Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
  • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
  • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
  • C07H19/23—Heterocyclic radicals containing two or more heterocyclic rings condensed among themselves or condensed with a common carbocyclic ring system, not provided for in groups C07H19/14 - C07H19/22
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
  • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
  • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
  • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
  • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
  • C07H19/14—Pyrrolo-pyrimidine radicals
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
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  • 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
• • 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
  • C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
  • C12Q2525/10—Modifications characterised by
  • C12Q2525/117—Modifications characterised by incorporating modified base
• • 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
  • C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
  • C12Q2563/107—Nucleic acid detection characterized by the use of physical, structural and functional properties fluorescence

Definitions

• FIELD [001] This specification relates to modified fluorescent nucleoside phosphates and their use to elucidate biological mechanisms.
• this specification discloses fluorescent nucleoside phosphates that are non-cytotoxic and therefore amenable to intracellular use.
• the phosphates spontaneously accumulate in cultured human cells following uptake via an energy-dependent pathway, in different cell localisations depending on the molecular structure of the nucleobase (some phosphates for example amassing preferentially in the nucleus, and some in the cytosol). This allows control of downstream cell endogenous labelling processes.
• this combination of properties has never been observed before for any other fluorescent nucleotides.
• this specification provides a non-invasive, non-genetic way to fluorescently label endogenous RNA. It can be used to visualise - in living cells - biochemical reactions that involve RNA production, transport, processing, secretion and protein interactions. This opens the door for new ways to study and develop novel nucleic acid-based therapies.
• a primary objective of the present specification is to provide modified nucleoside phosphates that can be used to conveniently and endogenously prepare fluorescently labelled RNA in-cellulo.
• this specification describes, in part, a compound of formula (I), a physiologically cleavable precursor or a salt thereof as claimed in claim 1.
• This specification also describes, in part, a process for preparing a compound of formula (I), a physiologically cleavable precursor or a salt thereof as claimed in claim 9.
• This specification also describes, in part, a composition for preparing a labelled RNA molecule as claimed in claim 18. [013] This specification also describes, in part, the use of a compound of formula (I), a physiologically cleavable precursor or a salt thereof to prepare a labelled RNA molecule as claimed in claim 19.
• This specification also describes, in part, a process for preparing a labelled RNA molecule in- vitro as claimed in claim 24. [015] This specification also describes, in part, a process for preparing a labelled RNA molecule in- cellulo as claimed in claim 25.
• a or “an” mean “at least one”. In any embodiment where “a” or “an” are used to denote a given material or element, “a” or “an” may mean one. In any embodiment where “a” or “an” are used to denote a given material or element, “a” or “an” may mean 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 100, 1000, 10000, 100000 or 1000000 (1 million).
• “Comprising” means that a given embodiment may contain other features.
• the given material may be formed of at least 10% w/w, at least 20% w/w, at least 30% w/w, or at least 40% w/w of the materials or elements (or combination of materials or elements).
• “comprising” may also mean “consisting of” (or “consists of”) or “consisting essentially of” (or “consists essentially of”).
• “consisting of” or “consists of” means the material or element is formed entirely of the material or element (or combination of materials or elements). In any embodiment where “consisting of” or “consists of” is mentioned the given material or element may be formed of 100% w/w of the material or element.
• “consisting essentially of” or “consists essentially of” means that a given material or element consists almost entirely of that material or element (or combination of materials or elements).
• the given material or element may be formed of at least 50% w/w, at least 60% w/w, at least 70% w/w, at least 80% w/w, at least 90% w/w, at least 95% w/w or at least 99% w/w of the material or element.
• R 1 is selected from hydro and R 2 is selected from cyano, or R 1 and R 2 together with the atoms to which they are attached form a 6-membered carboaromatic ring and R 3 is selected from -P(0)(OH) 2 , -P(0)(0H)-0-P(0)(0H) 2 , and -P(0)(0H)-0-P(0)(0H)-0-P(0)(0H) 2 .
• a "hydro" group is equivalent to a hydrogen atom. Atoms with a hydro group attached to them may be regarded as unsubstituted.
• R 1 and R 2 together with the atoms to which they are attached form a 6-membered carboaromatic ring
• this may mean a phenyl ring fused to the tetracyclic heteroaromatic system in the following manner:
• R 1 is hydro and R 2 is cyano, or R 1 and R 2 together with the atoms to which they are attached form a 6-membered carboaromatic ring and R 3 is selected from -P(0)(0H) 2 , -P(0)(0H)-0-P(0)(0H) 2 , and -P(0)(0H)-0-P(0)(0H)-0-P(0)(0H) 2 .
• R 1 is selected from hydro and R 2 is selected from cyano, or R 1 and R 2 together with the atoms to which they are attached form a 6-membered carboaromatic ring.
• R 1 is selected from hydro and R 2 is selected from cyano, or R 1 and R 2 together with the atoms to which they are attached form a 6-membered carboaromatic ring.
• R 1 is selected from hydro and R 2 is selected from cyano, or R 1 and R 2 together with the atoms to which they are attached form a 6-membered carboaromatic ring.
• R 1 and R 2 together with the atoms to which they are attached form a 6-membered carboaromatic ring.
• R 3 is selected from -P(0)(OH) 2 and -P(0)(0H)-0-P(0)(0H) 2 .
