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Glycosyl squaramides were synthesised and evaluated as low molecular weight gelators. Amphiphilic glycosyl squaramides 5 and 6, with a C-16 aliphatic chain, formed thermoreversible gels in polar organic solvents and 1 : 1 ethanol/water mixtures with high efficiency. Rheological analysis showed these gels achieve their structural stability 120 h after gelation and were robust, making them particularly suitable for biomedical applications. The interactions between solvent and gelator strongly influence SAFiN (Self-Assembled Fibrillar Network) formation, critical gelation concentration (CGC) and subsequent gel structure, as evidenced by SEM imaging of xerogels. Spectroscopic studies indicate that H-bonding is involved in the self-assembly of the glycosyl squaramides in organic solvents, while hydrophobic interactions are the major driving force for gel formation in the presence of water. The compounds described herein are the first reported examples of carbohydrate-squaramide conjugates capable of forming supramolecular gels.

We report the synthesis, photophysical characterization, and biological evaluation of four DNA-binding ruthenium(II) polypyridyl 4-nitro- and 4-amino-1,8-naphthalimide conjugates. A meta arrangement around the ring connecting the 1,8-naphthalimide to a bipyridine ligand creates a cleft, the result of which renders the shape of the complex complementary to that of DNA. We have demonstrated that each complex exhibits water solubility and a distinctive set of photophysical properties that has allowed the nature of their interaction with DNA to be probed by various ground- and excited-state titrations. Furthermore, by varying the ancillary ligands, we also demonstrate their ability to act as DNA photocleavers, where all compounds have been found to cleave supercoiled DNA with high efficiency. Detailed cellular uptake experiments revealed that the conjugates accumulate in the cytoplasm and nucleus of HeLa cells, showing characteristic red metal-to-ligand charge-transfer emission, and also exhibit photoactivated cytotoxicity within the cells upon irradiation at 450 nm. A comparison between the meta and para arrangements of the 1,8-naphthalimide moiety relative to the Ru(II) center suggests increased DNA binding in the case of the meta arrangement; however, bipyridine–4-amino-1,8-naphthalimide conjugates appear to show superior phototoxicity in comparison to their 4-nitro derivatives.

With this research we set out to develop a number of coumarin-based ‘AND’ logic fluorescence probes that were capable of detecting a chosen analyte in the presence of HCys. Probe JEG-CAB was constructed by attaching the ONOO� reactive unit, benzyl boronate ester, to a HCys/Cys reactive fluorescent probe, CAH. Similarly, the core unit CAH was functionalised with the nitroreductase (NTR) reactive p-nitrobenzyl unit to produce probe JEG-CAN. Both, JEG-CAB and JEG-CAN exhibited a significant fluorescence increase when exposed to either HCys and ONOO� (JEG-CAB) or HCys and NTR (JEG-CAN) thus demonstrating their effectiveness to function as AND logic gates for HCys and a chosen analyte.

The use of NIR-fluorescence imaging to demarcate tumour boundaries for real-time guidance of their surgical resection has a huge untapped potential. However, fluorescence imaging using molecular fluorophores, even with a targeting biomolecule attached, has a major shortcoming of signal interference from non-specific background fluorescence outside the region of interest. This poor selectivity necessitates prolonged time delays to allow clearance of background fluorophore and retention within the tumour prior to image acquisition. In this report, an innovative approach to overcome this issue is described in which cancer targeted off to on bio-responsive NIR-fluorophores are utilised to switch-on first within the tumour. Bio-responsive cRGD, iRGD and PEG conjugates have been synthesised using activated ester/amine or maleimide/thiol couplings to link targeting and fluorophore components. Their off to on emission responses were measured and compared with an always-on non-responsive control with each bio-responsive derivative showing large fluorescence enhancement values. Live cell imaging experiments using metastatic breast cancer cells confirmed in vitro bio-responsive capabilities. An in vivo assessment of MDA-MB 231 tumour imaging performance for bio-responsive and always-on fluorophores was conducted with monitoring of fluorescence distributions over 96 h. As anticipated, the always-on fluorophore gave an immediate, non-specific and very strong emission throughout whereas the bio-responsive derivatives initially displayed very low fluorescence. All three bio-responsive derivatives switched on within tumours at time points consistent with their conjugated targeting groups. cRGD and iRGD conjugates both had effective tumour turn-on in the first hour, though the cRGD derivative had superior specificity for tumour over the iRGD conjugate. The pegylated derivative had similar switch-on characteristics but over a much longer period, taking 9 h before a significant emission was observable from the tumour. Evidence for in vivo active tumour targeting was obtained for the best performing cRGD bio-responsive NIR-AZA derivative from competitive binding studies. Overall, this cRGD-conjugate has the potential to overcome the inherent drawback of targeted always-on fluorophores requiring prolonged clearance times and shows excellent potential for clinical translation for intraoperative use in fluorescence guided tumour resections.

