THE STRUCTURES OF GALACTOSYL-LACTOSE AND GALACTOBIOSYL- LACTOSE PRODUCED FROM LACTOSE BY SPOROBOLOMYCES SINGULARIS1

P. A. J. GOIIIX, J . F. T. SPENCEII, AND H. J . PHAFF~ iVational Research Coz~ncil of Canada, Prairie Regional Laboratory, Saskatoon, Saskatchewan

Received Decentber 12, 1963

ABSTRACT

Sporobolonzyces singzrlaris, when grown in a ~iledium containing lactose, utilized the glucose portlon as a carbon source and produced by tra~lsglycosplation as main products a trisaccharide 0-8-D-galactopyranosyl-(1 + 1)-0-13-D-galactoppranosyl-(1 --, 4)-D-glucose ( I ) and a tetra- saccharide O-13-~-galactopyra1losyl-(l --, 4)-0-~-~-galactopyra1losyi-(l + 4)-0-p-D-galacto- pyranosyl-(1 + 4)-D-glucose (11) (50% combined yield). Oligosaccharides of higher luolecular weight were not detected.

INTRODUCTION

Sporobolomyces sing~~laris was isolated by Phaff and do Carmo-Sousa (1) from frass of bark beetles attacking the western hemlock (Tsuga heterophylla). This yeast when grown on yeast nitrogen base, a chen~ically defined medium (Wickerham (2)), utilized lactose but not galactose. It was shown that the galactosyl moiety, instead of occurring free in the medium, was transferred to another lactose ~nolecule to yield a trisaccharide.

Phaff and do Carmo-Sousa showed that a yeast extract - lactose mediunl at pI-I 6 supported a more vigorous growth of the yeast, but did not result in any oligosaccharide formation (3). When the pH of the latter medium was adjusted initially to 3.75 the yield of the trisaccharide increased to substantial levels and a tetrasaccharide appeared. The tetrasaccharide is for~ned preferentially a t pH values slightly higher than that optimal for trisaccharide formation.

The oligosaccharides thus forrned were fractionated on a charcoal column to give a crystalline trisaccharide and an amorphous tetrasaccharide. The trisaccharide contained galactose and glucose. The latter was shown to be the reducing end-unit by acid hydrolysis before and after sodium borohydride reduction. Partial hydrolysis yielded 4-0-P-D-galacto- pyranosyl-D-galactose (4) and lactose, but in low yield. The 0-P-D-galactopyranosyl- (1 -+ 4)-0-P-D-galactopyranosyl-(1 -+ ~ ) - D - ~ ~ u c o s ~ (I) structure indicated by these data was confirmed by conversion to 0-P-D-galactopyranosyl-(I -+ 4)-0-P-D-galactopyranosyl- (I -+ 2)-D-erythritol (IV) by lead tetraacetate oxidation followed by sodium borohydride reduction ( 5 ) . This material consumed 4 moles of sodium periodate and released 1 mole of acid.

The tetrasaccharide contained galactose and a glucose reducing end-unit, which was linked in the 4-position as shown by its lead tetraacetate oxidation characteristics (6). The oxidation was carried out on a larger scale and the product reduced with sodiul-n borohydride. The resulting polyol appeared to be 0-D-galactopyranosyl-(I -+ 4)-0-D- galactopyranosyl-(1 -+ 4)-O-~-gala~t0pyran0~)il-(l -+ 2)-D-erythritol since it consumed 5 moles of sodium periodate with concoinitant forination of 1 mole of acid. Its specific rota- tion, which was close to that of galactobiosyl erythritol, suggested p-linkages. The identity of two of the three linkages in the tetrasaccharide was confirmed by prolonged oxidation

Canadian Journal of Chemistry. Volume 42 (19G4)

'Isszied as 1V.R.C. No . 7869. 2Departn~ent of Food Science azd Technology, University o j California, Davis, California.

