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In non-viscous aqueous solutions, the cyanine fluorescent dyes Cy3 and Cy5 have rather low fluorescence efficiency (the fluorescence quantum yields of Cy3 and Cy5 are 0.04 and 0.3, respectively [1, 2]) and short excited state lifetimes due to their structural features. In this work, we investigated the effect of solubility and rotational degrees of freedom on the fluorescence efficiency of Cy3 and Cy5 in several ways. We compared the fluorescence efficiencies of two cyanine dyes sCy3 and sCy5 with the introduction of a sulfonyl substituent in the aromatic ring as well as covalently bound to T10 oligonucleotides. The results show that because of the different lengths of the polymethine chains between the aromatic rings of the dyes, cis-trans-isomerization has a much greater effect on the Cy3 molecule than on the Cy5 molecule, while the effect of aggregation is also significant., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Ru-based catalysis results in highly unsaturated fatty acid (HUFA) ethyl esters (EE) deuterated to various extents. The products carry 2 H (D) mainly at their bis -allylic positions, where they are resistant to autoxidation compared to natural HUFA and are promising as neurological and retinal drugs. We characterized the extent of deuteration at each allylic position of docosa-4,7,10,13,16,19-hexaenoic acid deuterated to completion at bis -allylic and allylic positions (D-DHA) by two-dimensional (2D) and high-field (600 and 950 MHz) NMR. In separate experiments, the kinetics of docosahexaenoic acid (DHA) EE deuteration was evaluated using Paternò-Büchi (PB) reaction tandem mass spectrometry (MS/MS) analysis, enabling deuteration to be quantitatively characterized for isotopologues (D0-D14 DHA) at each internal allylic position. NMR analysis shows that the net deuteration of the isotopologue mixture is about 94% at the bis -allylic positions, and less than 1% remained as the protiated -CH 2 -. MS analysis shows that deuteration kinetics follow an increasing curve at bis -allylic positions with higher rate for internal bis -allylic positions. Percent D of bis -allylic positions increases linearly from D1 to D9 in which all internal bis -allylic positions (C9, C12, C15) deuterate uniformly and more rapidly than external bis -allylic positions (C6, C18). The mono -allylic positions near the methyl end (C21) show a steep increase of D only after the D10 isotopologue has been deuterated to >90%, while the mono -allylic position near the carboxyl position, C3, deuterates last and least. These data establish detailed methods for the characterization of Ru-catalyzed deuteration of HUFA as well as the phenomenological reaction kinetics as net product is formed.

Lipid peroxidation (LPO) plays a key role in many age-related neurodegenerative conditions and other disorders. Light irradiation can initiate LPO through various mechanisms and is of importance in retinal and dermatological pathologies. The introduction of deuterated polyunsaturated fatty acids (D-PUFA) into membrane lipids is a promising approach for protection against LPO. Here, we report the protective effects of D-PUFA against the photodynamically induced LPO, using illumination in the presence of the photosensitizer trisulfonated aluminum phthalocyanine (AlPcS 3 ) in liposomes and giant unilamellar vesicles (GUV), as assessed in four experimental models: 1) sulforhodamine B leakage from liposomes, detected with fluorescence correlation spectroscopy (FCS); 2) formation of diene conjugates in liposomal membranes, measured by absorbance at 234 nm; 3) membrane leakage in GUV assessed by optical phase-contrast intensity observations; 4) UPLC-MS/MS method to detect oxidized linoleic acid (Lin)-derived metabolites. Specifically, in liposomes or GUV containing H-PUFA (dilinoleyl-sn-glycero-3-phosphatidylcholine), light irradiation led to an extensive oxidative damage to bilayers. By contrast, no damage was observed in lipid bilayers containing 20% or more D-PUFA (D2-Lin or D10-docosahexanenoic acid). Remarkably, addition of tocopherol increased the dye leakage from liposomes in H-PUFA bilayers compared to photoirradiation alone, signifying tocopherol's pro-oxidant properties. However, in the presence of D-PUFA the opposite effect was observed, whereby adding tocopherol increased the resistance to LPO. These findings suggest a method to augment the protective effects of D-PUFA, which are currently undergoing clinical trials in several neurological and retinal diseases that involve LPO., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Arachidonic acid (ARA) is a major component of lipid bilayers as well as the key substrate for the eicosanoid cascades. ARA is readily oxidized, and its non-enzymatic and enzymatic oxidation products induce inflammatory responses in nearly all tissues, including lung tissues. Deuteration at bis-allylic positions substantially decreases the overall rate of ARA oxidation when hydrogen abstraction is an initiating event. To compare the effects of dosing of arachidonic acid (H-ARA) and its bis-allylic hexadeuterated form (D-ARA) on lungs in conventionally healthy mice and in an acute lung injury model, mice were dosed with H-ARA or D-ARA for six weeks through dietary supplementation and then challenged with intranasal lipopolysaccharide (LPS) for subsequent analysis of bronchoalveolar lavage fluid and lung tissue. Dosing on D-ARA resulted in successful incorporation of D-ARA into various tissues. D-ARA significantly reduced LPS-induced adverse effects on alveolar septal thickness and the bronchoalveolar area. Oral deuterated ARA is taken up efficiently and protects against adverse LPS-induced pathology. This suggests novel therapeutic avenues for reducing lung damage during severe infections and other pathological conditions with inflammation in the pulmonary system and other inflammatory diseases.

