Molecular recognition of DNA by rigid [n]-polynorbornane-derived bifunctional intercalators: synthesis and evaluation of their binding properties

We have exploited the concept of multivalency in the context of DNA recognition, using novel chemistry to synthesize a new type of bis-intercalator with unusual sequence-selectivity. Bis-intercalation has been observed previously, but design principles for de novo construction of such molecules are not known. Our compounds feature two aromatic moieties projecting from a rigid, polynorbornane-based scaffold. The length and character of the backbone as well as the identity of the intercalators were varied, resulting in mono- or divalent recognition of the double helix with varying affinity. Our lead compound proved to be a moderately sequence-selective bis-intercalator with an unwinding angle of 27 and a binding constant of about 8 M. 9-Aminoacridine rings were preferred over acridine carboxamides or naphthalimides, and a rigid [3]-polynorbornane scaffold was superior to a [5]-polynorbornane. The flexibility of the linker connecting the rings to the scaffold, although less influential, could affect the strength and character of the DNA binding.

Exploring the use of the tripeptide Gly–Gly–His as a selective recognition element for the fabrication of electrochemical copper sensors

The modification of electrodes with the tripeptide Gly–Gly–His for the detection of copper in water samples is described in detail. The tripeptide modified electrode was prepared by first self-assembling 3-mercaptopropionic acid (MPA) onto the gold electrode followed by covalent attachment of the tripeptide to the self-assembled monolayer using carbodiimide coupling. The electrodes were characterized using electrochemistry, a newly developed mass-spectrometry method and quantum mechanical calculations. The mass spectrometry confirmed the modification to proceed as expected with peptide bonds formed between the carboxylic acids of the MPA and the terminal amine of the peptide. Electrochemical measurements indicated that approximately half the MPA molecules in a SAM are modified with the peptide. The peptide modified electrodes exhibited high sensitivity to copper which is attributed to the stable 4N coordinate complex the peptide formed around the metal ion to give copper the preferred tetragonal coordination. The formation of a 4 coordinate complex was predicted using quantum mechanical calculation and confirmed using mass spectrometry. The adsorption of the copper to the peptide modified electrode was consistent with a Langmuir isotherm with a binding constant of (8.1 ± 0.4) 1010 M−1 at 25 °C.

Fluorescent and electrochemical sensing of (poly)phosphate nucleotides by ferrocene functionalised with two (ZnII-TACN-pyrene) complexes

The [Fc[BOND]bis{ZnII(TACN)(Py)}] complex, comprising two ZnII(TACN) ligands (Fc=ferrocene; Py=pyrene; TACN=1,4,7-triazacyclononane) bearing fluorescent pyrene chromophores linked by an electrochemically active ferrocene molecule has been synthesised in high yield through a multistep procedure. In the absence of the polyphosphate guest molecules, very weak excimer emission was observed, indicating that the two pyrene-bearing ZnII(TACN) units are arranged in a trans-like configuration with respect to the ferrocene bridging unit. Binding of a variety of polyphosphate anionic guests (PPi and nucleotides di- and triphosphate) promotes the interaction between pyrene units and results in an enhancement in excimer emission. Investigations of phosphate binding by 31P NMR spectroscopy, fluorescence and electrochemical techniques confirmed a 1:1 stoichiometry for the binding of PPi and nucleotide polyphosphate anions to the bis(ZnII(TACN)) moiety of [Fc[BOND]bis{ZnII(TACN)(Py)}] and indicated that binding induces a trans to cis configuration rearrangement of the bis(ZnII(TACN)) complexes that is responsible for the enhancement of the pyrene excimer emission. Pyrophosphate was concluded to have the strongest affinity to [Fc[BOND]bis{ZnII(TACN)(Py)}] among the anions tested based on a six-fold fluorescence enhancement and 0.1 V negative shift in the potential of the ferrocene/ferrocenium couple. The binding constant for a variety of polyphosphate anions was determined from the change in the intensity of pyrene excimer emission with polyphosphate concentration, measured at 475 nm in CH3CN/Tris-HCl (1:9) buffer solution (10.0 mM, pH 7.4). These measurements confirmed that pyrophosphate binds more strongly (Kb=(4.45±0.41)×106 M−1) than the other nucleotide di- and triphosphates (Kb=1–50×105 M−1) tested.

Truncation and mutation of a transferrin receptor aptamer enhance binding affinity

Aptamers are proving their utility in a number of applications. However, to be easily functionalized, their structure needs to be simplified. Therefore, we sought to truncate a 50-nucleotide aptamer specific to the transferrin receptor to its smallest functional unit using rational engineering of the predicted two-dimensional structure of the longer parent sequence. In addition, mutations were introduced into the binding loop to determine their effect on the selectivity of the aptamers. These base mutations enhanced the binding affinity of the aptamer, while retaining its specificity. The equilibrium dissociation constant (Kd) was reduced sixfold following the substitution of all four bases in the binding region. In addition, these aptamers were efficiently internalized into transferrin receptor-positive cells in a similar manner to the transferrin receptor antibody and demonstrated colocalization with this antibody. This study has shown that the smallest functional unit of this aptamer was 14 nucleotides. This small size will be advantageous for future applications, such as drug delivery or functionalization of other therapeutic modalities.