In order to investigate the relationship between bitterness and chemical structure of BPIa, eleven kinds of fragments and analogs of BPIa were synthesized. des–Gly2–BPIa and des-Pro4-BPIa exhibited extremely bitter taste. However, the pentapeptide (Arg–Gly–Pro–Pro–Phe) of BPIa possessed weak bitterness. The bitterness exhibition of BPIa probably derived from the spatial structure of its molecule.

In order to ascertain the validity of the structure of bitter peptide BPIa (H–Arg–Gly–Pro–Pro–Phe–Ile–Val–OH) isolated from casein hydrolyzate by Minamiura et al., the C-terminal octapeptide of β-casein (H–Arg–Gly–Pro–Phe–Pro–Ile–Ile–Val–OH) determined by Ribadeau Dumas et al., which is assumed to correspond to BPIa, was synthesized and compared with the synthetic BPIa which was confirmed to be identical with the natural one. The two synthesized peptides, however, did not match on thin-layer chromatography. The result indicates that the structure of BPIa need not be converted to Ribadeau Dumas’ formula. The octapeptide possessed an extremely bitter taste, with a threshold value of 0.004 mM (1 M=l mol dm−3). It is the most bitter peptide.

A bitter heptapeptide BPIa was synthesized and compared with the natural peptide, isolated by Minamiura et al. from casein hydrolyzate, by means of thin layer chromatography, paper electrophoresis, and carboxymethylcellulose column chromatography. All results for the two peptides matched very closely each other. The synthetic BPIa has an extremely bitter taste with its threshold value 0.05 mM. It is one of the most bitter compounds such as phenylthiourea and quinine.

In order to elucidate the relationship between chemical structure and bitter taste of BPIa, the analogs containing d-proline or glycine in place of the l-proline in the 3- or 4-positions were synthesized. The bitter taste of [d-Pro3,4]–BPIa (ld-d), [d-Pro3]–des–Pro3–BPIa (2d), and [Gly3]–des–Pro3–BPIa lacking the l-proline residue was much weaker than that of the peptides containing the l-proline, BPIa (1l-l), 1l-d, 1d-l, and 2l. The CD curves of these analogs and BPIa were measured in water. The results suggested that the bitter taste of BPIa is caused by the spatial structure attributed to the l-proline residue.

In order to elucidate the structure-taste relationship of bitter peptide BPIa (Arg–Gly–Pro–Pro–Phe–Ile–Val), cyclo-BPIa was synthesized. The result of both taste and CD examinations suggested that the bitterness of BPIa is caused by its spatial structure, which is analogous to that of cyclo-BPIa.