Tris(4-bromophenyl)aminium Hexachloroantimonate-Mediated Intermolecular C(sp2)-C(sp3) Free Radical Coupling of Vindoline with ß-Ketoesters and Related Compounds.

A powerful tris(4-bromophenyl)aminium hexachloroantimonate (BAHA) mediated regioselective intermolecular coupling reaction of vindoline with a wide range of substrates that include ß-ketoesters, ß-diketones, ß-ketoaldehydes, ß-ketonitriles, ß-ketolactones, ß-ketolactams, ß-cyanoesters, and malononitriles is detailed. The BAHA-promoted intermolecular sp3/sp2 coupling, representing a special class of selective C-H functionalization reactions with direct carbon-carbon bond formation, proceeds with generation of a quaternary center bound to the aryl C15 center of vindoline capable of accommodating of the vinblastine C16' methyl ester and functionalized for subsequent divergent heterocycle introduction. A comprehensive examination of the reaction scope, optimization of subtle reaction parameters, and key insights into the reaction mechanism are described. Contrary to what might be prevailing expectations, studies suggest the plausible mechanism entails initial single-electron oxidation of the substrate enolate, not vindoline, and subsequent regiospecific addition of the resulting electrophilic radical to vindoline. As such and beyond the new arylation reaction with vindoline, the studies define a host of new, previously unrecognized, applications of BAHA and related triarylaminium radical cations that arises from their ability to generate stabilized electrophilic radicals from ß-ketoesters and related substrates under nonreducing and metal-free conditions. Those exemplified herein include mediating stabilized enolate free radical arylation, dimerization, allylation, alkene addition, and α-oxidation reactions.

Isotopic analysis of otolith carbonates using laser heating: A fast method for obtaining high-resolution climate signal.

RATIONALE: The stable isotopic compositions of biogenic carbonates like fish otoliths (ear bones) are widely used for palaeoclimatic reconstruction. The conventional method using acid-digestion of micro-milled samples is a multi-step time-consuming process. Here we report a fast method based on laser heating of otolith carbonates to obtain accurate and high-resolution stable isotopic compositions. METHOD: Otoliths of catfish from the Gulf of Kutch were analysed to check the precision, accuracy and time-resolution of the isotope ratios. The CO2 , generated by heating otoliths with a 50 W CO2 laser, was analysed for its oxygen and carbon isotope ratio [δ18 O and δ13 C, with precision: 0.12 and 0.17‰ (1σ), accuracy: 0.13 and 0.25‰, respectively] using a continuous-flow isotope ratio mass spectrometer. The effect of laser power (0.7-2 W) was assessed for reproducible data. Samples were roasted and analysed to account for the effect of the inherent organic matter on the isotopic values. RESULTS: Roasting did not alter the δ18 O of the otoliths but increased the δ13 C slightly. High-resolution (125 µm) analysis of the right and left otolith of a fish yielded similar δ18 O and δ13 C values, suggesting the suitability of either of them for deriving the climate signal. An increase in δ18 O values from ~ -2‰ to ~ -1‰, observed across the ontogeny, is consistent with the known migratory behaviour of the catfish between freshwater and the sea. CONCLUSIONS: The otolith δ18 O value of an adult fish records the sea surface temperature (with ~3°C uncertainty) on a monthly scale. The otolith δ13 C values, with the knowledge of dietary δ13 C, provide the mean annual δ13 C value of dissolved inorganic carbon. The study provides a rapid method for retrieving high-resolution seasonal climate data from otoliths found aplenty in geological/archaeological records.

Synthesis, structure-activity relationship studies and evaluation of a TLR 3/8/9 agonist and its analogues.

A comprehensive SAR study of a putative TLR 3/8/9 agonist was conducted. Despite the excitement surrounding the potential of the first small molecule TLR3 agonist with a compound that additionally displayed agonist activity for TLR8 and TLR9, compound 1 displayed disappointing activity in our hands, failing to match the potency (EC50) reported and displaying only a low efficacy for the extent of stimulated NF-κB activation and release. The evaluation of >75 analogs of 1, many of which constitute minor modifications in the structure, failed to identify any that displayed significant activity and none that exceeded the modest activity found for 1.

A simple cryogenic method for efficient analysis of triple oxygen isotopes in silicates.

