Biochimica et Biophysica Acta, 378 (1975) 1--11 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 98186

RELATIONSHIP BETWEEN THE MUTAGENIC AND BASE-STACKING PROPERTIES OF HALOGENATED URACIL DERIVATIVES

THE CRYSTAL STRUCTURES OF 5-CHLORO- AND 5-BROMOURACIL

HELENE STERNGLANZ and CHARLES E. BUGG

Institute of Dental Research and Department of Biochemistry, University of Alabama in Birmingham, University Station, Birmingham, Ala. 35294 (U.S.A.) (Received July 23rd, 1974)

Summary

Three<limensional X-ray diffraction data were used to determine the crys- tal structures of 5-chlorouracil and 5-bromouracil , two mutagenic pyrimidine analogs that can substitute for thymine in DNA. Crystals of the two com- pounds are nearly isostructural. The space group is P2 , / c , with a = 8.450{6), b = 6.842(3), c = 11.072(16) £ , ~ = 123.53(19) ° for 5-chlorouracil, and a = 8.598(3), b = 6.886(1), c = 11.417(5) A, fl = 123.93(3) ° for 5-bromouracil . Intensi ty data were collected with an au tomated diffractometer . The structures were refined by full-matrix least-squares to R = 0.058 for 5-chlorouracil and R = 0.027 for 5-bromouracil . The analogs form planar, hydrogen-bonded ribbons that are nearly identical to those found in the crystal structure of thymine monohydra te . As in many other structures of 5-halogenated uracil derivatives, the bases assume a stacking pattern that permits intimate contacts between the halogen substituents and the pyrimidine rings of adjacent bases. This stacking pat tern involves halogen contacts tha t are significantly shorter than normal van der Waals interactions. The crystallographic results provide additional evidence that halogen substituents influence the stacking patterns of uracil derivatives, while exerting little direct effect on the hydrogen-bonding properties. The observed stacking patterns are consistent with the hypothesis that altered stacking interactions may account for the mis-pairing between 5-halogenated uracil bases and guanine residues within double-helical nucleic acids.

Introduction

The biological effects of 5-halogenated uracil derivatives have been studied in detail [1- -4] . Those derivatives with chloro, b romo, or iodo substituents at the 5-position can substitute for thymine residues in DNA. and 5-fluorouracil

can replace uracil residues in RNA. Halogenated uracil residues might be ex- pected to display hydrogen-bonding properties closely related to those of uracil and thymine, and therefore to form complementary Watson--Crick base-pairs with adenine in double-stranded nucleic acids£ However, nucleic acids that contain 5-halogenated uracil bases display unusual biological properties which may be a consequence of mis-pairing between the uracil derivatives and gua- nine. When incorporated into double-helical DNA, these halogenated bases pro- duce specific mutations that appear to arise from their occasional mis-pairing with guanine during genetic replication. Similarly, 5-fluorouracil residues in messenger RNAs produce coding errors, apparently by mis-pairing with guanine during translation processes.

Though it is generally accepted that 5-halogenated uracil residues in nu- cleic acids can mis-pair with guanine, there is little agreement as to the mecha- nism by which this occurs. Three models have been proposed to explain this phenomenon; they are the tautomer-shift, the ionization and the base-stacking models. According to the tautomer-shift model, the halogen substituents sta- bilize the enol tautomer-form of uracil, which can pair with guanine but not with adenine [5]. According to the ionization model, the electronegative halo- gen substituents induce ionization of the N(3) proton of uracil, thereby pro- ducing a configuration that is suitable for pairing with guanine [6]. According to the base-stacking model, halogen substituents affect base-stacking interac- tions within nucleic acids, thereby inducing conformational changes that lead to mis-pairing between 5-halogenated uracil residues and guanine [7--9]. Each of these models is supported by different physical studies. Spectroscopic inves- tigations indicate that halogen substituents at the 5-position of uracil enhance the stability of the enol tautomer-form [10]. Measurements of ionization con- stants demonstrate that 5-halogen substituents also markedly decrease the pK value at the N(3) position of uracil [11]. A number of studies have shown that halogen substituents alter base-stacking interactions between nucleosides in solution [12--14], and between the bases of single, double and triple stranded polynucleotides [15--21].

