On‐DNA Transfer Hydrogenolysis and Hydrogenation for the Synthesis of DNA‐Encoded Chemical Libraries

Abstract DNA‐encoded libraries (DELs) are an increasingly popular approach to finding small molecule ligands for proteins. Many DEL synthesis protocols hinge on sequential additions of monomers using split‐pool combinatorial methods. Therefore, compatible protecting group strategies that allow the unmasking of reactive functionality (e. g. amines and alcohols) prior to monomer coupling, or the removal of less desirable functionality (e. g., alkenes and alkynes) are highly desirable. Hydrogenation/hydrogenolysis procedures would achieve these ends but have not been amenable to DEL chemistry. We report a catalytic hydrogen transfer reaction using Pd/C, HCONH4 and the micelle‐forming surfactant, TPGS‐750‐M, which gives highly efficient conversions for hydrogenolysis of Cbz‐protected amines and benzyl protected alcohols and hydrogenation of nitros, halides, nitriles, aldehydes, alkenes and alkynes. Application to multicycle synthesis of an encoded compound was fully compatible with DNA‐amplification and sequencing, demonstrating its applicability to DEL synthesis. This method will enable synthetic DEL sequences using orthogonal protecting groups.


Solvents and Reagents
Chemicals were purchased from Fluorochem, Sigma-Aldrich and TCI, and used without further purification. Fmoc-NH-PEG4-COOH linker was purchased from Key Organics. TPGS-750-M was purchased from Sigma-Aldrich, concentrations of surfactant in water are quoted as percentages (by weight) as used by the supplier. All water used with DNA substrates was nuclease-free water purchased from ThermoFisher. DNA was purchased from Sigma-Aldrich as either solid supported crude material, or supplied as single strands after desalting.

Analytical Techniques
Calculated exact masses were quoted from ChemDraw Professional 15.0. DNA mass spectra were measured on an Agilent 6550 QTOF in negative mode, using standard 3200 m/z maximum and 2GHz extended dynamic range. Drying gas temperature was at 260 °C at 12 l/min, sheath gas temperature was 400 °C at 12 l/min, nebuliser at 45 psig, VCap voltage of 4000 V and nozzle voltage of 2000 V. 2 The LC was carried out on an Agilent 1260 Infinity 2 using an Agilent Advancedbio oligonucleotides column, 2.1x100 mm where the gradient was run at 0.8 ml/min from 10% MeOH to 50% MeOH over 4 mins against a 50 mM HFIP:15 mM DIPEA buffer solution. A 1 min flush at 95% MeOH preceded each run. Analysis of data was carried out using Agilent Qualitative Analysis version 7. Where appropriate a 1260 Infinity II Multiple Wavelength Detector was used and analysis was carried out at 260 nm.
The conversions were determined by integrating the peak areas in the total ion count chromatograms for the starting material and desired product and reported as a percentage. In cases where there were additional by-products formed the peak area for the desired product and those of all detectable products were used to determine the percentage of desired product relative to all detected components.
Gel electrophoresis was conducted using prepacked 4% E-Gel™ EX Agarose Gels on an Invitrogen™ E-Gel Power Snap Electrophoresis System, using Invitrogen™ Ultra Low Range DNA Ladders. DNA concentrations were calculated using a NanoDrop™ One/OneC Microvolume UV-Vis Spectrophotometer, pipetting 1 µl of sample on the loading plate.

Chromatography and Equipment
Preparative HPLC purification was carried on an Agilent 1260 infinity system using a Phenomenex Clarity 5 um Oligo-RP column, 21.2x250 mm, with a gradient run at 20 ml/min from 10% MeOH to 60% MeOH over 11 mins against a 200 mM HFIP:8 mM TEA buffer solution. Fractions were analysed at 260 nm wavelength.
PCR, ligation and phosphorylation were carried out using a Techne® Prime thermal cycler (5PRIMEG/02). qPCR was carried out using a Bio-Rad CFX96 real time system.

