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Studies of intermolecular interactions of nucleic acid bases in aqueous solutions by the proton magnetic resonance method
Biochimica et Biophysica Acta, 331 (1973) 9-20
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 97844
S T U D I E S OF I N T E R M O L E C U L A R I N T E R A C T I O N S OF N U C L E I C A C I D BASES I N A Q U E O U S S O L U T I O N S BY T H E P R O T O N M A G N E T I C RESONANCE METHOD S E L F - A S S O C I A T I O N OF 6 , 9 - D I M E T H Y L A D E N I N E A N D ITS P Y R A Z O L E ANALOGUE AND THEIR ASSOCIATION WITH METHYL-SUBSTITUTED CYTOSINE AND URACIL*
V. L. ANTONOVSKY, A. S. GUKOVSKAJA, G. V. NEKRASOVA, B. I. SUKHORUKOV and I. I, TCHERVIN Institute of Biochemistry and Physiology of Microorganisms, U.S.S.R. Academy of Sciences, Pustchino on the Oka, Moscow re#. (U.S.S.R.)
(Received May 15th, 1973)
SUMMARY To study intermolecular interaction the concentration and temperature dependence of chemical shifts of the protons of 6,9-dimethyladenine (I), its pyrazole analogue I-methylaminopyrazolo-3,4-d pyrimidine (II), as well as 1,4-dimethylcytosine and dimethyl derivatives of uracil, modelling the main and rare tautomeric forms of uracil: 1,3-; 1,4-; 2,4-dimethyluracils, both separately and in mixtures have been investigated. A conclusion has been made about homoassociation in solutions of I and I I as well as heteroassociation of I and II with pyrimidine derivatives into complexes, having the stacked structure. From the differences in values of the chemical shifts, which are dependent on concentration, for the protons of six-membered and five-membered cycles the authors came to conclusions about the conformation in the homoassociates of I and II. Association of studied compounds is not limited to the dimer formation, but is well described by the isodesmic model. Thermodynamic characteristics of association have been obtained and discussed. A correlation between equilibrium association constants and component polarizability has been revealed. Taking homoassociation of I and heteroassociation of I with IV as examples, it has been established, that in 2H20 stacking interaction of cation with cation and cation with neutral molecule is considerably weaker than the interaction between neutral molecules.
* Separate parts of the work were reported on the IH All-Union conference on the investigation of structures of organic compounds by physical methods, Kazan (1971) and on the IV International Biophysics Congress, Moscow (1972).
10
V . L . A N T O N O V S K Y et aL
INTRODUCTION
The investigation of the association of nucleic acid bases and their derivatives in solution is of great interest for the elucidation of the nature of forces stabilizing the DNA double helix 1. Hydrogen-bonded complexes of the nucleic acid bases are formed in organic solvents, whereas in aqueous media the association of the bases takes place due to interplanar stacking interaction which is favoured by strong solvent-solvent interactions. P.M.R. investigation of the chemical shift concentration dependence for nucleic acid-component protons in aqueous solutions has permitted the establishment of the stacked structure of the associates 2'3 as well as mutual orientation of the molecules in complexes of purine nucleosides and bases4' 5. With the help of the N.M.R. method, thermodynamic parameters of association have not been determined and the interaction of different groups and molecules in associates has been evaluated only qualitatively. The quantitative data of homoassociation of nitrous bases and their derivatives were obtained mainly by the vapour pressure osmometry method 4- s and the data of heteroassociation was obtained by solubility methods 9' lo. In the present work we have made an attempt to use the P.M.R. method for defining both the quantitative and qualitative characteristics of the nucleic acid bases stacking interaction. As the bases of nucleic acids may be subjected to tautomeric conversions which must considerably change the stacking interaction ability we have studied homoassociation of 6,9-dimethyladenine (I) modelling the main tautomeric form of adenine and heteroassociation of I with 1,4-dimethylcytosine (III) and uracil derivatives, modelling the main (diketo-) and rare (enol-) forms of uracil: 1,3-dimethyluracil (IV), 1,4-dimethyluracil (V), 2,4-dimethyluracil (VI). To evaluate the role of the isomeric conversion homoassociation of the pyrazole analogue of adenine: l-methyl,4-methylaminopyrazolo-[3,4-d]-pyrimidine (I1) and its heteroassociation with IV, V and V1 has been studied. The protonation of bases must influence their stacking interaction but up to now this question has not been studied. We have investigated the influence of medium acidity on the processes of self-association of I and heteroassociation of I with IV. Studies of the influence of isomerism, tautomerism and ionisation of the nucleic acid bases on their stacking interaction ability are also very important in connection with elucidation of the nature of spontaneous mutations. M A T E R I A L S A N D METHODS
The initial heterocyclic compounds have been synthesized and identified: I according to the method of mentioned in ref. 11; II, ref. 12; III, ref. 13; IV, ref. 14; V and VI, ref. 15. The medium pH values were measured directly in the N.M.R. spectrometer ampule on a LPU-01 potentiometer with the help of the glass electrode for micromeasurements, taking into account that p2H--pH-meter indication +0.4 (ref. 16). The measurements were carried out in buffer solutions having constant ionic strength 0.1. P.M.R. spectra were recorded on the "Varian" HA-100D spectrometer at the
P.M.R. STUDIES ON NUCLEIC ACID BASE INTERACTION
11
working frequency 100 MHz. The values of chemical shifts (iS) of resonance signals were expressed in Hz from hexamethyldisiloxane as an external reference. Shifts at the infinite dilution were found by graphical extrapolation of chemical shifts measured at 100 °C to zero base concentration. Shifts were corrected for bulk susceptibility by the application of both the external hexamethyldisiloxane and internal tert-butanol or acetone. The difference in values of the chemical shift concentration dependence between external and internal compartments does not exceed 4 Hz. The results are not due to an internal compartment base interaction because the temperature-dependent shifts of the bases were identical with added acetone or tert-butanol. RESULTS AND DISCUSSION Peak assionment Four peaks in the spectra of compounds studied have been observed. The peaks of III-VI were assigned to H-5 and H-6 according to ref. 17. The H-2 and H-8 peaks of I were differentiated on the basis of H-8 proton ability to exchange at elevated temperatures18: when heated to 100°C the intensity of the H-8 peak drops considerably. The exchange of the H-3 proton of II with 2HzO is already observed at 34 °C. Homo- and heteroassociation in aqueous solutions Concentration changes of P.M.R. spectra were studied at the interval of 0-0.4 M at temperatures from 34--100 °C. At a constant temperature proton resonances in I and II are all shifted to higher fields as the solute concentration is increased (Fig. 1). The same upfield shifts are noticed for pyrimidine-derivative protons in binary mixtures with I or II when the concentration of the latter increases (Fig. 2). When temperature increases the resonance signals of I and II protons as well as of pyrimidine-derivative protons in mixtures with I or II are shifted to low fields. The temperature dependence of 6 of I or II protons in one-component systems and of III-VI protons in the mixtures with I or II is a linear one. The observed concentration and temperature changes in P.M.R. spectra are manifested by ring current magnetic anisotropy effects and point out that: (1) both homoassociation of I and II and their association with pyrimidine derivatives take place in aqueous solution, (2) homo- and heteroassociates have a stacked structure 2,3,19. At the same time the concentration and temperature changes in solutions, containing only the pyrimidine derivatives, do not shift the resonance signals of III-IV protons more than of 4 Hz. The results obtained are conditioned by the low diamagnetic anisotropy of cytosine and uracil rings 2° and by the weak ability of pyrimidine derivatives for stacking interaction 6. The lack of a concentration effect in 2,4-dimethoxy-uracil spectra is evidently caused by the induction effect of the methoxy groups. Fig. I shows the convergence of H-2 and H-8 resonance signals in the spectra of I, while at the homoassociation of II peaks of H-6 and H-3 protons diverge. It should be noted that the values of concentration and temperature shifts of pyrimidine-ring protons, 6, are more than those of the imidazole ring for all
12
V. L, ANTONOVSKY e t a / . 850
810
~
77(] 75(1 420
380
N ;~" I~ X
790 ?80 770 ?60 7N)
E
sl°i
,~
600
1,3
~60
~'40I, '
H-6
~7o~
3310
o.1
0.2
03
0-4
0.~
0,2
0.3
M
M
Fig. 1. The concentration dependence of chemical shifts of the protons of 6,9-dimethyladenine ( 0 , H-2; ~ , H-8; [], -CH3) and I-methyl, 4-methylaminopyrazolo [3,4-d]-pyrimidine ( 0 , H-6, A, H-3, II, CH3) in 2H20 at 34 °C. GMDS, hexamethyldisiloxane. Fig. 2. Dependence of chemical shifts of the protons of 0,1 M 1,3-dimethyluracil upon concentration of 6,9-dimethyl-adenine in 2HzO at 34 °C.
