Advances in Molecular Relaxation and Interaction Processes, 17 (1980) 113-134 Elsevier Scientific Publishing Company, Amsterdam -Printed in Belgium

113

Solution and Solid State Studies on the Interactions of Protonated Cytosine Salts.

IV. Asymmetric Interbase Hydrogen Bonding and Interpyrimidine Base Stacking in

Triply Hydrogen-Bonded Cytosine Complexes. Crystal and Molecular Structure of

Bis[l-Methylcytosine, 1-Methylcytosinium] Hexafluorosilicate Dihydrate

THOMAS J. KISTENMACHER, MIRIAM ROSSI, CHIAN C. CHIANG, JOHN P. CARADONNA AND

LUIGI G. MARZILLI

Department of Chemistry, The Johns Hopkins University, Baltimore,

Maryland 21218, U. S. A.

(Received 30 January 1980 )

ABSTRACT

The crystal and molecular structuresof the complex bis(l-methylcytosine,

1-methylcytosinium) hexafluorosilicate dihydrate are reported. In the solid, a

1:l triply hydrogen-bonded complex consisting of one protonated and one neutral

1-methylcytosine base is observed. The hydrogen bonding in the complex cation is

asymmetric, and the asymmetry in the interbase hydrogen bonding is stimulated to a

large part by base stacking. The observed infinite, helical array of stacked

complexes, in which the base stacking is strong [mean stacking distance = 3.36A]

is such that the molecular overlap is between protonated and neutral l-methylcyto-

sine bases. The asymmetric interbase hydrogen bonding and the protonated/neutral

base stacking mode coexist in a synergistic interrelationship which maximizes the

molecular and crystal forces. The results found in this study are compared with a

variety of other systems in which asymmetrically hydrogen-bonded cytosine complexes

are observed, and it is found that although base stacking is also a predominant

feature of these complexes in the solid, the base stacking found in this study is

different.

0165.1919/80/0000~000/$0225 D 1980 Elsevier Scientific Publishing Company

114

INTRODUCTION

Protonation or deprotonation of nucleic acid bases has a critical effect on

their hydrogen bonding capabilities and often leads to non-Watson-Crick[l] base

pairing schemes. A particularly interesting example is the pyrimidine ribonucleo-

side cytidine. At neutral pH, the ring nitrogen atom N(3) of cytidine has an

available lone pair and acts as a hydrogen-bond acceptor, notably in the triply

hydrogen-bonded guanosine-cytidine base pairs in duplex regions of DNA and RNA.

At low pH, the N(3) site of cytidine bears a proton which acts as a hydrogen-bond

donor and the formation of guanosine-cytidine base pairing is precluded. However,

N(3)-protonated cytidine can form a triply hydrogen-bonded pair with a neutral

cytidine residue in a fashion exactly analogous to the guanosine-cytidine pair.

Such a cytidine-cytidineH+complex involves a base and its conjugate acid and, in

the absence of outside factors, one expects that the proton will be on either of

the N(3) sites in the pair with random probability[2].

Outside stimuli which can lead to the localization of the proton on one base

(and to the formation of an asymmetric N(3)-He **N(3) hydrogen bond) are most easily

studied in crystalline solids. Qualitatively, there are two attractive interaction

potentials which can be most easily utilized. First, advantage may be taken of the

difference in the hydrogen bonding affinities of the protonated and neutral

cytidine molecules. Second, it is conceivable that interacting dimers or higher

oligomers in which base-base stacking of the hydrogen-bonded pairs takes place

could provide an asymmetric environment conducive to proton localization.

We have previously reported an example of a triply hydrogen-bonded l-methyl-

cytosine, 1-methylcytosineH+complex in (1-methylcytosine, 1-methylcytosinium)

iodide monohydrate [3] . Association into dimers via a base stacking mode in which

a protonated base stacked with a neutral base and vice versa was taken to be an --

integral part of the potential which led to the localization of the proton and the

asymmetric interbase hydrogen bonding. The hemiprotonated form could be isolated

from a wide range of pH conditions and could be avoided only by removing the iodide

115

counterion by ion-exchange chromatography. In this paper, we wish to present an

analysis of the new complex bis(l-methylcytosine, 1-methylcytosinium) hexafluoro-

silicate dihydrate. This complex allows a further evaluation of the roles of

hydrogen bonding and base stacking in stimulating and stabilizing the localization

of the acidic proton on one of the hydrogen bonded bases.

