Four-color DNA sequencing with 3'-O-modified nucleotide reversible terminators and chemically cleavable fluorescent dideoxynucleotides.

DNA sequencing by synthesis (SBS) on a solid surface during polymerase reaction can decipher many sequences in parallel. We report here a DNA sequencing method that is a hybrid between the Sanger dideoxynucleotide terminating reaction and SBS. In this approach, four nucleotides, modified as reversible terminators by capping the 3'-OH with a small reversible moiety so that they are still recognized by DNA polymerase as substrates, are combined with four cleavable fluorescent dideoxynucleotides to perform SBS. The ratio of the two sets of nucleotides is adjusted as the extension cycles proceed. Sequences are determined by the unique fluorescence emission of each fluorophore on the DNA products terminated by ddNTPs. On removing the 3'-OH capping group from the DNA products generated by incorporating the 3'-O-modified dNTPs and the fluorophore from the DNA products terminated with the ddNTPs, the polymerase reaction reinitiates to continue the sequence determination. By using an azidomethyl group as a chemically reversible capping moiety in the 3'-O-modified dNTPs, and an azido-based cleavable linker to attach the fluorophores to the ddNTPs, we synthesized four 3'-O-azidomethyl-dNTPs and four ddNTP-azidolinker-fluorophores for the hybrid SBS. After sequence determination by fluorescence imaging, the 3'-O-azidomethyl group and the fluorophore attached to the DNA extension product via the azidolinker are efficiently removed by using Tris(2-carboxyethyl)phosphine in aqueous solution that is compatible with DNA. Various DNA templates, including those with homopolymer regions, were accurately sequenced with a read length of >30 bases by using this hybrid SBS method on a chip and a four-color fluorescence scanner.

Short synthesis of the C1-C14 stretch of discodermolide from building blocks prepared by asymmetric catalysis.

A convergent and stereoselective synthesis of the C1-C14 stretch of (+)-discodermolide demonstrates the utility of the "asymmetric catalysis approach" to complex polypropionates. The preparation of this complex synthon requires 15 steps in the longest linear sequence and 19 steps total from inexpensive materials.

3'-O-modified nucleotides as reversible terminators for pyrosequencing.

Pyrosequencing is a method used to sequence DNA by detecting the pyrophosphate (PPi) group that is generated when a nucleotide is incorporated into the growing DNA strand in polymerase reaction. However, this method has an inherent difficulty in accurately deciphering the homopolymeric regions of the DNA templates. We report here the development of a method to solve this problem by using nucleotide reversible terminators. These nucleotide analogues are modified with a reversible chemical moiety capping the 3'-OH group to temporarily terminate the polymerase reaction. In this way, only one nucleotide is incorporated into the growing DNA strand even in homopolymeric regions. After detection of the PPi for sequence determination, the 3'-OH of the primer extension products is regenerated through different deprotection methods. Using an allyl or a 2-nitrobenzyl group as the reversible moiety to cap the 3'-OH of the four nucleotides, we have synthesized two sets of 3'-O-modified nucleotides, 3'-O-allyl-dNTPs and 3'-O-(2-nitrobenzyl)-dNTPs as reversible terminators for pyrosequencing. The capping moiety on the 3'-OH of the DNA extension product is efficiently removed after PPi detection by either a chemical method or photolysis. To sequence DNA, templates containing homopolymeric regions are immobilized on Sepharose beads, and then extension-signal detection-deprotection cycles are conducted by using the nucleotide reversible terminators on the DNA beads to unambiguously decipher the sequence of DNA templates. Our results establish that this reversible-terminator-pyrosequencing approach can be potentially developed into a powerful methodology to accurately determine DNA sequences.

Scalable, catalytic asymmetric synthesis of syn, anti stereotriad building blocks for polypropionate antibiotics.

[reaction: see text] Asymmetric catalysis and chirality transfer by the 2,3-Wittig rearrangement were combined to provide a syn, anti stereotriad-containing olefinic alcohol in five steps from inexpensive starting materials. Development of this compound, a versatile intermediate for polypropionate synthesis, gave known building blocks for discodermolide.

Synthesis and herbicidal activity of 2-cyano-3-substituted-pyridinemethylaminoacrylates.

Two series of 2-cyano-3-substituted-pyridinemethylaminoacrylates, namely 12 new (Z)-2-cyano-3-methylthio-3-substituted-pyridinemethaneaminoacrylates and 10 new (Z)-2-cyano-3-alkyl-3-substituted-pyridinemethaneaminoacrylates, were synthesized as herbicidal inhibitors of photosystem II (PSII) electron transport. All of these compounds were confirmed by (1)H NMR, elemental, IR, and mass spectrum analyses. Their herbicidal activities were evaluated. Some compounds exhibited excellent herbicidal activities, even at a dose of 75 g/ha. A suitable substituent at the 2-position of the pyridine ring and the well-fit group at the 3-position of acrylate were essential for high herbicidal activity. 2-Cyanoacrylates containing a substituted pyridine ring provide higher herbicidal activities than parent compounds containing phenyl. These PSII inhibitor herbicides are safe to corn, which is a major crop in China.