Natural-abundance 17O NMR of monosaccharides

JOURNAL

OF MAGNETIC

RESONANCE

48,

43 l-446 (1982)

Natural-Abundance “0 NMR of Monosaccharides* IOANNIS P. GEROTHANASSIS AND J~~RGENLAUTERWEIN~ Institut de Chimie Organique, Universitt de Lausanne, 1005 Lausanne, Switzerland AND

NORMAN SHEPPARD School of Chemical Sciences, University of East Anglia. Norwich NR4 7TJ. England Received jarmary 5, 1982 Natural-abundance “high-resolution ” “0 NMR spectra of D-ghwSe, D-mannose, D-galactose, and some methoxy derivatives of D-&COSe were recorded in aqueous solution. The sensitivity and spectral resolution was improved by optimizing the accumulation and manipulation of data. The water solvent peak was suppressed through use of “O-depleted water or displaced by addition of paramagnetic shift reagents. With Dy3+ the “0 NMR spectrum of D-glucose remained unaltered, however, the water peak was shifted outside the carbohydrate spectral region. The “0 NMR resonances were assigned from earlier data for some specifically “O-enriched monosaccharide derivatives. The anomeric hydroxyl resonances could also be located because of their exchange with the “O-depleted water. Although the chemical shifts of the monosaccharides generally parallel the sequence of chemical shifts for simple primary and secondary alcohols and substituted ethers, several exceptions were found and discussed in terms of steric and electrostatic repulsive forces between oxygens. INTRODUCTION

Oxygen, one of the most important elements in organic chemistry, has found very limited applicability in NMR in comparison with the other common nuclei (‘H, *H, 13C, 19F, and “P). Three review articles have recently appeared on “0 NMR (1-3). In one of them (1) no mention has been made of future applications in organic chemistry despite the wide range of applications proposed in inorganic chemistry. In the second one (2) difficulties related the low sensitivity and large linewidths in “0 NMR, particularly when applied to larger organic molecules, are made clear. In the third one (3), despite a very comprehensive analysis of the available “0 organic data, no clear conclusion about future fields of applications was made. The difficulties in “0 NMR work result from the fact that, although the nuclear electric quadrupole moment is relatively small (4) the electric field gradient is * Presented in part at the 2nd European Symposium on Organic Chemistry, Stresa, Italy, June 1981. t Author to whom correspondence should be addressed. 431 0022-2364/82/090431-16$02.00/O CopyriSht 0 1982 by Academic Press, Inc. All ri&tts of rqcduction in any form raened.

432

GEROTHANASSIS,

LAUTERWEIN,

AND SHEPPARD CHOH

OH

OH

OH

II

III CHOH

CHOH

OH IV

VI

V

OH

VII

VIII

OH

OH

OH

IX

FIG. 1. Structure and configuration of the monosaccharides and methyl derivatives that have been measured by “0 NMR at natural abundance (I, III, V, VI, VII, VIII) (this paper) and after specific enrichment (II, III, IV, IX) (Ref. (12)). For I, VI, VII, and VIII only the (Y anomers are shown.

normally large, particularly for the covalently bound oxygen in organic compounds. Typical “0 quadrupolar coupling constants for the common organic functional groups are of the order of 7 to 13 MHz (5). This can lead to large linewidths at ordinary temperatures, particularly for molecules with long correlation times for tumbling. The extremely low natural abundance (0.037%) and the frequent occurrence of a strong rolling baseline (6, 7) attributable to experimental reasons cause additional problems. These facts present the major drawback to comprehensive “0 NMR studies in organic chemistry. Even so, however, Christ et al. (8), in their pioneering natural-abundance work in the early 196Os, discussed the pattern of “0 chemical shifts of over 100 simple organic compounds. They concluded that primary alcohols and ethers absorb close to the position of the water resonance and that branching of attached alkyl groups causes substantial deshielding effects. These results have been confirmed by recent natural-abundance FT measurements (2, 9-1 I). In addition, a reasonably clear distinction between the chemical shift ranges of primary alcohols (-2 to 10 ppm, except for methanol, -38 ppm), secondary alcohols (30 to 40 ppm), and tertiary alcohols (55 to 70 ppm) was observed. On the other hand, no compounds containing both primary- and secondary-type oxygens were investigated by these authors. Sugawara et al. (9) painted a rather gloomy picture for the future of “0 NMR spectroscopy as applied to molecules with molecular weight >150. Indeed, despite the large number of very important polyoxygen compounds which contain alcoholic and ether type oxygens, it was not until 1978 that Gorin and Mazurek (12) attempted the first application of “0 NMR to more complex organic

