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Synthesis of glycosyl diglycerides
CHEMISTRY AND PHYSICSOF LIPIDS 10 (1973) 267-285. NORTH-HOLLANDPUBL. CO.
SYNTHESIS OF GLYCOSYL DIGLYCERIDES V.I. SHVETS, A.I. BASHKATOVA and R.P. EVSTIGNEEVA * The Lomonosov Institute o f Fine Chemical Technology, Moscow, U.S.S.R.
Received July 5, 1972 Accepted August 1, 1972
Synthesis of mono- and diglycosyldiglycerideswith natural structure from 1,2-di-O-acyl-snglycerols, 1,2-O-isopropylidene-sn-glycerol,2,5-methylene-D-mannitolby the orthoester method of glycosylationis reported.
1. Introduction
Much attention is now devoted to the study of the structure, properties and biological functions of glycosyl diglycerides. These compounds are very common in the vegetable kingdom and isolated from bacterial cells and tissues of the central nervous system [ 1 - 3 ] . Glycosyl diglycerides are essentially derivatives of 1,2-di-O-acyl-sn-glycerols [ 1 - 3 ] . Their carbohydrates involve mono- and oligosaccharide structures (mostly derivatives of D-galactose, D-glucose, and D-mannose) [ 1 - 3 ] . Glycerol is esterified by saturated and unsaturated fatty acids C 15 - C 18 [ 1 - 3 ] . Glycosyl diglycerides from vegetable and animal tissues are characterised by 1,2-trans-glycoside bonds with bacterial cells containing in addition 1,2-cis-glycosides [ 1 - 3 ] . The bonds between monosaccharides have various configurations and are in different places [ 1 - 3 ] . Glycosyl diglycerides participate as components of cell membranes in many biological processes such as photosynthesis [4, 5], transport of carbohydrates through membranes [6], polysaccharide biosynthesis [7, 8], and so on. Isolation of glycosyl diglycerides from natural sources is effected through separation of lipid mixtures [ 1 - 3 ] . Yet, even chromatographically homogeneous samples of natural glycolipids of this class are not individual compounds, being a homologous mixture of diglyceride derivatives [ 1 - 3 ] . It is therefore of great importance to find a synthetic method that would enable the structure of glycosyl diglycerides to * Translated by A.L. Pumpiansky, Moscow.
268
KI. Shvets et al., Synthesis of glyeosyl diglycerides
be unambiguously determined and their biological role at the molecular level studied. To this end a number of structural problems concerning the spatial forms of glycosyl diglycerides should be discussed and stereospecific methods to construct glycoside bonds used. At present there are only several papers on this subject. They are all based on glycosylation according to Koenigs-Knorr [7, 9-12] which is not stereospecific [9] for the case under study. This study is concerned with a glycosyl diglyceride synthesis by the orthoester method of 1,2-trans-glycosylation [ 13]. This method has been chosen because of its stereospecificity and high yields as distinct from that of Koenigs-Knorr [13]. The only example previously reported in the literature is the glycosylation of glycerol derivatives by the orthoester method exemplified by 3-O-(/3-D-galactofuranosyl)-sn-glycerol [14]. Our synthetic schemes of mono- and diglycosyl glycerides are based on the interaction of orthoesters of mono- and disaccharides and alcohols (1,2-di-O-palmitoyl-sn-glycerol, 1,2-O-isopropylidene-sn-glycerol and 2,2'-O-bis-(1-Opalmit oyl-sn-glycerol).
2. Results and discussion
2.1. Synthesis of monoglycosyl diglycerides 2.1.1. Monoglycosyl diglycerides obtained from 1,2-diglycerides The synthesis was effected by direct and two-stage glycosylation (scheme 1, table 1). The first route [I 5, 16] was based on glycosylation of 1,2-di-O-palmitoyl-sn-glycerol (1) [17] with equimolar amounts of D-mannose, D-glucose, and D-galactose tert-butyl orthoesters (IIa-c, x = Bu-tert.) [16, 18, 19] in boiling chlorobenzene in the presence of a catalytic amount of 2,6-1utidinium perchlorate (0.02 mole per mole of orthoester II).* The optimum reaction time was found to be from 2.5 to 3 hr. The reaction products were chromatographed on silicic acid to give acetylated glycosyl diglycerides IIIa-c in a 37-41% yield. The configuration of the glycoside bond of compounds IIa-c was determined by IR and PMR spectra and o.r.d, curves. Thus the IR spectrum of mannoside Ilia showed the C - H absorption band at the anomeric centre to be at 840 c m - 1 , this points to the a-glycosydyl bond; in IR-spectrum of glycosides IIIb, c the same absorption was at 890 c m - 1, characteristic of/3-glycosides [20]. The same conclusion followed from PMR spectra, where in the case of mannoside IIIa the proton doublet at C 1 reveals a chemical shift characteristic of a-glycoside (6 4.84 p.p.m.) [20],
* The molar ratio of orthoester, alcohols and perchlorate will be further the same if not stated otherwise.
269
V.L Shvets et al., Synthesis of glyeosyl diglycerides
and a constant of spin-spin interaction (J1,2 = 1.9 Hz) between two equatorial protons [20]. CH2OH
R'-O-CH 2
I
R"_O-CH 2
f
H-C-OCOClsH31 + R-O-X --~
(II)
I
CH2OCOCIsH31 (I) "l
1
H-COCOClsH31
H-C-OCOClsH31-'+
I
l
/
CH20COCIsH3t
CH20COC15H31 (Ili)
~
R-O-CH 2
R'-O~CH2
H-C-OCOCIsHal
(IV)
R"-O-CH 2
H-C-OAc *---
I
I
I
CH20Ac (VI)
CH2OCOC15H31 (VII)
I
H-C-OH CH20H (V)
Here and below
AcOCH 2 R=
.Me
CH 20Ac
CH 20Ac
co/p-o\
a)
AcOO ~
b)
Ac
e)
Me
CH=OAc R'=
a)
•
Ac
CH2OAc
b)
CH= OAc
c)
Ac OAc
CH2OH R" =
OAc
CH~OH
CH2OH HO
a) HO ~
b)
)c-----O
e) H OH
X = 1) Bu-tert: 2)Et Scheme 1.
OH
270
V.Z Shuets
et al., Synthesis of glyeosyl diglycerides
TABLE 1 Physico-chemical constants of compounds prepared on synthesis of glycosyl diglycerides Compound
IIIa from Ia llIb from Ia
Yield (%)
4i .4 38.4
Melting point (°C) (solvent)
[c~] ~3°d
TLC
Rf
28.0-29.0(methanol) 80.0 80.5(methanol)
+24.2 7.2
0.6(1) 0.6(1)
llIc from Ia
37.0
45.0 46.0(methanol)
-0.61
0.6(I)
IVa from Ilia
92.3
108.0-1 lO.O(methanol)
+27.0
0.5(5) 0.55(7) O.85(8)
IVb from lllb
90.5
138.0-140.O(methanol)
-11.8
0.5(5) 0.63(7) O.86(8)
IVc from IIIc
82.7
141.0-142.0(methanol)
-6.53
O.5(5) 0.6(7) 0.82(8)
Vlla from I
49.4
68.0-69.0(hexane)
+11.27
0.3(2)
Vllb from I
40.2
85.0-86.0 (hexane)
+ 19.1
0.6(2)
Vllc from 1
41.4
35.0- 36.0(methanol,he xane)
+38.2
IXa from VIII
47.2
47.0-48.0(ether-hexane, 1 : 1 0 ) + 4 5 . 3
IXb from VIII
46.3
IX from Xllc b Xa from IXa Xb from IXb
68.4
Xc from IXc
86.2
- 5.9
0.5(4)
XIIc from VIII
50.2
+70.3
0.5(3)
-12.8
0.33(3)
45.5
- 6.3
0.33(3)
69.5
+37.48
0.5(4)
5.83
0.5(4)
Xllla from XVa c
85.3
XVa from XIV
43.8
102.0-103.0(ether -hexane 1:10)
0.6(2) 0.33(3)
lO0.O-lOl.O(ether)
+32.4
0.33(1)
35.0-37.0(methanol)
+22.0
0.31 (1)
XVIa from XVa
95.3
+14.0
0.6(1)
XVIIa from XVIa
91.2
108-O-109.0(methanol)
+24.0 e
0.5(5)
XX from XVII1
48.5
49.0-50.O(ether-hexane, 2:9)
+ 7.5
0.8(9)
XXI from I
80.1
109.0-110.O(methanol)
- 4.53
0.4(1)
XXII from XXI
92.2
224.0-225.0(methanol)
- 9.4
0.55(6)
a. Ilia c were also synthesised by isomerisation of V l l a - c in 40.0, 45.1, and 40.25% yield, respectively; l l l a - c was also obtained by acylating X to result in 93.6, 91,8, and 92.6% yield, respectively; Ilia was produced from Xllla, yield 93.2%. b. IXc was also obtained from VIII in a yield not exceeding 10%.. c. Xllla was prepared by esterification of Xa to yield 52.8%. d. 0.2-0.8% solution in chloroform.
V.I. Shvets et aL, Synthesis o f glycosyl diglycerides
PMR, 8 p,p.m. H1
Formula endo-C-Me
271
Calculated (%)
Found (%)
C
C
H
H
4.84(J1,2 = 1.9 Hz) 4.56(Jt, 2 = 6.9 Hz)
C49H86014 C49H86014
65.50
9.64
65.54
9.54
65.50
9.64
65.12
9.68
4"52(Jt,2 = 8.0 Hz)
C49H86014
65.50
9.64
65.27
9.68
4.93(J1, 2 = 1.9 Hz)
C41H78010
67.36
10.75
67.22
10.72
4"64(JI,2 = 5.7 Hz)
C41H78Olo
67.36
10.75
67.39
10.72
4.62(J1, 2 = 5.8 Hz)
C4tH78Olo
67.36
10.75
66.97
10.81
C49H86014 C49H86014 C49H86014
65.50
9.64
65.24
9.54
65.50
9.64
65.53
9.85
65.50
9.64
65.38
9.64
4.84(J1, 2 = 1.9 Hz)
C2oHaoO12
51.94
6.54
52.02
6.69
4"66(J1,2 = 6.9 Hz)
C2oH3oO12
51.94
6.54
51.90
6.49
4.52(Ji,2 = 7 Hz)
C,2oH3oO12
51.94
6.54
51.67
6.61
CITH26012I-L20
46.40
6.36
46.42
6.02
C17H26012.½H20
47.33
6.31
47.48
6.31
C17H26012
48.30
6.20
48.27
6.50
1.77 1.73 1.70
4"52(JI ,2 = 7 Hz)
1.70 C2oHa0012
51.94
6.54
51.75
6.40
4"93(J1,2 = 1.85 Hz)
C33Hs6013
59.95
8.52
60.18
8.73
4.87(Jl,2 = 2.0 Hz)
C67Hl12026
60.40
8.47
61.07
8.59
4"85(JI,2 = 1.5 Hz)
C51H88014
66.28
9.58
66.66
9.76
C43H8oO1o
68.21
10.65
68.59
10.91
C2sHaoOt s
50.71
6.08
50.81
6.06
C61H102022 C47H88015
61.69
8.65
61.15
8.81
63.20
9.93
63.16
10.03
1.78 4.52(3"1,2 = 7 Hz) 4.61(Ji, 2 = 6.4 Hz)
272
V.1. Shvets et al., Synthesis o f glyeosyl diglyeerides
With glucoside IIIb and galactoside IIIc the same doublet appears at 6 4.56 p.p.m. (JI,2 = 6.9 Hz) and 6 4.52 p.p.m. (J1,2 = 8 Hz) respectively, thus pointing to the/3-glycoside bond (spin-spin interaction between two axial protons) [20]. The shapes of the o.r.d, curves of glycosides IIIa-c were in accordance with the configuration suggested at C 1 [20]. Glycosyl diglycerides lIIa-c were also obtained through the scheme of two-stage glycosylation with intermediate isolation of orthoesters VIIa-c [21]. The reaction of 1,2-di-O-palmitoyl-sn-glycerol (1) with ethylorthoacetate monosaccharides (IIa-c, X = Et) [22, 23] was carried out conventionally by reesterification [24] in boiling dichloroethane in the presence of a catalytical amount of YsOH (0.02 mole per mole of orthoester VI). The optimum reaction time was found to be 2.5 hr. Orthoesters VIIa-c were isolated chromatographically on alumina or silicic acid in a 45-50% yield. The structure of orthoesters VIIa-c was proved by IR and PMR spectra, as well as by the hydrolytic probe for the orthoester grouping (0.1 N H2SO 4 in 90% aqueous acetone) [13]. The presence in the PMR spectrum ofa singlet 6 1.7-1.77 p.p.m. (C-Me) pointed to orthoesters VIIa-c predominantly in the form of endoisomers [25, 26]. Isomerisation of orthoesters VIIa-c into glycosides lIIa-c proceeds in weakly polar solvents in the presence of perchlorates of pyridine bases [24] with optimum conditions involving boiling dichloroethane, 0.02 mole of 2,2'-dipyridilium perchtorate per mole of orthoester VII and 1.5 hr reaction time. The physico-chemical properties, IR and PMR spectra and o.r.d, curves of the resulting IIIa-c glycosides chromatographycally obtained on alumina in a 40-45% yield were similar to those of glycosides IIIa-c prepared by direct glycosylation of I. From the preparative point of view preference should be given to direct glycosylation as it involves less stages and leads to higher glycoside yields (37-41% of IIIa-c from direct glycosylation and 16 25% from the two-stage glycosylation), as well as excludes the treatment of labile orthoesters VIIa -c. Deacetylation [12, 27] of compounds Illa-c was carried out by H2NNH 2 "H20 (5 moles per acetate group) in 85% ethanol or methanol boiled for a short time.* Crystallisation from ethanol gave 1,2-di-O-palmitoyl-3-O-(a-D-mannopyranosyl)-sn-glycerol (IVa), 1,2-di-O-palmitoyl-3-O-(/3-D-glucopyranosyl)-sn-glycerol (IVb) and 1,2-diO-palmitoyl-3-O-(~3-D-galactopyranosyl)-sn-glycerol (IVc) in 83- 92% yield. The structure of glycosides IVa-c and the configuration at C 1 were established by IR and PMR spectra and o.r.d, curves and found to be essentially similar to those of acetylated compounds IIta-c. To study the structural composition of glycosides IVa-c use was made of the acid and alkaline hydrolysis and the decomposition products were found by PC and TLC to involve on partial acid hydrolysis dipalmitin and corresponding monosaccharides, on alkaline hydrolysis mannosyl-, glucosyl- and galactosyl glycerols Va-c, * In other cases the reaction was effected similarly.
