WO2014016202A1 - Methods for catalytic alkylation of nucleic acids - Google Patents

Methods for catalytic alkylation of nucleic acids Download PDF

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Publication number
WO2014016202A1
WO2014016202A1 PCT/EP2013/065226 EP2013065226W WO2014016202A1 WO 2014016202 A1 WO2014016202 A1 WO 2014016202A1 EP 2013065226 W EP2013065226 W EP 2013065226W WO 2014016202 A1 WO2014016202 A1 WO 2014016202A1
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nucleic acid
catalyst
rhodium
copper
biological
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PCT/EP2013/065226
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French (fr)
Inventor
Dennis Gillingham
Kiril Tishinov
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Universität Basel
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Priority claimed from EP12177407.9A external-priority patent/EP2689847A1/en
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Publication of WO2014016202A1 publication Critical patent/WO2014016202A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F15/00—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic System
    • C07F15/0006—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic System compounds of the platinum group
    • C07F15/0073—Rhodium compounds
    • C07F15/008—Rhodium compounds without a metal-carbon linkage
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J31/00—Catalysts comprising hydrides, coordination complexes or organic compounds
    • B01J31/16—Catalysts comprising hydrides, coordination complexes or organic compounds containing coordination complexes
    • B01J31/22—Organic complexes
    • B01J31/2204—Organic complexes the ligands containing oxygen or sulfur as complexing atoms
    • B01J31/2208—Oxygen, e.g. acetylacetonates
    • B01J31/2226—Anionic ligands, i.e. the overall ligand carries at least one formal negative charge
    • B01J31/223—At least two oxygen atoms present in one at least bidentate or bridging ligand
    • B01J31/2239—Bridging ligands, e.g. OAc in Cr2(OAc)4, Pt4(OAc)8 or dicarboxylate ligands
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J31/00—Catalysts comprising hydrides, coordination complexes or organic compounds
    • B01J31/16—Catalysts comprising hydrides, coordination complexes or organic compounds containing coordination complexes
    • B01J31/22—Organic complexes
    • B01J31/2265—Carbenes or carbynes, i.e.(image)
    • B01J31/2269—Heterocyclic carbenes
    • B01J31/2273—Heterocyclic carbenes with only nitrogen as heteroatomic ring members, e.g. 1,3-diarylimidazoline-2-ylidenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F1/00—Compounds containing elements of Groups 1 or 11 of the Periodic System
    • C07F1/08—Copper compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2231/00—Catalytic reactions performed with catalysts classified in B01J31/00
    • B01J2231/30—Addition reactions at carbon centres, i.e. to either C-C or C-X multiple bonds
    • B01J2231/32—Addition reactions to C=C or C-C triple bonds
    • B01J2231/324—Cyclisations via conversion of C-C multiple to single or less multiple bonds, e.g. cycloadditions
    • B01J2231/327—Dipolar cycloadditions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2231/00—Catalytic reactions performed with catalysts classified in B01J31/00
    • B01J2231/40—Substitution reactions at carbon centres, e.g. C-C or C-X, i.e. carbon-hetero atom, cross-coupling, C-H activation or ring-opening reactions
    • B01J2231/42—Catalytic cross-coupling, i.e. connection of previously not connected C-atoms or C- and X-atoms without rearrangement
    • B01J2231/4277—C-X Cross-coupling, e.g. nucleophilic aromatic amination, alkoxylation or analogues
    • B01J2231/4283—C-X Cross-coupling, e.g. nucleophilic aromatic amination, alkoxylation or analogues using N nucleophiles, e.g. Buchwald-Hartwig amination
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2531/00—Additional information regarding catalytic systems classified in B01J31/00
    • B01J2531/02—Compositional aspects of complexes used, e.g. polynuclearity
    • B01J2531/0202—Polynuclearity
    • B01J2531/0208—Bimetallic complexes, i.e. comprising one or more units of two metals, with metal-metal bonds but no all-metal (M)n rings, e.g. Cr2(OAc)4
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2531/00—Additional information regarding catalytic systems classified in B01J31/00
    • B01J2531/10—Complexes comprising metals of Group I (IA or IB) as the central metal
    • B01J2531/16—Copper
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2531/00—Additional information regarding catalytic systems classified in B01J31/00
    • B01J2531/80—Complexes comprising metals of Group VIII as the central metal
    • B01J2531/82—Metals of the platinum group
    • B01J2531/822—Rhodium

Definitions

  • the present invention provides a method according to any of the preceding embodiments further comprising amplifying a nucleic acid sequence contained in a biological or an artificial composition, comprising (a) adding an oligonucleotide according to the above embodiments to the biological or an artificial composition, (b) amplifying the nucleic acid contained in the biological or an artificial sample using the oligonucleotide according to the above oligonucleotide embodiments.
  • said nucleic acid detection and amplification of the present invention is carried out in the presence of a mismatched and/or unpaired nucleobase and/or series of mismatched and/or unpaired nucleobases.
  • Figure 1A Illustrates proof of concept for rhodium-catalyzed NA alkylation.
  • Figure 3 Illustrates a catalyst linked to a guiding motif for modifying a nucleic acid.
  • a method of directly modifying a nucleic acid in a biological or an artificial composition comprises treating the composition directly with a catalyst (X) and substrate.
  • Direct modification or treating the composition directly according to the invention shall mean that the catalyst (X) contacts a native nucleic acid structure which has not been chemically treated pre- or post- synthetically to facilitate the modification.
  • the catalyst (X) according to this embodiment may not be linked to a guiding motif.
  • the guiding motif can be made of a nucleic acid, a locked nucleic acid, a peptide nucleic acid, or a polycationic peptide that binds to a nucleic acid according to the standard methods in art.
  • the guiding motif comprises a functional group for association with the catalyst (X).
  • the detection method comprises sequence- specific activation of the catalyst (X) via a guiding motif, which then alkylates a nucleic acid according to a method of the previous embodiments.
  • this method comprises (a) a catalyst (X) embedded in a stem-loop nucleic acid sequence or nucleic acid mimic sequence according to the previous embodiments which is held in an inactive state by a deactivating ligand at the opposite end of the said stem-loop sequence, (b) a biological or an artificial sample containing a target nucleic acid complementary to all or a portion of the stem-loop sequence binds and force open the loop creating a double- strand, thus removing the deactivating ligand from the the catalyst (X), (c) the catalyst (X) is then in an active state and reacts with added substrate or diazo compounds to generate a fluorescent signal, or to generate a molecule that can be detected enzymatically or immunohistochemically.
  • the detection step or method according to the above embodiments of the invention is performed by any means standard in the art.
  • detection is performed by fluorescence, radioactivity, coupled enzyme assay, or antibody detection (ELISA).
  • ELISA antibody detection
  • an oligonucleotide for amplifying a nucleic acid is provided.
  • the oligonucleotide is modified according to any of the preceding embodiments and is identical, is complementary, or hybridizes under stringent or highly stringent conditions to a nucleic acid.
  • an oligonucleotide set for amplifying a nucleic acid wherein the oligonucleotide set comprises a pair of oligonucleotides, and at least one of the pair of oligonucleotide is an oligonucleotide that is modified according to any of the preceding embodiments.
  • Lane 5 The alkylated primer is subjected to a click reaction with the rhodamine B azide substrate and then employed in the PCR.
  • Both key ⁇ -diazo carbonyl compounds Dz-1 and Dz-2 were synthesized from a common precursor, methyl 4-(2-bromomethyl)phenylacetate.
  • Dz-1 substitution of the bromine with dimethylamine and introduction of the a-diazo carbonyl function via nucleophilic diazo transfer afforded the target compound.
  • the preparation of Dz-2 included the introduction of an acetylene function for the subsequent 'click' chemistry reactions and a tetra(ethylene glycol) motif to improve the water solubility of the target diazo substrate.
  • the pellets were washed with 70 % (v/v) ethanol and dried under high vacuum.
  • the samples were further purified by micropreparative HPLC using method A.
  • the identity of all synthesized oligonucleotides was confirmed by ESI or MALDI TOF MS.
  • Rh 2 (OAc) 4 and the triacid were combined in a 25 mL round bottomed flask equipped with a stir bar. N,N-Dimethylaniline (5 mL) was added, a vigreux was attached and the reaction mixture was heated to 145 °C for 3 h, whereupon the mixture turned black. After 3.5 h a TLC (DCM/EtOAc 20:1) indicated 3 new spots.
  • the reaction mixture was poured into DCM/HCl 2M (15 mL each) and the organic phase was separated. The organic layer was extracted with 2M HC1 (2 x 15 mL) and the combined organics were then washed with water (2 x 20 mL).

Abstract

The present invention relates to catalysts, methods and kits for catalytic alkylation of nucleic acids, and more particularly to catalysts (X), methods and kits for selectively modifying nucleic acids in a biological or an artificial composition, wherein X is rhodium, copper, nickel, iron, or silver.

Description

METHODS FOR CATALYTIC ALKYLATION OF NUCLEIC ACIDS
FIELD OF THE INVENTION
The present invention relates to catalysts, methods and kits for catalytic alkylation of nucleic acids, and more particularly to catalysts (X), methods and kits for selectively modifying nucleic acids in a biological or an artificial composition, wherein X is rhodium, copper, nickel, iron, or silver. Further, the present invention relates to the modified nucleic acids in a biological or an artificial composition. In addition, the present invention relates to use of the said methods, nucleic acids, and/or kits.
BACKGROUNG ART
Deciphering the complex puzzle of nucleic acid chemistry and biology has proven incredibly difficult and continues unabated after more than a century. Chemists have begun to recognize that the extraordinary informational and structural properties that have made nucleic acids so fundamental in biology could be exploited in other contexts. Therefore, chemical strategies to modify nucleic acids would have a significant impact on studying their varied functions as well as repurposing their special abilities.
Although there are various chemical methods to modify nucleic acids, researchers typically resort to the effort of building a new phosphoramidite for solid-phase synthesis whenever an unnatural nucleic acid is required (for review see, e.g., Browne, K. A. Metal Ion-Catalyzed Nucleic Acid Alkylation and Fragmentation. Journal of the American Chemical Society 124, 7950-7962, (2002); Heetebrij, R. J. et al. Platinum (II)-Based Coordination Compounds as Nucleic Acid Labeling Reagents: Synthesis, Reactivity, and Applications in Hybridization Assays. ChemBioChem 4, 573-583, (2003); and Weisbrod, S. H. & Marx, A. Novel strategies for the site-specific covalent labeling of nucleic acids, Chemical Communications, 5675-5685 (2008).
In another method, the Cu-catalyzed azide-alkyne cycloaddition is used which allows a high degree of modularity from a single phosphoramidite (Rostovtsev, V. V., Green, L. G., Fokin, V. V. & Sharpless, K. B. A Stepwise Huisgen Cycloaddition Process: Copper(I)-Catalyzed Regioselective "Ligation" of Azides and Terminal Alkynes, Angewandte Chemie International Edition 41 , 2596-2599, (2002); and Torn0e, C. W., Christensen, C. & Meldal, M. Peptidotriazoles on Solid Phase: [1 ,2,3]-Triazoles by Regiospecific Copper(I)-Catalyzed 1 ,3-Dipolar Cycloadditions of Terminal Alkynes to Azides. The Journal of Organic Chemistry 67, 3057-3064,(2002).
Other chemical methods to achieve selective internal alkylation employ a reactive molecule linked to a guiding motif to direct specificity (see for example, Zhou, Q. & Rokita, S. E. A general strategy for target-promoted alkylation in biological systems. Proceeding of the National Academy of Sciences 100, 15452-15457, (2003); Onizuka, K., Taniguchi, Y. & Sasaki, S. Site-Specific Covalent Modification of RNA Guided by Functionality-Transfer Oligodeoxynucleotides. Bioconjugate Chemistry 20, (2009); and Song, C.-X. & He, C. Bioorthogonal Labeling of 5-Hydroxymethylcytosine in Genomic DNA and Diazirine-Based DNA Photo-Cross-Linking Probes, Accounts of Chemical Research, null-null, (2011).
Further, an enzymatic method has been developed for NA modification which co-opts the natural S-adenosylmethionine (SAM)/methyltransferase system by employing SAM analogues bearing functional groups that can undergo subsequent chemoselective transformation (see, Dalhoff, C, Lukinavicius, G., Klimasauskas, S. & Weinhold, E. Direct transfer of extended groups from synthetic cofactors by DNA methyltransferases. Nat Chem Biol 2, 31-32, (2006); and Motorin, Y. et al. Expanding the chemical scope of RNA: methyltransferases to site-specific alkynylation of RNA for click labeling, Nucleic Acids Research 39, 1943-1952, (2011).
DISCLOSURE OF THE INVENTION THE PROBLEMS AND THEIR SOLUTION
The major drawbacks the above-mentioned modification methods face is that they are unselective and/or labor-intensive. In addition, access to unnatural nucleic acids beyond the size limit of solid-phase synthesis is especially difficult. Furthermore, they require chemical manipulation of both the labeling group and the nucleic acid, in particular, the modification of nucleic acids occurs only after they have been altered chemically through solid-phase synthesis. Other modification methods involving a reactive molecule linked to a guiding motif to direct specificity are time-consuming, stoichiometric, and the guiding strand typically remains attached to the target after conjugation thus drastically changes nucleic acid properties. The enzymatic modification method is also complicated to establish. It can only accept one specialized structural motif (s-adenosylmethionine derivatives) and it is limited to those nucleic acid sequences and secondary structures that are naturally targeted by the enzyme. Therefore, it would be desirable to create alternative methods to achieve controlled modification of variety of nucleic acids in which modification occurs directly without the need of prior manipulation of the nucleic acid and without the need to synthesize complicated stoichiometric reagents for each new nucleic acid alkylation; are simple and user-friendly; avoid preparation of custom phosphoramidite; do not affect the ribophosphate backbone; are selective for exocyclic amine-containing nucleobases, in particular, are selective for unpaired nucleobases such as those present in single strands, mismatches, and bulge regions. The specificity of the method for unpaired nucleic acids sequences offers a new strategic tool for post- synthetic nucleic acid modification and may prove practical for chemical tailoring of DNA architectures in the burgeoning field of DNA nanotechnology (F. A. Aldaye, A. L. Palmer, H. F. Sleiman, Science 2008, 321 , 1795-1799). In addition, it would be desirable to create alternative methods to achieve sequence specific alkylation that are simple and use the guiding motif in catalytic amounts. The greatest drawbacks of current techniques employing a guiding strand to direct specificity are: the need for stoichiometric amounts, the time-consuming synthesis, and the covalent attachment of the guiding strand to the target nucleic acid. Using a simple tethering technique to attach the guiding strand to a catalyst capable of alkylating nucleic acids would solve all of these problems. Ultimately, it is desirable to improve diagnosis, prognosis, and monitoring of cancer or other diseases associated with genetic abnormality in nucleic acids and to improve prognosis of unwanted drug side effects, for differentiating of cell types, tissue, or for examination of cell differentiation.
