Incubation with the tau protein variants also led to a disappearance of the peak indicating the ribosome profile of the HeLa cell lysate and suppression of translation in the human in vitro translation system. The incubation of tau protein with the ribosomal RNA leads to the formation of tau-rRNA aggregates. The effect of K18 on the yeast ribosome can be mitigated in the presence of cellular polyanions like heparin and tRNA, thereby indicating the electrostatic nature of the aggregation process.The human tau is a microtubule-associated intrinsically unstructured protein that forms intraneuronal cytotoxic deposits in neurodegenerative diseases, like tauopathies. Recent studies indicate that in Alzheimer’s disease, ribosomal dysfunction might be a crucial event in the disease pathology. Our earlier studies had demonstrated that amorphous protein aggregation in the presence of ribosome can lead to sequestration of the ribosomal components. The present study aims at determining the effect of incubation of the full-length tau protein (Ht40) and its microtubule binding 4-repeat domain (K18) on the eukaryotic ribosome. Our  In addition to Alzheimer’s disease, abnormal aggregation of the tau protein has been linked to the pathogenesis of more than 20 other neurodegenerative disorders, collectively termed as tauopathies1. Although a link between pathological tau aggregation and cognitive impairment has been established, the mechanism by which tau aggregation causes neuronal dysfunction is unclear.Protein aggregate formation and their intracellular accumulation are associated with a wide range of neurodegenerative proteinopathies. The Alzheimer’s disease (AD) is a neurodegenerative disease characterised by formation of neurofibrillary tangles (NFTs) that are composed of hyperphosphorylated tau proteins2. More recent studies of the human tau interactome have also revealed a robust association of the tau protein with the ribonucleoproteome and these studies have indicated at the preferential association of the full-length tau protein Ht40 to the 80S ribosome3. Although aggregation of the tau protein is the primary event in AD, it has also been demonstrated that the impairment of cellular translation and ribosome dysfunction, due to tau-ribosome interaction, is an early event in the disease. Such disruption of protein synthesis machinery might contribute towards the neuronal loss which characterises the disease4. This is also in agreement with earlier studies which noted that a decline in protein synthesis and ribosome function is initiated during mild cognitive impairment (MCI) that develops into AD6. Although a profound loss in the ribosomal complexes in the affected regions of the brain have been shown to occur in parallel to the progression of the disease7, the reasons underlying the disappearance of the ribosomal peaks is not completely understood.Several lines of evidence suggest that the ribosome is a cellular interacting partner of the tau protein. Earlier immunohistochemical studies using tau specific antibody have demonstrated that, there is an association of the tau protein with both, the microtubule and the ribosome in the neuronal cells8. Hence, tau mediated neurodegeneration might arise due to either the loss of physiological function or the gain of toxic function. Tau aggregation would abolish its microtubule stabilizing function and hence, impair neuronal transport. However, the absence of significant neuronal abnormalities in tau knock-out mice9 have suggested that the loss of tau function may be less critical and the gain of toxic function in the process of tau aggregate formation might be the key factor in neurodegeneration. Several recent evidences also suggest that sequestration of cellular interacting partners (proteins or RNA) by protein aggregates could contribute to pathogenesis in a wide spectrum of neurodegenerative diseases10. The fact, that the intrinsically unstructured tau protein is capable of interacting with polyanionic cofactors like heparin11 and cellular RNAs12, raises the possibility that the tau protein is also capable of interacting with highly charged macromolecular complexes such as the ribosome. Our earlier studies had shown that the partial unfolding or amorphous aggregation of lysozyme and Bovine Carbonic Anhydrase II (BCAII), in the presence of empty non-translating prokaryotic or eukaryotic ribosome, could induce aggregation of ribosomal components13. Hence, based on these observations, the present study aims at determining the effect of incubation of the tau protein on the physical integrity of the eukaryotic 80S ribosome.The tau protein is a microtubule associated protein that promotes the assembly and stabilization of the microtubule structurein vitro experiments demonstrate that the incubation of tau variants (K18 and Ht40) with the eukaryotic yeast 80S ribosome leads to a dose dependent and progressive loss of ribosomal peak, that is indicative of the loss of ribosome population. A disappearance of the ribosome peak and decline in in vitro translation ability of the HeLa cell lysate containing the human ribosome is also observed upon incubation with the tau variants, indicating a similar loss of physical integrity of the human ribosome. The incubation of the tau variants with isolated yeast ribosomal RNA leads to the formation of large tau-rRNA containing aggregates. Studies have also been performed on the effect of the cellular polyanions like heparin and tRNA (that are known tau protein interactors) on the outcome of Ht40- and K18- ribosome co-aggregation.Our 14. Intrinsically unstructured proteins are also known to engage in non-native electrostatic interactions with their cognate cellular partners15. The electrostatic features of the tau protein variants used in our study and the ribosomes, which therefore might play a major role in the tau-ribosome interaction, is highlighted in Fig. 11. The full length tau protein has been colour coded based on the predominant charges of its corresponding domains and highlights that the Ht40 is a dipole with oppositely charged domains. The wide variability in the pI of the domains results in a considerable difference in their net charge at physiological pH16. This renders the N-terminal domain acidic (red), the central domain basic (blue) and the C-terminal domain neutral (green) as shown in Fig. 19, is shown in Fig. 20 and the rRNA has a major contribution in negative electrostatic potential of the ribosome21. A flowchart denoting the basic procedure of the in vitro experiments performed in the study discussed below is shown in Fig. Electrostatics have been shown to play a major role in determining the interactions of tau with its cellular partners22. However, no additional cellular polyanions that are necessary for inducing tau aggregation were present during the incubation. The centrifugal speed used in our study differs from the speed used earlier in polyA RNA induced tau aggregation studies23, for separation of monomeric tau from tau-RNA aggregates. At this speed , the ribosome or rRNA alone is pelleted irrespective of the presence of the tau proteins. Hence, at the centrifugal speed used in our studies , the larger aggregates would form the pellet (P) while the supernatant (S) might be constituted of the residual ribosomes and smaller aggregates. In our initial experiments the tau variants were incubated with the ribosome for 6 hours. The supernatant fractions obtained after Ht40-80S and K18-80S incubation were analysed by sucrose density gradient centrifugation (SDGC) and the A260 nm profile obtained was compared to the profile of equivalent amount of untreated yeast 80S ribosome. As shown in Fig. As stated in the introduction section, the effect of incubation of the tau protein on the eukaryotic 80S ribosome is the principal objective of the present study. As stated earlier, the flowchart in Fig. 24. In the presence of suitable controls, this method has also been used in our earlier studies as a semi-quantitative technique for following protein-ribosome aggregation process13. Similarly, analysis of the protein components of the pellet and the supernatant fractions by denaturing SDS-PAGE was also done which showed that upon incubation of the 80S ribosome with the tau variants K18 and Ht40 (for 6 hours), the presence of ribosomal proteins was observed, both in the supernatant as well as in the pellet . Control experiments were performed in which the yeast ribosome (0.1 μM) was incubated in the presence of the native bovine carbonic anhydrase II (nBCAII) protein (50 μM), under similar conditions, and centrifugation was performed as stated earlier. As shown in Fig. E. coli cells expressing the tau protein variants were lysed using the “direct boiling method” and the cellular extract obtained  was incubated with 0.1 µM yeast 80S ribosome for 6 hours and centrifuged. As shown in Fig. The constituents of the pellet and supernatant fractions were also analysed using electrophoretic methods. Agarose gel electrophoretic analysis of the pellet fractions Fig.  showed tlet Fig. . Control260 nm values. As shown in Fig. Surprisingly, it was noted from the SDS PAGE profile that both K18 and Ht40 appeared to be present in the supernatant fraction Fig. . This mi260 nm profile obtained by sucrose density centrifugation can reflect the total ribosomal population present in the lysate. Hence, in our subsequent experiment, the HeLa cell lysate containing 0.1 μM equivalent A260 nm units was incubated with 50 µM of tau proteins K18 or Ht40 for 6 hours, centrifuged and the A260 nm profile (obtained by SDGC) of the supernatant was compared to that of the untreated lysate. A significant alteration of the A260 nm profile  . This cell lysate represents a heterogeneous system that includes the translational machinery as one of its significant components and the Aile Fig.  and an a25) Fig.  and of p25) Fig. . In pres25) Fig. . However25) Fig.  and the 25) Fig.  that is in vitro and in vivo experiments need to be performed with purified human ribosomes and disease affected neurons respectively, our studies imply that tau-ribosome interaction could underlie the change in ribosome profile observed in diseased neurons. Further, based on the observed similarity in the effect of tau variants on both yeast and human ribosomes and the fact that yeast is established