Activation of carboxylic acids by pyrocarbonates. Synthesis of arylamides of N -protected amino acids and small peptides using dialkyl pyrocarbonates as condensing reagents

Activation of carboxylic acids was achieved via dialkyl pyrocarbonates (ROCO)₂O, (R = ethyl, isopropyl, sec-butyl, tert-butyl) in aprotic solvents in the presence of tertiary amines. A convenient one-pot procedure for the preparation of arylamides from N-protected amino acids including arginine and from di-tert-butyl pyrocarbonate in the presence of pyridine (Boc₂O-pyridine system) was reported. Analogously, diisopropyl, di-sec-butyl or diethyl pyrocarbonate could be used in the presence of N-methylmorpholine or triethylamine. A wide variety of N-protected amino acid arylamides were prepared in good yields.     

General procedure for preparing a reference mixture and determining the amount of dipeptide contaminant in N-alkoxycarbonylamino acids by high-performance liquid chromatography

The amount of N-alkoxycarbonyl-dipeptide contaminant in a sample of N-alkoxycarbonylamino acid is determined by reversed phase high-performance liquid chromatography on a μBondapak-C₁₈ column using aqueous methanol or acetonitrile as solvent and u.v.-absorbance monitoring. Reference mixtures of the N-substituted monomer and dipeptide are prepared by reaction of the symmetrical anhydride of the N-alkoxycarbonylamino acid with the sodium salt of the amino acid in aqueous dimethylformamide. The proportions of the two components in the reference mixture are established by ¹H-n.m.r. spectroscopy. N-Protected-dipeptide was detected (0.1–0.7%) in seven out of eight commercial benzyloxycarbonylamino acids examined.     

Racemization of α-amino acid esters by aliphatic ketones in the presence of carboxylic acids

Amino acid esters are racemized by dissolution in a mixture of aliphatic ketones and carboxylic acids. The racemization rate mainly depends on the structure of the amino acid and on the kind of ketone and carboxylic acid used, the best racemizing medium being acetone containing 15% acetic acid. The mechanism of the racemization and the practical consequences of this study in the optical resolution field are discussed.     

High-performance liquid chromatography of 2,4-disubstituted-5(4H)-oxazolones,anhydrides and other activated forms of N-acyl- and N-alkoxycarbonylamino acids

Normal phase high-performance liquid chromatography has been achieved of the common activated forms of valine on a LiChrosorb-CN or a μPorasil (underivatized silica) column using hexane containing 1.5 or 5%tert.-butanol as solvent. Compounds examined include the 2-alkoxy-5(4H)-oxazolones and symmetrical and mixed anhydrides of N-tert.-butoxycarbonyl-, N-benzyloxycarbonyl-, and N-9-fluorenylmethoxycar-bonylvaline, the chloride of the latter, a p-nitrophenyl ester, 2-methyl-4-isopropyl-5(4H)-oxazolone, valine-N-carboxyanhydride, and the N- and O-ethoxycarbonyl adducts of 1-hydroxybenzotriazole. The 5(4H)-ox-azolones from N-tert.-butoxycarbonylvaline and N-benzyloxycarbonylglycylvaline decomposed during chromatography on the μPorasil but not the LiChrosorb-CN column. The method allows direct monitoring of reactions involving generation or consumption of activated forms of amino acids.     

Racemization during aminolysis of mixed and symmetrical anhydrides of N-alkoxycarbonylamino acids by amino acid anions in aqueous dimethylformamide

Racemization attending the aminolysis of symmetrical (Sy) and mixed (Mx) anhydrides (An) of N-alkoxycarbonylamino acids by amino acid anions in aqueous dimethylformamide has been examined by reversed phase high-performance liquid chromatography analysis of the N-protected dipeptides produced. Racemization occurred in most cases when the anion was generated using 1 equiv. sodium hydrogen carbonate, in most cases for MxAn reactions when the base was sodium carbonate, and only in a few cases for SyAn reactions when the base was sodium carbonate. Less racemization was associated with MxAn reactions when the chloroformate of a secondary alcohol had been used for their generation. The change in chirality is explained on the basis of the formation and racemization of the 2-alkoxy-5(4H)-oxazolone, which is greater in the presence of the weaker base because of incomplete deprotonation of the amino acid zwitter-ion.     

