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| » Home » Membership » Awards » List of past recipients » CSJ Award » Aimoto
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Development of a Method for Protein Synthesis Using Peptide Thioesters as Building Blocks
By the 1980s a sufficient body of chemical knowledge was available to permit the synthesis of complicated peptides. However, many problems remained in the synthetic methodology related to the chemical synthesis of protein. In order to overcome these problems, Dr. Aimoto developed a method that combined the advantages of a solution method and a solid phase method for peptide synthesis by introducing a peptide thioester as a building block for protein synthesis. The developed method, called the thioester method, enabled the facile chemical synthesis of protein, in terms of time and skills required for completion. His research greatly contributed to creating the present trend in synthetic protein chemistry. |
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Prof. Saburo Aimoto
Institute for Protein Research,
Osaka University
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1. Development of a method for protein synthesis, in which peptide thioesters are used as building blocks
Dr. Aimoto found that a partially protected peptide thioester, prepared by a solid phase method, was a useful building block for protein synthesis. The thioester moiety is stable during HPLC purification, can be stored for long periods in a refrigerator, is generally well soluble in a polar organic solvent, and is selectively activated by silver ions to form a corresponding active ester in the presence of an active ester component. Thus, a peptide thioester with up to 50 amino acid residues can be used as a building block for the synthesis of a protein. Furthermore peptides and peptide thioesters containing S-protected cysteine residues can be used as building blocks under the standard coupling conditions without decomposition of cysteine residues. Thus the developed method is generally applicable to the synthesis of proteins including glycosylated or phosphorylated proteins.
2. Development of methods for peptide thioester synthesis
The bottleneck of the method that uses peptide thioesters as building blocks was the low efficiency of peptide thioester synthesis by a sold phase method. To overcome this problem Aimoto analyzed the factor(s) that contribute to the low yields of a peptide thioester when it was synthesized by a Boc solid phase method. Among the findings was the fact that the sulfur atom in the thioester accelerates the acid catalyzed cleavage of a peptide from a resin by a neighboring effect during TFA treatment used to remove the of Boc groups. Based on this finding he developed a resin that gave a peptide thioester in high yield simply by keeping the sulfur atom away from the resin by introducing a single amino acid residue between the resin and thioester moiety. Furthermore, he developed methods for peptide thioester synthesis by an Fmoc solid phase method, which is widely employed for peptide synthesis at present, especially for the synthesis of phosphorylated or glycosylated peptides. In initial experiments, he developed a reagent that contained a weak nucleophilic base for the direct synthesis of peptide thioesters. The reagent was sufficiently strong to cleave Fmoc groups while maintaining the thioester bond intact. However, an amino acid residue adjacent to a thioester moiety was partly epimerized during the peptide chain elongation cycles. To overcome this problem he developed an indirect method that resulted in negligible epimerization. In this method a peptide thioester is prepared via an efficient N to S acyl shift reaction mediated by a thiol-containing auxiliary group attached to a peptide bond after completion of the peptide chain elongation.
3. Development of a method to synthesize a protein by using an expressed protein segment
With the goal of developing a method that makes it possible to utilize an expressed protein segment, such as a protein segment that is uniformly labeled with 15N, as a building block for the thioester method, Aimoto developed a procedure to selectively introduce protecting groups to the side chain amino groups of the expressed protein segment based on Dixonfs idea for deleting the N-terminal one amino acid residue. (Dixon, H. B. F., Biochem. J., 90, 2C-3C (1964)). He demonstrated the utility of the method by synthesizing segmentally the 15N labeled phosphorylated p21Max protein (1-101) by using a chemically synthesized phosphorylated peptide thioester and an expressed15N labeled protein segment.
4. Design of a peptide that is spontaneously transformed to a peptide thioester in a neutral aqueous buffer
A reaction in which a peptide containing a cysteine residue is more or less transformed into an S-peptide at the cysteine residue via an N to S acyl shift reaction under acidic conditions is a well-known side reaction. Aimoto analyzed the reaction by 13C NMR and found that more than 80% of an original peptide was transformed into an S-peptide in a TFA-containing solvent at equilibrium. However, only a small amount of the S -peptide was detected by reversed phase HPLC. Based on these observations, he and his colleagues designed an attachment to the C-terminal of peptide that would trap the amino group of the cysteine residue, generated via an N to S acyl shift reaction, to stabilize the spontaneously generated peptide thioester. A peptide with a designed attachment, a Cys-Pro-ester unit, at the C-teminal was efficiently condensed with a cysteinyl peptide via a native chemical ligation reaction in a slightly basic buffer. The Cys-Pro-ester attached peptide is easily synthesized by standard Fmoc SPPS. Therefore this unique feature of peptide condensation indicates that this strategy will undergo further development and refinement in the future.
In summary Dr. Aimoto found that use of peptide thioester made it possible to overcome the difficulties involved with protein synthesis solely by a solution method or by a solid phase method. He also developed efficient synthetic methods for preparing peptide thioesters and greatly contributed to the establishment and development of the use of synthetic methods in protein synthesis, including phosphorylated or glycosylated proteins by the use of peptide thioesters. As a result of these achievements, he is deserving of the Chemical Society of Japan Award.
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