98
J. Phys. Chem. 1981, 85, 98-101
Thermodynamics of Syn-Anti Glycosyl Isomerization in Cyclic Adenosine 3’,5’-Monophosphate in Water and Aqueous Urea’ Paul R. Hemmes,” Leslie Oppenheimer, Frank Jordan; Carl A. Olson Chemlstty Laboratorles, Rutgers University, Newark, New Jersey 07102
and Sadakatsu Nlshlkawa Saga University, Saga, Japan (Received: May 8, 1980)
Ultrasonic relaxation measurements were performed on cyclic adenosine 3’,5’-monophosphate in water and in 7 M urea. The unimolecular relaxation was attributed to the glycosyl C-N conformational equilibrium. The apparent thermodynamics and kinetic properties, calculated from the relaxation data, were different in 7 M urea than in water. A detailed analysis of the results showed that the glycosyl conformational and stacking equilibria are strongly coupled but in 7 M urea the effects of base stacking no longer intervene. The magnitude and sign of the thermodynamic quantities calculated in 7 M urea can be rationalized by a solvation model in which the ribosephosphate moiety is less solvated in the syn than in the anti conformation. The conformational barrier in 7 M urea has a very low AHo of ca. 4 kJ/mol. The conformational equilibrium constant is about 4 at ambient temperatures.
Introduction It was demonstrated by Rhodes and Schimmel in 19712 and by this laboratory subsequently3p4that the kinetics of nucleoside and nucleotide C-N glycosyl isomerization processes can, in favorable cases, be monitored by ultrasonic relaxation techniques. Earlier, theory and methodology were presented to obtain thermodynamic parameters from purely relaxation data: and the thermodynamics of sp-anti isomerization in adenosine in aqueous medium was r e p ~ r t e d .Later, ~ evidence was presented for the existence of a two-state glycosyl conformational equilibrium in the second hormonal messenger cyclic adenosine 3’,5’monophosphate CAMP).^ Due to the importance of this compound in biology, a thorough understanding of its physical and chemical properties is desirable. Previous research aimed specifically at defining the glycosyl conformation of cAMP included an X-ray study demonstrating both an anti and a syn conformer to coexist in the unit cell,5a high-resolution lH NMR study suggesting a predominant anti conformer,6and lanthanide probe studies suggesting anti,’ syn; and rapidly equilibrating syn-anti mixtures? Studies by Lee and SarmalO showed that the (1) Taken in part from the Ph.D. Thesis of L.O.submitted to the Rutgers University Graduate Faculty, 1977. Supported in part by NIH Grant GM19338 from US. D.H.E.W.; the Rutgers University Research Council, The Busch Fund (at Rutgers University), and the Biomedical research support. (2) L. M. Rhodes and P. R. Schimmel, Biochemistry, 10,4426 (1971). (3) P. R. Hemmes, L. Oppenheimer, and F. Jordan, J.Am. Chem. SOC., 96, 6023 (1974). (4) P. R. Hemmes, L. Oppenheimer, and F. Jordan, J. Chem. SOC., Chem. Commun., 929 (1976). (5) K. Watenpaugh, J. Dow, L. H. Jensen, and S. Furberg, Science, 159, 206 (1968). (6) M. P. Schweizer and R. K. Robins in “Conformation of Biological Molecules and Polymers”, Proceedings of the Vth Jerusalem Symposium, Academic Press, 1973, p 329. (7) C. D. Barry, D. R. Martin, R. J. P. Williams, and A. V. Xavier, J. Mol. Biol., 84, 491 (1974). (8)D. K.Lavallee and A. H. Zeltmann, J.Am. Chem. Soc., 96,5552 (1974). ~--.
($G. V. Fazakerley, J. C. Russell, and M. A. Wolfe, J. Chem. SOC., Chem. Commun., 527 (1975). (10)C-H. Lee and R. H. Sarma, J. Am. Chem. SOC.,98, 3541 (1976).
