Chemical generation and modification of peptides containing multiple dehydroalanines† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c5cc05469a Click here for additional data file.

Development of an effective strategy to convert multiple cysteines into dehydroalanine residues within a single peptide using methyl dibromovalerate.


Reagents and Equipment
Unless otherwise stated, all reagents were purchased from Sigma Aldrich, Alfa Aesar, or Fisher Scientific and were used without further purification. Human plasma kallikrein was purchased from Molecular Innovations (HPKA-MIN), Z-Phe-Arg-AMC was purchased from Cambridge Bioscience. NMR data were collected using a Bruker Avance 500, Bruker DRX500, or Bruker DPX300 and analysed using MestReNova software. IR spectra were recorded using a Bruker Platinum-ATR. High resolution mass spectrometry was carried out on a Bruker maXis Impact or on a Bruker Daltonics micrOTOF using electrospray ionisation. Isotopic distributions in routine mass spectra were as expected. Liquid Chromatography Mass Spectrometry (LCMS) was performed on an Agilent 1200 series LC system comprising a Bruker HCT Ultra ion trap mass spectrometer and a Phenomenex Luna C18 50 × 2 mm column (5 m particle size) using a gradient of 5-90% MeCN over 1.8 min. LCMS data were obtained using a Bruker Ion Trap Mass Spectrometer. Mixtures of solvents, such as those used in column chromatography, are v/v and all column chromatography was carried out using Geduran Si 60 silica gel. Unless otherwise stated all non-aqueous reactions were performed under an atmosphere of nitrogen.

Synthesis of 2,5-dibromoadipamide (3)
2,5-dibromoadipamide was synthesised following a literature procedure. [1] Adipic acid (12.5 g, 85.6 mmol) was dissolved in SOCl 2 (37.5 mL), heated to 80 °C under reflux open to the air for 90 min then cooled to r.t. Carbon tetrachloride (50 mL) was added followed by NBS (34 g, 191 mmol) (NBS freshly recrystallized from H 2 O and dried). With vigorous stirring HBr (aq) (48 %, 5 drops) were added and the reaction heated to 80 °C. The reaction turned from red to black over the course of 2.5 h at which point it was cooled to r.t. and then to 0 °C. The precipitated solid was removed by filtration and the flask washed with ether (50 mL). The filtrate was concentrated in vacuo to give the crude acid chloride as a dark red viscous liquid.
Ammonium hydroxide solution (25%, 140 mL) was cooled to 0 °C and the crude acid chloride was added dropwise, turning the solution blue. The reaction was stirred for 1 h at 0 °C before the blue solid was isolated by filtration. The solid was suspended in MeOH-H 2 O (1:1, 100 mL) and heated to 60 °C for 30 min. Filtration and washing with methanol (100 mL) gave the product 9 (11.36 g, 44%), a mixture of diastereomers, as a grey solid. Synthesis of methyl 2,5-dibromovalerate (4) δ-Valerolactone (2.59 g, 25.9 mmol), bromine (1.99 mL, 38.9 mmol) and phosphorus tribromide (47 µL, 0.5 mmol) were combined and the reaction heated at 110 °C for 2.5 h. The reaction was then cooled to 0 °C before the addition of methanol (5.23 mL, 130 mmol) and p-toluenesulfonic acid monohydrate (47 mg, 0.25 mmol). The reaction was heated to 80 °C for 3 h before cooling and removal of excess methanol under reduced pressure. CH 2 Cl 2 (50 mL) was added and the lower organic layer obtained, washed sequentially with water (10 mL), NaOH (10%, 10 mL), and water (10 mL). Organics were dried (MgSO 4 ) and concentrated under reduced pressure. Product was further purified by column chromatography (1:4 EtOAc:hexane) to yield 4 (5.457 g, 77%) as a colourless oil.

General Procedure 2: Conversion of Cysteine to Dehydroalanine in Peptides using 3
The peptide was dissolved in water (0.4 mL) and TCEP (0.5 eq.) added. A solution of 2,5-dibromoadipamide (10 eq.) in DMF (0.1 mL) was added before the entire solution was transferred to a vial containing K 2 CO 3 (5 eq.). The vial was incubated in a 37 °C shaking incubator at 140 rpm. Reaction was monitored by LCMS.

LCMS optimisation of dehydroalanine formation using 3 and 4
Peptide (1.2 mg, 747 nmol) was pretreated with TCEP (0.09 mg, 373.5 nmol, 0.5 eq.) in water (300 µL), after half an hour at 37 °C this solution was used as the peptide stock solution. In a 96-well plate (polypropylene, Greiner Bio-one 651201), reactions were set up with the following conditions: peptide 6 0.623 mM, base (K 2 CO 3 or NH 4 HCO 3 , 62.3 mM), dibromo compound (3 or 4, ranging from 1.2 mM to 62.3 mM), 1:1 water:organic (DMF/DMSO/MeCN) total volume 20 µL. After incubation at 37 °C for 3 hours, the reactions were examined using LCMS with blank runs as spacers between samples. Ions corresponding to the product 7 and stapled by-products 8/9 were extracted using Bruker ESI Compass data analysis software and the extracted ion peaks integrated. The ratio of these MS peak integrals of 7 to 8/9 was plotted to estimate levels of both product and stapled byproduct.

Kallikrein Inhibition Assay
The inhibitory activity (IC 50 ) of each bicyclic peptide was determined by measuring the residual activity of HPK upon incubation for 30 min at room temperature with concentrations of bicyclic peptide ranging from 10 M to 0.05nM. The activity of HPK (2 × 10 -4 M) was measured using fluorogenic substrate Z-Phe-Arg-AMC (0.1 M) in Tris (10 mM, pH 7.4) supplemented with NaCl (150 mM), MgCl 2 (10 mM), CaCl 2 (1 mM), BSA (0.1%, w/v), Triton X-100 (0.01%, v/v) and DMSO (5%, v/v). Excitation at 355 nm, emission 460 nm. Measurements were taken every 30 seconds. A linear fit of the data points from 1.5 min to 30 min gave the rate of substrate cleavage. These were then converted to percentage kallikrein activity and plotted against the inhibitor concentration. A logistic fit was used to determine the IC 50 .