J . Phys. Chem. 1985,89, 230-234
230
Structural Studies on the Self-Association of Nucleosides in Aqueous Solutiont P. Martel Atomic Energy of Canada Limited, Chalk River Nuclear Laboratories, Chalk River, Ontario, KOJ 1JO, Canada (Received: May 17, 1984)
Small-angle and wide-angle neutron scattering were combined in a study of the self-association of A@,@-dimethyladenosine (dmA), 2’-deoxycytidihe (dCyt), and 6-methylpurine (6-Mepur) in aqueous solutions. Analysis of the small-angle neutron scattering indicated that the distributions of aggregate sizes were well described by a Gaussian function for concentrations of 0.13M or greater. The degree of self-association in solutions of dCyt was found to be much less than in solutions of dmA and 6-Mepur. Ultraviolet-irradiated and unirradiated solutions of the nucleosides were examined both with and without addition of 8-methoxypsoralen (8-MOP). Measurements were also carried out on mixed solutions of dmA and ethidium bromide (EB). The addition of 8-MOPand EB did not lead to any observable enhancement in aggregation. Upon cooling of concentrated solutions of dmA and 6-Mepur (but not dCyt), fine dendritic filaments were formed. Wide-angle scattering from these filaments was interpreted in terms of base stacking; the results were consistent with the hypothesis that the base stackin of nucleosides, unlike that of bare purine bases, does not lead to either perfect or nearly perfect parallel plane separations of 3.4
1.
Introduction
The self-association of nucleosides and D N A base derivatives in water has been a subject for study by many different experimental techniques. Some of the methods reported in the literature include vapor pressure osmometry,’ sedimentation equilibrium,2 optical absorption3 nuclear magnetic r e ~ o n a n c e dielectric ,~ permittivity response? and neutron diffractione6 Such measurements have indicated that the configurational stability of wet D N A depends not only on in-plane base-to-base bonding and sugarphosphate ‘backbone” connectivity but also on stacking forces acting between bases. Stacking forces are also of interest in themselves because they can serve as driving forces for the selfassembly of nucleic acid components in solution. In previous work it has been establi~hed,~ even with nucleosides, that base stacking interactions are the driving force for self-association in an aqueous environment. Since stacking occurs in water only, there can be no doubt that the process is somehow associated with the special properties of this important biological solvent. Nevertheless, not all methods have been sensitive to the role of water and so its precise function in the stacking process is still an open question. In spite of this, experiments in recent years have gone beyond the nucleoside stage to consider stacking interactions involving such complex molecules as adenosine triphosphate,* N6,P-dimethyladenyly1(3’-5’)N6,N6-dimethyladeno~ine,~and N6-dimethyl-
adenylyl(3’-5’)~ridine.’~ It should be pointed out that in many of the previous studies, nonlinear response with concentration was the factor which qualitatively determined that aggregation was occurring. The actual size of the aggregates was difficult to determine quantitatively and was often model dependent. In contrast, the neutron scattering method which we shall outline is fairly sensitive to aggregate sizes. The technique depends in part6 on the fact that DNA base stacking tends to produce a planar separation of -3.4 A which can be detected by diffraction. A possible disadvantage of the neutron method is that it requires large solute concentrations in order to obtain good statistics. However, in principle, this is not a barrier to obtaining relevant biological