Integrating circulating T follicular memory cells and autoantibody repertoires for characterization of autoimmune disorders.

Introduction: Autoimmune diseases are heterogeneous and often lack specific or sensitive diagnostic tests. Increased percentages of CD4+CXCR5+PD1+ circulating T follicular helper (cTfh) cells and skewed distributions of cTfh subtypes have been associated with autoimmunity. However, cTfh cell percentages can normalize with immunomodulatory treatment despite persistent disease activity, indicating the need for identifying additional cellular and/or serologic features correlating with autoimmunity. Methods: The cohort included 50 controls and 56 patients with autoimmune cytopenias, gastrointestinal, pulmonary, and/or neurologic autoimmune disease. Flow cytometry was used to measure CD4+CXCR5+ T cell subsets expressing the chemokine receptors CXCR3 and/or CCR6: CXCR3+CCR6- Type 1, CXCR3-CCR6- Type 2, CXCR3+CCR6+ Type 1/17, and CXCR3- CCR6+ Type 17 T cells. IgG and IgA autoantibodies were quantified using a microarray featuring 1616 full-length, conformationally intact protein antigens. The 97.5th percentile in the control cohort defined normal limits for T cell subset percentages and total number (burden) of autoantibodies. Results: This study focused on CD4+CXCR5+ T cells because CXCR5 upregulation occurs after cognate T-B cell interactions characteristic of autoimmune diseases. We refer to these cells as circulating T follicular memory (cTfm) cells to acknowledge the dynamic nature of antigen-experienced CXCR5+ T cells, which encompass progenitors of cTfh or Tfh cells as well as early effector memory T cells that have not yet lost CXCR5. Compared to controls, 57.1% of patients had increased CXCR5+CXCR3+CCR6+ cTfm1/17 and 25% had increased CXCR5+CXCR3-CCR6+ cTfm17 cell percentages. Patients had significantly more diverse IgG and IgA autoantibodies than controls and 44.6% had an increased burden of autoantibodies of either isotype. Unsupervised autoantibody clustering identified three clusters of patients with IgG autoantibody profiles distinct from those of controls, enriched for patients with active autoimmunity and monogenic diseases. An increased percentage of cTfm17 cells was most closely associated with an increased burden of high-titer IgG and IgA autoantibodies. A composite measure integrating increased cTfm1/17, cTfm17, and high-titer IgG and/or IgA autoantibodies had 91.1% sensitivity and 90.9% specificity for identifying patients with autoimmunity. Percentages of cTfm1/17 and cTfm17 percentages and numbers of high-titer autoantibodies in patients receiving immunomodulatory treatment did not differ from those in untreated patients, thus suggesting that measurements of cTfm can complement measurements of other cellular markers affected by treatment. Conclusions: This study highlights two new approaches for assessing autoimmunity: measuring CD4+CXCR5+ cTfm subsets as well as total burden of autoantibodies. Our findings suggest that these approaches are particularly relevant to patients with rare autoimmune disorders for whom target antigens and prognosis are often unknown.

Comparison of Patient Satisfaction Between Face-to-Face and Telehealth Modalities in Nephrolithiasis Nutritional Counseling.

INTRODUCTION: Following the COVID-19 pandemic, telehealth usage increased. Virtual visits minimize exposure risk while also addressing barriers to care. Telehealth offers the ability to increase patient access and provider efficiency. However, patient satisfaction with telehealth has not been fully determined. This study evaluated patient-perceived quality and satisfaction of virtual vs face-to-face visits during consultation with a dietician in the management of nephrolithiasis. METHODS: Ninety-six patients with previous diagnosis of nephrolithiasis underwent an initial, in-person nutrition consultation between May 2019 to February 2021. A follow-up with a dietician was randomized to in-person or telehealth. The telehealth group used an application called MDLive. The telehealth group used MDLive on a hospital computer during their follow-up with the urologist, whereas the in-person group had a separate appointment scheduled at a different location. Patient satisfaction following telehealth visits was assessed by the Telemedicine Satisfaction Questionnaire. Patient satisfaction following in-person visits was assessed with an 8-question modified Telemedicine Satisfaction Questionnaire, which lacked technology-related questions. RESULTS: Fifty patients were randomized to in-person follow-up and 46 to virtual follow-up. Within the virtual follow-up group more than 90% "agreed" or "strongly agreed" that they were satisfied with the quality of service provided through telemedicine. Greater than 82% reported intention to use telemedicine services again. There was no significant difference in patient satisfaction between telemedicine and face-to-face visits. Sixty-seven percent of patients in the telemedicine group reported better access to health care services and time saved and 89% reported independence accessing the telehealth system without assistance. CONCLUSIONS: This study supports the idea that telemedicine may be a successful alternative in the follow up of patients undergoing nutritional counseling for stone prevention. Future studies regarding telehealth use should evaluate which other urologic conditions are amenable to virtual management.