This work studies the impact of the electrostatic interaction between analyte molecules and silver nanoparticles (Ag NPs) on the intensity of surface-enhanced Raman scattering (SERS). For this, we fabricated nanostructured plasmonic films by immobilization of Ag NPs on glass plates and functionalized them by a set of differently charged hydrophilic thiols (sodium 2-mercaptoethyl sulfonate, mercaptopropionic acid, 2-mercaptoethanol, 2-(dimethylamino)ethanethiol hydrochloride, and thiocholine) to vary the surface charge of the SERS substrate. We used two oppositely charged porphyrins, cationic copper(II) tetrakis(4- N -methylpyridyl) porphine (CuTMpyP4) and anionic copper(II) 5,10,15,20-tetrakis(4-sulfonatophenyl)porphine (CuTSPP4), with equal charge value and similar structure as model analytes to probe the SERS signal. Our results indicate that the SERS spectrum intensity strongly, up to complete signal disappearance, correlates with the surface charge of the substrate, which tends to be negative. Using the data obtained and our model SERS system, we analyzed the modification of the Ag surface by different reagents (lithium chloride, polyethylenimine, polyhexamethylene guanidine, and multicharged metal ions). Finally, all those surface modifications were tested using a negatively charged oligonucleotide labeled with Black Hole Quencher dye. Only the addition of copper ions into the analyte solution yielded a good SERS signal. Considering the strong interaction of copper ions with the oligonucleotide molecules, we suppose that inversion of the analyte charge played a key role in this case, instead of a change of charge of the substrate surface. Changing the charge of analytes could be a promising way to get clear SERS spectra of negatively charged molecules on Ag SERS-active supports., (Copyright © 2021, Ranishenka et al.)

No general method currently is available for the quantitative determination of deuterium (D) at C positions along a hydrocarbon chain. Bis -allylic deuterated highly unsaturated fatty acids (D-HUFA) are a novel class of drugs stabilized against H-abstraction-mediated oxidation by deuteration at the most labile positions. Ru-based catalytic deuteration overcomes the limited scale of bis -allylic D-HUFA production by total organic synthesis; however, it produces a complex mixture of bis -allylic D isotopologues and isotopomers, requiring detailed sequencing for characterization. We report here adaptation and application of the Paternó-Büchi (PB) reaction of 2-acetylpyridine to a series of D-HUFA with analysis by shotgun lipidomics to determine position-specific quantitative D abundances. Sodiated PB D-HUFA result in diagnostic ions of high abundance upon collision-induced dissociation (CID) activation, enabling sensitive differentiation and quantification of D fraction at each bis - and mono -allylic position for each isotopologue. Catalytically deuterated isotopologues D5-7 linolenic acid (D5-7 LnA), D6-8 arachidonic acid (D6-8 ARA), D7-9 eicosapentaenoic acid (D7-9 EPA), and D9-11 docosahexaenoic acid (D9-11 DHA) incorporate 80-98, 95-100, 81-100, and 83-100% D at their bis -allylic positions, respectively. D-HUFA isotopologues having D number greater than or equal to bis -allylic sites (e.g., D10-DHA or D11-DHA) deuterated >95% at bis -allylic positions, except for D-LnA. The mono- allylic position near the methyl end deuterates to a much greater extent than the mono- allylic position near the carboxyl end, and both positions deuterate only when bis -allylic D is near-saturated. This method enables rapid, accurate characterization of position and isotopomer-specific D composition and enables sequencing along the chain.

An azido-derivative of a fluorescein bifluorophore was obtained and used for the synthesis of "molecular beacon"-type oligonucleotide fluorogenic probes for RT-PCR. Eight probe variants were synthesized based on an optimized sequence: with one or two quencher residues at the 3'-end, with a single or bifluorophore fluorescein label attached to 5'-end using modifying phosphoramidites (short linker) or "click reaction" (long linker). Comparison of probes in RT-PCR showed that probes with a doubled quencher (single fluorescein on a short linker) and doubled dye on a short linker (single dye) are somewhat superior in sensitivity to a standard probe (single quencher, single dye on a short linker) by the value of ΔC t = 1-2., Supplementary Information: The online version contains supplementary material available at 10.1134/S1068162021030055., Competing Interests: Conflict of InterestsThe authors declare they have no conflict of interest., (© The Author(s) 2021, ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2021, Vol. 47, No. 3, pp. 734–740. © The Author(s), 2021. This article is an open access publication.Russian Text © The Author(s), 2021, published in Bioorganicheskaya Khimiya, 2021, Vol. 47, No. 3, pp. 365–372.)

GalNAc oligonucleotide conjugates demonstrate improved potency in vivo due to selective and efficient delivery to hepatocytes in the liver via receptor-mediated endocytosis. GalNAc-siRNA and GalNAc-antisense oligonucleotides are at various stages of clinical trials, while the first two drugs were already approved by FDA. Also, GalNAc conjugates are excellent tools for functional genomics and target validation in vivo. The number of GalNAc residues in a conjugate is crucial for delivery as cooperative interaction of several GalNAc residues with asialoglycoprotein receptor enhances delivery in vitro and in vivo. Here we provide a robust protocol for the synthesis of triple GalNAc CPG solid support and GalNAc phosphoramidite, synthesis and purification of RNA conjugates with multiple GalNAc residues either to 5'-end or 3'-end and siRNA duplex formation.

