4472
D. SHAPIRO, E. S. RACHAMAN, A N D T. SHERADSKY
Me thy1
6-Deoxy-2,3-isopropylidene-a-~-mannoseenide .-
147-149", [ a l Z 3-28" ~ ( c 0.5, methanol-water, 2 : 1 , v . / v . ) in 0.6 g. yield. A n d . Calcd. for CsHlaOs (202.20): C , 53.41; H, 6.98. Found: C , 53.18; H, 7.07. The compound did not decolorize bromine water b u t retlucctl arnrnoriiacal silver nitrate solution. I t showed typical infrared carbonyl absorption. It formed a n amorphous his (p-nitrophenylhydrazone j. Amd. Calcd. for C ~ L H P ~ O(470.30): TSO C , 53.39; H , 5.12; S-, 17.78, Found: C, 53.11; H, S.62; S , 16.15. 6-Deoxy-~-gulitol.-i-Deoxy-2,3-isopropylidene-5-keto-~III~IInofuranose (0.10 g. I was reduced with an excess of sodium borohydride in water. .lfter standing 7 hr. a t room temperature, thc remaining borohydride was decomposed by addition i ~ f acetic acid. Sodium ions were removed by passing through il c(11umn of Dowes 5 0 H-, arid boric acid a n d acetic x i t i wcre driven off by repeatedly distilling absolute methanol f n ~ mthe residue. T h e remaining sirup crystallized fr~iin metlxiiiol; yield, 0.059 g . ( 7 3 5 of theory), m . p . 127', 111. n1.p. with authentic compound, 197-128". Muller and Reiclistein's reported m . p . 127'. T h e infrared spectrum of t h e coiiip~)u~itl was identical x i t h t h a t of a reference sample of l-deoxy-r~-glucit~~l (or 6-deox)--L-gulitol).26 A trace of a second B-deo\yIlesitrjl was detected by paper chrornatography. I t showed the same chrorriatographic properties as L-rhamnitol. This tie~iir~nstratcd the position of the keto group in B-deoxy-5-keto-2,3-is(1pr(i1,ylidene-D-mannofuranose.
Methyl 6-deoxy-6-iodo-a-~-mannoside ( I 9 . ) was heated 4 h r . under reflux in absolute methanol (50 m l . ) in which sodium (1.5 g . ) has been dissolved. Most (40 m l . ) of the methanol was removed by distillation under reduced pressure. The residue was taken up in chloroform (50 nil.), washed free of alkali with cold water, and dried over sodium sulfate. T h e chloroform was evaporated under reduced pressure and the remaining oil was distilled a t 0.001 mm. with a n oil bath temperature of 100110". T h e compound is extremely sensitive t o acid-catalyzed Iiydrolysis; even carbonic acid or exposure to air resulted in hydrolysis, rearrangement, a n d crystallization of 6-deoxy-2,3isopropylidene-5-keto-o-mannofuranose. This instability is responsible for an incorrect analysis. T h e compound, when freshly prepared, showed the typical infrared absorption for C=C and decolorized bromine water instantaneously. T h e yield of rnannoseenide was 0.50 g . , 80cC of theory, [LY]"D +51° ( r 0.4, methanol-water, 2 : 1 , v . / v . ) . In 0.01 -1: hydrochloric acid in methanol-water ( 2 : I , v . / v . ) coiitaining U.4c)7c of t h e compound, the specific rotation decreased during 16 h r . from +45 t o -23". Antzl. Calcd. for C I o H I G 0 5 :C, 56.54; H, 7.16. Found: C, 54.23; H, 7.41. T h e methyl 2,3-isopropylidene-n-D-mannoseenidewas also prepared in lower yield by using silver fluoride in pyridine9 for dehydroiodination of methyl 6-iodo-6-deouy-2,3-isopropylidenecu-n-tiiatinoside, 6-Deoxy-2,3-isopropylidene-5-keto-~-mannofuranose .--Methyl r,-deohyisc)propylidene-oi-D-Inannopyranoseenide ( 1 9 . ) was dissolved i n 0.01 .1; hydrochloric acid (methanol-water, 2 : 1 , v.;'v.) and left standing a t room temperature for 16 h r . T h e acid was neutralized with sodium bicarbonate and t h e solution was evaporated to dryness under reduced pressure. T h e residue was extracted with boiling ether and t h e conipourid was induced to crystallize by addition of petroleum ether. It was recrystallized from ether-petroleum ether t o give firm needles, m . p .
