- Email: [email protected]
Polysaccharide synthesis from mono- and oligo-saccharides by the action of phosphorus pentoxide in dimethyl sulphoxide
Polysaccharide synthesis from mono- and oligo-saccharides by the action of phosphorus pentoxide in dimethyl sulphoxide* Shigehiro Hiranot, Naoki Kashimura, Noboru Kosaka and Konoshin Onodera Department of Agricultural Chemistry, Kyoto University, Kyoto, Japan (Received 10 August 1971) Phosphorus pentoxide-dimethyl sulphoxide (PaOlo-DMSO) was found predominantly to catalyse the polymerization reaction of carbohydrates at below 35°C and the oxidation reaction at 60-65°C. A series of new synthetic polysaccharides were prepared from mono- and oligo-saccharides including 2-acetamido-2deoxy-D-glucose and hexuronic acids in up to 48% yield by the action of P4010DMSO at 10-25°C. These synthetic polysaccharides showed S2o,w0.68--1.34S, and the degree of polymerization fell in 15.3-4.7 monosaccharide units per polysaccharide chain on the basis of reducing end-group assay. A structural analysis by the methylation of the synthetic glucan (2) revealed a-1,4- and ~-l,6-glucosidic linkages as main chains with various branchings. The synthetic polysaccharides contained 1.3-15.9% phosphorus, but the linkages are unknown.
INTRODUCTION In previous years, a number of attempts have been made to synthesize naturally occurring polysaccharides and the related polymers of increasing interest from biochemical and industrial points of view 1. Fischer and Delbriick 2 first reported a procedure for the synthesis of an oligosaccharide from substituted monosaccharides by. the action of P4010. Since then, the simple principle in that a water molecule is eliminated with P4010 has been applied to the synthesis of various glycosides and polysaccharides from mono- and oligo-saccharides without significant success. In 1961, Micheel et al. 3 prepared a series of branched polysaccharides from mono- and oligo-saccharides by the action of P4010 in the presence of HCI or HBr as catalyst in dimethyl sulphoxide (DMSO). In the course of our studies on catalytic action of phosphorus compounds a-v, we found, in addition to the oxidation 4, s, the formation of polysaccharides (O-glycosides) and glycosylamines (Nglycosides) by the action of P4010 without any addition of catalyst in DMSO at below 35°C. More recently, Husemann and MiJller 9 prepared a cellulose-like polysaccharide * Presented at the annual meeting of the Agricultural Chemical Society of Japan, Tokyo, 1-4 April 1965, and a preliminary note of the present work appeared in a footnote of ref. 4 t To whom inquiries should be addressed. Present address: Department of Agricultural Chemistry, Tottori University, Tottori, Japan
190
POLYMER, 1972, Vol 13, May
from 2,3,6-tri-O-(N-phenylcarbamyl)-o-glucopyranose by the action of P4010 in DMSO-CHCIa. Independently Mizuno 10 confirmed P4010 as a good dehydrating agent for polysaccharide synthesis in DMSO and prepared a series of polysaccharides from monosaccharides. Partial structural analysis of the synthetic galactan and xylan indicated considerable branchings 11. The present work reports that P4010-DMSO catalyses polycondensation and oxidation depending on the reaction temperature. The synthesis of a series of new synthetic polysaccharides is described. These are prepared from mono- and oligo-saccharides including 2-acetamido2-deoxy-D-glucose and hexuronic acids by the action of P4010-DMSO at 10~-35°C. Furthermore, a structural analysis by the methylation of the synthetic glucan (2) is for the first time described. EXPERIMENTAL Analytical methods Hexoses and pentoses were analysed by the anthrone method lz, hexosamine by the Elson-Morgan method 13, hexuronic acids by the carbazole method 14, phosphorus by the Allen method 15, and reducing sugar values by both the ferricyanide16 and 3,5-dinitrosalicylic acid 17 methods. Sedimentation patterns were obtained with a synthetic b o u n d a r y cell at 59 780 rev/min with a Spinco Model E
Polysaccharide synthesis from mono- and oligo-saccharides by action of P401o in DMSO : Shigehiro Hirano et al. ultracentrifuge in ~ 1 ~ solution of the sample as dissolved in 0.15M KC1. Sedimentation constants are expressed as s20,w. Tiselius electrophoresis was carried out in two different buffer solutions at 4.0-4.5°C: (a) sodium borate (0.1M), pH 8.98, /z 0.15, 9.8mA; (b) acetic acid-sodium acetate (0.1 M), pH 5.0, t~ 0.10, 10.0 mA. Periodate oxidation was carried out at 4.0°C in the dark 18, and the periodate consumed is calculated in terms of the corresponding monosaccharide unit which is free of phosphorus. Infra-red (i.r.) spectra were recorded with a Shimadzu A R-6 spectrometer in Nujol or KBr pellet, and optical rotatory dispersion (o.r.d.) with a JASCO automatic recording spectropolarimeterwith 1~solution of the sample at 17 °C in water. Paper chromatography was carried out on Toyo Roshi No. 51 filter paper by descending method with a solvent: 1-butanol/ethanol/1 ~ ammonia (4 : 1 : 5, v/v), and the alkali silver nitrate reagent was utilized for the detection of reducing sugars. Enzyme digestion was carried out with c~-amylase originated from Asperigillus oryzae (Sigma Chemical Co.) at 35°C at pH 5-5, and with /3-amylase originated from barley malt (Sigma Chemical Co.) at 35°C at pH 5.0 tg. The change of specific rotation was measured during the mild acid hydrolysis of the synthetic polysaccharides: each of the polysaccharides was dissolved in 1.0~ concentration in both 0.01 N and 1.0N HCI and the solution was hydrolysed in a boiling water bath. Specific rotation was measured with a Yanagimoto direct reading polarimeter.
General procedure for polysaccharide synthesis To 50ml of anhydrous DMSO, 10g of P4Olo were added in small portions with stirring to produce a homogeneous solution in an ice bath. To the solution was added 10g of the corresponding carbohydrate or the mixture, and the solution was shaken at 15-35°C for 3-5 days. In some cases, 0"5 g of dried Dowex 50(H +) resin was additionally added as a catalyst tg the reaction mixture at the beginning of the reaction. The reaction mixture was poured into a large volume of acetone, and the mixture was stirred to produce an amorphous product. The supernatant solution was decanted and the residue was dis-
solved in ~ 300 ml of ice water. The solution was dialysed against running water through a cellophane tubing for two days and concentrated in vacuo at below 45 °C to give a syrupy product. Residual DMSO was removed from the syrup by evaporating three times after addition of 50 ml each of 1,4-dioxane. The polysaccharide thus obtained was precipitated by addition of five volumes of acetone. The mixture was kept at room temperature for 24 h. The precipitates produced were collected by centrifugation, washed with ether and dried over calcium chloride in a desiccator (see Table 1).
