Current approaches to reaction discovery focus on one particular transformation. Typically, researchers choose substrates based on their predicted ability to serve as precursors for the target structure, then evaluate reaction conditions for their ability to effect product formation. This approach is ideal for addressing specific reactivity problems, but its focused nature might leave many areas of chemical reactivity unexplored. Here we report a reaction discovery approach that uses DNA-templated organic synthesis and in vitro selection to simultaneously evaluate many combinations of different substrates for bond-forming reactions in a single solution. Watson-Crick base pairing controls the effective molarities of substrates tethered to DNA strands; bond-forming substrate combinations are then revealed using in vitro selection for bond formation, PCR amplification and DNA microarray analysis. Using this approach, we discovered an efficient and mild carbon-carbon bond-forming reaction that generates an enone from an alkyne and alkene using an inorganic palladium catalyst. Although this approach is restricted to conditions and catalysts that are at least partially compatible with DNA, we expect that its versatility and efficiency will enable the discovery of additional reactions between a wide range of substrates.

DNA-templated organic synthesis enables the translation of DNA sequences into synthetic small-molecule libraries suitable for in vitro selection. Previously, we described the DNA-templated multistep synthesis of a 13 824-membered small-molecule macrocycle library. Here, we report the discovery of small molecules that modulate the activity of kinase enzymes through the in vitro selection of this DNA-templated small-molecule macrocycle library against 36 biomedically relevant protein targets. DNA encoding selection survivors was amplified by PCR and identified by ultra-high-throughput DNA sequencing. Macrocycles corresponding to DNA sequences enriched upon selection against several protein kinases were synthesized on a multimilligram scale. In vitro assays revealed that these macrocycles inhibit (or activate) the kinases against which they were selected with IC50 values as low as 680 nM. We characterized in depth a family of macrocycles enriched upon selection against Src kinase, and showed that inhibition was highly dependent on the identity of macrocycle building blocks as well as on backbone conformation. Two macrocycles in this family exhibited unusually strong Src inhibition selectivity even among kinases closely related to Src. One macrocycle was found to activate, rather than inhibit, its target kinase, VEGFR2. Taken together, these results establish the use of DNA-templated synthesis and in vitro selection to discover small molecules that modulate enzyme activities, and also reveal a new scaffold for selective ATP-competitive kinase inhibition.

A judicious choice of photoreactive group is critical in successful photoaffinity labeling studies of small molecule-protein interactions. A set of carbohydrate-based photoaffinity probes was prepared to compare the effects of three major photoreactive groups on the efficiency and selectivity of crosslinking a binding protein with low affinity. We showed that, despite the low crosslinking yield, the diazirine probe displayed the high ligand-dependent reactivity consistent with the ideal mechanism of photoaffinity labeling. Moreover, we demonstrated that, among the three photoreactive groups, only the diazirine probe achieved highly selective crosslinking of a low-affinity binding protein in cell lysate.

FT-NIR spectra have been measured for various polypeptides and proteins with different secondary structures to find an NIR marker band for the structure of the proteins and polypeptides. Their FT-IR spectra have also been obtained to assist in the interpretation of the FT-NIR spectra. Comparison between the FT-NIR and FT-IR spectra shows that there is a clear correlation between the frequency of an NIR band near 4855 cm−1, assignable to a combination of amide A and amide II, and that of an IR band near 3300 cm−1 due to amide A for the polypeptides investigated. Therefore, the NIR band (hereafter, we identify it as amide A/II) may be used as a practical indicator for the strength of hydrogen bonds in the amide groups, as in the case of amide A. The frequency of amide A/II changes little with the secondary structure for both the polypeptides and proteins, and thus it is rather difficult to use this band as a marker band of the secondary structure. For the globular proteins such as hemoglobin, albumin, and lysozyme, irrespective of their secondary structure, both amide A/II and amide A appear in similar positions. However, collagen (Type I, Type IV, and Type V) gives them much higher frequencies, suggesting that the hydrogen bonds in collagen are much weaker than those in typical globular proteins. The amide A/II band shows a significant upward shift upon the thermal denaturation of pepsin. This observation indicates that amide A/II can also be used for monitoring the destruction of the hydrogen bonds in the amides induced by the denaturation.