Arbor: comparative analysis workflows for the tree of life.

We describe our efforts to develop a software package, Arbor, that will enable scientific research in all aspects of comparative biology. This software will enable developmental biologists, geneticists, ecologists, geographers, paleobiologists, educators, and students to analyze diverse types of comparative data at multiple phylogenetic and spatiotemporal scales using an intuitive visual interface. Arbor's user-defined workflows will be exported and shared so that entire analyses can be quickly replicated with new or updated data. Arbor will also be designed to easily and seamlessly expand to include novel analytical tools as they are developed. Here we describe the core components of Arbor, as well as provide details of one proposed test case to illustrate the software's key functionality.

Thermally-activated chemiluminescent squaraine rotaxane endoperoxide with green emission.

A squaraine rotaxane endoperoxide with a truncated squaraine chromophore undergoes a cycloreversion reaction and emits green light that can be transferred to red acceptor dyes. The results demonstrate that chemiluminescence emission for squaraine rotaxane endoperoxides comes from the excited squaraine inside the rotaxane.

Squaraine [2]catenanes: synthesis, structure and molecular dynamics.

Three squaraine [2]catenanes are synthesized and found to have bright, deep-red fluorescence and high chemical stability. The interlocked molecules undergo two large-amplitude dynamic processes, twisting of the squaraine macrocycle and skipping over the partner tetralactam.

Macrocycle breathing in [2]rotaxanes with tetralactam macrocycles.

The structural dynamics of two pairs of [2]rotaxanes were compared using variable-temperature NMR. Each rotaxane had a surrounding tetralactam macrocycle with either 2,6-pyridine dicarboxamide or isophthalamide bridging units. Differences were observed in two types of rotational processes: spinning of the phenylene wall units in the surrounding macrocycle of squaraine rotaxanes and macrocycle pirouetting in xanthone rotaxanes. The rotaxanes with macrocycles containing 2,6-pyridine dicarboxamide bridges exhibited higher rotational barriers due to a cavity contraction effect, which disfavored macrocycle breathing.

Storable, thermally activated, near-infrared chemiluminescent dyes and dye-stained microparticles for optical imaging.

Imaging techniques are a vital part of clinical diagnostics, biomedical research and nanotechnology. Optical molecular imaging makes use of relatively harmless, low-energy light and technically straightforward instrumentation. Self-illuminating, chemiluminescent systems are particularly attractive because they have inherently high signal contrast due to the lack of background emission. Currently, chemiluminescence imaging involves short-lived molecular species that are not stored but are instead generated in situ, and they typically emit visible light, which does not penetrate far through heterogeneous biological media. Here, we describe a new paradigm for optical molecular imaging using squaraine rotaxane endoperoxides, interlocked fluorescent and chemiluminescent dye molecules that have a squaraine chromophore encapsulated inside a macrocycle endoperoxide. Squaraine rotaxane endoperoxides can be stored indefinitely at temperatures below -20 °C, but upon warming to body temperature they undergo a unimolecular chemical reaction and emit near-infrared light that can pass through a living mouse.

Using the rotaxane mechanical bond to enhance chemical reactivity.

Rates of cycloreversion for squaraine rotaxane mono(endoperoxides) were enhanced by structural modifications that increased cross-component steric destabilization of the inward directed 9,10-anthracene endoperoxide group. The largest rate enhancements were obtained when the surrounding macrocycle contained two 2,6-pyridine dicarboxamide bridging units, which induced a cavity contraction effect. The precursor fluorescent, near-IR, squaraine rotaxanes are effectively photostable because the mono(endoperoxide) products, formed by reaction with photogenerated singlet oxygen, rapidly cyclorevert back to the original squaraine rotaxane.

Microwave-assisted slipping synthesis of fluorescent squaraine rotaxane probe for bacterial imaging.

Microwave heating accelerates quantitative squaraine rotaxane formation by slipping and facilitates production of a bacterial imaging probe with zinc dipicolylamine targeting ligands.

