The kinetics of many proton-coupled electron transfer (PCET) reactions cannot be adequately described by stepwise proton and electron transfer. Concerted electron− proton transfer (CPET) is another possibility, but examples exist where stepwise mechanisms are not viable yet there is no compelling evidence for CPET. This study investigates such a reaction, the oxidation of an NH-containing phenylenediamine radical cation, H 2 PD + , in the presence of pyridines in acetonitrile, using CV and UV/vis spectroelectrochemistry. As observed previously, the E 1/2 for the radical oxidation jumps to a considerably more negative potential upon addition of 1 equiv of pyridine. The CV wave broadens but stays chemically reversible. Further addition of pyridine leads to smaller E 1/2 shifts with continued reversibility. Different explanations have been put forth for this behavior; however, this study provides strong evidence that the E 1/2 shift can be completely explained by the overall reaction being H 2 PD + + pyr − e − → HPD + + Hpyr + . Classic stepwise proton−electron transfer cannot explain the reversibility, but it can be explained by a "wedge" scheme mechanism in which electron and proton transfer occurs in a stepwise fashion within the H-bond complex formed as an intermediate in proton transfer. This result points to the important role H-bonding may play in PCET even without CPET.

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In the human genome, heterozygous sites are genomic positions with different alleles inherited from each parent. On average, there is a heterozygous site every 1-2 kilobases (kb). Resolving whether two alleles in neighboring heterozygous positions are physically linked—that is, phased—is possible with a short-read sequencer if the sequencing library captures long-range information. TELL-Seq is a library preparation method based on millions of barcoded micro-sized beads that enables instrument-free phasing of a whole human genome in a single PCR tube. TELL-Seq incorporates a unique molecular identifier (barcode) to the short reads generated from the same high-molecular-weight (HMW) DNA fragment (known as ‘linked-reads’). However, genome-scale TELL-Seq is not cost-effective for applications focusing on a single locus or a few loci. Here, we present an optimized TELL-Seq protocol that enables the cost-effective phasing of enriched loci (targets) of varying sizes, purity levels, and heterozygosity. Targeted TELL-Seq maximizes linked-read efficiency and library yield while minimizing input requirements, fragment collisions on microbeads, and sequencing burden. To validate the targeted protocol, we phased seven 180-200 kb loci enriched by CRISPR/Cas9-mediated excision coupled with pulse-field electrophoresis, four 20 kb loci enriched by CRISPR/Cas9-mediated protection from exonuclease digestion, and six 2-13 kb loci amplified by PCR. The selected targets have clinical and research relevance (BRCA1, BRCA2, MLH1, MSH2, MSH6, APC, PMS2, SCN5A-SCN10A, andPKI3CA). These analyses reveal that targeted TELL-Seq provides a reliable way of phasing allelic variants within targets (2-200 kb in length) with the low cost and high accuracy of short-read sequencing.

The kinetics of many proton-coupled electron transfer (PCET) reactions cannot be adequately described by step-wise proton and electron transfer. Concerted electron-proton transfer (CPET) is another possibility, but examples exist where step-wise mechanisms are not viable yet there is no compelling evidence for CPET. This study investigates such a reaction, the oxidation of an NH-containing phenylenediamine radical cation, H2PD+, in the presence of pyridines in acetonitrile. H2PD+ is formed by a net one electron oxidation of 2,3,5,6-tetramethylphenylenediamine in acetonitrile via a rather complicated mechanism that likely proceeds through a H-bonded dimer. Once formed, it can be further oxidized at more positive potentials to the quinoidal dication, H2PD2+, which will be considerably more acidic than the radical cation. Indeed, the E1/2 for the radical oxidation jumps to a considerably more negative potential upon addition of 1 equivalent of the weak base pyridine. The CV wave broadens but stays chemically reversible. Further addition of pyridine leads to smaller E1/2 shifts with continued reversibility. This behavior could possibly be explained by stabilization of H2PD2+ through H-bonding or proton transfer to pyridine, however, UV-vis spectroelectrochemical experiments provide definitive evidence for proton transfer. Furthermore, CV studies with 4-substituted pyridine derivatives of weaker basicity show that the observed E1/2 with 1 equivalent of the pyridine depends in a Nernstian fashion on the pKa of the conjugate acid of the pyridine, indicating that all the pyridines studied deprotonate H2PD2+ to give the quinoidal cation, HPD+. The continued E1/2 shift observed upon further addition of the pyridines can then be completely explained by application of the Nernst equation to the overall reaction H2PD+ + pyr → HPD+ + Hpyr+ + e−. However, while this explains the thermodynamics, the classic step-wise proton-electron transfer mechanism for this reaction cannot explain the observed reversibility at high base concentration. In contrast, the reversibility can be explained by a “wedge” scheme mechanism in which electron and proton transfer occur within the H-bond complex formed as an intermediate in proton transfer. In this case, the electron-proton transfer could be concerted, however, the kinetics for the second oxidation in the presence of pyridine show no significant isotope effect. Furthermore, a new reduction peak that appears at faster scan rates suggests the presence of an intermediate in the electron-proton transfer. Both results strongly suggest that the electron-proton transfer is step-wise, not concerted, within the H-bond complex. This result points to the important role H-bonding may play in PCET even without CPET.

Ureidopyrimidones (UPy’s) are well-known to dimerize in weakly polar solvents via the formation of four strong, linear H-bonds. Application of heat or mechanical stress can be used to break these bonds, which has led to their use as a linker in self-healing supramolecular polymers. On-going research in our lab is focused on further increasing the versatility of the UPy system by creating electroactive UPy’s in which dimerization strength can also be controlled through oxidation and reduction. Previously, we have shown that dimers based on the ferrocene-UPy derivative, 1, break apart upon oxidation of the ferrocene to the ferrocenium form at mM concentrations in CH2Cl2. This could be due both to the creation of electrostatic repulsion and a decrease in H-bond strength due to the reduced H-donor ability of the O and N on the pyrimidone side. In this project, another Fc-UPy has been prepared, 2, in which the ferrocene is attached to the urea side of the molecule. In contrast to 1, oxidation of 2 shows a single reversible ferrocene CV wave in CH2Cl2 at mM concentrations. Under these conditions, 1H NMR indicates that 2 is fully dimerized, thus, it appears that, unlike with 1, oxidation has no significant effect on dimerization of 2. In both cases, oxidation would increase electrostatic repulsion. However, unlike in 1, oxidation of 2 should actually increase H-bond strength by making the urea NH a stronger H-donor. Therefore, it appears that the effect of oxidation on the H-donating or accepting ability is a more crucial factor for dimerization control than electrostatics in these systems. Figure 1