Indian Journal of Chemistry Vol. 39B, February 2000, pp. 103 - Ill

Semiempirical study of tautomerism in alloxan

Rita Kakkar*t & B K Sarma

Department of Chemistry, University of Delhi, Delhi II 0 007, India

Received 3 August 1998; accepted (revised) 8 March 1999

A comparative semiempirical study of tautomerism in alloxan is performed with a view to understand the areas of applicability and limitations of the three methods, MNDO, AM I and PM3 . The results are also compared with an ab initio study. It is found that the MNDO method overestimates the stability of lactim forms, while AM I method seems to underestimate their stabilities. All methods indicate that the tetraketo form is the most stable, followed by the monohydroxy forms. While the PM3 method seems to give the best results regarding relative stabilities of tautomers, it fails in reproducing experimental geometries and charge distributions and in explaining the chemical and biological reactivity of alloxan. The calculated MNDO and AM I charge densities agree with the results of an ab initio study and with experimental estimates, and are thus able to explain the absence of hydrogen bonding in the crystal structure of alloxan . Nucleophilic attack by water at 5-position leads to a significant increase in the stabilities of the tetraketo and 2-hydroxy forms . Thus, due to the absence of hydrogen bonding interactions in the crystal structure of alloxan, the MNDO geometries are in closest agreement with experiment, and the charge densities are also consistent with experimentally estimated ones.

The phenomenon of tautomerism is thought to play a major role in determining the chemical and biological activity of several compounds, particularly N­heterocycles, of which the nucleic acid bases are some examples. Another compound of similar structure, which is biologically active as it causes diabetes in experimental animals, is alloxan. The existence of several tautomeric equilibria in this compound is considered to determine its biological react1v1ty, as blockage of the lactim-lactam tautomerism by substitution at both nitrogens causes it to lose its biological activity'. As it is an unstable compound, very little is known about its physicochemical properties. It is thus an ideal compound for theoretical investigations.

We had previously reported2 Modified Neglect of Diatomic Overlap (MNOO) calculations on the tautomerism in alloxan wherein it was shown that the tetraketo (lactam) form is the predominant tautomer, followed by the monohydroxy forms. The predominance of the tetraketo form is found to be in agreement with X-ray crystallographic studies in the solid state3

.4.

Previous studies5 on tautomeric systems have shown that semiempirical methods upto the MNDO method fail to accurately determine the relative stabilities of tautomers, although the equilibrium

t(E-mail : kaks @del3 .vsnl.net.in)

geometries are correctly predicted. On the other hand, ab initio calculations below the level of 6-31 G** also suffer from the same drawback6

. In view of the large requirements of computational resources for such high level ab initio calculations for the generally large tautomeric systems of interest, it is not practical to carry out such calculations, and one has to resort to semiempirical methods of calculation. The most recent semiempirical methods, based on MNDO method, i.e., Austin Model 1, (AM1)7 and MNDO­Parametric Method 3, (MNDO-PM3)8

, are parameterized to reproduce data for systems in which intramolecular effects, particularly of the hydrogen bonding type, are present. These methods have been found to be adequate for the purpose of predicting tautomeric equilibria.

While the MNDO method generally overestimates the stabilities of the hydroxy forms , the AM 1 method is found to accurately predict relative energies for a number of systems, but fails in the case of cytosine9

and hydroxypyridazines 10·

11. The failure in the case of

hydroxypyridazines has been ascribed to the overestimation of stabilities of compounds with adjacent pyridine-like lone pairs 10

.13

. It also gives a preference to bifurcated hydrogen bonds rather than linear ones . The available data regarding the PM3 method also indicate that it is able to reproduce experimental tautomerization energies, except, again,

104 INDIAN J CHEM, SEC 8, FEBRUARY 2000

for compounds with two or more adjacent pyridine-l.k 1 . 14 1 e one patrs .

It is therefore necessary to study more systems and compare the data obtained by the several available methods in order to form a judgement regarding their capabilities and limitations, Accordingly, in the present work, we present the results of both AM 1 and PM3 calculations on the tautomeric equilibria in alloxan. A total of five tautomeric forms (see Figure 1) is possible, i.e., the tetraketo form (1), the

total of twelve systems were studied.

