New optically pure helicate-like architectures are extremely active against cancer cell lines, with IC50 values as low as 40 nM, but nearly three orders of magnitude less active against healthy cells. There is also low toxicity to microbes and amoeba. via highly diastereoselective self-assembly using readily accessible starting materials. Six pairs of enantiomers [Fe2L3]Cl4·nH2O (L = various bidentate ditopic ligands NN–NN) show very good water solubility and stability. Their activity against a range of cancer cell lines in vitro is structure-dependent and gives IC50 values as low as 40 nM. In an isogenic pair of HCT116 colorectal cancer cells, preferential activity was observed against cell lines that lack functional p53. Selectivity is also excellent, and against healthy human retinal pigment epithelial (ARPE19) and lung fibroblast (WI38) cells IC50 values are nearly three orders of magnitude higher. Cisplatin is unselective in the same tests. The compounds also appear to have low general toxicity in a number of models: there is little if any antimicrobial activity against methicillin-resistant Staphylococcus aureus and Escherichia coli; Acanthamoeba polyphaga is unaffected at 25 μg mL–1 (12.5 μM); Manduca sexta larvae showed clear evidence of systemic distribution of the drug, and rather than any observation of adverse effects they exhibited a significant mean weight gain vs. controls. Investigation of the mode of action revealed no significant interaction of the molecules with DNA, and stimulation of substantial cell death by apoptosis.A range of new helicate-like architectures have been prepared  This coincides with the resurgence of phenotypic drug discovery,7 where the targets of a drug are established after the observation of the useful biological effect. This strategy has led to a disproportionately high number of first-in-class drugs with novel mechanisms of action (1999–2008)9 The accompanying challenge for synthetic chemistry is to discover, perhaps without reference to some specific biomolecular target, new classes of drug candidates which are both potent and selective.The main purpose of current anticancer therapies is to eradicate tumour cells without damaging overall patient health. However, side effects limit the dosage of chemotherapeutic drugs which may be safely applied, and as a result, cancer cells often remain. This leads to poor outcomes in the clinic and the evolution of drug-resistant tumours.10 and this was borne out in early studies, particularly in the area of cancer.15 We have argued,16 however, that in order for helicates to be capable of translation to the clinic a number of criteria need to be addressed: optical purity and stability, solubility and chemical stability in water, availability on a practical scale, and synthetic diversity. Our recent work has attempted to address these matters17 using a new strategy whereby the absolute configurations of individual metal centres are controlled18 and linked together to form the prototype helicate-like architectures of 19 [Fe21L3]4+ contains a diamine linker while [Fe22aL3]4+ is based on a dialdehyde.19 Promising results were reported in a number of disease areas,21 including good activity against a range of cancer cell lines.20 Here we report the discovery of a new series of highly potent (40 nM) anticancer compounds of the dialdehyde class related to [Fe22aL3]4+ that preferentially kill cancer cells that lack functional p53, are nearly three orders of magnitude less toxic to healthy human cell lines tested and have low toxicity to microbes, amoeba and caterpillar larvae.Lehn recognized the potential of helicates in medicinal chemistry,While DNA does not appear to be the target, the compounds are triggering significant apoptotic cell death as part of their mode of action.via simple etherifications of 5-hydoxypicolinaldehyde.22The dialdehyde units of 4)2·6H2O and (R)-1-phenylethan-1-amine, in appropriate proportions, led to the rapid self-assembly of the bimetallic flexicates in acetonitrile solution at ambient temperature. For the majority of these new ZnII complexes NMR spectra indicated that within the limits of the experiment single diastereomers were formed (vide infra).Treatment with Zn behaved in a corresponding manner . These observations are consistent with the presence of two species – one of high-symmetry and one low – in thermodynamic equilibrium  but with the involvement of other related conformers particularly at higher temperatures. The processes leading to the observed NMR behaviour may correspond to exchange between these conformers, or indeed between isostructural low symmetry species. While the spectra are not sufficiently well resolved to determine kinetic parameters, we sought to investigate this molecular system by computational means.The sole exception was the 1,3-phenylene system Λ spectra . At 253 Zn-[Zn22eL3]4+ were located and minimised Cl4 (n = 2b–2e) were synthesised in high yield by heating the appropriate dialdehyde linker with either (R)- or (S)-1-phenylethan-1-amine and FeCl2 in methanol. 1H-NMR spectra were similar though slightly broader than the analogous ZnII perchlorate complexes and are consistent, along with 13C-NMR [22eL3]Cl4 exists as a similar mixture of conformers to the Zn analogue above. The complexes gave excellent electrospray mass spectrometry data e.g. ΛFe-[Fe22cL3]Cl4 gave a strong peak at m/z 420.17 Da for the tetracationic ion. The formula weights of the panel of complexes, including levels of hydration, were determined by correlation of NMR, IR, thermogravimetric and elemental analyses  π-stacking.23Absorbance spectra indicated that little decomposition of the flexicates occurred in water at pH 7 over weeks, but half-lives for decomposition could readily be recorded in hydrochloric acid (0.2 M) 24 (b) HCT116 p53+/+ and (c) HCT116 p53–/–.25 The HCT116 p53+/+ and HCT116 p53–/– cancer cells are human colorectal cancer cell lines that are genetically identical (isogenic) except for the presence or absence of functional p53.25 These were chosen to enable screening of the effects of p53 status as the loss of p53 function is common genetic event in patient tumours and is strongly associated with increased resistance to many conventional chemotherapeutic agents.26 In the cisplatin-sensitive (2.5 ± 0.5 μM) MDA-MB-468 cells, the new flexicates showed a range of activities [22bL3]Cl4 being very potent (0.2 ± 0.1 μM); an order of magnitude more so than cisplatin. Against HCT116 p53+/+ cells [e.g. [Fe22eL3]Cl4 (0.4 ± 0.1 μM), and some significant enantiomeric differences were observed. Against HCT116 p53–/– cells, cisplatin showed less activity (8.1 ± 1.8 μM) than for HCT116 p53+/+ cells (3.5 ± 1.5 μM) consistent with the increased resistance of cancer cells lacking p53 to many standard chemotherapeutic agents, while some of the flexicates were extremely active (2a annd 2c) with IC50 values in the nanomolar range e.g. ΔFe-[Fe22cL3]Cl4 (40 ± 3 nM) [Fe-[Fe22cL3]Cl4 and ΔFe-[Fe22cL3]Cl4 were both ∼9-fold more active against HCT116 p53–/– cancer cells than their genetically identical p53+/+ counterparts Cl4 SI = 836 ± 280. This excellent selectivity prompted us to investigate the toxicity of the compounds against a number of organisms.The most active flexicates in HCT116 p53s (WI38) . These aStaphylococcus aureus, USA300 JE2 .30 Kanamycin30 was used as a positive control.The compounds were screened against cultures of the gram-positive bacterium methicillin-resistant A300 JE2  (MRSA) a50 values observed in cancer cells.The new flexicates had very modest antimicrobial activity  or did nAcanthamoeba polyphaga. Concentrations of 2.5 μg mL–1 (1.25 μM) and 25 μg mL–1 (12.5 μM) showed no adverse effect on the amoeba development . Also we noted that larvae that ingested the flexicate solutions turned a bright purple colour over the course of the assay, suggesting that these compounds were persisting in the insect and not being rapidly metabolized or excreted.Oral and systemic toxicity of these flexicates was assessed in neonate larvae of ehaviour . Interesth instar larvae (n = 3). The cohorts were then allowed to continue feeding. Despite becoming purple, all larvae proceeded to develop into the pupal diapause stage as per the buffer control injections.Systemic toxicity was further tested by injection of 50 μg (0.25 μM) of the compounds directly into the hemocoel of cohorts of 5The mode or modes of action of such a new and different system will require intensive investigation and is likely to involve multiple targets and pathways. Here, we describe two preliminary studies towards this end.33 We investigated the effect that the new flexicates had on the denaturation temperature (Tm) of ct-DNA to screen for any indications of DNA binding.We have previously concluded that the induction of DNA damage is not involved in the mode of action of earlier flexicates, despite particular examples binding in a cell free environment.–1) was mixed with each flexicate (7.5 μM) in buffered conditions , to give 10 bases: 1 flexicate complex, and the absorbance at 260 nm between 25 °C and 90 °C was recorded (0.4 °C min–1). Tm for each experiment was calculated from the first derivative of a Boltzmann sigmoidal fit of the plot of absorbance versus temperature.Isolated ct-DNA  was measured to be 68.3 ± 0.5 °C. Most of the new flexicates had no significant effect on the denaturation of ct-DNA (T ca. 1 °C) reduction for 2eL enantiomers can be ascribed to an electrostatic effect.34 We are therefore satisfied that DNA is unlikely to be the target of this panel of compounds.f ct-DNA ; the sma35 One of the hallmarks of cancers is the evasion of apoptosis, thus enabling the long-term survival and proliferation of cancer cells.36 We thus investigated whether the most active flexicates are stimulating apoptotic death in cancer cells as part their mode of action.The chemosensitivity observed could be due to cytostatic or cytotoxic effects, and cell death can occur by several different mechanisms. These include programmed cell death by apoptosis, inflammatory necrosis, autophagy or ‘self-eating’, necroptosis and pyroptosis.+/+ cancer cells (24 h post-seeding) were incubated in fresh media containing flexicate or no flexicate (control) and were then analysed after 48 h for levels of apoptosis and necrosis. As cells start to undergo apoptosis, one of the first cellular changes is the externalisation of the membrane protein phosphatidylserine (PS). This can be detected and quantified by fluorescently labelled annexin V38 which can selectively bind externally exposed PS but is membrane-impermeable. This enables cells in the early stages of apoptosis to be distinguished from necrotic cells and cells in the late stages of apoptosis both of which have lost membrane integrity and will therefore also stain with the membrane-impermeable DNA stain propidium idodide.39HCT116 p53Fe-[Fe22aL3]Cl4 and ΔFe-[Fe22aL3]Cl4 were tested and both induced significant levels of apoptosis that were ∼2.6 fold (ΛFe-[Fe22aL3]Cl4) and ∼4.4 fold (ΔFe-[Fe22aL3]Cl4) above background control levels in the HCT116 cancer cells at 48h (Flexicates Λs at 48h . A signis at 48h . These pE)-5,5′-)dipicolinaldehyde  and (R)-1-phenylethan-1-amine  were dissolved in acetonitrile (10 mL) with ZnII perchlorate hexahydrate  and the solution was stirred at ambient temperature for 20 h. Ethyl acetate was added drop-wise to cause precipitation of a white crystalline solid, ΛZn-[Zn22cL3][ClO4]4·10H2O. Yield 0.214 g, 57%. 1H NMR  δH 8.06 , 7.49 , 7.36 , 7.14 , 7.09 , 6.95 , 6.64 , 6.12 , 5.38 , 4.64 , 1.61 . 13C{1H} NMR  δC 161.8 (CHN), 159.6, 142.0, 139.9, 139.4, 132.3 (Ar), 129.7 (CH), 129.4, 128.5, 126.4, 122.7 (Ar), 69.7 (CH2), 64.7 (CH), 23.6 (CH3). MS (ESI) m/z 411 [Zn2L3]4+. IR ν cm–1 2976 w, 1570 m, 1316 m, 1225 m, 1082 s, 762 w, 703 m, 653 m. Elemental analysis found  % C 51.08 (51.88), H 4.82 (5.26), N 7.38 (7.56).(E)-5,5′-)dipicolinaldehyde  and (R)-1-phenylethan-1-amine  were dissolved in methanol with FeII chloride . The solution was stirred at reflux (75 °C) for 48 h and all volatiles were removed under reduced pressure to yield a dark purple solid, ΛFe-[Fe22cL3]Cl4·9H2O. Yield 0.388 g, 97%. 1H NMR  δH 8.80 , 7.47 , 7.13 , 7.04 , 6.64 , 6.44 , 6.05 , 5.26 , 4.65 , 4.59 , 1.99 . 13C{1H} NMR  δC 171.0 (CHN), 158.8, 152.5, 144.8, 141.7, 131.2, 130.1, 129.8, 128.5, 125.5 (Ar), 121.0 (CH), 70.0 (CH2), 69.6 (CH), 26.1 (CH3). MS (ESI) m/z 406 [Fe2L3]4+, 505 [L + H].IR ν cm–1 3352 br s, 2970 br s, 1589 s, 1557 w, 1488 w, 1381 m, 1299 s, 1067 m, 1028 m, 760 m, 699 s, 562 w. Elemental analysis found  % C 59.92 (59.76), H 5.84 (5.96), N 8.63 (8.71).40 functional and the 6–31g* basis set, with convergence criteria of 0.0001 a.u. as implemented in the Firefly quantum chemistry package,41 which is partially based on the GAMESS(US) source code.42 Bimetallic systems were optimised using ligand field molecular mechanics (LFMM)43 as implemented in the DommiMOE program,44 before being annealed at 500 K for 1 ns prior to re-optimisation. Single point energy calculations of all structures were performed using the B3LYP-D3(BJ)40 functional and the deff2-TZVP basis set with energy convergence criteria of 0.0001 a.u. as implemented in the Firefly quantum chemistry package.41 The calculations were conducted by employing the RIJCOSX approximation with SCF convergence criteria set to ‘tight’, both of which are defined internally as part of the ORCA DFT quantum chemistry package.45 Where relevant, acetonitrile solvate correction was performed using the conductor-like screening model (COSMO)46 as implemented in ORCA.45Models of a number of possible conformers of Λ–1, 64 μM) complex in MH broth, diluted 2n μg mL–1, inoculated with each bacterial strain (103 cfu mL–1) were sealed and growth was monitored over 20 h at 37 °C with an iEMS 96-well plate reader  broth. 96-well plates  for 24 h at 37 °C, 5% CO2 prior to drug exposure. Compounds were added (100 μM to 5 nM in cell medium) for a further 96 h. 3--2,5-diphenyl tetrazolium bromide solution  was added for a final 4 h. Upon completion all solutions were aspirated, dimethyl sulfoxide (150 μl) was added and absorbance (540 nm) was recorded with a Thermo Scientific Multiskan EX microplate photometer.ICManduca sexta31 one-day-old neonate larvae with each flexicate  for 7 d at 28 °C and weighing to assess growth rate. Systemic toxicity assays47 were conducted by injecting an ethanol (70% v/v) swabbed region of first day fifth instar M. sexta larvae with each flexicate (0.5 mg mL–1 [0.25 μM] in PBS), before allowing them to continue feeding for 7 d at 28 °C, using physical stimulus to assess their status.Oral toxicity was established by feeding cohorts of –1, 7.5 × 10–5 per base, as determined by absorbance at 200 nm) with each complex (7.5 μM) in buffered conditions  to give 10 base: 1 complex. The absorbance at 260 nm as a function of temperature  was measured in a 1 cm masked quartz cuvette at a rate of 0.4 °C min–1 and run in triplicate. Tm was calculated from the first derivative of a Boltzmann sigmoidal fit of the plot of absorbance at 260 nm against temperature for each complex.Denaturation of ct-DNA was measured by mixing ct-DNA  for 24 h at 37 °C in 5% CO2, before treating with each flexicate (20 μM in fresh complete media for 48 h) or fresh media containing no drug (control). The supernatant containing any non-adhered, floating cells was then collected and pooled with cells harvested by trypsinisation. This pooled single cell suspension was washed twice with PBS and incubated with propidium iodide and Annexin-V-FLUOS (Roche) to stain apoptotic cells in accordance with the manufacturer's instructions. The proportion of early stage apoptotic cells and late stage apoptotic/necrotic cells were then quantified by flow cytometry as previously described.38Induction of apoptosis was determined by incubating HCT116 p5348 In contrast, in a conventional “helication” approach the bridging units are structure-determining, so a mechanism of stereoselection would need to be designed for each example. A further advantage of the flexicate platform is beginning to emerge in that we may be able to develop asymmetric molecules from symmetric ligands via the kinds of conformational abnormalities caused by bridges that partially oppose the predetermined stereochemistry e.g.2eL. We have already shown that asymmetric  optically pure assemblies are available using directional ligands.49 Further, this modular self-assembling system will allow us to probe the effects of peripheral functionality and lipophilicity.Our approach to metallohelix assembly has allowed us to generate a panel of biologically-compatible enantiomers incorporating various bridging groups. This was possible because in this so-called flexicate platform the stereochemistry of the metal complex units is predetermined very efficiently and largely independently of the bridges, and by a mechanism that also provides water-compatibility.Fe-[Fe22cL3]Cl4 shows a selectivity index  approaching 103, demonstrating superiority over the clinically used anticancer drug cisplatin in vitro (SI < 1). This selectivity is substantiated in tests with various models; bacteria and amoeba exposed to high concentrations were essentially unaffected, and in Manduca sexta larvae, where the systemic stability of the drug is evidenced, there is arguably a pro-biotic effect i.e. the insects appear to thrive.The activity of these new assemblies against cancer cells is strongly dependent on structure, with a range of potencies from 30 μM to as low as 40 nM. The most active compound Δ19 ([Fe21L3]4+, 33 there is no DNA damage akin to that induced by e.g. platinum drugs and alkylators.49 Instead, relevant examples of protein interaction and enzyme inhibition have been characterised.32 To achieve drug safety and cancer selectivity, mechanistic classes which do not involve induction of DNA damage are attractive, and this may well be the source of the excellent selectivities we describe in this manuscript. Mode of action studies indicate that these compounds can induce substantial cell death by apoptosis independent of any DNA damage. Extensive studies are now required to understand how this complex process, normally subverted in cancers, is induced by these compounds. The above observations of remarkable selectivity alongside very high potency and large enantiomeric differences are however all consistent with a subtle mechanism involving the targeting of oncogenic drivers.In respect of mechanism or mode of action, the lack of binding to DNA indicates that this is unlikely to be the general target in this panel. In fact only one early flexicate


Leishmania donovani, the causative agent of Indian visceral leishmaniasis has to face several barriers of the immune system inside the mammalian host for its survival. The complement system is one of the first barriers and consists of a well-balanced network of proteases including S1A family serine proteases (SPs). Inhibitor of serine peptidases (ISPs) is considered as inhibitor of S1A family serine peptidases and is reported to be present in trypanosomes, including Leishmania. In our previous study, we have deciphered the role of ISPs [LdISP1 and L. donovani inhibitor of serine peptidases 2 (LdISP2)] in the survival of L. donovani inside the sandfly midgut. However, the role of theses ISPs in the survival of L. donovani inside mammalian host still remains elusive. In the present study, we have deciphered the inhibitory effect of LdISPs on the host complement S1A serine peptidases, such as C1r/C1s and MASP1/MASP2. Our study suggested that although both rLdISP1 and rLdISP2 inferred strong interaction with C1complex and MBL-associated serine proteases (MASPs) but rLdISP2 showed the stronger inhibitory effect on MASP2 than rLdISP1. Moreover, we found that rLdISP2 significantly reduces the formation of C3, C5 convertase, and membrane attacking complex (MAC) by lectin pathway (LP) resulting in significant reduction in serum mediated lysis of the parasites. The role of LdISP2 on neutrophil elastase-mediated C5aR signaling was also evaluated. Notably, our results showed that infection of macrophages with ISP2-overexpressed Leishmania parasites significantly induces the expression of C5aR both at the transcript and translational level. Simultaneously, infection with ISP2KD parasites results in downregulation of host PI3K/AKT phosphorylation and increased in IL-12 production. Taken together, our findings clearly suggest that LdISP2 promotes parasite survival inside host by inhibiting MAC formation and complement-mediated lysis via LP and by upregulation of C5aR signaling. Leishmania sp., and transmitted to the human or other mammalian hosts by sandfly vector, affecting ~0.7 million of people worldwide  and AP (C3bBb3b),  . However3bBb3b), . C5c cle3bBb3b), .Leishmania uses different strategies/molecules to evade the host complement attack for its successful invasion of the host (Trypanosoma cruzi (T. cruzi) inhibits activation of classical and LP  and complement S1A peptidases has yet not been elucidated. The LdISPs might inhibit the SP of the complement channel and help the parasite to survive inside the host.The presence of the inhibitor of serine peptidases (ISPs) has been reported to be present in donovani  recentlydonovani , the pepdonovani . It has on of NE . Van denon of NE . HoweverL. donovani inhibitor of serine peptidases 2 (LdISP2) inhibits MASP2 activity leading to the reduction in MAC formation and parasite lysis by LP. Further, we have assessed the role of LdISP2 on NE and C5aR-mediated PI3K/AKT signaling. Our study suggested that LdISP2 promotes parasite survival inside host by inhibiting MAC formation and complement-mediated lysis via LP and by upregulation of C5aR signaling.In the present study, we have observed the inhibitory effect of LdISPs on the host complement serine peptidases C1r, C1s, MASPs (MASP1/MASP2) and its affector cross road complement molecule, i.e., C3c and C5c formation. We have found that Saccharomyces cerevisiae, penicillin G-sodium, streptomycin, human C4 molecule, anti-human C3b antibody, anti-human C4b antibody, NE from human leukocytes, substrate -N-α-Cbz-l-lysine thiobenzyl ester (Z-l-Lys-SBzl), VPR–7-amino-4 methyl coumarin, N-methoxy succinyl–Ala–Ala–Pro–Val-7-amino-4-methyl curamin, and DTNB  were purchased from Sigma-Aldrich (USA). Ficoll-Plaque Plus was purchased from GE Healthcare . Human IgM and C1 complex were purchased from Merk-Millipore (US). M199 media and fetal bovine serum (FBS) were purchased from Invitrogen (USA) and Gibco (USA), respectively. C3c and C5c ELISA kits were purchased from Life Science, Inc. (USCN). Antibody against human C5aR, C5b-C9, PI3K, AKT, p-AKT, and p-PI3K were purchased from Santa Cruz Biotechnology (USA). Complement C1q antibody, anti-human MBL antibody, Factor B antibody against human were purchased from Abcam . Secondary antibodies (HRP conjugated) were purchased from Jackson Laboratory (USA).THP1 cells, a human monocytic macrophage-like cell line was obtained from National Centre for Cell Science . Mannan from L. donovani promastigotes (AG83:MHOM/IN/1983/AG83) were used in our experiments. ISP2KD and ISP2 overexpressed (ISP2OE) Leishmania parasites were prepared in our lab as described previously (2 flasks in fresh Medium-199 supplemented with 10% FBS. The ISP2KD and ISP2OE cells were maintained in M199 media supplemented with neomycin (G418) at 200 µg/ml concentration , streptomycin (100 mg/ml), and 10% (v/v) FBS. The cells were maintained in 5% CO2 incubator at 37°C in tissue culture flask . The cells were subcultured at every 3–4 days to maintain the confluency of cells. For experimental infection, THP1cells (~106) were treated with 20 nM phorbol 12-myristate 13-acetate (PMA)  and incubated for 24 h to become adherent and matured. Prior to infection, cells were washed with RPMI media without FBS to remove non-adherent cells.THP1 cells, a human monocytic macrophage-like cell line was cultured in RPMI-1640 medium containing 10 mM HEPES, 1 mM sodium pyruvate supplemented with 4 mM NaHCO6 cells) in RPMI medium with 10% FBS in the presence of macrophage colony-stimulating factor (300 ng/ml) according to Mandal et al.  in coating buffer . Purified C1 complex (0–100 µg/ml) was diluted in PBS/1% w/v bovine serum albumin (BSA)/0.05% tween 20, whereas normal human serum (NHS) was diluted in veronal buffer saline (VBS)  in different dilution . Different concentration of C1 complex and serum was added to the antibody-coated plates or mannan-coated plates, respectively, incubate at 37°C for 2 h. Washing was performed throughout the assay with washing buffer, TBS (10 mM of NaCl)/0.05% Tween. Subsequently, after washing different concentration (0–50 µM) of rLdISP1 and rLdISP2 were added to the coated plates and incubated at 37°C for 2 h. Bound rLdISP1 and rLdISP2 was detected with the anti-ISP1 or anti-ISP2 antibody (1:500 dilutions) followed by addition of HRP conjugated anti-goat IgG-Ab and TMB substrate .ELISA was performed to study the interaction of rLdISP1 and rLdISP2 with C1 complex and MASPs according to Ferreira et al.  with min2, pH 7.4) and incubated for 2 h at 37°C. C1 and serum bound wells were washed with washing buffer according to Presanis et al. (l-lysine thiobenzyl ester (Z-l-Lys-SBzl) and DTNB  according to Keizer et al.  was bound to the antibody-coated plate as discussed previously. Mannan-coated wells were bound with 100 µl of serum (1:10 v/v) diluted in serum dilution buffer  was added to treated and untreated NE. Enzymatic hydrolysis of the substrate was monitored by spectrofluorometer by measuring the release of fluorescence . All the experiments were performed in triplicate, and the data expressed as means ± SD from three independent experiments.Additionally, the enzymatic assay was performed according to Morrisons et al.  to see t+ buffer . NHS pretreated with rLdISP1/rLdISP2 (0–50 µM) at 4°C for 1 h, or untreated NHS was added to the mannan-coated plates and incubated for 2 h at 37°C. The ELISA plate bound with serum-derived MASPs was used as a control. NHS pre-incubated with C1-inh (10 nM), the known inhibitor of classical and LP was used as the control inhibitor. C4 molecule (0.2 µg/ml) containing 20 mM HEPES, 140 mM NaCl, 5 mM CaCl2, pH 7.4, was added to the serum bound plate and incubated at 37°C for 2 h. Simultaneously, another plate was coated with C4b antibody (diluted in coating buffer) and incubated for 2 h. The cleaved C4 product was added to C4b antibody bound plate and further incubated for 2 h. The C4b antibody was added to the plate and incubated for another 2 h. The plates were washed in each step with TBS/0.05% Tween/5 mM of CaCl2, according to Presanis et al.  in modified veronal buffer . To determine the contribution of LP in C3c and C5c formation, lectin pathway-specific serum (LPSS) was prepared by incubating NHS (10% v/v) with “C1q antibody + Factor B antibody” , respectively, kept at 37°C for 2 h. The appropriate dilution of the antibody used in the experiment was standardized experimentally.Functional convertase assay was performed to monitor the C3c and C5c formation ntibody” . To blocLeishmania promastigotes (~2 × 106) were processed according to Okroj et al.  at 37°C for 2 h. Untreated NHS and LPSS were used as a control. j et al.  for the Leishmania parasites was detected by the deposition of C3b molecule on parasite surface and formation of MAC (Detail in SI) . Two experimental set up was made, in which an equal number of Leishmania promastigotes (~1 × 107) were taken. In one set, washed parasites were fixed in 4% paraformaldehyde and coated in ELISA plate for overnight at 4°C /0.05% tween 20 (3 times) then blocked with 3% BSA in PBS for 2 h at room temperature. For LP activation, different group of LPSS were added into the well and incubate for 1 h. Active formation of MAC via each group of serum was detected by using anti-human C5b-9 complex antibody , followed by incubation with HRP conjugated secondary antibody . In another set, parasites were either treated with heat inactive LPSS, active LPSS, LPSS pretreated with C1-inh (10 nM), LPSS pretreated with different concentration of rLdISP2 (5–50 µM). The treated parasites were used to analyze the complement-mediated lysis by counting the number of viable parasites in hemocytometer by trypan blue method. The parasites were stained with Giemsa for the microscopic examination of the different group of serum treated parasites.The comparative study of the complement pathway activation by l in SI) , 42. Addt at 4°C . The wel6) were used for two experimental setups. In one set, the adhered THP1 cells and hMDM were infected with either wild-type (WT), ISP2KD, or ISP2OE Leishmania parasites at parasite/macrophage multiplicities of 10:1. Cultures were kept at 37°C in 5% CO2 for 12 h. The unbound parasites were removed by washing with RPMI without FBS. The cells were harvested (12 h after infection) from each group, washed with PBS and kept at −80°C for RNA and protein isolation. In other set, macrophages (THP1 and hMDM) were pretreated with wortmannin (200 nM) for 3 h and then infected with WT parasites. Untreated macrophages were infected with WT, ISP2KD, and ISP2OE parasites. Infected cells were kept in incubator and cells were harvested (after 12 h) for protein isolation and the supernatant was collected for ELISA and kept at −80°C. The parasite load was measured both in THP1 cells and hMDM at 24 h after infection by counting the number of intracellular amastigotes per 100 macrophages and the rate of infection was also analyzed in both cells (THP1 and hMDM) by counting the percent infected macrophages after Giemsa staining.For infection experiment, equal number of THP1 and hMDM (~107) were treated with 20 nM PMA to obtain a macrophage-like cell line. Further to observe the effect of NE on the expression of C5aR, macrophages (~107) were treated with purified NE (10 µg/ml) and kept at 37°C for 2 h. Simultaneously, to evaluate the effect of the inhibitor on NE, NE was pre-incubated with ecotin (1 µM) or rLdISP1/rLdISP2 (10 µM) for 30 min at 4°C. These pretreated NE was used to treat the macrophages and kept at 37°C for 2 h. The macrophages were harvested for protein isolation and the protein samples were stored at −80°C for further study.THP1cells (~10UI macrophages (THP1 and hMDM) and macrophages infected with WT, ISP2OE and ISP2KD Ld parasites (for 12 h) were harvested and used for RNA isolation, using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. Simultaneously, RNA concentration was measured and cDNA was prepared by using 20 µg of RNA. cDNA was quantified and semi-quantitative PCR was performed to amplify C5aR (F-GAGGAGTACTTTCCACCAAAGG and R-AAATCGTGAGCGTGAGTAGAG). GAPDH was used as an endogenous control. The PCR was performed in thermocycler machine using thermocycling conditions -denaturation at 94°C for 5 min and 28 amplification cycles  followed by a final extension at 72°C for 5 min. The PCR products were run on the agarose gel (1.5%) and stained with ethidium bromide prior to analysis.The protein samples were prepared from NE treated macrophages to evaluate the C5aR expression. 12% SDS-PAGE was used for the separation of protein samples. Western blot was performed by transferring the gel onto PVDF membrane, followed by blocking with 3% BSA. The expression of C5aR was analyzed by using anti-human C5aR antibody . Alkaline phosphatase conjugated secondary antibody , followed by NBT/BCIP was used to detect the band. Additionally, macrophages (THP1 and hMDM) infected with WT, ISP2KD and ISP2OE Ld parasites were harvested and used for protein isolation and Western blot analysis using antibody against the C5aR. Furthermore, Western blot was performed to observe the expression of AKT, p-AKT, PI3K, and p-PI3K proteins in UI macrophages (THP1) or wortmannin pretreated Ld infected macrophages, macrophages infected with ISP2KD, ISP2OE, and WT parasites using anti -AKT, p-AKT, PI3K, p-PI3K primary antibody, respectively. Alkaline phosphatase conjugated secondary antibody  followed by addition of NBT/BCIP solution was used for detection. GAPDH was taken as an endogenous control.P ≤ 0.05 and a double asterisk (**) denotes P ≤ 0.001 when compared to control.The culture supernatant of the different group of infected macrophages (THP1 and hMDM) was used to measure cytokine production of IL-12, IL-10. The cytokines level was measured by sandwich ELISA using commercially available BD OptEIA ELISA kits . The experiments were performed in triplicate and the data are means ± SD from three separate experiments. An asterisk (*) denotes in silico protein–protein interaction was performed between LdISP1/LdISP2 and the peptidase involved in classical or lectin pathway of complement channels, such as C1r, C1s, MASP1, and MASP2. Our preliminary analysis revealed that predictive model of both LdISP1 and LdISP2 formed a stable complex with C1r, C1s, MASP1, and MASP2 via non-covalent hydrogen bonds (H-bonds). The different interacting amino acids of protein–protein interaction have been shown in Figure in silico analysis, it can be speculated that both LdISP1 and LdISP2 strongly interact with the complement serine peptidases C1r, C1s MASP1, and MASP2, where H-bond profile act as one of the most driving forces for protein–protein interaction stability.Nowadays, bioinformatics play a vital role in elucidating the possible protein–protein or protein–ligand interacting residues and their mode of binding , 43–45. −1 for rLdISP1and rLdISP2, respectively. This result showed that low range of rLdISPs (3.8–1.6 µM) was required for half of the saturation of C1 complex. A dose-dependent saturation curve was observed by increasing concentration of rLdISP1/rLdISP2 up to 50 µM  for MASPs than rLdISP1 (Ka-0.71 μM−1). The control protein sample containing only C1 complex or MASPs (no ISP), taken as the negative control did not show significant optical density. These results indicated that rLdISP1 and rLdISP2 have an efficient binding affinity for both C1 complex and serum-derived MASPs.It was reported previously that the strength of the protein–protein interaction could be indirectly estimated by some measured output signals that is directly proportinal to the receptor–ligand complex formation . TherefoIn order to investigate the effect of rLdISP1 and rLdISP2 on C1 complex and MASP1/MASP2 activity, specific enzyme assay was carried out Figure . No signIt was previously reported that MASP2 has the ability to cleave C4 molecule to produce C4a and C4b (Leishmania parasites to the active human serum lead to activate complement pathways resulting in the formation of C3 and C5c. The effect of the rLdISPs on C3 and C5c formation was assessed by using human serum or human serum pre-incubated with different concentration (0–50 µM) of rLdISP1 or rLdISP2. C3c and C5c formation via all complement pathways or individually via lectin pathway were accessed by sandwich ELISA. ~1.5- and ~2.5-fold reduction in C3c by all pathways was observed in presence of 10 and 50 µM of rLdISP2, respectively, when compared to control  (SI). As previous findings suggested that LdISP2 inhibits LP associated MASP2; therefore, MAC formation and complement-mediated lysis by LP was analyzed in presence of rLdISP2. ~1.5- and ~2.3-fold decrease in MAC formation was observed via LP in presence of 10 and 50 µM of rLdISP2 when compared to active LPSS (control)   formation and pathogen killing by complement-mediated lysis. Therefore, C3b deposition and MAC formation individually by the CP, LP, AP was analyzed in case of ) Figure A. Simultl Figure B. Micros) Figure C. HoweveThe activity of NE in presence of rLdISP1or rLdISP2 was analyzed by enzymatic assay using chromogenic substrate. ~49, ~60, and ~82% reductions in enzyme activity was observed in presence of 0.1, 1, and 10 µM of rLdISP2, respectively Figure A. HoweveThe expression of C5aR was analyzed at the transcript and protein level in macrophages (THP1 and hMDM) infected with WT, ISP2OE, and ISP2KD Ld parasites. Interestingly, at transcript level in case of THP1 cells, a ~5-fold increase in C5aR expression was observed in WT infected macrophages when compared to UI macrophages. However, a ~1.8-fold increase and a ~2.3-fold decrease in expression of C5aR was observed in ISP2OE and ISP2KD infected macrophages, respectively, when compared to WT infected macrophages   Figure A. IntereLeishmania parasites  infected with  Figures B,C. In c Figures Bi. Simil Figures Bii. More Figures Ci. Simil Figures Cii.Leishmania inside macrophages (THP1 and hMDM), parasite load and percentage of infectivity were measured in wortmannin pretreated macrophages infected with WT Ld and macrophages infected with WT, ISP2KD, ISP2OE Leishmania parasites for 24 h. Microscopic picture after Giemsa staining displaying different parasite load inside infected macrophages (THP1)  Figure A. In cas) Figure B. Parasi) Figure B. Moreov) Figure C. Simult) Figure D. Moreov) Figure E. TherefLeishmania from the sand fly to the mammalian host, the parasites are deposited in host skin through the regurgitation of promastigote secretory gel and migrate to the host blood stream  of C1r, C1s, MASP1, MASP2 and LdISPs (LdISP1/LdISP2). These complement proteins belong to S1A family and bear the catalytic domains having trypsin or chymotrypsin-like activity. According to Perona et al.  bound to the SP domain of MASP2 but not with C1r, C1s. Therefore, our findings evidence that LdISP2 inhibits MASP2 activity but not MASP1.Functional consequences of the interaction of rLdISP2 with C1 complex or MASPs was further assessed by enzymatic assay. The interaction and inhibition studies suggested that although, the strong interaction exists between rLdISP1/rLdISP2 and C1 complex but this interaction did not affect the functional activity of the C1 complex. Furthermore, the interaction of rLdISP2 with MASPs showed a significant inhibition in enzyme activity of serum-derived MASPs with MASP2 compared to MASP1. These evidences suggested that rLdISP2 inhibits MASP2 enzyme activity. The inhibitory property of rLdISP2 might be due to the presence of inhibition site, consisting of P1 reactive methionine that was found to be absent in LdISP1 , 24. OurPrevious findings by Ambrus et al.  and MegyT. cruzi inhibits the formation of the C3c by interacting with the complement component C2  formation. We have found that lectin pathway is the most activated pathway by L. donovani among all the pathways. Furthermore, we have also perceived that MAC formation and lysis of the parasites get significantly reduced in presence of rLdISP2. The possible reason might be that the presence of LdISP2 impaired the function of MASP2 that affects MAC formation, resulting in reduction in parasite lysis. Therefore, our study clearly indicates that Leishmania parasites escape from lysis mediated by lectin pathway using ISP2. A similar study was also performed by Ferreira et al.  that help in parasite clearance . The pervia impairment of C3 and C5c formation and leading to decrease in parasite lysis via reduced formation of MAC , Agamkuan, Patna, India. The institutional human ethical committee of RMRIMS , has approved all the protocol used in this study (Reference ID-04/IEC/2014).The experiment was designed by SV, AM, SD, and PD. The experiments were performed by SV, AM, and MYA. Data were analysed by SV, AM, and PD. The reagents, materials and analysis tools were provided by MYA, AjayK, KA, AKG, AK, VK, and PD. The paper was written by SV, AM, and PD.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest.


Rheum palmatum Linn, inhibited DPP4 activity with an in vitro IC50 of 5.76 µM without inhibiting either DPP8 or DPP9. A docking model revealed that emodin binds to DPP4 protein through Glu205 and Glu206, although with low affinity. Moreover, emodin treatment  in mice decreased plasma DPP4 activity in a dose-dependent manner. Our study suggests that emodin inhibits DPP4 activity and may represent a novel therapeutic for the treatment of type 2 diabetes.Historically, Chinese herbal medicines have been widely used in the treatment of hyperglycemia, but the mechanisms underlying their effectiveness remain largely unknown. Here, we screened a compound library primarily comprised of natural compounds extracted from herbs and marine organisms. The results showed that emodin, a natural compound from  Type 2 diabetes mellitus (T2DM) is a metabolic disease associated with insulin resistance and pancreatic β-cell failure . Therefo7-36) to inactive GLP-19-36 within minutes in vivo (Dipeptidyl peptidase 4 (DPP4), which was first identified by Hopsu-Havuand Glenner, rapidly degrades the active form of GLP-1  , vildaglRadix Astragali  has been used in the clinical treatment of diabetes and related complications for centuries . Radix Astragali  and Radihmanniae  are TCMsThe natural product library derived from Chinese herbs was purchased from Selleck Chemicals . Marine derived compounds were isolated and purified from marine organisms in our lab.The DPP4 screening assay was conducted using a DPP4 inhibitor screening assay kit , following the manufacturer’s protocol. Briefly, 30 µl of diluted assay buffer, 10 µl of diluted DPP4, and 10 µl of inhibitor were added to a 96-well plate. The reaction was initiated by adding 50 µl of diluted substrate solution to all of the wells, and this was followed by incubation with a plate cover at 37 °C for 30 min. After incubation, the fluorescence was read using an excitation wavelength of 360 nm and an emission wavelength of 460 nm.A DPP8 assay kit was purchased from BPS Bioscience (Cat# 800208), and the assay protocol was followed to test for inhibitory activity on DPP8. Briefly, DPP substrate 1 was diluted to make a 100 µM stock solution, and DPP8 protein was diluted in DPP assay buffer to 2 ng/µl (20 ng/reaction). For the tested compounds, 10 µl of diluted DPP8 protein, 5 µl of diluted DPP substrate 1, 84 µl of DPP assay buffer and 1 µl of inhibitor were addedinto the assay system for a total volume of 100 µl. The reaction mixtures were prepared in duplicate on a 96-well plate and incubated at room temperature for 10 min. The plate was read on an Envision plate reader  capable of excitation at 365 nm and emission detection at 460 nm.A DPP9 assay kit was purchased from BPS Bioscience (Cat# 800209), and the assay protocol was followed to test for inhibitory activity against DPP9. Briefly, DPP substrate 1 was diluted to make a 100 µM stock solution, and DPP9 protein was diluted in DPP assay buffer to 2 ng/µl (20 ng/reaction). For the tested compounds, 10 µl of diluted DPP9 protein, 5 µl of diluted DPP substrate 1, 84 µl of DPP assay buffer and 1 µl of inhibitor were added into the assay system for a total volume of 100 µl. The reaction mixtures were prepared in duplicate on a 96-well plate and incubated at room temperature for 10 min. The plate was read on an Envision plate reader  capable of excitation at 365 nm and emission detection at 460 nm.Docking of compounds to the DPP4 active site was modeled using the Glide package. The 3-dimensional model of DPP4 (PDB code: 2ONC) was used in the molecular modeling experiment . CompounDialysis assay was performed using Slide-A-Lyzer Dialysis Cassettes . Briefly, 2 mg DPP4 protein was incubated with emodin or dimethyl sulfoxide (DMSO) in 4 ml diluted assay buffer for 10 min at 37 °C. Mixed reaction solution was loaded onto a dialysis cartridge using a syringe and incubated at 4 °C for 8 h. The samples were removed from Dialysis Cassettes by syringes for DPP4 assay.Balb/c mice  and ob/ob (-/-) mice  were purchased from the Shanghai SLAC Laboratory Animal Co. Ltd.  and maintained in an air-conditioned room at 20–25 °C under a 12 h dark/light cycle and fed certified standard chow and tap water ad libitum. Experiments were conducted in compliance with the Guide for the Care and Use of Laboratory Animals. Mice were orally administered with emodin  and had their blood collected at 0.5, 1, 2, and 4 h after emodin treatment. The samples were subjected to plasma isolation immediately after collection. Plasma samples were tested for DPP4 activity with a DPP4-Glo assay kit  according to the manufacturer’s protocol. The experimental protocol was approved by Animal Care and Use Committee of Xiamen University(XM2015030514).p < 0.05.Results are presented as the mean ± standard error (SEM). Differences between the groups were analyzed using multiple variances (one-way ANOVA or two-way ANOVA) followed by Bonferroni’s test, with GraphPad Prism 5 software . Differences were considered to be statistically significant at To screen for novel DPP4 inhibitors from natural compounds, we established a natural compound library comprising 155 naturally derived compounds, in which 131 were isolated and purified from Chinese herbal medicines, and 24 were from marine organisms. DPP4 screening was first conducted on these 155 natural compounds by following a DPP4 screening assay kit protocol. All compounds (10 µM) were screened for DPP4 inhibitory activity. The results suggested that emodin showed greater than 50% inhibition in the DPP4 activity assay at 10 µM. Two other compounds were ruled out because of auto-fluorescence .in vitro with an IC50 of 5.76 µM and Ki of 0.85  . Our docRheum palmatum Linn. As a natural product and active ingredient of various Chinese herbs, emodin exerts its anti-diabetic effects partially by upregulating the expression of the pancreas L-type calcium channel in streptozotocin (STZ)-induced dyslipidemic diabetic rats (β-HSD1) activity in diet-induced obese (DIO) mice (in vivo via AMP-activated kinase (AMPK) activation. Emodin has also been shown to decrease blood glucose in rats with diabetes induced by low-dose STZ combined with high energy intake  mice . Xue, Diy intake . These din vivo, an animal experiment was conducted by oral administration of emodin  to Balb/c mice, followed by plasma collection at different time points to measure DPP4 activity in the blood. Plasma samples were collected at 0, 0.5, 1, 2 and 4 h after the oral dose of emodin, and plasma DPP4 activity was measured with a DPP4-Glo assay kit. The results suggested that emodin treatment  in mice decreased the plasma DPP4 activity in a dose-dependent manner  was administered on Balb/C mice (n = 5) or ob/ob (-/-) mice (n = 5). The DPP4 activity, blood glucoselevels and GLP-1 activity were tested 0, 1, 2, 4, 8 h after administration. The baseline level of DPP4 was higher in ob/ob (-/-) mice compared to Balb/C mice, and the data demonstrated a significant downregulation of DPP4 activity after emodin oral administration in both Balb/C mice and ob/ob (-/-) mice  mice . This do/-) mice . Meanwhi/-) mice , and thi/-) mice . These rin vitro and in vivo which may explain the anti-diabetes effects of this compound.Emodin has been detected in various Chinese herbs and is efficacious against inflammatory disorders and cancer  and livein vivo dose 30 mg/kg  is almost one ninth of the lowest dose used in these reports. However, this brings an attention to the chronic toxicity of emodin in the treatment of diabetes in the future. On the other hand, some reports have addressed that emodin isolated from rhubarb may have anti-cancer effects on a few human cancers  mice. Plasma DPP4 activity was inhibited by emodin administration in a dose-dependent manner, and the blood glucose levels were decreased in both mice strains.Based on the in vitro and in vivo. Emodin, as a novel anti-hypoglycemic compound, may stimulate new drug discovery for the treatment of type 2 diabetes.Together, these results suggest that emodin is a small molecule inhibitor of DPP4, showing activity both 10.7717/peerj.3283/supp-1Data S1Click here for additional data file.10.7717/peerj.3283/supp-2Data S2Click here for additional data file.10.7717/peerj.3283/supp-3Data S3Click here for additional data file.10.7717/peerj.3283/supp-4Data S4Click here for additional data file.10.7717/peerj.3283/supp-5Data S5Click here for additional data file.


We generated and characterized site-directed HA mutants on the genetic backbone of H5N1 clade 2.3.4 virus preferentially binding to α-2,3 receptors in order to identify the key determinants in hemagglutinin rendering the dual affinity to both α-2,3 (avian-type) and α-2,6 (human-type) linked sialic acid receptors of the current clade 2.3.4.4 H5NX subtype avian influenza reassortants. The results show that the T160A substitution resulted in the loss of a glycosylation site at 158N and led not only to enhanced binding specificity for human-type receptors but also transmissibility among guinea pigs, which could be considered as an important molecular marker for assessing pandemic potential of H5 subtype avian influenza isolates.The online version of this article (doi:10.1186/s13567-017-0410-0) contains supplementary material, which is available to authorized users. The highly pathogenic Asian-lineage avian influenza (HPAI) H5N1 viruses pose a serious pandemic threat due to their high virulence and mortality, and their increasingly expanding host range, as well as the significant ongoing evolution toward efficient transmission in mammalian models . As acknAccording to the literature, some particular amino acid substitutions in the vicinity of the HA receptor-binding domain (RBD) that contributed to the adaptation of avian influenza viruses to human hosts have been elucidated for the previous influenza pandemic subtypes, even if just one or two critical residues could be responsible for host switching since the RBD pocket is shallow. For instance, the E190D (H3 numbering throughout the paper) and G225D/E mutations in HA of the H1N1/1918 pandemic viruses, as well as the Q226L and G228S substitutions in HA of the H2N2/1957 and H3N2/1968 pandemic viruses, correlated with the conversion of binding preference to SA receptors from α-2,3 to α-2,6 type .In the last few years, natural reassortant HPAI H5NX viruses with various NA subtypes other than N1, at least including N2, N6 and N8, in the newly designated clade 2.3.4.4 affiliating to clade 2.3.4 have been circulating in many areas in China , 12. In Noteworthy, as compared with the H5N1 viruses in clade 2.3.4 showing complete α-2,3 receptor specificity, most of those above mentioned HPAI H5NX reassortant viruses can bind to both α-2,3 (avian-type) and α-2,6 (human-type) linked sialic acid receptors –22. HoweFirstly, the HA sequences of the previously reported H5NX clade 2.3.4.4 reassortants possessing dual receptor binding property were retrieved from the GenBank and GISAID public databases to be aligned with that of H5N1 clade 2.3.4 virus A/mallard/Huadong/S/2005 (HD/05) exhibiting just α-2,3 receptor specificity through the software MEGA (version 6.06). As shown in Table Subsequently, we introduced each of these six substitutions individually into the HA of HD/05 virus by a site-directed mutagenesis kit  to generate site-specific HA mutants via reverse genetics. Briefly, a mixture of 293T and MDCK cells was transfected with PHW2000-vectored plasmids encoding all eight influenza A virus genes using the PolyFect transfection reagent  as recommended by the manufacturer. After 48 h, the supernatant was harvested and inoculated into 10-day-old SPF embryonated chicken eggs for propagation of stock virus. All of the rescued viruses were confirmed by sequencing of their viral RNA, and no other amino acid changes in the viral genome apart from the expected mutated site in HA genes were observed, even when the rescued viruses were passaged for 5 generations in eggs.2SO4, and the absorbance at 450 nm was read for curve plotting.To evaluate the receptor binding property of the six rescued HA mutants, we conducted the solid-phase direct binding assay with corresponding α-2,3 and α-2,6 linked glycans as previously described . In shorAs shown in Figure 6 EID50 of the tested viruses in a volume of 300 µL (150 µL per nostril). Two animals from each group were humanely euthanized on day 3 post inoculation (pi) for virus titration from tissue samples of nasal wash, trachea, lung, kidney, spleen and brain. The remaining two animals were observed for signs of disease and death during a two-week period.An experiment was conducted in guinea pigs to further investigate whether the T160A substitution of HA affected the replication of the H5N1 virus. Groups of four serologically H5-negative female animals were initially anesthetized with pentobarbital sodium (50 mg/kg), and inoculated intranasally with 1010EID50/mL, whereas the wild type HD/05 viruses were just detected in the lungs. However, virus was not recovered from the brains, kidneys or spleens of any of the H5N1 inoculated animals. Although no obvious disease signs were observed during 2 weeks pi, seroconversion was detected in both of the animals challenged with the mutated HD-160A virus but not identified in the HD/05-inoculated group.From Table 6 EID50 and housed in a cage placed inside a negative-pressure isolator. Another three naive animals were put into the same cage 24 h later to act as the contact counterparts. Nasal washes were collected at 2-day intervals since day 2 pi (1 day post contact) and stored at −70 °C until use for virus titration in chicken embryonated in eggs. All exposed animals were checked for seroconversion at day 14 pi via determination of hemagglutinin inhibition (HI) antibodies. To prevent inadvertent mechanical transmission of virus from investigators, the contact guinea pigs were always treated first, and operating implements were changed between animals. These studies were performed under ambient conditions of 20–22 °C and 20–40% relative humidity. The airflow in the isolator was kept horizontal with a constant speed of 0.1 m/s.For viral contact transmission studies, three animals per group were infected intranasally at the dose of 10As shown in Figure Considering that the Asian lineage HPAI H5 subtype avian influenza viruses continuously expand both their geographical distribution and host range, whether they have gradually acquired efficient transmissibility between humans is of great concern. Although the natural isolates have still been deficient of such ability among people, two recent experimental studies report that reassortant H5N1 viruses with certain mutations in HA could gain the function of droplet transmission in a ferret model , 25. AccGlycosylation is an essential posttranslational modification for HA, actively involved in certain biological functions such as evasion of host immunity, HA cleavability and receptor binding . The addHere, we verified that the T160A mutation enabled H5N1 clade 2.3.4 virus to not only acquire binding affinity for human-type glycans but also transmit among guinea pigs. Although α-2,6 receptor binding property does not necessarily imply successful human transmission requiring additional synergy, mutants with high avidity for both α-2,3 and α-2,6 receptors may be intermediates during the evolution of novel H5 reassortants that could infect both poultry and humans to increase the risk of an influenza pandemic. Therefore, continued routine surveillance of H5 variants possessing dual receptor affinity is indispensable as an early warning system, and the presence or absence of the 158N glycosylation site could serve as an important molecular marker for assessing pandemic potential of H5 subtype avian influenza isolates.Additional file 1. Cartoon representation of the HA structure of A/mallard/Huadong/S/2005. The model was generated by automated homology modeling using SWISS-MODEL based on the template of PDB (ID: 4jul). The receptor-binding domain (RBD) constituting with amino acids 188-190, 134-138, 221-228, 98,153 and 183 of HA1 (H3 numbering), according to reference [9], were colored yellow for clarity. The six divergent amino acids discussed in the text were colored gray, of which simultaneously located in RBD were labeled orange. The six specific substitutions were shown as sticks in the magnified side pictures, respectively. ‘‘K’’ is lysine, ‘‘R’’ is arginine, ‘‘T’’ is threonine, “Q” isglutamine, ‘‘S’’ is serine, ‘‘N’’ is asparagine, ‘‘H’’ is histidine and ‘‘A’’ is alanine.


Dysregulation of blood sphingolipids is an emerging topic in clinical science. The objective of this study was to determine preanalytical biases that typically occur in clinical and translational studies and that influence measured blood sphingolipid levels. Therefore, we collected blood samples from four healthy male volunteers to investigate the effect of storage conditions , blood drawing , and sample preparation  on sphingolipid levels measured by LC-MS/MS. Our data show that sphingosine 1-phosphate (S1P) and sphinganine 1-phosphate (SA1P) were upregulated in whole blood samples in a time- and temperature-dependent manner. Increased centrifugation at higher speeds led to lower amounts of S1P and SA1P. All other preanalytical biases did not significantly alter the amounts of S1P and SA1P. Further, in almost all settings, we did not detect differences in (dihydro)ceramide levels. In summary, besides time-, temperature-, and centrifugation-dependent changes in S1P and SA1P levels, sphingolipids in blood remained stable under practically relevant preanalytical conditions. Besides their role in energy metabolism, several classes of lipids are considered as being “bioactive,” having numerous roles in cell biology, physiology, and disease. Among these lipids, sphingolipids, such as several dihydroceramides (dhCer) and ceramides (Cer), have been increasingly recognized as important mediators of several biological processes . As sphiThe goal of this study was to identify and characterize the most relevant factors occurring typically in clinical studies in order to define “standard operating procedures” for measuring sphingolipids in blood. We investigated the sphingolipid profile in human blood, serum, and plasma samples dependent on differences in blood drawing, sample preparation, freezing and storage conditions by high-performance liquid chromatography/tandem mass spectrometry (LC-MS/MS). Our results might be helpful for the design and comparison of future human prospective studies and to look more critically at the conclusions made from results of retrospectively analyzed samples, where the exact sampling conditions were not well defined.2 (mean ± SD). Blood laboratory parameters of the volunteers were as followed: cholesterol = 163 ± 31.6, triglycerides = 112 ± 28.5, high-density lipoproteins = 52.5 ± 10.3, low-density lipoproteins = 88.0 ± 32.7 .The characteristics of the four participants are shown in First, we evaluated whether there were changes in sphingolipid levels according to storage time and temperature. We incubated whole blood and serum samples at either +22 °C for different times or at +4 °C and +22 °C for up to 4 h. As shown in As shown in n = 3). By measuring the relative oxygen levels, we confirmed that we had collected venous and arterial blood ; mean ± SD; n = 3; Paired t-test; p = 0.04). As shown in Next, we analyzed the effects of the conditions that are typically used in the clinic for blood drawing, which might alter sphingolipid levels in blood. We first evaluated whether there were differences in the sphingolipid content in samples of venous and arterial sources taken from the same volunteers  under normal systolic blood pressure + 10 mmHg for either 1 min or 6 min in order to simulate long-term vein compression by a tourniquet since this can happen when blood sampling proves to be difficult. No changes were observed for S1P or SA1P levels B,D. As sg and 16,200× g significantly lowered the relative S1P and SA1P levels compared to the control (550× g), whereas Cer and dhCer levels were not altered . Again, no significant alterations were observed for all analyzed sphingolipids upon long-term storage .We hypothesized that sphingolipid concentrations in blood are subject to different systematic preanalytical factors, thus hampering the generalizability of results of sphingolipid analyses in blood samples under non-controlled conditions. Our data clearly define certain caveats that should be avoided when measuring sphingolipids in blood. In line with previous findings ,14, we fAs our study was not designed to explain the effects of temperature and time on sphingolipid concentrations, this needs further investigation. However, some preliminary conclusions could be drawn, e.g., the lack of an increase of these metabolites in isolated plasma suggests an involvement of the platelets or red blood cells. The effects of temperature and time on S1P and SA1P in whole blood samples could have several cellular causes. The most likely explanation is either the activation, destruction, or dysfunction of platelets or red blood cells or a combination of effects, as both cell types contain large amounts of both sphingolipids ,17. ConcFrom the clinical point of view, we showed that most of the relevant preanalytical variables did not influence S1P and SA1P levels in human blood samples. In addition, ceramide and dihydroceramide remained stable in almost all clinically important conditions, such as prolonged tourniquet during blood drawing, long-term storage, and several freeze–thaw cycles. The latter was also investigated by Hammad et al.  who checTaken together, our study gives important implications for future biomarker studies concerning the stability of sphingolipids. From our data, several recommendations can be extracted: Ceramides are surprisingly stable under the various conditions tested here. Besides this important implication for the reliability of studies about ceramides, we recommend especial caution if S1P or SA1P are in focus; study groups should avoid long processing times of blood samples, the use of different anticoagulants, and centrifugation steps. On the other hand, samples can be drawn from different types of vessels without any restrictions regarding vein compression time. As soon as plasma is isolated, also S1P and SA1P remain stable under various storage conditions.g for 5 min (4 °C). Thereafter, 20 µL aliquots were stored immediately at −80 °C until lipid extraction and LC-MS/MS measurement. The study was performed in accordance with the Declaration of Helsinki. Blood drawing was approved by the local ethics committee. All volunteers had signed a written informed consent.If not otherwise indicated, venous blood  and serum, 1 mL each) was used from four healthy non-fasting male volunteers. Plasma and serum (after coagulation) separation was performed by two-times centrifugation at 550× g, 5800× g, or 16,200× g; (6) Separated plasma samples (EDTA) were either snap-frozen in liquid nitrogen or frozen at −20 °C and −80 °C. After freezing, all aliquots were stored at −80 °C until lipid extraction; (7) Separated plasma (EDTA) was frozen at −80 °C for 1 h and completely thawed at +22 °C for 30 min. Freeze–thaw cycles were repeated 2–6 times. Plasma without additional freezing/thawing cycles served as the respective control; (8) Separated plasma (EDTA) was stored for 18 months at −80 °C. Freshly isolated plasma without long-term storage served as the respective control. After one-time thawing, aliquots were prepared for lipid extraction.The following experiments were performed: (1) Whole blood samples  were incubated at +4 °C and +22 °C for 4 h or at +22 °C for 0–8 h; (2) Separated plasma samples (EDTA) were incubated at +4 °C and +22 °C for 4 h. In both cases, immediately separated plasma and serum samples without incubation served as controls (0 h); (3) Venous  and arterial  blood samples (heparin) were collected and plasma was immediately separated; (4) Blood samples (EDTA) were collected under normal systolic blood pressure (RR) with compression +10 mmHg for either 1 min or 6 min to stimulate prolonged venous compression as it is often necessary when blood sampling proves to be difficult. Thereafter, plasma was immediately separated; (5) Plasma samples were separated from blood (EDTA) by 5 min centrifugation (+4 °C) at 550× v/v/v). The lower organic phase was evaporated at +45 °C under a gentle stream of nitrogen and reconstituted in 200 µL of tetrahydrofuran/water  with 0.2% formic acid and 10 mM ammonium formate. Afterward, amounts of sphingolipids were analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). An Agilent 1100 series binary pump  equipped with a Luna C8 column  was used for chromatographic separation. The column temperature was 35 °C. The HPLC mobile phases consisted of water with 0.2% formic acid and 2 mM ammonium formate (mobile phase A) and acetonitrile/isopropanol/acetone  with 0.2% formic acid (mobile phase B). For separation, a gradient program was used at a flow rate of 0.3 mL/min. The initial buffer composition 55% (A)/45% (B) was held for 0.7 min and then within 4.0 min linearly changed to 0% (A)/100% (B) and held for 13.3 min. Subsequently, the composition was linearly changed within 1.0 min to 75% (A)/25% (B) and then held for another 2.0 min. The total running time was 21 min and the injection volume was 15 μL. To improve ionization, acetonitrile with 0.1% formic acid was infused post-column using an isocratic pump at a flow rate of 0.15 mL/min. After every sample, sample solvent was injected for washing the column with a 12 min run. The MS/MS analyses were performed using a triple quadrupole mass spectrometer API4000  equipped with a Turbo V Ion Source operating in positive electrospray ionization mode. The MS parameters were set as follows: Ionspray voltage 5500 V, ion source temperature +500 °C, curtain gas 30 psi, collision gas 12 psi, nebulizer gas 40 psi, and heating gas 60 psi. The analysis was done in Multiple Reaction Monitoring (MRM) mode. Data acquisition was done using Analyst Software V 1.6 and quantification was performed with MultiQuant Software V 3.0 , employing the internal standard method (isotope dilution mass spectrometry). Variations in accuracy of the calibration standards were less than 15% over the whole range of calibration, except for the lower limit of quantification where a variation in accuracy of 20% was accepted.For lipid extraction, 10 µL human plasma and/or serum were mixed with 150 µL water, 150 µL extraction buffer , and 20 µL of the internal standard solution containing sphingosine-d7, sphinganine-d7 (200 ng/mL each), sphingosine 1-phosphate-d7, C17:0 Cer, C16:0 Cer-d31, C18:0 Cer-d3, C18:0 dhCer-d3 , and C24:0 Cer-d4  (400 ng/mL each). The mixture was extracted once with 1000 µL methanol/chloroform/hydrochloric acid  ± standard deviation (SD). Analysis were performed on relative values. For better comparability, time- and temperature-dependent effects on absolute SL concentrations are examplified in Table S2.Statistical analyses were performed with GraphPad Prism . Significant differences between two groups were evaluated by paired


For four related 1,1-disubstituted olefins, (DHQD)2PHAL-catalyzed asymmetric chlorocyclization delivers Cl+ uniformly to one π face, but cyclizes with strong but differing net syn vs. anti addition. 2PHAL-catalyzed chlorocyclizations of 1,1-disubstituted olefins show useful  asymmetric induction, stereochemically complete descriptions of these alkene additions have remained largely unknown. Herein, based on a combination of NMR, derivative, isotope labeling, and computational studies, we present detailed stereochemical analyses of chlorocyclizations of nucleophile-tethered 1,1-disubstituted styryl systems. The selectivities of the two asymmetric bond-forming processes, namely electrophilic chlorine attack and nucleophilic ring closure, are thus mapped out independently. Under the established optimal conditions, four related chlorocyclizations were subjected to this analysis. All showed a strong preference for Cl+ delivery from the same face of the alkene. However, depending on reaction conditions and substrate identity , the internal nucleophiles may close with a strong net preference for either syn or anti addition relative to the Cl atom. Studies of both uncatalyzed and (DHQD)2PHAL-catalyzed processes place new boundary conditions on the role of the catalyst in these reactions.Though (DHQD) With the advent of effective asymmetric catalytic methods, halocyclizations  have now emerged as useful tools for synthesis of chiral targets.2 symmetric seleno-THF analogs with high enantioselectivity,1Broadly, stereocontrol requires the reaction environment to be desymmetrized with a chiral catalyst that activates the reaction while guiding bond formation to prefer one face of the alkene. Many asymmetric halocyclizations of alkenes have now been described using a variety of chiral catalysts. Chiral phosphoric acid catalysts or related analogs were shown to catalyze bromo-amination and -etherification by Shi and coworkers;2PHAL.2 symmetric cinchona alkaloid derivative efficiently catalyzes the reaction of substrates bearing hydrogen-bond donor groups such as unsaturated amides, carbamates, naphthols and carboxylic acids. In all these asymmetric halocyclization reactions, the olefin undergoes electrophilic attack by a halenium ion (X+) donor with intramolecular ring closure by a pendant nucleophile. Mechanistically, halofunctionalization of alkenes has been extensively studied since their discovery. The exclusive formation of anti-products from olefin halogenation led Kimball in 1937 to propose a stepwise mechanism, with symmetrically bridged haliranium ions as putative intermediates.E3-type mechanism.E3-type concerted halofunctionalization. This nucleophile assisted alkene activation (NAAA)anti opening with a nucleophile.Our own and others' studies have uncovered several stereoselective halofunctionalizations based on the organocatalyst (DHQD)3 CH2Cl center lacked observable stereochemistry, leaving the face selectivity of chlorination unknown. To reveal the relative and absolute stereochemical outcomes of both the halenium ion attack and the nucleophilic closure in halocyclizations, we resorted to deuterium-labeling in the 1,1-disubstituted olefin substrates. In particular, we investigated the syn : anti selectivity of addition across the double bond, both for non-catalyzed reactions  and for their (DHQD)2PHAL-catalyzed analogues.With asymmetric halocyclizations empirically established as useful synthesis tools, we turned to mechanistic studies of reactions developed in our labs over the past six years. 1a, and those of amide 1b and carbamate 1c, should be noted at the outset. As earlier reported, (a) all these reactions employ (DHQD)2PHAL as the chiral catalyst and chlorinated hydantoins as the electrophilic chlorinating agents. (b) The configuration of the newly created stereogenic carbon (C5) in the product is nucleophile dependent. (c) In the case of the carbamate substrate 1c, the solvent can also modulate the C5 configuration.This study sought to uncover and understand the stereochemical relationships between the chlorenium ion delivery and the nucleophilic ring closure events that form the adducts shown in syn or anti halocyclization would shed light on the nature of the reaction path. (d) Is the overall reaction concerted or stepwise; and if the latter, what sort of intermediate  might be formed? (e) How is the ultimate enantioselectivity set?To understand these diverse behaviors, we asked the following questions: (a) is there a facial preference for electrophilic chlorine attack on the 1,1-disubstituted olefin? (b) If so, how strong is the preference, and how does it vary among substrates and conditions? (c) What patterns (if any) of stereochemical relationships are found between the chlorine atom and the nucleophile in the final adduct? Net 1a-D, 1b-D, and 1c-D leads to diastereomeric products that reveal not only the face selectivity of chlorenium ion attack on the olefin, but also the syn or anti relationship of addition between the delivered halogen and the captured nucleophile. The results of these studies show a diversity of relative and absolute stereochemical fates in both chlorenium ion delivery and ring closing processes. They also highlight the multiple ways that the catalyst can modulate reactivity.As in our previous mechanistic investigations of Reaction A,E-deuterated substrates 1b-D and 1c-D was accomplished in four steps  of the deuterated substrates 1b-D and 1c-D was taken into account by the mathematical treatment shown in the ESI.The synthesis of the ur steps . The deu2PHAL catalyzed chlorocyclizations of 1b-D and 1c-D (vide infra) led to mixtures of diastereomeric products. The absolute configuration of the deuterated chlorocyclized products 2b-D and 2c-D at C6 was determined via transformation of the major product of both deuterated and non-deuterated substrates to an epoxide with known configuration N-benzoyl β-amino alcohol 7-D  and Hb  with respect to the phenyl group. 1H NMR analysis of the epoxy amide 3b-D, obtained from the product of the chlorocyclization of 1b-Dvia Reaction B conditions, exhibits the peak at 3.10 ppm, establishing that the deuterium has a cis orientation with respect to the phenyl group. This leads to the assignment of R configuration for the carbon bearing the deuterium in epoxy amide 3b-D. Since the epoxy amide is formed through the SN2 closure of the corresponding chlorohydrin intermediate, the S configuration at C6 is assigned for amide 2b-D guration , top. Hyohol 7-D , top. Th1c-D under conditions denoted as Reaction C and C′ were determined ,3 mediated ring opening of the resulting chlorohydrin intermediate, gave 1,1-disubstituted epoxy sulfonamide 3c-D and ent-3c-D. The non-deuterated epoxy amide ent-3c was synthesized from 5S-2c following the same reaction protocols. ROESY analysis of epoxy amide ent-3c indicated that Ha (2.75 ppm) is cis to the phenyl group, while Hb (3.20 ppm) is trans. 1H NMR data obtained for the epoxides derived from the major products of Reaction C and C′ chlorocyclization reveal that the absolute configuration on the labeled C6 center of the cyclized products 5R-2c-D and 5S-2c-D is S for both  sluggishly in TFE at room temperature to afford an 85 : 15 mixture of the two diastereomers. Similarly, predominant anti addition is found for the uncatalyzed reaction of carbamate 1c-D with 1,3-dichloro-5,5-dimethylhydantoin (DCDMH), both in CHCl3-hexanes and n-PrOH solvent systems; there, anti : syn ratios were 84 : 16 and 97 : 3, respectively. Formation of significant quantities of the syn isomer serves as evidence that these reactions do not simply proceed via one path through a stereochemically-defined intermediate (e.g. a cyclic chloriranium ion) able then to dictate stereospecific cyclization to the anti isomer; at least two pathways must contribute to yield the two distinct diastereomers. The substantial shift in anti : syn selectivity (84 : 16 to 97 : 3) between the solvent conditions corresponds to an increase of ∼1 kcal mol–1 in the relative barrier for syn vs. anti closure; evidently carbamate 1c-D interacts fairly strongly with its surroundings, as expected for such a polar substrate.To understand the role of the catalyst in the enantiocontrolled reactions, the intrinsic diastereoselectivities of the uncatalyzed analogues of Reactions A–C and C′ were first investigated .24 All fanti : syn diastereoselectivity in the absence of catalyst, reaction parameters were systematically studied for chlorocyclization of the amidoalkene 1b-D. First, the effects of the chlorenium ion source were investigated  does show the lowest selectivity, no direct correlation is seen between chlorenium ion donor ability anti : syn ratios. Thus it appears that the departing moiety of the chlorine donor reagent remains involved at the point of diastereoselection.+ delivery followed by ring closure in an intermediate free of the leaving group from the chlorinating agent.To probe the factors that determine anti : syn ratios was next studied (3 and DCM gave high anti : syn ratios (98 : 2 and 97 : 3) whereas relatively polar, hydrogen bonding solvents eroded the anti selectivities. Likewise, running the reaction in a 1 : 1 mixture of CHCl3-hexanes gave a much higher 97 : 3 dr than in CHCl3 alone (86 : 14). These results suggest contributions from mechanisms that are differentially affected by solvent polarity and hydrogen bonding ability. For instance, nonpolar solvents would favor substrate self-association via hydrogen bonds between the amide moieties,anti products from reagent + substrate dimers , or when the amide substrate itself was present in concentrations as high as 0.5 M. Thus, any electrophilic intermediates, if formed, must have lifetimes shorter than trapping times in these neat nucleophilic solvents. And even the most aggressive chlorenium ion donors, expected to require little or no nucleophilic assistance, did not trigger intermolecular product formation.The effect of the reaction solvent on mers see . Polar,  complex . We notesyn product formation  molecules of olefin substrate and one molecule of the DCDPH reactant . Thus, (DHQD)2PHAL controls the facial selectivity of chlorenium ion attack, forming the major epimer with a 99 : 1 preference for the 6S configuration, but only moderate 6S selectivity in forming the minor (5S) product (73 : 27). The nucleophilic closure occurs with high selectivity (93 : 7 ratio) favoring the R configuration at C5. As such, the two bond-forming events appear to be independently controlled by the catalyst. Noteworthy is the fact that in Reactions A and B , the chlorenium ion delivery occurs to the same face of the olefin, but ring closure occurs on opposite faces of the cyclizing carbon . The overall process, therefore, is syn for chlorolactonization (Reaction A) and anti for chloroamidocyclization (Reaction B).Having probed the intrinsic diastereoselectivity of uncatalyzed Reaction B, we turned to the stereochemical analysis of the (DHQD)eference , top.22,nditions , bottom.1c shows a switch in the enantiopreference of (DHQD)2PHAL-catalyzed chlorocyclization depending on reaction conditions – primarily the reaction solvent.via deuterated probe 1c-D. Chlorocyclization of 1c-D catalyzed with (DHQD)2PHAL in 1 : 1 CHCl3 : hexane ; this behavior is thus stereochemically the same as for amide 1b-D. The chlorenium ion delivery to yield products with 6S configuration occurs with high selectivity (93 : 7) for the major (5R) diastereomers. As in the above cases, although the 6S selectivity is still predominant for the minor diastereomer, it occurs with reduced discrimination (61 : 39). Thus, the overall face selectivity for chlorination on the pro-S face of the  PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbdnyFC3" format="PBM" resolution="300" x-size="34" y-size="34" xml:space="preserve"> 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000	</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="16.000000pt" height="16.000000pt" viewBox="0 0 16.000000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 1440 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z M0 960 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z"/></g></svg>CHD site (i.e. 6S : 6R) is 91 : 9. This C6 stereoselectivity incidentally has the same numerical value as the enantioselectivity of the reaction (91 : 9 5R : 5S).In Reactions C and C′, carbamate n-PrOH and the catalyzed Reaction C in CHCl3-hexanes where anti additions predominate . Instead, the (DHQD)2PHAL catalyzed reaction in n-PrOH effects syn addition (net anti : syn = 10 : 90). However, as in all the other reactions discussed so far, chlorenium ion delivery occurs to the same face, forming the 6S epimers (with this substrate) in high (99 : 1) and good (85 : 15) enantioselectivity for the major and minor diastereomers, respectively. The net selectivity for chlorination of the C6 pro-S face of 1c-D is 98 : 2 whereas the C5 face selectivity is somewhat lower, at 90 : 10 pro-S.When applied to Reaction C′ , bottom,syn : anti addition ratios seen as a function of starting material and reaction conditions, it might be suggested that cis–trans isomerization in the starting olefin could explain the observed stereo-randomized products (see e.g. 99 : 1 in Reaction B), and (b) explicitly, by verification of the stereochemical integrity of labeled substrates 1b-D and 1c-D recovered during the course of Reactions B, C, and C′. Reactions quenched at various extents of conversion yield recovered alkenes with E/Z isomeric ratios identical within measurement uncertainties to the starting ratios for all three reactions 2PHAL organocatalyst, broad conformational searches on such 2 : 1 complexes were performed with the MMFF94  for anti and syn chlorocyclizations into one (DHQD)2PHAL pocket, together with a partner non-reacting substrate complexed to the other face. In these cases, the lengths of the partial (reacting) bonds , were held constant at the gas-phase TS values, but all other degrees of freedom were allowed to vary in the conformational search. Again, the lowest energy set of resulting conformations was reoptimized (still constrained) using PM6, after which full TS optimizations were completed. The resulting syn and anti TSs  were reevaluated via single-point B3LYP-D3/6-31G* energy calculations,n-PrOH solvents (dielectric constants of 4.8 and 20.3) computed via the C-PCM method.38Though a comprehensive quantum chemical simulation of interacting substrates, organocatalyst, reagents and solvents is beyond the scope of this report, limited simulation studies of the complexation and reaction processes of carbamate substrate e MMFF94  and Sybyvia the above procedures are displayed in anti and syn transition state structures correspond well with the fact that the difference between CHCl3 and n-PrOH media can switch the anti/syn addition selectivities of Reactions C/C′. Notably, the relaxed precursor complex 2PHAL-carbamate-DCDMH complex  with a small  free energy preference for syn. We suggest that the more intimately bound complexes in Energetically, the TS structures for  and syn  are calc complex  at the B3 stereocenter; syn and anti addition paths are in close competition. Conversely, a finding of poor enantioselectivity at C5 does not imply poor face-selectivity in the halogen-alkene bond formation.The work presented above highlights one of the fundamental challenges in developing highly enantioselective halocyclizations and more generally, additions across alkenes. Here, exceptional face-selectivity in the alkene chlorination is no guarantee of a strong enantiopreference for the newly created C5 spvia intramolecular versions of the AdE3 olefin addition.syn and anti paths can selectively occur among reactions A–C rules out bridged chloronium ions as stereocontrolling intermediates, as expected for these conjugated, 1,1-disubstituted alkenes.With the present array of data, our working mechanistic interpretation is that reaction occurs anti ring closure in contrast to the syn mode seen for carboxylic acids and for carbamates reacting in polar protic solvents. We propose that the amides and carbamates 1b and 1c  most readily fold “inward” to achieve the pro-anti conformation. In contrast, carboxylic acid 1a is less conformationally restricted and potentially capable of hydrogen bond formation, which enables a preference for it to fold outward and activate the alkene from the same face approached by the chlorine-donating hydantoin. Likewise, folding of the polar carbamate moiety of 1c out into the solvent could be supported by the more polar n-PrOH medium, favoring approach from the same face as the chlorenium ion delivery. The carbamate in n-PrOH (Reaction C′) thus behaves much like the carboxylic acid 1a, yielding mainly syn addition product.What remains difficult to explain is the observed variability in the ring closure stereochemistry for the four highlighted reactions. As noted above, the complexed alkenes in our calculated structures see  place th2PHAL, the relative and absolute stereochemical outcomes have been fully analyzed. In four distinct (DHQD)2PHAL—catalyzed processes—chlorocyclizations of carboxylic acid 1a, of amide 1b, and of carbamate 1csyn or anti selectivity depending on the nature of the nucleophile and the medium. Thus, the net stereoselectivities at the two new stereocenters appear to be related only in the sense that one of these concerted paths is strongly preferred over others in the optimized reactions. The resulting structural insights place boundary conditions on any mechanistic hypothesis proposed to further refine and generalize this synthetically versatile class of transformations. Detailed kinetic analyses and simulation efforts are ongoing to probe molecularity, catalyst-substrate-reagent binding, preferred conformations in different settings, and reaction rate effects.For a family of chlorocyclizations, both uncatalyzed and mediated by (DHQD)There are no conflicts to declare.Supplementary informationClick here for additional data file.