Gold(I)-catalysed synthesis of a furan analogue of thiamine pyrophosphate.

An analogue of thiamine having a furan ring in place of the thiazolium ring has been synthesised by a short and efficient route, involving gold(I)-catalysed cyclisation of an alkynyl alcohol to form the furan ring. The furan analogue of thiamine diphosphate (ThDP) was also made and tested for binding to and inhibition of pyruvate decarboxylase (PDC) from Zymomonas mobilis (overexpressed in E. coli with a N-terminal His-tag). It is a very strong inhibitor, with a K i value of 32.5 pM. It was also shown that the furan analogue of thiamine can be functionalised at the C-2 position, which will allow access to mimics of reaction intermediates of various ThDP-dependent enzymes.This work was supported by a studentship from the Cambridge Commonwealth Trust (A.I.)This is the final version. It was first published by the Beilstein-Institut at http://www.beilstein-journals.org/bjoc/single/articleFullText.htm?publicId=1860-5397-10-27

Revision in the first steps of the biosynthesis of the red antibiotic prodigiosin: use of a synthetic thioester to validate a new intermediate.

Funder: Frances and Augustus Newman FoundationFunder: Emmanuel College, University of CambridgeFunder: Cambridge Commonwealth TrustA biosynthetic pathway for the red-antibiotic, prodigiosin, was proposed over a decade ago but not all the suggested intermediates could be detected experimentally. Here we show that a thioester that was not originally included in the pathway is an intermediate. In addition, the enzyme PigE was originally described as a transaminase but we present evidence that it also catalyses the reduction of the thioester intermediate to its aldehyde substrate

Imaging cell surface glycosylation in vivo using "double click" chemistry.

Dynamic alterations in cell surface glycosylation occur in numerous biological processes that involve cell-cell communication and cell migration. We report here imaging of cell surface glycosylation in live mice using double click chemistry. Cell surface glycans were metabolically labeled using peracetylated azido-labeled N-acetylgalactosamine and then reacted, in the first click reaction, with either a cyclooctyne, in a Huisgen [3 + 2] cycloaddition, or with a Staudinger phosphine, via Staudinger ligation. The second click reaction was a [4 + 2] inverse electron demand Diels-Alder reaction between a trans-cyclooctene and a tetrazine, where the latter reagent had been fluorescently labeled with a far-red fluorophore. After administration of the fluorescent tetrazine, the bifunctional cyclooctyne-cyclooctene produced significant azido sugar-dependent fluorescence labeling of tumor, kidney, liver, spleen, and small intestine in vivo, where the kidney and tumor could be imaged noninvasively in the live mouse

Unexpected enzyme-catalysed [4+2] cycloaddition and rearrangement in polyether antibiotic biosynthesis

Enzymes that catalyse remarkable Diels–Alder-like [4+2] cyclizations have been previously implicated in the biosynthesis of spirotetronate and spirotetramate antibiotics. Biosynthesis of the polyether antibiotic tetronasin is not expected to require such steps, yet the tetronasin gene cluster encodes enzymes Tsn11 and Tsn15, which are homologous to authentic [4+2] cyclases. Here, we show that deletion of Tsn11 led to accumulation of a late-stage intermediate, in which the two central rings of tetronasin and four of its twelve asymmetric centres remain unformed. In vitro reconstitution showed that Tsn11 catalyses an apparent inverse-electron-demand hetero-Diels–Alder-like [4+2] cyclization of this species to form an unexpected oxadecalin compound that is then rearranged by Tsn15 to form tetronasin. To gain structural and mechanistic insight into the activity of Tsn15, the crystal structure of a Tsn15-substrate complex has been solved at 1.7 Å resolution

Metabolic glycan imaging by isonitrile-tetrazine click chemistry.

Seeing the sugar coating: N-Acetyl-glucosamine and mannosamine derivatives tagged with an isonitrile group are metabolically incorporated into cell-surface glycans and can be detected with a fluorescent tetrazine. This bioorthogonal isonitrile-tetrazine ligation is also orthogonal to the commonly used azide-cyclooctyne ligation, and so will allow simultaneous detection of the incorporation of two different sugars

18F-C2Am: a targeted imaging agent for detecting tumor cell death in vivo using positron emission tomography.

INTRODUCTION: Trialing novel cancer therapies in the clinic would benefit from imaging agents that can detect early evidence of treatment response. The timing, extent and distribution of cell death in tumors following treatment can give an indication of outcome. We describe here an 18F-labeled derivative of a phosphatidylserine-binding protein, the C2A domain of Synaptotagmin-I (C2Am), for imaging tumor cell death in vivo using PET. METHODS: A one-pot, two-step automated synthesis of N-(5-[18F]fluoropentyl)maleimide (60 min synthesis time, > 98% radiochemical purity) has been developed, which was used to label the single cysteine residue in C2Am within 30 min at room temperature. Binding of 18F-C2Am to apoptotic and necrotic tumor cells was assessed in vitro, and also in vivo, by dynamic PET and biodistribution measurements in mice bearing human tumor xenografts treated with a TRAILR2 agonist or with conventional chemotherapy. C2Am detection of tumor cell death was validated by correlation of probe binding with histological markers of cell death in tumor sections obtained immediately after imaging. RESULTS: 18F-C2Am showed a favorable biodistribution profile, with predominantly renal clearance and minimal retention in spleen, liver, small intestine, bone and kidney, at 2 h following probe administration. 18F-C2Am generated tumor-to-muscle (T/m) ratios of 6.1 ± 2.1 and 10.7 ± 2.4 within 2 h of probe administration in colorectal and breast tumor models, respectively, following treatment with the TRAILR2 agonist. The levels of cell death (CC3 positivity) following treatment were 12.9-58.8% and 11.3-79.7% in the breast and colorectal xenografts, respectively. Overall, a 20% increase in CC3 positivity generated a one unit increase in the post/pre-treatment tumor contrast. Significant correlations were found between tracer uptake post-treatment, at 2 h post-probe administration, and histological markers of cell death (CC3: Pearson R = 0.733, P = 0.0005; TUNEL: Pearson R = 0.532, P = 0.023). CONCLUSION: The rapid clearance of 18F-C2Am from the blood pool and low kidney retention allowed the spatial distribution of cell death in a tumor to be imaged during the course of therapy, providing a rapid assessment of tumor treatment response. 18F-C2Am has the potential to be used in the clinic to assess early treatment response in tumors

Prodrugs of Pyrophosphates and Bisphosphonates: Disguising Phosphorus Oxyanions

Pyrophosphates have important functions in living systems and thus pyrophosphate-containing molecules and their more stable bisphosphonate analogues have the potential to be used as drugs for treating many diseases including cancer and viral infections. Both pyrophosphates and bisphosphonates are polyanionic at physiological pH and, whilst this is essential for their biological activity, it also limits their use as therapeutic agents. In particular, the high negative charge density of these compounds prohibits cell entry other than by endocytosis, prevents transcellular oral absorption and causes sequestration to bone. Therefore, prodrug strategies have been developed to temporarily disguise the charges of these compounds. This review examines the various systems that have been used to mask the phosphorus-containing moieties of pyrophosphates and bisphosphonates and also illustrates the utility of such prodrugs.Kwong Mei Medhealt