Development of bridged nucleic acid analogues for antigene technology

In the last decade, increased efforts have been directed toward the development of oligonucleotide-based technologies for genome analyses, diagnostics, or therapeutics. Among them, an antigene strategy is one promising technology to regulate gene expression in living cells. Stable triplex formation between the triplex-forming oligonucleotide (TFO) and the target double-stranded DNA (dsDNA) is fundamental to the antigene strategy. However, there are two major drawbacks in triplex formation by a natural TFO: low stability of the triplex and limitations of the target DNA sequence. To overcome these problems, we have developed various bridged nucleic acids (BNAs), and found that the 2',4'-BNA modification of oligonucleotides strongly promotes parallel motif triplex formation under physiological conditions. Some nucleobase analogues to extend the target DNA sequence were designed, synthesized, and introduced into the 2',4'-BNA structure. The obtained 2',4'-BNA derivatives with unnatural nucleobases effectively recognized a pyrimidine-purine interruption in the target dsDNA. Some other examples of nucleic acid analogues for stable triplex formation and extension of the target DNA sequence are also summarized.     

Synthesis of difluorinated carbocyclic analogues of 5-deoxypentofuranoses

The synthesis of difluorinated carbocyclic analogues of 5-deoxypentofuranoses is described. The sequence involves an addition of PhSeCF₂TMS to carbohydrate-derived aldehydes followed by a radical cyclization, and provides a secure strategy for a future synthesis of pentofuranoses and nucleoside analogues.     

Soluble and polymer-supported 2- and 3-benzylated furans for the preparation of α,β-ethylenic carbonyl compounds

Soluble and polymer-supported 2- and 3-benzylated furans were subjected to a sequence involving a Diels-Alder reaction with α,β-acetylenic carbonyl compounds, a Michael addition, and a subsequent retro-Diels-Alder reaction to yield olefinic compounds. On solid support, this traceless strategy is advantageous since pure compounds were released in the thermal cycloreversion step. The fur-2-ylated resin allowed a highly diastereoselective synthesis.     

Skeletal and appendage diversity as design elements in the synthesis of a discovery library of nonaromatic polycyclic 5-iminooxazolidin-2-ones, hydantoins, and acylureas

Amino-acid derived cross-conjugated trienes were used as a starting point for the synthesis of a discovery library of over 200 polycyclic 5-iminooxazolidin-2-ones, hydantoins, and acylureas. The main feature of this library synthesis is a triple branching strategy, which provides efficient access to five skeletally diverse scaffolds. In addition, four sets of building blocks were applied in both a front end and a back end diversification strategy. Multiple fused rings were obtained by cyclization of diamides with phosgene and stereoselective Diels-Alder reactions with maleimides. The 5-iminooxazolidin-2-one scaffold was rearranged into the isomeric hydantoin scaffold through a sequence of ring-opening and ring-closing reactions.     

C−F Arylation of Polyfluorophenols by Means of Sigmatropic Dearomatization/Defluorination Sequence

Selective C−F arylation of polyfluorophenols with aryl sulfoxides has been accomplished by means of a sigmatropic dearomatization/defluorination sequence. This sequence consists of three processes: 1) interrupted Pummerer reaction to form S−O-tethered sulfonium salt; 2) C−C-forming [3,3] sigmatropic rearrangement with dearomatization; and 3) Zn-mediated defluorinative rearomatization. The present biaryl construction provides a facile access to polyfluorinated biaryls that is difficult to synthesize by other methods. The synthetic utility of the strategy is clearly demonstrated by the synthesis of a fluorinated analogue of Maxipost, a potassium channel modulator.     

Sequence Control in Polymer Chemistry through the Passerini Three‐Component Reaction

A new strategy to achieve sequence control in polymer chemistry based on the iterative application of the versatile Passerini three‐component reaction (P‐3CR) in combination with efficient thiol–ene addition reactions is introduced. First, stearic acid was used as a starting substrate to build up a sequence‐defined tetramer with a molecular weight of 1.6 kDa. Using an acid‐functionalized PEG allowed for an easier isolation of the sequence‐defined macromolecules by simple precipitation and led to a sequence‐defined pentamer in a block‐copolymer architecture. Importantly, this new strategy completely avoids protecting group chemistry. By following this strategy, a different side chain can be introduced to the polymer/oligomer backbone in a simple way and at a defined position within the macromolecule.     

