EP2880016A1 - Procedes de fonctionnalisation et reactifs utilises dans de tels procedes utilisant un anhydride aza-isatoïque ou un de ses derives, molecules biologiques ainsi traitees et kits - Google Patents

Procedes de fonctionnalisation et reactifs utilises dans de tels procedes utilisant un anhydride aza-isatoïque ou un de ses derives, molecules biologiques ainsi traitees et kits

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Publication number
EP2880016A1
EP2880016A1 EP13744493.1A EP13744493A EP2880016A1 EP 2880016 A1 EP2880016 A1 EP 2880016A1 EP 13744493 A EP13744493 A EP 13744493A EP 2880016 A1 EP2880016 A1 EP 2880016A1
Authority
EP
European Patent Office
Prior art keywords
rna
interest
group
molecule
binding molecule
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP13744493.1A
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German (de)
English (en)
French (fr)
Inventor
Thomas CAILLY
Frédéric FABIS
Ali Laayoun
Alain Laurent
Sylvain URSUEGUI
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Biomerieux SA
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Biomerieux SA
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Filing date
Publication date
Application filed by Biomerieux SA filed Critical Biomerieux SA
Publication of EP2880016A1 publication Critical patent/EP2880016A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/78—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D213/79—Acids; Esters
    • C07D213/80—Acids; Esters in position 3
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
    • C07D495/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D495/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6825—Nucleic acid detection involving sensors

Definitions

  • the present invention relates to novel methods including functionalization, labeling, capture or separation of biological molecules, and more specifically natural or synthetic ribonucleic acids (RNA) or synthetic chimeric DNA / RNA nucleic acids.
  • RNA ribonucleic acids
  • the term "functionalization” or the related terms (“functionalize” for example) will be used and may mean both labeling by a detectable molecule, addition of an adduct allowing capture, separation or separation. inhibition of biological molecules. It also relates to biological molecules thus treated or labeled, as well as kits that can be used in the field of diagnosis, in particular molecular diagnosis, using the detection and analysis of nucleic acids.
  • a first method is to set the interest group on the basis, whether natural or modified.
  • a second method proposes to set the interest group on sugar, again whether it is natural or modified.
  • a third method is for fixing the interest group on phosphate.
  • the group of interest on the basis was particularly used in the approach of labeling nucleic acids by incorporation of directly labeled nucleotides. In general, the functionalisation at the sugar level is much more neutral than that carried out on the phosphate or on the base, which impacts the specificity and the sensitivity.
  • diazo - based interest groups are not chemo - specific for RNA nor regio - specific for a particular position. They thus functionally undifferentiated DNA or RNA.
  • the diazo function is hardly compatible with conjugation with certain cyanines, such as Cy5 for example.
  • the regiospecific functionalization of the 3 'ends of the RNA can be obtained only from extremely specific enzymatic techniques.
  • Such techniques are used, for example, by Affymetrix (Santa Clara, USA) or Agilent Technologies (Santa Clara, USA) with the specific labeling of 3 'RNAs with the T4 RNA Ligase and a pCp-tagging substrate or by other enzymatic techniques described in some references:
  • RNAs or miRNAs short-chain non-coding RNAs, specific to eukaryotic cells
  • RNAs and in particular the 3 'end to minimize the effects of steric hindrance particularly marked on small duplexes are very poorly labeled by enzymatic techniques precisely because of their size as described by F. Natt in the reference: Nucleic Acids Research, 2007, Vol. 35, No. 7 e52.
  • diazo labeling technology is an excellent technique, the nature of the synthesis steps leading to these molecules is not compatible with the chemical nature of certain fluorophores (for example Cy5 which is unstable in the presence of hydrazine).
  • the diazo technology is therefore preferably used with biotin as a group of interest.
  • RNA vis-à-vis the DNA can also have applications in sample preparation also called “sample prep", such as the selection of the RNAs without using a DNAase, the selective capture of RNA in a medium containing DNA, decontamination, selective inhibition of amplification, etc.
  • Hiratsuka Hiratsuka BBA 1983 742 496-508 published one of the first articles on the reactivity of isatoic anhydride or isatoic anhydride methylated on free ribonucleosides (5 'triphosphate or 5'-OH). This is to synthesize fluorescent enzyme substrates for the study of these. Indeed, the isatoic anhydride, once opened, becomes fluorescent. However, to avoid any confusion, it is necessary to differentiate between the intrinsic fluorescence of the open isatoic anhydride, which becomes an anthranylate molecule (excitation: 335-350 nm and emission: 427-446 nm), and the fluorescence that it is provided by conjugation with a fluorophore.
  • RNA labeling with isatoic anhydride does not react on the exocyclic amines of bases, despite a nucleophilia usually described to be much higher than that of
  • isatoic anhydride is not known to react on the 5'-OH, isatoic anhydride reacts preferentially
  • Ovodov 1990 (Ovodov, FEBS 1990 270 111-114), by research based on work of Khorana 1963 (Stnark, Journal of the American Chemical Society 1963 75 2546 and Knorre Biokhimiya 1965 1218-1224), describes the acylation of messenger RNAs in an aqueous medium with acetic anhydride (5% DMF, 1M sodium acetate, pH 7, 2 to 3 hours at room temperature) to protect the RNA against the action of the RNases. It describes a level of acylation of 70-75% sufficient to inhibit the action of RNases.
