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International audience; We characterize a new class of crystallizing agent for soluble protein crystallization compatible with both pharmaceutical processes and high-resolution structure determination in biocrystallography. Poloxamers are amphiphilic nonionic multiblock polymers used in the cosmetic and pharmaceutical industries. Poloxamer P188 (EO75PO31EO75) is generally used as an emulsifier, solubilizer, and dispersing and wetting agent in the preparation of solid dispersions. Here, we divert the surfactant properties of poloxamer P188 at low concentrations into crystallizing properties at high concentrations of poloxamer P188 to crystallize urate oxidase and to control its crystal design.

The synthesis of a 28-residue peptide isolated from Ecballium elaterium of the Cucurbitaceae family which strongly inhibits trypsin activities (Ka = 8.10(-11)M), using BOP as the coupling reagent in a solid phase procedure is presented. This micro protein contains three disulfide bridges in its sequence and was obtained after oxidation of the six half-cystine residues either by air or with the use of carboethoxysulfenyl chloride. After purification by semi-preparative HPLC, the synthetic product was shown by trypsin inhibition tests to be identical with the trypsin inhibitor EETI II isolated from Ecballium elaterium.

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.

The syntheses of pseudo-tetrapeptides Boc-Trp-psi (CH2-NH)-Met-Asp-Phe-NH2 21 and Boc-Trp-Met-psi (CH2-NH)-Asp-Phe-NH2 20, representing the C-terminal tetrapeptide sequence of gastrin, in which amide bonds were replaced by CH2-NH bond, are described, as well as the syntheses of pseudo-peptide analogues Boc-Trp-psi (CH2-NH)-Nle-Asp-Phe-NH2 16, Boc-Trp-Nle-psi (CH2-NH)-Asp-Phe-NH2 11, and Boc-Trp-Nle-Asp-psi (CH2-NH)-Phe-NH2 5, in which the methionyl residue was replaced by a norleucyl residue. Pseudo-peptides 16 and 21, in which the amide bond between Trp and Met (or Nle) was substituted by a CH2-NH bond, stimulated gastric acid secretion in the rat in vivo. Pseudo-peptides 11 and 20, where the amide bond between Met (or Nle) and Asp was replaced by a CH2-NH bond, did not exhibit any activity on acid secretion in the rat in vivo but were potent inhibitors of pentagastrin-induced acid secretion. Peptides 11, 16, 20 and 21 all recognize the gastrin receptor on a mucosal cell preparation. Pseudo-peptide 5, in which the amide bond between Asp and Phe was replaced by a CH2-NH bond, was a less potent inhibitor of pentagastin-induced acid secretion and had a weaker affinity than the other pseudo-peptides.

International audience; The high diastereoselectivity of the hydrogenation of artemisinate by diazene to form dihydroartemisinate (diastereoselective ratio, dr, 97:3) necessary for efficient production of artemisin has been rationalized by state-of-the-art DFT calculations and identification of the noncovalent interactions by coupled ELF/NCI analysis. Remarkably, a single conformer of artemisinate is responsible for the high diastereoselectivity of the reaction. NMR studies confirm the preference for a single conformation that is found to be identical to that predicted by the calculations. The calculations and ELF/NCI analyses show that the hydrogenation of the exocyclic activated C═C double bond has a low energy barrier and that the lowest transition state and the preferred conformation of free artemisinate develop the same network of weak noncovalent interactions between the electron donor groups (oxygen and exocyclic C═C double bond) and CH bonds of the cis-decalene group of the artemisinate, which rationalize the high diastereoselectivity unusual for a strongly exothermic reaction.

Optically pure N-α-Fmoc-amino acid-N-carboxyanhydrides (Fmoc-AA-NCAs, Fmoc=9-fluorenylmethyloxycarbonyl) are proposed as precolumn reagents for the chiral analysis of asymmetric amines including α-amino acid alkyl esters. Separation of diastereomers arising from racemic amines is better achieved by liquid-solid adsorption chromatography on silica than by reversed-phase techniques. The sample preparation is easily performed while the properties of the Fmoc group allows high sensitivity by fluorescent detection. In this mode, picomolar limits of enantiomeric excess are detected.

