conformational properties of the osmium tetraoxide bispyridine ester of 1-methylthymine and a...

B101lV0RGAN.C CHEMISTR Y 6,347-364 (1976) 347

Conformational Properties of the Osmium

Tetraoxide Bispyridine Ester of 1 -Methylthymine

and a Comment on the

Linearity of the zrans O=Os=O Group

THOMAS J. KISTENMACHER, LUIGI G. MARZILLI, and MIRIAM ROSS1 Department of Chemistry, The Johns Hopkins University,

Baltimore, Matyland 21218

ABSTEUC’I’

The preparation and crystal and molecuhu structure of the osmium tetraoxide bispyridine ester of 1-methyhhymine are reported. The complex crystallizes in the trichnic system, space group Pi, with 4 = 1 l-493(6).4, b = 16_655(7)A, c = 6.082(2)A, a = 92.07(3)“. fl= 90.58 (3)“, -y = 71.36(4)“, I’ = 1102.4 A3, D, = 1.85(l) g cm-3, D, =

1.84 g crr~-~_ The unit cell contains 2 osmium tetraoxide bispyridine esters of 1-methylthymine, 2 waters of crystahiaation and 1 disordered pyridine of solvation. Intensities for 3814 independent reflections were collected by counter methods. The structure was solved by standard heavy-atom techniques and has been refined by fulkmatris least squares, based on F, to a final R value of 0.065. The osmium complex binds as a cis osmate ester to the C(5)C(6) bond of the methyhfed pyrimidine in a fashion which is expected to be similar to the binding of the complex to thymidine residues in nucleic acids. The conformation of the 1-methylthymine ester is that of a half chair with C(6) showing a substantial deviation, 0.55 A, from the best mean plane of the thymine moiety. The primary coordination sphere about the OS(W) atom is completed by 2 axiaI 0~0 bonds and the binding of the 2 pyridine Iigands in ciz positions in the equatorial plane containing the ester linkages. The O=Os=O group is substantiahy nonlinear, 164-O(5)“, and this nonlinearity is attributed to intracomples electronic effects.

INTRODUCTION

Osmate esters have been extensively studied both in small molecules and in attempts to obtain structural information on biopolymers by physical methods such as X-ray crystallography and electron microscopy [l- 171. The utility of

0 American Elsevier Publishing Company, Inc., 1976

348 T. J. KISTENMACHER, L. G. MARZILLI, AND M. ROSS1

such an approach has been demonstrated by the successful X-ray crystallographic study of yeast phenylalanine transfer RNA employing an osmium isomorph [ 133 and by the visualization of single osmium atoms attached to nucleic acid strands [IO] _ Rich and co-workers have prepared a bispyridine osmate ester of phenylalanine tRNA which is essentially a single site isomorph, and it is believed that the binding site is the free cis diol on the adenosine residue at the 3’OH end of the native molecule [ 13]_ Such an osmate ester will be referred to here as a (sugar) ester. Schevitz and co-workers have also prepared an essentiaIly single site, isomorphos bispyridine osmate ester of formylmethionine tRNA (151. It is believed that the binding site in this case is a cytidine residue [ 16]_ Moreover, Whiting and Ottensmeyer, employing dark field transmission electron microscopy, have been able to image single osmium atoms covalently attached to thymidine residues in single stranded DNA of bacteriophage +X174 [IO] _ In these latter two studies, the osmium is attached to the base by an ester linkage and such esters will be referred to here as (base) esters

Although a number of possibilities are available for attachment of osmium to biopolymers, most derivatives utilized thus far contain the osmate ester linkage in a compound of the type,

0 L II

‘OS/ 0

L’ll’O x

0

The ligand L is required to stabilize the osmate ester linkage towards hydrolysis and has been variously CN- [5], SCN- [5], pyridine or substituted pyridines [1,7,8,13,15,16] and also bipyridine(2L) [9] _

A recent study by Behrman has extended the chemistry of osmium labelling to isopentyladenosine [6] which occurs in several tRNAs. It is likely that the geometry about the osmium center in that study is similar to that in the thymidine or cytidine work described above.

