PHILIP A. J. GORIN and MYTOSK MAZUREK. Can. J. Chem. 53,1212(1975). The I3C signals of most of the more common sugars and their methyl glycosides have been assigned, based on the a-carbon and a-carbon effects that occur on deuterium substitution. For pyranoses having the same hydroxyl configuration, replacement of the CH,OH substituent at C-5 with CH,, CO,H, CO,Me, or H is accompanied by displacement of the C-4, C-5, and when applicable C-6 signals. The magnitudes of the displacements in the glucopyranose and mannopyranose series are almost identical and were of aid in assigning signals in the 13C spectrum of methyl (methyl a-D-mannopyranuronosid)uronate. The displacements, however, differ from corresponding ones observed in the galactopyranose series. Some assignments are made for the I3C signals of structurally related methyl aldofuranosides. PHILIP A. J. GORIN et MYTOSK MAZUREK. Can. J. Chem. 53,1212(1975. On a attribue les signaux en r.m.n. du 13C de la plupart des sucres usuels ainsi que de leurs glycosides de methyle; ces attributions ont Cte faites en se basant sur les effets que produisent les carbones a et des substitutions par du deuterium. Pour des pyrannoses ayant la m&me configuration des groupes hydroxyles, le remplacement d'un substituant C H 2 0 H en C-5 par CH3, C02H, C0,Me ou H s'accompagne par un dkplacement des signaux en '2-4, C-5 et lorsque c'est possible en C-6. L'amplitude de ces deplacements dans les series glucopyrannoses et mannopyrannoses sont presque identiques et a ete fort utile pour attribuer les signaux dans le spectre r.m.n. du 13C du (a-D-mannopyrannuronoside de m6thyle)uronate de mithyle. Les dCplacements different toutefois des deplacements correspondants observCs en serie galactopyrannose. On a fait quelques attributions pour des signaux de I3C dans des aldofurannosides de methyle qui leur sont relib d'une f a~o n structurale.[Traduit par le journal]In a previous study (I), the carbon-13 magnetic resonance (I3c.m.r.) spectra of deuterated derivatives of ap-D-glucose, ap-D-mannose, and ap-D-galactose were interpreted and discussed. With the aid of two suitable deuterated derivatives of each aldose it was possible to make an unambiguous assignment of each signal, since the signals of the 13C nucleus attached to the deuteron (a-carbon) disappeared and those of the adjacent P-carbons were displaced upfield by 0.02-0.10 p.p.m. (1). Since some of the assignments carried out elsewhere have proved to be incorrect, the studies in this laboratory have been alized by observing the expected a-carbon (2) and the P-carbon deuterium isotope effects (1) ( Table 1). Although, for the most part, the signal of the' a-carbon disappears, in the case of 5 -2~ derivatives of pentoses, or the 6-2H derivatives of 6-deoxyhexoses and methyl 6-deoxyhexopyranosides, the a-13C signals appear as triplets at 0.4 p.p.m. upfield (3). The P-carbon effect appeared as an upfield shift of 0.04-0.09 p.p.m. on monodeuteration, 0.07-0.12 p.p.m. on dideuteration, and 0.17 p.p.m. for the C-3 signal of p -~-a r a b i ...

