Efficient automated solid-phase synthesis of recognition-encoded melamine oligomers

Recognition-encoded melamine oligomers (REMO) are synthetic polymers with an alternating 1,3,5-triazine-piperazine backbone and side chains equipped with either a phenol or phosphine oxide recognition unit. Here, we describe an automated method for highly efficient solid-phase synthesis (SPS) of REMO of any specified length and sequence. These SPS protocols are amongst the most robust reported to date, as demonstrated by the synthesis of a mixed-sequence 42-mer, which was obtained in excellent crude purity on a 100 mg scale. Starting from loaded Wang resin and dichlorotriazine monomer building blocks, the SPS methods were automated and optimised on a commercial peptide synthesiser. Major side products were identified using LCMS, and the undesired side reactions were suppressed by the choice of resin, solvent and coupling conditions. REMO have been shown to form high-fidelity length- and sequence-selective H-bonded duplexes, analogous to nucleic acids, and automated synthesis will facilitate exploration of related functional properties, such as molecular replication and programmable self-assembly.


General Experimental Details
All reagents and materials used in the syntheses described were bought from commercial sources and used without prior purification.Dry solvents were obtained from a Grubbs PS-MD-5 solvent purification system and used with no further degassing.Thin layer chromatography (TLC) was carried out using silica gel 60F (Merck) on glass plates.LCMS analyses of samples were performed using a Waters Acquity H-class UPLC coupled with a single quadrupole Waters SQD2.Two different UPLC columns were used: an Acquity UPLC CSH C18 Column (130 Å, 1.7 µm, 2.1 mm x 50 mm), and an Acquity UPLC PRM PR BEH C4 Column (300 Å, 1.7 µm, 2.1 mm x 50 mm).
Purification of compounds by silica column chromatography were performed using an automated system (Combiflash® Rf+ or Combiflash® Rf+ Lumen) with pre-packaged silica cartridges (25 µm or 50 µm PuriFlash® columns).All NMR spectra were recorded using a Bruker 500 MHz Avance III Smart Probe Spectrometer, a Bruker 400 MHz Avance III HD Spectrometer, a Bruker 400 MHz Avance III HD Smart Probe Spectrometer, or a Bruker 400 MHz Neo Prodigy Spectrometer at 298 ± 0.1 K.The residual 1 H form of the solvent was used as the internal standard for referencing.In CDCl3, the 1 H spectra were referenced to δ 7.26 ppm and 13 C spectra referenced to δ 77.16 ppm.In DMSO-d6, the 1 H spectra were referenced to δ 2.50 ppm and 13 C spectra referenced to δ 39.52 ppm.Chemical shifts (δ) are quoted in ppm and coupling constants (J) quoted in Hz.Splitting patterns are reported as: s (singlet), bs (broad singlet), d (doublet), t (triplet), q (quartet) and m (multiplet).FT-IR spectra were collected with an ALPHA FT-IR Spectrometer from Bruker.HRMS spectra were recorded using a Waters SQD2 with Waters H-Class UPLC, equipped with a Waters Acquity UPLC BEH C18 Column (130 Å, 1.7µm, 2.1 mm x 50 mm).
Analytical reverse-phase HPLC was performed on an Agilent HP-1100 Series HPLC system.Preparative reverse-phase HPLC was performed on an Agilent HP-1100 Series preparative HPLC system.UV-vis spectra were collected on an Agilent Cary 60 UV-vis spectrophotometer controlled by Cary WinUV software.