information since the density of D N A bases in nucleic acids is very high. In our present study we examine self-association in solutions of 6-methylpurine (dMepur), N6,Z@-dimethyladenosine (dmA), and 2’-deoxycytidine (dCyt). The first of these three was previously studied by wide-angle neutron scattering (WANS). These studies6 indicated that water molecules form bridges between the edges of the stacked purines. The approximate size of the stacks at high solute concentration was also determined. Here we also present data obtained by small-angle neutron scattering (SANS). Results from dmA are analyzed in the light of other experimental Publication No. AECL-8615
0022-3654/85/2089-0230$01.50/0
findings which include an elegant ultrasonic absorption and dispersion study” and calorimetric measurements.12 Reference is made to a detailed study of the dielectric properties of cytidine in solution where evidence was found5 that water molecules near the solute may have a longer relaxation time than that for free water. Although cytidine is slightly different from deoxycytidine, the results of Solie and S ~ h e l l m a n indicate ’~ that deoxycytidine has a virtually identical equilibrium association constant to that of cytidine, thereby confirming the conclusion’ that modifications to sugars do not affect the stacking properties of nucleosides. Besides studying the stacking properties of pure nucleoside solutions, we have attempted to look for evidence that stacking might be enhanced by the presence in solution of large flat molecules which are known’e17 to intercalate between the bases of DNA. These molecules are 8-methoxypsoralen (8-MOP) and ethidium bromide (EB). Because of the low solubility of these molecules, our experiments amount to a search for evidence that they might serve as strong promoters for nucleation of aggregates. Photoreactive molecules like 8-MOP are used as dermal photosensitivity agents for the treatment of skin diseases such as psoriasis with ultraviolet (UV) irradiation. The first step in the biochemical pathway leading to photosensitivity is thought to be the intercalation of 8-MOP between adjacent DNA base pairs.” Pyrimidines are usually involved in the photoreactive formation of adducts which can be initiated by irradiation with UV lamps having maximal intensitia near 350 nm. In the present experiment a search was made for possible changes in self-association when
(1) Ts’o, P. 0. P.; Chan, S. I. J . Am. Chem. Soc. 1964,86, 4176. (2) van Holde, K. E.; Rossetti, G. P. Biochemistry 1967, 6, 2189. (3) Stepien, E.; Lisewski, R.;Wierzchowski, K. L. Acla Biochim. Pol. 1973, 20, 313. (4) Schweizer, M. P.; Broom, A. D.; Ts’o, P. 0. P.; Hollis, D.P.J . Am. Chem. Soc. 1968, 90, 1042. (5) Shepherd, J. C. W.; Schwarz, G. Biophys. Chem. 1977, 7, 193. (6) Martel, P. Eur. J . Biochem. 1979, 96, 213. (7) Ts’o, P. 0. P. “Basic Principles in Nucleic Acid Chemistry”; Ts’o, P. 0. P., Ed.; Academic Press: New York, 1974; Vol. I. (8) Lam, Y. F.; Kotowycz, G.Can. J . Chem. 1977, 55, 3620. (9) Tazawa, I.; Koike, T.; Inoue, Y. Eur. J . Biochem. 1980, 109, 33. (10) Hartel, A. J.; Lankhorst, P. P.; Altona, C. Eur. J . Biochem. 1982,129, 343. (1 1) Heyn, M. P.; Nicola, C. U.; Schwarz, G. J . Phys. Chem. 1977, 81, 1611. (12) Vickers, L. P.; Ackers, G.K. Arch. Biochem. Biophys. 1976,174,747. (13) Solie, T.N.; Schellman, J. A. J . Mol. Biol. 1968 33, 61. (14) Delbarre, A.; Gourevitch, M. I.; Gaugain, B.; LePeq, J. B.; Roques, B. P. Nucleic Acids Res. 1983, 11, 4467. (15) Mirau, P. A,; Kearns, D. R. Nucleic Acids Res. 1983, 11, 1931. (16) Sobell, H. M.; Tsai, C. C.; Jain, S. J.; Gilbert, S. G. J . Mol. Biol. 1977, 114, 333. (17) Shim, S . C.; Kim, Y. Z. Phorochem. Photobiol. 1983, 38, 265.
