Synthesis of regioselectively substituted cellulose derivatives and applications in chiral chromatography

Various cellulose-2,3-bis-arylcarbamate-6-O-arylesters and cellulose-2,3-bis-arylester-6-O-arylcarbamates, designed to test the possible combined effects of the known tris-arylcarbamate and tris-arylester classes, were synthesized with high regioselectivity at O-C(6), and their use as CSP s in liquid chromatography for enantiomeric separations was investigated. The separations obtained with the synthesized CSP s were compared to the separations achieved on a self-packed reference column, consisting of cellulose-tris-(3,5-dimethylphenyl-carbamate) as CSP standard. Among the synthesized, regioselectively substituted cellulose derivatives, 2,3-bis-O-(3,5-dimethylphenylcarbamate)-6-O-benzoate-cellulose and 2,3-bis-O-(benzoate)-6-O-(3,5-dichlorophenylcarbamate)-cellulose gave the best CSP s for the separation of the test racemates. CSP s from regioselectively substituted cellulose derivatives seem to exhibit higher selectivities than cellulose-tris-(3,5-dimethylphenylcarbamate) for certain classes of racemic compounds. Chirality 10:294–306, 1998. © 1998 Wiley-Liss, Inc.     

Chiral discrimination with regioselectively substituted cellulose esters as chiral stationary phases

Four kinds of cellulose derivatives, including two regioselectively substituted cellulose esters (6-O-acetyl-2,3-di-O-benzoyl cellulose and 2,3-di-O-acetyl-6-O-benzoyl cellulose), were synthesized so that the effects of their functional group distribution on their chiral discrimination ability could be examined. The degree of substitution by functional groups appeared to have a critical effect on the separation in most cases, but the type of the functional group at the C-6 position also significantly influenced chiral discrimination when a series of neutral arylalcohol derivatives were used as racemates. Copyright 2000 Wiley-Liss, Inc.     

TBAF and cellulose esters: unexpected deacylation with unexpected regioselectivity

Tetrabutylammonium fluoride has been found to catalyze the deacylation of cellulose esters. More surprisingly, the deacylation is highly regioselective. Even more remarkably, in contrast with the C-6 regioselectivity of other reactions of cellulose and its derivatives, this deacylation shows substantial selectivity for the removal of the acyl groups from the esters of the secondary alcohols at C-2 and C-3, affording cellulose-6-O-esters with high regioselectivity by a simple one-step process employing no protective groups.     

Synthesis of cellulose adipate derivatives

Cellulose esters containing adipates and other ester groups are synthesized by the reaction of commercially available cellulose esters in solution with the benzyl monoester of adipoyl chloride. The products, cellulose adipate esters in which the distal end of the adipate moiety is a benzyl ester, were easily converted to cellulose adipate derivatives by Pd-catalyzed hydrogenation. These cellulose adipate derivatives are promising biopolymers for drug delivery and other applications in which water-dispersion or swelling are desired.     

Facile synthesis of novel mutual derivatives of nucleosides and pyrimidines by regioselectively chemo-enzymatic protocol

We established a facile regioselectively chemo-enzymatic synthesis procedure for the preparation of mutual derivatives of nucleosides and pyrimidines by sequential Markovnikov addition and acylation. Firstly, pyrimidine derivatives containing vinyl ester group were synthesized from pyrimidines and divinyl esters through Markovnikov addition catalyzed by K₂CO₃ in DMSO at 80 °C, and the yields were ranged from 50% to 87%. Then regioselective acylation of ribavirin and cytarabine with pyrimidine vinyl ester was catalyzed by CAL-B (immobilized lipase from Candida antarctica) in anhydrous acetone. Reaction conditions of enzymatic acylation including enzyme resource and solvents were optimized. A series of mutual derivatives of nucleosides and pyrimidines were synthesized successfully and characterized with NMR, IR, and HRMS. This chemo-enzymatic protocol involving sequential Markovnikov addition and acylation provided a novel way of synthesizing complicated functional compounds regioselectively which was hard to be achieved either by chemical or by enzymatic methods.     

