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Rapid separation of acetylated oligosaccharides by reverse-phase high-pressure liquid chromatography
ANALYTICAL
BIOCHEMISTRY
97, 184- 190 (1979)
Rapid Separation of Acetylated Oligosaccharides by Reverse-Phase High-Pressure Liquid Chromatography GERALD B. WELLS ANDROBERT L. LESTER Department of Biochemistry, College of Medicine, University of Kentucky, Lexington, Kentucky 40536 Received January 8, 1979 Peracetylated saccharides were separated by chromatography on a reverse-phase support, eluting with mixtures of acetonitrile-water. Gradient elution for 2.5 h gave significant separations of all linear glucose oligomers containing up to 35 sugar residues. With isocratic elution retention was exponentially related to molecular mass and only slightly affected by linkage or anomeric configuration. The presence of glucosamine in various saccharides markedly reduced their retention.
The study of complex oligosaccharides, free or derived from glycolipids and glycoproteins, is often hampered by the lack of effective separation methods. Gel permeation chromatography with various supports (l-3) or paper chromatography (3,4) has been successfully employed but is often time consuming and/or does not yield effective separations of oligosaccharides containing more than lo-15 residues. The advantage of partition chromatography over gel permeation chromatography is that alterations in solvent composition result in separations over a wide range of partition coefficients. This report shows that reverse-phase chromatography on a highperformance support with a covalently bonded nonpolar phase is effective in the rapid separation of mixtures of acetylated glucose polymers and other saccharides. METHODS
For all the experimental data shown herein, chromatography was carried out with two water-jacketed 0.32 x loo-cm columns connected in series and packed dry with Vydac reverse-phase octadecyl support, 30-44 pm (The Separations Group, 0003-2697/79/l 10184-07$02.00/0 Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
Hesperia, Calif.) and maintained at 65°C. Two batches of Vydac support gave unsatisfactory results. Therefore other supports were tested and results equivalent to those presented were obtained with Bondapak CIs Corasil, 37-50 pm (Waters Associates, Milford, Mass.). A solvent gradient was delivered with two model 6000A pumps controlled by a model 660 programmer (Waters Associates) set at program No. 4. One pump delivered water and the other acetonitrile. The initial solvent composition was water:acetonitrile (9:l) and the final composition was water:acetonitrile (3:7). The concentration of acetonitrile (C) at time f is given C = C, + (C, - C,) (flf,)li3, where C, and C, are the initial and final acetonitrile concentrations, respectively, and tf is the total gradient time. The total gradient time was either 80 min at a flow rate of 2 ml/min (Condition A) or 160 min at a flow rate of 1 ml/min (Condition B). At a flow rate of 2 ml/min, the starting pressure was about 1500 psi and at 1 ml/ min the starting pressure was about 1000 psi; the pressure decreased with increasing acetonitrile concentration. After the gradient was completed, the column was prepared for the next sample by washing with 184
RAPID
SEPARATION
OF ACETYLATED
20 ml acetonitrile followed by 20 ml water: acetonitrile (9: 1) at a flow rate of 2 ml/min. Dry carbohydrate samples (1-25 mg) were acetylated by treatment with 1 ml pyridine:acetic anhydride (1: 1) at 100°C for 90 min. The reagents were removed in a stream of nitrogen and redried several times after addition of toluene. The acetylated products were dissolved in about 0.1-0.2 ml of acetonitrile. A total of 0.04 to 1.0 mg of carbohydrate was injected in 0.01-0.05 ml with a U6K injector (Waters Associates). A moving wire detector was employed (Model LCM2, Pye Unicam) to monitor all nonvolatile carbon compounds. Purified amylose (potato type III, Sigma Chemical Co.) was partially hydrolyzed at 100°C in 0.1 M H,SO, for 15-30 min. The hydrolysate was neutralized with AGl-X2HCO; resin (Bio-Rad Laboratories), centrifuged, and taken to dryness in vucuo several times after addition of ethanol. Deacetylation of acetylated oligosaccharides was carried out in 0.1 N KOH in methanol:toluene (3:l) for 60 min at room temperature. After neutralization with acetic acid, equal volumes of CHC& and water
were added and the product was recovered in the aqueous phase. End groups were reduced by incubating a solution containing 45 mM carbohydrate, 50 mM NaOH, and 0.23 M NaBH, overnight at 30°C. After neutralization with Dowex 50 (H+), the solution was filtered and taken to dryness several times in the presence of methanol to remove the boric acid. RESULTS
Partial hydrolysates of amylose were acetylated and subjected to isocratic elution with acetonitrile-water on a reversephase column. Each 0.74 increment in relative molecular mass (from one triacetylglucose unit) increased the logarithm of the capacity factor (k’) by about 0.52 for 30% acetonitrile-70% Hz0 and by about 0.206 for 50% acetonitrile-50% Hz0 (Table 1). This linear relationship between the molecular mass and the logarithm of the capacity factor, as well as the loss of theoretical plates with increased retention time (Table l), explains why elution with an 1
TABLE
SEPARATION OFACETYLATEDOLICOMERSFROMAN
AMYLOSEHYDROLYSATEWITH
30% CH,CN-70%
Degree of polymerization 1 2 3 4 5 6 7 8 9
10 11
Relative molecular weight 1 1.74 2.48 3.22 3.95 4.69 5.43 6.17 6.91 7.65 8.38
Theoretical plates 1493 1099 745
185
OLIGOSACCHARIDES
H,O
k’
log k’
0.269 0.866 2.91 9.75
-0.571 -0.063 0.464 0.989
ISOCRATIC ELUTION”
50% CH,CN-50%
Theoretical plates
1300 943 461
H,O
k’
log k’
0.231 0.373 0.604 0.970 1.55 2.49 4.00 6.46
-0.636 -0.428 -0.219 -0.013 0.191 0.397 0.602 0.810
n The flow rates were 2 ml/min for 30% acetonitrile and 1 ml/min for 50% acetonitrile. Other conditions as described under Methods. k’ = (total volume - nonsorbed volume) + nonsorbed volume. Nonsorbed volume = 6.7 ml.
