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Sulfur(VI) fluoride exchange (SuFEx) click chemistry has offered a facile and reliable approach to produce polysulfates and polysulfonates. However, the current SuFEx polymerization methods lack precise control of target molecular weight and dispersity. Herein, we report the first chain-growth SuFEx polycondensation process by exploiting the unique reactivity and selectivity of S–F bonds under SuFEx catalysis. Given the higher reactivity of iminosulfur oxydifluoride versus fluorosulfate, the chain-growth SuFEx polycondensation is realized by using an iminosulfur oxydifluoride-containing compound as the reactive chain initiator and deactivated AB-type aryl silyl ether-fluorosulfates bearing an electron-withdrawing group as monomers. When 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) was utilized as the polymerization catalyst, precise control over the polymer molecular weight and polydispersity was achieved. The resulting polymers possess great thermal stability but are easily degradable under mild acidic and basic conditions., The first chain-growth sulfur(VI) fluoride exchange polycondensation by exploiting the high reactivity of iminosulfur oxydifluorides over fluorosulfates is developed. The resulting polymers possess excellent thermal stability and full degradability under mild acidic and basic conditions.

Inhibition of acetylcholinesterase (AChE) by certain organophosphates (OPs) can be life-threatening and requires reactivating antidote accessibility to the peripheral and central nervous systems to reverse symptoms and enhance survival parameters. In considering dosing requirements for oxime antidotes in OP exposures that inactivate AChE, clearance of proton ionizable, zwitterionic antidotes is rapid and proceeds with largely the parent antidotal compound being cleared by renal transporters. Such transporters may also control disposition between target tissues and plasma as well as overall elimination from the body. An ideal small-molecule antidote should access and be retained in primary target tissues-central nervous system (brain), skeletal muscle, and peripheral autonomic sites-for sufficient periods to reactivate AChE and prevent acute toxicity. We show here that we can markedly prolong the antidotal activity of zwitterionic antidotes by inhibiting P-glycoprotein (P-gp) transporters in the brain capillary and renal systems. We employ the P-gp inhibitor tariquidar as a reference compound and show that tissue and plasma levels of RS194B, a hydroxyl-imino acetamido alkylamine reactivator, are elevated and that plasma clearances are reduced. To examine the mechanism, identify the transporter, and establish the actions of a transport inhibitor, we compare the pharmacokinetic parameters in a P-glycoprotein knockout mouse strain and see dramatic enhancements of short-term plasma and tissue levels. Hence, repurposed transport inhibitors that are candidate or Food and Drug Administration-approved drugs, should enhance target tissue concentrations of the zwitterionic antidote through inhibition of both renal elimination and brain capillary extrusion. SIGNIFICANCE STATEMENT: We examine renal and brain capillary transporter inhibition as means for lowering dose and frequency of dosing of a blood-brain barrier permanent reactivating antidote, RS194B, an ionizable zwitterion. Through a small molecule, tariquidar, and gene knockout mice, CNS antidote concentrations are enhanced, and total body clearances are concomitantly diminished. RS194B with repurposed transport inhibitors should enhance reactivation of central and peripheral OP-inhibited acetylcholinesterase. Activities at both disposition sites are a desired features for replacing the antidote, pralidoxime, for acute OP exposure.

The lack of efficient [18F]fluorination processes and target-specific organofluorine chemotypes remains the major challenge of fluorine-18 positron emission tomography (PET). We report here an ultrafast isotopic exchange method for the radiosynthesis of novel PET agent aryl [18F]fluorosulfate enabled by the emerging sulfur fluoride exchange (SuFEx) click chemistry. The method has been applied to the fully automated 18F-radiolabeling of 25 structurally and functionally diverse aryl fluorosulfates with excellent radiochemical yield (83-100%, median 98%) and high molar activity (280 GBq μmol-1) at room temperature in 30 s. The purification of radiotracers requires no time-consuming HPLC but rather a simple cartridge filtration. We further demonstrate the imaging application of a rationally designed poly(ADP-ribose) polymerase 1 (PARP1)-targeting aryl [18F]fluorosulfate by probing subcutaneous tumors in vivo.

Optimization of small-molecule probes or drugs is a synthetically lengthy, challenging, and resource-intensive process. Lack of automation and reliance on skilled medicinal chemists is cumbersome in both academic and industrial settings. Here, we demonstrate a high-throughput hit-to-lead process based on the biocompatible sulfur(VI) fluoride exchange (SuFEx) click chemistry. A high-throughput screening hit benzyl (cyanomethyl)carbamate (Ki = 8 μM) against a bacterial cysteine protease SpeB was modified with a SuFExable iminosulfur oxydifluoride [RN═S(O)F2] motif, rapidly diversified into 460 analogs in overnight reactions, and the products were directly screened to yield drug-like inhibitors with 480-fold higher potency (Ki = 18 nM). We showed that the improved molecule is active in a bacteria-host coculture. Since this SuFEx linkage reaction succeeds on picomole scale for direct screening, we anticipate our methodology can accelerate the development of robust biological probes and drug candidates.

Sulfur (Ⅵ) fluoride exchange (SuFEx) click chemistry has offered a facile and reliable approach to produce polysul-fates and polysulfonates. However, the current SuFEx polymerization methods lack precise control of target molecular weight and dispersity. Herein, we report the first chain-growth SuFEx polycondensation process by exploiting unique reactivity and selectivity of S-F bonds under SuFEx catalysis. Given the higher reactivity of iminosulfur oxydifluoride versus fluorosulfate, the chain-growth SuFEx polycondensation is realized by using an iminosulfur oxydifluoride-containing compound as the reactive chain initiator and deactivated AB-type aryl silyl ether-fluorosulfates bearing an electron-withdrawing group as monomers. When DBU was utilized as the polymerization catalyst, precise control over polymer molecular weight and polydispersity are achieved. The resulting polymers possess great thermal stability but are easily degradable under mild acidic and basic conditions.

