Addition of HOXt (X = A or B) improves the efficiency of phenol-based coupling reagents during peptide synthesis

In the course of comparing the effectiveness ofHATU, HBTU, and phenol-based coupling reagents, suchas the pentafluorophenyl, 2-nitrophenyl, and2,4,5-trichlorophenyl uronium salts by (a) formationof Fmoc-Ala-Val-OtBu, (b) (2+1) segment couplingand (c) stepwise solid phase peptide assembly oftypical model peptides such as the pentapeptideH-Tyr-Aib-Aib-Phe-Leu-NH₂ and ACP decapeptide(65–74), we found a striking improvement of the lesseffective phenol-based coupling reagents (HPyOPfp,HPyONp, and HPyOTcp), both with regard to reactionrate and extent of epimerization, when HOAt was addedand a clear superiority of HAPyU (in the presence andabsence of HOAt) relative to the compounds derivedfrom HOBt, HOPfp, HONp, and HOTcp.     

Synthesis of phosphopeptides by the Multipinast method: Evaluation of coupling methods for the incorporation of Fmoc- Tyr(PO3Bzl,H)-OH,Fmoc- Ser(PO3Bzl,H)-OH and Fmoc- Thr(PO3Bzl,H)-OH

Summary The efficiency of various coupling methods for the incorporation of the three monobenzyl phosphorodiesterprotected derivatives, Fmoc-Tyr(PO₃Bzl,H)-OH, Fmoc-Ser(PO₃Bzl,H)-OH and Fmoc-Thr(PO₃Bzl,H)-OH, was examined through the test synthesis of Ala-Ser-Gln-Gly-Xxx(PO₃H₂)-Leu-Glu-Asp-Pro-Ala-NH₂ (Xxx=Tyr, Ser, Thr) using the Multipin method of multiple peptide synthesis. The coupling methods examined were (1) PyBrop/DIEA; (2) BOP/HOBt/NMM; (3) BOP/HOBt/DIEA; (4) HBTU/HOBt/DIEA; (5) HATU/HOAt/DIEA; (6) HATU/DIEA; (7) DIC/HOBt; (8) DIC/HOBt/DIEA; (9)DIC/HOAt; (10) DIC/HOAt/DIEA. While all four DIC-based coupling procedures resulted in incomplete incorporation, both the HBTU/HOBt/DIEA and HATU/HOAt/DIEA coupling procedures provided most efficient incorporation of the three Fmoc-Xxx (PO₃Bzl,H)-OH derivatives. In the subsequent synthesis of the α-helical Tyr(P)-peptide, Glu-Thr-Gly-The-Lys-Ala-Glu-Leu-Leu-Ala-Lys-Tyr(PO₃H₂)-Glu-Ala-Thr-His-Lys-NH₂, analysis of the crude peptide by electrospray MS confirmed that several residue deletions had occurred but that complete incorporation of the Tyr(P)-residue had been accomplished using HBTU/HOBt/DIEA coupling of Fmoc-Tyr(PO₃Bzl,H)-OH. Multipin is a trademark of Chiron Technologies Pty. Ltd., Clayton, Victoria, Australia.     

Head-to-tail macrocyclization of cysteine-free peptides using an o-aminoanilide linker

A head-to-tail macrocyclization protocol for the preparation of cysteine-free cyclic peptides was investigated. The o-aminoanilide linker constructed in the peptide sequence by a standard Fmoc-based peptide synthesis procedure was subjected to nitrite-mediated activation under acidic conditions toward N-acyl benzotriazole as the active ester species. The subsequent cyclization smoothly proceeded by neutralization in the presence of additives such as 1-hydroxybenzotriazole (HOBt) and 1-hydroxy-7-azabenzotriazole (HOAt) to afford the expected cyclic pentapeptide, a CXCR4 antagonist. The cyclization efficiencies were dependent on the precursor open-chain sequence. The application of this step-wise activation-cyclization protocol to microflow reaction systems is also described.     

