Proc. Indian Acad. Sci. (Chem. Sci.), Vol. 93, No. 3, Apt'il 1984, pp. 261-269.
9 Printed in India.
Structures of isopropylidene nucleoside derivatives: implications for ribose ring flexibility under external cyclic constraints
M A V I S W A M I T R A and N G A U T H A M
Department of Physics and ICMR Centre on Genetics and Cell Biology, Indian Institute of Science, Bangalore 560012, India
Abstract. Crystal structures of six isopropylidene nucleoside derivatives are described. The results show that, under external cyclic constraints, the ribose assumes a variety of unusual conformations. In those compounds which possess a base-to-sugar cydization through the C(4') atom, the furanose pucker is predominantly C(4')-endo, O(4')-exo. The possible relevance of the sulphur geometry in two of the compounds to certain structural aspects of the action of the enzyme thymidylate synthetase is also pointed out.
Keywords. Crystal structure; isopropylidene nucleoside derivatives; ribose ring flexibility;
sulphur geometry; external cyclization
1. Introduction
N u m e r o u s crystallographic investigations have shown that nucleic acid constituents have a great deal o f structural flexibility (Viswamitra 1983; Viswamitra et al 1978). In particular the five-membered furanose ring in nucleosides and nucleotides is found to exist in a wide variety o f geometries (Sundaralingam 1973).
Crystal structures o f c o m p o u n d s where the furanose ring is under some constraint, such as external cyclization, are few in n u m b e r compared to those o f nucleosides containing the normal ribose ring. We review here the structures o f a series o f isopropylidene nucleoside derivatives where an isopropylidene group is attached to the 2' and 3' positions o f the ribose ring (figure 1). These compounds serve as models for studying the flexibility o f the furanose ring under external cyclic constraints.
2. Experimental
2.1 5'-Deoxy-5',6-epithio-5,6-dihydro-2',3'-O-isopropylidene-3-methyluridine. The con- formation is anti. Suoar pucker is O(4')-exo, C(4')-endo.
Crystals were grown by diffusion of water into a solution o f the substance in acetone.
They belong to the orthorhombic space g r o u p P2~2121 with a = 39-526(4), b = 6.607 (2) and c = 5.661 (2)A ( G a u t h a m et al 1982).
The dihydrouracil base is puckered with the C(5) and C(6) atoms displaced on opposite sides o f the ring plane, as shown in figure 2. XcN which defines the torsional angle C(6)-N(1)--C(1')-O(4') about the glycosidic bond N(1)--C(I'), is 32"8(6) ~
* To whom all correspondence should be addressed.
261
0 (5)~r~5
c . , / Rou.
Figure 1. Chemical structure of i=opropylidene nucleoside derivatives where an iso- propylidene group is attached to the 2' and 3' position of the ribose.
, • URACIL
( ~ - "g ctsl
Figure 2. Compound (i). The dihydrouraeil ring is puckered. The conformation is anti.
Sugar conformation is C(4')-e~k/o, O(l')-exo, sulphur is equatorial.
Cyclization of the base-sugar moiety constraints the molecule to this anti conforma- tion. The phase angle P of psuedorotation, which describes the puckering of the ribose ring is 253-7 ~ This brings the sugar geometry under the O(4')-exo, C(4')-endo conformation which is different from the C(2')/C(3')-endo geometry normally found in nucleosides and nucleotides (Altona and Sundaralingam 1972). The maximum amplitude of pucker, ~k= which indicates the deviation of atoms from the best ribose plane, is 28"8 ~ with the C(4') and 0(4') atoms displaced by about 0.2 A on either side of the plane. The dioxolane ring conformation is C(7)-endo. The conformation about the C(4')-C(5') bond is gauche-gauche. Thus the atom attached to C(5') (sulphur in the present case) is directly above the sugar ring. The molecular packing shows only van der Waals contacts, with the molecular coordination number being as high as 14 (figure 3).
2.2 5'-Deoxy-5',6-epithio-5,6-dihydro-2',3'-O-isopropylideneuridine. The conformation is anti. Sugar pucker is O(4')-exo, C(4')-endo.
This molecule is the unmethylated analogue of compound (i). Crystals were grown by evaporation of water/acetone solutions. They belong to the triclinic space group P1 with two independent molecules in a unit cell of dimensions a = 5.635(2), b = 11-077(2) and c = 11-582(2)A, ~ = 70-48(1), f l = 88"16(3) and ~, = 80"56(3) ~ (Gautham et a11983c). The bond lengths and angles involving the C(5) and C(6) atoms of the dihydrouracil base are considerably different from those in normal uracil bases because of the sp 3 hybridization (Gautham 1983). The glycosidic torsion XcN is 21.6 (9) and 29-4 (10) ~ in the two molecules respectively. The molecular conformation is anti (figure 4) as in compound (i). The sugar pucker is O(4')-exo, C(4')-endo in both molecules (P = 249.8 and 254"4~ ~,, 28.8 and 27"6~ The dioxolane ring conformation
Structure of isopropylidene 263
t~ 8
P,
ca
o
bo
~
gl
o
r4
g$
S C(E
C(5'~
Figure 4. Compound (ii). The tom,Formation is anti. Sugar pucker is C(4')-endo, O(l')-exo.
