Intermolecular O—H⋯O hydrogen bonds link these layers into a three-dimensional supramolecular framework.In a Ba coordination polymer incorporating 4-nitro-2,5,6-trioxo-1,2,5,6-tetrahydropyridin-3-olate, oxalate and aqua ligands, the complex monomers are connected into polymeric two-dimensional layers parallel to the  5HN2O6)(C2O4)0.5(H2O)2]n, the tenfold coordination of the Ba centre consists of four O atoms from the two 4-nitro-2,5,6-trioxo-1,2,5,6-tetrahydropyridin-3-olate (L) anions, three O atoms of two oxalate anions and three water molecules. The Ba—O bond lengths fall in the range 2.698 (3)–2.978 (3) Å. The L ligand chelates two Ba atoms related by a screw axis, leading to formation of fused five- and six-membered chelate rings. Due to the bridging function of the ligands and water molecules, the complex monomers are connected into polymeric two-dimensional layers parallel to the bc plane. Intermolecular O—H⋯O hydrogen bonds link these layers into a three-dimensional supramolecular framework.In the title coordination polymer, [Ba(C In the present paper, we report the molecular and crystal structure of a mixed-ligand barium complex containing 4-nitro-2,5,6-trioxo-1,2,5,6-tetrahydropyridin-3-olate (L) and oxalate anions as ligands.Mixed ligand coordination polymers containing bridging oxalate anions and 1,2-dicarbonyl carbocyclic or heterocyclic compounds exhibit high reactivity and different types of magnetism (C2O4)0.5(H2O)2]n, (I)L), the O7, O8 and O7A atoms of two oxalate anions, and the O9, O9A and O10 atoms of water molecules. The Ba—O bond lengths fall in the range 2.698 (3)–2.978 (3) Å, which is typical for ten-coordinate barium complexes containing oxalate anions  and 0.010 (3) Å, respectively, whereas the O1 and O3 atoms deviate from it by 0.171 (3) and 0.077 (3) Å, respectively. The plane of the nitro group is rotated by 11.9 (8)° with respect to the ring plane. The L ligand chelates two Ba atoms related by a screw axis forming fused chelate rings. The six-membered ring is almost planar (r.m.s. deviation = 0.0353 Å) and the five–membered ring is folded along the O1—O2 line by 19.0°. The geometry of the L anion in the Ba complex is close to that in the compounds studied earlier (H2O)2]+ form wide zigzag bands running along the screw axes in the b-axis direction Uiso(H) = 1.2Ueq of the parent atom. The crystal studied was a twin without superposition of reciprocal lattices. Apparently, an accidental overlapping of reflections of two domains is responsible for increased displacement ellipsoids of some atoms. The Uij components of atom O5 were restrained to approximate the isotropic behaviour.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989015006520/cv5485sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989015006520/cv5485Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989015006520/cv5485Isup3.molSupporting information file. DOI: 1057170CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Carbanions of phenylacetonitriles, benzyl sulfones, and dialkyl benzylphosphonates add nitroarenes at the ortho-position to the nitro group to form The online version of this article (doi:10.1007/s11030-015-9627-x) contains supplementary material, which is available to authorized users. A numbeortho-substituted benzene derivatives containing substituents suitable for cyclization to form a fused isoxazole ring (Scheme a) .Sc.Sc6 fro9, after 1,4-elimination of silanol anion, gives silylated oxime derivative 10. Intramolecular electrocyclization of the oxime ether 10 leads, after silanol elimination, to acridine 8. However, under action of the additional base, particularly t-BuOK desilylation of the oxime derivative 10 occurs leading to the nitroso (or oxime) anion 7 which undergoes intramolecular vinylic substitution of the cyano group. This process is facilitated by the presence of an oxygen nucleophile, i.e., in excess of t-BuOK, or trialkylsilanol anion generated by DBU or, in much lesser extent, by Formation of anthranils at a higher base/silylating agent ratio could be rationalized as follows. The bis-silylated 3a) in moderate yields . 4-Chloronitrobenzene reacted similarly as under Davis and Pizzini conditions (entries 3–5). To our delight, 4-fluoronitrobenzene successfully participates in the reaction to form expected 5-fluoroanthranils in satisfactory yields (entries 6–8). The observed reaction of 4-fluoronitrobenzene indicates that, at equally activated positions, substitution at the carbon bearing a hydrogen atom is faster than at a carbon bearing any other substituent, including readily replaceable fluorine atom [After choosing the most suitable reaction conditions Table . We focuine atom , 43, 45.3k and 3l when anions of benzyl sulphide (entry 11) or benzylphosphonate (entry 14) were used.As we expected, under the above conditions, the reaction was not limited to arylacetonitriles as nucleophile precursors. Interestingly, 4-nitroanisole furnished anthranils In the case of benzylphosphonate carbanion, the use of a DMF–THF mixture was beneficial, particularly when nitroarenes being weak electrophiles, such as 4-nitroanisole (entry 11), 4-nitro-N,N-dimethylaniline (entry 15), and 2,4-dimethoxynitrobenzene (entry 16) were used. These examples show how robust our new procedure is for the synthesis of anthranils, particularly since these nitroarenes are inactive in reactions with nucleophiles. In the literature, we found only one example of the VNS reaction of 4-nitro-N,N-dimethylaniline with chloromethyl phenyl sulfone (13 % yield)  and one 3j in moderate yield, and its formation is accompanied by products arising from an oxidation of para or ortho position to the nitro group, most probably 3-chloro-4-(or -2-)-nitrodiphenylacetonitrile.The reaction of 2-chloronitrobenzene with phenylacetonitrile (entry 10) deserves an additional comment. This reaction leads to the expected anthranil We have found that reactions of carbanions with nitroarenes in aprotic conditions using a strong base and silylating agent, 3-arylbenzisoxazoles are formed in good yields. The reaction is general with respect to both nitroarenes and C–H acids activated by groups of nucleofugal character. This reaction does not require a transition metal catalyst and thus can be attractive for use in the pharmaceutical industry.All reactions were run under argon atmosphere. Melting points are uncorrected. To a stirred solution of 4-nitrochlorobenzene , phenylacetonitrile  and diphenylsulfone  in THF (5 mL) cooled to To a stirred solution of nitroarene (3 mmol) and carbanion precursor (3 mmol) in THF (10 mL) cooled to Yellow solid, yield: 0.21 g (35 %). Mp. 48–50 J = 9.0, 6.5 Hz), 7.04–4.09 and 7.95–7.99 , 7.30 . m/z, %): 225 . Mp. 97–99 C; . M.p. 110–112 ∘C , 7.67–7.70 and 8.15–8.18 , 7.78 : 263 . M.p. 212–214 ∘C : 259 . M.p. 145–147 ∘C (lit.  143 -145J J J J J J J J J = 11.1 Hz). MS : 213 . M.p. }∘C lit.  96–97 \dJ J = 9.6, 4.8 Hz), 7.91 : 247 . M.p. 205–207 J J J J J J J = 9.3 Hz), 123.85 , 163.72 : 231 Pale yellow solid, yield: 0.24 g (35 %). M.p. 161–162 J J J J J m/z, %): 263 Yellow crystals, yield: 0.31 g (39 %). M.p. 116–121 J J m/z, %): 229 Yellow crystals, yield: 0.15 g (22 %). Mp. 105–J = 2.2 Hz), 7.06 : 259 Pale yellow crystals, yield: 0.37 g (48 %). M.p. 140–J J m/z, %): 225 Yellow crystals, yield: 0.24 g (35 %). M.p. 79–80 J m/z, %): 238 Yellow crystals, yield: 0.11 g (15 %). M.p. 113–115 J J m/z, %): 255 Yellow crystals, yield: 0.40 g (52 %). M.p. 151 Supplementary Material 1H and 13C spectra of compounds  3a-3n. (doc 6.23mb)


Their photophysical and electrochemical properties were revealed by UV–vis absorption and photoluminescence spectroscopy and cyclic voltammetry. Organic field-effect transistors (OFETs) were fabricated with these compounds as organic semiconductors, and their semiconducting properties were evaluated. OFETs with syn-DBBDF and syn-DNBDF showed typical p-type characteristics with hole mobilities of <1.5 × 10−3 cm2·V−1·s−1 and <1.0 × 10−1 cm2·V−1·s−1, respectively.Ladder-type π-conjugated compounds containing a benzo[2,1- Organic semiconductors have significantly been developed in the past two decades by virtue of their advantages, such as low weight, flexibility, large-area processability, which are different features from conventional silicon-based semiconductors. Organic semiconducting materials can be used as active layers in organic field-effect transistors (OFETs) –7, organb:4,5-b']difurans (BDFs) 1 and their application to OLEDs as hole-transporting materials  and studied their structure–property relationship difuran derivative 2 has been reported to demonstrate an excellent OFET mobility of 3.6 cm2·V−1·s−1 benzodifurans (anti-DBBDFs), which is also a π-extended homologue of BDF benzodifuran (anti-DNBDF 4) with a more extended π-conjugation afforded higher hole mobility of 0.33 cm2·V−1·s−1 [Furan-containing π-conjugated compounds have attracted less attention until recently –27. The aterials  32]. Th. Thb:4,5tionship –34. Furttionship –20. In p·V−1·s−1 . Previoue of BDF . The OFEoperties –37. For ·V−1·s−1 . Recentl·V−1·s−1 –41. Thesb:3,4-b']difuran skeleton, such as dibenzobenzodifuran (syn-DBBDF 5) and dinaphthobenzodifuran  gave boronate ester 7 in 57% yield. Then, terphenyl 9 was synthesized via palladium-catalyzed Suzuki–Miyaura cross coupling of boronate ester 7 with 2,3-difluoro-1,4-diiodobenzene (96% yield) and subsequent demethylation (95% yield). Finally, the desired syn-DBBDF 5 was successfully synthesized via the double intramolecular cyclization under basic conditions at high temperature (92% yield)  at longer wavelengths than syn-DBBDF 5 and syn-DNBDF 6, respectively , and the electrochemical properties were summarized in syn-DBBDF 5 exhibited two oxidation waves, and an onset potential of the first oxidation wave was determined to be 0.84 V (vs Fc/Fc+). Accordingly, the HOMO energy level was estimated to be −5.64 eV under the premise that the energy level of Fc/Fc+ is 4.8 eV below the vacuum level benzodifuran (syn-DNBDF 6). Based on the photophysical and electrochemical data, both compounds are expected to possess good air stability as organic semiconducting materials. The comparison with their anti-isomers revealed that the π-conjugation in syn-DBBDF 5 and syn-DNBDF 6 is less effective than those of their anti-isomers. OFETs based on these compounds were fabricated as bottom-gate top-contact devices, and their semiconducting properties were evaluated. All devices showed typical p-type transistor characteristics. The highest hole mobility of 1.0 × 10−1 cm2·V−1·s−1 was achieved when using syn-DNBDF-based OFET device.In summary, we investigated the synthesis and properties of ladder-type π-conjugated compounds, dibenzo[File 15–12, device fabrication/evaluation procedures, OFET characteristics, XRD patterns, and AFM images.General experimental procedures, synthetic procedures/characterization data of compounds


