Группа биоконъюгации

Научно-исследовательские подразделения

Руководитель: Коршун Владимир Аркадьевич, д. х. н.
v-korshun@yandex.ru+7(499)724-67-15

биоконъюгация, нуклеиновые кислоты, флуоресцентные красители

Группа занимается разработкой методов синтеза и изучением свойств конъюгатов природных и биологически активных веществ, биополимеров (преимущественно нуклеиновых кислот), органических флуоресцентных красителей, полициклических ароматических соединений, стабилизированных карбкатионов, неорганических люминесцентных нанокристаллов и кластеров.

Сотрудники Группы разрабатывают вещества с высокой противовирусной активностью на основе полициклического ароматического углеводорода перилена. Получено большое число таких соединений, как на основе нуклеозидов, так и имеющих ненуклеозидную природу. Эти вещества обладают широким спектром действия: они способны ингибировать репликацию многих оболочечных вирусов, например, вируса простого герпеса, гриппа, клещевого энцефалита, гепатита C и др.

Группа занимается созданием флуоресцентных красителей для ДНК-зондов. Получены функциональные производные уже известных и новых красителей, а также разработаны эффективные методы их введения в ДНК-зонды, например, флуорогенные ДНК-зонды типа Taqman или Molecular beacons для ПЦР в режиме реального времени. Предметом исследования являются не только конъюгаты с красителями, но и с белками, а также олигонуклеотид-олигонуклеотидные конъюгаты, которые могут служить структурными блоками для сборки ДНК-наноструктур.

На основе стабилизированных карбкатионов разрабатываются масс-спектрометрические метки для биомолекул в отщепляемом (масс-тэги) и не отщепляемом (дериватизация) вариантах. В первом случае детектируются собственно метки, которые проходят стадии аналитических манипуляций, будучи прикрепленными к биомолекуле, а затем отщепляются и детектируются в масс-спектрометре. Во втором случае ионогенные метки служат для улучшения ионизуемости анализируемых молекул; в масс-спектре детектируется конъюгат молекулы с меткой. Масс-метки применены для детекции мутаций в ДНК, определения аминогликозидных и гликопептидных антибиотиков, низкомолекулярных аминов и тиолов. Например, модификация низкомолекулярных летучих аминов позволяет легко детектировать их с помощью ВЭЖХ и уменьшить предел обнаружения таких аминов.

Группа сотрудничает с Лабораторией органического синтеза, Институтом полиомиелита и вирусных энцефалитов имени М.П. Чумакова РАН, Институтом микробиологии (Армения), НИИ по изысканию новых антибиотиков имени Г.Ф. Гаузе, Лабораторией молекулярной биологии в Кембридже (Великобритания), Оксфордским университетом (Великобритания), Институтом физико-органической химии НАН Беларуси, Центром нуклеиновых кислот (Дания), Университетом Альберты (Канада), Корнельским университетом (США), Индийским институтом технологии Дели (Индия) и др.

Группа биоконъюгации образована в 2010 г. в результате получения гранта Программы «Молекулярная и клеточная биология» Президиума РАН для новых групп.

  • Получение веществ с противовирусной активностью.

  • Создание флуоресцентных ДНК-зондов.

  • Разработка олигонуклеотидных конъюгатов в качестве блоков для ДНК-наноструктур.

  • Создание масс-спектрометрических меток для детекции биомолекул.

  • Обнаружено образование межцепочечного пиренового эксимера в большой бороздке ДНК (ChemBioChem, 2003, 4:841).
  • В качестве улучшенной эксимеробразующей флуоресцентной метки для нуклеиновых кислот предложен 1-фенилэтинилпирен (Nucleosides Nucleotides, 1997, 16:1461; Eur. J. Org. Chem., 2004, 1298; Bioconjugate Chem., 2007, 18:1972), использованный в различных типах ДНК-зондов (Mutation Res., 2006, 599:144; Meth. Mol. Biol., 2009, 578:209; Chem. Eur. J., 2008, 14:11010; Chem. Commun., 2010, 46:8362; Bioconjugate Chem., 2011, 22:533; Analyst, 2016, 141:1331).
  • Впервые был применен метод прямого присоединения флуоресцентной метки к нуклеиновому основанию с помощью тройной связи (Биоорган. химия, 1996, 22:923; ChemBioChem, 2006, 7:810). Среди таких соединений обнаружен новый класс противовирусных нуклеозидов, активных по отношению к вирусам простого герпеса тип 1, гриппа, гепатита С, клещевого энцефалита и другим вирусам, имеющим липидную оболочку (Биоорган. химия, 2003, 29:289; Вопросы вирусологии, 2006:34; Tetrahedron, 2006, 62:1279; Org. Biomol. Chem., 2006, 4:1091; PNAS USA, 2010, 107:17339; J. Virol., 2013, 87:3640; Med. Chem. Commun., 2016, 7:495).
  • Разработаны новые масс-спектометрические метки (Org. Biomol. Chem., 2008, 6:4593; Масс-спектрометрия, 2015, 12:253; Analyst, 2016, 141:3289) и на их основе предложен метод детекции однонуклеотидного полиморфизма с помощью MALDI масс-спектрометрии (Anal. Chem., 2008, 80:2342; Meth. Mol. Biol., 2009, 578:345) и метод анализа аминогликозидных антибиотиков (Acta Naturae, 2016, 8:128).
  • Изучено применение реакции циклоприсоединения азидов и алкинов для получения конъюгатов ДНК (Биоорган. химия, 2010, 36:437), в том числе блоков для сборки ДНК-наноструктур (Изв. АН, Сер. хим., 2006:1220; Tetrahedron, 2008, 64:1467; Chem. Commun., 2013, 49:511; Org. Lett., 2014, 16:4590; Tetrahedron, 2016, 72:2386) и эффективных ДНК-зондов для ПЦР в режиме реального времени (Anal. Bioanal. Chem., 2012, 404:59; Analyst, 2014, 139:2867; Analyst, 2016, 141:1331; Anal. Meth., 2016, 8:5826; Mol. Cell. Probes, 2016, 30:285).

