By comparing the sequences of DARPin with that of a peptide known for its high affinity for c‐Myc, we show that the recognition cannot be ascribed to sequence similarity but, instead, depends on the complementarity between the three‐dimensional arrangement of the molecular fragments involved: the α‐helix/loops domain of DARPin and the G4 backbone. Our results reveal that DARPins tertiary structure presents a charged hollow region in which G4 can be hosted, thus the more complementary the structural shapes, the more stable the interaction.We investigated the mechanisms leading to the specific recognition of Guanine Guadruplex (G4) by DARPins peptides, which can lead to the design of G4 s specific sensors. To this end we carried out all‐atom molecular dynamic simulations to unravel the interactions between specific nucleic acids, including human‐telomeric (h‐telo), Bcl‐2, and  DARPins are proteins able to specifically recognize G‐quadruplexes. Their small size combined with their easy design makes them a good competitor to antibodies for the identification and localization of G‐quadruplex. Using molecular dynamics calculations, we show that the selectivity of DARPins towards G‐quadruplexes is achieved by the recognition of a structural motif adopted by the DNA filament folding. In addition to the well‐known double helical arrangement, the important biological role of non‐canonical nucleic acid is nowadays widely recognized. Among the different non‐canonical DNA or RNA structures, guanine quadruplexes (G4 s) are highly studied and characterized.2, 3 FG4 s have been identified in cellular nucleic acids and have been associated to the control of key biological functions. As a matter of fact, G4 s are involved in gene regulations,5, 6 ic‐Myc oncogene promoter, and 2G10, which has a slight specificity for different G4 s.c‐MycBcl‐2 promoters.Understanding the factors underlying the specific recognition of G4 s by DARPins can facilitate the design of sensors able to discriminate the G4 s subcellular localization and their specific sequences. In this contribution, we model the DARPin/G4 interaction and, thus, unravel the specific recognition modes by combining molecular docking and long‐scale all atom molecular dynamics (MD) simulations. We focus on 2E4 DARPin, which is specific for the G4 present in the MD simulations shed light on the structural details underlying the specific DARPins/G4 interaction. Indeed, by sampling the conformational space through different initial interaction positions, it is possible to analyze whether the interaction is conserved, the binding of the G4 affects the flexibility, the nucleic acid rearranges to reach a more stable pose, or if the proposed DARPin/DNA complex is not stable and separates. In our case most of the G4 s/DARPin complexes are persistent and stable all along the MD and the peptides interact with the G4 s through regions composed of large loops and helices, which overlap well with the recognized canonical interaction zones of the DARPins.c‐Myc which in one of the poses leads to a very labile and mobile interaction between G4 and the protein as confirmed by clustering yielding two dominant structures, representing 41.71 % and 30.56 % of the trajectory, respectively. On the other hand, 2E4/h‐Telo (64.33 % of the trajectory) and 2G10/c‐Myc (75.10 %) yield dominant clusters that interacts only through the loop ends of DARPins and one or two nucleotides of the flexible G4 s loops. (All the clustered structures can be found in the Supporting Information).The only exceptions can be highlighted for 2E4/h‐Telo and eight amino acids of 2G10 can be identified. However, 2G10 interacts persistently through only five amino acids with c‐Myc and Bcl‐2 which in turn only bring a maximum of two or three nucleotides into persistent contact with the protein. Conversely, the 2E4/h‐Telo interaction appears to be driven by three nucleotides and six amino acids. For 2E4, the interaction gathers eight amino acids with both c‐Myc and Bcl‐2, yet a different number of nucleic acid residues is involved, that is, four for Bcl‐2 and seven for c‐Myc.Before exposing the structural details of the G4 s/DARPin complexes at the atomistic scale, it is interesting to consider the interaction at a residue‐level scale, and in particular classify the different interaction patterns in terms of the number of involved nucleic acid or protein residues. Figure This first analysis, at the residue level, already draws a general picture of the specific recognition of G4 by DARPins, confirming that the protein/nucleic acid recognition is favored by a high number of interacting residues. However, it needs to be completed identifying the exact nature of the interacting residues, their specific frequency, and the specific structural features.h‐Telo does not show any specific interacting region or hotspot with 2E4 or 2G10  a rather general interaction that does not involve any specific G4 region or sequence (h‐telo); 2) a non‐specific interaction involving particular G4 s regions, which are however recognized by all the proteins (Bcl‐2); 3) a specific interaction driven by a few nucleotides having very high contact frequencies with specific DARPin (c‐Myc).To improve the global scale analysis presented in the previous subsection we should identify a region of the G4 s that is selectively recognized by DARPins. As a matter of fact, Repeating the same analysis while focusing on the protein counterpart we identify the amino acids mostly involved in the recognition of the non‐canonical DNA structure. Similarly, to what has already been observed for G4 s, selectivity should correlate with few specific amino acids having high contact frequencies with G4 s. Conversely, for non‐selective recognition a more scattered distribution of the interaction frequencies should be observed.h‐Telo is also driven by amino acids whose contact frequency was low or zero for the other G4S. This case concerns mainly residues H107, L108, I111, R112, K133, F134, K136, and I141. However, caution should be taken to avoid overinterpretation of this result, since 2G10 is not showing any specificity for h‐Telo.c‐Myc residues Y45, R70, L75, S78, R79, and G80 develop persistent contacts, and hence could be regarded as potential hot‐spots for the selectivity of 2E4 towards this G4.The distribution of the interaction contacts of 2G10 with the three G4 s , which is able to preferentially recognize G4 in the c‐Myc promoter. Since 2E4 is also specific to c‐Myc, it is legitimate to ask whether there is a sequence similarity between DM102 and 2E4. It is also important to note that the artificial peptide DM102 has a hydrocarbon staple  between residues R8 and S15, which enforces a defined α‐helix. Hence, in addition to sequence similarity, one must also look for stable α‐helical secondary structure, which is, indeed, a structural motif frequently present in DARPins. Two algorithms were used: Clustal Omega and M‐coffee . Here it is important to specify that Clustal Omega is an individual method of alignment, while M‐coffee is an algorithm that combines results from several individual methods.Several examples of peptides able to selectively bind G4 s are reported in the literature. Usually, they are derived from the DHX36 helicase whose α‐helix provides the binding interface with the nucleic acid.38, 39c‐Myc contact frequency for the residues belonging to this region. Furthermore, from a structural point of view, the R70‐R79 region is organized in α‐helix motif and is located towards the canonical recognition zone of DARPins. Yet, this region is highly conserved among the DARPins designed by Scholz et al.Interestingly, the two algorithms show divergent results. Clustal Omega previews similarity mainly concerning the 2E4 region spanning M46 to V62. The representation of the 3D structure of this region  reveals a helical arrangement, which could partially support the 2E4 selectivity conditions. However, this region is also common to all DARPins, except for residue 58 ,c‐Myc’s by 2E4 does not necessarily involve sequence similarity between DM102 and 2E4. This is also supported by the fact that the mutations of the wild type sequence as performed by Scholz et al.Thus, the search for a conserved sequence between DM102 and 2E4, does not unambiguously justify the selectivity of the DARPin. Going a step further, this could suggest that the recognition of From our MD simulations two most important factors should be considered when analyzing the local structural arrangements of the DARPin/G4 the contact region. First, the DARPin canonical interaction zone is not consistently interacting throughout the whole MD simulation. Instead, as highlighted in Figure h‐Telo, which in the course of the MD simulation departs from the initial docking pose and slides over the DARPin surface until an interaction between its quartet and the peripheral loops is established at around 150 ns Bcl‐2 (6ZX7).Sequence alignment. DM102 peptide and DARPins sequences were aligned using the Clustal Omega on EBI serverDocking and selection of initial structures: The reconstructed DARPins and G4 were loaded onto the HADDOCK serverMolecular dynamics simulations: MD simulations has been performed for 2E4 and 2G10 interacting with c‐Myc, Bcl‐2 and h‐Telo. Three poses for each complex have been used as starting conditions. Each system was calculated in two independent replicates of 1 μs each, thus a total of 36 simulations of DARPin/G4 complexes have been performed. In addition. simulations of the free G4 s and DARPin have also been obtained as a control. All simulations have been run using the NAMD software[+ ions was added to assure charge equilibration. Hydrogen Mass Repartitioning (HMR)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.


