla-Arg + NH + NH + NH2 -Arg), 8.5 (d, 1H, 5-CH-Dnp), 8.7 (s, 1H, 3-CH-Dnp), 8.7 (s, 1H, 5-CH-Pyr) ppm. MS (MALDI-TOF): 533.3 [M+H]. HRMS (ES+): 532.2147 found, 532.2142 calculated. N␻ -(2,4-dinitrophenyl)-l-alanyl-l-alanyl-l-arginine diethylamide (Dnp-AlaAlaArg-DEA): Yellow solid. 1 H NMR (400 MHz, d6-DMSO): ı 0.9–1 (m, 6H, 2*CH -Et), 3 1.25–1.46 (m, 2H, ␣-CH2 -Arg), 1.2/1.4 (m, 2*CH3 -Ala), 1.7 (m, 1H, ␤-CH2 -Arg), 3.26 (m, 1H, ␥-CH2 -Arg), 3.3–3.4 (m, 4H, 2*CH2 -Et), 3.9 (m, 2H, 2*CH-Ala), 7.1 (d, 1H, 6-CH-Dnp), 7.7 (s, 7H, 3*NHAla-Ala-Arg + NH + NH + NH2 -Arg), 8.5 (d, 1H, 5-CH-Dnp), 8.7 (s, 1H, 3-CH-Dnp) ppm. MS (MALDI-TOF): 538.6 [M+H].

