Isosteric conversion of protein carboxyl groups into carboxamide groups. II. Application to lysozyme

For isosteric conversion of carboxyl groups of proteins into amide groups, ammonolysis of protein esters under mild conditions was attempted. Ammonolysis of methyl esters of lysozyme and bovine serum albumin proved to be incomplete. Highly reactive N-ethylsalicylamide esters of guanylated lysozyme were therefore prepared by subjecting the protein to reaction with N-ethylbenz-isoxazolium ion at pH 4.2, 0°. Per molecule, 5–7 ester groups were introduced, with concomitant decrease of activity of 80–90%. Only 0.3 tyrosine was modified. On hydrolysis at pH 9.2 the activity was completely restored. At pH 7.9 three classes of ester groups could be distinguished: one group of high rate of hydrolysis (k₁ = 1.5 min^-1), three groups of intermediate rate (k₂ = 0.13 min^-1) and two groups of low rate (k³ = 0.018 min^-1). The intermediate rate approximated the rate of hydrolysis of the model compound benzoylglycine N-ethyl-salicylamide ester (k = 0.15 min^-1). Ammonolysis at pH 9.2 in 2.0 M ammonia/ ammonium acetate provided complete conversion of the ester groups into amide groups without restoration of activity, confirming the essentiality of certain carboxyl groups. In particular, rearrangement of the ester groups into relatively stable imide groups by O–N acyl migration was found to be completely absent. When native lysozyme was esterified with N-ethylbenzisoxazolium ion the activity did not completely return on hydrolysis.     

NEW REACTION OF 4-CHLORO-7-NITROBENZOFURAZAN WITH AMINES AT HIGH LOCAL CONCENTRATIONS

The fluorogenic amine reagent 4-chloro-7-nitrobenzofurazan (CN,3Fz) reacts with amines to give one of two products, or a mixture, depending on the concentration of amine. At low concentration (e.g. 5 times 10^-4 M) of unprotonated amine the normal replacement of the chlorine occurs, giving Λ_max? 470 nm. At high concentration of unprotonated amine (e.g. 0.5M), a new product, Λ_max? 380 nm, is produced. At intermediate concentrations, both products are seen. These results were obtained with aqueous ammonia, methylamine and butylamine, and with butylamine in dry hexane. The 380 nm product also is obtained with high local concentrations of amines, such as with [Lys]_n at pH 8.5–10 and micellar dodecylamine at pH 9.5. These results are suggested to be analogous to those of very primitive proto-enzyme systems, by which clusters of small molecules can produce qualitative changes in “metabolism”. Reaction of CNBFz with gelatin gave qualitative evidence for cross-linking.     

FLUORESCENCE SPECTRA OF LYSOZYME EXCITED AT 305 NM IN PRESENCE OF UREA

Tryptophan 108 of hen egg white lysozyme was selectively excited at 305 nm and fluorescence spectra were recorded as a function of pH (2–9) and concentration of urea (0–8 M). Urea at low concentrations (1–4 M) quenches markedly the Trp 108 fluorescence around pH 7; the Λ_max, however, remains unaltered. The fluorescence quenching by urea is most likely due to local conformational changes around Trp 108 in the active site region of the enzyme. Substantial unfolding of the enzyme, however, was brought about by 4M urea below pH 3, and by 7 M urea at pH 10.3, as indicated by a marked red shift in the Λ_max of the fluorescence emission.     

