Screening of synthetic trehalose 6,6'-diesters and trehalose 6-monoesters as potential immunoreactants for the serodiagnosis of tuberculosis.

The absence of serological cross-reactivity between trehalose 2,3-diester (DAT, formerly SL-IV) and synthetic trehalose 6,6'-diesters and trehalose 6-monoesters was established by ELISA testing using polyclonal immune sera raised in rabbits sensitized with "DAT". From the screening of fifteen synthetic trehalose 6,6'- and 6-esters, "mirror" pseudo cord factor no. 1, "mirror" amides no. 5 and 6, cord factor analogues 7 and 8 and trehalose 6-monoesters 10 and 11 were selected for future, more extensive serological analysis. Paired comparisons of analogues among these fifteen substances showed that serodiagnostic discrimination power was more a function of the carbon chain length of their substituent groups--as well as of their position--than of the "mirror" constitution of the molecules. More exhaustive testing of these seven compounds is needed to select the synthetic product most efficient in the ELISA serodiagnosis of tuberculosis.

The structure of an antigenic glycolipid (SL-IV) from Mycobacterium tuberculosis.

The structure of an antigenic glycolipid isolated recently from cell walls of various strains of Mycobacterium tuberculosis and believed to be a sulfolipid consisting chiefly of 2,3-di-O-(hexadecanoyl/octadecanoyl)-alpha,alpha-trehalose 2'-sulfate (designated as SL-IV), was reinvestigated by mass spectrometry and nuclear magnetic resonance spectroscopy. The material proved to be a complex mixture of closely related components which are indeed 2,3-di-O-acyltrehaloses. However, no sulfate group was found in the antigen, and revision of its designation as a sulfolipid is therefore required. The lipid substituents comprise an estimated 20% of C14-C19 fatty acids, including tetradecanoic (myristic), 9-tetradecenoic (myristoleic), 9-hexadecenoic (palmitoleic), 9-octadecenoic (oleic), 10-methylhexadecanoic (tuberculopalmitic), and 10-methyloctadecanoic (tuberculostearic) acids in addition to hexadecanoic (palmitic) and octadecanoic (stearic) acids, and approximately 80% of higher, methyl-branched acids. Among the latter are, principally, 2,4-dimethyldocosanoic and 3-hydroxy-2,4,6- trimethyltetracosanoic acids, and a smaller proportion of 2,4,6-trimethyltetracos-2-enoic acid. Numerous further acids, related to those mentioned by methylene homology, are also present in small proportions.

Inhibition of interleukin-6 release and T-cell proliferation by synthetic mirror pseudo cord factor analogues in human peripheral blood mononuclear cells.

The effects of synthetic alkyl ((alkyl 6-deoxy-a-D-gluco-heptopyranosyluronate) 6-deoxy-a-D-gluco-heptopyranoside) uronates, a novel type of mirror pseudo cord factor, on the in vitro modulation of interleukin-6 production and T-cell proliferation in human peripheral blood mononuclear cells, were investigated. Synthetic mirror pseudo cord factors with alkyl chains ranging from C16 to C18 have very weak interleukin-6-inducing capacities and lack mitogenic activities for T-cell proliferation. However, they could inhibit IL-6 release induced by sonicated Bacillus Calmette-Guérin (S-BCG), bacterial endotoxin, and phytohaemagglutinin in a dose-dependent manner. Inhibition was observed not only with mononuclear cells but also with purified monocytes. Furthermore, these synthetic compounds could suppress T-lymphocyte proliferation stimulated by sonicated Mycobacterium tuberculosis H37Rv (S-H37Rv) antigens, S-BCG antigens, as well as by recombinant 65 kDa mycobacterial heat-shock protein. In contrast, these compounds failed to inhibit the phytohaemagglutinin-induced T-cell proliferation. We conclude that the inhibition of cytokine release and T-cell proliferation by synthetic mirror pseudo cord factors was due to direct blocking of the function and/or activity of monocytes or antigen-presenting cells.

