Mild, efficient, selective and "green" benzoylation of nucleosides using benzoyl cyanide in ionic liquid

Use of benzoyl cyanide (BzCN) for benzoylation of nucleosides has been studied, both in pyridine and in ionic liquid BzCN in 1-methoxyethyl-3-methylimidazolium methanesulfonate as ionic liquid has been found to be a "green "alternative compared to the pyridine-BzCN system. An efficient and selective benzoylation of nucleosides of both, the 2'-deoxy- and the ribo-series at ambient temperature was accomplished.     

MILD,EFFICIENT, SELECTIVE AND “GREEN” BENZOYLATION OF NUCLEOSIDES USING BENZOYL CYANIDE IN IONIC LIQUID

Use of benzoyl cyanide (BzCN) for benzoylation of nucleosides has been studied, both in pyridine and in ionic liquid. BzCN in 1-methoxyethyl-3-methylimidazolium methanesulfonate as ionic liquid has been found to be a “green” alternative compared to the pyridine-BzCN system. An efficient and selective benzoylation of nucleosides of both, the 2′-deoxy- and the ribo-series at ambient temperature was accomplished.     

High performance liquid chromatography of ceramides: application to analysis in human tissues and demonstration of ceramide excess in Farber's disease

Conditions have been determined for the benzoylation of ceramides containing nonhydroxy and hydroxy fatty acids, and a high performance liquid chromatography system for the separation and measurement of these derivatives has been devised that is capable of good resolution and high sensitivity. These methods have been used to determine quantitatively the levels of ceramides in human tissues, and in serum and urine, and to demonstrate elevated amounts of ceramide in Farber's disease urine and tissues.     

A high-performance liquid chromatographic procedure for the determination of disaturated phosphatidylcholine in human plasma

Plasma disaturated phosphatidylcholine (DSPC) concentration has been implicated as a risk factor for atherosclerosis. However, suitable methods for the estimation of these compounds in plasma are not available. In this paper, a method for the estimation of DSPC using argentation thin-layer chromatography and high-performance liquid chromatography is described. It is quantitative for the measurement of individual and total DSPC species and is not dependent on fatty acid chain length. The method employs hydrolysis of total plasma phosphatidyl choline by phospholipase C, followed by benzoylation of the diacylglycerols. The benzoates are then fractionated on silver nitrate-impregnated silica gel thin-layer chromatography plates, and the disaturated species separated and quantitated by high-performance liquid chromatography. The method is sensitive and reproducible and allows many samples to be done at once. With this method, the amounts of DSPC were found to be significantly higher in a group of normolipidemic diabetic subjects, compared to age-matched controls.     

Efficient large-scale synthesis of 5'-O-dimethoxytrityl-N4-benzoyl-5-methyl-2'-deoxycytidine

An efficient process to synthesize 5'-O-dimethoxytrityl-N4-benzoyl-5-methyl-2'-deoxycytidine in high yield and quality is described. Final benzoylation was improved by developing a method to selectively hydrolyze benzoyl ester impurities. This inexpensive approach was scaled up to multikilogram quantities for routine use in oligonucleotide therapeutics.     

Zinc triflate-benzoyl bromide: a versatile reagent for the conversion of ether into benzoate protecting groups and ether glycosides into glycosyl bromides

A simple and efficient method is developed for the chemoselective one-pot conversion of ethers (benzyl, TBDMS and acetal) to the corresponding benzoates by zinc triflate-catalyzed deprotection and benzoylation by benzoyl bromide. In the same reaction, methyl or p-methoxyphenyl glycosides are converted into glycosyl bromides that are useful in glycosylation reactions.     

Analysis and quantitation of free ceramide containing nonhydroxy and 2-hydroxy fatty acids, and phytosphingosine by high-performance liquid chromatography.