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C:\N.+IDI;IN J0URN.-IL OF CIIER.IISTRY. VOL. 42. 1964

SPOROBOLOMYCES L A C T O S E

S INGULARIS

1 . 4 MOLES I. 2 MOLES

P b ( O A c I 4

2. NoBH4

with lead tetraacetate to give a trialdehyde. This was reduced with sodium borohydride to a polyol (111) which was partly hydrolyzed (7) to 0-0-D-galactopyranosyl-(1 --t 4)-0-0- D-ga lac topyran~s~l - ( l --t 2)-~-erythritol (IV). I t was shown that the other linkage in the tetrasaccharide was 1,4 as follows. Oxidation of the galactotriosyl erythritol with periodic acid gave a product which was reduced with sodium borohydride. The polyol was hydro- lyzed to erythritol and glycerol, which were compared by gas-phase chromatography a s their acetates and shown to be present in equilllolar amounts. The tetrasaccharide fraction therefore contained, as a inajor component, the galactotriosyl glucose s t ruct~ire repre- sented by 11.

P-Transglycosyl activity of Sporobo~omy~es s ing~~ lar i s under these conditions appears t o be quite specific: a galactosyl n~oiety was transferred to the free 4-hydroxy group of lactose, the predonlinant sugar in the medium. The trisaccharide thus forined in turn con- tains a free 4-position which could be substituted with another galactose residue.

The final products have different structures from earlier reactions carried out using P-galactosidases. In general 0-fi-D-galactopyranosyl-(1 --t 6)-0-0-D-galact~pyranos~l-(1 --t

4)-D-glucose has been found to be the inail1 product. Pazur and co-workers (8, 9) have isolated this trisaccharide using a yeast lactase preparation. With culture filtrates or cell- free extracts of P. chrysogen~~m, appreciable quantities of this trisaccharide have been prepared by Ballio and Russi (10). When glucose was used as the acceptor, LVallenfels and co-workers (11) have shown with E. coli that the 1,6 link is fornled in preference to 1,3 and 1,4 links. The only report of preferential synthesis of galactosyl glucoses, other than the l , G form has been that of Wallenfels and Fischer (12), who showed that a calf intestine enzyme synthesized 1,3 and 1,4 links rather than the 1,6 link.

The enzyme responsible for the transgalactosylation reaction appears to be associated with the cells. This fact may be responsible for the more quantitative nature of the trnns- galactosylation reaction in this system than that studied with soluble lactase preparations. Particularly a t low pH values, i t may be presumed that the galactosyl-enzyme complex,

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GORIN ET AL.: G:\LACTOSYL- A N D GALACTOBIOS\iL-LACTOSE 1343

fornled after one molecule of lactose is cleaved, reillains intact until a second lnolecule of lactose approaches this site. At this stage the galactosyl nloiety is transferred to lactose rather than to water. A second galactosyl-enzyme complex could be responsible for transferring an additional galactosyl unit to the trisaccharide, thereby for~lling a tetrasaccharide.

EXPERIMENTAL Prod~lctiolr of Oligosaccl~arides

The organism was grown on a medium containing 10% lactose and 0.757; Difco yeast extract. Inoculum was grown in 500 n ~ l Erlenmeyer flaslcs containing 100 ml medium a t 24" C on a Gurnp shaker (240 r.p.m., 1 inch eccentricity). Cultures 24-28 hours old (100 1111 ~I~OCUIUIII) were used to inoculate a 5-liter New Bruns- wick stirred ferinentor containing 3 liters of medium a t pH 3.75. Initial adjustment of the pH was essential. At p l l values above 4, no oligosaccharides were obtained. The incubation tenlperature was 22" C, air flow rate 1 liter/minute and stirring speed 375 r.p.m. The progress of the fermentation was followed by means of paper chromatography, using ethyl acetate -acetic acid - water (G:k2 v/v) as solvent and the silver nitrate -alkali dip reagent (13) for detection of the sugars. The process was complete in 3-4 days. The super- natant liquid \\-as recovered by centrifugation and decolorized with activated charcoal (Darco G-60).