Published 1985 American Chemical Society
The Journal of Physical Chemistry, Vol. 89, No. 2, 1985 231
Self-Association of Nucleosides in Aqueous Solution mixtures of nucleosides and 8-MOP were irradiated with W light. Experiment
Materials. All substances were of analytical grade and were obtained commercially. Ethidium bromide, 6-methylpurine, 8methoxypsoralen, and M,N6-dimethyladenosine were purchased from Sigma Chemical Co. 2'-deoxycytidine monohydrate was purchased from Aldrich Chemical Co. ("Gold-Label" brand) and was specified as having a purity in excess of 99%. Aqueous solutions were made up with deuterium oxide ( D 2 0 ) having a purity in excess of 99.7% by weight. The pH of the D 2 0 as measured with an ordinary pH meter was 6.8; after mixing with dCyt and dmA, the measured value for both was 7.0. Apparatus and Procedures. Most of the specimens were contained in thin-walled cylindrical quartz chambers having inner diameters -5 mm. For some of the small-angle measurements a rectangular quartz container was used; it had a width greater than the beam width and a specimen thickness along the beam direction of 1.5 mm. The scattering chambers were sealed as soon as they were filled. During measurements temperatures were stable to f l 'C. The wide-angle diffraction measurements were carried out on a triple-axis spectrometer. No analyzer was used. The incident neutron wavelength, A, was 1.623 A, and the angular resolution at a Bragg angle, 0, of 13' was 0.8' full width at half-maximum. The scattering chambers were rotated six revolutions per counting interval. All data were corrected for quartz and air scattering. For the small-angle measurements with a rectangular chamber, a neutron wavelength of 2.5 A was used. These measurements were carried out using standard small-angle scattering procedures.ls In all cases hydrogen-deuterium exchange resulted in a large incoherent background. This background was found to be independent of angle at low angles by means of a neutron scattering run on pure HzO. UV irradiations were carried out with a bank of five General Electric lamps (Type F15T8-BLB) having an approximately Gaussian spectral output centered at 360 nm. Their spectral width was approximately 45 nm (full width at half-maximum). The specimens were irradiated in thin sealed quartz containers which presented a path length of 1.5 mm to the incoming light. The average lamp-to-specimen distance was 100 mm. Neutron Scattering Theory. In the case of small-angle neutron scattering the Debye expression for the intensity, Z(Q), as a function of wavevector transfer Q (= 4*/X sin 0) is
where lois a constant that depends on the difference in the neutron scattering length densities of the solute and solvent as well as the geometry of the e ~ p e r i m e n t . ' ~The function p(r) is a correlation function describing the average local fluctuations in scattering length density at points located a distance r apart. where a is a measure Often, p ( r ) can be approximated20 by of the size of local fluctuations. In this experiment we have utilized another correlation function (also suggested by Debye et alezo) which gave a good fit to data for nucleosides in solution; this function is p ( r ) = e-(rIa)2 If p ( r ) from eq 2 is inserted in (l), it follows that
(2)
+
Z(Q) = Z(0)e-af@/4 K (3) 4 . eq 3, Z(0) may be taken to be the where Z(0) = a 3 Z o ~ 1 / 2 / In intensity at Q = 0 for a given geometry and concentration, and K is a constant that accounts for background. For a two-component system of solute and solvent, p ( r ) represents the probability that a point at a distance r in an arbitrary direction from a given (18) Martel, P.; Kim, S. M.; Powell, B. M. Biophys. J . 1980, 31, 371. (19) Higgins, J. S.; Stein, R. S. J. Appl. Crystallogr. 1978, 1 1 , 346. (20) Debye, P.;Anderson Jr., H. R.; Brumberger, H. Appl. Phys. 1957, 28, 679.
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Q Figure 1. Small-angleneutron scattering profiles of (A) 6-methylpurine solution (0.7 M) and (B) N6,M-dimethyladenosine(0.13 M). The specimen temperature was 25 O C . The lines through the data represent least-squares fits using eq 3.