Synthesis of 1
To a solution of 3-hydroxybenzaldehyde (11.1 g, 90.9 mmol) in dry DMF (100 mL) under N2 at 0 °C was added imidazole (13.6 g, 199.8 mmol), then the mixture was stirred until a clear solution formed.Triisopropylsilyl chloride (21.5 mL, 19.3 g, 99.9 mmol) was added dropwise then the solution was stirred for 2 h at r.t.before it was quenched with water (100 mL).The aqueous layer was extracted with EtOAc (3x) before the combined organic layers were washed with water (3x), 5% LiCl soln.(3x) and brine.The organic phase was dried over MgSO4 and then the solvent was removed in vacuo to yield a pale-yellow oil (28.8 g).
Isobutylamine (9.94 mL, 7.31 g, 99.9 mmol) and molecular sieves (4 Å) were added to a solution of the crude product from the previous step (28.8 g) in DCM (150 mL) at r.t.The solution was stirred at r.t. until complete conversion of the aldehyde, which was monitored by 1 H NMR. The molecular sieves were removed, and the solvent was evaporated in vacuo to obtain a colourless oil.
MeOH (200 mL) was added, and the solution was cooled down to 0 ºC before sodium borohydride (3.78 g, 99.9 mmol) was added.The reaction mixture was stirred at r.t. until the disappearance of the imine intermediate, as monitored by 1 H NMR. The solvent was removed in vacuo and the residue was dissolved in 1 M NaOH solution (500 mL).The solution was extracted with DCM (3x).The organic phase was dried over MgSO4 and then the solvent was removed in vacuo to yield the crude product.The crude was purified by flash chromatography (SiO2, 0-10% gradient of MeOH in DCM) to yield the pure product 1 as a pale-yellow oil (28.82 g, 85.8 mmol, 94% over three steps).-1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 δ / ppm

Synthesis of 3
To a solution of 3-hydroxybenzaldehyde (9.0 g, 73.7 mmol) in dry DMF (50 mL) under N2 at 0 °C was added imidazole (11.0 g, 162.1 mmol), then the mixture was stirred until a clear solution formed.Triisopropylsilyl chloride (17.3 mL, 15.6 g, 81.0 mmol) was added dropwise then the solution was stirred for 2 h at r.t.before it was quenched with water (100 mL).The aqueous layer was extracted with EtOAc (3x) before the combined organic layers were washed with water (3x), 5% LiCl soln.(3x) and brine.The organic phase was dried over MgSO4 and then the solvent was removed in vacuo to yield a pale-yellow oil (20.5 g).
2-ethylhexylamine (13.3 mL, 10.5 g, 81.0 mmol) and molecular sieves (4 Å) were added to a solution of the crude product from the previous step (20.5 g) in DCM (150 mL) at room temperature.The solution was stirred at r.t. until complete conversion of the aldehyde, which was monitored by 1 H NMR. The molecular sieves were removed, and the solvent was evaporated in vacuo to obtain a colourless oil.
MeOH (175 mL) was added and the solution was cooled down to 0 ºC before sodium borohydride (3.06 g, 81.0 mmol) was added.The reaction mixture was stirred at r.t. until the disappearance of the imine intermediate, as monitored by 1 H NMR. The solvent was removed in vacuo and the residue was dissolved in 1 M NaOH solution (500 mL).The solution was extracted with DCM (3x).The organic phase was dried over MgSO4 and then the solvent was removed in vacuo to yield the crude product.The crude was purified by flash chromatography (SiO2, 0-10% gradient of MeOH in DCM) to yield the pure product 3 as a pale-yellow oil (24.83 g, 63.4 mmol, 86% over three steps).-1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 δ / ppm
The solution was stirred at -10 °C for 1 h before the solvent was removed in vacuo and the residue was dissolved in EtOAc.The solution was washed with 0.1 M HCl (3x) and brine before the organic phase was dried over MgSO4, then the solvent was removed in vacuo to yield the crude product.The crude was purified by flash chromatography (SiO2, 0-20% gradient of EtOAc in 40-60 petroleum ether) to yield the pure product 4 as a pale-yellow oil (22.76 g, 42.2 mmol, 87%).

Synthesis of 5
A solution of di-tert-butyl(chloro)phosphane (25 mL, 131.6 mmol), 37% aqueous formaldehyde (240 mL) and conc.HCl soln.(240 mL) was heated to 100 °C and refluxed overnight.The solution was cooled to 0 °C before being neutralised with NaOH (115 g) and NaHCO3 (40 g), then extracted with DCM (3x).The combined organic phase was washed with brine before being dried over MgSO4, then the solvent was removed in vacuo to yield the crude product.The crude was purified by recrystallisation with toluene to yield the pure product 5 as a white solid (16.82 g, 87.5 mmol, 66%).