To solve the problems mentioned above, the present invention provides such methods to modify a nucleic acid in a biological or an artificial composition.
Other features and advantages of the invention will be apparent from the following description and from the claims.
SUMMARY OF THE INVENTION
The present invention provides a catalyst (X) comprising a guiding motif, wherein X is selected from a group consisting of copper, rhodium, cobalt, nickel, iron, or silver; and wherein said guiding motif binds to a nucleic acid, whereby the catalyst (X) induces modification of the nucleic acid.
In one embodiment, the present invention provides a catalyst (X) comprising one or more ligands and a guiding motif, wherein X is selected from a group consisting of copper, rhodium, cobalt, nickel, iron, or silver; and wherein said guiding motif binds to a nucleic acid, whereby the catalyst (X) induces modification of the nucleic acid.
In one embodiment, X is copper and the ligand is N-heterocyclic carbene. In another embodiment, the X is rhodium, wherein said rhodium is rhodium (II) and is dimeric. Catalyst (X) comprising one or more ligands and a guiding motif, wherein X is selected from a group consisting of copper, rhodium, cobalt, nickel, iron, or silver; and wherein said guiding motif binds to a nucleic acid, whereby the catalyst (X) induces modification of the nucleic acid.
In another embodiment, the present invention provides a catalyst (X) for selectively modifying a nucleic acid in a biological or an artificial composition comprising a nucleic acid sequence or a nucleic acid mimic sequence, wherein the nucleic acid or the nucleic acid mimic is a stem-loop sequence, and wherein said stem-loop sequence comprises a deactivating ligand, and wherein X is selected from a group consisting of copper, rhodium, cobalt, nickel, iron, or silver.
In another embodiment, the present invention provides a method of selectively modifying a nucleic acid in a biological or an artificial composition, said method comprising the step of treating the composition with a catalyst (X) and a substrate, wherein X is rhodium, copper, cobalt, nickel, iron, or silver.
In further embodiment, the present invention provides a method of selectively modifying a nucleic acid in a biological or an artificial composition, said method comprising the step of treating the composition with a catalyst (X) comprising one or more ligands. Optionally, the ligands include a labile ligand set or a tight-binding ligand set, preferably, bipyridyl, N-heterocyclic carbene, or bidentate ligand and a substrate. In further embodiment said substrate is a diazo substrate.
In further embodiment, the present invention provides a method of selectively modifying a nucleic acid in a biological or an artificial composition, said method comprising the step of treating the composition with a rhodium-carbenoid.
In another embodiment, the present invention provides a method of selectively modifying a nucleic acid in a biological or an artificial composition, said method comprising the step of treating the composition with a catalyst (X) and a substrate, wherein said method comprising linking the catalyst (X) to a guiding motif and wherein the guiding motif is identical, is complementary, or hybridizes under stringent or highly stringent conditions to all or portion of the nucleic acid. In further embodiment, said guiding motif comprises a functional group for association with the catalyst (X). In further embodiment, said ligand comprising a functional group for association to said guiding motif.
In another embodiment, the present invention provides a method of selectively modifying a nucleic acid in a biological or an artificial composition, said method comprising the step of treating the composition with a catalyst (X) and a substrate, wherein the method further comprising the steps of (a) embedding the catalyst (X) in a nucleic acid sequence or a nucleic acid mimic sequence, wherein the nucleic acid or the nucleic acid mimic is a stem-loop sequence, (b) inactivating the catalyst (X) by a deactivating ligand at the opposite end of the said stem- loop sequence, (c) contacting the inactivated and embedded catalyst (X) with the nucleic acid in a biological or an artificial composition, wherein the nucleic acid is identical, is complementary, or hybridizes under stringent conditions to all or portion of the stem- loop sequence.
In another embodiment, the present invention provides a method of detecting a nucleic acid in a biological or an artificial composition, comprising (a) treating the composition comprising the nucleic acid with a catalyst (X) and a substrate according to any of the above embodiments; (b) contacting the treated nucleic acid in (a) with a probe specific for said treated nucleic acid to form a complex comprising said treated nucleic acid and said probe hybridized to said treated nucleic acid, and (c) detecting hybridization of said probe to said nucleic acid.
In another embodiment, the present invention provides a method of detecting a nucleic acid in a biological or an artificial composition, further to the above embodiment comprising (a) embedding the catalyst (X) in a nucleic acid sequence or a nucleic acid mimic sequence, wherein the nucleic acid is a stem- loop sequence, (b) inactivating the catalyst (X) by a deactivating ligand at the opposite end of the said stem-loop sequence, (c) contacting the inactivated and embedded catalyst (X) with the nucleic acid in a biological or an artificial composition, wherein the nucleic acid is identical, is complementary, or hybridizes under stringent or highly stringent conditions to all or portion of the stem-loop sequence, (d) contacting the modified nucleic acid in (c) with a probe specific for said modified nucleic acid to form a complex comprising said modified nucleic acid and said probe hybridized to said modified nucleic acid, and (e) detecting hybridization of said probe to said nucleic acid.
In another embodiment, the present invention relates to an oligonucleotide for amplifying a nucleic acid modified according to any of the preceding embodiments, which is identical, is complementary, or hybridizes under stringent or highly stringent conditions to a nucleic acid. In further embodiment, the present invention relates to an oligonucleotide set for amplifying a nucleic acid, wherein the oligonucleotide set comprises a pair of oligonucleotides, and at least one of the pair of oligonucleotide is an oligonucleotide according to above the above-mentioned embodiment.
In another embodiment, the present invention provides a method according to any of the preceding embodiments further comprising amplifying a nucleic acid sequence contained in a biological or an artificial composition, comprising (a) adding an oligonucleotide according to the above embodiments to the biological or an artificial composition, (b) amplifying the nucleic acid contained in the biological or an artificial sample using the oligonucleotide according to the above oligonucleotide embodiments. In further embodiment, said nucleic acid detection and amplification of the present invention is carried out in the presence of a mismatched and/or unpaired nucleobase and/or series of mismatched and/or unpaired nucleobases.
In another embodiment, the present invention provides a method according to any of the preceding embodiments further comprising amplifying a nucleic acid sequence contained in a biological or an artificial composition, comprising (a) adding an oligonucleotide according to the above embodiments to the biological or an artificial composition, (b) amplifying the nucleic acid contained in the biological or an artificial sample using the oligonucleotide according to the above oligonucleotide embodiments. In further embodiment, said nucleic acid detection and amplification of the present invention is carried out in the presence of a mismatched and/or unpaired nucleobase and/or series of mismatched and/or unpaired nucleobases.
In another embodiment, the present invention provides a method of determining the presence or absence of a genetic abnormality in a nucleic acid contained in a biological or an artificial composition, comprising (a) amplifying the nucleic acid contained in a biological or an artificial composition by a nucleic acid amplification method according to the above embodiments, (b) measuring a fluorescence intensity before and after amplifying the target nucleic acid sequence in (a), and, (c) detecting the presence or absence of a genetic abnormality by comparing the fluorescence intensities measured in (b). In further embodiment, the present invention provides a method of determining the presence or absence of a genetic abnormality in a nucleic acid contained in a biological or an artificial composition, wherein the genetic abnormality comprises a mismatched nucleobase, series of a mismatched nucleobases, an unpaired nucleobase, a series of unpaired nucleobases, or a series of repeated nucleobases expansions.
In another embodiment, the present invention provides a nucleic acid modified according to any of the preceding embodiments, and sequences complementary thereto. In further embodiment a nucleic acid modified according to the preceding embodiments is Short-hairpin RNA, double-stranded RNA, or microRNAs with 3'- overhangs.
In another embodiment, the present invention provides a kit for selectively modifying a nucleic acid in a biological or an artificial composition, wherein the kit comprises a catalyst (X), a substrate, and containers suitable for containing the said catalyst (X), a substrate and the biological or the artificial composition. In further embodiment, the present invention provides a kit wherein said catalyst (X) further comprising a guiding motif, wherein the guiding motif is identical, is complementary, or hybridizes under stringent or highly stringent conditions to all or portion of the nucleic acid. In further embodiment, said guiding motif comprising a functional group for association with the catalyst (X). In further embodiment, the catalyst (X) comprises a ligand that contains a functional group that facilitates association to said guiding motif. In further embodiment, the present invention provides a kit wherein said catalyst (X) comprises one ore more ligands and a guiding motif, wherein X is selected from a group consisting of copper, rhodium, cobalt, nickel, iron, or silver; and wherein said guiding motif binds to a nucleic acid, whereby the catalyst (X) induces modification of the nucleic acid.
In another embodiment, the present invention provides a kit for detecting and/or amplifying a nucleic acid in a biological or an artificial composition, wherein the kit comprising a rhodium catalyst, a substrate, an oligonucleotide, or an oligonucleotide set and/or a catalyst (X), a substrate.
Additional embodiments provide use of the methods, nucleic acids, oligonucleotides and/or kits for modifying, amplifying and detecting a nucleic acid in a biological or an artificial composition, for alkylating a RNA in vivo to alter protein production, RNA interference, and/or signaling between one or series of RNA, for generating chemical libraries of modified RNA for use in controlling or modulating RNA interference, or diseases of RNA misregulation, for detecting genetic abnormality in a nucleic acid, for diagnosis, prognosis, monitoring and/or classification of cellular proliferative disorders, and for prognosis of unwanted drug side effects.
DRAWINGS
Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.
Figure 1A. Illustrates proof of concept for rhodium-catalyzed NA alkylation.
Figure IB. Illustrates proof of concept for rhodium-catalyzed NA alkylation targeting unpaired bases. A variety of nucleic acids can be catalytically alkylated with rhodium- carbenoids generated from diazo compounds in aqueous buffer through an N-H insertion process. The method specifically targets unpaired bases such as those present in single strands, turn regions, and overhangs while leaving double-stranded sequences untouched.
Figure 2. Depicts application of the catalytic alkylation in PCR labeling.
Figure 3. Illustrates a catalyst linked to a guiding motif for modifying a nucleic acid.
Figure 4. Shows structural formulas of preferred diazo substrates.
Figure 5. Shows structural formulas of preferred bidentate catalyst.
Figure 6. Shows optimization of the concentration of the ascorbate (a) and the THPTA ligand (b) for the copper(I)-catalyzed oligonucleotide alkylation with a-diazo carbonyl compounds.
Figure 7. Depicts Cu(I) catalyst with N-heterocyclic carbene ligands where the ligands stabilize Cu(I) and suppress oxidative damage to the DNA or RNA
Figure 8. Illustrates synthesis of Rhodium- linked PNA sequence and its reaction with targeted nucleic acid.
Figure 9. Shows an overview of the auto-tandem catalytic process with Cu(I).
Figure 10. Illustrates the dependence of initial rate of diazo substrate disappearance on the aniline concentration.
DETAILED DESCRIPTION OF THE INVENTION In another preferred aspect of the invention, the method for modifying a nucleic acid in a biological or an artificial composition comprises the step of treating the composition with a catalyst (X) and a substrate, wherein X is rhodium, copper, cobalt, nickel, iron, silver, ruthenium, osmium, molybdenum, or palladium. More preferably, X is rhodium or copper. This method according to the invention can also be performed in vivo and in vitro. In one embodiment, the composition can be treated with a series of independently selected catalyst (X).
The biological or the artificial composition of the present invention is treated with a catalyst (X) and a substrate by any means standard in the art. The compositions according to the invention are preferably selected from mammalian blood, purified or partially purified blood components, blood plasma, serum, mammalian cell culture, nucleic acid sample, cell lysates, reconstituted cell components, prokaryotic cell cultures, or self-assembled nucleic acid structures.
The nucleic acid according to the present invention comprises an unpaired nucleobase, series of unpaired nucleobases, a mismatched nucleobase or series of mismatched nucleobases. Further, the nucleic acid according to the invention is preferably present within a DNA fragment, for example, genomic DNA, cDNA, and synthetic DNA fragment; within a RNA fragment, for example, whole RNA, mRNA, rRNA, siRNA, hnRNA, synthetic RNA, spliced RNA, and unspliced RNA fragment; and present in complexes with other proteins and/or with histones in chromatin. In further embodiment, the nucleic acid is Short-hairpin RNA, double-stranded RNA, or microRNAs with 3 '-overhangs.
In further embodiment of the invention, a method for modifying a nucleic acid in a biological or an artificial composition is provided wherein the method comprises a catalyst (X) and a substrate wherein the catalyst (X) comprises one or more ligands. Preferably, the ligand is a labile or tight-binding ligand set. A preferred labile ligand set is a bidentate ligand. A bidentate ligand preferably selected from any of the structural formulas as depicted in Figure 5 wherein R and R' can be the same or different and preferably selected, independently, from acridine, carboxlylate, alkyne, azide, ketone, aldehyde, and amide groups. An oxygen can be attached to either R or R' . The molar ratio between the said ligand and the rhodium catalyst or the catalyst (X) preferably is 1 or 2. A preferred tight-binding ligand is bipyridyl, preferably 2,2'-Bipyridyl, phenantroline, preferably 1 ,10-Phenantroline, and Tris(3- hydroxypropyltriazolylmethyl)amine (THPTA), or an N-heterocyclic carbene ligand. In further embodiment of the invention, the method for modifying a nucleic acid in a biological or an artificial composition comprises a catalyst (X) and a substrate wherein the catalyst (X) comprising a labile ligand set is pretreated with nucleic acid leading to substitution of the ligands with substituents on the nucleic acid. These nucleic acid- catalyst (X) construct can then be used to alkylate itself, or a complementary strand with a diazo substrate.
In the present invention, the catalyst (X), the catalyst (X) comprising a labile ligands, the substrates, including diazo substrates, and their method of manufacturing are known in the art (for example, see Bonar-Law, J. Chem. Soc, Dalton Trans., 2000, 4343-4347, and J. Am. Chem. Soc, 2004, 126 (47), pp 15378-15379).
In a further preferred embodiment of the invention said substrate is a diazo substrate. A preferred diazo substrate is selected from structural formula as described in Table 4 and depicted in figure 4 wherein R and R' preferably selected from a group consisting of alkyl chains of varying length, Fluorescent molecules, Biotin tag, digoxygenin, polyethylene glycol polymers of varying length, cationic peptides such as octa-arginine and related cell-penetrating peptides, nucleic acids or nucleic acid mimics. Preferred R groups are methyl, ethyl, ethylene glycol monomethyl ether and higher polyethyleneglycol (PEG) homologues. Preferred R' groups are dialkylbenzylamines, methylketones, aromatic rings, alkylhydroxylamines, alkylhydrazines, alkylazides, and (PEG)n where n = 1 , 2, 3.
In one embodiment, the present invention provides a guiding motif that is incorporated into the diazo substrate. In addition, the diazo substrates as shown in tables 3 and 4, for example, which incorporate an alkyne functional group provide a handle for further modification after the nucleic acid labeling. The substrate, preferably, the diazo substrate according to the present invention can be labeled.
In another embodiment of the invention, the composition is treated with a rhodium- carbenoid. The rhodium-carbenoid preferably generated from reacting a dirhodium tetracetate with a diazo substrate.
