The Circular Dichroism of Carbohydrates

ADVANCES IN CARBOHYDRATE CHEMISTRY AND BIOCHEMISTRY, VOL. 45

THE CIRCULAR DICHROISM OF CARBOHYDRATES BY W. CURTISJOHNSON,JR. Department of Biochemistry and Biophysics, Oregon State University, Coroallis, Oregon 97331 I. Introduction..

..........................................................

11. Measuring the Spectrum .................................................. 111. Unsubstituted Carbohydrates ...................... ....................

1. Monosaccharides .............................. .................... 2. Polysaccharides ... ........................................... 1V. Substituted Carbohydrataes ............................................... 1. Sulfate Derivatives .................................................... 2. h i d e Derivatives.. ................................................... 3. Carboxyl Derivatives.. ................................................. 4. Derivatives Having Mixed Substituents ................................... 5. Non-biological Derivatives .............................................

73 76 78 79 85 92 92 94 102 111 119

I. INTRODUCTION Circular dichroism (c.d.) spectroscopy measures the difference in absorption between left- and right-circularly polarized light by an asymmetric molecule. The spectrum results from the interaction between neighboring groups, and is thus extremely sensitive to the conformation of a molecule. Because the method may be applied to molecules in solution, it has become popular for monitoring the structure of biological molecules as a function of solvent conditions. Commercial instrumentation measures the c.d. of electronic absorption bands in the range of 1000 to 190 nm. Nucleic acids and proteins both have absorption bands in this region, and c.d. has been used extensively to study these molecules. Most sugars are transparent in this region, and so they have been relatively neglected. However, the diverse biological activities of sugars undoubtedly depend on their conformations. Thus, improvements in c.d. instrumentation for the short-wavelength region have stimulated interest in using this powerful technique for investigating the stereochemistry of sugar monomers, the configuration of intersaccharide linkages, the secondary structure of the polymers, and the interaction of sugars with themselves 73

Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

W. CURTIS JOHNSON, JR.

74

CHpOH HOG

OH

OH a-D- xy lopyranose

HO@OH

CH20H H O G O H

OH

HO

a-D-glucopyronose CH20H

HO@OH

a-D-idopyranose

YOOH CH20H

HO@OHHO

OH

Hb

Q-D-Or0 binopyronose

a-D-0 Itropyranose

GOH

a-o-goloctopyranose CH20H

HO

H

o HO

a-D-1 yxopyronose

a-D-rnonnopyranose CHpOH

HOG

O HC)

Q -D

H

HOO H O O OH

O

H

OH

"aoH

a-o-gulopyranose CH2OH

H

OH

- r i b o pyranose

O

a-D-01 lopyranose

a-D-talopyranose

FIG. l.--a-D-Mdo-pento- and -hexo-pyranoses.

and with other biological molecules. Although the research appears quite promising, work in this area has only begun, so that many problems remain to be solved. In this chapter, the ways in which modern c.d. instrumentation has been used to solve structural problems involving sugars are detailed. The discussion is limited to substituted and unsubstituted pyranoses, and will not cover complexes that can be formed with various chromophores. The structures of the a-D-aldo-pento and -hexo-pyranose monosaccharides are shown in Fig. 1. In all cases, these sugars will be studied as the six-membered-ring tautomer, as shown. The pyranose rings can adopt either of two different chair conformations' called 4C1and 'C4.Pyranoses usually adopt a chair conformation that puts the majority of bulky groups in the equatorial position, so that steric interactions are minimized. The 4Cl(D) conformation and the ring numbering system are shown in formula 1. (1) Eur. 1. Biochem., 111 (1980) 295-298.

THE CIRCULAR DICHROISM OF CARBOHYDRATES

a

75

l

1

a

The orientations of hydroxyl groups for the D-aldohexopyranoses in the 4 Cl(D) conformation are given in Table I. The C-1-0-5 bond is labile in aqueous solution, so that the pyranose exists as an equilibrium mixture of the (Y anomer [ 1-hydroxyl group axial for ‘ C 1 ( ~ )and ] p anomer [ 1-hydroxyl group equatorial for “C,(D)]with other forms. In general, the dissolved monosaccharide is involved in a complex equilibrium involving chair conformations, ring tautomerization, and anomerization. C.d. studies of such unsubstituted sugars are further complicated, because the absorption maxima for these saturated compounds fall below 190 nm. However, these difficulties can be overcome, as will be seen when the c.d. of unsubstituted sugars is discussed. Many biologically important sugars are derivatives having a chromophoric group that absorbs within the range of commercial instrumentation. Not only is the c.d. spectrum of such molecules easier to measure, but the interpretation of the spectrum is simplified, because only the chromophore is involved. Many laboratories have concentrated on the c.d. of such monomers and their polymers, and the results will be discussed. In some cases, derivatives of natural polysaccharides have been synthesized in order to facilitate their study with commercial c.d. instrumentation. Such non-biological derivatives have provided conformational information about the polysaccharide, and they are discussed in Section IV,5. TABLEI Orientation of Hydroxyl Groups for D-Aldopyranoses in the 4c,(D) Conformation Aldohexose

o-Glucopyranose D-Idopyranose D- Altropyranose D-GakCtOpyranOSe o-Mannopyranose D-GUIOpyraIIOSe D-Allopyranose D-Talopyranose

04

0-3

0-2

16

W. CURTIS JOHNSON, JR. 11. MEASURING THE SPECTRUM

C.d. is a special type of absorption spectroscopy. Thus a c.d. instrument is merely a normal absorption spectrometer with the optics to produce circularly polarized light and the electronics to detect the difference in absorption between left and right polarizations. Fig. 2 gives a block diagram for a typical c.d. instrument. An intense source of light is dispersed by the monochromator. The emerging monochromatic light is first linearly polarized, and the linearly polarized light is then converted into circularly polarized light by a quarter-wave retarder. Modern instruments use an isotropic plate that is stressed electronically to alternately produce light with each type of circular polarization. The light is partially absorbed by the sample, and the transmitted light produces a current in a photomultiplier. The magnitude of this current will have a small alternating-current (AC) component, because the stressed-plate modulator changes the polarization of the light sinusoidally, and the absorption of an asymmetric molecule depends on this polarization. Left- and right-circularly polarized light are produced at the two extremes of the sinusoidal modulation, so that the height of the AC signal as measured by the lock-in detector is a measure of the c.d. This AC signal is very small compared to the average magnitude of the transmitted light, which produces a direct current (DC) in the photomultiplier. To a very good approximation, the difference in absorption (A) between left- (L) and right (R)-circularly polarized light is proportional to the magnitude of the AC signal divided by the magnitude of the DC signal. AL-AR= AA = k(AC/DC)

The absorption of the sample will depend on the pathlength of the cell, 1, and the concentration of the sample, c, just as it does in normal absorption spectroscopy. According to Beer's Law, A(A) = E ( A ) l c ,

where the constant of proportionality, E, called the extinction coefficient, is a function of the wavelength of the incident radiation, A. The standard procedure is to express 1 in cm, and c in mol 1-', so the units of E are 1 cm-' * mol-'. This is the characteristic of the molecule obtained by measuring a normal absorption spectrum. In c.d., the characteristic of the molecule that is seen is the difference in extinction coefficient for the two types of circularly polarized light. Each type of light obeys Beer's Law, so that

-

EJA)-E~(A)=AE(A)=AA(A)/Ic.

Linearly polarized light passing through an asymmetric sample will become

71

THE CIRCULAR DICHROISM OF CARBOHYDRATES

source

+

monochromator

0 polarizer

modulating

retarder

power supply

lock-in

FIG. 2.-Block

J

chart recorder

Diagram of a Circular Dichroism Instrument.

elliptically polarized because of the c.d. Some workers report c.d. as the angle of ellipticity, i,b in degrees. This is related to the difference in absorption by

# = 32.98AA. The characteristic of the molecule is sometimes expressed as the molar ellipticity in deg . d l . mol-' dm-'. This is related to the difference in extinction coefficients by

-

[el = 3298 A&, where the factor of 100 enters for historical reasons. C.d. instruments are usually calibrated for the sign and magnitude of the constant in Eq. (1) by means of an aqueous solution of (+)-locamphorsulfonic acid (CSA). This compound has2 a A& of 2.36 for the c.d. maximum at 290.5 nm. A solution of 1 mg ml-' in a 1-mm pathlength cell has a AA of 1 . 0 2 ~ or a i,b value of 33.5 mdeg. The concentration of this hygroscopic material is readily determined by absorption spectroscopy. Because ~ ( 2 8 5 n m )is 34.5 for the anhydrous material, a solution of 1 mg * ml-' will give an A value of 0.743 in a 5-cm cell. CSA also has a negative c.d. band at 192.5 nm, with a AE of -4.8 for a convenient two-point calibration.

-

(2) G. C. Chen and J. T. Yang, Anal. Leu. 10 (1977) 1195-1205.

78

W. CURTIS JOHNSON, JR.

Because c.d. is measured as the small difference in absorption for the two types of circularly polarized light, these instruments always work at their limits of reliability, and the spectra tend to be “noisy.” The noise is statistical in nature, and proportional to the square root of the amount of light falling on the photomultiplier. As the absorbance of the sample is increased, the c.d. signal is increased proportionally, but the noise simultaneously increases because there is less transmitted light falling on the photomultiplier. An absorbance of 0.87 will give the best signal-to-noise ratio. In practice, 0.4 to 1.0 constitutes a practical range. Too little light falling on the photomultiplier can lead to artifacts. Workers should always measure the total absorbance of cell, solvent, and sample, in order to ensure that it does not exceed 1.0 over the range studied. It goes without saying that solvents should be transparent if the total absorbance is to be kept below 1.0. Furthermore, the wide variety of pathlengths, from 10 cm to 10 pm, that are commercially available to the experimentalist are particularly useful. A solvent that has an absorbance of 1.0 in a 1-mm cell will have an absorbance of only 0.1 in a 100-pm cell. Although the concentration of the sample will have to be increased as the pathlength is decreased, only 100 pL of solution are needed in order to fill the space between the windows in short-pathlength cells. Workers will find 100- and 50-pm cells particularly valuable for work at wavelengths shorter than 200 nm. Relatively long time-constants of 1 to 60 s are used in order to minimize the noise inherent in c.d. spectra. Thus, the scanning rate is low, and as c.d. instruments tend to drift with time, they should be calibrated daily, and baselines should be measured for each sample. 111. UNSUBSTITUTED CARBOHYDRATES Nucleic acids possess chromophoric bases that have absorption bands beginning at 300nm. The amide group of proteins has absorption bands with maxima at 215 and 190nm (and shorter wavelengths). However, unsubstituted sugars are saturated, and their chromophores, the CO and OH groups, have absorption maxima only below 185nm, which is the present limit of commercial c.d. instrumentation. Nevertheless, Listowsky and Englard3 successfully interpreted the long-wavelength tails of the first c.d. bands for a number of monosaccharides. All subsequent c.d. work has used instrumentation capable of making measurements far into the vacuum ultraviolet (u.v.). C.d. spectra of polysaccharides have been measured to 140 nm for films, and to 164 nm for aqueous solutions when extremely thin cells were used. (3) 1. Listowsky and S. Englard, Biochem Biophys. Res. Commun., 3 0 (1968) 329-332.

