Costanza Montis

Pierre Joseph

Chiara Magnani

Alejandro Marín-Menéndez

Francesco Barbero

Amalia Ruiz Estrada

Ridvan Nepravishta

Jesus Angulo

Alice Checcucci

Alessio Mengoni

Christopher J Morris
email: morris@uea.ac.uk

Debora Berti
email: debora.berti@unifi.it

MIVEGEC A M M Crcn

Multifunctional nanoassemblies target bacterial lipopolysaccharides for enhanced antimicrobial DNA delivery

Keywords: Nanomedicine, Antibiotic Resistance, Transcription Factor Decoys, Giant Unilamellar Vesicles, Biomimetic Membranes INTRODUCTION

The rising resistance of bacteria to antibiotics is recognized as a major healthcare issue that, together with the scarcity of new therapeutic approaches, mandates the development of new antimicrobials acting upon novel bacterial targets [START_REF] Holmes | Understanding the mechanisms and drivers of antimicrobial resistance[END_REF]. Recently, the World Health Organization (WHO) published a list of antibiotic-resistant "priority pathogens", a catalogue of 12 families of bacteria that pose the greatest threat to human health, for which immediate research effort is required [START_REF]Antibiotic Resistance[END_REF]. The list highlights the threat of resistant Gram-negative bacteria including Acinetobacter, Pseudomonas and various Enterobacteriaceae that are resistant to multiple antibiotics including last-resort agents such as carbapenems and third generation cephalosporins.

The inherent antimicrobial resistance of Gram-negative bacteria is related to their unique structure: differently from Gram-positive ones, cell walls of Gram-negative bacteria are characterized by the presence of an asymmetric outer membrane containing >70% w/w of lipopolysaccharides (LPSs), the general structure of which is sketched in Figure 1. LPS consists of an immunogenic lipid portion embedded in the outer leaflet of the lipid bilayer (Lipid A), which is bound to an oligosaccharide moiety (R-core) and to an outer polysaccharide portion protruding from the membrane (O-antigen). [START_REF] Schmidtchen | Peptide interactions with bacterial lipopolysaccharides[END_REF][START_REF] Clifton | An Accurate in Vitro Model of the E. coli Envelope[END_REF] This thick and highly anionic layer, strongly anchored to the underlying membrane, acts as an efficient xenobiotic permeability barrier [START_REF] Delcour | Outer membrane permeability and antibiotic resistance[END_REF].

Besides its effects on bacterial resistance, LPS (also called endotoxin), when present in the bloodstream, can trigger uncontrolled inflammatory responses of the host, eventually leading to sepsis and death from septic shock.

A rational approach to the development of effective therapeutics for systemic Gram-negative bacterial infections might include targeting of LPS for the dual purpose of enhancing cellular delivery of antimicrobial agents as well as blocking the immune response in advance of the onset of inflammation and sepsis. It has been recently demonstrated that the coagulation-related peptide, thrombin, binds and sequesters LPS, leading to accelerated aggregation and phagocytic clearance of E. coli bacteria by macrophages in vitro. [START_REF] Petrlova | Aggregation of thrombin-derived C-terminal fragments as a previously undisclosed host defense mechanism[END_REF] More generally, LPS targeting, binding and removal from the circulation is an innate anti-inflammatory strategy prevalent in species spanning invertebrates to higher mammals. For example, host defense peptides or antimicrobial peptides (AMPs) are an intrinsic component of the innate immune system and are characterized by a broad-spectrum antimicrobial and anti-inflammatory activity. [START_REF] Hancock | The immunology of host defence peptides: Beyond antimicrobial activity[END_REF] As such, these peptides have received much attention as potential alternatives to traditional antibiotics. [START_REF] Schmidtchen | Lipo)polysaccharide interactions of antimicrobial peptides[END_REF][START_REF] Singh | Importance of lipopolysaccharide aggregate disruption for the anti-endotoxic effects of heparin cofactor II peptides[END_REF] In previous studies we presented a nanostructured antimicrobial, made of a cationic bolaamphiphile (12-bis-THA, the structure of which is displayed in Figure 1a) associating with a Transcription Factor Decoy (TFD), an aptamer designed to interfere specifically with the gene transcription mechanisms of the bacteria, to form nanosized complexes (hereafter termed loaded nanoplexes, LNP). Delivery of therapeutic TFDs to pathogenic bacteria is contingent upon co-formulation with the self-assembling 12-bis-THA amphiphile, which binds to and efficiently protects from nuclease degradation the DNA component [START_REF] Marín-Menéndez | Antimicrobial Nanoplexes meet Model Bacterial Membranes: the key role of Cardiolipin[END_REF].

LNPs have been shown to be a promising antimicrobial system against both Gram-positive and Gram-negative bacteria [START_REF] Marín-Menéndez | Antimicrobial Nanoplexes meet Model Bacterial Membranes: the key role of Cardiolipin[END_REF][START_REF] Mamusa | Cationic liposomal vectors incorporating a bolaamphiphile for oligonucleotide antimicrobials[END_REF]. Their antibacterial efficacy is attributable to two distinct functions: first, a generic antimicrobial action of the cationic bolaamphiphile, interacting with and weakening cardiolipin-rich bacterial membranes; and secondly, a specific antimicrobial activity of the TFD towards selected genes involved in vital cellular functions of bacteria, such as metabolism and replication. [START_REF] Marín-Menéndez | Antimicrobial Nanoplexes meet Model Bacterial Membranes: the key role of Cardiolipin[END_REF] There is also an absence of known transcription factormediated resistance pathways, which is a clear advantage over many conventional antibiotic targets. Besides TFD complexation and delivery, the amphiphilic and cationic nature of 12-bis-THA, features shared with AMPs, makes these nanoplexes attractive as a possible LPS targeting species and for anti-inflammatory and antimicrobial purposes in the treatment of Gram-negative bacterial infections. In this report, we perform a fundamental study on the interaction of LNPs with biomimetic and in vivo bacterial membranes, in order to explore and understand their potential as multifunctional experimental therapeutics for the treatment of Gram-negative bacterial infections. In this respect, our aim is twofold: first, to specifically investigate 12-bis-THA-TFD nanoplexes as possible antimicrobial and anti-inflammatory systems for Gram-negative bacterial infections; second, to contribute, more generally, to the progress of the fundamental knowledge on the interaction of antimicrobials with Gram-negative bacteria, which will help in the design of more efficient multifunctional therapeutics. To this aim, we study the interaction of LNP with synthetic mimics of Gram-negative bacteria, from two complementary standpoints: (i) bacterial membrane destabilization to achieve an efficient delivery of the specific antimicrobial TFD into the bacterial cytoplasm (ii) LPS binding and sequestration for anti-inflammatory properties.

An ensemble of experimental techniques, spanning the colloidal length scale down to molecular scale interactions, were combined to demonstrate that LNPs are a versatile system that destabilizes the bacterial outer membrane, promoting cell entry of the antimicrobial TFD, as well as exerting anti-inflammatory action through LPS sequestration. Therefore, LNPs represent multifunctional, tunable, colloidal systems with potential therapeutic efficacy against Gram-negative bacterial pathogens. As such, they have the potential to offer tailored antiinfective treatment in healthcare settings that are increasingly challenged with antibioticresistant pathogens.

RESULTS AND DISCUSSION

Interaction of nanoplexes with biomimetic Gram-negative bacterial membranes:

Confocal Microscopy and Microfluidics

In order to examine the interaction of LNPs with bacterial cell models, we prepared Giant Unilamellar Vesicles (GUVs) of different and controlled compositions, mimicking either Gram-negative or Gram-positive bacteria and challenged them with LNP. GUVs are primitive mimics not able to fully reproduce the complexity of natural membranes, however, their synthetic nature, allowing a fine control over their composition, geometry and environment, makes them a valuable model to investigate cell-membrane-related phenomena in simplified conditions, both related to eukaryotic [START_REF] Dimova | Giant Vesicles and Their Use in Assays for Assessing Membrane Phase State, Curvature, Mechanics, and Electrical Properties[END_REF][START_REF] Laurencin | Interactions between giant unilamellar vesicles and charged core-shell magnetic nanoparticles[END_REF] and to bacterial cells [14,[START_REF] Anunthawan | Cationic amphipathic peptides KT2 and RT2 are taken up into bacterial cells and kill planktonic and biofilm bacteria[END_REF] ; here we employ GUVs containing the basic components of bacterial membranes, to assess the roles of individual lipid components in the cellular response to the nanoplexes. As bacterial membrane mimics, we prepared GUVs composed of POPC and cardiolipin (CL), including or excluding 17% w/w LPS from E. coli. While CL is a membrane-specific lipid present both in Gram-positive and Gram-negative bacteria, LPS is unique to Gram-negative membranes and is of paramount relevance in determining the inefficacy of many antibacterial treatments. Figure 2 shows some representative Confocal Microscopy (CLSM) images of the Gram-positive POPC-CL model (Figure 2a) and the Gram-negative POPC-CL-LPS model (Figure 2b) GUVs, containing 0.1% mol:mol Liss Rhod PE, a fluorescent lipid, embedded in the lipid bilayer (red). The GUVs' sections are visible as perfectly rounded circles, with Liss Rhod PE staining the lipid membrane shell. While the shape and size of the GUVs is similar in the absence and in the presence of LPS, a significant efficiency decrease of GUV's yield prepared with the usual electroformation procedure was observed in the latter case, probably due to the higher complexity of the lipid mixture and the increase of negatively charged species to be incorporated in the lipid assembly. In order to check the efficient incorporation of LPS in GUV membranes, we employed a fluorescently labeled LPS. Figure 2c-e Figure 3 displays some representative CLSM images of 120 μL GUVs (0.03 μg/μL lipid content, with or without LPS,), challenged with 100 μL of an aqueous dispersion of LNPs (0.18 mM 12-bis-THA and 10 μg/μL TFD), containing an AlexaFluor 488-labeled TFD (green). In this medium, LNPs have a size of 200 nm, and a zeta-potential in the order of +30 mV, similar to what previously shown [START_REF] Mamusa | Interaction between a cationic bolaamphiphile and DNA: The route towards nanovectors for oligonucleotide antimicrobials[END_REF]. When GUVs of POPC-CL were used, LNP docked at GUV membranes and fused with them after 2 h incubation (Figure 3a-c); in addition, part of the lipid probe originally staining the GUVs bilayer migrated into the aggregates (white arrows, Figure 3a). This proves a close interaction between the LNPs and the GUVs, also in the absence of lipopolysaccharides in the GUVs, which is expected considering the negative charge of CL (see Figure 1b), and the positive z-potential of the nanoplexes. However, even low concentrations of LNPs, when added to GUVs containing LPS (Figure 3d), displayed extensive docking and fusion with the GUV membranes. Especially noticeable were extended regions of colocalization of the red lipid label with the green TFD and a clustering of the GUVs with LNPs acting as intervesicular bridges (white arrows, Figure 3d). These interactions were more pronounced with higher concentration of the LNPs (Figure 3 e-g), with many GUVs completely disrupted and the remaining ones extensively deformed and collapsed.

To conclude, the presence of the LPS determines much more striking interactions between LNPs and the model lipid membranes. This effect is quite unexpected, considering that LPS act as a protective layer in Gram-negative bacteria, hindering the docking and penetration of exogenous species on bacterial membrane and, ultimately, their entry inside the bacterium.

In order to understand the dynamic aspects of this interaction and better elucidate the GUV membrane disruption, we shifted our focus from static to dynamic experiments, monitoring the LNP-GUV interaction kinetics with confocal microscopy imaging combined with a customized microfluidic configuration, designed to trap and challenge GUVs with a solution [START_REF] Magnani | Hybrid vesicles from lipids and block copolymers: Phase behavior from the micro-to the nano-scale[END_REF]. Briefly, a PDMS (polydimethylsiloxane) microfluidic chip consisting of two inlets was secured upon a coverglass. Through the first inlet we injected the GUV's dispersion, while the second one was for the subsequent injection of LNP dispersions (Figure 4a). From the first inlet, the injected GUVs flow through a microfluidic channel of 50 μm thickness and 350 μm width containing a series of U-shaped traps at defined channel positions. After GUVs trapping, LNPs stained with an Alexa 488-labeled TFD (green) were injected and the interaction monitored in real time. First, it should be highlighted that an optimal trapping of the GUVs inside the Ushaped obstacles was challenging due to adhesion effects of LNPs on the channel walls and on GUVs. Nevertheless, striking and unambiguous differences were observed in the absence and in the presence of LPS (compare the time evolution in Figures 4b andc). In the absence of LPS (Figure 4b), the docking of the LNPs at the GUV membrane was clearly visible, although no modifications in the shape of the GUVs were visible over a total LNP exposure time of 160 seconds, which is consistent with the results of static CLSM observations. Conversely, in the presence of LPS (Figure 4c,d), there was a high tendency of the GUVs to interact extensively with LNPs, eventually causing the shrinkage of GUVs (Figure 4c) and, for longer interaction times (Figure 4d), even the implosion of the GUVs, probably due to a lipid extraction effect of the LNPs aggregates (see Supplementary Movies). Interestingly, the shrinkage and implosion of GUVs was observed only when LPS was included in the membrane formulation: this suggests that LNPs bind to LPS and extract it from the membrane. Control experiments confirmed that GUVs prepared with and without LPS were both stable throughout the experimental timescale (data not shown).

These observations indicate that, contrary to current knowledge [START_REF] Nikaido | Molecular Basis of Bacterial Outer Membrane Permeability Revisited[END_REF], the presence of LPS favors, rather than inhibits, the interaction of nanoplexes with the target GUVs. This finding is particularly interesting for two reasons: first, the dramatic membrane perturbation and local disruption of GUVs suggests a dramatic increase in membrane permeability, which would favor the delivery of TFD into the GUV lumen and might, therefore, favor the antimicrobial effect of LNPs against Gram-negative bacteria; second, the microfluidic experiment points at a specific binding of 12-bis-THA nanoplexes to the LPS component of the lipid membrane and, possibly, its selective extraction, eventually leading to the complete shrinkage of the GUV. This latter effect suggests that 12-bis-THA-TFD nanoplexes could bind and sequester immunogenic LPS, with anti-inflammatory consequences.

Nanoplexes as antimicrobials against Gram-negative bacteria: LPS-dependent delivery of TFD

The antimicrobial activity of LNPs is dependent upon the delivery of TFD into the bacterial cytoplasm, so that vital cellular processes are repressed by competitive inhibition of transcription factor activity [START_REF] Mcarthur | Manipulating and understanding antibiotic production in Streptomyces coelicolor A3(2) with decoy oligonucleotides[END_REF]. To monitor this ability, we performed a Fluorescence Correlation Spectroscopy (FCS) investigation on the penetration of a fluorescent TFD both into Gram-positive and Gram-negative biomimetic GUV membranes. Figure 5a shows representative FCS profiles acquired for the Alexa 488-labeled TFD alone (blue filled circles) and after interaction with 12-bis-THA, to form LNPs (red empty circles). The FCS curve of the TFD could be analyzed with a two-component, three-dimensional normal diffusion model (see SI equation S5) [START_REF] Ries | Fluorescence correlation spectroscopy[END_REF][START_REF] Koynov | Fluorescence correlation spectroscopy in colloid and interface science[END_REF][START_REF] Schaeffel | Fluorescence correlation spectroscopy directly monitors coalescence during nanoparticle preparation[END_REF][START_REF] Montis | Size distribution of extracellular vesicles by optical correlation techniques[END_REF], where the fast component has a diffusion coefficient D1 of 370 ± 40 μm 2 s -1 ,while the slow component is characterized by D2 = 60 ± 20 μm 2 s -1 . The slower component is consistent with the diffusion of the dye conjugated to the oligonucleotide, while the fast component is clearly due to a small percentage of the dye not conjugated to the oligonucleotide, possibly through unavoidable nuclease contamination during non-sterile experimental procedures. However, through FCS these two components can be easily, reliably and precisely distinguished. In the presence of the bolaamphiphile, the TFD diffusion is clearly slowed down, due to the 12-bis-THA complexation with DNA and formation of nanoplexes. The diffusion coefficient of the complexed TFD, within the limits of accuracy of this experimental technique for objects of comparable size with respect to the size of the detection volume (see SI equation 3) [START_REF] Koynov | Fluorescence correlation spectroscopy in colloid and interface science[END_REF], can be evaluated as: D= 1.9 ± 1 μm 2 s -1 , consistent with nanoplexes of around 200 nm hydrodynamic diameter (SI equation S4).

The LNPs were next incubated with the model Gram-positive cytoplasmic membrane, POPC-CL (Figure 5b) and the model Gram-negative membranes, POPC-CL-LPS (Figure 5c) GUVs.

Figure 5b and 5c report some representative CLSM images, consistent with the previously discussed phenomena (see Figure 34). In brief, LNPs interact closely with the lipid membrane of GUVs, docking and eventually fusing into it. However, no TFD accumulation is observed inside the GUVs' lumen, and the LNPs' aggregates remain largely confined on the surface of the lipid membrane. FCS measurements were performed inside POPC-CL and POPC-CL-LPS GUVs in order to verify if TFD, in complexed or free form, is released inside the GUVs'.

Figure 5d shows the FCS curves acquired inside POPC-CL GUVs' (green filled markers) and inside POPC-CL-LPS GUVs' (purple empty markers), respectively. For POPC-CL GUVs the measured FCS profile can be interpreted according to a 1-component 3D fitting model, due to the sole presence of the free dye (D = 370 μm 2 s -1 ). For POPC-CL-LPS GUVs a second longer decay is present. Using a two-component model, in which the fast-diffusing component is fixed at D = 370 μm 2 s -1 , an estimate of the LNP intra-luminal diffusion coefficient was determined as D = 2.9 ± 0.6 μm 2 s -1 . These observations indicate that the presence of the LPS on the lipid membrane not only promotes the interaction of the LNPs with the GUVs' membrane, but also permits internalization of the nanoplexes smaller than the original ones (average hydrodynamic diameter 150 nm), suggesting a partial decomplexation when the LPS-containing membrane is crossed. In order to corroborate the data gathered on bacterial membrane mimics, we performed FCS on a live model Gram-negative bacterium, E. coli, to monitor the penetration of the fluorescently-labeled TFD into the bacterial cytoplasm. Clearly, FCS measurements on bacteria are particularly challenging due to the detection volume, which, in the z direction, is slightly larger than the bacterium size. This latter limitation does not allow a precise estimate of the diffusion coefficient inside the detection volume. E. coli bacteria were challenged with LNPs containing Alexa 488-labeled TFD. After 1h 30' incubation at r.t. the bacteria were extensively washed in order to exclude surface-bound LNPs from the analysis. Figures 6a,b show the CLSM images of E. coli after incubation with LNPs: the green fluorescence of the Alexa 488-labeled TFD inside the bacterial cytoplasm suggests the successful uptake of TFD.