[COSTRIBUTIOS F R O M
THE
Yol. 86
Acknowledgment.-LL,7e wish to express our appteciation to G. Cheniae, T. G. Traylor, P. Traylor, J . Bigeleisen, R . C. Fehey, and C . Perrin for helpful discussions and valuable advice. ( 2 5 ) H . hluller and T . Reichstein, Helu. C h i m . A d a . 21, 251 (19:38:. (26) Kindly provided by Dr. A . B . Foster, University of Birmingham.
DASIELSIEFF RESEARCH ISSTITUTE, THEW E I Z M A N N
Synthetic Studies on Sphingolipids.
X.'
ISSTITUTE O F SCIESCE,
REHOVOTH, ISRAEL]
Synthesis of Psychosine'
BY DAVIDSHAPIRO.ELIEZERS. RACHAMAN, AND TCVIA SHERADSKV RECEIVED JUSE 18, 1964 T h e synthesis of psychosine ( V I a ) is described. S-Dichloroacet?-l-3-O-'uenzoylsphingosine ( I V a ) was condeiisecl with acetobromogalactose, and t h e resulting product deacylated bl- sodium methovide t o the S-dichloroacetylcerebroside (\*a)which was hydrolyzed to \.Ia b y warming with 0.3 .V barium hydroxide a t 70-80' for 90 min By t h e same procedure glucnpsychositle and tiihydropsychosine were synthesized. T h e latter glycoside could also he ohtained h y hydrogenolysis of the S-henzylosycarbonyl derivative Yc.
Psychosine ( 1-D-sphingosyl p-D-galactoside, Via) is an alkaline hydrolysis product of the cerebrosides ( I ) in which the amino group forms an amide with a longchain fatty acid."." On the basis of results obtained froin enzymatic studies Cleland and Kennedy5 have recently postulated t h a t this galactoside is an intermediate in the biosynthesis of cerebrosides and other more complex glycosphingolipids. Lye wish to report the synthesis of psychosine and its dihydro derivative. In a recent communication6 we have demonstrated t h a t in order to assure an unequivocal synthesis of a sphingolipid involving substitution a t carbon 1 of the sphingosine molecule it is necessary to block both the amino and the secondary hydroxyl group. Thus. the synthesis of cerasine ( I ) and other natural cerebrosides' (1: For p a r t I X see D Shapiro and E
S
Rachamnn, . V n i u r e , 201, 878
Il:lt341
2 ; Taken f r o m p a r t of a thesis submitted by E S Rachaman t o t h e Senlite of t h e Hebrew University, Jerusalem, i n partial fulfillment of t h e requirements foi- t h e Ph.1) degree, April. 1964. .de and I< u' Humphrey. Biorhim. J . 18, 661 (1924). .dr and R XV,Humphrey. ibid , 2 0 , 82,; (1926). ' Cleland a n d P Kennedy, J Bioi. Chvm . 236, 4 5 (1960) ', See paoer in rei. 1.
*:
was achieved by glycosidation of the disubsti tuted sphingosine (11). The hydrolysis of cerebrosides of type I is known to proceed sluggishly giving psychosine in low yield and of poor quality.8 In contradistinction to the synthesis of cerebrosides in which the amide grouping is retained as part of the molecule, the protecting N-acyl in the synthesis of psychosine must he such as to undergo mild hydrolysis. On the other hand, hydrolysis should not take place prior to the removal of the benzoyl group, since in such a case it would be difhcult to avoid, during the course of the synthesis, 0-N benzoyl migration which proceeds rapidly a t a pH slightly above 7 . \Ye therefore undertook a systematic investigation of such protective groups which were likely to meet these requirements. T o this end we prepared the 3-0-, K-protected bases IVa-h as possible aglucons in the proposed synthesis. Some of these protective groups are well known in the chemistry of amino acids.g Sphingosine is, however, a much stronger 17) r) Shapiro and H 11,Flowers, J . A m . C h e m S O C ,83, 3327 ( l ! ) f i l ) 3) E. Klenk and R. Harle,
