2,2'-Dipyridyl diselenide: A chemoselective tool for cysteine deprotection and disulfide bond formation

Abstract

There are many examples of bioactive, disulfide-rich peptides and proteins whose biological activity relies on proper disulfide connectivity. Regioselective disulfide bond formation is a strategy for the synthesis of these bioactive peptides, but many of these methods suffer from a lack of orthogonality between pairs of protected cysteine (Cys) residues, efficiency, and high yields. Here, we show the utilization of 2,2'-dipyridyl diselenide (PySeSePy) as a chemical tool for the removal of Cys-protecting groups and regioselective formation of disulfide bonds in peptides. We found that peptides containing either Cys(Mob) or Cys(Acm) groups treated with PySeSePy in trifluoroacetic acid (TFA) (with or without triisopropylsilane (TIS) were converted to Cys-S-SePy adducts at 37 °C and various incubation times. This novel Cys-S-SePy adduct is able to be chemoselectively reduced by five-fold excess ascorbate at pH 4.5, a condition that should spare already installed peptide disulfide bonds from reduction. This chemoselective reduction by ascorbate will undoubtedly find utility in numerous biotechnological applications. We applied our new chemistry to the iodine-free synthesis of the human intestinal hormone guanylin, which contains two disulfide bonds. While we originally envisioned using ascorbate to chemoselectively reduce one of the formed Cys-S-SePy adducts to catalyze disulfide bond formation, we found that when pairs of Cys(Acm) residues were treated with PySeSePy in TFA, the second disulfide bond formed spontaneously. Spontaneous formation of the second disulfide is most likely driven by the formation of the thermodynamically favored diselenide (PySeSePy) from the two Cys-S-SePy adducts. Thus, we have developed a one-pot method for concomitant deprotection and disulfide bond formation of Cys(Acm) pairs in the presence of an existing disulfide bond.

Figure 1:. Removal of the 5-Npys group from Cys or Sec by ascorbolysis.

We previously found that Asc can reduce the Se–S bond of Sec(5-Npys) very easily, while the same reduction of the S–S bond of Cys(5-Npys) is very slow.

Figure 2:. Proposed reduction of the S–Se bond of Cys(PySe) by Asc.

We found that reduction of Sec-Se–SPy with Asc was fast. In order to gain the same chemoselective advantage with Cys residues, we “switched” the position of sulfur and selenium through the use of PySeSePy and derivatives so as to create a Cys-S–SePy adduct.

Figure 3:. Advantage of chemoselective reduction of Cys(PySe) adducts by Asc compared to conventional reducing agents.

A) Conventional reducing agents such as thiols will reduce each S–S or S–Se bond as shown. B) Asc chemoseletively reduces the S–Se bond of Cys(PySe) adducts. Note that R represents a covalent modification of Cys that can be removed by typical reducing agents.

Figure 4:. Deprotection of the Cys(Mob)-containing peptide using PySeSePy and Asc determined by HPLC and MS analyses.

A) HPLC chromatogram of the Cys(Mob)-containing peptide incubated in neat TFA for 4 h at 37 °C (control). B) HPLC chromatogram of the Cys(Mob)-containing peptide incubated in TFA/water (98:2) with 5-fold excess PySeSePy for 4 h at 37 °C. Quantitative conversion to the PySe-adduct is apparent. C) HPLC chromatogram of the peptide containing the Cys(PySe) adduct treated with 5-fold excess ascorbate in 100 mM ammonium acetate pH 4.5, for 4 h at room temperature. Panels D, E, and F are the mass spectra that correspond with panels A, B, and C, respectively. The arrow points to the observed m/z whereas the number inside the parentheses denotes the theoretical m/z value.

Figure 5:. Deprotection of the Cys(Acm)-containing peptide using PySeSePy and Asc determined by HPLC analysis.