Избранные публикации

  1. Topolyan A.P., Strizhevskaya D.A., Belyaeva M.A., Brylev V.A., Ustinov A.V., Formanovsky A.A., Korshun V.A. (2016). A triphenylcyclopropenylium mass tag: synthesis and application to ultrasensitive LC/MS analysis of amines. Analyst 141, 3289–3295 [+]

    Thiol adducts of triphenylcyclopropenylium undergo efficient heterolytic dissociation under conditions of both electrospray (ESI) and laser desorption ionization (LDI) mass spectrometry giving rise to a prominent signal of an aromatic C3Ph3(+) cation. A functionalized mass tagging reagent, an activated ester carrying an S-linked C3Ph3 unit, has been developed and used for the derivatization of amines and their subsequent HPLC/ESI-MS detection in low attomolar amounts.

    ID:1519
  2. Aparin I.O., Farzan V.M., Veselova O.A., Chistov A.A., Podkolzin A.T., Ustinov A.V., Shipulin G.A., Formanovsky A.A., Korshun V.A., Zatsepin T.S. (2016). 1-Phenylethynylpyrene (PEPy) as a novel blue-emitting dye for qPCR assay. Analyst 141 (4), 1331–1338 [+]

    An alkyl azide derivative of 1-phenylethynylpyrene (PEPy) dye was prepared and used in the functionalization of oligonucleotides via click chemistry. Spectral and photo-physical properties of the PEPy-modified oligonucleotides as a single strand, and in perfect or mismatched duplexes, have been studied. A series of PEPy-Dabcyl fluorogenic TaqMan probes were synthesized and tested in qPCR. PEPy proved to be a superior substitute for AMCA as a short wavelength fluorescent dye for qPCR probes. PEPy probes were shown to significantly reduce Cq (a fractional PCR cycle used for quantification) vs. AMCA labeled probes, thus improving on the reliability of detection. Moreover, a larger increase of fluorescence during amplification was observed in the case of PEPy probes that makes this dye very suitable for an end-point PCR technique. This study broadens the panel of fluorescent dyes suitable for the use in probes for quantitative real-time PCR.

    ID:1351
  3. Chistov A.A., Kutyakov S.V., Ustinov A.V., Aparin I.O., Glybin A.V., Mikhura I.V., Korshun V.A. (2016). 2-Ethynylperylene and improved synthesis of 3-ethynylperylene. Tetrahedron Lett. 57 (9), 1003–1006 [+]

    3-Ethynylperylene 1, an important precursor for fluorescent dyes, fluorescent nucleosides, and potent nucleoside-based antivirals, has been prepared from perylene on a multigram scale (3 steps, 55% overall yield). A simple synthesis of 2-ethynylperylene 2 from perylene (5 steps, 20% yield) has been developed. UV and fluorescence spectra of the ethynylperylenes 1 and 2 are compared to those of perylene.

    ID:1415
  4. Chistov , Ustinov , Korshun  (2016). Rigid amphipathic nucleosides suppress reproduction of the tick-borne encephalitis virus. Med. Chem. Commun. 7 (3), 495–499 [+]
    Rigid amphipathic fusion inhibitors (RAFIs), 5-arylethynyl uracil nucleosides with bulky aryl groups, appeared to have considerable activity against tick-borne encephalitis virus (TBEV) in cell culture. The rigid ethynyl linker and perylene residue are essential structural prerequisites for high activity, giving EC50 values of 18 and 24 nM for 5-(perylen-3-yl)ethynyl-arabino-uridine and 5-(perylen-3-yl)ethynyl-2′-deoxy-uridine, respectively, upon simultaneous mixing of the compounds, cells and virus.
    ID:1501
  5. Ponomarenko A.I., Brylev V.A., Nozhevnikova E.V., Korshun V.A. (2015). Recent advances in self-assembled fluorescent DNA structures and probes. Current Topics in Medicinal Chemistry 15 (13), 1162–78 [+]

    The combined efforts of chemistry, nanotechnology, and spectroscopy led to the development of self-assembled fluorescent DNA nanostructures, an inexhaustible source of refined and bizarre tools and powerful techniques for research and diagnostic applications. This multidisciplinary area has tremendous prospects for science and technology.

    ID:1327
  6. Топольян А.П., Стрижевская Д.А., Слюндина М.С., Беляева М.А., Иванова О.М., Коршун В.А., Устинов А.В., Михура И.В., Формановский А.А. (2015). Дериватизация первичных аминов катионом трис(2,6-диметоксифенил)метилия для анализа методом масс-спектрометрии МАЛДИ. Масс-спектрометрия 12 (4), 253–258 [+]

    Предложен новый способ дериватизации первичных аминов для их последующего анализа с помощью масс-спектрометрии с матрично-активированной лазерной десорбцией/ионизацией (МАЛДИ). Трис(2,6-диметоксифенил)метилий катион в мягких условиях реагирует с первичными аминами с образованием производных с фиксированным зарядом, обладающих высокой эффективностью десорбции/ионизации в МАЛДИ и поглощением в УФ-области. Подход опробован на ряде аминов, включая биологически активные соединения со стимулирующими и лекарственными свойствами.

    ID:1481
  7. Fomich M.A., Kvach M.V., Navakouski M.J., Weise C., Baranovsky A.V., Korshun V.A., Shmanai V.V. (2014). Azide phosphoramidite in direct synthesis of azide-modified oligonucleotides. Org. Lett. 16 (17), 4590–4593 [+]

    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.

    ID:1087
  8. Ryazantsev D.Y., Kvach M.V., Tsybulsky D.A., Prokhorenko I.A., Stepanova I.A., Martynenko Y.V., Gontarev S.V., Shmanai V.V., Zavriev S.K., Korshun V.A. (2014). Design of molecular beacons: 3' couple quenchers improve fluorogenic properties of a probe in real-time PCR assay. Analyst 139 (11), 2867–72 [+]

    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).

