Facile synthesis of 6-amino-6-deoxycellulose

6-Amino-6-deoxycellulose (4) was synthesized from cellulose by three reaction steps, namely bromination at C-6, displacement of bromine by azide ion, and reduction of the azide group to amino group, in 67% overall yield. The 13C NMR spectrum of compound 4 supports the expected structure for 6-amino-6-deoxycellulose. The degree of substitution of compound 4 was 0.96.     

A DFT study of the Al2Cl6-catalyzed Friedel–Crafts acylation of phenyl aromatic compounds

The reaction pathways of several Friedel–Crafts acylations involving phenyl aromatic compounds were studied using density functional theory. The reactions were related to the Friedel–Crafts polycondensation of polyaryletherketones. In particular, the acylation of benzene with benzoyl chloride to form benzophenone and variations on this reaction were investigated. The acylation of benzene by one molecule of terephthaloyl chloride or isophthaloyl chloride as well as acylations at the m-, o-, and p-positions of diphenyl ether with one molecule of benzoyl chloride were studied. Adding an additional acyl chloride group to the electrophile appeared to have little influence on the reaction pathway, although the activation energy for the C–C bond-forming steps that occurred when isophthaloyl choride was used was different to the activation energy observed when terephthaloyl chloride was used. Upon changing the nucleophile to diphenyl ether, the reactivity changed according to the trend predicted on based on the o-, p-directing effects of the ether group. The deprotonation step that restored aromaticity varied widely according to the reaction. The rate-determining step in all of the studied reactions was the formation of the acylium ion, followed in importance by either the formation of the Wheland intermediate or the abstraction of hydrogen, depending on the reactivity of the nucleophile.     

Structural determinants influencing the reaction of cysteine-containing peptides with palmitoyl-coenzyme A and other thioesters

Non-enzymatic thioesterification of specific cysteinyl peptides with fatty acyl-CoA has been previously demonstrated in both liposomes and aqueous medium. To identify the molecular basis for the differential reactivity of polypeptides in aqueous solutions, 26 synthetic cysteinyl peptides encompassing the palmitoylation sites of well known proteins (protein zero, proteolipid protein, beta-adrenergic receptor, p21(K-ras), transferrin receptor, CD-4 and SNAP-25) and six small thiol compounds were incubated separately with [3H]palmitoyl-CoA, [14C]acetyl-CoA and p-nitrophenyl thioacetate (NPTA). For each peptide, both the observed reaction rate constant at pH 7.5 and the pH-independent rate constant (k(2)) were calculated, and reactivity of the attacking sulfhydryl group was characterized using the Br?nsted equation (log k(2)=beta(nuc) pK(a)+C). In general, peptides bearing basic and aromatic amino acid residues showed the lowest thiol pK(a)s, and consequently displayed the highest acylation rates. Reaction with palmitoyl-CoA was complicated to analyze because of the variable partition of peptides in the acyl chain donor/detergent micelles. In contrast, a linear Br?nsted relationship was found for the reaction of the peptides with the water-soluble acetyl-CoA (beta(nuc)=0.59). A similar beta(nuc) value was obtained with the neutral NPTA, indicating that electronic effects other than those responsible for the acid-base properties of the thiol are less important. Thus, the concentration of the thiolate anion appears to be the major factor influencing the rate of the nucleophilic substitution reaction. These findings and the fact that the acylation sites in most proteins are surrounded by basic amino acids may partially explain the specificity of non-enzymatic palmitoylation regarding the acceptor sequences.     