as a model system for studies of neurodegenerative diseases27, all subsequent experiments in this study were performed with purified yeast 80S ribosome.Although, further In order to explore the time course followed by the tau-ribosome aggregation process, the tau-variants (50 µM) were incubated with the ribosome (0.1 µM) for different time periods and centrifuged. The supernatant and pellet fractions were analysed by SDGC and gel electrophoresis. The sucrose density gradient profiles of the supernatants in Fig. At each time point of incubation, the area under the 80S peak in the supernatant and the intensity of the rRNA band in the pellet provides a semi-quantitative measure for following the tau-ribosome aggregation process. Hence, the experiments in Fig. In the seeding experiments aliquots from the reaction mixture containing the ribosome and tau variants (K18 or Ht40) were withdrawn at different time points  was incubated with 50 µM of K18 or Ht40 for 48 hours at 37 °C and the change in light scattering intensity at 450 nm was measured. As shown in Fig. In order to further analyse the aggregates formed, the reaction mixture was centrifuged and the pellet and supernatant fractions were analysed using agarose gel electrophoresis and dot blot analysis . As shown in Fig. 31. A recent study with recombinant prion protein (rPrP) has showed that depending upon the protein: RNA stoichiometric ratio, the RNA is capable of inhibiting or stimulating protein aggregation. At a high protein: RNA ratio, rPrP aggregation28 like tau aggregation in our study, is stimulated, also leading to its co-aggregation with the RNA. Our experiments also collectively indicate that the rRNA component of the ribosome could play a significant role in the ribosome-tau aggregation phenomenon observed here.In this context, it should be noted that recent studies show that RNA can influence aggregation of disease-associated proteins14. Hence, further studies were performed to investigate the possibility of inhibition of tau initiated ribosome aggregation by electrostatic shielding agents in the form of competitor polyanions like heparin and tRNA. In these experiments the tau variants were incubated with the ribosome in presence of increasing concentrations  of tRNA or heparin, centrifuged and the aggregation process was followed by agarose gel electrophoresis of rRNA present in the insoluble tau-ribosome aggregates. The highest concentrations of tRNA and heparin used in these experiments were 10-fold higher than that of the 80S ribosome, as was used in our earlier studies13. Also, as reported in literature, the cellular stoichiometric ratio of tRNA with respect to the ribosome in yeast is approximately 10-fold32.Earlier studies have demonstrated the importance of electrostatic interactions in determining the interaction of tau with its cellular partners and that the behaviour of the tau protein can be modulated in presence of cellular polyanionsAs shown in Fig. We also analysed whether the time of addition of these polyanions could affect tau induced ribosome aggregation. As shown in Fig. From the selective effect of the polyanions, it can be concluded that under our experimental conditions, electrostatics plays a major role in determining the outcome of K18–80S aggregation process. Further reasons that might underlie the difference between K18 and Ht40–80S aggregation are discussed below and is depicted in the model Fig. .7. Our in vitro studies show that when full length tau protein (Ht40) and the K18 tau variants were incubated in vitro with isolated non-translating yeast ribosome under conditions that are conducive to tau aggregation (but in absence of polyanionic inducer), the aggregation of ribosomal components was induced. The tau protein variants had a similar effect on the ribosome profile of the HeLa cell lysate and suppression of translation by the human in vitro translation system was also observed in presence of the tau protein variants. The concentration and time dependence of tau mediated ribosome aggregation and the ineffectiveness of BCAII protein to induce 80S ribosome aggregation are indicative of a ribosome aggregation process that is subject to the specific presence of the tau proteins. Further in vivo experiments need to be performed with the disease affected neurons to confirm our observations made in vitro. Our studies imply that the tau proteins alone might mediate the change in ribosomal profiles observed in neurons of the Alzheimer’s affected regions of human brain7, which might be a vital determinant of tau aggregation toxicity.As stated in the introduction section, a significant change in the 80S ribosome profile occurs in the neurons of individuals afflicted with Alzheimer’s disease and a gradual decrease in the neuronal ribosome population is observed with the progression of the diseasede novo protein aggregation31. Since the eukaryotic ribosome is a large ribonucleoprotein particle, a large amount of tau- rRNA and tau-ribosomal protein aggregates are formed during the tau-ribosome aggregation process. As RNA molecules have been observed to contribute to the seeding behaviour of RNA-protein aggregates (as stated earlier), it might be suggested that the ribosome, majorly consisting of rRNA molecules, might rely on the rRNA component for this seeding behaviour. In addition, the large number of ribosomal proteins with intrinsically disordered regions can also play a significant role. Hence, further studies need to be performed to identify the component formed due to tau-ribosome co-aggregation that possesses the potential to self-perpetuate the ribosome aggregation process.The ability of tau-ribosome co-aggregates to seed the aggregation of ribosomes is another surprising phenomenon observed in this study. This study implies that the tau-ribosome aggregates formed can successfully propagate the aggregation of new ribosomes without requiring the presence of freshly added tau proteins. It might be noted that earlier studies have shown that when the K18 variant of the tau protein, rPrP and the p53 proteins were incubated with RNA, protein-RNA aggregates formed could act as seeds to nucleate 33 and co-translational protein folding34. Interestingly, an unrelated study has also implicated that the ribosome surface properties may limit the mobility of positively charged variants of recombinant GFP in E. coli21. The formation of aggregates upon incubation of the purified ribosomes with the positively charged GFP variant indicates at the tendency of a positively charged protein to induce ribosome aggregation. Our earlier studies have also suggested that electrostatics plays a role during co-aggregation of lysozyme  with the ribosome13. However, the contribution of net charge on a protein towards its ability to bring about ribosome aggregation needs further investigation. Recent studies have demonstrated that the disease associated intrinsically disordered proteins are highly charged and often rely on non-native electrostatic interactions for associating even with their appropriate cellular binding partners36. Such a tendency of engaging in promiscuous interactions might also lead to cytotoxicity. Indeed, it has been demonstrated that electrostatic interaction of the tau protein with anionic lipid membranes can induce tau aggregation and that the resultant membrane permeabilization may serve as a pathway by which tau protein aggregates exert their toxicity38. Earlier studies had demonstrated that cellular RNA (a major portion of which is rRNA) can induce tau fibrillization27 and RNA modulated prion aggregation is also widely reported in literature29.Another question that arises is what forms the basis for the tau-ribosome interaction that eventually culminates into the co-aggregation process reported in this study. Several recent studies have highlighted that the electrostatic interactions between the unfolded nascent polypeptide chain and the ribosome have diverse effects on translation rate39, in the tau-ribosome aggregation process, also requires to be further investigated. However, in light of present studies and taking into account the relative size and concentration of the eukaryotic ribosome and the tau variants, it is possible that the anionic surface of the ribosome, that is predominantly composed of rRNA, could present itself as a potential surface on which multiple tau molecules can associate . Whether such tau-ribosome interactions play a role in the observed co-aggregation of the tau proteins with the ribosome needs to be investigated.Our studies also show that the incubation of the tau variants with the rRNA isolated from the 80S ribosome could lead to the formation of tau-rRNA aggregates. The ability of the rRNA to induce rRNA-Tau aggregation implicated that the rRNA component of the ribosome plays a dominant role in this process, although, the participation of ribosomal proteins containing regions of intrinsic disorderate Fig. . Such asate Fig.  needs to3 and the differentially truncated forms of tau lose their affinity for microtubules, thus altering their subcellular localization and increasing the cytosolic tau concentration45. Studies on the abilities of other tau isoforms to initiate the tau-ribosome aggregation process also need to be performed. Preliminary studies conducted with the 3-repeat domain containing tau variant (K19) show that similar to K18 variant, K19 is able to aggregate the ribosome ome Fig. , althoug2, DTT, Tris-base, SP sepharose were purchased from Sigma Aldrich. Tau 4RD (K18), Tau 3RD (K19) and Ht40 clones were obtained as kind gifts from Dr.Jayant B. Udgaonkar’s Laboratory (NCBS). Polyallomer ultracentrifuge tubes were purchased from Beckman Coulter. Nitrocellulose filter was purchased from Bio-Rad, PVDF membrane was purchased from Millipore, mouse monoclonal anti-human Tau 4RD antibody was purchased from EMD Millipore (primary antibody for K18) , mouse monoclonal anti-mouse D8 antibody (primary antibody for Ht40)  and the goat-anti-mouse HRP conjugated antibody (Secondary antibody) were purchased from Santa-Cruz Biotechnology. The western chemiluminescence HRP substrate was purchased from Millipore (Immobilon). Yeast phenylalanine tRNA was purchased from Sigma-Aldrich. All other chemicals were local products of analytical grade. Experimental data analysis was performed using OriginPro8 , QuantityOne Bio-Rad and SIGMA-PLOT 14  software. PYMOL 2008  was used to display the Protein Data Bank files. Net charge calculations of human full-length tau (Ht40) and K18 were performed using PROTEIN CALCULATOR v3.4.Bovine Carbonic Anhydrase II (BCAII), NaCl, MgCl2 ; Buffer B for cell lysis: 50 mM PIPES NaOH pH 6.9,20 mM NaCl,1 mM EDTA,5 mM DTT, 1 mM PMSF22; Buffer C for protein purification22: 50 mM PIPES NaOH pH 6.9,20 mM NaCl; Buffer D for protein purification22: 50 mM PIPES NaOH, pH 6.9 500 mM NaCl; Buffer E for final buffer exchange and protein storage22: 25 mM Tris-HCl pH 7.5.Buffers used in this study were: Buffer A for aggregation reactions: 25 mM Tris-HCl pH 7.5, 50 mM NaCl, 5 mM MgClSaccharomyces cerevisiae ribosome (80S) was purified according to Chakraborty et al.49. Yeast 80S rRNA was extracted as mentioned in Piir et al.50. Tau K18 and Ht40 were purified using a two-step purification method: direct-boiling method52 and cation-exchange purification method22. The E. coli BL21(DE3) cells were transformed with Tau k18, k19, ht40 plasmids and cells were harvested for large scale purification by centrifugation. The cell pellets were resuspended and boiled in Buffer B for 20 minutes with gentle agitation after every 5 minutes. The boiled cell suspension was immediately chilled on ice for 15 minutes before centrifuging it at 4 °C and 16,000 g for 20 minutes. The supernatant was retained and incubated with SP sepharose for cation exchange chromatography and washed with increasing concentrations of NaCl (obtained my mixing Buffer C and Buffer D)22. Fractions containing pure protein were pooled together, concentrated and finally buffer exchanged into Buffer E and frozen at −80 °C until further use. The protein concentration was determined as mentioned in Ramachandran et al.22.Yeast et.al.22 with minor modifications. 50 µM Tau K18 or Ht40 was reduced in presence of 1 mM DTT for 2 hours at 37 °C in Buffer A, at the end of which 0.1 µM 80S or 1 µM 80S rRNA or 1 µM heparin or 1 µM tRNA or A260 nm units equivalent to 0.1 µM 80S (7.9 A 260 nm units) HeLa cell lysate was added and incubated for 6 hours at 37 °C or as mentioned. For aggregation reactions with increasing concentrations of tau, 10 µM, 25 µM and 50 µM of K18 and Ht40 was used. In control experiments 50 µM native BCAII (nBCAII) was incubated with 0.1 µM 80S for 6 hours at 37 °C. 80S (0.1 µM) was also incubated in the presence and absence of 1 mM DTT for 6 hours in buffer A and centrifuged. Aggregation reactions were centrifuged at 21,380 g for 45 minutes at 4 °C and the pellet and supernatant fractions separated. It should be noted that the centrifugal speed used in earlier studies on polyA RNA induced tau aggregation23 to separate monomeric tau and tau-RNA aggregates was 1,00,000 g which could not be used for our studies, since at this centrifugal speed the ribosome or rRNA alone is pelleted irrespective of the presence of the tau proteins. Hence, at the centrifugal speed of 21,380 g13, the larger aggregates formed the pellet (P), while the supernatant (S) constituted of the residual ribosomes or smaller aggregates. The constituents of the pellet fractions were analysed using 12% SDS-PAGE or 0.8% agarose gel electrophoresis and the supernatant fractions by sucrose density gradient centrifugation or SDS-PAGE after TCA precipitation. The SDS-PAGE were stained using Coomassie Brilliant Blue at 37 °C and the agarose gels were stained using ethidium bromide.The tau aggregation procedure and buffer composition were followed from Ramachandran 24. Briefly, the pellets had to be treated with 4 M urea and incubated for 20 minutes at room temperature (a treatment that was necessary to enable the RNA in the large aggregates to enter the gel), before loading on a 0.8% agarose gel for electrophoresis. The electrophoresis was performed in 1xTAE at 65 V for 10 min before visualizing under ultraviolet light using the GelDoc imaging system (MEGA BIO-PRINT 1100/20 M). In this experiment, the total RNA runs as a single band, in which the intensities of the rRNA bands were compared by densitometric analysis (QuantityOne Bio-Rad). This procedure had also been used in our earlier studies to follow the lysozyme ribosome co-aggregation process13. The total ribosome or rRNA used in the experiment was treated similarly and analysed. For the analysis of the protein constituents, the protein in the supernatant was TCA precipitated and the pellets were resuspended in Laemmli buffer containing 4 M urea and boiled before loading onto a 12% SDS-PAGE.Aggregation samples were centrifuged at 21,380 g for 45 minutes at 4 °C. The pellets and supernatant were analysed for their total rRNA content in a non-denaturing 0.8% agarose gel, using a procedure used earlier to study interaction of tau with cellular RNA (with minor modifications)Aggregation reactions were centrifuged at 21,380 g for 45 minutes at 4 °C and the supernatant fraction was layered on a 17–25% sucrose gradient. The gradient was centrifuged at 1,98,000 g  for 2.5 hours at 4 °C and 200 µl fraction volumes were collected from top to bottom and absorbance at 260 nm was measured for analysing the 80S ribosome profile.50 µM K18 or Ht40 was reduced in 1 mM DTT at 37 °C for 2 hours after which 0.1 µM 80S was added to it. The resultant reaction mixture was incubated at 37 °C for 24 hours and centrifuged at 21,380 g, 4 °C for 45 minutes. The supernatant and the resuspended (in 1 M Urea) pellet fractions were dotted on PVDF membrane. The membrane was blocked with 5% skimmed milk for 1.5 hours and then washed three times  with 1x PBST and incubated with specific primary antibody  at 4 °C for overnight. The membrane was then washed eight times  with 1x PBST and then incubated with secondary antibody  for 1.5 hours at room temperature. Then the membrane was washed with 1x PBST for eight times and incubated with chemiluminiscent HRP substrate and the signal was recorded using photographic plates.in vitro translation assay was done using 1-Step Human Coupled IVT Kit – DNA; 88881, ThermoFisher Scientific. K18 and Ht40 were added to the prescribed reaction mix to a final concentration of 50 µM. The positive control set contained no tau proteins and the negative control set did not contain the GFP (reporter gene) plasmid. All the reaction sets were incubated till 6 hours at 30 °C (as prescribed) and GFP (reporter gene) fluorescence was monitored at ex/em: 482/512 nm. The experiment was repeated three times.The 50 µM K18 or Ht40 was reduced at 37 °C for 2 hours after which 0.1 µM 80S was added. The resultant reaction mixture was incubated for 2 and 24 hours at 37 °C. For studies with rRNA, 1 µM 80S rRNA was incubated for 48 hours with 50 µM K18 or Ht40 reduced in 1 mM DTT as described above. Imaging of aggregation in the samples was done by using a transmission electron microscope (FEI Tecnai12BioTwin) with an acceleration voltage of 120 kV. Aliquots (5 µl) containing the aggregation mixture were placed on the copper grid coated with carbon film (300 meshes) and one drop of 2% uranyl acetate was placed on the grid. The excess water was removed carefully with filter paper and the grid was left to dry in air..50 µM K18 or Ht40 was reduced at 37 °C for 2 hours after which 0.1 µM 80S was added. The resultant reaction mix was centrifuged at 21,380 g for 45 minutes at 4 °C after 0 h, 0.5 h, 1 h, 1.5 h, 2 h, 4 h and 24 h of incubation for K18 and 0 h, 2 h, 4 h, 6 h, 8 h, 14 h and 24 h of incubation at 37 °C. The supernatant fractions were analysed using a 17–25% sucrose density gradient (as described below) and the pellets obtained were analysed using 0.8% agarose gel (as described below). The supernatant and pellet fractions obtained at 0.5 h, 2 h, 6 h and 24 h were also analysed using 12% SDS-PAGE as described above50 µM K18 was reduced in Buffer A for 2 hours at 37 °C, after which 0.1 µM 80S was added to it. 1 µl aliquots were drawn from this reaction mixture after 0, 1.5, 3, 4 hours of incubation (for K18 induced 80S aggregation) and 0, 2, 5, 8 hours of incubation (for Ht40 induced aggregation) added to 999 µl of fresh 0.1 µM 80S in Buffer A. This new reaction mixture was then incubated for 24 hours at 37 °C and then centrifuged at 21,380 g at 4 °C for 45 minutes. The insoluble fraction was resuspended in 4 M urea containing Buffer A and analysed using a 0.8% agarose gel electrophoresis.in vitro studies is heparin. In the presence of heparin at an appropriate stoichiometry the K18 aggregates much faster (24 hours)22 as compared to Ht40 (48–144 hours)53. Hence, the reaction was allowed to proceed till 48 hours in order for it to reach saturation. The supernatant and the resuspended (in 1 M Urea for 30 minutes at 37 °C) pellet fractions were dotted on PVDF membrane and the dot blot analysis was carried on as described above.50 µM K18 or Ht40 was reduced in 1 mM DTT at 37 °C for 2 hours after which 1 µM 80S was added to it. The resultant reaction mixture was incubated at 37 °C for 48 hours and centrifuged at 21,380 g, 4 °C for 45 minutes. The most commonly used Tau aggregation inducer for 50 µM of K18 or Ht40 was reduced in Buffer A with 1 mM DTT for 2 hours at 37 °C after which 1 µM of LiCl extracted 80SrRNA was added to the reaction mixture (t = 0 h) and further incubated for 48 hours at 37 °C. The light scattering of the solutions was measured at t = 0 h and t = 48 h and the increase was plotted in the form of bar diagrams. For control, the rRNA alone, K18 alone and Ht40 alone reaction sets were similarly incubated and the increase in their light scattering was measured at 48 hours. Before measuring the light scattering intensity, all the solutions were pipetted three times. The intensity was measured at excitation: 450 nm and emission: 450 nm in Hitachi F-2700 spectrofluoremeter.50 µM K18 was reduced in Buffer A for 2 hours at 37 °C in presence of 1 mM DTT, after which 0.1 µM 80S was added to it. 1 µM heparin or tRNA was added after 0, 1.5, 3 and 4 hours of addition of the ribosome in case of K18 and after 0, 2, 5 and 8 hours of addition of the ribosome in case of Ht40. The resultant reaction mixture was further incubated for 24 hours at 37 °C and centrifuged at 21,380 g at 4 °C for 45 minutes. The insoluble fraction was resuspended in 4 M urea containing Buffer A and analysed using a 0.8% agarose gel electrophoresis.Supplementary information.