THE CIRCULAR DlCHROlSM OF CARBOHYDRATES

79

I. Monosaccharides

The pyranoid monosaccharides provide a wide range of asymmetric molecules for study by the c.d. spectroscopist. However, these compounds are not without their difficulties. In aqueous solution, these compounds exist in a complex equilibrium involving the two possible chair conformers of the pyranoses, the furanoses, a and p anomers, and the acyclic form, as well as septanoses for aldohexoses and higher sugars. In order to minimize conformational problems, Nelson and Johnson4 chose D-xylose, D-glucose, and D-galactose for their studies. These sugars preferentially adopt the 4C, conformation of the pyranose form. Furthermore, the kinetics of anomerization are sufficiently slow that it was found possible to measure the c.d. of individual anomers. The results, given in Fig. 3, demonstrated that c.d. spectroscopy is indeed sensitive to differences among the monosaccharides. Fig. 3c shows that the first c.d. band for a-D-galactose is positive, whereas the first c.d. band for P-D-galactose is negative, and considerably more intense. This difference between the cad. spectra of the anomers demonstrates that it is not reasonable to make comparisons between c.d. spectra of pyranoses at anomeric equilibrium. The problems of anomeric equilibrium may be avoided by investigating 2-ketoses. Both a hydroxyl group and a hydroxymethyl group are attached to the anomeric carbon atom in such sugars, and the bulky hydroxymethyl group favors the equatorial position. These authors measured c.d. spectra for three ketoses, the 2-(hydroxymethyl) derivatives of a -L-xylose, a-Dxylose, and a-D-mannose, in aqueous solution. The anomeric hydroxyl group is replaced by a methoxyl group in the methyl glycosides. These compounds have stable ring-structures, so that equilibria among anomers and ring structures is not a problem. There is still an equilibrium between chair conformations, but, if sugars are chosen for which most of the groups are equatorial, one chair conformation will preponderate. Nelson and Johnson’ measured the c.d. spectra of 12 methyl pyranosides in aqueous solutions, and the results for 6 of them are given in Fig. 3. The c.d. spectra of these 21 monosaccharides that were studied contain a wealth of information, although proper analysis of the data is not always obvious. However, c.d.-difference spectra between pairs of sugars that differ at only one carbon atom can be used to simplify the analysis. Each of the chromophores in a monosaccharide (hydroxyl, methoxyl, hydroxymethyl, hemiacetal, and acetal) are symmetric and obtain their c.d. by interaction (4) R. G. Nelson and W. C. Johnson, Jr., J. Am. Chem. SOC.,98 (1976) 4290-4295. (5) R. G. Nelson and W. C. Johnson, Jr., J. Am. Chem. SOC.,98 (1976) 4296-4301.

W. CURTIS JOHNSON, JR.

80

2 I

o Ac -I

-2

0 -I

-2

-3 -4

I

I

I80

X (nml

I

200

I

180

X (nm)

I 200

FIG. 3.-Circular Dichroism Spectra of a (-) and @ ( - - - ) Pyranose Anomers of (a) D-Xylose; (b) D-Glucose; (c) D-Galactose; (d) Methyl D-Xyloside; (e) Methyl D-Glucoside; and ( f ) Methyl D-Galactoside. (Redrawn from Refs. 4 and 5.)

with other groups that are asymmetrically disposed about them. If it is assumed that the principle of pairwise interaction is valid, the c.d. will be given by the sum of pairwise interactions between the groups. C.d.-diff erence spectra then reflect changes in group interactions between the two molecules compared. Fig. 4 shows c.d.-difference spectra that are due to the presence of a hydroxymethyl group in one member of the pair. The pairs of pyranoses have the same configuration about each asymmetric carbon atom that is part of the ring. Three related pairs have the same configuration at the carbon atoms near the hydroxymethyl group and give very similar difference spectra. The similarity indicates that the rotameric distribution for the hydroxymethyl group is similar for each of these pairs. This is to be expected, as all three sugars have 4-hydroxyl groups oriented equatorially, but the relationship is certainly not obvious from the c.d. spectra themselves (see Fig. 3). In contrast, when the configuration near the hydroxymethyl group

THE CIRCULAR DICHROISM OF CARBOHYDRATES I

170

I

I

X

180

I

81

I

190

(nrnl

FIG. 4.-Difference-Circular Dichroism Spectra: a-D-manno- Heptulose minus a - D Tagatose (. . .); a-D-Glucose minusa-D-Xylose (- . - .); P-D-GhJCOSe minusp-D-Xylose (-); a-D-Sorbose minus a-D-Xylose (- - -). (Redrawn from Ref. 4.)

is different (as it is for the fourth pair), the c.d.-difference spectrum is very different. In general, difference spectra are similar when near neighboring groups are similar, but are different when near neighboring groups differ, demonstrating that c.d. is sensitive to configuration and conformation. These similarities reveal that the pyranoses and methyl pyranosides have more in common than is apparent from the c.d. spectra themselves, and where comparisons are possible, that the two types of sugar have the same conformation. Methyl D-pyranoside pairs have difference spectra that are similar to the corresponding pyranose pairs, even though the c.d. spectra are very different. For instance, the two methyl D-glucopyranoside and methyl Dxylopyranoside pairs show difference spectra that are similar to those shown for the pyranoses in Fig. 4, even though no such relationship is obvious from the c.d. spectra themselves. Because so many different chromophores are involved in the c.d. spectrum of a monosaccharide, fundamental interpretation of the spectrum is difficult. On the other hanil, similarities among the various diff erence-spectra having identical conformations in near neighboring groups suggest that a catalog

82

W. CURTIS JOHNSON, JR.

FIG. 5.-Circular Dichroism Fragment-Spectra (from Top to Bottom): Addition of a Hydroxymethyl Group at C-5, with the OH-4 Group Equatorial, Average for Five Pairs of Aldoses; 4-Hydroxyl Group Equatorial to Axial, One Pair of Aldoses; Addition of a Hydroxymethyl Group at C-5, with the 4-Hydroxyl Group Axial for a-D-Aldoses, Average for Two Pairs of Aldoses; and Addition of a Hydroxymethyl Group at C-5, with the 4-Hydroxyl Group Axial for P-D-AIdG. x,Average for Two Pairs of Aldoses. (Redrawn from Ref. 6.)

of c.d. “fragment” spectra might be compiled.6 A c.d. spectrum for a monosaccharide of given configuration and conformation could be predicted by algebraically summing such c.d. fragment-spectra. The c.d. spectra for the 21 monosaccharides that were studied provide the data for taking the first steps in compiling a catalog of fragment spectra. The c.d. spectra of the D-xylose monosaccharides, given in Figs. 3a and 3d, are difference spectra in the sense that the asymmetry of the sugar is due entirely to the functional group on the anomeric carbon atom, C-1. These four c.d. spectra provide the fundamental c.d. fragment-spectra in the catalog. Other examples of c.d. fragment-spectra are given in Fig. 5 for four functional (6) W. C. Johnson, Jr., Curbohydr. Res., 58 (1977) 9-20.

THE CIRCULAR DICHROISM OF CARBOHYDRATES

83

4

A€

2

0

-1

A€

0

-I

X

180

(nm)

200

X

180

200

(nm)

FIG. 6.-Measured Circular Dichroism Spectrum (-), Calculated Circular Dichroism Spectrum (- -), and Fragment Circular Dichroism Spectra (- - -) for the Corresponding DXylose and the Addition of a Hydroxymethyl Group at C-5: (a) a-D-
groups. The c.d. spectra of monosaccharides that have already been measured can be computed from a limited number of the fragment spectra in the catalog. As demonstrated in Fig. 6, the agreement between calculated and measured spectra is quite good. The c.d. fragment spectra may also be used to predict the c.d. spectrum of a sugar that has not yet been measured. The c.d. spectra of three monosaccharides are predicted in Fig. 7. These spectra were calculated in two different ways, and the agreement between the calculated spectra is fairly good. C.d. spectra measured for (-)-(S)-2-ethyltetrahydropyran' and ( S ) - ( + ) 1,2,2-trimethylpropyl ethyl ether* in solution demonstrate that the ether chromophore makes a substantial contribution, if not the total contribution, to the long-wavelength band for carbohydrates in aqueous solution. Solvent effects on ( S ) - (+)-1,2,2-trimethylpropyl ethyl ether show that the positions of the long-wavelength bands are sensitive to hydrogen bonding by the solvent,' and thus arise from excitation of the nonbonding pair of electrons. Lowering the temperature for this compound in a hydrocarbon solvent shifts the longest-wavelength band to the blue, testifying to its Rydberg (7) C. Bertucci, R. Lazzaroni, and W. C. Johnson, Jr., Carbohydr. Res., 132 (1984) 152-156. (8) C. Bertucci, R. Lazzaroni, P. Salvadori, and W. C. Johnson, Jr., J. Chem. SOC.,Chem. Commun., (1981) 590-591.

84

W. CURTIS JOHNSON, JR.

FIG. 7.-Predicted (- -) and Fragment (- - -) Circular Dichroism Spectra: P-L-Arabinose Calculated from (a) CY-D-XylOSe, and (b) Methyl P-L-Arabinoside; a-L-Arabinose Calculated from (c) P-D-XylOSe, and (d) Average Calculated for P-L-Arabinose; Methyl a-L-Arabinoside Calculated from (e) Methyl P-D-Xyloside, and (f) Methyl P-L-Arabinoside. (Redrawn from Ref. 6.)

character.'-'' On the other hand, an intrinsic, magnetic-transition dipole is necessary to account for the intensity of the c.d. for oxygen chromophores"p'2 such as hydroxyl groups and ethers, and this is present in a n + u* transition, but not in Rydberg's. The true transition is unE. H. Shaman, 0. Schnepp, P. Salvadori, C. Bertucci, and L. Lardicci, J. Chem. SOC., Chem. Commun., (1979) 1000-1001. M. B. Robin, Higher Excired States of Polyatomic Molecules, Academic Press, New York, 1974, Vol. 1, p. 265. P. A. Snyder and W. C. Johnson, Jr., J. Chem. Phys., 59 (1973) 2618-2628. P.A. Snyder and W. C. Johnson, Jr., J. Am. Chem. SOC.,100 (1978) 2939-2944.

THE CIRCULAR DICHROISM OF CARBOHYDRATES

85

doubtedly a mixture ( n + 3s/ a*) of the two idealized, one-electron assignment~.’~-’~ These assignments are consistent with the c.d. measurements. The c.d. spectra now measured for 21 monosaccharide^^^ together with the difference spectra, provide enough information for specific chromophores to be assigned the c.d. bands observed in the vacuum U.V. First, the configuration of the anomeric carbon atom of the methyl pyranosides is correlated with the sign of the second and third c.d. bands (counting from the longwavelength end of the spectrum). For instance, as may be seen in Fig. 2d, methyl a-D-xylopyranoside has a positive second band and a negative third band, whereas methyl fl -D-xylopyranoside has opposite signs for these two bands. This suggests that the methoxyl group is responsible for the 174-nm band and for at least part of the intensity of the 165-nm band in methyl pyranosides. Many c.d.-diff erence spectra indicate that the hydroxyl chromophore, which hydrogen-bonds well with the aqueous solvent, contributes intensity only below 175nm. The first band in the c.d. spectra of both the pyranoses and the methyl pyranosides is due to the ring-oxygen atom, as Listowsky and Englard3 suggested. This band is not obvious for either a- or fl-D-xylose, but it is significantly red-shifted when a hydroxymethyl group on C-5 or a methoxyl group on C-1 shields the ring-oxygen atom from the hydrogen-bonding ~ o l v e n t . ~The ” sign of the first band is then dependent on the configuration of groups about the ring-oxygen atom, particularly the orientation of the hydroxymethyl group on C-5. This assignment is consistent with assigning the methoxyl group lower energies than the hydroxyl group.

2. Polysaccharides C.d. spectra have now been measured for a number of unsubstituted polysaccharides. The structures for amylose and pustulan are given as formulas 2 and 3, respectively.

