636
V. S. R. RAOAND JOSEPH F. FOSTER
The Conformation of the Pyranose Rings in Mono-, Di-, and Polysaccharides at High pH by Proton Magnetic Resonance Studies
by V. S. R. Rao and Joseph F. Foster Department of Chemistry, Purdue University, Lafayette, IncEiana
(Received October 12, 106.4)
Starch and amylose as well as many model sugars and sugar derivatives show pronounced alterations of their specific optical rotation on passing from neutral to alkaline solution. I n the past, these effects have been attributed to modifications in ring conformation resulting from ionization of hydroxyl groups. By means of n.m.r. it is shown that the Dglucopyranose ring in all compounds examined exists in the C1 conformation in alkaline as well as in neutral aqueous solution. The changes in optical rotation of the free sugars D-glucopyranose and D-maltose are due to a shift of the anomeric equilibrium toward the p-form in alkali. The changes in rotation of methyl-p-maltoside and amylose are probably due to an increase in rotational freedom about the a-l74-glucosidic bond in alkaline solution, although the possibility of some contribution from a helix-coil transition in the case of amylose cannot be ruled out. The n.m.r. spectra of methyl P-L-arabinopyranoside in neutral and alkaline solution are virtually identical. The known change in specific rotation in this case must be due to slight distortions in the ring caused by ionization of the axial hydroxyl group.
Optical rotation studies have played an important role in conformational studies of carbohydrates. Reeves and Blouinl studied a number of methylglycosides and observed that in some cases the specific optical rotation is lower in alkaline than in neutral solution. It was observed further that these “alkalisensitive” glycosides have hydroxyl groups which would be in axial orientation in the assumed most stable C1 conformation. These changes in rotation in alkaline media were explained by assuming that axial ring hydroxyl groups have a tendency to assume an equatorial orientation on ionization. However, it was also noticed that some of the glycosides’ having axial hydroxyl groups in the C1 conformation are alkali stable. In the case of di- and polysaccharides, the situation is more complex. Changes in optical rotation in alkaline media were observed for methyl p-maltoside,2sucrose,1 and to a pronounced extent for amylose.2 The glucose units in these compounds would not possess any axial hydroxyl groups in the C1 conformation. Hence, Reet.es2proposed that some of the glucopyranose rings of both amylose and maltose exist in the B1 conformation (one axial OH) in neutral solution and shift to the The Journal of Physical Chemistry
3B conformation on ionization of hydroxyl groups. On the other hand, Hollo, et al.,a have observed a marked decrease in rotation in alkaline solutions for D-glucose and maltose as well as amylose and suggested that the changes in rotation are caused by a shift from the C1 to 3B conformation of the pyranose rings. The present authors4 earlier presented evidence that the D-glucopyranose units in these sugars exist exclusively in the C1 conformation in neutral solution. The specific explanation presented by Reeves is thus ruled out, but the transformation from C1 to 3B suggested by Hollo, et ab., remains a possibility. We have been most interested in this possibility in view of their suggestion that the C1 conformation would result in a natural tendency for amylose to exist in a helical configuration while the 3B conformation should lead to a flexible coil. Upon raising the pH of amylose solu(1) R. E. Reeves and F. A. Blouin, J . Am. Chem. Soc., 79, 2261 (1957). (2) R. E.Reeves, ibid., 76, 4595 (1954). (3) J. Hollo, J. Szejtli, and M. Toth, Staerke, 13, 222 (1961). (4) V. S. R. Rao and J. F. Foster, J . Phys. Chem., 67, 951 (1963).
CONFORMATION OF PYRANOSE RINGSBY P.M.R.STUDIES
637
Table I : Summary of N.m.r. Data Coupling const., JElEa,
Sample
Solvent
Methyl @-n-ghcopyranoside Methyl p-L-arabinopyranoside D-GlucOSe
DzO
1.5 N KOD Sucrose D-Maltose
2 N KOD Amylose
0.p.s.
...
Methyl a-D-glucopyranoside
2 N KOD
Chemicd shifts. ?-values
-Anomeric
2.6 7.7 7.4 1.7 2.0 3.0 7.0
... 6.7 3.0 3.1 3.0 8.0 2.8 7.2 2.4
Methyl protons
proton-
Other protona
HI,
E.3
..
5.2 5.14
..
.. ..
5.54 5.6
5.12 5.12
..
..
