External lipid membranes affect guest recognition properties of water-soluble hosts, enhancing molecular conformations and equilibria not observed in free solution. 19F or 13C nuclei that can be encapsulated inside the host, 2D EXSY NMR experiments can be used to analyze and compare the in/out guest exchange rates in aqueous solution, isotropically tumbling micelles, or magnetically ordered bicelles. These analyses show that embedding the deep cavitands in lipid bilayers slows the guest exchange rate, due to the lipids acting as a “compression sleeve” around the host, restricting guest egress. This effect also enhances guest conformations in the host that are not observed in free solution, such as axial cyclohexane conformers and ketone hydrates.The effects on the molecular recognition properties of water-soluble deep cavitand hosts upon embedding them in phosphocholine lipid bilayer environments have been studied by 2D NMR experiments. By employing suitable guests containing  Quantitation of the thermodynamics1H NMR spectroscopy. To maximize detection, these investigations are generally performed in controlled environments, in deuterated solvents and in the absence of NMR-visible additives and impurities.When analyzing the conformation and motion of molecules confined in small spaces, the predominant tool is NMR spectroscopy.1  spectroscopy of cavitand:supported lipid bilayer (SLB) aggregates1H NMR spectroscopy of these events has been limited to simple 1D experiments in fast tumbling micelles,Synthetic host molecules are capable of selective molecular recognition in far more complex and challenging environments than pure solvent, however. Hosts such as deep cavitand 1 2a,10 hav1 is soluble in water up to ∼20 mM, but is highly lipophilic, and is smoothly incorporated into a variety of lipid aggregates.3+) salts such as choline.2O solution, the association constants of guests such as 1-adamantanemethanol (Ka = 2.9 × 105 M–1), cyclohexanone (Ka = 1.6 × 105 M–1), choline (Ka = 2.6 × 104 M–1) and acetylcholine (Ka = 1.2 × 105 M–1) are relatively consistent, within an order of magnitude or so.5 M–1. The in/out kinetics of bound guests, as well as their motion while inside the cavity, have been extensively investigated in pure D2O by 2D NMR techniques.via an “SN1-like” mechanism, whereby the walls flex open to unfold the cavitand in the rate-determining step, followed by rapid guest exchange. In water, the energy barrier consists of three major components: the energy barrier to rotate around the C–O bonds in the cavitand walls–1), plus the energetic penalties of unfavorable solvation of the cavitand walls and the bound guest once unfolded. Depending on guest size, these barriers range from 16.0–17.2 kcal mol–1,–1 (adamantanol) to 14.6 s–1 (cyclohexane). All these investigations used 1H NMR for analysis, as the 1H peaks for bound guest are shifted strongly upfield, and are easily visible at negative ppm, unhindered by 1H peaks from the host or excess free guest.The tetracarboxylate cavitand What is not known is how embedding the cavitand in a lipid bilayer affects these molecular recognition properties. Other techniques have shown that host is capable of guest recognition in a bilayer, and that guest binding affinities are enhanced in some cases,13C, 15N or 31P. Unfortunately, observing individual proton signals via1H NMR analysis of the binding processes in those cases is complicated by the presence of multiple hydrocarbon peaks from the lipids. More sensitive NMR experiments are rendered impractical by line broadening, and so dynamic NMR experiments that are essential for analysis of guest motion and binding kinetics in synthetic cavity-containing hosts are challenging. Here we employ a variety of guest molecules with different detectable nuclei for molecular recognition in a deep, water-soluble host, and investigate the effect of embedding the host in biomimetic membrane environments on the guest dynamics, conformation and reactivity.NMR analysis of (bio)molecular structure and dynamics in membrane bilayers is well-studied,1H NMR analysis cannot be used, this introduces serious constraints on the nature of the guest. As such, we investigated a wide range of guest species . Also, the host:guest library must be paired with a suitable membrane environment for analysis. Obviously, a natural cell membrane is challenging to use, but a number of surrogates are known and used for NMR analysis of membrane-bound biomolecules. One of the most effective mimics is a magnetically oriented bicelle, which maintains the bilayer sheet form of natural membranes while aligning in the magnetic field to allow analysis by ssNMR techniques.NMR analysis of the molecular recognition of small molecules by deep cavitands in lipid environments is complicated by a number of factors. The guest must bind in the cavitand, and be detectable when bound inside the host in multiple different lipid environments. In addition, study of the in/out kinetics requires guests that are sufficiently soluble to display peaks for both free and bound guest. Study of internal motion or conformational bias requires guests that display multiple conformations or orientations while bound inside the cavity: if  species  for thei31P NMR analysis of the lipid system. The disordered micelles display only one averaged 31P phosphate peak , which are well-known to allow formation of both magnetically oriented bicelles and smaller disoriented micelles.31P NMR analysis of the lipid phosphate groups is an invaluable tool to confirm the structure of the lipid aggregates, but does not allow investigation of the host:guest properties of 1. We initially approached the host:guest studies using 1H NMR spectroscopy with the simplest, most optimal guest possible. Cyclooctane 2 is easily extracted into the cavity of 1, has an affinity >104 M–1 by NMR,1·2 in D2O was added to solutions of either DMPC:DHPC micelles (hereinafter denoted as PCm) or DMPC:DHPC bicelles (PCb) for a final [1] = [2] = 1.8 mM and the spectra acquired at 283 K and 308 K respectively  was applied.2 was only partially useful, we turned to guests 3 and 4, containing 19F nuclei. As 19F peaks are broadened in the solid state in a similar manner to 1H, we focused on fast-tumbling micelles in the 1·3/4 analysis, with a lower lipid concentration of 60 mg mL–1 for ease of measurement. To allow analysis of conformation and in/out guest exchange, the guest must contain 19F nuclei that are bound inside the cavity of 1, and display the characteristic chemical shift variations caused by the magnetic anisotropy of the host. Fortunately, both guests 3 and 4 are suitable guests for 1 in aqueous solution, albeit displaying weaker binding than the equivalent hydrocarbons. 19F NMR spectra of the host:guest complexes shows that the bound 19F nuclei are upfield shifted, Δδ ∼ –2 ppm, indicating that the difluorocyclohexanyl group is oriented to the cavity interior. Presumably, the OH and C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbdWZm6d" 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>O groups orient towards the external solvent to benefit from favorable H-bonding.As cyclooctane 3 and 4 show interesting and unexpected behavior when bound to 1, and provide excellent examples of the effect of embedding the host in the PC lipid environment. The 1H NMR spectrum of the 1·3 complex  separates the two fluorine peaks to reflect the distinct axial and equatorial positions (see Khyd(acetone) = 1.4 × 10–3, Khyd(fluoroacetone) = 0.11).4 in D2O in the absence of cavitand showed no obvious hydrate present. At the concentrations used, this indicates that <0.5% hydrate is present in solution. In contrast, when bound inside the cavity of 1, 13% of bound 4 exists in the hydrated form at 298 K. Embedding the host in PC lipids also biases the hydration equilibrium of 4, similar to the conformational bias seen in the binding of 3. When 4 was added to the 1·PCm system, 23% of the bound 4 was present in the hydrated form, compared to only 13% in 1·4 and <0.5% in free solution.Interestingly, the iol form . The 19F1 stabilizes normally unfavorable guest structures, and why this effect is enhanced when 1 is embedded in lipid aggregates. Cavitand 1 is well-known to display dual-mode recognition, whereby both the defined cavity and the upper rim groups can affect guest binding.1 than those with esters, ethers or ketones.3 evidently positions the OH group in closer proximity to the rim carboxylates, increasing the favorability of that conformer when bound. The hydrated gem-diol of 4 is capable of H-bonding with the carboxylates, whereas the ketone is not, hence the increased favorability of the bound hydrate , the same exchange conditions show only minimal crosspeaks for m1·3·PC  used to determine exchange rates; the full spectra are shown in the ESI.2OH axial. As such, the in/out exchange rates were determined by fitting the intensity of the four exchange peaks shown in τ ≥ 300 ms), the multiple methods of magnetization transfer in the system caused inaccuracies in the fitting, so the initial rate regions of the plot were used. For a detailed description of the fitting method, please see ESI.By taking the exchange spectra at multiple different mixing times, quantitation of the exchange rates was possible. The EXSY spectra of k–1 (or “koff”) for each guest, obtained at identical concentrations and temperatures for each guest in either aqueous solution (kfree) or in the micelle environment (kPC). Eyring analysisG‡. As expected, the rate is dependent on the nature of the guest, but most interestingly, it is also dependent on the external environment. The larger guest 3 shows a kfree = 4.2 s–1, comparable to that previously obtained for cyclooctanol.kPC = 1.8 s–1, corresponding to an additional 0.5 kcal mol–1 additional barrier conferred by the external environment surrounding the cavitand host. The same “compression sleeve” effect that enhanced the axial conformation of bound 3 slows the in/out exchange rate as well.4 showed that kfree = 8.7 s–1, similar to that to that previously obtained for cyclohexanone, as expected.kPC = 5.2 s–1. The retardation of exchange rate is slightly less in the case of the smaller guest 4, with a 0.3 kcal mol–1 additional barrier. Surprisingly, the EXSY spectrum of 1·4 allowed analysis of the in/out exchange of the hydrated gem-diol form of hyd4, as the crosspeaks were large enough to observe  = 5.7 s–1. Unfortunately, the equivalent crosspeaks in the PCm system were too small to accurately fit, so determination of kPC was unsuccessful in that case.EXSY analysis of guest  observe . The add1H to analyze the in/out exchange allows analysis of other, more biorelevant guests such as choline. While association constants for R-NMe3+ guests are easily determinable by ITC1 is susceptible to aggregation in free solution in the presence of lipophilic salts such as choline. Stable 1 : 1 complexes can be observed by NMR when substoichiometric amounts of guest are used,3+ salt causes aggregation and peak broadening, limiting analysis of the in/out rate by exchange NMR.5–8. 