Steven L. Cobb

Biochemistry: Biosynthesis of an organofluorine molecule

Although fluorine in the form of fluoride minerals is the most abundant halogen in the Earths crust, only 12 naturally occurring organofluorine compounds have so far been found, and how these are biosynthesized remains a mystery. Here we describe an enzymatic reaction that occurs in the bacterium Streptomyces cattleya and which catalyses the conversion of fluoride ion and S-adenosylmethionine (SAM) to 5′-fluoro-5′-deoxyfluoroadenosine (5′-FDA). To our knowledge, this is the first fluorinase enzyme to be identified, a discovery that opens up a new biotechnological opportunity for the preparation of organofluorine compounds.

Blending Gelators to Tune Gel Structure and Probe Anion-Induced Disassembly

Blending different low molecular weight gelators (LMWGs) provides a convenient route to tune the properties of a gel and incorporate functionalities such as fluorescence. Blending a series of gelators having a common bis-urea motif, and functionalised with different amino acid-derived end-groups and differing length alkylene spacers is reported. Fluorescent gelators incorporating 1-and 2-pyrenyl moieties provide a probe of the mixed systems alongside structural and morphological data from powder diffraction and electron microscopy. Characterisation of the individual gelators reveals that although the expected α-urea tape motif is preserved, there is considerable variation in the gelation properties, molecular packing, fibre morphology and rheological behaviour. Mixing of the gelators revealed examples in which: 1) the gels formed separate, orthogonal networks maintaining their own packing and morphology, 2) the gels blended together into a single network, either adopting the packing and morphology of one gelator, or 3) a new structure not seen for either of the gelators individually was created. The strong binding of the urea functionalities to anions was exploited as a means of breaking down the gel structure, and the use of fluorescent gel blends provides new insights into anion-mediated gel dissolution.

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.

A concise stereoselective synthesis of orthogonally protected lanthionine and β-methyllanthionine

Lantibiotics such as nisin are active against most Gram-positive bacteria and constitute an important class of antibacterial agents. These ribosomally synthesized peptides contain either one or both of the unusual amino acids meso-lanthionine (m-Lan) or beta-methyllanthionine (beta-MeLan). Nucleophilic ring opening of sulfamidates allows facile preparation of stereochemically pure derivatives of m-Lan and beta-MeLan with orthogonal protection for solid phase synthesis of lantibiotic analogues.

Excited State Dynamics of the Isolated Green Fluorescent Protein Chromophore Anion Following UV Excitation

A combined frequency-, angle-, and time-resolved photoelectron spectroscopy study is used to unravel the excited state dynamics following UV excitation of the isolated anionic chromophore of the green fluorescent protein (GFP). The optically bright S3 state, which is populated for hv > 3.7 eV, is shown to decay predominantly by internal conversion to the S2 state that in turn autodetaches to the neutral ground state. For hv > 4.1 eV, a new and favorable autodetachment channel from the S2 state becomes available, which leads to the formation of the neutral in an excited state. The results indicate that the UV excited state dynamics of the GFP chromophore involve a number of strongly coupled excited states.

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. Copyright

The Role of Phosphoglycans in the Susceptibility of Leishmania mexicana to the Temporin Family of Anti-Microbial Peptides

Natural product antimicrobial peptides (AMPs) have been proposed as promising agents against the Leishmania species, insect vector borne protozoan parasites causing the neglected tropical disease leishmaniasis. However, recent studies have shown that the mammalian pathogenic amastigote form of L. mexicana, a causative agent of cutaneous leishmaniasis, is resistant to the amphibian-derived temporin family of AMPs when compared to the insect stage promastigote form. The mode of resistance is unknown, however the insect and mammalian stages of Leishmania possess radically different cell surface coats, with amastigotes displaying low (or zero) quantities of lipophosphoglycan (LPG) and proteophosphoglycan (PPG), macromolecules which form thick a glycocalyx in promastigotes. It has been predicted that negatively charged LPG and PPG influence the sensitivity/resistance of promastigote forms to cationic temporins. Using LPG and PPG mutant L. mexicana, and an extended range of temporins, in this study we demonstrated that whilst LPG has little role, PPG is a major factor in promastigote sensitivity to the temporin family of AMPs, possibly due to the conferred anionic charge. Therefore, the lack of PPG seen on the surface of pathogenic amastigote L. mexicana may be implicated in their resistance to these peptides.

Investigating the Anti-leishmanial Effects of Linear Peptoids

Peptoids, a class of peptide mimetics, have emerged as promising anti‐infective agents against a range of bacterial infections. Herein we present the first study of the antiparasitic and specifically the anti‐leishmanial properties of linear peptoids. Peptoids were identified as having promising activity against Leishmania mexicana axenic amastigotes, a causative agent of cutaneous leishmaniasis.

Novel fluorinated lipopeptides from Bacillus sp. CS93 via precursor-directed biosynthesis

While attempting to improve production of fluoro-iturin A in Bacillus sp. CS93 new mono- and di-fluorinated fengycins were detected in culture supernatants by 19F NMR and tandem mass spectrometry, after incubation of the bacterium with 3-fluoro-l-tyrosine. The fluorinated amino acid was presumably incorporated in place of one or both of the tyrosyl residues in fengycin. Investigations to generate additional new fluorinated derivatives were undertaken using commercially available fluorinated phenylalanines and 2-fluoro- and 2,3-difluoro-tyrosine that were synthesised by Negishi cross-coupling of iodoalanine and fluorinated bromo-phenols. The anti-fungal activity of the fluorinated lipopeptides was assayed against Trichophyton rubrum and found to be similar to that of the non-fluorinated metabolites.

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.