CSF1 gene is frequently involved. In this article, we describe a newly identified gene fusion mediated by an inversion in a case of diffuse TSGCT. Multicolor‐fluorescence in situ hybridization (FISH) molecular karyotyping identified a pericentric inversion of chromosome 1 in 7 out of 17 analyzed cells 46,XX,inv(1)(p13.3q24.3) [7]/46,XX [10], and with interphase FISH the involvement the CSF1 locus was detected. After performing transcriptome sequencing analysis for fusion detection, only one out of five fusion gene algorithms detected a fusion involving the CSF1 gene product. The resulting chimera fuses a sequence from a human endogenous retrovirus (HERV) gene to CSF1 Exon 6 on chromosome 1, abrogating the regulatory element of the 3′ untranslated region of the CSF1 gene. This new translocation involving Exon 6 of the CSF1 gene fused to 1q24.1, supports the hypothesis that a mutated CSF1 protein is likely to play a vital role in the pathogenesis of TSGCT. The role of the HERV partner identified as a translocation partner, however, remains unclear. Our data add to the complexity of involved translocation partners in TSGCT and point to the potential difficulty of identifying fusion partners in tumor diagnostics using transcriptome sequencing when HERV or other repeat elements are involved.Tenosynovial giant cell tumors (TSGCTs) are rare tumors arising in tendons or the synoviae of joints and bursae. The localized type is benign while the diffuse type shows expansive growth leading to greater morbidity and is therefore considered locally aggressive. Typical recurrent chromosomal aberrations are found in the majority of TSCGT and the  Different translocations partners have been documented, most commonly 2q35–37, 5q22–31, 11q11–12, and 8q21–22 less frequently.CSF1 gene to the collagen 6A3 (COL6A3) promotor on 2q35–37 is believed to lead to deregulated expression of CSF1 through promoter swap.the COL6A3‐CSF1 fusion gene specifically has only been detected in 23%–33% of TSGCT cases.CSF1 gene locus in the majority (61%–77%) of TSGCT casesThere are typical recurrent chromosomal aberrations found in the majority of TSGCT, including trisomy of chromosomes 5 and 7 and translocations involving 1p11–13. The CSF1 Exon 6 on chromosome 1 abrogating the regulatory element of the 3′ region of the CSF1 gene. Our data add to the complexity of involved translocation partners in TSGCT and point to the potential difficulty of identifying fusion partners when HERV repeat elements are involved in tumor diagnostics using transcriptome sequencing.In this article, we describe a newly identified gene fusion mediated by an inversion in a case of diffuse‐type TSGCT using multicolor‐FISH molecular karyotyping and transcriptome sequencing. The resulting chimera protein fuses a sequence from a human endogenous retrovirus (HERV) gene to 22.1The tumor sample L4018 originated from the synovium of the knee of a 53‐year‐old female and was diagnosed as a diffuse‐type TSGCT. The diagnosis was histopathologically and clinically confirmed  and RP11‐96F24 (telomeric to CSF1) bracketing CSF1 locus enable the detection of translocation or inversion. Probe labeling and hybridization were performed according to previously described protocols.Custom‐designed split‐apart probe set bracketing the 2.3Total RNA was isolated from the L4018 frozen tissue sample using a standard Trizol isolation procedure.Isolated total mRNA was subjected to transcriptome sequencing via a commercial vendor  for sequencing with the HiSeq2000 platform .2.4CBL mutation status was checked from transcriptome sequencing results.The sample was pre‐processed using the BioWDL RNA‐seq pipeline (version 2.0.0). First, FastQC and MultiQC were used for quality control. Then, standard Illumina adapters were trimmed using Cutadapt and the quality control was performed again. Reads were mapped against the human genome GRCh38 and Ensembl gene annotation version 94 with Hisat2 and the gene expression quantification was performed with Stringtie. The ‐B flag was additionally set in Stringtie to be able to proceed with further analysis with Ballgown in R. Furthermore, the ‐e flag was set to create a count table with raw gene and transcript counts using Stringtie's prepDE.py script. The used gene fusion tools are STAR‐Fusion (version 1.6.0),2.5Total RNA from the L4018 frozen tissue sample was reverse transcribed with the Invitrogen Superscript III First‐Strand cDNA Synthesis Kit , according to the manufacturer's instructions using 1 μg total RNA for the reverse transcriptase reaction and oligodT primers.2.6CSF1 breakpoint identified by transcriptome sequencing. Based on the RNA sequence findings, primers were designed around the putative breakpoint in the CSF1 gene. Primers with positive PCR products are listed in Table CSF1‐S100A10, we used primers published by Panagopoulos et al.Real‐time quantitative PCR amplification of cDNA was performed to verify and analyze the The PCR amplifications were performed using the Thermo Fisher Applied Biosystems SYBR green real‐time PCR protocol, using 12.5 μl of SYBR® Green I master mix , 1.5 μl of the forward and reverse primers each, and 1 μl of cDNA (1:10 diluted). The PCR was run on CFX touch 96  with the cycling profile of initial denaturation for 3 min at 95°C followed by 40 cycles of 15 s at 95°C, 20 s at 55°C, and 60 s at 72°C.2.7The PCR product was size‐separated using electrophoresis and 1% (wt/vol) agarose gel in order to confirm the presence of an amplicon of the expected size.2.8The PCR products were submitted for Sanger sequencing .The BLAST softwareThe HERVd database33.1The tumor sample originated from the synovium of the knee of a 53‐year‐old female. She first presented with pain and swelling of the right knee and was diagnosed as a diffuse‐type TSGCT. In a time frame of approximately two decades, the patient underwent nine consecutive open arthroscopies to resect the tumor and its recurrences, and she received systemic treatment with tyrosine kinase inhibitors imatinib, nilotinib, and pexidartinib without an objective radiological response. At the age of 62, she underwent total knee replacement surgery after which she became pain‐free.3.2CSF1 and break‐apart FISH probes, bracketing the CSF1 locus showed a split signal (p13.3q24.3) [7]/46,XX [10] Figure . The brel Figure . FISH ar3.3CSF1 fusion using five different fusion detection tools, showed that the fusion was only detected by Squid, while all other fusion detection algorithms resulted in no reported fusion gene product. Squid reported 29 potential fusion events, from which CSF1 fusion with a gene desert observed with IGV viewer was scored the highest (>1000), other regions were not shown to be positive.Analysis of transcriptome sequencing for the CSF1 gene, visual inspection of the CSF1 region in IGV showed the presence of discordant reads after paired‐end read sequencing. The sequence mate reads mapped to the chromosome 1q region, compatible with the breakpoint identified at the chromosome level. The reads, however, were not mapped to any annotated gene region, but sitting in a gene desert region , that otherwise was not annotated in the reference genome.To confirm the fusion, primers were designed to amplify the chimeric RNA after a reverse transcriptase reaction Figure . A rever4CSF1 breakpoint at the 3′ of Exon 6 fused to an HERV element. Since this HERV translocation partner is not located in annotated genes, most of the standard fusion detection algorithms failed to report it. Only one tool we tested, Squid, is able to detect this particular event which is treated as a more general type of fusion event termed as transcriptomic structural variation.We identified a pericentric inversion inv(1)(p13.3q24.3) in a case of TSGCT by molecular karyotyping. Using transcriptome sequencing we identified a COL6A3‐CSF1 fusions were found in a significant part of TSGCT.CSF1 gene (1p13.2) in 20 out of 21 cases; however, only 3 out of 10 scorable cases involved the COL6A3 locus. The authors hypothesize that the COL6A3‐CSF1 fusion could bring CSF1 under the control of a strong COL6A3 promotor.Earlier studies showed a recurrent translocation t in 8 of 26 TSGCT cases.COL6A3‐CSF1 translocation could give rise to a functional chimeric protein leading to CSF1 overexpression since the part of the CSF1 gene encoding for the receptor‐binding site upstream of Exon 5 is lost due to a breakpoint downstream of Exon 5.CSF1‐S100A10 fusion gene carrying the translocation t. This breakpoint leads to the loss of the 3′ untranslated region (3′ UTR) of CSF1 Exon 9 that contains microRNA (miRNA) targets, a noncanonical G‐quadruplex and AU‐rich elements (AREs) controlling the expression of CSF1. In ovarian cancer models, different miRNA sites have been shown to downregulate the expression of CSF1, while binding of glyceraldehyde‐3‐phosphate dehydrogenase to the CSF1‐ARE regulates the stability and degradation of CSF1 mRNA.CSF1 breakpoints in 30 cases of whom 28 were confirmed by sequencing, all downstream of Exon 5 with the loss of the 3′ UTR as a consequence.However, other papers questioned whether the CSF1 and new research indicates that the self‐sustaining autocrine CSF1‐CSF1R signaling loop in neoplastic cells is lacking based on single‐cell sequencing.There is increasing evidence that shows the regulatory element or membrane domain loss by losing various proportions of the 3′ end of This article further supports the pathogenesis by a change in posttranscriptional regulation of TSGCT.CBL mutations were identified in 35% and 29% of studied TSGCT cases, respectively. CBL (11q23) codes for an E3 ubiquitin‐protein ligase that is involved in cell signaling and protein ubiquitination via receptor tyrosine kinases and mutations are associated with various hematological malignancies.CBL mutations could be the driving mutation in some cases of TSGCT; however, they could also merely be a secondary event. The CSF1 fusions and CBL mutations in these papers were not mutually exclusive, both variations were seen in 33% of cases, which could support the hypothesis that they are not driving mutations.CBL mutation was found.Tsuda et al.,CSF1 locus (1p13.2) and the 1q24.2 region on chromosome 1 in a gene desert. Blast sequence analysis of the fusion