:= CHzOH

HO

Ho 2 (13) (14) (15) (16) (17)

Ho’?EL Ho

Hgo3kL Ho

HO

3

J. Texter and E. S. Stevens, J. Chem. Phys., 69 (1978) 1680-1691. J. Texter and E. S. Stevens, J. Chem. Phys., 70 (1979) 1440-1449. J. Texter and E. S. Stevens, J. Org. Chem, 44 (1979) 3222-3225. B. K. Sathyanarayana, E. S. Stevens, and J. Texter, Biopolymers, 24 (1985) 1365-1383. G. A. Segal, K. Wolf and J. J. Diamond, J. Am. Chem. Soc., 106 (1984) 3175-3179.

86

W. CURTIS JOHNSON, JR.

.4

.L

A€ o

-. 2

-.4

I

180

I 200

I

X

I

c

10

(nm)

FIG. 8.-Circular Dichroism Spectra of a Solution of 20 mg of Pustulan mL at Day 0 (. . .), Day 7 (- * . -), Day 17 (- - -), Day 32 (- . -), and Day 38 (-). (Redrawn from Ref. 18.)

The configuration of each anomeric hydroxyl group that forms a polymer linkage is fixed, and the type of linkage has a significant effect on the physical properties of the polymer, as will be seen. Extensive studies have been performed on the (1 + 6)-P-~-glucan(pustulan)I6 and the (1 + 4)-cu-~-glucan(amylase).'* These are linear polysaccharides that may exist as helical polymers in aqueous solution, as demonstrated by c.d. spectros~opy.’~*’~ Characteristic of the helical structure of these glucans is a negative band at 182 nm, a crossover at 177 nm, and a more intensely positive band at shorter wavelengths (see Figs. 8 and 9). Stipanovic and Stevens” monitored the generation of the helical structure of pustulan by observing the formation of gels over a 38-day period. The authors attributed the initial observation of the c.d. band at 190nm (see Fig. 8) to formation of the helical structure, and the blue shift of the band with aging, to aggregation of the helices. Both Na+ and Ca2+ accelerate gelation, with Ca2+ proving to be about twice as effective as Na+. Because pustulan is not charged, the acceleration is presumably attributable to a decrease in the activity of the aqueous solvent. Fresh solutions of pustulan, which are presumably in a random-coil conformation, have a positive c.d. at 180 nm (see Fig. 8). The c.d. spectrum

-

-

(18) D. G. Lewis and W. C. Johnson, Jr., Biopolymers, 17 (1978) 1439-1449. (19) A. J. Stipanovic and E. S. Stevens, Inr. J. Biol. Macromol., 2 (1980) 209-212.

87

THE CIRCULAR DICHROISM OF CARBOHYDRATES

1

Ae

0 -1

-0

\

1 ;

‘1

/

/

I 170

............... 180 .....................190””

X (nm)

I 200

/

-2

FIG. 9.-Comparison Cyclomaltohexaose (-),

of the Circular Dichroism Spectra Observed for Amylose (. . .), and Methyl a-o-Glucopyranoside (- - -). (From Ref. 19.)

of the corresponding dimer, gentiobiose, is similar to that of the fresh solution of pustulan, but more intense. This indicates that some kind of chainlength dependence must exist. Lewis and Johnson’8 compared the c.d. spectra of amylose and cyclomaltohexaose, and showed that amylose is helical in aqueous solution. Cyclomaltohexaose is chromophorically equivalent to amylose, and it is known to assume a pseudohelix having zero pitch, and thus, no helical chirality. The conformation of amylose is clearly different from that of cyclomaltohexaose, as their c.d. spectra are very different (see Fig. 9). The difference in conformation was considered to be a matter of helical chirality. To confirm this, these workers measured the c.d. spectrum of an amylose-lbutanol complex presumed to have the V-form of helical conformation with the 1-butanol complexed in the channel of the helix.” The c.d. spectrum of the complex is identical to that of amylose in aqueous solution. In contrast to pustulan and gentiobiose, the amylose oligomers do not have a chainlength dependence.I8 Fig. 10 shows that the changes in c.d. with chainlength are progressive, with the blue shift of the 165-nm band uncovering the 182-nm band as the chainlength is increased. Pfannemuller and Ziegast” have shown that longer oligomers also fit into this series with (20) W. Banks and C. T. Greenwood, Polymer, 12 (1971) 141-144. (21) B. Pfannemuller and G . Ziegast, in D. A. Brant (Ed.), Solution PropertiesofPolysacchurides, Am. Chem. SOC.Symp. Ser., 150 (1981) 529-548.

W. CURTIS JOHNSON, JR.

88

A€

-31

'

I

170

I 180

X

I

I

190

I

I

200

(nm)

FIG.10.-Circular Dichroism Spectra of Amylose (-), Maltohexaose (- . -), Maltotetraose (- - -), Maltotriose (- . -), and Maltose (. . .) in Aqueous Solution at 10". (From Ref. 19.)

no abrupt change, indicating helix formation. This suggests that the conformation of the interior subunits is approximately the same in the oligomers and in amylose. All three c.d. spectra given in Fig. 9 (of amylose, cyclomaltohexaose, and methyl a-D-glucopyranoside) are due to interior Dglucopyranosyl units. However, the three c.d. spectra are very different from each other. This demonstrates once again the sensitivity of c.d. spectroscopy to differences in the conformation of sugars, probably at the glycosidic oxygen atom in this case. Stevens and coworkers measured the c.d. of a large number of Dg l u c a n ~ . ' ~The . ~ ~results, - ~ ~ given in Table 11, allowed them to reach certain generalizations about the spectra. First, gel formers display a negative band in the 180-190-nm region, as was discussed in detail for amylose and pustulan. They attributed this to local inflexibility in the polysaccharide chain. Second, all D-glucans studied exhibit a c.d. band in the 164-172-nm region. This band is positive for a-linkages and negative for p-linkages in the case of (1-+ 3)-and (1 + 4)-glucans, but uncorrelated with anomeric configuration for the (1+ 6)-glucans. Third, all of the D-glucans showed a (22) A. J. Stipanovic, E. S. Stevens, and K. Gekko, Macromolecules, 13 (1980) 1471-1473. (23) A. J. Stipanovic and E. S. Stevens, in Ref. 21, pp. 303-315. (24) L. A. Buffington, E. S. Stevens, E. R. Moms, and D. A. Rees, In?. J. Biol. Macromol., 2 (1980) 199-203. (25) A. J. Stipanovic and E. S. Stevens, Biopolymers, 20 (1981) 1183-1189.

THE CIRCULAR DICHROISM OF CARBOHYDRATES

89

band in the 145-150-nm region in their film spectra. With the exception of cellulose, this short-wavelength band is opposite in sign to that of the 164-177-nm band. Cellulose has only positive c.d. of strong intensity at short wavelengths, which is probably due to its special structural features in the solid state. TABLEI1 Circular Dichroism Bands for Unsubstituted Polysaccharides

D-Clucan

Branch linkages

Common name

(1+3)-a

-

Pseudonigeran

film

(1+3)-P-

-

Curdlan

gel film

(1+4)-a-

-

Amylose

solution

Form

gel film (1+4)-a-

-5% (1+6)-a-

Amylopectin

solution film

(1+4)-a-

-15% (1+6)-a-

Glycogen

solution film film

(1 + 4)-p-

-

Cellulose

(1+6)-a-

-5% ( 1 + 3 ) - a -

Dextran

solution film

Pustulan

solution gel

(1+6)-P-

(1+3)-a( 1 + 4)-a( 1 + 6)-a[(I +4)-a-i2

(om)

173 145 1175 172 145 182 1175 184 <175 184 168 150 182 <175 182 166 1150 182 <175 166 157 150 S 177 167 150 <177 184 <175

A%,, +0.82 -0.21 negative -0.42 positive -0.27 positive -0.27 positive --0.1 f0.36 negative -0.27 positive -0.06 +0.33 negative -0.14 positive +0.85 -3.9 negative +0.79 +0.49 negative positive -0.36 positive -0.08

film

180

164 164

f0.09

187 <180 166

-0.06 positive +1.0

-

Nigeran

film

-

Pullulan

solution film

+0.49

90

W. CURTIS JOHNSON, JR.

Texter and Ste~ens’~-’’ investigated the hydroxyl and ether chromophores theoretically, and concluded that each contributes n + a*/3s intensity in the 145-190-nm region. The precise transition wavelength will depend on the chromophore and its conditions, but the 180-190-nm band found for the gel formers is assigned to the ether oxygen atom of the linkage. Sathyanarayana and coworkers’6 calculated the c.d. of these polysaccharides when they are in the region still allowed by their flexibility, and obtained results in good agreement with experiment. Stevens and coworkers used their c.d. on the various D-glucans to assign, tentatively, the bands to specific chromophores. They found that derivatives of these polysaccharides that have all of their hydroxyl groups acetylated still exhibit the 177-nm band. They assigned this band (which occurs at somewhat shorter wavelengths for the helical polymers) to the ether of the acetal chromophore. This assignment is essentially consistent with the results obtained by Johnson and coworkers on unsubstituted monosaccharides. as films to 140nm. Three related galactomannans have been These polysaccharides have a (1 + 4)-P-~-mannopyranosyIbackbone with (1 + 6)-a-~-galactopyranosylsubstituents (see formula 4). H°CH OH

G? HO HO 4

The c.d. spectra were measured for the guar derivative containing 39% of a-D-galactopyranosyl groups; the tara derivative, 25% of a-D-galactopyranosyl; and the carob derivative, 19% of a-D-galactopyranosyl. The c.d. spectrum of guar as a solid film is given in Fig. 11. A positive c.d. band is observed at 169 nm, and a negative band at 145 nm. The intensity of these bands varies linearly with the content of D-galactopyranose, so that it is possible to extrapolate the measurements in order to find the intensities of these two bands for both D-mannan and D-galactan. At 0% of a-D-galactopyranose, the authors22found molar ellipticities of -8,000 and +80,000. At 100% of a-D-galactopyranose, the molar ellipticities are + 14,000 and -33,000. The longer-wavelength band was investigated in aqueous solution, and found to be similar to that observed for the films, but with substantially

THE CIRCULAR DICHROISM OF CARBOHYDRATES

91

.2

Ac

0

-. 2

150

160

X

170

180

190

(nm)

FIG.1 1.-Circular Dichroism Spectrum for a Solid Film of Guar Galactomannan. (Redrawn from Ref. 24.)

greater amplitude. The shorter-wavelength band in aqueous solution was predicted by measuring the optical rotatory dispersion (0.r.d.) and utilizing a Kronig-Kramers transform. The 0.r.d. could be accounted for by the observed band at 169 nm, and a predicted band of opposite sign at 145 nm, as observed in the film spectra. Agarose is closely approximated as the alternating copolymer of (1+ 3)-/3-~-galactopyranoseand (1+ 4)-3,6-anhydro-a-~-galactopyranose (see formula 5).

HoBow

/o

HO

5

0

The polymer is capable of forming left-handed, double helices, and undergoes a sol-gel transition where a network is formed through cooperative association of the helices. Fig. 12 shows the c.d. spectra of agarose in aqueous solution at various temperatures.26 As the temperature is increased, ( 2 6 ) J. N.Liang, E. S. Stevens, E. R. Morns, and D. A. Rees, Biopolyrners, 18 (1979) 327-333.

W. CURTIS JOHNSON, JR.

92 I

I

I

I

b

180

X

200

(nm)

FIG. 12.-Circular Dichroisrn Spectrum of Agarose Solution (1.5% w/v) at (a) 25", (b) 45", (c) 67", and (d) 78". (Redrawn from Ref. 26.)

the positive c.d. band decreases in intensity and shifts to longer wavelengths. This change in c.d. intensity is cooperative, but involves a hysteresis, as Fig. 13 shows. The authors considered that changes in intensity and position of the 180-nm c.d. band characterize helix formation and "melting" of the agarose, whereas the hysteresis monitors helix-helix aggregation. The vacuum-u.v. c.d. of an agarose film measured to 140nm has the positive band, observed in aqueous solution, at 180 nm and a more intense, negative band at 152 nm.

