FGF4) gene have previously been described in the domestic dog. An FGF4 retrocopy on chr18 is associated with disproportionate dwarfism, while an FGF4 retrocopy on chr12 is associated with both disproportionate dwarfism and intervertebral disc disease (IVDD). In this study, whole-genome sequencing data were queried to identify other FGF4 retrocopies that could be contributing to phenotypic diversity in canids. Additionally, dogs with surgically confirmed IVDD were assayed for novel FGF4 retrocopies. Five additional and distinct FGF4 retrocopies were identified in canids including a copy unique to red wolves (Canis rufus). The FGF4 retrocopies identified in domestic dogs were identical to domestic dog FGF4 haplotypes, which are distinct from modern wolf FGF4 haplotypes, indicating that these retrotransposition events likely occurred after domestication. The identification of multiple, full length FGF4 retrocopies with open reading frames in canids indicates that gene retrotransposition events occur much more frequently than previously thought and provide a mechanism for continued genetic and phenotypic diversity in canids.Two transcribed retrocopies of the fibroblast growth factor 4 ( Gene retrocopies, often previously referred to as processed pseudogenes, are formed through the mRNA-mediated gene duplication of cellular gene transcripts . In mammRetrocopies are more likely to come from highly expressed genes , with soFGF4) retrocopies have been described previously in dogs on chr18 [FGF4L1 (CFA18) and FGF4L2 (CFA12) in this study. Both FGF4L1 and FGF4L2 are associated with forms of disproportionate dwarfism that are common across many popular dog breeds, and there is evidence that these genes have been under selection owing their strong phenotypic effects [FGF4L2 has also been associated with canine chondrodystrophy, a disorder characterized by premature degeneration of the intervertebral discs, which predisposes affected dogs to intervertebral disc herniation [FGF4L2, indicating the possibility of alternative risk loci for the disorder [Two expressed, polymorphic fibroblast growth factor 4 gene  to identify the presence of an intron-less retrocopy, followed by inverse PCR to identify the site of insertion. Five additional FGF4 retrocopies were then identified, sequenced, and characterized.Because two recent, functional FGF4 retrocopies, and thus all reads coming from FGF4 retrocopies are aligned to the parental FGF4 gene locus. To identify any such novel FGF4 retrocopies, aligned paired end Illumina sequence data in the region surrounding the FGF4 gene  were downloaded from the Sequence Read Archive and analyzed. Sequencing files were viewed in Integrative Genomics Viewer [FGF4 gene retrocopy somewhere in the genome as retrocopies lack introns, while discordant paired end reads, wherein one mate maps to the FGF4 gene locus and the other mate maps to another region of the genome, are indicative of the putative insertion site for an FGF4 retrocopy  were utilized for this approach ,23,24,25s Viewer . DiscordFGF4 retrocopies in DNA samples. A total of 164 surgical cases that were previously shown to have 0 copies of FGF4L1 and FGF4L2 were used for novel FGF4 retrocopy discovery [FGF4 retrocopies was tested by amplifying the region between exon 1 and exon 3 of FGF4 , and fragments were then circularized by ligation at final concentrations varying between 1 and 10 ng/μL using T4 DNA ligase according to the manufacturer’s instructions for sticky end ligation . A set of inverted primers were designed that amplified circular DNA fragments containing the 5′ end of the FGF4 retrocopy insertion (http://bioinfo.ut.ee/primer3/) [Whole-genome sequence data were not available for any of the individuals treated by surgical decompression for presumed IVDD. Therefore, a molecular approach was developed to test for novel iscovery . The pre of FGF4 . The ideetrocopy . When anerse PCR  was thennsertion . PCR wasrimer3/) .FGF4 retrocopies came from the Bannasch Canine Repository and were obtained under UC Davis Animal Care and Use Committee protocol 18,561 [FGF4 retrocopies identified via discordant paired end reads or inverse PCR (FGF4 gene were observed in a dataset of 722 canids to determine which single-nucleotide variants (SNVs) were unique to FGF4 retrocopies [All canine DNA samples used for retrocopy sequencing and subsequent population genotyping of the l 18,561  (Supplemerse PCR . Entire erse PCR . Variantrocopies .FGF4 retrocopy insertion sites were defined using the 4-Way Multiz Alignment & Conservation track for CanFam2 on the UCSC genome browser [Evolutionarily conserved elements (ECR) near the  browser , which s browser .FGF4 retrocopy for population genotyping, as previously described [FGF4 retrocopy produces a different size amplicon when the retrocopy is present . Multivariable linear regression was performed in R studio using the generalized linear model function with sex and FGF4 genotype included to identify any association with height.Height was measured in selected cases to determine if FGF4 retrocopies, FGF4L1 and FGF4L2, evidence for four additional FGF4 retrocopies in canids was observed in the whole-genome sequence dataset (FGF4 retrocopies include a copy on CFA27 (FGF4L3) seen in three Nova Scotia Duck Tolling Retrievers (NSDTR); a copy on CFA22 (FGF4L4) seen in two Norwich Terriers; a copy on CFA13 (FGF4L5) seen in a Belgian Malinois and a Dutch Shepherd; and a copy on CFA36 (FGF4L6) seen in two red wolves. Sequence read archive (SRA) accession numbers for these individuals are available in In addition to the two known  dataset . The novFGF4 gene locus aligning to a partial FGF4 retrocopy insertion in the CanFam3 reference genome at chr7:68,372,263–68,373,442. To confirm whether this was a real FGF4 retrocopy fragment or a mistake in the reference assembly, primers were designed flanking the insertion and the region was amplified in six Boxers. Five were heterozygous for the CFA7 partial FGF4 retrocopy insertion, and Sanger sequencing confirmed the sequence matched the reference genome. Because this retrocopy only contains the 3′ UTR of the gene and has no ORF, this retrocopy fragment was not considered for further analysis.Discordant reads were also observed at the 3′ end of the FGF4L1 nor FGF4L2 was then tested for the presence of any FGF4 retrocopy using an exon–exon PCR assay. Four of these individuals tested positive for the presence of an FGF4 retrocopy. These samples were first tested for the other newly discovered FGF4 retrocopies. One sample, a Shetland Sheepdog, was heterozygous for FGF4L5. The medical history of this individual indicates that it received a hemilaminectomy to treat a mass that was not disc-related.A surgically treated population of 164 individuals that had neither FGF4 retrocopies were present in these individuals, indicating they contained a novel FGF4 retrocopy. Inverse PCR was then performed to discover the insertion site of the novel FGF4 retrocopies in these individuals, which was on CFA13 (FGF4L7) at approximately CFA13:25,020,600. The three dogs were all heterozygous for FGF4L7, and Sanger sequencing revealed that FGF4L7 is a full length FGF4 retrocopy.The three remaining dogs were all Pit Bull Terrier mixes that had received hemilaminectomies for IVDD at relatively young ages , and none of the newly discovered or previously defined FGF4 retrocopies were confirmed through PCR amplification and sequencing. The genomic location for the FGF4 retrocopies, their TSD, and genomic sequence surrounding the TSD are shown in FGF4L5 insertion site. Insertion sites for 6/7 of the FGF4 retrocopies had a low G/C content compared with the Canfam3 average of 41.3% . The number of evolutionarily conserved regions within 2.5 kb of the insertion sites is also reported in Novel of 41.3% . All FGFFGF4 retrocopy to the parental FGF4 sequence showed that each novel copy has a fully conserved ORF  were identified in either the ORF or the 5′ UTR of any of the retrocopies. However, six SNVs were identified in the 3′ UTR that differed from the reference genome FGF4 gene sequence. Analysis of a whole-genome sequencing variant calling dataset from 722 canids indicated that these SNVs are also present at the parental FGF4 gene  and an insertion . These indels were not identified in any canids other than the two red wolves in a whole-genome sequencing variant calling dataset, which included 46 gray wolves. The parental FGF4 locus was sequenced in seven red wolves to determine whether these variants also exist in the parental gene in red wolves or if they are unique to the retrocopy. While three individuals were heterozygous for the CFA18:48,416,575T>TA insertion at the parental FGF4 gene, CFA18:48,415,685CA C was not identified in any of the parental FGF4 sequences, indicating this variant may have occurred after retrotransposition and may