Reactions of the melatonin metabolite N1-acetyl-5-methoxykynuramine (AMK) with the tyrosine side-chain fragment, 4-ethylphenol

Abstract

The melatonin metabolite N¹-acetyl-5-methoxykynuramine (AMK) has previously been shown to interact with various free radicals. Using the ABTS cation radical [ABTS = 2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid)] as an electron abstracting reactant, which does not destroy the aromate, we found that the reactive intermediate derived from AMK strongly interacts with the benzene rings of other AMK molecules to form di- and oligomers. Since oligomerization is rather unlikely at physiological concentrations, we investigated reactions with other putative reaction partners. The incubation of tyrosine or several of its structural analogs with AMK in the presence of the ABTS cation radical led to numerous products, amongst which were compounds not detected when one of the educts was incubated with the ABTS cation radical alone. With tyrosine and most of its analogs, the number of products formed in the presence of AMK and ABTS cation radical was relatively high and included numerous oligomers. To optimize the yield of products of interest as well as their separation from other compounds, especially oligomers, we investigated the interaction with 4-ethylphenol, which represents the side chain of tyrosine lacking the carboxyl and amino residues of the amino acid, which otherwise can undergo additional reactions. A prominent product was chromatographically separated and analyzed by mass spectrometry [(+)-ESI-MS, (?)-ESI-MS, (+)-HRESI-MS], ¹H-NMR, and H,H-COSY correlations. The substance was identified as N-{3-[2′-(5″-ethyl-2″-hydroxyphenylamino)-5′-methoxyphenyl]-3-oxopropyl} acetamide. This chemically novel compound represents an adduct in which the amino nitrogen of AMK is attached to the C-2 atom of 4-ethylphenol, which corresponds to the C-3 atom in the benzene ring of tyrosine. This finding suggests that, upon interaction of AMK with an electron-abstracting radical, the kynuric intermediate may modify proteins at superficially accessible tyrosine residues. In fact, protein modification by an unidentified melatonin metabolite has been observed in an earlier study. The possibility of protein AMKylation may be of interest with regard to an eventual interference with tyrosine nitration or, more importantly, with tyrosine phosphorylation.     

Reactions of the melatonin metabolite N1-acetyl-5-methoxykynuramine (AMK) with the ABTS cation radical: identification of new oxidation products

The melatonin metabolite N1-acetyl-5-methoxykynuramine (AMK; 1), which was previously shown to be a potent radical scavenger, was oxidized using the ABTS cation radical [ABTS = 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid)]. Several new oxidation products were obtained, which were separated by repeated chromatography and characterized by spectroscopic methods such as mass spectrometry (ESI-MS and ESI-HRMS), 1H-NMR and 13C-NMR, HMBC, HSQC, H,H COSY correlations and IR spectroscopy. The main products were oligomers of 1 (3 dimers, 1 trimer and 2 tetramers). In all cases, the amino group N2 was involved in the reactions. Two of the dimers turned out to be cis (2a) and trans (2b) isomers containing an azo bond. In the other dimer (3a), the nitrogen atom N2 was attached to atom C5 of the second aromatic amine, with loss of the methoxy group. In the trimer (5), one N2 formed a bridge to C5 of unit B, as in the respective dimer, while this one of C had bridged to C6 of B. One of the tetramers (6) was composed of a trimer 5 attached to N2 of a fourth 1 molecule via an azo bond as in 2a/b. In the other tetramer (7), an additional C-C bond between rings B and C in 6 is assumed. Although oligomers of AMK may only attain low in vivo concentrations, the types of reactions observed shed light on the physiologically possible metabolism of AMK once reacted with a free radical. The displacement of a methoxy group, rarely seen in the oxidation of methoxylated biomolecules, underlines the reactivity of AMK (1). Preliminary data show that, in the presence of ABTS cation radicals, AMK (1) can interact with side chains of aromatic amino acids, a finding which may be crucial for understanding to date unidentified protein modification by a melatonin metabolite detected earlier in experiments with radioactively labeled melatonin.     