IV. SUBSTITUTED CARBOHYDRATES 1. Sulfate Derivatives

Many naturally occurring sugars have a sulfate group attached to one of the carbon atoms. Because the sulfate group does not have any transitions

THE CIRCULAR DICHROISM OF CARBOHYDRATES

I

20

I 40

I

60

T ("C)

I

80

93

0

FIG. 13.-The Intensity of the 188-nm Circular Dichroism Band of Agarose as a Function of Temperature. (Redrawn from Ref. 26)

within the range of commercial instrumentation, these sugars present the same problems that are encountered with unsubstituted sugars. The c.d. spectrum of b-carrageenan, a polymer of sulfate derivatives, has been measuredz7in the vacuum-u.v. region. As shown in formula 6, b-carrageenan is approximated as an alternating copolymer of 4-sulfato-/3-~-galactopyranose and 3,6-anhydro-2-sulfato-c~-~-galactopyranose.

6

The c.d. spectrum of a film of this polymer is shown in Fig. 14. It is similar, but opposite in sign, to the c.d. spectrum of agarose, with the shortwavelength band somewhat red-shifted.

(27) J. S. Balcerski, E. S. Pysh, G. C. Chen, and J. T. Yang, J. Am. Chem. Soc., 97 (1975) 6274-6275.

94

W. CURTIS JOHNSON, JR.

X

(nm)

FIG. 14.-Circular Dichroism of an &-CarrageenanFilm and a Sodium Hyaluronate Film (- - -). (Redrawn from Refs. 27 and 29.)

2. Amide Derivatives Amide derivatives have proved especially useful sugars for study by c.d. spectroscopy. The amide substituent is the same as the chromophore found in proteins, so that its optical properties have been extensively studied both experimentally and theoretically. 2-Acetamido sugars are found in many glycoproteins. The structure of 2-acetamido-2-deoxy-a-~-glucopyranose is given as an example in formula 7.

HO

7

The c.d. spectra of three common 2-acetamido derivatives in aqueous ' - ~ ~ measured such ~ o l u t i o n are ~ * given ~ ~ ~ in Fig. 15. Many l a b ~ r a t o r i e s ~have (28) C. A. Bush, in B. Pullman and N. Goldblum (Eds.), Excited States in Organic Chemistry and Biochemistry. Reidel, Dordrecht, Holland, 1977, pp. 209-220. (29) L. A. Buffington, E. S. Pysh, B. Chakrabarti, and E. A. Balazs, J. Am. Chem. Soc., 99 (1977) 1730-1734. (30) E. A. Kabat, K. 0. Lloyd, and S . Beychok, Biochemistry, 8 (1969) 747-756. (31) A. L. Stone, Biopolymers, 10 (1971) 739-751. (32) J.-P. Aubert, B. Bayard, and M.-H. Loucheux-Lefebvre, Carbohydr. Rer, 51 (1976) 263-268. (33) G . Keilich, Carbohydr. Res. 51 (1976) 129-134. (34) P. L. Coduti, E. C. Gordon, and C. A. Bush, Anal. Biochem., 78 (1977) 9-20. (35) H. R. Dickinson, P. L. Coduti, and C. A. Bush, Carbohydr. Res. 56 (1977) 249-257.

THE CIRCULAR DICHROISM OF CARBOHYDRATES

95

A€

L

I

180

I

I

200

X

I

I

220

I

I

240

(nm)

FIG. 15.-Circular Dichroism of 2-Acetamido-2-deoxy-~-glucopyranose (-), 2Acetamido-2-deoxy-~-galactopyranose (- - -), and 2-Acetamido-2-deoxy-~-mannopyranose (. . .) at Anomeric Equilibrium in Aqueous Solution (redrawn from Ref. 28). and 2-Acetamido2-deoxy-~-glucopyranose(- . -) at Anorneric Equilibrium in Aqueous Solution. (Redrawn from Ref. 29.)

spectra, and all agreed on the features of the long-wavelength band. Observed at -210nm for the anomeric mixtures, this band was assigned to the nr* transition of the amide. It is negative for 2-acetamido-2-deoxy-~glucose and 2-acetamido-2-deoxy-~-galactose,but positive for 2-acetamido2-deoxy-~-mannose.The transition is observed at wavelengths shorter than those typical for a-helical polypeptides, and thus is to be highly solvated. The a anomers of 2-acetamido-2-deoxy-~-glucoseand 2-acetamido-2deoxy-D-galactose have34 the same nr* c.d. bands in 1 : 1 methanol-water at 0" as the anomeric mixtures have in aqueous solution. This indicates that the anomeric configuration has little influence on the n r * c.d. band. However, workers do not agree as to the shape of the c.d. spectrum for these sugars at shorter wavelengths, as Fig. 15 demonstrate^.^^.^^ The correct spectrum still remains an open question, but the intense c.d. band expected at 190 nm for the amide r r * c.d. bands are of opposite sign for the two anomers and nearly cancel in the equilibrium mixture. Thus, differences in the anomeric mixtures could explain differences in the c.d. spectra. The amide rr* c.d. band is obvious for the anomeric mixture from 2-acetamido-

W. CURTIS JOHNSON, JR.

96

I

I

I

I

I

I

I

I

5

4 3 2 1

A€ c -1

-2 -3

-1

/

; I

180

I

X

200

I

220

(nm)

FIG. 16.-Circular Dichroism of Methyl 2-Acetamido-2-deoxy-a-~-galactopyranoside Methyl 2-Acetamido-2-deoxy-/3-~-galactopyranoside (- - -), Methyl 2-Acetamido-2deoxy-a -D-glucopyranoside (. . .), and Methyl 2-Acetamido-2-deoxy-~-~-glucopyranoside (- . -) in Aqueous Solution. (Redrawn from Ref. 38.) (-),

2-deoxy-~-galactose(see Fig. 15) and in the c.d. spectra of the methyl glycopyranosides (see Fig. 16). The c.d. expected from the n r * transition of the amide attached to C-2 of a monosaccharide has been calculated36in the one-electron approximation. The calculated results indicated that the sign of this c.d. band is independent of anomeric configuration, but depends on the relative position of the amide on C-2 and the hydroxyl group on C-3. This is in agreement with the experimental results presented in Figs. 15 and 16. Fig. 16 suggests that, although the n r * band is relatively insensitive to anomeric configuration, the intensity of the r r * band does depend on the orientation of this linkage oxygen atom. As will be seen, the linkage in oligomers can be studied by monitoring the r r * c.d. band of a component 2-acetamido sugar. 2-Acetamido-2-deoxy-~-glucose and 2-acetamido-2-deoxy-~-galactose, as well as their methyl glycopyranosides, have been studied in 1,1,1,3,3,3hexafluoro-2-propanol (HFIP). It was e ~ p e c t e d ’that ~ this solvent might so modify the dihedral angle between the 2-amide and the 3-hydroxyl group as to change the c.d. due to the n r * transition. Indeed, changes in intensity (36) D. Y. Yeh and C. A. Bush, J. Phys. Chem., 78 (1974) 1829-1833.

THE CIRCULAR DICHROISM OF CARBOHYDRATES

I

If -4

I

I

Jl

190

200

I

I

I

I

I

I

I

I

220

230

240

210

X

97

(nm)

FIG. 17.-Circular Dichroism of 2-Acetamido-2-deoxy-~-galactopyranose at Anomerk and in 1,1,1,3,3,3-HexafluoroisopropylAlcohol (- - -). (Redrawn Equilibrium in Water (-) from Ref. 35.)

were observed for the methyl pyranosides and even changes in sign for the pyranoses. Fig. 17 gives the results for 2-acetamido-2-deoxy-~-galactose. The 3-0-methyl derivative of methyl 2-acetamid0-2-deoxy-p-~glucopyranoside has the same c.d. spectrum in water and in fluorinated alcohols. This confirms that solvent binding to the 3-hydroxyl group is important in determining the orientation of the amide relative to the 3substituent. Buffington and Stevens37 measured the c.d. of 2-acetamido-2-deoxy-~glucose as a film cast from HFIP. The spectrum is considerably more intense than that observed by Dickinson and coworkers35 for a solution in HFIP, but shows the same general features shifted somewhat towards the red. This vacuum-u.v. c.d. spectrum (see Fig. 18) has, at 218 nm, an intense, positive band due to the nr*,an intense negative band due to the amide rr* at 200 nm, and a shoulder at 180 nm, but no other significant features down to 145nm. The c.d. spectra of three monosaccharides that have two amide substituents have been measured in the vacuum-u.v. The longwavelength portion of the spectra are in agreement with spectra measured on commercial instruments in earlier ~ o r k . ~These ~ , ~bis(acetamid0) ~ * ~ ~ (37) L. A. Buffington and E. S. Stevens, J. Am. Chem. Soc., 101 (1979)5159-5162. (38) A. Duben and C. A. Bush, Anal. Chem., 52 (1980)635-638. (39) C. A. Bush, A. Duben, and S. Ralapati, Biochemistry, 19 (1980)501-504. (40)T. Y. Shen, J. P. Li, C. P. Dorn, D. Ebel, R. Bugianesi, and R. Fecher, Carbohydr. Res., 23 (1972)87-102.

98

W. CURTIS JOHNSON, JR.

I

l

160

l

I

180

l

X

l

200

I

l

220

l

I

240

(nm)

FIG. 18.-The Circular Dichroism of a 2-Acetamido-2-deoxy-~-glucopyranose Film. (Redrawn from Ref. 37.)

sugars exhibit a low-intensity, positive band in the 240-200-nm region that can be attributed to the amide nr*.The PT* c.d. bands at shorter wavelength have about 10 times the intensity observed for the 2-acetamido sugars. As shown in Fig. 19 for 2-acetamido-1-N-( ~-aspart-4-oyl)-2-deoxy-p-~glucosylamine, exciton coupling between the amide chromophores presumably gives intense c.d. bands corresponding to the splitting of the electrically allowed, m r * transitions, in analogy with amide-amide interactions in proteins. Indeed, calculations on such bis(acetamid0) sugars4' showed that an exciton splitting of the two interacting, m r * transitions is expected. N.m.r. spectra of 2-acetamido-l-N-(~-aspart-4-oyl)-2-deoxy-~-~glucosylamine and the model compound 2-acetamido-1-N-acetyl-2-deoxyP-D-glucosylamine indicated that the amide protons are trans to their respective ring protons.39 The c.d. spectra of these two compounds are almost superposable, with an intense, positive band at 197 nm and an intense, negative band near 180 nm. Calculations of the c.d. expected from interaction of the amide mr* transitions, assuming the trans orientation indicated by the n.m.r. work, are consistent with the c.d. measured. Furthermore, these spectroscopic data are consistent with the proposal that the glucosylated L-asparagine residue in glycoproteins is involved in a type I p-turn involving three other adjacent amino acids in the protein. (41) C. A. Bush and A. Duben, J. Am. Chem. SOC.,100 (1978) 4987-4990.

THE CIRCULAR DICHROISM OF CARBOHYDRATES

180 FIG.

amine.

