Wisteria Floribunda agglutinin were consistently smaller than the DLZ/CA2 borders by ~100 μ on the CA1 or CA3 sides respectively. In addition, these patterns are dependent on position along the dorsal–ventral hippocampal axis such that PCP4 labeling often extended beyond the distal border of the DLZ into CA1. Finally, we found that, consistent with previous findings, mossy fibers innervate a subset of RGS14 positive neurons (~65%–70%) and that mossy fiber bouton number and relative size in CA2 are less than that of boutons in CA3. Unexpectedly, we did find evidence of some complex spines on apical dendrites in CA2, though much fewer in number than in CA3. Our results indicate that certain molecular markers may be better suited than others when defining the proximal and distal borders of area CA2 and that the presence or absence of complex spines alone may not be suitable as a distinguishing feature differentiating CA3 from CA2 neurons.Hippocampal area CA2 is a molecularly and functionally distinct region of the hippocampus that has classically been defined as the area with large pyramidal neurons lacking input from the dentate gyrus and the thorny excrescences (TEs) characteristic of CA3 neurons. A modern definition of CA2, however, makes use of the expression of several molecular markers that distinguish it from neighboring CA3 and CA1. Using immunohistochemistry, we sought to characterize the staining patterns of commonly used CA2 markers along the dorsal–ventral hippocampal axis and determine how these markers align along the proximodistal axis. We used a region of CA2 that stained for both Regulator of G‐protein Signaling 14 (RGS14) and Purkinje Cell Protein 4  as a reference. Here, we report that certain commonly used CA2 molecular markers may be better suited for drawing distinct boundaries between CA2/3 and CA2/1. For example, RGS14+ and STEP+ neurons showed minimal to no extension into area CA1 while areas stained with VGluT2 and  This finding was confirmed by Ishizuka et al., and others, with Lorente de Nó's nomenclature updated to include the absence or presence of thorny excrescences (TEs) characteristic postsynaptic structures associated with MF synapses  to label the specialized extracellular matrix called perineuronal nets , basic fibroblast growth factor (bFGF/FGF2), and alpha‐actinin 2 had been noted to be enriched in CA2  or effector proteins using Cre recombinase under the control of CA2‐specific promoters like Given the myriad of molecular tools available, we set out to further investigate the morphological features associated with the MF CA2 synapses and the distinct molecular characteristics of hippocampal area CA2, in relation to the neighboring CA subfields, to better understand both how best to define cellular heterogeneity in this region. In addition, we sought to gain a clearer understanding of the degree to which commonly used CA2 immunohistochemical markers colocalize in the proximal and distal borders of hippocampal area CA2.22.1All procedures were approved by the NIEHS Animal Care and Use Committee and were in accordance with the National Institutes of Health guidelines for care and use of animals. Wild‐type adult mice, either c57BL/6J (Jax 000664) or bred on a c57BL/6J background, were used for all experiments. Male and female adult mice (13–22 weeks old) were used for immunofluorescence experiments. Both male and female adult mice were used for viral injection experiments.2.2Adult mice were temporarily anesthetized using isoflurane (0.5% at 0.8 L/min) and then received and intraperitoneal injection of a ketamine/xylazine cocktail  at 0.1 cc/10 g body weight. Ophthalmic ointment was then applied to the eyes and the scalp was shaved and treated with two applications of betadine. Lidocaine (0.05 cc of 0.5% Marcaine) was injected subcutaneously. After being secured in the stereotaxic instrument, an incision was made along the midline exposing the skull surface and after removing the dura, pilot holes were unilaterally drilled above area CA2  and DG . Virus was infused into either CA2  or into the DG  using a Hamilton syringe position in a syringe pump (KD Scientific) and tube‐fed cannulas made in house from blunted 26G needles (BD). The cannula was left in position for a minimum of 8 min before a slow retraction. The scalp was then sutured, and Buprenorphine‐SR was administered subcutaneously  followed by the antisedant Atipamezole . Animals were allowed to recover on a warming pad until regaining consciousness and returning to the home cage. Animals remained in their home cages for a minimum of 2.5 weeks to allow for the proteins to express prior to tissue collection.2.3Mice were fully anesthetized with Fatal Plus  via intraperitoneal injection. Immediately after transcardial perfusion with 4% paraformaldehyde (pH 7.4) in phosphate buffered saline (PBS), brains were removed and post‐fixed in the same fixative overnight at 4°C. Brains were sectioned at 40 μm, at an angled horizontal plane .The following antibodies were used to label RGS14 and PCP4 in most experiments: mouse anti‐RGS14 , rabbit anti‐PCP4 , Alexa Fluor 568 secondary , and Alexa Fluor 488 secondary . Antibodies not mentioned here are summarized in Table 2.4t test assuming equal variance and a Gaussian distribution. If distributions proved to be unequal, a Welch's t test was calculated and reported as such in the text. Data are presented as means ± SEM.All images were acquired with an LSM 880 Zeiss confocal microscope. All quantifications were made on maximum intensity Z‐projected images with ImageJ  in CA1‐3. Dorsoventral depths corresponding approximately to the following figures from Paxinos and Franklin's mouse brain atlas  and other commonly used markers of CA2, 3D tile scans  were obtained at 0.35 μm (Z‐depth) increments to include all labeled RGS14 and PCP4 neurons of the stratum oriens (SO) border of distal CA1 is too diffuse to delineate objectively, the width of the SP was restricted throughout the more distal CA1 to match the width of the widest portion of the CA1 compact pyramidal cell layer.RGS14+/PCP4− and PCP4+/RGS14− cells were observed along the proximodistal hippocampal axis. Continuous immunohistochemical staining was defined as positively labeled cells being no more than 200 μm apart. Accordingly, imaging windows included at least 200‐μm beyond each border of continuous labeling. Measurements of each individual stain  were made using a magnification level of 150%, by an investigator blinded to just one stain. All counts for continuously labeled cell bodies were restricted to the SP and measured along the midline of SP in CA1‐3. We defined the CA1‐subiculum border as the distal‐most end of the compact pyramidal cell layer. Because the SP‐To determine the RGS14+/PCP4+ DLZ, the overlap of RGS14 and PCP4 was then visually inspected for co‐expression. Within the length of RGS14/PCP4 overlap, the borders of the DLZ were defined by the distal‐most and proximal‐most cells that show double‐labeling of RGS14 and PCP4.stratum lucidum. ImageJ was used to measure the area (μm2) of each hand‐drawn MFB. Large MFBs were characterized by a cross‐sectional area of at least 4 μm2, equivalent to the minimum volume (6 μm3) characteristic of giant MFBs  within the distal‐most 50 and 100 μm of the SL.For quantification of mossy fiber boutons (MFBs), 3D Z‐series stacks  were obtained at 0.64‐μm increments along the SL of the CA2‐CA3 axis. Two dorsal and two intermediate sections  each from all three animals were imaged 8.5‐μm thick maximum intensity projections centered on the SL of CA2 were generated and quantified at a magnification level of 75% in ImageJ. All mCherry‐positive MFBs within the SL were drawn manually, along the midline of the SL, as a function of distance from the distal end of 33.1Early investigations into hippocampal neuronal morphology by Lorente de Nó defined area CA2 as distinct region of Cornu Ammonis (CA) characterized by pyramidal neurons that lacked innervation from MF axons originating from granule cells of the DG, as well as an absence of complex spines, or thorns/TEs, seen on apical dendrites in CA3 neurons , towards CA1, and analyzed using ImageJ software , with a 166% reduction in boutons when comparing the CA3 region 500–600 μm from dSL, towards CA3, with the 100 μm adjoining the dSL . Last, identified boutons were divided into small (<2 μm2), medium (≥2 μm2 <4 μm2), and large (≥4 μm2) groupings. All three categories were equally represented in the distal‐most 100 μm of SL  with a large/small ratio of 1.2/1 , while a second injection containing (non‐Cre dependent) GCamp6f to label both CA2 and distal CA3 (CA3d) pyramidal neurons was targeted to rostrodorsal area CA2 Figure . MFBs weml-neuronbrowser.janelia.org). To further characterize the individual boutons of these distinct projections, we again labeled both presynaptic boutons (red) and postsynaptic neurons (green) and identified individual boutons in the horizontal plane  but then fell off dramatically in the more proximal regions of CA3 in the more ‘intermediate’ sections ventral to the viral injection site  in both dorsal sections  or mean bouton density . We note that no boutons were observed in CA3 at distances >400 μm from dSL in intermediate sections ventral to the viral injection site.Previous work has described an interesting phenomenon wherein MF axons originating from dorsal granule cells project in the horizontal plane to contact nearby CA3 and CA2 neurons but then continue ventrolaterally within CA2 and seem to target area CA2 exclusively . On average, the RGS14+ overlap with Znt3+ stratum lucidum measured 316.24 ± 7.58 μm in dorsal sections, which was significantly greater than in ventral hippocampus . Interestingly, the percent overlap of the two markers across the D‐V axis was similar at both levels implying that reduction in total RGS14 length is, on average, proportional to the reduction in RGS14+/Znt3+ overlap in dorsal and ventral sections . The remaining ~30% of RGS14+ neurons did not overlap with Znt3+, marking a subset of CA2 neurons that are unlikely to receive direct synaptic input from granule cells, and this fraction was also significantly greater than in ventral sections .In an effort to further characterize the relationship between MF axons and area CA2, we used immunohistochemistry (IHC) to label Zinc transporter 3 (Znt3) positive MFs and RGS14 positive neurons in both dorsal and ventral horizontal hippocampal sections Figure . When mestratum lucidum. Our interpretation of the imaged location was further aided by the RGS14+ measurements reported in Figure stratum lucidum towards CA3, though such excrescences uncommon  was that a single injection labeled both “off‐target” CA3 and targeted CA2 neurons, allowing us to visualize postsynaptic morphologies along the proximodistal CA3 → CA2 axis (towards CA1), including cells that both were and were not aligned with n Figure . Althoug3.2p = .006, dorsal vs. ventral; 526.04 ± 15.75 μm, p = .033, n = 59, 44, 42 sections from 17, 15, 13 animals, respectively). Conversely, PCP4+ labeling significantly