The route taken by this process resembles thymic T-cell development. However, it involves the re-organization of the Vδ1+ γδTCR into the αβTCR as a consequence of TCR-γ chain downregulation and the expression of surface Vδ1+Vβ+ TCR components, which we believe function as surrogate pre-TCR. This transdifferentiation process is readily detectable in vivo in inflamed tissue. Our study provides a conceptual framework for extrathymic T-cell development and opens up a new vista in immunology that requires adaptive immune responses in infection, autoimmunity, and cancer to be reconsidered.The lifelong generation of αβT cells enables us to continuously build immunity against pathogens and malignancies despite the loss of thymic function with age. Homeostatic proliferation of post-thymic naïve and memory T cells and their transition into effector and long-lived memory cells balance the decreasing output of naïve T cells, and recent research suggests that also αβT-cell development independent from the thymus may occur. However, the sites and mechanisms of extrathymic T-cell development are not yet understood in detail. γδT cells represent a small fraction of the overall T-cell pool, and are endowed with tremendous phenotypic and functional plasticity. γδT cells that express the Vδ1 gene segment are a minor population in human peripheral blood but predominate in epithelial (and inflamed) tissues. Here, we characterize a CD4 Stroma- and thymocyte-derived signals then induce their T-cell lineage commitment and the cells’ differentiation into either αβ or γδT cells through well-defined stages (DN1–DN4). In humans, these stages can be recognized by the expression of CD34, CD38, and CD1a surface proteins. The expression of functionally rearranged TCR-γ and TCR-δ chain genes in DN2/3 thymocytes leads to γδTCR complexes, which drive cellular proliferation and promote differentiation into γδT cells (+ and CD8+ expressing double-positive (DP) stages. In humans, this transition involves immature single-positive (ISP) CD4+ intermediates  are rare, phenotypically and functionally diverse cells that can give rise to all cell lineages of the immune system . T-cell es DN1–DN. In humamediates . DP T cemediates –10. TCR-mediates  until seIt is undisputed that the thymus provides the foremost source of naïve T cells and orchestrates normal T-cell lymphopoiesis to some degree throughout life , 13. How+ γδT cells are key players in the lymphoid stress-surveillance response. They constitute a minor T-cell population in the peripheral blood, but a major subset among tissue-residing and intraepithelial lymphocytes  and CLPs. Like thymus-seeding, early T (ETP), and DN1 progenitors, CD4+ Vδ1+ γδT cells express CD34 and CD38 but not CD1a (CD34+ CD38+ CD1aneg) on their surface; they also carry full-length transcripts of in-frame δ, γ, and β TCR gene rearrangements and express recombination-activating gene (RAG) and terminal deoxynucleotidyl transferase (TdT), which are typically found in DN2 and DN3 thymocytes. We show that CD4+ Vδ1+ γδT cells that lack thymus-homing properties but carry chemokine receptors (CCR) that direct circulating T cells to sites of inflammation, can develop into functional, mature CD4+ or CD8+ αβT cells in an inflammatory environment. In this study, we pinpoint the individual steps of this development, a process that is very similar to thymic T-cell development, but proceeds via a Vδ1+Vβ+ intermediate instead of a pre-TCR. We also show that the progenitors’ cellular intermediates are present in vivo in inflamed tissue and to a considerably lesser extent in peripheral blood of healthy individuals.In this study, we show that the rare and so far unappreciated entity of human CD4This fundamentally new role of γδT cells as an αβT-cell precursor contributes to the emerging concept of T-cell plasticity and recommends the reconsidering of adaptive immune responses in infection, autoimmunity, and cancer.+ Vδ1+ γδT cells. We generated CD4+ Vδ1+ γδT clones from the peripheral blood of 12 healthy individuals, from leukapheresis products (LPH) of GM-CSF-mobilized healthy stem-cell donors (n = 12), and also from the bone marrow of potential stem-cell donors (n = 10). Clones of this phenotype were extremely long-lived – they could be cultivated uninterruptedly ex vivo for up to more than a year under standard culture conditions. Importantly, over time, some clones could change their γδTCR into αβTCR. The morphology of the CD4+ Vδ1+ γδT-cell clones was similar to that of large granular lymphocytes (LGLs) . In contrast to most other Vδ1+ cells, their TCR-γ9+ chain  CD4+ Vδ1+ γδT-cell clones nevertheless uniformly expressed CD34lo (22/22), which is the common marker of most immature hematopoietic stem/progenitor cells. The clones also expressed C-X-C chemokine receptor type 4 (CXCR4), which maintains the quiescence of the HSC pool in bone-marrow niches   as well as CLP in human bone marrow, as well as linlo ETPs, and canonical DN1 in the thymus   than peripheral blood-derived clones did  , which is evidence for the presence of a more primitive precursor type in the bone marrow. Although CD4+ Vδ1+ γδT-cell clones did not initially express CD2 on their surface, they did so rapidly in the course of cultivation. This is additional evidence of the CD4+ Vδ1+ T-cell clones’ premature phenotype  . This suggests that the precursors found in the bone marrow are more primitive. GATA-3 was the major transcription factor while T-bet, RORc, and Foxp3 were only transcribed at very low levels  in standard culture conditions after stimulating the cells with PMA/ionomycin , as well as DN1–DN4 stage thymocytes and an ISP thymocyte, which in humans is CD4+.Thus, CD4+ Vδ1+ γδT cells did not express the chemokine receptors CCR9 (8/9) and CCR7 (9/9), which makes homing to the thymus less likely  and some expressed αβ instead of γδTCR , Vδ1+ clone P7: +3 months]  almost exclusively expressed surface TRBV11-2+ and -4.1+, and T-cell line K1.2 (K1.2-αβ+) almost exclusively TRBV27+ and -19+ . The clone P7-derived TCRαβ+ T-cell line did not express TCR Vβ families that are detectable with available TCR antibodies (data not shown). All CD4+ Vδ1+ T-cell clone-derived αβT cells were T10B9.1A-31+ (framework epitope of αβTCR heterodimer), WT31+ (CD3ε), CD3+, Vδ1neg, γδTCRneg, CD25lo/neg, CD28neg, and were also terminally differentiated effector-memory RA-re-expressing T cells (TEMRA)  phenotype (not shown). Furthermore, they did not transcribe TCR-δ segments, which is due to the deletion of the TCR-δ locus as a result of the recombination of the genes that encode the TCR-α chain. Nevertheless, the αβT-cell lines still transcribed rearranged γ chain segments /ionomycin stimulation with a (subtle) increase in IL-4 release and with the substantial de novo production of IFN-γ, demonstrating their functionality  of all Cβ+ Vδ1lo/neg. cells stained positive for TCRαβ , or CD8+ (8.37% ± 4.62 SD), or CD4+ (58.38% ± 11.21 SD), and in rare cases DN (3.5% ± 5.4 SD) .In order to study TCR re-organization during transdifferentiation, the expression of the constant region of the TCR chains, Cδ, Cγ, Cβ (BMA031) and Cα (as a TCR-αβ-associated framework epitope), in CD4γ Figure A. Vδ1lo/s Figure A. In ord+ Vδ1+ γδT-cell clones, we purified Vδ1+ γδT cells, including the DN, CD8+, and CD4+ subsets from peripheral blood mononuclear cells (PBMNCs) of healthy human donors. These cells (panVδ1+) were subsequently used to study whether inflammatory stimuli trigger transdifferentiation. For that two different inflammatory settings were compared: standard culture conditions were designated as mild inflammation, whereas stronger inflammatory stimuli were termed overt inflammation.As TCR change occurred in only one out of 50 established CD4+ T cells cultivated in the mild inflammatory environment  , as did the number of αβT cells generated from panVδ1+ cell pools   γδT cells  did . The transcription rate of pre-Tα was the same as in the controls with no template.PanVδ1) Figure A within + Figure A. The pel Figure B. The nu) Figure B. ConcomδT cells   created (more) Vδ1+ SP CD4+ cells and the novel phenotype of Vδ1+ CD4+ CD8+ DP T cells in five out of seven cultures . Interestingly, panVδ1+ T cells became weakly positive for Vβ chains on their surface (Vβ+); most of them expressed TRBV30 (n = 12) as shown by FACS analysis . It is worth noting that strong inflammation  , which correlated with the number of Vδ1+CD34dim T cells per Vδ1+ subset in the peripheral blood of diseased individuals, but was significantly different from their numbers in healthy PBMNCs , diseased 0.6–8.6 (n = 7)] , lupus erythematosus (n = 1), vitiligo (n = 1), and a viral infection associated with chronic fatigue syndrome (n = 1). The Vδ1+CD34dim precursors in the peripheral blood were mostly DN (52.08%) and CD8 (42.66%) (not shown), which is consistent with the finding that CD4+ is upregulated by inflammatory stimuli , we determined TCR-γ expression in vivo  and in native healthy bone marrow. Unexpectedly, TCR-γ chain (Cγ) expression was significantly higher in Vδ1+ than in Vδ2+ cells in the blood of healthy donors and in bone marrow. In contrast, there was a significantly higher expression of Cγ in the Vδ2+ than in the Vδ1+ cells in the blood of diseased volunteers and in bone marrow. When comparing absolute Cγ expression, the Vδ2+ subset showed a significant upregulation of TCR-γ chain expression during inflammation, whereas the Vδ1+ subset expressed the γ-chain constitutively in bone marrow and PBMNCs irrespective of inflammatory stimuli. Blood samples were taken from individuals suffering from uncharacterized viral infection (n = 3), pneumonia (n = 1), cystitis (n = 2), nephritis (n = 1);   Figure C.+ subset and the αβT-cell compartment in the peripheral blood of healthy and diseased individuals  and diseased  blood  than in the peripheral blood of healthy individuals   Figure C.