Oligonucleotides containing arylacetylene residues: Synthesis and post-synthetic modification via [3+2] cycloaddition

The synthesis of a phosphoramidite reagent for 5′-modification of oligonucleotides by introducing an arylacetylene residue has been described. Using the reaction with 3-(perylen-3-yl)propyl azide as an example, it was shown that the acetylene derivatives of oligonucleotides synthesized using this reagent undergo Cu^I-catalyzed [3+2] dipolar cycloaddition. Fluorescent conjugates were obtained in high yields and characterized by mass spectra. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 7, pp. 1220–1226, July, 2006.     

Synthesis of 1,2,3,4-tetrahydro-4-pyridinones by heterocyclization of β-amino-β-arylacryloyloxiranes

The heterocyclization of -alkyl(phenyl)aminocinnamoyloxiranes and their -bromo derivatives under base-catalysis conditions is a convenient method for the synthesis of 3-hydroxytetrahydro-4-pyridinones.Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 3, pp. 347–350, March, 1989.     

Highly fluorescent conjugated pyrenes in nucleic acid probes: (phenylethynyl)pyrenecarbonyl-functionalized locked nucleic acids

In recent years, fluorescently labeled oligonucleotides have become a widely used tool in diagnostics, DNA sequencing, and nanotechnology. The recently developed (phenylethynyl)pyrenes are attractive dyes for nucleic acid labeling, with the advantages of long-wave emission relative to the parent pyrene, high fluorescence quantum yields, and the ability to form excimers. Herein, the synthesis of six (phenylethynyl)pyrene-functionalized locked nucleic acid (LNA) monomers M(1)-M(6) and their incorporation into DNA oligomers is described. Multilabeled duplexes display higher thermal stabilities than singly modified analogues. An increase in the number of phenylethynyl substituents attached to the pyrene results in decreased binding affinity towards complementary DNA and RNA and remarkable bathochromic shifts of absorption/emission maxima relative to the parent pyrene fluorochrome. This bathochromic shift leads to the bright fluorescence colors of the probes, which differ drastically from the blue emission of unsubstituted pyrene. The formation of intra- and interstrand excimers was observed for duplexes that have monomers M(1)-M(6) in both complementary strands and in numerous single-stranded probes. If more phenylethynyl groups are inserted, the detected excimer signals become more intense. In addition, (phenylethynyl)pyrenecarbonyl-LNA monomers M(4), M(5), and M(6) proved highly useful for the detection of single mismatches in DNA/RNA targets.     

Simple reagent for the synthesis of oligonucleotides labeled with 3,3,3′,3′-tetramethyl-2,2′-indodicarbocyanine

Synthesis of a phosphoramidite reagent for the preparation of oligonucleotides labeled at the 5′-end with a fluorescent dye, 3,3,3′,3′-tetramethyl-2,2′-indodicarbocyanine, is described. The efficiency of this reagent is confirmed by the synthesis of several labeled oligonucleotides. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 1, pp. 154–158, January, 2006.     

Detection of point mutations using pyrene-labeled DNA probes

Pyrene-labeled oligodeoxyribonucleotide probes were shown to be suitable for the detection of point mutations. Reagents based on homochiral 2,4-dihydroxybutyramides were used to introduce pyrene residues at the 3"- and 5"- ends of oligonucleotide pairs. The oligonucleotide pair forms a tandem complex with a complementary target, giving rise to an excimer signal (_max 470—490 nm) in the fluorescence spectra when the pyrene residues come into close proximity. The maximum ratio of the intensity of the excimer signal to the monomer signal (_max 380—400 nm) is attained when (S)-N-(1-pyrenylmethyl)-3,3-dimethyl-2,4-dihydroxybutyramide is used to introduce the pyrene residue. The excimer fluorescence completely disappears with an increase in the distance between the pyrene residues (upon the introduction of an additional nucleotide in the target) or in the presence of a mismatch near the contact site of the probes.     

