1483
VICINALEFFECTS ON OPTICAL ACTIVITY
Vicinal Effects on the Optical Activity of Some Adenine Nucleosides by Daniel W. Miles, S. J. Hahn, Roland K. Robins, Morris J. Robins, and Henry Eyring Department of Chemistry and Institute for the Studv of Rate Processes, University of Utah, Salt Lake City, Utah 84112 (Received J u l y 80, 1967)
The optical rotatory dispersion, absorption, and, in some cases, the circular dichroism of some adenine nucleosides having diverse substituents in the carbohydrate ring have been examined. Solvent and temperature studies have also been performed when possible. Explanations for the changes in rotation with substituent are sought through theoretical means in correlation with available structural information. The changes in the 260-mfi Cotton effect lxoduced by the introduction of new s-electron systems can be accounted for quantitatively.
I. Introduction Concurrently with the study of chromophoric substituent eff ects1y2we have examined the optical rotatory dispersion (ORD), absorption spectra, and, in some cases, the circular dichroism (CD) of purine nucleosides having diverse substituents on the carbohydrate ring. Previous work in this area has shown that the replacement of the 2’-OH group by hydrogen or chlorine does not alter the sign of the characteristic 260-mp Cotton effect,3while, in contrast, a sulfur substituent at the 5’ position does reverse the sign,4 and the optical activity of 5’-mononucleotides and the corresponding nucleoside are not significantly d i f f e r e ~ ~ tThus . ~ ~ ~it appears that the phosphate group, despite its polar nature and T electrons, does not contribute significantly to the rotation. Recent interpretations7-9 of ORD and CD studies on dinucleoside phosphates and other model systems of RNA and DNA have used this fact to justify omitting all phosphate-base interactions. Meanwhile, several conflicting proposals, based mainly on ORD studies, have been made regarding the conformation of ribonucleoside^^^^ in solution. In this paper we describe the optical activity of some adenine nucleosides having, for the most part polar and moderate to weakly polarizable substituents. The optical rotatory dispersion of these derivatives are found to differ only slightly from one another. In addition we have measured the optical rotatory dispersion and circular dichroism of several derivatives having nonpolar and moderately polarizable groups, such as the carbon-carbon double bond and the thioether group. These substituents generally induce dramatic changes in the optical activity, with almost no effect on the absorption spectra down to 220 mp. Temperature studies demonstrate the presence of preferred conformations in the adenine nucleosides. A quantitative analysis based on the Kirkwood-Tinoco polarizability expression is carried out on representative compounds. All possible sugar-base conformations and several puckered conformations of the ribose moiety have been considered with the aid of a computer.
These results, intended to be but a first step toward the use of optical-rotation theory on the purine and pyrimidine nucleosides, demonstrate the dependence of optical rotation on the nature of the substituents, the sugar-base torsion angle,’O and changes in the puckered conformations of the ribose residue. While an analysis of these preliminary results in concert with other physicochemical evidence tend to suggest that an anti range of the torsion angle is preferred in adenine nucleosides, conclusive evidence awaits a more systematic study on suitable model adenine nucleosides.
11. Experimental Procedures Absorption and optical rotatory dispersion curves were determined on the Cary Model 14 spectrophotometer and the Cary Model 60 spectropolarimeter, respectively. The circular dichroism curves were measured on the Cary Model 6001 CD attachment for the Model 60. The CD unit was calibrated using the Cary Model 1401 circular dichroism attachment for the Model 14. The standard used was an aqueous solution of d-10-camphorsulfonic acid (J. T. Baker, Lot No. 9-361) with an EL - e ~ of. 2.2 at 290 mp. The Cary thermostatable sample cell of path length 1 cm was used (1) D . W. Miles, R . K. Robins, and €1. Eyring, Proc. Nat. Acad. Sei. U.S., 57, 1137 (1967). (2) D. W. Miles, R . K. Robins, and H . Eyring, J. Phys. Chem., 71, 3931 (1967). (3) T . R. Emerson, R . Swan, and T . L. V. Ulbricht, Biochem. Biophys. Res. Commun., 22, 505 (1966). (4) W. A . Klee and S . H . Mudd, Biochemistry, 6 , 988 (1967). (6) C. k’.Lin, D . W. Urry, and H . Eyring, Biochem. Biophys. Res. Commun., 17, 642 (1964). (6) J. T. Tang, T . Samejima, and P. K . Sarkar, Biopolymers, 4, 623 (1966). (7) M. ,M.Warshaw, C. A. Bush, and I. Tinoco, Biochem. Biophys. Res. Commun., 18, 633 (1965). (8) C. A. Bush and J. Brahms, J . Chem. Phys., 46, 79 (1967). (9) C. A . Bush and I. Tinoco, J . Mol. Biol., 23, 601 (1967). (10) J. Donohue and K. N. Trueblood, J . Mol. Biol., 2, 363 (1960). The torsion angle, $CN, is defined as the angle formed by the trace of the plane with the projection of the CI’-O bond when the projection is taken along the glycosidic bond. The angle is t,aken as zero when the carbohydrate ring oxygen is antiplanar to Cz of the purine or pyrimidine ring, and positive angles are taken as those measured in a clockwise direction when viewing the Ct’ to the ring nitrogen.