Method of Calculation All calculations were performed with the MNDO,

AMI and PM3 methods using the MOPAC 6.12 15 . program package . The molecular geometnes were

fully optimized with respect to the energy without any conformational or symmetry restrictions . The keyword PRECISE was used in all geometry optimizations.

4- and 2-hydroxy forms (II and Ill) and the 4,6- and Results and Discussion 2,4-dihydroxy forms (IV and V). Combined with the The following conclusions were dr<l;wn from our fact that each tautomer may have different rotamers, a MNDO calculations2

:

~ I? ' /H , NAN

~o, 011

! /H, N N NlN/H

~0~0 ~0 10 I 12 0

011 I o H IIC liB

o;'H, 1-t--...o

Ho---N~N H'---.._N~N

~0 " ~0 0

011 0

IliA IIIB

N1N

0

NlN N)lN

YH "Y y Hv--.o o_........ ' 'o o " I I I t

011 H HI

IVA IVB IVC

VA

Figure 1- Vari ous tautomers and rotamers of alloxan

KAKKAR eta!. : SEMIEMPIRICAL STUDY OF TAUTOMERISM rN ALLOXAN 105

(i) The tetraketo form is by far the most stable tautomer in the vapour state, and monohydroxy forms may also make some contribution .

. (ii) The variation in dipole moments suggests that, although the 4-hydroxy tautomer is more stable in the vapour phase, the 2-hydroxy tautomer may gain greater stability in aqueous solution. (iii) In general, the rotamer in which the migrating hydrogen is directed towards the nitrogen from which it originated, is more stable than the others, suggesting that no rotations about the C-0 single bonds occur after enolization. However, since, in most cases, the less stable rotamers have larger dipole moments, these may be the dominant ones in solution.

In this paper, these findings are reviewed in the light of the results of the present AM 1 and PM3 calculations.

Relative Tautomer Energies. Table I lists the calculated heats of formation of the tautomers (see Figure 1), along with the calculated dipole moments. All the three methods agree on the relative stabilities of the rotamers of a given tautomer, and, accordingly, the rotamers are labelled A-D in order of increasing energies (see Figure 1). It can be seen that both MNDO and PM3 predict the order of decreasing stabilities as I > II > III > V > IV, while AMI predicts the 4,6-dihydroxy form to be more stable than the 2,4-dihydroxy form (i.e., IV> V). However, all the three methods predict the tetraketo form to be the most stable, followed by 4-hydroxy and the 2-hydroxy tautomers. An ab initio study by Millefiori and Millefiori 16

, however, predicted the stability

order as I > III > II > V > IV. However, they had restricted themselves to a consideration of only the B rotamers of II and ill (see Figure 1), which, as we have shown above, are the less stable rotamers. In any case, all the methods predict the tetraketo form to be the most stable, followed by monohydroxy tautomers. The overwhelming stability of the tetraketo form with respect to the hydroxy tautomers is in agreement with X-ray crystallographic studies in the solid state3

.4.

Tautomeric equilibrium constants. The tautomeric equilibrium constants, KT, were also calculated at 300 K to estimate the molar concentrations of the hydroxy forms in the vapour phase. The free energy differences were estimated using enthalpies and entropies obtained from the calculated vibrational frequencies and rotational constants. Table II lists the logarithmic tautomeric constants (pKT values) calculated by the three methods. In each case, very small quantities of the hydroxy forms are indicated, except II A, which should be present to the extent of - 2.5%, and, to some extent, III A (- 0.2%) according to MNDO calculations. While the MNDO calculations give some importance to the hydroxy forms, the AMl method favours only the tetraoxo form, and the PM3 results are in between.

Solvent effects. Since less stable rotamers have higher dipole moments, it is possible that they gain stability in aqueous solution. To check this point, the interaction energies with water were calculated for all the tautomers and rotamers. The reaction field continuum model 17 was used, and the water-solute

Table 1-Heats of formation (kcallmol) arid dipole moments (Debyes) of alloxan tautomers

