Identification of a DNA N6-Adenine Methyltransferase Complex and Its Impact on Chromatin Organization.

DNA N6-adenine methylation (6mA) has recently been described in diverse eukaryotes, spanning unicellular organisms to metazoa. Here, we report a DNA 6mA methyltransferase complex in ciliates, termed MTA1c. It consists of two MT-A70 proteins and two homeobox-like DNA-binding proteins and specifically methylates dsDNA. Disruption of the catalytic subunit, MTA1, in the ciliate Oxytricha leads to genome-wide loss of 6mA and abolishment of the consensus ApT dimethylated motif. Mutants fail to complete the sexual cycle, which normally coincides with peak MTA1 expression. We investigate the impact of 6mA on nucleosome occupancy in vitro by reconstructing complete, full-length Oxytricha chromosomes harboring 6mA in native or ectopic positions. We show that 6mA directly disfavors nucleosomes in vitro in a local, quantitative manner, independent of DNA sequence. Furthermore, the chromatin remodeler ACF can overcome this effect. Our study identifies a diverged DNA N6-adenine methyltransferase and defines the role of 6mA in chromatin organization.

A Gut Microbial Mimic that Hijacks Diabetogenic Autoreactivity to Suppress Colitis.

The gut microbiota contributes to the development of normal immunity but, when dysregulated, can promote autoimmunity through various non-antigen-specific effects on pathogenic and regulatory lymphocytes. Here, we show that an integrase expressed by several species of the gut microbial genus Bacteroides encodes a low-avidity mimotope of the pancreatic ß cell autoantigen islet-specific glucose-6-phosphatase-catalytic-subunit-related protein (IGRP206-214). Studies in germ-free mice monocolonized with integrase-competent, integrase-deficient, and integrase-transgenic Bacteroides demonstrate that the microbial epitope promotes the recruitment of diabetogenic CD8+ T cells to the gut. There, these effectors suppress colitis by targeting microbial antigen-loaded, antigen-presenting cells in an integrin ß7-, perforin-, and major histocompatibility complex class I-dependent manner. Like their murine counterparts, human peripheral blood T cells also recognize Bacteroides integrase. These data suggest that gut microbial antigen-specific cytotoxic T cells may have therapeutic value in inflammatory bowel disease and unearth molecular mimicry as a novel mechanism by which the gut microbiota can regulate normal immune homeostasis. PAPERCLIP.

The architecture of a scrambled genome reveals massive levels of genomic rearrangement during development.

Programmed DNA rearrangements in the single-celled eukaryote Oxytricha trifallax completely rewire its germline into a somatic nucleus during development. This elaborate, RNA-mediated pathway eliminates noncoding DNA sequences that interrupt gene loci and reorganizes the remaining fragments by inversions and permutations to produce functional genes. Here, we report the Oxytricha germline genome and compare it to the somatic genome to present a global view of its massive scale of genome rearrangements. The remarkably encrypted genome architecture contains >3,500 scrambled genes, as well as >800 predicted germline-limited genes expressed, and some posttranslationally modified, during genome rearrangements. Gene segments for different somatic loci often interweave with each other. Single gene segments can contribute to multiple, distinct somatic loci. Terminal precursor segments from neighboring somatic loci map extremely close to each other, often overlapping. This genome assembly provides a draft of a scrambled genome and a powerful model for studies of genome rearrangement.

A small-molecule PI3Kα activator for cardioprotection and neuroregeneration.