    ID:1019
  9. Astakhova I.K., SanthoshKumar T., Campbell M.A., Ustinov A.V., Korshun V.A., Wengel J. (2013). Branched DNA nanostructures efficiently stabilised and monitored by novel pyrene-perylene 2'-α-l-amino-LNA FRET pairs. Chem. Commun. (Camb.) 49 (5), 511–3 [+]

    Novel pyrene-perylene α-l-LNA FRET pairs described herein effectively detect assembly of 2- and 3-way branched DNA nanostructures prepared by postsynthetic microwave-assisted CuAAC click chemistry. The fluorescent signalling of assembly by internally positioned FRET pairs is achieved with low to no fluorescence background signal, remarkably low limit of target detection values and stabilization of the resulting nanostructures.

    ID:754
  10. Colpitts C.C., Ustinov A.V., Epand R.F., Epand R.M., Korshun V.A., Schang L.M. (2013). 5-(Perylen-3-yl)ethynyl-arabino-uridine (aUY11), an arabino-based rigid amphipathic fusion inhibitor, targets virion envelope lipids to inhibit fusion of influenza virus, hepatitis C virus, and other enveloped viruses. J. Virol. 87 (7), 3640–54 [+]

    Entry of enveloped viruses requires fusion of viral and cellular membranes. Fusion requires the formation of an intermediate stalk structure, in which only the outer leaflets are fused. The stalk structure, in turn, requires the lipid bilayer of the envelope to bend into negative curvature. This process is inhibited by enrichment in the outer leaflet of lipids with larger polar headgroups, which favor positive curvature. Accordingly, phospholipids with such shape inhibit viral fusion. We previously identified a compound, 5-(perylen-3-yl)ethynyl-2'-deoxy-uridine (dUY11), with overall shape and amphipathicity similar to those of these phospholipids. dUY11 inhibited the formation of the negative curvature necessary for stalk formation and the fusion of a model enveloped virus, vesicular stomatitis virus (VSV). We proposed that dUY11 acted by biophysical mechanisms as a result of its shape and amphipathicity. To test this model, we have now characterized the mechanisms against influenza virus and HCV of 5-(perylen-3-yl)ethynyl-arabino-uridine (aUY11), which has shape and amphipathicity similar to those of dUY11 but contains an arabino-nucleoside. aUY11 interacted with envelope lipids to inhibit the infectivity of influenza virus, hepatitis C virus (HCV), herpes simplex virus 1 and 2 (HSV-1/2), and other enveloped viruses. It specifically inhibited the fusion of influenza virus, HCV, VSV, and even protein-free liposomes to cells. Furthermore, aUY11 inhibited the formation of negative curvature in model lipid bilayers. In summary, the arabino-derived aUY11 and the deoxy-derived dUY11 act by the same antiviral mechanisms against several enveloped but otherwise unrelated viruses. Therefore, chemically unrelated compounds of appropriate shape and amphipathicity target virion envelope lipids to inhibit formation of the negative curvature required for fusion, inhibiting infectivity by biophysical, not biochemical, mechanisms.

    ID:852
  11. Kvach M.V., Tsybulsky D.A., Shmanai V.V., Prokhorenko I.A., Stepanova I.A., Korshun V.A. (2013). Non-nucleoside phosphoramidites of xanthene dyes (FAM, JOE, and TAMRA) for oligonucleotide labeling. Curr Protoc Nucleic Acid Chem Chapter 4, Unit 4.55 [+]

    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.

    ID:851
  12. Osterman I.A., Ustinov A.V., Evdokimov D.V., Korshun V.A., Sergiev P.V., Serebryakova M.V., Demina I.A., Galyamina M.A., Govorun V.M., Dontsova O.A. (2013). A nascent proteome study combining click chemistry with 2DE. Proteomics 13 (1), 17–21 [+]

    To investigate the dynamic cellular response to a condition change, selective labeling of the nascent proteome is necessary. Here, we report a method combining click chemistry protein labeling with 2D DIGE. To test the relevance of the method, we compared nascent proteomes of actively growing bacterial cells with that of cells exposed to protein synthesis inhibitor, erythromycin. Cells were incubated with methionine analog, homopropargyl glycin, and their nascent proteome was selectively labeled with monosulfonated neutral Cy3 and Cy5 azides specially synthesized for this purpose. Following fluorescent labeling, the protein samples were mixed and subjected to standard 2D DIGE separation. The method allowed us to reveal a dramatic reduction of newly synthesized proteins upon erythromycin treatment, while the total proteome was not significantly affected. Additionally, several proteins, whose synthesis was resistant to erythromycin, were identified.

    ID:1063
  13. Ryazantsev D.Y., Tsybulsky D.A., Prokhorenko I.A., Kvach M.V., Martynenko Y.V., Philipchenko P.M., Shmanai V.V., Korshun V.A., Zavriev S.K. (2012). Two-dye and one- or two-quencher DNA probes for real-time PCR assay: synthesis and comparison with a TaqMan™ probe. Analytical and bioanalytical chemistry 404 (1), 59–68 [+]

    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.

    ID:753
  14. Tsybulsky D.A., Kvach M.V., Stepanova I.A., Korshun V.A., Shmanai V.V. (2012). 4',5'-Dichloro-2',7'-dimethoxy-5(6)-carboxyfluorescein (JOE): synthesis and spectral properties of oligonucleotide conjugates. J. Org. Chem. 77 (2), 977–84 [+]

    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.

    ID:656
  15. Kocherginskaya P.B., Romanova A.V., Prokhorenko I.A., Itkis D.M., Korshun V.A., Goodilin E.A., Tretyakov Y.D. (2011). Modification of quantum dots with nucleic acids. Russian Chemical Reviews 80 (12), 1209–1221 [+]

    The key principles and modern approaches to targeted modification of semiconductor colloidal nanoparticles, quantum dots, which exhibit unique photophysical properties and are a promising class of luminescent markers, are discussed. Attention is given to the preparation of their bioconjugates with nucleic acids, promising tools for biological microchips and resonance energy transfer sensors. The bibliography includes 80 references.

    ID:1066
  16. Astakhova I.V., Ustinov A.V., Korshun V.A., Wengel J. (2011). LNA for optimization of fluorescent oligonucleotide probes: improved spectral properties and target binding. Bioconjug. Chem. 22 (4), 533–9 [+]

    Mixmer LNA/DNA fluorescent probes containing the 1-(phenylethynyl)pyrene fluorophore attached to 2'-arabino-uridine were synthesized and studied. The conjugates displayed significantly higher hybridization affinity to target DNA, increased fluorescence quantum yields of single-stranded oligonucleotides and their duplexes, and improved ability to form an interstrand excimer compared to analogous non-LNA probes.