No.* System Heats of formation Di2ole moments MNDO AMI PM3 MNDO AMI PM3

Alloxan -135.1 -114.2 -139.1 1.23 1.71 1.32

IIA 4-hydroxy -132.5 -99.4 -127.8 1.69 1.61 1.26

liB -126.6 -95 .7 -125.8 2.68 2.16 2.20

IliA 2-hydroxy -130.5 -96.2 - 126.3 4.51 4.86 4.29

IIIB -123.2 -89.0 -119.5 5.80 6.00 5.43

IV A 4,6-dihydroxy -126.5 -80.9 -115.0 2.36 2.06 1.99

IVB -120.4 -76.8 -112.7 4.49 4.17 3.98

IV C -114.2 -72.4 -110.1 6.23 5.66 5.58

VA 2,4-dihydroxy -127.5 -79.9 -115.4 3.55 3.53 3.27

VB -125.9 -78.4 -113.8 5.89 5.77 5.41

vc -122.3 -76.7 -113.8 1.91 1.34 1.66

VD -121.4 -76.0 -113.0 4.54 3.98 4.12

* See Figure 1

106 INDIAN J CHEM, SEC 8 , FEBRUARY 2000

Table 11-Energies of interaction with water (kcaVmol) and logarithmic equilibrium constants in vapour (pKT<v>) and solution (pKT<s>)

No.* -Em, MNDO AMI PM3 MNDO

I 0.1 0.3 0.2

IIA 0.3 0.2 0.1 1.60

liB 0.7 0.4 0.4 5.63

IliA 1.8 2.1 1.6 2.65

IIIB 3.0 3.2 2.7 9.89

IVA 0.5 0.4 0.4 5.92

IVB 1.9 1.6 1.5 9.72

IVC 3.6 2.9 2.9 14.20

VA 1.0 0.9 1.1 4.88

VB 3.0 2.9 2.6 5.94

vc 0.3 0.2 0.2 8.57

VD 1.8 1.4 1.5 9.16

*See Table I and Figure 1

interaction energies are given in Table II . It can be seen that, although the interaction energies are fairly large for the rotamers with the larger dipole moments, the order of stabilities remains essentially unchanged. Because of the larger stabilization of III A in water due to its higher dipole moment (see Table I), it should be present to a higher extent in solution than form II A. As expected, the importance of the hydroxy tautomers, particularly III A, increases with temperature.

The foregoing discussion suggests that only the tetraoxo form is present in the vapour and solution phases, and the monohydroxy forms may be present to a small extent. The dihydroxy forms are too unstable to exist either in the vapour or the solution phase. Only the MNDO method predicts the existence of the monohydroxy forms. Experimental evidence 18

indicated the presence of a labile hydrogen in aqueous solution, as a pK. value of 7.2 had been observed 19

• This was thought to be the hydroxyl proton from the 4-hydroxy tautomer19

• Thus, only the MNDO calculations seem to be in agreement with this experimental evidence. Since no evidence for the existence of the dihydroxy forms exists (as the second pK. is too high), in the following, only the tetraoxo and the monohydroxy forms will be considered. It may be of interest to see that, while the MNDO method predicts the existence of the 4-hydroxy tautomer to the extent of 3% in aqueous solution, the AM I and PM3 methods predict its contribution as only 3x l0--9 % and lxi0-6 %, respectively, at 300 K. Experimentally, apart from the observation of the pK.

pKT(v) pKT(sl

AMI PM3 MNDO AMI PM3

10.46 7.89 1.51 10.49 7.90

13.15 9.36 5.25 13 .03 9.16

12.31 8.43 1.41 10.97 7.35

16.83 13.38 7.80 14.69 11.52

24.06 17.07 5.64 23 .98 16.92

26.70 18.54 8.47 25.74 17.59

29.89 20.71 11.69 27.94 18.73

24.40 16.4.5 4.23 23 .94 15.79

25.59 17.44 3.84 23 .65 15.66

26.84 17.72 8.45 26.93 17.66

27.30 18.32 7.94 26.46 17.33

value of 7.2 in aqueous solution, and that the N,N­dimethyl derivative, in which the possibility of tautomerism IS blocked, is nondiabetogenic 1

, very little evidence of tautomerism exists for alloxan. However, both these observations may be explained on the basis of ionization of the imine hydrogen in the tetraketo form, as has been found to be so in the related molecules uracil and barbituric acid.

Since the MNDO method is known to fa '/Our hydroxy forms, while the AM 1 method disfavours these forms, the PM3 results for relative tautomer stabilities, which are in-between the two values, may be considered most reliable. In addition, PM3 gives better values for heats of formation 14

. Hence, we may conclude that the monohydroxy tautomers are present to a small extelflt in solution and the vapour phase.

