Substrate activation of rat trypsins 1 and 2

Qualitative differences in the active center of rat trypsins 1 and 2 resulted in different ratios of K_cat for N¹-tosyl-l-arginine methyl ester vs K_cat for N¹-benzoyl-l-arginine ethyl ester. These ratios were 2.5 for trypsin 1 and 1.2 for trypsin 2.Substrate activation with N¹-tosyl-l-arginine methyl ester enhanced the catalytic rate constant of rat trypsin 1 2.5-fold and that of rat trypsin 2 only 1.5-fold. The increase in the catalytic rate constant found with N¹-benzoyl-l-arginine ethyl ester was the same (1.5-fold) for both trypsins. Consequently, at 20 mm substrate concentration, trypsin 1 catalyzed the esterolysis of N¹-tosyl-l-arginine methyl ester 4.5 times faster than that of N¹-benzoyl-l-arginine ethyl ester, while trypsin 2 was only 1.3 times more efficient with the first substrate.Furthermore, the activation of both rat enzymes by N-acetyl-l-tyrosine ethyl ester was even more effective than that obtained with the two cationic esters; the maximum rates of hydrolysis of this neutral substrate by trypsins 1 and 2 were enhanced 120- and 50-fold, respectively, by high concentrations of N-acetyl-l-tyrosine ethyl ester.     

An examination of the rate assay to determine the active site normality of papain

Data are presented which demonstrate that the α-N-benzoyl-l-argine ethyl ester rate assay procedure, based on a burst titration with N-benzyloxy-carbonyl-l-tyrosine p-nitrophenyl ester as previously desribed (1), is an accurate and reliable method for determining the normality of papain in solution.     

Synthesis of N-hydroxycinnamoyl amide derivatives and evaluation of their anti-oxidative and anti-tyrosinase activities

Twelve N-hydroxycinnamoyl amino acid amide ethyl esters (CAES) were synthesized by using l-amino acid ethyl ester hydrochloride and corresponding cinnamic acid (ferulic acid, acetylferulic acid and caffeic acid) as raw materials in the presence of a catalytic amount of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide-hydrochloride (EDC) and 1-hydroxybenzotriene (HOBt). The 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activities of CAES were evaluated. The anti-tyrosinase activities of N-feruloyl amino acid ethyl esters and the hydroxyl (OH) free radical scavenging activities of N-caffeoyl amino acid ethyl esters were also examined. DPPH free radical scavenging activity was shown in all CAES, of which N-caffeoyl amino acid ethyl esters demonstrated higher radical scavenging activity than N-feruloyl amide derivatives, and (E) -N-(caffeic acid)-l-glycinate ethyl ester (c₅) had the strongest ability to scavenge free radicals with an IC₅₀ value of 18.6 µM. The acetylferuloyl amino acid esters exhibited the highest tyrosinase inhibition activity among the tested amides.     

Synthesis and antiviral activity of novel glycyrrhizic acid conjugates with D-amino acid esters

Glycyrrhizic acid (GA) conjugates with methyl and ethyl esters of D-amino acids (D-Trp, D-Phe, D-Tyr, D-Val, D-Leu) have been synthesized by the activated esters method using mixtures of N-hydroxybenzotriazole or N-hydroxysuccinimide with N,N′-dicyclohexylcarbodiimide. GA conjugate with D-Trp ethyl ester exhibited antiviral activity against influenza viruses A/H3N2, A/H1N1/pdm09, A/H5N1, B (SI > 10–29), and HRSV (SI > 25). GA conjugate with D-Trp methyl ester inhibited influenza virus A/H1N1/pdm09 (SI > 30).     

