Solution phase synthesis of the 14-residue peptaibol antibiotic trichovirin I

Summary.  The 14-residue peptaibol antibiotic trichovirin I 4A of the structure Ac-Aib-L-Asn-L-Leu-Aib-L-Pro-L-Ala-L-Val-Aib-L-Pro-Aib-L-Leu-Aib-L-Pro-L-Leuol (Aib = α-aminoisobutyric acid, Leuol = leucinol) was synthesized by stepwise conventional solution phase synthesis using the Z/OtBu(OMe) strategy and HOBt/EDC as coupling reagents. Intermediates were fully characterized and the identity of the synthetic peptide with the component 4A of the natural, microheterogeneous peptide mixture was proven by electrospray mass spectrometry, HPLC, and bioassay. Received March 25, 2002 Accepted June 14, 2002 Published online December 18, 2002 RID="*" ID="*"  Dedicated to Prof. Dr. Günther Jung. Tübingen University, on the occasion of his 65^th anniversary. Authors' address: Prof. Dr. Hans Brückner, Interdisciplinary Research Center, Institute of Nutritional Science, Department of Food Sciences, Justus-Liebig-University of Giessen, Heinrich-Buff-Ring 26, D-35392 Giessen, Germany, Fax: +49-641-99-39149, E-mail: hans.brueckner@ernaehrung.uni-giessen.de Abbreviations: Amino acids are abbreviated according to three-letter-nomenclature; Aib, α-aminoisobutyric acid (2-methylalanine); Iva (isovaline, 2-ethylalanine); Leuol, L-leucinol [(S)-2-amino-4-methyl-1-pentanol]; AAA, amino acid analysis; EI-MS, electron impact mass spectrometry; ESI-MS, electrospray ionization mass spectrometry; HPLC, high performance liquid chromatography; Z, benzyloxycarbonyl; Fmoc, 9-fluorenylmethyoxycarbonyl; OtBu, tertiary butoxy (tert-butylester); OMe, methoxy (methyl ester); OBzl, benzyloxy (benzyl ester); TDM, N,N,N′,N′-tetramethyl-4,4′-diamino-diphenylmethane (Arnold's base); for other abbreviations see Experimental.     

Synthesis and use of <Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-di-Boc-glutamate γ-semialdehydes and related aldehydes

Summary.  This review article focuses on the synthesis and reactions of N,N-di-Boc glutamate and aspartate semialdehydes as well as related aldehydes. These building blocks are prepared according to various strategies from glutamic and aspartic acids and find interesting synthetic applications. In the first part, the methods for the synthesis of N,N-di-Boc-amino aldehydes are summarized. The applications of these chiral synthons for the synthesis of unnatural amino acids and other bioactive compounds are discussed in the second section. Received April 24, 2002 Accepted August 13, 2002 Published online January 30, 2003 Authors' address: Prof. Violetta Constantinou-Kokotou, Chemical Laboratories, Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece, E-mail: vikon@aua.gr Abbreviations: AcNH-TEMPO, 4-acetamido-2,2,6,6-tetramethyl-1-piperidinyloxy free radical; AIBN, 2,2′-azobis(2-methylpropionitrile); Aliquat, methyltrioctylammonium chloride; Bn, benzyl; Boc, tert-butoxycarbonyl; Bu^t, tert-butyl; m-CPBA, 3-chloroperoxybenzoic acid; DAST, diethylaminosulfur trifluoride; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; (R,R)-(+)-DET, (R,R)-(+)-diethyltartrate; DIBALH, diisobutyl aluminium hydride; DMAP, 4-dimethylaminopyridine; DMF, dimethylformamide; Et₃N, triethylamine; KHMDS, potassium bis(trimethylsilyl)amide; (S)-LLB, lanthanium-lithium-bis-metallic binaphthol catalyst; MsCl, methanesulfonyl chloride; NEM, N-ethylmorpholine; NMO, 4-methylmorpholine N-oxide; PPA, propylphosphonic acid anhydride; TBHP, tert-butyl hydroperoxide; TFA, trifluoroacetic acid; THF, tetrahydrofuran; TMSI, 1-(trimethylsilyl)imidazole; Trt, trityl.     