Antioxidant properties of the melatonin metabolite N1-acetyl-5-methoxykynuramine (AMK): scavenging of free radicals and prevention of protein destruction

In numerous experimental systems, the neurohormone melatonin has been shown to protect against oxidative stress, an effect which appears to be the result of a combination of different actions. In this study, we have investigated the possible contribution to radical scavenging by substituted kynuramines formed from melatonin via pyrrole ring cleavage. N1-Acetyl-5-methoxykynuramine (AMK), a metabolite deriving from melatonin by mechanisms involving free radicals, exhibits potent antioxidant properties exceeding those of its direct precursor N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) and its analog N1-acetylkynuramine (AK). Scavenging of hydroxyl radicals was demonstrated by competition with ABTS in a Fenton reaction system at pH 5 and by competition with DMSO in a hemin-catalyzed H2O2 system at pH 8. Under catalysis by hemin, oxidation of AMK was accompanied by the emission of chemiluminescence. AMK was a potent reductant of ABTS cation radicals, but, in the absence of catalysts, a poor scavenger of superoxide anions. In accordance with the latter observation, AMK was fairly stable in a pH 8 H2O2 system devoid of hemin. Contrary to AFMK, AMK was easily oxidized in a reaction mixture generating carbonate radicals. In an oxidative protein destruction assay based on peroxyl radical formation, AMK proved to be highly protective. No prooxidant properties of AMK were detected in a sensitive biological test system based on light emission by the bioluminescent dinoflagellate Lingulodinium polyedrum. AMK may contribute to the antioxidant properties of the indolic precursor melatonin.     

Antioxidant properties of the melatonin metabolite N1-acetyl-5-methoxykynuramine (AMK): scavenging of free radicals and prevention of protein destruction

Abstract

In numerous experimental systems, the neurohormone melatonin has been shown to protect against oxidative stress, an effect which appears to be the result of a combination of different actions. In this study, we have investigated the possible contribution to radical scavenging by substituted kynuramines formed from melatonin via pyrrole ring cleavage. N¹-Acetyl-5-methoxykynuramine (AMK), a metabolite deriving from melatonin by mechanisms involving free radicals, exhibits potent antioxidant properties exceeding those of its direct precursor N¹-acetyl-N²-formyl-5-methoxykynuramine (AFMK) and its analog N¹-acetylkynuramine (AK). Scavenging of hydroxyl radicals was demonstrated by competition with ABTS in a Fenton reaction system at pH 5 and by competition with DMSO in a hemin-catalyzed H₂O₂ system at pH 8. Under catalysis by hemin, oxidation of AMK was accompanied by the emission of chemiluminescence. AMK was a potent reductant of ABTS cation radicals, but, in the absence of catalysts, a poor scavenger of superoxide anions. In accordance with the latter observation, AMK was fairly stable in a pH 8 H₂O₂ system devoid of hemin. Contrary to AFMK, AMK was easily oxidized in a reaction mixture generating carbonate radicals. In an oxidative protein destruction assay based on peroxyl radical formation, AMK proved to be highly protective. No prooxidant properties of AMK were detected in a sensitive biological test system based on light emission by the bioluminescent dinoflagellate Lingulodinium polyedrum. AMK may contribute to the antioxidant properties of the indolic precursor melatonin.     

Melatonin and structurally-related,endogenous indoles act as potent electron donors and radical scavengers in vitro

Summary

The radical scavenging properties of melatonin, structurally-related indoles and known antioxidants were investigated in kinetic competition studies using the specific radical trapping reagent 2,2′-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS). In the presence of highly reactive radicals, ABTS is oxidized to the stable thiazoline cation radical, ABTS*⁺ which, due to its intense green color, can be measured photometrically at 420 nm absorbance. The indoles melatonin, 5-methoxytryptophol, 5-methoxyindole acetic acid and 5-methoxytryptamine as well as the phenolic and thiolic antioxidants ascorbic acid, Trolox, and glutathione inhibited ABTS cation radical formation and catalyzed ABTS radical cation reduction. Melatonin was the most potent radical scavenger and electron donor when compared with the methoxylated indole analogs and the other antioxidants tested. Melatonin, the methoxylated indole analogs and the other antioxidants tested acted as potent electron donors which scavenged initiating and propagating radicals and repaired oxidative damage due to electrophile intermediates.     

