We report asymmetric bioinspired total syntheses of the fungal metabolites emeriones A–C via stereoselective oxidations of two bicyclo[4.2.0]octadiene diastereomers. The central bicyclic scaffolds are prepared in an 8π/6π electrocyclization cascade of a stereodefined pentaene, which contains the fully assembled side chains of the emeriones. The anti‐aldol side chain is made using a Paterson‐aldol addition, and the epoxide of the dioxabicyclo[3.1.0]hexane side chain via ring‐closure onto an oxidized acetal. Our work has enabled the structural revision of emerione C, and resulted in the synthesis of a “missing” family member, which we call emerione D. DFT calculations identified two methyl groups that govern torquoselectivity in the 8π/6π cascade. The emeriones, three highly oxidized and densely functionalized polyketides, were synthesized from two enantioenriched fragments in a Stille‐8π/6π sequence, followed by late‐stage stereo‐ and chemoselective oxidations. This work enabled the structural revision of emerione C. E. nidulans,1) and B (2) share the same absolute configurations, while the bicyclo[4.2.0]octadieneoxide central scaffolds are enantiomeric with respect to each other. Emerione C has a bridging endoperoxide on the central core, and its proposed structure has a stereochemical configuration similar to emerione B.Natural products derived from polyenes that undergo cyclization/isomerization cascades initiated by an 8π electrocyclization have intrigued chemists for decades.3)4)s Figure , bottom,22, which can be prepared in four steps from propargyl alcohol (21) . The relative and absolute configuration of 25 was confirmed via X‐ray crystallography. Silyl protection of the hydroxyl group gave 26, followed by reductive removal of the chiral auxiliary.27) was converted to isopropyl ketone 28 via kinetic enolate formation and trapping with methyl iodide. [10] Removal of the silyl protecting group to give 29 could only be realized with HF⋅pyridine; other fluoride sources resulted in significant retro‐aldol reaction, and deprotection was sluggish under acidic conditions. Stille reaction of 29 with Me6Sn2 gave stannane 13.Our synthesis began with iodide alcohol 2 (Scheme 12 began with conversion of methyl angelate (30) into angelic aldehyde, which was found to be configurationally labile , Sharpless asymmetric dihydroxylation (SAD) proceeded with an improved dr of 86 : 14. Moreover, due to reagent control in the SAD reaction, 19 was isolated with 86 % ee (Scheme S1).19 proceeded with inversion of stereochemistry at C5 to give tetrahydrofuran 34, which contains the appropriate vicinal anti‐diol configuration for epoxide formation.34 to aldehyde 17 (Scheme S2), we hypothesized that the epoxide in 17 could be formed via oxidation of an acetal like 18.34 with aldehyde 35 under acidic conditions gave 36, whose absolute and relative configuration was confirmed via X‐ray crystallography. Noting that the preceding reaction is acid‐catalyzed, we developed a one‐pot procedure from triol 19 to acetal 18. In the event, after completion of the CSA‐catalyzed isomerization of 19 to 34, addition of acetal 37 produced 18, which could be crystallized to 96 % ee.While Upjohn oxidation of epoxide 18 with DDQ produced epoxide 39, presumably through the intermediacy of oxonium 38. To the best of our knowledge, this is the first synthesis of an epoxide from a 1,2‐diol using this approach.[39 gave alcohol 40, which oxidized to aldehyde 17 under Ley–Griffith conditions (TPAP/NMO). Wittig homologation of 17 produced aldehyde 42, which underwent Horner–Wadsworth–Emmons olefination and reduction to obtain alcohol 44. Manganese dioxide oxidation gave verrucosal (45),12.Pleasingly, oxidation of approach.19, 2012 and 13 were combined in a Stille coupling to give pentaene 9 (Scheme 2dba3/P(2‐furyl)3/CuI or the Liebeskind variant (CuTC/Pd(PPh3)4) both successfully delivered product. Interestingly, 9 could be purified via chromatography and fully characterized with no apparent isomerization or decomposition. Upon heating in toluene at 55 °C, 9 slowly (3 d) and cleanly isomerized into a roughly equimolar mixture of 10 and 11, as estimated by 1H‐NMR.9 proceeding with essentially no induced diastereocontrol to produce cyclooctatrienes 47 and 48. These diastereomers then each undergo highly torquoselective 6π disrotatory electrocyclization to 10 and 11, respectively. Pleasingly, 10 and 11 were chemo‐ and stereoselectively epoxidized with m‐CPBA at the least hindered of their three double bonds to give (−)‐emerione A (1) and (−)‐emerione B (2), respectively.To complete the synthesis of the emeriones, 2‐saturated dichloroethane solution of 11 with triplet sensitizer was irradiated , a single endoperoxide adduct (50) was formed. We expected 50 to be (−)‐emerione C; however, comparison of NMR spectra of 50 and literature data for emerione C  as it is depicted in Scheme 50, which may also be a natural product, (+)‐emerione D.When an Oy Figure C, and fo9 to 47 and 48, respectively, are nearly isoenergetic as are 47 and 48 . The two methyl groups in 47 and 48, which end up on the cyclobutane rings of 10 and 11, were found to have opposing influences on the torquoselectivity of the 6π electrocyclization. Replacing the bridgehead (green) methyl .To gain insight into the stereochemical outcome of the electrocyclization cascade, we employed density functional theory (DFT) calculations at the SMD(toluene)‐M06‐2X/Def2‐TZVP//M06‐2X/Def2‐SVP level of theory. The calculations reveal that the two transition states (TS1 and TS2) leading from In conclusion, we have completed an asymmetric bioinspired synthesis of all three emeriones, each with a longest linear sequence of 17 steps. Our synthesis has resulted in the reassignment of the structure of emerione C and the proposal of an additional family member, emerione D. As biological data of the emeriones is limited, current efforts in our lab aim to discover biological activities of these fascinating substances.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.Supporting InformationClick here for additional data file.


The repeatability of T1 and T2 values over 6 measurements in the International Society for Magnetic Resonance in Medicine/National Institute of Standards and Technology MRI system phantom was assessed to demonstrate intrascanner variation. The reproducibility between the 4 clinical scanners was assessed to demonstrate interscanner variation. The same‐day test–retest normal prostate mean T1 and T2 values from peripheral zone and transitional zone were also compared using the intraclass correlation coefficient and Bland–Altman analysis.MRF experiments were performed on 5 different 3 Tesla MRI scanners at 3 different institutions: University Hospitals Cleveland Medical Center , Brigham and Women's Hospital  in the United States, and Diagnosticos da America  in Brazil. Raw MRF data were reconstructed using a Gadgetron‐based MRF online reconstruction pipeline to yield quantitative T1 and 4.7% for T2 for relaxation values, within the range of 307.7 to 2360 ms for T1 and 19.1 to 248.5 ms for T2. Interscanner measurements showed that the T1 variation was less than 4.9%, and T2 variation was less than 8.1% between multicenter scanners. Both T1 and T2 values in in vivo prostatic tissue demonstrated high test–retest reliability (intraclass correlation coefficient > 0.92) and strong linear correlation (R2 > 0.840).The intrascanner variation of values measured using MRF was less than 2% for T1 and T2 are repeatable and reproducible between MRI scanners at different centers on different continents for the above measurement ranges.Prostate MRF measurements of T Click here for author‐reader discussions MRFMR FingerprintingPZPeripheral ZoneTZTransition ZoneISMRM/NISTInternational Society for Magnetic Resonance in Medicine/National Institute of Standards and TechnologyUHCMCUniversity Hospitals Cleveland Medical CenterBWHBrigham and Women's HospitalDASADiagnosticos da AmericaSVDSingular Value DecompositionCVCoefficient of VariationCIConfidence IntervalPI‐RADSProstate Imaging Reporting And Data System11 and T2 measurements in the prostate.1 and T2 values can be used to differentiate between normal peripheral zone (PZ) and prostate cancer,MR fingerprinting (MRF)1 and T2 measurements are also repeatable over time,In order to translate and use MRF meaningfully in clinical practice, the quantitative tissue properties measured with MRF must be repeatable and reproducible.1 and T2 estimates based on the MRF technique using the International Society for Magnetic Resonance in Medicine/National Institute of Standards and Technology (ISMRM/NIST) MRI system phantomThe purpose of this study was to evaluate multicenter repeatability and reproducibility of T22.12; matrix 400 × 400; flip angles 3.38–50°; TR 11.2–14.2 ms; slice thickness 5 mm; 3000 TRs, acquisition time 39 s/slice. A delay time of 5 s was inserted between measurements to ensure sufficient magnetization recovery before beginning the next experiment for both phantom and in vivo studies.This Health Insurance Portability and Accountability Act–compliant study was approved by the local institutional review board, and written informed consent was obtained for all in vivo scans. Experiments were performed on 5 different 3 Tesla MRI scanners  with different software versions  in 3 different institutions: University Hospitals Cleveland Medical Center (UHCMC) and Brigham and Women's Hospital (BWH) in the United States, and Diagnosticos da America (DASA) in Brazil. An MRF–fast imaging with steady‐state precession acquisition designed for use in the prostate2.21 is expected to range between 1000 and 2500 ms, and the T2 between 20 and 300 ms for 3 Tesla systems.1 values of  and T2 values of , denoted by min:step:max (ms), were used to balance between matching accuracy and MRF dictionary size. The dictionary had a total of 5970 entries.In order to efficiently