purine derivatives so far studied. This testifies to the predominant contribution of the associate conformations in which the H-2 proton is more shielded than H-8 (ref. 4). The average conformation of the homoassociate of I is apparently very close to the proposed one 4 for the homoassociate of adenosine. For I1 the concentration shift (A¢5) of the pyrazole ring proton (H-3) 6 is more considerable than that for the pyrimidine ring proton (H-6). It is due to the fact that isomery in the five-membered ring changes the associate geometry and as a result the five-membered rings of II are overlapped to a greater extent then in the homoassociate of I. At heteroassociation of I or II with III-VI (Table I) the value of the ~ upfield shift for the pyrimidine-derivative H-5 proton is 2-6 Hz higher, than that for the H-6 proton. We consider that the difference observed testifies to the order of heteroassociation, the main contribution to the interaction being made by the stack conformation in which A6 H-5 > A6 H-6. The chemical slaifts of I or II protons are displaced to low fields in mixtures of pyrimidine derivatives with I or II with a constant concentration of the latter when the concentration of the magnetically-inert pyrimidine derivative is increased. The dependence of the chemical shifts of I or II protons on the pyrimidine derivative concentration is linear (Fig. 3). The observed low-field displacement is caused by a decrease in the share of the homoassociates of I or II when the pyrimidine derivative concentration is increased.
Concentration o f l or H (mole/I )
0 0.4 0 0.4 0 0.4 0 0.4
Compounds
1,4-Dimethylcytosine 1,3-Dimethyluracil 1,4-Dimethyluracil 2,4-Dimethyluracil
H-6
CHa
613 767 360 580 740 346 612 783 364 586 761 350 642 810 417 620 789 406 682 848 420 647 817 400 H'omoassociation o f i
H-5 311 298 355 334 372 361 420 402
CHa
Chemical shift; H z , 34 °C
Association with I
6.2 4.5 4.3 6.0 8.2
6.0 4.3 9.2 40.0
--zlH kcal/ mole
10.0
K I/mol
T h e relative error o f K a n d K" d e t e r m i n a t i o n = 10 %, A H = 7 %.
21.0
15.7
11.5
11.7
16.5
--AS entropy units 1-[-6
CH3
612 783 364 591 767 354 642 810 417 614 785 503 682 848 420 647 818 401 H o m o a s s o c i a t i o n o f II
11-5
355 347 372 351 420 401
CH3
Chemical shift; H z , 34 °C
Association with I1
4.0 4.8 6.0 10.5
3.1 7.4 9.0 51.0
27.4
15.8
12.0
11.7
K" -- A H -- A S 1~mole kcal/ entropy mole units
DEPENDENCE OF PROTON CHEMICAL SHIFT VALUES FOR THE PROTONS OF URACIL AND CYTOSINE DERIVATIVES UPON T H E C O N C E N T R A T I O N O F 6 , 9 - D I M E T H ' Y L A D E N I N E (I) A N D I - M E T H Y L , 4 - M E T I ~ Y L A M I N O P Y R A Z O L O - [ 3 , 4 - d - ] P Y R I M I D I N E (II) I N 2/420 A N D T H E T H E R M O D Y N A M I C P A R A M E T E R S O F A S S O C I A T I O N
TABLE I
Z
,--t
,-t m
m
r~
>
© Z Z
~o
14
V . L . ANTONOVSKY et al.
810 j ul 8O0 7go o 78G
H
-
2
H-8
E
380 .¢:
330 m
.,o..,,..,.,,~,-,~CH5
320
JlO
M Fig. 3. Dependence of chemical shifts of the protons of 0.25 M 6,9-dimethyladenine upon concentration of 1,3-dimethyluracil in 2HzO at 34 °C. GMDS, hexamethyldisiloxane.
Calculation method for association constants For the quantitative analyses of association processes we considered the model of stepped association: K1 A2
Aa +B 1 K'~ A~ B~
A2 +As r~ A3
A2 + B , r'~ A2 B1
A , + A 1 K~n A,+I
A , + B 1 r'" ~_ A, B 1
A1 +At
Here A corresponds to I or II and B to III-V1. The interaction of the pyrimidine derivatives (B-B) may be neglected because it is weaker compared with A - A and A-B interactions. Let us consider two cases• (1) The complexes are formed with a number of molecules not more than two: K1 = K; t
KI=K
Kz = K3 . . . .
t.