EXPERIMENTAL

Crystals of the title compound, first erroneously assumed to be a hydrate of

I-methylcytosine.HF, were obtained by the slow evaporation of a solution of highly

purified l-methylcytosine(hereinafter l-MeC)[3] in 1M aqueous HF. Our X-ray

structure analysis, vide infra, yields the correct identity of the crystalline

material as bis(l-MeC, l-MeCH)SiF6.2H20, [C1002N6H15]2(SiF2).2H20. Chemical

analysis of a sample of the crystals is in quantitative agreement with the X-ray

formulation: Calcd.: C, 35.29; H, 5.04; N, 24.69. Found: C, 35.43; H, 4.42; N,

24.81. As all our manipulations were carried out in plastic ware, the hexafluoro-

silicate dianion was apparently already available in sufficient quantity in the

reagent grade HF utilized or generated from silica in the distilled water supply.

Whatever the source, we are confident of the presence of SiF6 2- in the crystalline

sample.

The crystals are unstable in humid air and to prevent decomposition were

coated with a thin layer of grease and placed in capillary tubes. The crystals

form as elongated prisms with [OlO] as the prism axis. The space group and

approximate cell dimensions were obtained from preliminary Weissenberg photographs.

The crystal system is monoclinic, with systematic absences (h + k = 2n + 1)

consistent with a C-centered cell and the space groups C2, Cm and C2/m. We have

employed the latter space group in all of our computations. The observed density,

measured by flotation in a mixture of cyclohexane and carbon tetrachloride,

indicated two formula units per cell. Precise unit-cell dimensions were obtained

from the setting angles for 15 reflections on a Syntex Pi automated diffractometer.

Complete crystal data are given in Table 1.

116

Table 1. Crystal data for [l-MeC, l-MeCH]2(SiF6).2HZ0

a = 23.754(5)A [C1602N6H15]2(SiF6)'2H20.

b = 6.719(2)A Mol wt. = 680.65

c = 9.258(2)A D = 1.543(S) g cm -3 - measd

B = 97.16(2)" D = 1.542 g cm -3 calcd

V = 1466.1 A3 z=2

X(MoKa) = 0.71069A Space Group C2/m

~(MoK~) = 1.9 cm -1

.~-____~ ~___---

The 4256 reflections in the +h hemisphere to 26 = 55" were surveyed on the

diffractometer, employing graphite monochromatized MoKa radiation. The crystal

used in data collection had the following well-developed faces and mean separations:

(loo)-(100) 0.07 mm, (001)-(001) 0.10 mm, (OlO)-(Oi?l) 0.18 mm. The crystal was

mounted with the b axis slightly tilted relative to the #I axis of the spectrometer

in order to reduce the possibility of multiple scattering. Intensity data were

collected in the 0-28 mode; individual scan speeds were determined from the number

of counts collected in a rapid scan at the calculated Bragg peak and the rate of

scanning (28) varied linearly from 1.5' min -L (less than 100 counts in the rapid

scan) to 12' min -1 (more than 1000 counts in the rapid scan). The intensities of

three standards were monitored after every 100 reflections and showed only

statistical variations over the course of the experiment. All reflection intensi-

ties were assigned observational variances based on the equation

02(I) = S + (BL + B,)(TS/ZT,)' + (PU2, where S, B1, and B2 are the scan and

extremum background counts, TS and TB are the scan and individual background

counting times (TN = TS/4 for all reflections), and p was taken to be 0.03 and

represents our estimate of the error proportional to the diffracted intensity[4].

Net intensities and their associated standard deviations were corrected for

Lorentz and polarization effects. No correction for the effects of absorption was

117

deemed necessary, as the neglect of such effects was expected to introduce errors

of less than 1%. An approximate absolute scale was determined by the method of

Wilson [S] , and a set of normalized structure factors, /E/Is, was obtained by a

standard procedure. Symmetry averaging (R = 0.028) of the data led to a set of

1835 independent intensities, of which 1488 had I > o(I) and have been employed in

the refinement of the structural parameters.

