10454 J. Phys. Chem. 1995, 99, zyxwvu 10454-10458 15N Chemical Shift Tensors of Uracil Determined from 15N Powder Pattern and lSN-l3C Dipolar NMR Spectroscopy Karen L. Anderson-Altmann,+Cu G. Phung, Stylianos Mavromoustakos, Zhiwen Zheng, Julio C. Facelli,' C. Dale Poulter, and David M. Grant* Department of Chemistry and Utah Supercomputing Institute, University of Utah, Salt Lake City, Utah 841 12 Received: July 12, 1994; In Final Form: February zyxwvu 27, 1995@ The "N chemical shift tensors of uracil are reported using I5N powder pattem techniques. The principal values of the I5N uracil tensors are obtained from the spectra of [l-'5N]uracil and [3-'jN]uracil, and the tensor orientations are determined from the spectrum of zyxwvu [ 1,3-'5N2,2-'3C]~ra~i1 by including the effects of the direct dipolar interaction in the spectral fitting routine. Ambiguities in the orientational assignments, which arise from the axial symmetry of the direct dipolar tensor, are resolved using molecular symmetry considerations and results of ab initio calculations of I5N chemical shielding tensors. The NI nitrogen has principal values of 196, 114, and 30 ppm and the N3 nitrogen 200, 131, and 79 ppm with respect to I5NH4N03. Assuming that the smallest (most shielded) chemical shift tensor components are oriented perpendicular to the molecular plane, the largest components are found to lie 18" and 9" off the NI-H and N3-H bonds, respectively, rotated toward CZ and Cq. These orientations are in good agreement with those calculated theoretically. In addition, inclusion of intermolecular hydrogen bond effects in the theoretical calculations significantly improves the correlation between the calculated and experimental principal values. Introduction Uracil is one of the four heterocyclic bases found as subunits of the nucleotides in RNA. The hydrogen bonding between uracil and adenine is important in the synthesis of mRNA from DNA and in the translation of the genetic code via tRNA. Guanine-uracil hydrogen-bonded base pairs are involved in the tertiary structure of tRNA and in codon-anticodon interactions. Nuclear magnetic resonance is a very useful tool for the study of hydrogen bonding, since the chemical shift of a magnetically active nucleus is sensitive to the surrounding electronic environ- ment. This is particularly true of the I5N nucleus, due to the lone pair of electrons. which provides a broader range of bonding possibilities and thus a greater sensitivity of chemical shielding to changes in electronic characteristics. Nitrogen nuclei play an active role in the hydrogen bonding of all of the base pairs. so I5N NMR should provide useful information about the extent of hydrogen bonding in these systems. The effects of A:U base pairing on the I5N chemical shifts of each species have been investigated in solution,'-3 where significant changes have been observed upon pair formation, even for the N! nucleus of adenine, which is not located at an interaction site. Using the technique of 'H-I5N multiple- quantum NMR, the A:U and G:U pairs in Escherichia coli zyxwvut 5s RNA have been identified by selectively labeling the N3 position of the uridines.' Information on the anisotropy of the chemical shielding interaction has the potential of providing an even better understanding of the electronic environment around a nucleus and how it is affected by hydrogen-bonding interactions, particularly if the orientation of the principal values of the chemical shift tensor can be determined. One way to obtain such orientational information is through the combined dipolar- chemical shift experiment,5.h which relies on the magnetic coupling of the nucleus of interest to another nucleus possessing ~ Current address: Chemiitry and Materials Branch. Code 474230D. Naval Air Warfare Center Weapons Division, China Lake. CA 93555. ' Abtract published in Adwince ACS Ah.rrructs, May 15. 1995. Utah Supercomputing Institute. 0022-365419512099- zyxwvuts 1 0454$09.0010 a spin. The resulting dipolar-chemical shift interaction affects the observed powder pattern. In order to understand the spectra of interacting base pairs, one must first obtain and interpret the spectra of the isolated, nonpairing molecules. In this paper, the I5N chemical shift tensors, including principal values and orientations, of the two nitrogens in uracil are reported. The only other complete determinations of I5N chemical shift tensors of aromatic nitrogens are for L-histidine hydrochloride monohyrate' and the tryptophan residue of fd bacteriophage,8 whereas the principal values alone have been determined for pyridine9 and histidine.I0 While previously calculated,' I this is the first experimental measurement of the I5N chemical shift anisotropies of ring nitrogens in any of the heterocyclic bases. Thus, this work provides a model for I5N chemical shift tensors in these types of systems. Experimental and Computational Methods Materials. Propiolic acid and polyphosphoric acid (PPA) were purchased from Aldrich Chemical Co. Unlabeled urea was purchased from Sigma Chemical Co. ['3C,'SNz]urea (99%) was purchased from Cambridge Isotope Laboratories. [ 1 -I5N]- uracil and [3-I5N]uracil,prepared using the procedure of Roberts and Poulter,'? were available from earlier studies in the Poulter laboratory. Synthesis of [ 1,3-15N,2-13C]Uracil. The procedure used was based on that originally described by Harada and Suzuk~.'~ In a 100 mL round-bottomed flask a mixture of 0.50 g of [I3C,l5N2]- urea (7.93 mol), 0.58 g of propiolic acid (8.28 mol), and 12 g of polyphosphoric acid was heated at 85 "C for 4 h with occasional stirring. The mixture was cooled in an ice-water bath, and water (24 mL) was added gradually as a precipitate formed. The mixture was maintained at approximately 5 "C for 24 h. The precipitate was collected by filtration and placed under high vacuum (0.01 " H g ) over P205 to give 0.73 g (80% yield) of a white solid. 'H, I3C, and "P NMR spectra showed the presence of a single product. 0 1995 American Chemical Society