The kinetics and stereochemistry of base hydrolysis of the seven isomers of [Co(dien)(ampy)Cl] and [Co(dien)(ibn)Cl]

The kinetics and stereochemistry for the base catalysed substitution reactions of all seven isomers (4 mer and 3 fac) of both [Co(dien)(ibn)Cl]²⁺ and [Co(dien)(ampy)Cl]²⁺ have been studied in detail, for water and azide ion as entering groups. The stereochemistry for the azide ion anation of some of the [Co(dien)(diamine)OH]²⁺ species have also been investigated. The mer isomers are of comparable reactivity and amongst the fastest reacting pentaaminechlorocobalt(III) complexes known. They are also much faster to hydrolyse than the fac species. In both the ibn and ampy systems, a common product stereochemistry is observed for the four reactant mer isomers (the product is a mixture of all four mer configurations), for both azide ion and water as nucleophiles, but not for the three fac reactants (H₂O as nucleophile). The kinetic and equilibrium distributions are quite different. For the mer isomer reactions, a common trigonal bipyramidal five-coordinate intermediate deprotonated at the sec-NH of the dien is overwhelmingly implicated. The substitution mechanisms are argued in detail. Other data reported include isomerisation rates and equilibrium distributions for some mer-hydroxo and a mer-aqua complex of exceptional reactivity, equilibrium distributions for the mer-phosphato complexes in the ampy system under different pH conditions, the crystal structure for the isolated m1-[Co(dien)(ampy)OP(OH)₃]Cl₃ · 2H₂O species, and a rationale for its predominance at neutral pH based on internal H-bonding.     

Differences in the chemical and catalytic characteristics of two crystallographically ''identical'' enzyme catalytic sites. Characterization of actinidin and papain by a combination of pH-dependent substrate catalysis kinetics and reactivity probe studies targeted on the catalytic-site thiol group and its immediate microenvironment.

The characteristics of actinidin (EC 3.4.22.14) and papain (EC 3.4.22.2), two cysteine proteinases whose catalytic-site regions appear to superimpose to a degree that approaches atomic co-ordinate accuracy of both crystal structures, were evaluated by determining (a) the pH-dependence in acid media of the acylation process of the catalytic act (k+2/Ks) using N alpha-benzoyl-L-arginine p-nitroanilide (L-Bz-Arg-Nan) as substrate and (b) the sensitivity of the reactivity of the catalytic-site thiol group and its pH-dependence to structural change in small, thiol-specific, two-protonic-state reactivity probes (2,2'-dipyridyl disulphide and methyl 2-pyridyl disulphide) where enzyme-probe contacts should be restricted to areas close to the catalytic site. Distortion of the catalytic sites of the two enzymes at pH less than 4 was evaluated over time-scales appropriate for both stopped-flow reactivity probe kinetics (less than or equal to 1-2 s) and steady-state substrate catalysis kinetics (3-5 min) by using the 2,2'-dipyridyl disulphide monocation as a titrant for non-distorted catalytic sites. This permitted a lower pH limit to be defined for valid kinetic analysis of both types. The behaviour of the enzymes at pH less than 4 requires a kinetic model in which the apparently biomolecular reaction of enzyme with probe reagent is separated from the process leading to loss of conformational integrity by a potentially reversible step. The acylation of actinidin with L-Bz-Arg-Nan in acidic media occurs in two protonic states, one produced by raising the pH across pKa less than 4 which probably characterizes the formation of -S-/-ImH+ ion pair (pKa approx. 3) and the other, of higher reactivity, produced by raising the pH across pKa 5.5, which may characterize rearrangement of catalytic-site geometry. The pH-dependence of the acylation of papain by L-Bz-Arg-Nan is quite different and is not influenced by protonic dissociation with pKa values in the range 5-6. The earlier conclusion that the acylation of papain depends on two protonic dissociations each with pKa approx. 4 was confirmed. This argument is now more firmly based because titration with 2,2'-dipyridyl disulphide permits the loss of conformational integrity to be taken into account in the analysis of the kinetic data at very low pH. Methyl 2-pyridyl disulphide was synthesized by reaction of pyridine-2-thione with methyl methanethiolsulphonate and its pKa at I = 0.1 was determined by spectral analysis at 307 nm to be 2.8.(ABSTRACT TRUNCATED AT 400 WORDS)     