General Procedures General MMT-Deprotection Method
The average loading of single stranded DNA attached to solid support was found by cleavage from solid support using the below method and repeating three times. Nanodrop concentration of cleaved DNA showed that 103 mg yielded 2 µmol of DNA.
The single-stranded DNA employed was a 14mer (GTCTTGCCGAATTC) modified with a 5' MMTamino C6 linker, bound to solid support at the 3' end. Solid supported DNA (103 mg, ca. 2 µmol) was washed with 3% trichloroacetic acid in DCM (10 x 500 µL). A yellow colour indicated that the deprotection was in progress. Once this colour subsided, the solid supported DNA was washed with DCM (3 x 500 µL) and left to air dry for 20 minutes before coupling to the headpiece.
General HP Synthesis and Cleavage from solid support to form 1 To a 1.5 mL microcentrifuge tube was added HATU (17 mg, 44 µmol), DIPEA (17 µL, 100 µmol) and DMF (1 mL). To this was added 12-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)dodecanoic acid (26 mg, 40 µmol), and the mixture was shaken for 20 minutes at room temperature. Deprotected solid supported DNA (ca. 2 µmol) was added, and the reaction was shaken at room temperature overnight. The mixture was then filtered and washed with DMF (3 x 500 µL), MeCN (3 x 500 µL), MeOH (3 x 500 µL) and DCM (3 x 500 µL), before being allowed to air dry for 20 minutes. 40% methylamine in water (500 µL) and 33% ammonia in water (500 µL) were mixed in a 1.5 mL microcentrifuge tube. The solid supported DNA was added and the mixture shaken overnight at room temperature. The mixture was then filtered and washed with water (3 x 500 µL), and the filtrate was concentrated to ca. 0.5 mL using a Genevac at 40 o C. The crude product was then purified by HPLC, fractions concentrated using a Genevac at 40 o C and dissolved in water (1 mL). The concentration of samples was then quantified by UV using a NanoDrop One by ThermoFisher. The usual amount was ca. 0.5-1 µmol of DNA after HPLC purification. The exact amount of the complimentary 14mer (GAATTCGGCAAGAC) was then added in water, and the solution was heated to 80 o C for 1 hour, then allowed to cool slowly. The double stranded DNA was concentrated using a Genevac at 40 o C until dry, and dissolved in water to form a 1 mM solution of the product. Figure S1. Chromatogram of HP-1

General Ethanol Precipitation Procedure for Purification of Intermediates and Products
To the reaction mixture was added 10% volume NaCl (5 M in water) and 3x volume cold EtOH. The mixture was allowed to sit for 1 hour at -78 o C, or overnight at -20 o C. The sample was then centrifuged at 13400 rpm for 10 minutes. The supernatant was decanted, and cold 70% EtOH was added. The mixture was centrifuged at 13400 rpm for a further 10 minutes and the supernatant was again decanted. The resulting pellet was allowed to air dry before being dissolved in water.

S5
Experimental Procedure and Characterisation of Amide Coupling to form 2-20, 22-26 HATU (5.7 mg, 15 µmol) and the appropriate carboxylic acid (15 µmol) were added to a 50 µL glass insert for a Para-dox TM 96-well micro photoredox plate. 5% TPGS-750-M (21 µL), H2O (4 µL) and HP 1 (5 µL, 1 mM in H2O) were added to the vial, followed by lutidine (6.92 µL, 60 µmol). Samples were vortexed for 30 seconds each, and then heated in a Para-dox TM 96-well micro photoredox plate at 45 o C for 16 hours. Samples were then diluted to 200 µL with H2O; DCM (2 x 400 µL) was added, and the samples were vortexed. The organic layer was discarded, the sample was filtered through a hydrophilic PTFE filter and analysed via mass spectrometry. Products were then precipitated according to the general ethanol precipitation procedure. Cbz-Protected amines  To a 200 µL PCR tube was added Headpiece 1 (20 µL, 1 mM in water), sodium borate buffer (20 µL, pH 9.4) and N-acryloxysuccinimide (8 µL, 200 mM in DMA). The solution was shaken at room temperature overnight. Samples were then diluted to 200 µL with H2O, filtered through a hydrophilic PTFE filter and analysed via mass spectrometry (94% conversion to desired product). 21 was then precipitated according to the general ethanol precipitation procedure.