match each measured signal timecourse to the appropriate combination of tissue property values, a precalculated MRF dictionary, which can be used as a look‐up table, was generated using Bloch equation simulations in MatLab . In the prostate region, the T2.31 and T2 values for each voxel. The Gadgetron reconstruction took 17.8 s for each slice.All map reconstruction was performed using a Gadgetron MRF implementation,2.41 and T2 values measured using MRF was validated using the T2 layer of ISMRM/NIST MRI system phantom with T1 values between 307.7 and 2360 ms and T2 values between 19.1 and 248.5 ms. The phantom was placed in the magnet for at least 20 min before the acquisition to reduce any errors due to motion of the water making up the phantom. Six single‐slice MRF measurements were then collected, with a delay of 5 s between measurements, on all 5 scanners. Following this acquisition, data for the same‐day test–retest study were collected on the UHCMC Verio 1, UHCMC Verio 2, UHCMC Skyra, and DASA Verio. The phantom was moved out of the magnet and placed again in the magnet, again allowed to settle for at least 20 min, and another set of 6 single‐slice MRF acquisitions was collected. Neither B0 nor B1 maps were collected in this study. The results from the MRF measurements were compared to the reference values measured and reported by NIST.The accuracy of the T2.5In addition to the phantom study, in vivo experiments were performed in 24 patients with suspected prostate cancer . The protocol used was the same as that described for the phantom study, with the following exceptions: No settling time was required for the in vivo prostate measurements. Also, instead of single‐slice measurements, 2 sets of 2‐slice MRF measurements with no slice gaps were acquired to assess same‐day test–retest reliability. The patients were removed from the scanner and then repositioned between the 2 MRF acquisitions.2.61 and T2 values was assessed using the coefficient of variation (CV), defined as the ratio of the SD to the mean of 6 measurements and expressed as a percentage:1 and T2 values between the 4 clinical scanners were calculated to demonstrate interscanner variation:For the ISMRM/NIST MRI system phantom study, the mean and SD for each sphere were calculated from a circular region of interest (70 pixels in size with a radius of 4.7 mm) that was manually drawn on the maps. For repeatability, intrascanner variation of Tl.k.b., with 13 years of radiology experience) in maps from both scans for all patients. Note that the ROIs from patients (10 pixels in size) were drawn in normal appearing regions (PI‐RADS 1 or PI‐RADS 2) with no specific findings. Mean T1 and T2 values were calculated for each region of interest. The intraclass correlation coefficientFor the in vivo subjects, regions of interest in the PZ and TZ were drawn by a radiologist  with the reference values. The bias for each vial  for each of the 5 scanners, is shown in Supporting Figure The means of the 6 measurements obtained from the ISMRM/NIST MRI system phantom on 5 scanners at the 3 different medical institutions are presented in Figure 1 values between 307.7 and 2360.0 ms and T2 values between 19.1 and 248.5 ms, as calculated by dividing the SD of the 6 repeat measurements by the mean of the 6 measurements (expressed as a percentage). Figure 1 and T2. The T1 estimates had a variation of 0.2% to 2.0%, and T2 estimates had a variation of 0.0% to 4.7%, with the exception of the vial with a T2 value of 19.1 ms, which showed a variation of 8.9%. The interscanner CVs over all 6 measurements are shown in orange in Figure 1 variation of 2.3% to 4.9% for T1 values between 307.7 and 2360.0 ms and T2 variation of 2.3% to 8.1% for T2 values between 40.5 and 248.5 ms. The variation increased in spheres with T2 values of lower than 28.8 ms. The difference between interscanner variations of multiple measurements and the variation in a single measurement is less than 2%.Figure 1 and T2 values in the ISMRM/NIST MRI system phantom were above 0.99 between repeated measurements made on the UHCMC Verio 1, UHCMC Verio 2, UHCMC Skyra, and DASA Verio  for limits of agreement when difference in measurements was plotted against the mean of the measurements  and acceptable agreement . The T2 values from PZ and TZ also showed a strong linear correlation  and acceptable agreement  with corresponding plots presented in Figure The Bland–Altman analysis revealed that 24 of 24 PZ T41 and T2 measurements on 5 different 3T MRI scanners with different software versions in 3 different medical institutions. It also demonstrates the use of a Gadgetron‐based online MRF reconstruction to generate quantitative maps rapidly at the scanner. This implementation enabled the same MRF reconstruction to be used on 5 different MRI scanners in 3 different locations where the personnel had technical expertise ranging from minimal to advanced. Additionally, the improvement in the workflow made possible through the use of the online reconstruction meant that quantitative maps could be provided immediately to the radiologist for annotation and analysis. Coupled with the results demonstrating repeatability and reproducibility, this work paves the way for a Gadgetron‐based MRF framework for quantitative mapping of the prostate to be distributed and used with a variety of MRI scanners around the world.This study assesses the repeatability and reproducibility of prostate MRF‐derived T1 and T2 values found in the ISMRM/NIST system phantom, intrascanner MRF T1 and T2 estimates showed small variations over 6 measurements. The interscanner measurements showed larger T1 and T2 variations between scanners at different institutions, which is similar to the results reported in Ref.1 CV < 2.0% and T2 CV < 4.7%) and reproducibility (T1 CV < 4.9% and T2 CV < 8.1%) for MRF T1 and T2 values in the phantom are similar to the reported prostate apparent diffusion coefficient values. However, T2 values lower than 30 ms and higher than 300 ms demonstrated larger variation. An underestimation of very high T2 values (> 300 ms) in the phantom study was observed as compared to reference values in Figure 2 are not expected to be clinically relevant in the prostate because cancer and prostatitis have much shorter measured T2. The T2 step size in the MRF dictionary was set to 10 ms from 160 to 200 and 50 ms from 250 to 500 ms because such high values were not originally expected to be encountered in vivo. Finer dictionary step size and higher maximum T2 values in the dictionary may improve the accuracy of high T2 values. Similarly, the higher CV seen for vials with a T2 value below 30 ms likely relates to dictionary coarseness (5 ms at this range), which is a substantial fraction of the measured values. A finer dictionary with smaller step sizes could result in an improved test–retest agreement and a lower CV. Other factors that may increase systematic variation of the measured T1 and T2 values ; National Institute of Biomedical Imaging and Bioengineering (P41 EB 015898); National Institutes of Health ; Siemens Healthineers.Wei‐Ching Lo is an employee of Siemens Medical Solutions. No potential conflicts of interest were disclosed by the other authors.Figure S1 Percentage bias in MRF‐FISP T1 (a) and T2 (b) values measured on the UHCMC Verio 1, UHCMC Verio 2, UHCMC Skyra, DASA Verio, and BWH Verio using the ISMRM/NIST MRI system phantom. The MRF‐FISP T1 and T2 values are compared to the reference values measured and reported by NIST.Figure S2 Bland‐Altman plots comparing same‐day test‐retest measurements using the ISMRM/NIST MRI system phantom on UHCMC Verio 1 (a and b), UHCMC Verio 2 (c and d), UHCMC Skyra (e and f), and DASA Verio (g and h).Figure S3 Demonstrative T1 and T2 maps generated using MRF‐FISP in the prostate collected on the UHCMC Verio 1, UHCMC Verio 2, UHCMC Skyra, DASA Verio, and BWH Verio. Values in the two zones were measured from ROIs like these shown here as black circles in the PZ.Click here for additional data file.


Here we report an additional, essential transfer factor of SGI1. This is a small RNA deriving from the 3′‐end of a primary RNA that can also serve as mRNA of ORF S022. The functional domain of this sRNA named sgm‐sRNA is encoded between the mpsA gene and the oriT of SGI1. Terminator‐like sequence near the promoter of the primary transcript possibly has a regulatory function in controlling the amount of full‐length primary RNA, which is converted to the active sgm‐sRNA through consecutive maturation steps influenced by the 5′‐end of the primary RNA. The mobilization module of SGI1 seems unique due to its atypical relaxase and the newly identified sgm‐sRNA, which is required for the horizontal transfer of the island but appears to act differently from classical regulatory sRNAs.The SGI1‐family elements that are specifically mobilized by the IncA‐ and IncC‐family plasmids are important vehicles of antibiotic resistance among enteric bacteria. Although SGI1 exploits many plasmid‐derived conjugation and regulatory functions, the basic mobilization module of the island is unrelated to that of IncC plasmids. This module contains the  oriT and two mobilization proteins, is unrelated to that of IncC plasmids. Here we report an additional, essential transfer factor encoded by SGI1, which is a small RNA. The mobilization module of SGI1 appears unique due to its atypical relaxase and the newly identified sRNA involved in the horizontal transfer of the island.The SGI1‐family mobilizable genomic islands and the IncA‐ and IncC‐family plasmids that specifically mobilize the SGI1‐like elements are major distributors of antibiotic resistance among enteric bacteria. The sophisticated interactions between SGI1 and its helper plasmid guarantee the efficient horizontal transfer and stability of SGI1 in the host cell. Interestingly, the basic mobilization module of SGI1, including  The most efficient