61

HRMS (ES+): 537.2664 found, 537.2659 calculated. N␻ -(benzyloxycarbonyl)-l-alanyl-l-alanyl-l-leucine (imidazol-1-yl)amide (Cbz-AlaAlaLeu-Im): White solid. 1 H NMR (400 MHz, d6-DMSO): ı 1.1–1.3 (m, 2H, ␻-CH -Lys), 1.2 2 (m, 6H, 2*CH3 -Ala), 1.4–2 (m, 3H, (CH2 )3 -Lys), 1.3 (m, 1H, CH-Lys), 2.8–3 (t, 2H, CH2 -Phe), 4–4.1 (m, 1H, CH-Ala), 4.3 (t, 1H, CH-Phe), 4.7 (t, 1H, CH-Lys), 5.0 (s, 2H, CH2 -Bn), 6.9 (d, 1H, 4-CH-Im), 7.7 (s, 5H, 3*NH), 7.3 (m, 5H, C6 H5 -Phe), 8.1 (d, 1H, 5-CH-Im), 8.8 (s, 1H, 2-CH-Im) ppm. MS (MALDI-TOF): 458.6 [M+H]. HRMS (ES+): 457.2321 found, 457.2325 calculated. N␻ -(benzyloxycarbonyl)-l-alanyl-l-alanyl-l-(O-methyl) glutamic acid (indol-1-yl)amide (Cbz-AlaAlaGlu(OMe)-Ind): White solid. 1 H NMR (400 MHz, d6-DMSO): ı 1.2–1.3 (6H, 2*CH -Ala), 3 2.1–2.3 (m, 4H, 2*CH2 , Glu), 3.6 (s, 3H, GluOCH3 ), 4.3–4.4 (m, 2H, 2*CH-Ala), 4.7–4.8 (m, 1H, CH-Glu), 5.1 (s, 2H, CH2 -Cbz), 6.9 (d, 1H, CH-Im), 7.3–7.4 (m, 5H, C6 H5 -Cbz), 7.6 (s, 3H, 3*NH), 6.2–8.2 (m, 6H, Ind) ppm. MS (MALDI-TOF): 537.3 [M+H]. HRMS (ES+): 536.2272 found, 536.2271 calculated. N␻ -(benzyloxycarbonyl)-l-alanyl-l-alanyl-l-leucine diethylamide (Cbz-AlaAlaLeu-DEA): Cream solid. 1 H NMR (400 MHz, d6-DMSO): ı 0.9–1 (m, 6H, 2*CH -Et), 3 3.3–3.4 (m, 4H, 2*CH2 -Et), 1.1–1.3 (m, 2H, ␻-CH2 -Lys), 1.2 (m, 6H, 2*CH3 -Ala), 1.4–2 (m, 3H, (CH2 )3 -Lys), 1.3 (m, 1H, CH-Lys), 2.8–3 (t, 2H, CH2 -Phe), 4–4.1 (m, 1H, CH-Ala), 4.3 (t, 1H, CH-Phe), 4.7 (t, 1H, CH-Lys), 5.0 (s, 2H, CH2 -Bn), 7.7 (s, 5H, 3*NH), 7.3 (m, 5H, C6 H5 -Phe) ppm. MS (MALDI-TOF): 495.5 [M+H]. HRMS (ES+): 494.2481 found, 494.2489 calculated. N␻ -(benzyloxycarbonyl)-l-alanyl-l-alanyl-l-glutamic acid (imidazol-1-yl)amide (Cbz-AlaAlaGlu(OH)-Im): White solid. 1 H NMR (400 MHz, d6-DMSO): ı 1.2–1.3 (6H, 2*CH -Ala), 3 2.1–2.3 (m, 4H, 2*CH2 , Glu), 4.3–4.4 (m, 2H, 2*CH-Ala), 4.7–4.8 (m, 1H, CH-Glu), 5.0 (s, 2H, CH2 -Cbz), 6.9 (d, 1H, CH-Im), 7.3–7.4 (m, 5H, C6 H5 -Cbz), 7.6 (s, 3H, 3*NH), 6.9, 8.1, 8.8 (3*s, 3H, 3*CH-Im), 6.2–8.2 (m, 6H, Ind) ppm. MS (MALDI-TOF): 491.6 [M+H]. HRMS (ES+): 490.2419 found, 490.2427 calculated. N␻ -(formyl)-l-alanyl-l-phenylalanyl-l-lysine (morpholin-1yl)amide (For-AlaPheLys-Mrf): White solid. 1 H NMR (400 MHz, d6-DMSO): ı 1.4–1.8 (m, 3H, (CH ) -Lys), 2 3 1,6 (s, 3H, CH3 -Ala), 2.6 (m, 2H, ␻-CH2 -Lys), 2.8–3 (t, 2H, CH2 -Phe), 3.2–3.6 (m, 8H, 4*CH2 -Mrf), 4–4.1 (m, 1H, CH-Ala), 4.3 (t, 1H, CHPhe), 4.7 (t, 1H, CH-Lys), 6.1 (s, 5H, 3*NH + NH2 ), 7.2 (m, 5H, C6 H5 Phe), 7.4 (s, 1H, For) ppm. MS (MALDI-TOF): 461.6 [M+H]. HRMS (ES+): 461.5545 found, 461.5512 calculated. N␻ -(formyl)-l-alanyl-l-phenylalanyl-l-lysine (piperidin-1yl)amide (For-AlaPheLys-Pip): White solid. 1 H NMR (400 MHz, d6-DMSO): ı 1.35–1.9 (m, 3H, (CH ) -Lys), 2 3 1.5 (s, 3H, CH3 -Ala), 1.5–1.6 (m, 6H, 3*CH2 -Pip), 2.5–2.6 (m, 2H, ␻CH2 -Lys), 2.7–3 (t, 2H, CH2 -Phe), 3.1–3.3 (m, 4H, 2*CH2 -Pip), 4–4.1 (m, 1H, CH-Ala), 4.3 (t, 1H, CH-Phe), 4.7 (t, 1H, CH-Lys), 6.1 (s, 5H, 3*NH + NH2 ), 7.2 (m, 5H, C6 H5 -Phe), 7.4 (s, 1H, For) ppm. MS (MALDI-TOF): 459.6 [M+H]. HRMS (ES+): 459.2820 found, 459.2846 calculated. N␻ -(formyl)-l-alanyl-l-phenylalanyl-l-lysine (indol-1-yl) amide (For-AlaPheLys-Ind): White solid.

