Anomeric configuration of nucleosides by application of the internal Nuclear Overhauser Effect

Vol. 48, No. 6, 1972 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ANOMERIC CONFIGURATION OF NUCLEOSIDES BY APPLICATION OF THE INTERNAL NUCLEAR OVERHAUSER EFFECT

R.J. Cushley*, B.L. Blitzer' and S.R. Lipsky

*Section of Physical Sciences Division of Health Science Resources Yale University School of Medicine New Haven, Connecticut 06510

Received July 31, 1972

Summary: -- Application of the internal Nuclear Overhauser Effect has been used to determine the anomeric configuration of purine and pyrimidine nucleosides. Irradiation of H4' of a-nucleosides produced an enhancement of the proton signals of the aglycone while irradiation of the 5'-protons did not. In the case of the B-nucleosides, irradiation of the 5'-protons produced an enhancement of the aglycone protons while irradiation of H4' produced no enhancement. Implications of these findings are discussed.

Because of the significance of nucleosides as building blocks of nucleic

acids, new information on the conformations of these molecules should prove of-

value. We wish to report o'z findings on the application of the internal

Nuclear Overhauser Effect (NOE)' to the determination of the anomeric con-

figuration of pyrimidine nucleosides. Previously, Cushley et al. 2,3 -- exploited

the anisotropy resulting from the 5,6-double bond of the aglycone of acetylated c

pyrimidine nucleosides to determine the anomeric configuration. Upon hydrogen-

ation of the 5,6-double bond a diamagnetic shift of the C2' acetoxy signal

was observed for pentofuranosyl pyrimidine nucleosides having a trans-Cl'- --

C2' relationship, and a paramagnetic (downfield) shift was observed for L

pentofuranosyl pyrimidine nucleosides having a cis-Cl'-C2' relationship.

Although the method requires but one conformer, it entails acetylation and

hydrogenation of the nucleoside. In addition, the method is not generally

applicable to purine nucleosides since selective hydrogenation of the pJrine

moiety is not possible, However, studies on model systems4 have shown the

technique might be extended to the purine series. These studies required

---

* Undergraduate Summer Fellow, 1969

Copyright 0 1972 by Academic Press, Inc. All rights of reproduction in any form reserved.

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a substantial population in a preferred anti" conformation wherein the 5,6-

double bond of pyrimidine or 28 of purine "sits over" the S-membered sugar ring

although it was clearly stated ".....pure conformers are not required". 3

Recently Hart and Davis6 have used internal NOE measurements to determine

the amount of 9 and anti conformer present in a number of nucleosides. ---

We, on the other hand, have used the internal NOE as a simple, effective

means of determining anomeric configuration of nucleosides. Only one anoner

is required and no chemical modification is necessary. The compounds chosen

for study include four my, B pairs, four 2'-deoxy sugars, 3 different pyrimidine

bases and 3 different purine bases.

Experimental: - ----- Pmr spectra were obtained using a Bruker HFX-3 high resolution

spectrometer operating at 21.5 kilogauss. Sources of the nucleosides are given

.I in Table I. Commercial samples were highest purity grade and used without

further purification. Samples were degassed using the alternate freeze-

pump-thaw technique to remove dissolved oxygen. Field frequency lock was

obtained on the references DSS (sodium-2,2-dimethyl-2-silapentane-5-sulfonate)

in H20, and TMS (tetramethylsilane) in DMSO-d6.

The Nuclear Overhauser Enhancements were determined from differences

in the integrated intensities of at least two sets of a minimum of 8 time-

averaged scans per set. While recording the first set, the appropriate

sugar proton was irradiated and the intensity of the aglycone proton measured.

Next, a control set was run with the decoupling frequency remaining on but

set downfield from the aglycone resonance signal a distance = VH sugar - H

Vaglycone from its value in the first set. This was to ensure enhancements

were due to NOE rather than the second rf. Finally, the second set of NOE values

* Donohue and Trueblood' have defined a torsion angle, $CN, between the Cl'-O-ring bond and the plane of the base with the projection along the Nl-C6 bond in pyrimidines and between the Cl'-O-ring bond and N9- C8 bond in purines. The anti conformation possesses an energy minimum in the range of -go to -65' when viewed along the Cl'-Nl bond and measured in a clockwise direction. The syn conformation will possess an energy minimum in the +126O to +180° range.

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was obtained in like manner to the first set. If the second set of enhance-

ments deviated from the first, both values were included in Table I.