Volume 72,h’umber 5 M a y 1968
1484
D. W. MILES,S. J. HAHN,R. K. ROBINS,11. J. ROBINS, AND H. EYRING
Table I : The ORD i\/leasuremen ts of Adenosine and Related Derivatives Compound
Structurea
2’,3’,5’-Trideoxyadenosine
I
2 ’,3’,5’-Trideoxy-2 ’,3’-didehydroadenosine Adenosine
I1
2’,3’-Dideox~-2’,3’-didehydroad enosine 6-Amino-9-(5 ’-S-ethyl5 ’-thio-
I11
IV
v
2’,3’,5’-trideoxy-2’,3’-didehydro-p-n-glycero-pentofuranosy1)purine Adenosine 5’-monophosphate
VI
2’,3 ’-Dideoxyadenosine
VI1
Adenosine 5’-monoacetate
VI11
Adenosine 5’-monopropionate
IX
2’-Deoxyadenosine
x
2’-Deoxyadenylic acid
XI
5’-Deoxyadenosine
XI1
2’,3’-O-Isopropylideneadenosine
XI11
Posn,b mr
W’I’
276 245 270 240 276 245 270 240 272 239
-1,400 +800 +5,400 -7,600 -2,700 +1,300 +5 ,000 -7,000 13,000 -24,000
272 246 273 246 274 246 274 246 272 245 270 250 274 246 276 246
*
haxv
+
Amplitude
mw
-2,200
260
14,600
+13,000
259.5
14,800
-4,000
260.5
14,900
+12,000
259.5
15,200
+37,000
259.5
14,900
-4,200
260
15,000
-2,600
259.5
14,600
-4,000
260
14,800
-4,100
260
14,900
-3,200
259.5
14,900
-3,400
260
15,000
-3,900
260
14,800
-3,800
260
14,900
-2,800 $1,400 -1,600 +1,000 -2,500 +1,500 -2,600 +1,500 -1,400 +1,800 -2,200 $1,200 -2,000 +1,900 -2,600 +1,200
a Structural formulas are given in Figure 1. Extremum positions of the ORD curves. t o all data reported in this paper using Pu’a D line values for n.
for all optical rotatory dispersion measurements so as to virtually eliminate the problem of base-line shifting (provided suitable care was exercised in manipulating the sample compartment). All measurements were triplicated and gave average values within f0.0004”. The temperature of the cell compartment was controlled by the P. M. Tamson circulation thermostate Model T-3, with the cooling spiral accessory for low-temperature studies. I n the temperature study, it was necessary to guard against heat-induced decomposition. All measurements were performed first a t the minimum temperature and then at the maximum temperature. The intervals between were measured as the sample was returned stepwise to the minimum temperature. In all reported data the two minimum curves were superimposable. Cell temperature was monitored by thermocouple techniques. All solvents were of spectral grade. All compounds were made and purified in the laboratory of Professor Robins or were commercial samples of high purity. Details of the preparation and characterization of these nucleosides are given elsewhere.“ The optical rotatory dispersion data are reported in terms of the reduced molecular rotation, [ A / ’ ] , defined as [M’] = 100a/cL(3/n2 2), where 01 is the observed
+
The Journal of Phz/sical Chemistry
emax
The Lorenz correction has been applied
rotation in degrees, c is the concentration in moles per liter of the solute, L is the path length in centimeters, and n should be the index of refraction of the solution at the wavelength that a! is measured. However,we have used only the Na D linevalue in the Lorentz correction factor for the internal electric field. Values of [ A l l ] were corrected for volume expansion upon heating.
