Intermediate Analogue Research Articles

Direct Current to Digital Converter (DIDC): A Current Sensor

This paper introduces a systematic approach to the design of Direct Current-to-Digital Converter (DIDC) specifically engineered to overcome the limitations of traditional current measurement methodologies in System-on-Chip (SoC) designs. The proposed DIDC addresses critical challenges such as high power consumption, large area requirements, and the need for intermediate analog signals. By incorporating a current mirror in a cascode topology and managing the current across multiple binary-sized branches with the Successive Approximation Register (SAR) logic, the design achieves precise current measurement. A simple comparator, coupled with an isolation circuit, ensures accurate and reliable sensing. Fabricated using the TSMC 180 nm process, the DIDC achieves 8-bit precision without the need for nonlinearity calibration, showcasing remarkable energy efficiency with an energy per conversion of 1.52 pJ, power consumption of 117 µW, and a compact area of 0.016 mm². This innovative approach not only reduces power consumption and area, but also provides a scalable and efficient solution for next-generation semiconductor technologies. The ability to conduct online measurements during both standard operations and in-field conditions significantly enhances the performance and reliability of SoCs, making this DIDC a promising advancement in the field.

Catalyst Control over S(IV)-stereogenicity via Carbene-derived Sulfinyl Azolium Intermediates.

Stereoselective synthesis utilizing small-molecule catalysts, particularly N-heterocyclic carbene (NHC), has facilitated swift access to enantioenriched molecules through diverse activation modes and NHC-bound reactive intermediates. While carbonyl derivatives, imines, and "activated" alkenes have been extensively investigated, the exploration of heteroatom-centered analogues of NHC-bound intermediates has long been neglected, despite the significant potential for novel chemical transformations they offer once recognized. Herein, we disclose a carbene-catalyzed new activation mode by generating unique sulfinyl azolium intermediates from carbene nucleophilic addition to in situ-generated mixed sulfinic anhydride intermediates. Combined experimental and computational mechanistic investigations pinpoint the chiral NHC-catalyzed formation of sulfinyl azolium intermediate as the enantio-determining step. The novel "S"-based carbene reactive intermediate imparts high efficiency for the catalytic construction of sulfur-stereogenic compounds, giving rise to sulfinate esters with high yields and enantioselectivities under mild conditions. Notably, distinct from most of the NHC-catalyzed enantioselective transformations focusing on the "C" central chiral products, our study realizes a unique carbene-catalyst control over chiral "S" stereocenters via direct asymmetric S-O bond formation for the first time. Furthermore, these sulfinyl-containing products could serve as versatile synthetic platforms for enantioenriched S-stereogenic functional molecules and exhibit remarkable antibacterial activities against rice plant pathogens, which is valuable for the development of novel agrochemical agents.

An inhibitory mechanism of AasS, an exogenous fatty acid scavenger: Implications for re-sensitization of FAS II antimicrobials.

Antimicrobial resistance is an ongoing "one health" challenge of global concern. The acyl-ACP synthetase (termed AasS) of the zoonotic pathogen Vibrio harveyi recycles exogenous fatty acid (eFA), bypassing the requirement of type II fatty acid synthesis (FAS II), a druggable pathway. A growing body of bacterial AasS-type isoenzymes compromises the clinical efficacy of FAS II-directed antimicrobials, like cerulenin. Very recently, an acyl adenylate mimic, C10-AMS, was proposed as a lead compound against AasS activity. However, the underlying mechanism remains poorly understood. Here we present two high-resolution cryo-EM structures of AasS liganded with C10-AMS inhibitor (2.33 Å) and C10-AMP intermediate (2.19 Å) in addition to its apo form (2.53 Å). Apart from our measurements for C10-AMS' Ki value of around 0.6 μM, structural and functional analyses explained how this inhibitor interacts with AasS enzyme. Unlike an open state of AasS, ready for C10-AMP formation, a closed conformation is trapped by the C10-AMS inhibitor. Tight binding of C10-AMS blocks fatty acyl substrate entry, and therefore inhibits AasS action. Additionally, this intermediate analog C10-AMS appears to be a mixed-type AasS inhibitor. In summary, our results provide the proof of principle that inhibiting salvage of eFA by AasS reverses the FAS II bypass. This facilitates the development of next-generation anti-bacterial therapeutics, esp. the dual therapy consisting of C10-AMS scaffold derivatives combined with certain FAS II inhibitors.