Averaging of each set of spectra was obtained by use of a Fabri-Tek

1064 Computer of Average Transients with 4096 data points. The SD-1

digitizer utilizes an input integrator for analog filtering. Such a filter

does not attenuate the incoming signal. Integration of the absorption signal

in the Fabri-Tek is calculated by:

X1=2-n ’ .e xi = 2-n (x0+x1----x..) 0

That is, in the integrate mode, the digital value in each memory

lo-ation equals the sum of the values in all previous locations. Therefore,

as long as the value n(n=O to 11) is the same for each run, integrals are

directly comparable. Although integrated intensities were accurate to l%,

NOE values were reproducible to only 4%. Therefore only values 14% can

be considered significant.

Power output for the decoupling frequency was determined to be 0.16-0.20

watts (50 fi ).

Results: __I- The integrated Overhauser enhancements are given in Table I. Each

value is the integrated intensity of at least 8 different scans (see experi-

mental). Examination of the Table clearly shows that irradiation at the

absorption frequency of H4' of the a-nucleosides II, IV, VI and VIII

produced a 9-16% enhancement of the H-6 protons of pyrimidines or H2 and/or

H8 of purines while irradiation of H5' produced no enhancement in the -

protons of the base. In all cases, the two 5'-protons were found to be

magnetically equivalent and were pseudodoublets.

With the compounds 1-(@-D-ribofuranosyl)-uracil(I), 1-6-D-2'-deoxyribo- = = furanosyl)-5-fluorouracil(III), 1-($-P-2'-deoxyribofuranosyl)-5-fluorocytidine

(V), 9-(B-D-2'-deoxy-2'-fluoroarabinofuranosyl)-adenine(VII), 9-(p-D-ribo- = = furanosyl)-hypoxanthine(IX) and 9-(3-z-2',3'-!J-isopropylidene)-guanine(X),

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Table I. NOE Enhancements in Nucleosides

Compound leference Solvent % enhancement H6 o f I ,yrimidines Cont.

b wt/vol HS' irradiated

5

0

6,7

-3

5

7,@

2

H4' irradiated

-.-a

13

0

9

1

15

15

15

15

10

20 16

of purines % enhancement HE,

H5' irradiated

I

H4' irradiated

H8 1 H2 H2

0

6

3-5C

j6-DM90

i6-DmO

Is-D&50

8

8

15

14

% enhancement HE of pwine

H5' irradiated H4' irradiated I

lod 2d x (8) e 16-DMSO

(a) NOE value not determined due to overlap of H2', H3' and H4'. (b) NOE values determined at 3dB more decmLpling power than nominal. (c) Total range of 4 sets of measurements. (d) Reference 6. (e) Cycle chemicals. (f) Wempen, I., Fox, J.J., in Methods in Enzymology, XIIA, edited by Gussman, L., and Moldive, K., Academic Press, N.Y., 1967, page 73. (g) Schwarz-Mann. (h) Hoffer, M. Duschinsky, R., Fox, J.J. and Yung, N.C., J. Amer. Chem. Sot., 81, 4112 (1959). (i) Wempen, I., Duschinsky, R., Kaplan, L. and Fox, Jx., J. Amer. Chem. Sot. 83, 4755 (1961). (j) Reference 10. Q Wright, J.A., Taylor, N.F., and Fox, J.J., J. Org. Chem., 34, 2632 (1969).

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R” R’

I; R = OH, R’= OH 10x0 form), R” = H

XC; R = H, R’=OH (oxoform), R”= F

Y; R = H, R’= NH2, R”= F

HO

If; R = OH, R’= OH (0x0 form), RI’= H

Ip; R= H, R’=OH(oxoform), RI’= F

XI; R = H, R’= NH2, RI’= F

R3

YE; R=OH, R’=R3=H, R2=F, R4=NH2 m; R=OH, R1=R3=H, R2=F, R4=NH2

1x ; R= R’=OH, R2=R3= H, R4= OH

X; R,R’= ‘“2 , ,R’=H, R3=NH2,R4=OH

H3C 0 CL

Legend to Figure 1. Structural formulas of the compounds used in this study. The conformations are shown in the anti form.

an enhancement of from 5% (compd I) to 17% (HB, compd IX) was observed when

H5' was irradiated with a second radiofrequency. in all cases but one

(compd IX) irradiation of H4' produced no enhancement. In the case of inosine,

IX, a small enhancement of H2 and H8 was found upon irradiation of H4'.