Chemoenzymatic modification of cell-surface glycan structures has emerged as a complementary approach to metabolic oligosaccharide engineering. Here, we identify Pasteurella multocida α2-3-sialyltransferase M144D mutant, Photobacterium damsela α2-6-sialyltransferase, and Helicobacter mustelae α1-2-fucosyltransferase, as efficient tools for live-cell glycan modification. Combining these enzymes with Helicobacter pylori α1-3-fucosyltransferase, we develop a host-cell-based assay to probe glycan-mediated influenza A virus (IAV) infection including wild-type and mutant strains of H1N1 and H3N2 subtypes. At high NeuAcα2-6-Gal levels, the IAV-induced host-cell death is positively correlated with haemagglutinin (HA) binding affinity to NeuAcα2-6-Gal. Remarkably, an increment of host-cell-surface sialyl Lewis X (sLeX) exacerbates the killing by several wild-type IAV strains and a previously engineered mutant HK68-MTA. Structural alignment of HAs from HK68 and HK68-MTA suggests formation of a putative hydrogen bond between Trp222 of HA-HK68-MTA and the C-4 hydroxyl group of the α1-3-linked fucose of sLeX, which may account for the enhanced host cell killing of that mutant., Glycan molecules can be modified directly on the cell surface via chemoenzymatic approaches. Here, the authors employ a set of four bacterial glycosyltransferases to develop a live cell-based killing assay to probe host cell glycan-mediated influenza A virus infection.

We detail here distinctive departures from lead classical cholinesterase re-activators, the pyridinium aldoximes, to achieve rapid CNS penetration and reactivation of AChE in the CNS (brain and spinal cord). Such reactivation is consistent with these non-canonical re-activators enhancing survival parameters in both mice and macaques following exposure to organophosphates. Thus, the ideal cholinesterase re-activator should show minimal toxicity, limited inhibitory activity in the absence of an organophosphate, and rapid CNS penetration, in addition to its nucleophilic potential at the target, the conjugated AChE active center. These are structural properties directed to reactivity profiles at the conjugated AChE active center, reinforced by the pharmacokinetic and tissue disposition properties of the re-activator leads. In the case of nicotinic acetylcholine receptor (nAChR) agonists and antagonists, with the many existing receptor subtypes in mammals, we prioritize subtype selectivity in their design. In contrast to nicotine and its analogues that react with panoply of AChR subtypes, the substituted di-2-picolyl amine pyrimidines possess distinctive ionization characteristics reflecting in selectivity for the orthosteric site at the α7 subtypes of receptor. Here, entry to the CNS should be prioritized for the therapeutic objectives of the nicotinic agent influencing aberrant CNS activity in development or in the sequence of CNS ageing (longevity) in mammals, along with general peripheral activities controlling inflammation.

Sulfur(vi) fluoride exchange (SuFEx) is a category of click chemistry that enables covalent linking of modular units through sulfur(vi) connective hubs. The efficiency of SuFEx and the stability of the resulting bonds have led to polymer chemistry applications. Now, we report the SuFEx click chemistry synthesis of several structurally diverse SOF4-derived copolymers based on the polymerization of bis(iminosulfur oxydifluorides) and bis(aryl silyl ethers). This polymer class presents two key characteristics. First, the [–N=S(=O)F–O–] polymer backbone linkages are themselves SuFExable and undergo precise SuFEx-based post-modification with phenols or amines to yield branched functional polymers. Second, studies of individual polymer chains of several of these new materials indicate helical polymer structures. The robust nature of SuFEx click chemistry offers the potential for post-polymerization modification, enabling the synthesis of materials with control over composition and conformation. Sulfur(vi) fluoride exchange (SuFEx)—a type of click chemistry that generates SVI-centred covalent linkages—has previously been used for polymer synthesis. Now, modular SuFEx polymerization using SOF4 has been used to generate helical polymers. Unlike previous examples of SuFEx polymerization, the backbone retains SVI–F motifs and therefore is able to undergo further SuFEx click reactions, enabling facile and efficient post-polymerization modification.

In the development of antidotal therapy for treatment of organophosphate exposure from pesticides used in agriculture and nerve agents insidiously employed in terrorism, the alkylpyridinium aldoximes have received primary attention since their early development by I. B. Wilson in the 1950s. Yet these agents, by virtue of their quaternary structure, are limited in rates of crossing the blood-brain barrier, and they require administration parenterally to achieve full distribution in the body. Oximes lacking cationic charges or presenting a tertiary amine have been considered as alternatives. Herein, we examine the pharmacokinetic properties of a lead ionizable, zwitterionic hydroxyiminoacetamido alkylamine in mice to develop a framework for studying these agents in vivo and generate sufficient data for their consideration as appropriate antidotes for humans. Consequently, in vitro and in vivo efficacies of immediate structural congeners were explored as leads or backups for animal studies. We compared oral and parenteral dosing, and we developed an intramuscular loading and oral maintenance dosing scheme in mice. Steady-state plasma and brain levels of the antidote were achieved with sequential administrations out to 10 hours, with brain levels exceeding plasma levels shortly after administration. Moreover, the zwitterionic oxime showed substantial protection after gavage, whereas the classic methylpyridinium aldoxime (2-pyridinealdoxime methiodide) was without evident protection. Although further studies in other animal species are necessary, ionizing zwitterionic aldoximes present viable alternatives to existing antidotes for prophylaxis and treatment of large numbers of individuals in terrorist-led events with nerve agent organophosphates, such as sarin, and in organophosphate pesticide exposure.