V.L Shvets et al., Synthesis of glycosyl diglycerides
273
respectively, and palmitic acid, on complete splitting including acid and alkaline hydrolysis palmitic acid as well as mannose, glucose and galactose, respectively. The products of alkaline hydrolysis soluble in water-glycosyl glycerols V a - c were isolated in a free state or as hexaacetates VIa-c obtained on treatment of compounds Va-c with acetyl chloride in carbon tetrachloride in the presence of pyridine. Glycosyl glycerols V, VIa-c were found to be identical to similar compounds obtained by deacylation of natural glycosyl diglycerides [28-30] and to those synthesised by the described methods [9, 11 ], which excluded racemisation at the glycerol chiral centre (table 2). Comparison of mannosyl-, glucosyl- and glactosyl dipalmitin IVa-c and similar natural glycosyl diglycerides [28-30] pointed to their identical physico-chemical and chromatographic properties, IR and PMR spectra and [a] 2D0. Mannosyl- and glucosyl glycerides IVa, b were the first synthetic compounds of this structure. Synthesis of galactosyl diglyceride IVc was previously reported [12], but the comparison of its characteristics (m.p. 56 °, [a] 20 absent; data for deacylation product-galactosyl glycerol - are: - m.p. 135-138 °, [a] 20 0 o) with respective constants of our synthetic 1,2-di-O2palmitoyl-3-O-(3-D-galactopyranosyl)-snglycerol (IVc, m.p. 141.0-142.0 o , [a] D zo -6.53 o ; data for the deacylatlon . product are: 3-O-3-D-galactosyl-sn-glycerol (Vc) - m.p. 136-137 ° , [a] 20 -7.9 ° ; 3-O-a-D galactosyl-sn-glycerol [9] - m.p. 150-152 °, [a] 20 +155 o) as well as with the characteristics of a similar compound (IVc, m.p. 152.0-154.0 °, [ct] 20 _2.04 o) [30] semi-synthesised by esterification of 3-O-3-D-galactosyl-sn-glycerol (Vc, m o 137 ° 20 o . . . "*" [0e] D --8.12 ) when isolated on deacylatlon of natural galactosyl dlglycerides, suggested this compound to involve a mixture of anomeric glycosides.
2.1.2. Monoglycosyl diglycerides obtained from 1,2-O-isopropylidene-sn.glycerol This synthetic route to glycosyl glycerides was chosen in order to avoid a number of difficulties arising in synthesis starting from 1,2-diglycerides. The proposed alternative [14, 15] overcame the main disadvantages of the former synthesis due to the comparative unavailability of 1,2-di-O-acyl-sn-glycerols (particularly with unsaturated acids) [31 ] and to the evolution of compounds Illa-c with properties rather similar to those of 1,2-diglycerides. The synthesis was based on the initial formation of glycosyl glycerols followed by introduction of fatty acids into the glycerol moiety of the molecule (scheme 2, table 1). Ethylorthoacetates of D-mannose, D-glucose, D-galactose (IIa-c, X = Et) were made to react with 1,2-O-isopropylidene-sn-glycerol (VIII) [17] under conditions of direct glycosylation of compound I. Glycosides IXa-c, chromatographically isolated on silicic acid were identified by chemical and physical methods, just as done previously, as 1,2-O-isopropylidene-3O-(2,3,4,6-tetra-O-acetyl-a-D-mannopyranosyl)-sn-glycerol(IXa, 47.2% yield), 1,2O-isopropylidene-3-O-(2,3,4,6-tetra-O-acetyl-3-D-glucopyranosyl)-sn-glycerol(IXb, 46.3% yield), and 1,2.O-isopropylidene-3-O-(2,3,4,6+tetra-O-acetyl-/3-D-galactopyranosyl)-sn-glycerol (IXc, < 10% yield).
274
KL Shvets et al., Synthesis o f glyeosyl diglyeerides
TABLE 2 Physico-chemical constants of glycosyl glycerols (V) and their hexacetates (VI)a Compound
Yield (%)
Va from IVa
Melting (°C) (solvent)
[a] ~0b
PC Rf
96.0
+ 42.5
Rfman 0.85(10)
Vb from IVb
94.2
- 32.3
0.33(11)
Vc from IVc
93.8
Via from Va
96.5
Vib from Vb
95.4
Vic from Vc
94.5
136.0-137.0(ethanol) -
7.9
0.34 (11)
+ 39.2 142.0-143.0(ethanol) 83.0-84.0(ethanol)
-
7.5 1.2
a. For compounds of a similar structure synthesised previously or isolated after deacylation of natural glAvcosyldiglycerides the following constants are reported in the literature : V a - [ a ] ~ " + 43.9°; 8 4.88 p.p.m. (Hi, Jl, 2 = 2 Hz) [ 2 8 ] ; V b - [ a l ~ ) ° - 32° [11]. The low yield of IXc and the direction o f the reaction to the f o r m a t i o n o f lower glycoside 1-O-ethyl-2,3,4,6-tetra-O-acetyl-3-D-galactopyranose might have been acc o u n t e d for b y the stereochemistry of the molecule of galactose and its derivatives. This suggestion was substantiated b y the stable yield of IXc, in contrast to that of similar derivatives of m a n n o s e and glucose IXa, b, w h e n the glycosylation condi-
> (V)
CH2OH
r
H-C-O~ [ _/CMe2 CH2 O..-
R'OCH 2
(VIII)
R'Br
(Xlc)
R'OCH 2
i
+ ROX -~ H-C-O_ [ ~CMe (II) CH20/"
I r
--, H - C - O H 2
] (x)
R-O-CH 2 [ H-C-O~ ] CMe2 CH2 0 /
R' -OCH 2
I
H-C-OH -
I
Scheme 2.
-
CH20COC I sH 31 (XIII)
X=Et
>(IIl
CH2OH
(IX)
(XIIc)
(Vl)
275
EL Shvets et al., Synthesis of glycosyl diglycerides
TLC
Rf
PMR, ~i p.p.m.
Formula
Calculated (%)
Found (%)
C
H
C
H
C9H1808
45.52
7.13
42.60
7.55
CgHt808
42.52
7.13
42.90
7.05
C9H1808
42.52
7.13
42.70
7.16
H~
0.55(3)
4.83(J1, 2 = 2 Hz)
C21H3oO14
49.80
5.97
49.79
6.14
0.55(3)
4.56(J1, 2 = 7 Hz)
C21H3oO14
49.80
5.97
50.59
5.89
0.55(3)
4.52(J1, 2 = 7 Hz)
C21H3oO14
49.80
5.97
49.86
6.08
Vc-m.p. 137.0°; [c~]~ ° - 8.12 ° [30] ; Vllb-m.p. 144°; l~[~)° - 4.4 ° [111; VIIc-m.p. 83.0-85.0"~; 8 4.43 p.p.m. (HI, J1,2 = 7 Hz) [42]. b. For Va-c 0.6-0.8% solution in water, for VIa-c 0.6-0.8% solution in chloroform. tions were changed (different solvent, another composition of starting compounds, changed duration of the process). Another possibility to obtain IXc was studied through the isomeric orthoesther XIIc to raise the yield. The latter was prepared from 1,2-O-isopropylidene-sn-glycerol (VIII)and 2,3,4,6-tetra-O-acetyl-a-D-galactopyranosyl bromide (IXc) [32] in a 50.2% yield in ethyl acetate in the presence of PbCO 3. The reaction product Xllc, chromatographically purified on alumina displayed a tendency to mild acid hydrolysis characteristic of orthoesters [13]. Its structure was confirmed by corresponding IR and PMR spectra. XIIc was isomerised to glycoside IXc in a 45.5% yield similar to orthoester VII conversion to glycosides III. Isopropylidene protection of mannoside IXa and glucoside IXb was removed by 10-20% aqueous solution of acetic acid at 100 ° (yield of Xa, b - 69.5% and 68.4%) and of IXc by 50% solution at 1 8 - 2 0 ° (yield of Xc - 86.2%). Compounds X a - c were chromatographically purified on silicic acid and shown by IR and PMR spectra and o.r.d, curves to be glycosides. Attempts made to use 1,2-O-ispropylidene-sn-glycerol (VIII) to synthesise glycosyl glycerols [9, 12] usually led to partial or complete racemisation at the glycerol chiral centre, apparently due to the acid formed on glycosylation. This process was also accompanied by partial anomerisation. In this respect the orthoester method was found.to have advantages over that of Koenigs-Knorr. First, the catalytic quantities of weakly acid catalysts (perchlorates of pyridine bases) used in these reactions were immediately consumed to give rise to orthester carbocation [ 13]. Hence the reaction medium was more strictly neutral and if some reasons tended the reaction products to become acidic the first to
276
V.I. Shvets et aL, Synthesis of glycosyl diglycerides
do so were glycosylating reagents, i.e. orthoesters (no glycoside formation), rather than the dioxalane ring of 1,2-O-isopropylidene-sn-glycerol (VIII). Secondly, the electrophylic centre of the orthoester carbocation was so strongly screened by the perchlorate ion that alcohol could attack only in one direction to form 1,2-transglycosides [13]. These considerations were confirmed when alkaline saponification of Xa-c gave glycosyl glycerols Va-c and their hexaacetates Via c, identical with respective compounds V and IVa-c prepared from glycosyl diglycerides IVa-c. Xa-c were acylated with palmitoyl chloride [17] in the presence of pyridine with the extent of esterification determined from the reaction temperature. It was shown that glucosyl- and galactosyt glycerols X b - c reacted in benzene at 5 5 - 6 0 ° to yield monoacylic derivatives XllIb, c. Acylation of mannosyl glycerol Xa gave an equal percentage of mono- and dipalmitins XIIla, Ilia, with the latter being identical with glycoside Ilia obtained from glyceride 1. Reactions in boiling toluene led to quantitative yields of diacyl derivatives IIIa-c similar to glycosyl dipalmitins IIIa-c prepared from compound I. Acylation of monopalmitoyl derivatives XIlla-c was effected under the same conditions. The step-wise acylation permitted various fatty acids to be introduced into glycosyl glycerol Xa-c and use of protection of monosaccharide hydroxylic groups by acetates did not hinder the formation of glycosyl diglycerides with unsaturated fatty acids. 2.1,3. Monoglycosyl diglycerides obtained from 2,5-O-methylene-D-mannitol The synthetic application of 1,2-O-isopropylidene-sn-glycerol (VIII) to obtain mixed glycosyl diglycerides was limited because the selective esterification of the hydroxyl group at glycerol C 1 due to its high reactivity could be accompanied by partial acylation of the hydroxyl group at C 2 leading to structurally heterogeneous glycosyl diglycerides. This limitation necessitated the use of a glycerol component with no acylation of the 2-OH-group on introduction of acid in the first position. Such a component was found to be 2,2'-O-methylene-bis-(1-O-acyl-sn-glycerol) prepared from D-mannitol through 2,5-O-methylene derivative [33]. The synthetic scheme [34] proposed was based on glycosylation of 2,2'-0methylene-bis-(1-O-palmituoyl-sn-glycerol) (XIV) [33 ] by ethylorthoacetate of D-mannose (lla, x = Et) and could be extended to obtain other glycosyl diglycerides as well (scheme 3, table 1). 1,2-trans-glycosylation of XIV with orthoacetate IIa was carried out in boiling chlorobenzene in the presence of 2,6-1uthidinium perchlorate, with the molar ratio of XIV, IIa and the catalyst of 1 : 1 : 0.05, during 1.5 hr. The reaction product, glycoside XVa, was chromatographically isolated on alumina in a 43.8% yield and characterised according to the general scheme used in this work. The rupture of the methylene bridge in glycoside XVa was effected by 50% methanol solution of acetic acid at 50-55 °. Mannosyl monoglyceride XIIIa, isolated chromatographically on silicic acid in a 85.3% yield was shown by physico-
277
El. Shvets et al., Synthesis of glycosyl diglycerides
HOCH2
CH20COC 1sH31
!
