WO2014124952A1 - Oligonucléotides alcyne-lna pouvant être traités par "chimie click" - Google Patents

Oligonucléotides alcyne-lna pouvant être traités par "chimie click" Download PDF

Info

Publication number
WO2014124952A1
WO2014124952A1 PCT/EP2014/052694 EP2014052694W WO2014124952A1 WO 2014124952 A1 WO2014124952 A1 WO 2014124952A1 EP 2014052694 W EP2014052694 W EP 2014052694W WO 2014124952 A1 WO2014124952 A1 WO 2014124952A1
Authority
WO
WIPO (PCT)
Prior art keywords
moiety
alkyne
oligonucleotide
lna
constitutes
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.)
Ceased
Application number
PCT/EP2014/052694
Other languages
English (en)
Inventor
Irina Kira ASTAKHOVA
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Syddansk Universitet
Original Assignee
Syddansk Universitet
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Syddansk Universitet filed Critical Syddansk Universitet
Publication of WO2014124952A1 publication Critical patent/WO2014124952A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

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

Definitions

  • the present invention relates to alkyne-LNA nucleotides, which can be efficiently derivatized by highly efficient copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC or "click") chemistry. In one embodiment, this allows preparation of fluorescent probes. In another embodiment, "click” chemistry allows preparation of peptide-oligonucleotide conjugates (POCs). Further derivatives of LNA nucleotides obtainable from "click” chemistry performed on alkyne-LNA nucleotides is also provided.
  • highly efficient copper (I)-catalyzed azide-alkyne cycloaddition CuAAC or "click” chemistry. In one embodiment, this allows preparation of fluorescent probes. In another embodiment, "click” chemistry allows preparation of peptide-oligonucleotide conjugates (POCs). Further derivatives of LNA nucleotides obtainable from "click” chemistry performed on alkyne-LNA nucleot
  • autoantibodies against single-stranded DNA have been thoroughly studied by Glick et al. (see, Biochemistry 2001, 40, 2911-2922; and Biochemistry 2003, 42, 30-41) who showed that binding requires a sequence-specific hydrogen bonding between T and G bases of the 20-25-mer oligonucleotide region and the autoantibody.
  • autoantibodies against double-stranded DNA a hallmark of the important autoimmune conditions Antiphospholipide syndrome and Systemic lupus erythematosus (SLE), are known to be non-sequence-specific and have not been studied in detail.
  • fluorescence assays require use of fluorophores that are photostable and chemically stable and simultaneously provide high fluorescence quantum yields.
  • fluorescent oligonucleotides containing bright cyanine and xanthene dyes are often applied in bioanalysis of nucleic acids and proteins, including antibodies.
  • the fluorescent probes have to bind target biomolecule with high affinity and specificity.
  • Affinity-enhancing locked nucleic acids 2'-amino-LNA containing fluorescent polyaromatic hydrocarbons (PAHs) at 2'-amino group meet these requirements and are therefore very promising for diagnostics and research on diverse nucleic acid targets.
  • Another appealing aspect of LNA/DNA probes is their very promising properties as aptamers in binding diverse protein targets.
  • POCs synthetic peptide-oligonucleotide conjugates
  • Novel POCs are described in which peptide chains are internally incorporated into oligonucleotides using a 2'- alkyne-2'-amino-LNA scaffold.
  • a range of useful biological tools is thereby provided, which can be synthesized in a rapid and efficient manner.
  • derivatization of said alkyne-LNA nucleotides with a fluorescent dye compound provides novel fluorescent probes.
  • the resulting oligonucleotide probes efficiently bind complementary nucleic acids and human autoimmune antibodies against double- stranded DNA, in both cases being monitored by a bright fluorescence response.
  • the fluorescence sensing approach is novel and simple since it allows efficient monitoring of diverse biomolecular interactions in solution following the same spectral principle and by a very simple fluorescence assay.
  • incorporation of the fluorescent LNAs into the probes brings additional binding selectivity into the developed aptasensor.
  • derivatization of said alkyne-LNA nucleotides with a peptide compound provides novel peptide-oligonucleotide conjugates (POCs) .
  • said alkyne-LNA nucleotides can be derived with a carbohydrate compound, a lipid compound or a protein compound.
  • the present invention provides an oligonucleotide comprising an alkyne-LNA nucleotide unit of formula (I) :
  • B is a purine or pyrimidine nucleobase; and A is selected from a single
  • Ci-Ci 0 alkyl or combinations thereof.
