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The [Tyr47, Nle53] preprorenin (47–60) peptide methyl ester, a tetradecapeptide related to the human renin prosegment, has been synthesized using a three-segment coupling strategy. Selective deprotection of the segments before coupling allowed an easy removal of the final tetradecapeptide side chain-protecting groups by acidolysis and an easy purification. Antibodies raised against this peptide bound the plasmatic inactive renin.

New α-amino ketones (5) derived from leucine have been synthesized by reaction of organometallics on N-methoxy-N-methylamide (4). The stereoselectivity of reduction of the α-amino ketones (5) was studied. The stereochemistry of the resulting α-amino alcohols (6) was established by transforming them into the oxazolidinones (8) by means of a new procedure.

The complete sequence of the structural gene coding for mouse submaxillary gland renin was recently reported and the amino acid sequence of preprorenin was deduced. This sequence includes a 45-amino acid peptide that corresponds to the prosegment of the renin precursor. To investigate whether peptides related to the renin prosegment are able to inhibit renin activity, we have synthesized four peptides having the following structures: Arg-Ile-Pro-OMe, butyloxycarbonyl(Boc)-Leu-Lys-Lys-Met-Pro-OMe, Boc-Arg-Ile-Pro-Leu-Lys-Lys-Met-Pro-OMe, and Boc-Glu-Arg-Ile-Pro-Leu-Lys-Lys-Met-Pro-OMe (corresponding to amino acids 12-14, 15-19, 12-19, and 11-19, respectively, of the renin prosegment). All four peptides were found to inhibit the activity of pure mouse submaxillary renin on a porcine synthetic tetra-decapeptide in vitro, and the most potent inhibitors exhibited IC50 values in the micromolar range. Enzymatic kinetic studies carried out using peptide 15-19 showed an uncompetitive or a mixed type of inhibition with a Ki value of 2.3 X 10(-6) M at 37 degrees C in 0.5 M citrate/phosphate buffer (pH 6.0).

The Michaelis–Arbuzov reaction involves the formation of dialkyl alkylphosphonates by heating alkyl halides with trialkyl phosphites, via an intermediate phosphonium salt. In recent years it has been found that phosphonium salts can be formed by reaction of a variety of trivalent phosphorus compounds with oxidizing electrophiles, and that the resulting phosphonium intermediates can be trapped with an alcohol to form alkoxyphosphonium cations. These cations can then undergo nucleophilic displacements, either with their own counterions or with added nucleophiles. This chapter is limited to species formed in situ from trivalent phosphorus compounds and various oxidants. The most commonly used trivalent phosphorus compounds are triphenyl phosphite, triphenylphosphine, and tris(dimethylamino)phosphine. The most commonly used oxidants are molecular halogens, alkyl halides, carbon tetrahalides, N-haloamides, N-haloamines, and diethyl azodicarboxylate (DEAD). Other oxidants that have been used include mercuric salts, peroxides, dithioesters, and sulfenamides. The more common combinations of phosphorus(III) compounds and oxidants are summarized. The replacement of hydroxyl groups by halogen, the most common use of oxyphosphonium intermediates, is the principal subject of this chapter. However, the replacement of hydroxyl groups by oxygen, nitrogen, sulfur, and carbon nucleophiles is also discussed. This review is restricted to alcoholic hydroxyl groups, and does not include a discussion of carboxylic hydroxyl groups. The principal combinations covered are triphenylphosphine–diethyl azodicarboxylate, triphenyl phosphite–methiodide, triphenylphosphine–halogen, triphenylphosphine–tetrahalomethane, triphenylphosphine–N-haloimide, and tris(dimethylamino)phosphine–tetrahalomethane. Some of these combinations have been discussed in recent reviews. Keywords: replacements; alcoholic hydroxyl groups; halogens; oxyphosphonium intermediates; triphenylphosphine; carbon tetrachloride; diethyl azodicarboxylate; carbonnitrogen bonds; esters; ethers; alcohol dehydration; scope; limitations; phosphoranes; halosuccinimide reagent; tris(dimethylamino)phosphine; anomeric position; tetrahalomethanes; haloamide; carbohydrates; selectivity; experimental procedures

Antibodies were raised against a synthetic tetradecapeptide which is a component of the non-renin portion (prosegraent) of human renin precursor. Inactive renin from human kidney and plasma strongly adsorbed to a gel coupled to iramunoglobulins purified from such an antiserum. These results suggest that renal and circulating inactive human renins contain in their structure the prosegment of prorenin.

