Supramolecular catalysis by recognition-encoded oligomers: discovery of a synthetic imine polymerase†

All key chemical transformations in biology are catalysed by linear oligomers. Catalytic properties could be programmed into synthetic oligomers in the same way as they are programmed into proteins, and an example of the discovery of emergent catalytic properties in a synthetic oligomer is reported. Dynamic combinatorial chemistry experiments designed to study the templating of a recognition-encoded oligomer by the complementary sequence have uncovered an unexpected imine polymerase activity. Libraries of equilibrating imines were formed by coupling diamine linkers with monomer building blocks composed of dialdehydes functionalised with either a trifluoromethyl phenol (D) or phosphine oxide (A) H-bond recognition unit. However, addition of the AAA trimer to a mixture of the phenol dialdehyde and the diamine linker did not template the formation of the DDD oligo-imine. Instead, AAA was found to be a catalyst, leading to rapid formation of long oligomers of D. AAA catalysed a number of different imine formation reactions, but a complementary phenol recognition group on the aldehyde reaction partner is an essential requirement. Competitive inhibition by an unreactive phenol confirmed the role of H-bonding in substrate recognition. AAA accelerates the rate of imine formation in toluene by a factor of 20. The kinetic parameters for this enzyme-like catalysis are estimated as 1 × 10−3 s−1 for kcat and the dissociation constant for substrate binding is 300 μM. The corresponding DDD trimer was found to catalyse oligomerisation the phosphine oxide dialdehyde with the diamine linker, suggesting an important role for the backbone in catalysis. This unexpected imine polymerase activity in a duplex-forming synthetic oligomer suggests that there are many interesting processes to be discovered in the chemistry of synthetic recognition-encoded oligomers that will parallel those found in natural biopolymers.


Experimental Procedures
The monomers and the oligomers used in this work were synthesised following the synthetic procedure that we recently reported. 1 The reagents and materials used in the synthesis were bought from commercial sources, without prior purification. Thin layer chromatography was carried out using with silica gel 60F (Merck) on aluminium. Flash chromatography was carried out on an automated system (Combiflash Companion, Combiflash Rf+ or Combiflash Rf Lumen) using prepacked cartridges of silica (25μ or 50μ PuriFlash® Columns). All NMR spectroscopy was carried out on a Bruker AVI250, AVI400, DPX400, AVIII400 spectrometer using the residual solvent as the internal standard. All chemical shifts (δ) are quoted in ppm and coupling constants given in Hz.
HPLC analysis were performed using a modular Agilent 1200 Series HPLC system composed of a HPLC high pressure binary pump, autosampler with injector programming capabilities, Peltier type column oven with 6 µL heat exchanger and a Diode Array Detector with a semi-micro flow cell (1.6uL, 6mm pathlength) to reduce peak dispersion when using short columns as in this case. The flow-path was connected using 0.12 mm ID stainless steel tubing to minimize peak dispersion. For the analysis of system ii, an HPLC method described in Section 3 was used.

NMR spectroscopy studies
All NMR kinetic experiments were performed using a Bruker 500 MHz AVIII HD Smart Probe spectrometers. Stock solutions of the selected monomers were freshly prepared in toluene-d8 at a known concentration. A known volume of aldehyde monomer and the monomeric or oligomeric complementary units were added to an NMR tube. Finally, a known volume of the aniline solution was added to the NMR tube, which was quickly sealed and vigorously shaken, before starting the spectra acquisition.  Figure S2. a) Kinetics of imine formation measured by integration of all imine and aldehyde signals in the 500 MHz 1 H NMR spectra (D 10 mM, N 20 mM, nBu3PO 10 mM). Total aldehyde signals (Ald tot, 10.51-10.26 ppm) and total imine (Imin tot, 9.25-8.92 ppm), together with monoimine (Imin 1, 8.97-8.93 ppm), monomeric bis imine (Imin 2, 9.10-9.00 ppm) and oligomeric imines (Imin oliogo, 9.22-9.12 ppm) are integrated and reported. b) Chemical structure of the formed imines.  S6 Figure S3. 500 MHz 1 H NMR spectra of a mixture of N (20 mM) and D (10 mM) in toluene-d8 in the presence of 5 mM AA. On the right side of the figure is reported the time (in minutes) after the addition of N. Figure S4. Kinetics of imine formation measured by integration of all imine and aldehyde signals in the 500 MHz 1 H NMR spectra (D 10 mM, N 20 mM, AA 5 mM). Total aldehyde signals (Ald tot, 10.51-10.26 ppm) and total imine (Imin tot, 9.25-8.92 ppm), together with monoimine (Imin 1, 8.97-8.93 ppm), monomeric bis imine (Imin 2, 9.10-9.00 ppm) and oligomeric imines (Imin oliogo, 9.22-9.12 ppm) are integrated and reported.  Figure S6. Kinetics of imine formation measured by integration of all imine and aldehyde signals in the 500 MHz 1 H NMR spectra (D 10 mM, N 20 mM, AAA 3.3 mM). Total aldehyde signals (Ald tot, 10.51-10.26 ppm) and total imine (Imin tot, 9.25-8.92 ppm), together with monoimine (Imin 1, 8.97-8.93 ppm), monomeric bis imine (Imin 2, 9.10-9.00 ppm) and oligomeric imines (Imin oliogo, 9.22-9.12 ppm) are integrated and reported. Peak Area (AU)

HPLC-MS studies
The chemical composition of the imine libraries was analysed by reverse phase HPLC using an HPLC samples were prepared using Jaytee vials screw clear 1.5 mL, using 0.2 mL (31x6 mm) VWR micro insert. For every experiment the last addition was a toluene solution of the required aniline, then the vial was closed, vigorously shaken and then immediately injected.
Imine hydrolysis on the HPLC column is minimal, as evidenced by the following observations: -imines are directly observed by mass spec of the HPLC peaks -comparison of Figures S6 and S79 shows that the rate of imine formation measured by NMR is similar to that measured by HPLC -the HPLC data in Figure 5 of the main text shows some aldehyde peaks, because it was recorded after 5 minutes, when imine formation is not complete. The HPLC data recorded after an hour in Figure S79 shows almost no aldehyde end groups, similar to what was observed directly by NMR.        Peak area percentage for reactions in the presence of AA (5 mM, red) and AAA (3.3 mM, green), relative to the corresponding peak area percentage in the presence of nBu3PO (10 mM). Figure S82. Total HPLC peak area as a function of the reaction time for a mixture of N (16 mM) and D (10 mM) in toluene, in the presence of nBu3PO (10 mM, blue circles) or AAA (3.3 mM, green circles). The decrease observed after 100 min for the AAA experiment is due to visible precipitation, presumably of longer oligomers (cf NMR experiments in Figure SS13).