AsotoOocter vinelandii produces a partly acetylated exocellular polysaccharide which consists mairily of ~-11lann~lrorIic acid ~~r~i t s and a small proportion of L-guluronic acid units. I t resenibles alginic acid since it contains 4-0-linked mannuronosyl residues and guluronosyl residues which arc 4-0-and (or) 5-0-linked. The specific rotation of bacterial sodium alginate is close to t h a t of its algal counterpart, thus suggesting t h a t the glycosidic configurations irre similar.Several species of Azotobacter are Itno\\-11 to form capsules, and some produce exocellular polysaccharides. Parilih and Jones have isolated exocellular polysaccharides froin Azotobacter indicum and, after fractionation, obtained an acidic component I\-11ich consisted predominantly of 0 -D -g l u c o p y r a i~u r o~~o s y l glycero-D-iiiannol~eptopyraiiosyl repeating units (I). The capsular polysaccharides ofAzolobacter clzroococcu?n \\-ere isolated by La~vson aiid Stacep (2) and metliylated, and a iieutral portion of the methylated polysaccharide \\las hydrolyzed to 2,3,6-and 2,4,6-tri-0-methyl-D-glucose, 2,3-di-0-inethyl-D-glucose, and 2,4,6-tri-0-methyl-~-g&ctose. Pro111 ail acidic mcthylated polysaccliaride fraction 2,4,K-tri-0-methyl-D-glucose, 2,3-di-0-methyl-D-glucuronic acid, and a partly methylated glucose derivative \\rere obtained. 'l'hese t\\-o iiiethylated fragments \\-ere also obtained from the acidic exocellular polysaccharide of Azotobacter indicztm, thus suggesting that the t\vo acidic polysaccharides may be siinilar in structure.These acidic polysaccl~arides differ from those forined exocellularlj-bj-Azotobacter vinelandii. Cohen and Johnstone reported that hydrolysis in aqueous acid gave rise to galacturonic acid, siualler aniounts of D-glucose and rhainnose, and possibly a trace of mannuronolactone (3). Another component \\.as later characterized bj-Claus as 2-keto-3-deoxygalactonic acid ( 4 ) . . q closely related Azotobacter species, A. agilzs, did iiot produce exocellular slinle, and it \\-as concluded that this difference streilgtheiled the designation of t\vo distinct species, A . agilis and A . vznelandz~ (,5). Tlie coniposition of the capsule of A . agills \\-as different from that of the exocellular polysaccharide of A . vinelandli and contained galactose aiid rhainnose units and a thiobarbituric acid positive moiet)-(3). The structure of a possible exocellular polygalacturonide formed bj-11. vinelandiz \\-as of interest and required further in\7estigation.A . vznelandi~ \\as gro\\-n on Burlt's nledium, and produced exocellular polysaccharides \\lhich \\-ere isolated by precipitation with ethanol. Hydrolysis of the precipitate I\-ith hot aqueous acid gave hexuronic acid and smaller amounts of glucose, arabinose, ribose, and rhaninose on paper chromatograms. This hydrolj.sis pattern suggested that a polysaccharide consisting exclusively of hexuronic acid residues \\-as present; purification \\.as effected by precipitation from the concentrated culture filtrate by acidification with hydrochloric a...

The oil forlned during fernlentation by a strain of Torldopsis nrag~roliae consisted 1nai111y of partly acetylated 2-0-0-D-glucopyranosyl-D-glucopyrano units attached 0-glycosidically to 17-L-hydroxyoctadec;unoic ancl 17-L-hydroxy-9-octadecenoic acids.A strain of osmophilic yeast, Torulopsis magnoliae, isolated fro111 SOW thistle petals, was found to produce (1) an extracellular, heavier than water oil. The main components of the oil are now shown to be glycolipids (I and 11) consisting of 2-0-/3-D-glucopyranosyl-D-glucopyranose units linked /3-glycosidically to 17-L-hydroxyoctadecanoic and 17-Lhydroxy-9-octadecenoic acids, the sugar moieties being partly acetylated.The oil was labile both to acid and alkali. I-Iot methanolic sodium methoxide removed the acetyl groups, which constituted about 1.5 substituent groups per mole (the positions of these substituents were not determined). The deacetylated product, after fractionation, yielded a solid (I and II),which contained allnost equal quantities of lactonic and acidic glycolipids, and it was on this mixture that the characterization studies of the sugar portion were carried out.Acid methanolysis of the deacetylated oil yielded 2 moles of methyl-a-D-glucopyranoside and 1 mole of a mixture