4.7
6.18 6.13 6.13 6.13 5.95 5.95 6.14
6.37 6.36 6.58 6.56 6.12 6.22 6.12 6.20 6.37 6.52
6.55 6.50 6.43 6.36 6.54 6.50
5.3
4.62(small) 5.2
6.12 6.55 5.75 5.9 6.1 6.15 6.25 6.35 6.0 6.15 6.25 6.3 6.4
5.3
6.13 6.32
5.2
6.14 6.38
4.52 4.52 4.75 4.58 4.78 4.7
tions, alterations in various hydrodynamic properties occur which can best be rationalized on the basis of a helix-coil transformation.6 I n the present work, it is shown by means of n.m.r. that the C1 conformation in D-glucose and simple glucosides as well as in maltose and higher saccharides is stable toward alkali and that various structural modifications must be invoked to account for the changes in optical rotation in the various cases.
Experimental The n.m.r. spectra were obtained on a precalibrated chart paper with a Varian A-60 proton magnetic resonance spectrometer. The position of 7 10 on this paper was checked with an external reference (tetramethylsilane in carbon tetrachloride). The impure water peak of DzO in simple DzO-sugar solutions was found to appear at 7 5.2. Optical rotation measurements were made with a Rudolph Model 220-80 spectropolarimeter equipped with a mercury-xenon source and rocking polarizer. The methyl a-D-glucoside and methyl p-D-glucoside were kindly provided by Dr. N. K. Richtmeyer of the National Institutes of Health. The corn amylose was prepared by butanol fractionation of acid-modified corn starch and was kindly supplied by Dr. T. J. Schoch of the Corn Products Refining Co. The pmethyl L-arabinoside (“C” grade) was obtained from the California Corporation for Biochemical Research and was recrystallized once from 95% ethanol. The other chemicals employed were C.P. grade.
6.10 6.30
Results and Discussion The n.m.r. data are summarized in Table I. I n Table I1 are given differences in specific rotation b e tween values measured in neutral and in alkaline solution. Reeves and Blouinl have classified the methylD-glucosides as “alkali stable” and methyl p-L-arabinoside as “alkali sensitive” by optical rotation studies. From Table I it is evident that the coupling constant J H ~ indicates H~ the protons a t C1 and Cz of methyl a-D-glucoside to be in axial-equatorial orientation. Table II: Difference in Specific Rotation [aInor [ a 3 4 3 6 in Neutral Solution and in 1 N NaOH or KOH Compound
Difference
Methyl ar-D-ghcoside Methyl @-D-glucoside Methyl @+arabinoside D-Glucose D-Xylose n-Ribose D-Mannose D-Lyxose Sucrose Maltose Methyl @-maltoside Amylose
0.0 -1.7 10.0 27.Oa 18.35 13.05 -9.85 -8.95 7.8 42.55 15.0 40.0
a
The difference in specific rotation at 436 m p .
( 5 ) V. S. R. Rao and J . F. Foster, Biopolymers, 1, 527 (1963).
Volume 69, Number 8 February 1966
V. S. R. R.AOAND JOSEPH F. FOSTER
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Similarly, the values of the coupling constants, 7.7 and 7.4 c.P.s., indicate that the protons a t C1 and CB of methyl p-D-glucoside are in axial-axial orientation, both in D20 and in strong alkaline media. These results are consistent with the C1 conformation and in agreement with Reeves' interpretation that a- and p-methyl D-glucosides exist in the same conformation in both the soIventsll~6i.e.,C1. Reeves and Blouinl observed a decrease in optical rotation, [ a ] ~of, about 10" for methyl p-L-arabinoside in 1 N XaOH as compared to neutral solution. These results were explained by assuming a shift in the ring conformation froin C1 to B2. It is clear from Table I that the values of the chemical shifts are virtually the same in both the solvents, indicating no significant change in the ring conformation. The slight change in the coupling constant J H , H ~ is not significant enough to suggest any change in ring conformation. If there were any major changes in ring conformation, drastic changes in n.m.r. spectra would be expected. The values of the coupling constants J H I H P , 1.7 and 2.0 c.P.s., indicate that the protons a t C1 and Cz are in equatorial-axial orientation and are consistent with the C1 conformation. These results are in disagreement with Reeves' suggestion. Methyl p-L-arabinoside has the C4 hydroxyl group in axial orientation in the C1 conformation. Since the hydroxyl probably requires greater space on ionization (see below), the C4hydroxyl would come closer to the axial protons on the same side of the ring and thereby might cause a slight distortion in the ring. Such a small distortion might explain the observed changes in optical rotation and might not be detectable in the n.m.r. spectrum. The optical rotations measured a t different pH values for D-glucose are shown in Figure 1. Similar to amylose, D-glucose also shows a decrease in rotation above approximately pH 10.5. The n.m.r. spectra of D-glucose in DzO and in 1.5 N KOD solutions are shown in Figure 2. Significant changes in the spectra are noticeable. I n alkaline solution (curve 11) the peak a t T 4.7, which has been assigned to the anomeric proton of a-D-glucose, is nearly absent. The peak a t T 6.37,' which has been attributed to the ring protons of a-glucose, is also missing. The coupling constant obtained from the peak splitting of the anomeric proton is virtually the same as that obtained for @-D-glucose (6.7 and 7.0 c.P.s.). I n all respects this spectrum is similar to that of p-D-glucose in D20. This means that a t high alkaline concentrations the equilibrium mixture of a-0-anomers of Dglucose shifts toward the p-anomer. Even in neutral solution the @-anomer of D-glucose predominates a t equilibrium; presumably, it has all hydroxyls in equaThe JOUT?UL~ of Physical Chemistry
- 250
- 235
Figure 1. Dependence of specific optical rotation maltose. at 436 mp on pH: 0 , D-glucose; 0,
'W I l r l l l l l l l , , , l l l i r , , , , , , I , , , ,
5.0
6.0
r
7.0
Values
Figure 2. N.m.r. spectra of D-glucose: I, in DzO; 11, in 1.5 N KOD.
