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Enzymes populate ensembles of structures necessary for catalysis that are difficult to experimentally characterize. We use time-resolved mix-and-inject serial crystallography at an x-ray free electron laser to observe catalysis in a designed mutant isocyanide hydratase (ICH) enzyme that enhances sampling of important minor conformations. The active site exists in a mixture of conformations, and formation of the thioimidate intermediate selects for catalytically competent substates. The influence of cysteine ionization on the ICH ensemble is validated by determining structures of the enzyme at multiple pH values. Large molecular dynamics simulations in crystallo and time-resolved electron density maps show that Asp 17 ionizes during catalysis and causes conformational changes that propagate across the dimer, permitting water to enter the active site for intermediate hydrolysis. ICH exhibits a tight coupling between ionization of active site residues and catalysis-activated protein motions, exemplifying a mechanism of electrostatic control of enzyme dynamics.

The first dual-function assay for human serine racemase (hSR), the only bona fide racemase in human biology, is reported. The hSR racemization function is essential for neuronal signaling, as the product, d-serine (d-Ser), is a potent N -methyl d-aspartate (NMDA) coagonist, important for learning and memory, with dysfunctional d-Ser-signaling being observed in some neuronal disorders. The second hSR function is β-elimination and gives pyruvate; this activity is elevated in colorectal cancer. This new NMR-based assay allows one to monitor both α-proton-exchange chemistry and β-elimination using only the native l-Ser substrate and hSR and is the most sensitive such assay. The assay judiciously employs segregated dual 13 C-labeling and 13 C/ 2 H crosstalk, exploiting both the splitting and shielding effects of deuterium. The assay is deployed to screen a 1020-compound library and identifies an indolo-chroman-2,4-dione inhibitor family that displays allosteric site binding behavior (noncompetitive inhibition vs l-Ser substrate; competitive inhibition vs adenosine 5'-triphosphate (ATP)). This assay also reveals important mechanistic information for hSR; namely, that H/D exchange is ∼13-fold faster than racemization, implying that K56 protonates the carbanionic intermediate on the si -face much faster than does S84 on the re -face. Moreover, the 13 C NMR peak pattern seen is suggestive of internal return, pointing to K56 as the likely enamine-protonating residue for β-elimination. The 13 C/ 2 H-isotopic crosstalk assay has also been applied to the enzyme tryptophan synthase and reveals a dramatically different partition ratio in this active site (β-replacement: si -face protonation ∼6:1 vs β-elimination: si -face protonation ∼1:3.6 for hSR), highlighting the value of this approach for fingerprinting the pyridoxal phosphate (PLP) enzyme mechanism.

Increasing utilization of nuclear power enhances the risks associated with industrial accidents, occupational hazards, and the threat of nuclear terrorism. Exposure to ionizing radiation interferes with genomic stability and gene expression resulting in the disruption of normal metabolic processes in cells and organs by inducing complex biological responses. Exposure to high-dose radiation causes acute radiation syndrome, which leads to hematopoietic, gastrointestinal, cerebrovascular, and many other organ-specific injuries. Altered genomic variations, gene expression, metabolite concentrations, and microbiota profiles in blood plasma or tissue samples reflect the whole-body radiation injuries. Hence, multi-omic profiles obtained from high-resolution omics platforms offer a holistic approach for identifying reliable biomarkers to predict the radiation injury of organs and tissues resulting from radiation exposures. In this review, we performed a literature search to systematically catalog the radiation-induced alterations from multi-omic studies and radiation countermeasures. We covered radiation-induced changes in the genomic, transcriptomic, proteomic, metabolomic, lipidomic, and microbiome profiles. Furthermore, we have covered promising multi-omic biomarkers, FDA-approved countermeasure drugs, and other radiation countermeasures that include radioprotectors and radiomitigators. This review presents an overview of radiation-induced alterations of multi-omics profiles and biomarkers, and associated radiation countermeasures., (© 2023 by Radiation Research Society. All rights of reproduction in any form reserved.)

Reaction discovery and catalyst screening lie at the heart of synthetic organic chemistry. While there are efforts at de novo catalyst design using computation/artificial intelligence, at its core, synthetic chemistry is an experimental science. This review overviews biomacromolecule-assisted screening methods and the follow-on elaboration of chemistry so discovered. All three types of biomacromolecules discussed─enzymes, antibodies, and nucleic acids─have been used as "sensors" to provide a readout on product chirality exploiting their native chirality. Enzymatic sensing methods yield both UV-spectrophotometric and visible, colorimetric readouts. Antibody sensors provide direct fluorescent readout upon analyte binding in some cases or provide for cat-ELISA (Enzyme-Linked ImmunoSorbent Assay)-type readouts. DNA biomacromolecule-assisted screening allows for templation to facilitate reaction discovery, driving bimolecular reactions into a pseudo-unimolecular format. In addition, the ability to use DNA-encoded libraries permits the barcoding of reactants. All three types of biomacromolecule-based screens afford high sensitivity and selectivity. Among the chemical transformations discovered by enzymatic screening methods are the first Ni(0)-mediated asymmetric allylic amination and a new thiocyanopalladation/carbocyclization transformation in which both C-SCN and C-C bonds are fashioned sequentially. Cat-ELISA screening has identified new classes of sydnone-alkyne cycloadditions, and DNA-encoded screening has been exploited to uncover interesting oxidative Pd-mediated amido-alkyne/alkene coupling reactions.