Water soluble squaramide macrocycles (MSQs) display high sulfate binding affinities in aqueous DMSO mixtures. The introduction of pyridine spacers into the macrocycles resulted in increased sulfate binding affinity in comparison to compounds with benzene spacers. [3]MSQ 6 was found to be a selective ligand for SO42− in highly competitive conditions and over a wide pH range (3.2–14).

The selective extraction of the hydrophilic sulfate ion from water is highly challenging because the high free energy of hydration of this ion makes it more difficult to extract than less hydrophilic ions such as chloride and nitrate. Lipophilic macrocyclic squaramide receptors 1 and 2 were synthesized. Receptor 2 efficiently extracted sulfate from aqueous sodium sulfate solutions into a chloroform phase, via exchange with nitrate ions, overcoming the Hofmeister bias. The resulting 2$SO4 2� complex was readily recycled through precipitation of BaSO4. Transport of sulfate across a bulk chloroform membrane by 2 was demonstrated across a wide pH range (pH 3.2–9.4) and in the presence of high concentrations of competing anions (chloride, nitrate and dihydrogenphosphate), opening the door to the use of 2 for the selective removal of sulfate from water across a range of applications.

With this research we set out to develop a coumarin-based novel fluorescent probe NTR-AHC for the detection of biological thiols and nitroreductase (NTR). Probe NTR-AHC was constructed by attaching the NTR trigger moiety (p-nitrobenzyl) and biothiol acceptor (maleic acid monoamide), to the core fluorophore AHC. In the presence of both glutathione (GSH) and NTR, probe NTR-AHC produced a >8-fold increase in fluorescence intensity at 463 nm.

The syntheses of two new squaramide-naphthalimide conjugates (SQ1 and SQ2) are reported where both compounds have been shown to act as selective fluorescence "turn on" probes for bromide in aqueous DMSO solution through a disaggregation induced response. SQ1 and SQ2 displayed a large degree of self-aggregation in aqueous solution that is disrupted at increased temperature as studied by 1H NMR and Scanning Electron Microscopy (SEM). Moreover, the fluorescence behavior of both receptors was shown to be highly dependent upon the aggregation state and increasing temperature gave rise to a significant increase in fluorescence intensity. Moreover, this disaggregation induced emission (DIE) response was exploited for the selective recognition of certain halides, where the receptors gave rise to distinct responses related to the interaction of the various halide anions with the receptors. Addition of F- rendered both compounds non-emissive; thought to be due to a deprotonation event while, surprisingly, Br- resulted in a dramatic 500-600% fluorescence enhancement thought to be due to a disruption of compound aggregation and allowing the monomeric receptors to dominate in solution. Furthermore, optical sensing parameters such as limits of detection and binding constant of probes were also measured toward the various halides (F-, Cl-, Br-, and I-) where both SQ1 and SQ2 were found to sense halides with adequate sensitivity to measure μM levels of halide contamination. Finally, initial studies in a human cell line were also conducted where it was observed that both compounds are capable of being taken up by HeLa cells, exhibiting intracellular fluorescence as measured by both confocal microscopy and flow cytometry. Finally, using flow cytometry we were also able to show that cells treated with NaBr exhibited a demonstrable spectroscopic response when treated with either SQ1 or SQ2.

