Infrared spectra of amino acid zwitterions isolated in alkali halide matrices
Introduction
Investigations of the spectroscopy of the amino acids have now been undertaken over several decades. The amino acids play a major role in the life of organisms on this planet, and as such have commanded our interest and attention. Because the amino acids may exist in the neutral or the dipolar (zwitterionic) forms, studies conducted in the vapor phase and in matrix isolation (MI) are of the neutral form while studies of aqueous solutions and the crystal refer to the dipolar form. In aqueous solutions and in the solid the zwitterions are subject to strong intermolecular interactions with neighboring molecules, either water or zwitterions. The theme of this paper concerns a new approach to vibrational studies of the zwitterions. In this approach, which is both theoretical and experimental, the zwitterions exist as monomers that are only weakly perturbed by their environment. A novel spray technique involving isolation in alkali halide matrices has been developed to achieve this. The method has similarities to the low-temperature, inert-gas, matrix isolation, where the perturbation by the matrix is considered to be minimal. It has the advantage of not requiring low-temperatures and sample vaporization. The role of the alkali halide is to provide a support to isolate and stabilize the amino acid in its dipolar, zwitterionic form.
The purpose of measuring the vibrational spectra of the zwitterions is to determine the frequencies of their fundamental vibrations, and their transition intensities. This information, together with the results of computational studies can be used to identify possible conformers and to predict molecular structures. Such an approach has been exploited with considerable success in the study of amino acids and dipeptides and has played a part in furthering our understanding of their structures and bonding.
There is a wide range of techniques that has been developed for the preparation of samples appropriate for the measurement of vibrational spectra. With reference to absorption spectra, the methods of sample preparation can be divided into two groups. In the first group, which includes vapors, beams, and low-temperature matrix isolation, the molecules experience minimum perturbation by their environment. In contrast, in the second group which includes solutions, mulls, and KBr pressed pellets, account must be taken of the strong intermolecular interactions. Examples of recorded spectra featuring these sampling techniques abound in the literature.
For the amino acids, essentially two types of spectra have been recorded corresponding to the two types of sample preparations described above. Those of the neutral, isolated molecule, as exemplified by the matrix-isolated spectrum of alanine (A) reported by Rosado and co-workers [1], and those of the strongly perturbed zwitterions as exemplified by the KBr pressed pellet spectrum of the dipeptide, alanyl-glycine (AG), and presented in Fig. 1. In the MI technique, spectra very similar to the vapor (neutral and monomolecular) are obtained but they are free of the band broadening associated with rotational structure. On the other hand, the pressed pellet or disk is essentially a dilute mixture of microcrystals of the sample with microcrystals of KBr, and so the spectrum of Fig. 1 is really that of the crystalline dipeptide. The crystal spectrum portrayed in the figure is typical of the crystal spectra of many molecules containing CH and NH bonds, and is characterized by broad and partly structured absorption extending from about 3500 to 2500 cm−1, and by a wealth of bands with varying widths below 1700 cm−1.
The infrared (IR) absorption spectrum of AG in KBr, prepared by our novel DSD (dissolution, spray, deposition) method, is presented in Fig. 2. It differs markedly from the KBr pressed pellet spectrum of Fig. 1 in that the broad absorption in the high frequency region is replaced by a small number of discrete, narrow bands, and the bands appearing in the region below 1700 cm−1 are fewer and better resolved. As such it marks a third type of amino acid spectrum. In common with the KBr pressed pellet, it records the spectrum of zwitterions, but of zwitterions only weakly perturbed by their environment.
The outstanding and unique feature of the DSD method is that monomeric zwitterions are obtained stabilized by the relatively much weaker intermolecular interactions with alkali halide neighbors. This is distinct from the crystal where a network of zwitterions characterized by strong intermolecular interactions is obtained, while in aqueous solutions a pattern of strong H-bonding between the solvent and the zwitterions occurs. One perspective on the difference can be gained from a comparison of the dielectric constants for water and the alkali halides, which are respectively 80, and 4–8 [2].
Section snippets
The method
The basic idea of our method is to separate and dilute the solid sample by dissolving it in a chosen solvent together with an alkali halide (matrix) in a high mass ratio of matrix to sample. In aqueous solution the sample molecules are well separated and are usually present as monomers. A DSD sample, corresponding to a room-temperature matrix isolation, is obtained by the controlled deposition of a very fine spray of a solution of sample and alkali halide onto an IR transparent window,
Infrared absorption spectra
In general, the IR absorption spectra of the amino acids and dipeptides are characterized by a number of prominent bands that are common to all of them. These bands correspond to the group vibrations of the amino acid chain. Thus, for the amino acids the spectra are characterized by prominent absorptions due to the asymmetric and symmetric stretches of the carboxyl group, and stretches and bends of the amino (NH3+) group. For the dipeptides the CO and NH stretches of the peptide linkage
Structures
Crystal structure determinations have been carried out for many of the amino acids and related molecules. These clearly identify the zwitterionic forms obtained in the presence of strong intermolecular interactions with like neighboring molecules. They are therefore only a poor guide to the neutral molecule structures obtained in matrix isolation and vapor, although they are a better, but still approximate representation of the structures in aqueous solutions. Similarly, they are only an
Other systems
Most of our DSD work has been restricted to amino acids and dipeptides. However, the DSD technique is not limited to these. Well-resolved DSD spectra have been recorded of purine, and of purine samples prepared from acid solution [3]. For the former a good match with theory is achieved for the N(9)H tautomer. The two DSD spectra are substantially different and more work is required to explore the nature of the purine in the KBr matrix, especially for purine prepared from acid solution [33].
The
Conclusions
The DSD sampling technique has been successful in producing well-resolved spectra of the amino acids and related substances that are quite distinct from the pressed pellet, aqueous solution and MI spectra. In essence, a new third category of spectra has been established.
The evidence which consists of:
- (i)
the highly resolved nature of the bands assigned to the NH stretching modes and the absence of the broad absorption extending from 3500 to 2500 cm−1;
- (ii)
the qualitative agreement noted between theory
Acknowledgements
Dr R Jacob, N. Cox, and M. Francis are thanked for their help in measuring some of the spectra.
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