1GS2
G. G. S. DUTTON A4NDK. HUNT
of Sciences of the U1:ited States of America. The model compounds used in the paper chromatography were kindly given by Dr. Carlton W. Dence
[CONTRIBUTION FROM 1HE
DEPARTMENT OF
Vol. 82
of this College, and initial exploratory work on this reaction was carried out by Mr. William Erby. SYRACUSE,
N. Y .
CHEMISTRY, rUIVERSIT1' O F
BRITISHCOLUMBIA]
The Constitution of the Hemicelluloses of Sitka Spruce (Picea sifchensis). 111. Structure of an Arabomethoxyglucuronoxylanl B Y G. G. S. DUTTON A N D I(.HUNT RECEIVED AUGUST10, 1959 Methylation of sitka spruce hemicelluloses obtained by potassium hydroxide extraction of a chlorite holocellulose greparation yielded a homogeneous sample after fractional precipitation Hydrolysis yielded 2,3,4-tri-0-methpl-~-xyfose( 1 mole), 2,3,5-tri-O-methyl-~-arabinose(0 4 mole), 2,3-di-O-methyl-~-xyl~ se (10 moles), 2-O-methyl-D-xj.lose ( 0 3 mole), 3-O-methylD-XyloSe (1 2 moles) and 2-0-(2,3,4-tri-0-methyl-~-glucurono~~l)-3-0-tilcth~l-D-xylose (3.4 moles). The structural features of the polysaccharide are discussed.
Previous papers in this series have reported on the nature of the aldobiouronic acid2 and the mannan portion3 of sitka spruce hemicelluloses. The present paper reports the constitution of an arabomethoxyglucuronoxylan. The current status of research on the constitution of hemicelluloses from coniferous trees has recently been summarized4 and is not repeated here. Sitka spruce sawdust was treated with sodium chlorite and the resulting holocellulose extracted with 5% potassium hydroxide.2 The mixed hemicelluloses were methylated, first with methyl sulfate and sodium hydroxide and finally with Purdie reagents. The partly methylated polysaccharide did not separate from the aqueous solution during the Haworth methylations and had to be recovered by dialysis and evaporation. The fully methylated product was fractionated frcin chloroform solution with petroleum ether and the results are shown in Table 11. Fraction 23b appeared to be homogeneous and was used in the subsequent experiments. XIethanolysis with 2% hydrogen chloride in methanol cleaved the polysaccharide which was separated into neutral and acidic components by the use of ion-exchange resins. The methyl ester of the acidic component was reduced with lithium aluminum hydride and yielded crystalline methyl 2-0(2,3,4-tri-0-methyl-~-glucoppranospl) -3 - 0 - mcthylD-xyloside identical with t h a t originally obtained from western hemlock6 and since obtained from sugar maple.6 The neutral sugars were resolved on a cellulose-hydrocellulose column7 using butanone-water azeotrope as the solvent.8 Three fractions were obtained and paper chromatography showed these to contain 2,3,5-tri-O-methyl-r.2,3-di-0arabinose, 2,3,4-tri-0-methy1-~-xylose, methyl-D-xylose and two monomethyl-D-xyloses. The first two have very similar Rf values and may (1) Presented a t t h e 136th A.C.S. Meeting, Atlantic C i t y , N . J . , Sept., 1959. (2) C. G. S. D u t t o n a n d K. H u n t , THISJ O U R N A L , 8 0 , 4-120 (1958). ( 3 ) C. G . S. D u t t o n a n d K . H u n t , ibid., 8 0 , 5697 (195s). (1) J. K N . Jones a n d T. J. Painter. J . Ciieni. SOC.,573 (1929) Dutton and F. S m i t h , THISJ O U R N A L , 78, 37.14 (19.:(i). mell, C a n . J . C h r m . , 3 7 , 8'33 (1959). ( 7 ) J . D. Geerdes, B. A. Lewis, It. SIontgomery and I;. S m i t h , Ariol. Chem., 26, 264 ( 1 9 5 4 ) . ( 8 ) I,. Boggs, L. S. Cuendet, I. E h r e n t h d , K . Koch and F . Smith, N u l u r e . 166, 320 (1950).