The difficulties inherent in obtaining precise geometrical parameters about the OS in the X-ray structure of phenylalanine tRNA prompted the study of the structure of the simpler bispyridine osmate (sugar) ester of adenosine [14]. Similarly, we felt that a structure of bispyridine (base) ester would be useful for both X-ray and electron microscopic studies of biopolymers. We have prepared in crystalline form and determined the structure of the bispyridine osmate (base) ester of 1-methylthymine. as a model for the binding to thymidine. After our study was essentially complete, we learned of a related study with thymine itself [17] - The hydrogen bonding scheme in the thymine ester utilizes the proton at N(l), which is not available in DNA or I-methylthymine. Since the coordinated hererocycle ligand L can participate in stacking interactions in osmate esters of monomers [ 141, dinucleoside phosphates [9] , and probabIy in tRNA [ 161, it is

OsO&y)a ESTER OF 1 -METHY LTHYMINE 349

particularly important to assess the relative importance of hydrogen bonding and stacking interactions in such compounds. The hydrogen bonding and stacking are in turn influenced by the conformation of the oxidized base. The disorder found in the molecular frameworks of the osmate esters in other studies [14,17] is, fortunately, not observed in the present study and, thus, a greater precision in the determination of the conformational features has been achieved_

EXPERIMENTAL

Preparation of the Bispyridine Osmate Ester of 1-methylthymine

The bispyridine osmate ester of I-methylthymine was prepared by dissolving 1.0 g (4 X 10-a moles) of 0s04 in 5 ml of pyridine followed by the addition of 055 g (4 X 10-S moles) of I-methylthymine in 17 ml of water. The reaction mixture was allowed to stand overnight, and the resulting dark brown crystals collected_

Collection and Reduction of the X-Ray Data

The bispyridine osmate ester of I-methylthymine crystallizes as slightly elongated parallelepipeds with c along the long axis_ Preliminary diffraction photographs showed onIy i symmetry, and the crystals were assigned to the tri- clinic system. Unit-cell dimensions for the Friedel reduced cell [I83 were derived from a least-squares fit to the 28, W, and x setting angles for 15 care- ful!y-centered reflections; the density was measured by the neutral buoyancy method in a mixture of bromoform and carbon tetrachloride and indicated 2 bispyridine osmate esters, 2 water molecules and 1 pyridine of solvation per unit cell. CompIete crystal data are collected in Table 1.

TABLE 1

Crystal Data

a = 11_493(6)A V= 1102.4A3 b = 16.655(7)A OSO,N,C~~H,~-H~O-O-~C~H~N c = 6.082(2)~ Mol. wt. 610.1 Q = 92_07(3)O Space group pi 13 = 90_58(3)” 2=2 y = 71.36(4)O d, = 1.85(l) g cme3 X(Mo KZi) = 0.7 1069-4 d, = 1.84 g cme3


The 4276 reflections in the th hemisphere to 28 = 50 o were measured on a SynoexPicomputer-controlled diffractometer; molybdenum graphite-monochro- mized radiation was employed. The crystal used in data collection had dimensions 0.45 X 050 X 055 mm; the long axis (c) was approximately aligned along the Q axis of the spectrometer. The intensity data were collected by the 8 - 28 scan technique; individual scan speeds were determined by a rapid scan at the calculated Bragg p;;ak, and the rate of scanning varied from 3 o min-1 (less than IO0 counts during the rapid scan) to 24 o min-l (more than 1000 counts during the rapid scan). Three standards were monitored after every 100 reflections, and their intensities showed no systematic variations over the course of the experi- ment (maximum deviation of any standard from its mean intensity of about 4%) The 4276 measured intensities, which included some symmetry-related reflections and standards, were reduced to a set of 38 14 independent intensities with I > a(f). All reflections were assigned observational variances based on the equation

o’(f) = S + (B1 + B2)(T$TB)” * @1)*,

where S, Br and B2 are the scan and individual extremum background counts, Ts and TB are the scan and individual background counting times (TB = I/4Ts for all reflections), and p was taken to be equal to 0.04 and represents the expected error proportional to the diffracted intensity [19] _ intensities and their standard deviations were corrected for Lorentz and polarization effects; no correction for absorption was applied. The squared structure factors were p!aced on en approximate absolute scale by the method of Wiison [20] _

