Peptides or small-size proteins are important substances for medicines, diagnosis, and molecular biology research. In organic synthesis, the peptide bonds formation is performed in an organic solution (liquid-phase peptide synthesis, LPPS), or on a resin (solid-phase peptides synthesis, SPPS). LPPS can prepare a high volume of peptides, but it is generally required long processes and high cost for peptides preparation and is not appropriate for long-chain peptides. SPPS can prepare long-chain peptides until 40 residues in a short time. However, it is difficult to obtain the pure peptides because of no purification of its intermediates. For a solution of these problems, Kent et al. reported native chemical ligation (NCL) method for the preparation of long-chain peptides. Because peptides with a long chain or difficult sequence formed β-sheet structure within a molecule, these peptides have high aggregability and low solubility, and their preparation and purification are generally difficult. Mutter et al. reported ‘pseudoproline’ method for difficult sequence-containing peptide preparation. We previously reported a series of prodrugs based on O-N intramolecular acyl migration. We reported ‘O-isoacylpeptide’ method for the preparation of difficult sequence-containing peptides using the prodrug strategy based on O-N intramolecular acyl migration.
- difficult sequence-containing peptide
- long-chain peptide
- O-N intramolecular acyl migration
- peptide synthesis
Peptides or small-size proteins are important substances for medicines, diagnosis, and molecular biology research, such as enzyme inhibitors, antagonists/agonists against receptors, antigenic peptides for antibody preparation, and peptide probes that detect a protein-peptide interaction. There are two general methodologies for peptide preparation—organic synthesis and genetic engineered synthesis. In organic synthesis, the peptide bonds formation is performed in an organic solution (liquid-phase peptide synthesis, LPPS), or on a resin (solid-phase peptides synthesis, SPPS). LPPS can prepare a high volume of peptides, but it is generally required long processes and high cost containing labor cost and is not appropriate for long-chain peptides. SPPS can prepare long-chain peptides until 40 residues in a short time. However, it is difficult to obtain the pure peptides because of no purification of their intermediates. For a solution of these problem, Kent et al. reported native chemical ligation (NCL) method for the preparation of long-chain peptides as shown in Figure 1A [1, 2, 3]. In NCL reaction, a peptide possessing thioester at the C-terminus and a peptide possessing Cys residue at the N-terminus are prepared by SPPS. Next, both peptides are condensed by nucleophilic attack of thiol group at the N-terminus Cys residue in an aqueous solution, and then the condensed peptide with a thioester bond is spontaneously transformed into the peptide in which both peptides are connected with an amide bond. Although NCL allowed to preparing the long-chain peptides, this method is only available for the preparation of peptides with one or more Cys residues. Yan and Dawson  reported a modified NCL method for preparation of the peptides with one or more Ala residues (Figure 1B, R1 = R2 = ▬H). In this reaction, the Cys-containing peptides that were obtained by NCL reaction are reduced into the Ala-containing peptides using Raney-Nickel catalyst. Haase et al.  reported other modified NCL method for preparation of the Val-containing peptides from peptides with one or more penicillamine (β-mercaptovaline) as shown in Figure 1B (R1 = R2 = ▬CH3) in a manner similar to Haase et al.
There are synthetic difficult peptides containing specific amino acid sequences. Because the difficult sequence-containing peptides and long-chain peptides formed β-sheet structure within a molecule, these peptides have high aggregability and low solubility in aqueous and organic solvents, and their preparation and purification are generally difficult. Although we can use various resin for preparation of difficult sequence-containing peptides, such as Tentagel™ (RAPP Polymere, Germany) with a PEG moiety on the polystyrene (PS) bead and HDODA resin with a flexible crosslinker , some game-changing technologies for the preparation of difficult sequence-containing peptides has been reported. Mutter et al. reported ‘pseudoproline’ method for the preparation of difficult sequence-containing peptides and long-chain peptides [7, 8] as shown in Figure 1C. Mutter et al. synthesized peptides on a resin using an oxazolidine-containing amino acid (pseudoproline) in which α-amino and hydroxyl groups of Ser or Thr are cyclized by acetonidation. The deprotection and cleavage of peptides on the resin by a strong acid can convert form peptides with cyclic amino acid residue into peptides with Ser or Thr residue. Because the cyclic amino acids have a structure similar to Pro and peptides with a cyclic amino acid residue have greatly different structure compared with original peptides, peptides containing a pseudoproline are prevented to form β-sheet structure within a molecule. Because the oxazolidine-containing amino acids are labile in acid media, the dipeptide units, which consist of Fmoc-protected amino acid and oxazolidine-containing amino acid are commercially available.
