Base-pairing configurations between purines and pyrimidines in the solid state

J. Mol. Biol. (1969) 43,407422

Base-pairing Configurations between Purines and Pyrimidines in the Solid State

IV. Crystal and Molecular Structure of two 1 :‘l Hydrogen-bonded Complexes, l-Methyl-5-bromouracil : 9-Ethyl-2-aminopurine

and l-Methyl-S-fluorouracil : 9-Ethyl-2-amiuopurine

FERNANDO Mmzat, HENRY M. SOBELL

Department of Chemistry, The University of Rochester Rocheder, N. Y. 24627, U.S.A.

and Department of Radiation Biology and Bkphysics

The University of Rochester School of Medicine and Dentistry Rochester, N. Y. 14620, U.S.A.

AND

GOPMATH MARTHA

Center for X-ray Crystilographic Research Roswell Park Memorial Institute

Buffalo, N.Y., U.S.A.

(Received 25 March 1969)

9.Ethyl-2-aminopurine forms 1: 1 hydrogen-bonded complexes with l-methyl- 5-bromouracil and with 1-methyl-5fluorouracil. Both structures have been determined by three-dimensional X-ray diffraction methods and refined by block diagonal least squares. 9-Ethyl-2-aminopurine: 1-methyl-5-bromouracil crystals are monoclinic, space group P2&, with a = 11.07 A, 6 = 12.34 A, c = 13.84 A, and ,B = 127’ 2’. The aminopurine and bromouracil bases form a planar complex, joined by two hydrogen bonds; an N-H . . . . 0 bond involving the amino group of 2-aminopurine to the carbonyl oxygen, O(2), of bromoumcil and an N-H . . . . N bond between the ring nitrogens N(1) of 2-aminopurine and N(3) of bromourecil. Adjacent base pairs are related by a center of symmetry and are connected by hydrogen bonds between aminopurine residues. This basic tetramer structure is repeated in the 9-ethyl-2-aminopnrine: I-methyl-5- fluorouracil crystalline complex in a completely different crystal lattice environ- ment. This second structure is monoclinic, space group P2,/c, with a = 8.56 A, b = 21.15 A, c = 7.63 A, and /l = 97” 48’. The base pairing configuration is of the Watson-Crick type in both structures, although certain geometric differences have been noted. These structures are discussed with respect to the preceding structures in this series of papers.

1. Introduction This is the fourth in a current series of papers describing crystal structure studies of purine-pyrimidine hydrogen bonded complexes. Previous papers in this series have described complexes between 9-ethyladenine : l-methyl-5-iodouraoil, g-ethyl- 8-bromoadenine : l-methyL5bromouraci1, 9-ethyl-2-aminoadenine : l-methyl-5 iodouracil, and 9-ethyI-2-aminoadenine : l-methylthymine (Sakore, Tavale & Sobell, 1969; Tavale, Sakore & Sobell, 1969; Sakore, Sobell, Mazza & Kartha, 1969).

t Present address: Centro Di Studio Per Lrt Strutturistica, Chimica, Del Consiglio Nazionale Delle Ricerche, Istituto do Chimica Farmaceutica, Citta Universitarie, Rome, Italy.

27 407

408 F. MAZZA, H. M. SOBELL AND G. KARTHA

These studies have suggested that halogen substitution on the uracil ring may alter

the stabilities of different purine-pyrimidine hydrogen bonded configurations which occur in solution prior to co-crystallization (Miller & Sobell, 1967; Kyogoku, Lord & Rich, 1967). This communication presents additional data in this regard and describes crystalline complexes between 9-ethyl-2-aminopurine : I-methyl-5-bromouracil and 9-ethyl-2-aminopurine : l-methyl-5-fluorouracil. In both cases, a Watson-Crick type base pairing configuration has been found, indicating that this hydrogen bonded configuration is a particularly stable type of association between these compounds. A preliminary communication describing a portion of this work has been published previously (Sobell, 1966).

2. Materials and Methods 9-Ethyl-2-aminopurine, 1-methyl-B-bromouracil and I-methyl-5-fluorouracil were syn-

thesized by the Cycle Chemical Company, Los Angeles, Calif. 1: 1 mixtures of these compounds were mixed in dimethylsulfoxide and allowed to evaporate slowly at room temperature, Both 9-ethyl-2-aminopurine: 1-methyl&bromouracil and 9-ethyl-2-aminopurine : I-methyl-5-fluorouracil complexes form plate-like crystals, and ultraviolet absorption spectra from solutions obtained from washed single crystals indicated 1: 1 complexes.

The unit cell constants and space groups for both structures were obtained initially from 30” precession photographs using CuKa radiation. The cell constants for 9-ethyl-2- aminopurine : I-methyl-5bromouracil were later refined using several high angle reflections measured on a General Electric XRD-5 manual diffractometer. The final crystal data for these structures are as follows:

9-ethyl-2-uminopurine : 1-methyl-5-bromourucil a = Il.07 + 0.004A b = 12.34 + 0.006 A c = 13.84 + 0.008 A /3 = 127” 2’ f 1’ Volume = 1510.7 A3 Space group: P2,/c Density (observed) = 1.60 g/cc. Density (calculated) = 1.58 g/cc.

9-ethyl-2-aminop&?ze: I-methyl-5-fluorouracil a = 8.56 f 0.010 A b = 21.15 + 0.015 A c = 7.63 + 0.010 A f9 = 97” 48’ + 2’ Volume = 1368.6 A3 Space group : P2,/c Density (observed) = 1.49 g/cc. Density (calculated) = 1.48 g/cc.