A new pepstatin derivative, pepstatinyl arginine methyl ester hydrochloride (pepstatinyl-Arg-O-Me), was synthesized with the aim of increasing water solubility without altering capacity to inhibit the renin-angiotensinogen reaction. Pepstatinyl-Arg-O-Me was shown to inhibit in vitro the reaction between either rat or purified hog renin and the natural rat renin substrate. In a fluorimetric assay, this derivative competitively inhibited the reaction between purified hog renin and a synthetic N-acetyl-tetradecapeptide renin substrate with a Ki of the same order of magnitude as that of pepstatin. In urethaneanesthetized ganglion-blocked rats, binephrectomized 24 hr before the experiments, both the plasma reinin concentration and the rise in blood pressure following intravenous injection of hog renin were inhibited by pepstatinyl-Arg-O-Me disappeared within 60 min. Pepstatinyl-Arg-O-Me inhibited the 1.4 mumoles/kg. The in vivo inhibitory effect of 1.4 mumoles/kg of pepstatinyl-Arg-O-Me disappeared within 60 min. Pepstatinyl-Arg-Op-Me inhibited the reaction of endogenous and exogenous renin with plasma substrate both in vitro and in vivo.

Pseudo-peptide analogues of the C-terminal tetrapeptide of gastrin, in which a peptide bond has been replaced by a CH2-NH bond, i.e. (tert-butyloxycarbonyl)-L-tryptophyl-psi (CH2-NH)-L-leucyl-L-aspartyl-L-phenylalanine amide (8), (tert-butyloxycarbonyl)-L-tryptophyl-L-leucyl-psi (CH2-NH)-L-aspartyl-L-phenylalanine amide (13), (tert-butyloxycarbonyl)-L-tryptophyl-L-leucyl-L-aspartyl-psi (CH2NH)-L-phenylalanine amide (20), were synthesized. The pseudo-peptides 8 and 13 were shown to have the same affinity as (tert-butyloxycarbonyl)-L-tryptophyl-L-leucyl-L-aspartyl-L-phenylalanine amide (21) for the gastrin receptor on isolated mucosal cells. The pseudo-peptide 20 exhibited lower affinity (IC50 congruent to 10(-5) M). The biological activity of these pseudo-peptides was studied on acid secretion in the anesthetized rat. Compound 8 stimulated acid secretion, identically with that of 21. Compound 13 did not exhibit any agonist activity but was able to antagonize the action of gastrin (ED50 = 0.3 mg/kg). Compound 20 did not show any agonist activity but was able to inhibit gastrin-induced acid secretion, with lower potency (ED50 = 15 mg/kg). The importance of the peptide bonds in the mode of action of gastrin is discussed, and a hypothetical approach of the mechanism of action is presented.

Four peptides related to human renin flap region have been synthesized. Two of them are ring closed through appropriately designed disulfide bridges. Structure analysis involving IR and NMR techniques and recognition by polyclonal human renin antibodies provides support for a beta-hairpin secondary structure of the cyclized peptides identical with that presented by the flap section in the speculative human renin model [Blundell, T., Sibanda, B. L., & Pearl, L. (1983) Nature (London) 304, 273-275; Sibanda, B. L., Blundell, T., Hobart, P. M., Fogliano, M., Bindra, J. S., Dominy, B. W., & Chirgwin, J. M. (1984) FEBS Lett. 174, 102-111].

Reduction of peptidyl N, O-dimethyl hydroxamates with lithium aluminium hydride in diethyl ether at 0 degree allowed the preparation of peptidyl aldehydes in excellent yield and optical purity. These aldehydic peptides are able to inhibit renin activity. They are the shortest renin inhibitors known to date.

Synthese des esters de succinimidyle, de trichloro phenyle et de perfluorophenyle des N-t-butoxycarbonyl et N-benzyloxycarbonyl α-aminoacides

The synthesis of Lys-Arg-Asn-Lys-Asn-Asn-Ile-Ala representing the C-terminal octapeptide of oxyntomodulin isolated from pig intestine is described. Its structure was confirmed by its 360-MHz 1H NMR spectra. The octapeptide was tested for its ability to inhibit pentagastrin-induced acid secretion, in the anaesthetized rat, in the conscious rat with chronic gastric fistula, and in the conscious cat with gastric chronic fistula. The octapeptide inhibits pentagastrin-induced acid secretion in all three models. Compared to oxyntomodulin, the parent hormone, the synthetic peptide was approximately 150 times less potent but has the same efficacy. Biological data are presented and discussed.

Activation of N-protected aminoacids by isopropenyl chloroformate leads to esters of primary, secondary and tertiary alcohols with 4-dimethylamino-pyridine as catalyst.