be unique to FGF4L6.The red wolf FGF4 retrocopies were identified was utilized to determine allele frequencies of the FGF4 retrocopies. A complete list of FGF4 retrocopy genotyping results is available in FGF4L3 was only observed in the NSDTR breed in the whole-genome sequencing dataset, and was thus tested in 100 randomly selected NSDTR. The allele frequency of FGF4L3 was 8.5% in the NSDTR.A targeted population genotyping approach based on the breeds in which FGF4L4 had an allele frequency of 16.7% in Norwich Terriers (n = 30). Further testing for FGF4L4 in related terrier breeds identified this retrocopy in Norfolk Terriers , Border Terriers , and Skye Terriers . Given the previous association of FGF4 retrogenes with skeletal dysplasia, FGF4L4 genotype was also compared to height at the withers in 24 Border Terriers using multiple linear regression. The regression analysis identified no significant association between FGF4L4 and height in Border Terriers , although only one homozygous wild type individual was included (included .FGF4L5 was not identified in any other Shetland Sheepdogs (n = 58) or Belgian Malinois (n = 14). Australian Shepherds (n = 19) and Anatolian Shepherd dogs (n = 5) also tested negative for FGF4L5. No additional Dutch Shepherd samples were available for population genotyping of FGF4L5 in the breed. FGF4L6 was tested in 14 red wolf samples, 5 of which were heterozygous .FGF4L7 (n = 201), and all tested negative for the retrocopy. Because FGF4L7 was identified in dogs treated for IVDD and could be contributing to the disorder, all mixed breed dogs from the Bannasch Canine Repository that had been treated surgically for IVDD were also tested for FGF4L7 (n = 55), all of which tested negative. However, two discordant reads mapping to the FGF4L7 were subsequently identified in the whole-genome sequence data of a single Chinese village dog (SRR7107669). Several breeds developed in Asia were then tested for FGF4L7, including Chow Chow (n = 22), Pugs (n = 9), Pekingese (n = 8), and Tibetan terriers (n = 6), none of which tested positive. However, FGF4L7 was identified in Chinese Shar-Pei .Pit Bull Terriers and Pit Terrier Mixes were then tested for FGF4 retrocopies exist in canids in addition to the previously identified FGF4L1 and FGF4L2. Novel retrocopies appear to be breed or breed group specific, contain intact ORFs, and have not accrued mutations that differentiate them from parental FGF4 gene haplotypes. The FGF4 retrocopies were retrotransposed from FGF4 genes with distinct haplotypes, indicating that the same copy has not been retrotransposed multiple times. It is unclear whether any of these novel copies are expressed retrogenes, or in what tissue types they could be expressed. FGF4L7 was identified in three dogs treated surgically for IVDD, however, the significance relative to IVDD is unknown. The majority of IVDD surgical cases examined in this study that were not explained by FGF4L2 were found to have no FGF4 retrocopies, indicating that there are risk factors other than FGF4 retrocopies that predispose dogs to IVDD.Multiple recently transposed FGF4L1 and FGF4L2 has indicated that the FGF4 retrocopies are capable of expression [FGF4 gene is GC-rich and contains many evolutionarily conserved transcription factor binding sites that were previously hypothesized to be conducive towards expression of the retrocopies [FGF4L3 retrocopy likely affects expression. It has also been reported that the expression of retrocopies is highly dependent on the genomic environment of the insertion sites [FGF4L1 and FGF4L2 have inserted into regions containing nearby evolutionarily conserved elements (ECRs). Similarly, ECRs at all but one of the FGF4 retrocopy insertion sites may be conducive towards expression. The different genomic context at the insertion sites for FGF4L1 and FGF4L2 could also explain the different phenotypes between the copies. If expressed, the novel FGF4 retrocopies may show unique expression profiles, resulting in phenotypic associations other than height and IVDD. FGF4 is involved in several cellular processes including cell growth, tissue repair, tumor growth and invasion, and is also a well-known proto-oncogene [Evidence for the expression of both pression ,15. The on sites . Both FGoncogene ,33.FGF4L2 has been shown to have a major association with IVDD [FGF4L2 retrogene, implicating alternate causative factors [FGF4 retrogenes are logical candidates for these FGF4L2 negative IVDD cases, however the additional FGF4 retrocopies identified in this study do not appear to provide a compelling explanation for this group of dogs owing to the limited frequency of the retrogenes in affected animals. Although FGF4L7 was identified in three dogs treated surgically for IVDD, it was not seen in any other breeds in the surgically treated data set, and the breed with the highest identified allele frequency  is not known to be among the breeds highly predisposed to IVDD [FGF4L4 retrogene. Interestingly, FGF4L7 inserted 5 Mb downstream from the HAS2 gene, a gene that has been implicated in the Shar-Pei wrinkled skin phenotype as well as Familial Shar-Pei Fever [FGF4L7 in the breed.Although ith IVDD ,19,34, c to IVDD . Similarei Fever . Strong FGF4L4 was not found to be associated with height in Border Terriers, the majority of Border Terriers tested had either one or two copies of FGF4L4, and only one individual with 0 copies was identified. If the retrocopy has a dominant effect on height in the breed, more homozygous wild type individuals will need to be measured to determine any effect. FGF4L4 was also found at low allele frequencies in other related terrier breeds, including the Skye, Norwich, and Norfolk Terriers, and may have originated in a common progenitor to the terrier breed group. As dog breeds are known to be highly inbred [FGF4L1 is also very common in Norwich and Norfolk Terriers [FGF4L1 and FGF4L2, making them the first breed to be identified with three FGF4 retrocopies.While y inbred ,38, a hiTerriers , and theFGF4 retrocopies in canids appear to have been very recently retrotransposed with no new mutations differentiating them from the parental FGF4 gene. Even the red wolf FGF4 retrocopy, FGF4L6, is nearly identical to the red wolf specific FGF4 haplotype. Dating the FGF4 retrocopy insertions is difficult owing to their short length (3.2 kbp) and sequence identity to the parental gene sequence; however, the FGF4 retrocopies are identical to canine-specific FGF4 gene haplotypes, which are distinct from modern wolf FGF4 haplotypes  and a hedgehog (Echinops telfairi) also have FGF4 retrocopies, although they are only 61.2% and 90.6% identical to the parental genes, indicating they are not recent [FGF4 retrocopies not found in their reference genomes. Another possibility is that L1 mediated gene retrotransposition in general is occurring more frequently in canids. If this was the case, recent, polymorphic retrocopies may be more common in canids in a greater number of genes than just FGF4.All the plotypes . This cotal gene ,39. HoweFGF4L1 and FGF4L2, retrocopies of other genes may have phenotypic consequences. As such, the possibility of retrocopy insertions should be considered when scanning critical intervals for disease trait associations. Recently inserted gene retrocopies can result in overexpression of the parental gene product, resulting in gain of function, which could be deleterious [FGF4 gene; a similar approach could be generalized to all genes to identify other polymorphic gene retrocopies in canids. Similar to the FGF4 retrocopies, other polymorphic retrocopies may play an important role in both breed health and phenotypic variation across dogs.While next generation sequencing allows for the detection of polymorphic gene retrocopies, they often go unidentified or misidentified by common variant calling methods . Howevereterious . In this