A peptide, corresponding to the entire sequence of the squash trypsin inhibitor EETI II (Ecballium elaterium trypsin inhibitor) in which the six cysteines, engaged in three disulphide bridges in native EETI II, have been replaced by six serines, has been synthesised. CD, Fourier-transform infrared spectroscopy (FTIR) and 1H-NMR studies of this peptide revealed that some secondary structures present in native EETI II are still populated in the absence of disulphide bonds. Native-like secondary structures were observed for segments 10-15 (helix), 16-19 and 22-25 (reverse turns) but no native tertiary interaction was detected. However, a non-native local interaction between the aromatic ring of Phe26 and the amide group of Gly28 was observed. It is hypothesised that the 10-15, 16-19 and 22-25 native-like local conformations could play a major role in the early folding of EETI II.

We describe a practical method to produce pure enantiomers of non-proteinogenic α-amino acids by pronase-catalyzed hydrolysis of their methyl esters. Each of the two pure enantiomers was obtained in high yield, while the reaction conditions were optimized with a view to large scale production. Part of this work was devoted to conceiving an analytical procedure especially designed to monitor the steric course of enzymatic reactions. © 1994 Wiley-Liss, Inc.

BackgroundRasburicase (Fasturtec® or Elitek®, Sanofi-Aventis), the recombinant form of urate oxidase from Aspergillus flavus, is a therapeutic enzyme used to prevent or decrease the high levels of uric acid in blood that can occur as a result of chemotherapy. It is produced by Sanofi-Aventis and currently purified via several standard steps of chromatography. This work explores the feasibility of replacing one or more chromatography steps in the downstream process by a crystallization step. It compares the efficacy of two crystallization techniques that have proven successful on pure urate oxidase, testing them on impure urate oxidase solutions.Methodology/principal findingsHere we investigate the possibility of purifying urate oxidase directly by crystallization from the fermentation broth. Based on attractive interaction potentials which are known to drive urate oxidase crystallization, two crystallization routes are compared: a) by increased polymer concentration, which induces a depletion attraction and b) by decreased salt concentration, which induces attractive interactions via a salting-in effect. We observe that adding polymer, a very efficient way to crystallize pure urate oxidase through the depletion effect, is not an efficient way to grow crystals from impure solution. On the other hand, we show that dialysis, which decreases salt concentration through its strong salting-in effect, makes purification of urate oxidase from the fermentation broth possible.ConclusionsThe aim of this study is to compare purification efficacy of two crystallization methods. Our findings show that crystallization of urate oxidase from the fermentation broth provides purity comparable to what can be achieved with one chromatography step. This suggests that, in the case of urate oxidase, crystallization could be implemented not only for polishing or concentration during the last steps of purification, but also as an initial capture step, with minimal changes to the current process.

The main product of N-methylated amino acid coupling, when oxybenzotriazole is present in the reagent (BOP, PyBOP, HBPyU) or as an additive (DCC/HOBt), is the weakly reactive benzotriazolyl ester. On the other hand, the corresponding new halogenated reagents PyBroP, PyCloP and PyClU give very good results and can be recommended.

Diesters of L-α-amino dicarboxylic acids were specifically hydrolysed by promise at their a carboxylic group while the corresponding D-enantiomers remained unchanged. This enabled easy obtention of side chain benzyl esters of L-α-amino dicarboxylic acids starting from both optically active and raceme dibenzyl ester derivatives.

The main product of N-methylated amino acid coupling, when oxybenzotriazole is present in the reagent (BOP, PyBOP, HBPyU) or as an additive (DCC/HOBt), is the weakly reactive benzotriazolyl ester. On the other hand, the corresponding new halogenated reagents PyBroP, PyCloP and PyClU give very good results and can be recommended.

Optical resolution of β-(1-naphthyl)alanine and β-(2-naphthyl)alanine have been efficiently carried out through enzymatic hydrolysis of their methyl ester and/or N-acetyl ester derivatives by immobilized enzymes. Difficulties related to the lipophilic character of these amino acids were overcome by using emulsions of n-butyl acetate–water as reaction medium. The use of an automatic recirculating apparatus allowed reproducible and repetitive use of the immobilized biocatalysts.