Discovery and early development of squaraine rotaxanes.

The chemical and photophysical properties of a fluorescent squaraine dye are greatly enhanced when it is mechanically encapsulated inside a tetralactam macrocycle. This feature article describes the synthesis, structure, and photophysical performance of first-generation squaraine rotaxanes, and shows how they can be used as fluorescent imaging probes and chemosensors.

Squaraine rotaxanes with boat conformation macrocycles.

Mechanical encapsulation of fluorescent, deep-red bis(anilino)squaraine dyes inside Leigh-type tetralactam macrocycles produces interlocked squaraine rotaxanes. The surrounding macrocycles are flexible and undergo rapid exchange of chair and boat conformations in solution. A series of X-ray crystal structures show how the rotaxane co-conformational exchange process involves simultaneous lateral oscillation of the macrocycle about the center of the encapsulated squaraine thread. Rotaxane macrocycles with 1,4-phenylene sidewalls and 2,6-pyridine dicarboxamide bridging units are more likely to adopt boat conformations in the solid state than analogous squaraine rotaxane systems with isophthalamide-containing macrocycles. A truncated squaraine dye, with a secondary amine attached directly to the central C(4)O(2) core, is less electrophilic than the extended bis(anilino)squaraine analogue, but it is still susceptible to chemical and photochemical bleaching. Its stability is greatly enhanced when it is encapsulated as an interlocked squaraine rotaxane. An X-ray crystal structure of this truncated squaraine rotaxane shows the macrocycle in a boat conformation, and NMR studies indicate that the boat is maintained in solution. Encapsulation as a rotaxane increases the dye's brightness by a factor of 6. The encapsulation process appears to constrain the dye and reduce deformation of the chromophore from planarity. This study shows how mechanical encapsulation as a rotaxane can be used as a rational design parameter to fine-tune the chemical and photochemical properties of squaraine dyes.

Cycloaddition to an anthracene-derived macrocyclic receptor with supramolecular control of regioselectivity.

N-Ethylmaleimide and maleic anhydride add to the interior face of one anthracene wall with unusual 1,4-regioselectivity, whereas singlet oxygen adds to both anthracene walls with 9,10-regioselectivity.

Synthesis and photophysical investigation of squaraine rotaxanes by "clicked capping".

Pseudorotaxane complexes of squaraine dyes and tetralactam macrocycles are converted into permanently interlocked rotaxane structures using copper-catalyzed and copper-free cycloaddition reactions with bulky stopper groups. The photophysical properties of the encapsulated squaraine depend on the structure of the macrocycle. In one case, squaraine rotaxanes are produced in near-quantitative yields and with intense near-IR fluorescence. In another case, squaraine fluorescence is greatly diminished upon macrocyclic encapsulation but the signal can be restored by dye displacement with anions.

Self-assembly of fluorescent inclusion complexes in competitive media including the interior of living cells.

Anthracene-containing tetralactam macrocycles are prepared and found to have an extremely high affinity for squaraine dyes in chloroform (log Ka = 5.2). Simply mixing the two components produces highly fluorescent, near-infrared inclusion complexes in quantitative yield. An X-ray crystal structure shows the expected hydrogen bonding between the squaraine oxygens and the macrocycle amide NH residues, and a high degree of cofacial aromatic stacking. The kinetics and thermodynamics of the assembly process are very sensitive to small structural changes in the binding partners. For example, a macrocycle containing two isophthalamide units associates with the squaraine dye in chloroform 400,000 times faster than an analogous macrocycle containing two 2,6-dicarboxamidopyridine units. Squaraine encapsulation also occurs in highly competitive media such as mixed aqueous/organic solutions, vesicle membranes, and the organelles within living cells. The highly fluorescent inclusion complexes possess emergent properties; that is, as compared to the building blocks, the complexes have improved chemical stabilities, red-shifted absorption/emission maxima, and different cell localization propensities. These are useful properties for new classes of near-infrared fluorescent imaging probes.