Glutamate 350 Plays an Essential Role in Conformational Gating of Long-Range Radical Transport in Escherichia coli Class Ia Ribonucleotide Reductase.

Escherichia coli class Ia ribonucleotide reductase (RNR) is composed of two subunits that form an active α2ß2 complex. The nucleoside diphosphate substrates (NDP) are reduced in α2, 35 Å from the essential diferric-tyrosyl radical (Y122•) cofactor in ß2. The Y122•-mediated oxidation of C439 in α2 occurs by a pathway (Y122 ⇆ [W48] ⇆ Y356 in ß2 to Y731 ⇆ Y730 ⇆ C439 in α2) across the α/ß interface. The absence of an α2ß2 structure precludes insight into the location of Y356 and Y731 at the subunit interface. The proximity in the primary sequence of the conserved E350 to Y356 in ß2 suggested its importance in catalysis and/or conformational gating. To study its function, pH-rate profiles of wild-type ß2/α2 and mutants in which 3,5-difluorotyrosine (F2Y) replaces residue 356, 731, or both are reported in the presence of E350 or E350X (X = A, D, or Q) mutants. With E350, activity is maintained at the pH extremes, suggesting that protonated and deprotonated states of F2Y356 and F2Y731 are active and that radical transport (RT) can occur across the interface by proton-coupled electron transfer at low pH or electron transfer at high pH. With E350X mutants, all RNRs were inactive, suggesting that E350 could be a proton acceptor during oxidation of the interface Ys. To determine if E350 plays a role in conformational gating, the strong oxidants, NO2Y122•-ß2 and 2,3,5-F3Y122•-ß2, were reacted with α2, CDP, and ATP in E350 and E350X backgrounds and the reactions were monitored for pathway radicals by rapid freeze-quench electron paramagnetic resonance spectroscopy. Pathway radicals are generated only when E350 is present, supporting its essential role in gating the conformational change(s) that initiates RT and masking its role as a proton acceptor.

Generation of a stable, aminotyrosyl radical-induced α2ß2 complex of Escherichia coli class Ia ribonucleotide reductase.

Ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside diphosphates to deoxynucleoside diphosphates (dNDPs). The Escherichia coli class Ia RNR uses a mechanism of radical propagation by which a cysteine in the active site of the RNR large (α2) subunit is transiently oxidized by a stable tyrosyl radical (Y•) in the RNR small (ß2) subunit over a 35-Å pathway of redox-active amino acids: Y122• ↔ [W48?] ↔ Y356 in ß2 to Y731 ↔ Y730 ↔ C439 in α2. When 3-aminotyrosine (NH2Y) is incorporated in place of Y730, a long-lived NH2Y730• is generated in α2 in the presence of wild-type (wt)-ß2, substrate, and effector. This radical intermediate is chemically and kinetically competent to generate dNDPs. Herein, evidence is presented that NH2Y730• induces formation of a kinetically stable α2ß2 complex. Under conditions that generate NH2Y730•, binding between Y730NH2Y-α2 and wt-ß2 is 25-fold tighter (Kd = 7 nM) than for wt-α2

Reverse Electron Transfer Completes the Catalytic Cycle in a 2,3,5-Trifluorotyrosine-Substituted Ribonucleotide Reductase.