Solution and Refmement of the Structure

Throughout the structure determination and refinement we have assumed that the correct space group is Pi;the successful refinement (vide infra) indicates that this was the correct choice. The positional coordinates of the osmium atom, its immediate coordination sphere, and one of the coordinated pyridine ligands were deduced from a three-dimensional Patterson synthesis. A subsequent structure factor-Fourier calculation allowed the positioning of the remaining heavy atoms in the osmate ester; a difference-Fourier map then revealed the position of the water of crystallization_ With all atoms included, the R value (= C iiF, I - I r”, Ii/C IF, I) stood at 0.19. Four cycles of isotropic least- squares refinement, minimizing the quantity Gw(] F0 \ - 1 F, I)* where w = 4F0*/o*(F0*), plus 1 cycle in which the heavy atoms were varied anisotropically lowered the R value to 0.076. At this stage, a second difference Fourier map was

OsO,(py), ESTER OF I-METHYLTHYMINE 351

calculated, and it revealed 2 features: (1) positions for the 18 hydrogen atoms on the osmate ester; (2) approximate positions for the 3 atoms of the disordered pyridine of solvation. The density maxima for the peaks attributed to the disordered pyridine (centered about the inversion center at 1, 0, I) ranged from 2.0 e/As to 15 e/A3 _ We have assumed the simplest type of disorder where only 1 of the 3 atomic sites is half occupied by a nitrogen and a carbon atom (the 2.0 e/A3 peak). No other peaks on this difference map exceeded 20.5 e/As.

The refinement was continued with: (a) a11 heavy atoms in the osmate ester and the oxygen atom of the water of crystallization refined anisotropically, (b) the atoms of the disordered pyridine of solvation restricted to isotropic refinement, (c) the inclusion, but no refinement, of the hydrogen atoms. Three cycles of refinement in this mode led to convergence (all shift/error less than 0.8) and to a final R value of 0.065. The final weighted R value [(Cw(l F, i -

I Fc l)2/Zwl F, 12)1'2] and goodness of fit [(Cw(l F, 1 - IF, 1)2/(N0 -

NW’=. where 1VO = 3814 independent observations and NV = 271 total variables] were 0.080 and 3.1, respectively. The relatively high value for the goodness of fit is probably reflective of the disorder in the crystal.

Neutral scattering factors for all of the nonhyrodgen atoms were taken from the compilation of Hanson, Herman, Lea and Skillman [2 l] ; the scattering curve for H was that of Stewart, Davidson and Simpson [22] _ The real part of the scattering curve for OS was corrected for anomalous dispersion effects [23] _ Final nonhydrogen parameters are given in Table 2, while the approximate parameters for the hydrogen atoms are collected in Table 3. A complete list of observed and calculated structure factor amplitudes is available [24] _

The crystallographic computations were performed with the following programs: structure factor-Fourier, X-RAY 67 [25] ; least-squares refinements. ORFLS [26] ; best planes, MEAN PLANE [27] ; illustrations, ORTEP [28]_ Calculations not cited were done with locally written programs_

RESULTS

The molecular structure of the bispyridine osmate ester of I-methylthymine is displayed in Fig. 1 with interactomic distances and angles given in Table 4. AS

expected the complex is 6 coordinate about the Os(V1) center with the equatorial plane defined by 2 N-bonded pyridine ligands in cis positions and the 2 Os-O(ester) linkages. The apical positions are occupied by the 2 mans Os=O

osmyl groups. The analysis shows that the osmium reagent has added cis across the C(5)C(6) bond of the I-methylthymine residue. That the ester linkages at C(5) and C(6) are cis is confirmed by the occurrence of both O(5) and O(6) on the same side of the saturated pyrimidine ring, Table 5.


3 u-2

-7

w z

z h

w

N -I-

z _ 2i 0

a C(1

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C(1

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C(1

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C(1

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C( 1

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3174

(14)

37

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3999

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51(1

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739(

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(15)

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329(

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108(

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(7)

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376(

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3015

(10)

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3100

( 12

) 27

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17)

2496

( 14

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6249

(22)

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2669

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105(

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5930

(34)

38

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3058

(29)

161(

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42(6

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1(16

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128(

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24(4

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4(12

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62(7

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304(

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383(

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266(

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496(

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680(

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632(

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(21)

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TABLE 3

Hydrogen Atom Parameters

Atom .ro Y Z Bb

515 416 480 3.0 333 337 -103 3-O 255 123 679 4.0 294 -17 774 5.0 391 -127 497 5.0 444 -89 160 5.0 401 53 86 4.0 I39 312 699 5.0

-56 354 858 6.0 -214 332 646 7.0 -170 249 309 8.0

24 229 150 7.0 642 221 -34 4.0 598 31s -125 4.0 537 245 -208 4.0 271 476 -229 4.0 205 520 -27 4-o 319 539 -114 4.0

a Fractional coordinates X 1 03. b Unscaled isotropic thermal parameters.