A formal synthesis of the antiviral drug (-)-oseltamivir (Tamiflu) has been accomplished starting from m -anisic acid via a dissolving metal or electrochemical Birch reduction. The correct absolute stereochemistry is efficiently set through enzyme-catalyzed carbonyl reduction on the resultant racemic α,β-unsaturated ketone. A screen of a broad ketoreductase (KRED) library identified several that deliver the desired allylic alcohol with nearly perfect facial selectivity at the new center for each antipodal substrate, indicating that the enzyme also is able to completely override inherent diastereomeric bias in the substrate. Conversion is complete, with d-glucose serving as the terminal hydride donor (glucose dehydrogenase). For each resulting diastereomeric secondary alcohol, O/N-interconversion is then efficiently effected either by synfacial [3,3]-sigmatropic allylic imidate rearrangement or by direct, stereoinverting N-Mitsunobu chemistry. Both stereochemical outcomes have been confirmed crystallographically. The α,β-unsaturation is then introduced via an α-phenylselenylation/oxidation/pyrolysis sequence to yield the targeted ( S )- N -acyl-protected 5-amino-1,3-cyclohexadiene carboxylates, key advanced intermediates for oseltamivir pioneered by Corey ( N -Boc) and Trost ( N -phthalamido), respectively.

Recent political unrest has highlighted the importance of understanding the short- and long-term effects of gamma-radiation exposure on human health and survivability. In this regard, effective treatment for acute radiation syndrome (ARS) is a necessity in cases of nuclear disasters. Here, we propose 20 therapeutic targets for ARS identified using a systematic approach that integrates gene coexpression networks obtained under radiation treatment in humans and mice, drug databases, disease-gene association, radiation-induced differential gene expression, and literature mining. By selecting gene targets with existing drugs, we identified potential candidates for drug repurposing. Eight of these genes (BRD4, NFKBIA, CDKN1A, TFPI, MMP9, CBR1, ZAP70, IDH3B) were confirmed through literature to have shown radioprotective effect upon perturbation. This study provided a new perspective for the treatment of ARS using systems-level gene associations integrated with multiple biological information. The identified genes might provide high confidence drug target candidates for potential drug repurposing for ARS.

A convenient synthetic route to α,α-difluoroalkylphosphonates is described. Structurally diverse aldehydes are condensed with LiF 2 CP(O)(OCH 2 CH═CH 2 ) 2 . The resultant alcohols are captured as the pentafluorophenyl thionocarbonates and efficiently deoxygenated with HSnBu 3 , BEt 3 , and O 2 , and then smoothly deblocked with CpRu(IV)(π-allyl)quinoline-2-carboxylate (1-2 mol %) in methanol as an allyl cation scavenger. These mild deprotection conditions provide access to free α,α-difluoroalkylphosphonates in nearly quantitative yield. This methodology is used to rapidly construct new bis-α,α-difluoroalkyl phosphonate inhibitors of PTPIB (protein phosphotyrosine phosphatase-1B).

How changes in enzyme structure and dynamics facilitate passage along the reaction coordinate is a fundamental unanswered question. Here, we use time-resolved mix-and-inject serial crystallography (MISC) at an X-ray free electron laser (XFEL), ambient-temperature X-ray crystallography, computer simulations, and enzyme kinetics to characterize how covalent catalysis modulates isocyanide hydratase (ICH) conformational dynamics throughout its catalytic cycle. We visualize this previously hypothetical reaction mechanism, directly observing formation of a thioimidate covalent intermediate in ICH microcrystals during catalysis. ICH exhibits a concerted helical displacement upon active-site cysteine modification that is gated by changes in hydrogen bond strength between the cysteine thiolate and the backbone amide of the highly strained Ile152 residue. These catalysis-activated motions permit water entry into the ICH active site for intermediate hydrolysis. Mutations at a Gly residue (Gly150) that modulate helical mobility reduce ICH catalytic turnover and alter its pre-steady-state kinetic behavior, establishing that helical mobility is important for ICH catalytic efficiency. These results demonstrate that MISC can capture otherwise elusive aspects of enzyme mechanism and dynamics in microcrystalline samples, resolving long-standing questions about the connection between nonequilibrium protein motions and enzyme catalysis., Competing Interests: The authors declare no competing interest.

Serine racemase (SR) is the first racemase enzyme to be identified in human biology and converts L-serine to D-serine, an important neuronal signaling molecule that serves as a co-agonist of the NMDA (N-methyl-D-aspartate) receptor. This overview describes key molecular features of the enzyme, focusing on the side chains and binding motifs that control PLP (pyridoxal phosphate) cofactor binding as well as activity modulation through the binding of both divalent cations and ATP, the latter showing allosteric modulation. Discussed are catalytically important residues in the active site including K56 and S84-the si- and re-face bases, respectively,-and R135, a residue that appears to play a critical role in the binding of both negatively charged alternative substrates and inhibitors. The interesting bifurcated mechanism followed by this enzyme whereby substrate L-serine can be channeled either into D-serine (racemization pathway) or into pyruvate (β-elimination pathway) is discussed extensively, as are studies that focus on a key loop region (the so-called "triple serine loop"), the modification of which can be used to invert the normal in vitro preference of this enzyme for the latter pathway over the former. The possible cross-talk between the PLP enzymes hSR and hCBS (human cystathionine β-synthase) is discussed, as the former produces D-serine and the latter produces H 2 S, both of which stimulate the NMDAR and both of which have been implicated in neuronal infarction pursuant to ischemic stroke. Efforts to gain a more complete mechanistic understanding of these PLP enzymes are expected to provide valuable insights for the development of specific small molecule modulators of these enzymes as tools to study their roles in neuronal signaling and in modulation of NMDAR function.