Escherichia coli class Ia ribonucleotide reductase is composed of two subunits (α and ß), which form an α2ß2 complex that catalyzes the conversion of nucleoside 5'-diphosphates to deoxynucleotides (dNDPs). ß2 contains the essential tyrosyl radical (Y122(•)) that generates a thiyl radical (C439(•)) in α2 where dNDPs are made. This oxidation occurs over 35 Å through a pathway of amino acid radical intermediates (Y122 → [W48] → Y356 in ß2 to Y731 → Y730 → C439 in α2). However, chemistry is preceded by a slow protein conformational change(s) that prevents observation of these intermediates. 2,3,5-Trifluorotyrosine site-specifically inserted at position 122 of ß2 (F3Y(•)-ß2) perturbs its conformation and the driving force for radical propagation, while maintaining catalytic activity (1.7 s(-1)). Rapid freeze-quench electron paramagnetic resonance spectroscopy and rapid chemical-quench analysis of the F3Y(•)-ß2, α2, CDP, and ATP (effector) reaction show generation of 0.5 equiv of Y356(•) and 0.5 equiv of dCDP, both at 30 s(-1). In the absence of an external reducing system, Y356(•) reduction occurs concomitant with F3Y reoxidation (0.4 s(-1)) and subsequent to oxidation of all α2s. In the presence of a reducing system, a burst of dCDP (0.4 equiv at 22 s(-1)) is observed prior to steady-state turnover (1.7 s(-1)). The [Y356(•)] does not change, consistent with rate-limiting F3Y reoxidation. The data support a mechanism where Y122(•) is reduced and reoxidized on each turnover and demonstrate for the first time the ability of a pathway radical in an active α2ß2 complex to complete the catalytic cycle.

Reversible, long-range radical transfer in E. coli class Ia ribonucleotide reductase.

Ribonucleotide reductases (RNRs) catalyze the conversionof nucleotides to 2'-deoxynucleotides and are classified on the basis of the metallo-cofactor used to conduct this chemistry. The class Ia RNRs initiate nucleotide reduction when a stable diferric-tyrosyl radical (Y•, t1/2 of 4 days at 4 °C) cofactor in the ß2 subunit transiently oxidizes a cysteine to a thiyl radical (S•) in the active site of the α2 subunit. In the active α2ß2 complex of the class Ia RNR from E. coli , researchers have proposed that radical hopping occurs reversibly over 35 Å along a specific pathway comprised of redox-active aromatic amino acids: Y122• ↔ [W48?] ↔ Y356 in ß2 to Y731 ↔ Y730 ↔ C439 in α2. Each step necessitates a proton-coupled electron transfer (PCET). Protein conformational changes constitute the rate-limiting step in the overall catalytic scheme and kinetically mask the detailed chemistry of the PCET steps. Technology has evolved to allow the site-selective replacement of the four pathway tyrosines with unnatural tyrosine analogues. Rapid kinetic techniques combined with multifrequency electron paramagnetic resonance, pulsed electron-electron double resonance, and electron nuclear double resonance spectroscopies have facilitated the analysis of stable and transient radical intermediates in these mutants. These studies are beginning to reveal the mechanistic underpinnings of the radical transfer (RT) process. This Account summarizes recent mechanistic studies on mutant E. coli RNRs containing the following tyrosine analogues: 3,4-dihydroxyphenylalanine (DOPA) or 3-aminotyrosine (NH2Y), both thermodynamic radical traps; 3-nitrotyrosine (NO2Y), a thermodynamic barrier and probe of local environmental perturbations to the phenolic pKa; and fluorotyrosines (FnYs, n = 2 or 3), dual reporters on local pKas and reduction potentials. These studies have established the existence of a specific pathway spanning 35 Å within a globular α2ß2 complex that involves one stable (position 122) and three transient (positions 356, 730, and 731) Y•s. Our results also support that RT occurs by an orthogonal PCET mechanism within ß2, with Y122• reduction accompanied by proton transfer from an Fe1-bound water in the diferric cluster and Y356 oxidation coupled to an off-pathway proton transfer likely involving E350. In α2, RT likely occurs by a co-linear PCET mechanism, based on studies of light-initiated radical propagation from photopeptides that mimic the ß2 subunit to the intact α2 subunit and on [(2)H]-ENDOR spectroscopic analysis of the hydrogen-bonding environment surrounding a stabilized NH2Y• formed at position 730. Additionally, studies on the thermodynamics of the RT pathway reveal that the relative reduction potentials decrease according to Y122 < Y356 < Y731 ≈ Y730 ≤ C439, and that the pathway in the forward direction is thermodynamically unfavorable. C439 oxidation is likely driven by rapid, irreversible loss of water during the nucleotide reduction process. Kinetic studies of radical intermediates reveal that RT is gated by conformational changes that occur on the order of >100 s(-1) in addition to the changes that are rate-limiting in the wild-type enzyme (∼10 s(-1)). The rate constant of one of the PCET steps is ∼10(5) s(-1), as measured in photoinitiated experiments.