2 P h y s r o l . C h e v . , 178, 221 (1928).
SYNTHESIS OF PSYCHOSINE
Oct. 20, 1964
C H3( C He) ,2
-CH =CH -CH
I -CI H -CH2 -0- ga Ia c tScheme ose CH
-
( C H 2)12 C H =C H -C
I OH N H I
I
I
co
-
co co
I V a-h -
I V a-e
I .acetobromo
-CH=CH
b, R =CH3(CH2)14
(Table I )
-
galactose, Hq(CN12
2. OH
I I C&(CH2)22CH3
a,R=CH3(CH2Il2
I 1 CgH5 R'
I l l a,b
I NH I
R-CH-CH-CH20H I I 0 NH I I
I cgH5
-
H -C H -C H 0 H
co co
Ii
I (CH2122CH3
R -CH-CH-CH20H I I 0 NH2
I
0
co
I
4473
R -C I H -C I H -C H2 -e OH
F
#
H
NH I
co I R'
V a-e(Table
a) OH
base, and its amides are expected to be more resistant to hydrolysis. Following our previous practice in this series, we first studied the approach to dihydropsychosine as model compound employing the sulfate of the saturated monosubstituted base I I I b as starting material. The latter compound as well as its unsaturated counterpart I I I a resulted from an acid-catalyzed ring scission of a substituted oxazoline previously described.' lo The compounds IVa-f could be prepared by direct acylation of I11 in aqueous medium using acid chlorides or anhydrides. In order to avoid a possible O+N benzoyl migration the reaction was carried out a t a pH of about 6 in a sodium acetate-acetic acid buffer solution. For the preparation of IVg p-nitrophenyl t-butyl carbonate was used as acylating agent in the presence of 40% potassium acetate solution, the optimum temperature being 60-65°. It is noteworthy that benzoyl migration took place, in part, under the influence of temperature even a t a pH below 7 . Approximately l5-20% of the sulfate were found to be converted into N-benzoyldihydrosphingosine whose quantity increased with elevation of temperature. Finally, IVh was obtained under anhydrous conditions (9) G. W . Anderson and A. C. McGregor, J . A m . Chem Soc., 79, 6180 (1957). (10) D. Shapiro, H. M . Flowers, and S. Spector-Shefer, ibid., 81, 4360 (19591,
by means of trifluoroacetic anhydride and sodium trifluoroacetate. The product gave, however, unsatisfactory analytical values, although its spectrum showed the expected bands. Initially we encountered difficulties in the preparation of IVa and IVb by direct acylation of 111. Since the dichloroacetyl group seemed the most promising one for masking the amine function, an effort was made to prepare IVb by a different route. It was finally synthesized by a sequence of reactions shown in Scheme 11. Treatment of dihydrosphingosine with methyl dichloroacetate gave the N-acyl derivative in almost quantitative yield. The primary hydroxyl was then protected by tritylation and finally the secondary hydroxyl was benzoylated. Removal of the trityl group with dilute acetic acid gave the desired disubstituted base. The high solubility of I X and X in most organic solvents made their purification difficult, and i t was found more advantageous to introduce the p nitrobenzoyl group in X . It is noteworthy that this scheme may be of interest in future synthetic work on sphingolipids, since i t provides the possibility of a direct application of crude natural sphingosine. T h e aglucons IV were condensed with tetraacetyla-D-galactosyl bromide in the presence of mercuric cyanide and in nitromethane-benzene as solvents.
, E. D. SHAPIRO
4474
s. R A C H A M A N , AND T. SHERADSKY
Vol. 86
SCHEME I1 Cln C H C 00 C Ha
R-CH-CH-CHgOH
(CeHsjaCC1
R-CH-CH-CHzOH I OH S H
pyridine
I
COCHClz \-I11 CeHaCOCI
u-
-+CcH5)3 -
R-CH-CH-CH2-O-C(
"-"-"-"-
I
; i; ;
I
1
OH
Dvridine ..
