Effect of protonation on electronic structure of guanosine and 5′-guanosine monophosphate and on glycosidic (CN) bond rotations

BIOPOLYMERS VOL. 13, 289-306 (1974)

Effect of Protonation on Electronic Structure of Guanosine and 5 '-Guanosine Monophosphate

and on Glycosidic (C-N) Bond Rotations

FRANK JORDAN, Department of Chemistry, Rutgers University, Newark, New Jersey 07106

synopsis Semiempirical molecular orbital studies were performed on 5'-guanosine monophos-

phate in its various states of base and phosphate ionization (employing the extended Huckel method) and on guanosine protonated on N-3 and on N-7 (employing CNDO/2). Semiempirical potential energy calculations (Lennard-Jones and Coulombic) failed to pinpoint the reasons for the recent experimental suggestion that guanine protonation increases the syn population in 2'- and 3'-guanosine monophosphates. Changes in electron densities upon base protonation are much in evidence and are a very sensitive function of the site of base protonation assumed. It is suggested that the CNDO/%type calculations when combined with '8C and 15N pHdependent chemical shifts can lead to assignment of the site of protonation in the DNA bases.

INTRODUCTION

We have recently reported calculations on the effect of base protonation on DNA base electronic structures and intermolecular interactions. Accord- ing to self-consistent field (SCF) methods (CND0/2 and MINDO), protonation affects not only all atomic electron densities (both u and r) but also influences ionization potentials. The protonated base invariably has a larger ionization potential than the neutral one.

Son et a1.2 reported that (based on 'H nuclear Overhauser enhancement measurements) protonation has an effect on the syn-anti conformational equilibrium in the guanosine monophosphates. This finding was of interest to us because of our previous work on the syn-anti conformational problem of nucleotides and nucle~sides.~-~

Studies on some adenosine mono phosphate^^ have shown that the base protonation effects are mostly confined to the base; there is essentially no charge transfer between the base and sugar phosphate.

While the present work was completed, two related studies appeared in the literature. Berthod and Pullman6 considered the syn-anti conformational problem in guanosine monophosphates (GMP) employing a molecular orbital method. Yathindra and S~ndaralingarn~ performed semiempirical potential barrier calculations on 5'-adenosine monophosphate employing a method also used in this paper. Neither of the above contribu-

289

@ 1974 by John Wiley & Sons, Inc.

290 JORDAN

tions considered the possible effects of base protonation or phosphate ionization on the barrier.

In a contribution on the 13C nmr spectra of purines (some of which were positively charged) the authors reported a good qualitative agreement between chemical shifts and CNDO/2 electron densities.* While in the solid state the site of base protonation is well e~tablished,~ in solution it has not been unequivocally assigned. In the present study an attempt is made to calculate the electronic structures of guanosine protonated at two likely sites to see if the site of protonation is reflected by atomic charge densities.

THEORETICAL METHODS

Due to the size of the molecules two different approaches had to be employed. Calculations on 5'-GMP as a function of base and phosphate ionization were performed using the Boyd and Lipscomblo parametrization of the extended Huckel theory. This approach has been shown to lead to dipole moments in reasonable agreement with experimental value^.^*^*'^ One may expect, however, that for charged species, an SCF theory is more satisfactory. This latter approach was applied to protonated guanosines in the form of the CNDO/2 theory."

The atomic charge densities are smaller according to CND0/2 than according to the extended Huckel procedure. The CNDO/2 net atomic charges when combined with monoatomic overlap charges (hybrid moments) lead to satisfactory dipole moments in the neutral DNA bases.'2 Furthermore, the more subtle effects of base protonation, such as those on the ionization potential, would not be reflected by the simpler non-SCF method.