This study evaluated the validity of the AP3 × ML3 reduction quality classification, which applies the concept of positive medial cortical support.A total of 120 trochanteric fractures classified as AO Foundation/Orthopedic Trauma Association 31A1 and A2 were retrospectively analyzed. The validity of the AP3 × ML3 classification was evaluated by comparison with the Baumgaertner reduction quality criteria. When using the AP3 × ML3 classification, reduction quality was divided into three classes based on the degree of anterior cortical contact between the proximal and distal fragments. Reduction quality was also divided into three classes when using the Baumgaertner criteria. The frequency of mechanical complications, including cutout, delayed union, and excessive migration of the lag screw, was retrospectively assessed. Intra-observer and inter-observer reliability was assessed using the intraclass correlation coefficient (ICC).Mechanical complications included 4 cases of cutout (3.3%) and 1 of delayed union (0.8%). Mechanical complications occurred for all levels of reduction quality in both classifications, except for the acceptable of the Baumgaertner criteria. When reduction quality was rated as good, acceptable, and poor the incidence of mechanical complications was 2.5%, 5.7%, and 16%, respectively, under the AP3 × ML3 classification and 3.3%, 0%, and 15.0%, respectively, under the Baumgaertner criteria. The ICC was 0.80 for intra-observer reliability and 0.57 for inter-observer reliability when using the AP3 × ML3 classification and 0.85 and 0.34, respectively, when using the Baumgaertner criteria.The AP3 × ML3 classification was reliable and easy to use compared with the widely used Baumgaertner reduction quality criteria.Level of evidence 4 Femoral trochanteric fractures are an important issue for orthopedic surgeons and are increasing in frequency with population aging . OptimalThe aim of this study was to evaluate validity of AP3 × ML3 classification by examining the hypothesis that the AP3 × ML3 classification would be useful and reliable compared with the Baumgaertner reduction quality criteria.This retrospective study examined all 128 consecutive femoral trochanteric fractures with AO Foundation/Orthopedic Trauma Association (AO/OTA) type 31A1 or A2 femoral trochanteric fractures treated at two institutions between January 2017 and December 2020. Eight fractures that were followed up for less than 3 months after surgery were excluded. The remaining 120 trochanteric fractures (in 117 patients) were included : 5659]. Informed consent was obtained via an opt-out procedure.The AP3 × ML3 classification and Baumgaertner criteria were compared using lateral AP and ML radiographs obtained in the standard way  and retrWhen using the Baumgaertner criteria, reduction quality was classified as good, acceptable, or poor Table  as in prFractures were classified according to the AO/OTA system, and the position of the lag screw was classified as good or poor as in an earlier study . A good Complications occurring up to the final follow-up visit were assessed retrospectively by review of the medical records. Mechanical complications included cutout, delayed union, and excessive migration of the lag screw. Clinical complications included infection, avascular necrosis, and fatal venous thromboembolism.p value of 0.05 was considered statistically significant. Continuous variables are reported as the mean (range). Independent-samples t tests were performed for all continuous variables and the chi-squared test or Fisher’s exact test for all categorical variables. Stepwise logistic regression analysis was performed to detect factors associated with mechanical complications. The following categorical variables were investigated as predictors of outcome: male sex, poor reduction according to AP3 × ML3 classification, poor reduction according to Baumgaertner criteria, poor positioning of the lag screw, and AO/OTA type A2.All statistical analyses were performed using JMP software . A Intra-observer reliability was measured for both the AP3 × ML3 classification and Baumgaertner criteria on two occasions separated by an interval of 7 days for each in 36 randomly selected patients. Inter-observer reliability was evaluated for two observers , each of whom worked independently. As in a previous study , the int p = 0.39). The average time of follow-up was 13.2 months (range 3−49 months).Mechanical complications occurred in 4 cases of cutout 3.3%) and 1 of delayed union (0.8%) (Table .3% and 1Table p = 0.675).Mechanical complications occurred for all levels of reduction quality under each classification, except for the acceptable of the Baumgaertner criteria Fig. . The incLogistic regression analysis of factors potentially associated with mechanical complications identified poor positioning of the lag screw to be a poor prognostic factor Table .Table 5The most important finding of this study was that the AP3 × ML3 classification was useful, easy to use, and reliable compared with the Baumgaertner reduction quality criteria.Fukuda et al.  stressedThe disadvantage of the Baumgaertner criteria is that they require measurement of the neck–shaft angle and distance. There has been some concern about how accurate the values of 4 mm and 20° are when ethnic and/or sex differences are taken into account . The ChaCutout is one of the most commonly reported mechanical complications of surgery for proximal femoral fractures . Bojan eThis study has several limitations. First, it had a retrospective design, which introduces potential selection bias. In addition, the average time of follow-up was relatively short. It was possible that complications were underestimated. Second, several types of cephalomedullary nails were used. The Gamma3 and InterTAN have been widely used, and there are no reports of a statistically significant difference in clinical outcome between these two types of nails . SimilarThe AP3 × ML3 classification was useful, easy to use, and reliable compared with the Baumgaertner reduction quality criteria.


We consider the basal (0001) surface plane of nitride MXenes with Mn+1Nn  stoichiometry and also compare to equivalent results for extended (001) and (111) surfaces of the bulk rock‐salt transition metal nitride compounds. The present results show that the composition of MXenes has a marked influence on the CO2‐philicity of these substrates, whereas the thickness effect is, in general, small, but not negligible. The largest exothermic activation is predicted for Ti‐, Hf‐, and Zr‐derived MXenes, making them feasible substrates for CO2 trapping. From an applied point of view, Cr‐, Mo‐, and W‐derived MXenes are especially well suited for CCS as the interaction with CO2 is strong enough but molecular dissociation is not favored. Newly developed kinetic phase diagrams are introduced supporting that Cr‐, Mo‐, and W‐derived MXenes are appropriate CCS substrates as they are predicted to exhibit easy capture at mild conditions and easy release by heating below 500 K.The interaction of CO Kinetic phase diagrams are introduced to predict the conditions at which nitride MXenes  capture CO2. The factors affecting CO2 interactions are analyzed together with the implications for carbon capture and storage. To properly compare to the MXene (0001) surfaces, only the M‐terminated layer perpendicular to the [111] direction is considered. By moving from 2D to extended surfaces of bulk TMNs we will be able to firmly establish the influence of thickness on the adsorption and subsequent activation of CO2.Extended surface models are employed to represent the basal (0001) plane of bare nitride MXenes with formula M2, we rely on first‐principles periodic DFT based calculations. From the obtained results we analyze the potential of these MXenes in CCS strategies. The total energy of the explored Mn+1Nn (0001) surfaces −M=Ti, Zr, Hf, V, Nb, Ta, Cr, Mo, and W− with n=1–3, as well TMN (001) and (111) ones without or with CO2 has been optimized using the Perdew‐Burke‐Ernzerhof (PBE)Ab Initio Simulation Package (VASP) code.p(3×3) supercell is used for the TMN (111) surfaces, and a c(2√2×2√2)P45° for the TMN(001) ones, as previously used in the past,.with 2 partial pressure (pCO2)at which rads=rdes. This dynamic equilibrium situation allows us to derive what we refer to as kinetic phase diagrams, a newly developed tool introduced previously by some of us,Following this strategy, multiscale simulations are carried with the aim to shed light on the conditions at which nitride MXenes may be appropriate for CCS. These are established by analyzing the crossover between CCS and non‐CCS crossover that is defined by a temperature and a CO22.12 adsorption energy with respect to the composition and thickness of nitride MXenes, see Figure 2 molecule. The most favorable conformations for CO2 adsorption on the investigated nitride MXenes are displayed in Figure 2 adsorption on the M‐terminated TMN (111) surfaces whereas different conformations were found on the TMN (001) surfaces as depicted in Figure S1 in the Supporting Information (SI).We start this section by analyzing the variation of the CO2 adsorption energies on bare M2N MXenes with values ranging from −1.03 (Mo2N) to −3.13 eV (Ti2N).d2‐MXenes  have the strongest adsorption (ca. −3.0 eV) followed by d3‐V, Nb, and Ta (ca. −1.8 eV), and d4‐Cr, Mo, and W (ca. −1.4 eV). Here, the case of Cr2N is an outlier being its behavior like a d3‐metal. These previous results together to those obtained here for the thicker M3N2 and M4N3 MXenes indicate that nitride MXenes with M3N2 stoichiometry have similar or even larger CO2‐philicity than the M2N counterparts, see Figure d2‐MXenes with M3N2 stoichiometry feature CO2 adsorption energies above −3.5 eV. Interestingly, no significant changes are found when moving to M4N3 nitride MXenes. The above results also show that the effect of the MXene thickness on the CO2 adsorption nitride MXenes is significantly larger than in carbide MXenes.2‐philicity in the Nb‐ and Ta‐derived nitride MXenes, slightly in the Ti‐, Zr‐, Hf‐ and Mo‐derived nitride MXenes, and no effect for the V‐ and Cr‐ ones. The case of nitride W‐MXenes follows an opposite trend as its affinity for CO2 decreases by increasing the thickness as further discussed later. Additional interesting insights emerges when comparing to (001) and (111) TMN surfaces. As expected from the trends in surface energy, the TMN (001) surface is clearly less active than the (111) one with a calculated adsorption energy below ca. −2.2 eV which is reasonable but not as high as the one corresponding to the TMN (111), behaving effectively as MXene surfaces. This indicates that the chemistry of the hard‐to‐prepare TMN (111) can be more easily studied using the corresponding nitride MXenes. Before closing the discussion regarding adsorption energies, it is worth mentioning that the presence of the CO2 on the (001) and (111) CrN, MoN, and WN surfaces promotes superficial deformations inducing reconstruction which has been attributed to the fact that the rock‐salt polymorph of these nitrides is not the most stable one.Previous DFT calculations using PBE‐D3 approach have recently reported highly exothermic CO2 on the (0001) nitride MXene surfaces is accompanied by a considerable charge transfer from the substrate towards CO2 which we estimate by adding the net Bader charge in the atoms of the adsorbed molecule. To explore whether a large adsorption energy is coupled to a large charge transfer with a concomitant CO2 activation we computed the topological Bader charge, Q, of the CO2 molecule and plotted it versus Eads, see Figure 2 adsorption. More in detail, the four set of data show that in the majority of the analyzed substrates, the Eads and Q values concentrate in the −2.5 to −1.0 eV and −1.75 to −0.9 e range, respectively. The set of TMN (001) surfaces does not follow any clear trend except that low Eads values are coupled to small Q on the CO2 molecule. This is a clear indication of the relatively stability (low reactivity) of the TMN (001) surfaces. As expected, the charge transfer induces structural changes on the CO2 molecule promoting a bending of the O−C−O angle; the activated CO2δ− adsorbate having angles in the 111.7–135.6° interval, depending on the substrate. The deviation from linearity is connected to Q but without clear trends. All in all, the results discussed so far support the claim that bare Mn+1Nn systems are appropriate for CO2 abatement.The large adsorption energy of CO2 adsorption energy, Eads, of nitride and carbide MXenes and its role on the activation of CO2. We have shown in an earlier work that, in the case of carbide MXenes, the thickness has a small or negligible effect on their interaction with CO2.2 adsorption, we compare the CO2 adsorption energy for the two families of MXenes. To this end, in Figure Eads (Mn+1Nn) versus Eads (Mn+1Cn) with the data for the nitride MXenes obtained in the present work and that of MXene carbides taken from previous work.2N and M4N3 MXenes feature a lower CO2‐philicity than their carbide counterparts (M2C and M4C3). On the other hand, M3N2 MXenes feature a larger