SH
CO
1
CHClz
IX
5. 5.
h
r
90% acetic acid
R-CH-CH-CH~-O-C(CsHs)~ I
1
1
1
0
NH
1
1
1
1
IVb
co co C6H5 CHCL
Lc
t.
c?
d
x
Exploratory glycosidation reactions demonstrated t h a t the compounds IVd-h could be eliminated as possible intermediates in the synthesis of psychosine or dihydropsychosine. Thus, the galactoside Vd was rather stable to hydrolysis and was recovered unchanged after refluxing with aqueous barium hydroxide for 8 hr. 'CYith Ve we had visualized the reduction of the benzoyl group with lithium aluminum hydride and a catalytic hydrogenation of the benzylamino derivative to dihydropsychosine. However, the amide proved to be stable to lithium aluminum hydride even on prolonged boiling in tetrahydrofuran. This is remarkable in view of the fact that N-benzoyldihydrosphingosine is reduced easily by this reagent." We have observad with other amides of this type a similar stability which must be attributed to an effect of the carbohydrate moiety. Glycosidation of IVg and IVh gave the corresponding galactosides only in negligible yields. However, whereas the trifluoroacetyl derivative could be hydrolyzed to dihydropsychosine, attempts to remove the t-butyloxycarbonyl group by means of trifluoroacetic acid under conditions described by Carpinol2 failed. At slightly elevated temperature splitting of the glycosidic bond occurred, apparently with simultaneous elimination of t-butyl alcohol and formation of an oxazolidone ring, as was indicated by the change in the infrared spectrum. The use of the dichloroacetyl group for protection of the amine function provided a smooth route to psychosine and dihydropsychosine. The latter could also be obtained by catalytic hydrogenation of Vc. The crude tetraacetyl galactosides resulting from the condensation of IVa and IVb, respectively, with acetobromogalactose were saponified directly to Va and Vb which were obtained in yields up to 7070. T o maintain these yields it was found essential to remove the mercury salts with hydrogen sulfide, z.e., under slightly (11) H. E. Carter. D Shapiro, and J. B. Harrison, J . A m . Chem. 1007 (1953). (12) L. A . Carpino, ibid., 79, 98 (1987).
5'06.
76,
SYNTHESIS OF PSYCHOSINE
Oct. 20, 1964
4475
TABLE I1
x-A%CTL-DL-SPHINGOSYL- (AND DIHYDROSPHISGOSYL-) Xp., Crystn. %I.[
4glucons V IVa IVb
a b
IX'C
c
IVd IVe
d e
a
O C .
solvent
172 1.55 12.5 148-130 138
Ethanol MeOH-acetonitrile (1:1) Chloroform hleOH-acetonitrile ( 1 : 1) MeOH
yo galactose
(pyridine)
+6.6' 4-1 6' +13 7' +4.6' +16 B o
&D-GALACTOPYRAKOSIDES
Formula
(Va-e), DERIVEDFROM
THE
Calcd.
CznHnKOaClz C , 54 54; C Z ~ H ~ Q X O ~CC, L5 4 . 3 6 , CazHasP;Oo C, 6 4 . 2 9 ; C,sHaiNOa C , 61.75; CaiHaaNOe C, 65.58;
AGLUCOSSI v Founda
H , 8 27, iT, 2 . 4 5 ; C1, 12 39 C , 54 60; H , 8 5 9 ; N , 2 44; C1, 1 2 . 3 4 C , 54 28; H , 9 27; N , 2 . 3 4 C, 63.69; H , 10 17; N , 2 . 7 7 C , 61 05; H, 9 41, N , 2 . 4 6 C , 64 9 8 ;
H , 8 . 3 5 ; N , 2 . 3 4 ; C1, 12 02 H , 8 74; N , 2 68, C1, 11 71 H , 9 30; S , 2 . 5 8 H, 10.03; N, 2.53 H , 9 27; N , 2 . 3 4
found: a , 29.5; b, 29.5; c, 30; d, 36; e, 30.
acidic conditions, prior to deacylation with sodium methoxide. The viscous oil remaining after evaporation of the reaction solvents formed a clear solution in petroleum ether although it was found to contain appreciable amounts of mercury. Direct treatment with alkali, even with sodium bicarbonate, reduced the yields considerably, apparently as a result of cleavage of the glycosidic linkage. \Ye have no explanation for this behavior, except the suggestion t h a t i t involves, a t least in part, a covalently bound mercury in t h e form of
8 I,
!