The potential function for rotating the base around the ribose phosphate is a composite one. It includes a Lennard-Jones (L-J) 6-12 type potential for the nonbonded potential energy along with a Coulombic term6J (i.e., Q = kq,q,/ertj). The latter contains the net atomic charges in the ribose and base, respectively, the distance rt5 separating the atoms i and j , the dielectric constant r , and a numerical constant k. The value of the dielectric constant employed is subject to argument. Following the suggestion of others, in a previous contribution B of 4 was used.5 Much to our surprise it was found that the electrostatic term had no significant effect on the shape of the potential energy curve for syn-anti equilibria in molecules as highly polar as the adenosine mononucleotide zwitterions (base protonated, phosphate monobasic) which possess a calculated dipole moment of 22-27 D.

In order to exaggerate the electrostatic effect, in the present study an r of 1 is employed. With a larger, perhaps more realistic, r , the Q contributions would become smaller.

Next, the question of the choice of the geometric model needs to be mentioned. The crystal structure of 5'-GMP has been determined.13 The ribose was found to have a C-3' endo c~nformation;'~ the conformation of the ribose and sugar phosphate with respect to the C-N glycosidic linkage is

GUANOSINE AND 5'-GUO 291

anti.15 The crystal structure of guanosine has also been determined.16 There are two molecules per unit cell. One (Guo A) has a C-2'endo ribose and nearly syn glycosidic bond; the other (Guo B) has a C-1' e m ribose and is in the anti conformation range. According to both molecular orbital and L-J calculations, the Guo A and Guo B models possess relatively small, rotational barriers and their most stable conformers have very similar energies in the neutral state. It has also been demonstrated that in solution ribose undergoes rapid conformational interconversion a t room temperature with purine nucleosides apparently favoring the C-2' endo conformer. 13*17

It appears likely that in solution the energy differences among various ribose conformers are much smaller than is the barrier to syn-anti rotation. All three geometric models listed above were employed in the potential barrier calculations.

RESULTS AND DISCUSSION

Electronic Structure of 5'-GMP

The extended Huckel calculations -were performed on four different ionization states of the molecule. At neutral pH the phosphate is mainly in the dianion form and the base is neutral (BOP2-, Figure 1). As the pH is lowered to below the phosphate pK218 (6.6 a t 25"C), the phosphate becomes a monoanion (BOP-, Figure 2) . Upon further lowering of the pH the base is protonated with a pK of near 2.4.18 This ionization state is designated B +P-, Figure 3. This zwitterionic structure is undoubtedly at equilibrium

Fig. 1. 5'-GMP; BOPZ-, extended Huckel, charges at atoms, overlap populations along bonds.

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0.79 (C r O j

0.52

Fig. 2. 5'-GMP; BOP-, extended Huckel, charges at atoms, overlap populations along bonds.

0.71 \0.58 10) v

Fig. 3. 5'-GMP; B+lP; extended Hiickel, charges at atoms, overlap populations along bonds.

with a certain, if unknown, amount of the neutral species BOP", Figure 4. Supposedly, upon further lowering of the pH the phosphate becomes protonated, then the base can pick up one or two other protons (e.g., B+Po, B2+Po, B3+P0). The electronic structures of these states were not calculated but the protonated guanosine probably well represents the B+Po state.

Since the crystal structure was reported for BOP2-, the other states were designed from this one. 0-H and N-H bond distances of 1.0 A were


-0.87

1.13 n JJ 9pp.-

0.77 -0.40

0.25 0.04

n FC?

-0.39 .O -0.89

-0.43 0 0.47

0.34 c 0.50

0.78 0.m

0.77 0.38 0.72 H 0.m

0.52 p ." \ r ( o,43

0.42 H -0.69 0

Fig. 4. 5'-GMP; BOP", extended Huckel, charges a t atoms, overlap populations along bonds.

assumed. Protons onto nitrogens were constructed along the negative of the C-N-C bond bisector vector.

As anticipated from the work on adenosine mono phosphate^,^ the effects of ionization are localized according to the extended Huckel method. The localization of the effect is more pronounced in the u portion (ribose phosphate) than in the ?r aromatic base using such methods.

