Enzymatic Esterification of Indole-3-acetic Acid to myo-Inositol and Glucose

Incubation of mature sweet corn kernels of Zea mays in dilute solutions of ¹⁴C-labeled indole-3-acetic acid leads to the formation of ¹⁴C-labeled esters of myo-inositol, glucose, and glucans. Utilizing this knowledge it was found that an enzyme preparation from immature sweet corn kernels of Zea mays catalyzed the CoA- and ATP-dependent esterification of indole-3-acetic acid to myo-inositol and glucose. The esters formed were 2-O-(indole-3-acetyl)-myo-inositol, 1-dl-1-O-(indole-3-acetyl)-myo-inositol, di-O-(indole-3-acetyl)-myo-inositol, tri-O-(indole-3-acetyl)-myo-inositol, 2-O-(indole-3-acetyl)-d-glucopyranose, 4-O-(indole-3-acetyl)-d-glucopyranose and 6-O-(indole-3-acetyl)-d-glycopyranose. An assay system was developed for measuring esterification of ¹⁴C-labeled indole-3-acetic acid by ammonolysis of the esters followed by isolation and counting the radioactive indole-3-acetamide.     

Structure of indole-3-acetic acid myoinositol esters and pentamethyl-myoinositols

GC-MS properties of three isomeric esters of indole-3-acetic acid and myoinositol, three esters of indole-3-acectic acid and myoinositol arabinoside and three esters of indole-3-acetic acid and myoinositol galactoside are presented. MS fragmentation patterns for the four possible pentamethyl myoinositols are also shown. These data indicated that the arabinose, and galactose of the glycosides were in the pyranose form and that C-1 of the sugar was linked to the 5 hydroxyl of myoinositol. Homologies in fragmentation patterns for the esters and the glycoside esters, together with knowledge of the properties of 2-O-indole-3-acetyl-myoinositol, permitted identification of one of the arabinosides as 5-O-l-arabinopyranosyl-2-O-indole-3-acetyl-myoinositol and one of the galactosides as 5-O-d- galactopyranosyl-2-O-indole-3-acetyl-myoinositol. The remaining two GLC peaks observed for the arabinoside were then, most likely, the two mixtures of diastereoisomers 1 d- and 1 l-5-O-l-arabinopryranosyl-1-O-indole-3-acetyl myoinositol and 1 d- and 1 l-5-O-l-arabinopyranosyl-4-O-indole-3-acetyl-myoinositol. The remaining two GLC peaks observed for the galactoside would then be the 1 d and 1 l-5-O-d-galactopyranosyl-1-O-indole-3-acetyl-myoinositol and 1 d- and 1 l-5-O-d- galactopyranosyl-4-O-indoleacetyl-myoinositol.     

Synthesis of N-[2-O-(2-acetamido-2,3-dideoxy-5-thio-d-glucopyranose-3-yl)-d-lactoyl]-l-alanyl-d-isoglutamine

N-[2-O-(2-Acetamido-2,3-dideoxy-5-thio-d-glucopyranose-3-yl)-d-lactoyl]-l-alanyl-d-isoglutamine, in which the ring-oxygen atom of the sugar moiety in N-acetylmuramoyl-l-alanyl-d-isoglutamine (MDP) has been replaced by sulfur, was synthesized from 2-acetamido-2-deoxy-5-thio-α-d-glucopyranose (1). O-Deacetylation of the acetylated acetal, derived from the methyl α-glycoside of 1 by 4,6-O-isopropylidenation and subsequent acetylation, gave methyl 2-acetamido-2-deoxy-4,6-O-isopropylidene-5-thio-α-d-glucopyranoside (4). Condensation of 4 with l-2-chloropropanoic acid, and subsequent esterification, afforded the corresponding ester, which was converted, viaO-deisopropylidenation, acetylation, and acetolysis, into 2-acetamido-1,4,6-tri-O-acetyl-2-deoxy-3-O-[d-1-(methoxycarbonyl)ethyl]-5-thio-α-d-glucopyranose (12). Coupling of the acid, formed from 12 by hydrolysis, with the methyl ester of l-alanyl-d-isoglutamine, and de-esterification, yielded the title compound.     