Vicinal diols and its derivatives can be exploited as model compounds for the investigation of radiation-induced free-radical transformations of hydroxyl-containing biomolecules such as carbohydrates, phospholipids, ribonucleotides, amino acids, and peptides. In this paper, for the first time, the prospects of isotope reinforcement approach in inhibiting free-radical transformations of hydroxyl-containing compounds in aqueous solutions are investigated on the example of radiolysis of 1,2-propanediol and 1,2-propanediol-2-d 1 aqueous solutions. At an absorbed dose rate of 0.110 ± 0.003 Gy·s -1 a profound kinetic isotope effect (KIE) is observed for the non-branched chain formation of acetone, which is a final dehydration product of predominant carbon-centred radicals CH 3 ·C(OH)CH 2 OH. In 0.1 and 1 M deaerated solutions at pH 7.00 ± 0.01, the values of KIE are 8.9 ± 1.7 and 15.3 ± 3.1, respectively. A rationale for the fact that a strong KIE takes place only in the case of chain processes, which may occur during free-radical transformations of vicinal diols, is also provided herein based on the results of 2-propanol and 2-propanol-2-d 1 indirect radiolysis. Lastly, the lack of KIE is shown in the case of 2-butanone formation from 2,3-butanediol or 2,3-butanediol-2,3-d 2 . This indicates that the type (primary, secondary) of the β-carbonyl radicals formed as a result of CH 3 ·C(OH)CH(OH)R (R = H, CH 3 ) dehydration determines the manifestation of the effect.

Rigid amphipathic fusion inhibitors are potent broad-spectrum antivirals based on the perylene scaffold, usually decorated with a hydrophilic group linked via ethynyl or triazole. We have sequentially simplified these structures by removing sugar moiety, then converting uridine to aniline, then moving to perylenylthiophenecarboxylic acids and to perylenylcarboxylic acid. All these polyaromatic compounds, as well as antibiotic heliomycin, still showed pronounced activity against tick-borne encephalitis virus (TBEV) with limited toxicity in porcine embryo kidney (PEK) cell line. 5-(Perylen-3-yl)-2-thiophenecarboxylic acid (5a) showed the highest antiviral activity with 50% effective concentration of approx. 1.6 nM., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Bright fluorescent probes with enhanced intensities in the fluorescein channel are of great value for plenty of biological applications. To design effective probes one should introduce as many as possible fluorophores to the biomolecule while leaving its native structure as intact as possible. To reach this compromise, we designed and synthesized fluorescein bifluorophores on the 3,5-diaminobenzoic acid scaffold, which allows for insertion of two fluorophores at one modification site of a biomolecule. Rigid structure of the branching linker group allows to minimize self-quenching the fluorophores. However, despite the structure similarities of fluorescein isomers (5-FAM and 6-FAM), different photophysical behavior was observed for the corresponding bifluorophores. Here we made efforts to get insight into these effects with the focus on the media viscosity impact.

In this work, we describe the synthesis of 5-(perylen-3-ylethynyl)uridine and its ability to effectively inhibit the replication of respiratory disease pathogens in cell culture, namely: influenza A virus (IVA); type 3 parainfluenza virus (PIV-3); and human respiratory syncytial virus (RSV). Related known compounds were also analyzed: 5-(perylen-3-ylethynyl)-2'-deoxy-uridine; 5-(perylen-3-ylethynyl)-arabino-uridine; and 1-carboxymethyl-3-pivaloyloxymethyl-5-(perylen-3-ylethynyl)uracil., Competing Interests: Conflict of InterestsThe authors declare that there is no conflict of interests., (© Pleiades Publishing, Ltd. 2020.)

Autoxidation of polyunsaturated fatty acids (PUFAs) damages lipid membranes and generates numerous toxic by-products implicated in neurodegeneration, aging, and other pathologies. Abstraction of bis-allylic hydrogen atoms is the rate-limiting step of PUFA autoxidation, which is inhibited by replacing bis-allylic hydrogens with deuterium atoms (D-PUFAs). In cells, the presence of a relatively small fraction of D-PUFAs among natural PUFAs is sufficient to effectively inhibit lipid peroxidation (LPO). Here, we investigate the effect of various D-PUFAs on the stability of liposomes under oxidative stress conditions. The permeability of vesicle membranes to fluorescent dyes was measured as a proxy for bilayer integrity, and the formation of conjugated dienes was monitored as a proxy for LPO. Remarkably, both approaches reveal a similar threshold for the protective effect of D-PUFAs in liposomes. We show that protection rendered by D-PUFAs depends on the structure of the deuterated fatty acid. Our findings suggest that protection of PUFAs against autoxidation depends on the total level of deuterated bi-sallylic (CD 2 ) groups present in the lipid bilayer. However, the phospholipid containing 6,6,9,9,12,12,15,15,18,18-d 10 -docosahexaenoic acid exerts a stronger protective effect than should be expected from its deuteration level. These findings further support the application of D-PUFAs as preventive/therapeutic agents in numerous pathologies that involve LPO., (© 2019 Federation of European Biochemical Societies.)