Harnessing the potential beneficial effects of kinase signalling through the generation of direct kinase activators remains an underexplored area of drug development1-5. This also applies to the PI3K signalling pathway, which has been extensively targeted by inhibitors for conditions with PI3K overactivation, such as cancer and immune dysregulation. Here we report the discovery of UCL-TRO-1938 (referred to as 1938 hereon), a small-molecule activator of the PI3Kα isoform, a crucial effector of growth factor signalling. 1938 allosterically activates PI3Kα through a distinct mechanism by enhancing multiple steps of the PI3Kα catalytic cycle and causes both local and global conformational changes in the PI3Kα structure. This compound is selective for PI3Kα over other PI3K isoforms and multiple protein and lipid kinases. It transiently activates PI3K signalling in all rodent and human cells tested, resulting in cellular responses such as proliferation and neurite outgrowth. In rodent models, acute treatment with 1938 provides cardioprotection from ischaemia-reperfusion injury and, after local administration, enhances nerve regeneration following nerve crush. This study identifies a chemical tool to directly probe the PI3Kα signalling pathway and a new approach to modulate PI3K activity, widening the therapeutic potential of targeting these enzymes through short-term activation for tissue protection and regeneration. Our findings illustrate the potential of activating kinases for therapeutic benefit, a currently largely untapped area of drug development.

Enteric tuft cells coordinate timely expulsion of the tapeworm Hymenolepis diminuta from the murine host by coordinating local but not systemic immunity.

Recognizing that enteric tuft cells can signal the presence of nematode parasites, we investigated whether tuft cells are required for the expulsion of the cestode, Hymenolepis diminuta, from the non-permissive mouse host, and in concomitant anti-helminthic responses. BALB/c and C57BL/6 mice infected with H. diminuta expelled the worms by 11 days post-infection (dpi) and displayed DCLK1+ (doublecortin-like kinase 1) tuft cell hyperplasia in the small intestine (not the colon) at 11 dpi. This tuft cell hyperplasia was dependent on IL-4Rα signalling and adaptive immunity, but not the microbiota. Expulsion of H. diminuta was slowed until at least 14 dpi, but not negated, in tuft cell-deficient Pou2f3-/- mice and was accompanied by delayed goblet cell hyperplasia and slowed small bowel transit. Worm antigen and mitogen evoked production of IL-4 and IL-10 by splenocytes from wild-type and Pou2f3-/- mice was not appreciably different, suggesting similar systemic immune reactivity to infection with H. diminuta. Wild-type and Pou2f3-/- mice infected with H. diminuta displayed partial protection against subsequent infection with the nematode Heligmosomoides bakeri. We speculate that, with respect to H. diminuta, enteric tuft cells are important for local immune events driving the rapidity of H. diminuta expulsion but are not critical in initiating or sustaining systemic Th2 responses that provide concomitant immunity against secondary infection with H. bakeri.

Assembly of a pangenome for global cattle reveals missing sequences and novel structural variations, providing new insights into their diversity and evolutionary history.

A cattle pangenome representation was created based on the genome sequences of 898 cattle representing 57 breeds. The pangenome identified 83 Mb of sequence not found in the cattle reference genome, representing 3.1% novel sequence compared with the 2.71-Gb reference. A catalog of structural variants developed from this cattle population identified 3.3 million deletions, 0.12 million inversions, and 0.18 million duplications. Estimates of breed ancestry and hybridization between cattle breeds using insertion/deletions as markers were similar to those produced by single nucleotide polymorphism-based analysis. Hundreds of deletions were observed to have stratification based on subspecies and breed. For example, an insertion of a Bov-tA1 repeat element was identified in the first intron of the APPL2 gene and correlated with cattle breed geographic distribution. This insertion falls within a segment overlapping predicted enhancer and promoter regions of the gene, and could affect important traits such as immune response, olfactory functions, cell proliferation, and glucose metabolism in muscle. The results indicate that pangenomes are a valuable resource for studying diversity and evolutionary history, and help to delineate how domestication, trait-based breeding, and adaptive introgression have shaped the cattle genome.

Macrophage immunotherapy: overcoming impediments to realize promise.

