Synthesis and characterization of poly-O-methyl-[n]-polyurethane from a d-glucamine-based monomer

Aminoalditol 1-amino-1-deoxy-D-sorbitol (1) was readily converted into 2,3,4,5-tetra-O-methyl derivative 5, a key precursor of a sugar-based [n]-polyurethane. For the polymerization, the free amino or primary hydroxyl groups of 5 were selectively activated and employed as starting monomers in two alternative procedures. Thus, the amino function of 5 was converted into the isocyanate derivative by treatment with di-tert-butyltricarbonate, and polymerized in situ in the presence of Zr(IV) acetylacetonate. The resulting poly(1-amino-1-deoxy-2,3,4,5-tetra-O-methyl-D-sorbitol)urethane (8) had a moderate molecular weight and showed the presence of urea units. The alternative synthesis of 8 involved the activation of the free hydroxyl group of 5 as the corresponding phenylcarbonate. The polymerization of this α-amino-ω-phenylcarbonate alditol monomer does not require a metal catalyst. The resulting material exhibited an improved molecular weight and higher purity than that obtained via the isocyanate. [n]-polyurethane 8 was highly soluble in water as well as in common organic solvents (chloroform, acetone, ethyl acetate, etc) and was obtained as an amorphous material which was characterized thermally and spectroscopically.     

Synthesis of the conjugation ready, downstream disaccharide fragment of the O-PS of Vibrio cholerae O:139

The linker-equipped disaccharide, 8-amino-3,6-dioxaoctyl 2,6-dideoxy-2-acetamido-3-O-β-d-galactopyranosyluronate-β-d-glucopyranoside (10), was synthesized in eight steps from acetobromogalactose and ethyl 4,6-O-benzylidene-2-deoxy-2-trichloroacetamido-1-thio-β-d-glucopyranoside. The hydroxyl group present at C-4^II in the last intermediate, 8-azido-3,6-dioxaoctyl 4-O-benzyl-6-bromo-2,6-dideoxy-2-trichloroacetamido-3-O-(benzyl 2,3-di-O-benzyl-β-d-galactopyranosyluronate)-β-d-glucopyranoside (9), is positioned to allow further build-up of the molecule and, eventually, construction of the complete hexasaccharide. Global deprotection (9→10) was done in one step by catalytic hydrogenolysis over palladium-on-charcoal.     

Synthesis of 1,3,4,6-tetra-O-acetyl-2-[3-alkyl-(aryl)-thioreido]-2-deoxy-α-d-glucopyranoses and their transformation into 2-alkyl(aryl)amino-(1,2-dideoxy-α-d-glucopyrano)[2,1-d]-2-thiazolines

1,3,4,6-Tetra-O-acetyl-2-deoxy-2-isothiocyanato-α-d-glucopyranose, produced from 1,3,4,6-tetra-O-acetyl-2-amino-2-deoxy-α-d-glucopyranose hydrochloride, thiophosgene, and calcium carbonate, was condensed with alkyl- and aryl-amines in ether to afford the crystalline 1,3,4,6-tetra-O-acetyl-2-[3-alkyl(aryl)-thioureido]-2-deoxy-α-d-glucopyranoses (2). Compounds 2 and the β anomers 3 were converted in high yield into 2-alkyl(aryl)amino-(3,4,6-tri-O-acetyl-1,2-dideoxy-α-d-glucopyrano)[2,1-d]-2-thiazoline hydrobromides (4) by hydrogen bromide-promoted cyclisation. The O-deacetylated thiazoline hydrobromide 5 was also isolated and converted into 2-[N-(4-methoxyphenyl)acetamido]-(3,4,6-tri-O-acetyl-1,2-dideoxy-α-d-glucopyrano)[2,1-d]-2-thiazoline (8). Conformational studies of 4 and 8 were made by ¹H-n.m.r. spectroscopy.     