• R 3 is selected from -P(0)(OH) 2 and -P(0)(0H)-0-P(0)(0H)-0-P(0)(0H) 2 . [043] In some embodiments R 3 is selected from -P(0)(0H)-0-P(0)(0H) 2 and -P(0)(0H)-0-P(0)(0H)- 0-P(0)(0H) 2 .
• R 3 is -P(0)(0H) 2 .
• R 3 is -P(0)(0H)-0-P(0)(0H) 2 . [046] In some embodiments R 3 is -P(0)(0H)-0-P(0)(0H)-0-P(0)(0H) 2 .
• a compound of formula (I) selected from ((2R,3S,4R,5R)- 5-(8-cyano-2,3,5,6-tetraazaaceanthrylen-2(6H)-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl dihydrogen phosphate 7a (compound 7a, 2CNqAMP), a physiologically cleavable precursor or a salt thereof, ((2R,3S,4R,5R)-5-(8-cyano-2,3,5,6-tetraazaaceanthrylen-2(6H)-yl)-3,4- dihydroxytetrahydrofuran-2-yl)methyl hydrogen triphosphate (compound 7, 2CNqATP), a physiologically cleavable precursor or a salt thereof and ((2R,3S,4R,5R)-5-(2,3,5,6- tetraazacyclopenta[de]tetracen-2(6H)-yl)-3,4-
• 2CNAqMP refers to the monophosphate described as compound 7a.
• 2CNAqDP refers to the diphosphate analogue of compound 7a (i.e. the analogous compound of formula (I) where R 3 is - P(0)(0H)-0-P(0)(0H) 2 ).
• 2CNAqTP refers to the triphosphate described as compound 7. Labelled residues derived from the incorporation of these compounds into RNA are "2CNqA labelled".
• pATP refers to the triphosphate described as compound 17.
• pAMP refers to the monophosphate analogue of compound 17 (i.e. the analogous compound of formula (I) where R 3 is - P(0)(OH) 2 ).
• pADP refers to the diphosphate of compound 17 (i.e. the analogous compound of formula (I) where R 3 is -P(0)(0H)-0-P(0)(0H) 2 ).
• Labelled residues derived from the incorporation of these compounds into RNA are "pA labelled".
• a compound of formula (I) selected from ((2R,3S,4R,5R)- 5-(8-cyano-2,3,5,6-tetraazaaceanthrylen-2(6H)-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl hydrogen triphosphate (compound 7, 2CNqATP) or a salt thereof and ((2R,3S,4R,5R)-5-(2,3,5,6- tetraazacyclopenta[de]tetracen-2(6H)-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl hydrogen triphosphate (compound 17, pATP) or a salt thereof.
• a compound of formula (I) which is ((2R,3S,4R,5R)-5-(8- cyano-2,3,5,6-tetraazaaceanthrylen-2(6H)-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl hydrogen triphosphate or a salt thereof.
• a compound of formula (I) or a salt thereof which is ((2R,3S,4R,5R)-5-(2,3,5,6-tetraazacyclopenta[de]tetracen-2(6H)-yl)-3,4-dihydroxytetrahydrofuran-2- yl)methyl hydrogen triphosphate or a salt thereof.
• a compound of formula (I) which is ((2R,3S,4R,5R)-5-(8- cyano-2,3,5,6-tetraazaaceanthrylen-2(6H)-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl hydrogen triphosphate.
• a physiologically cleavable precursor of a compound of formula (I) which is: (compound 7a, 2CNqAMP).
• a physiologically cleavable precursor of a compound of formula (I) which is: (compound 7a, 2CNqAMP).
• a salt of a compound of formula (I) which is: (compound 7a, 2CNqAMP).
• a compound of formula (I) which is: Or a physiologically cleavable precursor or salt thereof.
• a physiologically cleavable precursor of a compound of formula (I) which is:
• a physiologically cleavable precursor of a compound of formula (I) which is: (compound 7, 2CNqATP).
• a physiologically cleavable precursor of a compound of formula (I) which is: (compound 17, pATP).
• a salt of a compound of formula (I) which is: (compound 7, 2CNqATP).
• a salt of a compound of formula (I) which is: (compound 17, pATP).
• a compound of formula (I) which is: (compound 17, pATP).
• Atoms of the compounds and salts described in this specification may exist as their isotopes.
• Embodiments include all compounds of formula (I) where an atom is replaced by one or more of its isotopes (for example a compound of formula (I) where one or more carbon atom is an C or 13 C carbon isotope, or where one or more hydrogen atom is a 2 H or 3 H isotope).
• a physiologically cleavable precursor of a compound of formula (I) is for example one in which the mono-, di- or tri-phosphate group attached to the nucleoside portion of the molecule is masked with a suitable protecting group (for example a group bound to a phosphate group oxygen atom or phosphorus atom) that may be removed under physiological conditions.
• a suitable protecting group for example a group bound to a phosphate group oxygen atom or phosphorus atom
• physiologically cleavable precursors of a compound of formula (I) are converted to compounds of formula (I) (e.g. by metabolism), which can then take part in cellular processes (such as cellular localisation and RNA synthesis).
• a physiologically cleavable precursor is of a compound of formula (I) where R 3 is -P(0)(0H) 2 .