+ heterodimer, which must occur in the absence of the TCR-γ chain  inflammatory stimuli  BMA031. Using a set of mAbs that recognize the variable domain of 24 different human Vβ chains, we refined our analysis in order to demonstrate that peripheral Vδ1+ T cells of healthy individuals expressed the full spectrum of Vβ chains. While Miossec and other researchers showed that the Vδ1+ variable region can substitute Vα in functional T-cell receptor α-chains and thus serve as an agent for Vβ surface expression in the peripheral αβT cell subset , established from pleural effusion  on the surface of pre-B cells . The significantly higher in vivo TCR-γ expression on Vδ1+ T cells compared to Vδ2+ T cells in bone marrow and peripheral blood was unexpected  were identical to those observed in the Vδ2+ but not to those observed in the Vδ1+ subset in the blood of diseased individuals. In contrast, the CD4+ Vδ1+ clone fraction that underwent TCR change was identical to the peripheral Vδ1+ subset. This indicates that, in contrast to all other Vδ1+ cells, CD4+ Vδ1+ T cells have the capacity to modulate TCR expression, possibly due to the disulfide bond that links the constant region of Cδ to the Cγ1 segment of their TCR, a similarity they share with the Vδ2 TCR. In addition, they can downregulate TCR-γ chain expression during the process of transdifferentiation , or CD3/CD28, supports the assumption that selection accompanies transdifferentiation (not shown).Our finding that newly generated CD4+ cell cultures after the emergence of αβT cells , and recombinational accessibility is not downregulated by TCRβ chains . TRBV30 de novo α rearrangement in Vδ1+ precursor cells followed the exact hierarchical order of thymocytes that begin α-locus recombination with the 3′ end Vα and 5′ end Jα segments, from where the process proceeds to the distal ends of the locus . Moreover, our data are also consistent with the view that the TCR-α locus underlies tight regulation and that the TCR repertoire is not a vast and chaotic morass, but rather a patterned and perhaps even predictable system  exhausted the α-locus. The clone-derived αβT-cell line K1.1 changed TCR months after the establishment and showed rearranged TRAV-26, -25, -14 and -3, αβ T-cell line P7 rearranged TRAV-26-2, -26-1, -24, -19, -14, -10, and -2 segment.CD4 β chain  so that  β chain , 104. Li β chain  was repr+ Vδ1+-clone-derived TCRαβ+ T-cell lines K1.1 and P7 that lacked CD28 were examined in greater detail. These T-cell lines revealed an impaired proliferative response to mitogenic stimuli and carried public TCRs, as reported previously for viral and autoimmune diseases . They had no TRM phenotype, were negative for CD103 and were also CCR7lo or negative and expressed varying amounts of CD62L and CD28 (not shown). Thus, Vδ1+-derived T cells had diverse but distinct cell-surface phenotypes, a complex T-cell receptor repertoire, and produced diverse cytokines. They could thus be classified as “functional” Th type cells. The TCRs sequenced were identical to viral antigen-specific TCRs , which is in line with the observation that the number of Vδ1+ γδT lymphocytes is expanded in human diseases, including infections  immunology, namely, to understand how T-cell development is ultimately conducted in the absence of thymic function. The assignment of this fundamental role for γδT cells opens a new vista in immunology and requires reevaluation of adaptive immune responses in infection, autoimmunity and cancer.All procedures were carried out according to the Declaration of Helsinki and were approved by the Clinical Ethics Committee at the University of Tübingen (projects 38/2009B02 and 470/2013B02).Cells were pretreated with FcR Blocking Reagent (Miltenyi Biotec) according to the manufacturer’s instructions, resuspended in flow cytometry buffer  and incubated for 15 min  with one or more of the following monocolonal antibodies, all specific for human epitopes: FITC-anti-Vδ1, unconjugated anti-Vδ1 , APC-anti-TCRγδ , PE-anti-TCRγδ , PE-anti-TCRγδ  PE-anti-TCRαβ , FITC-anti-TCRαβ , murine anti-human TCR-αβ mAb . PE-anti-Vδ2 , FITC/PE/PerCP-anti-CD4  or Vioblue-anti-CD4 , FITC/PE/PerCP/APC-anti-CD3  APC-anti-CD3 , APC-anti-CD8β , and APC-H7-anti-CD8α . FITC/APC-anti-CD62L , PE/APC-anti-CD25 , PE-anti-CD135 , PE-anti-CD127 , and APC-anti-CD132 . All IgG isotypes and secondary antibody rat anti-mouse IgG 1-PE (X56) were purchased from BD Pharmingen. Cytokines were detected by intracellular staining using standard procedures, and with APC-conjugated anti-IL-2 (MQ-1-17H12), -IL-4 (MP4-25D2), -IL-5 (TRFK5), -IL-10(10JES3-19F1), -IL-13(JES10-5A2), -TNF-α (MAb11), -IFN-α (B27), all from BD Pharmingen, PE-anti-IL-17A from BioLegend, and biotinylated-anti-TGF-β (MAB240) from R&D Systems. Streptavidin conjugated with APC or PE (Invitrogen) was used for the detection of biotinylated antibodies; the cells were transferred into new vials after each washing step in order to reduce background and exclude false positive results.In order to exclude dead cells from being analyzed, all cells were subject to live/dead exclusion using the Alexa Fluor 350 NHS Ester kit. In order to exclude the possibility of contaminants from feeder cells, irradiated feeder cells were cultivated and analyzed as clone cultures and used as controls in all experiments performed. Cells were analyzed using LSR II or FACS Calibur systems, and the FACS Diva©software and CellQuest software programs were used for the acquisition and analysis of flow cytometric data.® Beta Mark Kit (Beckman Coulter), a multi-parametric analysis tool designed for the quantitative flow cytometric determination of the TCR Vβ repertoire of human T lymphocytes. Taking advantage of the fact that Vβ specificities may be grouped into mutually exclusive combinations, three Vβ expressions can be detected in the same tube using an innovative staining strategy that uses three mAb stained with two fluorophores only. One mAb is conjugated to a FITC molecule, the second to PE and the third one is a carefully balanced mixture of a PE- and a FITC-conjugated form.TRBV repertoires were analyzed with the IOTest+ CD4+ T-cell clones were harvested from culture dishes and washed twice in sterile 1 × PBS. For TCR-β chain expression analysis, cells were applied to the slides by cytospin, fixed in −20°C acetone/methanol , and stained with mouse-anti-human TCR Beta F1 (1:50) . Cy™ 3-goat-anti-mouse IgG (1:400) (Jackson ImmunoResearch) was used to visualize TCR-β chain expression. Slides were analyzed with an Axiophot microscope (Zeiss) and the AnalySIS® 3.2 software (Soft Imaging Systems).Vδ1RNA was isolated using the RNeasy Plus Mini Kit (Qiagen). cDNA was synthesized using the Superscript III First Strand Synthesis Super Mix formulation (Invitrogen). All cDNAs were tested for the expression of a 800 bp amplicon of β-actin.+ Vδ1+ clones and from panVδ1+ cultures were analyzed with ABI TaqMan primer/probe sets for PTCRA (Hs00300125_m1), GAPDH (Hs02758991_g1), and RORC (Hs01076122_m1), all purchased from Life Technologies. All other primers are self-designed and are available upon request. Invitrogen’s TaqMan assay reagent and BioRad’s IQ Master Mix were used for qPCR. Gene expression was calculated using the change-in-threshold method [ΔC(T)] and GAPDH as reference.cDNAs derived from CD4Done in accordance with Han et al. .Performed according to Gorski et al.  with minPerformed with discriminating primers obtained from Annik Lim and according to her protocol .The relevant PCR products required for spectratype analysis were reamplified with unlabeled C primer. Sequencing was performed with the 3130xl Genetic Analyzer (Applied Biosystems) using the BigDye Terminator v3.1 Sequencing Kit according to the manufacturer’s protocol. Translation of the cDNA sequence into the protein sequence was conducted with the EMBOSS Transeq software, which is available free of charge , 120.n = 12); healthy bone marrow were leftovers from HLA-typing of potential stem-cell donors (n = 10), aliquots of LPH were leftovers from quality control after graft generation (n = 12). All samples were obtained and handled according to the Declaration of Helsinki and the procedures were approved by the Clinical Ethics Committee at the University of Tübingen. PBMCs were isolated by density centrifugation (Ficoll-Hypaque). Up to 2 × 107 PBMCs were pretreated using FcR Blocking Reagent (Miltenyi Biotec) according to the manufacturer’s protocol, stained with FITC-anti-Vδ1  using 10 μl antibody per 1 × 107 PBMCs . Subsequently, the Anti-FITC MultiSort Kit (Miltenyi Biotec) was used for the isolation of “dim” cells. The cells were separated with columns (Miltenyi Biotec). FACS analysis usally had a purity of >99% Vδ1+ T cells and less than 0.16% αβTCR+ cells. Alternatively, Vδ1+ γδT cells were selected with the Anti-TCRγ/δ MicroBead Kit (Miltenyi Biotec).Informed consent was obtained from all volunteers. Sterile heparinized peripheral blood was collected from normal, healthy donors . All procedures were carried out according to the manufacturer’s instructions. Vδ1+ CD4enriched T cells were cloned using the limiting dilution procedure (0.1 cells/well) in round bottom 96-well plates in the presence of 200 IU/mL IL-2 (ImmunoTools), 1.0 ng/mL IL-7 (ImmunoTools), 0.25 μg/mL phytohemagglutinin (Oxoid), and 1 × 105 allogeneic, 80 Gy-irradiated feeder cells. After 21 days, Vδ1+ CD4+ T-cell clones were identified by FACS analysis and kept under standard culture conditions – which represents mild inflammation – until further manipulation and analysis. Cloning strategy routinely yielded 5–10% of initially seeded Vδ1+CD4+ T cells (0.1 cells/well). For panVδ1 culture assays, 1,000 Vδ1+ cells were seeded per well in round bottom 96-well plates and cultured in RPMI 1640 supplemented with 10% heat-inactivated FCS, L-glutamine (4 mM), and penicillin (50 U/mL)/streptomycin (50 μg/mL), all purchased from Biochrom GmbH.For the establishment of Vδ12, water-saturated atmosphere, 37°C). Non-adherent cells were subsequently removed along with the supernatant; adherent cells were washed twice with warm PBS, and new culture medium containing inflammatory cytokines was added. After 3 days, the monocytes were removed from the bottom of the culture flask with a cell scraper, counted, centrifuged, resuspended in conditioned medium, irradiated (80 Gy), and 1 × 105 monocytes/well added to panVδ1+ cell cultures.Overt inflammation was mimicked in panVδ1 cultures by adding the following cytokines to the standard culture medium in week 3: IL-1β , IL-18 , IL-6 , sIL-6R , and IL-12 . Monocytes that had been preactivated for 3 days in the presence of these inflammatory cytokines were also added to the culture. Monocytes were generated from PBMNCs by plastic adherence in cell-culture flasks for 2 h  and ionomycin . Brefeldin A (10 μg/mL) was added for the last 60 min of incubation. Cytokine production was measured by intracellular staining and FACS analysis as described above.αβT cells derived from transdifferentiated Vδ1t-test, error bars in the graphs depict the SEM. A p < 0.05 was considered as statistically significant.Statistical analyses were performed with the GraphPad Prism software V5.0 (GraphPad Software). Statistical differences were analyzed using the parametric student + CD4+ cells, proposed and identified Vδ1+ CD4+ cells as a CD34+ T cell precursor, planned, designed, supervised all experiments in vitro and ex vivo, interpreted data, identified transdifferentiation as the re-organization of the TCR-γδ into TCR-αβ via a Vδ+Vβ+ Vγneg.Vαneg. intermediate, established the developmental scheme, and wrote the manuscript.Hendrik Ziegler, Christian Welker, and Marco Sterk contributed equally to the work, and performed most of the experiments, contributed to and established methodology; Jan Haarer contributed and established methodology, and performed experimental work; Hans-Georg Rammensee and Rupert Handgretinger contributed to experimental design and Rupert Handgretinger provided essential material. Christian Welker prepared all figures and helped editing the manuscript; Karin Schilbach initiated all work on Vδ1The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.http://www.frontiersin.org/Journal/10.3389/fimmu.2014.00645/abstractThe Supplementary Material for this article can be found online at Click here for additional data file.Click here for additional data file.


