Studies on thermal stability reveal stark differences between N-ligands such as N-methyl imidazole and C-ligands such as tert-butyl isocyanide, including ligand exchange reactions and a surprising formation of white phosphorus. A milestone is the transformation of a nonmetal-bound isocyanide into phosphaguanidine or an acyclic bisaminocarbene bound to phosphorus; the latter is analogous to the chemistry of transition metal-bound isocyanides, and the former reveals the differences. This example has been studied via cutting-edge DFT calculations leading to two pathways differently favored depending on variations in steric demand. This study reveals the emergence of organometallic from coordination chemistry of a neutral nonmetal center.We describe nonmetal adducts of the phosphorus center of terminal phosphinidene complexes using classical C- and N-ligands from metal coordination chemistry. The nature of the L-P bond has been analyzed by various theoretical methods including a refined method on the variation of the Laplacian of electron density ∇ The scope of and knowledge about the chemistry of nonmetal-adducts remains very limited. Here, the authors describe nonmetal adducts of the phosphorus center of terminal phosphinidene complexes using classical C- and N-ligands from metal coordination chemistry. Initially, a large range of complexes MLn where the metal is coordinated by neutral and anionic ligands L such as water, ammonia and halides were explored  or side-on (η2) coordinated nitriles reported by Tilley for a cationic silylene ruthenium(II) complex24 and by Tokitoh for a neutral free silylene25. The same author also reported an example of a stable end-on isocyanide-to-silylene adduct using sterically demanding substituents at N27. This followed an early hypothesis by Weidenbruch, who had proposed such short-lived adducts as intermediates years before29. Tokitoh later demonstrated that the isocyanide ligand can be replaced by a different isocyanide, thus proving an equilibrium between the R2Si-CNR adduct and the free silylene and isocyanide30. Iwamoto and Kira also synthesized dialkylsilaketenimines without aromatic substituents at silicon, which possess a slightly bent cumulene structure in the solid state and a weak Si-C bond, i.e., dissociation occurs in solution above −30 °C31. Very recently, this field was expanded by Filippou who demonstrated that an isocyanide can be replaced in an oxidative fashion by reaction of (SIDipp)-Si(CNArMes) imidazolidin-2-ylidene, ArMes = 2,6-bisphenyl) with Ge(ArMes)Cl32. To the best of our knowledge, isosteric silylene adducts of carbon monoxide, R2Si-CO, have only been investigated by computation and/or detected in cryogenic matrices36. In 2002, Tokitoh reported the formation of azasilepines from the reaction of a silylene with pyridine or 4-(dimethylamino)pyridine (DMAP), with proposed Si-adducts as intermediates37. Recently, Inoue described the synthesis and isolation of stable DMAP-to-silylene adducts. Under elevated temperatures, these adducts undergo 1,1 additions of dihydrogen and 1,2 additions of ethene. In the absence of trapping reagents, the DMAP adducts decompose to disiletanes, azasilepines or cyclotrisilanes, depending on the substituents at Si38.A similar reactivity is highly uncharacteristic of main group metalloid and nonmetal compounds, save for some recent advances which will be briefly reviewed, focusing on group 13 and 14 metalloids and thereafter for nonmetals from group 15. Some rare but well investigated examples of neutral, low-coordinate metalloids include nitrile adducts of silylenes, including (transient) 43. But classical boranes, having all valence electrons involved in covalent bonding, can also form Lewis acid adducts with nitriles, isocyanides and carbon monoxide due to the vacant p-orbital. Of special interest is one report from B,C-cluster chemistry, in which a nitrile-to-carborane adduct reacts with secondary amines by nucleophilic addition to form acetamidine derivatives40.Adducts of low-coordinate boron centers representing group 13 metalloids seem to be rare1λ3-P-center, which was later expanded by the same author with studies on adduct formation at a (cationic) σ2λ2-phosphenium, including ligand substitution reactions55. Strained cyclic compounds such as (cationic) phosphirenium salts, can also undergo exchange reactions of the alkyne moiety, as demonstrated by Wild57.To the best of our knowledge, the chemistry of nonmetal main group element adducts at a low-coordinate phosphorus center started with a report by Burford on a (cationic) phosphadiazonium σend-on adducts of nitriles, carbonyls, thioketones, thiourea, imines, N-heterocyclic carbenes (NHCs), and some isolated examples of isocyanides and carbodiimides, to neutral electrophilic terminal phosphinidene complexes bearing a bulky P-substituent67. Later, Mathey proposed that N-methylimidazole (N-MeIm) can bind transiently end-on to electrophilic terminal phosphinidene molybdenum complexes69 which, finally, produced cyclo(oligo)phosphanes and [Mo(CO)3(N-MeIm)3] in the absence of trapping reagents70. Various families of end-on P-adducts of ligands  bound to phosphorus of terminal electrophilic phosphinidene complexes have recently been studied computationally73. The latest addition to this emerging field came from the group of Bertrand, who demonstrated that a sterically encumbered phosphanyl phosphinidene R2PP, accessed via a multistep synthetic protocol, can form a P-PR3 adduct. The latter can undergo replacement by stronger ligands, such as carbenes and isocyanides, resulting in mono-phosphacarbodiimides or phosphaalkenes, respectively74.Our group has described evidence for the formation of transient end-on bound) ligand into a different ligand coordinated to a nonmetal such as phosphorus, being similar to transition metal ligand transformations, has not yet been described.Despite these singular recent advances, the scope of and knowledge about the chemistry of such nonmetal-adducts remains very limited. This is not surprising because of the increasingly strong element-element bonds in second and third row group 15 to 17 elements, which make replacement reactions unfavorable. More importantly, a conversion of an , DMAP (2b), N-MeIm (2c) and, in addition, tert-butyl isocyanide (2d) as important representatives of classical C-ligands from transition metal coordination chemistry. All reactions of 1 with these ligands proceed smoothly in THF to afford the ligand adduct complexes 3a–d  which did not go to completion under ambient conditions . In some cases, small amounts of the common thermal decomposition products of 1 were observed. The isocyanide adduct 3d was obtained previously in lower yields from 3-iminoazaphosphiridine complexes via an exchange reaction67. The signal of the C2H proton of 3c appears at 6.41 ppm in the 1H NMR spectrum, hence shifted to high frequency compared to that of the free ligand 2c76 due to coordination to the phosphinidene complex. The same effect is also observed for the 15N{1H} NMR signals of the adducts that are found at −193.9 ppm (3b) and −183.9 ppm (3c) for the P-bound nitrogen nuclei while they occur at −112 ppm for 2b77 and −111.4 ppm for 2 c78. This shift to high frequency is similar to those induced by coordination to transition metals where coordination shifts Δδ(15N) are typically −50 to −100 ppm. For example, −65.3 ppm for pyridine upon coordination in [Pd(phen)(py)(C2H5)]CF3SO3 , in CD2Cl280. It was exciting that the N-MeIm-adduct structure 3c could also be determined by a single-crystal X-ray diffraction (SC-XRD) structure 5(PPh)] was previously described as a highly reactive intermediate by Mathey and coworkers70.We started out from our recent theoretical studies of pyridine , DMAP (2a), DMAP , N-MeIm 3b–d were stable at ambient temperature and all new compounds were fully characterized, except pyridine adduct 3a, including single crystal X-ray diffraction studies of 3b,c ° compared to Σ∠P = 321.9(5)° in 3c). The apparently shorter P-N bond of the N-MeIm adduct 3c (1.813(8) Å) compared to 3b (1.826(8) Å) is not statistically certain at the 99% confidence level. According to high-level DFT studies (CPCMtoluene/PWPB95-D3/def2-QZVPP(ecp)), the unusual adduct structure of complexes 3 is also reflected by a significant increase in electron density at phosphorus according to the variation of (Löwdin) electric charges  with respect to the electrophilic naked phosphinidene complex 4, thus reflecting an Umpolung of the terminal phosphinidene complexed P-center. This was confirmed by IR spectroscopic data for complexes 3b–d, which show increasing CO stretching frequencies for the A1-vibrational mode of the trans-CO ligands , thereby consistent with a diminished σ-donation capacity of the phosphorus ligand center in going from 3b to 3d.The adducts 3c: Fig. –115. The SI-Fig.  and show3a–d show significant 31P NMR signatures  complex adduct 3cCr was investigated electrochemically by interfacial voltammetry at ceramic screen-printed Pt composite electrodes (Ag/AgCl solid-dot reference electrode)  Fig. . The add3b–d all have a combination of the p-lone pair at phosphorus and metal d orbital character  character, the other adducts primarily display π*(W-C) character.The HOMOs of adducts 3d differing significantly from the rest. The experimentally obtained peak potentials 3cCr < 3b < 3c « 3d) for decreasing HOMO energy (3b > 3c > 3d), and increasing ease of reduction (3d < 3b < 3cCr < 3c) for increasing LUMO energy (3d < 3b < 3c).This difference in the redox molecular orbital topologies is mirrored by the cyclic voltammograms, with entclass1pt{minima3c,d was probed with a VT 31P{1H} NMR study to examine the thermal P-ligand dissociation and, hence, also the concomitant formation of the free terminal phosphinidene complex 4 , the intensity of the signal of 3c started to decrease, while only the formation of white phosphorus (8) could be observed; unfortunately, no evidence for formation of 4 was obtained. However, full conversion to 8 was only achieved by heating the reaction mixture at 90 °C for 16 hours. When complex 3d was heated to 100 °C, the decomposition to complex 9 was observed  at −520.6 ppm, 1% in solution). Upon changing the metal center from tungsten to chromium, using the same protocol, the isocyanide-to-phosphinidene complex adduct 3Crd was observed to decompose selectively to give 8 at 85 °C. However, the respective N-MeIm P-adduct 3Crc showed an additional product shifted to lower frequency at 569.4 ppm in the 