increased along the same axis with a mean length of 648.98 ± 21.42 μm in dorsal hippocampus, 774.23 ± 38.44 μm in intermediate sections and 852.96 ± 35.46 μm in ventral sections . Interestingly, the region of overlap, or DLZ, between these two CA2 markers, was significantly greater in dorsal sections compared with intermediate and ventral sections, similar to RGS14+ measures reported in Figure n = 51, 44, 42 sections, from 13 animals). While quantifying this overlap, we observed a pattern wherein these markers labeled neurons beyond the double labeled region, and so we aimed to quantify the extension of these hallmark CA2 markers beyond the DLZ in the direction of neighboring CA1  the degree to which RGS14 labeling colocalizes with another hallmark label of CA2, PCP4, and (2) whether that colocalization is dependent on dorsal–ventral location. For these experiments, we performed IHC to label both RGS14 and PCP4 in dorsal, intermediate, and ventral horizontal sections  though, at both levels, we found a higher percentage of quantified sections exhibiting RGS14+ staining beyond the DLZ and into CA3 . In ventral hippocampus, RGS14+/PCP4− straggler cells were less common and observed in only 26% of analyzed sections versus in 36% of sections with PCP4+/RGS14− cells . Taken together, we find reliable and continuous colocalization of RGS14 and PCP4 labeling across the dorsal–ventral axis, consistent with previous work, but observe heterogeneity among individually labeled neurons that extend beyond this double labeled region. Although RGS14 aligns well with the DLZ at the putative CA2/1 border, PCP4+ neurons were found to extend well into CA1 at all three DV levels and most abundantly at more ventral depths. Conversely, although we found both RGS14+ and PCP4+ neurons extending beyond the CA2/3 end of the DLZ, RGS14 staining was more likely to extend at both dorsal and intermediate depths and PCP4 being more likely to extend into ventral CA3.Regarding neuronal labeling proximal to the DLZ, towards CA3, we observed instances in which both RGS14+/PCP4‐ and PCP4+/RGS14− “straggler cells” extended beyond the DLZ. We found no significant difference in the mean length of these extensions of either labeled populations at any of the dorsoventral depths measured , though the difference was observed in dorsal‐most sections as well . Furthermore, the mean length of WFA staining is significantly longer in dorsal sections versus intermediate and ventral . This pattern is not biased to either the proximal (CA3) or the distal (CA1) end of the DLZ (data not shown). Given the fact that WFA is also known to label interneurons in the region, cells that were WFA but RGS14−/PCP4− were not included in WFA+ length measurements. Similarly, the mean length of immunopositivity for the PNN component ACAN was also significantly shorter at dorsal depths when compared with RGS14+ . Interestingly, when CA2 neurons were labeled with the fluorescent reporter GCaMP6f in combination with IHC staining of WFA and Znt3 , a region that we showed earlier was RGS14+ . This finding is most pronounced in intermediate and ventral sections  and the Vesicular Glutamate Transporter 2 (VGluT2) contribute to the distinct molecular profile of the CA2 region and label CA2 soma and SuM presynaptic terminals, respectively , the mean length of Amigo2‐GFP labeling is significantly longer than that of the DLZ in ventral levels  but extends beyond the DLZ in more ventral sections . With respect to the CA2/3 border region, GFP positive neurons extending beyond the CA3 border of DLZ are observed at all levels and increase in length along the dorsal‐intermediate‐ventral axis . This was especially true with respect to PCP4 extension into CA1, which as we have shown, increases in likelihood in more intermediate and ventral hippocampal sections.The CA2‐specific enrichment of the cell‐adhesion protein Amphoterin‐induced gene and open reading frame 2 (AMIGO2) has been employed by many labs as a genetically encoded marker that can be used to not only distinguish CA2 from CA1 and CA3 but also to manipulate CA2 neuronal activity in vivo during specific behavioral tasks  and medium (≥2 μm2 < 4 μm2) boutons remains relatively consistent along the CA2 → CA3 disto‐proximal axis we observe large MFBs becoming the predominate fraction beginning in the bin 200–300 μm from dSL and continuing through CA3 proper in more dorsal hippocampal sections. We also report that RGS14‐positive neurons extend, on average, ~300 μm from the end of stratum lucidum towards area CA3. Therefore, it is possible that the large MFBs that we observe in the 200 300 μm bin form synapses onto CA2 neurons near the CA2/CA3 interface. This idea that MF synapses in CA2 can in fact be comprised of presynaptic and postsynaptic compartments with morphologies similar to that of the much‐studied CA3 synapses is further supported by high resolution, high magnification confocal images that clearly show large, complex postsynaptic spines in stratum lucidum at distances <200 μm from the distal most end of the MF pathway  or CA2 neurons (Amigo‐2) in concert with immunohistochemical CA2 markers to build upon our reported characterization of complex spines along the apical dendrites of hippocampal pyramidal neurons.Consistent with previous work . Future work will also continue the characterization of immunohistochemical markers like STEP and ACAN to better understand how these markers align with the DLZ in more intermediate and ventral hippocampal sections.Immunohistochemical markers are valuable tools to label area CA2 in ex vivo and in vitro preparations but how consistently do they label the same population of neurons and how do these patterns persist along the dorsal–ventral hippocampal axis? To answer these questions, we compared the proximodistal lengths of commonly used molecular markers of CA2 to the region of RGS14+ and PCP4+ colocalization (DLZ) along the dorsal–ventral axis to gain insight into potentially useful patterns of expression. Throughout our analysis, RGS14 and STEP reliably labeled the distal end of the DLZ with little to no evidence of extension into CA1. PCP4+ staining, on the other hand, consistently extended beyond the DLZ and into CA1 with this observed pattern increasing in scale along the DV axis. Conversely, WFA, ACAN, and VGluT2 positive staining fell significantly short of the distal DLZ at all hippocampal depths. Taken together, PCP4, WFA, ACAN, and VGluT2 exhibit legitimate caveats if being used to confirm the border between areas CA2 and CA1. On the more proximal end of the DLZ, our analysis shows that VGlut2+ positive staining is useful for distinguishing the CA2/CA3 border in dorsal and intermediate sections but is less reliable in more ventral sections. Conversely, staining for STEP extended beyond proximal DLZ and into CA3 with a mean extension length of 247.4 ± 72 μm, while WFA staining mean length fell short of the proximal DLZ border at all measured depths. RGS14 and PCP4 exhibit more variability at the CA2/3 border with respect to position along the DV axis. Though both markers showed similar mean lengths of extension beyond the proximal DLZ, though in separate cells, PCP4 extension was observed in a lower percentage of sections when compared with RGS14 in dorsal and intermediate levels, while the opposite pattern was observed in ventral levels where RGS14 was less likely to extend into CA3 relative to PCP4. We also report that when using an Am2‐GFP mouse line to label CA2 neurons, we observed GFP signal extending beyond the DLZ. Furthermore, these GFP+ neurons were most commonly PCP4+/RGS14− though, in a few instances, we observed GFP+ neurons that were negative for both RGS14 and PCP4. While this may be an interesting finding, we note that, more likely, antibody penetration into the tissue is the limiting factor when comparing IHC staining with the expression of a genetically expressed marker, which will be seen throughout the tissue thickness. Further work would be necessary to confirm any lack of IHC‐derived signal in GFP+ neurons as an indicator of AMIGO2  Lateral view of hippocampus indicating dorsal, intermediate, and ventral depths for analysis. (a1–3) DAPI‐stained horizontal sections showing dentate gyrus anatomies of serial sections corresponding to dorsal (1), intermediate (2), and ventral (3) depths in (a). Dorsoventral depths correspond approximately to interaural depths  of Paxinos and Franklin's horizontal mouse brain atlas (2012).Click here for additional data file.FIGURE S2: Mossy fiber bouton (MFB) increase in number but not mean density along the distoproximal CA2 → CA3 axis. (a1) Reference overview image  of a dorsal horizontal section showing GCaMP6f‐expressing CA2/CA3 pyramidal neurons and mCherry‐expressing mossy fibers (MFs) originating from dorsal dentate gyrus. (a2–3) Expanded views  of insets from  showing hand‐drawn boundaries of isolated, well‐defined boutons in the distal‐most 0–100 μm of SL  and 500–600 μm from dSL . Inset in  shows example of a typical MFB in CA3. Scale bar = 3.5 μm (b) Total number of MFBs increases along the CA2 → CA3 axis of dorsal hippocampus . (c) Total MFB density in the 0–100 μm bin (relative to the end of stratum lucidum in CA2) is statistically similar in CA2 and CA3 .Click here for additional data file.FIGURE S3: Some CA2 pyramidal neurons display thorny excrescences on apical dendrites in stratum lucidum.  Z‐projected confocal images  of horizontal sections from dorsal hippocampus showing GCaMP6f‐expressing CA2 pyramidal neurons and mCherry‐expressing mossy fiber boutons originating from dorsal dentate gyrus granule cells. Apical dendrites of select CA2 pyramidal neurons display thorny excrescences (white arrows) located within 160 μm (a) and 90 μm (b) from dSL, respectively. Yellow asterisks indicate apical dendrites that lack thorny excrescences. Inset: Enlarged view showing thorny excrescences of a MF‐CA2 synapse.Click here for additional data file.FIGURE S4: Purkinje Cell Protein 4 (PCP4+)/Regulator of G‐protein Signaling 14 (RGS14−) extension beyond CA1 border of double‐labeled zone (DLZ) is most common in ventral levels.  Z‐projected confocal images showing PCP4 neuronal labeling in CA1 of horizontal sections from dorsal (a) and ventral (b) hippocampus. (c) Percentage of sections exhibiting PCP4+/RGS14− labeling beyond DLZ and into CA1 is largest in ventral hippocampus . (d) In the dorsal and intermediate hippocampus, the percentage of sections that exhibit RGS14+/PCP4− labeling beyond the DLZ, into CA3, is larger than the percentage of sections that exhibit PCP4+/RGS14− labeling in CA3. RGS14+/PCP4− straggler cells in CA3 are rare in the ventral hippocampus .Click here for additional data file.FIGURE S5: Dorsoventral differences in the labeling pattern of VGluT2. (a) VGluT2 labeling in the stratum oriens extends diffusely into proximal CA3 in ventral (right), but not dorsal (left) depths. (b) The mean fluorescence intensity of VGluT2 labeling in stratum oriens is greater in ventral levels . Scale bars = 100 μm.Click here for additional data file.