N-Acetyl-D and L-esters of 5′-AMP hydrolyze at different rates

Studies of the properties of aminoacyl derivatives of 5′-AMP are aimed at understanding the origin of the process of protein synthesis. Aminoacyl (2′,3′) esters of 5′-AMP can serve as models of the 3′-terminus of aminoacyl tRNA. We report here on the relative rates of hydrolysis of AC -D - and L -Phe AMP esters as a function of pH. At all pHs above 3, the rate constant of hydrolysis of the AC -L -Phe ester is 1.7 to 2.1 times that of AC -D -Phe ester. The D -isomer seems partially protected from hydrolysis by a stronger association with the adenine ring of the 5′-AMP. © 1993 Wiley-Liss, Inc.     

Polymers of malic acid and 3-alkylmalic acid as synthetic PHAs in the design of biocompatible hydrolyzable devices.

Poly(beta-malic acid) and poly(beta-3-alkylmalic acid) derivatives, as synthetic polyhydroxyalkanoates (PHAs), present several advantages as macromolecular materials for temporary biomedical applications. Indeed, such polymers, which can be synthesized through different chemical and biological routes, have cleavable ester bonds in their backbone for hydrolytic degradation, stereogenic centres in the monomers units for controlling the macromolecular structure. bioassimilable or non-toxic repeating units and lateral chemical functions which can be adapted to specific requirements. The strategy for building such complex architectures, with one or several specific pendant groups, is based on the anionic ring-opening polymerization or copolymerization of the large family of malolactonic and 3-alkylmalolactonic acid esters. Because we are able to control the monomer synthesis and the polymerization step, we have been able to prepare different degradable materials for the biomedical field, such as: degradable associating networks made up by the association of random copolyesters containing a small percentage of hydrophobic moieties and beta-cyclodextrin copolymers; degradable macromolecular micelles constituted by degradable amphiphilic block copolymers of poly(beta-malic acid) as hydrophilic segments and poly(beta-alkylmalic acid alkyl esters) as hydrophobic blocks; and degradable nanoparticles made up by hydrophobic poly(beta-malic acid alkyl esters) derivatives. We have also prepared a terpolymer which exhibits growth factor-like properties in vivo. Finally, poly(beta-malic acid) has been used as an additive in the preparation of peritoneal dialysis bags.     

Immobilization and chromatographic evaluation of novel regioselectively substituted amylose‐based chiral packing materials for HPLC

The regioselectively substituted amylose derivatives bearing a 4‐tert‐butylbenzoate or 4‐chlorobenzoate group at 2‐position, and 3,5‐dichlorophenylcarbamate and a small amount of 3‐(triethoxysilyl)propylcarbamate groups at 3‐ and 6‐positions were synthesized by a two‐step process based on the esterification of 2‐position of a glucose unit. The obtained derivatives were effectively immobilized onto macroporous silica gel by intermolecular polycondensation of triethoxysilyl groups. Their chiral recognition abilities were evaluated as chiral packing materials (CPMs) for high‐performance liquid chromatography. These CPMs showed high chiral recognition as well as the conventional coated‐type CPM, and can be used with the eluents‐containing chloroform and tetrahydrofuran. With the extended use of these eluents, improvement of chiral recognition and reversed elution orders were realized. For some racemates, the immobilized CPM exhibited ability comparable or better to the commercial immobilized amylose‐ or cellulose‐based columns, Chiralpak IA, IB, and IC. Chirality, 2011. © 2011 Wiley‐Liss, Inc.     

The synthesis of diazo, halo, and sulfoxy bile acid derivatives: potential affinity labels.