Synthesis of 5,6-dideoxy-3-O-methyl-5-C-(phenylphosphinyl)-d-glucopyranose and its 1,2,4-triacetate

Methyl phenylphosphonite or dimethyl phosphite underwent acid-catalyzed addition reactions with some hexofuranos-5-ulose 5-(p-tolylsulfonylhydrazones) (7, 9, and 16), to give the corresponding adducts, 17, 18, 19, and 21. The isomer ratios of the adducts were affected by a 3-substituent in the hydrazones. Treatment of adduct 21 with sodium borohydride and sodium dihydrobis(2-methoxyethoxy)-aluminate (SDMA), followed by acid hydrolysis, gave 5,6-dideoxy-3-O-methyl-5-C-(phenylphosphinyl)-d-glucopyranose (26), which was acetylated to give the 1,2,4-tri-O-acetyl derivatives 27a and 27b. Conformational analysis of compound 27a by X-ray crystallography revealed that the compound was 1,2,4-tri-O-acetyl-5,6-dideoxy-3-O-methyl-5-C-[(S)-phenylphosphinyl]-β-d-glucopyranose in the ⁴C₁(d) form having all substituents equatorial.     

Synthese der T-antigenen trisaccharide O-β-d-galactopyranosyl-(1→3)-O-(2-acetamido-2-desoxy-α-d-galactopyranosyl)-(1→6)-d-galactopyranose und O-β-d-galactopyranosyl-(1→3)-O-(2-acetamido-2-desoxy-α-d-galactopyranosyl)-(1→6)-d-glucopyranose und deren anknüpfung an proteine

The synthesis of the trisaccharides O-β-d-galactopyranosyl-(1→3)-O-(2-acetamido-2-deoxy-α-d-galactopyranosyl)-(1→6)-d-galactopyranose (15) and O-β-d-galactopyranosyl-(1→3)-O-(2-acetamido-2-deoxy-α-d-galactopyranosyl)-(1→6)-d-glucopyranose (27) is described and the synthesis of α-d-glycosides by reaction of 3,4,6-tri-O-acetyl-2-azido-2-deoxy-β-d-galactopyranosyl chloride with highly reactive hydroxyl groups is discussed. The trisaccharide 27 was coupled with serum albumin by formation of an imine intermediate and reduced to an amine, to yield a synthetic T-antigen. A similar coupling of 15 was unsuccessful.     

Hydrogenolysis of the isomeric 1,2:4,6-di-O-benzylidene-α-d-glucopyranose derivatives with the LiAlH4AlCl3 reagent

Both isomers of 1,2:4,6-di-O-benzylidene-α-d-glucopyranose (and their 3-O-acetyl and 3-O-benzyl derivatives) have been prepared and their ¹H- and ¹³C-n.m.r. spectra assigned. The mode of hydrogenolysis of the dioxolane ring in these isomers by the LiAlH₄AlCl₃ reagent is determined by the configuration at the acetal carbon and is independent of the electronic character of the two oxygen atoms.     

Synthesis of 2,6-di(acylamino)-2,6-dideoxy-3-O-(d-2-propanoyl-l-alanyl-d-isoglutamine)-d-glucopyranoses