13C-enriched R-NMe3+ guests were easily accessed via reaction of the corresponding dimethylamino precursor with enriched 13CH3I. As cavitand 1 is capable of binding numerous R-NMe3+ salts in lipid environment, we investigated a series of guests 5–8 with variable upper rim functionality. The bola-type bis-NMe3+ guests 5 and 8 were initially targeted to investigate the possibility of slowed tumbling in the cavity of 1, as opposed to in/out guest exchange. Encapsulation in 1 slows the up/down interconversion rate of hydrocarbons such as trans-decalin, but quantitation is challenging due to peak broadening. As 5 and 8 are symmetrical, it was envisaged that exchange would be observed between the two conformers in the m1·5·PC system via13C–13C EXSY NMR analysis. Unfortunately, neither guest 5 nor 8 are bound by 1 in either free aqueous solution or in the PCm environment. Evidently, the nature of the upper rim has a large effect on guest recognition, so we turned to guests that can display favorable H-bonds with the carboxylate groups, choline 6 and the dimethylamino-variant 7.The use of guest nuclei other than 6 and 7 was far more successful, and representative examples of the 13C–13C EXSY spectra are shown in 3+ guest (6 or 7) overlaps with peaks from the R-NMe3+ group in the phosphocholine lipids, as would be expected. Even though 6/7 are 13C-enriched, a significant proportion of 13C-PC is present due to the excess of lipids in the sample. Despite the interfering peaks for free guest, 13C peaks for both bound 1·6 and 1·7 are easily observable, with the characteristic upfield shift observed. The relative change in 13C δ upon binding is proportionally smaller than that for 1H, with Δδ ∼ –2 ppm, but this is easily enough to allow exchange analysis via13C–13C EXSY.Analysis of the unsymmetrical guests 13C–13C EXSY spectra with varying mixing times were obtained for samples of m1·6·PC and m1·7·PC at 298 K with 5.8 mM 1, 16 mM guest and 60 mg mL–1 lipid, as usual. The exchange rates were acquired via fitting the crosspeak intensities extracted as slices from the 2D spectra. In this case, the diagonal peak corresponding to free guest overlapped with peaks from the NMe3+ groups in the lipids. As the concentration of the lipids was constant, the signal from the lipids remained constant in the low mixing time experiments, and the accuracy of the fit for the off-rate was not compromised. The rates are shown in kPC (6) = 3.0 s–1 and kPC (7) = 5.7 s–1. The bulkier 7 exchanges more rapidly than choline 6, in contrast to the results for hydrophobic guests, where larger guests showed slowed exchange. It is likely that positioning the extra steric bulk at the upper rim lowers the affinity of 7 for the cavitand, as has been observed for other R-NMe3+ species, and a more rapid exchange rate is seen. The results from kfree for 6 and 7: if the “compression sleeve” effect of the micelle environment is assumed to be constant, then kfree for 6 and 7 would be on the order of ∼6 s–1 and ∼10 s–1, respectively.13C-labeled guests 6 and 7 were amenable to EXSY analysis in isotropically tumbling micelles, we next employed these guests towards detection of in/out exchange of host 1 in the more challenging, yet more relevant magnetically ordered bicelles. The bicelles were formed as described above , loaded into a 4 mm Bruker solid state rotor, and solutions of cavitand 1 and guests 6 and 7 were added. As might be expected, the larger concentration of lipids (and their overlapping phosphocholine groups) made analysis via 1D NMR challenging, even with 13C-enriched guests. Fortunately, the magnetic alignment at 308 K was good, and the presence of exchanging guest 7 was observable by 13C–13C EXSY analysis  were performed on a Bruker Avance NEO 400 9.4 T spectrometer with a 5 mm Prodigy CPP BBO BB-H&F z-gradient cryo-probe or a Bruker 14.1 T (600.01 MHz 1H) Avance I spectrometer equipped with a 5 mm BBO Z-grad probe. Micelle (PCm) experiments  were performed on a Bruker Avance NEO 400 9.4 T spectrometer with a 5 mm Prodigy CPP BBO BB-H&F z-gradient cryo-probe, a Bruker 14.1 T (600.01 MHz 1H) Avance I spectrometer equipped with a 5 mm BBO Z-grad probe or a Bruker Avance III 700 16.44 T spectrometer with a 5 mm CP TCI H–C/N-D z-gradient cryo-probe. Magnetically ordered bicelle (PCb) experiments  were performed at 9.4 T  on a Bruker AVIII spectrometer equipped with a double resonance, 4 mm MAS probe. Proton (1H) and carbon (13C) chemical shifts are reported in parts per million (δ) with respect to tetramethylsilane . Phosphorus (31P) chemical shifts are reported in parts per million (δ), and referenced internally with respect to 85% H3PO4. Fluorine (19F) chemical shifts are reported in parts per million (δ), and referenced internally with respect to CF3COOH. Deuterated NMR solvents were obtained from Cambridge Isotope Laboratories, Inc., Andover, MA, and used without further purification. Mass spectra were recorded on an Agilent 6210 LC TOF mass spectrometer using electrospray ionization with fragmentation voltage set at 115 V and processed with an Agilent MassHunter Operating System. All other materials , and diheptylphosphocholine (DHPC)) were obtained from Aldrich Chemical Company, St. Louis, MO, or TCI, Tokyo, Japan and were used as received. Solvents were dried through a commercial solvent purification system . Molecular modeling  was carried out using the AMBER force field1 was synthesized according to published procedures:1· choline complex.1D NMR experiments (q = 3.2 (q = long chain lipid/short chain lipid) were formed by mixing together DHPC and DMPC dissolved in chloroform. The chloroform was evaporated off under a stream of nitrogen and then the lipids were lyophilized for 4 h before the micelles were prepared to remove any residual TFA and chloroform. The solid lipids were dissolved in HEPES buffer, pH = 6.5 for a combined lipid concentration of 290 mg mL–1. NMR samples were prepared by dissolving 100 μL of the lipid mixture in 400 μL D2O, to a final combined lipid concentration of 60 mg mL–1, 1 mM HEPES. Cavitand 1 and guests were added to the mixture and equilibrated for 1 h before NMR analysis.Mixed lipid micelles with q = 3.2 (q = long chain lipid/short chain lipid) by mixing together DHPC and DMPC dissolved in chloroform. The chloroform was evaporated off under a stream of nitrogen, the lipids were lyophilized for 4 hours before the bicelles were prepared to remove any residual TFA and chloroform, and the solid lipids were dissolved in HEPES buffer, pH = 6.5 for a combined lipid concentration of 290 mg mL–1. This solution was subjected to 5 minutes of vortexing, followed by 30 minutes in a water bath at 45 °C, followed by 15 minutes in an ice bath. This process was repeated five times until the lipids were clear and non-viscous at 4 °C and milky-white and solid at room temperature, indicative of bicelle formation. NMR samples were prepared by dissolving 250 μL of the bicelles in 200 μL HEPES buffer, pH = 6.5, and 50 μL D2O for a lock, to a final combined lipid concentration of 150 mg mL–1, 2.5 mM HEPES. This solution was added to a 4 mm Bruker solid-state rotor. Cavitand 1 and guests were added to the mixture and equilibrated for 24 h before NMR analysis.Magnetically ordered bicelles were prepared according to literature procedures.19F–19F or 13C–13C NOESY pulse sequence supplied with the Bruker software. Each of the 512 F1 increments was the accumulation of 6 scans (19F) or 32 scans (13C). Before Fourier transformation, the FIDs were multiplied by a 90° phase-shifted sine square function in both the F2 and the F1 domain. 1 K _ 1 K real data points were used, with a resolution of 1 Hz per point.The 2D NOESY spectra of the cavitand:guest exchange processes were recorded at 298 K at either 400 MHz or 600 MHz with the phase sensitive 1H-X double resonance 4 mm MAS probe with no spinning. The 2D EXSY spectra of the cavitand:guest 7 exchange process in bicelles were recorded at 316 K at 9.4 T with a NOESY pulse sequence. Each of the 128 F1 increments was the accumulation of 128 scans with a relaxation delay of 3 s. Before Fourier transformation the FID was multiplied by a 90° sine square function in the F1 domain and an EM function in the F2 domain. 2 K (F2) _ 256 (F1) real data points were used with a resolution of 1 Hz per point.Bicelle samples containing guest 7 and cavitand were loaded in a 4 mm Bruker solid-state rotor, and experiments were performed on a Bruker AVIII spectrometer equipped with a 13C  dropwise. The reaction was stirred at ambient temperature for 16 hours, and the resulting precipitate was collected by vacuum filtration to obtain 220 mg of guest 5 as a white solid (88% yield). 1H NMR  δ 3.47 , 3.23  2.41 , 13C NMR  δ 62.3, 53.3, 17.4. ESI-MS m/z expected: 190.20, found [MH+] = 190.14.Tetramethyl-1,3-diaminopropane  was dissolved in 2 mL of acetonitrile at 0 °C followed by addition of iodomethane-13C  dropwise. The reaction was stirred at ambient temperature for 16 hours, and the resulting precipitate was collected by vacuum filtration to obtain 228 mg of a white solid (82% yield). 1H NMR  δ 3.92 , 3.39 , 3.07 . 13C NMR  δ 67.4, 55.6, 53.9. ESI-MS m/z expected: 105.16, found [M+] = 105.11.Dimethylethanolamine  dissolved in 1 mL of acetonitrile at 0 °C followed by addition of iodomethane-13C  dropwise. The reaction was stirred at ambient temperature for 16 hours and the resulting precipitate was collected by vacuum filtration to obtain 650 mg of white solid (76% yield). 1H NMR  δ 3.35 , 3.02 , 2.73 , 2.16 , 13C NMR  δ 53.8, 53.2, 44.1. ESI-MS m/z expected: 132.23, found [MH+] = 132.16.Tetramethylethylenediamine  was dissolved in 1 mL of acetonitrile at 0 °C followed by addition of iodomethane-N,N,N,N′,N′-Pentamethyl-1,2-ethanediaminium iodide-13C 7  was dissolved in 1 mL of acetonitrile at 0 °C followed by addition of iodomethane-13C  dropwise. The reaction was stirred at ambient temperature for 16 hours, and the resulting precipitate was collected by vacuum filtration to obtain 256 mg of a white solid (56% yield). 1H NMR  δ 4.09 , 3.35 , 13C NMR  δ 57.3, 53.7. ESI-MS m/z expected: 148.26, found [MH+] = 148.19.There are no conflicts to declare.Supplementary informationClick here for additional data file.