I H-C-O-CH2-O-C-H I I
H31CIsOCOCH2
R' -O -CH 2
+ (1 la) ~
CH2OH
H31ClsOCOCH2
(XIV)
I
H-C-OH
I
CH2OCOC15H31 (Xllla)
R'"COCI
CH2OR'
(XVa) R'OCH~
R'-OCH2
CH20COC 1sH31
1 I H-C-O-CH 2 - O - C - H I I
I
H-C-OCOR"'
i
CH2OCOClsH31 (IIIa, R'" = C15H31) (XVIa, R" = C17H33)
R"-O-CH 2 .,
L
H-C-OCOR"'
L
CH2OCOls H31 OVa, R'"= ClsH31) (XVIIa, R'" = ClTHaa)
Scheme 3. chemical constants, IR and PMR spectra and o.r.d, curve to be identical with 1-Opalmitoyl.3-O-(2,3,4,6-tetra-O-acetyl-a-D-mannopyranosyl)-sn-glycerol(XIIIa) prepared by selective acylation of mannosyl glycerol Xa. This suggested that monoesterification of glycosyl glycerols X a - c was more selective at the hydroxyl group at glycerol C 1 . Conversion of XIIIa to diacyl derivative III, XVIa was carried out by acylation with palmitoyl chloride and oleoyl chloride [35] in boiling toluene in the presence of pyridine. The reaction gave quantitative yields with Ilia being similar to glycoside Ilia prepared from 1,2-di-O-palmitoyl-sn-glycerol (1) and 1,2-O-isopropylidenesn-glycerol (VIII). Mannosyl dipalmitin Ilia and mannosyl diglyceride XVIa with residues of palmitic and oleic acids were found to be rather similar in their physico-chemical properties, chromatographic mobility and o.r.d, curves. Their difference (m.p., IR and PMR spectra) were caused by the double bond in the oleic acid of XVIa. Thus the IR spectrum of glycoside XVIa revealed a band-CH ---(t)ma x 3010 c m - 1 ) that was absent in the spectrum of Ilia. Deacetylation of XVIa resulted in 1-O-palmitoyl-2-O-oleoyl-3-O-(a-D-mannopyranosyl)-sn-glycerol (XVII) in a 91.2% yield. This product was the first unsaturated glycosyl diglyceride to be prepared synthetically. Identification of XV, XVI, XVIIa was effected by IR, PMR spectra and o.r.d, curves as in other cases. That method was more multistaged than the first two methods described and its apparent advantages in preparing mixed glycosyl diglycerides as compared with the preparation starting from 1,2-O-isopropylidene-sn-glycerol were found experimentally to be negligible.
278
KL Shvets et aL, Synthesis of glycosyl diglyeerMes
2.2. Synthesis of diglycosyl diglycerides
Having worked out synthetic routes to monoglycosyl diglycerides as exemplified by the synthesis of mannosyl-, glucosyl- and galactosyl diglycerides we proceeded to the synthetic study of oligoglycosyl diglycerides. It proved thereby possible to obtain diglycosyl diglyceride by binding together the oligosaccharide and diglyceride moieties of such a compound as/3-D-cellobioside-l,2-di-O-palmitoyl-3-O [/3-D-glucopyranosyl-(1 ~ 4)-/3-D-glucopyranosyl] -sn-glycerol (XXI1) [36] (scheme 4, table 1). The synthetic route used was that of direct glycosylation of glyceride 1 previously proposed for monoglycosyl diglycerides.
CH2OAc
CH2OAc
0 0CHA2OcAc~
AcOO A c ~ O O A ~ c OOAc OAc OAc (XVlll) CH2OAc CH2OAc
Ac
OAc OAc (XlX) CH2OAc CH:OAc
o ,O
Ac6~,--4"
OAc (XX)
O ACH2OAc c~ 1 O Br
+ (1)~ O-/---OEt Me 9
CH2OA¢
A~O~ = = f
O ~ OAc
H_(~_OOCCIsH31 OAc
,
(XXl) CNz0Ac
O~H o A----o ,--O-FH~ ~o.~/o. ~1 ._c-oocc,,.,,
OH (XXll)
OH
Scheme 4.
The synthesis was effected starting from 1,2-di-O-palmitoyl-sn-glycerol (1) and ethylorthoacetate of cellobiose XX. The latter was obtained from octaacetate XVIII [37] through acetobromocellobiose XIX on its interaction with ethanol in the presence of 2,6-1utidine in a 48.6% yield. The structure of orthoester XX was determined by physico-chemical methods previously used for structural identification of orthoesters VIIa-c and XIIc.
V.I. Shvets et al., Synthesis of glycosyl diglyeerides
279
Orthoester XX was introduced in the reaction of 1,2.trans-glycosylation with 1. It was found that an excess of I, 2 moles per mole of XX, and 0.1 mole of catalyst (2,6-1utinium perchlorate) increase the yield of glycoside XXI up to 80.1% as compared with 45.8% from the equimolar percentage of compounds I and XX. Glycoside XXI was isolated chromatographically on silicic acid and identified as above. Deacetylation of XXI gave a compound characterised by IR, PMR spectra and o.r.d, curve as being 1,2-di-O-palmitoyl-3-O-[/3-D-glucopyranosyl-(1 ~ 4)-/3-Dglucopyranosyl] -sn-glycerol (XXII) in a 92.2% yield.
1,2-dipalmitin
2. 3. Summary
The proposed synthetic routes to glycosyl diglycerides by orthester method of glycosylation can be used to prepare both saturated and unsaturated mono- or mixed 1,2-trans-glycosyl diglycerides with mono- and oligosaccharide residues in the carbohydrate moiety of the molecule. Of several alternatives involving 1,2-diglycerides, 1,2-O-isopropylidene-sn-glycerol- and 2,2'.O-methylene-bis-(1-O-acyl-sn-glycerol) the most suitable method is that of synthesis of glycosyl diglyceride from 1,2-O-isopropylidene-sn-glycerol.
3. Experimental part 3.1. Materials and methods
All m.p.s, were measured in open capillaries and not corrected. The reaction course, chromatographic separation and the properties of compounds were studied by TLC. The preparative and TLC were effected on silicic acid of the "aqueous" sort in such systems as ether-hexane 3 : 1 (1); ether-hexane, 1 : 1 (2); chloroform-acetone, 2 : 1 (4); chloroform-methanol-acetone, 40 : 5 : 5 (5); chloroform-methanol-water, 65 : 12 : 2 (6); on silica gel G in such solvent systems as chloroform-acetic acidwater, 50 : 35 : 3 (7) [38] ; chloroform-methanol-7 N NH4OH, 65 : 30 : 4 (8) [39] ; on alumina of the activity III after Brockman in such solvent systems as chloroform-acetone, 9 : 1 (9). Silicic acid and silica gel were preliminarily activated by heating at 1 2 0 - 1 4 0 ° for 4 hr. The compounds were revealed on chromatograms by sprinkling them with conc. H2SO 4 followed by carbonisation at 3 0 0 350 °. The descending chromatography* was effected on the paper "Leningradskaya bystraya" (PC) in such solvent systems as n-butanol-pyridine-0.1 N hydrochloric acid, 5 : 3 : 1 (10) [28] ; n-butanol-pyridine-water, 6 : 4 : 3 (11) [40, 41]. The products were identified by means of alkaline silver nitrate on heating up to 1 0 0 120 °. The o.r.d, curves and [a] 20 were measured on spectropolarimeter SPU-M. * Rfrna n is given in ratio of the length run of mannose.
280
V.1. Shvets et al., Synthesis of glyeosyl diglyeerides
IR spectra were recorded on spectrometer Perkin-Elmer, model 257 in nujol, PMR spectra on Varian HA-100D in ethanol-free chloform or in deuterochloroform, inner standard-TMS, with chemical shifts presented in the 8 scale. The compounds obtained all had a satisfactory elementary analysis (tables 1 and 2). 3.2. A c M hydrolysis
Glycosides IVa-c (3 rag) were heated in a sealed ampoule with 1 ml of 50% dioxane solution containing 5% of hydrogen chloride at 100 ° during 18 hr. The ampoule content was then concentrated by evaporation and the residue analysed by TLC in system 2 and PC in systems 10, 11 in the presence of samples of corresponding monosaccharides and of dipalmitin 1, palmitic acid and glycerol. 3.3. Alkaline hydrolysis
A mixture of compounds IVa-c (3 mg) in 0.07 N sodium methylate solution (3 ml) in methanol was kept at 18-20 ° during 20 hr and brought up to pH 4.5 by 37% hydrochloric acid. The mixture was then concentrated by evaporation and the residue analysed by TLC in system 2 and PC in systems 10, 11 in the presence of corresponding glycosyl glycerols Va c and palmitic acid. 3.4. Combined acM and alkaline hydrolysis
Glycosides IVa-c were completely split by consecutive acid and alkaline hydrolysis and then the reaction mixture was analysed by PC in systems 10, 11 in the presence of sample of mannose, glycose, galactose and glycerol. 1,2-Di-O-palmitoyl-3-O-(2,3,4,6-tetra-O-acetyl-a-D-mannopyranosyl)-sn-glycerol (!IIa). To prepare this compound a solution of 1.62 g (4 mmoles) of orthoester IIa, x = Bu-tert. [18], 2.3 g (4 mmoles) of 1,2-di-O-palmitoyl-sn-glycerol (1) [17], and 17 mg (0.08 mmole) of 2,6-1utidinium perchlorate in chlorobenzene (15 ml) was heated during 3 hr. The tert-butyl alcohol splitting off was simultaneously removed and the volume of the reaction mixture kept constant. When the reaction was over, chlorobenzene was concentrated by evaporation, the residue chromatographed on silicic acid, fractions of carbon tetrachloride washed away and ether gradually added from 2 to 20%. Fractions containing IIIa were concentrated by evaporation and crystallised from methanol. Yield: 1.5 g (41.4%). M.p. 28.0-29.0 °, [a] 20 +24.2 (0.76% solution in chloroform),Rf 0.6 (1); Umax: 1750 (C = 0 in COOR), 1135 (C-O at C1), 920 (pyranose ring), 840 (CH at C 1) cm--1; PMR, 8 2:05 (4 MeCO), 4.84 (H1,J 1 2 = 1.9 Hz). Similarly,' glycosylation of compounds I, VIII, XIV gave IIIb-c, IXa c, XVa, and XXI (table 1).
V.l. Shvets et aL, Synthesis of glycosyl diglyeerides
281
Deviations from this procedure concerning the solvents and catalysts used, the ratios of reacting compounds to the catalyst and the method of glycoside isolation were considered above when discussing the results obtained.