First, the addition of protons to the phosphate appears to have no effect on the ribose past the 0-5'atom. The phosphate dianion has seemingly two equivalent oxygens, confirming a resonance structure of the type 0-P-0, as shown by the similar bond lengths and the resulting similarity in net atomic populations and overlap population^.'^ The alkyl oxygen atom has very small negative charge and much smaller 0-P overlap populations compared to the other three 0-P bonds in the dianion.

Comparison of Figures 2 and 3 is informative in elucidating the effects of base protonation. According to the extended Huckel scheme the effect of N-7 protonation of the guanine base is confined to the imidazole portion of the ring. On the whole, the effect of protonation is more apparent on net atomic populations (charges) than on overlap populations. l9 Table I summarizes some other values for the various ionization states of 5'-GlhfP.

The absolute energy values are not very accurate and, of course, they refer to the gaseous state. Qualitatively, however, these values are sugges- tive. The zwitterion (B+P-) is predicted to be more inherently st,able than is the neutral state (BOP"). This was also found to be the case in adenosine-3'-5'-cyclic mon~phosphate.~ In the absence of solvation effects, the second phosphate ionization (P- + P") appears to be energetically less unfavorable than the first one (Po + P-), from either the zwitterion (loss of

294 JORDAN

TABLE I Calculated Properties of 5'-GMP

Orbital Dipole Ionizationc Ribose Ionization Energy8 Momentb Potential Base Phosphate

State (kcal/mole) (D ) (eV 1 Charged Charged

BOP*- - 62386 - 10.57 -0.22 -1.76 B"P- - 62485 - 10.57 -0.22 -0.77 B+P- - 62694 17.40 10.57 4-0.78 -0.77 BOPo - 62630 8.04 10.57 -0.22 +0.23

6 Sum over occupied molecular orbital energy levels multiplied by the occupation

b Based on net atomic populations.19 c Negative of the highest occupied molecular orbital energy level. d Sum of net atomic charges cleaving the molecule at the glycosidic C-N bond.

numbers.

proton from the base) or from the neutral specie (loss of proton from the phosphate).

The ionization potential appears to be independent of the ionization state, undoubtedly an artifact of the simple theory.

The dipole moment of the zwitterion B+P" is calculated to be twice as large than that of BOP". The percent increase in dipole moment on form- ing the zwitterion is much smaller than the one found in the adenosine mono phosphate^^ which showed an increase from 3 4 D in BOPo to 22-28 D in B+P-. It should be mentioned that the dipole moment of the 5'-GMP BOP" species (8.1 D) is close to all other theoretical values for these molecules and appears to be mainly due to the base3 (for example guanosine has a dipole moment of 7.9 D according to CND0/2). To this author's knowl- edge there is no experimental or theoretical suggestion in the literature concerning these enormous predicted moments for the B+P- species of both adenosine and guanosine monophosphates.

Electronic Structures of N-3-H+ and N-7-H+ Guanosines In a previous report the effects of base protonation at the base level were

discussed.' It was of interest to see what, if any, effect the ribose had on the electronic perturbations caused by the base protonation. It also appeared of interest to see if the site of proton attachment had any differential effect on base charges. Finally, the charges were needed for the Coulombic part of the barrier calculations.

There are five potential sites for base protonation in guanine: N-3, N-7, N-9, N-2 (amino), and the lactam oxygen. Based on the crystal structure data in the literature the N-2 amino and N-9 (site of ribose attachment) could be eliminated. Based on analogies with amides, the lactam oxygen probably requires a much stronger acid for protonation than the 2.4 pK would imply. N-3 and N-7 are probably the most likely sites, in fact N-7 is the only one with solid-state evidence for its exi~tence.~

Sta.rting with the crystallographic guanosine A geoometry, a proton was attached to N-3 and N-7, respectively, using 1.00 A for the N-H bond lengths .


n

-0.12

0.17 -0.20 0.00

I -0.21 0.14 I .

