Chloromethyl-triazole: a new motif for site-selective pseudo-acylation of proteins† †Electronic supplementary information (ESI) available: Synthesis and characterisation of compounds 3b, 4b, and peptides Pep1–3. Peptide and protein alkylation procedures. See DOI: 10.1039/c6cc06801d. Primary data fil

Chloromethyl triazoles are shown to be cysteine selective alkylation reagents for ‘near-native’ post-translational modification of protein and peptide substrates.

Mass spectra were obtained by electrospray (ESI) on a Bruker 12 T SolariX or Bruker microTOF II mass spectrometer. Mass-to-charge ratios (m/z) of all parent (molecular) ions ([M] +/-) and their intensities are reported, followed by (major) fragment or adduct ions and their intensities.
Melting points (mp) were determined on a Gallenkamp Electrothermal Melting Point apparatus and are uncorrected the temperature range and whether the substance undergoes decomposition (dec.) over this range is reported.
Rf values (Rf) were recorded using Merck Silicagel 60 F254 aluminium backed plates. Flash chromatography was carried out using Merck Kieselgel 60 (Merck 9385) under positive pressure. Eluent compositions are quoted as v/v ratios.
Electronic Supplementary Material (ESI) for ChemComm. This journal is © The Royal Society of Chemistry 2016

Procedures and Methods for Solid Phase Peptide Synthesis
For peptide synthesis, all amino acids and resins were purchased from either Novasyn (Merck) or Sigma-Aldrich and Fmoc-mPEG-OH was purchased from IRIS Biotech. All amino acids were N-Fmoc protected and side chains were protected with Boc (Lys); Trt (His, Cys); Pbf (Arg). Peptides were synthesised on Rink Amide AM resin with a loading capacity of 0.69 mmol g -1 or 2-chlorotrityl chloride resin with a loading of 1.0-1.5 mmol g -1 . Synthesis of peptides was performed manually using SPE tube and draining to a vacuum box. Agitation of the resin was carried out on a Stuart SB2 tube spinner at 20 rpm. Monitoring of coupling steps was performed using tandem chloranil and TNBS testing.

Method A: Resin Swelling
The required quantity of resin was weighed directly into an appropriate sized SPE tube equipped with filter and spigot. This was charged with DCM which was slowly allowed to drain from the tube. After several washes of the resin in this fashion the resin was suspended in DCM and left for 30 mins.

Method B: Deprotection of N-Fmoc Protecting Groups
N-Fmoc protecting groups were removed by the addition of piperidine (20% in DMF) to the resin and agitation (10 min), followed by washing of the resin (2 × DMF, 2 × DCM, 1 × DMF). This process was repeated a second time to ensure deprotection.

Method C: Tandem TNBS and chloranil testing
The TNBS test was employed to ensure deprotection and/or coupling of an amino acid residue. A small number of resin beads were placed into an Eppendorf and suspended in a small amount of DMF. To this was added several drops of DIPEA (10% in DMF) and 1 drop of TNBS solution (5% aq). The Eppendorf was then agitated (10 min) and the beads inspected, colourless beads indicated a successful coupling while bright red beads indicated a successful deprotection.
The chloranil test was run in parallel. A small number of resin beads were placed into an Eppendorf and suspended in a small amount of DMF. To this was added several drops of freshly made chloranil (2% in DMF) and several drops of acetaldehyde (2% in DMF). Immediate colour change indicated the successful deprotection of a secondary amine residue. The Eppendorf was then agitated (10 min) and the beads inspected, colourless beads indicated a successful coupling while blue beads indicated a successful primary amine deprotection.

Method D: Coupling of Amino acids
The desired amino acid (3.0 eq.) and OxymaPure® (3.0 eq.) were premixed in a small amount of DMF (5 min). To this was added DIC (3.0 eq.), the mixture was vortexed (1 min) and added to the resin. The resin was then agitated for 45 min and subsequently washed (2 × DMF, 2 × DCM, 1 × DMF). Coupling efficiency was tested (Method C): a negative result indicating complete coupling and a positive result requiring a repeat coupling of the amino acid. Following complete coupling, N-Fmoc deprotection of was achieved via Method B.

Method E: Resin Storage
The peptide was washed (4 × MeOH) and suspended in MeOH for 10 min to shrink the resin. After drainage the resin, tube and lid was dried using Et2O, tightly sealed and placed in a -20 °C freezer.
Method F: Peptide Capping 2-Napthoic acid was coupled using standard coupling conditions (Method D); coupling was confirmed using the TNBS test. FITC (5 eq) was added in minimal volume of DIPEA and agitated for 16 h. The resin was washed sequentially with DCM and DMF until the eluent turned colourless. Coupling was confirmed by performing a mini cleave and analysis by HPLC/MS.