distributors of resistance determinants are the conjugative plasmids and the integrative elements (IE), for example, the autonomous conjugative (ICE) and the nonautonomous mobilizable (IME) elements, which need a conjugative helper element (plasmid or ICE) for their horizontal transfer , followed by nicking either strand of the plasmid or IE DNA at the nic site of oriT by the relaxase. Subsequently, the relaxase and the covalently bound single‐stranded DNA are delivered to the type IV secretion system (T4SS) with the assistance of the membrane‐associated coupling protein (T4CP), which binds the cognate T4SS and the relaxosome complex. Finally, the transported DNA strand is recircularized and converted to the double‐strand form in the recipient cell  include HUH nucleases XK restriction enzymes, whereas the MOBT family enzymes belong to the Rep_trans nucleases , and TraI of the Neisseria genomic island GGI (MOBH) appear to be exceptional, which probably have adopted unique molecular mechanisms for DNA cleavage without forming covalent relaxase‐DNA intermediates , its variants, and related elements that form a large family of IMEs are often responsible for the MDR phenotype of human pathogens like Salmonella enterica serovars, Proteus mirabilis, Morganella morganii, Acinetobacter baumannii, Providencia stuartii, Enterobacter spp., Escherichia coli, or Klebsiella pneumoniae strains  and share a conserved backbone , the replication module     and several ORFs with unknown functions    , they are not essential for SGI1 transfer  have been identified  and the oriTSGI1, which is localized within the overlapping 3′ parts of S021 and S022  result was obtained as with SGI1‐CKOS021  deriving from pKK223‐3 and the resulting expression plasmid pJKI881 was used to complement the SGI1‐CKOS021 in a mating assay. For positive control, the entire mobSGI1 region was provided on the plasmid pMSZ949. In this assay, R55ΔTn6187, whose transfer functions are the same as that of the wt R55, was applied as a mobilization helper due to its reduced antibiotic resistance spectrum , which enabled us to apply the KmR complementing plasmids together with it. The results showed that SGI1‐CKOS021 could not be rescued by in trans expression of S021 protein, supporting the previous observations that S021 protein itself is not required for SGI1 transfer. In contrast, the entire mobSGI1 complemented the SGI1‐CKOS021 mutant, although its transfer frequency did not reach that of SGI1‐CWT  knocked out all the short ORFs that the KO S021 mutation might affect  had no deleterious effect as it caused only about twofold and fourfold reduction in the transfer rate of the complementing plasmid and SGI1‐CKOS021, respectively, compared with the positive control. This slight decrease was probably due to the partial withdrawal of MpsA and MpsB, which were now expressed only by SGI1 but utilized for the mobilization of both SGI1 and the approximately 15‐copy complementing plasmid. It is worth noting that this experiment proved again that the S021 KO mutation did not impair the expression of mpsAB operon as SGI1‐CKOS021 expressed MpsA and MpsB proteins in a sufficient amount for the transfer of both elements. Further extensive shortening of the 5′‐end of mobSGI1 fragment was not possible as deletion of mpsAB along with its 28 bp upstream sequence had no negative effect (pMNI71), but the removal of additional 34 bp (pMNI36) completely terminated the transfer of both the SGI1KOS021 and the complementing plasmid. On the other hand, longer DNA sequences could be deleted from the 3′‐end, which influenced the efficiency of complementation at various degrees. Removal of the last 169 bp of mobSGI1 had no significant effect (pMNI37), while larger deletions  abolished the mobilization of SGI1‐CKOS021, but the transfer of the complementing plasmids remained detectable (approximately 2 logs decrease in the transfer rate compared with that of pMNI37). The shortest fully active complementing segment (cloned in pMNI40) was deduced as 1,295–2,065 bp of mobSGI1. Its 5′‐end lies in the noncoding region (NCR) between mpsA and ORF S021 and the 3′‐end is 184 bp upstream of the START codon of S022 in the NCR between S022 and S023.The region of mob1 Figure . Since a2.3SGI1 fragment. Two promoter‐like motifs directed toward ORF S021 were found in the NCR upstream of S021 . In contrast, removal of the next promoter motif P5 caused almost 2 logs to decrease in the transfer rate, which could be fully restored by insertion of Pcat . Finally, the negative control plasmid pMNI41, which contained only the full‐length oriTSGI1, was not transferable. This confirmed again that MpsAB proteins along with the transfer apparatus of the helper plasmid are not enough for mobilization of oriTSGI1 and implied that the most important functional part of the required transfer factor is encoded in the region between mpsA and oriT. The results showed that the RNA factor is expressed mainly from P5 promoter, although P6 may also have a minor role. The 5′‐end of the fully active RNA lies in the sequence corresponding to the 1,966–2,012 bp region of mobSGI1; however, significant activity remains even after removing the large 5′ part of the RNA corresponding to the region between the promoter and oriT. In contrast, deletions affecting the sequences around the START of ORF S021  was inserted into a β‐galactosidase test plasmid pMSZ946 , while in SGI1‐C, the entire P5 was replaced with a cassette containing rrnBT1T2 terminators and the KmR gene (SGI1‐CΔP5)  Figure . The tra) Figure . The red) Figure  has a st2.5SGI1  or its 3′ truncated derivative lacking the downstream part of mobSGI1 from the 3′‐end of oriT (pJKI781). Both plasmids produced MpsAB proteins and contained intact ORF S021 (including the oriT), but in pJKI781, the P3–P6 region, the identified TSS and the sequence encoding the 5′‐end of the putative RNA was absent  or a truncated nonfunctional version of the presumptive RNA. The wt control pJKI1126 carried the mobSGI1 fragment identical to that of pMNI34, which proved previously transferable and could complement SGI1‐CKOS021, whereas pJKI1127 contained the slightly shorter fragment present in pMNI36, which was inactive in transfer and complementation . Every combination of the compatible test plasmids was introduced into the donor strain TG1Nal/R55ΔTn6187 and their transfer rate was measured in a mating assay. All plasmids (except c−) carried intact oriT, therefore, their transfer was detectable in the same mating. As expected, both plasmid partners were transferable (except c−), if the full‐length mobSGI1 was present in the p15A‐based plasmid pJKI780 (this part of the assay can be regarded as a positive control). The maximal transfer rate for pJKI780 was obtained in the presence of the nonmobilizable negative control (c−). This rate was slightly (but not significantly) reduced when pJKI780 was paired with pJKI1126 even though it could produce functional RNA. This observation can be explained by the competition between the two plasmids for MpsAB proteins and other components of the transfer apparatus that are provided by the low‐copy plasmid pJKI780 and the single‐copy helper plasmid, respectively. Titration of the mobilization proteins by oriT of the high‐copy plasmid leads to reduced levels of these components available for the low‐copy partner. Further decrease of pJKI780 transfer occurred when the high‐copy partner carried the 5′‐truncated nonfunctional mobSGI1 fragment (pJKI1127). In this case, competition exists not only for MpsAB and the transfer apparatus but also for the functional RNA produced only by the low‐copy pJKI780. The negative effect of the functional RNA deprivation was more obvious if the transfer of the high‐copy plasmids were compared: in the presence of pJKI780, the transfer rate of pJKI1127 producing the 3′‐truncated  RNA was about two orders of magnitude lower than that of pJKI1126  could only be mobilized if the longer, functional RNA was expressed by the high‐copy plasmid partner (pJKI1126). The transfer rate of pJKI781, however, did not reach that of wt mobSGI1‐bearing counterpart . These results indicated that deletions in the P5 promoter region and in the surroundings of the 5′‐end of S021 affect the same long RNA.The data accumulated so far not only excluded the participation of proteins in the establishment of the phenotype of S021 KO mutant but also explicitly suggested the existence of an RNA factor whose expression occurs mainly from P5 promoter located upstream of S022 Figures . The delt Figure . The pMBn Figure . The emp6 Figure . On the WT and the promoter mutant SGI1‐CΔP5 were compared. One of the probes was complementary to the 5′‐end of RNAs and hybridized near the TSS (5′‐probe), whereas the other overlapped the region affected by the KO S021 mutation and hybridized to RNAs that include sequences located at 470–720 bp from the TSS , confirming that the elimination of P5 promoter significantly decreased the transcription from this region. A 650–700‐base long RNA was detectable with both probes in the wt sample, supporting our results from the trans‐complementation test  can form a hair‐pin structure on the RNA and may be responsible for this effect.Therefore, the RNA species synthesized from P5 promoter region were analyzed by Northern hybridization, where the chromosomal SGI1‐Ct Figure . The 11‐repASGI1/SGI1‐CWT strain, and after joining the 5′‐ and 3′‐ends by T4 RNA ligase the circularized RNA was used as a template for reverse transcription. Then, the 5′–3′‐end junctions were amplified from the cDNA population with primers enclosing the ligation site , this assay suggested the existence of several alternative start sites. The 5′‐end of four RNAs . T4 RNA ligase can circularize only uncapped RNAs and the lack of 5′‐pyrophosphate cap makes RNA sensitive for degradation, which may explain the divergence observed in the 5′‐ends. The