Ab initio study of vibrational spectra of alanine in gas phase

The vibrational frequencies for the fundamental modes and the energies of low lying states of alanine in neutral and its zwitterionic form in gas phase have been calculated at RMP2 ab-initio level. The results so obtained have been compared with earlier theoretical calculations and experimental observations of IR and RAMAN spectrum. In the present study, it has been observed that our results are more nearer to the experimental observations than earlier Semi-empirical AM1 and ab-initio Restricted Hartree Fock and Density Functional Methods. It is also observed that unlike RHF/6-31G method of computation, Z-alanine could not maintain its zwitterion form with RMP2/6-31G method


INTRODUCTION
Amino acids are neutral in gas phase but have a zwitterionic structure in liquid or in solid phase.Zwitterionic structure allows hydrogen bonds to be formed, stabilizing the ionic conformation R-CH(COO -)NH 3 .Alanine is one of the 20 most common natural protienogenic amino acids i.e. the building blocks of proteins and is hydrophobic, with a methyl group side chain.L-alanine is the smallest naturally occurring chiral amino acid.It is also classified as a non-polar amino acid.D-alanine occurs in bacterial cell walls and in some peptide antibiotics.As alanine has both the active groups of an amine and a carboxylic acid they can be considered both acid and base.At a certain pH known as the isoelectric point, the amine group has a positive charge (is protonated) and the acid group a negative charge (is deprotonated).This ion is known as a zwitterion.The assignments for the fundamental vibrations of L, D, Z-alanine are Crystalline state serves as model zwitterion.The assignments for the fundamental vibrations of L, D, Z-alanine for an examination of a wide range of intermolecular interactions of importance in chemistry and biology 1 .Low frequency vibrations which contain information on weak interactions are of importance in enzyme reactions.Infrared and Raman spectra of á-alanine has been studied using different techniques.Robert et al 2 have reported the vibrational spectrum of single crystal of Lalanine.The temperature dependence of far infrared spectra of L-alanine has been investigated by Bandeker et al 3 .Csazar 4 has reported that out of the 13 possible conformations, five lowest energy conformers are within 6kj/mole of the lowest one.
Stepanian et al 5 studied the IR spectrum using matrix isolation technique.Barthes et al 6 have noted a structural instability in L-alanine while Migliori et al 7 have pointed out a nonlinear behavior of the low frequency Raman modes at 42 and 49 cm -1 .
Santosh Kumar et al 8 have studied the infrared, Raman and electronic spectra of alanine molecule in solid phase as well as in aqueous solution and calculated vibrational frequencies using AM1, RHF and DFT method with different basis sets and concluded that there is no significant difference between the corresponding frequencies of L and D-alanine in gas phase but frequencies are changed for alanine and Z-alanine in water.In the present work we have calculated the fundamental modes of vibrational frequencies for L, D and Z-alanine in gas phase using MP2 method 9 with different basis sets and compared with earlier theoretical calculations and experimental observations.The main purpose of our study would be therefore, to provide information on the spectra of alanine taken for the present study and on comparison with the experimental findings to see upto which extend there calculated results authenticate the adequacy and the reliability of the ab-initio method along with basis sets.

Computational details
In the ab-initio procedure, we first searched for the energy minima on the potential energy surface of alanine corresponding to the lowest energy conformer and then calculated the IR frequencies and intensities using harmonic approximation.Initially, geometry was optimized using AM1, RHF/6-31G, RMP2/6-31G and then this optimized structure is started as initial geometry for calculation of vibrational frequencies of L, D and Z-alanine (zwitterionic alanine) at RMP2/6-31G, RMP2/6-31+G* and RMP2/631++G** ab-initio level 10 .All calculation in the present work were carried out at the Department of Physics, Udai Pratap Autonomous College Varanasi on a Pentium IV PC using G 03 and GAUSS VIEW 4.1 VERSION 11 of ab-initio quantum mechanical program .

Relative stability and structure of alanine
The relative energies of different forms of alanine calculated at RMP2/6-31G, RMP2/6-31+G*, RMP2/6-31++G** and earlier calculated at RHF/6-31G and DFT level with various sets have been shown in table-1.From this table it is clear that one of the neutral form of alanine (L-alanine) is more stable as compared to its D-form and zwitterion in gas phase.Unlike RHF/6-31G level of calculation; the present study with RMP2/6-31G, RMP2/6-31+G*, RMP2/6-31++G** methods Z-alanine could not maintain its zwitterion form and it becomes neutral alanine with trans form of C-O-O-H.This trans form of neutral alanine is different only in the dihedral angle of C-O-O-H from the structure of the most stable form of alanine with cis form of C-O-O-H.