torial positions.8 The charged RO group would be more strongly solvated and have a greater effective volume than the neutral ROH groups. Hence, under ionizing conditions, there would be an enhanced destabilization of the a-form relative to the p-anomer. It appears certain that the observed decrease of the rotation of D-glucose with increasing pH above 10.5 is not due to a change in ring conformation but rather (6) R. E. Reeves, J. Am. Chem. SOC.,71, 215 (1949). (7) R. W.Lenz and J. P. Heeschen, J. Polgmer Sci., 51, 247 (1961). (8) R. E. Reeves, J. Am. Chem. Soc., 7 2 , 1499 (1950).
CONFORMATION OF PYRANOSE RINGSBY P.M.R.STUDIES
to a shift in the equilibrium mixture toward the panomer. Changes in optical rotation similar to that of Dglucose are observed for D-xylose and D-ribose. On the contrary, for D-mannose and D-lyxose it is observed that the specific rotation remains constant up to pH 10.5, but increases with further increase in pH. N.m.r. spectra on these latter two sugars, while of relatively poor quality, did show that the a-anomer increases in the equilibrium mixture in 1.5 N KOD solutions. It is known that the acetylated sugars of D-mannose and D-lyxose exist in the C1 conformation in neutral solution.9 I n this conformation for @-D-mannoseand pD-lyxose, the oxygen atoms a t C1, C2, and the ring oxygen come very close, which causes a particular instability. Hence, under ionizing conditions, even though the a-anomers of these compounds have one hydroxyl group more in axial orientation than the corresponding @-anomers, the equilibrium shifts toward the a-form. This further indicates that the rule that the anomer having more hydroxyl groups in the equatorial position will predominate in the equilibrium mixture is not completely general. Sucrose is also found to exhibit a decrease in specific rotation [ a ]of~ about 10" in 1N NaOH. It is known that the glucopyranose unit in sucrose exists in the C1 conformation in the crystalline state.l0 Since it has all the hydroxyl groups in equatorial positions, there is no reason to expect that the observed changes in rotation are due to changes in ring conformation from C1 to some boat form. The n.m.r. data for sucrose are given in Table I. The peak a t 4.5 is attributed to the anomeric proton of the glucopyranose unit. The value of the coupling constant J H ~3.0 H~ c.P.s., , obtained from the splitting of this peak, indicates the protons a t C1 and C2of the glucopyranose unit to be in equatorial-axial orientation, which is consistent with the C1 conformation. Minor changes in the n.m.r. spectrum a t high field have been observed in alkaline media. In the absence of a complete analysis of the spectrum it is difficult to interpret such changes. However, the position and the magnitude of the dihedral splitting of the peak due to the anomeric proton of the glucopyrancse unit are the same in both DzO and in 1.5 N KOD, indicating that no change has taken place in the conformation of this unit. The possibility of some alteration in the conformation of the fructose ring cannot be ruled out. Maltose also exhibits changes in specific rotation similar to D-glucose and amylose with increasing pH. In the spectrum of maltose in alkali (Table I) only two peaks corresponding to anomeric protons are observed, a t 4.8 and 5.2, as compared to three, a t r 4.6,4.7, and
639
5.3, in neutral solution. The peaks a t
r 4.7 and 5.3 have been attributed4 to the anomeric proton of the reducing unit and the peak a t r 4.6 to the anomeric proton of the nonreducing unit. In alkali, the peak at r 5.2 is enhanced in intensity, and the peak at. r 4.7 is missing, indicating that the reducing unit of maltose exists as the @-anomer,similar to the case of D-glucose. This is in agreement with the optical rotation data, since the value of [ ~ t ] 4 ~ , 3for D-maltose a t pH 13.7 is about the same as that of @-maltosein neutral solution. The peak a t r 4.8 undoubtedly is due to the anomeric proton of the nonreducing unit of maltose and is slightly shifted to the high-field side. Such a change is not observed in the position of the anomeric proton of the glucopyranose unit of sucrose. This might be due to the