Towards “bionic” proteins: replacement of continuous sequences from HIF-1α with proteomimetics to create functional p300 binding HIF-1α mimics

An extended sequence of α-amino acids in HIF-1α is replaced with a non-natural topographical mimic of an α-helix comprised from an aromatic oligoamide to reproduce its p300 recognition properties.


General Experimental
All commercial solvents and reagents were used without further purification unless stated otherwise. All non-aqueous reactions were performed under an atmosphere of nitrogen and using anhydrous solvents. Water-sensitive reactions were performed in oven-dried glassware, cooled under nitrogen before use, or flame dried and cooled, under vacuum if stated. Solvents were removed under reduced pressure using a Büchi rotary evaporator. Ether refers to diethyl ether and petrol refers to petroleum spirit (b.p. 40-60 °C). Flash column chromatography was carried out using silica (35-70 μm particles) or alumina (neutral, Brockman activity 1), with crude reaction mixtures loaded in the initial solvent system or its least polar constituent. Thin layer chromatography was carried out on commercially available silica pre-coated aluminium plates (Kieselgel 60 F254, Merck) or commercially available alumina pre-coated glass plates (neutral, Brockman activity 1). Strong cation exchange columns were carried out using SCX, 5.0 g pre-packed cartridge, Supelco.
Proton and carbon NMR spectra were recorded on a Bruker Avance 500, Avance DPX300 or DRX500 spectrophotometer with an internal deuterium lock. Carbon NMR spectra were recorded with composite pulse decoupling using the waltz 16 pulse sequence. Chemical shifts are quoted in parts per million downfield of tetramethylsilane, and coupling constants (J) are given in Hz. NMR spectra were recorded at 300 K unless otherwise stated. Infra-red spectra were recorded using a Perkin-Elmer Spectrum One FT-IR spectrophotometer. Melting points were determined using a Griffin and George melting point apparatus and are uncorrected. Nominal mass spectrometry was routinely performed on a Bruker HCT Ultra spectrometer using electrospray (+) ionization. Nominal and accurate mass spectrometry using electrospray ionisation was carried out by staff or the candidate in the School of Chemistry using a Micromass LCT-KA111, Bruker MicroTOF or Bruker MaXis Impact TOF mass spectrometer. Mass-directed HPLC purifications were run on an Agilent 1260 Infinity Preparative HPLC system equipped with a Waters XBridge™ Prep C18 19 × 100 mm column, 5 μm particle size, on an acetonitrile or methanol/water gradient (5-95% acetonitrile or methanol over 8 minutes) and an Agilent 6120 Quadrupole system equipped with a quadrupole MS detector, using electrospray ionisation (ESI).

Oligobenzamide Nomenclature
To simplify the numbering and NMR assignment of oligobenzamides, we have devised a sequential nomenclature, where each of the monomer building blocks is considered separately. The monomers are numbered from 1 to 3 starting from the N-terminal. Within each monomer, the numbering is the same: the carbons from the aminobenzoic acid are numbered using the standard system (the aromatic carbon bearing the carboxylic acid is C1, the one bearing the amine is C4). Then, the lateral chain is numbered: the carbon attached to the oxygen is the Cα, and the numbering of the aliphatic part of the side chain continues with Cβ, etc. In the case of aromatic side chains, the aromatic carbons are numbered CAr1, CAr2, etc. The numbering of the protons is based on the carbon numbering. To differentiate each individual carbon/proton, the monomer number is added as a prefix to the carbon/proton number representative examples are given above.

Preparation of Peptide-Oligobenzamide Hybrid, 2
Prepared following an adapted literature method. 1 Fmoc-monomer acid chlorides were prepared as previously described and loaded onto a CEM liberty peptide synthesiser. Glyloaded Wang resin was swelled in NMP for 30 minutes prior to loading onto the synthesiser.
The previously described methods were used to synthesise the oligobenzamide portion of the molecule, ending with the Fmoc-Gly-monomer. The peptide portion was then extended using standard Fmoc peptide synthesis.
The reaction mixture was then acidified with conc. HCl and extracted with ethyl acetate (3 × 5 ml). The combined organic layers were dried over MgSO 4 and concentrated in vacuo. The product was checked by LC-MS and used without further purification.

Peptide-PEG Conjugates
The C-terminal portion of the peptide was synthesised using standard Fmoc-SPPS as above on 0.3 mmol scale and checked by cleaving a small amount and performing LC-MS analysis.
The resin was then split into 3 equal portions.

Alternative PEG Linking Strategies
Initially, a one pot conjugation procedure was developed to allow the modular assembly of peptidic regions with linkers of various lengths thus negating the requirement to perform the peptide synthesis in triplicate. A route combining copper catalysed azide alkyne cycloaddition and thiol-ene reactions was envisioned. This was trialled with a model system as shown below. This reaction sequence progressed successfully in one pot as followed by LC-MS. By treating the commercially available NHS-PEG-Maleimide compound with a short azido-alkylamine followed by a thiol, the click handle and the first component can be

Clean progression by LC-MS
However, when the complete one pot procedure was attempted using the optimised conditions, no product was observed. At this stage it was decided to prepare the PEG-Peptide conjugates via linear peptide synthesis incorporating the PEG chain as with any other monomer.

Binding Assays
Binding assays and protein expression were carried out as previously reported. 2,3 The helix 2-3 peptide and the helix 2-extended peptide were purchased from Proteogenix, France and the sequences shown below. The competition assay data for the Helix 2-extended peptide is shown below.