affinity for CO2 than their carbide counterpart (M3C2), although the reasons for these behaviors deserve a more detailed analysis which is out of the scope of the present work. Noting that the dispersion of the results, we observe that it is larger in Eads (Mn+1Nn) than in Eads (Mn+1Cn) and moving also from d2‐ to d4‐MXenes which indicates that the non‐metallic element in the MXenes plays a non‐negligible role in the chemistry of these materials. According to the present results, one can conclude that the adsorption and activation of CO2 could be modulated somehow playing with the nature (carbide or nitride) and the thickness of MXenes which opens a window to optimize pressure and temperature to control the adsorption/desorption process which is very relevant for the possible use of these materials in CCS technologies. This is investigated in detail in the next section.At this point, it is interesting to compare the CO2.2Here we describe the results obtained from TST derived adsorption/desorption rates and the resulting kinetic phase diagrams. These display the pressure/temperature regions where adsorption or desorption prevails with the dashed lines indicating the situations where the two rates coincide leading to a dynamic equilibrium situation. Note that these are in general different from the phase diagrams that may be obtained from atomistic thermodynamics2N, M3N2, and M4N3 stoichiometries. We focus mainly on nitride MXenes with the lowest  and largest  CO2 adsorption energy, whereas the rest of cases including the TMN surfaces, with situations between the extreme situations shown in Figure i) one dominated by adsorption and relevant to CCS (dark orange) located at low T, ii) a transition zone where adsorption and desorption compete (light orange), and finally iii) the desorption dominated zone (white), where CO2 does not adsorb at all (non‐CCS zone). To provide error bars to the predictions, the border between the transition zones corresponds to the least favorable Eads values as predicted by the PBE functional, whereas the border between the CCS transition and the non‐CCS zones corresponds the Eads values including dispersion through the PBE‐D3 functional. This defines the light orange region which varies with the composition. Those substrates with a large variety of active sites with similar energetics expose a broader region than those ones whose adsorption energies are similar on different active sites. It must be noted that these limits are conservative because they include all plausible adsorption sites that are placed somewhere between them.Figure 2 partial pressure, pCO2, and on the temperature. Thus, three actionable pCO2conditions are considered in the present study: i) atmospheric pressure where pCO2=40 Pa,ii) a benchmark value corresponding to postcombustion exhaust gases and, hence, pCO2=15 kPa,iii) pure CO2 stream generation from a CCS system with pCO2=100 kPa (1 bar).2 activation occurs at room temperature (ca. 300 K). These conditions are essential to achieve efficient CO2 capture from air. Indeed, this is the main idea behind the technologies based on the Direct Air Capture (DAC).2N, Cr3N2, and W4N3 MXene (0001) exposed surfaces exhibit the lowest CO2‐philicity. Although all partial pCO2at 300 K are located inside the CCS zone, CO2 could be easily released just by increasing slightly the temperature at constant pressure. This scenario is especially highlighted on the Cr‐derived MXenes. On the other hand, the Ti2N, Hf3N2, and Hf4N3 MXene (0001) surfaces featuring the largest Eads are appropriate exclusively for CO2 trapping, as conditions are well in the CCS region and the shift towards the non‐CCS regime maintaining the pCO2constant requires high temperatures that could compromise somehow the stability of these MXene compounds. Note that the latter MXenes are highly reactive, and their surfaces are passivized by CO2 even under mild conditions.The CCS capability of a given surface depends on the CO2N, Cr3N2, and W4N3 nitride MXene emerge as promising DAC filters where CO2 is chemisorbed but can be easily released. Thus, once the MXene filters become saturated, heating at temperatures close to 500 K release CO2 for subsequent use or storage and regenerates the filter. The thus obtained CO2 feedstock can be employed in a second step to synthesis of fuels and chemicals based on the heterogeneously catalyzed CO2 hydrogenation via the reverse water gas shift (RWGS) or Fischer‐Tropsch reactions.2 making feasible the carbon capture and usage.From the preceding discussion CrEads at low coverage renders CCS suitable while those with smaller Eads may end up in being less useful. Note, however, that that the experimental uptake in the work of Persson et al.2C MXenes made using a similar low coverage situationBefore ending this section, we must point out that the present results have been obtained for a low coverage situation and that lateral interactions are likely to decrease the adsorption energy so MXenes with larger 3n+1Nn  stoichiometries and the (001) and (111) TMN surfaces were investigated as plausible substrates for CO2 capture. By means of a combination of first‐principles calculations and macroscopic simulations, the role of the thickness and the composition are revealed. The effect of the thickness on CO2 adsorption and activation is larger than in the case of carbide MXenes. For a given TM, all nitride MXenes expose similar CO2‐philicity but with small, yet noticeable, differences. On the contrary, the effect of the TM is very large, as in carbide MXenes, with Ti, Hf, and Zr‐derived nitride MXenes featuring the most exothermic adsorption followed by V, Nb, and Ta‐derived nitride MXenes and, finally, Cr, Mo, and W‐derived ones. The trends here reported for nitride MXenes of different thickness are somehow different to those reported for carbide MXenes with a much more marked influence of the nitride MXene thickness in the calculated CO2 adsorption energy. The analysis of the results also shows that the MXene (0001) surfaces behave as the (111) surface of bulk TMNs. These trends in adsorption energy are consistent with a significant charge transfer from the MXene (or TMN) substrate towards the CO2 molecule, a clear indication that, once chemisorbed on the MXenes, the CO2 molecule is highly activated which call for additional work to analyze its possible conversion to other useful chemicals. By comparing MXene carbides and nitrides, it is concluded that M2N and M4N3 MXenes have, in general, less affinity for CO2 than their carbide counterparts (M2C and M4C3), whereas the family M3N2 exposes larger affinities than M3C2. The present results suggest that the CO2 adsorption on MXenes can be monitored by selecting the appropriate composition and thickness of the material.The (0001) nitride MXene surfaces with M2. These diagrams provide unbiased information that can be useful to experimentalists and engineers working on CCS. We conclude that ‐derived MXenes constitute the most suitable materials to act as collector of CO2 due to the feasible CO2 capture at mild conditions and the affordable release by heating below 500 K. MXene can act as CO2 feedstock to heterogenous catalytic processes as the CO2 hydrogenation to obtain valuable fuels and chemicals.Finally, newly developed kinetic phase diagrams have been introduced that allow one to identify the conditions at which the MXene substrates are able to capture and release COThe authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.Supporting InformationClick here for additional data file.


These promising results pave the way to the design of high‐performing materials based on this new thienoacene, notably through the introduction of alkyl side‐chains.The charge transport of crystalline organic semiconductors is limited by dynamic disorder that tends to localize charges. It is the main hurdle to overcome in order to significantly increase charge carrier mobility. An innovative design that combines a chemical structure based on sulfur‐rich thienoacene with a solid‐state herringbone (HB) packing is proposed and the synthesis, physicochemical characterization, and charge transport properties of two new thienoacenes bearing a central tetrathienyl core fused with two external naphthyl rings: naphthothieno‐thienothienonaphthothiophene (DN4T) and naphthothieno‐thienothienonaphthothiophene are presented. Both compounds crystallize with a HB pattern structure and present transfer integrals ranging from 33 to 99 meV (for the former) within the HB plane of charge transport. Molecular dynamics simulations point toward an efficient resilience of the transfer integrals to the intermolecular sliding motion commonly responsible for strong variations of the electronic coupling in the crystal. Best device performances are reached with DN4T with hole mobility up to  2 V−1s−1 in polycrystalline films.The synthesis, physicochemical characterization, and charge transport of two new herringbone packing thienoacenes bearing a central tetrathienyl core fused with two external naphthyl rings are presented. Naphthothieno‐thienothienonaphthothiophene (DN4T) qualifies as one of the best conjugated core for organic field effect transistor applications, thanks to its resilience to dynamic disorder, affording a hole mobility of 2.1 cm Currently, such figures of merit are considerably higher being on the order of μ = 20 cm2 V−1s−1 and Ion/Ioff ratio = 106–107. However, mobility levels off and no significant improvements have been seen recently, raising the question of the ultimate performances of organic semiconductors, at room temperature. Charge transport is hampered by dynamic disorder that tends to localize charges, as evidenced by μ > 100 cm2 V−1s−1 at low temperature or at short time‐ and length‐scales. These encouraging results demonstrate that mobilities as high as μ ≈ 100 cm2 V−1s−1 could be reachable at room temperature, if one can design organic semiconductors that are resilient to dynamic disorder. The latter takes roots in thermal agitation, which perturbs the equilibrium position of atoms and molecules, causing a fluctuation of transfer integrals and site energies. Ultimately, a charge localization over a characteristic length occurs, as described by the transient localization scenario. Each degree of freedom bearing an energy of ½ kBT, there is no way to prevent molecules to vibrate around their equilibrium position, but strategies exist to prevent positional disorder from turning into dynamic disorder. First, rigid structures reduce large amplitude motions. Second, a weak dependency of transfer integrals on positional disorder is desirable. Third, 2D‐isotropic transfer integrals increase the number of π‐systems on which charges are delocalized. Not all intermolecular phonons are equal, low‐frequency, and large amplitude modes contribute more to dynamic disorder. Therefore, the design of materials requires both molecular and supramolecular engineering taking into account many requirements. In terms of molecular structures, for instance, large thienoacenes packing in herringbone (HB) motive leads to the highest charge carrier mobility values.Organic semiconductors for logic operations have generated considerable interest over the last four decades. The first organic field effect transistor (OFET) has been reported by Tsumura, in 1986.1 PerfFigureμ = 0.15 cm2 V−1s−1, and no crystal structure was given. The shape of thienoacenes is a key design element because it affects translational motions of molecules in crystal structures. In particular, motions along the longest molecular axis are suspected of causing important variations of transfer integrals in the case of HB packing materials. N‐shape molecular semiconductors, which deviate from linearity, even slightly such as dinaphthothieno‐thiophene (DNTT) and bis[1]benzothieno‐naphthodithiophene (BBTNDT) decrease such detrimental motions. A slight deviation from planarity also seems to favor high charge carrier mobility as evidenced in 3,11‐dioctyldinaphthobenzodithiophene (C8‐DNBDT‐NW) and 3,11‐didecyldinaphthobenzodithiophene (C10‐DNBDT‐NW) which are currently among the very best performing molecular semiconductors, with μ = 13 and 16 cm2 V−1s−1, respectively. DN4T differs only slightly from DNBDT with the central six‐membered ring being replaced by two fused thienyl rings. Based on this structural analogy, DN4T is also anticipated to lead to high charge carrier mobility in OFETs. A last but important design element comes from the fact that DN4T, isoDN4T, and BBTNDT (μ = 5.1 cm2 V−1s−1) are isomers, counting as much as 34 π‐electrons and differing from the position of fused phenyl and thienyl cycles. IsoDN4T accentuates the N‐shape whereas BBTNDT has a similar shape to DN4T. The comparison of their electronic performances, along with the crystal structure of L‐DBTTA that we also solved for the sake of comparison, will help to establish firm structure‐property relationships to further guide the design of high performing thienoacene‐based organic semiconductors.In this paper, we contribute to the general understanding of the links between molecular structure, crystal packing, and charge transport