Human glutathione transferase A1-1 demonstrates both half-of-the-sites and all-of-the-sites reactivity

A study of the kinetics of a heterodimeric variant of glutathione transferase (GST) A1-1 has led to the conclusion that, although the wild-type enzyme displays all-of-the-sites reactivity in nucleophilic aromatic substitution reactions, it demonstrates half-of-the-sites reactivity in addition reactions. The heterodimer, designed to be essentially catalytically inactive in one subunit due to a single point mutation (D101K), and the two parental homodimers were analyzed with seven different substrates, exemplifying three types of reactions catalyzed by glutathione transferases (nucleophilic aromatic substitution, addition, and double-bond isomerization reactions). Stopped-flow kinetic results suggested that the wild-type GST A1-1 behaved with half-of-the-sites reactivity in a nucleophilic aromatic substitution reaction, but steady-state kinetic analyses of the GST A1-D101K heterodimer revealed that this was presumably due to changes to the extinction coefficient of the enzyme-bound product. In contrast, steady-state kinetic analysis of the heterodimer with three different substrates of addition reactions provided evidence that the wild-type enzyme displayed half-of-the-sites reactivity in association with these reactions. The half-of-the-sites reactivity was shown not to be dependent on substrate size, the level of saturation of the enzyme with glutathione, or relative catalytic rate.     

Parallel solid-phase synthesis of a model library of 7alpha-alkylamide estradiol derivatives as potential estrogen receptor antagonists.

The C17-THP derivative of 7alpha-(11-azidoundecanyl)-estradiol (4) was synthesized and coupled to an aminomethyl resin via a photolabile o-nitrobenzyl linker. Reduction of the azide by the Staudinger reaction to its corresponding amine followed by acylation using four activated NFmoc protected amino acids gave a first level of diversity. Subsequent deprotection of the Fmoc followed by a second acylation with five activated carboxylic acids produced, after photocleavage, a model library of twenty antiestrogen-related 7alpha-alkylamide estradiol derivatives in acceptable overall yields and very good purities.     

Inhibition of chymotrypsin and pancreatic elastase by 4H-3,1-benzoxazin-4-ones

Eight 3,1-Benzoxazin-4-ones have been used to inactivate chymotrypsin and pancreatic elastase. Whereas 6,7-dimethoxy substitution only slightly decreased the acylation rate constant, the deacylation reaction was nearly unaffected. Bulky alkoxy groups in position 2 of the heterocyclic moiety were shown to increase enormously the acylation rate of chymotrypsin, but not that of elastase.     

Role of arginine in chemical cross-linking with N-hydroxysuccinimide esters

In order to clarify whether arginine has a promoting effect on the acylation of hydroxyl groups of serine, threonine, or tyrosine by homobifunctional cross-linking agents in aqueous solution, we carried out systematic experiments with model peptides, comparing relative reaction yields with covalently protected and unprotected arginines by MALDI-MS. The guanidinium group could be demonstrated to contribute to the reactivity of hydroxyl groups toward N-hydroxysuccinimide esters and catalyze the nucleophilic substitution, probably via hydrogen bonds.     

Disruption of nucleobase stacking to restore reactivity

Strong intermolecular interaction can prevent an organic molecule from dissolving in a reaction solution, thereby jeopardizing its reactivity and usefulness. Nucleobases and nucleosides (especially many purines and their derivatives) are notoriously difficult to dissolve in most organic solvents, generally attributed to their strong intermolecular interactions caused by the aromaticity, polarity and hydrogen-bonding. Guided by our computational study and prediction, to address this challenge, we have found that by doping the reaction solution with toluene (an inert aromatic compound), the added solvent molecules are capable of generating the stacking interaction with the solute molecules (e.g., purine derivatives) and disrupting the intermolecular stacking of the solute molecules. Thus, this inert doping can successfully address the insoluble challenge, dissolve the poorly soluble reactants (such as purine phosphoramidites), and restore the amidite reactivity for oligonucleotide synthesis. Our research has offered a simple strategy to efficiently synthesize labile oligonucleotides, via disrupting stacking interaction with inert aromatic molecules.     