Structural analysis by the methylation of the synthetic glucan (2) The synthetic glucan (2) (5-0 g) was dissolved in 200 ml of DMSO. The methylation was carried out by Hakomows procedure z°, twice by Purdie's procedure of continuous reftuxing for 5 days 21, and finally by Kuhn's procedure of treating for continuous 6 days 22. Further methylation did not give the increase of methoxyl content. No OH absorption was detected in 3000-3500cm -1 region in the i.r. spectrum (see Figure 1). Yield, 4.2g; [c~]~°+74 ° (c 1"0, methanol). Calcd for (CgH16Os)n: CH30, 45.6~. Found: CH30, 41.2~. The fully methylated product (3g) was refluxed in 200ml of 8 5 ~ HCOOH for 1 h and then refluxed in 200ml of 0.5N H2SO4 for 17h. After neutralizing with solid BaCO3, the filtrate was concentrated in vacuo to a syrup (2.8 g). Paper chromatography indicated nine spots of methylated o-glucoses. At the first step, the hydrolysate was separated into four fractions of mono-, di-, triand tetra-O-methylated D-glucoses by the preparative paper chromatography (Whatman 3MM). At the second step, the mono-O-methyl fraction was separated into three compounds, the di-O-methyl fraction into three compounds and tri-O-methyl fraction into two compounds, respectively by the second preparative paper chromatography (see Table 4).
Spot 1. The fraction was 2,3,4,6-tetra-O-methyl-Dglucose which was identified by mixture melting point and i.r. spectroscopy as 2,3,4,6-tetra-O-methyl-N-phenyl-Dglucopyranosylamine: m.p. 138-139°C; [c~]16+230° (c 0.5, acetone); reported 23 m.p. 137-138°C; [C~]D+228-239"5 ° (acetone). Calcd for C16H2sNOs: CH30, 39.9 ~. Found: CH30, 40.3 ~. Spot 2. The fraction was 2,3,4-tri-O-methyl-D-glucose which was identified by mixture melting point and i.r. spectroscopy as 2,3,4-tri-O-methyl-N-phenyl-D-glucopyranosylamine: m.p. 150 °C; [a]]5_ 103 ° (c 0.7, ethanol); reported 24 m.p. 150 °, HD--103 ° (ethanol). Calcd for ClsH~3NOs: CH30, 31.3~. Found: CHaO, 30.6~.
4000
2000
1500
I OOO
600
Cfl/-I Infra-red spectra at each step of the methylation of synthetic glucan (2). (a) Synthetic glucan (2); (b) after Hakomori's methylation (Found: CH30, 35:6%); (c) after Hakornori and Purdie's methylations (Found: CH30, 38.8%); (d) after Hakomori, Purdie and Kuhn's methylations (Found: CHaO, 41.7%)
Figure !
Spot 3. The fraction was identified by mixture melting point and i.r. spectroscopy as 2,3,6-tri-O-methyl-~-oglucopyranose: m.p. 120-122°C, H~7+83--* +20 ° (c 0.2, water); reported 25 m.p. 121-123°C, [a]D+90--* +70"5 ° (water). Calcd for CgHlsO6: CH30, 41.9~. Found: CH30, 41.3 ~. Spot 8. The fraction was identified by mixture melting point, i.r. and nuclear magnetic resonance (n.m.r.)spectroscopies as 3-O-methyl-~-D-glucopyranose: m.p. 161 °C, [a]])5 +85 ~ +56 ° (c 0.3, water); reported 26 m.p. 160-161°C, POLYMER, 1972, Vol 13, May
191
Polysaccharide synthesis from mono- and oligo-saccharides by action of P401o in DMSO : Shigehiro Hirano et al. Table 1 Polysaccharide synthesis from mono- and oligo-saccharides Synthetic
Reaction condition
polysaccharide
Carbohydrates used for polycondensation
Temperature (°C)
Time(h)
(1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17)
D-glucose* D-glucose D-glucose and c~-D-glucose-l-phosphate (K salt) (1000 : 1, w/w) D-glucose 2-acetamido-2-deoxy-D-glucose* 2-acetamido-2-deoxy-D-glucose L-arabinose D-xylose D-xylose and xylan (4 : 1, w/w) D-xylose and xylan (100 : 1, w/w) lactose* lactose maltose cellobiose 2-acetamido-2-deoxy-D-glucose and D-glucose (1 : 1, w/w) D-glucofuranuronolactone and D-glucose (1 : 1, w/w) 2-acetamido-2-deoxy-D-glucose and D-gluco ~uranuronolactone (1 : 1, w/w)
20-35 35 30-35 10-15 20-35 35 35 35 15 15 20-35 10-15 10-15 10-15 10-15 10-15 10-15
120 120 120 340 72 72 72 72 72 72 120 340 340 340 340 340 340
Yield
(%)
48 48 45 0.5 24 38 12 36 45 39 24 5 16 11 14 15 12
* Dry Dowex 50(H ÷) was used as a catalyst (see text)
[0~]D -]-104 ~ + 55 ° (water). 8(D20)3"61 (s 3H, MeO-3), 5.20 (d 1H, H-l, J1,2 3-0 Hz). Other spots could not be crystallized, but a tentative assignment of the spots was carried out by paper chromatography with the reference of reported Rtg values 27, 28 (Rtg; mobility rate on the paper chromatogram with reference to 2,3,4,6-tetra-O-methyl-D-glucose) (Table 4).
RESULTS AND DISCUSSION P4010-DMSO catalyses not only the oxidation but also the polycondensation of carbohydrates. Maximum yield of the oxidation products was obtained in the reaction at 60--65°C as reported in our previous papers 4,s. On the other hand, maximum yield of the synthetic polysaccharides was obtained in the reaction at 30-35°C (see Table 1). it was found that the reaction temperature influences significantly to the type of predominant reactions. The yield of the synthetic polysaccharides was up to 48 %, which is in agreement with a reported result 11. Catalytic action of Dowex 50(H +) was not significant in the polymerization reaction. This indicates that P4010 may act as dehydrating agent as well as acidic catalyst. The reaction with methyl-a-o-glucopyranoside, a-Dglucopyranose-l-phosphate and methyl-2-acetamido-2deoxy-~-o-galactopyranoside did not produce the corresponding polymer. This substantiates the view that the polysaccharides have mainly glycosidic linkages. It is of interest to examine an action of naturally occurring polysaccharides as a starter or an action of monosaccharide phosphates as an intermediate in the polycondensation reaction. The experiments were carried out in the polycondensation of o-xylose in the presence of natural xylan and in the polycondensation of D-glucose in the presence of c~-o-glucopyranose-l-phosphate (see Table 1). As a result, no significant action of these compounds was found in the yield and the physical properties of the products. The synthetic polysaccharides are soluble in water and DMSO, and the 15 % aqueous solution showed almost no viscosity in comparison with water.