血浆中去甲地西泮及代谢物奥沙西泮的HPLC测定方法和大鼠口服药代动力学

采用高效液相色谱法测定去甲地西泮(去甲安定)及其代谢产物奥沙西泮。以RP-C₁₈为固定相,乙腈—0.01mol·L^-1醋酸钠(pH3.8,33.3:66.6)为流动相,地西泮为内标物,紫外波长240nm处定量测定。去甲地西泮、奥沙西泮和内标物的保留时间分别为2.8min,4.85min和8.5min;绝对回收率分别为74%,86%和86%。奥沙西泮在35.3～2260ng·ml^-1,去甲地西泮在20～2560ng·ml^-1血浆浓度范围内线性关系良好,r=0.9997和r=0.9998。二药的最低检测浓度分别为10ng·ml、和7ng·ml^-1;日内和日间相对标准偏差(RSD)均分别小于6%和10%(n=5)。多种常用药物对样品的色谱峰无干扰。并用此法研究了大鼠单次口服去甲地西泮的药代动力学。     

Inhibitory Effects of N-acetylcysteine on Superoxide Anion Generation in Human Polymorphonuclear Leukocytes

It has been suggested that reactive oxygen species released by activated polymorphonuclear leukocytes (PMN) in man is one mechanism of tissue injury. Therapeutic action aimed at increasing antioxidant defence mechanisms is still a clinical challenge. This study examines the activity of N-acetylcysteine, a known antioxidant, in the protection of PMN exposed in-vitro to the chemoattractant peptide fMet-Leu-Phe (FMLP), the protein kinase C activator phorbol myristate acetate or the lipid peroxidation promoter t-butyl hydroperoxide. FMLP (3–300 nm) and phorbol myristate acetate (160 pm–160 nm) induced concentration-related superoxide anion generation. Pre-treatment with N-acetylcysteine (33–333 μm) resulted in concentration-related inhibition of superoxide production induced by FMLP (30 nm) or phorbol myristate acetate (16 nm); –log IC50 values were 3.97 ± 0.07 and 3–91 ± 0.10, respectively. Changes in intracellular calcium ion concentration ([Ca²⁺]_i) induced by FMLP (30 nm) were studied in fura-2-loaded human PMN. FMLP produced a transient calcium response, i.e. a peak followed by decay to a residual value above baseline. N-Acetylcysteine (333 μm) did not affect either basal [Ca²⁺]_i values or changes in [Ca²⁺]_i values after treatment with FMLP. Activation by phorbol myristate acetate caused a reduction in glutathione levels from 5.94 ± 0.86 (control) to 1.84 ± 0.51 nmol/3 × 10⁶ cells (P < 0.05 compared with control). Pre-treatment with N-acetylcysteine (333 μm) fully reversed the reduction in glutathione levels induced by phorbol myristate acetate (4.83 ± 0.68 nmol/3 × 10⁶ cells; P > 0.05 compared with control). Exposure to t-butyl hydroperoxide (0.5 mm, 30 min) markedly increased malondialdehyde levels (from 0.03 ± 0.02 to 0.73 ± 0.07 nmol/10⁶ cells), and index of lipid peroxidation. Malondialdehyde levels were significantly reduced in PMN treated with N-acetylcysteine (333 μm; 0.55 ± 0.04 nmol/10⁶ cells; P < 0.05 compared with untreated cells exposed to t-butyl hydroperoxide). In conclusion, N-acetylcysteine reduces superoxide generation in response to FMLP and phorbol myristate acetate and partially protects against lipid peroxidation in PMN from man. The protection afforded by N-acetylcysteine is not related to alteration of the intracellular calcium signal but might be effected by replenishment of the intracellular glutathione levels.     

Evidence for p-hydroxylation of N-ethyl-N-methylaniline

1. N,N-Dialkyl- and N-alkyl-4-aminophenols derived from N-ethyl-N-methylaniline have been isolated from incubation mixtures of rabbit hepatic microsomes fortified with an NADPH-generating system.

2. This is the first report of metabolic ring (4-C) hydroxylation in vitro of an N,N-dialkylaniline.

3. N-Ethyl-N-methyl-4-aminophenol was the major ring-hydroxylation product, whilst N-ethyl-4-aminophenol and N-methyl-4-aminophenol were formed in approx. equal amounts at about 15% of the tertiary compound.

4. An apparent V_max of 1.8 nmol/mg protein per?min and K_m of 1.7 × 10^?4M were determined for N-ethyl-N-methyl-4-aminophenol formation using an optimized incubation time of 10?min.     