Our laboratory has been particularly interested in the effects exerted by halogen substituents on base-stacking patterns within crystals of nucleic acid components [7--9,22]. Earlier studies have demonstrated that halogenated bases generally assume crystallographic stacking patterns that appear to be dominated by interactions involving tha halogen substituents. In crystal struc- tures of 5-halogenated uracil derivatives, these stacking patterns are often iden- tical to those originally proposed in the base-stacking model for mis-pairing [9]. However, it is not yet clear whether these solid-state stacking patterns actually represent the inherent properties of the bases, or if they are merely a consequence of hydrogen bonding and other crystallographic interactions.

In this paper we show that the crystal structures of 5-chlorouracil and 5-bromouracil display stacking patterns that are dominated by unusually strong interactions involving the halogen substituents. We present evidence that the halogen substituents have little direct effect on hydrogen-bonding schemes, and we discuss the relationship between these findings and the base-stacking model for the mis-pairing properties of 5-halogenated uracil derivatives.

Experimental

Clear lath-shaped crystals of 5~hlorouracil and of 5-bromouracil were obtained by slowly evaporating aqueous solutions of the compounds. Weissen- berg and oscillation photographs showed the crystals to be monoclinic, space group P21/c, as indicated by the systematic absence of reflections hOl with l odd and 0k0 with k odd. Similarities in the unit cell parameters and in the intensity patterns on Weissenberg films indicated that 5-chlorouracil and 5- bromouracil are isostructural (isomorphous); this was verified by the subse- quent structure determinations.

Data for 5-chlorouracil were obtained from a crystal with approximate dimensions of 0.30, 0.15 and 0.1 mm. Data for 5-bromouracil were obtained from a crystal with approximate dimensions of 0.20, 0.10 and 0.06 mm. All angular and intensity data were collected with a Picker FACS-1 X-ray diffrac- tometer by use of a scintillation counter and nickel-filtered copper radiation. Cell parameters were measured before and after intensity data were collected. Approximate cell parameters for use in collecting intensity data were calculated by a least-squares analysis of the angular settings for several medium-angle reflections (CuK~, k = 1.5418 A). Accurate values for cell parameters were determined immediately after data collection by a least-squares analysis of 20 values for high-angle reflections (CuKal , k = 1.54051 A). The final cell parame- ters, which were based on eight reflections for 5-chlorouracil and on twelve reflections for 5-bromouracil, were not significantly different from those ob- tained prior to data collection. Crystal data, including the final cell parameters, are given in Table I.

Intensity data were measured by use of a 0--20 scanning technique. The scanning speed was l ° /min , and a 20 s background measurement was per- formed at each terminus of the scans. For 5-chlorouracil the 888 symmetry- independent reflections with 20 < 128 ° were measured; for 5-bromouracil the 913 reflections with 20 < 127 ° were measured. Those reflections with scan counts below background levels were given their calculated negative intensity values and were retained in all subsequent calculations. Intensities were as- signed variances, o 2 (i), according to counting statistics plus a correctional term

TABLE I

CRYSTAL DATA k (CuK~) = 1.5418 A)

Reported standard deviations are double those obtained from the least-squares analyses. Densities (Do)

were measured by flotation in a mixture of l,l~2,2-tetrabromoethane and carbon tetrachloride. Crystal

data reported previously [23] are incorrect.

Stoichiometry C4H3N202CI C4H3N202Br

Space group P21/c P21/c a 8.450( 6) A 8.598 (3) A

b 6 . 8 4 2 ( 3 ) 6 . 8 8 6 (1) c 1 1 . 0 7 2 ( 1 6 ) 1 1 . 4 1 7 (5 )

fl 123.53 (19) ° 123.93 (3) ° D c 1.823 g'cm -3 2.262 g'cm -3

D O 1.82 2.26 5 5 . 8 c m - I 1 0 3 . 8 c m - I

(0.03S) 2 , S being the scan count. The intensities and their variances were corrected for Lorentz and polarization effects, absorption corrections were applied by using the program ORABS [24], and the data were scaled by Wilson plots [25].