Reaction of ceramides containing nonhydroxy fatty acids with benzoyl chloride in pyridine at 70 degrees C for 1 hr resulted in N-benzoylation to form N,N-acyl,benzoyl derivatives; O-benzoylation also occurred. However with ceramides containing 2-hydroxy fatty acids and phytosphingosine only O-benzoylation occurred even on prolonged treatment. Only O-benzoylation occurred on reaction with benzoic an hydride. However, the benzoylation of ceramides with phytosphingosine could not be achieved with benzoic anhydride and this benzoylation was performed by reaction with benzoyl chloride at 70 degrees C for 4 hr. Because N,N-acyl,benzoyl derivatives of ceramides containing nonhydroxy fatty acids produced by treatment with benzoyl chloride overlap methyl benzoate on high-performance liquid chromatography, benzoic anhydride was preferable for benzoylation of ceramides with nonhydroxy and 2-hydroxy fatty acids. On the other hand, the reaction with benzoyl chloride at 70 degrees C for 4 hr was used for quantitation of benzoylated ceramides containing 2-hydroxy fatty acids and phytosphingosine. 3-(p-Phenylbenzoyl)estrone was used as an internal standard for both reactions and values for ceramides containing 2-hydroxy fatty acids obtained by the two reactions were in good agreement. This procedure was applied to measurement of the ceramide levels in the brain, liver, and kidney of rats during development. The levels of ceramides containing nonhydroxy and 2-hydroxy fatty acids in the brain, liver, and kidney increased to the adult levels and then remained unchanged. Ceramide with phytosphingosine was detected in the liver and kidney, where its concentration gradually increased with age, but it was not found in the brain. The composition of nonhydroxy fatty acids were also analyzed.     

Selective 2''-benzoylation at the cis 2'',3''-diols of protected ribonucleosides. New solid phase synthesis of RNA and DNA-RNA mixtures.

5'-0-(Dimethoxytrityl)-2'-0-(benzoyl or 3,4,5-trimethoxybenzoyl)-base protected ribonucleosides have been prepared by selective benzoylation of the 2'-hydroxyl group. The isomerization of the 2'-benzoates to the 3'-benzoates was studied. The protected ribonucleosides have been converted to either methylphosphochloridites or methylphosphoamidites and used to synthesize oligoribonucleotides on silica gel solid support. The synthetic RNA were deprotected and isolated using conditions that minimize internucleotide cleavage. The use of 2'-benzoates as protecting groups for ribonucleosides has made it possible to easily prepare and isolate mixtures of DNA and RNA.     

Efficient Large-Scale Synthesis of 5′-O-Dimethoxytrityl-N 4-Benzoyl-5-Methyl-2′-Deoxycytidine

An efficient process to synthesize 5′-O-dimethoxytrityl-N⁴-benzoyl-5-methyl-2 ′-deoxycytidine in high yield and quality is described. Final benzoylation was improved by developing a method to selectively hydrolyze benzoyl ester impurities. This inexpensive approach was scaled up to multi-kilogram quantities for routine use in oligonucleotide therapeutics.     

Conversion of the beckman liquid phase sequencer to a gas-liquid phase sequencer

As an effective aid to extend the microsequencing capabilities the Beckman protein/peptide sequenator Series 890C has been successfully converted to a gas-liquid system, in which coupling buffer 25% trimethylamine was employed as a gas, and heptafluorobutyric acid as a liquid. The system has been found to be efficient for microsequencing (less than 100 pmol). The details of mechanical, plumbing, and other minor changes are described in this paper along with the results of sequencing proteins and peptides, directly and from blots.     