Synthesis of alpha,alpha-trehalose 2,3- and 2,3'-diesters with palmitic and stearic acid: potential immunoreactants for the serodiagnosis of tuberculosis.

Regioselective monoacylation, by the stannylation method, of 4,6:4',6'-di-O-benzylidene-alpha,alpha-trehalose with palmitoyl or stearoyl chloride afforded the 2-palmitate and 2-stearate of the diacetal, whereas partial diacylation led to the corresponding 2,3'-dipalmitate and 2,3'-distearate. Protection of the monoesters in the 2',3' positions by cyclizing silylation with 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane, followed by acylation of the silyl ethers, gave the fully protected 2,3-dipalmitate, 2,3-distearate, and 2-palmitate-3-stearate. Small proportions of other isomers and triesters were also produced in these reactions. Desilylation and debenzylidenation of the diesters finally furnished 2,3- and 2,3'-di-O-palmitoyl-2,3- and 2,3'-di-O-stearoyl-, and 2-O-palmitoyl-3-O-stearoyl-alpha,alpha-trehalose.

Evaluation of synthetic pseudo cord-factor-like glycolipids for the serodiagnosis of tuberculosis.

A number of glycolipids were evaluated in an ELISA test for their serodiagnostic usefulness in tuberculosis. One hundred and twelve (112) sera belonging to bacteriologically confirmed TB patients, patients with pathologies other than tuberculosis and healthy individuals were examined against several synthetic "mirror" pseudo cord factors (analogues of trehalose-6,6'-dimycolate or TDM) using natural cord factor and another recently described natural glycolipid (SL-IV) of Mycobacterium tuberculosis as control antigens. Analysis of the results shows that all synthetic "mirror" pseudo cord factors, except one with a short 8-carbon chain, were better recognized by the sera of tuberculosis patients than natural cord factor, with sensitivity and specificity values in the ELISA test similar to those reported for M. tuberculosis species-specific SL-IV. Of all antigens tested in this study, BDA. TDA, a bis(N,N-dioctadecylamide) of "trehalose dicarboxylic acid", [(alpha-D-glucopyranosyluronic acid) (alpha-D-glucopyranosiduranic acid)], showed the highest serodiagnostic discriminating power (93% sensitivity and specificity). We postulate that either these artificial molecules are cross-reactants of similarly structured native glycolipids of M. tuberculosis or that they bear closer resemblance to actual phagosome-lysosome-modified antigens than to native mycobacterial ones.

Synthesis of alkyl [(alkyl 6-deoxy-alpha-D-gluco-heptopyranosyl- uronate) 6-deoxy-alpha-D-gluco-heptopyranosid]uronates, a novel type of mirror pseudo cord factor.

The 1-octyl, 1-pentadecyl, 1-hexadecyl, 1-heptadecyl, and 1-octadecyl diesters of (6-deoxy-alpha-D-gluco-heptopyranosyluronic acid) 6-deoxy-alpha-D-gluco-heptopyranosiduronic acid, a new homolog of trehalosuronic acid, were prepared by two procedures. One procedure involved conversion of the peracetylated acid into its dichloride, reaction of the latter with the alkanols, and acid-catalyzed deacetylation of the products, whereas the other consisted of reaction of alkyl mesylates with the potassium salt of the unprotected acid.

Synthesis of L-gulose, L-galactose, and their acetylated aldehydo forms from 6-S-phenyl-6-thio-D-hexoses.