186
WELLS AND LESTER
exponential gradient would be required to give efficient separations of mixtures of acetylated oligosaccharides. Because of considerable peak sharpening observed at elevated temperatures, all chromatography was carried out at 65°C. Acetylated derivatives of glucose, lactose, raffinose, and stachyose are readily separated with an increasing ratio of acetonitrile to water and emerge in the order expected for reverse-phase chromatography (Fig. 1). The gradient employed in all of these experiments (lo-70% acetonitrile) separated a wide range of acetylated oligomers from partial hydrolysates of amylose (Figs. 2,3) as well as those in Karo corn syrup (Fig. 4). The initial peak at the solvent front also occurs in the reagent blank. In Fig. 2 a 160-min gradient with a flow rate of 1 ml/min discloses about 37 distinct peaks in the elution profile of a 15min hydrolysate of amylose. A useful profile can also be achieved in half the time (Fig. 3). Differences in the oligosaccharide size distribution can be readily observed and measured in the two amylose hydrolysates (Figs. 2,3) and the corn syrup sample. Above 0.2-0.4 pmol of sample, the resolution begins to deteriorate significantly. However, in order to be able to detect the higher oligomers in corn syrup and partially
0
I
0
I
16
Minutes
FIG. 1. Chromatography of carbohydrate standards. A mixture of acetylated glucose (I), lactose (2), raffinose (3), and stachyose (4) was chromatographed according to Condition A. Each standard was present at 0.2 Feq of hexose for the lCt+f volume injected.
hydrolyzed amylose (Figs. 2-4), the shorter oligomers were necessarily overloaded. Experiments with low molecular weight standards showed no evidence of partial acetylation or of multiple peaks from a single substance (with the exception noted below). Nonetheless, we could not rule out the possibility that some of the peaks in hydrolyzed amylose (Figs. 2,3) or in corn syrup (Fig. 4)
FIG. 2. Chromatography of partially hydrolyzed amylose. Oligosaccharides from a 15-min acid hydrolysate of amylose were acetylated and chromatographed according to Condition B. The degree of polymerization is indicated by numbers over the peaks. About 3.5 Feq of hexose was injected in 60 ~1.
RAPID SEPARATION
OF ACETYLATED
OLIGOSACCHARIDES
187
8
40 MINUTES
80
FIG. 3. Chromatography of partially hydrolyzed amylose. Oligosaccharides from a 30-min acid hydrolysate of amylose were acetylated and chromatographed according to Condition A. The degree of polymerization is indicated by numbers over the peaks. About 3.2 peq of hexose was injected in 30 ~1.
might not be different oligosaccharides. We collected the peaks from partially hydrolyzed amylose (Fig. 2) with putative degrees of polymerization 5, 10, and 15. The solvent
was removed and the materials were deacetylated (Methods). The ratio of hexose equivalents (5) to reducing equivalents (6) was measured calorimetrically. These ratios
0 I-0
, 20
40 MINUTES
4. Chromatography of acetylated oligosaccharides from Karo corn syrup. An acetylated sample of corn syrup was chromatographed according to Condition A. The degree of polymerization is indicated by numbers over the peaks. About 5 geq of hexose was injected in 50 ~1. FIG.