Inversion of chiral α-methylbenzylamine

More effective use of optical resolution processes can be obtained by increasing the overall yields after development of methods for inversion of the chiral centre of the unwanted isomer. The configuration of some optically active amines can be inverted in a three-step synthesis via the N,N-ditosylimides and a subsequent nucleophilic substitution by the azide ion. The azide product is reduced by hydrogenolysis. Low stereoselectivity caused by racemization to some extent was at first observed for the inversion of the benzylic substrate, (S)-α-methylbenzylamine ( 5a ). However, modified reaction conditions allowed increased stereoselectivity, a more rapid and almost complete inversion of this substate as well. © 1994 Wiley-Liss, Inc.     

Evidence that binding to the S2-subsite of papain may be coupled with catalytically relevant structural change involving the cysteine-25–histidine-159 diad. Kinetics of the reaction of papain with a two-protonic-state reactivity probe containing a hydrophobic side chain

A method is proposed by which site-specific reactivity probes that exhibit different reactivities in two ionization states can be used to detect association-activation phenomena that involve repositioning of acid/base groups in enzyme active centres. The pH-dependences of the apparent second-order rate constants (k) for the reactions of the thiol group of papain (EC 3.4.22.2) with a series of two-protonic-state reactivity probes are compared. The short-chain probes, 2,2'-dipyridyl disulphide and n-propyl 2-pyridyl disulphide, react at pH6 in adsorptive complexes and/or transition states with geometries that do not permit hydrogen-bonding of the pyridyl nitrogen atom with the active-centre imidazolium ion, as evidenced by the rate minima at pH6 and the rate maxima at pH4 provided by reagent protonation. Only when the probe molecule, e.g. 4-(N-aminoethyl 2'-pyridyl disulphide)-7-nitrobenzo-2-oxa-1,3-diazole [compound(III)], contains a long hydrophobic side chain is the reaction characterized by maximal rates at about pH6, as in the acylation step of the catalytic act (at pH6, k(compound III)/k(2,2'-dipyridyl disulphide) approximately 100). It is proposed that this striking difference in profile shape may result from binding of the hydrophobic side chain of compound (III) possibly in the S(2)-subsite of papain, which promotes a change in catalytic-site geometry involving repositioning of the imidazolium ion of histidine-159 and hydrogen-bonding with the N atom of the leaving group, as has been postulated to occur in the acylation step of substate hydrolysis.     

Synthesis and Biological Activity of the Mono- and Diamino Analogues of 2′-Deoxyadenosine,Cordycepin, 9-(3-Deoxy-α-D-Threo-Pentofuranosyl)-Adenine (A Structural Component of Agrocin 84) and 9-(2-Deoxy-α-D-Threo-Pentofuranosyl)Adenine

Abstract

The mono- and diamino analogues of 9-(2-deoxy-α-D-erythro-pen-tofuranosyl)adenine la, 9-(2-deoxy-α-D-threo-pentofuranosyl)adenine 4a, 9-(3-deoxy-α-D-erythro-pentofuranosyl)adenine 2a and 9-(3-deoxy-α-D-threo-pentofuranosyl)adenine 3a were synthesized by triphenylphosphine reduction of the corresponding azido compounds. The azido group was introduced by a substitution reaction with lithium azide on mesylates or, more directly, by reaction with lithium azide, triphenylphosphine and carbon tetrabromide. Of the newly synthesized compounds, only 3′-amino-2′,3′-dideoxyadenosine proved, albeit slightly, inhibitory to murine leukemia L1210 and mammary carcinoma FM3A, and human B-lymphoblast Raji, T-lymphoblast Molt/4F and T-lymphocyte MT-4 cell proliferation in vitro (50 % inhibitory dose : 43.1-323 μM). None of the compounds inhibited human immunodeficiency virus-induced cytopathogenicity in MT-4 cells.     