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1 H NMR (400 MHz, d6-DMSO): ı 1.4–2 (m, 3H, (CH ) -Lys), 1,6 2 3 (s, 3H, CH3 -Ala), 2.6 (m, 2H, ␻-CH2 -Lys), 2.8–3 (t, 2H, CH2 -Phe), 4–4.1 (m, 1H, CH-Ala), 4.3 (t, 1H, CH-Phe), 4.7 (t, 1H, CH-Lys), 6.1 (s, 5H, 3*NH + NH2 ), 7.2 (m, 5H, C6 H5 -Phe), 7.4 (s, 1H, For), 7.1–8.4 (m, 6H, Ind) ppm. MS (MALDI-TOF): 491.3 [M+H]. HRMS (ES+): 491.2525 found, 491.2533 calculated. N␻ -(formyl)-l-alanyl-l-phenylalanyl-l-lysine (DLhomoprolin-1-yl)amide (For-AlaPheLys-hP): White solid. 1 H NMR (400 MHz, d6-DMSO): ı 1.3–1.8 (mm, 9H, 3*CH 2 hP + (CH2 )3 -Lys), 1,6 (s, 3H, CH3 -Ala), 2.6 (m, 2H, ␻-CH2 -Lys), 2.8–3 (t, 2H, CH2 -Phe), 4–4.1 (m, 1H, CH-Ala), 4.3 (t, 1H, CH-Phe), 4.7 (t, 1H, CH-Lys), 5.37 (d, 1H, CH-hP), 7.0 (s, 5H, 3*NH + NH2 ), 7.0 (s, 1H, COOH-hP), 7.2 (m, 5H, C6 H5 -Phe), 7.4 (s, 1H, For) ppm. MS (MALDI-TOF): 503.4 [M+H]. HRMS (ES+): 503.2661 found, 503.2744 calculated. N␻ -(formyl)-l-alanyl-l-phenylalanyl-l-lysine diethylamide (For-AlaPheLys-DEA): White solid. 1 H NMR (400 MHz, d6-DMSO): ı 0.9, 3.4 (2*m, 6H, 2*Et), 1.4–2 (m, 3H, (CH2 )3 -Lys), 1,6 (s, 3H, CH3 -Ala), 2.6 (m, 2H, ␻-CH2 -Lys), 2.8–3 (t, 2H, CH2 -Phe), 4–4.1 (m, 1H, CH-Ala), 4.3 (t, 1H, CH-Phe), 4.7 (t, 1H, CH-Lys), 6.1 (s, 5H, 3*NH + NH2 ), 7.2 (m, 5H, C6 H5 -Phe), 7.4 (s, 1H, For) ppm. MS (MALDI-TOF): 447.4 [M+H]. HRMS (ES+): 447.2828 found, 447.2846 calculated.

2.5. Biological evaluation of synthesized compounds Biological activity of some synthesized compounds was demonstrated by in vivo and in vitro experiments. The haemostatic aldehyde For-Ala-Phe-Lys-al was used to compare the activities of peptamides and aldehydes. 2.5.1. Ki measurement The inhibition constants for some synthesized compounds were determined by standard procedure described by Drozd et al. [52]. The enzymes tested were plasmin and subtilisin. 2.5.2. In vitro experiments The peptide was dissolved in distilled water at a concentration of 0.01 M. The solution was placed in a bulb in which 0.5 ml of human blood plasma, 6.5 ml of water, 0.25 ml of caoline suspension and 0.18 ml of 1% acetic acid was added. The mixture was incubated at 37 ◦ C for 30 min. The reaction mixture was centrifuged and the euglobulin clot was separated. The clot was dissolved in 0.5 ml of Tris–HCl buffer with pH of 7.4. 0.5 ml of 0.27% CaCl2 solution was added and the reaction mixture was incubated at 37 ◦ C. The time of full clot dissolution was registered. Distilled water was used as control. 2.5.3. In vivo experiments The haemostatic effect of new peptamides was carried out in laboratory conditions using four ‘Shinshilla’ rabbits of 3–4 kg weight. The animals were narcotized with thiopental (6–8 ml of 1% solution intravenously and then 6–10 ml of 1,5% solution intraabdominally). Laparotomy was made using the dissection along the abdomen white line. The intestine and the front liver surface were elevated out of the wound and restricted by the tampons with warm physiologic solution. The liver was dissected to produce the round surface wound with square of about 1.5 cm2 and 0.3 cm depth. The capillary-parenchymatous bleeding was stopped by the application of tampon with peptamide solution directly on the wound. The water-wet tampon was used as control. The bleeding-stop time was measured and the blood loss was determined. Tranexamic and

O Xbb Xcc

Xaa PG

O

+ R1

3

N H 4

R2

acetonitrile, rt Subtilisin-72/Silochrom-C80 O Xbb Xaa Xcc PG 5

N

R1

R2

Scheme 1. Enzyme-catalyzed synthesis pf peptamides.

