The crystal structure of a uracil-acetone photoaddition product

377

Biochimica et Biophysica Acta, 407 (1975) 377--383 © Elsevier Scientific Publishing Company, Amsterdam

- - P r i n t e d in

The Netherlands

BBA 98431 THE CRYSTAL STRUCTURE OF A URACIL-ACETONE PHOTOADDITION PRODUCT*

MARTHA

T. STEIN**, H E L E N M. B E R M A N

and A.J. V A R G H E S E

The Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia,Pa. 19111 (U.S.A.) and Ontario Cancer Institute, Toronto, Ontario M 4 X I K9 (Canada) (Received May 20th, 1975)

Summary Crystals o f t h e a d d i t i o n p r o d u c t o f uracil and a c e t o n e , C7 H10 N2 0 3 , are triclinic, s p a c e g r o u p P1 w i t h Z = 2, F.W. 170 g / m o l , cell d i m e n s i o n s a = 5 . 9 0 8 (4), b = 1 2 . 1 0 2 (5), c = 6 . 2 5 3 (3) A, ~ = 9 4 . 0 8 (4), ~ = 1 1 3 . 4 5 (4), ~ = 9 6 . 8 4 (4) °, V = 4 0 3 . 8 (4) A 3 . T h e s t r u c t u r a l f o r m u l a is:

CH 3 H

T h e s t r u c t u r e was solved b y d i r e c t m e t h o d s a n d refined b y a f u l l - m a t r i x leastsquares p r o c e d u r e t o t h e final residual R = 0.069. E i g h t o f t h e t e n h y d r o g e n a t o m s w e r e l o c a t e d f r o m a d i f f e r e n c e m a p a n d w e r e r e f i n e d isotropically. B o t h the uracil ring and t h e o x e t a n e ring are nearly p l a n a r a n d t h e angle b e t w e e n these planes is 121 ° .

* S u p p l e m e n t a r y d a t a t o this s.rticle, giving details o f s t r u c t u r e f a c t o r s f o r u r a c i l - a c e t o n e a n d t h e p o s i t i o n a l a n d t h e t h e r m a l p s . r a m e t e r s o f a t o m s in u r a c i l - a c e t o n e are d e p o s i t e d w i t h , a n d c a n b e o b t a i n e d f r o m : Elsevier Scientific P u b l i s h i n g C o m p a n y , BBA D a t a D e p o s i t i o n , p.o. Box 1 5 2 7 , A m s t e r d a m , T h e N e t h e r l a n d s . R e f e r e n c e s h o u l d b e m a d e to B B A / D D / 0 2 8 / 9 8 4 3 1 / 4 0 7 ( 1 9 7 5 ) 3 7 7 . ** P r e s e n t address: D e p a r t m e n t o f Biology, M a s s a c h u s e t t s I n s t i t u t e o f T e c h n o l o g y , C a m b r i d g e , Mass. 0 2 1 3 9 , U.S.A.

378 Introduction Exposure of nucleic acids to small doses of ultraviolet light results in chemical changes primarily in the pyrimidine residues [1]. The type of photochemical change in a pyrimidine base depends on the conditions under which the irradiation is carried out [2]. Cyclobutane type dimers constitute a major product in DNA under a variety of conditions [3]. Of the m a n y possible isomers, only the cis-syn isomer is formed in natural DNA [4]. Other DNA photoproducts include pyrimidine-pyrimidine adducts [5] and t h y m i n y l 5,6-dih y d r o t h y m i n e [6]. There is evidence that ketones such as acetone, acetophenone and benzophenone add to pyrimidine bases and to DNA in the presence of near ultraviolet light [2]. Recently chemical identification of a photoaddition product of uracil and acetone has been reported [7]. It is obtained from uracil or cytosine irradiated in the presence of acetone with near ultraviolet light. This c o m p o u n d contains a uracil ring saturated at the C(5)-C(6) bond and a dimethyl oxetane ring at this C(5)-C(6) position. Crystallographic study of the p h o t o p r o d u c t was undertaken to confirm its structure and to determine the detailed stereochemistry of the two rings. This p h o t o p r o d u c t could be useful as a probe to study the mechanism of binding to the double helix of DNA or RNA, as will be discussed in detail later.