Pseudo-peptide analogues of the C-terminal tetrapeptide of gastrin, in which a peptide bond has been replaced by a CH2-NH bond, i.e. (tert-butyloxycarbonyl)-L-tryptophyl-psi (CH2-NH)-L-leucyl-L-aspartyl-L-phenylalanine amide (8), (tert-butyloxycarbonyl)-L-tryptophyl-L-leucyl-psi (CH2-NH)-L-aspartyl-L-phenylalanine amide (13), (tert-butyloxycarbonyl)-L-tryptophyl-L-leucyl-L-aspartyl-psi (CH2NH)-L-phenylalanine amide (20), were synthesized. The pseudo-peptides 8 and 13 were shown to have the same affinity as (tert-butyloxycarbonyl)-L-tryptophyl-L-leucyl-L-aspartyl-L-phenylalanine amide (21) for the gastrin receptor on isolated mucosal cells. The pseudo-peptide 20 exhibited lower affinity (IC50 congruent to 10(-5) M). The biological activity of these pseudo-peptides was studied on acid secretion in the anesthetized rat. Compound 8 stimulated acid secretion, identically with that of 21. Compound 13 did not exhibit any agonist activity but was able to antagonize the action of gastrin (ED50 = 0.3 mg/kg). Compound 20 did not show any agonist activity but was able to inhibit gastrin-induced acid secretion, with lower potency (ED50 = 15 mg/kg). The importance of the peptide bonds in the mode of action of gastrin is discussed, and a hypothetical approach of the mechanism of action is presented.

The synthesis of Lys-Arg-Asn-Lys-Asn-Asn-Ile-Ala representing the C-terminal octapeptide of oxyntomodulin isolated from pig intestine is described. Its structure was confirmed by its 360-MHz 1H NMR spectra. The octapeptide was tested for its ability to inhibit pentagastrin-induced acid secretion, in the anaesthetized rat, in the conscious rat with chronic gastric fistula, and in the conscious cat with gastric chronic fistula. The octapeptide inhibits pentagastrin-induced acid secretion in all three models. Compared to oxyntomodulin, the parent hormone, the synthetic peptide was approximately 150 times less potent but has the same efficacy. Biological data are presented and discussed.

The primary structure of human preprorenin has recently been determined from its cDNA sequence. It includes a 46-amino acid NH2-terminal prosegment. Six peptides corresponding to the entire prosegment (9-40), except for the NH2-terminal (1-8) and COOH-terminal (41-46) ends have been synthesized. These peptides were tested for their inhibitory effect on human plasma renin activity. Boc-Tyr-Thr-Thr-Phe-Lys-Arg-Ile-Phe-Leu-Lys-Arg-Met-Pro-OMe (where Boc represents t-butoxycarbonyl and OMe represents methoxy) (h Y(9-20) and its fragment Boc-Leu-Lys-Arg-Met-Pro-OMe h (16-20) were the most potent inhibitors with IC50 values of 2 X 10(-4) and 3 X 10(-4)M, respectively. Peptides located near the COOH-terminus were less inhibitory. The inhibitory capacity of h (16-20) was studied further on highly purified human renin acting on either pure human angiotensinogen or a synthetic human tetradecapeptide substrate. In both of these assays its inhibitory potency was about 10-fold greater than that found on plasma renin activity. Peptide h (16-20) was 3-6 times less potent in inhibiting human renin than its mouse counterpart m (15-19) was in inhibiting mouse renin. Kinetic studies carried out with h (16-20) showed a mixed type of inhibition. When human angiotensinogen was used as substrate, Ki and K'i values were 17.7 +/- 3.9 and 2.9 +/- 0.9 microM, respectively. These studies showed that human renin, like mouse renin and pepsin, can be inhibited by peptides derived from its prosegment. In addition, as in the case of pepsin, they suggest that the NH2-terminal part of the prosegment interacts more strongly with the active enzyme.

A chimeric peptide, 32 amino acids long, bearing two active sites, one inhibiting trypsin (Kd = 1.8 10(-9) M), one carboxypeptidase A (Kd = 3 10(-9) M), was designed and synthesized. It is a "squash inhibitor" (EETI II, 28 amino acids) elongated with the 4 amino acids from the C-terminus of the potato carboxypeptidase inhibitor. It has 3 disulfide bridges assembled in the particular knotted topology shared by the two inhibitors, by conotoxin omega, and possibly by E. coli enterotoxin ST1b.

