Benzylic C(sp3)-H fluorination.

The selective fluorination of C(sp3)-H bonds is an attractive target, particularly for pharmaceutical and agrochemical applications. Consequently, over recent years much attention has been focused on C(sp3)-H fluorination, and several methods that are selective for benzylic C-H bonds have been reported. These protocols operate via several distinct mechanistic pathways and involve a variety of fluorine sources with distinct reactivity profiles. This review aims to give context to these transformations and strategies, highlighting the different tactics to achieve fluorination of benzylic C-H bonds.

Pulsed electrolysis: enhancing primary benzylic C(sp3)-H nucleophilic fluorination.

Electrosynthesis is an efficient and powerful tool for the generation of elusive reactive intermediates. The application of alternative electrolysis waveforms provides a new level of control for dynamic redox environments. Herein, we demonstrate that pulsed electrolysis provides a favourable environment for the generation and fluorination of highly unstable primary benzylic cations from C(sp3)-H bonds. By introduction of a toff period, we propose this waveform modulates the electrical double layer to improve mass transport and limit over-oxidation.

Electrochemical Benzylic C(sp3)-H Direct Amidation.

Amide bonds are ubiquitous and found in a myriad of functional molecules. Although formed in a reliable and robust fashion, alternative amide bond disconnections provide flexibility and synthetic control. Herein we describe an electrochemical method to form the non-amide C-N bond from direct benzylic C(sp3)-H amidation. Our approach is applied toward the synthesis of secondary amides by coupling secondary benzylic substrates with substituted primary benzamides. The reaction has been scaled up to a multigram scale in flow.

Electrochemical Benzylic C(sp3)-H Acyloxylation.

The development of sustainable C(sp3)-H functionalization methods is of great interest to the pharmaceutical and agrochemical industries. Anodic oxidation is an efficient means of producing benzylic cations that can undergo subsequent in situ nucleophilic attack to afford functionalized benzylic products. Herein, we demonstrate the suitability of carboxylic acids as nucleophiles to yield benzylic esters. This method employs a series of secondary benzylic substrates and functionalized carboxylic acids and is demonstrated on a gram scale in flow.

Development and Clinical Validation of a Blood Test for Early Detection of Colorectal Adenomas and Cancer for Screening and Postpolypectomy Surveillance.

Background and Aims: There is a lack of convenient, sensitive, noninvasive strategies for screening and surveillance for colorectal neoplasia. An assay combining the results of circulating epithelial cells (CECs) and somatic mutations of cell-free DNA adjusting for age/sex using a unique algorithm is evaluated in patients requiring colonoscopy. Methods: A prospective single-site 458-subject study (asymptomatic: 43% screening/43% surveillance, enriched with 65 symptomatic subjects undergoing colonoscopy) was conducted. The test analyzed CECs and somatic mutations. The probability of advanced neoplasia (advanced adenoma [AA] and CRCs) was determined by logistic regression methods adjusted for expected CRC incidence rate, prior history of AA, and patient age and sex on a training subset. A linear predictor was developed to generate a score scaled from 0 to 100. The test performance was evaluated on an independent set of subjects using prespecified algorithms and cut point. Results: Based on a predefined clinical threshold and predictive model derived from the training set (n = 232), analysis of an independent asymptomatic validation set (n = 194) yielded 89% (lower exact one-sided 95% confidence interval [CI]: 80%) specificity and 100% (95% CI: 37%)/78% (95% CI: 61%) sensitivity for detection of CRC/AA. In a secondary analysis, excluding surveillance subjects, the 97-subject screening cohort yielded 91% (95% CI: 79%) specificity and CRC/AA sensitivity at 100% (95% CI: 37%)/83% (95% CI: 56%, 87% for advanced neoplasia 95% CI: 64%). Significant associations (P < .0001) were detected between FirstSight scores and adenoma size, number, and ordinally increasing pathology classification. Conclusion: A multimodal blood test that included CECs and somatic mutations with adjustment for age and sex demonstrated high sensitivity for the diagnosis of advanced colorectal neoplasia. The resulting score captures prognostic information for CRC progression of index adenoma size and number and has the potential to enable stratification of patients for screening or postpolypectomy surveillance colonoscopy.

