Synthesis of 1,2-diacyloxypropyl-3-(1′,2′-diacyl-sn-glycero)-phosphonate

The total synthesis of 1,2-diacyloxypropyl-3-(1′,2′-diacyl-sn-glycero)phosphonate is described. The 1,2-dipalmitoyloxypropyl phosphonic acid was prepared by an Arbusov reaction of 1,2-diacylglycerol bromohydrin with trimethyl phosphite; the final product was obtained by a coupling reaction involving the diacyloxypropyl-3-phosphonic acid and 1,2-dipalmitoyl-sn-glycerol, catalysed by tri-isopropylbenzene sulfonyl chloride. The resulting synthetic product was characterised by elemental analysis, phosphono-phosphorus determinations and IR spectroscopy.     

Synthesis of 1,2-dipalmitoyloxypropyl-3-(2-trimethylammoniumethyl)phosphinate

The total synthesis of 1,2-dipalmitoyloxypropyl-3-(2-trimethylammoniumethyl)phosphinate, the phosphinate analog of phosphatidylcholine, is described. The phosphinate analog has been essentially prepared by an Arbusov type reaction between 1,2-dipalmitoyl-sn-glycerolbromohydrin and 2-bromoethyl phosphonic acid dimethyl ester for 48 h at 170°C, followed by removal of the methyl ester with sodium iodide and reaction with aqueous trimethylamine to yield the final product. The phosphinate analog of phosphatidylcholine was characterized by elemental analysis, thin-layer chromatography (TLC), IR spectroscopy and phosphonophosphorus determinations.     

Bioactive sucrose esters from Bidens parviflora

An investigation on Bidens parviflora led to the isolation of three sucrose esters and a substituted truxillate. Their structures were elucidated as (6-O-(E)-p-coumaroyl)-beta-D-fructofuranosyl-(2-->1)-alpha-D-glucopyranoside, (6-O-(E)-p-coumaroyl)-beta-D-fructofuranosyl-(2-->1)-(6-O-(E)-p-coumaroyl)-alpha-D-glucopyranoside II, 6,6'-sucrose ester of (1alpha,2alpha,3beta,4beta)-3,4-bis(4-hydroxyphenyl)-1,2-cyclobutanedicarboxylic acid, dimethyl ester of (1alpha,2alpha,3alpha,4alpha)-2,4-bis(3,4-dihydroxyphenyl)-1,3-cyclobutanedicarboxylic acid on the basis of spectral and chemical evidence. These compounds were subjected to the following bioassays: the histamine release inhibition of rat mast cells induced by antigen-antibody reaction and the inhibitory activity of PGE(2) production by macrophages.     

Synthesis of phosphonate and ether analogs of rac-phosphatidyl-L-serine

The chemical synthesis of four phosphonate-containing phosphatidylserine analogs namely, L-serine (±)-[2,3-bis(hexadecyloxy) and 2,3-bis(Palmitoyloxy)-propyl] phosphonates, and L-serine (±)-[3,4-bis(hexadecyloxy and 3,4-bis(palmitoyloxy)-butyl]phosphonates is described. (±)-2,3-Bis(hexadecyloxy) and 2,3-bis(palmitoyloxy)-propylphosphonic acids and (±)-3,4-bis (hexadecyloxy)butylphosphonic acid were prepared by reaction of tris(trimethylsilyl) phosphite on the corresponding haloalkane. Condensation of the above phosphonic acids or (±)-3,4-bis (palmitoyloxy)butylphosphonic acid with N-carboxy-L-serine dibenzyl ester in the presence of trichloroacetonitrile or triisopropylbenzenesulfonyl chloride yielded the protected serine intermediates, which on hydrogenolysis gave the desired L-serine analogs. By a similar route, 1,2-dihexadecyl-rac-glycero-3-phosphoric acid was converted to 1,2-dihexadecyl-rac-glycerophospho-L-serine [L-serine (±)-2,3-bis(hexadecyloxy)propyl hydrogen phosphate(ester)].     

Isolation of hydroxy-Y base from rat liver tRNAPhe.

A Y-base derivative was isolated from rat liver tRNAPhe and its structure was assigned to be alpha-(carboxyamino)-beta-hydroxy-4,9-dihydro-4,6-dimethyl-9-oxo-1H-imidazol[1,2-a]purine-7-butyric acid dimethyl ester (hydroxy-Y), based on the results of mass spectrometry and chemical degradation. This modified base seems to be the major fluorescent component of rat liver tRNAPhe; the peroxy-Y base previously isolated from rat liver tRNAPhe and characterized by Nakanishi and his coworkers (1,2) was not present in our preparation.     