with the synthesis and characterization of two new large thienoacene cores: naphthothieno‐thienothienonaphthothiophene (DN4T) and naphthothieno‐thienothienonaphthothiophene (isoDN4T), displayed in 22.1 for the tetrathienoacene core. This synthetic pathway consists in naphthalene functionalization with thiomethyl and halide groups in view of a cross‐coupling with 2,5‐difunctionnalized thienothiophene, followed by subsequent oxidation of the thiomethyl groups into sulfoxides and cyclization into dinaphthotetrathienoacene. The synthesis of DN4T, depicted in Scheme m‐CPBA in a quantitative yield followed by subsequent cyclization in triflic acid containing 0.5 equivalent of phosphorus pentoxide and demethylation of the resulting triflate salt in refluxed pyridine according to the procedure of Dong et al. to give DN4T with an overall yield of 32%. The synthesis of isoDN4T takes a somewhat similar pathway. Regioselective C‐H methylthionation at the α‐ position of commercial β‐naphthol 6 was performed according to the procedure of Xiao et al. in the presence of sodium methylsulfinate and diiodine to obtain 1‐methylthio‐2‐naphthol 7 by electrophilic substitution through probable in situ Me‐S‐I formation. The end of the synthesis follows the strategy commonly used by Takimiya et al. with triflation of β‐hydroxy group to give 8. Stille cross‐coupling with 2,5‐bis‐trimethylstannylthienothiphene was performed in the presence of CuI as a ligand scavenger for Pd(PPh3)4 and LiCl to favor triflate insertion on the catalyst to obtain 9 in good yields. Like in DN4T, 2‐steps cyclization of disulfoxide 10 in TfOH/P2O5 mixture followed by demethylation in refluxed pyridine gave isoDN4T with an overall yield of 44%. Thanks to optimized reactions conditions, DN4T and isoDN4T were obtained with 31% and 47% yield, respectively, and in a quantity sufficient for further studies.The target compounds DN4T and isoDN4T were synthesized according to the strategy introduced by Hang et al.14 for2.2 and isoDNTT. The higher solubility of isoDN4T enabled us to perform a TOCSY 1D 1H NMR directly on this compound to assign its groups of protons to the corresponding 1H NMR signals   in the asymmetric unit , supporting the abovementioned considerations. Finally, a look at the fingerprint plots and relative contributions to the HS of DN4T, isoDN4T, and their first published isomer BBTNDT reveals an almost identical packing environment for the three molecules, with interestingly less S···S contributions to the HS in BBTNDT despite strong differences in terms of short contacts. Comparison with L‐DBTTA confirms the importance of the π···π‐stacking and edge‐to‐edge interactions resulting in its stacked packing structure, depicted by the much higher H···H, C···C, and S···S contributions than CH···π with respect to DN4T, isoDN4T, and BBTNDT  with isoDN4T (HB), even though all these considerations are still not enough to explain the preferred crystal packing of DNBDT (slip‐stacked) bearing a central phenyl ring instead of the central thienothienyl of DN4T.These results suggest that a smart balance between thienyl and phenyl rings, and particularly between face‐to‐face and face‐to‐edge interactions has to be reached when designing OSCs in view of obtaining a HB packing.36 The When plotting the values of each unit cell parameter  for each temperature point  were collected at low temperature (DN4T at 173 K and isoDN4T at 100 K), X‐ray powder diffraction patterns were collected at different temperatures from 93 to 303 K for DN4T and isoDN4T, indexed and refined to map the thermal expansion and possible phase transitions between low and room temperatures of DN4T and isoDN4T.37 Whe2.4 and transfer integrals were calculated from the experimental SCXRD results at the B3LYP functional and DZ basis set. The bonding–antibonding patterns of the HOMO orbitals of DN4T and isoDN4T look very similar to the corresponding data in DNTT and isoDNTT  which is consistent with the fact that phenyl rings can reduce λ more than thienyl ones. Electronic couplings in the crystal structure are overall larger in DN4T compared to isoDN4T resulting from the abundance of short contacts involving sulfur atoms , after geometry optimization in gas phase at the B3LYP 6–311G* level,38 andTableFigure rubrene: 34–43%, and DNTT: 30–70%). In isoDN4T, they represent around 25% of the mean value for the couplings in the blue dimers and 34% of the mean value for the coupling in the red dimer. As expected, the two dimers forming a HB angle along a given diagonal behave much alike since there is an inversion center in the unit cell of isoDN4T.Molecular dynamics simulations revealed that the standard deviations of transfer integral variations in DN4T are in the 10–16 meV range Table1, whic The detailed procedure as well as approximations and parameters used for this simulation can be found in Section S5.5, Supporting Information. The maps of the mobility anisotropy within the HB plane of DN4T and isoDN4T at the dynamic limit (defined as the maximum mobility the charge can reach in this framework) and at the equilibrium structure are depicted in FigureIn a first attempt to assess how the different connectivity in DN4T versus isoDN4T affects charge transport, we have performed kinetic Monte Carlo (kMC) simulations of hole transport assuming a purely hopping regime in the dynamic limit, in the framework of the Marcus–Levich–Jortner formalism.45 The2 V−1s−1) than in isoDN4T (≈0.8 cm2 V−1s−1). The highest mobility for DN4T is obtained at 180° and 0°  which corresponds to the 60 meV electronic coupling  are associated with the direction along the 99 and 33 meV dimers , and the minimum, to the 17 meV, b axis . PYS provided average values for the IEs, with DN4T (5.27 ± 0.02 eV) and isoDN4T (5.23 ± 0.01 eV), lying between the IEs of flat‐lying and standing molecular orientations as extracted from the UPS experiments (see Table  and DNBDT (5.45 eV) likely due to a higher π‐extension but still higher than their closest isomer BBTNDT (5.15 eV).Ionization energy (IE) values of DN4T and isoDN4T were measured by photoelectron yield spectroscopy (PYS) on powder samples under ambient conditions and by ultraviolet photoelectron spectroscopy (UPS) as well, at 10ee Table 12 andTo investigate the electrical performances of the DN4T isomers, thin‐film transistors (TFTs) with bottom‐gate top‐contact (hereinafter shortened as TC) and top‐gate bottom‐contact (hereinafter shortened as BC) configurations were fabricated through thermal evaporation in high vacuum on highly doped silicon wafer substrates at different temperatures .TableFigureBest performing devices were obtained with TC architecture with mobility values consistent with the BC ones . The main performance parameters of the TC devices in the linear regime are reported in 2 V−1s−1 and on/off current ratios (Ion/Ioff) of ≈105–106 could be extracted for DN4T, which is in the same order of magnitude as the reported mobility of BBTNDT. IsoDN4T devices reached mobilities of 3.0–4.2 × 10−3 cm2 V−1s−1 and Ion/Ioff between ≈102 and 103 with surprisingly the same lowering by three orders of magnitude in charge carrier mobility as reported from DNTT to isoDNTT devices, along with similar mobility values, despite the different molecular packing of the latter. The highest values were achieved for films produced using a substrate temperature of 100 °C during the deposition for both DN4T and isoDN4T, due to larger grain size. All thin films also exhibited the same single‐crystal phase as the solved one and no thin‐film polymorphism was observed through XRD characterization .Charge carrier mobilities ranging from 0.9 to 2.1 cm ratios Ion/Ioff 3Table a fair comparison should thus be limited to their orders of magnitude.The synthesis of DN4T and isoDN4T is as simple as that of DNTT, DNBDT, and BBTNDT due to their similar chemical structures. Both DN4T and isoDN4T can be synthesized in a few steps and in good overall yields, which enables their preparation in ≈0.5 g‐scale. These compounds are thermally stable up to 400 °C and can be sublimed under vacuum. They crystallize easily on a substrate upon deposition from vapor phase and show no thin‐film polymorphism. For ease of comparison, most of the properties having an influence on the charge carrier mobility of the thienoacene semiconductors of this work—except the sensitivity to dynamic disorder that will be discussed later—are presented in 2 V−1s−1. Regarding this aspect, the crystal structure comparison of L‐DBTTA with DN4T and isoDN4T shows that the lack of face‐to‐edge interactions like CH···π (in DN4T and isoDN4T) with respect to π···π stacking and edge‐to‐edge interactions (in L‐DBTTA) tends to pack the molecules into columns, thus leading to a strongly 1D transfer integrals distribution, detrimental to charge transport (as confirmed by its rather poor performances). This is nevertheless compensated in DN4T and isoDN4T through the replacement of the external phenyl rings of L‐DBTTA by naphthyl moieties that enables a favorable HB packing while improving the homogeneity of the transfer integrals distribution. In other words, the use of a central tetrathienyl core can lead to effective electronic coupling in the crystal, provided a HB crystal packing with several fused phenyl rings bringing enough CH···π interactions, and a central straight linear shape keeping high electron density on the sulfur atoms and lower reorganization energy. Further comparison of BTBT with DNTT, DN4T, and BBTNDT from Table π‐conjugation extension and preference of “acene” structures to “phene” ones. In addition, a smart balance between thienyl and phenyl rings as well as a careful linear arrangement must be applied to decrease λ. Moreover, the IE of these materials clearly scales with their ability to stabilize charges through delocalization (at least for HB compounds), which is directly related to the availability of a large and balanced transfer integral distribution. As the access to a low injection barrier is a requisite to high mobility, it is of course logical to observe materials with the lowest IE values providing the highest charge carrier mobilities.First of all, the data presented in Table crease λ.41 Mor We thus computed the variation of the electronic coupling between the three inequivalent dimers in the HB plane of DN4T and BBTNDT as a function of the displacement along the long axis of the molecules . It is also worth mentioning that the experimental value obtained for DN4T and isoDN4T is in good agreement with theoretical calculations. While the modeling correctly reproduces the trend going from DN4T to isoDN4T, the mobility values measured in isoDN4T are two orders of magnitude smaller than the theoretical results, a discrepancy likely reflecting strong scattering at grain boundaries, larger contact resistance or dynamic disorder, or a combination of these effects .Best charge transport properties in polycrystalline thin‐films are achieved for materials exhibiting large and balanced transfer integrals , namely DNTT, DN4T, and BBTNDT. These materials exhibit charge carrier mobility values of the same order of magnitude with slight fluctuations resulting from the delicate balance of crucial parameters (grain size, balance, and value of 4π‐extension of the aromatic core, preferring “acene” structures to “phene” ones, will also provide lower reorganization energies for the system. Finally, a quick evaluation of the impact of the long axis sliding motion suggests that a proper engineering of the HOMO shape aiming for a homogeneous spread over the full molecule through “acene” and “phene” moieties tends to favor a better resilience to dynamic disorder. Considering this aspect and the achievement of mobility values of the same order of magnitude for DN4T and BBTNDT within a thin‐film configuration, we conclude that DN4T qualifies as one of the best conjugated cores for OFET applications. Another distinct feature of DN4T is the central fused tetrathienyl moiety that was already present in L‐DBTTA, affording similar HOMO shapes, for both aromatic cores. However, the former forms a favorable HB packing, whereas the latter adopts a stacked motive that leads to unbalanced transfer integrals. Central fused tetrathienyl moiety appears thus an interesting molecular design element, provided that HB packing is maintained. Furthermore, the evaluation of the influence of the sliding modes of DN4T and BBTNDT on their transfer integrals tends to show that the central tetrathienyl core of DN4T helps reducing the impact of dynamic disorder on charge transport. On‐going modification with alkyl‐side chains will allow to tailor crystal structures for improved charge transport properties.From this body of work, it sounds thus logical that to climb the mobility ladder, one has to design a material exhibiting a HB packing with large and balanced transfer integrals. This will also further allow the material to stabilize charges hrough delocalization and hence access lower IE values, mandatory for efficient charge injection at the electrodes. Linear The authors declare no conflict of interest.Supporting InformationClick here for additional data file.