The trial structure of 5-chlorouracil was obtained by the heavy-atom method using chlorine as the heavy atom. A modified version of the full-matrix least-squares program ORFLS [26,27] was used to refine the trial structure. The quant i ty minimized was ~ w ( F o 2 _ Fc 2/k2 )2, with k as a scale factor and the weight w equal to 1/a 2 (Fo 2 ). Scattering factors for the nonhydrogen atoms were from the International Tables for X-Ray Crystallography [28], and anom- alous dispersion correction factors for these atoms were from Cromer and Liberman [29]. Hydrogen-atom scattering factors were from Stewart, Davidson and Simpson [30]. Refinement of the nonhydrogen atom positional and aniso- tropic temperature parameters for 5-chlorouracil converged at an R index (E liFo I -- IFc II/E IFo i) of 0.16. At this stage, a tom 0(4) displayed unusually large temperature parameters. A Fourier map was calculated by using phase angles derived from all the nonhydrogen atoms except atom 0(4) . Within cova- lent bonding distance of a tom C(6) this map revealed a peak with about one- half the electron density of that at the 0(4) position. This observation sug- gested that atom 0(4) might be disordered. The disordered molecule is appar- ently rotated 180 ° around the line defined by the halogen atom and atoms C(5), C(2) and 0(2) , so that two positions are possible for the carbonyl oxygen atom at 0(4) . Therefore, two positions, 0(4) and O(4'), the latter correspond- ing to the peak near C(6), were assigned to this oxygen atom. Occupancies of the two sites were included as parameters and least-squares refinement was resumed. Coordinates for the hydrogen atoms bonded to atoms N(1) and N(3) were determined from difference Fourier maps that were calculated during the latter stages of refinement. It was assumed that the hydrogen a tom bonded to atom C(6) must also be disordered. Coordinates for two partially occupied hydrogen sites H(C6) and H(C6'), which corresponded to hydrogen atoms bonded to C(6) and C(4), respectively, were calculated by assuming trigonal bonding and C--H bond distances of 0.95 A. Atoms H(C6) and H(C6') were assigned isotropic temperature parameters and were given occupancy parame- ters so that the total occupancies of 0(4) plus H(C6') and 0(4 ' ) plus H(C6) were about 1.0. Parameters for H(C6) and H(C6') were included in subsequent structure-factor calculations but not in the least-squares refinement. Final cycles of refinement included all other positional parameters, anisotropic tem- perature factors for the nonhydrogen atoms, isotropic temperature factors for the hydrogen atoms, occupancy parameters for O(4) and 0(4 ' ) , and Zacha- riasen's [31] isotropic extinction parameter g (as formulated by Coppens and Hamilton [32] ). During the last cycle of refinement no parameter shifted more than one-seventh of its standard deviation. The final R index for the 5-chlo- rouracil structure is 0.058, and the goodness-of-fit [ (Ew(Fo 2 - - F c 2 ) 2 / (rn - - s ) } 1/2, where m is the number of reflections used and s is the number of parameters refined] is 1.45. The occupancy parameters for 0(4) and 0(4 ' ) , which were not constrained to a total of 1.0, refined to 0.710(5) and 0.315(5), respectively. A final three-dimensional difference Fourier map showed no peaks or troughs that exceeded 0.36 e/A 3 in magnitude.

A trial structure for 5-bromouracil was obtained by the heavy-atom meth- od, using bromine as the heavy atom. As in the 5-chlorouracil structure, a tom 0(4) was found to be disordered. However, the major site for atom O(4) corresponded to the minor site (0(4')} in the 5-chlorouracil structure. The refinement procedure was identical to that described for 5-chlorouracil. The occupancy factors for 0(4) (the major site) and 0(4 ' ) (the minor site) refined to values of 0.890(8) and 0.118(9), respectively. During the last cycle of least- squares refinement no parameter shifted more than one-seventh of its standard deviation. The final R index is 0.027, and the goodness-of-fit is 1.51. A final difference Fourier map showed peaks and troughs with magnitudes as high as 0.5 e/A 3 in the immediate vicinity of the bromine a tom, but no other fluctua- tions that exceeded 0.30 e/A 3 in magnitude.