Amino acid–azetidine chimeras: synthesis of enantiopure 3‐substituted azetidine‐2‐carboxylic acids

Abstract: Azetidine‐2‐carboxylic acid (Aze) analogs possessing various heteroatomic side chains at the 3‐position have been synthesized by modification of 1‐9‐(9‐phenylfluorenyl) (PhF)‐3‐allyl‐Aze tert‐butyl ester (2S,3S)‐ 1 . 3‐Allyl‐Aze 1 was synthesized by regioselective allylation of α‐tert‐butyl β‐methyl N‐(PhF)aspartate 13 , followed by selective ω‐carboxylate reduction, tosylation, and intramolecular N‐alkylation. Removal of the PhF group and olefin reduction by hydrogenation followed by Fmoc protection produced nor‐leucine–Aze chimera 2 . Regioselective olefin hydroboration of (2S,3S)‐ 1 produced primary alcohol 23 , which was protected as a silyl ether, hydrogenated and N‐protected to give 1‐Fmoc‐3‐hydroxypropyl‐Aze 26 . Enantiopure (2S,3S)‐3‐(3‐azidopropyl)‐1‐Fmoc‐azetidine‐2‐carboxylic acid tert‐butyl ester 3 was prepared as a Lys–Aze chimera by activation of 3‐hydroxypropyl–Aze 26 as a methanesulfonate and displacement with sodium azide. Moreover, orthogonally protected azetidine dicarboxylic acid 4 was synthesized as an α‐aminoadipate–Aze chimera by oxidation of alcohol 26 . These orthogonally protected amino acid–Aze chimeras are designed to serve as tools for studying the influence of conformation on peptide activity.     

Racemization during aminolysis of activated esters of N-alkoxycarbonylamino acids by amino acid anions in partially aqueous solvents and a tactic to minimize it

Racemization during the aminolysis of activated esters of N-alkoxycarbonylamino acids by amino acid anions in aqueous dimethylformamide was examined by determining the epimeric products by high-performance liquid chromatography. Partial racemization occurred for a variety of esters, particularly when sodium hydrogen carbonate was used to generate the anion of d -valine. The racemization results from prolonged contact of unconsumed ester with the alkaline medium. Variation of the stoichiometry of reagents for reactions with N-benzyloxycarbonylphenylalanine (Z-Phe) 4-nitrophenyl ester revealed that racemization could be minimized by using Na₂CO₃ as base and a 50% excess of amino acid anion. An efficient synthesis of optically pure Z-l -Phe-D-Val-OH was achieved with a reaction time of 15 min.     

Coupling in the absence of tertiary amines: II

Formate salts of amino-components obtained by acidolysis of tert. butyloxy-carbonyl-derivatives with formic acid were converted to the tetrazolate salts which in turn were acylated with active esters. Addition of a tertiary base was not necessary for ready coupling. Further improvement was achieved by using a solution of tetrazole in formic acid for the removal of the tert.butyloxy-carbonyl group.     

Improved selectivity in the removal of the tert.-butyloxycarbonyl group

Removal of the tert.-butyloxycarbonyl group by trifluoroacetic acid in the presence of benzyloxycarbonyl groups, benzyl esters and benzyl ethers is rendered more selective by dilution with acetic acid. Trifluoroacetic acid-acetic acid mixtures, however, cause acetylation of hydroxyl groups and also some formation of tert.-butyl esters at free carboxyls. Hence, such mixtures are useful only for the deprotection of intermediates in which the hydroxyl and carboxyl groups are fully blocked. A search for a diluent without such inherent limitation led to the application of a mixture of phenol and p-cresol. Dilution of trifluoroacetic acid with phenols both improved the selectivity in the removal of the tert.-butyloxycarbonyl group and suppressed the alkylation of amino acid side chains as well. A 4:3:3 mixture of trifluoroacetic acid, phenol and p-cresol was found useful in the practical execution of partial deprotection.     