Regulatory T-cell dysfunction and cutaneous exposure to Staphylococcus aureus underlie eczema in DOCK8 deficiency.

BACKGROUND: Dedicator of cytokinesis 8 (DOCK8)-deficient patients have severe eczema, elevated IgE, and eosinophilia, features of atopic dermatitis (AD). OBJECTIVE: We sought to understand the mechanisms of eczema in DOCK8 deficiency. METHODS: Skin biopsy samples were characterized by histology, immunofluorescence microscopy, and gene expression. Skin barrier function was measured by transepidermal water loss. Allergic skin inflammation was elicited in mice by epicutaneous sensitization with ovalbumin (OVA) or cutaneous application of Staphylococcus aureus. RESULTS: Skin lesions of DOCK8-deficient patients exhibited type 2 inflammation, and the patients' skin was colonized by Saureus, as in AD. Unlike in AD, DOCK8-deficient patients had a reduced FOXP3:CD4 ratio in their skin lesions, and their skin barrier function was intrinsically intact. Dock8-/- mice exhibited reduced numbers of cutaneous T regulatory (Treg) cells and a normal skin barrier. Dock8-/- and mice with an inducible Dock8 deletion in Treg cells exhibited increased allergic skin inflammation after epicutaneous sensitization with OVA. DOCK8 was shown to be important for Treg cell stability at sites of allergic inflammation and for the generation, survival, and suppressive activity of inducible Treg cells. Adoptive transfer of wild-type, but not DOCK8-deficient, OVA-specific, inducible Treg cells suppressed allergic inflammation in OVA-sensitized skin of Dock8-/- mice. These mice developed severe allergic skin inflammation and elevated serum IgE levels after topical exposure to Saureus. Both were attenuated after adoptive transfer of WT but not DOCK8-deficient Treg cells. CONCLUSION: Treg cell dysfunction increases susceptibility to allergic skin inflammation in DOCK8 deficiency and synergizes with cutaneous exposure to Saureus to drive eczema in DOCK8 deficiency.

Multi-modal skin atlas identifies a multicellular immune-stromal community associated with altered cornification and specific T cell expansion in atopic dermatitis.

In healthy skin, a cutaneous immune system maintains the balance between tolerance towards innocuous environmental antigens and immune responses against pathological agents. In atopic dermatitis (AD), barrier and immune dysfunction result in chronic tissue inflammation. Our understanding of the skin tissue ecosystem in AD remains incomplete with regard to the hallmarks of pathological barrier formation, and cellular state and clonal composition of disease-promoting cells. Here, we generated a multi-modal cell census of 310,691 cells spanning 86 cell subsets from whole skin tissue of 19 adult individuals, including non-lesional and lesional skin from 11 AD patients, and integrated it with 396,321 cells from four studies into a comprehensive human skin cell atlas in health and disease. Reconstruction of human keratinocyte differentiation from basal to cornified layers revealed a disrupted cornification trajectory in AD. This disrupted epithelial differentiation was associated with signals from a unique immune and stromal multicellular community comprised of MMP12 + dendritic cells (DCs), mature migratory DCs, cycling ILCs, NK cells, inflammatory CCL19 + IL4I1 + fibroblasts, and clonally expanded IL13 + IL22 + IL26 + T cells with overlapping type 2 and type 17 characteristics. Cell subsets within this immune and stromal multicellular community were connected by multiple inter-cellular positive feedback loops predicted to impact community assembly and maintenance. AD GWAS gene expression was enriched both in disrupted cornified keratinocytes and in cell subsets from the lesional immune and stromal multicellular community including IL13 + IL22 + IL26 + T cells and ILCs, suggesting that epithelial or immune dysfunction in the context of the observed cellular communication network can initiate and then converge towards AD. Our work highlights specific, disease-associated cell subsets and interactions as potential targets in progression and resolution of chronic inflammation.

