- Email: [email protected]
Molecular orbital calculations on the conformation of nucleic acids and their constituents
Biochimica et Biophysica Acta, 331 (1973) 154-164
Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands BBA 97854
M O L E C U L A R ORBITAL CALCULATIONS ON THE CONFORMATION OF NUCLEIC ACIDS AND THEIR CONSTITUENTS VIII. CONFORMATIONS OF 2',3'- AND 3',5'-CYCLIC NUCLEOTIDES
ANIL SARAN, HELENE BERTHOD and BERNARD PULLMAN lnstitut de Biologie PhysicoChimique, Laboratoire de Bioehimie Thdorique, associd au C.N.R.S. 13, rue P. et M. Curie, Paris 5~ (France)
(Received June 21st, 1973)
SUMMARY The quantum mechanical PCILO method has been used for the determination of the conformational properties of 2',3'- and 3',5'-cyclic nucleotides. For the 2',3'compounds the calculations indicate an appreciable probability of both the gg and gt conformers about the exocyclic C(4')-C(5')bond with perhaps a predominance of gt for pyrimidines and gg for purines. The pyrimidine 2',3'-cyclic nucleotides should also show a preference for a syn arrangement about the glycosyl bond, while the purine derivatives have a global minimum at XCN == 270 °, at the borderline between syn and anti conformers. In the 3',5'- compounds, the conformation about the exocyclic C(4')C(5') bond is tg. The pyrimidine 3',5'-cyclic nucleotides should show a strong preference for the anti conformation about the glycosyl bond. The corresponding purine compounds exhibit a global energy minimum at XCN = 270 ° and a local one at 90 °, both thus at the borderline between syn and anti conformers, the first of which seems, however, to be more favorable for the syn conformations (Zcn < 270 o) and the other for the anti ones (ZcN < 90 °). The available experimental data from crystallographic and solution studies agree with these principal conclusions.
INTRODUCTION Continuing our quantum-mechanical PCILO (Perturbative Configuration Interaction using Localized Orbitals) studies on the conformation of nucleic acids and their constituents 1-7, we present in this paper the results of computations carried out on the conformation of 2',3'- and 3',5'-cyclic nucleotides. The torsion angles involved are shown schematically in Figs la and lb. Assuming conformations for the sugar and cyclic phosphate rings, the conformation of 2',3'-cyclic nucleotides (Fig. 1a) involves the evaluation of three torsional angles: (i) XCNabout the sugar-base glycosyl bond 8'9, (ii) ~c(~,)-c~s,) about the exocyclic C(4')-C(5') bond (whether gg, gt or tg) (ref. 4) and (iii) ~c(s,)-o(5,) about the C(5')-O(5') bond ¢. in the Y, 5'-cyclic nucleotides the conformation about the C(4')-C(5') bond is necessarily tg and the conforma-
CYCLIC NUCLEOTIDE CONFORMATION
H~ 5"~.~'
,
N1 (F:~/)
c Y- o5' / ~
n,
a' /
~
t
T2
1
63'
\
H',
---
"Hr
/15' VH4,("~/\"~'5, ~_~..2~--~ , ''H 0 s'
~
\
H ,---C--'~
\
31 /
z'b
/
o'F%,, (a)
~
N1 (Py) ~ "t'0 ~PN9(Pu)
~
H~,"t',~f'~(,...~'/-~:, ~3'
155
o
,,,)~
z'
2 -
I-I 0 ;:,'
o,
(b)
Fig. 1. Torsion angles involved in 2',3'- a n d Y,5'-cyclic nucleotides,
tion of these molecules involves the evaluation of only two torsion angles 7.cN and ~c(2,)-o(2') (Fig. lb) 4. A very limited amount of X-ray crystal data is available in the literature on these two classes of cyclic nucleotides. Two 2',3'-cyclic nucleotides: uridine 2',3'-O,Ocyclophosphorothioate lo,al and cytidine 2',3'-cyclic phosphate x2,13, and three 3',5'cyclic nucleotides :adenosine3',5'-cyclic phosphate 14, uridine Y,5'-cyclic phosphate15 - 17 and 5'-methylene adenosine 3',5'-cyclic monophosphonate is, ~9 have been studied crystallographically. Recently a number of N M R studies on 2',3'- and 3',5'cyclic monophosphates and analogs in solution has appeared in the literature. Smith and coworkers 2°-24 have utilized both aaC and 1H N M R techniques to study the conformation of 2',3'- and 3',5'-cyclic nucleotides, Schweizer and Robins 25 used proton magnetic resonance (PMR) to study the conformation of 3',5'-cyclic nucleotides and their analogs and Lavallee and Coulter 26, also utilizing the PMR technique, have investigated the conformation of pyrimidine 2',3'-cyclic nucleotides. The last two groups of authors 25'~6 have assigned a conformation of the bases about the glycosyl bond from their spectra, while Smith and co-workers 21'22 do not commit themselves with respect to this problem but evaluate the relative population of 9g, 9t or tg rotamers about the C(4')-C(5') bond in 2',3'-cyclic nucleotides. No theoretical computations, either in the classical empirical approach or in any of the available quantum mechanical approaches (EHT, CNDO, INDO etc.) have yet been reported on the conformation of 2',3'- and Y,5'-cyclic nucleotides. This is thus the first theoretical computation on these biologically very important class of nucleotides. PROCEDURE
(A) Method As stated earlier, the method employed in the present investigation is the
156
A. SARAN et al.
PCILO method, the details of which can be found in the original papers 27-3°, An outline of the principle of the method was presented in Part I of this series ~ (see also ref. 31). The detailed computer program may be obtained from Q.C.P.E. I,Quantum Chemistry Program Exchange) at the Chemistry Department of Indiana University, Bloomington, Ind., U.S.A. (B) Geometrical input data For cytidine 2',3'-cyclic phosphate the geometrical input data have been taken from the X-ray crystal structure investigated by Coulter 13. There are two molecules in the asymmetric unit, Molecule A with the ribose conformation O(l')-endo, and Molecule B with the ribose nearly planar, very slightly O(l')-exo. The computations have been carried out for both molecules. For uridine 2',3'-cyclic phosphate we have taken the crystallographic data of uridine 2',3'-O,O-cyclophosphorothioate 11 with the observed O(1 ')-exo conformation of the ribose, which, however, as will be seen later, leads to problems. As no X-ray data exist for purine 2',3'-cyclic nucleotides, we have adopted for the geometries of their ribose and cyclic phosphate those of cytidine 2',3'-cyclic phosphate in its two forms A and B. The geometries of the bases have been taken from the crystal structure studies of 5'-methylene adenosine 3',5'-cyclic monophosphate 19 and guanosine-5'-phosphate 32. It may be interesting to indicate here the information about the conformation of the ribose moiety as obtained from N M R studies. Smith and co-workers 2°'24 find that while pyrimidine 2',3'-cyclic nucleotides show a preference for the C(3')-endoC(2')-exo conformation of the ribose, adenosine 2',3'-cyclic phosphate tends (less) towards the C(2')-endo-C(3')-exo conformation and guanosine 2',3'-cyclic phosphate does not show preference for any of the two principal conformations of the ribose. These authors specify that such equilibria are restricted by the cyclic phosphate ring to smaller amplitudes of pucker than those associated with nucleosides. None of these conformations, however has been found yet in the known crystal structures of the 2',3'-cyclic nucleotides. The situation is simpler in the 3',5'-cyclic nucleotides because their ribose moieties are almost rigidly fixed in a narrow region of conformation. All the compounds whose crystal structures are known have their ribose moieties in the conformational range between C(3')-endo-C(4')-exo and C(4')-exo-C(3')-endo(3T4.-~4T3) 19. The PMR studies of Schweizer and Robins 25 on 3',5'-cyclic nucleotides and their analogs show that the ribose moieties of these nucleotides are in the C(3')-endo conformation. Another indication of the conformational rigidity in the riboses 3',5'cyclic nucleotides comes from the studies of Smith and his coworkers 2L22. Their combined PMR and 13C N M R studies show that the ribose conformation in adenosine 3',5'-cyclic phosphate and in dibutyryl 3',5'-cyclic adenosine phosphate is C(3')endo-C(4')-exo (3T4) and that in thymidine 3',5'-cyclic phosphate it is C(4')-exo (4E). Thus, the N M R studies 21 -23,25 although enlarging slightly the range of possible conformations (3E.--~4E), essentially support the rigidity of the riboses in 3',5'-cyclic nucleotides as observed in crystal structures 14- a9. We have adopted, in this investigation, the geometries of the ribose and the cyclic phosphate from the crystal structure of uridine 3',5'-cyclic phosphate Molecule B and kept them constant for carrying out the computations for all the nucleotides with different bases. The geometry"adopted
CYCLIC NUCLEOTIDE CONFORMATION
157
in each specific case for the base will be indicated in the text when the results of the computations are presented.