Sensitive quantification of mevalonate pathway intermediates and prediction of relative novel analogs by chemical derivatization-based LC-MS/MS

The mevalonate (MVA) pathway plays a crucial role in the occurrence and progression of various diseases, such as osteoporosis, breast cancer, and lung cancer, etc. However, determining all the MVA pathway intermediates is still challenging due to their high polarity, low concentration, chelation effect with metal compartments, and poor mass spectrometric response. In this study, we established a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method coupled with N2, N2, N4, N4-tetramethyl-6-(4-(piperazin-1-ylsulfonyl) phenyl)-1,3,5-triazine-2,4-diamine (Tmt-PP) labeling for the simultaneous analysis of all MVA intermediates in biospecimens. Chemical derivatization significantly improved the chromatographic retention, peak shape, and detection sensitivity of the analytes. Moreover, we employed a method named mass spectrum calculation to achieve the absolute quantification of the isomers, i.e., isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). The established method was fully qualified and applied to explore the difference of these metabolites in cisplatin-resistant non-small cell lung cancer (NSCLC) cells. Additionally, several MVA intermediate analogs, including isopentenyl monophosphate or dimethylallyl monophosphate (IMP/DMAMP), geranyl monophosphate (GMP), 5-triphosphomevalonate (MTP), and isopentenyl triphosphate or dimethylallyl triphosphate (ITP/DMATP), were identified for the first time using a knowledge-driven prediction strategy. We further explored the tissue distribution of these novel metabolites. Overall, this work developed a sensitive quantification method for all MVA intermediates, which will enhance our understanding of the role of this pathway in various health and disease conditions. The novel metabolites we discovered warrant further investigations into their biosynthesis and biological functions.

Engineering the Substrate Specificity of (S)-β-Phenylalanine Adenylation Enzyme HitB.

Adenylation enzymes catalyze the selective incorporation of aminoacyl building blocks in the biosynthesis of nonribosomal peptides and related natural products. Although β-amino acid units are one of the important aminoacyl building blocks in natural product biosynthesis, very little is known about the engineering of β-amino acid adenylation enzymes. In this study, we engineered the substrate specificity of the (S)-β-phenylalanine adenylation enzyme, HitB, involved in the biosynthesis of macrolactam polyketide hitachimycin. Based on the previously determined structure of HitB wild-type, we mutated Phe328 and Ser293, which are located near the meta and ortho position of the (S)-β-phenylalanine moiety, respectively. As a result, the HitB F328V and F328L mutants efficiently activated meta-substituted (S)-β-phenylalanine analogs, and the HitB T293G and T293S mutants efficiently activated ortho-substituted (S)-β-phenylalanine analogs. Structural analysis of the HitB F328L and T293G mutants with the corresponding nonhydrolyzable intermediate analogs revealed an enlarged substrate binding pocket for (S)-β-phenylalanine analogs, providing detailed insights into the structural basis for creating enzyme substrate promiscuity. Our findings may be useful for production of various β-amino acid-containing natural product analogs.

Synthetic Advances for Mechanistic Insights: Metal-Oxygen Intermediates with a Macrocyclic Pyridinophane System.

ConspectusMetalloenzymes, which are proteins containing earth-abundant transition-metal ions as cofactors in the active site, generate various metal-oxygen intermediates via activating a dioxygen molecule (O2) to mediate vital metabolic functions, such as the oxidative metabolism of xenobiotics and the biotransformation of naturally occurring molecules. By replicating the active sites of metalloenzymes, many bioinorganic chemists have studied the geometric and electronic properties and reactivities of model complexes to understand the nature of enzymatic intermediates and develop bioinspired metal catalysts. Among the reported model complexes, nonporphyrinic macrocyclic ligands are the predominant coordination system widely used in stabilizing and isolating diverse metal-oxygen intermediates, which allows us to extensively investigate the physicochemical characteristics of the analogs of reactive intermediates of metalloenzymes. In particular, it has been reported that the ring size of the macrocyclic ligands, defined by the number of atoms in the macrocyclic ring, drastically affects the identity of the metal-oxygen intermediate. Thus, systematic modification of the macrocyclic ligands has been a great subject being examined in various inorganic fields.In this Account, we describe synthetic advances of a macrocyclic ligand system by introducing pyridine donors into a 12-membered tetraazamacrocyclic ligand (12-TMC) that initially has 4 amine donors. Interestingly, the backbone of the pyridinophane ligand with 2 pyridine and 2 amine donors in a 12-membered ring is shown to be much more folded than in other macrocyclic ligands, thereby allowing the axial and equatorial donors to separately control the electronic structure of metal complexes. Then, we looked over independent electronic and steric effects on metal-oxygen species with thorough physicochemical analysis. The NiIII-peroxo complexes exhibit nucleophilic reactivity dependent on the steric hindrance of the second coordination sphere. Furthermore, the C-H bond strength of the second coordination sphere has also been an important factor in determining the stability of MnIV-bis(hydroxo) intermediates. Electronic tuning on CoIII-hydroperoxo intermediates results in a trend between the electron-donating abilities of para-substituents on pyridine in the pyridinophane ligand and electrophilic reactivities, from which mechanistic insights into the metal-hydroperoxo species have been gained. Importantly, the metal-oxygen intermediates supported by the pyridinophane ligand system have revealed quite challenging chemical reactions, including dioxygenase-like nitrile activation by CoIII-peroxo intermediates and the oxidation of aldehyde and aromatic compounds by manganese-oxygen intermediates. Based on the fine substitution of donors, we have addressed that those novel reactions originated from the unique framework of the pyridinophane system incorporating spin-crossover behavior and high redox potentials of the metal-oxygen intermediates. These results will be valuable for the structure-activity relationship of metal-oxygen intermediates, giving a better understanding on the enzymatic coordination system where amino acid ligands vary for specific chemical reactions.