OXYGEN-17

100

433

NMR OF MONOSACCHARIDES

50

0

-50

PPM

FIG. 2. “0 NMR spectrum of D-ghCOSe (I) in ordinary water at 80°C. Taal = 5 msec, NS = 460,000, total experimental time approximately 40 min. (A) normal spectrum. (B) after multiplication of the FID with a double-exponential Gaussian function (LB = -600 Hz, GB = 0.3). *, indicates resonance from the ordinary water. V, is a reflection of the water resonance attributable to quadrature detection.

compounds. They reported the chemical shifts and linewidths for almost 20 monosaccharide hydroxyl, ether, and acetate derivatives which had been enriched with “0 to approximately 2% in specific positions. In view of the recent advances in instrumentation and the considerable success achieved with smaller- and medium-sized phosphate molecules (13) we have attempted the first, to our knowledge, application of “0 NMR in natural abundance to a number of monosaccharides and their methoxy derivatives. Natural-abundance work has the advantage of avoiding the often very difficult and expensive preparation of labeled compounds. It is also usually a great advantage that all the oxygen sites can be studied simultaneously. EXPERIMENTAL

Two Bruker WH-400 spectrometers were used working at an “0 frequency of 54.24 MHz. Samples were run in cylindrical tubes of internal diameter 15 mm (University of Warwick, U.K.) or 10 mm (Spectrospin AG, Zurich). Data manipulations were carried out with an Aspect-2000 computer. The “0 FIDs were acquired during Tats = 5 to 7.5 msec after 90” pulses; spectral width = 20 kHz. A preacquisition delay At = 25 to 40 I,csec was usually applied which was efficient

434

GEROTHANASSIS,

LAUTERWEIN,

AND SHEPPARD

-OCH20H

- ~ _

.

.

,

100

.

.

.

.

.

50

.

.

.

.

.

0

.

’

. . ,

-50

PPM

FIG. 3. “0 spectrum of D-glucose (I) in “O-depleted water at 80°C. Tacq= 5 msec, NS = 350,000, total experimental time approximately 30 min. (A) normal spectrum. (B) after multiplication of the FID with a double-exponential Gaussian function (LB = -300 Hz, GB = 0.3; upper trace: LB = -800 Hz, GB = 0.1). 0, indicates resonance from the “O-depleted water.

in reducing the baseline distortions (the use of larger values of Af could introduce substantial sinusoidal baselines attributable to first-order linear phase correction (14)). The recycling time was chosen as ( Tacq + At). Each FID was zero-filled up to 16 K before FT. Gaussian convolution (15) was applied for two reasons, (a) to avoid the effects of truncation of the FID of the water and (b) to enhance resolution with minimum noise collection. The apodization-resolution enhancement function has the form exp(at-bt2), where a and b are adjustable parameters and are related to the Aspect parameters (LB) in hertz [(LB) c 0] and (GB) [0 < (GB) < l] as follows: a = -7&V) and b = a/2.(GB). Taq The spectra were run without field/frequency lock. The chemical shifts were determined relative to the resonance position of 1,4-dioxane, measured in a separate experiment. Dioxane is suggested to be a better reference material than water as the latter’s resonance is particularly temperature dependent (7). At 40°C the chemical shift of dioxane relative to water is +0.2 ppm. For the diamagnetic solutions the bulk magnetic susceptibility correction is estimated negligible relative to the accuracy of the chemical shift determination. The bulk susceptibility effects in the paramagnetic solutions (containing Co’+ or Dy3+) were determined by measuring the chemical shift difference of the water resonance in the normal cylindrical tube and in a spherical Pyrex bulb (16). (see Table 2).