Methylenetetrahydrofolate dehydrogenase (MTHFD2) is a mitochondrial enzyme involved in 1 C metabolism that is upregulated in various cancer cells, but absent in normal proliferating cells. Xanthine derivatives are the first selective inhibitors of MTHFD2 which bind to its allosteric site. Xanthine derivatives  were herein interrogated by molecular/induced‐fit docking, MM‐GBSA binding free energy calculations and molecular dynamics simulations in both MTHFD2 and MTHFD1 . The gained insights from our in silico protocol allowed us to study binding mode, key protein‐ligand interactions and dynamic movement of the allosteric inhibitors, correlating with their experimental binding affinities, biological activities and selectivity for MTHFD2. The reported conformational changes with MTHFD2 upon binding of xanthine derivatives were furthermore evaluated and confirmed by RMSF analyses of the MD simulation trajectories. The results reported herein are expected to benefit in the rational design of selective MTHFD2 allosteric inhibitors. Computational modeling of xanthine‐based inhibitors binding to the allosteric site of MTHFD2, with promising anticancer activity, was carried out. Using docking/IFD, MD simulations and binding free energy calculations, the binding modes in the MTHFD2 and MTHFD1 allosteric sites were analyzed, establishing a clear correlation with biological activity and selectivity. MD trajectories were furthermore analyzed to verify reported conformational changes in MTHFD2. Furthermore, MTHFD2 is believed to possess non‐metabolic functions involving RNA processing and epigenetic modification,[11],Methylenetetrahydrofolate dehydrogenase/cyclohydrolase (MTHFD2), which plays a key role in 1 C metabolism in purine and thymidine synthesis, is a bifunctional mitochondrial enzyme catalyzing the dehydrogenation of 5,10‐methylene‐THF (CH2−THF) with an NAD50 values of 96 nm and 663 nm for MTHFD1 and MTHFD2, respectively, and giving suppressed tumor growth in a mice xenograft model of colorectal cancer through intraperitoneal injection.Ki values in the nanomolar range.50 values of 254 nm, 11 nm and 47 nm, respectively, against MTHFD2 in the biochemical assays. All three inhibitors bind to the substrate binding site of MTHFD2 . The diaminopyrimidine‐based inhibitors were also tested in acute myeloid leukemia cells both in vitro and in vivo, and have shown to decrease replication fork speed, further facilitating replication stress and S‐phase arrest. Unfortunately, apart from the nanomolar MTHFD2 inhibitory activity, the three diaminopyrimidine‐based compounds inhibited MTHFD1 and MTHFD2L isoforms in the nanomolar range .50 of 1.6 μm and >18‐fold selectivity for MTHFD2. The best compound from the ‘DS’ series was reported as DS18561882 , compound 2 (PDB code: 7EHN), and compound 3 (PDB code: 7EHM), and coexisting with a folate‐based inhibitor . The ternary X‐ray structures confirmed that xanthine derivatives were bound to the allosteric site, while compound 5 occupied the substrate binding site of MTHFD2. Additionally, another X‐ray structure of MTHFD2 without the allosteric inhibitor, however, in complex with the folate‐based compound 5, cofactor NAD+ and pyrophosphate (Pi) was solved (PDB code: 7EHJ).Xanthine derivatives which bind selectivity to MTHFD2, were discovered by Lee et al.5, PDB code: 7EHJ), no conformational changes were detected. Furthermore, kinetic studies on MTHFD2 inhibition by compounds 1–3 were performed in order to explicate the mechanism of the enzymatic inhibition. The extent of MTHFD2 inhibition at any fixed concentration of the folate‐based inhibitor decreased on increasing the substrate concentration (tetrahydrofolate), which underlines that compound 5 underwent a competitive mode of MTHFD2 inhibition.2 and 3, demonstrating MTHFD2 inhibition in a non‐competitive manner. The outcomes from the kinetics studies of compounds 1–3 and compound 5 on MTHFD2 inhibition thus establish a good correlation with the aforementioned X‐ray structures, confirming a new and allosteric binding mode of xanthine derivatives, and featuring formation of catalytically inactive ternary complexes (enzyme‐substrate‐inhibitor).On comparing and superposing the four above‐mentioned X‐ray crystal structures , it was found that MTHFD2 went through various conformational changes when bound to the allosteric inhibitors, consequently impeding the binding of the cofactor and phosphate to MTHFD2. In particular, the βe‐αE loop (monomer A), the αD2′‐αD3′ loop (monomer B) and the αE′‐βf′ loop (monomer B) tend to move away from the allosteric site of MTHFD2 when the xanthine‐based inhibitors are bound  were analyzed by molecular dynamics (MD) simulations. The crystallographic binding mode, conformational dynamics and protein‐ligand interactions of compounds 1–3 were thoroughly investigated to explore the correlation with potent biological activity and selectivity for MTHFD2. In addition, we performed computational modeling of the most potent inhibitor; compound 4 , employing molecular docking and MD simulations since no previous crystallographic or docking data was available. Docking and MD simulations guided us to determine the putative binding modes of compounds 4 R and 4 S, correlating with the experimental binding affinity of the preferred enantiomer. The reported conformational changes induced by compounds 1–3, 4 R and 4 S at the allosteric site of MTHFD2 were verified from the MD trajectories and the corresponding RMSF analysis. Furthermore, as the above‐mentioned allosteric inhibitors of MTHFD2  are reported to be selective, we performed docking, induced‐fit docking and MD simulations on the co‐crystallized structure of MTHFD1 (PDB code: 6ECQ) to gain deeper insights on the cause of the poor binding to MTHFD1, influencing MTHFD2 selectivity. The selectivity preference of all compounds for MTHFD2 over MTHFD1 as suggested by the co‐crystallized/docking poses and MD simulations was further examined by MM‐GBSA binding free energy calculations on both the MTHFD2 and MTHFD1 isoforms.In the present work, we exclusively focused on the allosteric site of MTHFD2. The X‐ray structures of MTHFD2 in complex with the xanthine‐based allosteric inhibitors (compounds 1 (PDB code: 7EHV), compound 2 (PDB code: 7EHN) and compound 3 (PDB code: 7EHM) were co‐crystallized with MTHFD2 and each of them co‐exists with the folate‐based inhibitor  in the form of ternary complexes. The IC50 values of compound 1, 2 and 3 were found to be 4.0 μm, 0.69 μm and 0.78 μm, respectively, for MTHFD2.5 in the substrate binding site) in the absence of an allosteric inhibitor (PDB code: 7EHJ). Compound 4 R  and DS44960156 (PDB code: 6JIB) which bind selectively to the MTHFD2 isoform.: Molecular mechanics with generalized Born and surface area solvation (MM‐GBSA) is a largely used physics‐based method to estimate the binding free energy of a ligand bound to a protein.ComplexE, LigandE, and ReceptorE represent the energy calculations carried out in the Prime MM GBSA module for the optimized complex (complex), optimized free ligand (ligand), and optimized free receptor (receptor), respectively. The OPLS4 force fieldG Bind), the Coulomb energy contribution (ΔG Coulomb), the Lipophilic energy (ΔG Lipophilic) and the van der Waals energy  of all compounds are discussed in this work.where 1–3 in MTHFD2, docking poses and induced fit docking poses of all inhibitors in MTHFD1, protein‐ligand interaction histograms from the MD simulations of all compounds with respect to MTHFD2 and MTHFD1, and induced‐fit docking results of all inhibitors in MTHFD1.The enclosed Supporting Information contains crystallographic binding poses and redocking poses of compounds V. J. and L. A. E. formulated the study. V. J. performed the computations, analyzed the data and wrote the initial version of the manuscript. L. A. E. revised the text as final version of the manuscript.The authors declare no competing interests.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.