R-C-X-Hg-N-C-R'
I
0
(1
Amides are known to form complexes of this type which are easily split by sulfides.'3 LYith the hydrolysis of Va,b we encountered an interesting observation. Hydrolysis of natural cerebrosides is usually carried out by means of barium hydroxide in the presence of dioxane to facilitate solutio~~.'~ Following this method we found that the reaction was accompanied by a cleavage of the glycosidic bond giving a low yield of VI. This result was even more distinct with the trifluoroacetyl derivative. However, treatment of a suspension in aqueous barium hydroxide yielded S070 of V I . We attribute this behavior to the strong negative nature of the dichloroacetyl group which places these cerebrosides into the class of alkali-sensitive glycosides. I t is well established t h a t alkali sensitivity of glycosides is a function of the aglucon. It may withdraw electrons from the glycosidic bond as a result of activation by substituents in the P-position. T h e electron attraction inherent in the dichloroacetamido group may be expected to exercise such an effect. The molecule of a cerebroside consists of a hydrophobic and a hydrophilic part. In analogy to the behavior of higher fatty acids on water i t may form a solid and a liquid film consisting of a unimolecular layer of the lipid portion floating on the surface with the sugar moiety in the surface of water. Such a situation may arise in an aqueous suspension of VI. I t will impede ionic interaction between the two parts of the molecule and thus facilitate hydrolysis without affecting the glycosidic linkage. Psychosine is a potential key intermediate in the synthesis of fatty acid labeled cerebrosides and other glycosphingolipids, and i t seemed appropriate to study the acylation of its amino group. The reaction of dihydropsychosine with lignoceroyl chloride in the presence of sodium acetate under the conditions described for the preparation of the disubstituted sphingosines was accompanied by partial acylation of the hydroxyl groups and resulted in low yield. I t was (13) H . Ley and K . Shaefer, Be?., 36, 1309 (1902). (14) H . E. Carter and Y . Fujino, J . Bioi. Chem., 221, 879 (1956).
found that acylation could be effected conveniently and in a satisfactory yield by employing an activated ester such as p-nitrophenyl lignocerate. Experimental Acylation of 111. A . Preparation of IVa-f.-The racemic sulfate of I11 (4.5 g . ) obtained by a n acid-catalyzed ring scission of the corresponding substituted oxazoline' was dissolved in a mixture of tetrahydrofuran ( 7 5 ml.) and 2 -V acetic acid (15 m l . ) , T o the rapidly stirred solution were added simultaneously, during 20-30 min., 50% sodium acetate solution (80 m l . ) and the acylating agent dissolved in dry ether (ten volumes). Eight grains of the anhydride and 30% excess of t h e acid chloride were used, respectively, for t h e acylation of 0.01 mole of the sulfate. ..liter stirring for 4 h r . , the ethereal extract was washed several times, dried, and evaporated in ZJUCUO. T h e oily residue was recrystallized from methanol or acetonitrile. The physical properties and analytical d a t a are summarized in Table I.
B. 3-O-Benzoyl-N-t-butyloxycarbonyldihydrosphingosine (IVg).-To a solution of the sulfate of I I I b ( 9 9 . ) in tetrahydrofuran (150 ml.) and 1 iV acetic acid (50 nil.) were added p nitrophenyl t-butyl carbonate ( 3 g . ) and a 40% solutio11 of potassium carbonate (70 m l . ) . T h e mixture was stirred a t 55-65" for 8 h r . T h e upper layer was then poured into ice-water (300 ml.) containing 3 N sulfuric acid (50 ml.). T h e oily material which separated was taken up in ether and the ether was dried and evaporqted. T h e residue (10.5 9 . ) was digested with hot acetonitrile (100 rnl.). T h e insoluble part ( 3 8 . ) was unreacted sulfate. From t h e filtrate a product melting a t 71-76" ( 3 9 . ) precipitated upon cooling. It was dissolved in ether (90 ml.) and the insoluble part (1.5 9 . ) was filtered and crystallized from methanol. It melted a t 110" and was identified as S-benzoyldihydrosphingosine. T h e ethereal filtrate was evaporated and the residue mas crystallized from acetonitrile (40 nil.), yielding 3 . 1 5 g . , m.p. 89-90". Anal. Calcd. for C S ~ H ~ ~(505.7): NO~ C , 71.3; H , 10.2; h-,2.8. Found: C , 7 1 . 9 ; H, 10.01; S , 2 . 8 9 .