Scheme I: A p p r o x i m a t e C o n f o r m a t i o n a l Drawings f o r the S y n and A n t i Glycosyl C o n f o r m a t i o n o f c A M P “2
I
“2
I
ribosephosphate is rigid which result supports our contention that we are indeed observing glycosyl isomerization by ultrasonics. Lee and Sarma’O employing high-field, high-resolution ‘H NMR coupling constant and chelnical shift data also suggested the coexistence of two glycosyl conformational states of this molecule. Theoretical conformational energy calculations also suggested similar stabilities to the optimum syn and anti regions connected by a relatively small barrierall Over the years it has also become evident that the stacking of the nucleic base and the glycosyl conformational rotational degrees of freedom are interrelated.12-16 We have found that urea concentrations larger than 7 M effectively eliminate stacking in a model 6-methy1p~rine.l~Equipped with this information we have determined estimates of the thermodynamics of the unimolecular relaxation found in cAMP in H20 and in 7 M urea and here report the results of this study. The (11) F. Jordan, J. Theor. Biol., 41, 23 (1973). (12) T-D. Son, W. Guschlbauer, and M. Gueron, J. Am. Chem. SOC., 94. 7903 (1972). ’(13) hi. Sundaralingam, Ann. N.Y. Acad. Sci., 255, 3 (1975). (14) A. P. Zens,T. A. Bryson, R. B. Dunlap, R. R. Fisher, and P. D. Ellis, J. Am. Chem. Soc., 98, 7559 (1976). (15) C-h. Lee, F. S. Ezra, N. S. Kondo, R. H. Sarma, and S. S. Danyluk, Biochemistry, 15, 3627 (1976). (16) C. Chachaty, T. Yokono, T-D. Son, and W. Guschlbauer, B ~.O R-~ Y S . Chem., 6, 151 (1977). (17) This was demonstrated by studying the ultrasonic relaxation behavior of 6-methylpurine in Hz(rurea mixtures. The amplitude of the relaxation due to stacking, the only self-association procesa iikely to occur in 6-methylpurine, is reduced to 10% of ita value in water by addition of 7 M urea; see P. Hemmes, L.Oppenheimer, R. Rhinesmith, G. Anderle, D. Saar, and F. Jordan, J. Phys. Chem., 84,911 (1980).
0 1981 American Chemical Society
Glycosyl Isomerization in cAMP
The Journal of Physical Chemistty, Vol. 85,No. 1, 198 1 98
TABLE I: Ultrasonic Absomtion Data for cAMP Solutions sound velocity, temp, "C concn, M PH m /s fr" 1oi7~b 103(M,,/cT 40 ? 2 21.8 1.20 i 0 . 0 3 25 0.05 8.4 1500 22.0 1 . 1 3 t 0.03 8.7 1500 41 * 2 25 0.10 1.17 r 0.03 38 ? 2 25d 0.10 8.8 1525 37 * 2 24.8 0.97 i 0.03 25 0.21 8.9 1515 8.5 1510 45* 2 20.1 1.18 i 0.03 30 0.10 21.6 1.09 ? 0.03 8.9 1525 44i 2 30 0.21 35 0.10 8.8 1530 59 ? 2 17.5 1.17 + 0.03 86i 2 15.9 0.90 i 0.03 45 0.20 8.7 1540 a Relaxation frequency, Solvent value for elf". See definition in text. Resonator run. There can be small systematic differences between resonator results and pulse. The thermodynamics were determined by using pulse results only.