The polarized charges rapidly migrate to the electrodes from the Ti3C2Tx nanosheets by forming heterojunctions, achieving the maximum utilization of polarized charges and leading to enhanced piezoelectric output macroscopically. Optimally, PVDF/4wt%CsPbBr3/0.6wt%Ti3C2Tx‐PENG exhibits an excellent voltage output of 160 V under self‐polarization conditions, which is higher than other self‐polarized PENG previously. Further, the working principle and self‐polarization mechanism are uncovered by calculating the interfacial charge and electric field using first‐principles calculation. In addition, PVDF/4wt%CsPbBr3/0.6wt%Ti3C2Tx‐PENG exhibits better water and thermal stability attributed to the protection of PVDF. It is also evaluated in practice by harvesting the energy from human palm taps and successfully lighting up 150 LEDs and an electronic watch. This work presents a new idea of design for high‐performance self‐polarization PENG.Piezoelectric nanogenerator (PENG) for practical application is constrained by low output and difficult polarization. In this work, a kind of flexible PENG with high output and self‐polarization is fabricated by constructing CsPbBr 3–Ti3C2Tx heterojunctions in PVDF fiber. The polarized charges rapidly migrate from the Ti3C2Tx nanosheets to the electrodes by forming heterojunctions, achieving the maximum utilization of polarized charges and leading to enhanced piezoelectric output macroscopically. The PVDF/4 wt%CsPbBr3/0.6 wt% Ti3C2Tx‐PENG exhibits an excellent voltage output of 160 V.A flexible piezoelectric nanogenerator (PENG) with high output and self‐polarization is fabricated by constructing CsPbBr The first lead halide perovskite employed in the fabrication of PENG is MAPbI3, which exhibits output voltage and current density of 2.7 V and 140 nA cm−2, respectively. Later, FAPbBr3‐based PENG is developed with output voltage and current density of 8.5 V and 3.8 µA cm−2, respectively. Considering the stability of perovskites as well as flexible applications, composites of piezoelectric polymers and perovskites are gradually being mushroomed. Polyvinylidene fluoride (PVDF) and its copolymers are the most widely used in PENG such as PVDF/MAPbI3, PVDF/MAPbBr3, PVDF/FAPbBr3, PVDF/MAPbI3, PVDF/CsPbBr3, and PVDF/CsPbI3, because of their relatively high piezoelectric coefficients. However, the dipoles of composite materials are generally in a random arrangement, which obviously limits the improvement of piezoelectric properties. In addition, the buffering effect of elastic polymer PVDF causes the composite material to deform unevenly, creating a piezoelectric potential difference that makes a certain amount of polarized charge to be trapped in the material without contributing to the output current. These reasons lead to the unsatisfactory piezoelectric properties of composites.The use of mechanical motion as a natural source of power is gradually replacing battery or electrical power in driving smart electronics. In recent years, piezoelectric nanogenerator (PENG) based on piezoelectric materials has gained tremendous attention for its remarkable ability to convert mechanical energy into electrical energy.1 Lead However, materials with low breakdown voltages and large coercivity voltages are difficult to be polarized. In addition, the electric polarization of piezoelectric materials usually requires a long time of treatment under high temperatures and strong voltage, resulting in wasted energy and higher manufacturing cost. Meanwhile, the removal of the external electric field and heating condition causes depolarization of the material, which damages the stability of piezoelectric properties. The second approach is to control the orientation of materials . However, the special template and complex synthesis process of texturing cause it to be effective for 1D and 2D materials but not for granular materials. The third approach is the self‐polarization effect by introducing interfacial polarization between different components. By comparison, the self‐polarization method is more suitable for extensive industrial applications due to its low cost and simple synthesis process. Sultana et al. prepared PVDF/MAPbBr3 composites by electrospinning and found that the strong electric field and mechanical stretching of the electrospinning process caused in situ polarization of PVDF. According to our group's previous work, PVDF/CsPbBr3 composites can obtain high piezoelectric output (V = 33 V) without external electric field polarization. Regrettably, the performance of piezoelectric composites obtained by this method is still far from electric poling.At present, there are three strategies to enhance the dipole orientation toward outstanding piezoelectric performance. The first approach is to induce dipole alignment of piezoelectric materials by an electric poling process.14 How MXene, as a promising energy storage material, has attracted great interest due to its unique features such as excellent electronic conductivity, obvious security capability, environmental benignity, and excellent biocompatibility. Liu et al. synthesized the MXene/COF/Cu2O heterojunction for photocatalytic sterilization. Efficient photocatalysis is achieved due to the strong built‐in electric field generated at the heterojunction interface promoting rapid carrier migration. The as‐constructed MoS2@MXene@D‐TiO2 heterostructure in sodium‐ion batteries delivers admirable high‐rate reversible capacity due to the built‐in electric field between the non‐homogeneous phases that promotes the high Na+ transportation. Meanwhile, Agresti et al. suggested that the formation of heterojunctions between MXene and perovskite could regulate the arrangement of dipole moments. Therefore, the construction of MXene heterojunction with perovskite is expected to improve both the arrangement of dipole moments and the utilization of polarized charges.Synthesizing heterojunctions with a strong built‐in electric field can effectively transfer electrons which is an effective way to relieve trapped polarized charge.20 MXe3–Ti3C2Tx heterojunctions are first constructed in PVDF fibers, providing a path for the transfer of polarized charges inside the film and macroscopically enhancing the output performance of the devices. Meanwhile, the PVDF/CsPbBr3/Ti3C2Tx composite achieved self‐polarization without an additional electric field. Further, the mechanism of self‐polarization is explained by analyzing the interfacial charge and electric field using first‐principles calculation. Besides, the piezoelectric outputs of PENG under experimental and practical conditions are also investigated systematically.Herein, CsPbBr22.13/Ti3C2Tx composite fibers were fabricated through the electrospinning method, as schematically illustrated in Figure3C2Tx nanosheets were synthesized by HF/HCl etching and LiCl intercalation of Ti3AlC2 , oleic acid (OA), and PVDF were added to DMF solution and homogenized via stirring at 60 °C . Finally, the precursor was drawn under the high electric field to obtain fibers  pattern, the typical peak (104) of Ti3AlC2 disappears, and (002) shifts to a lower angle again confirming the successful preparation of ultrathin Ti3C2Tx nanosheets  image, the lateral size of Tim Figure. The almm Figure confirm 3/Ti3C2Tx composite fiber films are presented in Figure 3 and Ti3C2Tx in PVDF fibers. As shown in Figure 3C2Tx content. In electrospinning process, droplets are charged to produce jets that stretch to form fibers after overcoming the surface tension of the liquid. Therefore, the size of the fiber is mainly influenced by the Coulomb force and surface tension. Viscosity tests for each component precursor are listed in Figure 3C2Tx content, which represents an increase in the surface tension of the precursor. Theoretically, the fiber diameter increases with the increase of surface tension. Here, the regular decrease in fiber diameter is due to the addition of Ti3C2Tx improves the conductivity of the precursor, making the increase in Coulombic force greater than the effect of surface tension.SEM images of PVDF/CsPbBr3/Ti3C2Tx (0.6 wt% Ti3C2Tx) fibers in dark and bright fields. It can be seen that CsPbBr3 is uniformly dispersed inside the fibers, while the Ti3C2Tx is inserted into the fiber. High‐resolution transmission electron microscopy (HRTEM) images demonstrate that Ti3C2Tx nanosheets retain the hexagonal crystal structure of the parent Ti2AlC phase  crystal plane of Ti3C2Tx , (110), (200), (210), (211), and (220) crystal