-0.17

0.204 @ 0.18

0 .I8 0 .I8 0.19

/&y&20 -0.10

0.12 -0.07 0.08 p 0.26 \ - _- ..

-0.23 -0.23

-0.01 H -0.01 \;> 0.02

0.14

Fig. 5. CNDO/P atomic charges on guanosine A; N-3-H+ guanosine A and N-7-H+ guanosine A; from top to bottom.

Figure 5 presents the CND0/2 net atomic charges in N-3-H + Guo A and N-7-H+ Guo A. The charges for Guo A3 are also quoted for comparison. Several points need to be pointed out. First of all, delocalization of the effect of protonation is evident throughout the entire guanine base irrespec- tive of whether the pyrimidine portion (N-3) or imidazole portion (N-7) is protonated. That both the u and x charges are affected by the protonation of the guanine base was shown before.' While thc proton positive charges are uniformly increased upon protonation a t either N-3 or N-7, the C and N (and the single 0) base charges are often perturbed in different directions (decreased or increased) depending on the site of proton attachment. That is, the electron density is decreased at N-1, N-2, N-7, and C-2 by nearly equal amounts; at N-3, C-5,O-6, and C-8 by very di ferent amounts depending on the site of proton attachment. Curiously, a t C-4 and N-9 (and to some extent a t C-6) the electron density may be increased or decreased depending o n the site of protonation.

Zoltewicz ct aLZ0 proposed a mechanism for the hydrolysis of the glycosidic C-N linkage in guanosine. This mechanism strongly suggested that N-7 protonation enhanced the rate of hydrolysis (based on the hydrolysis pH-rate profile of unsubstituted guanosine compared to that of 7-methyl- guanosinium salts). The present results indicate that N-7 protonation of base decreases, and N-3 protonation increases, the electron density a t N-9. According to the proposed mechanism, electron deficiency at N-9 would accelerate the rate of heterolytic C-N bond breaking.20 The present results strongly suggest that N-7 is the predominant site of first base protonation in guanosine. The extended Huckel theory predicts decreased electron density at N-9 upon N-7 protonation (Figures 2 and 3).

296 JORDAN

The polarity of the C-8-H bond (from the difference in electron densities of the two atoms) appears to be enhanced by N-7 protonation and decreased by N-3 protonation. This further supports N-7 as the site of first base protonation, since the mechanism of H-D exchange at this position has been suggested to occur by a base catalyzed abstraction from a protonated species.21

The nucleoside also shows substantial increases in the ionization potential (given by the negative of the energy of the highest occupied molecular orbital according to Koopmans’ theorem) accompanying the protonation process. The calculated ionization potentials are 9.9 eV in Guo A, 15.9 eV in N-3-H + Guo A or N-7-H + GUO A.

Some very small perturbations in ribose charges are evident upon protonation at either of the two nitrogens.

Calculation on the Glycosidic Potential Barrier One of the motivations for the present study was the experimental nmr

result indicating that not only do the syn and anti populations coexist in aqueous solutions of 2‘, 3‘, and 5’-GMP with an equilibrium constant near unity but the synlantiratio increases for the 2’- and 3’-GMP’s as the acidity is lowered to pD near 1.2 The problem of glycosidic bond rotations in the neutral Guo A and Guo B molecules, has been disc~ssed.~J The results showed that inclusion of the Q term had no significant effects on the potential barrier height or shape, which was dominated by the L-J term. Quali- tatively, the situation is easy to assess. In the neutral nucleoside one finds that the ribose carries a net +0.2 charge, whereas the base has a net -0.2 charge when the molecule is cleaved along the glycosidic C-N bond. Since upon protonation the charges are redistributed to +0.8 on the base and +0.2 on the ribose, qualitatively, the syn conformation, in which the aver- age position of the ribose is closer to the base, becomes less stable due to Coulombic repulsions. This argument holds only roughly since specific atom-atom interactions may become important as will be shown later.