␧-aminocaproic acids were used as positive control (known and widely used agents). 3. Results and discussion 3.1. Novel route to the enzymatic synthesis of peptamides Now we wish to present the new simple, efficient and convenient method for the synthesis of peptide cyclic amides. Following our procedure, the catalyst was prepared by distributing of subtilisin-72 on the solid base Silochrom-C80 in an amount corresponding to the maximal value according to its adsorption isotherm on this silica derivative [46]. The enzyme was deposited as the solution in phosphate buffer (pH 7.5). After distribution the sorbent was thoroughly dried in a high vacuum over fresh phosphorus pentoxide. The catalyst could be efficiently used in the synthesis of peptide cyclic amides. Earlier we have shown that the reactions of subtilisin-catalyzed acylation of amino-acids could proceed better with the acylating agent containing the ester moiety [47a,b]. We performed the enzyme-catalyzed acylation of free secondary amine 4 with peptide esters 3 aiming to obtain N-protected peptide cyclic amide derivatives 5 (Scheme 1, where PG stands for protecting group; Xaa, Xbb and Xcc stand for amino acid residues in l-configuration; R1 and R2 stand for hydrocarbon radicals which can be substituted or taken together with the nitrogen atom form optionally substituted heterocycle). Common 2,4-dinitrophenyl (Dnp-), benzyloxycarbonyl (Cbz-), formyl (For-) and others can be used as protection groups. Amino acids Xaa, Xbb and Xcc could be chosen from l-amino acids which sequence is expected to manifest biological activity against desired enzyme; on the other hand the sequence Xaa–Xbb–Xcc should not contraverse the substrate specificity of the subtilisin. The secondary amine 4 could be chosen from diethylamine, piperidine, imidazole, indole, etc. (Table 3).

PGXaaXbbXccC(O)OCH3 + E-H

[PGXaaXbbXccC(O)OCH3][E-H]

3 H2O [PGXaaXbbXccC(O)][E][H2O]

[PGXaaXbbXccC(O)][E] + CH3OH NHR1R2 4

PGXaaXbbXccC(O)OH

[PGXaaXbbXccC(O)][E][NHR1R2]

6

[PGXaaXbbXccC(O)]NR1R2] 5 Scheme 2. Mechanism of enzyme-catalyzed interaction between acyl-donor and acyl-acceptor.

M.E. Sergeev et al. / Journal of Molecular Catalysis B: Enzymatic 80 (2012) 58–66

63

Table 2 Model syntheses on subtilisin-72 catalyzed acylation of piperidine with Dnp-AlaAlaLeu-OMe, investigation of reaction conditions. Run

Acyl donor 3a

Amine 4

Product 5

Solvent

pHb

Yield, %

Phase distribution %s/%c c

1 2 3 4 5

Dnp-AlaAlaLeu-OMe Dnp-AlaAlaLeu-OMe Dnp-AlaAlaLeu-OMe Dnp-AlaAlaLeu-OMe Dnp-AlaAlaLeu-OMe

Piperidine (Pip) Piperidine (Pip) Piperidine (Pip) Piperidine (Pip) Piperidine (Pip)

Dnp-AlaAlaLeu-Pip Dnp-AlaAlaLeu-Pip Dnp-AlaAlaLeu-Pip Dnp-AlaAlaLeu-Pip Dnp-AlaAlaLeu-Pip