( C) Notations The notations and definitions of the torsion angles involved in the present investigation are the same as in ref. 4, where they have been discussed in considerable length. The readers may thus refer to that paper for details. The three orientations of exocyclic C(5')-O(5') bond designated by the symbols gg, gt and tg refer to ¢c(4')-c(s,) which are equal to 60 °, 180 o and 300 °, respectively, and that the anti and syn ranges for the glycosyl torsion angles correspond to XcN = 0° :kg0 ° and XcN ----180 °:k90 °, respectively.
( D ) Construction of the conforrnational energy maps For 2',3'-cyclic nucleotides, the conformational energy maps have been constructed as a function of the torsion angles ~(cN and ~c(4,)-c(s,) with preselected values of ¢c(s,)-ots,). All hydrogen atoms have been taken into consideration in the computations, which have been carried out in 30 o increment of the angles. The presentation of the results on the conformational maps has been limited to the 5 kcals/mole isoenergy curve above the global minimum. The conformational energy maps for the 3',5'-cyclic nucleotides have been constructed as a function of the torsion angle ZcN with preselected values of (~C(2')-O(2')" The energies have been calculated with a 30 ° interval and are presented with the global minimum taken as energy zero, RESULTS AND DISCUSSION
( A ) Pyrimidine 2',Y-cyclic phosphates Fig. 2 shows the results of computations carried out for cytidine 2',3'-cyclic phosphate in the crystal form of Molecule A (ribose in the O(l')-endo conformation). Calculations have been carried out for three values of the rotation ~c~5')-ot5,), namely 180 °, 60 ° and 300 °. The three resulting maps being practically identical, we have reproduced here only the map corresponding ¢}c(5,)-o(5,) ---- 180 ° The results indicate that the most stable conformation should be syn with ZCN = 240 °. In Fig. 2 it is associated with #c(4,~-c~5,~ ~- 180 ° (gt). There exists, however, a very close energy minimum (0.5 kcal/mole above the global one) corresponding also to ZcN----240 ° but to #c~4,~-c~s,~ = 60 ° (gg). Another similar low energy local minimum exists also for XcN ~--90 o and gg conformation about the exocyclic bond. Calculations have also been performed for the crystal form of Molecule B (ribose nearly planar, slightly O(1 ')-exo) of cytidine 2',3'-cyclic phosphate. The results are not reproduced here being very similar to those of Fig. 2. The global energy minimum corresponds again to a syn conformation about the glycosyl bond (;(cN = 240 °), with, however, an inversion of the relative preference for the gg and gt conformation about the exocyclic bond. The computations account very satisfactorily for the observed crystal results, both molecules having the syn conformation about the glycosidic bond with XcN = 242.9 ° and 254.5 ° and one of them being gg and the other gt with respect to ~Pc(4,~-c(5,r The N M R study of cytidine 2',Y-cyclic phosphate 2. gives an evaluation of the
158
A. S A R A N et al.
~C4,-05, 36O
300
240
"-
180
120
60
0
60
120
180
240
500
360 XCN
Fig. 2. Conformational energy map for cytidine 2',3'-cyclic phosphate (Molecule A), Isoenergy
curves (kcal/mole) as a function Of ZcN and ~c(4')-c(5') with respect to the global energy minimum taken as energy zero. Calculations made with tPc(5,)_o(5,) -- 180°.
relative population of the gg, gt and t9 rotamers as 0.35, 0.46 and 0.19, respectively, in agreement with the order suggested by Fig. 2. It should be mentioned that the calculation of the relative population of g#, 9t and t9 rotamers from P M R studies, presupposes a preference for the 9t over the t9 conformer. On the other hand, computations carried out for uridine 2',3'-cyclic phosphate using the geometry of uridine 2',3'-O,O-cyclophosphorothioate as input data give very unsatisfactory results (a very limited conformationally allowed zone with a global minimum for 99 and ZcN = 90 °)which do not reproduce the X-ray observation (t9 and ZcN = --13 °) obviously because of the very unusual pucker of the ribose (O(l')-exo) which introduces very strong steric hindrances in the molecule. When computations are carried out for the same base with a modified geometry of the ribose, corresponding to that of Molecule A of cytidine cyclic phosphate the results are very similar t o those of Fig. 2. We are inclined to think that the above quoted crystal conformation of uridine 2',3'-O,O-cyclophosphorothioate must be due largely to the presence of a sulfur atom near the ribose moiety and to the crystal packing forces.
(B) Purine 2',Y-cyclic phosphates The conformational energy map of adenosine 2',3'-cyclic phosphate with the O ( l ' ) - e n d o conformation of the sugar is reported in Fig. 3. (The energy map being approximately independent of the 4~c(s,).o(s,) rotation, the figure corresponds to the value 180 ° for this angle.)
CYCLIC NUCLEOTIDE C O N F O R M A T I O N
159
¢c~,- c 5,
i 3 300
(
i
",J:/~
/jA I
240
f
//
~
')
'
'
:
0
60
120
180
240
i
300
360 XCN
Fig. 3. Conformational energy map for adenosine 2',3'-cyclic phosphate. Isoenergy curves (kcal/ mole) as a function of ZCN and ~¢(4')-c(5') with respect to the global energy minimum taken as energy zero. Calculations made with ~c(5')-o(5') = 180°.
