CONFORMATIONAL PROPERTIES, SPECTROSCOPY AND STRUCTURE OF ISATIN-(WATER)n COMPLEXES MILIND K SING, D M UPADHYA AND VIPIN B SINGH U P AUTONOMOUS COLLEGE VARANASI INDIA

Fig 1a: Isatin+W1a (Binding Energy: Kcal/mol at MP2/6-31G(d) level)

1. INTRODUCTION Oxyindoles are endogenous compounds found in mammalian body fluids and tissues, distributed in the central nervous system, that have shown an extensive range of biological effects. Particularly, Isatin (1H-indole-2, 3-Dione) an oxidation product of indigo is a biologically active molecule with a long history. Isatin is present in the brain and other tissues in humans and the substituted isatins are also found in some plants. The metabolism of isatin in humans is not yet well elucidated. The presence of two carbonyl groups in isatin makes it an attractive target. Therefore, the structure and properties of isatin remained a subject of great interest for a long time[1,2 ]. Despite this long-term interest only a few studies on the spectra and structure of isatin are available in the literature. Recently our group has carried out an extensive ab initio and Density Functional Theory (DFT) studies on the structures of isatin monomer and dimer isolated in the gas phase [ 3]. However, a molecular level understanding of the competing noncovalent interactions between isatin and a biologically relevant environment is essential to properly characterize the hydration of this bio-molecule. Isatin has numerous different receptor/binding sites and water molecules are likely to be present in these binding sites and may play a role in the way in which isatin interacts with water. Our goal is to probe the preferred binding sites for water molecules, and to determine how this binding affects the spectra and structure of isatin monomer. Therefore, in the present work we have investigated the structure and stability, vibrational characteristics and the vertical (electronic) excitation energies of isatin and isatin- water complexes using ab initio and DFT calculations. The results of this investigation are reported in this presentation. 1. M. Agostinha, R. Matos, M.S. Miranda, V.M.F. Morais and J.F.Liebman, Org. Biomol.Chem., 2003, 1, M. T. Silva and J. C. Netto-Ferreira, J. Photochemistry and Photobiology A: Chemistry, 2004,162, Milind K Singh and Ashutosh Gupta, Vipin B. Singh, J Mol. Struct. (Theochem) Accepted 2009

2. Methodology The optimized geometry, structure and stability of isatin and hydrogen bonded isatin-water complexes have been investigated using the second order Moller-Plesset (MP2) perturbation theory and the Density Functional Theory methods. The later method was also used to calculate the harmonic vibrational frequencies. The comparison of the calculated fundamental frequencies with the experimental results shows that the Density Functional Theory (DFT) is a reliable tool for the interpretation of IR spectra. In the DFT method, Becke’s 1988 exchange functional in combination with Becke’s three-parameter hybrid exchange functional and Lee, Yang, and Parr correlation functional (B3LYP) were employed. The 6-31+G (d) basis set is employed in the geometry optimization and vibrational frequency calculations. By adding the polarization and diffuse functions to the split valence set, wave numbers of carbonyl stretching modes of Isatin are, in general, found to be increased. The calculated values of vibrational frequencies were found to be more close to the experimental values; however, the scaling factors were necessary to find a good agreement. Stabilization energies (∆E Stab) of the isatin-water complexes have been calculated as follows: ∆E Stab = E Complex – (E Isatin + E Water) The vertical excitation energies from the ground state to the low lying excited states of isatin and Isatin- (water) n=1 complexes were also calculated by using the Time Dependent DFT methodology with the same B3LYP parameterization and 6-31+G(d) basis set. All the calculations were carried out using the Gaussian suite of program 28.

3. Results and discussion: A. Geometry and stabilization energies: The optimized structures of the different possible isomers of isatin-(water)1-3 clusters have been obtained from MP2 and DFT methods employing the basis set 6+31+G(d). The different initial geometries for the Isatin-(Water) 1 complex, in the vicinity of the two carbonyl oxygen atom and amide hydrogen, led to three stable geometries depicted in the Figure 1. The isomer Isatin-W1a, in which both isatin and water act as accepters as well as donors, is much more stable than the other two isomers of Isatin- (Water) 1 cluster. The larger stabilization energy for this isomer (W1a) arises from the additional stability of the cyclic structure with two hydrogen bonds between the amide moiety and water, as illustrated in figure 1a. The stabilization energy of W1a is found to be Kcal/mol at B3LYP/6-31+G (d) level, which is nearly equal to the stabilization energy of salicylic-water and formic-acid-water clusters probably due to resonance assisted hydrogen bonds. The hydrogen bond distances in this isomer are found to be Å (amide group acts as the hydrogen donor) and Å (water acts as the hydrogen donor) at the same level of theory. It seems that characteristics of H-bonds due to water are connected with the π-electron delocalization within the amide moiety and such delocalization affects the stability of the isatin-water complexes more significantly. The H- bond between the amide hydrogen and water’s oxygen in W1a isomer was found stronger as compared to corresponding H-bond in Indole-(Water)1 cluster.