• a physiologically cleavable precursor is of a compound of formula (I) where R 3 is -P(0)(0H)-0-P(0)(0H) 2 .
• a physiologically cleavable precursor is of a compound of formula (I) where R 3 is -P(0)(0H)-0-P(0)(0H)-P(0)(0H) 2 .
• a suitable physiologically cleavable precursor of a compound of formula (I) is for example any of the groups used to prepare nucleoside phosphate and/or phosphonate prodrugs in Pradere, U. et al., Chem. Rev. 2014, 114, 18, 9154-9218 (for example in Figure 3); Wiemer, A. J. et al., Top. Curr. Chem. 2015 (for example in Table 1); 360: 115-160 and/or Wiemer, A. J.; ACS Pharmacol. Transl. Sci. 2020, 3, 4, 613-626 (for example in Figure 2). The contents of these references are hereby incorporated by reference.
• a suitable salt of a compound of formula (I) is for example a base-addition salt.
• a base-addition salt is formed by bringing the compound of formula (I) into contact with a suitable organic or inorganic base.
• a base addition salt may be formed using a suitable organic base like a nitrogen base, for example ammonia or a trialkylamine like triethylamine.
• a base addition salt may also for example be formed using a suitable inorganic base like an alkali metal or rare earth hydroxide, for example potassium hydroxide, sodium hydroxide, magnesium hydroxide or manganese hydroxide.
• a compound of formula (I) which is a sodium, potassium, magnesium, or ammonium salt.
• a compound of formula (I) which is a sodium, potassium, or ammonium salt.
• a compound of formula (I) which is a sodium or ammonium salt.
• a compound of formula (I) which is a monopotassium, dipotassium, tripotassium, tetrapotassium, monosodium, disodium, trisodium, tetrasodium, monoammonium, diammonium, triammonium or tetraammonium salt.
• a compound of formula (I) which is a monosodium, disodium, trisodium, tetrasodium, monoammonium, diammonium, triammonium or tetraammonium salt.
• a compound of formula (I) which is a monosodium, disodium, trisodium or tetrasodium salt.
• a compound of formula (I) which is a trisodium salt
• a compound of formula (I) which is a monoammonium, diammonium, triammonium or tetraammonium salt.
• a compound of formula (I) which is a diammonum salt.
• a compound of formula (I) which is a triammonium salt.
• R 1 is selected from hydro and R 2 is selected from cyano, or R 1 and R 2 together with the atoms to which they are attached form a 6-membered carboaromatic ring
• PG 1 is a suitable protecting group
• iii Capping any remaining secondary alcohol groups with a suitable protecting group PG 2
• V Reacting the exposed primary alcohol group with a compound of formula (VI):
• R 4 is selected from a hydro group and a Ci_3alkyl group
• a process for preparing a compound of formula (I), a physiologically cleavable precursor or a salt thereof comprising: i. Providing a compound of formula (V) or a salt thereof:
• R 1 is selected from hydro and R 2 is selected from cyano, or R 1 and R 2 together with the atoms to which they are attached form a 6-membered carboaromatic ring
• PG 1 is a suitable protecting group
• R 4 is selected from a hydro group and a Ci_3alkyl group; vi. Oxidising the resultant phosphorus (III) compound to a phosphorus (V) compound; vii. Reacting the phosphorus (V) compound with a tetraalkylammonium pyrophosphate to generate a triphosphate; viii. Removing the protecting group PG 2 ; ix. Cleaving the resultant triphosphate from the support to generate a compound of formula (I) or salt thereof; and optionally xi. Forming a free acid, physiologically cleavable precursor or different salt of the compound of formula (I).
• a protecting group (“PG", for example PG 1 or PG 2 ) is any group suitable for temporarily protecting a reactive centre, for example a hydroxyl group. Suitable protecting groups for the reactive centres disclosed herein may be found for example in "Greene's Protective Groups in Organic Synthesis, Fourth Edition", Greene T. W., Wuts P. G. M.; John Wiley & Sons, Inc. 2007, doi: 10.1002/0470053488), the contents of all of which are herein incorporated by reference.
• Ci-3alkyl group is a straight chain or branched saturated alkyl group with the indicated number of carbons.
• Example Ci_3alkyl groups include methyl, ethyl, propyl and isopropyl.
• the secondary alcohols to be capped may be those on the ribose part of the molecule.
• step 1 unreacted nucleoside can be recovered, which minimizes loss of material
• the solid-supported nucleosides can be stored for up to 3 months without degradation;
• the route is compatible with automated synthesis
• R 4 may be a hydro group.
• R 4 may be a Ci-3alkyl group. It has been observed that when R 1 is a Ci_ 3alkyl group, the phosphoramidite reagent preparation is easier and higher yielding, but performs at least as well in step v above as when R 4 is a hydro group.
• R 4 may be methyl
• R 4 is a C1-3 alkyl group.
• the support may be a solid polymer.
• the support may be a solid polymer selected from controlled-porosity glass and polystyrene.
• the support may be polystyrene.
• the support may be controlled-porosity glass.
• the support may be functionalised with a primary amino group. This may form the reactive point of attachment to the support.
• the support may be controlled-porosity glass functionalised with a primary amino group (for example Amino-SynBaseTM).