111. Results The results of the ORD arid absorption measurements on the adenine ribonucleosides derivatives are summarized in Table I. The ORD curves of the adenine nucleosides display but a single Cotton effect above 230 mM centered at approximately 260 mp. The structural formulas of the compounds studied are contained in Figure 1. Figure 2 shows the optical rotatory dispersion mea(11) J. 11. McCarthy, Jr., M. J. Robins, L. B. Townsend, and It. K. Robins, J . Amer. Chem. Soc., 88, 1549 (1966); M. J. Robins, J. 11. McCarthy, Jr., and It. K. Robins, Biochemistry, 5, 224 (1966); M. J. Robius and It. K. Robins, J . Amer. Chem. Soc., 86, 3585 (1964); It. J. Rousseau, L. B. Townsend, and It. K. Robins, Biochemistry, 5 , 224 (1966); J. 11. McCltrthy, Jr., M. J. Robins, and R. K. Kobins, Chem. Commun., 536 (1967). The authors wish to thank G . B. Whitfield, Jr., of the Upjohn Co. for an autheritic sample of decoyinine (Anguutmycin A) used in this study.
VICIXAL
EFFECTS OS
1485
OPTICAL ACTIVITY
I
Ns Hoca IV
I11
I1
, I
1.“
OH
I
0
OH OH
vr
v
vrr
I
I -O-P-O-CH2
VI11
ca
@
II
0
HO
HO
HO X
N&J
XI
OH
XI1
I
Hoca, Hoca H2C+
“>Q
HO
CH,OH
HO
OH
OH
xv
XIV
H
2
C
HO
I G
OH
XVI
Angustmycin A Figure 1. The structural formulas of the compounds discussed in this paper.
sured in the uv region for 2’,3’,5’-trideoxyadenosine syladenirie nucleosides. The sign for all - is negative (I), 2’,3’,5’-trideoxy-2’,3 ’-didehydroadenosine (11), saturated furanosyladenine nucleosides and positive adenosine (111),2‘,3‘-dideoxy-2‘,3‘-didehydroadenosine for the unsaturated derivatives with the r-electron (IV), 6-amino-9-(5’-S-ethyl-5’-thio-2’,3’,5’-trideoxy-2’, system a, p, to C1’. The effect of a carbon-carbon 3’-didehydro-p-n-yZycero-pentofuranosyl)purine (V), double bond in this position is best demonstrated by and adenosine 5’-monophosphate (VI), which are comparing the ORD curves of structure I1 and its typical of the compounds studied and which will be reduced form, structure I. The substitution of a a emphasized in the Theory and Discussion sections. bond for 2’-H and 3’-H changes the amplitude from Among these compounds are three unsaturated furano-2200 to 13,000. The theoretical treatment of the Volume 72, Number 5 Mag 1368
1486
D. W. ~ L I L E S , S. J. HAHN,R. I X > VI1 > I. The 5’-OH in the unsaturated derivatives seems to make a negative contribution also (compare the amplitude of structure I1 with that of IV). Polar substituents with n-electrons systems, such as the phosphate or acetyl groups when attached to the 5’-carbon, are found to be just slightly more effective negative contributors to the rotation than the hydroxyl function. For example, compare the amplitude of adenosine, I11 (-4000) with the amplitude of adenosine 5’-monophosphate, VI (- 4200). On the other hand, a remarkable enhancement in the rotation is observed when a nonpolar substituent replaces a polar substituent at the 5’-carbon. For example, compare the amplitude of structure I1 (13,000) with that of structure V (37,000). These effects are even more dramatically revealed in the circular dichroism spectra of these compounds. Figure 3 shows that adenosine (111) and adenosine 5’-monophosphate (VI) give maxiThe Journal of Phgsical Chemistry
I
.16 I
I
220
240
260
A
I
1
280
(mu)
Figure 3. The circular dichroism ciirves of some adenosine ribonucleoside derivatives in aqueous solution a t p H 7 . These curves were used to obtain the rotational strengths of the 260-mp Cotton effects.