partner sequence against multiple reference databases showed similarities with sequences from a HERV. HERVs in the human genome are a heterogeneous group of viral elements that are believed to be derived from ancient germ‐line infections of exogenous retroviruses that became a fixed element in the human genome.gag, pro, pol, and env regions in between a long‐terminal repeat (LTR) at the 3′ and 5′. The LTRs regulate the activation and expression of HERV genes while these sequences contain main promotors, enhancers, and transactivation regions for transcription.The novel fusion event described in this article led to a novel fusion between the HERV families and HERV parts can be identified using the HERVd database,HERVs are associated with autoimmune disease and cancer, though the precise involvement and etiology remain unclear.HERV‐K_22q11.23 5′ ‐LTR‐UTR sequence upstream of the transcription factor ETS translocation variant 1 (ETV1) is reported, leading to enhanced expression of the ETV1 oncogene.CSF1.HERV‐K LTR/fibroblast growth factor receptor 1 (FGFR1) fusion gene.For example, in some cases of prostate cancer, a translocation of the FOS‐gene in an epithelioid hemangioma of bone. COBRA‐FISH karyotyping revealed a balanced translocation t in FOS, and further transcriptome sequencing showed a recurrent translocation breakpoint in the FOS gene fused to different partners in all three cases. The translocation leads to the insertion of a stop codon, leading to a truncated inactive FOS protein. This would not be picked up by next‐generation sequencing since the gene‐copy number is not altered and genes are expressed normally. Potentially, the raw data from the TSGCT case we describe in this report can be used for improving and benchmarking fusion detection algorithms that can address the limitation of gene annotation for developing better diagnostics.Difficulties with the detection of these HERVs exist given their non‐coding, non‐recurrent fusions that are not identified by existing bioinformatics algorithms. Many relevant fusion events involving HERVs can be missed due to this detection gap. An example of a relevant translocation that would also be missed by regular sequencing methods is the case in a previous report by Ijzendoorn et al.,CSF1 fusion product showed an early truncation of the translated protein in the ERV element. The relevance of the finding of this new chimeric CSF1‐HERV fusion gene is not known, however, one might hypothesize that in this case of TSGCT the transcriptional reactivation of the associated HERV sequence may play a role in its pathogenesis. However the loss of the 3′ end of CSF1 and abberant CSF1‐CSF1R signaling in neoplastic cells might be pathogenetically important as well.In silico analysis of the 5CSF1 gene fused to 1q24.2, a locus previously described as a HERV. Since CSF1 involvement is seen in a large part of TSGCTs, a mutation in the transcribed CSF1 protein is hypothesized to play a vital role in its pathogenesis. The loss of the 3′ UTR and regulatory processes involved, previously described in TSGCT cases, could explain the CSF1 overexpression leading to neoplastic growth. The translocation partner was identified as a known HERV sequence, however, its significance in the pathogenesis is unknown. Previously reported CSF1 fusion partners are so diverse and seem to lack any commonalities in function, therefore the role of the fusion partner could mainly lie in abrogating the negative regulatory sequence of 3′ UTR. The involvement of highly repetitive elements like ERV as driver mechanisms might be overlooked by the standard fusion reporting algorithms since the ERVs are not part of the standard gene annotation. Our results indicate the possibility of reanalysis of samples where no diagnosis has been made and a fusion event might be expected. Careful analysis of molecular data  is crucial in unusual cases as patients may become eligible for targeted therapy.We have described a new translocation in a case of TSGCT, involving a translocation breakpoint in Exon 6 of the


Molecular biology achieves control over complex reaction networks by means of molecular systems that translate a chemical input (such as ligand binding) into an orthogonal chemical output (such as acylation or phosphorylation). We present an artificial molecular translation device that converts a chemical input – the presence of chloride ions – into an unrelated chemical output: modulation of the reactivity of an imidazole moiety, both as a Brønsted base and as a nucleophile. The modulation of reactivity operates through the allosteric remote control of imidazole tautomer states. The reversible coordination of chloride to a urea binding site triggers a cascade of conformational changes in a chain of ethylene-bridged hydrogen-bonded ureas, switching the chain’s global polarity, that in turn modulates the tautomeric equilibrium of a distal imidazole, and hence its reactivity. Switching reactivities of active sites by dynamically controlling their tautomer states is an untapped strategy for building functional molecular devices with allosteric enzyme-like properties. Molecular biology achieves control over complex reaction networks by means of molecular systems that translate a chemical input into an orthogonal chemical output but the application of dynamic foldamers has been limited by the restriction to stereochemical changes that produce spectroscopic signals. Here, the authors present an artificial molecular translation device that converts a chemical input into an unrelated chemical output. Thus photoreceptor proteins7 induce membrane depolarisation as a result of a light signal, and G protein-coupled receptors regulate the interconversion of primary metabolites as a response to the binding of structurally unrelated hormones or environmental signals9.In living systems, complex networks of signalling pathways translate physical or chemical signals into biochemical function by modulation of the conformational and binding properties of receptors and other biomolecules. Allosteric effects15 and other supramolecular systems20. Dynamic foldamers are extended oligomeric structures that display well-defined global conformational preferences, which may be modulated by specific stimuli21 such as solvent polarity23, ligand25, light28 and pH31. However, the application of dynamic foldamers so far has been limited by the nature of their outputs, which have typically been restricted to stereochemical changes that produce spectroscopic (NMR or fluorescence) signals. Foldamers can also operate as catalysts33, but dynamic foldamers that translate chemical signals into the regulation of reactions have been limited to the switching of product stereochemistry35.Synthetic molecular translation devices that use allosteric changes to convert one type of chemical signal into an orthogonal output have been devised, in the form of dynamic foldamers36. Histidine plays an essential role in the ‘catalytic triad’ of active site residues in many enzymes, including trypsin, chymotrypsin, and acetylcholinesterase38, where its imidazole acts as both a general acid and a general base, allowing protons to shuttle between substrate and catalyst. Both natural functions and synthetic uses of imidazole employ the lone pair of the unprotonated nitrogen to achieve reactivity—the other (protonated) nitrogen is unreactive as a base and a nucleophile. Yet the interchange between the different reactivities at each nitrogen atom—inert or basic/nucleophilic—is in principle simply a matter of tautomerism. We reasoned that the imidazole tautomer states could be switched by exchanging an intramolecular hydrogen-bond donor for an intramolecular hydrogen-bond acceptor39. Switching the polarity of the hydrogen bond to the proximal nitrogen atom would activate the imidazole by switching its tautomer state, incorporating the imidazole NH as a hydrogen bond donor, and revealing a reactive nitrogen sp2 lone pair  was established by NMR spectroscopy  revealed that self-association was weak (Ka = 31 ± 2 M−1). Taken together, these data are consistent with the adoption by the prototype device 1 of the conformation shown, in which the terminal N-3,5-bis(trifluoromethyl)phenyl (BTMP) urea acts as a hydrogen bond donor, and the basic imidazole nitrogen atom acts as an intramolecular hydrogen-bond acceptor.A prototype device 1 Fig.  was desi2  and ωB97-XD/6-311 G(d)) the 13C NMR chemical shifts of the ring carbon atoms in these two alternative tautomer states , but have almost identical calculated shifts in the 5-alkyl-1H tautomer (green). Experimental support for the use of chemical shift separation between the 13C signals of C4 and C5 as a diagnostic indicator of tautomer state was provided by the empirical 13C NMR chemical shifts values of C4 and C5 for equivalent regioisomeric compounds 1,4-dimethylimidazole and 1,5-dimethylimidazole, in which an N-methyl group replaces the tautomeric NH  was titrated with 0–12 equiv. imidazole  = 4.0] by imidazole [pKa(H2O) = 6.95] to form imidazolium 2,4-dinitrophenolate44.The influence of induced tautomer preference on the reactivity of each ligated imidazole was first assayed by comparing the basicity of d 2 Fig. . In a pr2, with very similar results  with tetrabutylammonium chloride  SI5 in CD2Cl2 at 25 °C with Bu4NCl  significantly smaller than those recorded during the titration of foldamer 3 with similar amounts of ligand (CIS + 0.8 to +1.5 ppm). The titration of the longer tris(butylurea) SI6 in CD2Cl2 at 25 °C with Bu4NCl also led to much lower complexation induced shifts   . Fitting of the changes in NH chemical shift gives affinities for bromide and iodide that are similar to each other (Ka(Br−) = 170 ± 20 M−1; Ka(I−) = 150 ± 20 M−1) and around half the value for chloride. 