In one embodiment of the invention, a method of directly modifying a nucleic acid in a biological or an artificial composition is provided. The method comprises treating the composition directly with a catalyst (X) and substrate. Direct modification or treating the composition directly according to the invention shall mean that the catalyst (X) contacts a native nucleic acid structure which has not been chemically treated pre- or post- synthetically to facilitate the modification. The catalyst (X) according to this embodiment may not be linked to a guiding motif.
In one embodiment of the invention, the catalyst (X) is not conjugated to the substrate prior to treating the composition. One advantage of this embodiment is that the catalyst (X) only initiates the formation of a reactive substrate at the target nucleic acid. Therefore, the exposure of the cells and/or patients to potentially reactive and toxic substrates is reduced or eliminated. The nucleic acid can specifically be targeted via a guiding motif attached to the catalyst (X).
In another embodiment of the invention, a method of selectively modifying a nucleic acid according to any of the preceding embodiments is provided, wherein said method comprises linking a catalyst (X) to a guiding motif, wherein the guiding motif is identical, is complementary, or hybridizes under stringent or highly stringent conditions to all or portion of the nucleic acid. The term guiding motif generally refers to a molecular motif that is capable of binding to a nucleic acid target in a sequence-specific way.
Stringent hybridization conditions involve hybridizing at 68°C in 5x SSC/5x Denhardt's solution/ 1.0% SDS, and washing in 0.2x SSC/0.1% SDS at room temperature, or involve the art-recognized equivalent thereof (e.g., conditions in which a hybridization is carried out at 60°C in 2.5 x SSC buffer, followed by several washing steps at 37°C in a low buffer concentration, and remains stable). Moderately stringent conditions, as defined herein, involve including washing in 3x SSC at 42°C, or the art- recognized equivalent thereof. The parameters of salt concentration and temperature can be varied to achieve the optimal level of identity between the probe and the target nucleic acid. Guidance regarding such conditions is available in the art, for example, by Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, N.Y.; and Ausubel et al. (eds.), 1995, Current Protocols in Molecular Biology, (John Wiley & Sons, N.Y.) at Unit 2.10.
As depicted in Figure 3 and Figure 8, for sequence specific alkylation the rhodium or copper catalyst is linked to a guiding motif that is a nucleic acid or any other molecule capable of sequence- specific binding to nucleic acids. In another embodiment, the catalyst (X) is linked to a guiding motif that is a nucleic acid or any other molecule capable of sequence specific binding to nucleic acids. The guided catalyst (X) bind to the target nucleic acid and only then substrate or the diazo molecule is activated and the target nucleic acid is modified. The catalyst (X) can be linked to a guiding motif by any means standard in the art. It is preferred that alky lhydroxylamine/ke tone coupling be used as the linking strategy. The guiding motif can be made of a nucleic acid, a locked nucleic acid, a peptide nucleic acid, or a polycationic peptide that binds to a nucleic acid according to the standard methods in art. In further embodiment of the invention, the guiding motif comprises a functional group for association with the catalyst (X). The functional groups for linking the catalyst (X) with the guiding motif preferably are: (a) alkyl hydroxyl amine on the ligand of the rhodium catalyst and/or the catalyst (X) and a ketone or aldehyde incorporated within the guiding motif, (b) alkylhydroxlamine on one of the ends of the guiding motif and a ketone or aldehyde on one the ligands of the catalyst (X), (c) a sulfhydryl on both the catalyst (X) and the guiding motif that can be linked together via disulfide bond, (d) an azide on the guiding motif and an alkyne on one of the ligands of the catalyst (X), these can then be linked by copper-catalyzed azide alkyne Cycloaddition, (e) an alkyne on the guiding motif and an azide on one of the ligands of the catalyst (X), these can then be linked by copper-catalyzed azide alkyne Cycloaddition, and (f) a carboxylic acid on one or both ligands of the catalyst (X) can be linked with amide coupling techniques to a free amine incorporated within the guiding motif. It is further preferred that the ligand comprises a functional group for association to the guiding motif which are selected from alkylhydroxylamine, aldehyde, carboxylic acid, alkyne, azide and more preferably ketone.
In another preferred method of the present invention the method of selectively modifying a nucleic acid further comprising the steps of: (a) embedding the catalyst (X) in a nucleic acid sequence or a nucleic acid mimic sequence, wherein the nucleic acid or the nucleic acid mimic is a stem- loop sequence, (b) inactivating the catalyst (X) by a deactivating ligand at the opposite end of the said stem-loop sequence, (c) contacting the inactivated and embedded catalyst (X) with the nucleic acid in a biological or an artificial composition, wherein the nucleic acid is identical, is complementary, or hybridizes under stringent conditions to all or portion of the stem- loop sequence. The preferred guiding nucleic acid or nucleic acid mimic is peptide nucleic acid, morpholine nucleic acid, glycerol nucleic acid, and threose nucleic acid. It is preferred that said deactivating ligands for rhodium and X compounds are nitriles, dimethylanilines, sulfoxides, phosphines which are effective and reversible. It is further preferred that the guiding nucleic acid or nucleic acid mimic is identical, is complementary, or hybridizes under stringent conditions to all or portion of desired target nucleic acid sequence. More preferably, the guiding nucleic acid or nucleic acid mimic embodies a functional group that allows and facilitates association with the catalyst (X) in solution. The catalyst (X) of this embodiment preferably contains ligands with a functional group that facilitates association to said guiding motif. Preferably, this method comprises (a) a catalyst (X) embedded in a stem- loop nucleic acid sequence or nucleic acid mimic sequence according to the previous embodiments which is held in an inactive state by a deactivating ligand at the opposite end of the said stem- loop sequence, (b) a biological or an artificial sample containing a target nucleic acid complementary to all or a portion of the stem-loop sequence binds and force open the loop creating a double- strand, thus removing the deactivating ligand from the the catalyst (X), (c) the catalyst (X) is then in an active state and reacts with added diazo substrate.
In another aspect of the invention, the method of selectively modifying a nucleic acid according to any embodiments of the present invention, further comprises detecting a nucleic acid in a biological or an artificial composition, comprising (a) treating the composition comprising the nucleic acid with a catalyst (X) and a substrate, (b) contacting the treated nucleic acid in (a) with a probe specific for said treated nucleic acid to form a complex comprising said treated nucleic acid and said probe hybridized to said treated nucleic acid, and (c) detecting hybridization of said probe to said nucleic acid.
In a preferred embodiment, said detection further comprises (a) embedding the catalyst (X) in a nucleic acid sequence or a nucleic acid mimic sequence, wherein the nucleic acid is a stem- loop sequence, (b) inactivating the catalyst (X) by a deactivating ligand at the opposite end of the said stem- loop sequence, (c) contacting the inactivated and embedded catalyst (X) with the nucleic acid in a biological or an artificial composition, wherein the nucleic acid is identical, is complementary, or hybridizes under stringent or highly stringent conditions to all or portion of the stem- loop sequence, (d) contacting the modified nucleic acid in (c) with a probe specific for said modified nucleic acid to form a complex comprising said modified nucleic acid and said probe hybridized to said modified nucleic acid, and (e) detecting hybridization of said probe to said nucleic acid.
Inactivating the catalyst (X) in step (b) substantially eliminates the ability of the catalyst (X) to react with the substrate. Contacting the inactivated and embedded rhodium catalyst and/or catalyst (X) with the target nucleic acid forces the deactivating ligand to be removed from the rhodium catalyst and/or the catalyst (X).
In further embodiment of the invention, the detection method comprises sequence- specific activation of the catalyst (X) via a guiding motif, which then alkylates a nucleic acid according to a method of the previous embodiments. Preferably, this method comprises (a) a catalyst (X) embedded in a stem-loop nucleic acid sequence or nucleic acid mimic sequence according to the previous embodiments which is held in an inactive state by a deactivating ligand at the opposite end of the said stem-loop sequence, (b) a biological or an artificial sample containing a target nucleic acid complementary to all or a portion of the stem-loop sequence binds and force open the loop creating a double- strand, thus removing the deactivating ligand from the the catalyst (X), (c) the catalyst (X) is then in an active state and reacts with added substrate or diazo compounds to generate a fluorescent signal, or to generate a molecule that can be detected enzymatically or immunohistochemically.
The detection step or method according to the above embodiments of the invention is performed by any means standard in the art. Preferably, detection is performed by fluorescence, radioactivity, coupled enzyme assay, or antibody detection (ELISA). In another aspect of the invention, an oligonucleotide for amplifying a nucleic acid is provided. The oligonucleotide is modified according to any of the preceding embodiments and is identical, is complementary, or hybridizes under stringent or highly stringent conditions to a nucleic acid. In another embodiment, an oligonucleotide set for amplifying a nucleic acid is provided, wherein the oligonucleotide set comprises a pair of oligonucleotides, and at least one of the pair of oligonucleotide is an oligonucleotide that is modified according to any of the preceding embodiments.
It is preferable that the modified oligonucleotide for amplifying a nucleic acid according to the present invention is labeled. In further embodiment, the labeled modified oligonucleotide is identical, is complementary, or hybridizes under stringent or highly stringent conditions to a nucleic acid containing unpaired nucleobase, series of unpaired nucleobases, a mismatched nucleobase or series of mismatched nucleobases. In further embodiment, the modified oligonucleotide is identical, is complementary, or hybridizes under stringent or highly stringent conditions to a genetically abnormal nucleic acid. In further embodiment, the present invention provides a method according to any of the preceding embodiments for amplifying a nucleic acid sequence contained in a biological or an artificial composition. This method comprises (a) adding a modified oligonucleotide according to the above embodiments to the biological or an artificial composition, (b) amplifying the nucleic acid contained in the biological or an artificial composition using the modified oligonucleotide according to the above oligonucleotide embodiments.
The amplification method according to the invention is performed by any means in the art. It is preferred that the nucleic acid amplification method in (b) is performed via polymerase chain reaction (PCR), realtime PCR, quantitative PCR.
In further embodiment, said nucleic acid detection and amplification of the present invention is carried out in the presence of a mismatched and/or unpaired nucleobase and/or series of mismatched and/or unpaired nucleobases.
In another embodiment, the present invention provides a method of determining the presence or absence of a genetic abnormality in a nucleic acid contained in a biological or an artificial composition, comprising (a) amplifying the nucleic acid contained in a biological or an artificial composition by a nucleic acid amplification method according to the above embodiments, (b) measuring a fluorescence intensity before and after amplifying the target nucleic acid sequence in (a), and, (c) detecting the presence or absence of a genetic abnormality by comparing the fluorescence intensities measured in (b). In further embodiment, the present invention provides a method of determining the presence or absence of a genetic abnormality in a nucleic acid contained in a biological or an artificial composition, wherein the genetic abnormality comprises a mismatched nucleobase, series of a mismatched nucleobases, an unpaired nucleobase, a series of unpaired nucleobases, or a series of repeated nucleobases expansions.
In another embodiment, the present invention provides a nucleic acid modified according to any of the preceding embodiments, and sequences complementary thereto. In further embodiment a nucleic acid modified according to the preceding embodiments is Short-hairpin RNA, double-stranded RNA, or microRNAs with 3'- overhangs.
In another aspect of the invention a kit for selectively modifying a nucleic acid in a biological composition is provided. The kit comprises (a) a catalyst (X) or series of catalyst (X) (b) a substrate (c) containers suitable for containing the said catalyst (X), a substrate, and the biological composition.
In further embodiment, the kit comprises a catalyst (X) according to the preceding embodiments, wherein said catalyst (X) further comprises a guiding motif, wherein the guiding motif is identical, is complementary, or hybridizes under stringent or highly stringent conditions to all or portion of the nucleic acid. In further embodiment of the kit, said guiding motif comprising a functional group for association with the catalyst (X). In further embodiment of the kit, the catalyst (X) comprises a ligand that contains a functional group that facilitates association to said guiding motif. In further embodiment, the present invention provides a kit wherein said catalyst (X) comprises one ore more ligands and a guiding motif, wherein X is selected from a group consisting of copper, rhodium, cobalt, nickel, iron, or silver; and wherein said guiding motif binds to a nucleic acid, whereby the catalyst (X) induces modification of the nucleic acid.
In further embodiment of the invention, a kit for nucleic acid detection and amplification according is provided wherein the kit comprises (a) a catalyst (X) (b) a substrate (c) an oligonucleotide, or an oligonucleotide set.
In further embodiment of the invention, a kit for nucleic acid detection and amplification according is provided wherein the kit comprises (a) a rhodium catalyst (b) a catalyst (X) (c) a substrate (d) an oligonucleotide, or an oligonucleotide set.
The present invention provides novel uses of a catalyst (X) and a substrate for modifying, amplifying and detecting a nucleic acid in a biological or an artificial composition.
Further, the present invention provides novel uses of a method and/or a kit according to any of the preceding embodiments in alkylating RNA in vivo to alter protein production, RNA interference, and/or signaling between one or series of RNA. A nucleic acid modified according to the preceding embodiments, wherein nucleic acid is Short-hairpin RNA, double-stranded RNA, or microRNAs with 3 '-overhangs modified according to the preceding claims are used in RNA interference or antisense therapy. In another embodiment, the present invention provides novel uses of a method, an oligonucleotide and/or a kit according to any of the preceding embodiments in generating chemical libraries of modified RNA for use in controlling or modulating RNA interference, or diseases of RNA misregulation. The method preferably involves generating short hairpin RNAs or microRNAs or double- stranded RNAs according to established recombinant or synthetic techniques. These RNAs always have a 3'- overhang that would be freely available for reaction as described for example in Table 1 , entry 5. Libraries of diazo compounds are generated through diazo transfer with a variety of commercially available ester or ketone substrates that contain an enolizable alpha-proton. Other established methods for diazo synthesis could also be used to create libraries in one step from commercial compounds. Examples include condensing a hydrazine or protected hydrazine with a ketone and then oxidizing to obtain the diazo compound.
In another embodiment, the present invention provides novel uses of a method, an oligonucleotide and/or a kit according to any of the preceding embodiments in detecting genetic abnormality in a nucleic acid, in the diagnosis, prognosis, monitoring and/or classification of cellular proliferative disorders.
In further embodiment, the present invention provides a catalyst (X) comprising a guiding motif, wherein X is selected from a group consisting of copper, rhodium, cobalt, nickel, iron, or silver; and wherein said guiding motif binds to a nucleic acid, whereby the catalyst (X) induces modification of the nucleic acid.
In further embodiment, the catalyst (X) comprises one or more ligands and a guiding motif, wherein X is selected from a group consisting of copper, rhodium, cobalt, nickel, iron, or silver; and wherein said guiding motif binds to a nucleic acid, whereby the catalyst (X) induces modification of the nucleic acid. Optionally, said catalyst (X) comprises a labile or tight-binding ligand. Optionally, the X is copper. Optionally, the X is copper and the ligand is N-heterocyclic carbene. Optionally, X is rhodium and wherein said rhodium is rhodium (II) and is dimeric. Rhodium (II) means rhodium is in the +2 oxidation state.
