1205
ratio 0 6 6 / 0 3 5 is bracketed by the free and the eclipsed ratios. A preferentially eclipsed conformer would be indicated rather than staggered. The experimental ratio
is much closer to the free rotation model than the fix!d staggered model for a C-N bond length of 1.488 A, suggesting a low barrier to internal rotation in the ion.
The Crystal and Molecular Structure of a Derivative of 1,N'-Ethenoadenosine Hydrochloride. Dimensions and Molecular Interactions of the Fluorescent €-Adenosine(€Ado) System Andrew H.-J. Wang, Laurence G. Dammann, Jorge R. Barrio, and Iain C. Paul*
Contributionf r o m the Department of Chemistry, School of Chemical Sciences, University of Illinois, Urbana,Illinois 61801. Received September 14, 1973 Abstract: The crystal structure of 7-ethyl-3-P-~-ribofuranosylimidazo[2,1-i]purine hydrochloride monohydrate (3) has been determined both to establish the direction of addition of chloroacetaldehydeto adenine in the preparation of e-adenine derivatives and to probe the molecular interactions of the highly fluoresceat +adenosine derivative. The crystals of 3 are monoclinic, with a = 12.952 (3), b = 6.667 (2), and c = 9.679 (2)A, and = 101O 34' (1 '). There are two molecules of C14HlsN504+Cl-.H20 in the space group P21. The structure has been refined to an R factor of v.043 in 1373 nonzero reflections. The entire e-adenine moiety is near-planar with a maximum deviation of 0.028 A among the ring atoms. There are some minor differences in bond lengths in the adenine residue in 3 when compared to unbridged adenine rings, but the greatest differences involve the exocyclic angles at C(6). The arrangement about the glycosyl bond is syn (xCN= - 109.1 ") and the ribose ring exists in the C(2') endo-C(l ') exo conformation. The C(4')-C(5') exocyclic bond is in the trans-gauche arrangement. The N-H bond in the base, all three 0-H bonds in the sugar, and the water molecules are involved in hydrogen bonding in the monohydrate. The crystal can be divided into successive regions (in the a direction) of polar and nonpolar character. In the nonpolar regions, there are infinite stacks (in the b direction) of +adenine rings, e!ch of which overlap considerably with their neighbors with alternate ring-ring separations of 3.344 and 3.324 A. These overlaps are compared with those found in related molecules.
T
he reaction of chloroacetaldehyde with adenosine their capability for intermolecular interactions, we have and cytidine to produce fluorescent derivatives l s 2 carried out single crystal X-ray studies on a derivative is claiming increasing biochemical interest. Two of €Ado, and on eCyd, both as hydrochloride salts. For the formation of €Ado as the hydrochloride salt compounds that have been particularly useful as fluorescent probes in biochemical systems are 3-P-~-ribo- from chloroacetaldehyde and adenosine, a logical furanosylimidazo[2,l-i]purine(1 ,N6-ethenoadenosineor mechanistic sequence was suggested that first involved alkylation at N(l) (according to convention for num€Ado) and 5,6-dihydro-5-0~0-6-/3-~-ribofuranosylimidazo[l,2-~]pyrimidine (3,N4-ethenocytidine or bering in adenine) of the adenosine followed by ring closure of the aldehyde and 6-NHz groups with elimECyd), each shown as the hydrochlorides, 1 and 2, ination of water.2 A test of the direction of introduction of the etheno bridge can be made by utilizing the higher homologs, such as a-chlor~propionaldehyde~ or a-chloro-n-butyraldehyde. It has now been found that the latter reacts with adenosine more slowly than does chloroacetaldehyde but yields a pure ethyl-substituted €-adenosine that exhibits a fluorescent emission maximum at 440 nm with a quantum yield of 0.33 and a lifetime of 17 nsec under neutral conditions. In order to gain the additional information required, we chose the product of the reaction of adenosine with a-chloro1 2 n-butyraldehyde for the X-ray study of an example of an respectively. As an aid to understanding the fluoe-adenosine derivative. The X-ray analysis establishes rescent behavior of these compounds, in order to prothe product as 7-ethyl-3-/3-~-ribofuranosylimidazo[2,1vide detailed molecular dimensions, and to explore ilpurine hydrochloride monohydrate (EteAdo . HC1. H 2 0 ) (3), thereby proving that N(l) (rather than N(6)) (1) J. R. Barrio, J. A. Secrist 111, and N. J. Leonard, Biochem. Bioof adenosine reacts with the a carbon of the aldehyde. phys. Res. Commun., 46,597 (1972). (2) J. A. Secrist 111, J. R. Barrio, N. J. Leonard, and G. Weber, Biochemisrry, 11,3499 (1972). (3) J. R. Barrio, L. G. Dammann, L. H. ISirkegaard, R. L. Switzer, and N. J. Leonard, J . Amer. Chem. Soc., 95,961 (1973).