Results

Table II lists the atomic parameters and their estimated standard devia- t ions . .Est imated errors in positional parameters are about 0.001 A for C1, 0.000~ A for Br, 0.004 A for other nonhydrogen atoms, and 0.04 A for hydro- gen atoms. Tables of observed and calculated structure amplitudes have been deposited.*

The crystal-packing and hydrogen-bonding scheme for 5-bromouracil is depicted in Fig. 1. It is essentially the same as that for 5-chlorouracil except that atom 0(4) of 5-chlorouracil occupies the position of 0(4 ' ) in 5-bromour- acil, a tom N(1) is interchanged with N(3), and atom C(4) is interchanged with C(6). The uracil moieties are joined across crystallographic inversion cen- ters by pairs of N(1)--H • • • O(2) and N(3)--H • • • O(2) hydrogen bonds, result- ing in continuous hydrogen-bonded ribbons that run in the b direction. The hy- drogen-bonding scheme between the halogenated uracil moieties is nearly identi- cal to that found in the crystal structure of thymine monohydrate [34]. The hydrogen-bonded ribbons are depicted in greater detail in Fig. 2, where the hydrogen-bond dimensions are compared with those of thymine monohydrate . The ribbons of bases are stacked in the c direction. Fig. 3 shows the stacking patterns, as viewed perpendicular and 10 ° from parallel to the base planes. The stacked bases are alightly inclined with respect to each other and are separated by interplanar spacings of about 3.3 A and 3.4 A in 5-chlorouracil and 5- bromouracil, respectively. The stacking interactions involve close contacts be- tween the halogen substituents and a tom C(2): the C1 . . . . . . C(2) distance of 3.35 A and the Br . . . . . . C(2) distance of 3.45 A are about 0.2 A shorter than normal van der Waals contacts.

The type of disorder that occurs in the crystal structures of 5-chlorouracil and 5-bromouracil can be seen in Fig. 1. Since a tom 0(4) does not participate in hydrogen bonding, and since the disorder simply interchanges the N(1)--H and N(3)--H groups while leaving atom 0(2) unchanged, the hydrogen-bonding

* T h e s e tab les can b e o b t a i n e d o n r e q u e s t f r o m t h e Elsevier P u b l i s h i n g C o m p a n y BBA D a t a Depos i - t i o n , P. O. B o x 3 3 0 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 n u m b e r

B B A / D D / 9 8 1 8 6 1 3 7 8 ( 1 9 7 5 ) 1.

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57

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95

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57

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67

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71

9 (

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37

64

(4)

16

8

(12

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(8

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54

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7

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(4

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3)

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2

(29

)

Fig. 1. T h e c r y s t a l - p a c k i n g a n d h y d r o g e n - b o n d i n g s c h e m e fo r 5 - b r o m o u r a c i l as v i e w e d p e r p e n d i c u l a r t o t h e ab p l a n e . N i t r o g e n a t o m s a re n u m b e r e d ; b r o m i n e a t o m s a re r e p r e s e n t e d b y so l id c i rc les ; h y d r o g e n b o n d s a re d e p i c t e d b y d a s h e d l ines; O ( 4 ' ) a n d H ( C 6 ' ) , t h e m i n o r s i tes f o r t h e d i s o r d e r e d a t o m s , a r e given as d a s h e d circles . Essen t i a l ly t h e s a m e s c h e m e is f o u n d f o r 5 - c h l o r o u r a c i l , e x c e p t a t o m s 0 ( 4 ) a n d H ( C 6 ) w o u l d c o r r e s p o n d to t h o s e s h o w n f o r O ( 4 ' ) a n d H ( C 6 ' ) , N(1) a n d N(3) w o u l d b e i n t e r c h a n g e d , a n d C(4) a n d C(6) w o u l d b e i n t e r c h a n g e d . Th i s d r a w i n g a n d t h a t in Fig . 4 were p r e p a r e d us ing t h e p r o g r a m O R T E P [ 3 3 ] .

R R

I I er 2.e33 I I er 2-e09 I I i . .I. n,. ci 2.e26 I I ca ~-794 ~ [ J /N- -__N. \ T 2'842 -N N T 2~3e - " m N

I Cl 170" '% ~" • / • .,- s ~ . _ _ / I -r i ' / z . ~ ' ~ • [ , • ~ 4 I~r i f o ° / I~ . . . . U ~ 1 ~ U CI 175 * z • "~ I • 11.jI I T ' 7 ~ ° 0