Both structures contain four asymmetric units per unit cell, each asymmetric unit consisting of a 1: 1 9-ethyl-2-aminopurine: 1-methyl-&bromo- (or fluoro-) uracil base pair. Intensity data for the 9-ethyl-2-aminopurme: I-methyl-5-bromouracil crystal structure were collected on a General Electric XRD-5 diffractometer using the stationary crystal- stationary counter method (Furnas & Harker, 1955) with MoKa radiation, with the balanced filter (Zr against Y) technique. 2683 reflections were recorded to a maximum 26 angle of 50”, representing about 10% of the data theoretically accessible in the molybdenum sphere of reflection. An empirical absorption correction was applied which was a function of the 4 angle. Intensity data for the 9-ethyl-2-aminopurine: I-methyl-5-fluorouracil crystal structure were collected on an integrating Nonius Weissenberg camera and estimated visually using the multiple film technique. 2560 reflections were recorded, of which 2211 were non-zero. No absorption correction was applied. Computations were done on IBM 360-65 and 7040 digital computers.

2-AMINOPURINE-BROMO- AND -FLUOROURACIL COMPLEXES 401)

3. Results (a) 9-Ethyl-2-aminopurine: I-methyl-5-bromouracil

The 9-ethyl-2-aminopurine : 1-methyl-5-bromouracil structure was determined by the heavy atom method. An unsharpened Patterson function was calculated and this revealed interatomic vectors between bromine atoms between symmetry related molecules. A Fourier synthesis phased on the bromine atom positions suggested a trial structure, and this was subsequently confirmed by a second Fourier synthesis. The structure was then refined isotropically by block diagonal least squares to a

residual of 14.2%. The bromine atom positions were next refined anisotropically and this reduced the residual to 13h1o/o. Finally, all non-hydrogen atoms were given anisotropic temperature factors, and after several cycles of block diagonal least squares refinement the unweighted residual was 0.12, the weighted residual was O-13, reflections weighted according to a weighting scheme based on counting statistics, as described by Stout & Jensen, (1968). At this point, a difference Fourier synthesis was calculated, and an attempt was made to locate hydrogen atom positions. Al- though nine peaks were located in expected hydrogen atom positions, numerous other peaks were observed and it was therefore decided not to assign these as hydrogen atom positions. Tables 1, 2 and 3 show the final co-ordinates and temperature parameters obtained from this structure analysist.

TABLE 1

Final co-ordinates jar 1: 1 9-ethyl-2-aminopurine : I-methyl-5-bromouracil crystal structure after block diugonal lea&squares rejnement

Atom S-Ethyl-2-aminopurine

x/a Ylb +

N(l) C(2) NW N(3) C(4) C(5) C(6) N(7) C(8) NW C(9) C(lO)

N(1) C(l) C(2) w4 N(3) C(4) C(4) C(5) ‘76) Br(5)

0.2496(09) O-4756(06) O-0833(07) 0.3389( 12) 0.4766(08) 0~0453(09) 0.3965( 11) 0*5782(07) 0.0506(09) 0*3674(10) 0*3926(06) O.OOOS(OS) 0.3035(12) 0~3005(08) -0~0013(09) O-2127(12) 0.2875(08) 0*0378( 10) 0.1883(12) 0.3819(08) O-0785(09) O-1676( 11) 0.1832(07) 0.0242(09) 0.2259( 16) 0.1337(09) -0*0217(12) 0.3037( 11) 0.2033(06) -0.0438(09) 0.3747114) 0.1726(11) -0.1024(11) 0.5385(15) 0.1529(10) -0.0055(13)

1-Methyl-5-bromouracil 0*1996(10) 0*8280(06) 0*2222(08) 0.2359( 15) 0*9401(09) 0.2094( 13) 0.2390(11) O-7437(07) 0.1812(09) 0.3164(09) 0.7569(06) 0.1465(08) 0.1892(09) 0.6436(07) 0.1846(08) 0*1137(10) 0.6186(08) 0.2317(09) 0.0797(09) 0.5226(05) O-2304(07) 0.0811(10) 0.7088(08) O-2738(08) 0*1202(11) O-8046(08) 0.2664(09)

-0.0156(01) 0.6837(01) 0.3466(01)

The estimated standard deviations have been calculated from diagonal elements of the inverse matrix and are shown in parenthesis ( x 104).

t The observed and calculated structure factors for this structure and the following one have been microtilmed and stored at ASIS of Auxiliary Publication Service, 22 West 34th Street, New York, N.Y. 10001, under document number 00438.


TABLE 2

Anisotropic temperatureparametersfor the 9-ethyl-Z-aminopurin : I-methyl-5-bromouracil crystal structure after block diagonal least-squures re$nement

Atom B 11 Baa 9-Ethyl-2-aminopurine

833 B la B 13 B 23

N(l) 0*0170(13) 0~0051(06) cc3 0~0166(16) 0~0051(07) N(2) 0.0261(17) 0*0043(06) N(3) 0.0175(14) 0*0059(06) C(4) 0.0161(17) 0~0045(07) C(b) 0.0173(18) 0*0045(07) W) 0*0168(17) 0*0056(07) N(7) 0*0215(17) 0*0047(06) ‘78) 0*0262(25) O-0049(08) W-9 0*0190(16) 0~0040(05) C(9) 0.0182(20) 0*0103(11) WO) 0.0178(21) 0.0064(09)

N(1) 0.0183(14) C(l) 0*0228(23) C(2) O*OlOS( 14) O(2) 0*0220(14) N(3) 0*0134(12) C(4) 0.0107(13) G(4) 0.0235( 14) C(S) 0~0106(13) c(‘3) 0*0148(15) Be) 0*0167(02)

