ANNUAL REPORT No.55 2004

A. DEPARTMENT OF MOLECULAR GENETICS
A-d. Division of Nucleic Acid Chemistry - Saburo Aimoto Group

RESEARCH ACTIVITIES

(1) Development of a method for chemical synthesis of protein by using two different ligation methods

Saburo Aimoto (Institute for Protein Research, Osaka University)

--A thioester method and the native chemical ligation method are useful for protein synthesis. Though both methods use peptide thioesters as building blocks, no route was known to use the thioester method after the peptide bond formation reaction by the native chemical ligation method. In the thioester method, the SH groups of the cysteine residues have to be protected by a group that is stable in the presence of silver ions as an activator of thioester groups. On the other hand, the native chemical ligation method produces an SH group at the condensation site for chemoselective ligation is carried out at -X-Cys- site using the cysteine residue at the N-terminal of the C-terminal building block. In order to overcome this problem, we searched protecting groups for the thiol groups that are easily introduced to the free SH group in polypeptide under mild conditions and stable in the presence of silver ions. We examined several thiol protecting groups. Among them, thiosulfonate group was the most promising protecting group. This group, however, was revealed to partially decompose under alkaline conditions. Thus new protecting groups are still under investigation.

(2) Preparation of peptide thioesters by the Fmoc-solid phase method via an on-resin N-S acyl shift

Saburo Aimoto (Institute for Protein Research, Osaka University)

--Peptide thioesters are common intermediates in contemporary methods for protein synthesis. The peptide thioesters can be directly prepared either by Boc solid-phase peptide synthesis (SPPS) or by Fmoc SPPS using Fmoc(2-F)-amino acid derivatives. Peptide thioesters can be prepared by Fmoc SPPS indirectly via a safety catch resin, too. However each preparation method based on the Fmoc solid phase method has intrinsic difficulties such as low yields of products and recemization of the C-terminal amino acid residue. Therefore an innovative method was urgently requested. A new method under development is based on an on-resin N-S acyl rearrangement. Previously, we introduced the 2-mercapto-4, 5-dimethoxybenzyl (Dmmb) group as an auxiliary for extended chemical ligation. This auxiliary group can be removed by acid treatment following the ligation reaction. Unexpectedly, it was observed that N-S acyl rearrangement occurred, in part, during the acid treatment. Base on this finding, we searched for conditions to use this mechanism for preparation of peptide thioesters. For polypeptide synthesis, the prevention of a reverse acyl rearrangement is of prime importance in this context. A protected peptide was assembled on a resin using the Dmmb-group as a linker followed by Fmoc-based SPPS. After peptide assembly, the resin was treated with a reagent containing trifluoroacetic acid to result in the removal of the protecting groups. At the same time, the N-S acyl rearrangement occurred, resulting in resin-bound peptide thioester. Finally, free peptide thioester was obtained following treatment with 2-mercaptoethanesulfonic acid in the presence of a base and subsequent resin wash. This method permits us to prepare peptide thioesters by a Fmoc solid-phase method without racemization.

(3) Synthesis of post-translationally modified histone H3

Saburo Aimoto (Institute for Protein Research, Osaka University)

--Histones can undergo posttranslational modifications such as methylation, acetylation or/and phosphorylation. These modifications can play a role in gene regulation in an epigenetic manner. Focused on the N-terminal region of histone H3 we searched a strategy that would provide a synthetic procedure for post-translationally modified histones. First, synthesis of the N-terminal region of histone H3 peptides that contained Lys(Me₃) was examined by Boc and Fmoc SPPS. A desired product was obtained by the both methods though the Boc method gave better results than the Fmoc SPPS. Lys(Me₃)-containing peptide thioesters that were building blocks for histone H3 synthesis were prepared by Boc SPPS. Furthermore, two chemically synthesized building blocks covering [Lys(Me₃)⁴]-histone H3(1-12) and [Lys(Me₃)³⁶]-histone H3(13-43) were condensed by the thioester method to give [Lys(Me₃)^{4,
36}]-histone H3(1-43). Along with this totally chemical synthetic method, we are developing a method that uses a biologically expressed histone H3 segment for the total synthesis of modified histone H3. In the near future, a variety of modified histone H3 with a full sequence will be synthesized by condensing chemically synthesized modified-amino-acid-containing peptide thioesters and biologically expressed histone H3 segments.

(4) Synthetic studies of G protein-coupled receptor, opioid receptor like-1 (ORL-1)

Saburo Aimoto (Institute for Protein Research, Osaka University)

--About 30% of the human genome encodes membrane proteins. Among them G protein-coupled receptor family is the largest family of membrane receptor protein and is the target of most pharmaceuticals. Much of the information concerning the structure and function of these membrane proteins, however, remains to be uncovered because of the difficulties associated with biochemical sample preparation. As an alternative approach to obtaining membrane proteins, chemical synthesis represents a viable candidate. ORL-1 is a receptor of an opioid peptide, nociceptin. The strategy that is employed for the synthesis of the C-terminal region of ORL-1 is the combination of the native chemical ligation method and the thioester method. The first coupling between ORL-1(288-328) and ORL-1(329-370) was carried out in SDS solution by the native chemical ligation method. The coupling reaction conditions were thoroughly inspected in terms of pH of the solution, chemical composition of the reaction media. According to the defined conditions, ORL-1(288-370) that contained one transmembrane region and the C-terminal cytosolic tail region was obtained in the yield of 47%.⁴⁾ We are continuously searching the route to accomplish the total synthesis of ORL-1.