First very approximate calculations (EJ, neglecting freely rotating groups) were performed on the geometry derived from the 5’-GMP crystal structure. As Figure 6 indicates even removing all rotatable ribose groups (O-5’-phosphate1 0-3’-H, and 0-2’-H as well as the C-~’-H’S)~~ one gets a very large barrier to glycosidic rotation in the syn region of this molecule, which barrier is due to the unfavorable interaction between the C-3’ environment of the ribose and the pyrimidine portion of the base. It is essentially due to the ribose puckering (C-3’-endo) and undoubtedly cannot be overcome by even highly favorable phosphate-base interactions. Also, in solution the ribose conformation of 5’-GMP is much closer to a C-2l-endo 0118.13 Since the energy required to interconvert ribose conformers is low,” there is good reason to concentrate on only such conformers that allow for facile syn-anti interconversion. On the basis of molecular orbital results, Berthod and Pullmane claimed that phosphate-base interactions had no influence on the potential curves of 2’-GMP and 5’-GMP. Because of

GUANOSINE AND 5’-GUO 297

EMWY kcol/mole

loo

90

80

70

60

50

40

30

20

10 0

-10

-132 168 108 48 -12 -12 -72

‘CN

Fig. 6. L-J calculations on 5’-GMP; WR approximation.

these reasons the most extensive calculations were performed on Guo A and Guo B since in these molecules the barrier to rotation is fairly small5 and the phosphate has no interference with the potential curve.

The potential calculations were performed under three sets of conditions differing from each other in the types of atolhs included. The first follows the earlier work by Wilson and Rahman22 (WR), whose theoretical approach we employed for the L-J term. This approximation disregards all freely rotating groups on the ribose (i.e. 0-2’-H1 0-3’-H1 0-5’, O-S’H, C-5’-H’s). The next one (FJ) includes all ribose atoms except the 0-5‘-H and the C-5’-H’s. As an approximation to nucleotides this latter approach is more realistic in not allowing for an N-3-0-5’-H hydrogen bond often (but by no means always) accompanying a syn conformation. The third set includes all atoms (total )in the ribose. Since it has been suggested that the ribose conformation, the glycosidic geometry, and the dihedral angle

298 JORDAN

a - 50 - 4 0 -

30 -

10 2o I

Gwnafnc 8

- Q-WR,L-J-WR --- Q-FJ -.-.-Q-T&l \ A 1-J

9 0 -

8 0 - Gwnosinc A

70 .- 6 0 .

50 - 4 0 -

30 - 20 -

Fig. 7. Potential energy curves for N-3-H+ guanosines.

with respect to the C-4-C-5' bond are intimately related,' the last approximation would indicate whether a particular solid-state arrangement of the C-4'-C-5 region allows for relatively free glycosidic rotations.

Throughout the discussion the older definition of syn and anti ranges will be quoted23 (placing the syn near +150, the anti near -30" of XCN, rotational angle).

Figure 7 indicates the results of the calculations on N-3-H+ Guo A and N-3-H+ Guo B. Apparently, there is a very large barrier to syn-anti interconversion located near XCN = f 60", in what is nearer the anti center even in the WR approximation according to L-J. Interestingly, the Q


term favors the syn region once the 0-5' atom is included along with the other hydroxyl protons. Obviously, however, the Q term is very much smaller than the L-J term; hence this site of protonation would definitely hinder free rotation around the C-N bond.

Due to the crystallographic evidence as well as our own suggestion indicating the predominance of N-7 protonation, N-7-H + guanosines will be more extensively discussed. The system also has the advantage of having the attached proton far from the ribose, not interfering with the L-J calculations. Clearly, the L-J curves for neutral and N-7 protonated

I

Energy kco I/mole

30 '

2 8 .

26 .

I

I

I I

! ! I I

I I ! I I I I

I !