Synthesis of α-carboxyphosphinopeptides derived from norleucine

In the present study, we describe in detail the synthesis of a relatively rare class of phosphorus compounds, α-carboxyphosphinopeptides. We prepared several norleucine-derived α-carboxyphosphinic pseudopeptides of the general formula Nle-Ψ[PO(OH)]-Gly. These compounds could have important applications as transition state-mimicking inhibitors for methionine or leucine aminopeptidases or other enzymes. For the preparation of the key α-carboxyphosphinate protected precursors, we investigated, compared and improved two different synthetic methods described in literature: the Arbuzov reaction of a silylated N-protected phosphinic acid with a bromoacetate ester and the nucleophilic addition of a mixed O-methyl S-phenyl N-protected phosphonic acid or a methyl N-protected phosphonochloridate with tert-butyl lithioacetate. We also prepared two N-Fmoc protected synthons, Fmoc-Nle-Ψ[PO(OH)]-Gly-COOH and Fmoc-Nle-Ψ[PO(OAd)]-Gly-COOH, and demonstrated that these precursors are suitable building blocks for the solid-phase synthesis of α-carboxyphosphinopeptides.     

Synthesis of soluble poly(amide-ether-imide-urea)s bearing amino acid moieties in the main chain under green media (ionic liquid)

In this study, an optically active diamine, N,N′-(pyromellitoyl)-bis{N-[4(4-aminophenoxy)phenyl]-2-(4-methyl)pentanamide} (1) containing amino acid l-leucine was prepared in three steps. The step-growth polymerization of this chiral diamine with several diisocyanates in room temperature ionic liquid (IL), 1,3-dipropylimidazolium bromide as an environmentally friendly solvent and in a volatile organic solvent, is investigated. The polymerization yields and inherent viscosities of the resulting poly(amide-ether-imide-urea)s are compared in both solvents. The results show that the IL to be the superior polymerization media. All of the obtained polymers exhibited good solubility in some polar aprotic organic solvents such as N,N-dimethyacetamide, N,N-dimethyformamide, dimethyl sulfoxide while thermal stability was not disturbed based on thermogravimetric analysis and differential scanning calorimetry experiments. X-ray diffraction analysis of polymers shows that they are amorphous. The observation of optical rotation confirms the optical activity of prepared polymers.     

Synthesis of novel selenium containing sulfa drugs and their antibacterial activities

Synthesis of 3-[4-(N-substituted sulfamoyl)phenyl]-3,4-dihydro-4-oxo-7,9-dimethylpyri-do[3′,2′:4,5]selenolo[3,2-d]pyrimidines,7-[4-(N-substituted sulfamoyl)phenyl]-7,8-dihydro-8-oxo-3,4-diphenylpyrimido[4′,5′:4,5]selenolo [2,3-c]pyridazines and 1-[4-(N-substituted sulfamoyl)phenyl]-1,11-dihydro 11-oxo-4-methylpyrimido[4′,5′:4,5]selenolo[2,3-b]quinolines is reported. 4-Amino-N-pyrimidine-2-ylbenzene sulfonamide (a), 4-amino-N-(2,6-dimethylpyrimidin-4-yl)benzene sulfonamide (b), N-[(4-aminophenyl)sulfonyl] acetamide (c) with N-ethoxymethyleneamino of selenolo pyridine, selenolo pyridazine and selenolo quinoline derivatives respectively were obtained starting from 1-amino-N ⁴-substituted sulfanilamides. Spectroscopic data (IR, ¹H NMR, ¹³C NMR and Mass spectral) confirmed the structure of the newly synthesized compounds. Substituted pyrimidines, pyridazines and quinolines were screened for antibacterial activity against gram-positive and gram-negative bacteria. Selenolo derivative of N-[(4-aminophenyl)sulfonyl] acetamide (substitutent of sulfacetamide c) showed strong bactericidal effect against all the tested organisms. Selenolo[3,2-d]pyrimidin (substitutent a) showed a good bactericidal effect against Serratia marcescens, Staphylococcus aureus and Escherichia coli. Compounds selenolo[2,3-c]pyridazine (substitutent b), selenolo[2,3-b]quinoline(substitutents c)) exhibited a moderate bactericidal effect against Serratia marcescens. None of the synthesized seleno pyridazines has a considerable antimicrobial activity against the tested organisms. The minimum inhibitory concentration (MIC) of the most active compound-3-[4-(N-acetyl sulfamoyl)phenyl]-3,4-dihydro-4-oxo-7,9-dimethylpyrido[3′,2′:4,5]selenolo [3,2-d]pyrimidine was 10 mg ml⁻¹.     