In another embodiment, the present invention provides a catalyst (X) for selectively modifying a nucleic acid in a biological or an artificial composition comprising a nucleic acid sequence or a nucleic acid mimic sequence, wherein the nucleic acid or the nucleic acid mimic is a stem-loop sequence, and wherein said stem-loop sequence comprises a deactivating ligand, and wherein X is selected from a group consisting of copper, rhodium, cobalt, nickel, iron, or silver.
Further, the guiding motif, X, ligands, and composition of any of the preceding embodiments is also suitable, applicable and can be used for the catalyst (X) of the present invention. Experiments and results:
In an initial proof-of-invention experiment the simple tetradeoxynucleotide d(ATGC) was treated with 10 mol% Rh2(OAc)4 and the diazo substrate Dz-1 in aqueous buffer (Figure 1A). Under these conditions the Dz-1 substrate was completely consumed after 24 h and a number of new products were observed whose masses corresponded to singly- and doubly-modified d(ATGC). Tandem MS analysis of the monoalkylation products allowed unambiguous assignment of the purine bases as the sites of modification.
With the established feasibility of targeting NAs through rhodium-carbenoid catalysis, a more comprehensive investigation of the reaction with a series of hairpin sequences was performed (Table 1). Hairpins were chosen because they contain a number of common nucleic acid (NA) structural elements in a single molecule. The first hairpin we tested contained thymidines (Ts) in its turn region and was otherwise double- stranded (entry 1 , Table 1). It was established that Ts were unreactive from the experiment with d(ATGC) so this molecule allows an assessment of the susceptibility of double- stranded stretches to the alkylation. Interestingly this hairpin was completely unreactive, revealing the prospect of exploiting double strands as a type of shielding motif to enable the targeting of specific unpaired bases in a given NA. To test this possibility hairpins containing adenine as an overhang base (entries 2, 3, and 5) or in the turn-region (entry 4) were synthesized. As expected both of these motifs were viable substrates (entries 2, 3, and 5 with entry 4). The specificity of the process for unpaired NA sequences offers as described above a new strategic tool for postsynthetic NA modification and may prove practical for chemical tailoring of DNA architectures in the burgeoning field of DNA nanotechnology.
Short hairpin RNAs and short dsRNAs are molecules of high current interest because of their central role in the RNA interference (RNAi) pathway (J. Kurreck, Angew. Chem. Int. Ed. 2009, 48, 1378-1398). RNAs that participate in this pathway always contain 3'-overhangs, which are essential to trigger the assembly of the active ribonucleoprotein complex. The successful 3'-alkylation of a short hairpin RNA (entry 5, Table 1) is therefore especially significant as it establishes the method as a convergent approach to the synthesis of base-modified RNAs. Such molecules have been employed to study and modulate RNAi (H. Peacock, A. Kannan, P. A. Beal, C. J. Burrows, J. Org. Chem. 2011 , 76, 7295-7300; b) A. R. Hernandez, L. W. Peterson, E. T. Kool, ACS Chem. Biol. 2012), but thus far the diversity of the modified RNA pool has been limited due to the laborious linearity of solid-phase synthesis methods. The method according to the present invention alleviates many of the current problems. The preference for purine nucleobases observed for the alkylation of d(ATGC) (figure 1A) warranted a systematic evaluation of which features in the oligonucleotide primary structure were important in the modification. Homotetramers were analysed to probe the relative propensity of each base towards alkylation (entries 1-4, Table 2). Consistent with the results from the hairpin series homotetramers d(T)4 and r(U)4 were completely unmodified, indicating that neither the ribophosphate backbone nor T and U nucleobases are the site of reaction (The possibility that d(T)4 is simply an inhibitor of the catalyst was excluded by a control experiment where d(T)4 was added to the reaction of d(A)4 and no change was observed in its alkylation profile.
Additionally, MS/MS experiments confirm that trinucleotides containing C or A nested between Ts (entries 5-6, Table 2) were alkylated only at the C or A sites and never at the Ts (see, Figures 8-13). In contrast to the result with d(ATGC), where C was untouched, alkylation of d(C)4 proceeded efficiently (entry 4, Table 2), suggesting that purine bases are alkylated preferentially but that Cs can also be targeted. The remainder of the RNAs and DNAs shown in Table 2 provide further evidence of the generality of the alkylation process.
Diazo substrate that did not lead to NA alkylation was instead predominately converted to the corresponding hydroxyl insertion product (see header in Table 2). Despite this side-reaction a significant percentage of catalyst turnovers were directed to the oligonucleotide. Considering the enormous excess of water this indicates that N-H insertion is significantly faster than hydroxyl insertion (see Selectivity Factor, Table 2). It is tenable that this preference is simply a consequence of the increased nucleophilicity of the amine towards the intermediate rhodium carbenoid, but two observations speak to a more intimate role of the NA: First, while the alkylation of d(TGT) was slow and often led to the formation of a precipitate, a mixture of d(TAT) and d(TGT) led to smooth alkylation of both NAs. Second, while Dz-1 only slowly delivered hydroxyl insertion under the standard reaction conditions (<10% conv. in 24 h), when d(TAT) was present not only is the NA alkylated but the rate of the hydroxyl insertion side-reaction also increases significantly. The first step in the catalytic cycle for rhodium carbene formation is generally accepted to be the interaction of the negatively polarized carbon of the diazo molecule with the empty dz 2 orbital in the axial position of the rhodium complex (M. C. Pirrung, H. Liu, A. T. Morehead, /. Am. Chem. Soc. 2002, 124, 1014-1023). With this in mind it seems unlikely that a direct nitrogen coordination from the ON substrate to the catalyst is responsible for the selectivity observed as this would likely inhibit carbene formation (M. C. Pirrung, H. Liu, A. T. Morehead, /. Am. Chem. Soc. 2002, 124, 1014-1023). These results clearly demonstrate that certain NAs can affect the catalytic reaction and this augurs well for the potential of controlling the site of alkylation through the incorporation of particular NA sequences.
Given the unreactivity of Ts and Us, it was determined that the site of alkylation with the remaining nucleobases was the exocyclic N-H (C. J. Moody, Angew. Chem. Int. Ed. 2007, 46, 9148-9150). Alkylation of N3 of adenine and N7 of guanine were deemed unlikely based on analogy to the classic experiments of Maxam and Gilbert (A. M. Maxam, W. Gilbert, Proc. Natl. Acad. Sci. USA 1977, 74, 560-564). They established that alkylations at these positions— and not the exocyclic nitrogens— render the resulting modified bases highly susceptible to depurination: it was observed no such change in stability (Alkylated nucleotides were monitored at pH 5 and pH 8.5 for 24 h and their HPLC profiles were unchanged). More direct evidence for N-H insertion is garnered from MS3 experiments for the three trinucleotides d(TAT), d(TGT), and d(TCT). The ions observed after the fragmentation of these alkylated trinucleotides are in full agreement with those expected for exocyclic N-H insertion products, and are difficult to reconcile with any other potential reaction pathway (see, Figures 8-13) (S. S. Jensen, X. Ariza, P. Nielsen, J. Vilarrasa, F. Kirpekar, /. Mass Spectrom. 2007, 42, 49-57).
As the results collected in Table 3 indicate the process showed little dependence on the structure of the diazo substrate or reaction conditions. In terms of substrate scope maintaining water solubility at the concentrations 25-50 mM typically needed to achieve efficient alkylation. Efficient alkylation according to the invention means >20% conversion. If the nucleic acid concentration is low (<mM) then an increase in diazo concentration or multiple additions of diazo can still deliver efficient conversion. Although the substrate could be customized for any specific application, the propargyl- containing molecules Dz-4 and Dz-5 (entries 8-9, Table 3) should prove particularly versatile because they allow the Cu-catalyzed azide-alkyne cycloaddition to be employed to generate diverse structures from a single diazo precursor— a feature demonstrated by the 'click' reaction of both a rhodamine fluorophore and a biotin derivative (see bottom-right of Figure 2).
The practical potential of the process was highlighted through the generation of fluorescently labeled PCR amplicons. A modified T7 promoter primer (Figure 2) was first alkylated with Dz-5 and then used in a PCR reaction. We expected a PCR to be successful despite the unspecific alkylation profile with single-stranded NAs since double-helix formation and polymerase extension should only occur with those primers that get alkylated near their 5 '-end. Indeed this seems to be the case as robust PCR amplification is seen in all samples that employ alkylated T7 promoter primers (Fig. 2, Lanes 3-5). The experiment in lane 5 is particularly important as it highlights the potential of the technique for generating labeled genes; here the propargyl group is linked through a 'click' reaction to a rhodamine derivative prior to the PCR. The amplification product of this plasmid PCR is clearly visible with a standard UV lamp before the addition of ethidium bromide, indicating that labeled amplicons are accessible through this simple technique. Figure 2 depicts the following: top: The T7 promoter primer, which contains a short 5' extension, is alkylated under standard conditions with the propargyl-containing diazo substrate Dz-5. Gel: The gel is run without ethidium bromide to allow visualization of fluorescently labeled products (Lanes la-5a) and then soaked with ethidium bromide to visualize all NA species (Lanes lb-5b). Lane 1: 100 bp DNA ladder. Lane 2: Control reaction with unmodified T7 promoter primer and plasmid DNA. Lane 3: T7 promoter primer is alkylated with Dz-5 and this entire crude mixture is then employed in a PCR reaction with plasmid DNA. Lane 4: Same as lane 3 except the alkylated T7 promoter primer is purified before carrying out the PCR. Lane 5: The alkylated primer is subjected to a click reaction with the rhodamine B azide substrate and then employed in the PCR. Bottom- right: Both biotin- and rhodamine B-azide derivatives are viable substrates for the click reaction.
The methods according to the invention as disclosed above establish the feasibility of employing rhodium catalysis to achieve the direct alkylation of native NA structures. Moreover its predictable selectivity profile allows the strategic targeting of unpaired nucleobases such as those present in single strands, bulge regions, and overhangs. One of the key features of the present methods of the invention are their simplicity and directness: the catalyst is commercially available, the diazo compounds are readily prepared, and native NAs are viable substrates. The preference for N-H insertion observed here, as well as by others in the protein labeling field (J. M. Antos, J. M. McFarland, A. T. Iavarone, M. B. Francis, /. Am. Chem. Soc. 2009, 131 , 6301-6308) is intriguing. Particularly considering the myriad of other functional groups available and the enormous excess of water present in these reactions. The new catalytic alkylation methods according to the disclosed embodiments of the invention offer a powerful strategy in the labelling and modification of NA derivatives which can be applied toward tailoring of DNA architectures and in the labeling of NAs for biological study.
General Procedures
All reagents and solvents used were of analytical grade. Buffers were prepared with ultrapure water. All chemicals were purchased from Sigma- Aldrich, Fluka or Acros and used as received. Analytical TLC was performed on Silica gel 60 F254 pre-coated aluminium sheets. Flash chromatography was performed on Silica gel 60 40-63 mm (230-400 mesh) (SiliCycle, Quebec). Solid-phase oligonucleotide synthesis was carried out on an Expedite 8909 nucleic acid synthesis system (PerCeptive Biosystems). 1H and 13C NMR spectra were acquired on a Bruker AvanceIII+ (500 MHz) spectrometer at 298 K. Chemical shifts relative to TMS were referenced to the solvent's residual peak and are reported in ppm. Proton-decoupled 19F spectra were acquired on a Bruker AvanceIII+ (400 MHz) at 298 K. EI MS were obtained on a Finnigan MAT 95 device. ESI MS spectra were obtained on a Bruker Esquire3000plus spectrometer by direct injection in positive polarity of the ion trap detector. MALDI TOF analyses were carried out on a Bruker Microflex mass-spectrometer in linear negative mode using 3 hydroxypicolinic acid/ammonium citrate, pH 6.0 or 2,4,6 trihydroxyacetophenone/ ammonium citrate, pH 6.0 matrices. All HPLC procedures were carried out on an Agilent 1100 LC system equipped with a Chromolith Performance RP18e 4.6 x 100 mm column (Merck) using 100 mM triethylammonium acetate (pH 7.1)/acetonitrile gradients as a mobile phase. Elution was carried out at a flow rate of 1 ml/min using one of the following methods. Method A: 0-16 % acetonitrile in 12 min, 16-80 % acetonitrile in 3 min, 80 % acetonitrile in 2 min, or method B: 0-16 % acetonitrile in 12 min, 16-80 % acetonitrile in 15 min, 80 % acetonitrile in 2 min. Detection was carried out by monitoring the absorbance of the column effluent at 254 nm. Nucleic acids were obtained by any means standard in the art.
Chemical syntheses Diazo substrate synthesis strategy
Both key α-diazo carbonyl compounds Dz-1 and Dz-2 were synthesized from a common precursor, methyl 4-(2-bromomethyl)phenylacetate. In the case of Dz-1 substitution of the bromine with dimethylamine and introduction of the a-diazo carbonyl function via nucleophilic diazo transfer afforded the target compound. The preparation of Dz-2 included the introduction of an acetylene function for the subsequent 'click' chemistry reactions and a tetra(ethylene glycol) motif to improve the water solubility of the target diazo substrate.
Methyl 2-(4-(bromomethyl)phenyl)acetate (2). 4-(Bromomethyl)phenylacetic acid (0.2 g, 0.87 mmol) was mixed with dry methanol (2 ml) and dry benzene (2 ml) under nitrogen. Trimethylsilyldiazomethane (2 mol/1 in hexanes) was added drop-wise at continuous stirring until stable yellow color of the solution was obtained (the reaction vessel should be vented as gas evolution takes place). The mixture was stirred for 15 min at room temperature, then evaporated and dried under high vacuum to yield the pure compound as a colorless oil (0.21 g, 0.86 mmol, quant.). TLC (n-hexane:Et2O 4:1 v/v) Rf =0.36. 1H NMR (500.1 MHz, CDC13) d, ppm: 7.36 (d, J = 8.1 Hz, 2H), 7.26 (d, J = 8.2 Hz, 2H), 4.48 (s, 2H), 3.69 (s, 3H), 3.62 (s, 2H). 13C NMR (125.8 MHz, CDC13) d, ppm: 171.73, 136.68, 134.28, 129.75, 129.31 , 52.16, 40.86, 33.21. EI MS: 104.1 [M- Br-COOMe]+, 163.1 [M-Br]+, 183.0, 185.0 [M-COOMe]+, 242.0, 244.0 [M]+.
