Racemization studies in peptide synthesis through the separation of protected epimeric peptides by reversed-phase high performance liquid chromatography

Separation of protected epimeric peptides, Z-Gly-Xaa-Xbb-OMe (where Xaa and Xbb = chiral amino acid residues), by reversed-phase HPLC was utilized for studying racemization in peptide synthesis. Thus, the following factors which might affect the extent of racemization during the coupling by the carbodiimide method were investigated: the combination of amino acid residues to be coupled, coexisting tertiary amine salts, and the relative configuration of the amino acid residues. The following points were revealed: the combination of bulky residues at the coupling site results in extensive racemization in a polar solvent such as DMF, the amine hydrochlorides cause less racemization than the p-toluenesulfonates in DMF, and the influence of relative configuration differs depending on the solvent and the individuality of the amino components. Furthermore, the racemization-suppressing effect of some additives in the carbodiimide method was reevaluated by employing the same procedure.     

Effect of copper(II) chloride on suppression of racemization in peptide synthesis by the mixed anhydride and related methods

In segment couplings by the mixed anhydride method using isobutyloxycarbonyl chloride, the use of copper(II) chloride as an additive suppressed racemization completely in the same manner as in the carbodiimide method reported previously. This was confirmed by employing a number of couplings between Z-dipeptides and amino acid esters. The racemization-suppressing effect of other compounds were also evaluated by employing one of these model couplings to be at best only limitedly effective. Copper(II) chloride was effective also in the related method using EEDQ. Thus, in the couplings where a low level of racemization was observed without an additive, the addition of copper(II) chloride eliminated racemization even at ambient temperature where EEDQ is usually used. The effectiveness of copper(II) chloride was confirmed also in the BOP-C1 method. In the presence of HOBt racemization was reduced to a low but still detectable level, while it was suppressed completely by the addition of copper(II) chloride.     

Sensitivity to two major allergens (Cry j I and Cry j II) in patients with Japanese cedar (Cryptomeria japonica) pollinosis

Background: Japanese cedar (Cryptmeria japonica: CJ) pollinosis is one of the most important allergic diseases in Japan. Recently, the second major allergen (Cry j II) was isolated from CJ pollen. There have been no prevalence studies of sensitivity to Cry j I and Cry j II among a large number of patients with pollinosis. Objective: This study was conducted to evaluate the prevalence of sensitivity to Cry j I and Cry j II. We measured specific IgE antibodies to these allergens in the sera of 145 patients. Furthermore, comparison of the sensitivity to Cry j I and Cry j II was examined by the hisiamine release assay. Methods: Specific IgE antibodies to Cry j I and Cry j II were assayed by a fluorometric ELISA. Allergen-specific histamine release was measured by a radioimmunoassay kit, Results: More than 90% of 145 patients had specific IgE antibodies to both allergens. the remainder had specific IgE to either one or the other. There were seasonal changes in the level of specific IgE. The changes in the levels of anti-Cry j II IgE antibodies were parallel to those of anti-Cry j I IgE. The histamine release assay with leucocytes from the patients demonstrated that the allergenic potency of the two allergens is almost the same. Conclusion: Cry j II is an as important a major allergen as Cry j I.     

Oral Administration of Chymotrypsin Labile Peptide for a New Test of Exocrine Pancreatic Function (PFT) in Comparison with Pancreozymin-secretin Test

A new test using N-benzoyl-L-tyrosyl-p-aminobenzoic acid (N-BT-PABA) for an evaluation of exocrine pancreatic function was compared with a pancreozymin-secretin test in 38 subjects. Urinary recovery of PABA, which is absorbed from the intestine and conjugated in the liver after an oral administration of N-BT-PABA, depends mainly on chymotrypsin activity. The recovery rate of PABA in urine decreases in chronic pancreatitis, in which chymotrypsin activity in the duodenal juice is disturbed. The recovery rate of PABA in calcifying chronic pancreatitis was 40.2 +/- 15% and significantly less than 81.2 +/- 7.4% in normal subjects (P less than 0.01). The amount of PABA in urine during eight hours was correlated with parameters of volume output- bicarbonate concentration and amylase output stimulated by injections of pancreozymin and secretin (P-S test). The new test using N-BT-PABA is useful for the evaluation of exocrine pancreatic function in general practice.     

