A new diterpene synthase from the actinomycete Catenulispora acidiphila was identified and the structures of its products were elucidated, including the absolute configurations by an enantioselective deuteration approach. The mechanism of the cationic terpene cyclisation cascade was deeply studied through the use of isotopically labelled substrates and of substrate analogues with partially blocked reactivity, resulting in derailment products that gave further insights into the intermediates along the cascade. Their chemistry was studied, leading to the biomimetic synthesis of a diterpenoid analogue of a brominated sesquiterpene known from the red seaweed Laurencia microcladia. Catenulispora acidiphila and its products were identified. The enzyme mechanism was studied by isotopic labelling experiments and usage of substrate analogues with blocked reactivity, resulting in a series of derailment products. Their chemistry was studied, leading to the biomimetic synthesis of a diterpenoid analogue of a brominated sesquiterpene known from the red seaweed Laurencia microcaldia.A new diterpene synthase (CaCS) from  Catenulispora acidiphila and its mechanistic interrogation with isotopically labelled probes and with such GGPP analogues of limited reactivity.The fascinating skeletal diversity of terpenes is generated by terpene synthases (TSs) from a limited number of oligoprenyl diphosphates.2, 3 TC. acidiphila DSM 44928 caught our interest to study its TSs, because this strain was found to emit traces of a volatile diterpene hydrocarbon , suggesting that one of the TSs encoded in its genome should have DTS activity. From the two candidate enzymes returned by a BLAST search one appeared within a phylogenetic tree constructed from 3267 bacterial TS homologues close to several known STSs, while the second showed close phylogenetic relation to known DTSs and exhibited all highly conserved motifs expected for a functional enzyme , and was thus selected for further study. Gene cloning and expression followed by incubation of the purified protein  with GGPP resulted in the efficient formation of several diterpenes  with a 6‐6‐6 tricyclic system, and two hydrocarbons with the skeleton of a 6‐7‐5 tricyclic system, named isocatenula‐2,14‐diene (2) and isocatenula‐2(6),14‐diene (3). This identified the enzyme as Catenulispora acidiphilaCatenul‐14‐en‐6‐ol Synthase (CaCS). A BLAST search and amino acid sequence alignment with the four closest hits suggests that enzymes with the same function may be present in a few other actinomycetes .The actinomycete  Figure S, suggest1–3 . The conversion of all 20 isotopomers of (13C)GGPP, partly synthesised as reported previously and partly made accessible through enzymatic reactions by GGPP synthase (GGPPS) from Streptomyces cyaneofuscatus1–3 carrying the 13C‐label in the expected positions from all labelled substrates  revealed a strict stereochemical course for the geminal methyl groups C16 and C17 that did not show any scrambling of 13C‐labelling between these positions.The proposed cyclisation cascade for R)‐ and (S)‐(1‐2H)IPP,13C)FPP with GGPPS to (R)‐ and (S)‐GGPP, followed by cyclisation with CaCS . The hydride shift resulted in a direct 13C−2H connection in 1 when using (S)‐(1‐2H)IPP, as indicated by an upfield shifted triplet peak  for C11 in the 13C NMR, while a singlet with a minor upfield shift (−0.01 ppm) was obtained with (R)‐(1‐2H)IPP, in agreement with a deuterium located two positions away. The alternative of two sequential 1,2‐hydride shifts from A to B was excluded by incubation of GPP13C NMR as a result of deuterium bound at a carbon next to C11, indirectly giving further evidence for the 1,3‐hydride shift. We have previously shown that the analogous hydride shift occuring in the germacradienyl cation in the biosynthesis of many sesquiterpenes can be taken as an indicator for the absolute configurations of the products, as for 10S configuration the specific migration of the 1‐pro‐S hydrogen (HS) was observed, while for 10R configuration the 1‐pro‐R hydrogen (HR) was shifted.S in A may be taken as an indicator for 10S configuration and thus for the absolute configurations of 1–3 as shown in Scheme The 1,3‐hydride shift from C1 to C11 was followed by using (C to D was evident from an experiment with (2‐2H)DMAPP2H)GGPP, followed by cyclisation through CaCS. The mass spectra of 1–3  demonstrated the deprotonation step at C14 from C to D by the observed molecular ions at m/z=290 and 272, respectively. The reprotonation of D at C3 with an external proton from the medium was demonstrated by incubation of FPP and (3‐13C)IPP2O buffer, resulting in the typical upfield shifted triplet peak in the 13C NMR of 1 indicating a direct 13C−2H connection . Both experiments together also ruled out the hypothetical alternative of an intramolecular proton transfer from C14 to C3 in C that would bypass the neutral intermediate D. GC/MS analysis of the products obtained in the latter experiment simultaneously demonstrated the loss of the same deuterium in the deprotonation from G to 2 as taken up in the C3 reprotonation of D by a molecular ion at m/z=273 .The deprotonation at C14 from E to F was demonstrated using the labeled substrate FPP13C,7‐2H)‐2 for which a triplet peak was observed in the 13C NMR . Along similar lines, the 1,2‐hydride shift from F to G was evident from an upfield‐shifted triplet for C6 of 2 from substrates (2‐13C)FPPS)‐(2‐2H)IPP, generated in situ from (2‐2H)DMAPP with IDI from Escherichia coli,[F to yield 3 by a molecular ion at m/z=273 . If (2‐13C)FPP was replaced by (3‐13C)FPP,13C NMR spectrum , which further supported the 1,2‐hydride shift and ruled out a direct 1,3‐hydride shift from E to G in the biosynthesis of 2.The 1,2‐hydride shift from hia coli,24, 251–3 were determined using the substrate DMAPP together with the stereoselectively deuterated probes (E)‐ and (Z)‐IPP,S. coelicolor13C‐labelled GGPP . This reaction proceeds through a known stereochemical course with attack at C4 of IPP from the Si face.1–3 in which the deuterated carbon atoms serve as configurationally well defined stereogenic centres. The substitution of one of the two diastereotopic hydrogens by deuterium can be monitored with high sensitivity through HSQC spectroscopy. As a result of the 13C‐labelling, the signal for the remaining hydrogen is strongly enhanced, while the signal of the hydrogen replaced by deuterium has vanished. Together with the assignment of the relative orientation of the diastereotopic hydrogens with respect to the naturally present stereogenic centres in 1–3 by NOESY the absolute configurations of all three compounds could be concluded. Similar experiments were performed with (R)‐ and (S)‐IPP,1–3 . Herein, the attack at C1 of prenyl diphosphates is known to proceed with inversion of configuration.1–3  was stereoselectively deuterated, and all experiments simultaneously pointed to the same absolute configurations as shown in Scheme pro‐S hydrogen in A, discussed above as a potential indicator of a 10S configuration, was confirmed.The absolute configurations of A′ and the subsequent 1,3‐hydride shift to B′, the analogues of the natural intermediates A and B, are still possible, the next 1,14‐cyclisation cannot proceed as a consequence of the saturated C14−C15 bond  as biflora‐4,10(19)‐diene (5), the dihydro‐analogue of the natural diterpene biflora‐4,10(19),15‐triene (5 a) from Cubitermes umbratus soldiers,6). Their structures supported an initial 1,10‐cyclisation, but also showed an additional 1,6‐cyclisation not observed in the native enzyme products 1–3 obtained from GGPP. The cyclisation mechanisms for 5 and 6 were investigated by isotopic labelling experiments with 6,7‐dihydro‐GPP and all five isotopomers of (13C)IPP (Table S8), yielding doubly labelled isotopomers of 5 and 6 with GGPPS and CaCS. Similarly, the usage of 10,11‐dihydro‐FPP13C)IPPs, and of synthetic (1‐13C)‐ and (2‐13C)‐6,7‐dihydro‐GPP (Scheme S1) with IPP gave seven more singly labelled isotopomers of 5 and 6. Their 13C NMR spectra  indicated the origin for each carbon in the C1–C10+ C19 and C20 portion. Moreover, the 2JC,C couplings observed in the double labelling experiments with (1‐13C)IPP (labelling at C1/C5) and with (2‐13C)IPP (labelling at C2/C6) gave further evidence for the 1,6‐ring closure in 5 and 6.To further explore the cyclisation mechanism of CaCS, incubation experiments with substrate analogues with partially blocked reactivity were performed. For this purpose, 6,7‐dihydro‐GPP was synthesised (Scheme S1), elongated twice with IPP using GGPPS, and cyclised by CaCS. While the 1,10‐cyclisation to d Scheme . Therefo5 and 6 were determined by the enantioselective labelling strategy using 6,7‐dihydro‐GPP with (R)‐ and (S)‐IPP and with (E)‐ and (Z)‐IPP, or (R)‐ and (S)‐‐6,7‐dihydro‐GPP  with IPP . The experiments with (R)‐ and (S)‐IPP also demonstrated the specific migration of the 1‐pro‐S hydrogen from A′ to B′. The absolute configurations of 5 and 6 are analogous to that of natural 5 a.The absolute configurations of 5 a is also known as a degradation product of obscuronatin (4 a), a known diterpene alcohol from the soft coral Xenia obscuronata4). Therefore, we assumed 5 and 6 may likewise be degradation products of 4, and indeed their formation depended on the usage of MgSO4 during workup , suggesting that the 1,6‐cyclisation in 5 and 6 is not under enzyme control. Their formation can be explained by water elimination through cations H and I. Workup without drying the organic extract with MgSO4 allowed the isolation of 4, followed by structure elucidation through NMR spectroscopy , revealing the structure of an only 1,10‐cyclised enzyme product and thus further supporting the 1,10‐cyclisation as an early event of CaCS catalysis. Labelling of the carbons of the core structure strengthened the structural assignment, this time giving no 2JC,C couplings in the double labelling experiments with 6,7‐dihydro‐GPP and (1‐13C)IPP or (2‐13C)IPP in agreement with the macrocyclic ring, and allowed to follow the biosynthetic origin of the labelled carbons . However, the relative configuration of 4 with its distant stereogenic centres at C3 and C10/C11 could not independently be determined by NOESY. Therefore, the skeleton was rigidified in a reaction with NBS, resulting in the formation of 7 for which the full relative configuration could be determined by NMR‐based structure elucidation . Assuming that the stereogenic centres in 4 are not affected in the conversion to 7, these results allowed to deduce the full relative configuration for 4. Notably, this reaction may have some biosynthetic significance, as vanadium‐dependent haloperoxidases can catalyse similar transformations, which is especially important in the marine environment.[7 a is structurally closely related to 7 and is a known natural product from the seaweed Laurencia microcladia4 was determined through enantioselective deuteration .Compound D to E  and two 6‐7‐5 tricyclic compounds, isocatenula‐2,14‐diene (2) and isocatenula‐2(6),14‐diene (3). Extensive labelling studies gave detailed insights into the cyclisation cascade and the absolute configurations of all three products. Furthermore, substrate analogues with a saturated double bond were synthesised and enzymatically converted, leading for 14,15‐dihydro‐GGPP to a series of derailment products that further supported the mechanistic model for the cyclisation cascade. As substrate analogues may adopt a different conformation in the enzyme than the native substrate, the absolute configurations and cyclisation mechanisms to all derailment products were also investigated by labelling experiments, demonstrating that all compounds are formed from a common substrate fold. Some of the obtained derailment compounds showed structural similarities to known natural products from termites and soft corals and also allowed for a deeper understanding of their chemistry. However, the starting point of our study was the observed diterpene production by C. acidiphila, and although by the present work a diterpene synthase with interesting products has been discovered, none of these diterpenes showed an identical mass spectrum to the compounds observed in headspace extracts from C. acidiphila , suggesting that a different DTS may be responsible for its biosynthesis. Its discovery will be on the list of our future research.In summary, the diterpene synthase CaCS from 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.