Efficient route to (S)-azetidine-2-carboxylic acid

A new and efficient route to (S)-azetidine-2-carboxylic acid (>99.9% ee) in five steps and total yield of 48% via malonic ester intermediates was established. As the key step, efficient four-membered ring formation (99%) was achieved from dimethyl (S)-(1'-methyl)benzylaminomalonate by treating with 1,2-dibromoethane (1.5 eq) and cesium carbonate (2 eq) in DMF. Krapcho dealkoxycarbonylation of dimethyl (1'S)-1-(1'-methyl)benzylazetidine-2,2-dicarboxylate, the product of this cyclization procedure, proceeded with preferential formation (2.7:1, 78% total yield) of the desired (2S,1'S)-monoester, with the help of a chiral auxiliary which was introduced on the nitrogen atom. The undesired (2R,1'S)-isomer could be converted to that with proper stereochemistry, by a deprotonation and subsequent re-protonation step. Finally, lipase-catalyzed preferential hydrolysis of the (2S,1'S)-monoester and subsequent deprotection provided enantiomerically pure (S)-azetidine-2-carboxylic acid in a 91% yield from the mixture of (2S,1'S)- and (2R,1'S)-isomers.     

Synthesis of all-cis-1,3-diacylcyclopentane-1,2,3-triol-2-phosphate via acyl group migration in a cyclic diglyceride analog.

The acid-catalyzed isomerization of the diglyceride analog (1,2,3/0)-1,2-dipalmitoylcyclopentane-1,2,3-triol has been used to generate syn-syn-1,3-diacyl-cyclopentane-1,2,3-triol, a required intermediate in the synthesis of a symmetrical all-cis-1,2,3/0-2P cyclopentanoid phosphatidic acid analog. The all-cis cyclo-phosphatidic acid analog has therefore been obtained in the free acid form and as the diphenyl ester, dimethyl ester, and dipotassium salt derivatives. The compounds have been characterized by microanalysis and spectroscopic methods. The 1,2,3/0-2P analog is now available for comparative studies with the corresponding all-trans cyclophosphatidic acid (1,3/2-2P).     

Structure and enzymic activity of ribonuclease-A esterified at glutamic acid-49 and aspartic acid-53

The dimethyl ester of bovine pancreatic ribonuclease-A (dimethyl RNAase-A), the initial product of esterification of RNAase-A in anhydrous methanolic HCl, was isolated in a homogeneous form. The two carboxy functions esterified in this derivative are those of glutamic acid-49 and aspartic acid-53. There were no changes in the u.v.-absorption spectral characteristics, the accessibility of the methionine residues, the resistance of the protein to proteolysis by trypsin and the antigenic behaviour of RNAase-A as a result of the esterification of these two carboxy groups. Dimethyl RNAase-A exhibited only 65% of the specific activity of RNAase-A, but still had the same K_m value for both RNA and 2′:3′-cyclic CMP. However, the V_max. was decreased by about 35%. On careful hydrolysis of the methyl ester groups at pH9.5, dimethyl RNAase-A was converted back into RNAase-A. Limited proteolysis of dimethyl RNAase-A by subtilisin resulted in the formation of an active RNAase-S-type derivative, namely dimethyl RNAase-S, which was chromatographically distinct from dimethyl RNAase-A and had very nearly the same enzymic activity as dimethyl RNAase-A. Fractionation of dimethyl RNAase-S by trichloroacetic acid yielded dimethyl RNAase-S-protein and dimethyl RNAase-S-peptide, both of which were inactive by themselves but regenerated dimethyl RNAase-S when mixed together. Dimethyl RNAase-A-peptide was identical with RNAase-S-peptide. RNAase-S-protein could be generated from dimethyl RNAase-S-protein by careful hydrolysis of the methyl ester groups at pH9.5. The interaction of dimethyl RNAase-S-protein with RNAase-S-peptide appears to be about 4-fold weaker than that between the RNAase-S-protein and RNAase-S-peptide. Conceivably, the binding of the S-peptide `tail' of dimethyl RNAase-A with the remainder of the molecule is similarly weaker than that in RNAase-A, and this brings about subtle changes in the geometrical orientation of the active-site amino acid residues of these modified methyl ester derivatives. It is suggested that these changes could be responsible for the generation of the catalytically less-efficient RNAase-A and RNAase-S molecules (dimethyl RNAase-A and dimethyl RNAase-S respectively).     