Bile acid derivatives, with and without C-3 sulfate groups, and having either the diazo- or halomethylketone moieties, have been synthesized in good yield and purity. The synthetic sequence, COOH leads to COC1 leads to COCHN2 leads to COCH2X, was used with deoxycholic and cholic acids, which requires carefully controlled quench, work-up, and purification procedures, especially for the 3-sulfate esters (made from deoxycholic acid derivatives only). The pure title compounds are anticipated to be useful chemical probes (affinity labels), especially the completely water soluble sulfates, toward our studies of ileal active transport of bile salts. A new use for Sephadex LH-20 as a sulfate ester protecting group is reported. Also developed were the use of acetamide hydrochloride complex as a mild hydrochlorination reagent and a neutral desalting method for sulfate esters of deoxycholic acid derivatives.     

High-performance liquid chromatographic separation of fluorescent esters of hepoxilin enantiomers on a chiral stationary phase

Fluorescent anthryl (ADAM) derivatives of hepoxilins have been shown to possess good chromatographic properties affording good sensitivity for the high-performance liquid chromatographic analysis and detection of these compounds and related eicosanoids (12-hydroxyeicosatetraenoic acid) in biological samples. We report herein the separation of all possible stereoisomers of hepoxilins A₃ and B₃ as their methyl esters as well as their ADAM ester and acetate derivatives on a cellulose trisdimethylphenylcarbamate chiral stationary phase (Chiracel OD) in the normal-phase mode. This methodology is important to address the mechanistic route of biosynthesis of these products.     

High yielding synthesis of N-ethyl dehydroamino acids

Recently we reported the use of a sequence of alkylation and dehydration methodologies to obtain N-ethyl-α, β-dehydroamino acid derivatives. The application of this N-alkylation procedure to several methyl esters of β,β-dibromo and β-bromo, β-substituted dehydroamino acids protected with standard amine protecting groups was subsequently reported. The corresponding N-ethyl, β-bromo dehydroamino acid derivatives were obtained in fair to high yields and some were used as substrates in Suzuki cross-coupling reactions to give N-ethyl, β,β-disubstituted dehydroalanine derivatives. Herein, we further explore N-ethylation of β-halo dehydroamino acid derivatives using triethyloxonium tetrafluoroborate as alkylating agent, but substituting N,N-diisopropylethylamine for potassium tert-butoxide as auxiliary base. In these conditions, for all β-halo dehydroamino acid derivatives, reactions were complete and the N-ethylated derivative could be isolated in high yield. This method was also applied for N-ethylation of non-halogenated dehydroamino acids. Again, with all compounds the reactions were complete and the N-ethyl dehydroamino acid derivatives could be isolated in high yields. Some of these N-ethyl dehydroamino acid methyl ester derivatives were converted in high yields to their corresponding acids and coupled to an amino acid methyl ester to give N-ethyl dehydrodipeptide derivatives in good yields. Thus, this method constitutes a general procedure for high yielding synthesis of N-ethylated dehydroamino acids, which can be further applied in peptide synthesis.     

Synthesis of N-acetyl-muramyl-L-alanyl-D-glutamic-alpha-amide(MDP) or -alpha-methyl ester derivatives, bearing a lipophilic group at the C-terminal peptide end

We report the synthesis of nine lipophilic derivatives of N-acetyl-muramyl-L-alanyl-D-glutamic-alpha-amide (MDP) or -alpha-methyl ester in which the gamma-carboxyl function of the D-glutamyl residue is either esterified by a medium chain alcohol or substituted by an L-alanyl residue esterified by a medium or long chain alcohol. A new method is described which easily allows one to obtain derivatives of MDP, bearing a free or substituted amino-acyl or peptidyl residue on the gamma-carboxyl function.     