0~0033(06) 0.0062(07) 0*0045(06) 0.0063(06) 0~0066(06) 0*0058(07) 0.0044(04) 0*0049(07) 0.0031(06) 0~0071(01)

0*0097(08) -0.0024(14) 0*0085(10) -0~0018(17) 0.0170(12) 0.0002( 16) 0~0112(09) 0~0040(14) 0~0092(10) -0*0004( 17) 0*0104(11) -0.0012(17) O-0099( 10) -0*0003(17) 0*0134(11) 0*0003( 17) 0*0157(16) -0~0009(24) 0~0136(11) -0.0025(15) 0.0104(12) 0.0019(25) 0*0168(16) O~OOll(23)

I-Methyl-5-bromouraoil 0~0135(10) O.OOOS( 14) O-0226( 18) -0.0066(21) O-0076(09) -0.0037(16) 0~0136(10) -0*0016(15) 0.0094(09) 0*0018(14) 0~0089(10) 0.0030(16) 0~0173(10) 0*0006(13) 0.0063(08) 0*0029( 15) 0~0110(10) 0*0025( 16) 0~0115(01) 0*0018(02)

0.0217(19) 0.0181(22) 0.0368(27) 0*0227(21) 0*0192(24) 0.0221(25) 0.0210(23) 0.0280(25) 0.0318(36) O-0265(26) 0.0209(28) 0.0202(32)

0*0236(22) 0.0349(37) 0.0113(21) 0.0281(21) 0*0163(18) 0.0142(20) 0.0350(21) 0~0104(18) 0*0184(22) 0*0218(02)

-0~0033(11) -0~0013(13) -0~0026(13)

0*0020( 12) 0~0014(13) 0.0002(14) 0~0013(13) 0.0004(13)

-0~0008(18) -0*0037(12)

0*0025(20) 0.0034(20)

0*0002( 12) -0.0025(18) -0.0025(13) -0.0003(12) -0~0009(12)

0.0012(13) 0*0006( 11) 0.0020( 12)

-0~0017(13) 0~0000(02)

Estimeted standard deviations we shown in parenthesis ( x 104). The temperature parameters shown we coefficients in the expression,

T = exp{-UMa + B& -t Ma f B&k + 8& + B&Oh

TABLE 3 Principal azes of thermal ellipsoids for the 9-ethyl-2-aminopurine:

I-methyl-5-bromouracil crystal structure

Atom i u,(A) 9-Ethyl-2-aminopmke

CO%&2 COf%b costc

N(l) 1 2 3

C(2) 1 2 3

N(2) 1 2 3

N(3) 1 2 3

C(4) 1 2 3

0.16 - 0.7465 0.3786 0.5472 0.20 0.4560 0.8899 0.0063 0.28 0.4846 -0.2543 0.8370 0.17 0.8045 -0.0283 -0.5933 0.20 -0*1410 -0.9794 -0.1444 0.26 -0.6770 0.1998 -0.7919 0.16 -0.5247 0.7971 O-2989 0.22 -0.7353 -0.6013 0.3127 0.35 0.4290 -0.0558 0.9016 0.18 -0.8699 o-3497 o-3479 0*21 0.2032 0.8967 -0.3933 0.29 0.4496 0.2714 0.8510 0.16 0.6407 0.6282 -0.4414 0.20 0.6474 -0.7769 -0*3111 0.26 -0.5383 - 0.0423 -0.8417

2-AMINOPURINE-BROMO- AND -FLUOROURACIL COMPLEXES 411

TABLE 3-cmtinued

Atom i 9-Ethyl-2-aminopurinene-conlinued

u&Q CO% CO% CO&c

C(5)

C(6)

N(7)

W3)

N(9)

C(9)

C(lO)

N(l)

C(1)

w

WV

N(3)

C(4)

C(4)

(35)

C(‘3)

Br(51

1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3

1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3

0.16 0.7781 0.4614 - 0.4262 0.19 0.3805 -0.8861 - 0.2645 0.27 -0.4997 0.0436 -0.8651 0.17 0.7915 0.3650 -0*4901 0.21 0.3381 -0.9297 -0.1464 0.27 -0.5091 - 0.0498 -0.8593 0.19 -0.3919 -0.8916 0.2269 0.19 0.7920 - 0.4524 -0.4099 0.31 -0.4681 -0.0190 -0.8835 0.19 0.0057 -0.9988 -0.0491 0.23 0.8608 0.0299 -0.5081 0.33 -0.5090 o-0394 -0.8599 0.16 0.4387 -0.8383 -0.3236 0.19 -0.8348 -0.5135 0.1985 0.31 -0.3326 0.1831 -0.9251 0.20 0.8026 0.1291 -0.5823 0.26 0.5347 -0.5884 0.6066 0.29 0.2643 0.7982 0.5413 0.21 -0.0775 -0.9804 0.1813 0.26 -0.9176 - 0.0009 -0.3974 0.34 O-3898 -0.1972 -0.8995