As an essential component of immunity, macrophages have key roles in mammalian host defense, tissue homeostasis, and repair, as well as in disease pathogenesis and pathophysiology. A source of fascination and extensive research, in this Opinion we challenge the utility of the M1-M2 paradigm, and discuss the importance of accurate characterization of human macrophages. We comment on the application of single cell analytics to define macrophage subpopulations and how this could advance therapeutic options. We argue that human macrophage cell therapy can be used to alleviate many diseases, and offer a viewpoint on the knowledge gaps that must be filled to render such a therapeutic approach a reality and, ideally, a common future practice in precision medicine.

A locus at 19q13.31 significantly reduces the ApoE ε4 risk for Alzheimer's Disease in African Ancestry.

African descent populations have a lower Alzheimer disease risk from ApoE ε4 compared to other populations. Ancestry analysis showed that the difference in risk between African and European populations lies in the ancestral genomic background surrounding the ApoE locus (local ancestry). Identifying the mechanism(s) of this protection could lead to greater insight into the etiology of Alzheimer disease and more personalized therapeutic intervention. Our objective is to follow up the local ancestry finding and identify the genetic variants that drive this risk difference and result in a lower risk for developing Alzheimer disease in African ancestry populations. We performed association analyses using a logistic regression model with the ApoE ε4 allele as an interaction term and adjusted for genome-wide ancestry, age, and sex. Discovery analysis included imputed SNP data of 1,850 Alzheimer disease and 4,331 cognitively intact African American individuals. We performed replication analyses on 63 whole genome sequenced Alzheimer disease and 648 cognitively intact Ibadan individuals. Additionally, we reproduced results using whole-genome sequencing of 273 Alzheimer disease and 275 cognitively intact admixed Puerto Rican individuals. A further comparison was done with SNP imputation from an additional 8,463 Alzheimer disease and 11,365 cognitively intact non-Hispanic White individuals. We identified a significant interaction between the ApoE ε4 allele and the SNP rs10423769_A allele, (ß = -0.54,SE = 0.12,p-value = 7.50x10-6) in the discovery data set, and replicated this finding in Ibadan (ß = -1.32,SE = 0.52,p-value = 1.15x10-2) and Puerto Rican (ß = -1.27,SE = 0.64,p-value = 4.91x10-2) individuals. The non-Hispanic Whites analyses showed an interaction trending in the "protective" direction but failing to pass a 0.05 significance threshold (ß = -1.51,SE = 0.84,p-value = 7.26x10-2). The presence of the rs10423769_A allele reduces the odds ratio for Alzheimer disease risk from 7.2 for ApoE ε4/ε4 carriers lacking the A allele to 2.1 for ApoE ε4/ε4 carriers with at least one A allele. This locus is located approximately 2 mB upstream of the ApoE locus, in a large cluster of pregnancy specific beta-1 glycoproteins on chromosome 19 and lies within a long noncoding RNA, ENSG00000282943. This study identified a new African-ancestry specific locus that reduces the risk effect of ApoE ε4 for developing Alzheimer disease. The mechanism of the interaction with ApoEε4 is not known but suggests a novel mechanism for reducing the risk for ε4 carriers opening the possibility for potential ancestry-specific therapeutic intervention.

Utopia Point Bayesian Optimization Finds Condition-Dependent Selectivity for N-Methyl Pyrazole Condensation.

Utopia Point Bayesian Optimization (UPBO) was used to identify reaction conditions that are highly selective for the formation of N1 and N2-methyl-3-aryl pyrazole constitutional isomers. UPBO was used to explore a wide chemical space and identify basic reaction conditions for a typically acid-catalyzed Knorr pyrazole condensation. These studies revealed that selectivity in the reaction stems from a condition-dependent equilibrium of intermediates prior to dehydration. For the N2-methyl isomer reaction pathway, a hemiaminal intermediate was found to form reversibly under the reaction conditions, enabling a highly selective synthesis of the N2 isomer upon dehydrative workup. UPBO was able to successfully optimize conversion and selectivity simultaneously with search spaces of >1 million potential variable combinations without the need for high-performance computational resources.