Redox-linked changes to the hydrogen-bonding network of ribonucleotide reductase ß2.

Ribonucleotide reductase (RNR) catalyzes conversion of nucleoside diphosphates (NDPs) to 2'-deoxynucleotides, a critical step in DNA replication and repair in all organisms. Class-Ia RNRs, found in aerobic bacteria and all eukaryotes, are a complex of two subunits: α2 and ß2. The ß2 subunit contains an essential diferric-tyrosyl radical (Y122O(•)) cofactor that is needed to initiate reduction of NDPs in the α2 subunit. In this work, we investigated the Y122O(•) reduction mechanism in Escherichia coli ß2 by hydroxyurea (HU), a radical scavenger and cancer therapeutic agent. We tested the hypothesis that Y122OH redox reactions cause structural changes in the diferric cluster. Reduction of Y122O(•) was studied using reaction-induced FT-IR spectroscopy and [(13)C]aspartate-labeled ß2. These Y122O(•) minus Y122OH difference spectra provide evidence that the Y122OH redox reaction is associated with a frequency change to the asymmetric vibration of D84, a unidentate ligand to the diferric cluster. The results are consistent with a redox-induced shift in H-bonding between Y122OH and D84 that may regulate proton-transfer reactions on the HU-mediated inactivation pathway in isolated ß2.

STimulator of INterferon Genes Agonism Accelerates Antitumor Activity in Poorly Immunogenic Tumors.

The innate immune agonist STING (STimulator of INterferon Genes) binds its natural ligand 2'3'-cGAMP (cyclic guanosine-adenosine monophosphate) and initiates type I IFN production. This promotes systemic antigen-specific CD8+ T-cell priming that eventually provides potent antitumor activity. To exploit this mechanism, we synthesized a novel STING agonist, MSA-1, that activates both mouse and human STING with higher in vitro potency than cGAMP. Following intratumoral administration of MSA-1 to a panel of syngeneic mouse tumors on immune-competent mice, cytokine upregulation and its exposure were detected in plasma, other tissues, injected tumors, and noninjected tumors. This was accompanied by effective antitumor activity. Mechanistic studies in immune-deficient mice suggested that antitumor activity of intratumorally dosed STING agonists is in part due to necrosis and/or innate immune responses such as TNF-α activity, but development of a robust adaptive antitumor immunity is necessary for complete tumor elimination. Combination with PD-1 blockade in anti-PD-1-resistant murine models showed that MSA-1 may synergize with checkpoint inhibitors but can also provide superior tumor control as a single agent. We show for the first time that potent cyclic dinucleotides can promote a rapid and stronger induction of the same genes eventually regulated by PD-1 blockade. This may have contributed to the relatively early tumor control observed with MSA-1. Taken together, these data strongly support the development of STING agonists as therapy for patients with aggressive tumors that are partially responsive or nonresponsive to single-agent anti-PD-1 treatment by enhancing the anti-PD-1 immune profile.

Discovery and Optimization of Potent, Selective, and Brain-Penetrant 1-Heteroaryl-1H-Indazole LRRK2 Kinase Inhibitors for the Treatment of Parkinson's Disease.