The role of methionine and α-chymotrypsin-catalysed reactions

1. The reaction of α-chymotrypsin with sodium periodate at pH5·0 has been investigated. The enzyme consumes 2 moles of periodate/mole, and there is a concomitant fall in enzymic activity (with respect to l-tyrosine ethyl ester) to 55% of that of the native enzyme. After 3hr. no further change is observed in periodate uptake or in catalytic activity. 2. The oxidized enzyme is a homogeneous preparation of partially active chymotrypsin. 3. In the oxidized enzyme, one of the two methionine residues in the molecule has been converted into its sulphoxide. It is this reaction only that is responsible for the loss of activity. 4. The rate constants for the enzyme-catalysed acylation and deacylation reactions are unaltered by oxidation of the enzyme, both for a non-specific substrate (p-nitrophenyl acetate), and for three specific substrates: N-acetyl-l-tryptophan ethyl ester, N-acetyl-l-tryptophanamide and N-acetyl-l-valine ethyl ester. 5. The K_m values for the aromatic substrates with the oxidized enzyme are twice those with the native enzyme. No change in Michaelis constant is seen for the non-aromatic substrate N-acetyl-l-valine ethyl ester. 6. The evidence points to the oxidized methionine residue in the modified enzyme being situated in the locus of the active site at which aromatic (or bulky) side chains of the substrates are bound.     

The attachment of poly(ribitol phosphate) to lipoteichoic acid carrier

2-Acetamido-3,4,6-tri-O-acetyl-1-N-[N-(benzyloxycarbonyl)-L-aspart-1-oyl-(L-leucyl-L-threonyl-N²-tosyl-L-lysine p-nitrobenzyl ester)-4-oyl]-2-deoxy-β-D-glucopyranosylamine (21) and 2-acetamido-3,4,6-tri-O-acetyl-1-N-[N-(benzyloxycarbonyl)-L-aspart-1-oyl-(L-leucyl-L-threonyl-N²-tosyl-L-lysine p-nitrobenzyl ester)-4-oyl]-2-deoxy-β-D-glucopyranosylamine (22), 2-acetamido-3,4,6-tri-O-acetyl-1-N-[N-(benzyloxycarbonyl)-L-aspart-1-oyl-(glycine ethyl ester)-4-oyl]-2-deoxy-β-D-glucopyranosylamine, and 2-acetamido-3,4,6-tri-O-acetyl-1-N-[N-(benzyloxycarbonyl)-L-aspart-1-oyl-(phenylalanine methyl ester)-4-oyl]-2-deoxy-β-D-glucopyranosylamine were synthesized by condensation of 2-acetamido-3,4,6-tri-O-acetyl-1-N-[N-(benzyloxycarbonyl)-L-aspart-4-oyl]-2-deoxy-β-D-glucopyranosylamine with the appropriate protected amino acids and tri- and tetra-peptides. The amino acid sequences of 21 and 22 correspond to the protected amino acid sequences 34–37 and 34–38 of ribonuclease B that are adjacent to the carbohydrate-protein linkage.     

A study of the biologically active conformation of the cholecystokinin-4 dipeptide analogue GB-115

The biologically active conformation of N-(6-phenylhexanoyl)glycyl-tryptophan amide (GB-115), a highly active cholecystokinin-4 retro dipeptide analogue with the anxiolytic activity, has been studied using the conformational analysis by ¹H NMR spectroscopy in solution and the method of sterically restricted analogues. A study of the relationship between the preferable conformation in solution and the anxiolytic activity in the series of GB-115 derivatives showed that the biologically active conformation of this compound is the β-turn. Based on the data on the nuclear Overhauser effect ¹H NMR spectroscopy, this structure was identified as the β-turn of type II. Subsequent synthesis and study of the pharmacological activity of novel sterically restricted analogues of dipeptide GB-115: (2S)-2-{(3R)-3-[(6-phenylhexanoyl)amino]-2-oxopyrrolidine-1-yl}-3-(1H-indole-3-yl)propionic acid ethyl ester, N-(6-phenylhexanoyl)glycyl-N ^α-methyltryptophan ethyl ester, (2S)-2-[(10,11-dihydro-5H-dibenzo[b, f]azepin-5-ylcarbonyl)amino]-3-(1H-indole-3-yl)propionic acid methyl ester, and (2S)-2-[({3-[(ethoxycarbonyl)amino]-10,11-dihydro-5H-dibenzo[b, f]azepin-5-yl}carbonyl)amino]-3-(1H-indole-3-yl)propionic acid methyl ester confirmed that the β-turn of type II is the active conformation of GB-115.     