    ID:755
  17. Astakhova I.V., Lindegaard D., Korshun V.A., Wengel J. (2010). Novel interstrand communication systems within DNA duplexes based on 1-, 2- and 4-(phenylethynyl)pyrenes attached to 2'-amino-LNA: high-affinity hybridization and fluorescence sensing. Chem. Commun. (Camb.) 46 (44), 8362–4 [+]

    Functionalisation of 2'-amino-LNA oligonucleotides with 1-, 2- and 4-(phenylethynyl)pyrene fluorophores via a carbonyl linker (PEPyc) resulted in efficient interstrand communication systems in nucleic acid duplexes, providing effective tools for stabilization of nanostructures and fluorescence monitoring of DNA self-assembly.

    ID:756
  18. StVincent M.R., Colpitts C.C., Ustinov A.V., Muqadas M., Joyce M.A., Barsby N.L., Epand R.F., Epand R.M., Khramyshev S.A., Valueva O.A., Korshun V.A., Tyrrell D.L., Schang L.M. (2010). Rigid amphipathic fusion inhibitors, small molecule antiviral compounds against enveloped viruses. Proc. Natl. Acad. Sci. U.S.A. 107 (40), 17339–44 [+]

    Antiviral drugs targeting viral proteins often result in prompt selection for resistance. Moreover, the number of viral targets is limited. Novel antiviral targets are therefore needed. The unique characteristics of fusion between virion envelopes and cell membranes may provide such targets. Like all fusing bilayers, viral envelopes locally adopt hourglass-shaped stalks during the initial stages of fusion, a process that requires local negative membrane curvature. Unlike cellular vesicles, however, viral envelopes do not redistribute lipids between leaflets, can only use the energy released by virion proteins, and fuse to the extracellular leaflets of cell membranes. Enrichment in phospholipids with hydrophilic heads larger than their hydrophobic tails in the convex outer leaflet of vesicles favors positive curvature, therefore increasing the activation energy barrier for fusion. Such phospholipids can increase the activation barrier beyond the energy provided by virion proteins, thereby inhibiting viral fusion. However, phospholipids are not pharmacologically useful. We show here that a family of synthetic rigid amphiphiles of shape similar to such phospholipids, RAFIs (rigid amphipathic fusion inhibitors), inhibit the infectivity of several otherwise unrelated enveloped viruses, including hepatitis C and HSV-1 and -2 (lowest apparent IC(50) 48 nM), with no cytotoxic or cytostatic effects (selectivity index > 3,000) by inhibiting the increased negative curvature required for the initial stages of fusion.

    ID:757
  19. Ustinov A.V., Stepanova I.A., Dubniakova V.V., Zatsepin T.S., Nozhevnikova E.V., Korshun V.A. (2010). Modification of nucleic acids using [3+2] dipolar cycloaddition of azides and alkynes. Russ. J. Bioorgan. Chem. 36 (4), 401–445 [+]

    The use of the reaction of azide and alkyne cycloaddition for the synthesis of nucleic acid conjugates and DNA oligomer analogues is considered. The data on chemical and enzymatic techniques of azides and alkynes introduction into DNA are summarized.

    ID:657
  20. Astakhova I.V., Malakhov A.D., Stepanova I.A., Ustinov A.V., Bondarev S.L., Paramonov A.S., Korshun V.A. (2009). 1-Phenylethynylpyrene (1-PEPy) as refined excimer forming alternative to pyrene: case of DNA major groove excimer. Bioconjug. Chem. 18 (6), 1972–80 [+]

    1-Phenylethynylpyrene fluorochrome was studied as meta- and para-derivatives of arabino-uridine-2'-carbamates in ss and dsDNA. 1-PEPy showed red-shifted emission and increased fluorescence quantum yield compared to pyrene. Although 1-PEPy has very short excited lifetime (<2.5 ns), it is able to form inter- and intrastrand excimers on DNA, probably resulting from spatial preorganization of two dye molecules.

    ID:660
  21. Birikh K.R., Bernad P.L. Jr, Shmanai V.V., Malakhov A.D., Shchepinov M.S., Korshun V.A. (2009). SNP detection using trityl mass tags. Methods Mol. Biol. 578, 345–61 [+]

    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.

    ID:758
  22. Prokhorenko I.A., Astakhova I.V., Momynaliev K.T., Zatsepin T.S., Korshun V.A. (2009). Phenylethynylpyrene excimer forming hybridization probes for fluorescence SNP detection. Methods Mol. Biol. 578, 209–22 [+]

    Excimer formation is a unique feature of some fluorescent dyes (e.g., pyrene) which can be used for probing the proximity of biomolecules. Pyrene excimer fluorescence has previously been used for homogeneous detection of single nucleotide polymorphism (SNP) on DNA. 1-Phenylethynylpyrene (1-1-PEPy), a photostable pyrene derivative with redshifted fluorescence, is able to form excimers (emission maximum about 500-510 nm) and is well suitable for nucleic acid labeling. We have shown the utility of 1-1-PEPy in the excimer-forming DNA probes for detection of 2144A/G and 2143A/G transitions, and 2143A/C substitution in the 23S ribosomal RNA gene of Helicobacter pylori strains resistant to clarithromycin. The phenylethynylpyrene pair can be generated either from 1-1-PEPy pseudonucleoside 4-[4-(pyren-1-ylethynyl)phenyl]-1,3-butanediol or from 2'-O-(1-PEPy) modified nucleosides--2'-O-[3-(pyren-1-ylethynyl)benzyl]uridine and 2'-O-[4-(pyren-1-ylethynyl)benzyl]uridine.

    ID:759
  23. Ustinov A.V., Shmanai V.V., Patel K., Stepanova I.A., Prokhorenko I.A., Astakhova I.V., Malakhov A.D., Skorobogatyi M.V., Bernad P.L. Jr, Khan S., Shahgholi M., Southern E.M., Korshun V.A., Shchepinov M.S. (2008). Reactive trityl derivatives: stabilised carbocation mass-tags for life sciences applications. Org. Biomol. Chem. 6 (24), 4593–608 [+]

    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.