OXYGEN-17

100

435

NMR OF MONOSACCHARIDES

0

50

-50

PPM

FIG. 4. “0 NMR spectrum of D-ghCO% (I) in “O-depleted water (0) at 64°C. Tsq = 7.5 msec, NS = 45O,OO@total experimental time approximately 58 min. (A) normal spectrum. (B) after multiplication of the FID with a double-exponential Gaussian function (LB = -300 Hz, GB = 0.2).

All monosaccharides and their methoxy derivatives were purchased in anhydrous form from Sigma except for methyl a-D-glucopyranoside, which was obtained from Fluka. They were used without further purification. After drying over PIOSr 1.5 to 3 M solution of the carbohydrates were prepared in ordinary or 170-depleted water and kept for 4 hr at 65°C before recording the NMR spectra. 170-depleted water was obtained from Yeda-Stable Isotopes, Rehovot, Israel (I70 content approximately 3 X 10V3 o/o) or from Biogenzia Lemania, Lausanne (approximately 1 x 10e3 ?&Jo). CoCIZ and DyC13 - 6 HZ0 (Aldrich) were used without further purification. RESULTS

Choice of the Solvent Despite the considerable solubility of monosaccharides in water, I70 NMR spectroscopy appears difficult because of the intense water resonance which falls in the area of the sugar functional groups (12). Considering organic solvents, the solubility of the parent monosaccharides decreases in the order hexamethylphosphoramide > dimethylsulfoxide > sulfolane. Hexamethylphosphoramide was excluded because of its carcinogenic properties. The use of dimethylsulfoxide is limited because its 170 resonance falls in the same area as those of the monosaccharides. Sulfolane has its I70 absorption well away from those of the carbohydrates. However, it is

436

GEROTHANASSIS,

LAUTERWEIN,

AND SHEPPARD

CH20H Ho@oH

,,@H OH

OH \

Jr

CH20H

OH CHO

OH HOG OH

($0

& S’H co ‘OH

-H201T

C

‘OH

-V/T CHPH

CH?OH

H&$~H

,OOH

o

HOqH OH

OH

FIG. 5. Equilibrium between the a and fi forms of D-glucose and exchange of the anomeric oxygens with “O-depleted water (H200) via the free aldehyde form. O” denotes an oxygen atom with a depleted “0 concentration.

a poorer solvent than dimethylsulfoxide or water and it decomposes at higher temperatures. After these evaluations we returned to water as the solvent of choice, particularly since it was available in 170-depleted form to minimize the perturbing contribution of the solvent resonance (I 3). This choice appears also to be justified with regard to future biochemical applications of “0 NMR, e.g., the study of sugar-metal complexes (17). D-Ghcose Figure 2 illustrates the “0 spectrum of D-ghCOSe (I in Fig. 1) in ordinary water at 80°C. As previously recommended (7,13) a very short acquisition time was used in order to retain the optimum sensitivity for the monosaccharide resonances. Under these conditions, however, the FID of the solvent is truncated, and a strongly oscillating frequency spectrum was obtained after FT (Fig. 2A). Application of a double-exponential Gaussian apodization function (I 5) greatly reduced the sidelobes of the water resonance (Fig. 2B) and as a consequence, three resonances of D-glucose could be observed at 36.5, 47.9, and 63.7 ppm. Nevertheless, it was clear that further resonances of D-glucose were hidden beneath the strong water resonance. In Fig. 3. the considerable advantage of “O-depleted water is demonstrated. Using this solvent, two absorption lines near to the water resonance (-10.6 and 9.6 ppm) became very well defined, although they were scarcely observable when using ordinary water (Fig. 2B). We will discuss below why the two resonances at 36.5 and 47.9 ppm have disappeared in “O-depleted water (Fig. 3). Having obtained a good quality 170 spectrum of D-glucose at 80°C (Fig. 3) we

OXYGEN-17

I’ 100

‘.

‘I”‘, SO

NMR OF MONOSACCHARIDES

I’ 0

‘.