5′-Uridyl derivatives of N-glycosyl allophanic acid and biuret

(2′,3′-O-Isopropylidene-5′-uridyl) 4-(2,3,4,6-tetra-O-acetyl-β-d-glycopyranosyl)allophanates were obtained in the reactions of 2′,3′-O-isopropylidene-uridine and O-peracetylated β-d-gluco-, galacto- and xylopyranosylamines, and OCNCOCl. 2,3,4,6-Tetra-O-acetyl-β-d-glucopyranosyl isocyanate and N-(2′,3′-O-isopropylidene-5′-uridyl)urea gave 1-(2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl)-5-(2′,3′-O-isopropylidene-5′-uridyl)biuret. Deprotection of the β-d-gluco configured allophanate and biuret was carried out by standard methods.     

The Lectin from the Crustacean Liocarcinus depurator Recognizes O-acetylsialic Acids

A lectin that recognized sialic acids and agglutinated mouse erythrocytes was purified from hemolymph of the crab Liocarcinus depurator. It consisted of 38-kDa subunits and had a pI about 6.0. The specificity of the lectin was assayed by hemagglutination inhibition. N-acetylneuraminic acid (Neu5Ac) was a good inhibitor and its N-acetyl group at C-5 was critical for lectin-ligand interaction. Substitution of the C-9 hydroxyl on Neu5Ac with an O-acetyl group (9-O-Ac-Neu5Ac) increased the inhibitory potency of this molecule. Furthermore, O-acetyl substitution of all the hydroxyl groups yielded even better inhibitors (2,4,7,8,9-O-Ac-Neu5Ac and its 1-O-methyl ester). Removal of the hydroxyl or O-acetyl group connected to C-2 reduced the potency of these inhibitors. The lectin agglutinated and stimulated human but not mouse lymphocytes. It was also inhibited by Escherichia coli (O111:B4) lipopolysaccharide and agglutinated specific gram-negative bacteria. In vitro labeling with [³⁵S]methionine indicated that the lectin was synthesized in hepatopangreas of L. depurator. Immunofluorescence showed that among hemocytes it localized mainly in the large-granule population.     

Synthesis of methyl 2-acetamido-2-deoxy-3-O-(β-d-galactopyranosyl)-α-d-galactopyranoside from a corresponding thioglycoside derivative

The title disaccharide glycoside was synthesized by halide ion-promoted glycosidation, using methanol and the disaccharide bromide derived from methyl 2-azido-3-O-(2,3,4,6-tetra-O-benzoyl--d-galactopyranosyl)-4,6-O-benzylidene-2-deoxy-1-thio--d-galactopyranoside. This derivative in turn was prepared by silver triflate-promoted condensation of monosaccharide derivatives.     

Efficient synthesis of exomethylene- and keto-exomethylene-d-glucopyranosyl nucleoside analogs as potential cytotoxic agents

A novel series of exomethylene- and keto-exomethylene-d-glucopyranonucleosides with thymine, uracil, and 5-fluorouracil as heterocyclic bases have been designed and synthesized. Wittig condensation of the 3-keto glucoside 1 gave the corresponding 1,2:5,6-di-O-isopropylidene-3-deoxy-3-methylene-d-glucofuranose (2), which after hydrolysis and acetylation led to the precursor 1,2,4,6-tetra-O-acetyl-3-deoxy-3-methylene-d-glucopyranose (4).Compound 4 was condensed with silylated thymine, uracil, and 5-fluorouracil, respectively, deacetylated and acetalated to afford 1-(3′-deoxy-4′,6′-O-isopropylidene-3′-methylene-β-d-glucopyranosyl)pyrimidines 7a–c. Oxidation of the free hydroxyl group in the 2′-position of the sugar moiety led to the formation of the labile 1-(3′-deoxy-4′,6′-O-isopropylidene-3′-methylene-β-d-glucopyranosyl-2′-ulose)pyrimidines 8a–c. Finally, deisopropylidenation of the resulted derivatives 8a–c afforded the diol nucleosides 9a–c. The target keto-exomethylene analogs 9a–c were more cytostatic against a variety of tumor cell lines than the corresponding saturated-hydroxy-exomethylene derivatives 6. In particular, the 5-fluorouracil derivative 9c was highly cytostatic at an IC₅₀ (50% inhibitory concentration) ranging between 0.56 and 9.4 μg/mL, which was comparable to the free parental 5-fluorouracil base.     