Enzyme-catalyzed synthesis of sugar-containing monomers and linear polymers

Commercially available proteases and lipases were screened for their ability to acylate regioselectively sucrose and trehalose with divinyladipic acid ester. Opticlean M375 (subtilisin from Bacillus licheniformis) was observed to form sucrose 1'-O-adipate and trehalose 6-O-adipate in anhydrous pyridine. Novozym-435 (lipase B from Candida antarctica) catalyzed the synthesis of sucrose 6, 6'-O-divinyladipate and trehalose 6, 6'-O-divinyladipate in acetone. These diesters were then employed as monomers in polycondensation reactions with various diols (aliphatic and aromatic) catalyzed by Novozym-435 in organic solvents to yield linear polyesters with M(w)'s up to 22,000 Da. Spectroscopic analysis confirmed that only the vinyl end groups of sugar esters reacted in the enzymatic polymerization with the diol, and not the internal sugar-adipate linkages. The two-step enzymatic strategy to yield sugar-based polyesters, which is the first report of its kind, results in higher molecular weights and faster reaction times than one-step enzymatic polyester synthesis.     

Substrate specificity of an α-amino acid ester hydrolase produced by Acetobacter turbidans A.T.C.C. 9325

A partially purified preparation of an alpha-amino acid ester hydrolase was obtained from Acetobacter turbidans A.T.C.C. 9325, which catalyses synthesis of 7-(d-alpha-amino-alpha-phenylacetamido)-3-cephem-3-methyl-4- carboxylic acid (cephalexin) from methyl d-alpha-aminophenylacetate and 7-amino-3-deacetoxycephalosporanic acid. The enzyme preparation catalysed both cephalosprin synthesis from 7-amino-3-deacetoxycephalosporanic acid and suitable amino acid esters (e.g. methyl d-alpha-aminophenylacetate, l-cysteine methyl ester, glycine ethyl ester, d-alanine methyl ester, methyl dl-alpha-aminoiso-butyrate, l-serine methyl ester, d-leucine methyl ester, l-methionine methyl ester) and the hydrolysis of such esters. The substrate specificity of the enzyme preparation for the hydrolysis closely paralleled the acyl-donor specificity for cephalosporin synthesis, even to the reaction rates. Only alpha-amino acid derivatives could act as acyl donors. The hydrogen atom on the alpha-carbon atom was not always required by acyl donors. The hydrolysis rate was markedly diminished by adding 7-amino-3-deacetoxycephalosporanic acid to reaction mixtures, but no effect on the total reaction rate (the hydrolysis rate plus synthesis rate) was observed with various concentrations of 7-amino-3-deacetoxycephalosporanic acid. Both the hydrolytic and the synthetic activities of the enzyme preparation were inhibited by high concentrations of some acyl donors (e.g. methyl d-alpha-aminophenylacetate, ethyl d-alpha-aminophenylacetate). The enzyme preparation hydrolysed alpha-amino acid esters much more easily than alpha-amino acid derivatives with an acid-amide bond.     

Efficient synthesis of anacardic acid analogues and their antibacterial activities

Anacardic acid derivatives exhibit a broad range of biological activities. In this report, an efficient method for the synthesis of anacardic acid derivatives was explored, and a small set of salicylic acid variants synthesised retaining a constant hydrophobic element (a naphthyl tail). The naphthyl side chain was introduced via Wittig reaction and the aldehyde installed using directed ortho-metalation reaction of the substituted o-anisic acids. The failure of ortho-metalation using unprotected carboxylic acid group compelled us to use directed ortho-metalation in which a tertiary amide was used as a strong ortho-directing group. In the initial route, tertiary amide cleavage during final step was challenging, but cleaving the tertiary amide before Wittig reaction was beneficial. The Wittig reaction with protected carboxylic group (methyl ester) resulted in side-products whereas using sodium salt resulted in higher yields. The novel compounds were screened for antibacterial activity and cytotoxicity. Although substitution on the salicylic head group enhanced antibacterial activities they also enhanced cytotoxicity.     