As indicated above, our preliminary formulation of the crystalline specimens

was as a hydrate of l-MeC.HF, and all of our initial trial solutions were deduced

and analysed with that stoichiometry in mind. The presence of a very strong 020

reflection suggested high atomic occupancy in planes normal to the b axis and -

separated by b/2. Such a situation is readily consistent with space groups Cm and

C2/m, with the eight l-MeC residues per cell lying on the crystallographic mirror

planes normal to the b axis. Statistical analysis of the normalized structure . _

factors as well as the zero moment test of Howells, Phillips, and Rogers[6]

encouraged the employment of the centrosymmetric space group C2/m. Our early

attempts at deducing the structure focused on the utilization of direct methods;

while several tantalizing solutions were offered by various signed E-maps, we were

unable to extend any of these to a satisfactory model. The failure of direct

methods (in our hands, at least) is not particularly surprising in view of the

tremendous vector overlap observed in the Patterson synthesis.

We were, however, eventually able to utilize coherent vectors in an EL - 1

sharpened Patterson map and to effect a structural solution. Our analysis

hinged on three outstanding features of this well resolved synthesis: (1) as

expected, most of the vector density lies in the (U, 0 W) plane, and we confined

our attention there; (2) in this plane, recognition of two strong coherent

transforms was an essential aspect of the solution. One of these vectors, of

length 5.5A, was concluded to be the intracomplex cross vector between coplanar

molecules (one l-MeC and one 1-MeCH+) hydrogen bonded together. A second cross

vector, between hydrogen-bonded base pairs, was then deduced, and finally two

118

coherent symmetry vectors were identified and allowed the position and orientation

of each of the two independent l-MeC residues to be obtained. A structure

factor-difference Fourier sequence (R = Cl IF,I-IF,1 I/ZIFol = 0.63) revealed a

strong peak (-7 e/A3) at the 2/m symmetry site (0, 0, l/2); about this central peak

were six smaller peaks (-3 e/A3) at the vertices of a regular octahedron. We

immediately recognized and identified this set of peaks as resulting from an SiF6 2-

anion, consistent with the solution chemistry which yielded the crystalline

specimens, vide supra. Extending the model in this fashion lowered the R value to

0.48, and a difference-Fourier map showed a residual peak due to the now expected

water of crystallization. At a second glance, the Patterson synthesis had vector

density clearly in accord with the presence of the SiF6 2- group.

Refinement of the now complete (except for the location of the hydrogen atoms)

model was considerably more straightforward than the structural solution. Several

cycles of isotropic refinement, minimizing the quantity ZW(~F~/-IF~~)~ where

w = 4F02 / (F,*), quickly converged and lowered the R value [R = gIjFol-/Fc/l/gl~,I] o2

to 0.12. Two further cycles of refinement, employing anisotropic thermal parameters

for the nonhydrogen atoms, gave an R value of 0.09. A subsequent difference-Fourier

synthesis allowed the positioning of the hydrogen atoms, including the hydrogen atom

involved in the N(3)-11...N(3) hydrogen bond. This latter hydrogen atom is

asymmetrically positioned between the two independent N(3) atoms, yielding a base

pair formally made up of one protonated and one neutral l-MeC molecule. In the

remaining cycles of refinement, the positional and isotropic thermal parameters of

the hydrogen atoms were also allowed to vary. Furthermore, the 020 reflection was

given zero weight as its observed structure factor amplitude was -10% smaller than

its calculated amplitude; we attribute this disparity to a combination of secondary

extinction effects and counting losses. Three cycles in such a mode produced

convergence (all shift/error less than 0.5 for the nonhydrogen atom parameters and

less than 0.7 for the hydrogen atom parameters) and a final R value of 0.071. The

final weighted R value [R w = (sw(~F~]-/F~~)~/~w]F~~~)~'~] and goodness of fit

119

Table 2. Final atomic coordinates with their estimated standard deviations in

parenthesesa

Atom x Y z Atom x Y Z

(a) [SiF6]*-

Si Ob

F(1) 85(l)

F(2) 503(l)

(b) [l-M&H]+

O(*)A

N(llA

N(3lA

N(4lA

C(l)A

C(*lA

C(4)A

C(5)A

C(6)A

H(N3)A

H(5)A

H(6)A

H(1l)A

H(12)A

H(13)A

H(41)A

H(42)A

2496(l)

1539(l)

1966(l)

1463(l)

1583(3)

2030(l)

1458(l)

962(l)

1017(2)

234(2)

59(2)

70(l)

123(2)

193(2)

161(3)

118(2)

187(2)

0

0

1731(3)

0

0

0

0

0

0

0

0

0

0

0

0

64(8)

93(7)

-lll(ll)

0

0

l/2

3268(2)

5334(2)

1870(2)

1791(3)

-338(3)

-2603(3)

3392(4)

1167(3)

-1199(3)

-495(4)

956(4)