Bicarbonate enhances the hydroxylation, nitration, and peroxidation reactions catalyzed by copper, zinc superoxide dismutase. Intermediacy of carbonate anion radical

The effect of bicarbonate anion (HCO(3)(-)) on the peroxidase activity of copper, zinc superoxide dismutase (SOD1) was investigated using three structurally different probes: 5, 5'-dimethyl-1-pyrroline N-oxide (DMPO), tyrosine, and 2, 2'-azino-bis-[3-ethylbenzothiazoline]-6-sulfonic acid (ABTS). Results indicate that HCO(3)(-) enhanced SOD/H(2)O(2)-dependent (i) hydroxylation of DMPO to DMPO-OH as measured by electron spin resonance, (ii) oxidation and nitration of tyrosine to dityrosine, nitrotyrosine, and nitrodityrosine as measured by high pressure liquid chromatography, and (iii) oxidation of ABTS to the ABTS cation radical as measured by UV-visible spectroscopy. Using oxygen-17-labeled water, it was determined that the oxygen atom present in the DMPO-OH adduct originated from H(2)O and not from H(2)O(2). This result proves that neither free hydroxyl radical nor enzyme-bound hydroxyl radical was involved in the hydroxylation of DMPO. We postulate that HCO(3)(-) enhances SOD1 peroxidase activity via formation of a putative carbonate radical anion. This new and different perspective on HCO(3)(-)-mediated oxidative reactions of SOD1 may help us understand the free radical mechanism of SOD1 and related mutants linked to amyotrophic lateral sclerosis.     

Hispidin analogs from the mushroom Inonotus xeranticus and their free radical scavenging activity

Three new free radical scavengers were isolated from the methanolic extract of the fruiting bodies of Inonotus xeranticus (Hymenochaetaceae), along with the known compound davallialactone. Their structures were established as hispidin analogs by extensive NMR spectral data. Compounds 3 and 4 displayed significant scavenging activity against the superoxide radical anion, ABTS radical cation, and DPPH radical, while 1 and 2 exhibited potent antioxidative activity only against ABTS radical cation.     

Studies on Radiation Chemistry of Halogenophenols in Aqueous Solutions

Among the γ-radiolysis products of p-bromophenol in an aqueous solution, four new oligomers were obtained. By chemical and physical techniques their structures were elucidated as ortho-, meta- and para-terphenyl in which C-4, C-2′ and C-4″ are substituted by hydroxyl groups and C-5′ by a bromine atom and as 5-bromo-2,4′,4″-trihydroxy-m-terphenyl, respectively. These oligomers may be formed by the arylation of p-bromophenol or the dimeric product with an aryl radical intermediate resulted from debromination of p-bromophenol by some radiolysis product(s) of water.     

Structural effects on the reactivity 1,4-dihydropyridines with alkylperoxyl radicals and ABTS radical cation

A series of eight commercial C-4 substituted 1,4-dihydropyridines and other synthesized related compounds were tested for direct potential scavenger effect towards alkylperoxyl radicals and ABTS radical cation in aqueous Britton-Robinson buffer pH7.4. A direct quenching radical species was established. The tested 1,4-dihydropyridines were 8.3-fold more reactive towards alkylperoxyl radicals than ABTS cation radical, expressed by their corresponding kinetic rate constants. Furthermore, NPD a photolyte of nifedipine and the C-4 unsubstituted 1,4-DHP were the most reactive derivatives towards alkylperoxyl radicals. The pyridine derivative was confirmed by GC/MS technique as the final product of reaction. In consequence, the reduction of alkylperoxyl and ABTS radicals by 1,4-dihydropyridines involved an electron transfer process. Also, the participation of the hydrogen of the 1-position appears as relevant on the reactivity. Results of reactivity were compared with Trolox.     

Endogenous and dietary indoles: a class of antioxidants and radical scavengers in the ABTS assay

Indoles are very common in the body and diet and participate in many biochemical processes. A total of twenty-nine indoles and analogs were examined for their properties as antioxidants and radical scavengers against 2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) ABTS*+ radical cation. With only a few exceptions, indoles reacted nonspecifically and quenched this radical at physiological pH affording ABTS. Indoleamines like tryptamine, serotonin and methoxytryptamine, neurohormones (melatonin), phytohormones (indoleacetic acid and indolepropionic acid), indoleamino acids like L-tryptophan and derivatives (N-acetyltryptophan, L-abrine, tryptophan ethyl ester), indolealcohols (tryptophol and indole-3-carbinol), short peptides containing tryptophan, and tetrahydro-beta-carboline (pyridoindole) alkaloids like the pineal gland compound pinoline, acted as radical scavengers and antioxidants in an ABTS assay-measuring total antioxidant activity. Their trolox equivalent antioxidant capacity (TEAC) values ranged from 0.66 to 3.9 mM, usually higher than that for Trolox and ascorbic acid (1 mM). The highest antioxidant values were determined for melatonin, 5-hydroxytryptophan, trp-trp and 5-methoxytryptamine. Active indole compounds were consumed during the reaction with ABTS*+ and some tetrahydropyrido indoles (e.g. harmaline and 1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid ethyl ester) afforded the corresponding fully aromatic beta-carbolines (pyridoindoles), that did not scavenge ABTS*+. Radical scavenger activity of indoles against ABTS*+ was higher at physiological pH than at low pH. These results point out to structural compounds with an indole moiety as a class of radical scavengers and antioxidants. This activity could be of biological significance given the physiological concentrations and body distribution of some indoles.     