188
WELLS AND LESTER
for the putative (glucose),,:(glucose),,: (glucose), were 3.00:2.14:0.97; these are reasonably close to the expected 3:2:1 ratios, within the errors in the methods. In addition, various glucose oligomers or oligomer mixtures were obtained by chromatography of a corn syrup sample on a 0.45 x loo-cm Bio-Gel P-4 column (minus 400 mesh, Bio-Rad Laboratories) run at 0.1 ml/min at 65°C; the samples were dried, acetylated, and subjected to reverse phase chromatography (Condition B). The results were precisely as expected, that is, the oligomer (or oligomer mixtures) had the expected retention time(s) considering the retention volumes on the Bio-Gel column which we observed to have the usual exponential relationship to molecular weight. Thus, we conclude that the peaks observed in the chromatograms of acetylated amylose hydrolysate and acetylated corn syrup (Figs. 2-4) represent unique oligosaccharides with retention times proportional to the degree of polymerization and denoted by numbers above the peaks (Figs. 2-4). Various standard acetylated saccharides were chromatographed with a gradient elution (Condition A) and the retention data are given in Table 2. The cy-and P-anomers of glucose pentaacetate cannot be distinguished, and therefore one would suppose that it is even less likely that separations would occur of anomeric mixtures that might arise by acetylation of higher oligomers with free reducing ends. With the exception of glucosamine or glucosamine containing saccharides, the di-, tri-, and tetrasaccharides tested had retention times characteristic of each group. Disaccharides with 1-6 linkages were retained slightly longer than the corresponding I-4-linked glycosides, and the o-linked disaccharides had slightly longer retention times than their P-linked counterparts. Borohydride reduction of several disaccharides followed by acetylation would be expected to yield products with a 6.5% increase in molecular
TABLE RETENTION
2
OF VARIOUS ACETYLATED WITH GRADIENT ELUTION”
SACCHARIDES
Acetylated saccharides
k’
Mono- a-D-Glucose P-D-Glucose D-Glucose N-Acetyl-D-glucosamine
0.97 0.97 0.97 0.64
Di-
Maltose, glc(ol-4)glc Maltose, reduced Isomaltose, glc(Lyl-6)glc Isomaltose, reduced Lactose, gal@l-4)glc Lactose, reduced Cellobiose, glc(@l-4)glc Gentiobiose, glc(pl-6)glc Trehalose, glc(cul-la)glc Chitobiose, glcNAc(/S-4)glcNAc
1.64 1.68 1.70 1.72 1.52 1.60 1.54 1.58 1.66 0.77
Tri-
Maltotriose, glc(~l-4)glc(~l-4)glc Raffinose, g~(~i-6)glc(~l-2~)f~ glcNAc(ffl-4)glc(~l-2)myoinositol
2.41 2.47 1.86
Tetra-
Maltotetraose, (glccul-4), Stachyose, (galal-6),glc(crl-2P)fru Lacto-N-tetraose, gal(pl-3)glcNAc@l3)gal(/3 1-4)glc
3.24 3.37
Penta- Maltopentaose, (glc$l-4), Lacto-N-fucopentaose, fuc(~l-2)gal(~l3)~cNAc(~l-3)gal(~l-4)glc
2.09 4.18 3.16
a Except for the pen&acetates of Ly-and P-n-glucose, which were commercial products, all other saccharides were acetylated as described under Methods. Some saccharides were reduced with NaBH, prior to acetylation. Gradient elution was carried out according to Condition A (Methods). The capacity factor, k’, is defined in footnote a to Table 1. The total peak retention volume from the point of injection is given by 6.7 (k’ + 1)ml.
mass, and it can be seen that only a slight increase in retention time was observed for maltose, isomaltose, and lactose after reduction and acetylation. Glucosamine and glucosamine containing oligosaccha~des had much shorter retention times than their 2-O-acetyl counterparts (Table 2). The N-acetyl group would be expected to have a higher polarity and thus decrease the retention on the nonpolar reverse-phase support. Chitobiose has a
RAPID
SEPARATION
OF ACETYLATED
lower retention than glucose and tri-, tetra-, and pentasaccharides containing one glucosamine have retention times roughly equivalent to saccharides with one fewer hexose unit. In the case of lacto-l\r-tetraose, lacto-Nfucopentaose, and several other oligosaccharides from milk, generously supplied by Dr. Victor Ginsburg, NIH, we found a second peak with a longer retention time, equivalent to an additional hexose unit. This second peak had 5-15% of the detector response of the first peak and was also observed in samples reduced with borohydride. This peak increased in intensity with longer acetylation treatment and was not observed when acetylation was carried out at room temperature even for several days. The following experiment strongly suggests that the second peak results from partial di-l\r-acetylation yielding a less polar derivative. Lacto-N-tetraose was acetylated with [3H]acetic anhydride for 3 h at lOO”C, products were resolved by reversephase chromatography (Condition A), and fractions corresponding to peaks 1 and 2 were collected. Peak 2 yielded half as much 3H as peak 1. The material in each peak was subjected to alkali-catalyzed methanolysis (Methods) which would remove the Oacetyl groups and one (7) of the two N-acetyl groups. The resulting water-soluble products were passed through a column of AG- l-X2HCO,- (Bio-Rad Laboratories) to remove [3H]acetate and the radioactivity in the eluate was determined. Peak 1, the presumptive mono-l\r-acetyl derivative, would be expected to retain no radioactivity and 0.03 and 0.44% was found in two separate experiments. For peak 2, the presumptive di-l\r-acetyl derivative, one expects that there are 14 radioactive acetyl groups and that the deacetylation procedure would remove 13 0-acetyl groups and one-half of the two N-acetyl groups, leaving (0.5/14)100% = 3.57% of the radioactivity; we found 3.79 and 4.2% in two separate experiments. We also found that the remaining 3H in peak 2
