Discovering pathways through ribozyme fitness landscapes using information theoretic quantification of epistasis.

The identification of catalytic RNAs is typically achieved through primarily experimental means. However, only a small fraction of sequence space can be analyzed even with high-throughput techniques. Methods to extrapolate from a limited data set to predict additional ribozyme sequences, particularly in a human-interpretable fashion, could be useful both for designing new functional RNAs and for generating greater understanding about a ribozyme fitness landscape. Using information theory, we express the effects of epistasis (i.e., deviations from additivity) on a ribozyme. This representation was incorporated into a simple model of the epistatic fitness landscape, which identified potentially exploitable combinations of mutations. We used this model to theoretically predict mutants of high activity for a self-aminoacylating ribozyme, identifying potentially active triple and quadruple mutants beyond the experimental data set of single and double mutants. The predictions were validated experimentally, with nine out of nine sequences being accurately predicted to have high activity. This set of sequences included mutants that form a previously unknown evolutionary "bridge" between two ribozyme families that share a common motif. Individual steps in the method could be examined, understood, and guided by a human, combining interpretability and performance in a simple model to predict ribozyme sequences by extrapolation.

Rapid planning and analysis of high-throughput experiment arrays for reaction discovery.

High-throughput experimentation (HTE) is an increasingly important tool in reaction discovery. While the hardware for running HTE in the chemical laboratory has evolved significantly in recent years, there remains a need for software solutions to navigate data-rich experiments. Here we have developed phactor™, a software that facilitates the performance and analysis of HTE in a chemical laboratory. phactor™ allows experimentalists to rapidly design arrays of chemical reactions or direct-to-biology experiments in 24, 96, 384, or 1,536 wellplates. Users can access online reagent data, such as a chemical inventory, to virtually populate wells with experiments and produce instructions to perform the reaction array manually, or with the assistance of a liquid handling robot. After completion of the reaction array, analytical results can be uploaded for facile evaluation, and to guide the next series of experiments. All chemical data, metadata, and results are stored in machine-readable formats that are readily translatable to various software. We also demonstrate the use of phactor™ in the discovery of several chemistries, including the identification of a low micromolar inhibitor of the SARS-CoV-2 main protease. Furthermore, phactor™ has been made available for free academic use in 24- and 96-well formats via an online interface.

Emergent properties as by-products of prebiotic evolution of aminoacylation ribozymes.

Systems of catalytic RNAs presumably gave rise to important evolutionary innovations, such as the genetic code. Such systems may exhibit particular tolerance to errors (error minimization) as well as coding specificity. While often assumed to result from natural selection, error minimization may instead be an emergent by-product. In an RNA world, a system of self-aminoacylating ribozymes could enforce the mapping of amino acids to anticodons. We measured the activity of thousands of ribozyme mutants on alternative substrates (activated analogs for tryptophan, phenylalanine, leucine, isoleucine, valine, and methionine). Related ribozymes exhibited shared preferences for substrates, indicating that adoption of additional amino acids by existing ribozymes would itself lead to error minimization. Furthermore, ribozyme activity was positively correlated with specificity, indicating that selection for increased activity would also lead to increased specificity. These results demonstrate that by-products of ribozyme evolution could lead to adaptive value in specificity and error tolerance.

Development of copper-catalyzed deaminative esterification using high-throughput experimentation.

Repurposing of amine and carboxylic acid building blocks provides an enormous opportunity to expand the accessible chemical space, because amine and acid feedstocks are typically low cost and available in high diversity. Herein, we report a copper-catalyzed deaminative esterification based on C-N activation of aryl amines via diazonium salt formation. The reaction was specifically designed to complement the popular amide coupling reaction. A chemoinformatic analysis of commercial building blocks demonstrates that by utilizing aryl amines, our method nearly doubles the available esterification chemical space compared to classic Fischer esterification with phenols. High-throughput experimentation in microliter reaction droplets was used to develop the reaction, along with classic scope studies, both of which demonstrated robust performance against hundreds of substrate pairs. Furthermore, we have demonstrated that this new esterification is suitable for late-stage diversification and for building-block repurposing to expand chemical space.