RATIONALE: Oxygen isotopic ratios of silicates are excellent tools to reconstruct paleotemperature and isotopic composition of the precipitating fluid. However, the measurement of 17 O/16 O is difficult due to the low abundance of 17 O. The present study reports a simplified high-precision analytical technique for measuring the two oxygen isotope ratios, 17 O/16 O and 18 O/16 O, in silicates. METHODS: Silicate samples were ablated by a CO2 laser in a BrF5 environment. The released oxygen (O2 ) was then cryogenically trapped in a molecular sieve zeolite (MSZ). Associated contaminants such as BrF5 , F2 , NF3 etc. were cleaned by passing the gas through a NaCl trap followed by a cooled (-25°C) MSZ-packed U-tube trap. The purified O2 was analysed in a MAT 253 isotope ratio mass spectrometer for oxygen isotope ratios. RESULTS: The δδ17 O and δ18 O values of the working gas were calibrated by NBS-28 and crosschecked by inter-laboratory references UWG-2, SCO and IMAU-O2 . The average analytical precisions (using aliquots of NBS-28, UWG-2, SCO, and laboratory internal standards IIT-KGP-SQ quartz and IIT-KGP-NQ quartz) of the δ17 O, δ18 O and ∆'17 O values were 0.04‰, 0.08‰ and 4 per meg, respectively. CONCLUSIONS: A new cryogenic cleaning technique was developed that does not require GC but efficiently removes NF3 -contaminants from oxygen gas produced by laser fluorination of silicates. The technique is simple, quick and cost-effective and provides highly precise and accurate δ17 O, δ18 O and ∆'17 O values.

Exuberant Immobilization of Urease on an Inorganic SiO2 Support Enhances the Enzymatic Activities by 3-fold for Perennial Utilization.

Urease has been covalently immobilized on a 3-D networking silica gel (SG) using dimethyldichlorosilane (DMDCS) as second generation silane coupling reagent and m-nitroaniline as linker component in a robust methodology and subsequently characterized as [{Si(OSi)4(H2O)0.05}205.2] n=4{OSi(CH3)2-NH-C6H4-N═N-urease}·282.5H2O (molecular mass 263 445 g or 263.4 kDa). Selective coupling of tyrosine residue with an identifiable m-nitroaniline modified SG unit prevents enzyme-enzyme cross-linking leading to enhancement of enzymatic activity. The material worked at room temperature and its activity (luminescent and ammonia releasing efficiency) was enhanced by 3-fold (for both synthetic and real sample) compared to native enzyme values at neutral pH. Up to 30 days and 30 cycles, this 3-fold activity remains as such but reduces gradually to native enzyme level after 60 days and 60 cycles of reuse.

The Curtius rearrangement: mechanistic insight and recent applications in natural product syntheses.

The Curtius rearrangement is a versatile reaction in which a carboxylic acid can be converted to an isocyanate through an acyl azide intermediate under mild conditions. The resulting stable isocyanate can then be readily transformed into a variety of amines and amine derivatives including urethanes and ureas. There have been wide-ranging applications of the Curtius rearrangement in the synthesis of natural products and their derivatives. Also, this reaction has been extensively utilized in the synthesis and application of a variety of biomolecules. In this review, we present mechanistic studies, chemical methodologies and reagents for the synthesis of isocyanates from carboxylic acids, the conversion of isocyanates to amines and amine derivatives, and their applications in the synthesis of bioactive natural products and their congeners.

Whole tumor section quantitative image analysis maximizes between-pathologists' reproducibility for clinical immunohistochemistry-based biomarkers.

Pathologists have had increasing responsibility for quantitating immunohistochemistry (IHC) biomarkers with the expectation of high between-reader reproducibility due to clinical decision-making especially for patient therapy. Digital imaging-based quantitation of IHC clinical slides offers a potential aid for improvement; however, its clinical adoption is limited potentially due to a conventional field-of-view annotation approach. In this study, we implemented a novel solely morphology-based whole tumor section annotation strategy to maximize image analysis quantitation results between readers. We first compare the field-of-view image analysis annotation approach to digital and manual-based modalities across multiple clinical studies (~120 cases per study) and biomarkers (ER, PR, HER2, Ki-67, and p53 IHC) and then compare a subset of the same cases (~40 cases each from the ER, PR, HER2, and Ki-67 studies) using whole tumor section annotation approach to understand incremental value of all modalities. Between-reader results for each biomarker in relation to conventional scoring modalities showed similar concordance as manual read: ER field-of-view image analysis: 95.3% (95% CI 92.0-98.2%) vs digital read: 92.0% (87.8-95.8%) vs manual read: 94.9% (91.4-97.8%); PR field-of-view image analysis: 94.1% (90.3-97.2%) vs digital read: 94.0% (90.2-97.1%) vs manual read: 94.4% (90.9-97.2%); Ki-67 field-of-view image analysis: 86.8% (82.1-91.4%) vs digital read: 76.6% (70.9-82.2%) vs manual read: 85.6% (80.4-90.4%); p53 field-of-view image analysis: 81.7% (76.4-86.8%) vs digital read: 80.6% (75.0-86.0%) vs manual read: 78.8% (72.2-83.3%); and HER2 field-of-view image analysis: 93.8% (90.0-97.2%) vs digital read: 91.0 (86.6-94.9%) vs manual read: 87.2% (82.1-91.9%). Subset implementation and analysis on the same cases using whole tumor section image analysis approach showed significant improvement between pathologists over field-of-view image analysis and manual read (HER2 100% (97-100%), P=0.013 field-of-view image analysis and 0.013 manual read; Ki-67 100% (96.9-100%), P=0.040 and 0.012; ER 98.3% (94.1-99.5%), p=0.232 and 0.181; and PR 96.6% (91.5-98.7%), p=0.012 and 0.257). Overall, whole tumor section image analysis significantly improves between-pathologist's reproducibility and is the optimal approach for clinical-based image analysis algorithms.