The dimeric structure of [AriPr4PO2]2 was found to persist in solution through VT NMR spectroscopy and DOSY, supported by DFT calculations. The addition of DMAP to the 1,3‐dioxa‐2‐phospholane facilitates the loss of ethene to give AriPr4(DMAP)PO2 after days at room temperature, with this product also characterised by X‐ray crystallography and NMR spectroscopy. Replacement of the DMAP with pyridine induces ethene loss from the 1,3‐dioxa‐2‐phospholane to provide gram‐scale samples of [AriPr4PO2]2 in 75 % yield in 2 days at only 100 °C.Thermolysis of a 1,3‐dioxa‐2‐phospholane supported by the terphenyl ligand Ar Pyridines have been shown to mediate the release of ethene from a terphenyl supported 1,3‐dioxo‐2‐phospholane. This generates a dioxophosphorane dimer which is an oxygen analogue of Lawesson's and Woollins’ reagents. This compound was structurally characterised and its solution behaviour interrogated. Insight into the ethene release reaction is provided by the isolation of a DMAP‐coordinated dioxophosphorane These reagents have been intensively studied, and widely applied in the thiolation2 monomers which react with C=O bonds to form C=X and [ArP(O)X]n products, the latter containing strong P−O bonds which provide a reaction driving force. One such ArPX2 monomer  was reported by Appel and co‐workers in 1983.Chalcogens of phosphorus are widely utilised reactive molecules, finding applications in diverse fields such as desiccation,2‐O)2P(=O)Ar system has been structurally characterised. Instead, [ArPO2]3 trimers, as well as higher oligomers and polymers have been observed.2] fragment itself is highly reactive, likely a consequence of its unsaturated σ3λ5 structural arrangement and the limited steric demand provided by the lightest chalcogen, oxygen. In light of this potential, there have been forays into the generation of [RPO2] species. The heavy pnictogen analogues of nitromethane, MePO2,2,2 has begun to emerge as a generalisable route towards coordinated RPO2 fragments which are isolable under an inert atmosphere.In contrast to the heavier congeners, no ArP(=O)2 , has been reported to generate aryldioxophosphoranes via loss of ethene, although the harsh conditions involved often result in decomposition of the −PO2 moiety.5H4‐4‐N(CH3)2) and [PhPO2]3 to yield Ph(DMAP)PO2. This compound reacted with B(C6F5)3 to yield the “push‐pull” adduct Ph(DMAP)P(=O)O−B(C6F5)3.Alongside these oxidative methods, a number of other routes have been described to generate ArPOtert‐butylphenyl)‐2‐phospholane, the observed product was thought to be the result of activation of a flanking tert‐butyl group by the highly reactive dioxophosphorane.In the flash vacuum pyrolysis of 1,3‐dioxa‐2‐2, can induce the ejection of alkenes and generate LMgBO2(DMAP) }2]).2 moiety. We herein report the reaction of AriPr4P(OCH2)2 with either pyridine or DMAP affords the evolution of ethene at a reduced temperature relative to thermolysis and provides access to a light analogue of LR and WR, AriPr4P(=O)(μ2‐O)2P(=O)AriPr4.Over the last three decades, terphenyls have emerged as pre‐eminent ligands in the stabilisation of reactive main group centres as a consequence of their flanking steric bulk which serves to prevent access to the ligated main group atom without orienting groups directly within its coordination sphere. More recently, there has been an increasing awareness of the extent to which attractive dispersion forces between the ligands in terphenyl derivatives contributes to their structures and stability.iPr4P(OCH2)2, 1, via the reaction of ClP(OCH2)2 and AriPr4Li, the identity of which was confirmed by multinuclear NMR spectroscopy, mass spectrometry and single crystal X‐ray crystallography (See Supporting Information). The thermolysis of 1 was then investigated by differential scanning calorimetry. This analysis indicated an event consistent with melting at ca. 150 °C followed by a steady exothermic reaction which continues to ca. 270 °C containing a sharp endothermic kink. Notably, a second calorimetric run on this sample was bereft of any such features. We interpreted this as implication that an irreversible reaction had occurred, and rationalised the endothermic kink as a measurement artefact reflecting the loss of a gas .We began by synthesising Ar1 in an NMR tube at 150 °C hoping to identify the product of this reaction. After 36 h, the reaction mixture was dissolved in C6D6 and analysed by 1H and 31P NMR spectroscopy. The 1H NMR spectrum suggested the presence of one major new AriPr4 containing system as well as ethene, identified by a peak at 5.25 ppm  bridge of 1 were absent. The 31P NMR spectrum contained a major resonance at 17.5 ppm . Recrystallisation of the reaction mixture from C6D6 provided material suitable for single crystal X‐ray crystallography  Å) as well as a terminal P=O fragment on each phosphorus atom  Å) which are trans‐disposed across the P−O−P−O ring. The phosphorus chalcogen bonds are, unsurprisingly, shorter than those found in an unsubstituted analogue of LR2; S, 86.58(5);2]3, the P−O bond lengths were similar however, the P−O−P angles were significantly narrower, ([MesPO2]3 P−O−P=101.2° on average).Compound 2 Figure  containi2. Its 31P NMR spectrum, taken in d8‐toluene, was unchanged with varied temperature between 298 and 358 K . The 1H NMR DOSY of 2 provided a hydrodynamic radius of 6.14 Å which more closely correlated with the calculated radius of [AriPr4PO2]2 . These data were interpreted to indicate that 2 persisted as a dimer in solution .We then undertook studies to confirm the solution speciation of iPr4PO2 present in solution via the addition of Lewis acidic and basic reagents, as has been shown for Woollins’ reagent.6H6)/BS2//BP86/BS1, see Figure −1 higher in free energy than the dimer suggesting a very low concentration of this species, if any, was likely. Addition of an equivalent of B(C6F5)3 to a C6D6 solution of 2 provided no evidence of reaction, even with extended time and elevated temperature. In contrast, addition of an equimolar amount of DMAP to 2 provided two resonances in the 31P NMR spectrum; that of pure 2 and a resonance at 6.8 ppm PO2 (Compound 3). We interpret these data to suggest that rather than trapping free AriPr4PO2 that might be present in solution, the DMAP is capable of cracking the dimer in compound 2, as we would expect any free AriPr4PO2 to react readily with both B(C6F5)3 and DMAP. Furthermore, no reaction was observed between 2 and a diene,2 is unlikely and that, essentially, no free AriPr4PO2 exists in solutions thereof.Attempts were then made to trap any Ar2 and DMAP contained evidence of compound 2, free DMAP and compound 3 suggesting the former two compounds are in equilibrium with the latter. Nevertheless, repeated attempts to force the equilibrium completely towards compound 3 via heating were unsuccessful, yielding only mixtures of 2 and 3 according to the 31P NMR spectrum . In order to isolate an analytically pure sample of 3, we investigated the in situ generation of 2 in the presence of DMAP. Addition of DMAP to 1 showed signs of ethene release even at room temperature on an NMR scale PO2 by Manners and co‐workers.3, 1.4738(8) and 1.4781(8); Ph(DMAP)PO2, 1.4752(17) and 1.4742(14) Å) indicating a degree of multiple bonding and the geometrical parameters4: 3, 0.90; Ph(DMAP)PO2, 0.88) reflecting the similar, distorted tetrahedral configurations. Activation of the WR dimer by pyridine has also been reported to yield Ph(C5H5N)PSe2On a gram‐scale, an equimolar mixture of 2 and 3 . The ATR FTIR spectrum of 2 contains an absorption at 1182 cm−1 which was tentatively attributed to a P=O stretch, as well as a stretch at 799 cm−1 which was assigned as associated with the P−O−P−O ring based on its absence in the spectrum of 3. This can be compared to similar modes reported for [PhP(O)O]3 by Manners and co‐workers, which occurred at 974 cm−1.3 contained a number of stretches present for 2 implying some dissociation of the DMAP in the solid state. It also contained a new absorbance at 1025 cm−1 which, if reflecting a P=O stretching mode, would imply marginally weaker P=O bonding in 3 compared to 2, corresponding to the crystallographically defined P=O bond parameters.Infrared spectroscopy was applied to compounds 3 showed some evidence of DMAP decomplexation by NMR spectroscopy, through the reappearance of resonances associated with both 2 and free DMAP . We hoped that the replacement of DMAP by a less nucleophilic analogue, pyridine, might allow this transformation to occur in a more efficacious time frame but provide material which could be readily converted to 2. Hence, we were thus extremely gratified to find that reaction of 1 with an equivalent of pyridine in C6D6 provided a good yield of analytically pure 2 in 4 days at 100 °C . This reaction was scaled up to 1 g in toluene, providing an isolated yield of 75 %, with a reduction of reaction time to 2 days.Heating of 2 showed no propensity to react with boranes, we expected the terminal P−O moieties in 3 to readily undertake this reaction. Addition of one equivalent of B(C6F5)3 to a suspension of 2 in C6D6 gave rise to a number of resonances in the 1H, 19F and 31P NMR spectra . Full attribution of these peaks was not possible. These results are a contrast to Ph(DMAP)PO2 which cleanly formed an adduct, Ph(DMAP)P(=O)O−B(C6F5)3, with B(C6F5)3 and most likely reflect the extreme steric crowding in 3. As noted, 3 is in equilibrium with 2 and DMAP, and it is likely that the borane reacts not only with the P−O bonds but as a sequestering agent for the DMAP driving the equilibrium towards 2 and resulting in mixtures of compounds.Whilst 2] moieties (μ2‐O)2P(=O)AriPr4 (2), as well as its DMAP adduct, AriPr4(DMAP)PO2 (3). NMR spectroscopy supported by density functional theory calculations has provided evidence of the persistence of the dimeric nature of 2 in solution. We believe this new approach to generate dioxophosphoranes will enhance the range of such species accessible under mild conditions and lead, eventually, to the synthesis of an isolable monomeric RPO2 species as was established for a dithioxophosphorane almost forty years ago.In conclusion, we have developed a new method for the low‐temperature synthesis of RPO moieties1), 2149562 (for 2), 2149563 (for 3)2149561 (for  contain(s) the supplementary crystallographic data for this paper. These data are provided free of charge by the joint Cambridge Crystallographic Data Centre and Fachinformationszentrum Karlsruhe Access Structures service.Deposition Numbers The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.Supporting InformationClick here for additional data file.