Reaction based fluorescent probes for hydrogen sulfide

A reaction based fluorescence turn-on strategy for hydrogen sulfide (H(2)S) was developed. This strategy was based on a H(2)S-specific Michael addition-cyclization sequence. Other biological thiols such as cysteine and glutathione did not pursue the reaction and therefore did not turn on the fluorescence/consume the substrates. The probes showed good selectivity and sensitivity for hydrogen sulfide.     

Dual-labeled oligonucleotide probe for sensing adenosine via FRET: a novel alternative to SNPs genotyping

A novel FRET based strategy for DNA sequence analysis utilising base-discriminating fluorescence (BDF) nucleoside, (Py)U/(2-Ant)U, as donor in the dual-labelled oligonucleotide probe is reported; a selective/specific emission from acceptor, was observed upon excitation at the donor, only when the opposite base of the "smart" fluorescently labeled BDF nucleoside, (Py)U/(2-Ant)U, is adenine on the complementary target sequence.     

Sequence, structure, and active site analyses of p38 MAP kinase: exploiting DFG-out conformation as a strategy to design new type II leads

A new knowledge, structure, and sequence based strategy involving the effective exploitation of the DFG-out conformation is delineated. A comprehensive analysis of the structure, sequence, cocrystals, and active sites of p38 MAP kinase crystal structures present in Protein Data Bank (PDB) and the FDA approved MAP kinase drugs has been done, and the information is used for the design of type II leads. The 98 crystal structures, 138 cocrystals, and 31 FDA drugs comprise of 7 different sequences of 2 organisms viz., Homo sapiens and Mus musculus differing in sequence length, constituting both homo- and heterochains. Multiple sequence alignment with ClustalW showed >95% sequence similarity with highly conserved domains and a high propensity for mutations in the activation loop. The bound ligands were extracted, and their interactions with DFG in and out conformations were studied. These cocrystals and FDA drugs were fragmented on the basis of their binding interactions and their affinity to ATP and allosteric sites. The fragment library thus generated contains 106 fragments with overlapping drug fragments. A blue print constituting three main parts viz., head (ATP region), linker (DFG region), and tail (allosteric region) has thus been formulated and used to design 64 type II p38 MAP kinase inhibitors. The above strategy has been employed to design potent type II p38 MAP kinase inhibitors, which are shown to be very promising.     

Synthesis of polycyclic benzofused nitrogen heterocycles via a tandem ynamide benzannulation/ring-closing metathesis strategy. Application in a formal total synthesis of (+)-FR900482

A two-stage "tandem strategy" for the synthesis of benzofused nitrogen heterocycles is described that is particularly useful for the construction of systems with a high level of substitution on the benzenoid ring. The first stage in the strategy involves a benzannulation based on the reaction of cyclobutenones with ynamides. This cascade process proceeds via a sequence of four pericyclic reactions and furnishes a multiply substituted aniline derivative which can bear a variety of functionalized substituents at the position ortho to the nitrogen. In the second stage of the tandem strategy, ring-closing metathesis generates the nitrogen heterocyclic ring. This two-step sequence provides efficient access to highly substituted dihydroquinolines, benzazepines, benzazocines, and related benzofused nitrogen heterocyclic systems. The application of this chemistry in a concise formal total synthesis of the anticancer agents (+)-FR900482 and (+)-FR66979 is described.     

2‐Methoxy‐4‐methylsulfinylbenzyl: A Backbone Amide Safety‐Catch Protecting Group for the Synthesis and Purification of Difficult Peptide Sequences

The use of 2‐methoxy‐4‐methylsulfinylbenzyl (Mmsb) as a new backbone amide‐protecting group that acts as a safety‐catch structure is proposed. Mmsb, which is stable during the elongation of the sequence and trifluoroacetic acid‐mediated cleavage from the resin, improves the synthetic process as well as the properties of the quasi‐unprotected peptide. Mmsb offers the possibility of purifying and characterizing complex peptide sequences, and renders the target peptide after NH₄I/TFA treatment and subsequent ether precipitation to remove the cleaved Mmsb moiety. First, the “difficult peptide” sequence H‐(Ala)₁₀‐NH₂ was selected as a model to optimize the new protecting group strategy. Second, the complex, bioactive Ac‐(RADA)₄‐NH₂ sequence was chosen to validate this methodology. The improvements in solid‐phase peptide synthesis combined with the enhanced solubility of the quasi‐unprotected peptides, as compared with standard sequences, made it possible to obtain purified Ac‐(RADA)₄‐NH₂. To extend the scope of the approach, the challenging Aβ(1‐42) peptide was synthesized and purified in a similar manner. The proposed Mmsb strategy opens up the possibility of synthesizing other challenging small proteins.     