Direct electrochemical hydrodefluorination of trifluoromethylketones enabled by non-protic conditions.

CF2H groups are unique due to the combination of their lipophilic and hydrogen bonding properties. The strength of H-bonding is determined by the group to which it is appended. Several functional groups have been explored in this context including O, S, SO and SO2 to tune the intermolecular interaction. Difluoromethyl ketones are under-studied in this context, without a broadly accessible method for their preparation. Herein, we describe the development of an electrochemical hydrodefluorination of readily accessible trifluoromethylketones. The single-step reaction at deeply reductive potentials is uniquely amenable to challenging electron-rich substrates and reductively sensitive functionality. Key to this success is the use of non-protic conditions enabled by an ammonium salt that serves as a reductively stable, masked proton source. Analysis of their H-bonding has revealed difluoromethyl ketones to be potentially highly useful dual H-bond donor/acceptor moieties.

Semi-automated library preparation for high-throughput DNA sequencing platforms.

Next-generation sequencing platforms are powerful technologies, providing gigabases of genetic information in a single run. An important prerequisite for high-throughput DNA sequencing is the development of robust and cost-effective preprocessing protocols for DNA sample library construction. Here we report the development of a semi-automated sample preparation protocol to produce adaptor-ligated fragment libraries. Using a liquid-handling robot in conjunction with Carboxy Terminated Magnetic Beads, we labeled each library sample using a unique 6 bp DNA barcode, which allowed multiplex sample processing and sequencing of 32 libraries in a single run using Applied Biosystems' SOLiD sequencer. We applied our semi-automated pipeline to targeted medical resequencing of nuclear candidate genes in individuals affected by mitochondrial disorders. This novel method is capable of preparing as much as 32 DNA libraries in 2.01 days (8-hour workday) for emulsion PCR/high throughput DNA sequencing, increasing sample preparation production by 8-fold.

Detection of Circulating Tumor DNA with a Single-Molecule Sequencing Analysis Validated for Targeted and Immunotherapy Selection.

INTRODUCTION: Comprehensive genetic cancer profiling using circulating tumor DNA has enabled the detection of National Comprehensive Cancer Network (NCCN) guideline-recommended somatic alterations from a single, non-invasive blood draw. However, reliably detecting somatic variants at low variant allele fractions (VAFs) remains a challenge for next-generation sequencing (NGS)-based tests. We have developed the single-molecule sequencing (SMSEQ) platform to address these challenges. METHODS: The OncoLBx assay utilizes the SMSEQ platform to optimize cell-free DNA extraction and library preparation with variant type-specific calling algorithms to improve sensitivity and specificity. OncoLBx is a pan-cancer panel for solid tumors targeting 75 genes and five microsatellite sites analyzing five classes of NCCN-recommended somatic variants: single-nucleotide variants (SNVs), insertions and deletions (indels), copy number variants (CNVs), fusions and microsatellite instability (MSI). Circulating DNA was extracted from plasma, followed by library preparation using SMSEQ. Analytical validation was performed according to recently published American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology (AMP) guidelines and established the limit of detection (LOD), sensitivity, specificity, accuracy and reproducibility using 126 gold-standard reference samples, healthy donor samples verified by whole-exome sequencing by an external College of American Pathologists (CAP) reference lab and cell lines with known variants. Results were analyzed using a locus-specific modeling algorithm. RESULTS: We have demonstrated that OncoLBx detects VAFs of ≥ 0.1% for SNVs and indels, ≥ 0.5% for fusions, ≥ 4.5 copies for CNVs and ≥ 2% for MSI, with all variant types having specificity ≥ 99.999%. Diagnostic performance of paired samples displays 80% sensitivity and > 99.999% clinical specificity. Clinical utility and performance were assessed in 416 solid tumor samples. Variants were detected in 79% of samples, for which 87.34% of positive samples had available targeted therapy.