1-Methyl-5-bromouracil 0.16 -0.0966 0.9953 0.0064 0.22 -0.9572 -0.0947 0.2734 0.29 0,2727 0.0203 0.9619 0.17 0.4968 0.8660 0.0559 0.26 0.8663 -0.4911 -0.0911 0.38 -0.0514 0.0937 -0.9943 0.17 0.4203 0.8267 0.3741 0.20 -0.8597 0.2310 0.4555 0.23 0.2901 -0.5131 0.8078 0.20 -0.8348 -0.3446 0.4293 0.22 -0.2732 0.9364 0.2203 0.31 o-4779 -0.0667 0.8759 0.18 -0.8469 0.4533 0.2780 0.23 0.4401 0.8908 -0.1121 0.24 0.2985 -0.0274 0.9540 0.17 0.9157 -0.4015 -0.0150 0.21 0.3560 0.8281 -0.4329 0.24 0.1862 0.3911 0.9013 0.18 O-9348 -0.0561 -0.3507 0.18 -0.0443 -0.9981 0.0416 0.34 -0.3524 -0.0234 -0.9355 0.17 0.2683 -0~7800 0.5653 0.19 0.8224 -0.1202 -0.5561 0.22 -0.5017 -0.6141 -0-6092 0.13 0.4422 - 0.8842 -0.1507 0.22 -0.8785 -0.4608 0.1258 0.26 -0.1806 0.0768 -0.9805 0.19 -0.8951 0.2875 0.3408 0.24 0.2260 0.9514 -0.2091 0.28 0.3844 0.1101 0.9166

u! corresponds to the root-mean-squared displacement along the ith principal axis of the . elhpsold, and COB,,, cosIb and COB,,, the direction cosines which the ith axis makes with respect to the crystallographic axes, a, b and c*.


Figure 1 shows a clinographic view of the structure showing molecules in one unit cell and neighboring unit cells. Two 1 : 1 9-ethyl-2-aminopurine: l-methyl- 5-bromouracil dimers are hydrogen bonded together through aminopurine residues to form a tetramer structure shown in dark outline. Other symmetry-related molecules

FIG. 1. A olinographic view of the 9-ethyl-2-aminopurine: I-methyl-5-bromouracil crystal structure. One hydrogen-bonded tetramer structure is shown with dark lines, while lighter lines show symmetry related molecules. The largest circle represents the bromine atom.

are further behind and are shown with lighter lines. It is seen that the aminopurine- bromouracil complex is planar and involves two hydrogen bonds between bases ; an N-H. . . N bond connects the N(3) bromouracil ring nitrogen proton with the N(3) aminopurine ring nitrogen (2.80 A), and an N-H . , . 0 bond connects the amino group on aminopurine with the O(2) bromouracil carbonyl oxygen (2.97 A). Adjacent 2-aminopurine residues from weaker hydrogen bonds across a center of symmetry, as evidenced by the N-H . . . N bond length (3.11 A). The base-pairing configuration which is observed between these compounds is very similar to that occuring between 9-ethyl-2-aminopurine : l-methyl-5-fluorouracil, although the latter crystallizes in a completely different lattice structure. This suggests that this base pairing configuration is a particularly stable one between these compounds, and this will be discussed later.

Figure 2 shows a schematic diagram of the complex with bond distances and angles as obtained from this analysis. The estimated standard deviations are O-015 A for bond distances and 1” for bond angles involving light atomst. There is good

t These estimated standard deviations are calculated from co-ordinate standard deviations which were estimated from diagonal elements of the inverse matrix after anisotropic block diagonal lea&-squares refinement.


\ 1090

FIU. 2. A schematic diagrem of the 9-ethyl-2-aminopurine: 1-methyl-5-bromouraoil base pair showing bond distances and angles, aa well as other distances of interest. Dashed lines represent hydrogen bonds. A portion of a symmetry related 2-aminopurine residue is also shown.

agreement between the bond distances calculated for this structure and for the 9-ethyl-2-aminopurine: l-methyl-5-fluorouracil structure, described below.

Least-squares planes have been calculated for the individual purine and pyrimidine rings, as well as for the complex, and the results of these calculations are summarized in Table 4.

(b) 9-Ethyl-2-amin~urine: I-methyl-5,fEuorouracil

The 9-ethyl-2-aminopurine: 1-methyl-5-fluorouracil structure was determined by Patterson superposition methods. A three-dimensional sharpened Patterson function was calculated and this revealed sheets of hexagonal peaks in the (102) plane. A sheet-like structure whose molecular plane was oriented very close to the (102) plane was suggested by the magnitude of this reflection, and by its interplanar spacing (3.54 A). A trial model was constructed which closely resembled the base pairing configuration which was found in the previous structure. Adjacent dimers were related to each other by a center of symmetry, forming hydrogen bonds between the aminopurine residues. This tetramer structure was then oriented in the (102) plane, careful consideration being given to the orientation of hexagonal peaks surrounding the origin of the Patterson function. Inter-ring vectors between six- membered rings in aminopurine and fluorouracil could be predicted from the model


TABLE 4

Least-squares plane data for the 9-ethyl-2-aminopurine : I-methyki-bromouraeil crystal structure

Least-squares plane through the 9-ethyl-2aminopurinna molecule, excluding C( 10). -0.3456 X + O-2204 Y - 0.9121 2 - 0.3002 = 0

Distances of atoms from this least-squares plane:

Atom Distance from plane (A) N(1) -0.040 C(2) I 0.026 N(2) 0.008 N(3) 1 0.042 C(4) o-034 WY1 0.003 C(6) -0.054 N(7) 0.017 (-33) 0.044 NW - 0.007 C(9) -0.073

Least-squares plane through I-methyl-5-bromouracil molecule.