(4) N. K. Kochetkov, V. N. Shibaev, and A. A. Kost, Dokl. Akad. Nauk. SSSR, 205, 100 (1972). These authors did not establish the
structure of the product.
Paul, et al. / Deriuatiws of I,Ne-Ethenoadenosine Hydrochloride
1206
Figure 1. Stereoscopic view of the €Adocation. The percentage probability of the ellipsoids is 20 %.
In this paper we describe the crystal and molecular structures and molecular interactions of EteAdo HCI.HzO. We have also determined the structure of
Table I. Final Atomic Coordinates in Fractions of the Unit Cell Edgea X
eCyd HCI.5 In order to facilitate comparisons with related adenine molecules, the conventional atom numbering for adenine, as shown in 3, is used in this paper. e
0.3019 (1) 0.7411 (3) 0.0207 (3) 0.1280 (3) 0.1967 (3) 0.1541 (3) 0.0505 (3) -0.0226 (3) -0.1270 (3) 0.0366 (3) 0.1339 (4) 0.2102(3) -0.0632 (3) -0.1524 (3) -0.0418 (4) -0.1434 (4) 0.3226 (4) 0.3890 (4) 0,4955 (4) 0,4661 (4) 0,4773 (4) 0.3581 (3) 0.3827 (3) 0.5454 (3) 0.5840 (3) 0,729 (5) 0.692 (4) 0.157 (4) -0.180 (4) 0.163 (4) -0.227 (3) -0.001 (4) 0.007 (5) -0.205 (6) -0.146 (4) -0.191 (4) 0.337 (3) 0.353 (3) 0.538 (3) 0.504 (4) 0.441 (6) 0.433 (4) 0.427 (4) 0.604 (4) 0.613 (5)
H
HO OH 3,
2'
3
Experimental Section 7-Ethyl-3-P-~-ribofuranosylimidazo[2,1-i]purine Hydrochloride Monohydrate (EteAdo.HC1. H20) (3). The compound was made in a manner similar to the reaction of chloroacetaldehyde with adenosine.1,2 To 1.0 g of adenosine suspended in 20 ml of water was added 4.0 g of freshly prepared cu-chloro-n-butyraldehyde. The mixture was stirred at ca. 40", pH 4-4.5, until all of the adenosine had reacted as judged by tlc on cellulose in 1-butanol saturated with water. This required about 5.5 days. Ether (20 ml) was added, and the aqueous layer was separated and treated with decolorizing charcoal. The filtrate was evaporated to small volume and acetone was added. After refrigeration overnight, the colorless crystals were collected by filtration, washed with acetone, and (0.1 N HCI) 279 nm (E 11,2001, dried: yield 71 %; mp 149";, , ,A A,, 248 (4000); A:," 0.05 M phosphate 263 nm (sh), 270 (e 6200), 281 (6300), 302 (2900), Amin 251 (4100), 276 (5100), 290 (2800); , , ,A 0.1 N NaOH 263 nm (sh), 270 (e 8200), 280 (7800),, , ,A 253 (6700). 276 (7200); fluorescent emission max 440 nm, quantum yield 0.33, lifetime 17.2 nsec. Anal. Calcd for C,aH,aN;C1O~.H~O:C. 44.98; H. 5.39; N, 18.74. Found: C, 44.54; H, 5.54;-N, 18.46. Crystallographic Study of 3. The crystals of 3 are colorless, transparent, rectangular-shaped plates with well-developed (100) faces, and elongated in the b direction. Crystal Data. Cl4Hl8NSOI+C1-.H20, mol wt 372.8, monoclinic, a = 12.952 (3), fi = 6.667 (2), and c = 9.679 (2) A, P = 101 O 34 (I)', v = 818.9 A3,d,,eas = 1.50 g CW3, z = 2, dcaicd= 1.497 g ~ m - F(000) ~, = 392, ~ ( C K Ua ) = 24.1 cm-l. Systematic absences for OkO, when k = 2n 1, indicated that the space group is either P21 or PZl/m. Since the compound is optically active, the former must be the correct space group. The density was measured by flotation in a mixture of hexane and carbon tetrachloride and the cell data were obtained by a least-squares fit to the hand-centered settings for 12 reflections on a Picker FACS-1 diffractometer ( A C ~ K C = 1.5418 A).
+
( 5 ) A. H.4. Wang and I. C. Paul, in preparation.