HPLC法测定盐酸布桂嗪注射液的有关物质

目的:建立高效波相色谱法测定盐酸布桂嗪注射液中的有关物质.方法:采用C18柱,以甲醇-0.1 mol·L-1醋酸铵溶液(用氨试液调节pH值至7.0)(75:25)为流动相,检测波长为252 nm.结果:盐酸布桂嗪与其降解产物在该色谱条件下能够有效分离.结论:方法简便、专属性强,可用于测定盐酸布桂嗪注射液中有关物质.     

Fluorogenic substrates for chymotrypsin with new fluorescent markers

Several substrates for chymotrypsin Glt-Phe-NHR_x have been synthesized, where R_xNH₂ are the compounds 3-aminocoumarin (2), 6-aminocoumarin (3), 3-acetamido-6-aminocoumarin (4), 3-acetamido-8-aminocoumarin (5), 7-amino-4-methyl-2-quinolinone (AMQ), (6). The fluorescence properties of the new substrates and those of the corresponding free amines were examined. The compound 7-glutarylphenylalaninamido-4-methyl-2-quinolinone (Glt-Phe-AMQ), (15), provided a new suitable substrate for chymotrypsin determination. The enzymatic release of the fluorophore AMQ was measured at Λex = 360nm and Λem=435nm. The Km of 15 was 0.5mM and its kcat/Km ratio was 47 M^-1 s^-1. By using this substrate, the detection limit of chymotrypsin was 10 ng/ml.     

Inhibition of zaleplon metabolism by cimetidine in the human liver: in vitro studies with subcellular fractions and precision-cut liver slices

1. The effect of cimetidine on the metabolism of zaleplon (ZAL) in human liver subcellular fractions and precision-cut liver slices was investigated. 2. ZAL was metabolized to a number of products including 5-oxo-ZAL (M2), which is known to be formed by aldehyde oxidase, N-desethyl-ZAL (DZAL), which is known to be formed by CYP3A forms, and N-desethyl-5-oxo-ZAL (M1). 3. Human liver microsomes catalysed the NADPH-dependent metabolism of ZAL to DZAL. Kinetic analysis of three microsomal preparations revealed mean (± SEM) S₅₀ and V_max of 310 ± 24 µM and 920 ± 274 pmol/min/mg protein, respectively. 4. Human liver cytosol preparations catalysed the metabolism of ZAL to M2. Kinetic analysis of three cytosol preparations revealed mean (± SEM), K_m and V_max of 124 ± 14 µM and 564 ± 143 pmol/min/mg protein, respectively. 5. Cimetidine inhibited ZAL metabolism to DZAL in liver microsomes and to M2 in the liver cytosol. With a ZAL substrate concentration of 62 µM, the calculated mean (± SEM, n = 3) IC₅₀ were 596 ± 103 and 231 ± 23 µM for DZAL and M2 formation, respectively. Kinetic analysis revealed that cimetidine was a competitive inhibitor of M2 formation in liver cytosol with a mean (± SEM, n = 3) K_i of 155 ± 16 µM. 6. Freshly cut human liver slices metabolized ZAL to a number of products including 1, M2 and DZAL. 7. Cimetidine inhibited ZAL metabolism in liver slices to M1 and M2, but not to DZAL. Kinetic analysis revealed that cimetidine was a competitive inhibitor of M2 formation in liver slices with an average (n = 2 preparations) K_i of 506 µM. 8. The results demonstrate that cimetidine can inhibit both the CYP3A and aldehyde oxidase pathways of ZAL metabolism in the human liver. Cimetidine appears to be a more potent inhibitor of aldehyde oxidase than of CYP3A forms and hence in vivo is likely to have a more marked effect on ZAL metabolism to M2 than on DZAL formation. 9. The results also demonstrate that precision-cut liver slices may be a useful model system for in vitro drug-interaction studies.     

