Investigation of the intracellular delivery of fluoresceinated peptides by a host-[2]rotaxane

The development of methods to transport peptides into cells via a passive mechanism would greatly aid in the development of therapeutic agents. We recently demonstrated that an impermeable fluoresceinated pentapeptide enters the cytoplasm and nucleus of COS 7 cells in the presence of a host-[2]rotaxane by a mechanism that does not depend on an active cell-mediated process. In this report, we further investigate the ability of the host-[2]rotaxane to deliver peptides possessing a wide range of polarities (negatively charged, positively charged, polar, and apolar side chains) into live cells. Only in the presence of the host-[2]rotaxane were the Fl-peptides taken up by COS 7 and ES2 cells. Flow cytometry experiments demonstrated that the level of delivery is largely temperature and adenosine 5'-triphosphate (ATP) independent, and the membranes remain intact. Although the level of transport does depend upon the nature of the side chains, it does not correlate with calculated LogD values, indicating that an additional interaction with the host-[2]rotaxane is modifying the permeability properties of the peptide. The amount of Fl-peptides transported from an aqueous phase into a chloroform phase in the presence of the host-[2]rotaxane correlates with the intensity of cellular fluorescence. Extraction and U-tube studies show that the Fl-peptide can be released from its complex with the host-[2]rotaxane into an aqueous phase, and the host-[2]rotaxane can transport a greater than a stoichiometric amount of an Fl-peptide through a CHCl3 layer. These studies demonstrate the utility of the host-[2]rotaxane in delivering peptides of all polarities across a cell membrane.     

Dynamic covalent approach to [2]- and [3]roxtanes by utilizing a reversible thiol-disulfide interchange reaction

A dynamic covalent approach to disulfide-containing [2]- and [3]rotaxanes is described. Symmetrical dumbbell-shaped compounds with two secondary ammonium centers and a central located disulfide bond were synthesized as components of rotaxanes. The rotaxanes were synthesized from the dumbbell-shaped compounds and dibenzo-[24]crown-8 (DB24C8) with catalysis by benzenethiol. The yields of isolated rotaxanes reached about 90 % under optimized conditions. A kinetic study on the reaction forming [2]rotaxane 2 a and [3]rotaxane 3 a suggested a plausible reaction mechanism comprising several steps, including 1) initiation, 2) [2]rotaxane formation, and 3) [3]rotaxane formation. The whole reaction was found to be reversible in the presence of thiols, and thermodynamic control over product distribution was thus possible by varying the temperature, solvent, initial ratio of substrates, and concentration. The steric bulk of the end-capping groups had almost no influence on rotaxane yields, but the structure of the thiol was crucial for reaction rates. Amines and phosphines were also effective as catalysts. The structural characterization of the rotaxanes included an X-ray crystallographic study on [3]rotaxane 3 a.     

Efficient Intramolecular Energy Transfer between Two Fluorophores in a Bis‐Branched [3]Rotaxane

A bis‐branched [3]rotaxane, with two [2]rotaxane arms separated by an oligo(para‐phenylenevinylene) (OPV) fluorophore, was designed and investigated. Each [2]rotaxane arm employed a difluoroboradiaza‐s‐indacene (BODIPY) dye‐functionalized dibenzo[24]crown‐8 macrocycle interlocked onto a dibenzylammonium in the rod part. The chemical structure of the [3]rotaxane was confirmed and characterized by ¹H and ¹³C NMR spectroscopy and high‐resolution ESI mass spectrometry. The photophysical properties of [3]rotaxane and its reference systems were investigated through UV/Vis absorption, fluorescence, and time‐resolved fluorescence spectroscopy. An efficient energy‐transfer process in [3]rotaxane occurred from the OPV donor to the BODIPY acceptor because of the large overlap between the absorption spectrum of the BODIPY moiety and the emission spectrum of the OPV fluorophore; this shows the important potential of this system for designing functional molecular systems.     

Homo- and hetero-[3]rotaxanes with two pi-systems clasped in a single macrocycle

Here we present the first synthesis of a [3]rotaxane with two dumbbell components threaded through a single gamma-cyclodextrin macrocycle. This synthesis is carried out in two steps: first one dumbbell is synthesized threaded through the macrocycle to give a [2]rotaxane, then a second dumbbell is synthesized through the remaining cavity of the [2]rotaxane. We have synthesized a hetero- [3]rotaxane with one stilbene and one cyanine dye threaded through gamma-cyclodextrin, which exhibits quantitative energy transfer between the two encapsulated dyes. The stilbene [2]rotaxane intermediate in this synthesis has a remarkably high affinity for suitably shaped hydrophobic guests in aqueous solution, facilitating the synthesis of [3]rotaxanes and suggesting possible applications in sensors.     