192
POLYMER, 1972, Vol 13, May
The synthetic polysaccharides were hydrolysed with 2N H~SO4 at 100°C for 8-16h. Paper chromatographic examination indicated mainly the corresponding monosaccharide ( ~ 90 %) together with one or two minor spots which are considered to be oxidized products. The detailed structure of the minor spots is unknown. Partial acid hydrolysis (IN H2SO4 at 100°C for 30min) of the synthetic glucan (2) showed more than two spots of oligosaccharides in small quantity in addition to the large amount of D-glucose. One of the spots was identical with maltose on paper chromatogram. In the case of heteropolycondensation, (15) showed an equivalent proportion of the component sugars, but the proportion was changed in (16) and (17), with which low contents of uronic acid was observed. The synthetic polysaccharides contained 1.320.9% phosphorus. The phosphorus could not be removed by repeating the procedure of mild methanolysis2a, and the treatment was attended with partial degradation of some glycosidic linkages of the polysaccharides (see Table 2). The synthetic polysaccharides showed relatively narrow distribution in the molecular weight from behaviour in sedimentation patterns. The s20, w fell in 0.69-1.34S. Tiselius electrophoresis revealed that each of the polysaccharides consists of two or more components. These results indicate no typical difference of molecular weight with regard to the products, but there is some heterogeneous distribution of the phosphorus content. Reducing values of the products is relatively high in comparison with that of naturally occurring polysaccharides and the value indicates the degree of polymerization in 15.3-4.7 monosaccharide units per polysaccharide chain. The high reducing value may be due to the participation of new reducing groups produced in a small quantity during the polymerization by partial oxidation of the polymers 4, s or by decomposition of DMSO 6. The stability of the polymers towards dilute acid solutions was characteristic of pyranoside linkages more than furanoside ones or acyclic ones 3°-a2, and the change of specific rotation during acid hydrolysis suggests occurrence of the predominance of a-D- or fl-L-configuration. Moreover, the a-D-configuration was confirmed by both the positive plain curves in the o.r.d, analysis ~and the
Polysaccharide synthesis from mono- and oligo-saccharides by action of P40~o in DMSO : Shigehiro Hirano et al. Table 2
A n a l y s i s of the synthetic p o l y s a c c h a r i d e s [~,]~ Carbohydrate (%)
Reducing sugar value*
Sample
N (%)
P (%)
s2o, w
Method 1
Method 2
Periodate oxidation'l"
water) (degrees)
4.13 --
0.90 n.d. 0.80 1.34 0.69
14.7 13'2 12.6 17.6 13.3
9.3 10.4 9.6 n.d. n.d.
1.62 1.12 0.90 0.89 1.65
+87-5
---
7.0 7.7 6.9 3.6 12.0
40.0
--
--
15.9
n.d.
n.d.
6"5
n.d.
50-0 30'0 . . . . ----
---
---
-9.4 15.6
2.04 -3.68
11.0 20'9 4.7 6.4 1.3 3.5 3.5 6-5 2.3
n.d. n.d. 1.11 n.d. 1.27 1.00 0.86 0.83 0.89
n.d. n.d. n.d. n.d. 13'0 20.9 14.6 15.0 n.d.
n.d. n.d. 9.6 n.d. 10.8 n.d. n.d. n.d. n.d.
n.d. n.d. 0'73 n.d. 1.02 n.d. n.d. n.d. 10'0
Hexose
Hexosamine
Pentose
(2) (3) (6) (7)
69-0 72.0 70.7 ---
. . . 52.6 --
. . . -65.0
(8)
--
--
--76"5 66'8 83.9 47.6 49.3 41.5 --
--. . . . 40.6 -66'3
(1)
(9) (10) (11) (12) (13) (14) (15) (16) (17)
. . .
. . . .
Uronic acid
(c 1.0,
. . .
. . . .
+87.5 +88.0 +27.0 +20"0 +15.0 +10.0 +8.0 +24.0 +35.0 +95-0 +17-3 +6"1 +42.3 +12-0
* Method 1, the 3,5-dinitrosalicylic acid m e t h o d ; method 2, the ferricyanide method -j- Periodate c o n s u m e d during lt30h per m o n o s a c c h a r i d e unit (moles)
ments (Found: CHaO, 35.6%). As shown in Figure 1, successive methylations by Hakomori, Purdie and Kuhn's procedures gave a fully methylated product, which showed almost no OH absorption. Further additional methylations did not increase the methoxyl contents. The methoxyl content as observed is ~ 4 % less than that calculated. This may be due to the presence of oxymethylene bridges as originated from DMSO 6, partly oxidized products 4, a, and phosphorus. The recovery of total methylated derivatives was in 54 % yield. As shown in Table 4, molar ratio of tetra-, tri-, di- and mono-O-methyl-o-glucoses was in 4 : 5 : 2.5 : 1. In the tri-O-methyl fraction, the molar ratio of 2,3,4- and 2,3,6-tri-O-methyl-o-glucoses was in ~ 1 : ! . Taking a-o-anomeric configuration into consideration as described above, it is concluded that the synthetic glucan (2) has a-l,4- and a-l,6-glucosidic linkages as main chains with various branchings. Especially it is noteworthy that almost the same amount of tri-O-methyl fraction was tetra-O-methyl-D-glucose originating from the non-
850cm -1 absorption in the i.r. spectra. (7) showed a positive plain curve in the o.r.d, analysis, which supports /3-L-configuration (see Table 3). It is of interest to note that (1) was not hydrolysed with any of a- and fl-amylases, but (2) was hydrolysed with only a-amylase. On the other hand, both the enzymes hydrolysed (13). These observations were on the basis of increase of the reducing sugar values in the reaction mixture. This indicates a structural difference between the synthetic glucans (1) and (2). (1) was prepared in the presence of Dowex 50(H ÷) and (2) in the absence of the reagent. The selective oxidation of primary hydroxyl groups in (I) and (2) with nitrogen dioxide 3a did not produce any of uronic acids, which were examined by the carbazole reaction. The iodine reaction for starch was negative for (l), (2), (3), (4), and (13).
