5-Hydroxytryptophan as a building block in oligopeptides using Fmoc/tBu SPPS

Summary One of the initial steps in the development of therapeutic agents is the identification of lead compounds that bind to the target of interest. Use of combinatorial libraries allows the screening of thousands to millions of compounds. To optimize the characteristics of the first generation of compounds, many of their analogues need to be synthesized. In this sense, 5-hydroxytryptophan closely resembles tryptophan. Both, however, are sensitive to oxidation and alkylation during solid-phase peptide synthesis. In an effort to overcome these side reactions during oligopeptide synthesis, we prepared N -Fmoc-N ⁱⁿ-Boc-5-O-benzyl-5-hydroxytryptophan. To evaluate its properties, this building block was incorporated in a pentagastrin analogue using Fmoc/tBu chemistry. After deprotection and cleavage from the support, the products were purified and identified. The fully deprotected peptide was the main compound, contaminated by its 5-O-benzylated analogue. The relative amount of the latter decreased using longer deprotection times.This work will be reported in more detail elsewhere (Lescrinier, Th. et al., paper in preparation).     

Asymmetric catalysis of the Strecker amino acid synthesis by a cyclic dipeptide

Summary A novel cyclic dipeptide —cyclo[(S)-His-(S)-NorArg] — has been prepared which catalyzes an enantioselective version of the Strecker amino acid synthesis. The catalyst, when present in 2 mol % quantity in methanol solution, catalyzes the addition of hydrogen cyanide toN-alkylimines to afford-amino nitriles in high yield and high enantiomeric excess. Furthermore, acid hydrolysis ofN-benzhydryl--amino nitriles afforded the corresponding-amino acids directly. This methodology affords a variety of arylglycines in exceptionally high enantiomeric excess, but aliphatic amino acids were obtained with low enantioselectivity. Current efforts are underway to expand the scope of this reaction, as well as to elucidate the mechanism of catalysis and the roles played by substrate and catalyst in determining the stereochemical outcome of the reaction.A preliminary communication on this work has recently appeared in: Iyer MS, Gigstad KM, Namdev ND, Lipton MA (1996) J Am Chem Soc 118: 4910–4911.     

alpha,alpha'-disubstituted amino acids with silylated side chains as lipophilic building blocks for the synthesis of peptaibol analogues

New alpha,alpha'-disubstituted amino acids with silylated side chains have been synthesized in racemic form. Starting from a Schiff base of glycine tert-butyl ester, a large variety of alpha,alpha'-dialkylated amino acids has been obtained, depending on the alkylating reagents. The application of a hydrosilylation methodology enabled the synthesis of the same unnatural amino acids in an enantiomerically pure form. The ability of these bulky amino acids to be incorporated into peptides by solution-phase methodology has also been demonstrated, since constrained silylated dipeptides have been synthesized. These new lipophilic building blocks could be useful and innovative in the design of peptaibol analogues.     

Synthesis of building blocks for solid-phase introduction of diethylenetriaminepentaacetic acid (DTPA) to oligonucleotides and oligopeptides

Synthesis of building blocks that allow site-specific incorporation of diethylenetriaminepentaacetic acid (DTPA) to oligonucleotides and oligopeptides using phosphoramidite and Fmoc chemistries, respectively, is described.     

Parallel solid-phase synthesis of mucin-like glycopeptides

The glycopeptide, Ac-Pro-Thr(alpha-D-GalNAc)-Thr(alpha-D-GalNAc)-Thr(alpha-d-GalNAc)-Pro-Leu-Lys-NH(2) (1), which features three consecutive O-glycosylated Thr residues and mimics a portion of mucin 2, has been prepared by solid-phase synthesis. Seven related, partially glycosylated peptides (2-8) were synthesized as well. This suite of molecules allowed a systematic analysis of synthetic protocols. N(alpha)-(9-Fluorenylmethoxycarbonyl)-O-(3,4,6-tri-O-acetyl-2-azido-2-deoxy-alpha-D-galactopyranosyl)-L-threonine pentafluorophenyl ester [Fmoc-L-Thr(Ac(3)-alpha-D-GalN(3))-OPfp] was used as a building block that coupled efficiently when used in a relatively low molar excess, that is, approximately 1.5 equiv, with N,N-dimethylformamide (DMF) as the solvent. For conversion of the azido group to the N-acetyl function, direct treatment with thioacetic acid was preferred over a two-step procedure involving reduction with dithiothreitol (DTT) followed by N-acetylation. Effective O-deacetylation of 1-8 in solution was achieved by treatment with sodium methoxide (10-15 mM; approximately 5 equiv) in methanol. On-resin deacetylation techniques were also examined, using sodium methoxide (6-10 mM) in DMF-methanol (17:3) (for 4 and 11) or hydrazine (70 mM) in methanol (for 8). The more convenient on-resin technique in DMF-methanol gave yields similar to solution conditions, and promises to be widely useful for solid-phase glycopeptide synthesis. HPLC profiles showed that free glycopeptides elute earlier than the corresponding O-acetylated derivatives, and that retention times vary systematically with the number of sugar moieties. (1)H NMR studies carried out in water showed an increase in conformational organization of glycopeptides with increased density of glycosylation.     