Methyl 6-S-phenyl-6-thio-a-D-glucopyranoside, prepared in high yield from methyl a-D-glucopyranoside by the action of diphenyl disulfide and tributylphosphine in pyridine, was converted into 6-S-phenyl-6-thio-D-glucitol pentaacetate (7) by sequential hydrolysis, borohydride reduction, and acetylation. Oxidation of 7 with 3-chloroperoxybenzoic acid gave the corresponding S-epimeric sulfoxides, which underwent Pummerer rearrangement to 1-epimeric L-gulose S-phenyl monothiohemiacetal hexaacetates. Boron trifluoride-catalyzed reaction of the latter with thiophenol gave the analogous diphenyl dithioacetal, whereas base-catalyzed methanolysis led to free L-gulose. Treatment of 7 with N-chlorosuccinimide afforded 1-epimeric 1-chloro-1-S-phenyl-1-thio-L-gulitol pentaacetates, which were hydrolyzed to provide aldehydo-L-gulose pentaacetate. The same reaction sequences were performed with 6-S-phenyl-6-thio-D-galactose, synthesized in two steps from 1,2:3,4-di-O-isopropylidene-a-D-galactopyranose, furnishing ultimately L-galactose, its diphenyl dithioacetal pentaacetate, and aldehydo-L-galactose pentaacetate. Similar reaction sequences for the chain-terminal interchange of oxidation state in other omega-S-phenyl-omega-thioaldoses may prove useful for the preparation of less-common sugar derivatives.

Synthesis of a trehalose homolog, 6-deoxy-alpha-D-gluco-heptopyranosyl 6-deoxy-alpha-D-gluco-heptopyranoside, and the corresponding bis(heptosiduronic acid).

Crystalline 6-deoxy-alpha-D-gluco-heptopyranosyl 6-deoxy-alpha-D-gluco- heptopyranoside (8) and (6-deoxy-alpha-D-gluco-heptopyranosyluronic acid) 6-deoxy-alpha-D-gluco-heptopyranosiduronic acid (7) were synthesized from alpha, alpha-trehalose (1). Reaction of 2,3,4,2',3',4'-hexa-O-acetyl- 6,6'-di-O-tosyl-alpha, alpha-trehalose with sodium dicarbonylcyclopentadienyliron, followed by oxidative hydrolysis or methanolysis, gave the 2,3,4,2',3',4'-hexa-acetate of 7 or its dimethyl ester, respectively. O-Deacetylation (Zemplén) then gave 7 and its dimethyl ester. Reduction of the hexa-O-acetyldicarboxylic acid with borane-oxolane complex yielded 8. Alternatively, cyanide displacement of hexa-O-acetyl-alpha, alpha-trehalose 6,6'-ditriflate gave the dinitrile hexa-acetate of 7, which was O-deacetylated and then hydrolyzed with alkaline hydrogen peroxide to yield 7. 2,3,4,2',3',4'-Hexa-O-benzyl-alpha, alpha-trehalose 6,6'-ditriflate was similarly converted into the dinitrile, which was hydrolyzed to the corresponding diamide. Treatment of the 2,3,4,2',3',4'-hexa- O-acetyl-alpha, alpha-trehalosuronic acid 16 with thionyl chloride followed by diazomethane gave a crystalline bisdiazoketone which, however, failed to produce the expected bis(heptosiduronic acid) on attempted Wolff rearrangement.

Synthesis of 3-amino-2,3,6-trideoxy-2-fluoro-L-talose and -D-allose [(R)-2-fluoro-L-daunosamine and (R)-2-fluoro-D-ristosamine].