Results and Chromatograms
S20 Figure S26: Chromatogram and deconvoluted mass spectrum of 21 To a 50 µL glass insert for a Para-dox TM 96-well micro photoredox plate was added 10% wt Pd/C (5 µL of 8 mg in 200 µL water), 5% TPGS-750-M (12 µL), water (6 µL) and DNA (4 µL, 0.25 mM in water). Samples were vortexed for 30 seconds each, then ammonium formate (3 µL, 5.3 M in water) was added, and the samples were vortexed for a further 10 seconds. Reactions were then shaken at 1200 rpm, at room temperature in a PMS-1000i Microplate shaker for up to 2 hours. The samples were diluted to 200 µL with water, filtered through a hydrophilic PTFE filter and analysed via mass spectrometry. Products were precipitated according to the general ethanol precipitation procedure.

3% TPGS-750-M Procedure
To a 50 µL glass insert for a Para-dox TM 96-well micro photoredox plate was added 10% wt Pd/C (5 µL of 8 mg in 200 µL water), 5% TPGS-750-M (18 µL) and DNA (4 µL, 0.25 mM in water). Samples were vortexed for 30 seconds each, then ammonium formate (3 µL, 5.3 M in water) was added, and the samples were vortexed for a further 10 seconds. Reactions were then shaken at 1200 rpm, at room temperature in a PMS-1000i Microplate shaker for up to 2 hours. The samples were diluted to 200 µL with water, filtered through a hydrophilic PTFE filter and analysed via mass spectrometry. Products were precipitated according to the general ethanol precipitation procedure.  Experimental Procedure for Transfer Hydrogenation without TPGS-

750-M
To a 50 µL glass insert for a Para-dox TM 96-well micro photoredox plate was added 10% wt Pd/C (5 µL, 400 mM in H2O), water (18 µL) and DNA (4 µL, 0.25 mM in water). Samples were vortexed for 30 seconds each, then ammonium formate (3 µL, 5.3 M in water) was added, and the samples were vortexed for a further 10 seconds. Reactions were then shaken at 1200 rpm, at room temperature in a PMS-1000i Microplate shaker for up to 2 hours. The samples were diluted to 200 µL with water, filtered through a hydrophilic PTFE filter and analysed via mass spectrometry. Products were precipitated according to the general ethanol precipitation procedure.

Experimental Procedure for Transfer Hydrogenation without shaking
To a 50 µL glass insert for a Para-dox TM 96-well micro photoredox plate was added 10% wt Pd/C (5 µL, 400 mM in H2O), 5% TPGS-750-M (12 µL) water (6 µL) and DNA (4 µL, 0.25 mM in water). The sample was vortexed for 30 seconds, then ammonium formate (3 µL, 5.3 M in water) was added, and the sample was vortexed for a further 10 seconds. The reaction was then stood at room temperature for 1 hour. The sample was diluted to 200 µL with water, filtered through a hydrophilic PTFE filter and analysed via mass spectrometry. Products were precipitated according to the general ethanol precipitation procedure. To a 1 mM solution of DNA-derivative in water (5 nmol, 5 L) was added water (15 L), sodium borohydride (2 L, 400 mM in NMP) and palladium acetate (2 L, 50 mM in DMA). The reaction was shaken at rt for 1h. L-Cysteine (20 L, 300 mM in water) was then added and the suspension was shaken at rt for a further 4 hours. The solution was then diluted to 200 L with water, filter through a hydrophilic PTFE filter and precipitated according to the general precipitation procedure before being analysed via mass spectroscopy.    3.5% TPGS-750-M, 45 °C, 16 h, 58% yield over 2 steps. Yields determined by Nanodrop™ spectrophotometry.