-- - Coulmbic L-J -

-.- Total

-123 I77 117 57 -3 -63 -123

'CN (a)

Fig. 8 (continued)

300 JORDAN

guanosines should be essentially identical. Also, the Q term in the neutral molecule only shifted the zero points of the energy diagrams, leaving the shape and height of the barrier unchangedP

Figure 8 present8 the results on N-7-H+Guo A. Again, no rotation around the C-4'-C-5' bond was performed. Under optimal conditions one would have the WR results whereas the FJ results allow for inclusion of some other possibilities (e.g., 0-5' to base or 0-2'-H to base interactions). The barrier under optimal conditions (WR) can be rather small for this C-2'-endo ribose conformer (Figure 8c) with nearly equal energies of the best syn and anti XcN arrangement. The addition of the Coulombic term

-- Cwlanbie L-J Torot

- -.-.

177 117 57 -3 4 3 -I23 -123

'CN

( b )

Fig. 8 (colztinued)


EnerSy kal/mole

4

2

0

-2

-4

4

-8

-10

-12

-I4

-16

-18

-20

-22

- -- Cwlombic

-. -. - Total - L-J

-123 I77 117 57 -3 -63 -123

‘CN

(c )

Fig. 8. (a) N-7-H+ guanosine A; all atoms included. (b) N-7-H+ guanosine A; FJ approximation. (c) N-7-H + guanosine A; WR approximation.

of the protonated structure leads to the lowering of the anti energy range somewhat. The inclusion of the 0-5’ atom appears to introduce an L J repulsion as well as some electrostatic stabilization of the syn region (Figure 8b). The contrast of Figure 8b and c confirms the suggestion that if the ribose puckering and the C-4’-C-5’ dihedral angle can be ascertained in solution one can make reasonable predictions concerning the shape of the potential curve.’ Figure 8a also suggests that regardless of the C-4’-C-5’ dihedral angle,. some steric repulsions are bound to be introduced from the

302 JORDAN

C-5’ substituents in the syn region, the exact amount depending on the parameters employed in the simple potential calculation.

A major problem with all theoretical approaches is one’s ignorance of solvation effects. Most importantly, even with the inclusion of the Q term for the charged species, the relative energies of the most favorable syn and anti conformers are nearly equal. The Q term between maximal and mini- mal effects amounts to only 3 kcal/mole. In reality Q would be very much smaller, employing a dielectric constant very different from 1.

44

42

40

38 Ef=Fgy

36 kwl/mole

34

32

30

28

26

24

22

m 18

16

14

12

10

8

6

4

2

0

-2

-4

-61 136 76 16 -44 -104 -164 -44

XCN

(a 1 Fig. 9 (continued)


38

36 Energy

34 kwl/mole

32

30

28

26

Figure 9 presents the results for N-7-H+ Guo B, whose sugar conformation is C-l’ exo and has a gauche-gauche arrangement around the C-4’-C-5’ bond. In the WR approximation there is only a small barrier and the most stable syn and anti regions have nearly equal energies. The Coulombic term for the protonated species stabilizes the anti region. Introduction of the 0-5’ atom along with the other hydroxyl protons leads to repulsion in the anti region. This repulsion is due to a rotatable 0-2’-H as shown by a comparison of Figure 9b and the top of 9c (which neglects this atom). When both L-J and Q terms are included, L-J dominates even in the

. - - .

- -

40

24

22

20-

18

16

14

12

10

8 -

6 .

- -

- - *

- -

!

I I

I I i I

I

i

I I

’CN

(b 1 Fig. 9 (continued)

304

Energy kca l/rnojt

-22

-24

-26 2 ’

0 -

JORDAN

-

Coulombic -- - L-J -.- Total

3.- - - - - - - - - . --_-___ _-_ -/ ---- / \.

e .- __ . - .-. .\.-, -.-.-. / * \.-.-.- ,_.A .-‘ - .-\. - ., ’

:=

- 4

-6

-8

-10

-12

-14

- - .