Isolation and characterization of a membrane-bound proteinase from rat liver.

The proteinase previously found in chromatin prepared from a total rat liver homogenate was purified from the rat liver mitochondrial fraction. The membrane-bound enzyme is solubilized in either 0.6% digitonin or 0.5 m phosphate buffer. After a 1330-fold purification, the enzyme appears homogeneous by acrylamide-gel electrophoresis. Sucrose density gradient centrifugation indicated a molecular weight of 22,500, a molecular weight of 23,500 ± 10% has been estimated by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme showed a high substrate specificity. Among several proteins tested, only glucagon, nonhistone chromosomal proteins, and histones are good substrates. A limited proteolysis was found for the very-lysine-rich histone H1, which was split into a high molecular weight fragment (M_r 13,000). The highly phosphorylated histone H1 isolated from regenerating rat liver 24 h after partial hepatectomy exhibited the same susceptibility to the proteinase as H1 from normal liver. Large polypeptides of a nonhistone chromosomal protein fraction were degraded more rapidly than the small ones. N-Acetyl-l-tyrosine ethyl ester was used with alcohol dehydrogenase and NAD in a coupled enzyme assay for the proteinase. The apparent Michaelis constant for the hydrolysis of N-acetyl-l-tyrosine ethyl ester is 5.0 × 10^?3m. The proteinase has catalytic properties simlar to trypsin and chymotrypsin. The pH optimum was around 8, soybean trypsin inhibitor depressed the enzymatic activity, and the serine modifying reagents diisopropyl phosphofluoridate and phenylmethanesulfonyl fluoride inactivated the enzyme. The affinity reagent for chymotrypsin-like active sites, l-1-tosylamido-2-phenylethyl chloromethyl ketone, inactivated the proteinase.     

Chemical modification of ribonucleosides with N-acetyl-3,5-di[125I]iodotyrosyl hydrazide

Several 3,5-diiodotryrosyl derivatives have been synthesized by both sodium iodideiodine and the sodium iodide-iodic acid methods. Conditions optimizing yield and purity of the product have been established for the latter reaction. Under those conditions, treatment of N-acetyl-tyrosyl ethyl ester with sodium [¹²⁵I]iodide and iodic acid gave N-acetyl-3,5-di[¹²⁵I]iodotyrosyl ethyl ester (ADITEE) with high specific activity. Hydrazination of [¹²⁵I]ADITEE produces N-acetyl-3,5-di[¹²⁵I]iodotyrosyl hydrazide. This hydrazide has been successfully used to modify four different ribonucleoside dialdehydes.     

An Efficient Synthesis Of Acyclic N7- and N9-Adenine Nucleosides Via Alkylation With Secondary Carbon Electrophiles to Introduce Versatile Functional Groups At the C-1 Position of Acyclic Moiety

The introduction of versatile functional groups, allyl and ester, at the C-1 position of the acyclic chain in acyclic adenine nucleosides was achieved for the first time directly by alkylation of adenine and N⁶-protected adenine. Thus, the C-1′-substituted N⁹-adenine acyclic nucleoside, adenine-9-yl-pent-4-enoic acid ethyl ester (11), was prepared by direct alkylation of adenine with 2-bromopent-4-enoic acid ethyl ester (6), while the corresponding N⁷-regioisomer, 2-[6, (dimethylaminomethyleneamino)-purin-7-yl]-pent-4-enoic acid ethyl ester (10), was obtained in one step by the coupling of N,N-dimethyl-N′- (9H-purin-6-yl)-formamidine (9) with 2-bromopent-4-enoic acid ethyl ester (6). The functional groups, ester and allyl, were converted to the desired hydroxymethyl and hydroxyethyl groups, and subsequently to phosphonomethyl derivatives and corresponding pyrophosphorylphosphonates.     