Binding Energies (Kcal/mol), Dipole Moments (Debye) and Hydrogen bond distances (Å) of Isatin-(Water) n=1 Complexes S. N. Species Binding Energies (Kcal/mol) At B3LYP/ 6-31+(d) Binding Energies (Kcal/mol) At MP2/ (d) Dipole moment At B3LYP/ 6-31+(d) Dipole moment At MP2/ (d) 1Isatin Isatin+W1a Isatin+W1b Isatin+W1c

Isatin-(Water)1 In the isomer W1b, the two-carbonyl oxygen’s are interconnected with hydrogen atoms of water through double intermolecular homonuclear hydrogen bonds (O….H-O), in which water acts as the hydrogen donor. Interestingly the stabilization (binding) energy of this complex is found to be smaller ~6.43 Kcal/mol at B3LYP/6-31+G (d) level, than W1a. In spite of the double hydrogen bonds (O….H-O) the relatively less stability of this isomer arises probably due to the repulsion forces acting between the lone pair electrons of the two carbonyl oxygen’s. In the isomer W1c, a single hydrogen bond is formed between the ketonic carbonyl oxygen and water and the stabilization energy is found to be 7.21 Kcal/mol. Interestingly, the dipole moment of W1b complex is almost two times than the two other isomers. This can be understood as probably due to an accumulation of electronic charges at the nitrogen atom. The electronic charges at N-atom from ESP fit calculations are found to be -0.71, and for the isomers W1a,, W1b and W1c respectively, indicating a significant electronic charge displacement from N-atom to carbonyl (amide) C-atom in the complex W1b(See Table 1). This produces an increase in the overall charge separation, which results in unusual increase in the dipole moment in the complex W1b. Thus, the isatin complexes involving water at the W1b position show charge transfer character.

Table 1: Charges on the Atoms Atoms Isatin Isatin+1Wa Isatin +1Wb Isatin+1Wc C C H O O N

Isatin-(Water) 2, 3 Three different stable geometries are also investigated for Isatin-(Water)2 complex ( see Fig 2). The stabilization energies of the three isomers, Isatin+W2ab, Isatin+W2bc, and Isatin+W2ac are found to be 16.26, and Kcal/mole respectively at B3LYP/6-31+G(d) level. The dipole moments of W2ab and W2bc isomers in which the W1b position of water is involved, are high, whilst the dipole moment of the most stable W2ac isomer (of Isatin- (Water) 2 cluster), is relatively very small (Table 2). This indicates that while the W1b-position of water increases the dipole moment it decreases the binding energy of Isatin- (Water) 1, 2 clusters. It was already described in the earlier section that the W1b structure results in a significant electronic charge displacement from N-atom to carbonyl (amide) C-atom, resulting in an unusually large dipole moment (Table 1). The single stable geometries is investigated for Isatin+W3abc complex ( see Fig 3), which has the stabilization energy Kcal/mole at the B3LYP/6- 31+G(d) level. Thus the hydrogen bond strength of Isatin-(Water) n=1-3 clusters seems to be nearly additive. Interestingly the dipole moment of the complex W3abc was found to be approximately same as for isatin [See Table 1].

Fig 1b: Isatin+W1b (Binding Energy: -5.75Kcal/mol at MP2/6-31G(d) level)

Fig 1c: Isatin+W1c (Binding Energy: -5.98Kcal/mol at MP2/6-31G(d) level )

Fig 2a: Isatin+W2ab (Binding Energy: Kcal/mol at MP2/6-31G(d) level)

Fig 2b: Isatin+W2bc (Binding Energy: Kcal/mol at MP2/6-31G(d) level)

Fig 2c: Isatin+W2ac (Binding Energy: Kcal/mol at MP2/6-31G(d) level )

Fig 3: Isatin+W3abc (Binding Energy: Kcal/mol at MP2/6-31G(d) level)

B. Effect of Microsolvation on the IR spectra: The DFT calculations are used to obtain computed harmonic vibrational frequencies and infrared intensities for isatin and isatin- (water) n clusters. These, in turn, serve as a basis both for distinguishing, which structures would be observe experimentally and for analyzing the spectroscopic consequences of the hydrogen bonded structures. The frequency shift of the XH stretch vibration has been used as an indirect measure of the strength of an XH….Y H-bond. The magnitude of this frequency shift is correlated with a lengthening of the RXH bond, a shortening of the RXY distance and an increase in the H- bond binding energy. Selected vibrational frequencies for isatin and isatin water complex are described in Table 2.