Sulfuryl fluoride, SO2 F2 , has been found to derivatize phenols in all kinds of environments, even those in highly functional molecules. We now report that a solid fluorosulfuryl imidazolium triflate salt delivers the same "F-SO2 +" fragment to Nu-H acceptor groups in the substrates. However, this triflate salt is a far more reactive fluorosulfurylating agent than SO2 F2 and displays selectivity preferences of its own. Moreover, the new azolium triflate reagent reacts once with primary amines and anilines before the reaction stops. On the other hand, with triethylamine and two equivalents of the "F-SO2 +" donor present, it proceeds on to the bis(fluorosulfuryl)imides in good yield-two important conversions that we have never seen with sulfuryl fluoride as the electrophile.

Thionyl tetrafluoride (SOF4) is a valuable connective gas for sulfur fluoride exchange (SuFEx) click chemistry, that enables multidimensional linkages to be created via sulfur–oxygen (S–O) and sulfur–nitrogen (S–N) bonds. In this report, we expand the available SuFEx chemistry of SOF4 to include organolithium nucleophiles, and demonstrate, for the first time, the controlled projection of sulfur–carbon links at the sulfur center of SOF4-derived iminosulfur oxydifluorides (R1–N=SOF2). This protocol enables rapid and modular access to sulfonimidoyl fluorides [R1–N=SOFR2], another array of versatile SuFEx connectors with readily tunable reactivity of the S–F handle. Divergent connections derived from these valuable sulfonimidoyl fluoride units are also demonstrated, including the synthesis of sulfoximines, sulfonimidamides, and sulfonimidates.

In this work, we describe the SuFEx-based polycondensation between bisalkylsulfonyl fluorides (AA monomers) and bisphenol bis(t-butyldimethylsilyl) ethers (BB monomers) using [Ph3P=N-PPh3]+[HF2]− as the catalyst. The AA monomers were prepared via the highly reliable Michael addition of ethenesulfonyl fluoride and amines/anilines while the BB monomers were obtained from silylation of bisphenols by t-butyldimethylsilyl chloride. With these reactions, we were able to achieve a remarkable diversity of monomeric building blocks by exploiting readily available amines, anilines, and bisphenols as starting materials. The SuFEx-based polysulfonate formation reaction exhibited excellent efficiency and functional group tolerance, producing polysulfonates with a variety of side chain functionalities in >99% conversion within 10 min to 1 h. When bearing an orthogonal group on the side chain, the polysulfonates can be further functionalized via click-chemistry based post-polymerization modification.

A palladium-catalyzed fluorosulfonylvinylation reaction of organic iodides is described. Catalytic Pd(OAc)2 with a stoichiometric amount of silver(I) trifluoroacetate enables the coupling process between either an (hetero)aryl or alkenyl iodide with ethenesulfonyl fluoride (ESF). The method is demonstrated in the successful syntheses of eighty-eight otherwise difficult to access compounds, in up to 99 % yields, including the unprecedented 2-heteroarylethenesulfonyl fluorides and 1,3-dienylsulfonyl fluorides.

Sulfur(VI) fluoride exchange (SuFEx) is a new family of click chemistry based transformations that enable the synthesis of covalently linked modules via SVI hubs. Here we report thionyl tetrafluoride (SOF4 ) as the first multidimensional SuFEx connector. SOF4 sits between the commercially mass-produced gases SF6 and SO2 F2 , and like them, is readily synthesized on scale. Under SuFEx catalysis conditions, SOF4 reliably seeks out primary amino groups [R-NH2 ] and becomes permanently anchored via a tetrahedral iminosulfur(VI) link: R-N=(O=)S(F)2 . The pendant, prochiral difluoride groups R-N=(O=)SF2 , in turn, offer two further SuFExable handles, which can be sequentially exchanged to create 3-dimensional covalent departure vectors from the tetrahedral sulfur(VI) hub.

Acetylcholinesterase (AChE) is a pivotal enzyme in neurotransmission. Its inhibition leads to cholinergic crises and could ultimately result in death. A related enzyme, butyrylcholinesterase (BChE), may act in the CNS as a coregulator in terminating nerve impulses and is a natural plasma scavenger upon exposure to organophosphate (OP) nerve agents that irreversibly inhibit both enzymes. With the aim of improving reactivation of cholinesterases phosphylated by nerve agents sarin, VX, cyclosarin, and tabun, ten phenyltetrahydroisoquinoline (PIQ) aldoximes were synthesized by Huisgen 1, 3 dipolar cycloaddition between alkyne- and azide-building blocks. The PIQ moiety may serve as a peripheral site anchor positioning the aldoxime moiety at the AChE active site. In terms of evaluated dissociation inhibition constants, the aldoximes could be characterized as high-affinity ligands. Nevertheless, high binding affinity of these oximes to AChE or its phosphylated conjugates did not assure rapid and selective AChE reactivation. Rather, potential reactivators of phosphylated BChE, with its enlarged acyl pocket, were identified, especially in case of cyclosarin, where the reactivation rates of the lead reactivator was 100- and 6-times that of 2-PAM and HI-6, respectively. Nevertheless, the return of the enzyme activity was affected by the nerve agent conjugated to catalytic serine, which highlights the lack of the universality of reactivators with respect to both the target enzyme and OP structure.

The lack of simple, efficient [18F]fluorination processes and new target-specific organofluorine probes remains the major challenge of fluorine-18-based positron emission tomography (PET). We report here a fast isotopic exchange method for the radiosynthesis of aryl [18F]fluorosulfate based PET agents enabled by the emerging sulfur fluoride exchange (SuFEx) click chemistry. The method has been applied to the fully-automated 18F-radiolabeling of twenty-five structurally diverse aryl fluorosulfates with excellent radiochemical yield (83–100%) and high molar activity (up to 281 GBq µmol–1) at room temperature in 30 seconds. The purification of radiotracers requires no time-consuming high-performance liquid chromatography (HPLC), but rather a simple cartridge filtration. The utility of aryl [18F]fluorosulfate is demonstrated by the in vivo tumor imaging by targeting poly(ADP-ribose) polymerase 1 (PARP1).