Nucleic acids labeled with a fluorophore/quencher pair are widely used as probes in biomedical research and molecular diagnostics. Here we synthesized novel DNA molecular beacons double labeled with the identical dyes (R6G, ROX and Cy5) at 5'- and 3'-end and studied their photo physical properties. We demonstrated that fluorescence quenching by formation of the homo dimer exciton in such molecular beacons allows using them in homogeneous assays. Further, we developed and evaluated homo Yin-Yang DNA probes labeled with identical dyes and used them for detection of low copy HIV RNA by RT-qPCR. They demonstrated improved sensitivity (LLQ: 10 vs 30 copies mL -1 ) in comparison to commercially available Abbott RealTime HIV-1 kit based on VIC-BHQ dyes both for model mixtures (naive human plasma with added deactivated HIV-1 virus) and for preliminarily confirmed 36 clinical samples (4 vs 1 positive ones for low-copy samples)., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The synthesis of signal lipids, including eicosanoids, is not fully understood, although it is key to the modulation of various inflammatory states. Recently, isotopologues of essential polyunsaturated fatty acids (PUFAs) deuterated at bis-allylic positions (D-PUFAs) have been proposed as inhibitors of non-enzymatic lipid peroxidation (LPO) in various disease models. Arachidonic acid (AA, 20:4 n-6) is the main precursor to several classes of eicosanoids, which are produced by cyclooxygenases (COX) and lipoxygenases (LOX). In this study we analyzed the relative activity of human recombinant enzymes COX-2, 5-LOX, and 15-LOX-2 using a library of arachidonic acids variably deuterated at the bis-allylic (C7, C10, and C13) positions. Kinetic parameters (KM, V max ) and isotope effects calculated from kH/kD for seven deuterated arachidonic acid derivatives were obtained. Spectroscopic methods have shown that deuteration at the 13th position dramatically affects the kinetic parameters of COX-2 and 15-LOX-2. The activity of 5-LOX was evaluated by measuring hydroxyeicosatetraenoic acids (8-HETE and 5-HETE) using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Deuteration at the seventh and 10th positions affects the performance of the 5-LOX enzyme. A flowchart is proposed suggesting how to modulate the synthesis of selected eicosanoids using the library of deuterated isotopologues to potentially fine-tune various inflammation stages.

GalNAc conjugation is emerging as a dominant strategy for delivery of therapeutic oligonucleotides to hepatocytes. The structure and valency of the GalNAc ligand contributes to the potency of the conjugates. Here we present a panel of multivalent GalNAc variants using two different synthetic strategies. Specifically, we present a novel conjugate based on a support-bound trivalent GalNAc cluster, and four others using a GalNAc phosphoramidite monomer that was readily assembled into tri- or tetravalent designs during solid phase oligonucleotide synthesis. We compared these compounds to a clinically used trivalent GalNAc cluster both in vitro and in vivo. In vitro, cluster-based and phosphoramidite-based scaffolds show a similar rate of internalization in primary hepatocytes, with membrane binding observed as early as 5 min. All tested compounds provided potent, dose-dependent silencing, with 2-4% of injected dose recoverable from liver after 1 week. The two preassembled trivalent GalNAc clusters showed higher tissue accumulation and gene silencing relative to di-, tri-, or tetravalent GalNAc conjugates assembled via phosphoramidite chemistry.

Ferroptosis is a non-apoptotic form of regulated cell death caused by the failure of the glutathione-dependent lipid-peroxide-scavenging network. FINO 2 is an endoperoxide-containing 1,2-dioxolane that can initiate ferroptosis selectively in engineered cancer cells. We investigated the mechanism and structural features necessary for ferroptosis initiation by FINO 2 . We found that FINO 2 requires both an endoperoxide moiety and a nearby hydroxyl head group to initiate ferroptosis. In contrast to previously described ferroptosis inducers, FINO 2 does not inhibit system x c - or directly target the reducing enzyme GPX4, as do erastin and RSL3, respectively, nor does it deplete GPX4 protein, as does FIN56. Instead, FINO 2 both indirectly inhibits GPX4 enzymatic function and directly oxidizes iron, ultimately causing widespread lipid peroxidation. These findings suggest that endoperoxides such as FINO 2 can initiate a multipronged mechanism of ferroptosis.

Most antivirals target viral proteins and are specific for only one virus, or viral type. Whereas viral proteins are encoded in the plastic viral genome, virion lipids are not and their rearrangements during fusion are conserved among otherwise unrelated enveloped viruses. Antivirals that inhibit these lipid rearrangements could thus pose a high barrier to resistance and have broad-spectrum activity. Fusion occurs through a hemifusion stalk in which only the outer leaflets are fused and thus curved with a smaller radius for the polar heads than for the hydrophobic tails (negative curvature). Outer leaflets enriched in phospholipids with head groups of larger cross sections than their lipid tails ("inverted cone") disfavor negative curvature, inhibiting fusion. The rigid amphipathic fusion inhibitors (RAFIs) are synthetic compounds of inverted cone molecular geometry. They inhibit infectivity of otherwise unrelated enveloped viruses. The leading RAFI, aUY11, has an ethynyl-perylene hydrophobic and an uracil-arabinose polar moiety. aUY11 intercalates in viral envelopes and inhibits virion-to-cell fusion of a broad spectrum of otherwise unrelated enveloped viruses. Previous studies showed that amphipathicity, rigidity, and inverted cone molecular geometry were required. We propose that the inverted cone molecular geometry of the RAFIs increases the energy barrier for the hemifusion stalk, inhibiting fusion. Then, chemically distinct compounds with similar amphipathicity, rigidity, and inverted cone shape would have similar antiviral potencies, regardless of specific chemical groups. Alternatively, the perylene group exposed to visible light may induce viral lipid peroxidation. Then, the perylene group and absorbance at visible spectrum would be required. We now evaluated twenty-five chemically distinct RAFIs. The perylene moiety and absorption at visible spectrum were not required, but a minimum length of the hydrophobic moiety was, 10.3 Å. The arabino moiety could be modified or replaced by other groups. Cytidine was not tolerated. Bilayer intercalation was required but not sufficient. The vast majority of RAFIs had no overt cytotoxicity (CC 50  > 20 μM; TI > 250-1200). Carbonyl or butylamide substitutions for arabino, or cytidine replacement for uracil, increased cytotoxicity. Cytotoxicity was mainly determined by the polar moiety and there was no correlation between antiviral and cytostatic activities. The definition of the effects of shape and chemical groups of the RAFIs opens the possibility to the rational design of lipid-acting antivirals active against a broad spectrum of enveloped viruses., (Copyright © 2017 Elsevier B.V. All rights reserved.)