-82(4)

-116(4)

157(3)

377(4)

382(4)

385(8)

-318(4)

-306(4)

(c) [l-MeC]

OG’)B

N(l)B

N(3)B

N(4lB

C(l)B

C(*)B

C(4)B

C(5)B

C(6)8

H(5)B

H(6)B

H(11)B

H(12)B

H(13)B

H(41)B

H(42)B

(d) H20

W(1)

HWl)

2483(l)

3432(l)

3019(l)

3577(2)

3356(2)

2953(l)

3542(l)

4031(l)

3959(l)

440(l)

427(l)

301(2)

373(2)

328(3)

326(2)

392(2)

4631(l)

467(l)

0 -3687(2)

0 -3679(3)

0 -1478(3)

0 721(3)

0 -5282(4)

0 -2967(4)

0 -732(4)

0 -1464(4)

0 -2915(4)

0 -88(3)

0 -357(3)

93(g) -568(S)

59171 -567(4)

136(8) -559(6)

0 112(3)

0 124(3)

0

92(5)

2639(4)

324(3)

aNonhydrogen atoms x 104; hydrogen atoms x 103.

b Parameters devoid of esd's are fixed by symmetry.

120

[(Zw(iFo]-IFcl)'/(NO - NV)fl'2, where NO = 1487 observations and NV = 189 variables]

were 0.045 and 1.5, respectively. A final difference-Fourier synthesis was

featureless, except for peaks at + 0.3 e/A3 near the region of the SiF6'- anion.

Neutral scattering factor curves for the nonhydrogen[7] and hydrogen[8] atoms

were taken from standard sources. Anomalous dispersion corrections were applied

to the scattering curves for all the nonhydrogen atoms[9]. Final positional

parameters are collected in Table 2. Tables of thermal parameters and observed and

calculated structure-factor amplitudes have been deposited[lO]. The crystallo-

graphic computations were performed with a standard set of computer programs[ll].

RESULTS AND DISCUSSION

The [1-Methylcytosine, 1-Methylcytosinium] Cation. A projection view of the

[l-MeC, l-MeCH]+ cation is presented in Figure 1, with nonhydrogen-atom distances

Figure 1. An illustration of the asymmetric interbase hydrogen bonding in the

[l-MeC, I-MeCH]+ complex cation in the hexafluorosilicate salt.

121

and angles for both the formally neutral and protonated residues given in Table 3.

The complex cation exhibits a triply hydrogen-bonded base pairing scheme similar

to the Watson-Crick base pairing[l] between guanosine and cytidine in duplex

regions of DNA and RNA. The interbase hydrogen bonding is asymmetric, with one of

the l-MeC bases protonated at N(3), labeled A, and the other in its neutral form,

labeled B. In addition to the unambiguous location and refinement of the hydrogen

atom H(N3)A in the N(3)-H...N(3) hydrogen bond, the differences in the nonhydrogen-

atom geometry in the two l-MeC rings, Table 3, are in complete accord with the

presence of a protonated and a neutral ring system. We note in particular that the

highly sensitive endocyclic bond angle at N(3) of the pyrimidine ring is -4"

larger for the protonated base [C(Z)A-N(3)A-C(4)A = 124.5(2)'] than for the neutral

base [C(2)B-N(3)B-C(4)B = 120.1(2)"]. In addition, the observed differences,

Table 3, in several other molecular dimensions which are also sensitive to

protonation at N(3) [the bond lengths N(l)-C(2), N(l)-C(6), N(3)-C(2), N(3)-C(4),

and N(4)-C(4) and the bond angles 0(2)X(2)-N(l), N(l)-C(2)-N(3), N(3)-C(4)-C(S),

and N(4)-C(4)-C(S)] show exactly the same trend as noted on comparing the parameters

in neutral l-MeC[12] itself with several protonated salts of N(l)-substituted

cytosine derivatives [for example, protonated l-MeC chloride[l3], perchlorate[l4]

and protonated l-benzylcytosine nitrate[l4] and protonated cytidine nitrate[lS]].

Thus, it is well established in the solid that the present complex cation formally

contains a protonated and a neutral base and concomitantly an asymmetric interbase

hydrogen bond.