There is an increasing interest in targeting molecules to the mitochondrial matrix. Many proteins are naturally imported through the translocase complexes found in the outer and inner mitochondrial membranes. One possible means for importing molecules is therefore to use a mitochondrial pre-protein as a vector and assess what forms of molecules can be attached to the pre-protein as cargo. A major difficulty with this approach is to ensure that any chimaeric molecule does indeed access the mitochondrial matrix and does not merely associate with the mitochondrial membranes. We have recently demonstrated that click chemistry can be used both to demonstrate convincingly mitochondrial import of a peptide–peptide nucleic acid conjugate and also to quantify the mitochondrial uptake for specific synthetic conjugates. We now report an adaptation of the synthesis to facilitate simple quantification of multiple molecules and hence to calculate the efficiency of their mitochondrial import. In mammals, this small 16 569 bp) genome encodes just 13 polypeptides and all the RNA components (2 mt-rRNAs and 22 mt-tRNAs) required for their intra-mitochondrial synthesis. All 13 proteins are members of four of the five multi-subunit complexes of the mitochondrial inner membrane that couple oxidative phosphorylation (OXPHOS). Mutations in the mitochondrial genome can cause a wide variety of progressive disorders mostly affecting the muscle or nervous system [6 569 bp Over 20 years ago, we suggested that it might be possible to try and manipulate the level of heteroplasmy as a potential therapeutic intervention . We reasRecently, we have used the ClickIn strategy to show unequivocally that a mitochondrial pre-protein carrying a short peptide nucleic acid as cargo could be imported through the natural mitochondrial protein import machinery . To do t2.2.1.N-alpha-t-butyloxycarbonyl-l-glutamic amide (Boc-l-Glu-NH2) was obtained from Iris Biotech (Germany). Fmoc protected amino acids were obtained from AGTC Bioproducts (UK). All other reagents and chemicals were obtained from Sigma-Aldrich. Mass spectrometry was performed on an Applied Biosystems Voyager-DE PRO MALDI-TOF spectrometer in linear mode, and processed using mMass. HPLC purification was performed on a Varian 940-LC equipped with a Phenomenex Luna C18 column  using a flow rate of 4 ml min−1. For the chromatograms in 2O, B is 0.1% in acetonitrile). HPLC chromatograms of purified compounds are provided in the electronic supplementary material.Fmoc-PNA monomers were obtained from Link Technologies (UK). 2.2.N,N′-diisopropyl-carbodiimide (DIC) and ethyl (hydroxyimino)cyanoacetate (Oxyma) in a 1 : 1 : 1 ratio in dimethylformamide (DMF) unless otherwise specified.Peptides and PNAs were synthesized on a Liberty Blue peptide synthesizer using microwave irradiation on a Rink amide ChemMatrix support using Fmoc chemistry with five equivalents of Fmoc-amino acids, in vacuo for a minimum of 3 h. The peptides and PNAs were cleaved off the resin and deprotected by addition of an acidic cleavage cocktail for 60–90 min. Then, the solution was filtered off the resin, followed by washing of the resin (3 × 2 ml). The solution was evaporated using nitrogen flow, reducing the volume to 2 ml, after which 10 ml of cold diethyl ether was added, precipitating the peptide. The suspension was centrifuged and the supernatant was decanted. The pellet was resuspended in 10 ml of cold diethyl ether, followed by centrifugation, the ether was removed by decantation and the pellet dried in vacuo. The dried peptide was redissolved in 1 ml of Milli-Q water and purified on HPLC, where fractions with purity more than 95% (by MS) were selected.After synthesis, the resin was washed with DMF (3×), dichloromethane (3×) and diethyl ether (3×) and dried 2.3.2O  for 90 min. Purification of the peptide on HPLC was carried out using a 0–100% gradient : m/z 3255.64 observed .The peptide was synthesized on a 50 µmol scale. Fmoc-Cys(Trt)-OH and Fmoc-His(Trt)-OH were coupled at 50°C, other amino acids were coupled at 75°C for 10 min. Fmoc-Arg(Pbf)-OH was coupled using double couplings, as well as residues 1 to 12 (N-terminus). In total, 10 µmol of the peptide was deprotected and cleaved off the resin using 5 ml of TFA/DODT/TIS/H2.4.3)-OH), 75 µmol benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate and 150 µmol N,N-diisopropylethylamine dissolved in DMF was added to the resin. The resin was shaken for 90 min, after which the reaction mixture was filtered and the resin was washed with DMF (6 × 2 ml). The resin was loaded into the Peptide Synthesizer and the PNA was synthesized by automated peptide synthesis. Couplings of 30 min at 75°C were followed by a capping step  for 5 min at room temperature. Fmoc-deprotection was carried out in two treatments with 20% piperidine in DMF for 1 and 3 min at room temperature. The resin, now with the completed sequence, was washed with DMF, DCM and diethyl ether (3× each) and dried in vacuo.In total, 25 µmol resin was allowed to swell in DMF for 10 min, after which 75 µmol Fmoc-azido-lysine  and purified by HPLC on a 0–75% gradient : m/z 1376.10 observed .In total, 20 µmol he resin . The act2.6.2 was left to swell in DMF for 30 min. In a separate tube, 50 µmol DIC, HOAt and Boc-L-Glu-NH2 were mixed in 250 µl DMF and added without pre-activation to the resin and left to react for 90 min. The PNA was subsequently deprotected as described [2O  and purified by HPLC on a 0–75% gradient . m/z 1383.53 observed .In total, 5 µmol 2.7.4 was added 60 nmol MitoOct in MeOH, and the mixture was evaporated in a SpeedVac. The residue was redissolved in 200 µl MeOH and evaporated. This was repeated for five times, until completion of the reaction. The obtained product was purified by HPLC using a 0–75% gradient . m/z 1853.25 observed .To 50 nmol 2.8.3 was added 100 nmol 1 in 60 µl 20 mM ammonium bicarbonate buffer (pH 7.5) and left to react for 1 h at 37°C. The reaction was stopped by addition of 450 µl 0.1% TFA in H2O and the product was purified by HPLC using a 0–75% gradient , which was then lyophilized and dissolved in 50 µl of millipure water. m/z 4550.33 observed .To 50 nmol 2.9.−1 concentration in STE buffer  and stored for a maximum of 3 h on ice. Peptide–PNA 6 was dissolved at the indicated concentrations in 100 µl KCl buffer  supplemented with 10 mM potassium succinate, 4 µg ml−1 rotenone and 10 µM MitoOct. Where indicated carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone  was added to the buffer. The buffer was warmed for 5 min at 37°C after which the reaction was initiated by addition of 5 µl mitochondria suspension (100 µg protein). The mitochondria were incubated for 1 h at 37°C, after which the reaction was stopped by the addition of 3-phenyl-1,2,4,5-tetrazine . The mitochondria were pelleted by centrifugation at 16 000g for 1 min, and supernatant was removed. Next 100 µl 20% acetonitrile solution with 0.1% formic acid, 50 µM PhTet and 0.1 µM internal standard 5 was added to the pellet. The pellet was suspended by three cycles of freezing in solid CO2 (5 min) and sonication in a Grant XB2 ultrasonic bath. The obtained solution was centrifuged at 16 000g for 10 min. The sample was spotted onto an MALDI-plate using the bottom-layer method. Matrix  was spotted on the plate and 0.75 µl sample was mixed in. The spot was left to dry at room temperature, after which another layer of 0.75 µl of matrix was added. In total, 20 spectra with 10 shots each were collected per spot, using a minimum intensity of 1000 and a maximum of 10 000 as selection criterion, averages were taken of three spots per experiment. All experiments were performed in triplicate using different mitochondrial preparations and are presented as averages ±s.e.m.Rat liver mitochondria were prepared by differential centrifugation as previously described , and dil3.3.1.2 (1) to the bromo-acetylated PNA azide (3) in a 20 mM ammonium bicarbonate buffer rapidly drove the reaction to completion  and analysed by MALDI-TOF , and indicates higher concentrations of 6 might have a toxic effect on the mitochondria, as previously observed [Isolated rat liver mitochondria were incubated with various concentrations of peptide (0.1–10 µM) in import buffer, in the presence of MitoOct, for 1 h at 37°C. As previously described, the reaction was stopped by addition of PhTET and centrifugation. The pellet was extracted (20% acetonitrile, 0.1% formic acid, 50 µM PhTet, 0.1 μM 6 into 8 . Signifi to 5 µM c, and inobserved . It is iobserved , we see in organello. We now intend to tailor these methods to investigate the possibility of mitochondrial import of such antigenomic derivatives into whole cells.In summary, we report the optimization of a synthesis protocol that should allow for the ligation of a peptide pre-sequence with a library of diverse PNA or other nucleobase cargoes and confirm that our methods can reliably quantify the matrix import of cleaved molecules