Prostate-specific antigen changes as a result of chlormadinone acetate administration to patients with benign prostatic hyperplasia: A retrospective multi-institutional study

BACKGROUND: Antiandrogens used to treat benign prostatic hyperplasia (BPH) may affect the diagnosis of prostate cancer by decreasing serum prostate-specific antigen (PSA) values. We conducted a retrospective survey of BPH patients treated with chlormadinone acetate (CMA) and analysed the effect of CMA on their PSA values. METHODS: A chart-based, retrospective, multi-institutional survey of clinically diagnosed patients with BPH who had been treated with CMA (Prostal; 50 mg/day) was conducted. The patients were divided into three groups according to their baseline PSA values: <4 ng/mL, Group I; >or=4 ng/mL and <10 ng/mL, Group II; and >or=10 ng/mL, Group III. The changes in the PSA values from baseline after 16, 26, and 52 weeks of CMA therapy were predicted using a nonlinear regression model analysis. RESULTS: Data from 192 of the 303 enrolled patients were analysed. The predicted percent changes from baseline among all patients after 16, 26, and 52 weeks of treatment were -49.6%, -49.8%, and -49.9%, respectively. The corresponding values were -42.4%, -43.7% and -43.8% in Group I; -57.4%, -58.4% and -58.5% in Group II; and -50.6%, -50.6% and -50.6% in Group III, respectively. CONCLUSION: The PSA values reached a nadir, approximately -50% of the baseline, after 16 weeks of CMA administration and remained at the same level thereafter. When CMA is used to treat BPH patients, the possibility of abnormal increases in PSA associated with prostate cancer must be considered; if prostate cancer is suspected, prompt testing, including a biopsy, should be performed.     

Diagnostic Value of 201Tl-chloride Scintigraphy for Thyroid Tumors

Among 114 patients with thyroid disease examined by ²⁰¹Tl-chloridescintigraphy, 101 were given a histological diagnosis. Of 28cases of thyroid carcinoma, 22 (78.6%) were positive. In threeof the 22, an obvious accumulation of ²⁰¹Tl-chloride was revealedin the metastatic lymph nodes of the neck and mediastinum. Only 12 of 50 (24%) cases of thyroid adenoma were positive.In comparison, all cases of hyperthyroidism and chronic thyroiditiswere positive and all cases of subacute thyroiditis were negative.The histological types of thyroid tumor were examined. Of 22cases of papillary carcinoma, 18 (81.8%) were positive. In fourcases which revealed negative figures, the tumor was almostcompletely encysted in three and the tumor was small in theother. Of the adenomas, eight cases of tubular adenoma and threeof trabecular adenoma were all positive, but colloid adenomaand thyroid cyst cases were all negative. It was concluded that ²⁰¹Tl-chloride has no specific affinityfor malignant tumors and cannot be used to discriminate betweencarcinoma and adenoma, but surgery should be indicated for noduleswhich are positive scanning by ²⁰¹Tl-chloride.     

Effect of copper(II) chloride on suppression of racemization in peptide synthesis by the carbodiimide method

Copper(II) chloride was found to be an extremely efficient racemization-suppressing additive in the DCC method as compared with the hitherto known ones, by employing the model coupling Z-Gly-l -Val-OH +h-l -Val-OMe in DMF. Although some other copper salts also had a profound effect, copper(II) chloride was the best from the viewpoint of both racemization suppression and coupling efficiency. The effectiveness of copper(II) chloride was further confirmed by employing the EDC-mediated couplings of Z-Gly-containing dipeptides with amino acid esters or dipeptide esters, and those of Z-l -Ala (or l -Val)-l -Val-OH with amino acid esters or dipeptide esters. In almost all the cases studied, no detectable amount (< 0.1 %) of epimer was observed by the HPLC analysis in the presence of copper(II) chloride. This was also the case even with an extremely stringent coupling system Z-l -Pro-l -Val-OH + H-l -Pro-OMe. With reference to the mechanism of racemization suppression, it was found that copper(II) chloride has a strong ability to suppress the racemization of the 5(4H)-oxazolone, which may be formed from an activated carboxyl component during the coupling.     