OLIGOSACCHARIDES
189
material was stable to further methanolysis since a second round of treatment resulted in retention of 94.2% of the radioactivity in the sample that had retained 3.79% of the 3H on the first round of deacylation; thus, the total retention (3.79% x 0.942 = 3.57%) is exactly as predicted. We found that chitobiose and glucosamine do not give such appreciable yields of analogous products; it is known that di-l\r-acetylation procedures that work for some compounds inexplicably do not work for others (7). Since complete acetylation of oligosaccharide mixtures with pyridine:acetic anhydride (1: 1) was found to be difficult in some cases without heating and since partial diN-acetylation could occur with heating to give extra peaks, an alternative procedure stemming from the work of Hoffman et al. (8) was adopted. Acetylation was carried out overnight at room temperature by adding to the dry substance 2 ml formamide, 1 ml acetic anhydride, and 0.8 ml pyridine. If necessary, solubilization was aided by immersion of the tube in an ultrasonic bath. After addition of 5 ml of water, the mixture was extracted twice with 5-ml volumes of chloroform and the combined chloroform extracts were washed three times with 5-ml volumes of water. The chloroform extract was taken to dryness and redissolved in acetonitrile prior to column chromatography. DISCUSSION
Reverse-phase chromatography is an effective and rapid procedure for separating acetylated oligosaccharide mixtures. It can be seen that this technique can readily assess differences in oligosaccharide size distribution in various mixtures (Figs. 2-4). Used in conjunction with other chromatography techniques involving adsorption and gel permeation, it could facilitate hitherto difficult separations. With isocratic elution (Table 1) the decrease in theoretical plates observed with increased molecular weight is related to the
190
WELLS
AND LESTER
lower rate of mass transfer between mobile and stationary phases to be expected with increased molecular size and hence decreased diffusion coefficients (9). A programmed downward flow rate might be helpful for chromatography involving a larger span of molecular sizes. Since the presence of aminosugars in acetylated oligosaccharides shortens the retention time in reverse-phase chromatography, indicating a smaller apparent molecular size, and since the opposite obtains for gel permeation chromatography, these two techniques in combination appear promising for the separation and analysis of mixtures of oligosaccharides, some of which contain amino sugars. Acylation of the oligosaccharides with chromophoric or radioactive acyl groups should make this a very sensitive analytical technique. Since reduction with borohydride yields products with favorable chromatographic properties (Table 2), it is clear that use of [3H]borohydride for reduction could result in an extremely sensitive technique. We have found that a microparticulate reverse-phase packing (Partisi15 ODS, Whatman, Inc.) gives a higher column efficiency and loading factor. Since the moving wire detector we used is not in widespread use, we have explored detection by absorption of ultraviolet light. Monitoring absorbance at 205 nm with a Perkin-Elmer model LC-5.5
spectrophotometer, we can readily detect peaks containing 40 neq of acetylated hexose. The solvent gradient employed is useful for separating a wide molecular weight range of oligosaccharides. For any given pair of closely related oligosaccharides, we have found that isocratic elution or a very shallow gradient gives useful results. ACKNOWLEDGMENTS This work was supported in part by NIH Grant AI 12299, NSF Grant PCM 7609314, and a grant from the Research Corporation. We thank Dr. Victor Ginsburg of the National Institutes of Health for supplying milk oligosaccharides.
REFERENCES 1. John, M., Trenel, G., and Dellweg, H. (1969) J. Chromatogr. 42, 476-484. 2. Scobell, H. D., Brobst, K. M., and Steele, E. M. (1977) Cereal Chem. 54, 905-917. 3. Yamashita, K., Tachibana, Y., and Kobata, A. (1977) Arch. Biochem. Biophys. 182, 546-555. 4. Yamashita, K., Tachibana, Y., and Kobata, A. (1978) J. Eiol. Chem. 253, 3862-3869. 5. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., and Smith, F. (1956)Anal. Chem. 28,350356. 6. Nelson, J. (1944) J. Biol. Chem. 153, 375. 7. Inch, T. D., and Fletcher, H. G. (1%6) J. Org. Chem. 31, 1815-1820. 8. Hoffman, J., Lindberg, B., and Svensson, S. (1972) Acta Chem. Stand. 26,661-666. 9. Karger, B. L. (1971) in Modern Practice of Liquid Chromatography (Kirkland, J. J., ed.), pp. 2032, Wiley-Interscience, New York.
