Cleavage kinetics and anchor linked intermediates in solid phase peptide amide synthesis.

Kinetics and cleavage conditions of peptide amide synthesis were studied using the anchor molecules 5-(4'-aminomethyl-3',5'-dimethoxyphenoxy)valeric acid (4-ADPV-OH) and 5-(2'-aminomethyl-3'-5'-dimethoxyphenoxy) valeric acid (2-ADPV-OH). Unexpectedly the anchor amide alanyl-4-ADPV-NH2 was isolated and characterized as an intermediate during the cleavage with trifluoroacetic acid (TFA) of alanyl-4-ADPV-alanyl-aminomethyl-polystyrene to yield the alanine amide. As a matter of fact the NH--CH alpha bond of the alanyl spacer has to be cleaved to form this intermediate. Using TFA-dichloromethane (1:9) alanyl-4-ADPV-NH2 was obtained as a cleavage product in 50% yield within 60 min, whereas the isomeric alanyl-2-ADPV-NH2 was formed more slowly under these mild conditions. At high TFA concentration no difference between the 2- and 4-ADPV anchor was observed in the rate of formation of the free alanine amide. The presence of tryptophan amide in the cleavage mixture resulted in an anchor alkylated tryptophan amide, which remains stable in acidic solution but disappears rapidly in the presence of the resin. A low TFA/high TFA cleavage procedure is recommended for peptide amid synthesis applying the ADPV anchor.     

Trans-cis amide bond isomerization in fulleroprolines

The ¹H NMR study of fulleroproline derivative Ac-Fpr-OtBu and its Pro analogue Ac-l -Pro-OtBu over a range of temperatures in toluene-d₈ solution has enabled the comparison of their equilibrium and activation parameters for the trans/cis interconversion around the amide partial double bond. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Studies on the Selective Trifluoroacetolytic Scission of Native Peptaibols and Model Peptides Using HPLC and ESI‐CID‐MS

Representative members of a group of linear, N‐acylated polypeptide antibiotics (peptaibols) containing α‐aminoisobutyric acid (Aib) and, in part, isovaline (Iva), as well as proteinogenic amino acids and a C‐terminal‐bonded 2‐amino alcohol, were treated with anhydrous trifluoroacetic acid (TFA) at 37° for 0.5–26 h. The resulting fragments were separated by HPLC and characterized by electrospray ionization collision‐induced dissociation mass spectrometry (ESI‐CID‐MS). The following 16–20‐residue peptaibols were investigated: natural, microheterogeneous mixtures of antiamoebins and alamethicin F50, uniform paracelsin A, and synthetic trichotoxin A50/E. In the natural peptides, bonds formed between Aib (Iva) and Pro (Hyp) were rapidly and selectively cleaved within 0.5 h. Furthermore, TFA esters of the C‐terminal amino alcohols were formed. Depending on time, release of C‐terminal tri‐ and tetrapeptides as well as amino acids from the major fragments was observed. Synthetic homooligopeptides, namely Z‐ and Ac‐(Aib)₁₀‐O^tBu and Z‐(Aib)₇‐O^tBu, were analyzed for comparison. On treatment with TFA, a regular series of Z‐(Aib)_10–5‐OH from Z‐(Aib)₁₀‐O^tBu were detected within 0.5 h, and, after 3 h, release of a regular series of Z‐(Aib)_7–3‐OH from Z‐(Aib)₇‐O^tBu were observed. Moreover, concomitant release of the series of H‐(Aib)_10–3‐OH from the decapeptide occurred. From these data, a repetitive cleavage mechanism via intermediate formation of C‐terminal oxazolones on trifluoroacetolysis is proposed. Furthermore, their formation and stability in native peptaibols are correlated with subtle structural differences in protein amino acids linked to Aib. From the conspicuous concordance of the formation and abundance of regular series of trifluoroacetolytic fragments and of positive ions of the b‐series in CID‐MS, the generation of intermediate oxazolonium ions in both gas and liquid phase is concluded.     