FIG. 1. A perspective view of the bispyridine osmate ester of 1-methylthymine. The thermal ellipsoids are drawn at the 30% probability level.

OSO&~)~ ESTER OF 1 -METHYLTHYMINE 355

TABLE 4

Heavy-Atom Interatomic Distances (A) and Angles (deg.P

(a) Primary coordination sphere about the osmium atom

Bond lengths

OS-O(5) OS-O(~) Os-O(7)

O(S)-OS-O(~) 3(5)-Os-o(7) 0(5)-Os-O(8) 0(5)-Os-N(4) 0(5)-O+N(5) 0(6)-Os-O(7) O(6)-OS-O(S)

l-959(7) OS-O(8) 1.974(7) OS-N(~) l-732(7) OS-N(~)

Bond angles

84.4(4) O(6)-OS-N(~) 95.8(4) O(6)-OS-N(5) 95.7(4) 0(7)-Os-O(8) 93.2(S) O(7)-OS-N(~)

176.9(5) O(7)-OS-N(S) 96.9(4) O(8)-OS-N(~) 95_3(4) O(8)-OS-N(S)

N(4)-Os-N(5)

l-753(8) 2.165(9) 2.173(9)

177.4(S) 92.5(S)

164.0(S) 84.4(S) 84.4(5) 83.9(5) 84.7( 5) 89.8(S)

(b) A comparison of the I-methylthymine ester and 1-methylthymine

Bond lengths

N( 1 )-C(2) N(lkC(8) C(2)-0(2) C(2)-N(3) N(3)-C(4) C(4)-0(4) C(4K(5) C(5)-C(7) Ct5)-C(6) C(6)-N( 1)

ester

1.338( 13) I .452( 1.5) 1.339( 13) 1_390(13) i-369(13) 1.211(11) l-534(13) 1_526(15) 1.518(14) 1.447(13)

I-methylthymine

l-379(4) l-470(4) I .214(3) 1.379(4) l-375(4) 1.737(4) l-432(4) 1.49 7(4) l-346(4) l-383(4)

C(2)-N(l)C(6) 120.2(8) C(2)-N( 1 )X(8) 11 Y-5(8) (x6)-M 1 )-c(8) 118.3(8) N(1 )-C(2)-N(3) 116.7(8) N( 1 )S(2)-O(2) 124-i(8) N(3)<(2)-O( 2) 119-l(8) C( 2)-N(3)-C(4) 126.0(8) hT(3)-C(4)-C(5) 116.8(8)

Bond angles

ester I-methyIthymine

120.6(2) 118.2(Z) 12 1.2(2) I 15.4(Z) 133.3(Z) 121.3(2) 126_3(2) 116-l(2)


TABLE 4 (cont.)

N(3)-C(4)-0(4) C(.5)C(4kO(4) Ct4)C(5)-c(6) C(4)C(5)-0(5) cx4)C(5)C(7) C(6I-C(5)-C(7) C(6bC(Sj-OW N( 1 K(6FCW N( 1 )X(6)-0(6)

c(skc~6bo(6)

(cl The pyridine ligands

N(4)-c( 10) l-33(2) N(5)-C(:5j l-34(2) C(lO)-c(l I) l-39(2) C(15)-C(16) l-39(2) C(1 l)C(l2) I .38( 2) C(16)-C(17) I _33(3) C( 12)-c( 13) l-38(2) C(17)-C(18) l-37(3) C(13)-c(14) 1.37(Z) C(l8)-C(19) l-41(3) C( 14)-N(4) l-36(2) C(19)-N(5) l-33(2)

OS-N(4)-C( IO) OS-N(4)-C( 14) C( 1 O)-N(4)-c( 14) N(4)-C( i O)-c( 11) c(lOj-C(IIjC(I2) c(ll)-c(12)c(13) c(12)-c(13)-c(l4) C( 13)-C( 14)-N(4)