We developed a novel technique for the efficient conjugation of oligonucleotides with various alkyl azides such as fluorescent dyes, biotin, cholesterol, N-acetylgalactosamine (GalNAc), etc. using copper-catalysed alkyne-azide cycloaddition on the solid phase and CuI·P(OEt) 3 as a catalyst. Conjugation is carried out in an oligonucleotide synthesizer in fully automated mode and is coupled to oligonucleotide synthesis and on-column deprotection. We also suggest a set of reagents for the construction of diverse conjugates. The sequential double-click procedure using a pentaerythritol-derived tetraazide followed by the addition of a GalNAc or Tris-GalNAc alkyne gives oligonucleotide-GalNAc dendrimer conjugates in good yields with minimal excess of sophisticated alkyne reagents. The approach is suitable for high-throughput synthesis of oligonucleotide conjugates ranging from fluorescent DNA probes to various multi-GalNAc derivatives of 2'-modified siRNA.

Effects exerted by heavy isotope substitution in biopolymers on the functioning of whole organisms have not been investigated. We report on the decrease of permissive temperature of nematodes fed with bacteria containing 5,5-D2-lysine. We synthesized 5,5-dideuterolysine and, taking advantage of lysine being an essential amino acid, showed that C. elegans with modified lysine poorly develop from larvae into fertile adult hermaphrodites. This effect occurs only at high temperature within the permissible range for C. elegans (25 °C) and completely vanishes at 15 °C. The only known metabolic involvement of C5 in lysine is in post-translational modification through lysyl hydoxylases. Indeed, siRNA experiments showed that deficiency of let-268/plod lysyl-hydroxylase/glycosydase further amplifies the isotope effect making it apparent even at 20 °C, whereas control siRNAs as well as another lysyl-hydroxylase (psr-1/jmjdD) siRNA do not. We report for the first time that a site-specific deuteration may strongly affect the development of the whole animal organism especially under the conditions of deficiency of the corresponding enzyme. These findings provide the basis for our ongoing efforts to employ isotope effects for fine tuning of metabolic pathways to mitigate pathological processes.

The human DNA methyltransferase 3A (DNMT 3A) is responsible for de novo epigenetic regulation, which is essential for mammalian viability and implicated in diverse diseases. All DNA cytosine C5 methyltransferases follow a broadly conserved catalytic mechanism. We investigated whether C5 β-elimination contributes to the rate-limiting step in catalysis by DNMT3A and the bacterial M.HhaI by using deuterium substitutions of C5 and C6 hydrogens. This substitution caused a 1.59-1.83 fold change in the rate of catalysis, thus suggesting that β-elimination is partly rate-limiting for both enzymes. We used a multisite substrate to explore the consequences of slowing β-elimination during multiple cycles of catalysis. Processive catalysis was slower for both enzymes, and deuterium substitution resulted in DNMT 3A dissociating from its substrate. The decrease in DNA methylation rate by DNMT 3A provides the basis of our ongoing efforts to alter cellular DNA methylation levels without the toxicity of currently used methods., (© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Molecular beacons carrying JOE dye (4',5'-dichloro-2',7'-dimethoxy-6-carboxyfluorescein) on a rigid or flexible linker and one or two BHQ1 quenchers have been prepared and tested in real-time PCR using Fusarium avenaceum elongation factor 1α DNA template. The probes were different in their structures (loop size and stem length), linkers for dye attachment (6-aminohexanol or trans-4-aminocyclohexanol), quencher composition (single and double BHQ1) to elucidate the influence of all these features. Fluorogenic properties of the probes were studied and compared to those of FAM (fluorescein)-based probes. All the factors - stem length, JOE vs FAM, rigid vs flexible linker, single vs double quencher - appeared to play a considerable role in the probe's fluorescent properties and determine the usability of the probe at two different temperatures of fluorescence detection (55°С and 64°С)., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Racemization in proteins and peptides at sites of L-asparaginyl and L-aspartyl residues contributes to their spontaneous degradation, especially in the biological aging process. Amino acid racemization involves deprotonation of the alpha carbon and replacement of the proton in the opposite stereoconfiguration; this reaction is much faster for aspartate/asparagine than for other amino acids because these residues form a succinimide ring in which resonance stabilizes the carbanion resulting from proton loss. To determine if the replacement of the hydrogen atom on the alpha carbon with a deuterium atom might decrease the rate of racemization and thus stabilize polypeptides, we synthesized a hexapeptide, VYPNGA, in which the three carbon-bound protons in the asparaginyl residue were replaced with deuterium atoms. Upon incubation of this peptide in pH 7.4 buffer at 37 °C, we found that the rate of deamidation via the succinimide intermediate was unchanged by the presence of the deuterium atoms. However, the accumulation of the D-aspartyl and D-isoaspartyl-forms resulting from racemization and hydrolysis of the succinimide was decreased more than five-fold in the deuterated peptide over a 20 day incubation at physiological temperature and pH. Additionally, we found that the succinimide intermediate arising from the degradation of the deuterated asparaginyl peptide was slightly less likely to open to the isoaspartyl configuration than was the protonated succinimide. These findings suggest that the kinetic isotope effect resulting from the presence of deuteriums in asparagine residues can limit the accumulation of at least some of the degradation products that arise as peptides and proteins age., Competing Interests: M. S. Shchepinov declares a competing financial interest as the Chief Scientific Officer of Retrotope, Inc.