MeCN THF Dioxane MeCN MeCN

7.5 7.5 7.5 8.5 9.5

80 78 67 77 74

14/66 13/65 14/53 13/64 12/62

O NH Dnp

H N

5a

O

N

NH O

hydrolysis

H N O

O

80

60

40

0

NH Dnp

100

20

O NH

3a

O

N H 4a

O

Dnp

NH

We have tried to investigate if the solvent choice influenced target compound formation. According the literature data, tetrahydrofuran and dioxane could be used as appropriate reaction solvents for subtilisin-catalyzed reactions. It was found that the rate of product formation in THF was almost the same as in acetonitrile, but when the reaction was carried out in dioxane, the yields of target compounds decreased to about 40%. The formation of peptide cyclic amide 5a did not depend on the concentration of starting material in reaction mixture. Similar results were obtained using different amounts of acetonitrile and THF; the reaction yields were comparable (about 80–85%) in these cases. It was concluded that varying ratio of liquid to solid phase in general did not generally have an impact on the reaction course. The investigation of reaction optimal conditions is summarized in Table 2 using the synthesis of Dnp-AlaAlaLeu-Pip as a model. As far as the catalyst was prepared by depositing the enzyme as the solution in basic buffer, the experiments varying the pH of the buffer for the subtilisin distribution were conducted. It is a well-known fact that pH-optimum for subtilisin is about 8.5 [41]. We have found that usage of sodium phosphate (pH 7.5), Tris–Cl (pH 8.5) and sodium borate (pH 9.5) buffers did not have any significant influence on reaction course. The results were similar in all the cases, the yields of target compounds varied by 1–2% only. The further experiments were conducted using phosphate buffer (pH 7.5). The results of further syntheses with different amines and peptide esters are presented in Table 3. Referring to Moree and co-workers, some comparative experiments with primary amines were also carried out, but the reaction yields were not as good as in case of secondary amines [39]. Acylation of p-aminopyridine and 5-aminosalicylic acid was performed resulting in formation of the product in 56% and 6% yields, correspondingly. The efforts to obtain the derivatives of p-nitroaniline and 3-aminoquinoline failed; nothing but hydrolyzed peptide ester was recovered.

Yield, %Chromat.

Generally, enzyme-catalyzed reaction of amine acylation corresponds to the Scheme 2, where all the variable groups have the meanings designated above and E stands for enzyme. The model reactions were carried out using N-(2,4dinitrophenyl)-l-alanyl-l-alanyl-l-leucine methyl ester 3a (Dnp-Ala-Ala-Leu-OMe) with piperidine 4a (Pip) (Scheme 3) and N-(2,4-dinitrophenyl)-l-alanyl-l-alanyl-l-arginine methyl ester 3c (Dnp-Ala-Ala-Arg-OMe) with morpholine 4b (Mrf) (Scheme 4). It is well known that in organic media the enzyme’s activity can undergo significant changes [34]. Specific methods were developed to prevent the inactivation of enzyme-catalyst by organic media except for enzyme sorption, for instance, the enzyme can be lyophilized with amino-component or dispersed in solvent. Our attempts to use these methods were not successful; it turned out that the literature methods were not effective for the synthesis of peptide cyclic amides. We also tried procedures described by Rich ˜ and Montanez-Clemente [48,49]. It was found that the suggested method of lyophilization of amino component with the enzyme and the reaction in organic medium (THF) at 40 ◦ C did not lead to the formation of the desired amides 5a and 5b. Ester hydrolysis resulting to formation of free peptides 6a and 6b was observed in this case. The similar result was obtained when the reaction was carried out using the dispersed enzyme in organic medium at 37–45 ◦ C [31,50]. Finally, the synthesis of compound 5a was successively carried out using subtilisin-72 distributed on macroporous carrier Silochrom-C80 with acetonitrile as solvent. It was shown that the conversion of starting tripeptide 3a was almost quantitative and the desired product 5a was obtained in 80% yield. As can be seen from the kinetic curves presented in Fig. 2, the formation of desired peptamide and the hydrolysis of starting peptide ester begins immediately. In about 3 days the peptide acid started to form the reaction product with amino-component with the reaction rate increased significantly. It was discovered that the enzyme-catalyzed acylation reaction is possible only in case of tri-membered and more long peptides with the COOH moiety [39]. The optimal reaction time was determined; all the reactions thereafter were carried out for 5 days.

NH O

Scheme 3. Synthesis of Dnp-Ala-Ala-Leu-Pip.

0

H N O

6a

1

2

3

4

5

6

7

8

9

Time, d DnpAlaAlaLeuOMe DnpAlaAlaLeuPip

DnpAlaAlaLeuOH

OH Fig. 2. Kinetic curves of model reaction between Dnp-Ala-Ala-Leu-OMe and piperidine. X-axis: reaction time in time in days; Y-axis: yield determined by HPLC.