• PG 1 may be selected from trityl, dimethoxytrityl and trimethoxytrityl.
• PG 2 may be selected from acetyl, benzoyl, 2,2,2-trichloroethylcarbonyl, paramethoxybenzyl, methyl, tetrahydropyranyl, triethylsilyl, triisopropylsilyl, trimethylsilyl, tert- butyldimethylsilyl and methoxyethyl.
• PG 2 may be acetyl. Where an immobilised molecule is base labile, this allows for an efficient synthesis in which removal of the PG 2 group and cleavage from the resin may be accomplished in a single step.
• PG 1 may be dimethoxytrityl and PG 2 may be acetyl.
• immobilisation of the compound of formula (II) in step i) may occur mainly at the 2'-hydroxy position.
• immobilisation occurs mainly at the 2'-hydroxy position, this may be >50%, >60%, >70%, >80%, >90% or 100% of the total immobilisation (i.e. the total covalent binding of both secondary hydroxyl groups to the support).
• the tetraalkylammonium pyrophosphate may be tetrabutylammonium pyrophosphate.
• R 1 is selected from hydro and R 2 is selected from cyano, or R 1 and R 2 together with the atoms to which they are attached form a 6-membered carboaromatic ring, and PG 1 is selected from trityl, dimethoxytrityl and trimethoxytrityl; ii. Immobilising the compound of formula (V) or a salt thereof by linking one of its secondary alcohol groups to a controlled-porosity glass support; iii.
• a protecting group PG 2 selected from acetyl, benzoyl, 2,2,2-trichloroethylcarbonyl, paramethoxybenzyl, methyl, tetrahydropyranyl, triethylsilyl, triisopropylsilyl, trimethylsilyl, ferf-butyldimethylsilyl and methoxyethyl; iv. Removing the protecting group PG 1 ; v. Reacting the exposed primary alcohol group with a compound of formula (VI):
• R 4 is a Ci_3alkyl group
• a process for preparing a compound of formula (I), a physiologically cleavable precursor or a salt thereof comprising: i. Providing a compound of formula (V) or a salt thereof:
• R 1 is selected from hydro and R 2 is selected from cyano, or R 1 and R 2 together with the atoms to which they are attached form a 6-membered carboaromatic ring, and PG 1 is dimethoxytrityl; ii. Immobilising the compound of formula (II) or a salt thereof by linking one of its secondary alcohol groups to a controlled-porosity glass support; iii. Capping any remaining secondary alcohol groups with a protecting group PG 2 which is acetyl; iv. Removing the protecting group PG 1 ; v. Reacting the exposed primary alcohol group with a compound of formula (VI):
• R 4 is a methyl group
• optionally vii. Reacting the phosphorus (V) compound with tetrabutylammonium pyrophosphate to generate a triphosphate
• viii. Removing the protecting group PG 2
• ix. Cleaving the resultant phosphate from the support to generate a compound of formula (I) or salt thereof; and optionally x. Forming a free acid, physiologically cleavable precursor or different salt of the compound of formula (I).
• immobilising the compound of formula (II) or salt thereof in step ii) may be accomplished by a coupling reagent (for example succinic anhydride catalysed by dimethylaminopyridine when the support is functionalised with a primary amino group).
• a coupling reagent for example succinic anhydride catalysed by dimethylaminopyridine when the support is functionalised with a primary amino group.
• reaction of the exposed primary alcohol group with a compound of formula (III) may be accomplished using an activator (for example BTT activator or Activator 42 ® ).
• an activator for example BTT activator or Activator 42 ® .
• the phosphorus (III) compound in step vi) may be oxidised to a phosphorus(V) compound using aqueous pyridine and iodine.
• cleaving the triphosphate from the support may be accomplished using basic conditions (for example by treating with AMA). When there is a base-labile support and a base- labile protecting group is chosen for PG 2 , using these conditions allows simultaneous deprotection and cleavage.
• Compounds of formula (I) may be used as substrates for RNA synthesis along with other natural and synthetic RNA building blocks.
• composition for preparing a labelled RNA molecule comprising a compound of formula (I) and a natural ribonucleotide.
• a "natural ribonucleotide” comprises the appropriate natural ribonucleoside with a phosphate group (for example a monophosphate, diphosphate, or triphosphate group, such as those described by the definition of R 3 herein) bonded to the 5' hydroxy position.
• a "natural ribonucleotide” means a natural ribonucleoside triphosphate.
• a natural ribonucleotide (for example a natural ribonucleoside triphosphate) may be selected from cytidine 5' -triphosphate, uridine 5' -triphosphate, adenosine 5'- triphosphate and guanidine 5' -triphosphate.
• a composition of natural ribonucleotides (for example a composition of natural nucleoside triphosphates) may comprise combinations of varying amounts of these building blocks, in amounts sufficient to construct a target RNA molecule (for example as provided in NTP mix).
• RNA molecule for example a 2CNqA or pA labelled RNA molecule.
• a labelled RNA molecule comprises at least one modified fluorescent residue (for example a residue derived from a compound of formula (I) such that the modified residue is a 2CNqA or pA residue) but is otherwise similar to the natural RNA molecule (i.e. one with an unmodified adenosine residue at the same location as the 2CnqA or pA residue).