The title compounds were synthesized (as methyl glycosides) starting from 1,3,4,6-tetra-O-acetyl-2-deoxy-2-fluoro-beta-D-glucopyranose. Stereoselective methods of glycosylation gave, via the tri-O-acetylglycopyranosyl bromide, the methyl 2-deoxy-2-fluoro-alpha- and -beta-D-glucopyranoside triacetates. Each anomer was O-deacetylated and further transformed into the corresponding, 4,6-O-benzylidenated 3-triflate, and the triflates were converted by azide displacement into the 3-azido-2,3-dideoxy-2-fluoroglycosides having the D-allo configuration. Hanesssian-Hullar reaction then furnished the corresponding 6-bromo-6-deoxy-4-benzoates, which were dehydrobrominated to give the methyl 3-azido-4-O-benzoyl-2,3,6-tri-deoxy-2-fluoro-alpha- and -beta-D-ribo-hex-5-enopyranosides. Debenzoylation of the alpha-anomer, followed by catalytic hydrogenation, led to methyl 3-amino-2,3,6-trideoxy-2-fluoro-beta-L-talopyranoside [methyl (R)-2-fluoro-beta-L-daunosaminide], whereas the same sequence applied to the beta-anomer afforded methyl 3-amino-2,3,6-trideoxy-2-fluoro-beta-D-allopyranoside [methyl (R)-2-fluoro-beta-D-ristosaminide]. The overall yields for these 10-step sequences were 11-12 and 16%, respectively. The 1H- and 13C-n.m.r. data for the new fluoro sugar derivatives are discussed with respect to the dependence of JF,H and JF,C values on molecular geometry and substituent effects.

A facile formation of 2,5-anhydro sugars by ring contraction in methyl hexopyranoside 2-triflates under conditions of nucleophilic displacement.

Reaction of methyl 3,4-O-isopropylidene-2-O-(trifluoromethylsulfonyl)-alpha-L-fucopyr anoside and its beta anomer with a variety of nucleophilic reagents under mild conditions led to displacement of the triflic ester group, with migration of the ring-oxygen atom to C-2 and entry of the nucleophile at C-1, to give good yields of 2,5-anhydro sugar derivatives. Reagents employed were hydrogen fluoride-triethylamine complex, methanol in the presence of sodium hydrogencarbonate, sodium benzoate, sodium azide, potassium thiocyanate, and sodium borohydride. The same type of substitutive ring-contraction also occurred in methyl 4-O-benzyl-3-O-(4-methoxybenzyl)-2-O-(trifluoromethylsulfonyl)-alp ha-L- fucopyranoside and methyl 4,6-dideoxy-3-O-(4-methoxybenzyl)-2-O-(trifluoromethylsulfonyl)-al pha-L-xylo- hexopyranoside. The reaction is discussed in light of a literature survey of the chemical behavior of hexopyranoside 2-sulfonates in general, and 2-triflates in particular. Direct SN2 displacements, eliminations, and such different kinds of rearrangement as have previously been observed with structural analogs were not encountered in the present study. However, there is some precedent, in hexopyranoside 2-triflates, for the facile rearrangement that the four representatives here investigated have been found to undergo. The synthesis of these triflates from L-fucose is described.

Carbohydrate components for modified anthracyclines: synthesis of derivatives of 3-amino-3,4,6-trideoxy-L-lyxo- and -L-xylo-hexose, and attempts at fluorination of C-2.

Two new trideoxyglycosides, methyl 3,4,6-trideoxy-3-nitro-alpha-L-lyxo-hexopyranoside (7) and its alpha-L-arabino isomer, as well as the known alpha-L-xylo isomer (15), were synthesized from methyl 3,6-dideoxy-3-nitro-alpha-L-glucopyranoside (1) by methods involving elimination and reduction processes in mesylates prepared from 1. Catalytic hydrogenation of 7 and 15 gave the new and the known aminodeoxyglycosides, respectively, both of which were N-(trifluoroacetyl)ated and sub-subsequently O-(trifluoromethylsulfonyl)ated. Various attempts to effect displacement by fluoride ion in the N-protected 2-triflates so obtained, and also in a related 3-azido-2-triflate, were unsuccessful as far as fluorination at C-2 was concerned. Among other products, two new 2-enopyranosides resulting from elimination of triflic acid were obtained.

Nucleophilic ring-opening in a carbohydrate nitrocyclopropane: a stereospecific approach to chiral isoalkyl structures.