437

‘1’. -50

PPM

FIG. 6. “0 NMR spectra of methyl derivatives of D-ghCOSe in “O-depleted water (0) at 80°C. (A) methyl cY-D-glucopyranoside (III). T,,, = 7.5 msec, NS = 360,000, total experimental time approximately 46 min (LB = -300 Hz, GB = 0.25). (B) 3-O-methyl-D-glucose (VI). T,,es = 10 msec, NS = 2,700,000, overnight experiment (LB = -150 Hz, GB = 0.2).

made an attempt to lower the temperature in order to approach a biologically more relevant situation. Already at 64°C it became very difficult to observe the broad resonance at high frequencies (Fig. 4), although the two absorption lines near the water resonance were still observable. Therefore, in order to obtain a complete view of the “0 spectra of monosaccharides we were confined to work at elevated temperatures. For the present we assign the “0 resonances of D-glucose with the help of the chemical shift data of Gorin and Mazurek (12). Later we will give additional and more generally valid arguments for assignment (see Discussion). In Fig. 3 the chemical shift of the resonance at lowest frequency (-10.6 ppm) agrees well with the value of -6 ppm reported (12) for the primary alcohol group of methyl-a-lglycopyranoside-6-“0 (II in Fig. 1). Therefore, we tentatively assigned this resonance to the CHzOH group of D-glucose. The OH groups at the 2,3, and 4 positions are expected in the range between -6 and 6 ppm (12). We are thus led to assume that the intense absorption of D-glucose at 9.6 ppm (Fig. 3) occurs from overlapping resonances of these three hydroxyl groups. In “O-depleted water (Fig. 3) only one absorption of D-glucose was observed at high frequency (63.7 ppm). We assign this broad resonance to the ring oxygen of D-glucose. The chemical shift agrees well with that reported (12) for methyl cr-D-glucopyranoside-5’70 (III) (48 ppm) if the screening effect on the ring oxygen attributable to methyl substitution is taken into

438

GEROTHANASSIS,

LAUTERWEIN, TABLE

“0 CHEMICAL Compound I’ VII’ VIII III V VI’

SHIFTS OF DGLUCOSE AND METHYL DERIVATIVES

(VIII),

-CH20Hg

1-0Meb

9.6’

63.1

-10.6

-

6.3“ 1.3’

6.3’ -7.3”

52.6 50.5

-9.9 -7.3”

8.6” 9.6” -

8.6” 9.6”

50.4 55.4

10.8”

65.4

-11.4 -10.2 -10.7’

3-OH’

4-OHs

36.5 41.9

9.6’

9.6’

44.0’ 34.4 -50.5’

6.3k 1.3’ 8.6” 9.6” 10.gp

(VII), D-GALACTOSE (III, V, AND VI)b

-0-h

2-OH*

31.1 48.9

1

(I), D-MANNOSE OF D-GLUCOSE

1-OHd

-

AND SHEPPARD

-

3-OMeb

-

8.6” 9.6”

-

-

-10.7’

’ In parts per million relative to the resonance of external 1,Cdioxane. The chemical shift of dioxane relative to water is +2.1 ppm at 80°C. b 1.5 to 3 M aqueous solutions at 8O’C. ’ At equilibrium both (Y and /3 anomers are present (18). d Estimated error is +l ppm. ’ o and @anomers overlap to give a single resonance. ‘Resonance is overlapped by that of the ring oxygen. r Estimated error is f0.5 ppm. * Estimated error is +2 ppm attributable to the breadth of the resonances on a strong rolling baseline. ‘-’ Same letter indicates overlapping resonances.

account (see Discussion). Finally, this leaves the resonances at 36.5 and 47.9 ppm which are observable in ordinary water (Fig. 2), but disappear rapidly in “Odepleted water at high temperatures (Fig. 3). We assign these to the hemiacetal hydroxyl group at the carbon in the 1 position. Two resonances will occur since in aqueous solution the a-D-glucose ring opens and reforms by mutarotation (18) to give an important proportion of the anomeric configuration P-D-glucose (Fig. 5). At equilibrium at 40°C a 64:36 ratio of the /3 to a form is reported (19) which remains practically unchanged on going to 80°C as examined by j3C NMR. The a! and fl forms of the anomeric hydroxyl oxygen apparently occur in a characteristic chemical shift range as the I-OH resonance in 2,3,4,6-tetra-O-methyl-a-D-mannose-l-“0 (IV) was also observed at 40 ppm (ZZ). Figure 5 shows that the freealdehyde form, which is an intermediate in the equilibrium of the anomeric forms of D-glucose, can undergo reversible hydrolysis (29, 20) and thus exchange with “O-depleted water. The equilibrium of hydrate formation in monosaccharides depends both on steric and electronic factors (21). When exchange with “O-depleted water has taken place only negligible proportions of “0 will remain at the anomeric hydroxyl oxygens and as a consequence, the corresponding resonances will be very weak, if not beyond experimental observation (Fig. 3). Methyl