It is seen that the most stable conformation occurs for ZcN - 270 o, an intermediate position between the anti and syn orientations about the glycosyl bond and for the gg conformation about the exocyclic bond with two secondary minima, 0.5 kcal/mole above the global one, one for ZCN = 270 ° and gt conformation, the other for ZcN = 90 ° and gg conformation. We have also computed a conformational energy map for the same compound with a nearly planar conformation of the ribose. The results are very similar to those of Fig. 3. The global and the low-lying local energy minima correspond all to the gg conformations but there appears now also the possibility of a syn orientation about the glycosyl bond at ZCN = 180 o for the value of q~os,)-o(s,) equal to 60 ° The only experimental data with which we may compare our theoretical results come from N M R solution studies 24. The evaluation of the relative population of the go, gt and tg rotamers by this technique for the adenosine 2',3'-cyclic phosphate gives 0.60, 0.34 and 0.06, respectively, and points thus to a strong preponderance of the g9 conformer in good agreement with our results. We have constructed a few conformational energy maps for guanosine 2',3'cyclic phosphate. The overall features are similar to the corresponding maps for the adenosine compound. A typical map constructed with the same hypothesis as that of Fig. 3 is presented in Fig. 4. It resembles closely that of Fig. 3 indicating a preference for the gg conformation and for ZcN ~ 270 o with, however, close local energy minima for gg and ZCN = 90 o and gt and gcr~ = 270 °. This result is also in good agreement with the populations evaluated by the
160
A. S A R A N et al
<~Cz -Cb'
/
f
-
" ~
J
"
61
~o- : _ t : t ~ ) )> s , \ ! < 7: :~.... .//>.<::......\ . \ k 60
120
180
\
\
,,,J~,
~AY _-~
240
300
360 XCN
Fig. 4. C o n f o r m a t i o n a l energy m a p for g u a n o s i n e 2',3'-cyclic p h o s p h a t e . I s o e n e r g y curves (kcal/ mole) as a f u n c t i o n o f %cN a n d O c ( ¢ , ) . c ( s , ) w i t h respect to the global energy m i n i m u m t a k e n as energy zero. Calculations m a d e with ¢~¢(5')-o(s') = 180°.
N M R studies 24, which are 0.47, 0.34 and 0.19, respectively, for the gg, gt and tg conformations in the case of guanosine 2',3'-cyclic phosphate (sodium salts) and 0.48, 0.47 and 0.05 in the case of its pyridine salt.
( C) Pyrimidine 3',5'-cyclic phosphates Fig. 5 shows the results of computations carried out for uridine 3',5'-cyclic E
i
(kcll/mole) ,I
~2i
~0~I
8!
I
20
, 60
120
180
240
3'00
"~L 360 l
Fig. 5. C o n f o r m a t i o n a l energy m a p for uridine 3',5'-cyclic p h o s p h a t e , as a f u n c t i o n o f g c N. - ~/ic(2,).o(2, ) = 180°; - - -, £bc(2,)_0(2, ) = 60 °, with the global m i n i m u m taken as energy zero.
CYCLIC NUCLEOTIDE CONFORMATION
161
phosphate. The geometry of uracil has been taken from the crystal structure of uridine 3',5'-cyclic phosphate, Molecule B 15'16. Three preselected values of t~C(2,).O(2, ) equal to 60 °, 180 o and 300 ° have been used in the computations and the three curves have very similar shapes. We have reproduced in Fig. 5 two such curves, corresponding to ~ C ( 2 ' ) - O ( 2 ' ) ~ 6 0 o and 180 o. The global minimum for ~ C ( 2 ' ) - O ( 2 ' ) ~ 1 8 0 o is, respectively, 2.4 and 2.6 kcals/mole lower than that for t~C(2,)_O(2, ) equal to 60 ° and 300 °. It is seen from Fig. 5 that the global minimum associated with the (~C(2')-O(2') ~ 180 o curve occurs for XCN = 55 °-85 °, thus in the anti region. The syn region (ZcN ---180 °-270 °) is at least 3.3 kcals/mole higher. The curve associated with ~C(2')-O(2') ----60 ° predicts a global minimum at ZCN = 0 ° and the whole anti region for XCN-- 0 o to 90 ° is within 0.5 kcal/mole above this global minimum. The syn region for this curve is at least 4 kcals/mole above above the global minimum. Thus both curves predict a global minimum in the anti region of XCN"The crystalline asymmetric unit of uridine 3',5'-cyclic phosphate 16 contains two molecules with XCN ---- 77.0 ° (Molecule A) and 58.0 ° (Molecule B). As the hydrogen atom attached to 0(2') was not located in the crystal structure 16, the value of t~C(2,).O(2, ) cannot be given. The crystallographic conformation of uridine 3',5'-cyclic phosphate is, however, in excellent agreement with the predictions. The PMR results of Schweizer and Robins 25 also predict an anti conformation for uridine 3',5'-cyclic phosphate. For cytidine 3',5'-cyclic phosphate, the geometry of cytosine has been adopted from the crystal structure data of cytidine 2',3'-cyclic phosphate, Molecule A ~3. The results are very similar to those of Fig. 5 and predict the anti region (global minimum towards XCN = 60 °) to be more stable than the syn region by at least 4 kcals/mole. Unfortunately, neither crystal structure data nor PMR studies on cytidine 3',5'-cyclic phosphate are available. To complete the pyrimidine 3',5'-cyclic phosphate series we have extended our computations to thymidine 3',5'-cyclic phosphate. The geometry of thymine has been taken from the crystal structure studies of thymidine by Young, Tollin et a133. Since the O(2'-)-H group in thymidine is replaced by H, the O(2') of the ribose has been replaced by a hydrogen atom and there is no ~c(2,)-0(2,) torsion angle for this compound. The result of the computation shows a complete analogy to those obtained for uridine 3',5'cyclic phosphate and cytidine 3',5'-cyclic phosphate and will, therefore, not be reproduced here. The absence of the O(2'-)-H group in thymidine 3',5'-cyclic phosphate does not change the shape of the curve and again predicts the preference for an anti conformation over a syn one which in this case is about 4.4 kcals/mole more than the global minimum. No crystal structure data are available for this compound. Altogether it is thus clear that pyrimidine 3',5'-cyclic nucleotides show a strong preference for the anti conformation of the sugar-base orientation. ( D) Purine 3',5'-cyclic phosphates Fig. 6a shows the results of computations carried out for adenosine 3',5'-cyclic phosphate with preselected value of q~ct2,)-o(2,) ---- 180 °. As the published X-ray crystal results on adenosine 3',5'-cyclic monophosphate 14 do not contain detailed data on the geometry of the base we have used as input the geometry of the adenine ring in 5'-methylene adenosine 3',5'-cyclic monophosphonate 19. We have also carried out computations on this last compound itself by taking its crystallographic geometry
162 E
A. S A R A N et al. A
(~cal/moFe)
0
BO
120
180
240
300
560
XCN
Fig. 6. (a) C o n f o r m a t i o n a l energy m a p for adenosine Y,5'-cyclic p h o s p h a t e as a function o f ZCN. ~C(2")-O(2') = 180 °, a n d the global m i n i m u m taken as energy zero. ( b ) C o n f o r m a t i o n a l energy m a p for 5'-methylene adenosine Y,5'-cyclic m o n o p h o s p h o n a t e as a function o f ZCN. t~c(2')-o(2') ~ 44.2°, a n d the global m i n i m u m taken as energy zero.
and fixing t~C(2,).O(2, ) z 44.2 o in its crystallographic conformation. The results are shown in Fig. 6b. The two curves are practically identical in shape and point to no significant change in the conformational map due to the little changes in the ribose geometry. The representative curves show a global energy minimum towards ZcN ---- 270 °, thus at the borderline between syn and anti conformers and a secondary, local energy minimum towards ZCN ~ 90° about 2-3 kcals/mole above the global one, thus at the other borderline between the syn and anti conformers with, however, a shape of the potential well favoring in this case somewhat the anti conformations. Among the three crystallographic results available, two (adenosine 3',5'-cyclic phosphate, Molecule B and 5'-methylene adenosine 3',5'-cyclic monophosphonate/monohydrate) have a syn conformation, at ZcN ---- 258 o and 234.2 o, respectively, close to the global minimum of our map (0.4 and 0.8 kcal/mole above it, respectively). The third one, adenosine 3',5'-cyclic phosphate, Molecule A has an anti conformation, at ZCN --~ 50 o, corresponding obviously to the secondary energy minimum of our map. Solution data available for adenosine 3',5'-cyclic nucleotide are not clear cut. Schweizer and Robins 25 interpret their P M R results as indicating the existence of an anti conformation, while Klee and Mudd a4 deduce from the ORD studies that the compound exists in solution in the syn conformation. Finally, we have also carried out computations for guanosine 3',5'-cyclic phosphate. The geometry of guanine has been taken from the crystal structure of guanosine 5'-phosphate 32. The results are extremely similar to those of Fig. 6 with the global minimum again at ZCN = 270 ° with, however, a shape of the curve suggesting a preference for the neighbouring syn conformation (ZCN < 270 °) rather than for the anti ones (XCN 7> 270 o) and a local energy minimum around ZCN --- 90 ° of 3.2 kcals/mole above the global one. No X-ray crystal data are available as yet for this compound*. However, PMR data 25 on this compound in solution suggest that guanosine 3',5'-cyclic phosphate has the syn conformation. Altogether it appears thus that purine 3',5'-cyclic nucleotides exhibit two energy minima at the borderline between the syn and anti conformations with, however, a higher probability of the global minimum to be associated with the syn conformation. CONCLUSION
This exploration of the conformational possibilities of the cyclic nucleotides has brought into evidence a variety of patterns related to the two types of cyclic * See note added in proof.