¢
,
K2 =K
t 3
=
• • •
=0
=0
(2) There are complexes in solution which contain an arbitrary number of molecules. The constant of m o n o m e r attachment to the associate does not depend on the number of molecules in the complex (the isodesmic model)• For the first case the association constants are equal, respectively, K =
[A2] ([Ao] - 2[A2] - [AB]) 2 '
FA, B~]
K' =
(1)
([A0] -2[A2]- [A, B,])([Bo]- [As B,]) where [Ao] and [Bo] are the total concentrations of A and B. When B is absent the observed chemical shift of A proton (6 A) will be: 6A
A A A A = aM fr~ + ~D fl~ ;
l
or
[Ao]
_ _ -_ _- 3 A 2AA[AE]
(2)
P.M.R. STUDIES ON NUCLEIC
ACID BASE INTERACTION
15
where fA, foA, 6~ and 6D A are the molar fractions and the chemical shifts of protons in monomer (M) and dimer (D), respectively. AA
=
(~A_ (~A ;
AD A
A A = (~D - - 3M
Taking into consideration Eqn l, {-Ao] _
1 + 2([-go]- [-A2] ) 2KAAD AA
AA
(3)
When B is present we have correspondingly
[Ao] _ AB
1 + [Ao] + [Bo] - 2EA2] - [AB] K'A~u ABA~
(4)
K and d A are found from Eqn 3. Neglecting [AB] in Eqn 4 at the concentrations [Bo] < [Ao] and using [A2] from Eqn 2, we can calculate the approximate values of K' and ABAWThe accurate values can be found with te help of an iteration procedure by analogy with ref. 21. In the second case the stoichiometric molarity of A and B can be expressed as a sum of power series (when K[A1] < 1): ['Bo] = ~ [ a n B 1 ] = [ B I ] + K'[BI] ~ K n - ' [ a l ] n n=O
n=l
g[A,]
[
= [ a , ] 11 +
\
I
- -
K[All]
[Ao] -- ~ n([An]+[AnB,] ) = ~ nKn-I[Ai]n+K'['B,] ~ nKn-'EA,] n n=l
n=l
n=l
[AI](I + K'[BI]) (t-K[A,]) 2
(5)
Within the isodesmic model
t~A: ~AM J~rA..~ ~ t~A i'A..~~ (~AaB A JAnB 'A M An JAn n=2
n=l
e a 6 B = 6~ f~tB + ~~ 6A~B f~nB
(6)
n=l
6AAnand f A n represent the chemical shift and the molar fraction of A protons in A. (n >1 2) complex respectively; 6aAnS,f;.nBA,6A~Baand J)~.B -- the same for A and B protons in the A.B complex (n >/ 1). Let us assume that the chemical shift of a proton in associates does not depend on the number of A molecules in the stack:
aL
aL . . . . .
a~,~= a~=~:
aL . . . . . . .
~ " = ~AnB
=
....
~BAB
In addition as a result of B magnetic inertness: ~AAnB ----- 0An. "A
16
V.L.
ANTONOVSKY
et al.
Then zjB
=
co zjBBE n=l
B
.
AA
A A E (f;~ A_~_ f~.a) n=2
=
(7)
where AaAss
-A
A.
zjBB
B
B
Using Eqns 5 and 6 we transform Eqn 7: AA
----
l
([Ao](tK+(l-)K
'
h
)-t}A.~ _[Bo ]
(tK+(1-0K')[ao]
K't(l
--
A~ t)Aas
(tK + (1 - t)K')[Ao]
(8)
where t
=
3B/A]B
Unknown association constants and chemical shifts are found from the parameters of Eqn 8 at several values of [A0] and [Bo].
Thermodynamic parameters of association The calculation of association constants, based on the assumption that only bimolecular complexes are present in solution (Eqns 3 and 4), gives a considerable scattering ( > 100 %) in K and K' values, obtained from concentration dependences for different protons of each compound. It points out that the association of the compounds studied is not limited to the formation of dimers. The relative error for K and K' values calculated from 5 concentration dependences for different protons within the isodesmic model does not exceed +_ 10 % for all studied systems. Besides, for the pyrimidine derivatives we have studied, 58 does not change practically when [Bo] is changed at a constant [Ao]. In tis case Eqn 8 gives a linear dependence of Aa on [Bo] with a negative tangent. It is in good agreement with our experimental data (Fig. 3) and those obtained previously 19. Thus all results obtained show that the association of the studied compounds is well described by the isodesmic model. T s ' o 6 and Helmkamp 5 came to the same conclusion earlier investigating homoassociation of purine and its alkyl derivatives by the osmometry method. Recently Steiner 22, also using osmometry data, has shown that heteroassociation of purine and uridine is well described by the isodesmic model. However, most of heteroassociation constants known by this time have been calculated within the assumption of complex formation with a stoichiometry of 1:1. Directly from the N.M.R. data the association constants have not been determined at all. Calculated within the isodesmic model the homo- and heteroassociation constants for the compounds studied as well as the A H and AS values, obtained from the temperature dependence of the constants are given in Table I. The free energy of association in all cases does not exceed 2-3 RT. The thermodynamic characteristics of homoassociation of I obtained from the concentration changes in its P.M.R. spectra coincide within the experimental error with those determined independently by the osmometry method 1. From the data of ref. 8 the thermodynamic parameters of homoassociation of I are equal: A H ~ - - 8 . T k c a l / m o l e . 3 S = -21.6entropyunits. It should be noted that the homoassociation constants of I and lI are considerably higher than all those known by this time for nitrous heterocycles. The
P.M.R. S T U D I E S O N N U C L E I C A C I D BASE I N T E R A C T I O N
17
homoassociation constants of I and II are 4--4,5 times higher than those of their heteroassociation with III-VI. The result obtained confirmed once again that the introduction of methyl groups considerably increases the base ability for stacking interaction (cf. ref. 10). Highly negative values of A H and AS of association show that the association of nitrous heterocycles, unlike that of nonpolar hydrocarbons, cannot be explained by "hydrophobic" interations, which are characterized by small and often positive changes in AH and AS 23-a5. The calculations taking into consideration only hydrophobic interactions as well as stabilization of base stacks, owing to a decrease of the energy of water surface tension at the complex formation 26, fail to explain the dependence of stacking interaction on chemical structure a'27. Indeed, in spite of the fact that the surfaces of dimethyl derivatives of uracil are equal their association with 6,9-dimethyladenine occurs with different AF, AH and AS. Thus association of nitrous heterocycles cannot be explained solely by water-solute interaction. As was shown by quantum-chemical calculations the nucleic acid bases form associates already in vacuum due to Van der Waals-London-stacking interactions zS. A correlation between the values of association constants and component polarizabilities (~) is observed. The constants of association diminish in the sequence: I
I
+>
+
I
III
I >
+
IV
As follows from calculations of ref. 28, ~ of adenine > ~ of cytosine > 7 of uracil. As for dipole moments (p) similar correlation has not been found: # of cytosine > # of adenine ~ ~ of uracil 28. This comparison points out that dispersion forces and interactions of dipole-induced dipole type rather than dipole-dipole interactions make the main contribution to the formation of stacked associates in water. This conforms to the calculations of ref. 28. For the compounds studied the association constants change simultaneoulsy with the values of A6 of the associating molecule protons. The value of the association constant of IV, modelling the main uracil tautomeric form, with I is bigger than that for association of IV with II. On the contrary, the association constant of V - - the model analogue of rare tautomeric form of uracil - - with II is bigger than with I. At the same time the constant of interaction of IV with I is more than V with I. As it follows from ref. 5 changing the place of alkyl substition in the pyrimidine ring has little effect on the constant of purine homoassociation. The same seems to be valid for pyrimidine derivatives. That is why the cause of observed differences in thermodynamics of heteroassociation of IV-VI is probably due to the different ability for stacking interaction of individual tautomeric forms of uracil. The results obtained show that in spite of the low specificity of stacking interaction, tautomeric and isomeric conversions of nitrous bases lead to considerable changes in thermodynamic parameters of their association.