Evaluation of coupling conditions for the incorporation of Nα-Fmoc-Tyr(PO3H2)-OH in solid-phase peptide synthesis

Summary In order to minimise the formation of the pyrophosphate derivative of the target peptide when side-chain-unprotected phopshotyrosine is used in solid-phase peptide synthesis, this building block can be incorporated using benzotriazolyloxy-tris-(dimethylamino)phosphonium hexafluorophosphate/1-hydroxybenzotriazole/N-methylmorpholine (1:1:2.3) in the presence of a chaotropic salt (0.4 M LiCl in N-methyl-2-pyrrolidinone).Abbreviations BOP benzotriazolyloxy-tris-(dimethylamino)phosphonium hexafluorophosphate - DIEA diisopropylethylamine - Fmoc 9-fluorenylmethoxycarbony - HATU N-[(dimethylamino)1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmethan-aminium hexafluorophosphate N-oxide - HOBt 1-hydroxybenzotriazole - HPLC high-performance liquid chromatography - MALDI-TOF matrix-assisted laser-desorption ionization time-of-flight mass spectrometry - NMM N-methylmorpholine - NMP N-methyl-2-pyrrolidinone - Pmc 2,2,5,7,8-pentamethyl-chroman-6-sulfonyl - ® solid support - TFA trifluoroacetic acid - TPTU 2-(2-pyridon-1-yl)-1,1,3,3-tetramethyluroniumfluoroborate. Abbreviations used for amino acids follow the recommendations of the IUPAC-IUB Commission of Biochemical Nomenclature [Eur. J. Biochem., 138 (1984) 9]     

Effects of amounts of additives on peptide coupling mediated by a water-soluble carbodiimide in alcohols.

The optimal amounts of 1-hydroxybenzotriazole (HOBt), 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOOBt) and 1-hydroxy-7-azabenzotriazole (HOAt) for enhancement of peptide coupling mediated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) hydrochloride in alcoholic solvents were found to be less than equimolar against the carboxyl component or the carbodiimide. In comparison with the use of equimolar additives, the use of less equimolar ones was more effective in suppressing the competitive ester formation and in increasing the yield of desired peptides. EDC hydrochloride/around 0.1 equimolar HOAt or HOOBt were efficient reagents for peptide synthesis in the media.     

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     

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].     

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).     

Synthesis of phosphopeptides by the Multipinast method: Evaluation of coupling methods for the incorporation of Fmoc- Tyr(PO3Bzl,H)-OH, Fmoc- Ser(PO3Bzl,H)-OH and Fmoc- Thr(PO3Bzl,H)-OH

astMultipin is a trademark of Chiron Technologies Pty. Ltd., Clayton, Victoria, Australia.The efficiency of various coupling methods for the incorporation of the three monobenzyl phosphorodiester-protected derivatives, Fmoc- Tyr(PO3Bzl,H)-OH, Fmoc-Ser(PO3Bzl,H)-OH and Fmoc-Thr(PO3Bzl,H)-OH, was examined through the test synthesis of Ala-Ser-Gln-Gly-Xxx(PO3H2)-Leu- Glu-Asp-Pro-Ala-NH2 (Xxx = Tyr, Ser, Thr) using the Multipin method of multiple peptide synthesis. The coupling methods examined were (1) PyBrop/DIEA; (2) BOP/HOBt/NMM; (3) BOP/HOBt/DIEA; (4) HBTU/HOBt/DIEA; (5) HATU/HOAt/DIEA; (6) HATU/DIEA; (7) DIC/HOBt; (8) DIC/HOBt/DIEA; (9) DIC/HOAt; (10) DIC/HOAt/DIEA. While all four DIC-based coupling procedures resulted in incomplete incorporation, both the HBTU/HOBt/DIEA and HATU/HOAt/DIEA coupling procedures provided most efficient incorporation of the three Fmoc- Xxx(PO3Bzl,H)-OH derivatives. In the subsequent synthesis of the -helical Tyr(P)-peptide, Glu-Thr-Gly-The-Lys- Ala-Glu-Leu-Leu-Ala-Lys-Tyr(PO3H2)-Glu-Ala-Thr- His-Lys-NH2, analysis of the crude peptide by electrospray MS confirmed that several residue deletions had occurred but that complete incorporation of the Tyr(P)-residue had been accomplished using HBTU/HOBt/DIEA coupling of Fmoc- Tyr(PO3Bzl,H)-OH.     