Table 2: Theoretical IR spectra of Isatin and isatin clusters S.No. Description of Vibrations Calculated IR Wave No. at B3LYP/6-31+G(d) IsatinIsatin+W1a Isatin+W1b Isatin +W1cIsatin+W2ab Isatin+W2bcIsatin +W2ac Isatin +W3abc 1. C=O str 1816(418)1820(176)1809(365)1796(359)1814(168)1786(295)1795(428) 1790(384) 2. C=O str( Amide) 1836(270)1802(493)1826(271)1835(370)1795(447)1827(348)1808(271)1798(255) 3. N-H str3624(44)3490(207)3623(53) 3627(49) 3491(273)3625(55)3509(205)3492(291) 4. O-H sym. Str(Wa) (366) (239) (378)3630(266) 5. O-H asym. Str(Wa) (102) (111) (102)3843(108) 6. O-H sym. Str(Wb) (118) (174)3725(120) (194) 7. O-H asym. Str(Wb) (49) (67)3812 (59) (66) O-H sym. Str(Wc) O-H asym. Str(Wc) (342) 3831(118) (252) 3833(128) 3623(311) 3832(123) 3645(235) 3834(132)

Isatin-(Water) 1 The symmetric stretching mode of water was found to be red shifted by 160 and 117 cm-1 in the isomers Isatin- W1a and Isatin- W1c respectively whilst a red shift of only 20 cm-1 was found in the Isatin- W1b, indicating the relative strength of hydrogen bonding of corresponding isomers. The asymmetric stretching mode of water is very little affected due to hydrogen bonding, in general, however a significant red shift (53 cm-1) was found in the isomer Isatin- W1b. NH stretching is also significantly red shifted by 134 cm-1 in the isomer Isatin- W1a whereas the same stretching mode (in the similar condition) is shifted by 89 cm-1 in the indole-water cluster 10. This indicates stronger hydrogen bonding in isatin in comparison to indole at the NH site, which arises probably due to a higher electron density at N-atom in isatin. Carbonyl stretching vibration is also strongly affected by complexation of isatin with water. In the isomer W1a the ketonic C=O stretch is blue shifted (4cm-1) whereas the amide C=O stretch is red shifted by 34 cm-1.The order of the in phase and the out of phase carbonyl stretching modes of isatin is found to be reversed after complexation with water at the NH site.

Theoretical IR Spectra

Isatin-(Water) 2, 3 Again, the asymmetric stretching mode of water in Isatin+W2a & Isatin+W2c clusters is only slightly affected while the symmetric stretching mode is significantly red shifted due to hydrogen bonding. The NH stretching vibration of these complexes is red shifted by 133 and 105 cm-1 respectively. A significant red shift in asymmetric stretching mode of water was again found in the isomers involving Wb positions in comparison to other. The asymmetric and symmetric stretching modes of water are relatively less affected in the Isatin-(Water) 3 complex, however the NH stretching and carbonyl stretching modes are red shifted significantly.

C. Vertical Excitation energies: The four significant absorption peaks, reported in the UV-Visible spectra of isatin (in acetonitrile solution) are at 224, 232, 292 and 414 nm respectively ; these are well reproduced by our present calculations in the gas phase. The longest wavelength (broad) absorption peak of isatin is observed in benzene solution at 404 nm is predicted at 406 nm for the isatin monomer and is expected to involve excitation of a non bonding (lone pair) electron to an occupied π* orbital. Due to the small bathochromic shift observed for this peak was argued that this n-π* transition also has some charge transfer character.The sharp absorption peak observed at 292 nm, which is similar to the excitation of the Lb excited state in indole is attributed to π- π* transition. The two other absorption peaks observed at 232, and 224 nm are quite intense and are attributed to π- π* transitions.

Effect of Hydration on vertical excitation/electronic spectra The effect of single water hydration on these excitation energies is also investigated in the same approximation. The longest wavelength absorption peak of isatin (predicted at 406 nm) is seen to be significantly red shifted as observed experimentally when a single water molecule is added to isatin (Table 4). The π- π* electronic transitions earlier predicted at 232, and 224 nm (for isatin) are also shifted to longer wavelengths after addition of water. Interestingly, the higher energy π- π* transition at ~ 206 nm which is observed as a shoulder on the 224 nm absorption peak is seen to be blue shifted after monohydration of isatin.

4. Conclusion In the present report the structure, stability, vibrational characteristics and the vertical (electronic) excitation energies for isatin and Isatin- (water) n=1,2 complexes have been investigated using MP2 and DFT calculations. It has been shown that the cyclic hydrogen bond between amide moiety and water is the strongest among the all possible hydrogen bonds in the system. The hydrogen bond strength of Isatin-(Water) n=1-3 clusters is seems to be nearly additive. For a particular position of complexation of water, W1b, results in an unusual increase in the dipole moment. This is attributed to a significant amount of electronic charge displacement from the N atom to carbonyl (amide) C-atom which causes a large separation between the effective charges forming the dipole. These complexes are expected to show a small charge transfer character. The NH…OH hydrogen bonding is found to be relatively stronger in isatin as compared to indole. The experimentally observed electronic absorption peaks are reasonably reproduced by the TD-DFT vertical excitation energies. It was found that the longest wavelength absorption peak of isatin at 406 nm, is significantly red shifted after addition of a single water molecule. It is noted that, since the comparable experimental results are lacking, the present results can be used as a point of reference for future experimental work on the spectroscopy of Isatin-(Water) n clusters.