Effects of pH and Dose on Nasal Absorption of Scopolamine Hydrobromide in Human Subjects

Purpose. The present study was conducted to evaluate theeffects of formulation pH and dose on nasal absorption of scopolaminehydrobromide, the single most effective drug available for the prevention ofnausea and vomiting induced by motion sickness. Methods. Human subjects received scopolamine nasally at adose of 0.2 mg/0.05 mL or 0.4 mg/0.10 mL, blood samples were collected atdifferent time points, and plasma scopolamine concentrations were determinedby LC-MS/MS. Results. Following administration of a 0.2 mg dose, theaverage C_max values were found to be 262 ± 118, 419± 161, and 488 ± 331 pg/mL for pH 4.0, 7.0, and 9.0formulations, respectively. At the 0.4 mg dose the average C_maxvalues were found to be 503 ± 199, 933 ± 449, and 1,308± 473 pg/mL for pH 4.0, 7.0, and 9.0 formulations, respectively. At a0.2 mg dose, the AUC values were found to be 23,208 ± 6,824, 29,145± 9,225, and 25,721 ± 5,294 pg.min/mL for formulation pH 4.0,7.0, and 9.0, respectively. At a 0.4 mg dose, the average AUC value wasfound to be high for pH 9.0 formulation (70,740 ± 29,381 pg.min/mL)as compared to those of pH 4.0 (59,573 ± 13,700 pg.min/mL) and pH 7.0(55,298 ± 17,305 pg.min/mL) formulations. Both the C_maxand AUC values were almost doubled with doubling the dose. On the otherhand, the average T_max values decreased linearly with a decreasein formulation pH at both doses. For example, at a 0.4 mg dose, the averageT_max values were 26.7 ± 5.8, 15.0 ± 10.0, and 8.8± 2.5 minutes at formulation pH 4.0, 7.0, and 9.0, respectively. Conclusions. Nasal absorption of scopolamine hydrobromidein human subjects increased substantially with increases in formulation pHand dose.     

Synthesis of Potent Non-imidazole Histamine H3-Receptor Antagonists

Histamine has been converted into a non-imidazole H₃-receptor histamine antagonist by addition of a 4-phenylbutyl group at the N^α-position followed by removal of the imidazole ring. The resulting compound, N-ethyl-N-(4-phenylbutyl)amine, remarkably has a K_i = 1.3 μM as an H₃ antagonist. Using this as a lead compound, a novel series of homologous O and S isosteric tertiary amines was synthesised and structure-activity studies furnished N-(5-phenoxypentyl)pyrrolidine (K_i = 0.18 ± 0.10 μM, for [³H]histamine release from rat cerebral cortex synaptosomes) which, more importantly, was active in vivo. Substitution of NO₂ into the para position of the phenoxy group gave N-(5-p-nitrophenoxypentyl)pyrrolidine, UCL 1972 (K_i = 39 ± 11 nM), ED₅₀ = 1.1 ± 0.6 mg/kg per os in mice on brain tele-methylhistamine levels.     

LC-MS测定双氢青蒿素哌喹片中阿莫西林、头孢克洛和头孢克肟的残留量

目的 建立双氢青蒿素哌喹片中阿莫西林、头孢克洛、头孢克肟残留量的液质联用检测方法。方法 采用Thermo Gold C₁₈色谱柱(2.1 mm×50 mm,1.9μm);流动相为10 mmol·L^-1乙酸铵溶液(A)-乙腈(B)。阿莫西林、头孢克洛稀释剂为10 mmol·L^-1乙酸铵溶液(氨水调节pH值至7.2),流速为0.3 mL·min^-1,进样量为10μL;头孢克肟稀释剂为10 mmol·L^-1乙酸铵溶液(氨水调节pH值至11.0),流速为0.25 mL·min^-1,进样量为2μL。阿莫西林、头孢克洛和头孢克肟分别采用2种梯度洗脱条件。结果 阿莫西林、头孢克洛和头孢克肟线性良好,线性方程分别为y=3 734.53x–2 188.63(r=0.999 5),y=1 396.67x–266.554(r=0.999 8)和y=3 636.39x+2 277.48(r=0.999 7),重复性RSD分别为3.1%,2.8%,2.2%,回收率分别为107.1%...     