Synthesis of novel unnatural amino acid as a building block and its incorporation into an antimicrobial peptide

Considering the biological mechanism and in vivo stability of antimicrobial peptides, we designed and synthesized novel unnatural amino acids with more positively charged and bulky side chain group than lysine residue. The unusual amino acids, which were synthesized by either solution phase or solid phase, were incorporated into an antimicrobial peptide. Its effect on the stability, activity, and the structure of the peptide was studied to evaluate the potential of these novel unnatural amino acids as a building block for antimicrobial peptides. The incorporation of this unusual amino acid increased the resistance of the peptide against serum protease more than three times without a decrease in the activity. Circular dichroism spectra of the peptides indicated that all novel unnatural amino acids must have lower helical forming propensities than lysine. Our results indicated that the unnatural amino acids synthesized in this study could be used not only as a novel building block for combinatorial libraries of antimicrobial peptides, but also for structure–activity relationship studies about antimicrobial peptides.     

Helicobacter pylori utilises urea for amino acid synthesis

Abstract Helicobacter pylori has one of the highest urease activities of all known bacteria. Its enzymatic production of ammonia protects the organism from acid damage by gastric juice. The possibility that the urease activity allows the bacterium to utilise urea as a nitrogen source for the synthesis of amino acids was investigated. H. pylori (NCTC 11638) was incubated with 50 mM urea, enriched to 5 atom% excess ¹⁵N, that is the excess enrichment of ¹⁵N above the normal background, in the presence of either NaCl pH 6.0, or 0.2M citrate pH 6.0. E. coli (NCTC 9001) was used as a urease-negative control. ¹⁵N enrichment was detected by isotope ratio mass spectrometry. H. pylori showed intracellular incorporation of ¹⁵N in the presence of citrate buffer pH 6.0 but there was no significant incorporation of ¹⁵N in unbuffered saline or by E. coli in either pH 6.0 citrate buffer or unbuffered saline. The intracellular fate of the urea-nitrogen was determined by means of gas chromatography/mass spectrometry following incubation with ¹⁵N enriched 5 mM urea in the presence of either 0.2 M citrate buffer pH 6.0 or 0.2 M acetate buffer pH 6.0. After 5 min incubation in either buffer the ¹⁵n label appeared in glutamate, glutamine, phenylalanine, aspartate and alanine. It appears, therefore, that at pH and urea concentrations typical of the gastric mucosal surface, H. pylori utilises exogenous urea as a nitrogen source for amino acid synthesis. The ammonia produced by H. pylori urease activity thus facilitates the organism's nitrogen metabolism at neutral pH as well as protecting it from acid damage at low pH.     

Synthesis of an Fmoc-threonine bearing core-2 glycan: A building block for PSGL-1 via Fmoc-assisted solid-phase peptide synthesis

Selectins (L, E, and P) are vascular endothelial molecules that play an important role in the recruitment of leukocytes to inflamed tissue. In this regard, P-Selectin glycoprotein-1 (PSGL-1) has been identified as a ligand for P-Selectin. PSGL-1 binds to P-Selectin through the interaction of core-2 O-glycan expressing sialyl Lewis^x oligosaccharide and the three tyrosine sulfate residues. Herein, we report the synthesis of threonine-linked core-2 O-glycan as an amino acid building block for the synthesis of PSGL-1. This building block was further incorporated in the Fmoc-assisted solid-phase peptide synthesis to provide a portion of the PSGL-1 glycopeptide.     

Stimulation of renal amino acid reabsorption after treatment with triiodothyronine or dexamethasone in amino acid loaded rats

Summary In adult female anaestetized rats, the influence of triiodothyronine or dexamethasone on renal amino acid handling was investigated in leucine (20mg/100g b.wt.) or glutamine (45mg/100g b.wt.) loaded animals. Bolus injections of both amino acids were followed by temporary increase in fractional excretion of the administered amino acids as well of the endogenous amino acids which were not administered.Under load conditions (leucine and glutamine), dexamethasone treatment (60 g/100 g b.wt. for 3 days, i.p. once daily) was followed by a stimulation of renal amino acid reabsorption. The increase in fractional amino acid excretion after amino acid load was significantly lower than in untreated rats. The effect of triiodothyronine (20,g/1008 b.wt. for 3 days, i.p. once daily) was different in leucine and glutamine loaded animals: after leucine bolus injection a comparable stimulatory effect as shown for dexamethasone could be demonstrated, but after glutamine administration the stimulatory action of T3 was masked. T3 even increases fractional amino acid excretion in glutamine loaded rats as a sign of enhanced house-keeping in the renal tubular cells. These results confirm previous findings and indicate different effects of both hormones on the renal handling of amino acids.     