Inhibition of leucine-rich repeat kinase 2 (LRRK2) kinase activity represents a genetically supported, chemically tractable, and potentially disease-modifying mechanism to treat Parkinson's disease. Herein, we describe the optimization of a novel series of potent, selective, central nervous system (CNS)-penetrant 1-heteroaryl-1H-indazole type I (ATP competitive) LRRK2 inhibitors. Type I ATP-competitive kinase physicochemical properties were integrated with CNS drug-like properties through a combination of structure-based drug design and parallel medicinal chemistry enabled by sp3-sp2 cross-coupling technologies. This resulted in the discovery of a unique sp3-rich spirocarbonitrile motif that imparted extraordinary potency, pharmacokinetics, and favorable CNS drug-like properties. The lead compound, 25, demonstrated exceptional on-target potency in human peripheral blood mononuclear cells, excellent off-target kinase selectivity, and good brain exposure in rat, culminating in a low projected human dose and a pre-clinical safety profile that warranted advancement toward pre-clinical candidate enabling studies.

Kinetics of radical intermediate formation and deoxynucleotide production in 3-aminotyrosine-substituted Escherichia coli ribonucleotide reductases.

Escherichia coli ribonucleotide reductase is an α2ß2 complex and catalyzes the conversion of nucleoside 5'-diphosphates (NDPs) to 2'-deoxynucleotides (dNDPs). The reaction is initiated by the transient oxidation of an active-site cysteine (C(439)) in α2 by a stable diferric tyrosyl radical (Y(122)•) cofactor in ß2. This oxidation occurs by a mechanism of long-range proton-coupled electron transfer (PCET) over 35 Å through a specific pathway of residues: Y(122)•→ W(48)→ Y(356) in ß2 to Y(731)→ Y(730)→ C(439) in α2. To study the details of this process, 3-aminotyrosine (NH(2)Y) has been site-specifically incorporated in place of Y(356) of ß. The resulting protein, Y(356)NH(2)Y-ß2, and the previously generated proteins Y(731)NH(2)Y-α2 and Y(730)NH(2)Y-α2 (NH(2)Y-RNRs) are shown to catalyze dNDP production in the presence of the second subunit, substrate (S), and allosteric effector (E) with turnover numbers of 0.2-0.7 s(-1). Evidence acquired by three different methods indicates that the catalytic activity is inherent to NH(2)Y-RNRs and not the result of copurifying wt enzyme. The kinetics of formation of 3-aminotyrosyl radical (NH(2)Y•) at position 356, 731, and 730 have been measured with all S/E pairs. In all cases, NH(2)Y• formation is biphasic (k(fast) of 9-46 s(-1) and k(slow) of 1.5-5.0 s(-1)) and kinetically competent to be an intermediate in nucleotide reduction. The slow phase is proposed to report on the conformational gating of NH(2)Y• formation, while the k(cat) of ~0.5 s(-1) is proposed to be associated with rate-limiting oxidation by NH(2)Y• of the subsequent amino acid on the pathway during forward PCET. The X-ray crystal structures of Y(730)NH(2)Y-α2 and Y(731)NH(2)Y-α2 have been solved and indicate minimal structural changes relative to wt-α2. From the data, a kinetic model for PCET along the radical propagation pathway is proposed.

Incorporation of fluorotyrosines into ribonucleotide reductase using an evolved, polyspecific aminoacyl-tRNA synthetase.

Tyrosyl radicals (Y·s) are prevalent in biological catalysis and are formed under physiological conditions by the coupled loss of both a proton and an electron. Fluorotyrosines (F(n)Ys, n = 1-4) are promising tools for studying the mechanism of Y· formation and reactivity, as their pK(a) values and peak potentials span four units and 300 mV, respectively, between pH 6 and 10. In this manuscript, we present the directed evolution of aminoacyl-tRNA synthetases (aaRSs) for 2,3,5-trifluorotyrosine (2,3,5-F(3)Y) and demonstrate their ability to charge an orthogonal tRNA with a series of F(n)Ys while maintaining high specificity over Y. An evolved aaRS is then used to incorporate F(n)Ys site-specifically into the two subunits (α2 and ß2) of Escherichia coli class Ia ribonucleotide reductase (RNR), an enzyme that employs stable and transient Y·s to mediate long-range, reversible radical hopping during catalysis. Each of four conserved Ys in RNR is replaced with F(n)Y(s), and the resulting proteins are isolated in good yields. F(n)Ys incorporated at position 122 of ß2, the site of a stable Y· in wild-type RNR, generate long-lived F(n)Y·s that are characterized by electron paramagnetic resonance (EPR) spectroscopy. Furthermore, we demonstrate that the radical pathway in the mutant Y(122)(2,3,5)F(3)Y-ß2 is energetically and/or conformationally modulated in such a way that the enzyme retains its activity but a new on-pathway Y· can accumulate. The distinct EPR properties of the 2,3,5-F(3)Y· facilitate spectral subtractions that make detection and identification of new Y·s straightforward.