Cystathionine β-synthase (CBS) is a key regulator of sulfur amino acid metabolism, taking homocysteine from the methionine cycle to the biosynthesis of cysteine via the trans-sulfuration pathway. CBS is also a predominant source of H 2 S biogenesis. Roles for CBS have been reported for neuronal death pursuant to cerebral ischemia, promoting ovarian tumor growth, and maintaining drug-resistant phenotype by controlling redox behavior and regulating mitochondrial bioenergetics. The trans-sulfuration pathway is well-conserved in eukaryotes, but the analogous enzymes have different enzymatic behavior in different organisms. CBSs from the higher organisms contain a heme in an N-terminal domain. Though the presence of the heme, whose functions in CBSs have yet to be elucidated, is biochemically interesting, it hampers UV-vis absorption spectroscopy investigations of pyridoxal 5'-phosphate (PLP) species. CBS from Saccharomyces cerevisiae (yCBS) naturally lacks the heme-containing N-terminal domain, which makes it an ideal model for spectroscopic studies of the enzymological reaction catalyzed and allows structural studies of the basic yCBS catalytic core (yCBS-cc). Here we present the crystal structure of yCBS-cc, solved to 1.5 Å. Crystal structures of yCBS-cc in complex with enzymatic reaction intermediates have been captured, providing a structural basis for residues involved in catalysis. Finally, the structure of the yCBS-cc cofactor complex generated by incubation with an inhibitor shows apparent off-pathway chemistry not normally seen with CBS.

The importance of discovering new chemical transformations and/or optimizing catalytic combinations has led to a flurry of activity in reaction screening. The in situ enzymatic screening (ISES) approach described here utilizes biological tools (enzymes/cofactors) to advance chemistry. The protocol interfaces an organic reaction layer with an adjacent aqueous layer containing reporting enzymes that act upon the organic reaction product, giving rise to a spectroscopic signal. ISES allows the experimentalist to rapidly glean information on the relative rates of a set of parallel organic/organometallic reactions under investigation, without the need to quench the reactions or draw aliquots. In certain cases, the real-time enzymatic readout also provides information on sense and magnitude of enantioselectivity and substrate specificity. This article contains protocols for single-well (relative rate) and double-well (relative rate/enantiomeric excess) ISES, in addition to a colorimetric ISES protocol and a miniaturized double-well procedure. © 2017 by John Wiley & Sons, Inc., (Copyright © 2017 John Wiley & Sons, Inc.)

Herein we describe a formal thiocyanopalladation/carbocyclization transformation and its parametrization and optimization using a new elevated temperature plate-based version of our visual colorimetric enzymatic screening method for reaction discovery. The carbocyclization step leads to C-SCN bond formation in tandem with C-C bond construction and is highly stereoselective, showing nearly absolute 1,2- anti -stereoinduction (5 examples) for substrates bearing allylic substitution, and nearly absolute 1,3- syn -stereoinduction (16 examples) for substrates bearing propargylic substitution. Based upon these high levels of stereoinduction, the dependence of the 1,2-stereoinduction upon cyclization substrate geometry, and the generally high preference for the transoid vinyl thiocyanate alkene geometry, a mechanistic model is proposed, involving (i) Pd(ii)-enyne coordination, (ii) thiocyanopalladation, (iii) migratory insertion and (iv) β-elimination. Examples of transition metal-mediated C-SCN bond formation that proceed smoothly on unactivated substrates and allow for preservation of the SCN moiety are lacking. Yet, the thiocyanate functionality is of great value for biophysical chemistry (vibrational Stark effect) and medicinal chemistry (S,N-heterocycle construction). The title transformation accommodates C-, O-, N- and S-bridged substrates (6 examples), thereby providing the corresponding carbocyclic or heterocyclic scaffolds. The reaction is also shown to be compatible with a significant range of substituents, varying in steric and electronic demand, including a wide range of substituted aromatics, fused bicyclic and heterocyclic systems, and even biaryl systems. Combination of this new transformation with asymmetric allylation and Grubbs ring-closing metathesis provides for a streamlined enantio- and diastereoselective entry into the oxabicyclo[3.2.1]octyl core of the natural products massarilactone and annuionone A, as also evidenced by low temperature X-ray crystal structure determination. Utilizing this bicyclic scaffold, we demonstrate the versatility of the thiocyanate moiety for structural diversification post-cyclization. Thus, the bridging vinyl thiocyanate moiety is smoothly elaborated into a range of derivative functionalities utilizing transformations that cleave the S-CN bond, add the elements of RS-CN across a π-system and exploit the SCN moiety as a cycloaddition partner (7 diverse examples). Among the new functionalities thereby generated are thiotetrazole and sulfonyl tetrazole heterocycles that serve as carboxylate and phosphate surrogates, respectively, highlighting the potential of this approach for future applications in medicinal chemistry or chemical biology.

Developing specific chemical functionalities to deploy in biological environments for targeted enzyme inactivation lies at the heart of mechanism-based inhibitor development but also is central to other protein-tagging methods in modern chemical biology including activity-based protein profiling and proteolysis-targeting chimeras. We describe here a previously unknown class of potential PLP enzyme inactivators; namely, a family of quaternary, α-(1'-fluoro)vinyl amino acids, bearing the side chains of the cognate amino acids. These are obtained by the capture of suitably protected amino acid enolates with β,β-difluorovinyl phenyl sulfone, a new (1'-fluoro)vinyl cation equivalent, and an electrophile that previously eluded synthesis, capture and characterization. A significant variety of biologically relevant AA side chains are tolerated including those for alanine, valine, leucine, methionine, lysine, phenylalanine, tyrosine, and tryptophan. Following addition/elimination, the resulting transoid α-(1'-fluoro)-β-(phenylsulfonyl)vinyl AA-esters undergo smooth sulfone-stannane interchange to stereoselectively give the corresponding transoid α-(1'-fluoro)-β-(tributylstannyl)vinyl AA-esters. Protodestannylation and global deprotection then yield these sterically encumbered and densely functionalized quaternary amino acids. The α-(1'-fluoro)vinyl trigger, a potential allene-generating functionality originally proposed by Abeles, is now available in a quaternary AA context for the first time. In an initial test of this new inhibitor class, α-(1'-fluoro)vinyllysine is seen to act as a time-dependent, irreversible inactivator of lysine decarboxylase from Hafnia alvei. The enantiomers of the inhibitor could be resolved, and each is seen to give time-dependent inactivation with this enzyme. Kitz-Wilson analysis reveals similar inactivation parameters for the two antipodes, L-α-(1'-fluoro)vinyllysine (K i = 630 ± 20 μM; t 1/2 = 2.8 min) and D-α-(1'-fluoro)vinyllysine (K i = 470 ± 30 μM; t 1/2 = 3.6 min). The stage is now set for exploration of the efficacy of this trigger in other PLP-enzyme active sites.