The impact of time to death in donors after circulatory death on recipient outcome in simultaneous pancreas-kidney transplantation.

The time to arrest donors after circulatory death is unpredictable and can vary. This leads to variable periods of warm ischemic damage prior to pancreas transplantation. There is little evidence supporting procurement team stand-down times based on donor time to death (TTD). We examined what impact TTD had on pancreas graft outcomes following donors after circulatory death (DCD) simultaneous pancreas-kidney transplantation. Data were extracted from the UK transplant registry from 2014 to 2022. Predictors of graft loss were evaluated using a Cox proportional hazards model. Adjusted restricted cubic spline models were generated to further delineate the relationship between TTD and outcome. Three-hundred-and-seventy-five DCD simultaneous kidney-pancreas transplant recipients were included. Increasing TTD was not associated with graft survival (adjusted hazard ratio HR 0.98, 95% confidence interval 0.68-1.41, P = .901). Increasing asystolic time worsened graft survival (adjusted hazard ratio 2.51, 95% confidence interval 1.16-5.43, P = .020). Restricted cubic spline modeling revealed a nonlinear relationship between asystolic time and graft survival and no relationship between TTD and graft survival. We found no evidence that TTD impacts pancreas graft survival after DCD simultaneous pancreas-kidney transplantation; however, increasing asystolic time was a significant predictor of graft loss. Procurement teams should attempt to minimize asystolic time to optimize pancreas graft survival rather than focus on the duration of TTD.

Neuroimmunophysiology of the gastrointestinal tract.

Gut physiology is the epicenter of a web of internal communication systems (i.e., neural, immune, hormonal) mediated by cell-cell contacts, soluble factors, and external influences, such as the microbiome, diet, and the physical environment. Together these provide the signals that shape enteric homeostasis and, when they go awry, lead to disease. Faced with the seemingly paradoxical tasks of nutrient uptake (digestion) and retarding pathogen invasion (host defense), the gut integrates interactions between a variety of cells and signaling molecules to keep the host nourished and protected from pathogens. When the system fails, the outcome can be acute or chronic disease, often labeled as "idiopathic" in nature (e.g., irritable bowel syndrome, inflammatory bowel disease). Here we underscore the importance of a holistic approach to gut physiology, placing an emphasis on intercellular connectedness, using enteric neuroimmunophysiology as the paradigm. The goal of this opinion piece is to acknowledge the pace of change brought to our field via single-cell and -omic methodologies and other techniques such as cell lineage tracing, transgenic animal models, methods for culturing patient tissue, and advanced imaging. We identify gaps in the field and hope to inspire and challenge colleagues to take up the mantle and advance awareness of the subtleties, intricacies, and nuances of intestinal physiology in health and disease by defining communication pathways between gut resident cells, those recruited from the circulation, and "external" influences such as the central nervous system and the gut microbiota.

The natural history and genotype-phenotype correlations of TMPRSS3 hearing loss: an international, multi-center, cohort analysis.

TMPRSS3-related hearing loss presents challenges in correlating genotypic variants with clinical phenotypes due to the small sample sizes of previous studies. We conducted a cross-sectional genomics study coupled with retrospective clinical phenotype analysis on 127 individuals. These individuals were from 16 academic medical centers across 6 countries. Key findings revealed 47 unique TMPRSS3 variants with significant differences in hearing thresholds between those with missense variants versus those with loss-of-function genotypes. The hearing loss progression rate for the DFNB8 subtype was 0.3 dB/year. Post-cochlear implantation, an average word recognition score of 76% was observed. Of the 51 individuals with two missense variants, 10 had DFNB10 with profound hearing loss. These 10 all had at least one of 4 TMPRSS3 variants predicted by computational modeling to be damaging to TMPRSS3 structure and function. To our knowledge, this is the largest study of TMPRSS3 genotype-phenotype correlations. We find significant differences in hearing thresholds, hearing loss progression, and age of presentation, by TMPRSS3 genotype and protein domain affected. Most individuals with TMPRSS3 variants perform well on speech recognition tests after cochlear implant, however increased age at implant is associated with worse outcomes. These findings provide insight for genetic counseling and the on-going design of novel therapeutic approaches.