31P{1H} NMR spectrum in the range of 70 to 90 °C that appeared to be an intermediate, before the starting material fully decomposed to yield 8 at 100 °C diphosphene (6), which was previously reported by Schmutzler82. Furthermore, 31P{1H} NMR evidence for the butterfly-type P4 derivative 7 was obtained in the thermolysis using chromium complexes 3Crb and 3Crc. Formation of the DMAP tungsten complex 5b resulted under mild heating but was also detected under mass spectrometric conditions using the liquid injection field desorption ionization (LIFDI) technique.The stability of the P-adducts x 4 Fig. . Complex °C Fig. . The 31PN-MeIm adduct 3c as the most meaningful example to investigate. Adding DMAP (2b) or tert-butyl isocyanide (2d) to a THF solution of 3c led to a very fast ligand exchange, even at ambient temperature, thus yielding the P-adduct complexes 3b,d selectively . Upon heating to 70 °C a very unselective reaction was observed, thus leading to a complicated product mixture consisting of a multitude of products, several exhibiting AB-type spin systems, in the 31P{1H} NMR spectrum besides other products observed in the case of 2b, 2d, or in the thermal decomposition of 3c  and 10b and (36:64) with 31P NMR chemical shifts of −137.7 ppm  and −145.4 ppm , and −137.4 ppm  and −145.5 ppm  was observed, indicating a ligand dissociation prior to the rapid [2 + 1] cycloaddition of the transient terminal phosphinidene complex 4. At this point it should be also noted that π-systems such as alkenes can coordinate reversibly to phosphinidene complexes to form respective phosphirane complexes if terminal alkenes are used as solvent84.These preliminary results on the P-ligand bond strength prompted us to study ligand substitution reactions using the ely Fig. . Unfortu20. To test whether a P-bound ligand in complex 3d could behave similarly to a transition metal-bound ligand, the following reactions with primary amines were investigated.One of the cornerstone findings in transition metal organometallic chemistry is the addition of protic nucleophiles such as amines or alcohols to coordinated isocyanide to form carbene metal complexes as described by Richards and others3d was treated with methylamine in THF, a selective conversion gave the η1-phosphaguanidine-κP complex 11a at ambient temperature  fits very well with the rare η1-phosphaguanidine complex [W(CO)2(η5-C5H5){Ph2PC(N-p-tol)N(H)p-tol}Cl] (δ(31P) = − 25.6 ppm)85. The structure of 11a was confirmed by single crystal X-ray crystallography  bond. The phosphaguanidine complex 11b (−25.8 ppm) was formed as 1,2-addition product of the N-H to the P-C bond in 3d  was chosen, while keeping all reaction conditions constant. In the case of 3d, the reaction was drastically slowed so that a full conversion to the product 12 was not achieved at ambient temperature. Raising the reaction temperature to 40 °C did not lead to full conversion either and above 40 °C the reaction became increasingly unselective. The 31P NMR spectrum of the reaction solution showed a singlet for 12 and a value of −33.8 ppm which is slightly shifted to higher frequency compared to those of 11a,b, but without any P,H coupling. The drastically smaller 1JW,P coupling constant of 103.8 Hz in 12  indicates an increased negative charge density at phosphorus, similar to the zwitterionic complexes 3a–d but with a significantly different chemical shift. The resulting 1,2-C-N addition product 12 represents an example of an isocyanide-to-carbene ligand conversion at a neutral nonmetal center, analogous to transition metal coordination chemistry23.To test the selectivity of the addition reaction to 31P NMR chemical shifts  for the model phosphaguanidine complexes 16ap (−25.3 ppm) and 16 bp (−22.3 ppm) (vide infra) are in very good agreement with the aforementioned shift to low frequency observed for the experimentally obtained product in the reaction with isopropylamine 11b, compared to that of methylamine 11a 5{tBuP = C(NMe2)2}] (δ(31P) = −25.1 ppm, 1JW,P = 153.5 Hz)86 or [W(CO)5{PhP(IDipp)}] imidazol-2-ylidene) (δ(31P) = − 57.7 ppm, 1JW,P = 120 Hz)87.Computed  13 Fig. , the for88) were performed for two model reactions of the most labile N-methylimidazole adduct and using the simplified P-tert-butyl substituted pentacarbonyl-tungsten(0) complex model 3c” for the sake of computational efficiency .For a better understanding of the reactivity of the electrophilic phosphorus center both in ligand substitution reactions at the P atom and in the formation of metal ligated phosphiranes, quantum chemical calculations at the CCSD(T)/def2-TZVPP(ecp) level  and the C-N bond, various aspects of bonding were closely inspected by theoretical means at the same level and using the simplified P-tert-butyl substituted derivative 3d” derived from P-tert-butyl isocyanide  that readily undergoes a H shift over a moderate barrier to the very stable phosphaguanidine complex 16a. Rotamer 16ac (superscript c stands for a conformer) facilitates the kinetically hampered proton-transfer from phosphorus to the other N atom originally belonging to the isocyanide ligand, giving rise to a conformer (17ac) of the somewhat more stable diaminocarbene 17a. The latter could also be formed by N-to-N proton transfer in the Z-configured isomer 15aZ, although this route is disfavored compared to the above mentioned prototropy in 15aE. The experimentally observed product 11a in the reaction with methylamine  16ap, arising either via intermolecular (not studied) or intramolecular proton transfer from 17a. The sterically more demanding isopropylamine gives rise to similar or slightly higher energy model intermediates (not shown) 15bE , 16b  and the most stable final product 16 bp . This seems to indicate that the increase of steric bulk favors 16p over the initially formed intermediate 16 and, hence, points to a thermodynamic preference for the protomer also in case of reaction with methylamine when the true, more sterically crowded, triphenylmethyl group is used as P-substituent (3d).To understand the coordination vs. transition metal-like reactivity of the isocyanide ide Fig. . The nuctert-butylamine gives rise to a higher energy profile compared to methylamine  compared to the N-to-P one 15cE → 17c , which, after rotation, makes the diaminocarbene-adduct conformer 17cc the kinetically preferred product. This agrees with the experimental (31P NMR) observation in case of the reaction with tert-butylamine. The proposed zwitterionic structure for experimental (12) or model (17/17c) diaminocarbene-phosphinidene complex is supported by the similar values obtained for C-P bond strength parameters in 17cc compared to a reported model NHC-phosphinidene complex73.  = 0.1487/0.1473 au; ¼∇2ρ(r) = −0.0473/−0.0180 e·Å−5). Indeed, the electron transfer from the ligand to the phosphinidene complex fragment in model diaminocarbene-phosphinidene adduct 17cc (ΔqLöwdin = −0.35 e) is much higher than the values reported beforehand for 3a–d. Therefore, it indicates that the above-mentioned P-C(NHtBu)2 linkage in 17cc should be better considered as dative C → P bonding. It should be noted here that the calculations not only support the experimental results but also clearly reveal differences between the chemistry of isocyanide metal complexes and the P-adducts reported here. In the former case a 1,2-addition reactivity to the M-C(ligand) bond does not exist, while in case of the P-C(ligand) bond it is the preferred pathway for small primary amines. Only if sterics become important does the organometallic-like reactivity of the phosphorus center comes to the fore, i.e., the isocyanide-to-carbene ligand conversion via 1,2-addition to the C-N bond.As expected, the bulkier ine Fig. . The mos90 from their thermodynamic oxygen-transfer potentials (TOP)91, organyl-substituted phosphinidene κP-pentacarbonyltungsten(0) complexes, R-P-W(CO)5, possess highly electrophilic phosphorus centers. Consequently, they are more easily oxidized to give the respective terminal phosphinidene oxide complex as in case of the P-methyl-substituted phosphinidene complex , compared to ethyl isocyanide . At the much higher level of theory PWPB95-D3/def2-QZVPP(ecp)//RIJCOSX-B3LYP-D3/def2-TZVP(ecp), the computed TOP for R-P-W(CO)5 increase in the order CPh3 < Me < tBu   acidity of phosphinidene complexes R-P-W(CO)5 increases in the order CPh3 < Me < tBu   were also computed at the working level of theory  for common reference compounds BF3 (355.0), AlF3 (491.5), SiF4 (318.5), PF5 (390.8) and SbF5 (498.0), in good agreement with reported values94  computed at the CCSD(T)/CBS//PBEh-3c level. It should be noted here that the effect of low-coordinate element centers has not yet been thoroughly studied, but it seems to be apparent that the FIA value always increases with the coordination number/oxidation state94.The fluoride ion affinity (FIA) SI-Fig. . For com3a  and 12, is provided using Bader’s quantum theory of atoms in molecules (QTAIM). The Holthausen-Cowley criteria for dative bonding are fulfilled by the L → P bonds of adduct complexes 3a–c and 12  closer to the electron acceptor center (P in these cases)ii.2ρ valuewith vanishing ∇iii.97.displaying two valence-shell charge concentration (VSCC) regions (bands) located at the basin of the donor atom along the central part of the bond pathMoreover, additional evidence of the (varying) dative character for all herein described ligand-phosphinidene complex adducts  12 Fig. :i.existeVSCC, together with the small positive value of ∇2ρ at the BCP, was reported to allow unequivocal assignment to dative bonding73. The parameter τVSCC is defined as the product of the two (signed) VSCC positions, divided by the square of the bond path distance , here τVSCC = 0.0013. The positions of the minima for VSCCN/C and VSCCP  were obtained by deconvolution of the central part of the ∇2ρ plot into asymmetric Gaussian functions, as previously reported73.Most recently, the relative charge concentration bands position parameter, τ3a–c and 12 also show significant electron density transfers from the ligands to the phosphinidene complex units of ca. 0.5 e, and relatively high HOMO energy values above −6.12 eV (Table 12) exhibits a robust single C-P bond with a WBI close to unity and a rather large Gibbs free energy of complexation, whereas those arising from heteroaromatic N-donors (3a–c) display moderately weak N-P bonds and rather low complexation energies. Among them, 3b represents an interesting case because it constitutes the most labile isolable