Triosephosphate Isomerase: The Crippling Effect of the P168A/I172A Substitution at the Heart of an Enzyme Active Site.

The P168 and I172 side chains sit at the heart of the active site of triosephosphate isomerase (TIM) and play important roles in the catalysis of the isomerization reaction. The phosphodianion of substrate glyceraldehyde 3-phosphate (GAP) drives a conformational change at the TIM that creates a steric interaction with the P168 side chain that is relieved by the movement of P168 that carries the basic E167 side chain into a clamp that consists of the hydrophobic I172 and L232 side chains. The P168A/I172A substitution at TIM from Trypanosoma brucei brucei (TbbTIM) causes a large 120,000-fold decrease in kcat for isomerization of GAP that eliminates most of the difference in the reactivity of TIM compared to the small amine base quinuclidinone for deprotonation of catalyst-bound GAP. The I172A substitution causes a > 2-unit decrease in the pKa of the E167 carboxylic acid in a complex to the intermediate analog PGA, but the P168A substitution at the I172A variant has no further effect on this pKa. The P168A/I172A substitutions cause a 5-fold decrease in Km for the isomerization of GAP from a 0.9 kcal/mol stabilization of the substrate Michaelis complexes. The results show that the P168 and I172 side chains play a dual role in destabilizing the ground-state Michaelis complex to GAP and in promoting stabilization of the transition state for substrate isomerization. This is consistent with an important role for these side chains in an induced fit reaction mechanism [Richard, J. P. (2022) Enabling Role of Ligand-Driven Conformational Changes in Enzyme Evolution. Biochemistry 61, 1533-1542].

Study of Automatic Gain Control System in Broadband Radio Receiver

In modern times, almost all communication systems are digital. In the receivers of such communication systems, the analog signal is discretized and quantized using an analog-to-digital converter at an intermediate frequency. Automatic gain control is used to avoid overloading the analog-to-digital converter and to maintain intermediate frequency analog signals at a suitable fixed level. If the power of the received signals is much less than the load level of the analog-to-digital converter, the automatic gain adjustment increases the gain, reducing the quantization noise. If the received signal strength is high, the automatic gain adjustment lowers the gain so that the analog-to-digital converter is not overloaded. The accuracy of the adjustment of the transmission coefficient of the automatic adjustment of the digital amplification does not always satisfy the requirements given in real time. Based on the above, the article is devoted to the study of the digital automatic gain regulator of the broadband radio receiver.

Rh(iii)-catalyzed highly site- and regio-selective alkenyl C-H activation/annulation of 4-amino-2-quinolones with alkynes via reversible alkyne insertion.

3,4-Fused 2-quinolone frameworks are important structural motifs found in natural products and biologically active compounds. Intermolecular alkenyl C-H activation/annulation of 4-amino-2-quinolone substrates with alkynes is one of the most efficient methods for accessing such structural motifs. However, this is a formidable challenge because 4-amino-2-quinolones have two cleavable C-H bonds: an alkenyl C-H bond at the C3-position and an aromatic C-H bond at the C5-position. Herein, we report the Rh(iii)-catalyzed highly site-selective alkenyl C-H functionalization of 4-amino-2-quinolones to afford 3,4-fused 2-quinolones. This method has a wide substrate scope, including unsymmetrical internal alkynes, with complete regioselectivity. Several control experiments using an isolated key intermediate analog suggested that the annulation reaction proceeds via reversible alkyne insertion involving a binuclear Rh complex although alkyne insertion is generally recognized as an irreversible process due to the high activation barrier of the reverse process.