  • Servillo (Servillo Eur J.
  • RNA SHAPE chemistry reveals non-hierarchical interactions dominate equilibrium structural transitions in tRNA Asp transcripts. J. Am. Chem. Soc. 127, 4659-4667 (2005). EJ Merino, KA Wilkinson, JL Coughlan and KM Weeks, RNA structure analysis at single nucleotide resolution by Selective 2 '-Hydroxyl Acylation and Primer Extension (SHAPE). J. Am. Chem. Soc. 127, 4223-4231 (2005). SI Chamberlin and KM Weeks, local Mapping nucleotide flexibility by selective acylation of 2'-amine substituted RNA. J. Am. Chem. Soc. 122, 216-224 (2000) .e
  • the author uses derivatives of isatoic anhydride (N-methylated isatoic anhydride, isatoic anhydride, N-methylated nitro isatoic anhydride, nitro isatoic anhydride) which is reacted on transfer RNA or short model oligoribonucleotides (aqueous medium). pH 8, room temperature or 37 ° C for a few hours). Only the 2 '-OH which are not very constrained, that is to say they are not very involved in secondary or tertiary structures, are acylated because they are more accessible and less close to a diester phosphate skeleton. They become more reactive.
  • this partially acylated RNA is subjected to an in vitro transcription which generates DNA fragments more or less long, the elongation stopping each time a voluminous 2'-O-anthranylate, that is to say ie, an open isatoic anhydride molecule which is attached to the 2'-OH of the sugar, is encountered.
  • a voluminous 2'-O-anthranylate that is to say ie, an open isatoic anhydride molecule which is attached to the 2'-OH of the sugar
  • US-B-7, 244, 568 relates to the selective acylation, more or less partial, 2'-OH RNA with hydrophobic groups (butyryl or pentanoyl from the corresponding anhydrides).
  • the RNA thus becomes sufficiently hydrophobic to be selectively extracted with an organic solvent, or to be selectively immobilized (for example on a reverse phase, silica, a membrane, etc.).
  • a solid phase activated by an acid chloride or an anhydride which selectively immobilizes the RNA molecules relative to the DNA molecules. This phase can be carried out with immobilized isatoic anhydride or BCPB (immobilized benzyl chloride on polystyrene).
  • EP-B-1 966 631 proposes acylating agents compatible with a regiospecific acylation of RNA in an aqueous medium. These acylating agents must not bring a too much steric hindrance to maintain the hybridization properties of the RNA thus modified. These are mainly acylating agents allowing the introduction of an acetyl or formyl group which are used.
  • the thus partially modified RNA then serves as a template for a polymerization reaction, it can also serve as a probe in a Northern blot reaction.
  • the idea is to maintain the hybridization properties (the modified RNA remains the substrate of an elongation reaction), while destroying the secondary structures by the presence of the 2 'group and making the RNA thus modified. resistant to nucleases.
  • Example 22 indicates that the fluorescent labeling of the RNA is envisaged, but only by addition of the methylated isatoic anhydride, it is therefore an intrinsic fluorescence.
  • Patent EP-B-1.196.631 proposes a polynucleotide comprising mRNA, rRNA or viral RNA, for which more than 25% of the riboses are covalently modified at the 2 '-OH positions.
  • it relates to a method for producing double-stranded oligonucleotides and polynucleotides from a starting nucleic acid strand, from a plurality of mononucleotides (UTP, dTTP and / or dUTP, ATP and / or dATP). , GTP and / or dGTP, and CTP and / or dCTP), in the presence of polymerase and optionally primers allowing the formation of a nucleic acid strand complementary to the starting nucleic acid.
  • Patent application WO-A-2004/013155 describes the chemical modification of RNA in a mixture of feces, blood, etc., in order to differentiate DNA from RNA. It is acetic anhydride which is the reagent capable of doing this differentiation. The ester function is then hydrolyzed to regenerate the biologically active RNA.
  • this application proposes to protect the use of organic bases "Little" aggressive for RNA (lysine, diamines, etc.), in combination with a deprotection protocol.
  • isatoic anhydride In the state of the art, there are generally four different uses of isatoic anhydride. First, it is to exploit the intrinsic fluorescence properties of anthranyl esters to study the mechanism of certain enzymes. Secondly, the use of the acylating properties of isatoic anhydride to regioselectively inhibit the polymerization of the nucleic acids, it is therefore only necessary to introduce a bulky substituent at 2 'under the mildest possible conditions. and this in a more or less gentle way. Thirdly, the state of the art uses isatoic anhydride to prevent their degradation during an extraction step. Finally, and fourthly, it involves selectively extracting the acylated RNA relative to the non-acylated DNA. There is then an idea of the reversibility of the ester function so as to regenerate a free 2 'OH function and thus a functional RNA.
  • the conjugation of the isatoic anhydride to the group of interest must be done through atoms and bonds which can cause a very strong destabilization of the structure, a very great difficulty in the synthesis, a bad solubility in water, a loss of the physicochemical properties of the interest group by attenuation of the fluorescence, a very bad reactivity with regard to the RNA, a very great instability of the duplexes formed between the labeled RNA and the DNA (capture probes, for example, by a steric gene that is too important or because the functionalized RNA / DNA hybrids are no longer polymerase substrates).