123(l) 118(l) 119(l) 131(l) 120(l) 119(l) 119(l) l?‘(l) __

Bond angIes

ester

120_6(8) 122_5(8) 110.1(9) 109_4(8) 106_6(9) 111_4(9) 109.1(8) 112.8(S) 108_9(8) 109.5(8)

1-methylthymine

120-O(2) 123.9(2) 118.3(2)

119.3(Z) 122.4(2)

123.3(Z)

Bond lengths

Bond angles

OS-N( 5)-C( 15) OS-N(S)-C( 19) C(15)-N(5)-C(19) N(5)-C(15K(16) C(15)-C(16)-C(17) C(16)-C(17)-C(18) C( 17X( 18)C( 19) C(18)-C(l9)-N(5)

122(l) 121(I) 117(l) 123(l) 119(l) 121(l) 118(l) 122(l)

a Estimated standard deviations in the least significant figure are enclosed in parentheses_

DISCUSSION

Coordination Geometry about the OS(W) Center

The heavy-atom interatomic distances and angles about the OS(N) atom are collected in part (a) of Table 4. The absence of any disorder in the molecular

OsO&y)p ESTER OF 1 -METHYLTHYMINE 357

TABLE 5

Least-Squares Plane and the Deviation of IndividuaI Atoms from these Plane*

(a) The equatorial plane inciuding the osmium atom (-0.429X - 0.1881Y - 0.88332 = -411142A)

OS -0.007A N(5) -0.008A O(5) -0.010 O(7) --1_722* O(6) 0.013 O(8) I .729* N(4) 0.012 C(5) -o-275*

C(6) o-294*

(b) The I-methylthymine plane (0.8311X + 0.0652Y - 0.55242 = 5.6878A)

N(3) -0.044A C(6) -0_543* C(2) 0.043 O(4) O-148* N(1) -0.02 1 C(7) l-460* C(4) 0.022 C(8) o-090* C(5) 0.014 O(5) -o-795* O(2) o-215* O(6) - l-966*

(c) The pyridine ligands

(1) N(4), C(lO-14)(-0.9400X + 0.0491Y - 0.33772 = -4.6576A)

N(4) 0.006A C(12) 0.006A C(l0) -0.003 Ctl3) -0.003 C(l1) -0.003 C(l4) -0.003

OS o-027*

(2) N(S). C( 15-19)(0.0807X+0.9 111 Y - O-40422 = -2.88 14)

N(5) -0.009A C(17) 0.012A C(l5) 0.006 C(l8) -0.014 C(16) -0.008 C(l9) 0.013

OS 0.040*

o In each of the equations of the planes, X, Y, and Z are coordinates (A) referred to the orthogonal axes: X along the Q axis, Y in the ab plane and Z along the c* axis. Atoms designated by an asterisk were given zero weight in calculating the planes; the atoms used to define the plane were equally weighted.

framework of the osmate ester has allowed us to determine these parameters with reasonable precision. Unfortunately, this was not the case for the bispyridine osmate esters of adenosine [ 141 and thymine [ 17]_ In both of these deter- minations either data collection problems or disorder limited the accuracy of the determination of molecular distances and angles. In Table 6, we compare our molecular parameters to those observed in the osmate esters of thymine [ 171

358 T. J. KISTENMACHER, L. C. MARZILLI, AND M. ROSS1

TABLE 6

MolecuIar Parameters in a Variety of Os(V1) CompIexes Containing OS-O and Os=O Bonds

Compound

OS-O bond length(s)

os=o o=os=o bond length(s) bond angle

(a) Six Coordinate complexes

K~10sOaC1~1Q

K2 [Os%(OH), lb 2.03A (hyrodxyl)

~4[%?0t@02~41C 2.08 (bridging)

py2 -0~0~ - Adod

py2 -0~0~ -Te

pya l Cs04 l 1 -MeTf

(b) Five coordinate complexes

I 1.99(S) (ester) 1.9 l(5) (ester)

I

l-80(3) (ester)

2.00(3) (ester)

1.959(7) (ester) l-974(7) (ester)

l-873(7) (ester)

l-896(8) (ester)

I

1.92 (bridging)

1.87 (ester)

1.750(22)A 1 80°

1.77 180

1.79 160.5 1.79

l-78(4) 164(3) l-78(4)

l-82(3) 162(i) l-87(3)