C. 3-O-Benzoyl-N-trifluoroacetyldihydrosphingosine.--X mixture of the sulfate (4.05 g . ) , sodium trifluoroacetate (1.8 g . ) , and trifluoroacetic anhydride (15 m l . ) was stirred a t 40-50" for 2 h r . Most of the anhydride was then evaporated in vacuo and the residue was poured into ice-water and extracted with ether. T h e ether solution was washed with sodium bicarbonate solution until neutral and the solvent was dried and evaporated. T h e oily residue ( 6 8 . ) was dissolved in dry methanol and the solution was refluxed for 1 h r . dddition of ice-water precipitated t h e product which was dried and recrystallized from nitromethane. T h e pure compound weighed 2 . 5 g . and melted at
75O.
N-Dichloroacetyldihydrosphingosine(VIII).--A mixture of dihydrosphingosine ( 5 g . ) and methyl dichloroacetate (50 m l . ) was heated in a gently boiling water bath for 2 h r . The clear solution was cooled, petroleum ether (200 nil., b . p . 60-90') was added, and the precipitate was filtered and washed. Crystallization from methanol yielded 5 . 5 g. (SSL;;), m . p . 142-144'. T h e amide was identical with the product obtained by hydrolysis of IVb with sodium methoxide. Anal. Calcd. for C~oHaeClpXOs:C, 58.25; H , 9.59; C1, 17.20; N , 3.40. Found: C , 58.50; H , 9.44; C1, 17.09; S , 3.63. N-Trichloroacetyldihydrosphingosine, prepared similarly in 80% yield, melted a t 114-115". Anal. Calcd. for C2aHa8ClsSOa:C, 53.75; H, 8.57; C1, 23.80; N, 3.14. Found: C, 53.52; H, 8.33; CI, 23.14; S , 3.44. N-Dichloroacetyl-1-0-trityldihydrosphingosine(IX).-To a solution of N-dichloroacetyldihydrosphingosine ( 8 g . ) in dry tetrahydrofuran (160 ml.) were added freshly prepared trityl
4476
D. SHAPIRO, E. s. KACHAMAN, .4ND T. SHERADSKY
chloride ( 6 g.) dissolved in tetrahydrofuran ( 6 ml.) and d r y pyridine (1.6 g.). After stirring for 30 min., t h e mixture was allowed t o stand a t room temperature ( a b o u t 3 0 " ) for 48 h r . Most of t h e solvent was evaporated in vacuo under reduced pressure and t h e residue was taken u p iu ether. T h e ethereal solution was washed with dilute hydrochloric acid, then with water t o neutral, and was dried aud evaporated. T h e residue was dissolved in warm petroleum ether (50 ml.), and the solution was allowed t o stand a t room temperature for 2-3 hr. and filtered from trityl carbinol. T h e filtrate was cooled and the collected product was washed with a little cold petroleum ether, giving 9 g . (68c/o), m . p . 88-90". Two more recrystallizations a t room temperature from a little petroleum ether a n d from :i little niethanol, respectively, raised t h e melting point t o 94-95". Anal. Calcd. for C . ~ W H ~ : ~ C I ? SCO, I :71.53; H , 8.16; CI, 10.8%; N,2.14. Found: C , 71.40; H , 8 . 3 0 ; CI. 10.33; S , 1.94.