T ~.o4 - - N " " N \ N ~ \ N / -' ~.77

R R Fig. 2. R i b b o n s o f h y d r o g e n - b o n d e d bases in t h e c ry s t a l s t r u c t u r e s o f 5 - b r o m o u ~ a c i l , 5 ~ h l o r o u s a c i l a n d t h y m i n e m o n o h y d r a t e . A d j a c e n t bases a re r e l a t e d b y c r y s t a l l o g r a p h i c i nve r s ion cen t e r s . D o n o r - - a c c e p t o r d i s t a n c e s ( e s t i m a t e d S.D. = 0 . 0 0 5 A) , h y d r o g e n - - a c c e p t o r d i s t a n c e s ( e s t i m a t e d S .D. = 0 . 0 6 A) , a n d d o n o r - - h y d r o g e n - - a c c e p t o r ang les ( e s t i m a t e d S .D. = 4 °) a re g iven a n d l abe l l ed as Br , CI a n d T, w h i c h c o r r e s p o n d t o va lues f o r 5 - b r o m o u r a c l l , 5 - c h l o r o u r a c i ] a n d t h y m i n e m o n o h y d r a t e 0 r e s p e c t i v e l y .

0 H / H H

0 "Cl 335 -cI 0

/ \ : \ H H

~35

0

,=~ < -

545

(A) 5 CHLOROURACIL (B) 5 BROMOURACIL Fig. 3. S t a c k i n g p a t t e r n s fo r 5 -ch lorourac i l a n d 5 - b r o m o u r a c i l as v i e w e d ( t op ) p e r p e n d i c u l a r to t he p lanes

of t he bases t h a t are r e p r e s e n t e d by heavy l ines a n d ( b o t t o m ) 10 ° f r o m paral lel to t he p lanes o f t he bases . H a l o g e n . . . . . . C(2) i n t e r m o l e c u l a r d i s t ances ( in A) axe s h o w n .

scheme is unaffected by the disorder. The disorder should also have little effect on the base stacking interactions (Fig. 3).

Fig. 4 shows the bond lengths and thermal ellipsoids for 5-chlorouracil and 5-bromouracil. Bond angles are given in Table III. Corresponding bond lengths and angles of 5-chlorouracil, 5-bromouracil and thymine monohydra te [34] exhibit no appreciable differences. As in other crystal structures of py- rimidines, the ring systems are slightly nonplanar, but no ring atoms deviate from least-squares pyrimidine planes by more than 0.012 A for 5-chlorouracil and 0.017 A for 5-bromouracil. The chlorine a tom deviates by 0.043 A and the

5 7

....... ,~C(5) 4 ..~-., ~ C ( 5 ) ,,~,-x 0(4) , ~;...... !"~ 0 ( ) o(4) ~ (i.2s3)

0(4'; .~"<:,~lJ~ ~ -- ~ - 7 ~ ~

I - I

(A) 0 1 @ (B) 0 ( 2 ) ~

Fig. 4. Moleeular geometry and atomic numbering o f 5-chloroLuracg and 5-bromouracil. The n o n - hydrogen atoms are represented by thermal ellipsoids scaled to include 50% probabi l i ty . The hydrogen atoms are represented by spheres o f 0.1 A radius. 0 (4 ' ) and H(C6') , minor sites fo r the disordered atoms, are shown as dashed lines. Bond lengths are given m A; estimated standard devmt~ons are 0.004~0.006 A, except fo r the C(6)- -O(4 ' ) bond lengths, which have estimated standard deviations o f a b o u t 0.03 A.

TABLE III

BOND ANGLES INVOLVING NONHYDROGEN ATOMS OF 5-CHLOROURACIL AND 5-BROMOUR- ACIL

E s t i m a t e d standard dev ia t ions are ab ou t 0 . 3 ° , e x c e p t for angles involving O (4'), which are a b o u t 2 °.

A t o m s 5-Chlorouracfl 5-Bromouracil angle (o) angle (o)

C (2)--N(1)--C (6) 123.4 123.3 N(1)---C (2)---N(3) 115.4 115.2 N{1)---C (2)--O(2) 122.2 122.8 N(3)---C (2)--O(2) 122.5 122.0 C (2)--N(3)--C (4) 126.4 126.6 N(3)---C (4)--C (5) 114.9 114.1 N(3)--C (4)---0(4) 119.4 120.1 C (5)--C (4)--0(4) 125.7 125.8 C (4)---C (5)---C (6) 120.6 120.4 C (4)--C (5)--CI(Br) 118.8 119.2 C (6)--C (5)--CI(Br) 120.6 120.4 C (5)--C (6)---N(1) 119.3 120.4 N(1 )--C (6)---0(4') 117 116 C (5)--C (6)--0(4') 122 122

bromine atom by 0.062 £ . The 0(2) atoms are both less than 0.05 A from the pyrimidine planes. The disordered oxygen atoms, O(4) and O(4'), are displaced from the pyrimidine planes in 5-chlorouracil by 0.088 A and 0.152 A, respec- tively, and in 5-bromouracil by 0.05 h and 0.205 A, respectively.