Reversible 2D supramolecular organic frameworks encompassing branched viologen architectures and cucurbit[8]uril (CB[8]) were constructed and investigated. UV-vis investigation clearly indicates the formation and intermolecular dimerization of monocation radicals and their encapsulation into the hydrophobic CB[8] cavity which is further complemented by EPR (electron paramagnetic resonance) spectroscopy. Particle size measurements by dynamic light scattering method showed particle sizes in the range of several µm indicating larger aggregates. Zeta potential measurements suggested the instability of these particles and their tendency to form aggregates. TEM (transmission electron microscope) analysis further revealed the formation of supramolecular polymer  whose diameter were in the range of several µm as indicated by DLS measurements; however the oxidized form, i.e., the viologen dication with cucurbit[8]uril showed dotted spots in the range of sub nanometer level. The internal periodicities of the supramolecular polymers were analyzed by SAXs  measurements. Additionally, we have demonstrated that these supramolecular organic frameworks can be depolymerized by oxidation in air and again can be polymerized  by reduction under inert atmosphere demonstrating that these systems will be of broad interest. They typically involve a host and a guest molecule and based on their presence in a chemical entity, the interactions can be either intramolecular or intermolecular. Most of these interactions are concentration dependent and are usually probed using techniques such as NMR , cyclic voltammetry, UV-Vis, ITC , etc. These as formed supramolecular ensembles2 usually respond to external stimulus such as change in electrochemical potential, temperature, pH, solvent composition and light, etc3. Rational design and synthesis allows the preparation of variety of supramolecular polymers8, cross-linked gel materials10, hierarchical assemblies11 and metastable polymeric nanoparticles. In the last two decades, several supramolecular structures were designed by chemists using the concept of self-assembly12, templating and self-sorting strategies14. Complex-supramolecular architectures between electron deficient and electron rich moieties through non-covalent interactions were developed and were subjected to encapsulation upon addition of respective macrocyclic hosts such as crown ethers, cyclodextrins16, calixarenes17, cucurbiturils18 and pillararenes19. Cucurbit[n]uril (CB[n]), a condensed macrocyclic host molecule with n glyco-uril and n formaldehyde units have the ability to encapsulate different cationic and neutral guest molecules in aqueous medium20. Several studies were reported on the stable ternary inclusion complex formation between CB[8] host and viologen/different guests in different stoichiometric ratio21. Based on this unique property, CB[8] has been effectively employed as a linker in supramolecular assembly like supramolecular polymers, micelles, and hydrogels25. Recently some smart designed supramolecular polymers have been published by applying the strategy of homodimerization29 where chemically one electron reduced viologen species (V+•) undergo irreversible dimerization (V+•…•+V) and these dimerized species are subsequently included into CB[8] cavity via hydrophobic interactions. Gao et al. reported that 1,1′-binaphthyl inter-connected viologen based tweezer like molecules undergoes reversible modulation upon chemical or photochemical reduction30. Spectroelectrochemical investigations of different conjugated oligomeric viologens were reported by Zhao and coworkers. The authors illustrated that the viologen radical cations dimerized to linear supramolecular polymers31. Zhang et al. reported a two-dimensional viologen based supramolecular assembly formed by viologen radical dimerization which is enhanced by CB[8]27. Zhou et al. reported the construction of a three-dimensional viologen based cross-linked supramolecular assembly and they further discussed on the stability of radical dimer with preorganization of rigid building blocks28. Very recently Marchini et al. reported the pimerization of tetrahedrally arranged bipyridinium units wherein, reduced viologen units were intermolecularly dimerized, which stabilizedthe system as well asenchanced the supramolecular growth32. Following a similar strategy, herein we report the synthesis of star-shaped ethyl terminated viologen (ESV) and newly self-assembled network of different viologens  driven by radical–radical (V+•…•+V) dimerization. Further these dimerized species forms a supramolecular polymeric network in the presence of CB[8] and these assemblies were subjected to spectroscopic investigations.Supramolecular assembly encompasses molecular self-assembled structures, supramolecular polymers, molecular daisy chains, etc. that are governed through non-covalent interactions such as hydrogen bonding, hydrophobic interactions, electrostatic interactions, π-π interactions, etcFigure ESV) was synthesized in two steps following a reported procedure as shown in Fig. 16. The synthesis starts with the selective alkylation reaction of 4,4′-bipyridine with 1-bromoethane to obtain the key compound 1 (92%)  to obtain compound 2 (81%) in the first step. In the second step, compound 2 was reacted with well-known trimer core P03PF634 to afford ESV(65%). The newly synthesized star-shaped viologen was characterized by 1HNMR, 13CNMR and DEPT measurements and its purity was cross-checked by elemental analysis.Ethyl terminated star-shaped viologen  parameters for all experiments are; Model = Bruker EMX Plus, Modulation amplitude = 4.0 G, Modulation frequency = 100 KHz, Centre Field = 3480, Sweep Width = 1000 G and Conversion time = 15 ms. Zeta potential and size distribution measurements were carried out in Dynamic Light scattering (DLS) method using Nano ZS model ZEN 3600, Malvern Instrument and Microtrac-Nanotrac 250 instrument respectively. 2O. The working electrode was a glassy carbon disk  and the counter electrode was a Pt wire. The reference electrode was Ag/AgCl/KCl (3 M) electrode (Sinsil) separated by a salt bridge containing 0.1 M KCl/H2O. Prior to each experiment, the working electrode was carefully polished with alumina powder and rinsed with distilled water. Potentiostatic control was provided by CH-Instrument and formal potentials (E0′) were calculated from cathodic and anodic peak potentials in CV’s according to E0′ = (Epc + Epa)/2. The scan rate was 0.1 V/s.Absorbance and Electron Paramagnetic Resonance measurements were carried out in sodium phosphate buffer solution (0.1 M) containing sodium dithionite (50 mM) at RT. Cyclic voltammetry was carried out at room temperature in a standard three-electrode cell in aqueous medium. The electrolyte used was 0.1 M KCl/HCB[8] macro cyclic host molecule. For these analyses (0.1 M) sodium phosphate buffer solution containing (50 mM) sodium dithionite was used.The size distribution of the viologen radical dimerized species in the presence and absence of The morphological structures of the prepared samples were captured using Scanning Electron Microscope (SEM) of TESCAN, VEGA 3 with Bruker Detecter, Zeck Republic. Transmission Electron Microscope (TEM) (Tecnai 20 G2 (FEI make), Netherlands, was used to analyze the surface morphology.Small angle X-ray scattering experiments were conducted on Rigaku (Rigaku Smart Lab) using Cu Kα radiation, the power of X-ray source was operated at 50 mA and 40 kV and the d-spacing values were calculated by the formula d = 2π/q. For SAXs measurement, thin film of the respective polymer solution was dip coated on a glass plate and dried in glove box. The as dried thin films were then measured.CB[8]36 (17%).1H NMR: δ, ppm 5.80 , 5.52 , 4.20 .Reported procedure was followed to obtain 17.Reported literature is followed, for the sake of data completion, the procedure is presented here3CN and stirred at 80 °C. To this solution 1-bromo ethane  dissolved in (10 ml) CH3CN was added slowly over 8 h. The progress of the reaction was monitored by TLC (MeOH: HOAc: H2O –10:4:1). The reaction mixture was stirred for 24 h in total, and then cooled to room temperature. The reaction mixture was concentrated under reduced pressure and the resulting yellow precipitate was washed with excess diethyl ether to remove unreacted bipyridine. The bromide salt was then dissolved in minimal amount of water, precipitated with 3 M NH4PF6,filtered, washed and dried to yield the 1 as a white solid .1H NMR : δ, ppm 8.84 , 8.34 , 7.82–7.81 , 4.63 , 1.66 . 13C NMR : δ, ppm 154.7, 151.9, 145.5, 142.1, 126.8, 122.6, 118.2, 57.8, 16.4. Anal. Calcd for C12H13F6N2P: C, 43.65; H, 3.97; N, 8.48. Found C, 43.55; H, 3.86; N, 8.41.4,4ʹ-bipyridine  was dissolved in 20 ml CHp-xylylene dibromide  was dissolved in 10 mL CH3CN and heated to 80 °C under stirring. To this solution, 1  dissolved in CH3CN (10 mL) was added slowly over 8 h. The reaction mixture was stirred for 48 h in total, and then cooled to room temperature. The reaction mixture was concentrated under reduced pressure and the resulting yellow precipitate was washed with excess diethyl ether to remove unreacted dibromide. The bromide salt was then dissolved in minimal amount of water, precipitated with 3 M NH4PF6, filtered, washed and dried to yield compound 2 as a white solid .. 1H NMR : δ, ppm 8.94 , 8.37 , 7.57–7.47 , 5.81 , 4.66 , 4.61 , 1.64  13C NMR  δ, ppm 151.3, 150.8, 146.4, 146.2, 146.1, 141.2, 133.5, 131.0, 130.5, 128.3, 128.0, 85.0, 58.5, 33.4, 16.4. Anal. Calcd for C20H21BrF12N2P2: C, 36.44; H, 3.21; N, 4.25. Found C, 36.42; H, 3.09; N, 4.00.1  and p-xylylene dibromide  were dissolved in 25 ml CH3CN and stirred at 80 °C for 2 days. Then the reaction mixture was cooled to RT, and the precipitate was filtered and washed with CH3CN and dried. It was then dissolved in water, precipitated with NH4PF6, filtered, washed and dried to yield EDV . 1H NMR : δ, ppm 8.93 , 8.40–8.36 , 7.59 , 5.85 , 4.67 , 1.65 . 13C NMR  δ, ppm 151.6, 150.7, 146.6, 135.3, 131.3, 128.6, 128.3, 128.1, 65.0, 58.7, 16.5. Anal. Calcd for C32H38F24N4O2P4.3H2O: C, 35.24; H, 3.51; N, 5.14. Found. C, 35.18; H, 3.49; N, 5.08.Mixture of 1 were dissolved in 10 ml CH3CN and the reaction mixture was stirred at 80 °C for 24 h. Then the reaction mixture was cooled to RT, and the precipitate was filtered and washed with CH3CN and dried. It was then dissolved in water, precipitated with NH4PF6, filtered, washed and dried to yield the ETV . 1H NMR : δ, ppm 8.96–8.91 , 8.43–8.37 , 7.67–7.52 , 6.32 , 5.83 , 4.67 , 1.64 . 13C NMR  δ, ppm 151.8, 150.3, 146.7, 146.3, 132.8, 132.1, 128.4, 128.3, 128.1, 128.1, 64.5, 58.6, 16.5. Anal. Calcd for C45H48F36N6P6.: C, 35.03; H, 3.14; N, 5.45. Found C, 34.89; H, 3.06; N, 5.33.Mixture of 1,3,5-tris(bromomethyl)benzene  and  2  and P0 3PF634  were dissolved in 20 ml CH3CN, stirred at 80 °C for 4 days. The progress of the reaction was monitored by TLC. Then the reaction mixture was cooled to RT, filtered, washed with CH3CN and dried. It was then dissolved in water: MeOH (1:1) mixture, precipitated with 3 M NH4PF6, filtered, washed and dried to yield ESV. 1H NMR : δ, ppm 8.99–8.94 , 8.43–8.42 , 7.67 , 7.62 , 5.88–5.85 , 4.70 , 1.68 . 