A series of C-terminal peptide segments of gastrin, i.e., (tert-butyloxycarbonyl)-L-tryptophyl-L-methionyl-L-aspartic acid amide, (tert-butyloxycarbonyl)-glycyl-L-tryptophyl-L-methionyl-L-aspartic acid amide, (tert-butyloxy-carbonyl)-L-tyrosyl-glycyl-L-tryptophyl-L-methionyl-L-asp artic acid amide, and (benzyloxycarbonyl)-L-glutamyl-L-alanyl-L-tyrosyl-glycyl-L-tryptophyl-L -methionyl-L-aspartic acid amide were prepared and were shown to competitively inhibit the binding of labeled human gastrin to its receptors in an isolated gastric mucosal cell preparation and to antagonize the action of gastrin on gastric acid secretion (ED50 from 1.5 to 7 mg/kg) in vivo in the reperfused rat stomach, determined according to the method of Ghosh and Schild. From these studies, it could be concluded that the C-terminal phenylalanine residue, which is of primary importance for intrinsic biological gastrin-like activity, is not essential for binding to gastrin receptors.

Renin (EC 3.4.23.15), an aspartyl protease, is a rate limiting factor on angiotensin II production which is a potent vasoconstrictor peptide implicated in the regulation of blood pressure and electrolyte metabolism. So renin is an important target in the design of antihypertensive drugs. Different approaches in the inhibition of the renin-angiotensinogen system have been adopted, such as the use of substrate analogues (Szelke et al., 1982), statine-containing peptides (Boger et al., 1983, 1985; Guegan et al., 1986), peptides derived from the renin profragment sequence (Cumin et al., 1985) and specific monoclonal renin antibodies (Galen et al., 1984). Recently, specific blockade of the renin-angiotensinogen system has been obtained by active immunization against renin in normotensive marmoset (Michel et al., 1987) but this active immunization induces an autoimmune desease, so it was worthwhile to conduct experiments with synthetic peptides related to renin sequence and to try to determinate the major renin epitopes.

A successful synthesis of Asp1-Arg2-Val3-Tyr4-IIe5-His6-Pro7-Phe6-His9-Leu10-Leu11-Tyr12-Tyr13-Ser14 has been achieved by the classical stepwise solid-phase method using 1% crosslinked Merrifield resin and benzotriazolyloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP reagent) respectively as solid support and coupling reagent. The coupling protocol included in situ neutralization of the amino partners after Boc-deprotection and the coupling times seldom exceeded 30 min. For His6 and His9, Boc-His(Boc). DCHA was neutralized in situ and allowed to couple. The peptide, purified by reparative h.p.l.c., was obtained in 29% overall yield based on the first residue attached to the solid support.

Activation of N-protected α-amino acids and O-protected α-hydroxy acids with 2,2'-carbonylbis (3,5-dioxo-4-methyl-1,2,4-oxadiazolidine) 6 provide a stable activated intermediate 7 suitable for the synthesis of β-keto ester 8 . This activation was used in the preparation of the non-proteogenic units 1st 1 and Hip 4 present in the cyclodepsipeptides didemnins isolated from the tunicates Didemnidae.

Several protease inhibitors were found in the seeds of a Cucurbitacea, Ecballium elaterium, and were separated from one another by affinity and molecular sieve chromatography. Three main trypsin isoinhibitors were purified by ion-exchange chromatography and the sequence of the major one, EETI II, was elucidated and compared with other inhibitors of the squash family. It is a peptide of M.W. 3020 of strong inhibitory activity (Ka = 8 x 10(11) M-1) against trypsin, showing high Gly content, six half-cystine residues, but devoid of histidine, threonine, tryptophan, and tyrosine residues.

Ohne nahere Angaben wird kurz berichtet, das im Original die Darstellung der Salze (I)-(III) beschrieben ist, die - wie z.B. an (II) aufgefuhrt - mit Arenthiolen in einem SNZ-Prozes zu den Sulfiden wie (IV) reagieren, dagegen mit Ag-tosylat unter Retention zu den labilen Tosylglykosiden wie (V), die in Alkoholen ein Anomerengemisch der Glykoside (VI) liefern.

1H and 13C n.m.r. study of pseudo-peptide analogues of the C-terminal tetrapeptide of gastrin, obtained by replacing each peptide bond by a "reduced peptide bond", one at a time, e.g. Boc-Trp psi (CH2NH)Leu-Asp-Phe-NH2 2, Boc-Trp-Leu psi (CH2NH) Asp-Phe-NH2 3, Boc-Trp-Leu-Asp psi (CH2NH)Phe-NH2 4, were reported. The CH2NH bond was completely characterized. 1H and 13C spectroscopic data were reported. It appeared from the present work that the modifications produced by the replacement of a peptide bond by a CH2NH bond were localized around the CH2NH.