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M.E. Sergeev et al. / Journal of Molecular Catalysis B: Enzymatic 80 (2012) 58–66

NH2 Dnp O NH2 Dnp

HN

O

HN

NH

O 3b

O

HN

5b

NH

N H 4b

NH

NH

HN H N

O

O

N O

H N

hydrolysis NH2

O

O Dnp

O

HN

HN NH

O

NH 6b

H N O

OH

Scheme 4. Synthesis of Dnp-Ala-Ala-Arg-Mrf.

It was also found that in case of reactions with participation of arginine-containing peptide esters 3 the addition of at least 25% of dimethylsulfoxide into acetonitrile media is required due to low solubility of arginine derivatives 3 and 5 in acetonitrile alone. The best results for arginine derivatives were obtained using 35% DMSO/MeCN, when the decreased reaction time and better yields were observed. We believe that this could be due

to higher conformational flexibility of peptide in the presence of dimethylsulfoxide. These results are in agreement with the work of Yusupova and co-workers [50]. It should be pointed out that all the reactions using arginine-containing peptide esters resulted in moderate yields of the target amides as compared to the amine acylation with other peptides. It could be explained by the strong affinity of acidic arginine guanidine group to the silica-carrier,

Table 3 Subtilisin-72 catalyzed acylation of amines with protected peptide esters. Run

Acyl donor a

Amine

Product 5

Solvent

pHb

Yield, %

Phase distribution %s/%cc

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

Dnp-AlaAlaLeu-OMe Dnp-AlaAlaLeu-OMe Dnp-AlaAlaLeu-OMe Dnp-AlaAlaLeu-OMe Dnp-AlaAlaLeu-OMe Dnp-AlaAlaGlu(OMe)-OMe Dnp-AlaAlaGlu(OMe)-OMe Dnp-AlaAlaGlu(OMe)-OMe Dnp-AlaAlaGlu(OMe)-OMe Dnp-AlaAlaGlu(OMe)-OMe Dnp-AlaAlaGlu(OMe)-OMe Dnp-AlaAlaGlu(OMe)-OMe Dnp-AlaAlaArg-OMe Dnp-AlaAlaArg-OMe Dnp-AlaAlaArg-OMe Dnp-AlaAlaArg-OMe Dnp-AlaAlaArg-OMe Dnp-AlaAlaArg-OMe Cbz-AlaAlaLeu-OMe Cbz-AlaAlaGlu(OMe)-OMe Cbz-AlaAlaLeu-OMe Cbz-AlaAlaGlu(OMe)-OMe Cbz-AlaAlaGlu(OH)-OH For-Ala-Phe-Lys-OMe For-Ala-Phe-Lys-OMe For-Ala-Phe-Lys-OMe For-Ala-Phe-Lys-OMe For-Ala-Phe-Lys-OMe

Morpholine (Mrf) Diphenylamine (DPA) 5-Aminosalicylic acid (ASA) Imidazole (Im) 3-Aminoquinoline (AQ) Piperidine (Pip) Morpholine (Mrf) Diphenylamine (DPA) Diethylamine (DEA) 4-Aminopyridine (APy) Imidazole (Im) Indole (Ind) Morpholine (Mrf) Morpholine (Mrf) Morpholine (Mrf) Imidazole (Im) Pyrazole (Pyr) Diethylamine (DEA) Imidazole (Im) Indole (Ind) Diethylamine (DEA) Imidazole (Im) Imidazole (Im) Indole (Ind) Homoproline (hP) Diethylamine (DEA) Piperidine (Pip) Morpholine (Mrf)