To design a low‐cost, portable permanent magnet‐based MRI system capable of obtaining in vivo MR images within a reasonable scan time.A discretized Halbach permanent magnet array with a clear bore diameter of 27 cm was designed for operation at 50 mT. Custom‐built gradient coils, RF coil, gradient amplifiers, and RF amplifier were integrated and tested on both phantoms and in vivo.1‐weighted and T2‐weighted scans showed a good degree of tissue contrast. In addition, in vivo scans of the knee of a healthy volunteer were acquired at a spatial resolution of about 3 × 2 × 2 mm within 12 minutes to show the applicability of the system to extremity imaging.Phantom results showed that the gradient nonlinearity in the y‐direction and z‐direction was less than 5% over a 15‐cm FOV and did not need correcting. For the x‐direction, it was significantly greater, but could be partially corrected in postprocessing. Three‐dimensional in vivo scans of the brain of a healthy volunteer using a turbo spin‐echo sequence were acquired at a spatial resolution of 4 × 4 × 4 mm in a time of about 2 minutes. The TThis work has shown that it is possible to construct a low‐field MRI unit with hardware components costing less than 10 000 Euros, which is able to acquire human images in vivo within a reasonable data‐acquisition time. The system has a high degree of portability with magnet weight of approximately 75 kg, gradient and RF amplifiers each 15 kg, gradient coils 10 kg, and spectrometer 5 kg. However, even this system weights approximately 500 kg, which limits its transportability.A fourth approach to low‐field MRI, which has the advantages of the relative simplicity of a single fixed field strength magnet, no requirement for a magnet power supply, and increased portability, is to use/design a permanent magnet. Although the original commercial C‐arm or H‐arm systems were large and heavy (>1000 kg) with field strengths of between 0.1 T and 0.35 T,0 field that is oriented along the bore, more closely resembling conventional MRI systems.To reduce the weight of the magnet, a large number of distributed small magnets, rather than two very large disc magnets as in most permanent magnet designs, can be used. The most common geometry is a discretized version of a dipolar Halbach magnet,1‐weighted and T2‐weighted images form the essential anatomical sequences, with other sequences supplementing these based on the particular feature of the disease being studied. Although much higher spatial resolution can be achieved at high fields, the loss in SNR at low fields can be retrieved partially by the use of long echo trains in turbo spin‐echo sequences due to the long T2 value of CSF and the very low specific absorption rate intrinsic to low‐field MRI.Recently, our group described the design, construction, and characterization of a larger 27‐cm clear‐bore Halbach array magnet operating at 50 mT.In this paper we design a long echo‐train turbo spin‐echo sequence to image the CSF in healthy adult brain. The Cartesian k‐space coverage is restricted to an elliptical coverage to reduce the data acquisition time to less than 3 minutes for a 4 × 4 × 4 mm isotropic spatial resolution. We also show applications for extremity imaging, in which higher resolution (~3 × 2 × 2 mm) in vivo images of the human knee are captured in just over 10 minutes. Issues of temperature drift and image reconstruction in the presence of gradient nonlinearities are also discussed.22.13) per ring. The array has a clear bore of 27 cm and a length of 50 cm between the two outer rings. The ring diameters were designed using an optimization scheme to give the highest B0 homogeneity; the source code is available at https://github.com/LUMC‐LowFieldMRI/HalbachOptimisation. After measuring the field using a 3D robotic device, additional shimming was implemented by defining a 32.5‐cm‐diameter, 28‐cm‐long cylindrical grid of 15 rings of 60 potential magnet positions per ring. The filling of this grid was subsequently optimized using a genetic algorithm that has previously been shown to be suitable for creating tailored magnetic fields using permanent magnets,0 deviations located toward the edges of this diameter of spherical volume along the z‐axis. The total weight of the magnet, including all components, is about 75 kg.Details of the magnet design have been published previously2 aluminum extrusion profiles. Because the body can act as a very effective antenna, such as coupling to power lines in the surrounding walls,To minimize the environmental noise, the entire setup was placed inside a 62.5 × 62.5 × 85 cm Faraday cage constructed from 2‐mm‐thick aluminum sheets and 32 × 32 mm2.2For the phantom and knee imaging experiments, a 15‐cm‐long, 15‐cm‐diameter solenoid with 57 windings was used as a transmit/receive coil. The solenoid was segmented using three nonmagnetic capacitors  spaced evenly along the length of the solenoid to avoid self‐resonance effects. Impedance matching used an L‐network with 33 pF and 136 (2 × 68) pF capacitors in the parallel and series arms, respectively. There was a 4‐kHz shift downward in the resonant frequency when the knee was inserted. The Q value decreased from 54 to 47 when the coil was loaded with the knee, indicating that coil loss is dominant, as expected.For in vivo brain imaging, an elliptical solenoid was constructed with a width of 18.5 cm, height of 24.5 cm, and length of 20 cm, with 40 windings of 1‐mm diameter copper wire. It is segmented in three places with 82‐pf capacitors, with a 1‐pF to 30‐pF variable capacitor placed in parallel with one of the segmentation capacitors. Impedance matching used an L‐shaped matching network with 101 pF (68 pF + 33 pF) and 150 pF (82 pF + 68 pF) capacitors in the parallel and series arms, respectively. The resonance frequency increased 233 kHz when the coil was inserted into the magnet. The unloaded Q was 88, and loading the coil with a human head resulted in a 4‐kHz decrease in the resonance frequency of the coil and a decreased Q of 78.When either coil was used without the conductive sheet in place, the noise level increased by approximately a factor of 10 due to the “body antenna” effect. When the sheet was carefully wrapped around the entire body, and physical contact with the Faraday shield was present at several points, the noise level was essentially unchanged from a coil loaded with a tissue‐mimicking phantom within the solid Faraday enclosure.2.30 direction of the Halbach array. This results in y‐gradient and z‐gradient coils that are similar in design to a quadrupolar design, but an x‐gradient coil with significantly improved performance compared with a simple saddle design  on most clinical 1.5T and 3T superconducting whole‐body scanners have maximum gradient strengths in the range of 40‐80 mT/m, with slew rates of 150‐200 T/m/s. The GPAs for these systems are capable of simultaneously generating a driving current of 1 kA and voltage of 2 kV over a bandwidth of tens of kHz by cascading the outputs of several pulse‐width modulated stages. Typically the current signal is controlled by active feedback using high‐precision current sensors built into the circuit. Gain accuracy and linearity are typically below 0.05%, and total harmonic distortion below 0.25%.However, for lower field systems aimed at creating low‐cost, low‐maintenance MRI systems, such high gradient performance is not required. In this type of system, spin echo–based sequences with TEs on the order of tens of milliseconds indicate that very rapid switching and rise times (high slew rates) are not required. Spatial resolutions on the order of several millimeters indicate that frequency‐encoding gradient strengths on the order of tens of millitesla per meter are sufficient, and much more inexpensive linear GPA designs can be used. Wright et al developed a desktop MRI system capable of imaging objects approximately 2 cm in diameter. The GPAs used LM12 high‐power op‐amps capable of driving ±10 A into gradient coils with an efficiency of 0.45 G/cm/A.gd of the MOSFETs form two dominant poles. Simulation shows stable operation from 10 μH up to 500 μH. For simplicity, the circuit is shown as three separate, but connected, modules, each of which is described in detail subsequently.Because we do not need very high currents to achieve the desired spatial resolution ~3 mm,) high currents are not necessary, and the maximum voltage (15 V) is chosen such that simple air‐fan and heatsink cooling can be used. Each amplifier was constructed using a push‐pull configuration. The design for each channel is shown in Figure  mm, high18F]fluoroethyl)‐L‐tyrosines are capable of conducting current from the gradient coil back into the power supply. Their TO247 package has a large surface area that results in a low thermal resistance. L2 creates a phantom zero in the feedback loop that moves the poles of the amplifier to a better position for stability and sets the closed‐loop bandwidth of the amplifier to about 7 kHz. R22+C10 will damp the self‐resonance of the gradient coil. This helps stabilizing the amplifier further for a wide