Reduction of cysteine-S-protecting groups by triisopropylsilane

Abstract

Triisopropylsilane (TIS), a hindered hydrosilane, has long been utilized as a cation scavenger for the removal of amino acid protecting groups during peptide synthesis. However, its ability to actively remove S-protecting groups by serving as a reductant has largely been mischaracterized by the peptide community. Here, we provide strong evidence that TIS can act as a reducing agent to facilitate the removal of acetamidomethyl (Acm), 4-methoxybenzyl (Mob), and tert-butyl (Bu^t ) protecting groups from cysteine (Cys) residues in the presence of trifluoroacetic acid (TFA) at 37 °C. The lability of the Cys protecting groups in TFA/TIS (98/2) in this study are in the order: Cys(Mob) > Cys(Acm) > Cys(Bu^t ), with Cys(Mob) being especially labile. Unexpectedly, we found that TIS promoted disulfide formation in addition to aiding in the removal of the protecting group. Our results raise the possibility of using TIS in orthogonal deprotection strategies of Cys-protecting groups following peptide synthesis as TIS can be viewed as a potential deprotection agent instead of merely a scavenger in deprotection cocktails based on our results. We also tested other common scavengers under these reaction conditions and found that thioanisole and triethylsilane were similarly effective as TIS in enhancing deprotection and catalyzing disulfide formation. Our findings reported herein show that careful consideration should be given to the type of scavenger used when it is desirable to preserve the Cys-protecting group. Additional consideration should be given to the concentration of scavenger, temperature of the reaction, and reaction time.

Figure 1:

Extent of deprotection of Cys(Acm) by TIS at 37 ºC as measured by HPLC. (A) No reduction of the S-Acm bond in the Cys(Acm) test peptide was observed by HPLC after incubation in neat TFA for 12 h at 37 ºC. (B) In contrast, incubation of the test peptide with TFA/TIS (98/2) for 12 h at 37 ºC resulted in conversion of S-Acm to S-H (35%) and peptide disulfide (35%) as evidenced by the HPLC trace. (C) Addition of 5-fold excess TCEP to the reaction in (B) for 12 h resulted in nearly complete reduction of the disulfide to the reduced form. Traces D, E, and F are zoomed in views of traces A, B, and C, respectively.

Figure 2:

Mass chromatograms of the Cys(Acm) protected peptide under various conditions. The arrow points to the observed m/z whereas the number inside the parentheses denotes the theoretical m/z value. (A) Mass chromatogram of the Acm-test peptide after 12 h incubation in neat TFA at 37 ºC. The MS data show that nearly all of the peptide exists in the Cys(Acm) form, in agreement with HPLC analysis. (B) Mass chromatogram of the Acm-test peptide after 12 h incubation in TFA/TIS (98/2) at 37 ºC. The MS data show that inclusion of 2% TIS in the reaction mixture drives deprotection to the Cys-SH (m/z = 691.4) and disulfide (m/z = 690.46) forms in support of our HPLC analysis. Please note that the value m/z = 690.46 for the peptide disulfide correspond to the doubly charged ion (z = 2).

Figure 3:

Extent of deprotection of Cys(Acm) by TIS under standard deprotection conditions as measured by HPLC. (A) HPLC chromatogram of the Acm-test peptide after cleavage from the solid support using a cleavage cocktail containing TFA/TIS/H₂O (96/2/2) for 2 h at room temperature. (B) HPLC chromatogram of the Acm- test peptide after further incubation of the isolated peptide in neat TFA for 2 h at room temperature. (C) HPLC chromatogram of the Acm-test peptide after further incubation of the isolated peptide in TFA/TIS (98/2) for 2 h at room temperature. Little or no deprotection is observed as evidence by the absence of change in the HPLC chromatogram.

Figure 4:

Extent of deprotection of Cys(Acm) by commonly used SPPS scavengers at 37 ºC in TFA as measured by HPLC. (A) 2% H₂O. (B) 2% anisole. (C) 2% phenol. (D) 2% thioanisole. (E) 2% TES.

Figure 5:

Extent of deprotection of Cys(Mob) and Cys(Bu^t) by TIS at 37 ºC as measured by HPLC. (A) HPLC chromatogram of the Cys(Mob) test peptide after cleavage from the solid support using a cleavage cocktail containing TFA/TIS/H₂O (96/2/2) for 2 h at room temperature. (B) HPLC chromatogram of the Cys(Mob) test peptide after incubation in neat TFA for 12 h at 37 ºC. (C) HPLC chromatogram of the Cys(Mob) test peptide after incubation in TFA/TIS (98/2) for 12 h at 37 ºC. (D) HPLC chromatogram of the Cys(Bu^t) test peptide after cleavage from the solid support using a cleavage cocktail containing TFA/TIS/H₂O (96/2/2) for 2 h at room temperature. (E) HPLC chromatogram of the Cys(Bu^t) test peptide incubation in neat TFA for 12 h at 37 ºC. (F) HPLC chromatogram of the Cys(Bu^t) test peptide after incubation in TFA/TIS (98/2) for 12 h at 37 ºC.

Figure 6:

Mass chromatograms of the Cys(Mob)- and Cys(Bu^t) protected test peptides under various conditions. The arrow points to the observed m/z whereas the number inside the parentheses denotes the theoretical m/z value. (A) Mass spectrogram of the Cys(Mob) test peptide incubated in TFA for 12 h at 37 ºC. The peak at m/z = 827 corresponds to the oxidized Cys(Mob) peptide and the peak at m/z 793 corresponds to a dehydrated form of the Cys(Mob) peptide, (B) Mass spectrogram of the Cys(Mob) test peptide after incubation in TFA/TIS (98/2) for 12 h at 37 ºC. The inset shows a close up of the region between m/z 685 and m/z 695. The m/z values in the inset show the presence of the deprotected peptide and the peptide disulfide. (C) Mass spectrogram of the Cys(Bu^t) test peptide incubated in TFA for 12 h at 37 ºC. (D) Mass spectrogram of the Cys(Bu^t) test peptide incubated in TFA/TIS (98/2) for 12 h at 37 ºC.

Figure 7:

Potential mechanism of reduction of Cys(Acm) containing test peptide to Cys-SH by TIS.