be separated either by using a benzene-ethanolwater solventgor by taking advantage of the preferential furanoside formation of the arabinose derivative.6 The latter method of separation was used and the xylose derivative was characterized as the crystalline 2,3,4-tri-O-methyl-N-phenyl-~xylosylamine'O and the arabinose derivative by paper chromatography after hydrolysis of the furanoside. The 2,3-di-0-methyl-~-xylose,which formed the major component, was identified as the crystalline sugar.l1 The 2- and %O-methyl-~xyloses did not separate sufficiently well to enable crystalline derivatives to be prepared but enough of each sugar was obtained chromatographically pure to permit comparison with authentic samples. From the above experimental evidence i t is clear that the main structural feature of the heniicellulose is a chain of D-xylopyranose units linked through positions 1 and 4. Since hydrolysis involves an increase in rotation i t is presumed that /3linkages are involved. The isolation of the known crystalline disaccharide glycoside from the acidic component shows that the uronic acid occupies a terminal position and that the uronic acid is joined to CSof the main xylose chain thus confirming our previous results.2 The linkage in the aldobiuronic acid was judged to be a because of the high positive rotation and this now has been proved by lead tetraacetate degradation. Since arabinose was only isolated in the form of 2,3,5-tri-O-mcthyl-1~arabinose i t is clear t h a t the arabofuranose units must also occupy terminal positions and strongly suggests that the arabinose is an integral part of the molecule. No free uronic acid was observed after methanolysis and hydrolysis and thus the monomethylxylose units must represent branch points or artifacts caused by incomplete methylation or demethylation during hydrolysis. In view of the relative amounts of the two isomers obtained i t is possible that the arabinose is linked to position 3 of the main chain and that there is limited branching a t position 2. (9) G . A , Adams, Cnx. J . Chern., 33, 56 (1955). (IO) R. A. Laidlaw a n d E. C. V. Percival, J. Ciierit. S o ( - . , l i j O 0 (1949). (11) E. G . M e e k , ibid., 219 (192(i), (12) P. A. J. Gorin and A. S. Perliti, Can. J . C h r i n . , 36, 9!I9 (1H58).
April 5 , 1960
AN ARABOMETHOXYGLUCURONOXYLAN FROM SITKA SPRUCE
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It may be seen from Table I that the sitka spruce “xylan” has a repeating unit of about 16 xylose units to which are attached three .l-O-methyl-~glucuronic acid side chains a t position 2 and an occasional arabofuranose unit a t position 3. In this respect the sitka spruce arabomethoxyglucuronoxylan most closely resembles t h a t of western hemlock6 and apart from the presence of arabinose is similar to that from loblolly pine.4 European larch13 appears to have an essentially linear acidic “xylan” containing arabinose whereas t h a t from Norway spruce is linear but without arabinose. l 4 The presence of arabinose seems to be a minor and variable quantity and arabinose is absent entirely in the “xylans” from apple wood and cherry w00d.l~
solution from the final dialysis was concentrated to ca. 350 ml., cooled in ice and acidified with ice-cold hydrochloric acid ( 6 N ) . The curdy precipitate was dissolved in chloroform which was washed until free of chloride ion and then evaporated to a glassy solid (14 g.). The supernatant remaining after the precipitation of the polysaccharide was neutralized to PH 7 and extracted overnight with chloroform. A further quantity (1.5 9.) of polysaccharide was recovered which was not further examined. The partially methylated hemicellulose (14 9.) was dissolved in acetone (50 ml.) and methyl iodide (100 ml.) added. Silver oxide (25 g.) was added in 5 portions over a 7-hr. period to the stirred and refluxing solution. Two further methylations were carried out using the same quantity of reagents but without the addition of acetone. There was finally obtained a glassy solid (6.7 g.) which was fractionated. Fractionation of the Methylated Hemicellulose.