Authentic standards for the reductive-cleavage method. The positional isomers of partially methylated and acetylated or benzoylated 1,5-anhydroribitol

The eight positional isomers of methylated and benzoylated 1,5-anhydroribitol were generated simultaneously from 1,5-anhydroribitol by sequential partial methylation and benzoylation, and the four meso isomers and two enantiomeric pairs of isomers so-formed were isolated in chemically pure form by high-performance liquid chromatography. The corresponding acetates were obtained by debenzoylation of the pure isomers and acetylation. Reported herein are the 1H NMR spectra of the benzoates and the electron-ionization mass spectra of the acetates and the tri-O-methyl derivative. Also reported for the acetates and the tri-O-methyl derivative are their linear temperature-programmed gas-liquid chromatography retention indices on three different capillary columns.     

Authentic standards for the reductive-cleavage method: the positional isomers of partially methylated and acetylated or benzoylated 1,5-anhydro-L-arabinitol

Described herein is the synthesis of the eight positional isomers of methylated and acetylated or benzoylated 1,5-anhydro-L-arabinitol. The compounds were generated simultaneously from 1,5-anhydro-L-arabinitol by sequential partial methylation and benzoylation and isolated in pure from by high-performance liquid chromatography. The desired acetates were obtained by debenzoylation and acetylation of the pure isomers. Reported herein are the 1H NMR spectra of the benzoates and the electron-ionization mass spectra of the acetates and the tri-O-methyl derivative. Also reported for the acetates and the tri-O-methyl derivative are their linear temperature-programmed gas-liquid chromatography retention indices on three different capillary columns.     

农杆菌介导的大豆高频遗传转化

大豆(Glycine max(L.)Merrill)遗传转化目前常用的两种方法为农杆菌介导的子叶节转化系统和基因枪介导的体细胞胚转化,但这两种转化系统都存在转化频率低、难于重复及依赖于特定的基因型等问题.为了提高农杆菌介导的大豆子叶节的转化频率,采用了一种基于bar基因作为筛选标记基因的固体-液体筛选系统,与农杆菌共培养3d的大豆子叶节在MS添加2 mg/L 6-BA和5 mg/L的glufosinate的筛选培养基培养2周后,再转到含有0.01 mg/L TDZ和2mg/L glufosinate的液体培养基中筛选,并每周更换一次培养液.得到的再生芽首先经GUS分析为阳性后再转入生根培养基得到完整转化植株,然后通过Southern杂交分析证实外源基因整合到大豆基因组,转化植物含有1～2个基因拷贝数.该转化系统具有转化频率高、转化周期短以及不依赖于大豆基因型等优点,对影响该转化系统的一些因子进行了讨论.     

Mechanisms of quinacrine binding and fluorescence in nuclei and chromosomes

Summary The mechanism has been investigated whereby quinacrine binds to the DNA of nuclei and chromosomes in cytological preparations fixed in methanol-acetic acid. A variety of evidence is consistent with the idea that the quinacrine binds by intercalation. This is supported by a high value for the affinity of quinacrine for DNA, together with a saturation value of 0.2 quinacrine molecules/nucleotide; binding in the presence of strong salt solutions; and inhibition of fluorescence and banding by denaturation or depurination of DNA. At high quinacrine concentrations, weak binding of quinacrine to nuclei and chromosomes also occurs, but this is not relevant to the production of strong fluorescence or Q-banding patterns.A number of factors were tested which might have affected quinacrine fluorescence and banding. These included: pH; blocking protein amino groups by acetylation or benzoylation; introduction of hydrophobic groups by benzoylation; and dephosphorylation. All these treatments were without effect. However, comparison of the quinacrine fluorescence of human and onion nuclei, which differ substantially in the base composition of their DNa, shows that quinacrine fluorescence can be enhanced in cytological preparations by AT-rich DNA.In honour of Prof. P. van Duijn     

Preparation and analysis of benzoylated cerebrosides

The benzoylation of cerebrosides with benzoyl chloride and with benzoic anhydride is described, and the derivatives that contained hydroxy and nonhydroxy fatty acids were isolated by high pressure liquid chromatography (HPLC). Studies on the structures of these derivatives and the products formed by mild alkaline hydrolysis are reported. Reaction of cerebrosides containing nonhydroxy fatty acids with benzoyl chloride results in amide-acylation in addition to normal O-acylation. Mild alkali treatment of the N-diacyl derivative results in the formation of N-benzoyl psychosine. Derivatization with benzoic anhydride avoids amide-acylation. These derivatives are useful for the HPLC analysis of cerebrosides.     