1H NMR titration of 3 with Bu4N(NO3) also gave a clear CIS of +0.9 ppm in the BTMP urea NH protons. Fitting of these changes gave Ka(NO3−) = 1400 ± 600 M−1, greater than the value for chloride despite the smaller CIS. These binding constants approximate the reported relative affinities for ureas in organic solvents of all anions tested, albeit with relatively stronger binding to nitrate46. The strong downfield shift in NHD during the titration of 3 with each of bromide, iodide or nitrate is consistent with a switch of hydrogen bond directionality bringing the terminal NH into an internally hydrogen-bonded environment.Other anions induce a similar change in conformation. Comparable binding studies of 1  = 1600 ± 600 M−1. Applying a 1:2 binding model also gave an adequate fit but showed that the amount of higher order complexation would be small (for 1·Cl− + Cl−K12(Cl−) = 63 ± 8 M−1; Supplementary Fig. 1H NMR chemical shifts (CIS < 0.2 ppm) of the internal NH groups (which remain hydrogen bonded in both conformers) do not give diagnostic conformational information. The small upfield and downfield movements in the positions of these NH resonances during the titration could not be analysed quantitatively, but may result from the effect of chloride-induced changes in self-association of 1: dilution studies had shown that 1 weakly self-associates in CD2Cl2 (Kassoc = 31 ± 2 M−1), producing small downfield shifts in all NHs  was titrated into 1 (12 equiv.) in the presence of 2,4-dinitrophenol (1.1 mM in dry dichloromethane). As chloride was added, a yellow colour developed, alongside a progressive increase in absorbance between 330–470 nm carbonate 4  reacts with imidazole (1 or 3 equiv.) over a period of 13 h to give 4-nitrophenol and 4-nitrophenyl 1H-imidazole-1-carboxylate 5 as the sole products  = 7.07) under these conditions. UV-visible spectroscopy of the reaction showed a progressive increase in absorbance between 300–370 nm, and a decrease between 250–280 nm  was treated with 3 equiv. of either imidazole, 2,1, or 1 + Bu4NCl, and the reactions were monitored at 327 nm, the absorbance maximum of the respective acylated imidazole adducts  or 6 (from 2) being the sole products by 1H and 13C NMR spectroscopy and by HRMS . Data fitting shows the reaction of 4 with 1 in the presence of Bu4NCl is two-fold faster than reaction of 4 with 2, a foldamer with the imidazole covalently activated. Although this could be due to greater hydrogen bond polarisation in the longer foldamer48, the reaction of 4 with 2 in the presence of Bu4NCl is 1.4 times faster than with 2 alone . Despite binding more tightly to 3 than chloride, adding nitrate to 1 did not produce greater rate acceleration (k = 140 × 10−6 s−1) than chloride addition, although it was greater than that of the other halide anions tested. A control experiment using 1 with Bu4N(BPh4), which has a non-coordinating anion, produced a small increase in rate compared to 1 alone  perhaps due to an increase in solvent polarity effects.The data from each set of conditions were fitted to pseudo-first order kinetics to provide approximate relative reaction rates Fig. . This an4NCl induced increase in reactivity of 1, together with NMR data indicating the stoichiometric binding of chloride to 1 at the BTMP site produces a pronounced polarity switch, clearly indicates that the allosteric effect of chloride on the tautomer state of the imidazole is the major contributor to the overall rate increase. This switch-on of activity is clear upon delayed addition of Bu4NCl to the slow reaction of 1 with 4, which resulted in an immediate rate acceleration (Fig. k ~ 12 × 10−6 s−1, whereas the on-state after chloride addition showed a strong increase in the observed rate constant, calculated as kCl ~ 340 × 10−6 s−1. These values are in reasonable agreement with those calculated separately for each set of conditions (Fig. The magnitude of the Buion Fig. . The relons Fig. .1, inducing a shift in the tautomeric state of the imidazole terminus from an off state to an on state, activating the device as a Brønsted base and as a nucleophile. The low basicity and nucleophilicity of chloride reveals the ability of foldamer 1 to act as an artificial translation device, converting one form of chemical input into a functionally orthogonal chemical output. Modulating reactivities of active sites through the dynamic control of their tautomer states is an untapped strategy to build functional molecular devices. Future work will seek to integrate such components as control devices in more complex reaction networks.In conclusion, we have built a dynamic molecular device with allosteric enzyme-like properties. It operates by an original mechanism in which remotely induced switching between the tautomer states of an active site modifies reactivity. The binding of chloride reorganises the hydrogen-bonding network of 1–3, their spectroscopic characterisation and conformational analysis, their titration with anions, and kinetic studies of their reactions.The Supplementary Information provides details of methods for the synthesis of oligomers Supplementary InformationPeer Review File


Despite the widespread use of the Haber–Bosch process to industrially produce NH3, biological nitrogen fixation still accounts for half of the bioavailable nitrogen on Earth. An important feature of nitrogenase is that it operates under physiological conditions, where the equilibrium strongly favours ammonia production. This biological, multielectron reduction is a complex catalytic reaction that has perplexed scientists for decades. In this review, we explore the current understanding of the molybdenum nitrogenase system based on experimental and computational research, as well as the limitations of the crystallographic, spectroscopic, and computational techniques employed. Finally, essential outstanding questions regarding the nitrogenase system will be highlighted alongside suggestions for future experimental and computational work to elucidate this essential yet elusive process.Nitrogenase is the sole enzyme responsible for the ATP‐dependent conversion of atmospheric dinitrogen into the bioavailable form of ammonia (NH Nitrogenase performs a complex, multielectron reduction to convert dinitrogen into ammonia; this intricate catalytic reaction still perplexes scientists. In this review, we discuss the current understanding of the molybdenum nitrogenase system, limitations of contemporary approaches, and future directions for experimental and computational studies. ATP, adenosine triphosphateDF, density functionalEPR, electron paramagnetic resonanceFe protein, iron proteinFeMoCo, iron molybdenum cofactorMoFe protein, molybdenum iron proteinN2, dinitrogen2aseN, nitrogenaseNH3, ammonia2) to the fixed form of ammonia (NH3) is essential for life, as fixed nitrogen is a key component of many biological molecules  P cluster and a [7Fe:9S:C:Mo]‐R‐homocitrate FeMo‐cofactor (FeMoCo) cluster  and ultimately substrate reduction. Mo‐N2ase must undergo reduction of the FeMoCo active site for initial binding of substrates to the cofactor to occur. Notably, various substrates can be reduced by N2ase, including N2, C2H2, CN−, N3−, and SCN−. Unexpectedly, N2ase can also catalyse C–C bond formation during the reduction of certain substrates, including CH3NC and CO ) and 4Fe4S clusters of the Fe protein further complicate the analysis of spectroscopic data, as does the prediction that approximately half of the Mo‐N2ase N2 substrate intermediates will be even‐electron and thus “dark” when analysed with spin resonance techniques. Signal relaxation‐broadening above 25 K further confounds spectroscopic analysis of vital Fe–N species ) charge of −1, net E0 spin state of S = 3/2, atomic coordinates from the 1 Å resolution 3U7Q PDB X‐ray crystal structure , and mutagenesis and structural studies that implicate the Fe2‐Fe3‐Fe6‐Fe7 face as the catalytic reaction zone of FeMoCo [2 and His195 can provide hydrogen bonding to a Fe2‐coordinated substrate, N2 binding to the Fe2 position was simulated. Initially, FeMoCo was modelled to have a hydrogen bridging Fe2 and Fe6 and an additional hydrogen binding to S2B, the belt sulfur that connects Fe2 and Fe6. Upon equilibration of N2 within the ingress channel , N2 was found to bind Fe2 end‐on with a very small energy barrier (12 kJ·mol−1 when S = 1/2 or 16 kJ·mol−1S = 3/2), accompanied by dissociation of S2B‐H from Fe2 [2 molecule that initially binds Fe2 via the substrate channel may not be reduced, but instead could play a promotional role in expanding the reaction zone [2 enters via the ingress channel and binds to Fe6, where it is primed for intramolecular hydrogen transfer from atoms originating on S3B, culminating in reduction from N2 to NH3 [Dance  has revir et al. , 86, 87,r et al. , 89. andr et al. . Here, wf FeMoCo , 93, 94.from Fe2 . Notablyion zone . This me2 to NH3 .2ase mechanism of Siegbahn concerns the role of the interstitial carbide. By analysing models with 160–270 atoms using the B3LYP hybrid functional, Siegbahn has identified a mechanism in which Cc becomes hydrogenated to a methyl group that shifts to the periphery of the cluster core while bound to Fe6 (exo‐Fe6) [2 substrate binding and hydrogenation are proposed to proceed within the now empty central region. Importantly, formation of CH3 is categorized as an “activation phase” that is equivalent to the E0 state. The mechanism then proceeds via hydrogenation steps that mimic the Thorneley‐Lowe model, and E4‐H2/E4‐N2 exchange is included. The distinctive structural features of this Siegbahn hydrogenation cycle include the role of CH3 bound exo to Fe6, unhooking of S2B‐H from Fe6 during the hydrogenation and rehooking afterwards, as well as generation of five‐coordinate Mo due to partial dissociation of the homocitrate alcohol group [0) that resulted in loss of a sulfide (H2S3A) as opposed to protonation of Cc to a methyl group. This builds upon experimental studies that showed the inhibitor CO can replace S2B [2 binding revealed that the nitrogen substrate binds too weakly to force loss of a sulfide; however, CO binds significantly more strongly to FeMoCo and successfully displaces the sulfide in an almost thermoneutral step based on these DF studies. Overall, the four activation steps were found to result in a very weakly bound sulfide that could be released in a significantly exergonic step (−50 kJ·mol−1). This study found that the barrier for protonation of the carbide beyond CH was much higher than sulfur protonation. Additionally, after sulfide loss, the catalysis cycle was modelled to commence via N2 binding to Fe4, one of the most reduced Fe atoms in this system. Notably, sulfide loss was also