19.-Circular

200

X

220

99

240

(nrn)

Dichroism of 2-Acetamido-1-N-(~-aspart-4-oyl)-2-deoxy-~-~-glucosyl-

2,3-Bis(acetamido)-2,3-dideoxy-~-dideoxy-~-glucose has39 a single, intense, negative c.d. band at 198 nm. The two amide groups should give rise to a couplet of c.d. bands, but the short-wavelength band is not observed, and the reason for its absence is not yet clear. Kabat and studied amido-substituted sugars in their pioneering work on milk oligosaccharides, blood-group substances, and simpler model compounds. Their work was limited (by the commercial instrumentation then available) to measuring the amide nw* transition at wavelengths longer than 210 nm. Nevertheless, they were able to relate the intensity of this band to configurational features. The lowest intensity was found for oligosaccharides in which the 2-acetamido-2-deoxy-~glucopyranosyl residue is P-linked but unsubstituted. Intermediate intensity was found for oligosaccharides that have the 2-acetamido-2-deoxy-~glucopyranosyl residues substituted at 0 - 3or 0 - 4 by a P-D-galactopyranosyl group, or a-L-fucopyranose linked (1 + 2) to P-D-galactopyranose. The

-

(42) K. 0. Lloyd, S. Beychok, and E. A. Kabat, Biochemistry, 6 (1967) 1448-1454. (43) K. 0. Lloyd, S. Beychok, and E. A. Kabat, Biochemistry, 7 (1968) 3762-3765.

100

W. CURTIS JOHNSON, JR.

highest intensity was observed for oligosaccharides in which the 2acetamino-2-deoxy-~-glucopyranosy~ residue is disubstituted. investigated a number of oligosaccharides conAubert and taining 2-acetamido-2-deoxy-~-~-glucopyranosyl and -mannopyranosyl residues. Their c.d. measurements to 180 nm showed a negative c.d. band at -210 nm that is due to the amide nr* transition, and a positive c.d. band at -190nm due to the amide mr* transition. Although the spectra of all eight oligosaccharides studied were similar in shape, their magnitudes varied over a wide range. No correlation was found between the magnitude of the c.d. bands observed and the sequence, but the magnitude appeared to depend in a general way on the type of linkage involved. made extensive measurements on oligomers Bush and The c.d. spectra of the P-D-(1 + of 2-acetamido-2-deoxy-~-glucopyranose. 4)- and P-D-( 1 + 6)-linked dimers were measured in both water and HFIP. Except for the P-D-( 1 + 4)-linked dimer in alcohol, the c.d. resemble the average for the monomers in the same solvent, indicating that there is no interaction between the residues. In contrast, the P-D-( 1 + 4)-linked dimer shows a significant change in c.d. from the average of the monomers, suggesting that there may be an intramolecular hydrogen-bond between the 3-hydroxyl group on the reducing residue and the ring-oxygen atom of the nonreducing group. The c.d. spectra for the dimer, tetramer, and hexamer of 2-acetamido-2deoxy-D-glucopyranose having P-D-( 1+ 4) linkages (the chitin series of oligosaccharides) are shown in Fig. 20 on a per residue basis.34 All three oligomers have a negative nr* c.d. band at 210nm, and a positive m r * band at 193 nm. Although these spectra are similar to those of the monomer, indicating little interaction between the residues, there is a modest increase in intensity with chainlength. The c.d. spectrum of the trimer does not fit nicely into the series. Although the shape is the same, the c.d. is more intense than that observed for the tetramer.28Furthermore, the c.d. spectrum of the polymer (chitin), shown in formula 8, does not fall neatly into the series.

HO

H3CCNH

HO

II 0

H3CCNH

I1

8

0

The c.d. spectra of chitin, both in HFIP solution and as a film cast from HFIP, are shown3’ in Fig. 21. Chitin gels have a c.d. spectrum similar to

THE CIRCULAR DICHROISM OF CARBOHYDRATES I

I

190

200

101

1

I

I

I

210

220

230

2

1

A€

0

-1

-2

(-)

X (nmI

FIG. 20.-Circular Dichroism of Chitobiose (- * - .), Chitotetraose (- - -), and Chitohexaose in Aqueous Solution. (Redrawn from Ref. 34.)

A€

140

160

180

X

200

220

240

(nm)

FIG.21 .-Circular Dichroism of Chitin in 1,1,1,3,3,3-HexafluoroisopropylAlcohol Solution and as a Film Cast from 1,1,1,3,3,3-HexafluoroisopropylAlcohol (. . .). (Redrawn from Ref. 37).

(-),

102

W. CURTIS JOHNSON, JR.

that of the film in the accessible, longer-wavelength region. In contrast to the oligomers in aqueous solution, there appears to be, in the films and gels, a strong amide interaction that gives rise to the intense r r * transition observed at -200nm. The authors considered that the polymer forms “mats,” as observed by X-ray diffraction and infrared spectroscopy. Intermolecular hydrogen-bonds are formed between the amide substituents, and the amide is probably in the trans orientation so that the chains can form a sheet having each amide acting as both a hydrogen-bond donor and acceptor. This structure appears to be disrupted in the HFIP solvent.

3. Carboxyl Derivatives The c.d. spectra of glycuronic acids have been measured in a number of l a b ~ r a t o r i e s . ~These ~ ~ ~ - sugar ~ ’ derivatives, named after the corresponding aldohexose, have the exocyclic hydroxymethyl group on C-5 replaced by the carboxyl chromophore. The structure of P-D-mannopyranuronic acid is given as an example in formula 9, and the structures of the parent compounds in Fig. 1.

OH

HO 9

Listowsky and coworkers4s presented a particularly nice study of the c.d. of glycuronic acids. They measured D-glucuronic acid and D-galacturonic acid, as well as some methyl glycopyranosiduronic acids, in aqueous solution. Morris and coworkers4’ extended the experimental results to include both anomers of methyl pyranosides of all five naturally occurring glycuronic acids having the D-gluco, D-manno, D-galucto, D-gulo, and L-ido configurations. The c.d. spectrum of methyl a-D-mannopyranosiduronic acid as a function of pH is given in Fig. 22. In all cases, it was found that, in acid solution, the glycuronic acids having an equatorial 4-hydroxyl group exhibit a positive n r * transition at 208 nm and an “anomalous” negative n r * c.d. band at -233 nm. Measurements of the c.d. of D-glucuronic acid in 19: 1 1,Cdioxane-water solution, as well as those of the permethylated Dglucopyranosiduronic acid in water and hexane, demonstrated that the long-wavelength, n r * c.d. band gains its intensity at the expense of the (44) E. J. Eyring and J. T. Yang, Biopolymers, 6 (1968) 691-701. (45) 1. Listowsky, S. Englard, and G. Avigad, Biochemisfry, 8 (1969) 1781-1785. (46) B. Chakrabarti and E. A. Balazs, 1.Mol. Biol, 78 (1973) 135-141. (47) E. R. Moms, D. A. Rees, G. R. Sanderson, and D. Thorn, 1.Chem. SOC., Perkin Trans. 2, (1975) 1418-1425.

THE CIRCULAR DICHROISM OF CARBOHYDRATES I

.. .. ... ... ..__.. %

I

200

I

I

I

I

103

1

:

I

I

I

220

X

I

240

I

260

(nrn)

FIG. 22.-Circular Dichroism of Methyl a-D-Mannopyranosiduronic Acid at Various pH Values. (Redrawn from Ref. 47.)

shorter-wavelength, nr* band. However, D-galacturonic acid exhibits a single nr* band that is almost the same in water and in 19: 1 1,Cdioxanewater solution. These experiments indicated that two species are involved. “Anomalous” nT* c.d. bands having a component of opposite sign at long wavelengths are not peculiar to these sugars, but have been observed for many asymmetric acids and esters. Convincing arguments have been presented for a variety of origins of this effect, and various authors have attributed it to solvation species, conformational species, rotational isomers, and vibronic interactions. At present, the origin of the effect must still be considered an open question. The glyguronic acids having an axial 4-hydroxyl group show only a single, positive nr* c.d. band at -210nm, and that has a greater intensity than the band observed for glycuronic acids having an equatorial 4-hydroxyl group. However, the idoses, which have an axial 4-hydroxyl group when in the 4C,conformation, display a single, positive nr* band at 210 nm, as expected, and probably have a substantial contribution from the ‘C4confor-

W. CURTIS JOHNSON. JR.

104

mer in which the 4-hydroxyl group is equatorial. Of course, the 5-carboxyl group would be axial, not equatorial, in this conformation. The carboxyl chromophore is sensitive to pH, as Fig. 22 shows for the example of methyl a-D-mannopyranosiduronic acid?’ The particular sensitivity of this chromophore to its environment should prove useful in the investigation of uronic polymers. As the acid form is converted into the carboxylate anion, the c.d. spectrum changes significantly. Monitoring of these changes, at a single wavelength, as a function of the pH of the solution yielded a titration curve with pK values of the order of 3.3, in agreement with results derived by other methods. The isodichroic point evident in Fig. 22 demonstrates that two species are involved. Film spectra of D-glucuronic acid and sodium D-glucuronate (see Fig. 23), measured into the vacuum-u.v. region, added to our knowledge of the transitions involved.29The c.d. spectrum of D-glucuronic acid shows both nr* transitions at long wavelength and a c.d. band at 180 nm that is assigned to the carboxyl rr*.In addition to the rr* at 180 nm, sodium D-glucuronate shows two bands at longer wavelengths. Apparently, the carboxylate chromophore also has two c.d. bands of opposite sign that are associated with the n r * transition. The c.d. spectra of these two compounds in aqueous solution have been extended29to 185 nm. The c.d. spectrum of D-glucuronic acid appears to be the same as that measured for the film, but, whereas 1

I

I

I

I

I

I

I

I

I

0

0

AA

0 b I

180

I

200

X

220

240

(nrn)

FIG.23.-Circular Dichroism of (a) D-Glucuronic Acid and (b) Sodium D-Glucuronate as Films. (Redrawn from Ref. 29.)

THE CIRCULAR DICHROISM OF CARBOHYDRATES

105

both c.d. bands for the nn* transition are apparent for sodium D-glucuronate, the TT* c.d. band is now negative. Alginate is a copolymer of a-L-gulopyranuronic and fl -Dmannopyranuronic acid, the sugars being linked (1 + 4) (see formula 10). COzH

,O

10

The two types of residue are arranged in blocks, and in approximately alternating sequences. The negatively charged carboxylate chromophore binds with various cations to provide interesting properties for solutions, gels, and films of this polymer. The c.d. spectra of the alginate polymer have been measured on commercial i n s t r ~ m e n t s ~ ’and - ~ ~a study has been performed on vacuum-u.v. instrument^.^^ C.d. bands at 215 and 203 nm were assigned to the carboxylate nn* transition, and the c.d. band at 180 nm was assigned to the carboxylate m r * . Shorter-wavelength transitions, observed at 169 and 149 nm in film spectra, were assigned to transitions of the sugar in the polymer backbone. Intermolecular association in forming gels and films changes the intensity of both the short- and long-wavelength transition^.^^ The principal spectral changes are attributed to perturbation of the carboxylate chromophore by site-bound cations. Chelation of the divalent cation Ca2+to alginate chains has been studied e x t e n ~ i v e l y The . ~ ~ negative c.d. band at 205 nm for aqueous solutions of sodium poly-( L-gulopyranuronate) (called the m r * band rather than the n?r* band in this early paper) loses negative intensity as Ca2+is added (see Fig. 24). This change is equivalent to gaining positive c.d. intensity for a

-

E. R. Morris, D. A. Rees, D. Thorn, and J. Boyd, Carbohydr. Res., 66 (1978) 145-154. E. R. Morris, D. A. Rees, and G. Young, Carbohydr. Res., 108 (1982) 181-195. D. Thorn, G. T. Grant, E. R. Morris, and D. A. Rees, Carbohydr. Res., 100 (1982) 29-42. R. Seale, E. R. Morris, and D. A. Rees, Carbohydr. Res., 110 (1982) 101-112. E. R. Morris, D. A. Rees, G. Robinson, and G. A. Young, J. Mol. Biol.,138 (1980) 363-374. H. J. Jennings, R. Roy, and R. E. Williams, Carbohydr. Res., 129 (1984) 243-255. E. R. Moms, D. A. Rees, and D. Thorn, Carbohydr. Res., 81 (1980) 305-314. B. Stockton, L. V. Evans, E. R. Moms, and D. A. Rees, I n f . J. Biol. Macromol., 2 (1980) 176-178. B. Stockton, L. V. Evans, E. R. Moms, D. A. Powell, and D. A. Rees, Bof. Mar., 23 (1980) 563-567. J. N. Liang, E. S. Stevens, S. A. Frangou, E. R. Moms, and D. A. Rees, I n f . J. Biol. Macromol., 2 (1980) 204-208.

