Recent studies on the development and application of analytical methods in HPLC with spectrometric detection for bio-related compounds have been reviewed. First, new fluorescent derivatization reagents with benzofurazan skelton have been developed. In the course of the study, the relationship between fluorescent characteristics and substituent groups of benzofurazan compounds was investigated. The relationship enabled to design new fluorogenic reagents easily Secondly, the simultaneous determination method for the sequence and D/L configuration of peptides with fluorescent Edman reagent was developed. In this study, the racemization mechanism of amino acids in the cleavage/cyclization step was investigated and boron trifluoride was employed as cleavage/cyclization reagent in order to supress the racemization. Finally, sensitive and selective determination system for catecholamine with peroxyoxalate chemiluminescence detection after derivatization with ethylenediamine was developed. Utilizing this system, plasma catecholamine level of spontaneously hypertensive rats and Wistar-Kyoto rats after administration of calcium antagonists were measured, and the attenuation of the baroceptor reflex was investigated.
The recent development of molecular biology has been elucidating outlines of the cross-talk of biomolecules. The understanding of the function of these biomolecules from the viewpoint of chemistry is now demanded not only for the understanding of biological systems but also for the creation of novel functional molecules. Here two topics are described about peptide synthesis aiming at the elucidation and the creation of protein functions. The first topic is the development of approaches for the synthesis of Tyr (SO3H)-containing peptides. Tyrosine sulfation is one of the most popular protein post-translational modifications. Synthetic peptides are of great help for the elucidation of the biological significance of tyrosine sulfation. We have developed two approaches for the efficient synthesis of tyrosine sulfate [Tyr (SO3H)]-containing peptides. The first approach employs a dimethylformamide-sulfur trioxide (DMF-SO3) complex as a sulfating agent and safety-catch protecting groups for the selective sulfation of tyrosine in the presence of serine. The second approach employs the direct introduction of Tyr (SO3H) into the peptide chain in the form of Fmoc-Tyr (SO3Na) followed by deprotection at 4°C in trifluoroacetic acid. These approaches were successfully applied for the synthesis of cholecystokinin (CCK)-related peptides. The second topic deals with new approaches for the creation of artificial proteins through assembling α-helical peptides via selective disulfide or thioether formation. Approaches to assemble individual peptide segments on a peptide template were also developed. Four peptides corresponding to the transmembrane segments of the sodium channel (S4 in repeat I-IV) were assembled on a peptide template to give a protein having ion channel activity with rectification.
Influence of tableting conditions on sticking was investigated using two formulations, the one formulation containing ibuprofen (formulation A) and the other being the standard formulation (formulation B). Sticking was observed on formulation A, and was not observed on formulation B. The degree of sticking was decreased with increase in compression pressure, and increased with compression speed. The shape of punch face was influenced the degree of sticking (sugar concave < bebel < flat). It was thought that sticking was observed when the adhesion between tablet and punch was larger than that among tableting granules. We investigated the pressure placed on a scraper (SCR), the adhesion between granules and metal, and boring hardness of tablet. The results showed that sticking was not observed when the SCR and/or the adhesion were small enough or boring hardness of tablet was high enough. Furthermore, it was suggested that boring hardness distribution would influence sticking.
During the course of our studies on the chemical constituents of botanically identified Chinese licorice roots of various origins, we have identified 15 kinds of saponins and 49 kinds of flavonoids (including flavonoid glycosides). With these chemical constituents at hand, we have carried out the chemical evaluation studies of licorice roots available in Japan by means of high performance liquid chromatography (HPLC). In the HPLC analysis using an ODS reversed-phase column, a combination of 1% aqueous acetic acid and acetonitrile was used as the mobile phase in a gradient solvent system, and the eluate was monitored with UV absorption at 254 nm (for saponins with a diene or a conjugated carbonyl moiety, liquiritin, and liquiritin apioside) or at 350 nm (for other flavonoid constituents). By our analytical method described here, quantitative analyses of saponin and flavonoid constituents were effected simultaneously. We have found an interesting piece of information concerning the origin of parent plants and their characteristic constituents such as saponins and flavonoids.
Many diterpenoids containing mostly ent-kaurenoids have been isolated from the Rabdosia (Labiatae). The relation of the diterpenoids with its species in Japan has been studied. The ratio of diterpene types and the values of oxidation state in each plant were calculated. These species were found to be divided into four groups by the cluster analysis of the values of oxidation state of C-1 to C-20 in the diterpenoids of the Rabdosia.