Optimized Synthesis of a Key Intermediate of Nirmatrelvir

In this study, the development of a concise alternative process for synthesis of a key nirmatrelvir intermediate cyclic glutamine analog is described. The process proceedes via α-cyanomethylation of dimethyl N-BocGlu in the presence of NdCl3, followed by one-pot Raney nickel-catalyzed hydrogenation of the cyano group with concomitant cyclization and ammonolysis and subsequent deprotection of N-Boc to deliver the target intermediate cyclic glutamine analog in three steps with an 82% overall yield and 99.4 A% high-performance liquid chromatography (HPLC) purity. This study resulted in improved synthetic efficiency and stereoselectivity of α-cyanomethylation for production of a key nirmatrelvir intermediate cyclic glutamine analog.

Crystal structure of a type III Rubisco in complex with its product 3-phosphoglycerate.

The crystal structure of the complex of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) from Archaeoglobus fulgidus (afRubisco) with its products 3PGAs has been determined to a resolution of 1.7Å and is of the closed form. Type III Rubiscos such as afRubisco have 18 out of the 19 essential amino acid residues of canonical Rubisco; the 19th is Tyr rather than Phe. Superposition with the structure of a complex of the similar tkRubisco with the six-carbon intermediate analog 2CABP shows the same conformation of the 19 residues except for Glu46 and Thr51. Glu46 adopts a unique conformation different from that in other Rubiscos and makes two H-bonds with the ligand 3PGA. Similar to other closed state Rubiscos, the backbone of Thr51 is rotated and the side chain makes an H-bond with the ligand 3PGA. Two product 3PGA molecules are bound at the active site, overlapping well with the 2CABP of tkRubisco/2CABP. The positions of the P1 and P2 phosphate groups differ by 0.4 and 0.53 Å, respectively, between 2CABP and the two 3PGAs. This afRubisco/3PGA complex mimics an intermediate stage of the carboxylation reaction which occurs after the production of the two 3PGA products but before the reopening of the active site. The stability of this complex suggests that the Rubisco active site will not reopen before both 3PGA products are formed.

Cytosine as a root to a nonconventional layered hydroxide nanostructure

A self-assembled layered compound of formula {[Cu5(OH)8Cyt2]·2ClO4} n (1) was obtained from the direct reaction between copper perchlorate and cytosine. Thanks to its peculiar structural features, this coordination compound can be classified as an intermediate nonconventional analog of Layered Simple Hydroxides (LSHs) and Layered Double Hydroxides (LDHs), two fascinating classes of inorganic materials with variable chemical composition and structures. Like LDHs, 1 consists of positively charged layers of metal hydroxides and non-framework charge-balancing anions located between the layers. However, the hydroxide layers, similar to LSHs, contain only divalent copper(II) metal ions, supported by cytosine molecules with long contacts. Cytosine coordinates to the metal centers adopting an unusual µ3 coordination mode, the exocyclic oxygen atom simultaneously binding to three adjacent copper(II) centers. This coordination mode has not been observed with transition metal ions. This work further demonstrates that nucleobases and their derivatives, despite presenting a limited pool of molecules, can potentially afford a significantly expanded set of compounds with diverse nanostructures, owing to their multiple metal binding sites of different coordination properties and their ability to direct assembly of highly ordered structures. Their potential as building blocks should be further explored for the preparation of new bio-inorganic hybrid materials.

Organometallic μ-Nitridodiiron Complexes in Oxidation States Ranging from (III/III) to (IV/IV)