adduct (3a was not easily isolable in pure form) thus being the optimal candidate for ligand substitution reactions (vide infra). The most different situation is that observed for 3d’  that indicates mostly covalent character for the ligand-P bond. This is in line with low electron density transfer to the phosphinidene complex unit (0.33 e), because of π-backdonation to the isocyanide ligand, which is supported by the low-lying HOMO and the partial double-bond character, as indicated by the high WBI  and its valence tautomer with a planar P center corresponds to the vertex-type TS for the inversion at P, with a remarkably low barrier  compared to the other three-coordinated P(III) species98. The natural resonance theory (NRT) analysis of a very simplified model 3dMe,Me with methylisocyanide as ligand and a P-methyl substituent, reveals that the isocyanide ligand is linked by a formal P = C in a 28.7% extent, considering the twelve resonance structures contributing more than 2.1%  of [W(CO)5{P(CPh3)Cl2}], 18 μL  of 12-crown-4 and 0.08 mL  of a tert-butyllithium solution in 2 mL of THF at −80 °C. Afterwards, 0.030 g  of 4-dimethylaminopyridine (DMAP) (2b) was added at −50 °C. The reaction mixture was stirred for 18 h while it was allowed to slowly warm up to ambient temperature. All volatiles were removed in vacuo (<0.02 mbar) at ambient temperature. The product was extracted five times with 5 mL of diethyl ether using a filter cannula (Ø = 2 mm) with a glass microfiber filter paper (Whatman® GF/B) and a Schlenk frit . Afterwards, 40 mL of n-pentane were added to the solution. The obtained yellow suspension was stirred for 1 h at ambient temperature. The supernatant was filtered off using a filter cannula (Ø = 2 mm) with a Whatman® 595 filter paper at ambient temperature and the yellow solid residue was washed three times using 5 mL of n-pentane at ambient temperature. The product was obtained as yellow solid after drying for 1 h in vacuo (<0.02 mbar) at ambient temperature. Yield: 0.045 g ; yellow solid; mp: 141 °C (dec.); 1H NMR : δ 7.65 , 7.57–7.55 , 7.18–7.14 , 7.09–7.05 , 6.47–6.46 , 3.06 ; 13C{1H} NMR : δ 203.5 , 200.4 , 156.8, 151.5 , 147.4, 130.9 , 128.3, 125.9, 106.5, 63.9 , 39.4; 15N{1H} NMR : δ −193.9, −298.3; 31P NMR : δ 234.8 ; IR (ATR Diamond): 1917 (νCO), 1971 (νCO), 2054 (νCO); MS : m/z (%) = 598.0 (<1) [W(CO)5(PCPh3)]+, 569.9 (<1) [W(CO)4(PCPh3)]+, 541.9 (<1) [W(CO)3(PCPh3)]+, 514.0 (<1) [W(CO)2(PCPh3)]+, 274.0 (<1) [PCPh3]+, 243.0 (100) [CPh3]+, 121.0 (48) [DMAP-H]+; MS (LIFDI): m/z (%) = 721.2 (38) [M + H]+, 446.0 (10) [W(CO)5(dmap)]+, 243.2 (22) [CPh3]+; elemental analysis calcd (%) for C31H25N2O5PW: C 51.69, H 3.50, N 3.89; found: C 51.51, H 3.76, N 3.74.A solution of a Li/Cl phosphinidenoid tungsten(0) complex 1 was prepared using 1.425 g  of [W(CO)5{P(CPh3)Cl2}], 0.34 mL  of 12-crown-4 and 1.34 mL  of a tert-butyllithium solution in 22 mL of THF at −80 °C. Afterwards, 0.26 mL  of N-methylimidazole (2c) were added dropwise at −50 °C. The reaction mixture was stirred for 15.5 h while it was allowed to slowly warm up to ambient temperature. All volatiles were removed in vacuo (<0.02 mbar) at ambient temperature. The product was extracted six times with 30 mL of diethyl ether using a filter cannula (Ø = 2 mm) with a glass microfiber filter paper (Whatman® GF/B) and a P3 Schlenk frit . Residual product was extracted from the SiO2 using three times 40 mL of diethyl ether. Afterwards, 420 mL of n-pentane were added to the solution. The obtained yellow suspension was stirred for 1 h at ambient temperature. The supernatant was filtered off using a filter cannula (Ø = 2 mm) with a Whatman® 595 filter paper at ambient temperature and the yellow solid residue was washed three times using 20 mL of n-pentane at ambient temperature. The product was obtained as yellow solid after drying for 9 h in vacuo (<0.02 mbar) at ambient temperature. Yield: 0.805 g ; yellow solid; mp: 146 °C (dec.); 1H NMR : δ 7.75–7.73 , 7.14–7.09 , 7.00–6.94 , 6.41–6.40 , 6.33–6.32 , 5.11–5.10 , 1.83 ; 13C{1H} NMR : δ 203.2 , 200.4 , 147.4 , 142.1, 130.5 (br s), 130.3 , 128.4, 125.9, 119.8, 62.9 , 33.9; 15N{1H} NMR : δ −183.9, −215.2; 31P NMR : δ 199.2 ; IR (ATR Diamond): 1921 (νCO), 1966 (νCO), 2053 (νCO); MS (LIFDI): m/z (%) = 680.8 (38) [M]+, 243.1 (100) [CPh3]+; elemental analysis calcd (%) for C28H21N2O5PW: C 49.44, H 3.11, N 4.12; found: C 49.22, H 3.20, N 4.11.A solution of a Li/Cl phosphinidenoid tungsten(0) complex 1 was prepared using 0.67 g  of [W(CO)5{P(CPh3)Cl2}], 0.14 mL  of 12-crown-4 and 0.77 mL  of a tert-butyllithium solution in 30 mL of THF at −80 °C. Afterwards, 0.17 mL  of tert-butyl isocyanide (2d) was added dropwise at −50 °C. The reaction mixture was stirred for 16 h while it was allowed to slowly warm up to ambient temperature. All volatiles were removed in vacuo (<0.02 mbar) at ambient temperature. The product was extracted three times with 20 mL of diethyl ether. The solvent was removed in vacuo (<0.02 mbar) at ambient temperature and the product was obtained as yellow solid after drying under the same conditions for 2 h. Characterization data were consistent with literature values62. Yield: 0.56 g ; yellow solid; 1H NMR : δ 7.33–7.24 , 1.28 ; 1H NMR : δ 7.45–6.98 , 0.68 ; 13C{1H} NMR : δ 201.3 , 197.7 , 146.8 , 143.0 , 130.4 , 128.1, 126.8 , 61.5, 58.4 , 29.7; 15N{1H} NMR : δ −170.7; 31P : δ −50.5 ; IR (ATR diamond): 1889 (νCO), 1923 (νCO), 2060 (νCO), 2142 (νCN); MS : m/z (%) = 681 (0.1) [M]+, 244 (100) [CPh3 + H]+.A solution of a Li/Cl phosphinidenoid tungsten(0) complex 3c and 0.38 mL  of 1-pentene in 10 mL of benzene was stirred for 14 h at 70 °C. Afterwards, all volatiles were removed in vacuo (<0.02 mbar) at 70 °C. The obtained brown oil was further dried for 3 h at ambient temperature. The crude product was purified via column chromatography  using 200 mL of n-pentane. All volatiles were removed in vacuo (<0.02 mbar) at ambient temperature and the yellow oil was further dried for 1.5 h. Yield: 0.12 g ; pale-yellow oil; isomeric ratio: 34:66 (cis: trans); 1H NMR : cis−10a: δ 7.45–7.42 , 7.10–6.98 , 1.71–1.63 , 1.60–1.54 , 1.06–0.88 , 0.58 , −0.12– − 0.25 ; trans−10a: δ 7.45–7.42 , 7.10–6.98 , 1.71–1.63 , 1.45–1.17 , 0.81 , 0.78–0.75 ; 13C{1H} NMR : cis−10a: δ 198.3 , 197.2 , 142.6, 131.7 , 128.5, 127.7, 61.7 , 33.5 , 29.8 , 24.1 , 16.9 , 13.8; trans−10a: δ 197.4 , 196.8 , 142.1, 131.5 , 128.3, 127.7, 62.1 , 34.1 , 23.0 , 22.9 , 17.3 , 13.8; 31P NMR : cis−10a: δ −137.7 ; trans−10a: δ −145.4 ; IR (ATR diamond): 1913 (νCO), 1983 (νCO), 2071 (νCO), 2959 (νCH); MS (LIFDI): m/z (%) = 668.1 (100) [M]+, 243.1 (10) [CPh3]+.A solution of 0.233 g  of complex 3c and 0.40 mL  of 1-hexene in 12 mL of benzene was stirred for 16.5 h at 70 °C. Afterwards, all volatiles were removed in vacuo (<0.02 mbar) at ambient temperature. The crude product was purified via column chromatography  at ambient temperature using n-pentane. Yield: 0.12 g ; pale-yellow oil; isomeric ratio: 36:64 (cis: trans); 1H NMR : cis−10b: δ 7.45–7.43 , 7.10–7.05 , 7.03–6.97 , 1.74–1.65 , 1.62–1.53 , 1.28–1.18 , 1.16–1.09 , 1.03–0.92 , 0.70–0.67 , −0.09– −0.18 ; trans−10b: δ 7.45–7.43 , 7.10–7.05 , 7.03–6.97 , 1.74–1.65 , 1.44–1.32 , 1.28–1.18 , 1.03–0.92 , 0.85–0.83 , 0.80–0.76 ; 13C{1H} NMR : cis−10b: δ 198.3 , 197.2 , 142.6, 131.7 , 128.5, 127.7, 61.7 , 33.5 , 33.1 , 27.7 , 22.7, 17.1 , 14.0; trans−10b: δ 197.4 , 196.8 , 142.2, 131.5 , 128.3, 127.7, 62.1 , 32.0 , 31.8 , 23.1 , 22.8, 17.4 , 14.1; 31P NMR : cis−10b: δ −137.4 ; trans−10b: δ −145.5 ; IR (ATR diamond): 1904 (νCO), 1982 (w) (νCO), 2070 (νCO), 2957 (νCH); MS (LIFDI): m/z (%) = 682.3 (100) [M]+, 243.2 (5) [CPh3]+.A solution of 0.217 g  of complex 3d in 20 mL of THF. The solution was stirred for 21 h at ambient temperature. Afterwards, all volatiles were removed in vacuo (<0.02 mbar) at ambient temperature. The product was isolated as pale-yellow solid by column chromatography  using diethyl ether at ambient temperature. Yield: 0.03 g ; pale-yellow solid; mp: 131–132 °C (dec.); 1H NMR : δ 7.36–7.11 , 6.78 , 3.22 , 2.96 , 1.04 ; 13C{1H} NMR : δ 198.1 , 196.3 , 151.7, 143.9, 129.6, 128.4, 127.5, 66.0, 58.9 , 29.9, 28.3; 15N{1H} NMR : δ −144.6, −259.3; 31P NMR : δ −27.2 ; IR (ATR diamond): 1914 (νCO), 1988 (νCO), 2072 (νCO), 2372 (νPH), 3411 (νNH); MS : m/z (%) = 712.0 (0.1) [M]+, 684.0 (2) [M − CO]+, 628.0 (0.1) [M − 3CO]+, 599.9 (0.1) [M − 4CO]+, 572.1 (0.1) [M − 5CO]+, 542.9 (0.1) [M − 4CO-tBu]+, 514.9 (0.1) [M − 5CO−tBu]+, 440.9 (5) [M − CO−CPh3]+, 384.9 (2) [M − 3CO − CPh3]+, 356.8 (1) [M − 4CO − CPh3]+, 244.0 (100) [CPh3 + H]+, 166.0 (32) [CPh2]+, 113.0 (48) [C(NMe)N(H)tBu]+, 77.0 (2) [Ph+], 57.0 (65) [tBu]+; elemental analysis calcd (%) for C30H29N2O5PW: C 50.58, H 4.10, N 3.93; found: C 52.26, H 4.60, N 3.60.0.63 mL  of a methylamine solution was added to a solution of 0.17 g  of of complex 3d was dissolved in 13 mL  of isopropylamine at ambient temperature. The reaction mixture was stirred for 25 h at ambient temperature. All volatiles were removed in vacuo (<0.02 mbar) at ambient temperature. Yield: 0.19 g ; pale-yellow solid; mp: 147–148 °C (dec.); 1H NMR : δ 7.59–6.99 , 6.64 , 3.33 , 3.27 , 1.19 , 1.19 , 0.97 ; 13C{1H} NMR : δ 198.1 , 196.6 , 146.6, 144.0, 131.3–126.4 (m), 59.1 , 52.4 , 50.8 , 28.3, 24.8, 24.7; 15N{1H} NMR : δ −112.4, −260.0; 31P NMR : δ −25.8 ; IR (ATR diamond): 1915 (νCO), 1987 (νCO), 2070 (νCO), 2379 (νPH), 3408 (νNH); MS (LIFDI): m/z (%) = 740.3 (100) [M]+, 599.1 (3) [M − 5CO − H]+, 417 (16) [M − W(CO)5 + H]+, 243.2 (82) [CPh3]+; elemental analysis calcd (%) for C32H33N2O5PW: C 51.91, H 4.49, N 3.78; found: C 52.22, H 4.53, N 3.59.0.251 g  of complex Supplemenatry informationsPeer Review FileSource Data


Nephrology Dialysis Transplantation, Volume 38, Issue Supplement_1, June 2023, gfad063d_3739, https://doi.org/10.1093/ndt/gfad063d_3739This is a correction to: Nabil Hamouche and others, #3739 SHUNT PORTOSYSTEMIC COMPLICATED BY HYPERAMMONEMIA AND HEPATIC ENCEPHALOPATHY SUPPORTED BY DAILY DIALYSIS IN A CHRONIC HEMODIALYSIS PATIENT, Case Report section, 3rd paragraph, second sentence should read: “Pre-dialytic ammonia levels varied between 103 and 161 µmol/l as well as post-dialytic ammonia levels varied between 24 and 37 µmol/l, […]” instead of: “Pre-dialytic ammonia levels varied between 24 and 37 µmol/l, as well as post-dialytic ammonia levels varied between 103 and 161 µmol/l, […]”.In the originally published version of this manuscript, there was an transposition of level figures, in This error has been corrected in the article.