Transformation of N ′,N ′-dimethyl-N -(hydroxyphenyl)ureas by laccase from the white rot fungus Trametes versicolor

Transformation of N′,N′-dimethyl-N-(hydroxyphenyl)ureas was assayed in the presence of purified laccase produced by the fungus Trametes versicolor. The para- and ortho-hydroxyphenyl derivatives were enzymatically transformed, whereas the meta derivative was not. The performance of laccase-mediated transformation depended on the pH, with an optimum for the para-derivative degradation rate at pH 5. The pH also influenced the nature of the reaction products. The chemical was exclusively oxidised into p-benzoquinone at pH 3 and into mainly N′,N′-dimethyl-N-[(2,5-cyclohexadiene-1-one)-4-ylidene]urea at pH 6. The ortho- derivative was transformed essentially into insoluble purple compounds, probably appearing as polymers resulting from coupling of the parent compound. Received: 14 September 1998 / Received revision: 23 November 1998 / Accepted: 29 November 1998     

Glycopeptidolipids — a new class of artificial antigens with carbohydrate determinants. Synthesis of artificial antigen with type-specific oligosaccharide hapten fromNeisseria meningitidis group B

Synthetic lipopeptideN-palmitoyltyrosyl-seryl-seryl-asparaginyl-alanine, an analogue of B-mitogenic tripalmitoyl-pentapeptide fromEscherichia coli lipoprotein, was coupled with an oligosaccharide hapten fromNeisseria meningitidis lipooligosaccharide to give a glycopeptidolipid conjugate — the artificial antigen of a new type possessing the type-specific microbial determinant.Abbreviations iBu isobutyl - Bu^t t-butyl - Boc t-butoxycarbonyl - DCC N,N-dicyclohexylcarbodiimide - DMF N,N-dimethylformamide - DMSO dimethylsulfoxide - ONB N-hydroxy-5-norbornen-2,3-dicarboximide ester - ONp 4-nitrophenyl ester - Pal palmitoyl - TEMED N,N,N,N-tetramethylethylenediamine - Z benzyloxycarbonyl - KDO 2-keto-3-deoxyoctonic acid - Hep L-glycero-d-manno-heptose - TPP S-[2,3-bis(palmitoyloxy)-(2RS)propyl]-N-palmitoylcysteinyl-seryl-asparaginyl-alanine.     

Light-induced proton translocation through thylakoid and cytoplasmic membranes of Plectonema boryanum

Light-induced fluorescence changes of 9-aminoacridine, an indicator of proton gradient in energy-transducing membranes, were studied in Plectonema boryanum and other cyanobacteria. The fluorescence changes observed in cell suspensions resulted from a superposition of fluorescence quenching and enhancement as the analysis of the kinetic data shows. Both components of the fluorescence changes are abolished by 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) and m-chlorocarbonylcyanide phenylhydrazone. The inhibitory effect of DCMU is removed by 2,3,5,6- or N,N,N′,N′-tetramethyl-p-phenylenediamine. The fluorescence quenching sensitive to substrates and uncouplers of the photophosphorylation is only observed in membrane vesicles obtained by osmotic shock of P. boryanum spheroplasts. Presumably, light-induced quenching of the dye fluorescence in the cells of cyanobacteria is due to the proton transport from the cytoplasm in the inner space of thylakoids while fluorescence enhancement is due to the proton efflux from the cytoplasm into the incubation medium.     