We previously reported a series of prodrugs based on
O-Nintramolecular acyl migration and prodrugs
Previously, we reported a series of water-soluble prodrugs, such as human immunodeficiency virus type-1 (HIV-1) protease inhibitors and anti-cancer drugs [10, 11, 12, 15]. HIV-1 is a retrovirus that causes the acquired immunodeficiency syndrome (AIDS). Since HIV-1 encodes HIV-1 protease that is responsible for the processing of viral precursor proteins such as gag and gag-pol polyproteins to form mature structural proteins and some enzymes required in the production of infective viral particles, HIV-1 protease is an attractive target for the design of anti-AIDS drugs. HIV-1 protease is an aspartic protease that consists of a C2-symmetric homodimer, and its active site has some hydrophobic pockets. Thus most of the HIV-1 protease inhibitors that are optimized for the active site have high hydrophobicity and are sparingly water soluble. Many HIV-1 protease inhibitor formulations contain some solubilizers such as polyethylene glycol derivatives that often lead to unwanted side effects in clinical use. A water-soluble prodrug of amprenavir, fosamprenavir [16, 17], was approved by the US Federal Drug Administration (FDA) in 2003, and amprenavir was discontinued by the manufacture in 2004. Our previously reported HIV-1 protease inhibitors [18, 19, 20, 21] also showed poorly water-solubility similar to amprenavir. Hence, we designed a series of water-soluble prodrug of HIV-1 protease inhibitor using a novel prodrug strategy. An acyl migration on the β-hydroxy-α-amino acid residue such as Ser and Thr in strong acids was well-known in peptide chemistry . An
Next, we designed and synthesized water-soluble paclitaxel prodrug [12, 13]. Paclitaxel is an anti-cancer agent that was extracted from the Pacific yew tree
Since two natural amino acids, Ser and Thr, have a β-hydroxyl-α-amino acid structure, we designed the precursors of biomolecules, so-called ‘
Furthermore, we designed and synthesized photo cleavable-protected
4. Segment condensation of peptides using
Although NCL by Kent et al. allowed to preparing the long chain peptides, in general, conventional segment condensation other than NCL often involves the racemization of amino acid as shown in Figure 5A. Especially, it is a serious problem in SPPS because of no purification of intermediates. It is well-known that the urethane structure, such as Boc and Fmoc protecting group, on the α-amino group of amino acid can prominently reduce the racemization of amino acid in peptide bond coupling reaction. Hence, segment condensation between peptides other than NCL must not be in peptide chemistry. We noticed the protected
Recently, some important synthesis methods such as NCL and pseudoproline method for preparation of long chain and difficult sequence-containing peptides had been reported. Although these approaches allow to preparing some long chain peptides without a genetic engineered approach. However, these methodologies are not a panacea for a long chain and difficult sequence-containing peptides. We supply alternative solution for the long chain and difficult sequence-containing peptide preparation. Namely, we have developed the
This study was supported in part by the Grants-in-Aid for Scientific Research from MEXT (Ministry of Education, Culture, Sports, Science and Technology), Japan (KAKENHI No. 23590137, No. 26460163 and No.18K06562), and a donation from Mrs. Kazuko Fujita with the cherished desire of the late Tetsuro Fujita, Professor Emeritus of Kyoto University.
Conflict of interest
We confirmed independence from the funding source.