A) HPLC chromatogram of the Cys(Acm)-containing peptide incubated in neat TFA with 5-fold excess PySeSePy for 12 h at 37 °C. B) HPLC chromatogram of the Cys(Acm)-containing peptide incubated in TFA/TIS (98:2) with 5-fold excess PySeSePy for 12 h at 37 °C. C) HPLC chromatogram of the Cys(Acm)-containing peptide incubated in neat TFA for 12 h at 37 °C (control). D) HPLC chromatogram of the peptide containing the Cys(PySe) adduct treated with 5-fold excess ascorbate in 100 mM ammonium acetate pH 4.5, for 4 h at room temperature. The MS analysis that corresponds to each panel of Figure 5 is given in the Supporting Information as Figures S3.

Figure 6:. Deprotection of the Cys(Acm)-containing peptide using DTNP in the presence or absence of TIS determined by HPLC analysis.

A) HPLC chromatogram of the Cys(Acm)-containing peptide incubated in neat TFA for 12 h at 37 °C (control). B) HPLC chromatogram of the Cys(Acm)-containing peptide incubated in TFA/thioanisole (98:2) with 5-fold excess DTNP for 12 h at 37 °C. C) HPLC chromatogram of the Cys(Acm)-containing peptide incubated in TFA/thioanisole/TIS (96:2:2) with 5-fold excess DTNP for 12 h at 37 °C. The MS analysis that corresponds to panels 6A and 6B are given in the Supporting Information as Figure S4.

Figure 7:. HPLC chromatograms of guanylin peptide at various stages of synthesis.

A) Crude guanylin peptide after cleavage from the resin with concomitant removal of S-Trt protecting groups. Cys4 and Cys12 are in the free thiol form, while Cys7 and Cys15 remain S-Acm protected. B) HPLC trace of the guanylin intermediate after the formation of the first disulfide bond between Cys4 and Cys12 using air oxidation. C) HPLC trace of the mature guanylin peptide after the formation of the second disulfide bond between Cys7 and Cys15 using 1 eq. of PySeSePy, incubated for 8 h at 37 °C. D) HPLC trace of the guanylin standard purchased from CPC Scientific. MS analysis that corresponds to Figure 7 can be found in the Supporting Information as Figure S7.

Figure 8:. Possible mechanisms for 2,2´-dipyridyl diselenide-mediated disulfide formation.

(A) Cys(PySe) adducts results after Acm removal with PySeSePy. Asc is a one-electron reducing agent that can reduce the Cys-S–SePy adduct, resulting in a Cys-thiolate and a PySe• radical. The ejected PySe• radical can then combine with another PySe• radical, reforming PySeSePy. The Cys-thiolate then undergoes S_N2 attack onto the remaining Cys-S–SePy adduct to install the second disulfide bond. (B) An alternate mechanism has the PySe• radical formed after the reaction with Asc undergoing a S_RN2 reaction with the remaining Cys-S–SePy adduct forming PySeSePy and a Cys-S• radical. The Cys-S• radical then can undergo a rapid radical recombination to form the second disulfide bond. (C) Experimentally, we found that disulfide bond formation was independent of the addition of Asc. In the partially folded intermediate, the two Cys(PySe) adducts are close together in 3D-space. We postulate that PySeSePy spontaneously forms as shown due to the low redox potential of the resulting diselenide. This thermodynamic driving force pushes disulfide bond formation to completion.

Figure 9.. Mass spectra of guanylin with and without Asc treatment at pH 4.5 for 4 h at room temperature.

A) Mass spectrum of the synthesized guanylin peptide incubated in 100 mM ammonium acetate at pH 4.5 without Asc (control). B) Mass spectrum of the synthesized guanylin peptide incubated in 100 mM ammonium acetate at pH 4.5 with 5-fold excess Asc. C) Mass spectrum of the commercially available guanylin peptide incubated in 100 mM ammonium acetate at pH 4.5 with 5-fold excess Asc. As is evident, there is no change in mass upon reduction with Asc under these conditions. For all samples, the arrow points to the observed m/z whereas the number inside the parentheses denotes the theoretical m/z value.