Reinforcing the supply chain of umifenovir and other antiviral drugs with retrosynthetic software.

The global disruption caused by the 2020 coronavirus pandemic stressed the supply chain of many products, including pharmaceuticals. Multiple drug repurposing studies for COVID-19 are now underway. If a winning therapeutic emerges, it is unlikely that the existing inventory of the medicine, or even the chemical raw materials needed to synthesize it, will be available in the quantities required. Here, we utilize retrosynthetic software to arrive at alternate chemical supply chains for the antiviral drug umifenovir, as well as eleven other antiviral and anti-inflammatory drugs. We have experimentally validated four routes to umifenovir and one route to bromhexine. In one route to umifenovir the software invokes conversion of six C-H bonds into C-C bonds or functional groups. The strategy we apply of excluding known starting materials from search results can be used to identify distinct starting materials, for instance to relieve stress on existing supply chains.

Ultrahigh-Throughput Experimentation for Information-Rich Chemical Synthesis.

The incorporation of data science is revolutionizing organic chemistry. It is becoming increasingly possible to predict reaction outcomes with accuracy, computationally plan new retrosynthetic routes to complex molecules, and design molecules with sophisticated functions. Critical to these developments has been statistical analysis of reaction data, for instance with machine learning, yet there is very little reaction data available upon which to build models. Reaction data can be mined from the literature, but experimental data tends to be reported in a text format that is difficult for computers to read. Compounding the issue, literature data are heavily biased toward "productive" reactions, and few "negative" reaction data points are reported even though they are critical for training of statistical models. High-throughput experimentation (HTE) has evolved over the past few decades as a tool for experimental reaction development. The beauty of HTE is that reactions are run in a systematic format, so data points are internally consistent, the reaction data are reported whether the desired product is observed or not, and automation may reduce the occurrence of false positive or negative data points. Additionally, experimental workflows for HTE lead to datasets with reaction metadata that are captured in a machine-readable format. We believe that HTE will play an increasingly important role in the data revolution of chemical synthesis. This Account details the miniaturization of synthetic chemistry culminating in ultrahigh-throughput experimentation (ultraHTE), wherein reactions are run in ∼1 µL droplets inside of 1536-well microtiter plates to minimize the use of starting materials while maximizing the output of experimental information. The performance of ultraHTE in 1536-well microtiter plates has led to an explosion of available reaction data, which have been used to identify specific substrate-catalyst pairs for maximal efficiency in novel cross-coupling reactions. The first iteration of ultraHTE focused on the use of dimethyl sulfoxide (DMSO) as a high-boiling solvent that is compatible with the plastics most commonly used in consumable well plates, which generated homogeneous reaction mixtures that are perfect for use with nanoliter-dosing liquid handling robotics. In this way, DMSO enabled diverse reagents to be arrayed in ∼1 µL droplets. Reactions were run at room temperature with no agitation and could be scaled up from the ∼0.05 mg reaction scale to the 1 g scale. Engineering enhancements enabled the use of ultraHTE with diverse and semivolatile solvents, photoredox catalysis, heating, and acoustic agitation. A main driver in the development of ultraHTE was the recognition of the opportunity for a direct merger between miniaturized reactions and biochemical assays. Indeed, a strategy was developed to feed ultraHTE reaction mixtures directly to a mass-spectrometry-based affinity selection bioassay. Thus, micrograms of starting materials could be used in the synthesis and direct biochemical testing of drug-like molecules. Reactions were performed at a reactant concentration of ∼0.1 M in an inert atmosphere, enabling even challenging transition-metal-catalyzed reactions to be used. Software to enable the workflow was developed. We recently initiated the mapping of reaction space, dreaming of a future where transformations, reaction conditions, structure, properties and function are studied in a systems chemistry approach.

Kinetic sequencing (k-Seq) as a massively parallel assay for ribozyme kinetics: utility and critical parameters.