Isolation of Oli~osacckarides A portion of the hltered material corresponding to a starting weight of 35 g lactose was fractionated on a

column of Darco G-60:Celite 545 ('200 g:200 g) (14). This \\?as washed \\~it-h water, then 570 ethanol which eluted galactose and glucose (trace). Ethanol, 1070, eluted some lactose, then t\vo oligosaccharides moving a t a slightly faster rate than lactose on paper chromatograms. Later fractions contained the trisaccharide. Ethanol, 15%, removed the rest of the trisaccharide so that in all 12.0 g were obtained. Increasing the con- centration to 20% developed the tetrasaccharide (5.1 g) and later fractions were contaminated with a paper chromatograpl~ically slower-nloving sugar.

A more q~~an t i t a t ive analysis \\,as carried out by cellulose column chromatography. n-Butanol, one-half saturated with water, eluted galactose (0.25 g) and a trace of glucose. Acetone-water (4:l v/v) gave lactose and a disaccharide \vhich ran a little faster (0.64 g), trisaccharide (2.11 g), tetrasaccharide (1.17 g), and a material running slightly slower than the tetrasaccharide (0.21 g).

Characterization of the Trisaccl~aride ( I ) The trisaccharide crystallized from 90% aqueous methanol, yielding a first crop of 5.1 g and a second crop

of 2.0 g. Recrystallization from the same solvent gave material with m.p. 225-231" C and [ f f ] ~ +GS0 + + 45" (c, 1.0, HzO). Calculated for C18H3z016: C, 42.9; H , G.4. Found: C, 42.6; H, G.4570.

Hydrolysis a t 10O0 C for 3 hours in iV sulphuric acid gave galactose and glucose on a paper chron~atopram (solvent, butanol-pyridine-water 3: 1: 1 ; spray, p-anisidine hydrochloride (15)). A portion of the trisaccharide (5 ing) was reduced with sodium borohydride (5 mg) in water (5 cc) for 3 hours. The product on acid hpdro- lysis gave galactose only on a paper chromatogram with the reducing sugar spray.

The trisaccharide (1.0 g) was partially hydrolyzed in N sulphuric acid (5 ml) a t SO0 C for 45 minutes. The solution \\'as neutralized (BaCOy), filtered, and evaporated. Examination on a paper chro~natogra~n in ethyl acetate -acetic acid- water (9:2:2 v/v) showed the presence of t\vo disaccharides. These were separated on Whatman No. 3 paper sheets in an identical solvent to give lactose (72 mg) (1n.p. and mixed 1n.p. 197-201" C after two recrystallizations from methanol) and faster running 4-0-P-D-galactopyranosyl-D- galactose (80 mg). The latter was recrystallized twice from methanol-ethanol to give material (10 mg) which had an X-ray powder diagram identical with an authentic specimen (4).

Lead tetraacetate oxidation showed the reducing end of the trisaccharide to be linked in the 4-position (G), since 0.19, 0.48, and 1.08 moles of oxidant were consumed after 2, 5, and 15 minutes respectively.

The trisaccharide (1.0 g) was oxidized with lead tetraacetate (2 moles) in acetic acid (100 ml), containing water (2nd) . After 1 hour the reaction mixture was treated with oxalic acid (2 moles) in acetic acid. The lead osalate which formed was filtered off, and residual lead tetraacetate removed by addition of ethylene glycol (0.5 ml). Evaporation yielded a residue which was treated with sodium borohydride (1.0 g) in water (50 ml). After 1 hour excess reagent was destroyed with acetic acid and Amberlite IR120 was added to the solution. Filtration and evaporation gave a material fro111 which boric acid was removed by repeated addition and evaporation of methanol. The resulting polyol n ~ i x t ~ ~ r e was fractionated on a cellulose column using acetone-water (6:l v/v) as solvent. 0-P-D-Galactopyranosyl-(1 + 4)-0-P-D-galactopyranosyl-(1 + 2)-D- erythritol (0.37 g) was obtained and t\vo recrystallizations from methanol gave material with n1.p. 180-lS3" C and [ f f ] ~ +13" (c, 1.0, H20) . Calculated for CIGHaoOld: C, 43.05; H, 6.8. F o ~ ~ n d : C, 43.1; H , 6.8%. The polyo1 consumed 3.23, 3.70, 3.97, and 4.2G moles of sodium periodate releasing 0.85, 0.97, 1.07, and 1.19 moles of acid after 1, 3, 5, and 23 hours respectively.

