Keiko Hojo

Development of a method for environmentally friendly chemical peptide synthesis in water using water-dispersible amino acid nanoparticles

Due to the vast importance of peptides in biological processes, there is an escalating need for synthetic peptides to be used in a wide variety of applications. However, the consumption of organic solvent is extremely large in chemical peptide syntheses because of the multiple condensation steps in organic solvents. That is, the current synthesis method is not environmentally friendly. From the viewpoint of green sustainable chemistry, we focused on developing an organic solvent-free synthetic method using water, an environmentally friendly solvent. Here we described in-water synthesis technology using water-dispersible protected amino acids.

Amino acids and peptides. Part 39: A bivalent poly(ethylene glycol) hybrid containing an active site (RGD) and its synergistic site (PHSRN) of fibronectin

Fibronectin contains the active sequence Arg-Gly-Asp (RGD), along with its synergic site Pro-His-Ser-Arg-Asn (PHSRN). However, the PHSRN peptide does not show synergic activity when it is mixed with the RGD peptide, indicating that a spatial array between RGD and PHSRN in fibronectin may be necessary for synergic activity. Here, we have used an amino acid type poly(ethylene glycol) derivative (aaPEG) to design a bivalent PEG hybrid of fibronectin active peptides. We prepared the aaPEG hybrid peptides PHSRN-aaPEG, aaPEG-RGD, and PHSRN-aaPEG-RGD, and tested their biological activity. Whereas aaPEG-RGD promoted cell spreading activity, PHSRN-aaPEG had no activity. The PHSRN-aaPEG-RGD hybrid strongly promoted cell spreading compared with aaPEG-RGD. These results suggest that the PHSRN sequence in the PHSRN-aaPEG-RGD molecule synergistically enhances the cell spreading activity of the RGD sequence, and that the bivalent aaPEG hybrid method may be useful for conjugating functionally active peptides.

Development of a Method for the Solid-Phase Peptide Synthesis in Water

Various organic solvents are routinely used in peptide synthesis, safe disposal of which are now an important environmental problem. To circumvent this problem, during the last few years we focused on developing an organic solvent-free SPPS method using aqueous solvents. For the SPPS in water, we designed protected amino acids that could be used in the aqueous media. Here we described development of several types of water-soluble protected amino acids and their application to the SPPS in water, and a novel technology that uses water-dispersible protected amino acids for in-water peptide synthesis.

Facile synthesis of a chitosan hybrid of a laminin-related peptide and its antimetastatic effect in mice

Laminin, a cell adhesion protein, consists of three peptide chains (α, β and γ‐1). The β chain contains a Tyr‐Ile‐Gly‐Ser‐Arg (YIGSR) sequence that has been found to inhibit experimental metastasis in mice. We have prepared a hybrid of a water‐soluble chitosan and a laminin‐related peptide, and have examined its inhibitory effect on experimental metastasis in mice.

Development of a single-chain peptide agonist of the relaxin-3 receptor using hydrocarbon stapling

Structure-activity studies of the insulin superfamily member, relaxin-3, have shown that its G protein-coupled receptor (RXFP3) binding site is contained within its central B-chain α-helix and this helical structure is essential for receptor activation. We sought to develop a single B-chain mimetic that retained agonist activity. This was achieved by use of solid phase peptide synthesis together with on-resin ruthenium-catalyzed ring closure metathesis of a pair of judiciously placed i,i+4 α-methyl, α-alkenyl amino acids. The resulting hydrocarbon stapled peptide was shown by solution NMR spectroscopy to mimic the native helical conformation of relaxin-3 and to possess potent RXFP3 receptor binding and activation. Alternative stapling procedures were unsuccessful, highlighting the critical need to carefully consider both the peptide sequence and stapling methodology for optimal outcomes. Our result is the first successful minimization of an insulin-like peptide to a single-chain α-helical peptide agonist which will facilitate study of the function of relaxin-3.