  • RNAs by isatoic anhydride compounds in order to allow their capture by recognition molecules carried or not by a solid support.
  • WO-A-2012/076794 to use non-isatoic anhydride not for its intrinsic fluorescence but for its ability to bind specifically to the RNA and to allow binding with a group of interest, as defined below.
  • BOOM technology a technique of adsorption on silica in the presence of chaotropic salts
  • the technology is based on the particular affinity that is created between nucleic acids, silica and cations that would make the link between the phosphate groups and the silanol residues present on the surface of the silica as described in "Sample Preparation Techniques in Analytical Chemistry. Vol 162 Edited by S. Mitra Wiley-Interscience 2003 ".
  • EP-B-1.367.137 describes the improvement of BOOM technology by using magnetic silica particles for the extraction of nucleic acids, allowing much easier automation.
  • EP-B-1.476.550 discloses a method for purifying nucleic acids using a solid phase modified with thioethers and eluents consisting of lyotropic salts.
  • Patent EP-B-1,266,385 proposes ferromagnetic or ferrimagnetic silica particles for the purification of nucleic acids.
  • EP-B-1.762.618 discloses a solution for extracting nucleic acids from blood consisting of 15 to 35% guanidine and a detergent.
  • EP-B-1,771,562 discloses a novel method for extracting nucleic acids based on silica in the presence of organic adjuvants.
  • patent EP-B-1.589.105 proposes the extraction of nucleic acid at acidic pH on magnetic particles not covered with silica.
  • EP-B-1448799 discloses the more or less selective purification of nucleic acids on silica at alkaline pH and in the presence of various anions.
  • EP-A-1,510,577 attempts to protect the use of a mixture of chaotropic salts and hydrophilic additives to optimize DNA separation over RNA.
  • EP-B-1,479,769 describes and claims a selective separation between RNA and DNA. This is based on changes in temperature, ionic strength and chemical composition of some buffered solutions.
  • EP-B-1.869.179 proposes the use of ligands specific for RNA or DNA for the specific extraction of RNA with respect to DNA.
  • US-A-2007/0148651 discloses the specific extraction of RNA from a mixture of DNA and RNA by adsorbing RNA on magnetite particles in the presence of phosphate anions.
  • Patent application WO-A-2009/040444 describes the selective precipitation of genomic DNA on chitosan-coated magnetite particles from a DNA / RNA mixture. The RNA is then recovered in the supernatant by precipitation with alcohol. Finally, mention may be made of US-A-2005/0106576 and US-A-
  • NABP nucleic acid binding fragment
  • nucleic acids RNA or DNA
  • solid phase in the presence of various additives, which will modulate the adsorption more or less selectively.
  • the interactions involved during the adsorption of the nucleic acids are of a weak nature (electrostatic, ionic, hydrophobic, hydrogen bonds, etc.) in comparison with a covalent bond.
  • WO-A-2012/076794 The corresponding patent application, WO-A-2012/076794, is incorporated herein by reference.
  • the Applicant brought an early solution to the problem of separation between RNA and DNA from a complex medium. It was proposed to discriminate RNA from DNA on the basis of their chemical composition. Indeed, the only difference between RNA and DNA lies in the presence of a 2 'hydroxyl group of ribose.
  • the objective was therefore to take advantage of this function to specifically couple a binding molecule (an isatoic anhydride derivative) provided with a group of interest making it possible to perform a molecular sorting between the two macromolecules, based on the recognition specific to the interest group.
  • the reactive binding molecule had to be able to react under mild conditions (65 ° C., duration less than 1 hour and in an aqueous medium).
  • RNA captured and DNA-free can then be separated from its interest group directly on the solid phase so that it can in turn be specifically eluted and amplified. This method therefore makes it possible to sort and separate the RNA from the DNA with a very high selectivity, and consists of:
  • RNA 1-Making a covalent bond between the RNA and a binding molecule (isatoic anhydride derivative) bearing a group of interest.
  • the present invention allows:
  • RNA and aza-anthranylate groups have, after reaction of the binding molecule and cleavage of the linker, a "naked" RNA and without aza-anthranylate groups (in order to avoid any inhibition that would come from aza-anthranylate),
  • RNA an absolute resistance to nucleases by unscheduled functionalisation of the RNA
  • a group of interest is a molecule that:
  • electrophilic molecule / nucleophilic molecule polynucleotide / polynucleotide complement, hydrophobic ligand / hydrophobic solid phase, or
  • aza-isatoic anhydride By binding molecule, it is necessary to understand aza-isatoic anhydride or its derivatives. Note that when aza-isatoic anhydride is opened after reaction with a nucleophile, it is then called aza-anthranylate.
  • azo-isatoic anhydride By derivatives of azo-isatoic anhydride, it is necessary to include any organic compounds comprising a part corresponding to azo-isatoic anhydride and bearing on the aromatic part or on the heterocyclic part of the latter at least one radical, such as a chemical or organic group.