Molecular geometries. The optimized molecular geometries are given in Table III along with the experimental3 and ab initio 16 geometries for the tetraoxo isomer.

A comparison of the calculated geometries with the experimental geometries shows that, while the mean errors in the. bond lengths are - 0.02 A for all the three semiem'pirical methods, the errors in the bond angles are 0.5°, 1.4°, and 2.2°, respectively, for MNDO, AM 1 and PM3 calculations. The corresponding values are - 0.03 A and 1.5° for ab initio calculations. This indicates that the MNDO method gives geometries in closest agreement with experimental values. The PM3 values for the ring bond distances are larger than the experimental ones, while the C=O btmd distances are in better agreement

KAKKAR eta!.: SEMIEMPIRJCAL STUDY OF TAUTOMERlSM TN ALLOXAN 107

Table III- Optimized geometries of alloxan (bond lengths in A and angles in degrees)

Bond Parameter• MNDO AMI

C2Nl 1.410 1.407

C4N3 1.4 11 1.391

C5C4 1.528 1.520

H1N1 1.008 1.000

OxC2 1.225 1.245

OwC4 1.222 1.237

o11cs 1.218 1.222

N3C2N1 116.2 119.0

C4N3C2 126.5 124.0

CsC4N3 116.5 117.8

c6csc4 117.9 11 7.3

H7N1C2 116.0 116.7

OxC2N3 121.9 120.5

O wC4N3 119.0 120.4

o1 1csc4 121.0 121.4

01 2c6c s 124.5 121.8

C4N3C2N1 0.0 0.1

C5C4N3C2 -0.1 -0.1

H7N1C2N3 180.0 179.9

OxC2N3C4 180.0 -179.9

OwC4N3C2 179.9 179.9

OIIC5C4N3 -179.8 180.0

"See Figure 1; hFrom ref 16; cFrom ref 3

with experiment than the corresponding AM I bond di stances.

In all cases, the greatest discrepancy is in the C-N and C5-01 1 bond distances. This is because, in the crystal structure of alloxan, there are strong C5---0 10

and C4---0 10 interactions, rather than interactions of the hydrogen bonding type, which modify the corresponding bond distances.

Charge distributions. As mentioned in our previous paper2

, the lowest unoccupied molecular orbital (LUMO) of all oxan has a negati ve energy (-1.676, -1.838, and -1.744 eV, respectively, from MNDO, AM I and PM3 calculations), and the next few orbitals, too, have low or negati ve energies. Also the highest occupied molecular orbitals (HOMOs) have highly negative energies. Thi s is indicative of the fact that alloxan has a high ionization potential and electron affinity, and is thus a bad electron donor but a good electron acceptor. It, therefore , behaves as an electron acceptor in the Diel s-Alder reaction20

.

The same is true for the hydroxy tautomers.

PM3 Ab initioh Expt.c (ST0-3G)

1.426 1.425 1.388

1.420 1.423 1.364

1.526 1.546 1.521

1.001 1.021

1.220 1.218 1.219

1.214 1.219 1.213

1.204 1.222 1. 186

119.4 114.5 117.4

123.1 128.0 126.2

118.7 115.5 116.2

116.9 118.5 117.8

117.6 115.6

120.3 122.7 121.3

llfi.5 123.4

121.5 120.8 121.1

124.7 122.8 120.3

0.0 0.0

0.0 0.0

180.0 180.0

180.0 180.0

180.0 180.0

180.0 180.0

In fact, the partial charge densities on the various atoms, calculated by different methods (see Table IV), show that all the ring carbon atoms carry large positive charges. Only the nitrogens are negatively charged. The total ring charge, calculated by MNDO, AMI and PM3 methods, is 0 .55, 0.40 and 0.8 1, respectively. A point to note is that the PM3 calculation shows a smaller charge density at the nitrogens, which is consi stent neither with other calculations 16

, nor with the experimentally determined charge densities for the monohydrate21

.