A method for the facile solid-phase synthesis of gramicidin S and its analogs

Summary A simple method is described for the facile synthesis of gramicidin S and six other analogs, using standard solidphase synthetic technology and a single solution-phase cyclization step. The peptides were purified to homogeneity and characterized by plasma desorption time-of-flight mass spectrometry and NMR spectroscopy. Complete ¹H NMR assignments for all seven peptides (in aqueous solution) are presented. Unlike previous approaches, the presented method is simple, automatable, rapid (less than three days), high-yielding, requires no side-chain protection during cyclization, and appears to be generally applicable to the preparation of a variety of related head-to-tail cyclic peptides.Abbreviations Boc t-butyloxycarbonyl - BOP benzotriazoyl N-oxytris(dimethylamino)phosphonium hexafluorophosphate - Bzl benzyl - DCC N,N-dicyclohexylcarbodiimide - DCM dichloromethane - DIEA N,N-diisopropylethylamine - DMF N,N-dimethylformamide - DQF-COSY double-quantum-filtered correlation spectroscopy - DSS 2,2-dimethyl-2-silapentane-5-sulfonate, sodium salt - EDAC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide - HBTU 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate - HOBt 1-hydroxybenzotriazole - 4-MeBzl 4-methylbenzyl - NHS N-hydroxysuccinimide - NOESY nuclear Overhauser effect spectroscopy - PAM phenylacetamidomethyl (resin) - RP-HPLC reversed-phase high-performance liquid chromatography - TFA trifluoroacetic acid - TOCSY total correlation spectroscopy - Tos tosyl - Troc 2,2,2-trichloroethylcarbamate.     

Nα-Fmoc-O,O-(dimethylphospho)-L-tyrosine fluoride: A convenient building block for the solid-phase synthesis of phosphotyrosyl peptides

Summary Fmoc-O,O-(dimethylphospho)-l-tyrosine was converted into stable Fmoc-O,O-(dimethylphospho)-L-tyrosine fluoride by means of (diethylamino) sulfur trifluoride or cyanuric fluoride. This building block was used for efficient coupling of phosphotyrosine to the adjacent sterically hindered amino acid Aib or Ac₆c in, model peptide sequences as well as for the synthesis of the ‘difficult’ phosphotyrosine peptide Stat91^695–708. The phosphate methyl groups were cleaved on solid phase after peptide assembly by means of trimethylsilyl iodide in MeCN. Aib, α-aminoisobutyric acid Ac₆c, 1-amino-cyclohexyl-l-carboxylic acid; BOP, benzotriazol-l-yl-oxy-tris(dimethylamino) phosphonium hexafluorophosphate, CIP, 2-chloro-l, 3-dimethylimidazolidium hexafluorophosphate, DAST, (diethylamino)sulfur trifluoride; DBU 1,8-diazabicyclo[5.4.0]undec-7-ene; DCM, dichloromethane; DIEA, drisopropylethylamine; DMA dimethylacetamide; Fmoc, 9-fluorenylmethoxycarbonyl; HATU,O-(7-azabenzotriazol-l-yl)-1.1,3,3-tetramethyluronium hexafluorophosphate; HOAt, I-hydroxy-7-azabenzotriazole; HOBt,N-hydroxybenzotriazole; HPLC, high-performance liquid chromatography; MBHA, 4-methylbenzhydrylamine; MeCN, acetonitrile; NMP,N-methyl-2-pyrrolidinone; NMR, nuerear magnetic resonance; PS, polystyrene; PyBroP, bromotris(pyrrolidino)phosphonium hexafluorophosphate; Rink amide MBHA-PS, 4-(2′,4′-dimethoxyphenyl-Fmoc-aminophenyl)-phenoxyacetamido-norleucyl-MBHA-PS; TFA, trifluoroacetic acid; TMSI, trimethylsilyl iodide; TPTU, 2-(2-pyridon-l-yl)-1,1,3,3-tetramethyluroniumfluoroborate; t_R, retention time; UNCA, arethane-protected amino acidN-carboxy anhydride Abbreviations for amino acids and nomenclature of, peptide structures follow the recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature [Eur. J. Biochem., 138 (1984) 9].     