V O l . 86
Glycosidation of 3-O-(~-Nitrobenzoyl)-N-dichloroacetyldihydrosphingosine proceeded according t o t h e general metliotl except t h a t 8OC; of the aglucon was recovered unchanged, even after a re!jus of 18 h r . However, t h e yield of t h e cerehroride I-b was 50'; , based on t h e amount which entered the reaction. Psychosine (m-Sphingosyl 8-D-Galactopyranoside, Via),A suspension of t h e cerebroside \.a ( 1.15 9 , ) in 5f;; aqueous bariuni hydroxide sulutioii (40 m l . ) was rapidly stirred a t & - 7 0 ° for 1 hr. T h e temperature was then raised and rnaintaiiietl a t -,0--80" for an additional hour. T h e reaction mixture was cooled, filtered, and the precipitate \\-as washed rr-itli distilled Lvater. T h e dried product was dissolved in a inistiire of chloroform and methanol ( 2 :1 ) anti passed through a silicic acid column. Chloroform-metllano1 ( 4 . l), followed by a 2 : 1 mixture, reiiiovctl traces of unchanged cerebroside. Elution with methaiiol yieltletl pure psychosine 1930 rng., S O c ; ) . I t was recrystallized froin :i 3-O-Benzoyl-N-dichloroacetyl-1-trityldihydrosphingosine (X). little ethanol or from a mixture of methanol a n d acetonitrile -To a cooled solution of I S (6.65 g . ) in dry pyridine (70 m1.l ( I : 11. I t softened a t 130°, turned yelloiv a t ab(iut 150", a i i ( 1 was added benzoyl chloride ( 2 nil.) and the mixture was left a t melted with deconipwition a t 195-200", [ a ] % - 12.5" (pyritliiie j : room temperature overnight. T h e oily product which separated R cytolipin H = (1.4. upon addition of iceewater was takeii u p i n ether, t h e solution ' 4 n d . Calcd. for C 2 r H I ; S O ; , 0 . 3 H , 0 ; C , 61.21; €T, 10.28; was washed successively with cold 2 S hydrochloric acid, 5 ' ~ ; galactose, 38.3. F o u n d : C , 61.40; H , 10.10; galactose, 3 X 0 . sodium bicarbonate, and water, dried, and evaporated, T h e Psychosine Sulfate.-The base (120 mg. 1 was tiisscJlvetl in residue was recrystallized twice from absolute etliaiiol, yielding absolute ethanol t 4 n i l . ) aiid t h e solution \vas treated Ivitli 1 6 g . (go?), m . p . 72-74'. equiv. of 0 . 2 -T abii)lute alcoholic sulfuric acid, care being t:ikcn Anal. Calcd. for C46Hj;C12N04: C , 72.80; 11, 7 . 5 7 ; CI, t h a t i t is only ilightly acidic t o corigo red. Tlic mixture waq 9.34; K , 1.85. F o u n d : C , 72.42; H , 7.31; CI, 9.67; S ,1.67. allowcd to staiitl overiiight anti the precipitate \\:is filtered a i i d 3-0-(p-Nitrobenzoyl)-N-dichloroacetyl-l-trityldihydrosphingo- a.asheti \vith e t h e r . ;\fter drying in i'utuo over phosphorus sine.-To a cooled solution of p-tiitroberizo~.l chloride 1.2 g . 1 in pentoxide, the sulficte was recrystdlized from absolute e t l i a n ~ ~ l d r y chloroform (15 nil.) were added pyridine (0.6 nil.) and t h e ( 2 5 nd.). I t melted with decomposition a t 205~-210', after disubstituted base I S (3.6 g . ) . T h e mixture was stirred a t softening a t 1t5i)-1R00; [ aI 2 5 ~ 5 . i "(pyridine). room temperature until solution ensued anti kept for 15-16 lir. 4 u n l . Calctl. for CniHa;SO;.0.5H~S04.0.5H~O: C, a t 3 5 " . T h e oily residue obtained after \\-orking up as described H , 9.50. F o u n d : C, 5,5.30; H , 9.45. above was recrystallized from methanol (35 nil.) iii the cold and Dihydropsychosine \vas prepared either by hydrolysis of I'b as a second time from 40 nil. a t room temperature, yielding 2.6 described above or by hydrogenolysis of \-c with 10'; palladiuin g., m . p . 87-90". on charcoal a t 70 p.7.i. ;\fter recrystallization from et1i:iriol A n a l . Calcd. for C46H&lnSrOs: C , 68.66; H , 7.il8; C1, it melted a t l W O ,with softening a t 145'; [ C Y ] % - 7 " (pyridine). 