The voltammogram of [(Cp2Ti)3HATN(Ph)6] shows six oxidation and three reduction waves. Solution spectra of [(Cp2Ti)3HATN(Ph)6] and of the electrochemically formed oxidation products show electronic transitions in the UV, visible and the NIR ranges. Density functional theory (DFT) and linear response time‐dependent DFT show that the three formally titanium(II) centers transfer an electron to the HATN ligand in the ground state. The optically excited transitions occur exclusively between ligand‐centered orbitals. The charged titanium centers only provide an electrostatic frame to the extended π‐electronic system. Complete active self‐consistent field (CASSCF) calculation on a structurally simplified model compound, which considers the multi‐reference character imposed by the three titanium centers, can provide an interpretation of the experimentally observed temperature‐dependent magnetic behavior of the different redox states of the title compound in full consistency with the interpretation of the electronic spectra.Multinuclear transition metal complexes bridged by ligands with extended π‐electronic systems show a variety of complex electronic transitions and electron transfer reactions. While a systematic understanding of the photochemistry and electrochemistry has been attained for binuclear complexes, much less is known about trinuclear complexes such as hexaphenyl‐5,6,11,12,17,18‐hexaazatrinaphthylene‐tristitanocene [(Cp More complex?! The ground state of the three‐nuclear [(Cp2Ti)3HATN(Ph)6] complex is formed by spin states of different multiplicity and explains the rich electronic transition in the UV‐vis‐NIR ranges in different charge states. Contrary to common notion, IVCT transitions do not play a role in the NIR spectra. All steps are one electron processes (Table 2Ti)3HATN(Ph)6] allows the observation of even one more redox transition in the accessible potential range than for the previously reported related compoundThe voltammogram of [3HATN(Ph)6] and its oxidation and reduction products [(Cp2Ti)3HATN(Ph)6]3−, [(Cp2Ti)3HATN(Ph)6]2−, [(Cp2Ti)3HATN(Ph)6]1−, [(Cp2Ti)3HATN(Ph)6]1+ and [(Cp2Ti)3HATN(Ph)6]2+ show electronic transitions in the UV, vis and NIR spectral ranges , transitions from metal centered d‐type orbitals to ligand orbitals (MLCT) and of course transitions between purely ligand centered orbitals (in the visible region mainly π‐π*). In multinuclear complexes transitions between d‐type orbitals of different metal centers in different valence states can be observed as a fifth type of electronic transition, commonly referred to as IVCT. However, an IVCT is only possible in systems with extensive state mixing involving two d orbitals from the metal centers and one ligand orbital which provides strong electronic coupling between the metal centers.Different types of electronic transitions can be observed in transition metal complexes. In common literature they are usually visualized by molecular orbital (MO) diagrams, where MOs are combined from ligand orbitals Figure a and met6 ligand at least one electron is supposed to be transferred from the three Cp2Ti centers to the ligand. The cyclic voltammetric data of HATN(Ph)6 in Figure 6]2− was unstable in the electrolyte solution. The combined magnetic and electrochemical data made the ground state plausible with a negatively charged ligand, although the exact number of transferred electrons cannot be ascertained with the available methods.Experimental magnetic moments of the solid compound at elevated temperature suggest the existence of more than two unpaired electrons in the ground state of the neutral complex.The measured spectra displayed in Figure not provide a full explanation for the spectra of the oxidized species, i. e. there is disappearance of the expected CT bands in experimental NIR spectra. For the HATN complexes, this is not surprising because the applicability of the single electron picture and the appropriate definition of the ground state require more detailed quantum chemical calculation as provided in Sections 2.3. to 2.5. of this paper. For [(Cp2Ti)3HATN(Ph)6] studied here, one must suspect appearance of multiple spin states. Those scenarios prevent a meaningful applicability of a single electron picture in addition to the anyway very crude assumptions required to condense electrochemical and spectroscopic data in one picture. Therefore, we strongly discourage to derive theoretical quantities such as orbital energies from experimental data in case high quality quantum chemical calculations are available.While this assignment seemed plausible at first sight, it can 2.33h symmetry two degenerate HOMOs can be found 3HATN(Ph)6]. Generally, the bent titanocene(II) fragment is often used in the preparative chemistry in order to introduce electron transfer processes as starting point for a broad range of subsequent reactions.2Ti leaves this transfer as a valid option.Each Cpe Figure b. The el2Ti)3HATN(Ph)6] in the NIR range  give first insights into the excitations observed in the experimental NIR spectra. When the HATN molecule is charged, the calculation reproduces similar electronic excitations as found for the . In this compound three electrons were transferred to the ligand and caused absorptions at 800 nm and 920 nm. Calculation on the TD−B3LYP level confirmed that the observed spectra resulted from transitions from the highest occupied molecular orbital (HOMO) and a single occupied molecular orbital (SOMO) to the lowest unoccupied molecular orbital (LUMO). In this model compound the assignment was clear because no transition metals and d‐orbitals were involved in the coordination. However, up to six electrons of three TiCp2 complexes are available for a possible charge transfer in [(Cp2Ti)3HATN(Ph)6]. This significantly increases the complexity of the system comared to the system studied by Moilanen et al.This interpretation has precedence in literature. Moilanen et al.2Ti)3HATN(Ph)6] more precisely, four spin multiplicities  were calculated for the neutral species with PBE0. All multiplicities except S=3 show a strong spin contamination . The spin states S={0, 1, 2} are also energetically degenerate. The mixing of higher spin states leads to assumption of a strong multireference character produced by the titanium centers, which is reasonable for such a highly symmetrical system. As reported in the literature, high spin contamination of the ground state wavefunction can lead to even higher spin contamination in the excited states and therefore lead to prediction of unphysical excitations.In order to investigate the ground state of [(Cp2Ti)3HATN(Ph)6] can still be calculated with sufficient accuracy using TD‐DFT in the spin states S=1 and S=2. Also, the spin states S=0 and S=3 are energetically very close to each other but show no electronic excitations in the NIR range. Table However, the experimental spectrum of [3HATN(Ph)6], i. e. before any electronic excitation. Consequently, the titanium atoms are not involved in the electronic excitation process, but provide a charged frame for the excitation processes on the HATN(PH)6 ligand. Despite the good agreement of the theoretical excitations with experiment, the results are affected by spin degeneracy and spin contamination and therefore should be interpreted with caution. Both attributes indicate an intrinsic multi‐reference character. This character is caused by the titanium atoms and is not required to reproduce the observed NIR spectrum (cf. Section 2.5).Looking at the structure of the orbitals involved in the excitation process , it becomes clear that only ligand‐ligand excitations are observed. As hypothesized above, a charge transfer from the titanium atoms to the ligand takes place in the ground state of . All electron transfers are one electron processes clearly separated on the potential scale. The solution spectra of [(Cp2Ti)3HATN(Ph)6] and its oxidation and reduction products [(Cp2Ti)3HATN(Ph)6]3−, [(Cp2Ti)3HATN(Ph)6]2−, [(Cp2Ti)3HATN(Ph)6]1−, [(Cp2Ti)3HATN(Ph)6]1+ and [(Cp2Ti)3HATN(Ph)6]2+ show electronic transitions in the UV, vis and NIR spectral ranges. DFT calculations on TD‐DFT and CASSCF/NEVPT2 levels show that three electrons are transferred from the formally titanium(II) centers to the bridging ligand in the ground state. However, the complete spectra can only be explained when assuming that spin states of different multiplicity form the ground state of [(Cp2Ti)3HATN(Ph)6]. The observed electronic excitations in the UV, Vis and NIR ranges are all transitions between ligand levels. Contrary to common notion, LMCT or IVCT transitions do not play a role. The same assignment rules can also be reproduced by calculating the isolated and appropriately charged HATN ligand. The temperature‐dependent magnetic properties can be explained the hybridization of different electronic configurations of different total spin with one valence electron located at each of the three Ti centers and one electron in a SOMO of the ligand.Three reduction waves and six oxidation waves are observed in voltammograms of [(Cp2Ti)3HATN(Ph)6] was synthesized as reported.6), Sigma Aldrich, Steinheim, Germany), silver perchlorate . Acetonitrile (MeCN) and tetrahydrofurane (THF) were dried according to standard procedures2Ti)3HATN(Ph)6] was solved in dry THF to a concentration of 0.3 mM. complex and the pure HATN ligand.All calculations were performed using the program package ORCA in version 4.2.The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


It was highlighted that the original article  containeIncorrectThe returned similarity score is based on Jaccard similarity [18] of Morgan-style connectivity fingerprints [19] with a radius of up to 5.CorrectThe returned similarity score is based on Jaccard similarity [18] of Morgan-style connectivity fingerprints [19] with a radius of up to 3.