Polyunsaturated fatty acid (PUFA) peroxidation is initiated by hydrogen atom abstraction at bis-allylic sites and sets in motion a chain reaction that generates multiple toxic products associated with numerous disorders. Replacement of bis-allylic hydrogens of PUFAs with deuterium atoms (D-PUFAs), termed site-specific isotope reinforcement, inhibits PUFA peroxidation and confers cell protection against oxidative stress. We demonstrate that structurally diverse deuterated PUFAs similarly protect against oxidative stress-induced injury in both yeast and mammalian (myoblast H9C2) cells. Cell protection occurs specifically at the lipid peroxidation step, as the formation of isoprostanes, immediate products of lipid peroxidation, is drastically suppressed by D-PUFAs. Mitochondrial bioenergetics function is a likely downstream target of oxidative stress and a subject of protection by D-PUFAs. Pretreatment of cells with D-PUFAs is shown to prevent inhibition of maximal uncoupler-stimulated respiration as well as increased mitochondrial uncoupling, in response to oxidative stress induced by agents with diverse mechanisms of action, including t-butylhydroperoxide, ethacrynic acid, or ferrous iron. Analysis of structure-activity relationships of PUFAs harboring deuterium at distinct sites suggests that there may be a mechanism supplementary to the kinetic isotope effect of deuterium abstraction off the bis-allylic sites that accounts for the protection rendered by deuteration of PUFAs. Paradoxically, PUFAs with partially deuterated bis-allylic positions that retain vulnerable hydrogen atoms (e.g., monodeuterated 11-D1-Lin) protect in a manner similar to that of PUFAs with completely deuterated bis-allylic positions (e.g., 11,11-D2-Lin). Moreover, inclusion of just a fraction of deuterated PUFAs (20-50%) in the total pool of PUFAs preserves mitochondrial respiratory function and confers cell protection. The results indicate that the therapeutic potential of D-PUFAs may derive from the preservation of mitochondrial function., (Copyright © 2015 Elsevier Inc. All rights reserved.)

A rapid immunochromatographic assay was developed for the control of tetracycline (TC). The assay is based on the competition between immobilized TC-protein conjugate and TC in a tested sample for binding with polyclonal anti-TC antibodies conjugated to colloidal gold during the flow of the sample along a membrane strip with immobilized reactants. Conjugation of colloidal gold and the total immunoglobulin (IgG) fraction of polyclonal antibodies was used to increase the assay sensitivity to ensure low content of specific antibodies in the conjugate. This allowed effective inhibition of free TC and conjugate binding in the strip test zone. Photometric marker registration allows control of the reduction of binding, thereby enhancing detection sensitivity. The proposed assay allows TC to be detected at concentrations up to 20 ng/mL, exceeding the limit of detection of the known analogues, in a wide working range (more than two orders) of 60 pg/mL to 10 ng/mL, ensured through the use of polyclonal antibodies. The assay time is 10 min. The efficiency of the designed assay is shown to identify TC in milk; the degree of recovery of TC ranges from 90 to 112%. The precision of the concentrations measurements was no more than 10%.

Azide and phosphoramidite functions were found to be compatible within one molecule and stable for months in solution kept frozen at -20 °C. An azide-carrying phosphoramidite was used for direct introduction of multiple azide modifications into synthetic oligonucleotides. A series of azide-containing oligonucleotides were modified further using click reactions with alkynes.

Convenient preparation of fluorogenic hairpin DNA probes (molecular beacons) carrying a pair of FAM fluorophores (located close to 5'-terminus of the probe) or a pair of BHQ1 quenchers on 3'-terminus (with (BHQ1)2 or BHQ1-BHQ1 composition) is reported. These probes were used for the first time in a real-time PCR assay and showed considerable improvements in fluorogenic properties (the total fluorescence increase or signal-to-background ratio) in assay conditions vs. conventional one-FAM-one-BHQ1 molecular beacon probes as well as vs. hydrolyzable one-FAM-one-BHQ1 TaqMan probes. At the same time, such multiple modifications of the probe do not influence its Cq (a fractional PCR cycle used for quantification). The probe MB14 containing a BHQ1-BHQ1 pair showed a PCR fluorescence/background value of 9.6 which is more than two times higher than that of a regular probe MB2 (4.6). This study demonstrates prospects for the design of highly fluorogenic molecular beacon probes suitable for quantitative real-time PCR and for other potential applications (e.g. intracellular RNA detection and SNP/mutation analysis).