Optimization of brain-penetrant picolinamide derived leucine-rich repeat kinase 2 (LRRK2) inhibitors.

The discovery of potent, kinome selective, brain penetrant LRRK2 inhibitors is the focus of extensive research seeking new, disease-modifying treatments for Parkinson's disease (PD). Herein, we describe the discovery and evolution of a picolinamide-derived lead series. Our initial optimization efforts aimed at improving the potency and CLK2 off-target selectivity of compound 1 by modifying the heteroaryl C-H hinge and linker regions. This resulted in compound 12 which advanced deep into our research operating plan (ROP) before heteroaryl aniline metabolite 14 was characterized as Ames mutagenic, halting its progression. Strategic modifications to our ROP were made to enable early de-risking of putative aniline metabolites or hydrolysis products for mutagenicity in Ames. This led to the discovery of 3,5-diaminopyridine 15 and 4,6-diaminopyrimidine 16 as low risk for mutagenicity (defined by a 3-strain Ames negative result). Analysis of key matched molecular pairs 17 and 18 led to the prioritization of the 3,5-diaminopyridine sub-series for further optimization due to enhanced rodent brain penetration. These efforts culminated in the discovery of ethyl trifluoromethyl pyrazole 23 with excellent LRRK2 potency and expanded selectivity versus off-target CLK2.

An orally available non-nucleotide STING agonist with antitumor activity.

Pharmacological activation of the STING (stimulator of interferon genes)-controlled innate immune pathway is a promising therapeutic strategy for cancer. Here we report the identification of MSA-2, an orally available non-nucleotide human STING agonist. In syngeneic mouse tumor models, subcutaneous and oral MSA-2 regimens were well tolerated and stimulated interferon-ß secretion in tumors, induced tumor regression with durable antitumor immunity, and synergized with anti-PD-1 therapy. Experimental and theoretical analyses showed that MSA-2 exists as interconverting monomers and dimers in solution, but only dimers bind and activate STING. This model was validated by using synthetic covalent MSA-2 dimers, which were potent agonists. Cellular potency of MSA-2 increased upon extracellular acidification, which mimics the tumor microenvironment. These properties appear to underpin the favorable activity and tolerability profiles of effective systemic administration of MSA-2.

Use of 3-aminotyrosine to examine the pathway dependence of radical propagation in Escherichia coli ribonucleotide reductase.