In the asymmetrically hydrogen-bonded base pair, the central N(3)A_H(N3)A...N(3)B

hydrogen bond is strong [N(3)A. ..N(3)B = 2.833(4)A] and nearly linear [the bond angle

at H(N3)A is 174(2)"]. The other two hydrogen bonds in the complex involve the

exocyclic amino group of the protonated base acting as a donor and the carbonyl

group of the neutral base acting as the acceptor and vice versa. These two hydrogen --

bonds, Figure 1 and Table 4, are of different strength. The hydrogen bond between

the amino group of the protonated base and the carbonyl group of the neutral base

122

Table 3. Nonhydrogen atom bond lengths(A) and bond angles(deg)

(a) The [l-MeC, l-MeCH]+ complex

Bond

0(2)-C(2)

N(l)-C(1)

N(l)-C(2)

N(l)-C(6)

N(3)-C(2)

N(3)-C(4)

N(4)-C(4)

C(4)-C(5)

C(5)-C(6)

Bond angle

C(l)-N(l)-C(2)

C(l)-N(l)-C(6)

C(2)-N(l)-C(6)

C(2)-N(3)-C(4)

O(2)-C(2)-N(1)

O(2)-C(2)-N(3)

N(l)-C(2)-N(3)

N(3)-C(4)-N(4)

N(3)-C(4)-C(5)

N(4)-C(4)-C(5)

C(4)-C(5)-C(6)

N(l)-C(6)-C(5)

[l-MeCH];

1.212(3)

1.473(4)

1.365(3)

1.376(3)

1.383(3)

1.363(3)

1.301(3)

1.416(3)

1.333(3)

117.7(2)

121.0(Z)

121.3(2)

124.5(Z)

123.0(2)

121.2(2)

115.8(2)

117.7(2)

117.3(2)

124.9(Z)

118.7(2)

122.4(2)

[l-MeCIB

1.228(3)

1.473(4)

1.383(3)

1.360(3)

1.367(3)

1.343(3)

1.337(3)

1.416(3)

1.333(3)

118.5(2)

120.8(2)

120.8(2)

120.1(2)

119.2(2)

122.0(2)

118.8(2)

117.2(2)

121.0(2)

121.9(2)

118.2(2)

121.1(2)

123

Table 3, concluded.

(b) The SiF6'- dianion

Si-F(1) 1.641(Z) Si-F(Z) 1.66812)

F(l)-Si-F(2) 90.5(l) F(l)-Si-F(2)a 89.5(l)

F(2)-Si-F(2)b 88.4(l) F(Z)-Si-F(2)' 91.6(l)

Symmetry transforms: (a) -x, -y, 1 - 2

cc3 -x, Y, 1 - 7.

(b) x, -y, 2

Table 4. Comparison of hydrogen bonding parameters within a variety of

triply-hydrogen bonded base pairs containing cytosine derivatives a

D A D-H D-.*A H...A ID-H***A

(a) [I-MeC, 1-MeC11]2(SiF6).2H20

N(3A N(3)B l.O4(2)A 2.833(4)A l.SO(4)A 174(2)'

N(4)A 0(2)B 1.10(2) 2.736(4) 1.6414) 178(2)

N(4)B 0(2)A 0.89(2) 2.899(4) 2.01(4) 176(2)

(b) [l-MeC, l-MeCH]I*H20

N(3lA N(3)B 0.95(6) 2.836(7) 1.88(7) 177(4)

N(4)A O(Z)B 0.92(6) 2.755(7) 1.84(7) 180(4)

N(41B 0(23A O-91(6) 2.91717) 2.02(7) 174(4)

124

Table 4, concluded.

(cf [C, CH12(ZnC14f

N(3)A Nf3)B 0*94(S)

N(4)A 0(2)B 0.92(S)

N(4jB O(2)A 1.07(S)

(d) [C, CH] resorcylate*H20

N(3)A N(3)B 1.0

Nf4)A Of2)B 0.9

N(4)B 0(2V 0.9

(e) Cytosine-S-acetic acid

N(3)A,B N(3)B,A l-06(6)

N(4)kB O(2)BJ 0.92(6)

(f) Isocytosine

N(3)A N(3)B 0.95(5)

N(4)A OC2)B 0.89(S)

N(4)B 0(2P 0.95(5)

2.861(7)

2.784(7)

2.954(7)

1.9117) 176(4)

1.87(7) 178(4)

1.91(7) 167(4)

2.82(l) 1.9 157

2.78(l) 1.8 180

2*89(l) 2.0 170

2.823(8) 1.77(8) 174(3)

2.790(8) 1.88(8) 167(3)

2.908(9) 1.96(9) 178(3)

2.861(8) 1.97(8) 177(3)

2.904(8) 1.95(8) 175(3)

'The atomic numbering schemes other than for (a) and (b) have been altered from

the original papers to conform to that we have employed in (a) and (b).