A family of chemosensors based on the squaramidoquinoxaline moiety (cyclobuta[b]quinoxaline-1,2(3H,8H)-dione) (1–3) have been assembled by a simple room temperature one step synthesis. X-ray crystallographic analysis of 2 demonstrated both strong hydrogen bond donor ability and tendency to π stack in the solid state. Moreover, Compound 2, with its strongly electron withdrawing nitro-substituent, showed an obvious F− selective colour change (when measured against other halides) clearly visible to the naked eye from pink to green in DMSO solution. Further addition of F− led to a second colour change from green to yellow. Using TD-DFT, UV/Vis and NMR analysis we conclude that the observed colour changes are likely to be due to a two-step process involving two NH deprotonation steps. Colour changes of 1 and 3 were not as apparent in the presence of F− owing to the reduced acidity of the NH functionality of the squaramidoquinoxaline. The results of this study again demonstrate the use of squaramide derivatives as valuable building blocks in the field of anion recognition and demonstrate that the electron withdrawing aryl substituent is directly related to the sensing ability/acidity of the squaramide protons and can be used to tune their anion recognition behavior.

Large tumours contain regions with very low intracellular O2 concentrations. Known as hypoxia, this feature of tumours yields a highly reducing environment owing to the presence of numerous oxygen sensitive reductase enzymes. The development of new optical chemosensors for these various reductases presents an ideal approach to visualise areas of hypoxia or highly reducing environments. Critical to the success of such chemosensors is the design of probes containing a bioreductively activated moiety that either ensures the selective retention of fluorescence within a hypoxic tissue or a probe that irreversibly releases a reporter fluorophore. This Feature Article aims to summarise the fluorescent tools that have been developed to image tumour hypoxia and the various reductase enzymes associated with the bioreduction process.

The development of Ru(II) functionalized gold nanoparticles 1–3·AuNP is described. These systems were found to be mono-disperse with a hydrodynamic radius of ca. 15 nm in water but gave rise to the formation of higher order structures in buffered solution. The interaction of 1–3·AuNP with DNA was also studied by spectroscopic and microscopic methods and suggested the formation of large self-assembly structures in solution. The uptake of 1–3·AuNP by cancer cells was studied using both confocal fluorescence as well as transmission electron microscopy (TEM), with the aim of investigating their potential as tools for cellular biology. These systems displaying a non-toxic profile with favourable photophysical properties may have application across various biological fields including diagnostics and therapeutics.

A simple nitrobenzyl-umbelliferone (NCOU1) was synthesised containing a nitroreductase (NTR) trigger moiety. The presence of NTR, resulted in the fragmentation of the parent molecule and release of the highly emissive fluorophore umbelliferone via an NTR-catalyzed reduction of the nitro group. In the presence of the NTR enzyme, NCOU1 gave rise to a 5-fold increase in fluorescence intensity at 455 nm and was selective for NTR over other reductive enzymes. These results indicate that NCOU1 can be used as a simple assay for the detection of NTR. [Figure not available: see fulltext.]

Eight receptors 1–8 comprising an l-lysine scaffold modified at N- and C-termini with aliphatic alkyl chains and N,N′-alkyl amides, respectively, and bearing squaramide moieties on the amino acid side chain were synthesised by a combination of solid- and solution-phase chemistries and shown to complex various anions in 0.5% H2O in dimethyl sulfoxide-d6 solution. All of the receptors were found to bind Cl− , AcO− and BzO− via hydrogen-bond or acid–base interactions with the squaramide protons; however, 1 was found to bind to via hydrogen bonds formed between the anion and both the squaramide and amide NH moieties. Moreover, modification of both the N- and C-termini of the amino acids with different alkyl substituents had a negligible effect on their anion-binding properties while simultaneously conferring lipophilicities in a range that is optimal for molecules to behave as ‘drug-like’ systems as defined by Lipinski's rule of five. The results of this study demonstrate the versatility of such amino acid receptors as building blocks in the field of anion recognition.

The synthesis of six small peptide anion receptors based on thiourea and squaramide recognition moieties is described. These new receptors bind to tetrahedral sulfate anions with remarkable affinity and selectivity in aqueous so- lution as shown by NMR spectroscopy. Molecular modelling suggests that selectivity is mediated by a hydrogen bond network incorporating the amide backbone protons in a manner similar to that found in the sulfate-binding pro- tein.

The synthesis and photophysical properties of four squaramide based fluorescent anion sensors (1-4) are described. These luminescent compounds showed selectivity for Cl(-) over various other anions with concomitant changes in both their UV/visible and fluorescence properties upon Cl(-) addition, attributed to initial H-bonding followed by NH deprotonation in the presence of excess Cl(-), signaled by a color change. The nature of the electron withdrawing aryl substituents is directly related to the H-bonding ability/acidity of the squaramide protons and can be used to tune the deprotonation behavior.