3,4,6~Tri~-acetyl-~2~( ~2-di~-palmit~yl~sn~gly~eryl-3-~-~rth~acetyl)-~-D-mannopyranose (VIIa). To prepare this compound a solution of 3 g (8 mmoles) of orthoester IIa, x = Et [22], 4.5 g (8 mmoles) of 1,2-diglyceride I and 27.6 mg (0.16 mmole) of YsOH in dichloroethane (50 ml) was heated during 2.5 hr. The ethanol splitting off was simultaneously removed and the volume of the reaction mixture kept constant. When the reaction was over, dichloroethane was concentrated by evaporation, the residue chromatographed on silicic acid with benzene used as eluent whose ether content was gradually raised from 5 to 30%. Fractions containing orthoester VIIa were concentrated by evaporation and crystallised from 10 ml of hexane. Yield: 3.5 g (49.4%). M.p. 68.0-69.0 °, [a] 20 +11.27 ° (0.40% solution in chloroform), R f 0.3 (2); Vmax: 1740 (C = 0 in COOR), 1170 ( C - 0 at C1), 900 (pyranose ring) c m - 1 ; PMR, 6 1.77 (endo-C-Me), 2.0 (3MeCO)). VIIb, c were obtained similarly except for using alumina rather than silicic acid in chromatography (table 1). The samples of orthoesters VIIa-c were hydrolysed completely by O. 1 N H2SO 4 in 90% aqueous acetone [13]. 1,2-Di-O-palmitoyl-3-O-(2,3,4,6-tetra-O-acetyl-a-D-mannopyranosyl)-sn-glycerol (IIIa). To prepare this compound a solution of 1.0 g (0.11 mmole) of orthoester VIIa and 7.9 mg (0.022 mmole) of 2,2'-dipyridilium perchlorate in dichloroethane (20 ml) was boiled during 1.5 hr and then kept at 18-20 ° for 12-14 hr. The glyceride precipitate was separated, the filtrate concentrated by evaporating and the solid dissolved in 30 ml of 0.1 N H2SO 4 in 90% aqueous acetone and left standing for 25 hr. The mixture was neutralised with 10% NaHCO 3 and extracted with chloroform (2 × 30 ml). The extract was dried by CaC12, concentrated to 5 ml and passed through a column over alumina. The resulting product was eluated with chloroform and after crystallisation from methanol (2 ml) gave pure crystalline Ilia. Yield: 0.4 g (40.0%). M.p. 30.0-31.0 °, [a] 20 +22.3 ° (0.33% solution in chloroform), R.f 0.6 (I), IR an PMR spectra were identical with those of glycoside Ilia obtained from I. Similarly, isomerisation of VIIb, c resulted in glycosides IIIb, c, that of XIIc in IXc (table 1). 1,2-Di-O-palmitoyl-3-O-(a-D-mannopyranosyl)-sn-glycerol (IVa). This compound was prepared from a mixture of 0.68 g (0.75 mmole) of IIIa, 0.3 ml (15 mmoles) of H2NNH2"H20 in 85% ethanol (45 ml) was boiled for 25 min, then kept at 18-20 ° for 15 hr and 1 hr at 0 °. The precipitate was filtered off, washed with 85% ethanol and recrystallised from methanol (1 ml). Yield: 0.52 g
282
V.I. Shvets et aL, Synthesis of glycosyl diglycerides
(92.3%). M.p. 108.0-1 t0.0 °, [o~]20 +27.0 (0.84% solution chloroform). R f 0.5 (5), 0.55 (7), 0.85 (8); Vmax: 3600-3100(OH), 1730 (C = O in COOR), a number of bands over the range of 1350-1200 (CH 2 in C15H31), 915 (pyranose ring), 840 (C H at C1) cm-1;PMR, 6 4.93 (H 1,J1,2 = 1.9 Hz). Similarly, Illb, c, XVIa, XXI gave IVb, c, XVIIa, XII (table 1).
3-O-a-D-mannopyranosyl-sn-glycerol (Va). To prepare this compound a mixture of 0.5 g (0.7 mmole) of mannosyl diglyceride IVa and 35 ml (3.5 mmoles) of 0.1 N NaOH was heated at 100 ° for 1 hr, cooled and neutralised by adding Dowex-50 (H+). The resin was removed and the product washed with water (10 ml). The combined filtrates were extracted with ether (2 × 20 ml) and the aqueous layer concentrated by evaporation to an amorphous form. Yield: 0.17 g (96%). [a] 20 +42.5 ° (0.62% solution in water)Rfman 0.82 (10); Vmax: 3600-3100 (OH), 1100 1000 (C-O), 920 (pyranose ring) 840 (C-H at C1) cm -1. Similarly, IVb, c gave Vb, c (table 2).
1,2-Di-O-acetyl-3-O-(2,3, 4,6-tetra-O-acetyl-a-D-mannopyranosyl)-sn-glycerol (Via). To prepare this compound 0.13 g (0.51 mmole) of mannosyl glycerol Va was suspended in carbon tetrachloride (4 ml) and 1 ml (12 mmoles) of pyridine, 2 ml (25 mmoles) of acetyl chloride in carbon tetrachloride (2 ml) added. The mixture was left standing at 18-20 ° for 6 hr. The precipitate was separated, washed with carbon tetrachloride and the substrate concentrated by evaporation to result in an oily substance that was then analytically purified by preparative TLC in system 9. Yield: 0.25 g (96.5%). [c~]20 +39.2 ° (0.35% solution in chloroform), R f 0.65 (9); Vmax: 1750 )C = O in OCOMe), 1220 (C-O in OCOMe), 1130 (C-O at C1), 1080, 1050 (C-O), 915 (pyranose ring), 835 (C-H at C1) cm-1;PMR, 6 2.2 (6 MeCO), 4.83 (H l, Jl,2 = 2 Hz). Similarly, Vb, c gave hexaacetates VIb, c (table 2).
3•4•6•Tri-•-acety•-••2••-(1•2-•-is•pr•pylidene-sn.g•ycery•-3••••rth•acety•)-a-Dgalactopyranose (Xllb). To prepare this compound a mixture of 6.5 g (16 mmoles) of 2,3,4,6-tetra-Oacetyl-a-D-galactopyranosyl bromide (XIc) [32], 10.5 g (75 mmoles) of CaSO4, and 14 g (46 mmoles) of PbCO 3 in ethylacetate (150 ml) was boiled for several minutes. Then 1.0 g (7.6 mmoles) of 1,2-O-isopropylidene-sn-glycerol (VIII) [ 16] was added and the mixture boiled for 3 hr. Another 1.Og (7.6) mmoles of VIII) was subsequently added and the mixture boiled for 10 hr more. The suspension was cooled down to 18-20 °, 6.0 g (27 mmoles) of Ag20 and 37 ml of 95% aqueous acetone introduced and the mixture left standing for 48 hr. The reaction mixture was then filtered off, concentrated by evaporation and the residue separated on a column with alumina by washing with chloroform. Fractions containing the product were concentrated by evaporation to give an oily substance. Yield: 3.22 g
V./.
Shvets et al., Synthesis of glycosyl diglycerides
283
(50.2%). [ct] 20 +70.3 (0.6% solution in chloroform), Rf 0.5 (3); Vrnax: 1750 (C = O in OCOMe), 1370 (C-Me), 1220 (C-O in OCOMe), 1150 (C-O at CI), 910 (pyranose ring) cm-1; PMR, 6 1.36 (C-Me2), 1.70 (endo-C-Me). The sample of orthoester XXc was completely hydrolysed by 0.1 N H2SO 4 in 90% aqueous acetone [13].
4-0-(2,3,4,6-Tetra-O-acetyl-(3-D-glucopyranosyl)-3,6-di-O-acetyl-l,2-O-ethyl-ortho. aeetyl-~-D-glueopyranose (XX). To prepare this compound, to a solution of 3.0 g (4.9 mmoles) oft~-D-cellobiose octaacetate XVIII [37] in chloroform (15 ml) were added 10 ml of glacial acetic acid saturated with HBr (100 mmoles) at 0 ° and 1 ml of acetic anhydride. The mixture was diluted with chloroform (25 ml) and washed with glacial water (2 X 10 ml). The organic layer was dried with CaCI2 and concentrated by evaporation. The residue (3.02 g) - crude bromide )(IX - was dissolved in chloroform (10 ml), 5 ml of 2,6-1utidine, 15 ml of ethanol were added and the reaction carried out at 18-20 ° during 96 hr. The reaction mixture was dissolved in chloroform (20 ml) and washed with glacial water (2 × 15 ml). The organic layer was dried with MgSO4, concentrated by evaporation, and the residue chromatographed over alumina with the compounds being eluated by chloroform. The fractions containing orthoester XX were concentrated by evaporation and crystallised from 15 ml of hexane-ether mixture (9 : 2). Yield: 1.42 g (48.5% on XVIII). M.p. 49-50 °, [ct] 20 + o 7.5 (0.43% solution in chloroform), Rf 0.8 (9); Vmax: 1750 (C = O in OCOMe), 1220 (C-O in OCOMe), 1160 (C-O at C1), 900 (pyranose ring); PMR, 8 1.78 (endo-C-Me), 2.0-2.15 (6 MeCO). The sample of orthoester XX was completely hydrolysed by 0.1 N H2SO 4 in 90% aqueous acetone [13].
3-O-(2,3,4,6-tetra-O-acetyl-a-D-mannopyranosyl).sn.glycerol(Xa). To prepare this compound a solution of 0.55 g (1.2 mmole) of mannoside IXa in 10% acetic acid (25 ml, 42 mmoles) was heated at 100 ° for 1 hr, cooled and neutralised with saturated aqueous NaHCO 3. The mixture was diluted with water (50 ml) and extracted with chloroform (3 X 50 ml). The combined extracts were dried with MgSO4, concentrated by evaporation and the product isolated by pre20 parative TLC in system 4 to obtain an oily substance. Yield: 0.32 g (69.5%). [~] D +37.48 (0.71% solution in chloroform), Rf 0.5 (4); Vmax: 3700-3100 (OH), 1750 (C = O in OCOMe), 1230 (C-O in OCOMe), 1140 (C-O at CI) , 1100-1000 (C-O), 915 (pyranose ring) cm-1. Almost similarly (see above, §II) IXb, c gave Xb, c (table 1).
1-O-Palmitoyl-3-O-(2,3,4,6-tetra-O-acetyl-a.D.mannopyranogyl).sn.glycerol(XIIIa) and 1,2-di-O-palrnitoyl-3-O-(2,3,4,6-tetra-O-acetyl.a.D.mannopyrasonyl).sn.glycerol (IIIa). To prepare these compounds, to a solution of 0.17 g (0.4 mmole) of glycoside
v.1. Shvets et aL, Synthesis of glyeosyl diglyeerides
284
Xa and 0.1 ml (1.2 mmole) pyridine in benzene (15 ml) 0.214 g (0.8 mmole) of palmitoyl chloride was added with stirring at 00 and the mixture was left standing for 3 hr. Then the temperature was raised up to 5 5 - 6 0 ° and the reaction carried on 6 - 7 hr. The precipitate was filtered off, the solvent concentrated by evaporation and the residue, consisting of XIIla and Ilia separated by preparative TLC in system 1. Yield of Xllla (an oily substance): 0.15 g (55.5% on Xa), [c~]20 +31.4 ° (0.21% chloroform solution), R f 0.33 (I); Vmax: 3450 (OH), 1750 (C = O in COOR), 1140 (C-O at C1), 920 (pyranose ring) c m - l : PMR, 6 2.05 (4 MeCO), 4.93 (H 1, J1 2 = 1.85 Hz). Yield of Ilia: 0.19 g (52.8% on Xa). M.p. 2 8 - 2 8 .3 o , [cq 20 +23.4 ~ (0.35% chloroform solution), R f 0.6 (1). IR and PMR spectra were identical with those of glycoside Illa synthesised from I. Similar acylation of Xb, c gave only monopalmitoyt derivatives XIIlb,c (table 1).
1,2-Di-palmi toyl-3-O-( 2,3, 4, 6-tetra-O-acety l-a-D-manno pyranosyl )-sn-gly cerol (Ilia). To prepare this compound a mixture of 0.1 g (0.24 mmole) of glycoside Xllla and 0.13 g (0.48 mmole) of palmitoyl chloride was boiled for 6 hr in the presence of pyridine (0.08 ml, 0.001 mmole) and toluene (10 ml). The reaction mixture was filtered, concentrated by evaporation and the residue crystallised from 2 ml of methanol. Yield: 0.14 g (93.2%). M.p. 28.0-29.0 °, [a] 2D0+23.9 ° (0.76% chloroform solution), R f 0.6 (I). IR and PMR spectra were identical with those of glycoside Ilia, synthesised from I. Similarly X a - c gave llIa-c. The same conditions also resulted in conversion of XIIIa to XVIa (table 1).
1.O.Palmitoyl-3.0-( 2,3, 4,6-tetra-O-acetyl-a-mannopyranosyl)-sn-glycerol (XIIIa). To prepare this compound a solution of 0.6 g (0.45 mmole) of glycoside XVa in 50% methanol solution of acetic acid (20 ml, 170 mmoles) was heated at 50-55 ° during 1 hr. The reaction mixture was dissolved with chloroform (100 ml), washed with water (50 ml) and saturated aqueous solution of NaHCO 3 (50 ml), then repeatedly with water (30 ml) and finally dried with CaC12. The product was isolated by TLC in system 1 in the form of an oily substance. Yield: 0.55 g (85.3%). [a] 20 + 32.4 ° (0.2% solution in chloroform), R f 0.33 (I). IR and PMR spectra were identical with those of XIIla obtained from Xa.