Iron complexes with nitrido ligands are of interest as molecular analogues of key intermediates during N2-to-NH3 conversion in industrial or enzymatic processes. Dinuclear iron complexes with a bridging nitrido unit are mostly known in relatively high oxidation states (III/IV or IV/IV), originating from the decomposition of azidoiron precursors via high-valent Fe≡N intermediates. The use of a tetra-NHC macrocyclic scaffold ligand (NHC = N-heterocyclic carbene) has now allowed for the isolation of a series of organometallic μ-nitridodiiron complexes ranging from the mid-valent FeIII-N-FeIII (1) via mixed-valent FeIII-N-FeIV (type 4) to the high-valent FeIV-N-FeIV (type 5) species that are interconverted at moderate potentials, accompanied by axial ligand binding at the FeIV sites. Magnetic measurements and electron paramagnetic resonance spectroscopy showed the homovalent complexes to be diamagnetic and the mixed-valent system to feature an S = 1/2 ground state due to very strong antiferromagnetic coupling. The bonding in the Fe-N-Fe moiety has been further probed by crystallographic structure determination, 57Fe Mössbauer and UV-vis spectroscopies, as well as density functional theory computations, which revealed high covalency and nearly identical Fe-N distances across this redox series. The latter has been rationalized in terms of the nonbonding nature of the combination of Fe dz2 atomic orbitals from which electrons are successively removed upon oxidation, and these redox processes are best described as being metal-centered. The tetra-NHC-ligated μ-nitridodiiron series complements a set of related complexes with single-atom μ-oxido and μ-phosphido bridges, but the Fe-N-Fe core exhibits a comparatively high stability over several oxidation states. This promises interesting applications in view of the manifold catalytic uses of μ-nitridodiiron complexes based on macrocyclic N-donor porphinato(2-) or phthalocyaninato(2-) ligands.

Structure and function of an N-acetyltransferase from the human pathogen Acinetobacter baumannii isolate BAL_212.

Acinetobacter baumannii is a Gram‐negative bacterium commonly found in soil and water that can cause human infections of the blood, lungs, and urinary tract. Of particular concern is its prevalence in health‐care settings where it can survive on surfaces and shared equipment for extended periods of time. The capsular polysaccharide surrounding the organism is known to be the major contributor to virulence. The structure of the K57 capsular polysaccharide produced by A. baumannii isolate BAL_212 from Vietnam was recently shown to contain the rare sugar 4‐acetamido‐4,6‐dideoxy‐d‐glucose. Three enzymes are required for its biosynthesis, one of which is encoded by the gene H6W49_RS17300 and referred to as VioB, a putative N‐acetyltransferase. Here, we describe a combined structural and functional analysis of VioB. Kinetic analyses show that the enzyme does, indeed, function on dTDP‐4‐amino‐4,6‐dideoxy‐d‐glucose with a catalytic efficiency of 3.9 x 104 M−1 s−1 (±6000), albeit at a reduced value compared to similar enzymes. Three high‐resolution X‐ray structures of various enzyme/ligand complexes were determined to resolutions of 1.65 Å or better. One of these models represents an intermediate analogue of the tetrahedral transition state. Differences between the VioB structure and those determined for the N‐acetyltransferases from Campylobacter jejuni (PglD), Caulobacter crescentus (PerB), and Psychrobacter cryohalolentis (Pcryo_0637) are highlighted. Taken together, this investigation sheds new insight into the Type I sugar N‐acetyltransferases.

Explorations of the nonheme high-valent iron-oxo landscape: crystal structure of a synthetic complex with an [FeIV2(μ-O)2] diamond core relevant to the chemistry of sMMOH.

Methanotrophic bacteria utilize methane monooxygenase (MMO) to carry out the first step in metabolizing methane. The soluble enzymes employ a hydroxylase component (sMMOH) with a nonheme diiron active site that activates O2 and generates a powerful oxidant capable of converting methane to methanol. It is proposed that the diiron(II) center in the reduced enzyme reacts with O2 to generate a diferric-peroxo intermediate called P that then undergoes O-O cleavage to convert into a diiron(IV) derivative called Q, which carries out methane hydroxylation. Most (but not all) of the spectroscopic data of Q accumulated by various groups to date favor the presence of an FeIV2(μ-O)2 unit with a diamond core. The Que lab has had a long-term interest in making synthetic analogs of iron enzyme intermediates. To this end, the first crystal structure of a complex with a FeIIIFeIV(μ-O)2 diamond core was reported in 1999, which exhibited an Fe⋯Fe distance of 2.683(1) Å. Now more than 20 years later, a complex with an FeIV2(μ-O)2 diamond core has been synthesized in sufficient purity to allow diffraction-quality crystals to be grown. Its crystal structure has been solved, revealing an Fe⋯Fe distance of 2.711(4) Å for comparison with structural data for related complexes with lower iron oxidation states.