Derivatives of D-Glucose

Figure 6A shows the spectrum of methyl ar+-glucopyranoside (III) in 170-depleted water. The general absorption pattern is very similar to that in Fig. 3 and

OXYGEN-17

C

l-OH

. ..,;-:I----

-oA

------Y 1..

‘1.

100

‘.

50

439

NMR OF MONOSACCHARIDES

1’.

0

“I“

-50

PPM

FIG. 7. “0 NMR spectrum of D-mannose (VII) at 80°C. (A) in ordinary water (*). Tsq = 5 msec, NS = 550,000, total experimental time 47 min (LB = -300 Hz, GB = 0.3; upper trace: LB = -800 Hz, GE = 0.35). (B) in “O-depleted water 0. T.,, = 7.5 msec, NS = 1,400,000, total experimental time 2.9 hr (LB = -150 Hz, GB = 0.3; upper trace: LB = -800 Hz, GB = 0.2).

we assigned the ether and alcohol oxygens as described before for D-glucose. The ring oxygen absorption at 50.4 ppm agreed well with the value of 48 ppm reported for methyl cu-D-glucopyranoside-5-“0 (12). No separate resonance was resolved for the O-methyl group; however, from the increased intensity of the absorption at 8.6 ppm one may infer that this group is overlapping with the hydroxyl groups in the 2, 3, and 4 positions. A similar spectrum was obtained for methyl P-Dglucopyranoside (V). Again, the O-methyl resonance could not be resolved but was tentatively assigned by comparison of the relative signal intensities. Table 1 lists the assignments and chemical shift data. Figure 6B shows the spectrum of 3-O-methyl-D-glucose (VI) in “O-depleted water. The absorption pattern at high frequencies is similar to that of D-glucose in ordinary water (Fig. 2B). Obviously, in this case the a! and @ anomeric hydroxyl groups have not undergone complete exchange with water. The 3-O-methyl resonance is assumed to overlap with the resonance of the primary alcohol group at -10.7 ppm since the intensity of the latter absorption is strongly enhanced (Fig. 6). o-Mannose

and D-Galactose

Figure 7 shows the spectrum of D-mannose (VII) in ordinary and “O-depleted water. In ordinary water (Fig. 7A) the resonances from the ring and anomeric hydroxyl oxygens are not resolved but appear to be superposed to give an asym-

440

GEROTHANASSIS,

LAUTERWEIN,

AND SHEPPARD

B

100

50

0

-50

PPM

FIG. 8. “0 NMR spectrum of D-galactose (VIII) at 8O’C. (A) in ordinary water (*). Taes= 5 msec, NS = 600,000, total experimental time 51 min (LB = -300 Hz, GB = 0.25). (B) in “O-depleted water 0. Tscq= 5 msec, NS = 450,000, total experimental time approximately 39 min (LB = -300 Hz, GB = 0.3).

metrically shaped absorption band. Application of a strong double-exponential Gaussian function confirmed the presence of at least two resonances. If we tentatively assign the line at highest frequency to the ring oxygen, then the intense line is to be attributed to the overlapping resonances from the (Y and @ anomeric oxygens. This assignment is confirmed by the spectrum in “O-depleted water (Fig. 7B), where the intensity of the anomeric oxygen resonances has considerably decreased resulting from solvent exchange (Fig. 5). In Fig. 7B two absorption lines at low frequencies are also apparent which in analogy to D-glucose can probably be assigned to the resonances from the 2,3,4,-OH groups (6.3 ppm) and the CH20H group (-9.9 ppm) (Table 1). The spectrum of D-galactose (VIII) in ordinary water (Fig. 8A) shows tivo resonances at high frequency, one of them (34.4 ppm) being considerably less intense. It is therefore most probable that the latter resonance is one of the two expected anomeric hydroxyl oxygens, the other being overlapped by the ring oxygen absorption at 50.5 ppm (cu and /3 anomers of VIII are distributed 27:73 at 40°C (19)). The spectrum of VIII at low frequency in “O-depleted water is peculiar in that the absorption at -7.3 ppm has strongly increased in intensity relative to that of the combined line from the secondary OH groups. It seems therefore that in Dgalactose one of the latter resonances is shifted to lower frequency relative to Dglucose and D-mannose to give an overlapping signal with that of the CHIOH group (see Discussion).