  • ribonucleic acid By “functionalization of at least one molecule of ribonucleic acid” is meant the action of grafting a group of interest - by covalent or non-covalent bonding - on said ribonucleic acid molecule.
  • the term “functionalization” or the terms Related (“functionalize”, for example) means the direct or indirect labeling of said ribonucleic acid molecule by a detectable molecule, the addition of a moiety to said ribonucleic acid molecule for capture, separation or isolation. inhibition of the latter etc.
  • connecting arm is defined a spacer arm of organic nature, such as a single covalent bond, for connecting the binding molecule and the group of interest. It may have a function that can be cleaved by a physicochemical, photochemical, enzymatic, chemical, thermal, etc.
  • RNA is a natural or synthetic polymer consisting of at least two successive modified or unmodified ribonucleotide units.
  • DNA is defined by a natural or synthetic polymer consisting of at least two successive modified or unmodified deoxyribonucleotide units.
  • RNA segment consisting of at least one DNA segment and at least one RNA segment.
  • inhibition is meant the inability of the RNA excessively functionalized by an isatoic anhydride derivative to be amplified by genetic material amplification technology (NASBA, PCR, etc.)
  • NASBA genetic material amplification technology
  • PCR PCR
  • sugar is a compound ribose or deoxyribose.
  • the solution is said to be heterogeneous, for example when it contains a solid support optionally having RNAs fixed on its surface.
  • the pyridinyl group is selected based on the fact that, in addition to solubility, its ability to attract electrons increases the reactivity of the carbonyl function making it more suitable for nucleophilic attack of RNA hydroxyl groups. .
  • the present invention relates to a method of functionalizing at least one ribonucleic acid (RNA) molecule contained in a liquid sample, which comprises the following steps:
  • binding molecule constituted by an azoisatoic anhydride or a derivative thereof
  • a linking arm connecting the binding molecule with the group of interest, b) reacting the anhydride function of the binding molecule with at least one hydroxyl group carried: at the 2 'position of the ribose of one of the nucleotides of the RNA, and / or
  • the present invention also relates to a method for labeling at least one ribonucleic acid (RNA) molecule contained in a liquid sample, which comprises the following steps:
  • binding molecule consisting of an aza-isatoic anhydride or a derivative thereof, having an intrinsic fluorescence
  • the present invention also relates to a method for capturing or separating at least one ribonucleic acid (RNA) molecule which comprises the following steps:
  • binding molecule constituted by an azoisatoic anhydride or a derivative thereof
  • a group of interest consisting of a ligand complementary to an anti-ligand
  • RNA molecules thus captured or separated and use the remainder of the sample enriched in DNA.
  • the linker is associated with the linker molecule before said linker is associated with the group of interest.
  • the arm The binding molecule is associated with the binding molecule after said linker is associated with the group of interest.
  • the binding molecule is previously associated with the RNA.
  • the present invention still relates to a method of selective capture of at least one RNA molecule using at least one binding molecule, a group of interest consisting of a ligand complementary to an anti-ligand, and a linker arm connecting the binding molecule with the group of interest, the binding molecule being constituted by an aza-isatoic anhydride or a derivative thereof, which is covalently attached at the level of a hydroxyl group carried:
  • the present invention finally relates to a method for separating RNA molecules from other biological constituents, in particular DNA molecules consisting of:
  • RNA and DNA undifferentiated nucleic acids
  • the present invention also provides a functionalization reagent, of formula 1):
  • R 1 and R 2 represent, independently of one another, H or a group of interest, knowing that at least one of the radicals R 1 and R 2 is represented by a group of interest, and
  • the grouping of interest can be:
  • a marking marker or precursor at. a marking marker or precursor, or b. a ligand recognizable by a recognition molecule or surface, or particle, etc., to form a stable complex
  • X 1 and X 2 each represent, independently of one another, a linking arm which links the group of interest to the binding molecule
  • radicals Z 1 , Z 2 , Z 3 and Z 4 are constituted by a nitrogen atom (N), the other radicals consist of a carbon atom (C).
  • the invention provides a functionalization reagent of formula (2):
  • R 1 , R 2 and R 3 represent, independently of one another, H or a group of interest, given that at least one of the radicals R 1 , R 2 and R 3 is represented by a group of interest , and
  • a marking marker or precursor at. a marking marker or precursor, or b. a ligand recognizable by a recognition molecule or surface, or particle, etc., to form a stable complex
  • X 1 , X 2 and X 3 each represent, independently of one another, linking arms which connect the group of interest to the binding molecule
  • radicals Z 1 , Z 2 and Z 3 are constituted by a nitrogen atom (N), the other radicals consist of a carbon atom (C).
  • Y represents Z 4 or the group CX 3 -R 3 ,
  • the interest group is selected from:
  • a marking marker or precursor or d. a ligand recognizable by a recognition molecule or surface, or particle, etc., to form a stable complex
  • the capture or separation means is constituted by a solid support, such as particles of polymer or silica, magnetic or not, or a filter or by the inner wall of a container.
  • the link arm X is an arm organic spacer for connecting the binding molecule and the group of interest, such as a simple covalent bond between the binding molecule and the group of interest or a single carbon atom, optionally substituted, a sequence of at least two carbon atoms, optionally containing aromatic structures and / or hetero atoms (oxygen, sulfur, nitrogen, etc.).
