Christopher J. Hayes

Identification of Internal Ribosome Entry Segment (IRES)-trans-Acting Factors for the Myc Family of IRESs‡

ABSTRACT The proto-oncogenes c-, L-, and N-myc can all be translated by the alternative method of internal ribosome entry whereby the ribosome is recruited to a complex structural element (an internal ribosome entry segment [IRES]). Ribosome recruitment is dependent upon the presence of IRES-trans-acting factors (ITAFs) that act as RNA chaperones and allow the mRNA to attain the correct conformation for the interaction of the 40S subunit. One of the major challenges for researchers in this area is to determine whether there are groups of ITAFs that regulate the IRES-mediated translation of subsets of mRNAs. We have identified four proteins, termed GRSF-1 (G-rich RNA sequence binding factor 1), YB-1 (Y-box binding protein 1), PSF (polypyrimidine tract binding protein-associated splicing factor), and its binding partner, p54nrb, that bind to the myc family of IRESs. We show that these proteins positively regulate the translation of the Myc family of oncoproteins (c-, L-, and N-Myc) in vivo and in vitro. Interestingly, synthesis from the unrelated IRESs, BAG-1 and Apaf-1, was not affected by YB-1, GRSF-1, or PSF levels in vivo, suggesting that these three ITAFs are specific to the myc IRESs. Myc proteins play a role in cell proliferation; therefore, these results have important implications regarding the control of tumorigenesis.

Redirection of carotenoid metabolism for the efficient production of taxadiene [taxa-4(5),11(12)-diene] in transgenic tomato fruit

The taxanes are a group of polycyclic diterpenes produced by various species of yew. The potent anticancer drug paclitaxel (marketed as Taxol™) is the commercially most important taxane with annual sales in 2000 exceeding

The ‘Hirao reduction’ revisited: a procedure for the synthesis of terminal vinyl bromides by the reduction of 1,1-dibromoalkenes

1.6 billion. Paclitaxel is currently obtained either by direct extraction from yew trees or by the extraction of the precursor 10-deactilbaccatin III, which is then converted to paclitaxel by semi-synthesis. Apart from cost, one of the main draw backs to taxol in cancer treatment is the development of resistance by tumours, commonly due to the expression of ABC transporter efflux pumps which remove the drug from the target cell. A number of natural taxanes and semisynthetic derivates, have recently been shown to act as potent inhibitors of ABC transport proteins. These compounds have no effect upon microtubule polymerization (the normal target of paclitaxel), but have the ability to restore drug sensitivity when given in combination with paclitaxel to resistant cell lines. In work to be described elsewhere, we sort to carry out a structure function analysis of the ability of novel oxidised taxanes to act as ABC transporter inhibitors. For this study 100 mg or more of taxadiene [taxa-4(5),11(12)-diene], the first taxane in the paclitaxel pathway, was required as starting material from which to synthesize these compounds. Taxadiene is synthesised directly from geranylgeranyl diphosphate (GGPP), which is found in most plant tissues where it serves as a common precursor for many metabolites. The synthesis and use of GGDP are tightly regulated in most vegetative organs, however, in tomato fruit it is used almost exclusively for the production of coloured carotenoids which accumulate to high levels in the plastid as lycopene crystals. Expressing taxadiene synthase in a yellow-fruited tomato line that lacks the ability to utilise GGPP for carotenoid synthesis allowed GGPP normally utilised for making carotenoids to be re-routed for the production of taxadiene, allowing the facile extraction of 160 mg of highly pure taxadiene from 1 kg of freeze dried fruit.

Rapid access to α-alkoxy and α-amino acid derivatives through safe continuous-flow generation of diazoesters.

Abstract A convenient procedure for the synthesis of vinyl bromides is described, which involves the selective reduction of the corresponding 1,1-dibromoalkenes with dimethylphosphite and triethylamine. The 1,1-dibromoalkenes are obtained in excellent yields from the corresponding aldehydes via olefination with carbon tetrabromide and triphenylphosphine.

Enantioselective Total Syntheses of Omuralide, 7-epi-Omuralide, and (+)-Lactacystin