They were obtained by the reaction of excited Group‐9 metal atoms with NF3 and their IR spectra, isolated in solid rare gases (neon and argon), were recorded. Aided by the observed 14/15N isotope shifts and quantum‐chemical predictions, all four stretching fundamentals of the novel complexes were safely assigned. The F−N stretching frequencies of the fluoronitrenoid complexes FNCoF2 (1056.8 cm−1) and FNRhF2 (872.6 cm−1) are very different and their N−M bonds vary greatly. In FNCoF2, the FN ligand is singly bonded to Co and bears considerable iminyl/nitrene radical character, while the N−Rh bond in FNRhF2 is a strong double bond with comparatively strong σ‐ and π‐bonds. The anticipated rearrangement of FNCoF2 to the nitrido CoVI complex is predicted to be endothermic and was not observed.The fluoronitrenoid metal complexes FNCoF 2 and FNRhF2 as well as the first ternary RhVI and IrVI complexes NIrF3 and NRhF3 are reported. They were obtained by the reaction of excited Group‐9 metal atoms with NF3 and their IR spectra, isolated in solid rare gases, were recorded.The fluoronitrenoid metal complexes FNCoF In the case of neutral, covalently bound ligands, no polarization of bonding electrons towards the ligand and no or only little charge transfer from the metal to the ligand is generally to be expected. Additionally, not long‐ago a few examples were reported where an iminyl radical (2NR.) is coordinated to a transition metal.2−); iminyl, M(2RN.); nitrene, M(RN); and triplet nitrene (3RN..)In this bonding Scheme the anionic NRomplexes.4, 5 A2, M=Co, Rh, Ir. These simple nitrene complexes should allow a rigorous experimental and quantum‐chemical comparison of the electronic properties of the CoIV complex with those of its heavier congeners. Fluoronitrenoid‐metal complexes show more complex nitrenoid‐metal binding modes and new reactivities. The fluoronitrene ligand shares some similarities with the oxygen molecule, since the nitrogen 2p electrons involved in metal‐nitrogen bonding are accommodated in a degenerate pair of π*(F−N) orbitals. Hence any metal‐to‐ligand charge transfer in a fluoronitrene complex will increase the occupancy of these π*(F−N) orbital, rendering the N−F stretching frequency a highly sensitive probe for the polarity and the strength of the N=M bond: a weak nitrogen‐metal bond in a metal‐nitrene complex result in a strong fluorine‐nitrogen bond and vice versa. Utilizing the F−N functionality and relying upon a high metal‐fluorine bond energy we have targeted the synthesis of high‐valent nitridometal trifluorides N≡MF3, starting from the fluoronitrene complexes by an oxidative F−N to M−F fluorine migration, by which the formal metal oxidation state will be increased by two units. As far as we know, molecular nitridometal trifluorides, NMF3, are known only for the early transition metals of group IV  is rare, with IrO3, Ir(η2‐O2)O2, IrF6, Rh(η2‐O2)O2 and RhF6 as the only examples.4]+ or in the tricoordinated cationic bis(nitrene) cobalt complex [(IMes)Co(NDipp)2]+.+.6, 14 2 , we have studied the gas‐phase reaction of the laser‐ablated free metal atoms with NF3 seeded in a 1:1000 excess of neon or argon. The reaction products were deposited on a gold‐plated copper mirror cooled to 5 and 12 K and IR‐spectroscopically investigated . According to preliminary calculations at the DFT‐B3LYP and BP86 levels of theory the direct insertion of the excited metal atoms into the F−N bond of NF3 to F2N−MF, and the subsequent fluorine migration from nitrogen to the metal center to yield the desired FN=MF2 is highly exothermic for all three metals ), and becomes strongly exothermic for the iridium congener , Table S1), which rendered the detection of the iridium nitrene complex difficult if not impossible.To obtain the group 9 metal difluorides, F−N=MF3 precursor . Our CCSD(T) calculations for this species yield two imaginary frequencies which likely are caused by a close‐lying excited electronic state which interferes with the calculation of displaced steps during the numerical hessian calculation where the symmetry is lowered to C1. However, the structure obtained at the B3LYP level of theory is close to the one obtained at the CCSD(T) level  indicating a strong vibrational coupling between these two modes. Analyzing the N−M and F−N stretching frequencies of FNCoF2 and FNRhF2 we found surprisingly large differences in the bonding of the fluoronitrene ligand. In general, the two singly occupied anti bonding π*(F−N)‐orbitals of the FN ligand form a σ and a π bond to these metal centers, as depicted in the qualitative molecular orbital (MO) interaction diagram shown in Figure 2 (1056.8 cm−1) appeared red‐shifted by 62.6 cm−1 from the absorption of free, neutral FN (1119.4 cm−1),2 (872.6 cm−1) is much stronger red‐shifted by 246.8 cm−1.The assignment of the IR spectra obtained after co‐depositing evaporated rhodium and diluted NF3 Figure . As desc2 (586.1 cm−1) is most likely associated with a N−Co single bond, while the N−Rh frequency of FNRhF2 (761.4 cm−1) can be attributed to a N=Rh double bond. These experimental observations are consistent with calculated atomic charges for the FN fragment in FNCoF2  and FNRhF2 , as well as Mayer and Wiberg bond orders for the N−M bonds listed in Table 2π2δ1 π*0σ*0) associated with the qualitative MO Scheme shown in Figure 2A′′ ground state of FNCoF2, followed by states with significant weights which contain single and double π→π* excitations (Table S4). For FNRhF2 a much smaller extend of non‐dynamic correlation was determined, since the most dominant configuration as depicted in Figure 2A′ electronic ground state. As a consequence of these correlation effects significant higher σ* and π* populations  were found for FNCoF2 compared to FNRhF2 . The effective bond orders (EBO)2 , 159 (RhN); for comparison: triple‐bond additive covalent radii: 160 pm (IrN) and 160 pm (RhN),d3 configuration and a Jahn–Teller distorted 2A′ electronic ground state was determined for both species. The N−Ir stretching frequency in neon of 1150.4 cm−1 is moderately higher than that observed for diatomic IrN embedded in solid neon (1111.1 cm−1).−1) of 221.2 cm−1 occurred compared to diatomic RhN embedded in argon (891.4 cm−1).−1 in NRhF3 from 580 N m−1 in RhN indicates a significant strengthening of the nitrogen‐metal bond induced by the fluorine ligands. This fluorine effect is less pronounced for the already strong triple bond in IrN, where force constants increase from 950 to 1020 N m−1 for IrN and NIrF3, respectively.For the nitrido complexes NRhF2 and FNRhF2 and of the nitrido complexes NRhF3 and NIrF3 by the reaction of the free group 9 metal atoms with NF3. The IR spectra of these compounds isolated in solid rare gases (neon and argon) were recorded and, aided by the observed 14/15N isotope shifts and quantum‐chemical predictions, all four stretching fundamentals of these complexes were safely assigned. Neither nitrido nor other ternary complexes of RhVI and IrVI have yet been reported. The anticipated rearrangement of FNCoF2 to the nitrido CoVI complex was not observed, because this reaction is endotherm. The covalently bound FN ligand in these high‐valent metal complexes is almost neutrally charged, and the formal picture of an FN2− ligand bound to a MF2 fragment is a rather coarse approximation for these fluoronitrenoid complexes. However, the bonding of the FN ligand in FNCoF2 and FNRhF2 was found to be strikingly different. In FNCoF2 the FN ligand is singly bonded to Co and bears considerable iminyl/nitrene radical character, while the N=Rh double bond in FNRhF2 shows comparatively strong σ‐ and π‐bonds. The stretched N‐Co bond and the poor overlap especially between the ligand π‐ and the metal 3d‐orbitals can likely be attributed to a repulsion between the ligand orbitals and the outermost core 3s,3p shell of cobalt.In summary, we described the exothermic formation of the fluoronitrenoid complexes FNCoFThe authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


Dynamical aspect of such large responses is an important issue to be clarified for future THz data processing. Here the THz anomalous Hall conductivity in Mn3Sn thin films is investigated by polarization-resolved spectroscopy. Large anomalous Hall conductivity 3Sn, and will also open new avenue for studying nonequilibrium dynamics in Weyl antiferromagnets.Antiferromagnetic spin motion at terahertz (THz) frequencies attracts growing interests for fast spintronics, however, their smaller responses to external field inhibit device application. Recently the noncollinear antiferromagnet Mn Antiferromagnets promise great potential for spintronic applications due to their spin excitations at terahertz frequencies, however, read out of the spin state is hindered by the small response to external fields. Here the authors demonstrate all optical readout of the spin state of antiferromagnetic Mn3Sn at THz frequency. Therefore, optical control of the antiferromagnets have been intensively investigated in the past few decades2. Nonlinear interaction with THz magnetic field has been also studied3, which leads to direct spin manipulation for future data processing in nonvolatile memory devices. Readout of the magnetization information in antiferromagnets is, however, still very difficult since they are generally not sensitive to external field because of the much smaller net magnetization than in ferromagnets, which has made their practical application challenging. On the other hand, recent discovery of Weyl semimetals with inversion or time-reversal symmetry breaking have attracted tremendous interests4 not only for fundamental physics as condensed matter analog of massless Weyl fermions but also for highly intriguing response functions that reflect the broken symmetry such as nonreciprocal or second-order nonlinear responses6. In particular, recent reports in the broken time-reversal symmetry Weyl semimetal candidate noncollinear antiferromagnet Mn3X 9 have opened new pathways for utilizing these materials as functional spin-based devices at room temperature. In spite of the vanishingly small net magnetization in the antiferromagnetic phase, these materials show large anomalous Hall effect (AHE)9, anomalous Nernst effect11, magneto-optical Kerr effect12, and magnetic spin Hall effect13 in a stark contrast to conventional antiferromagnets. These large responses comparable to ferromagnets have been attributed to the Berry curvature in momentum space18, which is in particular enhanced at the Weyl nodes. Such Weyl semimetals, or Weyl (antiferro)magnets, are of great interest since the magnetic ordering can be controlled by temperature or external field, as small coercive field of 0.05 T has been reported for bulk Mn3Sn7. A recent neutron scattering experiment has also revealed THz spin excitation in Mn3Sn19. Therefore, deep understanding of the dynamical properties of Mn3Sn in the THz frequency range would contribute to developing novel devices based on fast motion of antiferromagnetic spins and large response to external field.The