Five 2,6-di(acylamino)-2,6-dideoxy-3-O-(d-2-propanoyl-l-alanyl-d-isoglutamine)-d-glucopyranoses (lipophilic, muramoyl dipeptide analogs) were synthesized from benzyl 2-(benzyloxycarbonylamino)-3-O-(d-1-carboxyethyl)-2-deoxy-5,6-O-isopropylidene-β-dglucopyranoside (1). Methanesulfonylation of 3, derived from the methyl ester of 1 by O-deisopropylidenation, gave the 6-methanesulfonate (4). (Tetrahydropyran-2-yl)ation of 4 gave benzyl 2-(benzyloxycarbonylamino)-2-deoxy-3-O-[d-1-(methoxycarbonyl)ethyl]-6-O-(methylsulfonyl)-5-O-(tetrahydropyran-2-yl)-β-d- glucofuranoside, which was treated with sodium azide to give the corresponding 6-azido derivative (6). Condensation of benzyl 6-amino-2-(benzyloxycarbonyl-amino)-2,6-dideoxy-3-O-[d-1-(methoxycarbonyl)ethyl]-5-O-(tetrahydropyran-2-yl)-β-d-glucofuranoside, derived from 6 by reduction, with the activated esters of octanoic, hexadecanoic, and eicosanoic acid gave the corresponding 6-N-fatty acyl derivatives (8–10). Coupling of the 2-amino derivatives, obtained from compounds 8, 9, and 10 by catalytic reduction, with the activated esters of the fatty acids, gave the 2,6-(diacylamino)-2,6-dideoxy derivatives (11–15). Condensation of the acids, formed from 11–15 by de-esterification, with the benzyl ester of l-alanyl-d-isoglutamine, and subsequent hydrolysis, afforded benzyl 2,6-di(acylamino)-2,6-dideoxy-3-O-(d-2-propanoyl-l-alanyl-d-isoglutamine benzyl ester)-β-d-glucofuranosides. Hydrogenation of the dipeptide derivatives thus obtained gave the five lipophilic analogs of 6-amino-6-deoxymuramoyl dipeptide, respectively, in good yields.     

Methyl derivatives of 2-acetamido-2-deoxy-3-O-(β-d-glucopyranosyluronic acid)-d-glucose (hyalobiouronic acid) from methylated hyaluronic acid

Methanolysis of methylated hyaluronic acid, followed by acetylation, gave, in 70% yield, crystalline methyl 2-acetamido-2-deoxy-4,6-di-O-methyl-3-O-(methyl 4-O-acetyl-2,3-di-O-methyl-β-d-glucopyranosyluronate)-α-d-glucopyranoside. Removal of the O-acetyl and methyl ester groups gave compounds that are useful in the investigation, by ¹H-n.m.r. spectroscopy, of interaction within chains of hyaluronic acid in solution.     

Effects of gibberellic Acid on endogenous indole-3-acetic Acid and indoleacetyl aspartic Acid levels in a dwarf pea

Two-week-old dwarf peas (Pisum sativum cv Little Marvel) were sprayed with gibberellic acid (GA₃), and after 3 or 4 days the upper stem and young leaf samples were analyzed for indole-3-acetic acid (IAA) and indole-3-acetyl aspartic acid by an isotope dilution high performance liquid chromatography method. GA₃ increased IAA levels as much as 8-fold and decreased indole-3-acetyl aspartic acid levels.     

Synthesis of 5,6-dideoxy-3-O-methyl-5-C-(phenylphosphinyl)-d-glucopyranose and its 1,2,4-triacetate

Methyl phenylphosphonite or dimethyl phosphite underwent acid-catalyzed addition reactions with some hexofuranos-5-ulose 5-(p-tolylsulfonylhydrazones) (7, 9, and 16), to give the corresponding adducts, 17, 18, 19, and 21. The isomer ratios of the adducts were affected by a 3-substituent in the hydrazones. Treatment of adduct 21 with sodium borohydride and sodium dihydrobis(2-methoxyethoxy)-aluminate (SDMA), followed by acid hydrolysis, gave 5,6-dideoxy-3-O-methyl-5-C-(phenylphosphinyl)-d-glucopyranose (26), which was acetylated to give the 1,2,4-tri-O-acetyl derivatives 27a and 27b. Conformational analysis of compound 27a by X-ray crystallography revealed that the compound was 1,2,4-tri-O-acetyl-5,6-dideoxy-3-O-methyl-5-C-[(S)-phenylphosphinyl]-β-d-glucopyranose in the ⁴C₁(d) form having all substituents equatorial.     