Intraoperative hypotension in ambulatory surgery centers.

STUDY OBJECTIVES: To measure the incidence of intraoperative hypotension (IOH) during surgery in ambulatory surgery centers (ASCs) and describe associated characteristics of patients and procedures. DESIGN: Retrospective analysis. SETTING: 20 ASCs. PATIENTS: 16,750 patients having non-emergent, non-cardiac surgery; ASA physical status 2 through 4. INTERVENTIONS: None. MEASUREMENTS: We assessed incidence of IOH using the definition from the Centers for Medicare & Medicaid Services (CMS) Merit-based Incentive Payment System (MIPS)-mean arterial pressure (MAP) < 65 mmHg for at least 15 cumulative minutes-and three secondary definitions: minutes of MAP <65 mmHg, area under MAP of 65 mmHg, and time-weighted average MAP <65 mmHg. MAIN RESULTS: 30.9% of ASC cases had a MAP <65 mmHg for at least 15 min. The incidence of IOH varied significantly, and was higher among younger adults (age 18-39; 36.2%), females (35.2%), and patients with ASA physical status 2 (32.8%). IOH increased with increasing surgery length, even when time-weighted, and was higher among low complexity (30.6%) than moderate complexity (28.8%) procedures, and highest among high complexity procedures (44.1%). CONCLUSIONS: There was substantial occurrence of IOH in ASCs, similar to that described in academic hospital settings in previous literature. We hypothesize that this may reflect clinician preference not to intervene in perceived healthy patients or assumptions about ability to tolerate lower blood pressures on behalf of these patients. Future research will determine whether IOH in ACSs is associated with adverse outcomes to the same extent as described in more complex hospital-based surgeries.

Incidence of intraoperative hypotension during non-cardiac surgery in community anesthesia practice: a retrospective observational analysis.

BACKGROUND: Intraoperative hypotension (IOH) is well-described in the academic setting but not in community practice. IOH is associated with risk of postoperative morbidity and mortality. This is the first report of IOH in the community setting using the IOH measure definition from the Centers for Medicare and Medicaid Services Merit-based Incentive Payment System program. OBJECTIVES: To describe the incidence of IOH in the community setting; assess variation in IOH by patient-, procedure-, and facility-level characteristics; and describe variation in risk-adjusted IOH across clinicians. METHODS: Design Cross-sectional descriptive analysis of retrospective data from anesthesia records in 2020 and 2021. Setting Forty-five facilities affiliated with two large anesthesia providers in the USA. Participants Patients aged 18 years or older having non-emergent, non-cardiac surgery under general, neuraxial, or regional anesthesia. Cases were excluded based on criteria for the IOH measure: baseline mean arterial pressure (MAP) below 65 mmHg prior to anesthesia induction; American Society of Anesthesiologists (ASA) physical status classification of I, V, or VI; monitored anesthesia care only; deliberate induced hypotension; obstetric non-operative procedures; liver or lung transplant; cataract surgery; non-invasive gastrointestinal cases. Main outcomes IOH, using four definitions. Primary definition: binary assessment of whether the case had MAP < 65 mmHg for 15 min or more. Secondary definitions: total number of minutes of MAP < 65 mmHg, total area under MAP of 65 mmHg, time-weighted average MAP < 65 mmHg. RESULTS: Among 127,095 non-emergent, non-cardiac cases in community anesthesia settings, 29% had MAP < 65 mmHg for at least 15 min cumulatively, with an overall mean of 12.4 min < 65 mmHg. IOH was slightly more common in patients who were younger, female, and ASA II (versus III or IV); in procedures that were longer and had higher anesthesia base units; and in ambulatory surgery centers. Incidence of IOH varied widely across individual clinicians in both unadjusted and risk-adjusted analyses. CONCLUSION: Intraoperative hypotension is common in community anesthesia practice, including among patients and settings typically considered "low risk." Variation in incidence across clinicians remains after risk-adjustment, suggesting that IOH is a modifiable risk worth pursuing in quality improvement initiatives.