fact that when glucose units are connected through an a-1,4 linkage, space conflict restricts somewhat the rotation about glucosidic bonds connecting the units. Such a steric hindrance to freedom of rotation might change on ionization of hydroxyls, which in turn might affect the shielding of the anomeric proton, causing a change in the chemical shift. The value of the coupling constant J H l ~ obtained 2 from the peak splitting of the anomeric proton of the nonreducing unit is 2.4 C.P.S. This indicates that the protons on C1 and C2 carbons of the nonreducing unit of maltose are in equatorial-axial or equatorial-equatorial orientation. The 3B conformation would demand an axialaxial orientation of these two protons with an expected coupling constant J H ~ofHabout ~ 6-9 C.P.S. Some changes in the nature of the spectra would also be expected in the region r 6-6.5, since the CH2OH group would be in axial orientation in the 3B conformation. The magnitude of the coupling constant, the position of the peak due to the anomeric proton of the nonreducing unit of maltose, and the pattern of the spectra in the region r 6-6.5 all indicate that the conformation of the nonreducing unit of maltose in alkali is the same as that in neutral s o l ~ t i o nnamely ,~ C1. Moreover, if the nonreducing unit of maltose is in the C1 conformation in neutral solution, there is no reason for the ring to change its conformation in alkali, since it does not have any axial hydroxyls to cause further instability on ionization. The present results show that the change in rotation with pH above 10.5 is due primarily to a shift in the a-p equilibrium toward the @-anomer. There may be a small additional contribution from an alteration of the geometry of the a-1,4glucosidic bond, possibly an increase in rotational
(9) R. U. Lemieux, R. K. Kullnig, H. J. Bernstein, and W. G. Schneider, J. Am. Chem. SOC.,79, 1005 (1957). (10) C. A. Beevers and W. Coohran, Nature, 157, 872 (1946).
Volume 69, Number 3 February 1966
V. S. R. RAOAND JOSEPH F. FOSTER
640
freedom. Probably this is the major contributing factor in the case of methyl-p-D-maltoside where the decrease is much smaller (15"). Because of the poor solubility of amylose in DzO, it has not been possible to obtain good n.m.r. spectra in neutral solution. Low molecular weight amylose was dissolved in 2 N KOD solution. The n.m.r. data obtained on such solutions are shown in Table I. The peaks are broad because of the high polymeric nature of the compound. The nature of the spectra and the approximate positions of these peaks are in agreement with those of a-D-glucose. The single peak on the low-field side (T 4.7) also indicates that the glucopyranose units exist in a single conformation. The data are consistent with the X-ray result that all the glucopyranose units in alkali amylose exist in the single conformation, C1.ll This result, together with the known alkali stability of the methyl glucosides, virtually eliminates the possibility that the decrease in specific rotation of amylose in alkali is due to any alteration in ring conformation. In this case, the decrease must result from a change in the polymer configura-
The Journal of Physical Chemistry
tion, for example a helix-coil transition, or possibly from an increase of rotational freedom about the glycosidic linkages. In this connection it is of interest that the obsewed decrease in specific rotation of methyl p-maltoside in alkali (15.0') leads to the prediction of a change of 33" for an infinitely long amylose polymer assuming the effect to be due to a change in rotational freedom and to be the same in all a-1,4-glucosidic bonds. In other words, the observed change of 40" in the case of amylose can be almost completely accounted for on this basis without the need of invoking special effects in the polymer such as a helix-coil transition.
Acknowledgments. The authors wish to express their appreciation to the Corn Industries Research Foundation for financial support of this work, and to Drs. T. J. Schoch and N. K. Richtmeyer for furnishing some of the carbohydrate samples. (11) F, R. Senti and (1948).
L. P. Witnauer, J . Am. Chem. Soc., 70, 1438