Structural studies of 4-O-acetyl-α-N-acetylneuraminyl-(2→3)-lactose, the main oligosaccharide in echidna milk

The main oligosaccharide (50%) in the milk of the Australian echidna (Tachyglossus aculeatus) has been identified unequivocally as 4-O-acetyl-α-N-acetylneur-amínyl-(2→3)-lactose. The 4-O-acetyl substituent of the sialic acid residue was characterised by g.l.c.-m.s. of the isolated (after mild, acid hydrolysis) and trimethyl-silylated/esterified sialic acid, and by m.s. (after derivatisation) and 500-MHz, ¹H-n.m.r. spectroscopy of the intact oligosaccharide. Information about the glycosidic bonds was obtained by methylation analysis and 500-MHz, ¹H-n.m.r. spectroscopy. This animal species is the third one known to produce 4-O-acetylated sialic acid.     

Diazomethane-catalysed rearrangement of α-d-glucopyranosyl esters of N-acylamino acids into 2-O-acylaminoacyl-α-d-glucopyranoses

Both anomers of 1-O-[N-(tert-butoxycarbonyl)-L-α-glutamyl]-d-glucopyranose (2) were converted into the unprotected 1-esters, characterised as the trifluoroacetate salts 3α and 3β. On esterification with diazomethane and acetylation, the N-acetylated derivative of 3β and 2β gave the peracetylated 1-O-[5-methyl N-acetyl- and -tert-butoxycarbonyl-L-glutam-1-oyl]-β-d-glucopyranoses (4β and 6β), respectively. Similar treatment of 2α and 3β led to acyl migration, to yield 1,3,4,6-tetra-O-acetyl-2-O-[5-methyl N-(tert-butoxycarbonyl)-L-glutam-1-oyl]-α-d-glucopyranose (7α,64%) with traces of 7β, and a mixture (≈2:1:0.2) of the N-acetyl analogue of 7α (8α), 4α, and 8β, respectively. Treatment of 1-O-[5-methyl N-(tert-butoxycarbonyl)-L-glutam-1-oyl]-α-d-glucopyranose (10) and the corresponding glutam-5-oyl isomer 12 in N,N-dimethylformamide with diazomethane for 1 h resulted in 1 → 2 O-acyl transfer to give, upon acetylation, 7α and the fully acetylated 2-O-[1-methyl N-(tert-butoxy- carbonyl)-L-glutam-5-oyl]-α-d>-glucopyranose in yields of 70 and 90 %, respectively; in the absence of diazomethane, 10 and 12 remained unchanged. Similar experiments with α-d-glucopyranosyl esters of N-acetylglycine, N-acetylalanine, and N-(tert-butoxycarbonyl)phenylalanine yielded the 2-O-acyl derivatives in high yields and with high retention of anomeric configuration. The structures of the rearrangement products were proved both spectroscopically and chemically. The results imply that diazomethane functions as a base in the migration process.     

Synthesis and antibacterial activities of a novel alkylide: 3-O-(3-aryl-2-propargyl) and 3-O-(3-aryl-2-propenyl)clarithromycin derivatives

A series of novel 3-O-(3-aryl-E-2-propenyl)clarithromycin derivatives 8 and 3-O-(3-aryl-2-propargyl)clarithromycin derivatives 11 were designed, synthesized, and evaluated for their in vitro antibacterial activities. Compared with 8c and 11c (Ar was 5-pyrimidyl), 3-O-(3-(5′-pyrimidyl)-Z-1-propenyl) counterpart 6c displayed 4- to 64-fold more potent activities against erythromycin-susceptible Staphylococcus aureus and Streptococcus pneumoniae. Moreover, the activities of 6c, 8c, and 11c against erythromycin-resistant S. aureus and S. pneumoniae were in general 4-fold higher than those of the reference compound, clarithromycin and azithromycin.     

Michael Heidelberger as a Carbohydrate Chemist

The deoxyaldaric acids corresponding in structure to the 3-deoxy-2-C-(hydroxymethyl)aldonic (isosaccharinic) acids have been identified as products of treatment of various carbohydrates with alkali and oxygen-alkali. The structures of the acids were determined from the mass spectra of their Me₃Si derivatives on the basis of previously known, specific fragmentation reactions. The g.l.c.-m.s. technique was used, and g.l.c. retention data are given. The identified species are 2-deoxy-3-C-(hydroxymethyl)tetraric, 3-deoxy-2-C-hydroxymethyl-erythro-pentaric, 3-deoxy-2-C-hydroxymethyl-threo-pentaric, 2-methyltartronic, 2-(2-hydroxyethyl)tartronic, and 2-(2,3-dihydroxypropyl)tartronic acids. Their formation from 4-O-substituted uronic and ulosonic acids is briefly discussed.     