Escherichia coli ribonucleotide reductase (RNR), an alpha2beta2 complex, catalyzes the conversion of nucleoside 5'-diphosphate substrates (S) to 2'-deoxynucleoside 5'-diphosphates. alpha2 houses the active site for nucleotide reduction and the binding sites for allosteric effectors (E). beta2 contains the essential diferric tyrosyl radical (Y(122)(*)) cofactor which, in the presence of S and E, oxidizes C(439) in alpha to a thiyl radical, C(439)(*), to initiate nucleotide reduction. This oxidation occurs over 35 A and is proposed to involve a specific pathway: Y(122)(*) --> W(48) --> Y(356) in beta2 to Y(731) --> Y(730) --> C(439) in alpha2. 3-Aminotyrosine (NH(2)Y) has been site-specifically incorporated at residues 730 and 731, and formation of the aminotyrosyl radical (NH(2)Y(*)) has been examined by stopped-flow (SF) UV-vis and EPR spectroscopies. To examine the pathway dependence of radical propagation, the double mutant complexes Y(356)F-beta2:Y(731)NH(2)Y-alpha2, Y(356)F-beta2:Y(730)NH(2)Y-alpha2, and wt-beta2:Y(731)F/Y(730)NH(2)Y-alpha2, in which the nonoxidizable F acts as a pathway block, were studied by SF and EPR spectroscopies. In all cases, no NH(2)Y(*) was detected. To study off-pathway oxidation, Y(413), located 5 A from Y(730) and Y(731) but not implicated in long-range oxidation, was examined. Evidence for NH(2)Y(413)(*) was sought in three complexes: wt-beta2:Y(413)NH(2)Y-alpha2 (a), wt-beta2:Y(731)F/Y(413)NH(2)Y-alpha2 (b), and Y(356)F-beta2:Y(413)NH(2)Y-alpha2 (c). With (a), NH(2)Y(*) was formed with a rate constant that was 25-30% and an amplitude that was 25% of that observed for its formation at residues 731 and 730. With (b), the rate constant for NH(2)Y(*) formation was 0.2-0.3% of that observed at 731 and 730, and with (c), no NH(2)Y(*) was observed. These studies suggest the evolution of an optimized pathway of conserved Ys in the oxidation of C(439).

Structural examination of the transient 3-aminotyrosyl radical on the PCET pathway of E. coli ribonucleotide reductase by multifrequency EPR spectroscopy.

E. coli ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to deoxynucleotides, a process that requires long-range radical transfer over 35 A from a tyrosyl radical (Y(122)*) within the beta2 subunit to a cysteine residue (C(439)) within the alpha2 subunit. The radical transfer step is proposed to occur by proton-coupled electron transfer via a specific pathway consisting of Y(122) --> W(48) --> Y(356) in beta2, across the subunit interface to Y(731) --> Y(730) --> C(439) in alpha2. Using the suppressor tRNA/aminoacyl-tRNA synthetase (RS) methodology, 3-aminotyrosine has been incorporated into position 730 in alpha2. Incubation of this mutant with beta2, substrate, and allosteric effector resulted in loss of the Y(122)* and formation of a new radical, previously proposed to be a 3-aminotyrosyl radical (NH(2)Y*). In the current study [(15)N]- and [(14)N]-NH(2)Y(730)* have been generated in H(2)O and D(2)O and characterized by continuous wave 9 GHz EPR and pulsed EPR spectroscopies at 9, 94, and 180 GHz. The data give insight into the electronic and molecular structure of NH(2)Y(730)*. The g tensor (g(x) = 2.0052, g(y) = 2.0042, g(z) = 2.0022), the orientation of the beta-protons, the hybridization of the amine nitrogen, and the orientation of the amino protons relative to the plane of the aromatic ring were determined. The hyperfine coupling constants and geometry of the NH(2) moiety are consistent with an intramolecular hydrogen bond within NH(2)Y(730)*. This analysis is an essential first step in using the detailed structure of NH(2)Y(730)* to formulate a model for a PCET mechanism within alpha2 and for use of NH(2)Y in other systems where transient Y*s participate in catalysis.

Impacting Global Health Through Equipment Repurposing: Measurable Progress Through the Accumulation of Many Tiny Victories.

There is an active and growing effort occurring in laboratories throughout Africa to research the underpinnings of endemic communicable diseases, many of which are considered "neglected tropical diseases" as defined by the World Health Organization. Across the continent, scientists, doctors, health care workers, and students investigate the in vitro activity of pharmacologically active extracts against known pathogens in hope of discovering new treatments for the diseases that affect the local population. During the summer of 2014, I had the opportunity to visit laboratories in 3 different countries engaged in this area of research through participation in the Merck Fellowship for Global Health (Merck is known as Merck, Sharp & Dohme outside of the United States and Canada.), in which Merck sponsors employees on a short-term sabbatical to work with a global health-focused nonprofit organization. This commentary describes the objectives of the fellowship program, the specific project to which my co-fellow and I contributed, and the story of a subsequent equipment donation effort that was inspired by my individual fellowship experience. It also captures a few of the more notable challenges and opportunities for the scientists in the laboratories we visited. Finally, for the reader who may be curious as to how she or he can contribute, I hope to move you to action by highlighting some of the opportunities for researchers to positively and creatively impact global health from their "home" lab benches and hoods.