1.732( 7) 164.0(S)

l-753(8)

1_670(12)

l-675(8)

u Ref [ 29]_ The equivalence of the 2 Os=O bond lengths and the linearity of

the O=Os=O bond angle are fixed by crystallographic symmetry (D4h)- b Ref. [30]_ The equivalence of the 4 OS-OH bond lengths and the 2 Os=O

bond lengths and the linearity of the O=Os=O bond angle are fixed by crystallo-

graphic symmetry (C&$h )- c Ref. 1311. d Ref. [141. e Ref. [171_ f This work.

s Ref_ [II]. h Ref. [12].

0sO&.~y)~ ESTER OF I -METHYLTHYMINE 359

and adenosine [14] and to a variety of other Os(VI) compIexes which contain Os=O or OS-O bonds. We have included in this list 2 five coordinate osmate esters of simple organic compounds [ 11 ,121.

The average parameters in the present determination are as follows: Os-O(ester) bond length = l-97( l)A, Os=O bond length = l-74( l)A, Os-N(pyridine) bond length = 2_17(1)A. These values are in reasonable agreement with those obtained by averaging over the osmate esters of adenosine and thymine: Os-O(ester) bond length = 1_93(2)A, Os=O bond length = 1.8 1(5)A, Os-N(pyridine) bond length = 2_16(4)A. Furthermore, our average value for the Os=O bond length is in quite good agreement with those found in KZ [OsO&l,] [29], 1.75(2)A, Kz [OsOz (OH),] [30] , 1_77A, and K4 (Os,06(N0&] [3 1 1 , 1.79A. The Os-O(ester) and Os=O bond lengths in the 6 coordinate complexes are both considerably longer than in the 5 coordinate complexes [Os-O(ester)AvE = 1.SS(I)A, Os=OXVE = 1_67(1)A] [ 11,121 as is expected owing to the increase in coordination from number 5 to 6.

A point of considerable interest that emerges from a study of Table 6 is that in a significant number of the 6 coordinate complexes, there is a rather dramatic deviation of the O=Os=O bond angle from 180 O_ it should first be noted that the O=Os=O linkage is nonlinear in only those cases, Table 6, where the equatorial plane is occupied by unequivalent ligands (e.g., in K2 [Os02Cl,] [293 and Kz [Os02(OH),] [30] the O=Os=O bond angles are required by symmetry to be 180 “)_ A pertinent point is that in the Scoordinate complexes the OS atom is significantly out of the equatorial plane [ 11,12]_ In the more symmetric 6- coordinate complexes, the OS atom is essentially in the equatorial plane, see Table 5, plane (a), for example. Skapski and co-workers [ 11,123 have attributed the deviation of the OS atom out of the plane to electronic effects involving the strong n-bonding Os=O group and the OS-O bonds in the equatorial plane. It is probable that the nonlinearity of the O=Os=O linkage in the 6-coordinate complexes arises from similar electronic considerations. It is unlikely in the 6- coordinate complexes, because of the presence of 2 tram Os=O bonds, that the OS will be significantIy out of the equatorial plane as is seen above_ what is likely is that the O=Os=O bonds will bend away from the shortest OS-X (equatorial) bonds to minimize interbond electronic interactions. Certainly, in the osmate ester of I-methylthymine this has taken place. A study of Table 4 reveals thal all of the N(pyridine)-Os=O bond angles are significantly less than 90 o [ave angle = 84.4(3) “1 while all of the O(ester)-Os=O bond angles are significantly greater than 90 o [ave O(ester)-Os=O bond a&e = 95_9(7) “I_ These trends are consistent with the considerably shorter Os=O(ester) bond length of 197( l)A than the Os-N(pyridine) bond length of 2_17(1)A_ The general-conclusion is that the O=Os=O linkage, because of its strong n-bonding characteristics, will minimize interbond electronic repulsions by going nonlinear in those cases where the equatorial plane is occupied by ligands with unequivalent OS-L bond lengths.