Novel 1-O-indole-3-acetyl-β-d-glucose-dependent acyltransferase transferring indoleacetyl moiety to some mono- di- and oligosaccharides

1-O-(indole-3-acetyl)-β-d-glucose: sugar indoleacetyl transferase (1-O-IAGlc-SugAc) is a novel enzyme catalyzing the transfer of the indoleacetyl (IA) moiety from 1-O-(indole-3-acetyl)-β-d-glucose to several saccharides to form ester-linked IAA conjugates. 1-O-IAGlc-SugAc was purified from liquid endosperm of Zea mays by fractionation with ammonium sulphate, anion-exchange, Blue Sepharose chromatography, affinity chromatography on Concanavalin A-Sepharose, adsorption on hydroxylapatite and preparative PAGE. The obtained enzyme preparation indicates only one band of R _f 0.67 on 8% non-denaturing PAGE consisting of two polypeptides of 42 and 17 kDa in SDS/PAGE. Highly purified 1-O-IAGlc-SugAc shows maximum transferase activity with monosaccharides (mannose, glucose, and galactose), lower activity with disaccharides (melibiose, gentobiose) and trisaccharide (raffinose) and minimal enzymatic activity with oligosaccharides from the raffinose family as well. The novel acyltransferase exhibits, besides its primary indoleacetylation of sugar, minor hydrolytic and disproportionation activities producing free IAA and supposedly 1,2-di-O-(indole-3-acetyl)-β-glucose, respectively. Presumably, 1-O-IAGlc-SugAc, like 1-O-indole-3-acetyl-β-d-glucose-dependent myo-inositol acyltransferase (1-O-IAGlc-InsAc), is another member of the serine carboxypeptidase-like (SCPL) acyltransferase family.     

O-Benzylated thioglucoses, intermediates for the synthesis of (1→6)- and (1→4)-linked oligosaccharides: S-benzylation, coupling, and thioglycoside cleavage

The use of the chloroacetyl group as a protecting group has been studied for a 2-methylglyco-[2′,1′:4,5]-2-oxazoline. The reaction of chloroacetyl chloride or chloroacetic anhydride with 2-acetamido-1,3,4-tri-O-acetyl-2-deoxy-β-d-glucopyra-nose provided 2-acetamido-1,3,4-tri-O-acetyl-6-O-(chloroacetyl)-2-deoxy-β-d-glucopyranose which, on treatment with anhydrous ferric chloride in dichloromethane, produced the desired oxazoline. The glycosylating capability of the oxazoline has been investigated with aglycon hydroxides, to give the corresponding 2-acetamido-2-deoxy-β-d-glucopyranosides. The chloroacetyl group can be selectively removed by treatment with thiourea, and migration of O-acetyl groups was not observed under these conditions.     

Synthetic studies on amino-alpha-glycosides of aminocyclitols. Synthesis of kanamycin analogues

A diastereoisomer of Kanamycin C has been synthesized by a modified Koenigs—Knorr reaction of 3,4,6-tri-O-acetyl-2-(2,4-dinitroanilino)-2-deoxy-α-D-glucopyranosyl bromide with 4-O-(3-acetamido-2,4,6-tri-O-benzyl-3-deoxy-α-D-glucopyranosyl)-N,N′-di[(benzyloxy)carbonyl]-2-deoxystreptamine. Several Kanamycin analogues were synthesized by a similar condensation reaction. Each of the condensed products was isolated as its crystalline tetra-N-acetyl derivative and was proved by n.m.r. spectroscopy in D₂O to have the α-configuration.     

