A comparison of with illustrates how crucial the addition of amine base in the StBu deprotection step of the precursor peptide is to the final outcome of the synthesis. The addition of base to the deprotection step results in a significantly simplified chromatogram and greatly increased yield of the desired product. While this modified procedure did improve the synthesis, the desired oxidized product was only present as 44% of the crude mixture. The remaining peaks were identified as oligomeric side products and a large amount of impurity identified in the chromatogram as Peak A (representing 41% of the total area). The HPLC chromatogram clearly showed this peak having an enhanced absorbance at 254 nm, and once isolated exhibited a deep yellow color. Both of these facts are consistent with the existence of a 5-Npys-conjugated impurity.
Our initial attempts at using the previously established procedure of Galande for synthesizing peptide PTVTGCCG(ox) (peptide I in ) resulted in the formation of a complex product mixture (). Careful MALDI analysis of the analytical HPLC peaks in the chromatogram showed minimal cyclic, monomeric-oxidized product, with most of the chromatogram populated by oligomers, StBu adducts, and other uncharacterized components, implying an inefficient removal of the StBu protecting group at the beginning of the reaction sequence. Further, we found that the StBu group was recalcitrant to removal even after extended incubation with excess βME (). It was hypothesized that by carrying out the βME deprotection step in 0.1 NMM/DMF, the process would be accelerated due to the higher proportion of βME thiolate at the increased pH. Incubation of a small amount of resin with cleavage cocktail after 90 min of treatment using these augmented conditions showed complete removal of the StBu group from Cys as demonstrated by MALDI-MS analysis ().
In this procedure, two (non-vicinal) Cys side chains in the peptide disulfide precursor are orthogonally protected as their StBu and Mmt conjugates. The synthetic approach begins with treating the peptide attached to the solid support with a 20% βME/DMF solution to reductively remove the StBu group. The free sulfhydryl is then treated with excess DTNP in DCM to convert the Cys residue into a Cys(5-Npys) derivative. Treatment of the resulting resin with a 1% TFA/DCM solution serves the dual purpose of Mmt removal from the attacking sulfhydryl group as well as to activate the mixed disulfide bond of the electrophilic Cys(5-Npys) group by protonating the pyridyl nitrogen atom, resulting in directed disulfide formation.
In order to do the type of enzymatic assay for this study, we needed two different types of peptides: intramolecular cyclic disulfides/selenylsulfides and intermolecular acyclic disulfides/selenylsulfides. Although Kamber  has previously reported synthesizing a vicinal disulfide peptide containing a pair of Cys(Acm) derivatives using I2 as the oxidant, we have found that I2 is not a good reagent for peptides containing Sec because of a high degree of deselenization that occurs during oxidation. In order to achieve the highest purity and yield upon cleavage from the solid-support, we envisioned an on-resin oxidation/deprotection procedure.
H-U(Mob)G-OH was synthesized using standard Fmoc protocol from 2-chlorotrityl chloride resin similarly to the C(5-Npys)G fragment described above, using Fmoc-U(Mob) as the second residue instead of Boc-C(5-Npys). Following α-Fmoc removal, the dipeptide fragment was released from the resin using 1% TFA/DCM and concentrated. This fragment was utilized immediately in the following coupling procedure without further purification.
This modified procedure was also extended to the synthesis of peptide PTVTGCUG(ox) (peptide II). Here the nucleophile is the selenium atom of a Sec(Mob) group, and though the selenium atom is a stronger nucleophile than sulfur, the requirement for addition of an organic base to the deprotection solution is also present as shown by the results in . If NMM is omitted from the deprotection solution, the result is incomplete removal of the StBu group from the adjacent Cys residue promoting significant dimer formation and other side reactions. The neighboring nucleophile will prefer to attack the highly electrophilic, mixed disulfide bond of a Cys(5-Npys) group (on a nearby chain) in comparison to the neighboring mixed disulfide formed in a Cys(StBu) derivative. This effect is most likely enhanced by the difficulty in making a vicinal disulfide bond ().