复方氨基酸脂质凝胶对实验动物皮肤创伤的修复作用

目的 观察复方氨基酸脂质凝胶对实验动物皮肤创伤的修复作用。方法 复制大鼠烫伤模型、豚鼠皮肤切割伤模型及家兔皮肤切割伤伴感染模型,测定皮肤愈合率。结果 复方氨基酸脂质凝胶对大鼠皮肤烫伤有促进恢复作用,对豚鼠皮肤切割伤有促愈合作用,在家兔皮肤切割伤伴感染模型显示脂质凝胶对家兔切割伤后不滴加细菌与滴加金黄色葡萄球菌或绿脓杆菌的皮肤愈合均有促进作用,其创面愈合百分率显著高于基质组。结论 复方氨基酸脂质疑胶能促进实验性创伤动物的创面修复。     

LC-MS/MS法同时测定人血浆中氯吡格雷及其羧酸代谢物的浓度

目的建立快速测定人血浆中氯吡格雷及其羧酸代谢物SR26334含量的LC-MS/MS法。方法乙醚-正己烷(4:1,V:V)2次液-液提取法(中性和酸化条件下),采用Teknokroma C_(18)色谱柱,以那格列奈和吡格列酮为内标,同时测定血浆中氯吡格雷和SR26334的浓度。流动相:甲醇-0.1%甲酸(80:20,V:V);流速:0.2mL·min~(-1);以多反应离子监测方式检测:氯吡格雷[M+H]~+,m/z 322.1→212.1;那格列奈[M+H]~+,m/z 318.3→166.2;氯吡格雷羧酸代谢物SR26334[M+H]~+,m/z 308.1→q98.1;吡格列酮[M+H]~+,m/z 357.2→134.2。结果氯吡格雷和内标那格列奈的保留时间分别在4.4和3.7min,SR26334和内标吡格列酮的保留时间分别在1.3和1.7min,氯吡格雷的线性范围为5～5000ng·L~(-1);SR26334的线性范围为20～2500μg·L~(-1)。提取回收率大于75%,方法回收率大于90%,日内、日间RSD小于10%(n=5)。结论本方法简便快速,适用于氧吡格雷制剂的新药临床研究和临床长期治疗病人血药浓度的常规监测。     

Synthesis and X‐ray study of the 6‐(N‐pyrrolyl)purine and thymine derivatives of 1‐aminocyclopropane‐1‐carboxylic acid

Abstract: The novel purine and pyrimidine derivatives of 1‐aminocyclopropane‐1‐carboxylic acid 1 and 2 were obtained by alkylation of 6‐(N‐pyrrolyl)purine and thymine with methyl 1‐benzamido‐2‐chloromethylcyclopropanecarboxylate. X‐ray crystal structure analysis shows that the cyclopropane rings in 1 and 2 posses Z‐configuration. The cyclopropane ring atoms and attached atoms of the benzamido and methoxycarbonyl moiety of both molecules are disposed perpendicularly to each other. The carbonyl oxygen of the methoxycarbonyl moiety adopts in both compounds a synperiplanar conformation with respect to the midpoint of the distal bond of the cyclopropane ring. The torsion angles φ and ψ for the 1‐aminocyclopropane‐1‐carboxylic acid residue in 1 and 2 correspond to a folded conformation, while the torsion angles ω define antiperiplanar conformation. Intermolecular hydrogen bonds connect the molecules of 1 into dimers. Each dimer is hydrogen‐bonded with four ethanol molecules, thus forming discrete unit. On the contrary, intermolecular hydrogen bonds link the molecules of 2 generating three‐dimensional network.     

Solid-phase synthesis of chicken vasoactive intestinal peptide by a mild procedure using Nα-9-fluorenylmethyloxycarbonyl amino acids

The octacosapeptide amide corresponding to the entire amino acid sequence of chicken vasoactive peptide (VIP) was assembled on a p-benzyloxybenzylamine resin support using the base-labile 9-fluorenylmethyloxycarbonyl as N^α-protecting group, cleaved by mild acid treatment, and purified by gel-filtration and ion-exchange chromatography. The symmetrical anhydride coupling was employed and monitored by two independent methods, and acetic anhydride termination was incorporated to minimize formation of deletion peptides. The homogeneity of the final product, obtained in 18% yield, was assessed by t.l.c., disc electrophoresis, amino-terminal amino acid analysis, and amino acid analyses of acid and enzyme hydrolysates. The purified chicken VIP was shown to be active on gastric acid secretion and on pancreatic blood flow. Previously reported ring closure of the Asp-Asn unit seemed to be at a minimum, owing to the mild basic and acid treatments.     

