Orthogonal functionalisation of α-helix mimetics† †Electronic supplementary information (ESI) available: Additional binding data and fluorescence characterisation. Experimental procedures and characterisation of all new compounds. See DOI: 10.1039/c4ob00915k Click here for additional data file.

We present methodology to modify N-alkylated aromatic oligoamide α-helix mimetics using ‘click’ chemistry.

lie on the same face of the molecule, these matched the position of Phe19, Trp23 and Leu26, which are located at the i, i + 4, i + 7 positions of the p53 helix respectively. All the conformations within 1.5 kJ/mol from the lowest energy conformation were superimposed with the p53 α-helix and a mean value of the RMSD (Root Mean Square Deviation) resulting from the superimposition of the nitrogen of the helix mimetic scaffold and the α carbon of the key amino acids of the p53 helix was calculated for conformers with matched side chain orientation.

Dihedral Angle Analysis
The energy of the lowest energy conformer determined in the conformational search as the central Naryl bond was rotated was calculated in the software Macromodel® using the MMFFs (Merk Molecular Force Fields) method. Water was chosen as implicit solvent. The data was plotted as relative energy calculated from the maximum and minimum energy.

Docking
The protein structure for hDM2 (PDB ID: 1ycr) was prepared using the protein preparation wizard within Maestro (Schrodinger) and the docking grid prepared by selecting the binding groove of the p53 helix using Glide (Schrodinger). Conformer libraries of compounds were generated using Omega (Openeye Scientific) and prepared for docking using Ligprep (Schrodinger). Docking was performed using Glide (Schrodinger) allowing flexible ligands but penalising non-planar amide bonds.

Fluorescence Anisotropy Direct Binding
Fluorescence anisotropy assays were performed in 384-well plates (Greiner Bio-one). Each experiment was run in triplicate and the fluorescence anisotropy measured using a Perkin Elmer EnVisionTM 2103 MultiLabel plate reader with excitation at 480 nm and emission at 535 nm (5 nm bandwidth). The experiments were performed in 40 mM phosphate buffer at pH 7.5 containing 200 mM NaCl and 0.02 mg ml -1 bovine serum albumin (BSA).

Thermophoresis Direct Binding
The experiments were performed in 40 mM phosphate buffer at pH 7.5 containing 200 mM NaCl and 0.02 mgml -1 bovine serum albumin (BSA). hDM2 17-126 was diluted into buffer across 16 eppendorfs from 400 μM to 12 nM. The fluorescently tagged trimer was then added giving final protein concentrations from 200 μM to 6 nM and a fixed trimer concentration of 50 nM. The themophoresis of each sample was measured using a Nano Tempter Monolith NT.115 Microscale Thermophoresis. Ligand bound was calculated from Eq. 3 using λ = 1. K d was calculated from Eq. 4.

Fluorescein-N-(N-(N-(Benzyl-4-aminobenzoyl)-N-naphth-1-yl-4-aminobenzoyl)-N-isobutyl-4aminobenzoyl)-glycine, 5
The trimer on glycine-loaded Wang resin (74.8 mg, 0.036 mmol) was suspended in THF: H 2 O (1:1, 1 ml) and azido-fluorescein (18 mg, 0.036 mmol) was added followed by CuSO 4 .5H 2 O (0.9 mg, 3.6 x 10 -3 mmol) and sodium ascorbate (1.4 mg, 7.2 x 10 -3 mmol). The suspension was then mixed overnight on a spinner at room temperature. The solution was then drained and the resin washed sequentially with H 2 O, CH 2 Cl 2 and Et 2 O. The product was then cleaved from the resin TFA: CH 2 Cl 2 (1:1, 1 ml). The solvent was then removed affording the desired trimer as a dark orange solid (53.   An HPLC trace of Compound 5 was not obtained as the fluorescein label caused extensive broadening of the signal and traces of the compound proved difficult to remove from the column so further attempts were not made.

Synthesis of Coumarin and Fluorescein Azides
Scheme S1 -Synthesis of azide-functionalised fluorophores

Determination of the binding of FITC-NOXA-B to Mcl-1
The procedure followed was adapted from our previous paper on O-alkylated aromatic oligoamides. 1 Briefly: Mcl-1 was serially diluted (446 pM -5 μM) into a solution of FITC-NOXA-B (25 nM) (40 mM Sodium phosphate buffer pH 7.54, 200 mM NaCl, 0.02 mg/ml of bovine serum albumin) -the total volume of each well was 100 μL, the plates were allowed to incubate at room temperature for 45 minutes. Each experiment was run in triplicate and the fluorescence anisotropy measured using a Perkin Elmer EnVisionTM 2103 MultiLabel plate reader, with excitation at 480 nm and emission at 535 nm (30 nM bandwidth) and the intensity (Eq. 1) was calculated for each point. This was used to calculate anisotropy (Eq. 2) and plotted to a sigmoidal fit in Origin 7 to determine the minimum and maximum anisotropies (r min and r max ). Using equation 3, the data for the anisotropy was converted to fraction bound and multiplied by the FITC-NOXA-B concentration then fitted in origin 7 (Eq. 4) to give the dissociation constant K d = 18.7 ± 0.9 nM. From a plot of intensity (Eq. 1) against concentration of protein λ was calculated to be 1.  A reverse titration was also performed whereby the protein concentration was held constant and FITC-NOXA-B was serially diluted. The intensity and anisotropy was calculated for each point as before and converted to a fraction bound using the same value of r min and r max . As saturation approaches, the anisotropy begins to drop, whilst the intensity increases, so the asymptote is not reached in this experiment. The fraction bound was then multiplied by the total FITC-NOXA-B concentration at each point and fit (Eq. 4) in origin 7, where y is the fraction bound times the tracer concentration and x is the concentration of added tracer, to give the dissociation constant K d = 13 ± 2 nM.    The observed increase in anisotropy is likely to be the result of aggregation of the trimer to the BAK tracer and Bcl-x L complex. The fluorescent peptide is not displaced from the binding site by addition of the helix mimetic.

Direct Binding Assay Development
hDM2 17-126 was diluted into buffer across the 24 wells from 400 μM to 36 nM. The fluorescently tagged trimer was then added giving final protein concentrations from 200 μM to 18 nM and a fixed trimer concentration of 50 nM. For control wells the fluorescent trimer was replaced with the equivalent volume of buffer. In all experiments the G factor, or ratio between the efficiency of the S(ame) and P(erpendicular) channels was set to 1. Ligand bound was calculated from Eq. 3 using λ = 2.06. K d was calculated from Eq. 4. Figure S9 -Change of intensity during addition of hDM2 used to calculate λ.
Mcl-1 172-327 was diluted into buffer across the 24 wells from 200 μM to 18 nM. The fluorescently tagged trimer was then added giving final protein concentrations from 100 μM to 9 nM and a fixed trimer concentration of 50 nM. For control wells the fluorescent trimer was replaced with the equivalent volume of buffer. In all experiments the G factor, or ratio between the efficiency of the S(ame) and P(erpendicular) channels was set to 1. Ligand bound was calculated from Eq. 3 using λ = 2.31. K d was calculated from Eq. 4.