NMR (Pro)chiral Discrimination Using Polysaccharide Gels

Abstract

Residual dipolar couplings (RDCs) are valuable for structure determination by solution-state NMR spectroscopy; this useful attribute has led to the development of several classes of alignment media such as liquid crystals, bicelle and phage suspensions, and compressed polyacrylamide gels.[1] Chiral media such as stretched gelatin and collagen have also been shown recently to resolve enantiomers by quasi diastereomeric complexation.[2] The degree of alignment in the magnetic field that guest molecules inside these media experience is generally a function of the concentration of the latter down to a minimum anisotropic concentration for that particular medium. However it would be very advantageous if one could reversibly adjust the resulting RDCs within a wide range without having to change the sample because of overlapping and unresolved spectral resonances. In addition it would be even better to be able to switch the sign of the dipolar coupling constant D when needed. Recently we described a simple device that achieves this by using a gel of gelatin as the chiral alignment medium. [3,4] The method is based on the gel containing polar guest molecules in an elastic silicone rubber tube that allows the rapid and reversible adjustment of the degree of nuclear alignment inside the NMR samples (starting from zero alignment) by merely stretching the rubber tube. [4,5] Stretched samples can be quickly and reversibly converted into compressed ones by melting the stretched gel by heating it at 37 8C, letting the gel reset by cooling, and then (partially) releasing the now isotropic sample; this compresses the gel and changes the sign of D. Although gelatin has been an extremely versatile alignment medium, there are drawbacks to its use: the relatively high protein concentration needed to resolve mixtures may limit its use for structure determination of proteins, and the need to retain samples below 20 8C does not allow kinetic studies at physiological temperatures.Here, we describe two new chiral anisotropic gels that can be reversibly adjusted just like gelatin, but need much lower gel concentrations, and can be used at 37 8C. Some unanticipated properties of these gels (see below) provide new opportunities in studies of (pro)chiral molecular structures. These gels are commercially available polysaccharides prepared from seaweed: i-and k-carrageenan. As solutes prochiral glycine, alanine enantiomers, and prochiral [D 6 ]DMSO were used.The i-and k-carrageenan gels were prepared in concentrations between 3-10 % (w/v). Both gels were very sensitive to the concentration of NaCl, and generally became more rigid and brittle than identical gels prepared without added NaCl. i-Carrageenan formed the more elastic gel. Gelation occurred upon heating to 90 8C for several minutes, but we were also able to prepare i gels by restricting heating to 42 8C; this is a useful property if guest molecules, such as peptides and proteins, which readily denature in temperatures over 50 8C, are to be included in the sample. The guest molecules c...

1H and 13C NMR studies of glycine in anisotropic media: Double-quantum transitions and the effects of chiral interactions

“…In our hands these phenomena were able to be recorded because of the large interaction between glycine and the anisotropic electric field-gradient tensor (shielding tensor) in stretched and compressed polymer gels, particularly in gelatin gels. The ''hidden'' scalar coupling of the enantiotopic methylene protons of glycine can only be measured by using chiral anisotropic media which in addition to their proven potential in enantiodiscrimination is another advantage of chiral alignment media [1, 10,19]. The accuracy of estimates of values of any assumed NMR parameters can be quickly checked by simulating the experimental data from stretched and compressed states of the same sample; this is a reversible process which inverts the signs of dipolar coupling constants D while leaving the signs of the scalar coupling constants J unchanged.…”

“…Gelatin gels (Gelita Asia Pacific, West Krugersdorp South Africa) and carrageenans gels (Sigma-Aldrich, St Louis, MO, USA) were prepared as described previously [9,10]. Cromolyn sodium salt (Aldrich) was dissolved in aqueous solutions of neutral pH (b).…”

“…3b). Stretched i-carrageenan gel although chiral as well did not yield this effect [10]. The additional signals could be correlated with the central resonance(s) by 13 C phase-sensitive COSY experiments.…”

“…These interactions are important in characterising ordered tissues such as cartilage and tendons, and potentially in other biological tissues such as muscle. In the present work we recorded z-spectra for glycine (an important amino acid constituent of connective tissue proteins) and its 13 C isotopomer glycine-2-13 C. Since chirality is an integral part of biological tissues, we dissolved glycine in chiral gelatin and carrageenan gels [9,10], as well as into achiral cromolyn liquid crystal [11] for comparisons.…”

Variable angle NMR spectroscopy and its application to the measurement of residual chemical shift anisotropy

“…The numerous applications range from improved assignment [5,6], discrimination of enantiomers [7][8][9][10][11][12][13][14][15], the indirect measurement of J-couplings [16], enhanced resolution by RDCs [17], relative configuration [18][19][20][21][22][23][24][25][26][27][28][29], and the 3D-conformation of molecules [30][31][32][33][34][35][36]. Next to liquid crystalline alignment media [37,38], the full power in structure determination is reached by stretched gels, which are now available for the major NMR solvents [21,26,[39][40][41][42][43][44][45][46][47], with no lower limit in alignment strength.…”