W. CURTIS JOHNSON, JR.

106

I

-. 8

I

200

I

I

X

220

I

I

240

(nm)

FIG. 24.-Circular Dichroism of Sodium Poly-(L-gulopyranuronate)as a Function of Stoichiometry of Added Ca2+:0% (-), 25% (- . -), 50% (- - -), 75% (- . . -), and 100% (. . .). (Redrawn from Ref. 48.)

band at -207 nm. The stoichiometry, investigated by c.d. and equilibrium dialysis, shows one Caz+bound for each four L-gulopyranuronate residues. This is consistent with dimerization of the chains into an “egg box” structure. Two residues from each of the dimer chains form a nest of oxygen atoms which satisfies the criteria for Ca2+chelation. Similar experiments on alginate indicated occurrence of this type of dimerization for the poly-(Lgulopyranuronate) blocks found in these natural polysaccharides. Furthermore, c.d. and 0.r.d. have been combined to eliminate interference from the poly-(D-mannopyranuronate) and alternating sequences in such studies.49 In a similar manner, c.d. has been used to study gelation of alginates through the addition of various divalent cations.” Monovalent cations have also been studied extensively, and, although the c.d. changes are less spectacular, they indicate association of the poly-( L-gulopyranuronate) blocks.” Finally, competitive inhibition between various polysaccharides has been used to study specific, intermolecular associati~n.’~ As expected, short poly-( L-gulopyranuronate) blocks can abolish alginate gelation, whereas poly-( D-mannopyranuronate) has little effect.

THE CIRCULAR DICHROISM OF CARBOHYDRATES

107

C.d. spectroscopy is now being applied to more complicated polysaccharides. The 3-deoxy-~-manno-2-octulopyranosylonic acids found in Escherichia coli LP1092 have been definitely assiglled the 0-D configuration.53 The negative nrr* c.d. band exhibited by this polysaccharide correlates with the negative c.d. of methyl 3-deoxy-a-~-rnanno-2octulopyranosidonic acid rather than the positive c.d. band exhibited by acid. methyl 3-deoxy-~-~-manno-2-octulopyranosidonic The utility of c.d. spectroscopy is well illustrated in its application to C.d. spectra for determine alginate composition and bl~ck-structure.~'~~~-~~ the three types of idealized blocks [poly-( a-L-gulopyranuronic acid), poly(P-D-mannopyranuronic acid), and the alternating polymerIs4 are given in Fig. 25. The c.d. for the alternating polymer is different from the average of spectra for the two homopolymers, showing that the c.d. is sensitive to nearest-neighbor residues. As an additional benefit, this means that the c.d. spectra of natural alginates can be analyzed for the three types of block structure. These workers used a least-squares fitting, together with the

I

!

I

X

(nm)

FIG. ZS.-Circular Dichroism for Blocks of Poly( L-gulopyranuronate) (- -), Poly(omannopyranuronate) (- - -), and Alternating Sequences (- . -) as Found in Alginate. (Redrawn from Ref. 54.)

108

W. CURTIS JOHNSON, JR.

constraint that the sum of the three structures must equal 100%, to analyze alginates from various source^.^^-^^ Because the carboxyl chromophore is sensitive to pH, their solutions were carefully neutralized to pH 7.0. Furthermore, divalents must be strictly excluded, because these ions cause the poly-( a-L-gulopyranuronic acid) blocks to associate and thus change their c.d. spectrum. The analysis of material extracted from the stipes of Alaria esculenta, gathered at Cullercoats on the British east coast, is illustrated in Fig. 26, where the composition is 18% of poly-( P-D-mannopyranuronic acid), 51% of poly-( cu-L-gulopyranuronic acid), and 31Yo of the alternating polymer.55 It is also possible to estimate the proportion of each sugar by dividing the intensity of the negative trough that falls between 218 and 208 nm by the difference between the trough and the peak at -200nm. As long as the overall D-mannopyranuronic acid level is below 60% (corresponding to an all-negative c.d. spectrum), the ratio of D-mannopyranuronic acid to Lgulopyranuronic acid is approximately twice this ratio. The ratio is proportional to the level of D-mannopyranuronic acid when this sugar is at high levels. In this case, the percentage of D-mannopyranuronic acid is -27 times the ratio +40%. Applying these empirical relationships to the c.d.

x

I

200

I

X

I

220

I

I

240

I

(nm)

FIG. 26.-Circular Dichroism of Alginate from the Stipes of Alaria esculenta Gathered at and the Component Analysis for Poly(L-gulopyranuronate)(- - -), Po~Y(DCullercoats (-) mannopyranuronate) (. . .) and Alternating Sequences (- . -). (Redrawn from Ref. 35.)

THE CIRCULAR DICHROISM OF CARBOHYDRATES

109

spectrum of Alaria esculenta (shown in Fig. 25) gives a value of 34% of D-mannopyranuronic acid, in good agreement with values inherent in a block c o m p ~ s i t i o n . ~ ~ The sodium salt of poly(D-galacturonic acid) has a positive c.d. band due to the nm* transition at 208 nm that decreases in amplitude and blue shifts as the polymer gels.58Ravanat and R i n a ~ d conducted o~~ a particularly extensive study on oligo( D-galactopyranuronic acid) in the acid form and as sodium or calcium salts. Dissolved in aqueous solution in the absence of a salt, the c.d. band due to the n r * transition increases somewhat with the degree of polymerization (d.p.) from the dimer through the polymer. As the anionic form is converted into the undissociated form with the addition of hydrochloric acid, the intensity of this c.d. band decreases in all cases. The authors considered that, under these conditions, they observed an acidic form that would be stabilized by hydrogen bonding, and postulated a helix with three-fold screw symmetry, such as has been described for sodium pectinates in the solid state.60The same results are observed if the equilibrium is shifted to the associated form by decreasing the dielectric constant. Additions of sodium hydroxide to afford the sodium salt decrease the intensity of this band and shift it to shorter wavelength^,'^ as shown for the dimer and the polymer in Fig. 27. It may be noted the isodichroic point lies at 198 nm. In contrast, observation of the c.d. with the addition of Ca(OH)*, as a function of d.p., demonstrated that terminal and central units react differently towards Ca2+. This is illustrated in Fig. 27 for the dimer and the polymer. Again, the intensity of the c.d. band decreases as the polymer binds calcium and begins to gel. Results for both salt forms are attributed to a helix having a two-fold screw-symmetry, in analogy with calcium pectates6’ The gelling would then involve a multi-chain association, with crosslinking by the calcium ions to form an “egg box” s t r u ~ t u r e . ~ ~ * s ’ ~ ~ ~ Liang and Stevens6* extended the c.d. spectra of poly(D-galactopyranuronic acid) and the sodium and calcium salts into the vacuum-u.v. region. In addition to the nr* c.d. band at -208 nm, these authors observed a negative band at -170 nm and a positive band at -145 nm for both poly( D-galactopyranuronic acid) and its sodium salt. The c.d. of the calcium salt could be measured only to 170 nm, with the indication of the second negative c.d. band. They observed these two shorter-wavelength bands for (58) G. T. Grant, E. R. Morris, D. A. Rees, P. J. C. Smith, and D. Thorn, FEES Lerr., 32 (1973) 195. (59) G. Ravanat and M. Rinaudo, Biopolymers, 19 (1980) 2209-2222. (60) K. J. Palmer and M. B. Hartzog, J. Am. Chem. Soc., 67 (1945) 2122-2127. (61) C. Sterling, Biochim. Biophys. Acta, 26 (1957) 186-197. (62) J. N. Liang and E. S. Stevens, In?. Biol. Macromol., 4 (1982) 316-317.

W. CURTIS JOHNSON. JR.

110

1.5

A€

1

.5

200

200 220 240

220 240

X

(nm)

FIG. 27.-Circular Dichroism Spectra of Galactopyranuronic Acid for d.p. = 2 (First and Third Panels) and d.p. = 340 (Second and Fourth Panels) as a Function of Percent of Neutralization with NaOH (First and Second Panels) and Ca(OH), (Third and Fourth Panels): 0% (-), 20% (- - -), 40% (- . -), 60% * .), 80% (- . . -), and 100% (- -). (Redrawn from Ref. ( 9

59.)

a number of polysaccharides, and assigned them to ring transitions. In this case, the carboxylate mr* transition probably also contributes to the 170-nm band. The results for a poly(D-galactopyranuronic acid) film are shown in Fig. 28.

AA

140 FIG. 28.-Circular from Ref. 62.)

160

180

200

X (nm)

220

240

Dichroism of a Film of Poly(D-galactopyranuronic Acid). (Redrawn

THE CIRCULAR DICHROISM OF CARBOHYDRATES

111

The main component of pectins is poly( D-galactopyranuronic acid) in which the carboxyl chromophore may be esterified to a variable extent. The polymer has a positive c.d. band at -210 nm for the carboxyl, carboxylate, and acetate c h r o m o p h o r e ~ .The ~ ~ *c.d. ~ ~ has been used to investigate the effect of carboxyl ionization, carboxyl esterification, conformational changes, and association, for variations in pH, temperature, and ionic strength.63A linear change wa5 observed upon esterification, indicating that there is no fundamental change in the polysaccharide. On ionization of the carboxyl chromophore, there is a sharp decrease in intensity of the c.d. as the carboxylate anion is formed. Changing the solvent conditions can cause the pectin to gel, and this association gives rise to an increase in the c.d. intensity. Increasing the temperature decreases the association, as witnessed by a decrease in the intensity of the c.d. bands. Gelation by various divalent cations has also been extensively studied by c.d. spectroscopy. 4. Derivatives Having Mixed Substituents

N-Acetylneuraminic acid is a common group in glycoproteins, and it contains both the amide and carboxyl chromophores. As shown in formula 11, this nine-carbon sugar derivative has an equatorial amido group on C-5 and both a hydroxyl group and a carboxyl group on the anomeric carbon atom.

11

The carboxyl chromophore is axial for the a anomer and equatorial for the p anomer. The sugar was studied as the carboxylate anion as it has a (low) p K of 2.6, and the compound is degraded in acidic solution. The c.d. spectrum of this compound contains contributions from the carboxylate nT* at -217 nm, the amide nr* at -210 nm, and the amide w r * at -190 nm. Apparently, all of these bands are positive, giving rise to a c.d. spectrum a maximum at 199 nm and a shoulder at 210 nm. (see Fig. 29) The c.d. spectra of a number of derivatives confirmed these assignments. (63) I. G. Plaschina, E. E. Braudo, and V. 9. Tolstoguzov, Carbohydr. Res., 60 (1978) 1-8. (64) H. R. Dickinson and C. A. Bush, Biochemistry, 14 (1975) 2299-2304. (65) G. Keilich, R. Bossmer, V. Eschenfelder, and L. Holmquist, Carbohydr. Res., 40 (1975) 255-262. (66) L. D. Melton, E. R. Morns, D. A. Rees, and D. Thom, J. Chem. SOC.,Perkin Trans. 2, (1979) 10-17.

W. CURTIS JOHNSON. JR.

112

5

-

c 200

l

220

X

240

260

I

(nrn)

FIG.29.-Circular Dichroism of N-Acetylneuraminate (. . .), and Its Methyl a- (-) and Methyl /3- (- - -) D-Ketopyranosides in Aqueous Solution at -pH 3.2. (Redrawn from Ref. 65.)