similar variance was detected in the other four clones, which started around the 1,957 bp position . The next group of transcripts started at or near 1,911 bp . The −10 box of the nearest predicted promoter P6 is located at 1,986–1,993 bp, thus it seems impossible that it could initiate transcription at a distance of 30 and 75 bp downstream. Even though the sequence of these clones supported the existence of two additional TSSs, promoters could not be found by in silico methods at the appropriate positions. Thus, these putative “TSSs” are formed rather by cleavage of longer transcripts at specific sites susceptible to the attack of endo‐RNases. Interestingly, two RNAs started upstream of the P5 TSS suggesting that weak upstream promoters could also initiate transcription with lower frequency. Nevertheless, the results clearly indicated that many transcripts initiated in the upstream region of S022 terminate in the critical region surrounding the START of ORF S021. These results also suggested that the primary transcripts undergo maturation steps, by which the biologically active transfer factor, called sgm‐sRNA , may be formed. It is worthy of note that the long primary RNA transcripts include the entire S022 ORF, which has an unambiguous SD‐box making its translation possible. Consequently, these RNAs can also serve as mRNAs for S022. Although the production and function of S022 protein were not directly examined, the involvement of this protein in SGI1 transfer was excluded , can be deleted from or replaced in the sgm‐sRNA without negative effects on SGI1 transfer  completely abolished pMNI41 transfer. In contrast, deletions directed from oriT toward the TSS did not, suggesting that the same sgm‐sRNA remained functional in these cases. SGI1‐CΔ7 containing the largest deletion, which removed the oriT, the entire S022, and its 5′‐UTR, was also active in complementation. However, it was about one to three orders of magnitude and less efficient compared with that of SGI1‐CΔoriT or SGI1‐CΔ2 . Considering these and the previous results  of the primary transcript. Mobilization of the oriT‐bearing pMNI41 by the inner deletion mutants of SGI1‐C  shows the best match to the σs Figure . This wa5 Figure , and the Figures  and S5.  Figures . Thus, wSGI1—that are per se not transferable—indicated that the deletion mutations affecting the 5′ and 3′ parts of mobSGI1 are in linkage, which anticipated an RNA spanning the downstream part of mobSGI1 . This sequence motif can form a stem‐loop structure on the RNA and may act as a termination signal or a cleavage site for endo‐RNases  have no serious effect on SGI1 transfer  terminates about 70–80 base from TSS of P5 Figure  and is pHybridization with the 5′‐probe revealed the presence of several further RNA species, whose length ranges between approximately 350 and 600 base and appear similar or less abundant than the 650–700 base long primary transcript. These derivatives can be formed also by early termination or site‐specific RNA cleavage causing 3′ truncation of the transcript and leading to the partial or entire removal of the core region required for the activity of sgm‐sRNA. Thus, most of these RNAs are probably not functional. In contrast, the 3′‐probe covering the core domain of sgm‐sRNA  noncoding RNAs synthesized in intergenic regions from their own promoters, but many of them are generated from 5′‐ or 3′‐UTRs or even from the coding regions of mRNAs by transcription or posttranscriptional processing  of the full‐length primary transcript possibly via posttranscriptional maturation, that is, RNase cleavages  was not necessary for the function, removal of the 1,877–1,966 bp of mob region caused a decrease in SGI1 transfer . This is consistent with the importance of sgm‐sRNA in the mobilization of SGI1‐like elements. SGI1‐B2 from Proteus mirabilis strain PmSC17 carries a large IS26‐based transposon inserted at the proximal end of the core domain of sgm‐sRNA , for example, between the 1,502 and 1,572 bp positions, where the 5′‐end of sgm‐sRNA was localized . The SmR/SpRSulRApSCmSFloSTcS segregant of ST1375, named ST21S/1 emerged during a passage experiment  according to  of donor and recipient strains grown in LB under selection for the chromosomal resistance markers and markers of all additional components  present in the donor strains were mixed, centrifuged for 1 min, washed with 0.5 ml 0.9% NaCl, and spread onto LB agar plates, which were incubated for 6 hr at 37°C. For compensation of the lower fitness of the donors (mainly based on the incompatibility of SGI1 and IncC helper plasmid and the genetic load due to the plasmid content), 3:1 donor/recipient ratio was applied in the matings. At the end of incubation, the bacterial lawn was suspended in 4 ml 0.9% NaCl solution, and 5 μl of serial dilutions was dropped onto selective LB plates to determine the titers of donor, recipient, and transconjugant cells. In cases of low transfer frequency , rare transconjugants were detected by spreading 100 μl (instead of dropping 5 μl) of undiluted bacterial suspension obtained from the mating LB plates. In cases where no transconjugants were detected, the detection limit was calculated as 1 transconjugant/ml divided by the donor or the recipient titer and this fraction was regarded as a threshold value of the detectable minimal transfer frequency in that assay. Titers of recipients and donors were determined by selection for their sole chromosomal marker  except in case of using the E. coli strain Tuner . The transconjugants were selected for the chromosomal marker of the recipient and the transferred marker of SGI1‐C or the test plasmid (TcSp or TcKm). The transfer frequencies were calculated as ratios of transconjugant/recipient and transconjugant/donor CFUs from at least four independent parallels (n ≥ 4).In the mating assays, r Figure  where, i4.3SGI1 in pFOL1372—delS021for‐delS021rev; SGI1‐CΔ2—deloriTfor‐delS022rev; SGI1‐CΔ5—delRNA3for‐deloriTrev; SGI1‐CΔ6—delRNA4for‐deloriTrev; and SGI1‐CΔ7—deloriTfor‐delS022upstream_rev  followed by religation in the case of pFOL1372 (resulting in pJKI773). In the KO mutant ORFs S019–S022, 83‐bp sequences near the 5′‐end of each ORF were replaced with an 84‐bp sequence deriving from the PCR template plasmid pKD3 after Flp‐induced deletion of the resistance marker.All KO mutagenesis were carried out according to the one‐step gene inactivation method . Then, SGI1‐CΔP5 was mobilized by R55ΔTn6187 into TG1Nal::repASGI1 host strain through the TcR TG90 strain in a two‐step mating process.For the generation of TG1Nal::SGI1‐C4.4E. coli TG1 harboring pMSZ946 or pJKI990 (as a negative control) using RNeasy Mini Kit (QIAgen) according to the manufacturer's recommendations. For Northern analysis, RNA was isolated from strain TG1Nal::repASGI1  was used for the extension, while Sequenase version 2.0 DNA sequencing kit (USB) was applied to generate sequence ladder for the test plasmid pMSZ946. Both kits were used as recommended by the manufacturers. The primers lacZoutE and pUCfor24 used for both reactions were labeled as described by Murányi et al. . Product4.6Five micrograms of total RNA samples were separated on a 5% or 8% denaturing TBE‐polyacrylamide gel containing 8 M urea 200 V (~12 mA). Gels were stained for 20 min in TBE buffer containing 0.5 μg/ml ethidium bromide, photographed next to a gel ruler under UV light, then electro‐transferred  and crosslinked onto Hybond‐N+ nylon membranes (Amersham) using the LKB 2117 Multiphore electroblotting unit and Amersham ultraviolet crosslinker, respectively. The template DNA for generation of the 3′‐ and 5′‐end hybridization probes were amplified using primer pairs T3_SGI_17741for–T7_SGI_17994rev and T3_SGI_18219for–T7_SGI_18628rev, respectively. The RNA probes were synthesized with T3 polymerase and the blots were hybridized and developed using the DIG Northern Starter Kit (Roche) according to the manufacturer's protocol.4.7repASGI1 , 40 units of RNase OUT (Invitrogen) in 1× T4 Ligase buffer (Thermo Fisher) in a final volume of 25 μl at 37°C for 1 hr. The enzymes were inactivated at 92°C for 10 min and the RNA was precipitated by 96% ethanol and dissolved in 10 μl nuclease‐free water. For reverse transcription, 20 pmole of SGI_S022promseq primer was added to 8 μl of the circularized RNA and annealed at 70°C for 5 min, then the mixture was cooled on ice for 1 min and used as a template for reverse transcription. The reaction was carried out in a final volume of 20 μl using the RevertAid H Minus First‐Strand cDNA Synthesis kit (Fermentas) according to the manufacturer's protocol. For the amplification of the 5′–3′‐end junctions, S022_promfor_Nc and S021promseq primers were used. The reaction mix contained 1 μl of cc. or 10× diluted reaction mix from reverse transcription or, as a negative control, 1 μl of the 4× diluted circularized RNA sample, 1μl dNTP mix (10 mM each), 10 pmol of primers, 1× DreamTaq reaction buffer (ThermoFisher), and 1.5 u DreamTaq polymerase supplemented to 2.5 mM MgCl2 in a final volume of 25 μl. The cycling conditions were as follows: initial denaturation at 94°C for 2 min, amplification in 35 cycles at 94°C for 20 s, 55°C for 30 s, 72°C for 40 s, and a final synthesis at 72°C for 7 min. The 250–300 bp range of the PCR product was isolated from the gel, digested with NcoI, and ligated into the NcoI‐HincII‐digested pGEM‐5Zf(+) (Promega). Individual clones were sequenced on ABI Prism 3100 Genetic Analyzer (Perkin–Elmer).This experiment was based on the method described by Kuhn and Binder . Five mi4.8Promoter motifs were predicted by BPROM (Solovyev & Salamov, J.K. conceived the project. I.N., J.K., M.S., and A.H. designed and carried out the experiments and analyzed the data. J.K. prepared the figures and wrote the paper and all authors reviewed the manuscript.Supplementary MaterialClick here for additional data file.Fig S8Click here for additional data file.