Synthesis of peptides containing α,β-didehydroamino acids. Scope and limitations

Summary α, β-Didehydroamino acids, which are key components of both natural andde novo peptides, are frequently encountered in naturally occurring peptides — mostly of microbial and fungal origin and/or from marine organisms. Herein, we report on a reappraisal of the use of the water-soluble carbodiimide/CuCl method for the preparation of this kind of peptide in both solution and solid-phase modes and describe some side reactions encountered during the process. Abbreviations: Alloc, allyloxycarbonyl; Barlos resin, 2-chlorotrityl chloride resin, Boc,tert-butoxycarbonyl; Boc₂O, di-tert-butyl dicarbonate; Bzl, benzyl; DABCO, 1,4-diazabicyclo[2.2.2]octane; DAST, (diethylamino)sulfur trifluoride; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; DDAAs, α, β-didehydroarnino acids; DEAD, diethyl azodicarboxylate; Dha, Didehydroalanine, (Z)-Dhb, Z-Didebydroaminobutyric acid; (Z)-Dhp, Z-Didehydrophenylalanine; DSC, disuccinimidyl carbonate; DDP, α, β-didehydropeptides; EDC, WSC, 3-(3′-dimethylaminopropyl)-1-ethylearbodiimide; Fmoc, fiuorenylmethoxycarbonyl; HATU, N-{(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridino-1-ylmethylene}-N-methylmethanaminium hexafluorophosphate; HOAt, 1-hydroxy-7-azabenzotriazole; (β-OH)Phe, phenylserine or β-hydroxyphenylalanine; Ph₃P, triphenylphosphine; PyAOP, (7-azabenzotriazol-1-yloxy)-tris(pyrrolidino)phosphonium hexafluorophosphate; TEA, triethylamine; TFA, trifluoroacetic acid. Amino acid symbols denote thel-configuration unless stated otherwise. All solvent ratios are volume/volume unless stated otherwise.     

Comparison of the stepwise and convergent approaches in the solid-phase synthesis of rat Tyr0-atriopeptin II

Summary Tyr^o-Atriopeptin II was synthesized on a 2-chlorotrityl resin by both, the stepwise and the convergent approach. For both methods an Fmoc/tBu(Trt)-based protection scheme was used. The convergent methodology utilizes the sequential condensation of four protected peptide fragments. These were chosen so that after every condensation reaction, the amino-terminal region of the newly formed resin-bound peptide did not contain a β-turn. This ‘designed’ convergent synthesis gave the target peptide in much higher yield and purity than the conventional stepby-step synthesis. HOAc, acetic acid; Boc,tert-butyloxycarbonyl; DCC, dicyclohexylcarbodiimide: DCM, dichloromethane; DIC, diisopropylcarbodiimide; DIEA,N,N-diisopropylethylamine; DMFN,N-dimethylformamide; DMSO, dimethylsulfoxide; EDT. ethanedithiol; FAB-MS, fast atom bombardment mass spectrometry; Fmoc, 9-luorenylmethoxycarbonyl; HOBt, 1-hydroxybenzotriazole; HPLC, high-performance liquid chromatography; i-PrOH, isopropanol; Mmt, 4-methoxytrityl; PEG-PS, polvethyleneglycol grafted polystyrene; Pme, 2,2,5,7,8-pentamethylchroman-6-sulfonyl; RP, reversed phase; rt, room temperature; SPPS, solid phase peptide synthesis;tBu,tert-butyl; TFA, trifluoroacetic acid; TFE, trifluoroethanol; TLC, thin layer chromatography; Trt, triphenylmethyl, trityl. Abbreviations used for amino acids follow the rules of the IUPAC-IUB Commission of Biochemical Nomenclature [J. Biol. Chem. 247 (1972), 977]. All amino acids are of the L-configuration.     