associated with hydrides preferring a bridging position, which had a stabilizing effect on the overall cluster [exo Fe‐CH3 group would likely be hydrolytically unstable and a detailed mechanism for regeneration of the E0 resting state has yet to be clearly outlined [A critical feature in the Nexo‐Fe6) , 97, 98.ol group . Wei andlace S2B . Simulat cluster . It shououtlined .2ase reduction mechanism using computational studies, with recent work focusing on the potential role of the μ2‐bridging S2B position in substrate binding and reduction [4 structures. This full N2ase mechanism is proposed to begin with N2 binding in a “half‐bridging” mode within the empty S2B site. N2H2 is then produced using intramolecular hydrogen transfer from protons already present on the cluster with the first protonation requiring approximately 17 kJ·mol−1 of energy. Interestingly, Cao and Ryde suggest N2 hydrogenation most favourably proceeds from intermediate NNH2 bound end‐on to H2NNH in a side‐on bridging conformation (Fe2/Fe6). Formation of H2NNH2 (hydrazine) was then reported to be the most stable subsequent structure, and the next reduction step facilitated facile cleavage of the NN bond resulting in NH3 bound solely to Fe2 and NH2− stabilized by a bridging conformation between Fe2 and Fe6. The final mechanistic step involves NH3 dissociation and protonation of NH2− to NH3, but only after S2B rebinds to the cluster. Overall, this work proposed a hybrid mechanism, in which E5‐E7 states favour an alternating mechanism (with the exception of NNH2 being reported as the most stable N2H2 state), while other steps in the mechanism suggest a distal approach, such as favourable formation of NNH3. This computational work builds upon experimental studies to suggest N2 could bind to an empty S2B site and coordination of nitrogen substrate between Fe2 and Fe6 atoms may facilitate the formation of two NH3 molecules via an energetically favourable mechanism.Ryde has explored the Neduction , 102. In2ase system have provided important insights ranging from identification of likely substrate ingress and egress channels to proposed detailed mechanisms for the entire reduction process; however, computational analyses are also not without limitations. Each of these studies involves constraining the parameters of the nitrogenase system based upon the focus of that particular study, such as deciding the number of atoms used (model size), choice of functional and basis set, defining the overall model charge, and choice of solvation dielectric constant. Defining the functional choice, particularly hybrid vs. pure, has been found to especially influence which FeMoCo structures and bonds are energetically and geometrically favoured with wide variations reported (> 200 kJ·mol−1) [Computational studies avoid many of the limitations inherent with experiments and can provide novel insights into the unprecedented structure and coordination chemistry of FeMoCo. As detailed above, various theoretical analyses of the NJ·mol−1) . As such2ase system include:2 fixation? Current models have singularly focused on a general mechanism where N2 reduction proceeds through a sequence of proton and electron transfers, but it must be noted that no definitive evidence for any on‐path, partially reduced NN intermediate has been presented.What are the mechanistically relevant intermediates for N6 core remain rigid throughout N2 reduction?Does the cofactor rearrange during turnover? In particular, how do the belt sulfurs exchange/interchange during turnover? Does the CFeDoes each cycle of reduction by the Fe protein result in the coupled transfer of one proton and one electron to the FeMoCo, as typically modeled?What is the exact role of His195 within the substrate reduction mechanism? Can this residue supply multiple hydrogens to the cofactor?2/N2 exchange step accompanied by hydrogenation of N2? Is this a unique reaction associated with N2 reduction, or do other substrates also display this property?Is the HWhat are the roles of homocitrate in the substrate reduction mechanism?Advances in experimental and computational techniques have aided our understanding of several facets of biological nitrogen fixation. Given the complexity of the metallocluster active site and current inability to trap mechanistically relevant intermediates, outstanding questions regarding the N2ase experimental and computational work, as well as evaluations of the Fe‐N2ase and V‐N2ase systems, in order to advance our knowledge of this process. There are a number of promising approaches to further illuminate steps in the N2ase mechanism. Experimentally, strategies for the site‐specific labelling of various atoms in the FeMo‐cofactor, including iron [n states for analysis. The recent revolution in electron microscopy is opening up structural studies of solutions of nitrogenase in defined states [2 [2ase mechanism for biological nitrogen fixation.These queries represent only a few of the unresolved details regarding this system, and future experiments will need to combine findings from Mo‐Ning iron , molybdeing iron , carbon ing iron , 106, suing iron , 57, 62,ing iron  will alling iron , 109 or ing iron , 111 to d states , 114, asd states , 116, asd states  and parttates [2 . Ultimat


CA‐RE) reaction is a “click‐like” protocol for facile synthesis of donor‐acceptor chromophores from an alkyne and tetracyanoethylene. Herein we shed light on the mechanism of this reaction by detailed kinetics studies using 1H NMR spectroscopy. By considering several experiments simultaneously, a variety of mechanistic models was evaluated. Surprisingly, a model in which the final 1,1,4,4‐tetracyanobuta‐1,3‐diene product promoted the first step was the only one that described well the experimental data. This autocatalysis model also involved a non‐concerted, stepwise formation of the cyclobutene cycloaddition adduct. By proper choice of conditions, we were able to generate the transient cyclobutene in sufficient amount to verify it as an intermediate using 13C NMR spectroscopy. For its final retro‐electrocyclization step, simple first‐order kinetics was observed and only minor solvent dependence, which indicates a concerted reaction.The [2+2] cycloaddition ‐ retro‐electrocyclization ( The “click‐like” [2+2] cycloaddition ‐ retro‐electrocyclization (CA‐RE) reaction was followed under a variety of conditions using NMR spectroscopy. Several mechanistic models were evaluated by considering multiple experiments simultaneously. Surprisingly, only a model in which the final 1,1,4,4‐tetracyanobuta‐1,3‐diene product promoted the first step described well all the data. The CA‐RE reaction is hence an autocatalytic reaction. CA‐RE) reaction between the strong electron acceptor tetracyanoethylene (TCNE) and an alkyne containing an electron‐donating substituent group (EDG) is an efficient and atom‐economic protocol for the synthesis of 1,1,4,4‐tetracyanobuta‐1,3‐diene derivatives with no side‐products being formed phenylacetylene and para‐substituted benzylidenemalononitriles in polar solvents followed bimolecular, second‐order kinetics.N,N‐dimethylamino)phenylacetylene and TCNE was inconclusive as to whether the reaction is first or second order, and the actual mechanism for this reaction is still puzzling.The [2+2] cycloaddition ‐ retro‐electrocyclization 3 end‐group was discarded due to its bulkiness. Upon treatment with TCNE, 1 should form the presumed intermediate 2 in a cycloaddition that may occur either concerted (as shown) or asynchronously : the alkyne substrate 1, the presumed cyclobutene intermediate 2, and the product 3; when using 13C NMR spectrosocopy, TCNE can also be detected. The species are simplistically also labelled with letters A (=1), B (TCNE), C (=2), and P (=3) in the concerted model (Model 1) shown in Scheme C1 (corresponding to the zwitterion) and C2 .We chose the anilino‐substituted alkyne 3CN, CDCl3, C6D6, and C6D5CD3. Yet, in CD3CN and other polar solvents, protodesilylation was found to occur readily, providing the product 4 . Interestingly, both the aryl, NH2 and to a lesser extent trimethylsilyl proton resonances of 1, 2, and 3 move slightly during the reaction progress, which is clearly evident when expanding the spectra . Figure ortho to the amino group of product 3 during the reaction, after adding more reactants, and finally after diluting the product sample. The evident changes suggest the presence of some fast exchanging species on the NMR timescale with a weight‐averaged chemical shift . It is well‐known that aniline and TCNE can form charge‐transfer complexes,λmax 584 nm. From a series of measurements at different concentrations, we obtain an association constant for the aniline•TCNE complex of 2 M−1 . For comparison, the association constant in dichloromethane has previously been determined to 5.4 M−1 (ref. λmax 610 nm) and to 2–3 M−1 . The spectra were recorded with an internal standard (cyclohexane) in a known concentration, from which the concentrations of all species were calculated using the most upfield aryl signals ; the corresponding protons used for analysis are labelled in Scheme The reaction progress was followed by A, C, and P for Model 1 and A, 1C+2C, and P for Model 2 is as small as possible. A detailed explanation and walk‐through of the program is described in Supporting Information, p. S2, and a separate article describing the code will be published in a suitable journal at a later stage. Importantly, the advanced fitting takes all four experiments into account simultanenously. Considering data from each experiment individually could generate better, near‐perfect individual fits, but it would give non‐identical rate constants between the experiments. A couple of selected fits are shown in Figure 1 and TCNE into account in our fitting, but this model would not either give a good result.By fitting the simple Models 1 and 2 outlined in Scheme C (=2) builds up slower than the models 1 and 2 are accounting for. To figure out the reason for this, we performed systematic studies where conditions were slightly changed, except for keeping the initial concentrations of the two starting materials close to identical (unless otherwise specified). An overview of all the experiments are provided in