Physicochemical and formulation developability assessment for therapeutic peptide delivery--a primer.

Peptides are an important class of endogenous ligands that regulate key biological cascades. As such, peptides represent a promising therapeutic class with the potential to alleviate many severe disease states. Despite their therapeutic potential, peptides frequently pose drug delivery challenges to scientists. This review introduces the physicochemical, biophysical, biopharmaceutical, and formulation developability aspects of peptides pertinent to the drug discovery-to-development interface. It introduces the relevance of these properties with respect to the delivery modalities available for peptide pharmaceuticals, with the parenteral route being the most prevalent route of administration. This review also presents characterization strategies for oral delivery of peptides with the aim of illuminating developability issues with the drug candidate. A brief overview of other routes of administration, including inhaled, transdermal, and intranasal routes, is provided as these routes are generally preferred by patients over injectables. Finally, this review presents formulation techniques to mitigate some of the developability obstacles associated with peptide delivery. The authors emphasize opportunities for the thoughtful application of pharmaceutical science to the development of peptide drugs and to the general advancement of this promising class of pharmaceuticals.

Unnatural amino acids: better than the real things?

Considerable effort has been dedicated to the development of technology for the site-specific incorporation of unnatural amino acids into proteins, with nonsense codon suppression and expressed protein ligation emerging as two of the most promising methods. Recent research advances in which these methods have been applied to study protein function and mechanism are briefly highlighted, and the potential of the methods for efficient, widespread future use in vitro and in vivo is critically evaluated.

Gold(I)-catalyzed regioselective cyclizations of silyl ketene amides and carbamates with alkynes.

The gold(I)-catalyzed regioselective cyclizations of silyl ketene amides or carbamates with alkynes were utilized to construct cyclopentanes or dehydro-delta-lactams.

Phosphate triester hydrolysis promoted by an N2S(thiolate)Zn complex: mechanistic implications for the metal-dependent reactivity of peptide deformylase.

The zinc(II) complex (PATH)ZnOH, where PATH is an N2S(thiolate) ligand, has been investigated for its ability to promote the hydrolysis of the phosphate triester tris(4-nitrophenyl) phosphate (TNP). The hydrolysis of TNP was examined as a function of PATH-zinc(II) complex concentration, substrate concentration, and pH in a water/ethanol mixture (66:33 v/v) at 25 degrees C. The reaction is first order in both zinc(II) complex and substrate, and the second-order rate constants were derived from linear plots of the observed pseudo-first-order rate constants versus zinc complex concentration at different pH values. A pH-rate profile yielded a kinetic pK(a) of 8.52(5) for the zinc-bound water molecule and a pH-independent rate constant of 16.1(7) M(-1) s(-1). Temperature-dependent studies showed linear Eyring behavior, yielding the activation parameters DeltaH++ = 36.9(1) kJ mol(-1) and DeltaS++ = -106.7(4) J mol(-1) K(-1). Interpretation of the kinetic data leads to the conclusion that hydrolysis of TNP takes place through a hybrid mechanism, in which the metal center plays a dual role of providing a nucleophilic hydroxide and activating the substrate through a Lewis acid effect. The synthesis and structural characterization of the related nickel(II) and iron(II) complexes [(PATH)2Ni2]Br2 (2) and (PATH)2Fe2Cl2 (3) are also described. Taken together, these data suggest a possible explanation for the low reactivity of the zinc(II) form of peptide deformylase as compared to the iron(II) form.