Aqueous synthesis of N,S-dialkylthiophosphoramidates: design, optimisation and application to library construction and antileishmanial testing.

We recently reported the use of PSCl3 for the thiophosphorylation of alkylamines where the resulting N-thiophosphoramidate ions could be readily S-alkylated (Chem. Commun., 2011, 47, 6156-6158.). Herein we report the development of this methodology using amino acid, amino sugar, aminonucleoside and aniline substrates. The hydrolysis properties of N-thiophosphoramidate ions and their reactivities towards alkylating agents are also explored. In addition, we demonstrate the application of our approach to the preparation of a small library of compounds, including quinoline-based N,S-dialkylthiophosphoramidates which were tested for antileishmanial activity.

Studies on the antileishmanial properties of the antimicrobial peptides temporin A, B and 1Sa.

Given the paucity and toxicity of available drugs for leishmaniasis, coupled with the advent of drug resistance, the discovery of new therapies for this neglected tropical disease is recognised as being of the utmost urgency. As such antimicrobial peptides (AMPs) have been proposed as promising compounds against the causative Leishmania species, insect vector-borne protozoan parasites. Here the AMP temporins A, B and 1Sa have been synthesised and screened for activity against Leishmania mexicana insect stage promastigotes and mammalian stage amastigotes, a significant cause of human cutaneous disease. In contrast to previous studies with other species the activity of these AMPs against L. mexicana amastigotes was low. This suggests that amastigotes from different Leishmania species display varying susceptibility to peptides from the temporin family, perhaps indicating differences in their surface structure, the proposed target of these AMPs. In contrast, insect stage L. mexicana promastigotes were sensitive to two of the screened temporins which clearly demonstrates the importance of screening AMPs against both forms of the parasite.

Bifunctional up-converting lanthanide nanoparticles for selective in vitro imaging and inhibition of cyclin D as anti-cancer agents.

Inhibition of the CDK4/cyclin D complex through the substrate recruitment site on the cyclin positive regulatory subunit is recognised as being a promising anti-cancer target. Specific peptide sequences can be used to selectively disrupt this target, but the development of peptides as anti-tumor agents in vitro/in vivo presents several obstacles. Poor cell internalization, low sensitivity towards enzymatic degradation in vivo, and ineffectiveness in monitoring via indirect screening are all issues which must be overcome. Herein, we describe the surface functionalization of lanthanide nanoparticles with cyclin D-specific peptides to prepare novel nanomaterials (UCNPs-P1) which can target the CDK4/cyclin D complex. The nanomaterials prepared (UCNPs-P1) are cell permeable and they display parallel emission spectra in vitro and in an aqueous biological environment. They can also be used in low dose concentrations under harmless NIR excitation and emission via upconversion. Uniquely, in addition to acting as a bioimaging probe, UCNPs-P1 also exhibits promising cytotoxicity towards cancer cells. In light of the aforementioned properties, the prepared functionalized nanomaterials (UCNPs-P1) offer the first real dual acting system for cyclin D imaging and simultaneous inhibition of cancer cell division.

Real time detection of cell cycle regulator cyclin A on living tumor cells with europium emission.

Six water-soluble europium complexes (Eu-L1-P(n) and Eu-L2-P(n), n = 1, 2 and 3) with one antenna chromophore, two different linkers (L1 and L2) and three proposed cyclin A specific peptides (P1: -GAKRRLIF-NH2; P2: -GGAKRRLIF-NH2; P3: -Hex- GAKRRLIF-NH2) have been synthesized. With structural information available, comparisons of the cyclin grooves of cyclin A and the six europium complexes have been made, and insights have been gained into the determinants for peptide binding and the foundation of differential binding. Experiment-wise, the linear and two-photon induced photophysical properties of these conjugates were monitored in aqueous solution. Numerous in situ/in vitro biological assays have been carried out, such as responsive emission changes in situ/in vitro, Western blot and cellular uptake. As imaging agents, complexes with peptides P3: -Hex-GAKRRLIF-NH2 showed high selectivity to cyclin A in numerous cancer cells. When it comes to responsive optical signal changes, complex Eu-L2-P3 exhibited a threefold emission enhancement upon binding with cyclin A (100 nM cyclin A, ϕ = 8% to 21%, log KB = 5.83, detection limit = 5 nM), and this could be initiated by the shortened distance between the antenna and the lanthanide after they bind/get into cyclin A. It is promising that our compounds (especially compound Eu-L2-P3) could serve as the template for structure-guided efforts to develop potential imaging therapeutics on the basis of selective imaging of CDK2/cyclin A activity.

Evaluation of a solid-supported tagging strategy for mass spectrometric analysis of peptides.

We have explored two divinylbenzene cross-linked polystyrene supports for use in a solid-supported N-terminal peptide tagging strategy. Resin-bound tags designed to be cleaved in a single step at the N-terminus of peptides have been devised and explored as peptide N-terminal tagging reagents (constructs) for subsequent mass spectrometric analysis. While the brominated tagging approach shows promise, the use of these specific solid supports has drawbacks, in terms of tagging reaction scale, for real applications in proteomics.

Two-photon induced responsive f-f emissive detection of Cyclin A with a europium-chelating peptide.

Responsive linear and two-photon induced europium emissive probes have been synthesised with a tailor made peptide for the detection of Cyclin A, the hypersensitive Eu emission (Eu-2) gave the real time signalling and also enhanced the two-photon absorption cross section from 12 GM to 68 GM after Cyclin A binding.