OXYGEN-17

Application

441

NMR OF MONOSACCHARIDES

of Shift Reagents

In spite of the effective use of “O-depleted water in reducing the intensity of the solvent water resonance, the residual peak may still overlap some sugar resonances. We therefore explored the use of Co’+ (22) and Dy3+ (26, 23, 24) as “0 shift reagents. The paramagnetic metal ions induce characteristic chemical shifts for both sugar and solvent oxygen resonances (Table 2). The “0 spectrum at a molar ratio of CoClz to D-glucose of 0.042 is shown in Fig. 9. The Co2+ ion shifts all resonances in the same direction, with magnitudes of the order H20 % CH20H > CHOH > ring 0. The chemical shift pattern of D-glucose probably reflects the degree of steric accessibility of Co’+ to the lone pairs of the oxygen atoms. Because of the high-frequency shift and the greater sensitivity of the water molecules to Co’+ an overlap of the water, and the secondary and ring oxygen resonances of Dglucose was observed at cobalt to glucose molar ratios < 0.04. It is also a disadvantage that all oxygen resonances broaden through their interaction with Co*+. However, because the effect is largest for the water resonance, a negligible truncation of the FID was obtained (Fig. 9A). TABLE EFFECT

OF PARAMAGNETIC

Salt

COCI,

DyCl,. 6H20

Metal/glucose molar ratio

SHIFT

REAGENTS

Chemical

shift*

2

ON THE “0

I-OH

CHEMICAL

SHIFTS

OF D-GLUCOSE

2,3,4-OH

-0-

-CH,OH

36.5 47.9

9.6

63.7

-10.6

(I)

H,O

-0.6

0.028

6,

39.4 50.8

13.8

c

1.9

68.9

0.042

62

40.6 53.0

17.3

65.9

8.3

108.0

(arscd

4.1 5.1

7.7

2.1

18.9

108.6

(6,~6,)

7.6 8.6

11.6

5.6

22.4

112.1

0.033

6,.

44.3 56.9

17.6

12.2

0.060

62

53.4 66.9

21.4

81.8

6.3

-36.4

(a,.&)

16.9 19.0

17.8

18.1

16.9

-35.8

1.5 3.6

2.4

2.7

1.5

-51.2

’ 3 M aqueous solution at 80°C. b In parts per million relative to the ’ After correction for magnetic bulk d After correction for magnetic bulk ’ Resonance is overlapped by that of

resonance of external 1,Cdioxane. susceptibility (+3.5 ppm). susceptibility (- 15.4 ppm). the water.

-14.6

442

GEROTHANASSIS,

LAUTERWEIN,

AND SHEPPARD

B

200

150

100

50

0

-50

PPM

FIG. 9. “0 NMR spectrum of a 3 M solution of glucose (I) in ordinary water at 90°C containing 0.13 Au CoCl*. T.cq = 6 msec, NS = 250,000, total experimental time 25 min. (A) normal spectrum. (B) after multiplication of the FID with a double-exponential Gaussian function (LB = -750 Hz, GE = 0.3). The chemical shift scale is represented after bulk susceptibility correction (see Table 2).