range of inductances. The instrumentation amplifier (AD8221ARZ) is a classic three op‐amp design, with 80‐dB common mode rejection ratio up to 10 kHz (unity gain) and a 2‐V/μs slew rate, with 8 nV/root Hz noise at 1 kHz.The input stage consists of an operational amplifier, which provides the necessary voltage gain sufficient to drive the MOSFETS. The output stage consists of a MOSFET rail‐to‐rail common source stage. It consists of two power MOSFETs arranged in a push‐pull configuration. The output MOSFETs are capable of delivering more than 90A and have a RDS ON of 25 mΩ. The fast intrinsic diodes of these O‐(2‐[c = 7 kHz) were placed on the gradient lines to reduce RF noise introduced by the gradient amplifiers and gradient lines entering the Faraday cage.The current slew rate is a function of the maximum voltage (15 V), maximum current used , and coil inductance and resistance, and was measured to be approximately 30 T/m/s. Each gradient amplifier produces an output gain of 3.3 A per 1‐V input from the ±10 V 16‐bit digital‐to‐analogue gradient drivers of the MR console. Figure A Magritek Kea2 spectrometer  was used to generate gradient wave forms and RF pulses as well as digitizing the generated echoes. The RF pulses generated by the spectrometer were amplified by a custom‐built 1‐kW RF amplifier  with the center of the tubes spaced 17 mm apart. Data were acquired using a 2D spin‐echo projection sequence with the following scan parameters: FOV = 256 × 256 mm, 128 × 128 data points, TR/TE = 6000 ms/100 ms, and pulse duration = 100 µs. Images were acquired with the main axis of the phantom in the +y  and +x  axes, both perpendicular to the B2.6All images were acquired from healthy volunteers giving informed consent according to the policies of the local ethics committee.0 homogeneity over the brain, measured as the FWHM of the Fourier transform of the FID signal, was approximately 600 Hz.For brain imaging, all data were acquired with a bandwidth of 20 kHz, a 90º pulse length of 100 μs with a power of 47 dBm, and a 180º pulse length of 100 μs with a power of 50 dBm. Phase cycling of successive 180º pulses in the turbo spin‐echo train followed the regular Carr‐Purcell‐Meiboom‐Gill modulation. k‐Space acquisition was restricted to an elliptical coverage in the two phase‐encoding directions, to reduce scan time. All data were filtered using a sine‐bell squared filter. B2‐weighted sequence with long effective TE. Based on a series of low‐resolution inversion‐recovery images with TI varying between 100 ms and 2800 ms, the T1 of CSF was estimated to be about 1750 ms, with the T2 value assumed to be only slightly lower, as it is well‐known that at very low field T1 ≈ T2 for mobile liquids. Both the T1 and T2 relaxation times of brain tissue are much shorter, being reported on the order of 500 ms and 250 ms, respectively.2‐weighted for bright CSF, T1‐weighted for a rapid anatomical image (CSF dark), and an inversion‐recovery sequence that produces good contrast but measurable signal intensities from both CSF and brain tissue. The spatial resolution was chosen to fulfill the relatively coarse requirements for pediatric imaging  within a very short imaging time of 2 minutes per 3D data set.Because the aim of the work is to map areas of CSF, seen mostly as bright areas on a darker background, we used a T1‐weighted imaging, FOV = 192 × 192 × 256 mm, acquisition matrix = 48 × 48 × 64, TR/TE = 500 ms/10 ms, echo train length (ETL) = 6, and acquisition time = 2 minutes 23 seconds (k‐space data were acquired on a center‐out trajectory); for T2 weighted imagine: FOV = 160 × 192 × 256 mm, acquisition matrix = 40 × 48 × 64, TR/TE = 3000 ms/20 ms, ETL = 40, effective TE = 410 ms, and acquisition time = 1 minutes 48 seconds (k‐space data were acquired on a low‐high linear trajectory); and inversion recovery: FOV = 160 × 192 × 256 mm, acquisition matrix = 40 × 48 × 64, TI/TR/TE = 100 ms/3000 ms/20 ms, ETL = 40, acquisition bandwidth = 20 kHz, and acquisition time = 1 minutes 48 seconds (k‐space data were acquired using a center‐out trajectory).The sequence parameters for the sequences are as follows. For TIn addition to these relatively low‐resolution images, we also acquired a higher resolution image to demonstrate that the SNR is sufficient to acquire images at this resolution . k‐Space data were acquired on a center‐out trajectory.1 values were 200, 140 and 70 ms, and T2 values were 47, 84 and 20 ms, respectively. To obtain signal from all tissues (with no attempt to optimize contrast between tissues), data were acquired using a 3D T1‐weighted turbo spin‐echo sequence with the following parameters: FOV = 256 × 256 × 256 mm, acquisition matrix = 128 × 128 × 128, TR/TE = 115 ms/15 ms, ETL = 3, acquisition bandwidth = 20 kHz, and RF pulse duration = 100 µs. k‐Space data were acquired on a center‐out trajectory and filtered using a sine‐bell squared filter. B0 homogeneity over the knee, measured as the FWHM of the Fourier transform of the FID signal, was about 400 Hz.For an illustrative case of knee imaging, in which higher spatial resolution is desirable and imaging times can be increased somewhat, we imaged the knee of a healthy volunteer. Relaxation data for muscle, fat, and cartilage were estimated from previously published formulas and measurements.2.7Gx,Gy, and Gz are simulated gradient field maps ; and t is the readout time. This approach to gradient nonlinearity correction is analogous to that used in conjugate phase reconstruction,Python 3.7 and Numpy 1.17. The optimized einsum function of Numpy was used to reduce memory consumption and reconstruction time. Reconstruction time for a 128 × 128 × 128 voxel data set was 43 seconds on an Intel Core i7‐6700 quad‐core CPU with 32 GB RAM. The algorithm was also adapted to run on a GPU using the CuPy Python library. Reconstruction time for a 128 × 128 × 128 voxel data set when executed on a Nvidia GTX 1660 super with 6 GB on‐board RAM was less than 1 second.Fourier‐based image encoding assumes a linear relationship between space and gradient magnitude. Because of the relatively low length‐to‐diameter ratio (~1:1) possible for our gradient coils, there are significant image distortions arising from gradient nonlinearity, particularly along the x‐axis . A basic distortion correction is implemented by replacing the standard inverse fast Fourier transform by3Figure Figure Figure Figure Figure 4Being able to design a permanent magnet array with readily available commercial small magnets that are inexpensive ;Being able to form the housing for such an array with readily available and inexpensive plastics, which can be readily machined with acceptable tolerances;Potentially being able to repair the magnet very simply by replacing individual magnets or rings of magnets should damage occur;Having each of the electronics components be run from standard power outlets, and ultimately be battery/solar/diesel engine powered; andDesigning gradient and RF amplifiers that operate for the specific tasks required  rather than being multipurpose, much more expensive and heavy units.We have designed and constructed a low‐cost, portable, low‐field MRI system operating at 50 mT for in vivo imaging. We would characterize the novelties of the problem of compactness, low cost, and sustainability as follows:In terms of power consumption and requirements, the RF amplifier runs off standard 110/240 V main supply and has to supply about 500 W at a duty cycle of less than 1%. The gradient amplifier also runs off a standard 110/240 V main supply and has to provide 300 VA at a duty cycle of about20%. In terms of heating, the RF amplifier and gradient amplifier both contain heat sinks and have simple air fan cooling. A thermal camera indicated that the maximum component temperature reached in the gradient amplifier was approximately 60°C.In vivo images of the brain and the knee have been acquired. For the brain scans, our ultimate aim is to image pediatric hydrocephalus in resource‐limited settings. As discussed by Obungoloch et al,This type of system can also be used for applications in which the imaging time can be increased, and could be used as a point‐of‐care device. As an example, we showed that we can acquire in vivo images of the human knee with a nominal 2 × 2 × 2 mm resolution with a SNR of about 20:1 within 12 minutes.0 inhomogeneity over the imaging region of interest with respect to the strength of the frequency encoding gradient, and T2 decay during the turbo spin‐echo train. Taking each in turn, temperature‐induced B0 drift causes by heat radiated from the body  were measured for the knee scans to be approximately 160 Hz over the 12‐minute imaging session, which corresponds to a FWHM of the point spread function in the frequency‐encoding dimensions of around 1.9 mm for the 240‐mm FOV and 20‐kHz bandwidth used. However, these are very easily corrected by applying a corresponding phase shift to the k‐space data. The NMR‐measured inhomogeneity over the knee corresponded to a FWHM of about 400 Hz: Imaging with a pixel bandwidth of 300 Hz/pixel gives a point spread function