Characterization of the Tetrasaccl~aride (11) Using methods identical with those used with the trisaccharide, the tetrasaccharide was sho\vn to contain

galactose and a glucose-reducing end. The tetrasaccharide had [el" +43" (c, 1.2, H?O) and when oxidized

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1344 CANADIAN JOURXAL OF CHEMISTRY. VOL. 43. 19Gi

with lead tetraacetate it c o ~ ~ s u ~ n e d 0.47,0.86, and 1.50 moles of oxidant after 3, 5, and 15 minutes respectively. The gradual uptalce showed that the reducing end was 1,4-linked.

The tetrasaccharide was converted to its corresponding erythritol deri\,ative by lead tetraacetate oxidation followecl by sodiu~n boroh~dr ide reduction by the method described above. For fractionation of the product on a cellulose column acetone-water (5 : l v/v) eluant \vas used. The product (0.41 g from 0.80 g s t a r t i ~ ~ g material) was an~orphous and had [a]" +19" (c, 1.0, H2O). 011 periodate oxidation it consumed 3.50, 4.40, 4.58, and 5.07 moles of oxidant after 1, 2.5, 4, and 23 hours respectively with concomitant formation o[ 0.55, 1.06, 1.13, and 1.23 nioles of acicl. Since these data showed that the t\vo central units of the tetra- saccharide can be linlced either 1,2 or 1,4 the following experiment \vas carried out.

The erythritol derivative (30 ~ n g ) was oxidized overnight in water (5 ml) containing periodic acid (100 mg). The reagent \\;as then clestroyed with barium carbonate and the solution filtered. The oxidation product was then reduced, with sodium borohydride (100 mg), and the reaction mixture worl<ed up as previously described in this section. The resulting polyol was hydrolyzed by heating for 3 hours a t 100' C in iV sulphuric acid (1 rnl). The acid was neutralized (BaCOa) and the so l~~ t ion filtered and evaporated. The product con- tained erythritol and glycerol, but no hexose. The relative proportion of these polyols was co~npared by acetylation (hot NaOAc-Ac?O method) and gas-liquid chronlatography (G.L.C.) of the acetates a t 150' C 011 a col~unn of 2% LAC-IR206 on Chromosorb I\'. In terms of peal; areas the glycerol and erythritol acetates \\rere present in the ratio 1.67:1, respectively. UThen a similar experiment mas carried out with galnctobiosyl erythritol the polyol nlixture 011 acetylation and G.L.C. examination gave peal; areas of 3.63:l for the glycerol and erythritol acetates. The erythrito1:glycerol ratio was 2.2 times that obtained from the galactobiosyl erythritol mixture. This shows the central units of the tetrasaccharide to be 1,4-linked and the structure of the tetrasaccharide to be mainly 0-D-galactopyranosyl-(1 + 4)-0-D-galactopyranosyl-(1 + 4)-0-D-galacto- pyranosyl-D-glucose.

0-8-~-Galactopyranosyl-(1 + 4)-0-6-D-galaclopyra?zosyl-(1 + 2)-D-erytlzritol (I v) fronz 0-6-~-Gu/actopyranosyl- (1 -, 4)-0-8-D-galactopyrano~yl-(1 + 4)-0-6-D-gala~t0pyra?t0~yl-(~ + 4)-D-glz~cose (11)

The tetrasaccharide (0.90 g) \\,as dissolved in water (5 ml) and treated with lead tetraacetate (3.G g) in acetic acid (250 1111). After 15 hours ethylene glycol (0.5 tnl) was added, follo\ved by oxalic acid (0.9 equiva- lents) in acetic acid (25 1111). The precipitate mhich formed was removed by liltration and the filtrate evap- orated to dryness. X solution of sodium borohydride (1.0 g) in water (25 rill) was added and the reduction allowed to proceed for 2 hours. Excess reagent was then destroyed with acetic acid and the reduction product (111) \vas obtained by the method described previously in this section. On a paper chro~natogran~ it ran slightly faster than galactobiosyl erythritol (solveilt, acetone-water 4 : l v / \ ; spray, ammoniacal silver nitrate). Only a small amount of galactotriosyl erythritol was present.