plane of CsPbBr3(PDF# 54–0752), confirming the formation of CsPbBr3 nanoparticles within the polymer fiber. However, due to the low loading weight percent and uniform dispersion of Ti3C2Tx, the peak representing Ti3C2Tx did not appear in XRD spectra. The crystalline forms of composite films are further explored by the fourier transform infrared spectroscopy (FTIR) as shown in Figure β phase content is exposed in the Supporting Information. With the addition of 4 wt% CsPbBr3, the β phase content increased from 78.7% to 94.6%  are investigated by X‐ray photoelectron spectroscopy (XPS). Figure  F1s has a single peak at 688.1 eV, which is consistent with the organic fluorine , which is proportional to the dielectric permittivity and the remnant polarization. Here, the dielectric permittivity and dielectric loss of composites are measured in Figure 3C2Tx to the highest value of 58 for 1.0 wt% Ti3C2Tx at the frequency of 100 Hz. The addition of Ti3C2Tx increases the interfacial polarization, which is confirmed in the calculation section, leading to an increase in the dielectric coefficient. The dielectric loss is two orders of magnitude smaller than the dielectric constant. Due to the low‐frequency oscillation, more attention is paid to the data pattern after 1000 Hz. However, the dielectric loss increases with the increase of Ti3C2Tx because the increase of Ti3C2Tx forms a conducting path. The increase in dielectric loss is detrimental to the piezoelectric performance. Finally, a quasi‐static d33 meter was used to measure the d33 of PVDF/CsPbBr3/Ti3C2Tx films as 36.1 pC/N. The detailed test method and procedure are shown in Figure The performance of PENG is directly dependent on its piezoelectric constant d33, whichFurther, the topography, amplitude, and phase images of piezoresponse force microscope (PFM) are shown in Figure 2.2Figure2.To investigate the piezoelectric properties of composite films, the top–bottom configuration of PENG is prepared, where polydimethylsiloxane (PDMS) is a surface adhesion and passivation layer. The detailed fabrication procedure of PENG is illustrated in Isc) density and open‐circuit voltage (Voc) of composite are measured under palm tap  was selected for subsequent tests. The performance of PVDF/CsPbBr3/Ti3C2Tx at different frequencies was examined in Figure Isc and Voc remain stable in the range of 0.2–1.5 Hz, indicating that they can perform energy harvesting and conversion tasks in a wide frequency range. Table3/Ti3C2Tx has a significant output voltage. A switching‐polarity test was performed as shown in Figure The piezoelectric output of short‐circuit current  is first calculated by Equation Eb3C2Tx–CsBr, Ti3C2Tx–PbBr, and Ti3C2Tx–PVDF are calculated and displayed in Figure0−3 reflect the charge depletion and accumulation region, respectively. There is a large amount of charge transfer and exchange between Ti3C2Tx and PbBr. It can be seen that the charge exchange is mainly concentrated between the Pb and O atoms.ρAB, ρA, and ρB are the charge density of interfaces AB, A, and B, respectively. Based on ∆q, the charge displacement curve is calculated by Equation  polarization . In addition, CsPbBr3 also induces space charge polarization (Ps) at the interface of the polymer matrix  using HF/HCl etchant. The etching solution was prepared by mixing 6 mL deionized water (DI water), 12 mL hydrochloric acid , and 2 mL hydrofluoric acid . 1 g Ti3AlC2 was slowly added to the etchant solution for about 5 min and then stirred at 400 rpm for 24 h at 35 °C. The as‐obtained multilayer Ti3C2Tx was washed with DI water and centrifuged at 3500 rpm (5 min per time) until pH ≥ 6. The precipitate was collected and redispersed into 20 mL DI water by shaking, and then added to a solution of 40 mL DI water containing 1 g LiCl and stirred at 400 rpm for 4 h at 35 °C. The obtained Ti3C2Tx dispersions were washed by centrifugation at 8000 rpm using DI water until the pH exceeded 6. Further, the sediment was redispersed in 35 mL DMF solution to probe sonicated in a cold bath for 20 min (power: 600 w). The supernatant was collected by centrifugation at 3500 rpm for 1 h. For the quantitative analysis of Ti3C2Tx content in DMF, 2 mL of the solution was dried under a vacuum at 60 °C.Ti3C2Tx suspension (solvent was DMF) was added to the DMF solution and sonicated for 30 min to obtain a uniformly dispersed solution. Subsequently, CsBr and PbBr2 were added to the above solution and stirred for 20 min to dissolve it fully. The OAm and oleic acid (OA) were added and stirred magnetically for 20 min at 500 rpm. Finally, PVDF (molecular weight 1 200 000) was added to obtain the precursor solution by magnetically stirring at 60 °C for 4 h with a weight concentration of 14%. The prepared precursor was drawn into a syringe with a 0.5 mm inner diameter stainless‐steel needle for electrospinning. Electrospinning fibers were collected at a stainless‐steel rotating cartridge collector with a speed of 3000 rpm. The distance from the needle to the collector was 18 cm. The flow rate was maintained at 0.04 mm min−1. The applied voltage was set as 15 kV. Inside the electrospinning machine, the temperature was set to 30 °C, and the humidity was adjusted to 60%. Then, the electrospinning film was dried for 2 h in a normal oven at 80 °C. PENG was the top‐bottom–electrode configuration.The composite nanofibers were prepared by the electrospinning method. Initially, the TiFirst, electrospinning fiber film was cut to the appropriate size (1 cm × 1 cm) as an active layer. Then the aluminum foil serves as the top and bottom electrodes bonded to both sides of the active layer via PDMS. Finally, it was encapsulated by polyimide (PI) tape.3C2Tx and PVDF/CsPbBr3 fiber was performed by TEM, selected area electron diffraction (SAED), and HRTEM on Tecnai G2 F30 S‐TWIN. Atomic Force Microscope (AFM) was utilized to test the morphology and thickness of Ti3C2Tx nanosheets. Besides, the local piezoelectric properties were investigated with PFM based on an AFM system (Bruker Multimode 8). The dielectric properties were measured by a precision LCR meter (Agilent 4294A) at room temperature. The absorbance of Ti3C2Tx in DMF solution was measured by a UV–vis spectrophotometer . The viscosity of the precursor was obtained by a viscometer (DV2T‐LV). The phase structure was characterized using XRD on Ultima IV and FTIR on Nicolet IS10. The surface electronic states of PVDF/CsPbBr3 fiber film were measured by XPS using AXI ULTRA with a monochromatic Al Kα source. Electrical analysis was accomplished by applying an external force on the PENG through a linear motor  and recorded by a source measure unit (Keithley Instruments). The quasi‐static d33 measuring instrument (ZJ‐3AN) was used to measure d33. The value of the palm tap force was measured by a pressure sensor (SIMBATOUCH‐SBT961M).The surface morphology of the samples was characterized by a field emission scanning electron microscope on Gemini Sigma 300/VP. Microstructure of Ti The exchange–correlation functional was presented by the generalized gradient approximation in the form of Perdew–Burke–Ernzerhof. The core–electron interaction was treated by the projector‐augmented wave method. The long‐range weak van der Waals interactions were described by Grimme's DFT‐D3 dispersion correction. The cutoff energy was set as 460 eV. The convergency criteria of energy and Hellman–Feynman forces on each atom were set as 1 × 10−4 eV per atom and 0.05 eV Å−1, respectively. The Brillouin zone was sampled by a k‐point mesh 4 × 4 × 3. Based on the optimized CsPbBr3 crystal, the slab models with seven octahedron layers were constructed by slicing the CsPbBr3 crystal along (110) lattice plane. The slab terminated by Cs—Br and Pb—Br were both considered and a vacuum layer with the size of 20 Å was added along z‐axis to avoid interactions between mirror slabs. The dipole correction along z‐axis of slab was implemented to correct the errors introduced by the periodic boundary conditions. Considering the limited computational resource, a section of the PVDF was selected to investigate the interaction between CsPbBr3‐PVDF and Ti3C2Tx‐PVDF.The first‐principles calculation was performed in the framework of density functional theory (DFT) via Vienna ab‐initio simulation package code.41 TheThe authors declare no conflict of interest.Supporting InformationClick here for additional data file.Supplemental Video 1Click here for additional data file.Supplemental Video 2Click here for additional data file.


