Benzoylarginine

The same enzyme was used for the hydrolysis ofp-nitrophenyl-P-D-galactopyrano-side to D-galactose (Scheme 4.97) by Kanno et al. [409,410] in a PMMA microreactor. Quantitative hydrolysis was reported and the reaction was about five times faster than the batch reaction. This unexpected rate enhancement is one of the few examples in which a difference was observed in kinetics between batch and microscale. A second example was reported by Maeda and coworkers [411], They described the trypsin-catalyzed hydrolysis of benzoylarginine-p-nitroanilide and found that the rate of reaction seemed to be 20 times greater than the batchwise system (Scheme 4.98). 

The separation of the substrate from the reaction product benzoylarginine was carried out by reverse phase HPLC on a /tBondapak CN column with a mobile phase of 0.05 Af ammonium acetate-methanol (85 15). The column was eluted isocratically, and the eluent was monitored at 254 nm. 

The copolymei(L-Cys, L-Glu) hydrolyzed benzoylarginine derivatives(ethyl ester and amides, 19) with an optimum pH of 6.1 and optimum temperature of 40 C. This pH value corre onds to one of the two optimum pH in the hydrolysis of PNPA. The urease activity was also noted for this copolymer, and increased witii air oxidation of some SH groups (i2<5). 

G. Fleck and S. J. Singer, unpublished experiments. Enzymatic activity was measured by the rate of alkali consumption by the hydrolysis of benzoylarginine ethyl ester at pH 8 in a phosphate-NaCl buffer (Inagami and Sturtevant, 1960). 

Figure 1 shows the influence of the solution pH on the yield and initial rate of synthesis of CBZ-Lys-Gly-OMe at a temperature of 25°C and [CBZ-Lys] = [Gly-OMe] = 20 mM. The maximum yield is achieved for pH values around 6-6.5. Under thermodynamically controlled conditions, the peptide synthesis occurs between the non-ionic forms of the acyl-donor (CBZ-Lys) and the nucleophile (Gly-OMe). The concentration of these nonionic forms depends on the pH, since an intermediate value between both pK (pHopt = V IpKa +pKb]) is needed in order to achieve high synthetic yields. On the other hand, the reaction rate increases up to pH 7, which is in agreement with the results obtained in the synthesis of the peptide benzoylarginine-leucinamide catalyzed by immobilized trypsin (10), where the authors suggest the nucleophihc attack of the non-ionic form of the nucleophile on the acyl-enzyme complex as the controlling step of the peptide reaction. 

The activity of trypsin is determined by comparing the rate at which it hydrolyzes benzoylarginine ethyl ester hydrochloride R with the rate at which trypsin BRP hydrolyzes the same substrate in the same conditions. 

Store the solutions at 0-5°C. Warm 1 mL of each solution to about 25°C over 15 min and use 50 xL of each solution for each titration. Carry out the titration in an atmosphere of nitrogen. Transfer 10.0 mL of borate buffer solution pH 8.0 (0.0015 M) R to the reaction vessel and, while stirring, add 1.0 mL, of a freshly prepared 0.686% m/V solution of benzoylarginine ethyl ester hydrochloride R. 

Benzoylarginine Ethyl Ester Hydrochloride [C15H23CIN4O3 (Mr 342.8) AA-benzoyl-L-arginine ethyl ester hydrochloride ethyl (S)-2-benzamido-5-guanidinovalerate hydrochloride]... 

The enzyme works optimally at pH > 9 with N-protected amino acid esters as acyl donors, and amino acids or amino acid amides as nucleophiles (acceptors). At that pH value it has high esterase activity an amino acyl-enzyme complex will be formed rapidly which transfers the acyl residue to the acceptor molecule (transpeptidation), and since at alkaline pH the rate of peptide cleaving is minimal, the product formed will be released in excellent yield. As an example the last coupling step in a synthesis of the opioide peptide Met-enkephalin that started with benzoylarginine ethylester may be shown (Fig. 8). 

The ferrocene 44 bearing only one tetrapeptide chain (-Gly-Gly-L-Tyr-L-Arg-OH) is designed to bind to papain [147]. The ferrocene 44 acts as an efficient competitive papain inhibitor for AT-benzoylarginine ethyl ester hydrolysis, with an inhibition constant Ki of 9 xM at pH 6.2. Binding of papain to the ferrocene receptor 44 causes an electrochemical response, resulting in a small cathodic shift of the redox potential of the ferrocene moiety of 44. 

Quite recently, additional thiol proteinases have been found in lyso-somes. Cathepsin T, which catalyzed conversion of multiple forms of tyrosine aminotransferase, was demonstrated by Gk>hda and Pitot (20, 21). This enzyme does not cleave benzoylarginine-2-naphthylamide, a substrate for cathepsin B and cathepsin H. Melloni et al. (22, 23) reported the presence of a thiol peptidyl peptidase in a lysosomal membrane fraction of rabbit liver. In addition, cathepsin N with strong collagenolytic activity was found in bovine spleen and human placenta, although its lysosomal origin has not yet been demonstrated (24-26). 

Limited experimental data for liquid-soUd mass transfer are available. Oliver and Hoon [47] measured heat transfer in two-phase flow in capillaries using very viscous liquids and hence with thick lubricating layers. The best data set is by Horvath et al. [48], who measured the hydrolysis of N-benzoylarginine ethyl ester in a 1.2 m long tube coated with the immobilized enzyme trypsin. The intrinsic rate of this reaction was high enough that mass transfer from the liquid was limiting. The experimental data are reported as Sh versus Is/dh with the Reynolds number Re as a parameter and Sh versus Re with the aspect ratio Is/dh as a parameter. 

In the field of organic synthesis, it was reported that the catalytic hydrolysis of umbelliferone esters (7-acetoxycoumarin) to 7-hydroxycoumarinby porcine pancreatic lipase covalently immobilized on microchannel reactors almost completed within 1 min, to be compared with 4 min in a normal batch reaction [86]. The same group demonstrated an improvement in the yield of trypsinmicrochannel reactor with a lower enzyme concentration but a 20-fold higher reaction rate than in a batch reactor [87]. 

Elastase (EC 3.4.21.11) an endopeptidase specific for the Elastic (see) in animal elastic fibers. Its inactive precursor, proelastase, is formed in the vertebrate pancreas and converted in the duodenum to elastase by the action of trypsin. The natural substrate of E. is elas-tin, an insoluble protein rich in valine, leucine and isoleucine. E. attacks the peptide bond adjacent to a nonaromatic, hydrophobic amino acid. The best synthetic substrates are therefore acetyl-Ala-Ala-Ala-OCHj and benzoylalanine methyl ester. Benzoylarginine ester (a trypsin substrate), and acetyltyrosine ester (a chymotrypsin substrate) are not attacked by E. 

Trypsin, chymotrypsin, and carboxypeptidase, long thought to be specific only for the hydrolysis of peptide bonds also display a specific esterase activity. 2 Trypsin, for example, splits ammonia from benzoylargininamide and as well splits ethanol from benzoylarginine ethyl ester. No activity, however, is shown toward ethyl butyrate, a substrate for an aliphatic esterase. 

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