C59A variant exhibited an increased production of cattleyene and other diterpenes with diverse skeletons. Its structure showed a widened active site cavity explaining the relaxed selectivity. Isotopic labeling experiments revealed a remarkable cyclization mechanism involving several skeletal rearrangements for one of the novel diterpenes.The crystal structures of cattleyene synthase (apo‐CyS), and CyS complexed with geranylgeranyl pyrophosphate (GGPP) were solved. The CyS C59A mutant generated diterpenes with novel carbon skeletons. The remarkable cyclization mechanism of one of these products was characterized by isotopic labeling experiments.The crystal structure of the diterpene synthase CyS was solved, providing the structural basis for cattleyene biosynthesis. A CyS Modellings in conjunction with site‐directed mutagenesis and additional labeling experiments are discussed that provide a deeper understanding of cattleyene production by CyS.We recently discovered the cattleyene  synthase2+ were obtained by soaking and the structure was solved at 1.87 Å using the apo‐CyS structure as template. CyS‐GGPP‐Mg2+ is the first structure of a terpene synthase (TS) in complex with the native substrate GGPP, providing an ideal opportunity for analyzing GGPP binding and interactions in the active site.High‐quality crystals of purified CyS  were obtained and the structure of apo‐CyS was solved at 2.00 Å, using the structure of SvS89DVHCD94) is located on helix C and the NSE triad (D232DLFS236YGKE240) on helix H. The CyS‐GGPP‐Mg2+ structure shows a similar fold to apo‐CyS  and two β strands (β1 and β2) close to the active site become ordered .Apo‐CyS adopts the classical α‐helical fold of class I TSs, with ten core (A‐J) and three short α helices , four aliphatic  and three polar residues . The intermediates A–G are stabilized by cation‐π interactions and van der Waals forces with aromatic residues , in an equivalent position to the effector G182 in selinadiene synthase.1  to produce isopentenyl (IPP) and dimethylallyl pyrophosphate (DMAPP) was used . The F62A and W318A enzyme variants showed a substantially decreased or completely abolished production of 1, confirming the important roles of F62 and W318 in the biosynthesis of 1. The W81A variant only produced minor amounts of 1. Considering the interaction of W81 with GGPP, this residue may be required to keep the substrate and the intermediates in suitable conformations.The above modellings show how CyS catalyzes the biosynthesis of s Figure . For thi2, while the exchanges of F86A and W160A resulted in decreased levels of 1 with simultaneous production of several new compounds (3–6). Diterpene 3 was only observed in trace amounts, and yields of 4, 5 and 6 were about 30 %, 20 % and 20 % of that of 1, with an overall higher production by the W160A in comparison to the F86A variant. Gratifyingly, the C59A variant gave a similar product profile, but with a much better production (about 6‐fold in comparison to the F86A variant except for 3). Exchanges of other active site residues  led to no significant product change compared to wild‐type CyS .The variations of A190G and A191G caused the formation of an additional product 2–6 were isolated from large scale fermentations of engineered E. coli expressing the CyS A190G or C59A variety, and their structures were elucidated by NMR spectroscopy. All five diterpenes exhibit different carbon skeletons  and 5 originate from alternative deprotonations of C , its crystal structure was solved at 2.30 Å. The structure is highly similar to the apo‐CyS and CyS‐GGPP‐Mg2+ structures  and renders it towards the active site, while in CySC59A this interaction is disrupted. As a result, F86 moves a bit away from the active site and the phenyl ring rotates 24° clockwise GGPP, prepared enzymatically from 13C‐labeled FPP, GPP or IPP precursors (Table S8), were enzymatically converted with CySC59A, followed by extraction with C6D6 and analysis of the product mixture through 13C NMR . All 20 experiments resulted in the detection of the labeled carbons of the six products, with one signal matching the NMR data of 6 in each experiment. The results revealed a remarkable mechanism for its formation with the first steps towards C being the same as for the other products, but then branching out through I to O with involvement of multiple ring closures, 1,2‐hydride shifts, and skeletal rearrangements FPPC59A, resulting in a slightly upfield shifted triplet for C‐7 of 6  due to a direct 13C‐2H bond in the product. The 1,2‐hydride shifts from K to L and from N to O represent a forward and backward movement of the same hydrogen. Consequently, when using GGPPC59A the deuterium atom will end up in its starting position, in agreement with the observed minor upfield shift for the signal of C‐3 of 6, indicating a deuterium atom in a neighbouring position . The stereoselectivity of the final deprotonation was investigated by conversion of DMAPP and (E)‐ or (Z)‐IPPC59A, showing loss of deuterium from (E)‐IPP and retainment from (Z)‐IPP, i.e. loss of the α‐oriented proton in O .Compound 2+, representing the first example of a TS in complex with its native substrate, were solved. Intermediate modelling and site‐directed mutagenesis gave detailed insights into the biosynthesis of 1. Based on the structure, several CyS variants were designed, and especially CySC59A showed a strongly altered product profile with formation of several new compounds. The C59A exchange widens the active site cavity, resulting in a relaxed product selectivity, with a series of unprecedented ring closures and skeletal rearrangements towards 6. Previous site‐directed mutageneses revealed critical TS residues for functionality, while exchanges of other residues lead to changed product profiles.In summary, the structures of CyS and CyS‐GGPP‐MgThe authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.Supporting InformationClick here for additional data file.


Identify protein contact points between TP53 and minichromosome maintenance (MCM) complex proteins 2, 3, and 5 with high resolution allowing for potential novel Cancer drug design.A next‐generation sequencing‐based protein–protein interaction method developed in our laboratory called AVA‐Seq was applied to a gold‐standard human protein interaction set. Proteins including TP53, MCM2, MCM3, MCM5, HSP90AA1, PCNA, NOD1, and others were sheared and ligated into the AVA‐Seq system. Protein–protein interactions were then identified in both mild and stringent selective conditions.Known interactions among MCM2, MCM3, and MCM5 were identified with the AVA‐Seq system. The interacting regions detected between these three proteins overlap with the structural data of the MCM complex, and novel domains were identified with high resolution determined by multiple overlapping fragments. Fragments of wild type TP53 were shown to interact with MCM2, MCM3, and MCM5, and details on the location of the interactions were provided. Finally, a mini‐network of known and novel cancer protein interactions was provided, which could have implications for fundamental changes in multiple cancers.We provide a high‐resolution mini‐interactome that could direct novel drug targets and implicate possible effects of specific cancer mutations. Using a next‐generation sequencing‐based protein‐protein interaction method, we identified interactions among MCM2, MCM3, and MCM5. While many of the interacting regions we detected overlap with existing structural data of the complex, we also recovered novel domains with higher resolution. Additionally, we were also able to identify, for the first time, that wildtype TP53 interacts with MCM2, MCM3, and MCM5 and provide the location of the interactions. The novelty in this approach affords protein interactions to be mapped with a higher resolution in a high‐throughput manner; millions of protein fragment pairs can be screened simultaneously. The system also can titrate the interaction strength via the competitive inhibitor (3‐AT) concentration in the media. By increasing the 3‐AT concentration, the user can select for stronger protein–protein interactions. Here, the use of 2 and 5 mM 3‐AT concentrations allowed us to detect a range of interaction strengths.The all‐vs‐all sequencing (AVA‐Seq) method has been detailed previously.2.32 fold change of the normalized read counts of greater than or equal to 1 and a False Discovery Rate of <10%.Data were collected from an Illumina NGS platform using standard library and sequencing chemistry methods for paired 150 bp read lengths per the manufacturers' recommended protocol. Fastq files of paired‐end sequences were aligned to a sequence database of the human protein interaction reference set and read counts for each fragment pair were normalized and scaled using methods as described previously.2.4UniProt.org.Using recent structural data (PDB 6xtx), protein contact points between MCM proteins were identified using 5 Å distance33.1The AVA‐Seq method recovered 10 interacting fragment pairs between MCM2 and MCM5 Figure .46 TheThe average fragment length tested in this study is 150 residues3.2The MCM2|MCM3 interaction Figure  shows mo3.3MCM5|MCM3 is a physiological interaction referenced in the literature and detected with our system. The interacting fragments between MCM5 and MCM3 and the enrichment in protein contact points were identified from the structure (PDB 6xtx) Figure . The fra3.4All five MCM2|TP53 interacting fragment pairs were recovered in the mild selective pressure conditions Table .Interacting fragment pairs for TP53|MCM3 are predominantly localized to the C‐terminal disordered region of MCM3 Figure  as seen TP53|MCM5 interacting fragment pairs recovered with this study are shown in Figure Table 3.5Figure 3.6Interactions such as ORC2|MCM2,4This study provided an opportunity to identify physiologically relevant interacting fragments using the AVA‐Seq method. This was done by overlapping interacting fragment pairs between MCM2, MCM3, and MCM5 with cryo‐EM protein contact points Figures , 2, 3. HMCM2 in Figure Interacting fragments near the C‐terminus of MCM3 are enriched in the more robust selective conditions (green fragment pairs) in both the MCM2|MCM3 interaction Figure .Not surprisingly, interacting fragment pairs appear to localize differently when comparing interactions recovered exclusively in the 2 mM 3‐AT condition (gray colored fragments) and fragments recovered in both 2 and 5 mM or exclusively in 5 mM conditions (green colored fragments) in Figures d = ~1–150 μM) but show high specificity.The C‐terminal region of TP53 has at least 40 different binding partners and utilizes the intrinsically “disordered region for mediating and modulating” PPIs.With respect to transient interactions––work by others showed strong interactions between certain MCM proteins and GOF TP53 mutants but failed to detect an interaction with wild type until in vivo methods capable of detecting transient interactions were used. We have recovered many interacting fragment pairs between the MCM2, MCM3, and MCM5 proteins with wild type TP53. Interestingly, there were no interacting fragment pairs between MCM2|TP53 in the 5 mM 3‐AT conditions. This could indicate a more transient interaction between MCM2|TP53 when compared to the TP53 interactions with MCM3 or MCM5. It would be of interest to further investigate the preferences of TP53 binding with regards to the MCM2‐7 proteins especially given their similar domain structures.We observe interactions of the MCM proteins with TP53 localizing outside of the MCM box domains in 5 mM 3‐AT, while the fragment pairs recovered in only 2 mM seem to have a preference to contain the MCM box domains Figure . This isThere are multiple lines of evidence including in vivo and in vitro experiments that both mutant TP53 and wild type TP53 interact with the MCM proteins Table . AdditioA double positive association exists between mutant TP53 R175H and all MCM2‐7 complex proteins in both the cytosol and nucleus as determined via proteomic enrichment analysis.It is worth noting that the TP53 construct from this study contains two mutations (P72R and P278A). The P72R is a common mutation in 40% of many populations and is associated with some diseases but is predicted to be benign.One explanation for the AVA‐Seq method being able to identify these interactions could be the increased sensitivity of the assay. By this, we mean if the binding affinity of wild type TP53 to MCM proteins is higher (weaker or more transient interaction) than the gain‐of‐function mutation R273H with a given substrate, there would be an increased challenge in detecting interactions. The power of AVA‐Seq is it allows us to look beyond the expected interactions to determine other exciting and potentially novel biologically relevant interactions that warrant further evaluation. It would be worth exploring the range of binding affinities detectable by AVA‐Seq by using proteins with known binding affinity and making a “standard curve” by increasing competitive inhibition ranging from 0 to 5 mM 3‐AT, for example.In the future, we plan to screen wild type TP53 and a library TP53 mutants against the human genome, allowing researchers to select potentially subtle differences in protein–protein interactions to examine further. TP53 gain‐of‐function mutations can inherently change the pool of interacting proteins, resulting in different biological effects.In conclusion, we overlayed protein fragment interaction pairs with known protein contact points from a recent cryo‐EM structure of the MCM complex allowing us to obtain a high resolution view of the interacting domains. We show the intrinsically disordered wild type TP53 interacts with MCM2, MCM3, and MCM5. We postulate these interactions may not have been identified previously due to the weaker binding of a wild type TP53 compared to a gain‐of‐function TP53 mutant. Finally, we generated a mini‐cancer interactome highlighting known and novel protein interactions between TP53, MCM2, MCM3, MCM5, NOD1, SMAD1, SMAD4, ORC2, ORC4, and HSP90AA1. We believe these will provide an excellent starting point for potential novel drug design.The authors declare that there are no conflict or competing interests.S.S.‐R. conceived the idea and designed the study; S.S.‐R. and N.M.A. collected the data; S.S.‐R., J.A., and J.A.M. analyzed the data; S.S.‐R. and J.A.M. wrote the manuscript.This research was supported by funding from Qatar Foundation to Weill Cornell Medicine in Qatar in the form of the BMRP2 grant.All analyses were based on previous studies; thus, no ethical approval and patient consent are required.Table S1–S5Click here for additional data file.