Background Urate oxidase (EC 1.7.3.3 or UOX) catalyzes the conversion of uric acid and gaseous molecular oxygen to 5-hydroxyisourate and hydrogen peroxide, in the absence of cofactor or particular metal cation. The functional enzyme is a homo-tetramer with four active sites located at dimeric interfaces. Results The catalytic mechanism was investigated through a ternary complex formed between the enzyme, uric acid, and cyanide that stabilizes an intermediate state of the reaction. When uric acid is replaced by a competitive inhibitor, no complex with cyanide is formed. Conclusion The X-ray structure of this compulsory ternary complex led to a number of mechanistic evidences that support a sequential mechanism in which the two reagents, dioxygen and a water molecule, process through a common site located 3.3 Å above the mean plane of the ligand. This site is built by the side chains of Asn 254, and Thr 57, two conserved residues belonging to two different subunits of the homo-tetramer. The absence of a ternary complex between the enzyme, a competitive inhibitor, and cyanide suggests that cyanide inhibits the hydroxylation step of the reaction, after the initial formation of a hydroperoxyde type intermediate.

In this paper, solubility and interactions in solution of the recombinant urate oxidase from Aspergillus flavus, rasburicase, are studied both in the absence and in the presence of salt at a pH close to the pI. An intense salting-in effect is demonstrated first by an increased solubility when various salts are added. Thus, merely adding salt does not induce rasburicase crystallization. Second virial coefficient measurements also confirm this effect by exhibiting repulsive interactions over a large range of salt concentrations. Therefore, the salting-in effect enables the stabilization of rasburicase solution at high concentrations. Moreover, it enables crystals of improved size and habit to be grown when polymer is added to a solution of rasburicase concentrated with salt, or when salt is removed from it. We also show, with the example of high pressure macromolecular crystallography, that salt enables the stabilization of the desired polymorph under the highly concentrated polyethylene glycol conditions required by this technique.

Microproteins with proteinase inhibitory activity, 28 to 30 amino acids long, with 3 disulfide bridges have been isolated from Ecballium elaterium seeds. A peptide (EETI II) was isolated and behaved as a powerful trypsin inhibitor (Kd = 10(-11) to 10(-12) M). It was sequenced, chemically synthesized and the 3-D structure determined by 2-D 1H NMR. The information gained in the process enabled us to synthesize modified derivatives with inhibitory activity towards pancreatic elastase, chymotrypsin and human leucocyte elastase (Kd = 10(-7) to 10(-9) respectively). The most striking characteristic that appeared during the synthetic approach was the unfailing ability of the 28 amino acid peptides to refold and correctly close the 3 disulfide bridges, giving in each case an active compound. These disulfide bridges are assembled in a particular knotted structure, shared by few other bioactive peptides and called the 'knottin' structure. Molecular modeling of the peptide and a comparison with the other active molecules with similar topology allowed the synthesis of a chimaeric peptide, bearing 1 active site against a seryl-protease (trypsin), and 1 against a metallo-protease (carboxypeptidase A). The bis-headed peptide was able to inhibit both enzymes separately and concomitantly.

Autolysis of porcine pepsin at pH 4 affords a derivative possessing intrinsic proteolytic activity. This derivative was isolated by alumina pseudo-affinity chromatography and gel-filtration and was found to result from the tight association of two identical molecules, 135 amino acids long, emerging from the N-terminal domain of pepsin. This finding emphasizes a similarity with the only aspartyl-proteases known to act as dimers, the retroviral proteases.

PyBOP® (benzotriazolyloxy-tris[pyrrolidino]-phosphonium hexafluorophosphate), an analog of BOP where dimethylamino groups are replaced with pyrrolidino, is the only analog exhibiting equivalent properties in peptide bond formation. It can be used instead of BOP for the sake of safety.

BroP (bromo tris(dimethylamino) phosphonium hexafluorophosphate) is a particularly suitable reagent for coupling N-methylated amino acids. It is stable and gives very high yields in short reaction times. Dipeptides are obtained without appreciable epimerization.

Urate oxidase from Aspergillus flavus catalyzes the degradation of uric acid to [S]-allantoin through 5-hydroxyisourate as a metastable intermediate. The second degradation step is thought either catalyzed by another specific enzyme, or spontaneous. The structure of the enzyme was known at high resolution by X-ray diffraction of I222 crystals complexed with a purine-type inhibitor (8-azaxanthin). Analyzing the X-ray structure of urate oxidase treated with an excess of urate, the natural substrate, shows unexpectedly that the active site recaptures [S]-allantoin from the racemic end product of a second degradation step.