Enamine and pyrrole formation from 2-amino-2-deoxy-D-glucose and dimethyl acetylenedicarboxylate

Addition of 2-amino-2-deoxy-β-D-glucopyranose to dimethyl acetylenedicarboxylate afforded an almost quantitative yield of amorphous 2-deoxy-2-(1,2-dimethoxycarbonylvinyl)amino-D-glucose (5). Acetylation of this adduct gave crystalline 1,3,4,6-tetra-O-acetyl-2-deoxy-2-[(Z)-1,2-dimethoxycarbonylvinyl]amino-α-D-glucopyranose (6a); the corresponding β-D anomer (6b) was obtained by addition of 1,3,4,6-tetra-O-acetyl-2-amino-2-deoxy-β-Dglucopyranose to dimethyl acetylenedicarboxylate. O-Deacetylation of tetra-acetate 6a with barium methoxide in methanol occurred selectively at C-1, yielding enamine 6c derived from 3,4,6-tri-O-acetyl-2-amino-2-deoxy-α-D-glucopyranose. Conversion of the crude adduct 5 into 3-methoxycarbonyl-5-(D-arabino-tetrahydroxybutyl)-2-pyrrolecarboxylic acid (7) took place by heating in water or in slightly basic media in yields up to 83%. Acetylation of 7 gave the tricyclic derivative 8, and its periodate oxidation afforded 5-formyl-3-methoxycarbonyl-2-pyrrolecarboxylic acid (9). Oxidation of 9 with alkaline silver oxide yielded 3-methoxy-carbonyl-2,5-pyrroledicarboxylic acid (10).     

Limonoids from the fruits of Melia toosendan

Four (1–4) new and seven known limonoids were isolated from the EtOH extract of the fruits of Melia toosendan. The structures of the new compounds were established on the basis of spectroscopic methods to be 12-O-methyl-1-O-deacetylnimbolinin B (1), 12-O-methy-1-O-tigloyl-1-O-deacetylnimbolinin B (2), 12-O-ethylnimbolinin B (3), and 1-O-cinnamoyl-1-O-debenzoylohchinal (4). Additionally, two new tirucallane-type triterpenoids, named meliasenins S (5) and T (6), were obtained from the same fractions during purification of the limonoids.     

Biotransformation of gallic acid by <Emphasis Type="Italic">Beauveria sulfurescens</Emphasis> ATCC 7159

Preparative-scale fermentation of gallic acid (3,4,5-trihydroxybenzoic acid) (1) with Beauveria sulfurescens ATCC 7159 gave two new glucosidated compounds, 4-(3,4-dihydroxy-6-hydroxymethyl-5-methoxy-tetrahydro-pyran-2-yloxy)-3-hydroxy-5-methoxy-benzoic acid (4), 3-hydroxy-4,5-dimethoxy-benzoic acid 3,4-dihydroxy-6-hydroxymethyl-5-methoxy-tetrahydro-pyran-2-yl ester (7), along with four known compounds, 3-O-methylgallic acid (2), 4-O-methylgallic acid (3), 3,4-O-dimethylgallic acid (5), and 3,5-O-dimethylgallic acid (6). The new metabolite genistein 7-O-β-D-4″-O-methyl-glucopyranoside (8) was also obtained as a byproduct due to the use of soybean meal in the fermentation medium. The structural elucidation of the metabolites was based primarily on 1D-, 2D-NMR, and HRFABMS analyses. Among these compounds, 2, 3, and 5 are metabolites of gallic acid in mammals. This result demonstrated that microbial culture parallels mammalian metabolism; therefore, B. sulfurescens might be a useful tool for generating mammalian metabolites of related analogs of gallic acid (1) for complete structural identification and for further use in investigating pharmacological and toxicological properties in this series of compounds. In addition, a GRE (glucocorticoid response element)-mediated luciferase reporter gene assay was used to initially screen for the biological activity of the 6 compounds, 2–6 and 8, along with 1 and its chemical O-methylated derivatives 9–13. Among the 12 compounds tested, 11–13 were found to be significant, but less active than the reference compounds of methylprednisolone and dexamethasone.     