• the labelled RNA molecule may be a 2CNqA or pA labelled mRNA
• RNA molecule labelled with 2CNqA In one embodiment there is provided the use of a compound of formula (I), a physiologically cleavable precursor or a salt thereof where R 1 is selected from hydro and R 2 is selected from cyano to prepare an RNA molecule labelled with 2CNqA. [145] In one embodiment there is provided the use of a compound of formula (I), a physiologically cleavable precursor or a salt thereof where R 1 and R 2 together with the atoms to which they are attached form a 6-membered carboaromatic ring to prepare an RNA molecule labelled with pA. [146] In one embodiment there is provided the use of a compound of formula (I), a physiologically cleavable precursor or a salt thereof to enzymatically prepare a labelled RNA molecule.
• a process for preparing a labelled RNA molecule in-vitro comprising: i. Providing a DNA template to a composition comprising a compound of formula (I), a physiologically cleavable precursor or a salt thereof and a natural ribonucleotide; ii. Treating the resultant mixture with an RNA polymerase; optionally iii. Monitoring the labelled RNA molecule using microscopy; and optionally iv. Isolating the labelled RNA molecule.
• a process for preparing a labelled RNA molecule in-vitro comprising: v. Providing a DNA template to a composition comprising a compound of formula (I), a physiologically cleavable precursor or a salt thereof and a natural ribonucleotide; vi. Treating the resultant mixture with an RNA polymerase; vii. Monitoring the labelled RNA molecule using microscopy; and viii. Isolating the labelled RNA molecule.
• microscopy may be confocal laser scanning fluorescence microscopy.
• a process for preparing a labelled RNA molecule in-vitro may be carried out in the presence of transcription buffer (e.g. 5X transcription buffer), magnesium salt (e.g. magnesium(ll) chloride) and/or an RNase inhibitor (e.g. Ribolock).
• transcription buffer e.g. 5X transcription buffer
• magnesium salt e.g. magnesium(ll) chloride
• RNase inhibitor e.g. Ribolock
• kits for preparing a labelled RNA molecule comprising: i. A compound of formula (I); ii. A composition of natural ribonucleotides; iii. An RNA polymerase; optionally iv. A DNA template; and optionally v. Instructions for use.
• RNA molecule in one embodiment there is provided the use of a compound of formula (I), a physiologically cleavable precursor or a salt thereof in live cells to prepare an endogenously labelled RNA molecule.
• a compound of formula (I), a physiologically cleavable precursor or a salt thereof in one embodiment there is provided the use of a compound of formula (I), a physiologically cleavable precursor or a salt thereof to prepare a labelled RNA molecule in-cellulo.
• a process for preparing a labelled RNA molecule in- cellulo comprising: i. Providing a compound of formula (I), a physiologically cleavable precursor or a salt thereof to a eukaryotic or prokaryotic cell; ii. Allowing the organism to spontaneously internalise the compound; iii. Allowing the organism to prepare a labelled RNA molecule; optionally iv. Monitoring the labelled RNA molecule using microscopy; and optionally v. Isolating the labelled RNA molecule.
• a process for preparing a labelled RNA molecule in- cellulo comprising: vi. Providing a compound of formula (I), a physiologically cleavable precursor or a salt thereof to a eukaryotic or prokaryotic cell; vii. Allowing the organism to spontaneously internalise the compound; viii. Allowing the organism to prepare a labelled RNA molecule; ix. Monitoring the labelled RNA molecule using microscopy; and x. Isolating the labelled RNA molecule.
• a eukaryotic cell may be comprised in c. elegans or a zebra fish.
• a labelled RNA molecule may comprise >10%, >20%, >30%, >40%, >50%, >60%, >70%, >80%, >90% or 100% of modified residues (for example derived from a compound of formula (I) such that the modified residue is a 2CNqA or pA residue) in place of unmodified adenosine residues.
• modified residues for example derived from a compound of formula (I) such that the modified residue is a 2CNqA or pA residue
• a labelled RNA molecule may comprise 10%-20%, 10%-30%, 10%-40%, 20%-50%, 30%-60%, 40%-70%, 50%-80% or 50%-90% of modified residues (for example derived from a compound of formula (I) such that the modified residue is a 2CNqA or pA residue) in place of unmodified adenosine residues.
• modified residues for example derived from a compound of formula (I) such that the modified residue is a 2CNqA or pA residue
• a labelled RNA molecule may be selected from mRNA and ribosomal RNA.
• Figure 2 Preparation of pA nucleoside.
• Figure 3 Cytotoxicity assessment.
• A Cell viability measured as reduction in metabolic activity using the alamarBlue assay
• B Cell membrane integrity assessment measured by the lactate dehydrogenase (LDH) leakage assay. Fluh-7 cells were treated with the compounds for 24 hours at the indicated dose. Error bars represent standard deviation of three independent experiments.
• Figure 4 Confocal fluorescence microscopy images of live Fluh-7 cells exposed to 2.5 mM 2CNqATP or pATP in complete cell culture medium for (A) 20 h at 37 °C (B) 1.5 h at 37 °C or (C) 1.5 h at 4°C.