The E2 state has been proposed to contain a hydride but direct spectroscopic evidence is still lacking. The E2 state can, however, relax back the E0 state via a H2 side‐reaction, implying a hydride intermediate prior to H2 formation. This E2→E0 pathway is one of the primary mechanisms for H2 formation under low‐electron flux conditions. In this study we present an exploration of the energy surface of the E2 state. Utilizing both cluster‐continuum and QM/MM calculations, we explore various classes of E2 models: including terminal hydrides, bridging hydrides with a closed or open sulfide‐bridge, as well as models without. Importantly, we find the hemilability of a protonated belt‐sulfide to strongly influence the stability of hydrides. Surprisingly, non‐hydride models are found to be almost equally favorable as hydride models. While the cluster‐continuum calculations suggest multiple possibilities, QM/MM suggests only two models as contenders for the E2 state. These models feature either i) a bridging hydride between Fe2 and Fe6 and an open sulfide‐bridge with terminal SH on Fe6 (2‐hydE) or ii) a double belt‐sulfide protonated, reduced cofactor without a hydride (2‐nonhydE). We suggest both models as contenders for the E2 redox state and further calculate a mechanism for H2 evolution. The changes in electronic structure of FeMoco during the proposed redox‐state cycle, E0→E1→E2→E0, are discussed.The iron‐molybdenum cofactor (FeMoco) is responsible for dinitrogen reduction in Mo nitrogenase. Unlike the resting state, E Hydride formation versus Fe reduction: Hybrid quantum mechanics / molecular mechanics calculations were used to explore the the E2 redox state of the iron‐molybdenum cofactor of nitrogenase. Two stable isomers were found: a bridging hydride model (with an open belt sulfide‐bridge) and a non‐hydride model (with two belt sulfides protonated and reduced Fe). A path back to the resting state, by H2 evolution via a hydride‐proton reaction was found. Such spin‐vector diagrams were popularized by Noodleman and coworkers to describe the electronic structure of iron‐sulfur clusters.3, Fe4 and Fe6 being spin‐down. The remaining 8 electrons deserve further attention. Three of them belong to the Mo(III) ion  and are curiously in an unusual non‐Hund configuration as previously described.2 models compared to E0 and E1, we will not discuss the Mo electrons further here. The 5 remaining electrons are Fe‐based and are shown as minority‐spin vectors in Figure 0 redox state (two in the Fe4S3C subcubane and one in the MoFe3S3C subcubane) that also features BS7 class determinants as lowest in energy. Compared to E0 we have added two additional electrons to the cofactor; one additional electron into the MoFe3S3C subcubane 2Fe(III) with the two Fe(III) ions located at Fe4 and Fe5 positions, being antiferromagnetically aligned. The position of the Fe(III)‐Fe(III) pair at Fe4 and Fe5 positions can be rationalized as the super‐exchange pathway should be stronger at this non‐protonated belt‐sulfide position leading to stronger antiferromagnetic coupling in the Fe(III)‐Fe(III) pair.By utilizing localized orbital analysis on the BS7‐346 broken‐symmetry determinant the complex electronic structure of the 43 unpaired electrons of this [MoFe2‐hydE model features a rather different electronic structure as shown in Figure 2 and Fe6. The rest of the electronic structure of 2‐hydE resembles more the E0 redox state, formally 3Fe(II)4Fe(III). Different to E0, however, the mixed‐valence pairs, Fe2‐Fe3 and Fe6‐Fe7, are not as delocalized as previously found[6‐Fe7 mixed‐valence pair localizing on the sulfhydryl‐bound Fe6 ion while the minority‐spin electron in the Fe2‐Fe3 pair localizes on Fe3. The bridging hydride in the 2‐hydE model shows some asymmetry, having a shorter Fe2‐H bond (1.64 Å) than an Fe6−H bond (1.72 Å).The sly found12, 142‐hydE, features a bridging hydride between Fe2 and Fe6 and a proton on the terminal S2B in close proximity. Such a structure immediately suggests a possible mechanism for H2 evolution, via direct hydride and proton combination, leading to H2 formation and the reformation of the S2B sulfide bridge between Fe2 and Fe6, resulting in an E0 redox state of FeMoco. We note that we tried stabilizing a possible η2‐H2 intermediate that might form prior to H2 elimination but this was found not to occur. Instead, a saddle point on the minimum energy path towards H2 formation was located starting from the 2‐hydE model. These calculations were performed using the BS7‐235 solution with the α‐195His residue in either the Nδ or Nϵ protonation states for the QM/MM models or with the minimal cluster‐continuum model. The resulting saddle point geometries and activation barriers are compared in Figure The hydride model, His‐Nϵ(H)) or 22.8 kcal/mol (α‐195His‐Nδ(H)). The slightly lower energy barrier for the α‐195His‐Nϵ(H) model is likely due to a stabilizing α‐195His‐Nϵ‐H⋯S2B hydrogen‐bond. Interestingly, the barrier is much lower, 11.6 kcal/mol, when calculated using the minimal cluster model, where the protein environment (including α‐195His) is absent. This lower barrier seemingly arises due to a more favorable saddle point geometry in the minimal cluster model where the hydride is closer to the sulfide‐proton, resulting in a ‘later’ transition state. This may be enabled by increased flexibility in the minimal cluster model, allowing an increased Fe2‐Fe6 distance, seemingly not possible in the QM/MM model, though we note that it's also possible that the protein environment might be capable of rearrangement to further reduce the barrier, something not captured by our current computational model (which is biased towards the resting state E0 structure). Zero‐point vibrational corrections to the barrier were calculated  for the α‐195His‐Nϵ(H) form. This reduces the activation barrier from 20.6 kcal/mol to 18.5 kcal/mol. The zero‐point vibrational energy also allows one to calculate the kinetic isotope effect (KIE) for H/D substitution for H2 formation. The KIE was calculated to be 3.21 which is in good agreement with the experimental KIE estimates .The QM/MM calculations predict relatively high activation barriers of either 20.6 kcal/mol , the stability of a bridging‐hydride structure with an open sulfide bridge which in turn results in a higher activation barrier for H2 formation may thus be beneficial for the enzyme in slowing down the H2 relaxation and allowing build‐up of more reduced states E3 and E4, to which N2 will eventually bind.The saddle point geometries reveal partial H−H bond formation between the hydride  and the sulfide‐proton at the same time as the sulfide bridge reforms between Fe2 formation, starting from a bHCBS(S2B) model, using a minimal cluster‐continuum model approach (where the homocitrate was modelled as glycolate) and the BP86 functional. Such a closed sulfide‐bridge hydride model is predicted to be rather high in energy  compared to our favored 2‐hydE model ‐OBS(6)) at our TPSSh‐QM/MM level of theory. Attempts to locate a similar closed‐bridge saddle point were not successful , likely due to the different representation of the protein environment and the different theory level.We note that in the study by Khadka et al. the authors also located a saddle point for H2 formation saddle point in shown in Figure 0 state and interestingly the minority‐spin electron between Fe2 and Fe3 is localized on Fe2 instead of Fe3 as in the 2‐hydE structure in Figure 0 BS‐state the electron is more delocalized6‐Fe7 pair, the minority‐spin electron instead shows more localization on Fe7. The localization of the electron at Fe2 likely contributes to H−H bond formation by increasing the hydricity. The ability of the minority‐spin electrons in the mixed‐valence pairs of FeMoco to delocalize or localize depending on the situation as seen here, may be behind some of the interesting reactivity that this cofactor exhibits.A localized orbital analysis of the H2 redox state of FeMoco in terms of hydride and non‐hydride isomers while accounting for multiple broken‐symmetry states for each model with a detailed explicit treatment of the protein environment. We demonstrate that for hydride models there is a strong thermodynamic preference for opening up the protonated belt sulfide bridge, that is, to convert a bridging, μ‐SH group to a terminal SH group on one of the Fe atoms. This appears to stabilize the bridging hydride, similarly to what we previously found for bridging hydrides of E4 models.2 and Fe6 and with a terminal sulfhydryl group residing on Fe6.We have presented a detailed exploration of the potential energy surface of the E2‐nonhydE model where 2 electrons have reduced the Fe ions of the cofactor and 2 H+ are present on sulfides S2B and S5A. We note in this context that EPR studies under turnover conditions have revealed two distinct S=3/2 signals (known as 1b and 1c) with notably different g‐tensors  and with an estimated energy difference of ∼1–2 kcal/mol (1b being more stable).0(H+) state .2‐hydE and 2‐nonhydE models suggested in this work. The study by Lukoyanov et al.2 redox level based on the known photoreactivity of metal hydrides. It was furthermore shown by photolysis of 1b that a photogenerated version of 1c (1c*) can form that was suggested to be a different hydride isomer. While a non‐hydride isomer for 1c was deemed unlikely by the authors of the study since a non‐hydride E2 isomer had been previously captured by photolysis of the E4 state , this may simply mean that multiple E2 photoproducts may form in these experiments.Surprisingly, however, it is as energetically favorable to not form a hydride at all, as in the 2 redox state under turnover conditions. Future calculations of EPR g‐tensors of QM/MM models may be one possibility; however, this is highly challenging, even for mononuclear complexes.[57Fe isotope labelling of FeMoco becomes possible.57Fe nuclear resonance vibrational spectroscopy  with a fairly delocalized electronic structure, to our favored model of the 1‐electron reduced 1E state (with a reduced Fe in the MoFe3S3C sub‐cubane and a protonated S2B belt sulfide) that exhibits a slightly more localized electronic structure. From the Fe‐reduced, S2B‐protonated 1E model one can reduce the Fe ions again, while protonating S5A, which leads to the 2‐nonhydE model with an even more localized electronic structure, probably best described as 5Fe(II)2Fe(III). Alternatively, the 2‐hydE state can form where the electrons are instead stored as part of the bridging hydride with the rest of the cofactor remaining rather similar to the E0 state . From the 2‐hydE model a direct path towards H2 evolution is easily imagined and we were able to locate a saddlepoint (2‐hyd‐TSE) for H2 formation and relaxation of the cofactor back to the 0E state.Our preliminary proposal for an 2‐nonhydE model is geometrically much closer to the proposed 1(S2B)E model (requiring only Fe reduction and proton transfer to S5A) and hence it seems possible that upon reduction of the E1 state, this non‐hydridic E2 state could form first, before relaxing to the slightly more stable hydride species, 2‐hydE (and relaxing back to E0 via H2 evolution). Details about the nature of electron/proton transfer pathways in nitrogenase are scarce, however. With these two E2 models being close in energy this further begs the question whether an E3 state might initially form from a hydridic E2 state or a non‐hydridic E2 state. Formation of E3 from the non‐hydridic state, however, seems unlikely as both the Fe sub‐cubane and the Mo sub‐cubane are in their reduced forms. With the two electrons being primarily localized on the hydride in 2‐hydE (likely to apply to other hydride models as well) this should make reduction of the Mo sub‐cubane a more likely reduction to reach the EPR‐silent E3 state.The 2 model using the QM/MM or minimal‐cluster model approach. Bond lengths, bond orders and Hirshfeld charges of selected E2 models. Localized orbital analysis for E0, E1 and E2 models. Information on hydrogen orientations for E2 models. Cartesian coordinates for all optimized structures.Relative energies and Mulliken spin populations of all BS states calculated for each EThe 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.Supporting InformationClick here for additional data file.


