Single-molecule methods have matured into central tools for studies in biology. Foerster resonance energy transfer (FRET) techniques, in particular, have been widely applied to study biomolecular structure and dynamics. The major bottleneck for a facile and general application of these studies arises from the need to label biological samples site-specifically with suitable fluorescent dyes. In this work, we present an optimized strategy combining click chemistry and the genetic encoding of unnatural amino acids (UAAs) to overcome this limitation for proteins. We performed a systematic study with a variety of clickable UAAs and explored their potential for high-resolution single-molecule FRET (smFRET). We determined all parameters that are essential for successful single-molecule studies, such as accessibility of the probes, expression yield of proteins, and quantitative labeling. Our multiparameter fluorescence analysis allowed us to gain new insights into the effects and photophysical properties of fluorescent dyes linked to various UAAs for smFRET measurements. This led us to determine that, from the extended tool set that we now present, genetically encoding propargyllysine has major advantages for state-of-the-art measurements compared to other UAAs. Using this optimized system, we present a biocompatible one-step dual-labeling strategy of the regulatory protein RanBP3 with full labeling position freedom. Our technique allowed us then to determine that the region encompassing two FxFG repeat sequences adopts a disordered but collapsed state. RanBP3 serves here as a prototypical protein that, due to its multiple cysteines, size, and partially disordered structure, is not readily accessible to any of the typical structure determination techniques such as smFRET, NMR, and X-ray crystallography.

As a part of our continuing studies on 'Polyamines and their role in human disease' we are investigating how polyamines, and especially how novel polyamine conjugates, interact with DNA. We are studying how these conjugates interact with circular plasmids in order to produce nanometre-sized particles suitable for transfecting cells. Our considerations of structure--activity relationships (SAR) within naturally occurring and synthetic polyamines have shown the significance of the inter-atomic distances between the basic nitrogen atoms. As these atoms are typically fully protonated under physiological conditions, they exist in equilibrium as polyammonium ions. The covalent addition of a lipid moiety, typically one or two alkyl or alkenyl chains, or a steroid, allows much greater efficiency in DNA condensation and in the cellular transfection achieved. Thus efficient DNA condensation and subsequently drug delivery (i.e. with DNA as the drug) can be brought about using novel polyamine conjugates. Taking further advantage of the functionalization of specific steroids (e.g. cholesterol and certain bile acids), we have designed and prepared novel fluorescent molecular probes as tools to throw light on the problematic steps in non-viral gene delivery which still impede efficient gene therapy. Thus, the current aims of our research are to understand, design and prepare small-molecule lipopolyamines for non-viral gene therapy (NVGT). The rational design and practical preparation of non-symmetrical polyamine carbamates and amides, based on steroid templates of cholesterol and the bile acid lithocholic acid as the lipid moiety, provides fluorescent molecular probes that condense DNA. These novel lipopolyamine conjugates mimic the positive charge distribution found in the triamine spermidine and the tetra-amine spermine alkaloids. After optimizing their SAR, these fluorescent probes will be useful in monitoring gene delivery in NVGT.

A new series of ligands has been synthesized where the cinnamoyl group of the 14-cinnamoylamino morphinones has been introduced to the 7α-substituent of the 6,14-bridged oripavine series. In vitro the compounds were mostly low efficacy partial agonists or antagonists with some selectivity for the mu opioid receptor, with evidence of mu efficacy in vivo. The similarity in SAR between these 6,14-bridged oripavines and the 14-cinnamoylamino series suggests a similar mode of interaction with the mu opioid receptor.

Ligands from the naltrexamine series have consistently demonstrated agonist activity at kappa opioid receptors (KOR), with varying activity at the mu opioid receptor (MOR). Various 6β-cinnamoylamino derivatives were made with the aim of generating ligands with a KOR agonist/MOR partial agonist profile, as ligands with this activity may be of interest as treatment agents for cocaine abuse. The ligands all displayed the desired high affinity, non-selective binding in vitro and in the functional assays were high efficacy KOR agonists with some partial agonist activity at MOR. Two of the new ligands (12a, 12b) have been evaluated in vivo, with 12a acting as a KOR agonist, and therefore somewhat similar to the previously evaluated analogues 3–6, while 12b displayed predominant MOR agonist activity.