point in the solute will itself also be in the solute. In terms of eq 2 an average of this distance, ( r ) ,can be defined to be given by
Comparison of a for different solutions can yield a measure of relative degrees of aggregation. Thus, if a1and cy2 are the CY'S obtained for two different solutions, the ratio of the probable numbers of aggregates possessing the length segment r is proportional to
In the analysis of SANS data Z(Q) was convoluted with the spectrometer resolution function as described in ref 18. In all fitting procedures, x2 was minimized, where
Here, Ci is the calculated value at the point i, Ei is the experimental value at i, N is the number of data points, and P is the number of parameters. Wi = (1 /Ei)2,
Results In an earlier publication6 use was made of Schemer's theory for broadening of wide-angle peaks to show that at high concentrations the number of stacked monomers was approximately nine in solutions of 6-methylpurine at 28 O C . In applying this theory it was assumed that the stacked monomers lay in embryonic crystallites. In the upper panel of Figure 1 we show the results of a SANS measurement of a 6-Mepur solution at a concentration of 91 g / L ( ~ 0 . 7M) and a temperature of 25 OC. The sample chamber for this experiment had rectangular geometry. The line through the data represents a computer fit using eq 3. From the fitting, a = 8.7 f 0.6 A. When this solution was diluted by a factor of 2, a was found to be 8.1 f 1.0 A. At 0.7 M osmometric measurements1 suggest that there is a distribution of stacks as
232
The Journal of Physical Chemistry, Vol. 89, No. 2, 1985
-r
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28 Figure 2. Neutron diffraction profiles: (A) upper curve, scattering from a 0.9 M heavy water solution of 2'-deoxycytidine at 29 OC; lower curve, heavy only. (B) upper curve, scattering from a D20solution of N6fl-dimethyladenosinc (0.13 M) at 30 O C ; lower curve, D20only. (C) Same water (D20) specimen as in (B) after cooling for 48 h at 5 "C. Scan data were obtained with the specimen at 30 OC, with filaments present. The peak near 28 = 1 3 O corresponds to a plane separation of 7.2 A.
follows: 20% dimer, 20.5% trimer, 18.7% tetramer, 16% pentamer, and 13.2% hexamer. The S A N S measurements do not directly yield absolute percentages of the various n-mers. If a = 9 %., the probability that there are lar e numbers of hemmers with r values spanning -3.4 X 5 = 17 seems small since p(17) < 0.03. However, detailed model calculations would be necessary to verify this conclusion. The lower panel (B) of Figure 1 shows SANS data obtained from a solution of dmA (39 g/L = 0.13 M). The solid line in panel B represents the same type of computer fit as in A. For dmA, a = 9.2 f 0.4 A. It is apparent from the fits that our Gaussian approximation for p ( r ) is satisfactory. Combinations of wide- and small-angle scattering results are illustrated in Figure 2. These scans are typical of most of the results obtained in this experiment. For these measurements the samples were contained in cylindrical quartz chambers having inner diameters =5 mm. A neutron wavelength of 1.623 A was employed, and the angular range was 1.4 I 28 I60' in steps of 0.10'. The lower curves in panels A and B represent the scattering intensities for D20and serve as reference data for dCyt (250 g/L
1
= 0.9 M, upper data in panel A) and dmA (39 g/L 0.13 M, upper data in panel B). The upper data in panel C were obtained with the same specimen as that used in B after cooling for 48 h a t 5 OC. This cooling procedure changed the sample from a transparent liquid to one which contained a dense agglomeration of fine, white, hairlike filaments. These filaments subsequently remained stable at 25 OC and permitted the upper scan data in C to be obtained. The lower data in C are for DzO.A similar cooling of the dCyt solution did not produce any filamentary structure. The upper curve in Figure 3 illustrates the wide-angle scattering from filaments formed from 6-Mepur in a solution which was originally 1.75 M. These filaments were produced on cooling the solution to 15 'C for 40 min. Note that whereas the dmA filaments produced no peak in the diffraction for a spacing of 3.4 A, the 6-Mepur filaments yielded a strong peak corresponding to this spacing. A qualitative survey of Figure 2 reveals several features about the self-associative characteristics of dCyt and dmA. In the first place the change in the position of the water structure factor peak from that of D20at 28 = 30' is very slight in either dCyt or dmA
The Journal of Physical Chemistry, Vol. 89, No. 2, 1985 233
Self-Association of Nucleosides in Aqueous Solution
6000
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28 Figure 3. Scattering from a 1.75 M heavy water solution of 6-methylpurine containing dendritic filaments (upper curve). The neutron wavelength was 2.24 A. The lower curve represents scattering from pure DzO.The arrows indicate peak positions corresponding to 3 . 4 4 (28 = 39') and 6.8-A plane separations. The peak near 28 = 18' corresponds to a plane separation of 7.2 A.