Dnp-AlaAlaLeu-Mrf Dnp-AlaAlaLeu-DPA Dnp-AlaAlaLeu-ASA Dnp-AlaAlaLeu-Im Dnp-AlaAlaLeu-AQ Dnp-AlaAlaGlu(OMe)-Pip Dnp-AlaAlaGlu(OMe)-Mrf Dnp-AlaAlaGlu(OMe)-DPA Dnp-AlaAlaGlu(OMe)-DEA Dnp-AlaAlaGlu(OMe)-APy Dnp-AlaAlaGlu(OMe)-Im Dnp-AlaAlaGlu(OMe)-Ind Dnp-AlaAlaArg-Mrf Dnp-AlaAlaArg-Mrf Dnp-AlaAlaArg-Mrf Dnp-AlaAlaArg-Im Dnp-AlaAlaArg-Pyr Dnp-AlaAlaArg-DEA Cbz-AlaAlaLeu-Im Cbz-AlaAlaGlu(OMe)-Ind Cbz-AlaAlaLeu-DEA Cbz-AlaAlaGlu(OMe)-Im Cbz-AlaAlaGlu(OH)-Im For-Ala-Phe-Lys-Ind For-Ala-Phe-Lys-hP For-Ala-Phe-Lys-DEA For-Ala-Phe-Lys-Pip For-Ala-Phe-Lys-Mrf

MeCN MeCN MeCN MeCN MeCN MeCN MeCN MeCN MeCN MeCN MeCN MeCN 25% DMSO/MeCN 35% DMSO/MeCN 35% DMSO/MeCN 35% DMSO/MeCN 35% DMSO/MeCN 35% DMSO/MeCN MeCN MeCN MeCN MeCN MeCN MeCN MeCN MeCN MeCN MeCN

7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 8.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5

66 0 14 87 0 74 78 0 69 56 73 70 45 31 35 46 49 36 82 75 83 96 88 76 68 89 77 64

12/54 0/0 6/8 81/6 0/0 18/56 14/64 0/0 57/12 9/47 51/22 3/67 19/26 10/21 11/24 19/27 18/31 5/31 56/26 27/48 64/19 71/25 70/18 16/60 22/46 81/8 66/11 60/4

a Standard three-letter abbreviations used to designate l-amino acid residues; Dnp- stands for 2,4-dinitrophenyl; Cbz- stands for benzyloxycarbonyl; starting peptide esters were obtained by conventional methods of peptide synthesis; such as EDCI/HOBt coupling and activated-esters coupling of amino acids. In case of tripeptide Ala-Ala-Glu; the dimethyl ester of glutamic acid was used. b Phosphate buffer (pH 7.5), Tris buffer (pH 8.5) and borate buffer (pH 9.5) used. c Designates percentage of product in liquid (%s) and solid (%c) phases.

M.E. Sergeev et al. / Journal of Molecular Catalysis B: Enzymatic 80 (2012) 58–66 Table 4 Inhibition data for synthesized compounds.

0.65 0.6

Absorbance, A410

65

0.55 0.5 0.45 0.4 0.35

Run

Inhibitor

Ki plasmin, ␮M

1 2 3 4 5

For-Ala-Phe-Lys-ala,b For-Ala-Phe-Lys-Pipb For-Ala-Phe-Lys-Mrfb Z-Ala-Ala-Leu-DEAc Z-Ala-Ala-Leu-Mrfc

23 19.2 19.1

a b

0.3

c

0.25 0.2 1

1.5

2

2.5

3

3.5

4

Ki subtilisin, nM

9.4 8

Lit. data by Potetinova et al. [37]. Chromogenic substrate ForAlaPheLyspNA used. Chromogenic substrate ForAlaAlaLeupNA used.

Table 5 Euglobulin factor XIIa-dependent lysis time.

Time, days Native enzyme

E/C-80, dry

E/C-80+MeCN

E/C-80+MeCN+inhibitor

Fig. 3. Activity of native enzyme and enzyme under conditions of reaction. X-axis: reaction time in days; Y-axis: absorbance at  410 nm (A410 ).

