Flow Asymmetric Propargylation: Development of Continuous Processes for the Preparation of a Chiral ß-Amino Alcohol.

The development of a flow chemistry process for asymmetric propargylation using allene gas as a reagent is reported. The connected continuous process of allene dissolution, lithiation, Li-Zn transmetallation, and asymmetric propargylation provides homopropargyl ß-amino alcohol 1 with high regio- and diastereoselectivity in high yield. This flow process enables practical use of an unstable allenyllithium intermediate. The process uses the commercially available and recyclable (1S,2R)-N-pyrrolidinyl norephedrine as a ligand to promote the highly diastereoselective (32:1) propargylation. Judicious selection of mixers based on the chemistry requirement and real-time monitoring of the process using process analytical technology (PAT) enabled stable and scalable flow chemistry runs.

Chemoselective hydrosilylation of hydroxyketones.

A chemoselective method for the hydrosilylation of ketones has been developed, using the combination of triphenylsilane and a catalyst prepared from Ni(COD)2 and the simple N-heterocyclic carbene IMes. The most notable feature of this method is that free hydroxyls are largely unaffected, thus providing a simple one-step procedure for the conversion of hydroxyketones to mono-protected diols, wherein the protecting group is exclusively installed on the ketone-derived hydroxyl. The process is typically high yielding with both simple ketones and more complex hydroxyketone substrates.

A streamlined strategy for aglycone assembly and glycosylation.

Multipurpose sugars: Carbohydrate-derived silane reagents are utilized as the reductant for nickel-catalyzed aldehyde-alkyne reductive coupling reactions and as the glycosyl donor for subsequent intramolecular glycosylation. The approach enables the assembly of the carbon-carbon framework and stereochemical features of an aglycone while simultaneously establishing the site of glycosylation.

Synthesis of 7-oxo-dihydrospiro[indazole-5,4'-piperidine] acetyl-CoA carboxylase inhibitors.

Synthesis of oxo-dihydrospiroindazole-based acetyl-CoA carboxylase (ACC) inhibitors is reported. The dihydrospiroindazoles were assembled in a regioselective manner in six steps from substituted hydrazines and protected 4-formylpiperidine. Enhanced regioselectivity in the condensation between a keto enamine and substituted hydrazines was observed when using toluene as the solvent, leading to selective formation of 1-substituted spiroindazoles. The 2-substituted spiroindazoles were formed selectively from alkyl hydrazones by ring closure with Vilsmeier reagent. The key step in the elaboration to the final products is the conversion of an intermediate olefin to the desired ketone through elimination of HBr from an O-methyl bromohydrin. This methodology enabled the synthesis of each desired regioisomer on 50-75 g scale with minimal purification. Acylation of the resultant spirocyclic amines provided potent ACC inhibitors.

Truncation of MBD4 predisposes to reciprocal chromosomal translocations and alters the response to therapeutic agents in colon cancer cells.

We previously identified a novel genomic instability phenotype of multiple reciprocal chromosomal translocations in a MLH1-defective, microsatellite unstable (MSI) colon cancer cell line (HCA7) and, further, showed that it was unlikely to be directly caused by the mismatch repair (MMR) defect in this cell line. To gain insight into the molecular basis to this novel translocation phenotype, we examined coding and splice-site nucleotide repeat tracts in DNA repair genes for mutations by direct sequencing together with RT-PCR expression analysis of the associated transcript. The material was a selected panel of 8 MSI cell lines including HCA7. A strong candidate identified through this approach was MBD4 as it showed a homozygous truncating mutation associated with substantial loss of the transcript in HCA7 not seen in the other lines. In previous published studies, heterozygous MBD4 mutations were observed in up to 89% of sporadic MSI microdissected colon tumor foci. Using MFISH, we show that over-expression of the truncated MBD4 (+MBD4(tru)) in DLD1, a MSH6 defective, MSI human colon carcinoma cell line predisposed these cells to acquire structural chromosomal rearrangements including multiple reciprocal translocations after irradiation, reminiscent of those seen in HCA7. We also show that over-expression of MBD4(tru) in DLD1 alters the colony survival after exposure to cisplatin or etoposide. These data suggest a wide role for MBD4 in DNA damage response and maintaining chromosomal stability.