Supporting Information, p. S5 and S6. Here we shall only discuss selected ones. First, we tested whether a spinning or non‐spinning sample would make a difference. A comparison between experiments with a spinning sample and non‐spinning sample can be seen in Supporting Information, p. S55; there is no noticeable difference between the two. We then tested whether the presence of water would have an effect; samples of different (1/TCNE) ratios of 1 : 1, 1 : 2 and 2 : 1 in anhydrous C6D6 and in C6D6 saturated with water  were studied. The accumulation of the intermediate saw no difference between dry and wet samples, but protodesilylation to compound 4 occurred in all wet samples and to a smaller degree in the anhydrous 2 : 1 ratio experiment. We note that actual isolation of product 3 failed as it is readily desilylated to furnish 4 after exposure to air for a short period of time. Based on the wet/dry experiments together with our failed attempt to isolate 3, we conclude that 3 is highly sensitive to water and will form 4 if water is present, but this sensitivity is not critical for studying the actual CA‐RE reaction of interest here.Our experimental data indicate that the intermediate 3N, 1,8‐diazabicyclo[5.4.0]undec‐7‐ene (DBU), and a spatula tip of Na2CO3 in separate experiments. Both Et3N and DBU resulted in a black tar of decomposed TCNE, most likely resulting from a nucleophilic attack. The addition of Na2CO3 gave no apparent change of the outcome of the reaction.A range of acid/base additive experiments was also conducted. As base additives, we used a drop of Et4d as the solvent for the reaction. This actually gave a massive increase in reaction rate but we reckon that this result originates from an increased polarity of the solvent rather than the acidic properties of AcOD‐4d. The polarity influence could indicate a stepwise cycloaddition mechanism involving a transient zwitterion. In actual fact, the acid properties of acetic acid retard the RE step (see below), but this step is not rate‐determining. The reaction was also found to be much faster in CD2Cl2 relative to benzene, but quantitative experiments were difficult to perform reliably on account of the volatility of this solvent. The reaction progress from an experiment using CD2Cl2 as solvent can be seen in Supporting Information, p. S100. The intermediate is formed almost instantaneously. An immediate color change to blue is also observed when mixing together the reactants in this solvent, ascribed to formation of charge‐transfer complexes . We tested a model using H+ as a catalyst for the first step, but once again no good fit could be obtained based on all four experiments. The motivation for investigating such a model was that both the Si and the NH2 group could potentially promote protonation of the carbon carrying the SiMe3 group; Si via stabilization of the resulting carbocation at the neigboring carbon (hyperconjugation) and NH2 by forming via its lone pair electrons an allene resonance form that could engage in the cycloaddition. Lastly, we used a buffer system with NH4OAc in C6D6 but it neither inhibited nor enhanced the reaction in any way and only resulted in formation of 4.Next, we added trifluoroacetic acid (TFA) in various equivalents, but such additions would always result in immediate deprotection of the trimethylsilyl group. A comparison of the effect of addition of instead AcOH in various equivalents can be seen in Supporting Information, p. S87. These experiments showed only slightly faster intermediate formation with more equivalents of AcOH. To further investigate the effect of acetic acid, we also used AcOD‐1/TCNE/aniline. This resulted in a noticable slower formation of intermediate 2 (C) compared to conditions using a ratio of 1 : 2 (1/TCNE), but still faster than conditions using a ratio of 1 : 1 (1/TCNE). A smaller build‐up of C suggests that competing charge‐transfer complexes greatly inhibit the reaction. The effect can be seen in Supporting Information, p. S98.As discussed earlier, TCNE can form charge‐transfer complexes with aniline, and we therefore performed an experiment with aniline as additive in a 1 : 2 : 1 ratio of AB) as well as with product (complex ABP) are taken into account, using either a concerted first step or a stepwise formation of the cyclobutene product. These two models were now used to fit the experimental data, and the results are shown in Figure C1) seemed to perform slightly but noticeably better. This result is in line with previous suggestions based on studies of other substrates.[C1 into the cyclobutene intermediate C2 (with ratio of rate constants k2/k5=20.4), hence being almost concerted. The rate constants at 300 K for all the steps of the two models are listed in Table 1/k−1) for complex AB, we obtain 0.04 M−1 (Model 3) and 0.03 M−1 (Model 4), and for complex ABP (k3/k−3), we obtain 3.4×106 M−1 (Model 3) and 1.2×106 M−1 (Model 4). The complex formation between AB and P thus seems remarkably strong, while the complex formation between A and B is 2 orders of magnitude smaller than we determined for the complex between aniline and TCNE (which is reasonable considering the stronger donor strength of unsubstituted aniline). The complex ABP transforms much faster into intermediate C or C1 (and released P) than does complex AB: k4/k2=118 (Model 3); k4/k2=62 (Model 4).The color changes occurring during reaction indicate both intramolecular (donor‐acceptor chromophore product) and intermolecular charge‐transfer absorptions. Together with the fact that NMR spectroscopy showed that chemical shifts of specific protons in the alkyne substrate, intermediate and products changed during the entire reaction progress, this seems to indicate that we have some associations between the species. This observation stimulated us to perform experiments where the product would be present from the very beginning of the reaction in various concentrations. Somewhat surprisingly, this presence strongly enhanced the build‐up of intermediate as shown in Figure bstrates.4i, 5 2Cl2 as solvent resulted in intermediate 2 being by far the highest in concentration after 15 minutes of reaction time . This conveniently enables us to look at the last step of the proposed mechanism, the retroelectrocyclization (RE) step. Thus, we decided to study any solvent polarity influence. We initiated two identical experiments in CD2Cl2 but added C6D6 to one of the samples after 8 minutes of reaction time (increasing the volume by a factor of 1.5) to reduce the polarity. Figure 2Cl2 and in a mixture of CD2Cl2 and C6D6. The experimental data were well described by straight lines in both cases, indicating clear first‐order kinetics of the last step. Moreover, the slopes of the trend lines are quite similar in the two cases. These results offer strong evidence for a concerted last step of the reaction, the retro‐electrocyclization, with no polar intermediates involved. From the experiment in CD2Cl2/C6D6, we obtain a rate constant of 2.3×10−4 s−1, and in neat CD2Cl2 a rate constant of 2.8×10−4 s−1. These rate constants are close to those obtained from the fits of Models 3 and 4, namely 2.35×10−4 s−1 (Model 3) and 2.54×10−4 s−1 (Model 4). Yet, our previously mentioned experiment in pure AcOD‐4d gave a rate constant of 1.6×10−4 s−1. It appears that the acid slightly retards the reaction, presumably by protonating the aniline. Thus, the presence of an electron‐donating aryl group, that is a neutral aniline, does seem of some importance for promoting the RE step.Using CD2 (=C=C2). Thus, by using a (1/TCNE) ratio of 1 : 5, we are able to get the highest possible intermediate concentration and by timing a measurement for 13C NMR spectroscopy when the intermediate is at its highest concentration, we managed to obtain a detailed 13C NMR spectrum of 2 with the least interfering signals of 1 and 3. Figure 13C NMR spectrum with partial assignments. The chemical shifts are listed in Table 2 with HSQC  and HMBC . Due to the short lifetime of intermediate 2, the signal to noise ratio was less than desireable but we were still able to assign carbon 6 resonating at 139.00 ppm from the HMBC spectrum and carbon atoms 2 and 3 resonating at 114.61 and 128.60 ppm, respectively, from the HSQC spectrum. For comparison, the sp2‐carbon atoms of cyclobutene resonate at 162.3 ppm,2, the signals for these carbon atoms move significantly downfield, 183.13 and 168.74 ppm (C5 and C6 of 3). The signal at −2.35 ppm of compound 2 we have confidently assigned to the aliphatic carbon 7. The signals at 38.34 and 41.82 ppm are assigned to carbon atoms X1 and X2 but we cannot say which signal belongs to which carbon. Similarly, the two cyano signals cannot be distinguished. The remaining signals corresponding to carbon atoms 1, 4 and 5 cannot be individually assigned to these three atoms by any of our experiments.Our systematic studies allow us to use conditions that can provide additional characterization of the transient cyclobutene intermediate 29Si NMR spectroscopy to follow the conversion. With a (1/TCNE) ratio of 1 : 5, a reaction path including three species was observable on the NMR time scale . The starting material 1 showed the 29Si resonance at −18.37 ppm , and the product 3 at 5.53 ppm . The 29Si NMR spectroscopic study thus revealed the same patterns as the 1H NMR spectroscopic study, with three observable species each behaving as expected, namely one species decreasing in intensity in time, another one appearing and later disappearing, and the last one appearing and persisting.As the compounds contain a trimethylsilyl group, we also turned to 1/TCNE (1 : 5) with concentrations of 0.04 M/0.2 M in benzene. Over the course of 8 h, multiple IR spectra (in the range 3000 cm−1 to 650 cm−1) of the reaction mixture were recorded.Finally, the reaction progress was followed using in situ IR spectroscopy Figure . We used−1 are rather weak in both the experimental and calculated spectra. Calculations provided peaks for the C=C of the new cyclobutene ring formed in 2 at 1577, 1598 and 1615 cm−1 .A computational study was performed for comparison. Thus, the two substrates, the cyclobutene intermediate and the final product were optimized at the B3LYP/6‐31+G level using the Gaussian 03 