Biotransformation of nitrosobenzene,phenylhydroxylamine, and aniline in the isolated perfused rat liver

1. Haemoglobin-free single-pass perfusion of isolated rat liver with [¹⁴C]aniline, [¹⁴C]phenylhydroxylamine, and [¹⁴C]nitrosobenzene was carried out.

2. Perfusion with aniline revealed apparent enzyme kinetics for 4-aminophenol formation with K_m = 144μM, V_max = 51 nmol/min per g liver wet; for 2-aminophenol K_m = 144μM, V_max = 16nmol/min per g; for acetanilide K_m = 33μM, V_max = 25 nmol/min per g. Formation of phenylhydroxylamine and nitrosobenzene was observed at a rate of 1.5 nmol/min per g provided that these metabolites had been trapped within red cells.

3. Perfusion with phenylhydroxylamine displayed a metabolic pattern similar to aniline with apparent phenylhydroxylamine reduction kinetics of K_m = 260μM and V_max = 600 nmol/min per g. In addition an acid-labile phenylhydroxylamine glucuronide was formed.

4. Perfusion with nitrosobenzene showed very rapid reduction to phenylhydroxylamine and to the metabolites observed with phenylhydroxylamine. In postmicrosomal supernatant, enzymic reduction of nitrobenzene by NADH and NADPH showed K_m = 12μM nitrosobenzene and V_max = 5000nmol/min per g.

5. Three per cent of nitrosobenzene was irreversibly bound to liver proteins. After 20min perfusion with nitrosobenzene, 0.95 μmol of liver glutathione was lost per 10μmol nitrosobenzene infused; 0.16μmol of glutathione was released with effusate and bile, 0.46μmol of glutathionesulphinanilide was produced, the rest, 0.33μmol, may have formed mixed disulphides.     

Determination of adrenergic agonists from extracts and herbal products of Citrus aurantium L. var. amara by LC

The purpose of this study was to set up a HPLC method to separate adrenergic amines (dl-octopamine, dl-synephrine and tyramine) and to determine their content in fruits, extracts and herbal products of Citrus aurantium L. var. amara. A rapid method for the quantitative analysis of these amines is described, based on their separation by RP-HPLC technique with UV detection. The analysis were conducted on a Lichrospher RP-18 column at room temperature, using a mobile phase consisting of 0.02 M citric acid–0.02 M NaH₂PO₄ (7:3 v/v) and adjusted to a final pH of 3. The detection was at 220 nm. Since some of these amines are chiral compounds and their enantiomers showed different pharmacological activity, the direct separation of synephrine enantiomers was carried out with HPLC on a β-cyclodextrin stationary phase. The mobile phase consisted of methanol–NaH₂PO₄ 25 mM pH 3.5 (20:80 v/v) and tetrabutylammonium hydrogen sulfate 10 mM in ratio of 30:70 v/v in isocratic condition and the detection was at 220 nm. The two proposed methods were applied to the analysis of fruits, extracts and herbal products of C. aurantium L. var. amara. Taking into account that some authors have reported that l-synephrine may be converted into its d-form by high temperature, this optical isomerization was monitored by the same HPLC method used for the separation of enantiomers.     