Cu(OBt)2 and Cu(OAt)2, copper(II)-based racemization suppressors ready for use in fully automated solid-phase peptide synthesis.

The complexes Cu(OBt)2 and Cu(OAt)2, which are derived from copper(II) and HOBt and HOAt, respectively, are shown to be more effective in suppressing racemization during solid-phase peptide synthesis (SPPS) than are those compounds currently being used for this purpose. These compounds can readily be used in conjunction with the commonly applied coupling reagents in fully automated systems for solid-phase peptide chemistry.     

Practical protocols for stepwise solid-phase synthesis of cysteine-containing peptides.

This study details a series of conditions that may be applied to ensure 'safe' incorporation of cysteine with minimal racemization during automated or manual solid-phase peptide synthesis. Earlier studies from our laboratories [Han et al. (1997) J. Org. Chem. 62, 4307-4312] showed that several common coupling methods, including those exploiting in situ activating agents such as N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU), N-[1H-benzotriazol-1-yl)-(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HBTU), and (benzotriazol-1-yl-N-oxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP) [all in the presence of N-methylmorpholine (NMM) or N,N-diisopropylethylamine (DIEA) as a tertiary amine base], give rise to unacceptable levels (i.e. 5-33%) of cysteine racemization. As demonstrated on the tripeptide model H-Gly-Cys-Phe-NH(2), and on the nonapeptide dihydrooxytocin, the following methods are recommended: O-pentafluorophenyl (O-Pfp) ester in DMF; O-Pfp ester/1-hydroxybenzotriazole (HOBt) in DMF; N,N'-diisopropylcarbodiimide (DIPCDI)/HOBt in DMF; HBTU/HOBt/2,4,6-trimethylpyridine (TMP) in DMF (preactivation time 3.5-7.0 min in all of these cases); and HBTU/HOBt/TMP in CH(2)Cl(2)/DMF (1:1) with no preactivation. In fact, several of the aforementioned methods are now used routinely in our laboratory during the automated synthesis of analogs of the 58-residue protein bovine pancreatic trypsin inhibitor (BPTI). In addition, several highly hindered bases such as 2,6-dimethylpyridine (lutidine), 2,3,5,6-tetramethylpyridine (TEMP), octahydroacridine (OHA), and 2,6-di-tert-butyl-4-(dimethylamino)pyridine (DB[DMAP]) may be used in place of the usual DIEA or NMM to minimize cysteine racemization even with the in situ coupling protocols.     

Microwave-assisted solid-phase peptide synthesis of the 60–110 domain of human pleiotrophin on 2-chlorotrityl resin