Catalysis of electron transfer across phospholipid bilayers by iron-porphyrin complexes

Phospholipid vesicles containing K₃Fe(CN)₆ were prepared from egg yolk phosphatidylcholine. Hemin dimethyl ester was incorporated into these vesicles during preparation in ratios of phospholipid to hemin dimethyl ester that varied from 200 : 1 to 45000 : 1. Electron transfer across the bilayer was measured anaerobically after injecting the vesicles into a solution containing reduced indigotetrasulfonic acid. Vesicles containing hemin dimethyl ester exhibited high rates of electron transfer (240 electrons/molecule hemin dimethyl ester per min). Conditions could be selected where the rate-limiting step for catalysis was either the bimolecular reaction between ferric hemin dimethyl ester and reduced indigotetrasulfonic acid or the movement of hemin dimethyl ester from interface to interface. The hemin dimethyl ester-catalyzed electron transfer went to completion within a few seconds, completely oxidizing the reduced indigotetrasulfonic acid. Valinomycin (in the presence of potassium) and carbonyl cyanide p-trifluoromethoxyphenylhydrazone were without effect on catalyzed electron transport. Thus, the electron transport is not electrogenic but is a coupled, neutral system. By specific assay, neither phosphate nor cyanide was significantly transported during electron transfer but evidence is provided to suggest that a coordinated hydroxide accompanies movement of Fe(III) hemin dimethyl ester from the inside surface to the outside surface of the bilayer. It was also demonstrated in a bulk phase transport system that hemin dimethyl ester readily catalyzes transfer of S¹⁴CN^? through a chloroform layer separating two aqueous phases. Another more hydrophobic iron-porphyrin complex, Fe(III) tetraphenylporphyrin, was found to be twice as effective as hemin dimethyl ester. Other porphyrin complexes were also tested as control systems. No significant catalysis was found for metal-free protoporphyrin IX dimethyl ester or Ni(II) tetraphenylporphyrin. The results are discussed in comparison with in vivo electron transport and the future usefulness of this model system.     

Incorporation of phosphonic acid diesters into lipid model membranes. Part II. X-ray and neutron diffraction studies.

Mixtures of egg phosphatidylcholine and phosphonic acid diethyl or dibutyl esters of the general type RP(O)(OR')2 with R = hexane or dodecane were studied at room temperature in the fluid lamellar state by X-ray and by neutron diffraction. Generally a molar ratio of lipid and ester of 1:0.5 was used. Additionally an equimolar lipid/ester mixture of hexane phosphonic acid diethyl ester was studied. Depending on the ester used and its concentration a single L alpha-phase was observed above a certain water content which changes to an L alpha + water two phase system at high water concentration. Despite the large amounts of the amphiphilic ester molecules incorporated in the membrane and their high molecular asymmetry, the mixtures qualitatively show the typical hydration and swelling behaviour of non-charged lipid membranes. However, the incorporation of the esters induces a higher hydration capacity, a lateral extension and a decrease in membrane thickness. The position of the ester molecules and their orientation in the membrane were determined by neutron diffraction using partially deuterated esters. The esters were found to be located with their phosphonic moiety near or in the lipid/water interface. The lamellar structure contradicts this location of the cone-shaped ester molecules which should increase the tendency to form hexagonal structures. However, the experimental findings can be understood if one considers a partial interdigitation of the last hydrocarbon groups of the lipid chains accompanied by a larger disorder in the hydrophobic centre of the membrane. In the case of hexane phosphonic acid dibutyl ester, a vertical translocation of the ester takes place below a certain water content where it is distributed between two locations at the lipid water interface and the centre of the membrane.     