Reactivity of horseradish peroxidase compound II toward substrates: kinetic evidence for a two-step mechanism

Transient kinetic analysis of biphasic, single turnover data for the reaction of 2,2'-azino-bis[3-ethylbenzthiazoline-6-sulfonic acid] (ABTS) with horseradish peroxidase (HRPC) compound II demonstrated preequilibrium binding of ABTS (k(+5) = 7.82 x 10(4) M(-)(1) s(-)(1)) prior to rate-limiting electron transfer (k(+6) = 42.1 s(-)(1)). These data were obtained using a stopped-flow method, which included ascorbate in the reaction medium to maintain a low steady-state concentration of ABTS (pseudo-first-order conditions) and to minimize absorbance changes in the Soret region due to the accumulation of ABTS cation radicals. A steady-state kinetic analysis of the reaction confirmed that the reduction of HRPC compound II by this substrate is rate-limiting in the complete peroxidase cycle. The reaction of HRPC with o-diphenols has been investigated using a chronometric method that also included ascorbate in the assay medium to minimize the effects of nonenzymic reactions involving phenol-derived radical products. This enabled the initial rates of o-diphenol oxidation at different hydrogen peroxide and o-diphenol concentrations to be determined from the lag period induced by the presence of ascorbate. The kinetic analysis resolved the reaction of HRPC compound II with o-diphenols into two steps, initial formation of an enzyme-substrate complex followed by electron transfer from the substrate to the heme. With o-diphenols that are rapidly oxidized, the heterolytic cleavage of the O-O bond of the heme-bound hydrogen peroxide (k(+2) = 2.17 x 10(3) s(-)(1)) is rate-limiting. The size and hydrophobicity of the o-diphenol substrates are correlated with their rate of binding to HRPC, while the electron density at the C-4 hydroxyl group predominantly influences the rate of electron transfer to the heme.     

Hydrogen atom abstraction by Cu(II)- and Zn(II)-phenoxyl radical complexes, models for the active form of galactose oxidase

The Cu(II) and Zn(II) complexes of phenoxyl radical species [M(II)(L1*)(NO3)]+ (M=Cu or Zn, L1H: 2-methylthio-4-tert-butyl-6-[[bis[2-(2-pyridyl)ethyl]amino]methyl]phenol ) and [M(II)(L2*)(NO3)]+ (M=Cu or Zn, L2H: 2,4-di-tert-butyl-6-[[bis[2-(2-pyridyl)ethyl]amino]methyl]phenol) are prepared as model complexes of the active form of galactose oxidase (GAO). Hydrogen atom abstraction of 1,4-cyclohexadiene and tert-butyl substituted phenols by the GAO model complexes proceeds very efficiently to give benzene and the corresponding phenoxyl radical or its C-C coupling dimer as the oxidation products, respectively. Kinetic analyses on the oxidation reactions have shown that the hydrogen atom abstraction of the phenol substrates is significantly enhanced by the coordinative interaction of the OH group to the metal ion center of the complex, providing valuable insight into the enzymatic mechanism of the alcohol oxidation. Details of the substrate-activation process have been discussed based on the activation parameters (deltaH* and deltaS*) of the reactions.     

Synthesis of 5,10-seco analogs of testosterone

A number of 5,10-seco analogs of testosterone has been synthesized starting from products of the radical oxidation of 3beta,17beta-diacetoxy-5alpha-androstan-5alpha-ol. The obtained compounds possess a flexible 10-membered ring with substituents (O, -OH) at C-3 and C-5. Similar derivatives with an (E)- and (Z)-Delta(1(10))-double bond have been prepared also. X-ray analysis and a combination of NMR experiments have been used for their structure elucidation and conformation analysis.     