BIOCHEMISTRY
97, 184- 190 (1979)
Rapid Separation of Acetylated Oligosaccharides by Reverse-Phase High-Pressure Liquid Chromatography GERALD B. WELLS ANDROBERT L. LESTER Department of Biochemistry, College of Medicine, University of Kentucky, Lexington, Kentucky 40536 Received January 8, 1979 Peracetylated saccharides were separated by chromatography on a reverse-phase support, eluting with mixtures of acetonitrile-water. Gradient elution for 2.5 h gave significant separations of all linear glucose oligomers containing up to 35 sugar residues. With isocratic elution retention was exponentially related to molecular mass and only slightly affected by linkage or anomeric configuration. The presence of glucosamine in various saccharides markedly reduced their retention.
The study of complex oligosaccharides, free or derived from glycolipids and glycoproteins, is often hampered by the lack of effective separation methods. Gel permeation chromatography with various supports (l-3) or paper chromatography (3,4) has been successfully employed but is often time consuming and/or does not yield effective separations of oligosaccharides containing more than lo-15 residues. The advantage of partition chromatography over gel permeation chromatography is that alterations in solvent composition result in separations over a wide range of partition coefficients. This report shows that reverse-phase chromatography on a highperformance support with a covalently bonded nonpolar phase is effective in the rapid separation of mixtures of acetylated glucose polymers and other saccharides. METHODS
For all the experimental data shown herein, chromatography was carried out with two water-jacketed 0.32 x loo-cm columns connected in series and packed dry with Vydac reverse-phase octadecyl support, 30-44 pm (The Separations Group, 0003-2697/79/l 10184-07$02.00/0 Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
Hesperia, Calif.) and maintained at 65°C. Two batches of Vydac support gave unsatisfactory results. Therefore other supports were tested and results equivalent to those presented were obtained with Bondapak CIs Corasil, 37-50 pm (Waters Associates, Milford, Mass.). A solvent gradient was delivered with two model 6000A pumps controlled by a model 660 programmer (Waters Associates) set at program No. 4. One pump delivered water and the other acetonitrile. The initial solvent composition was water:acetonitrile (9:l) and the final composition was water:acetonitrile (3:7). The concentration of acetonitrile (C) at time f is given C = C, + (C, - C,) (flf,)li3, where C, and C, are the initial and final acetonitrile concentrations, respectively, and tf is the total gradient time. The total gradient time was either 80 min at a flow rate of 2 ml/min (Condition A) or 160 min at a flow rate of 1 ml/min (Condition B). At a flow rate of 2 ml/min, the starting pressure was about 1500 psi and at 1 ml/ min the starting pressure was about 1000 psi; the pressure decreased with increasing acetonitrile concentration. After the gradient was completed, the column was prepared for the next sample by washing with 184
RAPID
SEPARATION
OF ACETYLATED
20 ml acetonitrile followed by 20 ml water: acetonitrile (9: 1) at a flow rate of 2 ml/min. Dry carbohydrate samples (1-25 mg) were acetylated by treatment with 1 ml pyridine:acetic anhydride (1: 1) at 100°C for 90 min. The reagents were removed in a stream of nitrogen and redried several times after addition of toluene. The acetylated products were dissolved in about 0.1-0.2 ml of acetonitrile. A total of 0.04 to 1.0 mg of carbohydrate was injected in 0.01-0.05 ml with a U6K injector (Waters Associates). A moving wire detector was employed (Model LCM2, Pye Unicam) to monitor all nonvolatile carbon compounds. Purified amylose (potato type III, Sigma Chemical Co.) was partially hydrolyzed at 100°C in 0.1 M H,SO, for 15-30 min. The hydrolysate was neutralized with AGl-X2HCO; resin (Bio-Rad Laboratories), centrifuged, and taken to dryness in vucuo several times after addition of ethanol. Deacetylation of acetylated oligosaccharides was carried out in 0.1 N KOH in methanol:toluene (3:l) for 60 min at room temperature. After neutralization with acetic acid, equal volumes of CHC& and water
were added and the product was recovered in the aqueous phase. End groups were reduced by incubating a solution containing 45 mM carbohydrate, 50 mM NaOH, and 0.23 M NaBH, overnight at 30°C. After neutralization with Dowex 50 (H+), the solution was filtered and taken to dryness several times in the presence of methanol to remove the boric acid. RESULTS
Partial hydrolysates of amylose were acetylated and subjected to isocratic elution with acetonitrile-water on a reversephase column. Each 0.74 increment in relative molecular mass (from one triacetylglucose unit) increased the logarithm of the capacity factor (k’) by about 0.52 for 30% acetonitrile-70% Hz0 and by about 0.206 for 50% acetonitrile-50% Hz0 (Table 1). This linear relationship between the molecular mass and the logarithm of the capacity factor, as well as the loss of theoretical plates with increased retention time (Table l), explains why elution with an 1
TABLE
SEPARATION OFACETYLATEDOLICOMERSFROMAN
AMYLOSEHYDROLYSATEWITH
30% CH,CN-70%
Degree of polymerization 1 2 3 4 5 6 7 8 9
10 11
Relative molecular weight 1 1.74 2.48 3.22 3.95 4.69 5.43 6.17 6.91 7.65 8.38
Theoretical plates 1493 1099 745
185
OLIGOSACCHARIDES
H,O
k’
log k’
0.269 0.866 2.91 9.75
-0.571 -0.063 0.464 0.989
ISOCRATIC ELUTION”
50% CH,CN-50%
Theoretical plates
1300 943 461
H,O
k’
log k’
0.231 0.373 0.604 0.970 1.55 2.49 4.00 6.46
-0.636 -0.428 -0.219 -0.013 0.191 0.397 0.602 0.810
n The flow rates were 2 ml/min for 30% acetonitrile and 1 ml/min for 50% acetonitrile. Other conditions as described under Methods. k’ = (total volume - nonsorbed volume) + nonsorbed volume. Nonsorbed volume = 6.7 ml.