(5) Design of the inhibitors to human T-cell leukemia virus type-1 protease

Saburo Aimoto (Institute for Protein Research, Osaka University)

--Human T-cell leukemia virus type 1 (HTLV-1), a retrovirus associated with a number of human diseases, was the first human retrovirus isolated from patients with adult T-cell leukemia/ lymphoma by Gallo et al. An HTLV-1 gene codes an asparatic protease (PR), which processes its own polyproteins, which are transcripted owing to three reading frames. As the result of a series of cis processing, a set of proteins is produced, which are necessary for viral replication. Thus, HTLV-1 PR plays a key role in the duplication of HTLV-1 in a manner analogous to the human immunodeficiency virus type-1 protease in acquired immunodeficiency syndrome. In the design of potent protease inhibitors for this virus, the knowledge of the characteristics of HTLV-1 PR itself and its substrate specificities is critical. Then, we synthesized HTLV-1 PR and examined its substrate specificities. Based on the obtained data we designed HTLV-1 protease inhibitors of an olefin-containing cyclic peptide.¹⁾

PUBLICATIONS

Papers
1. Bang, J.K., Hasegawa, K., Kawakami, T., Aimoto, S. and Akaji, K. (2004). Synthesis of an olefin-containing cyclic peptide using the solid-phase Honer-Emmons reaction. Tetrahedron Lett., 45, 99-102.
2. Sekine, S., Kataoka, K., Tanaka, M., Nagata, H., Kawakami, T., Akaji, K., Aimoto, S. and Shizukuishi, S. (2004). Active domains of salivary statherin on apatitic surfaces for binding to Fusobacterium nucleatum cells. Microbiology. 150, 2373-9.
3. Yamada, H., Sasaki, T., Niwa, S., Oishi, T., Murata, M., Kawakami, T. and Aimoto, S. (2004). Intact Glycation End Products Containing Carboxylmethyl-Lysine and Glyoxal Lysine Dimer Obtained from Synthetic Collagen Model Peptide, Bioorg. Med. Chem. Lett., 14, 5677-80.
4. Sato, T., Saito, Y. and Aimoto, S. (2004). Synthesis of the C-terminal Region of Opioid Receptor Like 1 in an SDS Micelle by the Native Chemical Ligation: Effect of Thiol Additive and SDS Concentration on Ligation Efficiency. J. Peptide Sci., Epub. Dec. 20.
5. Onoda, A., Yamamoto, H., Yamada, Y., Lee, K., Adachi, S., Okamura, T. A., Yoshizawa-Kumagaye, K., Nakajima, K., Kawakami, T., Aimoto, S. and Ueyama, N. (2005). Switching of turn conformation in an aspartate anion peptide fragment by NH . . . O(-) hydrogen bonds. Biopolymers, Epub., Jan 4.

ORAL PRESENTATIONS

1. 川上徹、相本三郎「拡張型ペプチドライゲーション―光により除去できる補助基―」日本化学会第84春期年会、兵庫県、2004年3月
2. 伊藤麻里、竹嶋明子、長谷川功紀、嶋田直子、川上徹、相本三郎「酸化的修飾タンパク質の検出を目的とした蛍光標識試薬の開発」日本化学会第84春期年会、兵庫県、2004年3月
3. 斎藤泰宏、佐藤毅、松下修門、西村典子、川上徹、相本三郎「膜蛋白質の合成法の開発」日本化学会第84春期年会、兵庫県、2004年3月
4. 方正奎、仲裕美、川上徹、相本三郎、赤路健一「オレフィン含有ペプチドの固相合成法の開発とプロテアーゼ阻害剤合成への応用」日本化学会第84春期年会、兵庫県、2004年3月
5. Sato, T., Saito, Y., Nishimura, N., Kawakami, T. and Aimoto, S. Synthetic Studies of Membrane Protein Based on Ligation Chemistry. The 10th Akabori Conference, Awaji, Japan, April, 2004.
6. Sato, T., Saito, Y., Matsushita, N., Nishimura, N., Kawakami, T. and Aimoto, S. Synthetic Studies of Membrane Proteins: Synthesis of the C-terminal Region of opioid receptor Like 1. The 8th International Chinese Peptide Symposium, Kunming, China, July, 2004.
7. Sato, T., Saito, Y., Nishimura, N., Kawakami, T. and Aimoto, S. Synthetic studies of membrane protein based on ligation chemistry. The 3rd International Peptide Symposium, Plague, Czech Republic, September, 2004.
8. Sato, T., Saito, Y., Nishimura, N. and Aimoto, S. Chemical synthesis of the C-terminal region of the opioid receptor like 1. The 3rd International Peptide Symposium, Plague, Czech Republic, September, 2004.
9. Kawakami, T. and Aimoto, S. Extended chemical ligation for polypeptide synthesis by using a photoremovable auxiliary. The 3rd International Peptide Symposium, Plague, Czech Republic, September, 2004.
10. Sumida, M., Kawakami, T., Vorherr, T. and Aimoto, S. Preparation of Peptide Thioesters by the Fmoc-Solid Phase Method via an On-resin N-S Acyl Shift. The 1st Asian Pacific International Peptide Symposium, Fukuoka, Japan, November, 2004.
11. Saito, Y., Sato, T., Matsushita, N., Nishimura, N., Kawakami, T. and Aimoto, S. Development of strategy for membrane protein synthesis. The 1st Asian Pacific International Peptide Symposium, Fukuoka, Japan, November, 2004.

POSTER PRESENTATIONS

EDUCATION

1. Dr. S. Aimoto was invited to give a lecture at Kyoto University, January, 2005.

SOCIAL CONTRIBUTIONS AND OTHERS

1. Dr. S. Aimoto organized a joint symposium entitled “Protein Researches in Bioscience and Bioengineering", between Institute for Protein Research, Osaka University and Seoul National University, Osaka, June, 2004.
2. 相本三郎,日本ペプチド学会評議員.