A fast and efficient microwave-assisted solid phase peptide synthesis (MW-SPPS) of a 51mer peptide, the main heparin-binding site (60–110) of human pleiotrophin (hPTN), using 2-chlorotrityl chloride resin (CLTR-Cl) following the 9-fluorenylmethyloxycarbonyl/tert-butyl (Fmoc/tBu) methodology and with the standard N,N′-diisopropylcarbodiimide/1-hydroxybenzotriazole (DIC/HOBt) coupling reagents, is described. An MW-SPPS protocol was for the first time successfully applied to the acid labile CLTR-Cl for the faster synthesis of long peptides (51mer peptide) and with an enhanced purity in comparison to conventional SPPS protocols. The synthesis of such long peptides is not trivial and it is generally achieved by recombinant techniques. The desired linear peptide was obtained in only 30 h of total processing time and in 51% crude yield, in which 60% was the purified product obtained with 99.4% purity. The synthesized peptide was purified by reversed phase high performance liquid chromatography (RP-HPLC) and identified by electrospray ionization mass spectrometry (ESI-MS). Then, the regioselective formation of the two disulfide bridges of hPTN 60–110 was successfully achieved by a two-step procedure, involving an oxidative folding step in dimethylsulfoxide (DMSO) to form the Cys⁷⁷–Cys¹⁰⁹ bond, followed by iodine oxidation to form the Cys⁶⁷–Cys⁹⁹ bond.     

Unexpected racemization of proline or hydroxy-proline phenacyl ester during coupling reactions with Boc-amino acids.

When L-proline or O-benzyl-trans-4-hydroxy-L-proline phenacyl ester was coupled with Boc-amino acids in dimethylformamide using water-soluble carbodiimide (WSCI) in the presence of anhydrous 1-hydroxybenzotriazole (HOBt) as coupling reagents, extensive racemization was observed at the C alpha of the proline or hydroxy-proline residue. The extent of racemization was measured by HPLC after the coupling with Boc-L-Leu-OH in the presence or absence of HOBt. The extent of racemization increased when HOBt was added to the reaction mixture, but greatly decreased when it was not, indicating that HOBt was needed for inducing racemization. Almost no racemization was observed when the coupling reaction was carried out by the mixed anhydride procedure in tetrahydrofuran or by the carbodiimide method in dichloromethane without using HOBt. In the case of coupling reactions with ordinary L-amino acid phenacyl esters, no racemization was observed. Examination of some model systems yielded sufficient evidence to prove that HOBt is an efficient catalyst for racemizing proline or hydroxy-proline phenacyl ester not only in the stage of cyclic intermediate formation but also in the opening of the ring structure. Thus, the racemization reaction was found to be closely related to the formation of the cyclic carbinol-amine derivative.     

Solid-phase synthesis and characterization of N-methyl-rich peptides.

A library of peptides required for a project investigating the factors relevant for blood-brain barrier transport was synthesized on solid phase. As a result of the high N-methylamino acid content in the peptides, their syntheses were challenging and form the basis of the work presented here. The coupling of protected N-methylamino acids with N-methylamino acids generally occurs in low yield. (7-azabenzotriazol-1-yloxy)-tris(pyrrolidino)phosphonium hexafluorophosphate (PyAOP) or PyBOP/1-hydroxy-7-azabenzotriazole (HOAt), are the most promising coupling reagents for these couplings. When a peptide contains an acetylated N-methylamino acid at the N-terminal position, loss of Ac-N-methylamino acid occurs during trifluoroacetic acid (TFA) cleavage of the peptide from the resin. Other side reactions resulting from acidic cleavage are described here, including fragmentation between consecutive N-methylamino acids and formation of diketopiperazines (DKPs). The time of cleavage is shown to greatly influence synthetic results. Finally, high-performance liquid chromatography (HPLC) profiles of N-methyl-rich peptides show multiple peaks because of slow conversion between conformers.     

Studies on sensitivity to racemization of activated residues in couplings of N-benzyloxycarbonyldipeptides.

