226

Biochimica et Biophysica Acta, 418 (1976) 226--231

@)Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 98484

T H E C R Y S T A L AND M O L E C U L A R S T R U C T U R E OF AN OSMIUM BISPYRIDINE ADDUCT OF THYMINE

s. NEIDLE and D.I. STUART Department of Biophysics, King's College, 26--29 Drury Lane, London, WC2B 5RL (U.K.)

(Received July 25th, 1975)

Summary The bispyridine osmium adduct of t h y m i n e has been crystallised and subjected to an X-ray diffraction analysis. It crystallises in the triclinic space group P1, with cell dimensions a = 7.975(3), b = 10.381{3), c = 11.036(3) A, a = 82.73(2) °, fl = 77.22(3) °, 7 = 101.75(3), and with t w o molecules in the unit cell. The analysis has shown t ha t the osmium reagent has added cis across the 5,6 t h y m i n e bond.

Introduction There has been considerable recent interest in the interaction of osmium(VI) complexes with nucleic acids. Schevitz et al. [ 1] have obtained an osmium derivative o f yeast f o r m y l m e t h i o n i n e transfer RNA, which is isomorphous with the native tRNA, and has the osmium a t om b o u n d to essentially a single site. Rosa and Sigler [2] have now established that the site of covalent a t t a c h m e n t is a h y d r o g e n - b o n d e d cytidine at position 38, with presumably the pyrimidine base itself being bonde d to the osmium atom. This heavy-atom derivative has been of considerable help in the structure analysis of this t R N A (Sigler, P.B., personal c o m m u n i c a t i o n ) . The reagent used in the above studies was a bispyridine com pl ex o f osm i u m tetroxide. This is k n o w n to react with bot h olefins themselves [ 3 ] , and substituted olefins such as nucleic acid bases [ 4 ] , as well as with cis diols. Kim et al. [5,6] have utilised this latter reaction to p r o d u c e an osmium bispyridine ester o f adenosine, which t h e y have used as a heavy-atom derivative marker in the structure analysis of yeast phenylalanine tRNA. A crystal structure analysis of the adenosine adduct itself has c o n f i r m e d the cis diol ligand binding to osmium [ 7 ] . It has been shown [4,8] that t h y m i n e is the m ost reactive nucleic acid base to the bispyridine osmium t e t r o x i d e reagent, and this p r o p e r t y has been

227 suggested as a potential m e t h o d of selectively labelling nucleic acids for electron microscopic analysis and sequencing. We have obtained the osmium • thymine adduct using a published procedure [4], and have subjected single crystals to an X-ray structure analysis to establish the geometry of the complex. Methods The complex was prepared in aqueous solution, from osmium tetroxide and t h y m i n e in approximately equimolar quantities, together with a considerable excess of pyridine [4]. The black amorphous mass obtained was recrystallised from water, producing black lustrous prisms. These were checked for purity by means of thin-layer chromatography on cellulose plates. Weissenbergand oscillation photographs revealed the crystals to have triclinic symmetry. Cell dimensions were obtained from measurements on a G.E. XRD6 diffractometer. These were determined to be a = 7.975(3), b = 10.381(3), c = 11.036(4)A, a = 82.73(2) °, 13 = 77.22(3) ° , 7 = 101.75(3) °. The crystal density, as measured by flotation, was 2.10(2) g • cm -3, compared with a calculated density of 2.083 g • cm -3 for two molecules of C1 s Hi 6 N4 06 Os per unit cell. The assumed space group was P1, with one molecule per asymmetric unit;

TABLE I FINAL POSITIONAL P A R A M E T E R S FOR THE NON-HYDROGEN ATOMS (×104 ) AS FRACTIONS OF THE UNIT-CELL EDGES Standard deviations are in parentheses. Atom

x

y

z

Os O1 02 03 04 NI' C2' C3' C4' C5' C6' NI" C2" C3" C4" C5" C6" N1 C2 N3 C4 C5 C6 07 08 C9

817(2) --81(50) 2895(39) --346(42) 1578(36) 2272(35) 1707(52) 2659(53) 4362(66) 5003(65) 3895(55) --1566(42) --1398(61) --2987(65) --4564(65) --4726(64) --3118(51) 9(53) --303(58) 342(45) 1707(43) 2317(43) 800(46) --1601(44) 2362(38) 3932(51)

2234(2) 2257(40) 1486(26) 510(31) 2724(33) 4194(26) 5014(39) 6297(39) 6727(48) 5974(48) 4630(40) 2854(31) 3928(44) 4175(48) 3233(48) 2193(48) 2086(38) --1598(41) --2164(37) --1410(33) --267(32) 83(31) --445(34) --3340(33) 383(29) --446(37)