  • the linker X comprises a function or a linkage capable of being cleaved by a physico ⁇ chemical , photochemical, thermal, enzymatic and / or chemical means allowing the separation of the molecule from binding with respect to the RNA under particular light, temperature, enzymatic or chemical conditions.
  • the invention also provides a functionalized RNA biological molecule obtainable by one of the methods mentioned above.
  • the invention proposes to also cover a kit for detecting a target RNA molecule comprising a reagent, as described above.
  • the invention relates to a method of functionalization, which comprises the following additional step between steps a) and b) of hydrolyzing the terminal monophosphate group at the 3 'position of each strand of ARN to be functionalized.
  • a linking arm substitutes the pyridynyl group.
  • the cleavable function is designed and positioned so that it can form after cleavage a cyclic heterocycle of 5 to 8 members with the carbonyl function of the RNA aza-anthranylate and thus release a naked RNA, having its 2 '- OH free (2), usable in any mode of amplification, detection or protection of the RNA and where it is necessary to absolutely avoid any potential inhibition that would be due to aza-anthranylate.
  • This last embodiment is preferably based on the formation of a spontaneously forming aza-thi-lactone during the cleavage of the cleavable disulfide function (-SS-) of the linker arm carried by the position 5.
  • a spontaneously forming aza-thi-lactone during the cleavage of the cleavable disulfide function (-SS-) of the linker arm carried by the position 5.
  • Such self-immolatable systems have never been described in aza-isatoic series and for the purpose of releasing an RNA.
  • This latter embodiment also makes it possible to avoid the release of by-products during hydrolysis with DTT, the thiolactone remaining on the support.
  • Figure 1 shows the principle of sorting RNA using aza-isatoic anhydride according to a first embodiment.
  • FIG. 2 presents the principle of sorting RNA in the presence of an azoisatoic anhydride with a group inducing the release of the "naked" RNA molecule in a second embodiment, called aza-isatoic anhydride. -immolable.
  • Figure 3 describes the synthesis of an aza-isatoic derivative with a biotinylated group of interest.
  • Figure 4 presents a graph on the study of the stability in solution of an aza-isatoic compound compared to that of an isatoic compound.
  • Figure 5 shows a graph on functionalization tests of a 27-sea ORN with derivatives of isatoic anhydride and aza-isatoic anhydride.
  • Figure 6 proposes to highlight the chemoselectivity of the aza-isatoic series for RNA over that of AD.
  • RNA ribonucleic acid
  • Biot-peg 4 -COPFP 3- (2- (2- (3-biotin-dPEG 3 -propanamido) ethyl) disulfanyl) propanoic acid ester and pentafluoro phenol
  • Biot-peg 4 -SS-azaIAMe Molecule A of the present application (1- ⁇ 5- [(3aS, 6aR) -2-oxo-hexahydro-1H-thieno [3,4-d] imidazolidin-4- yl] pentanamido ⁇ -N- (2 - ⁇ [2- (3- ⁇ 1-methyl-2,4-dioxo-1H, 2H, 4H-pyrido [2,3-d] [1,3] oxazin-6 ⁇ yl ⁇
  • Biot-peg 4 -SS-IAMe 5- (3- (2- (2- (biotin-dPEG 3 -propanamido) ethyl) disulfanyl)) propanamido) isatoic anhydride, as described in Example 1-10 of WO-A-2012/076794, ⁇ TLC: thin layer chromatography,
  • MilliQ water ultrapure water (Millipore, Molsheim, France)
  • EDC 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide
  • NP1 PI nuclease
  • ODN Oligo-deoxyribonucleotide
  • TEAAc Triethyl ammonium acetate
  • the LC-MS analyzes were performed with a WATERS Alliance 2795 HPLC chain equipped with a PDA 996 diode array detector (Waters), a ZQ 2000 mass spectrometry detector (Waters), an Empower version software 2 and a WATERS XTerra MS C18 column (4.6 x 2.5 ⁇ m) used at a flow rate of 1 ml / min at 30 ° C (detection at 260 nm or max plot).
  • the ZQ 2000 mass spectrometer has an Electrospray ionization source. Ionizations were performed in positive mode with a cone voltage of 20V and a voltage at the level of the capillary of 3.5kV.
  • condition 1 The conditions used for the HPLC analyzes are as follows (conditions 1):
  • Example 1 Synthesis of a Derivative of Aza-Isatoic Anhydride Conjugated to a Group of Interest Introduction: A general description of the synthesis of the compounds which will be described in Example 1.
  • the conjugation of the azo-isatoic anhydride or its derivatives to a group of interest presupposes a chemical reaction between the aza-isatoic anhydride, equipped with a reactive function, and the molecule of interest, also being provided with a reactive function. It should be noted that it is particularly important to preserve the integrity of the aza-isatoic anhydride portion during this coupling. Those skilled in the art know a multitude of ways to conjugate two molecules together to obtain a new molecule with properties common to both.
  • the strategy chosen for this synthesis is based on the iodination in position 6 of chloro nicotinic acid, protected in the form of terbutyl ester 2, to obtain the compound
  • This then allows the introduction into position 6 of a precursor of a carboxylic acid function (compound 5), said function will be the point of attachment for the introduction of a linker and then a group of interest via pseudo-peptide couplings.
