**3.** *O***-isoacylpeptide method**

Since two natural amino acids, Ser and Thr, have a β-hydroxyl-α-amino acid structure, we designed the precursors of biomolecules, so-called '*O*-isoacylpeptides', using our prodrug strategy *via O*-*N* intramolecular acyl migration [14–16]. *O*-isoacylpeptides that have an *O*-acyl ester bond instead of amide bond in the Ser/ Thr residue of biomolecules are promptly converted to the corresponding biomolecules under physiological conditions. It is known that some biomolecules are aggregate in aqueous solutions because of their β-sheet structure formation. Among them, Aβs show strong water-insolubility and aggregability, making their handling in biochemical research potentially complicated. Hence, chemical synthesis on resins and purification of Aβs, especially Aβ1-42, is particularly challenging. To solve these problems, we designed *O*-isoacyl-Aβ1-42 that has an ester bond instead of the amide bond at Gly25-Ser26 in Aβ1-42. Synthesis of Aβ1-42 using *O*-isoacylpeptide method showed in **Figure 3A**. Peptide bonds formation on the PS-resin was performed by 9-fluorenylmethoxycarbonyl (Fmoc)-based SPPS using diisopropylcarbodiimide (DIPCDI) as a coupling reagent in the present of 1-hydroxybenzotriazole (HOBt). The hydroxy group in N-terminal Ser residue of the protected Aβ27-42 that was prepared on a resin was esterified by Fmoc-Gly-OH using DIPCDI in the presence of catalytic amount of *N,N*-dimethyl-4-aminopyridine (DMAP). Next, remaining peptide bonds formation using the Fmoc-based SPPS similar to the former

*Peptide Synthesis*

alkaline media *via O-N* intramolecular acyl migration (**Figure 2A**). Since our HIV-1 protease inhibitors contain a β-hydroxy-α-amino acid residue, (2S,3S)-3-amino-2-hydroxy-4-phenylbutyric acid (Apns), we designed and synthesized the *O*-acyl isomer of KNI-727 as a water-soluble prodrug. The prodrug of KNI-727 was stable as an HCl salt in unbuffered aqueous solutions and in strong acidic solution such as gastric juice, and could be rapidly converted to the parent compound, KNI-727, *via O-N* intramolecular acyl migration reaction under the physiological condition

*Prodrugs based on O-N intramolecular acyl migration (A) O-N intramolecular acyl migration. (B) Prodrug of HIV-1 protease inhibitor, KNI-727. (C) Paclitaxel prodrug. (D) HPLC profile of the O-N intramolecular acyl migration of KNI-727 prodrug in PBS (pH 5.5) at 37°C. (E) Time course of the migration reaction of* 

**34**

**Figure 2.**

*KNI-727 prodrug.*

#### **Figure 3.**

*O-isoacylpeptide method. (A) Aβ1-42 synthesis via O-N intramolecular acyl migration. (B) Aβ1-42 synthesis using an dipeptide unit.*

procedure formed the protected *O*-isoacyl-Aβ1-42, and then *O*-isoacyl-Aβ1-42 was obtained by deprotection and cleavage from a resin. *O*-isoacyl-Aβ1-42 was stable in acidic aqueous solution and unbuffered aqueous solution, and could rapidly release the native Aβ1-42 peptide. Because *O*-isoacylisopeptides showed good stability in acidic media, *O*-isoacylisopeptides can be easily isolated and purification in acidic solution [23]. In this manner, *O*-isoacylpeptide method has the advantage over the 'pseudoproline' method. *O*-isoacyl-Aβ1-42 could be easily synthesized on a resin and purified by preparative HPLC using a reverse phase C18 column in acidic eluent solvents, and could release native Aβ1-42 that consists of Aβ1-42 monomers. However, because ester bond formation on a resin often involves a racemization of protected

**37**

**Figure 4.**

*Photo-click peptide. (A) Synthesis of photo-click Aβ1-42. (B) Photo-click reaction.*