Synthesis of 6
To a solution of 5 (16.82 g, 87.5 mmol) and triethylamine (18.3 mL, 13.3 g, 131.1 mmol) in dry DCM (200 mL) at 0 °C was added mesyl chloride (10.1 mL, 15.0 g, 131.1 mmol) dropwise under N2.The reaction mixture was stirred at r.t.overnight, after which it was diluted with DCM (200 mL), washed with water (2x) and brine and then dried over MgSO4.The solvent was removed in vacuo, and the crude was used in the next step without further purification.
The crude product obtained in the previous step was dissolved in isobutylamine (52.1 mL, 38.4 g, 524.4 mmol) and the reaction mixture was stirred at 120 °C in a microwave reactor for 2 h.The excess of isobutylamine was removed in vacuo and the residue was dissolved in EtOAc.The solution was washed with a sat.solution of Na2CO3 (3x) and then dried over MgSO4 before the solvent was removed in vacuo to yield the product 6 as a yellow oil (20.7 g, 83.7 mmol, 96% over two steps).
The crude product was used in the next step without further purification.The spectroscopic data matches that previously reported in the literature at: Troselj, P., Bolgar, P., Ballester, P., Hunter, C.A.

Synthesis of 8
To a solution of 5 (1.55 g, 8.06 mmol) and triethylamine (1.68 mL, 1.22 g, 12.1 mmol) in dry DCM (20 mL) at 0 °C was added mesyl chloride (0.99 mL, 1.39 g, 12.1 mmol) dropwise under N2.The reaction mixture was stirred at r.t.overnight, after which it was diluted with DCM (20 mL), washed with water (2x) and brine and then dried over MgSO4.The solvent was removed in vacuo, and the crude was used in the next step without further purification.
The crude product obtained in the previous step was dissolved in 2-ethylhexylamine (9.12 mL, 7.19 g, 55.6 mmol) and the reaction mixture was stirred at 120 °C in a microwave for 2 h.The residue was dissolved in EtOAc and the resultant solution was washed with sat.solution of Na2CO3 (3x) then dried over MgSO4 before the solvent was removed in vacuo to yield the crude product.The excess 2-ethylhexylamine was removed by flash chromatography (SiO2, 0-10% gradient of MeOH in DCM) to yield the product 8 as a yellow oil (2.31 g).
The product contained some impurities and was used in the next step without further purification.Figure S13: 13 C NMR (101 MHz, chloroform-d) spectrum of 9.

Synthesis of 10
A solution of 1-Boc-4-bromopiperidine (400 mg, 1.51 mmol) and sodium azide (197 mg, 3.03 mmol) in DMF (5 mL) was stirred overnight at 60 °C under N2 atmosphere.The mixture was diluted with water (20 mL) then extracted with EtOAc (3x).The combined organic layers were washed with water (3x), 5% LiCl soln.(3x) and brine.The organic phase was dried over MgSO4 and then the solvent was removed in vacuo to obtain a colourless oil.
The obtained product was dissolved in DCM (6 mL) before TFA (2 mL) was added and the mixture was stirred at r.t. for 30 mins.The solvent and reagent were removed by flushing the mixture under N2 to obtain a colourless oil.
To a solution of cyanuric chloride (419 mg, 2.27 mmol) in THF (40 mL) at -78 °C was added dropwise a solution of the crude obtained in the previous step and DIPEA (1.05 mL, 781 mg, 6.04 mmol) in THF (10 mL).The solution was stirred at -78 °C for 1 h.The solvent was removed in vacuo and the residue was dissolved in EtOAc.The solution was washed with 0.1 M HCl (3x) and brine before the organic phase was dried over MgSO4, then the solvent was removed in vacuo to yield the crude product.The crude was purified by flash chromatography (SiO2, 0-20% gradient of EtOAc in 40-60 petroleum ether) to yield the pure product 10 as a white solid (316 mg, 1.15 mmol, 76% over three steps).