The molecules of each compound pack together in the solid state with offset face-to-face π-stacking, and intermolecular C—H⋯Nnitrile and C—H⋯Npyridine interactions. 4-Chloropyridine-2-carbonitrile, (I), exhibits pairwise centrosymmetric head-to-head C—H⋯Nnitrile and C—H⋯Npyridine interactions, forming one-dimensional chains, which are π-stacked in an offset face-to-face fashion. The intermolecular packing of the isomeric 6-chloropyridine-2-carbonitrile, (II), which differs only in the position of the chloro substituent on the pyridine ring, exhibits head-to-tail C—H⋯Nnitrile and C—H⋯Npyridine interactions, forming two-dimensional sheets which are π-stacked in an offset face-to-face fashion. In contrast to (I), the offset face-to-face π-stacking in (II) is formed between molecules with alternating orientations of the chloro and nitrile substituents.The two title compounds are isomers of C The pyridine heterocycle features prominently in many valuable synthetic compounds (Bull N-oxide with trimethylsilanecarbonitrile (TMSCN) N-oxide hydrochloride with sodium cyanide et al., 2002et al., 1971viz. C4—Cl and C6—Cl of 1.740 (3) Å in (I)et al., 19994-Chloropyridine-2-carbonitrile, (I)Both (I)nitrile and C—H⋯Npyridine interactions, however, the packing motifs are unique, and also different than those found in the related structure 2-chloropyridine-4-carbonitrile (CSD refcode LOBVIJ). For a discussion of weak C—H⋯X interactions, see Desiraju & Steiner in Fig. 3. The chain Fig. 3. This π-via head-to-tail C—H⋯Nnitrile and C—H⋯Npyridine interactions to form two-dimensional sheets that are parallel to the (001) plane ne Fig. 5. As in  al. 1994 and Jels al. 2015.4-Chloropyridine-2-carbonitrile (97%) and 6-chloropyridine-2-carbonitrile (96%) were purchased from Aldrich Chemical Company, USA. 4-Chloropyridine-2-carbonitrile was recrystallized from 95% ethanol.Uiso(H) = 1.2Ueq(C) of the aryl C atoms.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989015011767/rz5161sup1.cifCrystal structure: contains datablock(s) global, I, II. DOI: 10.1107/S2056989015011767/rz5161Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989015011767/rz5161IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S2056989015011767/rz5161Isup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989015011767/rz5161IIsup5.cmlSupporting information file. DOI: 1407613, 1407612CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The structures of 2- and 3-substituted tertiary carboxamides and analogous pyrrolidine structures are compared. 17H13NO3, N-methyl-4-oxo-N-phenyl-4H-chromene-3-carboxamide (1), and the chromone carbonyl pyrrolidine derivatives, C14H13NO3, 3-(pyrrolidine-1-carbonyl)-4H-chromen-4-one (3) and 2-(pyrrolidine-1-carbonyl)-4H-chromen-4-one (4) have been determined. Their structural features are discussed and compared with similar compounds namely with respect to their MAO-B inhibitory activities. The chromone carboxamide presents a –syn conformation with the aromatic rings twisted with respect to each other [the dihedral angle between the mean planes of the chromone system and the exocyclic phenyl ring is 58.48 (8)°]. The pyrrolidine derivatives also display a significant twist: the dihedral angles between the chromone system and the best plane formed by the pyrrolidine atoms are 48.9 (2) and 23.97 (12)° in (3) and (4), respectively. Compound (3) shows a short C—H⋯O intramolecular contact forming an S(7) ring. The supramolecular structures for each compound are defined by weak C—H⋯O hydrogen bonds, which link the molecules into chains and sheets. The Cambridge Structural Database gave 45 hits for compounds with a pyrrolidinecarbonyl group. A simple statistical analysis of their geometric parameters is made in order to compare them with those of the molecules determined in the present work.The crystal structures of the 3-substituted tertiary chromone carboxamide derivative, C Previous data acquired on the development of new MAO-B inhibitors allowed us to conclude that 2-substituted chromones carboxamides based on the N-phenyl-4-oxo-4H-2-chromone carboxamide (6) skeleton have no significant IMAO-B activity whereas 3-substituted carboxamides based on the N-phenyl-4-oxo-4H-3-chromone carboxamide (5) core have been shown to be potent and selective inhibitors  have very similar conformations and indicate that the displayed IMAO-B activity is mostly dependent on electronic factors modulated by the nature and position of the substituent group attached to the exocyclic phenyl substituent  and its isomer N-methyl-4-oxo-N-phenyl-4H-chromene-2-carboxamide (2), both tertiary carboxamides, as opposed to the secondary carboxamides (5) and (6) and 3-(pyrrolidine-1-carbonyl)-4H-chromen-4-one (3) and 2-(pyrrolidine-1-carbonyl)-4H-chromen-4-one (4), which instead of the carboxamide have a carbonyl pyrrolidine linked to the chromone (see Scheme). Compounds (2), (5) and (6), N-methyl-4-oxo-N-phenyl-4H-chromene-2-carboxamide, N-phenyl-4-oxo-4H-3-chromone carboxamide and N-phenyl-4-oxo-4H-2-chromone carboxamide, have previously been characterized by X-ray diffraction  is the building block of a large family of natural and synthetic compounds of the utmost importance in medicinal chemistry (Gaspar 2), (5) and (6) have previously been characterized. The ellipsoid plots for the remaining structures, e.g. for (1), (3) and (4), are given in Figs. 15) exhibits significant IMAO-B activity. Its isomer (6) is much less active while the remaining ones are inactive towards MAO-B, suggesting that substitution on position number 3 of the chromone is required and it must be a secondary carboxamide. As will be discussed, the presence of a tertiary amide induces significant conformational changes to the compounds that can explain the lack of activity for those compounds.As mentioned above, the compounds discussed in this work are presented in the Scheme. Compounds (1) is a phenyl chromone carboxamide similar to (5) where the amidic hydrogen atom has been replaced by a methyl substituent. Since the nitrogen atom of the amide tends to be planar due to the partial sp2 hybridization of the C—N bond and, owing to the high rotational barrier around that bond, amides often exhibit –anti/–syn conformations with respect to the C–N rotamer. The inactive chromone carboxamides (1) and (2) present –syn conformations whereas chomone (5) (active) and (6) (inactive) are in the –anti form. In (5) and (6) the aromatic rings are roughly co-planar  and (4) [1.340 (3) Å] shows that these values are comparable with those presented for the corresponding bonds in the carboxamides (1) [1.361 (2) Å] and (2) [1.3528 (14) Å], indicating partial sp2 hybridization of the nitrogen atom in (3) and (4). Furthermore, a search made in the Cambridge Structural Database  shows that the C—N distances range between 1.294 and 1.361 Å [the mean value is 1.335 (2) Å], suggesting that in carbonyl pyrrolidines the C—N bond displays partial hybridization  and (2). This contrasts with what occurs in (5) and (6) where, due to the presence of the imidic nitrogen atom, the molecules display N—H⋯O intramolecular S(6) rings and, due to the –anti configuration, they present weak Carom—H⋯O hydrogen bonds (the carbonyl group of the amide acting as acceptor for the ortho-carbon atom of the benzyl ring), resulting in second S(6) rings  there is a short intramolecular contact C312—H312⋯O4 in which the pyrollidine carbon atom acts as a donor to the carbonyl oxygen atom, O4, of the chromone, forming an S(7) ring. The search of the CSD  the C2—H2⋯O4 and C5—H5⋯O1 hydrogen bonds link the molecules into a axis, Fig. 4x − 1, y + 1, z) hydrogen bond, Fig. 5In compound (3) the molecules are linked by C—H⋯O interactions and by C—H⋯π interactions. The C8—H8⋯O4 chains running parallel to the c axis which are propagated by the twofold screw axes at (z) and (z), respectively, Figs. 7bc plane, Fig. 9x + y + z + 1) interaction links the molecules into C(5) chains running along the a axis propagated by the twofold screw axis at  chains running along the b axis which are propagated by the twofold screw axis at  there is a short contact between C214—H21C and O4 chain which runs along the c axis, propagated by the twofold screw axis at (z), Fig. 12x + y − z) but in this case the angle at H8 is 121° and so this interaction will be relatively weak. It forms a C(7) chain parallel to the a axis propagated by the glideplane at b axis, Fig. 13In compound (R ≤ 0.10 (see supplementary data for the search fragment). The mean value for the C—O bond length was 1.235 (2) Å with a range of 1.209–1.282 Å. The values for (3) and (4) are 1.239 (4) and 1.230 (2) Å, respectively. The mean C—N bond length is 1.335 (2) Å with a range of 1.294–1.361Å. The values for (3) and (4) are 1.337 (4) and 1.340 (3) Å, respectively. The values for these compounds are close to the mean values in each case.A search of the Cambridge Structural Database  [−0.5 (5) and 171.9 (3)°] and those for (4) [1.1 (3) and −175.3 (2)°] are well within the ranges specified above.The torsion angles around the C(carbonyl) and N(pyrrolidine) bond involving the carbonyl O atom lie in ranges between −9.15 and 8.023° with a mean value of close to zero and between −161.33 and 166.71° with a mean value close to 180° for both the C atoms attached to the N atom within the pyrolidine group. The respective torsion angles for (3) are found in five compounds in the CSD: LISLAB, 1-(1-pyrrolidinylcarbonyl)cyclopropyl sulfamate -4,5-bis(pyrrolidinylcarbonyl)-2,2-dimethyl-1,3-dioxolane  rings are formed. In QIBBEJ and SINHAZ, an S(7) ring similar to that in (2) is formed. In PEQHAU, an S(8) ring is formed, Fig. 14Intramolecular C—H⋯O short contacts similar to that in  was synthesized in a low yield (10%) by a one-pot reaction using 4-oxo-4H-chromene-3-carboxylic acid as starting material. The activation of the carboxylic acid was obtained by the coupling reagent bromotripyrrolidinophosphonium hexafluorophosphate (PyBrOP) and the amide obtained by reacting the ester derivative with N-methylaniline. The crude product was purified by flash chromatography (ethyl acetate and ethyl acetate/ CH2Cl2 in an 4:1 ratio). Crystals suitable for X-ray diffraction were obtained from ethyl acetate.3-(Pyrrolidine-1-carbonyl)-4H-chromen-4-one, (3) and 2-(pyrrolidine-1-carbonyl)-4H-chromen-4-one, (4) were synthesized in moderate yields, 57% and 45%, by a one-pot reaction using 4-oxo-4H-chromene-3-carboxylic and 4-oxo-4H-chromene-2-carboxylic acids, respectively, as starting materials. The synthetic strategy encompasses the activation of the chromone carboxylic acids by reaction with phosphorus(V) oxychloride with formation in situ of an acid chloride intermediate. The acid chlorides react with pyrrolidine giving the desired amides. Crystals suitable for X-ray diffraction for both compounds were obtained from a solution of CH2Cl2/n-hexane solvent 1:1, m.p. for (3): 421–426K; m.p. for (4): 382–386K.2) = 0.99 Å with Uiso = 1.2Ueq(C), and C—H(methyl) = 0.98Å with Uiso = 1.5Ueq(C). The methyl hydrogen atoms were generated in idealized positions and checked on a final difference map.Crystal data, data collection and structure refinement details are summarized in Table 410.1107/S2056989015017958/lh5791sup1.cifCrystal structure: contains datablock(s) general, 1, 3, 4. DOI: 10.1107/S2056989015017958/lh57911sup2.hklStructure factors: contains datablock(s) 1. DOI: 10.1107/S2056989015017958/lh57913sup3.hklStructure factors: contains datablock(s) 3. DOI: 10.1107/S2056989015017958/lh57914sup4.hklStructure factors: contains datablock(s) 4. DOI: Click here for additional data file.10.1107/S2056989015017958/lh57911sup5.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989015017958/lh57913sup6.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989015017958/lh57914sup7.cmlSupporting information file. DOI: 1427459, 1427458, 1025355CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


A copper-catalyzed cascade reaction of α,β-unsaturated esters with keto esters is reported. It features a copper-catalyzed reductive aldolization followed by a lactonization. This method provides a facile approach to prepare γ-carboxymethyl-γ-lactones and δ-carboxymethyl-δ-lactones under mild reaction conditions. Paraconic acid and its analogues, sharing the similar structure of a γ-lactone with an attached β-carboxylic acid, are widely found in natural products. Related syntheses have been actively explored due to the potential antitumor and antibiotic activities of these compounds –4. The sA, which is subjected to further lactonization to form the lactone. Lam’s group has furnished a cobalt-catalyzed conjugate reductive aldolization–lactonization cascade reaction using diethylzinc as a reductant [Alternatively, the lactone is also formed through the reaction of a carbonyl with an enolate that can be generated from a metal-catalyzed conjugate addition of an α,β-unsaturated diester. As shown in eductant . Floch eeductant –11, whiceductant . Bertraneductant . To addreductant  and imineductant , respectAlthough syntheses of β-carboxy-γ-lactones have been studied extensively, synthetic methods for γ-carboxymethyl-γ-lactones have been scantly explored. Taking into account the known bioactivities of the β-carboxy-γ-lactone class, its close isomer γ-carboxymethyl-γ-lactone should be of pharmaceutical interest as well. To date we have found only one example of the synthesis of the γ-carboxymethyl-γ-lactone involving the reaction of α-bromocarboxylate with zinc and a γ-oxocarboxylate . To thisA in B as described in B possesses a γ-alkoxide unit and an ester unit as that in intermediate A, and therefore, it is expected to afford a γ-carboxymethyl lactone as the final product. Furthermore, change of the linker’s length is expected to yield δ-carboxymethyl-δ-lactones.Considering that the reported conjugate addition–aldolization–lactonization cascade reactions proceed via the key intermediate Copper-catalyzed reductive intermolecular aldolization reactions have been discussed previously –19. Cons1a) was employed as a copper hydride acceptor. In the presence of methyl 4-oxo-4-phenylbutanoate (2a), as expected it underwent the proposed cascade reaction to furnish 3aa, a γ-lactone with a γ-exo-ester group, in 90% yield  with 2a and PMHS in toluene at 25 °C gave two diastereomers which were detected by GC, however inseparable by column chromatography. Formation of the anti-isomer was slightly favored at 25 °C  was used to replace methyl acrylate, and several biphosphorous ligands were screened for improving the diastereoselectivity phenylpentanoates In conclusion, a novel copper-catalyzed three-component cascade reaction among α,β-unsaturated esters, keto esters and a silane was developed. The reaction involves conjugate addition of copper hydride, generated in situ, to α,β-unsaturated esters, followed by the aldolization with a keto ester and lactonization. It gives γ-carboxymethyl-γ-lactones and δ-carboxymethyl-δ-lactones in high yields, and provides a promising synthetic method for ester-functionalized γ-lactones and δ-lactones.A typical procedure: Under a N2 atmosphere, a solution of CuF(PPh3)3·2MeOH , and Xantphos  in toluene (1 mL) in a Schlenk tube was stirred for 15 min. PMHS (0.040 mL) was added to the solution, and the resulting solution was stirred for 30 min. A solution of 1a  and 2a  in toluene (1 mL) was added slowly to the tube. The progress of the reaction was monitored by TLC. When most of 2a was consumed, the reaction was quenched by the addition of a NH4F solution in methanol/H2O (10 mL). The mixture was filtered. The filtrate was separated, and the aqueous phase was extracted with dichloromethane (3 × 10 mL). The combined organic phase was washed with brine, dried and concentrated in vacuo. The conversion of 2a was determined by GC. Column chromatography afforded methyl 2-methyl-2-(5-oxo-2-phenyltetrahydrofuran-2-yl)propanoate (3aa)  as colorless solid. mp 81–83 °C. Rf = 0.34 (EA/PE = 20:80). 1H NMR  δ 7.38–7.29 , 3.67 , 3.00–3.15 , 2.63–2.46 , 2.38–2.23 , 1.26 , 1.16 ; 13C NMR  δ 176.22, 175.32, 140.31, 128.16, 128.03, 126.48, 91.31, 52.18, 50.05, 31.02, 28.88, 22.01, 21.27; LRMS–EI (70 ev), m/z (%): 203 (3), 161 (100), 143 (5), 133 (47), 115 (39), 105 (80), 91 (17), 77 (51), 55 (10), 51 (10); HRMS–FAB calculated for C15H18O4 + NH4+, 280.1543; found, 280.1550.For those products as diastereomers, the conversion of keto esters and diastereoselectivities of lactones were determined by GC analyses.File 1Characterization data of the products.