13 C NMR : δ, ppm 151.5, 151.4, 151.3, 150.6, 146.6, 146.5, 146.2, 146.1, 135.7, 135.2, 135.1, 132.6, 131.2, 128.3, 128.0, 127.3, 123.4, 64.8, 64.4, 58.5, 16.4. Anal. Calcd for C99H96F72N12P12.2H2O: C, 36.82; H, 3.12; N, 5.20. Found C, 36.75; H, 2.96; N, 5.09.Mixture of +•) shows an absorption maxima at 400 and 600 nm respectively37. This monocation radical has the tendency to dimerize i.e., formation of intramolecular charge transfer complex between the reduced monocation radicals and there is an equilibrium between the monocation radical and its dimeric form. A typical UV-Vis measurement shows absorption maxima at 400, 600 nm corresponding to the monocation radical, upon complexation the wavelength shifts to 550 nm (blue shift)38 corresponding to the monocation radical and an additional 800–1100 nm corresponding to the dimer is observed39. The ratio between these two bands indicates the degree of dimerization.In UV-Vis, dialkyl bipyridinium salts (viologens) typically show an absorption maxima at 250 nm approx. corresponding to the dication, and its one electron reduced monocation radical (VEV) is used as a model compound in this work. UV-vis spectra of four different ethyl terminated viologen compounds  were recorded in aqueous phosphate buffer (pH = 7) solution containing excess sodium dithionite (50 mM) and are displayed in Fig. 2+) to monocation radical (V+•)]. All the viologen radical cations , ESV6(+•) except EV(+•)) displayed broad and strong absorption bands. EDV2(+•) showed absorption maxima at 525 and 1020 nm, ETV3(+•) at 525 and 990 nm, ESV6(+•) at 590 and >1100 nm corresponding to the monocation radical and its dimer respectively. In the case of EV(+•), an absorption maxima centered around 600 nm corresponding to the monocation radical was observed , all the other branched viologens (EDV2(+•), ETV3(+•), ESV6(+•)) undergo dimerization. In the presence of CB[8], the affinity to form radical dimer (V+•)2 increases as the dimer can be easily encapsulated into the hydrophobic cavity of CB[8] which is additionally stabilized by ion-dipole interactions between the oxygen atoms (16 nos) and radical cation40. On the other hand, the possibility for intramolecular stacking between radical cations of peripheral viologen units in the projected viologen derivatives is fewer as the intramolecular radical dimerization is mainly distant dependent16. Further the presence of rigid benzene core moiety and unfavorable steric interactions suppress the possibilities of intramolecular dimerization.Ethyl viologen , EDV2(+•), ETV3(+•) & ESV6(+•). The concentration of the monocation radical (0.50 mM) and CB[8] (0.25 mM) were kept constant throughout the experiment. EPR spectra were recorded in sodium phosphate buffer solution (0.1 M) containing sodium dithionite (50 mM). The radical cation of the model compound (EV(+•)) showed a sharp EPR signal with a g-factor value 2.0046 31 and this is in agreement with the reported values for simple viologen monocation radical indicating the very organic feature of the radical32. The sharp hyperfine splitting of respective viologen radical cations (EV(+•), EDV2(+•), ETV3(+•)& ESV6(+•)) indicated that the radical cations (here after denoted as (V+•)) exclusively gets dimerized (V+•)2 as reported by Zhang et al.27. However, all these molecules EV(+•), EDV2(+•), ETV3(+•)& ESV6(+•) displayed a different behavior than the molecules reported by Zhang et al. EPR spectra of all these radicals displayed hyperfine splitting with high resolution corresponding to a dimer, and we could not detect any weak broad hyperfine signal corresponding to the monocation radical very closer to the radical dimer except in the case of ESV6(+•)  before the addition of CB[8]27. These results clearly indicate that the monocation radicals exclusive dimerize under our experimental conditions. Nevertheless, in the case of EV, these results are in conflict with the UV-Vis measurements and this could be related to the equilibration time. Probably the EV(+•) needs more time for the complex formation and equilibration time under UV-Vis measurement conditions is insufficient.Electron paramagnetic resonance spectroscopy (EPR) gives clear insight on the material with unpaired electrons. Since we reduced the viologen dication to monocation radical and encapsulated them into the CB[8] cavity, EPR analysis can probe the nature of the radicals and their interaction with CB[8]. In this work, we have investigated the paramagnetic properties as well as the dimerization tendency of four different viologen radical cations such as 046 Fig.  closer t+•)2. These broad hyperfine structures specify the encapsulation of viologen dimeric species into the hydrophobic cavity of CB[8]. As a result of encapsulation, electron-electron exchange interaction between the corresponding viologen radicals becomes reduced (dipole-dipole interactions becomes weak)38. The strength of the broad hyperfine structure increases in the order EDV2(+•) > EV(+•) > ETV3(+•) > ESV6(+•) 2 upon complexation with CB[8]. All these results clearly indicate the existence of intermolecular- interactions between the radical dimers (V+•)2 which is further enhanced in the presence of CB[8] as indicated by UV-Vis and EPR measurements. Intermolecular interactions in these structure leads to molecular self-assembly in a 2D (EDV2(+•), ETV3(+•), ESV6(+•)) orientation. Addition of CB[8] further enhances the formation of inclusion complexes leading to the construction of a well-defined 2D supramolecular polymers as reported by Zhang et al.27 and Marchini et al.32. These self-assembled architectures are guided through non-covalent interactions and their consequent movements and rotations indicate that the supramolecular network should be polydisperse and hence the order of these polydisperse supramolecular polymers may be ideal or randomly arranged 2D structures26.Under identical conditions CB[8] was added to the solution containing radical dimer, equilibrated and EPR spectrum was recorded as displayed in Fig. +•) Fig.  indicati2+ ↔ V+•) and monocation radical to neutral bipyridinylidene (V+• ↔ V0) respectively. The electrochemical potential required for the redox process of viologens solely depends on N-substitution16.Viologens or 4,4′-bipyridinium salts exhibit excellent redox behavior and undergoes two consecutive one electron reduction to form the monocation radical and neutral bipyridinylidene. A standard dialkyl viologen shows redox peaks at −0.4 V and −0.8 V corresponding to the reduction of dication to monocation radical  and their inclusion complexation with CB[8] were monitored by cyclic voltammetry (CV)  displayed two reduction peaks at a potential of −0.62 V and −0.89 V. Further the positive and negative potential shifts for the rest of the three viologen compounds were compared with redox potential values of EV. Typically the first reduction, V2+ ↔ V+• occurs easily, while the second reduction (V+• ↔ V0) becomes more difficult, as more electrical stimuli is required for reducing the monocation radical (V+•) [there is an equilibrium between the monocation radical and its dimerized form (V+• ↔ V+•- V+•)]. Thus the difficulty in the second reduction i.e., larger shift in the electrochemical potential can be attributed to the dimerization of the monocation radical. The electrochemical potential difference between the two reduction and two oxidation waves in ethyl terminated viologens were investigated by CV, and the potential differences were denoted as ∆Ep, ∆Ep increases with increase in 4,4′-bipyridinium units in the respective viologen architectures .The electrochemical properties of four different ethyl terminated viologens CV) Fig.  in the pEV, 0.33 V for EDV, 0.35 V for ETV and 0.46 V for ESV and these shift in potential differences enhance the stability of their respective dimer (V+•)2. On the other hand, the anodic potential increased in the order EV (0.21 V) < EDV (0.24 V) < ETV (0.29 V) < ESV (0.30 V) and such increments can be accounted for the additional stabilization of the dimer. It is noteworthy that in the absence of CB[8], all the four viologens displayed similar CV profile and diffusion behavior. In the presence of CB[8], viologens compounds displayed different behavior, EDV and ESV showed same number of redox waves with obvious positive and negative redox potential shifts. Particularly EDV showed larger ∆Ep values of 0.42 V and 0.66 V for the corresponding anodic and cathodic potential differences in the presence of CB[8]. These large difference in the reduction potential could be ascribed to the higher tendency of radical dimerization (V+•)2 in the presence of CB[8] and further stacking of such inclusion assembly as probed by particle size measurements. Noteworthy is the very low diffusion current of the ESV-CB[8] complex (approx. 5–7 times lesser) compared to that of the parent compound (ESV) depicting the fact that it forms large molecular aggregates which limits its self-diffusion towards the electrode surface. In all the other cases decrease in diffusion current of approx. 1.5–2 times was observed indicating the formation of inclusion complex. With and without CB[8], the first oxidation peak in all the four cases depicts preferential adsorption of the complex over the electrode surface . The other viologens such as the model compound EV and the trimer ETV showed three different reduction waves, wherein the first two reduction peaks are ascribed to the EV2+ ↔ EV+• and EV2+ + EV+• → (EV+•)2 processes27. The third reduction peak is attributed to the conversion of radical cation to its neutral form EV+• ↔ EVo at the potentials of −0.89 (EV) and −0.79 (ETV). The slight potential shifts on both anodic as well as cathodic side is a result of an improved stability of (V+•)2 after encapsulation into hydrophobic cavity of CB[8].Notably, the reduction potential differences were 0.27 V for  with CB[8]. To infer more details into the supramolecular framework, particle size measurements were done for viologen compounds without and with CB[8] in aqueous solutions. These supramolecular framework were constructed by self-stacking the respective viologen radical cations (V+•) and they exhibit certain hydrodynamic diameter (DH) (particle size) in water. The DH value of supramolecular entity formed by (V+•) before the addition of CB[8] was found to be 0.66 μm for EV, 0.53 μm for EDV, 0.85 μm for ETV and 0.35 μm for ESV  produced aggregates with the DH value 1.35 μm which was twice when compared with the value (0.66 μm) without CB[8], denoting that 2:2 or 1:1 complex was formed in the mixture. Further the solution containing 0.2 mM CB[8] and EDV2(•+) formed aggregates with the DH value of 0.72 μm which is slightly larger compared to the value (DH = 0.53 μm) without CB[8]. This smaller difference in particle size indicates the formation of 2:1 complex between CB[8] and EDV2(•+) in water. On the other hand in the presence of 0.3 mM CB[8], the solution containing dimerized species of ETV3(•+), ESV6(•+) produced aggregates with the DH values of 1.04 μm and 1.03 μm respectively. These DH values of un-complexed ETV3(•+) and ESV6(•+) species were 0.85 μm and 0.35 μm. These particle size differences indicates the formation of 2:1 and 2:3 complexes of ETV3(•+) and ESV6(•+) with CB[8] respectively.UV-Vis, EPR and cyclic voltammetric studies clearly revealed the possibility of formation of intermolecular dimerized supramolecular network where the arrangement on 2D depends on the nature of the viologen precursor EV(•+), EDV2(•+), ETV3(•+) and ESV6(•+) respectively. All these values indicate high instability/rapid aggregation of these molecules. It is noteworthy that in the absence of CB[8], the zeta potential values were negative and as soon as the intermolecular dimer gets encapsulated in CB[8] cavity, the value becomes positive.In addition, zeta potential measurements were carried out to study on the stability of these monocation radicals in the absence and presence of CB[8] Figure . The staETV3(•+) with cucurbit[8]uril) whose diameter were in the range of several µm as indicated by DLS measurements; however the oxidized form, i.e., the viologen dication with cucurbit[8]uril showed dotted spots in the range of sub nanometer level  analysis of viologen trimer further revealed the formation of supramolecular polymer .HR-TEM images of the samples were given in supporting information Figures  and S11.