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1.
Acc Chem Res ; 53(6): 1244-1256, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32441091

RESUMO

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 high-performance 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 intracellularly, and some of the most advanced SCNPs engage in concurrent and tandem catalysis with a naturally occurring biocatalyst. By tracing our own efforts, this Account provides a few entry points into the broader literature and also points to both the remaining challenges and overall promising future envisioned for this unique class of functional macromolecules.


Assuntos
Anticorpos/metabolismo , Materiais Biomiméticos/química , Enzimas/metabolismo , Polímeros/química , Materiais Biomiméticos/síntese química , Dendrímeros , Impressão Molecular , Polímeros/síntese química , Relação Estrutura-Atividade
2.
J Am Chem Soc ; 140(42): 13695-13702, 2018 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-30192530

RESUMO

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.

3.
Biochem J ; 450(1): 231-42, 2013 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-23216060

RESUMO

In the present paper we show a comprehensive in vitro, ex vivo and in vivo study on hydrolytic detoxification of nerve agent and pesticide OPs (organophosphates) catalysed by purified hBChE (human butyrylcholinesterase) in combination with novel non-pyridinium oxime reactivators. We identified TAB2OH (2-trimethylammonio-6-hydroxybenzaldehyde oxime) as an efficient reactivator of OP-hBChE conjugates formed by the nerve agents VX and cyclosarin, and the pesticide paraoxon. It was also functional in reactivation of sarin- and tabun-inhibited hBChE. A 3-5-fold enhancement of in vitro reactivation of VX-, cyclosarin- and paraoxon-inhibited hBChE was observed when compared with the commonly used N-methylpyridinium aldoxime reactivator, 2PAM (2-pyridinealdoxime methiodide). Kinetic analysis showed that the enhancement resulted from improved molecular recognition of corresponding OP-hBChE conjugates by TAB2OH. The unique features of TAB2OH stem from an exocyclic quaternary nitrogen and a hydroxy group, both ortho to an oxime group on a benzene ring. pH-dependences reveal participation of the hydroxy group (pKa=7.6) forming an additional ionizing nucleophile to potentiate the oxime (pKa=10) at physiological pH. The TAB2OH protective indices in therapy of sarin- and paraoxon-exposed mice were enhanced by 30-60% when they were treated with a combination of TAB2OH and sub-stoichiometric hBChE. The results of the present study establish that oxime-assisted catalysis is feasible for OP bioscavenging.


Assuntos
Butirilcolinesterase/metabolismo , Substâncias para a Guerra Química/metabolismo , Organofosfatos/metabolismo , Oximas/química , Paraoxon/metabolismo , Sarina/metabolismo , Animais , Catálise , Substâncias para a Guerra Química/toxicidade , Feminino , Humanos , Concentração de Íons de Hidrogênio , Inativação Metabólica , Cinética , Camundongos , Camundongos Endogâmicos , Organofosfatos/toxicidade , Oximas/metabolismo , Paraoxon/toxicidade , Sarina/toxicidade
4.
J Biol Chem ; 287(15): 11798-809, 2012 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-22343626

RESUMO

We present a systematic structural optimization of uncharged but ionizable N-substituted 2-hydroxyiminoacetamido alkylamine reactivators of phosphylated human acetylcholinesterase (hAChE) intended to catalyze the hydrolysis of organophosphate (OP)-inhibited hAChE in the CNS. Starting with the initial lead oxime RS41A identified in our earlier study and extending to the azepine analog RS194B, reactivation rates for OP-hAChE conjugates formed by sarin, cyclosarin, VX, paraoxon, and tabun are enhanced severalfold in vitro. To analyze the mechanism of intrinsic reactivation of the OP-AChE conjugate and penetration of the blood-brain barrier, the pH dependence of the oxime and amine ionizing groups of the compounds and their nucleophilic potential were examined by UV-visible spectroscopy, (1)H NMR, and oximolysis rates for acetylthiocholine and phosphoester hydrolysis. Oximolysis rates were compared in solution and on AChE conjugates and analyzed in terms of the ionization states for reactivation of the OP-conjugated AChE. In addition, toxicity and pharmacokinetic studies in mice show significantly improved CNS penetration and retention for RS194B when compared with RS41A. The enhanced intrinsic reactivity against the OP-AChE target combined with favorable pharmacokinetic properties resulted in great improvement of antidotal properties of RS194B compared with RS41A and the standard peripherally active oxime, 2-pyridinealdoxime methiodide. Improvement was particularly noticeable when pretreatment of mice with RS194B before OP exposure was combined with RS194B reactivation therapy after the OP insult.


Assuntos
Acetamidas/química , Antídotos/química , Reativadores da Colinesterase/química , Oximas/química , Acetamidas/farmacocinética , Acetamidas/toxicidade , Acetilcolinesterase , Animais , Antídotos/farmacocinética , Antídotos/toxicidade , Encéfalo/metabolismo , Inibidores da Colinesterase/química , Inibidores da Colinesterase/toxicidade , Reativadores da Colinesterase/farmacocinética , Reativadores da Colinesterase/toxicidade , Avaliação Pré-Clínica de Medicamentos/normas , Feminino , Humanos , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Dose Letal Mediana , Camundongos , Estrutura Molecular , Organofosfatos/química , Organofosfatos/toxicidade , Oximas/farmacocinética , Oximas/toxicidade , Ligação Proteica , Padrões de Referência , Relação Estrutura-Atividade , Distribuição Tecidual
5.
J Biol Chem ; 286(34): 29718-24, 2011 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-21730071

RESUMO

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 bio-scavenging. 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 aging-prone alkyl phosphate inhibitors.