Hypotensive and Vasorelaxant Effects of Citronellol,a Monoterpene Alcohol,in Rats

Abstract: Citronellol is an essential oil constituent from the medicinal plants Cymbopogon citratus, Cymbopogon winterianus and Lippia alba which are thought to possess antihypertensive properties. Citronellol‐induced cardiovascular effects were evaluated in this study. In rats, citronellol (1–20 mg/kg, i.v.) induced hypotension, which was not affected by pre‐treatment with atropine, hexamethonium, N^ω‐nitro‐l ‐arginine methyl ester hydrochloride or indomethacin, and tachycardia, which was only attenuated by pre‐treatment with atropine and hexamethonium. These responses were less than those obtained for nifedipine, a reference drug. In intact rings of rat mesenteric artery pre‐contracted with 10 μM phenylephrine, citronellol induced relaxations (pD₂ = 0.71 ± 0.11; E_max = 102 ± 5%; n = 6) that were not affected by endothelium removal, after tetraethylamonium in rings without endothelium pre‐contracted with KCl 80 mM. Citronellol strongly antagonized (maximal inhibition = 97 ± 4%; n = 6) the contractions induced by CaCl₂ (10^?6 to 3 × 10^?3 M) and did not induce additional effects on the maximal response of nifedipine (10 μM). Finally, citronellol inhibited the contractions induced by 10 μM phenylephrine or 20 mM caffeine. The present results suggest that citronellol lowers blood pressure by a direct effect on the vascular smooth muscle leading to vasodilation.     

Small intestinal sulphoxidation of Albendazole

1. The in vitro sulphoxidation of Albendazole (ABZ) by rat intestinal microsomes has been examined. The results revealed intestinal sulphoxidation of ABZ by intestinal microsomes in a NADPH-dependent enzymatic system. The kinetic constants for sulphoxidase activity were V_max = 46 pmol/min/mg protein and Michaelis constant K_m = 6·8 μM.

2. The possible effect of inducers (Arochlor 1254 and ABZ pretreatment) and inhibitors (erythromycin, methimazole, carbon monoxide and fenbendazole), was also studied. In rat pretreated with Arochlor 1254, V_max was 52 pmol/min/mg protein, whereas oral administration of ABZ increased the intestinal sulphoxidation of the drug, V_max being 103 pmol/min/mg protein.

3. Erythromycin did not change the enzymatic biconversion of ABZ, but methimazole and carbon monoxide inhibited the enzyme activity by approximately 60 and 30% respectively. Fenbendazole (a structural analogue of ABZ) was a competitive inhibitor of the sulphoxidation process, characterized by a K_i or 69 μM.

4. These data demonstrate that the intestinal enzymes contributing to the initial sulphoxidation of ABZ may be similar to the hepatic enzymes involved in the biotransformation process by the P450 and FMO systems, a conclusion that needs to be further established.     

Concentration-dependent metabolism of diazepam in mouse liver

Previous mouse liver studies with diazepam (DZ),N-desmethyldiazepam (NZ), and temazepam (TZ) confirmed that under first-order conditions, DZ formed NZ and TZ in parallel. Oxazepam (OZ) was generatedvia NZ and not TZ despite that preformed NZ and TZ were both capable of forming OZ. In the present studies, the concentration-dependent sequential metabolism of DZ was studied in perfused mouse livers and microsomes, with the aim of distinguishing the relative importance of NZ and TZ as precusors of OZ. In microsomal studies, theK _ms andV _maxs, corrected for binding to microsomal proteins, were 34 μM and 3.6 nmole/min per mg and 239 μM and 18 nmole/min per mg, respectively, forN-demthylation andC ₃-hydroxylation of DZ. TheK _ms andV _maxs forN-demethylation andC ₃-hydroxylation of TZ and NZ, respectively, to form OZ, were 58 μM and 2.5 nmole/min per mg and 311 μM and 2 nmole/min per mg, respectively. The constants suggest that at low DZ concentrations, NZ formation predominates and is a major source of OZ, whereas at higher DZ concentrations, TZ is the important source of OZ. In livers perfused with DZ at input concentrations of 13 to 35 μM, the extraction ratio of DZ (E{DZ}) decreased from 0.83 to 0.60. NZ was the major metabolite formed although its appearance was less than proportionate with increasing DZ input concentration. By contrast, the formation of TZ increased disporportionately with increasing DZ concentration, whereas that for OZ decreased and paralleled the behavior of NZ. Computer simulations based on a tubular flow model and thein vitro enzymatic parameters provided a poorin vitro-organ correlation. TheE{DZ}, appearance rates of the metabolites, and the extraction ratio of formed NZ (E{NZ, DZ}) were poorly predicted; TZ was incorrectly identified as the major precursor of OZ. Simulations with optimized parameters imporved the correlations and identified NZ as the major contributor of OZ. Saturation of DZN-demethylation at higher DZ concentrations increased the role of TZ in the formation of OZ. The poor aqueous solubility (limiting the concentration range of substrates usedin vitro), avid tissue binding and the coupling of enzymatic reactions in liver, favoring sequential metabolism, are possible explanations for the poorin vitro-organ correlation. This work emphasizes the complexity of the hepatic intracellular milieu for drug metabolism and the need for additional modeling efforts to adequately describe metabolite kinetics. This work was supported by the Medical Research Council of Canada (MA-9104).     