(i)EDV showed a broad scattering peak with a d-spacing value of 4.4 nm corresponding to {200} facet41.(ii)ETV showed relatively less scattered broad peak with a d-spacing value of 5.7 nm corresponding to {100} facet(iii)ESV showed a sharp peak with a d-spacing value of 6.8 nm corresponding to {100} facet41.The internal periodicities of the samples were measured by SAXs  measurements and are shown in Fig. 27. This is further confirmed by SAD pattern measured from the TEM analysis.The internal periodicities of these samples clearly reflect the fact that they form 2D architectureBased on UV-Vis studies, SAXs measurements and particle size measurements, we propose the following scheme to illustrate the supramolecular self-assembly process as shown in Fig. 2 gas.It is well-known that the viologen dication undergoes two electron reductions to produce monocation radical and a neutral species and such reduced species can be reoxidized chemically/electrochemically. Throughout this study, we employed reduced monocation radical to study its dimerization and subsequent formation of supramolecular organic framework. Hence it is possible to reoxidize this species by aerial oxidation or by passing O+•/V(+•)2 with CB[8] was oxidized by bubbling O2 and the spectra was recorded which showed typical viologen dication (V2+) absorption at 220–260 nm. This solution was again degassed with N2 and reduced with sodium dithionite. The corresponding UV-Vis spectra showed typical bands for the monocation radical and the dimerized species. Likewise, the experiment was repeated for five consecutive cycles and the system was found to be reversible. Thus, the supramolecular polymerization can be reversed by bubbling oxygen and can be formed again by degassing with N2 and reducing with sodium dithionite. Under our experimental conditions, we were able to repeat this cycle for at least five times without much decrease in the redox activity and hence we believe that these supramolecular polymerization can be reversed i.e., depolymerized. Once oxidized, the dicationic viologens exhibit electrostatic repulsion and hence they unwind from CB[8] cavity, where only one viologen unit remain encapsulated in the CB[8] cavity. Thus the system becomes monomeric. Again the reduction with sodium dithionite under inert atmosphere yields monocation radical which can undergo facile intermolecular dimerization into the CB[8] cavity forming supramolecular polymeric network. Thus oxidation leads to depolymerization of the network and subsequent reduction yields the supramolecular polymeric network.To probe more on the reversibility of supramolecular polymerization, we performed five subsequent cycles of UV-V is measurements by consecutive oxidation and reduction of the same solution Fig. . The ful and CB[8] and the interactions were probed spectroscopically in detail by employing UV-Vis spectroscopy, cyclic voltammetry, EPR spectroscopy and particle size measurements. The mechanism of self-assembly was: (i) strong stacking of viologen radical cations (V+•) in aqueous/phosphate buffer solution containing excess of sodium dithionite, (ii) the dimerized species (V+•)2 can be included in to hydrophobic cavity of CB[8] which additionally stabilizes the (V+•)2 species. UV-vis investigation clearly proves that the radical cations were involved in intermolecular dimerization and the dimerized species were encapsulated into CB[8] cavity. This inclusion behavior was nicely supplemented by UV-Vis spectroscopy in the region of 800–1100 nm. EPR studies also revealed that the addition of CB[8] enhanced the dimerization. Particle size measurements showed a particle size in the range of several µm indicating larger aggregates and strongly supported the formation of 2D single -assembly as reported by Zhou et al. and Zhang et al. However, our results partly vary from their previous reports in terms of particle size. Zeta potential measurements suggested that these particles are highly unstable and have the tendency to form aggregates readily, and notably zeta potential value changed from negative to positive upon encapsulation with CB[8]. In addition, we have demonstrated that these supramolecular organic frameworks can be depolymerized by oxidation in air and again can be polymerized  by reduction under inert atmosphere. Such reversible systems are of broad interest as they show advantages over irreversible systems. A key point of these newly–assembled processes are high stability of radical cations with formation of larger aggregates leading to the formation of 2D assembly as indicated by TEM. SAXs profile of the supramolecular polymer samples showed broad scattering peaks indicating the internal periodicity of the supramolecular polymer. Thus the dimerization of radical cations can be considered as a new strong non-covalent force to construct highly ordered supramolecular ensembles for wide range of applications. Future studies will focus on the photophysical and spectroelectrochemical investigations on these systems towards reversible electron/charge storage properties.In summary, we reported the 2D supramolecular organic framework formed between the different viologen guest molecules Supplementary Information


Cancer immunotherapy offers a promising approach in cancer treatment. The adenosine A2A receptor (A2AR) could protect cancerous tissues from immune clearance via inhibiting T cells response. To date, the role of A2AR in head and neck squamous cell carcinoma (HNSCC) has not been investigated. Here, we sought to explore the expression and immunotherapeutic value of A2AR blockade in HNSCC.The expression of A2AR was evaluated by immunostaining in 43 normal mucosae, 48 dysplasia and 165 primary HNSCC tissues. The immunotherapeutic value of A2AR blockade was assessed in vivo in genetically defined immunocompetent HNSCC mouse model.+Foxp3+ regulatory T cells (Tregs), which partially expressed A2AR, was observed in an immunocompetent mouse model that spontaneously develops HNSCC. Pharmacological blockade of A2AR by SCH58261 delayed the tumor growth in the HNSCC mouse model. Meanwhile, A2AR blockade significantly reduced the population of CD4+ Foxp3+ Tregs and enhanced the anti-tumor response of CD8+ T cells.Immunostaining of HNSCC tissue samples revealed that increased expression of A2AR on tumor infiltrating immune cells correlated with advanced pathological grade, larger tumor size and positive lymph node status. Elevated A2AR expression was also detected in recurrent HNSCC and HNSCC tissues with induction chemotherapy. The expression of A2AR was found to be significantly correlated with HIF-1α, CD73, CD8 and Foxp3. Furthermore, the increased population of CD4These results offer a preclinical proof for the administration of A2AR inhibitor on prophylactic experimental therapy of HNSCC and suggest that A2AR blockade can be a potential novel strategy for HNSCC immunotherapy.The online version of this article (doi:10.1186/s12943-017-0665-0) contains supplementary material, which is available to authorized users. Head and neck squamous cell carcinoma (HNSCC), arising in the oral cavity, larynx, hypopharynx and oropharynx, is the 6th most common cancer worldwide . Despite+ Foxp3+ regulatory T cells (Tregs) [Solid tumors are often infiltrated by immune cells, predominantly T lymphocytes and myeloid cells . More T  (Tregs) . As comp (Tregs) . Moreove (Tregs) , 11. Tre (Tregs) , 13. The (Tregs) .+ Foxp3+ cells [Immunosuppressive adenosine 3′5’-monophosphate (cAMP)-mediated pathway, signaling through adenosine A2A receptor (A2AR), can inhibit T lymphocytes and natural killer (NK) cells in hypoxic, inflamed, and cancerous microenvironment . A2AR in3+ cells . These rIn the present study, we aimed to identify the correlation between A2AR expression and clinicopathological characteristics in HNSCC tissue microarrays. In vivo, we sought to investigate the anti-tumor effect induced by A2AR pharmacological blockade in genetically defined immunocompetent HNSCC mouse model.Human tissue microarrays (TMAs) include 43 histologically confirmed normal oral mucosae, 48 dysplasia (Dys), 165 primary HNSCC (PH), 12 recurrent HNSCC, and 17 HNSCC with induction chemotherapy . HPV infection was determined by immunohistochemistry staining of p16 and further confirmed by HPV DNA in situ hybridization as previously described . The patTgfbr1/Pten double conditional knockout (2cKO) mice  were bred as previously described [Tgfbr1flox/flox/Ptenflox/flox mice  from same cage were separated as wide type (WT) control. All the mice were bred in the FVBN/CD1/129/C57 mixed background.The time inducible tissue-specific escribed . The micTgfbr1/Pten 2cKO mice were given tamoxifen by oral gavage for 5 consequent days [n = 6) and A2AR antagonist SCH58261 treatment group (n = 6). A week later, SCH58261 (1 mg/kg) or vehicle was intraperitoneally injected into Tgfbr1/Pten 2cKO mice every other day until the end point. The endpoint was determined according to a systematic evaluation by the veterinarian. Photographs of tumor-bearing mice were taken at day 19 and day 34. Body weight and the tumor volumes were measured every other day. All mice were euthanized at the end of the study.The ent days . And theSingle cell suspension was isolated from spleen, lymph nodes, peripheral blood and tumors according to a standardized protocol . Cells f+ T cells were purified from freshly isolated tumor infiltrated lymphocytes of the 2cKO mice from vehicle group or