69 research outputs found
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
Biosynthesis of the antifungal haterumalide, oocydin A, in Serratia, and its regulation by quorum sensing, RpoS and Hfq.
Polyketides represent an important class of bioactive natural products with a broad range of biological activities. We identified recently a large trans-acyltransferase (AT) polyketide synthase gene cluster responsible for the biosynthesis of the antifungal, anti-oomycete and antitumor haterumalide, oocydin A (ooc). Using genome sequencing and comparative genomics, we show that the ooc gene cluster is widespread within biocontrol and phytopathogenic strains of the enterobacteria, Serratia and Dickeya. The analysis of in frame deletion mutants confirmed the role of a hydroxymethylglutaryl-coenzyme A synthase cassette, three flavin-dependent tailoring enzymes, a free-standing acyl carrier protein and two hypothetical proteins in oocydin A biosynthesis. The requirement of the three trans-acting AT domains for the biosynthesis of the macrolide was also demonstrated. Expression of the ooc gene cluster was shown to be positively regulated by an N-acyl-L-homoserine lactone-based quorum sensing system, but operating in a strain-dependent manner. At a post-transcriptional level, the RNA chaperone, Hfq, plays a key role in oocydin A biosynthesis. The Hfq-dependent regulation is partially mediated by the stationary phase sigma factor, RpoS, which was also shown to positively regulate the synthesis of the macrolide. Our results reveal differential regulation of the divergently transcribed ooc transcriptional units, highlighting the complexity of oocydin A production.This research was supported by the EU Marie-Curie Intra-European Fellowship for Career Development (FP7-PEOPLE-2011-IEF) Grant No. 298003. The Salmond laboratory is supported by funding through the Biotechnology and Biological Sciences Research Council, BBSRC (UK). Work with plant pathogens was carried out under DEFRA Licence No. 50864/197900/1.This is the final version of the article. It first appeared from Wiley via http://dx.doi.org/10.1111/1462-2920.1283
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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
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Substrate flexibility of the flavin-dependent dihydropyrrole oxidases PigB and HapB involved in antibiotic prodigiosin biosynthesis
In the biosynthesis of the tripyrrolic pigment prodigiosin, PigB is a predicted flavin-dependent oxidase responsible for formation of 2-methyl-3-amylpyrrole (MAP) from a dihydropyrrole. To prove which dihydropyrrole is the true intermediate, both possibilities, 5a (resulting from transamination of the aldehyde of 3-acetyloctanal) and 6 (resulting from transamination of the ketone), were synthesised. Only 5a restored pigment production in a strain of Serratia sp. ATCC 39006 blocked earlier in MAP biosynthesis. PigB is membrane-associated and inactive when its transmembrane domain was deleted, but HapB, its homologue in Hahella chejuensis, lacks the transmembrane domain and is active in solution. Two colorimetric assays for PigB and HapB were developed, and the HapB-catalysed reaction was kinetically characterised. Ten analogues of 5a were synthesised, varying in the C2 and C3 side-chains, and tested as substrates of HapB in vitro and for restoration of pigment production in Serratia ΔpigD in vivo. All lengths of side-chain tested at C3 were accepted but only short side-chains at C2 were accepted. The knowledge that 5a is an intermediate in prodigiosin biosynthesis and the ease of synthesis of analogues of 5a makes a range of prodigiosin analogues readily available by mutasynthesis.We acknowledge the Frances and Augustus Newman foundation, the Cambridge Commonwealth Trust, Emmanuel College, Cambridge, and the B.B.S.R.C. (award codesBB/N008081/1 and BB/K001833/1) for funding this research
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
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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
Investigating intermediates in 6-methylsalicylic acid biosynthesis
6-Methylsalicylic acid (6-MSA) is one of the oldest known polyketides. It is synthesised in vivo by the polyketide synthase 6-methylsalicylic acid synthase (6-MSAS), a multifunctional enzyme which uses its active sites iteratively. The stereochemistry of the hydroxyl produced from the single ketoreduction, as well as the order of dehydration, cyclisation and aromatisation steps, remain cryptic, despite extensive study. Holo 6-MSAS was heterologously expressed in E. coli and purified in two steps. A non-hydrolysable carba(dethia)malonyl-N-acetylcysteamine analogue was synthesised and used to off-load enzyme-bound intermediates from 6-MSAS. In assays with acetyl-CoA and acetoacetyl-CoA alone, diketide and triketide intermediates were off-loaded and detected by HPLC-HR-ESI-MS. In the presence of NADPH, the off-loaded triketide was reduced by the ketoreductase domain of 6-MSAS. A potential dehydrated intermediate was also observed. The dehydratase domain of 6-MSAS has recently been reassigned as a thioester hydrolase. To test this theory, the catalytic histidine residue in 6-MSAS was mutated to an alanine and the abolition of production of 6-MSA in vivo was observed. Mutated 6-MSAS was still able to produce the shunt product triacetic acid lactone. Incubation of mutated 6-MSAS with acetyl-CoA, malonyl-CoA, NADPH and carba(dethia)malonyl-N-acetylcysteamine saw only the off-loading of diketide and triketide analogues. To investigate the stereochemistry of ketoreduction in 6-MSA biosynthesis, steps were made to synthesise the resolved diastereomeric reduced-triketide CoAs which would be the substrates for the ketoreductase domain. Attempts to phosphopantetheinylate apo 6-MSAS in vitro with three different phosphopantetheinyltransferases were unsuccessful. Limited proteolysis of both holo and apo 6-MSAS found that the apo synthase rapidly lost a C-terminal fragment while holo 6-MSAS was much more stable under the same conditions. Attempts were made to express the acyl carrier protein domain from 6-MSAS to overcome these problems. These experiments represent the first use of the non-hydrolysable analogue methodology in a Type I iterative polyketide synthase and provide a framework for future experiments investigating intermediates in the biosynthesis of 6-MSA.EThOS - Electronic Theses Online ServiceGBUnited Kingdo
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