A micropreparative chromatographic strategy for the purification and sequence analysis of murine granulocyte-colony stimulating factor (mG-CSF)

Summary A micropreparative chromatographic strategy using short (<5 cm) 1 and 2 mm ID HPLC columns for preparing pure mG-CSF and its peptide fragments is described. The proteins and peptides are used to obtain both N-terminal and internal amino acid sequence data for the purpose of constructing sequence specific oligonucleotide probes for use in the isolation of the gene coding for the protein of interest, thereby ultimately enabling amplified amounts of protein to be generated using prokaryotic expression systems.     

MALDI-TOF/TOF de novo sequence analysis of 2-D PAGE-separated proteins from Halorhodospira halophila, a bacterium with unsequenced genome

Because protein identifications rely on matches with sequence databases, high-throughput proteomics is currently largely restricted to those species for which comprehensive sequence databases are available. The identification of proteins derived from organisms with unsequenced genomes mainly depends on homology searching. Here, we report the use of a simplified, gel-based, chemical derivatization strategy for de novo sequence analysis using a MALDI-TOF/TOF mass spectrometer. This approach allows the determination of de novo peptide sequences of up to 20 amino acid residues in length. The protocol was applied on a proteomic study of 2-D PAGE-separated proteins from Halorhodospira halophila, an extremophilic eubacterium with yet unsequenced genome. Using three different homology-based search algorithms, we were able to identify more than 30 proteins from this organism using subpicomole quantities of protein.     

Mixed-element capture agents: a simple strategy for the construction of synthetic,high-affinity protein capture ligands

Demonstration of a simple strategy to generate synthetic high-affinity protein capture agents of practical utility for protein-detecting microarrays. The model study highlights capture of the MBP-Mdm2 fusion protein on a solid support by a linear sequence of peptides that bind to the two individual polypeptide chains.     

Sequence confirmation of chemically modified RNAs using exonuclease digestion and matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

A broadly applicable, robust, and rapid method for complete sequence confirmation of highly modified oligonucleotides containing a mixture of 2′‐deoxy, 2′‐fluoro, 2′‐o‐methyl, abasic and ribonucleotides is presented. The passenger (sense) and guide (antisense) strands from synthetic short interfering RNA duplexes (siRNA) were digested individually using both 5′‐ and 3′‐exonucleases and the resulting ladders were analyzed using matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectrometry. Conditions for enzymatic digestion and MALDI‐TOF mass analysis were investigated and optimized, and the digestion pattern and sequence coverage of each strand was discussed. Complete sequence confirmation for the antisense strands of four synthetic RNA duplexes was obtained, whereas a three‐base sequence gap in the 5′‐end was observed for all four sense strands. A general strategy is proposed for routine sequence confirmation of highly modified oligonucleotides, and the potential for complete automation of the method is also discussed. Copyright © 2009 John Wiley & Sons, Ltd.     

Novel strategy for the design of a new zinc finger: creation of a zinc finger for the AT-rich sequence by alpha-helix substitution

In this communication, a novel strategy for the design of a zinc finger peptide on the basis of alpha-helix substitution has been demonstrated. Sp1HM is a helix-substituted mutant for the wild-type Sp1(zf123) and its alpha-helix of each finger is replaced by that of fingers 4-6 of CF2-II. The circular dichroism spectrum of Sp1HM suggests that Sp1HM has an ordered secondary structure similar to that of Sp1(zf123). From the analyses of the DNA binding affinity and specificity by gel mobility shift assay, it is clearly indicated that Sp1HM specifically binds to the AT-rich sequence (5'-GTA TAT ATA-3') with 3.2 nM dissociation constants. Moreover, the zinc finger peptides for the sequence alternating between the AT- and GC-rich subsites can also be created by the alpha-helix substitution. This strategy is evidently effective and is also more convenient than the phage display method. Consequently, our design method is widely applicable to creating zinc finger peptides with novel binding specificities.     