Catalytic properties of cross-linked enzyme crystals in organic media

The activity and the enantioselectivity of cross-linked enzyme crystals (CLECs) of subtilisin in the transesterification between N-acetyl-l-phenylalanine ethyl ester and n-propanol have been examined in various organic solvents. The activity of CLECs of subtilisin in decane was 780 times greater than that in triethylamine. CLECs of subtilisin preferred l-enantiomer in the transesterification between N-acetyl-phenylalanine ethyl ester and n-propanol, and the (k_cat/K_M)_L/(k_cat/K_M)_D ratio was 20 000 in cyclohexane.     

Purification and Partial Characterization of Human C1-Esterase

C1-Esterase was purified from the euglobulin fraction of human plasma by successive column chromatography on DEAE-cellulose, hydroxylapatite and TEAE-cellulose. The final product, purified 3500-fold with respect to serum, hydrolyzed 1,155 μmoles of N^α-acetyl-l-tyrosine ethyl ester per milligram of protein at pH 7.4 and 37°C in 15 min. The homogeneity of the purified C1-esterase was confirmed by ultracentrifugation and disc-electrophoresis. Its s_20,w value was 4.3 and its molecular weight was determined as 113,000 by gel filtration on Sephadex G–200.

Cl-Esterase possesses esterolytic activity for both N^α-acetyl-l-tyrosine ethyl ester and N^α-tosyl-l-arginine methyl ester, and acts on human kininogen I and II releasing kinin very slowly.     

The phosphorylation region of lysine-rich histone in dividing cells

N-Bromosuccinimide cleavage of in vivo ³²P-labelled lysine-rich histone isolated from rapidly dividing cells has been studied. N-Bromosuccinimide cleaves F₁-histone into two fragments, a small N-terminal piece and a larger C-terminal portion. The phosphate-induced microheterogeneity and associated radioactivity which has been linked to cell replication, is found in the carboxyterminal fragment. No phosphorous is found associated with the amino-terminal fragment when histone phosphorylation is associated with cell division. The specific tryptic phosphopeptides obtained from in vivo labelled F₁ are clearly different from those obtained from in vitro incubations of free F₁-histone and cytoplasmic protein kinase.     

Growth of Kluyveromyces marxianus and formation of ethyl acetate depending on temperature

Conversion of lactose into ethyl acetate by Kluyveromyces marxianus allows economic reuse of whey-borne sugar. The high volatility of ethyl acetate enables its process-integrated recovery by stripping. This stripping is governed by both the aeration rate and the partition coefficient, K _EA,L/G. Cultivation at elevated temperatures should decrease the K _EA,L/G value and thus favor stripping. K. marxianus DSM 5422 as a potent producer of ethyl acetate was cultivated aerobically in whey-borne media for studying temperature-dependent growth and ester formation. Shake flask cultivation proved thermal tolerance of this yeast growing from 7 to 47 °C with a maximum rate of 0.75 h^?1 at 40 °C. The biomass yield was 0.41 g/g at moderate temperatures while low and high temperatures caused distinct drops. The observed μ-T and Y _X/S-T dependencies were described by mathematical models. Further cultivations were done in an 1-L stirred reactor for exploring the effect of temperature on ester synthesis. Cultivation at 32 °C caused significant ester formation (Y _EA/S?=?0.197 g/g) while cultivation at 42 °C suppressed ester synthesis (Y _EA/S?=?0.002 g/g). The high temperature affected metal dissolution from the bioreactor delivering iron for yeast growth and preventing ester synthesis. Cultivation at 32 °C with a switch to 42 °C at the onset of ester synthesis allowed quick and efficient ester production (Y _EA/S?=?0.289 g/g). The high temperature lowered the K _EA,L/G value from 78 to 44 L/L which heightened the gas-phase ester concentration (favoring ester recovery) without increasing the liquid-phase concentration (avoiding product inhibition).     

A support for affinity chromatography that covalently binds amino groups via a cleavable connector arm.