mum ellipticities of -2100 and - 2200, respectively,12 at 260 mp, whereas compounds I1 (with the C2’-Ca’ double bond) and compound V (with both the Cz’-C3’ double bond and the 5’-thioether group) give large positive ellipticities at 260 mp of 11,000 and 30,000, respectively. Figure 4 shows the circular dichroic spectra of angustmycin A (XV) and 4’-exocyclic methylene adenosine (XVI). Both these compounds have a Cq’-C5’ double bond. These compounds, in contrast to compounds 11, IV, and V, give a small negative 260-mp CD band. Adequate interpretation of the ORD and CD data of adenine nucleosides should include explanations of the low amplitudes exhibited by adenosine 5’-monophosphate, augustmycin A, and 4‘-exocyclic methylene adenosine, and the remarkable effect of the 5’-thioether group and the Cz’-C3’ double bond. A more detailed discussion of these effects will follow the development of the theory. Figure 5 contains the results of temperature and solvent studies on the nucleosides containing the Cz’-Ca’ double bond. The measured amplitudes of the 260-mp Cotton effect are given as a function of temperature in water, methanol, acetonitrile, and dioxane. Decreases in amplitude of 30-35y0 are observed over a temperature range of 80”. The unsaturated nucleoside with the thioether function (V) is found to undergo a 50% loss of amplitude by the combination of temperature (12) The CD curves of structure I11 and V I in Figure 3 appear t o be superimposable because of choice of coordinates.
1487
VICINALEFFECTS ON OPTICALACTIVITY
Angustrqcin A (XV)
-.- . -
4',5'-Exocyclic methylene adenosine (XVI)
+4
-
.t4
jl
il*I
c 2
-2
*t2
!I \I
0
+?4
- - --
$.,)'
-
+*'
*++
\+, '
A
H
," /
Fr -Y\"\
+? -2
'%'
0 -L
. -4
-6
- -6
L
L
I
220
1
240
and solvent effects. The temperature effects on compound I1 and IV (see Figure 5 ) , which cannot be ascribed to changes in solvent perturbation with temperature since the circular dichroism curves of the rigid cyclonucleosides are invariant to temperature changes over this temperature range, l 3 definitely support the idea of R preferred range of torsion angle.14 For example, the amplitude of the 260-mp Cotton effect of I1 decreases from 14,000 to 9000 over the temperature range 0-80". In Figure 6 we show the ORD of 3-deazaadenosine (XIV) at neutral and acidic pH values. The complexity in the 220-250-mp region for the pH 7 curve has been attributed to the n 4 T* transition in this region, which is blue shifted upon protonation.16 The 260-mp Cotton effect is negative and slightly smaller than the 260-mp Cotton effect of adenosine (the Lorentz correction factor has not been applied to Figure 6). This compound is included in this study because the absence of the nitrogen at position 3 eliminates the electrostatic repulsion between the oxygen in the carbohydrate ring and the nitrogen. This interaction has been considered as one of the factors3 that stabilize the adenosine derivatives in the anti conformation. However, the implications of Figure 6 are complicated by the fact that removal of the N-3 nitrogen is predicted to shift the Bz,-transition moment by approximately 30" (see Figure 13 and discussion of ref 2).