186
WELLS AND LESTER
exponential gradient would be required to give efficient separations of mixtures of acetylated oligosaccharides. Because of considerable peak sharpening observed at elevated temperatures, all chromatography was carried out at 65°C. Acetylated derivatives of glucose, lactose, raffinose, and stachyose are readily separated with an increasing ratio of acetonitrile to water and emerge in the order expected for reverse-phase chromatography (Fig. 1). The gradient employed in all of these experiments (lo-70% acetonitrile) separated a wide range of acetylated oligomers from partial hydrolysates of amylose (Figs. 2,3) as well as those in Karo corn syrup (Fig. 4). The initial peak at the solvent front also occurs in the reagent blank. In Fig. 2 a 160-min gradient with a flow rate of 1 ml/min discloses about 37 distinct peaks in the elution profile of a 15min hydrolysate of amylose. A useful profile can also be achieved in half the time (Fig. 3). Differences in the oligosaccharide size distribution can be readily observed and measured in the two amylose hydrolysates (Figs. 2,3) and the corn syrup sample. Above 0.2-0.4 pmol of sample, the resolution begins to deteriorate significantly. However, in order to be able to detect the higher oligomers in corn syrup and partially
0
I
0
I
16
Minutes
FIG. 1. Chromatography of carbohydrate standards. A mixture of acetylated glucose (I), lactose (2), raffinose (3), and stachyose (4) was chromatographed according to Condition A. Each standard was present at 0.2 Feq of hexose for the lCt+f volume injected.
hydrolyzed amylose (Figs. 2-4), the shorter oligomers were necessarily overloaded. Experiments with low molecular weight standards showed no evidence of partial acetylation or of multiple peaks from a single substance (with the exception noted below). Nonetheless, we could not rule out the possibility that some of the peaks in hydrolyzed amylose (Figs. 2,3) or in corn syrup (Fig. 4)
FIG. 2. Chromatography of partially hydrolyzed amylose. Oligosaccharides from a 15-min acid hydrolysate of amylose were acetylated and chromatographed according to Condition B. The degree of polymerization is indicated by numbers over the peaks. About 3.5 Feq of hexose was injected in 60 ~1.
RAPID SEPARATION
OF ACETYLATED
OLIGOSACCHARIDES
187
8
40 MINUTES
80
FIG. 3. Chromatography of partially hydrolyzed amylose. Oligosaccharides from a 30-min acid hydrolysate of amylose were acetylated and chromatographed according to Condition A. The degree of polymerization is indicated by numbers over the peaks. About 3.2 peq of hexose was injected in 30 ~1.
might not be different oligosaccharides. We collected the peaks from partially hydrolyzed amylose (Fig. 2) with putative degrees of polymerization 5, 10, and 15. The solvent
was removed and the materials were deacetylated (Methods). The ratio of hexose equivalents (5) to reducing equivalents (6) was measured calorimetrically. These ratios
0 I-0
, 20
40 MINUTES
4. Chromatography of acetylated oligosaccharides from Karo corn syrup. An acetylated sample of corn syrup was chromatographed according to Condition A. The degree of polymerization is indicated by numbers over the peaks. About 5 geq of hexose was injected in 50 ~1. FIG.