The real‐time analyses reveal an amorphous Na–SexSy intermediate phase appears during the direct conversion from SeS2 to Na2S, and a reverse reaction succeeds at 100 °C with a prior formation of Se. The absence of polysulfides and a much lower desodiation temperature in contrast to Na–S nanobatteries demonstrate that the Se incorporation significantly lowers the conversion reaction barrier. According to these findings, the ASS SeS2 batteries using a Na3SbS4 solid electrolyte (SE) are assembled using various SE:C ratios in the composite cathodes to investigate the effect of the ion and electron transport on the electrochemical properties, including the effective transport properties, MacMullin number, and the tortuosity factor. The obtained results in turn confirm the findings from the in situ TEM. These findings are applicable to optimize other S‐based active materials and improve their utilization.All‐solid‐state (ASS) Na–S batteries are promising for a large‐scale energy‐storage system owing to numerous merits. However, the high conversion reaction barrier impedes their practical application. In this work, the basic mechanism on how Se catalyzes the conversion reaction in the Na–S batteries is unraveled. The sodiation/desodiation of Na–SeS xSy intermediate, promoting the transformation from SeS2 to Na2S without the formation of polysulfides. The reverse desodiation accomplishes at a low temperature of 100 °C.Se incorporation lowers the energy barrier in a Na–S battery and thereby catalyzes the reversible conversion reaction. The real‐time transmission electron microscopy discovers an amorphous Na–Se Typically, Na–S batteries work at room temperature with a higher specific capacity and lower energy consumption compared to their high temperature counterparts are highly anticipated. Nevertheless, similar to the Li–S batteries, the Na–S batteries also suffer the polysulfide shuttle effect, which causes the bottleneck issue regarding the irreversible capacity loss and rapid capacity degradation during electrochemical cycling. One of the methods to eliminate the shuttle effect is to use ASS Na–S batteries with a SE as the ion conductor.The full utilization of intermittent renewable energies requires large‐scale energy storage systems with high safety and reliability.1 Na–S Among them, Se is particularly attractive, not only because Se and S are in the same group in the periodic table, but also because Se doping effectively accelerates the reaction dynamics of S, and alleviates the formation of polysulfides, as is extensively studied in the liquid‐electrolyte‐based batteries. For various Se–S ratios, SeS2 gains a high ionic conductivity and decent electronic conductivity, thereby an optimal composition for ASS Li–S batteries with good rate performance. In addition, SeS2 has a high theoretical capacity of 1342 mAh g−1.Another drawback of the Na–S batteries is the low electronic and ionic conductivities within the elemental S active material, especially in ASS batteries using SE with solid–solid point contacts. The intrinsic modification and the optimization of cathode components are required for properly utilizing S. Accordingly, high conductivity elements such as Fe, Ti, Se, Te are usually introduced for S to improve the electrochemical activity.5 Amon In situ TEM equipped with charging/discharging device can not only monitor the real‐time phase transition during the electrochemical processes, but also analyze the reaction kinetics by calculating the migration rate of the reaction front (RF). Generally, various carbon materials such as spherical mesoporous carbon, grapheme, and carbon nanotube were used as S/Se carrier for carrying out the electrochemical processes. The in situ TEM studies on the charging/discharging process of the ASS Na–S nanobatteries reveal multistep sodiation transformations from S to Na2S via polysulfides , and the Na2S sodiation product cannot be reversibly oxidized until ≈300 °C. The discovery of the intermediate phases and the evaluation of the RF migration are important to understand the redox reaction kinetics during the electrochemical processes.However, the fundamental science for the role of Se in Na–S system is still unclear so far. One of the reasons lies in the limitation of the characterization method due to the moisture sensitive nature of Na‐ and S‐based products. In view of this point, real‐time characterizations such as, in situ Raman, X‐ray diffraction (XRD), transmission electron microscopy (TEM) etc., filled with inert protective atmosphere are particularly suitable for evaluating ASS Na–S batteries to provide valuable and reliable information.9 In s Such an optimization is more important in ASS batteries due to the heterogeneous three‐phase disperse system in the composite cathode, especially for S‐based active materials with low electron and ion conductivities that need carbon and SE conducting additives to provide sufficient electron/ion transport. In addition to a proper compositional proportion, the corresponding quantitative evaluation of ion/electron transport in the composite cathode is worth to make clear, which is also a key factor to improve the battery properties.The component optimization in the cathode is significant for realizing high capacity and good rate performance for ASS batteries. The quantification of the effective charge transport and the tortuosity factor have been used to assess the ion/electron transport bottleneck in an electrode for the batteries using liquid organic electrolyte, and thereby optimizing the component design of the electrode.13 Suc2 batteries and reveals the significant influence of Se incorporation in the S‐based active material. We use SeS2 rather than SeaS1‐a to avoid elemental Se and S and other impurities. The amorphous Na–SexSy intermediate phase generated during the conversion reaction is observed for the first time, and demonstrates a notable feature to lower the reaction energy barrier. Moreover, the comprehensive in situ TEM investigation figures out the dominant factor of the electrochemical conversion in the Na–SeS2 nanobatteries. The TEM findings guide the instruction toward the component design of the cathode. The ASS Na–SeS2 battery with an optimized composite cathode delivers a high reversible capacity and excellent rate capability. The corresponding evaluation of the reaction kinetics through the galvanostatic intermittent titration technique (GITT) and in situ electrochemical impedance spectra (EIS) in turn verify the TEM result, demonstrating a reasonable optimization strategy regarding Na–S batteries.In this work, a study of in situ TEM implemented with nanobatteries gives the fundamental insight into understanding the structural and chemical evolutions on the ASS Na–SeS22  as cathode. Figure2@CNT  during the sodiation process at room temperature. When SeS2@CNT was attached initially with Na, there is no reaction on the contact surface , the 2nd RF is observed (marked with a black triangle), indicating the formation of new phases, as shown in Figure 2@CNT can be distinctly observed. The diameters of the three selected regions representing before, partial, and complete sodiation were measured, respectively, as 210, 260, and 310 nm, as shown in Figure 2S and Na2Se. The volume expansions of SeS2 are calculated to be 53% and 118%, which are smaller than the theoretical value of 170% due to the confinement of the CNT. The diameter variation and volume expansion of various SeS2 for the nanobatteries at different voltages are summarized in Table The nanobatteries for in situ TEM observations were constructed using the carbon‐nanotube‐encapsulated SeSe Figure . When a e Figure , the 1st2 is identified from the EDP. Remarkably, in region #2, an amorphous instead of crystalline phase was probed, as indicated by the amorphous halos. This demonstrates that an amorphous Na–Se–S intermediate phase first forms during the sodiation, which is designated as Na–SexSy, though its detailed composition is unknown yet. Region #3 coexists amorphous and crystalline phases. The diffraction rings are associated with (111), (220), and (311) of Na2S, indicating that the first formed crystalline product is Na2S. This is different from the previous result that Na2Se first generated in the Na–SeS2 batteries using liquid organic electrolyte. The EDP analysis at region #4 exhibits that (111) and (311) diffraction rings belong to Na2Se appear in addition to Na2S. Hence, the discharging of single‐crystalline SeS2 is supposed to follow the sequence of SeS2→Na–SexSy→Na2S→Na2S/Na2Se  were collected at four different regions from intact to fully reacted (sodiation), marked with red (#1), blue (#2), yellow (#3), and green (#4) circles, as illustrated in Figure e Figure .2 as an active material was carried out in the same test conditions, as shown in Figure 2 during the sodiation process are the same as those of single‐crystalline SeS2, demonstrating a generalizability of the phase evolutions during the sodiation process in Na–SeS2 batteries, regardless of the crystallinity of SeS2. The high‐resolution transmission electron microscope (HRTEM) image on region #3 displays only the amorphous phase and crystalline Na2S, as shown in Figure 2S. No sodium polysulfides is observed. The HRTEM result confirms that the crystalline Na2S was generated directly from the amorphous Na–SexSy phase.To ensure the reliability and generalizability of the TEM result, the reproducible experiment using amorphous replacing crystalline SeS2 at 20 and 38.5 eV are identified from the spectrum of region #1  were conducted at these four regions to distinguish the element variation during the different sodiation stages, as shown in Figure 1 Figure . By incr2 evolution states. The single crystalline SeS2 evolved to amorphous Na–SexSy at the beginning of sodiation. Then the amorphous phase directly generated the crystalline Na2S. The sodiation eventually forms the crystalline Na2Se in addition to Na2S. The operando observations are in accordance with the results from the in situ measurements on the four selected regions shown in Figure 2 nanobattery, i.e., SeS2→Na–SexSy→Na2S and finally Na2S/Na2Se. This is significantly different from the sodiation process in Na–S nanobatteries, which exists step‐by‐step transitions with the intermediate phases including Na2S5, Na2S4, and Na2S2. The different scenarios indicate a lower sodiation‐reaction‐energy barrier for SeS2, which can be attributed to the formation of Na–SexSy by Se incorporation.To monitor the evolution process during sodiation at a fixed position, operando EDPs Figure  were per2 nanobattery, the reaction kinetics were investigated systematically with the in situ TEM technique for the sodiation and desodiation processes. First, the sodiation was performed at different bias voltages of −1 and −3 V, respectively, as shown in FigurexSy) is measured from the distance between the 1st and 2nd RF. The velocity of the RF migration can be used to evaluate the sodiation reaction kinetics. Figure I–V curve applied on the Na–SeS2@CNT nanobatteries. The current increases with increasing bias voltage, indicating an accelerated electron transport. Hence, the