International audience; Urate oxidase from Aspergillus flavus (uricase or Uox; EC 1.7.3.3) is a 135 kDa homotetramer with a subunit consisting of 301 amino acids. It catalyses the first step of the degradation of uric acid into allantoin. The structure of the extracted enzyme complexed with a purine-type inhibitor (8-aza-xanthin) had been solved from high-resolution X-ray diffraction of I222 crystals. Expression of the recombinant enzyme in Saccharomyces cerevisiae followed by a new purification procedure allowed the crystallization of both unliganded and liganded enzymes utilizing the same conditions but in various crystal forms. Here, four different crystal forms of Uox are analyzed. The diversity of the Uox crystal forms appears to depend strongly on the chemicals used as inhibitors. In the presence of uracil and 5,6-diaminouracil crystals usually belong to the trigonal space group P3(1)21, the asymmetric unit (AU) of which contains one tetramer of Uox (four subunits). Chemical oxidation of 5,6-diaminouracil within the protein may occur, leading to the canonical (I222) packing with one subunit per AU. Coexistence of two crystal forms, P2(1) with two tetramers per AU and I222, was found in the same crystallization drop containing another inhibitor, guanine. Finally, a fourth form, P2(1)2(1)2 with one tetramer per AU, resulted fortuitously in the presence of cymelarsan, an additive. Of all the reported forms, the I222 crystal forms present by far the best X-ray diffraction resolution (similar to1.6 Angstrom resolution compared with 2.3-3.2 Angstrom for the other forms). The various structures and contacts in all crystalline lattices are compared. The backbones are essentially conserved except for the region near the active site. Its location at the dimer interface is thus likely to be at the origin of the crystal contact changes as a response to the various bound inhibitors.

High-resolution X-ray structures of the complexes of Aspergillus flavus urate oxidase (Uox) with three inhibitors, 8-azaxanthin (AZA), 9-methyl uric acid (MUA) and oxonic acid (OXC), were determined in an orthorhombic space group (I222). In addition, the ligand-free enzyme was also crystallized in a monoclinic form (P2(1)) and its structure determined. Higher accuracy in the three new enzyme-inhibitor complex structures (Uox-AZA, Uox-MUA and Uox-OXC) with respect to the previously determined structure of Uox-AZA (PDB code 1uox) leads to a reversed position of the inhibitor in the active site of the enzyme. The corrected anchoring of the substrate (uric acid) allows an improvement in the understanding of the enzymatic mechanism of urate oxidase.

The three iodothyronine selenodeiodinases catalyze the initiation and termination of thyroid hormone effects in vertebrates. Structural analyses of these proteins have been hindered by their integral membrane nature and the inefficient eukaryotic-specific pathway for selenoprotein synthesis. Hydrophobic cluster analysis used in combination with Position-specific Iterated BLAST reveals that their extramembrane portion belongs to the thioredoxin-fold superfamily for which experimental structure information exists. Moreover, a large deiodinase region imbedded in the thioredoxin fold shares strong similarities with the active site of iduronidase, a member of the clan GH-A-fold of glycoside hydrolases. This model can explain a number of results from previous mutagenesis analyses and permits new verifiable insights into the structural and functional properties of these enzymes.

[55985-85-8] C21H20Br3O2P (MW 574.93) InChI = 1S/C21H19O2P.Br3/c22-21(23)16-17-24(18-10-4-1-5-11-18,19-12-6-2-7-13-19)20-14-8-3-9-15-20;1-3-2/h1-15H,16-17H2;/q;-1/p+1 InChIKey = YYZOBYLMPAYJGX-UHFFFAOYSA-O (bromine transfer agent for selective α-bromination of ketones1 and ketone acetals2 in the presence of double bonds,1-3 activated aromatic rings,2 esters1 or another free enolic position1, 4) Alternate Name: 2-carboxyethyltriphenylphosphonium tribromide. Physical Data: mp 139–41 °C. Solubility: sol THF (25 g/100 mL), dichloromethane; insol alcohol. Form Supplied in: orange solid; not widely available; purity ≥98%. Analysis of Reagent Purity: identification in phosphonium salt mixtures by TLC/SIMS.5 Preparative Methods: Ph3P (26.2 g, 0.1 mol), acrylic acid (7.2 g, 0.1 mol) and 49% HBr (50 mL) were stirred at rt until a homogeneous solution was obtained, then heated at 95–100 °C for 10 min, whereupon the mixture was cooled and glacial HOAc (240 mL) was added; bromine (9.8 mL) was added slowly with stirring to this mixture at 0 °C; after 0.5 h, the product was filtered and recrystallized from MeCN to give the perbromide (52.5 g, 91%);1 a two step procedure involving bromination of the isolated intermediate monobromide can also be used. Handling, Storage, and Precautions: irritant and moisture sensitive solid; store in a dry place. This reagent should be handled in a fume hood.