Structure of the synthetic glucan (2) Hakomori's methylation procedure did not give the fully methylated product of (2) even after twice treatTable 3
Infra-red and o.r.d, data of the synthetic polysaccharides Nujol ?max (cm-1) *
Sample
o.r.d. (c 1.0, water, 17°C) ( d e g r e e s )
COO-
-CONH-
P=O
Anomeric
700
600
(1)
--
--
1240w
850w
+38
+72
+158
+303
+418
+638
(2)
--
--
1240w
850w
n.d.
------. . ----1630-1650w 1640-1670 s
-1640-1670 s ----. . -----1640-1670 s
1240 w -1230w 1240w 1260w 1260w
850 w ------
+ 66
+ 154
+ 307
+ 430
+ 655
+18 +10
+33 +18
+58 +29
+75 +33
+84 +48
+ 25
+ 32
+ 52
+ 67
+ 100
1240w -1240w -1240w --
-850 w -850w 850w 850 w
+ 31 n.d. +17 +10 n.d, n.d. + 20 n.d. + 70 +2 +5 +24 n.d,
+ 90 +16 +14 +39
+ 130 +30 +45 +66
+ 210 +62 +65 +130
+ 295 +93 +76 +171
+ 425 +148 +108 +249
(3) (6) (7) (8) (9) (10) (11 ) (12) (13) (14) (15) (16) (17)
500
400
350
300nm
* In addition to these absorptions, strong a b s o r p t i o n of HO at 3300-3400cm -1 appeared in all s a m p l e s s, s t r o n g ; w, w e a k
POLYMER,
1972,
Vol
13, M a y
193
Po/ysaccharide synthesis from mono- and o/igo-saccharides by action of P4010 in DMSO : Shigehiro Hirano et aL Table 4 O-Methyl derivatives of D-glucose as isolated from the acid hydrolysate of the fully methylated product of synthetic glucan (2)
Spot
Paper chromatography (Rtg)* Solvent 1
Solvent 2
Assignment of O-methylD-glucose
(rag)
ratio
Yieldt Molar
1
1"00
2,3,4,6-
550
4" 0
2 3 4 5 6 7 8 9
0" 79 0" 72 0"54 (0"57) 0.65 (0"65) 0'49(0"51) 0.50(0"51) 0'44(0"46) 0.62(0.61) 0"37 0.44 0.32 0.31 0.41
2,3,42,3,62,3-~ 2,6-::I: 2,4-:1: Not assigned 3Not assigned
647
5" 0
301
2'5
133
1.0
* Rtg, mobility rate on the paper chromatograms with reference to £,3,4-tetra-O-methyI-D-glucose. Solvent 1, 1-butanol/ethanol/l% ammonia (4:1 : 5, v/v). Solvent 2, 1-butanol/ethanol/water (4:1 : 5,
v/v) t The synthetic glucan (2) (5.0g) was used for the methylation. The recovery of the methylated fractions was in 54~ yield ~LTentative assignment on the basis of paper chromatography
reducing end group. The presence of a large amount of the tetra-O-methyl fraction may be because of low molecular weight of the synthetic glucan. This is also in agreement with the high reducing value.
REFERENCES 1 Goldstein, I. J. and Hullar, T. L. Adv. Carbohydr. Chem. 1966, 21,431 2 Fischer, E. and Delbriick, K. Ber. Dtsch. Chem. Ges. 1909, 42, 2776 3 Micheel, F., BtJckmann, A. and Meckstroth, W. Makromol. Chem. 1961, 48, 1 40nodera, K., Hirano, S. and Kashimura, N. J. Am. Chem. Soc. 1965, 87, 4651 50nodera, K., Hirano, S. and Fukumi, H. Agric. BioL Chem. 1964, 28, 173
194
POLYMER, 1972, Vol 13, May
60nodera, K., Hirano, S., Kashimura, N. and Yajima, T. Tetrahedron Lett. 1965, p 4327 70nodera, K., Hirano, S., Kashimura, N., Masuda, F., Yajima, T. and Miyazaki, N. J. Org. Chem. 1966, 31, 1291 80nodera, K., Hirano, S. and Kashimura, N. Carbohydr. Res. 1968, 6, 276 9 Husemann, E. and Miiller, G. J. Makromol. Chem. 1966, 91,212 10 Mizuno, T. Nippon Nogeikagaku KaisM 1967, 41,189 11 Mizuno, T. Nippon Nogeikagaku Kaishi 1967, 41, 195 12 Scott, T. A., Jr. and Melvin, E. H. Anal. Chem. 1953, 25, 1656 13 Elson, L. A. and Morgan, W. T. J. Biochem. J. 1933, 27, 1824 14 Dische, Z, J. Biol. Chem. 1947, 167, 189 15 Park, J. T. and Johnson, M. J. J. Biol. Chem. 1949, 181, 149 16 Bruner, R. L. Methods in Carbohydr. Chem. 1964, 4, 67 17 Allen, R. J. L. Biochem. J. 1940, 34, 858 18 Guthrie, R. D. Methods Carbohydr. Chem. 1962, 1,435 19 Whelan, W. J. Methods Carbohydr. Chem. 1964, 4, 252, 261 20 Hakomori, S. J. Biochem., Tokyo 1964, 55. 205 21 Purdie, T. and lrvine, J. C. J. Chem. Soc. 1903, 83, 1021 22 Kuhn, R., Trischmann, H. and L~Sw,1. Angew. Chem. 1955, 67, 32 23 Onuki, M. Nippon Nogeikagaku Kaishi 1933, 9, 90 24 Geerdes, J. D., Lewis, B. A. and Smith, F. J. Am. Chem. Soc. 1957, 79, 4209 25 Irvine, J. C. and Hirst, E. I. J. Chem. Soc. 1922, 121, 1213 26 lrvine, J. C. and Hogg, T. P. J. Chem. Soc. 1914, 105, 1386 27 Durand, H. W., Dull, M. F. and Tipson, R. S. J. Am. Chem. Soc. 1958, 80, 3691 28 Hirst, E. L., Hough, L. and Jones, J. K. N. J. Chem. Soc., 1949, p 928 29 Kantor, T, G. and Schubert, M. J. Am. Chem. Soc. 1957, 79, 152 30 Micheel, F. and Gresser, W. Bet. Dtsch. Chem. Ges. 1958, 91, 1214 31 Kent, P. W. Biochem. J. 1953, 55, 361 32 Mora, P. T., Wood, J. W., Maury, P. and Young, B. G. J. Am. Chem. Soc. 1958, 80, 693 33 Schachman, H. K. Methods Enzymol. 1957, 4, 32 34 Gibbons, R. A. in 'Glycoproteins', (Ed. A. Gottschalk), Elsevier Publishing Co., Amsterdam, 1966, 1st edn, p 61 N o t e added in p r o o f - - A further p r o o f o f p o l y m e r was o b t a i n e d by the molecular weight analysis according to the A r c h i b a l d m e t h o d aa ( t i m e = 38 min, T = 2 9 9 ° C , oJ2= 1.3633 x 10B). M o l e c u l a r weights o f (3), (6) and (l l) are calculated as 7600, 16000 and 9500, respectively, by assuming the partial specific volumes '~4of(3) and (I I ) to be 0.613 and that 34 o f (6) to be 0.666.
