Background: Contemporary oxime antidotes to organophosphate poisoning cannot penetrate CNS to reactivate inhibited acetylcholinesterase. Results: Structural, in vitro optimization of ionizable hydroxyiminoacetamido amine acetylcholinesterase reactivators produced superior antidotal responses for VX-, sarin-, paraoxon-, and tabun-exposed mice. Conclusion: Ionizable hydroxyiminoacetamido amines are promising centrally active acetylcholinesterase reactivators. Significance: A mechanism-based iterative refinement of acetylcholinesterase reactivation kinetics coupled with pharmacokinetic analyses yields efficient CNS penetrating antidotes.

A major challenge in performing reactions in biological systems is the requirement for low substrate concentrations, often in the micromolar range. We report that copper cross-linked single-chain nanoparticles (SCNPs) are able to significantly increase the efficiency of copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) reactions at low substrate concentration in aqueous buffer by promoting substrate binding. Using a fluorogenic click reaction and dye uptake experiments, a structure-activity study is performed with SCNPs of different size and copper content and substrates of varying charge and hydrophobicity. The high catalytic efficiency and selectivity are attributed to a mechanism that involves an enzyme-like substrate binding process. Saturation-transfer difference (STD) NMR spectroscopy, 2D-NOESY NMR, kinetic analyses with varying substrate concentrations, and computational simulations are consistent with a Michaelis-Menten, two-substrate, random-sequential enzyme-like kinetic profile. This general approach may prove useful for developing more-sustainable catalysts and agents for biomedicine and chemical biology.

We describe here the synthesis and activity of a new series of oxime reactivators of cholinesterases (ChEs) that contain tertiary amine or imidazole protonatable functional groups. Equilibration between the neutral and protonated species at physiological pH enables the reactivators to cross the blood-brain barrier and distribute in the CNS aqueous space as dictated by interstitial and cellular pH values. Our structure-activity analysis of 134 novel compounds considers primarily imidazole aldoximes and N-substituted 2-hydroxyiminoacetamides. Reactivation capacities of novel oximes are rank ordered by their relative reactivation rate constants at 0.67 mM compared with 2-pyridinealdoxime methiodide for reactivation of four organophosphate (sarin, cyclosarin, VX, and paraoxon) conjugates of human acetylcholinesterase (hAChE). Rank order of the rates differs for reactivation of human butyrylcholinesterase (hBChE) conjugates. The 10 best reactivating oximes, predominantly hydroxyimino acetamide derivatives (for hAChE) and imidazole-containing aldoximes (for hBChE) also exhibited reasonable activity in the reactivation of tabun conjugates. Reactivation kinetics of the lead hydroxyimino acetamide reactivator of hAChE, when analyzed in terms of apparent affinity (1/K ox ) and maximum reactivation rate (k 2 ), is superior to the reference uncharged reactivators monoisonitrosoacetone and 2,3-butanedione monoxime and shows potential for further refinement. The disparate pH dependences for reactivation of ChE and the general base-catalyzed oximolysis of acetylthiocholine reveal that distinct reactivator ionization states are involved in the reactivation of ChE conjugates and in conferring nucleophilic reactivity of the oxime group.It has become increasingly apparent that efficient reinstatement of CNS acetylcholinesterase (AChE) 2 activity inhibited in organophosphate (OP)-intoxicated individuals is required for sustained symptom recovery. In particular, nerve agent OPs already used by terrorists, but also active metabolites of OP-based pesticides, readily cross the bloodbrain barrier (BBB). The exposure to OP doses close to lethality results in initial severe motor convulsions and epileptic seizures. Accumulating evidence points to these seizure events being linked to irreversible long term compromise of cognitive functions and alteration of CNS electrical excitability. Once accumulated into hydrophobic sites, OPs that do enter the CNS are retained and partition slowly back into the circulation. For example, victims of Tokyo subway nerve gas attack in 1995 were found to suffer from both short and long term symptoms of OP exposure (1-4). Accordingly, comprehensive protection from and treatment of OP intoxication to minimize the longer term consequences require administration of antidotes capable of reactivating OP-inhibited AChE in the CNS. Current therapy directed to reactivating inhibited AChE is limited to the peripheral circulation because commonly used quaternary pyridinium aldoxime reactivators do not cross the BBB at ...