    ID:659
  24. Astakhova I.V., Korshun V.A., Jahn K., Kjems J., Wengel J. (2008). Perylene attached to 2'-amino-LNA: synthesis, incorporation into oligonucleotides, and remarkable fluorescence properties in vitro and in cell culture. Bioconjug. Chem. 19 (10), 1995–2007 [+]

    During recent years, fluorescently labeled oligonucleotides have been extensively investigated within diagnostic approaches. Among a large variety of available fluorochromes, the polyaromatic hydrocarbon perylene is an object of increasing interest due to its high fluorescence quantum yield, long-wave emission compared to widely used pyrene, and photostability. These properties make perylene an attractive label for fluorescence-based detection in vitro and in vivo. Herein, the synthesis of 2'- N-(perylen-3-yl)carbonyl-2'-amino-LNA monomer X and its incorporation into oligonucleotides is described. Modification X induces high thermal stability of DNA:DNA and DNA:RNA duplexes, high Watson-Crick mismatch selectivity, red-shifted fluorescence emission compared to pyrene, and high fluorescence quantum yields. The thermal denaturation temperatures of duplexes involving two modified strands are remarkably higher than those for double-stranded DNAs containing modification X in only one strand, suggesting interstrand communication between perylene moieties in the studied 'zipper' motifs. Fluorescence of single-stranded oligonucleotides having three monomers X is quenched compared to modified monomer (quantum yields Phi F = 0.03-0.04 and 0.67, respectively). However, hybridization to DNA/RNA complements leads to Phi F increase of up to 0.20-0.25. We explain it by orientation of the fluorochrome attached to the 2'-position of 2'-amino-LNA in the minor groove of the nucleic acid duplexes, thus protecting perylene fluorescence from quenching with nucleobases or from the environment. At the same time, the presence of a single mismatch in DNA or RNA targets results in up to 8-fold decreased fluorescence intensity of the duplex. Thus, distortion of the duplex geometry caused by even one mismatched nucleotide induces remarkable quenching of fluorescence. Additionally, a perylene-LNA probe is successfully applied for detection of mRNA in vivo providing excitation wavelength, which completely eliminates cell autofluorescence.

    ID:762
  25. Birikh K.R., Korshun V.A., Bernad P.L. Jr, Malakhov A.D., Milner N., Khan S., Southern E.M., Shchepinov M.S. (2008). Novel mass tags for single nucleotide polymorphism detection. Anal. Chem. 80 (7), 2342–50 [+]

    A new method suitable for single nucleotide polymorphism detection and other applications based on oligonucleotide probe extension has been developed. 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. A new family of sulfur-linked laser-cleavable trityl labels with vastly improved flying abilities is implemented in this study. Corresponding reagents compatible with automated oligonucleotide synthesis are presented. Utility of this method for SNP genotyping is demonstrated.

    ID:765
  26. Kvach M.V., Ustinov A.V., Stepanova I.A., Malakhov A.D., Skorobogatyi M.V., Shmanai V.V., Korshun V.A. (2008). A convenient synthesis of cyanine dyes: reagents for labeling of biomolecules. Eur. J. Org. Chem. 12, 2107–2117 [+]

    Four tetramethylindo(di)carbocyanine-derived carboxylicacids have been prepared using a modified one-pot procedure for the dye assembly. The acids were converted into oxysuccinimide esters and phosphoramidite reagents. The efficiency of the reagents in the labeling of oligonucleotides and proteins has been demonstrated.

    ID:666
  27. Astakhova I.V., Korshun V.A., Wengel J. (2008). Highly fluorescent conjugated pyrenes in nucleic acid probes: (phenylethynyl)pyrenecarbonyl-functionalized locked nucleic acids. Chemistry 14 (35), 11010–26 [+]

    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.

    ID:760
  28. Filichev V.V., Astakhova I.V., Malakhov A.D., Korshun V.A., Pedersen E.B. (2008). 1-, 2-, and 4-ethynylpyrenes in the structure of twisted intercalating nucleic acids: structure, thermal stability, and fluorescence relationship. Chemistry 14 (32), 9968–80 [+]

    A postsynthetic, on-column Sonogashira reaction was applied on DNA molecules modified by 2- or 4-iodophenylmethylglycerol in the middle of the sequence, to give the corresponding ortho- and para-twisted intercalating nucleic acids (TINA) with 1-, 2-, and 4-ethynylpyrene residues. The convenient synthesis of 2- and 4-ethynylpyrenes started from the hydrogenolysis of pyrene that has had the sulfur removed and separation of 4,5,9,10-tetrahydropyrene and 1,2,3,6,7,8-hexahydropyrene, which were later converted to the final compounds by successive Friedel-Crafts acetylation, aromatization by 2,3-dichloro-5,6-dicyano-1,4-benzoquinone, and a Vilsmeier-Haack-Arnold transformation followed by a Bodendorf fragmentation. Significant alterations in thermal stability of parallel triplexes and antiparallel duplexes were observed upon changing the attachment of ethynylpyrenes from para to ortho in homopyrimidine TINAs. Thus, for para-TINAs the bulge insertion of an intercalator led to high thermal stability of Hoogsteen-type parallel triplexes and duplexes, whereas Watson-Crick-type duplexes were destabilized. In the case of ortho-TINA, both Hoogsteen and Watson-Crick-type complexes were stabilized. Alterations in the thermal stability were highly influenced by the ethynylpyrene isomers used. This also led to TINAs with different changes in fluorescence spectra depending on the secondary structures formed. Stokes shift of approximately 100 nm was detected for pyren-2-ylethynylphenyl derivatives, whereas values for 1- and 4-ethynylpyrenylphenyl conjugates were 10 and 40 nm, respectively. In contrast with para-TINAs, insertion of two ortho-TINAs opposite each other in the duplex as a pseudo-pair resulted in formation of an excimer band at 505 nm for both 1- and 4-ethynylpyrene analogues, which was also accompanied with higher thermal stability.