Characterizing genotype-phenotype relationships of biomolecules (e.g. ribozymes) requires accurate ways to measure activity for a large set of molecules. Kinetic measurement using high-throughput sequencing (e.g. k-Seq) is an emerging assay applicable in various domains that potentially scales up measurement throughput to over 106 unique nucleic acid sequences. However, maximizing the return of such assays requires understanding the technical challenges introduced by sequence heterogeneity and DNA sequencing. We characterized the k-Seq method in terms of model identifiability, effects of sequencing error, accuracy and precision using simulated datasets and experimental data from a variant pool constructed from previously identified ribozymes. Relative abundance, kinetic coefficients, and measurement noise were found to affect the measurement of each sequence. We introduced bootstrapping to robustly quantify the uncertainty in estimating model parameters and proposed interpretable metrics to quantify model identifiability. These efforts enabled the rigorous reporting of data quality for individual sequences in k-Seq experiments. Here we present detailed protocols, define critical experimental factors, and identify general guidelines to maximize the number of sequences and their measurement accuracy from k-Seq data. Analogous practices could be applied to improve the rigor of other sequencing-based assays.

Microbial predictors of healing and short-term effect of debridement on the microbiome of chronic wounds.

Chronic wounds represent a large and growing disease burden. Infection and biofilm formation are two of the leading impediments of wound healing, suggesting an important role for the microbiome of these wounds. Debridement is a common and effective treatment for chronic wounds. We analyzed the bacterial content of the wound surface from 20 outpatients with chronic wounds before and immediately after debridement, as well as healthy skin. Given the large variation observed among different wounds, we introduce a Bayesian statistical method that models patient-to-patient variability and identify several genera that were significantly enriched in wounds vs. healthy skin. We found no difference between the microbiome of the original wound surface and that exposed by a single episode of sharp debridement, suggesting that this debridement did not directly alter the wound microbiome. However, we found that aerobes and especially facultative anaerobes were significantly associated with wounds that did not heal within 6 months. The facultative anaerobic genus Enterobacter was significantly associated with lack of healing. The results suggest that an abundance of facultative anaerobes is a negative prognostic factor in the chronic wound microbiome, possibly due to the increased robustness of such communities to different metabolic environments.

A map of the amine-carboxylic acid coupling system.

Chemical transformations determine the structure of a product, and therefore its properties, which in turn affect complex macroscopic functions such as the metabolic stability of pharmaceuticals or the volatility of perfumes. Therefore, reaction selection can influence the success or failure of a candidate molecule to meet a functional objective. The coupling of an amine with a carboxylic acid to form an amide bond is the most popular chemical reaction used for drug discovery1. However, there are many other ways to connect these two common functional groups together. Here we show computationally that amines and acids can couple via hundreds of hypothetical yet plausible transformations, and we demonstrate experimentally the application of a dozen such reactions. To investigate the contribution of chemical transformations to properties, we developed a string-based notation and used an enumerative combinatorics approach to produce a map of conceivable amine-acid coupling transformations, which can be charted using chemoinformatic techniques. We find that critical physicochemical parameters of the products, such as partition coefficient and polar surface area, vary considerably depending on the transformation chosen. Data mining the amine-acid coupling system produced here should enable reaction discovery, which we demonstrate by developing an esterification reaction found within the mapped space. Complex molecules with distinct property profiles can also be discovered within the amine-acid coupling system, as we show here via the late-stage diversification of drugs and natural products.

Palladium-Catalyzed Direct C(sp2)-H ortho-Arylation of Anilides Using 2-Aminophenylpyrazole as the Directing Group.

Palladium-catalyzed ortho-arylation of anilides was achieved using 2-aminophenyl-1H-pyrazole (2-APP) as a new directing group. Using Pd(OAc)2 as the catalyst and AgO as the promoter, mono- and diarylation of anilides were realized in up to 89% isolated yield. Further manipulation of the arylation product may be accomplished by a 2-step sequence involving an acidic hydrolysis of the methylated amide. More interestingly, in the presence of K2CO3, tandem C-C/C-N cyclization products were obtained for a couple of substrates.

Helically Chiral Peptides That Contain Ferrocene-1,1'-diamine Scaffolds as a Turn Inducer.