Challenges and Opportunities for Paving the Road to Global Health Equity Through Implementation Science.

Implementation science focuses on enhancing the widespread uptake of evidence-based interventions into routine practice to improve population health. However, optimizing implementation science to promote health equity in domestic and global resource-limited settings requires considering historical and sociopolitical processes (e.g., colonization, structural racism) and centering in local sociocultural and indigenous cultures and values. This review weaves together principles of decolonization and antiracism to inform critical and reflexive perspectives on partnerships that incorporate a focus on implementation science, with the goal of making progress toward global health equity. From an implementation science perspective, wesynthesize examples of public health evidence-based interventions, strategies, and outcomes applied in global settings that are promising for health equity, alongside a critical examination of partnerships, context, and frameworks operationalized in these studies. We conclude with key future directions to optimize the application of implementation science with a justice orientation to promote global health equity.

SGLT1 contributes to glucose-mediated exacerbation of ischemia-reperfusion injury in ex vivo rat heart.

Hyperglycaemia is common during acute coronary syndromes (ACS) irrespective of diabetic status and portends excess infarct size and mortality, but the mechanisms underlying this effect are poorly understood. We hypothesized that sodium/glucose linked transporter-1 (SGLT1) might contribute to the effect of high-glucose during ACS and examined this using an ex-vivo rodent heart model of ischaemia-reperfusion injury. Langendorff-perfused rat hearts were subjected to 35 min ischemia and 2 h reperfusion, with variable glucose and reciprocal mannitol given during reperfusion in the presence of pharmacological inhibitors of SGLT1. Myocardial SGLT1 expression was determined in rat by rtPCR, RNAscope and immunohistochemistry, as well as in human by single-cell transcriptomic analysis. High glucose in non-diabetic rat heart exacerbated reperfusion injury, significantly increasing infarct size from 45 ± 3 to 65 ± 4% at 11-22 mmol/L glucose, respectively (p < 0.01), an association absent in diabetic heart (32 ± 1-37 ± 5%, p = NS). Rat heart expressed SGLT1 RNA and protein in vascular endothelium and cardiomyocytes, with similar expression found in human myocardium by single-nucleus RNA-sequencing. Rat SGLT1 expression was significantly reduced in diabetic versus non-diabetic heart (0.608 ± 0.08 compared with 1.116 ± 0.13 probe/nuclei, p < 0.01). Pharmacological inhibitors phlorizin, canagliflozin or mizagliflozoin in non-diabetic heart revealed that blockade of SGLT1 but not SGLT2, abrogated glucose-mediated excess reperfusion injury. Elevated glucose is injurious to the rat heart during reperfusion, exacerbating myocardial infarction in non-diabetic heart, whereas the diabetic heart is resistant to raised glucose, a finding which may be explained by lower myocardial SGLT1 expression. SGLT1 is expressed in vascular endothelium and cardiomyocytes and inhibiting SGLT1 abrogates excess glucose-mediated infarction. These data highlight SGLT1 as a potential clinical translational target to improve morbidity/mortality outcomes in hyperglycemic ACS patients.

Representation Matters: Trust in Digital Health Information Among Black Patients With Prostate Cancer.