Two variations of the procedures are also presented that include either 12C or 13C at the C8 position of adenosine, and 13C at either the C8 or C2 position of guanosine. These 13C tags permit the incorporation of two 15N‐labeled nucleosides into an RNA strand while ensuring that their nuclear magnetic resonance (NMR) signals can be distinguished from each other by the presence or absence of C‐N coupling. While the major application of these specifically 15N‐labeled nucleosides is NMR, the additional mass makes them useful in mass spectrometry (MS) as well. The procedures can also be adapted to synthesize the labeled deoxynucleosides. The Support Protocol describes the synthesis of 7‐methylguanosine. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC.This article describes the specific incorporation of Basic Protocol 1: Syntheses of ‐ and adenosineSupport Protocol: Synthesis of 7‐methylguanosineBasic Protocol 2: Synthesis of ‐ and guanosine Basic Protocol 11). The first 15N label is introduced by a direct nitrosation/reduction to give 2. This is followed by ring closure using either diethoxymethyl acetate in dimethylformamide (DMF) to give 3a with a 12C at the C8 position, or [13C]sodium ethyl xanthate to give 3b with a 13CSH at the C8 position. Removing the thiol group(s) with Raney nickel forms hypoxanthine (4a/b), which can readily be converted to 6‐chloropurine (5a/b), which are excellent substrates for enzymatic transglycosylation. The second 15N label is then introduced into the nucleoside by displacement of the chloride by 15NH3, which is generated in situ to give the labeled adenosines 7a/b.As shown in Figure CAUTION: The procedures in this article use a number of highly toxic and dangerous reagents. Raney nickel is pyrophoric when dry and may burst into flames if not kept wet. Phosphorous oxychloride (POCl3) is very reactive and hydrolyzes to hydrochloric and phosphoric acids, which are both highly corrosive to skin and tissue. Cyanogen bromide is very dangerous; improper use of this reagent can cause death. All reactions must be done with great care in an appropriate chemical fume hood.1; also called 6‐amino‐2‐thioxo‐1,2‐dihydro‐4(3H)‐pyrimidinone; MilliporeSigma)4‐Amino‐6‐hydroxy‐2‐mercaptopyrimidine monohydrate Sodium ethyl xanthate , for 13C synthesis only[NaOH50% aqueous Raney 2800 nickel (RaNi) slurry (MilliporeSigma)Dipotassium salt of EDTA3)Phosphorous oxychloride 5% (v/v) NHEthyl acetateEthyl ether1 M HCl7‐Methylguanosine (see Support Protocol)2HPO40.02 M K3 M NaOHPurine nucleoside phosphorylase (MilliporeSigma)15N]Ammonium chloride  sodium ethyl xanthate to give 13C at the 8 position (steps 16b to 22b).The synthesis is continued using either diethoxymethyl acetate in DMF to give 16a2 from the funnel into a 100‐ml round‐bottom flask. Rinse funnel with portions of 96% formic acid and add them to the flask to give a final volume of 25 ml.Scrape out most of 17aAdd a stir bar, attach a condenser, and reflux the solution 1 hr to make the formate salt.18aConcentrate to dryness using a rotary evaporator and scrape down the sides of the flask with a spatula, if necessary.19aInsert a rubber septum and displace the air with nitrogen.20a20 ml anhydrous DMF;1.63 ml DEMA ;0.24 ml of 96% formic acid .Use syringes to add the following through the septum:21aHeat mixture 3 hr in an oil bath set at 130°C. Follow reaction by HPLC.The flask is lifted from the oil bath and allowed to cool briefly, and then a small syringe with a long, dry needle is used to get a sample for HPLC.22aCool flask, concentrate solution to a solid using a rotary evaporator, and loosen it with a spatula if necessary.23aAdd 15 ml acetonitrile to the flask, attach a condenser, and reflux 10 min using the 130°C oil bath.24aCool flask to room temperature, add 10 ml acetonitrile, and then chill in an ice bath.25a3a by vacuum filtration and wash twice with 5 ml cold acetonitrile.Collect 26a3a over P2O5 in the vacuum desiccator overnight. Proceed to step 27.Without removing it from the funnel, dry The yield is usually >95%.16b2 from the funnel into a 100‐ml round‐bottom flask and add 0.80 g (5.5 mmol) [13C]NaSCSOEt.Scrape out most of 17bInsert a condenser into the flask, attach a nitrogen line and vent needle, and displace air for 5 min.18bAdd 15 ml DMF and reflux mixture under nitrogen ∼3 hr, using HPLC to monitor the reaction.19b3b.Cool mixture in an ice bath and add 50 ml cold acetonitrile to precipitate 20b3b by vacuum filtration and wash twice with 5 ml cold acetonitrile. Save filtrate and both washes.Collect the solid 21bConcentrate filtrate and washes, and purify by preparative reversed‐phase chromatography.22b3b over P2O5 in a vacuum desiccator overnight. Continue with step 27.Dry the combined portions of The yield is usually >95%.273a/b from the funnel into a 100‐ml round‐bottom flask.Scrape out most of 28Rinse funnel with portions of water and add them to the flask to give a final volume of 30 ml.29Add a stir bar and 2 ml of 96% formic acid.30Weigh 4.5 g of 50% aqueous RaNi slurry into a small glass vial or beaker.CAUTION: RaNi is pyrophoric (will spontaneously burst into flames) if it is allowed to dry out. To weigh it, shake the bottle and immediately transfer some of the suspended RaNi to the vial. Continue adding the suspension (shaking the bottle each time) until 4.5 g of the 50:50 mixture of RaNi/water has been measured out.31Using a dropper, transfer the RaNi suspension over 5 min to the reaction flask and add 1.5 g dipotassium salt of EDTA.Traces of remaining RaNi in the vial and dropper should be destroyed with 6 M HCl.32Connect a condenser to the flask. Using the 130°C oil bath, reflux ∼2 hr while monitoring the reaction for completeness by HPLC.33Remove flask from the oil bath and allow it to cool only briefly.34Remove condenser from the flask and carefully filter the hot reaction mixture to remove the RaNi. Rinse the flask and then the funnel with three 10‐ml portions of boiling water, adding these washes to the filtrate.To destroy the RaNi remaining in the funnel, the funnel should be transferred to a large beaker and portions of 6 M HCl should be slowly added until no black particles can be observed.35Concentrate filtrate and washes to dryness in a 100‐ml round‐bottom flask using the rotary evaporator.364a/b over P2O5 in the vacuum desiccator overnight.Dry 4a/b can be purified by reversed‐phase chromatography if desired.The yield is usually >95%. 374a/b into a very dry 100‐ml round‐bottom flask.Weigh 0.69 g (5.0 mmol) 383 and 2 ml (16 mmol) N,N‐dimethylaniline.Add 20 ml  POCl3; it is very reactive.Use great care with POCl39Attach a condenser and reflux 20 min under nitrogen.The resulting solution should be black and homogeneous. It is essential for this reaction to remain anhydrous.40Monitor reaction for completeness by HPLC and continue refluxing the mixture for ≤30 min more.41N,N‐dimethylaniline and continue the evaporation.Concentrate mixture to a very small volume using a rotary evaporator, first with an aspirator and then with a vacuum pump protected with a dry‐ice trap. Add 10 ml 3, or localized heating from later neutralization with NH3 may cause the reaction to reverse. Be extremely careful to dry the evaporator condenser and trap prior to use, to keep dry ice in the traps throughout this process, and then to pour the collected POCl3 into a plastic container of ice to decompose it.It is necessary to remove all traces of POCl423 to dissolve the black gum.Cool flask in an ice bath and very slowly add 30 ml of 5% NHAs a neutral compound, 6‐chloropurine is insoluble in water but under basic conditions it will ionize and therefore dissolve.433 if necessary) and then pour it into a separatory funnel. Wash it first with 30 ml ethyl acetate and then twice with 30 ml ethyl ether. Check the layers by HPLC.Make sure the pH of the solution is >10  7‐methylguanosine and 0.78 g (5 mmol) 54Add 250 units of purine nucleoside phosphorylase. Insert a septum and heat the mixture ∼3 days in an oven at 30°C with gentle agitation. Monitor reaction for completeness each day by HPLC.55Pour mixture into 10 ml DMF and stir ∼1 hr at room temperature.56Filter suspension to remove most of the solid 7‐methylguanine. Suspend solid in 5 ml fresh DMF, stir 15 min, and filter.57Concentrate combined filtrates to a small volume using a rotary evaporator and add 15 ml water (more if necessary to dissolve the solid).586a/b by preparative reversed‐phase HPLC and dry it over P2O5 in the vacuum desiccator overnight.Purify the crude The yield is usually 85% to 95%.596a/b into a clean Teflon liner of a bomb and add 0.54 g  of [15N]NH4Cl and 7 ml anhydrous DMSO.Place 1.44 g (5 mmol) 603 and immediately seal the bomb.Add 1.5 g  KHCO61Heat the bomb 3 days in an 80°C oven, swirling the mixture once or twice each day.62Cool the bomb to room temperature and then to –20°C for ≥30 min. Open it carefully and dilute mixture with 10 ml water. Adjust the pH to 7 with glacial acetic acid.2 will have been generated.Care should be taken when opening the bomb because CO63Check the reaction by HPLC.647a/b by preparative reversed‐phase HPLC and dry over P2O5 in the vacuum desiccator overnight.Purify The yield is usually 80% to 90%. See Tables Although 7‐methylguanosine can be purchased from MilliporeSigma, it is quite easy to make. Commercial 7‐methylguanosine is very expensive and the purity may not be as high.GuanosineN,N‐DimethylacetamideNitrogen gas sourceDimethyl sulfate3Concentrated aqueous NHAcetone, 4°C95% (v/v) ethanolEthyl ether250‐ml round‐bottom flaskRubber septum10‐ml syringesCAUTION: Dimethyl sulfate is very dangerous because it is a potent alkylating agent; wear gloves and use caution.1Place 9.91 g (35 mmol) guanosine in a 250‐ml round‐bottom flask along with a stir bar.2N,N‐dimethylacetamide as a solvent, insert a rubber septum, and displace the air with nitrogen gas.Add 80 ml 3Carefully withdraw 7.0 ml  dimethyl sulfate with a 10‐ml syringe and add it to the suspension.4Stir mixture ∼6 hr and monitor reaction for completeness by HPLC.53 and then check the pH using pH paper. Continue to add more NH3 slowly, frequently checking the pH, until the mixture is pH 10.Slowly add 10 ml concentrated aqueous NHThis step is essential and quenches the excess dimethyl sulfate.6Slowly add the mixture to 300 ml cold acetone in an ice bath to precipitate the product.7Collect the white precipitate by vacuum filtration.8Check the acetone filtrate by HPLC for product. If there is a significant amount, concentrate the filtrate to a small volume, chill, and collect the additional product by vacuum filtration. Combine it with the rest.9Suspend solid crude product in 300 ml of 95% ethanol, stir 5 min, and collect by filtration.10Suspend solid product in 300 ml ethyl ether, stir 5 min, and collect by filtration.112O5 in a vacuum desiccator overnight.Dry pure product over PThe yield is usually 80% to 90%. It is very important to keep this compound dry. It should be transferred to a bottle with a tight lid, the air should