The oxidi~ed tetrasaccharide derivative \vas hydrolyzed a t 60' C overnight in 0.1 N hydrochloric acid (25 ml). The solution was then neutralized ( A ~ P C O ~ ) , filtered, and the filtrate evaporated to a crust. This was fractionated on a cellulose column using acetone-water (4: 1 v/v) as s o l v e ~ ~ t . Galactobiosyl erythritol (0.30 g) was obtained and this was recrystallized twice fro111 aqueous n~ethanol to yield a product with m.p. 186-188" C and [ a ] ~ +l i0 (c, 1.0, H20). Calculated for C16H30011: C, 43.05; H , 6.8. Found: C, 42.9; H , 6.87;. The X-ray diffraction patterns of the product and authentic material were identical.

ACKSO\\;LEDGMESTS

The a u t h o r s wish to thank ;\Iiss E. B a r k e r of the Departnlent of Food Sc ience and T e c h n o l o g y , U n i v e r s i t y of Ca l i fo rn ia , Davis, a n d M r . N. R. G a r d n e r f o r t e c h n i c a l ass is t -

ance. T h a n l t s are d u e also to AIr. ;\/I. A I a z u r e k for c a r r y i n g o u t c a r b o n and hydrogen a n a l y s e s , and A h . W. C. Haid f o r p r e p a r i n g X-ray c r y s t a l p a t t e r n s .

REFEREXCES

1. I-I. 1. PHAFF and L. DO C.4R~lo-So~sh. Antorlie \:an Lceu\ve~~hoel<, J. Microbial. Serol. 28, 103 (10G.2). 2. L. J. \\,.~CKEILIIA>I. Taxononly of yeasts. U.S. Dept. Xgr. Tech. Bull. 1029 (1951). 3. H. J. P H ~ F F and L. DO CAIIIIO-SOUSA. Unpublished \vork. 4. J . I<. GILLFI~UI, A. S. PERLIX, and T. E. T I ~ E L L . Can. J. Chem. 36, l i 4 l (1958). 5. A. J . CHARLSOT, P. A. J . GORIN, and A. S. PERLIX. Can. J . Chem. 34, 1811 (1956).

A. J . CHARLSOT and A. S. PERLIS. Can. J . Chem. 34, 1200 (1056). : I . J . GOLDSTEIK, G. W. HAY, B. 2. Lrivrs, and F. SIIITH. iibstracts of Papers, 135th Meeting of the American Chemical Society, Boston, Mass. April, 1950. p. 3D.

8. J . H. P..zzu~~. Science, 117, 355 (1953). 9. J . H. PAZUI~ . J . Biol. Chem. 208, 439 (1954).

10. A. BALLIO and S. Russr. Tetrahedron. 9. 125 (1060). , , ~~

11. I<. ~VALLENFELS, D. BECK, and J . L E H ~ A N K . l31;~ublished work. Cited Advan. Carbohydrate Chem. 16, 255 (1961).

12. I<. WALLEXPELS and J. FISCHER. Z. I'hysiol. Che~n. 321, 223 (1060). 13. v\'. E. TREVELYAN, D. P. ~ ' ~ ~ O C T E R , and J . S. HARRISON. hTature, 166, 444 (1950). 14. R. I,. ~VHISTLBR and D. F. Dullso. J. Am. Chem. Soc. 72, 677 (1950). 15. L. HOGGH, J. I<. N. JONES, and W. H. \YADIIAS. J. Chem. Soc. l i 02 (1950).

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