Their chemistry in aqueous solution was explored by UV/vis spectroscopy, conductivity, cyclic voltammetry, and electron paramagnetic resonance studies. Their DNA binding was analyzed by electronic spectroscopy , circular dichroism, and viscosity methods. The cytotoxicity of the complexes was assessed on human cancer cell lines MDA-MB-231, MCF-7 , A549  and A2780cis , and non-tumor cell lines MRC-5 (lung) and MCF-10A (breast). The major species are ternary, in solution and solid state. Complexes are highly cytotoxic as compared to Cisplatin. Complexes containing bam and phen are interesting candidates to study their in vivo activity in triple-negative breast cancer treatment.Coordination complexes may act as anticancer agents. Among others, the formation of the complex may facilitate the ligand uptake by the cell. Searching for new copper compounds with cytotoxic activity, the complex Cu-dipicolinate was studied as a neutral scaffold to form ternary complexes with diimines. A series of [Cu(dipicolinate)(diimine)] complexes (where diimine: Phenanthroline, phen, 5-NO Cancer causes about 19 million new cases and 10 million deaths yearly (worldwide data for 2020) .Developing new metal coordination compounds (“complexes”) with potential anticancer activity offers a broad range of possibilities, from varying the metal centers and ligands to combining their properties, that could lead to new metallo-pharmaceuticals. Several metal centers have been identified to give rise to complexes that present antitumor activity, such as Pt, Ru, Ga, Au, and Cu, among others ,4. This Diimines, such as bipyridine (bipy) and phenanthroline (phen) derivatives , exhibitDifferentiating the biological effects of metals versus their coordinated ligands can be difficult in coordination compounds. A current issue is whether the cytotoxic activity of diimine coordination compounds results from the intact complex, i.e., the metal bound to the organic molecule (the ligand), or if the metal transfers the ligand into the cell, acting as a delivery moiety. Both phenomena seem to coexist in the present-day state of knowledge . The met2-phenanthroline, 5nitro-phen, neocuproine, neo, 3,4,7,8-tetramethyl-phenanthroline, tmp, bathophenanthroline, batho and L-dipeptide: Ala-Gly, Ala-Phe, Phe-Ala, and Phe-Phe among others)  compounds were developed  ,10,11,122(dipic)2(tmp)2]·7H2O. As many metallic complexes bind to DNA as a part of their mechanism of action, its interaction with isolated Calf Thymus-DNA (CT-DNA) was studied by circular dichroism (CD), electronic spectroscopy , and viscosity methods. The cytotoxicity of the complexes was assessed against MDA-MB-231, MCF-7 , MCF-10A , A549 , MRC-5 , and A2780cis (Cisplatin-resistant human ovarian carcinoma).Aiming at identifying compounds with improved activity against cancer cells, we studied other neutral Cu-ligand scaffolds for diimine incorporation to ascertain their influence on cytotoxic activity . In thisAll chemical compounds  and solvents were used as commercially available without further purification.To a suspension of dipicolinic acid 0.15 mmol) in 10 mL of water basic copper(II) carbonate was added in 100% excess and allowed to react with constant stirring at 60 °C for 30 min ·3H2(dipic)2(bipy)2]·12H2O. (2) Blue solid. Yield 32%. Elemental analysis Calc. for CuC17H23N3O10/Exp. %C 41.42/41.79, %N 8.52/8.37, %H 4.70/4.29. Purity 98%. FT-IR: 2900–3500 cm−1 ν(C-H), ν(O-H), ν(N-H); 1631 cm−1 ν(COO−)as; 1528 cm−1 ν(C-C), ν(C-N); 1446 cm-1 ν(C-C), ν(C-N), ν(COO−)s; 1159 and 1088 cm−1 ρ(C-H); 910 cm−1 δ(C-C-C), δ(C-C-N); 857 cm−1 δoop(C-H), δ(C-C-C), δ(C-C-N); 731 cm−1 ρ(C-H); 537 and 414 cm−1 ν(Cu-N/O). ΛM (25 °C): 15 Scm2 mol−1.·7H2O)]·2H2O. (4) Blue solid. Yield 31%. Elemental analysis Calc. for CuC19H17N3O7/Exp. %C 49.30/49.73, %N 9.08/9.33, %H 3.70/3.44. Purity 97%. FT-IR: 2900–3500 cm−1 ν(C-H), ν(O-H), ν(N-H); 1624 cm−1 ν(COO−)as; 1520 cm−1 ν(C-C), ν(C-N); 1427 cm-1 ν(C-C), ν(C-N), ν(COO−)s; 1145 and 1083 cm−1 ρ(C-H); 910 cm−1 δ(C-C-C), δ(C-C-N); 851 cm−1 δoop(C-H), δ(C-C-C), δ(C-C-N); 723 cm−1 ρ(C-H); 544 and 442 cm−1 ν(Cu-N/O). ΛM (25 °C): 16 Scm2 mol−1.·2.5H2O)]·7.5H2O. (6) Blue solid. Yield 43%. Elemental analysis Calc. for CuC19H27N4O14.5/Exp. %C 37.60/37.75, %N 9.23/9.28, %H 4.48/4.15. Purity 99%. FT-IR: 2900–3500 cm−1 ν(C-H), ν(O-H), ν(N-H); 1622 cm−1 ν(COO−)as; 1521 cm−1 ν(C-C), ν(C-N); 1427 cm-1 ν(C-C), ν(C-N), ν(COO−)s; 1185 and 1087 cm−1 ρ(C-H); 918 cm−1 δ(C-C-C), δ(C-C-N); 856 cm−1 δoop(C-H), δ(C-C-C), δ(C-C-N); 735 cm−1 ρ(C-H); 519 and 443 cm−1 ν(Cu-N/O). ΛM (25 °C): 11 Scm2 mol−1.·3H2O)]·H2O. (8) Blue solid. Yield 37%. Elemental analysis Calc. for CuC31H23N3O6/Exp. %C 62.36/62.58, %N 7.04/7.12, %H 3.88/3.49. Purity 98%. FT-IR: 2900–3500 cm−1 ν(C-H), ν(O-H), ν(N-H); 1624 cm−1 ν(COO−)as; 1495 cm−1 ν(C-C), ν(C-N); 1427 cm-1 ν(C-C), ν(C-N), ν(COO−)s; 1152 and 1089 cm−1 ρ(C-H); 913 cm−1 δ(C-C-C), δ(C-C-N); 851 cm−1 δoop(C-H), δ(C-C-C), δ(C-C-N); 738 cm−1 ρ(C-H); 549 and 443 cm−1 ν(Cu-N/O). ΛM (25 °C): 13 Scm2 mol−1.[Cu(dipic)(batho)(H2(dipic)2(tmp)2]·7H2O. (9) Blue solid. Yield 28%. Elemental analysis Calc. for CuC23H26N3O7.5/Exp. %C 52.32/52.26, %N 7.95/7.83, %H 4.96/4.58. Purity 98%.FT-IR: 2900–3500 cm−1 ν(C-H), ν(O-H), ν(N-H); 1621 cm−1 ν(COO−)as; 1528 cm−1 ν(C-C), ν(C-N); 1425 cm-1 ν(C-C), ν(C-N), ν(COO−)s; 1157 and 1081 cm−1 ρ(C-H); 910 cm−1 δ(C-C-C), δ(C-C-N); 870 cm−1 δoop(C-H), δ(C-C-C), δ(C-C-N); 724 cm−1 ρ(C-H); 552 and 443 cm−1 ν(Cu-N/O). ΛM (25 °C): 10 Scm2 mol−1. [The lipophilicity of the complexes was studied by Thin Layer Reverse Chromatography using ALUGRAMb) were determined using the Benesi–Hildebrand method [obs as a function of 1/[DNA] plot.Absorption titration measurements in the UV were carried out keeping the complex concentration constant at 10–15 µM in 5 mM buffer Tris/HCl pH = 7.5 and 50 mM of NaCl while varying the concentration of Calf Thymus-DNA (CT-DNA) from 0 to 250 μM. The intrinsic binding constants  and presence (η) of complexes was calculated as: (η/η0) = t − t0/tDNA − t0, where t0 and tDNA are the flow times of the buffer and DNA solution, respectively, while t is the flow time of the DNA solution in the presence of copper complexes. Data are presented as (η/η0)1/3 versus the ratio [complex]/[DNA] ·yH2O were obtained . Among those, six yielded single crystals suitable for X-ray diffraction studies. Five of the obtained crystals were tested and corresponded to previously reported crystal structures, complexes (1) ·12H2O and [Cu2(dipic)2(tmp)2]·7H2O a carboxylic oxygen atom from the dipic, coordinated in the equatorial plane, acts as a bridge between the two metal centers.The complexes present five or six coordination indexes where the position of the donor atoms varies depending on the geometry of the metal center. The dipic is situated in the equatorial plane for five coordinated compounds, and the diimine is approximately perpendicular to it, as previously reported for [Cu(dipeptide)(phen)] complexes ,10,11,129) is a dinuclear one. Each copper(II) center presents an octahedral coordination geometry with tetragonal distortion, and the dipic acts as a tridentated ditopic ligand connecting both metal centers (The complex containing tmp ( centers . Due to  centers .Bond lengths and angles around the copper(II) center are presented in The Cu-Cu distance is 3.317 Å. The dinuclear moiety is further stabilized through intramolecular π···π interactions where the tmp molecules are parallel to each other with inter-centroid distances of 3.753 Å .9) can participate, a Full Interaction Map (FIM) was constructed using Mercury x] family to verify in the bulk samples the number of solvent molecules that are hydration or coordinated, and, therefore, the degrees of hydration. This allowed us to assess which compounds are hexacoordinated in the solid state, including complexes (5) and (6), where no crystal structure is available.TGA and DSC measurements were carried out for complexes of the ·2.5H2O and [Cu(dipic)(5nitro-phen)(H2O)]·2H2O, respectively, confirming 5 and 6 coordinated copper(II) centers.In the case of complexes where the crystal structure was determined, TGA results are consistent with what is expected from the structure, confirming that the bulk’s identity is the same as in crystals. As for complexes  and (9)   are charmax and the donor atoms coordinated equatorially to the Cu(II)  complex measured in DMF where λmax is 760 nm , in, in1/2 ves (ROS) .The lipophilicity of the ligands follows the order tetra > batho > bam > 4metil-phen ≈ 5nitro-phen ≈ neo > phen > dmb > bipy . Taking 1), (4), (7), and (8), all of which correspond to structures containing discrete mononuclear units  and lipophilicity were explored. To that end, Hirshfeld surface analysis was performed on data from complexes (DNA binding is considered part of the mechanisms of action for copper(II) complexes containing planar heterocyclic ligands . In thisAll recorded spectra and Benesi–Hildebrand linearization curves are available in Kb and circular dichroism spectra.2O)x] are in the range 1 and 16 × 103 M−1, presenting similar results to those observed for the ternary copper(II) complex containing dipicolinate and 3-amino-1H-1,2,4-triazole 2+ complexes, including Cu-neo as detected by fiber EPR studies ·7H2O. In them, the diimine was coordinated perpendicular to the dipic, with the diimine influencing the coordination polyhedra. The major complex species in solution were hexacoordinated, ternary ones, with dimeric complexes hydrolyzing to monomeric ones.Four new copper complexes were obtained, including a new crystalline structure of  were potent cytotoxic agents, more active than Cisplatin on the studied cell lines, and even active on cells resistant to Cisplatin. Despite that, complexes were also cytotoxic to non-tumor cells. The complexes containing the diimines bam (diimine)] with other [Cu(ligand)(diimine)] complexes of the same diimine.


