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).     

Hexafluoroacetone as protecting and activating reagent: Site-selective functionalization of iminodiacetic acid

Summary. Hexafluoroacetone was applied as a bidentate protecting and activating agent for the syntheses of RGD-peptide mimetics starting from iminodiacetic acid in solution and on solid phase.     

Evaluation of COMU as a coupling reagent for in situ neutralization Boc solid phase peptide synthesis

Benzotriazole‐based coupling reagents have dominated the last two decades of solid phase peptide synthesis. However, a growing interest in synthesizing complex peptides has stimulated the search for more efficient and low‐cost coupling reagents, such as COMU which has been introduced as a nonexplosive alternative to the classic benzotriazole coupling reagents. Here, we present a comparative study of the coupling efficiency of COMU with the benzotriazole‐based HBTU and HCTU for use in in situ neutralization Boc‐SPPS. Difficult sequences, such as ACP(65–74), Jung–Redeman 10‐mer, and HIV‐1 PR(81–99), were used as model target peptides on polystyrene‐based resins, as well as polyethylene glycol‐based resins. Coupling yields obtained using fast in situ Boc‐SPPS cycles were determined with the quantitative ninhydrin test as well as via LC‐MS analysis of the crude cleavage products. Our results demonstrate that COMU coupling efficiency was less effective compared to HBTU and HCTU with HCTU ≥ HBTU > COMU, when polystyrene‐based resins were employed. However, when the PEG resin was employed in combination with a safety catch amide (SCAL) linker, more comparable yields were observed for the three coupling reagents with the same ranking HCTU ≥ HBTU > COMU. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Improved solid phase synthesis of luteinizing hormone releasing hormone analogues using 9-fluorenylmethyloxycarbonyl amino acid active esters and catalytic transfer hydrogenation with minimal side-chain protection and their biological activities

Using mainly 9-fluorenylmethyloxycarbonyl amino acid 2, 4, 5-trichlorophenyl esters in the presence of 1-hydroxybenzotriazole and the solid supportp-alkoxybenzyl alcohol resin, synthesis of luteinizing hormone releasing hormone analogues was carried out with minimal side-chain protection. Catalytic transfer hydrogenation was employed for removal of NO₂ and Z-groups from Arg and < Glu respectively avoiding the use of HF and this led to good yields. An aromatic, hydrophilic amino acid, D-(p-hydroxyphenyl) glycine was incorporated into luteinizing hormone releasing hormone molecule along with other modifications. The agonistic as well as antagonistic activities of all the peptides have been studied     

Convergent solid‐phase and solution approaches in the synthesis of the cysteine‐rich Mdm2 RING finger domain

The RING finger domain of the Mdm2, located at the C‐terminus of the protein, is necessary for regulation of p53, a tumor suppressor protein. The 48‐residues long Mdm2 peptide is an important target for studying its interaction with small anticancer drug candidates. For the chemical synthesis of the Mdm2 RING finger domain, the fragment condensation on solid‐phase and the fragment condensation in solution were studied. The latter method was performed using either protected or free peptides at the C‐terminus as the amino component. Best results were achieved using solution condensation where the N‐component was applied with the C‐terminal carboxyl group left unprotected. The developed method is well suited for large‐scale synthesis of Mdm2 RING finger domain, combining the advantages of both solid‐phase and solution synthesis. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis and application of N‐[1‐(4‐(4‐fluorophenyl)‐2,6‐dioxocyclohexylidene)ethyl] (Fde)‐protected amino acids for optimization of solid‐phase peptide synthesis using gel‐phase 19F NMR spectroscopy