Figure 6c displays five representative FCS curves acquired inside the bacterial cytoplasm of different E. coli cells. The clearly visible correlation decay is due to the diffusion of the TFD inside the bacterial cytoplasm. However, the FCS profiles were generally noisy, due to the complex and heterogeneous intracellular environment, united with a low fluorophore concentration. From these data, the reliable determination of a diffusion coefficient (and possibly a hydrodynamic size) is not possible. However, by fitting the experimental curves as due to a single component 3D diffusion, a diffusion coefficient of about 5 ± 2 μm 2 s -1 was inferred. This value is one order of magnitude higher than the diffusion coefficient of a lipidated Bodipy probe (molecular mass ~ 500 Da) sequestered in the cytoplasmic membrane of E. coli [START_REF] Nenninger | Independent mobility of proteins and lipids in the plasma membrane of Escherichia coli[END_REF] and determined through FRAP (D= 0.6 ± 0.2 μm 2 s -1 ). This supports the claim that the fluorescent species diffuses in the cytoplasm, rather than the cell membrane, which has a considerably higher effective viscosity. In addition, if we compare this result with the diffusion coefficient obtained inside the LPS-containing GUVs (D = 2.9 ± 0.6 μm 2 s-1) and take into account that the bacterial cytoplasm is characterized by a higher viscosity with respect to water [START_REF] Nenninger | Independent mobility of proteins and lipids in the plasma membrane of Escherichia coli[END_REF][START_REF] Kumar | Mobility of cytoplasmic, membrane, and DNAbinding proteins in Escherichia coli[END_REF][START_REF] Mika | Molecular sieving properties of the cytoplasm of Escherichia coli and consequences of osmotic stress[END_REF], we can conclude that inside the bacteria the TFD is, at least partially, decomplexed.

Overall, these experiments prove the significant role of LPS in promoting membrane permeabilization and subsequent TFD delivery inside cells' cytoplasm, upon interaction with LNPs.

Nanoplexes as anti-inflammatory agents: LPS binding and sequestration.

The observations highlighted above prompted us to investigate the mechanism of LPS sequestration by 12-bis-THA and its biological consequences. Using saturation transfer difference (STD) NMR spectroscopy [START_REF] Mayer | Characterization of ligand binding by saturation transfer difference NMR spectroscopy[END_REF] we sought to examine, at the molecular level, the extent and specificity of 12-bis-THA binding to liposomes of DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) in the absence and in the presence of LPS, taken as model membranes. STD NMR is appropriate for studying small-molecule binders of weak-medium affinity to large target molecules [START_REF] Mayer | Group epitope mapping by saturation transfer difference NMR to identify segments of a ligand in direct contact with a protein receptor[END_REF]. Through saturation of the target "receptor" molecule (which in this study is LPS bound to a liposome surface) and by studying the transfer to a bound ligand (here represented by 12-bis-THA), a map of ligand-receptor binding sites can be constructed. No STD signals were observed for samples containing 12-bis-THA and pure DOPC liposomes (Figure 7a, blue spectrum) indicating the absence of interaction in this system. In sharp contrast, many STD peaks were observed with DOPC liposomes containing LPS (Figure 7a, red spectrum), indicating extensive interaction between LPS-decorated liposomes and 12-bis-THA (reference spectrum for which is shown in green, Figure 7a). The STD NMR technique affords an exquisite insight into the ligand domains that bind to a target macromolecule through the comparison of the extents of STD for different resonances after irradiation at different "receptor" frequencies [START_REF] Monaco | Differential Epitope Mapping by STD NMR Spectroscopy To Reveal the Nature of Protein-Ligand Contacts[END_REF]. For example, irradiation of LPS at different frequencies such as -4 ppm for the lipophilic core, or 4.9 ppm and 5.2 ppm for the glucosamine rings GlcN-B and GlcN-A of LPS (Figure 1b), permits the construction of an epitope binding map, detailing the proximity of 12-bis-THA protons to the LPS binding site (Figure 7b). Careful analysis identified the orientation of the protons labeled here as C, E, I, F and K toward GlcN-A and GlcN-B (highlighted in Fig 1b). This region presents a positive charge located on the Nitrogen atom, which presumably makes contact with the negative charge located on the GlcN-A orienting the head protons H, G, A, B, D away from this contact point (details of the binding orientation are in the Supplementary Information). It is also interesting to note that the protons labeled as M and N located in the center of the symmetrical linker, are oriented toward the aliphatic chains of DOPC/LPS liposome (Figure 7b). Following these arguments, one may conclude that the binding region of the 12-bis-THA on the LPS is centered on the GlcN-A negative charge, establishing close proximity with the positively charged Nitrogen atom at the polar head of 12-bis-THA, while the central protons of the lipophilic linker interact closely with the aliphatic region of the DOPC/LPS liposome. These data indicate that the interaction between 12-bis-THA and LPS is specific and not solely attributable to charge compensation. Functional neutralization of LPS by 12-bis-THA was confirmed by in vitro studies with mouse RAW 264.7 macrophage cells. We challenged macrophages with E. coli LPS in the presence or absence of escalating concentrations of 12-bis-THA or polymyxin B (PxB), which was deployed as a gold standard LPS binder [START_REF] Paracini | Liquid crystalline bacterial outer membranes are critical for antibiotic susceptibility[END_REF] used in the clinic as both an antibiotic [START_REF] Falgas | Colistin: The Revival of Polymyxins for the Management of Multidrug-Resistant Gram-Negative Bacterial Infections[END_REF] and endotoxin removal tool [START_REF] Cruz | Effectiveness of polymyxin Bimmobilized fiber column in sepsis: A systematic review[END_REF]. LPS-only controls consistently increased the nitrite concentration of the culture medium to > 10 M. The co-application of 12-bis-THA with LPS caused a concentration-dependent decrease in nitrite concentration, indicating reduced nitric oxide by macrophage cells i.e. an anti-inflammatory effect mediated through LPS sequestration.

Strikingly, 12-bis-THA assemblies and PxB were of comparable potency at inhibiting nitric oxide release from macrophages in response to LPS (Figure 7c) with IC50 values of 5.1 and 0.5 M, respectively. The viability of macrophage cells was not compromised across the 10 nM -100 μM concentration range tested, as judged by MTT assay (Supplementary Figures). These results indicate that the extracellular binding of LPS by 12-bis-THA is sufficient to prevent the acute pro-inflammatory response mediated through the cell surface toll-like receptor 4 receptor complex and the downstream inducible nitric oxide synthase (iNOS). Confiscation of bloodcirculating LPS in septic patients is a key research priority in light of the increasing number of Gram negative bacterial sepsis deaths worldwide [START_REF] Chopra | Treatment of health-care-associated infections caused by Gram-negative bacteria: a consensus statement[END_REF]. The neutralization of LPS is a highly desirable characteristic that has been pursued by a number of groups who have employed a range of different scaffolds spanning the small and macromolecular size ranges [START_REF] Chen | Topomimetics of amphipathic β-sheet and helix-forming bactericidal peptides neutralize lipopolysaccharide endotoxins[END_REF][START_REF] David | Towards a rational development of anti-endotoxin agents: Novel approaches to sequestration of bacterial endotoxins with small molecules[END_REF][START_REF] Sestito | Amphiphilic Guanidinocalixarenes Inhibit Lipopolysaccharide (LPS)-and Lectin-Stimulated Toll-like Receptor 4 (TLR4) Signaling[END_REF][START_REF] Kumar | Piscidin-1analogs with double L-and D-lysine residues exhibited different conformations in lipopolysaccharide but comparable anti-endotoxin activities[END_REF][START_REF] Ong | Short synthetic β-sheet forming peptide amphiphiles as broad spectrum antimicrobials with antibiofilm and endotoxin neutralizing capabilities[END_REF].