2676(2) 1299(43) 2044(34) 3269(36) 4074(35) 1840(34) 999(48) 336(50) 486(59) 1343(58) 1879(50) 3611(39) 4090(55) 4941(58) 5089(57) 4328(59) 3673(50) 3436(48) 2743(58) 1198(41) 1057(44) 2196(43) 3366(44) 2396(38) 15(36) 2228(48)

228 this was confirmed by the structure analysis. The absorption coefficient for the crystal using Cu-Ka radiation (~ = 1.5418 £ ) was 146.0 cm -~ . Intensity data were collected photographically using the equi-inclination Weissenberg m e t h o d and multi-film packs. The layers h k O - 6 and O k l were photographed using nickel-filtered Cu-Ka radiation. The intensity films were scanned by the Science Research Council microdensitometer service, with an Optronics P-1000 Photoscan. The agreement index (defined as F, II--IiL/F, Ii, where i is the mean of a number of measurements of the same I i reflection) for various measurements of the same reflection upon successive films of a pack varied between 10 and 12%; this reflects the relatively poor quality of the crystal, and parallels experience with crystals of the osmium bispyridine ester of adenosine [7]. The data were merged and sorted to a c o m m o n scale, and absorption corrections applied to the 1453 unique observed reflections. The structures were solved by the heavy-atom method, using osmium as the heavy atom, and refined by full-matrix, least-squares techniques, to a final agreement index R w of 0.097 ( R w = Y,w liFo I--IFc il/F,w iFo l, with weights w set to I / ( o 2 (F) + 0.0071Fi 2 }). Hydrogen atoms were not located. Anisotopic thermal parameters were refined only for t h e osmium and its four bonded oxygen atoms. Table I gives the final atomic positional parameters; listings of thermal parameters, as well as observed and calculated structure factors, can be obtained on request. Computer programs used in the analysis and refinement were written by Dr. G.M. Sheldrick, and run on a CDC 7600 computer. Results and Discussion Two projections of the molecule are shown in Fig. 1; Fig. 2 details the intramolecular bond distances and angles. Estimated standard deviations in

04

~

~

Fig. 1. Two views o f t h e c o m p l e x , d i s t i n g u i s h e d by a 45 ° r o t a t i o n a b o u t t h e b axis.

229

1"29 \. 122° }121°

1-45

114°

2° .39

1"39

2.00

. ~14.1 ~1~ 1o 0 / ~

11-49

1.51 108/ 1.51

b112 °

) 1"4(~

1-22

' 1"06 148°

Fig. 2. Intramolecular bond lengths and angles. O t h e r a n g l e s are: O1-OS-O3, 91°; O1-OS-O2, 99°; O3-OS-O4, 102°; N11-OS-NI I I 94°; N J I . o s . o I , 8 6 ° ; N I I . O S . O 3 , 175°;N11 l . o s . o 2 ' 1 7 2 ° ; N I 1 I.OS. O4, 82 °; O2-C5-C4, 109°; C6-C5-C9,114 ° .

bond lengths range from a b o u t 0.03A for osmium-oxygen and -nitrogen bonds, to 0.04--0.07£ for other bonds; standard deviations in angles range from 1 ° for the former to between 2 ° and 4 ° for non-osmium angles. These high standard deviations (by contemporary standards of crystal structure analysis), are believed to reflect the poor crystal quality of the specimen used particularly in respect of high mosaic spread and thermal diffuse scattering. As mentioned above, the analysis of the osmium bispyridine ester of adenosine was reported [7] to have suffered from similar problems; indeed similar poor estimates of bond length and angle reliability were obtained in this analysis. In the present study, it has been observed that the geometry of part of the thymine ring is particularly poorly defined, with anomalous bond lengths and angles. Difference electron density maps strongly suggest structural disorder in this region of the molecule; it was not possible to satisfactorily account for this disorder. Introduction of anisotropic thermal parameters for the relevant atoms did not improve the model at all, and did not significantly lower the agreement index Rw. Therefore, in view of the poor quality of the geometric data, any structural deductions must be made with care. The analysis has confirmed the prediction [4] that the osmium tetroxide bispyridine reagent has attacked the 5,6 double bond of thymine, to form an O2, 0 3 cyclic ester. This ester is cis-fused to the thymine ring. As expected, the cyclic ester is markedly non-planar. (Fig. lb). The average osmium-ester oxygen and osmium-ligand distances agree, within experimental error, with other find-