of 1.3 pixels, or 2.6 mm in the frequency‐encoding dimension. The echo train length of three echoes with an effective TE of 15 ms and estimated T2 of muscle of about 200 ms means that the point spread function due to T2 decay during the echo train is subpixel0 shimming and including knowledge of the B0 field distribution in the image reconstruction. Similar calculations can be applied to the brain data, but because these are acquired at a coarser digital resolution of 4 × 4 × 4 mm, there is a correspondingly smaller effect. Several methods can also potentially be used to reduce the total imaging time, such as simple half Fourier, compressed sensing and parallel imaging, using multiple receivers.To estimate the true resolution, there are several components in addition to digital resolution that must be considered. These include frequency drifts due to temperature changes (in vivo and gradient induced), the degree of BμV for a 100‐kHz bandwidth , corresponding to 0.7 nV/square root Hertz, which is within experimental error of the theoretical limit of 0.9 nV/square root Hertz. This level remains unchanged when the 50‐Ω load is located at the end of a long coaxial cable, which is passed through the Bayonet Neill–Concelman interface to the inside of the Faraday cage. When the impedance‐matched RF coil is connected, the noise level is unchanged. Connecting the RF amplifier introduces no additional noise, but when the gradient amplifier is turned on, the noise level increases to 0.33 μV. This indicates that some degree of coupling exists between the gradient coils and RF coil. The first‐order gradient filter reduces this by approximately 10%, but there is still room for improvement in reducing the noise level. We also note that there are a series of harmonic spikes that occur every 200 kHz, arising from the gradient amplifier; however, given the small imaging bandwidth used, this is not a problem with the current setup.Noise measurements performed with each of the components of the system present and absent indicate that there are some improvements still to be made in terms of SNR. With a 50‐Ω terminated input to the Magritek spectrometer, the noise level is 0.18 We also anticipate that the Halbach magnet geometry can be expanded for future applications, moving away from the very simple cylindrical symmetrical structure illustrated here, such as hemisphericalFIGURE S1 Schematic of a custom‐built 1‐kW RF amplifier with the three main stages of the amplifier labeledClick here for additional data file.FIGURE S2 A 3D model of the x‐gradient coil wire pattern designed using the target field method described in Krishnan et alClick here for additional data file.FIGURE S3 A 3D model of the y‐gradient coil wire pattern designed using the target field method described in Krishnan et alClick here for additional data file.FIGURE S4 A 3D model of the z‐gradient coil wire pattern designed using the target field method described in Krishnan et alClick here for additional data file.


Streptomyces mobaraensis encoding the first cases of a bacterial geranylfarnesyl diphosphate synthase and a type I sesterterpene synthase was identified. The structures of seven sesterterpenes produced by these enzymes were elucidated, including their absolute configurations. The enzyme mechanism of the sesterterpene synthase was investigated by extensive isotope labeling experiments.A biosynthetic gene cluster from  2=52=XXV. Not the answer to everything, but the number of carbons in a sesterterpene, for which we describe the first bacterial type I terpene synthase and geranylfarnesyl diphosphate synthase encoded by a gene cluster from Streptomyces mobaraensis. Seven sesterterpenes were obtained with the recombinant enzymes and their biosynthesis was analyzed by isotope labeling experiments.11001 KIB 015 and the venezuelaenes from Streptomyces venezuelae.[Bacillus clausii that converts GFPP and hexaprenyl diphosphate into β‐geranylfarnesene (1) and β‐hexaprene, respectively,Streptomyces somaliensis, a membrane protein belonging to the UbiA superfamily, that converts GFPP into somaliensenes A (2) and B (3)  is spata‐13,17‐diene synthase (SpS) from Streptomyces xinghaiensis, but this enzyme functions naturally as a diterpene synthase.5) synthase from Aspergillus clavatus, a few StTSs from fungi[Streptomyces mobaraensis NBRC 13819 and characterization of the encoded enzymes as unprecedented bacterial representatives of a GFPPS and a type I StTS (SmTS1). The structures of seven products made by SmTS1 and the experimentally verified cyclization mechanism to these compounds are presented.The bacterial farnesyl diphosphate (FPP) synthases (FPPS) from ades ago.8, 9 MS. mobaraensis released several sesterterpenes . A BLAST search revealed that this bacterium encodes at least 10 type I TSs , two of which showed close homology to the characterized 2‐methylisoborneol synthase and geosmin synthase from Streptomyces coelicolor.[E. coli and protein purification that was successful for SmTS1  and SmTS6–SmTS10 (SmTS4 and SmTS5 were insoluble proteins). All soluble proteins were tested with GFPP as substrate that was only converted by SmTS1, but not by the other enzymes, into a mixture of sesterterpene hydrocarbons and one sesterterpene alcohol , while geranyl diphosphate (GPP), FPP and GGPP were not accepted by SmTS1. Notably, the in vitro reaction with GFPP yielded all sesterterpenes observed in the headspace extracts, in addition to a few more compounds that were only obtained from the enzymatic reaction; they represent the less volatile compounds with higher retention times and one trace compound. A genome analysis through antiSMASHE. coli, followed by incubation of the purified enzymes  with DMAPP, GPP, FPP and GGPP together with IPP confirmed this hypothesis by the observation of sesterterpenes in all cases .Volatiles can efficiently be analyzed by trapping on charcoal filters using a closed‐loop stripping apparatus, followed by extraction of the filters and GC/MS analysis of the obtained extracts.elicolor.32, 333 impregnated silica gel chromatography and structure elucidation by one‐ and two‐dimensional NMR spectroscopy  resulted in the structures of compounds 6–12 shown in the boxes of Scheme 6–11) and sestermobaraol (12). Their common biosynthesis can be explained from GFPP by 1,15‐14,18‐cyclization to cation A, followed by a 1,5‐hydride shift to B. These steps are also proposed for the cyclizations by some fungal and plant StTSs, but in most cases different stereoisomers of A and B are relevant.[Penicillium brasilianum sesterbrasiliatriene synthase (PbSS) the cyclization cascade proceeds through the same intermediates A and B.10) with its unusual C3=C25 double bond localization can arise from B by a 1,5‐proton shift to C and deprotonation. Alternatively, B may undergo a 6,10‐cyclization to D, which is the direct precursor to sesterbrasiliatriene (13) in P. brasilianum (in brackets),13 was not observed as a product of the sestermobaraene synthase SmTS1. A 1,2‐hydride shift to E, 6,11‐2,12‐cyclization to F and deprotonation results in sestermobaraene F (11). From D in a folded conformation a 7,12‐cyclization to G and a 3,11‐cyclization to H are possible. In this secondary cation that may not be thermodynamically favored the hydrogen H12 is located directly underneath the cationic center which can thus undergo a possibly concerted 1,4‐hydride shift to J. Deprotonation then leads to the main product sestermobaraene A (6). From H an alternative (concerted) 1,2‐methyl migration to K and attack of water yields sestermobaraol (12). A 1,2‐hydride transfer from G to L, skeletal rearrangement to M, 2,7‐cyclization to N and deprotonation leads to sestermobaraene D (9). Cation L can also react by 2,12‐cyclization to O that yields sestermobaraene C (8) by deprotonation, or sestermobaraene B (7) by 1,4‐hydride shift to P and loss of a proton. This biosynthetic hypothesis was deeply investigated by enzymatic conversion of various isotopically labeled terpene precursors (Table S10). Specifically, all 25 isotopomers of (13C)GFPP, enzymatically prepared with GFPPS from the corresponding labeled GPP, FPP, GGPP or IPP isotopomers that were synthesized as shown in Scheme S6 or as reported previously,[13C‐label in the expected positions of compounds 6–12 in all cases . These experiments supported the overall model, especially the proposed skeletal rearrangements from H to K and from L to M. The products obtained from (20‐13C) and (21‐13C)GFPP revealed a strict stereochemical course regarding the fate of the geminal methyl groups, without any distribution of labeling between these carbons, i. e. the 1,5‐hydride shift from A to B proceeds with high face selectivity at C19.A large scale incubation, followed by extensive compound purification by HPLC, standard and AgNOrelevant.25, 266–12, experiments with stereoselectively deuterated and 13C‐labeled compounds were performed. The interpretation of the results made use of the known stereochemical course of the prenyltransferase reaction with inversion of configuration at C1 of the elongated oligoprenyl