A series of peptides that contain homo- and heterochiral Ala-Pro sequences attached to the turn-inducing ferrocene-1,1'-diamine scaffold were synthesized. The effects of the backbone chirality and the N-terminal group (Boc/Ac) on the conformational properties of the novel peptidomimetics were thoroughly explored by IR, NMR, and CD spectroscopy and the experimental observations were corroborated by DFT studies in solution. The most stable conformers of the homochiral peptides adopted the interstrand hydrogen-bond patterns, realized through ten- and thirteen-membered rings. The common feature of the most stable conformers of the heterochiral peptides was the adoption of the turn-like structures that feature the simultaneous intra- (seven-membered) and interstrand (sixteen-membered) hydrogen-bonded rings. An exchange of two N-terminal groups had a somewhat larger influence on the distribution of the hydrogen-bond patterns in homochiral than in heterochiral derivatives. The homochiral peptides that contain pyridine moieties as metal coordination sites formed 1:1 complexes with divalent metal ions, which included Zn2+ , Cd2+ , Cu2+ and Fe2+ .

An ultrasensitive fluorescent probe for rapid determination of thiophenols.

An ultrasensitive fluorescent probe TPP was developed for the highly selective detection of thiophenols based on excited-state intramolecular proton transfer (ESIPT) mechanism, its synthesis through a simple straightforward combination of HBT fluorophore with 2,4-dinitrobenzene functional group. A kinetic study of TPP towards thiophenol displayed a fast response time (~150s) and significant turn-on fluorescence enhancement (~60 fold). Selective and competitive experiments exhibited an excellent selectivity of TPP toward thiophenols over biothiols (Cys, GSH) and other aliphatic thiols or nucleophiles. Using this ultrasensitive probe (LOD, 1.05×10-8M), we have successfully monitored and quantified highly toxic thiophenols in aqueous media and real-water samples.

Achieving High Drug Loading and Sustained Release of Hydrophobic Drugs in Hydrogels through In Situ Crystallization.

Inadequate drug loading of hydrophobic drugs is a classic problem when hydrogels are utilized as sustained-release carriers of drugs. Herein, a strategy to load plenty of hydrophobic drugs is presented. The antitumor drug 10-hydroxycamptothecin in the thermogel of poly(d,l-lactic acid-co-glycolic acid)-b-poly(ethylene glycol)-b-poly(d,l-lactic acid-co-glycolic acid) is employed. The drug is soluble in an alkaline medium, yet insoluble in a neutral/acidic medium. The crystallization is triggered after adding an alkaline drug solution into an acidic copolymer solution. The concentrated copolymer aqueous solution undergoes a sol-gel transition upon heating, faster than the crystallization. As a result, plenty of evenly dispersed drug microcrystals are formed. The in vitro and in vivo experiments indicate both high drug loading and sustained release with enhanced antitumor efficacy and reduced adverse effects. The system resolves the challenge in formulation of hydrophobic drugs in hydrogels, and is stimulating for encapsulating drugs with a soluble-insoluble transition into a material environment.

2-Aminophenyl-1H-pyrazole as a Removable Directing Group for Copper-Mediated C-H Amidation and Sulfonamidation.

2-Aminophenyl-1H-pyrazole was discovered as a removable bidentate directing group for copper-mediated aerobic oxidative C(sp(2)-H) bond amidation and sulfonamidation. When Cu(OAc)2 was employed as the copper source and 1,1,3,3-tetramethylguanidine as an organic base, the reaction, optimally carried out overnight in DMSO at 80 °C in open air, produced a variety of amides and sulfonamides in moderate to excellent yields. This directing group has proven to be particularly efficient in C-H sulfonamidation.

Imaging of living cells and zebrafish in vivo using a ratiometric fluorescent probe for hydrogen sulfide.

We have developed a novel colorimetric and ratiometric fluorescence probe for the selective and sensitive monitoring of hydrogen sulfide based on a dicyanoisophorone platform. An excellent linear relationship of fluorescence intensity ratio (I637/I558) (R(2) = 0.9867) versus hydrogen sulfide concentration in the range of 1-12 µM was obtained. This probe exhibited a remarkable fluorescence response to hydrogen sulfide over other physiological thiols or biological species, which fluoresces in the red region with a large Stokes shift (172 nm). This probe was successfully utilized to monitor H2S under in vitro physiological conditions and for imaging H2S in living cells and living zebrafish in vivo.