INTRODUCTION In previous years, a number of attempts have been made to synthesize naturally occurring polysaccharides and the related polymers of increasing interest from biochemical and industrial points of view 1. Fischer and Delbriick 2 first reported a procedure for the synthesis of an oligosaccharide from substituted monosaccharides by. the action of P4010. Since then, the simple principle in that a water molecule is eliminated with P4010 has been applied to the synthesis of various glycosides and polysaccharides from mono- and oligo-saccharides without significant success. In 1961, Micheel et al. 3 prepared a series of branched polysaccharides from mono- and oligo-saccharides by the action of P4010 in the presence of HCI or HBr as catalyst in dimethyl sulphoxide (DMSO). In the course of our studies on catalytic action of phosphorus compounds a-v, we found, in addition to the oxidation 4, s, the formation of polysaccharides (O-glycosides) and glycosylamines (Nglycosides) by the action of P4010 without any addition of catalyst in DMSO at below 35°C. More recently, Husemann and MiJller 9 prepared a cellulose-like polysaccharide * Presented at the annual meeting of the Agricultural Chemical Society of Japan, Tokyo, 1-4 April 1965, and a preliminary note of the present work appeared in a footnote of ref. 4 t To whom inquiries should be addressed. Present address: Department of Agricultural Chemistry, Tottori University, Tottori, Japan
190
POLYMER, 1972, Vol 13, May
from 2,3,6-tri-O-(N-phenylcarbamyl)-o-glucopyranose by the action of P4010 in DMSO-CHCIa. Independently Mizuno 10 confirmed P4010 as a good dehydrating agent for polysaccharide synthesis in DMSO and prepared a series of polysaccharides from monosaccharides. Partial structural analysis of the synthetic galactan and xylan indicated considerable branchings 11. The present work reports that P4010-DMSO catalyses polycondensation and oxidation depending on the reaction temperature. The synthesis of a series of new synthetic polysaccharides is described. These are prepared from mono- and oligo-saccharides including 2-acetamido2-deoxy-D-glucose and hexuronic acids by the action of P4010-DMSO at 10~-35°C. Furthermore, a structural analysis by the methylation of the synthetic glucan (2) is for the first time described. EXPERIMENTAL Analytical methods Hexoses and pentoses were analysed by the anthrone method lz, hexosamine by the Elson-Morgan method 13, hexuronic acids by the carbazole method 14, phosphorus by the Allen method 15, and reducing sugar values by both the ferricyanide16 and 3,5-dinitrosalicylic acid 17 methods. Sedimentation patterns were obtained with a synthetic b o u n d a r y cell at 59 780 rev/min with a Spinco Model E
Polysaccharide synthesis from mono- and oligo-saccharides by action of P401o in DMSO : Shigehiro Hirano et al. ultracentrifuge in ~ 1 ~ solution of the sample as dissolved in 0.15M KC1. Sedimentation constants are expressed as s20,w. Tiselius electrophoresis was carried out in two different buffer solutions at 4.0-4.5°C: (a) sodium borate (0.1M), pH 8.98, /z 0.15, 9.8mA; (b) acetic acid-sodium acetate (0.1 M), pH 5.0, t~ 0.10, 10.0 mA. Periodate oxidation was carried out at 4.0°C in the dark 18, and the periodate consumed is calculated in terms of the corresponding monosaccharide unit which is free of phosphorus. Infra-red (i.r.) spectra were recorded with a Shimadzu A R-6 spectrometer in Nujol or KBr pellet, and optical rotatory dispersion (o.r.d.) with a JASCO automatic recording spectropolarimeterwith 1~solution of the sample at 17 °C in water. Paper chromatography was carried out on Toyo Roshi No. 51 filter paper by descending method with a solvent: 1-butanol/ethanol/1 ~ ammonia (4 : 1 : 5, v/v), and the alkali silver nitrate reagent was utilized for the detection of reducing sugars. Enzyme digestion was carried out with c~-amylase originated from Asperigillus oryzae (Sigma Chemical Co.) at 35°C at pH 5-5, and with /3-amylase originated from barley malt (Sigma Chemical Co.) at 35°C at pH 5.0 tg. The change of specific rotation was measured during the mild acid hydrolysis of the synthetic polysaccharides: each of the polysaccharides was dissolved in 1.0~ concentration in both 0.01 N and 1.0N HCI and the solution was hydrolysed in a boiling water bath. Specific rotation was measured with a Yanagimoto direct reading polarimeter.
General procedure for polysaccharide synthesis To 50ml of anhydrous DMSO, 10g of P4Olo were added in small portions with stirring to produce a homogeneous solution in an ice bath. To the solution was added 10g of the corresponding carbohydrate or the mixture, and the solution was shaken at 15-35°C for 3-5 days. In some cases, 0"5 g of dried Dowex 50(H +) resin was additionally added as a catalyst tg the reaction mixture at the beginning of the reaction. The reaction mixture was poured into a large volume of acetone, and the mixture was stirred to produce an amorphous product. The supernatant solution was decanted and the residue was dis-
solved in ~ 300 ml of ice water. The solution was dialysed against running water through a cellophane tubing for two days and concentrated in vacuo at below 45 °C to give a syrupy product. Residual DMSO was removed from the syrup by evaporating three times after addition of 50 ml each of 1,4-dioxane. The polysaccharide thus obtained was precipitated by addition of five volumes of acetone. The mixture was kept at room temperature for 24 h. The precipitates produced were collected by centrifugation, washed with ether and dried over calcium chloride in a desiccator (see Table 1).