CYCLIC NUCLEOTIDE CONFORMATION
163
structures studied (2',3' and 3',5') and the nature of the base type (purine or pyrimidine) considered. In the 2',3'-cyclic nucleotides two torsion angles are fundamental which define the orientations of the exocyclic C(5')-O(5') bond and of the base with respect to the sugar. The calculations seem to indicate a preference for both the gg and the gt conformations about the exocyclic C(4')-C(5') bond with perhaps a predominance of gt in the case of the pyrimidine nucleotides and of gg in the case of the purine nucleotides. The energy of the tg conformers is appreciably higher than those of the gt and gg ones. The pyrimidine 2',3'-cyclic nucleotides also show a distinct preference for a syn arrangement of the base with respect to the sugar, while the theoretical results for the purine derivatives place the global energy minimum at the borderline between the syn and anti conformers. In the 3',5'-cyclic nucleotides the orientation of the exocyclic C(5')-O(5') bond is necessarily tg and the essential degree of freedom relates to ZcN. The calculations indicate that the pyrimidine bases should manifest a strong preference for the attti conformation (ZcN = 60 °-90 °). The purine derivatives ,on the other hand exhibit a global energy minimum at ~(cN -- 270 o and a local one at ZcN = 90 °, both at the borderline between the syn and anti conformations. The available crystallographic and solution data seem to confirm the plausibility of the theoretical results. In the last above-quoted case of the purine 3',5'-cyclic nucleotides they seem to indicate moreover that the global minimum at ZcN - 270 ° is more favorable than syn conformations with a torsion angle slightly smaller than this value, while the local minimum at ZcN =: 90 ° favors the anti conformations with the torsion angles towards somewhat smaller values. To the already-quoted references we may add a recent one relevant to calorimetric investigations of crystalline 3',5'-cyclic nucleotides 35 which seems also to confirm the existence of at least some of the purine compounds in two conformational arrangements within the unit cell while the pyrimidines manifest only one such arrangement. NOTE ADDED IN PROOF (Received November 1st, 1973) Such data have just been published 36. The experimental ZcN = 258 ° is in excellent agreement with our prediction. ACKNOWLEDGEMENTS The authors thank Dr I. C. P. Smith for communication of data prior to publication. This work was supported by the A.T.P. No. A655-2303 of the C.N.R.S. REFERENCES 1 2 3 4 5
Berthod, t-I. and Pullman, B. (1971) Biochim. Biophys. Acta 232, 595-606 Berthod, I-L and Pullman, B. (1971) Biochim. Biophys. Acta 246, 359-364 Pullman, B., Perahia, D. and Saran, A. (1972) Biochim. Biophys. Acta 269, 1-14 Saran, A., Pullman, B. and Perahia, D. (1972) Biochim. Biophys. Acta 287, 211-231 Pullman, B. and Berthod, H. (1973) in Conformations of Biological Molecules and Polymers, 5th Jerusalem Syrup. Proc., pp. 209-224, Academic Press, New York
164 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
A. SARAN et al.
Saran, A., Pullman, B. and Perahia, D. (1973) Biochim. Biophys. Acta 299, 497-499 Saran, A., Perahia, D. and Pullman, B. (1973) Theor. Chim. Acta Berlin, 30, 31-44 Sundaralingam, M. (1969) Biopolymers 7, 821-860 Donohue, J. and Trueblood, K. N. (1960) J. Mol. BioL 2, 363-371 Saenger, W. and Eckstein, F. (1969) Angew. Chem. Int. Engl. Edn. 8, 595-596 Saenger, W. and Eckstein, F. (1970) J. Am. Chem. Soc. 92, 4712-4718 Coulter, C. L. and Greaves, M. L. (1970) Science 169, 1097-1098 Coulter, C. L. (1973) J. Am. Chem. Soc. 95, 570-575 Watenpaugh, K., Dow, J., Jensen, L. I-L and Furberg, S. (1968) Science 159, 206-207 Coulter, C. L. (1968) Science 159, 888-889 Coulter, C. L. (1969) Acta Crystallogr. B25, 2055-2065 Coulter, C. L. (1970) Acta Crystallogr. B26, 441-441 Sundaralingam, M. and Abola, J. (1972) Nat. New Biol. 235,244-245 Sundaralingam, M. and Abola, J. (1972) J. Am. Chem. Soc. 94, 5070-5076 Smith, I. C. P., Mantsch, H. H., Lapper, R. D., Deslauriers, R. and Schleich, T. (1973) in Conformations o f Biological Molecules and Polymers, 5th Jerusalem Syrup. Proc., pp. 381-402, Academic Press, New York Lapper, R. D., Mantsch, H. H. and Smith, I. C. P. (1972) J. Am. Chem. Soc. 94, 6243-6244 Blackburn, B. J., Lapper, R. D. and Smith, I. C. P. (1973) J. Am. Cem. Soc. 95, 2873-2878 Lapper, R. D., Mantsch, H. lrI. and Smith, I. C. P. (1973) J. Am. Chem. Soc. 95, 2878-2880 Lapper, R. D. and Smith, I. C. P. (1973) J. Am. Chem. Soc. 95, 2880-2884 Schweizer, M. P. and Robins, R. K. (1973) in Conformation o f Biological Molecules and Polymers, 5th Jerusalem Syrup. Proc., pp. 329-343, Academic Press, New York Lavallee, D. K. and Coulter, C. L. (1973) J. Am. Chem. Soc. 95, 576-581 Diner, S., Malrieu, J. P. and Claverie, P. (1969) Theor. Chim. Acta Berlin 13, 1-17 Malrieu, J. P., Claverie, P. and Diner, S. (1969) Theor. Chim. Acta Berlin 13, 18-45 Diner, S., Malrieu, J. P., Jordan, F. and Gilbert, M. (1969) Theor. Chim. Acta Berlin 15, 100110 Jordan, F., Gilbert, M., Malrieu, J. P. and Pincelli, U. (1969) Theor. Chim. Acta Berlin 15, 211-224 Pullman, B. and Pullman, A. (1973) Adv. Protein Res., in the press Murayama, W., Nagashima, N. and Shimuzu, Y. (1969) Acta Cystallogr. B25, 2236-2245 Young, D. W., Tollin, P. and Wilson, I-[. R. (1969) Acta Crystallogr. B25, 1423-1432 Klee, W. A. and Mudd, S. H. (1967) Biochemistry 6, 988-998 Bryan, A. M. and Olafsson, P. G. (1973) Biopolymers 12, 229-235 Chang, A. K. and Sundaralingam, M. (1973) Nature New Biology 244, 136-137
Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands BBA 97854
M O L E C U L A R ORBITAL CALCULATIONS ON THE CONFORMATION OF NUCLEIC ACIDS AND THEIR CONSTITUENTS VIII. CONFORMATIONS OF 2',3'- AND 3',5'-CYCLIC NUCLEOTIDES
ANIL SARAN, HELENE BERTHOD and BERNARD PULLMAN lnstitut de Biologie PhysicoChimique, Laboratoire de Bioehimie Thdorique, associd au C.N.R.S. 13, rue P. et M. Curie, Paris 5~ (France)
(Received June 21st, 1973)
SUMMARY The quantum mechanical PCILO method has been used for the determination of the conformational properties of 2',3'- and 3',5'-cyclic nucleotides. For the 2',3'compounds the calculations indicate an appreciable probability of both the gg and gt conformers about the exocyclic C(4')-C(5')bond with perhaps a predominance of gt for pyrimidines and gg for purines. The pyrimidine 2',3'-cyclic nucleotides should also show a preference for a syn arrangement about the glycosyl bond, while the purine derivatives have a global minimum at XCN == 270 °, at the borderline between syn and anti conformers. In the 3',5'- compounds, the conformation about the exocyclic C(4')C(5') bond is tg. The pyrimidine 3',5'-cyclic nucleotides should show a strong preference for the anti conformation about the glycosyl bond. The corresponding purine compounds exhibit a global energy minimum at XCN = 270 ° and a local one at 90 °, both thus at the borderline between syn and anti conformers, the first of which seems, however, to be more favorable for the syn conformations (Zcn < 270 o) and the other for the anti ones (ZcN < 90 °). The available experimental data from crystallographic and solution studies agree with these principal conclusions.