Analysis of trace amino acid neurotransmitters in hypothalamus of rats after exhausting exercise using microdialysis

A simple but effective coupling of microdialysis and capillary electrophoresis with laser induced fluorescence detection technique was applied to analysis of amino acid neurotransmitters in the hypothalamus of rats after acute exhausting exercise. The separation of amino acids was achieved using an uncoated fused-silica capillary (57 cm×75 μm I.D.) with a buffer of 10 mM disodium tetraborate at pH 10 and an applied voltage of 12.5 kV. The detection limit was 10⁻¹⁰ M for each amino acid. It is sufficiently sensitive and rapid for the determination of amino acids in a 5-μl Microdialysate. In comparison to pre-exercise, a significant increase in the levels of six hypothalamic amino acids (arginine, glycine, lysine, glutamic acid, alanine, γ-amino-n-butyric acid) was found after exercise. These results demonstrate that the increase of metabolic amino acids in the hypothalamus of rats can be induced by exhausting exercise and suggests that amino acid neurotransmitters may play functional roles in the central effects of exercise.     

Influence of triiodothyronine and dexamethasone on renal amino acid handling in rats loaded with various amino acid mixtures

Summary In adult female rats, the influence of dexamethasone or triiodothyronine on renal amino acid handling was investigated in amino acid loaded animals. Amino acids were administered intravenously as two mixtures, each containing four amino acids to overload amino acid reabsorption capacity. Bolus injections of both mixtures were followed by temporary increase in fractional excretion of the administered amino acids as well of the amino acids which were not covered in the mixtures. The administration of the two mixtures was followed by different interactions between various amino acid carriers.After dexamethasone pretreatment (60µg/100g b.wt. for 3 days, once daily) a stimulation of the renal amino acid handling could be shown. Triiodothyronine (20µg/100g b.wt. for 3 days, once daily) did not increase tubular reabsorption capacity for amino acids. It even increased fractional amino acid excretion in amino acid loaded rats as a sign of enhanced amino acid metabolism in the kidney and/or increased amino acid uptake into the tubular cells from the luminal site.     

Facile synthesis of glycosylated Fmoc amino acid building blocks assisted by microwave irradiation

The synthesis of glycosylated Fmoc amino acids by reaction of mono- and disaccharide peracetates with Fmoc amino acids having free carboxyl groups was rapidly promoted by Lewis acids (SnCl₄, BF₃·Et₂O) under microwave irradiation. The products are useful building blocks for the synthesis of glycopeptides.     

Thia-analogues of amino acids synthesis of peptide derivatives containing 3-thia-analogues of amino acids

Summary N-Protected dipeptides containing L-3-thia-analogues of phenylalanine, p-nitro-phenylalanine, lysine and leucine respectively were prepared applying an enantioselective enzymatic reaction step. Racemic synthetic intermediates of the type acyl-NH-CH(R¹)-CO-D,L-NH-CH(S-R²)-COOBzl were selectively deprotected at the C-terminus by enzymatic hydrolysis using thermitase or trypsin.Abbreviations Ac acetyl - AcOEt ethyl acetate - AcOH acetic acid - Boc tert.-butyloxycarbonyl - Bz benzoyl - Bzl benzyl - DMF dimethyl-formamide - EtOH ethanol - THF tetrahydrofuran - Z benzyloxycarbonyl Dedicated to Prof. D. Cavallini at the occasion of his 75th birthday.     

Peptide deformylase as biocatalyst for the synthesis of enantiomerically pure amino acid derivatives

Peptide deformylases (PDFs) catalyze the removal of the N-terminal formyl group from nascent polypeptides. In spite of the vast amount of literature on PDF, no information whatsoever is available on its use in organic synthesis. To be able to explore the potential of E. coli PDF (EcPDF) in biocatalytic applications, a simple and efficient purification procedure was developed. This method, which was based on one affinity chromatographic step, furnished about 200 mg of pure EcPDF from 1 L of E. coli culture. The enzyme combined a good activity (tof ≥5 s⁻¹) with an almost complete enantioselectivity (E ratio >500) in the resolution of N-formylated α- and β-amino acids, α-amino acid amides and α-aminonitriles. N-Formyl derivatives of non-functionalized amines and β-amino alcohols were hydrolyzed with low to moderate activity and enantioselectivity. EcPDF was also successfully applied in the enantioselective formylation of α-aminonitriles, yielding, e.g. (S)-N-formyl-phenylalanine nitrile with >99.5% ee. The enzyme also proved very suitable for the mild and selective deprotection of N-formyl peptides as was shown for N-formyl-Leu-Tle-NHCH₃. This deprotection increased the diastereomeric excess of the dipeptide, which was unsatisfactory because of racemization of the N-terminal amino acid in the chemical peptide coupling step.     