The first SuFEx click chemistry synthesis of SOF4-derived copolymers based upon the polymerization of bis(iminosulfur oxydifluorides) and bis(aryl silyl ethers) is described. This novel class of SuFEx polymer presents two key characteristics: First, the newly created [-N=S(=O)F-O-] polymer backbone linkages are themselves SuFExable and primed to undergo further high-yielding and precise SuFEx-based post-modification with phenols or amines to yield branched functional polymers. Second, studies of individual polymer chains of several of these new materials indicate the presence of helical polymer structures, which itself suggests a preferential approach of new monomers onto the growing polymer chain upon the formation of the stereogenic linking moiety.

Sulfur fluoride exchange (SuFEx) has emerged as the new generation of click chemistry. We report here a SuFEx-enabled, agnostic approach for the discovery and optimization of covalent inhibitors of human neutrophil elastase (hNE). Evaluation of our ever-growing collection of SuFExable compounds toward various biological assays unexpectedly revealed a selective and covalent hNE inhibitor: benzene-1,2-disulfonyl fluoride. Synthetic derivatization of the initial hit led to a more potent agent, 2-(fluorosulfonyl)phenyl fluorosulfate with IC 50 0.24 μM and greater than 833-fold selectivity over the homologous neutrophil serine protease, cathepsin G. The optimized, yet simple benzenoid probe only modified active hNE and not its denatured form.

Drug candidates that form covalent linkages with their target proteins have been underexplored compared with the conventional counterparts that modulate biological function by reversibly binding to proteins, in part due to concerns about off-target reactivity. However, toxicity linked to off-target reactivity can be minimized by using latent electrophiles that only become activated towards covalent bond formation on binding a specific protein. Here we study sulfuramidimidoyl fluorides, a class of weak electrophiles that undergo sulfur(VI) fluoride exchange chemistry. We show that equilibrium binding of a sulfuramidimidoyl fluoride to a protein can allow nucleophilic attack by a specific amino acid side chain, which leads to conjugate formation. We incubated small molecules, each bearing a sulfuramidimidoyl fluoride electrophile, with human cell lysate, and the protein conjugates formed were identified by affinity chromatography-mass spectrometry. This inverse drug discovery approach identified a compound that covalently binds to and irreversibly inhibits the activity of poly(ADP-ribose) polymerase 1, an important anticancer target in living cells.

Since the development in the 1950's of 2-PAM (Pralidoxime), an antidote that reactivates organophosphate conjugated acetylcholinesterase in target tissues upon pesticide or nerve agent exposure, improvements in antidotal therapy have largely involved congeneric pyridinium aldoximes. Despite seminal advances in detailing the structures of the cholinesterases as the primary target site, progress with small molecule antidotes has yet to define a superior agent. Two major limitations are immediately apparent. The first is the impacted space within the active center gorge, particularly when the active center serine at its base is conjugated with an organophosphate. The reactivating nucleophile will have to negotiate the tortuous gorge terrain to access the phosphorus atom with its most nucleophilic form or ionization state, the oximate anion. A second limitation stems from the antidote crossing the blood-brain barrier sufficiently rapidly, since it is well documented that central acetylcholinesterase inhibition gives rise to cardiovascular and respiratory compromise. The associated hypoxia then leads to a sequelae of events, including poor perfusion of the brain and periphery, along with muscle fasciculation, tremors and eventually seizures. We consider both the barriers confronting and further achievements necessary to enhance efficacy of antidotes.

A Heck-Matsuda process for the synthesis of the otherwise difficult to access compounds, β-arylethenesulfonyl fluorides, is described. Ethenesulfonyl fluoride (i.e., vinylsulfonyl fluoride, or ESF) undergoes β-arylation with stable and readily prepared arenediazonium tetrafluoroborates in the presence of the catalyst palladium(II) acetate to afford the E-isomer sulfonyl analogues of cinnamoyl fluoride in 43-97 % yield. The β-arylethenesulfonyl fluorides are found to be selectively addressable bis-electrophiles for sulfur(VI) fluoride exchange (SuFEx) click chemistry, in which either the alkenyl moiety or the sulfonyl fluoride group can be the exclusive site of nucleophilic attack under defined conditions, making these rather simple cores attractive for covalent drug discovery.

A selection of heteroaryl fluorosulfates were readily synthesized using commercial SO2F2 gas. These substrates are highly efficient coupling partners in the Suzuki reaction. Through judicious selection of Pd-catalysts, the fluorosulfate functionality is differentiated from bromide and chloride; the order of reactivity being: -Br > -OSO2F > -Cl. Exploiting this trend allowed the stepwise chemoselective synthesis of a number of polysubstituted pyridines, including the drug, Etoricoxib.

Tabun represents the phosphoramidate class of organophosphates that are covalent inhibitors of acetylcholinesterase (AChE), an essential enzyme in neurotransmission. Currently used therapy in counteracting excessive cholinergic stimulation consists of a muscarinic antagonist (atropine) and an oxime reactivator of inhibited AChE, but the classical oximes are particularly ineffective in counteracting tabun exposure. In a recent publication (Kovarik et al., 2019), we showed that several oximes prepared by the Huisgen 1,3 dipolar cycloaddition and related precursors efficiently reactivate the tabun-AChE conjugate. Herein, we pursue the antidotal question further and examine a series of lead precursor molecules, along with triazole compounds, as reactivators of two AChE mutant enzymes. Such studies should reveal structural subtleties that reside within the architecture of the active center gorge of AChE and uncover intimate mechanisms of reactivation of alkylphosphate conjugates of AChE. The designated mutations appear to minimize steric constraints of the reactivating oximes within the impacted active center gorge. Indeed, after initial screening of the triazole oxime library and its precursors for the reactivation efficacy on Y337A and Y337A/F338A human AChE mutants, we found potentially active oxime-mutant enzyme pairs capable of degrading tabun in cycles of inhibition and reactivation. Surprisingly, the most sensitive ex vivo reactivation of mutant AChEs occurred with the alkylpyridinium aldoximes. Hence, although the use of mutant enzyme bio-scavengers in humans may be limited in practicality, bioscavenging and efficient neutralization of tabun itself or phosphoramidate mixtures of organophosphates might be achieved efficiently in vitro or ex vivo with these mutant AChE combinations.