Structural analysis by the methylation of the synthetic glucan (2) The synthetic glucan (2) (5-0 g) was dissolved in 200 ml of DMSO. The methylation was carried out by Hakomows procedure z°, twice by Purdie's procedure of continuous reftuxing for 5 days 21, and finally by Kuhn's procedure of treating for continuous 6 days 22. Further methylation did not give the increase of methoxyl content. No OH absorption was detected in 3000-3500cm -1 region in the i.r. spectrum (see Figure 1). Yield, 4.2g; [c~]~°+74 ° (c 1"0, methanol). Calcd for (CgH16Os)n: CH30, 45.6~. Found: CH30, 41.2~. The fully methylated product (3g) was refluxed in 200ml of 8 5 ~ HCOOH for 1 h and then refluxed in 200ml of 0.5N H2SO4 for 17h. After neutralizing with solid BaCO3, the filtrate was concentrated in vacuo to a syrup (2.8 g). Paper chromatography indicated nine spots of methylated o-glucoses. At the first step, the hydrolysate was separated into four fractions of mono-, di-, triand tetra-O-methylated D-glucoses by the preparative paper chromatography (Whatman 3MM). At the second step, the mono-O-methyl fraction was separated into three compounds, the di-O-methyl fraction into three compounds and tri-O-methyl fraction into two compounds, respectively by the second preparative paper chromatography (see Table 4).
Spot 1. The fraction was 2,3,4,6-tetra-O-methyl-Dglucose which was identified by mixture melting point and i.r. spectroscopy as 2,3,4,6-tetra-O-methyl-N-phenyl-Dglucopyranosylamine: m.p. 138-139°C; [c~]16+230° (c 0.5, acetone); reported 23 m.p. 137-138°C; [C~]D+228-239"5 ° (acetone). Calcd for C16H2sNOs: CH30, 39.9 ~. Found: CH30, 40.3 ~. Spot 2. The fraction was 2,3,4-tri-O-methyl-D-glucose which was identified by mixture melting point and i.r. spectroscopy as 2,3,4-tri-O-methyl-N-phenyl-D-glucopyranosylamine: m.p. 150 °C; [a]]5_ 103 ° (c 0.7, ethanol); reported 24 m.p. 150 °, HD--103 ° (ethanol). Calcd for ClsH~3NOs: CH30, 31.3~. Found: CHaO, 30.6~.
4000
2000
1500
I OOO
600
Cfl/-I Infra-red spectra at each step of the methylation of synthetic glucan (2). (a) Synthetic glucan (2); (b) after Hakomori's methylation (Found: CH30, 35:6%); (c) after Hakornori and Purdie's methylations (Found: CH30, 38.8%); (d) after Hakomori, Purdie and Kuhn's methylations (Found: CHaO, 41.7%)
Figure !
Spot 3. The fraction was identified by mixture melting point and i.r. spectroscopy as 2,3,6-tri-O-methyl-~-oglucopyranose: m.p. 120-122°C, H~7+83--* +20 ° (c 0.2, water); reported 25 m.p. 121-123°C, [a]D+90--* +70"5 ° (water). Calcd for CgHlsO6: CH30, 41.9~. Found: CH30, 41.3 ~. Spot 8. The fraction was identified by mixture melting point, i.r. and nuclear magnetic resonance (n.m.r.)spectroscopies as 3-O-methyl-~-D-glucopyranose: m.p. 161 °C, [a]])5 +85 ~ +56 ° (c 0.3, water); reported 26 m.p. 160-161°C, POLYMER, 1972, Vol 13, May
191
Polysaccharide synthesis from mono- and oligo-saccharides by action of P401o in DMSO : Shigehiro Hirano et al. Table 1 Polysaccharide synthesis from mono- and oligo-saccharides Synthetic
Reaction condition
polysaccharide
Carbohydrates used for polycondensation
Temperature (°C)
Time(h)
(1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17)
D-glucose* D-glucose D-glucose and c~-D-glucose-l-phosphate (K salt) (1000 : 1, w/w) D-glucose 2-acetamido-2-deoxy-D-glucose* 2-acetamido-2-deoxy-D-glucose L-arabinose D-xylose D-xylose and xylan (4 : 1, w/w) D-xylose and xylan (100 : 1, w/w) lactose* lactose maltose cellobiose 2-acetamido-2-deoxy-D-glucose and D-glucose (1 : 1, w/w) D-glucofuranuronolactone and D-glucose (1 : 1, w/w) 2-acetamido-2-deoxy-D-glucose and D-gluco ~uranuronolactone (1 : 1, w/w)
20-35 35 30-35 10-15 20-35 35 35 35 15 15 20-35 10-15 10-15 10-15 10-15 10-15 10-15
120 120 120 340 72 72 72 72 72 72 120 340 340 340 340 340 340
Yield
(%)
48 48 45 0.5 24 38 12 36 45 39 24 5 16 11 14 15 12
* Dry Dowex 50(H ÷) was used as a catalyst (see text)
[0~]D -]-104 ~ + 55 ° (water). 8(D20)3"61 (s 3H, MeO-3), 5.20 (d 1H, H-l, J1,2 3-0 Hz). Other spots could not be crystallized, but a tentative assignment of the spots was carried out by paper chromatography with the reference of reported Rtg values 27, 28 (Rtg; mobility rate on the paper chromatogram with reference to 2,3,4,6-tetra-O-methyl-D-glucose) (Table 4).
RESULTS AND DISCUSSION P4010-DMSO catalyses not only the oxidation but also the polycondensation of carbohydrates. Maximum yield of the oxidation products was obtained in the reaction at 60--65°C as reported in our previous papers 4,s. On the other hand, maximum yield of the synthetic polysaccharides was obtained in the reaction at 30-35°C (see Table 1). it was found that the reaction temperature influences significantly to the type of predominant reactions. The yield of the synthetic polysaccharides was up to 48 %, which is in agreement with a reported result 11. Catalytic action of Dowex 50(H +) was not significant in the polymerization reaction. This indicates that P4010 may act as dehydrating agent as well as acidic catalyst. The reaction with methyl-a-o-glucopyranoside, a-Dglucopyranose-l-phosphate and methyl-2-acetamido-2deoxy-~-o-galactopyranoside did not produce the corresponding polymer. This substantiates the view that the polysaccharides have mainly glycosidic linkages. It is of interest to examine an action of naturally occurring polysaccharides as a starter or an action of monosaccharide phosphates as an intermediate in the polycondensation reaction. The experiments were carried out in the polycondensation of o-xylose in the presence of natural xylan and in the polycondensation of D-glucose in the presence of c~-o-glucopyranose-l-phosphate (see Table 1). As a result, no significant action of these compounds was found in the yield and the physical properties of the products. The synthetic polysaccharides are soluble in water and DMSO, and the 15 % aqueous solution showed almost no viscosity in comparison with water.