    ID:761
  29. Astakhova I.V., Korshun V.A. (2008). 2- and 4-phenylethynylpyrenes--new fluorescent labels for DNA. Bioorg. Khim. 34 (4), 570–2 [+]

    It was shown by the example of 1-, 2-, and 4-phenylethynylpyrene 2'-arabino-carbamate derivatives of uridine that the position of pyrene substitution substantially affects the spectral and photophysical properties of the fluorophore and its ability to interact with the nucleic base.

    ID:763
  30. Lindegaard D., Madsen A.S., Astakhova I.V., Malakhov A.D., Babu B.R., Korshun V.A., Wengel J. (2008). Pyrene-perylene as a FRET pair coupled to the N2'-functionality of 2'-amino-LNA. Bioorg. Med. Chem. 16 (1), 94–9 [+]

    Detection of nucleic acid hybridization via fluorescence resonance energy transfer (FRET) using pyren-1-ylmethyl and perylen-3-ylmethyl N2'-functionalized 2'-amino-LNA nucleosides incorporated into oligonucleotides exhibited a clear distance dependence of the FRET efficiency, ranging from below 10% when the fluorophores were approximately 40A apart to approximately 90% when the fluorophores were in close proximity.

    ID:766
  31. Ustinov A.V., Dubnyakova V.V., Korshun V.A. (2008). A convenient “click chemistry” approach to perylene diimide–oligonucleotide conjugates. Tetrahedron 64 (7), 1467–1473 [+]

    Terminal alkyne-containing phosphoramidite and solid support were prepared and used for the synthesis of 5′- and 3′-alkyne-modified oligonucleotides. The copper-catalyzed Huisgen [3+2] cycloaddition reaction in water–DMSO was used for the efficient preparation of perylene diimide–oligonucleotide conjugates suitable for constructing various types of PDI-modified DNA duplexes.

    ID:1067
  32. Kvach M.V., Stepanova I.A., Prokhorenko I.A., Stupak A.P., Bolibrukh D.A., Korshun V.A., Shmanai V.V. (2007). Practical synthesis of isomerically pure 5- and 6-carboxytetramethylrhodamines, useful dyes for DNA probes. Bioconjug. Chem. 20 (8), 1673–82 [+]

    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.

    ID:658
  33. Kvach M.V., Tsybulsky D.A., Ustinov A.V., Stepanova I.A., Bondarev S.L., Gontarev S.V., Korshun V.A., Shmanai V.V. (2007). 5(6)-carboxyfluorescein revisited: new protecting group, separation of isomers, and their spectral properties on oligonucleotides. Bioconjug. Chem. 18 (5), 1691–6 [+]

    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.

    ID:661
  34. Prokhorenko I.A., Malakhov A.D., Kozlova A.A., Momynaliev K., Govorun V.M., Korshun V.A. (2006). Phenylethynylpyrene-labeled oligonucleotide probes for excimer fluorescence SNP analysis of 23S rRNA gene in clarithromycin-resistant Helicobacter pylori strains. Mutat. Res. 599 (1-2), 144–151 [+]

    The use of phenylethynylpyrene excimer forming pair in the design of specific fluorescent probes for determination of A2144G (A2143G and/or A2143C) mutations in 23S rRNA gene of Helicobacter pylori is described. Analysis of fluorescence spectra of model duplexes revealed optimal positions of fluorophore residues in the probe sequences for maximum efficiency of SNP detection. Application of excimer forming probes for analysis of DNA samples isolated from natural bacterial strains of H. pylori was demonstrated.

    ID:1068
  35. Ustinov A.V., Korshun V.A. (2006). Oligonucleotides containing arylacetylene residues: synthesis and post-synthetic modification via [3+2] cycloaddition. RUSSIAN CHEMICAL BULLETIN 55 (7), 1268–1274 [+]

    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 CuI-catalyzed [3+2] dipolar cycloaddition. Fluorescent conjugates were obtained in high yields and characterized by mass spectra.

    ID:1069
  36. Skorobogatyi M.V., Malakhov A.D., Pchelintseva A.A., Turban A.A., Bondarev S.L., Korshun V.A. (2006). Fluorescent 5-alkynyl-2'-deoxyuridines: high emission efficiency of a conjugated perylene nucleoside in a DNA duplex. Chembiochem 7 (5), 810–6 [+]

    Four fluorescent 5-alkynyl-2'-deoxyuridines were studied in DNA oligonucleotides and their duplexes. The fluorescence response to hybridization differs dramatically for nucleosides containing a perylene fluorochrome either conjugated or not conjugated to the nucleobase. The conjugated nucleoside, 5-(perylen-3-ylethynyl)-2'-deoxyuridine, shows enhanced long-wavelength emission in the DNA duplex, in contrast to the blue fluorescence of perylene on a flexible linker (in 5-[(perylen-3-yl)methoxyprop-1-ynyl]-2'-deoxyuridine), which is quenched upon duplex formation.

    ID:767
  37. Skorobogatyi M.V., Ustinov A.V., Stepanova I.A., Pchelintseva A.A., Petrunina A.L., Andronova V.L., Galegov G.A., Malakhov A.D., Korshun V.A. (2006). 5-Arylethynyl-2'-deoxyuridines, compounds active against HSV-1. Org. Biomol. Chem. 4 (6), 1091–6 [+]

    Three new 5-arylethynyl-2'-deoxyuridines containing bulky aryls have been prepared and tested against HSV-1 in Vero cells. The introduction of a substituent in the phenyl group of an inactive compound, 5-phenylethynyl-2'-deoxyuridine, leads to the appearance of anti-HSV properties. The most active compounds are those containing a polycyclic aromatic hydrocarbon residue attached to the 5 position of 2'-deoxyuridine through a rigid triple bond.