Synthesis and characterization of organotin Schiff base chelates

The synthesis and characterization of five organotin compounds containing Salophen(^tBu) [Salophen(^tBu)=N,N′-phenylene-bis(3,5-di-tert-butylsalicylideneimine)], Salomphen(^tBu) [Salomphen(^tBu)=N,N′-(4,5-dimethyl)phenylene-bis(3,5-di-tert-butylsalicylideneimine)] and Phensal(^tBu) [Phensal(^tBu)=3,5-di-tert-butylsalicylidene(1-aminophenylene-2-amine)] ligands is described. These compounds include the monomeric complexes LSnCl₂ (where L=Salophen(^tBu), L=Salomphen(^tBu)), L(ⁿBu)SnCl (where L=Salophen(^tBu), Salomphen(^tBu)), L(ⁿBu)SnCl₂ (where L=Phensal(^tBu)). Spectroscopic techniques including ¹¹⁹Sn NMR and X-ray crystallography were used in the characterization of the compounds.     

Synthesis and reactivity of imido niobium complexes containing the functionalized (dichloromethylsilyl)cyclopentadienyl ligand

The niobium complex [NbCp^ClCl₄] (Cp^Clη⁵-C₅H₄(SiCl₂Me)) (1) with a functionalized (dichloromethylsilyl)cyclopentadienyl ligand was isolated by the reaction of [NbCl₅] with C₅H₄(SiCl₂Me)(SiMe₃). Complex 1 was a precursor for the imido silylamido derivative [NbCp^NCl₂(NtBu)] (Cp^Nη⁵-C₅H₄[SiClMe(NHtBu)]) (2) after addition of LiNHtBu, which subsequently gave the dichlorosilyl compound [NbCp^ClCl₂(NtBu)] (3) when reacted with SiCl₃Me. Addition of LiNHtBu to complex 2 gave the niobium amido complex [NbCp^NCl(NHtBu)(NtBu)] (4), which slowly evolved with exchange of the niobium-amido and the silicon-chloro groups to give the dichloroniobium complex [NbCp^NNCl₂(NtBu)] (Cp^NNη⁵-C₅H₄[SiMe(NHtBu)₂]) (5). Reaction of 2 with excess LiNHtBu gave the silyl-η-amido constrained geometry complexes [Nb{η⁵-C₅H₄[SiMe(NHtBu)(-η-NtBu)]}(NHtBu)(NtBu)] (6) and [Nb{η⁵-C₅H₄[SiClMe(-η-NtBu)]}(NHtBu)(NtBu)] (7), whereas addition of one equimolecular amount of LiNHtBu to 5 in C₆D₆ afforded complex [NbCp^NNCl(NHtBu)(NtBu)] (8). All of the new complexes were characterized by ¹H, ¹³C and ²⁹Si NMR spectroscopy.     

Preparation of an unprotected phosphotyrosine building block and its application in solid-phase synthesis of phosphopeptides

Summary A new and facile synthesis of tyrosine phosphorylated peptides has been developed.N ^α-Fmoc-Tyr(tBu)-OPfp was treated with TFA, phosphorylated with phosphorous oxychloride and the resulting phosphoric acid dichloride was hydrolysed to giveN ^α-Fmoc-Tyr(PO₃H₂)-OPfp1 in an overall yield of 98%. Compound1 was used in solid-phase peptide synthesis of phosphopeptides2, 3 and4, which are fragments of murine adipocyte lipid binding protein. The advantage of using the Pfp ester was the absence of pyrophosphates and other byproducts.     

Inversion of 310-helix screw sense in a (D-αMe)Leu homotetrapeptide induced by a guest D-(αMe)val residue

The terminally blocked tetrapeptide pBrBz-[D -(αMe)Leu]₂-D -(αMe)Val-D -(αMe)Leu-OtBu is folded in the crystal state in a left-handed 3₁₀-helical structure stabilized by two consecutive 1 ← 4 C?O ?H? N intramolecular H-bonds, as determined by X-ray diffraction analysis. A CD study strongly supports the view that this conformation is also that largely prevailing in MeOH solution. A comparison with the published conformation of pBrBz-[D -(αMe)Leu]₄-OtBu indicates that incorporation of a single internal β-branched (αMe)Val guest residue into the host homo-tetrapeptide from the γ-branched (αMe)Leu residue is responsible for a dramatic structural perturbation, i.e. an inversion of the 3₁₀ screw sense from right to left-handed.     