• FIG. 5 Measured mean fluorescence intensity (MFI) of 2CNqATP or pATP inside single living cells after exposure to 2.5 pM pATP or 2CNqATP after the indicated time. Cells were washed, trypsinized and analysed using flow cytometry with excitation at 405 nm. A) MFI distribution of the measured cell samples for non-treated cells, and cells treated with 2CNqATP and pATP. B) Mean MFI of 2CNqATP in single living cells plotted against the exposure time. C) Mean MFI of pATP in single living cells plotted against the exposure time. Lines are to guide the eyes.
• MFI mean fluorescence intensity
• FIG. 6 Dose response of cell uptake measured as normalized fluorescence intensity in cell lysates harvested from Fluh-7 cell cultures exposed to different concentrations of A) 2CNqATP or B) pATP for 24 h. 2CNqATP was excited at 355 nm and emission detected using 460 nm bandpass filter. For pATP bandpass filter for excitation at 380 nm and emission at 410 nm were used. Error bars represent standard deviation.
• Figure 7 Normalized mean fluorescence intensity of live Fluh-7 cells after exposure to 2.5 pM 2CNqATP (upper graph) or pATP (lower graph) in presence of increasing concentrations of ATP (black, solid connecting line) or adenosine (grey, dotted connecting line). Cells were exposed for 4 h, washed and analysed by flow cytometry using 405 nm laser for excitation. Lines are to guide the eyes; error bars represent standard deviation of three independent experiments. Lines are to guide the eyes.
• Figure 8 Fluorescence emission spectra of cell-extracted and purified RNA from Fluh-7 cells treated with 2.5 pM of (A) 2CNqATP or (B) pATP for 24 h showing that cell machinery is active and can incorporate certain nucleotide analogues into endogenously produced RNA.
• Black solid lines represent cell-extracted labelled RNA; grey dotted lines represent the following controls: (light black, dashed) compound added to cell-lysate of non-treated cells prior to RNA purification; compound added to RNA prior to final column purification (dark grey, dotted); and compound added directly to the RNA purification column (light grey, dashed).
• the spectra are normalized to the corresponding absorption at 260 nm, reflecting the total RNA concentration in the solutions.
• Figure 9 Spectral comparison of extracted RNA from 2CNqATP-treated Huh-7 cells to 2CNqATP and in-vitro 2CNqA-modified RNA strands.
• FIG. 1 Cytotoxicity assessment using 2CNqAMP.
• A Cell viability measured as reduction in metabolic activity using the alamarBlue assay
• B Cell membrane integrity assessment measured by the lactate dehydrogenase (LDH) leakage assay. Huh-7 cells were treated with the compounds for 24 hours at the indicated dose. Error bars represent standard deviation of three exposures.
• Figure 11 Confocal fluorescence microscopy images of live Huh-7 cells exposed to 2.5 pM 2CNqAMP or DPBS (as control) in complete cell culture medium for (A) 24 h at 37 °C (B) 1.5 h at 37 °C or (C) 1.5 h at 4°C.
• Figure 12 Measured mean fluorescence intensity (MFI) of 2CNqAMP inside single living cells after exposure to 2.5 pM 2CNqAMP after the indicated time. Cells were washed, trypsinized and analysed using flow cytometry with excitation at 405 nm. Mean MFI of 2CNqAMP in single living cells plotted against the exposure time. Shown are two independent experiments (indicated as empty/filled squares).
• Figure 13 Spectroscopic readout of cell-extracted and purified RNA from Huh-7 cells treated with 2.5 pM 2CNqAMP for 24 h at 37 °C showing that cell machinery is active and can incorporate certain nucleotide analogues into endogenously produced RNA.
• A Spectral comparison of extracted RNA from 2CNq AMP-treated Huh-7 cells to 2CNqATP, RNA from 2CNqATP-treated Huh-7 cells, and in- vitro 2CNqA-modified RNA strands.
• Amino-SynBaseTM CPG 500/110 (LCAA) 2 from LinkTech (Nu. 1397-C025, 1 g, 0.08 mmol) was activated by shaking in trichloroacetic acid 3% in DCE (8 mL, 0.08 mmol) for 18 h.
• the activated support was then filtered off and washed with 9:1 triethylamine:diisopropylethylamine (20 mL), dichloromethane (20 mL) and diethyl ether (20 mL).
• the activated support was dried under vacuum for 2 days before use.
• N,N-diisopropyl-4H-benzo[d][l,3,2]dioxaphosphinin-2-amine 5 (345 mg, 1.36 mmol) was dissolved in 4.8 mL ACN and reacted portionwise with the support (3 equal couplings with reaction times 60 s - 60 s - 90 s respectively).
• BTT activator (2.4 mL) was also added. The support was subsequently washed with ACN (3x5 mL).
• Oxidation Pyridine/Water/Iodine (9/1/12.7 v/v/w, 5mL) for 45 s, followed by ACN wash (3x5 mL) and drying of the support in an argon flow.
• Triphosphorylation Two injections of bis(tetrabutylammonium) dihydrogen diphosphate 6 (0.5 M, 5 ml) for 15 min and 18 hours, respectively. The support was subsequently rinsed with DMF (5 mL), water (3x5 mL), ACN (5 mL) and then dried in an argon flow.