Assembly of an electronically switchable rotaxane on the surface of a titanium dioxide nanoparticle

This paper describes the self-assembly of a heterosupramolecular system consisting of a tripodal [2]rotaxane adsorbed at the surface of a titanium dioxide nanoparticle. The tripodal [2]rotaxane consists of a dumbbell-shaped molecule, incorporating two electron-poor viologens, threading an electron-rich crown ether. The [2]rotaxane also incorporates a bulky tripodal linker group at one end and a bulky stopper group at the other end. The [2]rotaxane is adsorbed, via the tripodal linker group, at the surface of a titanium dioxide nanoparticle. The structure and function of the resulting hetero[2]rotaxane have been studied in detail by (1)H NMR spectroscopy and cyclic voltammetry. A key finding is that it is possible to electronically address and switch the above hetero[2]rotaxane.     

High-yield diastereoselective synthesis of planar chiral [2]- and [3]rotaxanes constructed from per-ethylated pillar[5]arene and pyridinium derivatives

Planar chiral [2]- and [3]rotaxanes constructed from pillar[5]arenes as wheels and pyridinium derivatives as axles were obtained in high yield using click reactions. The process of rotaxane formation was diastereoselective; the obtained [2]rotaxane was a racemic mixture consisting of (pS, pS, pS, pS, pS) and (pR, pR, pR, pR, pR) forms of the per-ethylated pillar[5]arene (C2) wheel, and other possible types of the [2]rotaxane did not form. Isolation of the enantiopure [2]rotaxanes with one axle through (pS, pS, pS, pS, pS)-C2 or (pR, pR, pR, pR, pR)-C2 wheels was accomplished. Furthermore, pillar[5]arene-based [3]rotaxane was successfully synthesized by attachment of two pseudo [2]rotaxanes onto a bifunctional linker. [3]Rotaxane formed in a 1:2:1 mixture with one axle threaded through two (pS, pS, pS, pS, pS)-C2, one (pS, pS, pS, pS, pS)-C2 and one (pR, pR, pR, pR, pR)-C2 (meso form), or two (pR, pR, pR, pR, pR)-C2 wheels. The [3]rotaxane enantiomers and the meso form were successfully isolated using appropriate chiral HPLC column chromatography. The procedure developed in this study is the starting point for the creation of pillar[5]arene-based interlocked molecules.     

Host-[2]rotaxane: advantage of converging functional groups for guest recognition

A host-[2]rotaxane was constructed by converting a diaminophenylcalix[4]arene into a [2]rotaxane using the DCC-rotaxane method (Zehnder, D.; Smithrud, D. B. Org. Lett. 2001, 16, 2485-2486). N-Ac-Arg groups were attached to the dibenzo-24-crown-8 ring of the rotaxane to provide a convergent functional group. To demonstrate the advantage provided by the rotaxane architecture for recognition of guests that contain a variety of functional groups, association constants (K(A)) for N-Ac-Trp, indole, N-Ac-Gly, fluorescein, 1-(dimethylamino)-5-naphthalenesulfonate, and pyrene bound to the [2]rotaxane were determined by performing (1)H NMR and fluorescence spectroscopic experiments. The host-[2]rotaxane had the highest affinity for fluorescein with a K(A) = 4.6 x 10(6) M(-)(1) in a 98/2 buffer (1 mM phosphate, pH 7)/DMSO solution. A comparison of K(A) values demonstrates that both the aromatic pocket and ring of the host-[2]rotaxane contribute binding free energy for complexation. Association constants were also derived for the same guests bound to the diaminophenylcalix[4]arene and to a diphenylcalix[4]arene that contained arginine residues displayed in a nonconvergent fashion. The host-[2]rotaxane provides higher affinity and specificity for most guests than the host with divergent N-Ac-Arg groups of the one that only has an aromatic pocket. For example, the K(A) for the complex of the host-[2]rotaxane and fluorescein in the DMSO/water mixture is more than 2 orders of magnitude greater than association constants derived for the other hosts.     