-The methylated hemicellulose (6.7 g.) was dissolved in chloroform (100 ml.) and diluted with ethyl ether (100 ml.). A small amount of inorganic material was removed by centrifugation TABLE 1 and the resulting clear orange solution fractionated by the HYDROLYSIS PRODUCTS OF SITKA SPRUCEMETHYLATEDaddition of petroleum ether (30-60’). Methanolysis of Methylated Hemicellulose.-Fracticn XYLAN 23b was considered to be homogeneous and the methoxyl Mole value indicated a fully methylated pentosan. Accordingly Wt., ratio 0.66 g. was dissolved in methanol (30 ml.) containing 2% Sugar derivative mg. (approx.) hydrogen chloride and the solution refluxed until the rota2,3,4-Tri-O-methyl-~-xylose 28 1 tion was constant (9 hr.). The solution was neutralized 2,3,5Tri-O-methyl-~-arabinose 12 0.4 (AgzC03) and evaporated to give a sirup consisting of methyl 2,3-Di-O-methyl-~-xylose 256 10 glycosides (0.70 g.). The sirup was dissolved in saturated barium hydroxide solution (20 ml.), allowed to stand over3-O-Methyl-~-xylose 28 1.2 ni5ht a t room temperature and finally heated for 1 hr. a t 8 0.3 2-0-Methyl-~-xylose 60 . The cooled aqueous solution was then continuously 2-0-(2,3,4-Tri-0-methyl-~-glucuronosyl)extracted with petroleum ether (30-60”, cu. 200 ml.) for 20 3-O-methyl-~-xylose 195 3.4 hr. The excess barium hydroxide was neutralized with Dry Ice, the solution was filtered and the filtrate passed through It is of interest to note t h a t in all cases but one a column of Amberlite IR-120 resin to remove barium ions. Separation and Identification of the Acidic Component.in which the structures of xylose hemicelluloses acidic eluate was passed through a column of Duolite Ahave been reported the uronic acid has occupied 4The resin which removed the acidic component. The neutral a terminal position and it has invariably been iso- eluate was evaporated to a sirup (0.26 g . ) , The acidic com-
lated as an aldobiuronic acid because of the difficulty of hydrolyzing the glycosidic bond. The one exception so far noted is t h a t of hemicellulose B from ground nut shells.16 It is claimed that in this instance the L-arabinose units are linked to position 4 of the glucuronic acid which in turn is glycosidically linked to the main xylose chain. This exception is important since i t suggests an alternative biosynthetic pathway and the structure of this hemicellulose is a t present being reinvestigated by us. Experimental’’
Isolation of Sitka Spruce Hemicellulose.-Chlorite holocellulose was extracted with 5y0 potassium hydroxide and the hemicelluloses precipitated by the addition of ethanol after acidification with acetic acid. From 300 g. of sawdust there was obtained 23.9 g. of hemicellulose having- .[ C. ~ I ~ -8’ (c, 0.6 in water). Methylation of Sitka Spruce Hemicellulose .-Hemicellulose (18g.) was dissolved ih water (175 ml.) and solid sodium hydroxide (50 9.) added. Methyl sulfate (150 mi.) and sodium hydroxide solution (350 ml.) were added with vigorous stirring over a 3-hr. period and acetone was added periodically to control the foaming. During this period the temperature was maintained a t 50’ and finally was raised to 90’ for 0.5 hr. Since the partly methylated polysaccharide did not separate, the cooled solution was dialyzed overnight against running water and the polysaccharide recovered by evaporation to a thick sirup. The methylation was repeated five more times and dialysis was necessary each time. The (13) G. 0. Aspinall and J. E. McKay, J . Chem. SOL.,1059 (1958). (14) G. 0. Aspinall and M. E. Carter, i b i d . , 3744 (1956). (16) G. G. S. Dutton, S. A. McKelvey and G . Murata, to be published. (16) B. Radhakrishnamurty and V. R . Srinivasan, Proc. I n d i a n Acad. Sci., 49, 98 (1959). (17) All evaporations were carried out at ca. 15 mm. and at a bath temperature not exceeding 40°.
TABLEI1
FRACTIONATION OF METHYLATED HEMICELLULOSE Fraction
Total petroleum ether added, ml.
Weight, g.
[aIz5D
(CHCld
Ohle,
%
..
1 190 0.67 -30.8’ 2 220 .53” -28.9 38.8 3 260 .53 -29.4 39.2 4 310 .83 -24.1 .. 5 410 i0 -20.1 .. 6” .. 1.42 5.4 .. 7b .. 0.42 .... 23a 40 0.14 -32.9 .. 23b 55 .66 -37.5 39.4 23c 80 .10 -22.6 .. ~D By precipitation of mother liquor from fraction 5 into an excess of petroleum ether. * By evaporation of mother liquor from fraction 6. Amalgamate fractions 2 and 3; refractionate to give 23a, b, c.