Acetylcholinesterase (AChE), an enzyme that degrades the neurotransmitter acetylcholine, when covalently inhibited by organophosphorus compounds (OPs), such as nerve agents and pesticides, can be reactivated by oximes. However, tabun remains among the most dangerous nerve agents due to the low reactivation efficacy of standard pyridinium aldoxime antidotes. Therefore, finding an optimal reactivator for prophylaxis against tabun toxicity and for post-exposure treatment is a continued challenge. In this study, we analyzed the reactivation potency of 111 novel nucleophilic oximes mostly synthesized using the CuAAC triazole ligation between alkyne and azide building blocks. We identified several oximes with significantly improved in vitro reactivating potential for tabun-inhibited human AChE, and in vivo antidotal efficacies in tabun-exposed mice. Our findings offer a significantly improved platform for further development of antidotes and scavengers directed against tabun and related phosphoramidate exposures, such as the Novichok compounds.

Sulfur-Fluoride Exchange (SuFEx) is the new generation click chemistry transformation exploiting the unique properties of S-F bonds and their ability to undergo near-perfect reactions with nucleophiles. We report here the first SuFEx based protocol for the efficient synthesis of pharmaceutically important triflones and bis(trifluoromethyl)sulfur oxyimines from the corresponding sulfonyl fluorides and iminosulfur oxydifluorides, respectively. The new protocol involves the rapid exchange of the S-F bond with trifluoromethyltrimethylsilane (TMSCF3) upon activation with potassium bifluoride in anhydrous DMSO. The reaction tolerates a wide selection of substrates and proceeds under mild conditions without need for chromatographic purification. A tentative catalytic mechanism is proposed supported by DFT calculations, involving formation of the free trifluoromethyl anion followed by nucleophilic displacement of the S-F through a five-coordinate intermediate. The preparation of a benzothiazole derived bis(trifluoromethyl)sulfur oxyimine with cytotoxic selectivity for MCF7 breast cancer cells demonstrates the utility of this methodology for the late-stage functionalization of bioactive molecules.

Sulfur-Fluoride Exchange (SuFEx) is a new generation click chemistry transformation that exploits the unique properties of S-F bonds and their ability to undergo near-perfect reactions with nucleophiles. We report here the first SuFEx based protocol for the efficient late-stage synthesis of pharmaceutically important trifluoromethyl sulfones and bis(trifluoromethyl)sulfur oxyimines from the corresponding sulfonyl fluorides and iminosulfur oxydifluorides, respectively. The new protocol involves the rapid exchange of the S-F bond with trifluoromethyltrimethylsilane (TMSCF3, Ruppert’s reagent), upon activation with catalytic potassium bifluoride in anhydrous DMSO. The reaction tolerates a wide selection of substrates and proceeds under mild conditions without need for chromatographic purification. DFT calculations provide the first reported mechanism of anhydrous SuFEx reactivity, which confirms catalytic bifluoride behaviour with a five-coordinate sulfur intermediate. The preparation of a benzothiazole derived bis(trifluoromethyl)sulfur oxyimine with cytotoxic selectivity for MCF7 breast cancer cells demonstrates the utility of this methodology for the late stage functionalization of bioactive molecules.

Sulfur(VI) Fluoride Exchange (SuFEx) is a new family of click chemistry transformations which relies on readily available materials to produce compounds bearing the SVI—F motif. The potential of SuFEx in drug discovery has just started to be explored. We report the first method of SuFEx chemistry for the conversion of phenolic compounds to their respective arylfluorosulfate derivatives in situ in 96-well plates. This method is compatible with automated synthesis and screening to quickly assess the biological activities of the in situ generated, crude products. U sing this method, we perform late-stage functionalization of a panel of known anticancer drugs to generate the corresponding arylfluorosulfates. These in situ generated arylfluorosulfates are directly tested in a cancer-cell growth inhibition assay in parallel with their phenolic precursors. We discover three arylfluorosulfates that exhibit improved anticancer cell proliferation activities compared to their phenol precursors. Among these three compounds, the fluorosulfate derivative of Fulvestrant possesses significantly enhanced activity to down-regulate estrogen receptor (ER) expression in ER+ breast cancer cell line M CF-7 and the fluorosulfate derivative of Combretastatin A 4—a general anticancer drug currently being evaluated under clinical trials—exhibits a 70-fold increase in potency in the drug resistant colon cancer cell line HT-29.

Aryl fluorosulfates were prepared by a simple method and employed as coupling partners in the Suzuki-Miyaura reaction. The cross-coupling reactions were performed in water under air at room temperature without ligands or additives such as surfactants or phase-transfer reagents and proceeded smoothly to give excellent yields. Aryl fluorosulfates could also be used as alternatives to halides or triflates in other coupling reactions.