An efficient approach to Aspidosperma alkaloids via [4 + 2] cycloadditions of aminosiloxydienes: stereocontrolled total synthesis of (+/-)-tabersonine. Gram-scale catalytic asymmetric syntheses of (+)-tabersonine and (+)-16-methoxytabersonine. Asymmetric syntheses of (+)-aspidospermidine and (-)-quebrachamine

Described is a concise, highly stereocontrolled strategy to the Aspidosperma family of indole alkaloids, one that is readily adapted to the asymmetric synthesis of these compounds. The strategy is demonstrated by the total synthesis of (+/-)-tabersonine (rac-1), proceeding through a 12-step sequence. The basis for this approach was provided by a highly regio- and stereoselective [4 + 2] cycloaddition of 2-ethylacrolein with 1-amino-3-siloxydiene developed in our laboratory. Subsequent elaboration of the initial adduct into the hexahydroquinoline DE ring system was accomplished efficiently by a ring-closing olefin metathesis reaction. A novel ortho nitrophenylation of an enol silyl ether with (o-nitrophenyl)phenyliodonium fluoride was developed to achieve an efficient, regioselective introduction of the requisite indole moiety. The final high-yielding conversion of the ABDE tetracycle into pentacyclic target rac-1 relied on intramolecular indole alkylation and regioselective C-carbomethoxylation. Our approach differs strategically from previous routes and contains built-in flexibility necessary to access many other members of the Aspidosperma family of indole alkaloids. The versatility of the synthetic strategy was illustrated through the asymmetric syntheses of the following Aspidosperma alkaloids: (+)-aspidospermidine, (-)-quebrachamine, (-)-dehydroquebrachamine, (+)-tabersonine, and (+)-16-methoxytabersonine. Of these, (+)-tabersonine and (+)-16-methoxytabersonine were synthesized in greater than 1-g quantities and in enantiomerically enriched form ( approximately 95% ee). The pivotal asymmetry-introducing step was a catalyzed enantioselective Diels-Alder reaction, which proceeded to afford the cycloadducts in up to 95% ee. Significantly, the synthetic sequence was easy to execute and required only four purifications over the 12-step synthetic route.     

A real-time decoding sequencing based on dual mononucleotide addition for cyclic synthesis

We propose a real-time decoding sequencing strategy in which a template is determined without directly measuring base sequence but by decoding two sets of encodings obtained from two parallel sequencing runs. This strategy relies on adding a mixture of different two-base pair, A + G, C + T, A + C, G + T, A + T or C + G (abbreviated as AG, CT, AC, GT, AT, or CG), into the reaction each time. When a template is cyclically interrogated twice with any two kinds of dual mononucleotide addition (AG/CT, AC/GT, and AT/CG), two sets of encodings are obtained sequentially. The two sets of encodings allow for the bases to be sequentially decoded, moving from first to last, in a deterministic manner. This strategy applies fewer cycles to obtain longer read length compared to the traditional real-time sequencing strategy [1]. Partial rnpB gene was applied to verify the applicability of the decoding strategy via pyrosequencing. The results indicated that the sequence could be reconstructed by decoding two sets of encodings. Moreover, streptococcal strains could be differentiated by comparing signal intensity in each cycle and encoding size of each template. This strategy is likely to be applied to differentiate nucleic acid sequence as encoding size and signal intensity in each cycle vary with the base size and composition. Furthermore, it has the potential in building a promising strategy that could be utilized as an alternative to conventional sequencing systems.     

Directed Metalation?Cross‐Coupling Strategies. Total Syntheses of the Alleged and the Revised Phenanthrene Natural Product Gymnopusin

The total synthesis of gymnopusin ( 2 ) is described. The originally assigned structure for gymnopusin 1a was found to be incorrect by total synthesis using the Directed ortho‐Metalation (DoM)? Cross‐Coupling? Directed remote Metalation (DreM) sequence, a demonstrable key strategy for the regioselective construction of the 9‐phenanthrol core. The revised structure of gymnopusin ( 2 ) was confirmed by synthesis by adopting the same strategy but involving a key remote anionic Fries‐rearrangement step. Both routes highlight methodologies and concepts which may be of value in the regiocontrolled synthesis of phenanthrenoids specifically and in complex polycyclic aromatics in general.     

A unified synthetic strategy toward oroidin-derived alkaloids premised on a biosynthetic proposal

Details of the evolution of a synthetic strategy toward the spirocyclic chlorocyclopentane core of oroidin-derived alkaloids, including the axinellamines and potentially adaptable to palau'amine, are described. A proposed refinement of the Kinnel-Scheuer biosynthetic proposal for palau'amine is posited. Studies undertaken to improve the regioselectivity and efficiency of a key Diels-Alder reaction utilizing a novel protecting group strategy, microwave chemistry, and other strategies are described. Further insights regarding the suitability of different protecting groups during the epoxidation/chlorination/ring contraction sequence are disclosed. Several interesting by-products from this reaction sequence are reported. These studies have led to a unified synthetic strategy to the axinellamines and palau'amine.