Radical intermediates in the catalytic oxidation of hydrocarbons by bacterial and human cytochrome P450 enzymes

Cytochromes P450cam and P450BM3 oxidize alpha- and beta-thujone into multiple products, including 7-hydroxy-alpha-(or beta-)thujone, 7,8-dehydro-alpha-(or beta-)thujone, 4-hydroxy-alpha-(or beta-)thujone, 2-hydroxy-alpha-(or beta-)thujone, 5-hydroxy-5-isopropyl-2-methyl-2-cyclohexen-1-one, 4,10-dehydrothujone, and carvacrol. Quantitative analysis of the 4-hydroxylated isomers and the ring-opened product indicates that the hydroxylation proceeds via a radical mechanism with a radical recombination rate ranging from 0.7 +/- 0.3 x 10(10) s(-1) to 12.5 +/- 3 x 10(10) s(-1) for the trapping of the carbon radical by the iron-bound hydroxyl radical equivalent. 7-[2H]-alpha-Thujone has been synthesized and used to amplify C-4 hydroxylation in situations where uninformative C-7 hydroxylation is the dominant reaction. The involvement of a carbon radical intermediate is confirmed by the observation of inversion of stereochemistry of the methyl-substituted C-4 carbon during the hydroxylation. With an L244A mutation that slightly increases the P450(cam) active-site volume, this inversion is observed in up to 40% of the C-4 hydroxylated products. The oxidation of alpha-thujone by human CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 occurs with up to 80% C-4 methyl inversion, in agreement with a dominant radical hydroxylation mechanism. Three minor desaturation products are produced, with at least one of them via a cationic pathway. The cation involved is proposed to form by electron abstraction from a radical intermediate. The absence of a solvent deuterium isotope effect on product distribution in the P450cam reaction precludes a significant role for the P450 ferric hydroperoxide intermediate in substrate hydroxylation. The results indicate that carbon hydroxylation is catalyzed exclusively by a P450 ferryl species via radical intermediates whose detailed properties are substrate- and enzyme-dependent.     

Novel spironolactone-analogs as impurities in spironolactone

Six novel spironolactone-analogs steroids (3-8) were isolated from spironolactone by using various chromatographic methods. Their structures were elucidated by spectrometric analysis. Two of the analogs (3 and 7) were confirmed by X-ray crystallography. The A-ring of compounds 3-7 is opened at C-2C-3 bond, and compound 7 is an organic polysulfide, which has a rare, nine-membered ring with a five sulfur atom bridge.     

Oxidation of aromatic alcohols by laccase from Trametes versicolor mediated by the 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) cation radical and dication

Oxidation of aromatic alcohols, such as non-phenolic lignin model compounds, by oxidised species of 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) has been investigated. The cation radical and dication formed from ABTS were both capable of oxidising aromatic alcohols to aldehydes. The reactions terminated at the level of the aldehyde and no acids were formed. The cation radical and dication worked in a cycle as an electron-transfer compound between an oxidant and alcohol. In addition to the oxidation of the primary benzyl-hydroxyl group, an oxidation of the secondary α-hydroxyl group to the ketone by the dication was possible. All distinguishing features of these reactions corresponded to the results of the oxidation performed by the laccase of Trametes versicolor in the presence of ABTS. The decomposition products from the dication alone and ABTS with laccase confirmed the supposition that the dication was involved in the laccase mediator system. A reaction mechanism based on deprotonation of the alcohol cation radical was predicted to play a key role in the irreversible followup reaction and to be the driving force of the process. Received: 8 June 1998 / Received revision: 23 September 1998 / Accepted: 2 October 1998     

Formation of Phenolic and Indolic Compounds by Anaerobic Bacteria in the Human Large Intestine