Metrics & MoreArticle Recommendations CONSPECTUS: Cross-linking of polymers significantly alters their physical properties, greatly expanding their everyday utility. Indeed, the polymeric networks resulting from linkages between polymer chains are found in everyday materials from soft contact lenses and automobile tires to enamel coatings and highperformance adhesives. In contrast, intramolecularly cross-linked polymers have received far less attention until recent years, in large part because they are synthetically more challenging to prepare. In this Account, we trace our own efforts to develop the chemistry of intramolecularly cross-linked macromolecules, starting with dendrimers. Dendrimers provided an excellent starting point for investigating intramolecular cross-linking because they are single molecular entities. We showed that the end groups of dendrimers can be extensively cross-linked using the ring-closing metathesis reaction and that the discrete structure of the dendrimer provides unique opportunities for characterizing the number and location of the cross-links as well as some physical properties of the macromolecule such as its size and rigidity. Increasing the number of ring-closing metathesis reactions correlated with a reduction in size and an increase in rigidity. The general strategy applied to dendrimers was extended to star polymers and hyperbranched polyglycerols. Each of these macromolecules has a core or an initiating group from which the branches emanate. Linking the end groups or branches of these polymers presents a unique opportunity to chemically remove the core of the cross-linked macromolecule in a process that is reminiscent of that used to produce covalent molecular imprinted polymers. Recognizing this analogy, we sought a compelling application for cross-linked dendrimers, the first example of unimolecular imprinting, where a single polymer contains a single molecular imprint. The quality of the imprinting was mixed but pointed to an alternative general strategy for molecular imprinting in polymers. The effort also focused attention on synthetic antibodies and the general biomimicry provided by this class of macromolecules. Indeed, cross-linking of polymers either covalently or non-covalently bears a loose resemblance to folding of proteins into defined three-dimensional shapes. The synthesis and study of cross-linked linear polymers, often called single-chain nanoparticles (SCNPs), has emerged as a very active area of research in the past few years. Our experience with the cross-linking of branched polymers combined with an interest in performing organic synthesis within living cells led us to develop copper-containing SCNPs as artificial clickases. These polymeric clickases exhibit all of the hallmarks of enzymatic catalysis. One clickase containing a polyacrylamide backbone performs low-concentration copper-assisted alkyne−azide click reactions at unprecedented rates. Another performs click reactions within living cells. Other organic transformations can be performed intracel...

The cholinesterases, acetylcholinesterase (AChE) and butyrylcholinesterase, are primary targets of organophosphates (OPs). Exposure to OPs can lead to serious cardiovascular complications, respiratory compromise, and death. Current therapy to combat OP poisoning involves an oxime reactivator (2-PAM, obidoxime, TMB4, or HI-6) combined with atropine and on occasion an anticonvulsant. Butyrylcholinesterase, administered in the plasma compartment as a bio-scavenger, has also shown efficacy but is limited by its strict stoichiometric scavenging, slow reactivation, and a propensity for aging. Here, we characterize 10 human (h) AChE mutants that, when coupled with an oxime, give rise to catalytic reactivation and aging resistance of the soman conjugate. With the most efficient human AChE mutant Y337A/F338A, we show enhanced reactivation rates for several OP-hAChE conjugates compared with wild-type hAChE when reactivated with HI-6 (1-(2-hydroxyiminomethyl-1-pyridinium)-3-(4-carbamoyl-1-pyridinium)). In addition, we interrogated an 840-member novel oxime library for reactivation of Y337A/F338A hAChE-OP conjugates to delineate the most efficient oxime-mutant enzyme pairs for catalytic bioscavenging. Combining the increased accessibility of the Y337A mutation to oximes within the space-impacted active center gorge with the aging resistance of the F338A mutation provides increased substrate diversity in scavenging potential for agingprone alkyl phosphate inhibitors.