Experimental Light amber crystals of the photoaddition product were grown which were in space group P1, Z = 2 with cell dimensions a = 5.908 (4), b = 12.102 (5), c = 6.253 (3) A, ~ = 94.08 (4), fl = 113.45 (4), ~/ = 96.84 ° . A needle crystal, 0.65 X 0.13 X 0.09 mm, was used to collect three-dimensional data on a Syntex automated diffractometer with m o n o c h r o m a t e d CuK~ radiation and the variable 0-20 scan technique. Intensities were measured for 3320 reflections. The raw data were corrected for intensity loss by means of a linear plot derived from the change in intensity of measured standard reflections as a function of time o f exposure (9.7% over 93 h). Values for o(F) were derived from counting statistics and measured instrumental uncertainties. The formula used was o(F) = (F/2){o 2 (I)/P + 62 }v, where o(I) is derived from counting statistics alone and 5 is the measured instrumental uncertainty. The data were reduced to a unique set of 1663 independent reflections, corrected for Lorentz and polarization effects and converted to structure amplitudes. A Wilson plot of the data produced estimated temperature and scale factors. Structure determination and refinement The structure was determined by direct methods by means of the program MULTAN [8] using the 177 reflections with E* greater than 1.5 and 125 reflections with zero intensity which constitute the 4o set of reflections. A * E is t h e n o r m a l i z e d s t r u c t u r e f a c t o r w h i c h w a s i n t r o d u c e d b y J. K a r l e a n d H. H a u p t m a n n ( 1 9 5 6 ) ( A c t a C r y s t . 9, 6 3 5 ) b e c a u s e o f i t s m a t h e m a t i c a l c o n v e n i e n c e f o r u s e i n p r o b a b i l i t y r e l a t i o n s h i p s . I t is r e l a t e d t o F , t h e s t r u c t u r e f a c t o r f o r e a c h d i f f r a c t i o n o b s e r v a t i o n .

379 previous attempt to find a solution without using the 4o set had been unsuccessful. The initialE m a p revealed nine non-hydrogen atoms and this fragment was used to calculate a Fourier synthesis using as coefficients E values greater than 1.0. The remaining three non-hydrogen atoms were immediately apparent and the R value with this trial structure was 0.34. Three cycles of isotropic refinement and one subsequent cycle of anisotropic refinement were calculated. The positions of eight of the ten hydrogen atoms were derived from a difference Fourier synthesis at this stage. The remaining hydrogen atom positions (HN3 and H8") were calculated assuming ideal trigonal and tetrahedral geometries, respectively. The structure was then refined anisotropically (eight hydrogen atoms isotropically and two held fixed) to conventional R of 0.069 (Rw = 0.082). N o extinction or absorption corrections were made. The atomic scattering factors used for oxygen, nitrogen and carbon atoms were those in International Tables for X-ray Crystallography [9] and for hydrogen atoms those of Stewart et al. [10]. All programs used were from the CRYSNET package [11]. Reflections with F o ~< 2o(F) were assigned zero weights. Initiallythe data with F o > 2a(F) were given unit weights in the least squares refinement. In the final cycles (below R = 0.12) the weights were derived from an analysis of variance. The two linearfunctions useclfor o were: for 20(F) < Fo < 212 for Fo/> 212

a = 0.343 Fo + 66.5 o = 0.167 Fo + 105.0

The final atomic parameters and the structure factors have been deposited in the BBA data bank. Discussion The molecule has the cis-syn configuration. Drawings of the single molecules with the proper atom labels and the distances and angles are shown in Fig. 1.

The uracil ring As with other pyrimidine rings which are saturated at C(5)-C(6), this ring is n o t planar. The puckering is small compared with that found in many other similar structures. The maximum deviation from the least squares plane (Table I) is 0.08 A for C(5) which is similar to that for the trans-anti-thymine dimer (0.06 A) [12]. Typical deviation from planarity for other pyrimidine dimers is several tenths of an Angstrom. The equation of the best plane for the uracil p h o t o p r o d u c t appears in Table I, and the distances of the atoms from that plane are illustrated in Fig. 2b. Torsion angles around the pyrimidine ring are reported in Fig. 2a. The distances and angles of the uracil ring are comparable to those found in other uracil p h o t o p r o d u c t s [13,14]. The oxetane ring The oxetane ring configuration in this structure appears to be unique among the three oxetane crystal structures so far reported. In the 3,3-dimethyl(N-trimethylammonium methylene) oxetane cation [15] and in 2,3-bis(4-

380

1204 ~

~20.9

116.9 124.1

897

'17,2

f26.9

119.5

Fig. 1. U r a c i l - a c e t o n e p h o t o p r o d u c t w i t h the c o r r e c t a t o m labels, b o n d d i s t a n c e s a n d angles. T h e estim a t e d s t a n d a r d d e v i a t i o n s f r o m the d i s t a n c e s are 0 . 0 0 5 A f o r t h e C-O a n d C-N, 0 . 0 0 7 for t h e C-C. T h e e s t i m a t e d s t a n d a r d d e v i a t i o n s f o r t h e angles are 0.4 °. T h e 5 ( S ) , 6 ( R ) f o r m is illustrated.