Synthesis of carbohydrate analogs (positional,configurational, and optical) of N-acetylmuramoyl-l-alanyl-d-isoglutamine,and their immunoadjuvant activities

2-Acetamido-2-deoxy-4- and -6-O-(d-2-propanoyl-l-alanyl-d-isoglutamine)-d-glucopyranose, 2-acetamido-2-deoxy-3-O-(d-2-propanoyl-l-alanyl-d-isoglutamine)-d-allopyranose, -d-gulopyranose, -d-galactopyranose, -d-mannopyranose, and -l-idopyranose, and 3-O-(d-2-propanoyl-l-alanyl-d-isoglutamine)-d- and -l-glucopyranose were synthesized, in order to clarify the structural requirements for the immunoadjuvant activity of the carbohydrate moiety in N-acetylmuramoyl-l-alanyl-d-isoglutamine. Immunoadjuvant activity of the N-acetylmuramoyl-dipeptide analogs was examined in guinea-pigs.     

Bifunctional indole-3-acetyl transferase catalyses synthesis and hydrolysis of indole-3-acetyl-myo-inositol in immature endosperm of Zea mays

1- O -(indole-3-acetyl)- β - d -glucose: myo -inositol indoleacetyl transferase (IA- myo -inositol synthase) is an important enzyme in IAA metabolism. This enzyme catalyses the transfer of the indole acetyl (IA) moiety from 1- O -(indole-3-acetyl)- β - d -glucose to myo -inositol to form IA- myo- inositol and glucose. IA- myo -inositol synthase was purified to an electrophoretically homogenous state from maize liquid endosperm by fractionation with ammonium sulphate, anion-exchange, adsorption on hydroxylapatite, affinity chromatography on ConA-Sepharose, preparative PAGE and isoelectric focusing. We thus obtained two enzyme preparations which differ in their R _f on 8% polyacrylamide gel. The preparation of R _f 0.36 contained a single 56.4 kDa polypeptide, whereas the preparation of R _f 0.39 consisted of two polypeptides of 56.4 and 53.5 kDa. Both purified preparations of IAInos synthase also exhibited the activity of an IAInos hydrolase, showing that the dual activity was associated with a single protein. Results of gel filtration and analytical SDS-PAGE suggest that the native enzyme exists as both a monomeric (65 kDa) and homo- or heterodimeric form (110–130 kDa). Analysis of peptide maps and amino acid sequences of two 21 amino-acid peptides showed that polypeptides of 56.4 and 53.5 kDa have the same primary structure and that the 3 kDa difference in molecular mass is probably caused by different glycosylation levels. Comparison of this partial and internal amino acid sequence with sequences of other plant acyltransferases indicated similarity to several proteins which belonged to the serine carboxypeptidase-like (SCPL) acyltransferase family.     

The synthesis of oligosaccharide-asparagine compounds. V. O- -D-mannopyranosyl-(1 leads to 6)-O-(2-acetamido-2-deoxy- -D-glucopyranosyl)-(1 leads to 4)-2-acetamido-N-(L-aspart-4-oyl)-2-deoxy- -D-glucopyranosylamine

O-α-d-Mannopyranosyl-(1→6)-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-(1→4)-2-acetamido-N-(l-aspart-4-oyl)-2-deoxy-β-d-glucopyranosylamine (12), used in the synthesis of glycopeptides and as a reference compound in the structure elucidation of glycoproteins, was synthesized via condensation of 2,3,4,6-tetra-O-acetyl-α-d-mannopyranosyl bromide with 2-acetamido-4-O-(2-acetamido-3-O-acetyl-2-deoxy-β-d-glucopyranosyl)-3,6-di-O-acetyl-2-deoxy-β-d-glucopyranosyl azide (5) to give the intermediate, trisaccharide azide 7. [Compound 5 was obtained from the known 2-acetamido-4-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-d-glucopyranosyl)-3,6-di-O-acetyl-2-deoxy-β-d-glucopyranosyl azide by de-O-acetylation, condensation with benzaldehyde, acetylation, and removal of the benzylidene group.] The trisaccharide azide 6 was then acetylated, and the acetate reduced in the presence of Adams' catalyst. The resulting amine was condensed with 1-benzyl N-(benzyloxycarbonyl)-l-aspartate, and the O-acetyl, N-(benzyloxycarbonyl), and benzyl protective groups were removed, to give the title compound.