Synthesis of 11
To a solution of 1-Boc-4-ethynylpiperidine (200 mg, 0.96 mmol) in DCM (1 mL) was added TFA (3 mL) before the mixture was stirred at r.t. for 30 mins.The solvent and reagent were removed by flushing the mixture under N2 to obtain a colourless oil.
To a solution of cyanuric chloride (264 mg, 1.43 mmol) in THF (40 mL) at -78 °C was added dropwise a solution of the crude obtained in the previous step and DIPEA (0.33 mL, 247 mg, 1.91 mmol) in THF (10 mL).The solution was stirred at -78 °C for 1 h.The solvent was removed in vacuo and the residue was dissolved in EtOAc.The solution was washed with 0.1 M HCl (3x) and brine before the organic phase was dried over MgSO4, then the solvent was removed in vacuo to yield the crude product.The crude was purified by flash chromatography (SiO2, 0-20% gradient of EtOAc in 40-60 petroleum ether) to yield the pure product 11 as a white solid (238 mg, 0.93 mmol, 97% over two steps).-1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 δ / ppm

Synthesis of 12
To a solution of 3 (2.00 g, 5.11 mmol) in DCM (20 mL) at 0 °C was added Fmoc chloride (1.45 g, 5.62 mmol), then triethylamine (0.79 mL, 0.57 g, 5.62 mmol) was added dropwise.The reaction was stirred for 2 h at r.t.before the mixture was washed with 1 M K2CO3 solution and brine.The organic phase was dried over MgSO4 and then the solvent was removed in vacuo to yield the crude product as a yellow oil.The crude was purified by flash chromatography (SiO2, 0-10% gradient of EtOAc in 40-60 petroleum ether) to yield the product 12 (3.04g, 4.95 mmol, 97%) as a colourless oil.
The NMR spectra are consistent with the presence of two slowly exchanging rotamers in solution.Where rotamers are distinguishable, the corresponding peaks are listed together.-1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 δ / ppm 12 (3.00g,4.89 mmol) was dissolved in THF (40 mL) and the solution was acidified to pH 3 by adding 1 M AcOH.TBAF (9.78 mL, 9.78 mmol, 1 M in THF) was added dropwise and the reaction mixture was stirred at r.t. until complete conversion of the starting material, as monitored by LCMS (approx. 2 h).After completion, the reaction was quenched with 5% aq.soln.HCl and extracted with EtOAc (3x) followed by washing with 5% aq.soln.HCl (3x) and brine.The organic phase was dried over MgSO4 and the solvent was removed in vacuo to yield the crude product.The crude was purified by flash chromatography (SiO2, 0-40% gradient of EtOAc in 40-60 petroleum ether) to yield the pure product 13 (1.85 g, 4.05 mmol, 83%) as a colourless foam.
The reaction was stirred for 2 h at r.t.before the mixture was washed with 1 M K2CO3 solution and brine.The organic phase was dried over MgSO4 and then the solvent was removed in vacuo to yield the crude product as a yellow oil.The crude was purified by flash chromatography (SiO2, 0-20% gradient of EtOAc in 40-60 petroleum ether) to yield the product 14 (1.16 g, 2.08 mmol, 97%) as a colourless oil.
Quantification of resin loading: Functionalised Wang resin was treated with a solution of DBU in DMF (2 mL, 2 vol.%) and agitated for 30 mins. 1 mL of the solution was removed from the resin and diluted with 4 mL acetonitrile.A 1 mL aliquot of the resultant solution was taken and diluted to 12.5 mL with acetonitrile.The absorbance of the DBUfulvene adduct (λ = 304 nm, ε = 9254 M -1 cm -1 ) was measured to estimate the resin loading (0.36 mmol g -1 ).