results demonstrate a coupling of the stacking and syn-anti glycosyl conformational equilibria for cAMP (Scheme I) and suggest that the latter is strongly environment dependent. The results also complement our recent report in which we demonstrated a change in glycosyl conformational barrier in transferring 2'-deoxyadenosine from a self-stacked to a heterostacked (i.e., to ethidium bromide or indole-3-acetic acid) environment.l8 Experimental Section Adenosine and cAMP were obtained from Aldrich Chemical Co. and were employed without further purification. All inorganic chemicals were of highest purity. Distilled, deionized, and sonicated water was employed. The latter procedure was dictated by the need for gas-free water as bubbles tended to interfere with the ultrasonic measurements. The pH was measured on a Radiometer pHm 26 meter. The temperature was maintained with better than f O . l OC accuracy by Haake circulating baths or a Lauda K2/R circulating bath equipped with a compressor to maintain lower temperatures. Pulse ultrasonic measurements were taken at 15-300 MHz with equipment described e1~ewhere.l~ A swept-frequency interferometer (resonator) was employed in the measurements at 1-37 MHz. The instrumentation resembled the one described by Eggers and Funcka20 I t consisted of a Pegelmessplatz PSM-5 (spectrum analyzer) manufactured by Wandel and Goltermann of Reutlingen, Germany. Accurate frequency measurement was made by a Tektronix 153 instrument interface. Results CAMPin H20. Pulse ultrasonic relaxation experiments were performed from 15 to 255 MHz with a 5-MHz transducer. The resonator was also used at one concentration and temperature, duplicating the results of the pulse instrument. Results for cAMP at various concentrations and temperatures are summarized in Table I. The agreement between the two instrumental approaches is satisfactory. The relaxation frequency, f,, was found to be constant over a fourfold concentration range. Hence the process can be concluded to be unimolecular. The quantity pmar/CT(pmaxis defined below, CT is total nucleotide concentration), however, exhibits a slight concentration dependence believed to be outside the experimental error. The data suggest that the concentration of the species undergoing the unimolecular relaxation (isomerization) is being depleted by a concentration-dependent react ion. Assuming temperature-dependent variation in pmar/CT, we performed a thermodynamic analysis on all data3 (18) F. Jordan, S. Nishikawa, and P. Hemmes, J. Am. Chem. SOC.,102, 3913 (1980). (19) H-C. Wang and P. Hemmes, J.Am. Chem. SOC.,95,5115 (1973). (20) F. Eggers and Th. Funck, Reu. Sci. Instrum., 44, 969 (1973).
TABLE 11: Apparent Thermodynamic and Kinetic Results of CAMP in Water at AV" = 0.0 cm3/mol Kb = typ, (syn)/ x X C AHoTa (anti) k f , sei k,, s-' 2.2 2.2 11.0 53.9 2.4 2.1 53.5 11.5 2.7 1.7 16.0 (1) 53.1 2.6 1.5 17.5 (2) 52.7 1.6 21.5 3.5 (1) 53.1 5.3 1.2 44.3 53.9 (1) Note that the uncertainty of AV" is a A F T in kJ/mol. at least *1 cm3/mol due to a flat minimization of the data: AH"^ = 53.1 kJ/mol (rmd = 7.8 ppt), ASS = 53.1 kJ/mol (std dev = 0.41), AS"= 199 J/deg mol, AH^* = 31 kJ/mol, (1)pulse method, ( 2 ) resonator. The ultrasonic method determines the magnitude of AG"(K). It cannot distinguish whether this number denotes (syn/anti) of (antilsyn). 25 25 30 30 35 45
(1)
(2)
presented in Table I. A very broad minimum of the quality of data fitting parameter *To - A H S O , occurred at AVO = 0.0 cm3/mol and resulk with this AVO are given in Table 11. The symbols employed3 are defined as follow: p = (a- a& is the experimentally determined quantity, a the sound absorption coefficient of the solution, a. the sound absorption coefficient of the solvent, X the wavelength of sound, pmaris the maximum value of p which occurs when the frequency f equals the relaxation frequency f,, AVO the the volume change for the syr-anti interconversion, enthalpy change for this process calculated at each temp e r a t ~ r eA, ~ H S " the enthalpy calculated from a In K vs. i / T plots3 cAMP in Aqueous Urea. Base stacking has been observed in the ultrasonic region for P , P - d i m e t h y l adenine,21NGjNG-dimethyladenosine, and 6-rnethylp~rine.~~ In this laboratory the ultrasonic absorption spectrum of 6-methylpurinewas determined at a concentration of 0.075 M and 25 "C and both pmaxand f, (2.1 X at 35 MHz) were found to bel7 in accord with literature values.22a Addition of 7 M urea caused at least an 85% decrease in the acoustic effect and indicated that urea disrupts the stacks.17 Addition of 7 M urea to solutions of adenosine and CAMP,on the other hand, caused a ca. 40% increase in p-/CT. Apparently, stacking is coupled to the glycosyl equilibrium in such a fashion that some of the monomers in the stack are incapable of glycosyl isomerization. The ultrasonic relaxation spectrum of CAMPin 7 M urea was determined as a function of concentration at 25 "C and at 15,20, and 30 "C, employing both pulse and resonator techniques. The solution pH was always 8.5 f 0.25 p
aTo
(21) D. Porschke and F. Eggers, Eur. J. Biochern., 26, 490 (1972). (22) (a) F. Garland and R. C. Patel, J. Phys. Chem., 78, 848 (1974); (b) F. Garland and S. D. Christian, ibid., 79, 1247 (1975).