Solid-phase synthesis of two glycopeptides containing the amino acid sequence 5 to 9 of somatostatin

2,3,4,6 Tetra-O-acetyl-1-N-[N-(tert-butyloxycarbonyl)-l-aspart-4-oyl]-d-glucopyranosylamine and 2-acetamido-3,4,6-tri-O-acetyl-1-N-[N-(tert-butyloxycarbonyl)-l-aspart-4-oyl]-2-deoxy-d-glucopyranosylamine were introduced, respectively, by the solid-phase procedure in the amino acid sequence 5 to 9 of somatostatin. The two resulting glycopeptides β-d-Glcp-(1→4)- and β-d-GlcpNAc-(1→4)-Asn-Phe-Phe-Trp-Lys-OH were homogeneous on examination by t.l.c. and l.c., and their structures were confirmed by m.s. of the N-acetyl, permethyl derivatives.     

A large-scale synthesis of somatostatin for clinical use by a novel alternating solution/solid-phase procedure

A large-scale synthesis of somatostatin was developed. A stepwise C→N approach in solution was used, employing N(α)-t-butoxycarbonyl amino acid active esters. The scheme of semipermanent protection utilized 2-(methylsulfonyl)-ethoxycarbonyl for the -amino group of lysine; acetamidomethyl for the β-thiol groups of cysteine; the orange-colored 2-[4-(phenylazo)-phenylsulfonyl]-ethoxy group for the C-terminal carboxy group of cysteine. All condensations and N(α)-deprotections were carried out in homogeneous solution, while isolation and purification of peptides carrying the colored group was achieved by precipitation and washing of the solid products. Thus, the “alternating solution/solid-phase peptide synthesis” combines advantages of both the classical solution synthesis and the Merrifield solid-phase technique. The overall yield was 5%, or 16 g of somatostatin from 100 g of the novel amino acid derivative, N(α)-t-butoxycarbonyl-S-acetamidomethyl- -cysteine 2-[4-(phenylazo)-phenylsulfonyl]-ethyl ester. An improved method for the preparation of S-acetamidomethyl- -cysteine, free of thiazolidine carboxylic acid, is described.     

3-(1-Piperidinyl)alanine formation during the preparation ofC-terminal cysteine peptides with the Fmoc/t-Bu strategy

Summary Several side reactions have been detected for cysteine-containing peptides. During the synthesis ofC-terminal cysteine peptides, a base-catalyzed elimination of the sulfhydryl-protected side-chain to afford the dehydroalanine derivative followed by a nucleophilic addition to the alkene was observed. MALDI-TOF analysis was a useful analytical technique to determine this phenomenon.Abbreviations Acm acetamidomethyl - Boc tert-butyloxycarbonyl - t-Bu tert-butyl - DBU 1,8-diazabicyclo[5.4.0]undec-7-ene - DIEA N,N-diisopropylethylamine - DMF N,N-dimethylformamide - Fmoc 9-fluorenylmethyloxycarbonyl - HATU N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-yl-methylene]-N-methylmethanaminium hexafluorophosphateN-oxide - HBTU N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphateN-oxide - HOAt 1-hydroxy-7-azabenzotriazole - HOBt 1-hydroxybenzotriazole - HPLC high-performance liquid chromatography - MALDI-TOF matrix-assisted laser desorption/ionization time-of-flight mass spectrometry - PAC 4-hydroxymethylphenoxyacetic acid handle - PAL 5-(4-(9-fluorenylmethyloxycarbonyl)aminomethyl-3,5-dimethoxy-phenoxy)valeric acid handle - PEG-PS polyethylene glycol-polystyrene graft supports - PS polystyrene - Reagent R TFA/thioanisole/1,2-ethanedithiol/anisole (90:5:3:2) - S-t-Bu S-tert-butyl mercapto - TFA trifluoroacetic acid - Trt triphenylmethyl. Amino acid symbols denote thel-configuration, unless indicated otherwise     

The synthesis of isomeric 4-prolinylamines and 4,4′-diprolinylamines

Starting from the previously describedtert-butyl esters of 4-epimericN-benzyloxycarbonyl-4-hydroxyprolines andN-benzyloxycarbonyl-4-trans- and 4-cis-trifluoroacetaminoprolinetert-butyl esters, the corresponding uprotected 4-aminoprolines and a number of their partially protected derivatives were synthesized via the intermediate 4-O-mesyl and 4-azide derivatives. The reductive amination ofN-benzyloxycarbonyl-4-oxoprolinetert-butyl ester with ammonium acetate led toN-benzyloxycarbonyl-4-cis-4′-cis- and 4-cis-4′-trans-diprolinylamines. The¹H NMR and CD spectra of the synthesized compounds are described.     