The use of Dy3+ proved advantageous for several reasons (Fig. 10). The water resonance is moved to low frequency, whereas the resonances of D-glucose are shifted to higher frequencies by approximately equal amounts. Indeed, one can show that after correction for the bulk magnetic susceptibility (see Experimental) the chemical shifts of the glucose oxygens are negligible (Table 2). Therefore, the water resonance can readily be shifted by Dy3+ without overlapping the noninteracting resonances from D-glucose. In addition, Dy3+ does not lead to appreciable broadening of the resonances (Fig. 10). DISCUSSION

From the chemical shift data of specifically “O-labeled monosaccharide derivatives (12) the “0 spectrum at natural abundance of glucose was proposed to consist of two signals, one at -6 to 6 ppm corresponding to the oxygen nuclei at the 2, 3, 4, and 6 positions and the other at 40 to 48 ppm corresponding to those at the 1 and 5 positions. Although this prediction is essentially confirmed by the present work, we have obtained more refined “high-resolution” spectra by applying

OXYGEN-17

NMR

OF

443

MONOSACCHARIDES

high magnetic fields, “O-depleted water, and sophisticated data treatment. As seen in Figs. 2 and 3, altogether five signals from D-ghCOSe could be resolved, including the two signals from the (Y and /3 anomeric hydroxyl oxygens. Although this may still appear meagre in terms of spectral resolution when compared to ‘H and 13C NMR, the reasonably short experimental time and the valuable information contained in the “0 chemical shift and linewidth data lead us to predict a more intensive future application of “0 NMR to carbohydrates and other intermediatesized molecules. I70 Chemical Shifts The data of Table 1 can be summarized to give the following chemical shift pattern for the various oxygen containing functional groups: primary OH’s at C(6) between -7 and -12 ppm; secondary OH’s at C(2), C(3), and C(4) between 6 and 11 ppm (except for D-galactose, see below); secondary OH’s at C( 1) at approximately 36 and 47 ppm, depending on the (Yor /3 anomeric configuration; ring oxygens between 50 and 66 ppm; methoxy groups shifted by 20 to 35 ppm toward low frequencies relative to the corresponding hydroxyl groups. In view of the small number of sugar derivatives studied so far, the above chemical shift ranges can be only preliminary and will have to be extended to accommodate further examples. 2,3,kOH

__ rI’. 100 FIG. 10. “0

.

.

I“. 50

.

1.. 0

“I‘

” -50

PPM

NMR spectrum of a 3 M solution of ~-glucose. (I) in ordinary water at 80°C containing 0.18 A4 DyCI,* 6 H20. T.es = 5 msec, NS = 680,000, total experimental time 57 min (LB = -300 Hz, GB = 0.3; upper trace: LB = -600 Hz, GB = 0.3). The chemical shift scale is represented after bulk susceptibility correction (see Table 2).

444

GEROTHANASSIS,

LAUTERWEIN,

AND

SHEPPARD

The assignments in Table 1 were made essentially with the help of specifically “O-enriched monosaccharides derivatives (I 2). However, the same assignments could have been made as well in an independent manner on the basis of the “0 chemical shifts of simple alcohols and ethers (8, 9) which increase from primary alcohols (-7 to 10 ppm) to secondary alcohols (30 to 40 ppm) to substituted ether groups (64 ppm for diisopropyl ether (10)). This sequence of chemical shift ranges is in accordance with the 8 deshielding effect attributable to alkyl branching (2, 3, 8). In contrast, replacing the hydrogen directly bonded to oxygen by a methyl group (III, V, VI) induces a shielding of this oxygen (Table l), and this is in agreement with earlier results (3, 12). It is also noted that methylation of the anomeric oxygen (III, V) produces a shielding of the ring oxygen by 8 to 13 ppm. As expected this does not occur for methylation of the oxygen in the 3 position (VI). The secondary hydroxyl at C( 1) exists in both the (Y and p anomeric configurations in aqueous solution. It is distinguished from the other hydroxyls by exchange with water (Fig. 4). Therefore, in “O-depleted water the observed decrease in signal intensity (Figs. 3,6B, 7B) leads to an unequivocal assignment of the anomeric hydroxyl resonances. The a! and j3 anomers could not be assigned individually since the preponderance of either anomeric form of I and VI (the only cases where two individual resonances were detected) was insufficient to be visualized from the “0 signal intensities. The simple alcohols and ethers provide only an approximate guide to the chemical shifts of the same functional groups in monosaccharides. Obviously, oxygens separated by several bonds may strongly influence each other if they are spatially close. The exceptionally low chemical shift of one of the secondary OH groups in D-galactose (Table 1) is probably attributable to the 4-OH group which is cis with respect to both adjacent OH and CHIOH substituents. A similar low chemical shift (-8 ppm) was found (12) for the 3-OH group in methyl @-D-allopyranoside3-“0 (IX). Thus, “0 chemical shifts, as for 13C (25), appear extremely sensitive to molecular geometry. This can be rationalized in terms of steric or electrostatic repulsion or hydrogen-bonding interactions between pairs of adjacent cis- or transOH ring substituents. Since the anomeric hydroxyl oxygen and the ring oxygen are separated by only one carbon atom additional mutual interactions are expected through lone-pair repulsions. They appear to be responsible for the large deshielding of the I-OH group and its sensitivity to epimerisation (Table 1). It is noted that gem-diols also give high chemical shifts of approximately 60 ppm (26). “0 Linewidths