Study on the Phenomenon of Locational Variation in the Speed of Discoloration During the Endoscopic Congo Red Test The Second Report: Ultrastructural Studies on Parietal Cell Populations

Both quantitative and morphological studies of parietal cells were done in order to investigate the cause of the phenomenon of locational variation in the speed of discoloration during the endoscopic Congo red test. The endoscopic Congo red test was performed in twelve patients with a peptic ulcer and/ or chronic gastritis. In each case, at the times when spotty discoloration was observed, five biopsy specimens were obtained from both the quickly discolored portion (already discolored portion) –Q and the slowly discolored portion (not yet discolored portion) –S of the greater curvature of the middle body at the same time and were separated into two groups (the Q-group and the S–group). Quantitative studies of the parietal cells were performed using the cell isolation method described in our first report. A morphological study was performed with an electron microscope. Two specimens were fixed in glutaraldehyde and osmium tetroxide, and finally embedded in Epon resin. Ultra-thin secretions contrasted with uranyl acetate and lead citrate were viewed with a JEOL-1200EX electron microscope. Each parietal cell was classified according to the classification of ultrastructual stages put forward by Black et al. The number of parietal cells per milligram wet weight of the quickly discolored portion was significantly larger than that of the slowly discolored portion similar to the results of our first report. However the ultrastructural aspects of the parietal cells were not found to be different between the Q–group and S–group portion. It is concluded that the locational variation of the speed of discoloration during the endoscopic Congo red test may be due to the unequal distribution of parietal cells and that the sensitivity and responsiveness of parietal cells to the stimulation may be almost the same in all of the acid secreting area.     

NUCLEAR MAGNETIC RESONANCE STUDY ON SOLVENT DEPENDENCE OF SIDE CHAIN CONFORMATIONS OF TYROSINE AND TRYPTOPHAN DERIVATIVES

The stereoselectively β-deuterated species of Ac-Ar-NHMe, Ac-Ar-OH and Ac-Ar-OEt (Ar = Tyr and Trp) and H-Trp-NHMe were synthesized. 270-MHz ¹H n.m.r. spectra of the normal and deuterated species of these aromatic amino acid derivatives were analyzed. For most of the tyrosine and tryptophan derivatives in various polar and nonpolar solvents, the lower-field and higher-field β-proton signals were found to be due to the pro-S and pro-R protons, respectively. However, the alternative assignments apply for H-Trp-NHMe in aqueous solution and for Ac-Tyr-NHMe and Ac-Trp-NHMe in nonpolar solvents. Such alternative assignments of β-proton signals were also found for H-Phe-NHMe, Ac-Phe-NMe₂ and Ac-Phe-OtBu. From the analyses of the ¹H n.m.r. spectra of tyrosine and tryptophan derivatives, the vicinal coupling constants and rotamer populations about the C_α-C_β bond were determined in various solvents. The rotamer populations of H-Trp-NHMe, Ac-Ar-NHMe, Ac-Ar-OH and Ac-Ar-OEt depend significantly on solvent polarity. Each of these four types of derivatives exhibits specific solvent-polarity dependences of rotamer populations. The solvent dependences of rotamer populations were substantially reduced on replacement of α-carbonyl group by methylene group, suggesting the effect of this carbonyl group in the solvent dependence of rotamer populations.     

Nuclear magnetic resonance analyses of side chain conformations of histidine and aromatic amino acid derivatives

Stereoselectively β-deuterated species were synthesized of Ac-His-NHMe, Ac-His-OEt, Ac-His-OH and H-His-NHMe, which are useful as models of histidine residues in peptides. From the spectral comparison of ¹H n.m.r., the β-proton resonances of the normal species were unambiguously assigned. In (C²H₃)₂SO, C₂²H₅O²H, C²H₃O²H, and C₅²H₅N solution and in aqueous solution, the lower-field and higher-field components of β-proton resonances of the four histidine derivatives are assigned to the pro-R and pro-S protons, respectively. The alternative assignments apply for Ac-His-NHMe, Ac-His-OEt and Ac-His-OH in non-polar solvents such as C²HCl₃. Vicinal coupling constants ³J_αβS and ³J_αβR were obtained for calculating the fractional populations of rotamers about the C_α–C_β bond. The rotamer populations depend little on the ionization states of the α-amino and carboxyl groups or the imidazole ring. The rotamer populations depend significantly on the solvent polarity, similar to those of Phe, Tyr and Trp derivatives. For the two β-proton resonances of His, Phe, Tyr, and Trp derivatives in a variety of solvents, linear relationships are found between the differences in chemical shifts and the differences in vicinal coupling constants.