Journal of the American Chemical Society
96:4
Y
0 . 25Wb 0.1286 (8) 0.1382 (8) 0.1379 (10) 0.1394 (8) 0.1394 (9) 0.1374 (9) 0.1351 (9) 0.1345 (7) 0.1406 (8) 0.1447 (9) 0.1422 (7) 0.1404 (10) 0.1376 (9) 0.1511 (11) 0.1594 (11) 0.1506 (9) 0.0005 (9) 0.1034 (9) 0.3234 (9) 0.4090 (9) 0.3409 (6) -0.1958 (6) 0.0648 (7) 0.4208 (7) 0.004 (11) 0.191 (9) 0.142 (13) 0.124 (10) 0.166 (9) 0.152 (8) 0.022 (9) 0.287 (12) 0.025 (13) 0.193 (9) 0.268 (11) 0.142 (8) 0.010 (7) 0.049 (8) 0.394 (8) 0.361 (14) 0.552 (9) -0.197 (8) 0.032 (8) 0,503 i7j
0.1777 (1) 0.6328 (4) 0.3803 (4j 0.3857 (5) 0.5024 (4) 0.6200 (5) 0.6290 (4) 0.4993 ( 5 ) 0.4603 (4) 0.7671 (4) 0.8387 ( 5 ) 0.7595 (4) 0.2616 (5) 0.3131 (5) 0.1143 (5) 0.0051 (5) 0.8127 (5) 0.7498 (5) 0.7829 (5) 0.7634 (5) 0.6215 (6) 0.7776 (4) 0.8036 (4) 0.9258 (3) 0.6091 (4) 0.677 (6) 0.625 (5) 0.307 (6) 0.520 (5) 0.957 (5) 0.268 (4) 0.102 (5) 0,101 (6) 0,010 (7) -0.107 (6) 0.010 (6) 0.918 (4) 0.643 (4) 0.722 (4) 0.832 (5) 0.544 (8) 0.598 (5) 0.875 (5) 0.927 (5) 0.674 (5)
'
a Standard deviations in parentheses. The y coordinate of the C1 ion was held constant to define the origin in the b direction.
A crystal 0.5 mm in the b direction and 0.2 X 0.06 mm in cross section was used for data collection. The general procedures for data collection were as described previously.6 The a t a n t s of (6) J. K. Frank and I. C. Paul, J . Amer. Chem. Soc., 95, 2324 (1973).
February 20, 1974
1207
\
1329915)
C(41 114 o(3)" \0(3)'
Lil i
1048(4P /j24(8) 116 215)"
151818)
102 5(4)" 1104(5)' /l098(5)' c(3')
l429(6)
O(3'l
1
100 5(3P l173(4~\1717) C(2') 112413)"
I516(7)
b
O(2')
Figure 2. (a) Bond distances and angles involving non-hydrogen atoms in the eAde base. (\I Bond distances and angles in the ribose ring The range of C-H distances is 0.88 (6) to 1.20 (8) A and of 0-H distances is 0.75 (5) to 0.96 (7) A. data hkl and t?kl were measured to 20 = 130". No evidence for crystal deterioration or loss of intensity was noted. Of 1526 possible unique reflections, 1373 were considered to be significantly above background, using a 2u criterion based on counting statistics. The data were corrected for Lorentz and polarization effects, but not for absorption. The structure was solved by the heavy atom method based on chlorine. Full-matrix least-squares refinement varying positional and isotropic temperature factors for the nonhydrogen atoms gave an R factor of 0.104 and R1,[.Zw(lFoI -. ~ F o 1 ) 2 / Z w ~ F o 1 2of] 10.122. /2, All reflections were given weights using a program written by Dieterich,? which uses essentially the method described by Corfield, Doedens, and Ibers* and the quantity minimized was Zw(lF,I 1 Fc/1*. Introduction of anisotropic temperature factors for the nonhydrogen atoms into the refinement reduced the R factor to 0.065 and a difference map was calculated at this stage which showed clearly the positions of all the hydrogen atoms. When the model included positional parameters for all atoms, anisotropic thermal parameters for the non-hydrogen atoms, and isotropic thermal parameters for the hydrogen atoms, full-matrix least-squares refinement gave final values of R and R2 of 0.043 and 0.045, respectively, on all observed reflections, A final difference map contained no peaks greater than 0.3 e/A3. The final values for the positional parameters are listed in Table I.Q (7) D. A. Dietrich, Ph.D. Thesis, University of Illinois, 1973. (8) P. W. R. Corfield, R. J. Doedens, and J. A. Ibers, Inorg. Chem., 6 , 197 (1967). (9) See paragraph at end of paper regarding supplementary material.
