Activity guided fractionation of a commercially available yeast extract led to the identification of (S)-N2-(1-carboxyethyl)guanosine 5′-monophosphate, a nucleotide exhibiting pronounced umami taste enhancing activity. Applying model reaction systems, this compound was found to be formed upon Maillard reaction of 5′-GMP with short chained carbohydrates. Consequently, systematic studies were undertaken to better understand thermal transformation reactions of the purine ribonucleotide resulting in two structurally diverse groups of Maillard-modified 5′-GMP derivatives which were evaluated with regard to structure–activity relationships in human sensory studies. Results suggested that substitution of the nucleotide’s exocyclic amino function with small, unpolar residues has a beneficial effect on taste enhancement. In addition, selected representatives of the 5′-GMP derivatives were tested in a cell-based taste receptor assay, underlining the human sensory results.