Heterotrimeric GTP-binding proteins (G-proteins) play an important role in mediating signal transduction generated by neurotransmitters or hormones. Go, a member of the Gi/Go subfamily, is the most abundant G-protein found in the brain. Recently, the alpha subunit of Go (Gαo) was characterized as an inducer of neuronal differentiation. However, its underlying molecular mechanisms have remained unclear to date, since the downstream effectors of Gαo are ambiguous.in vitro and in vivo. Necdin interacted directly and preferentially with activated Gαo, compared to wild-type protein. Interestingly, Gαo did not interact with Gαi, despite high sequence homology between the two proteins. We subsequently analyzed whether Gαo modulates the cellular activities of Necdin. Notably, expression of Gαo significantly augmented Necdin-mediated cellular responses, such as proliferation and differentiation. Moreover, activation of type 1 cannabinoid receptor (CB1R), a Gi/oα-coupled receptor, augmented cell growth suppression, which was mediated by Gαo and Necdin in U87MG cells containing CB1R, Gαo, and Necdin as normal components.A neurally differentiated embryonal carcinoma-derived protein (Necdin) was isolated as an interacting partner for Gαo from a mouse brain cDNA library using yeast two-hybrid screening. Interactions between the proteins were confirmed with several affinity binding assays, both These results collectively suggest that Necdin is a candidate downstream effector for Gαo. Our findings provide novel insights into the cellular roles of Gαo and its coupled receptor. Heterotrimeric GTP-binding proteins (G-proteins) mediate signaling from G protein-coupled receptors (GPCRs) to intracellular downstream effectors . BindingTo date, 21 Gα subunits encoded by 16 genes have been identified –5]. Pre. Pre5]. Cannabis sativa Linnaeus (marijuana), have recently received considerable attention as potential therapeutic agents, owing to their various pharmacological actions, including pain control, tumor regression, neurogenesis, neuroprotection, and anti-inflammatory effects . Tw. TwCanna effects , predomi effects , and (2) effects , mainly  effects .Neurally differentiated embryonal carcinoma-derived protein (Necdin) was originally isolated from P19 embryonic carcinoma cells . Necdin,in vitro and in vivo, were further confirmed with several affinity binding assays. Furthermore, activation of Gαo enhanced Necdin activity. Our findings collectively indicate that Necdin is a candidate downstream effector for Gαo.In this study, we performed yeast two-hybrid screening to identify downstream effectors for Gαo using a constitutively active form of Gαo as bait from a mouse brain cDNA library. Consequently, Necdin was identified as a Gαo-interacting protein. Interactions between Gαo and Necdin, both in vivo. As shown in Figure in vitro as well as in vivo.We performed yeast two-hybrid screening to identify Gαo-interacting partners from a mouse brain cDNA library. Necdin protein was isolated using the constitutively active mutant of Gαo as bait, and interactions confirmed with several affinity binding assays. Beads charged with bacterially expressed GST or GST-Gαo proteins were incubated with soluble proteins obtained by detergent extraction of 293T cells transfected with FLAG-tagged Necdin (FLAG-Necdin), and the reaction mixtures probed with antibodies against FLAG. GST-Gαo specifically interacted with FLAG-Necdin Figure A. To detS-transferase (GST)-tagged Necdin (GST-Necdin) proteins in the presence or absence of AlF4−, an activator of Gα protein. After glutathione-Sepharose beads were added to the reactions, beads were analyzed with antibodies against Gαo and Necdin. Necdin bound directly to Gαo in the presence of AlF4− -tagged-Necdin (HA-Necdin). Cell lysates were incubated with antibodies against Necdin, and the immunocomplexes analyzed with immunoblot analysis using the indicated antibodies  293T and Neuro2a cells transfected with plasmids encoding Gα and Necdin, and (2) U87MG cells endogenously expressing CB1R, which triggers the Gi/o-mediated signaling pathway upon activation. In transfection experiments, the cellular activities of Necdin were significantly enhanced by Gαo, but not Gαo/i. As shown in Figures Several lines of evidence support the finding that Gαo modulates neuronal differentiation. Gαo is the most abundant Gα protein in brain tissue and one of the major membrane components of growth cones ,29],,,31]. Gα. Gα31]. We postulate that Necdin-mediated neuronal differentiation is attributable to its activity as a strong growth suppressor. Necdin exhibits anti-proliferative activity in various cells and is highly expressed in normal tissues but downregulated in tumor cells ,33]. Ne. Ne33]. Considerable evidence supports the theory that cannabinoids are potential therapeutic agents for cancer. Cannabinoids have been shown to inhibit tumor cell proliferation in various cell types, including glioma, pheochromocytoma, prostate, and leukemia cells –42]. Ca. Ca42]. Several assay systems are available to measure the cellular activities of Gα proteins. For example, Gαq activity can be measured based on accumulation of inositol phosphate, which is modulated by phospholipase C, a downstream effector of Gαq. The amount of accumulated cAMP represents the activity of adenylyl cyclase, a downstream effector of Gαi and Gαs. However, an effective assay system to measure Gαo activity has not been developed to date owing to the ambiguity of its downstream effectors. Our results showing that Necdin is a candidate downstream effector of Gαo independent of Gαi may facilitate the development of a Necdin-specific assay system for Gαo.We have identified a novel downstream effector molecule and signaling pathway for Gαo. Activation of Gαo enhances Necdin-E2F1 interactions, which, in turn, modulate cell differentiation and proliferation. Our current findings present an additional novel signaling pathway to explain the diverse roles of Gαo and cannabinoid receptor.NotI and XbaI, and ligating the fragment into the corresponding sites of pcFLAG. pcHA-Necdin encoding HA-Necdin was constructed by digesting pcFLAG-Necdin with NotI and XbaI and ligating the fragment into the corresponding restriction sites of pcHA. pGEX-5X-Necdin, an expression plasmid for GST-Necdin, was generated by digesting pcFLAG-Necdin with EcoRI and cloning into the corresponding restriction sites of pGEX-5X . To generate psi-RNA-hH1ZeoG2-Necdin, Necdin-shRNA plasmid, pcFLAG-Necdin was amplified using the primers 5’-GTA CCT CGC CCG AAG AAC GGA TAG AAG ATC AAG AGT CTT CTA TCC GTT CTT CGG GCT TTT TGG AAA-3’ and 5’-AGC TTT TCC AAA AAG CCC GAA GAA CGG ATA GAA GAC TCT TGA TCT TCT ATG CGT TCT TCG GGC GAG-3’. Amplified PCR products were inserted into psi-RNA-hH1ZeoG2 plasmid  using HindIII and ACC65I restriction enzymes.The pRC/CMV-Necdin plasmid was a generous gift from Dr. K. Yoshikawa . pcDNA3-E2F1 and pGL3-E2F4B-Luc reporter plasmids were kindly provided by Dr. J. Cheong . Plasmids pRC/CMV-GαoWT and pRC/CMV-GαoQ205L encode GαoWT and GαoQ205L of Gαo, respectively . To geneThe bait plasmid (pHybTrp/Zeo-GαoQ205L) was transformed into the yeast reporter cell line, L40, with the mouse brain cDNA library  as recommended by the manufacturer. The methods used for isolation of positive clones are described in a previous report .2, 1 mM EDTA, 1% Triton X-100, 5 μg/ml aprotinin, 10 μg/ml leupeptin, 2 μg/ml pepstatin A, and 2 mM phenylmethylsulfonyl fluoride) for 1 h at 4°C with gentle rotation.Human embryonic kidney cell line, 293T, mouse neuroblastoma cell line, Neuro2a, and human glioblastoma cell line, U87MG, were maintained in DMEM supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin and 100 μg/ml streptomycin. 293T and Neuro2a cells were transiently transfected with the indicated concentrations of plasmids using calcium phosphate and polyethylenimine, respectively. For affinity binding assays, after 48 h of transfection, cells were harvested and extracted with PBTX buffer , as indicated, in HEMNDL buffer  for 1 h at 30°C. GST-Necdin was added to the reaction at a final concentration of 200 nM, and incubated for an additional 20 min at 20°C. Following the addition of glutathione-Sepharose 4B beads, reactions were incubated for 1 h at 4°C with gentle rotation. Beads charged with proteins were analyzed with the indicated antibodies.Fusion proteins, His-Gαo and GST-Necdin, were purified from BL21 cells using glutathione-Sepharose 4B beads and HisTrapectively . His-Gαo6 cells/dish) were plated on 100 mm tissue culture dishes and transiently transfected with the appropriate combinations of expression plasmids. U87MG cells (1 × 105 cells/dish) were plated on 100 mm tissue culture dishes. After 24 h, cells were serum-starved with 2% FBS for 20 h. Cells were treated with 20 μM Win 55,212-2, a CB1R agonist, for 1 h in the absence or presence of 30 ng/ml pertussis toxin , a Gi/oα inhibitor. Pretreatment with PTX was performed for 20 h prior to Win 55,212-2 application. Newborn rat brain extracts were prepared as reported previously [293T and Neuro2a cells (1.5 × 103 cells/coverslip) were plated on coverslips and transfected with expression plasmids for Gαo (0.1 μg) and FLAG-Necdin (0.25 μg). After 48 h, cells were fixed with 4% paraformaldehyde in PBS for 15 min and incubated for 1 h at room temperature with antibodies against Gαo and FLAG. Cells were washed with PBS and incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG and Alexa Fluor 568-conjugated goat anti-mouse IgG  for 30 min at room temperature. After washing with PBS and counterstaining with DAPI , cells were observed under a LSM510 confocal laser scanning microscope .293T cells (1 × 10Pregnant mice (C57BL/6 J) were killed via cervical dislocation and E15.5 embryos were placed immediately in ice-cold 4% PFA. After overnight fixation, tissues were embedded in paraffin and sectioned to 5-μm thickness. Antigens were retrieved by boiling in 10 mM sodium citrate (pH 6.0) in a microwave oven, the sections were blocked in PBS with 5% normal serum, and probed with primary antibodies against Necdin  and Gαo (1:400). The sections were then incubated with Alexa Fluor 488- or 594-conjugated anti-IgG secondary antibodies and counterstained with Hoechst 33258 . Fluorescent images were acquired using a Zeiss LSM710 confocal microscope (Carl Zeiss).4 cells per well. 293T cells were transfected with the appropriate combinations of indicated plasmids. To identify transfected cells, we co-transfected with the GFP expression plasmid in all experiments. U87MG cells were serum-starved and pretreated with 30 ng/ml PTX. After 20 h, cells were treated with 3 μM Hu210 , a CB1R agonist for 6 h. Both cell lines were labeled with 10 μM BrdU (Sigma-Aldrich), 12 h before harvest. Next, cells were washed with PBS and fixed in 4% paraformaldehyde in PBS for 15 m. After fixation, cells were subjected to immunofluorescence staining against BrdU [293T and U87MG cells were plated on 6-well plates at a density of 2 × 10nst BrdU .4 cells per well. Cells were transfected with the indicated combinations of plasmids, and 100 ng of pEGFP. After 24 h, cells were serum-starved for 36 h. Fluorescence images were