control of magnetism has been a key issue for modern data processing and recording in spintronic devices. From the viewpoint of manipulation speed, antiferromagnets are promising materials since spin precession motion occurs typically at terahertz (THz) frequencies, a few orders of magnitude higher than ferromagnetsσxy(ω) at THz frequencies has been investigated in ferromagnets with the perspective to reveal the microscopic origin of the AHE22. In general, the AHE could arise from the intrinsic Berry curvature determined by the electronic band structure24 or the extrinsic skew scattering or side jump with impurities26. If it is intrinsic, the AC anomalous Hall conductivity spectrum shows peculiar resonant structures due to interband transition as presented in infrared or THz spectroscopy22. The AHE in THz frequency has been also observed in a ferromagnetic semiconductor27 or quantum anomalous Hall state in a topological insulator28. For Weyl semimetals, however, the THz anomalous Hall conductivity remains unexplored experimentally, although a number of theoretical efforts have been devoted to investigating the AC AHE across the Weyl nodes33. A recent angle-resolved photoemission spectroscopy (ARPES) for Mn3Sn has captured the Weyl-like dispersion at several meV above the Fermi energy EF34. Therefore, low-energy THz polarimetry will provide deep insight into the microscopic picture of the large AHE in the Weyl semimetal.Previously, the anomalous Hall conductivity spectrum 3Sn thin films showing the large AHE comparable to the bulk35, we perform THz time-domain spectroscopy (THz-TDS) with precise polarization resolution to reveal the THz anomalous Hall conductivity. Polarization rotation is clearly observed at room temperature and zero magnetic field, which quantitatively agrees with the large AHE previously reported in the DC resistivity measurement. The results also demonstrate small dissipation in the AHE up to THz frequencies, which is consistent with an intrinsic origin of it. The temperature dependence of the Hall conductivity is also discussed with regards to macroscopic time-reversal symmetry in the spin order.In this work, using Mnx3+Snx1− thin films  deposited on SiO2 or thermally oxidized Si substrates by DC magnetron sputtering35. Figure c-axis of magnetic structure, respectively. Figure x3+Snx1− (x = 0.02) on the SiO2 substrate obtained by the grazing angle XRD measurement. All the peaks can be indexed by the hexagonal D019 Mn3Sn structure and no additional peaks coming from plausible impurity phases are observed, which is consistent with that in the films on a thermally oxidized Si substrate35.Samples used in this work are polycrystalline Mnab plane. Such a noncollinear antiferromagnetic spin in the Kagome bilayer is characterized by cluster magnetic octupole moments18, which macroscopically breaks time-reversal symmetry and is expected to result in the appearance of Weyl nodes18. The small net magnetization occurs in the ab plane by slight canting of the 120° order. Note that the large AHE is not explained by the small net magnetization but rather attributed to the cluster magnetic octupole12. An external magnetic field is applied normal to the film surface to align the cluster magnetic octupole along z-direction as well as the small magnetization vector. Thus, the electric field parallel to the sample surface (x-direction) induces the anomalous Hall conductivity Figure xx(ω) from 2 to 10 meV (ω/2π = 0.5 to 2.5 THz) were obtained in the thin-film approximation. Figure σxx(ω) in Mnx3+Snx1− thin films at various temperatures (x = 0.02). We fit the data by using the Drude model σ0 is DC conductivity and τ is carrier scattering time. The solid curves in Fig. τ as a function of temperature is shown in the inset to Fig. 3Sn thin film36 and is discussed later.By conventional transmission THz-TDS (see Methods), THz longitudinal conductivity spectra σσxx(ω) in Fig. σxy(ω) is also in the DC limit. This is because the microscopic mechanisms are different between σxx(ω) and σxy(ω). σxx(ω) is well described with Drude model where any kinds of momentum scattering are involved. On the other hand, σxy occur without any scattering process or only with impurity-induced scattering, depending on its intrinsic or extrinsic origin. In particular, in addition to the Weyl semimetallic bands, Mn3Sn also has other metallic bands34 which dominates the longitudinal conductivity. Since the intrinsic AHE origantes from the integrated Berry curvature of the occupied states over the entire Brillouin zone, the frequency dependence of σxy is not obvious even if the scattering time in σxx is known and therefore to be investigated experimentally.Note that, although the THz longitudinal conductivity 40. WGP1 defines the polarization of the incident THz electric field as x direction, and WGP3 is set to block the x-component THz field before detection. By using two types of configurations for WGP2  and Ey(ω) are obtained. In the small-angle limit, the rotation-angle and ellipticity-angle spectra, θ(ω) and η(ω), are expressed as y-component electric field is maximized41 (see details in Methods). To evaluate the precision of our measurement, without samples we performed 1 scan and 10 scans of THz-TDS with the configs. 1 and 2, respectively, which gives one data set of the θ(ω) and η(ω) spectra within 60 s. With increasing the number of the data sets, the standard deviation of θ(ω) is plotted as a function of frequency in Fig. Figure x3+Snx1− thin films were performed at zero magnetic field after the samples are magnetized under field of 5 T, where the magnetization vector M is normal to the film surface. Figure θ(ω) and η(ω) spectra, respectively, with different film thicknesses of d = 50, 200, and 400 nm at room temperature (x = 0.02). With using a bare SiO2 substrate as a reference, the polarization rotation of ~4 mrad is observed for a 50-nm sample, and the rotation angle increases as the film thickness increases. The broken curves in Fig. M), which corresponds to the reverse of the cluster octupole moment. Upon flipping, the signs of the rotation angle are reversed. The filled circles in Fig. θ(ω) averaged over this energy range calculated by the following formZ0 = 377 Ω and the substrate refractive index ns = 1.92. With the longitudinal conductivity σxx = 3800 Ω−1cm−1 and the Hall conductivity σxy = 19 Ω−1 cm−1, which was evaluated for a 200-nm-thick film in DC resistivity measurements, the calculation reasonably reproduces our THz experimental data (solid line). In the small thickness regime (d ≪ 50 nm), θ is proportional to the film thickness d. In the large thickness regime (d ≫ 50 nm), θ is saturated to be ~σxy/σxx. Slight difference of the magnitudes in θ could be attributed to the difference of geometry; the DC resistivity is measured with electrodes attached on the film surface while the THz conductivity is measured in transmission. These results clearly indicate that the large AHE in Mn3Sn also appears in the THz frequency range as a polarization rotation. It may be also noteworthy that the DC AHE is usually measured under magnetic field by changing its sign to avoid creation of magnetic domains under zero field and to detect difference of Hall resistivity between positive and negative field with removing artifact such as contact resistance. In the present experiment, the Hall conductivity can be clearly observed as light polarization rotation in a noncontact fashion without using magnetic field. We also confirmed that the polarization rotation was unchanged even several months after the sample fabrication and magnetization, exhibiting robustness of magnetic information in the Mn3Sn thin films35.The polarization-resolved THz-TDS measurements for Mnη(ω) is negligibly small for all the samples at room temperature. It is Kramers–Kronig relation consistent with the fact that θ(ω) is flat with a value expected from DC. Using Eq. (xy(ω) and plotted in Fig. σxy(ω) at ω/2π = 1 THz (~4 meV) is as small as ~0.4 Ω−1 cm−1 and become much smaller for lower frequency in contrast to the large anomalous Hall conductivity σxy(ω) indicates the dissipative part of the Hall current24, this result provides a direct evidence for dissipationless feature of the anomalous Hall current in Mn3Sn up to the THz frequency scale, which is also consistent with the intrinsic nature of the AHE driven by the large Berry curvature in momentum space. A recent study of thermal transport in Mn3Sn has also reported negligible inelastic scattering with phonons in the AHE42.Figure sing Eq. , we obtax3+Snx1− (x = 0.08) on a Si substrate at room temperature by using (110) GaP crystals and 20-fs laser pulses with 1-kHz repetition (see Methods). The precision of our broadband measurement was evaluated in the same way as Fig. σxy(ω) spectra up to ~7 THz, in which data with a flipped sample was used as a reference. The broadband data are smoothly connected to the low-frequency measurement. The gradual growths of the dissipative-part Hall conductivity Im σxy(ω) with increasing photon energy can be attributed to a broad optical transition across the band-crossing (or anticrossing) points as discussed for ferromagnets22. While the onset of the interband transition coincides with twice the chemical potential for the model proposed in a ferromagnet22, Im σxy(ω) in Weyl semimetals is expected to show a different spectrum associated with anisotropic cone dispersion32 which is inherent to a pair of Weyl nodes. A recent ARPES study for Mnx3+Snx1− (x = 0.03) has revealed the Weyl-like dispersions at ~8 meV above EF with strong band renormalization by a factor of 5 in comparison with the first-principle calculation34. Notably these Weyl nodes are type-II34, where both electron and hole pockets exist with a strongly anisotropic dispersion as illustrated in Fig. 32. In the present experiment, onset of the interband transition was not clearly discerned, perhaps because it could be obscured by the room temperature thermal energy.To investigate in broader frequency range, we also performed the same polarization-resolved THz-TDS for a 50-nm Mnx3+Snx1− films (x = 0.02) on a SiO2 substrate. The sample was magnetized at 300 K in advance and cooled down under zero magnetic field. Figure σxy(ω) and Im σxy(ω) at higher temperature (300–200 K) and lower temperatures (160–10 K). Figure σxy(ω) averaged between 2 and 10 meV as a function of temperature. As the temperature decreases, Re σxy(ω) is sharply suppressed around 250 K. The similar sharp reduction of the AHE as well as magnetization has been also observed in the DC measurement35. Such a spin-reorientation phase transition has been studied by neutron scattering experiments, which have revealed that the 120° spin order in each ab plane rotates to form a helical spin ordering along the c-axis below 250 K43. Interestingly, although the inverse triangular spin structure in the ab plane of the Kagome bilayer below 420 K breaks macroscopic time-reversal (T) symmetry, the helical spin ordering that develops along the c-axis below 250 K