Substitution of -CD2- at the reactive centers of linoleic and linolenic acids reduces the rate of abstraction of D by a tocopheryl radical by as much as 36-fold, compared to the abstraction of H from a corresponding -CH2- center. This H atom transfer reaction is the rate-determining step in the tocopherol-mediated peroxidation of lipids in human low-density lipoproteins, a process that has been linked to coronary artery disease. The unanticipated large kinetic isotope effects reported here for the tocopherol-mediated oxidation of linoleic and linolenic acids and esters suggests that tunneling makes this process favorable.

This unit describes the preparation of 5- and 6-carboxy derivatives of the xanthene fluorescent dyes fluorescein (FAM), 4',5'-dichloro-2',7'-dimethoxy-fluorescein (JOE), and tetramethylrhodamine (TAMRA) as individual isomers, and their conversion to non-nucleoside phosphoramidite reagents suitable for oligonucleotide labeling. The use of a cyclohexylcarbonyl (Chc) protecting group for blocking of phenolic hydroxyls facilitates the chromatographic separation of isomers of carboxy-FAM and carboxy-JOE as pentafluorophenyl esters. Acylation of 3-dimethylaminophenol with 1,2,4-benzenetricarboxylic anhydride gave a mixture of 4-dimethylamino-2-hydroxy-2',4'(5')-dicarboxybenzophenones, easily separable into individual compounds upon fractional crystallization. Individual isomeric benzophenones are precursors of 5- or 6-carboxytetramethylrhodamines. The dyes were converted into 6-aminohexanol- (JOE), 4-trans-aminocyclohexanol- (FAM and JOE), and hydroxyprolinol-based (TAMRA) phosphoramidite reagents., (© 2013 by John Wiley & Sons, Inc.)

Polyunsaturated fatty acids (PUFAs) undergo autoxidation and generate reactive carbonyl compounds that are toxic to cells and associated with apoptotic cell death, age-related neurodegenerative diseases, and atherosclerosis. PUFA autoxidation is initiated by the abstraction of bis-allylic hydrogen atoms. Replacement of the bis-allylic hydrogen atoms with deuterium atoms (termed site-specific isotope-reinforcement) arrests PUFA autoxidation due to the isotope effect. Kinetic competition experiments show that the kinetic isotope effect for the propagation rate constant of Lin autoxidation compared to that of 11,11-D(2)-Lin is 12.8 ± 0.6. We investigate the effects of different isotope-reinforced PUFAs and natural PUFAs on the viability of coenzyme Q-deficient Saccharomyces cerevisiae coq mutants and wild-type yeast subjected to copper stress. Cells treated with a C11-BODIPY fluorescent probe to monitor lipid oxidation products show that lipid peroxidation precedes the loss of viability due to H-PUFA toxicity. We show that replacement of just one bis-allylic hydrogen atom with deuterium is sufficient to arrest lipid autoxidation. In contrast, PUFAs reinforced with two deuterium atoms at mono-allylic sites remain susceptible to autoxidation. Surprisingly, yeast treated with a mixture of approximately 20%:80% isotope-reinforced D-PUFA:natural H-PUFA are protected from lipid autoxidation-mediated cell killing. The findings reported here show that inclusion of only a small fraction of PUFAs deuterated at the bis-allylic sites is sufficient to profoundly inhibit the chain reaction of nondeuterated PUFAs in yeast., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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.

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.

Lysyl oxidase (LOX) is implicated in several extracellular matrix related disorders, including fibrosis and cancer. Methods of inhibition of LOX in vivo include antibodies, copper sequestration and toxic small molecules such as β-aminopropionitrile. Here, we propose a novel approach to modulation of LOX activity based on the kinetic isotope effect (KIE). We show that 6,6-d(2)-lysine is oxidised by LOX at substantially lower rate, with apparent deuterium effect on V(max)/K(m) as high as 4.35 ± 0.22. Lys is an essential nutrient, so dietary ingestion of D(2)Lys and its incorporation via normal Lys turnover suggests new approaches to mitigating LOX-associated pathologies., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The facile abstraction of bis-allylic hydrogens from polyunsaturated fatty acids (PUFAs) is the hallmark chemistry responsible for initiation and propagation of autoxidation reactions. The products of these autoxidation reactions can form cross-links to other membrane components and damage proteins and nucleic acids. We report that PUFAs deuterated at bis-allylic sites are much more resistant to autoxidation reactions, because of the isotope effect. This is shown using coenzyme Q-deficient Saccharomyces cerevisiae coq mutants with defects in the biosynthesis of coenzyme Q (Q). Q functions in respiratory energy metabolism and also functions as a lipid-soluble antioxidant. Yeast coq mutants incubated in the presence of the PUFA α-linolenic or linoleic acid exhibit 99% loss of colony formation after 4h, demonstrating a profound loss of viability. In contrast, coq mutants treated with monounsaturated oleic acid or with one of the deuterated PUFAs, 11,11-D(2)-linoleic or 11,11,14,14-D(4)-α-linolenic acid, retain viability similar to wild-type yeast. Deuterated PUFAs also confer protection to wild-type yeast subjected to heat stress. These results indicate that isotope-reinforced PUFAs are stabilized compared to standard PUFAs, and they protect coq mutants and wild-type yeast cells against the toxic effects of lipid autoxidation products. These findings suggest new approaches to controlling ROS-inflicted cellular damage and oxidative stress., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Simple and scalable synthesis of 5- and 6-carboxytetramethylrhodamines (TAMRAs) is reported. Acylation of 3-dimethylaminophenol with 1,2,4-benzenetricarboxylic anhydride afforded a mixture of 4-dimethylamino-2-hydroxy-2',4'(5')-dicarboxybenzophenones, which can be easily separated into individual compounds upon recrystallization from methanol and acetic acid. Individual benzophenones were reacted with 3-dimethylaminophenol to give 5- or 6-carboxytetramethylrhodamines. The dyes were converted into hydroxyprolinol-based phosphoramidite reagents suitable for oligonucleotide synthesis. 5- and 6-TAMRA isomers on oligonucleotides showed similar absorption and emission spectra. Fluorescence quantum yield of the dyes correlates with the presence of dG nucleosides in the adjacent region of oligonucleotide sequence. Several energy transfer primers containing on their 5'-termini (6-FAM)dT(n)(6-TAMRA) dye system (n = 0, 2, 4, 6, 8, 10, 12, 14) were prepared, and their spectral properties were studied.