observed with a IX71 fluorescence microscope , and the percentage of neurite-bearing cells among the GFP-positive population calculated. Cell processes greater than cell body in length were counted as neurites.Neuro2a cells were plated on 6-well tissue culture dishes at a density of 3 × 10Total RNA was extracted from cultured cells with the Easy-spin total RNA extraction kit , according to the manufacturer’s instructions. First-strand complementary DNA (cDNA) was synthesized using 1 μg of total RNA as the template, 500 ng of oligo (dT), and AccuPower™ RT-Premix  in a total volume of 20 μl, according to the manufacturer’s recommendation. The relative mRNA expression of Necdin was assessed using TOPreal™ qPCR premix  on the CFX96™ PCR System . Primer sequences used were as follows: Necdin, 5’-GCT CAT GTG GTA CGT GTT GG-3’; and 5’-TGC TTC TGC ACC ATT TCT TG-3’; GAPDH, 5’-TGG GCT ACA CTG AGC ACC AG-3’; and 5’-GGG TGT CGC TGT TGA AGT CA-3’. The Necdin mRNA values were normalized to the amount of GAPDH that was measured.4 cells per well. Cells were transfected with the indicated combination of plasmids, together with 0.1 μg of pGL3-E2F4B-Luc reporter plasmids containing four consensus E2F binding sites [4 cells per well and transfected with 0.3 μg of pGL3-E2F4B-Luc reporter plasmids using Lipofectamine 2000 (Life Technologies). After 24 h, cells were serum-starved for 16 h and treated with the indicated amounts of Hu210 for 6 h in the absence or presence of 30 ng/ml PTX. Pretreatment with PTX was performed for 1 h before Hu210 application. For knockdown experiment, U87MG cells transfected with pGL3-E2F4B-Luc (0.3 μg) and Necdin-shRNA (0.2 μg) were treated with indicated amount of Win 55,212-2 for 6 h.Neuro2a cells were plated on 6-well tissue culture dishes at a density of 3 × 10ng sites . For norBrdU: BromodeoxyuridineCB1R: Type 1 cannabinoid receptorCB2R: Type II cannabinoid receptorFBS: Fetal bovine serumGαo: alpha subunit of GoGαoQ205L: Constitutively activated mutant of GαoGαoWT: Wild-type of GαoGFP: Green fluorescence proteinGPCR: G protein-coupled receptorsS-transferaseGST: Glutathione-Necdin: Neurally differentiated embryonal carcinoma-derived proteinPKA: Protein kinase APTX: Pertussis toxinRit: Ras-like protein in all tissuesRb: Retinoblastoma proteinThe authors declare that they have no competing interests.HJ, SL, SK and SG designed experiment, HJ, SL and SK performed experiments, and SG and HJ wrote the manuscript. All authors read and approved the final manuscript.Gαo enhances cell growth suppression induced by Necdin. 293T cells were transfected with plasmids encoding various types of Gα (0.5 μg) and FLAG-Necdin (1 μg), as indicated. To identify transfected cells, we co-transfected with the pEGFP (100 ng). After 24 h of transfection, cells were labeled with 10 μM BrdU for 12 h and stained with antibodies against BrdU and GFP. Scale bar, 20 μm.Click here for fileGαo promotes Necdin-induced neurite outgrowth. Neuro2a cells were transfected with plasmids encoding various types of Gα (0.5 μg) and FLAG-Necdin (1 μg). To identify transfected cells, we co-transfected with the pEGFP (100 ng). After 24 h of transfection, cells were serum-starved and observed 30 h later. Scale bar, 50 μm.Click here for fileEffect of E2F1 and Necdin on E2F4B-luciferase reporter gene activity. Neuro2a cells were transfected with the indicated combinations of plasmids encoding FLAG-Necdin (0.25 μg), E2F1 (0.03 μg), E2F4B-Luc reporter gene (0.1 μg), and β-galactosidase (0.3 μg). The total amount of plasmid DNA used for transfection was maintained by adding pcDNA3. After 48 h, cells were subjected to luciferase and β-galactosidase assays. Luciferase activity was normalized to that of β-galactosidase. Data are presented as the average ± SE of at least three independent experiments. *, p < 0.001.Click here for file


Bacillus subtilis K1, produces a complex mixture of lipopeptides exhibiting potent antifungal activity. These lipopeptides were purified by high-performance liquid chromatography and analyzed using MALDI-TOF-MS as well as liquid chromatography coupled with ESI-MS. A heterogenous mixture of lipopeptides belonging to three different families of cyclic lipopeptides, viz., fengycins, iturins and surfactins, was detected in the cell-free extracellular extract of B. subtilis K1. The detailed mass spectrometric characterization revealed the presence of four variants of iturin A and three variants of iturin C varying in the β-amino fatty acid chain length from C13 to C17. The MS/MS of monovalent alkali metal ion adducts (Na and K) of iturin suggested the Glu4 as a binding site for metal ion. The LC-ESI-MS/MS analysis of surfactins enabled the identification of seven surfactin variants with the variations in Val/Ile/Leu at position 4 and C13–C17 β-hydroxy fatty acids. This study demonstrates the application of tandem mass spectrometry in identification of closely related lipopeptides from a heterogenous mixture obtained from a natural source. Furthermore, this is the first report of an endophytic bacillus strain co-producing so many variants of surfactins and iturins.The banyan endophyte, The online version of this article (doi:10.1007/s13205-013-0151-3) contains supplementary material, which is available to authorized users. B. subtilis K1, isolated from the aerial roots of banyan tree, was found to inhibit growth of Aspergillus niger, Alternaria brunsii, Fusarium oxysporum, Cladosporium herbarum 1112, Candida albicans and Lasiodiplodia theobromae ABFK1 , which in turn is peptide bonded through its α-COO group to N-terminal amino acid. The members of iturin family exhibit heterogeneity at 1, 4, 5, 6 and 7 amino acid position/s in the peptide moiety as well as in the βAA length, which varies from 14 to 17 carbons. On the basis of variation of amino acids in peptide moiety, iturins have been classified as: iturin A, iturin C, iturin D, iturin E, bacillomycin D, bacillomycin F, bacillomycin L, bacillomycin Lc and mycosubtilin  medium in 1,000-mL flask to an initial OD(600 nm) of ~0.05 and incubated at 30 °C for 72 h on orbital shaker (150 rpm).The B. subtilis K1 was centrifuged at ~10,000×g at 4 °C for 20 min. The supernatant was collected and its pH was lowered to pH 2 using 6 N HCl and incubated at 4 °C for 4 h in order to precipitate lipopeptides. The acid precipitates were recovered by centrifugation at ~10,000×g at 4 °C for 20 min, and pellet thus obtained was solubilized in anhydrous methanol  using methanol/water/0.1 % trifluroacetic acid solvent system. The elution of lipopeptides was done using a gradient of 80–95 % methanol (v/v) for 50 min, 95 % methanol (v/v) for 5 min and 95–80 % for 5 min . The lipopeptide fractions were mixed with equal volume of α-cyano-4-hydroxy-cinnamic acid matrix and spot applied on the target plate. The mass spectrometric (MS and MS/MS) data acquisition was carried out as per the method described previously   using MeOH/H2O/0.1 % (v/v) formic acid on an Agilent 1100 HPLC system, coupled with HCT Ultra ETD II  ion trap mass spectrometer in positive ion mode. A gradient  pumped at a constant flow rate of 0.2 mL/min was used with a run time of 60 min. The data were acquired in an auto MS2 mode using collision-induced dissociation method for fragmentation and data were analyzed by Data Analysis software ver. 4.0 .LC–ESI–MS spectra were acquired over the mass range of A. niger 40211, A. flavus, A. parasiticus, F. oxysporum 1072, Chrysosporium indicum, Candida albicans, Trichosporon sp. 1110, Alternaria brunsii (2), and Cladosporium herbarum 1112. Fungal spore suspension was prepared by harvesting spores into sterile distilled water and the spore counts were determined using haemocytometer. In case of Candida albicans and Trichosporon sp. 1110, culture suspension was prepared by cultivating them in 50-mL potato dextrose broth under agitated condition (150 rpm) at 30 °C for 10–12 h. The cell numbers were determined using a hemocytometer and adjusted to 1 × 106 cells/mL by appropriate dilution with sterile distilled water. The MIC of pure lipopeptides were determined by double dilution technique against susceptible fungal cultures in sterile 96-well microtiter plates with each well containing 100 μL of potato dextrose broth. After dilution ~102 spores of test fungus were inoculated into each well. To control wells, corresponding aliquot of MeOH instead of sample was added. The plates were incubated for 24–48 h at 30 °C and MIC values for each fraction were determined against susceptible test fungi based on the highest dilution showing no growth.Stock solutions (1 mg/mL) of iturin A2, iturin A2/A3/A4 and fengycin were prepared by dissolving lyophilized fractions in MeOH and analyzed for activity against B. subtilis K1 revealed the presence of compounds with molecular mass ions in the range, m/z 1,028–1,109, m/z 994–1,065 and m/z 1,421–1,566, which were assigned as iturins, surfactins and fengycins, respectively , 1,043.5 (P3), 1,057.5 (P4), 1,071.5 (P5) and 1,072.5 (P6)  were also detected which were putatively assigned as sodium adducts  and potassium adducts  of corresponding iturins , m/z 339.6 (βAA-N), m/z 502.9 (βAA-N-Y-), m/z 616.6 (βAA-N-Y-N), m/z 744.9 (βAA-N-Y-N-Q) and m/z 201.7 (S-N), m/z 298.8 (S–N-P), m/z 426.6 (S-N-P-Q), m/z 541.0 (S-N-P-Q-N), m/z 703.7 (S-N-P-Q-N-Y-), m/z 817.6 (S-N-P-Q-N-Y-N) were found in the MS2 spectrum of m/z 1,043.5. This fragment series suggested yet another ring cleavage site, i.e. amide bond between βAA and Ser1 of iturin homolog. The ion at m/z 183.7 seemed to have formed due to loss of 41 Da (–CH2–C=O)+ from βNC14 and was assigned as βNC12. The lipopeptide sequences derived from the MS/MS data for the molecules at m/z 1,029.5, 1,043.5, 1,057.5 and 1,071.5 were assigned as iturin A homologs, based on published literature , Y (m/z 136.5) and P (m/z 70.3) could be identified in MS/MS spectra of all these three iturins while the ion at m/z 339.7 could be assigned as an internal ion of tripeptide −NPQ  based on the heterogeneity of βAA   with same molecular mass of 1,057.5 and thus, m/z 1,057.5 may be one of these three iturin A isomers, which cannot be distinguished by mass spectrometry. Along with these four protonated iturin A species, fifth species with m/z 1,072.5 of iturin was also detected. The molecular mass ion at m/z 1,072.5 differed from 1,071.5 by 1 Da and it is difficult to separate the species varying by 1 Da in MS spectrum. Moreover, in MALDI-MS/MS spectrum of m/z 1,072.5 most of the fragmentation belonging to iturin A species was at m/z 1,071.5 therefore, to determine the identity of iturin species at m/z 1,072.5, LC-ESI-MS/MS method was used. In the ESI-MS spectrum of molecular mass ions ranging within the m/z 1,040 to m/z 1,080 (eluted within the time range of 9–23 RT), three isotopic peak clusters with mono isotopic masses at m/z 1,043.5, 1,057.5, 1,072.5 were observed . The isotopic distribution in these peak clusters seemed to be abnormal as the intensity of second isotopic peak  in each cluster is higher than the first isotopic peak. Moreover, these compounds are falling in the m/z range where normal isotopic distribution is anticipated. This type of isotopic distribution in the