Utilization of a Crown Ether/Amine‐Type Rotaxane as a Probe for the Versatile Detection of Anions and Acids by Thin‐Layer Chromatography

A crown ether/amine‐type [2]rotaxane was synthesized and utilized as a probe for the detection of acids and anions. The addition of acids to the amine‐type [2]rotaxane solution generated corresponding crown ether/ammonium‐type [2]rotaxanes, which were purified by silica gel column chromatography as ammonium salts. The isolated yields of the [2]rotaxanes, possessing a variety of anions, depended on the acidity and polarity of the counter anions. The behaviours of the ammonium‐type [2]rotaxanes on thin‐layer chromatography (TLC) silica gel reflected the properties of the counter anions. The treatment of the amine‐type [2]rotaxane with acids afforded the corresponding ammonium‐type [2]rotaxanes bearing several different anions. The ammonium‐type [2]rotaxanes behaved similarly to the purified [2]rotaxanes on the TLC silica gel. Furthermore, we succeeded in the analysis of anions using mixtures of the amine‐type [2]rotaxane and salts in an appropriate solvent. We demonstrated the detection of anions by the combination of TLC and the utilization of the [2]rotaxane probe.     

Cyclodextrin-Based [c2]Daisy Chain Rotaxane Insulating Two Diarylacetylene Cores

A [c2]daisy chain rotaxane with two diarylacetylene cores was efficiently synthesized in 53 % yield by capping a C₂-symmetric pseudo[2]rotaxane composed of two diarylacetylene-substituted permethylated α-cyclodextrins (PM α-CDs) with aniline stoppers. The maximum absorption wavelength of the [c2]daisy chain rotaxane remained almost unchanged in various solvents, unlike that of the stoppered monomer, indicating that the two independent diarylacetylene cores were insulated from the external environment by the PM α-CDs. Furthermore, the [c2]daisy chain rotaxane exhibited fluorescence emission derived from both diarylacetylene monomers and the excimer, which implies that the [c2]daisy chain structure can undergo contraction and extension. This is the first demonstration of a system in which excimer formation between two π-conjugated molecules within an isolated space can be controlled by the unique motion of a [c2]daisy chain rotaxane.     

Self-assembly and dynamics of [2]- and [3]rotaxanes with a dinuclear macrocycle containing reversible Os-N coordinate bonds

With a dinuclear macrocycle 2 that contains weak reversible OsVI-N coordinate bonds, self-assembly and equilibrium dynamics of [2]- and [3]rotaxanes have been investigated. When the macrocycle 2 was mixed together with threads 4a-e, which all contain an adipamide station but different sizes of end groups, [2]pseudorotaxane- and rotaxane-like complexes were immediately formed with large association constants of >7 x 103M(-1) in CDCl3 at 298 K. Exchange dynamics, explored by 2D-EXSY experiments, suggest that assembly and disassembly of complexes occur through two distinct pathways, slipping or clipping, and this depends on the size of the end groups. The slipping pathway is predominant with smaller end groups that give pseudorotaxane-like complexes, while the clipping pathway is observed with larger end groups that yield rotaxane-like complexes. Under the same conditions, exchange barriers (deltaG++) were 14.3 kcalmol(-1) for 4a and 16.7 kcalmol(-1) for 4d, and indicate that the slipping process is at least one order of magnitude faster than the clipping process. Using threads 13a and 13b that contain two adipamide groups, more complicated systems have been investigated in which [2]rotaxane, [3]rotaxane, and free components are in equilibrium. Concentration- and temperature-dependent 1H NMR spectroscopic studies allowed the identification of all possible elements and the determination of their relative distributions in solution. For example, the relative distribution of the free components, [2]rotaxane, and [3]rotaxane are 30, 45, and 25 %, respectively, in a mixture of 2 (2mM) and 13a (2mM) in CDCl3 at 10 degrees C. However, [3]rotaxane exists nearly quantitatively in a mixture of 2 (4 mM) and 13 a (2 mM) in CDCl3 at a low temperature - 10 degrees C.     

Synthesis of [2]- and [3]rotaxanes through Sonogashira coupling

The synthesis of new [2]- and [3]rotaxanes through Sonogashira coupling has been accomplished in the aim to built oligo(phenyleneethynylene) (OPE) encircled by crown ethers. Optimization of the Sonogashira coupling for the formation of the [2]rotaxane (capping reaction) is presented and the best method has been applied for the synthesis of the longer [3]rotaxane.     