-

%N

( c )

Fig. 9. (a) N-7-H+ guanosine B; all atoms included. (b) N-7-H+ guanosine B; (c) Guanosine B; WR approximation on atoms included, bottom FJ approximation.

portion; WR and 0-5’ and 0-3’-H top portion.

-I6

-18

-20

charged species. Very importantly, the comparison of the charged versus the uncharged guanosine models strongly suggests that for the two models employed (C-2’ endo and C-1’ ezo sugars) the barrier to glycosidic rotation is small, i.e., at room temperature facile interconversion should be possible regardless of the state of base protonation.

What is clear from the calculations is that the very subtle effects observed in the nuclear Overhauser effect (NOE) experiments on 2’-GMP, 3’-GMP, and 5‘-GRIIP may result from a number of small differences. As the pH is changed, the changes in the ratio of synlanti populations involve much less

--. _-- - c-----_cc--d ,’.+---.-.-.- __/ +---

- ._._. /---- \ ‘Z. -.-. -. -./ e.-. /. \ .- .- -.-.-.-_


than 1 kcal/mole AH differences. This type of change may be due to a small change in ribose conformation, or in the C-4‘-C-5’ dihedral angle arrangement. Assuming that these two parameters are the same in all three nucleotides, in the absence of the phosphate the syn region is probably very slightly favored in terms of base-sugar interactions. The fact that near neutral pH the synlunt i ratio is larger in 2’-GMP and 3’-GMP than in 5’-GMP may be due to the lack of phosphate in the 5’ position in the former ones. As opposed to Coulombic attractions, such a substituent would also introduce nonbonded repulsions in a gauche-gauche C-4’-C-5’ arrangement, not present in 2’-GMP and 3’-GMP. That protonation increases the synlunt i ratio in 2’-GMP is perhaps due to Coulombic attraction between the phosphate and the protonated imidazole portion of the guanine base according to our molecular models.

CONCLUSIONS It is evident that the molecular orbital calculations can be applied to a

variety of chemical problems in nucleic acid research. The results on protonated versus neutral nucleotides and guanosines

indicate that the changes in charges are reasonably reliable and may become useful in a number of areas. Pugmire et aL8 in the assignment of 13C chemical shifts of purines and Hug and T i n o ~ o ~ ~ in their recent theoretical studies on DNA base electronic spectra also showed the applicability of the CNDO/2 method.

The sites of protonation of DNA bases can be assigned by a combined application of results reported in this paper and l3C and 15N nmr measurements, since the net atomic charges on C and N are a very sensitive function of the assumed site of protonation.

The molecular orbital and other semiempirical potential barrier calculations lead to valuable predictions concerning the existence of major barriers, their approximate magnitude, and the regions of allowed conformations. None of the methods so far employed to study the syn-anti conformational problem (extended Huckel,3 PCIL0,G or L e n n a r d - J o n e ~ ~ ~ 5 ~ ~ ~ ~ ~ ) are capable of giving correct absolute energies for various conformers. Instead, the barriers are calculated as differences between two large numbers, both of which are themselves subject to dispute. In fact, the experimental energy of a molecule is one of the most difficult properties to reproduce the- oretically. For the same geometrical input, however, the regions of allowed conformations are well defined by the various approaches, hence the methods are useful in polymer mode1 building.

With the present state of the art, it is probably premature to attempt to explain subtle effects that may be due to any number of small changes alone or in combination. The Coulombic term has only a minor contribution to the L-J term, even in charged systems.

Computer time was generously provided by the Rutgers University Center for Com- puter and Information Services.

306 JORDAN

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a recent review of the evidence.

SOC., 92, 1741 (1970).

Received June 27, 1973 Revised September 10, 1973

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Effect of protonation on electronic structure of guanosine and 5′-guanosine monophosphate and on glycosidic (CN) bond rotations