    ID:662
  38. Skorobogatyi M.V., Pschelintseva A.A., Petrunina A.L., Stepanova I.A., Andronova V.L., Galegov V.A., Malakhov A.D., Korshun V.A. (2006). 5-Alkynyl-2′-deoxyuridines, containing bulky aryl groups: evaluation of structure–anti-HSV-1 activity relationship. Tetrahedron 62 (6), 1279–1287 [+]

    Four 5-alkynyl-2′-deoxyuridines containing different bulky substituents and flexible linkers between the triple bond and the aromatic residue have been prepared and tested against HSV-1 in Vero cells. Two nucleosides containing carbonyl groups, 5-(4-benzoylphenoxypropyn-1-yl)-2′-deoxyuridine and 5-(estron-3-yloxypropyn-1-yl)-2′-deoxyuridine, showed low cytotoxicity and moderate antiviral activity. The flexible linker appears not to be favorable for antiviral properties of 5-alkynyl-2′deoxyuridines: 5-[(perylen-3-yl)methoxypropyn-1-yl]-2′-deoxyuridine showed considerable cytotoxicity and no antiviral activity in contrast to the active and nontoxic 5-(perylen-3-ylethynyl)-2′-deoxyuridine, a nucleoside with a rigid triple-bond-connection of the aromatic system to the nucleobase.

    ID:664
  39. Kvach M.V., Gontarev S.V., Prokhorenko I.A., Stepanova I.A., Shmanai V.V., Korshun V.A. (2006). A simple reagent for the synthesis of oligonucleotides labeled with 3,3,3′,3′-tetramethyl-2,2′-indodicarbocyanine. Russ. Chem. Bull. Int. Ed. 55 (1), 159–163 [+]

    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.

    ID:665
  40. Bernad P.L. Jr, Khan S., Korshun V.A., Southern E.M., Shchepinov M.S. (2005). S(O)-pixyl protecting group as efficient mass-tag. Chem. Commun. (Camb.)  (27), 3466–8 [+]

    We report herein the design, preparation and first applications of novel trityl tags with adjustable stability, efficient as protecting groups or MS analytes.

    ID:1070
  41. Khan S., Bernad P.L. Jr, Korshun V.A., Southern E.M., Shchepinov M.S. (2005). Synthesis of S-pixyl derivatives for mass spectrometric application. Synlett  (16), 2453–2456 [+]

    Synthesis of novel S-pixyls based on the thioxanthyl skeleton is described. Thioxanthone derivatives were prepared by a regioselective intramolecular Friedel-Crafts acylation reaction and converted into S-pixyl derivatives by Grignard synthesis. S-Pixyl carbocations stabilised by electron donating groups on the thioxanthyl backbone produced exceptional mass spectra under (MA)LDI conditions (laser desorption ionisation, both with and without ­matrix), and can be detected down to femtomol levels.

    ID:1072
  42. Dioubankova N.N., Malakhov A.D., Shenkarev Z.O., Korshun V.A. (2004). Oligonucleotides containing new fluorescent 1-phenylethynylpyrene and 9,10-bis(phenylethynyl)anthracene uridine-2′-carbamates: synthesis and properties. Tetrahedron 60 (21), 4617–4626 [+]

    The synthesis of two novel fluorescent uridine-2′-carbamate phosphoramidites is described. The reagents carrying fluorescent polyaromatic hydrocarbons 1-phenylethynylpyrene (PEPy) or 9,10-bis(phenylethynyl)anthracene (BPEA) are suitable for oligonucleotide synthesis. Prepared oligonucleotide conjugates show strong dye emissions at 401 and 485 nm, but low FRET rate when located in the oligonucleotide duplex. The dyes show considerable compensation of the usual carbamate duplex destabilization. The possible explanation of both effects is binding of PEPy and BPEA to the minor groove of the DNA duplex.

    ID:1073
  43. Malakhov A.D., Skorobogatyi M.V., Prokhrenko I.A., Gontarev S.V., Kozhich D.T., Stetsenko D.A., Stepanova I.A., Shenkarev Z.O., Berlin Yu.A., Korshun V.A. (2004). 1-Phenylethynylpyrene and 9,10-bis(phenylethynyl)anthracene, useful fluorescent dyes for DNA labeling: excimer formation and energy transfer. Eur.J.Org.Chem  (6), 1298–1307 [+]

    A series of novel modifying reagents, including phosphoramidites and solid supports, have been synthesized, and used for the introduction of 1-(phenylethynyl)pyrene (PEPy) and 9,10-bis(phenylethynyl)anthracene (BPEA) fluorescent dyes into predetermined positions of synthetic oligonucleotides. These two fluorophores have been shown to constitute an energy donor−acceptor pair, and can be used as such in fluorescent oligonucleotide probes, designed for the detection and structural studies of nucleic acids. The sensitivity of the probe to duplex formation is demonstrated. The formation of the PEPy and BPEA excimers is reported for the first time on nucleic acids.

    ID:663
  44. Dioubankova N.N., Malakhov A.D., Stetsenko D.A., Korshun V.A. (2004). Detection of point mutations using pyrene-labeled DNA probes. RUSSIAN CHEMICAL BULLETIN 53 (2), 463–470 [+]

    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 (λmax 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 (λmax 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.

    ID:1074
  45. Dioubankova N.N., Malakhov A.D., Stetsenko D.A., Gait M.J., Volynsky P.E., Efremov R.G., Korshun V.A. (2003). Pyrenemethyl ara-uridine-2'-carbamate: a strong interstrand excimer in the major groove of a DNA duplex. Chembiochem 4 (9), 841–7 [+]

    The synthesis of new nucleoside derivatives, ara-uridine-2'-carbamates, and their incorporation into synthetic DNA oligomers is described. The modification directs ligands into the major groove of duplex DNA and somewhat destabilizes the duplexes of modified oligonucleotides with complementary DNA or RNA. In the case of pyrenemethyl carbamate modification in DNA-DNA duplexes, the destabilization is considerably reduced. The pyrenemethyl derivative also shows remarkable spectral properties: a "reversed" absorbance change for pyrene at 350 nm in the course of denaturation of the DNA duplex, as compared to the change seen in the nucleotide absorbance at 260 nm. This derivatization also causes pronounced sequence-dependent excimer formation in the major groove.