Mechanisms of quinacrine binding and fluorescence in nuclei and chromosomes

The mechanisms has been investigated whereby quinacrine binds to the DNA of nuclei and chromosomes in cytological preparations fixed in methanol-acetic acid. A variety of evidence is consistent with the idea that the quinacrine binds by intercalation. This is supported by a high value for the affinity of quinacrine for DNA, together with a saturation value of 0.2 quinacrine molecules/nucleotide; binding in the presence of strong salt solutions; and inhibition of fluorescence and banding by denaturation or depurination of DNA. At high quinacrine concentrations, weak binding of quinacrine to nuclei and chromosomes also occurs, but this is not relevant to the production of strong fluorescence or Q-banding patterns. A number of factors were tested which might have affected quinacrine fluorescence and banding. These included: pH; blocking protein amino groups by acetylation or benzoylation; introduction of hydrophobic groups by benzoylation; and dephosphorylation. All these treatments were without effect. However, comparison of the quinacrine fluorescence of human and onion nuclei, which differ substantially in the base composition of their DNA, shows that quinacrine fluorescence can be enhanced in cytological preparations by AT-rich DNA.     

Selective benzoylation of some N-acetyl-N-aryl-β-D-xylopyranosylamines

Selectivity in the benzoylation of N-acetyl-N-p-methoxyphenyl-, -p-bromophenyl-, and -p-chlorophenyl-β-D-xylopyranosylamines has been demonstrated. The structures of the products was shown by using periodate oxidation and n.m.r. spectroscopy. The relative reactivity of the hydroxyl groups was HO-3 ≈ HO-4 > HO-2. The β-D-xylopyranosylamine derivatives were shown to possess the ⁴C¹ conformation.     

In vitro somatic embryogenesis and plant regeneration of cassava

An efficient and reproducible plant regeneration system, initiated in somatic tissues, has been devised for cassava (Manihot esculenta Crantz). Somatic embryogenesis has been induced from shoot tips and immature leaves of in vitro shoot cultures of 15 cassava genotypes. Somatic embryos developed directly on the explants when cultured on a medium containing 4–16 mg/l 2,4-D. Differences were observed with respect to the embryogenic capacity of the explants of different varieties. Secondary embryogenesis has been induced by subculture on solid or liquid induction medium. Long term cultures were established and maintained for up to 18 months by repeated subculture of the proliferating somatic embryos. Plantlets developed from primary and secondary embryos in the presence of 0.1 mg/l BAP, 1mg/l GA₃, and 0.01 mg/l 2,4-D. Regenerated plants were transferred to the field, and were grown to maturity.     

Authentic standards for the reductive-cleavage method. The positional isomers of partially methylated and acetylated or benzoylated methyl 2-(acetylmethylamino)-2-deoxy-beta-D-glucopyranoside

Described herein is the synthesis of eight positional isomers of methylated and acetylated or benzoylated methyl 2-(acetylmethylamino)-2-deoxy-beta-D-glucopyranoside. The compounds were generated simultaneously from methyl 2-(acetylmethylamino)-2-deoxy-beta-D-glucopyranoside by sequential partial methylation and benzoylation and isolated in pure form by high-performance liquid chromatography (HPLC). The desired acetates were obtained by debenzoylation and acetylation of the pure isomers. Reported herein are the 1H NMR spectra of the benzoates and the electron-ionization mass spectra of the acetates and the tri-O-methyl derivative. Also reported for the acetates and the tri-O-methyl derivative are their linear temperature-programmed gas-liquid chromatography (GLC) retention indices on three different capillary columns.