The relative orientation of the 2 pyridine ligands to the rest of the complex seems to be dependent on 2 features: (1) the self-stacking of N(4)-py with itself about an inversion center (see below) and (2) weak interactions between N(S)-py and the osyml oxygens, C( IQ-H( 15) --- O(7) distance =2.29A and C( 19)-H{ 19) ---

O(8) distance = 2_23A_ As a result, the N(S)-py ligand approximately parallels the 0(7)=Os=O(8) line and the N(4)-py ligand is approximately perpendicular to the 0(7)=Os=O(8) line; the dihedral angle between the mean planes of N(4).-py and N(S)-py is 84-O(3) O_ The higher thermal motion in N(S)-py, see Fig. 1, is consistent with its collection of weak interactions_ The bond lengths and angles in the coordinated pyridine ligands are normal, Table 4.

Conformational Properties of the Saturated Pyrirnidine Ring

The formation of the 2 osmate ester bonds at C(5) and C(6), Fig. 1, leads to a saturated pyrirnidine ring system A similar situation occurs in the simple hydrogenation of pyrimidines to yield 5,6_dihydropyrimidine derivatives. A point of interest is a comparison of the conformational features of the saturated pyrimidine molecuies in each of these cases. Plane (b) of TabI& 5 shows the deviations of individual atoms in the pyrimidine ester from the itandard mean plane defined by N(l), C(2), N(3) and C(4). As can be seen, the observed conformation of the pyrimidine ester is that of a half-chair with C(5) essentially in the plane (deviation = 0.014A) and C(6) substantially out of the plane (deviation = -0543A)_ This can be contrasted to the dihydropyrimidines where the conformation is almost uniformly observed to be that of a twist half chair with the atoms C(5) and C(6) both showing substantial deviations from the standard mean plane [dihydrouracil [32], C(5) 0.14-4, C(6) -0_45A; dihydrouridine (molecule A) [33], C(5) 0_17A, C(6) -0.47A; dihydrothymine (molecule A) 1341, C(S) -0_42A, C(6) -0.3lA: dihydrothymine (molecule B) (341, C(5) -0_43A, C(6) 0.32A; dihydrothymidine [35], C(5) 0_27A, C(6) -0_41A] _

The conformational flexibility in the dibydropyrimidines is underscore’d by the occurrence of configurational isomers in the structure of dihydrothymine [34] and conformational isomers in the structure of dihydrouridine 1331; in fact, the second molecule in the structure of dihydrouridine [33] has conformational parameters very similar to those we have determined here for the osmate ‘ester of I-methylthymine with C(5) lying in the plane, deviatkn = -O.OOlA, and C(6) substantially out of the plane, deviation = -0.65A. _

It is expected that the conformational differences in the osmate ester of I-methylthymine and the dihydropyrimidines may be directly attributable to the presence of the relatively inflexible OsOz-ester system. If this is the case, the conformational characteristics of the osmate esters of thymine and I-methyl- thyrnine shotlld be essentially identical_ The fact that they are not (the osmate ester of thymine [17] shows a twist half-chair conformation with deviations at

OsO,(py), ESTER OF I-METHYLTHYMINE 361

C(5) and C(6) equal to 0.26A and -0_34A, respectively) may be a consequence of the disorder of the thymine residue in that osmate ester, i-e_, the disorder in

the thymine structure may preclude an accurate display of the conformational properties of the saturated ring [ 17]_

A further point of conformational interest is the deviations of the exocyclic substituents from the plane of the I-methylthymine residue. The substituents on the planar part of the residue, C(B)Ha, O(2) and O(4), are all situated on the same side of the plane with deviations of O.O9A, 0.22A and O-ISA, respectively_ What affect, in particular, the deviation from the mean plane of the carbonyl oxygen atom O(4) might have on the ability of such osmate esters to form hydrogen bonds with adenosine residues in duplex regions of nucleic acids is unclear from this study alone. It is known, however, that the binding of 5,6- dihydrouracii to 9-ethyladenine is much weaker than the binding of I-cyclo- hexyluracil [36]. This result is probably due to the same type of conformational disturbances we have noted above.

