Studies on deprotection of cysteine and selenocysteine side-chain protecting groups

Abstract

We present here a simple method for deprotecting p-methoxybenzyl groups and acetamidomethyl groups from the side-chains of cysteine and selenocysteine. This method uses the highly elecrophilic, aromatic disulfides 2,2'-dithiobis(5-nitropyridine) (DTNP) and 2,2'-dithiodipyridine (DTP) dissolved in TFA to effect removal of these heretofore difficult-to-remove protecting groups. The dissolution of these reagents in TFA, in fact, serves to 'activate' them for the deprotection reaction because protonation of the nitrogen atom of the pyridine ring makes the disulfide bond more electrophilic. Thus, these reagents can be added to any standard cleavage cocktail used in peptide synthesis.The p-methoxybenzyl group of selenocysteine is easily removed by DTNP. Only sub-stoichiometric amounts of DTNP are required to cause full removal of the p-methoxybenzyl group, with as little as 0.2 equivalents necessary to effect 70% removal of the protecting group. In order to remove the p-methoxybenzyl group from cysteine, 2 equivalents of DTNP and the addition of thioanisole was required to effect removal. Thioanisole was absolutely required for the reaction in the case of the sulfur-containing amino acids, while it was not required for selenocysteine. The results were consistent with thioanisole acting as a catalyst. The acetamidomethyl group of cysteine could also be removed using DTNP, but required the addition of > 15 equivalents to be effective. DTP was less robust as a deprotection reagent. We also demonstrate that this chemistry can be used in a simultaneous cyclization/deprotection reaction between selenocysteine and cysteine residues protected by p-methoxybenzyl groups to form a selenylsulfide bond, demonstrating future high utility of the deprotection method.

Figure 1

(A) Analytical HPLC chromatogram of peptide C(StBu)U(Mob)G-NH₂ that was treated with a cleavage cocktail containing TFA/H₂O (95/5). The peak labeled as 1 (37.9% of total peak area) is the fully deprotected and cyclic peptide. The material in peak 2 (17% of total peak area) was found to have a m/z of 831.7 and corresponds to a dimeric form of the target peptide in which the Mob groups have been removed from each monomer. Peak 3 is the fully protected peptide with a m/z of 537.6 and is 37.8% of the total peak area. (B) Analytical HPLC chromatogram of peptide C(Trt)U(Mob)G-NH₂. that was treated with a cleavage cocktail containing TFA/EDT/H₂O (95/2.5/2.5). One major peak predominates (peak 2 – 94.5% of peak area) and MS analysis identified it as peptide CU(Mob)G-NH₂ (the trityl group is removed while the Mob group remains on Sec). Peak 1 was found to be the fully deprotected, cyclic peptide with a m/z of 327 and was 5.5% of the total peak area.

Figure 2

Our postulated mechanism for the spontaneous deprotection/cyclization reaction (on-resin) for peptide Cys(StBu)-Sec(Mob)-Gly-PAL-Resin. The reaction is dependent on high concentrations of acid, which would protonate the sulfur atom of the StBu group. Concomitant attack by the Se atom on the neighboring sulfur would result in a trivalent selenonium cation that would quickly break down in the presence of scavenger. This scavenger could be the released StBu group or water present in the TFA. The shaded circule represents the resin bead.

Figure 3

Our postulated mechanism for spontaneous deprotection/cyclization (on-resin) of a Sec(Mob) residue when a highly reactive Cys(5-Npys) residue is in the adjacent position. The propinquity of the highly electrophilic disulfide bond allows for easy attack by the Se atom onto the central S atom. This attack results in a trivalent selenonium cation that quickly breaks down to the cyclized product. This mechanism is nearly identical the one in Figure 2, except that the peptidyl-disulfide bond is highly activated for nucleophilic attack by the presence of the 5-Npys group. The shaded circle represents the resin bead.

Figure 4

In (A) exogenous DTNP can be added to a peptide dissolved in TFA containing a Sec(Mob) group. The highly acidic solution activates DTNP by protonation of the nitrogen atom in the pyridine ring. This creates a very good electron sink and enhances the electrophilicity of the disulfide bond of DTNP. The highly nucleophilic selenium atom attacks this disulfide bond, resulting in a trivalent selenonium cation that breaks down with scavenger to form a Sec(5-Npys) residue. As shown in (B) a Cys(Mob) residue can also be deprotected by DTNP, but it requires the addition of thioanisole. Thioanisole most likely forms a reactive, trivalent sulfonium cation when it attacks the disulfide bond of DTNP. This trivalent species is much more reactive than DTNP alone and we believe that it is this trivalent species that allows for the less nucleophilic sulfur atom (compared to selenium) to attack the resulting, reactive disulfide bond, causing deprotection and formation of a Cys(5-Npys) residue. This 5-Npys group can be removed by simple reduction with reagents such as dithiothreitol or β-mercaptoethanol.

