Selective and Potent Proteomimetic Inhibitors of Intracellular Protein–Protein Interactions**

Inhibition of protein–protein interactions (PPIs) represents a major challenge in chemical biology and drug discovery. α-Helix mediated PPIs may be amenable to modulation using generic chemotypes, termed “proteomimetics”, which can be assembled in a modular manner to reproduce the vectoral presentation of key side chains found on a helical motif from one partner within the PPI. In this work, it is demonstrated that by using a library of N-alkylated aromatic oligoamide helix mimetics, potent helix mimetics which reproduce their biophysical binding selectivity in a cellular context can be identified.


General Considerations
All chemicals and solvents were purchased and used without further purification. 1 H, 13 C and 2D NMR Spectra were recorded with a Bruker DRX 500 MHz or DPX 300 MHz spectrometer. 1 H NMR spectra are referenced to tetramethylsilane (TMS) and chemical shifts are given as parts per million downfield from TMS. Coupling constants are reported to the nearest 0.1 Hz. Melting points were determined using a Griffin D5 variable temperature apparatus and are uncorrected. Microanalyses were obtained on a Carlo Erba Elemental Analyser MOD 1106 instrument. IR spectra were recorded with a Perkin-Elmer FTIR spectrometer and samples were analysed in the solid phase. Mass spectra (HRMS) were obtained with a Bruker maxis impact 3000 spectrometer using electrospray ionisation. LC-MS experiments were run on a Waters Micromass ZQ spectrometer. Analytical HPLC analysis was carried out on an Agilent Technologies 1260 Infinity on a gradient of 95-5% acetonitrile in water. Analytical TLC was performed on 0.2 mm silica gel 60 F254 precoated aluminium sheets (Merck) and visualised by using UV irradiation or, in the case of amine intermediates, by staining with ninhydrin solution. Flash chromatography was carried out on silica gel 60 (35-70 micron particles, FluoroChem). The convention used to assign the spectroscopic data and for naming compounds for this series of aromatic oligoamides has been described previously. [1] Additional side chain functionalities were assigned in the NMR as 2-3 O denoting the central aromatic ring functionalised at the 3-position on an oxygen atom.

Synthetic Chemistry
Helix mimetics and associated monomer building blocks were synthesized following previously reported methodology according to Scheme S1 and is described in the following section whilst characterization is described from pg 40 and 1 H NMR spectra and LC-MS analyses are provided from pg 71. Helix mimetics used in biophysical and cell based screens described in this work are given in table S1. Helix mimetics labelled with biotin or fluorescein are annotated as such and given the parent compounds number from which they were derived.
Scheme S1 Synthesis of helix mimetics on solid-phase.
To label mimetics with a biotin probe, a synthesis of an appropriate tag was developed as illustrated in Scheme S2.
Scheme S2 Synthesis of a novel biotin azide for use in solid phase 'click' chemistry.
It was also necessary to obtain three additional alkyne tagged monomers (Fig. S1). These were synthesized using established methods. Figure S1 -Structures of the alkyne-functionalised monomers used in 'click' chemistry reactions.

Solid-Phase Trimer Synthesis
Amino acid-loaded Wang resin (0.1 mmol) was swelled in anhydrous DMF (5 ml) 15 minutes prior to reaction. The monomers (0.5 mmol) were each dissolved in anhydrous CHCl 3 (10 ml) and pre-activated for coupling with Ghosez's reagent (315 μl, 20% in CHCl 3 , 0.48 mmol) for 1 hour at room temperature. The coupling reactions were carried out on a CEM Liberty microwave assisted automated peptide synthesiser. A small sample was removed and cleaved from the resin with TFA: CH 2 Cl 2 (1:1, 1 ml) and analysed by LC-MS to confirm formation of the desired trimer; coupling reactions were assumed to have gone to completion.

'Click' Chemistry
The trimers on amino acid-loaded Wang resin were suspended in THF: H 2 O (1:1, 1 ml) and the azide was added (1 equivalent) along with CuSO 4 .5H 2 O (10 mol%) and sodium ascorbate (20 mol%). The reaction mixture was stirred overnight at room temperature. The resin was then washed with H 2 O (1 ml, 5 mins) and subjected to the cleavage protocol.

Cleavage
The cleavage step was carried out manually, in 1.5 ml 'Extract-Clean' polypropylene reservoirs fitted with 20 μm polyethylene frits (Alltech). The resin was transferred to the reservoir and washed with CH 2 Cl 2 (1 ml, 5 mins) and diethyl ether (1 ml, 5 mins). A 1:1 mixture of TFA: CH 2 Cl 2 was added and the mixture was stirred for 30 mins at room temperature and the contents collected and the procedure repeated. The resulting solution was concentrated affording the target compound.

Protein Expression and Fluorescence Anisotropy
Expression of hDM2 and fluorescence anisotropy assays were performed as described previously. [2] Expression of Bcl-x L and fluorescence anisotropy assays were performed as described previously. [3] Expression of Mcl-1 and fluorescence anisotropy assays were performed as described previously. [4] Direct binding assays were performed as described previously. [4] Fluorescence anisotropy assays were performed in 384-well plates (Greiner Bio-one). Each      this is not too surprising given that 75 has a naphthyl group as the C-terminal monomer but, as the isoleucine side chain might also participate in binding it is possible that the aromatic naphthyl group effectively mimics the tryptophan on p53 resulting in the observed activity. Supporting this hypothesis, dimer 77 also contains a naphthyl but was synthesized using glycine loaded Wang resin so does not possess enough side chains to mimic all of the 'hot-spot' residues on p53

Synthetic Procedures and Characterisation Monomers
Fmoc-protected monomers a, c, k, m, o, and s; [6] e, f, g, h, l, n, q, r and t; [1] j and u [4] were synthesised as described previously.