188
WELLS AND LESTER
for the putative (glucose),,:(glucose),,: (glucose), were 3.00:2.14:0.97; these are reasonably close to the expected 3:2:1 ratios, within the errors in the methods. In addition, various glucose oligomers or oligomer mixtures were obtained by chromatography of a corn syrup sample on a 0.45 x loo-cm Bio-Gel P-4 column (minus 400 mesh, Bio-Rad Laboratories) run at 0.1 ml/min at 65°C; the samples were dried, acetylated, and subjected to reverse phase chromatography (Condition B). The results were precisely as expected, that is, the oligomer (or oligomer mixtures) had the expected retention time(s) considering the retention volumes on the Bio-Gel column which we observed to have the usual exponential relationship to molecular weight. Thus, we conclude that the peaks observed in the chromatograms of acetylated amylose hydrolysate and acetylated corn syrup (Figs. 2-4) represent unique oligosaccharides with retention times proportional to the degree of polymerization and denoted by numbers above the peaks (Figs. 2-4). Various standard acetylated saccharides were chromatographed with a gradient elution (Condition A) and the retention data are given in Table 2. The cy-and P-anomers of glucose pentaacetate cannot be distinguished, and therefore one would suppose that it is even less likely that separations would occur of anomeric mixtures that might arise by acetylation of higher oligomers with free reducing ends. With the exception of glucosamine or glucosamine containing saccharides, the di-, tri-, and tetrasaccharides tested had retention times characteristic of each group. Disaccharides with 1-6 linkages were retained slightly longer than the corresponding I-4-linked glycosides, and the o-linked disaccharides had slightly longer retention times than their P-linked counterparts. Borohydride reduction of several disaccharides followed by acetylation would be expected to yield products with a 6.5% increase in molecular
TABLE RETENTION
2
OF VARIOUS ACETYLATED WITH GRADIENT ELUTION”
SACCHARIDES
Acetylated saccharides
k’
Mono- a-D-Glucose P-D-Glucose D-Glucose N-Acetyl-D-glucosamine
0.97 0.97 0.97 0.64
Di-
Maltose, glc(ol-4)glc Maltose, reduced Isomaltose, glc(Lyl-6)glc Isomaltose, reduced Lactose, gal@l-4)glc Lactose, reduced Cellobiose, glc(@l-4)glc Gentiobiose, glc(pl-6)glc Trehalose, glc(cul-la)glc Chitobiose, glcNAc(/S-4)glcNAc
1.64 1.68 1.70 1.72 1.52 1.60 1.54 1.58 1.66 0.77
Tri-
Maltotriose, glc(~l-4)glc(~l-4)glc Raffinose, g~(~i-6)glc(~l-2~)f~ glcNAc(ffl-4)glc(~l-2)myoinositol
2.41 2.47 1.86
Tetra-
Maltotetraose, (glccul-4), Stachyose, (galal-6),glc(crl-2P)fru Lacto-N-tetraose, gal(pl-3)glcNAc@l3)gal(/3 1-4)glc
3.24 3.37
Penta- Maltopentaose, (glc$l-4), Lacto-N-fucopentaose, fuc(~l-2)gal(~l3)~cNAc(~l-3)gal(~l-4)glc
2.09 4.18 3.16
a Except for the pen&acetates of Ly-and P-n-glucose, which were commercial products, all other saccharides were acetylated as described under Methods. Some saccharides were reduced with NaBH, prior to acetylation. Gradient elution was carried out according to Condition A (Methods). The capacity factor, k’, is defined in footnote a to Table 1. The total peak retention volume from the point of injection is given by 6.7 (k’ + 1)ml.
mass, and it can be seen that only a slight increase in retention time was observed for maltose, isomaltose, and lactose after reduction and acetylation. Glucosamine and glucosamine containing oligosaccha~des had much shorter retention times than their 2-O-acetyl counterparts (Table 2). The N-acetyl group would be expected to have a higher polarity and thus decrease the retention on the nonpolar reverse-phase support. Chitobiose has a
RAPID
SEPARATION
OF ACETYLATED
lower retention than glucose and tri-, tetra-, and pentasaccharides containing one glucosamine have retention times roughly equivalent to saccharides with one fewer hexose unit. In the case of lacto-l\r-tetraose, lacto-Nfucopentaose, and several other oligosaccharides from milk, generously supplied by Dr. Victor Ginsburg, NIH, we found a second peak with a longer retention time, equivalent to an additional hexose unit. This second peak had 5-15% of the detector response of the first peak and was also observed in samples reduced with borohydride. This peak increased in intensity with longer acetylation treatment and was not observed when acetylation was carried out at room temperature even for several days. The following experiment strongly suggests that the second peak results from partial di-l\r-acetylation yielding a less polar derivative. Lacto-N-tetraose was acetylated with [3H]acetic anhydride for 3 h at lOO”C, products were resolved by reversephase chromatography (Condition A), and fractions corresponding to peaks 1 and 2 were collected. Peak 2 yielded half as much 3H as peak 1. The material in each peak was subjected to alkali-catalyzed methanolysis (Methods) which would remove the Oacetyl groups and one (7) of the two N-acetyl groups. The resulting water-soluble products were passed through a column of AG- l-X2HCO,- (Bio-Rad Laboratories) to remove [3H]acetate and the radioactivity in the eluate was determined. Peak 1, the presumptive mono-l\r-acetyl derivative, would be expected to retain no radioactivity and 0.03 and 0.44% was found in two separate experiments. For peak 2, the presumptive di-l\r-acetyl derivative, one expects that there are 14 radioactive acetyl groups and that the deacetylation procedure would remove 13 0-acetyl groups and one-half of the two N-acetyl groups, leaving (0.5/14)100% = 3.57% of the radioactivity; we found 3.79 and 4.2% in two separate experiments. We also found that the remaining 3H in peak 2