program package.CA‐RE reaction on alkynes where the p‐NH2 group of 1 is replaced by either p‐NH(CO)CH3, p‐OMe, p‐CN, or m‐NH2 . At room temperature, none of these alkynes reacted with TCNE. This is in line with previous findingsN,N‐dimethylaniline require forcing conditions to react, that is, elevated temperatures (70 °C), and that alkynes with electron‐withdrawing substituents are unreactive.We also attempted the 1H NMR spectroscopy have revealed that the first and rate‐determining step of the CA‐RE reaction cannot be described as a simple second‐order reaction. By fitting various models to a large set of experimental data, we found that the mechanism involves autocatalysis. Thus, the final product of the reaction was found to promote generation of the cyclobutene intermediate, most likely on account of formation of charge‐transfer complexes. It is speculative at the moment how such complex formations actually promote the cycloaddition, but the product may act as a template organizing the reacting species in the right manner as schematically suggested in Figure 13C NMR spectrum of the intermediate and were able to assign several of its signals unequivocally, verifying it as an intermediate in the CA‐RE reaction. The three species (in fast exchange with complexes) observed by 1H NMR spectroscopy were also observed by 29Si NMR spectroscopy.Detailed kinetics studies using CA‐RE reaction may inspire and lead to future development of simple catalysts for this “click‐like” reaction  added to the alkyne solution. Aliquots from each of these solutions, corresponding to the desired substrate ratio, were mixed in an NMR tube right before the sample was subjected to NMR spectroscopy. The experiments were set up with one measured sample followed by a “ghost measurement” that corresponds to the waiting time until the next utilized measurement. This procedure was repeated for as many hours as needed or until no change in concentration of product was observed. From the NMR spectroscopic data, the concentrations of species could then be calculated by using the integral from the internal standard (cyclohexane) as a reference with a known constant concentration by comparing all other integrals towards this reference. A custom‐made kinetics program was used to fit models to the recorded data as described in the Supporting Information.6D6 or toluene‐d8 were performed by adding pure alkyne 1 and TCNE to the NMR tube followed by adding solvent with a reference (cyclohexane). The sample was then measured in the same way as described above and concentrations of species calculated in the same manner.All NMR experiments run in other solvents than CIR Spectroscopic Studies: The IR spectroscopic measurements were performed with a 6.3‐mm AgX fiber conduit with a wetted Au, diamond, C22 IR probe equipped to an in situ IR‐measuring device; IR spectra were recorded at meaningful intervals of 5–10 minutes. The reaction mixture itself was prepared by dissolving 1  in benzene (5 mL) in a 10 mL, 2‐necked, pear‐shaped flask. The IR probe was then lowered into the mixture and a single spectrum of compound 1 was measured. The probe was then removed shortly before the next measurement, and TCNE  was added and the probe inserted again. The reaction mixture was stirred for 30 s, whereafter the first measurement of the reaction was recorded. The reaction mixture would not be stirred for the rest of the experiment.UV‐Vis Absorption Spectroscopic Studies: The UV‐Vis spectroscopic measurements were performed in a 1‐cm path length cuvette. Absorption spectra of 1 : 1 mixtures of aniline and TCNE at different concentrations were measured. The absorbance of the charge‐transfer absorption band at each concentration was used to calculate the association constant.Computations – IR Frequencies: Compounds were optimized at the B3LYP/6‐31+G level using the Gaussian 03 program package.The authors declare no conflict of interest.1As 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.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.


Nature Communications 10.1038/s41467-023-42685-6, published online 02 November 2023Correction to: The original version of this Article contained an error in Fig. 1. In panels a and b the 3 black stars in each panel identifying the patients infected by a single strain were missing.  These have now been added in panel a and b in Fig 1.In addition, in the Result section of the main text the tabs labelled “Table 1” and “Table 2” in Supplementary Data 6 were incorrectly referred to as “Supplementary Table 1” and Supplementary Table 2”; these are now referred to as “Table 1 in Supplementary Data 6” in the first and fourth sentence of the fifth paragraph, and “Table 2 in Supplementary Data 6” in the fifth sentence of the seventh paragraph.These errors have been corrected in both the PDF and HTML versions of the article.


In comparison to the traditional anthraquinone method, photosynthesis is a green and energy‐saving process for H2O2 production. To improve the stability and practical application value of the H2O2 synthesized by photocatalysis, the H2O2 photosynthesis should be conducted in pure water without involving any sacrificial reagents. In this regard, organic semiconducting catalysts pose as a suitable candidate for photocatalytic H2O2 synthesis owing to their metal‐free nature to prevent H2O2 decomposition by the metal ions. In this Perspective, the H2O2 photosynthesis history is firstly introduced, followed by a review of the organic semiconductor photocatalysts reported to date. Finally, the main problems to thwart the advances of current pure H2O‐to‐H2O2 photosynthesis are discussed, followed by proposed solutions to address these issues in order to pave new ways for the development of highly efficient metal‐free organic photocatalysts for sustainable pure H2O‐to‐H2O2 conversion.Hydrogen peroxide (H 2O2Photocatalysis for H: Metal‐free organic semiconductors are next‐generation photocatalysts for the synthesis of H2O2 from water and molecular oxygen. Photoabsorption ability, separation efficiency of photogenerated charge carriers, reaction active sites, and crystallinity of the catalysts are the major factors to affect the yields of H2O2 photosynthesis. This work provides a useful guidance to the researchers in this field to synthesize highly efficiently organic photocatalysts. However, organic semiconductors often exhibit poor separation of photogenerated charge carriers, and hence, sacrificial reagents such as ethanol and methanol are usually added to consume the photoinduced holes to thwart the electron–hole pairs recombination for enhanced photocatalytic efficiency. However, the residual sacrificial reagents will severely affect the stability and purity of the H2O2 produced, thereby affecting the practical application value of the H2O2. Therefore, many organic semiconducting polymers,3N4), resins, covalent triazine frameworks (CTFs), and covalent organic framework (COFs), were modified by constructing opportune active sites at the molecular level to photosynthesize high concentration of H2O2 from pure water without any sacrificial reagents. In this Perspective, the state‐of‐the‐art organic semiconductor photocatalysts with various modification approaches are reviewed  efficiency. In 2014, Shiraishi et al. firstly reported metal‐free g‐C3N4 photocatalysts for H2O2 photosynthesis,3N4 suffers from intrinsic low photogenerated charge carrier separation efficiency, narrow light absorption range, low crystallinity, and exhibits single active sites, which greatly limits the photocatalytic performance of H2O2 synthesis. Therefore, finding new metal‐free photocatalysts and the relevant modification methods to further improve the photocatalytic performance is the topic of current study.Solar light‐driven production of H3N4 is an organic layered polymer composed of C and N elements with high thermal and chemical stability.3N4 was first studied for H2O2 photosynthesis in ethanol solution.3N4, the formed internal peroxides prevented one‐electron reduction of O2. However, the H2O2 yield over g‐C3N4 was still very low, only 30 μmol after 12 h of light illumination. The low yield could be attributed to several reasons: (1) the intrinsic weak absorption of visible light; (2) low separation efficiency of the photogenerated charge carriers; (3) low crystallinity of g‐C3N4 inhibiting charge transfer. Therefore, incessant research efforts were devoted to improving the efficiency of g‐C3N4 for H2O2 photosynthesis. In order to enhance the water oxidation activity for H2O2 photosynthesis in pure water, g‐C3N4 was compounded with homophthalic diimide (PDI).2O2, while ensuring that the conduction band position can still effectively reduce oxygen, thereby forming a dual pathway to generate H2O2 from pure water  was further hybridized with C3N4/PDI,3N4/PDI/rGO system, the g‐C3N4/PDI/rGO/BN composite exhibited an average SCC efficiency of 0.27 %.3N4, while promoting the oxygen reduction reaction (ORR), which could produce 2.0 mm h−1 H2O2 in the presence of household LED.g‐C3N4 for H2O2 photosynthesis from pure water  was doped in the RF resin.2O2 solution with an SCC efficiency of 1.0 %. In addition, monodisperse RF resin microspheres (MRFs) was also studied, in which the mesoporous microspheres can effectively modulate the charge distribution in the nano space to result in an SCC efficiency of 1.1 %.3N4, H2O2 can only be directly generated from the O2 reduction through this pathway, whereas no H2O oxidation could take place as there is no suitable active site to oxidize H2O directly into H2O2.Compared to g‐C3N4. Meanwhile, organic water oxidation sites can also be artificially introduced into CTF through structural modification. Hence, CTF may be an ideal material for H2O2 photosynthesis via 2e− dual channels. It was reported that the introduction of acetylene and diacetylene can serve as the active sites for WOR  with spatially separated redox centers by rationalizing the structure at the molecular level.2 reduction. From photocatalytic experiments, it exhibited excellent photocatalytic efficiency with an average SCC efficiency of 0.76 %, and the H2O2 yield over CHF‐DPDA (diphenyldiacetylene) was measured to be 69 μmol h−1, which was much higher than