8.8; 5 , 3 . 4 8 . F o u n d : C , 68.59; H , 6.96; C1,8.82; S , 3 . 8 0 . Anul. Calcd. for C?rH?!,SO 62.17; H , 10.65; S , 3.02. 3-0-Benzoyl-N-dichloroacetyldihydrosphingosine( IVb) by Fo~iiid:C , 62.19; H , 10.42; S Detritylation of X.--.l suspension of S ( 7 . 5 g . ) iii BO", acetic N-Dichloroacetyl-I-DL-Sphingosylp-L-glucopyranoside w a c acid (75 nil.) was warmed on a boiling water b a t h wit11 occasional prepared by t h e general procedure, 1n.p. lii0" with previous shaking for 10-15 rnin. T h e clear solution was poured into sintering a t 130c; [ O ! ' ~ Df 5 " (pyridine). ice-water (200 ml. ), a n d t h e precipitate was filtered anti washed A n u i . Calcd. for C26H4;CI?S0,.H?O: C , 58.87; H , 8.36; with water. T h e d r y product \vas dissolved i t i hot h e i m c (40 CI, 12.05. F o ~ i i i d :C , 52.52; H,8.45; C1, 12.17. m l . ) , the solution was cooled, and t h e separated tritylcnrbinol Glucopsychosine ( I-oI--sphingosyl 0-L-glucopyranoside), prewas filtered off. T h e filtrate was concentrated in u z m o and t h e pared by hydrolysis of t h e preceding cerebroside with aqueous residue was crystallized from ethanol (20 nil. ), yielriiiig 3.1 g. barium hydroiide, melted a t 190" with decomposition; [ a ]% (Sic,), m . p . 71-72', -5.7". Anal. Calcd. for C?;H4:XI?S04: C , 62.79; H , 8.39; C1, Anal. Calcd. for C?4H4:SO;.H.0: C , 60.09; €I, 10.29. 13.73; S , 2.71. F o u n d : C , 62.78; H , 8 . 3 2 ; CI, 13.4%; S ,2.56. 3-0-(p-Nitrobenzoyl)-N-dichloroacetyldihydrosphingosine Found: C , 60.14; H , 10.20. N-Benzyloxycarbony1dihydrosphingosine.-To a cooled soluwas prepared sirnilarly from t h e p-nitro derivative of S; yield tion of 3-O-beiizoyl-S-ber1zylosycarbonyldihy~lrospliingosi11e 8 6 5 , m . p . 107-108O. (I\.c, 0.1 g . ) in methanol (9 in1.i was added 1 S sodium liy.-lnczl. Calcd. for C P : H ~ ~ C I ? S ? OC~, : 57.75; H , 7.4: C1, droxide ( 1 i n l . ) and t h e inisture was alloir-ed t o stand :it room 12.64; S,4.99. F o u n d : C , 38.02; H,7.63: Cl, 12.29; S , temperature for 3 h r , T h e crystalline product which separated 5.19. was washed with cold inethariol-water ( 1 : 1j and recrystallized G1ycosidation.-.% solution of t h e benzoylcer:iinide I\' (0.004 from ethyl acetate ( 4 ( i i i i 1 . 1 : yield 70 rug. ( 8 0 ( ; ) ,I I I . ~ . 110mole) in d r y nitromethane (60 t n l . ) and d r y benzene (A0 r n l . ) 11"". was heated with stirring a n d exclusion of moisture t o 100-110" .-1nnl. Calcd. for C2GHISS04: C , 71.7; 11, 10.30; S , 3.21. until 25-30 ml. of t h e solvent distilled off slowly. T h e mixture F o u n d : C , 71.44; H , 10.28; S , 3 . 1 3 . was allowed t o cool t o room teiiiperature and tetraacetylgalx2-Phenyl-4-hydroxymethyl-5-pentadecyl-2-oxazolidone .tosyl bromide (0.004 inole) and mercuric cyanide (0.004 inole) .A solution of 3-O-benzoy1-S-be1izyl~xycarborlyldihy~lrosphi1ig~~were added quickly. T h e weighed materials had becii dried sine 1 I l - e , 1 9 . ) in methanol (ti0 nil.) was treated with 1 ,T soin t ~ a c u oovernight. T h e solution was stirred a t 80" for 8-10 dium hydroxide ( 10 n i l . ) , T h e prccipitate formed w~ dissolved hr., cooled, a n d , after addition of ether, sh:iken several times by slight tr-arming anti t h e solution \\-asleft overnight a t room with cold saturated hydrogen sulfide solution. T h e black preternperature. ;\ddition of water ( 4 0 nil.) coinpleted the precipitate was filtered off and the filtrate was shaken several times cipitatiori. T h e raw product (0.62 g., 9 j r '
