Andrew N. Bigley

The first two enzymes recognized to be PhoK-type phosphatases were from Sphingobium sp. TCM1 (Sb-PhoK) and Sphingomonas sp. BASR1 (Sm-PhoK) which were utilized in bioremediation of organophosphate flame-retardants and heavy metal contamination, respectively. The PhoK-type phosphatases are members of the nucleotide pyrophosphatase/phosphodiesterase (NPP) family of diesterases that have evolved a phosphatase activity. These enzymes were noted for very high activity with model compounds compared to other alkaline phosphatases, but very little was known about their substrate specificity or the activity with phosphomonoesters derived from flame-retardants or other environmental phosphoesters. Bioinformatics analysis has been utilized to identify PhoK homologues from a large group of Sphingomonadaceae family members including additional species that are known or suspected to utilize the organophosphate flame-retardants as nutrient sources. Nine homologues were selected for kinetic characterization using a synthesized library of organophosphate monoesters derived from flame-retardants, environmental phosphoesters, and biological monophosphates. The Sphingomonadaceae PhoK enzymes were found to have high enzymatic efficiency against a broad range of substrates. Against phenyl phosphate Sm-PhoK has a k _cat of 1100 s^–1 and a k _cat/K _m of 1.8 × 10⁶ M^–1 s^–1. The best overall activity was observed with the homologue from Sphingobium yanoikuyae (Sy-PhoK), another species known to degrade organophosphate flame-retardants. This enzyme hydrolyzed all tested substrates with an efficiency greater than 3 × 10⁴ M^–1 s^–1. The high catalytic activity and remarkably broad substrate specificity make the Sphingomonadaceae PhoK enzymes particularly suited for bioremediation as well as commercial applications where high turnover will be advantageous.

Nucleic acid nanoparticles (NANPs) fabricated by using the DNA origami method have broad utility in materials science and bioengineering. Their site-specific, heterovalent functionalization with secondary molecules such as proteins or fluorophores is a unique feature of this technology that drives its utility. Currently, however, there are few chemistries that enable fast, efficient covalent functionalization of NANPs with a broad conjugate scope and heterovalency. To address this need, we introduce synthetic methods to access inverse electron-demand Diels-Alder chemistry on NANPs. We demonstrate a broad conjugate scope, characterize application-relevant kinetics, and integrate this new chemistry with strain-promoted azide-alkyne cycloaddition chemistry to enable heterovalent click reactions on NANPs. We applied these chemistries to formulate a prototypical chemical countermeasure against chemical nerve agents. We envision this additional chemistry finding broad utility in the synthetic toolkit accessible to the nucleic acid nanotechnology community.

Organophosphate flame retardants have been widely used in plastic products since the early 2000s. Unfortunately, these compounds leach out of the plastics over time and are carcinogenic, developmental toxins, and endocrine disruptors. Due to the high usage levels and stable nature of the compounds, widespread contamination of the environment has now been observed. Despite their recent introduction into the environment, bacteria from the Sphingomonadaceae family have evolved a three-step hydrolytic pathway to utilize these compounds. The second step in this pathway in Sphingobium sp. TCM1 is catalyzed by Sb-PDE, which is a member of the polymerase and histidinol phosphatase (PHP) family of phosphatases. This enzyme is only the second case of a PHP-family enzyme capable of hydrolyzing phosphodiesters. Bioinformatics analysis has now been used to identify a second PHP diesterase from Novosphingobium sp. EMRT-2 (No-PDE). Kinetic characterization of Sb-PDE and No-PDE with authentic organophosphate flame-retardant diesters demonstrates that these enzymes are true diesterases with more than 1000-fold selectivity for the diesterase activity seen in some cases. Synthesis of a wide array of authentic flame-retardant diesters has allowed the substrate specificity of these enzymes to be determined, and mutagenic analysis of the active site residues has identified key residues that give rise to the high levels of diesterase activity. Despite high sequence identity, No-PDE is found to have a broader substrate specificity against flame-retardant derived diesters, and k_cat/K_m values greater than 10⁴ M^-1 s^-1 are seen with the best substrates.