The crystal structure of the title compound obtained from the reduction of its nitro precursor, methyl 3-methoxy-4-nitroisoxazole-5-carboxylate, is described. 6H8N2O4, a new derivative of isoxazole, has been synthesized and structurally characterized. The crystal structure shows the molecule to be almost planar (r.m.s. deviation for the non-hydrogen atoms = 0.029 Å), this conformation being supported by an intramolecular N—H⋯O hydrogen bond. In the extended structure, the molecules are linked by N—H⋯O hydrogen bonds into chains propagating along [010].The title compound, C After 14 h stirring at room temperature, the mixture was poured into an ice-cold aqueous solution of HCl  and extracted into Et2O (5 × 80 ml). The combined organic layers were washed with a saturated aqueous solution of Na2CO3 (80 ml), dried over MgSO4, filtered and concentrated under reduced pressure to afford a light yellow crystalline solid, which was purified by silica gel column chromatography , affording methyl 3-methoxyisoxazole-5-carboxylate  as a colourless crystalline solid.The starting material, methyl 3-methoxyisoxazole-5-carboxylate, was prepared according to the previously described literature procedure with minor modifications  to afford methyl 3-methoxy-4-nitroisoxazole-5-carboxylate  as yellowish oil: Rf = 0.41 ; 1H , δ: (p.p.m): 4.14 , 4.02 ; 13C , δ: (p.p.m): 164.0, 157.4, 155.0, 127.7, 58.9, 54.2; HRMS m/z (APCI+), found: [M + H]+ 203.0295, C6H7N2O6 requires [M + H]+ 203.0299.Triflic anhydride  was added to a solution of tetramethylammonium nitrate  in DCM (3 ml) at room temperature. The suspension was stirred for 2 h, then a solution of methyl 3-methoxyisoxazole-5-carboxylate  in dichloromethane (DCM) (10 ml) was added. After 48 h stirring under reflux, the mixture was cooled to room temperature and partitioned between water (30 ml) and DCM (40 ml). The organic layer was separated and washed with water (50 ml). The aqueous layer was extracted with DCM (3 × 50 ml). The combined organic layers were washed with brine (50 ml), dried over MgSOSynthesis of methyl 4-amino-3-methoxyisoxazole-5-carboxylate2O (AcOH = acetic acid) . After stirring at 50°C for 2 h, the solution was cooled to room temperature and the solvent was removed under reduced pressure. The residue was partitioned between water (20 ml) and ethyl acetate (EtOAc) (20 ml). The mixture was basified with a saturated aqueous solution of Na2CO3 and further extracted with EtOAc (3 × 20 ml). The combined organic layers were washed with brine (20 ml), dried over MgSO4, filtered and concentrated under reduced pressure to afford a pale-yellow solid, which was purified by silica gel column chromatography , affording methyl 4-amino-3-methoxyisoxazole-5-carboxylate  as a colourless crystalline solid: Rf = 0.74 ; m.p. 111–112°C; 1H , δ: (p.p.m): 4.15 , 4.05 , 3.92 ; 13C , δ: (p.p.m): 164.5, 159.1, 138.4, 125.6, 57.5, 51.9; HRMS m/z (ESI+), found: [M + Na]+ 195.0373, C6H8N2O4Na requires [M + Na]+ 195.0382.Iron powder  was added to a solution of methyl 3-methoxy-4-nitroisoxazole-5-carboxylate  in AcOH/HCrystal data, data collection and structure refinement details are summarized in Table 210.1107/S2414314623006235/hb4437sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2414314623006235/hb4437Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2414314623006235/hb4437Isup3.molSupporting information file. DOI: Click here for additional data file.10.1107/S2414314623006235/hb4437Isup4.cmlSupporting information file. DOI: 2281787CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Fraxinus hubeiensis is a plant endemic to China and widely used as folk medicine to treat various diseases. However, its chemical constituents have never been reported sufficiently. Thus, the primary objective of this study was to investigate the phytochemical constituents and biological activities of F. hubeiensis leaves. Hence, combined column chromatographic and spectroscopic techniques were used to identify and characterize the secondary metabolites such as a pair of 3-keto-glycoside epimers (1) and (2), along with five known compounds (3 ~ 7). The results of α-glucosidase inhibitory activity exhibited that 1 and 2 had moderate activity with IC50 values of 359.50 and 468.43 µM, respectively, compared to a positive control acarbose with the IC50 value of 164.08 µM. However, Compounds 1–6 were shown to be inactive against the tested microbes.The online version contains supplementary material available at 10.1186/s13065-023-01070-6. Fraxinus hubeiensis belongs to the family Oleaceae which comprises about 27 genera and more than 400 species in the world + peak at m/z 229.0683  and supported by the 13 C-NMR data ; two methylene groups at δH 3.80 , 3.86 , and 3.54 , 3.78 ; four methine group at δH 5.16 , 4.39 , 4.22  and 3.69 , combined with its DEPT and HSQC spectrum. All eight carbons were well resolved in the 13 C NMR spectrum, which indicated the presence of a carbonyl carbon in the downfield region. The connectivity of CH (4)- CH (5)-CH2 (6) could be well deduced from the cross-peaks of H-4/H-5/H-6 in the 1 H-1 H COSY spectrum , supporting the α-glycosidic configuration. Thus, the structure of 1 was elucidated as α-D-ribo-hex-3-ulopyranoside as shown in Fig. The molecular formula of ranoside  was deteranoside  was deteβ-D-ribo-hex-3-ulopyranoside (2), was also obtained as colorless gum. The molecular formula was determined to be C8H14O6 based by HR-ESI-MS peak at [M + Na]+ peak at m/z 229.0697 . Interesting that, the 1 H and 13 C-NMR signals were different from those of its enantiomer 1, even when they were mixed together. Meanwhile, the HMBC cross-peaks confirmed the structure of 2  of the anomeric proton indicated it had β-glycosidic configuration. Therefore, the structure of 2, as a new natural product, was determined as β-D-ribo-hex-3-ulopyranoside.ranoside , was als3) , was purchased from Shanghai Luwei Technology Co., Ltd.). Escherichia coli , Pseudomonas aeruginosa  and Candida albicans  and Bacillus subtilis (B. subtilis), were all purchased from Shanghai Bioresource Collection Center.α-glucosidase and acarbose from F. hubeiensis were collected in the Bonsai garden of Jingzhou in China, in June 2017. Plant materials were identified by one of the authors (Prof. Qing-Lai Wu). A voucher specimen (No. DJBL201706) was deposited at TCM and Ethnomedicine Innovation & Development International Laboratory in Hunan University of Chinese Medicine.The dried leaves of F. hubeiensis (5.0 kg) were pulverized and immersed in 40 L 95% aqueous EtOH for three times at room temperature, each time 10 days. The solvent was evaporated under vacuum to obtain a crude extract (1.01 g), and the extract was suspended in 2 L H2O and partitioned successively with petroleum ether  and ethyl acetate (EtOAc), to yield PE (311.0 g), EtOAc (189.0 g), and water (420.1 g) fractions. The EtOAc fraction was isolated by column chromatography (CC) using silica gel (200–300 mesh) and eluted with PE/CH2Cl2 (9:1), PE/EtOAc (9:1), CH2Cl2/EtOAc (9:1), CH2Cl2/MeOH (9:1), CH2Cl2/MeOH (1:1) gradientally, to yield eight fractions (Fr.A-H).The dried leaves of 2Cl2/EtOAc (1:0 ~ 0:1) to obtain twelve subfractions (Fr. A1-A12), and subfraction Fr. A9 was absorbed to CC with Sephadex LH-20, eluting with CH2Cl2/MeOH (1:1) to obtain fourteen subfractions and yield compound 4 (4.0 mg) and 6 (5.2 mg). Fr. C (5.1 g) was absorbed to silica gel CC  to obtain eight major subfractions (Fr.C1-C8). And then Fr.C5 was loaded to a Sephadex LH-20 CC  and recrystallized to yield compound 5 (7.0 mg).Fr. A (2.1 g) was loaded to silica gel CC and eluted with CH2Cl2/EtOAc (1:0 ~ 0:1) to get ten subfractions (Fr. G1-G10), and Fr.G9 (1.2 g) was loaded on a Sephadex LH-20 CC, eluting with MeOH to obtain eight subfractions (Fr.G9.1-G9.8). Fr. G9.4 was purified on Sephadex LH-20 CC, eluting with MeOH to yield compound 3 (5.3 mg). Fr. G9.2 was purified by semi-preparative HPLC  to yield a mixture of 1 and 2 , exhibiting a single peak, whose value of optical rotation was close to zero. Therefore, the mixture of 1 and 2 was separated on a Chiral NQ (2)-RH Column  to obtain 1(1.5 mg), tR1:4.7 min and 2 (2.1 mg), tR2:5.2 min.Fr. G (40.3 g) was isolated by CC on silica gel, eluting with CH1): white, amorphous gum; c 0.06, MeOH); UV (MeOH) λmax (log ε): 198 (1.43) ,282 (0.38) nm; IR (KBr) νmax3512, 2958, 1652, 1506, 1032, 1018 cm−1; 1 H and 13 C NMR data , see Table m/z 229.0683 [M + Na]+ .Compound : white, 2): white, amorphous gum; c 0.08, MeOH); UV (MeOH) λmax (log ε): 198 (1.19), 286 (0.13) nm; IR (KBr) νmax3512, 2958, 1652, 1506, 1032, 1018 cm− 1; 1 H and 13 C NMR data , see Table m/z 229.0697 [M + Na]+ .Compound (mentclass2pt{minim2CO3 (0.2 mol/L). The absorbance of each well was measured at a wavelength of 405 nm, and the data were recorded and measured in parallel for three times. In this experiment, acarbose was used as the positive control, PBS buffer was used as the blank group, and DMSO solution was used as the negative control group. Other reagents were consistent with the sample experimental group. The absorbance at 405 nm was measured by microplate reader, and the inhibition rate was calculated.The α-glucosidase inhibition assay was carried out on the basis of the method reported with minor modifications , 15. BriThe α-glucosidase inhibitory activity was expressed as percent inhibition and was calculated as follows:1–6 were screened for antimicrobial activities against the tested microbes  by the previously reported method [1–6 exhibited no activities. Perhaps for these isolates, the rang of strains should be expanded to test for the activity. However, because of no sufficient of these compounds isolated from this plant, it was not able to screen them more broadly for antimicrobial activities.Compounds d method , 17. RegF. hubeiensis including two ketoglycosides, one methylinsitol, three coumarins and salicylic acid. All compounds except 5 were isolated from F. hubeiensis for the first time. This study is the first reported of successful isolation of a pair of 3-ketoglycoside isomers by chiral-phase HPLC, which are undescribed compounds from nature. The results of α-glucosidase inhibitory indicated that 1 and 2 possessed moderate activities. It was worth mentioning that the activity of 1 with α-configuration was better than that of 2 with β-configuration, so the subsequent work on structural modification based on the ketose-type skeleton and their structure-activity relationship deserves further study. According to traditional uses of the plant, compounds 1–6 were evaluated for antimicrobial effect. Unfortunately, these isolates had no activity. Therefore, more in-depth phytochemistry and pharmacological research is worth continuing in the future.In summary, seven compounds were isolated from the leaves of Below is the link to the electronic supplementary material.Supplementary Material 1