SCH58261 treated group by immunomagnetic sorting using the mouse CD8+ T cell isolation kit and following the manufacturer’s instructions (Miltenyi Biotech). The purity of the isolated CD8+ T cells was measured by surface staining with anti-CD8 mAb. The overall purity of the resulting cells was 85.3% ± 1.2%. Cell viability was >90% as measured by trypan blue exclusion.CD8+ T cells were cultured in RPMI medium at a concentration of 1 × 106 for 8 h. The supernatants were collected for IFN-γ and TNF-α measurement. The levels of IFN-γ and TNF-α were determined by enzyme-linked immunosorbent assay (ELISA) (BD Pharmingen and R&D System).Freshly isolated CD8Briefly, the human HNSCC tissue sections were hydrated and antigen retrieval. Then sections were blocked with goat serum and incubated with rabbit polyclonal antibody against A2AR (Abcam) at 4 °C overnight, followed by incubation with fluorochrome conjugated secondary antibodies  and DPAI (Vector Laboratories). The images were observed and taken using C2+ confocal microscope system (Nikon).Paraffin sections of human HNSCC tissue microarrays or mouse HNSCC section were rehydration in graded alcohol. The antigen retrieval was performed in boiled sodium citrate. All the sections were incubated in 3% hydrogen peroxide for endogenous peroxidase blockade. Goat serum or rodent block (for mouse section) was used to block the non-specific binding at 37 °C for 1 h. Next, sections were incubated with antibody for A2AR (Abcam 1:200), HIF-1α (Abcam 1:200), CD73 (Genetex 1:200), Foxp3 (Abcam 1:100), CD8 , CD8α  at 4 °C for 12 h. On the day 2, sections were incubated with secondary biotinylated immunoglobulin G antibody solution and an avidin-biotin-peroxidase reagent. Then, the section stained with DAB kit (Mxb Bio) and the sections lightly counterstained with haematoxylin . Negative control with primary antibody replaced by PBS, isotype control and commercial available positive control for each antibody were set in parallel.n = 6, respectively) and stored at −80 °C. All samples were lysed with RIPA Lysis Buffer (Beyotime), which contains protease inhibitors and phosphatase inhibitors. Lysates were denatured in loading buffer (Beyotime) at 95 °C for 5 min. Protein samples were separated by 12% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore). Rabbit A2AR antibody , Rabbit CD73 antibody  and Rabbit HIF-1α antibody (Abcam 1:1000) were used as primary antibody, GAPDH  was used as loading control. Western blot staining was performed by using enhanced chemiluminescence detection kit (Advansta). All Western blots were repeated at least 3 times.The mouse tumor tissues were carefully dissected  with background subtraction. The histoscore of each slice was quantified by Aperio Quantification System (Version 9.1). The histoscore of pixel quantification was calculated as the total intensity/total cell member.+ T cells, MDSCs and TAMs in spleen, lymph nodes and peripheral blood of different groups and the spleen index. Unpaired t test was used to analyze the difference of A2AR expression in tumor size (T1 + T2 vs T3 + T4), lymph node metastasis (N0 vs N1 + N2), HPV infection status (HPV+ vs HPV-), primary HNSCC and recurrence HNSCC (primary vs recurrence), primary HNSCC and TPF chemotherapy specimen (primary vs post TPF), population change of CD4+ Foxp3+ Tregs and CD4+ Foxp3+ A2AR+ cells from each group, the immunohistochemical staining of Foxp3+ and CD8+ cells from each group and the increased body weight. The data are presented as the Mean ± SEM, and statistical significance was determined as P < 0.05. The correlation of A2AR and HIF-1α, CD73, Foxp3, and CD8 was analyzed by two-tailed Pearson’s statistics. The Kaplan-Meier method and the log-rank test were applied to analyze the overall survival rate (OS) between A2AR high group and A2AR low group. The median of A2AR histoscore was chosen as cut-off. For the hierarchical cluster, the histoscore were converted into −3 to 3 using Microsoft excel. Then, the results were imported and hierarchical analysis was performed with Cluster 3.0. The heatmap was visualized with Java TreeView 1.0.5.Statistical analysis was performed GraphPad Prism 5.0 (Graph Pad Software Inc). One-way ANOVO followed Tukey test was applied to analyze the difference in A2AR expression in normal oral mucosa, oral epithelial dysplasia and HNSCC, pathological grades, the population change of CD8ADORA2A, encoding human A2AR, in Estilo’s tongue squamous cell carcinoma (SCC) dataset, a publicly available cancer database [P < 0.001, Grade I vs. Grade III, P < 0.001), larger tumor size , and positive lymph node status . These data clearly indicated that the increased expression of A2AR in HNSCC was correlated with advanced disease stage. To further explore the prognostic value of A2AR in HNSCC, Kaplan-Meier analysis was carried out and the median of A2AR histoscore was selected as a cutoff point. Higher expression of A2AR indicated a rather poorer outcome as compared with lower A2AR expression cohort  and HNSCC tissues with induction chemotherapy (P < 0.01) was significantly increased  and CD73 (P < 0.001 r = 0.2751). This correlation indicated the synergic activation of the hypoxia-CD73-A2AR pathway in human HNSCC.It has been reported that hypoxia could induce the expression of HIF-1α and subsequently activated CD73-A2AR adenosine pathway, which benefited to the immune escape of cancer cells . On the + T cells anergy, thus exerting a potent immunosuppressive function [P < 0.01 r = 0.2285), and negatively correlated with CD8 . The relationship among these molecules was determined by Hierarchal clustering analysis in primary HNSCC  mouse model by crossing K14-CreERtam; Tgfbr1flox/flox (Tgfbr1 cKO) mice with Ptenflox/flox mice, which spontaneously develops HNSCC with full penetration, and is suitable for preclinical intervention, especially for cancer immunotherapy research [Loss of an HNSCC . On the research . In ordeTgfbr1 in epithelia induces an enhanced paracrine effect of TGF-β1 on tumor stroma [+ Foxp3+ Tregs in spleen, lymph nodes and peripheral blood from wild type mice or 2cKO tumor bearing mice respectively by quantitative flow cytometric analysis. As compared with the wild type, significant increase of CD4+ Foxp3+ Tregs population was found in the spleen, lymph nodes and peripheral blood of 2cKO tumor bearing mice . The tumor growth was mainly restricted to the head and neck area of mice  and MDSCs (CD11b+ Gr-1+) were significantly increased in vehicle group as compared with wild type mice. However, SCH58261 treatment could not significantly reduce the frequency of TAMs and MDSCs . We found that SCH58261 treatment significantly increased the production of IFN-γ and TNF-α , we constructed a spontaneous immune competent HNSCC mouse model, which was suggested as an appropriate pre-clinical animal model for HNSCC research. The emerging tumors were not only pathologically indistinguishable from the human HNSCC, but also presented major molecular alternations in human HNSCC [Tgfbr1 and Pten in mouse head and neck epithelia activated hypoxia-CD73 pathway and consequently induced the elevation of A2AR on the immune cells, including CD4+ Foxp3+ Tregs and CD8+ T cells. Additionally, deletion of Tgfbr1 in murine head and neck epithelia resulted in enhanced paracrine effect of TGF-β1 in tumor stroma, which facilitated the immunosuppressive status and promoted the tumor progression [+ Foxp3+ Tregs in the tumor of 2cKO mice. It has been reported that A2AR stimulation enhanced the proliferation of Tregs [+ Foxp3+ Tregs in 2cKO tumor bearing mice, emphasizing the potential role of A2AR signaling in regulating the expansion or functions of Tregs in HNSCC. These findings also provided us with the rationale for decreasing Tregs by A2AR antagonist. Indeed, pharmacological blockade of A2AR by antagonist repressed the tumor growth of 2cKO mice and reduced the population of CD4+ Foxp3+ Tregs. Meanwhile, an enhanced anti-tumor response of CD8+ T cells was observed in 2cKO tumor bearing mice treated with A2AR antagonist SCH58261, indicating the improvement of the immunosuppressive status. This result was partially in accordance with the study that A2AR protected tumor cells from anti-tumor CD8+ T cells [Alterations of PTEN/PI3K/AKT/mTOR pathway and TGF-β1 are the most frequent molecular events during HNSCC tumorigenesis and progression . In the an HNSCC . In the gression . Given tgression , we founof Tregs . In the  T cells . It has  T cells . In the Cytotoxic chemotherapeutic agents are widely employed in the war for fighting against cancer . NeverthTgfbr1 and Pten in murine epithelia activated HIF-1α, CD73 and subsequently induced A2AR overexpression in tumor infiltrating immune cells, accumulating immunosuppressive CD4+ Foxp3+ Tregs in the stroma of tumors. Furthermore, pharmacological blockade of A2AR in vivo by antagonist SCH58261 repressed the tumor growth, inducing a reduction of CD4+ Foxp3+ Tregs and an enhanced anti-tumor response of CD8+ T cells. Hence, our results provided preclinical evidence that A2AR probably be a potential immunotherapeutic target for treatment of HNSCC.In summary, our study revealed the elevated expression of A2AR in human HNSCC tissues, which was correlated with the advanced pathological grade, larger tumor size, positive lymph node status and poor prognosis of HNSCC patients. Moreover, deletion of Additional file 1: Fig. S1.ADORA2A mRNA level in human tongue squamous cell carcinoma. ADORA2A mRNA level in Estilo’s tongue squamous cell carcinoma (SCC) dataset (P = 0.001) (JPEG 200 kb)Additional file 2: Fig. S2.a Represent immunohistochemistry image of p16 and A2AR in HPV positive (HPV+) and HPV negative (HPV-) sample. b The expression of A2AR was not related to HPV infection status  (JPEG 690 kb)The relationship of A2AR expression and HPV status. Additional file 3: Fig. S3.n = 165) (JPEG 404 kb)Hierarchical clustering of HIF-1α, CD73, A2AR, CD8 and Foxp3 histoscore in primary HNSCC. The relationship among HIF-1α, CD73, A2AR, CD8 and Foxp3 was determined by Hierarchal clustering analysis in primary HNSCC  in spleen, lymph node (LN), peripheral blood and tumor from wild type mice (WT) and 2cKO tumor bearing mice treated with DMSO (vehicle group) or with SCH58261. b Quantification of TAMs (CD11b+ F4/80+) in spleen, lymph node (LN), peripheral blood and tumor from WT mice and 2cKO tumor bearing mice treated with DMSO or with SCH58261  (JPEG 288 kb)A2AR blockade is unable to influence the population of MDSCs and TAMs.