The chemical and enzymatic oxidation of hematohemin IX. Identification of hematobiliverdin IX alpha as the sole product of the enzymatic oxidation

Oxidative cleavage of hematohemin IX in pyridine solution in the presence of ascorbic acid (coupled oxidation), followed by esterification of the products with boron trifluoride/methanol produced the four possible hematobiliverdin dimethyl esters in 11.1% overall yield. Transetherifications took place simultaneously with the esterification reaction and resulted in the formation of the dimethyl ester of hematobiliverdin IX gamma 8a,13a-dimethyl ether (1.8%), the dimethyl ester of hematobiliverdin IX beta 13a,18a-dimethyl ether (1.9%), the dimethyl ester of hematobiliverdin IX delta 8a-monomethyl ether (1.4%), and the dimethyl ester of hematobiliverdin IX alpha 18a-monomethyl ether (0.4%). The latter was the sole product obtained after the enzymatic oxidation of hematohemin with heme oxygenase, after esterification of the reaction product with boron trifluoride/methanol. When the esterification step was omitted hematobiliverdin IX alpha was obtained from the enzymatic oxidation. The structures of the hematobiliverdin derivatives were secured by their NMR and mass spectra data. Saponification of the dimethyl esters afforded the hematobiliverdin methyl ethers, which were excellent substrates of biliverdin reductase and were readily reduced to the corresponding bilirubins. Hematobiliverdin IX alpha was also a good substrate of biliverdin reductase. It is concluded that the enzymatic oxidation of hematohemin IX by heme oxygenase is alpha-selective, while biliverdin reductase shows no selectivity in the reduction of the four hematobiliverdin isomers.     

Preparation of protohemin monomethyl ester

A new and simple method for the preparation of protohemin monomethyl ester was described. The method is involved with the direct esterification of a propionic acid group of hemin, followed by column chromatography on silica gel. The physical properties (ir, solubility, tlc, etc.) of the product was intermediate between those of protohemin and protohemin dimethyl ester.     

Oxidative ozonolysis of 6-oxo-PGF1α, 11,15-diacetate, methyl acetal, methyl ester

Ozonolysis of 6-oxo-PGF_1α, 11,15-diacetate, methyl acetal, methyl ester followed by oxidative workup and treatment with diazomethane gave 3-acetoxy-5-hydroxy-2-(methoxycarbonyl) cyclopentane acetic acid, γ-lactone and dimethyl 3-acetoxy-5[[−(methoxycarbonyl)valeryl] oxy]-1,2-cyclopentane dicarboxylate as two of the major products. The mass spectral properties of the latter compound were identical with those previously published by other investigators.     

Bioisosteres of 9-carboxymethyl-4-oxo-imidazo[1,2-a]indeno-[1,2-e]pyrazin-2-carboxylic acid derivatives. Progress towards selective, potent in vivo AMPA antagonists with longer durations of action

A novel series of 2- and 9-disubstituted heterocyclic-fused 4-oxo-indeno[1,2-e]pyrazin derivatives was synthesized. One of them, the 9-(1H-tetrazol-5-ylmethyl)-4-oxo-5,10-dihydroimidazo[1,2-a]indeno[1,2-e]pyrazin-2-yl phosphonic acid 4i exhibited a strong and a selective binding affinity for the AMPA receptor (IC50 = 13 nM) and demonstrated potent antagonist activity (IC50 = 6nM) at the ionotropic AMPA receptor. This compound also displayed good anticonvulsant properties against electrically-induced convulsions after ip and iv administration with ED50 values between 0.8 and 1 mg/kg. Furthermore, a strong increase in potency was observed when given iv 3 h before test (ED50 = 3.5 instead of 25.6 mg/kg for the corresponding 9-carboxymethyl-2-carboxylic acid analogue). These data confirmed that there is an advantage in replacing the classical carboxy substituents by their bioisosteres such as tetrazole or phosphonic acid groups.     

海洋链霉菌Streptomyces lusitanus SCSIO LR32中芳酰胺类代谢产物的研究

采用硅胶柱色谱和半制备反相高效液相色谱分离方法,对南海海洋放线菌Streptomyces lusitanus SCSIOLR32的次级代谢产物进行了研究,分离得到四个酰胺类化合物,经MS1、H和13 C NMR波谱分析鉴定为二甲基甲苯2,4-二氨基甲酸甲酯(1),甲苯2,4-二氨基甲酸乙酯(2),甲苯2,6-二氨基甲酸甲酯(3)和甲苯2,6-二氨基甲酸乙酯(4)。运用X-单晶衍射确定了1的结构式。其中化合物2和4是首次从自然界中分离得到。采用16S分子生物学方法鉴定该菌株为链霉菌属放线菌。     

The First Synthesis of 4′-Branched 5′-Deoxycarbocyclic 9-Deazaadenosine and Phosphonic Acids as Antiviral Agents

The first synthetic route to 4′-trifluoromethylated 5′-deoxycarbocyclic-9-deazaadenosine analog and its phosphonic acid derivatives was described from α-trifluoromethyl-α,β-unsaturated ester. The C–C bond connection between cyclopentane and base moiety was accomplished using Knoevenagel type condensation from ketone derivative 11. Synthesized nucleoside and phosphonic acid analogs were tested for anti-HIV activity as well as cytotoxicity.     