The IL-4Rα Q576R polymorphism is associated with increased severity of atopic dermatitis and exaggerates allergic skin inflammation in mice.

BACKGROUND: Atopic dermatitis (AD) is characterized by TH2-dominated skin inflammation and systemic response to cutaneously encountered antigens. The TH2 cytokines IL-4 and IL-13 play a critical role in the pathogenesis of AD. The Q576->R576 polymorphism in the IL-4 receptor alpha (IL-4Rα) chain common to IL-4 and IL-13 receptors alters IL-4 signaling and is associated with asthma severity. OBJECTIVE: We sought to investigate whether the IL-4Rα R576 polymorphism is associated with AD severity and exaggerates allergic skin inflammation in mice. METHODS: Nighttime itching interfering with sleep, Rajka-Langeland, and Eczema Area and Severity Index scores were used to assess AD severity. Allergic skin inflammation following epicutaneous sensitization of mice 1 or 2 IL-4Rα R576 alleles (QR and RR) and IL-4Rα Q576 (QQ) controls was assessed by flow cytometric analysis of cells and quantitative RT-PCR analysis of cytokines in skin. RESULTS: The frequency of nighttime itching in 190 asthmatic inner-city children with AD, as well as Rajka-Langeland and Eczema Area and Severity Index scores in 1116 White patients with AD enrolled in the Atopic Dermatitis Research Network, was higher in subjects with the IL-4Rα R576 polymorphism compared with those without, with statistical significance for the Rajka-Langeland score. Following epicutaneous sensitization of mice with ovalbumin or house dust mite, skin infiltration by CD4+ cells and eosinophils, cutaneous expression of Il4 and Il13, transepidermal water loss, antigen-specific IgE antibody levels, and IL-13 secretion by antigen-stimulated splenocytes were significantly higher in RR and QR mice compared with QQ controls. Bone marrow radiation chimeras demonstrated that both hematopoietic cells and stromal cells contribute to the mutants' exaggerated allergic skin inflammation. CONCLUSIONS: The IL-4Rα R576 polymorphism predisposes to more severe AD and increases allergic skin inflammation in mice.

Small cell lung cancer recurring in the retina.

Intraocular metastases are rare, usually isolating to the uveal tract. Retinal metastases are less common still, presenting a unique diagnostic and therapeutic challenge. This case observes a 66-year-old female ex-smoker presenting with unilateral painful vision loss 2 months from completion of definitive chemoradiotherapy for early-stage small cell lung cancer. A retinal lesion was discovered on examination and initially treated as viral retinitis without improvement. Two vitrectomy procedures were unable to confirm the diagnosis. Eventual retinal biopsy under general anaesthesia confirmed small cell lung cancer oligometastasis (7 months from completion of definitive chemoradiotherapy), without evidence of other sites of disease on imaging. She received local radiation to retina with resolution of eye pain. She eventually developed intrathoracic relapse of disease and was treated with palliative chemotherapy (14 months from completion of definitive chemoradiotherapy).

Benzocyclobutadienes: An Unusual Mode of Access Reveals Unusual Modes of Reactivity.

The reaction of an aryne with an alkyne to generate a benzocyclobutadiene (BCB) intermediate is rare. We report here examples of this reaction, revealed by Diels-Alder trapping of the BCB by either pendant or external electron-deficient alkynes. Mechanistic delineation of the reaction course is supported by DFT calculations. A three-component process joining the benzyne first with an electron-rich and then with an electron-poor alkyne was uncovered. Reactions in which the BCB functions in a rarely observed role as a 4π diene component in Diels-Alder reactions are reported. The results also shed new light on aspects of the hexadehydro-Diels-Alder reaction used to generate the benzynes.