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SYNTHESIS OF GLYCOSYL DIGLYCERIDES V.I. SHVETS, A.I. BASHKATOVA and R.P. EVSTIGNEEVA * The Lomonosov Institute o f Fine Chemical Technology, Moscow, U.S.S.R.
Received July 5, 1972 Accepted August 1, 1972
Synthesis of mono- and diglycosyldiglycerideswith natural structure from 1,2-di-O-acyl-snglycerols, 1,2-O-isopropylidene-sn-glycerol,2,5-methylene-D-mannitolby the orthoester method of glycosylationis reported.
1. Introduction
Much attention is now devoted to the study of the structure, properties and biological functions of glycosyl diglycerides. These compounds are very common in the vegetable kingdom and isolated from bacterial cells and tissues of the central nervous system [ 1 - 3 ] . Glycosyl diglycerides are essentially derivatives of 1,2-di-O-acyl-sn-glycerols [ 1 - 3 ] . Their carbohydrates involve mono- and oligosaccharide structures (mostly derivatives of D-galactose, D-glucose, and D-mannose) [ 1 - 3 ] . Glycerol is esterified by saturated and unsaturated fatty acids C 15 - C 18 [ 1 - 3 ] . Glycosyl diglycerides from vegetable and animal tissues are characterised by 1,2-trans-glycoside bonds with bacterial cells containing in addition 1,2-cis-glycosides [ 1 - 3 ] . The bonds between monosaccharides have various configurations and are in different places [ 1 - 3 ] . Glycosyl diglycerides participate as components of cell membranes in many biological processes such as photosynthesis [4, 5], transport of carbohydrates through membranes [6], polysaccharide biosynthesis [7, 8], and so on. Isolation of glycosyl diglycerides from natural sources is effected through separation of lipid mixtures [ 1 - 3 ] . Yet, even chromatographically homogeneous samples of natural glycolipids of this class are not individual compounds, being a homologous mixture of diglyceride derivatives [ 1 - 3 ] . It is therefore of great importance to find a synthetic method that would enable the structure of glycosyl diglycerides to * Translated by A.L. Pumpiansky, Moscow.
268
KI. Shvets et al., Synthesis of glyeosyl diglycerides
be unambiguously determined and their biological role at the molecular level studied. To this end a number of structural problems concerning the spatial forms of glycosyl diglycerides should be discussed and stereospecific methods to construct glycoside bonds used. At present there are only several papers on this subject. They are all based on glycosylation according to Koenigs-Knorr [7, 9-12] which is not stereospecific [9] for the case under study. This study is concerned with a glycosyl diglyceride synthesis by the orthoester method of 1,2-trans-glycosylation [ 13]. This method has been chosen because of its stereospecificity and high yields as distinct from that of Koenigs-Knorr [13]. The only example previously reported in the literature is the glycosylation of glycerol derivatives by the orthoester method exemplified by 3-O-(/3-D-galactofuranosyl)-sn-glycerol [14]. Our synthetic schemes of mono- and diglycosyl glycerides are based on the interaction of orthoesters of mono- and disaccharides and alcohols (1,2-di-O-palmitoyl-sn-glycerol, 1,2-O-isopropylidene-sn-glycerol and 2,2'-O-bis-(1-Opalmit oyl-sn-glycerol).
2. Results and discussion
2.1. Synthesis of monoglycosyl diglycerides 2.1.1. Monoglycosyl diglycerides obtained from 1,2-diglycerides The synthesis was effected by direct and two-stage glycosylation (scheme 1, table 1). The first route [I 5, 16] was based on glycosylation of 1,2-di-O-palmitoyl-sn-glycerol (1) [17] with equimolar amounts of D-mannose, D-glucose, and D-galactose tert-butyl orthoesters (IIa-c, x = Bu-tert.) [16, 18, 19] in boiling chlorobenzene in the presence of a catalytic amount of 2,6-1utidinium perchlorate (0.02 mole per mole of orthoester II).* The optimum reaction time was found to be from 2.5 to 3 hr. The reaction products were chromatographed on silicic acid to give acetylated glycosyl diglycerides IIIa-c in a 37-41% yield. The configuration of the glycoside bond of compounds IIa-c was determined by IR and PMR spectra and o.r.d, curves. Thus the IR spectrum of mannoside Ilia showed the C - H absorption band at the anomeric centre to be at 840 c m - 1 , this points to the a-glycosydyl bond; in IR-spectrum of glycosides IIIb, c the same absorption was at 890 c m - 1, characteristic of/3-glycosides [20]. The same conclusion followed from PMR spectra, where in the case of mannoside IIIa the proton doublet at C 1 reveals a chemical shift characteristic of a-glycoside (6 4.84 p.p.m.) [20],
* The molar ratio of orthoester, alcohols and perchlorate will be further the same if not stated otherwise.
269
V.L Shvets et al., Synthesis of glyeosyl diglycerides
and a constant of spin-spin interaction (J1,2 = 1.9 Hz) between two equatorial protons [20]. CH2OH
R'-O-CH 2
I
R"_O-CH 2
f
H-C-OCOClsH31 + R-O-X --~
(II)
I
CH2OCOCIsH31 (I) "l
1
H-COCOClsH31
H-C-OCOClsH31-'+
I
l
/
CH20COCIsH3t
CH20COC15H31 (Ili)
~
R-O-CH 2
R'-O~CH2
H-C-OCOCIsHal
(IV)
R"-O-CH 2
H-C-OAc *---
I
I
I
CH20Ac (VI)
CH2OCOC15H31 (VII)
I
H-C-OH CH20H (V)
Here and below
AcOCH 2 R=
.Me
CH 20Ac
CH 20Ac
co/p-o\
a)
AcOO ~
b)
Ac
e)
Me
CH=OAc R'=
a)
•
Ac
CH2OAc
b)
CH= OAc
c)
Ac OAc
CH2OH R" =
OAc
CH~OH
CH2OH HO
a) HO ~
b)
)c-----O
e) H OH
X = 1) Bu-tert: 2)Et Scheme 1.
OH
270
V.Z Shuets
et al., Synthesis of glyeosyl diglycerides
TABLE 1 Physico-chemical constants of compounds prepared on synthesis of glycosyl diglycerides Compound
IIIa from Ia llIb from Ia
Yield (%)
4i .4 38.4
Melting point (°C) (solvent)
[c~] ~3°d
TLC
Rf
28.0-29.0(methanol) 80.0 80.5(methanol)
+24.2 7.2
0.6(1) 0.6(1)
llIc from Ia
37.0
45.0 46.0(methanol)
-0.61
0.6(I)
IVa from Ilia
92.3
108.0-1 lO.O(methanol)
+27.0
0.5(5) 0.55(7) O.85(8)
IVb from lllb
90.5
138.0-140.O(methanol)
-11.8
0.5(5) 0.63(7) O.86(8)
IVc from IIIc
82.7
141.0-142.0(methanol)
-6.53
O.5(5) 0.6(7) 0.82(8)
Vlla from I
49.4
68.0-69.0(hexane)
+11.27
0.3(2)
Vllb from I
40.2
85.0-86.0 (hexane)
+ 19.1
0.6(2)
Vllc from 1
41.4
35.0- 36.0(methanol,he xane)
+38.2
IXa from VIII
47.2
47.0-48.0(ether-hexane, 1 : 1 0 ) + 4 5 . 3
IXb from VIII
46.3
IX from Xllc b Xa from IXa Xb from IXb
68.4
Xc from IXc
86.2
- 5.9
0.5(4)
XIIc from VIII
50.2
+70.3
0.5(3)
-12.8
0.33(3)
45.5
- 6.3
0.33(3)
69.5
+37.48
0.5(4)
5.83
0.5(4)
Xllla from XVa c
85.3
XVa from XIV
43.8
102.0-103.0(ether -hexane 1:10)
0.6(2) 0.33(3)
lO0.O-lOl.O(ether)
+32.4
0.33(1)
35.0-37.0(methanol)
+22.0
0.31 (1)
XVIa from XVa
95.3
+14.0
0.6(1)
XVIIa from XVIa
91.2
108-O-109.0(methanol)
+24.0 e
0.5(5)
XX from XVII1
48.5
49.0-50.O(ether-hexane, 2:9)
+ 7.5
0.8(9)
XXI from I
80.1
109.0-110.O(methanol)
- 4.53
0.4(1)
XXII from XXI
92.2
224.0-225.0(methanol)
- 9.4
0.55(6)
a. Ilia c were also synthesised by isomerisation of V l l a - c in 40.0, 45.1, and 40.25% yield, respectively; l l l a - c was also obtained by acylating X to result in 93.6, 91,8, and 92.6% yield, respectively; Ilia was produced from Xllla, yield 93.2%. b. IXc was also obtained from VIII in a yield not exceeding 10%.. c. Xllla was prepared by esterification of Xa to yield 52.8%. d. 0.2-0.8% solution in chloroform.
V.I. Shvets et aL, Synthesis o f glycosyl diglycerides
PMR, 8 p,p.m. H1
Formula endo-C-Me
271
Calculated (%)
Found (%)
C
C
H
H
4.84(J1,2 = 1.9 Hz) 4.56(Jt, 2 = 6.9 Hz)
C49H86014 C49H86014
65.50
9.64
65.54
9.54
65.50
9.64
65.12
9.68
4"52(Jt,2 = 8.0 Hz)
C49H86014
65.50
9.64
65.27
9.68
4.93(J1, 2 = 1.9 Hz)
C41H78010
67.36
10.75
67.22
10.72
4"64(JI,2 = 5.7 Hz)
C41H78Olo
67.36
10.75
67.39
10.72
4.62(J1, 2 = 5.8 Hz)
C4tH78Olo
67.36
10.75
66.97
10.81
C49H86014 C49H86014 C49H86014
65.50
9.64
65.24
9.54
65.50
9.64
65.53
9.85
65.50
9.64
65.38
9.64
4.84(J1, 2 = 1.9 Hz)
C2oHaoO12
51.94
6.54
52.02
6.69
4"66(J1,2 = 6.9 Hz)
C2oH3oO12
51.94
6.54
51.90
6.49
4.52(Ji,2 = 7 Hz)
C,2oH3oO12
51.94
6.54
51.67
6.61
CITH26012I-L20
46.40
6.36
46.42
6.02
C17H26012.½H20
47.33
6.31
47.48
6.31
C17H26012
48.30
6.20
48.27
6.50
1.77 1.73 1.70
4"52(JI ,2 = 7 Hz)
1.70 C2oHa0012
51.94
6.54
51.75
6.40
4"93(J1,2 = 1.85 Hz)
C33Hs6013
59.95
8.52
60.18
8.73
4.87(Jl,2 = 2.0 Hz)
C67Hl12026
60.40
8.47
61.07
8.59
4"85(JI,2 = 1.5 Hz)
C51H88014
66.28
9.58
66.66
9.76
C43H8oO1o
68.21
10.65
68.59
10.91
C2sHaoOt s
50.71
6.08
50.81
6.06
C61H102022 C47H88015
61.69
8.65
61.15
8.81
63.20
9.93
63.16
10.03
1.78 4.52(3"1,2 = 7 Hz) 4.61(Ji, 2 = 6.4 Hz)
272
V.1. Shvets et al., Synthesis o f glyeosyl diglyeerides
With glucoside IIIb and galactoside IIIc the same doublet appears at 6 4.56 p.p.m. (JI,2 = 6.9 Hz) and 6 4.52 p.p.m. (J1,2 = 8 Hz) respectively, thus pointing to the/3-glycoside bond (spin-spin interaction between two axial protons) [20]. The shapes of the o.r.d, curves of glycosides IIIa-c were in accordance with the configuration suggested at C 1 [20]. Glycosyl diglycerides lIIa-c were also obtained through the scheme of two-stage glycosylation with intermediate isolation of orthoesters VIIa-c [21]. The reaction of 1,2-di-O-palmitoyl-sn-glycerol (1) with ethylorthoacetate monosaccharides (IIa-c, X = Et) [22, 23] was carried out conventionally by reesterification [24] in boiling dichloroethane in the presence of a catalytical amount of YsOH (0.02 mole per mole of orthoester VI). The optimum reaction time was found to be 2.5 hr. Orthoesters VIIa-c were isolated chromatographically on alumina or silicic acid in a 45-50% yield. The structure of orthoesters VIIa-c was proved by IR and PMR spectra, as well as by the hydrolytic probe for the orthoester grouping (0.1 N H2SO 4 in 90% aqueous acetone) [13]. The presence in the PMR spectrum ofa singlet 6 1.7-1.77 p.p.m. (C-Me) pointed to orthoesters VIIa-c predominantly in the form of endoisomers [25, 26]. Isomerisation of orthoesters VIIa-c into glycosides lIIa-c proceeds in weakly polar solvents in the presence of perchlorates of pyridine bases [24] with optimum conditions involving boiling dichloroethane, 0.02 mole of 2,2'-dipyridilium perchtorate per mole of orthoester VII and 1.5 hr reaction time. The physico-chemical properties, IR and PMR spectra and o.r.d, curves of the resulting IIIa-c glycosides chromatographycally obtained on alumina in a 40-45% yield were similar to those of glycosides IIIa-c prepared by direct glycosylation of I. From the preparative point of view preference should be given to direct glycosylation as it involves less stages and leads to higher glycoside yields (37-41% of IIIa-c from direct glycosylation and 16 25% from the two-stage glycosylation), as well as excludes the treatment of labile orthoesters VIIa -c. Deacetylation [12, 27] of compounds Illa-c was carried out by H2NNH 2 "H20 (5 moles per acetate group) in 85% ethanol or methanol boiled for a short time.* Crystallisation from ethanol gave 1,2-di-O-palmitoyl-3-O-(a-D-mannopyranosyl)-sn-glycerol (IVa), 1,2-di-O-palmitoyl-3-O-(/3-D-glucopyranosyl)-sn-glycerol (IVb) and 1,2-diO-palmitoyl-3-O-(~3-D-galactopyranosyl)-sn-glycerol (IVc) in 83- 92% yield. The structure of glycosides IVa-c and the configuration at C 1 were established by IR and PMR spectra and o.r.d, curves and found to be essentially similar to those of acetylated compounds IIta-c. To study the structural composition of glycosides IVa-c use was made of the acid and alkaline hydrolysis and the decomposition products were found by PC and TLC to involve on partial acid hydrolysis dipalmitin and corresponding monosaccharides, on alkaline hydrolysis mannosyl-, glucosyl- and galactosyl glycerols Va-c, * In other cases the reaction was effected similarly.