-0.3992 X + 0.1111 Y - 0.9101 2 - 1.2368 = 0


Atom Distance from plane (A) N(l)1 0.004 C(l)], - 0.075 WV 0.018 O(2) 0.109 N(3) -0.042 C(4) -0.024 G(4) -0.062 C(5) - 0.009 ‘33) -0.017 W5) 0.088

Dihedral angle between this plane and previous plane: 7.0”

Least-squares plane through complex (excluding C(10)): -0.3928 X + 0.1791 Y - 0.9020 2 - 0.6088 = 0

The planes were calculated according to the method of Blow (1960). X, Y and Z directions correspond to the a, b and c* crystallographic directions.

structure, and heavy peaks corresponding to these were observed in the Patterson function. Further consideration of the Patterson function revealed that this basic dimer structure could explain the majority of the peaks and rings in the (102) three- dimensional Patterson section, and it was decided to calculate structure factors based on this trial model. The structure was refined isotropically using full matrix least squares to a residual of O-18 (Sobell, 1966). Anisotropic re6nement was then carried out using block diagonal least squares and this reduced the residual to 0.12%. At this point, a difference Fourier synthesis was calculated and this clearly revealed the positions of all hydrogen atoms (see Fig. 3). When these hydrogen atoms were included in the final structure-factor calculation, the residual was O~ll”/o. Tables 6, 6, 7 and 8 show the final co-ordinates and temperature parameters which have been obtained from this structure analysis.


a

Fro. 3. A difference Fourier synthesis showing hydrogen atom electron density contours for the 9-ethyl-2-aminopurine: 1-methyl-5.fluorouracil crystal structure. Contours have been drawn at approximately 0.3 electron/A3 intervals.

TABLE 5

Find co-ordinates for 1: 1 9-ethyl-Z-aminopurine : I-methyL5,fluorouracil crystal structure after block diagonal least-squares re$nement

Atom x/a 9-Ethyl-2-aminopurine

ylb Z/C

N(l) C(2) N(2) N(3) C(4) C(5) C(6) N(7) C(8) NM C(9) WO)

N(1) (31) cc3 O(2) N(3) C(4) O(4) (25) f-36) F(5)

0.2678(06) 0.1031(02) 0*3190(10) 0.1569(08) 0.0671(03) 0.3857( 12) 0*1758(07) 0.0042(03) 0.3767( 11) 0.0328(06) 0.0883(02) 0.4575(09) 0.0235(08) 0*1509(03) 0.4598( 12) 0*1287(08) 0.1929(03) 0.3944( 12) 0.2525(08) 0.1659(03) 0.3244( 13) 0.0825(07) 0.2553(02) 0.4244(10)

-0.0409(09) 0.2496(03) 0*4957(12) -0.0881(07) 0.1869(02) 0.5239(09) -0*2193(09) 0.1627(03) 0.6118(13) -0.3564( 10) 0.2069(04) 0.5957( 17)

1-Methyl-5-fluorouracil 0*6886(06) -O-0473(02) 0.1665(10) O-6996(10) -0~1170(03) 0.1635(14) 0.5620(08) -0*0202(03) 0.2282( 13) 0.4589(06) -0.0519(02) 0.2806(09) 0*5560(06) 0.0446(02) 0.2220( 10) 0*6648(08) 0.0843(03) 0.1614(13) 0.6467(06) 0.1415(02) 0.1599(09) 0.7924(08) 0*0510(03) 0*1017(13) 0*8015(08) -0~0109(03) 0.1062(13) 0.9031(05) O.OSSS(O2) 0.0402(08)

The estimated standard deviations have been calculated from diagonal elements of the inverse matrix and are shown in parenthesis (X 104).


TBLE~ Anisotropic temperature parameters for the 9-ethyl-Z-aminopurine:

I-methyl-5-jluorouracil crystal structure after block diugonul least-squares rejinement

9.Ethyl-2-aminopurine B 33 B 12 B 13 B 23

N(l) cm NW N(3) C(4) C(5) C(0) N(7) C(8) N(9) C(9) C(lO)

N(1) (31) C(2) O(2) N(3) C(4) O(4) C(5) C(6) F(5)

0.0117(08) 0~0140(11) 0.0152( 10) 0*0116(08) 0*0126(10) 0~0141(11) 0~0115(11) 0.0174(10) 0.0188(14) 0*0146(09) 0*0174(13) 0.0208(15)

0.0112(08) 0.0180(14) 0*0127(11) 0*0138(08) 0~0110(08) 0.0132(12) 0*0188(10) 0~0129(11) 0.0124( 12) 0*0166(08)

0~0019(01) 0*0014(01) 0*0016(01) 0~0014(01) 0~0014(01) 0*0016(01) 0~0020(02) 0~0014(01) 0~0015(01) 0~0014(01) 0.0018(02) 0.0023(02)

0~0016(01) 0*0016(01) 0*0017(01) 0~0019(01) 0.0016(01) 0~0017(01) 0~0010(01) 0*0023(02) 0~0026(02) 0*0028(01)

0*0434(21) -0*0003(05) 0.0401(25) 0~0004(06) 0*0514(23) 0*0013(05) 0.0408( 19) -0~0010(05) O-0384(24) -0~0009(06) O-0376(25) -0~0012(06) 0.0444(27) -0~0010(06) 0.0417(20) -0~0013(05) 0.0349(23) -0~0004(07) 0.0390(19) -O.OOOS(OS) 0.0413(27) 0*0002(07) 0.0652(40) 0*0028(09)

1-Methyl-5-fluorourecil 0*0408(20) O~OOlS(O5) 0*0492(31) 0*0027(07) 0.0433(27) 0~0009(07) 0.0519(19) 0~0004(05) 0.0432(20) 0~0019(06) 0.0427(27) 0~0011(07) O-0535(21) 0~0007(05) 0*0430(28) 0~0000(07) 0*0424(27) 0~0030(07) 0.0538( 18) -0~0010(05)