There is currently great interest in human serine racemase, the enzyme responsible for producing the NMDA co-agonist d-serine. Reported correlation of d-serine levels with disorders including Alzheimer's disease, ALS, and ischemic brain damage (elevated d-serine) and schizophrenia (reduced d-serine) has further piqued this interest. Reported here is a structure/activity relationship study of position Ser 84 , the putative re -face base. In the most extreme case of functional reprogramming, the S84D mutant displays a dramatic reversal of β-elimination substrate specificity in favor of l-serine over the normally preferred l-serine- O -sulfate (∼1200-fold change in k cat / K m ratios) and l (l-THA; ∼5000-fold change in k cat / K m ratios) alternative substrates. On the other hand, the S84T (which performs l-Ser racemization activity), S84A (good k cat but high K m for l-THA elimination), and S84N mutants (nearly WT efficiency for l-Ser elimination) displayed intermediate activity, all showing a preference for the anionic substrates, but generally attenuated compared with the native enzyme. Inhibition studies with l- erythro -β-hydroxyaspartate follow this trend, with both WT serine racemase and the S84N mutant being competitively inhibited, with K i = 31 ± 1.5 μm and 1.5 ± 0.1 mm, respectively, and the S84D being inert to inhibition. Computational modeling pointed to a key role for residue Arg-135 in binding and properly positioning the l-THA and l-serine- O -sulfate substrates and the l- erythro -β-hydroxyaspartate inhibitor. Examination of available sequence data suggests that Arg-135 may have originated for l-THA-like β-elimination function in earlier evolutionary variants, and examination of available structural data suggests that a Ser 84 -H 2 O-Lys 114 hydrogen-bonding network in human serine racemase lowers the p K a of the Ser 84 re -face base., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

A generalized strategy is presented for the rapid assembly of a set of bivalent ligands with a variety of linking functionalities from a common monomer. Herein, an array of phosphatase-inert mannose-6-phosphonate-presenting ligands for the cation-independent-mannose 6-phosphate receptor (CI-MPR) is constructed. Receptor binding affinity varies with linking functionality-the simple amide and 1,5-triazole(tetrazole) being preferred over the 1,4-triazole. This approach is expected to find application across chemical biology, particularly in glycoscience, wherein multivalency often governs molecular recognition.

The gaseous neuromodulator H2S is associated with neuronal cell death pursuant to cerebral ischemia. As cystathionine β-synthase (CBS) is the primary mediator of H2S biogenesis in the brain, it has emerged as a potential target for the treatment of stroke. Herein, a "zipped" approach by alkene cross-metathesis into CBS inhibitor candidate synthesis is demonstrated. The inhibitors are modeled after the pseudo-C 2-symmetric CBS product (l,l)-cystathionine. The "zipped" concept means only half of the inhibitor needs be constructed; the two halves are then fused by olefin cross-metathesis. Inhibitor design is also mechanism-based, exploiting the favorable kinetics associated with hydrazine-imine interchange as opposed to the usual imine-imine interchange. It is demonstrated that the most potent "zipped" inhibitor 6S reduces H2S production in SH-SY5Y cells overexpressing CBS, thereby reducing cell death. Most importantly, CBS inhibitor 6S dramatically reduces infarct volume (1 h post-stroke treatment; ∼70% reduction) in a rat transient middle cerebral artery occlusion model for ischemia.

This study introduces new methods of screening for and tuning chiral space and in so doing identifies a promising set of chiral ligands for asymmetric synthesis. The carbafructopyranosyl-1,2-diamine(s) and salens constructed therefrom are particularly compelling. It is shown that by removing the native anomeric effect in this ligand family, one can tune chiral ligand shape and improve chiral bias. This concept is demonstrated by a combination of (i) x-ray crystallographic structure determination, (ii) assessment of catalytic performance, and (iii) consideration of the anomeric effect and its underlying dipolar basis. The title ligands were identified by a new mini version of the in situ enzymatic screening (ISES) procedure through which catalyst-ligand combinations are screened in parallel, and information on relative rate and enantioselectivity is obtained in real time, without the need to quench reactions or draw aliquots. Mini-ISES brings the technique into the nanomole regime (200 to 350 nmol catalyst/20 μml organic volume) commensurate with emerging trends in reaction development/process chemistry. The best-performing β-d-carbafructopyranosyl-1,2-diamine-derived salen ligand discovered here outperforms the best known organometallic and enzymatic catalysts for the hydrolytic kinetic resolution of 3-phenylpropylene oxide, one of several substrates examined for which the ligand is "matched." This ligand scaffold defines a new swath of chiral space, and anomeric effect tunability defines a new concept in shaping that chiral space. Both this ligand set and the anomeric shape-tuning concept are expected to find broad application, given the value of chiral 1,2-diamines and salens constructed from these in asymmetric catalysis.

Described are the synthesis and application of α-t-butyldimethylsilyl-α-methoxyacetaldehyde as a formal methoxyvinyl cation equivalent. Addition of Grignard reagents to the title aldehyde, followed by treatment of the intermediate β-hydroxysilanes with KH, gives good yields of large Z -methoxyvinylated products. Assuming a Peterson-like elimination mechanism, one can infer that the Grignard addition proceeds with high syn selectivity. These results are consistent with a chelation control model involving coordination to the α-methoxy group in the title aldehyde rather than an alternative stereoelectronic Felkin-Anh-type model. It must be noted that a steric Felkin-Anh model also accounts for the observed stereochemistry. All told, the title reagent can be employed to efficiently append a Z -configured methoxyvinyl group to an appropriate R-M species, in two steps.