V.L Shvets et al., Synthesis of glycosyl diglycerides
273
respectively, and palmitic acid, on complete splitting including acid and alkaline hydrolysis palmitic acid as well as mannose, glucose and galactose, respectively. The products of alkaline hydrolysis soluble in water-glycosyl glycerols V a - c were isolated in a free state or as hexaacetates VIa-c obtained on treatment of compounds Va-c with acetyl chloride in carbon tetrachloride in the presence of pyridine. Glycosyl glycerols V, VIa-c were found to be identical to similar compounds obtained by deacylation of natural glycosyl diglycerides [28-30] and to those synthesised by the described methods [9, 11 ], which excluded racemisation at the glycerol chiral centre (table 2). Comparison of mannosyl-, glucosyl- and glactosyl dipalmitin IVa-c and similar natural glycosyl diglycerides [28-30] pointed to their identical physico-chemical and chromatographic properties, IR and PMR spectra and [a] 2D0. Mannosyl- and glucosyl glycerides IVa, b were the first synthetic compounds of this structure. Synthesis of galactosyl diglyceride IVc was previously reported [12], but the comparison of its characteristics (m.p. 56 °, [a] 20 absent; data for deacylation product-galactosyl glycerol - are: - m.p. 135-138 °, [a] 20 0 o) with respective constants of our synthetic 1,2-di-O2palmitoyl-3-O-(3-D-galactopyranosyl)-snglycerol (IVc, m.p. 141.0-142.0 o , [a] D zo -6.53 o ; data for the deacylatlon . product are: 3-O-3-D-galactosyl-sn-glycerol (Vc) - m.p. 136-137 ° , [a] 20 -7.9 ° ; 3-O-a-D galactosyl-sn-glycerol [9] - m.p. 150-152 °, [a] 20 +155 o) as well as with the characteristics of a similar compound (IVc, m.p. 152.0-154.0 °, [ct] 20 _2.04 o) [30] semi-synthesised by esterification of 3-O-3-D-galactosyl-sn-glycerol (Vc, m o 137 ° 20 o . . . "*" [0e] D --8.12 ) when isolated on deacylatlon of natural galactosyl dlglycerides, suggested this compound to involve a mixture of anomeric glycosides.
2.1.2. Monoglycosyl diglycerides obtained from 1,2-O-isopropylidene-sn.glycerol This synthetic route to glycosyl glycerides was chosen in order to avoid a number of difficulties arising in synthesis starting from 1,2-diglycerides. The proposed alternative [14, 15] overcame the main disadvantages of the former synthesis due to the comparative unavailability of 1,2-di-O-acyl-sn-glycerols (particularly with unsaturated acids) [31 ] and to the evolution of compounds Illa-c with properties rather similar to those of 1,2-diglycerides. The synthesis was based on the initial formation of glycosyl glycerols followed by introduction of fatty acids into the glycerol moiety of the molecule (scheme 2, table 1). Ethylorthoacetates of D-mannose, D-glucose, D-galactose (IIa-c, X = Et) were made to react with 1,2-O-isopropylidene-sn-glycerol (VIII) [17] under conditions of direct glycosylation of compound I. Glycosides IXa-c, chromatographically isolated on silicic acid were identified by chemical and physical methods, just as done previously, as 1,2-O-isopropylidene-3O-(2,3,4,6-tetra-O-acetyl-a-D-mannopyranosyl)-sn-glycerol(IXa, 47.2% yield), 1,2O-isopropylidene-3-O-(2,3,4,6-tetra-O-acetyl-3-D-glucopyranosyl)-sn-glycerol(IXb, 46.3% yield), and 1,2.O-isopropylidene-3-O-(2,3,4,6+tetra-O-acetyl-/3-D-galactopyranosyl)-sn-glycerol (IXc, < 10% yield).
274
KL Shvets et al., Synthesis o f glyeosyl diglyeerides
TABLE 2 Physico-chemical constants of glycosyl glycerols (V) and their hexacetates (VI)a Compound
Yield (%)
Va from IVa
Melting (°C) (solvent)
[a] ~0b
PC Rf
96.0
+ 42.5
Rfman 0.85(10)
Vb from IVb
94.2
- 32.3
0.33(11)
Vc from IVc
93.8
Via from Va
96.5
Vib from Vb
95.4
Vic from Vc
94.5
136.0-137.0(ethanol) -
7.9
0.34 (11)
+ 39.2 142.0-143.0(ethanol) 83.0-84.0(ethanol)
-
7.5 1.2
a. For compounds of a similar structure synthesised previously or isolated after deacylation of natural glAvcosyldiglycerides the following constants are reported in the literature : V a - [ a ] ~ " + 43.9°; 8 4.88 p.p.m. (Hi, Jl, 2 = 2 Hz) [ 2 8 ] ; V b - [ a l ~ ) ° - 32° [11]. The low yield of IXc and the direction o f the reaction to the f o r m a t i o n o f lower glycoside 1-O-ethyl-2,3,4,6-tetra-O-acetyl-3-D-galactopyranose might have been acc o u n t e d for b y the stereochemistry of the molecule of galactose and its derivatives. This suggestion was substantiated b y the stable yield of IXc, in contrast to that of similar derivatives of m a n n o s e and glucose IXa, b, w h e n the glycosylation condi-
> (V)
CH2OH
r
H-C-O~ [ _/CMe2 CH2 O..-
R'OCH 2
(VIII)
R'Br
(Xlc)
R'OCH 2
i
+ ROX -~ H-C-O_ [ ~CMe (II) CH20/"
I r
--, H - C - O H 2
] (x)
R-O-CH 2 [ H-C-O~ ] CMe2 CH2 0 /
R' -OCH 2
I
H-C-OH -
I
Scheme 2.
-
CH20COC I sH 31 (XIII)
X=Et
>(IIl
CH2OH
(IX)
(XIIc)
(Vl)
275
EL Shvets et al., Synthesis of glycosyl diglycerides
TLC
Rf
PMR, ~i p.p.m.
Formula
Calculated (%)
Found (%)
C
H
C
H
C9H1808
45.52
7.13
42.60
7.55
CgHt808
42.52
7.13
42.90
7.05
C9H1808
42.52
7.13
42.70
7.16
H~
0.55(3)
4.83(J1, 2 = 2 Hz)
C21H3oO14
49.80
5.97
49.79
6.14
0.55(3)
4.56(J1, 2 = 7 Hz)
C21H3oO14
49.80
5.97
50.59
5.89
0.55(3)
4.52(J1, 2 = 7 Hz)
C21H3oO14
49.80
5.97
49.86
6.08
Vc-m.p. 137.0°; [c~]~ ° - 8.12 ° [30] ; Vllb-m.p. 144°; l~[~)° - 4.4 ° [111; VIIc-m.p. 83.0-85.0"~; 8 4.43 p.p.m. (HI, J1,2 = 7 Hz) [42]. b. For Va-c 0.6-0.8% solution in water, for VIa-c 0.6-0.8% solution in chloroform. tions were changed (different solvent, another composition of starting compounds, changed duration of the process). Another possibility to obtain IXc was studied through the isomeric orthoesther XIIc to raise the yield. The latter was prepared from 1,2-O-isopropylidene-sn-glycerol (VIII)and 2,3,4,6-tetra-O-acetyl-a-D-galactopyranosyl bromide (IXc) [32] in a 50.2% yield in ethyl acetate in the presence of PbCO 3. The reaction product Xllc, chromatographically purified on alumina displayed a tendency to mild acid hydrolysis characteristic of orthoesters [13]. Its structure was confirmed by corresponding IR and PMR spectra. XIIc was isomerised to glycoside IXc in a 45.5% yield similar to orthoester VII conversion to glycosides III. Isopropylidene protection of mannoside IXa and glucoside IXb was removed by 10-20% aqueous solution of acetic acid at 100 ° (yield of Xa, b - 69.5% and 68.4%) and of IXc by 50% solution at 1 8 - 2 0 ° (yield of Xc - 86.2%). Compounds X a - c were chromatographically purified on silicic acid and shown by IR and PMR spectra and o.r.d, curves to be glycosides. Attempts made to use 1,2-O-ispropylidene-sn-glycerol (VIII) to synthesise glycosyl glycerols [9, 12] usually led to partial or complete racemisation at the glycerol chiral centre, apparently due to the acid formed on glycosylation. This process was also accompanied by partial anomerisation. In this respect the orthoester method was found.to have advantages over that of Koenigs-Knorr. First, the catalytic quantities of weakly acid catalysts (perchlorates of pyridine bases) used in these reactions were immediately consumed to give rise to orthester carbocation [ 13]. Hence the reaction medium was more strictly neutral and if some reasons tended the reaction products to become acidic the first to
276
V.I. Shvets et aL, Synthesis of glycosyl diglycerides
do so were glycosylating reagents, i.e. orthoesters (no glycoside formation), rather than the dioxalane ring of 1,2-O-isopropylidene-sn-glycerol (VIII). Secondly, the electrophylic centre of the orthoester carbocation was so strongly screened by the perchlorate ion that alcohol could attack only in one direction to form 1,2-transglycosides [13]. These considerations were confirmed when alkaline saponification of Xa-c gave glycosyl glycerols Va-c and their hexaacetates Via c, identical with respective compounds V and IVa-c prepared from glycosyl diglycerides IVa-c. Xa-c were acylated with palmitoyl chloride [17] in the presence of pyridine with the extent of esterification determined from the reaction temperature. It was shown that glucosyl- and galactosyt glycerols X b - c reacted in benzene at 5 5 - 6 0 ° to yield monoacylic derivatives XllIb, c. Acylation of mannosyl glycerol Xa gave an equal percentage of mono- and dipalmitins XIIla, Ilia, with the latter being identical with glycoside Ilia obtained from glyceride 1. Reactions in boiling toluene led to quantitative yields of diacyl derivatives IIIa-c similar to glycosyl dipalmitins IIIa-c prepared from compound I. Acylation of monopalmitoyl derivatives XIlla-c was effected under the same conditions. The step-wise acylation permitted various fatty acids to be introduced into glycosyl glycerol Xa-c and use of protection of monosaccharide hydroxylic groups by acetates did not hinder the formation of glycosyl diglycerides with unsaturated fatty acids. 2.1,3. Monoglycosyl diglycerides obtained from 2,5-O-methylene-D-mannitol The synthetic application of 1,2-O-isopropylidene-sn-glycerol (VIII) to obtain mixed glycosyl diglycerides was limited because the selective esterification of the hydroxyl group at glycerol C 1 due to its high reactivity could be accompanied by partial acylation of the hydroxyl group at C 2 leading to structurally heterogeneous glycosyl diglycerides. This limitation necessitated the use of a glycerol component with no acylation of the 2-OH-group on introduction of acid in the first position. Such a component was found to be 2,2'-O-methylene-bis-(1-O-acyl-sn-glycerol) prepared from D-mannitol through 2,5-O-methylene derivative [33]. The synthetic scheme [34] proposed was based on glycosylation of 2,2'-0methylene-bis-(1-O-palmituoyl-sn-glycerol) (XIV) [33 ] by ethylorthoacetate of D-mannose (lla, x = Et) and could be extended to obtain other glycosyl diglycerides as well (scheme 3, table 1). 1,2-trans-glycosylation of XIV with orthoacetate IIa was carried out in boiling chlorobenzene in the presence of 2,6-1uthidinium perchlorate, with the molar ratio of XIV, IIa and the catalyst of 1 : 1 : 0.05, during 1.5 hr. The reaction product, glycoside XVa, was chromatographically isolated on alumina in a 43.8% yield and characterised according to the general scheme used in this work. The rupture of the methylene bridge in glycoside XVa was effected by 50% methanol solution of acetic acid at 50-55 °. Mannosyl monoglyceride XIIIa, isolated chromatographically on silicic acid in a 85.3% yield was shown by physico-
277
El. Shvets et al., Synthesis of glycosyl diglycerides
HOCH2
CH20COC 1sH31
!