Synthesis and characterization of a metallocycle-based molecular shuttle

Kinetically stable metallocycle-based molecular shuttles of [2]rotaxanes 4a and 4b, along with [3]rotaxanes 5a and 5b, have been prepared using the rhenium(I)-bridged metallocycle 2 and the dumbbell components containing two stations, 3a and 3b. The rotaxanes were self-assembled by hydrogen bonding interactions upon heating a Cl(2)CHCHCl(2) solution containing their components at 70 degrees C. Each rotaxane was isolated in pure form by silica gel chromatography under ordinary laboratory conditions and fully characterized by elemental analysis and various spectroscopic methods. The (1)H NMR signals for the amide NH and the methylene -(CH(2))(4)- of the station were considerably changed when occupied by the metallocycle. In [2]rotaxane 4b, which has a larger naphthyl spacer, the occupied and unoccupied stations gave widely separated signals in the (1)H NMR spectroscopy at room temperature, but averaged signals of two stations were observed in [2]rotaxane 4a, which has a smaller phenyl spacer. This is attributed to the shuttling of the metallocycle between two stations. The coalescence temperature experiment gave a shuttling rate of approximately 670 s(-)(1) at 19 degrees C in CDCl(3), corresponding to an activation free energy (DeltaG()) of 13.3 kcal/mol. With respect to the relative position of the chloride in the rhenium(I) center, two diastereomers are possible in the [2]rotaxane and three diastereomers are possible in the [3]rotaxane. In fact, the rotaxanes exist as diastereomeric mixtures in nearly equal amounts of all possible diastereomers on the basis of the amide NH signals of the station in the (1)H NMR spectroscopy.     

Use of Cleavable Coordinating Rings as Protective Groups in the Synthesis of a Rotaxane with an Axis that Incorporates More Chelating Groups Than Threaded Macrocycles

A new methodology allowing preparation of a linear “unsaturated” [3]rotaxane consisting of an axis incorporating more coordination sites than threaded rings was developed. It was based on the preliminary synthesis of a “saturated” [5]rotaxane consisting of a four‐chelating site axis threaded through four macrocyclic components, two of them being cleavable rings incorporating a lactone function and the two others being “secure” non‐cleavable rings. The stoppering reaction was based on click chemistry. Subsequently, cleavage and removal of the two lactone‐containing macrocycles from the [5]rotaxane in basic medium afforded the desired “unsaturated” [3]rotaxane in quantitative yield.     

Synthesis and characterization of a persistent paramagnetic rotaxane based on alpha-cyclodextrin

The synthesis and spectroscopic properties of a novel paramagnetic [2]rotaxane is described. This rotaxane is made from molecules having an alkyl chain flanked by 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) groups. Complexation of sebacoyl chloride by alpha-cyclodextrin followed by reaction with a bulky aminonitroxide resulted in the trapping of the cyclodextrin, threaded by the alkyl chain, thus generating the rotaxane structure. The structure of the paramagnetic [2]rotaxane was fully characterized by ESI-MS, 1D and 2D NMR and ESR spectroscopy.     

Organogel formation by a cholesterol-stoppered bistable [2]rotaxane and its dumbbell precursor

The switching properties, gelation behavior, and self-organization of a cholesterol-stoppered bistable [2]rotaxane containing a cyclobis(paraquat-p-phenylene) ring and tetrathiafulvalene/1,5-dioxynaphthalene recognition units situated in the rod portion of the dumbbell component have been investigated by electrochemical, spectroscopic, and microscopic means. The cyclobis(paraquat-p-phenylene) ring in the [2]rotaxane can be switched between the tetrathiafulvalene and 1,5-dioxynaphthalene recognition units by addressing the redox properties of the tetrathiafulvalene unit. The organogels can be prepared by dissolving the [2]rotaxane and its dumbbell precursor in a CH2Cl2/MeOH (3:2) mixed solvent and liquified by adding the oxidant Fe(ClO4)3. Direct evidence for the self-organization was obtained from AFM investigations which have shown that both of the [2]rotaxane and its dumbbell precursor form linear superstructures which we propose are helical in nature.     

Templated conversion of a crown ether-containing macrobicycle into [2]rotaxanes

A crown ether-containing macrobicycle was used as the wheel component in a templated synthesis of a [2]rotaxane with an acetal-containing axle. The molecular structures of the macrobicycle and the [2]rotaxane were characterized by NMR spectroscopy and X-ray crystallography. The chloride-binding ability of the macrobicycle, either free in solution or when it is part of a [2]rotaxane, is quite weak as determined by NMR titration experiments. A second analogous [2]rotaxane, with a longer axle, was synthesized, and its solvent-dependent co-conformation was characterized by 2D NMR spectroscopy. The position of the wheel along the axle can be controlled by the solvent polarity, however, attempts to use metal cations such as Na(+), K(+), Ba(2+), and Ag(+) to switch the wheel position in polar solvents were unsuccessful.     