  • the formation of azosatoid anhydride is by intromolecular cyclization of the tert-butyl amino ester 11 or 12 in the presence of phosgene, as is well represented in FIG. work on stable precursors throughout the synthesis.
  • the reaction mixture is evaporated to dryness, taken up in 200 mL of a 5% aqueous K 2 CO 3 solution and then extracted with dichloromethane (3 ⁇ 150 mL). The organic phases are then combined, dried over anhydrous sodium sulphate, filtered and finally evaporated. The final product is obtained as an oil with a yield of 88% (5.97 g, 27.94 mmol).
  • the reaction mixture is evaporated to dryness, taken up in 75 ml of water and then extracted with dichloromethane (3 ⁇ 75 ml). The organic phases are then combined, dried over anhydrous sodium sulphate, filtered and finally evaporated.
  • the oil obtained is then purified on a column of silica gel using an eluent gradient (EP then EP / Et 2 ⁇ 0.95: 0.5). The final product is obtained as an oil with a yield of 85% (2.35 g, 11.28 mmol).
  • tert-butyl 2- (methylamino) nicotinate 3 (21.61 mmol, 1 eq) are dissolved in 30 ml of a dichloromethane / acetic acid mixture (6: 1). 5.83 g of N-iodosuccinimide (25.93 mmol, 1.2 eq) are added to the reaction medium at room temperature. The reaction is stirred magnetically at room temperature for 30 minutes.
  • the reaction mixture is then neutralized with 7 ml of a saturated aqueous solution of sodium thiosulphate, taken up in 100 ml of a 5% aqueous K 2 CO 3 solution and then extracted with dichloromethane (3 ⁇ 100 ml).
  • the organic phases are then combined, dried over anhydrous sodium sulphate, filtered and finally evaporated.
  • the solid obtained is then purified on a column of silica gel using an eluent gradient (EP then EP / Et 2 ⁇ 0 9: 1).
  • the final product is obtained in the form of a yellow powder with a yield of 96% (6.92 g, 20.71 mmol).
  • the reaction mixture is evaporated to dryness, taken up in 50 ml of dichloromethane and then filtered through Celite. The volume of 200 mL of water is added and the mixture is extracted with dichloromethane (3x150 mL). The organic phases are then combined, dried over anhydrous sodium sulphate, filtered, and finally evaporated. The resulting solid is then purified on a silica gel column using an eluent gradient (EP at EP / Et 2 ⁇ 0 7: 3). The final product is obtained in the form of a yellow powder with a yield of 76% (3.33 g, 11.39 mmol).
  • the reaction mixture is evaporated to remove tetrahydrofuran.
  • the pH of the aqueous solution obtained is adjusted to 6 with acetic acid.
  • This solution is then extracted with ethyl acetate (4x50 mL).
  • the organic phases are then combined, dried over anhydrous sodium sulphate, filtered and finally co-evaporated with toluene.
  • the final product is obtained in the form of a yellow powder with a yield of 92% (4.25 g, 15.16 mmol).
  • cystamine 26.64 mmol, 1 eq
  • cystamine 26.64 mmol, 1 eq
  • a solution of 5.814 g of Boc 2 0 (26, 64 mmol; 1 eq) and 11.14 mL of TEA (79.12 mmol; 3 eq) in 40 mL of methanol is then added dropwise to the cystamine solution for 45 minutes, with magnetic stirring at room temperature.
  • the reaction medium is then evaporated to dryness until a white solid is obtained.
  • the solid obtained is taken up in 70 ml of a 1M Na3 ⁇ 4PO 4 solution.
  • the mixture is then extracted with diethyl ether (3 x 90 mL).
  • the aqueous phase is alkalized at pH 9 using a 1M NaOH solution.
  • the mixture is then extracted with ethyl acetate (6 x 50 mL).
  • the organic phases are combined, dried over MgSO 4 and then evaporated under vacuum.
  • the product is obtained as an oil with a yield of 42% (2.82 g, 11.19 mmol).
  • the reaction mixture is evaporated to dryness, taken up in 75 mL of saturated aqueous sodium bicarbonate solution and then extracted with ethyl acetate (4x75 mL). The organic phases are then combined, dried over anhydrous sodium sulphate and filtered. The solid obtained is then purified on a silica gel column using an eluent gradient (DCM / AcOEt 8/2 with DCM / AcOEt 2/8). The final product is obtained in the form of a white powder with a yield of 94% (5.2 g, 10.10 mmol).
  • reaction mixture is evaporated to dryness, taken up in 50 ml of saturated aqueous sodium bicarbonate solution and then extracted with ethyl acetate (4x50 ml). The organic phases are then combined, washed with water (2 ⁇ 50 mL), dried over anhydrous sodium sulphate and filtered. The final product is obtained as an oil with a quantitative yield (1.6 g, 3.86 mmol).
  • IR (KBr): ⁇ 3378 (NH), 2977, 2931, 1682 (CO), 1612, 1578, 1520, 1368, 1304, 1228, 1160, 1131, 1095, 910, 732 cm -1 .