Zaitzevia Champion, 1923 are described from China, namely Zaitzeviasichuanensissp. nov. and Zaitzeviafengtongzhaiensissp. nov. from Sichuan Province, and Zaitzeviayingzuijieensissp. nov. from Hunan Province. Habitus and diagnostic features of the new species are illustrated. A checklist of all known Chinese Zaitzevia species is given, and a key and distributional map of Zaitzevia species from the Chinese mainland are provided.Three new riffle beetles of the genus  Macronychini genus Zaitzevia Champion, 1923 includes 24 valid species that are distributed from East, Southeast, and Central Asia to North America .The material examinedduring this work is deposited in the Institute of Entomology, Guizhou University, Guiyang, China  (or PARATYPE (yellow)), ♂, Habitus images were taken using a Canon 5D Mark IV digital camera with an MP-E 65 mm f/2.8 1–5× macro lens. A Godox MF12 flash was used as the light source. Images of the morphological details were taken using a Canon 5D Mark IV digital camera in conjunction with a Mitutoyo Plan NIR 10 lens and a Godox MF12 flash was used as the light source or a Nikon SMZ25 stereoscopic microscope with a Nikon DS-Ri2 camera. Zerene Stacker (v. 1.04) was used for image stacking. All images were improved and grouped into plates in Adobe Photoshop CS5 Extended.HL—length of head from the anterior epistomal margin to the occipital constriction;HW—width of head across compound eyes;PL—length of pronotum along the midline;PW—maximum width of pronotum;EL—length of elytra along the suture;EW—maximum width of elytra;CL—the sum of PL + EL.Morphological terminology and the format for the descriptions follow those of Taxon classificationAnimaliaColeopteraElmidaeJiang & Chensp. nov.7786F923-FE6B-5994-9C3E-4849230656ADhttps://zoobank.org/7E78CB26-B54D-447B-B110-CCCEACA0635A Holotype. : China: ♂, labeled ‘China: Sichuan, Ya’an City (雅安市), Muping Town (穆坪镇), Lengmugou Geopark (冷木沟地质公园), an unnamed stream, 30°22′25′′N, 102°48′52′′E; H: 935m, 25.07.2022, R.-X. Jiang & F.-E. Li leg.’ (GUGC). Paratype: 1 ♂, 2 ♀♀; 1 ♀, with the same label data as the holotype; 1 ♂, 1 ♀, labeled ‘Sichuan, Chengdu City (成都市), Dayi County (大邑县), Xiling Town (西岭镇), Xilingxueshan (西岭雪山), Jiaoziping (椒子坪), an unnamed stream, 30°40′12′′N, 103°15′07′E; H: 1350m, 21.07.2022, R.-X. Jiang & F.-E. Li leg.’ (GUGC).Body elongately ellipticalMale.   and the wrinkled elytra. However, the new species can be well distinguished from Z.chenzhitengi by the following characters: 1) elytra weakly wrinkled (cf. distinctly wrinkled in Z.chenzhitengi); 2) different form of elytra ; 3) different form of aedeagus ; 4) different modification of areas between the lateral margins and the sublateral carinae . The new species can be distinguished from Z.muchenae by the much longer and wider longitudinal impression of pronotum and the obviously different form of aedeagus.Taxon classificationAnimaliaColeopteraElmidaeJiang & Chensp. nov.9D122626-BC16-5C9A-BB17-AB875196363Ehttps://zoobank.org/A32F5E97-91DB-41D0-9A7E-F243CC0B86AD Holotype. : China: ♂, labeled ‘China: Sichuan, Ya’an City (雅安市), Baoxing County (宝兴县), Fengtongzhai Township (蜂桶寨乡), Fengtongzhai N. R. (蜂桶寨国家级自然保护区), Dashuigou (大水沟), an unnamed stream, 30°35'18′′N, 102°52′24′′E; H: 1505m, 23.07.2022, R.-X. Jiang & F.-E. L. leg.’ (GUGC). Paratype: 1 ♀, with the same label data as the holotype (GUGC).Body elongately ellipticalMale.   different body size ; 2) elytra weakly wrinkled (cf. distinctly wrinkled in Z.chenzhitengi); 3) obviously different form of aedeagus . Compared with Z.sichuanensis sp. nov., areas between lateral margins and sublateral carinae not convex in the new species, and distinctly convex in Z.sichuanensis sp. nov., these two species also can be distinguished by the obviously different from of aedeagus  aedeagus of the new species is similar to Z.xiongzichuni Jiang & Wang, 2020, both species with a pair of crescent-shaped sclerotizations near apex of median lobe. However surface of Z.xiongzichuni shiny and not wrinkled. Median lobe of Z.xiongzichuni much slender, and weakly narrowed at middle, apical margin of sternite IX with a tuft of short setae, while median lobe of Z.fengtongzhaiensis sp. nov. is shorter and not narrowed at middle, and the apical margin of sternite IX is without setae.see Fig. . ZaitzevTaxon classificationAnimaliaColeopteraElmidaeJiang & Chensp. nov.DCFEC690-3CCC-5814-97AB-E0F3766544E7https://zoobank.org/57149AEE-921A-4AF3-9DAC-8BDC9BF37239 Holotype. : China: ♂, labeled ‘China: Hunan, Huaihua City (怀化市), Huitong County (会同县), Yingzuijie N. R. (鹰嘴界国家级自然保护区), light trap, H: ~400m, 02.08.2022, J.-H. Huang leg.’ (GUGC). Paratype: 6 ♂♂, 1 ♀, with the same label data as the holotype (GUGC).Body elongately ellipticalMale.   and a very short, longitudinal impression on the pronotum. Z.yingzuijieensis sp. nov. can be distinguished from Z.yaeyamana by the following characters: 1) punctures on elytra much denser (vs sparser in Z.yaeyamana); 2) punctures on elytra much large (vs punctures on elytra very thin in Z.yaeyamana); 3) median lobe of aedeagus long and slender, about three times as long as phallobase . Zaitzeviayingzuijieensis sp. nov. can be distinguished from Z.tangliangi by the distinctly asymmetric apex of the median lobe of the aedeagus, while the apex of the median lobe is nearly symmetrical in Z.tangliangi. The sclerotizations near the apex of the median lobe is rounded at one end in the new species but pointed in Z.tangliangi.The new species shares a similar habitus with several other species including the following:


Correction: Neurocrit Care 10.1007/s12028-023-01690-9g2)” has been corrected to “temporal intensity autocorrelation curve (g2)” in Fig. 6 caption.In the original article, the phrase “electric field intensity temporal autocorrelation curve (


However, the lack of specific agonists for all α1 subtypes has limited our understanding of the physiological roles of different α1-AR subtypes, and led to the stagnancy in agonist-based drug development for these receptors. Here we report cryo-EM structures of α1A-AR in complex with heterotrimeric G-proteins and either the endogenous common agonist epinephrine or the α1A-AR-specific synthetic agonist A61603. These structures provide molecular insights into the mechanisms underlying the discrimination between α1A-AR and α1B-AR by A61603. Guided by the structures and corresponding molecular dynamics simulations, we engineer α1A-AR mutants that are not responsive to A61603, and α1B-AR mutants that can be potently activated by A61603. Together, these findings advance our understanding of the agonist specificity for α1-ARs at the molecular level, opening the possibility of rational design of subtype-specific agonists.α α1-adrenergic receptors (α1- AR) play critical roles in the cardiovascular and nervous systems. Here, the authors report molecular insights into the mechanisms underlying the discrimination between α1A-AR and α1B-AR by the agonist A61603. They function through nine distinct human adrenergic receptor (AR) subtypes: α1-type , α2-type  and β-type 4. Due to the lack of selective pharmacological agonists for α1-ARs, the therapeutic potential of α1-ARs has been largely unexplored9. However, mouse genetic studies with individual gene deletions have demonstrated distinct but overlapping physiological functions of α1-AR subtypes in the regulation of blood pressure, cardiac hypertrophy, vascular smooth muscle contraction, neurotransmission, learning and memory, and metabolism17. For example, the α1A-AR subtype is a vasopressor expressed in resistance arteries and is required for normal arterial blood pressure regulation11. In addition, α1B-AR in human coronary endothelial cells mediates vasodilation19.Epinephrine and norepinephrine are neurotransmitters of the sympathetic nervous system, and hormones secreted by the adrenal medulla1-ARs couple to the Gq family of G-proteins, leading to the stimulation of phospholipase C-β to cleave phosphatidylinositol-4,5-bisphosphate into inositol-1,4,5-trisphosphate and 2-diacylglycerol20. The former promotes the release of Ca2+ from intracellular stores, while the latter activates protein kinase C22. While some pharmacological compounds exist that can target α1-ARs, there are only two structurally related α1A-specific agonists, and no α1B-AR and α1D-AR selective agonists have been reported17. A61603 is a high affinity, selective α1A-AR agonist which shows almost no activity at α1B-AR and α1D-AR23. Furthermore, there are no structures available for the active states of α1-ARs. In this paper, we use cryo-EM to determine the structures of α1A-AR/Gq signaling complexes with epinephrine or A61603. These structures reveal the molecular basis for the binding specificity of A61603 for α1A-AR, and the different conformations of epinephrine in interacting with α-ARs versus β-ARs. Gaussian accelerated molecular dynamics (GaMD) simulations and functional studies provide further insights into the mechanisms of specificity, ultimately enabling the validation of key sites that determine the ability of A61603 to specifically activate α1A-AR but not α1B-AR25.