The preparation of a new succinimidyl ester agarose derivative (SEPE-Agarose) is described. This agarose derivative can be used for covalently linking proteins and other ligands containing amino groups to agarose via phenyl ester linkages that can later be broken under mild conditions which should not alter other groups which may be present in proteins such as cystinyl residues and glycosyl residues. SEPE-Agarose is prepared by the reaction of bis[4-[2-(N-succinimidoxycarbonyl)ethyl]phenyl]succinate with an aminoethylcarbamylmethyl derivative of agarose. Studies of the covalent binding and release of trypsin and myoglobin to SEPE-Agarose indicate that gels containing 0.1 to 0.6 μmol protein/ml of gel are obtained by reacting protein (0.5–5 mg/ml) with the N-succinimidyl ester groups in SEPE-Agarose. Protein-linked gel is reasonably stable in dilute phosphate buffers (pH ≤ 7.4, ≤ 25 °C). Protein is released from the gel, however, by treatment at 25 °C with solutions containing nucleophiles such as 1 m imidazole-glycine, pH 7.4, for 4 h, or 1 m hydroxylamine, pH 7, for 10 min. Protein is also released from the gel by treatment with 1 m Tris pH 8.2 for 24 h. SEPE-Agarose should prove useful in affinity chromatography and immunoabsorption when it is difficult or impractical to elute material bound to conventional affinity supports.     

Chemical modification of the carbohydrate moiety in N-acetylmuramoyl-l-alanyl-d-isoglutamine,and the immunoadjuvant activity

Five carbohydrate analogs of N-acetylmuramoyl-l-alanyl-d-isoglutamine have been synthesized from benzyl 2-acetamido-2-deoxy-3-O-[d-1-(methoxycarbonyl)ethyl]-α-d-glucopyranoside (1) and the corresponding 6-O-benzoyl derivative (2). Chlorination of 1 and 2 with triphenylphosphine in carbon tetrachloride gave the 4,6-dichloro compound 3 and the 6-O-benzoyl-4-chloro compound (4), which were treated with tributyltin hydride, to yield benzyl 2-acetamido-2,4,6-trideoxy-3-O-[d-1-(methoxycarbonyl)ethyl]-α-d-xylo-hexopyranoside (6) and benzyl 2-acetamido-6-O-benzoyl-2,4-dideoxy-3-O-[d-1-(methoxycarbonyl)ethyl]-α-d-xylo-hexopyranoside (7), respectively. Methanesulfonylation of 8, derived from 7 by debenzoylation, gave the 6-methanesulfonate, which underwent displacement with azide ion to afford benzyl 2-acetamido-6-azido-2,4,6-trideoxy-3-O-[d-1-(methoxycarbonyl)ethyl]-α-d-xylo-hexopyranoside (10). Hydrolysis of the methyl ester group in compounds 3, 5 (debenzoylated 4), 6, 8, and 10 gave the corresponding free acids, which were coupled with l-alanyl-d-isoglutamine benzyl ester, to yield the dipeptide derivatives in excellent yields. Hydrogenation of the dipeptide derivatives thus obtained gave the five carbohydrate analogs of N-acetylmuramoyl-l-alanyl-d-isoglutamine, respectively, in good yields. The immunoadjuvant activity of the N-acetylmuramoyl-dipeptide analogs was examined.     

The secretory nature of the excretory gland cells of Stephanurus dentatus. II. Presence of hydrolytic enzymes

Enzymes catalyzing the hydrolysis of casein, N-benzoyl-l-tyrosine ethyl ester, p-nitrophenyl acetate, and l-leucyl-β-naphthylamine hydrochloride were found in extracts of the excretory gland cells of Stephanurus dentatus.     