be displaced with nitrogen, and it should be stored at –20°C. It can be kept for up to 3 months.Basic Protocol 27a/b) to the N1 oxide (8a/b), which is followed by a one‐flask set of reactions without purification to give 12c‐f. Labeled cyanogen bromide is generated in situ from labeled potassium cyanide and bromine, and its reaction with 8a/b gives the oxadiazolidines 9c‐f. Treatment with triethylamine opens the oxadiazolidine ring, allowing the N1 oxide to be methylated by methyl iodide to give 10c‐f. Aqueous sodium hydroxide then opens the pyrimidine ring, which allows rotation around the C5‐C6 bond of 11c‐f. This rotation brings the cyano group near the deformylated 3‐amino group to facilitate ring closure upon neutralization and heating to give 12c‐f. Enzymatic deamination then gives the final products, 13c‐f. The reagent combinations to convert labeled adenosines to labeled guanosines are summarized in Figure As shown in Figure 2‐15N2]Adenosine (7a) or adenosine  methanol3‐Chloroperoxybenzoic acid (MCPBA), purified (see recipe)Ethyl ether2O5)Phosphorous pentoxide adenosine (7b) into a 100‐ml round‐bottom flask. Add a stir bar and 50 ml of 50% methanol.Weigh 1.35 g (5.0 mmol) of cyanogen bromide or [15N]cyanogen bromide, as appropriate for the desired labeling pattern, stir 2 hr, and monitor reaction for completeness by HPLC.Add 7.5 mmol freshly prepared potassium cyanide dissolved in 20 ml water, and stir 30 min. Draw the solution into a syringe in order to add it to a reaction.Place 3 ml water into a small pear‐shaped flask and add 1.2 g  bromine. Chill the flask in an ice bath, slowly add 0.50 g  cyanogen bromide (see recipe) but use [15N]potassium cyanide instead of potassium cyanide.Prepare as for [13C]carbon disulfide . Cover the flask with aluminum foil and stir the solution overnight at room temperature. Concentrate to dryness and dry over P2O5 in a vacuum desiccator overnight. Store up to 6 months at –20°C.Dissolve 0.40 g (10 mmol) NaOH in 40 ml absolute ethanol. Add 0.77 g  NH3 can be done using much milder conditions. Also, the lipophilicity of the 6‐chloro group helps in the purification of the nucleoside. The purine nucleoside phosphorylase uses 7‐methylguanosine as a sugar donor and both generates the ribose‐α‐1‐phosphate and couples it to the 6‐chloropurine. The reaction is driven to completion by precipitation from solution of the very insoluble 7‐methylguanine. The final amination has been optimized so that it only requires two equivalents of [15N]NH4Cl with KHCO3. Although not described here, adenosine can be converted to adenosine . The order of addition during the formation of cyanogen bromide is also important. In the final enzymatic deamination to make 13c‐f, it is important that 12c‐f be purified for the enzymatic reaction to work satisfactorily.In the chlorination that converts max data from an HPLC system with a diode array detector and mass data from an LCMS being particularly helpful.The early steps in these protocols generally give high yields, even for inexperienced workers. The later steps are more challenging, and, with experience, the stated yields can be obtained. Because most of these compounds are fairly polar, the use of TLC to monitor reactions is not very convenient. Reversed‐phase HPLC is much more informative, with UV λThe total time for Basic Protocol Roger A. Jones: Conceptualization, project administration, supervision, writing original draft, writing review and editing; Barbara L. Gaffney: Investigation, project administration, supervision, writing original draft, writing review and editing.The authors declare no conflict of interest.


While the fabricated photodetector exhibits good sensitivity (3.1 mS W−1per □) and fast photoresponse (31 µs), the bias‐, polarization‐, and spatial‐resolved measurements point to an intrinsic photoconductive mechanism. By increasing the incident power density from 1.5 to 200 µW µm−2, the photocurrent power dependence varies from sublinear to superlinear. At higher illuminating conditions, prominent superlinearity is observed with a giant power exponent of γ = 1.5. The unusual photoresponse can be explained by a two‐recombination‐center model where density of states of the recombination centers (RC) effectively closes all recombination channels. The photodetector is integrated into camera for taking photos with enhanced contrast due to superlinearity. This work provides an effective route to enable higher optoelectronic efficiency at extreme conditions.Photodetector based on two‐dimensional (2D) materials is an ongoing quest in optoelectronics. 2D photodetectors are generally efficient at low illuminating power but suffer severe recombination processes at high power, which results in the sublinear power‐dependent photoresponse and lower optoelectronic efficiency. The desirable superlinear photocurrent is mostly achieved by sophisticated 2D heterostructures or device arrays, while 2D materials rarely show intrinsic superlinear photoresponse. This work reports the giant superlinear power dependence of photocurrent based on multilayer Ta γ = 1.5 due to suppression of recombination channel. The photodetector is integrated into camera, showing enhanced imaging contrast due to the superlinearity.2D photodetectors generally suffer recombination processes, which result in the sublinear power dependence of photoresponse. Here, the article reports giant superlinear power dependence of photocurrent with power exponent reaching  The photoconductive detector is one of the most stable optoelectronic devices with broad working bandwidth, high responsivity, and high gain. The photoresponse of this device is mainly determined by material properties due to the simple structure and physical mechanism. When semiconductor material absorbs incident photons, whose energy is equal to or greater than the bandgap, photon‐generated electrons and holes will be separated in opposite directions and collected by the electrodes with an external bias. The photocurrent (Iph) increases as a function of incident power (P) following a power‐law dependence of Iph∝Pγ. The power exponent (γ) varies between different materials because of electron–hole generation, trapping, recombination process, and other mechanisms.Optoelectronic devices based on two‐dimensional (2D) materials have attracted intense research attention owing to their excellent performances of high sensitivity,1, 2γ = 1) since the photocurrent is solely determined by the photogeneration of electron–hole pairs. In most 2D‐based devices, Iph exhibits a sublinear power dependence under high‐intensity illumination due to dominating contribution from defects and impurities. As light intensity increases, those defects serve as effective recombination centers (RC) and capture more photocarriers which lead to the saturation of photocurrent (γ < 1) and decreased responsivity. As for superlinear power dependence (γ > 1), it is found in comparatively rare cases and features increased photoresponsivity with power. Recently, the superlinear power‐dependent photocurrent was reported in a series of artificial 2D structures such as graphene/h‐BN, graphene/WSe2, WS2/MoS2 heterojunctions, and sheet array. The typical origin of superlinearity from heterostructure devices is the photothermionic effect, where hot carriers are injected from the gate side to overcome the Schottky barrier exponentially as external injection bias increases, resulting in significantly extended spectral bandwidth and responsivity. Meanwhile, the multicenter Shockley–Read–Hall process also contributes to the superlinear response in arrayed structures such as printed MoS2 and GaTe transistor arrays because the array structure keeps photocarriers from massive recombination at high luminous power.For the ideal case, a linear increase of photocurrent with incident power is expected  is desired which potentially allows for stronger optoelectronic efficiency at the high power regime and enables better performance if integrated into the discussed sophisticated structures.The desired superlinear photoresponse is mainly achieved by sophisticated 2D heterostructures and arrays.29 How2NiS5 at ambient condition. The photodetector manifests itself with a simple metal‐Ta2NiS5‐metal structure. Bias‐dependent and spatial scanning photocurrent measurements suggest the photoconductive origin of the photoresponse so that the photocurrent is determined by the intrinsic material property of Ta2NiS5. The photoconductive devices feature a fast response of 31 µs, along with good sensitivity of 3.1 mS W−1per □, and polarization‐sensitive anisotropy. At the low intensity regime (1.5 − 15 µW µm−2), photocurrent shows conventional sublinear power dependence. Upon increasing the power density (15 − 200 µW µm−2), the photocurrent becomes weakly superlinear. With illuminating power density higher than 200 µW µm−2, strong superlinear power dependence is found with a giant power exponent γ = 1.5 for the homogeneous 2D material. Different from the previous report where the capture cross‐section plays a major role in determining the weak superlinearity, here, the unusual strong superlinearity requires the presence of RC with distinct density of states. We present a two‐RC model to capture the main finding of the experiments which is further quantitatively proved by the multiparameter fitting. The fabricated Ta2NiS5 device is tested for taking photographs. The image contrast is clearly enhanced due to the superlinearity of the device. Our work sheds light on the superlinear photocurrent which allows enhanced optoelectronic performance of photoconductive devices at high illuminating power.In this work, we report the prominent and intrinsic superlinear power dependence of photocurrent based on homogeneous Taus report32 whe22NiS5 crystallizes in the orthorhombic system  as shown in Figureb‐axis. Each layer consists of the periodically arranged [TaS6]2 chains and NiS4 chains. The armchair structure runs along the a‐axis leading to the quasi‐one‐dimensional structure along with the resultant anisotropic electronic and optical characteristics. The high‐quality Ta2NiS5 crystals are prepared by chemical vapor transport method  plane. The extracted lattice constant b is 12.11 Å. The inset presents the full width at half‐maximum (FWHM) of 0.16°. The lattice properties and anisotropic characteristics can be further examined by Raman microscope. The randomly polarized Raman spectrum is shown in Figure −1 correspond to the 2Ag and 3Ag phonon modes, respectively. Angle‐resolved polarized Raman spectra are carried out in both parallel and perpendicular polarization configurations. Figure x, y, z coincides with the crystal direction a, b, c, respectively. The excitation beam propagates in y direction and the polarization is controlled by a half‐wave plate. More details are provided in the Experimental Section and Section g modes in Ta2NiS5 is given byTad Figure  with tems Figure  are founa, b, and c are the amplitude of Raman tensor elements. The