The effect of protonation on the base ability for stacking interaction Upon acidification the 6 concentration dependence for the protons of I becomes weaker while at pEH < 2 it disappears. Evidently in a strong acid medium the homoassociates are not formed because of the mutual repulsion of cations. For
18
V.L. ANTONOVSKY
et al.
determination of quantitative characteristics of the interaction of protonated molecules with neutral ones we assumed that the equilibrium protonation constants of free and associated bases are equal. In this case the observed chemical shift of a proton is: 3 = 6Af + ( l - - f )fAn+
(9)
where A and AH + represent neutral and protonated forms of the base and f represents the fraction of neutral molecules which is equal to antilg(pZH-pK) . f = l+antilg(p2H_pK),
K -
[A-I[2H +] [_A2H+]
(10)
The value of protonation pK of I found from the sigmoid curve of" 3 dependence on pZH, taking into account the influence of ionic strength according to Debye-Hiickel, is equal to 4.0. Figs 4 and 5 give the comparison of 3 experimental values for the protons of I at different pZH values from those calculated by Eqn 9 on the assumption that the complexes of protonated molecules with neutral ones were not formed. In this case for the solution with a given pZH value and total base concentration Co, fia should be equal to the chemical shift of the corresponding proton in neutral solution having the base concentration f . Co. 3A-Values in neutral medium were taken from the concentration dependences at p2H = 8 (Fig. 1) for I. Figs 4 and 5 show that the experimental data coincidence with the calculated ones within the experimental error. The similar coincidence of experimental and calculated g-values was also found for purine 29 ranging from 0-6 p2H units, at the concentration interval of" 0.075-0.4 mole/1. i i
T
i
l
l
870 860 IJO
s~o 830 ~E '-~ 820 810 -~ 800 420: 410 400
l
~
i
I
I
i
I
l
870
H-2
-rt,n 850
~
H-8
Z E
~
'
~
830
CH3 8~0
0,9.5
~0 CH3 i
2 34
t
J
56
i
J
o J
7 8
"-d 790
g
I
2
[
plI pD Fig. 4. Dependence of chemical shifts of the protons of 0.05 M 6,9-dimethyladenineupon p2}{ (pD) in 2FIzO, at 34 °C. Points, experimental; curves, calculated. Fig. 5. Dependence of chemical shifts of H-2 proton of 6,9-dimethyl adenine upon pZH (pD) in ZH20 at 34 °C. C), 0.05 M; A, 0.25 M; points, experimental; curves, calculated. GMDS, hexamethyldisiloxane.
P.M.R. STUDIES ON NUCLEIC ACID BASE INTERACTION
19
The resonance signal position for IV in the range of 0-10 p2H units remains constant and corresponds to the molecular form of the heterocycle*. In the presence of I the 6 dependence for the protons of IV on p2H has the shape of a sigmoid curve. The inflexion point of the curve corresponds to the p2H value equal to the value of protonation pK of I. In a mixture of IV (B) with I (A), fib of the protons of IV is determined by: ~B
B
B
B
B
B
B
(11)
where ~B+B, ~ + B
are the chemical shift and the fraction of B proton in the A+B complex, respectively. On the assumption that fA+B = 0 the 6B value for the solution with a given p2H and total I concentration Co should be equal to the chemical shift of the corresponding IV proton in a neutral mixture with total I concentration fco. So calculated curves coincide with the experimental data within the error of the experiment (Fig. 6). I
I
i
i
,
~ 780 "~ ~
770~.
~c~
~.~ b~O o
d~
600I
~'~-~o
. . . . . . 2 5 4 5
6
p]) Fig. 6. Dependence of chemical shifts of the protons of 0.1 M 1,3-dimethyl uracil in the presence of 0.25 M 6,9 -dimethyladenine upon p'H. (pD) in 2HzO at 34 °C. Points, experimental; curves, calculated. GMDS, hexamethyldisiloxane.
Another evidence for the independence of the ionization and association processes is the coincidence of pK values obtained from ionization curves at different concentrations in the studied concentration range with an accuracy of ~0.1 l-mole -1 (Fig. 5). Thus the obtained data show that in aqueous solutions of compounds studied the stacking interaction of neutral molecules with cations is considerably weaker than between the neutral molecules themselves. Protonation causes redistribution of electronic density in the molecule, which may result in the weakening of the dispersion forces which are primarily responsible for the stacking interaction of nitrous heterocycles in vacuurn 3 o. Unfortunately at the present time there are no experimental data and calculations permitting description of electronic density distribution in cations of the nucleic acid bases. In water, the diminution of the coordination number both for a stack formed from neutral molecules and that containing a cation is approximately equal. At the * Ionization pK of IV as determined spectrophotometrically is equal to --2.07.
20
v . L . ANTONOVSKY et aL
s a m e t i m e t h e i n t e r a c t i o n o f w a t e r m o l e c u l e s w i t h a c a t i o n is s t r o n g e r t h a n w i t h a n e u t r a l h e t e r o c y c l e a n d c o n s e q u e n t l y the e n t h a l p y c o m p o n e n t d o e s n o t f a v o u r s t a c k f o r m a t i o n o f a s s o c i a t e - c o n t a i n i n g c a t i o n s a n d n e u t r a l m o l e c u l e s (see refs 31 a n d 32).