diphosphate and attack at C4 of IPP from the Si face.A to B was investigated by conversion of (7‐13C)GPP and (E)‐ or (Z)‐IPP6–8 the specific migration of deuterium from (E)‐IPP to C19 was evident from an upfield shifted triplet by 13C‐2H spin coupling in the 13C‐NMR spectrum . For the minor compounds 9–12 no such triplet signals were observable , because for 13C connected to deuterium the transversal relaxation time for spin‐spin relaxation is increased, resulting in strongly reduced signal intensities in comparison to the intensities for carbons connected to protium. Furthermore, the nuclear quadrupole moment of deuterium causes line broadening. Indirect evidence for 9–12 was obtained by the clearly observable singlet for C19 in the experiment with (Z)‐IPP that was not detected with (E)‐IPP as a result of deuterium binding, suggesting that the 1,5‐hydride shift is also relevant for these compounds.To follow the hydrogen migrations in the biosynthesis of B to C towards compound 10 was investigated using (R)‐ and (S)‐GPP13C)IPP,S)‐GPP a singlet was observed for C2 in the 13C‐NMR spectrum, no signal was detected with (R)‐GPP . Analysis of the enzyme products by GC/MS revealed a strongly diminished production of 10 from (R)‐GPP caused by a deuterium kinetic isotope effect. Product analysis by the more sensitive HSQC spectroscopy in comparison to 13C NMR demonstrated that with (S)‐GPP the crosspeak for H13 was vanished, while with (R)‐GPP no crosspeak for H2β was detected. Taken together, these data support the 1,5‐proton shift from B to C with specific migration of the 1‐pro‐R proton of GPP into the H2 position of 10. The same experiment also demonstrated the specific loss of the 1‐pro‐S proton of GPP in the deprotonation step to 6, as was concluded based on GC/MS and 13C‐NMR data .The 1,5‐proton shift from D to E was shown by conversion of GGPP11 in the 13C‐NMR . Along similar lines, the 1,4‐hydride transfer from H to J towards 6 was shown by a two‐step enzymatic transformation first of GPP and (Z)‐(4‐2H)IPPStreptomyces coelicolor FPPS,13C)IPP,6 . The formation of other isotopomers, for example, from unreacted GPP after the first step (Scheme S4 b), could not completely be suppressed and gave rise to additional 13C‐NMR signals for C2 of 6.The 1,2‐hydride shift from G to L in the biosynthesis of 8 and 9 (Scheme S5) was followed by incubation of GPP and IPP, synthesized as in Scheme S7, with GFPPS and SmTS1. For C11 of compound 8 an upfield shifted triplet was observed, while the signals for the other labeled carbons were clearly visible and appeared as doublets because of 13C‐13C spin coupling . The upfield shift of these signals is a result of two deuterium atoms bound to neighboring carbons. For the minor product 9 it was impossible to detect a triplet for C11. For compound 7, the same hydrogen is passed on in the 1,4‐hydride shift from O to P, as was shown by the triplet for C3 obtained from the same incubation experiment . Here the unusually large upfield shift is caused by the simultaneous effect of one directly bound deuterium atom and two deuterium atoms in the neighboring position.The 1,2‐hydride migration from 6–12 were determined by an enantioselective labeling strategy with enzymatic conversion of GPP and (R)‐ or (S)‐IPP13C‐labels (black dots), the introduced stereoselective deuterations at C1, C5 and C9 can efficiently be monitored by HSQC spectroscopy . The relative orientation of the natural stereogenic centers in 6–12 to the stereogenic anchors at the deuterated carbons can be deduced by NOESY and gives rise to the absolute configurations of all seven sesterterpenes (red and blue arrows show key NOESY correlations). Similar experiments with GPP and (E)‐ or (Z)‐IPP6–12.The absolute configurations of S. mobaraensis encoding the showcase representatives of a bacterial GFPPS and a type I StTS. The remarkable structures of seven new sesterterpenes were elucidated, including the absolute configurations by an enantioselective deuteration approach. The complex cyclization mechanism from GFPP to all seven identified products was investigated in a series of labeling experiments that allowed to follow every elementary step including skeletal rearrangements, hydride and proton migrations, and a stereospecific deprotonation. Whether the polyketide synthase encoded by a gene adjacent to the StTS and GFPPS genes is functionally related and for example, involved in the biosynthesis of a bacterial meroterpenoid, an important natural product class in Streptomyces,S. mobaraensis cultures as in the in vitro incubations performed in this study disfavors this hypothesis.In summary, we have identified a biosynthetic gene cluster in 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.


At the cellular level, the latest studies demonstrate a complex disturbance of metabolomics involving peroxisomal function and lipid homeostasis in the patients. Our study delineates a case of two severely affected siblings with a new hemizygous variant c.221T>C (p.L74P) in ATP6AP1 gene, who both died due to liver failure before reaching 1 year of age. We bring novel pathobiochemical observations including the finding of increased reactive oxygen species in the cultured fibroblasts from the older boy, a striking copper accumulation in his liver, as well as describe the impact of the mutation on the protein in different organs, showing a tissue‐specific pattern of ATP6AP1 level and its posttranslational modification.Congenital disorders of glycosylation (CDG) represent a wide range of >140 inherited metabolic diseases, continually expanding not only with regards to the number of newly identified causative genes, but also the heterogeneity of the clinical and molecular presentations within each subtype. The deficiency of ATP6AP1, an accessory subunit of the vacuolar H ApoC‐IIIapolipoprotein C‐IIIBiPbinding‐immunoglobulin proteinCDGcongenital disorders of glycosylationCHXcycloheximideDHEdihydroethidiumEndo Hendoglycosidase HERendoplasmic reticulumERADendoplasmic reticulum‐associated degradationICCimmunocytochemistryICP‐MSinductively coupled plasma mass spectrometryPNApeanut agglutinin (lectin)PNGase Fpeptide: N‐glycosidase FROSreactive oxygen speciesTFtransferrintuntunicamycinVLCFAvery long chain fatty acidsWESwhole‐exome sequencing1ATP6AP1 was identified by exome sequencing in 11 male patients from 6 families.ATP6AP1 codes for an accessory subunit of a vacuolar H+‐ATPase or V‐ATPase, which normally pumps cytosolic protons into the lumen of endocytic and secretory organelles and thus maintains the acidic pH to ensure their correct function (including Golgi glycosylation), as well as serves to acidify the extracellular space in specific cells.ATP6V0A2, ATP6V1A, ATP6V1E1, and ATP6AP2) have been found to underlie a glycosylation disorder in humans.In 2016, a novel inherited metabolic disorder due to mutations in X‐linked 2ATP6AP1, who presented with a very severe phenotype resulting in early death due to liver failure, while they showed no signs of immunodeficiency or neurologic involvement like the majority of the 13 previously described ATP6AP1‐CDG cases.Here we outline a case of two male siblings . Peroxisomal perturbance as indicated by abnormal serum VLCFA levels in both patients was supported by ICC analysis of catalase, which showed a visibly decreased fluorescence signal localised to peroxisomes . The staining with LAMP2 antibody pointed to an increased abundance of lysosomes , concurring with autophagy disruption, which has been well described in ATP6AP2 defect.G,H). Our preliminary results have revealed variably elevated ROS also in other CDG subtypes, namely PMM2‐CDG, ALG8‐CDG, RFT1‐CDG, and SLC10A7‐CDG  using PNA lectin Figure , which is Figure C,D. Thes Figure E,F, conG Figure , showing4ATP7A or ATP7B have been found in P1 by WES. It is known that the copper transporting enzymes ATP7A/ATP7B reside mainly in the trans‐Golgi network, and presumably this specific environment, defined by lower pH, facilitates the release of Cu from these ATPases.Another characteristic aspect of ATP6AP1‐CDG pathophysiology is the altered copper metabolism reflected by low serum copper and ceruloplasmin in the patients  extra N‐glycan(s) on the mutant protein is an early event in ATP6AP1 biosynthesis  pathway. Considering the recognised role of STT3B in ER quality control, where it adds N‐glycans onto severely misfolded proteins to facilitate an alternative protein degradation pathway via N‐glycan‐dependent ERAD,Western blot analysis of ATP6AP1 was carried out in the available tissues from P1, uncovering a significantly decreased amount of the full‐length 62‐kDa form in his frontal cortex and liver Figure . A surpry Figure . Twenty  Figures . Very in6Available in Supporting Information.The authors have no conflict of interest to declare.The study was performed in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the General University Hospital in Prague.All blood and tissue samples were analysed with informed consent from the parents of the patients.Data S1. Supporting informationClick here for additional data file.


