Signature of Glycylglutamic Acid Structure

Glutamate (Glu) is of great interest in biomedical research. It is considered a biomarker in diabetes, which may potentially contribute to the development of autism in genetically vulnerable human populations, and it is found in relation to advanced glycation end products (AGEs) [1]. Additionally, Glu plays an active role in the function of ligand-gated ion channel glutamate receptors, chloride channels capable of filtering glutamate, as well as Potassium (K⁺)-channel [2]. Glu attains α [3] and β [4] crystal forms and Cβ-CH2 show asymmetric ¹H signal pattern in NMR spectra.

The current study was undertaken to understand the signal patterns of Cβ-CH₂ in Glu of the smallest dipeptide, Glycylglutamic Acid (GlyGlu), as well as the order, and planarity of the amide bond in the molecule.

NMR spectra of GlyGlu were measured in D₂O to deduce ¹H and ¹³C chemical shifts and coupling constants. GlyGlu was crystallized from MeOH and the structure was determined by single crystal X-ray diffraction techniques.

The sidechain of Glu in the dipeptide dissimilates the β form. The amino group of Gly (Glycine) is protonated and exhibits hydrogen bonding with the main chain carboxylate group of a symmetry-related Glu that is deprotonated in the crystal packing of GlyGlu. The deprotonated main chain carboxylate of Glu is also in hydrogen-bonding distance from the side chain carboxylic acid group that is in the protonated form of a symmetry-related Glu of the dipeptide. The Cβ-CH₂ geminal protons on the side chain of Glu have different chemical shifts and splitting pattern in ¹H NMR reflecting their dissymmetric environment.

The results reported will be useful for monitoring changes that Glu and/or molecules in connection to Glu may undergo in in vivo, in situ, and in vitro conditions. This provides a valuable metric which will enable the examination of the metabolites relevant to the detection and diagnosis of disease or developmental conditions, as well as scrutinizing the effectiveness of treatment options.