INTRODUCTION Continuing our quantum-mechanical PCILO (Perturbative Configuration Interaction using Localized Orbitals) studies on the conformation of nucleic acids and their constituents 1-7, we present in this paper the results of computations carried out on the conformation of 2',3'- and 3',5'-cyclic nucleotides. The torsion angles involved are shown schematically in Figs la and lb. Assuming conformations for the sugar and cyclic phosphate rings, the conformation of 2',3'-cyclic nucleotides (Fig. 1a) involves the evaluation of three torsional angles: (i) XCNabout the sugar-base glycosyl bond 8'9, (ii) ~c(~,)-c~s,) about the exocyclic C(4')-C(5') bond (whether gg, gt or tg) (ref. 4) and (iii) ~c(s,)-o(5,) about the C(5')-O(5') bond ¢. in the Y, 5'-cyclic nucleotides the conformation about the C(4')-C(5') bond is necessarily tg and the conforma-
CYCLIC NUCLEOTIDE CONFORMATION
H~ 5"~.~'
,
N1 (F:~/)
c Y- o5' / ~
n,
a' /
~
t
T2
1
63'
\
H',
---
"Hr
/15' VH4,("~/\"~'5, ~_~..2~--~ , ''H 0 s'
~
\
H ,---C--'~
\
31 /
z'b
/
o'F%,, (a)
~
N1 (Py) ~ "t'0 ~PN9(Pu)
~
H~,"t',~f'~(,...~'/-~:, ~3'
155
o
,,,)~
z'
2 -
I-I 0 ;:,'
o,
(b)
Fig. 1. Torsion angles involved in 2',3'- a n d Y,5'-cyclic nucleotides,
tion of these molecules involves the evaluation of only two torsion angles 7.cN and ~c(2,)-o(2') (Fig. lb) 4. A very limited amount of X-ray crystal data is available in the literature on these two classes of cyclic nucleotides. Two 2',3'-cyclic nucleotides: uridine 2',3'-O,Ocyclophosphorothioate lo,al and cytidine 2',3'-cyclic phosphate x2,13, and three 3',5'cyclic nucleotides :adenosine3',5'-cyclic phosphate 14, uridine Y,5'-cyclic phosphate15 - 17 and 5'-methylene adenosine 3',5'-cyclic monophosphonate is, ~9 have been studied crystallographically. Recently a number of N M R studies on 2',3'- and 3',5'cyclic monophosphates and analogs in solution has appeared in the literature. Smith and coworkers 2°-24 have utilized both aaC and 1H N M R techniques to study the conformation of 2',3'- and 3',5'-cyclic nucleotides, Schweizer and Robins 25 used proton magnetic resonance (PMR) to study the conformation of 3',5'-cyclic nucleotides and their analogs and Lavallee and Coulter 26, also utilizing the PMR technique, have investigated the conformation of pyrimidine 2',3'-cyclic nucleotides. The last two groups of authors 25'~6 have assigned a conformation of the bases about the glycosyl bond from their spectra, while Smith and co-workers 21'22 do not commit themselves with respect to this problem but evaluate the relative population of 9g, 9t or tg rotamers about the C(4')-C(5') bond in 2',3'-cyclic nucleotides. No theoretical computations, either in the classical empirical approach or in any of the available quantum mechanical approaches (EHT, CNDO, INDO etc.) have yet been reported on the conformation of 2',3'- and Y,5'-cyclic nucleotides. This is thus the first theoretical computation on these biologically very important class of nucleotides. PROCEDURE
(A) Method As stated earlier, the method employed in the present investigation is the
156
A. SARAN et al.
PCILO method, the details of which can be found in the original papers 27-3°, An outline of the principle of the method was presented in Part I of this series ~ (see also ref. 31). The detailed computer program may be obtained from Q.C.P.E. I,Quantum Chemistry Program Exchange) at the Chemistry Department of Indiana University, Bloomington, Ind., U.S.A. (B) Geometrical input data For cytidine 2',3'-cyclic phosphate the geometrical input data have been taken from the X-ray crystal structure investigated by Coulter 13. There are two molecules in the asymmetric unit, Molecule A with the ribose conformation O(l')-endo, and Molecule B with the ribose nearly planar, very slightly O(l')-exo. The computations have been carried out for both molecules. For uridine 2',3'-cyclic phosphate we have taken the crystallographic data of uridine 2',3'-O,O-cyclophosphorothioate 11 with the observed O(1 ')-exo conformation of the ribose, which, however, as will be seen later, leads to problems. As no X-ray data exist for purine 2',3'-cyclic nucleotides, we have adopted for the geometries of their ribose and cyclic phosphate those of cytidine 2',3'-cyclic phosphate in its two forms A and B. The geometries of the bases have been taken from the crystal structure studies of 5'-methylene adenosine 3',5'-cyclic monophosphate 19 and guanosine-5'-phosphate 32. It may be interesting to indicate here the information about the conformation of the ribose moiety as obtained from N M R studies. Smith and co-workers 2°'24 find that while pyrimidine 2',3'-cyclic nucleotides show a preference for the C(3')-endoC(2')-exo conformation of the ribose, adenosine 2',3'-cyclic phosphate tends (less) towards the C(2')-endo-C(3')-exo conformation and guanosine 2',3'-cyclic phosphate does not show preference for any of the two principal conformations of the ribose. These authors specify that such equilibria are restricted by the cyclic phosphate ring to smaller amplitudes of pucker than those associated with nucleosides. None of these conformations, however has been found yet in the known crystal structures of the 2',3'-cyclic nucleotides. The situation is simpler in the 3',5'-cyclic nucleotides because their ribose moieties are almost rigidly fixed in a narrow region of conformation. All the compounds whose crystal structures are known have their ribose moieties in the conformational range between C(3')-endo-C(4')-exo and C(4')-exo-C(3')-endo(3T4.-~4T3) 19. The PMR studies of Schweizer and Robins 25 on 3',5'-cyclic nucleotides and their analogs show that the ribose moieties of these nucleotides are in the C(3')-endo conformation. Another indication of the conformational rigidity in the riboses 3',5'cyclic nucleotides comes from the studies of Smith and his coworkers 2L22. Their combined PMR and 13C N M R studies show that the ribose conformation in adenosine 3',5'-cyclic phosphate and in dibutyryl 3',5'-cyclic adenosine phosphate is C(3')endo-C(4')-exo (3T4) and that in thymidine 3',5'-cyclic phosphate it is C(4')-exo (4E). Thus, the N M R studies 21 -23,25 although enlarging slightly the range of possible conformations (3E.--~4E), essentially support the rigidity of the riboses in 3',5'-cyclic nucleotides as observed in crystal structures 14- a9. We have adopted, in this investigation, the geometries of the ribose and the cyclic phosphate from the crystal structure of uridine 3',5'-cyclic phosphate Molecule B and kept them constant for carrying out the computations for all the nucleotides with different bases. The geometry"adopted
CYCLIC NUCLEOTIDE CONFORMATION
157
in each specific case for the base will be indicated in the text when the results of the computations are presented.
