J . Org. Chem. 1993,58, 5513-5517
6613
( l‘-13C)-2‘-Deoxyribonucleosides:Structural and Conformational
Insights Derived from 13C-lH Spin Coupling Constants Involving C1‘ Tapasree Bandyopadhyay, Jian Wu, and Anthony S. Serianni’ Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556 Received March 9, 1993 (Revised Manuscript Received May 17,1993)
2’-Deoxyadenosine (l),%’-deoxycytidine(2), and thymidine (3)have been prepared with Wenrichment at C1’ (99 atom% 13C) and studied by lH NMR spectroscopy at 500 MHz in 2Hz0. VCH, VCH and 3&H values were measured between C1’ and several protons in the furanose and base moieties of 1-3 and compared to related values observed in corresponding ribonucleosides and erythronucleosides. Results are consistent with the expected shift to south conformers on conversion of ribonucleosides to 2’-deoxyribonucleosides. The observation that C1’ of 1-3 couples more strongly to H2’S (-5.7 Hz) than to H2’R (10.4Hz) has been explained using model compounds that mimic the C1’42’H2’R and Cl’-C2’-H2’S coupling pathways in pure north and south conformers of 1-3. Results may be a useful probe of N/S equilibria in 2’suggest that the difference, 1z&1t,H2jSl - l2Jci~,~y~1, deoxyribonucleosides in 2Hz0 solution. Model compounds have also been used to probe the effect of ring conformation on the chemical shifts of H2’R and H2’S in 1-3. For simple, unphosphorylated 2-deoxy-~-~-ribofuranosyl rings in aqueous solution, the difference, 6 ~ - y~ H Z~S may , be correlated with N/S distribution.
Introduction In recent years stable isotopes have played a key role in the development of NMR spectroscopy as an integral tool to assess the conformations of macromolecules and their complexes in solution. For example, the combined use of 1%- and/or lSN-1abeled proteins with multidimensional NMR methods has assisted in the collection and interpretation of otherwise complex spectra by providing enhanced sensitivityand/or spectral editing features.’ The binding of small substrates to protein receptors has been studied by labeling either partner, usually uniformly with 13C, to permit, via several approaches, the discrimination between the signals of the substrate and those of the receptor.2a-fWhile these applications are now commonly appreciated in the study of protein structure, related approaches to the study of nucleic acids have not been as rigorously developed, although a few studies of uniformly 1%-labeled RNA and DNA have appeared recently.%-’ The paucity of applications in these latter systems likely results from the unavailability of 13C- and ‘SN-labeled nucleosides, and a limited knowledge of W-lH and l3Cl3C spin coupling constants in these biomolecules. This latter information is critical to the proper implementation of many multipulse NMR methods used for sensitivity enhancement and/or spectral editing. Nucleicacids are homophosphofuranose polysaccharides composed of conformationallyflexiblemonomers (e.g., &Dribofuranose 5-phosphate, 2-deoxy-b-~-erythro-pentofuranose5-phosphate)that are substituted at the anomeric carbon with nitrogen-containing heterocycles. Thus, Author to whom correspondence should be addressed. (1) (a) Kay, L. E.; Clore, G. M.; Bax, A.; Gronenbom, A. M. Science 1990,249,411. (b)Zuiderweg, E. R. P.; Fesik, 5.W. Biochemistry 1989, 28,2387. (2) (a) Fesik, 9. W.; Luly, J. R.; Erickeon, J. W.; Abad-Zaptero, C. Biochemistry 1988,27,8297. (b) Wider, G.; Weber, C.; WQthrich,K. J. Am. Chem. SOC.1991,113,4676. (c) Fesik, S.W. Nature 1988,332,865. (d) G.;W M c h , K. Q. Rev. Biophys. 1990,23,39. (e) h e n , M. K.; M i h c k , S. W.; Wandleae,T. J.; Schreiber,S. L. J. Org. Chem. 1991, 66,6262. (0Fesik, 5.W. J. Med. Chem. 1991,34,2937. (9) Michnicka, M. J.; Harper, J. W.; King, 0. C. Biochemistry 1993, 32, 395. (h) Nikonowia, E. P.; Pardi, A. J. Am. Chem. SOC.1992, 114, 1082. (i) Nikonowicz, E. P.; Pardi, A. Nature 1992,355,184.
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0022-3263/93/1958-5513$04.00/0
studies of 13C-lH spin couplingswithin the furanosylrings of these polymers are logicalextensionsof previous studies of carbohydrate systems in general? and furanose rings in ~articular.~ Using methods described previously,&&three 2‘-deoxyribonucleosides (2’-deoxyadenosine (1),2’-deoxycytidine (2), thymidine (3)(Scheme I) have been prepared with ‘SC-enrichment at Cl’, and W-lH spin couplings involving C1’ have been examined within the furanosyl ring and across the N-glycoside linkage. These couplings have been compared to corresponding couplings in the ribonucleosidess* and erythronucleosides6bin order to validate their utility as conformationalprobes. The effect of furanose ring conformation on the chemical shifts of the C2 protons of 2-deoxyaldofuranosyl ringshas also been examined with the use of model compounds.