that of C3N4 and CTF‐BDDBN . However, most of the triazine organic semiconductors are in amorphous form with low crystallinity that largely limits the transportation of photogenerated charge carriers. Therefore, it is necessary to search for other organic semiconductor materials with high crystallinity along with the presence of multiple active sites to further improve the photocatalytic efficiency.CTFs own a large number of oxygen‐reducing active sites (C=N), which is similar to g‐C2O2 photosynthesis with ethanol as a sacrificial reagent in 2020.2O2 with a superior SCC efficiency of 1.08 %.2O2 whereas COF‐TfpDaaq  altered the redox pathway to a two‐step single‐electron process by changing the active sites. In addition, based on in‐situ Fourier‐transform infrared (FTIR) spectroscopy, the high photoactivity of COF‐TfpBpy is attributed to the protonation of bipyridine monomer, which promotes the rate‐limiting reaction (2e− WOR) and improves Yeager‐type oxygen adsorption to accelerate 2e− one‐step oxygen reduction. In order to enhance the activity of COFs for H2O2 photosynthesis, highly polar ionic sites were introduced in COFs materials to promote the charge carrier separation.Compared to other organic semiconductor materials, COF with high crystallinity has great advantages in the field of photocatalysis Figure . It was 2O2 experiments were performed in a two‐phase system. However, the solubility of O2 in water is low (0.25 μmol cm−3 for oxygen‐saturated water), which greatly limits the H2O2 photocatalytic synthesis activity. In addressing this problem, three‐phase reaction interface was established with COFs, in which the H2O2 generated at the three‐phase interface was 2.9 mmol g−1 h−1,2O2 from organic semiconductor materials is still in the nascent stage, and there is still much room for development. The search for suitable materials and efficient synthetic approaches to achieve stable and highly efficient photocatalytic production of H2O2 from pure water at high yields requires the joint efforts of researchers from different fields.Presently, most of the photocatalytic synthesis of H2O2. In comparison to the inorganic materials for photocatalytic production of H2O2, organic polymeric semiconductor materials that do not contain any metal are more suitable for photocatalytic H2O2 production, and, ideally, the process should not involve any organic reagents as sacrificial reagents. This method has gradually developed into one of the most effective H2O2 synthesis methods with an aim of replacing the traditional anthraquinone oxidation. However, there is still a big chasm difficult to cross from laboratory extension to daily application. In order to solve this, a series of in‐depth studies from the aspects of molecular‐level design of organic polymeric semiconductor materials for improving the photocatalytic performance is necessary . Therefore, to solve the problem ofstability, it is necessary to improve the stability of the material itself and the stability of H2O2 production. And improving the crystallinity of organic semiconductors, and then improve the efficiency of carrier separation of materials, the formation of multi‐electron double channel selectivity, to a greater extent to inhibit the generation of oxygen free radical, thus improving the stability of H2O2 production. Also increasing the degree of crystallinity of organic semiconductors will improve the stability of the material itself.Till date, most of the studies focus on the search for suitable photocatalysts or center on the modifications of the catalysts to improve the overall efficiency. However, the stability of the generated H2O2 could not be excluded. Therefore, visible to near‐infrared response activities of the organic semiconductors are equally deserving of much attention due to the potential photothermal effect.According to the empirical rule of Van't Hoff, the reaction rate increases by two‐to‐four times for every 10 K increase in the reaction temperature. The experimental studies demonstrated that the photocatalytic efficiencies increase gradually with the increase in temperature for both resins and COFs.2O2 can be obtained by oxygen reduction or water oxidation via photocatalytic reactions. There are three ways for the oxygen reduction or water oxidation: 1e− process to reactive oxygen species , 2e− process to H2O2, and 4e− process to either H2O (oxygen reduction) or O2 (water oxidation). Evidently, the dual‐channels 2e− paths are more conducive to the generation of H2O2. However, there are still very limited organic semiconductor materials with dual‐channels 2e− paths for the photosynthesis of H2O2. Since the difference in reaction pathways is closely related to the active sites in the catalysts, the active sites of different catalysts should be identified. For instance, the C=N (imine bond) in g‐C3N4, CTFs, and COFs is supposed to be the active site. Therefore, to improve the efficiency of photocatalytic generation of H2O2, it can be achieved by constructing more C=N bonds or investigating other possible active sites for O2 reduction, such as thiophene, anthraquinone, pentacenne‐dichromene‐dione, triazine, and benzene groups. Meanwhile, since the rate of WOR determines the overall performance of photocatalytic synthesis of H2O2, it is meaningful to introduce more active sites for WOR, such as acetylene, diacetylene, mellitic triimide, and perylene imides group.As mentioned previously, H2O2, in situ spectral characterization techniques and theoretical calculations should be the most effective methods. The in situ spectral characterization such as in situ IR and Raman spectroscopies were generally used to confirm the conformational changes of the catalysts and the intermediate species in the reaction. However, at present, gas–solid in situ technology is commonly used, which is far from the actual liquid–solid reaction environment. Therefore, with an aim of revealing the process of photocatalytic synthesis of H2O2 more accurately, liquid–solid in situ spectral technology should be introduced to understand the interaction mechanism between H2O and O2. In addition, theoretical calculations including band structures, differential charge density mappings, adsorption energies, and possible reaction pathways can also be used as supplementary evidence to support the results from in situ spectral characterization. With these, the reliability of the photocatalytic synthesis of H2O2 mechanisms will be greatly improved.The findings from the theoretical study can provide reliable technical supports for designing efficient and stable organic photocatalysts. In the process of photocatalytic synthesis of H2O2 over organic polymer semiconductors in terms of their stability, synthesis temperature, photocatalytic mechanisms, and active sites. In addition to the above‐mentioned aspects, there are still many factors waiting for the researchers to explore and study in order to develop an organic photocatalytic system with remarkable efficiency.All in all, this perspective summarizes the current problems for photocatalytic synthesis of HThe authors declare no conflict of interest.1


The PAAD values obtained by employing the equations of molar entropy, scaled molar enthalpy, scaled molar Gibbs free energy and isobaric molar heat capacity are 1.2919%, 1.5639%, 1.5957% and 2.4041%, respectively, from the experimental data of the KBr (X 1Σ+) molecule. The results for the potential energies, bound-state energy spectra, and thermodynamic functions are in good agreement with the literature on diatomic molecules.In this study, a diatomic molecule interacting potential such as the deformed Schiöberg oscillator (DSO) have been applied to diatomic systems. By solving the Schrödinger equation with the DSO, analytical equations for energy eigenvalues, molar entropy, molar enthalpy, molar Gibbs free energy and constant pressure molar heat capacity are obtained. The obtained equations were used to analyze the physical properties of diatomic molecules. With the aid of the DSO, the percentage average absolute deviation (PAAD) of computed data from the experimental data of the  The potential energy function is the medium through which a quantum mechanical system interacts with particles and molecules in its neighborhood. Examples of interaction potentials include the Tietz potential, Schiöberg potential, Hua potential, Rosen-Morse potential and Woods-Saxon potential6. Oscillators are modeled using molecular spectroscopic parameters such as the vibrational–rotational coupling coefficient (αe), equilibrium harmonic vibrational frequency (ωe), equilibrium bond length (re), and equilibrium dissociation energy (De). These parameters are usually determined experimentally or by ab initio calculations.A diatomic molecule oscillator is a potential energy function used to describe the rotational-vibrational states of a molecular system. To qualify as a diatomic molecule oscillator, a potential energy function must satisfy some prescribed conditions also known as the Varshni conditions21. Some of the techniques used to obtain the bound state solutions of the wave equations are the asymptotic iteration method22, quantization rules24, the supersymmetric quantum mechanics approach25, the Nikiforov-Uvarov method and its parametric versions27, and the Laplace transformation method28.The solutions of the Schrödinger equation (SE) have been obtained analytically with different potential energy models. Expressions for the bound-state energies have been successfully used to investigate the thermodynamic, optical, magnetic and other physical properties of substances30. The thermoplastic property, transition and melting points of nanostructures have also been investigated through measurements of heat capacity32. In a very recent advancement, the Gibbs free energy equation developed from the well-known Fu-Wang-Jia (FWJ) oscillator has been used to establish the equilibrium constant for the water gas shift reaction33.Thermodynamic functions have immense applications in science and technology. For instance, enthalpy and entropy measurements have been used to determine the melting points of organic molecules, and to detect diseases in plantsp) exist in the literature, some examples can be found in