Fmoc deprotection of resin:
The functionalised resin was swollen with DCM for 30 mins then washed with DMF (5x) before being treated with a solution of piperidine in DMF ( 2x 5mL, 20 vol.%) and agitated for 10 mins.The resin was then washed alternatingly with DMF (5x) and DCM (5x).Quantification of resin loading: Functionalised Wang Resin was treated with a solution of DBU in DMF (2 mL, 2 vol.%) and agitated for 30 min. 1 mL of the solution was removed from the resin and diluted with 4 mL acetonitrile.A 1 mL aliquot of the resultant solution was taken and diluted to 12.5 mL with acetonitrile.The absorbance of the DBU-fulvene adduct (λ = 304 nm, ε = 9254 M -1 cm -1 ) was measured to estimate the resin loading (0.14 mmol g -1 ).HPLC Purification: Analytical reverse-phase HPLC was performed on an Agilent HP-1100

General methods using CEM Liberty
Series HPLC system composed of a high-pressure binary pump, an autosampler with injector programming capabilities and a diode array detector with a semimicro flow cell (6 mm path length, 5 µL volume
for Optimised Automated Solid-Phase SynthesisTentaGel Wang Resin (90 µm mesh) (3.00 g, 0.72 mmol based on advertised loading) was swollen in dry DCM for 30 min before a solution of 15 (0.578 g, 1.44 mmol) and triphenylphosphine (0.377g, 1.44 mmol) in dry DCM was added to the resin.A solution of diisopropyl azodicarboxylate (0.284 mL, 0.291 g, 1.44 mmol) was diluted 5-fold in dry DCM then added dropwise to the resin.The resin was agitated at r.t.overnight, and then washed alternatingly with DCM (5x) and DMF (5x) to yield Resin 2.
Blue Automated Synthesiser: Heated automated Solid-Phase Organic Synthesis (SPS) was performed on a CEM Liberty Blue automated synthesizer on a 50 µmol scale.Solutions of piperazine (0.7 M) in DMF, 2 (0.125 M) in DMF, 7 (0.125 M) in DMF, 10 (0.125 M) in DMF and DIPEA (0.5 M) in DMF were prepared for coupling.General synthetic protocols performed were:Fmoc deprotection: The loaded Wang resin was agitated in a solution of piperazine in DMF (7 mL, 0.7 M, 2 x 10 min).The deprotection solution was then drained and the resin was washed with DMF (4 x 5 mL).Coupling cycle:The resin-bound oligomer was first agitated in a solution of 2, 7 or 10 (0.1 M, 10 eq.) and DIPEA (0.1 M, 10 eq.) in DMF (5 mL) for 10 or 15 mins at 90 °C.The 1 st coupling solution was drained, and the resin was washed with DMF (4 x 5 mL).The resinbound oligomer was then agitated in a solution of piperazine (5 mL, 0.7 M) in DMF for 10 or 90 °C.The 2 nd coupling solution was drained, and the resin was washed with DMF (4 x 5 mL).4-Ethynylpiperidine coupling (manual MW-assisted SPS):The resin-bound oligomer was swollen in DMF for 15 min before being agitated in a solution of 4-ethynylpiperidine trifluoroacetate (0.1 M) and DIPEA (1 M) in DMF (5 mL) for 30 mins at 90 °C.The coupling solution was drained, and the resin washed with DMF (4 x 5 mL).TIPS deprotection:The resin-bound oligomer was swollen in THF for 15 min before being agitated in a solution of TBAF (5 mL, 1 M in THF) at r.t. for 1 h.The solution was drained, and the resin washed with MeOH and THF alternatingly (4 x 5 mL) before being subjected to another deprotection cycle.Resin Cleavage:The resin was agitated in a mixture of TFA:TIS:DCM (90:5:5 v/v/v) at r.t. for 2 h.The resin was filtered and washed with DCM (5 x 5 mL) and then subjected to another cleavage cycle.The combined filtrates from both cleavage cycles were concentrated under N2 flow before drying in vacuo.