Methyl 2-diazo-2-(4-((dimethylamino)methyl)phenyl)acetate (3, Dz-1). Compound 2
(0.2 g, 0.82 mmol) was dissolved in 2 ml of absolute ethanol, dimethylamine in absolute ethanol (2 ml, 5.6 mol/l) was added and the mixture was stirred at room temperature for 30 min. The volatiles were then removed under vacuum and the residue was partitioned between ethyl acetate and saturated sodium bicarbonate solution. The organic phase was washed with brine, dried with anhydrous sodium sulfate and evaporated under vacuum. The remaining yellow oil was dried under high vacuum, and then mixed with p-acetamidobenzenesulfonyl azide (pABSA, 0.216 g,
0.90 mmol) in 5 ml of dry acetonitrile under nitrogen. DBU (0.187 g, 1.23 mmol) was added to the solution with continuous stirring and the mixture was stirred for 24 h at room temperature. The mixture was then evaporated under vacuum and the residue purified by flash chromatography on Si60 in DCM/methanol to afford a yellow-orange oil (0.082 g, 0.35 mmol, 43 %). TLC (DCM:methanol 10:1 v/v) Rf=O.33. ¾ NMR (500.1 MHz, CDCI3) d, ppm: 7.43 (d, J = 8.5 Hz, 2H), 7.33 (d, J = 8.5 Hz, 2H), 3.86 (s, 3H), 3.41 (s, 2H), 2.23 (s, 6H). 13C NMR (125.8 MHz, CDC13) d, ppm: 165.76, 136.63, 129.74, 124.07, 123.94, 63.87, 52.01 , 45.33. ESI MS: Calcd for C12H16N3O2 + [M+H]+ 234.1 , found 234.1. tert-Butyl4-(2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl)piperazine-l-carboxylate (4b). 1-Boc-piperazine (0.362 g, 1.9 mmol) was dissolved in 15 ml of dry acetonitrile under nitrogen. K2CO3 (1.0 g, 7.0 mmol) was added, the mixture was heated up to reflux and compound 4a (prepared as previously described1) (0.5 g, 1.9 mmol) as a solution in 5 ml of dry acetonitrile was introduced with continuous stirring. The mixture was refluxed for 18 h, then filtered and the filtrate evaporated under vacuum. The residue was purified by flash chromatography on Si60 in DCM/methanol to afford the target product as a colorless oil (0.551 g, 1.5 mmol, 80 %). TLC (DCM:methanol 20:1 v/v) Rf =0.14. 1H NMR (500.1 MHz, CDC13) d, ppm: 3.68 (ddd, J = 4.5 Hz, J = 3.5 Hz, J = 2.0 Hz, 2H), 3.55-3.64 (m, 12H), 3.39-3.41 (m, 4H), 2.55 (dd, J = 5.7 Hz, J = 5.7 Hz, 2H), 2.40-2.42 (m, 4H), 1.41 (s, 9H). 13C NMR (125.8 MHz, CDC13) d, ppm: 154.69, 79.55, 72.70, 70.58, 70.24, 70.22, 68.64, 61.59, 57.85, 53.32, 43.96, 42.93, 28.40. ESI MS: Calcd for C17H34N2NaO6 + [M+Na]+ 385.2, found 385.3. tert-Butyl4-(2-(2-(2-(2-(tosyloxy)ethoxy)ethoxy)ethoxy)ethyl)piperazine-l-carboxylate (4c). Compound 4b (0.550 g, 1.5 mmol) was mixed with triethylamine (0.307 g, 3.0 mmol) and 4-dimethylaminopyridine (0.019 g, 0.16 mmol) in 30 ml of dry acetonitrile under nitrogen. p-Toluenesulfonyl chloride (0.318 g, 1.7 mmol) as a solution in 10 ml of dry acetonitrile was introduced at continuous stirring. The mixture was stirred at room temperature for 48 h, then evaporated under vacuum and residue was purified by flash chromatography on Si60 in DCM/methanol to yield the target product as a colorless oil (0.517, 1.0 mmol, 66 %). TLC (DCM:methanol 20:1 v/v) Rf =0.28. 1H NMR (500.1 MHz, CDC13) d, ppm: 7.78 (d, J = 8.3 Hz, 2H), 7.33 (d, J = 8.5 Hz, 2H), 4.14 (ddd, J = 4.5 Hz, J = 4.0 Hz, J = 3.0 Hz, 2H), 3.67 (ddd, J = 5.0 Hz, J = 4.0 Hz, J = 3.5 Hz, 2H), 3.57-3.61 (m, 10H), 3.40-3.42 (m, 4H), 2.58 (dd, J = 6.0 Hz, J = 6.0 Hz, 2H), 2.41-2.43 (m, 7H), 1.44 (s, 9H). 13C NMR (125.8 MHz, CDC13) d, ppm: 154.75, 144.81 , 132.99, 129.84, 127.99, 79.58, 70.77, 70.63, 70.52, 70.37, 69.23, 68.86, 68.70, 57.81 , 53.37, 44.02, 43.00, 28.43, 21.67. ESI MS: Calcd for C24H41N2O8S+ [M+H]+ 517.3, found 517.2 tert-butyl4-(2-(2-(2-(2-bromoethoxy)ethoxy)ethoxy)ethyl)piperazine-1-carboxylate (4d). LiBr (0.695 g, 8.0 mmol) was mixed with dry acetone (12 ml) and heated up to reflux under nitrogen. Compound 4c (0.517 g, 1.0 mmol) as a solution in 3 ml of dry acetone was added with continuous stirring and the mixture was refluxed until the reaction was complete as judged by TLC (2 h). The solution was then cooled to room temperature and evaporated under vacuum. The residue was mixed with 30 ml of DCM and the white precipitate that formed was removed by filtration. The filtrate was taken up to dryness and the residue was purified by flash chromatography on Si60 in DCM/methanol to afford the target product as a colorless oil (0.419 g, 1.0 mmol, 98 %). TLC (DCM:methanol 20:1 v/v) Rf =0.28. 1H NSOM4R (500.1 MHz, CDC13) d, ppm: 3.81 (dd, J = 6.3 Hz, J = 6.3 Hz, 2H), 3.61-3.69 (m, 10H), 3.47 (dd, J = 6.3 Hz, J = 6.3 Hz, 2H), 3.42-3.44 (m, 4H), 2.60 (dd, J = 5.9 Hz, J = 5.9 Hz, 2H), 2.43-2.45 (m, 4H), 1.45 (s, 9H). 13C NMR (125.8 MHz, CDC13) d, ppm: 154.77, 79.59, 71.23, 70.70, 70.62, 70.58, 70.42, 68.91 , 57.84, 53.38, 40.25, 30.31 , 28.44. ESI MS: 425.2, 427.1 [M+H]+, 447.1 , 449.1 [M+Na]+. ESI MS: Calcd for C17H34BrN2O5 + [M+H]+ 425.2, 427.2, found 425.2, 427.1; Calcd for C17H33BrN2NaO5 + [M+Na]+ 447.2, 449.2, found 447.1 , 449.1. tert-Butyl4-(2-(2-(2-(2-(piperazin- 1 -yl)ethoxy)ethoxy)ethoxy)ethyl)piperazine- 1 - carboxylate (4e). Compound 4d (0.400 g, 0.93 mmol) was dissolved in 5 ml of dry acetonitrile and added to a stirring mixture of piperazine (0.404 g, 4.7 mmol) and K2CO3 (0.650 g, 4.7 mmol) in 15 ml of dry acetonitrile. The mixture was refluxed for 18 h under nitrogen. After cooling to room temperature the solids were filtered off, the filtrate was evaporated under vacuum and the residue was purified by flash chromatography on Si60 in DCM/methanol containing 0.5 % triethylamine. The final product was evaporated two times from toluene and dried under high vacuum to give a colorless oil (0.285 g, 0.66 mmol, 71 %). TLC (DCM:methanol 5:1 v/v containing 0.5 % Et3N) R/ =0.22. ¾ NMR (500.1 MHz, CDC13) d, ppm: 3.56-3.62 (m, 12H), 3.39- 3.41 (m, 4H), 2.87 (dd, J = 5.0 Hz, J = 5.0 Hz, 4H), 2.55 (ddd, J = 11.0 Hz, J = 5.5 Hz, J = 5.0 Hz, 4H), 2.38-2.46 (m, 9H), 1.42 (s, 9H). 13C NMR (125.8 MHz, CDC13) d, ppm: 154.72, 79.55, 70.58, 70.39, 70.38, 68.69, 68.82, 58.33, 57.81 , 54.71 , 53.36, 45.88, 44.00, 43.10. ESI MS: Calcd for C21H43N4O5 + [M+H]+ 431.3, found 431.3; Calcd for C21H42N4NaO5 + [M+Na]+ 453.3, found 453.2; Calcd for C21H42KN4O5 + [M+K]+ 469.3, found 469.2.
Methyl2-(4-((4-(2-(2-(2-(2-(4-(prop-2-yn-1-yl)piperazin-1-yl)ethoxy)ethoxy) ethoxy)ethyl)pi-perazin-1-yl)methyl)phenyl)acetate (4f). tert-Butyl 4-(2-(2-(2-(2- (piperazin-1-yl)ethoxy)-ethoxy)ethoxy)ethyl)piperazine-l-carboxylate 4e (0.10 g, 0.23 mmol) was mixed with K2CO3 (0.20 g) in dry acetonitrile (5 ml) under nitrogen. Methyl 4-(bromomethyl)phenylacetate 2 (0.056 g, 0.23 mmol) as a solution in 5 ml of acetonitrile was added with continuous stirring. The mixture was stirred at room temperature until the reaction was complete as judged by TLC (2 h). The solids were then filtered off, the filtrate was evaporated to dryness under vacuum and the remaining colorless oil was dissolved in 5 ml of dry DCM. 2,6-Lutidine (0.093 g, 0.87 mmol) was added and the mixture was cooled in an ice- water bath under nitrogen. TMSOTf (0.111 g, 0.5 mmol) was introduced drop- wise with continuous stirring. The ice bath was removed and the mixture was stirred at ambient temperature until complete as judged by TLC (1 h). The solvent was removed under vacuum and the remaining oil was dissolved in dry acetonitrile (5 ml), K2CO3 (0.26 g) was added and the mixture was stirred for 10 min under nitrogen. Propargyl bromide (0.056 g, 0.36 mmol) as a solution in toluene was then added and the mixture was stirred at room temperature until complete as judged by TLC (1 h). The solvent was taken up to dryness under vacuum and the residue was purified by flash chromatography on Si60 in DCM/methanol to afford the desired product as a colorless oil (0.098 g, 0.18 mmol, 80 %). TLC (DCM:methanol 10:1 v/v) R^O.10. ¾ NMR (500.1 MHz, CD3CN) d, ppm: 7.27 (d, J = 8.2 Hz, 2H), 7.23 (d, J = 8.2 Hz, 2H), 4.53 (br s, 1H), 3.63 (s, 3H), 3.53-3.62 (m, 16H), 3.27 (d, J = 2.5 Hz, 2H), 2.50-2.76 (m, 21 H). 13C NMR (125.8 MHz, CD3CN) d, ppm: 172.89, 137.38, 134.53, 130.23, 130.08, 79.79, 74.39, 70.78, 70.74, 70.69, 68.15, 67.59, 62.47, 58.08, 57.89, 53.96, 53.89, 52.53, 52.38, 51.63, 46.88, 40.90. ESI MS: Calcd for C29H47N4O5 + [M+H]+ 531.4, found 531.3; Calcd for C29H46N4NaO5 + [M+Na]+ 553.3, found 553.3; Calcd for C29H46KN4O5 + [M+K]+ 569.3, found 569.3.
Methyl2-diazo-2-(4-((4-(2-(2-(2-(2-(4-(prop-2-yn-1-yl)piperazin-1-yl)ethoxy) ethoxy) ethoxy) ethyl)piperazin-1-yl)methyl)phenyl)acetate (4, Dz-2). Compound 4f (0.090 g,
0.17 mmol) was mixed with p-acetamidobenzensulfonyl azide (0.062 g, 0.26 mmol) in 5 ml of dry acetonitrile under nitrogen. DBU (0.103 g, 0.68 mmol) was introduced drop- wise with continuous stirring. The mixture was stirred for 48 h at room temperature, then evaporated to dryness under vacuum. Purification of the residue by flash chromatography on Si60 in DCM/methanol yielded the target product as an orange-yellow oil (0.028 g, 0.05 mmol, 30 %). TLC (DCM:methanol 10:1 v/v) Rf=0.22. 1H NMR (500.1 MHz, CD3CN) d, ppm: 7.44 (d, J = 8.5 Hz, 2H), 7.33 (d, J = 8.5 Hz, 2H), 3.81 (s, 3H), 3.49-3.54 (m, 12H), 3.44 (s, 2H), 3.22 (d, J = 2.5 Hz, 2H), 2.39-2.49 (m, 21H). 13C NMR (125.8 MHz, CD3CN) d, ppm: 166.48, 137.53, 130.49, 125.19, 124.83, 80.19, 74.15, 71.11 , 71.01 , 69.53, 69.47, 62.90, 58.57, 58.51 , 54.42, 54.24, 53.92, 52.61 , 52.52, 47.12. ESI MS: Calcd for C29H45N6O5 + [M+H]+ 557.3, found 557.4; Calcd for C29H44N6NaO5 + [M+Na]+ 579.3, found 579.3; Calcd for C29H44KN6O5 + [M+K]+ 595.3, found 595.3.
Rhodamine B 2,3,4,5,6-pentafluorophenyl ester (5). Rhodamine B (0.600 g, 1.35 mmol), 2,3,4,5,6-pentafluorophenol (0.250 g, 1.36 mmol) and 4-(N,N- dimethylamino)pyridine (0.021 g, 0.17 mmol) were dissolved in 20 ml of dry DCM under nitrogen and Ν,Ν' -dicyclohexylcarbodiimide (0.327 g, 1.59 mmol) as a solution in 2 ml of DCM was added with continuous stirring. The mixture was stirred at room temperature until complete as judged by TLC (1.5 h). It was then evaporated and the residue purified by column chromatography on Si60 in DCM/methanol to afford the target product as a deep-purple solid (0.610 g, 1.0 mmol, 74 %). TLC (DCM:methanol 10:1 v/v) Rf=0.20. 1H NMR (500.1 MHz, CD3CN) d, ppm: 8.53 (dd, J = 8.0 Hz, J = 1.0 Hz, 1H), 8.02 (ddd, J = 7.6 Hz, J = 7.6 Hz, J = 1.3 Hz, 1H), 7.93 (ddd, J = 7.8 Hz, J = 7.8 Hz, J = 1.3 Hz, 1H), 7.53 (dd, J = 7.6 Hz, J = 1.0 Hz, 1H), 7.12 (d, J = 9.5 Hz, 2H), 6.98 (dd, J = 9.6 Hz, J = 2.5 Hz, 2H), 6.82 (d, J = 2.5 Hz, 2H), 3.62 (q, J = 7.2 Hz, 8H), 1.23 (t, J = 7.1 Hz, 12H). 13C NMR (125.8 MHz, CD3CN) d, ppm: 162.47, 158.67, 157.44, 156.63, 142.91 (m, CF), 140.91 (m, CF), 139.88 (m, CF), 137.93 (m, CF), 135.96, 135.79, 133.05, 131.90, 131.82, 131.77, 127.45, 115.47, 114.14, 96.95, 46.66, 12.72. 19F NMR (376.5 MHz, CD3CN) d, ppm: -154.76 (d, J = 16.8 Hz), -159.47 (t, J = 21.0 Hz), -164.16 (dd, J = 16.9 Hz, J = 21.9 Hz). ESI MS: Calcd for C34H30F5N2O3 + [M]+ 609.2, found 609.3. l-(2-Azidoacetyl)-4-rhodamine B piperazine (6). Compound 5 (0.035 g, 0.06 mmol) was dissolved in 5 ml of dry acetonitrile and N-(2-azidoacetyl)piperazine2 (0.014 g, 0.08 mmol) as a solution in 1 ml of acetonitrile was added with continuous stirring under nitrogen. The mixture was stirred for 24 h at room temperature, then evaporated under vacuum and the residue purified by column chromatography on Si60 in DCM/methanol to afford a deep purplish-violet waxy solid (0.016 g, 0.03 mmol, 48 %). TLC (DCM:methanol 5:1 v/v) Rf=0.32. 1H NMR (500.1 MHz, DMSO-d6) d, ppm: 7.71-7.78 (m, 3H), 7.52-7.53 (m, 1H), 7.10-7.16 (m, 4H), 6.95 (d, J = 2.3 Hz), 4.11- 4.13 (m, 3H), 3.66 (q, J = 7.2 Hz, 8H), 3.39-3.41 (m, 3H), 3.22-3.24 (m, 1H), 2.65-2.69 (m, 3H), 1.20 (t, J = 7.2 Hz, 12H). 13C NMR (125.8 MHz, DMSO-d6) d, ppm: 166.07, 165.77, 157.05, 155.54, 155.11 , 135.13, 131.77, 130.82, 130.74, 130.71 , 130.45, 129.82, 127.54, 114.30, 113.02, 95.90, 49.67, 45.40, 45.36, 45.15, 45.08, 42.31 , 12.47. ESI MS: Calcd for C34H40N7O3 + [M]+ 594.3, found 594.4. l-(2-Azidoacetyl)-4-(D-biotinyl)piperazine (7). D-Biotin 4-nitrophenyl ester (0.068 g, 0.19 mmol) was suspended in 5 ml of dry acetonitrile and N-(2-azidoacetyl)piperazine2 (0.042 g, 0.25 mmol) as a solution in 1 ml of acetonitrile was added with continuous stirring under nitrogen. The mixture was stirred for 24 h at room temperature, then evaporated under vacuum and the residue purified by column chromatography on Si60 in DCM/methanol to afford a colorless oil which was crystallized from chloroform (0.062 g, 0.16 mmol, 84 %). TLC (DCM:methanol 10:1 v/v) Rf=0.23. 1H NMR (500.1 MHz, CD3CN) d, ppm: 5.42 (br s, 1H), 5.15 (br s, 1H), 4.41 (dddd, J = 6.5 Hz, J = 5.0 Hz, J = 2.5 Hz, J = 1.0 Hz, 1H), 4.24 (ddd, J = 8.0 Hz, J = 4.5 Hz, J = 2.0 Hz, 1H), 3.99 (s, 2H), 3.47-3.56 (m, 6H), 3.28-3.34 (m, 2H), 3.17 (ddd, J = 7.9 Hz, J = 6.9 Hz, J = 4.5 Hz, 1H), 2.88 (dd, J = 12.7 Hz, J = 5.0 Hz, 1H), 2.63 (d, J = 12.7 Hz, 1H), 2.35 (dd, J = 8.1 Hz, J = 7.0 Hz, 2H), 1.65-1.72 (m, 1H), 1.53-1.63 (m, 3H), 1.38-1.44 (m, 2H). 13C NMR (125.8 MHz, CD3CN) d, ppm: 172.37, 167.13, 163.83, 62.30, 60.71 , 56.30, 50.99, 45.75, 45.40, 45.11 , 42.63, 42.32, 41.74, 41.66, 41.12, 33.19, 29.10, 29.04, 25.75. ESI MS: 418.2 [M+Na]+. ESI MS: Calcd for C16H25N7NaO3S+ [M+Na]+ 418.2, found 418.2.
Oligonucleotide syntheses and purification
Solid-phase oligonucleotide synthesis was carried out on l-μιηοΐ CPG columns using standard phosphoramidite chemistry with 0.3 M 5-benzylthio-l-H-tetrazole as activator. The DNA oligonucleotides were cleaved from the support with 32 % (v/v) aqueous ammonia for 2 h at room temperature and deprotected for 18 h at 55 °C, then freeze-dried and purified by micropreparative HPLC using method A. The RNA oligonucleotides were synthesized using TBDMS protection strategy for the 2' -OH group. Cleavage from the support was done in 32 % aqueous ammonia/ethanol 3:1 (v/v) for 2 h at room temperature followed by deprotection for 12 h at 55 °C. The samples were freeze-dried and the TBDMS groups were cleaved with triethylamine/triethylamine trihydrofluoride/N-methylpyrrolidine 1.5:2:3 (v/v/v) for 2 h at 65 °C. The crude oligoribonucleotides were isolated by precipitation with 3 M NaOAc (1/10 volume) and n-butanol (4 volumes) for 2 h at -78 °C followed by centrifugation (15,000 x g, 10 min). The pellets were washed with 70 % (v/v) ethanol and dried under high vacuum. The samples were further purified by micropreparative HPLC using method A. The identity of all synthesized oligonucleotides was confirmed by ESI or MALDI TOF MS.
Rh(II)-Catalyzed oligonucleotide modification using a-diazocarbonyl compounds A number of a-diazocarbonyl substrates were screened as potential candidates for the target reaction of oligonucleotide modification (Table 4). Compounds 1-6 all proved to be efficient in modifying various oligonucleotide substrates. Water solubility, however, posed an issue as only entries 1 (Dz-1), 2 and 5 (Dz-2) were sufficiently water-soluble under the conditions of the analysis. Increasing the bulkiness of the ester moiety appeared to have a negative effect on the yields of modified oligonucleotide as for entry 2 the efficacy of modification was reduced twice in comparison to the methyl ester counterpart (entry 1) and with the compound from entry 7 only trace amounts of modified oligonucleotide were isolated.
General procedure for Rh2(OAc)4-catalyzed oligonucleotide modification using a- diazocarbonyl compounds. Typically 10 or 20 ml reaction mixtures containing 5 mM oligonucleotide, 500 mM Rh2(OAc)4 and 50 mM a-diazocarbonyl compound in 100 mM MES buffer, pH 6.0 were kept at 20 °C for 24-72 h. Analysis of the reaction products was carried out by micropreparative HPLC-separation of 5-ml aliquots of the reaction mixtures using method A. The collected peak fractions were further assessed by ESI-MS or MALDI TOF.
Modification of DNA hairpin with a 5 '-adenine overhang. The hairpin-forming oligonucleotide d(ACG GAA TTC CGT TTT TCG GAA TTC CG) containing a restriction site for EcoRI and a 5 '-adenine overhang was alkylated using diazo compound Dz-1 according to the general procedure. The reaction mixture (10 ml) was separated by micropreparative HPLC using method A and the isolated peak fractions were analyzed by MALDI TOF and freeze-dried. The two singly-modified fractions obtained, Ml and M2, were digested with EcoRI (Roche, 30 units per mg oligonucleotide) in 1xSuRE/Cut Buffer H for restriction enzymes (Roche) for 4 h at 20°C. ESI MS and MS/MS of the obtained fragments confirmed that the alkylation takes place at the 5 '-adenine overhang in both cases.
Oligonucleotide labeling by Rh2(OAc)4-catalyzed acetylene-tagging followed by Cu(I)- catalyzed click chemistry. The oligonucleotide d(TTT ATT TGT TTC TTT) was alkylated using diazo compound Dz-2 according to the general procedure. The reaction mixture (20 ml) was separated by micropreparative HPLC using method A and the isolated peak fractions were freeze-dried. The main fraction of modified oligonucleotide was further tagged with the Rhodamine B azide or Biotin-azide (see Fig. 2) via Cu(I)-catalyzed click chemistry according to the general protocol of Hong et al. (Hong, V., Presolski, S. I., Ma, C. & Finn, M. G. Analysis and Optimization of Copper-Catalyzed Azide- Alkyne Cycloaddition for Bioconjugation. Angewandte Chemie International Edition 48, 9879-9883, doi:10.1002/anie.200905087 (2009). The reaction mixtures contained 250 mM acetylene-tagged oligonucleotide, 10 mM azide, 500 mM THPTA ligand, 100 mM CuS04 and 10 mM sodium ascorbate in 100 mM potassium phosphate buffer, pH 7.0. The reactions were carried out at 20 °C. The reaction progress was checked by analytical HPLC using method B. Upon completion of the reaction (typically 1.5-2 h) 10-ml aliquots were separated by micropreparative HPLC and the components of the peak fractions were identified by MALDI TOF.
Fluorescent labeling of PCR primers by Rh2(OAc)4-catalyzed acetylene-tagging followed by Cu(I)-catalyzed click chemistry. The 5 '-extended T7 promoter primer d(CCC CTA ATA CGA CTC ACT ATA GGG) was alkylated using Dz-2 according to the general procedure. The reaction mixture (20 ml) was separated by micropreparative HPLC using method A and the isolated peak fractions were freeze-dried. The total fraction of modified oligonucleotide was further tagged with compound 12 as described previously for oligonucleotide d(TTT ATT TGT TTC TTT). Upon completion of the reaction (2 h) 10-ml aliquots were separated by micropreparative HPLC and the total fraction of fluorescently-labeled primer was freeze-dried.
PCR test experiment. The PCR amplification of the 213-488 region of the pET-19b vector with a 170-bp sequence cloned between the Ndel and Xhol sites was tested with unmodified, acetylene-tagged, and fluorescently-labeled primer. The final concentration of the unmodified and the acetylene-tagged primers in the PCR mixtures was 2 mM, and 10 mM for the fluorescently-labeled one. The amplification products were analyzed by gel electrophoresis in 2% agarose in TAE buffer without ethidium bromide. The gels were visualized by transillumination with UV-light. In the absence of ethidium bromide only the band corresponding to the fluorescently labeled primer was visible. The gels were then soaked in TAE buffer containing ethidium bromide to visualize all nucleic acid containing species.
Example of methods of manufacturing the catalyst comprising a labile ligand:
Figure imgf000032_0001
Procedure based on: Bonar-Law, J. Chem. Soc, Dalton Trans., 2000, 4343-4347 Scale: 0.113 mmol of Rh2(OAc)4
Rh2(OAc)4 and the triacid were combined in a 25 mL round bottomed flask equipped with a stir bar. N,N-Dimethylaniline (5 mL) was added, a vigreux was attached and the reaction mixture was heated to 145 °C for 3 h, whereupon the mixture turned black. After 3.5 h a TLC (DCM/EtOAc 20:1) indicated 3 new spots. The reaction mixture was poured into DCM/HCl 2M (15 mL each) and the organic phase was separated. The organic layer was extracted with 2M HC1 (2 x 15 mL) and the combined organics were then washed with water (2 x 20 mL). The combined organics were dried over MgSO4 , filtered and concentrated to give 20 mg of a green semisolid. Since the amount was too low, the still greenish water phase was extracted with DCM/MeCN (4:1 , 40 mL) whereupon the color of the organic phase changed to purple (Rh-MeCN interaction). The organics were dried over MgSO4, filtered and concentrated to give 131 mg of a green semisolid. Both portions showed the same 3 spots on the TLC so they were combined to give 151 mg of a crude green semisolid. Purified by flash chromatography: dry loaded on Silica in DCM. 15/1 cyclohexane/EtOAc, then 7/1 , then 1/1 , then pure EtOAc. Some green residue stayed on top of silica column. Back isolation by washing it out from the top with EtOAc. Silica was then washed with EtOAc and filtered. Washed with pure MeCN (purple), later MeOH. Solvent evaporated to give 32 mg of a crude purple solid, 55 mg of a green solid obtained after MeOH wash of the silica. Both analyzed by ESI (product inside with dimethylaniline as Ligand). Therefore all combined and MeCN added, extracted with 2 M HC1 (see Reference), organic phase separated, dried over Na2SO4 and concentrated to give 38 mg of a dark green solid consistent with desired product according to NMR and Mass- spectroscopy.
Second ligand substitution:
Figure imgf000034_0001
Procedure from: J. Am. Chem. Soc, 2004, 126 (47), pp 15378-15379, Supporting Scale: 0.059 mmol of mono-ligated complex
The non purified Rh2-complex from first ligand substitution (35 mg) was transferred into a 4 mL microwave vial equipped with a stirring bar and 1.5 mL of 1 ,2- dichloroethane were added (sonication). To this solution was added a portion of diacid (7 mg) and the vial was closed with the suited cap and heated up in an oil bath (130°C). After 35 min the vial was removed from the oil bath and cooled down to rt (5 min, waterbath). Another portion of diacid (7 mg) was added and the vial was sealed and heating at 130°C was resumed. After another 35 min the vial was again removed from the oil bath, cooled down and another portion of diacid (5 mg) was added. The vial was sealed again and heating at 130°C was resumed for 35 min. After cooling down the vial to rt in a water bath, a TLC (1:1 Cyhex/EtOAc) indicated new spots and remaining keto-diacid. The reaction was cooled to rt (let it stand) whereupon a green solid setteled. The supernatant was separated and evaporated to give 30 mg of a green solid. ESI-MS (neg mode) indicated only non reacted keto-diacid, Rh2(OAc)4+N,N-
Dimethylaniline and Starting Material. The remaining suspension was evaporated to dryness and resulted in 26 mg of a dark green solid. ESI-MS (neg mode) indicated the desired product with some other masses. TLC analysis (4:1 cyhex/EtOAc) of the solid indicated 3 different products. The crude (26 mg) was dissolved in MeOH (dry load) and subjected to a 5 g silica prepacked column equilibrated in (4:1 cyhex/EtOAc). The mixture was eluted with 100 mL (4:1 cyhex/EtOAc), 50 mL (2:1), 50 mL (1:1), 50 mL (1:2) and 50 mL pure EtOAc. The remaining colored material was eluted with pure MeOH.
F7-10 turquoise solid, 4 mg, dirhodium with two keto-diacid ligands
F15-23 green solid, 12 mg, desired product Experiment for sequence specific alkylation:
Synthesizing a short nucleic acid complementary to a 9-base pair region of the target sequence and then coupling the last base at the 5'-end has a 5'-amine, a-hydroxylamine acetic acid to the amine before cleaving from the solid support. Mixing a target nucleic acid sequence with the guiding strand in a 5:1 ratio to allow duplex formation. Adding the rhodium complex containing a ketone on one of the aromatic rings of the bidentate ligand followed by the diazo substrate. The catalyst self-assembles through oxime formation between catalyst ketone and the 5'-hydroxylamine of the guiding strand. Adding the diazo which alkylates the target strand after forming a carbene with the catalyst. The catalyst dissociates from the target strand and binds with a different molecule of the target strand for further alkylation.
Experiment for nucleic acid triggered catalyst activation and detection:
Synthesizing a nucleic acid incorporating a stem-loop structure with an a- hydroxylamine acetic acid at the 5 '-end and a dimethylaniline at the 3 '-end. Adding the rhodium complex containing a ketone on one of the aromatic rings of the bidentate ligand and a complex forms through oxime formation with the 5 '-terminal hydroxylamine. The dimethylaniline coordinates to the vacant axial orbital of the rhodium complex thereby deactivating the catalyst. Adding the Diazo substrate but no reaction occurs due to the deactivating effect of the tethered ligand. Adding a nucleic acid which is complementary to the loop region of the stem- loop sequence. The stem- loop is therefore unfolded to allow the double-helix to form between the loop and the added nucleic acid, consequently the deactivating ligand is dissociated from the catalyst and the catalyst can now react with the diazo substrate to generate a fluorescent product. The diazo substrate is a substituted phenol derivative which undergoes cyclization to a fluorescent coumarin product upon rhodium carbene formation. The appearance of the fluorescent signal therefore confirms the presence of the target nucleic acid.
Copper catalyst and its advantages
The method of selectively modifying nucleic acid in a biological or an artificial composition with copper catalyst and a substrate as disclosed above has a number of advantages with respect to previously reported strategies, as well as to the previously reported dirhodium-based method. Copper is cheap and readily available. Also copper has a greater preference for nucleic acid alkylation. With previous catalysts an undesirable side-reaction is the O-H insertion with water. Copper catalysts deliver predominately attack on the nucleic acid and very little undesired side-reactions. Several examples for copper(I)-catalyzed alkylation of short DNA oligonucleotides (ON) are shown in Table 5.
Copper catalysis through the Cu(I) oxidation state, as is performed here, is also valuable because it allows seamless integration with a large body of literature on the Cu-catalyzed azide-alkyne Cycloaddition (CuAAC) (El-Sagheer, A. H.; Brown, T., Click chemistry with DNA. Chem. Soc. Rev. 2010, 39 (4), 1388-1405). This reaction also uses the same conditions as the present invention and so the two reactions can be run in tandem with the same catalyst system. The two different substrates required for the respective reactions could be added sequentially in any order, or at the same time. Since the conditions are the same for both catalytic reactions both processes will occur concurrently.
Copper delivers a different alkylation profile than the above described rhodium system with certain nucleic acids. Therefore these systems are complementary and taken together they could constitute a set of catalysts that can be selected to modify or selectively modify nucleic acids in any desired way, where the reactivity is controlled by the precise combination of catalyst and ligand. In addition they could be used together in one vessel to modify nucleic acids at different positions.
In further embodiment, the present invention demonstrate that copper, in particular, Cu(I)-catalysis can deliver N-H insertion (NHI) with, for example, a-diazocarbonyl compounds in aqueous media. Despite being carried out in water only trace amounts of O-H insertion are seen, indicating the catalyst's overwhelming preference for NHI. According to the present invention, Cu-catalyzed NHI is highly efficient and selective in aqueous media with both simple (Table 8) and complex (Tables 7, 9, & 10) aniline- type substrates. The NHI process is fully compatible with the Cu-catalyzed azide- alkyne cycloaddition (CuAAC) (see for example, V. V. Rostovtsev, L. G. Green, V. V. Fokin and K. B. Sharpless, Angew. Chem. Int. Ed., 2002, 41, 2596-2599; and C. W. Torn0e, C. Christensen and M. Meldal, /. Org. Chem., 2002, 67, 3057-3064) and indeed both processes can be run simultaneously with the same catalyst (formally this is an auto-tandem catalytic reaction(D. E. Fogg and E. N. dos Santos, Coord. Chem. Rev., 2004, 248, 2365-2379.)) Importantly, the reaction is not only efficient with small molecules, but can also be used to directly modify complex biomolecular structures such as DNA and RNA (Figure 9).
Methods for DNA and RNA tailoring are crucial for understanding their biological role as well as adapting them for diagnostic or therapeutic use. In this sense the CuAAC is a mainstay of nucleic acid (NA) chemical biology as it enables the efficient and straightforward introduction of a variety of functionally important tags and reporter groups into NAs under mild conditions. However, a major obstacle in applying the CuAAC with NAs is the need to introduce the alkyne or azide moiety in large complex structures. This is usually done with modified monomer units for solid-phase synthesis, modified deoxynucleoside triphosphates for PCR, or by enzyme-catalyzed 3 '-terminal transfer of an azido-derivatized nucleotide. As disclosed in the preceding embodiments, the methods of the present invention also relates on an N-H insertion (NHI) reaction of a rhodium(II)- stabilized carbenoid directly into the exocyclic amino groups of the nucleobases. Combining the Rh-catalyzed method of present invention with the CuAAC reaction, however, still required separate catalysts and reaction vessels.
The initial examination of the catalytic NHI started with the short oligonucleotide d(ATGC) (see Table 7) in aqueous MES buffer at pH 6. Further refinement of the reaction conditions, including testing different ligands and copper sources, led to a convergence with the CuAAC conditions for bioconjugation developed by Finn (Table 7) (See V. Hong, S. I. Presolski, C. Ma and M. G. Finn, Angew. Chem. Int. Ed., 2009, 48, 9879-9883). The key components of the Finn conditions are sodium ascorbate and the tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) ligand. Ascorbate insures the Cu(I) oxidation state is maintained even in the presence of oxygen (diazo compounds can also mediate the Cu(II)→Cu(I) reduction, but the process is much slower). A consequence of Cu(I) in the presence of oxygen, however, is the formation of reactive oxygen species (ROS).
The present invention further outlines the scope of the method with other simpler nucleophiles like anilines, alkyl amines, alcohols, and thiols (see Table 8). Indeed using the optimized conditions from Table 7, anilines were transformed with uniformly high efficiency (entries 1-9, Table 8). The reactions proceeded smoothly at as low as 1 mol% catalyst loading, giving complete conversion of the starting material and excellent isolated yields of the monoalkylation products. Little dependence on the electronic nature of the aniline was seen, since comparable results were obtained with electronically dissimilar anilines (entries 3-7, Table 8). A detailed analysis of the minor side-products seen in the crude reaction mixtures revealed the formation of a small amount of doubly alkylated aniline, as well as trace amounts of O-H insertion products with water and ascorbate. The observation of O-H insertion products prompted us to test 1-butanol (entry 17, Table 8) but it proved completely unreactive.
In contrast to Cu(I) catalysis in organic solvents (see for example M. E. Morilla, M. M. Diaz-Requejo, T. R. Belderrain, M. C. Nicasio, S. Trofimenko and P. J. Perez, Chem. Comm., 2002, 2998-2999; M. R. Fructos, T. R. Belderrain, M. C. Nicasio, S. P. Nolan, H. Kaur, M. M. Diaz-Requejo and P. J. Perez, /. Am. Chem. Soc, 2004, 126, 10846- 10847) other types of nucleophiles were unreactive (entries 10-17, Table 8). In the case of the nitrogen-based nucleophiles a complete selection for anilines over aliphatic amines and hydroxylamines was observed. At least in the case of anilines vs. alkyl amines this astonishing selectivity is likely attributable to the different protonation states of the substrates due to the differences in pKa of the respective conjugate acids. From a practical standpoint this dramatic solvent-dependent reactivity offers a simple strategy to select the site of reaction in molecules that contain both aryl and alkyl amines.
A kinetic analysis of the aniline NHI did not display Michaelis-Menten saturation kinetics. Instead the decrease in the initial velocities of diazo consumption at high aniline concentration (Figure 10) suggests substrate inhibition of the catalyst. This finding is consistent with the lack of reactivity observed with strongly Lewis-basic substrates such as imidazole, thiols, and hydroxyl amines (entries 10-16, Table 8). Furthermore, conversion of the two pyridyl- aniline substrates (entries 8 and 9, Table 8) were substantially slower than the other anilines.
Armed with a better understanding of the key reaction parameters, delineating the reaction scope with NAs were analyzed (Table 9). The alkylation products of the three trimers, d(TAT), d(TGT), and d(TCT) have previously been characterized by NMR. This unambiguous characterization provides a set of MS and HPLC standards to allow assignment of the sites of reactivity in the present invention. Based on these comparisons, those reaction products from entries 2-4 of Table 9 were identified that result from NHI with the exocyclic amino groups of the A, G, or C bases (comparison by ESI-MS, MS/MS, and HPLC). For d(TGT) and d(TCT) a second set of products were observed; these likely come from the modification of the endocyclic nitrogen. In the case of d(TGT), it is discovered a peak corresponding to double- alkylation of the G nucleobases - a result indicative of the potency of the Cu(I) catalyst system.
The tetramer d(TTTT) reacted poorly delivering only 19% conversion, an observation consistent with our previous studies on rhodium-carbenoids where nucleobases lacking readily available polar X-H bonds (T and U) were poorly alkylated. The mixture of minor products was not characterized but likely arises from targeting the phosphate oxygens or the terminal 3'- or 5'-hydroxyl groups in the absence of other more potent nucleophiles.
RNA also proved to be a viable substrate for carbene insertion (Table 9, entry 7). Its extensive alkylation produced a number of product species highlighting the potential of the Cu(I)-system for RNA-tagging and dense functionalization.
The propensity of double- stranded DNA motifs to react was tested on three hairpin structures (entries 8-10, Table 9). All three hairpins were significantly less reactive than the single-stranded NAs tested, delivering only modest yields of modified DNA at extended reaction times. As expected the hairpin containing only Ts in the loop (entry 8, Table 9) proved to be the least reactive with only 8% conversion. In comparison the remaining two hairpins (entries 9 and 10, Table 9) exhibit a two-fold increase in reactivity. These contain an unpaired A as either a 3 '-overhang (entry 9) or in the loop region (entry 10), indicating that copper preferentially targets nitrogens that are not involved in Watson-Crick base-pairing.
With the scope outlined, optionally, the culmination of the present invention was to combine the CuAAC with NHI in a single-catalyst process. The concept was tested with a collection of different amines, alkynes, diazo compounds, and azides as shown in Table 10. Entries 1 and 2 present small molecule substrates and in these cases the reactions proceeded smoothly affording the desired N-aryl triazole/NHI products in good yields. Furthermore, changing the position of the propargyl function had no effect on the tandem process (cf. entries 1 , 2, and 3, Table 10). This multi-component catalytic process should prove useful for medicinal and combinatorial chemists since it provides a robust strategy to quickly assemble complex molecular scaffolds.
Guided by the results with small molecules, the simultaneous CuAAC/NHI with more challenging DNA-based substrates were performed. With DNA, a bifunctional propargylated a-diazocarbonyl compound was employed to serve as a connecting element between the NA and the azide-bearing tag (see entries 3, 4, and 5, Table 10). A number of alkylation products were observed for each DNA with moderate (27%) to good (59-82%) conversion of the DNA starting material. Tandem MS analysis of the d(ATG) modification products revealed that the guanine base is the primary alkylation site, undergoing two consecutive modifications before the adenine is modified. The efficacy of the CuAAC reaction assured that all modified NAs bore the triazole tag. The potency of the Cu(I) system facilitates the introduction of multiple alkylations on a single NA when the reaction is run to high conversions of the starting NA. Therefore, in its present form the auto-tandem catalytic approach to NA alkylation is best suited to applications in labelling or dense functionalization of NAs. Mutiply- alkylated NAs are important in a variety of applications including for DNA and RNA detection probes and for the construction of functional DNA architectures. The methods of the present invention is orthogonal and complementary to established approaches based on solid- phase synthesis or enzymatic polymerization (S. H. Weisbrod and A. Marx, Chem. Comm., 2008, 5675-5685; E. T. Kool, Curr. Opin. Chem. Biol., 2000, 4, 602-608). Therefore, the present invention further discloses an approach for molecular conjugation based on the discovery that Cu(I)-carbenoid chemistry is viable in water. The efficacy of Cu(I) for catalyzing both CuAAC and arylamine NHI results in combining both reactions in a one-pot operationally simple process. The substrate range includes simple arylamines as well as the nucleobases in DNA and RNA.
Other Metals
Included in the table 6 are results with other metals. As it is shown, copper is the most active metal; however, activity is also seen with cobalt (Lu, H.; Dzik, W. I.; Xu, X.; Wojtas, L.; de Bruin, B.; Zhang, X. P., Experimental Evidence for Cobalt(III)-Carbene Radicals: Key Intermediates in Cobalt(II)-Based Metalloradical Cyclopropanation. J. Am. Chem. Soc. 2011, 133 (22), 8518-8521.) nickel, iron, and silver. The activity seen with these metals is promising since screening for appropriate activating ligands could greatly increase the efficiency. Further metals such as ruthenium (Ho, C.-M.; Zhang, J.-L.; Zhou, C.-Y.; Chan, O.-Y.; Yan, J. J.; Zhang, F.-Y.; Huang, J.-S.; Che, C.-M., A Water-Soluble Ruthenium Glycosylated Porphyrin Catalyst for Carbenoid Transfer Reactions in Aqueous Media with Applications in Bioconjugation Reactions. J. Am. Chem. Soc. 2010, 132 (6), 1886-1894.), osmium, molybdenum, and palladium are known in the art for carbene formation with diazo substrates and therefore they can also be employed in nucleic acid alkylation with diazo substrates according to the present invention.
Ligands and Conditions
A variety of ligands and metal sources have been tested as shown on table 7. The concentrations of the ligand and the ascorbate needed for the in situ generation of the catalytically- active copper(I) species have been optimized (Figure 6a and b) to give the best reaction conditions: 5 mM oligonucleotide, 0.5 mM CuSO4, 2.5 mM Tris(3- hydroxypropyltriazolylmethyl)amine (THPTA), 50 mM methyl 2-diazo-2-(4- ((dimethylamino)methyl)phenyl)acetate (Dz1), 10 mM sodium ascorbate, 100 mM MES, pH 6. A number of ligands give good alkylation profiles. The bipyridyl ligand or N-heterocyclic carbene ligands are preferable because it would offer a simple method to immobilize the copper onto a guiding group and then copper could be used to achieve sequence- specific nucleic acid alkylation. In particular, N-heterocyclic carbene ligands are ideal partners for copper catalysis according to the present invention (see Figure 7). These ligands almost completely suppress the oxidative damage to DNA and RNA that is often caused by copper(I). Other common bidentate ligands for copper could also be used to achieve this end.
OTHER EMBODIMENTS
The detailed description set-forth above is provided to aid those skilled in the art in practicing the present invention. However, the invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed because these embodiments are intended as illustration of several aspects of the invention. The embodiments set-forth above can be performed and combined with other disclosed embodiments according to the invention. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description which do not depart from the spirit or scope of the present inventive discovery. Such modifications are also intended to fall within the scope of the appended claims.
Figure imgf000043_0001
Figure imgf000044_0001
Figure imgf000045_0001
Figure imgf000046_0001
Figure imgf000047_0001
Figure imgf000048_0001
Figure imgf000049_0001
Figure imgf000050_0001
Figure imgf000051_0001

Claims

Claims
1) Catalyst (X) comprising one or more ligands and a guiding motif, wherein X is selected from a group consisting of copper, rhodium, cobalt, nickel, iron, or silver; and wherein said guiding motif binds to a nucleic acid, whereby the catalyst (X) induces modification of the nucleic acid.
2) The catalyst (X) according to claim 1 , wherein X is copper.
3) The catalyst (X) according to claim 1 , wherein X is copper and the ligand is N- heterocyclic carbene.
4) The catalyst (X) according to claim 1 , wherein X is rhodium and wherein said rhodium is rhodium (II) and is dimeric.
5) Catalyst (X) for selectively modifying a nucleic acid in a biological or an artificial composition comprising a nucleic acid sequence or a nucleic acid mimic sequence, wherein the nucleic acid or the nucleic acid mimic is a stem-loop sequence, and wherein said stem-loop sequence comprises a deactivating ligand, and wherein X is selected from a group consisting of copper, rhodium, cobalt, nickel, iron, or silver.
6) A method of selectively modifying a nucleic acid in a biological or an artificial composition, said method comprising the step of treating the composition with a catalyst (X) and a substrate, wherein X is selected from a group consisting of copper, rhodium (II), cobalt, nickel, iron, or silver.
7) The method of selectively modifying a nucleic acid according to claim 6, wherein said catalyst (X) is not conjugated to the substrate prior to treating the nucleic acid to be modified.
8) The method of selectively modifying a nucleic acid according to claim 6 or 7, wherein X is copper.
9) The method of selectively modifying a nucleic acid according to claim 6 or 7, wherein X is rhodium (II) and is dimeric.
10) The method of selectively modifying a nucleic acid according to any of the claims 6-9, wherein said catalyst (X) further comprises one or more ligands.
11) The method of selectively modifying a nucleic acid according to claim 10,
wherein the ligand is N-heterocyclic carbine, and wherein the X is copper. 12) The method of selectively modifying a nucleic acid according to any of claims 6- 11 , wherein said method comprising linking the catalyst (X) to a guiding motif, wherein the guiding motif is identical, is complementary, or hybridizes under stringent or highly stringent conditions to all or portion of the nucleic acid.
13) A method of selectively modifying a nucleic acid according to any of claims 6-12, further comprising the steps of:
a. embedding the catalyst (X) in a nucleic acid sequence or a nucleic acid mimic sequence, wherein the nucleic acid or the nucleic acid mimic is a stem-loop sequence;
b. inactivating the catalyst (X) by a deactivating ligand at the opposite end of the said stem-loop sequence; and
c. contacting the inactivated and embedded catalyst (X) with the nucleic acid in the biological or an artificial composition, wherein the nucleic acid is identical, is complementary, or hybridizes under stringent conditions to all or portion of the stem- loop sequence.
14) A kit for selectively modifying a nucleic acid in a biological or an artificial
composition, wherein the kit comprises (a) a catalyst (X), wherein X is copper, cobalt, nickel, iron, or silver, (b) a substrate, and (c) containers suitable for containing said catalyst (X), the substrate, and the composition.
15) A kit for selectively modifying a nucleic acid in a biological or an artificial
composition, wherein the kit comprises (a) a catalyst (X) according to any of the claims 1-5, (b) a substrate, and (c) containers suitable for containing said catalyst (X), the substrate, and the composition.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019063803A1 (en) * 2017-09-29 2019-04-04 Baseclick Gmbh Click based ligation
CN112844258A (en) * 2019-11-27 2021-05-28 中国科学院大连化学物理研究所 Cysteine-modified nano core-shell silica gel material and preparation and application thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20010005588A1 (en) * 1999-12-27 2001-06-28 Fuji Photo Film Co., Ltd. DNA chip and its preparation
WO2001083482A1 (en) * 2000-05-03 2001-11-08 The Scripps Research Institute Dna alkylating agent and activation thereof
US20110263816A1 (en) * 2009-11-23 2011-10-27 Rice University Metallopeptide catalysts

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20010005588A1 (en) * 1999-12-27 2001-06-28 Fuji Photo Film Co., Ltd. DNA chip and its preparation
WO2001083482A1 (en) * 2000-05-03 2001-11-08 The Scripps Research Institute Dna alkylating agent and activation thereof
US20110263816A1 (en) * 2009-11-23 2011-10-27 Rice University Metallopeptide catalysts

Non-Patent Citations (43)

* Cited by examiner, † Cited by third party
Title
"Current Protocols in Molecular Biology", 1995, JOHN WILEY & SONS
A. M. MAXAM; W. GILBERT, PROC. NATL. ACAD. SCI. USA, vol. 74, 1977, pages 560 - 564
A. R. HERNÁNDEZ; L. W. PETERSON; E. T. KOOL, ACS CHEM. BIOL., 2012
BONAR-LAW, J. CHEM. SOC., DALTON TRANS., 2000, pages 4343 - 4347
BROWNE, K. A.: "Metal Ion-Catalyzed Nucleic Acid Alkylation and Fragmentation", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 124, 2002, pages 7950 - 7962
C. J. MOODY, ANGEW. CHEM. INT. ED., vol. 46, 2007, pages 9148 - 9150
C. W. TORNØE; C. CHRISTENSEN; M. MELDAL, J. ORG. CHEM., vol. 67, 2002, pages 3057 - 3064
CHRISTINE G. ESPINO ET AL: "Expanding the Scope of C-H Amination through Catalyst Design", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 126, no. 47, 1 December 2004 (2004-12-01), pages 15378 - 15379, XP055049078, ISSN: 0002-7863, DOI: 10.1021/ja0446294 *
COPELAND KIMBERLY D ET AL: "DNA hydrolysis and oxidative cleavage by metal-binding peptides tethered to rhodium intercalators", BIOCHEMISTRY, AMERICAN CHEMICAL SOCIETY, US, vol. 41, no. 1, 8 January 2002 (2002-01-08), pages 343 - 356, XP002480894, ISSN: 0006-2960, [retrieved on 20011211], DOI: 10.1021/BI011793K *
D. E. FOGG; E. N. DOS SANTOS, COORD. CHEM. REV., vol. 248, 2004, pages 2365 - 2379
DALHOFF, C.; LUKINAVICIUS, G.; KLIMASAUSKAS, S.; WEINHOLD, E.: "Direct transfer of extended groups from synthetic cofactors by DNA methyltransferases", NAT CHEM BIOL, vol. 2, 2006, pages 31 - 32, XP055022779, DOI: doi:10.1038/nchembio754
E. T. KOOL, CURR. OPIN. CHEM. BIOL., vol. 4, 2000, pages 602 - 608
EL-SAGHEER, A. H.; BROWN, T.: "Click chemistry with DNA", CHEM. SOC. REV., vol. 39, no. 4, 2010, pages 1388 - 1405
ERKKILA K E ET AL: "Recognition and Reaction of Metallointercalators with DNA", CHEMICAL REVIEWS, AMERICAN CHEMICAL SOCIETY, WASHINGTON, DC, US, vol. 99, no. 9, 1 January 1999 (1999-01-01), pages 2777 - 2796, XP002465366 *
F. A. ALDAYE; A. L. PALMER; H. F. SLEIMAN, SCIENCE, vol. 321, 2008, pages 1795 - 1799
GRAMLICH PHILIPP M E ET AL: "Postsynthetic DNA modification through the copper-catalyzed azide-alkyne cycloaddition reaction", ANGEWANDTE CHEMIE. INTERNATIONAL EDITION, WILEY VCH VERLAG, WEINHEIM, vol. 47, no. 44, 20 October 2008 (2008-10-20), pages 8350 - 8358, XP009120328, ISSN: 1433-7851, [retrieved on 20080922], DOI: 10.1002/ANIE.200802077 *
H. PEACOCK; A. KANNAN; P. A. BEAL; C. J. BURROWS, J. ORG. CHEM., vol. 76, 2011, pages 7295 - 7300
HEETEBRIJ, R. J. ET AL.: "Platinum (II)-Based Coordination Compounds as Nucleic Acid Labeling Reagents: Synthesis, Reactivity, and Applications in Hybridization Assays", CHEMBIOCHEM, vol. 4, 2003, pages 573 - 583, XP009127381, DOI: doi:10.1002/cbic.200200498
HO, C.-M.; ZHANG, J.-L.; ZHOU, C.-Y.; CHAN, O.-Y.; YAN, J. J.; ZHANG, F.-Y.; HUANG, J.-S.; CHE, C.-M.: "A Water-Soluble Ruthenium Glycosylated Porphyrin Catalyst for Carbenoid Transfer Reactions in Aqueous Media with Applications in Bioconjugation Reactions", J. AM. CHEM. SOC., vol. 132, no. 6, 2010, pages 1886 - 1894
HONG, V.; PRESOLSKI, S. I.; MA, C.; FINN, M. G.: "Analysis and Optimization of Copper-Catalyzed Azide-Alkyne Cycloaddition for Bioconjugation", ANGEWANDTE CHEMIE INTERNATIONAL EDITION, vol. 48, 2009, pages 9879 - 9883, XP055016858, DOI: doi:10.1002/anie.200905087
J. AM. CHEM. SOC., vol. 126, no. 47, 2004, pages 15378 - 15379
J. KURRECK, ANGEW. CHEM. INT. ED., vol. 48, 2009, pages 1378 - 1398
J. M. ANTOS; J. M. MCFARLAND; A. T. LAVARONE; M. B. FRANCIS, J. AM. CHEM. SOC., vol. 131, 2009, pages 6301 - 6308
JAWALEKAR A M ET AL: "Conjugation of Nucleosides and Oligonucleotides by [3+2] cycloaddition", THE JOURNAL OF ORGANIC CHEMISTRY, AMERICAN CHEMICAL SOCIETY [NOT]ETC., vol. 73, no. 1, 4 January 2008 (2008-01-04), pages 287 - 290, XP002579220, ISSN: 0022-3263, [retrieved on 20071204], DOI: 10.1021/JO702023S *
KAZUMITSU ONIZUKA ET AL: "Site-Specific Covalent Modification of RNA Guided by Functionality-Transfer Oligodeoxynucleotides", BIOCONJUGATE CHEMISTRY, vol. 20, no. 4, 15 April 2009 (2009-04-15), pages 799 - 803, XP055049077, ISSN: 1043-1802, DOI: 10.1021/bc900009p *
KIRIL TISHINOV ET AL: "Structure-Selective Catalytic Alkylation of DNA and RNA", ANGEWANDTE CHEMIE INTERNATIONAL EDITION, vol. 51, no. 48, 22 October 2012 (2012-10-22), pages 12000 - 12004, XP055048795, ISSN: 1433-7851, DOI: 10.1002/anie.201205201 *
LU, H.; DZIK, W. I.; XU, X.; WOJTAS, L.; DE BRUIN, B.; ZHANG, X. P.: "Experimental Evidence for Cobalt(III)-Carbene Radicals: Key Intermediates in Cobalt(II)-Based Metalloradical Cyclopropanation", J. AM. CHEM. SOC., vol. 133, no. 22, 2011, pages 8518 - 8521
M. C. PIRRUNG; H. LIU; A. T. MOREHEAD, J. AM. CHEM. SOC., vol. 124, 2002, pages 1014 - 1023
M. E. MORILLA; M. M. DIAZ-REQUEJO; T. R. BELDERRAIN; M. C. NICASIO; S. TROFIMENKO; P. J. PEREZ, CHEM. COMM., 2002, pages 2998 - 2999
M. R. FRUCTOS; T. R. BELDERRAIN; M. C. NICASIO; S. P. NOLAN; H. KAUR; M. M. DIAZ-REQUEJO; P. J. PEREZ, J. AM. CHEM. SOC., vol. 126, 2004, pages 10846 - 10847
MOTORIN, Y. ET AL.: "Expanding the chemical scope of RNA: methyltransferases to site-specific alkynylation of RNA for click labeling", NUCLEIC ACIDS RESEARCH, vol. 39, 2011, pages 1943 - 1952, XP055230450, DOI: doi:10.1093/nar/gkq825
ONIZUKA, K.; TANIGUCHI, Y.; SASAKI, S.: "Site-Specific Covalent Modification of RNA Guided by Functionality-Transfer Oligodeoxynucleotides", BIOCONJUGATE CHEMISTRY, 2009, pages 20
ROSTOVTSEV, V. V.; GREEN, L. G.; FOKIN, V. V.; SHARPLESS, K. B.: "A Stepwise Huisgen Cycloaddition Process: Copper(I)-Catalyzed Regioselective ''Ligation'' of Azides and Terminal Alkynes", ANGEWANDTE CHEMIE INTERNATIONAL EDITION, vol. 41, 2002, pages 2596 - 2599, XP002524189, DOI: doi:10.1002/1521-3773(20020715)41:14<2596::AID-ANIE2596>3.0.CO;2-4
S. H. WEISBROD; A. MARX, CHEM. COMM., 2008, pages 5675 - 5685
S. S. JENSEN; X. ARIZA; P. NIELSEN; J. VILARRASA; F. KIRPEKAR, J. MASS SPECTROM., vol. 42, 2007, pages 49 - 57
SAMBROOK ET AL.: "Molecular Cloning, A Laboratory Manual", 1989, COLD SPRING HARBOR PRESS
SCHATZSCHNEIDER U ET AL: "Bifunctional rhodium intercalator conjugates as mismatch-directing DNA alkylating agents", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, ACS PUBLICATIONS, US, vol. 126, no. 28, 21 July 2004 (2004-07-21), pages 8630 - 8631, XP002472998, ISSN: 0002-7863, DOI: 10.1021/JA048543M *
SONG, C.-X.; HE, C.: "Bioorthogonal Labeling of 5-Hydroxymethylcytosine in Genomic DNA and Diazirine-Based DNA Photo-Cross-Linking Probes", ACCOUNTS OF CHEMICAL RESEARCH, 2011
TORNØE, C. W.; CHRISTENSEN, C.; MELDAL, M.: "Peptidotriazoles on Solid Phase: [1,2,3]-Triazoles by Regiospecific Copper(I)-Catalyzed 1,3-Dipolar Cycloadditions of Terminal Alkynes to Azides", THE JOURNAL OF ORGANIC CHEMISTRY, vol. 67, 2002, pages 3057 - 3064, XP002567967, DOI: doi:10.1021/jo011148j
V. HONG; S. I. PRESOLSKI; C. MA; M. G. FINN, ANGEW. CHEM. INT. ED., vol. 48, 2009, pages 9879 - 9883
V. V. ROSTOVTSEV; L. G. GREEN; V. V. FOKIN; K. B. SHARPLESS, ANGEW. CHEM. INT. ED., vol. 41, 2002, pages 2596 - 2599
WEISBROD, S. H.; MARX, A.: "Novel strategies for the site-specific covalent labeling of nucleic acids", CHEMICAL COMMUNICATIONS, 2008, pages 5675 - 5685
ZHOU, Q.; ROKITA, S. E.: "A general strategy for target-promoted alkylation in biological systems", PROCEEDING OF THE NATIONAL ACADEMY OF SCIENCES, vol. 100, 2003, pages 15452 - 15457, XP055049075, DOI: doi:10.1073/pnas.2533112100

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