Despite the wide synthetic potential of diazo compounds (X H insertion, ylide formation, cyclopropanation, cycloaddition etc.), concerns over the hazards associated with their preparation, isolation, and use have hindered their full exploitation in both academic and industrial laboratories. A few diazo compounds are commercially available (e.g. ethyl and butyl diazoacetate, TMS-diazomethane and diazodimedone), but safe and convenient access to a wider range of useful functionalized diazo species is still desirable. Diazo transfer can be used to access a-diazocarbonyls, but this only partially addresses the safety concerns associated with the diazo species, as the use of equally hazardous azidebased diazo-transfer reagents is still required. Ideally, it would be beneficial if the diazo species could be generated and consumed in situ so that handling of the hazardous diazo compound is avoided altogether. Recent work by Ley, Jamison, Kappe, and others has shown that highly reactive diazo and azido compounds can be used in lab-scale continuous-flow reactors to achieve a number of very useful synthetic transformations, and indeed work from our own laboratory has shown that ethyl diazoacetate can be used in-flow to access b-keto esters. We therefore wondered if it was possible to actually generate a-diazocarbonyl compounds under flow conditions and then use these materials directly in further synthetic manipulations, thus minimizing exposure to any potentially hazardous material. In effect, could we develop a continuous-flow diazo generator and then demonstrate its use to prepare a range of useful a-alkoxy (3 a–i) and aamino acid (4 a–i) derivatives through O H and N H insertion (Scheme 1)? At the outset we were aware that in order to provide an acceptable solution to the problem, we needed to identify a way to access the diazo compounds of interest (2 a–i) from starting materials that showed an acceptable safety profile, that is, the precursor molecules and reagents should be safer to prepare and handle than the diazocarbonyl compounds being produced. Of the methods available for the generation of a-diazocarbonyl compounds, we were particularly attracted to the Bamford–Stevens reaction as it uses readily accessible arylsulfonylhydrazones (e.g., 1 a–i) as starting materials, with the corresponding diazocarbonyls being generated upon exposure to relatively weak base at moderate reaction temperatures. Thermal stability studies (DSC and TGA) were conducted on the tosylhydrazone 1 b and its corresponding methyl diazoester 2 b 16] in order to determine if a safe window of operation could be identified for the continuous-flow process (see the Supporting Information). The results clearly show that the rate of initial mass loss from diazoester 2 b peaks at 125 8C, which corresponds to a significant exotherm. In comparison tosylhydrazone 1 b has a rate of mass loss which peaks at 221 8C, indicating that it is substantially more thermally stable. We therefore concluded that there would be significant safety benefits in adapting the Bamford–Stevens reaction for use in-flow to produce diazoesters, which in turn could be utilized immediately in subsequent transformations without needing to be isolated or purified. Reassured by these data, a wider range of arylsulfonylhydrazones 1 a–i was readily prepared from simple and inexpensive starting materials (Scheme 2). [a] Dr. H. E. Bartrum, Prof. Dr. C. J. Moody, Prof. Dr. C. J. Hayes School of Chemistry, University of Nottingham University Park, Nottingham, NG7 2RD (UK) Fax: (+44) 115-951-3564 E-mail : c.j.moody@nottingham.ac.uk chris.hayes@nottingham.ac.uk [b] Dr. D. C. Blakemore Pfizer Global Research and Development Ramsgate Road, Sandwich, Kent, CT13 9NJ (UK) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201101590. Scheme 1. Proposed flow process for diazoester synthesis. CFC =convection flow coil reactor, BPR=back-pressure regulator, oct=octanoate.

An enantioselective formal synthesis of the proteasome inhibitor (+)-lactacystin

An alkylidene carbene 1,5-CH insertion has been used as a key step in an enantioselective total syntheses of omuralide, its C7-epimer, and (+)-lactacystin. An additional noteworthy feature of the synthesis is the use of a novel oxidative deprotection procedure, utilizing DMDO, for the conversion of a late-stage benzylidene acetal into a primary alcohol and a secondary benzoate ester.

Synthetic studies towards phomactin A. Concise synthesis of the novel tricyclic furanochroman system

Abstract An enantioselective formal synthesis of the proteasome inhibitor (+)-lactacystin has been achieved using an alkylidene carbene 1,5-CH insertion reaction as a key step. The key cyclisation precursor was synthesised in high diastereomeric excess using a combination of known procedures, with the two key asymmetric centres being introduced via a Sharpless asymmetric epoxidation reaction. KHMDS induced 1,5-CH insertion produced a 3-pyrroline product, which was oxidised to the corresponding 3-pyrrolin-2-one using (1) TPAP/NMO; (2) NaClO 2 ; (3) NaBH 4 . The formal synthesis was then completed with a few standard functional group interconversions.

Synthesis of β-Keto Esters In-Flow and Rapid Access to Substituted Pyrimidines

A concise synthesis of the tricyclic furanochroman unit 2 found in the unusual PAF antagonist phomactin A 1 is described. The synthesis is based on elaboration of the novel dihydrofuran enol ether 3 as key intermediate via the iodopyran 6 or the dihydrofuran 20. Treatment of 3 with dimethyldioxirane in acetone-water then gave the furanochroman 2.

High-content screening identifies small molecules that remove nuclear foci, affect MBNL distribution and CELF1 protein levels via a PKC-independent pathway in myotonic dystrophy cell lines

We have developed an in-flow process for the synthesis of β-keto esters via the BF(3)·OEt(2)-catalyzed formal C-H insertion of ethyl diazoacetate into aldehydes. The β-keto esters were then condensed with a range of amidines to give a variety of 2,6-substituted pyrimidin-4-ols.

Enantioselective total syntheses of (−)-clasto-lactacystin β-lactone and 7-epi-(−)-clasto-lactacystin β-lactone

Myotonic dystrophy (DM) is a multi-system neuromuscular disorder for which there is no treatment. We have developed a medium throughput phenotypic assay, based on the identification of nuclear foci in DM patient cell lines using in situ hybridization and high-content imaging to screen for potentially useful therapeutic compounds. A series of further assays based on molecular features of DM have also been employed. Two compounds that reduce and/or remove nuclear foci have been identified, Ro 31-8220 and chromomycin A3. Ro 31-8220 is a PKC inhibitor, previously shown to affect the hyperphosphorylation of CELF1 and ameliorate the cardiac phenotype in a DM1 mouse model. We show that the same compound eliminates nuclear foci, reduces MBNL1 protein in the nucleus, affects ATP2A1 alternative splicing and reduces steady-state levels of CELF1 protein. We demonstrate that this effect is independent of PKC activity and conclude that this compound may be acting on alternative kinase targets within DM pathophysiology. Understanding the activity profile for this compound is key for the development of targeted therapeutics in the treatment of DM.