Synthesis of 3-Aminoimidazo[4,5-c]pyrazole Nucleoside via the N-N Bond Formation Strategy as a [5:5] Fused Analog of Adenosine

3-Amino-6-(β-D-ribofuranosyl)imidazo[4,5-c]pyrazole (2) was synthesized via an N-N bond formation strategy by a mononuclear heterocyclic rearrangement (MHR). A series of 5-amino-1-(5-O-tert-butyldimethylsilyl-2,3-O-isopropylidene-β-D-ribofuranosyl-4-(1,2,4-oxadiazol-3-yl)imidaz-oles (6a-d), with different substituents at the 5-position of the 1,2,4-oxadiazole, were synthesized from 5-amino-1-(β-D-ribofuranosyl)imidazole-4-carboxamide (AICA Ribose, 3). It was found that 5-amino-1-(5-O-tert-butyldimethylsilyl-2,3-O-isopropylidene-β-D-ribofuranosyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)imidazole (6a) underwent the MHR with sodium hydride in DMF or DMSO to afford the corresponding 3-acetamidoimidazo[4,5-c]pyrazole nucleoside(s) (7b and/or 7a) in good yields. A direct removal of the acetyl group from 3-acetamidoimidazo[4,5-c]pyrazoles under numerous conditions was unsuccessful. Subsequent protecting group manipulations afforded the desired 3-amino-6-(β-D-ribofuranosyl)imidazo[4,5-c]pyrazole (2) as a 5:5 fused analog of adenosine (1).     

Synthesis of unsaturated aldehydes by hydroboration of propargyl alcohols and from aldehydo sugar precursors

Propargyl acetates obtained by ethynylation of aldehydo sugar derivatives, followed by acetylation, can be converted by hydroboration with bis(isoamyl)borane and subsequent treatment with hydrogen peroxide into α,β-unsaturated aldehydes; the latter may also be obtained by treating the original aldehydo sugar derivative with formylmethylenetriphenylphosphorane. By these two routes the aldehydo sugars 2,3-O-isopropylidene-aldehydo-D-glyceraldehyde (1), 2,3,4,5-tetra-O-acetyl-aldehydo-D-arabinose (5), and 2,3:4,5-di-O-isopropylidene-aldehydo-D-arabinose (9) have been converted with 2-carbon chain-extension into the corresponding trans-unsaturated aldehydes 3, 7, and 11, respectively. Likewise, by the acetylene route, 1,2:3,4-di-O-isopropylidene-6-aldehydo-α-D-galacto-hexodialdo-1,5-pyranose (13) was converted into the C₈ unsaturated aldehyde 15, although the Wittig route was unsuccessful in this instance, as it was with methyl 2,3-di-O-acetyl-4-deoxy-6-aldehydo-β-L-threo-hex-4-enodialdo-1,5-pyranoside (16).     

Synthesis of 4-O- and 6-O-(2′-iodoethyl)-d-glucose

O-Allylation of 1,2,3,6-tetra-O-acetyl- -glucopyranose followed by an ozonation/reduction sequence gave the 4-hydroxyethyl derivative. This hydroxyethyl substituent was also introduced at C-6, starting from 1,2:3,5-bis(O-methylidene)-α- -glucofuranose using an alkylation/reduction sequence. These 4- and 6-O-hydroxyethyl derivatives were then converted to the title compounds by iodination followed by deprotection. Noteworthy is the particular stability of the carbon–iodine bond in these ethers, a prerequisite for their potential use in Single Photon Emitted Computed Tomography medical imaging (SPECT).     

1,4-anhydride formation of solvolysis of 1,6-disubstituted derivatives of 2,3,4,5-tetra-O-acetylallitol

The 6-O-mesyl, 6-O-tosyl, 6-bromo-6-deoxy, and 6-deoxy-6-iodo derivatives of 1,4-anhydro-DL-allitol were obtained by treatment of the corresponding 1,6-di-substituted derivatives (2, 3, 6, 4) of 2,3,4,5-tetra-O-acetylallitol with hot, methanolic hydrogen chloride. Compounds 2 and 3 were prepared by the acetolysis of the 1,6-di-O-mesyl and 1,6-di-O-tosyl derivatives (8 and 11) of di-O-benzylideneallitol. Iodide displacement on 2 gave 4, and detritylation-bromination of 2,3,4,5-tetra-O-acetyl-1,6-di-O-tritylallitol (5) gave 6. The acetal residues of di-O-benzylideneallitol have been shown to span the secondary carbon atoms.     