solution. With a concentrated 6-Mepur solution: it was possible to detect a second peak a t slightly lower angles caused by diffraction from embryonic crystallites having purine stacking at a spacing of 3.4 A. In the present experiment such a plane spacing would have produced extra intensity at 20 = 27.6O. When dendritic filaments formed in 6-Mepur solution (see Figure 3), a sharp peak corresponding to a 3.4-A spacing was observed. In the case of N6,prdimethyladenine6overlapping peaks were observed in the 3.4-A region but no single peak predominated as in the case of 6-Mepur. A strong peak did occur in N6,p-dimethyladenine corresponding to a plane spacing of 1.2 A; by reference to single-crystal diffraction studies2' we conclude that this 7.2-A peak was due to A-B-B-A stacking caused by tilting of A and B purines with respect to one another by 11O. In the case of nucleosides22 base tilting is even more probable because of the steric hindrance offered by the sugar moieties. Thus, the peak in C of Figure 2 2 is probably due to a tilting of a t 20 = 13' (spacing ~ 7 . A) adjacent bases which doubles the repeat distance for diffraction. Before considering quantitative analyses of the results, another interesting feature of the neutron scattering should be pointed out by reference to Figure 2. This feature is the increase in intensity at low angles for both dCyt and dmA solutions (upper curves in panels A and B, respectively). It may be seen that the rate of increase is significantly less for dCyt (panel A). As we shall see, this difference in small-angle scattering is an indication that the average size of aggregates in dCyt solution is smaller than it is in dmA solution. In the case of dmA solution (panel B) the small-angle portion of the scattering from 1.4 to 9' (20) was analyzed by using eq 3. The fitting procedure yielded a value for a of 8.8 f 0.2 A, in satisfactory agreement with the value 9.2 f 0.4 %I obtained by analyzing the data in Figure 1B. It was concluded that dmA data from cylindrical specimens could be analyzed in this angular range to yield values of a. Since the small-angle region for dCyt seemed to extend to larger values of 20, the SANS for these solutions was analyzed for 1.4 5 20 5 15'. A previously described procedure6 was used in an attempt to extract a nucleosidic intensity component from WANS near the peak position in the D 2 0 scattering. N o second peak was resolvable although slight shifts to lower angles of the peak position were found with nucleoside present. Quantitative results for SANS from solutions of dmA and dCyt are presented in Tables I and 11, respectively. The parameters were obtained by the least-squares-fitting procedure defined above. Any increase in a implies that more large-sized aggregates are present. If intercalating agents were serving as centers for nucleation, a might increase. However, the values for a for the three solutions in Table I all lie within overlapping error limits. (21) Sternglanz, H.; Bugg, C. E. J . Cryst. Mol. Struct. 1978, 8, 263. (22) Broom, A. D.; Schwcizer, M.P.; Ts'o, P.0.P.J . Am. Chem. SOC. 1967,89, 3612.