Macrocyclic squaramide derivatives bind sulfate ions with high selectivity and affinity in aqueous mixtures., A number of macrocyclic squaramide-containing receptors (MSQs) have been designed and synthesised and their interaction with a range of inorganic anions was studied in solution by 1H NMR spectroscopy and ESI-HRMS. The binding data revealed remarkable binding of sulfate in aqueous mixtures from 0.5 to 50% v/v H2O/DMSO-d6. The larger [3]-MSQs were found to better match the size and shape of the sulfate ion than the [2]-MSQs, providing high affinity and selectivity for sulfate while other tetrahedral divalent anions such as selenate, phosphate species and chromate have substantially lower binding affinities. In mixtures of anions mimicking the composition of either nuclear waste or plasma, the [3]-MSQs were still able to bind sulfate ions with high affinity.

The synthesis, spectroscopic characterisation and biological evaluation of mono- and bis-1,8-naphthalimide-conjugated ruthenium(ii)-polypyridyl complexes is presented. Spectroscopic DNA titrations, together with denaturation studies, show strong binding of both species to DNA through the naphthalimide arms. Linear and circular dichroism (LD and CD) spectroscopy reveal close association of the Ru(bpy)3(2+) core with DNA in the case of the mono-naphthalamide complex, [Ru(bpy)2(bpy-NAP)](2+). Significantly, binding by the second naphthalimide arm in the [Ru(bpy)2(bpy-NAP2)](2+) complex is found to displace the Ru(bpy)3(2+) centre from the DNA backbone. This 'negative allosteric effect' is found to have a dramatic influence on the photoinduced damage of plasmid DNA, and the viability of HeLa cancer cells upon photoactivation. Overall the study clearly maps and correlates the relationship between molecular structure, in vitro binding and activity, and in cellulo function.

An oxothiosquaramide was shown to bind to chloride through hydrogen bonding interactions in DMSO and found to exhibit pH switchable chloride transport across phospholipid bilayers via an antiport transport mechanism.

A series of side-chain modified peptides have been prepared via an iterative sequence of peptide couplings and azide–alkyne cycloadditions (‘click’ reactions) using Fmoc-solid phase peptide synthesis. This efficient modular synthetic route allows the systematic and sequential incorporation of a variety of side-chain modifications onto short peptides. The versatility of this approach was demonstrated by the synthesis of a series of short peptides with appended anion recognition motifs and fluorescent indicators.

A family of organic molecules containing the DNA intercalating chromophores, 4-nitro- and 4-amino 1,8-naphthalimide, conjugated to a diquat derivative by an ‘orthogonal’ phenyl spacer have been prepared and characterised. Their binding interactions with double-stranded DNA were studied by a variety of spectroscopic techniques. These charged organic compounds are found to exhibit excellent binding affinities to DNA with binding constants comparable to those exhibited by metal complexes. SB. 15/05/2013

The new Ru(II) polypyridyl complex 1 was synthesised using microwave irradiation from the new polypyridyl ligand 2 ‘DipyTAP’, and its photophysical properties, and DNA binding abilities were investigated using various spectroscopic techniques; and 1 was shown to act as a ‘NIR molecular light switch’ for DNA with an emission window between 680 and 860 nm.

The synthesis and photophysical and biological investigation of Ru(II)-polypyridyl stabilized water-soluble, luminescent gold nanoparticles (AuNPs) are described. These structures bind to DNA and undergo rapid cellular uptake, being localized within the cell cytoplasm and nucleus within 4 h.

The quaternarized pdppz derivative 1 was shown to bind strongly to DNA with concomitant changes in its ground and excited state photophysical properties. Furthermore, the compound also showed rapid cellular uptake, and induced apoptosis upon light irradiation in various cancer cell lines after 24 hours of incubation. AMS. No Keywords.

The synthesis and photophysical properties of 1 and 2, two Ru(II)-polypyridyl based-1,8-naphthalimide Tröger's bases, are described; these were found to stabilize double stranded DNA, undergo rapid cellular uptake, displaying good luminescence without affecting cell viability even after 24 hours of incubation.