OLIGOSACCHARIDES
189
material was stable to further methanolysis since a second round of treatment resulted in retention of 94.2% of the radioactivity in the sample that had retained 3.79% of the 3H on the first round of deacylation; thus, the total retention (3.79% x 0.942 = 3.57%) is exactly as predicted. We found that chitobiose and glucosamine do not give such appreciable yields of analogous products; it is known that di-l\r-acetylation procedures that work for some compounds inexplicably do not work for others (7). Since complete acetylation of oligosaccharide mixtures with pyridine:acetic anhydride (1: 1) was found to be difficult in some cases without heating and since partial diN-acetylation could occur with heating to give extra peaks, an alternative procedure stemming from the work of Hoffman et al. (8) was adopted. Acetylation was carried out overnight at room temperature by adding to the dry substance 2 ml formamide, 1 ml acetic anhydride, and 0.8 ml pyridine. If necessary, solubilization was aided by immersion of the tube in an ultrasonic bath. After addition of 5 ml of water, the mixture was extracted twice with 5-ml volumes of chloroform and the combined chloroform extracts were washed three times with 5-ml volumes of water. The chloroform extract was taken to dryness and redissolved in acetonitrile prior to column chromatography. DISCUSSION
Reverse-phase chromatography is an effective and rapid procedure for separating acetylated oligosaccharide mixtures. It can be seen that this technique can readily assess differences in oligosaccharide size distribution in various mixtures (Figs. 2-4). Used in conjunction with other chromatography techniques involving adsorption and gel permeation, it could facilitate hitherto difficult separations. With isocratic elution (Table 1) the decrease in theoretical plates observed with increased molecular weight is related to the
190
WELLS
AND LESTER
lower rate of mass transfer between mobile and stationary phases to be expected with increased molecular size and hence decreased diffusion coefficients (9). A programmed downward flow rate might be helpful for chromatography involving a larger span of molecular sizes. Since the presence of aminosugars in acetylated oligosaccharides shortens the retention time in reverse-phase chromatography, indicating a smaller apparent molecular size, and since the opposite obtains for gel permeation chromatography, these two techniques in combination appear promising for the separation and analysis of mixtures of oligosaccharides, some of which contain amino sugars. Acylation of the oligosaccharides with chromophoric or radioactive acyl groups should make this a very sensitive analytical technique. Since reduction with borohydride yields products with favorable chromatographic properties (Table 2), it is clear that use of [3H]borohydride for reduction could result in an extremely sensitive technique. We have found that a microparticulate reverse-phase packing (Partisi15 ODS, Whatman, Inc.) gives a higher column efficiency and loading factor. Since the moving wire detector we used is not in widespread use, we have explored detection by absorption of ultraviolet light. Monitoring absorbance at 205 nm with a Perkin-Elmer model LC-5.5
spectrophotometer, we can readily detect peaks containing 40 neq of acetylated hexose. The solvent gradient employed is useful for separating a wide molecular weight range of oligosaccharides. For any given pair of closely related oligosaccharides, we have found that isocratic elution or a very shallow gradient gives useful results. ACKNOWLEDGMENTS This work was supported in part by NIH Grant AI 12299, NSF Grant PCM 7609314, and a grant from the Research Corporation. We thank Dr. Victor Ginsburg of the National Institutes of Health for supplying milk oligosaccharides.
REFERENCES 1. John, M., Trenel, G., and Dellweg, H. (1969) J. Chromatogr. 42, 476-484. 2. Scobell, H. D., Brobst, K. M., and Steele, E. M. (1977) Cereal Chem. 54, 905-917. 3. Yamashita, K., Tachibana, Y., and Kobata, A. (1977) Arch. Biochem. Biophys. 182, 546-555. 4. Yamashita, K., Tachibana, Y., and Kobata, A. (1978) J. Eiol. Chem. 253, 3862-3869. 5. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., and Smith, F. (1956)Anal. Chem. 28,350356. 6. Nelson, J. (1944) J. Biol. Chem. 153, 375. 7. Inch, T. D., and Fletcher, H. G. (1%6) J. Org. Chem. 31, 1815-1820. 8. Hoffman, J., Lindberg, B., and Svensson, S. (1972) Acta Chem. Stand. 26,661-666. 9. Karger, B. L. (1971) in Modern Practice of Liquid Chromatography (Kirkland, J. J., ed.), pp. 2032, Wiley-Interscience, New York.