( C) Notations The notations and definitions of the torsion angles involved in the present investigation are the same as in ref. 4, where they have been discussed in considerable length. The readers may thus refer to that paper for details. The three orientations of exocyclic C(5')-O(5') bond designated by the symbols gg, gt and tg refer to ¢c(4')-c(s,) which are equal to 60 °, 180 o and 300 °, respectively, and that the anti and syn ranges for the glycosyl torsion angles correspond to XcN = 0° :kg0 ° and XcN ----180 °:k90 °, respectively.
( D ) Construction of the conforrnational energy maps For 2',3'-cyclic nucleotides, the conformational energy maps have been constructed as a function of the torsion angles ~(cN and ~c(4,)-c(s,) with preselected values of ¢c(s,)-ots,). All hydrogen atoms have been taken into consideration in the computations, which have been carried out in 30 o increment of the angles. The presentation of the results on the conformational maps has been limited to the 5 kcals/mole isoenergy curve above the global minimum. The conformational energy maps for the 3',5'-cyclic nucleotides have been constructed as a function of the torsion angle ZcN with preselected values of (~C(2')-O(2')" The energies have been calculated with a 30 ° interval and are presented with the global minimum taken as energy zero, RESULTS AND DISCUSSION
( A ) Pyrimidine 2',Y-cyclic phosphates Fig. 2 shows the results of computations carried out for cytidine 2',3'-cyclic phosphate in the crystal form of Molecule A (ribose in the O(l')-endo conformation). Calculations have been carried out for three values of the rotation ~c~5')-ot5,), namely 180 °, 60 ° and 300 °. The three resulting maps being practically identical, we have reproduced here only the map corresponding ¢}c(5,)-o(5,) ---- 180 ° The results indicate that the most stable conformation should be syn with ZCN = 240 °. In Fig. 2 it is associated with #c(4,~-c~5,~ ~- 180 ° (gt). There exists, however, a very close energy minimum (0.5 kcal/mole above the global one) corresponding also to ZcN----240 ° but to #c~4,~-c~s,~ = 60 ° (gg). Another similar low energy local minimum exists also for XcN ~--90 o and gg conformation about the exocyclic bond. Calculations have also been performed for the crystal form of Molecule B (ribose nearly planar, slightly O(1 ')-exo) of cytidine 2',3'-cyclic phosphate. The results are not reproduced here being very similar to those of Fig. 2. The global energy minimum corresponds again to a syn conformation about the glycosyl bond (;(cN = 240 °), with, however, an inversion of the relative preference for the gg and gt conformation about the exocyclic bond. The computations account very satisfactorily for the observed crystal results, both molecules having the syn conformation about the glycosidic bond with XcN = 242.9 ° and 254.5 ° and one of them being gg and the other gt with respect to ~Pc(4,~-c(5,r The N M R study of cytidine 2',Y-cyclic phosphate 2. gives an evaluation of the
158
A. S A R A N et al.
~C4,-05, 36O
300
240
"-
180
120
60
0
60
120
180
240
500
360 XCN
Fig. 2. Conformational energy map for cytidine 2',3'-cyclic phosphate (Molecule A), Isoenergy
curves (kcal/mole) as a function Of ZcN and ~c(4')-c(5') with respect to the global energy minimum taken as energy zero. Calculations made with tPc(5,)_o(5,) -- 180°.
relative population of the gg, gt and t9 rotamers as 0.35, 0.46 and 0.19, respectively, in agreement with the order suggested by Fig. 2. It should be mentioned that the calculation of the relative population of g#, 9t and t9 rotamers from P M R studies, presupposes a preference for the 9t over the t9 conformer. On the other hand, computations carried out for uridine 2',3'-cyclic phosphate using the geometry of uridine 2',3'-O,O-cyclophosphorothioate as input data give very unsatisfactory results (a very limited conformationally allowed zone with a global minimum for 99 and ZcN = 90 °)which do not reproduce the X-ray observation (t9 and ZcN = --13 °) obviously because of the very unusual pucker of the ribose (O(l')-exo) which introduces very strong steric hindrances in the molecule. When computations are carried out for the same base with a modified geometry of the ribose, corresponding to that of Molecule A of cytidine cyclic phosphate the results are very similar t o those of Fig. 2. We are inclined to think that the above quoted crystal conformation of uridine 2',3'-O,O-cyclophosphorothioate must be due largely to the presence of a sulfur atom near the ribose moiety and to the crystal packing forces.
(B) Purine 2',Y-cyclic phosphates The conformational energy map of adenosine 2',3'-cyclic phosphate with the O ( l ' ) - e n d o conformation of the sugar is reported in Fig. 3. (The energy map being approximately independent of the 4~c(s,).o(s,) rotation, the figure corresponds to the value 180 ° for this angle.)
CYCLIC NUCLEOTIDE C O N F O R M A T I O N
159
¢c~,- c 5,
i 3 300
(
i
",J:/~
/jA I
240
f
//
~
')
'
'
:
0
60
120
180
240
i
300
360 XCN
Fig. 3. Conformational energy map for adenosine 2',3'-cyclic phosphate. Isoenergy curves (kcal/ mole) as a function of ZCN and ~¢(4')-c(5') with respect to the global energy minimum taken as energy zero. Calculations made with ~c(5')-o(5') = 180°.
It is seen that the most stable conformation occurs for ZcN - 270 o, an intermediate position between the anti and syn orientations about the glycosyl bond and for the gg conformation about the exocyclic bond with two secondary minima, 0.5 kcal/mole above the global one, one for ZCN = 270 ° and gt conformation, the other for ZcN = 90 ° and gg conformation. We have also computed a conformational energy map for the same compound with a nearly planar conformation of the ribose. The results are very similar to those of Fig. 3. The global and the low-lying local energy minima correspond all to the gg conformations but there appears now also the possibility of a syn orientation about the glycosyl bond at ZCN = 180 o for the value of q~os,)-o(s,) equal to 60 ° The only experimental data with which we may compare our theoretical results come from N M R solution studies 24. The evaluation of the relative population of the go, gt and tg rotamers by this technique for the adenosine 2',3'-cyclic phosphate gives 0.60, 0.34 and 0.06, respectively, and points thus to a strong preponderance of the g9 conformer in good agreement with our results. We have constructed a few conformational energy maps for guanosine 2',3'cyclic phosphate. The overall features are similar to the corresponding maps for the adenosine compound. A typical map constructed with the same hypothesis as that of Fig. 3 is presented in Fig. 4. It resembles closely that of Fig. 3 indicating a preference for the gg conformation and for ZcN ~ 270 o with, however, close local energy minima for gg and ZCN = 90 o and gt and gcr~ = 270 °. This result is also in good agreement with the populations evaluated by the
160
A. S A R A N et al
<~Cz -Cb'
/
f
-
" ~
J
"
61
~o- : _ t : t ~ ) )> s , \ ! < 7: :~.... .//>.<::......\ . \ k 60
120
180
\
\
,,,J~,
~AY _-~
240
300
360 XCN
Fig. 4. C o n f o r m a t i o n a l energy m a p for g u a n o s i n e 2',3'-cyclic p h o s p h a t e . I s o e n e r g y curves (kcal/ mole) as a f u n c t i o n o f %cN a n d O c ( ¢ , ) . c ( s , ) w i t h respect to the global energy m i n i m u m t a k e n as energy zero. Calculations m a d e with ¢~¢(5')-o(s') = 180°.