13C and 1H NMR studies of helix-coil transition of poly(β-benzyl-L-aspartate) and poly(γ-benzyl-L-glutamate): Behavior in nonprotonating solvent mixtures,and origin of solvent-induced chemical shift

From the results of ¹³C-nmr measurement of poly(β-benzyl-L -aspartate) and its model compounds in dimethyl sulphoxide/deuterated chloroform mixtures, it was found that the side chain of poly(β-benzyl-L -aspartate) is solvated by dimethyl sulphoxide in the region more than dimethyl sulphoxide 20% (v/v), where the backbone maintains the α-helix. The chemical shift differences in the benzyl group carbons of poly(γ-benzyl-L -glutamate) (trifluoroacetic acid/deuterated chloroform) accompanied by the helix-coil transition, originate from the interaction between the ester group of the side chain and trifluoroacetic acid. The chemical shift difference in the ester carbon is similar. On the other hand, the chemical shift differences of the side-chain carbons in the alkyl portion (C^β, C^γ) originate not only from the interaction between the ester group of the side chain and trifluoroacetic acid, but also from some other unknown factors. The chemical shift differences of the side-chain carbons of poly(β-benzyl-L -aspartate) originate from the interaction between the ester group of the side chain and trifluoroacetic acid.     

Stereochemical assignment of naturally occurring 2,3-epoxy-2-methylbutanoate esters

A method to determine the absolute configuration of 2,3-epoxy-2-methylbutanoate ester residues in natural products is presented, based on (i) the reduction of the ester function to yield a 2-methyl-1,2-butanediol, (ii) esterification of the obtained primary alcohol with either (R)-(+)- or (S)-(-)-Mosher's acid to afford the corresponding Mosher's ester, and (iii) 1H-NMR spectral comparison of the final product with that of the Mosher's esters prepared from 2-methyl-1,2-butanediols of known stereochemistry.     

两种化感物质对茄子幼苗光合作用及叶绿素荧光参数的影响

采用基质栽培,研究不同浓度的2,6-二叔丁基苯酚及邻苯二甲酸二甲酯对茄子幼苗光合作用及叶绿素荧光参数的影响.结果表明,两种化感物质使茄子叶片叶绿素含量、光合速率（P_n）、气孔导度（G_s）下降,细胞间隙CO₂浓度（C_i）先降低后升高;使茄子叶片的初始荧光(F_o)增加,PSⅡ最大光化学效率(F_v/F_m)、光系统Ⅱ光合电子传递量子效率(Ф_PSⅡ)、光化学猝灭系数(q_P)、天线转化效率(F_v^' /F_m^′)降低.2,6-二叔丁基苯酚使茄子叶片非光化学猝灭系数(q_N)先升高后降低,邻苯二甲酸二甲酯使q_N低于对照,对茄子叶片光合机构造成了伤害.     

Enzymatic synthesis of lysophosphatidic acid and phosphatidic acid

Immobilised 1,3-specific lipase from Rhizopus arrhizus was used as catalyst for the esterification of -glycero-3-phosphate and fatty acid or fatty acid vinyl ester in a solvent-free system. With lauric acid vinyl ester as acyl donor, a_w<0.53 favored the synthesis of lysophosphatidic acid (1-acyl-rac-glycero-3-phosphate, LPA1) and the spontaneous acyl migration of the fatty acid on the molecule. Subsequent acylation by the enzyme resulted in high phosphatidic acid (1,2-diacyl-rac-glycero-3-phosphate, PA) formation and high total conversions (>95%). With oleic acid, maximum conversions of 55% were obtained at low water activities. Temperatures below melting point of the product favored precipitation and resulted in high final conversion and high product ratio [LPA/(PA+LPA)]. Thus, LPA was the only product with lauric acid vinyl ester as acyl donor at 25°C. Increased substrate ratio ( -glycero-3-phosphate/fatty acid) from 0.05 to 1 resulted in a higher ratio of LPA to PA formed, but a lower total conversion of -glycero-3-phosphate. Increased amounts of enzyme preparation did not result in higher esterification rates, probably due to high mass-transfer limitations.