A series of 24 peptides Z-Gly-Xaa(R)-OH where Xaa = 15 different residues and R = H, NH2, tBu, Bzl, Trt, Mtr, and StBu were coupled with valine benzyl ester in dimethylformamide or dichloromethane at +5 degrees. The accompanying racemization was determined by analysis of the epimeric products by normal phase high-performance liquid chromatography (HPLC) for Xaa(R) = Met, Cys(StBu) and Lys(Z) and by reversed-phase HPLC after removal of benzyl-based protecting groups for Xaa(R) = Ser(tBu), Thr(tBu) and Arg(Mtr). The coupling methods examined included mixed anhydride (MxAn) at -5 degrees, and N,N'-dicyclohexylcarbodiimide (DCC), benzotriazol-1-yl-tris(dimethylamino)phosphonium hexafluorophosphate (BOP) and O-benzotriazol-1-yl-N,N,N',N'-tetramethyluroniumhexafluorophosp hate (HBTU) in the presence of 1-hydroxybenzotriazole (HOBt). Very few couplings gave stereochemically pure products. The order of sensitivity to racemization of residues depended on the method of coupling and the solvent. It varied most when comparing MxAn to HOBt-assisted reactions; it varied moderately when comparing HOBt-assisted reactions. There was less variation in comparing BOP and HBTU reactions that are initiated by the same mechanism. Leu, Nle, Phe, Asn, Lys(Z) and Asp(OBzl) are identified as the residues least sensitive to racemization. DCC-HOBt generally led to less epimerization than the other methods.     

Convergent Solid-phase Peptide Synthesis Performed in a CHCl3-phenol Mixed Solvent: Synthesis of Amyloid β-peptides as Examples with a Difficult Sequence

Convergent solid-phase peptide synthesis (CSPPS) involving the coupling of protected peptide segments on a solid support performed in a β-sheet disrupting solvent consisting of a mixture of CHCl₃ and phenol (v/v, 3/1), proceeded smoothly without danger of epimerization or of significant phenyl ester formation with the carboxyl component when diisopropylcarbodiimide (DIC) was used in the presence of 1-hydroxy-7-azabenzotriazole (HOAt) or 6-chloro-1-hydroxybenzotriazole (Cl-HOBt). In particular, this synthetic strategy using the CHCl₃ and phenol mixed solvent proved to be essential for coupling sparingly soluble segments even with difficult sequences. The present approach was successfully applied to the synthesis of amyloid β-peptide (Aβ) (1-40) and also its reversed Aβ (40-1) as an inactive control peptide.     

Characterization of synthetic peptide byproducts from cyclization reactions using on-line HPLC-ion spray and tandem mass spectrometry

Summary The cyclization of a linear dynorphin A (Dyn A) analogue to give the lactam derivative cyclo[d-Asp², Dap⁵]Dyn A(1–13)NH₂ (where Dap=,-diaminopropionic acid) was studied to evaluate the usefulness of different coupling reagents for side chain to side chain lactam formation. This cyclization proved to be difficult and yielded substantial byproducts that varied depending upon the activating reagent used. On-line HPLC-ion spray mass spectrometry was more practical and useful than conventional HPLC alone for characterizing the products of these cyclization reactions. Peptide byproducts could be identified from the series of multiply charged ions observed, even when some of these peptides eluted from the HPLC with similar retention times. In addition to the desired cyclic peptide, the peptide byproducts observed following the cyclization using BOP (benzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate) were the linear peptide, the cyclic dimeric peptide and the linear peptide resulting from aspartimide rearrangement. The peptide byproducts obtained following cyclization using HATU (O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) and HAPyU (O-(7-azabenzotriazol-1-yl)-1,1,3,3-bis(tetramethylene)uronium hexafluorophosphate) were predominantly linear tetramethylguanidinium (Tmg) and dipyrrolidinylguanidinium (Dpg) derivatives resulting from alkylation of the side chain of Dap by HATU and HAPyU, respectively; in addition to monomeric guanidinium derivatives, dimeric and aspartimide-containing peptides were also produced. Peptide sequencing by ion spray tandem mass spectrometry was performed to confirm the structure of both pure peptides and peptide byproducts in the crude samples. A unique fragmentation for the ,-bond of the Dap side chain was demonstrated and could be used to identify linear peptide byproducts. The distinctive fragment ions from this cleavage were also observed for the peptides containing the Tmg and Dpg functionalities on the Dap side chain.     

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.     

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.