High‐resolution X‐ray structures and experimental charge density analyses supported by periodic quantum‐chemical calculations provide insight into the influence of the crystalline environment on the structure of these [Cl3]− anions as well as into the progress of the bond formation between a dichlorine molecule and a Cl− anion.So far, several publications have discussed the bonding concepts in polyhalides on a theoretical basis. In particular, the trichlorine monoanion is of great interest because its structure should be symmetrical and show two equidistant Cl−Cl bonds. However, apart from matrix‐isolation studies, only asymmetric trichlorine anions have been reported so far. Herein, the trichlorine monoanions in 2‐chloroethyltrimethylammonium trichloride  A highly symmetric [Cl3]− monoanion has so far only been observed in a matrix‐isolation study3 enabled us to conduct the first topological analysis of an experimental charge‐density of a 22 valence electron system. This analysis not only allows a comparison of a quantum‐chemically computed ED with an experimental one from the crystalline state as probed by XRD, it also provides an insight into intermolecular interactions posed by the crystalline environment. In addition, the high‐resolution X‐ray structure and charge density analysis for two asymmetric trichlorine monoanions [NMe3EtCl][Cl3], 1, and [NMe4][Cl3], 2, provide insight into the influence of the crystalline environment on the Cl−⋅⋅⋅Cl2 interaction as well as into the progress of the bond formation between a dichlorine molecule and a Cl− anion as visualized by the experimental ED of the reactants  calculations at the B3LYP/def2‐TZVP level for periodic crystalline solids and isolated [Cl3]− and Cl2 molecules. The electron density at the bond critical point ρ(rBCP) of the Cl−Cl bonds ranges from 0.27 to 0.84 e Å−3. The shorter Cl1−Cl2 bond in 1 and 2 has approximately twice the electron density of the longer Cl2⋅⋅⋅Cl3 bond, while the Cl–Cl distances of 3 show, as expected, an intermediate ρ(rBCP) value of 0.6 e Å−3. The calculated ρ(rBCP) values agree well with the experimental ones and are consistent with those for isolated [Cl3]−, which decay exponentially with the bond lengths , the calculated values increase for short bond lengths, reach a maximum for the equilibrium bond length of [Cl3]−, and decay again for larger bond lengths. The experimentally determined ∇2ρ(rBCP) values deviate from this trend for short bonds in 1 and 2, as known also from other experimental measurements.Table S23 lists the most important QTAIM22, 23ρ(rBCP) as well as the negative total electronic energy density H(rBCP) and the ratio of potential to kinetic energy density (|V|(rBCP)/ G(rBCP)).1 and 2, show the highest ratio, although it still belongs to the intermediate covalent/ionic range. Also the ELFBCP values confirm that the bonds explored here have minimal covalent content. In symmetric [Cl3]− the electrons appear to be as delocalized as in a homogeneous electron gas of equal density . When elongating one bond and shortening the other, the ELFBCP value for the former decreases further—indicating the ionic character—while the ELFBCP value of the latter increases, approaching the value in Cl2. However, even in dichlorine, ELFBCP is much smaller than 1.0, reflecting its charge shift character.Consistent with the VB description [Eq. (1)] the Cl−Cl bonds are predominantly ionic with minute covalent contributions. This is indicated by the values for ρrBCP as we1–3. As expected for an ionic interaction, the valence shell of Cl3 in 1 and 2 is much less polarized towards Cl2 than that of Cl1 or that in 3. The valence‐shell charge concentration (VSCC) at the atoms Cl2 and Cl3 in 1 and 2 is characteristic for an ion‐like interaction, although in compound 2, a stronger polarization along the bond path indicates a slightly higher covalent content , while in 1 and 2, starting to resemble the ELF plot of isolated Cl2 . But the two ELF maxima from the lone pairs of Cl3—unlike in the Laplacian—dissolve into a spherical area of high ELF value, precisely as one would expect for the valence shell of a chloride ion engaged in a non‐shared interaction. In all systems, the valence shell is clearly separated from the core region by a sphere of nearly zero ELF values. For all Cl1 (Cl1′) and Cl2, ELF shows two maxima  with ELF values close to 1.0, representing the lone pairs of these atoms. Between these atoms in 1 and 2, ELF reaches values of about 0.65 (cf. Table S23). This is significantly lower than 1.0, which one would expect for a genuine covalent bond, reflecting these bonds’ charge‐shift nature. For the symmetric trichlorine ion 3, ELF is again smaller, indicating a more delocalized character of the electrons in these bonds. Finally, for the longest bonds in 1 and 2, ELF decreases further, indicating a vanishing covalent contribution. Previous studies on halogen−halogen bonding[11]−, [Cl12]2−, and [Cl13]−,1, exceed twice the van der Waals radius of chlorine, and the corresponding ρ(rBCP) values are too small (0.03 e Å−3) to be associated with a significant Cl−Cl interaction (cf. Table S21).As in the Laplacian, the two lone pairs of Cl2 in the ELF plots move towards Cl3 in n bonding6, 7 a1 and 2. In the solid‐state they are commonly[3]− anions from equidistance. In Figure 3]− ions and their related cations are illustrated by colour‐coded Hirshfeld surfaces.1 and 2 and the equidistant ones in 3 are apparent. The shortest contacts (2.629 Å) are observed for the most asymmetric [Cl3]− anion in 1. In 2, the [Cl3]− anion is exposed to more short C−H⋅⋅⋅Halogen contacts, but they are on average 0.04 Å longer. Anyhow, in both systems, these contacts are sufficient to stabilize the—at least in vacuum—less stable asymmetric [Cl3]− anion. The higher symmetry of 3 arises from even weaker interacting [NnPr4]+ cations.There is a significant number of C−H⋅⋅⋅halogen contacts with distances below the sum of the van der Waals radii, especially for  commonly12, 29In summary, for the first time, the 3c–4e bond of a symmetric trichlorine anion was characterized by the topological analysis of the experimental density distribution and compared to two asymmetric trichlorine ions. Quantum‐chemical DFT calculations for both periodic crystals and isolated molecules/ions do not fully agree for the Laplacian but support the qualitative analysis of bond properties. There appears to be a smooth transition from the asymmetric to the symmetric compound. For the asymmetric trichlorine ions, short Cl⋅⋅⋅H contacts were detected, contributing to the considerable structural differences of these trichlorine anions.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.


Halide and phenyl substituted germaborenes were shown to react with azides at room temperature and transfer a borylene moiety to give iminoboranes. This iminoborane synthesis based on a borylene transfer route was investigated computationally in the case of the phenyl substituted germaborene. Germaborenes act as borylene transfer agents to azides in the synthesis of iminoboranes. In borylene transfer chemistry sources of borylenes are almost exclusively transition metal borylene complexes.1, 2 described previously were reacted at rt with organoazides RN3   Å  which can be compared with other B–N triple bonds [1.190(4)–1.265(7) Å] were found in both molecular structures.. The signal in the 31P NMR spectrum of 3 was found at −9.6 ppm [4: −10.5 ppm] and in the 11B NMR spectrum of 3 at 2.8 ppm [4: 1.6 ppm]. The 31P and 11B NMR spectra of compounds 3’ and 4’ are shown in the Supporting Information.Halide substituted germaborenes ructures.3c, 6 3 the formation of germylene Lewis pair A was observed by 31P NMR spectroscopy  followed by reduction with Mg/anthracene. Following this straightforward procedure phenyl substituted germaborene 5 was isolated in high yield . An ORTEP of the molecular structure of 5 is placed in the Supporting Information. As in the case of halidegermaborenes 1 and 2 phenylgermaborene 5 exhibits an intramolecular photochemically activated reversible [2+2] cycloaddition with a phenyl moiety of the terphenyl substituent .5 in hands the transfer of phenylborylene was studied . Phenylborylene which was transferred in the described examples was characterized in 2006 as a highly reactive species in inert gas matrices.After addition of MeA and organoazide two equivalents of azide were used in the reactions of phenylgermaborene.9 and 9’ of these side reactions were placed in the Supporting Information.Due to a Staudinger‐type side reaction between germylene phosphine Lewis pair 3)3SiN3 was investigated computationally for derivative 5 at the B3LYP‐D3BJ/6‐31G*G=−28.7 kcal mol−1) concerted 1,3‐dipolar cycloaddition of (CH3)3SiN3 to the Ge=B double bond. This reaction has a low Gibbs free energy of activation ΔG≠ of 12 kcal mol−1. The resulting five‐membered triazole corresponds to a very shallow minimum on the PES as the classical barrier for fragmentation to the iminoborane (PhBNSi(CH3)3), N2, and A is only 1.1 kcal mol−1, even disappears on the ΔG scale.The transfer of PhB: to 3SiN3 and suggest a very facile fragmentation of the reactive intermediate.Overall, the borylene transfer reaction is strongly exergonic, ΔTo conclude, halide and phenyl substituted germaborenes react as main group element based borylene sources in reactions with organoazides. Thus, a straightforward synthetic room temperature route to iminoboranes was presented.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.


An implicit account of the solvent effect can be carried out using traditional static quantum chemistry calculations by applying an external electric field to the studied molecular system. This approach allows one to distinguish between the effects of the macroscopic reaction field of the solvent and specific solute–solvent interactions. In this study, we report on the dependence of the simulation results on the use of the polarizable continuum approximation and on the importance of the solvent effect in nonpolar solvents. The latter was demonstrated using experimental data on tautomeric equilibria between the pyridone and hydroxypyridine forms of 2,6-di-tert-butyl-4-hydroxy-pyridine in cyclohexane and chloroform. Noncovalent interactions allow our world to exist. Their specific interaction energies ,2,3,4, tThe situation in which the properties of the molecular system under consideration depend on its interaction with the solvent is most common. The best method for such cases is the molecular dynamics approach ,30,31. E3N···HF complex under the action of an external electric field has been studied in the gas-phase and PCM approximations /(ε + 0.5) b. In bot3N···H and N···F distances of H3N···HF under the effect of an external electric field at the gas-phase (ε = 1) and PCM (ε = 2 and 100) approximations.3N···HF complex is no exception +···F−, the difference becomes much larger.It is shown that the AuF approach can simulate the dissociation of alkali metal halides. For small perturbation of these molecules, the use of the PCM approximation is optional. In contrast, for large disturbances and especially close to the dissociation it is strongly recommended to use the PCM approximation. Similar conclusions can be drawn for proton transfer in HThe AuF approach may be used to model solvent effects in nonpolar solvents. The integral effect can be estimated using the gas-phase approximation. By comparing these results with those obtained in the PCM approximation, we can distinguish between the effects of the macroscopic reaction field of the solvent and the specific solute–solvent interactions. In this work, such an analysis was carried out using experimental data on tautomeric equilibria between pyridone and hydroxypyridine in cyclohexane and chloroform. Although this analysis cannot determine the nature of the interactions taking place in the solution, it still allows us to evaluate the influence of these interactions on the parameters of the system under study and the need to take them into account when interpreting experimental data.


