Preparation of protected peptidyl thioester intermediates for native chemical ligation by Nα‐9‐fluorenylmethoxycarbonyl (Fmoc) chemistry: considerations of side‐chain and backbone anchoring strategies,and compatible protection for N‐terminal cysteine*,†

Abstract: Native chemical ligation has proven to be a powerful method for the synthesis of small proteins and the semisynthesis of larger ones. The essential synthetic intermediates, which are C‐terminal peptide thioesters, cannot survive the repetitive piperidine deprotection steps of N^α‐9‐fluorenylmethoxycarbonyl (Fmoc) chemistry. Therefore, peptide scientists who prefer to not use N^α‐t‐butyloxycarbonyl (Boc) chemistry need to adopt more esoteric strategies and tactics in order to integrate ligation approaches with Fmoc chemistry. In the present work, side‐chain and backbone anchoring strategies have been used to prepare the required suitably (partially) protected and/or activated peptide intermediates spanning the length of bovine pancreatic trypsin inhibitor (BPTI). Three separate strategies for managing the critical N‐terminal cysteine residue have been developed: (i) incorporation of N^α‐9‐fluorenylmethoxycarbonyl‐S‐(N‐methyl‐N‐phenylcarbamoyl)sulfenylcysteine [Fmoc‐Cys(Snm)‐OH], allowing creation of an otherwise fully protected resin‐bound intermediate with N‐terminal free Cys; (ii) incorporation of N^α‐9‐fluorenylmethoxycarbonyl‐S‐triphenylmethylcysteine [Fmoc‐Cys(Trt)‐OH], generating a stable Fmoc‐Cys(H)‐peptide upon acidolytic cleavage; and (iii) incorporation of N^α‐t‐butyloxycarbonyl‐S‐fluorenylmethylcysteine [Boc‐Cys(Fm)‐OH], generating a stable H‐Cys(Fm)‐peptide upon cleavage. In separate stages of these strategies, thioesters are established at the C‐termini by selective deprotection and coupling steps carried out while peptides remain bound to the supports. Pilot native chemical ligations were pursued directly on‐resin, as well as in solution after cleavage/purification.     

Chemical and enzymatic treatment of endothelin

Endothelin-1 (ET), the most potent vasoconstrictor yet discovered, is a peptide containirig 21 amino acids with two intrachain disulfide bridges. With the aim of obtaining two-chain derivatives, Et was submitted to chemical and enzymatic treatments. Reaction of ET with CNBr in 70% HCOOH gave, in addition to the expected [Hse⁷ lactone]-7,8-seco-ET and unreacted material, a by-product whose molecular weight was 25 m.u. greater than that of ET. When the reaction mixture, after lyophilisation, was immediately quenched with NH3-saturated dry MeOH, two products could be recovered in a 5:1 ratio, both obtained by nucleophilic attack of the homoserine lactone: the expected [Hse⁷-NH₂]-7,8-seco-ET and [Hse⁷]ET, resulting from competitive intramolecular reaction of the deprotonated α-amino group of the Asp⁸ residue. The Lys⁹-Glu¹⁰ bond turned out to be very resistant to enzymatic attack both by Lys-C-endopeptidase and trypsin. The 9,10-seco-ET derivative could be obtained by treatment with Lys-C-endopeptidase only by using a high enzyme/ET ratio and after a prolonged incubation time. Cleavage of the Lys⁹-Glu¹⁰ bond could not be achieved by treatment with trypsin, even with a high enzyme/substrate ratio. The main product was 13, 14-seco-ET, deriving from the action of chymotripsin (present as an impurity in the trypsin preparation) on Tyr¹³. The structure of these peptides was confirmed by amino-acid sequence analysis and fast atom bombardment mass spectrometry (FAB-MS). Nicking of the ET structure at different positions had different impact on the biological properties of the resulting derivatives. © Munksgaard 1995.     

Trypsin-mediated semisynthesis of salmon calcitonin

Salmon calcitonin, CT(1-32)·NH₂, was synthesised by the trypsin-mediated coupling of the peptide fragments CT(1-24) and CT(25-32)·NH₂, prepared by conventional Fmoc solid-phase chemistry. Optimal conditions regarding reaction time course, pH, proportion of catalyst, substrate concentration and composition of the reaction medium were determined from initial studies on the coupling of CT(1-11) to CT(12-24) and of CT(12-24) to CT(25-32)·NH₂. For the final successful semisynthesis, we found that it was unnecessary to protect lysine residues not involved in the coupling, and that secondary hydrolysis at these sites could be prevented by increasing the pH of the reaction medium. The reaction achieved equilibrium after 30-45 min, with overall conversion of around 30% of the initial amount of CT(1-24) substrate into product. Yields were depressed due to cyclisation of the CT(1-24) substrate via air-oxidation of the Cys¹ and Cys⁷ residues.     