Figure 5

Plot of percent deprotection of a Sec(Mob) residue in target peptide VTGGU(Mob)G-OH versus equivalents of DTNP in the TFA cocktail. 100% deprotection can be achieved with less than stochiometric addition of DTNP (0.5 equivalents).

Figure 6

A catalytic role for DTNP is proposed here based on the results in Figure 5 that show high levels of Mob removal can be achieved with less than 1 equivalent of DTNP. The free 2-thio-5-nitro-pyridine that is released in the deprotection reaction could cleave the Se-S bond of the Sec(5-Npys) derivative by attack on the sulfur atom resulting in a peptide with a free selenol and regenerating DTNP. Excess DTNP alters this equilibrium process resulting in the selenol being functionalized as the 5-Npys derivative. The thione form of 2-thio-5-nitro-pyridine most likely predominates in solution as is shown here [25].

Figure 7

Progress of removal of the Acm group of the cysteine residue in test peptide VTGGC(Acm)A followed by analytical HPLC. In (A), the peptide has been treated with 2% thioanisole and TFA only as a control. Peak 1 is the Acm-protected peptide, and peak 2 is thioanisole. In (B) the peptide has been treated with 3 equivalents of DTNP in TFA with 2% thioanisole in the cocktail. A new peak is clearly visible in the trace at 35 minutes (labeled as peak 3). This peak was identified by MALDI-TOF MS as the 5-Npys protected peptide (VTGGC(5-Npys)A with m/z = 658). In (C) the peptide was treated with 15 equivalents of DTNP in TFA with 2% thioanisole in the cocktail. Near complete deprotection is observed, but there is still some Acm-protected peptide present. In all traces, detection at 214 nm is represented by the solid line and detection at 254 is represented by the dashed line.

Figure 8

A plot of percent deprotection of the Mob group from Cys (closed squares) or the Acm group of Cys (open circles) versus equivalents of DTNP added to the peptide dissolved in TFA with 2% thioanisole. The test peptides here were either VTGGC(Acm)G-OH or VTGGC(Mob)G-OH. The Cys(Mob)-containing peptide can be fully deprotected with 2 eq. of DTNP, while ∼90% deprotection is achieved for the Cys(Acm)-containing peptide with 15 eq.

Figure 9

A plot of percent deprotection of the Mob group from Cys (closed squares) or the Acm group of Cys (open circles) versus equivalents of DTP added to the peptide dissolved in TFA with 2% thioanisole. The test peptides here were either VTGGC(Acm)G-OH or VTGGC(Mob)G-OH. As can be seen in the plot, DTP is a less effective deprotection reagent than DTNP under the same conditions (compare the plot in Figure 9 with the plot in Figure 8).

Figure 10

Selective deprotection of a Sec(Mob) residue in the presence of a Cys(Mob) residue, followed by formation of a selenylsulfide bond. In (A) the purified peptide AEAU(Mob)GPC(Mob)K-OH is treated with TFA and incubated for 1 h at RT and then injected onto the HPLC column. A large peak at 33 minutes is present corresponding to the fully-protected peptide [Sec(Mob) and Cys(Mob)]. In (B), the peptide has been treated with 1 equivalent of DTNP in TFA for 1 h at RT, followed by injection onto the HPLC column. A new peak centered at 13 minutes is present after treatment with DTNP. In (C) an aliquot of the peptide sample in B was taken after 1 h of reaction and an additional equivalent of DTNP plus 2% thioanisole was added to the aliquot. This aliquot was allowed to react for an additional hour and then injected onto the HPLC column. The peak at 13 minutes has grown larger while the peak at 33 minutes has nearly disappeared. MALDI-TOF MS analysis identified the peak at 13 minutes as the fully deprotected and cyclized peptide (m/z = 824.4) as shown in Figure 11. The solid line is the absorbance at 214 nm and the dashed line is the absorbance at 254 nm.

Figure 11

A postulated mechanism for selective deprotection of a Sec(Mob) residue in the presence of a Cys(Mob) residue followed by cyclization to form a selenylsulfide bond. There are two likely mechanisms for the cyclization reaction to occur. In (A) the Sec residue is deprotected with DTNP to form a Sec(5-Npys) residue. Since there is a nearby Cys(Mob) residue, the sulfur atom of Cys(Mob) can attack the highly reactive, neighboring selenylsulfide bond, with concomitant loss of the Mob group. In (B) some of the Sec residues will exist as the free selenol. The selenol would then be able to attack the disulfide of the neighboring Cys(5-Npys) residue that forms upon addition of a second equivalent of DTNP and thioanisole. In either case, the result is fully cyclic, deprotected peptide.