1H and 13C NMR Assignments for (N-methyl)-(−)-(α)-Isosparteinium Iodide and (N-methyl)-(−)-Sparteinium Iodide

(‒)-Sparteine (1) and (–)-(α)-isosparteine (2) are members of the lupine alkaloid family.[1-2] Sparteine has found extensive use in asymmetric organic transformations, including lithiations[3] and Pd-catalyzed oxidations.[4-7] (α)-Isosparteine, which can be made from sparteine, has been utilized as a chiral ligand for a limited number of stereoselective reactions.[8-9] The two compounds differ in that 1 displays an exo-endo arrangement of the bridgehead hydrogens at C-11 and C-6, respectively, while 2 retains an exo-exo arrangement of these atoms (Figure 1). This study is focused on assigning 1H chemical shifts and coupling constants and 13C chemical shifts for N-methyl derivatives of sparteine and isosparteine, both of which have been fully characterized by X-ray crystallography. X-ray analysis of (N-methyl)-(–)-sparteinium iodide (3) revealed a chair-chair-boat-chair conformation (Figure 1),[10-11] and its 1H and 13C NMR chemical shift assignments were reported by Duddeck and co-workers in 1995.[12] An X-ray analysis of (N-methyl)-(α)-isosparteinium iodide (4) showed an all-chair conformation in which the N-CH3 group is positioned in close proximity to the transannular nitrogen lone pair, resulting in a +NCH•••N hydrogen bond.[13] Our group has harnessed the bridging geometry in 4 with an equilibrium isotope effect to investigate 1H and 3H chemical shift differences in (N-CH2D) and (N-CHDT) isotopologs of 4.[14-15] Simeonov, Duddeck, and co-workers have previously reported 1H and 13C NMR chemical shift assignments for 4 dissolved in DMSO-d6.[16] We noticed discrepancies between our 1H and 13C assignments for 3 and 4 and values reported in the earlier studies. This was especially true for the 1H data for 4, where 16 out of 27 assignments differ from the previously reported values. Spectral assignments for 3 and 4 are also compared with quantum-mechanically computed 13C and 1H NMR chemical shifts[17-21] to further validate the assignments reported here

Vibrational Analysis of a Rate-Slowing Conformational Kinetic Isotope Effect

An enthalpy-entropy approach to analyzing a rate-slowing conformational kinetic isotope effect (CKIE) in a deuterated doubly-bridged biaryl system is described. The computed isotope effect (kH/kD = 1.075, 368 K) agrees well with the measured value (kH/kD = 1.06, 368 K). The rateslowing (normal isotope effect) nature of the computed CKIE is shown to originate from a vibrational entropy contribution defined by the twenty lowest frequency normal modes in the ground state and transition state structures. This normal entropy contribution is offset by an inverse vibrational enthalpy contribution, which also arises from the twenty lowest frequency normal modes. Zero point vibrational energy contributions are found to be relatively small when all normal modes are considered. Analysis of the HZPE, Hvib, and Svib energy terms arising from the low frequency vibrational modes reveals their signs and magnitudes are determined by larger vibrational energy differences in the labeled and unlabeled ground state structures

Calcium Bistriflimide-Mediated Sulfur(VI)–Fluoride Exchange (SuFEx): Mechanistic Insights toward Instigating Catalysis

We report a mechanistic investigation of calcium bistriflimide-mediated sulfur(VI)–fluoride exchange (SuFEx) between sulfonyl fluorides and amines. We determine the likely pre-activation resting state─a calcium bistriflimide complex with ligated amines─thus allowing for corroborated calculation of the SuFEx activation barrier at ∼21 kcal/mol, compared to 21.5 ± 0.14 kcal/mol derived via kinetics experiments. Transition state analysis revealed: (1) a two-point calcium-substrate contact that activates the sulfur(VI) center and stabilizes the leaving fluoride and (2) a 1,4-diazabicyclo[2.2.2]octane additive that provides Brønsted-base activation of the nucleophilic amine. Stable Ca–F complexes upon sulfonamide formation are likely contributors to inhibited catalytic turnover, and a proof-of-principle redesign provided evidence that sulfonamide formation is feasible with 10 mol % calcium bistriflimide

Automating Data Analysis for Two-Dimensional Gas Chromatography/Time-of-Flight Mass Spectrometry Non-Targeted Analysis of Comparative Samples