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     

Stability of the O-octanoyl group of rat ghrelin during chemical synthesis: Counter-ion-dependent alteration of an ester bond breakage

Summary Rat ghrelin, a 28-amino acid residue peptide with an octanoyl group at the side chain of Ser3, was synthesized chemically by applying Fmoc/ ^t Bu strategy. An ester linkage between octanoic acid and the hydroxyl function of Ser3 was found to be maintained without serious damage during the final deprotection with trifluoroacetic acid (TFA). The most notable finding was the counter-ion-dependent stability change of the octanoyl moiety in the molecule. After consolidation of the counter-ion to TFA (TFA form), the octanoyl group persisted stably upon dissolution in water, whereas in the case of the acetate-form peptide, both de-octanoylation and dehydration (formation of the dehydro-Ala residue) occurred in aqueous solution at the same Ser3 residue. The amounts of these degraded products varied with factors such as solvent, temperature and times of lyophilization. These experimental findings lay the basis for performing the bioassay of ghrelin, which has an octanoyl moiety involved in its numerous biological activities thus far revealed.     

Syntheses of Group 14 bis(tert-butylamido)cyclodiphosph(III)azane dichlorides, and the solid-state structures of the silicon and tin derivatives

To test the synthetic utility of bis(tert-butylamido)cyclodiphosph(III)azanes as ligands we extended the coordination chemistry of these diamides from Group 4 to Group 14. The syntheses of compounds of the formula cis-[^tBuNP(μ-^tBuN)₂PN^tBu]ECl₂, E = Si (1), Ge (2), Sn (3) and the solid-state structures of 1 and 3 are reported. Silicon tetrachloride reacted with dilithiobis(tert-butylamido)cyclodiphosph(III)azane to cleanly produce cis-[^tBuNP(μ-^tBuN)₂PN^tBu]SiCl₂, but for the germanium and tin analogues the interaction of GeCl₄ or SnCl₄ with the diazastannylene cis-[^tBuNP(μ-^tBuN)₂PN^tBu]Sn proved to be a better method. Single-crystal X-ray studies on both 1 and 3 revealed that they had C_s-symmetric structures, the central element being coordinated by two amide nitrogens and two chlorides, in addition to being weakly coordinated by one of the cyclodiphosph(III)azane ring nitrogens. Using structural comparisons between crystallographically-independent 1a and 1b, between 1 and 3, and between 3 and its isomorphous zirconium analogue, the nature of this donor bond is discussed.     

Conformational Characterization of the 1-Aminocyclobutane-1-carboxylic Acid Residue in Model Peptides

A series of N- and C-protected, monodispersed homo-oligopeptides (to the dodecamer level) from the small-ring alicyclic C^α,α-dialkylated glycine 1-aminocyclobutane-1-carboxylic acid (Ac₄c) and two Ala/Ac₄c tripeptides were synthesized by solution methods and fully characterized. The conformational preferences of all the model peptides were determined in deuterochloroform solution by FT-IR absorption and ¹H-NMR. The molecular structures of the amino acid derivatives Z-Ac₄c-OH and Z₂-Ac₄c-OH, the tripeptides Z-(Ac₄c)₃-OtBu, Z-Ac₄c-(L -Ala)₂-OMe and Z-L -Ala-Ac₄c-L -Ala-OMe, and the tetrapeptide Z-(Ac₄c)₄-OtBu were determined in the crystal state by X-ray diffraction. The average geometry of the cyclobutyl moiety of the Ac₄c residue was assessed and the τ(N–C^α–C′) bond angle was found to be significantly expanded from the regular tetrahedral value. The conformational data are strongly in favour of the conclusion that the Ac₄c residue is an effective β-turn and helix former. A comparison with the structural propensities of α-aminoisobutyric acid, the prototype of C^α,α-dialkylated glycines, and the other extensively investigated members of the family of 1-aminocycloalkane-1-carboxylic acids (Ac_nc, with n=3, 5–8) is made and the implications for the use of the Ac₄c residue in conformationally constrained peptide analogues are briefly examined. © 1997 European Peptide Society and John Wiley & Sons, Ltd     