TABLE I: SANS Results for N6,N6-DimethytadenosineSolutions
sample 10-31(0) A 10-3~ (1) 4:l volumetric mixture of 2.0 f 0.1 8.7 f 0.3 4.9 f 0.1 1.27 dmA (0.13 M) and pure D20(irradiated 34 h) (2) 4:l volumetric mixture of 2.0 f 0.1 8.2 f 0.3 4.4 f 0.1 1.31 dmA (0.13 M) and 8-MOP (9.2 X lod M) (irradiated 29 h) (3) 4:l volumetric mixture of 1.8 f 0.1 8.2 f 0.3 3.8 f 0.1 1.26 dmA (0.13 M) and ethidium bromide (3.8 X M) (unirradiated) TABLE 11: SANS Results for 2'-Deoxycytidine Solutions
sample 10-31(0) a,A 10-3~ 4.6 f 0.3 8.5 f 0.1 1.37 (1) 0.9 M dCyt 1.0 f 0.1 5.0 f 0.3 8.4 f 0.1 1.18 (2) 0.9 M dCyt (irradiated 1.0 f 0.1 30 h) (3) 1:l volumetric mixture 1.05 f 0.05 4.6 f 0.3 8.7 f 0.1 1.26 of 1.8 M dCyt and 8-MOP (9.2 X lod M) (irradiated 20 h) k-6;
4
D EOXY CYTl 0I NE
N6, N6
DIMETHYLADENOSINE
Figure 4. Skeletal models of p,p-dimethyladenoine and deoxycytidine drawn to scale. The circles were drawn with radii equal to a,where CY
is a relative measure of the sizes of aggregates formed in aqueous solution (see text).
Upon irradiation of a sample of dmA containing %MOP, no visually detectable change in the transparency of the solution (sample 2, Table I) was apparent. Similarly, when the pure dCyt solution (sample 2, Table 11) was irradiated, it also remained transparent. However, UV irradiation of the dCyt solution containing 8-MOP (sample 3, Table 11) led to a deep yellow coloration of the sample. Nevertheless, no statistically significant change in a was measured in the latter case.
J . Phys. Chem. 1985, 89, 234-239
234
It should be noted that in all cases the cy value for dCyt is significantly less than its value for dmA. This suggests that self-association in dCyt solutions is much less than in dmA solutions. Our inability to produce filaments by cooling concentrated dCyt solutions to 5 OC is in accord with this result. Figure 4 shows skeletal models of molecules of dmA and dCyt which have been drawn to scale. The circles about these have radii approximately equal to their respective cy's (9 A for dmA and 4.6 A for dCyt). It is apparent from this figure that there is much more room available for aggregation of dmA within its a than there is in the case of dCyt. This conclusion is in accord with previous studies which indicated a large equilibrium stacking constant of 34 M-l for dmA solutions at 25 OC,I1 while that for dCyt is only 0.91 M-'.13 Our result is also in accord with the general conclusion' that purine derivatives tend to associate much more than pyrimidine derivatives. According to Shepherd and Schwarz5 the mean aggregation number (including monomers in the average) is 1.3 in 0.5 M cytidine, while in 0.18 M dmA," ~25% of the dmA molecules are in aggregates containing more than six nucleosides. If one assumes an intermolecular separation of 3.4 A, six nucleosides would span =17 A. For r = 9.0 A, the ratio ofp(r)'s for dmA and dCyt is calculated to be ~ 1 7if , a for dmA and dCyt is taken to be 9 and 4.6 A, respectively.