Peptide synthesis ‘in water’ by a solution-phase method using water-dispersible nanoparticle Boc-amino acid

Regulatory pressure has compelled the chemical manufacturing industry to reduce the use of organic solvents in synthetic chemistry, and there is currently a strong focus on replacing these solvents with water. Here, we describe an efficient in‐water solution‐phase peptide synthesis method using Boc‐amino acids. It is based on a coupling reaction utilizing suspended water‐dispersible nanoparticle reactants. Using this method, peptides were obtained in good yield and with high purity. Copyright

A conjugate from a laminin-related peptide, Tyr-Ile-Gly-Ser-Arg, and chitosan: efficient and regioselective conjugation and significant inhibitory activity against experimental cancer metastasis,

A laminin-related antimetastatic peptide was conjugated with chitosan, and antimetastatic activity of the peptide–chitosan conjugate was assayed. Chitosan was converted to its organosoluble derivative, 6-O-trityl-chitosan, in 3 steps, and then coupled with the peptide portion, Ac-Tyr-Ile-Gly-Ser-Arg-βAla-OH (βAla; β-alanine), which contains a spacer amino acid at the carboxy-terminus. The product was treated with CHCl2CO2H to afford the desired conjugate, Ac-Tyr-Ile-Gly-Ser-Arg-βAla–chitosan. The peptide was introduced to every 6.3 glucosamine residues. The conjugate proved to have higher inhibitory activity against experimental lung metastasis of B16BL6 melanoma cells in mice than did the parent peptide.

Preparation of a Chitosan Hybrid of an Antimetastatic Laminin‐related Peptide

A hybrid of chitosan and an antimetastatic laminin-related peptide was prepared. Acetyl-Tyr-Ile-Gly-Ser-Agr-βAla-OH (Ac-YIGSRβA-OH) was prepared by a solid-phase method and reacted with a water-soluble chitosan. Chitosan amino groups did not react with the peptide using diphenylphosphoryl azide, diisopropylcarbodiimide/1-hydroxybenzotriazole, water-soluble carbodiimide/1-hydroxy-benzotriazole, phosphazo, or 2-(1 H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) methods. A small spacer was therefore put between the peptide and the chitosan. tert-Butyloxycarbonyl-Gly (spacer) was reacted with chitosan by the TBTU method. After removal of the protecting group, the Gly-chitosan was coupled with Ac-YIGSRβA-OH by the water-soluble carbidiimide method to give Ac-YIGSRβAG-chitosan. The inhibitory effect of the peptide-chitosan hybrid on experimental metastasis in mice was not reduced, but actually potentiated, suggesting that chitosan may be used as a drug carrier for peptides.

Aqueous microwave-assisted solid-phase peptide synthesis using fmoc strategy. II. Racemization studies and water based synthesis of cysteine-containing peptides.

We have developed a microwave (MW)-assisted peptide synthesis using Fmoc-amino acid nanoparticles in water previously. It is an organic solvent-free, environmentally friendly method for peptide synthesis. In this study, we have investigated the racemization of cysteine during an aqueous based coupling reaction with MW irradiation. Under our MW-assisted protocol using WSCI and DMTMM, the coupling reaction can be performed with low levels of racemization of cysteine. We also demonstrated the synthesis of the nonapeptide oxytocin analogue, Cys(Acm)-Tyr-Ile-Gln-Asn- Cys(Acm)-Pro-Leu-Gly-NH2 using our water based MW-assisted protocol with Fmoc-amino acid nanoparticles.

Preparation of a Tat-Related Transporter Peptide for Carrying the Adenovirus Vector into Cells

We synthesized a Tat-related peptide acetyl-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Pro-Gln-Gly-Cys amide, Ac-Tat(48-60)-Gly-Cys-NH(2), having high intracellular permeability, and conjugated this peptide to adenovirus vector to enhance gene transfer efficiency of adenovirus vector into cells. The peptide was prepared by the solid-phase peptide synthesis method and a bifunctional crosslinker 6-maleimidohexanoic acid N-hydroxysuccinimide ester was used to conjugate the peptide to adenovirus vector containing luciferase gene. The novel conjugate of adenovirus vector and Ac-Tat(48-60)-Gly-Cys-NH(2) peptide exhibited excellent gene transfer efficacy in B16BL6 cells.