enhanced driving force at −3 V during sodiation can be ascribed to the elevated electronic transport capability. The length of the amorphous Na–SexSy region is shown in Figure To evaluate the different influences of the ion and electron transport on the redox reaction of the Na–SeS2S oxidation. Two approaches including an increase of the bias voltage and temperature were adopted to systematically investigate the desodiation process. First, the Na–SeS2 nanobattery was discharged at a voltage of −3 V and sustained for 118, 408, 910, and 1609 s  process, the desodiation process is more complicated because of the higher energy barrier for the NaT Figure  changed T Figure  was appln Figure . Howevers Figure  were cars Figure  captured2S and Na2Se was thus investigated at different temperatures. The presodiation on the Na–SeS2 nanobattery was performed at −1 V for 2364 s, as shown in Figure FigurexSy intermediate phase  for Na–SeS2 compared with that for Na–S ASS battery can be attributed to the existence of Se, which can provide an enhanced ionic conductivity for lowering the decomposition barrier of Na2S.Generally, a higher temperature enable faster ion transport. The desodiation of Nae Figure  and Na2Se Figure . The temC Figure . An obviC Figure  induced s Figure  indicates Figure , the nans Figure  are obses Figure , indicat2S and Na2Se. In contrast, elevating the ionic transport capability attains the decomposition of Na2S and Na2Se, demonstrating that the sluggish ion transport is the dominated factor to hamper the oxidation of Na2S and Na2Se. Second, the sodiation process of the ASS Na–SeS2 nanobattery follows the sequence of SeS2→amorphous Na–SexSy→Na2S and finally Na2S/Na2Se. That is, the crystalline Na2S is generated from amorphous Na–SexSy, but not from SeS2 directly, nor from sodium polysulfides. Third, the decomposition temperature of Na2S for the Na–SeS2 nanobattery is intensively reduced from ≈300 to 100 °C, which is attributed to the lowered decomposition energy barrier by Se doping. The corresponding conversion processes are schematically displayed in Figure Based on the above‐mentioned in situ TEM analyses, several conclusions can be given. First, elevating the electronic transport capability facilitates the sodiation process, but contributes little for the oxidation of Nam(SE)/m(C) ratios were investigated. m(SE)/m(C) represents the mass ratio of the solid electrolyte (SE) to carbon additive (C). The values for each component and corresponding designations  were summarized in Figure2 active material was fixed to 30 wt%. The m(SE)/m(C) ratios are in the range of 1.33–6, enabling various ionic and electronic conductivities for the composite cathodes. The X‐ray diffraction (XRD) patterns of the cathodes are shown in Figure 3SbS4 phase. The diffraction peak at 26.4° indexed to carbon is observed in 3‐4‐3 and 3‐5‐2, because of their relatively high carbon‐additive contents.The above conclusions can be used to guide the design of the composite cathodes in ASS batteries, which in turn can verify the correctness of the results based on the nanobatteries. Accordingly, the electronic and ionic conductivities were involved into the fabrication of composite cathodes. The composite cathodes with different −1, as shown in Figure 2Se and Na2S, respectively. Obviously, the oxidation currents at these peaks increase with increasing the SE fraction in the cathode. It is notable that the oxidation peaks are very weak for 3‐4‐3, indicating that a high C (30 wt%) and low SE content (40 wt%) cannot overcome the energy barrier to realize the reversibility of the active material. This confirms the in situ TEM results that the improvement of ion transport can effectively accelerate the decomposition of Na2S and Na2Se, promoting the utilization of the active materials. That is, inadequate ion transport in the composite cathode would result in impeded oxidation of Na2S/Na2Se.To analyze the reversibility of the discharge products, cyclic voltammetry (CV) tests were carried out for the 3‐4‐3, 3‐5‐2, 3‐6‐1 composite cathodes in a voltage window of 0.5–2.8 V with a scan rate of 0.1 mV s2 (1342 mAh g−1). The 3‐6‐1 battery delivers the highest reversible discharge capacity (494 mAh g−1), while 3‐4‐3 shows almost no reversible capacity. For further exploration of the reason for the difference, the representative 3‐4‐3 and 3‐6‐1 batteries were selected for the galvanostatic intermittent titration technique (GITT) tests, as illustrated in Figure 2S and Na2Se discharging products cannot be decomposed because of an insufficient ionic conductivity. The hindered ion transport for the 3‐4‐3 cathode is confirmed by the in situ electrochemical impedance spectra (in situ EIS), as shown in Figure 2Se/Na2S.The rate performance for different cathodes was conducted with current densities of 0.1, 0.2, 0.3, 0.4, 0.5 C, and then set back to 0.1 C, as shown in Figure 2 active material is 1.34 mg cm−2. At the second cycle, the 3‐4, 3‐5‐2, 3‐5.5‐1.5, and 3‐6‐1 batteries deliver discharge capacities of 71, 205, 451, and 608 mAh g−1, respectively. Even subjected to a higher current density of 0.3 C, the 3‐6‐1 battery still delivers a capacity of ≈330 mAh g−1 with good cycling retention, as shown in Figure −2 based on SeS2  polarization method, which is relevant to the volume fractions of SE (ϕ(SE)) and C (ϕ(C)) in the composite cathode. The volume fractions were calculated from the theoretical density and mass fraction of each component, which are listed in Table σeff) is related to the conductivity of SE or carbon (σ0), the volume fraction (ϕ), and the tortuosity factor (τ2) of transport pathways in the composite cathodeTo gain a deeper insight into the relationship between the ion/electron transport and the fraction of the conducting phases, the quantitative transport analyses for the composite cathodes in ASS Na–SeSσeff, ion) and the effective total electronic conductivity  were measured from the composite cathodes, as shown in Figure 3SbS4  was measured using EIS  was measured using the DC polarization method  and σeff, el−ϕ(C) curves are, respectively, shown in Figureσ0, ion (0.72 mS cm−1) and σ0, el (9334 mS cm−1) values. σeff, ion increases almost linearly with increasing ϕ(SE), indicating faster ion transport regarding a higher ϕ(SE). By contrast, the increase of σeff, elec becomes very slow when ϕ(C) higher than 0.2, indicating that a low volume fraction of C is enough to meet the demand for constructing effective electronic pathways.Herein, the effective total ionic conductivity σeff, ion NM was introduced to further compare the effective electron and ion transport performance with the formulaThe MacMullin number NM thus indicates severer impeded transport in the cathode. As shown in Figure NM values of the ion transport  are in a wide range of 57–2490, significantly broader than those of electron transport . This demonstrates that the ion tranport is the bottleneck limiting the cathode performance, because good electron conducting networks can be constructed by a small amount of carbon additives. Generally, the NM, ion values for a liquid electrolyte are below 20, as liquid can infiltrate the voids among the electrodes. However, the solid–solid point contacts in ASS batteries cause a large amount of voids even pressed under a high pressure. This is another reason to make it a challenge to construct a good ion conducting network in ASS batteries.A larger The tortuous degree of the the charge transport in a composite cathode should be also taken into account for the compositional design. To quantify the tortuous degree, the Bruggeman model is introduced as follows21, and the C additive (acetylene black) were used for preparing the composite cathodes. The three materials with a weight of 0.5 g were mixed in a ball milling device (Fritsch Pulverisette 7) with a rotation speed of 200 rpm for 2 h, followed by a rotation speed of 500 rpm for 1 h. The component design of the composite cathodes is listed in Table The preparations of composite cathodes are as follows. The Narly work.22 The2@CNT refers to Huang’ s method. Initially, an anodic aluminum oxide (AAO) template with the pore size of about 200 nm was heated to 700 °C for depositing a carbon layer with a thickness about 15 nm via C3H6 decomposition. Then the carbon‐coated AAO was submerged in a molten selenium sulfide under 150 °C for the percolation of selenium sulfide into the carbon‐coated AAO. The exceed selenium sulfide was removed by elevating the temperature to 250 °C. Finally, the SeS2@CNT was obtained by etching the AAO template with hydrofluoric acid.The preparation of SeSs method.23 Iniα, 40 kV 200 mA) in the range of 10°–70°. In situ TEM characterizations were carried out in a Cs‐corrected TEM . The electrochemical properties of the ASS batteries were studied on a Neware system. The cyclic voltammetry (CV) tests were conducted on a Princeton 4000 electrochemical workstation by applying a scan rate of 0.1 mV s−1 in the voltage range of 0.5–2.8 V. The GITT tests were charged/discharged at a current density of 0.017 C for 1 h then resting for 2 h. The in situ EIS were conducted in a frequency range of 1 Hz to 5 MHz at voltages of 1.4, 1.2, 1.0, 0.8, and 0.5 V during discharging process and 1.8, 2.0, 2.2, 2.4, 2.6, and 2.8 V during charging process at room temperature.XRD was conducted using Rigaku D/MAX‐2500/PC  at 100 MPa, then 40 mg Na3SbS4 was pressed on both sides of the composite cathode at 200 MPa. Finally, 30 mg Na15Sn4 was pressed on both sides of the as‐prepared Na3SbS4ǀǀSeS2‐SE‐CǀǀNa3SbS4 cell at 300 MPa. For the latter, SeS2‐SE‐C was pressed at 300 MPa and the stainless steel foils were attached on both sides to block the ions.The electron‐ and ion‐blocking cells for DC polarization tests were respectively assembled by using the multilayer Na2@CNT working electrode, metallic Na counter/reference electrodes, and Na2O SE naturally formed on the Na surface. For assembling, SeS2@CNT was dispersed in ethanol, then dropped to an aluminum rod and dried. The metallic Na was scratched onto another side of the aluminum rod. The assembled nanobatteries were constructed on a microelectrochemical systems (MEMS) heating device, which comprises of an electric circuit and a heating circuit. Subsequently, the device was inserted into a Cs‐corrected environmental TEM  for observation.The nanobattery setup consists of a SeS2 batteries were assembled in a home‐made mold with a diameter of 10 mm. The as‐prepared composite cathodes with an area loading of 1.34 mg cm−2, Na3SbS4 electrolyte (100 mg), and Na15Sn4 (50 mg) powders were cold‐pressed under a pressure of 350 MPa for rate and cycling characterizations.The ASS Na–SeSR2 values exceed 0.98, and the p values are less than 0.05, indicating a valid fitting.The Origin software (OriginLab trail version) was utilized for data fitting/analyzing. Due to the heavy workload for sample preparations, only one sample (composite cathode) was prepared for each data point to get a rough comparison. Five data points were collected for each fitting. For all fitting, the The authors declare no conflict of interest.Supporting informationClick here for additional data file.