0.0084(21) 0.0061(27) 0.0227(24) 0.0053(20) 0.0034(24) 0.0017(26) 0.0081(27) 0.0059(23) 0.0042(28) 0.0049(20) 0~0131(30) 0.0253(40)

0*0041(20) O-0072(23) O-0042(28) 0*0149(19) 0.0082(20)

-0.0011(28) 0.0132(23) 0.0076(28) O-0096(28) 0.0226(19)

0*0032(08) 0~0017(09) O*OOOO(OS) 0*0000(07)

-0~0007(09) 0~0020(09) 0.0025(10) 0*0003(08)

-0~0013(09) -0~0010(07) -0~0004(11)

0~0014(15)

-O.OOOl(OS) 0*0017(11)

-O*OOOS(lO) 0~0006(07) 0*0006(08) 0.0012( 10) 0.0006(08) 0~0001(11) 0*0012(11) 0*0036(07)

Estimated standard deviations are shown in parenthesis ( x 104). The temperature parameters shown are coefficients in the expression,

T = exp{ - (I%&~ + FM2 + ha + B&k + A& + B&4 1.

TABLE 7 Positional parameters for hydrogen atoms in 9-ethyl-Z-aminopurine :

I-methyl-5-jluorouracil crystal structure as obtained from diflerence Fourier synthesis

9-Ethyl-2-aminopurine Atom da ylb

HP4 0.2503 -0~0093 ‘Wb) 0.1339 -0.0234 H(6) 0.3142 0.1914 H(8) -0.0885 0.2891 JWW -0.2585 0.1266 W’b) -0.1879 o-1429 H(lOa) -0.3781 0.2448 H( lob) -0.4101 O-1829 H(lOc) -0.3286 0.2436

I-Methyl-5-fluorourail HP4 0.6994 -0.1398 Htlb) 0.7751 -0.1247 H(lc) 0.5946 -0.1344 H(3) 0.4803 0.0617 H(6) 0.8765 -0.0304

0.3437 0.4693 0.2449 0.5616 0.5283 0.7189 0.5247 0.6791 0.7107

0.2700 0.1152 0.0920 0.2303 0.0485


TABLE 8

Principal axe+s of thermal ellipsoids for the g-ethyl- Z-aminopurine : 1-methyl-5-jluorouracil crystal structure

Atom 9.Ethyl-2-aminopurine

i %@) cwa CO% COSIC

N(l)

C(2)

N(2)

N(3)

C(4)

C(6)

N(7)

CR’)

N(9)

C(9)

N(l)

C(l)

w

1 2 3

1 2 3

1 2 3

1 2 3

1 2 3

1 2 3

1 2 3

1 2 3

1 2 3

1 2 3

1 2 3

1 2 3

1 2 3

1 2 3

1 2 3

o-19 - 0.5799 0.21 0.8134 0.36 0.0447

0.17 - 0.0606 0.22 0.9943 0.34 -0.0875

0.18 - 0.4135 0.22 0.8960 o-39 0.1618

0.17 0.21 0.34

- 0.3700 - 0.9239

0.0974

0.17 -0.2443 0.22 -0.9588 0.33 o-1449

0.18 -0.2899 0.23 -0-9328 0.33 0.2142

0.19 -0.7658 0.22 0.6405 0.36 0.0568

0.17 0.1765 0.25 0.9769 0.35 -0.1207

0.18 0.0416 0.26 0.9782 0.32 - 0.2034

0.17 -0*1118 0.23 -0.9864 0.34 0.1204

0.20 - 0.0605 0.25 0.9945 0.35 0*0850

0.21 -0.4628 0.27 0.8767 0.44 0.1310

- 0*8090 0.0954 -0.5687 0.1223 -0.1486 - 0.9879

0.9946 - 0*0838 0.0675 0.0822 0.0784 0.9931

0.9084 0.0615 0.4180 -0.1499 0.0069 O-9868

- 0.9284 - 0.0329 0.3715 -0.0918

-0.0036 -0.9952

-0.9678 - 0.0610 0.2505 -0.1339 0.0257 -0.9891

- 0.9554 0.0558 0.2690 -0.2399

-0-1216 - 0.9692

-0.6421 0.0333 - 0.7573 0.1277 -0.1191 -0.9913

0.9843 -0.0033 -0.1748 0.1232

0.0250 0.9924

0.9955 0*0848 - 0.0579 0.1993 -0.0747 0.9762

- 0.9919 -0.0594 0.1180 -0.1143 0.0458 -0.9917

0.9979 0.0233 0.0622 -0.0837

-0~0180 0.9962

0.8863 0.0140 0.4600 -0.1405 0.0528 0.9900

I-Methyl-5-fluorouracil 0.17 0.5885 -0.8064 0.22 - 0.7991 -0.5911 0.35 0.1229 0.0179

0.18 - 0.3304 0.9403 0.26 0.9394 0.3359 0.38 -0.0909 0.0541

0.19 -0.3614 0.9324 0.22 0.9232 0.3687 0.36 -0.1306 - 0.0446

0.0583 -0.1096 - 0.9923

-0.0813 0.0676 0.9944

- 0.0056 0.1379 0.9904


TABLE S-continued

Atom 1-Methyl-5-fluorouracil-coru%~ed

i 4‘Q CO%. CO%lJ CO%

(32) 1 0.20 -0.2686 0.9631 -0.0133 2 0.22 0.9625 0.2679 -0.0434 3 0.39 0.0383 0.0244 0.9990