α1-AR, we solved cryo-EM structures of human α1A-AR bound to A61603 (a synthetic specific agonist for α1A-AR) at 2.6 Å or epinephrine  at 3.0 Å, both in complex with its cognate signaling Gq heterotrimer (mini-GαqGβ1Gγ2) 26. We thus compared our active state structures of α1A-AR with this inactive state structure of α1B-AR. This comparison revealed characteristic conformational changes associated with class A GPCR activation     denotes the opening of TM6 in class A GPCRs for G-protein engagement27 , and W6.48 to form new contacts  Fig. , and an 53) Fig. . In addi1A-AR–Gq, the para-hydroxyl group of the catechol ring of epinephrine forms H-bond with the hydroxyl side-chain of S1885.43  and N3127.38 (in β2-AR) interacted through hydrogen bonds with the β-carbon hydroxyl and the N-methyl group of epinephrine. In α1A-AR, it is F3127.38 at the equivalent position, and its side-chain did not form hydrogen bonds with epinephrine  in the methanesulfonamide group of A61603 in the GaMD simulations , α1A-AR(A189S), and α1A-AR(M292L) by mutating these residues to the corresponding residues in α1B-AR. Since these residues are involved in hydrophobic interactions with A61603 . Since α1A-AR is coupled to Gq, we used calcium responses as a functional readout  reduced the apparent affinity of the A61603 response by more than 1000-fold , α1A-AR(A189S), α1A-AR(M292L), and α1A-AR , α1A-AR(M292L), and α1A-AR relative to the starting cryo-EM structure were 4.62 ± 1.68 Å, 3.21 ± 1.05 Å, and 3.99 ± 1.13 Å, respectively , α1A-AR(M292L), and α1A-AR reduced the binding of A61603 to the receptor  exhibited a smaller RMSD with lower fluctuations in the GaMD simulations , α1A-AR(A189S), α1A-AR(M292L), and α1A-AR were 4.25 ± 0.98 Å, 3.43 ± 0.47 Å, 4.13 ± 1.33 Å, and 4.38 ± 1.27 Å, respectively , α1A-AR(M292L), and α1A-AR, but still maintained in α1A-AR(A189S), during the simulations  and −11.52 ± 2.47 kcal/mol for α1A-AR, compared to −26.73 ± 0.22 kcal/mol for the wild-type α1A-AR. α1A-AR(A189S) exhibited a similar binding free energy of A61603  as the wild-type α1A-AR. However, an exception was observed in α1A-AR(M292L), where the binding free energy was −27.88 ± 1.53 kcal/mol. This likely resulted from the exclusion of the entropy and MM/GBSA free energy calculations of GaMD simulation frames without the energetic reweighting36. In general, the systems with a stronger functional response to A61603-binding exhibited more favorable binding free energy compared to those with weaker or no functional responses. Therefore, the GaMD simulation findings correlated well with the calcium response data.In parallel, GaMD simulations were performed on αα1A-ARA18S, α1A-AR, α1A-ARM92L, and 1B-AR from non-responsive to responsive to A61603. This type of exercise is usually difficult given that many residues are typically involved in ligand interactions. However, based on our above data, most of the agonist interacting residues are the same between α1A-AR and α1B-AR, except for the three residues . Wild-type α1B-AR did not respond to A61603 at concentrations up to 10 μM, while it was responsive to epinephrine  responded to A61603 in a dose-dependent manner with an EC50 around 10 nM , α1B-AR(S208A) or α1B-AR(L314M) alone did not respond to A61603 , and α1B-AR. In wild-type α1B-AR and α1B-AR(S208A), A61603 exhibited large RMSDs relative to the starting conformation, with reduced interactions between A61603 and α1B-AR  exhibited low RMSD at ~2.5 Å, forming stable interactions with residues D1253.32 and S2075.43 in the receptor  showed the strongest binding affinity , followed by α1B-AR(S208A)  and wild-type α1B-AR  , α1A-AR(V185A), α1A-AR(A189S), α1A-AR(M292L), and α1A-AR were 1.46 ± 0.10 Å, 2.36 ± 0.52 Å, 1.10 ± 0.088 Å, 1.74 ± 0.39 Å, and 2.58 ± 0.33 Å, respectively (Supplementary Table 1A-AR(A189S)  exhibited a similar RMSF value as wild-type α1A-AR, while the other α1A-AR mutants  showed higher A61603 fluctuations. The RMSF values of A61603 in α1B-AR, α1B-AR(S208A) and wild-type α1B-AR were 0.98 ± 0.21 Å, 3.55 ± 1.97 Å, and 4.28 ± 0.30 Å, respectively , one low-energy state of A61603 with a narrow conformational space was sampled. Conversely, the other mutant α1A-ARs sampled larger conformational space with at least two distinct low-energy states. Among the α1B-AR systems, α1B-AR sampled only one low-energy state, while α1B-AR(WT) and α1B-AR(S208A) sampled at least two low-energy states with much larger conformational space  of A61603 in different receptor systems. In this context, larger RMSF values indicate weaker binding of A61603. The calculated RMSF values of A61603 in α, α1A-ARM92L, and 1-ARs are one of the three major subfamilies of ARs. Due to the lack of specific agonists, the physiological functions of α1-ARs are still not completely revealed, and their therapeutic potentials in treating cardiovascular, neurological, neuropsychiatric, and inflammatory disorders are not fully exploited. Here we have investigated the structural basis of activation and agonist interactions with α1-AR. Our data shows that M2926.55 and V1855.40 of α1A-AR define the specificity of A61603 for α1A-AR. Hydrophobic M2926.55 in α1A-AR corresponds to hydrophobic L6.55 in α1B-AR and α1D-AR, while aromatic Y6.55 in α2-AR and polar N6.55 in β-ARs  revealed that they share some conserved interactions while also displaying certain unique interactions  residues 228-255, and C-terminal residues 351-466. A hemagglutinin (HA) signal peptide, a FLAG tag, and a T4 lysozyme were fused to its N-terminus, and a PreScission protease cleavage site, an eGFP, and an 8xHis tag were added to its C-terminus. This construct was subcloned into pFastBac1 vector for expression in Spodoptera frugiperda Sf9 insect cells. Sf9 cells were grown to 2 to 3 million cells per ml in ESF 921 protein-free medium (Expression Systems) before 50 ml of baculoviruses were added for infection. 72 h later, cells were harvested by centrifugation, flash frozen in liquid nitrogen, and stored at −80 °C until use. For α1A-AR purification, thawed cell pellets were lysed with 1% n-Dodecyl-β-d-Maltopyranoside  in a buffer containing 25 mM HEPES (pH 7.0), 350 mM NaCl, 100 μM A61603  or epinephrine , supplemented with protease inhibitors  at 4 °C. Insoluble fractions were removed by ultracentrifugation at 142,000 × g for 1 h at 4 °C. The supernatant was then incubated with house-made GFP nanobody beads for at least 2 h, and washed with a buffer containing 25 mM HEPES (pH 7.0), 350 mM NaCl, 0.05% DDM, and 100 μM A61603 or epinephrine before PreScission protease was added to elute α1A-AR from GFP nanobody beads overnight at 4 °C. Eluted α1A-AR was further purified by size-exclusion chromatography using a Superdex 200 Increase 10/300 column  equilibrated with a buffer containing 25 mM HEPES (pH 7.0), 150 mM NaCl, 0.02% Lauryl Maltose Neopentyl Glycol , and 100 μM A61603 or epinephrine. Peak fractions were pooled and concentrated for complex assembly.The human αiN–Gβ1–Gγ2 and scFv16 were kindly provided by Dr. Bryan L Roth (University of North Caroline at Chapel Hill)37. For the expression of heterotrimeric mini-GαqiN–Gβ1–Gγ2, a single virus that encodes all three subunits, including mini-GαqiN, Gβ1, and Gγ2, with a 6xHis tag fused to the N-terminus of Gβ1, was used to infect Sf9 cells. 48 h post infection, cells were harvested by centrifugation, flash frozen in liquid nitrogen, and stored at −80 °C. Cell pellets were thawed in a lysis buffer containing 20 mM HEPES (pH 7.5), 100 mM NaCl, 2 mM β-mercaptoethanol, 1 mM MgCl2, 0.1 mM GDP, 0.2% (v/v) Triton X-100, supplemented with protease inhibitors  at 4 °C and insoluble fractions were removed by centrifugation at 142,000 × g for 1 h. Supernatant was collected and incubated with HisPur Ni-NTA Resin (Thermo Fisher Scientific) for 2 h at 4 °C. Resin was then washed with 20 mM imidazole in the lysis buffer, and eluted with 250 mM imidazole in the lysis buffer. Eluted heterotrimeric G-protein was further purified by size-exclusion chromatography using a Superdex 200 Increase 10/300 column  equilibrated with a buffer containing 20 mM HEPES (pH 7.5), 100 mM NaCl, 0.02% LMNG, 0.1 mM TCEP, 1 mM MgCl2, and 10 μM GDP. Peak fractions were pooled and concentrated for complex assembly.The expression constructs of heterotrimeric mini-Gαq37. 72 h after infection, Sf9 cell culture media expressing scFv16 was collected and PH balanced to pH 8.0 by addition of Tris buffer. 10 mM calcium chloride was then added to quench the chelating agents with stirring at room temperature for 1 h. The precipitate was removed by centrifugation at 8000 × g for 30 min. The supernatant was incubated with HisPur Ni-NTA Resin with stirring for 4 h at 4 °C. The Ni-NTA resin was then collected with centrifugation at 800 g and washed with 20 mM imidazole in a buffer containing 20 mM HEPES (pH 7.5) and 100 mM NaCl before 200 mM imidazole in the same buffer was used to elute the protein. The C-terminal 8xHis tag was removed by the treatment of PreScission protease overnight at 4 °C. The protein was then further purified by size-exclusion chromatography using a Superdex 200 Increase 10/300 column  equilibrated with a buffer containing 20 mM HEPES (pH 7.5) and 100 mM NaCl. Peak fractions were pooled and concentrated for complex assembly.scFv16 was expressed and purified from Sf9 cells as a secreted protein1A-AR–Gq–scFv16 complex, α1A-AR, and heterotrimeric Gq were mixed at a 1:1.5 molar ratio and incubated for 30 min at room temperature followed by the treatment of 0.4 U Apyrase (New England Biolabs) for another 30 min at room temperature. A 1.5 molar access of scFv16 was then added to the mixture and incubated overnight at 4 °C. The next day, the mixture was centrifuged at 16,000 × g for 10 min to remove any precipitants. The supernatant was then loaded onto a Superdex 200 Increase 10/300 column equilibrated with 10 mM HEPES pH 7.0, 100 mM NaCl, 0.1 mM TCEP, 0.02% LMNG, and 30 μM A61603 or epinephrine. The elution fractions from a single peak containing pure α1A-AR–Gq–scFv16 complex concentrated to 2.5 mg/ml and used directly for making cryo-EM grids.To assemble the α1A-AR–Gq–scFv16 complex at a concentration of 2.5 mg/ml was applied to glow-discharged 400 mesh gold Quantifoil R1.2/1.3 holey carbon grids (Quantifoil Micro Tools) and vitrified using a Vitrobot Mark IV (Thermo Fisher Scientific/FEI) at 22 °C and 