Assuntos
Acetilcolinesterase/química , Envelhecimento , Organofosfatos/química , Oximas/química , Compostos de Piridínio/química , Acetilcolinesterase/genética , Acetilcolinesterase/metabolismo , Domínio Catalítico/fisiologia , Humanos , Mutação de Sentido Incorreto , Oximas/metabolismo , Compostos de Piridínio/metabolismo
6.
J Biol Chem ; 286(22): 19422-30, 2011 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-21464125

RESUMO

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.


Assuntos
Acetilcolinesterase/química , Butirilcolinesterase/química , Reativadores da Colinesterase/química , Oximas/química , Inibidores da Colinesterase/química , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Organofosfatos/química , Fosforilação
7.
Chem Commun (Camb) ; 58(7): 985-988, 2022 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-34935784

RESUMO

We report a modular approach in which a noncovalently cross-linked single chain nanoparticle (SCNP) selectively binds catalyst "cofactors" and substrates to increase both the catalytic activity of a Cu-catalyzed alkyne-azide cycloaddition reaction and the Ru-catalyzed cleavage of allylcarbamate groups compared to the free catalysts.


Assuntos
Nanopartículas/química , Elementos de Transição/química , Alcinos/química , Compostos Alílicos/química , Compostos Alílicos/metabolismo , Azidas/química , Catálise , Cobre/química , Reação de Cicloadição , Glutationa/química , Rutênio/química
8.
Chem Biol Interact ; 203(1): 67-71, 2013 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-22975155

RESUMO

A library of more than 200 novel uncharged oxime reactivators was used to select and refine lead reactivators of human acetylcholinesterase (hAChE) covalently conjugated with sarin, cyclosarin, VX, paraoxon and tabun. N-substituted 2-hydroxyiminoacetamido alkylamines were identified as best reactivators and reactivation kinetics of the lead oximes, RS41A and RS194B, were analyzed in detail. Compared to reference pyridinium reactivators, 2PAM and MMB4, molecular recognition of RS41A reflected in its Kox constant was compromised by an order of magnitude on average for different OP-hAChE conjugates, without significant differences in the first order maximal phosphorylation rate constant k(2). Systematic structural modifications of the RS41A lead resulted in several-fold improvement with reactivator, RS194B. Kinetic analysis indicated K(ox) reduction for RS194B as the main kinetic constant leading to efficient reactivation. Subtle structural modifications of RS194B were used to identify essential determinants for efficient reactivation. Computational molecular modeling of RS41A and RS194B interactions with VX inhibited hAChE, bound reversibly in Michaelis type complex and covalently in the pentacoordinate reaction intermediate suggests that the faster reactivation reaction is a consequence of a tighter RS194B interactions with hAChE peripheral site (PAS) residues, in particular with D74, resulting in lower interaction energies for formation of both the binding and reactivation states. Desirable in vitro reactivation properties of RS194B, when coupled with its in vivo pharmacokinetics and disposition in the body, reveal the potential of this oxime design as promising centrally and peripherally active antidotes for OP toxicity.


Assuntos
Acetilcolinesterase/metabolismo , Reativadores da Colinesterase/farmacologia , Oximas/farmacologia , Acetamidas/química , Acetamidas/farmacologia , Acetilcolinesterase/química , Inibidores da Colinesterase/toxicidade , Reativadores da Colinesterase/química , Avaliação Pré-Clínica de Medicamentos , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/metabolismo , Humanos , Cinética , Modelos Moleculares , Organofosfatos/toxicidade , Compostos Organofosforados/toxicidade , Oximas/química , Paraoxon/toxicidade , Sarina/toxicidade
9.
Chem Biol Interact ; 187(1-3): 238-40, 2010 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-20382137

RESUMO

Organophosphates (OPs) exert their toxicity by inhibiting primarily acetylcholinesterase (AChE) and to a lesser extent butyrylcholinesterase (BChE). Binary mixtures of mammalian AChE and oximes of varying structure have been recently considered for treatment of OP poisoning as catalytic bioscavengers. In this study wild type human AChE and human AChE with residue mutations D134H, D134H_E202Q and D134H_F338A were characterized and investigated for inhibition by OPs and consequent oxime reactivation of phosphylated enzymes. The rationale for selecting these substitution positions was based on D134H being a naturally occurring single nucleotide polymorphism (SNP) in humans and that E202Q and F338A mutations slow aging of OP inhibited AChEs. Inhibition of D134H by paraoxon and analogues of cyclosarin was 2-8 times slower than inhibition of wild type (wt), while reactivation of the paraoxon inhibited enzyme by 2PAM was 6 times faster. Both inhibition and reactivation of D134H_E202Q and D134H_F338A double mutants were up to two orders of magnitude slower than the wt indicating that introduction of the active center substitutions abolished fully the effect of the peripherally located D134H. These results indicate that selected residues outside the active center influence inhibition, reactivation and catalysis rates through longer range interactions.


Assuntos
Acetilcolinesterase/química , Acetilcolinesterase/metabolismo , Reativadores da Colinesterase/farmacologia , Ativação Enzimática/efeitos dos fármacos , Mutação , Compostos Organofosforados/farmacologia , Oximas/farmacologia , Acetilcolinesterase/genética , Linhagem Celular , Inibidores da Colinesterase/farmacologia , Humanos , Polimorfismo de Nucleotídeo Único
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