The oxygen atoms carry large negative charges. This dipolar nature of the C=O groups shows why, unlike most pyrimidines, the interactions present in the solid state are of the C=O---C=O type rather than of the hydrogen bonding type. Due to the charge separation in the C=O bonds, the ring bonds weaken, and the total ring bond orders, as calculated by the MNDO, AM I and PM3 methods, are 5.56, 5.70 and 5.84, respectively, instead of the expected value of 6.0.

The electron defi ciency of the alloxan ring is al so responsible for its susceptibi lity to nucleophil ic

108 INDIAN J CHEM, SEC B, FEBRUARY 2000

Table IV -Calculated partial charge densities and bond orders

Atom/ MNDO AMI PM3 Ab initiob Expt.c Bond•

N1 -0.406 -0.369 -0.021 -0.378 -0.504

c 2 0.493 0.409 0.23 1 0.417 0.164

c 4 0.330 0.268 0.193 0.290 0.086

c5 0.210 0.192 0.238 0.160 0.064

H7 0.229 0.280 0.130 0.226 0.336

Ox -0.331 -0.323 -0.351 0.271 -0.044

Ow -0.261 -0.254 -0.279 -0.219 -0.113

011 -0.154 -0.128 -0.163 -0.144 -0.004

C2N1 0.966 0.996 1.02 1 0.700

C4N3 0.975 1.022 1.047 0.690

c5c4 0.839 0.834 0.850 0.682

H1N1 0.900 0.868 0.937 0.694

OsC2 1.812 1.759 1.770 0.882

0 10C4 1.898 1.851 1.858 0.882

o 11C5 2.015 2.012 2.012 0.876

•see Figure 1; bFrom ref 16; cFrom ref 21

attack. In our previous paper2, by charting the electrostatic potential (ESP) maps and by studying the nature of LUMOs, we had shown why, although C2 carries a larger negative charge, it is the C5 atom, amongst all ring carbons, which is the most susceptible to nucleophilic attack.

Also, due to its susceptibility to nucleophilic attack, alloxan is found to exist as a monohydrate, a white solid, from which the yellow anhydrous compound can be obtained by heating in vacuo. Although the white solid is described in most text­books as alloxan .H20 , the name monohydrate is a misnomer, as its actual structure is 5,5-dihydroxy barbituric acid, as was suggested by Baeyer22. This is formed by the nucleophilic attack of water at C5. The so-called monohydrate has been used as the starting material for the synthesis of several cyclic urea derivatives, and also as a powerful diabetes inducer in pharmacological studies23

. It is now well known by X-ray investigation that in anhydrous alloxan the molecule is planar with C2v symmetry3

, while in the hydrate, both -OH and -CO groups participate in intermolecular hydrogen bonding, the molecule losing its planaritl4

. All the three methods predict significant asymmetry, particularly of the two -OH groups. Al so, the puckering of the ring is most significant for the PM3 geometry (see Table V).

The formation of this monohydrate significantly stabilizes the system, i.e. by -8 kcal/mol, but does not lead to any significant change in the positive charge

Table V --Optimized geometries of alloxan monohydrate (bond lengths in A and bond angles in degrees)

Parameter MNDO AMI PM3 Ex pt.•

C2NI 1.408 1.407 1.425 1.374

C4N3 1.409 1.387 1.417 1.379

c5c4 1.570 1.543 1.552 1.539

H1N1 1.009 1.000 1.001 0.980

OsC2. 1.226 1.246 1.221 1.221

OIOC4 1.222 1.237 1.216 I 209

o11C5 1.397 1.405 1.403 1.394

HI3011 0.949 0.971 0.95 1 0.940

N3C2N1 11 6.4 119.2 119.5 117.2

C4N3C2 127.0 124.2 123 .3 126.7

C5C4N3 117.4 118.4 11 9.9 117.2

c 6c 5c 4 114.8 115.6 114.3 11 3.9

H7N1C2 115 .9 116.5 117.6 115.4

OsC2N1 121.8 120.4 120.3 12 1.7

OwC4N3 118.9 121.1 116.6 121.3

01 1c 5c4 110.4 110.2 Ill. I 111.3

HI3ollc5 114.7 107.3 108.5 111.4

C4N3C2N1 0.4 1.1 0.5

C5C4N3C2 -0.3 -1.8 -0.3

H1N1C2N3 179.7 179.0 179. 1

0 8C2N3C4 - !70. 7 - 178.9 -179.6

0 10C4N3C2 178.7 176.0 178.0

011CsC4N3 118.3 122.2 120.0

H13o11csc4 60.6 55.6 53 .7

•From ref24

on the ring. (However, the MNDO method predicts a slight increase in the positive charge on the ring and the ring bond order) . There is not much change in the ring geometry, either, except in the C5-C4 and C5-C6

bonds, which lengthen slightly. The LUMO still has a large negative energy.