Nickel-Catalyzed Enantioselective Reductive Cross-Coupling of Styrenyl Aziridines.

A Ni-catalyzed reductive cross-coupling of styrenyl aziridines with aryl iodides is reported. This reaction proceeds by a stereoconvergent mechanism and is thus amenable to asymmetric catalysis using a chiral bioxazoline ligand for Ni. The process allows facile access to highly enantioenriched 2-arylphenethylamines from racemic aziridines. Multivariate analysis revealed that ligand polarizability, among other features, influences the observed enantioselectivity, shedding light on the success of this emerging ligand class for enantioselective Ni catalysis.

Development of N2O-MTV for low-speed flow and in-situ deployment to an integral effect test facility.

A molecular tagging velocity (MTV) technique is developed to non-intrusively measure velocity in an integral effect test (IET) facility simulating a high temperature helium-cooled nuclear reactor in accident scenarios. In these scenarios, the velocities are expected to be low, on the order of 1 m/s or less, which forces special requirements on the MTV tracer selection. Nitrous oxide (N2O) is identified as a suitable seed gas to generate NO tracers capable of probing the flow over a large range of pressure, temperature, and flow velocity. The performance of N2O-MTV is assessed in the laboratory at temperature and pressure ranging from 295 to 781 K and 1 to 3 atm. MTV signal improves with a temperature increase, but decreases with a pressure increase. Velocity precision down to 0.004 m/s is achieved with a probe time of 40 ms at ambient pressure and temperature. Measurement precision is limited by tracer diffusion, and absorption of the tag laser beam by the seed gas. Processing by cross-correlation of single shot images with high signal-to-noise ratio reference images improves the precision by about 10% compared to traditional single shot image correlations. The instrument is then deployed to the IET facility. Challenges associated with heat, vibrations, safety, beam delivery, and imaging are addressed in order to successfully operate this sensitive instrument in-situ. Data are presented for an isothermal depressurized conduction cool-down. Velocity profiles from MTV reveal a complex flow transient driven by buoyancy, diffusion, and instability taking place over short (<1 s) and long (>30 min) time-scales at sub-meter per second speed. The precision of the in-situ results is estimated at 0.027, 0.0095, and 0.006 m/s for a probe time of 5, 15, and 35 ms, respectively.

Multifunctional biomaterials from the sea: Assessing the effects of chitosan incorporation into collagen scaffolds on mechanical and biological functionality.

UNLABELLED: Natural biomaterials such as collagen show promise in tissue engineering applications due to their inherent bioactivity. The main limitation of collagen is its low mechanical strength and somewhat unpredictable and rapid degradation rate; however, combining collagen with another material, such as chitosan, can reinforce the scaffold mechanically and may improve the rate of degradation. Additionally, the high cost and the risk of prion transmission associated with mammal-derived collagen has prompted research into alternative sources such as marine-origin collagen. In this context, the overall goal of this study was to determine if the incorporation of chitosan into collagen scaffolds could improve the mechanical and biological properties of the scaffold. In addition the study assessed if collagen, derived from salmon skin (marine), can provide an alternative to collagen derived from bovine tendon (mammal) for tissue engineering applications. Scaffold architecture and mechanical properties were assessed as well as their ability to support mesenchymal stem cell growth and differentiation. Overall, the addition of chitosan to bovine and salmon skin-derived collagen scaffolds improved the mechanical properties, increasing the compressive strength, swelling ratio and prolonged the degradation rate. Mesenchymal stem cell (MSC) attachment and proliferation was most improved on the bovine-derived collagen scaffold containing a 75:25 ratio of collagen:chitosan, and when MSC osteogenic and chondrogenic potential on the scaffold was assessed, a significant increase in calcium production (p<0.001) and sulfated glycosaminoglycan (sGAG) production (p<0.001) was observed respectively. Regardless of chitosan content, the bovine-derived collagen scaffolds out-performed the salmon skin-derived collagen scaffolds, displaying a larger pore size and higher percentage porosity, more regular architecture, higher compressive modulus, a greater capacity for water uptake and allowed for more MSC proliferation and differentiation. This versatile scaffold incorporating the marine biomaterial chitosan show great potential as appropriate platforms for promoting orthopaedic tissue repair while the use of salmon skin-derived collagen may be more suitable in the repair of soft tissues such as skin. STATEMENT OF SIGNIFICANCE: Collagen is commonly used in tissue engineering due to its biocompatibility; however, it has low mechanical strength and an unpredictable degradation rate. In addition, high cost and risk of prion transmission associated with mammalian-derived collagen has prompted research into alternative collagen sources, namely, marine-derived collagen. In this study, scaffolds made from salmon-skin collagen were compared to the more commonly used bovine-derived collagen with a focus on orthopaedic applications. To improve the mechanical properties of these scaffolds, another marine biomaterial, chitosan, was added to produce scaffolds with increased mechanical stability. The collagen-chitosan composites were also shown to support mesenchymal stem cell differentiation towards both bone and cartilage tissue. This multi-functional scaffold therefore has potential in both bone and cartilage regeneration applications.