Construction of 1x1 Library
The following code abbreviations for each DNA section have been used: Sequences involved in the first ligation included adapter (A), primer (P), overhangs (OH) and building block 1 (BB1). Prior to ligations, the 5' terminus of any DNA strands that required ligating was phosphorylated; for cycle 1 this refers to sequences P (AGGTCGGTGTGAACGGATTTG), OH1BB1 (GTATGCACACGC) and A'P'OH1' (ATAC CAAATCCGTTCACACCGACCT GAATTCGGCAAGAC). To the DNA strands (50 µM, 1000 pmol in overall reaction media of 20 µL) was added PNK reaction buffer (2 µL, 500 mM Tris-HCl [pH 7.6 at 25 o C], 100 mM MgCl2, 50 mM DTT, 1 mM spermidine), ATP (2 µL, 10 Mm, Thermo Scientific), T4 Polynucleotide Kinase (1 µL, 10U/µL. Thermo scientific) and nuclease free water (up to 20 µL). The reaction was conducted at 37 o C for 1 hour, followed by heating to 75 o C for 10 minutes. DNA was used in the subsequent ligation without purification or preparation.
Ligations were performed in 200 µL PCR tubes, using 20 µL of each phosphorylation reaction mixture, alongside 10 µL of 0.1 mM solutions of non-phosphorylated A (GTCTTGCCGAATTC) and OH2'BB1' (TAGG GCGTGTGC). To the DNA strands was added 10X T4 DNA ligase buffer (9 µL, 400 mM Tris-HCl, 100 mM MgCl2, 100 mM DTT, 5 mM ATP), water (up to 90 µL) and T4 DNA Ligase (3 µL, 30 Weiss U/µL). Ligations were conducted at 25 o C for 16 hours, followed by heating to 75 o C for 10 minutes. Product 51 was then visualised by gel electrophoresis, showing a band at ca. 50 base pairs in length (expected 47 and 51 base pairs), before being purified by EtOH precipitation. The resulting pellet was then reconstituted in water to form at 0.1 mM solution.
Ligations were performed in 200 µL PCR tubes, using 20 µL of each phosphorylation reaction mixture, alongside 10 µL of a 0.1 mM solution of OH3'BB2' (CGTA GTACATGC). To the DNA strands was added 10X T4 DNA ligase buffer (9 µL, 400 mM Tris-HCl, 100 mM MgCl2, 100 mM DTT, 5 mM ATP), water (up to 90 µL) and T4 DNA Ligase (3 µL, 30 Weiss U/µL). Ligations were conducted at 25 o C for 16 hours, followed by heating to 75 o C for 10 minutes. The product was then visualised by gel S46 electrophoresis, showing a band at ca. 60 base pairs in length (expected 59 and 63 base pairs), before being purified by EtOH precipitation. The resulting pellet was reconstituted in water (9 µL) for the initial amide coupling.
HATU (5.7 mg, 15 µmol) and Cbz-Valine-OH (3.73 mg, 15 µmol) were added to a 50 µL glass insert for a Para-dox TM 96-well micro photoredox plate. 5% TPGS-750-M (21 µL) and DNA (9 µL, 0.11 mM in H2O) were added to the vial, followed by lutidine (6.92 µL, 60 µmol). Samples were vortexed for 30 seconds each, and then heated in a Para-dox TM 96-well micro photoredox plate at 45 o C for 16 hours. Samples were then diluted to 200 µL with H2O; DCM (2 x 400 µL) was added, and the samples were vortexed. The organic layer was discarded, the sample was filtered through a hydrophilic PTFE filter and then precipitated according to the general ethanol precipitation procedure, and reconstituted in water (10 µL) for subsequent Cbz-deprotection.
To a 50 µL glass insert for a Para-dox TM 96-well micro photoredox plate was added 10% wt Pd/C (5 µL, 400 mM in H2O), 5% TPGS-750-M (12 µL), amide coupling product (10 µL, 0.1 mM in water). Samples were vortexed for 30 seconds each, then ammonium formate (3 µL, 5.3 M in water) was added, and the samples were vortexed for a further 10 seconds. Reactions were then shaken at 1200 rpm, at room temperature in a PMS-1000i Microplate shaker for 2 hours. The samples were diluted to 200 µL with water, filtered through a hydrophilic PTFE filter and precipitated according to the general ethanol precipitation procedure. The pellet was reconstituted in water and further purified through a 10000 Da MW filter to yield 53 (0.59 nmol).

Quantitation of DNA by qPCR
A three building block headpiece was prepared (see S43-S47). This was divided equally into two and one sample was subjected to the hydrogenation conditions (2% TPGS-750-M, procedure S20). Each sample was resuspended in 100 L of H2O. 2 L of each sample was taken and made up to 200 L with H2O. The two samples were subjected to quantitative PCR analysis using triplicates using a Bio-Rad CFX96 real time system.
For each well contained 10 L of ThermoFischer SYBR green PCR master mix, 0.2 L of forward primer (10 M), 0.2 L of reverse primer (10 M), 7.6 L H2O 7.6 L and 2 L sample mixture.