Kumiko Yoshizawa-Kumagaye

Synthesis and biological properties of antiparallel and parallel dimers of α-human atrial natriuretic peptide

To obtain antiparallel and parallel dimers of a-human atrial natriuretic peptide(α-hANP), two fully protected peptides I and II having the same amino acid sequence as a-hANP with different protective groups at the cysteinyl residues were synthesized, the former having Acm and Npys and the latter MeBzl and Acm. Equivalent amounts of peptides I and II were mixed and subjected to HF deprotection. Next, the first disulfide bond was linked between the remaining Npys group in I and the liberated SH group in II to form a mono-disulfide dimer. The second disulfide bond was formed within the newly formed dimer between the remaining Acm groups by treatment with iodine, giving an antiparallel dimer. The parallel dimer of α-hANP was synthesized similarly starting from the protected peptide II. These dimers could be clearly segregated on HPLC. The retention time on HPLC of the antiparallel dimer was identical with that of natural β-hANP. Both dimers showed biological activities as high as one third to one sixth of α-hANP in smooth muscle spasmolytic activity, and almost the same level of natriuretic activity as α-hANP at a high dose (10 nmol/kg) but about one fifth the activity at low dose (1 nmol/kg). In these assay systems, the antiparallel dimer showed a slower onset and a tendency of longer duration than α-hANP.

Biological properties of adrenomedullin conjugated with polyethylene glycol

Adrenomedullin (AM) is a vasodilator peptide with pleiotropic effects, including cardiovascular protection and anti-inflammation. Because of these beneficial effects, AM appears to be a promising therapeutic tool for human diseases, while intravenous injection of AM stimulates sympathetic nerve activity due to short-acting potent vasodilation, resulting in increased heart rate and renin secretion. To lessen these acute reactions, we conjugated the N-terminal of human AM peptide with polyethylene glycol (PEG), and examined the biological properties of PEGylated AM in the present study. PEGylated AM stimulated cAMP production, an intracellular second messenger of AM, in cultured human embryonic kidney cells expressing a specific AM receptor in a dose-dependent manner, as did native human AM. The pEC50 value of PEGylated AM was lower than human AM, but no difference was noted in maximum response (Emax) between the PEGylated and native peptides. Intravenous bolus injection of 10nmol/kg PEGylated AM lowered blood pressure in anesthetized rats, but the acute reduction became significantly smaller by PEGylation as compared with native AM. Plasma half-life of PEGylated AM was significantly longer than native AM both in the first and second phases in rats. In summary, N-terminal PEGylated AM stimulated cAMP production in vitro, showing lessened acute hypotensive action and a prolonged plasma half-life in comparison with native AM peptide in vivo.

Problems associated with use of the Nin-cyclohexyloxycarbonyl (Hoc) group for tryptophans

Abstract During HF reaction in the presence of anisole or p -cresol, a side reaction was observed associated with use of the N in -cyclohexyloxycarbonyl (Hoc) group for tryptophan (Trp). The structure of the modified Trp and the HF conditions for suppressing this side reaction are described.

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

Rat ghrelin, a 28-amino acid residue peptide with an octanoyl group at the side chain of Ser3, was synthesized chemically by applying Fmoc/tBu 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.

Switching of turn conformation in an aspartate anion peptide fragment by NH . . . O- hydrogen bonds.

Aspartic acid protease model peptides Z–Phe–Asp(COOH)–Thr–Gly–Ser–Ala–NHCy (1) and AdCO–Asp(COOH)–Val–Gly–NHBzl (3), and their aspartate anions (NEt4)[Z–Phe–Asp(COO−)–Thr–Gly–Ser–Ala–NHCy] (2) and (NEt4)[AdCO–Asp(COO−)–Val–Gly–NHBzl] (4), having an invariant primary sequence of the Asp–X(Thr,Ser)—Gly fragment, were synthesized and characterized by 1H‐NMR, CD, and infrared (IR) spectroscopies. NMR structure analyses indicate that the Asp Oδ atoms of the aspartate peptide 2 are intramolecularly hydrogen‐bonded with Gly, Ser, Ala NH, and Ser OH, supporting the rigid β‐turn‐like conformation in acetonitrile solution. The tripeptide in the aspartic acid 3 forms an inverse γ‐turn structure, which is converted to a β‐turn‐like conformation because of the formation of the intramolecular NH · · · O− hydrogen bonds with the Asp Oδ in 4. Such a conformational change is not detected between dipeptides AdCO–Asp(COOH)–Va–NHAd (5) and (NEt4)[AdCO–Asp(COO−)–Val–NHAd] (6). The pKa value of side‐chain carboxylic acid (5.0) for 3 exhibits a lower shift (0.3 unit) from that of 5 in aqueous polyethyleneglycol lauryl ether micellar solution. NMR structure analyses for 3 in an aqueous micellar solution indicate that the preorganized turn structure, which readily forms the NH · · · O− hydrogen bonds, lowers the pKa value and that resulting hydrogen bonds stabilize the rigid conformation in the aspartate anion state. We found that the formation of the NH · · · O− hydrogen bonds involved in the hairpin turn is correlated with the protonation and deprotonation state of the Asp side chain in the conserved amino acid fragments.