φa, φb, and φc are the phases of the elements, and φca = φc − φa. θ denotes the angle between the polarization vector of incident light eI and the a‐axis of the crystal. The angle‐dependent phonon intensity can be well fitted by the Raman tensor as shown in Figure ei∥es), I∥(Ag) reaches the global maximum along the armchair direction and local maximum along the zigzag direction. Meanwhile, both Ag modes present fourfold symmetry in the perpendicular configuration (ei⊥es). The polarized Raman spectra agree with the theoretical prediction and help to identify the crystal direction. Based on our infrared spectroscopy measurement, a direct band gap of 273 meV is extracted for the as grown Ta2NiS5 which agrees with the general consensus of Ta2NiS5 being a narrow gap semiconductor. More details are given in Section The anisotropic Raman response can be quantitatively derived as2NiS5, the as‐grown single crystals are exfoliated by mechanical method, and device fabrications are performed by a home‐built lithography system with lift‐off procedures. Figure2NiS5 is transferred to the SiO2/Si substrate and contacted by electrodes (5 nm Cr/70 nm Au). The photocurrent is measured under the ambient condition with illumination of 632.8 nm laser. Due to the narrow gap nature of Ta2NiS5[36], the photoresponse is expected to be insensitive to the wavelength of visible lasers, but the laser beam with lower wavelength is found capable of damaging the sample at moderate intensity. Figure p = 0.324 mW µm−2 (defined as incident power per unit area). The edge of the spot size is defined by the position with 1.5 standard deviations. The photocurrent Iph is defined as Iph≡Iillumination−Idark, which describes the difference between current with and without laser illumination. The measured photocurrent presents symmetric and linear bias dependence and goes through the origin of the plot. The photocurrent is extracted as Iph = 5.35 µA under bias voltage of U = 1 V and incident power density of p = 0.324 mW µm−2. The photoresponsivity reaches a reasonable value of Rλ=IphP=2.5mAW−1 in small‐gap semiconductor. The Rλ does not reflect the intrinsic property of the device and material since it varies with the bias. A more proper physical parameter is the photoconductive responsivity Rσ extracted as 3.1 mS W−1per □. Figure  The observed bias dependence suggests the photoconductive origin rather than the photovoltaic mechanism of the measured device. Otherwise, the Schottky barrier or other built‐in potential results in the nonlinear response in both Idark − U and Iph − U test. Meanwhile, the negligible photocurrent at U = 0 V is also against the photovoltaic mechanism. Figure 2NiS5 flake. The photoconductive origin is further proved by spatial‐resolved experiments as shown in Figure U = 1 V and p = 0.324 mW µm−2. The FWHM of the laser spot is 1.44 µm Iph(0)=11+(2πωτ)2. The best fitting of the experimental results gives a fast photoresponse time of τ = 31.1 µs. The comparative fast photoresponse suggests finite influence from the dopants. Meanwhile, the Ta2NiS5 crystal is known to be anisotropic, and we studied the photoresponse by illuminating the device with linear‐polarized light. The angle‐dependent photocurrent is shown in Figure The optoelectronic property of the photoconductive device is further examined by switching, time‐dependent, and polarization‐dependent experiments. Figure n Figure 44Iph The bolometric mechanism behaves similarly in bias‐ and spatial‐resolved experiments, but the response speed of the Ta2NiS5 device is much faster than the general bolometric devices with a typical response time of 1 − 100 ms. In addition, the absorption rate of the Ta2NiS5 is found to be independent of the incident light power. Meanwhile, the conductivity of Ta2NiS5 increases linearly with temperature. These facts, combined with the observation of superlinear power dependence, further validate that the bolometric effect does not contribute to the observed photoresponse. More details are given in Section The photoconductive origin of the photocurrent is evident by the discussed photocurrent measurements with multiple tuning knobs. The photovoltaic and photothermoelectric mechanisms are firstly ruled out because of the linear bias‐dependent and spatial origin. The working frequency of our device also precludes the Dyakonov–Shur mechanism which is usually observed in THz regime.45 TheFigureIph − U curves under different incident light intensities. The incident laser spot is kept at the center of the sample. The higher incident power is expected to result in larger photocurrent due to the increased photogenerated electron–hole pairs in the Ta2NiS5. Lower intensity data is not shown because of the overlapping with other low intensity curves. We extract the photocurrent at U = 1 V, as exhibited in Figure Rσ is observed at high illumination power, indicating counterintuitive higher optoelectronic efficiency. To better resolve that, we plot the photocurrent in different incident power regimes and perform the power‐law fitting in Figure Iph∝pγ. With incident power density lower than 0.015 mW µm−2, a sublinear power dependence of the photocurrent is observed with γ = 0.53 ± 0.03. The error scale is given by the fitting error. By increasing the illuminating power, the power dependence of the photocurrent experiences a transition from sublinear to superlinear. Within the power regime of 0.015 − 0.2 mW µm−2, the photocurrent becomes weakly superlinear with γ = 1.15 ± 0.01. As light intensity further increases, strong superlinear dependence is found at the high incident power regime with the power exponent of γ = 1.5 ± 0.1. A similar trend can also be found in the linear fit of log–log plot  feature distinct parameters. Among them, the most critical two are the density of states (iN) and the capture cross‐sections for electrons (Sni). Sni describes the ability of RCi to capture the electron. Considering the described system at equilibrium, upon absorbing incident photons, electron–hole pairs are generated across the band gap (process A). The solid and hollow dots denote electrons and holes, respectively. Before those carriers are collected by the electrodes, there is a certain probability (mainly determined by Sni) for the photogenerated electrons to be captured by RC1 (process B) or RC2 (process C). Similarly, RC might also capture the photogenerated holes from valence band (process E and G). Meanwhile, it is possible for the captured electron on RC2 to be thermally emitted to the conduction band (process D) before recombined with holes, while the captured holes might experience the similar procedure (process F). All those procedures influence the carrier population of the states and in turn vary the probability of each procedure. The probability between different processes varies dramatically with different orders of magnitude. For example, the cross‐sections of process B, E, H (labeled in dashed line) are negligibly low due the large energy difference between initial and final states. All processes are considered in the model calculation despite its probability. It is worth to note that process A is the only one originating from the photoelectric transition. All remaining processes denote pure electric processes including thermal excitation, trapping, and nonradiative recombination. Other photoelectric transitions and occupation conditions are further discussed in Section n in the conduction band. Therefore, the photocurrent reads as Iph = nqµES, where q is electronic charge, µ is mobility, E is electric field, and S is cross‐sectional area of the channel. Without light illumination, the Fermi level in our model lies near the center of gap. This is further supported by the excitation energy extracted by the transport measurement  is much smaller than RC2 (Sn2), the recombination channel on the RC1 is effectively closed , −nvSn2(N2−n2), n2P2, and −S′v′np term corresponds to the procedure A, B, C, D, and H, respectively; n, n1, n2 represents the electron density of conduction band, RC1, and RC2, respectively; p represents the hole density of valence band; v denotes thermal velocity of the carriers which is assumed to be equal for simplicity; F denotes the density of electron–hole pairs created by optical excitation per second which is determined by light intensity, quantum efficiency, and absorption rate; Sn1, Sn2 denotes the electron capture cross‐section of RC1 and RC2, respectively; S′ denotes recombination cross‐section between free electrons and free holes; P2 denotes the probability per unit time for the thermal ejection of an electron in RC2 into the CB. For all other energy levels, similar equations can be derived by fully considering related carrier procedures which ultimately achieve equilibrium. The overall equations are provided in Section 2. For parameter settings with high N1N2 (green curve), the electron concentration is intensely saturated at high incident power. In contrast, such a saturation feature is weakened for lower values setting of N1N2. The difference in the electron density has a profound influence on the electron density of conduction band through process C and process D. This is further supported by the calculated photocurrent in Figure N1N2 setting, all curves exhibit a superlinear feature due to the negligible Sn1. However, a higher N1N2 leads to a more prominent superlinear power dependence of the photocurrent which agrees with the discussed picture.The 2NiS5 device which is well‐fitted by the model (red line). The critical fitting parameters are extracted as N1/N2 = 16.06, Sn1/Sn2=0.87×10−4  is deposited as electrodes and Ohmic contact has been proved by the I − U measurement.Multilayer TaPhotodetectors were excited by 632.8 nm laser through a 50×, NA = 0.8 objective. The FWHM of the focal spot was 1.44 µm. Bias‐, angle‐, spatial‐, time‐ and power‐dependent photocurrent were measured by Keithley 2450 using two‐terminal method. Spatial photocurrent scanning was carried out by an additional piezo‐actuated stage. The photoswitching test was performed with a periodically switched shutter. The modulation frequency‐dependent measurement was carried out by Stanford Research SR‐860 lock‐in amplifier with a chopper.The authors declare no conflict of interest.2NiS5 single crystals; J.M. and Y.D. performed the crystal characterization including XRD and infrared spectrum with help from W.W. and Z.S. under the supervision of C.Z., C.D., and Z.S.; X.D., J.W., B.L., and Y.M. conducted the device fabrication under the supervision of F.Y., N.Z., P.X., and C.D.; X.M. conducted the photocurrent experiments with help from Y.D. and Z.S.; N.B.J. performed the DFT calculation under the supervision of A.N.; X.Y., X.M., Y.D., C.Z., and J.C. wrote the paper with the help of all authors.X.M., Y.D., and W.W. contributed equally to this work. X.Y. conceived the idea and supervised the overall research. X.M. carried out the growth of the TaSupporting InformationClick here for additional data file.