Electrocatalysts exposed to ~3–30 ALD cycles of TiO2 exhibited overpotentials at 10 mA cm–2 of geometric current density that were several hundred millivolts lower than uncoated catalysts, with correspondingly higher specific activities. For example, the deposition of TiO2 onto IrO2 yielded a 9-fold increase in the OER-specific activity in 1.0 M H2SO4 . The oxidation state of titanium and the potential of zero charge were also a function of the number of ALD cycles, indicating a correlation between oxidation state, potential of zero charge, and activity of the tuned electrocatalysts.We report that TiO Broader context2 emissions future depends on the electrochemical production of fuels and commodity chemicals. In the absence of a substantial carbon tax, electrochemical production of these materials must be cost competitive with conventional production. The levelized cost of electrochemically produced chemicals depends heavily on operational expenses  and the balance of systems costs, and depends relatively less on the price of the catalyst.1 Therefore, one pathway to low cost electrochemical fuel and commodity chemical production is to reduce the OpEx by fabricating highly active catalysts. Current methods to enhance catalytic activity are limited or rely on computationally-expensive calculations. Simple tools that can be used to enhance the catalytic activity for a variety of chemical reactions, such as tuning catalysts through atomic layer deposition as presented here, are essential to developing low-cost electrochemical systems that can meet global energy and chemical demands.Realizing a low anthropogenic CO3 Despite potential advantages , the industrial use of many heterogeneous electrocatalysts is currently limited in part by suboptimal catalytic activity and/or selectivity. In addition, there are limited methods to tune the selectivity and activity of heterogeneous electrocatalysts.2 Methods and design tools such as doping, inducing strain, and mixing metal oxides have been used to improve the catalytic activity of heterogeneous electrocatalysts.7 The activity of heterogeneous electrocatalysts can also be tuned by applying thin layers of another material, leading to an altered surface charge density on the resulting composite material relative to the bulk charge density of either individual material.13 This approach has been widely used to alter the catalytic and electronic properties of core/shell nanoparticles, although additional tuning of the particle support structure is necessary to create an efficient heterogeneous electrocatalyst.15 Density functional theory calculations have shown that a single atomic layer of TiO2 on RuO2 should lead to enhanced selectivity for the chlorine-evolution reaction (CER) relative to the oxygen-evolution reaction (OER).9 Enhanced catalytic activity for the OER has been reported for WO3 photocatalysts coated with 5 nm of alumina, with the activity increase ascribed to an alteration in the electronic surface-state density.16 Enhanced catalytic activity has also been observed at the interface between TiO2 and RuO2, with charge transfer between RuO2 and TiO2 resulting in a mixed phasewithanintermediatechargedensity.5Highly active electrocatalysts are required for the cost-effective generation of fuels and commodity chemicals from renewable sources of electricity.2, RuO2, and F-doped SnO2 (FTO) were tuned and evaluated for the chlorine-evolution reaction (CER) and the oxygen-evolution reaction (OER). The CER provides a promising approach to infrastructure-free wastewater treatment as well as for the production of chlorine, an important industrial chemical whose global annual demand exceeds seventy million metric tons.18 The OER is the limiting half-reaction for water splitting that could provide hydrogen for transportation and could also provide a precursor to energy storage via thermochemical reaction with CO2 to produce an energy-dense, carbon-neutral fuel.19Herein, atomic layer deposition  has been used to tune the surface charge density, and consequently tune the catalytic activity, of electrocatalytic systems in a fashion consistent with estimates based on group electronegativity concepts . To test these predictions, the activities of the known electrocatalysts, IrOχ) relative to the group electronegativity of RuO2 (χ ≈ 2.72), the most active catalyst for the OER in the benchmarking literature  as well as the most active catalyst for the CER.20 IrO2 (χ ≈ 2.78) and FTO (χ ≈ 2.88) were also investigated because they have higher electronegativities than RuO2, and therefore using ALD to overcoat these catalysts with TiO2 (χ ≈ 2.62) is expected to shift their surface electronic properties and catalytic activities towards that of RuO2, the optimal single metal oxide catalyst. These materials were also chosen because TiO2, IrO2, RuO2, and other materials are commonly used to form mixed metal oxide electrodes, most notably the dimensionally stable anode (DSA), in which TiO2 increases the anode’s stability, but does not confer enhanced activity to the aggregated material.21Each material tested was selected based on its theoretical group electronegativity  were determined for IrO2, RuO2, and FTO as a function of the successive number of TiO2 ALD cycles  for the OER at 10 mA (cmgeo)–2 in 1.0 M H2SO4 and for the CER at 1 mA (cmgeo)–2 in 5.0 M NaCl adjusted to pH 2.0 with HCl. Current densities were chosen to produce >95% measured Faradaic efficiency for each catalyst , and current–potential data were corrected for the solution resistance (<2.0 mV correction) as measured by electrochemical impedance spectroscopy (see ESI† for details). The three catalysts were prepared on substrates that had very low roughness to minimize effects in geometric overpotential measurements due to surface area differences. Specifically, electrocatalyst samples consisted of a ~300 nm metal–oxide film sputter deposited on a (100)-oriented Si substrate, in the case of IrO2 and RuO2, or commercially available TEC 15 FTO glass substrates, in the case of FTO-based electrocatalysts. TiO2 overlayers were then deposited on top of the electrocatalysts. The microstructure of a typical IrO2-based electrocatalyst is shown in the cross-sectional scanning electron microscopy (SEM) image in Overpotentials (geo)–2 for bare RuO2 and IrO2 agreed well with values reported for catalysts prepared on similarly flat surfaces. We are unaware of comparable OER data for FTO or for CER catalysts.22 The overpotentials for IrO2 and FTO, for both the OER and CER, initially showed an improvement  with increasing ALD cycle number, before exhibiting an inflection point due to an increase in overpotential at higher ALD cycle numbers ) is a standard quantity for comparing the OER activity of heterogeneous electrocatalysts . For IrO2 and RuO2 catalysts, the OER specific activities of the uncoated catalysts were in good agreement with previously reported values.20 We are unaware of reported specific activities for FTO for the OER or for any catalyst for the CER. The specific activities for the OER and CER were characterized by volcano-type relationships as a function of the TiO2 ALD cycle number  in 0.5 M H2SO4. To compare these catalysts, we measured the roughness of our catalysts using AFM . For our catalysts, bare IrO2 exhibited a Tafel slope of ~60 mV dec–1 in good agreement with previously reported OER catalysts.24 As the activity of our IrO2 based catalyst increased from bare IrO2 to 10 TiO2 ALD cycles, the Tafel slope remained constant at ~60 mV dec–1 while the exchange current density (i0)increased from ~1 × 10–7 to ~2 × 10–5 mA (cmAFMSA)–2. Initially the IrOx/SrIrO3 catalyst also had an OER Tafel slope of ~60 mV dec–1 and an i0 of ~7 × 10–6 mA (cmAFMSA)–2. For the IrOx/SrIrO3, however, after a period of activation the Tafel slope improved dramatically to ~40 mV dec–1, which indicates a previously unknown OER mechanism, while the i0 deteriorated to ~3 × 10–7 mA (cmAFMSA)–2 . In our case, IrO2 coated with 10 ALD cycles of TiO2 exhibited lower overpotentials than the freshly prepared IrOx/SrIrO3 catalyst at current densities <1 mA (cmAFMSA)–2 and lower overpotentials than the activated IrOx/SrIrO3 catalyst at <0.02 mA (cmAFMSA)–2, but substantially higher overpotentials at the more industrially relevant current densities of >10 mA (cmAFMSA)–2.25 Further discussion on surface roughness, including AFM, and SEM sample characterization is presented in the ESI† .The specific activity –2. For thinly coated catalysts (3–10 cycles) the OER stability improved from about 1 h to about 4 h, while for thicker TiO2 coatings (>30 cycles) the OER stability increased to >9 h . The loss in activity for the OER for TiO2 coated samples was associated with a loss in the TiO2 coating as illustrated in X-ray photoelectron spectroscopy (XPS) measurements of the Ti 2p core level before and after electrochemical stability testing . For the CER, all catalysts were relatively stable over the 24 h testing period except for the FTO-based catalysts which followed the same trend as the OER, with thicker TiO2 coatings stabilizing the electrodes. XPS measurements of the stable CER catalysts indicated that the TiO2 overcoating was still present even after 24 h of continuous operation . These results indicate that, as prepared here, these catalysts are not long-term stable, and substantial work is needed to obtain an industrially relevant catalyst. Similarly prepared catalysts exhibit enhanced stability by making the catalyst material thicker, annealing the catalyst, or mixing SbxOy, TiO2, TaxOy, or SnO2 into the catalyst.28 It is possible that similar techniques could be used to enhance the stability of the catalysts presented in this work.To test the longevity of the enhanced catalytic performance with TiO2 is not readily explained by surface morphological changes of the electrocatalyst. Deposition of TiO2 does not substantially affect the electrochemically active surface area, a metric believed to be related to active site density, and changes in the surface area alone do not account for the magnitude of the enhancement in the specific activity . Furthermore, while high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) images and STEM electron dispersive X-ray spectroscopy (EDS) maps of IrO2 samples with 10 cycles of TiO2 ,32 consistent with expectations