Recent applications of bifunctional trityl groups

Triphenylmethyl derivatives represent an important class of dyestuffs as well as a useful family of protecting groups widely used in organic synthesis to transiently block various functional moieties. These applications are well documented and have been a subject of a number of reviews. Here we focus instead on some novel applications of a trityl which make good use of its ability to easily form a stabilised cation in combination with additional peripheral functionalities. Topics covered include applications in bioconjugation, cross-linking, mass-spectrometry, fluorescence and optics.     

Photosensitizing Antivirals

Antiviral action of various photosensitizers is already summarized in several comprehensive reviews, and various mechanisms have been proposed for it. However, a critical consideration of the matter of the area is complicated, since the exact mechanisms are very difficult to explore and clarify, and most publications are of an empirical and “phenomenological” nature, reporting a dependence of the antiviral action on illumination, or a correlation of activity with the photophysical properties of the substances. Of particular interest is substance-assisted photogeneration of highly reactive singlet oxygen (¹O₂). The damaging action of ¹O₂ on the lipids of the viral envelope can probably lead to a loss of the ability of the lipid bilayer of enveloped viruses to fuse with the lipid membrane of the host cell. Thus, lipid bilayer-affine ¹O₂ photosensitizers have prospects as broad-spectrum antivirals against enveloped viruses. In this short review, we want to point out the main types of antiviral photosensitizers with potential affinity to the lipid bilayer and summarize the data on new compounds over the past three years. Further understanding of the data in the field will spur a targeted search for substances with antiviral activity against enveloped viruses among photosensitizers able to bind to the lipid membranes.     

Two-dye and one- or two-quencher DNA probes for real-time PCR assay: synthesis and comparison with a TaqMan? probe

A typical TaqMan? real-time PCR probe contains a 5'-fluorescent dye and a 3'-quencher. In the course of the amplification, the probe is degraded starting from the 5'-end, thus releasing fluorescent dye. Some fluorophores (including fluorescein) are known to be prone to self-quenching when located near each other. This work is aimed at studying dye-dye and dye-quencher interactions in multiply modified DNA probes. Twenty-one fluorogenic probes containing one and two fluoresceins (FAM), or a FAM-JOE pair, and one or two BHQ1 quenchers were synthesized using non-nucleoside reagents and "click chemistry" post-modification on solid phase and in solution. The probes were tested in real-time PCR using an ~300-bp-long natural DNA fragment as a template. The structural prerequisites for lowering the probe background fluorescence and increasing the end-plateau fluorescence intensity were evaluated and discussed.     

Tris(2,6-dimethoxyphenyl)methyl carbenium ion as a charge derivatization agent for the analysis of primary amines by MALDI mass spectrometry

A novel derivatization method for the analysis of primary amines by MALDI mass spectrometry is proposed. Tris(2,6-dimethoxyphenyl) methyl carbenium cation reacts smoothly with primary amines, forming permanently charged adducts with the mass increment +359 Da and absorbance in the UV region. The approach was tested on a number of amines, including biologically active compounds and therapeutic agents.     

4',5'-Dichloro-2',7'-dimethoxy-5(6)-carboxyfluorescein (JOE): synthesis and spectral properties of oligonucleotide conjugates

A convenient procedure for the preparation of the fluorescent dye 4',5'-dichloro-2',7'-dimethoxy-5(6)-carboxyfluorescein (JOE) is reported; the overall yield achieved starting from isovanillin is 10 times higher (40% vs 4%) compared to the known procedure. Isomers (5- and 6-) are easily chromatographically separable as pentafluorophenyl esters of 3',6'-O-bis(cyclohexylcarbonyl) derivatives. Four non-nucleoside JOE phosphoramidites based on 5- and 6-isomers and flexible 6-aminohexanol (AH) or rigid 4-trans-aminocyclohexanol (ACH) linkers have been prepared and used for oligonucleotide labeling. Spectral and photophysical properties of 5'-JOE-modified oligonucleotides have been studied. Fluorescence quantum yield of the dye correlates with the nature of the linker (rigid vs flexible) and with the presence of dG nucleosides in close proximity to a JOE residue.