The c.d. spectra of a number of other derivatives have been measured as models for the linkages found in o l i g o m e r ~ .Spectra ~ ~ * ~ ~of the methyl aand P-D-ketopyranosides of N-acetylneuraminic acid are given in Fig. 29. They do not differ substantially from the c.d. spectra for the fundamental sugars, except that the a-ketopyranoside has a positive c.d. band of low intensity at 250 nm. These types of bands occur from time to time for still obscure reasons, as discussed for carboxyl derivatives. The c.d. spectra are also available for a few oligosaccharides that contain N-acetylneuraminic a ~ i d . ~ These ~ * ~spectra ~ * depend ~ ~ ~ on ~ the ~ -intersac~ ~ charide linkages and the state of ionization of the carboxyl group, but no systematic scheme has yet been set up to derive configurational information from the c.d. spectra. Of particular interest is the c.d. spectrum of beef ganglioside:' which fully differentiates the amide nr*,amide r r * , and carboxylate nr* c.d. bands. In muramic acid, the 2-hydroxyl group of D-glucose is replaced by an amine group, and a lactic acid group is attached to OH-3. The structure of muramic acid [2-amino-3-0-( ~-l-carboxyethyl)-2-deoxy-~-glucopyranose] is given in formula 12.

qq;. C H ,OH

HO HsC

'H

12

(67) A. L. Stone and E. H. Koludny, Chern. Phys. Lipids, 6 (1971) 274-279. (68) H. I. Jennings and R. E. Williams, Carbohydr. Res., 50 (1976) 257-265.

THE CIRCULAR DICHROISM OF CARBOHYDRATES

113

Listowsky and coworkers showed69that the c.d. of this sugar derivative is due entirely to lactic acid, and confirmed that this chromophore is in the D configuration for muramic acid. N-Acetylmuramic acid, in which the amino group is replaced by an amido group at C-2, has a c.d. spectrum that is roughly a linear combination of the lactic acid in muramic acid and the amide in 2-acetamido-2-deoxy-~-glucose. This indicates that the amide chromophore and the lactic acid chromophore in N-acetylmuramic acid behave independently. Hyaluronic acid is an important biological polysaccharide found in the intracellular matrix of most connective tissues. Fundamentally, it is a linear copolymer of p -D-glucopyranosyluronic acid and 2-acetamido-2-deoxy-PD-glucopyranosyl residues. The D-glucuronic acid is P-D-(1 + 3)-linked to 2-acetamido-2-deoxy-~-glucopyranose, and the 2-acetamido-2-deoxy-~glucopyranose is P-D(1 + 4)-linked to the D-glucopyranuronic acid, to give an alternating copolymer (see formula 13). CO,H H3CCNH HO

II 0

HO 13

~ t ~ ~ ~ 3 1 , 6first 7 , 7 0measured the c.d. of hyaluronic acid. Chakrabarti and coworker^^^^^'-^^ extensively studied the c.d. of this polysaccharide on commercial instruments, and collaborated with Buffington and Stevensz9in a c.d. study in the vacuum-u.v. region. The c.d. spectra of hyaluronic acid at pH 2.5, and as the hyaluronate anion at pH 6.9, are given in Fig. 30. Only minor differences are observed at the two pH values, although the spectrum of the anion is somewhat more intense than that of the acid. Changes in interaction or configuration must occur on forming the polymer, as the negative band for hyaluronic acid is too intense to be accounted for by averaging the monomer ~pectra.’~ On the other hand, the c.d. bands expected for the acetamido and carboxylate m r * transitions between 180 and 190nm are not observed. (69) (70) (71) (72) (73) (74)

1. Listowsky, G. Avigad, and S. Englard, Biochemistry, 9 (1970) 2186-2189. A. L. Stone, Biopolymers, 7 (1969) 173-188. J . W. Park and B. Chakrabarti, Biopolymers, 16 (1977) 2807-2809. J. W. Park and B. Chakrabarti, Biochim. Biophys. Acra, 544 (1978) 667-675. J. W. Park and B. Chakrabarti, Biopolymers, 17 (1978) 1323-1333. J. W. Park and B. Chakrabarti, Biochim. Biophys. Acra, 541 (1978) 263-269.

114

W. CURTIS JOHNSON, JR.

6t +

-1

-6 O

I b 180

220

200

X

24 0

(nm)

FIG.30.-Circular Dichroism of (a) Hyaluronic Acid at pH 2.5 and (b) Sodium Hyaluronate at pH 6.9. (Redrawn from Ref. 29.)

Cowman and coworker^^^*^^ investigated oligosaccharides featuring both types of linkage found in sodium hyaluronate, in an attempt to ascertain why the two m r * transitions are not observed. An increase in intensity at 210nm was found to result from the conformational changes due to the from 2-acetamido-2-deoxy-/?-~(1 + 4)-/?-~-glycosidic linkage glucopyranose to sodium /?-~-glucopyranosyluronate.~~ In the case of the 190-nm region (see Fig. 31), c.d. spectra of the oligomers showed that the two linkages give rise to W ~ T *c.d. bands of opposite sign, so that there is an accidental cancellation of these spectral contributions, and the expected band is absent for sodium h y a l ~ r o n a t e . ~ ~ Films of sodium hyaluronateZ9have a significantly different c.d. spectrum, as shown in Fig. 14. The intense, negative c.d. band observed at 195nm was assigned to the amide m d ' transition. The authors considered that the amide group participates in an intramolecular hydrogen-bond, to form a solid-state, helical structure, and the resulting decrease in rotational freedom gives rise to the large, negative, rotational strength. (75) M. K. Cowman, E. A. Balms, C. W. Bergmann, and K. Meyer, Biochemistry, 20 (1981) 1379-1385. (76) M. K. Cowman, C. A. Bush, and E. A. Balms, Biopolymers, 22 (1983) 1319-1324.

THE CIRCULAR DICHROISM OF CARBOHYDRATES

115

X (nm) FIG.31.-Circular Dichroism of a Hyaluronic Acid Segment Comprising -18 Disaccharide Units (-), the Tetrasaccharide Featuring P - D - (+ ~ 3) Linkages for the End Residues (. . .), and the Tetrasaccharide Featuring /3-~-(1-4)Linkages for the End Residues (- - -). (Redrawn from Ref. 76.)

The work of Chakrabarti and ~ ~ ~ o r k e showed r ~ ~ that ~ -hyaluronic ~ ~ * ~ ~ , ~ ~ acid in aqueous ethanol solvent at pH 2.5, and the Cu(I1)-hyaluronate complex at pH 6.8, apparently have the same intense, negative, c.d. band observed for the hyaluronate film (see Fig. 14), although spectra had not been measured at <205 nm (see Fig. 32). The transition is readily monitored in solution, and has been found to be cooperative with respect to temperature, solvent composition, and pH. Methyl hyaluronate does not show this c.d. change, confirming that the protonated carboxyl group is involved in the transition for the mixed-solvent system. These workers considered that the four-fold, helical model which allows hydrogen bonding between the amide hydrogen atom and the carboxyl oxygen atom may be the appropriate conformation for all three systems, where helical structures go into an aggregated state. Chondroitin is quite similar to hyaluronic acid, with the exception that the 4-hydroxyl group of the N-acetylglycopyranosylamine is now axial instead of equatorial (see formula 13 for hyaluronic acid). This polysaccharide may be sulfated at C-6 of the N-acetylgalactopyranosylamine, to form chondroitin 6-sulfate. Chondroitin and chondroitin 6-sulfate have (77) N. Figueroa and B. Chakrabarti, Biopolyrners, 17 (1978) 2415-2426. (78) B. Chakrabarti, N. Figueroa, and J. W. Park, in J. D. Gregory and R. W. Jeanloz (Eds.), FTOC.Int. Syrnp. Glycoconjugotes, 4th,(1979) 119-124.

W. CURTIS JOHNSON, JR.

116

A€

FIG. 32.-Circular Dichroism of Hyaluronic Acid in 20% Ethanol at pH 2.5 (-) pH 6.5 (- * -), and the Difference Spectrum (- - -). (Redrawn from Ref. 73.)

and at

similar c.d. spectra in aqueous solution.25.73.15 In the acid form, at pH -2.5, both polysaccharides have a negative c.d. band at 210 nm, and show another negative band that peaks below 170nm. At neutral pH, the uronic acid residue is ionized and, in addition to a negative c.d. band at 210 nm, there

X

(nrn)

FIG. 33.-Circular Dichroism of Chondroitin 6-Sulfate in D20 at p D -7 (- - -) and pD -2.5 (-). (Redrawn from Ref. 25.)

THE CIRCULAR DICHROISM OF CARBOHYDRATES

117

-

is a positive band at 190 nm. The results for chondroitin 6-sulfate'' are shown in Fig. 33. There is little sign of interaction, as the c.d. of the polysaccharides is nearly the same as that of the constituent monosaccharides. Stone's c.d. spectra3' of glycosaminoglycans that contain substituents other than the chromophoric amide group are given in Fig. 34. This pioneering work demonstrated that c.d. spectroscopy could be used to determine the type of sugar polymer, and could probably be used to monitor configuration and conformation. The negative nr* c.d. band at longer wavelengths is common to all of the polymers studied. The shorter-wavelength c.d. band, which is often incompletely measured, depends on the polymer structure. The (1 + 4)-linked amino sugars show a positive m r * amide transition at 190 nm. The (1 + 4)-linked amino sugars have a negative m r * band that generally lies below the limits of the commercial instruments used. Stone

-

w 0

4

2

A€ 0 -2 -4

7I I I I I I I I I I I 200 250

2 0 0 250

200

250

X (nrn)

FIG. 34.-Circular Dichroism of Glycosaminoglycans: (a) Hyaluronic Acid; (b) Heparan Sulfate from Normal Mammalian Tissue; (c) Chondroitin 4-Sulfate; (d) Dermatan Sulfate; (e) Chondroitin 6-Sulfate; (f) Shark Sulfated-Keratan Sulfate (-) and Mammalian Keratan Sulfate (. . .); and ( 9 ) Heparin. (Redrawn from Ref. 31.)

118

W. CURTIS JOHNSON, JR.

and coworker^'^ used these optical properties to monitor glycosaminoglycans in the urine of patients with Hurler’s syndrome, and Chung and EllertonEomonitored the binding of Cu(I1) with heparin by using the c.d. of the amide chromophore. The extracellular polysaccharides from the Xanthornonas species of bacteria are interesting materials that find industrial application. The polymer has a cellulose-like backbone with trisaccharide side-groups on alternate residues. The side groups consist of an a-D-mannopyranosyl residue with a 6-acetate substituent, a p-D-glucopyranuronic acid residue, and a p-Dmannopyranosyl residue with a 4,6-pyruvic acetal substituent on 1/3rd of the residues. Thus, although the cellulose-like backbone absorbs only in the vacuum-u.v. region, the side groups contain acetate and carboxylate chromophores that absorb light within the range of commercial c.d. instruments. The c.d. spectrum of the polysaccharide xanthan from the Curnpestris species (see Fig. 35) confirmed the results of ‘H-n.m.r.-spectral, viscosity, and monochromatic optical-rotation studies.” All of these methods indicated that the polysaccharide forms, in aqueous solution, an ordered structure that can be “melted” to a disordered structure by increasing the temperature. The intensity of the 205-nm, positive c.d. band diminishes linearly with temperature, but the intensity of the 220-nm, negative c.d. band increases with temperature, and this shows a cooperative melting comparable to that disclosed by the optical-rotation studies. There is no dependence on concentration, indicating that only a single chain is involved in forming the ordered structure. Furthermore, the ordered structure is increasingly stable at higher ionic strength. On the basis of all of these physical measurements taken in combination, the authors proposed that the side groups fold down, and interact with the backbone to form the ordered structure. The complex oligosaccharides comprising the T“ and “T” antennae of three glycoproteins have been studied by c.d. spectroscopy.’‘ Five antennae were studied in all, because two of the proteins have two closely related forms. The c.d. for the three fundamental oligosaccharides are given in Fig. 36. These are asparagine-linked glycopeptides, and the intense, conservative c.d. band that results from the interaction between the two amides has already been discussed in Section III,2. Fig. 36 shows that the c.d. spectra

-

(79) A. L. Stone, G . Constantopoulos, S. M. Sotsky, and A. Dekaban, Biochim. Biophys. Acta, 222 (1970) 79-89. (80) M. C. M. Chung and N. F. Ellerton, Biopolymers, 15 (1976) 1409-1423. (81) E. R. Morris, D. A. Rees, G . Young, M. D. Walkinshaw, and A. Darke, J. Mol. Biol., 110 (1977) 1-16. (82) C. A. Bush, V. K. Dua, S. Ralapati, C. D. Warren, G. Spik, G . Strecker, and J. Montreuil, J. Biol. Chem., 257 (1982) 8199-8204.