Drug candidates are generally discovered using biochemical screens employing an isolated target protein or by utilizing cell-based phenotypic assays. Both noncovalent and covalent hits emerge from such endeavors. Herein, we exemplify an "Inverse Drug Discovery" strategy in which organic compounds of intermediate complexity harboring weak, but activatable, electrophiles are matched with the protein(s) they react with in cells or cell lysate. An alkyne substructure in each candidate small molecule enables affinity chromatography-mass spectrometry, which produces a list of proteins that each distinct compound reacts with. A notable feature of this approach is that it is agnostic with respect to the cellular proteins targeted. To illustrate this strategy, we employed aryl fluorosulfates, an underexplored class of sulfur(VI) halides, that are generally unreactive unless activated by protein binding. Reversible aryl fluorosulfate binding, correct juxtaposition of protein side chain functional groups, and transition-state stabilization of the S(VI) exchange reaction all seem to be critical for conjugate formation. The aryl fluorosulfates studied thus far exhibit chemoselective reactivity toward Lys and, particularly, Tyr side chains, and can be used to target nonenzymes (e.g., a hormone carrier or a small-molecule carrier protein) as well as enzymes. The "Inverse Drug Discovery" strategy should be particularly attractive as a means to explore latent electrophiles not typically used in medicinal chemistry efforts, until one reacts with a protein target of exceptional interest. Structure-activity data can then be used to enhance the selectivity of conjugate formation or the covalent probe can be used as a competitor to develop noncovalent drug candidates. Here we use the "Inverse Drug Discovery" platform to identify and validate covalent ligands for 11 different human proteins. In the case of one of these proteins, we have identified and validated a small-molecule probe for the first time.

Polysulfates and polysulfonates possess exceptional mechanical properties making them potentially valuable engineering polymers. However, they have been little explored due to a lack of reliable synthetic access. Here we report bifluoride salts (Q+[FHF]–, where Q+ represents a wide range of cations) as powerful catalysts for the sulfur(VI) fluoride exchange (SuFEx) reaction between aryl silyl ethers and aryl fluorosulfates (or alkyl sulfonyl fluorides). The bifluoride salts are significantly more active in catalysing the SuFEx reaction compared to organosuperbases, therefore enabling much lower catalyst-loading (down to 0.05 mol%). Using this chemistry, we are able to prepare polysulfates and polysulfonates with high molecular weight, narrow polydispersity and excellent functional group tolerance. The process is practical with regard to the reduced cost of catalyst, polymer purification and by-product recycling. We have also observed that the process is not sensitive to scale-up, which is essential for its future translation from laboratory research to industrial applications. Both click chemistry and polymer synthesis require reliable transformations with high selectivity, efficiency and fidelity. Now, bifluoride salts can be used as powerful catalysts for the sufur(VI) fluoride exchange (SuFEx) click reaction and they are applied to the synthesis of polysulfates and polysulfonates in a practical and scalable manner.

Intoxication by organophosphate (OP) nerve agents and pesticides should be addressed by efficient, quickly deployable countermeasures such as antidotes reactivating acetylcholinesterase or scavenging the parent OP. We present here synthesis and initial in vitro characterization of 14 imidazole aldoximes and their structural refinement into three efficient reactivators of human butyrylcholinesterase (hBChE) inhibited covalently by nerve agent OPs, sarin, cyclosarin, VX, and the OP pesticide metabolite, paraoxon. Rapid reactivation of OP–hBChE conjugates by uncharged and nonprotonated tertiary imidazole aldoximes allows the design of a new OP countermeasure by conversion of hBChE from a stoichiometric to catalytic OP bioscavenger with the prospect of oral bioavailability and central nervous system penetration. The enhanced in vitro reactivation efficacy determined for tertiary imidazole aldoximes compared to that of their quaternary N-methyl imidazolium analogues is attributed to ion pairing of the cationic imidazolium with Asp 70, altering a reactive alignment of the aldoxime with the phosphorus in the OP–hBChE conjugate.

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.

Molecules that bind selectively to a given protein and then undergo a rapid chemoselective reaction to form a covalent conjugate have utility in drug development. Herein a library of 1,3,4-oxadiazoles substituted at the 2 position with an aryl sulfonyl fluoride and at the 5 position with a substituted aryl known to have high affinity for the inner thyroxine binding subsite of transthyretin (TTR) were conceived of by structure-based design principles and were chemically synthesized. When bound in the thyroxine binding site, most of the aryl sulfonyl fluorides react rapidly and chemoselectively with the pKa-perturbed K15 residue, kinetically stabilizing TTR and thus preventing amyloid fibril formation, known to cause polyneuropathy. Conjugation t50s range from 1 to 4 min, ~ 1400 times faster than the hydrolysis reaction outside the thyroxine binding site. X-ray crystallography confirms the anticipated binding orientation and sheds light on the sulfonyl fluoride activation leading to the sulfonamide linkage to TTR. A few of the aryl sulfonyl fluorides efficiently form conjugates with TTR in plasma. A few of the TTR covalent kinetic stabilizers synthesized exhibit fluorescence upon conjugation and therefore could have imaging applications as a consequence of the environment sensitive fluorescence of the chromophore.

The copper catalyzed azide alkyne cycloaddition (CuAAC) reaction – the quintessential ‘click’ reaction – was used to synthesise dimers of the neuraminidase inhibitor zanamivir in high yields. The effect upon anti-viral activity of varying the linker length and the number of triazole units was explored. All dimers were tested for anti-viral activity against influenza A/Sydney/5/97 and B/Harbin/7/94 in a cytopathic effect (CPE) assay.