Modified Two-Layer Preservation Method (M-Kyoto/PFC) Improves Islet Yields in Islet Isolation

Islet allotransplantation can achieve insulin independence in patients with type I diabetes. Recent reports show that the two-layer method (TLM), which employs oxygenated perfluorochemical (PFC) and UW solution, is superior to simple cold storage in UW for pancreas preservation in islet transplantation. However, UW solution has several disadvantages, including the inhibition of Liberase activity. In this study, we investigated the features of a new solution, designated M-Kyoto solution. M-Kyoto solution contains trehalose and ulinastatin as distinct components. Trehalose has a cytoprotective effect against stress, and ulinastatin inhibits trypsin. In porcine islet isolation, islet yield was significantly higher in the M-Kyoto/PFC group compared with the UW/PFC group. There was no significant difference in ATP content in the pancreas between the two groups, suggesting that different islet yields are not due to their differences as energy sources. Compared with UW solution, M-Kyoto solution significantly inhibited trypsin activity in the digestion step; moreover, M-Kyoto solution inhibited collagenase digestion less than UW solution. In conclusion, the advantages of M-Kyoto solution are trypsin inhibition and less collagenase inhibition. Based on these data, we now use M-Kyoto solution for clinical islet transplantation from nonheart-beating donor pancreata.     

Changes in the ratio of urinary α1-microglobulin to ulinastatin levels in patients with Alzheimer-type dementia and vascular dementia

Abstract Relationships between urinary levels of α1-microglobulin (α1M) and ulinastatin (UT) in patients with dementia were investigated. There were no significant differences in α1M and UT levels and α1M: UT ratios among three groups: age-matched control subjects, patients with either Alzheimer-type senile dementia (ATD) or vascular dementia (VD). Although a positive correlation was established between α1M and UT levels in these groups, the regression of the demented patients differed significantly from that of controls ( P <0.05). A tendency towards a negative correlation between α1M: UT ratios and the levels of severity or duration of the disease was displayed in the ATD group, whereas a tendency toward a positive correlation between α1M: UT ratios and the levels of severity was observed in the VD group. These results suggest that changes in the relationships between urinary levels of α1M and UT may provide a useful biochemical index for diagnoses of ATD and VD.     

血清α_1——抗胰蛋白酶在原发性肝癌诊断中的意义

作者采用火箭免疫电泳方法(ELA)分别对200例正常健康人、32例原发性肝癌及105例肝炎,肝硬化病例作了血清α_1—抗胰蛋白酶(α_1AT)定量测定。正常健康人及肝癌病人含量分别为2.510±0.280g/L(250.96±28.02mg/dl)、3.210±0.403g/L(321.01±40.26mg/dl),二者有显著性差异(P<0.01);3例αFP阴性的肝癌病例,α_1AT含量均高于正常,提示α_1AT对原发性肝癌的诊断有一定意义,尤其是α_1FP阴性病例。     

Mouse monoclonal antibodies to the human C3b receptor

Mouse monoclonal antibodies were raised against the human C3b receptor (CR1) molecule that had been purified from solubilized erythrocytes membranes. Four hybridomas were selected, cloned and expanded because their supernatants reacted strongly with insolubilized CR1 by ELISA and intensely stained B-dependent areas of the spleen and glomerular podocytes by indirect immunofluorescence. The four monoclonal antibodies, named J3D3, J8B10, J3B11 and J7C2, were IgG1 immunoglobulins. J3D3 immunoprecipitated two protein bands of apparent mol. wts 200,000 and 220,000 from 125I-surface-labeled human erythrocytes, which correspond to the two major allotypic forms of CR1. By indirect immunofluorescence, monoclonal antibodies stained polymorphonuclear leucocytes (PMN), most peripheral blood B-cells and a small subset of peripheral blood T-cells. J3D3 bound to CR1 on erythrocytes, PMN and lymphocytes with an affinity of 1-3 X 10(9) M-1 and recognized 170-1330 antigenic CR1 sites with an average of 740 sites/erythrocyte in 100 healthy individuals, approx. 50,000 sites/PMN and 15,000 sites/lymphocyte. There was a bimodal distribution of CR1 numbers on erythrocyte in the normal population. The four monoclonal antibodies similarly inhibited CR1-mediated decay of preformed cell-bound alternative- and classical-pathway C3 convertase sites. Two antibodies, J3D3 and J3B11, inhibited C3b-dependent rosette formation with lymphocytes, although much less efficiently than F(ab')2 polyclonal anti-CR1 antibody. Differences that were observed in the relative capacity of the antibodies to inhibit some of the functions of CR1 and in their ability to compete for binding of 125I-J3D3 to CR1 on erythrocytes, suggested that they are directed against different epitopes on CR1. Monoclonal antibodies provide useful means to assess and analyze the biological and immunoregulatory functions of the C3b receptor.     

Trypsin activity in chyme and juxtamural layer of mucus in experimental blocking of the Brunner's glands region of the rat duodenum

Department of Histology and Department of Operative Surgery, I. M. Sechenov Moscow Medical Academy Laboratory of Electron Microscopy, Institute of Nutrition, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR I. V. Sudakov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 112, No. 11, pp. 542–545, November, 1991.     

Preparation of a proteolytic enzyme mixture containing trypsin,elastase, and chymotrypsin for use in tissue disaggregation

Summary We describe a purification method for tissue culture-grade trypsin that yields an enzyme mixture with reproducible activities of trypsin, elastase, and chymotrypsin and eliminates amylase and lipase.