I H-C-O-CH2-O-C-H I I
H31CIsOCOCH2
R' -O -CH 2
+ (1 la) ~
CH2OH
H31ClsOCOCH2
(XIV)
I
H-C-OH
I
CH2OCOC15H31 (Xllla)
R'"COCI
CH2OR'
(XVa) R'OCH~
R'-OCH2
CH20COC 1sH31
1 I H-C-O-CH 2 - O - C - H I I
I
H-C-OCOR"'
i
CH2OCOClsH31 (IIIa, R'" = C15H31) (XVIa, R" = C17H33)
R"-O-CH 2 .,
L
H-C-OCOR"'
L
CH2OCOls H31 OVa, R'"= ClsH31) (XVIIa, R'" = ClTHaa)
Scheme 3. chemical constants, IR and PMR spectra and o.r.d, curve to be identical with 1-Opalmitoyl.3-O-(2,3,4,6-tetra-O-acetyl-a-D-mannopyranosyl)-sn-glycerol(XIIIa) prepared by selective acylation of mannosyl glycerol Xa. This suggested that monoesterification of glycosyl glycerols X a - c was more selective at the hydroxyl group at glycerol C 1 . Conversion of XIIIa to diacyl derivative III, XVIa was carried out by acylation with palmitoyl chloride and oleoyl chloride [35] in boiling toluene in the presence of pyridine. The reaction gave quantitative yields with Ilia being similar to glycoside Ilia prepared from 1,2-di-O-palmitoyl-sn-glycerol (1) and 1,2-O-isopropylidenesn-glycerol (VIII). Mannosyl dipalmitin Ilia and mannosyl diglyceride XVIa with residues of palmitic and oleic acids were found to be rather similar in their physico-chemical properties, chromatographic mobility and o.r.d, curves. Their difference (m.p., IR and PMR spectra) were caused by the double bond in the oleic acid of XVIa. Thus the IR spectrum of glycoside XVIa revealed a band-CH ---(t)ma x 3010 c m - 1 ) that was absent in the spectrum of Ilia. Deacetylation of XVIa resulted in 1-O-palmitoyl-2-O-oleoyl-3-O-(a-D-mannopyranosyl)-sn-glycerol (XVII) in a 91.2% yield. This product was the first unsaturated glycosyl diglyceride to be prepared synthetically. Identification of XV, XVI, XVIIa was effected by IR, PMR spectra and o.r.d, curves as in other cases. That method was more multistaged than the first two methods described and its apparent advantages in preparing mixed glycosyl diglycerides as compared with the preparation starting from 1,2-O-isopropylidene-sn-glycerol were found experimentally to be negligible.
278
KL Shvets et aL, Synthesis of glycosyl diglyeerMes
2.2. Synthesis of diglycosyl diglycerides
Having worked out synthetic routes to monoglycosyl diglycerides as exemplified by the synthesis of mannosyl-, glucosyl- and galactosyl diglycerides we proceeded to the synthetic study of oligoglycosyl diglycerides. It proved thereby possible to obtain diglycosyl diglyceride by binding together the oligosaccharide and diglyceride moieties of such a compound as/3-D-cellobioside-l,2-di-O-palmitoyl-3-O [/3-D-glucopyranosyl-(1 ~ 4)-/3-D-glucopyranosyl] -sn-glycerol (XXI1) [36] (scheme 4, table 1). The synthetic route used was that of direct glycosylation of glyceride 1 previously proposed for monoglycosyl diglycerides.
CH2OAc
CH2OAc
0 0CHA2OcAc~
AcOO A c ~ O O A ~ c OOAc OAc OAc (XVlll) CH2OAc CH2OAc
Ac
OAc OAc (XlX) CH2OAc CH:OAc
o ,O
Ac6~,--4"
OAc (XX)
O ACH2OAc c~ 1 O Br
+ (1)~ O-/---OEt Me 9
CH2OA¢
A~O~ = = f
O ~ OAc
H_(~_OOCCIsH31 OAc
,
(XXl) CNz0Ac
O~H o A----o ,--O-FH~ ~o.~/o. ~1 ._c-oocc,,.,,
OH (XXll)
OH
Scheme 4.
The synthesis was effected starting from 1,2-di-O-palmitoyl-sn-glycerol (1) and ethylorthoacetate of cellobiose XX. The latter was obtained from octaacetate XVIII [37] through acetobromocellobiose XIX on its interaction with ethanol in the presence of 2,6-1utidine in a 48.6% yield. The structure of orthoester XX was determined by physico-chemical methods previously used for structural identification of orthoesters VIIa-c and XIIc.
V.I. Shvets et al., Synthesis of glycosyl diglyeerides
279
Orthoester XX was introduced in the reaction of 1,2.trans-glycosylation with 1. It was found that an excess of I, 2 moles per mole of XX, and 0.1 mole of catalyst (2,6-1utinium perchlorate) increase the yield of glycoside XXI up to 80.1% as compared with 45.8% from the equimolar percentage of compounds I and XX. Glycoside XXI was isolated chromatographically on silicic acid and identified as above. Deacetylation of XXI gave a compound characterised by IR, PMR spectra and o.r.d, curve as being 1,2-di-O-palmitoyl-3-O-[/3-D-glucopyranosyl-(1 ~ 4)-/3-Dglucopyranosyl] -sn-glycerol (XXII) in a 92.2% yield.
1,2-dipalmitin
2. 3. Summary
The proposed synthetic routes to glycosyl diglycerides by orthester method of glycosylation can be used to prepare both saturated and unsaturated mono- or mixed 1,2-trans-glycosyl diglycerides with mono- and oligosaccharide residues in the carbohydrate moiety of the molecule. Of several alternatives involving 1,2-diglycerides, 1,2-O-isopropylidene-sn-glycerol- and 2,2'.O-methylene-bis-(1-O-acyl-sn-glycerol) the most suitable method is that of synthesis of glycosyl diglyceride from 1,2-O-isopropylidene-sn-glycerol.
3. Experimental part 3.1. Materials and methods
All m.p.s, were measured in open capillaries and not corrected. The reaction course, chromatographic separation and the properties of compounds were studied by TLC. The preparative and TLC were effected on silicic acid of the "aqueous" sort in such systems as ether-hexane 3 : 1 (1); ether-hexane, 1 : 1 (2); chloroform-acetone, 2 : 1 (4); chloroform-methanol-acetone, 40 : 5 : 5 (5); chloroform-methanol-water, 65 : 12 : 2 (6); on silica gel G in such solvent systems as chloroform-acetic acidwater, 50 : 35 : 3 (7) [38] ; chloroform-methanol-7 N NH4OH, 65 : 30 : 4 (8) [39] ; on alumina of the activity III after Brockman in such solvent systems as chloroform-acetone, 9 : 1 (9). Silicic acid and silica gel were preliminarily activated by heating at 1 2 0 - 1 4 0 ° for 4 hr. The compounds were revealed on chromatograms by sprinkling them with conc. H2SO 4 followed by carbonisation at 3 0 0 350 °. The descending chromatography* was effected on the paper "Leningradskaya bystraya" (PC) in such solvent systems as n-butanol-pyridine-0.1 N hydrochloric acid, 5 : 3 : 1 (10) [28] ; n-butanol-pyridine-water, 6 : 4 : 3 (11) [40, 41]. The products were identified by means of alkaline silver nitrate on heating up to 1 0 0 120 °. The o.r.d, curves and [a] 20 were measured on spectropolarimeter SPU-M. * Rfrna n is given in ratio of the length run of mannose.
280
V.1. Shvets et al., Synthesis of glyeosyl diglyeerides
IR spectra were recorded on spectrometer Perkin-Elmer, model 257 in nujol, PMR spectra on Varian HA-100D in ethanol-free chloform or in deuterochloroform, inner standard-TMS, with chemical shifts presented in the 8 scale. The compounds obtained all had a satisfactory elementary analysis (tables 1 and 2). 3.2. A c M hydrolysis
Glycosides IVa-c (3 rag) were heated in a sealed ampoule with 1 ml of 50% dioxane solution containing 5% of hydrogen chloride at 100 ° during 18 hr. The ampoule content was then concentrated by evaporation and the residue analysed by TLC in system 2 and PC in systems 10, 11 in the presence of samples of corresponding monosaccharides and of dipalmitin 1, palmitic acid and glycerol. 3.3. Alkaline hydrolysis
A mixture of compounds IVa-c (3 mg) in 0.07 N sodium methylate solution (3 ml) in methanol was kept at 18-20 ° during 20 hr and brought up to pH 4.5 by 37% hydrochloric acid. The mixture was then concentrated by evaporation and the residue analysed by TLC in system 2 and PC in systems 10, 11 in the presence of corresponding glycosyl glycerols Va c and palmitic acid. 3.4. Combined acM and alkaline hydrolysis
Glycosides IVa-c were completely split by consecutive acid and alkaline hydrolysis and then the reaction mixture was analysed by PC in systems 10, 11 in the presence of sample of mannose, glycose, galactose and glycerol. 1,2-Di-O-palmitoyl-3-O-(2,3,4,6-tetra-O-acetyl-a-D-mannopyranosyl)-sn-glycerol (!IIa). To prepare this compound a solution of 1.62 g (4 mmoles) of orthoester IIa, x = Bu-tert. [18], 2.3 g (4 mmoles) of 1,2-di-O-palmitoyl-sn-glycerol (1) [17], and 17 mg (0.08 mmole) of 2,6-1utidinium perchlorate in chlorobenzene (15 ml) was heated during 3 hr. The tert-butyl alcohol splitting off was simultaneously removed and the volume of the reaction mixture kept constant. When the reaction was over, chlorobenzene was concentrated by evaporation, the residue chromatographed on silicic acid, fractions of carbon tetrachloride washed away and ether gradually added from 2 to 20%. Fractions containing IIIa were concentrated by evaporation and crystallised from methanol. Yield: 1.5 g (41.4%). M.p. 28.0-29.0 °, [a] 20 +24.2 (0.76% solution in chloroform),Rf 0.6 (1); Umax: 1750 (C = 0 in COOR), 1135 (C-O at C1), 920 (pyranose ring), 840 (CH at C 1) cm--1; PMR, 8 2:05 (4 MeCO), 4.84 (H1,J 1 2 = 1.9 Hz). Similarly,' glycosylation of compounds I, VIII, XIV gave IIIb-c, IXa c, XVa, and XXI (table 1).
V.l. Shvets et aL, Synthesis of glycosyl diglyeerides
281
Deviations from this procedure concerning the solvents and catalysts used, the ratios of reacting compounds to the catalyst and the method of glycoside isolation were considered above when discussing the results obtained.