192
POLYMER, 1972, Vol 13, May
The synthetic polysaccharides were hydrolysed with 2N H~SO4 at 100°C for 8-16h. Paper chromatographic examination indicated mainly the corresponding monosaccharide ( ~ 90 %) together with one or two minor spots which are considered to be oxidized products. The detailed structure of the minor spots is unknown. Partial acid hydrolysis (IN H2SO4 at 100°C for 30min) of the synthetic glucan (2) showed more than two spots of oligosaccharides in small quantity in addition to the large amount of D-glucose. One of the spots was identical with maltose on paper chromatogram. In the case of heteropolycondensation, (15) showed an equivalent proportion of the component sugars, but the proportion was changed in (16) and (17), with which low contents of uronic acid was observed. The synthetic polysaccharides contained 1.320.9% phosphorus. The phosphorus could not be removed by repeating the procedure of mild methanolysis2a, and the treatment was attended with partial degradation of some glycosidic linkages of the polysaccharides (see Table 2). The synthetic polysaccharides showed relatively narrow distribution in the molecular weight from behaviour in sedimentation patterns. The s20, w fell in 0.69-1.34S. Tiselius electrophoresis revealed that each of the polysaccharides consists of two or more components. These results indicate no typical difference of molecular weight with regard to the products, but there is some heterogeneous distribution of the phosphorus content. Reducing values of the products is relatively high in comparison with that of naturally occurring polysaccharides and the value indicates the degree of polymerization in 15.3-4.7 monosaccharide units per polysaccharide chain. The high reducing value may be due to the participation of new reducing groups produced in a small quantity during the polymerization by partial oxidation of the polymers 4, s or by decomposition of DMSO 6. The stability of the polymers towards dilute acid solutions was characteristic of pyranoside linkages more than furanoside ones or acyclic ones 3°-a2, and the change of specific rotation during acid hydrolysis suggests occurrence of the predominance of a-D- or fl-L-configuration. Moreover, the a-D-configuration was confirmed by both the positive plain curves in the o.r.d, analysis ~and the
Polysaccharide synthesis from mono- and oligo-saccharides by action of P40~o in DMSO : Shigehiro Hirano et al. Table 2
A n a l y s i s of the synthetic p o l y s a c c h a r i d e s [~,]~ Carbohydrate (%)
Reducing sugar value*
Sample
N (%)
P (%)
s2o, w
Method 1
Method 2
Periodate oxidation'l"
water) (degrees)
4.13 --
0.90 n.d. 0.80 1.34 0.69
14.7 13'2 12.6 17.6 13.3
9.3 10.4 9.6 n.d. n.d.
1.62 1.12 0.90 0.89 1.65
+87-5
---
7.0 7.7 6.9 3.6 12.0
40.0
--
--
15.9
n.d.
n.d.
6"5
n.d.
50-0 30'0 . . . . ----
---
---
-9.4 15.6
2.04 -3.68
11.0 20'9 4.7 6.4 1.3 3.5 3.5 6-5 2.3
n.d. n.d. 1.11 n.d. 1.27 1.00 0.86 0.83 0.89
n.d. n.d. n.d. n.d. 13'0 20.9 14.6 15.0 n.d.
n.d. n.d. 9.6 n.d. 10.8 n.d. n.d. n.d. n.d.
n.d. n.d. 0'73 n.d. 1.02 n.d. n.d. n.d. 10'0
Hexose
Hexosamine
Pentose
(2) (3) (6) (7)
69-0 72.0 70.7 ---
. . . 52.6 --
. . . -65.0
(8)
--
--
--76"5 66'8 83.9 47.6 49.3 41.5 --
--. . . . 40.6 -66'3
(1)
(9) (10) (11) (12) (13) (14) (15) (16) (17)
. . .
. . . .
Uronic acid
(c 1.0,
. . .
. . . .
+87.5 +88.0 +27.0 +20"0 +15.0 +10.0 +8.0 +24.0 +35.0 +95-0 +17-3 +6"1 +42.3 +12-0
* Method 1, the 3,5-dinitrosalicylic acid m e t h o d ; method 2, the ferricyanide method -j- Periodate c o n s u m e d during lt30h per m o n o s a c c h a r i d e unit (moles)
ments (Found: CHaO, 35.6%). As shown in Figure 1, successive methylations by Hakomori, Purdie and Kuhn's procedures gave a fully methylated product, which showed almost no OH absorption. Further additional methylations did not increase the methoxyl contents. The methoxyl content as observed is ~ 4 % less than that calculated. This may be due to the presence of oxymethylene bridges as originated from DMSO 6, partly oxidized products 4, a, and phosphorus. The recovery of total methylated derivatives was in 54 % yield. As shown in Table 4, molar ratio of tetra-, tri-, di- and mono-O-methyl-o-glucoses was in 4 : 5 : 2.5 : 1. In the tri-O-methyl fraction, the molar ratio of 2,3,4- and 2,3,6-tri-O-methyl-o-glucoses was in ~ 1 : ! . Taking a-o-anomeric configuration into consideration as described above, it is concluded that the synthetic glucan (2) has a-l,4- and a-l,6-glucosidic linkages as main chains with various branchings. Especially it is noteworthy that almost the same amount of tri-O-methyl fraction was tetra-O-methyl-D-glucose originating from the non-
850cm -1 absorption in the i.r. spectra. (7) showed a positive plain curve in the o.r.d, analysis, which supports /3-L-configuration (see Table 3). It is of interest to note that (1) was not hydrolysed with any of a- and fl-amylases, but (2) was hydrolysed with only a-amylase. On the other hand, both the enzymes hydrolysed (13). These observations were on the basis of increase of the reducing sugar values in the reaction mixture. This indicates a structural difference between the synthetic glucans (1) and (2). (1) was prepared in the presence of Dowex 50(H ÷) and (2) in the absence of the reagent. The selective oxidation of primary hydroxyl groups in (I) and (2) with nitrogen dioxide 3a did not produce any of uronic acids, which were examined by the carbazole reaction. The iodine reaction for starch was negative for (l), (2), (3), (4), and (13).
Structure of the synthetic glucan (2) Hakomori's methylation procedure did not give the fully methylated product of (2) even after twice treatTable 3
Infra-red and o.r.d, data of the synthetic polysaccharides Nujol ?max (cm-1) *
Sample
o.r.d. (c 1.0, water, 17°C) ( d e g r e e s )
COO-
-CONH-
P=O
Anomeric
700
600
(1)
--
--
1240w
850w
+38
+72
+158
+303
+418
+638
(2)
--
--
1240w
850w
n.d.