The G-type nerve agents, sarin (GB), soman (GD), and cyclosarin (GF), are among the most toxic compounds known. Much progress has been made in evolving the enzyme phosphotriesterase (PTE) from Pseudomonas diminuta for the decontamination of the G-agents; however, the extreme toxicity of the G-agents makes the use of substrate analogues necessary. Typical analogues utilize a chromogenic leaving group to facilitate high-throughput screening, and substitution of an O-methyl for the P-methyl group found in the G-agents, in an effort to reduce toxicity. Till date, there has been no systematic evaluation of the effects of these substitutions on catalytic activity, and the presumed reduction in toxicity has not been tested. A series of 21 G-agent analogues, including all combinations of O-methyl, p-nitrophenyl, and thiophosphate substitutions, have been synthesized and evaluated for their ability to unveil the stereoselectivity and catalytic activity of PTE variants against the authentic G-type nerve agents. The potential toxicity of these analogues was evaluated by measuring the rate of inactivation of acetylcholinesterase (AChE). All of the substitutions reduced inactivation of AChE by more than 100-fold, with the most effective being the thiophosphate analogues, which reduced the rate of inactivation by about 4–5 orders of magnitude. The analogues were found to reliably predict changes in catalytic activity and stereoselectivity of the PTE variants and led to the identification of the BHR-30 variant, which has no apparent stereoselectivity against GD and a k_cat/K_m of 1.4 × 10⁶, making it the most efficient enzyme for GD decontamination reported till date.

The COVID-19 pandemic threatens to overwhelm healthcare systems around the world. The only current FDA-approved treatment, which directly targets the virus, is the ProTide prodrug remdesivir. In its activated form, remdesivir prevents viral replication by inhibiting the essential RNA-dependent RNA polymerase. Like other ProTide prodrugs, remdesivir contains a chiral phosphorus center. The initial selection of the (S_P)-diastereomer for remdesivir was reportedly due to the difficulty in producing the pure (R_P)-diastereomer of the required precursor. However, the two currently known enzymes responsible for the initial activation step of remdesivir are each stereoselective and show differential tissue distribution. Given the ability of the COVID-19 virus to infect a wide array of tissue types, inclusion of the (R_P)-diastereomer may be of clinical significance. To help overcome the challenge of obtaining the pure (R_P)-diastereomer of remdesivir, we have developed a novel chemoenzymatic strategy that utilizes a stereoselective variant of the phosphotriesterase from Pseudomonas diminuta to enable the facile isolation of the pure (R_P)-diastereomer of the chiral precursor for the chemical synthesis of the (R_P)-diastereomer of remdesivir.

The organophosphate flame-retardants are used to inhibit combustion and increase plasticity in plastics and durable foams. While not neurotoxic, these compounds are potential carcinogens, endocrine disrupters and developmental toxins. The phosphotriesterase from Sphingobium sp. TCM1 (Sb-PTE) is unique among phosphotriesterase enzymes for its ability to hydrolyze these compounds, and its ability to hydrolyze any one of the three different ester bonds within a given substrate. In some cases, the hydrolysis of a methyl ester exceeds that of a p-nitrophenyl ester within a single substrate. There is a stereochemical component to this hydrolysis where the two enantiomers of chiral substrates give different product ratios. To investigate the stereoselectivity for the product distribution of Sb-PTE, a series of 24 phosphotriesters was synthesized with all possible combinations of methyl, cyclohexyl, phenyl and p-nitrophenyl esters. Prochiral compounds were made chiral by differential isotopic labeling using a chemo/enzymatic strategy, which allowed the differentiation of hydrolysis for each ester in all but two compounds. The rate equations for this unique enzymatic mechanism were derived, the product ratios were determined for each substrate, and the individual kinetic constants for hydrolysis of each ester within each substrate were measured. The findings are consistent with the rate limiting step for substrate hydrolysis catalyzed by Sb-PTE being the formation of a phosphorane-like intermediate and the kinetic constants and product ratios being dictated by a combination of transition state energies, inductive effects, and stereochemical constraints.