PURPOSE: Although the majority of US adults obtain health information on the internet, the quality of information about prostate cancer is highly variable. Black adults are underrepresented in online content about prostate cancer despite a higher incidence of and mortality from the disease. The goal of this study was to explore the perspectives of Black patients with prostate cancer on the importance of racial representation in online content and other factors influencing trust. MATERIALS AND METHODS: We conducted 7 virtual focus groups with Black patients with prostate cancer in 2022 and 2023. Participants completed an intake questionnaire with demographics followed by a group discussion, including feedback on purposefully selected online content. Transcripts were independently analyzed by 2 investigators experienced in qualitative research using a constant comparative method. RESULTS: Most participants use online sources to look for prostate cancer information. Racial representation is an important factor affecting trust in the content. A lack of Black representation has consequences, including misperceptions about a lower risk of prostate cancer and discouraging further information-seeking. Other key themes affecting trust in online content included the importance of a reputable source of information, professional website structure, and soliciting money. CONCLUSIONS: Underrepresentation of Black adults in prostate cancer content has the potential to worsen health disparities. Optimal online communications should include racially diverse representation and evidence-based information in a professional format from reputable sources without financial conflict.

Microbial dietary protein metabolism regulates GLP-1 mediated intestinal transit.

Depletion of gut microbiota is associated with inefficient energy extraction and reduced production of short-chain fatty acids from dietary fibers, which regulates colonic proglucagon (Gcg) expression and small intestinal transit in mice. However, the mechanism by which the gut microbiota influences dietary protein metabolism and its corresponding effect on the host physiology is poorly understood. Enteropeptidase inhibitors block host protein digestion and reduce body weight gain in diet-induced obese rats and mice, and therefore they constitute a new class of drugs for targeting metabolic diseases. Enteroendocrine cells (EECs) are dispersed throughout the gut and possess the ability to sense dietary proteins and protein-derived metabolites. Despite this, it remains unclear if enteropeptidase inhibition affects EECs function. In this study, we fed conventional and antibiotic treated mice a western style diet (WSD) supplemented with an enteropeptidase inhibitor (WSD-ETPi), analyzed the expression of gut hormones along the length of the intestine, and measured small intestinal transit under different conditions. The ETPi-supplemented diet promoted higher Gcg expression in the colon and increased circulating Glucagon like peptide-1 (GLP-1) levels, but only in the microbiota-depleted mice. The increase in GLP-1 levels resulted in slower small intestinal transit, which was subsequently reversed by administration of GLP-1 receptor antagonist. Interestingly, small intestinal transit was normalized when an amino acid-derived microbial metabolite, p-cresol, was supplemented along with WSD-ETPi diet, primarily attributed to the reduction of colonic Gcg expression. Collectively, our data suggest that microbial dietary protein metabolism plays an important role in host physiology by regulating GLP-1-mediated intestinal transit.

Grazing preferences of three species of amoebae on cyanobacteria and green algae.

Twenty species/isolates of cyanobacteria and green algae were isolated from cyanobacterial bloom samples in lakes associated with the upper Qu'Appelle River drainage system in southern Saskatchewan, Canada. Three amoebae species (Cochliopodium sp., Vannella sp. and Vermamoeba vermiformis) were also isolated from one of these samples, and were subjected to grazing assays to determine which species of cyanobacteria or algae could potentially serve as a food source. Amoeba grazing rates were quantified based on the diameter of the plaque after 12 days on agar plate assays, and by estimation of the amoeba population growth rate from the rate of increase of plaque area. The common cyanobacterial bloom-formers Dolichospermum sp. and Aphanizomenon flos-aquae supported high growth rates for all three amoebae, while green algae, with the exception of one green alga/amoeba combination, did not support growth of the tested amoebae. Many of the cyanobacterial and algal isolates that did not support amoebae growth were ingested, suggesting that ingestion did not determine grazing success. Overall, while the cyanobacteria Dolichospermum sp. and Aphanizomenon flos-aquae were suitable food sources for the amoebae, the other cyanobacteria were grazed in an unpredictable manner, with some species/strains grazed by some amoebae and some species not grazed at all.

Shop Talk: A Qualitative Study to Understand Peer Health-related Communication Among Black Men at the Barbershop.