------. . ----1630-1650w 1640-1670 s
-1640-1670 s ----. . -----1640-1670 s
1240 w -1230w 1240w 1260w 1260w
850 w ------
+ 66
+ 154
+ 307
+ 430
+ 655
+18 +10
+33 +18
+58 +29
+75 +33
+84 +48
+ 25
+ 32
+ 52
+ 67
+ 100
1240w -1240w -1240w --
-850 w -850w 850w 850 w
+ 31 n.d. +17 +10 n.d, n.d. + 20 n.d. + 70 +2 +5 +24 n.d,
+ 90 +16 +14 +39
+ 130 +30 +45 +66
+ 210 +62 +65 +130
+ 295 +93 +76 +171
+ 425 +148 +108 +249
(3) (6) (7) (8) (9) (10) (11 ) (12) (13) (14) (15) (16) (17)
500
400
350
300nm
* In addition to these absorptions, strong a b s o r p t i o n of HO at 3300-3400cm -1 appeared in all s a m p l e s s, s t r o n g ; w, w e a k
POLYMER,
1972,
Vol
13, M a y
193
Po/ysaccharide synthesis from mono- and o/igo-saccharides by action of P4010 in DMSO : Shigehiro Hirano et aL Table 4 O-Methyl derivatives of D-glucose as isolated from the acid hydrolysate of the fully methylated product of synthetic glucan (2)
Spot
Paper chromatography (Rtg)* Solvent 1
Solvent 2
Assignment of O-methylD-glucose
(rag)
ratio
Yieldt Molar
1
1"00
2,3,4,6-
550
4" 0
2 3 4 5 6 7 8 9
0" 79 0" 72 0"54 (0"57) 0.65 (0"65) 0'49(0"51) 0.50(0"51) 0'44(0"46) 0.62(0.61) 0"37 0.44 0.32 0.31 0.41
2,3,42,3,62,3-~ 2,6-::I: 2,4-:1: Not assigned 3Not assigned
647
5" 0
301
2'5
133
1.0
* Rtg, mobility rate on the paper chromatograms with reference to £,3,4-tetra-O-methyI-D-glucose. Solvent 1, 1-butanol/ethanol/l% ammonia (4:1 : 5, v/v). Solvent 2, 1-butanol/ethanol/water (4:1 : 5,
v/v) t The synthetic glucan (2) (5.0g) was used for the methylation. The recovery of the methylated fractions was in 54~ yield ~LTentative assignment on the basis of paper chromatography
reducing end group. The presence of a large amount of the tetra-O-methyl fraction may be because of low molecular weight of the synthetic glucan. This is also in agreement with the high reducing value.
REFERENCES 1 Goldstein, I. J. and Hullar, T. L. Adv. Carbohydr. Chem. 1966, 21,431 2 Fischer, E. and Delbriick, K. Ber. Dtsch. Chem. Ges. 1909, 42, 2776 3 Micheel, F., BtJckmann, A. and Meckstroth, W. Makromol. Chem. 1961, 48, 1 40nodera, K., Hirano, S. and Kashimura, N. J. Am. Chem. Soc. 1965, 87, 4651 50nodera, K., Hirano, S. and Fukumi, H. Agric. BioL Chem. 1964, 28, 173
194
POLYMER, 1972, Vol 13, May
60nodera, K., Hirano, S., Kashimura, N. and Yajima, T. Tetrahedron Lett. 1965, p 4327 70nodera, K., Hirano, S., Kashimura, N., Masuda, F., Yajima, T. and Miyazaki, N. J. Org. Chem. 1966, 31, 1291 80nodera, K., Hirano, S. and Kashimura, N. Carbohydr. Res. 1968, 6, 276 9 Husemann, E. and Miiller, G. J. Makromol. Chem. 1966, 91,212 10 Mizuno, T. Nippon Nogeikagaku KaisM 1967, 41,189 11 Mizuno, T. Nippon Nogeikagaku Kaishi 1967, 41, 195 12 Scott, T. A., Jr. and Melvin, E. H. Anal. Chem. 1953, 25, 1656 13 Elson, L. A. and Morgan, W. T. J. Biochem. J. 1933, 27, 1824 14 Dische, Z, J. Biol. Chem. 1947, 167, 189 15 Park, J. T. and Johnson, M. J. J. Biol. Chem. 1949, 181, 149 16 Bruner, R. L. Methods in Carbohydr. Chem. 1964, 4, 67 17 Allen, R. J. L. Biochem. J. 1940, 34, 858 18 Guthrie, R. D. Methods Carbohydr. Chem. 1962, 1,435 19 Whelan, W. J. Methods Carbohydr. Chem. 1964, 4, 252, 261 20 Hakomori, S. J. Biochem., Tokyo 1964, 55. 205 21 Purdie, T. and lrvine, J. C. J. Chem. Soc. 1903, 83, 1021 22 Kuhn, R., Trischmann, H. and L~Sw,1. Angew. Chem. 1955, 67, 32 23 Onuki, M. Nippon Nogeikagaku Kaishi 1933, 9, 90 24 Geerdes, J. D., Lewis, B. A. and Smith, F. J. Am. Chem. Soc. 1957, 79, 4209 25 Irvine, J. C. and Hirst, E. I. J. Chem. Soc. 1922, 121, 1213 26 lrvine, J. C. and Hogg, T. P. J. Chem. Soc. 1914, 105, 1386 27 Durand, H. W., Dull, M. F. and Tipson, R. S. J. Am. Chem. Soc. 1958, 80, 3691 28 Hirst, E. L., Hough, L. and Jones, J. K. N. J. Chem. Soc., 1949, p 928 29 Kantor, T, G. and Schubert, M. J. Am. Chem. Soc. 1957, 79, 152 30 Micheel, F. and Gresser, W. Bet. Dtsch. Chem. Ges. 1958, 91, 1214 31 Kent, P. W. Biochem. J. 1953, 55, 361 32 Mora, P. T., Wood, J. W., Maury, P. and Young, B. G. J. Am. Chem. Soc. 1958, 80, 693 33 Schachman, H. K. Methods Enzymol. 1957, 4, 32 34 Gibbons, R. A. in 'Glycoproteins', (Ed. A. Gottschalk), Elsevier Publishing Co., Amsterdam, 1966, 1st edn, p 61 N o t e added in p r o o f - - A further p r o o f o f p o l y m e r was o b t a i n e d by the molecular weight analysis according to the A r c h i b a l d m e t h o d aa ( t i m e = 38 min, T = 2 9 9 ° C , oJ2= 1.3633 x 10B). M o l e c u l a r weights o f (3), (6) and (l l) are calculated as 7600, 16000 and 9500, respectively, by assuming the partial specific volumes '~4of(3) and (I I ) to be 0.613 and that 34 o f (6) to be 0.666.