Rapid and high-resolution stable isotopic measurement of biogenic accretionary carbonate using an online CO2 laser ablation system: Standardization of the analytical protocol.

RATIONALE: The elaborate sampling and analytical protocol associated with conventional dual-inlet isotope ratio mass spectrometry has long hindered high-resolution climate studies from biogenic accretionary carbonates. Laser-based on-line systems, in comparison, produce rapid data, but suffer from unresolvable matrix effects. It is, therefore, necessary to resolve these matrix effects to take advantage of the automated laser-based method. METHODS: Two marine bivalve shells (one aragonite and one calcite) and one fish otolith (aragonite) were first analysed using a CO2 laser ablation system attached to a continuous flow isotope ratio mass spectrometer under different experimental conditions (different laser power, sample untreated vs vacuum roasted). The shells and the otolith were then micro-drilled and the isotopic compositions of the powders were measured in a dual-inlet isotope ratio mass spectrometer following the conventional acid digestion method. RESULTS: The vacuum-roasted samples (both aragonite and calcite) produced mean isotopic ratios (with a reproducibility of ±0.2 ‰ for both δ18 O and δ13 C values) almost identical to the values obtained using the conventional acid digestion method. As the isotopic ratio of the acid digested samples fall within the analytical precision (±0.2 ‰) of the laser ablation system, this suggests the usefulness of the method for studying the biogenic accretionary carbonate matrix. CONCLUSIONS: When using laser-based continuous flow isotope ratio mass spectrometry for the high-resolution isotopic measurements of biogenic carbonates, the employment of a vacuum-roasting step will reduce the matrix effect. This method will be of immense help to geologists and sclerochronologists in exploring short-term changes in climatic parameters (e.g. seasonality) in geological times.

An enantioselective enzymatic desymmetrization route to hexahydro-4H-furopyranol, a high-affinity ligand for HIV-1 protease inhibitors.

An enantioselective synthesis of (3aS,4S,7aR)-hexahydro-4H-furo[2,3-b]pyran-4-ol, a high-affinity nonpeptide ligand for a variety of potent HIV-1 protease inhibitors is described. The key steps involved a highly enantioselective enzymatic desymmetrization of meso-diacetate, an efficient transacetalization, and a highly diastereoselective reduction of a ketone. This route is amenable to large-scale synthesis using readily available starting materials.

Heat Shock Protein 90 Associates with the Per-Arnt-Sim Domain of Heme-free Soluble Guanylate Cyclase: IMplications for Enzyme Maturation.

Heat shock protein 90 (hsp90) drives heme insertion into the ß1 subunit of soluble guanylate cyclase (sGC) ß1, which enables it to associate with a partner sGCα1 subunit and mature into a nitric oxide (NO)-responsive active form. We utilized fluorescence polarization measurements and hydrogen-deuterium exchange mass spectrometry to define molecular interactions between the specific human isoforms hsp90ß and apo-sGCß1. hsp90ß and its isolated M domain, but not its isolated N and C domains, bind with low micromolar affinity to a heme-free, truncated version of sGCß1 (sGCß1(1-359)-H105F). Surprisingly, hsp90ß and its M domain bound to the Per-Arnt-Sim (PAS) domain of apo-sGC-ß1(1-359), which lies adjacent to its heme-binding (H-NOX) domain. The interaction specifically involved solvent-exposed regions in the hsp90ß M domain that are largely distinct from sites utilized by other hsp90 clients. The interaction strongly protected two regions of the sGCß1 PAS domain and caused local structural relaxation in other regions, including a PAS dimerization interface and a segment in the H-NOX domain. Our results suggest a means by which the hsp90ß interaction could prevent apo-sGCß1 from associating with its partner sGCα1 subunit while enabling structural changes to assist heme insertion into the H-NOX domain. This mechanism would parallel that in other clients like the aryl hydrocarbon receptor and HIF1α, which also interact with hsp90 through their PAS domains to control protein partner and small ligand binding interactions.