The H-C(5-Npys)G-OH fragment was synthesized using standard Fmoc protocol from 2-chlorotrityl chloride resin (1.40 mmol/g). The Fmoc-Gly-OH fragment (40 μmol) was incubated with a ~5-fold excess of resin in 0.1 NMM/DCM for 40 min, followed by MeOH capping of any unreacted attachment sites as described above. Boc-Cys(5-Npys) was then coupled using the previously-described protocol. Removal of the dipeptide fragment from the resin was effected by treatment of the resin with neat TFA for 20 min. Following evaporation of the TFA solvent, the crude isolate was dissolved in 4 mL H2O and injected directly into preparative HPLC for purification.
Interchain peptide linked PTVTGC/CG was synthesized via solution fragment coupling of H-PTVTGC(SH)-OH with H-C(5-Npys)G-OH. The peptide H-PTVTGC(SH)-OH was synthesized via standard Fmoc protocol from 2-chlorotrityl chloride resin (1.40 mmol/g), in which a ~5-fold excess of resin was treated with 40 μmol of Fmoc-Cys(Trt)-OH in 0.1 NMM/DCM for 40 min. Following this initial loading step, the remaining attachment sites on the resin were capped via treatment with 1:0.5:8.5 MeOH/NMM/DCM for an additional 40 min. The remaining amino acid residues were attached using the previously described standard Fmoc protocol. Following peptide elongation, α-Fmoc removal, and acidolytic cleavage, preparatory HPLC purification of this fragment was carried out as previously described.
All peptides for this study were synthesized on a Symphony™ multiple peptide synthesizer (Protein Technologies Inc., Tuscon, AZ). via Fmoc protocol, utilizing either 2-chlorotrityl chloride resin, PAL-PEG resin, or Fmoc-Gly-PEG-PS resin. Double coupling using standard HBTU activation was employed for peptide elongation. A typical on-resin coupling procedure is as follows: 20% piperidine/DMF (2 × 10 min); DMF washes (6 × 30 sec); 5 eq. Fmoc amino acid and HBTU in 0.4 NMM/DMF (2 × 30 min); DMF washes (3 × 30 sec). Cleavage of peptides from their respective resins was accomplished through treatment of the dried resin with 96:2:2 TFA/TIS/H2O for 2 h. Following filtration of the resin, the cleavage supernatant was evaporated to one fifth its original volume in a stream of nitrogen, followed by precipitation into cold anhydrous diethyl ether.
Interchain selenylsulfide linked peptide PTVTGC/UG was synthesized via solution fragment coupling of H-PTVTGC(5-Npys)-OH with H-U(Mob)G-OH in neat TFA. The peptide fragment H-PTVTGC(5-Npys)-OH was synthesized from a resin-bound Fmoc-PTVTGC(StBu) precursor via standard Fmoc protocol from 2-chlorotrityl chloride resin as described above. Following peptide elongation, the α-Fmoc protecting group was converted to a Boc group via treatment of the resin with 20% piperidine/DMF (2 × 10 min) followed by incubation with Boc2O (10 eq.) in 5 mL 0.1 NMM 1:1 DMF/DCM for 40 min. After removing the StBu protecting group by treatment with βME as described above, the resulting free sulfhydryl was converted to a 5-Npys derivative by treatment of the resin with 10 eq. DTNP in 5 mL DCM for 1 h. Following thorough washing of the resin with DCM, the peptide was cleaved from the resin with neat TFA and immediately purified by preparatory HPLC as previously above.
Fmoc-protected amino acids and HBTU were purchased from Synbiosci Corporation (Livermore, CA). Resins for solid phase peptide synthesis (SPPS) were purchased from Novabiochem (San Diego, CA) and Applied Biosystems (Foster City, CA). Boc-Cys(5-Npys)-OH was purchased from Bachem (King of Prussia, PA). Fmoc-Sec(Mob)-OH was manufactured using a procedure previously reported [–]. Solvents for peptide synthesis and all other required reagents were purchased from Fisher Scientific (Pittsburgh, PA). DNA primers were purchased from IDT (Coralville, IA).