Meeting of Drug Metabolism Group Extrahepatic Metabolism

1. Four non-acidic primary metabolites of N-(5-pyrrolidinopent-3-ynyl)succinimide (BL 14) were identified and quantified using g.l.c. and mass spectrometry. The metabolites are α-hydroxy-N-(5-pyrrolidinopent-3-ynyl)succinimide (A), N-(5-(2-oxopyrrolidino)-pent-3-ynyl)succinimide (B), N-(2-hydroxy-5-pyrrolidinopent-3-ynyl)succinimide (C) and N-(5-pyrrolidinopent-3-ynyl)succinimide N'-oxide (E), the latter analysed after reduction to the parent amine.

2. In rat liver preparations, all metabolites are formed by microsomal, NADPH-dependent enzyme systems, but with different characteristics. The response to inhibitors such as CO and SKF 525A indicates participation of cytochrome P-450 enzymes in the formation of all metabolites. Phenobarbital pretreatment markedly enhances propynylic hydroxylation (C) but has little or no effect on the other metabolic pathways. Succinimide hydroxylation (A) exhibits a pH optimum at 7±0, while the formation of metabolites B and C increases at pH values between 6±4 and 7±7.

3. Kinetic studies on the formation of metabolites A-C revealed differences in the Michaelis constant, while the V_max values were similar. Succinimide hydroxylation (A) is most efficient with a K_m of 3±7 × 10^?5 M, compared with a K_m of 1±7 × 10^?3 M for propynylic hydroxylation (C).

4. The formation of metabolites B and E conforms to the corresponding mechanisms for lactam and N-oxide formation for other xenobiotics. The formation of metabolites A and C represents two extremities, reflected in their different responses to phenobarbital pretreatment, pH changes and in their different K_m values. Although little can be discerned about the mechanisms from the literature, the enzymes cataly sing both reactions appear to be cytochromes.     

Rigid conformation in tert.-butyloxycarbonyl-l-alanine ortho-nitrophenyl ester

Ortho-nitrophenyl esters of blocked amino acids have significantly higher aminolysis rates than the corresponding para-nitrophenyl esters. This difference prompted a series of investigations that led to the present study. Single crystal X-ray diffraction analysis of tert.-butyloxycarbonyl-l -alanine o-nitrophenyl ester has shown a rather rigid molecular conformation with very restricted rotation about the ester bond owing to the wedging of one of the nitro oxygen atoms between the two oxygens of the ester group. A para nitro group can not interact in a similar fashion and consequently can have many more rotamers. The C₁₄H₁₈N₂O₆ ester crystallizes in space group P2₁, with a = 5.180 Å, b = 9.726 Å, c = 15.616 Å and β= 92.36 Å. There is only one intermolecular hydrogen bond, N(1)H…O(0) at 3.125 Å, that links neighboring molecules into infinite chains. There are no intramolecular hydrogen bonds.