Over the past two decades, the domains of both frontline synthetic organic chemistry and process chemistry and have seen an increase in crosstalk between asymmetric organic/organometallic approaches and enzymatic approaches to stereocontrolled synthesis. This review highlights the particularly auspicious role for dehydrogenase enzymes in this endeavor, with a focus on dynamic reductive kinetic resolutions (DYRKR) to "deracemize" building blocks, often setting two stereocenters in so doing. The scope and limitations of such dehydrogenase-mediated processes are overviewed, as are future possibilities for the evolution of enzymatic DYRKR.

Described is an efficient stereocontrolled route into valuable, densely functionalized fluorinated phosphonates that takes advantage of (i) a Clostridial enzyme to set the absolute stereochemistry and (ii) a new [3,3]-sigmatropic rearrangement of the thiono-Claisen variety that is among the fastest sigmatropic rearrangements yet reported. Here, a pronounced rate enhancement is achieved by distal fluorination. This rearrangement is completely stereoretentive, parlaying the enzymatically established β-C-O stereochemistry in the substrate into the δ-C-S stereochemistry in the product. The final products are of interest to chemical biology, with a platform for Zn-aminopeptidase A inhibitors being constructed here. The enzyme, Clostridium acetobutylicum (CaADH), recently expressed by our group, reduces a spectrum of γ,δ-unsaturated β-keto-α,α-difluorophosphonate esters (93-99% ee; 10 examples). The resultant β-hydroxy-α,α-difluorophosphonates possess the "L"-stereochemistry, opposite to that previously observed for the CaADH-reduction of ω-keto carboxylate esters ("D"), indicating an unusual active site plasticity. For the thiono-Claisen rearrangement, a notable structure-reactivity relationship is observed. Measured rate constants vary by over 3 orders of magnitude, depending upon thiono-ester structure. Temperature-dependent kinetics reveal an unusually favorable entropy of activation (ΔS(‡) = 14.5 ± 0.6 e.u.). Most notably, a 400-fold rate enhancement is seen upon fluorination of the distal arene ring, arising from favorable enthalpic (ΔΔH(‡) = -2.3 kcal/mol) and entropic (ΔΔS(‡) = 4 e.u., i.e. 1.2 kcal/mol at rt) contributions. The unusual active site plasticity seen here is expected to drive structural biology studies on CaADH, while the exceptionally facile sigmatropic rearrangement is expected to drive computational studies to elucidate its underlying entropic and enthalpic basis.

The glmS riboswitch is unique among gene-regulating riboswitches and catalytic RNAs. This is because its own metabolite, glucosamine-6-phosphate (GlcN6P), binds to the riboswitch and catalytically participates in the RNA self-cleavage reaction, thereby providing a novel negative feedback mechanism. Given that a number of pathogens harbor the glmS riboswitch, artificial actuators of this potential RNA target are of great interest. Structural/kinetic studies point to the 2-amino and 6-phosphate ester functionalities in GlcN6P as being crucial for this actuation. As a first step toward developing artificial actuators, we have synthesized a series of nine GlcN6P analogs bearing phosphatase-inert surrogates in place of the natural phosphate ester. Self-cleavage assays with the Bacillus cereus glmS riboswitch give a broad SAR. Two analogs display significant activity, namely, the 6-deoxy-6-phosphonomethyl analog (5) and the 6-O-malonyl ether (13). Kinetic profiles show a 22-fold and a 27-fold higher catalytic efficiency, respectively, for these analogs vs glucosamine (GlcN). Given their nonhydrolyzable phosphate surrogate functionalities, these analogs are arguably the most robust artificial glmS riboswitch actuators yet reported. Interestingly, the malonyl ether (13, extra O atom) is much more effective than the simple malonate (17), and the "sterically true" phosphonate (5) is far superior to the chain-truncated (7) or chain-extended (11) analogs, suggesting that positioning via Mg coordination is important for activity. Docking results are consistent with this view. Indeed, the viability of the phosphonate and 6-O-malonyl ether mimics of GlcN6P points to a potential new strategy for artificial actuation of the glmS riboswitch in a biological setting, wherein phosphatase-resistance is paramount.

In this issue of Chemistry & Biology, Arrendale and coworkers demonstrate a new prodrug strategy for intracellular delivery of an α, α-(difluoromethylene)phosphonate phosphoserine mimic. The deprotected pseudo-phosphopeptide releases the pro-apoptotic FOXO3a-transcription factor from its 14-3-3-adaptor protein complex, resulting in leukemic cell death., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

A streamlined entry into the sesquiterpene lactone (SQL) cores of linearifolin and zaluzanin A is described. Stereochemistry is controlled through transformations uncovered by ISES (In Situ Enzymatic Screening). Absolute stereochemistry derives from kinetic resolution of 5-benzyloxypentene-1,2-oxide, utilizing a β-pinene-derived-Co(III)-salen. Relative stereochemistry (1,3-cis-fusion) is set via formal halometalation/carbocyclization, mediated by [Rh(O(2)CC(3)F(7))(2)](2)/LiBr. Subsequent ring-closing metathesis (RCM-Grubbs II) yields the title exomethylene-δ-lactone SQL cores. In complementary fashion, RCM with Grubbs-I catalyst provides the oxabicyclo[3.3.1]nonyl core of xerophilusin R and zinagrandinolide.

An NADP-dependent alcohol dehydrogenase from Clostridium acetobutylicum (CaADH) has been expressed and characterized. CaADH enantioselectively reduces aromatic α-, β- and γ-keto esters to the corresponding D-hydroxy esters and provides a building block for the Taxotère side chain (95% yield, 95% de, 99% ee) by dynamic reductive kinetic resolution (DYRKR).