Design,synthesis and biological evaluation of amino acids-oleanolic acid conjugates as influenza virus inhibitors

Viral entry inhibitors are of great importance in current efforts to develop a new generation of anti-influenza drugs. Inspired by the discovery of a series of pentacyclic triterpene derivatives as entry inhibitors targeting the HA protein of influenza virus, we designed and synthesized 32 oleanolic acid (OA) analogues in this study by conjugating different amino acids to the 28-COOH of OA. The antiviral activity of these compounds was evaluated in vitro. Some of these compounds revealed impressive anti-influenza potencies against influenza A/WSN/33 (H1N1) virus. Among them, compound 15a exhibited robust potency and broad antiviral spectrum with IC₅₀ values at the low-micromolar level against four different influenza strains. Hemagglutination inhibition (HI) assay and docking experiment indicated that these OA analogues may act in the same way as their parent compound by interrupting the interaction between HA protein of influenza virus and the host cell sialic acid receptor via binding to HA, thus blocking viral entry.     

Chemoselectively addressable HCan building blocks in peptide synthesis: L-homocanaline derivatives

(S-2-amino-5-(aminooxy)pentanoic acid (L -homocanaline, HCan), a structural analogue of lysine, contains a reactive alkyloxyamine side chain and is therefore considered to react chemoselectively with carbonyl compounds by forming a kinetically stable oxime bond. The chemical synthesis of L -homocanaline starting from protected glutamic acid derivatives is described. Two orthogonally protected homocanaline derivatives were synthesized and their use in standard SPPS procedures was exemplified for the synthesis of a chemoselectively addressable cyclic peptide for use in TASP design. Moreover, the wide range of applications of this unique building block was demonstrated for the chemoselective ligation of an unprotected disaccharide to a HCan containing model peptide resulting in a chimeric glycopeptide structure. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Design and synthesis of bile acid-based amino sterols as antimicrobial agents

New bile acid-based amino sterols were synthesized in good yields from C-3β-oxiranes as key intermediates. These derivatives were evaluated for their in vitro antimicrobial properties against human pathogens. These compounds showed better antibacterial activity as compared to antifungal activity. Compounds 21 and 22 showed comparable antibacterial activity to gentamicin against Staphylococcus aureus with IC₅₀ values of 5.14 and 4.46 μg/mL. This is the first report for the synthesis of C-3β-oxiranes on the steroids having A/B cis ring junction and these oxiranes have been used for the synthesis of amino sterols 17, 18, 21, and 22.     

Novel O-glycosyl amino acid mimetics as building blocks for O-glycopeptides act as inhibitors of galactosidases

As potential lead structures for a new class of glycosidase inhibitors the novel O-glycosyl amino acid mimetics 3'-O-[2,6-anhydro-D-glycero-L-gluco-heptitol-1-yl]-L-serine 3 and-L-threonine 4 were synthesized, employing regio- and stereoselective aziridine ring opening methodology. They proved to be stable in the presence of glycosidases and showed competitive inhibition of alpha-galactosidase from Aspergillus niger.     

Purification of fatty acid ethyl esters by solid-phase extraction and high-performance liquid chromatography

We have developed a two-step method to purify fatty acid ethyl esters (FAEE) using solid-phase extraction (SPE), with a recovery of 70±3% (mean±S.E.M.) as assessed using ethyl oleate as a recovery marker from a standard lipid mixture in hexane. The first step of the SPE procedure involves application of a lipid mixture to an aminopropyl-silica column with simultaneous elution of FAEE and cholesteryl esters from the column with hexane. Gas chromatographic analysis of FAEE without interference from cholesteryl esters may be performed using the eluate from the aminopropyl-silica column, thus eliminating the need for an octadecylsily (ODS) column in this case. The FAEE can then be separated from the cholesteryl esters, if necessary, by chromatography on an ODS column and elution with isopropanol-water (5:1, v/v). Both the aminopropyl-silica and ODS columns were found to be effective for up to four uses. To permit isolation of specific FAEE species following isolation of total FAEE by the two-step SPE method, we have also developed a purification scheme for individaal FAEE by high-performance liquid chromatography (HPLC). Thus, this simple method allows for reproducible isolation of total FAEE by SPE and isolation of individual FAEE species by HPLC.