100
The Journal of Physical Chemistry, Vol. 85, No. 1, 1981
TABLE 111: Experimental Results for CAMP-7 M Urea Solutionsa
103 x
20 0.15 1.7 34 25 0.30 1.6 35 25 0.15 1.6 35 30 0.15 1.5 36 a Combined pulse and resonator results. tion in text, Relaxation frequency.
Hemmes et al.
TABLE V : Thermodynamic and Kinetic Results for CAMP-7 M Urea Solutions at A v" = 3.0 cm3/mola
sound velocity, m/s 1640 1638 1636 1634 See defini-
Kb = temp, "C
RT, kJ/mol
15 20 25 30 a AH" = 4.6 5 28 J/(K mol).
(synj/ (anti)
10-;kf, S-
lO-'k,, S-'
3.8 4.2 1.67 4.02 4.3 4.3 1.75 4.05 4.8 4.4 1.8 4.07 5.2 4.6 1.9 4.05 1.6 kJ/mol, AHf* = 10 t 6 kJ/mol, A S " = See footnote b in Table 11.
Scheme I1 TABLE IV: Solvent Properties of a 10.25 M Urea Solution calcd values temp, concn, density, "C M g/mL
C
caf/'g
lit. values density,a g/mL
A"-B,..,-A'
(or A'-B,-,-A'
or A'-B,-,-A")
cal/g
0 7.08 1.1159 0.1123 5 7.06 0.7759 0.77062 10 7.05 0.7194 0.77562 15 7.035 20 1.02 0.7859 0.18348 25 7.00 1.10033 1.1024 30 6.98 1.1005 1.1003 40 6.95 1.0953 0.7964 0.7870 50 6.905 1.0884 Density from ref 23b. C,, values from ref 24. It should be noted that the expression for CPowas in error in ref 24. The fourth term has the incorrect sign.
to ensure that the state of ionization remained constant, Le., monoanionic. A potentiometric titration of cAMP in 7 M urea showed that the pK, does not differ significantly from its value in water. Table I11 lists the ultrasonic results. Both pmagand f, are independent of concentration at 25 "C up to 0.25 M, hence in 7 M urea the rotational isomerization process is no longer coupled to a concentrationdependent reaction. At high concentrations of cAMP (0.30 M in urea) hmax decreases. Addition of MgClz causes the observed relaxation to vanish. We conclude that formation of ion pairs influences the syn-anti process by shifting the syn-anti reaction to one side. Prior to the determination of thermodynamic properties of the syn-anti process, solvent properties such as the coefficient of thermal expansion of 7 M urea had to be calculated (assuming that the static solution properties of dilute nucleoside and nucleotide solutions are very similar to those of aqueous urea solutions) by employing literature data23,24 for density. The calculated solution properties are quoted in Table IV. The final minimization yielded three different values of AVO, -2.0,0, and +3 cm3/mol. Only the last one of these gave a nonnegative rotational barrier within experimental error, hence this must be the acceptable value. Thermodynamic and kinetic values obtained at 3 cm3/mole are presented in Table V. Adenosine in H 2 0 and Aqueous Urea. Extensive results in water were reported from this laboratory earlier.3 On the basis of the results on cAMP and 6-methylpurine, adenosine was reinvestigated in 7 M urea. At 25 OC lo3. (pmm/CT)and f , are 3.0 and 33 MHz in H20 and 4.25 and 34 MHz (according to the resonator method) in 7 M urea. (23) (a) R. H. Stockes, Austr. J . Chem., 20, 2087 (1967); (b) F. T. Gucker, Jr., F. W. Gage, and C. E. Moser, J. Am. Chem. SOC.,60, 2582 (1938). (24) F. T. Gucker and F. D.Ayes, J. Am. Chem. SOC.,59,2152 (1937).