Refs.58. Different potential functions have been employed in the literature to construct analytical model equations66.The solution canonical partition function is a prelude to obtaining statistical-mechanical models  for the calculation of thermo-chemical properties of gaseous molecules. The partition function takes into account; the vibrational, rotational and translational effects of the diatomic system. Analytical equations for the prediction of the molar entropy (S), enthalpy (H), Gibbs free energy (H), and isobaric heat capacity  = coshq (αr)/sinhq (αr), coshq (αr) = ½ (eαr + eαr−), sinhq (αr) = ½ (eαr−eαr−), r is the interparticle separation, U0 is the depth of the potential well, q, α and σ are potential parameters. Evidently,  is constructed by employing the Varshni conditions6r, the speed of light is designated c, and μ is the reduced mass of a molecule. Inserting Eq. −2, γ = αre. The next step is to determine the potential screening parameter, α. The αe-ωe relationship given in publication64 can be used, vize = ħ/4πcM0re2, ħ = h/2π, h being the Planck constant. U″(re) and U‴(re) are obtained from Eq. (Equation  is a diamentclass2pt{minimmentclass2pt{minimPutting Eq.  into 4)4) and si28. However, owing to the simplicity of the parametric Nikiforov-Uvarov (PNU) technique27, it is considered in this work.In this section, an analytical equation for the energy spectra is derived by solving the radial SE confined by the DSO. Different analytical methods for solving the SE exist in the literature27αj  are constant coefficients, n = 0, 1, 2, … is the vibrational  quantum number and ℓ = 0, 1, 2, … is the rotational  quantum number. The quantization condition leading to energy spectra is written as27The PNU method gives that with the aid of a suitable coordinate transformation, a second-order differential equation of the hypergeometric-type can be expressed asM0 moving in a radial potential field, U (r) is given byJ = ℓ (ℓ + 1) is the angular momentum of the system, unℓ (r) is the radial wave function and Enℓ is the bound-state energy eigenvalue. Owing to the presence of the factor r-2 in the centrifugal term, expression . Nevertheless, an approximate analytical solution can be obtained with the help of approximation models. For small values of r, a Pekeris-type approximation scheme can be written for r-2 as followsdj  are deduced by the procedures outlined in Ref.71 asThe radial SE for a particle of mass Inserting Eqs.  and 11)11) into z-1 = 1−q-1 eαr2, Eq. ,,7), one s in Eq.  gives α4τ1, τ2 and τ3 in  = 0, substituting  = ZvibZrotZtra, where T is the temperature of the system, Zvib, Zrot, and Ztra are the vibrational, rotational and translational partition functions, respectively55. The vibrational partition function depends on the oscillator used to model the diatomic system, it is given as34β = 1/(kBT), kB is the Boltzmann constant. Putting Eq. }, followed by the mapping x = (z2 + 2ςκ2)½ to evaluate the integral, the summation in  of the system can be evaluated from the relation53The molar entropy (J molT) = ZvibZrotZtra into (31) and using Eqs.  an anT) = Zing Eqs.  in the r−1) of the DSO can be deduced from the expression54The molar enthalpy (J molT) = ZvibZrotZtra and Eqs.  an anT) = Zand Eqs.  and 34)T) = Zvib45298.15 is given by 31) into 45For the purpose of relating to observed data, the scaled Gibbs free energy is defined as−1 K−1) is evaluated from 40. Substituting expression (The constant pressure (isobaric) molar heat capacity (in J molpression  into thi by Eqs.  and 41)−1 K−1) i7Li2 (2 3Πg), NaBr (X 1Σ+), KBr (X 1Σ+) and KRb (B 1Π) molecules. The model parameters for these molecules are given in Table De, re, ωe and αe were obtained from publications79. The values of the potential parameters also listed in Table In this section, the equation derived for the energy levels and thermochemical functions are applied to diatomic substances including , NaBr X Σ+, KBr  of the predicted results from the observed data for the molecule is employed as accuracy indicator. The PAAD values are interpreted according to Lippincott condition for the applicability of a model equation. The Lippincott criterion requires that the PAAD value of the predicted data from the observed data is at most 1% of the experimental results. The smaller the PAAD value, better the model equation. The PAAD value is written in compact form asmin, Umax) for different vales of r . The results obtained are given in Tables 79, and the multireference configuration interaction (MRCI) data78 for the molecules are also included in the tables. The inclusion of the RKR and MRCI data is to allow for comparison of the predicted values of the potential energies with the observed data for the molecules. The variation in potential energy of the molecules as a function of interparticle separation is given in Figs. Utilizing the spectroscopic parameters in Table De in Eq. (7Li2 (2 3Πg), NaBr (X 1Σ+), KBr (X 1Σ+) and KRb (B 1Π) molecules, respectively. Therefore, based on the Lippincott criterion, it can be inferred that the DSO could satisfactorily reproduce the experimental RKR and MRCI results for the selected diatomic molecules.The accuracy of the DSO to model the experimental RKR data can be determined by letting X = RKR, Y = U and Z = e in Eq. . With th1 = r−2 is plotted as a function of interparticle separation. On the same scale and axes, the approximation function F2 = d1 + d2 cothq (αr) + d3 cothq2 (αr) is also plotted. The graphical plots depicting F1 and F2 for the diatomic molecules are shown in Figs. r chosen for the interparticle separations, the Pekeris approximation F2 is a good representation of the observed function F1. The implication of the result is that based on the parameters of the diatomic molecules considered in this study, the Pekeris approximation model F2 could be used to eliminate the function F1 to analytically solve the SE , NaBr (X 1Σ+), KBr (X 1Σ+) and KRb (B 1Π) molecules, respectively. Therefore, based on the Lippincott requirement for the applicability of a model equation, the present formula for the pure vibrational state energies could satisfactorily predict the experimental data for the NaBr (X 1Σ+) molecule, and marginally model the results for 7Li2 (2 3Πg) and KRb (B 1Π) molecules. The PAAD value obtained for the KRb (B 1Π) molecule is relatively high (≈ 4% of the observed data), suggesting that the present energy levels equation could not satisfactorily predict the observed data for the KRb (B 1Π) molecule.With the aid of Eq. , pure vi81. The NIST data is available for the gaseous NaBr and KBr molecules only. Thus, our discussions are restricted to these two molecules. PAAD values computed in the temperature range 300–6000 K are used to gauge the accuracy of the model equations.In this section, the thermodynamic functions developed for the DSO are used to analyze the thermochemical properties of pure substances. To substantiate the accuracy of the model equations, numerical data are obtained analytically and the results are compared with the literature on gaseous substances. The experimental results were retrieved from the National Institute of Standards and Technology (NIST) databaseNIST), (HNIST), (GNIST) and (CpNIST). Graphical plots of the thermochemical equations versus temperature are represented in Figs. Tables NIST and Z = S. The PAAD values deduced are 0.5401% and 1.2919%, for NaBr and KBr molecules, respectively. The obtained PAAD values are within the Lippincott error limit. This means that molar entropy equation proposed for the DSO could satisfactorily predict the NIST data for the gaseous NaBr and KBr molecules.Figure NIST and Z = Hscaled in Eq. . Based on the Lippincott condition, it can be inferred that the molar Gibbs free energy model for the DSO could satisfactorily predict the Gibbs free energy of the selected diatomic molecules.The variations in molar Gibbs free energy with temperature is graphically represented in Fig. In Fig. pNIST, and Y = Cp, the PAAD values deduced for the molecules are 2.9770% and 2.4041% for the ground state NaBr and KBr, respectively. The results clearly suggest that the isobaric molar heat capacity could not accurately predict the experimental results for the NaBr and KBr molecules. Nevertheless, the results in the tables suggest that the model could be used to obtain the molar heat capacity of the molecules within the low temperature range.Taking X = Z = C7Li2 (2 3Πg), NaBr (X 1Σ+), KBr (X 1Σ+) and KRb (B 1Π) molecules. The percentage average absolute deviation (PAAD) of the predicted data from the experimental data of the molecules is used as the goodness-of-fit indicator. The PAAD values obtained with the DSO are 1.3319%, 0.2108%, 0.2359% and 0.8841% for the molecules. The equation of bound state energy levels gave PAAD of 1.0956%, 0.2935%, 3.8667% and 1.4629% from the experimental data of the 7Li2 (2 3Πg), NaBr (X 1Σ+), KBr (X 1Σ+) and KRb (B 1Π) molecules. PAAD values were also obtained using the expression for molar entropy, scaled molar enthalpy, scaled molar Gibbs free energy and constant pressure molar heat capacity models. The results obtained for NaBr (X 1Σ+) molecule are 0.5401%, 1.9428%, 0.8164% and 2.9770%. The corresponding results for KBr (X 1Σ+) are 1.2919%, 1.5639%, 1.5597% and 2.4041% from the NIST data. The results obtained are in good agreement with theoretic data reported in existing literature and available experimental data on diatomic systems. The results obtained in this study could have practical applications in the many fields of physics and engineering such as solid-state physics, chemical physics, chemical engineering and molecular physics.In this work, the necessary conditions for a diatomic molecule oscillator were used to construct an improved version of the deformed Schiöberg oscillator (DSO). By employing the parametric Nikiforov-Uvarov solution recipe to solve the radial SE with the DSO, analytical expressions for energy spectra and canonical partition function were obtained. Using the obtained partition function, thermodynamic properties such as molar entropy, enthalpy, Gibbs free energy and isobaric heat capacity were developed for the DSO. The obtained equations were used to analyze the physical properties of diatomic substances including