A low molecular weight protease inhibitor peptide found in ovaries of the desert locust Schistocerca gregaria (SGPI-2), was purified from plasma of the same locust and sequenced. It was named SGCI. It was found active towards chymotrypsin and human leukocyte elastase. SGCI was synthesized using a solid-phase procedure and the sequence of its reactive site for chymotrypsin was determined. Compared with an inhibitor purified earlier from another locust species, the total sequence of SGCI showed 88% identity. In particular, the sequence of the reactive site of these inhibitors was identical. Our search for a closely related peptide in an insect species far removed from locusts, the lepidopteran Spodoptera littoralis, was unfruitful but a different chymotrypsin inhibitor, belonging to the Kazal family, was found whose mass is greater than that of SGCI (20 vs 3.6 kDa). Its N-terminal sequence shares 80% identity with that of a chymotrypsin inhibitor purified earlier from the haemolymph of another lepidopteran. Conservation of the amino acid sequence in the reactive site seems to be an exception among protease inhibitors.

The gene coding for urate oxidase, an enzyme that catalyzes the oxidation of uric acid to allantoin, is inactivated in humans. Consequently, urate oxidase is used as a protein drug to overcome severe disorders induced by uric acid accumulation. The structure of the active homotetrameric enzyme reveals the existence of a small architectural domain that we call T-fold (for tunnelling-fold) domain. It assembles to form a perfect unusual dimeric alpha 8 beta 16 barrel. Urate oxidase may be the archetype of an expanding new family of tunnel-shaped proteins that now has three members; tetrahydropterin synthase, GTP cyclohydrolase I and urate oxidase. The structure of the active site of urate oxidase around the 8-azaxanthine inhibitor reveals an original mechanism of oxidation that does not require any ions or prosthetic groups.

Tert-butyl fluorocarbonate (Boc-F) is efficiently used for the synthesis of tert-butyl esters of N-protected amino acids. The reaction proceeds at room temperature and under mild conditions in the presence of triethylamine and 4-dimethylamino-pyridine.

Activation of Boc-amino acids and Boc-N-methyl amino acids leads to the corresponding NCA. This side reaction explains the low yields obtained when coupling N-methyl amino acids. It was not observed with the Z-or Fmoc-protective groups.

p-Hydroxyphenyl propionic resin (Table I, compound 4) was used to prepare the 1–6 protected fragment of LH-RH which was then condensed with “BOP” (benzotriazolyl N-oxytrisdimethylaminophosphonium hexafluorophosphate) as coupling reagent to the 7–10 residue synthesized on the same resin. Peptidylresin was divided into two aliquots in order to obtain LH-RH after aminolysis and treatment with liquid hydrogen fluoride, LH-RH-COOH after saponification followed by hydrogenation, or treatment with liquid hydrogen fluoride. The resulting hormones were rapidly purified by the sole means of two gel filtrations.

Polyoxyethylene perfluoroalkyl surfactants are synthesized from polyoxyethylene glycols using an appropriate intermediate monooxyphosphonium salt. In contrast with methods involving the polyaddition of ethylene oxide to a fluoroalcohol, the present procedure allows us to obtain surfactant molecules with a definite number of oxyethylene units, i.e. with a well-defined hydrophile lipophile balance (HLB). Fluorinated microemulsions could thus be prepared for the first time by mixing the surfactant of appropriate HLB to water and a fluorocarbon.

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.

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

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.