  • Example 1.10 Synthesis of 5- [2- ( ⁇ 2 - [(2- ⁇ 5 - [(3aS, 6aR) -2-oxo-hexahydro-1H-thieno [3,4-d] imidazolidin-4-yl] tert-Butyl [ ⁇ ] -benzoyl (ethyl) disulfanyl] ethyl] carbamoyl) ethyl] -2- (methylamino) nicotinate
  • the reaction mixture was evaporated to dryness and directly purified on a C18 grafted silica gel column, using an eluent gradient (3 ⁇ 40 to 3 ⁇ 40 / ACN 3/7).
  • the final product is obtained in the form of a white powder with a yield of 70% (545 mg, 0.85 mmol).
  • Example 1.11 Synthesis of 5- [(3aS, 6aR) -2-oxo-hexahydro-1H-thieno [3,4-d] imidazolidin-4-yl] -N- (2- ⁇ [2- (3- ⁇ 1-methyl-2,4-dloxo-1H, 2H, 4H-pyrido [2,3-d] [1,3] oxazin-6-yl ⁇ propanamido) ethyl] disulfanyl ⁇ ethyl) entanamide:
  • reaction mixture is evaporated to dryness and directly purified on a C18 grafted silica gel column, using an eluent gradient (3 ⁇ 40 to 3 ⁇ 40 / ACN 4/6).
  • the final product is obtained in the form of a white powder with a yield of 42% (200 mg, 0.33 mmol). Mp: 125 ° C.
  • IR (KBr): ⁇ 3299 (NH), 2927, 1785 (CO), 1735 (CO), 1704 (CO), 1641 (CO), 1612, 1488, 1326, 1232, 1179, 1069, 1045, 979, 787 , 745, 674 cm -1 .
  • the reaction mixture is evaporated to dryness and directly purified on a C18 grafted silica gel column, using an eluent gradient (3 ⁇ 40 to 3 ⁇ 40 / ACN 4/6).
  • the final product is obtained in the form of a white powder with a yield of 85% (730 mg, 0.822 mmol).
  • Example 1.13 Synthesis of 1- ⁇ 5- [(3aS, 6aR) -2-oxohexahydro-1H-thieno [3,4-d] imidazolidin-4-yl] entanamido ⁇ -N- (2- ⁇ [2 - (3- ⁇ 1-methyl-2,4-dioxo-1H, 2H, 4H-pyrido [2,3-d] [1,3] oxazin-6-yl ⁇ propanamido) ethyl] disulfanyl ⁇ ethyl) -3 , 6,9, 12-tetraoxapentadecan-15-amide:
  • IR (KBr): ⁇ , 3426 (NH), 2926, 2875, 1782 (CO), 1729 (CO), 1641 (CO), 1550, 1490, 1369, 1330, 1093, 788, 746, 677 cm -1 .
  • the precipitate obtained is then filtered on sintered, then washed with MeOH (3x30 mL) and diethyl ether (3x90 mL).
  • the product is obtained in the form of a white powder with a yield of 70% (2.46 g, 7.21 mmol).
  • Example 3 Demonstration of the Reactivity at Room Temperature of an Aza-Isatoic Compound with respect to a Model 27-Basin Oligonibonucleotide (ORN) - Measurement of the Functionalization Ratio: Objective: In order to demonstrate the increase in the aza-isatoic series reactivity of the 27-position ORN functionalization tests at room temperature and at 65 ° C were carried out for the reference isatoic derivative, Biot-peg 4 -SS-IAMe and for the aza-isatoic derivative Biot-peg 4 - SS-azaIAMe (14).
  • the ORN population (labeled and unlabeled) is separated from the excess label by acetone / lithium perchlorate precipitation. Then, the ORNs thus obtained are subjected to hydrolysis with PI nuclease (Aldrich, Saint Louis, USA) and with alkaline phosphatase (Aldrich, Saint Louis, USA) in order to hydrolyze the diester phosphate bonds of the ORN.
  • PI nuclease Aldrich, Saint Louis, USA
  • alkaline phosphatase Aldrich, Saint Louis, USA
  • the rate of functionalization of the ORN is evaluated by measuring: the area corresponding to the mono-nucleoside terminal adducts (functionalized at 2 'and 3') and the area corresponding to the acylated di-nucleotides
  • the pellet is dried with acetone and evaporated at the centrifugal evaporator (RCT 60, Jouan, St Herblain, France).
  • the residue is then taken up in 20 ⁇ l of a H 2 O / DMSO 85/15 solution and an HPLC injection (conditions 1) is performed to verify the absence of functionalization reagent.
  • 2 ⁇ l of PI nuclease (ref .: N8630 Sigma-Aldrich, St.
  • the degree of functionalization is evaluated in UV at 260 nm, by integration of the corresponding mass of acylated dinucleotides and acylated nucleosides, relative to the masses corresponding to the four ribonucleosides.
  • the functionalization rate values presented in the context of this invention do not take into account the correction factor that must be applied as a function of the molar extinction coefficient at 260 nm of each of the nucleosides. act of nucleosides or dinucleotides.
  • FIG. 5 shows the results obtained during functionalization tests on 27-mer ORNs with the BiotEGg 4 -SS-IAMe derivatives, as described in FR 1060102 and Biot-peg 4 -SS-azalAMe 1, at room temperature and at 65 ° C.