The larger amplitude observed in aqueous urea indicates that in water the glycosyl isomerization is coupled to a concentration-dependent phenomenon: stacking.
Discussion Addition of 7 M urea to solutions of cAMP and adenosine yields an approximately 40% increase in p m a r / Cas ~ would be predicted from the decoupling of the concentration-dependent effect. Proton transfer can be discounted (see results in adenosine) in the pH range studied. Hydrogen bonding between the bases is unlikely in water based on all available evidence (A-A interaction at best leads to two intermolecular hydrogen bonds which would "melt" in water well below ambient temperatures). Therefore the increase in absorption resulting from the addition of urea must result from the decoupling of the stacking interacti~nsl~ from the glycosyl conformational isomerizations. Base stacking has been observed at concentrations less than 0.01 M.2192"27 These associations proceed to a significant extent in nucleosides and their phosphate derivatives. Some of the associated molecules must also participate in glycosyl isomerization according to the magnitude of the change in absorption induced by 7 M urea. It is then likely that the stacks contain some species capable of glycosyl isomerization and the energy required for this isomerization is similar to that required for the unstacked species. A possible scheme accounting for the observed behavior is shown in Scheme 11, where A' and A" are glycosyl isomers of the monomer at the ends of the stack capable of glycosyl isomerization, and B are stacked species incapable of glycosyl isomerization. In 7 M urea, pmax/CT is independent of nucleoside concentration indicating that stacking is no longer coupled to the glycosyl isomerization process. With the realization of the coupling of stacking and glycosyl conformationalequilibria, and due to the lack of information concerning the aggregation number within the stacks and the temperature dependence of such aggregation numbers in water, the thermodynamics of the glycosyl equilibration in 7 M urea solutions were determined. The magnitude of the thermodynamic parameters obtained in 7 M urea, but not those in water, appear to be in reasonable accord with thermodynamic data on other isomerization processes. These values may include a contribution from the ion pairing process with sodium ion. (25) J. M. Delabar and W. Guschlbauer,J. Am. Chem. SOC.,96, 5729 (1973). (26) T-D. Son and C. Chachaty, Biochim. Biophys. Acta, 336,l(1973). (27) F. Jordan and H. Niv, Biochim. Biophys. Acta, 476, 265 (1977).
Glycosyl Isomerization in cAMP
The Journal of Physical Chemistty, Vol. 85, No. 1, 1981 101
Thus the values are only semiquantitative. The results in 7 M urea are as follow: for K > 1; AHo > 0; AS' > 0, and AVO < 0. It is important to compare the results in Table I1 with those in Table V to see the effect which neglect of the stacking equilibrium has on the apparent thermodynamic properties in water. The basic method used to evaluate these terms, as described in ref 3, is to use the temperature dependence of the magnitude of the excess absorption (pmw). The quantity pu,,/CT should be independent of concentration for an isomerization. This is not the case. What is true, however, is that ~,,JCmonomeris independent of temperature. Cmonomer, however, is a strongly temperature dependent quantity due to destacking. The apparent thermodynamic quantities therefore include a contribution from stacking equilibria. Since, in fact, the true thermodynamic quantities for the syn-anti process (as found in aqueous urea) are small compared to the quantities for stacking, the latter dominate the calculation. Thus the apparent quantities should approximate those for stacking rather than isomerization. Since stacking also occurs in adenosine our previous conclusions for that system3as well as Rhodes and Schimmel's estimate2 are in error. The proposed scheme and the known influence of urea on stacking also accounts for another anomaly in the data, namely, the fact that AH" is greater for the aqueous system than for the urea solutions. The relaxation time for a simple isomerization is given by T-' = kf k,. If this process is coupled to a concentration-dependent term, say for simplicity a dimerization, we predict two relaxation times. The faster of these is approximately 7-l = kl + kl+ 4kzA + k-z
+
in which kl + klare the forward and reverse rate constants for isomerization,kz k2are the forward and reverse rate Iis the monomer constants for the dimerization, and ; concentration. It is clear that the activation enthalpy for the latter case will be greater than for the simple case since k2 and k2will contribute as will the temperature-dependent A plausible explanation of the results in 7 M urea invokes a solvation model. As the glycosyl equilibrium constant is greater than unity in the endothermic direction, the process must be entropy driven. This can be explained if, for example, solvation of the group on the 5' position of the sugar (hydroxy or cyclic phosphate) plays a vital role in the glycosyl equilibrium. Consider as a possibility anti.nH20 + syn-(n - y)HzO + y H 2 0
+
A.
i.e., the base interferes with solvation of the 5' group more in the syn than in the anti conformation. Solvation of the cyclic phosphate (or of the hydroxy group) should be exothermic. It should also represent ordering and electrostriction of solvent molecules (ASo and AVO both negative). Hence the process anti syn will be endothermic but entropically favored with a positive AVO. This model predicts that the syn conformer will predominate
-
at high temperatures and the value of K is chosen accordingly. This resolves the problem with ultrasonic methods that only the magnitude of AGO can be determined. Since the rotation has a very low barrier (less than 4 kJ/mol) other processes will affect this conformational degree of freedom. As the concentration increases base stacking will occur. Nuclear Overhauser enhancement studies also suggest that the anti/syn conformer ratio increases in the stacked aggregates.12 If our further assumption above is correct the low temperature stacked preference is for the anti conformation, as found in most crystallographic studies.13 'H NMR spin-lattice relaxation measurements on adenosine mononucleotides demonstrated that the anti conformation is induced by intermolecular associations (i.e., stacking in water) and that there is flexibility around the glycosyl C-N bond.14 Very high-field 'H NMR studied6and spin-lattice relaxation studied6on dinucleoside monophosphates also suggested an interrelation between glycosyl conformational preferences and stacking interactions. The latter study also suggested that even in the dinucleoside monophosphates glycosyl rotation flexibility still exists.16
Conclusions It was demonstrated here and elsewhere4J7that the glycosyl conformational and stacking equilibria are interdependent in water. In 7 M urea, solutions of adenosine and cAMP give rise to a concentration-independent ultrasonic relaxation strictly reflecting the glycosyl equilibrium. The work on cyclic AMP has further substantiated our earlier claim (and that of Rhodes and Schimmel) that ultrasonic relaxation spectroscopy is a rather unique tool that can be used (without the necessity of extensive data fitting required by either nuclear Overhauser enhancement or relaxation magnetic resonance methods) to demonstrate the presence of two or multistate glycosyl conformational equilibria in nucleosides and nucleotides. A more laborious treatment allows determination of the thermodynamic and kinetic properties of this conformational equilibrium. Very recently, Gerlt et al.28proposed that the solvation of cAMP plays an important role in the biochemical activity of this molecule. The model here proposed to explain our results suggests that solute-solvent interactions help determine the position of the conformational equilibrium in this molecule. Taken together these hypotheses suggest that the glycosyl conformation found in a receptor site for cAMP may depend on whether or not the site is hydrophobic. Acknowledgment. We are especially thankful to the General Medical Science Institute of the National Institutes of Health for financial support of this research. The invaluable help of Mr. Robert Rhinesmith in the construction of the resonator instrument is also gratefully acknowledged. (28)J. S.Gerlt, N. I. Gutterson, R. E. Drews, and J. A. Sokolow, J. Am. Chem. SOC.,102, 1665 (1980).