Further Studies on the Side Reactions Associated with Use of Nπ- Benzyloxymethylhistidine

The use of N(alpha)-tert.-butyloxycarbonyl-N(pi)-benzyloxymethylhistidine [Boc-His(Bom)] in peptide synthesis results in a serious level of side products arising from the generation of formaldehyde during the HF cleavage reaction. In particular, when treating a His(Bom)-containing peptide having Cys at the N-terminus by HF, this leads to almost complete conversion of the Cys-peptide to thiazolidyl (Thz)-peptide unless precautions are taken. Also, the reaction of formaldehyde with the N-terminal Trp and the N-methylanthranyl (Nma) group was found to produce tetrahydro-beta-carboline and dihydroquinazolin derivatives, respectively, upon isolation from HF mixtures. The addition of cysteine as a scavenger in HF proved to be effective for suppressing modification arising from the generation of formaldehyde.

Easy‐to‐Attach/Detach Solubilizing‐Tag‐Aided Chemical Synthesis of an Aggregative Capsid Protein

A solubilizing Trt-K10 tag was developed for the effective chemical preparation of peptides/proteins with low solubility. The Trt-K10 tag comprises a hydrophilic oligo-Lys sequence and a trityl anchor, and can be selectively introduced to a side chain thiol of Cys of deprotected peptides/proteins with a trityl alcohol-type introducing reagent Trt(OH)-K10 under acidic conditions. Significantly, the ligation product in the reaction mixture of a thiol-additive-free native chemical ligation can be modified directly in a one-pot manner to facilitate the isolation of the product by high-performance liquid chromatography. Finally, the Trt-K10 tag can be readily removed with a standard trifluoroacetic acid cocktail. Using this easy-to-attach/detach tag-aided method, a hepatitis B virus capsid protein that is usually difficult to handle was synthesized successfully.

O-Acyl isopeptide method: development of an O-acyl isodipeptide unit for Boc SPPS and its application to the synthesis of Aβ1-42 isopeptide

The O‐acyl isopeptide method was developed for the efficient preparation of difficult sequence‐containing peptide. Furthermore, development of the O‐acyl isodipeptide unit for Fmoc chemistry simplified its synthetic procedure by solid‐phase peptide synthesis. Here, we report a novel isodipeptide unit for Boc chemistry, and the unit was successfully applied to the synthesis of amyloid β peptide. Combination of Boc chemistry and the isodipeptide unit would be an effective method for the synthesis of many difficult peptides. Copyright

Non-pretreated O-acyl isopeptide of amyloid β peptide 1-42 is monomeric with a random coil structure but starts to aggregate in a concentration-dependent manner.

An isopeptide of amyloid β peptide 1-42 (isoAβ42) was considered as a non-aggregative precursor molecule for the highly aggregative Aβ42. It has been applied to biological studies after several pretreatments. Here we report that isoAβ42 is monomeric with a random coil structure at 40 μM without any pretreatment. But we also found that isoAβ42 retains a slight aggregative nature, which is significantly weaker than that of the native Aβ42.

Efficient HF Conditions for Suppressing Trp Modification Associated with Use of the N in-Cyclohexyloxycarbonyl (Hoc) Group

We have recently demonstrated the utility of a combined solid-phase and solution approach for protein synthesis [1]. The procedure consists of the preparation of protected segments by solid-phase with Boc chemistry and subsequent segment condensation in solution, followed by final deprotection using HF. Each fully protected segment with a free α-carboxyl group is detached from an N-[9-(hydroxymethyl)-2-fluorenyl] succinamic acid (HMFS) linker [2] by treatment with 20% morpholine/DMF. When employing a base-labile linker such as HMFS, the side-chain protecting groups must be completely stable under the basic conditions used to detach the segments from the resin. For this purpose, the cyclohexyloxycarbonyl (Hoc) group was developed as a base-resistant side-chain protecting group for the Trp residue suited to our synthetic strategy. However, side products of MW 108 higher than that of the desired product were observed, when the Hoc group was exposed to HF in the presence of p-cresol, and these side products were identified as two steric trans-isomers of 2-[2’-(p-cresol)]-2,3-dihydrotryptophan (Figure 1) [3]. The side reaction arising from modification of the Trp residue is inherent to the carbamate-type N in-protecting groups when performing HF treatment in the presence a scavenger. In the present study, we investigated the HF conditions applicable to the practical peptide synthesis for suppressing the modification of the Trp residue. Furthermore, application of this side reaction was examined to develop a facile procedure for introducing aromatic ring systems at the 2-position of the indole moiety.