BACKGROUND: While successful health promotion efforts among Black men have been implemented at barbershops, the focus has largely been on outcomes as opposed to the processes by which outcomes are produced. An understanding of processes can be leveraged in the design and implementation of future efforts to improve the health of Black men. PURPOSE: The objectives of the present study were to: (i) understand peer-derived sources of health-related support at the barbershop and (ii) understand the role of the barbershop in promoting health among Black men. METHODS: Seven focus groups were conducted at barbershops used predominately by Black men. Each focus group lasted between 45 and 60 min. Using a thematic approach, each focus group was independently coded by two coders using a codebook derived from an inductive and deductive approach. The results were confirmed with members of the community advisory board. RESULTS: Three themes emerged: (i) dynamic and candid exchange of health-related support at the barbershop; (ii) tailored forms of health-related and judgment-free communication that provide encouragement and increase motivation; and (iii) characteristics of a supportive environment at the barbershop that facilitate health-related communication. CONCLUSIONS: The findings of the present study offer a potential pathway for public health efforts seeking to improve health among Black men. Those interested in designing and implementing these efforts can create tailored programs for Black men by recognizing and leveraging the unique dynamics of health-related conversations at the barbershop.

For many Black men, barbershops are more than just a place for a haircut. Barbershops are community hubs that have transformed into safe places for difficult conversations about health. The goal of our study was to understand how Black men communicate about health at the barbershop. To address this goal, we conducted focus groups among the true experts­Black men. We asked them: (i) how do Black men communicate about health at the barbershop? (ii) what do Black men communicate about health at the barbershop? and (iii) what about the barbershop facilitates these conversations? These men indicated that barbershops are a place where Black men can openly and dynamically provide health support to one another through role modeling, passing of wisdom, and passive testimonials. They said their conversations about health are often judgment-free and tailored to provide encouragement and motivation. Finally, they said that the supportive atmosphere of the barbershop facilitates these types of conversations. Although barbershops have been sources of health-related support for Black men for generations, the findings from this study can be used by those developing health promotion programs (in partnership with barbershops) to promote health among Black men.

N1-Selective Methylation of Pyrazoles via α-Halomethylsilanes as Masked Methylating Reagents.

The development of a highly selective N-methylation of pyrazole heterocycles using commercially available, bench-stable α-halomethylsilanes as masked methylating reagents is described. Sterically bulky α-halomethylsilanes significantly improve the selectivity of N-alkylation over traditional methylating reagents and readily undergo protodesilylation in the presence of a fluoride source and water to give N-methyl pyrazoles. Selectivities of 92:8 to >99:1 N1/N2 regioisomeric ratios were achieved with a range of pyrazole substrates in good yields.

Umpolung Flow Chemistry for the Synthesis of a 3-Oxo-3H-spiro[benzofuran-2,4'-piperidine] Building Block.

An efficient and scalable route to tert-butyl 3-oxo-3H-spiro[benzofuran-2,4'-piperidine]-1'-carboxylate, a central prochiral intermediate in the synthesis of SHP2 inhibitor GDC-1971 (migoprotafib), was achieved. Preparation of the title compound from readily available 2-fluorobenzaldehyde included formation of a modified Katritzky benzotriazole hemiaminal, which, upon deprotonation by n-butyllithium, participated in umpolung reactivity via 1,2-addition to tert-butyl 4-oxopiperidine-1-carboxylate (N-Boc-4-piperidone). Most notably, this reaction was developed as a robust plug-flow process that could be executed on multiple kilograms without the need for pilot-scale reaction vessels operating at low cryogenic temperatures. Treatment of the resulting tetrahedral intermediate with oxalic acid resulted in collapse to the corresponding 4-(2-fluorobenzoyl)-4-hydroxypiperidine, which was isolated as a solid via crystallization. The synthesis concluded with an optimized intramolecular SNAr reaction and final crystallization to generate tert-butyl 3-oxo-3H-spiro[benzofuran-2,4'-piperidine]-1'-carboxylate as a stable, high-quality intermediate suitable for further functionalization toward GDC-1971.