N‐[1‐(4‐(4‐fluorophenyl)‐2,6‐dioxocyclohexylidene)ethyl] (Fde) protected amino acids have been prepared and applied in solid‐phase peptide synthesis monitored by gel‐phase ¹⁹F NMR spectroscopy. The Fde protective group could be cleaved with 2% hydrazine or 5% hydroxylamine solution in DMF as determined with gel‐phase ¹⁹F NMR spectroscopy. The dipeptide Ac‐L ‐Val‐L ‐Val‐NH₂ 12 was constructed using Fde‐L ‐Val‐OH and no noticeable racemization took place during the amino acid coupling with N,N′‐diisopropylcarbodiimide and 1‐hydroxy‐7‐azabenzotriazole or Fde deblocking. To extend the scope of Fde protection, the hydrophobic nonapeptide LLLLTVLTV from the signal sequence of mucin MUC1 was successfully prepared using Fde‐L ‐Leu‐OH at diagnostic positions. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis and application of Nα‐Fmoc‐Nπ‐4‐methoxybenzyloxymethylhistidine in solid phase peptide synthesis

The 4‐methoxybenzyloxymethyl (MBom) group was introduced at the N^π‐position of the histidine (His) residue by using a regioselective procedure, and its utility was examined under standard conditions used for the conventional and the microwave (MW)‐assisted solid phase peptide synthesis (SPPS) with 9‐fluorenylmethyoxycarbonyl (Fmoc) chemistry. The N^π‐MBom group fulfilling the requirements for the Fmoc strategy was found to prevent side‐chain‐induced racemization during incorporation of the His residue even in the case of MW‐assisted SPPS performed at a high temperature. In particular, the MBom group proved to be a suitable protecting group for the convergent synthesis because it remains attached to the imidazole ring during detachment of the protected His‐containing peptide segments from acid‐sensitive linkers by treatment with a weak acid such as 1% trifluoroacetic acid in dichloromethane. We also demonstrated the facile synthesis of Fmoc‐His(π‐MBom)‐OH with the aid of purification procedure by crystallization to effectively remove the undesired τ‐isomer without resorting to silica gel column chromatography. This means that the present synthetic procedure can be used for large‐scale production without any obstacles. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Evaluation of acid‐labile S‐protecting groups to prevent Cys racemization in Fmoc solid‐phase peptide synthesis

Phosphonium and uronium salt‐based reagents enable efficient and effective coupling reactions and are indispensable in peptide chemistry, especially in machine‐assisted SPPS. However, after the activating and coupling steps with these reagents in the presence of tertiary amines, Fmoc derivatives of Cys are known to be considerably racemized during their incorporation. To avoid this side reaction, a coupling method mediated by phosphonium/uronium reagents with a weaker base, such as 2,4,6‐trimethylpyridine, than the ordinarily used DIEA or that by carbodiimide has been recommended. However, these methods are appreciably inferior to the standard protocol applied for SPPS, that is, a 1 min preactivation procedure of coupling with phosphonium or uronium reagents/DIEA in DMF, in terms of coupling efficiency, and also the former method cannot reduce racemization of Cys(Trt) to an acceptable level (<1.0%) even when the preactivation procedure is omitted. Here, the 4,4′‐dimethoxydiphenylmethyl and 4‐methoxybenzyloxymethyl groups were demonstrated to be acid‐labile S‐protecting groups that can suppress racemization of Cys to an acceptable level (<1.0%) when the respective Fmoc derivatives are incorporated via the standard SPPS protocol of phosphonium or uronium reagents with the aid of DIEA in DMF. Furthermore, these protecting groups significantly reduced the rate of racemization compared to the Trt group even in the case of microwave‐assisted SPPS performed at a high temperature. © 2013 The Authors. European Peptide Society published by John Wiley & Sons, Ltd.     

An HPLC‐ESMS study on the solid‐phase assembly of C‐terminal proline peptides

DKP formation is a serious side reaction during the solid‐phase synthesis of peptide acids containing either Pro or Gly at the C‐terminus. This side reaction not only leads to a lower overall yield, but also to the presence in the reaction crude of several deletion peptides lacking the first amino acids. For the preparation of protected peptides using the Fmoc/tBu strategy, the use of a ClTrt‐Cl‐resin with a limited incorporation of the C‐terminal amino acid is the method of choice. The use of resins with higher loading levels leads to more impure peptide crudes. The use of HPLC‐ESMS is a useful method for analysing complex samples, such as those formed when C‐terminal Pro peptides are prepared by non‐optimized solid‐phase strategies. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.