We have previously reported a general method for the deracemisation of racemic chiral amines (primary, [1] secondary [2] and tertiary [3] ) by using variants of the enzyme monoamine oxidase N (MAO-N) from Aspergillus niger. This deracemisation process employs a combination of an enantioselective enzymatic oxidation of the amine to afford the corresponding imine or iminium ion, together with a non-selective chemical reduction of the imine or iminium ion back to the racemic starting material (Scheme 1). The use of an (S)-selective MAO-N enzyme leads to accumulation of the (R)-amine in high enantiomeric excess and yield through several rounds of oxidation and reduction.Previously, we have applied this chemo-enzymatic approach to the deracemisation of the alkaloid (AE)-crispine A. [4] Crispine A (1) was first isolated from extracts of the plant Carduus crispus (welted thistle), along with the cytotoxic crispine B (2, Figure 1) and three other bicyclic isoquinoline alkaloids. [5] Although the deracemisation of (AE)-crispine A resulted in the generation of the R enantiomer in > 97 % ee, the reaction required 40 h to proceed to completion with a MAO-N-5 variant in an overall yield of 48 %. This previous study also highlighted that less-functionalised analogue 3 was more reactive with the same variant, taking only 6 h to reach completion (> 97 % ee), thus indicating that the two methoxy groups of racemate (AE)-1 resulted in lower activity. We reasoned that this drop in activity was due to steric interference between these two groups and residues within the active site of the enzyme. As a result, we employed a combination of molecular modelling and a rational re-design of the MAO-N-5 variant to identify and develop potential new MAO-N variants that could have enhanced activity towards enantiomer (S)-1. In addition, we sought to improve the efficiency of the overall synthesis of enantiomer (R)-1 by utilising the microwave synthesis of the racemic amine coupled with the enhanced deracemisation that is brought about by changes to the MAO-N enzyme.To identify MAO-N variants that have improved activity towards compound (AE)-1, we modelled (S)-crispine A into the active site of the MAO-N-5 enzyme (PDB code 2VVM). [6] Four residues (Phe210, Leu213, Met242 and Met246), which are located at the entrance to the active site channel, were identified as providing possible steric interactions with the methoxy groups of crispine A (1). To optimise these residues, two randomised libraries were created: The first library targeted amino acids Phe210 and Leu213 (library A) and the second library targeted Met242 and Met246 (library B). Both libraries were screened against compound (AE)-1 by using our previously reported solid-phase assay [7] (Figure 2); sixteen active "hits" were collected from each of the two libraries (A and B), which were then subjected to a second round of screening to eliminate any false positives. After the second round of screening, the two MAO-N variants from each library that had the highest activity, as judged by t...

This research describes the effects of structural variation and medium effects for the novel split-oligonucleotide (tandem) probe systems for exciplex-based fluorescence detection of DNA. In this approach the detection system is split at a molecular level into signal-silent components, which must be assembled correctly into a specific 3-dimensional structure to ensure close proximity of the exciplex partners and the consequent exciplex fluorescence emission on excitation. The model system consists of two 8-mer oligonucleotides, complementary to adjacent sites of a 16-mer DNA target. Each probe oligonucleotide is equipped with functions able to form an exciplex on correct, contiguous hybridization. This study investigates the influence of a number of structural aspects (i.e. chemical structure and composition of exciplex partners, length and structure of linker groups, locations of exciplex partner attachment, as well as effects of media) on the performance of DNA-mounted exciplex systems. The extremely rigorous structural demands for exciplex formation and emission required careful structural design of linkers and partners for exciplex formation, which are here described. Certain organic solvents (especially trifluoroethanol) specifically favour emission of the DNA-mounted exciplexes, probably the net result of the particular duplex structure and specific solvation of the exciplex partners. The exciplexes formed emitted at approximately 480 nm with large Stokes shifts ( approximately 130-140 nm). Comparative studies with pyrene excimer systems were also carried out.

Natural myo-inositol phosphate antioxidants containing the 1,2,3-trisphosphate motif bind Fe(3+) in the unstable penta-axial conformation.

myo-Inositol phosphates possessing the 1,2,3-trisphosphate motif share the remarkable ability to completely inhibit iron-catalysed hydroxyl radical formation. The simplest derivative, myo-inositol 1,2,3-trisphosphate [Ins(1,2,3)P(3)], has been proposed as an intracellular iron chelator involved in iron transport. The binding conformation of Ins(1,2,3)P(3) is considered to be important to complex Fe(3+) in a 'safe' manner. Here, a pyrene-based fluorescent probe, 4,6-bispyrenoyl-myo-inositol 1,2,3,5-tetrakisphosphate [4,6-bispyrenoyl Ins(1,2,3,5)P(4)], has been synthesised and used to monitor the conformation of the 1,2,3-trisphosphate motif using excimer fluorescence emission. Ring-flip of the cyclohexane chair to the penta-axial conformation occurs upon association with Fe(3+), evident from excimer fluorescence induced by pi-pi stacking of the pyrene reporter groups, accompanied by excimer formation by excitation at 351 nm. This effect is unique amongst biologically relevant metal cations, except for Ca(2+) cations exceeding a 1 : 1 molar ratio. In addition, the thermodynamic constants for the interaction of the fluorescent probe with Fe(3+) have been determined. The complexes formed between Fe(3+) and 4,6-bispyrenoyl Ins(1,2,3,5)P(4) display similar stability to those formed with Ins(1,2,3)P(3), indicating that the fluorescent probe acts as a good model for the 1,2,3-trisphosphate motif. This is further supported by the antioxidant properties of 4,6-bispyrenoyl Ins(1,2,3,5)P(4), which closely resemble those obtained for Ins(1,2,3)P(3). The data presented confirms that Fe(3+) binds tightly to the unstable penta-axial conformation of myo-inositol phosphates possessing the 1,2,3-trisphosphate motif.

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