Described is an efficient heterologous expression system for Sulfolobus solfataricus ADH-10 (Alcohol Dehydrogenase isozyme 10) and its use in the dynamic reductive kinetic resolution (DYRKR) of 2-arylpropanal (Profen-type) substrates. Importantly, among the 12 aldehydes tested, a general preference for the (S)-antipode was observed, with high ee's for substrates corresponding to the NSAIDs (nonsteroidal anti-inflammatory drugs) naproxen, ibuprofen, flurbiprofen, ketoprofen, and fenoprofen. To our knowledge, this is the first application of a dehydrogenase from this Sulfolobus hyperthermophile to asymmetric synthesis and the first example of a DYRKR with such an enzyme. The requisite aldehydes are generated by Buchwald-Hartwig-type Pd(0)-mediated alpha-arylation of tert-butyl propionate. This is followed by reduction to the aldehyde in one [lithium diisobutyl tert-butoxyaluminum hydride (LDBBA)] or two steps [LAH/Dess-Martin periodinane]. Treatment of the profenal substrates with SsADH in 5% EtOH/phosphate buffer, pH 9, with catalytic NADH at 80 degrees C leads to efficient DYRKR, with ee's exceeding 90% for 9 aryl side chains, including those of the aforementioned NSAIDs. An in silico model, consistent with the observed broad side chain tolerance, is presented. Importantly, the SsADH-10 enzyme could be conveniently recycled by exploiting the differential solubility of the organic substrate/product at 80 degrees C and at rt. Pleasingly, SsADH-10 could be taken through several "thermal cycles," without erosion of ee, suggesting this as a generalizable approach to enzyme recycling for hyperthermophilic enzymes. Moreover, the robustness of this hyperthermophilic DH, in terms of both catalytic activity and stereochemical fidelity, speaks for greater examination of such archaeal enzymes in asymmetric synthesis.

Prior evidence supporting the direct observation of phosphorane intermediates in enzymatic phosphoryl transfer reactions was based on the interpretation of electron density corresponding to trigonal species bridging the donor and acceptor atoms. Close examination of the crystalline state of beta-phosphoglucomutase, the archetypal phosphorane intermediate-containing enzyme, reveals that the trigonal species is not PO-3 , but is MgF-3 (trifluoromagnesate). Although MgF-3 complexes are transition state analogues rather than phosphoryl group transfer reaction intermediates, the presence of fluorine nuclei in near-transition state conformations offers new opportunities to explore the nature of the interactions, in particular the independent measures of local electrostatic and hydrogen-bonding distributions using 19F NMR. Measurements on three beta-PGM-MgF-3 -sugar phosphate complexes show a remarkable relationship between NMR chemical shifts, primary isotope shifts, NOEs, cross hydrogen bond F...H-N scalar couplings, and the atomic positions determined from the high-resolution crystal structure of the beta-PGM-MgF--3 -G6P complex. The measurements provide independent validation of the structural and isoelectronic MgF--3 model of near-transition state conformations.

This overview focuses on the (alpha,alpha-difluoromethylene)phosphonate mimic of phosphoserine (pCF(2)Ser) and its application to the study of kinase-mediated signal transduction-pathways of great interest to drug development. The most versatile modes of access to these chemical biological tools are discussed, organized by method of PCF(2)-C bond formation. The pCF(2)-Ser mimic may be site-specifically incorporated into peptides (SPPS) and proteins (expressed protein ligation). This isopolar, dianionic pSer mimic results in a "constitutive phosphorylation" phenotype and is seen to support native protein-protein interactions that depend on serine phosphorylation. Signal transduction pathways studied with this chemical biological approach include the regulation of p53 tumor suppressor protein activity and of melatonin production. Given these successes, the future is bright for the use of such "teflon phospho-amino acid mimics" to map kinase-based signaling pathways.

The use of NADH- and NADPH-dependent ketoreductases to access enantioenriched pharmaceutical building blocks is reported. Seven structurally diverse synthons are obtained, including those for atomoxetine (KRED 132), talampanel (RS1-ADH and CPADH), Dolastatin (KRED 132), and fluoxetine (KRED 108/132). Ethanol may be used as stoichiometric reductant, regenerating both nicotinamide cofactors, particularly under four-electron redox conditions. Its favorable thermodynamic and economic profile, coupled with its advantageous dual cosolvent role, suggests a new application for biomass-derived ethanol.

On the one hand, owing to its electronegativity, relatively small size, and notable leaving group ability from anionic intermediates, fluorine offers unique opportunities for mechanism-based enzyme inhibitor design. On the other, the "bio-orthogonal" and NMR-active 19-fluorine nucleus allows the bioorganic chemist to follow the mechanistic fate of fluorinated substrate analogues or inhibitors as they are enzymatically processed. This article takes an overview of the field, highlighting key developments along these lines. It begins by highlighting new screening methodologies for drug discovery that involve appropriate tagging of either substrate or the target protein itself with (19)F-markers, that then report back on turnover and binding, respectively, via an the NMR screen. Taking this one step further, substrate-tagging with fluorine can be done is such a manner as to provide stereochemical information on enzyme mechanism. For example, substitution of one of the terminal hydrogens in phosphoenolpyruvate, provides insight into the, otherwise latent, facial selectivity of C-C bond formation in KDO synthase. Perhaps, most importantly, from the point of view of this discussion, appropriately tailored fluorinated functionality can be used to form to stabilized "transition state analogue" complexes with a target enzymes. Thus, 5-fluorinated pyrimidines, alpha-fluorinated ketones, and 2-fluoro-2-deoxysugars each lead to covalent adduction of catalytic active site residues in thymidylate synthase, serine protease and glycosidase enzymes, respectively. In all such cases, (19)F NMR allows the bioorganic chemist to spectrally follow "transition state analogue" formation. Finally, the use of specific fluorinated functionality to engineer "suicide substrates" is highlighted in a discussion of the development of the alpha-(2'Z-fluoro)vinyl trigger for amino acid decarboxylase inactivation. Here (19)F NMR allows the bioorganic chemist to glean useful partition ratio data directly out of the NMR tube.