IV. Theory Several recent p a p e r ~ ~16v1' * * *have ~ > utilized the general
I
260
5;x
Q
, *
I
280
I
500
treatment of optical rotation devised in 1962 by Tinoco. l8 Tinoco's derivation retains the formalism of the one-electron model as originated by Condon, Altar, and Eyringlg and Gorin, Walter, Iiauzmann, and Eyring20,21 which focused attention on the contribution of individual electronic transitions. The Kirkwood model is revised by the Tinoco treatment so that it too emphasizes the individual rotational strengths. For Cotton effects of strong absorption bands interacting with vicinal groups containing nonpolar but fairly polarizable substituents, the coupled oscillator model of Iiirkwood should account for most of the rotational strength. The rotational strength of the 260-mp adenine transition arising from each base-sugar (13) D. W. Miles, 11. K. Robins, and H. Eyring, unpublished data. (14) That changes in rotation with temperature does not ensue from changes in the conformational equilibrium involving 3'-endo, 3'-ezo, or 2'-endo puckered conformations of the ribose ring is indicated by our calculations (see next sections). The extreme situation involving 100% depopulation of one puckered state in favor of There another generally affects the calculations by only lO-ZO%. is no evidence that more drastic changes in ribose conformation can be induced by temperature. (15) D. W. Miles, Ph.D. Thesis, University of Utah, Salt Lake City, Utah, 1967. (16) R. W. Woody and I. Tinooo, J . Chem. Phys., 46, 4927 (1967). (17) E. S. Pysh, J . Mol. Biol., 23, 587 (1967). (18) I. Tinoco, Advan. Chem. Phys., 4, 113 (1962). (19) E. U. Condon, W. Altar and H. Eyring, J. Chern. Phys., 5 , 753 (1937). (20) E. Gorin, J. Walter, and H. Eyring, ibid., 6, 824 (1938). (21) W. J. Kausmann, J. Walter, and €1. Eyring, Chem. Rev., 26, 339 (1940). Volume 72, Number 5 M a y 1068
D. W. MILES,S. J. HA”, R. K. ROBINS, M. J. ROBINS, A N D H. EYI~ING
1488
40
35
30
C
25
2
I x
20
A(mp1
Figure 6. The optical rotatory dispersion and absorption spectra of 3-deaxaadenosine a t neutral and acidic pH values.
15
10
Table I1
-200
I
I
00
200
,
I
LOO
60°
c-c
800
0-H
Figure 5 . The amplitude of the 260-rnw Cotton effect as a function of temperature and solvent of the unsaturated adenine nucleosides. Roman numerals refer to the structures of Figure 1.
Rj
=
a ~ , v o ~ ~ c i (a33 oa~ - 4 , ( G F ) , C(VO*
0.75 0.43 0.30 2.14 0.00
c-0
Temperature
bond interaction is given by the IGrkwood-Tinocol8 expression
1024(aaa- a , , ) , cc
Bond
c=c C-H
10- 1 5 ~ 0 , 8ec - 1
2.3 2.0 2.0 1.8 2.3
which are parameters characteristic of the 260-mp transition of adenine) give for the specific case where the subscript j refers to a C-C, C=C, C-0, or 0-H bond (in biotsZ4)
- v,’)
Rc-c
67O(Gl?)c=c
Rc-c = 19O(GF)c-c Rc-o = 130(GF)c-o where the symbols and the center of the adenine ring used in the calculation of r Z jare as defined in ref 2. Equation 1 has been used in several recent calculations to account for the rotational strength arising from the interaction of strong electric dipole transitions with the ~~~ far-uv transitions of the vicinal g r ~ u p s . ~ ~The emphasis on bond polarizabilities rather than group polarizabilities eliminates the arbitrary division of the molecule into groups. For a given i , j pair, the rotational strength is seen to be a function of the distance vt,, their mutual orientation, and the anisotropy - all) of the j t h bond. The polarizability ellipsoid for each bond is localized at the midpoint of each bond. The anisotropy of bond polarizabilities (taken from the work of LefevreZ2)and vo (see ref 2 ) to be used in eq 1 are given in Table 11. These values when substituted into eq 1 (along with v, = 1.15 X 10l6sec-’, bios = 4DQ y = 45” (transition-moment directionz3) The Journal of Phusical Chemistry
R0-H = SO(GF)o-II
In the above expressions the geometrical factor, GE’, must be expressed in Bngstrom units. The geometrical factor can be evaluated once the atomic coordinates of ~J* (22) R. J. Mi. LeFevre and C. G. LeFevre, J . Chem. Soc., 3549 (1956); It. J. W. LeFevre, A. Sundaram, and 11. K. l’ierens, ibid., 479 (1963): M. Aroney and It. J. W. LeFevre, ibid., 3002 (1958); R. Bramley, C. G. LeFevre, It. J. W. LeFevre, and B. 1’. Itao, ibid.,
1183 (1959). (23) Stewart and Jensen (It. F. Stewart and L. H. Jensen, J . Chem. Phys., 40, 2071 (1964)) have found two orientations ( y = 45 or - 3 O relative to an axis from C4-G and measured positive toward CS) for the Bzu transition of the adenine chromophore which are consifitent with the crystal symmetry and dichroic ratio. We have chosen the former value because it is more consistent with theoretical means of estimating polarization directions and because this orientation gave such good agreement in a recent theoretical treatment of cycloadenosine.2 (24) This unit has a value of cgs (L. Velluz, M. Legrand, and M. Grosjean, “Optical Circular Dichroism,” Academic Press Inc., New York, N. II., 1965, p 75.