Enantioselective Syntheses of (-)-Alloyohimbane and (-)-Yohimbane by an Efficient Enzymatic Desymmetrization Process.

Enantioselective syntheses of (-)-alloyohimbane and (-)-yohimbane was accomplished in a convergent manner. The key step involved a modified mild protocol for the enantioselective enzymatic desymmetrization of meso-diacetate. The protocol provided convenient access to an optically active monoacetate in multi-gram scale in high enantiomeric purity. This monoacetate was converted to (-)-alloyohimbane. Reductive amination of the derived aldehyde causes the isomerization leading to the trans-product and allows the synthesis of (-)-yohimbane.

Control of spin coupling through a redox-active bridge in a dinickel(II) porphyrin dimer: step-wise oxidations enable isolations of a chlorin-porphyrin heterodimer and a dication diradical with a singlet ground state.

A dinickel(II)porphyrin dimer has been used here in which the redox-active pyrrole-moiety, similar to the tryptophan residue in diheme enzymes such as MauG and bCcP, has been placed between two Ni(II)porphyrin centers connected via a flexible, but unconjugated methylene bridge. This arrangement provides a large physical separation between the two metal centers and thus displays almost no communication between them through the bridge. Upon treatment with DDQ as an oxidant, the dinickel(II) porphyrin dimer slowly gets converted into an indolizinium-fused chlorin-porphyrin heterodimer. However, oxidations of the dinickel(II) porphyrin dimer up to two oxidizing equivalents using oxidants such as AgSbF6 and FeCl3 resulted in the formation of a dication diradical complex. Interestingly, in order to stabilize such a highly oxidized dication diradical, two non-conjugated methylene spacers undergo facile 2e-/-2H+ oxidation to make the bridge fully π-conjugated for promoting through-bond communication. Through the oxidized and conjugated bridge, two porphyrin π-cation radicals display considerable communications leading to an efficient intramolecular spin coupling to form a singlet state. Interestingly, the redox-active nature of the bridge controls the electronic communication just by simple oxidation or reduction, and thereby, acts as a molecular switch for efficient magnetic relay.

Stereoselective synthesis of hydroxy stilbenoids and styrenes by atom-efficient olefination with thiophthalides.

The synthesis of stilbenoids and styryl carboxylic acids is accomplished with high E-stereoselectivity by olefination of aldehydes with thiophthalides under basic conditions. The olefination is highly atom-efficient as it only loses elemental sulfur during the reaction. This olefination, in conjunction with retro Kolbe-Schmitt reaction, allows facile synthesis of E-hydroxystilbenoids with minimal employment of protecting groups. This study also discloses two important findings: formation of i) 4-methylsulfanyl isocoumarins and ii) an 2-arylindenone.

A novel plant lectin, NTL-125, interferes with SARS-CoV-2 interaction with hACE2.

COVID-19 caused by SARS-CoV-2 virus has had profound impact on the world in the past two years. Intense research is going on to find effective drugs to combat the disease. Over the past year several vaccines were approved for immunization. But SARS-CoV-2 being an RNA virus is continuously mutating to generate new variants, some of which develop features of immune escape. This raised serious doubts over the long-term efficacy of the vaccines. We have identified a unique mannose binding plant lectin from Narcissus tazetta bulb, NTL-125, which effectively inhibits SARS-CoV-2 replication in Vero-E6 cell line. In silico docking studies revealed that NTL-125 has strong affinity to viral Spike RBD protein, preventing it from attaching to hACE2 receptor, the gateway to cellular entry. Binding analyses revealed that all the mutant variants of Spike protein also have stronger affinity for NTL-125 than hACE2. The unique α-helical tail of NTL-125 plays most important role in binding to RBD of Spike. NTL-125 also interacts effectively with some glycan moieties of S-protein in addition to amino acid residues adding to the binding strength. Thus, NTL-125 is a highly potential antiviral compound of natural origin against SARS-CoV-2 and may serve as an important therapeutic for management of COVID-19.

Evaluation of physicochemical and biological properties of nonreconstituted MYL-1401O vials, reconstituted MYL-1401O suspension in vial, and diluted MYL-1401O suspension in infusion bags (0.9% saline) for extended duration.