Synthesis of Neu5Ac- and Neu5Gc-α-(2→6′)-lactosamine 3-aminopropyl glycosides

In order to prepare 3-aminopropyl glycosides of Neu5Ac-α-(2→6′)-lactosamine trisaccharide 1, and its N-glycolyl containing analogue Neu5Gc-α-(2→6′)-lactosamine 2, a series of lactosamine acceptors with two, three, and four free OH groups in the galactose residue was studied in glycosylations with a conventional sialyl donor phenyl [methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio- -glycero-α- and β- -galacto-2-nonulopyranosid]onates (3) and a new donor phenyl [methyl 4,7,8,9-tetra-O-acetyl-5-(N-tert-butoxycarbonylacetamido)-3,5-dideoxy-2-thio- -glycero-α- and β- -galacto-2-nonulopyranosid]onates (4), respectively. The lactosamine 4′,6′-diol acceptor was found to be the most efficient in glycosylation with both 3 and 4, while imide-type donor 4 gave slightly higher yields with all acceptors, and isolation of the reaction products was more convenient. In the trisaccharides, obtained by glycosylation with donor 4, the 5-(N-tert-butoxycarbonylacetamido) moiety in the neuraminic acid could be efficiently transformed into the desired N-glycolyl fragment, indicating that such protected oligosaccharide derivatives are valuable precursors of sialo-oligosaccharides containing N-modified analogues of Neu5Ac.     

Synthesis and antileishmanial evaluation of 1-aryl-4-(4,5-dihydro-1H-imidazol-2-yl)-1H-pyrazole derivatives

A series of 1-aryl-4-(4,5-dihydro-1H-imidazol-2-yl)-1H-pyrazoles (4a–g) and 5-amino-1-aryl-4-(4,5-dihydro-1H-imidazol-2-yl)-1H-pyrazoles (5a–g) were synthesized and evaluated in vitro against three Leishmania species: L. amazonensis, L. braziliensis and L. infantum (L. chagasi syn.). The cytotoxicity was assessed. Among the derivatives examined, six compounds emerged as the most active on promastigotes forms of L. amazonensis with IC₅₀ values ranging from 15 to 60 μM. The reference drug pentamidine presented IC₅₀ = 10 μM. However, these new compounds were less cytotoxic than pentamidine. Based on these results, the more promising derivative 5d was tested further in vivo. This compound showed inhibition of the progression of cutaneous lesions in CBA mice infected with L. amazonensis relative to an untreated control.     

Hydroxylation of steroids by Fusarium oxysporum, Exophiala jeanselmei and Ceratocystis paradoxa

The potential of Fusarium oxysporum var. cubense UAMH 9013 to perform steroid biotransformations was reinvestigated using single phase and pulse feed conditions. The following natural steroids served as substrates: dehydroepiandrosterone (1), pregnenolone (2), testosterone (3), progesterone (4), cortisone (5), prednisone (6), estrone (7) and sarsasapogenin (8). The results showed the possible presence of C-7 and C-15 hydroxylase enzymes. This hypothesis was explored using three synthetic androstanes: androstane-3,17-dione (9), androsta-4,6-diene-3,17-dione (10) and 3α,5α-cycloandrost-6-en-17-one (11). These fermentations of non-natural steroids showed that C-7 hydroxylation was as a result of that position being allylic. The evidence also pointed towards the presence of a C-15 hydroxylase enzyme.The eleven steroids were also fed to Exophialajeanselmei var. lecanii-corni UAMH 8783. The results showed that the fungus appears to have very active 5α and 14α-hydroxylase enzymes, and is also capable of carrying out allylic oxidations.Ceratocystis paradoxa UAMH 8784 was grown in the presence of the above-mentioned steroids. The results showed that monooxygenases which effect allylic hydroxylation and Baeyer–Villiger rearrangement were active. However, redox reactions predominated.     