MUC5AC) and mucin‐2 (MUC2) in CRC cells by NOTCH3 and SMARCA4. Furthermore, NOTCH3 was found to control the expressions of MUC5AC and MUC2 in a SMARCA4‐dependent manner. MUC5AC and MUC2, which encode two secreted mucins, are located on chromosome 11p15.5, and are linked to the development of MCA. This finding suggests that the interaction between NOTCH3 and SMARCA4 may be involved in MCA differentiation by jointly targeting MUC5AC and MUC2. Patients with MCA are often treated in accordance with CRC guidelines. Determining the relationship between NOTCH3 and SMARCA4 by demonstrating their interactions in the pathophysiology of MCA could provide novel therapeutic targets and help identify potential prognostic markers for MCA.Adenocarcinoma is the most prevalent histological subtype of colorectal cancer (CRC), with mucinous colorectal adenocarcinoma (MCA) being a unique form. Although the mucinous subtype is known to elicit a worse response to chemotherapy and immunotherapy than the nonmucinous subtype, its pathogenesis remains poorly understood. Neurogenic locus notch homolog protein 3 (NOTCH3), a member of the NOTCH subfamilies, is highly expressed in CRC. In the past three decades, many studies have been performed evaluating the biological role of NOTCH3 in CRC. However, the precise activities of NOTCH3 in MCA, as well as the mechanisms involved in its transcriptional control, are yet to be elucidated. Our finding showed that the critical transcriptional regulatory factor transcription activator BRG1 (SMARCA4) directly binds to the intracellular domain of NOTCH3 to control transcriptional regulation. Moreover, RNA‐sequencing results indicated a common targeting effect on the transcriptional activity of mucin‐5AC ( MUC5AC and MUC2 by recruiting transcription activator BRG1 (SMARCA4) to regulate the growth, invasion, and migration of CRC cells, which involving in the differentiation and development of mucinous colorectal adenocarcinoma (MCA), indicating a novel molecular mechanism of occurrence of MCA mediating by NOTCH3.Neurogenic locus notch homolog protein 3 (NOTCH3) is highly expressed in colorectal cancer (CRC). NOTCH3 may target  In addition to the aging population and the deterioration of people's dietary habits, obesity, smoking, and lack of physical exercise exacerbate the risk for CRC . CRC patIn recent decades, CRC treatment has become more individualized owing to the advancement in genomics and molecular pathology of cancer biomarkers, which warrants the refinement of the cancer subtype classification according to its histological and genetic characteristics for strategizing an effective treatment plan. Adenocarcinoma is the most prevalent histological subtype of CRC, with mucinous adenocarcinoma being a unique subtype defined by a significant number of mucinous components accounting for more than 50% of the tumor size . StatistThe excessive activation of the NOTCH pathway is involved in the high occurrence and development of CRC. The pathway is implicated in the regulation of cell‐to‐cell communication, and the dynamic control of  eukaryotic proliferation, differentiation, and apoptosis of the cell in various tissues, especially in CRC tissues and cells , 14, 15.Previous research has confirmed that the activation of the NOTCH pathway necessitates the involvement of many transcription‐regulating factors . MoreoveMUC5AC) and mucin 2 (MUC2) in CRC cells. The secretory mucin coding genes MUC5AC and MUC2 have been linked to the presence of MCA on chromosome 11p15.5   and His‐tagged human NOTCH3 intracellular domain (aa 1665–2321)  were mixed and incubated on an ice bath for 3 h. Subsequently, the mixture was incubated with Streptavidin Magnetic Beads . After washing five times with the washing buffer, the proteins were eluted with the wash buffer supplemented with 15 mm of reduced glutathione. The elutes were separated using 12% SDS/PAGE and transferred onto the PVDF membranes , and then finally probed with anti‐GST (Proteintech) and anti‐His (Proteintech) antibodies. GST from Wuhan Genecreate  was used as the negative control.Two purified recombinant human glutathione 2.112 incubator for the indicated times . After adding 10 μL of CCK‐8 solution to each well, the cells were incubated in an incubator for 30 min. The medium without cells and the CCK‐8 solution group were used as blank controls. Finally, the absorbance at 450 nm was measured with a microplate reader .The following experiments were carried out according to the instructions of the CCK‐8 cell proliferation and cytotoxicity detection kit . The cells were inoculated into 96‐well plates. Different groups of cells were pretreated according to the experimental conditions. Cells were incubated in a 37 °C, 5% CO2.12The colon cancer cells were transfected in groups for 24 h, and the clone formation experiment was performed. For this purpose, 2000 cells were inoculated into each well of a six‐well plate, with three in each group, and the liquid was changed every 2–3 days. Two weeks later, clone formation was calculated. The supernatant was discarded, and the cells were washed twice with 1 × PBS for 10 min and then fixed with paraformaldehyde. Then, the fixed solution was discarded and dyed for 10 min with an appropriate amount of crystal violet, followed by 1 × PBS cleaning, counting, and taking pictures. Clone formation in this assay is calculated as follows:Clone Formation Rate (%)=the number of clones/the number of inoculated cells)×100%2.13−1 with a serum‐free medium, followed by pre‐cooling at 4 °C on an ice bath. At the center of the bottom of the chamber, 100 μL of diluted Matrigel was added vertically and then incubated at 37 °C for 4–5 h to make it dry and gelatinous. The CRC cells (1 × 104 cells/well) were treated under different conditions in the upper chamber, and the cell culture medium was added to the lower chamber, followed by incubation with 5% CO2 at 37 °C and under 95% humidity for 24 h, stained with crystal violet, and finally fixed. The cells from randomly selected fields were counted under the microscope. The procedure for the invasion and migration assay was repeated thrice.After 36–48 h of transfection, Transwell analysis was performed in a modified Boyden chamber equipped with 8‐μm pore filters. The invasion assay was coated with Matrigel and then thawed overnight at 4 °C and diluted to obtain a final concentration of 1 mg·mL2.145 cells/well) in triplicate. A few cells were scratched from the center of each well with a small sterile pipette tip. The collected cells were gently washed with 1 × PBS thrice, the scribed cells were removed, and a fresh culture medium was added. The cells were incubated at 37 °C under a 5% CO2 humidified chamber for 48 h. The wound areas were visualized under a microscope  at different time points . Wound closure was then calculated using the following formula: Wound width (%) =  × 100%. The procedure for the wound healing assay was repeated thrice.The cells from different treatment groups were inoculated at an equal density into six‐well plates (5 × 102.155 cells/well). Different groups of cells were pretreated according to experimental conditions. After discarding the medium, trypsinize the cells with EDTA‐free trypsin, add cell culture medium to terminate the digestion, and collect the digested cells into a centrifuge tube. The cells were centrifuged at 106 g for 5 min, and the supernatant was discarded. Add 1 mL of 4 °C pre‐chilled 1 × PBS to resuspend the cells, and repeat the centrifugation step once. Dilute the Binding Buffer with deionized water at a 1 : 9 dilution ratio. Resuspend cells in 1 × binding buffer to maintain a cell concentration of 1–5 × 106 cells·mL−1. Then take 100 μL of cell suspension and 5 μL Annexin V/FITC solution, mix well, and incubate at room temperature for 5 min in the dark. Finally, 5 μL of propidium iodide solution  and 400 μL of PBS were added and mixed, and flow cytometry was performed immediately.The following experiments were carried out according to the instructions of the Annexin V‐FITC/PI Apoptosis Detection Kit . The cells were inoculated into six‐well plates  was used to purify double‐stranded cDNA. The purified double‐stranded cDNA was repaired, A‐tailed, and connected to the sequencing connector, and then the fragment size was selected, and finally, the PCR amplification was performed. After the constructed library passed the quality test of Agilent 2100 Bioanalyzer , it was sequenced by Illumina HiSeqTM 2500 sequencer  to produce double‐terminal data of 125 or 150 bp. After passing the quality inspection, the Illumina sequencer was used for sequencing, and the sequencing data were obtained for follow‐up bioinformatics analysis.The total RNA of the sample was extracted and digested with deoxyribonuclease I (DNase I) , then the mRNA was enriched with Oligo (dT) magnetic beads [Dynabeads™ Oligo (dT)2.17The total RNA was extracted from the CRC cells and transfected in different groups with the TRIzol reagent (Invitrogen Life Technologies). Quantitative real‐time PCR (qRT‐PCR) was performed with specific primers  and the SYBR‐Green PCR Master Mix Kit  on the ABI 7500 System . GAPDH was used for normalizing the gene expression. All primers used in this study were synthesized by Sangong Biotech . The primers used in this study have been listed in Table 2.18The slides were soaked in 1 × PBS for 3 min, 4% paraformaldehyde was used to fix the slides for 30 min, and soak the slides for 3 min each time with 1 × PBS. PBS was sucked dry using an absorbent paper, and the slides were sealed with normal goat serum at room temperature for 30 min. After blocking, the cells were incubated with the corresponding primary antibodies mouse monoclonal anti‐MUC2  and rabbit monoclonal anti‐MUC5AC  overnight at 4 °C, followed by treatment with Goat anti‐Mouse IgG (H+L) Cross‐Adsorbed‐Alexa Fluor 488 Secondary Antibody and Goat anti‐Rabbit IgG (H+L) Cross‐Adsorbed‐Alexa Fluor 594 Secondary antibody, respectively, for 1 h at room temperature, after which the sections were washed with 1 × PBS thrice for 3 min each time. The nucleus was stained with 4′, 6‐diamidinophenyl‐indole (DAPI) . The sealing was performed using the sealing liquid containing an anti‐fluorescence quenching agent, and the slides were finally observed under a confocal microscope .2.19g for 20 min at 4 °C. The samples or the standard working solution were added to the micro ELISA plate well