Chemical reactions to open the cyclopropane ring in (1R)-1,2-dideoxy-3,4:5,6-di-O-isopropylidene-1,2-C-methylene-1-nitro-D-m annitol (1) were investigated. Catalytic hydrogenation over Pd-C produced the corresponding 1-amino compound, isolated as its N-acetyl derivative, but failed to cleave the ring. However, ring opening succeeded by nucleophilic addition of sodium thiophenoxide to 1, giving 1,2-dideoxy-3,4:5,6-di-O-isopropylidene-1-nitro-2-C- (phenylthio)methyl-D-mannitol. The latter reacted further with thiophenoxide to furnish phenyl 2-deoxy-3,4:5,6-di-O-isopropylidene-2-C-(phenylthio)methyl-D- mannothiohydroximate. Raney nickel converted both of these thio sugar derivatives into the same product, namely, 1-acetamido-1,2-dideoxy-3,4:5,6-di-O-isopropylidene-2-C-methyl-D-mannito l. The use of these transformations for the design of stereospecific syntheses of chiral isoalkyl structures is proposed.

Two syntheses of 3-amino-3-deoxy-alpha-D-altropyranosyl 3-amino-3-deoxy-alpha-D-altropyranoside, a new analog of alpha,alpha-trehalose, involving reduction of a diazide and reductive amination of a diketone.

A new diamino sugar, 3-amino-3-deoxy-alpha-D-altropyranosyl 3-amino-3-deoxy-alpha-D-altropyranoside (5) was synthesized by two routes starting from alpha,alpha-trehalose. The first route involved reduction and deprotection of a previously described, benzylidenated diazido analog. The second approach proceeded from the known 2,2'-di-O-benzyl-4,6;4',6'-bis-O-benzylidene derivative of alpha-D-altropyranosyl alpha-D-altropyranoside, to the corresponding 3,3'-diketone, which was subjected to reductive amination with sodium cyanoborohydride and ammonium acetate. The major product, separated in 39% yield from by-products after N-acetylation, was deprotected to give 5. Four by-products were isolated in low yields and determined to be monoaminated analogs which comprise two epimeric, 3'-hydroxy structures and two 3'-epimeric, 3'-cyano-3'-hydroxy structures in their non-aminated residues. A number of observations concerning the 13C- and 1H-n.m.r. spectra of the products are discussed, especially with regard to chemical-shift dependencies for certain ring and substituent protons, and attention is drawn to some inter-residue shielding phenomena.

Synthesis of (S)-2-fluoro-L-daunosamine and (S)-2-fluoro-D-ristosamine.

Derivatives of (S)-2-fluoro-L-daunosamine and (S)-2-fluoro-D-ristosamine were synthesized, starting ultimately from 2-amino-2-deoxy-D-glucose which was converted, according to the literature, into methyl 2-benzamido-4, 6-O-benzylidene-2-deoxy-3-O-(methylsulfonyl)-alpha-D-glucopyranoside (2). Treatment of 2 with tetrabutylammonium fluoride gave a 63% yield of (known) methyl 3-benzamido-4,6-O-benzylidene-2,3-dideoxy-2-fluoro-alpha-D-altropyran oside (4), together with a 6% yield of its 2-benzamido-2,3-dideoxy-3-fluoro-alpha-D-gluco isomer. From 4, the corresponding 6-bromo-2,3,6-trideoxyglycoside 4-benzoate (6) was obtained by Hanessian-Hullar reaction. Dehydrobromination of 6, followed by catalytic hydrogenation of the resulting 5-enoside, and subsequent debenzoylation and N-trifluoroacetylation, afforded the fluorodaunosaminide, methyl 2,3,6-trideoxy-2-fluoro-3-trifluoroacetamido-beta-L-galactopyranos ide. Reductive debromination of 6, followed by debenzoylation and N-trifluoroacetylation, gave the fluororistosaminide, methyl 2,3,6-trideoxy-2-fluoro-3-trifluoroacetamido-alpha-D-altropyran oside. The 1H-n.m.r. spectra of the new aminofluoro sugars are discussed with respect to the effects of neighboring amino and acylamido substituents on geminal and vicinal 1H-19F coupling constants, in comparison with the reported effects of oxygen substituents.