fengycins has been previously described and has been attributed to the presence of two fengycins differing by 1 Da in same isotopic cluster . The MS/MS spectra of ions at m/z 1044.5, 1,072.5 also yielded similar peptide sequences with variation of βAA fatty acids chain length. Using LC-MS/MS approach, molecular mass ions at m/z 1,044.5, 1,058.5 and 1,072.5 were identified as iturin C homologues with C14 to C16 βAA fatty acids  were found to be same except b6 and b7 ion. The b6 and b7 ions of m/z 1,065.0 and 1,079.7 varied from each other by 14 Da, which may be attributed to the difference in the chain length of βAA by single –CH2.The precursor ions at m/z 1,081.5 was assigned as [M+K]+ ion of iturin A. To confirm its identity, tandem mass spectrometric analysis was carried out. Figure m/z 1,081.5 with a-, a′-, b-, b′-, y- and y′-fragment ions. Along with b- and y-ions some internal ions were also observed. The ions at m/z 337.6, 464.9, 562.8, 655.4, 677.4, 838.8, 1,046.2 and 1,064.1 could be assigned as [S-N-P+K]+, [S-N-P-Q+K]+, [βAA-S-N-P+K]+, [N-P-Q-N-Y+K]+, [N-βAA-S-N-P+K]+, [Y-N-βAA-S-N-P+K]+, [M+K-CONH2]+ and [M+K-NH3]+, respectively. On the basis of the fragmentation pattern observed, two ring cleavage sites: (1) between Asn6-Ser7 and (2) βAA-Ser7 were predicted. The sequences derived from the fragment ion series corresponding to the predicted ring cleavages were N-P-Q-N-Y-N-βAA-S and βAA-N-Y-N-Q-P-N-S, respectively  and potassium adducts , suggesting that possible site of metal interaction was Glu4 in iturin A homologues (Papayannopoulos 4-Pro5 peptide bond in the sodiated and potassiated iturins. It has been reported that the complexation of iturin homologues with sodium/potassium ions confers the extra-stability to the otherwise highly susceptible bond (X-Pro bond)  and mycosubtilin (C-16) differ in peptide sequence but have same mass  Leu/Ile2-Leu/Ile3-Leu/Ile4-Asp5-Leu/Ile6-Leu/Ile7(OH)+H]+ and [(H)Leu/Ile2-Leu/Ile3-Val4-Asp5-Leu/Ile6-Leu/Ile7(OH)+H]+ with a gain of 18 Da, which is in agreement with observations reported by Hue et al. +H+Na]+, which is equivalent to y4 + H2O. The common peaks at m/z 707.5 and 594.3 were observed in this sodium adduct which could be assigned as internal peptide fragment ions [Leu2-Leu3-Val4-Asp5-Leu6-Leu7(OH)+H+Na]+ and [Leu3-Val4-Asp5-Leu6-Leu7(OH)+H+Na]+, respectively, which are equivalent to y6 and y5-ions with addition of water molecule. The presence of these intense fragment ions in ESI-MS/MS spectra of surfactin is a result of cleavage of peptide bond after double hydrogen transfer +H+K]+ and [Leu3-Val4-Asp5-Leu6-Leu7(OH)+H+K]+, respectively. The ion at m/z 495.3 could be assigned as [Val4-Asp5-Leu6-Leu7(OH)+H+K]+  were assigned as homologues with 13-, 14- and 16-C β-OH FA, respectively while potassium adduct m/z 1,060.7 was assigned as surfactin homologue with 14-C β-OH FA. The heterogeneity in the Val/Leu/Ile at positions 2, 4 and 7 in the peptide sequence of surfactins is well established  for purified iturin A homologues and crude antifungal extract were determined using double dilution method in 96-well microtitre plate. Table n et al. . The ituh et al.  also rep13–C16 β-amino fatty acid variants of iturin A as well as iturin C and C13–C17 β-OH fatty acid isoforms of surfactin exhibiting variation in Val/Ile/Leu at position 4. Seven different variants of iturins as well as of surfactins produced by B. subtilis K1 could be identified by tandem mass spectrometry. The MIC of crude extract was lower than purified iturin fractions, suggesting synergism amongst cyclic lipopeptides co-produced by B. subtilis K1 in inhibiting the growth of fungi. Further, it would be worth exploring the role of these isoforms in biology of producing bacilli strain.The paper describes antifungal activity and in detail mass spectrometric investigation of cyclic lipopeptides such as CSupplementary material 1 (DOCX 613 kb)Below is the link to the electronic supplementary material.


ΔΔΔΔΔΔΔΔ- and ΛΛΛΛΛΛΛΛ-Pd6(RuL3)8 MOCs is accomplished by pre-resolution of the Δ/Λ-Ru-metalloligand precursors. The obtained Pd–Ru bimetallic MOCs feature in large D4-symmetric chiral space imposed by the predetermined Ru(II)-octahedral stereoconfigurations, which are substitutionally inert, stable, water-soluble and are capable of encapsulating a dozen guests per cage. Chiral resolution tests reveal diverse host–guest stereoselectivity towards different chiral molecules, which demonstrate enantioseparation ability for atropisomeric compounds with C2 symmetry. NMR studies indicate a distinctive resolution process depending on guest exchange dynamics, which is differentiable between host–guest diastereomers.Absolute chiral environments are rare in regular polyhedral and prismatic architectures, but are achievable from self-assembly of metal–organic cages/containers (MOCs), which endow us with a promising ability to imitate natural organization systems to accomplish stereochemical recognition, catalysis and separation. Here we report a general assembly approach to homochiral MOCs with robust chemical viability suitable for various practical applications. A stepwise process for assembly of enantiopure  Homochiral molecular capsules offer potential applications in chiral separation and stereospecific catalysis. Here, by pre-resolution of Δ/Λ-metalloligand precursors, the authors are able to assemble enantiopure supramolecular cages capable of stereoselective host-guest behaviour. T-symmetry1415161718O-symmetry or higher were proved to be more formidable because of more possible stereoisomers and the demand to transmit single chirality from more subcomponents202223ΔΔΔΔ- or ΛΛΛΛ-cages based on the stereogenic tris-chelate metal centres15161516The design and synthesis of discrete nanoscale metal–organic cages/containers (MOCs) with specific configurations and cavities applying directional bridging ligands and geometrically prefixed metals is emerging as an appealing topic in recent supramolecular coordination chemistry124567891012D3-symmetric [Ru(bpy)3]2+- or [Ru(phen)3]2+-type compounds, which are widely explored in DNA interactions, asymmetric catalysis and supramolecular chiral assemblies363738393]2+-type metalloligand for homochiral MOC self-assembly14151617181941434445An alternative way to construct stable and robust homochiral MOCs based on the stereogenic metal centres is to design a metalloligand27282930313334Δ/Λ-Pd6(RuL3)8 MOCs racemate , which show the shape of an octahedron (defined by Pd6 centres) or a rhombic dodecahedron (defined by Pd6Ru8 centres)MOC-16 integrating the same handed Δ- or Λ-3 enantiomers to display either ΔΔΔΔΔΔΔΔ or ΛΛΛΛΛΛΛΛ homoconfigurations, indicative of strong cooperative stereochemical coupling between the metal centres1415161718192223Δ- orΛ-MOC-16. However, thus assembled chiral Δ- andΛ-MOCs co-crystallize simultaneously to give racemic products that are not ready for practical applications.We have previously assembled heteronuclear Δ/Λ-[Ru(phen)3]2+ precursors and developed a pair of enantiomeric triangular metalloligands incorporating fixed chiral octahedral Ru(II) centres and pyridyl (Py) terminals ready for assembly of enantiopure Δ- and Λ-Pd6(RuL3)8 MOCs separately. As shown in Δ/Λ-[Ru(phen)3]2+ was first resolved into a pair of enantiomers (Δ- and Λ-1) in good yields using K2[Sb2{(+)-tartrate}2]·3H2O as chiral induction agent, and then oxidized into Δ- and Λ-[Ru(Phendione)3]2+ (Δ- and Λ-2). With the aid of chiral shift reagent Eu((+)tfc)3, the enantiopurity was tested to be 94.8% for Δ-1 and 95.3% for Λ-1 , and, finally, the coordination assembly of Δ- and Λ-3 enantiomers with Pd2+ ions unambiguously resulted in a pair of homochiral Δ- and Λ-Pd6(RuL3)8 cages, namely Δ-MOC-16 and Λ-MOC-16, respectively. 1H NMR spectra of two optically pure Δ-/Λ-MOCs-16 enantiomers give well-resolved proton patterns basically identical to previously reported racemic rac-Δ/Λ-MOCs-16 (1H-1H-COSY and high-resolution mass spectrometry (HR-ESI-TOF-MS) have also been performed to verify formation of Pd6(RuL3)8 cage structures 8 cage with the ΔΔΔΔΔΔΔΔ homoconfigurations 8 cage with the ΛΛΛΛΛΛΛΛ homoconfigurations .Careful examination of the crystal structures of symmetry . If regaD4 group . Furthers cavity  probablyΔ-/Λ-[Ru(phen)3]2+ precursors were well preserved all the way down (Δ-/Λ-[Ru(phen)3]2+ mainly presents three absorption peaks at 225, 265 and 450 nm  transition between Ru2+ and phen ligands. All three absorption bands are reflected in the corresponding CD spectra of the resolved Δ-1 and Λ-1 with the middle peak at 265 nm, giving the most prominent CD signal. Taking Δ-1 as an example, the same tendency of first negative and second positive Cotton effect from longer to shorter wavelength in the three CD bands is in accordance with the Δ-type octahedral chirality established for the Ru2+ coordination centrevice versa for the Λ-1 compound. For the rest three pairs of enantiomers, because of the cutoff effect of the solvents (dimethylsulphoxide (DMSO) or H2O), the CD signals corresponding to the absorption at 225 nm were not fully presented; however, the other bands were clearly detected in the whole synthetic process, preserving the same chirality attributes for the same series of enantiomers . Furthermore, because of the accumulation effect (eightfold in Δ- or Λ-MOC-16 compared with Δ- or Λ-3) of multiple chiral Ru centres in one entity in the final enantiopure Δ-/Λ-MOCs-16, a remarkable increase in CD signal intensities was observed for Δ- or Λ-MOC-16 (Δɛ=∼720 M−1 cm−1) in comparison with Δ- or Λ-3 (Δɛ=∼120 M−1 cm−1). Optical rotation tests also manifested the absolute configurations in Δ- and Λ-MOCs-16 . From these CD studies we see that the stereochemistry of octahedral Ru centres is robust enough to survive all reaction conditions, exactly in agreement with the observations of chirality preservation in crystallographic study. The stereochemical stability of Δ- and Λ-MOCs-16 has also been testified against heating and longtime stay in solution  spectra were also employed to monitor the whole synthetic and assembly processes to confirm that the absolute chirality of the starting way down . In MeCNd 450 nm , with thsolution , confirmΔ-/Λ-MOC-16 cages, we selected two types of racemic organic molecules, one carrying a chiral C* centre and the other characteristic of C2-symmetric chirality  averaged from three parallel experiments  makes it convenient to implement enantioseparation either in a homogeneous two-phase way (Method I: organic-water transfer as shown in Method II: solid-solution transfer as described in Methods). In comparison with the normally insoluble metal–organic frameworks for chiral separation, the water solubility of MOCs-16 offers advantages by using the hydrophobic effect to transfer water-insoluble organic guests into the aqueous phase, and the guest transformation between the organic-water phases is easy to accomplish. Extraction of the resolved chiral guests from the water phase of Δ-/Λ-MOCs-16 readily leads to recovery of the empty cages, which can be reused for the next runs of chiral resolution without a significant loss of the enantioseparation ability as tested by four cycles of R/S-6-Br-BINOL