The target structures were characterized by steady‐state UV/Vis absorption, fluorescence and transient absorption spectroscopy, as well as cyclic and differential pulse voltammetry. Both donor–acceptor dyads show ultrafast Förster Resonance Energy Transfer and photoinduced electron transfer, thereby leading to extremely low fluorescence quantum yields even in the lowest polarity cyclohexane solvent.Two macrocyclic architectures comprising oligothiophene strands that connect the imide positions of a perylene bisimide (PBI) dye have been synthesized via a platinum‐mediated cross‐coupling strategy. The crystal structure of the double bridged PBI reveals all  The synthesis and properties of a perylene bisimide dye bridged by one or two quinquethiophene units are described. The compounds show panchromatic light absorption in the UV/Vis spectrum, multiple reversible redox processes, and a highly efficient charge separation by photoinduced electron transfer. These include purely aesthetic reasons,ectively.4c, 9 5T‐PBI and 2‐PBI(5T) started with the imidization of perylene bisanhydride 1 with sterically hindered amines2 and 3 which could only be accomplished using microwave irradiation as well as elevated temperatures yielding PBIs 4 and 5 (Scheme n‐BuLi and tin source Sn(C4H9)3Cl. Upon Sn–Pt exchange of 6 and 7 with Pt(COD)Cl2 the precursors 8 and 9 could be obtained. At this point, it should be noted that the compounds 4, 6 and 8 prevail as mostly inseparable syn/anti rotameric mixtures in solution. In case of 5T‐PBI, precursor molecules 8 exhibiting a syn‐conformation are a prerequisite for the desired macrocyclization to proceed and accordingly elevated temperatures in order to overcome the calculated inversion barrier of 114 kJ mol−1  were applied for the final reaction.paraphenylenes,5T‐PBI and 2‐PBI(5T) in yields of 30 % and 4 %, respectively (Scheme 2‐PBI(5T) in comparison to 5T‐PBI (vide infra). Target molecules 5T‐PBI and 2‐PBI(5T) were characterized by nuclear magnetic resonance (NMR) spectroscopy and high‐resolution mass spectrometry (HRMS). In comparison to their linear phenyl end‐capped α‐quinquethiophene counterpart 5T (Scheme 5T‐PBI and 2‐PBI(5T) adopt a more rigid all‐syn configuration in solution. Two doublet and two singlet proton signals in the aromatic region of the 1H NMR spectrum for both constrained oligothiophenes confirm the high symmetry . Interestingly, both sets of oligothiophene signals for 5T‐PBI and 2‐PBI(5T) barely differ in the chemical shift suggesting a very similar chemical proton environment, which is also true for the PBI subunit proton signals when compared to Ref‐PBI (Scheme Our synthesis of macrocycles 2‐PBI(5T) in chlorobenzene. While the small set of signals in the aromatic region of the 1H NMR (SI) suggested a very high degree of symmetry (Dh2) in solution, contrarily in the crystal of 2‐PBI(5T) two different manifestations of the molecule are present, presumably due to packing effects in the solid state. One motif has a crystallographic inversion center . Together with the slightly extended interring thiophene‐thiophene bond lengths (1.453(3) Å to 1.477(3) Å) the above discussed factors add up to a strained macrocycle which also leads to electronic consequences (vide infra). Due to the fact that the central PBI chromophore only exhibits a small twist between the two naphthalene subunits of about 3° it can be concluded that the oligothiophene bridges compensate for the major part of the macrocyclic strain energy of 30.6 kJ mol−1 for 2‐PBI(5T) which is slightly more than two‐fold higher as for 5T‐PBI . In comparison to similarly sized macrocycles such as [12]cycloparaphenylene (117 kJ mol−1)−1)2‐PBI5T) can be considered as moderately strainedFigure 5T‐PBI) in dichloromethane (CH2Cl2) with Bu4NPF6 as supporting electrolyte , differential pulse voltammetry (DPV) and spectroelectrochemistry , mark the formation of an anionic  PBI chromophore, respectively. Compared to Ref‐PBI  these reduction events are at about 0.1 V more negative potential which might be due to a macrocyclic ring‐strain induced destabilization of the anionic states that are characterized by a reduced length of the PBI unit along its long axis.2‐PBI(5T) . Due to the nodes which are located at the imide nitrogen atoms, conjugation of PBI to the residues is interrupted. Likewise, due to the meta‐connectivity, the two oligothiophene units are rather independent π‐conjugated units. This interpretation is supported by the almost invariant redox properties as well as by time‐dependent density functional theory (TDDFT) calculations (Table S2), where the oscillator strengths for HOMO–LUMO transitions were determined to be close to zero, which agrees well with the observed UV/Vis absorption spectra (vide infra), that show now charge transfer (CT) band.Two reversible reduction waves for both structures, I Figure  suggest 5T‐PBI the oxidation waves also reflect one‐electron processes , whereas two bridge moieties are simultaneously oxidized in 2‐PBI(5T). Accordingly, the two oxidation potentials Eox,1=+0.39 V and Eox,2=+0.56 V each represent the transfer of two electrons . Therefore, due to the installation of a second semi‐oligothiophene circle in 2‐PBI(5T) tri‐and tetracationic states are accessible. With a better resolution of the peaks in DPV  can be observed. Thus, upon formation of (2‐PBI(5T))2+ the increased quinoidal character of the oligothiophene chain calls for an almost stepwise oxidation to the tricationic (2‐PBI(5T))3+ and tetracationic (2‐PBI(5T))4+ state due to severe Coulomb interactions between the charges in both bridging units. This can be interpreted as an electronic intramolecular cation‐cation communication between the bridged thiophene chains.2‐PBI(5T) do not resemble those of structurally closely related and fully conjugated α‐cyclo[10]thiophene of Bäuerle and co‐workers2‐PBI(5T) also in this structure both oligothiophenes show two reversible oxidation events of which the high energy oxidation process splits in to two CV waves. Thus, the multi‐oxidation process for 2‐PBI(5T) involving four electrons presumably occurs stepwise. Furthermore, the oxidations are in the same range compared to the linear all‐anti oligothiophene analogue 5T .5T‐PBI and 2‐PBI(5T) are nearly a sum of the reference structures Ref‐PBI and 5T. Thus, as envisioned, by connecting both PBI's imide positions with oligothiophene chains a covalently linked donor‐acceptor dyad with independent subunits has been be achieved.While all reduction waves correspond to one‐electron processes of the PBI subunit, the waves for the oxidations of both macrocycles have to be differentiated. For V Figure  a broade5T‐PBI the PBI segment is independently reduced and spectral changes occur only for this part of the spectrum (450–550 nm) while the oligothiophene region (300–450 nm) mainly remains unchanged. In contrast, electrochemical oxidation leads to the selective emerging of oligothiophene cation and dication related bands. The spectral signature for anion and dianion of 5T‐PBI depicted in Figure .−5T‐PBI  due to the two additional phenyl units and the consequent enhanced conjugation length. This band shape similarity of both macrocyclic subunits in comparison with the free donor and acceptor analogues further underlines the fact that both π‐systems have to be regarded as electronically decoupled.This lack of electronic communication between donor and acceptor is further illustrated in the SEC Figures  and f. Um Figure  look muc5T‐PBI and 2‐PBI(5T) were performed in CH2Cl2  the absorption maxima of the macrocyclic PBIs in 5T‐PBI and 2‐PBI(5T) experience a slight bathochromic shift of 6 nm (215 cm−1) and 11 nm (390 cm−1) with an additional decrease of their respective molar extinction coefficients . While only one oligothiophene absorption maximum can be found for the latter macrocycle  the monobridged analogue shows two distinct maxima at 369 nm and 420 nm, respectively. According to the absorption spectrum of 2‐PBI(5T) both bridges are not in full conjugation but rather have to be considered as photophysically independent. Further, the spectral signatures with most intense absorption bands at rather high energy indicate a loss in π‐conjugation due to the rotational twists of the oligothiophene backbone. While Ref‐PBI exhibits fluorescence quantum yields (Φfl) close to unity in many solvents5T is a strong emitter with 53 %5T‐PBI and 2‐PBI(5T) show no emission in polar solvents such as dichloromethane , which favor photoinduced electron transfer .Compared to the structureless absorption of the phenyl end‐capped α‐oligothiophene Φfl below 0.1 % were determined . Due to the distinguishable spectral regions of the oligothiophene bridge donor and PBI core acceptor both subunits can be excited separately. Upon selective excitation of the PBI acceptor at 480 nm in cyclohexane, typical PBI emission with mirror‐image behavior can be observed for 5T‐PBI, whereas 2‐PBI(5T) shows similar but broadened vibronic patterns . Donor chain excitation at 340 nm and 310 nm for both macrocycles 5T‐PBI and 2‐PBI(5T) affords likewise a PBI emission which is attributed to a fast Förster Resonance Energy Transfer to the PBI acceptor unit.Even in cyclohexane only extremely low 2Cl2 . The thereof derived evolution associated difference spectra (EADS) reveal a similar behavior for 5T‐PBI and 2‐PBI(5T) −1 and (0.2 ps)−1 for 5T‐PBI and 2‐PBI(5T), respectively. Thus, the rate of the 2‐PBI(5T)’s charge separation is two times faster which is presumably due to the second oligothiophene bridge which increases the charge separation probability and therefore also the rate. In closely related but less rigid and non‐cyclic PBI dyads functionalized with oligothiophenes the charge separation process of a PBI‐octithiophene dyad5T‐PBI and 2‐PBI(5T). To the best of our knowledge, the unique cyclic molecules 5T‐PBI and 2‐PBI(5T) allow for one of the fastest PET in an imide functionalized PBI‐oligothiophene dyad known in literature which is probably due the rigid structure and the extremely close proximity of donor and acceptor subunits.For a full understanding of the excited state dynamics, transient absorption (TA) spectra were measured upon excitation at 530 nm in CHI Figure . Thus, wm Figure  and f. Tm Figure  within 8In summary we synthesized two novel types of dyads comprising a central accepting PBI core which is bridged by one or two oligothiophene donor chains. Single crystal X‐ray analysis provided unambiguous proof for the complete enclosure of the PBI acceptor by the 12‐membered donor macrocycle. Rigidification and bending of each quinquethiophene semicircle leads to distinct high energy bands in the absorption spectra of both macrocycles in comparison to the linear phenyl‐end‐capped oligothiophene analogue. The imide functionalization of the PBI with oligothiophene(s) ensures the intended electronic and photophysical independency of the PBI and oligothiophene units. Upon photo‐excitation both macrocyclic dyads showed an ultrafast photoinduced electron transfer from the oligothiophene to the perylene bisimide subunit to afford a charge separated state for which both the radical anionic PBI and the radical cationic oligothiophene could be assigned by the comparison of the transient absorption spectra with those obtained by spectroelectrochemistry.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.Supporting InformationClick here for additional data file.