The topography and electronic structure of Dy2ScN@C80 adsorbed on Au(111) were studied by STM. X‐ray magnetic CD studies show that the Dy2ScN@C80 monolayers exhibit similarly broad magnetic hysteresis independent on the substrate used, but the orientation of the Dy2ScN cluster depends strongly on the surface. DFT calculations show that the extent of the electronic interaction of the fullerene molecules with the surface is increasing dramatically from MgO to Au(111) and Ag(100). However, the charge redistribution at the fullerene‐surface interface is fully absorbed by the carbon cage, leaving the state of the endohedral cluster intact. This Faraday cage effect of the fullerene preserves the magnetic bistability of fullerene‐SMMs on conducting substrates and facilitates their application in molecular spintronics.Magnetic hysteresis is demonstrated for monolayers of the single‐molecule magnet (SMM) Dy 2ScN@C80Monolayers of Dy exhibit magnetic hysteresis on Au, Ag, and MgO|Ag substrates. The substrates influence the ordering of the endohedral cluster, but do not affect the width of the hysteresis. The carbon cage protects the endohedral magnetic cluster and thus ensures that it sustains magnetic bistability. The molecules were deposited under ultra‐high‐vacuum conditions (p≤10−9 mbar) via organic molecular beam epitaxy using a home build evaporator. Typical evaporation conditions were 20 minutes at 450–460 °C. The substrate was kept at room temperature during deposition, and STM studies were also performed at room temperature using Omicron VT‐STM/AFM microscope.To ensure the suitability of the evaporated thin films of Dy2 anchored to the edges of the substrate step ,2ScN@C80 molecules are organized in a hexagonal close‐packed (hcp) structure with a lattice parameter of 1.15±0.05 nm  surface.STM measurements revealed that at these evaporation conditions the surface of the substrate is covered with ca. 0.5 ML of fullerene molecules . The endohedral fullerenes diffuse across the terraces of Au(111) and self‐assemble into closed monolayer islands of several 10×10 nm2ScN@C80 on Au(111) was studied by scanning tunneling spectroscopy (STS). The spectra acquired in the field of view presented in Figure 2ScN@C80 molecules on the Au(111) surface.eff=1.7±0.1 eV if the shoulder at +0.7 V is assigned to the LUMO or Δeff=2.05±0.1 eV if only peak maxima are used.The electronic structure of Dy2ScN@C80. At the HOMO energy of −1.035 eV below the Fermi level, regions of supposed Au(111) fcc‐like termination in between the topographically brighter double stripes in Figure 2ScN@C80 molecules on Au(111) vary following the altered reconstruction from quasi‐epitaxial regions (in between the double stripes) to incommensurate double stripe areas, which affects the local electronic structure of the fullerene|metal interface.The normalized differential conductance map Figure  e measur2ScN@C80 monolayers were studied by Dy‐M4,5 XMCD at the X‐Treme beam line at the Swiss Light Source, Paul Scherrer Institut.2 atmosphere (10−6 mbar) while keeping the substrate at 645 K. Dy2ScN@C80 evaporation conditions onto the Au(111) substrate were adopted from the ex situ studies described above. In situ characterization by STM confirmed formation of similar monolayer islands . The same evaporation conditions were then used for the growth on Ag(100) and MgO|Ag(100), for which in situ STM characterization was not possible during the beamtime. The coverage of the Ag(100) and MgO|Ag(100) substrates by Dy2ScN@C80 was lower than for Au(111) (as estimated from XAS intensity), which ensures that all XMCD measurements were performed in the submonolayer regime. At the base temperature of the cryostat the temperature at the sample was near 2 K for the Au(111) crystal as estimated from separate measurements of ErThe magnetic properties of DyM5 XAS and XMCD spectra of Dy2ScN@C80 sub‐monolayers measured at T≈2 K in the magnetic field of 6.5 T are shown in Figure 2ScN@C80 is slightly stronger in the grazing (30°) than in the normal (90°) incidence. On Ag(100), the difference between the two orientations is enhanced, with much stronger XMCD response at the grazing incidence. Finally, no difference can be seen between the spectra measured at 30° and 90° on the MgO|Ag(100) substrate. These results are further corroborated by the angular dependence of XMCD asymmetry  and MgO|Ag(100) shows that the Dy2ScN cluster is randomly oriented in the Dy2ScN@C80 monolayers on these substrates. On the other hand, the anisotropy of the XMCD in the Dy2ScN@C80 monolayer on Ag(100) indicates that the endohedral cluster preferentially adopts a parallel orientation with respect to the surface. Parallel alignment of Dy2ScN was also observed for the Dy2ScN@C80 monolayer on Rh(111).As magnetic moments of Dy ions are strongly anisotropic and are locked to the direction of the Dy−N bonds Figure  a, the a3+ ions in Dy2ScN@C80 can be estimated using XMCD sum rules.2ScN@C80 on Au(111), the sum rule analysis gives the moment of 5.4±0.5 μB per Dy3+ ion at 90° and 6.1±0.5 μB at 30°. On Ag(100), the moment increases from 2.9±0.5 μB at 90° to 5.7±0.5 μB at 30°. These values are significantly smaller than 10 μB, the ground‐state magnetic moment of Dy3+ ion in the axial ligand field. However, when magnetic moments are anisotropic, the measured moment corresponds to the projection of the magnetic field onto the easy axis. As a result, in powder samples of Dy2ScN@C80 with disordered Dy2ScN clusters, the apparent magnetic moment of Dy3+ ions is reduced to 5 μB.3+ ions in Dy2ScN@C80 on the Au(111) substrate are thus close to the value expected for the disordered cluster, which agrees with the lack of the angular dependence of the XMCD. For Dy2ScN@C80 on the Ag(100) substrates, the small magnetization perpendicular to the surface agrees well with the preferential in‐plane alignment of the endohedral cluster.The magnetic moment of DyM5 edge was used to study magnetization curves of Dy2ScN@C80 monolayers. Magnetic hysteresis with a coercive field of ca 0.4 T is observed for Dy2ScN@C80 monolayers on all three substrates near 2 K  and Ag(100) substrates , Ag(100), and MgO surfaces were performed at the PBE‐D level with PAW 4f‐in‐core potentials using the VASP 5.0 code.2ScN cluster may adopt different orientations inside the carbon cage, it is important to have a comprehensive sampling of possible structural configurations. Using recently proposed Fibonacci sphere sampling,2ScN cluster were generated for a fullerene molecule on each substrate, and their structures were optimized. For an isolated Dy2ScN@C80 molecule this approach gave only three unique conformers with the relative energies of 0, 41, and 47 meV. But a substantially different situation is found for Dy2ScN@C80 molecule on a substrate  and Ag(100), the lowest‐energy conformers are grouped at θ=5–30°, which corresponds to nearly parallel orientations of the cluster to the substrate. These results agree well with the observation of the in‐plane ordering of the Dy2ScN cluster on the Ag(100) surface, but do not fully capture the experimental finding of the weaker ordering effect on the Au(111) substrate. Presumably, the Au(111) surface reconstructions may decrease the ordering of the cluster in adsorbed fullerenes, but computational description of such effects is not feasible at this moment. For the MgO substrate, calculations show the grouping of the most stable conformers near the angles of 35–40° and 75–85°, but the overall energy spread of the conformers is smaller than on metals. Although XMCD measurements are performed 2 K, it is reasonable to expect that the distribution of the conformers in the experimental samples should be different from the equilibrium for 2 K. When the sample is cooled down from room temperature, at certain point above the base temperature the angular distribution of the conformers will be frozen. Effectively, this situation can be modelled by considering conformers within a certain energy cut‐off. We simulated XAS and XMCD spectra of different conformers with MULTIX codeFor DyEdisp).Edef necessary to distort the structures of the isolated Dy2ScN@C80 molecule and the substrate to those they adopt in the interacting system, is found to be 366 meV for Au(111), 157 meV for Ag(100), and 39 meV for MgO, to which fullerene contributions are 132, 46, and 12 meV, respectively. The remaining terms of 0.64 eV for Au(111), 0.47 eV for Ag(100), and −0.13 eV for MgO are due to the electronic contribution ECoul/cov, which includes both Coulomb and covalent terms. Though 4–5 times smaller than dispersion, these interactions are responsible for the changes in the electronic structure at the interface and hence are considered further in more details. Note also that the variation of the relative energy for different cluster orientations .The PBE‐D binding energy of the fullerene molecule to the surface is 2.91 eV for Au(111), 2.53 eV for Ag(100), and 1.43 eV for MgO (Table Qmol) accumulated on the surface. Calculations of atomic charges with the Bader code2ScN@C80 transfers 0.2–0.3 e to the Au(111) substrate, but acquires a negative charge of −(0.2–0.3) e on Ag(100) and −(0.09–0.14) e on MgO. Importantly, despite the considerable variation of the net fullerene charge in dependence on the substrate and the cluster orientation . The role of the Faraday cage that the fullerene plays for the endohedral cluster2ScN@C80 are weakly affected by the substrate.Interaction of a fullerene molecule with the substrate leads to a change in the electronic distribution of both. The net