The scattering curves for C1-, C, N, and 0 used in the analysis were taken from the compilation by Cromer and Mann,l0 and the one for hydrogen was that calculated by Stewart, et nL1l
Results and Discussion Molecular Dimensions of the €-Adenine Ring. A stereoscopic view of the EteAdoH+ cation, looking normal to the plane of the eAde moiety, is shown in Figure 1. The bond lengths and angles involving nonhydrogen atoms are shown in Figure 2 . A rigid body analysis12 of the thermal motion in the €-adenine moiety, while showing that the ring system could be tzeated as a good rigid body (rms A of U(z,j) is 0.0027 resulted in corrections to the bond lengths of 0.003 A or less. In subsequent discussions, the uncorrected values are used. The site of protonation in the EteAdo cation is the N(6) position. Other purine nucleosides that have been investigated as monocations in halide salts include formycin hydrobromide,I3 aristeromycin
ta2),
(10) D. T. Cromer and J. B. Mann, A c f a Crysfallogr., Sect. A , 24, 321 (1968). (11) R. F. Stewart, E. R. Davidson, and W. T. Simpson, J . Chem. Phys., 42,3175 (1965). (12) V. Schomaker and K. N. Trueblood, Acta Crystaiiogr., Sect. B, 24,63 (1968). (13) H. Koyama, K. Maeda, and H. Umezawa, Tetrahedron L e f t . , 597 (1966).
Paul, et al.
Derivatives of I ,Ne-Ethenoadenosine Hydrochloride
1208
hydrobromide,14 2',3'-O-isopropylideneadenosine hydroiodide, l 5 5 '-methylammonium-5 '-deoxyadenosine iodide monohydrate, l6 and adenosine hydrochloride. l 7 While hydrogens atoms were not included in several of these analyses, there is no evidence that the sugar rather than the nucleic acid base was the site of protonation. Since the N(6) and N(l) atoms are linked by the 1,N6-etheno bridge and constrained in a five-membered ring, some differences in the bond lengths and angles involving these two atoms can be anticipated when dimensions are compared with those of an unbridged adenine moiety, whether neutral or protonated. Table I1 lists the average values of the dimensions in the pyTable 11. Comparison of the Bond Lengths (A) and Angles (deg) of the Pyrimidine Ring in EteAdo.HCl(3), with Those in 3-Methyl-3H-imidazo[2,1-i]purin-8(7H)-one (4), and the Averaged Values of 12 Protonated Adenine Moieties Bond lengths N(l)-C(2) N( 1)-C(6) ~(2)-~(3) N(3)-C(4) C(4)-C(5) C(5)-C(6) C(6)-N(6) Bond angles C(2)-N(l)-C(6) N(l)-C(Z)-N(3) C(2)-N(3)-C(4) N(3)-C(4)-C(5) C(4)-C(5)-C(6) C(5)-C(6)-N(l) N(l)-C(6)-N(6) N(6)-C(6)-C(5)
3a
4*
A~ctd
1 , 3 8 0(5) 1.379 (5) 1.291 (6) 1.360 (6) 1.362(6) 1.413(6) 1 ,329 ( 5 )
1.363 (3) 1.381 (3) 1.308 (3) 1.358 (3) 1.383(3) 1.404(3) 1 ,326 (3)
1.356 10.012 1.368 f 0.012 1.307 & 0.012 1.358 + 0.010 1.373xt0.015 1.414f0.017 1 ,312 f 0,012
122.9 (4) 123.0(5) 114.1 (4) 128.6(5) 115.9 (5) 115.4(4) l08.9(4) 135.6(5)
124.9 (1) 123.2(2) 112.9 (1) 128.3 (2) 117.1 (2) 113.6(1) 114.6(2) 131.8(1)
123.8 i.0 . 9 1 2 6 . 0 10 . 9 1 1 1 . 2 10 . 9 128.021 1.1 117.7 f 1 . O 113.62I 0 . 8 121.1 2I 1 . 2 125.5f 1.3
a This work. Reference 27. c Averaged values among adenosine hydrochloride,17 S'-AMP,'* 3'-ALMP,19 A(2'-5')U,20 3'-deoxy-3'-(dihydroxyphosphinylmethyl)adenosine,~1 5'methyleneadenosine 3 ',5'-cyclic monophosphonate monohydrate, 1 2 puromycin dihydrochloride pentahydrate,23 AdeH+-C3-Nic+,24and uridylyl(3'-5')-adenosine hemihydrate.25,20 d The crystals of uridylyl(3'-5')-adenosine hemihydrate have two independent molecules in the asymmetric unit and were studied by two different gr0ups.~~26 Thus, four sets of values for this molecule were included in the average.
rimidine ring of a protonated adenine in 12 different molecules. 18--26 The values for another somewhat similar, although neutral, compound, 3-methyl-3Himidazo[2,1-i]purin-8(7H)-one (4), 27 are also included in (14) T. Kishi, M. Muroi, T. Kusaka, M. Nishikawa, K. Kamiya, and K. Mizuno, Chem. Comun., 852 (1967); Chem. Pharm. Bull., 20,940 (1972). (15) J. Zussman, Acta Crystallogr., 6, 504 (1953). (16) W. Saenger, J . Amer. Chem. Soc., 93,3035 (1971). (17) I