A new method suitable for single nucleotide polymorphism (SNP) detection using differential oligonucleotide probe extension has been developed. Sulfur-linked laser-cleavable trityl labels are implemented in this protocol. The method is based on mass spectrometry and utilizes a single surface for affinity purification of extended probes and matrix-independent desorption-ionization of the cleavable labels. The usefulness of this method for SNP genotyping is demonstrated.

The rational design of novel triarylmethyl (trityl)-based mass tags (MT) for mass-spectrometric (MS) applications is described. We propose a "pK(R+) rule" to correlate the stability of trityl carbocations with their MS performance: trityls with higher pK(R+) values ionise and desorb better. Trityl blocks were synthesised that have high pK(R+) values and are stable in conditions of MS analysis; these MTs can be ionised by matrix as well as irradiation with a 337 nm nitrogen laser. (13)C-Labelled tags were prepared for MS quantitation applications. Moreover, the tags were equipped with a variety of functional groups allowing conjugation with different functionalities within (bio)molecules to enhance the MS characteristics of the latter. The MS behaviour of model polycationic trityl compounds with and without the matrix was studied to reveal that poly-trityl clusters are always singly charged under the (MA)LDI-TOF conditions. Several peptide-trityl conjugates were prepared and comparisons revealed a beneficial effect of trityl tags on the conjugate detection in MS. Trityl compounds containing para-methoxy- and dimethylamine groups, as well as a xanthene fragment, showed considerable enhancement in MS detection of model peptides; thus they are promising tools for proteomic applications. Dimethoxytrityl derivatives allow one to distinguish between Arg- and Lys-containing peptides. Maleimido trityl derivatives are suitable for the efficient derivatisation of thiol-containing peptides in pyridine.

Pentafluorophenyl esters of 5- and 6-carboxyfluorescein-3',6'-O-dipivalate can be easily separated in multigram quantities by column chromatography. The individual isomers were converted into stable phosphoramidites suitable for oligonucleotide synthesis. The use of the cyclohexylcarbonyl (Chc) protecting group instead of pivaloyl (Piv) facilitates the separation of isomers. The fluorescence spectra of 5- and 6-carboxyfluoresceins on oligonucleotides were compared.

Energy transfer between different fluorescent 5-alkynyl-2' -deoxyuridines in complementary and mismatched duplexes was studied.

New reagents (CPGs and phosphoramidites) for automatic solid phase synthesis of modified oligonucleotides were designed. Three oligonucleotides carrying fluorescent label at the 5'-terminus and an anchor group at the 3'-terminus were prepared and their immobilization in orthogonal conditions on solid supports was studied.

Background: Hydrophobic polystyrene is the most common material for solid phase immunoassay. Proteins are immobilized on polystyrene by passive adsorption, which often causes considerable denaturation. Biological macromolecules were found to better retain their functional activity when immobilized on hydrophilic materials. Polyacrylamide is a common material for solid-phase carriers of biological macromolecules, including immunoreagents used in affinity chromatography. New macroformats for immunoassay modified with activated polyacrylamide derivatives seem to be promising., Results: New polymeric matrices for immunoassay in the form of 0.63-cm balls which contain hydrazide functional groups on hydrophilic polymer spacer arms at their surface shell are synthesized by modification of aldehyde-containing polystyrene balls with hydrazide derivatives of poly(meth)acrylic acid. The beads contain up to 0.31 micromol/cm2 active hydrazide groups accessible for covalent reaction with periodate-oxidized antibodies. The matrices obtained allow carrying out the oriented antibody immobilization, which increases the functional activity of immunosorbents., Conclusions: An efficient site-directed antibody immobilization on a macrosupport is realized. The polymer hydrophilic spacer arms are the most convenient and effective tools for oriented antibody coupling with molded materials. The suggested scheme can be used for the modification of any other solid supports containing electrophilic groups reacting with hydrazides.

The effect of pH of the blocking solution on the non-specific sorption of peroxidase conjugates to the 0.63-cm formylated polystyrene beads was studied. Anti-G-HRP and BSA-HRP sorption was shown to dramatically depend on pH of blocking protein solutions and was minimum at pH=pI. Sorption of G-HRP weakly depended on pH of the blocking buffer. The blocking efficiency of proteins on unmodified beads, as well as of Tween-20 on formylated beads appeared to be pH independent. The optimized blocking step resulted in a multiple decrease in the non-specific sorption of conjugates on formylated beads.