Non-targeted analysis of environmental samples, using comprehensive two‐dimensional gas chromatography coupled with time-of-flight mass spectrometry (GC × GC/ToF-MS), poses significant data analysis challenges due to the large number of possible analytes. Non-targeted data analysis of complex mixtures is prone to human bias and is laborious, particularly for comparative environmental samples such as contaminated soil pre- and post-bioremediation. To address this research bottleneck, we developed OCTpy, a Python™ script that acts as a data reduction filter to automate GC × GC/ToF-MS data analysis from LECO® ChromaTOF® software and facilitates selection of analytes of interest based on peak area comparison between comparative samples. We used data from polycyclic aromatic hydrocarbon (PAH) contaminated soil, pre- and post‐bioremediation, to assess the effectiveness of OCTpy in facilitating the selection of analytes that have formed or degraded following treatment. Using datasets from the soil extracts pre- and post‐bioremediation, OCTpy selected, on average, 18% of the initial suggested analytes generated by the LECO® ChromaTOF® software Statistical Compare feature. Based on this list, 63–100% of the candidate analytes identified by a highly trained individual were also selected by OCTpy. This process was accomplished in several minutes per sample, whereas manual data analysis took several hours per sample. OCTpy automates the analysis of complex mixtures of comparative samples, reduces the potential for human error during heavy data handling and decreases data analysis time by at least tenfold

Carbodiimide and Isocyanate Hydroboration by a Cyclic Carbodiphosphorane Catalyst

We report hydroboration of carbodiimide and isocyanate substrates catalyzed by a cyclic carbodiphosphorane catalyst. The cyclic carbodiphosphorane outperformed the other Lewis basic carbon species tested, including other zerovalent carbon compounds, phosphorus ylides, an N-heterocyclic carbene, and an N-heterocyclic olefin. Hydroborations of seven carbodiimides and nine isocyanates were performed at room temperature to form N-boryl formamidine and N-boryl formamide products. Intermolecular competition experiments demonstrated the selective hydroboration of alkyl isocyanates over carbodiimide and ketone substrates. DFT calculations support a proposed mechanism involving activation of pinacolborane by the carbodiphosphorane catalyst, followed by hydride transfer and B−N bond formation

Peroxiredoxin Catalysis at Atomic Resolution

Peroxiredoxins (Prxs) are ubiquitous cysteine-based peroxidases that guard cells against oxidative damage, are virulence factors for pathogens, and are involved in eukaryotic redox regulatory pathways. We have analyzed catalytically active crystals to capture atomic resolution snapshots of a PrxQ-subfamily enzyme (from Xanthomonas campestris) proceeding through thiolate, sulfenate, and sulfinate species. These analyses provide structures of unprecedented accuracy for seeding theoretical studies, and show novel conformational intermediates giving insight into the reaction pathway. Based on a highly non-standard geometry seen for the sulfenate intermediate, we infer that the sulfenate formation itself can strongly promote local unfolding of the active site to enhance productive catalysis. Further, these structures reveal that preventing local unfolding, in this case via crystal contacts, results in facile hyperoxidative inactivation even for Prxs normally resistant to such inactivation. This supports previous proposals that conformation-specific inhibitors may be useful for achieving selective inhibition of Prxs that are drug targets

A bacterial inflammation sensor regulates c-di-GMP signaling, adhesion, and biofilm formation