Giardia lamblia is an important and ubiquitous cause of diarrheal disease. The primary agents in the treatment of giardiasis are nitroheterocyclic drugs, particularly the imidazoles metronidazole and tinidazole and the thiazole nitazoxanide. Although these drugs are generally effective, treatment failures occur in up to 20% of cases, and resistance has been demonstrated in vivo and in vitro . Prior work had suggested that side chain modifications of the imidazole core can lead to new effective 5-nitroimidazole drugs that can combat nitro drug resistance, but the full potential of nitroheterocycles other than imidazole to yield effective new antigiardial agents has not been explored. Here, we generated derivatives of two clinically utilized nitroheterocycles, nitrothiazole and nitrofuran, as well as a third heterocycle, nitropyrrole, which is related to nitroimidazole but has not been systematically investigated as an antimicrobial drug scaffold. Click chemistry was employed to synthesize 442 novel nitroheterocyclic compounds with extensive side chain modifications. Screening of this library against representative G. lamblia strains showed a wide spectrum of in vitro activities, with many of the compounds exhibiting superior activity relative to reference drugs and several showing >100-fold increase in potency and the ability to overcome existing forms of metronidazole resistance. The majority of new compounds displayed no cytotoxicity against human cells, and several compounds were orally active against murine giardiasis in vivo . These findings provide additional impetus for the systematic development of nitroheterocyclic compounds with nonimidazole cores as alternative and improved agents for the treatment of giardiasis and potentially other infectious agents.

Vancomycin has long been considered a drug of last resort. Its efficiency in treating multiple drug-resistant bacterial infections, particularly methicillin-resistant Staphylococcus aureus (MRSA), has had a profound effect on the treatment of life-threatening infections. However, the emergence of resistance to vancomycin is a cause for significant worldwide concern, prompting the urgent development of new effective treatments for antibiotic resistant bacterial infections. Harnessing the benefits of multivalency and cooperativity against vancomycin-resistant strains, we report a Click Chemistry approach towards reengineered vancomycin derivatives and the synthesis of a number of dimers with increased potency against MRSA and vancomycin resistant Enterococci (VRE; VanB). These semi-synthetic dimeric ligands were linked together with great efficiency using the powerful CuAAC reaction, demonstrating high levels of selectivity and purity.

A selection of heteroaryl fluorosulfates were readily synthesized using commercial SO2F2 gas. These substrates are highly efficient coupling partners in the Suzuki reaction. Through judicious selection of Pd catalysts the fluorosulfate functionality is differentiated from bromide and chloride; the order of reactivity being: -Br> -OSO2 F> -Cl. Exploiting this trend allowed the stepwise chemoselective synthesis of a number of polysubstituted pyridines, including the drug Etoricoxib.

Ligand binding sites on acetylcholinesterase (AChE) comprise an active center, at the base of a deep and narrow gorge lined by aromatic residues, and a peripheral site at the gorge entry. These features launched AChE as a reaction vessel for in situ click-chemistry synthesis of high-affinity TZ2PA6 and TZ2PA5 inhibitors, forming a syn-triazole upon cycloaddition within the gorge from alkyne and azide reactants bound at the two sites, respectively. Subsequent crystallographic analyses of AChE complexes with the TZ2PA6 regioisomers demonstrated that syn product association is accompanied by side chain reorganization within the gorge, freezing-in-frame a conformation distinct from an unbound state or anti complex. To correlate inhibitor dimensions with reactivity and explore whether in situ cycloaddition could be accelerated in a concentrated, crystalline template, we developed crystal-soaking procedures and solved structures of AChE complexes with the TZ2PA5 regioisomers and their TZ2/PA5 precursors (2.1-2.7 Å resolution). The structures reveal motions of residue His447 in the active site and, unprecedentedly, residue Tyr341 at the gorge mouth, associated with TZ2 binding and coordinated with other side chain motions in the gorge that may guide AChE toward a transient state favoring syn-triazole formation. Despite precursor binding to crystalline AChE, coupling of rapid electric field fluctuations in the gorge with proper alignments of the azide and alkyne reactants to form the triazole remains a likely limiting step. These observations point to a prime requirement for AChE to interconvert dynamically between sequential conformations to promote favorable electrostatic factors enabling a productive apposition of the reactants for reactivity.

The acetylcholine-binding proteins (AChBPs), which serve as structural surrogates for the extracellular domain of nicotinic acetylcholine receptors (nAChRs), were used as reaction templates for in situ click-chemistry reactions to generate a congeneric series of triazoles from azide and alkyne building blocks. The catalysis of in situ azide-alkyne cycloaddition reactions at a dynamic subunit interface facilitated the synthesis of potentially selective compounds for nAChRs. We investigated compound sets generated in situ with soluble AChBP templates through pharmacological characterization with α7 and α4β2 nAChRs and 5-hydroxytryptamine type 3A receptors. Analysis of activity differences between the triazole 1,5-syn- and 1,4-anti-isomers showed a preference for the 1,4-anti-triazole regioisomers among nAChRs. To improve nAChR subtype selectivity, the highest-potency building block for α7 nAChRs, i.e., 3α-azido-N-methylammonium tropane, was used for additional in situ reactions with a mutated Aplysia californica AChBP that was made to resemble the ligand-binding domain of the α7 nAChR. Fourteen of 50 possible triazole products were identified, and their corresponding tertiary analogs were synthesized. Pharmacological assays revealed that the mutated binding protein template provided enhanced selectivity of ligands through in situ reactions. Discrete trends in pharmacological profiles were evident, with most compounds emerging as α7 nAChR agonists and α4β2 nAChR antagonists. Triazoles bearing quaternary tropanes and aromatic groups were most potent for α7 nAChRs. Pharmacological characterization of the in situ reaction products established that click-chemistry synthesis with surrogate receptor templates offered novel extensions of fragment-based drug design that were applicable to multisubunit ion channels.

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.

This invention provides a method for preparing a 1,2,3-triazole compound comprising contacting an organic azide with a 2-substitued-1-haloalkyne, in the presence of a copper catalyst and a copper-coordinating ligand (preferably a tertiary amine) in a liquid reaction medium, thereby forming a 1,4,5-substituted-1,2,3-triazole compound including a halo substituent at the 5-position of the triazole, the organic portion of the organic azide at the 1-position of the triazole, and the substituent of the 1-iodoalkyne at the 4-position of the triazole. A method for preparing 1-iodoalkynes is also provided.