Mechanism of the Intramolecular Hexadehydro-Diels-Alder Reaction.

Theoretical analysis of the mechanism of the intramolecular hexadehydro-Diels-Alder (HDDA) reaction, validated against prior and newly measured kinetic data for a number of different tethered yne-diynes, indicates that the reaction proceeds in a highly asynchronous fashion. The rate-determining step is bond formation at the alkyne termini nearest the tether, which involves a transition-state structure exhibiting substantial diradical character. Whether the reaction then continues to close the remaining bond in a concerted fashion or in a stepwise fashion (i.e., with an intervening intermediate) depends on the substituents at the remaining terminal alkyne positions. Computational modeling of the HDDA reaction is complicated by the significant diradical character that arises along the reaction coordinate, which leads to instabilities in both restricted singlet Kohn-Sham density functional theory (DFT) and coupled cluster theory based on a Hartree-Fock reference wave function. A consistent picture emerges, however, from comparison of broken-symmetry DFT calculations and second-order perturbation theory based on complete-active-space self-consistent-field (CASPT2) calculations.

Health outcomes associated with smoke exposure in Albuquerque, New Mexico, during the 2011 Wallow fire.

OBJECTIVE: This study examined the association between PM2.5 levels and emergency department (ED) visits for selected health outcomes in Albuquerque, New Mexico, during the Wallow fire of 2011. DESIGN: Measurements of 24-hour average concentrations of PM2.5 obtained from the City of Albuquerque were used to calculate wildfire smoke exposure in Albuquerque. Daily ED visits were collected by the New Mexico Department of Health from individual nonfederal licensed facilities in the Albuquerque area. Poisson regression was used to assess the relationship between ED visits for selected respiratory and cardiovascular conditions and varying levels of PM2.5 exposure. SETTING: Albuquerque, New Mexico. PARTICIPANTS: Patients visiting an ED for select conditions before, during, and after the wildfire. MAIN OUTCOME MEASURE: Relative increase in ED visits for selected conditions during the wildfire period. RESULTS: Analysis of PM2.5 exposure data and ED visits in Albuquerque before and during the Wallow fire indicated that compared with the period prior to the fire, there was an increased risk of ED visits for some respiratory and cardiovascular conditions during heavy smoke conditions, and risk varied by age and sex. The population of 65+ years was especially at risk for increased ED visits. There was a significantly increased risk of ED visits among the 65+ population for asthma (RR [relative rate] = 1.73, 95% confidence interval [CI] = 1.03-2.93) and for diseases of the veins, lymphatic and circulatory system (RR = 1.56, 95% CI = 1.00-2.43). For the age group of 20 to 64 years, there was a statistically significant increase in ED visits for diseases of pulmonary circulation (RR = 2.64, 95% CI = 1.42-4.9) and for cerebrovascular disease (RR = 1.69, 95% CI = 1.03-2.77). CONCLUSIONS: High levels of PM2.5 exposure due to the Wallow fire were associated with increased ED visits for respiratory and cardiovascular conditions in Albuquerque. More effective and targeted preventive measures are necessary to reduce morbidity rates associated with wildfire smoke exposure among vulnerable populations.