N(3) 1 0.17 - 0.5983 0~8000 -0.0441 2 0.22 0.8002 0.5995 0.0192 3 0.35 -0.0418 0.0238 0.9988

C(4) 1 0.19 0.4383 -0.8874 0.1431 2 0922 -0.8674 -0.4593 -0.1914 3 0.36 0.2355 -0.0402 -0.9710

O(4) 1 0.19 0*0913 -0.9956 0.0194 2 0.26 -0.9958 -0.0912 0.0077 3 0.39 - 0.0059 -0~0200 -0.9998

C(5) 1 0.22 0.9982 o-0099 0.0596 2 0.23 -0.0095 0.9999 -0~0070 3 0.35 -0.0597 0.0064 0.9982

C(6) 1 0.19 0.8597 - 0.5093 0.0401 2 0.26 -0.5108 -0.8574 0.0622 3 0.35 0.0027 -0.0739 -0.9973

F(5) 1 0.22 0.8473 0.4963 -0.1892 2 0.26 -0.5130 0.8570 -0.0492 3 0940 0.1377 0.1387 0.9807

U, corresponds to the root-mean-squared displacement along the ith principal axis of the ellipsoid, and COQ, coatb and cosCo the direction cosines which the ith axis makes with respect to the crystallographic axes, a, b and c*.

= ~,Pd,e ----- Bottom

FIG. 4. The 9-ethyl-2-aminopurine: I-methyl-5-fluorouracil crystal structure viewed perpendicular to the (102) plane showing overlapping of molecules in adjacent sheets. Dashed lines indicate hydrogen bonds.


The structure has already been described in some detail (Sobell, 1966). Adjacent base pairs are related across centers of symmetry and are connected together by hydrogen bonds between aminopurine residues to form a tetramer structure. This tetramer structure then packs together to form an interlocking sheet structure. There is little direct stacking between purine and pyrimidine rings in adjacent sheets. This is shown clearly in Figure 4, which shows the structure viewed perpendicular to the (102) plane. Except for the nucleoside complexes which display stacking between adjacent purine residues and pyrimidine residues (Haschemeyer & Sobell, 1963, 196&z; Haschemeyer & Sobell, 1964, 1965b), the alkylated purine-pyrimidine complexes generally show little base stacking, perhaps indicating the relative magnitude of stacking forces compared with hydrogen bonding in stabilizing these crystal structures.

Figure 5 schematically shows the complex with the bond distances and angles as obtained from this analysis. The estimated standard deviations are 0.01 A for bond

FIG. 6. A schematic diagram of the 9-ethyl-2aminopurine: I-methyl-5-fluoroumcil base pair showing bond distcmces and angles, 8~ well as other distances ofinterest. Dashed lines represent hydrogen bonds. A portion of 8 symmetry related 2-aminopurine residue is also shown.

distances and O-8’ for bond angles involving light atoms. The bond distances and angles are in good agreement with those calculated for the preceeding structure.

Least-squares planes have been calculated for the individual purine and pyrimidine rings, as well as for the complex, and the results of these calculations are summarized in Table 9.


TABLE 9

Least-squares plane data for the 9-ethyl-Z-aminopurine: l-methyl-5;1Zuorouracil crystal structure

Least-squares plane through the 9-ethyl-2aminopurine molecule, excluding C( 10).

-0.4438 X - 0.0130 Y - O-8961 2 - 3.0472 = 0


Atom Distance from plane (A) N(1) 0.013 C(2) 0.003 NW 0.000 N(3) -0.010 C(4) -0.013 C(5) -0.015 C(6) 0.006 N(7) 0.016 C(8) -0.004 NW -0.023 C(9) 0,027

Least-squares plane through I-methyl-5-fluorouraoil molecule.

-0.3871 X - 0.0372 Y - 0.9213 2 - 3.3266 = 0


Atom Distance from plane (A) N(1) 0.011 C(l) -0.027 WI 0.018 O(2) - 0.005 N(3) 0*008 C(4) 0.002 O(4) -0.023 C(5) 0.007 C(f-3 0.011 F(5) -0.002

Dihedral angle between this plane and previous plane: 3.8”

Least-squares plane through complex, excluding C(10) :

-0.3755 X - 0.0325 Y - 0.9263 2 - 3.2300 = 0

The planes were calculated according to the method of Blow (1960). X, Y and 2 directions correspond to the a, b and c* crystallographic directions.

4. Discussion

It has previously been suggested that halogen substitution on the uracil ring could be an important factor altering the stabilities of various purine-pyrimidine hydrogen bonded configurations which exist in solution prior to co-crystallization (Sakore, Tavale t Sobell, 1969; Tavale et al., 1969; Sakore, Sobell, Mazza & Kartha, 1969). This study was undertaken to see whether similar base-pairing changes occur when the closely related adenine analog compound, 9-ethyl-2-aminopurine, is co- crystallized with different halogenated and non-halogenated uracil derivatives. Since it has not yet been possible to form crystalline complexes between g-ethyl- 2-aminopurine and 1-methylthymine (or 1-methyluracil) however, no statement can be made concerning the possible perturbing effect which halogen substitution


FIG. 6. A schematic diagram which shows the slight difference in base pairing cotigurcttions for both structures. The base pair outlined with heavy lines represents the 9-ethyl-2aminopurine : I-methyl-5-bromouracil complex, while the lighter lines represent the 9-ethyl-2-aminopurine: 1-methyl-5-fluorouracil structure. See text for discussion.