.
-
ponent was eluted with sodium hydroxide (15 ml., 1 N ) and the deep yellow solution passed through a fresh column of Amberlite IR-120 resin. The colorless eluate was strongly acidic and was evaporated to dryness (0.195 g.). The acidic component was dissolved in methanol (15 ml.) and treated with ethereal diazomethane until a green color persisted. The ester (0.186 g.) was recovered by evaporation and was reduced with lithium aluminum hydride as previously described.6 There was thus obtained methyl 2 - 0 - (2,3,4 - tri-0-methyl-~-glucosy~)-3-O-methyl-~-xy1os~de, on seeding with an authentic sample, having m.p. and mixed m.p. 164-167” and [ a ] 2 3 D+84” (c 0.8 in water). Separation and Identification of the Neutral Components.-The petroleum ether extract (0.12 g.) and the neutral eluate from the Duolite A 4 resin (0.26 9.) were combined and the mixed glycosides hydrolyzed by heating with sulfuric acid (30 ml., 1 N)on a steam-bath until the rotation was
1684
JOHN
constant (8 hr.).
H. FRIED, GLEN E. -ART11
The solution was neutralized (BaC03)
;mtl evaporated t o :L sirup which was extracted once with mcthanol to remove a sinal1 amount of inorganic niatter. A mixture of the methylated sugars (0.353 g.) was dissulverl it1 butanone-water azeotrope ( 2 nil.) cuntaininga few drops o f methanol and added to the top of a cellulose-hydrocellulose column (40 X 2.8 cm. i.d.). Two drops of Sudan I\' was added to mark the front and the column developed with
butanone-water azeotrope. The column was jacketed a t 30" and t h e front time was 3.5 hr. with a rate of flow of 25 ml./hr. Tubes 1 (from the dye)-50 were collected every 15 min. and thereafter a t 30-min. intervals, Tyith the results shown in Table 111.
TABLE I11 SEPARATION OF XEUTRAL SUGARS Tube iiiinilrcr
Component niirnlrt~
3-11
1
13-27 60-85
2 3
Identity
2,3,5-Tri-O-methyl-~-arabiiiose 2,3,4-Tri-O-methyl-~-xylose 2,3-Di-O-methyl-~-xylose 2- and 3-0-171ethgl-~-sylc,se
Component 1.-The sirup (40.3 mg.), which had a very slight positive rotation, was shown readily by paper chromatography (butanone-water) t o be a mixture. Using the values [.ID -38.5' (c 1 in methanol) for 2,3,5-tri-O-methyIL-arabinose and [ a ]18.5" ~ (c 1 in methanol) for 2,3,4-trI0-methyl-D-xylose, i t was concluded from the rotation of the sirup that there were present ca. 13 mg. of the arabinose derivative and 27 mg. of the xylose derivative. mrhen the sirup was dissolved in methanol containing 1% HCI, the solution became strongly positive in rotation and reached a constant value in 3.5 hr. -4fter neutralization
[CONTRIBUTION FROM
THE
MERCK, S H A R P
-1ND
LEWISH.S h R E T T
l7Ol. 82
(.4g*c03) the mixture of neutral sugar arid furanoside was separated (111 the cellulose -hydrocellulose coluiiiii . 'Tile tlcutral sugar r w s dctectctl witli p-:iilisidinc > p r : ~ yi l l t1Ilws 10-31 (10 i n i l l . frartirrris) a t i d the fiirwosidr i l l 1u1w- 1 S (positive hlolisch : i d negative p-anisitline). Component la. 2,3,Ei-Tri-O-methylsirup obtained in tubes 1-8 was hydrol acid and oii chromatographic examinatit consist of a single component and t o Ijehavc in the same n a y as an authentic sample of 2,3,5-tri-0-niethyl-r-arabi11~~se. Component 1b. 2,3,4-Tri-O-methyl-~-xylose.-The sirup from tubes 10-21 (20 rng.) partly crystallized on seeding anti was characterized as the crystalline 2,3,i-tri-O-metliyl-Nphenyl-D-xylosylamine, m .p. and mised m.p. 98-100'. Component 2. 2,3-Di-O-methyl-~-xylose.-This component (256 ing.) crystallized on seeding and after rccryst:illization had m.p. and mixed m.p. 7 5 - i i o . Component 3. Mixture of 2- and 3-O-Methyl-~-xyloses. --The sirup (36 rng.) had [ o ] * ~ D +19.8" and was shown by paper chrornatography for 20 hr. in hutanone-water t(Jbe :t mixture of monometh~lu~-loses.Froin tuhes (30-62 and 81.8.5 it mas possible to obtain chrom:~tograpliicallypurc saniples corresponding to 3- and 2-O-methyl-~-xylose, respectively, but not in sufficicnt quantity to prepare any crystalline derivatives. From t h e rotation of the sirup i t was judged t o contain ca. 78%; 3-0-ineth~i-D-sylosearid 22%, 20-methyl-n-xylose.