• DMF diMF
• ACN ACN
• N,N-diisopropyl-4FI-benzo[d][l,3,2]dioxaphosphinin-2-amine 5 (345 mg, 1.36 mmol) was dissolved in 4.8 mL ACN and reacted portionwise with the support (3 equal couplings with reaction times 60 s - 60 s - 90 s respectively).
• BTT activator (2.4 mL) was also added. The support was subsequently washed with ACN (3x5 mL).
• reaction mixture was allowed to cool to RT, concentrated in vacuo, absorbed onto Celite and purified by flash-chromatography (KP-Sil, 330 g, Flept:EtOAc, 95:5 to 70:30 to yield the target compound (10, 23.5 g, 76 %) as a white solid.
• reaction mixture was concentrated in vacuo, absorbed on Celite and purified by flash-chromatography (KP-Sil 330 g, Hept:EtOAc 90:10 to 40:60) to yield 6 (18.2 g, 75 %) as a yellow solid.
• the crude product was purified by flash chromatography (KP-Sil 100 g, DCM:MeOH 100:0 to 95:5) which yielded a mixture of products consisting of Boc- and de-Boc protected product (7a and 7b, 3.1 g).
• the material obtained was used in the next step without further purification.
• the mixture of compound 7a and 7b (2.85 g) was dissolved in MeCN (32 mL) and sodium methanolate (3.9 mL, 21.3 mmol) was added. The reaction mixture was stirred at RT for 1 h.
• succinylated support 3 400 mg, 82 pmol/g, 0.03 mmol
• DMAP 0.008 g, 0.07 mmol
• DIC 203 mI, 1.31 mmol
• N,N-diisopropyl-4H-benzo[d][l,3,2]dioxaphosphinin-2-amine 5 (345 mg, 1.36 mmol) was dissolved in 4.8 mL ACN and reacted portionwise with the support (3 equal couplings with reaction times 60 s - 60 s - 90 s respectively).
• BTT activator (2.4 mL) was also added. The support was subsequently washed with ACN (3x5 mL).
• Oxidation Pyridine/Water/Iodine (9/1/12.7 v/v/w, 5mL) for 45 s, followed by ACN wash (3x5 mL) and drying of the support in an argon flow.
• Triphosphorylation Two injections of bis(tetrabutylammonium) dihydrogen diphosphate 6 (0.5 M, 5 ml) for 15 min and 18 hours, respectively. The support was subsequently rinsed with DMF (5 mL), water (3x5 mL), ACN (5 mL) and then dried in an argon flow.
• DMF diMF
• ACN ACN
• Cleavage of the triphosphate was done in 2 h at room temperature with AMA (50/50 v/v mix of 23 % aq. NFUOH and 40 % aq. methylamine, 5 mL). After 2 hours, the AMA filtrate was purged in a round-bottom flask and the support was rinsed 3 times with 23% aq. NFUOH solution.
• pATP 17 can also be made by a slightly modified route wherein the coupling step (b above) is carried out with a modified phosphoramidite such as 6-chloro-N,N-diisopropyl-4-methyl-4FI- benzo[d][l,3,2]dioxaphosphinin-2-amine 8 (compound (Ilia) above).
• a modified phosphoramidite such as 6-chloro-N,N-diisopropyl-4-methyl-4FI- benzo[d][l,3,2]dioxaphosphinin-2-amine 8 (compound (Ilia) above).
• Equation 1 The spectra of molar absorptivities (e in M _1 cm _1 ) were calculated using Equation 1, with A being the measured absorption, ⁇ 4260 and e260 the absorption or molar absorptivity, respectively, at 260 nm.
• Huh-7 human liver cell line was used.
• Huh-7 is a well-differentiated human hepatic cell line with epithelial-like morphology. 43
• the cells were cultured at 37 °C under 5% CO2 in Dulbecco's modified Eagle medium (DMEM GlutaMax with added phenol red, Gibco) containing 4.5 g/l glucose with an addition of 10% foetal bovine serum (FBS, Gibco, origin Brazil), 2 mM L-Glutamine, and 1 mM sodium pyruvate (hereafter referred to as CCM, complete cell culture medium).
• DMEM GlutaMax Dulbecco's modified Eagle medium
• FBS foetal bovine serum
• FBS foetal bovine serum
• CCM complete cell culture medium
• the adherent cells were washed twice with DPBS, containing no calcium or magnesium, and detached with 0.25% trypsin ethylenediaminetetraacetic acid lx (trypsin-EDTA, Gibco, with phenol red).
• trypsin-EDTA trypsin ethylenediaminetetraacetic acid lx
• the cells were counted after trypsin neutralization, diluted to the desired number of cells (Table 2) and thereafter incubated at 37 °C with 5% CO2 for 24 h before experiments.
• Example 5 Cytotoxicity [214] To investigate the cytotoxicity of compounds 7 and 17 Fluh-7 cells were seeded in 96-well plates as described above. Prior to treatment, the conditioned medium was removed from the Fluh-7 cells, and the compounds (stock solution in DPBS diluted in CCM) were added to the cells for different exposure times. Unexposed cells were treated the same way, with the same amount of added DPBS to CCM, instead of the compounds. Two different cytotoxicity assays (alamarBlue to measure metabolic activity and LDH leakage to measure cell membrane integrity) were performed in parallel using the cells and the culture medium from each sample, respectively. Treatments were done in triplicates and the experiment was repeated twice.