BACKGROUND: MYL-1401O; trastuzumab-dkst (Ogivri™; Mylan Inc.) is a biosimilar to the trastuzumab reference product (Herceptin®; Genentech, USA). Assessment of physicochemical stability and biological activity for the non-reconstituted, reconstituted, and infused solution over an extended, clinically relevant duration is critical for ensuring optimal patient outcomes and health resource utilization. METHODS: The physicochemical and biological stability of MYL-1401O was assessed in non-reconstituted vials stored at 25 °C ± 2 °C/60% ± 5% relative humidity (RH) for 6 months, reconstituted 21 mg/mL solution in vials stored at 2 °C to 8 °C for 10 days, and diluted in 0.9% saline-containing infusion bags at 0.3 mg/mL and 4.0 mg/mL stored for 77 days at 2 °C to 8 °C, plus an additional 2 days at 25 °C ± 2 °C/60% ± 5% RH. RESULTS: At all storage conditions tested, MYL-1401O was physicochemically and biologically stable for extended duration and under various temperature and humidity conditions. CONCLUSIONS: MYL-1401O retained its physicochemical and biological stability under different storage conditions, which supports advanced preparation of MYL-1401O, better efficiency, less wastage, and cost-savings for better patient management.

Local conformational flexibility provides a basis for facile polymer formation in human neuroserpin.

Neuroserpin is a regulator of neuronal growth and plasticity. Like other members of the serpin family, neuroserpin undergoes a large conformational change as part of its function. Unlike other serpins such as α(1)-antitrypsin, wild-type neuroserpin will polymerize under near-physiological conditions, and will spontaneously transition to the latent state. To probe the origins of this conformational lability, we have performed hydrogen exchange measurements and molecular-dynamics simulations on human neuroserpin. Hydrogen exchange indicates that neuroserpin has greater flexibility in the breach region and in ß-strand 1C compared with α(1)-antitrypsin. Molecular-dynamics simulations show that the distance between the top of ß-strands 3 and 5A averages 4.6 Å but becomes as large as 7.5 Å in neuroserpin while it remains stable at ∼3.5 Å in α(1)-antitrypsin. Further simulations show that the stabilizing S340A mutation suppresses these fluctuations in neuroserpin. The first principal component calculated from the simulations shows a movement of helix F away from the face of ß-sheet A in neuroserpin while no such movement is evident in α(1)-antitrypsin. The increased mobility of these regions in neuroserpin relative to α(1)-antitrypsin provides a basis for neuroserpin's increased tendency toward the formation of polymers and/or the latent state.

Effects of glycosylation on the stability and flexibility of a metastable protein: the human serpin α(1)-antitrypsin.

Protein glycosylation commonly stabilizes proteins thereby increasing protein half-lives and protecting against denaturation or proteolytic degradation. While generally beneficial, such stabilization is potentially disadvantageous in the case of inhibitory serpins. These protease inhibitors are metastable and a conformational transition to a more stable form is key to their function. Instability is therefore essential for these inhibitory serpins and mutagenesis has demonstrated that substantial stabilization results in compromised function. We have used optical spectroscopy and hydrogen/deuterium exchange and mass spectrometry to investigate the effects of glycosylation on the human serpin alpha-1 antitrypsin (α(1)-AT). Previous studies found that unglycosylated recombinant α(1)-AT populates a molten globule at low denaturant and that the ability to populate this state is correlated with efficient protease inhibition. Further, a high degree of conformational flexibility was found in several important regions. Guanidine hydrochloride denaturation monitored by circular dichroism indicates that plasma α(1)-AT, which is glycosylated at 3 sites, is substantially stabilized relative to the unglycosylated form. However, hydrogen exchange reveals complete loss of protection in plasma α(1)-AT above 1 M GuHCl, similar to what is seen for the recombinant form. Sugars therefore appear to stabilize the compact denatured state of α(1)-AT without significant stabilization of the folded state. Native state hydrogen exchange reveals minor perturbations to native flexibility, but high flexibility in key regions such as the f helix is conserved. ß-strand 1c is stabilized in plasma α(1)-AT, which may confer increased resistance to forming pathogenic polymers. Overall, our results indicate that glycosylation of inhibitory serpins does not interfere with either native state flexibility or the native instability that is required for efficient function, though it may confer resistance to degradation by proteases and thus extend the half-life of circulating serpins.

Total Synthesis of Meayamycin and O-Acyl Analogues.

A short, scalable total synthesis of meayamycin is described by an approach that entails a longest linear sequence of 12 steps (22 steps overall) from commercially available chiral pool materials (ethyl l-lactate, BocNH-Thr-OH, and d-ribose) and introduces the most straightforward preparation of the right-hand subunit detailed to date. The use of the approach in the divergent synthesis of a representative series of O-acyl analogues is exemplified.