The calculated geometries (see Table V), particularly the AMI geometry, are in good agreement with the experimental geometry. Again the greatest discrepancies in the PM3 geometries are in the ring bond distances.

The calculated charge densities indicate that this hydrate is formed by the donation of a small amount of charge density (- 0. I 5) by water to alloxan. Also, the ionization potential reduces slightly, while the electron affinity reduces drastically on hydrate formation.

In our previous paper2, we had used the ESP maps to explain why the site of nucleophilic attack by water

KAKKAR eta!.: SEMIEMPIRICAL STUDY OF TAUTOMERISM IN ALLOXAN 109

and other nucleophiles is C5. The ESP. maps also indicated the following:

(i) In all planes parallel to the molecular plane, there are no areas of negative potential near the nitrogens, signifying that the nitrogen lone pairs, which are perpendicular to the molecular plane, are thoroughly delocalized with the 1t electron cloud.

(ii) Because of this, these nitrogens cannot participate in intermolecular hydrogen bonding. Rather, the large positive potential near C5 and the negative potentials at 0 10 and 0 12 imply that there can be interactions of the C=O---C=O type.

It was found2 that in the anhydrous compound, the potential minimum in the plane parallel to the average plane and at a distance of 1.4 A, which is half the distance of 'close contact' in the alloxan crystae, is at 0 8, while the ·maximum is at C5• There are also secondary minima at C4 and C6. Hence, the predominant interactions are of the C2=0s---Cs=011 or C2=0s---C4=010 type. An unusually large crystal density characterizes alloxan, and the carbon-oxygen nonbonding distances in the crystal (2.77 A) are much shorter than the actual sums of their van der Waals radii . This is due to the strong interactions between the two atoms.

However, as noted before, in the hydrate, the interactions are of the hydrogen bonding kind. Thus, it is -instructive to examine the reasons for the differe~ces in interactions.

In the case of anhydrous molecule, the HOMO was found2 to be the oxygen nonbonding orbital (08, 0 10

and On), while the LUMO is the C5-011 1t* orbital. In the monohydrate, however, since there is no C5-011 1t

bond, the LUMO is a mixture of the carbonyl 1t

bonds, viz. C4-010, C6-0 12 and Cr08 bonds. Therefore, the intermolecular interactions in the monohydrate are expected to be different from those in the anhydrous compound. It will not be out of place to mention here that, as is apparent from the charge density distributions (see Table IV), the orbital compositions of the HOMO and LUMO, as calculated by the PM3 method, are entirely different from those calculated by MNDO and AMI methods. Thus, according to the PM3 calculations, the nitrogen atoms should be neutral, while all other methods, including ab initio16 an<;J experiment21 indicate otherwise. The nitrogens are negatively charged as they accept electronic charge from the Cr08 carbonyl bond2. Thus, while both MNDO and AMI calculations place the oxygen nonbonding orbitals

(010, 0 11 and 0 12 in equal amounts) as the HOMO, the PM3 HOMO is a mixture of the nitrogen 2pz orbitals (lone pairs), which are not delocalized as in the MNDO and AM 1 cases. The same is true of the orbital composition of the monohydrate. It may be pointed out that the same phenomenon, i.e. the tendency of the PM3 method to give a different ordering of orbitals, was also observed by Fabian14.

It is also instructive to examine whether hydration also stabilizes the hydroxy tautom~rs to the same extent. While all three methods predict the hydration energies to be similar for the three tautomers, there are differences in the magnitudes of the calculated hydration energies. AMl predicts the largest enthalpy of hydration (- -19 kcaJJmol), while the MNDO and PM3 values are- -8 and- -13 kcaJJmol, respectively. The 4-hydroxy hydrate is stabilized to the smallest extent due to the proximity of the three hydroxy groups. Thus, the possibility of tautomerism is decreased further on hydration, and only a small amount of the 4-hydroxy tautomer may exist in equilibrium with the tetraoxo form (pKT=1.95, 7.86 and 5.69, respectively from MNDO, AMI and PM3 calculations). Experimental evidence from X-ray24·25 , 1H NMR26, and 14N NQR27 investigations on the monohydrate gave no evidence of tautomerism.