resolution with Δ-MOCs-16 .The resolution results unveil that the homochiral t MOC-16 . However1H NMR titration was performed in an attempt to acquire association constantsMOC-16 cage has a huge molecular size (3.3 × 3.3 nm) and cavity  where a large amount of guests could be hosted 505152Λ-MOC-16 (or Δ-MOC-16) with R- and S-BINOL guests of C2 symmetry undergoes remarkably different host–guest interaction processes, showing distinguishable guest inclusion behaviours for R- and S-BINOL atropisomers as demonstrated in 1H NMR enantiodifferentiation experiments because of the formation of a pair of host–guest diastereomers R-BINOL⊂Λ-MOC-16 and S-BINOL⊂Λ-MOC-16. In contrast, titration of Δ- and Λ-MOC-16 cages with the same C*-chiral S-1-(1-naphthyl)ethanol guest, which should also give a pair of diastereomers S-1-(1-naphthyl)ethanol⊂Δ-MOC-16 and S-1-(1-naphthyl)ethanol⊂Λ-MOC-16, just results in rather similar 1H NMR chemical shift patterns (Λ-MOC-16 (or Δ-MOC-16) cage exhibits the same guest inclusion behaviours for R- and S-enantiomeric guests carrying C* stereocentres.To further understand the host–guest interactions for insight into the resolution mechanism, patterns , indicatR-BINOLs by Λ-MOC-16 at 298 K causes inverse chemical shifts of cage protons, with those inside the cage moving upfield while those outside the cage moving downfield . It is notable that the guest signals display a continuously downfield shift, characteristic of fast guest exchange. These NMR observations imply more dynamic host–guest interactions for S-BINOL⊂Λ-MOC-16 inclusion compared with R-BINOL⊂Λ-MOC-16 inclusion at the room temperature. Broadening of H resonance is indicative of slow and restricted molecular rotation and tumbling54S-BINOL⊂Λ-MOC-16 system, faster guest exchange dynamics may present, showing averaged influence on host protons either inside or outside. When the cage cavity is getting fulfilled (∼12 guest per cage), the overall host–guest dynamics is slowed down to make NMR unable to discriminate resonating frequency. In contrast, guest exchange in the R-BINOL⊂Λ-MOC-16 system is slow enough at room temperature, thus showing distinguishable impact on host protons inside and outside. Such a guest dynamic difference between two host–guest diastereomers may account for the intrinsic factor that determines the enantioseparation ability of homochiral Δ- or Λ-MOCs-16 towards racemic R/S-BINOLs. On the contrary, titration of Δ- and Λ-MOCs-16 with S-1-(1-naphthyl)ethanol guests shows a similar guest exchange dynamics 3]2+ precursors, giving rise to the assembly of enantiopure D4-symmetric Δ- and Λ-MOC-16 cages separately, which feature in high guest inclusion capacity and substantial stereochemical stability. The single-crystal diffraction analyses of the individual Δ- and Λ-MOC-16 cages verified the formation of absolute ΔΔΔΔΔΔΔΔ- and ΛΛΛΛΛΛΛΛ homoconfigurations, respectively, in corresponding Pd6(RuL3)8 cages, and the crystallization of optically pure cage products. The stereoselective inclusion of chiral molecules has been tested for two kinds of organic racemates, that is, classic chiral compounds having C* centres and atropisomeric compounds characteristic of C2 symmetry, with the phase transformation resolution processes. Successful enantioseparation of atropisomers has been accomplished by the use of these homochiral Δ- and Λ-MOCs-16, manifesting an unprecedented dynamic resolution process based on the kinetically driven guest exchange. The possible resolution mechanism has been investigated by the means of 1H NMR titration, 1H NMR enantiodifferentiation experiments as well as variable-temperature 1H NMR study. In general, this kind of assembly process may provide a new platform to study the stereochemical transmission of optically stable metal centres to versatile homochiral entities in coordination chemistry, and the dynamic resolution behaviour imposed by stereoconfiguration of metal centres might be useful in various chiral resolution of synthetic and industrial significance.In conclusion, pre-resolution of a pair of enantomeric Unless otherwise stated, all commercial reagents and solvents were used as commercially purchased without additional purification. The NMR spectra were recorded on Bruker AVANCE III 400 (400 MHz). Circular dichroism spectra and ultraviolet–visible absorption spectra were measured with a JASCO J-810 spectropolarimeter. Specific rotations were recorded on ADP440+B+S. HR-ESI-TOF mass spectra were tested on Bruker Maxis 4G, and data analyses were processed with the Bruker Data Analysis software. HPLC spectra were measured on Agilent-2000. Diffraction data for the single crystals were collected on an Agilent SuperNova X-ray diffractometer using micro-focus dual X-ray sources . SynthesI422 space group, a=32.2284(4) Å, c=38.2801(7) Å, V=39,760.5(13) Å3, Mr=9,727.82, Dx=0.813 g cm−3, Z=2, μ=2.599 mm−1, 16,396 independent reflections, of which 9,021 observed (I>2σ(I)), R1=0.0711, wR2=0.2375, S=1.008, Flack parameter=0.131(13).I422 space group, a=32.5722(6) Å, c=38.7258(9) Å, V=41,086.1(19) Å3, Mr=9,727.82, Dx=0.786 g cm−3, Z=2, μ=2.515 mm−1, 17,111 independent reflections, of which 7,718 observed (I>2σ(I)), R1=0.0913, wR2=0.2680, S=1.038, Flack parameter=0.139(16).P41 space group, a=25.5619(2) Å, c=12.5769(2) Å, V=8,217.88(18) Å3, Mr=2,037.48, Dx=1.647 g cm−3, Z=4, μ=0.556 mm−1, 19,736 independent reflections, of which 17,221 observed (I>2σ(I)), R1=0.0678, wR2=0.1759, S=1.050, Flack parameter=0.00(4).P43 space group, a=25.5802(1) Å, c=12.5709(1) Å, V=8,225.73(9) Å3, Mr=2,037.48, Dx=1.645 g cm−3, Z=4, μ=0.556 mm−1, 20,142 independent reflections, of which 17,755 observed (I>2σ(I)), R1=0.0408, wR2=0.1071, S=1.025, Flack parameter=−0.033(8).P212121 space group, a=13.8114(2) Å, b=14.0525(2) Å, c=20.7957(3) Å, V=4,036.13(10) Å3, Mr=1,028.64, Dx=1.693 g cm−3, Z=4, μ=5.107 mm−1, 7,890 independent reflections, of which 7,536 observed (I>2σ(I)), R1=0.0438, wR2=0.1195, S=1.025, Flack parameter=−0.015(4).P212121 space group, a=13.7734(2) Å, b=14.0148(2) Å, c=20.7100(3) Å, V=3,997.68(10) Å3, Mr=1,022.59, Dx=1.699 g cm−3, Z=4, μ=5.156 mm−1, 7,980 independent reflections, of which 7,737 observed (I>2σ(I)), R1=0.0409, wR2=0.1084, S=1.032, Flack parameter=−0.011(3).R/S-BINOL, R/S-3-Br-BINOL, R/S-6-Br-BINOL and R/S-naproxen molecules, Method I based on a solution–solution transfer was applied to avoid host–guest precipitation ethanol and R/S-benzoin molecules, Method II based on a solid-solution transfer was applied directly. The powder of guest racemate was suspended in the aqueous solution of Δ-or Λ-MOC-16. The mixture was stirred vigorously at room temperature. After centrifugation, the filtrate was collected and extracted with CHCl3. The extractants were combined and the solvent was removed by rotary evaporator to afford white solid as resolved guest. The ee analysis is the same as in Method I.Two kinds of methods were used to resolve racemic guests depending on whether the guest inclusion leads to precipitation. For racemic pitation . An aqueAccession codes: The X-ray crystallographic coordinates for structures reported in this Article have been deposited at the Cambridge Crystallographic Data Centre (CCDC), under deposition number CCDC 1432349–1432354. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.How to cite this article: Wu, K. et al. Homochiral D4-symmetric metal–organic cages from stereogenic Ru(II) metalloligands for effective enantioseparation of atropisomeric molecules. Nat. Commun. 7:10487 doi: 10.1038/ncomms10487 (2016).Supplementary Figures 1-29, Supplementary Tables 1-9 and Supplementary MethodsCrystallographic Information Files


In the crystal, molecules are linked via C—H⋯O and C—H⋯N hydrogen bonds, forming layers parallel to (101).The title compound, C For example, Le Corre et al.  inhibitory activities -one moiety (N1–N3/C1–C10), are essentially planar (r.m.s. deviation = 0.023 Å), with the maximum deviation from the mean plane being 0.036 (2) Å for atom C8. The nitrobenzene ring (C12–C17) is inclined to this mean plane by 6.8 (1)°, while the nitro group (N4/O2/O3) is inclined to the benzene ring by 15.0 (3)°.In the molecular structure of the title compound Fig. 1, the thrvia C—H⋯O and C—H⋯N hydrogen bonds, forming layers lying parallel to (101); see Fig. 2ca 3.4 Å, but there are no significant interlayer interactions present -one), no hits were obtained. For substructure S2 , seven hits were found. Two of these compounds have substructure S3 -one), viz 9-fluoropyridopyrimidoindole-5,11-dione  and p-nitrobenzaldehyde  was added acetic acid . This mixture was refluxed in an oil bath (ca. 423-433 K) for 5 h after which it was left to stand for 24 h. During this time a yellow precipitate formed. It was filtered and washed with distilled water, giving yellow crystals of the title compound . Yellow block-like crystals suitable for X-ray analysis were grown from a solution of ethanol:water (2:1) by slow evaporation at room temperature. The title product is insoluble in benzene, chloroform, acetic acid, acetone, DMF, and DMSO, but soluble in trifluoroacetic acid. 1H NMR : 3.15 , 4.16 , 7.44 , 7.60 , 7.83 , 7.98 , 8.63 , 9.00 . Rf = 0.47 .To a mixture of 2,3-trimethylenepyrido[2,3-Uiso(H) = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989016003583/su5283sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989016003583/su5283Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989016003583/su5283Isup3.cmlSupporting information file. DOI: 1456732CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In the crystal, molecules are linked  21H16N2O, has an almost planar (r.m.s. deviation = 0.0074 Å) 1,2-dibenzylidenehydrazine backbone with an approximately orthogonal almost planar (r.m.s. deviation = 0.0368 Å) phenylethanone substituent on one of the imine C atoms. The dihedral angle between the two mean planes is 76.99 (4)°. In the crystal, molecules are linked via C—H⋯O hydrogen bonds and C—H⋯π contacts, forming a three-dimensional structure with molecules stacked along the a-axis direction.The title compound, C As a result of their fascinating physical and chemical properties, azines and their derivatives have been utilized extensively in areas such as dyes -2-[(E)-1-(2-hydroxyphenyl)methylidene]hydrazono}-1,2-diphenylethan-1-one benzylidenehydrazono]ethan-1-one, with a pyridyl ring in the same position -1,2-diphenylethanone skeleton in the Cambridge Structural Database : C 80.75, H 5.16, N 8.97%; found: C 80.73, H 5.17, N 9.01%.A mixture of benzaldehyde , benzil  and hydrazine hydrate  in 50 ml of ethanol containing 2 drops of acetic acid was refluxed for about 2 h. The reaction was monitored by TLC until completion. Excess solvent was evaporated under vacuum and the resulting yellow solid product was recrystallized from absolute ethanol to afford yellow needles of the title compound . Analysis calculated for CUiso = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989014026358/su5029sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989014026358/su5029Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989014026358/su5029Isup3.cmlSupporting information file. DOI: 1036846CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: