N-terminal lipophilic membrane-insertive elements and then conjugated to vancomycin. These modified lipoglycopeptides, the ‘vancapticins’, possess enhanced membrane affinity and activity against methicillin-resistant Staphylococcus aureus (MRSA) and other Gram-positive bacteria, and retain activity against glycopeptide-resistant strains. Optimised antibiotics show in vivo efficacy in multiple models of bacterial infection. This membrane-targeting strategy has potential to ‘revitalise’ antibiotics that have lost effectiveness against recalcitrant bacteria, or enhance the activity of other intravenous-administered drugs that target membrane-associated receptors.The public health threat posed by a looming ‘post-antibiotic’ era necessitates new approaches to antibiotic discovery. Drug development has typically avoided exploitation of membrane-binding properties, in contrast to nature’s control of biological pathways via modulation of membrane-associated proteins and membrane lipid composition. Here, we describe the rejuvenation of the glycopeptide antibiotic vancomycin via selective targeting of bacterial membranes. Peptide libraries based on positively charged electrostatic effector sequences are ligated to  The antibiotic vancomycin inhibits bacterial cell wall synthesis by binding to a membrane-associated precursor. Here, Blaskovich et al. synthesize vancomycin derivatives containing lipophilic peptide moieties that enhance membrane affinity and in vivo activities against glycopeptide-resistant strains. Methicillin-resistant Staphylococcus aureus (MRSA), a major cause of community and hospital-acquired infections, leads to significant morbidity and mortality7. Vancomycin is often used to treat MRSA infections, but clinical isolates that are resistant are increasingly common6: VISA – vancomycin-intermediate S. aureus: MIC (minimum inhibitory concentration) 4–8 μg mL−1; hVISA – heteroresistant VISA: predominantly susceptible but with a resistant subpopulation; and, more rarely7, VRSA – vancomycin-resistant S. aureus: MIC ≥ 16 μg mL−1. Even moderate reductions in vancomycin susceptibility (MIC shift from ≤1 to 1.5–2.0 µg mL−1) can lead to significantly worse clinical outcomes9. In the last 15 years antimicrobial agents such as daptomycin and linezolid have emerged as approved therapeutic compounds for vancomycin-resistant bacteria10, but resistance to both of these antibiotics was identified in vancomycin-resistant enterococci (VRE) and MRSA shortly after their approval12.Antibiotic resistant bacteria pose a grave threat to human health and there now is an urgent need to develop new antibiotics13, but rarely assesses membrane-binding properties14. In contrast, nature exploits subtle changes in the composition of biological membranes to control signal transduction, vesicular transport and receptor recycling with membrane-associated proteins such as Ras15, Src16, Rab17, HIV-1 GAG18 and MARCKS19. In these proteins, N-terminal and/or C-terminal lipophilic groups  and clusters of basic amino acids act as an electrostatic ‘switch’ to control translocation between the cytosol and membrane surface via selective phosphorylation of serine and tyrosine residues21. We postulated that appending such motifs to drugs acting on membrane-associated targets could enhance membrane binding and concomitantly increase drug concentration at the target site20. The potential also exists to discriminate between different types of membranes to increase drug selectivity. Given the alarming rise of multi-drug resistant bacteria and paucity of new antibiotics in the pipeline, we validated this approach using the glycopeptide antibiotic vancomycin.Drug development traditionally focuses on optimising target affinity, selectivity and protein binding in the context of drug pharmacodynamicsC-terminus of vancomycin. The vancapticins display Gram-positive activity, including activity against vancomycin-resistant strains. The vancapticins were highly efficacious in multiple infections models in mice, with a low propensity for innate and induced resistance, and a pharmacokinetic (PK) profile consistent with once daily dosing in humans. Taken together, these results suggest vancapticins could be candidates for further development as a promising new therapy for the treatment of antibiotic-resistant Gram-positive bacterial infections.In this study, we designed and synthesised a series of vancomycin derivatives, designated vancapticins, by appending membrane targeting motifs onto the C-terminal tripeptide (l-Lys-d-Ala-d-Ala) of the peptidoglycan precursor Lipid II, preventing cross-linking by bacterial transglycosylases and transpeptidases. A ‘back-to-back’ dimerisation enhances antimicrobial activity via a surface-templated chelate effect with membrane-bound Lipid II23. In developing a membrane-targeting vancomycin analogue, it was important not to occlude the peptide binding site, nor disrupt interactions at the dimer interface. Previous derivatisations of vancomycin have utilised the vancosamine sugar amine or heptapeptide free carboxyl group;24 other modification sites are possible, but more synthetically challenging. In order to attach basic electrostatic effector peptide segments containing lipophilic membrane-insertive elements (MIE), using chemistry compatible with unprotected functionality, we amidated vancomycin with pyridyldithioethylamine, then selectively ligated unprotected basic peptides containing a C-terminal Cys residue under mild conditions via disulphide exchange  Streptococcus pneumoniae, and more than 100-fold more active than vancomycin against VanA Enterococcus faecium (VRE) , and led to improved activity for a minimal basic KKK motif.Vancomycin blocks cell wall synthesis by binding to the nge Fig. –3. The i2, Supplementary Figs. −1) against other Gram-positive strains, including daptomycin-resistant MRSA, MDR S. pneumoniae, S. pyogenes and S. epidermidis, but were inactive against Gram-negative Escherichia coli (data not shown). Importantly, these new compounds were stable when exposed to plasma    of 9.4 × 10−10 against MRSA (ATCC 43300) (Supplementary Table 17 and 24 induced low levels of resistance compared to daptomycin when MRSA (ATCC 43300) cultures were serially cultured over 20 days in the presence of increasing sub-lethal antibiotic concentrations , while E. faecium exhibited greater sensitivity toward longer alkyl groups >C12 (Table 25 vs. 19) highlighting the importance of the insertive element for antimicrobial potency. Variation of the charged region from zero to three Lys residues (18–20)  was selected as optimum. Enantiomeric all d-amino acid effector sequences or linkers were equipotent with the natural L-amino acids, as were replacements of Lys with Orn or Dab, suggesting that no chiral molecular recognition was involved  for over 12 h following a single 10 mg kg−1 subcutaneous (SC) dose  dose of 19 was equivalent to a single 200 mg kg−1 dose of vancomycin, with a 6-log reduction compared to the saline control at 24 h  infection model using bioluminescent methicillin-sensitive S. aureus (MSSA) Xen-29, with imaging at 9 h post infection again showing good efficacy for 19, but a reduced activity for 24  differ from vancomycin by acting primarily via transglycosylase inhibition33. The remarkable activity of our compounds could be due to a number of factors: (i) an increased affinity for the target terminal d-Ala-d-Ala dipeptide of Lipid II, (ii) enhanced dimerisation leading to higher avidity for membrane-bound Lipid II34, (iii) an additional or different mode of action or target than vancomycin (such as membrane permeabilisation), and/or (iv) membrane localisation resulting in an enhanced drug concentration at the membrane surface, which could serve to enhance any of the above.Vancomycin acts by inhibiting the transpeptidase step of peptidoglycan synthesis, although there is also evidence of blocking the previous transglycosylase step17 and 24 inhibited peptidoglycan formation in a cell free radiolabelled synthesis assay that monitors the incorporation of 14C-labelled N-acetylglucosamine [14C-GlcNAc] into Lipid II and then into peptidoglycan, with Lipid II accumulating at tenfold lower concentration of compound compared to vancomycin , required a 100-fold molar excess of Ac2Kaa to ablate the activity of vancomycin. In contrast, the new compounds required over 10,000-fold molar excess to neutralise antibiotic action, also confirming that the new analogues still targeted Lipid II and peptidoglycan synthesis  studies. There was a propensity to bind more strongly to anionic  over zwitterionic  lipid bilayers  was found to exceed 100 µM for selected compounds 17, 19 and 23. Furthermore, compounds were generally non-haemolytic against human erythrocytes in whole blood at 1600 µg mL−1, with any haemolysis observed not correlating with lipophilicity  to a similar or greater degree than the control antibiotic oritavancin 538, as measured by the fluorescence output of diSC3(5) , whereas vancomycin, daptomycin and dalbavancin showed no effect under the conditions of the assay. Similar effects were observed against MSSA (ATCC 29213) ], which is prevented from partitioning to the surface of polarised cells during disruption of membrane potential, causing dye release into the media(5) Fig. . All com ATCC 292  was incubated with selected antibiotic compounds at 16 µg mL−1 for 1 h  followed by exposure to propidium iodide  , which stains nuclear chromatin upon cell membrane disruption, resulting in fluorescence enhancementide Fig. . Again, 1. Over the past four decades the rational improvement of existing classes of antibiotics has proven to be a very successful strategy. Vancomycin is a logical candidate for improvement via specific membrane-targeting, given its mode of action and the length of time it took (more than 20 years) for significant clinical resistance to arise.Whilst there is a current perception that Gram-positive antibiotics are less urgently required than Gram-negative therapies, in reality the number of drug-resistant Gram-positive infections, and resulting deaths, far outnumber those from resistant Gram-negative bacteriaMembrane-ligand interactions are an underexplored area of drug design. We created a series of membrane-targeted vancomycin derivatives with significantly improved activity against a range of Gram-positive bacteria, including vancomycin-resistant strains. The modular nature of their structure Fig. , with a d-Ala-d-Ala ligand in free solution required to antagonise vancapticin antimicrobial activity against live bacteria -ria Fig. .17 and 24 eliciting a comparable response as oritavancin, but fourfold to sixfold less than daptomycin. Vancapticin 19 and dalbavancin were much less active, while vancomycin had no effect. The increased membrane activity of the vancapticins appears to enhance their potency, but does not translate into increased killing kinetics: compound 24 shared a similar slowly bactericidal profile with vancomycin  or HypoGel hydroxymethylbenzoic acid (HMBA) resin (Iris Biotech GmbH), respectively. Peptides bearing free C-terminal carboxylic acids were assembled on Fmoc-Cys(Trt) or Fmoc-Lys(Boc) Wang polystyrene resin. 9-Fluorenylmethyloxycarbonyl (Fmoc)-l-amino acids utilised standard side-chain protecting groups: tert-Butyl , Trityl (Cys), tert-Butoxycarbony (Boc) or 4-methyltrityl (Mtt) or 1--3-methylbutyl (ivDde)  and were purchased from Iris Biotech GmbH, as were the coupling reagents O-(benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate (HBTU) and O-(benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBop). Azabenzotriazol-1-yl-N,N,N’,N’-tetramethyluronium hexafluorophosphate (HATU) was sourced from GenScript Corporation (USA), and (S)-pyridylthio cysteamine.HCl from Toronto Research Chemicals Inc. Vancomycin.HCl 1 was purchased from Hallochem Pharma Co. Ltd, daptomycin 4 from Molekula (Cat # 64342447) and oritavancin diphosphate 5 (Cat # 317101), and dalbavancin.HCl 3 (Cat # 317136) from MedKoo Biosciences. Dalbavancin 3 was also synthesised internally, as was telavancin 2. Analytical liquid chromatography / mass spectrometry (LC/MS) was performed using reverse-phase high-performance liquid chromatograph (RP-HPLC) on an Eclipse XDB-Phenyl column  coupled to an Agilent 1200 series system with detection by a single quadrupole mass spectrometer (6110), a UV detector operating at 210 nm and an evaporative light scattering detector (ELSD). Preparative HPLC was performed using reverse-phase HPLC on an Eclipse XDB-Phenyl column  coupled to an Agilent 1260 Infinity system with detection by UV (210 nm). Elution was effected using appropriate gradients with 0.05% formic acid  or 0.1% trifluoroacetic acid (TFA) (preparative) in water (solvent A) and 0.05% formic acid  or 0.1% TFA (preparative) in acetonitrile (solvent B). (+)-electrospray ionisation mass spectrometry/mass spectrometry (ESI–MS/MS) were obtained using an API QSTAR™ Pulsar Hybrid LC-MS/MS System using the following conditions: column oven 40 °C, MS parameters: CUR: 20, IS: 5300, TEM: 450, GS1: 45, GS2: 45, ihe: ON, DP 60. HPLC Column: Waters Atlantis® T3 5 µm 2.1 × 50 mm with Atlantis® T3 5 µm 2.1 × 10 mm guard column. Flow rates and solvent: 0.4 mL min−1, solvent A: 0.1% formic acid in H2O, solvent B: 0.1% formic acid in acetonitrile; isocratic 0% B from 0→1 min, gradient 0%→100% B from 2→9 min, isocratic 100% from 5→12 min, gradient 100%→0% B from 12→12.5 min and isocratic 2% B from 12.5→17.5 min. High resolution mass spectrometry (HRMS) was performed on a Bruker Micro time-of-flight (TOF) mass spectrometer using (+)-ESI calibrated to NH4OAc.1H (600 MHz), 13C (150 MHz) and 2D NMR spectra were obtained in d6-dimethylsulphoxide (DMSO) calibrated to TMS resonances  using a Bruker Avance-600 spectrometer equipped with a TXI cryoprobe.l-Lys(ivDde)-OH or Fmoc-l-Orn(ivDde)-OH (5 eq), hydroxybenzotriazole (5 eq) in anhydrous DMF (2.5 mL per 1 mmol amino acid) was treated with 1,3-diisopropylcarbodiimide (DIC) (5 eq) and 4-dimethylaminopyridine (0.3 eq). The resulting solution was added to the resin and the resin was shaken at room temperature overnight. The resin was drained, and successively washed with DMF, methanol (MeOH) and dichloromethane (DCM) (3 × each). A solution of acetic anhydride (4 eq) and diisopropylethylamine (DIPEA) (8 eq) in DMF (~10 mL per 1 gram of resin) was added to the resin to block any unreacted sites. After shaking for 1 h, the resin was drained and washed with DMF, MeOH and DCM (3 × each) and dried in vacuo.HypoGel HMBA resin was washed with anhydrous dimethylformamide (DMF) (3×) under an inert atmosphere. A mixture of Fmoc-The starting resin was washed with anhydrous DMF (3 × ). Fmoc-deprotection was performed by shaking the resin with piperidine in DMF  for 30 min. The resin was drained and washed successively with DMF, MeOH, DCM and DMF (3 × each). A solution of Fmoc-protected amino acid or fatty acid (4 equiv) in DMF (~0.4 M) was treated with HBTU (4 equiv) and DIPEA (16 equiv). After 10 min, the activated amino acid solution was added to the resin. The resin was agitated at room temperature (RT) for 1 h, drained, washed successively with DMF, MeOH and DCM (3 × each), and dried in vacuo. Coupling efficiency was assessed using the Kaiser test and/or by cleavage of 2–3 mg of resin and analysis by LCMS. If required, the coupling reaction was repeated.2O). The material was then used in the next step without further purification (intermediate) or purified by preparative HPLC .Crude lipopeptide was treated with an excess of hydrazine hydrate  at RT. Reaction progress was monitored by LC/MS. Upon completion, the solution was concentrated in vacuo and the resulting crude product was lyophilised (ACN/HThe Mtt-protected vancomycin derivative was treated with a solution of TFA/triethylsilane (TES)/DCM  at room temperature for 1 h and concentrated under reduced pressure. The process was repeated if deprotection was incomplete as determined by LCMS analysis. Upon completion, the solution was concentrated in vacuo and the crude product was purified by preparative HPLC.2O). (ii) HMBA hypogel resin: C-terminal N-methyl amides. Prior to cleavage, the resin was washed with tetrahydrofuran (THF) (3×) and DIPEA (2×) and then treated with methylamine  with overnight agitation at RT. The resin was drained, and washed with THF, DCM and ACN (3× each). The combined filtrates were removed under reduced pressure to afford crude product.(i) Rink-amide, Wang- and 2-CTC-resins. Peptides incorporating cysteine were cleaved from the resin by agitation with TFA/1,2-ethanedithiol (EDT)/triisopropylsilane (TIPS)/water . Non-cysteine containing peptides were cleaved from the resin by agitation with TFA/TIPS/water . Resins were filtered and washed with TFA (3×). The combined filtrates were concentrated in vacuo and the crude peptides were precipitated with cold diethyl ether, isolated by centrifugation and lyophilised (ACN/H43 (1.5 equiv) in acetonitrile/water  was added. After standing at room temperature for 1 h, the reaction mixture was concentrated by lyophilisation, and the resulting crude product was purified by preparative RP-HPLC.GPeptides incorporating cysteine were dissolved in acetonitrile/water , and the pH was adjusted (pH 8–9) by addition of DIPEA (ca. 5 eq). A solution of O-(7-Azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate (HATU) (1.3 eq) and vancomycin hydrochloride (1.2 eq) in anhydrous DMF (~ 30 mL per 1 mmol of peptide) was cooled to 0 °C. After 30 min, DIPEA was added (10 eq), and stirring was continued for 1 h at room temperature. The solvent was evaporated in vacuo and the residue was lyophilised from ACN/H2O. The crude product was purified by preparative RP-HPLC.A stirred solution of peptide (1 eq), m/z 144, which corresponded to the loss of the vancosamine. This and other fragments showed that the two sugars, vancosamine and glucose, remained intact throughout the reaction sequence. Sequential losses of the MIE and EEPS fragments could also be observed in the MS/MS spectra. 1H, 13C and 2D  NMR experiments were used to confirm the structure of 19 (Supplementary Table Compound purity was determined using LC-(+)-ESI-MS/UV/ELSD and their molecular formulae determined by (+)-HR-ESI-MS. (+)-ESI-TOF-MS/MS analysis was also undertaken and representative examples of fragmentation are given in Supplementary Fig. 1  in dry dimethylsulphoxide (DMSO)  was added a solution of (S)-pyridylthio cysteamine hydrochloride  in dry DMF . PyBOP  in dry DMF  was added followed by DIPEA . The resulting solution was stirred at room temperature overnight and the solvent was removed under reduced pressure. The crude product was precipitated with diethyl ether and collected by vacuum filtration as a white solid, 10.8 g. The crude product was used for the disulphide exchange reaction without further purification. A small amount of crude product (100 mg) was purified by HPLC to give 43 as white solid , 47 mg, 71% yield. HPLC purity > 95%, tR 6.4. (ES) m/z (MH22+) 808.7, (MH33+) 540.0. HRMS exact mass (ESI microTOF-LC): calcd for C73H85Cl2N11O23S2 808.7314 (MH22+), found 808.7348.To a solution of vancomycin hydrochloride tR 4.8. (ES) m/z (MH33+) 1150.3, (MH44+) 863.0 (MH55+) 690.7, (MH66+) 575.8, (MH77+) 493.7.HPLC purity > 95%, nC11CO-GSSKSPSKKKKKKPGDC(SCH2CH2NH-Vancomycin)-NH2(7): HPLC purity > 95%, tR 5.0. (ES) m/z (MH33+) 1159.4, (MH44+) 870.2 (MH55+) 696.3, (MH66+) 580.5, (MH77+) 497.6. HRMS exact mass (ESI microTOF-LC): calcd for C155H241Cl2N35O47S24+ 869.6585 (MH44+), found 869.6595.Myristoyl-GSSKSPSKKKKKKPGDC-NH2(8a): HPLC purity > 95%, tR 5.5. (ES) m/z (MH22+) 1000.0, [M + 3H]3+ 667.2, [M + 4 H]4+ 500.7, [M + 5 H]5+ 400.8, HRMS exact mass (ESI microTOF-LC): calcd for C89H167N25O24S4+ 500.5579 (MH44+), found 500.5645.nC13CO-GSSKSPSKKKKKKPGDC(SCH2CH2NH-Vancomycin)-NH2(8): HPLC purity > 95%, tR 5.0. (ES) m/z (MH33+) 1168.9, (MH44+) 877.2 (MH55+) 702.0, (MH66+) 585.2, (MH77+) 501.7. HRMS exact mass (ESI microTOF-LC): calcd for C157H248Cl2N35O47S27+ 501.3839 (MH77+), found 501.3819.nC15CO-GSSKSPSKKKKKKPGDC(SCH2CH2NH-Vancomycin)-NH2(9): HPLC purity > 95%, tR 5.3. (ES) m/z (MH33+) 1178.2, (MH44+) 884.1 (MH55+) 707.5, (MH66+) 589.8, (MH77+) 505.7. HRMS exact mass (ESI microTOF-LC): calcd for C159H252Cl2N35O47S27+ 505.3884 (MH77+), found 505.3859.4-Ph-Bz-GSSKSPSKKKKKKPGDC(SCH2CH2NH-Vancomycin)-NH2(10): HPLC purity > 95%, tR 4.8. (ES) m/z (MH33+) 1159.2, (MH44+) 869.6, (MH55+) 695.8, (MH66+) 580.1, (MH77+) 497.3.nC13CO-GSSKSPSKKKPGDC(SCH2CH2NH-Vancomycin)-NH2(11): HPLC purity > 95%, tR 6.1. (ES) m/z (MH33+) 1040.8, (MH44+) 781.0 (MH55+) 625.0, (MH66+) 521.0. HRMS exact mass (ESI microTOF-LC): calcd for C139H211Cl2N29O44S26+ 520.7325 (MH66+), found 520.7328.Myristoyl-GSSKSPSKKKPC-NH2(12a): HPLC purity > 95%, tR 5.7. (ES) m/z (MH22+) 721.9, [M + 3 H]3+ 481.7, [M + 4 H]4+ 361.5. HRMS exact mass (ESI microTOF-LC): calcd for C65H121N17O17S2+ 721.9418 (MH22+), found 721.9427.nC13CO-GSSKSPSKKKPC(SCH2CH2NH-Vancomycin)-NH2(12): HPLC purity > 95%, tR 5.2. (ES) m/z (MH22+) 1474.1, (MH33+) 983.6, (MH44+) 738.0 (MH55+) 590.2, (MH66+) 492.4. HRMS exact mass (ESI microTOF-LC): calcd for C133H202Cl2N27O40S25+ 590.2679 (MH55+), found 590.2701.Myristoyl-GSKKKC-NH2(13a): HPLC purity > 95%, tR 6.1. (ES) m/z (MH+) 859.4, (MH22+) 430.3, [M + 3 H]3+ 287.3. HRMS exact mass (ESI microTOF-LC): calcd for C40H80N10O8S2+ 430.2935 (MH22+), found 430.3061.nC13CO-GSKKKC(SCH2CH2NH-Vancomycin)-NH2(13): HPLC purity > 95%, tR 5.8. (ES) m/z (MH22+) 1182.4, (MH33+) 789.0, (MH44+) 592.1 (MH55+) 473.9. HRMS exact mass (ESI microTOF-LC): calcd for C108H160Cl2N20O31S24+ 591.7589 (MH44+), found 591.7585.Myristoyl-GSKKKC-OH (14a): HPLC purity > 95%, tR 6.3. (ES) m/z (MH+) 860.4, (MH22+) 430.8, (MH33+) 287.6. HRMS exact mass (ESI microTOF-LC): calcd for C40H78N9O9S+ 860.5638, found 860.5802.nC13CO-GSKKKC(SCH2CH2NH-Vancomycin)-OH (14): HPLC purity > 95%, tR 5.9. (ES) m/z (MH22+) 1184.7, (MH33+) 789.7, (MH44+) 592.4 (MH55+) 474.1. HRMS exact mass (ESI microTOF-LC): calcd for C108H158Cl2N19O32S24+ 789.0041 (MH44+), found 789.0006.Myristoyl-KKKC-OH – disulphide dimer (15a): HPLC purity > 95%, tR 6.4. (ES) m/z (MH+) 1429.8, (MH22+) 715.5, (MH33+) 477.4, (MH44+) 358.4 (MH55+) 286.9. HRMS exact mass (ESI microTOF-LC): calcd for C70H138N14O12S22+ 715.5025, found 715.5058.nC13CO-KKKC(SCH2CH2NH-Vancomycin)-OH (15): HPLC purity > 95%, tR 5.8. (ES) m/z (MH22+) 1110.8, (MH33+) 741.5, (MH44+) 556.4 (MH55+) 445.4. HRMS exact mass (ESI microTOF-LC): calcd for C103H149Cl2N17O29S22+ 1110.9757 (MH22+), found 1110.9783.nC13CO-GSKKK-K(Vancomycin)-OH (16): HPLC purity > 95%, tR 6.1. (ES) m/z (MH33+) 772.9, (MH44+) 579.8, (MH55+) 464.1. HRMS exact mass (ESI microTOF-LC): calcd for C109H159Cl2N19O22+ 1158.0382 (MH22+), found 1158.0396.nC13CO-KKK-K(Vancomycin)-OH (17): HPLC purity > 95%, tR 5.9. (ES) m/z 1086.9 (MH22+), (MH33+) 724.8, (MH44+) 543.8. HRMS exact mass (ESI microTOF-LC): calcd for C104H153Cl2N17O294+ 543.5094 (MH44+), found 543.5109.nC10CO-KKK-K(Vancomycin)-OH (18): HPLC purity > 95%, tR 5.5. (ES) m/z (MH22+) 1065.8, (MH33+) 710.7, (MH44+) 533.4. HRMS exact mass (ESI microTOF-LC): calcd for C101H146Cl2N17O293+ 710.3278 (MH33+), found 710.3245.nC10CO-KK-K(Vancomycin)-OH (19): HPLC purity > 95%, tR 4.5. (ES) m/z (MH22+) 1001.9, (MH33+) 668.4, (MH44+) 501.3. HRMS exact mass (ESI microTOF-LC): calcd for C95H135Cl2N15O284+ 500.9739 (MH44+), found 500.9753.nC10CO-K(Vancomycin)-OH (20): HPLC purity > 95%, tR 7.1. (ES) m/z (MH22+) 873.5, (MH33+) 582.9. HRMS exact mass (ESI microTOF-LC): C83H110Cl2N11O263+ 582.2328 (MH33+), found 582.2322.nC10CO-KK-K(Vancomycin)-NH2 (21): HPLC purity > 95%, tR 5.4. (ES) m/z (MH22+) 1000.3, (MH33+) 667.8, (MH44+) 501.0. HRMS exact mass (ESI microTOF-LC): calcd for C95H136Cl2N16O274+ 500.7279 (MH44+), found 500.7270.nC10CO-KK-K(Vancomycin)-NHMe (22): HPLC purity > 95%, tR 5.6. (ES) m/z (MH22+) 1007.8, (MH33+) 672.3, (MH44+) 504.5. HRMS exact mass (ESI microTOF-LC): calcd for C96H138Cl2N16O274+ 504.2318 (MH44+), found 504.2315.POP2K-KK-K(Vancomycin)-OH (23): HPLC purity > 95%, tR 6.7. (ES) m/z (MH22+) 1177.3, (MH33+) 785.3, (MH44+) 589.4. HRMS exact mass (ESI microTOF-LC): calcd for C116H141Cl2N17O323+ 1176.9647 (MH33+), found 1176.9644.POB2K-KK-K(Vancomycin)-NHMe (24): HPLC purity > 95%, tR 6.1. (ES) m/z (MH22+) 1185.2, (MH33+) 789.9, (MH44+) 592.6. HRMS exact mass (ESI microTOF-LC): calcd for C117H144Cl2N18O312+ 1183.4806 (MH22+), found 1183.4811.Ac-KK-K(Vancomycin)-OH (25): HPLC purity > 95%, tR 4.6. (ES) m/z (MH22+) 939.7, (MH33+) 626.0, (MH44+) 469.8. HRMS exact mass (ESI microTOF-LC): calcd for C86H117Cl2N15O284+ 469.4387 (MH44+), found 469.4365.POB2K-K-Dap-K(Vancomycin)-NHMe (27): HPLC purity > 95%, tR 5.3. (ES) m/z (MH22+) 1162.4, (MH33+) 775.8, (MH44+) 582.1. HRMS exact mass (ESI microTOF-LC): calcd for calcd for C114H139Cl2N18O313+ 775.3071 (MH33+), found 775.3041.POB2K-Dap-K-K(Vancomycin)-NHMe (28): HPLC purity > 95%, tR 5.2. (ES) m/z (MH22+) 1163.8, (MH33+) 775.8, (MH44+) 582.1. HRMS exact mass (ESI microTOF-LC): calcd for C114H139Cl2N18O313+ 775.3071 (MH33+), found 775.3042.POB2K-Dap-Dap-K(Vancomycin)-NHMe (29): HPLC purity > 95%, tR 5.4. (ES) m/z (MH22+) 1142.7, (MH33+) 761.9, (MH44+) 571.6. HRMS exact mass (ESI microTOF-LC): calcd for C111H133Cl2N18O313+ 761.2915 (MH33+), found 761.2932.POB2K-K-Dab-K(Vancomycin)-NHMe (30): HPLC purity > 95%, tR 5.2. (ES) m/z (MH22+) 1169.4, (MH33+) 780.5, (MH44+) 585.7. HRMS exact mass (ESI microTOF-LC): calcd for C115H141Cl2N18O313+ 779.9790 (MH33+), found 779.9766.POB2K-Dab-K-K(Vancomycin)-NHMe (31): HPLC purity > 95%, tR 5.4. (ES) m/z (MH22+) 1169.9, (MH33+) 780.5, (MH44+) 585.6. HRMS exact mass (ESI microTOF-LC): calcd for C115H141Cl2N18O313+ 779.9790 (MH33+), found 779.9761.POB2K-Orn-Orn-K(Vancomycin)-NHMe (34): HPLC purity > 95%, tR 5.1. (ES) m/z (MH22+) 1170.7, (MH33+) 779.9, (MH44+) 585.6. HRMS exact mass (ESI microTOF-LC): calcd for C115H141Cl2N18O313+ 779.9790 (MH33+), found 779.9788.POB2K-K-Orn-K(Vancomycin)-NHMe (32): HPLC purity > 95%, tR 5.1. (ES) m/z (MH22+) 1176.8, (MH33+) 785.0, (MH44+) 589.2. HRMS exact mass (ESI microTOF-LC): calcd for C116H144Cl2N18O314+ 588.7400 (MH44+), found 588.7418.POB2K-Orn-K-K(Vancomycin)-NHMe (33): HPLC purity > 95%, tR 5.2. (ES) m/z (MH22+) 1177.8, (MH33+) 785.2, (MH44+) 589.2. HRMS exact mass (ESI microTOF-LC): calcd for C116H143Cl2N18O313+ 784.6509 (MH33+), found 784.6490.POB2K-KK-Orn(Vancomycin)-NHMe (35): HPLC purity > 95%, tR 6.1. (ES) m/z (MH22+) 1177.8, (MH33+) 785.2, (MH44+) 589.1. HRMS exact mass (ESI microTOF-LC): calcd for C116H143Cl2N18O313+ 784.6509 (MH33+), found 784.6497.nC9CO-KKK-K(Vancomycin)-OH (36): HPLC purity > 95%, tR 5.2. (ES) m/z (MH22+) 1059.2, (MH33+) 706.2, (MH44+) 529.8. HRMS exact mass (ESI microTOF-LC): calcd for C100H145Cl2N17O29 529.4937 (MH44+), found 529.4933.nC11CO-KKK-K(Vancomycin)-OH (37). HPLC purity > 95%, tR 5.6. (ES) m/z (MH22+) 1071.9, (MH33+) 715.0, (MH44+) 536.9. HRMS exact mass (ESI microTOF-LC): calcd for C102H147Cl2N17O29 1071.9958 (MH22+), found 1072.0001.nC12CO-KKK-K-(Vancomycin)-OH (38): HPLC purity > 95%, tR 5.7. (ES) m/z (MH22+) 1078.9, (MH33+) 719.9, (MH44+) 540.3. HRMS exact mass (ESI microTOF-LC): calcd for C103H150Cl2N17O29 719.6715 (MH33+), found 719.6710.nC10CO-GSKKK-K-(Vancomycin)-OH (39): HPLC purity > 95%, tR 5.4. (ES) m/z (MH22+) 1136.9, (MH33+) 758.7, (MH44+) 569.1. HRMS exact mass (ESI microTOF-LC): calcd for C106H154Cl2N19O32 758.3456 (MH33+), found 758.3477.POB-KKK-K-(Vancomycin)-OH (40): HPLC purity > 95%, tR 5.0. (ES) m/z (MH22+) 1079.3, (MH33+) 720.2, (MH44+) 540.4. HRMS exact mass (ESI microTOF-LC): calcd for C103H133Cl2N17O302+1078.9385 (MH22+), found 1078.9326.POB-KK-K-(Vancomycin)-OH (41): HPLC purity > 95%, tR 5.9. (ES) m/z (MH22+) 1015.3, (MH33+) 677.3, (MH44+) 508.3. HRMS exact mass (ESI microTOF-LC): calcd for C97H123Cl2N15O294+ 507.9492 (MH44+), found 507.9498.POB2K-KKK-K-(Vancomycin)-OH (42): HPLC purity > 95%, tR 6.0. (ES) m/z (MH22+) 1241.3, (MH33+) 828.3, (MH44+) 621.5. HRMS exact mass (ESI microTOF-LC): calcd for C122H155Cl2N19O334+ 621.0097 (MH44+), found 621.0068.S. aureus , Streptococcus pneumoniae (MDR ATCC® 700677™) and Enterococcus faecium (MDR VanA ATCC® 51559™). ATCC strains were sourced from the American Type Culture Collection and NRS strains from NARSA  via BEI Resources (www.beiresources.org). All compounds were prepared to 160 µg mL−1 solution in water from a stock solution of 1 mM concentration.Antimicrobial activity of compounds was tested against a number of bacterial strains by broth microdilution (BMD) assay with MIC determination, including −1 and compounds from 8 to 0.003 µg mL−1 with final assay volumes of 100 µL per well. Gram-positive bacteria were cultured in Mueller Hinton broth (MHB)  at 37 °C overnight. A sample of each culture was then diluted 40-fold in fresh MHB and incubated at 37 °C for 2–3 h. The resultant mid-log phase cultures were added to the compound-containing 96-well plates to give a final cell density of 5 × 105 colony forming unit (CFU) mL−1. All the plates were covered and incubated at 37 °C for 24 h. MICs were the lowest concentration showing no visible growth.The compounds, along with standard antibiotics were serially diluted twofold across the wells of 96-well non-binding surface (NBS) polystyrene plates (Corning 3641). , but for lipoglycopeptide antibiotics  where Clinical & Laboratory Standards Institute (CLSI) guidelines recommend addition of 0.002% polysorbate 80 for MIC determination, values obtained in NBS plates with no additive match those obtained in untreated polystyrene plates with addition of 0.002% polysorbate 80—Supplementary Table The compounds, along with standard antibiotics, tested in BMD-MIC assay as above, in the presence of a mixture of 50% human serum (Sigma-Aldrich) and 50% MHB.−1) in MHB.The compounds, along with standard antibiotics, were tested in BMD-MIC assay as above, in the presence of 1 and 5% Survanta (beractant) suspension , in duplicate. The tripeptide N,N΄-diacetyl-l-Lys-d-Ala-d-Ala was serially diluted twofold across the compound-containing wells from 6400 to 3 µg mL−1. To this the resultant mid-log phase cultures were diluted to the final concentration of 5 × 105 CFU mL−1, then added to wells of the compound-containing plates. All the plates were covered and incubated at 37 °C for 24 h. The molar excess was determined as the ratio of [ligand]/[antibiotic] in the well containing the lowest ligand concentration that completely suppressed inhibition after 24 h incubation.All compounds were dissolved in HS. aureus ATCC® 43300™ (MRSA) was cultured in MHB  at 37 °C overnight. A sample was then diluted 40-fold in fresh MHB and incubated at 37 °C for 2–3 h until the culture reached early exponential phase (OD600 of 0.2–0.4). Vancapticin 24  and vancomycin  were serially diluted twofold across the wells of 96-well microtitre plates  with a final volume of 50 µL, then combined with 50 µL of bacterial inoculum  and incubated at 37 °C. Serial dilutions of the cultures were plated at each desired time point, using a multichannel pipette as follows: in row A of a 96-well plate, 50 µL of sterile activated charcoal suspension (25 mg mL−1) was added, while 90 µL of 0.9% sterile saline per well was added to the other rows. At selected time-points, 50 µL aliquots were transferred from the time-kill assay plate to the first row containing the charcoal suspension and mixed well. The wells were further diluted 1 in 10  for the appropriate number of dilutions and 10 μL of each dilution was spotted in duplicate onto Luria-Bertani (LB) agar, then incubated overnight at 37 °C. The colonies in each spot were counted and used to calculate the number of viable CFU mL−1 remaining in the original culture by considering the dilution factors .19 was determined following literature procedures42.The resistance frequency for Compound 19 was prepared to 160 µg mL−1 solution in water from the given stock solution of 1 mM concentration. Vancomycin was prepared to 1.28 mg mL−1 solution in water.Compound −1) with swirling to ensure that the bottom of the plate was coated equally. An additional 80 mL of fresh agarose was added to the remaining 80 mL of agarose [A] sample to achieve a twofold dilution to provide agarose sample [B]. Once again, 80 mL of agarose sample [B] was plated in four petri plates. This procedure was repeated until a range of concentrations of the antibacterial compound above the expected MIC was obtained. This method was done individually for every antibacterial compound under study. Agarose plate MIC values used for this study were determined to be 1 µg mL−1 for vancomycin and 0.06 µg mL−1 for compound 19.Tryptic soy broth (TSB) with 1% agarose and 0.002% Tween 80 was maintained at 45 to 50 °C in a water bath. 160 mL of agarose was transferred to 200 mL glass bottle, maintaining the remaining agarose in the water bath. The appropriate amount of compound  was added to the 160 mL agarose sample [A]. A volume of 80 mL of agarose sample [A] was plated in four petri plates  were used to determine the titre of the culture. An aliquot of 100 µL (10 µL in some cases) of these three dilutions were added to compound-free agar petri plates. This was done in duplicate and the plates were incubated overnight at 37 °C. Resultant separated visible colonies were counted; those colonies that grow together were not included to determine the titre of the culture. An aliquot of 100 µL of the neat (undiluted) stationary-phase culture and 10−1 dilution was plated separately onto the agar plate containing each concentration of antibacterial compound. The plates were incubated overnight at 37 °C.Bacteria were subcultured at 37 °C overnight with shaking at 220 rpm. The bacterial culture was serially diluted in MHB to achieve a 10The colonies were counted after 24 h. The resistance frequency is determined by dividing the number of colonies obtained on the plates containing the antibacterial agent by the number of cells plated.17 and compound 2443.A resistance induction assay was conducted for vancomycin, daptomycin, compound S. aureus ATCC®43300™ was grown in Mueller Hinton broth supplemented with 50 µg mL−1 of CaCl2. The bacterial culture was incubated at 37 °C in a shaker at 195 rpm.S. aureus growth culture was serially diluted and plated on a solid LB agar plates in quadruplicates and incubated at 37 °C overnight to determine viable colony count.The mid log −1 to 0.08 µg mL−1 for daptomycin, 32 µg mL−1 to 0.06 µg mL−1 for vancomycin and 0.5 µg mL−1 to 0.001 µg mL−1 for both vancapticins with 100 µL of the dilutions added to the plate. MRSA culture in mid log growth with 0.6 optical density at 600 nm (OD600) was diluted to an equivalence of 105 CFU mL−1 and 100 µL was added to the antimicrobial containing plate. The plate was then incubated at 37 °C overnight.The initial determination of the compound MICs were taken from the literature and empirically measured. Doubling MIC dilutions were prepared in Cation-adjusted Mueller-Hinton broth (Ca-MHB) in NBS 96-well plates  with columns 11 and 12 allocated for bacterial culture without the antimicrobial and Ca-MHB media. The starting MIC ranges were 20 µg mLS. aureus (MRSA) culture was determined by reading at OD600 on the Epoch microplate spectrophotometer and 0.1 OD600 was the chosen cut-off determining growth vs. no growth. The sample selection of S. aureus and preparation was carried out as previously described43 with 10 µL of the sub-lethal MIC S. aureus cultures (i.e. the maximum antibiotic concentration with OD600 ≥ 0.1) diluted in 10 mL of Ca-MHB for the following day’s MIC plate. Doubling dilutions were supplemented with intermediate MICs to reduce the MIC difference and provide a ladder for non-susceptible isolates for the 20 day period with an average overnight incubation of 19 h at 37 °C. The non-susceptible isolates were grown overnight at 37 °C with shaking before being preserved in 20% glycerol and as a pellet (stored frozen at −80 °C).After overnight incubation, the growth of the ®HB-8065 liver hepatoceullular carcinoma) and HEK293  human cell lines using standard −2,5-diphenyltetrazolium bromide (MTT) assay protocol with slight changes. In brief, HepG2 and HEK293 cells  were seeded as 1.5 × 104 cells per well in a clear 96-well tissue culture treated plate in a final volume of 100 μL in Dulbecco’s Modified Eagle Medium (DMEM)(GIBCO-Invitrogen #11995-073), in which 1% of FBS was added. Cells were incubated for 24 h at 37 °C, 5% CO2 to allow cells to attach to the plates. Compounds prepared at 100 µM to 0.046 µM in threefold dilutions were added into each well and incubated for 24 h at 37 °C, 5% CO2. After the incubation, 0.4 mg mL−1 MTT −2,5-diphenyltetrazolium bromide) (Invitrogen) was added to each well. The plates were then incubated for 2 h at 37 °C, 5% CO2. The media was removed, and purple formazan crystals were resuspended in 60 μL of DMSO. The absorbance was read at 570 nm using a Polarstar Omega plate reader. The data was then analysed by Prism software. Results are presented as the average percentage of control ± S.D. for each set of duplicate wells using the following equation: percent viability = (ABSTEST – ABSPOSITIVE CTRL / ABSUNTREATED –ABS POSITIVE CTRL)×100.Compound cytotoxicity was evaluated against HepG2  to the final volume of 80 μL and incubated at 37 °C for 3 h on a horizontal shaker at 200 rpm. For negative and positive controls, human whole blood in 0.9% NaCl solution (Ablank) and in sdH2O (Awater) were used, respectively. After incubation, both test and Ablank were mixed with 1 mL 0.9% NaCl solution, or sdH2O for the complete hydrolysis Awater. After centrifugation of samples at 2500 rpm (700 × g) for 5 min, the release of haemoglobin was monitored by measuring the absorbance (Asample) of the supernatant at 540 nm using BMG Labtech PolarStar Omega multimode reader. The percentage of haemolysis was calculated according to the equation:Haemolytic activity was measured as previously described with slight modifications46 with a higher throughput modification. Briefly, a cascade dilution of each antibiotic was performed in a low binding polypropylene V-bottom shaped 96-well plates in the presence of 0.2 M NaCl solution across 12 concentrations ranging from 1000 to 0.1 μM, except the control melittin (100 to 0.01 μM). An identical cascade dilution was performed for the neutral detergent Triton X-100 and the anionic detergent sodium dodecyl sulphate (SDS) starting from 0.1% down to 0.00001% (vol:vol for Triton X-100 and wt:vol for SDS). An aliquot of 6-diphenyl-1,3,5-hexatriene (DPH) in DMSO was added in each well to yield the final DPH concentration of 5 μM. The reaction mixtures were mixed for 30 s and transferred in horizontal triplicate (25 μL per well) into a black falcon 384-well plate. The 384-well plate was then sealed, wrapped up in aluminium foil and incubated for 30 min at room temperature. The fluorescence intensity was monitored in real time using a BMG Labtech PolarStar Omega multimode reader fitted with 360-10 and 430-10 excitation and emission filters respectively, at an excitation wavelength of 360 nm and an emission wavelength of 430 nm.CMCs were determined as previously described37. Briefly, early exponential phase cells were pelleted and washed twice in 10 mM HEPES buffer (pH 7.4) containing 50 µg mL−1 of CaCl2 and 5 mM Glucose, and then re-suspended in the same buffer (2 × 108 CFU mL−1). diSC3(5) was added to a final concentration of 1.5 µM and incubated at room temperature in the dark to enable dye uptake and fluorescence quenching. After 30 min, cells were diluted 50-fold in assay buffer and 45 µL of cells were added to a 384-well black walled polystyrene plate   in the presence of untreated cells, no compound). Note that the MIC values for oritavancin and compound 17 increase to 1 µg mL−1, and compound 24 to 2 µg mL−1, in polystyrene plates. Each sample was tested in quadruplicate and independent assays were performed twice showing similar results. Data were analysed and presented using Prism software.Antibiotic-induced bacterial cytoplasmic membrane depolarisation was determined using the fluorescent dye 3,3-dipropylthiacarbocyanine diSC3(5)  as previously described−1 of CaCl2 and 5 mM glucose, and then re-suspended in the same buffer (4 × 108 CFU mL−1). Cell suspension was added to a 384-well black walled polystyrene plate  containing compounds, giving a final concentration of 16 µg mL−1. After 1 h incubation at 37 °C, 5 µg mL−1 of PI was added and fluorescence was monitored for 90 min using a Tecan Infinite® m1000 Pro Multi-mode reader (excitation/emission 535/620 nm). Data were corrected by subtraction of fluorescence signal arising from untreated cells in the presence of PI. Note that the MIC values for oritavancin and compound 17 increase to 1 µg mL−1, and compound 24 to 2 µg mL−1, in polystyrene plates. Each sample was tested in quadruplicate and independent assays were performed twice showing similar results. Data was analysed and presented using Prism software.To evaluate the ability of the compounds to disrupt the cytoplasmic membrane integrity, the membrane impermeable fluorescent DNA intercalating dye propidium iodide (PI) was used. Early exponential phase cells were pelleted and washed twice in 10 mM HEPES buffer (pH 7.4) containing 50 µg mL−1 reference power and stirring speed of 1000 rpm. All samples were prepared in 0.1 M NaOAc, pH 5.0 buffer. Each experiment consisted of sequential injections into the calorimeter cell (0.2 mL) with 240 s equilibration intervals. Corrections of small heat effects from separate blank titrations were made prior to analysis. Data were processed using the MicroCal Origin 7.0 software package provided with the instrument to fit a single site binding model or dissociation model to estimate the association constant (Ka), enthalpy (ΔH), entropy (ΔS) and stoichiometry (N). The Gibbs free energy change (∆G) was calculated using the Gibbs–Helmholz thermodynamic equation, , at 298 K, with a 10 μcal sR is the ideal gas constant (8.31 J mol−1 K−1) and T is the temperature in Kelvin.Where Ligand binding experiments consisted of 12 sequential injections  of ligand  into the calorimeter cell containing the antibiotic (monomer solution 25 µM for vancomycin and 40 µM for vancapticins). Antibiotic concentration was selected to avoid self-dimerisation.Dissociation experiments involved 13 sequential injections  of concentrated (3.0 mM) antibiotic solutions titrated to buffer only. Dissociation experiments involving ligand maintained a constant concentration of excess ligand (9.0 mM) dissolved in buffer in both the calorimeter cell and titration syringe to ensure that antibiotic molecules were predominantly in their ligand-complexed form.sn-glycero-3-phosphocholine) and DMPG  were purchased from Avanti Polar Lipids. Small unilamellar vesicles (SUV) were prepared in phosphate buffered saline (PBS) (pH 7.4) by sonication and extrusion. In brief, lipids were dissolved in chloroform in 25 mL round-bottom flasks, and then deposited as a thin film by removal of the solvent (chloroform) under reduced pressure on a rotary evaporator and dried under high vacuum for at least 2 h. PBS was then added into each flask to give a suspension, which was sonicated for 5 cycles of 5 min each. The suspension was passed 17 × through a 50-nm polycarbonate filter in an Avestin Lipofast Basic extrusion apparatus to give a translucent solution of vesicles, which should possess a mean diameter of 50 nm. The SUVs were injected across the L1 sensor chip for 30 min at a flow rate of 2 mL min−1 to form a supported lipid bilayer. The coverage of the lipid bilayer was determined from the extent of non-specific binding of bovine serum albumin . A series of concentrations of vancomycin and vancapticins were passed sequentially over the different phospholipid-containing flow cells at a flow rate of 30 µL min−1 for 180 s. The amount of antibiotic bound at equilibrium just before the end of the injection was corrected by subtraction of the bulk-refractive index difference observed at the beginning and the end of each injection. After each injection cycle, the lipid surface was regenerated with 20 mM 3-[(3-cholamidopropyl)dimethylammonio]−1-propanesulfonate (CHAPS) and a fresh lipid bilayer was loaded as described above. All assays were carried out at 25 °C in triplicate. The equilibrium binding response values obtained were normalised by dividing the average observed response (RU) by the molecular weights (MW) of each compound, i.e. RUadjusted = 100 × RU/MW.SPR experiments were performed using a BIAcore 3000  instrument with a Biacore vesicle capture L1 sensor chip. DMPC  was used to catalyse the polymerisation of peptidoglycan from UPD-linked precursors in vitro following literature procedures47. B. megaterium was grown in the medium containing 1% (w/v) tryptone (Difco-Bacto), 0.5% (w/v) yeast extract (Difco-Bacto), 0.25% (w/v) K2HPO4 and 0.5% (w/v) glucose with pH 7.2. Bacteria were harvested when the density had reached to 0.4 mg dry weight mL−1 and washed in the buffer 0.05 M Tris-HCl (pH 7.8) containing 10 mM MgCl2 by centrifugation at 6000 × g for 10 min at 4 °C. All subsequent manipulations were carried out on ice. The bacteria were resuspended in the same buffer at a density of 50 mg dry weight mL−1, and then subjected to three freeze/thaw cycles. Bacteria were then homogenised by using a glass homogeniser at 4 °C (2 × 10 min). The broken cell suspension was centrifuged at 6000 × g for 10 min at 4 °C. The supernatant (Supernatant 1) which contained the unbroken cells and the majority of the cell walls was collected. The pellet was resuspended in the same buffer and re-centrifuged at 6000 × g for 10 min at 4 °C, and the supernatant (Supernatant 2) was collected. The two supernatants (Supernatant 1 and Supernatant 2) were combined and centrifuged at 20,500 rpm  for 1 h at 4 °C. The pellet, consisting mainly of membrane fragments together with some cell wall material, was washed once with the same buffer and finally resuspended in 0.05 M Tris-HCl (pH 7.8) containing 10 mM MgCl2. In vitro peptidoglycan synthesis was performed by mixing the following contents in a final volume of 20 µL: 50 mM Tris-HCl (pH 7.8), 10 mM MgCl2, B. megaterium membrane faction (around 40 to 50 µg protein), 0.4 mM uridine 5’-diphospho N-acetyl muramoyl-l-alanyl-d-γ-glutamyl-l-lysyl-d-alanyl-d-alanine , 6.5 µM [14C]UDP-N-acetylglucosamine  and vancapticins at final concentrations of 0, 0.03, 0.1, 1, 3 and 10 µg mL−1. The reaction mixtures were incubated at RT for 3 h and placed in a boiling water bath for 3 min to inactivate the enzymes. 5 µL of each sample was separated using TLC on silica gel plates  for 2 h in isobutyric acid /1 M NH4OH . The TLC plates were then dried and exposed to a phosphorimaging screen  for 1 week and scanned using a Typhoon 8600 . Integrated density value (IDV) of each band on silica gel was analysed using AlphaEase FC software (AlphaImager 2200). The change of peptidoglycan or Lipid II were calculated as percent from the IDV values of the control (without any antibiotic or compound).The cell-free particulate fraction of 48, with modifications. Briefly, test compounds (20 μM) were prepared from a 0.2 mM H2O stock solution (40 μL in 400 μL assay volume) except eucatropine, which was 300 μg mL−1 in 100% DMSO . The precipitating solution was prepared by adding 5 μL of 10 mM carbutamide in DMSO to 100 mL acetonitrile . The human plasma sample  and phosphate-buffer saline  were pre-heated at 37 °C for 30 min. 40 μL of test compounds (0.2 mM) were added to protein-low binding Eppendorf tubes, 160 μL of buffer was added, and the mixture was vortexed. For eucatropine, 10 μL of eucatropine (300 μg mL−1) was added to a protein-low binding Eppendorf tube, 190 μL of buffer was added, and the mixture was vortexed. In each case, 200 μL of plasma was added and the resultant mixture vortexed. For the initial time point (reaction time t = 0 h), 50 μL of sample was immediately transferred into a protein-low binding Eppendorf tube and quenched with 150 μL of the ice-cold precipitating solution, followed by vortexing and subsequent storage at 4 °C. For the remaining time points, the plasma solution was incubated and shaken (150 rpm) at 37 °C. 50 μL aliquots were collected at time points of 0, 1, 3, 6 and 24 h and quenched with 150 μL the ice-cold precipitating solution. All samples were placed in a 4 °C fridge for 30 min then centrifuged at 14,000 × g for 8 min at 4 °C. The supernatant (100 μL) was transferred to a glass vial insert and stored at 4 °C until LC-MS/MS analysis. The percentage of test compounds remaining at the individual time points relative to sample at time point 0 h were reported.Compound stability in human plasma was performed as previously reportedThe stability of compounds in the presence of physiological concentrations of glutathione was assessed according to the protocol below: 20 µL of a 1 mM stock solution of the test compound was added to 180 µL of glutathione (reduced form) PBS solution within a plastic HPLC insert, providing a solution with a final concentration of 100 µM of compound and either 5 mM or 0.5 mM in glutathione. The sample was placed in a HPLC sampling rack and sampled at hourly intervals up to 10 h. Care was taken to prepare the sample immediately before injection. The UV area was then plotted for the loss of compound against time. Percentages were plotted relative to total vancomycin derivative at 0 h. Results are shown in Supplementary Fig. Performed by CDCO, Monash University, Australia.−1 protein concentration. The metabolic reaction was initiated by the addition of a nicotinamide adenine dinucleotide phosphate (NADPH)-regenerating system (i.e. NADPH is the cofactor required for cytochrome P450-mediated metabolism) and quenched at various time points over the 60 min incubation period by the addition of acetonitrile. Control samples (containing no NADPH) were included  to monitor for potential degradation in the absence of cofactors. Concentrations of each test compound in quenched samples were determined by ultra performance liquid chromatography mass spectrometry (UPLC-MS) (Waters/Micromass Xevo triple quadrupole mass spectrometer). Note: Due to the minimal degradation of test compounds in this assay, a metabolite search was not conducted. In addition, glucuronidation was not considered in this study and may require further assessment using alternative test systems.The solubility of test compounds and their recovery from the incubation matrix were confirmed prior to the metabolic assay. The metabolic stability assay was performed by incubating each test compound (1 μM) with human  and mouse  liver microsomes at 37 °C and 0.4 mg mLk). Each substrate depletion rate constant was then used to calculate: (1) a degradation half-life, where t1/2 = ln(2) k−1; (2) an in vitro intrinsic clearance value, where CLint,in vitro = k [microsomal protein content (0.4 mg mL−1)]−1; (3) a predicted in vivo hepatic intrinsic clearance value, where CLint = CLint,in vitro × [liver mass (g)/body weight (kg)] × [microsomal protein (mg) / liver mass (g)]; and (4) a predicted in vivo hepatic extraction ratio, where EH = CLbloodQ−1 = Clint [Q + Clint]−1.Test compound concentration vs. time data were fitted to an exponential decay function to determine the first-order rate constant for substrate depletion. In cases where clear deviation from first-order kinetics was evident, only the initial linear portion of the profile was utilised to determine the degradation rate constant  20.7 mL min−1 kg body weight−151, mouse liver mass 87.5 g liver / kg body weight51, mouse microsomal protein 45 mg g−1 liver mass51, mouse hepatic blood flow (Q) 90 mL min−1 kg body weight−1 51.Scaling parameters assumed were: human liver mass 20 g liver / kg body weight−1 with a dose volume of 2 mg mL−1, providing a dose of 2 mg mL−1 (based on free base concentration). An additional three mice were dosed subcutaneously (SC) administered to each animal via subcutaneous bolus on each animals’ back per facility SOPs using test article formulated in deionised water at 2 mg mL−1 with a dose volume of 5 mg mL−1, providing a dose of 10 mg mL−1. All animals were killed at the last study time point .Performed by WuXi AppTec (Shanghai) Co., Ltd. Seven to 9-week-old male CD1 mice  weighing 25–35 g were acclimated for approximate 3 days before being used in the study. Animals are group housed during acclimation and in-life study in compliance with the National Research Council 'Guide for the Care and Use of Laboratory Animals.' The animal room environment is controlled  with temperature and relative humidity monitored daily. Animals were deprived of food for approximately 16 h before formulation administration then allowed access to Certified Rodent Diet  ad libitum 4 h post dosing. Water was autoclaved before being provided to the animals ad libitum. Formulations of compounds were prepared on the morning of the dosing day. The formulation for the IV group was filtered with filter of 0.22 μm before being dosed to animals. After dose formulation preparation, duplicate 50 μL aliquots were removed from each dose formulation for use in dose validation. For each compound studied, three mice were dosed intravenously (IV) administered to each animal via tail vein per facility SOPs using test article formulated in deionised water at 1 mg mLg) 4 °C for about 10 min. After centrifugation, plasma was transferred into another pre-labelled and pre-chilled polypropylene microcentrifuge tubes, then quick-frozen over dry ice and stored at −70 ± 10 °C until LC/MSMS analysis.Plasma samples were collected as the following target times after each dose administration: IV Sampling Time points (hours post dosing): 0, 0.083, 0.25, 0.5, 1, 2, 4, 8 and 24. SC Sampling Time points (hours post dosing): 0, 0.25, 0.5, 1, 2, 4, 8 and 24. Approximately 30 μL blood was obtained via submandibular or saphenous vein for the first several time points. For the last time point, samples were collected via cardiac puncture while the mouse was under anaesthesia. All blood samples were transferred into pre-chilled plastic microcentrifuge tubes containing 2 μL of ethylenediaminetetraacetic acid dipotassium salt (K2-EDTA) (0.5 M) as anticoagulant and placed on wet ice until centrifugation. Harvested blood samples were centrifuged within 30 min of collection at 7000 rpm . The sample analysis was performed concurrently with a set of calibration standards and two sets of QC samples using the LC-MS/MS method.LC-MS/MS methods for the quantitative determination of the test article in mouse plasma were developed with an internal standard. Benchtop stability of the compound in mouse plasma was determined at mid QC concentrations in triplicate at 0, 2 h at room temperature. The stability was determined using mean peak area ratio of T2/T0 sample. If the mean peak area ratio is within 80%~120%, the test article in the plasma is considered stable for 2 h at room temperature. A standard curve consists of eight non-zero calibration standards for the LC-MS/MS method with a target lower limit of quantification (LLOQ) at ≤ 3 ng mLA minimum of six calibration standards is back calculated to within ± 20% of their nominal concentrations; and a minimum of 4 out of 6 QC samples is back calculated to within ± 20% of their nominal concentrations.The mean calculated concentration in the single blank matrix should be ≤ 0.5 times the LLOQ.the mean calculated carry-over concentration in the single blank matrix immediately after the highest standard injection should be ≤ LLOQ.C0, T½, CL, Vdss, Cmax, Tmax, AUC0-last, AUC0-inf, %F, MRT0-last, MRT0-inf and graphs of plasma concentration vs. time profile were derived.Plasma concentration vs. time data from individual animals was analysed by WinNonLin non-compartmental model . Pharmacokinetic parameter 52.Performed at the University of Queensland, following similar previous literature procedures5 CFU MRSA (Strain ATCC® 43300™) was injected intramuscularly into both left and right thighs of all mice. Two hours after initiation of infection, saline, vancomycin or vancapticin compounds were injected subcutaneously in the lower back region. After an additional 2 h, a 50 µL sample of blood was obtained from the tail bleed to analyse for presence of antibiotic compound. At 24 h following infection, mice were killed and an additional blood sample collected for compound analysis. Thighs were then removed, weighed and homogenised in a fixed volume of saline. The homogenate solution was filtered, diluted and seeded onto agar plates, which wre incubated overnight at 37 °C. Colony counts were used to establish the CFU in the thigh homogenates, the CFU thigh−1 and the CFU g−1 of thigh.Summary: Adult (8-week-old) female CD1 mice were made neutropenic by two injections of cyclophosphamide 4 days and 1 day prior to infection. An inoculum of 10−1. Likewise, vancomycin (Sigma) and vancapticins were also dissolved in sterile saline to a final concentration of 60 mg mL−1and 18.5 mg mL−1 respectively. All compounds were prepared in low binding Eppendorf tubes and kept at −20 °C until used.Cyclophosphamide monohydrate (Sigma) was dissolved in sterile saline to a concentration of 30 mg mL®43300™) was taken from the storage at −80 °C and freshly seeded on agar plates for overnight growth. From the overnight culture preparation, a single colony was diluted into 10–12 mL of MHB and incubated overnight at 37 °C. A log-phase subculture was obtained by adding 100 μL of overnight subculture in 10 mL MHB and incubated for a further 2–3 h. Finally, the OD600 value of the bacterial suspension was determined and the colony forming units per millilitre (CFU mL−1) extrapolated. A full dilution of the bacterial cell suspension in saline was achieved by washing (3220 × g for 10 min) and the OD600 in saline determined. The suspension was then diluted out accordingly in order to achieve a 2 × 106 CFU mL−1 solution (105 CFU in 50 μL thigh−1).An MRSA subculture bacterial isolate  and 1 day (100 mg kg−1) prior to experimental infection. The infection model using MRSA was established by intramuscular injection of 50 µL of early-log-phase bacterial MRSA suspension (around 2 × 106 CFU mL−1) in saline into both thigh muscles. After 2 h, a single dose of vancomycin (200 mg kg−1) or compound of this invention (25 mg kg−1) was administered by a subcutaneous injection over the interscapular (area at back of the neck). Untreated animals received equivalent volume of saline (Baxter). The mice were monitored for signs of normal behaviour  during and following dosing. Two h after saline/antibiotic treatment, 0.05 mL of blood was collected by tail incision. 24 h after MRSA infection, mice were killed and blood collected from the heart by cardiac puncture  or by tail incision (vancomycin and vancapticin treated groups). For each mouse, both thighs were collected aseptically by cutting the leg at the hip and knee, placed in 10 mL of cold sterile saline and the individual weight of each thigh recorded.Eight-week-old female outbred CD1 mice (UQBR-AIBN) were rendered neutropenic by injecting two doses of cyclophosphamide intraperitoneally 4 days (150 mg kgg for 15 min. Plasma was collected and kept at −80 °C until used.Blood samples were taken at 2 h (tail incision) and 22 h (cardiac puncture or tail incision) post saline/vancomycin/vancapticin treatment using Lithium-Heparin Microvette® (Sardest) or using heparin coated syringes. All samples were kept at 4 °C and spun down at 10,000 × −1 and the CFU g−1 of thigh calculated based on the plate count and dilution factor.Thighs were homogenised at 20,000 rpm for 15 s using a Polytron MR2500E using a 200 mm probe (Kinematica). Homogenate solutions were filtered using a 100 µm pore size filter (BD) and 1 mL of filtrate solution placed on ice and serial dilutions promptly done (1:10 and 1:100) and seeded onto appropriate nutrient agar plates (Bactolaboratories) and incubated at 37 °C overnight. Colonies were counted the next day and CFU thigh53.Performed by Eurofins, Taiwan, following similar previous literature proceduresS. pneumoniae (ATCC® 6301™). Male ICR mice were inoculated intratracheally (IT) with S. pneumoniae at an inoculum size of 2.96 × 106 CFU per mouse (20 µL per mouse). Vancapticins 19 and 24 were administered at 25 mg kg−1 to separate groups of mice by subcutaneous (SC) injection 2 h post-infection. The reference standard, vancomycin 1, was dosed at 25 mg kg−1 qd SC at 2 h after IT inoculation. Mortality was observed for 10 days. An increase in survival rate of 50 percent or more ( ≥ 50%) of the treated animals relative to the vehicle group indicates significant antimicrobial activity. Subcutaneous injection (SC) of 19 and 24 at 25 mg kg−1 was associated with pronounced increase in the 10-day survival rate relative to the vehicle group, indicating significant antimicrobial activity. The vancomycin treated group, 25 mg kg−1 SC qd, also exhibited significant antimicrobial activity  relative to the vehicle control.The study objective was to evaluate the antimicrobial activity of test articles in a lung infection model with −1 were each administered subcutaneously (SC) to test animals 2 h after inoculation. The dosing volume was 10 mL kg−1.Test substances at 25 mg kgMale ICR mice weighing 22 ± 2 g were provided by BioLasco Taiwan (under a Charles River Laboratories Technology Licensee). Space allocation for 5 animals was 29 × 18 × 13 cm. Mice were housed in animal cages and were maintained in a controlled temperature (20–23 °C) and humidity (50−80%) environment with 12 h light/dark cycles for at least three days at Eurofins Panlabs Taiwan, Ltd. laboratory prior to use. Free access to standard lab chow for mice ) and tap water in bottle were granted. All aspects of this work including housing, experimentation and disposal of animals were performed in general accordance with the Guide for the Care and Use of Laboratory Animals .S.pneumoniae (ATCC® 6301™) was obtained from the American Type Culture Collection . The bacterial culture was diluted in brain–heart infusion broth containing to achieve the target inoculum size of 1.0–5.0 × 106 CFU per 20 µL that would be inoculated intratracheally into each animal.Bacto agar , brain heart infusion broth , fetal bovine serum , sodium chloride , vancomycin  and water for injection (WFI) .Animal cage , biokinetic reader , biological safety cabinet , centrifuge , laminar flow , orbital shaking incubator , Pipetman P100 , refrigerated incubator  and ultra-low temperature freezer .S. pneumoniae (ATCC® 6301™), Infected Lung Model. Groups of 10 male specific-pathogen-free ICR mice weighing 22 ± 2 g were used. Acute pneumonia was induced by intratracheal inoculation with a LD90–100 dose (2.96 × 106 CFU mouse−1) of S. pneumoniae (ATCC® 6301™) suspended in 20 μL of BHI. Vehicle (10 mL kg−1), vancomycin and test substances at 25 mg kg−1 were each administered subcutaneously 2 h post-infection. Mortality was recorded daily for 10 days following inoculation. Increase of 50 percent or more (≥ 50%) in survival rate relative to the vehicle control group indicates significant anti-infective activity.Assay # 608100 27. Xen-29 , a methicillin-sensitive S. aureus (MSSA) strain which contains a copy of the modified luxABCDE operon of Photorhabdus luminescens was taken from the storage at −80 °C and freshly seeded on agar plates for overnight growth at 37 °C. From the overnight culture preparation, a single colony (tested for bioluminescence) was diluted into 10 mL of MHB and incubated overnight at 37 °C. A mid log-phase subculture was obtained by adding 100 μL of overnight subculture in 10 mL MHB and incubated for a further 2–3 h. Finally, the OD600 value of the bacterial suspension was determined and the colony forming units per millilitre (CFU mL−1) was extrapolated. The suspension was then diluted out accordingly in order to achieve a 2.5 × 108 CFU mL−1 solution (2.5 × 107 CFU in 100 μL). Seven-week-old female outbred CD1 mice (UQBR-UQCCR) were rendered neutropenic by injecting two doses of cyclophosphamide (Sigma-Aldrich) intraperitoneally 4 days (150 mg kg−1) and 1 day (100 mg kg−1) prior to experimental infection. The peritonitis infection model using bioluminescent Xen-29 was established by intraperitoneal injection of 100 µL of the adjusted bacterial Xen-29 suspension (around 2.5 × 107 CFU) in saline. 30 min after bacterial inoculation, a single dose of daptomycin (50 mg kg−1), vancomycin (200 mg kg−1) or vancapticin (25 mg kg−1) was administered by a subcutaneous injection over the interscapular area (area at back of the neck). Untreated animals received equivalent volume of saline (Baxter). Mice were monitored for signs of normal behaviour  during and following dosing. Blood samples (50 µL) were taken 5.5 h post Xen-29 inoculation by tail bleed using Lithium-Heparin Microvettes® (Sardest). All samples were kept at 4 °C and spun down at 10,000 × g for 15 min. Plasma samples were collected and kept at −80 °C until used; while the pellets were re-suspended in 100 µL of sterile saline, serial diluted and spread on agar plates for overnight incubation at 37 °C. Xen-29 colonies were then counted, with the CFU mL−1 blood calculated. After killing, each mouse was opened and intraperitoneal swabs were taken for CFU determination. Individual spleens were also harvested aseptically, homogenised in 100 µL of sterile saline by crushing the tissue with a syringe plunger against a 50 µm filter, serial diluted and plated on agar plates for O/N incubation at 37 °C. Xen-29 colonies from intraperitoneal swabs and spleens were then verified. Luminescent images were acquired using the Xenogen IVIS-200 Optical In Vivo Imaging System (PerkinElmer). Luminescent images were quantified with the IVIS Living Image software where the total flux (number of photons/second) was calculated by a user defined region of interest (ROI) covering the infection sites.Performed at the University of Queensland, following similar previous literature proceduresAll relevant data are available in this article and its Supplementary Information files, or from the corresponding authors on request.Supplementary InformationPeer Review File


II cations, three benzoate (Bnz) and one pyridine-3-carbonitrile (CPy) molecule; the Bnz anions act as bidentate ligands through the carboxylate O atoms, while the Cpy anion acts as a monodentate N(pyridine)-bonding ligand. The complete centrosymmetric trinuclear complex thus comprises a linear array of three ZnII cations. In the crystal, the Bnz anions link to the Cpy N atoms via weak C—H⋯N hydrogen bonds, forming a two-dimensional network. The Hirshfeld surface analysis confirms the role of H-atom contacts in establishing the packing.The asymmetric unit of the title complex contains one half of the complex molecule, one and a half Zn 3(C7H5O2)6(C6H4N2)2], contains one half of the complex molecule, i.e. one and a half ZnII cations, three benzoate (Bnz) and one pyridine-3-carbonitrile (Cpy) molecule; the Bnz anions act as bidentate ligands through the carboxylate O atoms, while the Cpy ligand acts as a monodentate N(pyridine)-bonding ligand. The complete centrosymmetric trinuclear complex thus comprises a linear array of three ZnII cations. The central ZnII cation shows an octahedral coordination and is bridged to each of the terminal ZnII cations by three Bnz anions. By additional coordination of the CPy ligand, the terminal ZnII cations adopt a trigonal–pyramidal coordination environment. In the crystal, the Bnz anions link to the Cpy N atoms via weak C—H⋯N hydrogen bonds, forming a two-dimensional network. C—H⋯π and π–π interactions [between the benzene and pyridine rings of adjacent molecules with an intercentroid distance of 3.850 (4) Å] help to consolidate a three-dimensional architecture. The Hirshfeld surface analysis confirms the role of H-atom contacts in establishing the packing.The asymmetric unit of the title complex, [Zn II complexes of benzoic acid derivatives change depending on the nature and position of the substituent groups on the benzene ring, the nature of the additional ligand molecule or solvent, and the pH and temperature of synthesis bis(pyridine-3-carbonitrile-κN)trizinc(II), [Zn3(C7H5O2)6(C6H4N2)2], and report herein its crystal and molecular structures as well as a Hirshfeld surface analysis.The structure–function–coordination relationships of the arylcarboxylate ion in ZnII cations, which are coordinated by six benzoate anions and two neutral pyridine-3-carbonitrile ligands. The middle ZnII cation occupies a special position and lies on a crystallographic inversion centre. The benzoate anions act as bidentate ligands, bridging two pairs of ZnII cations. The pyridine-3-carbonitrile ligands are monodentately coordinated through the pyridine N atoms ms Fig. 1.i and O3i) of the two symmetry-related, bidentately coordinated benzoate anions around the Zn1 atom form a slightly distorted square-planar arrangement, while the slightly distorted octahedral coordination sphere is completed by the two carboxylate O atoms (O5 and O5i) of the two symmetry-related, bidentately coordinated benzoate anions in the axial positions   of 3.1845 (2) Å in the structurally related transition metal(II) complex [Zn3(benz)6(nia)2] (where benz = benzoate and nia = nicotinamide) z] Fig. 1. On the z] Fig. 1. The sumA, C2—C7; B, C9—C14; C, C16—C21) rings by 6.4 (3), 26.5 (3) and 5.1 (3)°, respectively, while the benzene and pyridine  rings are oriented at dihedral angles of A/B = 76.2 (2), A/C = 82.9 (2), A/D = 6.2 (2), B/C = 89.2 (2), B/D = 70.0 (2) and C/D = 83.0 (2)°.The Zn1 and Zn2 atoms lie [0.7337 (1) and −0.1793 (6) Å], [1.0911 (1) and −0.2676 (6) Å] and [1.3428 (1) and 0.0520 (7) Å] above and/or below of the planar (O1/O2/C1), (O3/O4/C8) and (O5/O6/C15) carboxylate groups, respectively. The (O1/O2/C1), (O3/O4/C8) and (O5/O6/C15) carboxylate groups are twisted away from the attached benzene (Bnz⋯NCpy (Bnz = benzoate and Cpy = pyridine-3-carbonitrile) hydrogen bonds   in H2O (25 ml) and pyridine-3-carbonitrile  in water (25 ml) with sodium benzoate  in water (100 ml) at room temperature. The mixture was filtered and set aside to crystallize at ambient temperature for several days, giving colourless single crystals .The title compound was prepared by the reaction of ZnSOUiso(H) = 1.2Ueq(C)].Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989017016899/nk2240sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989017016899/nk2240Isup2.hklStructure factors: contains datablock(s) I. DOI: 1587257CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


II cations to form polymeric chains running along the a axis.The 1,2-bis(pyridin-4-yl)ethene molecules bridge Fe 12H10N2)2(H2O)4](CH3C6H4SO3)2·2CH3OH}n, the FeII cation, located on an inversion centre, is coordinated by four water molecules in the equatorial positions and two 1,2-bis(pyridin-4-yl)ethene molecules in the axial positions. This results in a distorted octahedral geometry for the [N2O4] coordination polyhedron. The 1,2-bis(pyridin-4-yl)ethene molecules bridge the FeII cations, forming polymeric chains running along the a-axis direction. Stabilization of the crystal structure is provided by O—H⋯O hydrogen bonds; these are formed by coordinated water molecules as donors towards the O atoms of the methanol molecules and tosylate anions as acceptors of protons, leading to the formation of a three-dimensional supramolecular network. Weak C—H⋯O hydrogen bonds are also observed in the crystal.In the title polymeric complex, {[Fe(C Spin crossover (SCO), sometimes referred to as a spin transition or a spin equilibrium behaviour, is a phenomenon that occurs in some metal complexes wherein the spin state of a compound changes due to the influence of external stimuli such as temperature, pressure, light irradiation, magnetic field or guest effects  observed for spin-crossover complexes ethene in which FeII cation has a distorted octahedral coordination environment [FeN2O4], formed by two N atoms of 1,2-bis(pyridin-4-yl)ethene and by four O atoms of four water molecules  and Fe1—O2 = 2.077 (2) Å. The small difference in the lengths of the Fe—O bonds of 0.037 Å could be associated with a different participation of the water hydrogen atoms in hydrogen bonding. The metal-to-ligand distances clearly indicate the high-spin nature of the complex described herein.The Fees Fig. 1. Two 1,2II octahedral distortion parameter  is Σ|90 − Θ| = 28.15 (8), where Θ is the cis-N—Fe—O and cis-O—Fe—O angles in the coordination environment of the FeII atom. This value indicates a significant polyhedral distortion, which can be explained by the Jahn–Teller effect and the presence of different types of ligands.The FeII cations into polymer chains that propagate along the a-axis direction. Stabilization in the crystal structure is ensured by O—H⋯O hydrogen bonds ethene fragments with Feds Fig. 2: (i) H ark Fig. 2. In addiII cation has a distorted octahedral coordination environment [FeN2O4], formed by two N atoms of 1,2-bis(pyridin-4-yl)ethene and by four O atoms of four water molecules; however, it contains 2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-olate as the anion and crystallizes in the orthorhombic Pbcn space group. In this analogue, Fe1—N1 = 2.2304 (2), Fe1—O2 = 2.1030 (2) and Fe1—O4 = 2.0908 (2) Å   in water (2.5 ml), the second was a mixture of water/methanol  and the third layer was a solution of 1,2-bis(pyridin-4-yl)ethene  in methanol (2.5 ml). After two weeks, red crystals grew in the second layer; these were collected and maintained under the mother solution until measured.Crystals of the title compound were prepared by the slow diffusion method between three layers in a 10 ml tube. The first layer was a solution of [Fe(OTs)Uiso(H) = 1.2Uiso(C). Methyl H atoms were placed in their expected calculated positions (C—H = 0.98 Å) and refined as rotating groups with Uiso(H) = 1.5Ueq(C). Hydrogen atoms of the water molecules were assigned based on the difference-Fourier map, and the O—H distances and the H—O—H angles were constrained using DFIX (O—H = 0.84 Å) and DANG (H—H = 1.34 Å) instructions. The hydrogen H atom of the solvent methanol molecule was assigned based on the difference-Fourier map, and the O—H distance was constrained using a DFIX (O—H = 0.96 Å) instruction.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989017017054/xu5910sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2056989017017054/xu5910Isup2.hklStructure factors: contains datablock(s) I. DOI: 1587771CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


As a typical by-product, small amounts of the sterically crowded pyrazabole derivative trans-4,8-bis-1,3,5,7-tetraisopropylpyrazabole, C36H62B2H8 or trans-{HB}2, 2, were formed. Both title compounds have been structurally characterized through single-crystal X-ray diffraction. In 1, two isopropyl groups are each disordered over two orientations with occupancy ratios of 0.574 (10):0.426 (10) and 0.719 (16):0.281 (16). In 2, one isopropyl group is similarly disordered, occupancy ratio 0.649 (9):0.351 (9).The reaction of EuI Ln(C5Me5)2 . An unusual structural feature of the unsolvated lanthanide sandwich complexes Ln(C5Me5)2  is their bent metallocene structure in the solid state. This opens up the coordination sphere around the central divalent lanthanide ions and accounts for the very high reactivity of these compounds borate. The homoleptic divalent lanthanide complexes Ln(TpMe2)2  have been found to adopt a highly symmetrical, trigonal–antiprismatic coordination comprising an almost linear B⋯Ln⋯B arrangement 2 is the result of the much larger cone angle of TpMe2 (239°) as compared to that of the C5Me5 ligand (142°)  2 . Computational studies indicated that steric repulsion between the isopropyl groups forces the TpiPr2 ligands apart and permits the formation of unusual interligand C—H⋯N hydrogen-bonding interactions that help to stabilize the structure 2 also stands out due to its bright-yellow photoluminescence, which has been investigated in great detail 2 was easily prepared in 83% yield by treatment of the bis-THF adduct of europium(II) diiodide, EuI2(THF)2, with 2 equiv. of KTpiPr2 in THF solution  mono(scorpionate) complex, [HB]2EuIII (1), in addition to a frequently observed by-product, the sterically crowded 4,8-bis(pyrazolyl)pyrazabole derivative trans-{HB}2 (2). Both products have been structurally characterized through single-crystal X-ray diffraction.The organometallic chemistry of divalent lanthanides provides fascinating structures such as the sandwich complexes )2 Fig. 1b. Compu2(THF)2 was prepared from Eu metal and 1,2-diiodoethane using an established literature procedure 2 with 1.5 equiv. of KTpiPr2 in THF produced a fluorescent, neon-yellow solution and a white precipitate of potassium iodide. Crystallization from n-pentane solvent afforded bright-yellow, air-sensitive crystals, which turned out to be the unexpected europium(II) mono(scorpionate) complex (TpiPr2)2EuIII (1). The 78% isolated yield of 1 was surprisingly high. The coordinated neutral 3,5-diisopropylyrazole ligands clearly resulted from fragmentation of the TpiPr2 ligand. Ln-induced fragmentation of substituted Tp ligands is well documented -derived polysulfide complexes (iPr2pz)]+, while a peak at m/z 616 corresponds to the ion [Eu(TpiPr2)]+.The starting material EuI1 by addition of a large volume of non-polar hexamethyldisiloxane (HMDSO) resulted in the formation of well-formed, colorless, cube-like crystals in low yield. These turned out to be another ligand fragmentation product typical for lanthanide Tp chemistry, namely the 4,8-bis(pyrazolyl)pyrazabole derivative trans-{HB}2 (2). The parent pyrazabole, {H2B(μ-pz)}2 has been known since 1966 when it was reported by Trofimenko contemporaneously with the discovery of Tp ligands pyrazaboles in which two hydrogen atoms at boron are replaced by pyrazolyl moieties HB(μ-pz)}2, has been achieved by thermolysis of the free acid of the hydrotris(pyrazolyl)borate anion °. The three Eu—N bonds cover the range 2.581 (2)–2.633 (2) Å, which resembles that observed in the corresponding homoleptic EuII complex Eu(TpiPr2)2 . In 1, the coordination of the iodido ligand relative to the (TpiPr2)− ligand is slightly tilted [I—Eu⋯B = 151.49 (5)°], and an almost linear arrangement of the iodido ligand and one of the TpiPr2 N-donor atoms is realized [I—Eu—N2 = 165.92 (5)°]. A strongly distorted octahedral coordination is completed by the two neutral  ligands, with coordination angles of 138.80 (7)° (N4—Eu—N8) and 137.43 (7)° (N6—Eu—N10). The corresponding Eu—N bond lengths  are slightly longer than those to the (TpiPr2)− ligand, which may be due to the absence of negative ligand charge. The NH⋯N distances between the two pyrazole NH moieties and potential hydrogen-acceptor atoms  are in the range 2.512 (2)–2.610 (2) Å, but the groups are not in a proper orientation for efficient hydrogen bonding [N—H⋯N 115.0 (2)–122.0 (2)°]. Consequently, stabilization of the molecular structure by intramolecular hydrogen bonding is presumably of less importance.Both title compounds it Fig. 2. The Tpi2 exists as a centrosymmetric dimer in the crystal, which formally results from two HB2 monomers  moieties, resulting in a planar, six-membered B2N4 ring. The B—N bonds within this ring are virtually equal at 1.554 (2) Å (B—N1) and 1.557 (2) Å (B—N2′), and therefore similar to that within the (TpiPr2)− ligand in 1. In contrast, the B—N bond to the terminal  moiety (B—N3) is slightly shortened to 1.532 (2) Å. The B atoms in 2 exhibit a virtually ideal tetrahedral coordination with bonding angles in the narrow range 108.3 (1)–110.7 (1)°. The molecular structure of 2 is very similar to that of the 3,5-dimethylpyrazolyl analog, trans-{HB}2 +, 616 (92) [Eu(TpiPr2)]+, 477 (85), 321 (100), 302 (55) [EuBH(iPr2pz-CH3)]+, 152 (21) [iPr2pz]+, 137 (63).2: Analysis calculated for C36H62B2N8, M = 628.56 g mol−1: C 68.79, H 9.94, N 17.83%. Found: C 68.50, H 10.10, N 17.53%. M.p. 553 K. IR: ν 3176 w, 3094 w (ν C—H pyrazolyl), 2966 s, 2928 m, 2869 m, 2825 w (ν CH3), 2467 w (ν B—H), 1576 w, 1541 m, 1497 m, 1461 m, 1369 m, 1301 s, 1233 vs, 1169 vs, 1134 vs, 1090 s, 1063 s, 1041 m, 1015 m, 982 s, 919 w, 879 w, 832 s, 788 s, 751 m, 723 m, 675 m, 566 m, 508 m, 473 w, 365 m, 302 m, 246 m, 137 m, 106 m, 75 m cm−1. 1H NMR : δ 11.0 , 5.75 , 2.87–2.91 , 1.15–1.27  ppm. 13C NMR : δ 160.7 , 97.5 (C—H pyrazolyl), 28.5 (C—H iPr), 23.5 (CH3iPr). 11B NMR : δ −4.3  ppm. MS: m/z (%) 627 (62) [M]+, 476 (100) [C27H46B2N6]+, 461 (75) [C26H43B2N6]+, 325 (66) [C18H31B2N4]+, 152 (74) [C6H2B2N4]+, 137 (89) [pz2].Uiso(H) = 1.5Ueq(C) for methyl H atoms and Uiso(H) = 1.2Ueq for all others. In 1, two isopropyl groups are each disordered over two orientations with occupancy ratios of 0.574 (10):0.426 (10) and 0.719 (16):0.281 (16). In 2, one isopropyl group is similarly disordered, occupancy ratio 0.649 (9):0.351 (9).Crystal data, data collection and structure refinement details are summarized in Table 110.1107/S2056989017016498/zl2718sup1.cifCrystal structure: contains datablock(s) 1, 2. DOI: 10.1107/S2056989017016498/zl27181sup2.hklStructure factors: contains datablock(s) 1. DOI: 10.1107/S2056989017016498/zl27182sup3.hklStructure factors: contains datablock(s) 2. DOI: 1585878, 1585877CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Here we report two pediatric T-ALL cases and present their clinical features, histology, immunophenotypes, cytogenetics, and molecular diagnostic findings. The first patient is a two-year-old girl with leukocytosis, circulating lymphoblasts, and a cryptic insertion of a short-arm segment at 10p12 into the long-arm segment of 11q23 resulting in an MLL and AF10 fusion transcript, which may be the first reported in γδ T-ALL. She responded to the chemotherapy protocol poorly and had persistent diseases. Following an allogeneic bone marrow transplant, she went into remission. The second patient is an eleven-year-old boy with a normal white cell count, circulating blasts, and a normal karyotype, but without any immature cellular markers by flow cytometric analysis. He responded to the chemotherapy well and achieved a complete remission. These cases demonstrate the diverse phenotypic, cytogenetic, and molecular aspects of γδ T-ALL. Early T-precursor- (ETP-) ALL and their differential diagnosis from other mature γδ T-cell leukemia/lymphomas are also discussed.Gamma delta ( T-cell acute lymphoblastic leukemia (T-ALL) with expression of alpha beta (αβ) or γδ TCR has been previously described in about 35% T-ALL cases, while γδ T-ALL cases represented 9–12% of T-ALL including children and adults [γδ expression include hepatosplenic T-cell lymphoma, skin and mucosal γδ T-cell lymphoma, and γδ T-cell large granular lymphocytic (T-LGL) leukemia [γδ T-cell lymphoma [γδ T-cell lymphomas, other than hepatosplenic T-cell lymphoma and primary cutaneous γδ T-cell lymphoma, are often classified within subcategories of T-cell lymphomas. γδ T-ALL is uncommon with only 2% of all acute lymphoblastic leukemia (ALL) cases showing expression of the γδ TCR [γδ TCR expression and T-cell differentiation are not frequently reported in T-ALL [γδ T-ALL and discuss the differential diagnosis of other types of γδ T-cell leukemia/lymphoma.Gamma delta . Bone marrow evaluation showed 90% lymphoblasts, exhibiting slightly open chromatin and irregular nuclear contours , cryp ins, +17 . Of interest, the centromeric 5′ AF10 signal is inserted into the 11q23 region ( MLL/MLLT10 (MLL/AF10) fusion transcript  at 118.8 thousand/contours . Flow cy and MPO . Cytogen12), +17 . FISH as3 region . By FISH3 region . The reaanscript . The patAt day 15 following induction, flow cytometry revealed residual disease with 35% blasts in bone marrow. Following reintensification I therapy, the patient had significantly decreased blast percentages but remained persistently positive for minimal residual disease (MRD), with the last MRD before bone marrow transplant (BMT) at 0.018% by flow cytometry. She eventually underwent allogeneic BMT. She had been on day +126 after transplant, remained MRD negative, and had been followed up at the LSU hospital.γδ T-cell lymphoblasts. After maintenance on intravenous fluid and allopurinol therapy, he was transferred to SJCRH for chemotherapy. His bone marrow showed 89% blasts that were small to medium in size with a high nuclear to cytoplasmic ratio, fine chromatin, round to slightly irregular nuclei, and scant cytoplasm  leukemia, and γδ T-ALL [γδ T-cell lymphoma is a subtype of peripheral T-cell lymphoma, occurring mainly in skin and mucosal regions, often harboring cytotoxic activity. In the skin, γδ T-cell lymphoma can be divided into mycosis fungoides-like and primary cutaneous γδ T-cell lymphoma, presenting with Sézary syndrome in blood involvement [γδ T-cell lymphoma may occur in the nasopharynx, lung, gastrointestinal tracts, and other organs, with intestinal γδ T-cell lymphoma being type II enteropathy associated [The differential diagnosis of these two pediatric cases includes skin and mucosal γδ T-ALL , 4, 6. Polvement , 4, 7. Msociated , 6, 8, 9γδ T-cell lymphoma with extranodal and systemic involvement [γδ and negative for CD4, CD5, CD8, and TCR αβ. Although the first patient showed hepatosplenomegaly with a γδ T-cell phenotype, she had marked leukocytosis at the beginning of her disease course, with lymphoblastic appearing neoplastic cells positive for CD5 and CD34 and negative for CD2. Thus, the diagnosis of hepatosplenic T-cell lymphoma is not supported.Hepatosplenic T-cell lymphoma is a type of olvement , 10, 11.olvement . The tumolvement . Phenotyαβ type, there are rare cases of γδ T-LGL leukemia [αβ counterpart, γδ T-LGL leukemia patients are prone to having rheumatoid arthritis, lower absolute neutrophil counts, more severe thrombocytopenia, and a higher probability of CD4 and CD8 double negativity. However, both groups of T-LGL leukemia frequently have anemia, an indolent clinical course, and a similar overall survival [In spite of the fact that the majority of T-LGL leukemia cases are of leukemia , 13. In survival , 13.γδ T-ALL is similar to αβ T-ALL in the majority of clinical and hematological aspects [αβ or TCR γδ in association with different differentiating stages are rarely reported [ NOTCH1, TAL1, and HOX1 [γδ T-ALL is a rare variant of T lineage lymphoblastic leukemia/lymphoma. Compared to αβ T-ALL, γδ T-ALL tends to present with lower hemoglobin concentrations in children, more frequent splenomegaly and higher WBC in adults, and higher percentages of the CD45RA−/CD45RO+ phenotype in both children and adults [γδ T-ALL usually shows TCR gamma and TCR delta chain gene rearrangements, TCR beta chain and biclonal rearrangements involving both Vδ1 and Vδ2 segments have also been reported [γδ T-ALL patients were likely in medullary (mature) T-ALL stage at presentation with variable surface CD3 expression. Neither of our patients has ETP-ALL due to the strong CD5 expression in the first patient and lack of immature or myeloid markers in the second patient. The cryptic insertion involving MLL and AF10 resulting in the expression of the fusion transcript has rarely been reported in T-ALL [ MLL/MLLT10 fusion may be the first reported in γδ T-ALL. Previous studies have shown a higher percentage of γδ T-ALL, at 9~12%, in comparison with the very low proportion of γδ T-cells in the normal thymus, which is at 1% [γδ T-cells have a higher chance of progression to malignancy compared to αβ T-cells or a subset of γδ T-ALL may originate from extrathymic tissues [γδ T-cells are more commonly double negative for CD4 and CD8, a significant percentage of γδ T-ALL may exhibit CD4, CD8, or both CD4 and CD8, which may be due to antigen evolution during malignant transformation [γδ T-ALL may represent a subcategory of acute lymphoblastic leukemia with slightly distinctive clinical and laboratory features. More studies are needed to further investigate such subtypes of T-ALL, including their pathological diagnosis and clinical management.T-ALL comprises approximately 20% of all ALL cases , 2, 14. and HOX1 , 14. γδ in T-ALL . To our