*Isoacylpeptide Method for Long-Chain and Difficult Sequence-Containing Peptide Preparation*

amino acid, *O*-isoacyl-dipeptide units such as Boc-Ser(Fmoc-Gly)-OH have been commercial available from some chemical suppliers. *O*-isoacylpeptide method using an *O*-isoacyl-dipeptide unit was shown in **Figure 3B**. Use of Fmoc-*O*-isoacyldipeptide allows to synthesize the isoacylpeptides by the conventional Fmoc-based

Furthermore, we designed and synthesized photo cleavable-protected *O*-isoacylpeptide, so called 'photo-click peptide'. Because *O*-isoacylpeptides are rapidly converted to the biomolecules by stimuli such as pH changes and photo irradiation, as with the click of a button, we term 'click peptide'. Click peptides that can

*DOI: http://dx.doi.org/10.5772/intechopen.84248*

SPPS without use of DMAP catalyst.

*Isoacylpeptide Method for Long-Chain and Difficult Sequence-Containing Peptide Preparation DOI: http://dx.doi.org/10.5772/intechopen.84248*

amino acid, *O*-isoacyl-dipeptide units such as Boc-Ser(Fmoc-Gly)-OH have been commercial available from some chemical suppliers. *O*-isoacylpeptide method using an *O*-isoacyl-dipeptide unit was shown in **Figure 3B**. Use of Fmoc-*O*-isoacyldipeptide allows to synthesize the isoacylpeptides by the conventional Fmoc-based SPPS without use of DMAP catalyst.

Furthermore, we designed and synthesized photo cleavable-protected *O*-isoacylpeptide, so called 'photo-click peptide'. Because *O*-isoacylpeptides are rapidly converted to the biomolecules by stimuli such as pH changes and photo irradiation, as with the click of a button, we term 'click peptide'. Click peptides that can

**Figure 4.** *Photo-click peptide. (A) Synthesis of photo-click Aβ1-42. (B) Photo-click reaction.*

*Peptide Synthesis*

**36**

**Figure 3.**

*using an dipeptide unit.*

procedure formed the protected *O*-isoacyl-Aβ1-42, and then *O*-isoacyl-Aβ1-42 was obtained by deprotection and cleavage from a resin. *O*-isoacyl-Aβ1-42 was stable in acidic aqueous solution and unbuffered aqueous solution, and could rapidly release the native Aβ1-42 peptide. Because *O*-isoacylisopeptides showed good stability in acidic media, *O*-isoacylisopeptides can be easily isolated and purification in acidic solution [23]. In this manner, *O*-isoacylpeptide method has the advantage over the 'pseudoproline' method. *O*-isoacyl-Aβ1-42 could be easily synthesized on a resin and purified by preparative HPLC using a reverse phase C18 column in acidic eluent solvents, and could release native Aβ1-42 that consists of Aβ1-42 monomers. However, because ester bond formation on a resin often involves a racemization of protected

*O-isoacylpeptide method. (A) Aβ1-42 synthesis via O-N intramolecular acyl migration. (B) Aβ1-42 synthesis* 

generate bioactive molecules in situ *via* a 'click' appear to be useful tools for chemical biology research. Synthesis of photo-click peptide was shown in **Figure 4A**. Photo-click Aβ1-42 could prepare on a resin in a similar manner showed in **Figure 3A**, using photo-cleavable protected amino acid instead of Boc-Ser-OH. Photo-cleavable protected amino acid, 6-nitroveratryloxycalbonyl (Nvoc)-Ser-OH, was coupled to protected Aβ27-42 on the resin after Fmoc-deprotection, and then Fmoc-Gly-OH was coupled on the hydroxyl group of Ser26 using DIPCDI in the presence of catalytic amount of DMAP. The remaining peptide bonds formation using the Fmoc-based SPPS formed the protected *O*-isoacyl-Aβ1-42. Deprotection and cleavage from resin released *O*-isoacyl-Aβ1-42. Photo-click Aβ1-42 could rapidly release the native Aβ1-42 peptide by photo-irradiation and subsequent *O*-*N* intramolecular acyl migration under physiological condition (**Figure 4B**).