The reactive oxygen species produced during inflammation through the neutrophilic respiratory burst play profound roles in combating bacterial pathogens and regulating the microbiota. Among these, the neutrophilic oxidant bleach, hypochlorous acid (HOCl), is the most prevalent and strongest oxidizer and kills bacteria through non-specific oxidation of proteins, lipids, and DNA. Thus, HOCl can be viewed as a host-specific cue that conveys important information about what bacterial physiology and lifestyle programs may be required for successful colonization. Nevertheless, bacteria that colonize animals face a molecular challenge in how to achieve highly selective detection of HOCl due to its reactive and transient nature and chemical similarity to more benign and non-host-specific oxidants like hydrogen peroxide (H2O2). Here, we report that in response to increasing HOCl levels E. coli regulates biofilm production via activation of the diguanylate cyclase DgcZ. We show the molecular mechanism of this activation to be specific oxidation of a conserved cysteine that coordinates the zinc of its regulatory chemoreceptor zinc-binding (CZB) domain, forming a zinc-cysteine redox switch 685-fold more sensitive to oxidation by HOCl over H2O2. Dissection of the signal transduction mechanism through quantum mechanics, molecular dynamics, and biochemical analyses reveal how the cysteine redox state alters the delicate equilibrium of competition for Zn++ between the CZB domain and other zinc binders to relay the presence of HOCl through activating the associated GGDEF domain to catalyze c-di-GMP. We find biofilm formation and HOCl-sensing in vivo to be regulated by the conserved cysteine, and point mutants that mimic oxidized CZB states increase production of the biofilm matrix polymer poly-N-acetylglucosamine and total biofilm. We observe CZB-regulated diguanylate cyclases and chemoreceptors in phyla in which host-associated bacteria are prevalent and are possessed by pathogens that manipulate host inflammation as part of their colonization strategy. A phylogenetic survey of all known CZB sequences shows these domains to be conserved and widespread across diverse phyla, suggesting CZB origin predates the bacterial last universal common ancestor. The ability of bacteria to use CZB protein domains to perceive and thwart the host neutrophilic respiratory burst has implications for understanding the mechanisms of diseases of chronic inflammation and gut dysbiosis

A bacterial inflammation sensor regulates c-di-GMP signaling, adhesion, and biofilm formation

Bacteria that colonize animals must overcome, or coexist, with the reactive oxygen species products of inflammation, a front-line defense of innate immunity. Among these is the neutrophilic oxidant bleach, hypochlorous acid (HOCl), a potent antimicrobial that plays a primary role in killing bacteria through nonspecific oxidation of proteins, lipids, and DNA. Here, we report that in response to increasing HOCl levels, Escherichia coli regulates biofilm production via activation of the diguanylate cyclase DgcZ. We identify the mechanism of DgcZ sensing of HOCl to be direct oxidation of its regulatory chemoreceptor zinc-binding (CZB) domain. Dissection of CZB signal transduction reveals that oxidation of the conserved zinc-binding cysteine controls CZB Zn2+ occupancy, which in turn regulates the catalysis of c-di-GMP by the associated GGDEF domain. We find DgcZ-dependent biofilm formation and HOCl sensing to be regulated in vivo by the conserved zinc-coordinating cysteine. Additionally, point mutants that mimic oxidized CZB states increase total biofilm. A survey of bacterial genomes reveals that many pathogenic bacteria that manipulate host inflammation as part of their colonization strategy possess CZB-regulated diguanylate cyclases and chemoreceptors. Our findings suggest that CZB domains are zinc-sensitive regulators that allow host-associated bacteria to perceive host inflammation through reactivity with HOCl

MIDA boronates are hydrolysed fast and slow by two different mechanisms

MIDA boronates (N-methylimidodiacetic boronic acid esters) serve as an increasingly general platform for small-molecule construction based on building blocks, largely because of the dramatic and general rate differences with which they are hydrolysed under various basic conditions. Yet the mechanistic underpinnings of these rate differences have remained unclear, which has hindered efforts to address the current limitations of this chemistry. Here we show that there are two distinct mechanisms for this hydrolysis: one is base mediated and the other neutral. The former can proceed more than three orders of magnitude faster than the latter, and involves a rate-limiting attack by a hydroxide at a MIDA carbonyl carbon. The alternative 'neutral' hydrolysis does not require an exogenous acid or base and involves rate-limiting B-N bond cleavage by a small water cluster, (H2O)n. The two mechanisms can operate in parallel, and their relative rates are readily quantified by (18)O incorporation. Whether hydrolysis is 'fast' or 'slow' is dictated by the pH, the water activity and the mass-transfer rates between phases. These findings stand to enable, in a rational way, an even more effective and widespread utilization of MIDA boronates in synthesis

Mechanism and Chemoselectivity for HOCl-Mediated Oxidation of Zinc-Bound Thiolates

Quantum mechanical calculations reveal the preferred mechanism and origins of chemoselectivity for HOCl‐mediated oxidation of zinc‐bound thiolates implicated in bacterial redox sensing. Distortion/interaction models show that minimizing geometric distortion at the zinc complex during the rate‐limiting nucleophilic substitution step controls the mechanistic preference for OH over Cl transfer with HOCl and the chemoselectivity for HOCl over H2O2