1459
VICINALEFFECTS ON OPTICALACTIVITY
I - -. - * Adenosine (?‘-ex01 ,..- ... - ..,I+, -exocyclic methylene adenosine (3’-endo) [WI] 1
I
*
Adenosine (J‘-endo)
i
2’
I 1 -- - I/-‘ \ / / I
,>’ , 5 ‘-trideoxy-2 ’ -,J ’ -didohydroodenosine () ’ -endo)
I-
[ I1
\
1-
\
-6
-
I
-6
I l
,
I
,
I
~
I
I
I
IJ
#
I
,
1
,
l
I
I
_
Figure 7 . The calculated rotational strengths of adenosine (3’-endo), adenosine (3’-ero), 4’-exocyclic methyleneadenosine (3’-endo), and 2’,3’,5’-trideoxy-2’,3’-didehydroadenosine (3’-endo) as a function of the torsion angle. The calculations were based on eq 1 of the text summed over all i,j pairs.
the system are known. I n this study, the coordinates for adenosine in the 3’-endo conformation are taken from the X-ray data.25 Drieding and Fiesers’ molecular models were used to estimate the coordinates of the other molecules. Using eq 1 we have calculated via computer the rotational strengths of the 260-mp adenine band for all values of the sugar-base torsion angle. The calculations were made with the ribose moiety in the 3‘-endO conformation, Le., C’-3 is out of the plane defined by C’-1, the furanose ring oxygen, and C’-4 and is lying on the same side of the sugar plane as C’-5 and the ring of the base. Calculations were also made with the ribose moiety in the 3’-exo and 2’-end0 conformations. The results for the calculations performed on adenosine (3’-endo), adenosine (3’-exo), and 4‘-exocyclic methyleneadenosine (3’-endo) are displayed graphically in Figure 7 . I n comparing experiment with theory, we shall be concerned more with explaining relative changes in the rotation as a function of substituents and changes in the orientation of sugar and base rather than attaining actual numerical agreement. We proceed with the initial assumption that the same preferred range of torsion angle is favored by all adenine nucleosides at room temperature in neutral aqueous solution. This range will be taken from 10 to - 60” or from 25 to - 5 5 O , in correlation with the structural information available in Table 111. The information summarized in Table 111 is taken from the
Table I11 : Summary of Structural Information for Adenine Nucleosides and Nucleotidesa
Adenine structure
Out-ofplane atom
Allowed range, deg
Torsion angle, deg
+cw
Deoxyadenosine
C’-3 exo
25 to -85
-6
Adenosine 5 ‘-phosphate
C’-3 endo
10 to -60 -85 to -140
-20
Adenosine 3’-phosphate
C’-3 endo
-10 to -60 -85 to -140
Adenosine in 5-bromouridine-adenosine complex Adenosine in the coenzyme of vitamin Blz
c’-3 C’-3 endo
a Taken from Table I of ref 26. from Table I11 of ref 26.
-4
-10 to -60 -85 t o -140
-11
-10 to -60 -86 to -140
- 69
* Allowed +CN
range is taken
recent analysis of steric interactions present in nucleoside systems by Haschemeyer and Rich.26 I n Figure 7 it is seen that the rotational strength of each derivative changes slowly and in a regular manner as the torsion angle is varied. This is found to be true (25) J. Kraut and L. H. Jensen, Acta Crystallogr., 16, 79 (1963). (20) A. E. A. Haschemeyer and A. Rich, J . Mol. B i d , 27, 369
(1967). Volume 72,Number 6
M a y 1968
D. W. MILES,S. J. HAHN,R. I