125

is notably stronger [N(4)A*..O(Z)B = 2.736(4)A] than the hydrogen bond between the

amino group of the neutral base and the carbonyl group of the protonated base

tN(4)B *a-O(2)A = 2.899(4)A]. Moreover, we note that the parameters found here are

virtually identical with those we recently determined in the asymmetrically hydrogen-

bonded base pair in the compound [l-MeC, l-MeCH]I*H20[3]. The only noticeable

difference is that the two hydrogen-bonded rings are not coplanar in the iodide

complex (with a dihedral angle of 4,9(4)O)[3], whereas the two rings are required

to be coplanar here in the hexafluorosilicate salt as a consequence of positioning

the two bases on a crystallographic mirror plane.

The Hexafluorosilicate Dianion. A perspective view of the hexafluorosilicate

dianion is presented in Figure 2; the SiF6 2- anion is crystallographically required

to possess at least Z/m(C,,) molecular symmetry and, in fact, the observed molecular

symmetry, as commonly found, is nearly m3m(Oh), Table 3. The two independent Si-F

bond lengths are Si-F(lf = 1.641(2)A and Si-F(2) = 1.668(2)A. For comparison, me

take the expected mean Si-F distance in an idealized SiF6 2- dianion to be 1.695(8)A,

Figure 2. A perspective view of the hexafluorosilicate dianion. The site

symmetry is Z/m(C,h).

126

a value we deduce from the average of ten Si-F distances reported earlier and

corrected for the effects of thermal motion[l6]. The two Si-F bond lengths reported

here are 0.04-0.05A shorter than this deduced value. It is clearly seen in Figure

2, however, that the SiF ‘- 6 group in the present case is undergoing large ‘apparent’

thermal motion. In particular, the root-mean-square displacements parallel to the

b axis are 0.433A and 0.345A, respectively, for F(1) and F(2). The employment of a

very simple, riding-model-like correction[l7] for the thermal motion suggests an

extension of the Si-F lengths in each bond to about 1.70A, in good qualitative

agreement with the expected value given above. Whether such a correction is com-

pletely justified,given the possible rotational disorder [either static or dynamic]

of the SiF62- group, is questionable. In fact, we attempted a disorder model in

space group C2/m, with each of the anisotropic F atoms replaced by two isotropic

atoms displaced along the large axis of each thermal ellipsoid. This model was

slightly less successful than the fully anisotropic model and we did not pursue it

further nor have we attempted a more sophisticated description of the SiF6 2- group

motion.

Crystal Packing. A projection view of the crystal packing for

[l-MeC, I-MeCH] (SiF6)*2H20 is given in Figure 3. The view direction is approxi-

mately normal to the (010) plane, with a rotation of 10’ about the a axis to reveal -

more clearly the integrity of the components of the unit cell. The

[l-MeC, l-MeCH]+ cations and the Si and F(1) atoms of the SiF62- octahedron lie in

the crystallographic mirror planes.

We will first examine several hydrogen-bonding interactions in the structure.

In addition to the hydrogen bonding within the [l-MeC, l-MeCH]+ cation, there are

several interactions involving the cationic complexes, the SiF6 2- dianions, and the

water molecule of crystallization. The amino group of the protonated l-MeC base

acts as a donor in a bifurcated hydrogen-bond system with the acceptors being two

symmetry-related F(2) atoms of the SiF6 2- anion. The parameters in this hydrogen-

bond system are as follows: N(4)A.. .F(2) [x, y, -1 + z and x, -y, -1 + z] = 3.019(4)A,

127

/ Z

F)

X

A

Figure 3. The crystal packing in [I-MeC, I-MeCH]2(SiF6)-2H20. The view direction

is approximately along b. Shaded components lie at y = 1/2; unshaded

components lie at y = 0. Thin lines denote hydrogen bonding interac-

tions, see the text.