Biotransformation of alkyl and aryl carbonates: enantioselective hydrolysis

Summary N-(Benzyloxycarbonyl)-l-asparty-l-phenylalanine methyl ester, the precursor of the synthetic sweetener aspartame, was continuously synthesized in an immobilized thermolysin plug-flow type reactor at 25° C with the substrates (N-benzyloxycarbonyl-l-aspartic acid and l-phenylalanine methyl ester) dissolved in ethyl acetate. The immobilized enzyme was quite stable in ethyl acetate containing 2.5% 0.01 M 2-(N-morpholino)ethanesulphonic acid-NaOH buffer, pH 6.0, and 20 mM CaCl₂ with or without the substrate at 25° C. By periodically washing the column, we could conduct a continuous reaction for over 500 h with an average yield of 95% and a space velocity of 1.85 h ^–1.Offprint requests to: K. Nakanishi     

Synthesis, characterization and X-ray crystal structure of [Re(L4)(CO)3]Br · 2CH3OH (L4 = N,N-bis[(2-diphenylphosphino)ethyl]methoxyethylamine): A model compound for novel cationic Tc(I)-tricarbonyl radiotracers useful for heart imaging

This report describes synthesis and evaluation of cationic complexes, [^99mTc(CO)₃(L)]⁺ (L = N-methoxyethyl-N,N-bis[2-(bis(3-ethoxypropyl)phosphino)ethyl]amine (L1), N-[(15-crown-5)-2-yl]-N,N-bis[2-(bis(3-ethoxypropyl)phosphino)ethyl]amine (L2) and N-[(18-crown-6)-2-yl]-N,N-bis[2-(bis(3-ethoxypropyl)phosphino)ethyl]amine (L3)) as potential radiotracers for heart imaging. Preliminary results from biodistribution studies in female adult BALB-c mice indicated that the cationic ^99mTc(I)-tricarbonyl complex, [^99mTc(CO)₃(L2)]⁺, has a significant localization in the heart at 60 min post-injection. To understand the coordination chemistry of these bisphosphine ligands with the ^99mTc(I)-tricarbonyl core, we prepared [Re(CO)₃(L4)]Br (L4: N,N-bis[(2-diphenylphosphino)ethyl]methoxyethylamine) as a model compound. [Re(CO)₃(L4)]Br has been characterized by elemental analysis, IR, ESI-MS, NMR (¹H, ¹³C, ¹H-¹H COSY, and ¹H-¹³C HMQC) methods, and X-ray crystallography. In solid state, [Re(CO)₃(L4)]⁺ has a distorted octahedron coordination geometry with PNP occupying one facial plane. The chelator backbone adopts a “chair” conformation with phosphine-P atoms at equatorial positions and the amine-N at the apical site. In solution, [Re(CO)₃(L4)]⁺ is able to maintain its cationic nature with no dissociation of carbonyl ligands or any of the three PNP donors.     

Solvent effect on the stereoselective addition reaction of optically active amines to N-methylphenylalanine N-carboxyanhydride

The effect of solvent on stereoselectivity in the nucleophilic addition reaction of various optically active amines to N-methylphenylalanine N-carboxyanhydride has been investigated. In m-dimethoxybenzene as solvent, (S)-valine, (S)-leucine, and (S)-phenylalanine ethyl esters reacted preferentially with (R)-N-methylphenylalanine N-carboxyanhydride, but the stereoselectivity decreased considerably in nitrobenzene and dimethylacetamide as solvents. In the latter solvents, the dipolar interactions between an amine and an N-carboxyanhydride and the orientation of the substituent of N-carboxyanhydride were seriously affected, hence the stereoselectivity decreased. As a consequence, the enantiomer selection by the terminal amine of a growing chain in the nucleophilic addition-type polymerization of α-amino acid N-carboxyanhydride can be controlled by the choice of solvent. (S)-Proline ethyl ester and (S)-α-phenylethylamine reacted preferentially with (S)-N-methylphenylalanine N-carboxyanhydride in m-dimethoxybenzene, and this type of selectivity did not change in nitrobenzene. But in dimethylacetamide the stereoselectivity decreased. In the transition state of the reaction of these amines and N-methylphenylalanine N-carboxyanhydride dipolar interactions are operating, which should be destroyed by dimethylacetamide but may not be affected by nitrobenzene.