recovers the macroscopic T-symmetry again, which results in the disappearance of the net Berry curvature. Therefore, the drastic reduction of THz AHE at low temperature as well as DC AHE is ascribed to the spin-reorientation phase transition to the helical structure46. In addition to the polarization rotation, a peculiar temperature dependence is also found in the scattering time τ obtained from σxx(ω) in the inset of Fig. 36. This result might be also related to the appearance of the Weyl nodes around which backscattering could be suppressed.We also measured the temperature dependence of the THz AHE for Mnσxy(ω) increases slightly below 150 K. The Re σxy(ω) spectra at the low temperatures in Fig. σxy(ω) spectra in Fig. c-axis48. Note that our THz measurement was performed at zero magnetic field on cooling after the demagnetization process (we first applied a field of 5 T perpendicular to the film surface and decrease the field down to 0 T) at 300 K. In such a situation the macroscopic magnetic moment is much smaller than that in case of field cooling due to random orientation of ferromagnetic domains. The peculiar Hall conductivity spectra at low temperatures in Fig. Figure 3Sn thin films, we observed the large anomalous Hall conductivity of Mn3Sn in THz frequency with negligibly-small dissipation at room temperature. Such a large THz response in the antiferromagnets is desirable and paves the way for ultrafast readout of spin with THz current on spintronic device. The far-field THz polarimtry presented in this work is possible only for large area due to the diffraction limit. For more practical application, THz anomalous Hall conductivity must be measured in much smaller regions. Recently the light-induced THz AHE of a tiny graphene flake has been detected via contacted electrodes combined with optical pulses and THz current generation on chip49. Our demonstration of the THz AHE will lead to such a readout of the spin information on integrated devices.In summary, by using the polarization-resolved THz spectroscopy for the Mn3Sn is highly demanded for ultrafast writing. If the external strong magnetic field is applied to the in-plane opposite direction to the ground state, all of the spins on the Kagome bilayer change their directions simulataneously, resulting in the flip of the cluster magnetic octupole. In terms of the spin precession, it corresponds to the damping of the acoustic collective mode, which could occur at picosecond timescale due to the exchange interaction in the antiferromagnetic metal.Importantly, our all-optical approch in the noncontact way with picosecond time resolution can be extended to pump–probe measurements for the study of nonequilibrium dynamics. THz control of the cluster magnetic octupole in Mn50. Even in static regime, further investigation of THz responses with suppressing thermal energy in another noncollinear antiferromagnetic compound Mn3Ge, where the inverse-triangular spin ordering survives at low temperature9, will clarify interband transition around the Weyl nodes. Higher-frequency infrared polarimetry would be also important for direct comparison with first principle calculation to identify the energies of the multiple Weyl nodes from the Fermi surface.From fundamental point of view, optical control of Weyl antiferromagnetis is also highly intriguing as recently the THz field control of Weyl nodes has been demonstrated in a noncentrosymmetric Weyl semimetal3Sn polycrystalline films (50–400 nm) were fabricated as reported in ref. 35 on thermally oxidized Si (Si/SiO2) substrates and quartz (SiO2) substrates by DC magnetron sputtering from a Mn2.7Sn target in a chamber with a base pressure of <5 × 10−7 Pa. The Mn3Sn layer was deposited at room temperature, and it was subsequently annealed at 500 °C for 1 h. The sputtering power and Ar gas pressure were 60 W and 0.4–0.6 Pa, respectively. The compositions of the Mn3Sn films were determined by scanning electron microscopy-energy dispersive X-ray spectrometry. A hexagonal D019 Mn3Sn phase was confirmed by XRD measurement in Mnx3+Snx1− thin films . The data for 50-nm-thick Mnx3+Snx1− film (x = 0.02) on a SiO2 substrate is shown in Fig. 7, ensuring quality of the thin film.Mn2 plates and then the pulse width is compressed down to 20 fs by chirped mirrors. Broadband THz-TDS up to 7 THz (~28 meV) is realized by optical rectification and EO sampling with 300-μm-thick (110) GaP crystals.As a light source for THz-TDS, we used a mode-locked Ti:Sapphire laser with 800-nm central wavelength, 100-fs pulse duration, and the 76-MHz repetition rate . THz pulses were generated from an interdigitated photoconductive antenna on a GaAs substrate with 50-V biased voltage and 50-kHz modulation frequency. The transmitted THz pulses were detected by electro-optical (EO) sampling in a 2-mm-thick (110) ZnTe crystal with another sampling pulse. For broadband THz-TDS in Fig. t(ω). In the thin-film approximation, the THz longitudinal conductivity spectrum Im σxx(ω) is obtained from the relationZ0 is the vacuum impedance, d is the film thickness, ns is the refractive index of the substrate, and ds is the substrate thickness.In our experiment, we measured the sample and a bare substrate as a reference and then obtained the complex amplitude transmittance x-direction by the first WGP1. The x- and y-components of the THz field just after transmitting the sample are defined as Ex and Ey, respectively. After the sample, other two WGP (WGP2 and WGP3) are inserted before the EO sampling. The angle of WGP3 is fixed to block the x-component THz field, and the ZnTe crystal is set to maximize the detection efficiency of the y-component field. Such a sensitive detection of the tiny y-component THz field is important for high resolution of polarization41. The rotational angle of the WGP2 is controllable during the measurement. We define ϕ as the angle from the x-direction to the transmission direction of polarization for the WGP2. The signal F measured in the EO sampling is expressed asR(ϕ) is the rotation matrix. TWGP2 is the complex Jones matrix of the WGP2, which is expressed ast|| and t⊥ are the complex transmittances of the WGP2 for THz field polarization parallel and perpendicular to the wires. The Jones matrix TWGP2 is used to consider the effect of the finite extinction ratio of the WGP2 for accurate polarization measurement. As shown in the inset of Fig. ϕ = 0°) and config. 2 (ϕ = 45°), and then we obtained the signals F1(t) and F2(t) and their Fourier components F1(ω) and F2(ω), respectively. By solving Eq. (Ex(ω) and Ey(ω) are obtained asSchematic of our polarization-resolved spectroscopy setup is shown in Fig. Ex(ω) and Ey(ω), the rotation-angle and ellipticity-angle spectra, θ(ω) and η(ω), are expressed asBy using θ(ω) and η(ω) are simply described asIn the small-angle limit, xy(ω) is obtained from the relationω → 0). The σxy(ω) spectra of the thin film at low temperature in Fig. In the thin-film approximation, the anomalous Hall conductivity spectrum σmentclass2pt{minimPeer Review File


Simple and robust assays to monitor enzymatic ATP cleavage with high efficiency in real‐time are scarce. To address this shortcoming, we developed fluorescently labelled adenosine tri‐, tetra‐ and pentaphosphate analogues of ATP. The novel ATP analogues bear — in contrast to earlier reports — only a single acridone‐based dye at the terminal phosphate group. The dye's fluorescence is quenched by the adenine component of the ATP analogue and is restored upon cleavage of the phosphate chain and dissociation of the dye from the adenosine moiety. Thereby the activity of ATP‐cleaving enzymes can be followed in real‐time. We demonstrate this proficiency for ubiquitin activation by the ubiquitin‐activating enzymes UBA1 and UBA6 which represents the first step in an enzymatic cascade leading to the covalent attachment of ubiquitin to substrate proteins, a process that is highly conserved from yeast to humans. We found that the efficiency to serve as cofactor for UBA1/UBA6 very much depends on the length of the phosphate chain of the ATP analogue: triphosphates are used poorly while pentaphosphates are most efficiently processed. Notably, the novel pentaphosphate‐harbouring ATP analogue supersedes the efficiency of recently reported dual‐dye labelled analogues and thus, is a promising candidate for broad applications. Novel fluorescently labelled ATP analogues for direct monitoring of ubiquitin activation: Fluorescently modified adenosine tri‐, tetra‐ and pentaphosphates (ATP) analogues changing their fluorescence properties upon enzymatic turnover have been synthesised and applied to both E1s found in humans. Pentaphosphates were found to have superior processing and with the used acridone fluorophore the read‐out showed both fluorescence intensity and lifetime, making this assay versatile in application. Indeed, we identified a fluorescent dye with an acridone core structure that is efficiently quenched by the canonical nucleobase adenine.While triphosphates were poorly processed, we found that also in this case acceptance is improved by elongation of the phosphate chain, that is, tetraphosphates are better accepted than triphosphates. In fact, elongation of the phosphate chain to pentaphosphate resulted in even better analogues enabling us to follow the activation of Ub by both enzymes, UBA1 and UBA6, in real‐time.Recently, we developed γ‐modified ATP analogues that are suitable as model compounds for monitoring enzymatic ATP consumption by fluorescence lifetime readout. Compared to other probes, these compounds contain only one instead of two fluorophores, which promises better enzymatic acceptance. Fluorescence lifetime changes between the intact and the cleaved ATP analogues are caused by the quenching of fluorescence by the nucleobase adeninePET‐based quenchers that have previously been employed in biological assays are guanine In order to overcome this shortcoming, we now investigated fluorescent dyes with long fluorescent lifetimes. Acridone and quinacridone as well as their derivatives are interesting candidates for our purpose, especially because they are extremely photostable,N‐alkylated fluorescent dyes were treated with hot sulphuric acid, which yielded Sacridone‐COOH (2.1) and S2quinacridone‐COOH (4), respectively. For disulfonation of the functionalized acridone, we used 65 % SO3 in H2SO4 to yield S2acridone‐COOH (2.2).Both 2.1, 2.2 and 4 in hand, we performed PESA to obtain insights into the HOMO energies in air. To be efficiently quenched by adenine, the HOMO energies of the fluorescent dyes should be lower than that of ATP (−5.78±0.01 eV)With the water‐soluble compounds 1, 2.1 and 2.2) possess lower HOMO energies than ATP, whereas the sulfonated S2quinacridone (4) is higher in energy. For the acridone derivatives (1–2.2), we measured HOMO energies of −5.91±0.05 eV (1), −5.93±0.03 eV (2.1) and −6.03±0.02 eV (2.2). These findings nicely confirmed our expectation that the HOMO energies are decreasing, the more electron‐withdrawing groups are introduced. Since