T H E CIRCULAR DICHROISM OF CARBOHYDRATES

I1

200

I

I

220

I

I

240

I

I

260

X (nm)

I

I

280

I

119

I

3C

FIG. 35.-Circular Dichroism of the Xanthan from Xanthomonas campestris at Various Temperatures. (Redrawn from Ref. 81.)

are sensitive to the different types of antennae. By using model oligosaccharides, these workers showed that each of these c.d. spectra is the algebraic sum of the c.d. due to the linkage plus the c.d. of the other chromophores in the system. This means that there are no other interactions between the chromophores in the system, indicating that the antennae have an “open” conformation. 5. Non-biological Derivatives Unsubstituted polysaccharides absorb light only in the vacuum-u.v. region, but derivatives of such polysaccharides can often be synthesized that absorb light within the range of commercial instruments. Bittinger and K e i l i ~ synthesized h~~ carbanilyl derivatives of a number of a-and p-glycans. Derivatives of the P-glycans all displayed a weak, negative c.d. band at -240nm. In contrast, all derivatives of the a-glycans showed a strong, (83) H. Bittiger and G. Keilich, Biopolymers, 7 (1969) 539-556.

120

W. CURTIS JOHNSON, JR.

A€

180

X

200

220

(nm)

FIG. 36.-Circular Dichroism of Antennae Found in Glycoprotein STF-A from Serum and LTF-D from Lactotransferrin (- - -). Transferin (. . .), OTF-C from Ovotransferin (-), (Redrawn from Ref. 82.)

negative c.d. band at -240 nm and a strong, positive c.d. band at -225 nm. Such a c.d. couplet is characteristic of exciton interaction, implying that all of the a glycans studied assume a helical conformation in the 1,4-dioxane solvent used for these studies. An almost planar arrangement was proposed for the p-glycans. Pfannemuller and Bergs4 studied carbanilated and tritylated derivatives of amylose and cellulose in 1,Cdioxane. As shown in Fig. 37, 2,3-di-Ocarbanilylamylose had an especially large exciton interaction between the derivative groups, suggesting a helical structure. The c.d. spectra for 2,3,6-tri0-carbanilylamylose and 2,3-di-O-carbanilyl-6-O-tritylamylose showed somewhat less-intense exciton bands. In contrast, 6- 0-tritylamylose showed only a weak c.d. band, indicating an unordered structure in 1,Cdioxane. Cellulose was also studied as the 2,3,6-tri-O-carbanilyl and 2,3-di-0carbanilyl-6-0-trityl derivatives. A single, intense c.d. band was observed, suggesting a viable secondary structure without exciton interaction. Acetates have been particularly useful for studies conducted with commercia1 c.d. instruments,'s~25~s5~as and they are usually examined in the par(84) B. Pfannemuller and A. Berg, Makromol. Chem., 180 (1979) 1201-1213. (85) S. Mukherjee, R. H. Marchessault, and A. Sarko, Biopolymers, 11 (1972) 291-301.

(86) S. Mukherjee, A. Sarko, and R. H. Marchessault, Eiopolymers, 11 (1972) 303-314. (87) J. W.-P. Lin and C. Schuerch, 1. Polym. Sci., 10 (1972) 2045-2060. (88) A. Sarko and C. Fischer, Biopolymers, 12 (1973) 2189-2193.

THE CIRCULAR DICHROISM OF CARBOHYDRATES

121

A€

X

(nm)

FIG.37.-Circular bichroism of 2,3-Di-O-carbanilylamylose (-), 2,3,6-Tri-O-carbanilyl(. . .). (Redrawn from Ref. 84.) amylose (- - -), and 2,3-Di-O-Carbanilyl-6-O-tritylamylose

ticularly transparent solvent 2,2,2-trifluoroethanol (TFE). Acetates of xylan, cellulose, dextran, mycodextran, (1 + 6)-cu-~-mannan, and (1 + 6 ) 4 D-galactan show no conformational effects, and assume, presumably, a random coil in solution. However, amylose triacetate shows" two c.d. bands in this region, a positive band at -235 nm and a negative band at -205 nm; this suggests that the amylose assumes a helical conformation. The c.d. spectra of the oligomers confirmed this interpretation. The monomer and dimer display the normal nr* c.d. band expected for the isolated acetate chromophore, whereas the c.d. of the trimer is similar to that of amylose triacetate, indicating acetate-acetate interaction. Furthermore, the c.d. spectrum of amylose triacetate varies with temperature (see Fig. 38), as would be expected for a change in conformation. Films of amylose triacetate have the normal nr* c.d. band of the acetate chromophore, but the c.d. spectrum changes to that for amylose triacetate in solution when the films are annealed to afford a crystalline structure. Crystalline amylose has been found to have a helical conformation. All of these data, taken together, indicate that amylose triacetate in TFE is present in a helical conformation.

122

W. CURTIS JOHNSON,JR. .06 -

I

I

I

I

I

FIG. 38.-Circular Dichroism of Amylose Triacetate in l,l,l-Trifluorethanol at Various Temperatures. (Redrawn from Ref. 88.)

Stipanovic and Stevens25extended the c.d. of acetylated glucans into the vacuum-u.v. region. They studied derivatives for both a- and p - ( l + 3)-, -( 1 + 4)-, and -(1 + 6)-linked glucans in TFE and as films. All show a negative c.d. band near 190nm for the ~ T T transition, * which is independent of configuration and conformation. The expected exciton splitting for the m r * transition of amylose triacetate was not observed. However, the 170nm band observed for films of all of these triacetates did display an exciton splitting, with particularly high intensity bands of opposite sign in the case of cellulose triacetate. Amylose and dextran have been in aqueous solution as the xanthate derivatives. Dextran xanthate has no observable c.d., but amylose xanthate in aqueous solution has a complex c.d. that indicates an organized structure. Benzyl derivatives of (1 + 6)-a-~-glucan,(1 + 6)-a-~-mannan,and (1 + 6)-a-~-galactan have been studied in lY4-dioxane.These derivatives have complex and interesting c.d. spectra due to the m r * transition of the chromophore with resolved vibrational structure.89 However, a conformational interpretation of these interesting spectra is not possible at this time. A carboxymethyl derivative of dextran has been prepared in order to study the effect of charge density, concentration, degree of neutralization, (89) J.-P. Merle and A. Sarko, Carbohydr. Res., 30 (1973) 390-394.

THE CIRCULAR DICHROISM OF CARBOHYDRATES

123

and added salts on the c.d. spectrum.g0 Here, the carboxyl chromophore that is present in a number of biologically important sugars is involved; it has already been discussed in that regard. Because the chromophore herein is far removed from an asymmetric center, the magnitude of the c.d. is lower than for the biological sugars, but the same types of bands are displayed. For the carboxylate anion, there is a single, negative c.d. band at -213 nm due to the nr* transition. For the carboxylic acid at low pH, there are two c.d. bands, one negative, at -207 nm, and one positive, at -227 nm. Both of these bands are probably due to the nr* transition. The author found that an increase in charge density caused an increase in the magnitude of the c.d., with concomitant shift of the c.d. bands to the red. The magnitude of the c.d. was found to be independent of the concentration of the polymer if a salt was present, but there was a concentration dependence in aqueous solution in the absence of a salt. C.d. spectra were recorded for systematic variations in the concentration and type of counter-ion. Ethylene dithioacetals and diethyl dithioacetals have been investigated for a number of monosa~charides.~' The c.d. spectra show one, or two, c.d. band(s) of low intensity between 235 and 250 nm, and a third band of low intensity that peaks below 220 nm. These workers found no overall relationship between the configurational pattern of the monosaccharide and the sign of these bands. However, there does appear to be a correlation between the configurations of C-2, C-3 and C-4 and the sign of the c.d. band that peaks below 220 nm. Sallam investigated 2-phenyl-1,2,3-osotriazole derivatives linked to the anomeric carbon atom for a large number of m o n o s a ~ c h a r id e sHe .~ ~showed that the sign of the c.d. band that occurs at -260nm depends on the anomeric configuration. The a-Dor p-Lconfiguration exhibits a positive c.d. band, whereas the p-Dor a- configuration exhibits a negative c.d. band. Nakanishi and coworkers developed a particularly useful system for determining the configuration and conformation of sugars using di-pbromobenzoate derivative^.^^-^^ The strong absorption caused by each of the two benzoate groups attached to the sugar will interact, giving rise to an exciton splitting which results in two intense, c.d. bands of opposite K. Gekko, Biopolymers, 18 (1979) 1989-2003. M. K. Hargreaves and D. L. Marshall, Carbohydr. Rex, 29 (1973) 339-344. M. A. E. Sallam, Curbohydr. Res., 129 (1984) 33-41. N. Harada, H. Sato, and K. Nakanishi, Chem. Commun., (1970) 1691-1693. N. Harada and K. Nakanishi, J. Am. Chem. SOC.,91 (1969) 3989-3991. N. Harada and K. Nakanishi, Circular Dichroic Spectroscopy-Exciton Coupling in Organic and Eioorganic Stereochemistry, Univ. Science Books, Mill Valley, CA, 1982. (96) K. Nakanishi, M. Kuroyanagi, H. Nambu, E. M. Oltz, R. Takeda, G . L. Verdine, and A. Zask, Pure Appl. Chem., 56 (1984) 1031-1048.

(90) (91) (92) (93) (94) (95)

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W. CURTIS JOHNSON,

JR.

sign. The sign of the long-wavelength band is the sign of the chirality as defined by these workers, and, using their methods can be related to the relative configuration of the two groups or the favored conformation. The difference in intensity between the two extrema of the two c.d. bands can be used to determine the configuration. For a di-p-bromobenzoate, the values in AE units are: 1,2-ee, 62; 1,2-ea, 62; 1,2-aa, 6; 1,3-ee, 0; and 1,3-ea, 16. For tri-p-bromobenzoates, the observed difference in intensity for the two extrema will be the algebraic sum of the three pairwise interactions possible.96Because the extinction coefficients for these sugars are known? concentrations are easily determined. The method can also be used to determine the branching points in o l i g ~ s a c c h a r i d e s . This ~ ~ * ~work ~ demonstrated the power inherent in c.d. spectroscopy for investigating the structure of sugar monomers and polymers.

ACKNOWLEDGMENTS This work was supported by National Science Foundation grant DMB-8415499 from the Biophysics program, and Public Health Service grant GM-21479 from the Institute of General Medical Sciences.

(97) H.-W. Liu and K. Nakanishi, 1. Am. Chem. Soc., 103 (1981) 5591-5593. (98) H.-W. Liu and K. Nakanishi, 1. Am. Chem. SOC.,103 (1981) 7005-7006.