Conclusions Small-angle neutron scattering indicates that aggregation tends to be significantly greater in purine solutions (6-Mepur, dmA) than in pyrimidine solution (dCyt). The probability that a given length segment will exist in any aggregate was found be Gaussian to a good approximation for all concentrations employed in this experiment. Our results indicate that known intercalators do not serve as strong promoters for nucleation of nucleoside aggregates in solution. In spite of the UV-induced discoloration of dCyt solution containing 8-MOP, no changes in aggregation were observed. In the case where EB was added to dmA solution, no enhancement in the aggregate size was observed, in spite of the known affinity14 of EB for nucleotides. In another study,I5 it was shown that high levels of EB, more than 1 molecule per 20 base pairs, were required to inhibit structural changes in DNA. In our experiment the number of E B s per dmA was of the order of 1/35. Since 8-MOP is even less soluble, the number of 8-MOP molecules per nucleoside
was much smaller (= l/104). However, the ratio of 8-MOP molecules to water molecules in our solution is similar to that in humans undergoing phototherapy for psoriasis. Wide-angle scattering studies of nucleoside filaments indicate that the stacking repeat distance for diffraction is not dominated by a 3.4-A planar separation as it is with a simple purine (6Mepur). The observation of a 7.2-A repeat in wet filaments of N6,i@-dimethyIadenosine is ascribed to tilting of adjacent bases relative to one another because of the steric hindrance caused by ribose sugars. The prime cause of base association is undoubtedly London dispersion forces' between the hydrophobic faces of these planar molecules. However, interbase water bridging along the external edges of stacks is also likely to be a contributory factor, since recrystallization from aqueous solution has been observed to stabilize a 3.4-A spacing in crystalline hydrates of bases. Furthermore, single-crystal X-ray diffraction from DNA fragments23 has shown that water can bridge across the edges of DNA bases; when this occurs, there is a stabilization of the B structure, where the bases are stacked at a separation of 3.4 A. Recently, ultrasonic measurements by Hemmes et al.24have also indicated stacking with a hydration shell around nucleosidic aggregates which is highly structured. Many studies of self-association of DNA bases have assumed perfect stacking of bases one above another with water ordered along the stack edges. It seems likely, even in the absence of a sugar phosphate backbone, that complicated molecules such as nucleosides will try to satisfy the stacking force; however, steric hindrances and interbase water links along directions other than the stack may lead to large deviations from perfect stacking at a separation of 3.4 A. In any case our measurements suggest that there is a strong tendency toward aggregation even when London dispersion forces are unfavorably affected by geometrical constraints. Registry No. dmA, 2620-62-4; dCyt, 95 1-77-9;6-Mepur, 2004-03-7; 8-MOP, 298-81-7; EB, 1239-45-8. (23) Kopka, M. L.; Fratini, A. V.; Drew, H. R.; Dickerson, R. E. J . Mol. Biol. 1983, 163, 129. (24) Hemmes. P.; Mayevski, V. A,; Buckin. V. A,: Sarvazyan, A. P. J . Phys. Chem. 1980, 84, 699.
Vibrational Spectra of Nickel( I I ) Monoformyideuteroporphyrins Deborah L. Willems and David F. Bocian*' Department of Chemistry, University of California, Riverside, California 92521 (Received: June 20, 1984)
Infrared and visible region (B and Q state) resonance Raman (RR) spectra of Ni(2-FDP) and Ni(4-FDP) are reported and compared to those of Ni(2,4-FDP) (FDP = formyldeuteroporphyrinIX dimethyl ester). Virtually all of the in-plane porphyrin skeletal modes and a number of out-of-plane porphyrin modes and internal vibrations of the formyl groups are assigned. The vibrational frequencies, E,mode splittings, and RR enhancements are essentially identical for Ni(2-FDP) and Ni(CFDP), indicating that substitution of the formyl group at the two different porphyrin ring positions results in nearly an identical perturbation of the *-electronic structure of the macrocycle. The Cb-formyl group stretching motion and the formyl hydrogen bending mode are kinematically coupled to one another. For Ni(2,4-FDP), the in-phase and out-of-phase combinations of each type of motion are substantially different in energy due to large differences in kinematic coupling between various phase combinations of the stretching and bending vibrations.
Naturally occurring porphyrins typically contain substituents such as vinyl or formyl groups which can conjugateinto the s-electronic structure of the porphyrin macrocycle.z It has been
proposed that the effects of protein interactions with these substituents could be transmitted to the metal center through the porphyrin *-system to mediate ligand-binding affinities or redox potentials.3-" Consequently, there has been interest in charac-
1982-1984.
(2) Smith, K.M. In "Porphyrins and Metalloporphyrins"; Smith, K. M., Ed.; Elsevier: Amsterdam, 1975; pp 3-58.
Introduction
(1) Alfred P. Sloan Fellow,
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0 1985 American Chemical Society