the measured HOMO energies are lower than that of ATP, we moved on to the synthesis of a γ‐modified ATP analogue bearing disulfonated acridone. Here, we followed a well‐established synthesis strategy. In brief, commercially available ATP was alkylated,2acridone‐COOH (2.2) with N,N,N’,N’‐tetramethyl‐O‐(N‐succinimidyl)uronium tetra‐fluoroborate (TSTU) in situ which was finally coupled without purification to the free amine to yield Ap3‐Dye (5)  in the intact and cleaved state. As a model enzyme for cleavage, we used the ATP hydrolysing phosphodiesterase I from Crotalus adamanteus . Before cleavage, we measured a fluorescence lifetime value of 8.71±0.10 ns. By addition of SVPD, the triphosphate is cleaved and the quencher adenine is released as AMP and separated from the fluorophore. Here, we measured a value of 15.64±0.25 ns, corresponding to an absolute lifetime change of 6.93±0.27 ns. With typical experimental errors in fluorescence lifetime determination in the range of 100 ps, this difference is well suited to use lifetimes for quantification of ATP analogue cleavage. Additional absorption and emission spectra recorded before and after cleavage with SVPD did not reveal a spectral shift assuming a low interaction between the sulfonated dye and the nucleobase adenine (data are shown in the Supporting Information).As a first proof of concept, we determined the fluorescence lifetimes of Ap2acridone‐NHS (Ap4‐Dye (6), Scheme 4‐Dye (6) we found a fluorescence lifetime of 8.74±0.03 ns, which is similar to that of Ap3‐Dye (5) (8.71±0.10 ns).Encouraged by these results, we next focused on the synthesis of ATP analogues with longer phosphate chains to determine their acceptance by UBA1. From our previous experiments on the enzymatic turnover of ATP analogues by UBA1, we knew that modified δ‐tetraphosphates are superior substrates compared to modified γ‐triphosphates in UBA1‐mediated activation of Ub.ϵ‐modified alkylated pentaphosphate. Described synthesis routes of alkylated pentaphosphates that follow PV‐N activation and subsequent substitution often lead to unwanted side‐products.5‐Dye. Diisopropylamino dichlorophosphine (7) is reacted first with pyrophosphate to form a cyclic pyrophosphoryl P‐amidite (8) which is coupled to 6‐azidohexyl phosphate by subsequent oxidation to form 1‐(6‐azido)hexyl phosphoryl cyclotriphosphate (9). The cyclic trimetaphosphate is then opened by adding adenosine monophosphate as nucleophile and MgCl2 to yield ϵ‐O‐6‐azidohexyl)‐adenosine‐O5’‐pentaphosphate (10) in a one‐pot reaction with an overall yield of 11 %. Reduction of the azide followed by coupling of the activated S2acridone using its NHS ester yielded the desired compound (Ap5‐Dye (11), 28 % over two steps, Scheme 5‐Dye 11, the fluorescence lifetime of 9.86±0.19 ns is slightly higher than that observed for the tri‐ and tetraphosphates 5 and 6 . The same fluorescence lifetime of the free dye was measured when 10 was treated with SVPD as for (5+6) (15.64±0.25 ns). The reduced quenching for longer distances between dye and quencher pairs is in accordance to the literature where efficient PET‐quenching takes place on a sub‐nanometre scale.Findings from other groups showed that modified pentaphosphates are even better substrates for nucleic acid polymerases than shorter congenersn‐Dye). This ensures that the observed activity is due to the Apn‐Dye analogue and does not originate from potential contaminations of natural ATP. After preincubation, rSAP was inactivated by heating the mixture to 65 °C for 5 minutes. In the first lane in Figure n) and a concurrent decrease of free Ub and non‐modified E6AP, respectively. The results obtained clearly indicate that Ap3‐Dye (5) is poorly processed by UBA1, while the extension of the phosphoanhydride chain increases activity. In fact, Ap5‐Dye (11) was found to be an excellent substrate for UBA1, superior also to Ap4‐Dye (6).With all three compounds in hand, we tested them towards their performance in an E6AP auto‐ubiquitination assay that was previously shown to be well‐suited to qualitatively evaluate the acceptance of ATP analogues by UBA1 Figure .15, 43 An was neither observed with Ap3‐Dye (5) nor with Ap4‐Dye (6) whereas the pentaphosphate analogue (11) was accepted as substrate. However, the efficiency appeared to be lower than for UBA1.Similar to UBA1, UBA6 is known to support E6AP autoubiquitination.2acridone was followed. The same picture as already seen in the E6AP auto‐ubiquitination assay described above was observed. With Ap3‐dye (5) no increase in fluorescence intensity and thus no activated Ub was detected whereas the tetraphosphate and the pentaphosphate are processed by UBA1 and UBA6. Again, the pentaphosphate 11 shows its superior properties over Ap4‐Dye (6). With UBA1 it is able to adenylate Ub about 2 times faster  and with UBA6 even 6 times faster .Next, we investigated whether real‐time experiments are feasible by using UBA1 as well as UBA6 . A standard multiwell plate reader was used for this purpose and the increase in intensity at 450 nm of S4‐Cy3) (for structure see Supporting Information). Interestingly, at high Ub concentration we observed for all three compounds a reaction inhibition , while at low substrate concentration, Michaelis–Menten kinetics were observed. A possible explanation for this observation is that the UBA1‐SH binding site is non‐covalently occupied by a second Ub upon increasing Ub concentration, thus, inhibiting the transfer of adenylated Ub to the active‐site cysteine of UBA1 by forming a thioester with the C‐terminal glycine carboxylate of Ub. Therefore, we fitted our data  μm for Ap5‐Dye (11) which is more than 7 times lower than the KM for Ap4‐Dye (6) (29.1±3.0) μm and almost 20 times lower than the doubly labelled Cy5‐Ap4‐Cy3 (76.1±6.7) μm. However, the KM for Ub with natural ATP is even lower (0.2 μm) suggesting that our analogues may somewhat interfere with Ub binding.The high affinity binding site is described by 3‐dye (5) as cofactor, while elongation of the phosphate chain rescues activity as already seen in the SDS‐PAGE analysis. Ap4‐Dye (6) and Ap5‐Dye (11) are both linearly processed which shows once more the suitability of our setup. Moreover, the extension of the tetraphosphate chain to Ap5‐Dye (11) increases acceptance of both UBA1 and UBA6 significantly. When all cognate enzymes are present, UBA1 activates Ub approximately 2.9 times faster with Ap5‐Dye (11) as ATP source compared to Ap4‐Dye (6). This tendency is also seen with UBA6 where the activation with Ap5‐Dye (11) is even 4.9 times faster than with Ap4‐Dye (6).Finally, we measured the activation of Ub by UBA1 and UBA6 under conditions, where both thioesters (i.e. UBA1 ∼ Ub and UBA6 ∼ Ub) are unloaded. To do so, we added UbcH5b and E6AP in the same concentration as for the SDS‐PAGE experiment Figure . As showIn conclusion, we developed and explored novel fluorescently labelled adenosine tri‐, tetra‐, and pentaphosphates. The ATP analogues bear a single acridone‐based dye at the terminus of the phosphate chain. Most importantly, the dye's fluorescence is quenched by the adenine residue of the ATP analogue. Fluorescence is restored upon cleavage of the phosphate chain and dissociation of the dye from the adenosine moiety. Thereby the activity of ATP cleaving enzymes can be followed.In comparison to our earlier approaches, in this approach only one dye modification is appended to the ATP analogue. This has several advantages. Obviously, the synthesis towards the probes is simplified making these sensors more readily available. Another advantage is the absence of any modification at the nucleobase thereby rendering the analogues to be superiorly processed by the enzymes investigated here. This might be due to the fact that upon usage by the adenylate‐forming enzymes investigated here, the formed reactive adenylated ubiquitin species is identical to that with natural ATP.5‐Dye (11) best. We could also show that the herein presented analogues can be readily used in real‐time assays to follow Ub activation by UBA1 and UBA6. Notably, using the developed disulfonated acridone, the read‐out can be both fluorescence intensity and lifetime. These characteristics make the herein developed ATP analogues versatilely applicable for future uses, for example, in the high‐throughput screening for effectors of E1 enzymes.By elongation of the phosphate chain, we were able to increase acceptance and reaction velocity significantly and demonstrated this for UBA1 and UBA6 that accept ApThe authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


The electrostatic interaction between these axial hydrogen atoms and the ‐OMe oxygen is stabilising. The effect is explored using high‐level ab initio and DFT calculations in the framework of NBO, QTAIM and NCI analysis across a range of derivatives, and experimentally (19F{1H}‐NMR at −80 °C) for some illustrative examples. The effect is significant in energy terms for a weak interaction, and illustrates a new stereoelectronic aspect attributed to selective fluorine substitution in organic chemistry.We report counter‐intuitive axial preferences in non‐stereochemically biased, selectively fluorinated methoxycyclohexanes. These pseudo‐anomeric effects are apparent when electronegative CF Fluorine chemistry: Selective fluorination in methoxycyclohexanes induces an electrostatic 1,3‐diaxial interaction favouring a counter‐intuitive axial preference. This is in line with diminishing anomeric stabilisation in solvents of increasing polarity. A ‘double bond/no bond’ analysis emerged a decade later which was in line with bond lengths emerging from crystallography and theory.ax1, hyperconjugative interactions between a lone pair of the ring oxygen and the σ*C−O antibonding orbital of the methoxyl group (endo anomeric effect) stabilises 1ax. Subsequently a reciprocal exo‐anomeric was argued recognising hyperconjugation between the methoxyl oxygen and the σ*C−O orbital of the ring oxygen.et al.,n(lone pair) interactions stabilising 1ax and also the axial conformers of 1,3‐dioxolanes such as 2ax, and have argued that such effects contribute to anomeric stabilisation in carbohydrates too. Wiberg et al.,The anomeric effect: It′s complicated’. In that paper, evidence is presented to support a significant role for 1,3‐diaxial CH⋅⋅⋅n stabilisation, for example by exemplifying a significantly increased anomeric preference for 1,3‐dioxolanes 3 over 4, due to the remote electronegative trifluoromethyl groups of 3 which increase polarisation of the axial hydrogens. Thus, the more recent discussion has tended to raise the profile of Coulombic forces and de‐emphasise that of the hyperconjugative rationale when exploring the origin of the anomeric effect. In this paper we show how the difluoromethylene group (CF2) can act as a surrogate endo‐O or can be placed as a substituent to promote axial stability in cyclohexane systems, by fully exploiting electrostatic 1,3‐diaxial CH⋅⋅⋅nO interactions.The first interpretations for the anomeric effect argued destabilising