Microwave-assisted synthesis and characterization of optically active poly (ester-imide)s incorporating l-alanine

Pyromellitic dianhydride (1) was reacted with L-alanine (2) to result [N,N′-(pyromellitoyl)-bis-l-alanine diacid] (3). This compound (3) was converted to N,N′-(pyromellitoyl)-bis-l-alanine diacyl chloride (4) by reaction with thionyl chloride. The microwave-assisted polycondensation of this diacyl chloride (4) with polyethyleneglycol-diol (PEG-200) and/or three synthetic aromatic diols furnish a series of new PEIs and Co-PEIs in a laboratory microwave oven (Milestone). The resulting polymers and copolymers have inherent viscosities in the range of 0.31–0.53 dl g⁻¹. These polymers are optically active, thermally stable and soluble in polar aprotic solvents such as DMF, DMSO, NMP, DMAc, and sulfuric acid. All of the above polymers were fully characterized by IR spectroscopy, ¹H NMR spectroscopy, elemental analyses, specific rotation and thermal analyses. Some structural characterizations and physical properties of these optically active PEIs and Co-PEIs have been reported.     

Aqueous-phase quantitative NMR determination of amino acid enantiomer ratio by 13C-NMR using chiral neodymium shift reagent

A neodymium-(S)-PDTA (PDTA = N,N,N′,N′-tetrakis[(hydroxycarbonyl)methyl]-1,2-diaminopropane) complex was found exceptionally useful in the quantitative determination of enantiomer ratios of water-soluble natural amino acids by ¹³C-NMR. The method is demonstrated on mixtures of l- and d-enantiomers of various amino acids. The interactions of the chiral shift reagent with the amino acid molecules were rationalized by molecular orbital calculations.     

Synthesis and physicochemical characterization of the lanthionine analog of somatostatin[1–14]

Summary The synthesis of the lanthionine analog of somatostatin[1–14] on a Kaiser-oxime resin is described. The 12-residue peptide segment [3–14] was assembled and cyclized on the resin by using the method of peptide cyclization on an oxime resin (PCOR); the product was obtained with good yield (41%) and purity (94%). The Fmoc protecting group on the N-terminus was cleaved with DBU, followed by a 2+12 segment condensation in solution. The chromatographic (HPLC, CZE) and spectral (UV, NMR) properties of the lanthionine and the natural somatostatins have been studied and compared. Preliminary biological tests show that the lanthionine and the natural somatostatins exhibit similar binding affinities to somatostatin receptor SSTR2.Abbreviations Ala_L one end of a lanthionine unit - Boc tert-butyloxycarbonyl - BOP benzotriazol-l-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate - Bzl benzyl - Cbz benzyloxycarbonyl - DQF-COSY double-quantum-filtered correlated NMR spectroscopy - CZE capillary zone electrophoresis - DBU 1,8-diazabicyclo[5.4.0]undec-7-ene - DCC N,N-dicyclohexylcarbodiimide - DCM dichloromethane - DIEA N,N-diisopropylethylamine - DMF N,N-dimethylformamide - DMSO-d₆ hexadeuterated dimethylsulfoxide - EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide - Fmoc 9-florenylmethoxycarbonyl - For formyl - HMPA hexamethylphosphoramide - HOBt N-hydroxybenzotriazole - HOHAHA homonuclear Hartmann-Hahn experiment - HPLC high-performance liquid chromatography - ROESY rotating frame nuclear Overhauser enhancement spectroscopy - TFA trifluoroacetic acid - PCOR peptide cyclization on an oxime resin - Tmac₂O trimethylacetic or pivalic anhydride - Tos p-toluenesulfonyl     

Coupling of iminodiacetic acid with amino acid derivatives in solution and solid phase