Differential scanning calorimetry (DSC) as a tool for probing the reactivity of polyynes relevant to hexadehydro-Diels-Alder (HDDA) cascades.

The differential scanning calorimetry (DSC) behavior of a number of alkyne-rich compounds is described. The DSC trace for each compound exhibits an exothermic event at a characteristic onset temperature. For the tri- and tetraynes whose [4 + 2] HDDA reactivity in solution has been determined, these onset temperatures show a strong correlation with the cyclization activation energy. The studies reported here exemplify how the data available through this operationally simple analytical technique can give valuable insights into the thermal behavior of small molecules.

Rates of hexadehydro-Diels-Alder (HDDA) cyclizations: impact of the linker structure.

The rates of the hexadehydro-Diels-Alder (HDDA) reaction of substrates containing, minimally, a 1,3,8-triyne subunit are reported. Several series of related substrates, differing in the nature of the three-atom tether that links the 1,3-diyne and diynophile, were examined. Seemingly small changes in substrate structure result in large differences in cyclization rate, spanning more than 8 orders of magnitude. The reactivity trends revealed by these studies should prove useful in guiding substrate design and choice of reaction conditions in future applications.

Dichlorination of (hexadehydro-Diels-Alder generated) benzynes and a protocol for interrogating the kinetic order of bimolecular aryne trapping reactions.

The efficient dichlorination of benzynes prepared by the hexadehydro-Diels-Alder (HDDA) reaction is reported. Cycloisomerization of a triyne substrate in the presence of dilithium tetrachlorocuprate is shown to provide dichlorinated products A by capture of the benzyne intermediate. A general strategy for discerning the kinetic order of an external aryne trapping agent is presented. It merely requires measurement of the competition between bimolecular vs unimolecular trapping events (here, dichlorination vs intramolecular Diels-Alder (IMDA) reaction to give A vs B, respectively) as a function of the concentration of the trapping agent.

Alkane desaturation by concerted double hydrogen atom transfer to benzyne.

The removal of two vicinal hydrogen atoms from an alkane to produce an alkene is a challenge for synthetic chemists. In nature, desaturases and acetylenases are adept at achieving this essential oxidative functionalization reaction, for example during the biosynthesis of unsaturated fatty acids, eicosanoids, gibberellins and carotenoids. Alkane-to-alkene conversion almost always involves one or more chemical intermediates in a multistep reaction pathway; these may be either isolable species (such as alcohols or alkyl halides) or reactive intermediates (such as carbocations, alkyl radicals, or σ-alkyl-metal species). Here we report a desaturation reaction of simple, unactivated alkanes that is mechanistically unique. We show that benzynes are capable of the concerted removal of two vicinal hydrogen atoms from a hydrocarbon. The discovery of this exothermic, net redox process was enabled by the simple thermal generation of reactive benzyne intermediates through the hexadehydro-Diels-Alder cycloisomerization reaction of triyne substrates. We are not aware of any single-step, bimolecular reaction in which two hydrogen atoms are simultaneously transferred from a saturated alkane. Computational studies indicate a preferred geometry with eclipsed vicinal C-H bonds in the alkane donor.

Synthesis of complex benzenoids via the intermediate generation of o-benzynes through the hexadehydro-Diels-Alder reaction.

The hexadehydro-Diels-Alder (HDDA) cascade enables the synthesis of complex benzenoid products with various substitution patterns through aryne intermediates. The first stage of this cascade involves the generation of a highly reactive ortho-benzyne intermediate by a net [4+2] cycloisomerization of a triyne substrate. The benzyne can be rapidly 'trapped' either intramolecularly or intermolecularly with myriad nucleophilic or π-bond-donating reactants. As a representative example of a general procedure for synthesizing highly substituted benzenoids, this protocol describes the synthesis of a typical triyne substrate and its use as the reactant in an HDDA cascade to form a phthalide. The synthetic procedure detailed herein (four chemical reactions) takes 16-20 h of active effort over a period of several days for the preparation of the triyne precursor and ∼2 h of active effort over a 3-d period for the generation and trapping of the benzyne and isolation of the phthalide product.