dipole–dipole repulsion between the oxygen lone pairs in the equatorial anomer.2H for hydroxyl (OH)2‐) for oxygen (‐O‐) has had some currency. The flagship arena for this replacement has been the difluoromethylene phosphonate (RCF2P(O)OH2) motif as a hydrolytically stable phosphate (ROP(O)OH2) analogue, a replacement that has been successfully explored in a range of circumstances.The high electronegativity of fluorine has resulted in a rich literature, particularly in medicinal and bioorganic chemistry, using fluorine to tune electronic profiles.exo‐anomeric effect was partially (≈50 %) restored in difluoromethylcyclohexane 6 relative to pyran 5  analogue. NBO analysis of 6 indicated lone pair donation from the exocyclic oxygen into the σ*C−C(F2) antibonding orbital, and a weaker back donation of a lone pair of the axial fluorine into the exocyclic σ*C−O orbital. The study demonstrated too that the solution structure of the maltose analogue 7 was also influenced by an exo‐anomeric preference, an effect that was lost when the CF2 group was replaced by CH2. Understanding fluorine effects is a subject of wide interest, and given this isolated study on regaining the exo‐anomeric effect, it seemed appropriate to explore the endo‐anomeric effect itself with an endo CF2. This required a simpler molecule, one where ring interconversion is not biased by the stereochemistry associated with the additional hydroxyl functionality found with the equatorial substituents in 5 and 6. Therefore in this study a focus was placed on analogues of 2‐methoxypyran 1 where the oxygen atoms are replaced sequentially by difluoromethylene for 8 and 9 and then a double replacement in 10. Indeed an axial preference for 8 has already been reported.11, where the fluorines are located at C4 remote from the methoxyl substituent.In the context of such a replacement it was previously reported that an 5 Figure .18 The pG = eq−ax) for anomers of 8–11 are summarised in Figure 8  and unexpectedly 11  have an axial preference. On the other hand, compounds 9 and 10 have an equatorial preference.Theory analysis was carried out in the gas phase and the resultant free energy differences , and their solution conformations explored by 1H and 19F{1H} NMR  the equatorial conformer dominated8 and 11 at room temperature, ring interconversion was too rapid to resolve the axial and equatorial conformers; however, when the samples were cooled to −80 °C, then the individual AB‐signals of the fluorines in each conformer, were resolved. Hexane was selected as the least polar NMR solvent available to most closely approximate the gas phase calculations, and samples were also run in more polar dichloromethane. For cyclohexane 8 only the axial anomer 8ax is observed in hexane. However, in DCM both anomers are apparent with a slight bias in favour of 8eq. It is well‐known that more polar conformers will be better accommodated in a more polar solvent,μ“; Table S21 in the Supporting Information) indicate that 8eq (μ=3.38 D) is more polar than 8ax (μ=1.86 D). However, the result that 8ax is exclusively observed in hexane is consistent with an inherent axial bias as suggested by gas‐phase calculations . The anomer ratio in DCM is reproduced when the calculations are carried out in a solvent dielectric continuum. NMR experiments were carried out for 11 at low temperature (−80 °C) in both DCM and hexane. It proved difficult to assign the signals for the 11ax and 11eq conformers by standard NMR methods. The alpha C1 hydrogen signal was very broad and featureless in the 1H NMR spectrum at low temperature, and no useful coupling constant information could be extracted to secure assignments. The 19F{1H} NMR AB‐signals were readily resolved; however, the remote nature of the fluorine atoms to the anomeric centre did not offer any discriminating coupling constants to differentiate conformers. In the end, assignments were made by theory, calculating the relative 19F NMR chemical shifts of the anomers at the mPW1PW91/6‐31G(d) level,11ax have chemical shifts within the signals of the AB system for 11 equiv. The theory outcome matches experiment very closely and aided the assignment of the chemical shifts of the anomers of both compounds 8 and 11. From this assignment it is clear that the axial conformer dominates in hexane in a ratio of 11ax/11eq≈10:1, which is in excellent agreement with the calculated Gibbs free energy of 1.34 kcal mol−1 in the gas phase . This ratio is reduced to 11ax:11eq≈3:2 in dichloromethane, which is again in excellent agreement with the calculated Gibbs free energies of 0.27 kcal mol−1 . Interestingly in this case it is actually the more polar conformer 11ax, (μ=3.14) which dominates in hexane and the less polar 11eq (μ=2.85 D) which increases in the more polar solvent. This is counter‐intuitive, but consistent with the 11ax being stabilised by local intramolecular electrostatic interactions, which will be stronger in hexane and weakened by increasing the polarity of the solvent.In order to support these studies experimentally, samples of compounds R Figure . In the 11 cannot be accounted for by nO→σ*C−C(F2) hyperconjugation due to the remoteness of the fluorines. Also dipole/dipole relaxation is inconsistent with the preference of 11ax over 11eq, as 11ax is the more polar of the two anomers. In order to explore further and assess a role for 1,3‐diaxial C−Oaxδ−⋅⋅⋅Haxδ+−C interactions, theory studies on a wider range of methoxycyclohexane combinations were carried out. These explored for example, placing CF2 groups at C‐2 and C‐4 as in 12 and C‐2, C‐4 and C6 as in 13. The outcomes are presented in full in Table S21 (see Supporting Information) and selected examples are highlighted in Figure 12 and 13 the location of the CF2 groups significantly increases the axial preference and particularly so for cyclohexane 13, where the predicted magnitude of the axial preference  is striking. The arrangement of three electronegative CF2 groups in 13 maximises the electropositive nature of the C‐3 axial hydrogens, increasing intramolecular electrostatic stabilisation. When CF2 groups are placed at C‐3, and C‐3 & C‐5 as in 14 and 15 respectively, then these systems revert to a strong equatorial preference due to electrostatic repulsions between 1,3‐diaxial O and F atoms which significantly destabilise axial conformers 14ax and 15ax.The axial preference for E(T) of each conformer into its Lewis ΔE(L) and non‐Lewis ΔE(NL) contributions by NBO analysisE(L) contribution represents the hypothetical energy of the conformer without hyperconjugative stabilization, and ΔE(NL) the energy of stabilization by hyperconjugation. The ΔE(L) energy, which represents classical steric/electrostatic interactions, showed a higher weight than ΔE(NL) for the preference of the axial geometry. Thus, the total steric energy of each conformer was obtained by Natural Steric Analysis ΔE(NSA)E(NCE),ENCE=ΣA,BQAQB/RAB) and atomic charges derived from the Natural Population Analysis (NPA).That electrostatic interactions are the most important driving force for the preference for the axial geometry was evidenced by deconstructing the total energy ΔE(NCE) energy showed a higher contribution than ΔE(NSA) for axial stabilisation, mainly for molecules that had the ability to form strong C‐Oaxδ−⋅⋅⋅Haxδ+‐C interactions, for example, 13ax has a total 28.3 kcal mol−1 higher electrostatic stabilization than 13eq. The total electrostatic stabilization was further deconstructed to atom‐atom electrostatic interactions and those that make the axial geometry more stable are shown to be mainly Haxδ+−Oδ− attractive interactions (see Tables S2–S19 in the Supporting Information for full details). Such interactions were further studied using the quantum theory of atoms in molecules (QTAIM)Accordingly, the Δq(A), atomic energy E(A), first intramolecular atomic dipole moment μ1(A) and the atomic volume V(A) are fulfilled to indicate non‐classical C−Oaxδ−⋅⋅⋅Haxδ+−C stabilising long range interactions (Table S20 in Supporting Information). The NCI method shows isosurfaces that indicate the formation of stabilising C−Oaxδ−⋅⋅⋅Haxδ+−C interactions .Although QTAIM does not show a bond critical point for these 1,3‐diaxial interactions , all of the atomic properties used by QTAIM to characterize a conventional hydrogen bond  and for 13ax this increases to −18.1 kcal mol−1, the system studied here with the highest axial preference . Similar trends are found for 11ax and 12ax. These electrostatic interactions become stronger with the number of electronegative CF2 groups vicinal to the axial hydrogen atoms.Of note are the calculated NPA charges and electrostatic interactions for representative molecules 11ax is more polar than 11eq.They also weaken with increasing polarity as evinced by calculations simulating solvents of increasing dielectric constants such as cyclohexane, chloroform, dichloromethane and acetone (Table S21 in Supporting Information). Consistent with this analysis, the axial preference decreases with increasing media polarity, even though e.g., 9ax, 10ax, 14ax and 15ax have either smaller local electrostatic stabilising interactions between the Oδ‐ax or Fδ‐ax and Hδ+ax atoms, such as −7.1 to −8.7 kcal mol−1 in 9ax and −6.9 to −11.4 kcal mol−1 in 10ax and, consequently, a preference for an equatorial geometry  or for 14ax and 15ax where they have strong C−Faxδ−⋅⋅⋅Oaxδ− destabilising electrostatic interactions  analyses and NMR spectroscopy. A similar NBO analysis was explored for 8ax and 11ax. Rotational energy profiles  for 8ax. The outcome for 8ax mirrors that for carbohydrate analogues 6 and 7,C−C(F2) antibonding orbital. This hyperconjugative interaction for 8ax is illustrated in the orbital image in Figure 11ax is clear and consistent with the remote location of the fluorine atoms.As previously discussed, carbohydrate analogues 8, 12 and 13, the axial preferences are similar or up to three times that of prototype 1. These observations add to the discussion on factors that contribute to the anomeric effect itself and they clearly support a role for intramolecular 1,3‐diaxial attraction between polarised C−H(ax)δ+ hydrogen atoms and the anomeric oxygen δ−OR. NBO does offer some support for exo‐hyperconjugative interactions in 8ax. Given that 11ax, which cannot accommodate such hyperconjugation has more than half the free energy  preference for the axial conformer than 8ax , then it is clear that although hyperconjugation plays a role, electrostatic forces dominate.In conclusion we have been able to demonstrate pseudo‐anomeric effects by placing difluoromethylene groups vicinal to axial hydrogen atoms at C‐3 in methyoxycyclohexanes and in cases, such as The observations here illustrate another aspect of the stereoelectronic influence of fluorine in small organic molecules and offers a design feature for influencing molecular conformation in organic materials or bioactives, and they contribute to the ongoing discussion on the origins of the anomeric effect.The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.