and incubated at 37 °C for 90 min. After discarding the liquid from the plate, 100 μL of biotinylated antibody working solution was added immediately, mixed, and incubated at 37 °C for 60 min. After washing each well thrice with 350 μL of the washing solution, 100 μL of HRP conjugate working solution was added to each well, and the plate was incubated at 37 °C for 30 min. After discarding the liquid in the plate, each well was washed five times with the washing solution. Then, 90 μL of the substrate reagent was added to each well, and the samples were incubated at 37 °C for 15 min. After the stop solution was added, the optical density  of each well was measured at 450 nm using the SynergyH1 Microplate Reader . The concentrations of MUC5AC and MUC2 were directly proportional to the OD value at 450 nm. Finally, the concentrations of MUC5AC and MUC2 in the sample were calculated by drawing the standard curve.The expression levels of MUC5AC and MUC2 in HCT116 cell culture supernatant were measured by ELISA for MUC5AC  and anti‐MUC2 , respectively. The samples were prepared according to the manufacturers' instructions. Briefly, HCT116 cell supernatant was collected under each condition and centrifuged at 1000 2.20ConSite service based on the JASPAR datasets for this purpose. As is already known, the transcription factor CSL is the key effector of canonical NOTCH signaling. Accordingly, the transcription binding sites prediction of NOTCH3‐CSL to the MUC5AC and MUC2 promoter were performed as described elsewhere [MUC5AC: 5′‐GGAGGGAGAGTCTAGCCACA‐3′ (sense) and 5′‐GAAGCTGTTGACTGGTCCGA‐3′ (antisense); MUC2 was: 5′‐CCTGTGTGCCTGATTCCGTA‐3′ (sense) and 5′‐GCTCATGGAGCTGTGTCAGA‐3′ (antisense). The specific antibody information is provided in Table The CHIP Assay Kit  was used to perform the CHIP assay as instructed by the manufacturer. Rabbit monoclonal anti‐NOTCH3 antibodies  or rabbit anti‐IgG antibodies  were used for precipitation, and the IP was purified on protein A/G immunoprecipitation beads. The DNA purified from CHIP was ligated with the adapter and subjected to PCR amplification, as per the manufacturer's instructions (Illumina). We used the lsewhere . The pri2.21MUC5AC and MUC2 promoter luciferase reporter plasmids containing the predictive binding sites of NOTCH3‐CSL were constructed with support from GeneCreate Biological Engineering Co., Ltd. The CSL/RBPJκ (CSL) overexpression plasmid was constructed from Hanheng . The two reporter plasmids were then co‐transfected with the CSL overexpression plasmid, NICD3 overexpression plasmid, and NC‐shRNA or SMARCA4‐shRNA into HCT116 cells using Lipofectamine™ 3000 (Thermo Scientific). At 48 h after transfection, firefly and renilla luciferase activities were measured using the Dual Luciferase@ Assay System . The luciferase activities were normalized by determining the renilla luciferase activity.2.22graphpad prism 6.01 Software . The data were expressed as mean ± standard errors of the mean (SEM). A two‐way analysis of variance (ANOVA) was used for multiple experimental group analyses. Differences between two groups were analyzed using Student's t‐test. The correlation analysis was evaluated by Pearson's correlation test. Survival analysis was estimated using the Kaplan–Meier method with the log‐rank test. P < 0.05 was considered to be statistically significant.Statistical analyses were performed using the 33.1http://cbioportal.org) were analyzed . TCGA database was used to analyze the expression of NOTCH3 and SMARCA4 in the CRC tissues and the adjacent normal tissues, and the results revealed that both of them showed higher expression in cancer tissues when compared to the adjacent tissues  and STRING (https://string‐db.org/)  Fig. , which ig/) Fig.  in CRC, 3.2To further clarify the correlation between NOTCH3 and SMARCA4, IHC was performed using the CRC and paracancerous tissue array to explore the correlation between NOTCH3 and SMARCA4. The results suggested that the expression of NOTCH3 and SMARCA4 in CRC tissues was significantly higher than that in the paracancerous tissues at the protein level, this difference was statistically significant Fig. . MoreoveTo investigate the common features of the signaling pathway regulation between NOTCH3 and SMARCA4 in colon cancer cells, the CRC HT29 and HT115 cells  was used to predict the domains in which SMARCA4 may interact with other proteins. The results showed that SMARCA4 contained two domains (http://smart.embl.de/smart/show_motifs.pl?ID=SMCA4_HUMAN) that may interact with other proteins, namely the SMART BRK domain (aa 612–656) and SMART BROMO domain (aa 1455–1566). Furthermore, His‐NICD3, GST‐612 (BRK domain), and GST‐1455 (BROMO domain) were expressed and purified using Escherichia coli and subjected to GST pulldown assays. The results showed that His‐NICD3 was pulled down by GST‐612 and GST‐1455  among the NC‐siRNA and NOTCH3‐siRNA or SMARCA4‐siRNA group were identified by DESeq2 . P < 0.0MUC5AC, LOC730268, HLA‐DRA, RGPD5, MUC2, DDT, and FOXJ1, and the increased genes were PRR9, GFPT2, PSAT1, VGF, U2AF1L5, IFNL3, IFNL2, and SKOR1, while the expression of ALB increased after the NOTCH3 knockdown and decreased after the SMARCA4 knockdown. In recent years, several studies have shown that mucins play an important regulatory role in the progression of CRC, especially MUC2 and MUC5AC [Compared with the NC‐siRNA group, the NOTCH3‐siRNA group had 54 differential genes meeting the above conditions, and the SMARCA4‐siRNA group had 50 differential genes meeting the above conditions. The intersection of these differential genes was considered, and 16 differential genes were reported from both groups Fig. . The 16 d MUC5AC , 52. Mord MUC5AC , 38, 39.MUC5AC and MUC2 were promoted through the JASPAR datasets and then verified the same by CHIP‐qPCR experiments. We discovered that NOTCH3 could bind to the MUC5AC and MUC2 promoter fragments that included the CSL binding sites using CHIP assays  was further analyzed  were observed in mammals, all of which were found to regulate cell homeostasis by participating in intercellular communication and regulating cell proliferation, differentiation, and apoptosis . The carIt has been confirmed that the activation of the NOTCH pathway requires a variety of transcriptional regulators , and theIn this study, the cBioPortal for Cancer Genomics database analysis, TCGA database analysis, and IHC experiments revealed a strong correlation between NOTCH3 and SMARCA4 in CRC Figs  and 2. OPrevious studies on the NOTCH signaling pathway have demonstrated that NOTCH are essential for cell proliferation, invasion, metastasis, and apoptosis of CRC , 66, 67.MUC5AC and MUC2 by recruiting SMARCA4 (Fig. In addition, the possible targeting factors of NOTCH3 and SMARCA4 in colon cancer cells were screened through RNA‐seq, and, for the first time, we identified that MUC5AC and MUC2 may be the targeted factors for the co‐regulation of NOTCH3 and SMARCA4 interaction Fig. . NOTCH3 CA4 Fig. . FurtherCA4 Fig. . Early sPatients with MCA are now treated using the same conventional treatment recommendations as those with CRC. However, patients with mucinous adenocarcinoma require specific, customized treatment because of their poor response to standard chemotherapy and immunotherapy. Furthermore, multicolor fluorescence immunohistochemistry has revealed that the positive rate of cells co‐expressed with NOTCH3 and SMARCA4 was markedly higher in the MUC5AC and MUC2 co‐overexpression positive group and that there was a higher positive correlation between NOTCH3 and SMARCA4 in CRC patients with the co‐overexpression of MUC5AC and MUC2 Fig. . These rIn‐depth investigation of NOTCH3 and SMARCA4's interaction mechanism and biological functions in transcriptional regulation and their participation in the regulation of downstream target factors MUC5AC and MUC2 are expected to help understand the molecular mechanisms involved in the differentiation and development of MCA. Meanwhile, relevant intervention treatment will be performed for NOTCH3 and its related targets, and relevant joint molecular diagnostic criteria will be formulated for the early clinical diagnosis of MCA. The establishment of staging evaluation and subtype refinement criteria will be able to provide a more accurate theoretical basis for interpreting the pathogenesis of MCA and improving the current prevention and treatment strategies of CRC, which has a broad clinical application prospect.5The present study revealed that SMARCA4 interacts with NICD3 directly to regulate the growth, invasion, and migration of CRC cells. In addition, NOTCH3 may target MUC5AC and MUC2 by recruiting SMARCA4, which has possible involvement in the differentiation and development of MCA. Our results may improve the understanding of the specific molecular mechanism of occurrence and development of MCA mediating by NOTCH3. We hope to provide a better basis for clinical patients with optimized individualized treatment, which is of great significance for improving the rate of clinical diagnosis, prognosis, and survival rate of MCA patients.The authors declare no conflict of interest.XY, YC, XZ and YH performed the experiment and drafted the manuscript. KX and JR conducted the clinicopathological data collection and analysis. YH, BW and YY analyzed the data. XY, YC, XZ, HH, WH and WW interpreted the results of the experiments. WW and WH conceived the research and finalized the manuscript. All authors have approved the final version of the manuscript and agree to be accountable for all aspects of the work.https://publons.com/publon/10.1002/1878‐0261.13296.The peer review history for this article is available at Fig. S1. Screening of specific small‐interference RNA and overexpression adenovirus validation.Fig. S2. The prognostic model analysis by the expression of NOTCH3 and SMARCA4 based on a CRC patient study cohort and the TCGA database.Fig. S3. NOTCH3 regulates the progression of SW480 cells in a SMARCA4‐dependent manner.Fig. S4. Changes in the migration and invasion abilities of HCT116 cells in different groups.Table S1. Clinical characteristics of patients with CRC used for general IHC analysis.Table S2. Clinical characteristics of patients with CRC used for multicolor Manual IHC analysis.Table S3. Antibodies used in related experiments.Table S4. The siRNA sequence used in the present study.Table S5. Primer sequences used in qRT‐PCR.Click here for additional data file.