REFERENCES 1 Molecular Association in Biology (1968) (Pullman, B., ed.), Academic Press, Paris 2 Chan, S. I., Schweizer, M. P., Ts'o, P. O. P. and I-Ielmkamp, G. K. (1964) Jr. Am. Chem. Soc. 86, 4182 3 Jardetzky, O. (1964) Biopolymers Symposia I, 50l 4 Broom, A. D., Schweizer, M. P. and Ts'o, P. O. P. (1967) J. Am. Chem. Soc. 89, 3612 5 Helmkamp, G. K. and Kondo, N. S. (1967) Biochim. Biophys. Acta 145, 27 6 Tso, P. O. P., Melvin, I. S. and Olson, A. C. (1963) J. Am. Chem. Soc. 85, 1289 7 Gill, S. I., Downing, M. and Sheats, G. F. (1967) Biochemistry 6, 272 8 POrschke, D. and Eggers, F. (1972) Eur. J. Biochem, 26, 190 9 Ts'o, P. O. P. and Chan, S. I. (1964) J. Am. Chem. Soc. 86, 4176 10 Nakano, N. [. and Igrashy, S. I. (1970) Biochemistry 9, 577 11 Robbins, R. K. and Lin, H. H. (1957) J. Am. Chem. Soe. 79, 490 12 Taylor, E. C. and Loeffler, P. K. (1960) J. Am. Chem. Soc. 82, 3147 13 Kenner, G. W., Reese, C. B. and Todd, A. R. (1955) J. Chem. Soc., 855 14 Davidson, D. and Baudish, O. (1926) J. Am. Chem. Soc. 48, 2379 15 Hilbert, G. E. and Johnson, T. B. (1930) J. Am. Chem. Soc. 52 2001 16 Bates, R. G. (1964) Determination o f p H , John Wiley and. Sons Inc., New York 17 Bullock, F. J. and Jardetzky, O. (1964) J. Or#. Chem. 29, 1968 18 Matsura, S. and Goto, T. (1963) Tetrahedron Lett. 1499 19 Schweizer, M. P., Chan, S. I. and Ts'o, P. O. P. (1965) J. Am. Chem. Soc. 87, 5241 20 Giessner-Prettre, C. and Pullman, B. (1965) C.R. Acad. Sci. Paris 261, 2521 21 Nakano, M., Nakano, N. and 14iguchi, T. (1967) J. Phys. Chem. 21, 3955 22 Steiner, R. F. (1971) Biochim. Biophys. Acta 247, 22 23 Frank, 14. S. and Evans, M. W. (1945) J. Chem. Phys. 13, 507 24 Kauzman, W. (1959) Adv. Protein Chem. 14, 1 25 Nemethy, G, and Scheraga, 14. A. (1962) J. Phys. Chem. 66, 1773 26 Sinanoglu, O. and Abdulnur, S. (1965) Fed. Proc. 24, Suppl. 15, S-12 27 Crothers, D. M. and Ratner, D. I. (1968) Biochemistry 7, 1823 28 Pullman, B. (1965) J. Chem. Phys. 43,233 29 Gukovskaya, A. S., Tchervin, I. I., Sukhorukov, B. I. and Antonovsky, V. L. (1973) Biophisika 18, 557 30 De Voe, 14. and Tinoco, I. (1962) J. MoL BioL 4, 500 31 Sinanoglu, O. and Abdulnur, S. (1964) Photochem. PhotobioL 3, 333 32 Claverie, P. and Pullman, B. (1966) J. Theor. BioL 12, 419
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 97844
S T U D I E S OF I N T E R M O L E C U L A R I N T E R A C T I O N S OF N U C L E I C A C I D BASES I N A Q U E O U S S O L U T I O N S BY T H E P R O T O N M A G N E T I C RESONANCE METHOD S E L F - A S S O C I A T I O N OF 6 , 9 - D I M E T H Y L A D E N I N E A N D ITS P Y R A Z O L E ANALOGUE AND THEIR ASSOCIATION WITH METHYL-SUBSTITUTED CYTOSINE AND URACIL*
V. L. ANTONOVSKY, A. S. GUKOVSKAJA, G. V. NEKRASOVA, B. I. SUKHORUKOV and I. I, TCHERVIN Institute of Biochemistry and Physiology of Microorganisms, U.S.S.R. Academy of Sciences, Pustchino on the Oka, Moscow re#. (U.S.S.R.)
(Received May 15th, 1973)
SUMMARY To study intermolecular interaction the concentration and temperature dependence of chemical shifts of the protons of 6,9-dimethyladenine (I), its pyrazole analogue I-methylaminopyrazolo-3,4-d pyrimidine (II), as well as 1,4-dimethylcytosine and dimethyl derivatives of uracil, modelling the main and rare tautomeric forms of uracil: 1,3-; 1,4-; 2,4-dimethyluracils, both separately and in mixtures have been investigated. A conclusion has been made about homoassociation in solutions of I and I I as well as heteroassociation of I and II with pyrimidine derivatives into complexes, having the stacked structure. From the differences in values of the chemical shifts, which are dependent on concentration, for the protons of six-membered and five-membered cycles the authors came to conclusions about the conformation in the homoassociates of I and II. Association of studied compounds is not limited to the dimer formation, but is well described by the isodesmic model. Thermodynamic characteristics of association have been obtained and discussed. A correlation between equilibrium association constants and component polarizability has been revealed. Taking homoassociation of I and heteroassociation of I with IV as examples, it has been established, that in 2H20 stacking interaction of cation with cation and cation with neutral molecule is considerably weaker than the interaction between neutral molecules.
* Separate parts of the work were reported on the IH All-Union conference on the investigation of structures of organic compounds by physical methods, Kazan (1971) and on the IV International Biophysics Congress, Moscow (1972).
10
V . L . A N T O N O V S K Y et aL
INTRODUCTION
The investigation of the association of nucleic acid bases and their derivatives in solution is of great interest for the elucidation of the nature of forces stabilizing the DNA double helix 1. Hydrogen-bonded complexes of the nucleic acid bases are formed in organic solvents, whereas in aqueous media the association of the bases takes place due to interplanar stacking interaction which is favoured by strong solvent-solvent interactions. P.M.R. investigation of the chemical shift concentration dependence for nucleic acid-component protons in aqueous solutions has permitted the establishment of the stacked structure of the associates 2'3 as well as mutual orientation of the molecules in complexes of purine nucleosides and bases4' 5. With the help of the N.M.R. method, thermodynamic parameters of association have not been determined and the interaction of different groups and molecules in associates has been evaluated only qualitatively. The quantitative data of homoassociation of nitrous bases and their derivatives were obtained mainly by the vapour pressure osmometry method 4- s and the data of heteroassociation was obtained by solubility methods 9' lo. In the present work we have made an attempt to use the P.M.R. method for defining both the quantitative and qualitative characteristics of the nucleic acid bases stacking interaction. As the bases of nucleic acids may be subjected to tautomeric conversions which must considerably change the stacking interaction ability we have studied homoassociation of 6,9-dimethyladenine (I) modelling the main tautomeric form of adenine and heteroassociation of I with 1,4-dimethylcytosine (III) and uracil derivatives, modelling the main (diketo-) and rare (enol-) forms of uracil: 1,3-dimethyluracil (IV), 1,4-dimethyluracil (V), 2,4-dimethyluracil (VI). To evaluate the role of the isomeric conversion homoassociation of the pyrazole analogue of adenine: l-methyl,4-methylaminopyrazolo-[3,4-d]-pyrimidine (I1) and its heteroassociation with IV, V and V1 has been studied. The protonation of bases must influence their stacking interaction but up to now this question has not been studied. We have investigated the influence of medium acidity on the processes of self-association of I and heteroassociation of I with IV. Studies of the influence of isomerism, tautomerism and ionisation of the nucleic acid bases on their stacking interaction ability are also very important in connection with elucidation of the nature of spontaneous mutations. M A T E R I A L S A N D METHODS