N M R studies 24, which are 0.47, 0.34 and 0.19, respectively, for the gg, gt and tg conformations in the case of guanosine 2',3'-cyclic phosphate (sodium salts) and 0.48, 0.47 and 0.05 in the case of its pyridine salt.
( C) Pyrimidine 3',5'-cyclic phosphates Fig. 5 shows the results of computations carried out for uridine 3',5'-cyclic E
i
(kcll/mole) ,I
~2i
~0~I
8!
I
20
, 60
120
180
240
3'00
"~L 360 l
Fig. 5. C o n f o r m a t i o n a l energy m a p for uridine 3',5'-cyclic p h o s p h a t e , as a f u n c t i o n o f g c N. - ~/ic(2,).o(2, ) = 180°; - - -, £bc(2,)_0(2, ) = 60 °, with the global m i n i m u m taken as energy zero.
CYCLIC NUCLEOTIDE CONFORMATION
161
phosphate. The geometry of uracil has been taken from the crystal structure of uridine 3',5'-cyclic phosphate, Molecule B 15'16. Three preselected values of t~C(2,).O(2, ) equal to 60 °, 180 o and 300 ° have been used in the computations and the three curves have very similar shapes. We have reproduced in Fig. 5 two such curves, corresponding to ~ C ( 2 ' ) - O ( 2 ' ) ~ 6 0 o and 180 o. The global minimum for ~ C ( 2 ' ) - O ( 2 ' ) ~ 1 8 0 o is, respectively, 2.4 and 2.6 kcals/mole lower than that for t~C(2,)_O(2, ) equal to 60 ° and 300 °. It is seen from Fig. 5 that the global minimum associated with the (~C(2')-O(2') ~ 180 o curve occurs for XCN = 55 °-85 °, thus in the anti region. The syn region (ZcN ---180 °-270 °) is at least 3.3 kcals/mole higher. The curve associated with ~C(2')-O(2') ----60 ° predicts a global minimum at ZCN = 0 ° and the whole anti region for XCN-- 0 o to 90 ° is within 0.5 kcal/mole above this global minimum. The syn region for this curve is at least 4 kcals/mole above above the global minimum. Thus both curves predict a global minimum in the anti region of XCN"The crystalline asymmetric unit of uridine 3',5'-cyclic phosphate 16 contains two molecules with XCN ---- 77.0 ° (Molecule A) and 58.0 ° (Molecule B). As the hydrogen atom attached to 0(2') was not located in the crystal structure 16, the value of t~C(2,).O(2, ) cannot be given. The crystallographic conformation of uridine 3',5'-cyclic phosphate is, however, in excellent agreement with the predictions. The PMR results of Schweizer and Robins 25 also predict an anti conformation for uridine 3',5'-cyclic phosphate. For cytidine 3',5'-cyclic phosphate, the geometry of cytosine has been adopted from the crystal structure data of cytidine 2',3'-cyclic phosphate, Molecule A ~3. The results are very similar to those of Fig. 5 and predict the anti region (global minimum towards XCN = 60 °) to be more stable than the syn region by at least 4 kcals/mole. Unfortunately, neither crystal structure data nor PMR studies on cytidine 3',5'-cyclic phosphate are available. To complete the pyrimidine 3',5'-cyclic phosphate series we have extended our computations to thymidine 3',5'-cyclic phosphate. The geometry of thymine has been taken from the crystal structure studies of thymidine by Young, Tollin et a133. Since the O(2'-)-H group in thymidine is replaced by H, the O(2') of the ribose has been replaced by a hydrogen atom and there is no ~c(2,)-0(2,) torsion angle for this compound. The result of the computation shows a complete analogy to those obtained for uridine 3',5'cyclic phosphate and cytidine 3',5'-cyclic phosphate and will, therefore, not be reproduced here. The absence of the O(2'-)-H group in thymidine 3',5'-cyclic phosphate does not change the shape of the curve and again predicts the preference for an anti conformation over a syn one which in this case is about 4.4 kcals/mole more than the global minimum. No crystal structure data are available for this compound. Altogether it is thus clear that pyrimidine 3',5'-cyclic nucleotides show a strong preference for the anti conformation of the sugar-base orientation. ( D) Purine 3',5'-cyclic phosphates Fig. 6a shows the results of computations carried out for adenosine 3',5'-cyclic phosphate with preselected value of q~ct2,)-o(2,) ---- 180 °. As the published X-ray crystal results on adenosine 3',5'-cyclic monophosphate 14 do not contain detailed data on the geometry of the base we have used as input the geometry of the adenine ring in 5'-methylene adenosine 3',5'-cyclic monophosphonate 19. We have also carried out computations on this last compound itself by taking its crystallographic geometry
162 E
A. S A R A N et al. A
(~cal/moFe)
0
BO
120
180
240
300
560
XCN
Fig. 6. (a) C o n f o r m a t i o n a l energy m a p for adenosine Y,5'-cyclic p h o s p h a t e as a function o f ZCN. ~C(2")-O(2') = 180 °, a n d the global m i n i m u m taken as energy zero. ( b ) C o n f o r m a t i o n a l energy m a p for 5'-methylene adenosine Y,5'-cyclic m o n o p h o s p h o n a t e as a function o f ZCN. t~c(2')-o(2') ~ 44.2°, a n d the global m i n i m u m taken as energy zero.
and fixing t~C(2,).O(2, ) z 44.2 o in its crystallographic conformation. The results are shown in Fig. 6b. The two curves are practically identical in shape and point to no significant change in the conformational map due to the little changes in the ribose geometry. The representative curves show a global energy minimum towards ZcN ---- 270 °, thus at the borderline between syn and anti conformers and a secondary, local energy minimum towards ZCN ~ 90° about 2-3 kcals/mole above the global one, thus at the other borderline between the syn and anti conformers with, however, a shape of the potential well favoring in this case somewhat the anti conformations. Among the three crystallographic results available, two (adenosine 3',5'-cyclic phosphate, Molecule B and 5'-methylene adenosine 3',5'-cyclic monophosphonate/monohydrate) have a syn conformation, at ZcN ---- 258 o and 234.2 o, respectively, close to the global minimum of our map (0.4 and 0.8 kcal/mole above it, respectively). The third one, adenosine 3',5'-cyclic phosphate, Molecule A has an anti conformation, at ZCN --~ 50 o, corresponding obviously to the secondary energy minimum of our map. Solution data available for adenosine 3',5'-cyclic nucleotide are not clear cut. Schweizer and Robins 25 interpret their P M R results as indicating the existence of an anti conformation, while Klee and Mudd a4 deduce from the ORD studies that the compound exists in solution in the syn conformation. Finally, we have also carried out computations for guanosine 3',5'-cyclic phosphate. The geometry of guanine has been taken from the crystal structure of guanosine 5'-phosphate 32. The results are extremely similar to those of Fig. 6 with the global minimum again at ZCN = 270 ° with, however, a shape of the curve suggesting a preference for the neighbouring syn conformation (ZCN < 270 °) rather than for the anti ones (XCN 7> 270 o) and a local energy minimum around ZCN --- 90 ° of 3.2 kcals/mole above the global one. No X-ray crystal data are available as yet for this compound*. However, PMR data 25 on this compound in solution suggest that guanosine 3',5'-cyclic phosphate has the syn conformation. Altogether it appears thus that purine 3',5'-cyclic nucleotides exhibit two energy minima at the borderline between the syn and anti conformations with, however, a higher probability of the global minimum to be associated with the syn conformation. CONCLUSION
This exploration of the conformational possibilities of the cyclic nucleotides has brought into evidence a variety of patterns related to the two types of cyclic * See note added in proof.