ethoxyphenyl)3,3-dimethyl oxetane [16] the oxetane ring is puckered by 26 ° and by 16 °, respectively. However, in the p h o t o p r o d u c t , the ring is virtually fiat (Table I and Fig. 2a). The torsion angles around the ring range between +2 ° and --2 ° . The four-membered ring of cyclobutane shows a similar ring puckering variation as illustrated by trans-l,3-cyclobutanedicarboxylic acid [17]. When TABLE I PLANES IN THE URACIL-ACETONE PHOTOPRODUCT

Atom

D i s t a n c e f r o m p l a n e (A)

(a) Uracil ring: 0 . 5 3 9 X + 0 . 8 1 6 Y - - 0 . 2 0 8 Z + 3 . 1 8 8 = 0 0(2) C(2) N(1)

C(6) C(5) C(4) 0(4) N(3)

+0.019 --0.002 --0.034 --0.038 +0.081 +0.007 --0.051 +0.018

(b) Oxctane ring: --0.698 X + 0.050 Y + 0.713 Z -- 1.200 = 0 C(5) C(6) O(10) C(7)

+0.014 --0.013 --0.014 +0.013

381

~05

B

G(5}

0(2)

/ 0(4) F i ~ 2. (a) A v i e w d o w n the 0 ( 5 ) - 0 ( 6 ) b o n d with the torsion angles a r o u n d the uracil ring indicated as well as t h e d i h e d r a l a n g l e b e t w e e n the uracil and o x e t a n e rings. (b) A v i e w o f the u r a c i l r i n g s h o w i n g deviations f r o m the best plane, magnified b y a factor o f 15.

crystallized alone, the ring is planar. However, when crystallized with sodium salt of the acid, the ring is puckered by 25 ~. (The anion itself has a planar cyclobutane ring.) Adman and Margulis [17] believe that this variation in ring puckering arises primarily from crystal packing forces. The out-of-plane ring vibration energy for cyclobutane is a b o u t 400 cm -1 or 1.15 kcal/mol [18]. In contrast, oxetane has a ring puckering vibration energy of only 15 cm -1 or 43 cal/mol [19]. Gaseous oxetane (trimethylene oxide) appears to be planar [20]. This study for the unsubstituted ring indicates that the oxetane ring is more readily deformed than is cyclobutane. (Crystallographic support for the higher barrier to puckering in cyclobutane is that observed torsion angles cluster around zero and 25--35 ° rather than varying smoothly up to 26 ° for oxetane). Crystal packing forces and the steric restriction from attachment to uracil may contribute to the planarity of the oxetane ring in this p h o t o p r o d u c t . The dihedral angle between the uracil and oxetane rings is 121 ° . In certain thymine and uracil phot0dimers, where cyclobutane is the ring between the heterocyclic rings, the dihedral angles are 114 ° [12] and 140 ° [14]. Apparently considerable variation in this angle is possible. Steric interactions would be expected to be greater in the cis dimer.

The hydrogen bonding There are two unique hydrogen bonds in this system. N(3) donates a hydrogen to O(2) of an inversion related molecule to form a hydrogen-bonded

382

0

Fig. 3. T h e h y d r o g e n - b o n d e d

dimer formed between two centrosymmetricaUy related molecules.

dimer (Fig. 3). N(1) donates a hydrogen to 0 ( 4 ) in a translationally related molecule to form a hydrogen-bonded chain.