Vibrational spectroscopy
The infrared region of the electromagnetic spectrum extends from 14,000cm -1 to10cm -1 .The region of most interest for chemical analysis is the mid-infrared region(4,000cm -1 to 400cm -1 ) which corresponds to changes in vibrational energies within molecules.We have used MP2 optimized geometries as the starting point to calculate vibrational frequencies of L, D and Z-alanine.A scaling factor 0.9434 common to all frequencies is used to adjust the theoretical frequencies before comparison with earlier experimentally observed and theoretically calculated values of IR and RAMAN spectrum .The no. of bonds in alanine are 12 therefore there are 12 stretching vibrations and 21 bending vibrations according to formula (N-1) and 3N-6-(N-1) where N is no. of atoms.The assignments for calculated frequencies of L-alanine at RMP2/6-31G++g** are enlisted in table- (2).The optimized structures, IR and RAMAN spectrum of L, D and Z-alanine calculated at RMP2/6-31++G** level are shown in Fig. 1. (a to i ).A typical infrared spectrum is usually divided into two regions.The left half below 1000cm -1 is finger print region and right half above 1000 to 4000cm -1 is functional group region.The right half, above 2000cm -1 usually contains relatively few peaks, but some very diagnostic information can be found here.A very broad peak in this region between 3100cm -1 and 3600cm -1 indicates the presence of exchangeable protons, typically from amine or carboxylic acid groups.The calculated IR at RMP2/6-31++G** in this region is 3569 cm -1 is assigned to O-H stretching, which is greater frequency than expected for O-H stretching, which must be nearer to 3300 cm -1 this is because acid form strong bonds than alcohol, and their O-H bonds are more strongly polarized as -O -ä -H + ä this exceptional bonding stretch can also be explained on the basis of large contribution of the ionic resonance structure.The most characteristic vibrations of COOH is C=O Table1: Energies (a.u.),relative stabilities ("E) , zero point vibrational energies (ZPV), total energies (TE) including zero (ZPE)(a.u.) of L-alanine ,D-alanineand Z-alanine using rhf/  The calculated frequency 1080 cm -1 at RMP2/6-31++G is attributed C-O stretching vibrations.Here the hydrogen bonding as well as resonance weaken the C=O bond, as a result absorption takes place at lower frequency than that of aldehyde, ketone and alcohol.The calculated IR of L-Alanine at RMP2/ 6-31++G** is 3460 cm -1 is assigned to NH 2 antisymmetric stretching and the peak at 3364 cm -1 is assigned to NH 2 symmetric stretching.As oxygen is more electronegative than Nitrogen, results in a greater change in dipole moment than in N-H stretching hence O-H stretching vibration is observed at higher frequency.The observed IR and Raman frequencies at 1114 cm -1 and 1131 cm -1 is assigned to C-N stretching corresponding In the IR spectrum containing a large no. of peaks, it is noted that the region 1650-400cm -1 contain peaks due to bending vibrations but it is rarely possible to assign a specific peak to a specific group in the experimental IR spectrum.Few of the peaks observed in this region are, 1595cm -1 in IR and at 1615cm -1 in the RAMAN spectrum is assigned to NH 2 bending corresponding theoretical values of L, D by RMP2/6-31G and Z-alanine by RMP2/6-31+G* are 1605cm -1 , 1602cm -1 and 1616cm -1 respectively.
The peak observed at 1454cm -1 in IR and 1469 cm -1 in RAMAN spectrum is assigned to the CH 2 (C 5 -H 9 -H 7 ) bending , corresponding theoretical values of L, D and Z-alanine by RMP2/6-31++G** are 1453cm -1 , 1454cm -1 and 1459cm -1 respectively.Also the peak observed at 1307cm -1 in IR and 1338cm -1 in RAMAN spectrum is assigned to C 3 -H 4 bending corresponding theoretical values of Lalanine by RMP2/6-31++G** , D-alanine by RMP2/ 6-31G and Z-alanine by RMP2/6-31+G* are 1312cm -1 ,1310cm -1 and 1312cm -1 respectively.From the comparison of vibrational frequencies and energies calculated at MP2 level, with earlier theoretical calculations and experimental observations of the IR and RAMAN spectrum we conclude that L-alanine is the stable form of alanine among all other forms in solid phase and we get vibrational frequencies at par with experimental values.We have given assignments to all the frequencies which explain all possible aspects of molecular structure of alanine and these aspects serve as powerful tool in explaining the various enzymatic reactions e.g.alanine is the nonessential amino acid remains an important element of human muscle tissue, and is integral in the development of proteins throughout our bodies.
First isolated in 1879, alanine is processed from glutamate in muscle cells through a process defined as transamination.Transamination is a complex process in which there is a transfer of an amine group (organic compounds containing nitrogen) of a particular acid to one molecule (ketone acid) on another acid.This process takes place in alanine, resulting in the creation of pyruvic and glutamic acids.Alanine is also important because of the liver's ability to transform this simple amino acid into pyruvate.
Pyruvate is the beginning compound responsible for starting the Krebs cycle.This energy cycle is critical in producing ATP (energy) from specific chemical and enzymatic activities.Pyruvate may also provide benefits in cellular respiration and aid in the inhibition of body fat.Alanine is also transferred to a-ketoglutarate, and like pyruvate, is an organic compound that provides for a variety of critical processes in the body.A-ketoglutarate may provide a beneficial effect on the body's anabolic proper ties, hormones, and immune system response.