Effect of ultrasound on enzymatic acylation of konjac glucomannan

A comparative study was made of enzymatic acylation of konjac glucomannan with vinyl esters under ultrasonic irradiation and shaking in organic solvent tert-butanol. Among the 13 enzymes selected, Novozym 435 exhibited the highest acylation activity towards KGM whether under ultrasonic irradiation or shaking. The application of ultrasonic irradiation instead of shaking during the acylation led to improvement in the initial reaction rate, yield and degree of substitution of the modified KGM. Appropriate ultrasound power (100 W) and water activity (0.75) were found to accelerate enzymatic reaction. The acceleration effect of ultrasound on Novozym 435-catalyzed acylation decreased with an increase in the chain length of the acyl donors from C2 to C18. Moreover, the acylation of KGM in tert-butanol was proved to be a regioselective one, with C6-OH being acylated. Compared with shaking, ultrasound did not change regioselectivity of Novozym 435 in the acylation.     

Studies on the origin of stereoselectivity in the synthesis of 1,2-trans glycofuranosyl azides

The stereoselectivity of the 1,2-trans directed, Lewis acid-catalysed azidation of peracylated furanoses was found to depend on the reactivity of the azide donor (azide nucleophilicity) and the configuration at the anomeric centre relative to the neighbouring 2-O-acyl group. Reactions of 1,2-trans glycosyl esters with highly nucleophilic azide donors, generated from SnCl4 and Me3SiN3, were stereospecific. The results are interpreted in terms of the rapid reaction of the azide species with bicyclic 1,2-acyloxonium (1,2-O-alkyliumdiyl-D-glycofuranose) ions, which were the primarily formed reactive intermediates. When using 1,2-cis glycosyl esters as starting materials the selectivity was reduced (90-94% de); the same is true with 1,2-trans counterparts if less nucleophilic Me3SiN3 in combination with Me3SiOTf catalyst was used. This occurred due to the appearance of the more reactive but less selective oxocarbenium (glycofuranoxonium) ions either as primarily formed reactive intermediates in the former case or after equilibration with acyloxonium ions in the latter case. Protected 1,2-trans beta-D-glycofuranosyl azides with ribo, xylo and 3-deoxy-erythro-pento configurations were best prepared from the corresponding glycosyl esters using 0.05 equivalents of SnCl4, i.e., under anomerization-free conditions. Azidation of methyl glycofuranosides proceeds with inferior (80-90% de) and less predictable selectivity irrespective of the starting anomeric configuration.     

Diazo- and azido-functionalized glutaraldehydes as cross-linking reagents and potential fixatives for electron microscopy

The synthesis of diazo and perfluorophenyl azide (PFPA) functionalized glutaraldehydes 7 and 13a-d as new cross-linking reagents for bioconjugation and potential fixatives for electron microscopy is reported. A key step is the generation of the 1,5-dialdehyde structures by oxidative cleavage of the corresponding cyclopentene epoxide using HIO4 in aqueous tetrahydrofuran. A model reaction between 3-substituted glutaraldehyde 14 and 6-aminohexanoic acid resulted in the formation of pyridinium ion containing products with UV spectra comparable to those observed with glutaraldehyde itself. Thus modification of glutaraldehyde in the 3-position most probably did not significantly change its reactivity with amines under chemical-fixation conditions. Fixation of red blood cells by 7 demonstrates that as a fixative, 7 is comparable to glutaraldehyde.     