This small enhancement was no doubt due to partial irradiation of H2' or H3*

due to their proximity to H4' in the spectrum of IX since, when the decoupling

power was decreased by 5dB, H4' irradiation gave 0% enhancement for H2 and H8

while H5' irradiation showed 5% and 7% enhancements, respectively. Never-

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theless, the large intensity gains of 14-17% for H8 and 7-9% for H2, when H5'

was irradiated,clearly distinguish the 3- configuration by NOE. The sig-

nificant enhancement of H2 in compound IX when H5' is irradiated indicates a.

substantial amount of _syn (H2 above pyranose ring) as well as the anti isomer ---

present. This is in keeping with the predictions for purine nucleosides

of kinetic energy calculations. 5,7-g

On the other hand, there is an overwhelming body of evidence indicating

that the aglycone of pyrimidine nucleosides exists predominantly or exclusively

in the anti form. -- Thus, long-range proton-fluorine coupling in 15 5-fluoro-

pyrimidine nucleosides was interpreted in terms of a major population in the

anti conformation. 10 Similarly, circular dichroism studies on 38 pyrimidine

nucleosides, 11

chemical shifts of sugar protons due to anisotropy of the

2-carbonyl, 12 and carbon-13-proton coupling between Hl' and C2

13 have all

been interpreted in terms of only an anti conform.ation. --

On the other hand Hart and Davis 6,14 have stated that the anti confor-

mation for cytidine is unimportant since there was no observed interaction

between H5' and H6. Lack of interaction between H6 and HS' in cytidine is

at odds with the findings presented herein as a test of the anomeric configur-

ation.

Clearly, lack of an NOE is insufficient evidence to rule o-it an anti --

form since, due to critical dependence on r6 in the equatio!i5governing

dipole-dipole relaxation,3 , in a system of like spins j:

j.k= A. g 21, < q;> J

slight changes in the furanose conformation might twist C5' away from the aglycone

and an NOE might not occur even though the aglycone were anti. Recent evidence

suggests the furanose moiety exists as one of the following equilibria: C2'

endowC3' exo; C2' exoWC3' endo; C2' endo-C3' exofSC2' exo-C3' endo.16)17

Hart and Davis6y14 reported large NOE values on H6 for cytidine upon double

irradiation of H2' and H3'. These interactions can only occur when the two

sugar protons are syn to the aglycone as in a $-D configuration. -- =

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For those cases in which the H2', H3' and H4' chemical shifts are nearly

identical and selective decoupling of H4' is difficult, advantage may be taken

of the well established fact that 0-acetylation of sugar hydroxyls produces ~ -

a downfield shift of+O.Sppm in the directly at-tached ring proton (i.e. H2'

and H3'). Thus, we feel that the NOE method should prove of value for de-

termining the anomeric configuration of nucleosides.

Acknowled*nents: --- --- We wish to thank Dr. J.J. Fox for generous supply of some of the compounds used in this study. Work supported by grant RR 00356 from the Biotechnology Resources Branch of the National Institutes of Health.

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17.

References ----

The internal NOE phenomenon is succinctly described in the first communication on its application: Anet, F.A.L., Bourn, A.J.R., J. Amer. Chem. Sot., 87, 5250 (1955). Cushley, R.J., Watanabe, K.A. and Fox, J.J., Chem. Commun., 598 (1966). Cushley, R.J., Watanabe, K.A. and Fox, J.J., J. Amer. Chem. Sot., 89 394 (1967).

--'

Cushley, R.J., McMurray, W.J., Lipsky, S.R. and Fox, J.J., Chem. Commun., 1611 (1968). Donohue, J. and Trueblood, K.N., J. Mol. Biol., 2, 363 (1960). Hart, P.A. and Davis, J.P., J. Amer. Chem. Soc.,=z, 512 (1969); Biochem. Biophys, Res. Commun., 34, 733 (1969). Haschemeyer, A.E.V. and Rich, A., J. Mol. Biol., 27, 369 (1967). Lakshminarayanan, A.V. and Sasisekharan, V., Biochim. Biophys. A., 204, 49 (1970). -7- Wilson, H.R. and Rahman, A., J. Mol. Biol., 56, 129 (1971). Cushley, R.J., Wempen, I. and Fox, J.J., J. %ier. Chem. Sot., 90, 709 (1958). Miles, D.W., Townsend, L.B., Robbins, M.J., Robbins, R.K., Inskeep, W.H. and Eyring, H., ibid 93, 1603 (1971). Schweizer, M.P., Witozgki, ;S.T. and Robbins, R.K., ibid 93, 277 (1971). Lemieux, R.U., Nagabhushan, T.L. and Paul, B., Can. J. CEm., SO_, 773 (1972). Hart, P.A. and Davis, J.P., J. Amer. Chem. Sot., 2, 753 (1971). Abragam, A., The Principals of Nuclear Magnetism, Oxford University Press, London, 1951, Chapter VIII. key, A.A., Smith, I.C.P. and Hruska, F.E., J. Amer. Chem. Sot., 93, 1765 (1971). Dugas, H., Blackburn, B.J., Robbins, R.K., Deslauriera, R. and Smith, 1.C.P ibid 93, 3468 (1971). --

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Anomeric configuration of nucleosides by application of the internal Nuclear Overhauser Effect