To block the renin-substrate reaction by immunological tools is a long-standing dream. Since 1951 active immunization and the passive transfer of antirenin antisera have been successfully carried out in different species and in different experimental models of hypertension and normotension. These studies indicated that renin is a powerful immunogenic protein, capable of breaking down self-tolerance in different species. In this initial period the most significant results were obtained with hog renin. Passive transfer of antisera raised against hog renin or active immunization with hog renin was able to decrease blood pressure in renovascular or essential hypertension in dogs. Renin was semipurified and injected without adjuvant, however, since there was no method for determining plasma renin activity. Recently, complete purification of murine and human renin has allowed an extension of this approach, using the passive transfer of antirenin polyclonal antisera or monoclonal antibodies. Active immunization against pure human renin was successful in normotensive marmosets. This immunization with a nearly homologous renin in a primate model induced a significant decrease in blood pressure, associated with a complete disappearance of plasma renin activity. Unfortunately this powerful immunization was associated with an autoimmune disease that is specific for the kidney, related to self-recognition of the production site of renin by antibodies and lymphocytes. Similar results were reported with the use of mouse submandibular gland renin as an immunogen in spontaneously hypertensive rats (SHR). This manipulation decreased blood pressure in SHR to a level near that of normotensive Wistar-Kyoto control rats. However, again the animals showed a severe autoimmune disease of the kidney.(ABSTRACT TRUNCATED AT 250 WORDS)

Two peptides were synthesized, corresponding to the sequences (−19 to −7) and (−26 to −17) of the prorenin prosegment. Monoclonal antibodies were raised to these sequences and used to characterize human plasma inactive renin. Only anti (−19 to −7) reacted with inactive renin, as measured by direct assay or affinity chromatography. The data were used to evaluate two possible inactive renin stuctures: (i) plasma inactive renin is a truncated prorenin lacking the prosegment N-terminal portion; (ii) its spatial conformation masks the N-terminal extremity, preventing interaction of this region with specific antibodies.

The title compound (VIb) and the remaining analogues (VI) are prepared starting from the condensation of the protected amino acids (I) with Meldrum's acid (III).

Polyclonal and monoclonal antibodies were raised against pure human renin, but nothing was known about the regions against which they were directed. Using a three-dimensional model of mouse submandibular renin, we selected seven peptide sequences as belonging to potential epitopes. The main criteria for their choice were the location of the peptide sequences near the catalytic region and on the surface of the renin molecule and their hydrophilicity. After transposition of the regions to the 340-amino acid sequence of human renin, the seven peptides (corresponding to amino acids 50-60, 63-71, 81-90, 118-126, 162-169, 247-255, and 287-295) were synthesized, coupled to bovine serum albumin, and injected into rabbits. Five of these peptides elicited antibodies, and 50-68% binding of the corresponding iodinated peptide was obtained with a 1:25 dilution of antiserum. The antisera titers ranged from 1:5,000 to 1:100,000 when tested by enzyme-linked immunosorbent assay. The same antisera bound 15-65% of labeled pure human renin at a final dilution of 1:2.5, the highest percentage being obtained with peptide 81-90 antiserum. At a 1:5 dilution, the five antisera inhibited renin activity by 23-68% in human plasma with a high renin activity (40 ng of angiotensin I/h/ml). At a final dilution of 1:50, peptide 81-90 antiserum was still capable of producing 25% inhibition. Purified IgG (0.6 mg) from this antiserum inhibited pure human renin activity by up to about 40%, as measured by its reaction with pure synthetic human tetradecapeptide substrate. Antigenic peptides that mimic a part of the human renin sequence, especially peptide 81-90 representing the "flap" covering the cleft between the two renin lobes, constitute promising tools for the development of a synthetic antirenin vaccine.

A series of tri- and tetrapeptide derivatives, analogues of the gastrin C-terminal region with no phenylalanine residue, were synthesized. These peptides were tested for their ability to inhibit gastrin-stimulated acid secretion in vivo as well as binding of [125I]-(Nle11)-HG-13 to gastric mucosal cell receptors in vitro. Most of the peptides tested exhibited gastrin antagonist activity in vivo and in vitro. Most active derivatives were 20-30 times more potent than the well-known gastrin antagonist derivatives proglumide and benzotript and had 20-200 times more binding affinity. The smallest fragment exhibiting antagonist activity was the tripeptide Boc-L-tryptophyl-L-methionyl-L-aspartic acid amide.