Synthesis and activity of polyacetylene substituted 2-hydroxy acids,esters, and amides against microbes of clinical importance

A series of novel polyacetylene substituted 2-hydroxy acids and derivatives were prepared and characterized. Alkylation of butane-2,3-diacetal (BDA) protected glycolic acid with iodoalkyl substituted polyacetylene compounds gave the corresponding diacetal protected polyacetylene substituted 2-hydroxy acids. Diacetal deprotection through acid mediated hydrolysis, transesterification or aminolysis afforded the 2-hydroxy-polyacetylenic acid, ester or amide derivatives. Twenty one of these novel compounds were tested against 10 microbes of clinical importance and several of them showed good antimicrobial activity, in particular against Pseudomonas aeruginosa.     

Easy Access to Enantiomerically Pure Nonproteinogenic Amino Acids

The protease from Bacillus licheniformis (alcalase) shows a remarkable broad substrate tolerance and high enantioselectivity against nonproteinogenic racemic amino acid derivatives. N‐acetyl protected amino acid esters of mono‐, di‐ or tri‐substituted phenyl alanines and even tert.‐leucine were hydrolyzed with high enantioselectivity. The obtained mixtures of (S)‐N‐acetyl amino acid and (R)‐N‐acetyl amino acid ester can easily be separated. The R‐ or S‐amino acids were obtained by acidic cleavage of the optically pure derivatives or the (R)‐ester was racemized by treatment with potassium t‐butylate.     

Inhibition of trypsin and urokinase by Cbz-amino(4-guanidinophenyl)methanephosphonate aromatic ester derivatives: the influence of the ester group on their biological activity

The urokinase plasminogen activator is a trypsin-like serine protease, important in tumor development. Here, we report the synthesis and biochemical evaluation of selective and potent diaryl esters of phosphonic-type inhibitors for urokinase. We have found that the substituted phenyl ester ring has a strong influence on the inhibitory activity of these compounds. This led to the most potent phosphonic inhibitor for uPA synthesized to date.     

Lipase catalyzed reaction of L-ascorbic acid with cinnamic acid esters and substituted cinnamic acids

To perform the lipase-catalyzed synthesis of L-ascorbic acid derivatives from plant-based compounds such as cinnamic and ferulic acid under mild reaction conditions, the activities of immobilized Candida ntarctica lipase with different cinnamic acid esters and substituted cinnamic acids were compared. As a result, immobilized C. ntarctica lipase was found to prefer vinyl cinnamic acid to other esters such as allyl-, ethyl-, and isobutyl cinnamic acids as well as substituted cinnamic acids such as p-coumaric acid, caffeic acid, ferulic acid, and sinapic acid. Based on these results, large-scale synthesis of 6-O-cinnamyl-L-ascorbic acid ester was performed using immobilized C. ntarctica lipase in dry organic solvent, resulting in 68% yield (493 mg) as confirmed by ¹³C-NMR.     

Neutral, acidic, and basic derivatives of anthranilamide that confer different formal charge to reducing oligosaccharides

To facilitate the use of oligosaccharides as analytical tools in biological studies, we have designed, synthesized, and conjugated to maltosaccharides a novel series of homologous small fluorescent moieties that differ in formal charge. These moieties are amide derivatives of anthranilic acid: uncharged N-(2-aminobenzoyl)glycinamide (ABGlyAmide; 2), acidic N,N-dimethyl-N(')-(2-aminobenzoyl)ethylenediamine (ABGlyDIMED; 3), and basic N-(2-aminobenzoyl)glycine (ABGly; 1). Routes for synthesis and optimal reaction conditions for glycoconjugation by conventional reductive amination are presented, as is the compatibility of these adducts with common analytical and preparative chromatographic methods, including RP-HPLC and HPAEC-PAD. These novel anthranilic acid derivatives confer both fluorescence and defined charge to oligosaccharides, and so enhance the repertoire of chromatographic and analytical methods for which anthranilic acid can be used. Furthermore, because glucosaccharides have rigid solution structure, these small fluorescent adducts with different formal charge are ideal tools for molecular sizing studies of membrane pores.