Hybrid Beamformer Exploiting Multistream per User Transmission for Millimeter-Wave NOMA Communications

This treatise investigates multistream hybrid beamforming (HBF) for a millimeter-wave non-orthogonal multiple access (NOMA) system deployed in a downlink of an urban microcell environment. Maximization problem is developed to optimize sum-rate. For the sake of ensuring high correlation of users’ channels, user’s clustering and ordering are based on the angle of arrivals and users’ channel-weights, respectively. An optimized analog combiner of each user is obtained from the first <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$N_{s}$ </tex-math></inline-formula> column vectors of the left unitary matrix derived from the singular value decomposition of the channel of each user. Analog beamformer is matched to the phase of the strong users ’ composite intermediate analog channel to maximize the beamforming gain. Both the analog combiner and precoder for sub-connected structure (SCS) are formulated via a novel dominant sub-array matrix elements’ extractor. Conventional zero-forcing processing is contemplated for digital precoding. Memory space complexity is evaluated to corroborate the simplicity of the proposed schemes’ computational complexity. Results obtained from the simulation exhibit that HBF-NOMA attain superior sum-rate than HBF-orthogonal multiple access and conventional multiuser schemes in line of sight link. Lastly, the proposed SCS-HBF-NOMA precoding scheme performs higher than fully connected counterpart in terms of energy efficiency.

Enantioenriched BCP Benzylamine Synthesis via Metal Hydride Hydrogen Atom Transfer/Sulfinimine Addition to [1.1.1]Propellane.

The stereoselective synthesis of bicyclo[1.1.1]pentane (BCP) benzylamine derivatives from [1.1.1]propellane and mesityl sulfinimines via metal hydride hydrogen atom transfer (MH HAT) is reported. Medicinally relevant heterocyclic BCP methanamines are prepared with high diastereoselectivity. The strategic impact of the method is demonstrated via the streamlined synthesis of the BCP analogue of a key levocetirizine intermediate. Mechanistic evidence for a competitive H2 evolution pathway and the importance of controlled silane addition during reaction initiation are disclosed.

Amino Acid Based Antimicrobial Agents - Synthesis and Properties.

Structures of several dozen of known antibacterial, antifungal or antiprotozoal agents are based on the amino acid scaffold. In most of them, the amino acid skeleton is of a crucial importance for their antimicrobial activity, since very often they are structural analogs of amino acid intermediates of different microbial biosynthetic pathways. Particularly, some aminophosphonate or aminoboronate analogs of protein amino acids are effective enzyme inhibitors, as structural mimics of tetrahedral transition state intermediates. Synthesis of amino acid antimicrobials is a particular challenge, especially in terms of the need for enantioselective methods, including the asymmetric synthesis. All these issues are addressed in this review, summing up the current state‐of‐the‐art and presenting perspectives fur further progress.

The Phospha-Bora-Wittig Reaction

We report the phospha-bora-Wittig reaction for the direct preparation of phosphaalkenes from aldehydes, ketones, esters, or amides. The transient phosphaborene Mes*P=B–NR2 reacts with carbonyl compounds to form 1,2,3-phosphaboraoxetanes, analogues of oxaphosphetane intermediates in the classical Wittig reaction. 1,2,3-Phosphaboraoxetanes undergo thermal or Lewis acid-promoted cycloreversion, yielding phosphaalkenes. Experimental and density functional theory studies reveal far-reaching similarities between classical and phospha-bora-Wittig reactions.

Caught in Action: X-ray Structure of Thymidylate Synthase with Noncovalent Intermediate Analog.

Methylation of 2-deoxyuridine-5'-monophosphate (dUMP) at the C5 position by the obligate dimeric thymidylate synthase (TSase) in the sole de novo biosynthetic pathway to thymidine 5'-monophosphate (dTMP) proceeds by forming a covalent ternary complex with dUMP and cosubstrate 5,10-methylenetetrahydrofolate. The crystal structure of an analog of this intermediate gives important mechanistic insights but does not explain the half-of-the-sites activity of the enzyme. Recent experiments showed that the C5 proton and the catalytic Cys are eliminated in a concerted manner from the covalent ternary complex to produce a noncovalent bisubstrate intermediate. Here, we report the crystal structure of TSase with a close synthetic analog of this intermediate in which it has partially reacted with the enzyme but in only one protomer, consistent with the half-of-the-sites activity of this enzyme. Quantum mechanics/molecular mechanics simulations confirmed that the analog could undergo catalysis. The crystal structure shows a new water 2.9 Å from the critical C5 of the dUMP moiety, which in conjunction with other residues in the network, may be the elusive general base that abstracts the C5 proton of dUMP during the reaction.