Fluorometric calcium measurements have revealed presynaptic residual calcium (Ca(res)) to be an important regulator of synaptic strength. However, in the mammalian brain, it has not been possible to monitor Ca(res) in fibers that project from one brain region to another. Here, we label neuronal projections by injecting dextran-conjugated calcium indicators into brain nuclei in vivo. Currently available dextran conjugates distort Ca(res) due to their high affinity for calcium. Therefore, we synthesized a low-affinity indicator, fluo-4 dextran, that can more accurately measure the amplitude and time course of Ca(res). We then demonstrate the utility of fluo-4 dextran by measuring Ca(res) at climbing fiber presynaptic terminals. This method promises to facilitate the study of many synapses in the mammalian CNS, both in brain slices and in vivo.

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Hydrogen-bonded tapes comprised of monomeric molecular precursors are used to define structural parameters for the design of related oligomers encoded with predetermined modes of assembly. Application of this "covalent casting" strategy vis-à-vis the one-dimensional H-bonding motif expressed by 2-amino-4,6-dichlorotriazine has enabled the design of high-affinity duplex molecular strands. Dimeric, trimeric, and tetrameric duplex oligomers are prepared through an iterative synthetic protocol involving sequential homologation of the oligo(aminotriazine). The mode of assembly and interstrand affinity of homologous oligomers are established in solution by (1)H NMR dilution experiments, isothermal titration calorimetry (ITC), vapor pressure osmometry (VPO), cross-hybridization experiments involving the analysis of dye-labeled strands via thin-layer chromatography (TLC), and in the solid state by X-ray crystallographic analysis. Binding free energy per unimer (-Delta G degrees/n) increases significantly upon extension from monomer to dimer to trimer, signifying a strong positive cooperative effect. Nanomolar binding affinity (K(d) = 1.44 +/- 0.50 nM) was determined for the duplex trimer by ITC in 1,2-dichloroethane at 20 degrees C. In-register duplex formation is not observed for the tetramer, which appears to adopt an alternative binding mode. These data give insight into the structural and interactional features of the oligomers required for high-affinity, high-specificity binding and define a platform for the design of second-generation systems and related duplex strands for use in nanoscale assembly.

A major theme in the development of the chemical sciences resides in improving the capability to negotiate issues of selectivity in the organization of matter on increasingly greater-length scales. 1 While molecular synthesis has advanced to an art through the precise control of chemo-, regio-, stereo-, and enantioselectivity, the identification and orchestration of persistent noncovalent binding motifs is extending synthetic technology to the nanoscopic regime by allowing the construction of supramolecular architectures through the self-assembly of instructed molecular components under equilibrium conditions. 2 Along these lines, the H-bond-mediated self-assembly of molecular precursors represents a powerful strategy for the logic-driven retrosynthesis and construction of nanoscopic materials in structurally homogeneous form. 2e Nevertheless, the nominal stability of most noncovalent aggregates detracts from their usefulness. To address this deficiency, template-directed syntheses involving the covalent capture of discrete noncovalent superstructures 3 and the polymerization of organized assemblies 4,5 have been described. Robust nanoscale assemblies may also be obtained through the stabilization of kinetically labile systems by the preorganization of binding sites and accumulation of multiple binding interactions. 6 Macromolecular systems are well-suited to this latter strategy, and recently much attention has been given to the preparation of polymers incorporating H-bonding moieties 7 and the self-assembly of dendritic macromolecules 8 and block copolymers. 5a,9,10 As part of a program involving the development of synthetic methodologies for the synthesis of nanostructured materials via self-assembly of macromolecular precursors, we herewith report preliminary studies on the design and de novo synthesis of synthetic polymer strands capable of duplex formation through the "covalent casting" of 1-dimensional H-bonding motifs. Excluding systems that borrow from naturally occurring superstructural motifs (e.g., homo-DNA 11 ), to the best of our knowledge this report represents the first efforts toward unnatural polymer duplexes assembling through the action of inter-strand H-bonds. 12 Cooperative binding via preorganization of multiple complementary binding sites enhances association through the reduction of entropic terms for the formation of discrete objects through receptor-substrate interactions. 2e,13 For the application of this principle to 1-dimensional superstructures, such as the H-bonded tape I, it was hypothesized that preorganization of the molecules comprising the 1-dimensional superstructure could be achieved by introduction of a covalent linker as shown schematically for the partially cast H-bonded tape II. Introduction of a second linking group fully casts the H-bonded tape to afford a covalentnoncovalent ladder material, polymer duplex III (Scheme 1).In accord with this strategy, I should be comprised of molecules capable of functioning as a platform for subsequent elaboration to III. Trichloro-...