The initial heterocyclic compounds have been synthesized and identified: I according to the method of mentioned in ref. 11; II, ref. 12; III, ref. 13; IV, ref. 14; V and VI, ref. 15. The medium pH values were measured directly in the N.M.R. spectrometer ampule on a LPU-01 potentiometer with the help of the glass electrode for micromeasurements, taking into account that p2H--pH-meter indication +0.4 (ref. 16). The measurements were carried out in buffer solutions having constant ionic strength 0.1. P.M.R. spectra were recorded on the "Varian" HA-100D spectrometer at the
P.M.R. STUDIES ON NUCLEIC ACID BASE INTERACTION
11
working frequency 100 MHz. The values of chemical shifts (iS) of resonance signals were expressed in Hz from hexamethyldisiloxane as an external reference. Shifts at the infinite dilution were found by graphical extrapolation of chemical shifts measured at 100 °C to zero base concentration. Shifts were corrected for bulk susceptibility by the application of both the external hexamethyldisiloxane and internal tert-butanol or acetone. The difference in values of the chemical shift concentration dependence between external and internal compartments does not exceed 4 Hz. The results are not due to an internal compartment base interaction because the temperature-dependent shifts of the bases were identical with added acetone or tert-butanol. RESULTS AND DISCUSSION Peak assionment Four peaks in the spectra of compounds studied have been observed. The peaks of III-VI were assigned to H-5 and H-6 according to ref. 17. The H-2 and H-8 peaks of I were differentiated on the basis of H-8 proton ability to exchange at elevated temperatures18: when heated to 100°C the intensity of the H-8 peak drops considerably. The exchange of the H-3 proton of II with 2HzO is already observed at 34 °C. Homo- and heteroassociation in aqueous solutions Concentration changes of P.M.R. spectra were studied at the interval of 0-0.4 M at temperatures from 34--100 °C. At a constant temperature proton resonances in I and II are all shifted to higher fields as the solute concentration is increased (Fig. 1). The same upfield shifts are noticed for pyrimidine-derivative protons in binary mixtures with I or II when the concentration of the latter increases (Fig. 2). When temperature increases the resonance signals of I and II protons as well as of pyrimidine-derivative protons in mixtures with I or II are shifted to low fields. The temperature dependence of 6 of I or II protons in one-component systems and of III-VI protons in the mixtures with I or II is a linear one. The observed concentration and temperature changes in P.M.R. spectra are manifested by ring current magnetic anisotropy effects and point out that: (1) both homoassociation of I and II and their association with pyrimidine derivatives take place in aqueous solution, (2) homo- and heteroassociates have a stacked structure 2,3,19. At the same time the concentration and temperature changes in solutions, containing only the pyrimidine derivatives, do not shift the resonance signals of III-IV protons more than of 4 Hz. The results obtained are conditioned by the low diamagnetic anisotropy of cytosine and uracil rings 2° and by the weak ability of pyrimidine derivatives for stacking interaction 6. The lack of a concentration effect in 2,4-dimethoxy-uracil spectra is evidently caused by the induction effect of the methoxy groups. Fig. I shows the convergence of H-2 and H-8 resonance signals in the spectra of I, while at the homoassociation of II peaks of H-6 and H-3 protons diverge. It should be noted that the values of concentration and temperature shifts of pyrimidine-ring protons, 6, are more than those of the imidazole ring for all
12
V. L, ANTONOVSKY e t a / . 850
810
~
77(] 75(1 420
380
N ;~" I~ X
790 ?80 770 ?60 7N)
E
sl°i
,~
600
1,3
~60
~'40I, '
H-6
~7o~
3310
o.1
0.2
03
0-4
0.~
0,2
0.3
M
M
Fig. 1. The concentration dependence of chemical shifts of the protons of 6,9-dimethyladenine ( 0 , H-2; ~ , H-8; [], -CH3) and I-methyl, 4-methylaminopyrazolo [3,4-d]-pyrimidine ( 0 , H-6, A, H-3, II, CH3) in 2H20 at 34 °C. GMDS, hexamethyldisiloxane. Fig. 2. Dependence of chemical shifts of the protons of 0,1 M 1,3-dimethyluracil upon concentration of 6,9-dimethyl-adenine in 2HzO at 34 °C.
purine derivatives so far studied. This testifies to the predominant contribution of the associate conformations in which the H-2 proton is more shielded than H-8 (ref. 4). The average conformation of the homoassociate of I is apparently very close to the proposed one 4 for the homoassociate of adenosine. For I1 the concentration shift (A¢5) of the pyrazole ring proton (H-3) 6 is more considerable than that for the pyrimidine ring proton (H-6). It is due to the fact that isomery in the five-membered ring changes the associate geometry and as a result the five-membered rings of II are overlapped to a greater extent then in the homoassociate of I. At heteroassociation of I or II with III-VI (Table I) the value of the ~ upfield shift for the pyrimidine-derivative H-5 proton is 2-6 Hz higher, than that for the H-6 proton. We consider that the difference observed testifies to the order of heteroassociation, the main contribution to the interaction being made by the stack conformation in which A6 H-5 > A6 H-6. The chemical slaifts of I or II protons are displaced to low fields in mixtures of pyrimidine derivatives with I or II with a constant concentration of the latter when the concentration of the magnetically-inert pyrimidine derivative is increased. The dependence of the chemical shifts of I or II protons on the pyrimidine derivative concentration is linear (Fig. 3). The observed low-field displacement is caused by a decrease in the share of the homoassociates of I or II when the pyrimidine derivative concentration is increased.
Concentration o f l or H (mole/I )
0 0.4 0 0.4 0 0.4 0 0.4
Compounds
1,4-Dimethylcytosine 1,3-Dimethyluracil 1,4-Dimethyluracil 2,4-Dimethyluracil
H-6
CHa
613 767 360 580 740 346 612 783 364 586 761 350 642 810 417 620 789 406 682 848 420 647 817 400 H'omoassociation o f i
H-5 311 298 355 334 372 361 420 402
CHa
Chemical shift; H z , 34 °C
Association with I
6.2 4.5 4.3 6.0 8.2
6.0 4.3 9.2 40.0
--zlH kcal/ mole
10.0
K I/mol
T h e relative error o f K a n d K" d e t e r m i n a t i o n = 10 %, A H = 7 %.
21.0
15.7
11.5
11.7
16.5
--AS entropy units 1-[-6
CH3
612 783 364 591 767 354 642 810 417 614 785 503 682 848 420 647 818 401 H o m o a s s o c i a t i o n o f II
11-5
355 347 372 351 420 401
CH3
Chemical shift; H z , 34 °C
Association with I1
4.0 4.8 6.0 10.5
3.1 7.4 9.0 51.0
27.4
15.8
12.0
11.7
K" -- A H -- A S 1~mole kcal/ entropy mole units
DEPENDENCE OF PROTON CHEMICAL SHIFT VALUES FOR THE PROTONS OF URACIL AND CYTOSINE DERIVATIVES UPON T H E C O N C E N T R A T I O N O F 6 , 9 - D I M E T H ' Y L A D E N I N E (I) A N D I - M E T H Y L , 4 - M E T I ~ Y L A M I N O P Y R A Z O L O - [ 3 , 4 - d - ] P Y R I M I D I N E (II) I N 2/420 A N D T H E T H E R M O D Y N A M I C P A R A M E T E R S O F A S S O C I A T I O N
TABLE I
Z
,--t
,-t m
m
r~
>
© Z Z
~o
14
V . L . ANTONOVSKY et al.
810 j ul 8O0 7go o 78G
H
-
2
H-8
E
380 .¢:
330 m
.,o..,,..,.,,~,-,~CH5
320
JlO
M Fig. 3. Dependence of chemical shifts of the protons of 0.25 M 6,9-dimethyladenine upon concentration of 1,3-dimethyluracil in 2HzO at 34 °C. GMDS, hexamethyldisiloxane.