Summary The IR studies for the preactivation step of N-protected iminodiacetic acid with different coupling reagents (TCFH, TFFH, HATU, HBTU, HSTU) were reported here and showed the formation of an anhydride as an active intermediate in case of TCFH and TFFH. The formation of a mixture of an anhydride and an active ester (-OBt,-OAt or-OSu) were observed for HBTU, HATU or HSTU coupling reagent. Dependent on the coupling conditions, acylation of N-protected iminodiacetic acid with amino acid ester or amide derivatives in solution phase gave monoor di-substituted iminodiacetic acid derivatives. Coupling of N-protected iminodiacetic acid with an amino acid or peptide attached to a solid support (PAL-PEG-PS or Wang resin) gave only the monosubstituted iminodiacetic acid derivatives. Abbreviations: HBTU, N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate N-oxide; Boc,t-butyloxycarbonyl; DCC, N,N′-dicyclohexylcarbodiimide; DIC, N,N′-diisopropylcarbodiimide; DIEA, diisopropylethylamine; HATU, N-[(dimethylamino)-1H-1,2,3,-triazolo[4,5-b]pyridin-1-yl-methylene]-N-methylmethanaminium hexafluorophosphate N-oxide; DMF, N,N-dimethylformamide; Bsmoc, 1,1-dioxobenzo[b]thiophene-2-ylmethoxycarbonyl; Fmoc, 9-fluorenylmethyloxycarbonyl; HOAt, l-hydroxy-7-azabenzotriazole; HOBt, l-hydroxybenzotriazol; IDA, iminodiacetic acid; HSTU, O-(succinimidyl)-tetramethyluronium hexafluorophosphate; TCFH; 1,1,3,3-tetramethyl-2-chloroformamidinium hexafluorophosphate; TFFH, 1,1,3,3-tetramethyl-2-fluoroformamidinium hexafluorophosphate; TMS-Cl, trimethylchlorosilane. Amino acids and peptides are abbreviated and designated following the rules of the IUPAC-IUB Commission of Biochemical Nomenclature (J. Biol. Chem., 247 (1972) 997).     

Putative primary involvement of <Emphasis Type="Italic">Arabidopsis</Emphasis> phosphoinositide-specific phospholipase C1 within abscisic acid-induced stomatal closing

Stomatal closing to abscisic acid (ABA) was studied in leaf epidermal peels of a dexamethasone (Dex)-inducible transgenic line expressing the phospholipase C AtPLC1 antisense in the Columbia genetic background. In the absence of Dex, the Ca²⁺ buffer, ethylene glycol-bis(b-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) and the phopholipase C inhibitor, 1-[6-{[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl]-1H-pyrrole-2,5-dione (U73122) specifically inhibited the response to 20 μM ABA, whereas the Ca²⁺ buffer, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) inhibited the response to 20 or 30 μM ABA. Neither EGTA nor BAPTA increased the U73122 effect. Applying 30 μM Dex specifically affected 20 μM ABA-induced stomatal closing through reducing its magnitude as well as suppressing the EGTA, BAPTA and U73122 inhibitory effects. Neither Dex nor U73122 changed the specific inhibitory effects of both the antagonist of cyclic ADP-ribose synthesis, nicotinamide and the GTP-binding protein (G protein) modulators, pGlu-Gln-D-Trp-Phe-D-Trp-D-Trp-Met-NH₂ (GP Ant-2) and mas17 on 30 μM ABA-induced stomatal closing. When tested in combination, substituting nicotinamide for mas17, but not for GP Ant-2, enhanced their inhibitory effect to an extent that BAPTA did not increase. These results supported that AtPLC1 primarily mediates the Ca²⁺-dependent stomatal closing response to 20 μM ABA as much as 30 μM Dex did not affect 20 μM ABA-induced stomatal closing when tested on the wild type Columbia-4 ecotype. Furthermore, the present study suggested that Ca²⁺ mobilization did not involve any dependency between AtPLC1 and a putative G protein-coupled ADP-ribosyl cyclase at the tested ABA concentrations.