Abstract Batch culture incubations were used to investigate the effects of pH (6.8 or 5.5) and carbohydrate (starch) availability on dissimilatory aromatic amino acid metabolism in human fecal bacteria. During growth on peptide mixtures, tyrosine and phenylalanine fermentations occurred optimally at pH 6.8, while individual metabolic reactions were inhibited by up to 80% in the presence of 10 g l⁻¹ starch. Tryptophan metabolites were not detected in these experiments. When free amino acids replaced peptides, phenol production was increased during carbohydrate fermentation, although formation of p-cresol, another tyrosine metabolite was strongly inhibited. Phenylpropionate, which is produced from phenylalanine, was unaffected by starch. Tryptophan was fermented in these studies, although indole production was reduced in the starch fermentors. The importance of different fermentation substrates (casein, peptide mixtures, free amino acids) on aromatic amino acid metabolism was investigated in incubations of material taken from the proximal bowel. The phenylalanine metabolites, phenylacetate and phenylpropionate, were the principal phenolic compounds formed from all three substrates. Phenol was the major tyrosine metabolite produced in casein and peptide fermentations, while hydroxyphenylpropionate was a more important tyrosine product from free amino acids. Indole was the sole product of tryptophan metabolism, but was formed only from the free amino acid. Bacterial metabolism of individual phenolic and indolic compounds was also investigated. Phenol, p-cresol, phenylacetate, phenylpropionate, 4-ethylphenol, indole, indoleacetate, and indolepropionate were not metabolized by colonic bacteria. However, hydroxyphenylacetate was hydrolyzed to p-cresol, while hydroxyphenylpropionate was transformed into phenylpropionate. Indolepyruvate was either converted to indoleacetate or metabolized into indole. Indolepropionate, and to a lesser degree indoleacetate were produced from indolelactate. These data show that human colonic anaerobes are able to extensively degrade either free or peptide-bound aromatic amino acids, with the concomitant formation of toxic metabolic products. These processes are controlled to a significant degree by environmental factors such as pH and carbohydrate availability, and this ultimately influences the types and amounts of fermentation products that can be formed in different regions of the large bowel. Received: 25 January 1996; Accepted: 8 May 1996     

A simple, post-additional antioxidant capacity assay using adenosine triphosphate-stabilized 2,2'-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) radical cation in a G-quadruplex DNAzyme catalyzed ABTS-H2O2 system

The scavenging of 2,2'-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) radical cation (ABTS(+)) by antioxidants has been widely used in antioxidant capacity assay. Because of ABTS(+) disproportionation, however, this radical cannot be prepared on a large scale and stored long-term, making it unsuitable for high-throughput detection and screening of antioxidants. We developed a modified "post-additional" antioxidant capacity assay. This method possessed two remarkable features: First, instead of natural peroxidases, an artificial enzyme, G-quadruplex DNAzyme, was used for the preparation of ABTS(+), thus greatly reducing the cost of the assay, and eliminating the strict demand for the storage of enzymes. Second, an ABTS(+) stabilizer, adenosine triphosphate (ATP), was used. In the presence of ATP, the disproportionation of ABTS(+) was effectively inhibited, and the lifetime of this radical cation was prolonged about 6-fold (12 days versus 2 days), making the large-scale preparation of ABTS(+) possible. Utilizing this method, the antioxidant capacities of individual antioxidants and real samples can be quantified and compared easily. In addition, this method can be developed as a high-throughput screening method for antioxidants. The screening results could even be judged by the naked eye, eliminating the need for expensive instruments.     

Specificity of acceptor binding to Leuconostoc mesenteroides B-512F dextransucrase: binding and acceptor-product structure of alpha-methyl-D-glucopyranoside analogs modified at C-2, C-3, and C-4 by inversion of the hydroxyl and by replacement of the hydroxyl with hydrogen

The specificity of acceptor binding to the active site of dextransucrase was studied by using alpha-methyl-D-glucopyranoside analogs modified at C-2, C-3, and C-4 positions by (a) inversion of the hydroxyl group and (b) replacement of the hydroxyl group with hydrogen. 2-Deoxy-alpha-methyl-D-glucopyranoside was synthesized from 2-deoxyglucose; 3- and 4-deoxy-alpha-methyl-D-glucopyranosides were synthesized from alpha-methyl-D-glucopyranoside; and alpha-methyl-D-allopyranoside was synthesized from D-glucose. The analogs were incubated with [14C]sucrose and dextransucrase, and the products were separated by thin-layer chromatography and quantitated by liquid scintillation spectrometry. Structures of the acceptor products were determined by methylation analyses and optical rotation. The relative effectiveness of the acceptor analogs in decreasing order were 2-deoxy, 2-inverted, 3-deoxy, 3-inverted, 4-inverted, and 4-deoxy. The enzyme transfers D-glucopyranose to the C-6 hydroxyl of analogs modified at C-2 and C-3, to the C-4 hydroxyl of 4-inverted, and to the C-3 hydroxyl of 4-deoxy analogs of alpha-methyl-D-glucopyranoside. The data indicate that the hydroxyl group at C-2 is not as important for acceptor binding as the hydroxyl groups at C-3 and C-4. The hydroxyl group at C-4 is particularly important as it determines the binding orientation of the alpha-methyl-D-glucopyranoside ring.