Experimental Section Materials. 2’-Deoxyadenosine, 2’-deoxycytidine,thymidine, 2-deoxy-D-glucose (2-deoxy-~-arabho-hexose)and 2-deoxy-Dallose (2-deoxy-~-ribo-hexoe)were purchased from Sigma Chemical Co. [l’-W]-2’-Deoxyadenosine,[l’-W]-2’-deoxycy-
tidine, [1’-1SC]thymidine, and [l’-lgClribothymidine(99atom%) (3) (a) Bock, K.; Pedersen, C. Acta Chem. Scand. 1977, B31,354. (b) Schwarcz, J. A.; Cyr, N.; Perlin, A. S. Can. J. Chem. 1976,53, 1872. (c) Cyr, N.; Hamer, G. K.; Perlin, A. S. Can. J. Chem. 1978,56, 297. (d) Schwarn, J. A.; Perlin, A. 5.Can. J. Chem. 1972,50,3667. (e) Bock, K.; Lundt, I.; Pedersen, C. Tetrahedron Lett. 1973, 1037. (0Bock, K.; Pedersen, C. J. Chem. SOC.Perkins Trans. 2 1974, 293. (B) Bock, K.; Pedersen, C. Acta Chem. Scand. 1975, B29,258. (4) (a) Cyr, N.; Perlin,A. S. Can. J. Chem. 1979,57,2504. (b)Serianni, A. S.;Barker, R. J. Org.Chem. 1984,49,3292. (c) Snyder, J. R.; Serianni, A. S. J. Org. Chem. 1986, 51, 2694. (d) Vuorinen, T.; Serianni, A. S. Carbohydr.Res. 1990,209,13. (e) Snyder,J.R.; Serianni,A. 5.Carbohydr. Res. 1987, 163, 169. (5)(a) Kline, P. C.; Serianni, A. S. J. Am. Chem.SOC.1990,112,7373. (b)Kline,P. C.;Serianni,A. S.J. Org.Chem. 1992,57,1772. (c)Crystalline labeled nucleosideswere analyzed by high-resolution maee spectrometry (Finnigan-MAT 8430),giving the following results: [l’-~sC]-O’-deo.yadenosine, Cld-IlaOaNs, m/e 252.1052 calcd, 252.1070 found; [l’-’sCI-2’m/e 228.0940 calcd, 228.0935 found; I1’deoxycytidine, C&~O&, lsClthymidine, C$Ilfi&., m/e 243.0936 calcd, 243.0942 found; [l’Wlribothymidine, C1&IIsJ~O~, m/e 259.0885 calcd,259.0876 found. (6) (a) Kline, P. C.; Serianni,A. S. Magn. Reson. Chem.1990,28,324. (b) Austin, P. W.; Hardy, F. E.; Buchanan, J. C.; Baddiley, J. J. Chem. SOC.1963, 5360. (c) Hodge, J. E.; Hofreiter, B. T. Meth. Carbohydr. Chem. 1962, I , 380. (d) Bock, K.; Pedersen, C. Ado. Carbohydr. Chem. Biochem. 1983,41, 27-66.
0 1993 American Chemical Society
6614 J. Org. Chem., Vol. 58, No. 20, 1993
Bandyopadhyay et al.
Scheme I
Table I. 1H-lH and W-lH Spin Coupling Constants. in
HO.
k5hHSR
s
H
s S H2
HO
2 1 ~B-adenine B-cytosine ~
~
~
3 B-thymine
HPR
were prepared as described previously.kcL Deuterium oxide PH20, 99 atom% 2H) was obtained from Cambridge Isotope Laboratories. The methyl a- and @-pyranosidesof 2-deoxy-D-glucoae (2deoxy-D-arabino-hexose)were prepared by Fischer glycosidation. 2-Deoxy-D-glucose(0.5 g) was dissolved in anhydrous methanol (30 mL), Dowex HCR-W2 (H+)ion-exchange resin (1.0 g) was added, and the mixture was refluxed for 24 h. After cooling and removal of the resin by filtration, the glycoside mixture was chromatographed on a 70 X 3.5 cm column containing Dowex 1x2 (200-400 mesh) resin in the hydroxide form,Bbusingdistilled water as the solvent. Fractions (20 mL) were collected and assayed with phenol-sulfuric acid.& The a-pyranoside eluted before the @-pyranoside,at elution volumes of -280 mL and -400 mL, respectively. The pyranosides were identifiedby their characteristic lSC chemical shifta.“ Instrumentation. High-resolution lH NMR spectra were obtained at 500 MHz. Probe temperature was regulated at 30 OC, and sample solutions (0.6 mL, 10-20 mM in 2H20)were analyzed in 5-mm NMR tubes. Spectra were obtained with sufficient digital resolution to permit the use of resolutionenhancement to improve the detection of small couplings. Computer simulation of 500-MHz 1H NMR spectra was performed using the LAOCN5 program as implemented in the FTNMR program (VAX version) available from Hare Research, Inc. of Woodinville, WA. The computationswere conducted on a Digital VaxStation 3200 minicomputer equipped with a Tektronix CX4107 graphics terminal. Results
coupled nuclei*
Hl‘,H2’ Hl’,H2” H2‘, H2” H2’, H3’ H2”, H3’ H3’, H4’ H4’,H5’ H4’,H5” H5‘, H5” H5,H 6 Hl‘,H3’ Hl’,H 5 H6,CHd
2’-Deoxyribonuclsosider 1-8 compound 1 2 7.7 -6.5 6.3 -6.7 -14.1 -14.2 6.1 4.1 3.3 -6.7 -3.1 -4.0 3.3 3.6 4.3 5.3 -12.7 -12.5 7.6 0.6 0.4
30
6.8 6.7 -14.2 4.1 6.7 3.9 3.9 5.2 -12.5 -0.5
1.3 170.8 (170.3) 170.1 (169.0) 0 -0 5.7‘ -5.7e Cl’,H3’ 4.5 (3.0) 4.6 (3.7) Cl’,H4’ 2.4 (0.6) 2.2 (