may have in the base pairing interaction. Both 9-ethyl-2-aminopurine: l-methyl- 5-bromouracil and 9-ethyl-2-aminopurine : l-methyl-5-fluorouracil complexes in- volve Watson-Crick type base-pairing configurations and form similar planar tetramer structures in different crystal lattice environments. This suggests that this base-pairing configuration is a particularly stable interaction between these compounds, more long range lattice forces being only of secondary importance in this regard. Although the geometry of both base pairs is very similar, it is worthwhile to note that there are significant differences between them. The interglycosidic separation (that is, the C(1) - C(9) distance) for the 9-ethyl-2-aminopurine: l-methyl- ii-bromouracil base pair is 1090 A, while it is 10.75 A for the 9-ethyl-2-aminopurine : l-methyl-5-fluorouraoil base pair. This reflects the slight difference in purine- pyrimidine ring orientations in these complexes (shown in Fig. 6) and is due to a shortening in the N-H . . . N hydrogen bond length and a lengthening of the N-H . . . 0 hydrogen bond length in the aminopurine-bromouracil complex compared with the aminopurine-fluorouracil complex (2.80, 2.94 A; 2.97, 2+38 A). This also explains the differences in angles between the glycosidic bonds and the C(1) - C(9) interatomic vector, i.e. 49.4”, 46.5”; 52-P, 54.0”. The shortness of the N-H . . . N hydrogen bond in the aminopurine-bromouracil structure probably reflects strong association between these compounds, and this has been observed between similar compounds in solution (Miller & Sobell, 1967). There is at present no data concerning the aminopurine-fluorouracil solution interaction; however, the crystal data suggest that this interaction may be considerably weaker and infrared solution studies would be of interest in this regard.

1.Methyl-5bromouracil and l-methyl-5-fluorouracil have been shown to form crystalline complexes with 9-ethyladenine (Katz, Tomita & Rich, 1965; Tomita, Katz & Rich, 1967). In addition, 9-methyladenine and 9-ethyl-8-bromoadenine form complexes with l-methyl-5-bromouracil (Baklagine, Volkenshtein t Kondrashev,


1966; Tavale et aZ., 1969). Of these structures, only 9-ethyl-Sbromoadenine: l-methyl- 5-bromouracil involves base pairing on the N(1) adenine side, forming a “reversed” Watson-Crick type base pair. Hydrogen bonding in this structure partly involves the N(6) adenine amino group interacting with the O(2) carbonyl oxygen on bromouracil. In the 9-ethyl-2-aminopurine : 1-methyl-5.bromouracil structure, one sees a similar interaction involving the N(2) amino group on aminopurine and the O(2) bromouracil carbonyl oxygen. Although one can attempt to relate these pairing differences with the inductive effect accompanying halogen substitution on the pyrimidine ring, an effect such as this cannot explain all base pairing configurations which have been observed (see, for example, Sobell, 1966; Tomita et al., 1967). More complete quantum mechanical calculations are required to give quantitative explanations of the forces involved in these base pairing studies.

This work has been supported in part by the National Institutes of Health, U.S. Public Health Service, the National Science Foundation, the American Cancer Society, and an institutional grant to the University of Rochester from the American Cancer Society. Computations were done on the IBM 360-65 computer at the Computation Center at the University of Rochester, and the IBM 7040 computer at the Roswell Park Memorial Institute, Center for X-ray Crystallographic Research, for which we thank Dr David Harker. Facilities for part of this work were provided by the U.S. Atomic Energy Com- mission at the University of Rochester Atomic Energy Project and this paper has been assigned Report no. UR-49-1096. One of us (F.M.) gratefully acknowledges a travel grant from the Italian Minister0 degli Esteri and Consiglio Nazionale delle Ricerche (Rome) to come to the United States. Two of us (H.M.S. and G.K.) are recipients of Research Career Development Awards from the National Institutes of Health, U.S. Public Health Service.

REFERENCES

Baklagine, Y. G., Volkenshtein, M. V. & Kondrashev, Y. D. (1966). Zhuvnal Strukurnoi Khimii, 7, 399.

Blow, D. M. (1960). Acta Cry&. 13, 168. Furnas, T. & Harker, D. (1955). Rev. Sci. In&rum. 26, 449. Haschemeyer, A. E. V. & Sobell H. M. (1963). Proc. Nat. Acad. Sci., Wash. 50, 872. Hsschemeyer, A. E. V. & Sobell, H. M. (1964). Nature, 202, 969. Haschemeyer, A. E. V. & Sobell, H. M. (1965a). Actu Cry& 18, 525. Haschemeyer, A. E. V. & Sobell, H. M. (19655). Actu Cry&. 19, 125. Katz, L., Tomita, K. & Rich, A. (1965). J. Mol. Biol. 13, 340. Kyogoku, Y., Lord, R. C. & Rich, A. (1967). Proc. Nat. Acad. Sci., Wash. 57, 250, Miller, J. H. & Sobell H. M. (1967). J. Mol. BioZ. 24, 345. Sakore, T. D., Sobell, H. M. Mazza, F. & Kartha, G. (1969). J. Mol. BioZ. 43, 385. Sakore, T. D., Tavale, S. S. & Sobell, H. M. (1969). J. Mol. BioZ. 43, 361. Sobell, H. M. (1966). J. Mol. BioZ. 18, 1. Stout, G. H. & Jensen, L. H. (1968). X-ray Structure Determination. A Practical Guide.

New York : Macmillan. Tavale, S. S., Sakore, T. D. & Sobell, H. M. (1969). J. Mol. Biol. 43, 375. Tomita, K., Katz, L. & Rich, A. (1967). J. Mol. BioZ. 30, 545.

Documents

Base-pairing configurations between purines and pyrimidines in the solid state