Calculation method for association constants For the quantitative analyses of association processes we considered the model of stepped association: K1 A2
Aa +B 1 K'~ A~ B~
A2 +As r~ A3
A2 + B , r'~ A2 B1
A , + A 1 K~n A,+I
A , + B 1 r'" ~_ A, B 1
A1 +At
Here A corresponds to I or II and B to III-V1. The interaction of the pyrimidine derivatives (B-B) may be neglected because it is weaker compared with A - A and A-B interactions. Let us consider two cases• (1) The complexes are formed with a number of molecules not more than two: K1 = K; t
KI=K
Kz = K3 . . . .
t.
¢
,
K2 =K
t 3
=
• • •
=0
=0
(2) There are complexes in solution which contain an arbitrary number of molecules. The constant of m o n o m e r attachment to the associate does not depend on the number of molecules in the complex (the isodesmic model)• For the first case the association constants are equal, respectively, K =
[A2] ([Ao] - 2[A2] - [AB]) 2 '
FA, B~]
K' =
(1)
([A0] -2[A2]- [A, B,])([Bo]- [As B,]) where [Ao] and [Bo] are the total concentrations of A and B. When B is absent the observed chemical shift of A proton (6 A) will be: 6A
A A A A = aM fr~ + ~D fl~ ;
l
or
[Ao]
_ _ -_ _- 3 A 2AA[AE]
(2)
P.M.R. STUDIES ON NUCLEIC
ACID BASE INTERACTION
15
where fA, foA, 6~ and 6D A are the molar fractions and the chemical shifts of protons in monomer (M) and dimer (D), respectively. AA
=
(~A_ (~A ;
AD A
A A = (~D - - 3M
Taking into consideration Eqn l, {-Ao] _
1 + 2([-go]- [-A2] ) 2KAAD AA
AA
(3)
When B is present we have correspondingly
[Ao] _ AB
1 + [Ao] + [Bo] - 2EA2] - [AB] K'A~u ABA~
(4)
K and d A are found from Eqn 3. Neglecting [AB] in Eqn 4 at the concentrations [Bo] < [Ao] and using [A2] from Eqn 2, we can calculate the approximate values of K' and ABAWThe accurate values can be found with te help of an iteration procedure by analogy with ref. 21. In the second case the stoichiometric molarity of A and B can be expressed as a sum of power series (when K[A1] < 1): ['Bo] = ~ [ a n B 1 ] = [ B I ] + K'[BI] ~ K n - ' [ a l ] n n=O
n=l
g[A,]
[
= [ a , ] 11 +
\
I
- -
K[All]
[Ao] -- ~ n([An]+[AnB,] ) = ~ nKn-I[Ai]n+K'['B,] ~ nKn-'EA,] n n=l
n=l
n=l
[AI](I + K'[BI]) (t-K[A,]) 2
(5)
Within the isodesmic model
t~A: ~AM J~rA..~ ~ t~A i'A..~~ (~AaB A JAnB 'A M An JAn n=2
n=l
e a 6 B = 6~ f~tB + ~~ 6A~B f~nB
(6)
n=l
6AAnand f A n represent the chemical shift and the molar fraction of A protons in A. (n >1 2) complex respectively; 6aAnS,f;.nBA,6A~Baand J)~.B -- the same for A and B protons in the A.B complex (n >/ 1). Let us assume that the chemical shift of a proton in associates does not depend on the number of A molecules in the stack:
aL
aL . . . . .
a~,~= a~=~:
aL . . . . . . .
~ " = ~AnB
=
....
~BAB
In addition as a result of B magnetic inertness: ~AAnB ----- 0An. "A
16
V.L.
ANTONOVSKY
et al.
Then zjB
=
co zjBBE n=l
B
.
AA
A A E (f;~ A_~_ f~.a) n=2
=
(7)
where AaAss
-A
A.
zjBB
B
B
Using Eqns 5 and 6 we transform Eqn 7: AA
----
l
([Ao](tK+(l-)K
'
h
)-t}A.~ _[Bo ]
(tK+(1-0K')[ao]
K't(l
--
A~ t)Aas
(tK + (1 - t)K')[Ao]
(8)
where t
=
3B/A]B
Unknown association constants and chemical shifts are found from the parameters of Eqn 8 at several values of [A0] and [Bo].
Thermodynamic parameters of association The calculation of association constants, based on the assumption that only bimolecular complexes are present in solution (Eqns 3 and 4), gives a considerable scattering ( > 100 %) in K and K' values, obtained from concentration dependences for different protons of each compound. It points out that the association of the compounds studied is not limited to the formation of dimers. The relative error for K and K' values calculated from 5 concentration dependences for different protons within the isodesmic model does not exceed +_ 10 % for all studied systems. Besides, for the pyrimidine derivatives we have studied, 58 does not change practically when [Bo] is changed at a constant [Ao]. In tis case Eqn 8 gives a linear dependence of Aa on [Bo] with a negative tangent. It is in good agreement with our experimental data (Fig. 3) and those obtained previously 19. Thus all results obtained show that the association of the studied compounds is well described by the isodesmic model. T s ' o 6 and Helmkamp 5 came to the same conclusion earlier investigating homoassociation of purine and its alkyl derivatives by the osmometry method. Recently Steiner 22, also using osmometry data, has shown that heteroassociation of purine and uridine is well described by the isodesmic model. However, most of heteroassociation constants known by this time have been calculated within the assumption of complex formation with a stoichiometry of 1:1. Directly from the N.M.R. data the association constants have not been determined at all. Calculated within the isodesmic model the homo- and heteroassociation constants for the compounds studied as well as the A H and AS values, obtained from the temperature dependence of the constants are given in Table I. The free energy of association in all cases does not exceed 2-3 RT. The thermodynamic characteristics of homoassociation of I obtained from the concentration changes in its P.M.R. spectra coincide within the experimental error with those determined independently by the osmometry method 1. From the data of ref. 8 the thermodynamic parameters of homoassociation of I are equal: A H ~ - - 8 . T k c a l / m o l e . 3 S = -21.6entropyunits. It should be noted that the homoassociation constants of I and lI are considerably higher than all those known by this time for nitrous heterocycles. The
P.M.R. S T U D I E S O N N U C L E I C A C I D BASE I N T E R A C T I O N
17
homoassociation constants of I and II are 4--4,5 times higher than those of their heteroassociation with III-VI. The result obtained confirmed once again that the introduction of methyl groups considerably increases the base ability for stacking interaction (cf. ref. 10). Highly negative values of A H and AS of association show that the association of nitrous heterocycles, unlike that of nonpolar hydrocarbons, cannot be explained by "hydrophobic" interations, which are characterized by small and often positive changes in AH and AS 23-a5. The calculations taking into consideration only hydrophobic interactions as well as stabilization of base stacks, owing to a decrease of the energy of water surface tension at the complex formation 26, fail to explain the dependence of stacking interaction on chemical structure a'27. Indeed, in spite of the fact that the surfaces of dimethyl derivatives of uracil are equal their association with 6,9-dimethyladenine occurs with different AF, AH and AS. Thus association of nitrous heterocycles cannot be explained solely by water-solute interaction. As was shown by quantum-chemical calculations the nucleic acid bases form associates already in vacuum due to Van der Waals-London-stacking interactions zS. A correlation between the values of association constants and component polarizabilities (~) is observed. The constants of association diminish in the sequence: I
I
+>
+
I
III
I >
+
IV
As follows from calculations of ref. 28, ~ of adenine > ~ of cytosine > 7 of uracil. As for dipole moments (p) similar correlation has not been found: # of cytosine > # of adenine ~ ~ of uracil 28. This comparison points out that dispersion forces and interactions of dipole-induced dipole type rather than dipole-dipole interactions make the main contribution to the formation of stacked associates in water. This conforms to the calculations of ref. 28. For the compounds studied the association constants change simultaneoulsy with the values of A6 of the associating molecule protons. The value of the association constant of IV, modelling the main uracil tautomeric form, with I is bigger than that for association of IV with II. On the contrary, the association constant of V - - the model analogue of rare tautomeric form of uracil - - with II is bigger than with I. At the same time the constant of interaction of IV with I is more than V with I. As it follows from ref. 5 changing the place of alkyl substition in the pyrimidine ring has little effect on the constant of purine homoassociation. The same seems to be valid for pyrimidine derivatives. That is why the cause of observed differences in thermodynamics of heteroassociation of IV-VI is probably due to the different ability for stacking interaction of individual tautomeric forms of uracil. The results obtained show that in spite of the low specificity of stacking interaction, tautomeric and isomeric conversions of nitrous bases lead to considerable changes in thermodynamic parameters of their association.