Candida albicans and Aspergillus fumigatus are essentially identical and reveal N- and C-terminal Rossmann fold domains that form the glucose-6-phosphate and UDP-glucose substrate binding sites, respectively. These Tps1 structures with substrates or substrate analogues reveal key residues involved in recognition and catalysis. Disruption of these key residues severely impaired Tps1 enzymatic activity. Subsequent cellular analyses also highlight the enzymatic function of Tps1 in thermotolerance, yeast-hypha transition, and biofilm development. These results suggest that Tps1 enzymatic functionality is essential for the fungal stress response and virulence. Furthermore, structures of Tps1 in complex with the nonhydrolyzable inhibitor, validoxylamine A, visualize the transition state and support an internal return-like catalytic mechanism that is generalizable to other GT-B-fold retaining glycosyltransferases. Collectively, our results depict key Tps1-substrate interactions, unveil the enzymatic mechanism of these fungal proteins, and pave the way for high-throughput inhibitor screening buttressed and guided by the current structures and those of high-affinity ligand-Tps1 complexes.The disaccharide trehalose is critical to the survival of pathogenic fungi in their human host. Trehalose-6-phosphate synthase (Tps1) catalyzes the first step of trehalose biosynthesis in fungi. Here, we report the first structures of eukaryotic Tps1s in complex with substrates or substrate analogues. The overall structures of Tps1 from  Candida albicans and Aspergillus fumigatus in complex with substrates, substrate analogues, and inhibitors. These structures reveal key protein-substrate interactions, providing atomic-level scaffolds for structure-guided drug design of novel antifungals that target Tps1.Invasive fungal diseases have emerged as major threats, resulting in more than 1.5 million deaths annually worldwide. This epidemic has been further complicated by increasing resistance to all major classes of antifungal drugs in the clinic. Trehalose biosynthesis is essential for the fungal stress response and virulence. Critically, this biosynthetic pathway is absent in mammals, and thus, the two enzymes that carry out trehalose biosynthesis, namely, trehalose-6-phosphate synthase (Tps1) and trehalose-6-phosphate phosphatase (Tps2), are prominent targets for antifungal intervention. Here, we report the first eukaryotic Tps1 structures from the pathogenic fungi  Cryptococcus, Candida, and Aspergillus, cause significant burdens to the health care system  have increased tremendously in immunocompromised patients over recent decades. Two million cases of IFDs are reported annually, with an overall mortality rate higher than 50% . Pathogee system –4. Even  system –. The abs system –.Candida albicans to adapt or overcome cellular stresses like growth at high temperatures and oxidative stress  as the search model. The structure of Tps1 in complex with UDP and G6P was determined by molecular replacement using the Tps1-UDPG structure as the search model. Both structures take the hexagonal space group P64 with two subunits in the asymmetric unit. Selected data collection and refinement statistics are listed in C. albicans Tps1 adopts the typical GT-B fold of the retaining glycosyltransferase family, which is characterized by two modified Rossmann fold domains  of 1.8 Å for 452 corresponding Cα atoms. Furthermore, the structural superposition using the DALI pairwise comparison server reveals a highly conserved active site , Cryptococcus neoformans (accession number Q6IVK9), A. fumigatus (accession number Q4WHW0-Tps1B), and E. coli (accession number P31677) are aligned. The sequences are annotated as Tps1_CA, Tps1_CN, Tps1_AF, and Tps1_EC, respectively. The secondary structure of C. albicans Tps1 is shown above the sequences, whereby α-helices are depicted by arrowheads and β strands by rectangles. Identical residues found in all four Tps1 proteins are contained in blue boxes, while those identical in 3 of 4 proteins are contained in light blue boxes. The secondary structure elements are colored as described in the legend to FIG S1, DOCX file, 1.2 MB.Structure-based sequence alignment of Copyright © 2017 Miao et al.2017Miao et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.00643-17.6TABLE S1 C. albicans Tsp1 data collection and refinement statistics. Download TABLE S1, DOCX file, 0.1 MB.Selected Copyright © 2017 Miao et al.2017Miao et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the C. albicans Tps1. Adding to the substrate recognition and binding affinity is the ribose ring of UDP, as the O2 and O3 hydroxyl groups form hydrogen bonds with the side chain of residue E387. With regard to the phosphate moiety of UDP, one hydrogen bond is made between its α phosphate group and the backbone amide group of residue L383. A conserved Arg-Lys pair (residues R280 and K285) interacts with the β phosphate. These interactions are significant for positioning the UDPG in the correct conformation for the activity and subsequent stabilization of the UDP leaving group  substrates/product in the structure of the Tps1-UDPG and Tps1-(UDP+G6P) complexes.  2FCopyright © 2017 Miao et al.2017Miao et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the In order to locate the G6P binding site, the structure of Tps1 in complex with UDP and G6P was solved to 2.37-Å resolution. The most notable interactions between G6P and Tps1 are between the phosphate moiety and side chains of residues Y89 and R318 . AnotherC. albicans was aligned with those of E. coli, Cryptococcus neoformans, and A. fumigatus , K285A, D379A, and E387A. Compared with that of the wild-type protein, no enzymatic activity was detected for these mutants. Thus, the identity of these substrate-binding residues is essential for Tps1 activity. Additionally, these mutations were introduced into C. albicans by replacing the wild-type allele in the tps1Δ/TPS1 strain with the TPS1 gene carrying the designated nucleotide changes conferring the changes in codons. At 37°C, a tps1Δ/tps1Δ deletion strain and the strains with K285A, D379A, and E387A substitutions but not the strain with the Y89F substitution showed temperature sensitivity , and Tps1-(UDP+VDM), clearly reveals retention of the anomeric configuration upon catalysis  mechanism, with UDPG deprotonating G6P. This mechanism has been proposed and experimentally characterized for OtsA, the E. coli homologue of Tps1 . Both structures were determined in complex with UDP and VDM to obtain accurate information on the substrate stereochemistry and active-site residue–substrate interactions.In order to test our hypothesis that the Tps1 proteins from other pathogenic fungi employ the same enzymatic mechanism as that observed for the A. fumigatus Tps1A-(UDP+VDM) complex was determined to 2.80-Å resolution by molecular replacement using the structure of C. albicans Tps1-UDPG as the search model. Two molecules were present in the asymmetric unit, and electron density was not visible for residues 1 to 11, 35 to 39, 56 to 63, 68 to 70, and 480 to 515 of chain B. The presence of a disordered C terminus (residues 480 to 515) is consistent with secondary structure prediction by PSIPRED  of 0.52 Å for 452 corresponding Cα atoms  complex structure, is also seen between the β phosphate oxygen atom and the N-glycosidic bond of VDM. This interaction further supports the idea that Tps1 utilizes an SN-i mechanism  complexes strongly support an internal return-like mechanism (SN-i) for catalysis, which is utilized by E. coli OtsA and likely applies to other prokaryotic and eukaryotic GT-B fold retaining glycosyltransferases. Thus, our current results have deepened significantly our understanding of trehalose biosynthesis in pathogenic fungi and its importance in cell viability and have provided the foundation for designing antifungal inhibitors with potentially low or no off-target effects.In summary, we report here the structures of the first fungal and, indeed, the first eukaryotic Tps1 proteins in complex with substrates, product, substrate analogues, or post–transition-state analogues. These structures, together with the results of enzymatic activity assays, highlight key residues for Tps1 activity. Subsequent genetic and e strain , a TPS1 e strain . This ise strain . Therefotps1 gene from C. albicans strain SC5314 and tps1A and tps1B genes from A. fumigatus strain Af293 were codon optimized for expression in E. coli (GenScript). These codon-optimized genes were cloned into a pET-28a kanamycin-resistant vector via restriction sites NdeI and SacI. These vectors contained an N-terminal hexahistidine affinity tag followed by a thrombin cleavage site. Each vector was transformed into BL21(DE3)pLysS cells  and induced by the addition of 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG)  at 15°C for 16 h when the optical density at 600 nm (OD600) reached 0.6. The Tps1 proteins were purified by Ni2+-nitrilotriacetic acid (NTA) affinity column chromatography followed by thrombin cleavage. Each protein was purified further using size exclusion chromatography via a Superdex S200 column  in buffer containing 20 mM Tris, pH 8.0, 200 mM NaCl, 5% glycerol, 5 mM MgCl2, and 1 mM β-mercaptoethanol (βME). The resulting purified Tps1 enzymes were concentrated using Amicon Ultra concentrators . To prepare singly and doubly mutated Tps1, a standard site-directed mutagenesis protocol was employed  400.The employed . For allA. fumigatus Tps1A and Tps1B were concentrated using an Amicon Ultra concentrator  to 12 mg/ml. Tps1A and Tps1B were cocrystallized with 10 mM UDP and VDM. Crystals were grown at 25°C by the hanging-drop vapor diffusion method. Diffraction-quality crystals of the Tps1A-(UDP+VDM) complex appeared after 1 week from solutions of 0.1 M sodium malonate, pH 8.0, 0.1 M Tris, pH 8.0, and 30% PEG 1000. Tps1B-(UDP+VDM) crystals appeared after 3 weeks from solutions of 0.2 M lithium sulfate monohydrate, 0.1 M Tris, pH 8.5, and 20% PEG 3350.Purified d-glucose. Then, 1 M ammonium hydroxide (20 ml) was used to release validoxylamine A from IR-120 resin. After filtration, the solvent was removed to yield validoxylamine A  as an off-white solid. 1H nuclear magnetic resonance (NMR) : δ 6.08 to 5.91 , 4.30 to 4.08 , 3.76 to 3.56 , 3.39 , 3.31 to 3.24 , 2.03 to 1.83 , 1.31 . Mass spectrometry (MS) (electrospray ionization [ESI]): [M+H]+ 336.The title compound was synthesized according to the procedure described by Iwasa et al. . ValidamC. albicans Tps1 structure with UDPG was determined by molecular replacement using Phaser (1UQU)  ID-22 line and were indexed and scaled using HKL 2000 . The C. g Phaser  and withr (1UQU) . The oth in Coot  and impr in Coot  and rebu2, and 2 mM dithiothreitol (DTT). Final concentrations of 3 μM, 1 mM, and 1 mM were utilized for Tps1, UDPG, and G6P, respectively. Activity assays were performed in Greiner 96-well plates, and the decreases in absorbance at 340 nm were recorded using a SpectraMax M5 plate reader (Molecular Devices). The initial enzymatic activity velocities were plotted and compared between WT protein and mutant proteins.The catalytic activity of Tps1 was measured utilizing a continuous enzyme coupled assay as previously reported . BrieflyC. albicans strains used in this study are listed in C. albicans strains were grown in YPD  medium. Cultures were maintained at 30°C unless otherwise specified. The deletion mutant and strains carrying point mutations in TPS1 were constructed using the Clox system , and set to a concentration of 1 × 10To assess hypha formation in liquid, strains were grown overnight in YPD at 30°C and transferred to YPD containing 10% fetal bovine serum as described in reference g for 5 min at 4°C. The supernatant was removed, and the cell pellet was resuspended in fresh PBS. Cells were washed three times, and then the cells were resuspended in PBS to achieve a final concentration of 1 × 107 CFU/ml. Individual contact lenses (Acuvue) were transferred to a 12-well tissue culture plate. Contact lenses were submerged in PBS for 15 min before being transferred to a new 12-well plate. Two contact lenses per strain were used. To each contact lens, 4 ml of the respective Candida cell suspension was added and allowed to incubate (without shaking) at 37°C for 90 min to permit cell attachment. Following attachment, each contact lens was carefully transferred to a new 12-well plate containing 4 ml of YNBD  medium. Plates containing the lenses were then incubated for 22 h at 37°C with gentle shaking at 50 rpm.Biofilms were grown on contact lenses according to a protocol published previously . BrieflySamples were fixed with 4% paraformaldehyde (Electron Microscopy Sciences) in PBS for 45 min at room temperature. Samples were rinsed twice with PBS and then dehydrated through a series of ethanol washes . The samples were subsequently coated with gold and imaged with a field emission scanning electron microscope (FESEM) (FEI XL30) in high-vacuum mode at 5 kV.C. albicans Tps1 in complex with UDPG, UDP and G6P, and UDP and VDM have been deposited in the Protein Data Bank under accession codes 5HUT, 5HUU, and 5HVL. The coordinates and structure factors of A. fumigatus Tps1A and Tps1B bound to UDP and VDM have been deposited in the Protein Data Bank under accession codes 5HVM and 5HVO.The coordinates and structure factors of