100% humidity. Micrographs were collected on a 300 kV Titan Krios electron microscope (Thermo Fisher Scientific/FEI) with a Gatan K3 direct electron detector  in super-resolution mode at a nominal ×81,000 and ×64,000 magnification for the A61603 and epinephrine-bound complexes, respectively. For the A61603-bound complex, 7,773 movies in the defocus range of −1.0 to −1.8 μm were recorded with a total accumulated dose of 50 e−/Å2. For the epinephrine-bound complex, 10,486 movies in the defocus range of −1.0 to −1.8 μm were recorded with a total accumulated dose of 52 e−/Å2.3.5 µL of α44. The effects of contrast-transfer function were estimated with CTFFIND v4.1.845. For the A61603 dataset, Relion 2D template-based auto-picking was used to pick particles and the resulting particle stacks were two-times Fourier cropped and processed through 2D classification in CryoSparc v3.3.1 to remove false positives, receptor alone, or G-proteins alone particles46  and 219,834 (epinephrine) particles obtained by 3D variability analysis that yielded higher resolution in the transmembrane region were selected and non-uniform refinement of these particles in CryoSparc yielded maps with 2.6 Å-resolution (A61603) and 3.0 Å-resolution (epinephrine) using the 0.143 Fourier Shell Correlation criterion. These consensus stacks were also subjected to focused refinement of α1A-AR and Gαq region in CryoSparc. The focused refinement map showed significant improvement in the α1A-AR region compared to the consensus map. Models were built starting from AlphaFold predicted structure of inactive α1A-AR (AF-P35348-F1) and scFv16 bound Gq heterotrimer (PDB code 6WHA) in Coot v0.9.1, which combined with consensus and focused refinement maps, were used to generate composite maps in Phenix dev-469448. The resulting maps were super-sampled in Coot to 0.856 Å per pixel (A61603) and 0.8608 Å (epinephrine) with 320-voxel boxes. The A61603 and epinephrine bound α1A-AR–Gq–scFv16 models were real-space refined against the composite maps in Phenix, and a work/free half-map pair was used to ensure against over-fitting  were added by SWISS-MODEL51. The initial models of the A61603-bound mutants of α1A-AR(V185A), α1A-AR(A189S), α1A-AR(M292L), and α1A-AR were built by VMD based on the A61603–α1A-AR–Gq cryo-EM structure. All six simulation systems were prepared with the CHARMM-GUI web server for using the membrane protein input generator52. The receptor was inserted into a palmitoyl-oleoyl-phosphatidyl-choline (POPC) bilayer. All chain termini were capped with neutral patches (acetyl and methylamide). All the disulfide bonds in the complexes that were resolved in the cryo-EM structures were maintained in the simulations. The systems were solvated in 0.15 M NaCl solution at temperature 310 K. The AMBER ff14SB and AMBER LIPID 21 parameter sets were used for the receptor and lipids54. The GAFF2 parameters with RESP charges were used for epinephrine and A6160355. For each of the complex systems, initial energy minimization, thermalization, and 20 ns cMD equilibration were performed before GaMD simulations. A cutoff distance of 9 Å was used for the van der Waals and short-range electrostatic interactions and the long-range electrostatic interactions were computed with the particle-mesh Ewald summation method56. A 2-fs integration time step was used for all MD simulations. The SHAKE algorithm was applied to all hydrogen-containing bonds. With all other atoms fixed, the lipid tails were energy minimized for 5000 steps using the conjugate gradient algorithm and melted with a constant number, volume, and temperature (NVT) run for 0.5 ns at 310 K. The six systems were further equilibrated using a constant number, pressure, and temperature (NPT) run at 1 atm and 310 K for 1 ns with 1.0 kcal/(mol Å2) harmonic position restraints applied to the protein and ligand atoms. Then, conventional MD simulations were performed on each system for 20 ns at 1 atm pressure and 310 K with a constant ratio constraint applied on the lipid bilayer in the X-Y plane. The GaMD module implemented in the GPU version of AMBER22 was then applied to perform the simulations57. GaMD simulations included an 8-ns short cMD simulation used to collect the potential statistics for calculating GaMD acceleration parameters, and a 48-ns equilibration after adding the boost potential. Finally, three independent 500-ns GaMD simulations with randomized initial atomic velocities were performed for the epinephrine–α1A-AR–Gq, A61603–α1A-AR–Gq, and A61603-bound mutant α1A-AR–Gq complexes. The average and SD of the system potential energies were calculated every 800,000 steps (1.6 ns). All GaMD simulations were run at the “dual-boost” level by setting the reference energy to the lower bound. One boost potential was applied to the dihedral energetic term and the other to the total potential energetic term. The upper limit of the boost potential SD, σ0 was set to 6.0 kcal/mol for both the dihedral and the total potential energetic terms.The epinephrine–α58. Root-mean-square fluctuations (RMSFs) were calculated for the agonist A61603, averaged over three independent GaMD simulations. Interactions between epinephrine and the receptor were measured by the distance between the CG atom of D106 and N1 atom of epinephrine, and the OG atom of S188 and O2 atom of epinephrine. Interactions between A61603 and the receptor were measured by the distance between the CG atom of D106 and the N2 atom of A61603, the N atom of A189 and the O2 atom of A61603, the OG atom S188 and the N atom of A61603, the OG atom of S188 and the O atom of A61603, and the OG atom of S188 and the O2 atom of A61603. Time courses of these reaction coordinates obtained from the GaMD simulation were plotted in Fig. CPPTRAJ was used to analyze the GaMD simulations2 at 37oC in humidified incubator. HEK293T cells were seeded on poly-L-lysine coated glass coverslips (18 mm) one day before transfection. GCaMP 6 f (0.2 μg per well) and α1A-AR plasmid (0.5 µg per well) were used to transfect cells using Lipofectamine 2000 in 12-well plates. Cells were protected with 10 μM Prazosin 6–8 hours after transfection. Calcium imaging experiments were performed 24 hours after transfection. Cells were placed on an inverted Nikon Eclipse Ti2-E microscope equipped with an Andor Zyla 5.5 sCMOS camera, Lumencor SOLA-SE II light engine, and CFI Nikon Plan Apo Lambda ×20 with 0.75 NA. GCaMP6f was excited using 488 nm SOLA LED light. Cells were continuously perfused with extracellular (EX) solution (containing (in mM): 135 NaCl, 5.4 KCl, 10 HEPES, 2 CaCl2, 1 MgCl2, pH = 7.4) utilizing a gravity-driven perfusion system at room temperature. Drugs (A61603 or epinephrine) were dissolved in EX solution. Calcium imaging was analyzed using ImageJ 1.52a and NIS-Elements Advance Research 5.2.6. Each single cell was manually selected as region of interest and the fluorescence intensity was normalized to the baseline before agonist application. An averaged trace (60–200 cells per recording) was used to quantify maximum fluorescence amplitude after drug application. Dose–response curve was done using nonlinear fit in GraphPad Prism.HEK293T cells were purchased from ATCC , authenticated by Bio-Synthesis, Inc. and routinely tested negative for mycoplasma. Cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) in 5% CO2+ assays were repeated three times, and the data are represented as mean ± SD of the three independent experiments. The analysis was done using the log(agonist) vs. response function of Prism 9 (GraphPad) as indicated in the figure legends. Cryo-EM data collection and refinement statistics are listed in Supplementary Table In Fig. Further information on research design is available in the Supplementary InformationPeer Review FileReporting SummarySource Data


The title one-dimensional coordination polymer features alternating 26- and 16-membered rings. II coordination polymer, namely, polynickel(II)], [Ni(C12H14O4)(C16H18N4)]n or [Ni(adc)(bmib)]n, (I)  was synthesized and characterized. It exhibits a one-dimensional extended structure built up from alternating [Ni2(bmib)2] 26-membered rings and [Ni2(adc)2] 16-membered rings. The nickel atom lies on a crystallographic twofold axis and both ligands are completed by mirror symmetry. The solid-state luminescence spectra of (I) and the bmib ligand show strong emissions at 442 and 410 nm, respectively.An Ni These Ni—O and Ni—N bond lengths are typical and show no deviations from those in other distorted octahedral NiII coordination polymers °. In the extended structure, two bmib ligands bridge two NiII atoms and construct a [Ni2(bmib)2] 26-membered ring with an Ni⋯Ni distance of 12.100 (2) Å. Two carboxylate groups of one adc ligand exhibit an O,O-chelating mode such that two adc ligands link two NiII atoms and construct an [Ni2(adc)2] 16-membered ring with Ni⋯Ni = 8.0978 (16) Å. The NiII atoms are alternately connected by the bridging bmib and adc moieties, resulting in a chain containing alternative [Ni2(bmib)2] and [Ni2(adc)2] loops propagating along the b-axis direction ] Table 1. Atoms Oon Fig. 2.3.n chain is surrounded by six further chains (adc)]ns Fig. 3. There a4.et al., 2014The solid-state luminescence spectra of (I)5.et al., 2016) revealed only the four coordination polymers noted in the Chemical context section.The bmib ligand is widely used in coordination chemistry but for Ni–bmib compounds, a search of the Cambridge Structural Database 2.6H2O (0.28 mmol), H2adc (0.22 mmol), NaOH (0.38 mmol) and H2O (14.0 ml) was added to a 20.0 ml Teflon-lined stainless steel autoclave, which was then sealed and heated to 393 K for 5 d. Green crystals of (I)A mixture of bmib (0.22 mmol), Ni were placed geometrically (C—H = 0.93–0.98 Å) and refined using a riding model with Uiso(H) = 1.2Ueq(C) for CH and CH2 or 1.5Ueq(C) for CH3 groups.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989023006059/hb8041sup1.cifCrystal structure: contains datablock(s) . DOI: 10.1107/S2056989023006059/hb8041Isup3.hklStructure factors: contains datablock(s) I. DOI: 2280450CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: