Development of 4-fluorophenyl 3-nitro-2-pyridinesulfenate as a new Npys protecting reagent and its application to efficient disulfide formation

概要

Introduction
　The 3-nitro-2-pyridinesulfenyl (Npys) group was discovered as a widely-used functional group for the protection of amino (-NH2 ), hydroxy (-OH) and thiol (-SH) groups particularly in peptide chemistry. Npys-protected cysteine (Cys(Npys)), which prepare from reaction of conventional SH-protected cysteines with Npys chloride (Npys-Cl), can selectively react with another free SH to form a disulfide bond. Focused on this chemistry, we developed a one-pot Solid-Phase Disulfide Ligation (SPDSL) strategy with a key-point on the Npys-Cl resin, which includes two steps: (1) the t-Bu protected Cys-containing compound A is loaded onto the Npys-Cl resin via an active disulfide bond, and (2) compound A on the resin is readily transferred to another unprotected SH-containing compound B by a rapid and selective disulfide exchange reaction, resulting in the formation of a new disulfide compound (A-S-S- B) (Figure 1). This strategy was successfully applied to synthesize cyclic peptides and peptide-drug conjugates. However, Npys-Cl has some obvious defects, such as: instability issues with light or moisture and its tendency to dimerize with itself even at low temperatures. Especially, in SPDSL strategy, laborious preparation of the Npys-Cl resin is a dragging step, which needed performing immediately prior to use.

　To overcome these defects, in this thesis study, as new surrogates of Npys-Cl, 4- fluorophenyl 3-nitro-2-pyridinesulfenate (Npys-OPh(pF)) and its resin were developed. These compounds were applicable to i) Npys protections of amino, hydroxyl and thiol groups,
ii) synthesis of mono-cyclic disulfide peptide oxytocin and disulfide-linked glycoconjugate in SPDSL strategy, and iii) total synthesis of disulfide-linked protein analogue.

Chapter 1. Preparation and stability evaluation of Npys sulfenates as protecting reagents
　Firstly, the synthesis of Npys phenoxides was investigated by coupling various phenols with Npys-Cl. Among these derivatives, Npys-OPh(pF) (1) was the best sulfenate in terms of the efficiency of its preparation and consequently 1 was mainly used for further optimization of the sulfenate preparation process. As a result of optimization, 1 can be obtained with a yield of 73% in the presence of 1,2-dichloroethane (1,2-DCE) by using 1.2 equivalents of 4-fluorophenol and 4 equivalents of N , N - di i s opr opyl et hyl am i ne (DIPEA) (Scheme 1).

　Next, the Npys protection of functional groups of amino acids was examined using Npys- OPh(pF) (1). 1 successfully introduced an Npys group to the -amino group of HCl·H-Ala- OtBu and H-Leu-OtBu, the corresponding Npys-Ala-OtBu and Npys-Leu-OtBu were obtained with a yield of 91% and 87%, respectively. Next, the -amino group of Fmoc-Lys-OH·HCl was also protected and Fmoc- Lys(Npys)-OH was synthesized with a yield of 85%. Moreover, the less nucleophilic hydroxy groups in Boc-Ser-OMe and Z-Tyr-OMe were also protected, giving the corresponding compounds with yields of 30% and 44%, respectively. The carboxy group of Fmoc-Gly-OH failed to react with 1. The reaction with the unprotected thiol group of Z-Cys-OEt gave the protected compound, Z- Cys(Npys)-OEt in 14% yield (Table 1). Thus, 1 has the ability to protect amino, hydroxy and thiol groups.

　Finally, the stability of 1 was also examined, both during storage and in solution. NMR analysis showed that 25% of Npys-Cl was converted to the dimer after 3 weeks under 4 ºC. In contrast, no decomposition of 1 was observed after 2 months. Additionally, 1 was stable for more than 20 h but Npys-Cl was unstable in DMSO. Therefore, it is suggested that Npys- OPh(pF) (1) would be a practical agent of choice for Npys-based chemistry.

Chapter 2. Use of solid-supported Npys-OPh(pF) (7) in the construction of disulfide- linked hybrid molecules
　Npys-OPh(pF) (1), a newly developed prospective surrogate of Npys-Cl, could provide possibility to meet the dawn of discovering a new resin, which used as a stable surrogate of the conventional Npys- Cl resin in SPDSL strategy. In chapter 2, firstly, Fmoc-Cys(Npys)-OH was synthesized by coupling Fmoc-Cys(t-Bu)-OH with a variety of Npys-OR1s under acid conditions. The results are shown in Scheme 2, using 1, a satisfactory yield of Cys(Npys) was obtained with 96%. On the other hand, Npys-OPh(pF) (1) has good storage and solution stability compared to Npys-Cl. Thus, 1 was chose as a representative compound.

　Next, to apply the chemistry of Npys-OPh(pF) (1) to the SPDSL strategy, the Npys- OPh(pF) resin (7) was successfully developed and obviated a laborious activation step necessary when using the Npys-Cl resin. This new resin (7) was applied to synthesize asymmetric disulfide bond consisted of two thiol fragments. Resin (7) was applicable to the synthesis of the intermediate disulfide peptide of oxytocin from two peptide fragments and disulfide-linked glycoconjugate (Scheme 3).

　Finally, to demonstrate the usefulness of Npys-OPh(pF) resin (7), its stability was investigated under storage conditions. The conventional Npys-Cl resin must be used instantly after synthesis. By contrast, resin (7) is stable after 1 day although it gradually decomposed in one week. And surprisingly, resin (7) is stable under -20 ºC more than 3 months. These results indicated that the stable Npys-OPh(pF) resin (7) can contribute to create a disulfide- conjugate not only between peptides but also between peptide and sugar, and presumably, drugs, polynucleotides and proteins.

Chapter 3. Modular chemical synthesis of the human immunodeficiency virus type 1 protease analog via serial disulfide-bond formation
　Npys-protected cysteine (Cys(Npys)), prepared from the reaction between conventional SH-protected cysteines and Npys-OPh(pF) (1), can selectively react with another free SH to form the disulfide bonds. On the other hand, the intrinsic structures of proteins that have been developed during evolution have not always been optimized for their functions to an ideal and perfect state. Therefore, it may be possible to diversify the function of proteins by converting the unique amide bonds of proteins to other bonds. In chapter 3, converting them to more flexible disulfide bonds were considered and firstly a human immunodeficiency virus type 1 protease (HIV-1 PR) analogue was synthesized with two disulfide bonds in the main chain was synthesized by the disulfide-ligation method. HIV-1 PR analogue composed of 115aa residues was divided into three fragments 3-5 (Scheme 4) and the first disulfide ligation was performed between fragment 3 with Cys(Npys) at the C-terminus and fragment 4 with free SH at the N-terminus. Then, resultant fragment 6 was converted to the Cys(Npys) form 7 using Npys-OPh(pF) (1), and directly ligated with fragment 5 to form the second disulfide. The final deprotection of Mts gave the double disulfide-linked HIV-1 protease monomer analogue 9. The results suggest that Npys-based disulfide ligation can be expand to the synthesis of various protein analogues.

Conclusion
　This thesis study demonstrated that the development of a new Npy sulfenate reagent, Npys-OPh(pF) (1), is a stable surrogate of the Npys-Cl and its resin in liquid- and solid-phase synthesis. This new reagent could protect amino, hydroxy and thiol groups in various amino acids. And in SPDSL strategy, Npys-OPh(pF) resin (7) was applicable to the disulfide formation in oxytocin and glycoconjugate. The new Npys-OPh(pF)-based disulfide ligation was used to synthesize a disulfide-linked HIV-1 protease analogue. These results indicate that Npys-OPh(pF) (1) can contribute to the construction of various disulfide-linked molecules in peptide/protein chemistry, chemical biology and drug development.