of fatty acid methyl esters. Gas -liquid phase chromatography of the esters on a silicone column showed one principal peak a t the C20 saturated position with a shoulder suggesting partial resolution of saturated and unsaturated esters (2). However, on a butanediol succinate colulnn (3) no peaks appeared after 2 hours (normal emergence time for a C20 ester was 30 minutes). The properties suggested that the major con~pounds present were C18 esters containing a polar substituent, such as a llydroxyl group (4) and the presence of the latter group was confirmed by infrared spectroscopy. From the quantity of permanganate-periodate reagent (5) consumed, the mixture was shown to contain 65% of nlonoolefinic ester. Fractional crystallization of the methyl esters also yielded 65% unsaturated and 35% saturated ester. However, later fermentations contained only about 10yo of the saturated material. Catalytic hydrogenation showed that the unsaturated c 1 8 ester (IV) contained one double bond and the product obtained was identical with the saturated Cl8 con~ponent (111). The position of the hydroxyl group in these compounds was shown to be a t C17, by the formation of 17-0x0-stearic acid (V) when the saturated acid was oxidized with chromium trioxide. An authentic specimen of V was synthesized by the method of Bergstrom et al. (6) from 15-bromopentadecanoic acid. The latter was prepared from 15-hydroxypentadecanoic acid which was in turn obtained from the 15,lB-dihydroxypalinitic acid described by Leinieux (7).The unsaturated component (IV) was shown to be 17-hydroxy-9-octadecenoic acid methyl ester since the acid on oxidation with permanganate-periodate (5) yielded azelaic 1Manz4script received Decevlber SO, 1960.

Yeast forms of Paracoccidioides brasiliensis grown in liquid medium produced exocellular components. Immunodiffusion reactions and immunoprecipitations of 1311-radiolabeled antigenic components with sera from patients having paracoccidioidomycosis (PCM) were used to monitor the isolation of specific constituents. Components having the main antigenic activity (fCon A) were isolated by exclusion from a Bio-Gel P30 column, followed by successive binding of eluted material to a Sepharose-concanavalin A column, and elution. The product contained, from sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, a minor 43,000molecular-weight (MW) component (gp43), a polydisperse high-MW glycoconjugate, and a diffusely migrating 55,000-MW glycoprotein (gp55). Other components, including a 72,000-MW glycoprotein, were irregularly expressed. The high-MW glycoconjugate complex contained, on the basis of methylation and 13C nuclear magnetic resonance data, a branched structure of mainly mannopyranosyl units. These were nonreducing ends, 6-0-, 2-0-, and 2,6-di-O-substituted, and the specific rotation of +160 indicated that the glycosidic configurations of the units were a and 0i in a ratio of ca. 1:1 (concanavalin A binding indicated that nonreducing ends or 2-0-substituted units or both of ot-D-mannopyranose were present). A small proportion of nonreducing end units of D-galactopyranose were also present in this polysaccharide. gp55 is a glycoprotein containing a complex carbohydrate moiety with fucose, mannose, galactose, and glucose, either as terminal nonreducing units or substituted in positions indicated by methylation data. Both PCM and normal human sera precipitated the high-MW glycoconjugate from 131I-labeled fCon A preparations, whereas gp55 was unreactive with human sera. gp43 was a specific antigenic component of P. brasiliensis culture filtrates which could be isolated in a pure form by gel filtration column chromatography (Sephadex G150) or by Sepharose-patient immunoglobulin G affinity chromatography. 1311-labeled gp43 reacted equally well with 10 PCM sera and hyperimmune rabbit serum against the band E antigen of Yarzabal at a 10-3 dilution. At the same dilution, no reaction was detected with sera from normal individuals and from patients with other mycoses. Similarly, only PCM sera and the hyperimmune anti-E serum gave precipitin lines with gp43 in the less sensitive immunodiffusion tests. gp43 consisted of three components, with pl 6.7, 6.4 and 6.2, all of which reacted with PCM serum.

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