that addition of TiO2 does not fundamentally change the mechanism or the potential determining step for either reaction. Hypothesized mechanisms generally involve coordination of either OOH or OCl groups to unsaturated sites on the metal oxide in the potential determining reaction steps.35 FTO based catalysts exhibited very large overpotentials for both the CER and OER and had correspondingly high Tafel slopes in excess of 190 mV dec–1, potentially indicating a different, much less efficient mechanism than the process that controls the reactivity of the more active catalysts.The enhancement in catalytic performance observed with deposition of TiO of TiO2  indicateous film  and cataEZC) of the electrocatalysts were measured as a function of TiO2 thickness  and (S3) and Fig. S12–S15 for details and discussion on handling thin TiO2 layers in EZC measurements). EZC thus yields insight into the strength of the bonds on the catalyst surface.37EZC is also qualitatively very similar to group electronegativity which describes how difficult it is for molecules to gain electrons and is correlated to OER activity . Additionally, EZC of metal electrodes has been correlated with metal–oxygen single bond strengths which is also qualitatively similar to computationally derived oxygen binding energies which have long been correlated with electrocatalytic activity.38EZC, group electronegativity, and oxygen binding energies each have their strengths and weaknesses. EZC is measurable, but it is not easy to predict. Electronegativity is completely theoretical and very simple to calculate, but does not take into account more complex qualities of materials like edge sites. Oxygen binding energies are strongly theoretically grounded and can take into account complexities of materials like edge sites, but they are also relatively difficult to calculate. These strengths and weaknesses show that all these descriptors may be used complimentarily to predict and understand catalytic activity . Measured EZC values for bare RuO2 and IrO2  were consistent with previously reported values for Ru and Ir.39 We are unaware of reported EZC values for FTO. As the RuO2 and IrO2 samples were coated with increasing ALD cycles of TiO2 the EZC shifted from lower to higher potentials in both cases and eventually reached the value for bulk TiO2. This behavior is consistent with the expected trends for equilibrated group electronegativities. The EZC for bare FTO (450 mV vs. SCE) was less than that for bulk TiO2 and greater than bare IrO2 or RuO2. The FTO EZC decreased with increasing TiO2 cycles up to 10 cycles and as the TiO2 cycles increased beyond 10 the EZC increased until it reached the bulk value of TiO2 at large cycle numbers. The overall trend of the FTO EZC increasing to higher values with increasing TiO2 cycle number is consistent with group electronegativity arguments. However, the intermediate behavior where the EZC decreases and then increases is not well explained by group electronegativity and could, in part, arise from the complicated behavior of the F dopant atoms (further discussion on the limits of group electronegativity are found in the ESI†). For all catalysts, the EZC continued to shift even beyond the point where TEM data indicated that the film is continuous (40 ALD cycles). This suggests that the exposed metal oxide is not fully responsible for the shift in EZC and that the surface TiO2 is likely responsible in part for the EZC shift. Shifts in EZC with incremental TiO2 deposition suggest that ALD can be used to tune the catalytic performance. These data reveal that the catalysts with the highest activity for the CER have EZC values between 50 and 75 mV vs. SCE  for the CER. Additionally, active OER and CER catalysts for all systems investigated have EZC values between 25 and 200 mV vs. SCE with the best OER catalysts having a somewhat higher EZC (~110 mV vs. SCE) than the best CER catalysts (~60 mV vs. SCE).To investigate the electrocatalysts’ surface electronic properties the potentials of zero charge , stacked from bottom to top, for increasing ALD TiO2 thickness, with 0 cycles indicating the bare catalyst substrate. Deposition of low cycle numbers of ALD TiO2 on IrO2 and RuO2 produced Ti core-level peaks that were at ~456.6 eV and ~457.6 eV, which is consistent with previously reported binding energies for Ti3+ states.41 As the ALD cycle number increased, the Ti oxidation state for these samples gradually increased to its bulk oxidation state (~+4), and signals indicative of bulk TiO2 were eventually observed  and the thin TiO2 film, in which the chemical nature of the phase produces a more oxidized metal, with the mixed phase most likely dominated by Ti4+ sites.To further understand the surface states of the catalysts, X-ray photoelectron spectroscopy was used to measure the Ti oxidation state. observed . In the 2 cycles was accompanied by a peak shift of the Ti 2p3/2 peak relative to the bulk TiO2 peak position . The Ti 2p3/2 peak of the IrO2-and RuO2-based catalysts shifted from reduced, lower binding energies to the more oxidized, higher binding energies typical of bulk TiO2. The FTO-based Ti 2p3/2 peak shifts from more oxidized, high binding energies at low TiO2 cycles to lower binding energies for intermediate TiO2 cycles (10–40 cycles) before increasing again to higher binding energies at large TiO2 thicknesses (>60 cycles). The Ti 2p3/2 peak shift is qualitatively consistent with the variation in EZC with TiO2 cycle number suggesting that the change in the surface charge density is correlated with a change in the Ti oxidation state.The variation in the Ti oxidation state with ALD TiO2 thickness can be explained by charge transfer from the underlying metal oxide substrate. In this scenario, a more reduced Ti species present at low deposited cycles of TiO2 on IrO2 and RuO2 would be accompanied by a more oxidized metal oxide substrate. To confirm this hypothesis, we measured the Ir 4f, Ru 3d, and Sn 3d core-level photoemission . Unlike in the case of the Ti 2p spectra, the Ir 4f, Ru 3d, and Sn 3d core-level photoemission exhibited very small changes between the bare metal oxide substrate and those with varying thicknesses of TiO2. This was reflected in the peak shifts of the main peak for the Ir 4f, Ru 3d, and Sn 3d spectra with TiO2 thickness relative to that of the bare substrate , which were an order of magnitude lower than those for the Ti 2p core-level photoemission and mostly within the error of the measurement (±0.1 eV). While peak fitting (see the ESI† for details) of these spectra indicates that initial deposition of TiO2 leads to a slightly more oxidized Ir and Ru state, and a slightly more reduced Sn state for FTO, no trend with thickness was observed for any of the substrates, and changes in the oxidation state of the underlying catalyst are likely below the detection limit for the techniques used in this study .The variation in the Ti oxidation state with TiO2 can tune surface electron densities of the catalyst in a direction consistent with predictions from group electronegativity concepts . Given that concomitant changes in electrochemical activity were observed with deposition of TiO2, these data indicate that ALD may be useful to tune the activity of other catalysts for diverse reactions, including those critical for renewable energy storage and wastewater treatment.In summation, surface characterization suggests that atomic layer deposition of low cycle numbers of TiO


This paper describes the impact of ligandacidity on the formation of VO2 nanocrystals from the solvothermalreaction of vanadyl acetylacetonate (VO(acac)2) with stoichiometricamounts of water. Carboxylic acids examined herein favor the formationof the monoclinic VO2(B) phase over the tetragonal VO2(A) phase as the concentration of water in the reaction increases.However, the threshold concentration of water required to obtain phase-pureVO2(B) nanocrystals increases as the pKa of the carboxylic acid decreases. We also observe thatincreasing the concentration of VO(acac)2 or the concentrationof acid while keeping the concentration of water constant favors theformation of VO2(A). Single-crystal electron diffractionmeasurements enable the identification of vanadyl carboxylate speciesformed in reactions that do not contain enough water to promote theformation of VO2. Increasing the length of the carbon chainon aliphatic carboxylic acids did not impact the phase of VO2 nanocrystals obtained but did result in a change from nanorod tonanoplatelet morphology. These results suggest that inhibiting therate of hydrolysis of the VO(acac)2 precursor either bydecreasing the ratio of water to VO(acac)2 or by increasingthe fraction of water molecules that are protonated favors the formationof VO2(A) over VO2(B).Vanadium dioxide (VO The most stable bulk phase, VO2(R), with a rutilestructure, is well known for its low-temperature metal-to-insulatortransition to VO2(M) and is used for smart window applications.2 Like VO2(M), the metastable tetragonal phase known asVO2(A) has also been investigated as a material for opticalswitches,4 while the metastable monoclinic phase knownas VO2(B) has been used as a cathode material in lithium-and sodium-ion batteries.11 All of these applications benefit from the solution processabilityafforded by nanocrystalline morphologies. Given the variation in theapplication of multiple crystal polymorphs of VO2, thereis considerable motivation to understand how to control which polymorphsform under various reaction conditions.Vanadium dioxide (VO2(A) andVO2(B) are typically synthesized using solvothermal methodsat elevated pressure.18 These methods often utilize V2O5 and a reductantin water, and control over the crystal phase is usually achieved byvarying the reaction temperature. VO2(A) is the major productobtained at reaction temperatures between 220 and 270 °C, andVO2(B) is the major product obtained at temperatures between180 and 200 °C.15 Varying the pressure by varying the volume of thereaction mixture within the pressure-sealed reaction vessel19 also impacts the phase of the resulting product.One study showed that a mixture containing V2O5 and oxalic acid reacting at 180 °C in a pressure-sealed autoclavecould result in phase-pure VO2(B) after 24 h and phase-pureVO2(A) after 7 days.20 However,this result could