Synthesis of octyl S-glycosides of tri- to pentasaccharide fragments related to the GPI anchor of Trypanosoma brucei

The three oligosaccharide octyl-S-glycosides Man-α1,6-Man-α1,4-GlcNH₂-α1,S-Octyl (19), Man-α1,6-(Gal-α1,3)Man-α1,4-GlcNH₂-α1,S-Octyl (27) and Man-α1,2-Man-α1,6-(Gal-α1,3)Man-α1,4-GlcNH₂-α1,S-Octyl (37), related to the GPI anchor of Trypanosoma brucei were prepared by a stepwise and block-wise approach from octyl 2-azido-2-deoxy-3,6-di-O-benzyl-1-thio-α-d-glucopyranoside (8) and octyl 2-O-benzoyl-4,6-O-(1,1,3,3-tetraisopropyl-1,3-disiloxane-1,3-diyl)-1-thio-α-d-mannopyransoside (9). Glucosamine derivative 8 was obtained from 1,3,4,6-tetra-O-acetyl-2-azido-2-desoxy-β-d-glucopyranose (1) in five steps. Mannoside 9 was converted into the corresponding imidate 12 and coupled with 8 to give disaccharide octyl-S-glycoside 13 which was further mannosylated to afford trisaccharide 19 upon deprotection. Likewise, mannoside 9 was galactosylated, converted into the corresponding imidate and coupled with 8 to give trisaccharide 25. Mannosylation of the latter afforded tetrasaccharide 27 upon deprotection. Condensation of 25 with disaccharide imidate 35 gave, upon deprotection of the intermediates, the corresponding pentasaccharide octyl-S-glycoside 37. Saccharides 19, 27 and 37 are suitable substrates for studying the enzymatic glycosylation pattern of the GPI anchor of T. brucei.     

An Improved Synthesis of 2′-Deoxy-9-deazaadenosine and an N-7 Blocked Derivative Useful for the Synthesis of Modified Oligonucleotides Containing 2′-Deoxy-9-deazaadenosine

Abstract

As an epimerization resistant synthon in the synthesis of oligo-nucleotides consisting of C-nucleoside analogues, hitherto unknown 5-benzyloxy-methyl-3-(2-deoxy-β-D-erythro-pentofuranosyl)pyrrolo[3,2-d]pyrimpyrimidine (7-benzyloxymethyl-2′-deoxy-9-deazaadenosine) was prepared in seven steps from the known 3-amino-2-cyano-4-(2,3-O-isopropylidene-5-O-trityl-β-D-ribofuranosyl)-pyrrolpyrrole (1). Treatment of 1 with benzyl chloromethyl ether in the presence of potassium t-butoxide and 18-crown-6 afforded the N-protected pyrrole 2, which was converted into the 9-deazapurine derivative 3 in high yield by heating in EtOH. 7-Benzyloxymethyl-9-deazaadenosine 4 was obtained from 3 by acid hydrolysis in 2.5% methanolic hydrogen chloride. After protection of the hydroxyl groups of 4 with Markievicz's reagent, the product 5 was converted into the 2′-O-phenoxythiocarbonyl derivative 6. Reduction of 6 with butyltin hydride in the presence of 2,2′-azobis(2-methylpropionitrile), followed by desilylation with triethylammonium fluoride, afforded the desired 7-benzyloxymethyl-2′-deoxy-9-deazaadenosine (8) in high overall yield. The benzyloxymethyl group of 8 was removed by hydrogenolysis over palladium hydroxide (Degussa type) to give 2′-deoxy-9-deazaadenosine (9) in quantitative yield. The structure of 9 is discussed.     

Synthesis of 1,6-thioanhydro-D-glucitol

Treatment of 2,4-O-benzylidene-1,6-di-O-tosyl-D-glucitol (1) with potassium thiolbenzoate afforded the 6-S-benzoyl compound 2 and its 5-benzoate 4, the structure of which was proved chemically. When 1 was acetylated and then treated with the thiolate, the acetylated 6-S-benzoyl compound 19 was obtained in good yield in addition to some 1,6-di-S-benzoyl derivative 21. Treatment of 19 with acetic anhydride-acetic acid-sulfuric acid afforded 2,3,4,5-tetra-O-acetyl-6-S-acetyl-1-O-tosyl-D-glucitol (26), which was converted by sodium methoxide into a mixture of 1,5-anhydro-6-thio-D-glucitol (28) and 1,6-thioanhydro-D-glucitol (29). These two compounds were isolated as their acetates (30 and 31) by column chromatography, or by converting 28 into its S-trityl derivative (32).