  • the uncorrected functionalization rate was evaluated at 13.5% for the aza-isatoic derivative 1 against 7.5% for the reference compound, Biot-peg 4 -SS-IAMe.
  • the reactivity towards an oligoribonucleotide was therefore increased by a factor of 1.8 in aza-isatoic series at 65 ° C.
  • This example demonstrates a conservation of the chemo-selectivity in aza-isatoic series for an ORN compared to an ODN.
  • the presence of 2 '-OH groups specific for ORN, is at the origin of the chemo-specific reaction on the ORN.
  • EXAMPLE 5 Selective Extraction of HIV RNA Transcripts from a Solution Containing a Mixture of HIV RNA Transcripts and Genomic DNA: Objective: The aim is to demonstrate that the concept of RNA enrichment of a biological solution, containing a mixture of RNA and DNA using as functionalizing reagent the azasatoic derivative 1 at room temperature is possible. For this, two biological models of nucleic acids were chosen, transcribed HIV for RNA and genomic calf DNA. In order to compare the efficacy of the azasatoic derivative 1, these tests were also performed on the reference compound Biot-peg 4 -SS-IAMe at 65 ° C. and at room temperature. Procedure: The nucleic acids used in this example are as follows:
  • the final concentrations of TEAAc and functionalizing reagent are respectively 250 mM and 1.5 mM.
  • the three tubes are incubated for 1 hour at room temperature or at 65 ° C in a heated rack.
  • a volume of 900 ⁇ L of lysis buffer (Easy Mag buffer, 280134, bioMérieux, Marcy l'Etoile, France) and 50 ⁇ L of magnetic silica particles (EasyMAG silica, ref 280133, bioMérieux, Marcy l'Etoile, France ) are added to each tube.
  • the latter are immediately stirred by vortex effect after adding the silica, and then incubated for 10 minutes at room temperature.
  • the supernatants are removed by aspiration using a pipette.
  • the tubes are still vortexed and magnetized to remove the supernatant.
  • a first wash is carried out with 500 ⁇ l of washing buffer 1 (Easy Mag buffer, reference 280130, bioMérieux, Marcy l'Etoile, France). Two washes are then carried out with 900 ⁇ L and then 500 ⁇ L of washing buffer 2 (Easy Mag buffer, reference 280131, bioMérieux, Marcy l'Etoile, France). Finally, a final washing step is carried out with 500 ⁇ l of elution buffer 3 (Easy Mag buffer, reference 280132, bioMérieux, Marcy l'Etoile, France) at room temperature.
  • Elution of the nucleic acids is carried out with 20 ⁇ l of elution buffer 3 stirred in a heating stirrer (1400 rpm) at 70 ° C. After 5 minutes, the tubes are vortexed and magnetized to recover the supernatants. These are dosed with the Qubit® Fluorometer instrument, ref. Q32857, Invitrogen (Carlsbad, California, United States of America), and using the Quant-iT RNA Assay Kit Kit 5-100 ng (ref Q32855, Invitrogen, Carlsbad, California, United States of America), and Quant-iT dsDNA HS Assay Kit 0.2-100 ng (Ref Q32854, Invitrogen, Carlsbad, California, USA) of America). These assay kits make it possible to determine an RNA / DNA ratio in a mixture.
  • step 2 15 ⁇ l of nucleic acids purified in step 2 and then 5 ⁇ l of 4% PBS + 0.4% SDS are added to each pellet.
  • the tubes are incubated for 10 minutes with gentle agitation (vortex stirrer at the minimum speed) at room temperature. After magnetic separation, the supernatants are recovered using tapered tips. This supernatant is dosed as before with the Qubit® Fluorometer.
  • Each pellet is suspended in 8 ⁇ l of a 100 mM DTT solution in PBS IX. Each test is then vortexed and incubated for 1 hour at 40 ° C, 300 RPM. After magnetic separation, the supernatant is recovered using tapered tips. This supernatant is assayed with the Qubit® Fluorometer instrument (Invitrogen, Carlsbad, California, USA) as previously (about 100 ng RNA is collected).
  • RNA extraction extraction [ purified flml / [RNA] RNA at V stapel.
  • RNA enrichment process from a selective functionalization of RNA with the aza-isatoic derivative 1 and at room temperature, is demonstrated.
  • the DNA / RNA ratio thus passes from 90/10 at the end of the purification step, to a ratio of 4/96 after the complete process (test 3). This result demonstrates the efficacy of the azasatoic derivative 1 at room temperature for the selective extraction of RNA.
  • this derivative makes it possible to enrich the mixture with RNA by performing the functionalization step at room temperature.
  • the intermediate selectivity is increased by a factor of 5 to ambient temperature relative to the reference compound under the same conditions at 65 ° C.
  • the aza-isatoic derivative 1 4 makes it possible to functionalize the ribonucleic acids at room temperature, and consequently to increase the selectivity of the extraction process with respect to the RNA.
  • Example 6 Demonstration of the concept of self-immolatable group associated with a biotinylated aza-isatoic anhydride. Synthesis of an aza-isatoic derivative with a disulfide group on the aromatic part

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