Discussion

The base-stacking patterns of 5-chlorouracil and 5-bromouracil appear to be largely dominated by interactions involving the halogen substituents. As depicted in Fig. 3, the bases stack in a pattern that permits intimate contact between the halogen substituent of one uracil moiety and the ring system of the adjacent base. In both crystal structures the stacking patterns display halo- gen . . . . . . C(2) distances that are appreciably shorter than normal van der Waals contacts, suggesting that the halogen interactions may be of major impor- tance in stabilizing the observed patterns. These stacking patterns are character- istic of those found in many crystal structures of halogenated purines and pyrimidines [7--9,22]. In contrast, the stacking pattern of thymine monohy- drate shows N(3) of one molecule positioned above the pyrimidine ring of its neighbor and the methyl substituent at C(5) far removed from the adjacent molecule. Like 5-chlorouracil and 5-bromouracil, other halogenated bases gen- erally assume crystallographic stacking patterns that involve intimate contacts between halogen substituents and ring systems of adjacent bases. These findings indicate that halogen substituents affect solid-state stacking interactions. There is evidence that halogen substituents alter base-stacking interactions in aqueous solution also. For example, in water, 5-bromouridine stacks more extensively than either uridine or thymidine [12--14]. Furthermore, 5-halogenated uracil and cytosine residues enhance the conformational stabilities of single-stranded, double-helical, and triple-helical polynucleotides in aqueous solution, and these

10

effects have been at tr ibuted to the influence exerted by the halogenated py- rimidines on base-stacking within polynucleotides [15--21] . Therefore, it ap- pears that halogen substituents affect base-stacking interactions in a variety of crystalline and noncrystalline environments.

Although the halogen substituents seem to influence base-stacking interac- tions in the crystal structures of 5-chlorouracil and 5-bromouracil, they appear to have little effect on the hydrogen-bonding properties of the uracil moiety. As shown in Fig. 2, 5-chlorouracil and 5-bromouracil form the same type of hydrogen-bonded ribbons as those found in the crystal structure of thymine monohydrate , and the hydrogen bond dimensions are no t significantly altered when the methyl group at the 5-position of thymine is replaced by a chlorine or bromine substituent. These findings are consistent with earlier conclusions that the crystallographic hydrogen-bonding properties of uracil moieties are not significantly altered by halogen substituents at the 5-position [35] .

Crystallographic studies of 5-halogenated uracil derivatives have also failed to provide any direct evidence that halogen substituents alter the preferred tautomer form of uracil moieties, or result in stable ionized states that can be isolated in crystalline form. To date, almost thir ty crystal structures that con- taln 5-halogenated uracil derivatives have been reported, including free pyrimi- dines, nucleosides, and complexes of the uracil derivatives with various purines and pyrimidines [9] . Despite exposure of uracil derivatives to a variety of hydrogen-bonding environments in these crystal structures, no enol tautomer forms or ionized N(3)--H groups have been found. Thus, the available crystallo- graphic data fail to provide any direct support to either the tautomer-shift or the ionization model for mis-pairing. On the other hand, nearly all crystal structures of 5-halogenated uracil derivatives display stacking patterns that, like those of 5-chlorouracil and 5-bromouracil, involve close halogen . . . . . . base contacts. The base-stacking model for mis-palring is based upon the simple premise that halogenated uracil derivatives tend to form stacking patterns that permit interactions between the halogen substituents and adjacent bases within nucleic acids. This premise is clearly consistent with the crystallographic data that are now available.

Acknowledgements

We thank Miss Catherine Sims and Miss Mary Ann Comer for assistance with the preparation of this manuscript. This work was supported by N.I.H. grants CA-12159, DE-02670 and RR-145.

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