Sulphur is axial in one molecule amd equatorial in the other.
Figure 5. Compound (ii). The molecules show self base-pairing through N(3)-H . .. 0(2) hydrogen bonds.
is O(2')-endo in one molecule and C(7)-endo in the other. The conformation about the C(4')-C(5') bond is gauche-gauche in both molecules. In the crystal structure the molecules show self base-pairing through N ( 3 ) - - H . . . 0(2) hydrogen bonds (figure 5).
2.2a Sulphur geometry in compounds (i) and (ii) and relevance to certain structural aspects of enzyme action Compounds (i) and (ii) were prepared by Drs D M Brown and S A Salisbury of the University of Cambridge as models for understanding certain aspects of the action o f thymidylate synthetase enzyme. The structure analysis was taken up as the position of the sulphur atom with respect to the dihydrouracil ring plane was of particular interest in this context.
In compound (i), sulphur is found equatorial with respect to the dihydrouracil base, i.e. almost in the plane o f the base. In compound (ii) which has two crystallographically independent molecules the sulphur position is equatorial in only one. It is found to
Structure of isopropylidene 265 possess an axial geometry in the other i.e. almost perpendicular to the base.
In the action of thymidylate synthetase one of the steps involves the conversion o f the uracil base to a dihydrouracil derivative with the addition of sulphur at C(6) and CH3 at C(5). This addition reaction is generally supposed to be trans-diaxial in character i.e.
both substituents are added along the axial direction. However, dTMP, which is the final product of the enzymic reaction has CH3 in the equatorial position. A conformational inversion about the C(5)-C(6) bond may therefore be necessary during the reaction to take both CH3 and S from axial to equatorial positions. The present structures show that, at least energetically, both the equatorial and axial conformations for sulphur are equally possible (Gautham et al 1983c).
2.3 2',3'-O-isopropylideneuridine. The conformation is anti. Sugar pucker is C(3')-exo, C(4')-endo.
Crystals o f the native compound and the 5-bromo analogue of 2',3'-O-isopropylidene- uridine were obtained by evaporation o f water/acetone solutions of the compounds.
They belong to the orthorhombic space group P212121. They are isostructural with the unit cells differing mainly in the b-axis dimension. The cell parameters are: Native a
= 5.236 (1), b = 12.789 (2), c = 19"890(5)A (Katti et al 1981); 5-bromo analogue a
= 5.251 (4), b = 14.962 (5), c = 19-112 (5)A (Gautham et al 1983a).
The nucleoside has the common anti conformation with ZCN = 3"4 and 14"2 ~ in the native compound and Br analogue respectively (figure 6). The furanose pucker is C(3')-exo, C(4')-endo (Native: P = 216.3 ~ ~m = 23"7~ Br: P = 217.7 ~ r = 19"6~
The geometry about the C(4')-C(5') bond is gauche-gauche. The intramolecular contact between C(6) o f the base and O(4') of the ribose is 2.71 A. However the geometry is not suitable for postulating a C ( 6 ) - H . . . 0(4') hydrogen bond (angle C ( 6 ) - H . . . 0(4') is 96.4~ Two intramolecular hydrogen bonds, N ( 3 ) - H . . . 0(2) and O(5')-H . . . 0(4) stabilize the crystal structure (figure 7). Both the molecular conformation and packing interactions are very similar in both the structures.
Br
Figure 6. Compound (iii). The molecular con- formation is anti, gauche-gauche. Sugar pucker is C(3')-exo, C(4')-endo.
Q
0 ( 4 ) ~ ~''~
Figure 7. Compound (iii). Two intermolecular hydrogen b o n d s (N(3)-H... 0(2) and O(5')-H... 0(4)) stabilize the crystal structure.
Sugar pucker is C(3')-endo.
Crystals were grown by diffusion of water into a solution of the compound in acetone. They belong to the trigonal space group P32 with a = b = 13.385(4), c = 9.900 (5)A (Gautham et al 1983b).
The molecule has the syn conformation (Xcs = - 115.9 (7) ~ unlike in the previous three structures. The exocyclic 0(2) atom o f the base is directly above the ribeysr ring (figure 8) instead o f being away from it as in the compounds with the anti conformation.
It may be mentioned that syn uracils are rare, the preferred orientation lacing anti. The sugar pucker is C(Y)-endo. (P = 14.1~ a geometry not seen previously in iso- propylidene derivatives. The C(3') atom is displaced by (Y322 A from the ribosr ring plane (~/m = 21"7~ The conformation about the C(4')-C(5') bond is gauche-trans with the 0(5') atom turned away from the ribose as shown in figure 8. This is unlike the gauche-gauche geometry observed in the previous three compounds. The crystal structure has the molecules packed in helices around the 32 screw axis with the uracil bases parallel to the helix axis (figure 9). The structure is stabilized by a possible bifurcated hydrogen bond between N(3) as donor and 0(2) and 0(4) as acceptors. A striking feature of the crystal structure is the complete absence of stacking interactions.