Substitution reactions of a water-soluble metalloporphyrin with azide and 1,1,3,3-tetramethyl-2-thiourea

The substitution reactions of tetrakis-(4-N-methylpyridyl)porphinecobalt (III) (CoIIITMpyP) with azide and with 1,1,3,3-tetramethyl-2-thiourea (TMTU) have been studied as a function of pH at 25 degrees and an ionic strength of 0.5 M. The mechanistic pathway proposed for thiocyanate [1] and pyridine [2] is applicable to these ligands as well once allowance is made for two attacking forms of azide, N3- and HN3. A TMTU axial substituent has about the same influence on the rate of further ligand substitution as does SCN- and a much larger influence than does azide. Similar behavior between bound SCN- and bound TMTU is also shown in electron-transfer reactions with Ru(NH3)62+. Whereas both sulfur-containing ligands enhance the rate relative to the diaquo complex, the azide complex undergoes reduction an order of magnitude more slowly than does the diaquo complex.     

Enzymatic acylation of polar dipeptides: Influence of reaction media and molecular environment of functional groups

The enzymatic acylation of polar dipeptides was investigated. First, the Novozym435^®-catalyzed acylation of Lys-Ser, HCl exhibiting three potential acylable sites was carried out in organic media (2-methyl-2-butanol, oleic acid) and in an ionic liquid ([Bmim]⁺[PF₆]^?). In these reactions, the chemo-selectivity of the acylation was exclusively in favour of the N?-lysine acylation and the efficiency (substrate conversion) was demonstrated to be under control of the peptide solubility. The use of [Bmim]⁺[PF₆]^? permitted to significantly improve the dipeptide solubility, and to enhance both substrates conversion and initial rates of acylation reaction. In the three reaction media used, the O-acylated derivative of the dipeptide was never detected suggesting a weak reactivity of the serine hydroxyl group due to its molecular environment and particularly to the presence of a free carboxylic group known for its electroattractor property.Last, the acylation of a natural dipeptide (carnosine), exhibiting a very low solubility in organic solvents, was also performed. Carnosine was successfully N-acylated in 2-methyl-2-butanol, and a yield of 39% was obtained when improving the substrate solubility: a better dispersibility was obtained by application of a high pressure on the reaction medium just before starting the reaction.     

The pH dependence of tyrosine cyanuration in proteins

The pH, temperature, and reagent concentration dependences of the cyanuration of tyrosines within proteins are compared with those of the free amino acid. The results indicate that the mechanism of the cyanuration reaction is a nucleophilic aromatic substitution of the SN 2 type, where the tyrosine oxygen acts as a nucleophile and the displacement takes places at a carbon atom which is part of the six-membered ring of the s-triazene. This reaction must compete with the hydrolysis of cyanuric fluoride in aqueous media. These competing reactions generate curves for the pH dependence of the reactivity of tyrosine with cyanuric fluoride whihc vary in a predictable manner with the extent of ionization of tyrosine and the concentration of cyanuric fluoride. In the case of proteins, the dependence of the degree of tyrosine reactivity on pH and temperature within the pH range of full reactivity of the free amino acid reflects constraints imposed by the microenvironment of these groups within the three-dimensional structure of the macromolecule.     

Metabolism of chrysene, 5-methylchrysene, 6-methylchrysene and 5,6-dimethylchrysene in rat liver cytosol, in vitro, and in rat subcutaneous tissue, in vivo

The polynuclear aromatic hydrocarbon chrysene undergoes a bioalkylation substitution reaction in vitro, in rat liver cytosol preparations, and in vivo, in rat dorsal subcutaneous tissue to yield 6-methylchrysene as a metabolite. In addition, both 5-methyl- and 6-methylchrysene were found to undergo a dealkylation reaction in these tissues to yield chrysene as well as both a biooxidation reaction to yield the corresponding hydroxyalkyl substituted chrysene and a bioalkylation reaction to give a dimethyl substituted chrysene. 5-Methylchrysene enzymatically cyclized to the 4,5-methylenechrysene derivative, an analog of benzo[a]pyrene in these tissues. 5,6-Dimethylchrysene was metabolized to monomethyl chrysenes, chrysene, and the hydroxyalkyl substituted chrysenes. The results suggest that chemical or biochemical substitution of a methyl group at the center of highest biochemical reactivity may be a necessary step in the metabolic activation and carcinogenicity of these compounds and their methylene bridged metabolites.