The high-resolution “0 NMR spectra were normally obtained after treatment of the FID by a double-exponential Gaussian function (15). As a result the true linewidths cannot be read off directly from the modified spectra. Nevertheless, approximations about relative linewidths are possible (a computer program for quantitative analysis is in preparation). In all the, carbohydrates studied the resonance of the ring oxygen was considerably broader than that of the primary

OXYGEN-17

NMR

OF

MONOSACCHARIDES

445

hydroxyl group or even the composite signal of the 2, 3, and 4-OH groups. This is what would be expected from the rates of reorientation of the various field gradient axes which induce quadrupolar relaxation (3). They depend on the degrees of rotational freedom which are greatest for the CH20H group with internal rotation axes about C-O and C-C bonds, intermediate for the CHOH groups with rotation about the C-O bond, and least for the more rigidly held ring oxygen. CONCLUSION

From the results reported in this paper it is clear that despite earlier doubts, good quality “0 NMR spectra of monosaccharides in natural abundance can be obtained within reasonable experimental time for molar concentrations in aqueous solution at 80°C. This necessitates the combined application of (a) a high-field superconducting spectrometer, (b) fast pulsing and a double-exponential Gaussian apodization function, together with (c) high-quality 170-depleted water and/or (d) lanthanide shift reagents (Dy3’). On the other hand, it is also recognized that one of the drawbacks of the presently available techniques is the high temperatures needed which limits “0 NMR to stable carbohydrates and makes applications to biochemical problems difficult. ACKNOWLEDGMENTS

The authors thank Dr. H. P. Kellerhals, Spectrospin A. G. for technical help on the WH-400; Drs. 0. Howarth and E. H. Curzon for assistance obtaining the initial “0 spectra on the WP-400 at the University of Warwick; Drs. A. H. Haines and R. Hunston for helpful discussions; the Greek Scholarship Foundation for a grant to one of us (I.P.G.); and the Swiss National Science Foundation for financial support to another (J.L.). REFERENCES I. W. G. KLEMPERER, Angew. Chem. Int. Ed. 17, 246 (1978). 2. C. RODGER AND N. SHEPPARD, in “NMR and the Periodic Table” (R. K. Harris and B. I. Mann, Eds.), pp. 383-400, Academic Press, New York/London, 1978. 3. J. P. KINTZINGER, in “NMR Basic Principles and Progress” (P. Diehl, E. Fluck, and R. Kosfeld, Eds.), Vol. 17, Springer-Verlag, Berlin, 1981. 4. R. K. HARRIS in “NMR and the Periodic Table” (R. K. Harris and B. I. Mann, Eds.), pp. 6-7, Academic Press, New York/London, 1978. 5. C. P. CHENGANDT. L. BROWN, J. Am. Chem. Sot. 101, 2327 (1979); 102, 6418 (1980). 6. D. CANET, C. COULSON-GINET, AND J. P. MARCHAL, J. Magn. Reson. 22, 537 (1976). 7. I. P. GEROTHANASSIS, PH.D. thesis, University of East Anglia, Norwich, England, 1980. 8. H. A. CHRIST, P. DIEHL, H. R. SCHNEIDER, AND H. DAHN, Helv. Chim. Acta 44, 865 (1961). 9. T. SUGAWARA, Y. KAWADA AND H. IWAMURA, Chem. Left., 1371 (1978). 10. T. SUGAWARA, Y. KAWADA, M. KATOH, AND H. IWAMURA, Bull. Chem. Sot. Jpn. 52, 3391 (1979). 11.

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