CYCLIC NUCLEOTIDE CONFORMATION
163
structures studied (2',3' and 3',5') and the nature of the base type (purine or pyrimidine) considered. In the 2',3'-cyclic nucleotides two torsion angles are fundamental which define the orientations of the exocyclic C(5')-O(5') bond and of the base with respect to the sugar. The calculations seem to indicate a preference for both the gg and the gt conformations about the exocyclic C(4')-C(5') bond with perhaps a predominance of gt in the case of the pyrimidine nucleotides and of gg in the case of the purine nucleotides. The energy of the tg conformers is appreciably higher than those of the gt and gg ones. The pyrimidine 2',3'-cyclic nucleotides also show a distinct preference for a syn arrangement of the base with respect to the sugar, while the theoretical results for the purine derivatives place the global energy minimum at the borderline between the syn and anti conformers. In the 3',5'-cyclic nucleotides the orientation of the exocyclic C(5')-O(5') bond is necessarily tg and the essential degree of freedom relates to ZcN. The calculations indicate that the pyrimidine bases should manifest a strong preference for the attti conformation (ZcN = 60 °-90 °). The purine derivatives ,on the other hand exhibit a global energy minimum at ~(cN -- 270 o and a local one at ZcN = 90 °, both at the borderline between the syn and anti conformations. The available crystallographic and solution data seem to confirm the plausibility of the theoretical results. In the last above-quoted case of the purine 3',5'-cyclic nucleotides they seem to indicate moreover that the global minimum at ZcN - 270 ° is more favorable than syn conformations with a torsion angle slightly smaller than this value, while the local minimum at ZcN =: 90 ° favors the anti conformations with the torsion angles towards somewhat smaller values. To the already-quoted references we may add a recent one relevant to calorimetric investigations of crystalline 3',5'-cyclic nucleotides 35 which seems also to confirm the existence of at least some of the purine compounds in two conformational arrangements within the unit cell while the pyrimidines manifest only one such arrangement. NOTE ADDED IN PROOF (Received November 1st, 1973) Such data have just been published 36. The experimental ZcN = 258 ° is in excellent agreement with our prediction. ACKNOWLEDGEMENTS The authors thank Dr I. C. P. Smith for communication of data prior to publication. This work was supported by the A.T.P. No. A655-2303 of the C.N.R.S. REFERENCES 1 2 3 4 5
Berthod, t-I. and Pullman, B. (1971) Biochim. Biophys. Acta 232, 595-606 Berthod, I-L and Pullman, B. (1971) Biochim. Biophys. Acta 246, 359-364 Pullman, B., Perahia, D. and Saran, A. (1972) Biochim. Biophys. Acta 269, 1-14 Saran, A., Pullman, B. and Perahia, D. (1972) Biochim. Biophys. Acta 287, 211-231 Pullman, B. and Berthod, H. (1973) in Conformations of Biological Molecules and Polymers, 5th Jerusalem Syrup. Proc., pp. 209-224, Academic Press, New York
164 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
A. SARAN et al.
Saran, A., Pullman, B. and Perahia, D. (1973) Biochim. Biophys. Acta 299, 497-499 Saran, A., Perahia, D. and Pullman, B. (1973) Theor. Chim. Acta Berlin, 30, 31-44 Sundaralingam, M. (1969) Biopolymers 7, 821-860 Donohue, J. and Trueblood, K. N. (1960) J. Mol. BioL 2, 363-371 Saenger, W. and Eckstein, F. (1969) Angew. Chem. Int. Engl. Edn. 8, 595-596 Saenger, W. and Eckstein, F. (1970) J. Am. Chem. Soc. 92, 4712-4718 Coulter, C. L. and Greaves, M. L. (1970) Science 169, 1097-1098 Coulter, C. L. (1973) J. Am. Chem. Soc. 95, 570-575 Watenpaugh, K., Dow, J., Jensen, L. I-L and Furberg, S. (1968) Science 159, 206-207 Coulter, C. L. (1968) Science 159, 888-889 Coulter, C. L. (1969) Acta Crystallogr. B25, 2055-2065 Coulter, C. L. (1970) Acta Crystallogr. B26, 441-441 Sundaralingam, M. and Abola, J. (1972) Nat. New Biol. 235,244-245 Sundaralingam, M. and Abola, J. (1972) J. Am. Chem. Soc. 94, 5070-5076 Smith, I. C. P., Mantsch, H. H., Lapper, R. D., Deslauriers, R. and Schleich, T. (1973) in Conformations o f Biological Molecules and Polymers, 5th Jerusalem Syrup. Proc., pp. 381-402, Academic Press, New York Lapper, R. D., Mantsch, H. H. and Smith, I. C. P. (1972) J. Am. Chem. Soc. 94, 6243-6244 Blackburn, B. J., Lapper, R. D. and Smith, I. C. P. (1973) J. Am. Cem. Soc. 95, 2873-2878 Lapper, R. D., Mantsch, H. lrI. and Smith, I. C. P. (1973) J. Am. Chem. Soc. 95, 2878-2880 Lapper, R. D. and Smith, I. C. P. (1973) J. Am. Chem. Soc. 95, 2880-2884 Schweizer, M. P. and Robins, R. K. (1973) in Conformation o f Biological Molecules and Polymers, 5th Jerusalem Syrup. Proc., pp. 329-343, Academic Press, New York Lavallee, D. K. and Coulter, C. L. (1973) J. Am. Chem. Soc. 95, 576-581 Diner, S., Malrieu, J. P. and Claverie, P. (1969) Theor. Chim. Acta Berlin 13, 1-17 Malrieu, J. P., Claverie, P. and Diner, S. (1969) Theor. Chim. Acta Berlin 13, 18-45 Diner, S., Malrieu, J. P., Jordan, F. and Gilbert, M. (1969) Theor. Chim. Acta Berlin 15, 100110 Jordan, F., Gilbert, M., Malrieu, J. P. and Pincelli, U. (1969) Theor. Chim. Acta Berlin 15, 211-224 Pullman, B. and Pullman, A. (1973) Adv. Protein Res., in the press Murayama, W., Nagashima, N. and Shimuzu, Y. (1969) Acta Cystallogr. B25, 2236-2245 Young, D. W., Tollin, P. and Wilson, I-[. R. (1969) Acta Crystallogr. B25, 1423-1432 Klee, W. A. and Mudd, S. H. (1967) Biochemistry 6, 988-998 Bryan, A. M. and Olafsson, P. G. (1973) Biopolymers 12, 229-235 Chang, A. K. and Sundaralingam, M. (1973) Nature New Biology 244, 136-137