Infections with the diarrheagenic pathogen, Giardia lamblia, are commonly treated with the 5-nitroimidazole (5-NI) metronidazole (Mz), and yet treatment failures and Mz resistance occur. Using a panel of new 2-ethenyl and 2-ethanyl 5-NI derivatives, we found that compounds with a saturated bridge between the 5-NI core and a pendant ring system exhibited only modestly increased antigiardial activity and could not overcome Mz resistance. By contrast, olefins with a conjugated bridge connecting the core and a substituted phenyl or heterocyclic ring showed greatly increased antigiardial activity without toxicity, and several overcame Mz resistance and were more effective than Mz in a murine giardiasis model. Determination of the half-wave potential of the initial one-electron transfer by cyclic voltammetry revealed that easier redox activation correlated with greater antigiardial activity and capacity to overcome Mz resistance. These studies show the potential of combining systematic synthetic approaches with biological and electrochemical evaluations in developing improved 5-NI drugs.

In situ click chemistry is a target-guided synthesis technique for discovering potent protein ligands by assembling azides and alkynes into triazoles inside the affinity site of a target protein. We report the rapid discovery of a new and potent inhibitor of bacterial chitinases by the use of in situ click chemistry. We observed a target-templated formation of a potent triazole inhibitor of the chitinase-catalyzed chitin hydrolysis, through in situ click chemistry between a biologically active azide-containing scaffold and structurally unrelated alkyne fragments. Chitinase inhibitors have chemotherapeutic potential as fungicides, pesticides and antiasthmatics. Argifin, which has been isolated and characterized as a cyclopentapeptide natural product by our research group, shows strong inhibitory activity against chitinases. As a result of our efforts at developing a chitinase inhibitor from an azide-bearing argifin fragment and the application of the chitinase template and a library of alkynes, we rapidly obtained a very potent and new 1,5-disubstituted triazole inhibitor against Serratia marcescens chitinase (SmChi) B. The new inhibitor expressed 300-fold increase in the inhibitory activity against SmChiB compared with that of argifin. To the best of our knowledge, our finding of an enzyme-made 1,5-disubstituted triazole, using in situ click chemistry is the second example reported in the literature.

The emerging field of global mass-based metabolomics provides a platform for discovering unknown metabolites and their specific biochemical pathways. We report the identification of a new endogenous metabolite, N(4)-(N-acetylaminopropyl)spermidine and the use of a novel proteomics based method for the investigation of its protein interaction using metabolite immobilization on agarose beads. The metabolite was isolated from the organism Pyrococcus furiosus, and structurally characterized through an iterative process of synthesizing candidate molecules and comparative analysis using accurate mass LC-MS/MS. An approach developed for the selective preparation of N(1)-acetylthermospermine, one of the possible structures of the unknown metabolite, provides a convenient route to new polyamine derivatives through methylation on the N(8) and N(4) of the thermospermine scaffold. The biochemical role of the novel metabolite as well as that of two other polyamines: spermidine and agmatine is investigated through metabolite immobilization and incubation with native proteins. The identification of eleven proteins that uniquely bind with N(4)-(N-acetylaminopropyl)spermidine, provides information on the role of this novel metabolite in the native organism. Identified proteins included hypothetical ones such as PF0607 and PF1199, and those involved in translation, DNA synthesis and the urea cycle like translation initiation factor IF-2, 50S ribosomal protein L14e, DNA-directed RNA polymerase, and ornithine carbamoyltransferase. The immobilization approach demonstrated here has the potential for application to other newly discovered endogenous metabolites found through untargeted metabolomics, as a preliminary screen for generating a list of proteins that could be further investigated for specific activity.

In this three-component reaction, alkynes undergo a copper(I)-catalyzed cycloaddition with sodium azide and formaldehyde to yield 2-hydroxymethyl-2 H-1,2,3-triazoles, which are useful intermediates that can be readily converted to polyfunctional molecules. The hydroxymethyl group can also be removed, providing convenient access to N H-1,2,3-triazoles. The reaction is experimentally simple and readily scalable.

1,2,3-Triazole-based polymers generated from the Cu(I)-catalyzed cycloaddition between multivalent azides and acetylenes are effective adhesive materials for metal surfaces. The adhesive capacities of candidate mixtures of azide and alkyne components were measured by a modified peel test, using a customized adhesive tester. A particularly effective tetravalent alkyne and trivalent azide combination was identified, giving exceptional strength that matches or exceeds the best commercial formulations. The addition of Cu catalyst was found to be important for the synthesis of stronger adhesive polymers when cured at room temperature. Heating also accelerated curing rates, but the maximum adhesive strengths achieved at both room temperature and high temperature were the same, suggesting that crosslinking reaches the same advanced point in all cases. Polytriazoles also form adhesives to aluminum, but copper is bound more effectively, presumably because active Cu(I) ions may be leached from the surface to promote crosslinking and adhesion. © 2007 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 45: 5182–5189, 2007

Tyrosine O-sulfation is a common protein post-translational modification that regulates many biological processes, including leukocyte adhesion and chemotaxis. Many peptides with therapeutic potential contain one or more sulfotyrosine residues. We report a one-step synthesis for Fmoc-fluorosulfated tyrosine. An efficient Fmoc-based solid-phase peptide synthetic strategy is then introduced for incorporating the fluorosulfated tyrosine residue into peptides of interest. Standard simultaneous peptide-resin cleavage and removal of the acid-labile side-chain protecting groups affords the crude peptides containing fluorosulfated tyrosine. Basic ethylene glycol, serving both as solvent and reactant, transforms the fluorosulfated tyrosine peptides into sulfotyrosine peptides in high yield.