230 ings [7,9]. The complex has a distorted octahedral co-ordination, with the osmate ester oxygens and the t w o pyridine ligand nitrogen atoms approximately coplanar. The two Os = O bonds are trans. It is likely that the thymine ring is distorted from planarity, since bond C5-C6 in the complex has no longer any double bond character, with consequent loss of thymine aromaticity. However, the precise nature and extent of the deviations from planarity cannot be c o m m e n t e d upon, because of the large uncertainties in some of the relevant atomic positions. In spite of the apparent long C2-O7 bond length, it is unlikely that enolisation at C2 has occurred since N1 is probably hydrogen bonded to O4 in a centro-symmetrically related molecule with a separation of 2.75A (Fig. 3), strongly suggesting that N1 has a proton attached. The analyses of 5,6-dihydrothymine [ 1 0 ] , 5,6-dihydrouracil [11] and 5,6-dihydrouridine [ 1 2 ] , have all shown that C5-C6 bond saturation has markedly puckered the six-membered pyrimidine rings, and that C2-O7 remains a carbonyl bond. As expected, both C5 and C6 were observed to be out of the N1, C2, N3, C4 plane in all three structures; in both 5,6-dihydrouracil and 5,6-dihydrouridine, the carbonyl oxygen atoms also deviated from this plane (by as much as 0.3A for 0 7 of one of the t w o independent dihydrouridine molecules). The structure analysis of a mercury(II)-dihydrouracil complex [13] also shows a similar pattern of ring and carbonyl substituent out-of-plane deformation. It is likely that attachment of the osmium atom and its ligands to the thymine ring has altered the latter's potential for Watson-Crick and other types of hydrogen-bonded association. An infrared spectral study of the association between 1-cyclohexyluracil and 9-ethyladenine, compared with that using the substituted 5,6-dihydrouracil has shown the former to be much stronger [ 1 4 ] . This is thought to be mainly due to differences in N3 pK values, b u t certainly

I

f

Fig.

3. The

a axis

I

¢

i

p r o j e c t i o n o f t h e crystal s t r u c t u r e ; d a s h e d lines r e p r e s e n t h y d r o g e n b o n d s .

231

the dihydropyrimidine ring pucker would play a part in weakening the hydrogen bonds. Thus, formation of the osmium adduct described here could well interfere with nucleic acid structure and conformation, at least in the localised region of binding, for example in tRNA [2], or in DNA itself. The packing of molecules in the unit cell is shown in Fig. 3. The structure consists of hydrogen-bonded dimers, held together by partial parallel sta~kings of pyridine rings. It is perhaps of interest that Rosa and Sigler [2] have speculated that the osmium bispyridine adduct of yeast initiator tRNA has enhanced stability due to functional groups in addition to attachment at cytidine-38; this enhancement may well be due, at least in part, to stacking interactions between pyridine ligands and adjacent tRNA bases. Acknowledgements We are grateful to Drs. P.B. Sigler and M. Spencer for discussions, the Cancer Research Campaign for support, and the Science Research Council microdensitometer service for providing film scanning facilities. References 1 Schevitz, R.W., Navia, M.A., Bantz, D.A., Cornick, G., Rosa, J.J., Rosa, M.D.H. and Sigler, P.B. (1972) Science 177,429--431 2 Rosa, J.J. and Sigler, P.B. (1974) Biochemistry 13, 5102--5110 3 Subbaraman, L.R., Subbaraman, J. and Beh~man, E.J. (1972) Inorg. Chem. 11, 2621--2627 4 Subbaraman, L.R., Subbaraman0 J. and Behrman, E.J. (1971) Bioinorg. Chem. 1, 35---55 5 Kim, S.H., Quigley, G., Suddath, F.L., McPherson, A., Sneden, D., Kim, J.J., Weinzierl, J., Blattmann, P. and Rich, A. (1972) Proc. Natl. Acad. Sci. U.S. 69, 3746--3750 6 Kiln, S.H., Quig|ey, G.J., Suddath, F.L., McPherson, A., Sneden, D., Kim, J.J., Weinzierl, J. and Rich, A. (1973) Science 179, 285--288 7 Corm, J.F., Kim, J.J., Suddath, F.L., Blattmann, P. and Rich, A. (1974) J, Am. Chem. Soc. 96, 7152--7153 8 Highton, P.J., Mutt, B.L., Shafa, F. and Beer, M. (1968) Biochemistry 7,825---833 9 CoUin, R., Griffith, W.P., Phillips, F.L. and Skapski, A.C. (1974) Biochim. Biophys. Acta 354, 152-154 10 Furberg, S. and Jensen, L.H. (1968) J. Am. Chem. Soc. 90, 470--474 11 Rohrer, D.C. and Sundaralingam, M. (1970) Acta Crystanogr. B26, 546--553 12 Suck, D., Saenger, W. and Zechmeister, K. (1972) Acta CrystaUogr. B28, 596--605 13 Carrabine, J.A. and Sundaralingam, M. (1971) Biochemistry 10, 292--299 14 Kyogoku, Y., Lord, R.C. and Rich, A. (1967) Proc. Natl. Acad. Sci. U.S. 57, 250--257

The crystal and molecular structure of an osmium bispyridine adduct of thymine.

The bispyridine osmium adduct of thymine has been crystallised and subjected to an X-ray diffraction analysis. It crystallises in the triclinic space ...
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