only be obtained with a filling ratio of 80/100mL in the pressure-sealed vessel: a filling ratio of 60/100 mL onlyyielded VO2(B) with no phase change present after 7 days.Nanocrystals of themetastable polymorphs VO2 nanocrystals. Specifically, we showed that decreasingthe concentration of water present in the solvothermal reaction mixtureof vanadyl acetylacetonate (VO(acac)2) and lauric acidin toluene to 4 equiv or less per vanadium center produces phase-pureVO2(A) nanocrystals, whereas using 20 equiv or more ofwater produces phase-pure VO2(B) nanocrystals.21 This approach has the added advantage of enablingaccess to smaller nanocrystals with dimensions less than 500 nm. Incontrast, reactions that use water as the solvent produce very largeparticles, usually nanorods that are several microns long.10 We hypothesize that the mechanismby which the concentration of water impacts the crystal phase of VO2 nanocrystals is through controlling the relative rates ofprecursor hydrolysis and condensation. Decreasing the concentrationof water slows hydrolysis and may allow nanocrystal nucleation viacondensation of partially hydrolyzed species. We suspect that condensationof these partially hydrolyzed species favors the formation of VO2(A) over VO2(B).Recently, we demonstrated that tuning the chemical rather thanphysical reaction conditions enables control over the crystal phaseof VOKa and steric bulk of organic carboxylic acid ligandsto vary the rate of precursor hydrolysis. We find that replacing lauricacid with a stronger acid, namely trifluoroacetic acid, expands therange of water concentrations that lead to the formation of VO2(A) nanocrystals by a factor of almost four, up to 15 equivof water per vanadium center. Increasing the concentration of trifluoroaceticacid and/or the VO(acac)2 precursor relative to water alsofavors the formation of VO2(A) over VO2(B).These experiments indicate that reaction conditions that suppressthe rate of precursor hydrolysis  favor the formationof VO2(A).21 In contrast tovarying the acidity of carboxylic acid, changing the chain lengthof aliphatic carboxylic acids does not alter the crystal phase ofthe product. This observation indicates that the steric bulk of theseligands does not alter the relative rates of precursor hydrolysisand condensation significantly enough to impact which crystal phaseof VO2 nucleates.Here, we test this hypothesisby tuning the p2 nanocrystals from VO(acac)2. We varied two properties of these ligands—their acidityand their steric bulk—to determine which property has the largestinfluence on the resulting nanocrystals. Our previous work demonstratesthat the amount of water present in a solvothermal reaction with VO(acac)2 and lauric acid in toluene determines both the crystal phaseof VO2 synthesized, as well as the length of the resultingnanorods.21 Using this synthesis methodology,we varied the amount of water present in the reaction from 0.5 to20 mmol (2–80 equiv per vanadium) in the presence of 4 equiv(1 mmol) of a selection of acids with varying pKa’s shown in 2(A) products (or product mixtures containingVO2(A)) to phase-pure VO2(B) products as theamount of water present in the reaction increases past a particularthreshold concentration. This observation is consistent with our previouslyreported study in which the threshold concentration of water requiredfor the formation of phase-pure VO2(B) in the presenceof lauric acid (pKa = 5.3) was determinedto be 5 mmol (20 equiv per vanadium).21 Here we observe that decreasing the pKa of the carboxylic acid increases the threshold concentration ofwater required to obtain VO2(B) instead of VO2(A). For example, trifluoroacetic acid (pKa = 0.23) has a threshold concentration of 15 mmol (60 equiv per vanadium).Notably, the threshold water concentration observed in the absenceof acidic ligands is 3 mmol (12 equiv per vanadium). This observationsuggests that the minimum concentration of water required to formphase-pure VO2(B) is 12 equiv per vanadium center. Additionof carboxylic acids increases this threshold.We set out to investigate therole of carboxylic acid ligands inthe synthesis of VO2(A) or VO2(B)  of water by electron diffraction suggests that these productsare the corresponding vanadyl carboxylate compounds—VO(benzoate)2 and VO(4-nitrobenzoate)2—and that thesecompounds also crystallize as one-dimensional chains of octahedrallycoordinated vanadium ions with a structure that is entirely analogousto VO(acetate)2 (Supporting Information). The powder X-ray diffractionpattern of the product obtained from the reaction of VO(acac)2 with 4 equiv of trifluoroacetic acid and 4 equiv of waterresembles that of the other confirmed vanadyl carboxylate species.We therefore strongly suspect that this pattern corresponds to VO(trifluoroacetate)2. These data indicate that carboxylic acids can displace acetylacetonate,and 1 mmol (4 equiv) of water is insufficient to hydrolyze these speciesto form VO2. In a solvothermal reaction utilizing VO(benzoate)2 as a precursor in the absence of water and benzoic acid,the VO(benzoate)2 precursor is recovered. VO(benzoate)2 also forms upon reaction of 4 equiv of benzoic acid withVO(acac)2 in the absence of water under the same solvothermalconditions . This observation indicates that ligand exchangeto form the VO(carboxylate)2 species does not require thepresence of water.We also observe that in the presence of very low concentrationsof water (1 mmol or less), the addition of acetic, benzoic, 4-nitrobenzoic,and trifluoroacetic acid results in products whose powder X-ray diffractionpatterns do not match that of either VOr VO2(B) 2. Inthe2, VO(benzoate)2, and VO(4-nitrobenzoate)2 state that these species are insoluble in most solvents,which isconsistent with our observations.29 Although VO(trifluoroacetate)2 has been reported previously as a soluble monomeric complex,30 the species we isolated from the reaction ofVO(acac)2 in the presence of 4 equiv each of trifluoroaceticacid and water is not soluble in polar or nonpolar solvents. We thereforesuspect that this species is also a coordination polymer. VO(benzoate)2 and VO(4-nitrobenzoate)2 are reported to crashout immediately upon addition of vanadyl sulfate to an aqueous solutionof the corresponding carboxylate,29 indicatingthat monomeric carboxylate complexes cannot be isolated even in thepresence of excess acid. Titration of acetic acid into aqueous solutionsof the vanadyl ion VO2+ achieves a maximum of 1.5 boundacetates per solvated vanadium center,32 indicatingthat stable solutions in which all monomeric species are bound totwo acetate ligands cannot be achieved. Based on these previous reports,we conclude that monomeric vanadyl acetate, benzoate, or 4-nitrobenzoatespecies are highly unlikely to exist in nonpolar toluene solutionscontaining 4 equiv of carboxylic acid per vanadium, such as thoseused here.The earliest reports of coordination polymers ofVO(acetate)2 in the absence of water produces no reaction—therecoveredreaction mixture still contains lauric acid and VO(acac)2 (see Supporting Information). Furthermore,we note that formation of VO2(A) occurs in the presenceof lauric acid and 0.5 or 1 mmol water, whereas at least 2 mmol water(8 equiv) is required to observe the formation of any VO2 species in the presence of acetic, benzoic, 4-nitrobenzoic, or trifluoroaceticacid 2 and lauric acid are intactin the mixture recovered from a solvothermal reaction run in the absenceof water, we cannot completely rule out the formation of some monomericvanadyl laurate species in solution or the presence of a small fractionof such species in the recovered reaction mixture.In contrast to benzoic acid, reaction of lauric acidwith VO(acac)ticacid 1. Wesus2 from the solvothermal reaction of VO(acac)2 involves hydrolysis of VO(acac)2 to form [VO(H2O)5]2+ followed by condensation of this speciesto generate VO2.21 This vanadyl aquo complex is stable under neutral aqueous conditions34 and has a pKa of 5.3–6.0.35 We proposed in our previous work that the relativerate of hydrolysis versus condensation controls whether VO2(A) or VO2(B) nuclei form. Slow hydrolysis enables condensationof partially hydrolyzed species, whereas faster hydrolysis promotesthe formation of the fully hydrolyzed species before significant condensationoccurs. We hypothesized that condensation of the fully hydrolyzedspecies favors the formation of VO2(B) nuclei, while condensationof partially hydrolyzed species favors the formation of VO2(A) nuclei. Here, we observe that addition of stronger carboxylicacids increases the threshold concentration of water required to obtainphase-pure VO2(B) nanocrystals instead of VO2(A). This observation is consistent with our hypothesis that therate of hydrolysis controls the crystal phase of VO2 becauseincreasing the strength of the organic acid should inhibit hydrolysisof VO(acac)2. More acidic reaction conditions result ina higher ratio of positively charged hydronium ions to water molecules,which lowers the concentration of neutrally charged water availableto hydrolyze the VO(acac)2. Hydronium ions are much lessnucleophilic than neutral water and therefore less reactive towardhydrolysis.Previousreports have posited that the mechanism for the formationof VO2(A) over VO2(B) by conductinga series of reactions in which we varied the relative concentrationsof VO(acac)2, water, and trifluoroacetic acid (2(A) becomes the favored product as the concentration of either VO(acac)2 or trifluoroacetic acid increases relative to the concentrationof water. 2 from 0.25 to 1mmol while keeping the concentration of water constant at 15 mmolresults in a transition from VO2(B) products to VO2(A) products. Obtaining VO2(B) from a reactioncontaining 1 mmol of VO(acac)2 and 1 mmol trifluoroaceticacid requires increasing the amount of water present to at least 30mmol, whereas VO2(B) can be obtained using only 15 mmolof water in the presence of 0.25 mmol of VO(acac)2 and1 mmol trifluoroacetic acid. 2 is kept constant at 0.25 mmol and theratio of trifluoroacetic acid to water is increased by either decreasingthe concentration of water or increasing the concentration of acid.In both cases, higher ratios of trifluoroacetic acid to water favorthe formation of VO2(A).We further tested our hypothesis that slow hydrolysispromotesthe formation of VOtic acid 3. VO2(A)2(A) and VO2(A) is a more thermodynamicallystable phase than VO2(B), we investigated whether strongcarboxylic acids could mediate the conversion of VO2(B)nanocrystals to VO2(A). Previous reports show that conversionfrom VO2(B) to VO2(A) is possible under solvothermalconditions at high temperatures.9 For example, Zhang,et al. obtained VO2(A) nanocrystals upon reacting a solutioncontaining VO2(B) nanocrystals and water at 260 or 280°C for 48 h.3 Additionally, anotherreport also heated VO2(B) nanocrystals to 280 °C underhydrothermal conditions for 48 h to convert them to VO2(A).9 We investigated the possibilitythat lowering the temperature and using an acidic reaction environmentcould also instigate this transformation. 