when arginine-containing peptide bound to solid support and became more sterically hindered for enzyme to be able to form acyl–enzyme complex, which in turn led to lowered yields of the final amides. It was found that the reaction product distributed nonuniformly between the reaction phases (see Table 2 for details). It was shown that imidazole and diethylamine derivatives were mostly dissolved in the reaction solvent and the other amide-derivatives were better sorbed on silochrom. In case of arginine-amides all the reaction products are mostly distributed on the solid carrier. We believe that such a distribution patterns are related to the hydrophobicity of the whole target molecule. In most cases the work-up of only one phase was sufficient for the isolation of target peptamide in pretty good yield. It was of great interest to us to find out why subtilisin was capable of catalyzing the synthesis of an inhibitor for itself and all the while it did not become inactivated when the concentration of target inhibitor in reaction mixture increased. Schechter and Ziv reported that competitive inhibitors can activate proteases to catalyze the synthesis of peptide bonds [51]. They also showed that acyl–enzyme complex formed in reaction medium served as initiator of tripartite reaction and the process became cyclic. In addition to already reported data we demonstrated that, contrary to the common process of enzyme inhibition in solution, the inhibition of subtilisin distributed on silochrom in acetonitrile did not take place. The comparative experiments showed that the carrier-distributed subtilisin was slightly less active than the native enzyme, but the addition of subtilisin inhibitor Cbz-Ala-Ala-Leu-Mrf activated the enzyme in time-dependent manner. The results are shown in Fig. 3. As can be seen, the activity of the enzyme distributed on silochrom (E/C-80, dry composition) and the activity of distributed enzyme under acetonitrile layer (E/C-80 + MeCN) decreased slowly during the course of experiment. The activity of the enzyme distributed on the carrier and treated with inhibitor in acetonitrile (Z/C-80 + MeCN + inhibitor) dropped dramatically at first, but over time the activity grew strongly and eventually became almost as high as that in above mentioned cases. This is in agreement with the data of Ziv and Schechter and gives the answer for the posed question. Biological evaluation of some of synthesized compounds was started by measuring the binding constants (Ki) against plasmin and subtilisin (Table 4). The Ki data show that morpholinamide and piperidinamide with the amino-acid sequence Ala-Phe-Lys (specific for plasmin) possess the similar Ki value when compared with previously reported aldehyde [37]. Diethylamide and morpholinamide with the sequence Ala-Ala-Lys (specific for subtilisin) have

Run

Inhibitor

Lysis time, min

1 2 3 4 5 6 7

For-Ala-Phe-Lys-al For-Ala-Phe-Lys-Pip For-Ala-Phe-Lys-Mrf For-Ala-Phe-Lys-Ind For-Ala-Phe-Lys-hP For-Ala-Phe-Lys-DEA Control

3.8 21.9 22.6 9.5 8.3 7.5 3

± ± ± ± ± ± ±

0.3 1.2 0.9 0.6 0.6 0.4 0.8

Table 6 Bleeding-stop time on liver surface wound. Run

Inhibitor

Bleeding-stop time, s

1 2 3 4 5 6

For-Ala-Phe-Lys-al For-Ala-Phe-Lys-Pip For-Ala-Phe-Lys-Mrf ␧-Aminocaproic acida Tranexamic acida Control

83 60 70 140 98 237

a

± ± ± ± ± ±

5 1 7 7 15 13

Commercial positive control, common drug for bleeding termination.

also shown good Ki values against subtilisin. With these in mind, we made the in vitro and in vivo experiments to establish the activity of newly synthesized compounds in bleeding termination. For the in vitro experiment we used the euglobulin factor XIIadependent lysis model (Table 5). The results show that some of synthesized amides with plasmin-specific sequence Ala-Phe-Lys are capable of significant prolongation of euglobulin clot in comparison with previously described aldehyde and control experiment. Piperidinamide and morpholinamide were found to be the best agents in this model and were evaluated for in vivo activity in rabbits. The experiments with animals were made with acute bleeding model (Table 6). This model simulates the general surgical problem of termination of strong bleeding of inner organs and includes the acute capillary-parenchymatous bleeding of liver-wound. Our investigation has shown that compounds For-Ala-Phe-Lys-Pip and For-Ala-Phe-Lys-Mrf have the bleeding-terminating activity almost twice higher than standard commercial agents tranexamic and ␧aminocaproic acids. Previously described For-Ala-Phe-Lys-al was evaluated too and has shown the activity similar to amides, but its instability in aqueous media restricts the application of this aldehyde in surgical area. 4. Conclusions A convenient and high-yield approach was suggested for the synthesis of peptidyl secondary amides. This involves the application of subtilisin-72 distributed on macroporous silica carrier as catalyst for the reaction of acylation of secondary amines with peptidyl esters in organic medium. The applied method allows to obtain peptide secondary amides in high yields under mild conditions. Thus obtained peptide amide derivatives could be efficiently used

66

M.E. Sergeev et al. / Journal of Molecular Catalysis B: Enzymatic 80 (2012) 58–66

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