Bond Lengths and Angles in the Saturated I-Methylthymine Ester

The saturation of the C(5)-C(6) bond in 1-methylthymine by the formation of the cis ester, besides enducing the conformational changes noted above, has produced the expected alteration in the electronic structure of the pyrimidine framework and correspondingly significant alterations are seen in many bond lengths and angles. A comparison of the molecular dimensions in the bispyridine osmate ester of I-methylthymine and I-methylthymine itself [37] appears in part (b) of Table 4. The formation of the cis ester has produced 2 major bond length changes: (1) the expected elongation of the C(5)-C(6) bond length from its pseudoaromatic value of 1_346(4)A in I-methylthymine [37] to a nearly, albeit somewhat short, parafinic bond length of 1.5 lg(14)A. The observed value compares favorably with the C(5)C(6) distances found in the dihydropyrimidines: 152(4)A In dihydrothymine [34] , 1 SO( in dihydrothymidine [35], 1_507(6)A in dihydrouracil [32] and I .514(5)A in dihydrouridine 1331; and (2) the elongation of the C(4)C(5) bond length of 1_432(4)A in l-methyithymine to l_534(13)A in the ester. Furthermore, there are minor, but possibly significant, perturbations in the bond .lengths at N(l), N( 1)-C(2) being about 0.04A less and N(l)-C(6) about 0.06A longer than the corresponding values in I-methylthymine. These latter 3 differences are also in accord with the results found in the dihydropyrimidines.

The change in hybridization at C(5) and C(6) is also rather dramatically reelected in the bond angles at these sites, Table 4. In general, the bond angles are within the expected range for tetrahedral carbons with nonequivalent substituents, with the smallest angfe being C(4)C(5)-C(7) at 106.6(9) o and the largest at N(l)-C(6)-C(S) at 112.8(S) O_ There are also some significant, but


FIG. 2. b stereoview of the crystal packing. The view direction is down c with the b axis horizontal and the Q axis vertical.

less dramatic, differences in the bond angles at N(l), consistent with the alterations in the N( 1)-C(2) and N( l)C(6) bond lengths noted above, see Table 4_

Crystal Packing

The crystal packing is dominated by 2 principal types of interactions: (1) formation of hydrogen bond dimers about the inversion center at l/2, l/2, l/2 via the I-methylthymine moiety of the ester involving hydrogen bonds of the type N(3)-H(3) --- O(2); and (2) stacking of 1, N(4)-py, of the coordinated pyridine ligands about the center of inversion at l/2,0, l/2, see Fig- 2_

The parameters in the N(3)-H(3) --- 0(2)[1 -x, 1 --y_ 1 -z] hydrogen bond system are as follows: N(3)-H(3), 0.86A; N(3) --- O(Z), 2.90(2)A; H(3) --- O(2), 2_05A, and the angle N(3)-H(3) --- O(2) is 169 O. These parameters are consistent with a reasonably strong hydrogen bond [38 ] _ This hydrogen bond system accounts for a substantial fraction of the hydrogen bonding capability of the complex, H(3) being the ordy good hydrogen bond donor and C(2)*(2) being 1 of the 2 good acceptor carbonyl groups The carbonyl C(4)=0(4) does not par- take in any formal hydrogen bonding_

The stacking of the coordinated pyridine, N(4)-py, about the center of inversion at l/2, 0, l/2, Fig- 3, is essentially a direct overlap of the 6-membered rings with a mean separation of 3.49A and short contacts as follows: N(4) --- C(lZ)[l - x, -y. 1 - z], 3.53(2)A; C(l0) --- C(l3)[1 - x, -y, 1 - z], 3.5 l(2)A; C(11) --- Cj14)[ 1 - x, -y, 1 - z] ,3.52 (2)A. A very similar stacking of 1 of the coordinated pyridine ligands occurs in the bispyridine osmate ester of thymine [17], mean separation 3_4A, and in the bispyridine osmate ester of adenosine one of the coordinated pyridine Iigands stacks with a symmetry- related adenosine moiety, resulting in a mean stacking distance 3SA [14]_ The ubiquity of the stacking interactions involving one of the coordinated pyridine ligands in the above 3 structures implies that this interaction is quite determina- tive in the crystallization process and probably rivals the hydrogen bonding in each of these structures in terms of crystal stabilization.

OSO&~)~ ESTER OF I-METHYLTHYMINE 363

This investigation WQS supported by the National Institutes of Health through Public Health Service Grant No. GM 20544. We thank Miss G. Pavlovitz and Mr. B. E. Hanson for experimental asstitance and Drs_ Neidle and Behrman for preprints.

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Receised 23 January I9 76; revised I3 iWay I 9 76

conformational properties of the osmium tetraoxide bispyridine ester of 1-methylthymine and a...

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