A set of bivalent mannose 6-phosphonate 'molecular rulers' has been synthesized to examine ligand binding to the M6P/IGF2R. The set is estimated to span a P-P distance range of 16-26A (MMFF energy minimization on the hydrated phosphonates). Key synthetic transformations include sugar triflate displacement for phosphonate installation and Grubbs I cross-metathesis to achieve bivalency. Relative binding affinities were tested by radioligand displacement assays versus PMP-BSA (pentamannosyl phosphate-bovine serum albumin). These compounds exhibit slightly higher binding affinities for the receptor (IC(50)'s=3.7-5 microM) than the parent, monomeric mannose 6-phosphonate ligand and M6P itself (IC(50)=11.5+/-2.5 microM). These results suggest that the use of an alpha-configured anomeric alkane tether is acceptable, as no significant thermodynamic penalty is apparently paid with this design. On the other hand, the modest gains in binding affinity observed suggest that this ligand set has not yet found true bivalent interaction with the M6P/IGF2R (i.e., simultaneous binding to two distinct M6P-binding pockets).

Etoposide is one of the most successful chemotherapeutic agents used for the treatment of human cancers. The drug kills cells by inhibiting the ability of topoisomerase II to ligate nucleic acids that it cleaves during the double-stranded DNA passage reaction. Etoposide is composed of a polycyclic ring system (rings A-D), a glycosidic moiety at the C4 position, and a pendent ring (E-ring) at the C1 position. Although drug-enzyme contacts, as opposed to drug-DNA interactions, mediate the entry of etoposide into the topoisomerase II-drug-DNA complex, the substituents on etoposide that interact with the enzyme have not been identified. Therefore, saturation transfer difference [1H]-nuclear magnetic resonance spectroscopy and protein-drug competition binding assays were employed to define the groups on etoposide that associate with yeast topoisomerase II and human topoisomerase IIalpha. Results indicate that the geminal protons of the A-ring, the H5 and H8 protons of the B-ring, and the H2' and H6' protons and the 3'- and 5'-methoxyl protons of the pendent E-ring interact with both enzymes in the binary protein-ligand complexes. In contrast, no significant nuclear Overhauser enhancement signals arising from the C-ring, the D-ring, or the C4 glycosidic moiety were observed with either enzyme, suggesting that there is limited or no contact between these portions of etoposide and topoisomerase II in the binary complex. The functional importance of E-ring substituents was confirmed by topoisomerase II-mediated DNA cleavage assays.

This report presents an overview of the family of naturally occurring 'vinylic' amino acids, namely those that feature a C-C double bond directly attached to the α-carbon, along the side chain. Strategies that have been brought to bear on the stereocontrolled synthesis of these olefinic amino acids are surveyed. The mechanistic diversity by which such 'vinylic triggers' can be actuated in a PLP (pyridoxal phosphate) enzyme active site is then highlighted by discussions of vinylglycine (VG), its substituted congeners, particularly AVG [4E-(2'-aminoethoxy)vinylglycine], and a naturally occurring VG-progenitor, SMM (S-methylmethionine).

Pd(II)-mediated rearrangement of allylic N-PMP (p-methoxyphenyl) trifluoroacetimidates provides the first formal sigmatropic route to quaternary, alpha-vinylic amino acids, potential suicide substrates for PLP enzymes. The amino acid side chains enter via transition-metal-mediated C-C bond constructions, including (i) Cu(I)-mediated conjugate addition (Ala); (ii) Pd(0)/AsPh3-mediated Stille coupling (allyl-Gly, Phe, DOPA, m-Tyr); and (iii) Pd(0)/Pt-Bu3-mediated Negishi coupling (Leu). In the synthesis of the DOPA decarboxylase inactivator, alpha-vinyl-m-tyrosine, the new N-PMP trifluoroacetimidate rearranges much more efficiently than the corresponding trichloroacetimidate.

We report a new continuous spectrophotometric assay for human cystathionine beta-synthase (hCBS). This assay relies upon the finding that hCBS will take cysteamine in place of L-homocysteine, thereby producing thialysine. Thialysine is, in turn, decarboxylated by lysine decarboxylase, releasing CO2 that is monitored by the sequential action of phosphoenolpyruvate carboxylase and L-malate dehydrogenase. The decrease in absorbance at 340 nm is monitored as reduced nicotinamide adenine dinucleotide is consumed. Using this four-enzyme couple, we find that Km(app) = 1.2+/-0.2 mM for L-serine and 5.6+/-2.2 mM for cysteamine, with kcat = 1.3+/-0.1s(-1) for the formation of thialysine by hCBS. For comparison purposes, the same hCBS reaction was monitored via a radioactive single time point assay using 14C-(C-1)-labeled L-serine and cysteamine as substrates, counting the thialysine product, following ion exchange chromatography. This assay yielded Km(app) = 2.2+/-0.5 mM for L-serine and 6.6+/-2.2 for cysteamine, with kcat = 2.5+/-0.4 s(-1). These numbers indicate that, although it possesses a shortened carbon chain and lacks a carboxyl group, cysteamine displays a catalytic efficiency (kcat/Km) with hCBS that is within an order of magnitude of that observed with its natural thiol cosubstrate, L-homocysteine.