CONCLUSIONS

In this study we investigated the interaction of the nanostructured, antimicrobial 12-bis-THA-TFD nanoplexes (LNPs) with Gram-positive and Gram-negative bacterial biomimetic membranes. We found that the interaction between the LNPs and Gram-negative bacteria critically involves LPS binding to 12-bis-THA nanoplexes which favors the docking of LNPs to the bacterial membrane. These LNP-LPS interactions lead to two phenomena, both of paramount importance to future application of LNPs as efficient and multifunctional therapeutics for Gram-negative infections: (i) they induce membrane destabilization and increase membrane permeability, allowing the efficient delivery of antimicrobial TFDs into the bacterial cytoplasm; (ii) they afford LPS binding and sequestration, with its associated antiinflammatory effects, comparable with the golden standard PxB. These results demonstrate that the combination of 12-bis-THA and the TFD into nanoplexes represents a promising strategy to tackle Gram-negative bacterial infections, with a synergistic, multifunctional approach. In addition, the mechanistic insights on the interaction between nanoplexes and Gram-negative bacterial membrane models can be exploited to predict the antimicrobial-antiinflammatory behavior of different formulations based on cationic amphiphiles and nucleic acids and, therefore, will provide useful basic information for the design of novel effective therapeutics against antimicrobial resistant Gram-negative bacterial infections. Key mutations in LPS biosynthesis, such as sugar phosphorylation, lipid A modification or O-antigen deletions have been identified in Gram negative bacteria such as Pseudomonas aeruginosa isolated from chronically infected cystic fibrosis patients [START_REF] Maldonado | Lipopolysaccharide modification in gram-negative bacteria during chronic infection[END_REF]. Binding between 12-bis-THA and mutant LPS from antimicrobial resistant Gram-negative bacteria as well as antibacterial activity is the subject of ongoing research and will be reported elsewhere. The concomitant anti-inflammatory activity of LNPs contributes a second, highly attractive attribute to this new drug delivery platform, making it an important tool in the fight against antibiotic-resistant pathogens. Web Server (University at Albany, USA). The lyophilized powder was first suspended in filter sterilised Milli-Q water to a concentration of 1 mg/ml. Once in dilution, the samples were incubated 5 minutes at 95°C and then let to cool down at room temperature to allow the complementary sequences to anneal. Then, there were kept at -20°C until used.

EXPERIMENTAL SECTION

Materials

12-bis-THA-TFD nanoplexes (LNP) preparation

The dispersion of 12-bis-THA with chloride counter ion (0.18 mM) in aqueous solution was obtained by first dissolving the compound in milliQ-water. The homogenous dispersion in water was then obtained by vigorous stirring with vortex, leading to the formation of empty nanoplexes (ENPs). Similarly, loaded nanoplexes (LNPs) were obtained by adding the bolaamphiphile dispersion (0.18 mM) to a solution containing the TFD in milliQ-water (10 μg/mL) and stirring the mixture with vortex for 30 seconds. When required, Alexa Fluor 488labelled TFD was used.

Membrane models preparation

Giant Unilamellar Vesicles were prepared through electroformation, as described elsewhere [START_REF] Montis | Model lipid bilayers mimic non-specific interactions of gold nanoparticles with macrophage plasma membranes[END_REF]. Lipid composition was 90% w/w POPC and 10% w/w Cardiolipin (plus 17% w/w LPS for LPS containing GUVs) and 0.1% (for confocal microscopy imaging) or 0.01% (for Fluorescence Correlation Spectroscopy experiments) with respect to the total lipid amount of the fluorescent dyes (β-Bodipy or Liss Rhod PE) was added. The dispersions of GUVs in 50 mM Sucrose were employed within 24 hours after preparation for CLSM and FCS experiments.

Further details are in the SI.

For NMR experiments, 20 mg/mL dispersions of DOPC and DOPC:LPS (50% w/w) liposomes in D2O, were prepared according to a standard protocol of dry lipid film hydration, freeze-thaw and sonication [START_REF] Montis | Nucleolipid bilayers: A quartz crystal microbalance and neutron reflectometry study[END_REF], which is described in details in the SI.

Confocal Laser Scanning Microscopy

CLSM experiments were carried out with a laser scanning confocal microscope Leica TCS SP8 (Leica Microsystems GmbH, Wetzlar, Germany) equipped with a 63x water immersion objective. [START_REF] Baglioni | Polymer Film Dewetting by Water/Surfactant/Good-Solvent Mixtures: A Mechanistic Insight and Its Implications for the Conservation of Cultural Heritage[END_REF] The 488 nm laser line was employed to detect β-Bodipy fluorescence and Alexa 488-labeled TFD (λ excitation 488 nm, λ emission 498 nm -530 nm); the 561 nm laser line was employed to detect Liss Rhod PE dye inside the GUVs' membrane or Alexa 568-labeled LPS (λexcitation 561 nm, λ emission 571 nm -650 nm). Samples containing LNPs and GUVs were incubated for one hour before the acquisition of the images. Further details are in the SI.

Microfluidics experiments

Polydimethylsiloxane (PDMS) -glass microfluidic chips were fabrication is reported in the SI. 50 µL of the solution obtained by electroformation was diluted to 1 mL with a 15 mM aqueous sucrose solution, filtered at 0.2 µm. The obtained solution was injected in one inlet of a Yshaped microfluidic channel (Figure 4), while a diluted (1:10) LNPs standard solution with Alexa-488-labelled TFD was injected in the other inlet. The flow rates of the injected GUVs and LNPs, ranging from 0.1 to 1 μL/min, were controlled thanks to two syringes connected to a syringe pump (Nemesys, Cetoni GmbH) by standard tubing (1/16'' OD, 0.81mm ID). In a microfluidic experiment first GUVs were injected at 1 μL/min and LNPs at 0.1 μL/min, to achieve efficient GUVs trapping without significant exposure to LNPs, then the flow rate was reversed to 0.1 μL/min for GUVs and 1 μL/min for LNPs to expose GUVs to LNPs. Chips were put under vacuum for at least 20 min before the experiments to reduce bubble trapping during channel filling. Characterization was achieved by imaging, by Confocal Laser Scanning Microscopy CLSM (Leica TCS SP8).

Fluorescence Correlation Spectroscopy

FCS measurements were carried out using a Leica TCS SP8 confocal microscope (Leica Microsystems GmbH, Wetzlar, Germany) equipped with a PicoQuant FCS modulus (PicoQuant, Berlin, Germany). Measurements were performed using a 63x water immersion objective. The fluorescently-labeled TFD and Rhodamine 110 for calibration of the confocal volume were excited using the 488 nm-laser line of an Ar laser and the fluorescence emission was acquired using a Hybrid SMD detector in the 498-530 nm range.

For experiments on TFD release inside GUVs 120 µL GUVs dispersion in Sucrose was put in measurement wells (Lab-Tek Chambered 1.0 Borosilicate Coverglass System, Nalge Nunc International, Rochester, NY USA) and diluted with 80 μL Glucose 50 mM. 100 μL (for POPC-CL GUVs) or 20 μL (for POPC-CL-LPS GUVs) of LNPs standard solution prepared as previously described, with Alexa-labelled TFD, were added to the dispersion. Samples containing LNPs and GUVs were incubated for one hour before the acquisition of FCS curves inside GUVs lumen.

For FCS experiments on E. coli a bacterial dispersion in H2O was incubated with the same volume of LNPs (prepared with Alexa 488-labeled TFD). To visualize the bacteria in confocal microscopy and to carry out FCS experiments, the bacteria were layered on the bottom of appropriate wells, which were pre-treated with polylysine, to favor bacterial adhesion on the coverglass. The dispersion of bacteria or bacteria and LNPs was allowed to interact with the polylysine layer for 1h at r.t. in a humidity-controlled chamber, then the supernatant was removed and the bacterial layer inside the wells was extensively washed with H2O and then diluted with 300 µL H2O. FCS experiments in bacteria were carried out by focusing with CLSM the confocal volume in the center of cells' cytoplasm and then acquiring FCS curves.

All details on FCS data analysis are reported in the SI.

Saturation Transfer Difference NMR spectroscopy

1 H NMR Saturation transfer difference experiments were acquired on a Bruker AVANCE 500 MHz at 298K. To a solution of 12-bis-THA 400 µM in D2O was added a suspension of LPS/DOPC liposomes or only DOPC liposomes in D2O at the final concentration of 2 µM of LPS considering a MW of 10 kDa for the LPS. Shortly after that, the sample was transferred in 5 mm NMR tubes for spectra acquisition. The saturation transfer difference (STD) experiment was acquired using the Bruker library pulse stddiffesgp with a SW of 16 ppm a TD of 32K a relaxation delay (D1) of 6 seconds and a variable time for the train of the selective saturating 90° Gaussian pulse, from 0.5 to 6 s. The spectra were acquired with 32 or 64 scans.

The saturation pulse (ON) was positioned in three different regions of the spectra, at -4.0 ppm, 4.9 ppm and 5.2 ppm. These resonances were chosen as representative of the lipophilic core of the LPS/DOPC liposome, GlcN-B and GlcN-A of LPS respectively. The OFF pulse was always positioned at 40 ppm and the spectra obtained were used as a reference. With the aim to obtain insight of the binding orientation of the 12-bis-THA to the LPS the epitope mapping protocol was used. The saturation received from the proton of the ligand is calculated considering the STD build-up curve in a time course experiment using a variable time for the saturation pulse from 0.5 to 6 seconds. The data were fitted to the equation STD=STDmax * (1-exp(-K*t) from where the STD0 was calculated as STDmax*K = STD0. [START_REF] Mayer | NMR-Based Characterization of Phenothiazines as a RNA Binding Scaffoldt[END_REF]

Figure 1 .

 1 Figure 1. Systems under investigation. (Panel a) Antimicrobial nanoplexes: structure of the 12-bis-THA cationic bolaamphiphile (whose assemblies are named empty nanoplexes, ENP); representative structure of a hairpin Transcription Factor Decoy (TFD); scheme sketching TFD and 12-bis-THA interaction to form loaded nanoplexes (LNPs). (Panel b) Membrane models: structure of the lipids employed in the membrane models: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and Cardiolipin (CL); general structure of a Lipopolysaccharide (LPS), with the polysaccharide skeleton and the Lipid A moieties highlighted. Hep: Heptulose (ketoheptose); Kdo: 3-deoxy-α-Dmannooctulosonic acid; P: phosphate.

Figure 2 .

 2 Figure 2. Biomimetic bacterial model membranes. (a-e) Confocal microscopy images of GUVs made of: (a) POPC with 10% w/w CL and (b-e) POPC with 10% w/w CL, plus 17% w/w LPS: (a, b) representative images of GUVs containing the fluorescent tag Liss Rhod PE (red); (c-e) representative images of GUVs of POPC with 10% w/w CL, plus 17% w/w LPS containing the fluorescent lipid tags Bodipy (green) and Alexa-568-conjugated LPS (red) (the separated red (c) and green (d) channels and channel overlays (e) are displayed); (f) spatially resolved emission spectra at λexc 488 nm (green curve, excitation/emission of Bodipy) and λexc 561 nm (red curve, excitation/emission of Alexa-568conjugated LPS) acquired on the same GUV membrane, highlighting the colocalization of the fluorescent tags. Scale bars 30 μm.

  display representative CLSM images of GUVs containing both the lipid fluorescent probe Bodipy (green) and Alexa 568-labeled LPS (red) shown both as separate (2c, d) and overlaid (2e) channels. The colocalization of the fluorescence emission of the two probes confirms the successful inclusion of LPS in the GUVs, corroborated by the spatially resolved emission spectra (Figure 2f) acquired on the same GUV lipid membrane regions.

Figure 3 .

 3 Figure 3. Interaction of LNPs with biomimetic membrane models: CLSM images of GUVs fluorescently labeled with Liss Rhod PE (red) of: (a-c) POPC with 10% w/w CL and (d-g) with the same composition, plus 17% w/w LPS; 120 μL GUVs prepared as described in the experimental section, upon incubation with 20 μL (a-d) and 100 μL (e-g) of LNPs standard solution containing a fluorescent TFD (green); both separate channels and overlayed channels are displayed, with fluorescence colocalization areas seen in yellow. Scale bars 30 μm.

Figure 4 .

 4 Figure 4. Interaction of LNPs with target membranes: Microfluidics. (a) Experimental set-up for the microfluidics experiment; (b-d) representative CLSM time lapse images of (b) POPC-CL and (c, d) POPC-CL-LPS GUVs labeled as described in the main text in a 160 s (b, c) and 400 s (d) timescale; the overlay of Liss Rhod PE and Alexa 488 fluorescence, with colocalization area highlighted (yellow), and bright field (greyscale) are displayed. Scale bars 10 μm.

Figure 5 .

 5 Figure 5. Interaction of LNPs with target membranes: FCS. (a) Representative FCS curves of Alexa 488-labelled TFD (blue filled circles) and of LNPs containing Alexa 488-labelled TFD (red empty circles), curve fitting of the experimental curves (continuous lines); (b, c) representative CLSM images of (b) POPC-CL and (c) GUVs after two hours incubation with LNPs; (d) Representative FCS curves of LNPs containing Alexa 488-labelled TFD measured for POPC-CL GUVs (green filled circles) and for POPC-CL-LPS GUVs (purple empty circles), inside GUVs' lumen (as highlighted with colored spots in (b,c)). The continuous lines display the curve fitting of the experimental curves.

Figure 6 .

 6 Figure 6. Interaction of LNPs with live bacteria: FCS. (a, b) Representative CLSM images of E. coli bacteria upon incubation with LNPs containing Alexa 488-labelled TFD, Alexa 488 fluorescence (green) and bright field (greyscale) overlay; (c) five representative FCS curves of LNPs containing Alexa 488-labelled TFD measured inside E. coli cytoplasm (empty and filled markers), curve fit of the experimental data (continuous lines). The inset of (c) shows a representative brightfield CLSM image of E. coli cytoplasm where FCS has been measured.

Figure 7 :

 7 Figure 7: Binding of 12-bis-THA to LPS and its biological effects. (a) Structural formula of Lipid A (b) STD-NMR spectra of 12-bis-THA (400 μM) alone (green), in the presence of DOPC liposomes (blue) or DOPC:LPS liposomes (red). (c) Structural formula of one half of the symmetrical 12-bis-THA molecule overlaid with colored circles indicating proton orientation in LPS-containing model membranes. Green are protons oriented toward GlcN-A; magenta are protons oriented toward GlcN-B; yellow are protons oriented toward the aliphatic chains of DOPC/LPS liposome. (d) Dose-dependent effects of 12-bis-THA (empty circles) and PxB (filled circles) on nitric oxide release by RAW 264.7 macrophages, determined by Griess assay of media nitrite levels. Data are mean of 3 independent experiments. Error bars are omitted for clarity.

POPC ( 1 -

 1 palmitoyl-2-oleoyl-sn-glycero-3-phospho-choline), DOPC (1,2-dioleoyl-snglycero-3-phosphocholine), Cardiolipin (sodium salt from bovine heart) and Lipopolysaccharide from Escherichia coli were acquired from Sigma Aldrich (St. Louis MO). 12-bis-THA (1,10-(dodecane-1,12-diyl)-bis-(9-amino-1,2,3,4-tetrahydroacridinium) chloride, MW = 635.6 g/mol) was synthesized by Shanghai Chempartners co. (Denmark) with >98% purity. The TFD and its Alexa-488-labelled version were kindly provided to us by Procarta Biosystems Ltd. (Norwich, UK). Both non-labelled and Alexa 488-labelled oligonucleotide were specifically design to finally obtain a hairpin secondary structure according to the oligo analyser tool (version 3.1) from IDT-DNA free software and confirmed with the DINAMelt

Inhibition of nitric oxide production by macrophage cells

The murine macrophage RAW 264.7 cell line (American Type Culture Collection, USA) was maintained at 37 °C in DMEM supplemented with 10% heat inactivated fetal bovine serum, in a 5% CO2 incubator. Cells were seeded onto 96 well plates (10 5 per well) and grown overnight.

Culture medium was removed and replaced with 0.1 mL fresh medium supplemented with 500 ng E. coli LPS alone or LPS combined with escalating concentrations of 12-bis-THA or PxB.

After 24 h, media was removed, centrifuged briefly (200 x g, 5 min) to remove any cellular material and then subjected to a Griess Assay [START_REF] Bryan | Methods to detect nitric oxide and its metabolites in biological samples[END_REF] to determine media nitrite concentration as an indicator of nitric oxide production by iNOS.


Dr Justine Mansot

Jean-Jacques Vasseur

Dr Stellios Arseniyadis
email: s.arseniyadis@qmul.ac.uk

Michael Smietana
email: michael.smietana@umontpellier.fr

Prof. Dr J.-J Vasseur

MINIREVIEW ,-Unsaturated 2-Acyl-Imidazoles in Asymmetric Biohybrid Catalysis

Keywords: Biohybrid catalysis, DNA, Acyl imidazole, Artificial metalloenzyme

Me

Cu

Introduction

The conjugate addition of nucleophiles to electron-deficient alkenes is a common strategy for the formation of CC and Cheteroatom bonds which dates back to the pioneering work of Komnenos in 1883 [1] and Michael in 1887. [2] After more than a century of improvements devoted to the optimization of all the reaction parameters, catalytic asymmetric conjugate additions have become highly selective and, most importantly, highly reliable transformations leading to a broad range of optically active compounds with use in fields such as natural product synthesis and medicinal chemistry. [3] Over the years, it has been demonstrated that both organometallic and non-organometallic nucleophiles could be involved. [4] This later class emerged in the early 80's with the description of a diamine/Co(II) complex [5] and later by using chiral bisoxazoline type ligands [6] able to coordinate a variety of metals. [7] It soon emerged that bidentate substrates were ideally fitted to provide the necessary synergistic effect to reach high catalytic performance through an efficient coordination with the chiral metallic complex. Over the years, a number of ,-unsaturated acyl derivatives (compounds 1-7, Figure 1) were evaluated in a variety of synthetic transformations including Michael and Mukaiyama-Michael type additions, Diels-Alder and hetero-Diels-Alder cycloadditions as well as Friedel-Crafts alkylations. [8][9][10][11][12][13][14][15][16] However, all the reactions involving the aforementioned substrates required cryogenic conditions to reach high levels of enantioselectivity ultimately limiting the scope. To overcome this drawback, Evans and coworkers introduced the use of ,-unsaturated 2-acyl-imidazole 8 as a new bidentate substrate. [17] The latter showed an increased reactivity attributed to a lower steric demand and a higher electophilicity of the enone when compared to the ,-unsaturated-acyl-pyrazoles

(1), oxazolidinones

, thiazolidine-2-thiones (4), phosphonates (5), benzimidazoles (9), thiazoles (10) and pyridines (11). [18] In addition, the chiral Lewis acid-catalysed conjugate addition of various indoles in the presence of (PyBox)-Sc(III) led to the corresponding Friedel-Crafts alkylation products with excellent levels of enantioselectivity.

The synthesis of ,-unsaturated 2-acyl-imidazoles is relatively trivial and usually relies on either the introduction of the imidazole ring on an ,-unsaturated acyl precursor or on the formation of the double bond via some sort of olefination reaction (Figure 2). Okamoto et al. were, to the best of our knowledge, the first to describe the synthesis of ,-unsaturated acyl imidazoles such as 8 through the addition of the deprotonated N-methylimidazole 13 onto ,-unsaturated-Nacyl-pyrrolidine 12 (Figure 2). [19] Other syntheses involve the direct addition of the deprotonated form of 13 on an ,-unsaturated ester (14), a carboxylic acid (15), an acyl chloride (16) or on a Weinreb amid (18). Similarly, the addition of 13 on an enal (17) followed by an oxidation of the resulting alcohol intermediate can also be envisioned. [21] Ultimately, a tandem aldol/crotonization between aldehyde 19 and N-methyl-2acylimidazole 20 [22,23] or a Wittig reaction with the appropriate phosphorous ylide 21 [17] also leads to the title compound. Finally, the phosphorous ylide can be replaced by a phosphonate derivative such as 22, which can readily undergo a Horner-Wadsworth-Emmons type olefination. [24] In addition to improving catalytic performances, the imidazole moiety presents the advantage of being very versatile. Indeed, it can be readily converted to a wide variety of useful chemical functionalities such as ketones, aldehydes, [25] amides, esters, [26] and carboxylic acids through simple synthetic transformations (Figure 3). [25,27]

DNA-based asymmetric catalysis

Natural DNA-helix

The concept of DNA-based asymmetric catalysis, first introduced by Roelfes and Feringa in 2005, [31] relies on a transfer of chirality from the DNA double helix to a pro-chiral substrate using an achiral transition metal catalyst imbedded within the DNA scaffold (Figure 4). The resulting supramolecular architecture provides the necessary chiral microenvironment to induce sufficient enantiodiscrimination. [32] This concept was initially tested on a Diels-Alder reaction between azachalcone 11 and cyclopentadiene 23 in the presence of commercially available DNA extracted from salmon testes (st-DNA). The first generation of ligands that were used were derived from the 9-aminoacridine intercalant, which was covalently bound to a Cu(II) binding diamine. High endo/exo selectivities were obtained (up to 98/2 in favour of the endo product) albeit moderate enantioselectivities (up to 53% ee). Nonetheless, these results demonstrated that the DNA double helix could be used as a powerful source of chirality in the context of asymmetric catalysis. [33] Roelfes and co-workers were later able to improve the selectivities by simply replacing the pyridine moiety by an imidazole. [34][35] By doing so, -aryl-substituted ,-unsaturated acyl imidazoles could be converted to the corresponding endo bicyclic product in up to 98% ee (Figure 5). The best results were obtained when using commercially available 4,4'-dimethyl-2,2'-bipyridine L3 as ligand, also known to be a DNA groove binder. This study also revealed that the catalyst loading could be lowered to 5 mol% without any loss of either enantioselectivity or diastereoselectivity.

These results prompted Roelfes and co-workers to evaluate other CC bond forming reactions in presence of DNA. [36] Hence, the addition of nitromethane or dimethyl malonate to 8 allowed the formation of the corresponding Michael addition products with ees up to 94% and 99%, respectively (Figure 6). Mechanistically, the formation of the major enantiomer was proposed to result from the Si face attack of the complexed acylimidazole substrate as shown in Figure 7. The nature of the Michael donor was later extended to malonitrile by Li et al. [37] Hence, after a thorough screening of the reaction conditions, the use of L3/Cu(II) in conjunction with st-DNA was shown to afford excellent conversions and high levels of selectivity (up to 72% ee, Figure 8). Interestingly, the addition of dissymmetric cyanoacetate derivatives generated the two diastereoisomers in roughly the same ratio in up to 84% ee. Another benchmark in the field concerned the optimization of the enantioselective Friedel-Crafts alkylation with indoles. [38] Hence, through binding affinity studies, Roelfes and co-workers were able to show that only 16% of the catalyst was bound to DNA, while at the same time a 30-fold rate enhancement could be observed when the reaction was run in the presence of DNA, thus suggesting that the background reaction was negligible. As a general trend, the reactions all led to full conversions and ees reaching up to 83% (Figure 9). As a general trend, the best selectivities were obtained with substrates bearing an alkyl substituent at the  position of the enone. Most importantly, the authors were able to show that reaction was scalable and that the catalysts could be recycled without any noticeable loss of reactivity or selectivity. Finally, pyrroles were also shown to be compatible with ees reaching up to 76%.

A few years later, Sugiyama et al. reported an intramolecular version of the Friedel-Crafts alkylation using the bifunctional acylimidazoles 36 (Figure 10). [39] The reactions catalysed by a st-DNA/5,6-dimethyl-1,10-phenanthroline L4/Cu(II) complex proved highly selective as up to 78% ee were obtained with a 5-methoxy group on the indole ring. In sharp contrast, substrates substituted on the nitrogen afforded much lower selectivities. The influence of the co-solvent in the Friedel-Crafts alkylation and the Michael addition was also evaluated. [40] It was notably shown that up to 10% v/v of acetonitrile in water did not affect the binding constant of the catalyst to DNA while allowing an efficient scale-up of the reactions. Furthermore, an acceleration rate of the reaction was noted and attributed to a fast dissociation constant of the product.

The catalytic enantioselective oxa-Michael addition was also reported by Roelfes and co-workers with up to 86% ee (Figure 11). [41] In these experiments, methanol but also ethanol and isopropanol were used in 40 v/v%, however the conversions dropped when increasing the size of the alkyl group at the -position of the enone, while no reactivity was observed with -aromatic substituents. Also worth noting is the formation of the hydration side product in all cases, however this could be minimized by lowering the temperature to 18 °C.

The first example of a non-enzymatic syn-hydration was reported by Roelfes and co-workers in 2010 using st-DNA in association with the same L2/Cu(II) metallic co-factor used for the oxa-Michael addition (Figure 12). [42] Under these conditions, ,-unsaturated 2-acyl imidazoles could be readily converted to the corresponding -hydroxy ketones in up to 72% ee. In all these experiments, only the -methyl substituted acyl imidazole led to full conversions albeit with low ees, while substrates bearing a -t-butyl moiety led to higher ees but lower conversions. Surprisingly, replacing the methyl by an i-propyl group on the imidazole ring led to a racemic mixture of products, thus highlighting the impact of the steric hindrance induced by the substrate as well as the coordination sphere environment on the stereoselectivity of the reaction.

Synthetic DNA-helix 2.2.1 Supramolecular approach

The results obtained with natural DNA clearly highlight the importance of the binding between the metallic co-factor and the double helix. This prompted the community to next evaluate the influence of specific sequences on the reaction rate and ultimately on the enantioselectivity. A first set of experiments was run on the Diels-Alder cycloaddition between acylpyridine 11 (Figure 1) and cyclopentadiene using various L3/Cu(II)/DNA complexes. [43] The results showed an interesting correlation between the high affinity of the L3/Cu(II) metallic-co-factor for the GC-rich 12-mer sequence d(TCAGGGCCCTGA)2, ODN1, which is higher than that for natural st-DNA), and the increase of both the reaction rate (100-fold) and the selectivity.

A similar study was performed on the Friedel-Crafts alkylation using this time ,-unsaturated 2-acyl imidazole 8 as a model substrate. [38] Similarly to the Diels-Alder cycloaddition, ODN1 led to usually higher ees and conversions than st-DNA, while AT-rich oligonucleotides were found to induce higher ees.

Interestingly, in the case of the intramolecular Friedel-Crafts alkylation with 2-acyl imidazole 36, Sugiyama and co-workers showed that AT-rich duplexes gave rise to higher enantioselectivities. [39] To understand these results a theoretical investigation was undertaken by modelling 28 conformations of ODN2 d(CAAAAATTTTTG)2 with L4/Cu(II). The simulations revealed a higher binding energy of ODN2 toward the pro-(S) conformation of 2-acylimidazole 36. Moreover, a shorter distance between the two carbons involved in the newly formed CC bond was observed in the case of the pro-S complex more inline with the expected transitional state. [44] This sequence-selectivity was also evaluated in the case of the syn-hydration, where higher selectivities were obtained with sequences bearing an AT-rich central segment, however the structure of the ligand was also shown to be crucial. [42b] Finally, in the case of the oxa-Michael addition, AT-rich ODN2 gave rise to higher ees [conv. 82%; 43% ee] than the GC-rich sequence ODN1 [conv. 68%; 19% ee] (Figure 11), albeit still lower than st-DNA. [41] These results prompted our group to devote our first efforts in the field of biohybrid catalysis toward the control of the enantioselective outcome. While many studies evaluated various parameters including the influence of the metallic co-factor to selectively access one specific enantiomer, the inversion of selectivity were generally partial, often very ligand-specific and thus unpredictable. [31,39,45,46] Taking into account the best selectivities obtained so far in the Friedel-Crafts alkylation of ,-unsaturated 2-acyl imidazole 8, we synthesized the mirror image of ODN1, ODN4, using L-nucleotide phosphoramidites. As expected, the use of the left-handed L-d(TCAGGGCCCTGA)2 ODN4 sequence in the presence of the L3/Cu(II) complex afforded the opposite enantiomer with the exact the same enantioselectivity than the one obtained with ODN1 (Figure 13). [22] The method was also validated on the conjugate addition of dimethyl malonate and nitromethane, thus showcasing the generality of the method.

We later applied this concept to short double-stranded RNA sequences, [47] which are known to adopt a different helical shape (A-form helix) with a large and shallow minor groove and a deeper and tighter major groove. Although the enantioselectivities achieved were lower than the ones obtained with DNA, we found that the GC rich 16-mer ORN1, L-(CAGUCAGUACUGACUG)2, provided the best selectivities with up to 54% ee and an expected inversion of selectivity depending on the D or L nature of the sequence (Figure 14).

L3

Achieving sequence-specificity was another challenge we decided to tackle. Hence, instead of evaluating the sequence dependency of a given reaction, we envisioned to design sequence-specific catalysts. The well-known minor groove binder Hoechst-33258, recognized for its strong affinity for AT-rich regions of DNA, was selected and modified to incorporate a Cu(II) binding site (Figure 15). [45] Among all the ligands that were synthesized and evaluated in the Cu(II)-catalysed Friedel-Crafts alkylation of ,-unsaturated 2-acyl imidazole 8 with 5-methoxyindole 32, ligand L5 provided the best selectivities (up of 47% ee with ct-DNA). Although moderate, this result reinforced the indisputable correlation between affinity and selectivity as the highest selectivities were obtained with the ligand having the highest binding affinity with DNA.

Sequence specific targeting of DNA was also at the centre of the recent work published by Qiao and co-workers. [46] A four unit pyrrole-polyamide groove binder known for its high affinity toward DNA was selected and covalently modified with a 4,4′-dimethyl-2,2′-bipyridine (L6, Figure 16).

The Cu(II)-catalysed

Friedel-Crafts alkylation of ,-unsaturated 2-acyl imidazole 8 with 5-methoxyindole was again selected as a model reaction. A screening of both selfcomplementary AT and GC-rich oligonucleotides identified ODN9, d(TCGGGGCCCCGA)2, as the lead sequence (up to 48% ee). Circular dichroism spectra and singular value decomposition analyses suggested that different binding modes could co-exist depending on the nature of the sequence therefore affecting the enantioselectivity.

Covalent approach

The anchoring strategy used to link the transition metal complex to DNA is a critical parameter that influences the nature of the second coordination sphere provided by the bio-scaffold. The supramolecular anchoring approach of a metallic-cofactor to DNA allows a modular assembly of the biohybrid catalyst and permits a rapid optimization of the reaction conditions. In contrast, the covalent anchoring approach allows to specifically position the metallic co-factor independently of its binding affinity to DNA but requires multiple synthetic steps to assemble each biohybrid catalyst.

Taking advantage of the reactivity of bifunctional platinum complexes towards DNA, Gjonaj and Roelfes synthesized a bipyridine-platinum complex and covalently attached it to st-DNA. After addition of Cu(NO3)2, the new DNA-based catalyst L7 was evaluated in the benchmark Friedel-Crafts alkylation of ,-unsaturated 2-acyl imidazole 8 with 5-methoxyindole 32 (Figure 17). [48] Although the ees were slightly lower than the ones obtained with the typical supramolecular approach, this new biohybrid catalyst proved particularly robust as showcased by a series of 10 recycling experiments that led to no noticeable erosion of either the reactivity or the selectivity. Park and Sugiyama later developed various synthetic DNA-based hybrid catalysts containing an intrastrand bipyridine ligand. The latter were prepared by incorporating a 2,2′-bipyridine-5,5′-dicarboxylic acid moiety within a specific 13-mer (ODN11) or an auto-complementary single-strand 29-mer (ODN14) sequence (Figure 18). [49] The modified 13-mer oligonucleotide was then associated with a complementary strand containing either a G or a C nucleobase against the ligand and the resulting biohybrid catalysts were evaluated in the intramolecular Friedel-Crafts alkylation of acyl imidazole 36. As a general trend, the catalysts containing a cytosine facing the bipyridine led to higher selectivities (84% ee with ODN11/ODN13 and 86% ee with the hairpin ODN16 vs 17% ee with ODN11/ODN12). Figure 18. DNA-based asymmetric intramolecular Friedel-Crafts alkylation using an intrastrand bipyridine ligand.

These results prompted the authors to investigate further the role of the cytosine within the catalytic pocket. Hence, various counter-strands incorporating a triethylene glycol (ODN15a) or an alkyl linker (ODN15b and ODN15c) opposite to cytosine were prepared and evaluated in the Diels-Alder reaction between both,-unsaturated 2-acyl imidazole 8 and ,-unsaturated 2-acyl pyridine 11 and cyclopentadiene (23). The best results were obtained with the triethylene glycol linker which afforded ees ranging between 88 and 92% (Figure 19). [50] Figure 19. DNA metalloenzyme-based asymmetric Diels-Alder.

Solid-Supported DNA helix

The use of solid-supported DNA for asymmetric catalysis was first introduced by Park and Sugiyama based on a noncovalent immobilization of DNA on ammonium-functionalized silica beads. [51] The method proved highly effective with selectivities reaching up to 94% ee in the Diels-Alder cycloaddition between ,-unsaturated 2-acyl pyridine 11 and cyclopentadiene. Following these results, the authors also developed DNA-silica minerals made from cationic Mg(II) ions, N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride (TMAPS), tetraethoxysilane (TEOS) and a Cu/ligand complex. [52] Considering the crucial importance of heterogeneous catalysis for industrial applications, our group has also embarked on the development of an eco-friendly and recyclable biohybrid catalyst. Our approach, which relied on the use of commercially available cellulose-supported (CS) DNA in conjunction with a Cu(II)/L3 complex, led to high levels of selectivity in both the Friedel-Crafts alkylation and the Michael addition on ,-unsaturated 2-acyl imidazole 8 (Figure 20). [53] Moreover, we demonstrated that the CS-ct-DNA-Cu(II)/L3 biohybrid catalyst could be recycled up to 10 times without any loss of reactivity or selectivity and even adapted to a single-pass continuous-flow process.

G-quadruplex

Supramolecular approach

G-Quadruplex (G4DNA) are secondary structures composed of four-stranded G-rich DNA sequences. The core consists of four guanines bases associated through Hoogsteen hydrogen bonds forming planar square arrangements. These structures are stabilized by monovalent cations (preferably K+) that sit in the centre and interact with eight guanines. Depending on their unimolecular or bimolecular nature, G4DNA are distinguished by their topological variety which include mainly parallel, antiparallel or '3+1' hybrids (Figure 21). [54][55][56][57] Considering these different arrangements and their respective ability to bind to a large variety of ligands, G4DNA also appeared as privileged bioscaffolds for asymmetric catalysis. [58] In the case of G4DNA-based biohybrid catalytic systems, both the supramolecular and covalent anchoring strategies were evaluated on the benchmark Diels-Alder cycloaddition, the Friedel-Crafts alkylation and the Michael addition.

The first example of a G4-based asymmetric catalysis was reported by Moses and co-workers in 2010 on the standard Diels-Alder cycloaddition between azachalcone 11 and cyclopentadiene. [59] G4DNA forming sequences such as h-Tel d(AGGGTTAGGGTTAGGGTTAGGG) and c-Kit d(AGGGAGGGCGCTGGGAGGAGG) were selected and evaluated in the presence of Cu(II) and ligand L3. Despite an excellent endo/exo diastereoselectivity and high conversions (up to 85%), the ees were rather moderate (up to 34%). Nonetheless, these results demonstrated the ability of G4DNA to act as good chiral scaffolds. Interestingly, Li et al. later demonstrated that G4DNA/Cu(II) could catalyse the reactions in the absence of ligand in up to 74% ee. [60] Moreover, the importance of the conformation was emphasized by the observation of an inversion of selectivity between parallel and anti-parallel G-quadruplexes. This reversal of selectivity was also observed with h-tel-Cu(II) in the presence of different alkali metal ions (Figure 22). [61] Hence, the enantioselectivity could be switched from () 58% to (+) 67% by simply changing the nature of the alkali metal ion form Na + to K + . This inversion was attributed to a structural transformation from an antiparallel to a hybrid-type G-quadruplex structure. [62] Figure 22. h-Tel G4DNA-catalysed asymmetric Diels-Alder.

The same group also prepared a terpyridine L8/Cu(II) complex having a high affinity to h-tel (Scheme 23). The resulting biohybrid catalyst induced a 73-fold rate acceleration compared to L8 and ees up to 99%. Interestingly, a comparison between substrates 8 and 11 confirmed the prevalence of the acylimidazole scaffold over the acylpyridyl. [63] The importance of the conformation of the G4DNA was also confirmed in both the Friedel-Crafts alkylation [64] and the Michael addition. [65] Hence, the selectivity outcome of a given reaction can easily be set by fine-tuning the conditions (choice of the alkali metal ions, the ligands, the co-solvents…). On the down side, the ees obtained with G4DNA catalysts never exceeded 75%, far below the selectivities generally obtained in the field.

In order to improve the performances of the G4DNA, a covalent anchoring strategy was also evaluated, thus circumventing the issues related with the binding affinity of the ligand.

Covalent approach

The covalent positioning of a bipyridine ligand into the G4DNA was first reported by Jäschke and co-workers. [66] New G4DNA biohybrid catalysts were obtained by covalently attaching a L3/Cu(II) complex via different linkers of varying length. The evaluation of these new G4DNA catalysts on the asymmetric Michael addition of dimethyl malonate on ,-unsaturated 2-acyl imidazole 8 revealed the importance of the positioning of the ligand, the length of the linker and the topology of the G4DNA. Interestingly, a stunning inversion of the selectivity was observed when using ODN17, which carries the modification at position 12 compared to ODN18 which bears the exact same ligand at position 10 (Figure 24). Also worth pointing out is the catalyst recyclability, which was later demonstrated by reusing the catalyst over 10 times without any erosion of the selectivity. [67] Figure 24. G4DNA-based asymmetric Michael addition through a covalent modification.

Peptide and Protein-based asymmetric catalysis 3.1 Artificial metalloenzymes

The design of artificial metalloenzymes incorporating a synthetic transition-metal catalyst into a protein-binding site also represents an emerging approach to engineer biohybrid catalysts. Similar to DNA-based biohybrid catalysts, covalent, dative, or non-covalent anchoring strategies can be applied to polypeptidic scaffolds. In this respect, Roelfes et al. selected the bovine Pancreatic Polypeptide bPP, [65,66] a 36 residue polypeptide hormone as a new biohybrid scaffold. [67] The natural peptidic hormone was truncated to 31 residues and grafted at the Tyr7 position with an active site by introducing a nonproteinogenic amino acid capable of binding a Cu(II) ion. The bPP variants were evaluated in the Cu(II)-catalysed Diels-Alder and Michael addition with ,-unsaturated substrates 8 and 11 (Figure 25). The results showed that peptides containing L-3-pyridylalanine led to good enantioselectivities on both the Diels-Alder (up to 83% ee) and the Michael addition (up to 86% ee). While this latter biohybrid catalyst was obtained by solid phase synthesis, the same group reported a few years later the in vivo incorporation of a metal-binding non-proteinogenic amino acid into the transcription factor Lactoccocal multidrug resistance Regulator (LmrR). [68] The in-vivo incorporation enabled the generation of LmrR mutant variants that permit a rapid optimization and a precise positioning of the bipyridine ligand (BpyAla) inside the LmrR. Application of these artificial metalloenzymes in the catalytic enantioselective Friedel-Crafts alkylation of ,-unsaturated acyl imidazole 8 with various indoles led to ees up to 83% and paved the way for directed evolution of artificial metalloenzymes (Figure 26).

The concept was later extended to three other proteins (QacR, CgmR and RamR) belonging to the TetR family of Multidrug Resistance Regulators. [69] Among them, one QacR mutant induced up to 94% ee in the Cu(II)-catalysed enantioselective Friedel-Crafts alkylation with a reverse of selectivity compared to other mutants including LmrR-based artificial metalloenzymes.

The supramolecular assembly of an LmrR-based artificial metalloenzyme was also envisioned by combining a LmrR variant with a Cu(II) complex of phenanthroline ligand L4 (Figure 27). [70] The rationale behind the study was based on the ability of planar aromatic ligands to bind to the LmrR hydrophobic pocket. The supramolecular approach proved to be superior to the covalent approach with ees up to 94% in the Cu(II)-catalysed enantioselective Friedel-Crafts alkylation. These results prompted the authors to design a metal regulated LmrR-based artificial metalloenzyme combining both supramolecular and covalent anchoring strategies. The catalytic site was composed of the Cu(II)/L4 ligand described above, while the regulatory site was introduced covalently by standard site directed mutagenesis technique [71] resulting in the formation of a dimeric protein containing two metal binding moieties. (Figure 28). [72] In the absence of Fe 2+ ions, the 2-2'-bipyridine ligand covalently linked to LmrR binds to the catalytically active Cu(II)/L4 complex and deactivates the artificial metalloenzyme. The addition of Fe 2+ ions traps the 2-2'-bipyridine ligands and frees the active Cu 2+ complex, which subsequently reactivates the catalyst. Hence, when applied to the Friedel-Crafts alkylation between ,-unsaturated acyl imidazole 8 and 2-methyl indole, selectivities up to 88% ee were obtained thus demonstrating the synthetic potential of programmable artificial biohybrid catalysts.

Peptide

Recently, Hermann and co-workers identified cyclic peptides as a novel metallopeptide design containing only natural amino acids. The peptide scaffold was assembled by solid-phase peptide synthesis and cyclized by an intramolecular disulfide linkage between cysteines at the N-and C-termini. To determine the importance of a particular residue, a small library of 15 cyclic nonapeptides was synthesized and optimized using the alanine scanning technique. [73] The ability of the histidine to complex Cu 2+ ions allowed to avoid the use of additional binding ligands.

These peptides were first evaluated on the Cu(II)-catalysed Diels-Alder cycloaddition, which led to the selection of shorter peptides that were ultimately used in the asymmetric Cu(II)-catalysed Friedel-Crafts alkylation. Interestingly, the truncated hexapeptide CGIARC afforded the highest selectivities with ees up to 99% in the Diels-Alder cycloaddition and 86% in the Friedel-Crafts alkylation (Figure 29).

Conclusion

While the use of acyl-imidazole in classical organic media are described by Campagne and co-workers in the back-to back accompanying mini-review, biohybrid catalysis has emerged recently as a particularly powerful tool for the enntioselective synthesis of chiral building blocks and ,-unsaturated acyl imidazoles have undoubtedly been a key to this success. Indeed, these substrates have not only been found to be highly versatile, they were also shown to exhibit interesting reactivity patterns and a high compatibility with a large variety of bioscaffolds. While various highly enantioselective CC and CO bond forming reactions have been unveiled, the use of ,-unsaturated acyl imidazoles in the context of asymmetric biohybrid catalysis is clearly still in its infancy and a plethora of new reactions are expected in the years to come. REVIEW ,-Unsaturated acylimidazoles have become particularly useful building blocks in asymmetric catalysis. Surprisingly however, their use in asymmetric biohybrid catalysis as bidendate substrates able to interact with artificial metalloenzymes has only recently emerged. This review highlights the current efforts made in the field. appear as leading synthons for the asymmetric cons bonds. This Minireview highlights the current and increasing interest of these key building blocks in the context of asymmetric biohybrid catalysis with the aim to stimulate further research into their still unexploited potential.

Figure 1 .

 1 Figure 1. Enone commonly used in asymmetric catalysis (R1 = Alkyl, Aryl, R2 = Alkyl, R3 = Alkyl).

Figure 3 .

 3 Figure 3. Post-transformation of the imidazole moiety.

Figure 4 .

 4 Figure 4. DNA-based asymmetric Diels-Alder.

Figure 5 .

 5 Figure 5. DNA-based asymmetric Diels-Alder.

Figure 6 .

 6 Figure 6. DNA-based asymmetric Michael.

Figure 7 .

 7 Figure 7. Schematic representation of the complexed acylimidazole substrate in DNA.

Figure 8 .

 8 Figure 8. DNA-based conjugate addition of malonitrile derivatives.

Figure 9 .

 9 Figure 9. DNA-based asymmetric Friedel-Crafts alkylation.

Figure 10 .

 10 Figure 10. DNA-based intramolecular Friedel-Crafts alkylation.

Figure 11 .

 11 Figure 11. DNA-based asymmetric oxo-Michael addition.

Figure 12 .

 12 Figure 12. DNA-based asymmetric syn-hydration.

Figure 14 .

 14 Figure 14. RNA-based asymmetric Friedel-Crafts alkylation. [ORN1 L-(CAGUCAGUACUGACUG)2].

2 Figure 13 .

 213 Figure 13. Mirror image DNA-based asymmetric catalysis

Figure 15 .

 15 Figure 15. DNA-based asymmetric Friedel-Crafts alkylation with minor groove-binding ligands. [ODN6: 5'-d(CGAATTCGTT TTCGAATTCG)-3', ODN7: 5'-d(CGTATACGTTTTCGTATACG)-3'].

Figure 16 .

 16 Figure 16. DNA-based asymmetric Friedel-Crafts alkylation using pyrrole-polyamide L6.

Figure 17 .

 17 Figure 17. DNA-based asymmetric Friedel-Crafts alkylation using a cisplatin ligand.

Figure 20 .

 20 Figure 20. CS-ct-DNA-based asymmetric Friedel-Crafts alkylation and Michael addition.

Figure 21 .

 21 Figure 21. G4DNA conformations.

Figure 25 .

 25 Figure 25. Bovine Pancreatic Polypeptide bPP as scaffold for asymmetric transformations.

Figure 26 .

 26 Figure 26. QacR-based asymmetric Friedel-Crafts alkylation.

Figure 27 .

 27 Figure 27. Supramolecular approach for the LmrR-based asymmetric Friedel-Crafts alkylation.

Figure 29 .

 29 Figure 29. Cyclic peptide-based asymmetric Diels-Alder and Friedel-Crafts alkylation.

Acknowledgements

The Agence Nationale de la Recherche is gratefully acknowledged for funding project D-CYSIV (ANR-2015-CE29-0021-01).