The effect of protonation on the base ability for stacking interaction Upon acidification the 6 concentration dependence for the protons of I becomes weaker while at pEH < 2 it disappears. Evidently in a strong acid medium the homoassociates are not formed because of the mutual repulsion of cations. For
18
V.L. ANTONOVSKY
et al.
determination of quantitative characteristics of the interaction of protonated molecules with neutral ones we assumed that the equilibrium protonation constants of free and associated bases are equal. In this case the observed chemical shift of a proton is: 3 = 6Af + ( l - - f )fAn+
(9)
where A and AH + represent neutral and protonated forms of the base and f represents the fraction of neutral molecules which is equal to antilg(pZH-pK) . f = l+antilg(p2H_pK),
K -
[A-I[2H +] [_A2H+]
(10)
The value of protonation pK of I found from the sigmoid curve of" 3 dependence on pZH, taking into account the influence of ionic strength according to Debye-Hiickel, is equal to 4.0. Figs 4 and 5 give the comparison of 3 experimental values for the protons of I at different pZH values from those calculated by Eqn 9 on the assumption that the complexes of protonated molecules with neutral ones were not formed. In this case for the solution with a given pZH value and total base concentration Co, fia should be equal to the chemical shift of the corresponding proton in neutral solution having the base concentration f . Co. 3A-Values in neutral medium were taken from the concentration dependences at p2H = 8 (Fig. 1) for I. Figs 4 and 5 show that the experimental data coincidence with the calculated ones within the experimental error. The similar coincidence of experimental and calculated g-values was also found for purine 29 ranging from 0-6 p2H units, at the concentration interval of" 0.075-0.4 mole/1. i i
T
i
l
l
870 860 IJO
s~o 830 ~E '-~ 820 810 -~ 800 420: 410 400
l
~
i
I
I
i
I
l
870
H-2
-rt,n 850
~
H-8
Z E
~
'
~
830
CH3 8~0
0,9.5
~0 CH3 i
2 34
t
J
56
i
J
o J
7 8
"-d 790
g
I
2
[
plI pD Fig. 4. Dependence of chemical shifts of the protons of 0.05 M 6,9-dimethyladenineupon p2}{ (pD) in 2FIzO, at 34 °C. Points, experimental; curves, calculated. Fig. 5. Dependence of chemical shifts of H-2 proton of 6,9-dimethyl adenine upon pZH (pD) in ZH20 at 34 °C. C), 0.05 M; A, 0.25 M; points, experimental; curves, calculated. GMDS, hexamethyldisiloxane.
P.M.R. STUDIES ON NUCLEIC ACID BASE INTERACTION
19
The resonance signal position for IV in the range of 0-10 p2H units remains constant and corresponds to the molecular form of the heterocycle*. In the presence of I the 6 dependence for the protons of IV on p2H has the shape of a sigmoid curve. The inflexion point of the curve corresponds to the p2H value equal to the value of protonation pK of I. In a mixture of IV (B) with I (A), fib of the protons of IV is determined by: ~B
B
B
B
B
B
B
(11)
where ~B+B, ~ + B
are the chemical shift and the fraction of B proton in the A+B complex, respectively. On the assumption that fA+B = 0 the 6B value for the solution with a given p2H and total I concentration Co should be equal to the chemical shift of the corresponding IV proton in a neutral mixture with total I concentration fco. So calculated curves coincide with the experimental data within the error of the experiment (Fig. 6). I
I
i
i
,
~ 780 "~ ~
770~.
~c~
~.~ b~O o
d~
600I
~'~-~o
. . . . . . 2 5 4 5
6
p]) Fig. 6. Dependence of chemical shifts of the protons of 0.1 M 1,3-dimethyl uracil in the presence of 0.25 M 6,9 -dimethyladenine upon p'H. (pD) in 2HzO at 34 °C. Points, experimental; curves, calculated. GMDS, hexamethyldisiloxane.
Another evidence for the independence of the ionization and association processes is the coincidence of pK values obtained from ionization curves at different concentrations in the studied concentration range with an accuracy of ~0.1 l-mole -1 (Fig. 5). Thus the obtained data show that in aqueous solutions of compounds studied the stacking interaction of neutral molecules with cations is considerably weaker than between the neutral molecules themselves. Protonation causes redistribution of electronic density in the molecule, which may result in the weakening of the dispersion forces which are primarily responsible for the stacking interaction of nitrous heterocycles in vacuurn 3 o. Unfortunately at the present time there are no experimental data and calculations permitting description of electronic density distribution in cations of the nucleic acid bases. In water, the diminution of the coordination number both for a stack formed from neutral molecules and that containing a cation is approximately equal. At the * Ionization pK of IV as determined spectrophotometrically is equal to --2.07.
20
v . L . ANTONOVSKY et aL
s a m e t i m e t h e i n t e r a c t i o n o f w a t e r m o l e c u l e s w i t h a c a t i o n is s t r o n g e r t h a n w i t h a n e u t r a l h e t e r o c y c l e a n d c o n s e q u e n t l y the e n t h a l p y c o m p o n e n t d o e s n o t f a v o u r s t a c k f o r m a t i o n o f a s s o c i a t e - c o n t a i n i n g c a t i o n s a n d n e u t r a l m o l e c u l e s (see refs 31 a n d 32).
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