Charged species .The high value of the calculated electron affinity shows that' alloxan should readily gain an electron to form the corresponding radical anion. This expectation is supported by studies28·29 in aqueous and aprotic media. Experimental evidence also28 shows that the radical anion can be protonated at low pH values. We carried out calculations on the radical anion and found a significant decrease in all the ring bond distances, except the C4-N3 and C6-N1 distances, which increase by about 0.8 A. The magnitude of spin density is maximum at C5 ( -0.490) and 0 11 (0.539). As noted earlier2, the LUMO in neutral alloxan is the C5-0 11 antibonding 1t* orbital, and thus the unpaired electron is distributed among these two atoms . The si te of protonation of the radical anion is expected to be the oxygen carrying the largest negative charge29'30

. Thi s is the 0 8 atom, which carries a negative charge of -0.471 , while all other oxygens carry equal negative charges ( -0.406). However, the structure of the HOMO shows that it is the oxygen nonbonding orbital, and that contains the largest contribution from 0 11 . Also, as expected from the composition of the LUMO of neutral all oxan , the

110 fNDIAN J CHEM, SEC B, FEBRUARY 2000

unpaired electron in the C5-0 11 antibonding 1t* orbital reduces the C5-011 bond order from 2.016 in neutral alloxan to 1.547 in the radical anion. It is this carbonyl bond that suffers the maximum decrease in bond order and hence the site of protonation of the radical anion should be 0 11 • This is in agreement with the conclusions based on ESR experiments31

•32

•

Alloxan is also expected to undergo deprotonation to form the corresponding dianion. As noted earlier2

,

even in the dianion the ring is almost neutral, the negative charge being distributed among the oxygens . The negative charge is least at 0 11 . In this case, the carbonyl bond orders that decrease the most are the C4-0IO and C6-0, 2 bond orders.

Conclusions Our comparative studies of tautomerization

energies have indicated the following : (i) The calculated optimized geometries are in

closest agreement with the experimental geometries for MNDO calculations. The deviations are particularly large for the PM3 and ab initio case.

(ii) While all calculations agree that the most stable tautomer is the tetraketo form and that the monohydroxy forms (4-hydroxy and 2-hydroxy) are next in order of energy, only the MNDO method indicates the presence of any of the tautomers, except the tetraketo form, at room temperature. It is also found that the amount of the monohydroxy tautomers increases with temperature and on dissolution in polar solvents. However, due to the tendency of the MNDO method to overestimate the stabilities of hydroxy forms and the tendency of the AM I method to underestimate their stabilities, it may be concluded that PM3 results are most reliable for estimating relativ'e tautomer energies.

(iii) The calculated charge densities show a large charge separation in the carbonyl groups. This shows that, in the crystal structure of alloxan, the predominant interactions should be C=O---C=O. However, the charge densities calculated from the PM3 method are at variance with all theoretical calculations as well as experimental estimations.

All these findings show that, in this particular case, the MNDO method gives results in closest agreement with experiment. Thi s can be ascribed to the absence of hydrogen bonding in the crystal structure of alloxan due to the carbon-oxygen interactions and, hence, the AM I and PM3 methods, which are parameterized to reproduoe data for systems in which

intramolecular hydrogen bonding interactio11s are present, fail in this case.

These conclusions emphasise the need for exercising caution when using the semiem)?irical methods for determining relative tautomer stabilities. !n cases such as alloxan (and parabanic acid) in which no hydrogen bonding is present, the MNDO method gives superior results. This is particularly true of the PM3 method, for which the calculated charge densities do not match with any other method.

The calculated electrostatic potentials, using the MNDO charges, as well as the orbital composition of the highest molecular orbitals also indicate that, in anhydrous alloxan, the nitrogen lone pair is thoroughly delocalized with the carbonyl 1t system, and is hence not available for hydrogen bonding.

The site of protonation of the radical anion is also correctly predicted on the basis of the MNDO results.

All these findings are in accord with experimental data.

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