Solid-phase synthesis of tyrosyl H-phosphonopeptides and methylphosphonopeptides

Phosphopeptides are a useful tool for the investigation of phosphorylation as a reversible post-translational modification. There is a growing interest in using mimics of phosphoamino acids involved in phosphorylation in order to study the enzymes concerned in these processes. These mimics should contain a non-hydrolysable or isoelectrically modified phosphate moiety to be used as a specific inhibitor of phosphatases and kinases. We introduce solid-phase synthesis of H- and methylphosphonopeptides as a new class of mimics of phosphotyrosyl peptides. The peptides were synthesized on solid phase using the standard fluorenyl-methyloxycarbonyl (Fmoc) strategy. Tyrosine residues were incorporated as allyl-protected derivatives, which were selectively deprotected on the resin by treatment with Pd(PPh₃)₄. The peptide resin carrying the side-chain unprotected tyrosine of the model peptide Gly-Gly-Tyr-Ala was phosphonylated with di-tert-butyl-N,N-diethyl-phosphoramidite in the presence of ¹H-tetrazole, yielding H-phosphonopeptides after trifluoroacetic acid (TFA) cleavage. Alternatively, phosphonylation of the unprotected tyrosine with O-tert-butyl-N,N-diethyl-P-methylphosphonamidite catalysed by ¹H-tetrazole and followed by oxidation led to the methylphosphonopeptides after TFA cleavage. We obtained both the H-phosphonopeptides and the methylphosphonopeptides of the tetrapeptide in high yields and purities above 90%, according to reversed-phase high-performance liquid chromatography (RP-HPLC). To investigate the general applicability of our new methodology, we synthesized phosphonopeptides up to 13 amino acids long, corresponding to recognition sequences of tyrosine kinases. After cleavage and deprotection, all phosphonopeptides were obtained in high yields and purities of about 90%, as shown by mass spectrometry. The only by-product found was the unmodified peptide. © 1997 European Peptide Society and John Wiley & Sons, Ltd.     

Convenient solid phase synthesis method for the preparation of cysteine C-terminally derivatized peptides

Summary This paper describes a novel solid phase peptide synthesis method for the systematic C-terminal modification of cysteine-containing peptides. In this method, cysteine is linked to chloromethylated polystyrene resin by its thiol functionality, followed by protection of the N-terminus and derivatization of the carboxylic acid to esters or amides. We report here on examples of the methodology and its application to the synthesis of Ac-Asp-cyclo(Cys-Gly-Pro-Cys)-NHBzl, a cyclic peptide amide. The method has been applied to the synthesis of complex esters as well as amides.Abbreviations Ac acetyl - AcN acetonitrile - Ac₂O acetic anhydride - AcOH acetic acid - Boc t-butyloxycarbonyl - BOP benzotriazol-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate - Bzl benzyl - cHex cyclohexyl - DBU 1,8-diazabicyclo[5.4.0]-undec-7-ene - DCC N,N-dicyclohexylcarbodiimide - DCM dichloromethane - DIEA diisopropylethylamine - DMF dimethylformamide - DMS dimethylsulfide - HOB 1-hydroxybenzotriazole - MBzl 4-methyl benzyl - MeOH methanol - TEA triethylamine - TEAP triethylammonium phosphate - TFA trifluoroacetic acid     

Defect peptide chemistry: Perturbations in the structure of a homopentapeptide induced by a guest residue interrupting side-chain regularity

The fully blocked pentapeptide Tfa-(Deg)₂-L -Abu-(Deg)₂-OtBu (Tfa:triflouroacetyl; Deg: C^α,α-diethylglycine; OtBu: tert-butoxy) adopts in the crystal state a regular, right-handed 3₁₀-helical structure stabilized by three N ? H …? O ? C intramolecular 1 ← 4 (or C₁₀) H bonds, as determined by an x-ray diffraction analysis. However, a Fourier transform ir absorption and ¹H-nmr study strongly supports the view that in deuterochloroform solution the four Deg residues at both termini of the peptide main chain are involved in successive, fully extended C₅ forms. A comparison with the stable, fully developed, multiple C₅ conformation of Tfa-(Deg)₅-OtBu indicates that incorporation of an Abu guest residue, interrupting the side-chain uniformity of the host (Deg)₅ homopeptide, while altering only marginally the conformation in a solvent of low polarity, is responsible for a dramatic perturbation of the crystal-state structure. © 1994 John Wiley & Sons, Inc.     

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.