The packing The structure is aligned so that there are stacked layers of pyrimidine rings, rows of oxetanes, and a strongly hydrophobic region of methyl groups. Biological aspects o f the structure Although this uracil photoaddition product contains two asymmetric carbon atoms, C(5) and C(6), only the cis-syn isomer is formed and in the centrosymmetric crystal both enantiomers are present. According to the CahnIngold-Prelog convention, these isomers are designated 5(S), 6(R) and 5(R),6(S). The 5(S),6(R) form is illustrated in Fig. 1. It has the oxetane ring above and to the right of the plane of the uracil ring when the N(1) atom is at the b o t t o m . The 5(R),6(S) form would have the oxetane ring below the uracil ring and the N(1) atom again at the b o t t o m . Because this molecule is an analog of a naturally occurring base of nucleic acids, it is important to examine the effect of incorporating it into a nucleic acid polymer. Model building suggests that the replacement of thymine by the 5(S),6(R) form would be sterically incompatible with the ribose phosphate backbone of either A-DNA or B-DNA*. It would probably cause the helix to unwind in order to a c c o m m o d a t e the change. This deformation would occur in both purine-pyrimidine and pyrimidine-pyrimidine stacking sequences. Such a change in the base stacking distances might p r o m o t e intercalation or might increase the probability of mistakes in replication. The other form (5(R),6(S)) can replace thymine in the double helix w i t h o u t substantial conformational change. Both pyrimidinepurine and pyrimidine-pyrimidine sequences could a c c o m m o d a t e this change, b u t the methyl groups and the oxetane ring would be likely to interfere with the binding of protein to the major groove in both A-DNA and B-DNA and in RNA. Therefore, this optical isomer may serve as a useful structural probe for the binding of proteins to the major groove of the double helix if it can be incorporated into DNA. * A - D N A a n d B - D N A are t h e t w o ~.~ajor c o n f o r m a t i o n a l classes f o r n a t i v e , o r d e r e d D N A . R N A is r e l a t e d t o t h e A - D N A s t r u c t u r a l t y p e . 6 o t h classes h a v e parallel b a s e s b u t d i f f e r in t h e tilt o f t h e b a s e s to t h e h e l i x axis, t h e n u m b e r o f b a s e s p e r t u r n , a n d t h e c o n f o r m a t i o n o f t h e s u g a r ring. (See A r n o t t , S., D o v e r , S.D. a n d W o n a c o t t , A . J . ( 1 9 6 9 ) A c t a C r y s t . B 2 5 , 2 1 9 2 f o r a d e t a i l e d s t r u c t u r a l comparison.)

383 Acknowledgements This work was supported by U.S.P.H.S. grants GM-21589, CA-06927 and RR-05539 from the National Institutes of Health, grant AG-370 from the National Science Foundation, grants from the Medical Research Council of Canada and the National Cancer Institute of Canada, and an appropriation from the Commonwealth of Pennsylvania. We are grateful to David Zacharias for assistance in data collection, H.L. Carrell for help in computing, Peter Young for technical assistance, and Nadrian Seeman for helpful discussions. References 1 McLaren, A.D. and Shugar, D. (1964) Photochemistry of Protein and Nucleic Acids, McMinan, New York 2 Vs.rghese, A.J. (1972) P h o t o p h y s i o l o g y VII, 208--274 3 Setlow, R.B. (1968) Progr. Nucleic Acid Res. Mol. Biol. 8, 257--295 4 Varghese, A.J. and Wang, S.Y. (1967) Nature 2 1 3 , 9 0 9 - - 9 1 0 5 Va~rghese, A.J. and Patrick, M.H. (1969) Nature 2 2 3 , 2 2 9 - - 3 0 9 6 Varghese, A.J. (1970) Biochem. Biophys. Res. Commun. 38, 484--490 7 Varghese, A.J. (1975) Photochem. Photobiol. 21, 147--152 8 Germain, G., Woolfson, M.M. and Main, P. (1971) Acta Crystalogr. A 2 7 , 3 6 8 - - 3 7 6 9 I ntern ational Tables for X-ray Crystallography (1962) Vol. III, Kynoch Press, Birmingham 10 Stewart, R.F., Davidson, E.R. and Simpson, W.T. (1965) J. Chem. Phys. 42, 3 1 7 5 - - 3 1 8 7 11 Meyer, Jr, E.F., Morimoto, C.N., Villarreal, J., Berman, H.M., Carrell, H.L., Stodola, R.K., Koetzle, T.F., Andrews, L.C., Bernstein, F.C. and Bernstein. H.J. (1974) CRYSNET, Fed. Proc., Vol. 33, No. 12, 2 4 0 2 - - 2 4 0 5 12 Camerman, N. and Nyburg, S.C. (1969) Acta Crystalogr. B25, 388--394 13 Konnert, J. and Karle, IC (1971) J. Cryst. Mol. Struct. 1, 107--114 14 Adman, E. and Jensen, L.H. (1970) Acta Crystalogr. B26, 1326--1334 15 McGandy, E.L. and Fasiska, E.J. (1970) Am. Chem. Assoc. Meet. August, p. 53 (H2) 16 Holan, G., Kowala, C. and Wunderlich, J.A. (1973) Chem. Commun. 34 17 Adman, E. and Margulis, T.N. (1968) J. Am. Chem. Soc. 90, 4517--4521 18 Ruthjens, G.W., Freeman, N.K., Gwinn, W.D. and Pitzer, K.S. (1953) J. Am. Chem. Soc. 75, 5634-5642 19 Kydd, R.A., Wieser, H. and Danyluk, M. (1972) J. Mol. Spectrosc. 44, 14--17 20 Kiefer, W., Bernstein, H.J., Danyluk, M. and Wieser, H.W. (1972) J. Mol. Spectxosc. 4 3 , 3 9 3 - - 4 0 0