Esterification of N-protected α-amino acids was achieved via isopropenyl chlorocarbonate (IPCC) activation. In situ alcoholysis of the unstable mixed anhydride intermediate was catalyzed by 4-(dimethylamino)pyridine (DMAP). Competing isopropenyl ester formation was negligible when using methylene chloride as the solvent. A variety of esters from primary and secondary alcohols were obtained with good yields (60 to 96 %), and even the more hindered tertiobutyl alcohol gave acceptable yields under more drastic conditions. The improvement in depsipeptide synthetic methodology is illustrated by preparation of the antibiotic valinomycin, using IPCC for ester bond formation, and BOP reagent for peptidic coupling and the last-step cyclization.

Mono-esters of N-protected di-acids aspartic and glutamic are prepared by a one-pot activation with alkyl chloroformates or isopropenyl chloroformate and an additionnal alcohol. This process involves the intermediate internal anhydride formation.

The solution conformation of synthetic Ecballium elaterium trypsin inhibitor II, a 28-residue peptide with 3 disulfide bridges, has been studied by {sup 1}H 2D NMR measurements. Secondary structure elements were determined: a miniantiparallel {beta}-sheet Met 7-Cys 9 and Gly 25-Cys 27, a {beta}-hairpin 20-28 with {beta}-turn 22-25, and two tight turns Asp 12-Cys 15 and Leu 16-Cys 19. A set of interproton distance restraints deduced from two-dimensional nuclear Overhauser enhancement spectra and 13 {phi} backbone torsion angles restraints were used as the basis of three-dimensional structure computations including disulfide bridges arrangement by using distance geometry calculations. Computations for the 15 possible S-S linkage combinations lead to the proposal of the array 2-19, 9-21, 15-27 as the most probably structure for EETI II.

Five peptides related to human renin flap region have been synthesized. Two of them are ring closed through properly designed disulfide bridges. Structure analysis involving IR, CD and NMR techniques and recognition by antibodies raised against human renin support our assumption that in the protein, the flap region adopts a β and γ turn leading to the carbonyl group of residue 83 (Tyr) interacting with the NH's of residues 85 (Thr) and 86 (Gly).

A predictive equation based on extrathermodynamic assumptions is proposed, that allows the prediction of the degree of epimerization in tripeptide model reactions of condensation of peptide segments as a function of the primary structure. The experimental validation of this equation is presented in the field of the 125 tripeptides containing alanine, leucine, phenylalanine, valine and isoleucine, the prediction requires 13 epimerization measurements on reference reactions. Sixteen statistically chosen predictions are checked experimentally showing a good fit in spite of the drastic basic assumptions needed to set up the equation.

“Extended X-Ray Absorption Fine Structure” (EXAFS) spectroscopy has been used for probing the environment of the Zn2+ cation in three 3-propenamid-Zn tetraphenyporphyrin complexes. The lack of evidence for a short axial bond of the zinc atom to the sulfur atom of the cysteine terminal residue of the lateral peptidic chain is discussed, together with the few indications which suggest that a Zn-S interaction, if any, can only be weak and probably long range (4.1 A ?).

The carboxamidomethyl esters (CAM esters) are proposed for carboxyl protection in peptide synthesis. Amino acid CAM ester derivatives were easily prepared and showed good stability in the deblocking conditions of other common protecting groups used in peptide synthesis. The CAM esters were selectively and rapidly hydrolyzed in an alcaline medium.

The action of carbon tetrachloride and tris(dimethylamino)phosphine at low temperature on 2,3:4,6-di-O-isopropylidene-α-D-mannopyranose affords the α anomeric form of the corresponding alkoxytris(dimethylamino)phosphonium chloride. The condensation of aromatic thiols with this salt leads to β-thiomannosides by an SN2 process and the condensation of alcohols, preceded by the action of silver salts in order to avoid the formation of the glycosyl chlorides, yields a mixture of α- and β-mannosides.

Oxyntomodulin is a peptide isolated from porcine intestine which consists of the whole glucagon sequence extended at its C-terminal part by a basic octapeptide. The analogue (Nle-27)-oxyntomodulin of the human sequence has been synthesized by solid-phase peptide synthesis, purified by HPLC and identified. Its biological activities are the same as those of the natural hormone.

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.

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).