3,4,6~Tri~-acetyl-~2~( ~2-di~-palmit~yl~sn~gly~eryl-3-~-~rth~acetyl)-~-D-mannopyranose (VIIa). To prepare this compound a solution of 3 g (8 mmoles) of orthoester IIa, x = Et [22], 4.5 g (8 mmoles) of 1,2-diglyceride I and 27.6 mg (0.16 mmole) of YsOH in dichloroethane (50 ml) was heated during 2.5 hr. The ethanol splitting off was simultaneously removed and the volume of the reaction mixture kept constant. When the reaction was over, dichloroethane was concentrated by evaporation, the residue chromatographed on silicic acid with benzene used as eluent whose ether content was gradually raised from 5 to 30%. Fractions containing orthoester VIIa were concentrated by evaporation and crystallised from 10 ml of hexane. Yield: 3.5 g (49.4%). M.p. 68.0-69.0 °, [a] 20 +11.27 ° (0.40% solution in chloroform), R f 0.3 (2); Vmax: 1740 (C = 0 in COOR), 1170 ( C - 0 at C1), 900 (pyranose ring) c m - 1 ; PMR, 6 1.77 (endo-C-Me), 2.0 (3MeCO)). VIIb, c were obtained similarly except for using alumina rather than silicic acid in chromatography (table 1). The samples of orthoesters VIIa-c were hydrolysed completely by O. 1 N H2SO 4 in 90% aqueous acetone [13]. 1,2-Di-O-palmitoyl-3-O-(2,3,4,6-tetra-O-acetyl-a-D-mannopyranosyl)-sn-glycerol (IIIa). To prepare this compound a solution of 1.0 g (0.11 mmole) of orthoester VIIa and 7.9 mg (0.022 mmole) of 2,2'-dipyridilium perchlorate in dichloroethane (20 ml) was boiled during 1.5 hr and then kept at 18-20 ° for 12-14 hr. The glyceride precipitate was separated, the filtrate concentrated by evaporating and the solid dissolved in 30 ml of 0.1 N H2SO 4 in 90% aqueous acetone and left standing for 25 hr. The mixture was neutralised with 10% NaHCO 3 and extracted with chloroform (2 × 30 ml). The extract was dried by CaC12, concentrated to 5 ml and passed through a column over alumina. The resulting product was eluated with chloroform and after crystallisation from methanol (2 ml) gave pure crystalline Ilia. Yield: 0.4 g (40.0%). M.p. 30.0-31.0 °, [a] 20 +22.3 ° (0.33% solution in chloroform), R.f 0.6 (I), IR an PMR spectra were identical with those of glycoside Ilia obtained from I. Similarly, isomerisation of VIIb, c resulted in glycosides IIIb, c, that of XIIc in IXc (table 1). 1,2-Di-O-palmitoyl-3-O-(a-D-mannopyranosyl)-sn-glycerol (IVa). This compound was prepared from a mixture of 0.68 g (0.75 mmole) of IIIa, 0.3 ml (15 mmoles) of H2NNH2"H20 in 85% ethanol (45 ml) was boiled for 25 min, then kept at 18-20 ° for 15 hr and 1 hr at 0 °. The precipitate was filtered off, washed with 85% ethanol and recrystallised from methanol (1 ml). Yield: 0.52 g
282
V.I. Shvets et aL, Synthesis of glycosyl diglycerides
(92.3%). M.p. 108.0-1 t0.0 °, [o~]20 +27.0 (0.84% solution chloroform). R f 0.5 (5), 0.55 (7), 0.85 (8); Vmax: 3600-3100(OH), 1730 (C = O in COOR), a number of bands over the range of 1350-1200 (CH 2 in C15H31), 915 (pyranose ring), 840 (C H at C1) cm-1;PMR, 6 4.93 (H 1,J1,2 = 1.9 Hz). Similarly, Illb, c, XVIa, XXI gave IVb, c, XVIIa, XII (table 1).
3-O-a-D-mannopyranosyl-sn-glycerol (Va). To prepare this compound a mixture of 0.5 g (0.7 mmole) of mannosyl diglyceride IVa and 35 ml (3.5 mmoles) of 0.1 N NaOH was heated at 100 ° for 1 hr, cooled and neutralised by adding Dowex-50 (H+). The resin was removed and the product washed with water (10 ml). The combined filtrates were extracted with ether (2 × 20 ml) and the aqueous layer concentrated by evaporation to an amorphous form. Yield: 0.17 g (96%). [a] 20 +42.5 ° (0.62% solution in water)Rfman 0.82 (10); Vmax: 3600-3100 (OH), 1100 1000 (C-O), 920 (pyranose ring) 840 (C-H at C1) cm -1. Similarly, IVb, c gave Vb, c (table 2).
1,2-Di-O-acetyl-3-O-(2,3, 4,6-tetra-O-acetyl-a-D-mannopyranosyl)-sn-glycerol (Via). To prepare this compound 0.13 g (0.51 mmole) of mannosyl glycerol Va was suspended in carbon tetrachloride (4 ml) and 1 ml (12 mmoles) of pyridine, 2 ml (25 mmoles) of acetyl chloride in carbon tetrachloride (2 ml) added. The mixture was left standing at 18-20 ° for 6 hr. The precipitate was separated, washed with carbon tetrachloride and the substrate concentrated by evaporation to result in an oily substance that was then analytically purified by preparative TLC in system 9. Yield: 0.25 g (96.5%). [c~]20 +39.2 ° (0.35% solution in chloroform), R f 0.65 (9); Vmax: 1750 )C = O in OCOMe), 1220 (C-O in OCOMe), 1130 (C-O at C1), 1080, 1050 (C-O), 915 (pyranose ring), 835 (C-H at C1) cm-1;PMR, 6 2.2 (6 MeCO), 4.83 (H l, Jl,2 = 2 Hz). Similarly, Vb, c gave hexaacetates VIb, c (table 2).
3•4•6•Tri-•-acety•-••2••-(1•2-•-is•pr•pylidene-sn.g•ycery•-3••••rth•acety•)-a-Dgalactopyranose (Xllb). To prepare this compound a mixture of 6.5 g (16 mmoles) of 2,3,4,6-tetra-Oacetyl-a-D-galactopyranosyl bromide (XIc) [32], 10.5 g (75 mmoles) of CaSO4, and 14 g (46 mmoles) of PbCO 3 in ethylacetate (150 ml) was boiled for several minutes. Then 1.0 g (7.6 mmoles) of 1,2-O-isopropylidene-sn-glycerol (VIII) [ 16] was added and the mixture boiled for 3 hr. Another 1.Og (7.6) mmoles of VIII) was subsequently added and the mixture boiled for 10 hr more. The suspension was cooled down to 18-20 °, 6.0 g (27 mmoles) of Ag20 and 37 ml of 95% aqueous acetone introduced and the mixture left standing for 48 hr. The reaction mixture was then filtered off, concentrated by evaporation and the residue separated on a column with alumina by washing with chloroform. Fractions containing the product were concentrated by evaporation to give an oily substance. Yield: 3.22 g
V./.
Shvets et al., Synthesis of glycosyl diglycerides
283
(50.2%). [ct] 20 +70.3 (0.6% solution in chloroform), Rf 0.5 (3); Vrnax: 1750 (C = O in OCOMe), 1370 (C-Me), 1220 (C-O in OCOMe), 1150 (C-O at CI), 910 (pyranose ring) cm-1; PMR, 6 1.36 (C-Me2), 1.70 (endo-C-Me). The sample of orthoester XXc was completely hydrolysed by 0.1 N H2SO 4 in 90% aqueous acetone [13].
4-0-(2,3,4,6-Tetra-O-acetyl-(3-D-glucopyranosyl)-3,6-di-O-acetyl-l,2-O-ethyl-ortho. aeetyl-~-D-glueopyranose (XX). To prepare this compound, to a solution of 3.0 g (4.9 mmoles) oft~-D-cellobiose octaacetate XVIII [37] in chloroform (15 ml) were added 10 ml of glacial acetic acid saturated with HBr (100 mmoles) at 0 ° and 1 ml of acetic anhydride. The mixture was diluted with chloroform (25 ml) and washed with glacial water (2 X 10 ml). The organic layer was dried with CaCI2 and concentrated by evaporation. The residue (3.02 g) - crude bromide )(IX - was dissolved in chloroform (10 ml), 5 ml of 2,6-1utidine, 15 ml of ethanol were added and the reaction carried out at 18-20 ° during 96 hr. The reaction mixture was dissolved in chloroform (20 ml) and washed with glacial water (2 × 15 ml). The organic layer was dried with MgSO4, concentrated by evaporation, and the residue chromatographed over alumina with the compounds being eluated by chloroform. The fractions containing orthoester XX were concentrated by evaporation and crystallised from 15 ml of hexane-ether mixture (9 : 2). Yield: 1.42 g (48.5% on XVIII). M.p. 49-50 °, [ct] 20 + o 7.5 (0.43% solution in chloroform), Rf 0.8 (9); Vmax: 1750 (C = O in OCOMe), 1220 (C-O in OCOMe), 1160 (C-O at C1), 900 (pyranose ring); PMR, 8 1.78 (endo-C-Me), 2.0-2.15 (6 MeCO). The sample of orthoester XX was completely hydrolysed by 0.1 N H2SO 4 in 90% aqueous acetone [13].
3-O-(2,3,4,6-tetra-O-acetyl-a-D-mannopyranosyl).sn.glycerol(Xa). To prepare this compound a solution of 0.55 g (1.2 mmole) of mannoside IXa in 10% acetic acid (25 ml, 42 mmoles) was heated at 100 ° for 1 hr, cooled and neutralised with saturated aqueous NaHCO 3. The mixture was diluted with water (50 ml) and extracted with chloroform (3 X 50 ml). The combined extracts were dried with MgSO4, concentrated by evaporation and the product isolated by pre20 parative TLC in system 4 to obtain an oily substance. Yield: 0.32 g (69.5%). [~] D +37.48 (0.71% solution in chloroform), Rf 0.5 (4); Vmax: 3700-3100 (OH), 1750 (C = O in OCOMe), 1230 (C-O in OCOMe), 1140 (C-O at CI) , 1100-1000 (C-O), 915 (pyranose ring) cm-1. Almost similarly (see above, §II) IXb, c gave Xb, c (table 1).
1-O-Palmitoyl-3-O-(2,3,4,6-tetra-O-acetyl-a.D.mannopyranogyl).sn.glycerol(XIIIa) and 1,2-di-O-palrnitoyl-3-O-(2,3,4,6-tetra-O-acetyl.a.D.mannopyrasonyl).sn.glycerol (IIIa). To prepare these compounds, to a solution of 0.17 g (0.4 mmole) of glycoside
v.1. Shvets et aL, Synthesis of glyeosyl diglyeerides
284
Xa and 0.1 ml (1.2 mmole) pyridine in benzene (15 ml) 0.214 g (0.8 mmole) of palmitoyl chloride was added with stirring at 00 and the mixture was left standing for 3 hr. Then the temperature was raised up to 5 5 - 6 0 ° and the reaction carried on 6 - 7 hr. The precipitate was filtered off, the solvent concentrated by evaporation and the residue, consisting of XIIla and Ilia separated by preparative TLC in system 1. Yield of Xllla (an oily substance): 0.15 g (55.5% on Xa), [c~]20 +31.4 ° (0.21% chloroform solution), R f 0.33 (I); Vmax: 3450 (OH), 1750 (C = O in COOR), 1140 (C-O at C1), 920 (pyranose ring) c m - l : PMR, 6 2.05 (4 MeCO), 4.93 (H 1, J1 2 = 1.85 Hz). Yield of Ilia: 0.19 g (52.8% on Xa). M.p. 2 8 - 2 8 .3 o , [cq 20 +23.4 ~ (0.35% chloroform solution), R f 0.6 (1). IR and PMR spectra were identical with those of glycoside Illa synthesised from I. Similar acylation of Xb, c gave only monopalmitoyt derivatives XIIlb,c (table 1).
1,2-Di-palmi toyl-3-O-( 2,3, 4, 6-tetra-O-acety l-a-D-manno pyranosyl )-sn-gly cerol (Ilia). To prepare this compound a mixture of 0.1 g (0.24 mmole) of glycoside Xllla and 0.13 g (0.48 mmole) of palmitoyl chloride was boiled for 6 hr in the presence of pyridine (0.08 ml, 0.001 mmole) and toluene (10 ml). The reaction mixture was filtered, concentrated by evaporation and the residue crystallised from 2 ml of methanol. Yield: 0.14 g (93.2%). M.p. 28.0-29.0 °, [a] 2D0+23.9 ° (0.76% chloroform solution), R f 0.6 (I). IR and PMR spectra were identical with those of glycoside Ilia, synthesised from I. Similarly X a - c gave llIa-c. The same conditions also resulted in conversion of XIIIa to XVIa (table 1).
1.O.Palmitoyl-3.0-( 2,3, 4,6-tetra-O-acetyl-a-mannopyranosyl)-sn-glycerol (XIIIa). To prepare this compound a solution of 0.6 g (0.45 mmole) of glycoside XVa in 50% methanol solution of acetic acid (20 ml, 170 mmoles) was heated at 50-55 ° during 1 hr. The reaction mixture was dissolved with chloroform (100 ml), washed with water (50 ml) and saturated aqueous solution of NaHCO 3 (50 ml), then repeatedly with water (30 ml) and finally dried with CaC12. The product was isolated by TLC in system 1 in the form of an oily substance. Yield: 0.55 g (85.3%). [a] 20 + 32.4 ° (0.2% solution in chloroform), R f 0.33 (I). IR and PMR spectra were identical with those of XIIla obtained from Xa.
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