Reactive fragments targeting carboxylate residues employing direct to biology, high-throughput chemistry

The screening of covalent or ‘reactive’ fragment libraries against proteins is becoming an integral approach in hit identification, enabling the development of targeted covalent inhibitors and tools. To date, reactive fragment screening has been limited to targeting cysteine residues, thus restricting applicability across the proteome. Carboxylate residues present a unique opportunity to expand the accessible residues due to high proteome occurrence (∼12%). Herein, we present the development of a carboxylate-targeting reactive fragment screening platform utilising 2-aryl-5-carboxytetrazole (ACT) as the photoreactive functionality. The utility of ACT photoreactive fragments (ACT-PhABits) was evaluated by screening a 546-membered library with a small panel of purified proteins. Hits identified for BCL6 and KRASG12D were characterised by LC-MS/MS studies, revealing the selectivity of the ACT group. Finally, a photosensitised approach to ACT activation was developed, obviating the need for high energy UV-B light.


Solvents, reagents and consumables
Solvents were anhydrous and reagents purchased from commercial suppliers were used as received.
Protein stock solutions used: • Milli-Q phosphate-buffered saline (PBS) (pH 7.2) • 50 mM HEPES (pH 7.4) Plates used: • Greiner 384 white low volume plates (#784075) • Low pH: Sample was eluted using a gradient shown in Table S3 with a flow rate of 1.0 mL/min. Solvent  High pH: Sample was eluted using a gradient shown in Table S4 with a flow rate of 1.0 mL/min. Solvent A (10 mM ammonium bicarbonate in water adjusted to pH 10 with ammonia solution) and solvent B (acetonitrile). Negative Electrospray on a Waters SQD2 instrument, with a scan range of 100-1000 Da and a scan frequency of 5 Hz. A high pH (HpH) method was used and the sample was eluted using a gradient shown in Table S4 with a flow rate of 1.0 mL/min. Solvent A (10 mM ammonium bicarbonate in water adjusted to pH 10 with ammonia solution) and solvent B (acetonitrile).

Mass directed automated preparative HPLC (MDAP)
MDAP HPLC was carried out on a Waters® Xselect instrument equipped with a CSH C18 column High pH: C18 column (150 mm × 30 mm, 5 μm packing diameter, 40.0 mL/min flow rate) using a gradient elution at ambient temperature using mobile phases of water with 10 mM ammonium bicarbonate adjusted to pH 10 with ammonia solution (solvent A) and acetonitrile (solvent B).
The gradient of acetonitrile required to elute the product was determined by the LC-MS retention time. The methods were selected dependent on the retention time of desired material and are shown below (Table S5) and an exemplar gradient for Method B (Table S6).  (Table S7).
High pH: Gradient elution using mobile phases of water with 10 mM ammonium bicarbonate adjusted to pH 10 with ammonia solution (solvent A) and acetonitrile (solvent B) (Table S8).  the summed scans were deconvoluted (using a maximum entropy algorithm) over a mass to charge ratio (m/z) range dependent on the protein (Table S10).   ≥2 substituents α to nitrogen) and cyclic/non-cyclic. Based on our previous findings, primary unhindered, primary hindered and secondary cyclic unhindered amines achieved the highest conversions in the HTC reactions and so only amines with these annotations were chosen, the rest were filtered out. 7 A small number (<5) of positive controls were also included which belonged to the one other categories of amines (e.g. secondary cyclic hindered, 3e). This left a selection of 546 amines which were available from GSK compound stores.

Time (min) Flow rate (mL/min) Solvent A (%)
From our previous report 7 these selected amines also fulfilled the following criteria: • Unstable compounds and compounds with other liabilities had been removed using The selected amines (10 mM, 20 µL) were ordered from GSK's solution stores in Greiner 384 PP Fbottom plates (#781201). Following small molecule LC-MS directly from the plates to assess the amine's initial purity the HTC reactions were undertaken (SI section 2.5).

ACT-PhABit synthesis
Two Greiner PP 384-well plates were charged with 546 amines (

Protein addition and irradiation protocol
Protein samples were prepared for screening according to the following protocols: • For solid proteins (carbonic anhydrase I (CAI), carbonic anhydrase II (CAII) and myoglobin), the solid was weighed out (typically ~1-1.5 mg) and dissolved in the appropriate volume of buffer (PBS or HEPES) such that its concentration was 100 µM. This stock solution was then further diluted in either PBS/HEPES to afford a 1 µM protein solution.
Digested samples were injected on an Easy-nLC 1000 UHPLC system (Thermo Scientific). The nanoLC was interfaced to a Q-Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Scientific).
The crosslinking versus wavelength ±photosensitiser at 10 and 60 minutes was plotted using R Studio software (Version 3.6.3, ggplot), each data point was recorded in duplicate and the mean and SD were calculated and plotted.  Scheme S1

methyl N-(tert-butoxycarbonyl)-O-(4-fluorophenyl)homoserinate (4d)
To a stirring solution of 4c (402 mg, 1.21 mmol), triphenylphosphine (475 mg, 1.81 mmol) and 4fluorophenol (135 mg, 1.21 mmol) in THF (8 mL) at 0 °C under nitrogen was added a solution of DIAD (0.35 mL, 1.81 mmol) in THF (2 mL), dropwise over 10 min. The resultant solution was stirred under nitrogen for a further 22.5 h, allowing the reaction to warm to rt. The reaction mixture was concentrated in vacuo to afford a yellow oil. The oil was stirred in TBME (4 mL) at 0 °C for 0.5 h. The solid that precipitated was filtered off. The filtrate was concentrated in vacuo to afford a yellow oil.
The oil was dissolved in CH2Cl2 and purified by flash column chromatography (silica, 0-100% EtOAc in cyclohexane). The relevant fraction(s) were concentrated in vacuo to afford the desired product as a colourless oil. Some fractions overlapped with 4-fluorophenol were also concentrated in vacuo to afford a colourless oil. In this case, the crude oil was dissolved in CH2Cl2 (10 mL) and 2M aq. NaOH (10 mL) before shaking and separating. The organic layer was dried (hydrophobic frit) and concentrated in vacuo to afford the desired product as a colourless oil.