H(41)A...F(2) = 2.29(4)A, angle N(4)A-H(41)A-.-F(2) = 149(2) °. The water molecule

of crystallization plays the role of an intermediary in a hydrogen-bond network

the neutral I-MeC molecule and the SiF62- anion. The oxygen atom of the coupling

water molecule accepts a hydrogen bond from the exocyclic amino group of the neutral

I-MeC molecule [N(4)B...W(1) = 2.883(4)A, H(42)B.-.W(1) = 1.99(4)A,

angle N(4)B-H(42)B..~W(~) = 172(2)°], while donating hydrogen bonds to two symmetry-

related F(2) atoms [W(1)-.-F(2)(I/2 - x, 1/2 - y, 1 - z and 1/2 - x, -1/2 + y,

1 - z) = 2.932(3)A, H(WI).--F(2) = 2.13(5)A, angle W(1)-H(WI)..-F(2) = 162(3)°].

128

In contrast to the extensive hydrogen-bonding interactions observed for F(2), the

F(1) atom of the SiF6 2- group has its principal interactions with the hydrogen atoms

off the carbon atoms C(5)A and C(6)A of the protonated l-MeC base [H(5)A.. *F(l) =

2.37(4)A, H(6)A...F(l) = 2.28(4)A]. These interactions are expected to be favorable,

but weak.

The water-bridged hydrogen bond system between the neutral l-MeC molecule and

the SiF62- dlanion provides a reasonable degree of coupling between the atomic

planes at y = 0 and y = l/2, but the major interaction between these layers may well

be produced by the substantial base-base stacking of the complex cationsi Figure 3).

This base stacking arises from applying the two-fold screw axis operation parallel

along the b axis to the complexes; this screw axis intersects the (010) plane near -

to the center of mass of the complex cations. There are three significant conse-

quences of applying the two-fold screw axis to produce the molecular overlap between

base pairs: (1) there is a set of strongly overlapping cations in which the

protonated base of one hydrogen-bonded pair overlaps with the neutral molecule of

the symmetry-related base pair and vice versa; -- (2) the mean separation between

these overlapping base pairs is small (3.36A = b/2) and suggestive of a strong

interaction; (3) finally, the set of overlapping base pairs is not restricted to a

dimeric interaction, as in [l-MeC, l-MeCH]I.H20[3], but is an infinite helical array

of uniformly spaced base pairs with their molecular planes exactly normal to the

helix axis.

Moreover, we believe here, as in other such systems, that the asymmetry in the

base stacking (neutral/protonated and protonated/neutral) may well be the predominant

force responsible for the localization of the proton in the interbase N(3)-H**.N(3)

hydrogen bond. In particular, we note that the asymmetry observed in the base

pairing and in the neutral/protonated stacking arrangement is a synergistic inter-

relationship which allows for maximum molecular and crystal stability[l8].

Furthermore, we have previously argued that the highly favorable, asymmetrically

hydrogen-bonded and stacked sequence observed in the dimeric structure of the iodide

129

A 0

C D

Figure 4. Interbase hydrogen bonding and base-base stacking in (A) cytosine-5-

acetic acid; (8) the [I-MeC, I-MeCH] + cation in [I-MeC, I-MeCH]I.H20,

D = 3.22A; (C) the [C, CH] + cation in [C, CH]2(ZnCI4), D = 3.26A;

(D) the [I-MeC, I-MeCH] + cation in [I-MeC, I-MeCH]2(SiF6)'2H20,

D = 3.36A.

130

salt and repeated here in polymeric form in the hexafluorosilicate salt may well

provide a model for elements of the hemiprotonated form of polycytidylic acid[l9].

The hemiprotonated form of polycytidylic acid is known to exist in solution as a

stacked, hydrogen bonded structure[l9]. We anticipate that this hemiprotonated

polymer will avail itself in some way of the same highly-favorable asymmetric base

pairing and base stacking we have observed in the iodide and now the hexafluoro-

silicate salt of [l-MeC, l-MeCH]+.

Comparison with Other Triply Hydrogen-Bonded Cytosine Systems. Five other struc-

turally characterized systems in addition to the SiF6 2- salt reported here contain

a set of triply hydrogen-bonded base pairs of cytosine derivatives. The first such

species, cytosine-5-acetic acid[20], Figure 4A, partially exists (50% of the time)

in an inner salt or zwitterionic form, such that one half of the time the carboxylate

proton is bound to N(3) of one of the pyrimidine bases. Inversion-related

zwitterions pair into ‘symmetrically’ hydrogen bonded complexes, Figure 4A. In this

complex, the proton in the N(3)-H. **N(3) hydrogen bond is asymmetrically located and

equally distributed over the two sites near each of the symmetry-related N(3) atoms,

Figure 4A and Table 4. No base-base stacking is observed in this zwitterionic

species, as charge repulsion effects mitigate against a base stacking interaction.