HPLC法测定野菊花水提取液中有机酸类药效组分的含量

目的建立同时测定野菊花中三种有机酸类药效组分含量的方法。方法采用HPLC法测定。色谱条件：Diamonsil C18色谱柱（4.6×250 mm,5μm）;流动相：A相为0.5%磷酸水溶液,B相为乙腈,进行梯度洗脱,在0-10 min,B相从10%线性改变至20%,10-30 min,B相从20%线性改变至30%;检测波长为326 nm;柱温为室温。结果绿原酸的线性范围为0.095 2-1.904μg（r=0.999 86）,咖啡酸的线性范围为0.0372-0.744μg（r=0.99988）,3,5-二咖啡酰奎尼酸的线性范围为0.186-3.72μg（r=0.999 91）。回收率分别为98.07%、98.73%和98.12%,RSD分别为1.96%、0.71%、0.99%（n=5）。结论HPLC法可同时测定野菊花中绿原酸、咖啡酸和3,5-二咖啡酰奎尼酸三个有机酸类药效组分,该方法具有准确、快速、重现性好的特点。     

Effect of acute lindane and alcohol intoxication on serum concentration of enzymes and fatty acids in rats.

This study examines possible synergistic effects of lindane and ethanol on inducing liver injury and serum fatty acid derangement in adult male Wistar rats. When administered together, ethanol and lindane-induced even more pronounced increase of alanine aminotransferase (165 +/- 10 U/L) and gamma-glutamyltranspeptidase activity (10.3 +/- 0.6 U/L) than after isolated administration of either substance. In addition, separate administration of lindane and ethanol was followed by a significant decrease of linoleic acid level in the serum (301 +/- 38 mg/L, 276 +/- 35 mg/L vs. 416 +/- 48 mg/L). However, when ethanol administration was followed by lindane injection, serum linoleic acid was at the similar level found in the control group (516 +/- 62 mg/L). Ethanol-treated rats that received lindane 30 min after ethanol administration have shown a marked increase of palmitic (421 +/- 27 mg/L) and linolic acid level (43 +/- 5 mg/L) in comparison with rats that have been treated only with ethanol (316+/-26 mg/L for palmitic and 32 +/- 2 mg/L for linolic acid) or lindane (295 +/- 26 mg/L for palmitic and 301 +/- 38 mg/L for linolic acid). Linolic acid level was significantly greater in comparison with control group (29 +/- 1 mg/L). In conclusion, this study found enough evidence to support the hypothesis that acute ethanol intoxication potentiates lindane-induced liver injury and enhances lipid derangement.     

Cellular function of neuropathy target esterase in lysophosphatidylcholine action

Neuropathy target esterase (NTE) plays critical roles in embryonic development and maintenance of peripheral axons. It is a secondary target of some organophosphorus toxicants including analogs of insecticides and chemical warfare agents. Although the mechanistic role of NTE in vivo is poorly defined, it is known to hydrolyze lysophosphatidylcholine (LPC) in vitro and may protect cell membranes from cytotoxic accumulation of LPC. To determine the cellular function of NTE, Neuro-2a and COS-7 cells were transfected with a full-length human NTE-containing plasmid yielding recombinant NTE (rNTE). We find the same inhibitor sensitivity and specificity profiles for rNTE assayed with LPC or phenyl valerate (a standard NTE substrate) and that this correlation extends to the LPC hydrolases of human brain, lymphocytes and erythrocytes. All of these LPC hydrolases are therefore very similar to each other in respect to a conserved inhibitor binding site conformation. NTE is expressed in brain and lymphocytes and contributes to LPC hydrolase activities in these tissues. The enzyme or enzymes responsible for erythrocyte LPC hydrolase activity remain to be identified. We also show that rNTE protects Neuro-2a and COS-7 cells from exogenous LPC cytotoxicity. Expression of rNTE in Neuro-2a cells alters their phospholipid balance (analyzed by liquid chromatography-mass spectrometry with single ion monitoring) by lowering LPC-16:0 and LPC-18:0 and elevating glycerophosphocholine without a change in phosphatidylcholine-16:0/18:1 or 16:0/18:2. NTE therefore serves an important function in LPC homeostasis and action.