2(B) nanocrystalscan indeed be converted to VO2(A) when heated in toluenein the presence of 4 equiv of trifluoroacetic acid and 20 equiv ofwater at 230 °C over the course of 120 h. This conversion isindicated by the coalescence of two pairs of diffraction peaks at2θ = 14.4 and 15.3° and 2θ = 29 and 30.2° intosingle peaks at 2θ = 15 and 30°, respectively. The resultingpeaks correspond to the (110) and (220) planes of the VO2(A) structure. The overall decrease in the number of diffractionpeaks signals an increase in symmetry as the monoclinic VO2(B) structure converts to the tetragonal VO2(A) structure.The VO2(A) nanocrystals obtained from this reaction aresmaller than the initial VO2(B) nanocrystals (2(B) nanocrystals and re-nucleation as VO2(A), possibly using VO(trifluoroacetate)2 as areaction intermediate. We note that in reactions containing VO(acac)2, lauric acid, and 2 or 20 equiv of water, we do not observeany indications of phase interconversion over the course of a 24 hreaction period. Examining products present in these reaction mixturesafter various reaction times reveals that only one type of crystallineproduct is observed per reaction condition: VO2(A) forthe reaction containing 2 equiv of water and VO2(B) forthe reaction containing 20 equiv of water (see Supporting Information). This observation implies that forreactions starting from VO(acac)2, the final VO2 product phase nucleates directly from condensation of hydroxylatedspecies formed upon precursor hydrolysis.Since the presence of strong carboxylic acids favorsthe formationof VOcrystals 4. This c2 nanocrystals, we next investigatedthe impact of tuning the steric bulk of the carboxylic acid ligandon the solvothermal synthesis of VO2 nanocrystals. A selectionof aliphatic carboxylic acids with varying carbon-chain lengths waschosen, specifically butyric (4C), hexanoic (6C), heptanoic (7C),decanoic (10C), lauric (12C), and stearic (18C) acid. All of thesecarboxylates have relatively similar pKa’s of ∼4.8–5.3.36 Figure S14in the Supporting Information shows powderX-ray diffraction spectra obtained from reactions of VO(acac)2 in the presence of 4 equiv of each of these acids and 20equiv of water. These data demonstrate that VO2(B) is obtainedin every case. Likewise, the morphology of the nanocrystals remainsconstant with variation of the ligand chain length, except for stearicacid, as shown in Figure S14. Reactionscontaining aliphatic carboxylic acids with chain lengths less than18 carbons produce VO2(B) nanorods of average length 110–150nm, while stearic acid yields nanoplatelets in addition to nanorods.Overall, since only one of the aliphatic carboxylic acids resultedin a difference in morphology, we conclude that varying the lengthof aliphatic carboxylic acids does not strongly influence the hydrolysisand growth of VO2(B) nanocrystals.After establishing that the acidity of the carboxylicacid ligandsimpacts the phase of VO2(B) nanocrystalssynthesized with carboxylic acids of various pKa’s. Since all of the acids produce phase-pure VO2(B) with 60 equiv of water, these products were analyzed byscanning electron microscopy  nanocrystals from VO(acac)2 in thepresence of polyvinylpyrrolidone (PVP)18 and from V2O5 in the presence of oxalic acid.7 The first study discovered that changing theconcentration of PVP in the reaction impacted the morphology of theresulting nanocrystals. Nanorods formed in the absence of PVP, whileaddition of 3 mg/L PVP formed nanoflowers, and 54 mg/L PVP formednanocarambolas.18 Thus, the change in morphologyfrom nanoflowers to nanocarambolas occurred after an increase in PVPconcentration by a factor of 18. The second study found that using0.06 mol/L oxalic acid produced nanorods, while 0.12 mol/L oxalicacid produced a mixture of nanobundles and nanocarambolas.7 Both of these studies concluded that the interactionof ligands with the surface of the nanocrystal during growth impactsthe final morphology.38Finally, the reaction containing trifluoroaceticacid producednanocarambolas, which are elongated aggregates of stacked nanosheetswhose cross section resembles a six-pointed star.Supporting Information). At early reaction times (3 h), we observe nanorods with shortrod-like protrusions growing perpendicular to the long axis. After6 h, we observe fully formed nanocarambolas. We hypothesize that theshort protrusions observed at early times expand along the long axisof the original nanorod, forming sheets that result in the 6-armednanocarambola structure. Unlike other carboxylic acids used here,trifluoroacetic acid has two moieties that can engage in strong interactionswith the nanocrystal surface: the carboxylate group and the fluorineatoms. Carboxylic acid groups are well known to bind to the surfacesof metal oxide nanoparticles.40 In the case of TiO2, trifluoroacetic acid ligands were found to have a similarstructure-directing effect as the addition of HF—both additivesresulted in the stabilization of {001} facets and the formation ofnanosheets in the case of HF and a mixture of nanosheets and truncatedoctahedrons in the case of trifluoroacetic acid.41 The authors also observed that aliphatic carboxylic acids,such as oleic acid, form truncated octahedrons, indicating that thecarboxylate group also stabilizes {001} facets but not to the sameextent as fluoride ions. The authors attributed the intermediate structure-directingeffect of trifluoroacetic acid to its partial degradation under solvothermalreaction conditions to form fluoride ions. We suspect that similardegradation of a fraction of the trifluoroacetic acid molecules maybe responsible for the formation of VO2(B) nanocarambolas.To explore the mechanism by which the unusual nanocarambolamorphologyforms, we varied the duration of the reactions run in the presenceof trifluoroacetic acid (see Ka and increasing the concentration of carboxylicacid ligands present in the reaction mixture while keeping the concentrationof water constant favors the formation of VO2(A), whereasincreased pKa and decreased acid concentrationfavor the formation of VO2(B). Furthermore, increasingthe concentration of the precursor relative to the concentration ofwater leads to VO2(A), whereas VO2(B) formsfrom reaction mixtures containing lower precursor-to-water ratios.These observations are consistent with our hypothesis that the rateof precursor hydrolysis relative to condensation controls which crystalphase nucleates. We identified VO(carboxylate)2 speciesas likely reaction intermediates formed prior to nucleation of theVO2 species. Finally, our results suggest that acidityalone likely does not have a strong impact on nanocrystal morphology.Instead, interactions involving substituents on the carboxylic acidligands, such as intermolecular π–π interactionsor interactions between fluoride ions formed upon degradation of −CF3 groups and the nanocrystal surface, may play a significantrole in determining the morphology of VO2(B) nanocrystals.Overall, this work provides important insight into how carboxylicacids impact the crystal phase and morphology of VO2 nanocrystals.Our work demonstrates the effects of theacidity of the reactionmixture on the crystal phase and morphology of vanadium dioxide nanocrystalsobtained under solvothermal reaction conditions. Decreasing the p2, stearic acid, decanoic acid, heptanoicacid, hexanoic acid, and butyric acid purchased from Sigma-Aldrichand lauric acid purchased from Millipore were pumped into the gloveboxovernight in an evacuated antechamber before use. Benzoic acid and4-nitrobenzoic acid purchased from Oakwood, acetic acid purchasedfrom Fisher Scientific, and trifluoroacetic acid purchased from Sigma-Aldrichwere used without further purification.Prior to the addition of waterto the reaction mixtures, all manipulations were carried out in anMBraun inert atmosphere glovebox under a nitrogen atmosphere unlessotherwise stated. Autoclave reactors and Teflon liners were pumpeddown in an evacuated antechamber overnight prior to use in the glovebox.Molecular sieves  were activated by heatingat 200 °C under vacuum overnight. Toluene was dried over activatedsieves (20% by weight) for at least 24 h, then purged with nitrogenfor at least 1 h before being transferred via a cannula to a Schlenkflask. Toluene was then pumped into the glove box overnight in anevacuated antechamber and stored over activated 3 Å molecularsieves. VO(acac)Trifluoroacetic acid is bothvolatile and corrosive and should therefore be handled exclusivelyin a fume hood. Autoclave reactors become pressurized at elevatedtemperatures and should be handled with care. No attempts should bemade to open autoclave reactors unless they are at room temperature.2 and10 mL of toluene inside a nitrogen-filled glovebox. The autoclavereactor was sealed and removed from the glovebox. In ambient air,the autoclave reactor was opened, and the desired amount of waterwas added (3–20 mmol). A carboxylic acid (1 mmol) was addedto the reaction mixture either before  or after  removal from the glovebox.Tables S1 and S4 in the Supporting Information contain detailed lists of the masses and volumes of the variousreagents used in these reactions. After the addition of water, theautoclave reactor was sealed again and heated in an oven at 200 °Cfor 24 h before it was removed and allowed to cool to room temperature.The black solid product was collected by centrifugation under ambientconditions and washed with ethanol. The product was rotovapped todryness and stored in the glovebox under nitrogen.A 25 mL Teflon-lined autoclavereactor was charged with 0.25 mmol (0.068 g) VO(acac)2 (0.075–3 mmol) and 10 mL of toluene. The autoclave reactorwas sealed and removed from the glovebox. In ambient air, the autoclavereactor was opened and the desired amounts of water (0.5–20mmol) and trifluoroacetic acid (0.5–4 mmol) were added . The autoclavereactor was sealed again and heated in an oven at 200 °C for24 h before it was removed from the oven and allowed to cool to roomtemperature. The black solid product was collected by centrifugationunder ambient conditions and washed with ethanol. The product wasrotovapped to dryness and stored in the glovebox under nitrogen. Vanadylcarboxylate products were collected and purified using proceduresidentical to those used for the vanadium dioxide nanocrystals.A 25 mL Teflon-lined autoclavereactor was charged with the desired amount of VO(acac)2 nanocrystals. Single nanocrystals of VO(benzoate)2 and VO(4-nitrobenzoate)2 were structurally analyzedusing a Rigaku XtaLAB Synergy-ED electron diffractometer equippedwith a HyPix-ED detector (see Supporting Information for details).Powder X-ray diffractionmeasurements were performed using a Rigaku XtaLAB Synergy-S Dualflexsingle-crystal diffractometer operating in the powder collection modeusing Cu Kα radiation. Nanocrystalline powders were affixedto a Nylon loop (0.1 mm inner diameter) with a light coating of viscousoil and mounted on a goniometer for data collection. An FEI TecnaiF20 G2 scanning transmission electron microscope operated at 200 kVand a Zeiss Auriga scanning electron microscope with an InLens detectoroperated at 5–20 kV were used to analyze the morphology ofthe VO