C(B) (
N (1) ~ " ~ . , ~ ' ~ C (2 } .X. ,.. w f .
0{2) 0(/-')
0(5')
Fig-are 8. Compound (iv). The molecular conformation is syn, oauche-trans. Sugar pucker is C(Y)-eado.
Structure of isopropylidene 267
Figure 9. Compound (iv). The molecules pack in helices around the 32 screw axis with a possible bifurcated hydrogen bond N(3)-H... O(2)/N(3)--H... 0(4).
Figure 10. Compound (v). The molecular conformation is syn, gauche-trans as in com- pound (iv). Sugar pucker is C(2')-exo, C(3')-endo.
2.5 5'-O-acetyl.2',3'-O-isopropylideneuridine. The molecular conformation is syn. Sugar pucker is C(2')-exo, C(3')-endo.
Large crystals were grown by evaporation o f acetone solutions o f the compound.
They belong to the monoclinic space group P21 with a = 6-510(2), b = 8.432(2), c = 15-114 (2) A, fl = 101.42 (3) ~ (Seshadri et al 1983).
The molecular conformation is syn (XcN = - 103"9(3~ as in c o m p o u n d (iv) (figure 10). The ribose has the twist conformation C(2')-exo, C(3')-endo ( P = 350-8 ~
~km = 17"2~ Besides the present compounds, (iv) and (v), four other uracil nucleosides (Saenger and Sheit 1970; Depmeier et al 1977; Suck et a11972, 1974) have been reported to have a syn conformation, two of which have the same ribose pucker as in the present case. The conformation about the C(4')-C(5') bond is gauche-trans. This orientation (Chem. Sci.)- 6
were to be gauche-gauche. The crystal structure is stabilized by the presence of N(3)-H . . . 0 ( 4 ' ) hydrogen bonds ( N - H . . . O = 2.870A; N - I q . . . O = 169.3 ~ , figure 11). The involvement o f the ribose oxygen O (4') in such hydrogen bond formation is rarely seen in the crystal structures of nucleosides and nucleotides. It is generally found to take part in strong stacking interactions with bases (Bugg et al 1971).
2.5a ]VMR studies show syn conformation for compounds (iv) and (v) also in solution. In order to investigate whether the syn orientation found in compounds (iv) and (v) was an effect of the crystal environment, a 270 M H z proton NMR study of these compounds in DMSO solution was performed using the nuclear Overhauser effect (NOE). NOE o f the order o f 10 % was observed between the protons attached to C(6) and to C ( l ' ) atoms.
Little or no NOE was observed between the protons attached to C(6) and those attached to C(2') and C(3') atoms. Thus there is a clear indication that H(6) is closer to H ( l ' ) than
~
0(2)~ ~,%_C (2'}
C (1')~c(2~ 169\.~ .~.,,~
C 1 6 ) ~
CYS~ ~'O(4J
Figure
11. Compound (v). Detail of the crystal structure showing N(3)-H... O(4') hydrogen bond.(35018} ~
j(14.11)
(326.6, ... ~ . . ~ ~ j ( 2 6 . 6 , (267"4)~' ~ 0 ~ ~ ~ ~ ( 3 7 . 9 ,
(255-8)~
( 2 5 0 - 4 1 ~ ~ {a) ( 2 & 9.8 )*--.."~ \ 90
(245.5)
( 2 1 7 7 ) ~ . J ~-~(115.8)
(215.8) ~
(214"8, p(.;
(bJ
NO.~ 10 20 30 40
9 rn (*)
Figure
12. Pseudorotation parameters for ribose for isopropylidene derivatives. Eachpoint on the circle represents a specific value of phase angle P (given in parantheses). * Base Sugar cyclized nucleosides. [] Pyrimidines, [] Purines.Structure of isopropylidene 269 to H(2') or H(3') which is possible only if the compounds are present almost entirely in the syn conformation in solutions (Gautham et al 1984).
3. Implications for ribose ring conformational flexibility
A comparison of the present results with other isopropylidene nucleoside crystal structures (Zussman 1953; Delbaere and James 1974; Fujii et al 1976; Yamagata et al 1980, 1981; Sprang et a11978; Bode and Shenk 1977) shows that the ribose ring assumes a variety of conformations. Figure 12a, which is a plot of the phase angle P on the pseudorotation wheel, indicates this clearly. Figure 12b gives the distribution of ~km, the maximum amplitude of pucker. As can be seen from the figure, ~,,~ shows no preference for any particular range. It varies from 4 ~ (i.e. the furanose ring is almost flat) to nearly 49 ~ (i.e. the ring is highly puckered with one of the ring atoms displaced by more than 0.5 A from the mean plane). However it is of interest to note that in those compounds where there is a base-to-sugar cyclization through the C(4') atom, as in compounds (i) and (ii), the furanose pucker is restricted to the C(4') -endo, O(4')-exo region of the psuedorotation wheel.
Acknowledgement
We thank the Department of Science and Technology for financial support.
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