Ketone hydrosilylation with sugar silanes followed by intramolecular aglycone delivery: an orthogonal glycosylation strategy.

Gettin' a little sugar-no alcohol required: A procedure for the direct glycosylation of ketones without a hydroxy intermediate enables the site-selective glycosylation of hydroxyketones at the ketone or the alcohol functionality without the use of protecting groups on the aglycone (see scheme). Site selectivity is controlled by the catalyst structure in hydrosilylation and dehydrogenative silylation reactions with sugar silanes. Bn=benzyl.

The identification of a novel alternatively spliced form of the MBD4 DNA glycosylase.

Methyl-CpG binding protein 4 (MBD4) is a mismatch-specific G:T and G:U DNA glycosylase. During an analysis of MBD4 expression in HeLa cells we noted the presence of an unexpectedly short reverse transcribed product. This cDNA lacked the region encoding the methyl-binding domain and exon 3 of MBD4 but retained the glycosylase domain. Sequence comparison indicates the existence of a previously unreported cryptic splice site in the MBD4 genomic sequence thus illuminating a mechanism whereby a glycosylase acquired a methyl-binding capacity, thus targeting potential mutagenic CpG sites. In vitro assays of this highly purified species, refolded in arginine rich conditions, confirmed that this unique, short version of MBD4 possessed uracil DNA glycosylase but not thymine DNA glycosylase activity. We conclude that the identification of a transcript encoding a short version of MBD4 indicates that MBD4 expression may be more complex than previously reported, and is worthy of further investigation.

MBD1, MBD2 and CGBP genes at chromosome 18q21 are infrequently mutated in human colon and lung cancers.

The genes MBD1 and MBD2 encode methyl-CpG binding proteins that suppress transcription from methylated promoters. In contrast, CGBP encodes a protein that binds promoters containing unmethylated CpG and stimulates transcription. All three are located on human chromosome 18q21, a region of frequent loss of heterozygosity in several cancers. These genes therefore represent candidate tumour suppressor genes, whose loss of function could affect the normal regulation of gene expression, whether by lack of complete suppression of genes normally silenced (via loss of MBD1 and MBD2) or by some loss of activation of genes normally expressed (via loss of CGBP), either way contributing to the tumorigenic phenotype. We have confirmed by fluorescent in situ hybridization that MBD1 and MBD2 bracket the DCC locus giving a gene order of MBD1/CGBP-DCC 5'-DCC 3'-MBD2. Mutation analyses by single-stranded conformation polymorphism in colon and lung cancer cell lines and primary tumours revealed a small number of mutations, suggesting only a limited role of these genes in human tumorigenesis.

Positional and functional mapping of a neuroblastoma differentiation gene on chromosome 11.

BACKGROUND: Loss of chromosome 11q defines a subset of high-stage aggressive neuroblastomas. Deletions are typically large and mapping efforts have thus far not lead to a well defined consensus region, which hampers the identification of positional candidate tumour suppressor genes. In a previous study, functional evidence for a neuroblastoma suppressor gene on chromosome 11 was obtained through microcell mediated chromosome transfer, indicated by differentiation of neuroblastoma cells with loss of distal 11q upon introduction of chromosome 11. Interestingly, some of these microcell hybrid clones were shown to harbour deletions in the transferred chromosome 11. We decided to further exploit this model system as a means to identify candidate tumour suppressor or differentiation genes located on chromosome 11. RESULTS: In a first step, we performed high-resolution array CGH DNA copy-number analysis in order to evaluate the chromosome 11 status in the hybrids. Several deletions in both parental and transferred chromosomes in the investigated microcell hybrids were observed. Subsequent correlation of these deletion events with the observed morphological changes lead to the delineation of three putative regions on chromosome 11: 11q25, 11p13-->11p15.1 and 11p15.3, that may harbour the responsible differentiation gene. CONCLUSION: Using an available model system, we were able to put forward some candidate regions that may be involved in neuroblastoma. Additional studies will be required to clarify the putative role of the genes located in these chromosomal segments in the observed differentiation phenotype specifically or in neuroblastoma pathogenesis in general.