This protocol was applied to the release of a phosphorylatedserine derivative and the nucleotide analogue AZT monophosphate. Nucleotiderelease in the presence of ATP and a kinase provides a diphosphate,demonstrating that this method can be applied to biological processes.Phosphate mono- anddiesters can be liberated efficiently fromboryl allyloxy (BAO) and related phosphotriesters by H Other phosphate prodrugs5 employ the esterase labile pivaloylmethyl and S-acyl-2-thioethyl groups, while cyclic phosphotriestergroups are activated by acidic hydrolysis and cytochrome P450 oxidation.Phosphate prodrugs addressthis problem 2, as see6 Elevated levels of H2O2 and its abilityto initiate chemical reactions create the potential for its use indrug release, illustrated by the release of fluorophores7 and cytotoxins8 fromboronates in cells and animals. Our prior studies in which boronateoxidation initiates fragmentation reactions that release polar groupsthrough the generation of boron enolates from boryl allyloxy (BAO)groups9 or hemiacetal intermediates fromα-boryl ethers10 led us to explorethe potential of peroxide-mediated phosphate release. Herein we describethe development of a new phosphate group-release strategy based onthe peroxide-mediated oxidation of borylated phosphoesters  followed bythe addition of excess H2O2·urea (30 equiv)in D2O at 37 °C. The final concentration of the substratewas 2 mM with a CD3CN:D2O ratio of 7:3 (v/v).The resulting H2O2 concentration is higher thancellular levels,11 though prior relatedstudies10 show that this concentrationpredicts cellular responses. Product concentrations were quantifiedagainst an internal standard. The products of these reactions werevalidated by comparison to independently prepared and characterizedphosphates.127 and 8 areshown in 7 proceededrapidly and efficiently via the intermediate boron enolate, with 94%of the starting material being consumed within 18 min and phosphate 9 being produced in an equal amount. This experiment showeda pseudo-first-order rate constant of 2.3 × 10–3 L mol–1 sec–1 and a half-lifeof 301 s.12 The reaction with 8 was more complex since two cleavage events are required. Spectraloverlap prohibited monitoring the formation of the phosphomonoester,though acrolein production is easily detected. The reaction showeda rapid consumption of 8, with the concentration of themonocleavage product 10 increasing and then ultimatelybecoming negligible at 24 min. Subsequent NMR analysis confirmed thatthe product was phosphate 11. Acrolein was formed inan 88% yield based on the two equiv that are expected from the cleavageof two BAO groups. The second BAO cleavage is significant, since itshowed that phosphate dianion release is faster than boron enolateprotonation. Additionally, the absence of a significant buildup ofthe monocleavage product indicates that the second cleavage is notsubstantially slower than the first. The pinacol esters partiallyhydrolyze to boronic acid intermediates prior to oxidation, althoughthis does not impact cleavage efficiency. Phosphate release was notobserved in the absence of peroxide or solely in the presence of urea.12Time courses for theoxidative cleavage reactions of Serveral substrates were synthesized and servedto highlight thescope of the process 1. The re12 releases phenylphosphate 13 somewhat more slowly than the breakdownof 8 (entry 1). While 90% of the starting material wasconsumed after18 min, dicleavage product was formed in 63% yield, and 27% of thecorresponding monocleavage product remained. This proceeded to 70%and 23% of the respective products after 25 min.12 We speculate that the rate arises from solubility issues,as turbidity was observed during the course of the reaction. The phosphorothioate 14 releases thiophosphate 15 in 79% yield after18 min and 94% yield after 49 min, with turbidity again being observedas a potential source of the slowed release. Delivering thiophosphates,which are substantially more stable toward enzymatic cleavage thanphosphates,13 is significant, regardlessof the release rate. Phosphodiesters that contain an aryl and an alkylgroup cleave selectively as shown in the conversion of 16 to 17. Aryl phosphates can be cleaved enzymaticallyto release monoalkyl phosphates.14 Compound 18, in which the BPin group is replaced by the recently reportedand more easily handled EPin group,15 releases 9 extremely efficiently, albeit somewhat more slowly. Consumptionwith quantitative product release did not occur until 32 min. Boronate 18 is significantly more hydrolytically stable than 7, which could be useful for applications in which the boronicacid analogue does not readily traverse a cell membrane. The diminishedrate of release will be valuable when a slower phosphate productionis therapeutically beneficial.The phosphotriester 16 leadingto concerns about its formation duringprodrug cleavage, though the release of a glutathione scavenger couldaugment the potency of these agents in numerous applications.17 Although our studies with deliberate acroleinrelease through the cleavage of BAO-containing molecules showed notoxicity,12 the potential for a competitivebiological response inspired us to prepare oxidatively cleavable boronateanalogues that mitigate byproduct electrophilicity. The α-borylphosphate 19 reacts with peroxide to form 9 within 12 min, indicating that this group reacts even faster thanthe BAO group. Boronates 20 and 22 (secBAO)are homologues of the original vinyl boronate and release methyl vinylketone and crotonaldehyde, respectively, in addition to 21. These byproducts are significantly less electrophilic than acrolein.18 The phosphates were prepared by the additionof the alcohols to dimethylphosphoryl chloride, since the phosphoramiditeprotocol was unsuccessful. Both of these substrates release dimethylphosphate in the presence of H2O2, though 20 showed an alternate, slower mechanism for release in theabsence of peroxide, making structurally similar phosphates unsuitablefor biological applications.12 This studyshows that a range of options exist should byproduct release leadto a competitive biological response.Acrolein is an inhalation toxinthat can sequester glutathione,23 served as a model for the conditionalrelease of phosphoserine. The process was conducted with the initialconcentration of 23 being 0.2 mM in biologically relevantPBS buffer, which is an order of magnitude more dilute than the previousstudies to observe the capacity for oxidative cleavage at low substrateand oxidant concentrations. Serine phosphate 24 was formedin 89% yield after 60 min and quantitatively after 140 min as determinedby HPLC, showing that phosphorylated amino acid units can be releasedunder conditional control in peptides and proteins.19 Insterestingly, replacing the BAO groups of 8 with p-borylbenzyl groups8 resultedin oxidation but only monocleavage.12 Thus,the BAO group is substantially more effective for releasing dianionicphosphates.20Serine derivative 25 in which the boronic acid(HO-BAO group) proved desirable for isolation purposes (211H NMR studies showed that 25 reacts withH2O2 as expected to release AZT monophosphate(AZTMP) 26 in 91% yield after 25 min. Moreover, the negativecontrol phosphate 27 proved to be completely inert underthese conditions.12We explored the potentialfor nucleotide release through the preparationof AZT-derived phosphate purposes 3.211H NM26 in the presence of ATP to form AZT diphosphate 28 and ADP , ATP (10 μM),and TMPK. Hydrogen peroxide (30 equiv) was added for the oxidativerelease studies of 25. After 1 h CellTiter-Glo reagentwas added, and luminescence was measured after 5 min. The resultsare shown in 28. The negative controls 27 and H2O2-free 25 failed to initiateATP consumption. The combination of 25 and H2O2 showed a level of ATP consumption equivalent to thatof the positive control 26, confirming that oxidativecleavage provides a nucleotide analogue that can engage in enzymaticreactions.Prior to conducting phosphorylationstudies, we showed that ATPis stable to H2O2. The reactions are rapid and efficient, evenat low substrate and peroxide concentrations. Applying the protocolto AZT monophosphate release demonstrated that the released nucleotideis a suitable substrate for enzymatic phosphorylation, validatingthe compatibility of the process with biomolecular transformations.The elevated levels of H2O2 in numerous diseasestates indicate that this approach will be applicable to the developmentof site-selective phosphate-containing prodrugs.We have shown that a range of organoboronates canbe incorporatedinto phosphotriesters to release phosphoesters upon exposure to H


APOL1 gene variants prevalent in 13% of this population contributes to the genetic component of the excess risk for nondiabetic kidney failure , Writing – Review & Editing).