    ID:768
  46. Andronova V.L., Skorobogatyi M.V., Manasova E.V., Berlin Y.A., Korshun V.A., Galegov G.A. (2003). Antiviral activity of some 5-arylethynyl 2'-deoxyuridine derivatives. Russ. J. Bioorgan. Chem. 29 (3), 262–266 [+]

    5-(3-Perylenylethynyl)-2'-deoxyuridine was prepared by cross-linking 5-iodo-2'-deoxyuridine derivatives with 3-ethynylperylene followed by deprotection. 5-(1-Perylenylethynyl)-, 5-(3-perylenylethynyl)-, and 5-[4-(2-benzoxazolyl)phenylethynyl]-2'-deoxyuridine were found to inhibit in Vero cells the replication of type 1 herpes simplex virus and its drug-resistant strains.

    ID:1071
  47. Shchepinov M.S., Korshun V.A. (2003). Recent applications of bifunctional trityl groups. Chem. Soc.Rev. 32 (3), 170–80 [+]

    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.

    ID:769
  48. Korshun V.A., Stetsenko D.A., Gait M.J. (2003). Uridine 2'-carbamates: facile tools for oligonucleotide 2'-functionalization. Curr Protoc Nucleic Acid Chem Chapter 4, Unit 4.21 [+]

    A facile method for preparation of uridine 2'-carbamate derivatives based on reaction of 3',5'-disilyl-protected uridine with 1,1'-carbonyldiimidazole followed by treatment with an aliphatic amine is presented. A phosphoramidite monomer suitable for automated oligonucleotide synthesis is obtained in a few steps. The compounds are useful for the introduction of various labels and modifications into an oligonucleotide chain. Although 2'-carbamate modification is somewhat destabilizing for DNA-DNA and DNA-RNA duplexes, it is suitable for the direction of ligands into the minor groove of duplexes or at non-base-paired sites (e.g., loops and bulges) of oligonucleotides. Pyrene-modified oligonucleotide 2'-carbamates show a considerable increase in fluorescence intensity upon hybridization to a complementary RNA (but not DNA).

    ID:764
  49. Dioubankova N.N., Malakhov A.D., Stetsenko D.A., Korshun V.A., Gait M.J. (2002). (R)-2,4-Dihydroxybutyramide seco-pseudonucleosides: new versatile homochiral synthons for synthesis of modified oligonucleotides. Org. Lett. 4 (26), 4607–4610 [+]

     Two series of seco-pseudonucleoside synthons were synthesized from (R)-(+)-alpha-hydroxy-gamma-butyrolactone and (R)-(-)-pantolactone by aminolysis, side-chain protection, dimethoxytritylation, and phosphitylation or solid-phase attachment. The phosphoramidites and solid supports were used in automated DNA synthesis to prepare oligonucleotides modified with one or more 2,4-dihydroxybutyramide units bearing side-chain reporter groups. These new oligonucleotide modification reagents allow the introduction of a label into any desired position within an oligonucleotide chain during solid-phase assembly.

    ID:1075
  50. Balakin K.V., Korshun V.A., Mikhalev I.I., Maleev G.V., Malakhov A.D., Prokhorenko I.A., Berlin Y.A. (1998). Conjugates of oligonucleotides with polyaromatic fluorophores as promising DNA probes. Biosens Bioelectron 13 (7-8), 771–778 [+]

    Conjugates of pyrene and perylene with oligodeoxynucleotides were synthesized and tested as hybridisation probes. A 13-mer containing a 3-peryleneacetic acid residue attached to the 5' end through a hexamethylenediamine linker showed no response in the fluorescent spectrum upon hybridisation to the complementary sequence. At the same time, pyrene-labelled probes are sensitive to duplex formation. A pyrene pseudonucleotide unit based on 4-(1-pyrenyl)-1,3-butanediol can be introduced into any predetermined position(s) of the oligonucleotide chain. The probes polylabelled in this fashion displayed considerable changes in the excimer-to-monomer fluorescence intensity ratio after duplex formation. The internal location of two pyrene residues in the probe provides a drastic enhancement of excimer fluorescence (approximately 470 nm) upon hybridisation. When two pyrene units were brought into close proximity to two pyrenes in the complementary strand upon duplex formation, strong excimer emission at approximately 450 nm was detected. This effect provides a basis for a sensor construction designed to detect nucleic acid hybridisation.

    ID:1076
  51. Korshun V.A., Pestov N.B., Birikh K.R., Berlin Y.A. (1992). Reagent for introducing pyrene residues in oligonucleotides. Bioconjug. Chem. 3 (6), 559–62 [+]

    A novel pyrenyl-containing phosphoramidite reagent, N-[4-(1-pyrenyl)butyryl]-O1-(4,4'-dimethoxytrityl)-O2- [(diisopropylamino)(2-cyanoethoxy)phosphino]-3-amino-1 ,2-propanediol (5), has been synthesized from 4-(1-pyrenyl)butanoic acid in four steps with the 52% overall yield and used to incorporate pyrene residue(s) into oligonucleotides. Oligonucleotides 6 and 7, bearing one or two pyrenes at the 5'-terminus, have been prepared by means of that reagent, characterized with fluorescence spectra, and successfully used as primers in a polymerase chain reaction.

    ID:1077

Коршун Владимир Аркадьевич

  • Москва, ул. Миклухо-Маклая, 16/10 — На карте
  • ИБХ РАН, корп. 34, комн. 323
  • Тел.: +7(499)724-67-15
  • Эл. почта: v-korshun@yandex.ru

Отчётная кампания по достижениям подразделений в 2015 г.

Обобщены данные о самособирающихся ДНК-наноструктурах и структурированных флуоресцентных ДНК- зондах (Ponomarenko A.I., Brylev V.A., Nozhevnikova E.V., Korshun V.A. Recent advances in self-assembled fluorescent DNA structures and probes. Curr. Top. Med. Chem., 2015, 15 (13), 1162–1178).

Предложена масс-спектрометрическая метка для аминов (Топольян А.П., Стрижевская Д.А., Слюндина М.С., Беляева М.А., Иванова О.М., Коршун В.А., Устинов А.В., Михура И.В., Формановский А.А., Борисов Р.С. Дериватизация первичных аминов катионом трис(2,6-диметоксифенил)метилия для анализа методом масс-спектрометрии МАЛДИ. Масс-спектрометрия, 2015, 12 (4), 253–258).