effect of this redistribution can be evaluated via the charge of the fullerene molecule  surface, density depletion and accumulation regions are formed near the fullerene and near/at the upper gold metal atoms, respectively. For the fullerene on the Ag(100) surface, the regions of the density accumulation and depletion are intertwined in a complex manner. The spatial extension of the density affects at least two layers of Au and Ag metal atoms and small changes can be seen down to the fourth layer. For the fullerene on the MgO, the difference density has the least extended profile, and changes in the electron density are visible only in the upper layer of the substrate.A closer look into the interfacial charge transfer is provided by the difference electron density Δρ correlates with the angular dependence of the relative energy of the Dy2ScN@C80 conformers. Apparently, the Dy2ScN cluster tends to avoid the parts of the fullerene π‐system interacting with the metallic surface. This can be best achieved in the parallel configuration of the Dy2ScN cluster, in which all three metal atoms do not interact with the surface‐perturbed parts of the fullerene cage. For the MgO substrate, the situation is different because the ionic substrate has strongly inhomogeneous charge distribution, and here the electrostatic interactions between the substrate and the carbon cage play an important role. As the fragments of the fullerene cage coordinated by the endohedral metals have pronounced variation of the electrostatic potential distribution,The spatial extension of Δ2ScN@C80 molecule  shows that interaction with the MgO has only a minor effect on the fullerene‐projected DOS. The highest‐occupied states are dominated by the carbon cage, whereas the lowest‐energy unoccupied states have noticeable contributions from the endohedral cluster. The LUMO of the fullerene has a distinct peak in the DOS at 0.85 eV above the Fermi level. The DFT‐predicted gaps for the isolated Dy2ScN@C80 molecule and the molecule on the MgO are 1.65 and 1.63 eV, respectively. The interaction with the Au(111) surface results in more pronounced changes of the fullerene DOS   of the isolated Dy2ScN@C80 fulfills this criterion. XMCD studies of the Dy2ScN@C80 submonolayers on Au(111), Ag(100), and MgO|Ag(100) revealed a distinctive influence of the substrates on the ordering of the endohedral cluster. On Ag(100), the Dy2ScN units are preferentially aligned parallel to the surface, on Au(111) there is only a slight preference of the parallel alignment, whereas on MgO|Ag(100) no ordering is found at all. However, despite the strong influence of the surface on the structural ordering, the magnetic behavior of Dy2ScN@C80 molecules does not show a noticeable dependence on the substrate. Magnetic hysteresis with the coercivity of ca 0.4 T is found near 2 K in submonolayers of Dy2ScN@C80 on all three substrates. DFT calculations showed that the charge redistribution at the metal–fullerene interface is confined within the contact region. The electron transfer affects only the carbon cage, whereas the charge state of the endohedral cluster remains intact. Thus, the fullerene acts as a Faraday cage protecting the electronic and magnetic properties of the endohedral species on conducting substrates.Realization of magnetic bistability in monolayers of SMMs in contact with an electrode is a necessary step towards making use of the single‐molecule nature of their magnetism in spintronic devices. In this work we showed that DyThe 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.


Z,E‐configured dienoic acid amide derivatives which are difficult to make otherwise. Control experiments as well as the isolation and crystallographic characterization of a (tricarbonyl)iron pyridone complex suggest that the active iron catalyst generated in situ exhibits high affinity to the polarized diene system embedded into the heterocyclic ring system of the substrates, which likely serves as the actual recognition element.In the presence of simple iron salts, 2‐pyridone derivatives react with Grignard reagents under mild conditions to give the corresponding 1,6‐addition products; if the reaction medium is supplemented with an aprotic dipolar cosolvent after the actual addition step, the intermediates primarily formed succumb to ring opening, giving rise to non‐thermodynamic  Keep it closed or lock it open: The unique ability of simple iron salts to catalyze the 1,6‐addition of Grignard reagents to 2‐pyridones is likely due to the affinity of the active iron species generated in situ to the polarized diene subunit of the substrates. When the reaction medium is complemented with an aprotic dipolar cosolvent, C−C bond formation is followed by opening of the heterocyclic ring. A undergo an unusual transformation when treated with Grignard reagents in the presence of catalytic Fe(acac)3 at low temperature 3 (5 mol %), gave product 2 a in which the heterocyclic ring is intact 2 and FeCl3 work almost equally well,3 or in the absence of an iron precatalyst.2‐Pyridones are obvious and relevant candidates:14, 156 derived from 1‐benzyl‐5‐bromopyridin‐2(1H)‐one: the fact that the bromide substituent persists under the chosen reaction conditions implies that the iron catalyzed addition reaction to the heterocyclic ring is even faster than a regular iron catalyzed cross coupling, although the latter process is known to proceed very rapidly at low temperatures;[Li⋅(OEt2)]2 (entry 10),0 olefin complexes , fragment.[12 or 16 with either [CpFe(C2H4)2][Li(tmeda)][2H4)2][5 served this purpose well. In this context, it is important to note that (tricarbonyl)iron complexes of 2‐pyrones had previously been found susceptible to nucleophilic attack at the carbonyl group rather than at the enol ether site,[4‐bound heterocyclic ligand is manifest in the massive elongation of the C4−C5 bond (1.418(5) Å) in pyrone complex 23 versus 1.340(1) Å in unbound pyrone 12; the effect is even stronger in the pyridone complex 24 (C4−C5 1.436(2) Å in versus 1.343(2) Å in free 16). As expected, the C3−C4 bond is somewhat contracted (1.403(5) Å in 23 versus 1.427(1) Å in the 12; 1.397(2) Å in 24 versus 1.420(3) Å in 16). Since neither 2‐pyrones nor 2‐pyridones show significant aromatic character, these heterocycles are torsionally flexible. In fact, the lactone (lactam) moiety is forced out of co‐planarity with the olefinic π‐system; as a result, orbital overlap is disrupted and the enol ether (enamide) character lost, at least in part. The incipient carbenoid nature of C5, which carries a carbon‐metal bond and a leaving group, is unmistakable in the structures of complexes 23, 24 and related complexes[23 or 24, because the C2−C3 bond of the pyrone (pyridone) ligand has already lost much of its former double bond character (1.435(6) Å in 23 versus 1.358(1) Å in 12; 1.438(2) in 24 versus 1.351(3) Å in 16) and a carbon‐metal bond is also looming. Hence, adducts of this type seem to be ideally set up for regioselective transfer of a hydrocarbyl residue from a loaded iron center to the heterocyclic ligand framework.Although such coordination to an Fe(+2) center certainly activates the carbonyl group and also enhances the vinylogous Michael acceptor properties of the pyrone, it is unlikely that this canonical Lewis acid/base interaction renders the observed 1,6‐additon so astoundingly facile. Extensive screening in this laboratory showed that iron is almost uniquely capable in catalyzing this transformation. 14, 15),37, 4016 and 25 to give 18 a and 27, respectively, would be in line with the torquoselective preference of this substituent, but the exclusive inward orientation of the methyl group in 26 violates the established selectivity rules of electrocyclic processes;18 a and 27 is hence more likely thermodynamic in origin (the structure of 27 in the solid state is contained in the SI). Product 29, the double bond geometry of which mirrors the stereostructure of the substrate, is arguably an even more stringent case:C would hardly entail formation of a single stereoisomer since the two relevant substituents (Me versus Et) have the same electronic character and are similar in size. Rather, this outcome suggests that some stereochemical communication between the breaking C−O bond and the metal center is operative in the stereodetermining step, as tentatively drawn in D. We appreciate, however, that further experimental and computational scrutiny is needed to either confirm or disprove this view.The available experimental evidence is less clear with regard to the subsequent ring opening step. Yet, two pieces of evidence seem to advocate for an ionic rather than electrocyclic mechanism: as outlined above, the addition of DMF is necessary to entail ring cleavage in the pyridone series; pericyclic processes in general, however, are not expected to show such a strong solvent dependence.47, 48The present study extends the structural space covered by iron catalyzed C−C‐bond formation in general and the rather unconventional formal ring opening/cross coupling in particular. Although not all mechanistic questions could be answered in full detail, the available data suggest that the high affinity of low‐valent iron catalysts to (polarized) dienes constitutes a formidable driving force which can likely be exploited in different context too. Further efforts at leveraging this and other as yet untapped opportunities of homogeneous iron catalysis for organic synthesis are underway and will be reported in due course.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.