• Lactate Dehydrogenase (LDH) Leakage Assay To test for released LDH, the CyQUANT LDH Cytotoxicity Assay kit (Invitrogen) was used. Reaction mixes and enzymatic control (LDH, pure enzyme) were prepared according to the manufacturer ' s instructions. Maximum LDH release were determined by treating cells with a 1:10 dilution of lysis buffer in serum free medium for 30 min at 37 °C and 5% C0 2 .
• Time-lapse Imaging To image the uptake of compounds 7 and 17 over time, the time-lapse setup of the NIS software was used. For every compartment of the dish, one field of view was chosen, from which images were captured every 15 minutes in the first 2 hours, and then every hour over 18 hours (i.e. total time of 20 hours). Exposure time started by adding pre-warmed (37 °C) CCM containing 2.5 mM of FBA-TP to the cells. The fresh CCM for the control cells contained an equal volume of DPBS instead of compound solution.
• 2CNqATP (compound 7) is seemingly evenly distributed across the cytosol and cell nuclei. The bright spots inside of individual nuclei, further indicate an accumulation also in nucleoli. By contrast, pATP accumulates in the cytosol, but not in the cell nucleus. Moreover, its distribution in the cytoplasm is not even, instead it appears to localise with some preference to intracellular structures near the nuclei, which could be part of the endoplasmic reticulum or Golgi.
• Control samples were CCM, containing an equal volume of DPBS instead of FBA-TP. All treatments were done in triplicate. The experiment was setup such that all samples could be harvested at the same time at the end of the experiment. The FBA-TP solutions where therefore added at different time points. At the end of the experiment, the cells were washed twice with DPBS, and thereafter harvested by addition of 0.25% trypsin-EDTA. The trypsin was neutralized by adding DPBS with 2% added FBS and samples were transferred to a 96 U-bottom well plate for high throughput readout.
• Luminex CellStream flow cytometer with a high throughput sampler (FITS) connected to the CellStream Acquisition software was used.
• Excitation wavelength was 405 nm, with the emission passing through a 456/51 nm bandpass filter.
• the pATP uptake displays a lag phase during the initial 15 min and is then internalised at a near constant rate during the following 3 h ( Figure 5 C).
• the pATP uptake does not reach saturation within the 4 h experiment time frame. This finding clearly shows different uptake kinetics of compounds 7 and 17.
• Results The results are shown in Figure 7.
• the cellular (or lysate) fluorescence of the compounds is decreased with increasing (super-stoichiometric) additions ATP or adenosine as competitors, but the response is different.
• 2CNqATP competes with both ATP and adenosine with a stronger competition effect for ATP at low concentrations.
• the uptake of pATP also competes with ATP and adenosine, but the effect is considerably weaker.
• RNA Extraction Cells were seeded in 12-well plates as described above. Treatment solutions were prepared by diluting compounds 7 and 17 to a concentration of 2.5 mM or 5 pM in CCM. Conditioned medium was removed from the cells and treatment solutions were added. For control cells CCM alone was added. Cells were incubated for 24 h at 37 °C and 5% CO2. For RNA extraction and purification, a QIAGEN RNeasy Mini Kit was used, following the manufacturer ' s protocol. Briefly, the cells were washed with DPBS, lysed, homogenized by 12 times passing it through a 20-gauge needle (0.9 mm outer diameter), and added to an equal volume of ethanol.
• the resulting solution was transferred to a RNeasy spin column and centrifuged. Then, binding buffer was added on top of the column, where after it was centrifuged again. The column-bound RNA was washed five times with washing buffer, with a centrifugation step between each addition. To elute the extracted RNA, 30 pL of the provided RNase-free water was applied to the column and centrifuged. This step was repeated twice to increase the yield of extracted RNA.
• RNA extracted from the unexposed cells were determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific) and FBA-TP was added to reach a final concentration of 2.5 pM in the solution. All control samples were applied to RNeasy spin columns and treated the same way as the compound- exposed cell samples, following the purification protocol described above.
• RNA samples and controls were recorded on a Cary 4000 spectrometer (Agilent Technologies) between 200 nm and 600 nm. Emission spectra were measured on a SPEX Fluorolog 3 spectrofluorimeter (Jobin Yvon Horiba). 2CNqA was excited at 350 nm and pA was excited at 370 nm. The excitation and emission bandpass were 2 nm and 3 nm respectively. For excitation spectra the emission wavelength was set to 443 nm.
• pATP and 2CNqATP accumulate in different intracellular locations. pATP accumulates preferentially in the cytosol (with some marked localisation at intracellular structures around the nuclei), while 2CNqATP is seemingly evenly distributed across the cytosol and nuclei. This means that 2CNqATP, beside passing the cellular membrane via an active mechanism, is also effectively retained in the nucleus with a particular accumulation in the nucleoli. Nucleoli are sites of ribosome biogenesis (ribosomal RNA transcription, formation, and maturation; see Fladjiolov, A. A. "The Nucleolus and Ribosome Biogenesis" vol. 12 (Springer Vienna, 1985) suggesting that 2CNqATP, when spontaneously incorporated into cell-synthesized RNA, could be used to fluorescently label ribosomal RNA.

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