Nonnutritive sweeteners: taste-structure relationships for some new simple dihydrochalcones.

Six sweet and five nonsweet simple nonglycosidic dihydrochalcones were prepared, two of which have properties comparable to those of the intensely sweet neohesperidin dihydrochalcone.

Development of rebiana, a natural, non-caloric sweetener.

Rebiana is the common name for high-purity rebaudioside A, a natural non-calorie sweetener 200-300 times more potent than sucrose. It provides zero calories and has a clean, sweet taste with no significant undesirable taste characteristics. It is functional in a wide array of beverages and foods and can be blended with other non-calorie or carbohydrate sweeteners. It is stable under dry conditions, and has much better stability than aspartame or neotame in aqueous food systems. Studies undertaken for the development of a purification process and for the full characterization of the properties of rebiana are reported here.

Diterpenoid sweeteners. Synthesis and sensory evaluation of stevioside analogues with improved organoleptic properties.

Congeneric series of stevioside (1) and rebaudioside A (3) analogues have been prepared. It was found that the bitter-taste component endogenous in the natural compounds 1 and 3 may be eliminated by increase in molecular hydrophilic character. Through the series of compounds prepared, bitter-taste character was correlated with k', a chromatographic indicator of gross hydrophilicity. An analogue (11) of stevioside, shown chromatographically to be of increased hydrophilicity, was prepared and found to exhibit no bitter-taste character. Similarly an analogue (13) of rebaudioside A, having increased polarity, was prepared and found not to exhibit any bitter taste. The rebaudioside A analogue 13 was determined to have higher potency than 11 is suggested as a potential non-nutritive sweetener for food applications.

Understanding the mechanism of sweet taste: synthesis of ultrapotent guanidinoacetic acid photoaffinity labeling reagents.

Azido-functionalized analogs of potently sweet guanidinoacetic acids have been synthesized for use as sweetener receptor photoaffinity labeling reagents. These compounds have been synthesized using readily available starting materials. One of the azido-labeled guanidinoacetic acids has been evaluated in an electrophysiological model in the Rhesus monkey. We found that the photoaffinity-labeling reagent caused irreversible inhibition in electrophysiological response to sweeteners upon exposure of the monkey tongue to a combination of the reagent and UV light.

N,N'-disubstituted guanidine high-potency sweeteners.

The role and function of the aryl group in the highly potent trisubstituted guanidine sweeteners 7a-d was investigated. Four disubstituted guanidines, lacking the aryl group, were prepared. These guanidines contain a hydrophobic substituent on one nitrogen and a carboxymethyl group substituted on one of the other nitrogens. They were found to be tasteless or to have a significantly lower sweetness potency than the corresponding trisubstituted compounds. Possible rationales for the effects of structure on the sweet taste activity are discussed.

Dihydrochalcone sweeteners. A study of the atypical temporal phenomena.

Neohesperidin dihydrochalcone (NHDHC), known since 1963 as an intensely sweet compound, is determined to be 340 +/- 60 (p less than 0.05) times more potent than sucrose. The unusual temporal properties of this material are hypothesized as being due to the effects of metabolism, conformation, chelation, or hydrophobicity. Forty-four analogues are synthesized to test the four hypotheses, none of which are strongly supported. A method of quantitation of temporal characteristics of tastant molecules is developed so as to allow comparison of taste appearance time (AT) and extinction time (ET) of experimental compounds. Four of the new compounds, 40 and 43-45, exhibit high sweetness potencies, ranging from 280 and 440 times sucrose, and may be useful in selected food systems. The temporal taste characteristics remain unimproved over NHDHC, however.

Taste in chimpanzee: I. The summated response to sweeteners and the effect of gymnemic acid.

The purpose of this study was to investigate further with electrophysiological and behavioral techniques the similarity between the sense of taste of humans and chimpanzees, especially with regard to the effects of gymnemic acid. Gymnemic acid (GA) is a powerful suppressor of sweet taste in humans but lacks this ability in nonprimates and lower primates. The summated taste responses from the chorda tympani nerve were recorded in nine adult chimpanzees, Pan troglodytes. The results show that all tested compounds that taste to humans elicited nerve responses in chimpanzees. GA suppressed or abolished the response to all sweeteners, but had no effects on the responses to the nonsweet compounds. The suppression varied from complete abolishment (aspartame, saccharin), to about 50% reduction (xylitol). When the effect of GA was tested on concentration series, 20% remained of the response to sucrose, whereas the responses to aspartame and saccharin were basically abolished. Higher concentrations of GA suppressed more. The effects of GA developed also in the presence of saccharin, but seemed less pronounced. The behavioral results were obtained with a one-bottle preference technique before and after GA. The results demonstrated that after exposure of the tongue to GA, the animals' liking for sweet diminished. These results parallel psychophysical and electrophysiological findings in humans. The way GA suppressed sweet taste in chimpanzees added one more characteristic to those that set chimpanzees apart from monkeys and close to humans.

Recognition of superpotent sweetener ligands by a library of monoclonal antibodies.

Monoclonal antibodies (mAb) made to the superpotent guanidino sweet tasting ligand, N-(p-cyanophenyl)-N'-(diphenylmethyl)-guanidineacetic acid were examined for their molecular recognition specificities using 14 different sweetener analogues in a competitive radioimmunoassay. The effects of variations in pH on ligand binding was also examined by radioimmunoassay. Photoaffinity labelling of the binding site was accomplished using a radiolabelled azido-derivative of the parent ligand, and L-chain or H-chain labelling was easily identified in several different mAb. For two of the mAb examined in this study (NC6.8 and NC10.14), the analogue binding studies are in agreement with the known Fab-ligand crystal structures. Monoclonal antibodies to this family of sweet tasting compounds may be useful probes for the study of sweet taste chemistry and identification of novel sweet taste ligands from combinatorial chemical libraries.

Identification of residues in monoclonal antibody NC10.8 that bind to the sweetener N-(p-cyanophenyl)-N'-(diphenylmethyl)guanidinoacetic acid by using radioligand binding, absorption and fluorescence spectroscopy, computer-aided molecular modeling, and site-directed mutagenesis.

The interactive residues for mouse mAb NC10.8, which binds a superpotent guanidinium sweetener N-(p-cyanophenyl)-N'-(diphenylmethyl)guanidinoacetic acid with high affinity (Kd = 5 nM), were examined by using radioligand competitive binding, photoaffinity labeling, absorption and fluorescence spectroscopy, computer-aided molecular modeling, and site-directed mutagenesis. Competitive ligand analogue binding data revealed important structural features and a pH sensitivity for ligand binding. Spectroscopy of the sweetener-mAb complex revealed ligand-induced fluorescence quenching and the presence of a charge-transfer band. Site-directed mutagenesis of L:96W abolished the ligand-induced fluorescence quenching and reduced Ab affinity. The apparent Kd increased from 5 nM to more than 200 nM after such modification. A theoretical model of the Fv region was generated with use of a knowledge-based algorithm, and this model was used to identify the locations of key residues in the complementarity determining regions. These experimental and theoretical studies support the prediction that the sweetener ligand coordinates with the following residues: L:34H contacts the cyanophenyl ring, L:27DR forms a salt bridge with the acetic acid moiety, L:96W forms a pi-pi interaction with the cyanophenyl ring, and H:95E contacts the positively charged aryl nitrogen. These studies are important to our understanding of Ab-ligand specificity and may also shed light on the important chemical motifs responsible for elevated levels of sweetness potency in organic compounds.