Nucleoside analogs are among the most common medications given for the treatment of viral infections and cancers. The therapeutic effectiveness of nucleoside analogs can be dramatically improved by phosphorylation. The ProTide approach was developed using a phosphorylated nucleoside that is masked by esterification with an amino acid and phenol forming a chiral phosphorus center. The biological activity of the ProTides depends, in part, on the stereochemistry at phosphorus and thus it is imperative that efficient methods be developed for the chemical synthesis and isolation of diastereomerically pure ProTides. Chiral ProTides are often synthesized by direct displacement of a labile phenol (p-nitrophenol or pentafluorophenol) from a chiral phosphoramidate precursor with the appropriate nucleoside analog. The ability to produce these chiral products is dictated by the synthesis of the chiral phosphoramidate precursors. The enzyme phosphotriesterase (PTE) from Pseudomonas diminuta is well known for its high stereoselectivity and broad substrate profile. Screening PTE variants from enzyme evolution libraries enabled the identification of variants of PTE that can stereoselectively hydrolyze the chiral phosphoramidate precursors. The variant G60A-PTE exhibits a 165-fold preference for hydrolysis of the RP-isomer, while the variant In1W-PTE has a 1400-fold preference for hydrolysis of the SP-isomer. Using these mutants of PTE, the SP- and RP-isomers were isolated on a preparative scale with no detectable contamination of the opposite isomer. Combining the simplicity of the enzymatic resolution of the precursor with the latest synthetic strategy will facilitate the production of diastereometrically pure nucleotide phosphoramidate prodrugs.

The bacterial enzyme phosphotriesterase (PTE) is noted for its ability to hydrolyze many organophosphate compounds including insecticides and chemical warfare agents. PTE has been the subject of multiple enzyme evolution attempts, which have been highly successful against specific insecticides and the G-type nerve agents. Similar attempts targeting the V-type nerve agents have failed to reach the same degree of success. Enzyme evolution is an inherently complex problem, which is complicated by synergistic effects, the need to use analogs in high throughput screening, and a lack of quantitative data to direct future efforts. Previous evolution experiments with PTE have assumed an absence of synergy and minimally screened large libraries, which provides no quantitative information on the effects of individual mutations. Here a systemic approach has been applied to a 28,800 member six-site PTE library. The library is screened against multiple V-agent analogs, and a combination of sequence and quantitative activity analysis is used to extract data on the effects of individual mutations. It is demonstrated that synergistic relationships dominate the evolutionary landscape of PTE, and that analog activity profiles can be used to identify variants with high activity for substrates. Using these approaches, multiple variants improved more than 1500-fold in kcat/Km for the hydrolysis of VX were identified, including one variant that is 9200-fold improved relative to wild-type PTE and is specific for the SP-enantiomer of VX. Multiple variants highly active for SP-VR were identified, the best of which is 13,400-fold improved in kcat/Km relative to wild-type PTE.

Organophosphorus flame retardants are stable toxic compounds used in nearly all durable plastic products and are considered major emerging pollutants. The phosphotriesterase from Sphingobium sp. TCM1 (Sb-PTE) is one of the few enzymes known to be able to hydrolyze organophosphorus flame retardants such as triphenyl phosphate and tris(2-chloroethyl) phosphate. The effectiveness of Sb-PTE for the hydrolysis of these organophosphates appears to arise from its ability to hydrolyze unactivated alkyl and phenolic esters from the central phosphorus core. How Sb-PTE is able to catalyze the hydrolysis of the unactivated substituents is not known. To interrogate the catalytic hydrolysis mechanism of Sb-PTE, the pH dependence of the reaction and the effects of changing the solvent viscosity were determined. These experiments were complemented by measurement of the primary and secondary 18-oxygen isotope effects on substrate hydrolysis and a determination of the effects of changing the pK_a of the leaving group on the magnitude of the rate constants for hydrolysis. Collectively, the results indicated that a single group must be ionized for nucleophilic attack and that a separate general acid is not involved in protonation of the leaving group. The Brønsted analysis and the heavy atom kinetic isotope effects are consistent with an early associative transition state with subsequent proton transfers not being rate limiting. A novel binding mode of the substrate to the binuclear metal center and a catalytic mechanism are proposed to explain the unusual ability of Sb-PTE to hydrolyze unactivated esters from a wide range of organophosphate substrates.

A catalytic nanoscavenger with long blood residence time provides long-term prophylactic protection against nerve agents in animal models.