Acknowledgment.-The authors are grateful to the National Research Council of Canada for continued financial support and to Dr. J. E=. N. Jones F.R.S. for the gift of crystalline 2,."s-tli-O-~riethyl-Dxylose. T'ASCOUVER,
AVD
E.e.,CASAl).3
DOHMERESEARCH LABORA rORIE'3l
Alkylated Adrenal Hormones. The Synthesis of 5-Methylated Pregnanes BY JOHN H. FRIED, GLEXE. ARTH AND LEWISH. S.IRETT RECEIVED AUGUST14, 1959 The synthesis of 5-methylpregnane-llP,17o1,2l-triol-3,20-dione acetate and 5-methyl-l-pregtietie-l1B,l~o1,21-triol-3,20dione acetate via angular niethylation of a suitably protected 6-ketone I1 is described.
Although there is no evidence that allo-dihydro- ing material for the projected synthesis. Although hydrocortisone exhibits anti-inflammatory activity I1 possesses carbonyl functions a t C-6 and C - l l l when administered systemically to animals, local ac- selective alkylation of the 6-ketone was judged to tivity has been demonstrated with this compound.' be the more likely possibility due to the relatively The possibility that the lack of systemic activity unreactive character of saturated 1l-ketosteroids4a; could be due t o rapid reduction of the 3-ketone furthermore, on the basis of the stabilitiesdb of the function, prompted us t o prepare the sterically enolates involved and in the absence of over-riding hindered 5-methyl analog. This paper describes steric effects, preferential methylation of I1 at C-5 The two possible the synthesis of 5-methylpregnane-11P,17aJ2l-appeared to be indicated.5 triol-3,20-dione 2 1-acetate and 5-methyl- 1- p e g - enolic forms of the C-G ketone have double bonds nene-1 lp,17a,21-triol-3,20-dione 21-acetate. Chu- between carbons five and six and six and seven. Turner6 has shown that in the cholestane series the man2 has described the preparation of &-methylcholestane-3@,6P-diol by reaction of cholesteryl-@- former is La. 1.5 kcal. more stable than the latter. oxide and methylmagnesium iodide. Attempts to This is in agreement with the initial formation of a utilize this reaction with 17aJ20,20,21-bismethyl- C-5 substituted bromide as the result of brominaenedioxy - 3 - ethylenedioxy - 5,6p - oxidopregnane- tion of 6-ketosteroids.' Initial attempts to methylate I1 using either po110-01(I) did not give the desired 5a-methyl product tassium t-butoxide in t-butyl alcohol or sodium hybut a compound of unknown constitution. However, 17a,20,20,21-bismethylenedioxy-3-dride in benzene were unsuccessful; however, addiethylenedioxy-allo-pregnane-6,1l-dione(II), re(4) (a) Private comrnunicntion from Dr. R. E. Rcyler; cf., howcently r e p ~ r t e d ,appeared ~ t o be a suitable start- ever, E . R . H. Jones, G. D 3,.leakins and J . S. Stephenson, J . Chein. (1) J. 1.. Hollander, E. AI. Brown, Jr., R. A . Jessar, L. Udell, S . Smukler and M. A . Bowie, A n i t . Rherimalir Diseases, 13, 297 (1954). (2) 31 C h u m a n , J . C h e m . .Tor. J n f i n i i . P z i ~ eC h e w Sect., 7 0 , 2:,8 ( 1 8.49). J C J I ~ R U A I . .81, f X ) J. 11. Pried, G. R. Arth : m i I.. €1. S a r e t t , THIS I%.'iR (l!l,i!i)
.So
