Hajime Hibino

Synthetic procedure for N-Fmoc amino acyl-N-sulfanylethylaniline linker as crypto-peptide thioester precursor with application to native chemical ligation.

N-sulfanylethylanilide (SEAlide) peptides 1, obtainable using Fmoc-based solid-phase peptide synthesis (Fmoc SPPS), function as crypto-thioesters in native chemical ligation (NCL), yielding a wide variety of peptides/proteins. Their acylating potential with N-terminal cysteinyl peptides 2 can be tuned by the presence or absence of phosphate salts, leading to one-pot/multifragment ligation, operating under kinetically controlled conditions. SEAlide peptides have already been shown to be promising for use in protein synthesis; however, a widely applicable method for the synthesis of N-Fmoc amino acyl-N-sulfanylethylaniline linkers 4, required for the preparation of SEAlide peptides, is unavailable. The present study addresses the development of efficient condensation protocols of 20 naturally occurring amino acid derivatives to the N-sulfanylethylaniline linker 5. N-Fmoc amino acyl aniline linkers 4 of practical use in NCL chemistry, except in the case of the proline- or aspartic acid-containing linker, were successfully synthesized by coupling of POCl(3)- or SOCl(2)-activated Fmoc amino acid derivatives with sodium anilide species 6, without accompanying racemization and loss of side-chain protection. Furthermore, SEAlide peptides 7 possessing various C-terminal amino acids (Gly, His, Phe, Ala, Asn, Ser, Glu, and Val) were shown to be of practical use in NCL chemistry.

4-Methoxybenzyloxymethyl group, a racemization-resistant protecting group for cysteine in Fmoc solid phase peptide synthesis.

The 4-methoxybenzyloxymethyl (MBom) group was introduced for sulfhydryl protection of Cys in combination with Fmoc chemistry. The MBom group proved to substantially suppress racemization of Cys during its incorporation mediated by phosphonium or uronium reagents. Furthermore, this group was found to significantly reduce racemization of the C-terminal Cys linked to a hydroxyl resin during repetitive base treatment, in comparison with the usually used trityl (Trt) and acetamidomethyl (Acm) groups.

Big angiotensin-25: a novel glycosylated angiotensin-related peptide isolated from human urine.

The renin-angiotensin system (RAS), including angiotensin II (Ang II), plays an important role in the regulation of blood pressure and body fluid balance. Consequently, the RAS has emerged as a key target for treatment of kidney and cardiovascular disease. In a search for bioactive peptides using an antibody against the N-terminal portion of Ang II, we identified and characterized a novel angiotensin-related peptide from human urine as a major molecular form. We named the peptide Big angiotensin-25 (Bang-25) because it consists of 25 amino acids with a glycosyl chain and added cysteine. Bang-25 is rapidly cleaved by chymase to Ang II, but is resistant to cleavage by renin. The peptide is abundant in human urine and is present in a wide range of organs and tissues. In particular, immunostaining of Bang-25 in the kidney is specifically localized to podocytes. Although the physiological function of Bang-25 remains uncertain, our findings suggest it is processed from angiotensinogen and may represent an alternative, renin-independent path for Ang II synthesis in tissue.

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.

Synthesis of 3-Methoxyellipticine and Ellipticine by Friedel-Crafts Reaction of Indole-2,3-dicarboxylic Anhydride and Selective Demethylation

Reaction of 1-benzylindole-2,3-dicarboxylic anhydride with 2,4,6-trimethoxypyridine in the presence of a Lewis acid gave 1-benzyl-3-(2,4,6-trimethoxynicotinoyl)indole-2-carboxylic acid as the sole product in high yield, which could be changed to 1-benzyl-3-(2,4,6-trimethoxynicotinoyl)indole. 1-Benzyl-3-(2,4,6-trimethoxynicotinoyl)indole was converted to 3-methoxy-ellipticine and ellipticine by selective demethylation and triflation of the methoxy group.

Bromination of Dimethyl 1-Substituted Indole-2,3-dicarboxylates

Treatment of dimethyl indole-2,3-dicarboxylate with pyridinium hydrobromide perbromide or bromine in the presence of Lewis acid gave dimethyl 5-bromoindole-2,3-dicarboxylate as the sole product. In a similar manner, dimethyl 1-benzyl- and l-benzenesulfonyl-indole-2,3-dicarboxylates provided a mixture of the corresponding 5-bromoindole and 6-bromoindole derivatives. However, methyl l-trifluoromethanesulfonylindole-2,3-dicarboxylate gave methyl 6-bromo-1-trifluoromethanesulfonylindole-2,3-dicarboxylate as a major product.

Postsynthetic modification of unprotected peptides via S-tritylation reaction.

Tritylation using trityl alcohol (Trt-OH) in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) is a convenient and efficient procedure that can offer S-protection of the Cys located in fully unprotected peptides. The procedure simply requires Trt-OH and HFIP to selectively promote S-tritylation in the presence of peptide nucleophilic functionalities.

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

The 4‐methoxybenzyloxymethyl (MBom) group was introduced at the Nπ‐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 Nπ‐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