Marsh and coworkers[21] subsequently studied the structure of the pyrimidine

base isocytosine. Isocytosine can exist in either of two tautomers depending on

which of the ring nitrogen atoms bears a hydrogen substituent. In the solid, these

tautomers (1,4-dihydro-2-amino-4-oxo-pyrimidine and 3,4-dihydro-2-amino-4-oxo-

pyrimidine) are present in equal amounts and hydrogen bond together in exactly the

fashion depicted in Figure 1. Details of the interbase hydrogen bonding in this

asymmetric complex are given in Table 4. Germane to our discussion here is the

existence in the crystal of dimeric packs of roughly parallel base pairs, Table 5,

with a mean separation of 3.36A. In this dimeric arrangement, the base-base overlap

is substantial and such that one tautomer overlaps the second tautomer and

vice versa[21]. --

131

More recently, Tamura, Sato and Hata[22], while studying drug-related molecular

complexes containing aromatic organic acids, determined the structure of a 2:l

complex of cytosine and resorcylic acid. In the solid, a proton from the planar

organic acid is transferred to one of the cytosine residues and employed in producing

a monomeric pair of triply hydrogen-bonded cytosine bases; here again the hydrogen

bonding is asymmetric, Table 4, and stabilized, we believe, by base stacking inter-

actions involving the hydrogen-bonded cytosine base pair and the resorcylate anion.

A similar example of cytosine, cytosinium base pairing is available in the

compound bis(C, CH)ZnC14[23]. The interbase hydrogen bonding is asymmetric, Table

4, and dimeric base pairs with C/CH+ molecular overlap are observed, Figure 4C.

The mean separation between the essentially coplanar base pairs is 3.26A.

Lastly, we present in Tables 4 and 5 and Figures 4B and 4D, the hydrogen-bond

parameters and base-base overlap diagrams we have found in the iodide and here in

hexafluorosilicate salt of [l-MeC, l-MeCH]+. We note in particular that the degree

of base-base overlap in the dimeric units found in bis(C, CH)ZnC14 and

Table 5. Comparison of base pair-base pair stacking distances within a variety

stacked base pairs containing cytosine derivatives

Compound Dihedral angle between Mean stacking distance

bases in the base pair between base pairs

(a) [l-MeC, l-MeCH]2(SiF6).2H20 o.oO 3.36A

(b) [l-MeC, l-MeCH]I.ZH20 4.9(4) 3.22

(c) [C, W2(ZnC14) 2.4(6) 3.26

(d) Isocytosine 8.9(S) 3.36

132

[l-MeC, l-MeCH]I*2H20 are virtually identical (Figures 4B and 4C), but different

from that found in the polymeric, columnar arrangement in the structure of the

hexafluorosilicate salt, Figure 4D. In the dimeric arrays, the molecular overlap

largely involves the placement of the carbonyl group of the protonated base over

the heterocyclic ring of the neutral molecule. In the polymeric array, the degree

of overlap is noticeably larger and involves a mode in which the N-CH3 bonds of both

neutral and protonated bases lie over heterocyclic ring centers. It is expected

that both observed overlap modes are highly favorable[24], with perhaps that dis-

played in the polymeric arrangement of the hexafluorosilicate salt being slightly

more so.

Finally, the observation of triply hydrogen-bonded base pairs in a number of

protonated cytosine salts suggests that such an entity is competitive with the

formation of fully protonated bases for which the hydrogen bonding and stacking

interactions are solely with the anionic counter ion. Initially, for anions such

as Cl-, Br-, C104- and NO ^ 3 , the preference (for crystallizable materials) seemed to

lie with full protonation and strong association of the cations and anions through

extensive hydrogen bond networks. Counter examples (with the anions resorcylate,

I-, ZnC14'- and SiF o*-) now exist, however, in which hemiprotonated, interbase

hydrogen-bonded complexes are present. One suspects that the hydrogen bonding

affinity of the counter ion plays some role in determining the degree of protonation

of the cytosine residues, since the known examples of hemiprotonated cytosine base

pairs are all found with weak-to-poor hydrogen-bond acceptor anions. Beyond that,

it would seem to be a delicate balance amongst a variety of 'crystal packing' forces

as to whether cytosine derivatives crystallize from acidic solutions as fully

protonated or hemiprotonated salts.

ACKNOWLEDGIMEN'IS

Support of this research was provided by the National Institutes of Health

through Public Health Service Grant No. GM 20544.

133

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