Reversing a rotaxane recognition motif: threading oligoethylene glycol derivatives through a dicationic cyclophane

An already well-established recognition motif-namely one in which the NH2+ centers in the rod sections of the dumbbell components of rotaxanes are encircled by macrocyclic polyether components-has been turned simultaneously outside-in and inside-out, a fact that has been proved beyond any doubt by the stoppering of both ends of a [2]pseudorotaxane to give a stable [2]rotaxane. The [2]pseudorotaxane is formed in nitromethane when a benzylic dibromide, obtained after reacting an excess of 1,4-bis(bromomethyl)benzene with hexaethylene glycol, is added to an equimolar amount of a dicationic cyclophane in which two -CH2OCH2- chains link a pair of dibenzylammonium ions through the para positions on their phenyl rings. When the [2]pseudorotaxane is reacted in nitromethane with triphenylphosphine, a [2]rotaxane and the corresponding free dumbbell compound are isolated in 58 and 31% yields, respectively. The structure of the [2]rotaxane is established by using mass spectrometry (FABMS and ESMS) and NMR (1H and 13C) spectroscopy in nitromethane-d3. The [2]rotaxane exhibits quite dramatic changes in the 1H chemical shifts of the signals for its CH2N+ and CH2O protons compared with those in the free dumbbell compound. The 1H NMR spectrum of the [2]pseudorotaxane shows many similar features. Titration experiments with three of the six different CH2O probes give an average Ka value of 2900 +/- 750 M-1 in nitromethane-d3. The new recognition motif for the template-directed synthesis of rotaxanes can now be exploited at both the molecular and macromolecular levels of structure with numerous potential applications in sight.     

Quantitative conformational study of redox-active [2]rotaxanes, part 1: Methodology and application to a model [2]rotaxane

This paper reports a novel methodology for the conformational analysis of [2]rotaxanes. It combines NMR spectroscopic (COSY, NOESY and the recently reported paramagnetic line-broadening and suppression technique) and electrochemical techniques to enable a quantitative analysis of the co-conformations of interlocked molecules and the conformations of their components. This methodology was used to study a model [2]rotaxane in solution. This [2]rotaxane consists of an axle that incorporates an electron-poor, doubly positively charged viologen that threads an electron-rich crown ether. It has been shown that the axle of the [2]rotaxane in its dicationic state adopts a folded conformation in solution and the crown ether is localised at the viologen moiety. Following a one-electron reduction of viologen, the paramagnetic radical cation of the [2]rotaxane retains its folded conformation in solution. The data also demonstrate that in the radical cation the crown ether remains localised at the viologen, despite its reduced affinity for the singly reduced viologen. The combined quantitative NMR spectroscopic and electrochemical characterisation of the electromechanical function of the model [2]rotaxane in solution provides an important reference point for the study of switching in structurally related bistable [2]rotaxanes, which is the subject of the second part of this work.     

Translational isomerism in a [3]catenane and a [3]rotaxane

[structure: see text] Post-assembly covalent modification using Wittig chemistry of [2]rotaxane ylides, wherein NH(2)(+) centers in the dumbbell-shaped components are recognized by dibenzo[24]crown-8 (DB24C8) rings, has afforded a [3]catenane and a [3]rotaxane with a precise and synthetically prescribed shortage of DB24C8 rings. The nondegenerate pairs of translational isomers present in both of these interlocked molecular compounds provide the fundamental platform on which to construct sensory devices and nanochemomechanical systems.     

Palladium‐Templated Subcomponent Self‐Assembly of Macrocycles,Catenanes, and Rotaxanes

The reaction of 2,6‐diformylpyridine with diverse amines and Pd^II ions gave rise to a variety of metallosupramolecular species, in which the Pd^II ion is observed to template a tridentate bis(imino)pyridine ligand. These species included a mononuclear complex as well as [2+2] and [3+3] macrocycles. The addition of pyridine‐containing macrocyclic capping ligands allows for topological complexity to arise, thereby enabling the straightforward preparation of structures that include a [2]catenane, a [2]rotaxane, and a doubly threaded [3]rotaxane.