Described is a new method for the screening of an array of catalysts, in situ, to estimate enantioselectivity and relative rates. We term this approach "double-cuvette ISES (in situ enzymatic screening)". The Co(III)-salen mediated hydrolytic kinetic resolution (HKR) of (+/-)-propylene oxide is used as a model reaction to demonstrate proof of principle. In two parallel cuvettes, a lower CHCl3-based organic layer is loaded with the epoxide and the chiral salen catalyst. Aqueous reporting layers, containing distinct "reporting enzymes" and their nicotinamide cofactors, are layered above the organic layers. The 1,2-propanediol enantiomers formed by the chiral catalyst diffuse into the aqueous layer and are oxidized there by the reporting enzymes at rates dependent upon the diol concentration, the R:S ratio of the diol, and the enantioselectivity of the reporting enzymes. A focused chiral salen library was constructed from seven chiral 1,2-diamines, derived from amino acid, terpenoid, and carbohydrates skeletons, and seven salicylaldehyde derivatives. Double-cuvette ISES identified a couple of interesting combinatorial hits in this salen array, wherein either the sense or magnitude of enantioselection for a given chiral diamine depends significantly upon the choice of "salicylaldehyde" partner. A comparison of predicted ee's and relative rates using this new screening tool with those independently measured is provided.

[reaction: see text] Mannose 6-phosphate mimics locked into the alpha-configuration and bearing hydrolase-resistant phosphate surrogates were synthesized and evaluated for binding affinity to the mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R). Affinity increases as the phosphate surrogate is varied in the order malonyl ether < malonate < phosphonate. An alkene cross-metathesis approach to sought-after bivalent M6P-bearing ligands is also described. These compounds were designed to map onto biantennary sectors of high-mannose-type oligosaccharides carried by glycoprotein M6P/IGF2R ligands.

An in situ enzymatic screening (ISES) approach to rapid catalyst evaluation recently pointed to Ni(0) as a new candidate transition metal for intramolecular allylic amination. This led to further exploration of chiral bidentate phosphine ligands for such transformations. Herein, a variety of P,N-ligands are examined for this Ni(0)-chemistry, using a model reaction leading into the vinylglycinol scaffold. On the one hand, an N , N -bis(2-diphenylphosphinoethyl)alkylamine ('PNP') ligand proved to be the fastest ligand yet seen for this Ni(0)-transformation. On the other, phosphinooxazoline (PHOX) ligands of the Pfaltz-Helmchen-Williams variety gave the highest enantioselectivities (up to 51% ee) among P,N-ligands examined.

An ISES (in situ enzymatic screening) lead pointed to conditions (PMP N-protecting group, Ni(cod)(2) catalyst precursor) under which chiral, bidentate phosphines could promote Ni(0)-mediated allylic amination. Therefore, bidentate phosphines bearing central, axial, and planar chirality were examined with two model substrates of interest for PLP-enzyme inhibitor synthesis. In the best case, with (R)-MeO-BIPHEP, vinylglycinol derivative 2 was obtained in 75% ee (97% ee, one recrystallization) from 1. Further manipulation provided a Ni(0)-mediated entry into l-vinylglycine. [reaction: see text]

Protected alpha-formyl amino acids, themselves available from the corresponding alpha-vinyl amino acids, are stereoselectively transformed into the (Z)-configured alpha-(2'-fluoro)vinyl amino acids via a three-step sequence. The route employs McCarthy's reagent, diethyl alpha-fluoro-alpha-(phenylsulfonyl)methyl phosphonate, and proceeds via the intermediate (E)-alpha-fluorovinyl sulfones and (E)-alpha-fluorovinylstannanes. The latter may either be exploited as novel cross-coupling partners for fluorovinyl branch extension or be globally deprotected, to provide the title compounds. [structure: see text]

To identify a selective inhibitor of mammalian agmatinase, screening was performed on four analogues of agmatine with modifications directly to the guanidine group, six analogues with modifications to the carbon-amine chain, and one analogue with modifications at both ends of the molecule. Control compounds were aminoguanidine and 7-nitroindazole, known inhibitors of the three isoforms (i, e, n) of nitric oxide synthase (NOS), and arcaine, a known inhibitor of the glutamate NMDA receptor. These compounds were compared for inhibition of rat agmatinase and arginine decarboxylase (ADC) activities. Results were studied by ab initio Hartee-Fock descriptors based on optimized geometries and van der Waals radii. Linear correlations were obtained using various geometric and electronic descriptors of the carbon (C), nitrogen (N), and hydrogen (H) atoms in the guanidine moiety. The best fit equation for percent activity remaining of rat agmatinase was = 0.3225 D + 72.76 D1916 + 64.97 D1920 - 192.58 H21 - 253.09 (r = 0.89), where D is the calculated dipole moment, D1916 and D1920 are the N19-N16 and N19-N20 distances, respectively, and H21 is the charge on H21. This agmatinase equation is distinct from the equations fit for ADC, the three NOS isoforms, and inhibition of NMDA receptor binding.

A pair of diastereomeric (4S,5S)- and (4S,5R)-4-methoxycarbonyl-5-phenylselenomethyl-2-phenyl oxazolines, derived from L-vinylglycine, serve as precursors to protected, quaternary, L- and D-α-(2-tributylstannyl)vinyl amino acids, respectively, in three steps {(i) alkylative side chain installation, (ii) eliminative ring-opening and (iii) vinyl selenide to vinyl stannane interconversion}. The title compounds may be protodestannylated to the corresponding free, quaternary L- and D-vinyl amino acids. Alternatively, the 2-stannylvinyl α-branch (or the derivative 2-iodovinyl branch) may be exploited to access novel quaternary, L- and D-β,γ-unsaturated amino acids via a range of transition metal-mediated cross coupling reactions.

[reaction: see text] Treatment of primary alkyl triflates or iodides with the potassium salt of diethyl (alpha-fluoro-alpha-phenylsulfonylmethyl)phosphonate yields (alpha-fluoro-alpha-phenylsulfonylalkyl)phosphonates. These can be cleanly desulfonated, in a matter of minutes, with Na(Hg) in MeOH/THF/NaH(2)PO(4). This two-step procedure complements previously reported triflate displacement approaches to alpha-nonfluorinated and alpha,alpha-difluorinated phosphonates.