  • the invention also provides fluorescent LNA oligonucleotides, comprising a fluorescent-LNA nucleotide monomer of formula (II), as well as methods for their synthesis:
  • B is a purine or pyrimidine nucleobase, preferably a pyrimidine nucleobase, more preferably thymine (T) .
  • Q constitutes a peptide moiety.
  • Figures 1 and 2 show fluorescence detection of DNA/RNA ( Figure 1 ) and monoclonal autoantibodies ( Figure 2) in a medium salt buffer at 19 °C using 1.0 ⁇ and 0.5 ⁇ probes, respectively.
  • the probes in each case are ssON7, ON7: DNA, ON7 : RNA, ssON8, ON8: DNA, ON8: RNA.
  • Figures 3A-3D show gel electrophoresis of 5'- 32 P-labelled oligonucleotides incubated with HS.
  • Locked nucleic acid is a nucleotide analogue in which the sugar ring is constrained as part of a bicyclic system. This locks the furanose in the C3'-endo conformation, thus mimicking the conformation found in RNA.
  • 2'-amino-LNA is an analogue of LNA, which has a higher affinity for complementary DNA/RNA strands than unmodified LNA.
  • the invention provides an alkyne-LNA oligonucleotide.
  • the oligonucleotide comprises an alkyne-LNA nucleotide unit of formula (I) :
  • B is a purine or pyrimidine nucleobase, such as a pyrimidine nucleobase, e.g. thymine (T) .
  • A suitably consists of a combination of 2-5 of the above- mentioned groups.
  • Ci-Cio alkyl is intended to mean a linear, cyclic or branched hydrocarbon group having 1 to 10 carbon atoms, respectively, such as methyl, ethyl, propyl, /so-propyl, cyclopropyl, butyl, /so-butyl, tert-butyl, cyclobutyl, pentyl, cyclopentyl, hexyl, and cyclohexyl.
  • Ci_Ci 0 alkyl as a part of the group A are Ci-C 6 -alkyl, such as Ci-C 4 -alkyl and C 2 -C 4 -alkyl, such as C 2 alkyl.
  • Ci-Cio-alkyl the term “optionally substituted” is intended to mean that the group in question may be substituted one or several times, preferably 1-3 times, with group(s) selected from hydroxy, Ci- 6 -alkoxy (I.e. Ci. 5 -alkyl-oxy), amino, mono- and di(Ci_ 5 -alkyl)amino, -N(Ci. 4 -alkyl) 3 + , cyano, nitro, Ci_ 5 - alkylthio, and halogen .
  • halogen includes fluoro, chloro, bromo, and iodo.
  • Ci-Ci 0 -alkyl (and any variants hereof) as a part of the group A is unsubstituted.
  • nucleobase is intended to encompass purine and pyrimidine nucleobases, such as a pyrimidine nucleobases like thymine (T), uracil (U) and cytosine (C), and purine nucleobases like guanine (G) and adenine (A) .
  • Alkyne-LNA oligonucleotides in which B thymine are indicated as M 1 in this specification.
  • the alkyne-LNA oligonucleotide suitably comprises between 5-50 nucleotides, preferably between 5-25 nucleotides. At least one of these is the alkyne-LNA nucleotide monomer of formula (I) . It may be advantageous to include more than one, e.g. 2 or more, 3 or more, 5 or more alkyne-LNA nucleotide monomer of formula (I) in the alkyne-LNA oligonucleotides of the invention.
  • alkyne-LNA oligonucleotides are those comprising monomer M 1 , in which B is thymine, as shown in Schemes 1 and 2. Further details of the synthesis and characterisation of the alkyne-LNA nucleotide monomer with the structure M 1 is to be found in the
  • alkyne-LNA oligonucleotides are those listed in SEQ ID. 1-4.
  • Especially interesting structural motif includes multiple incorporation (2-4) of alkyne groups separated by 2-5 nucleotides, such as those in SEQ ID. No. 2, 3 or 4.
  • Azide-alkyne cycloaddition using a copper (Cu) catalyst to form a triazole is known as the "click" reaction.
  • This reaction exhibits high yield, good atom economy, high selectivity for terminal alkynes, and functions in vitro and in vivo.
  • the alkyne-LNA nucleotides of the invention can be readily derivatized by highly efficient copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC or "click") chemistry. Having a 2'- amino-LNA scaffold, the LNA-oligonucleotides disclosed herein demonstrate beneficial thermal stabilities of complementary complexes with DNA/RNA compared to those with a 2'-uridine scaffold .
  • Fluorescent bioconjugates must be stable, easy to design and prepare, adaptable for both homogeneous and solid-phase detection and nano-assembly formats. They must also provide a robust and easily interpretable fluorescence signal.
  • PAHs polyaromatic hydrocarbons
  • the LNA skeleton provides excellent selectivity and stability of binding DNA/RNA targets.
  • CuAAC reaction was recently demonstrated as an ideal method for bioconjugation giving stable fluorescent 1,2,3-triazole products both in vitro and in vivo, and also for attachment of peptides to nucleic acids.
  • spectral properties of PAHs can be remarkably improved by attaching to nucleotide via 1,2,3-triazole moiety.
  • Scheme 1 Modified monomer M 1 for CuAAC preparation of the fluorescent probes targeting DNA/RNA and autoimmune antibodies.
  • the alkyne-LNA oligonucleotides of formula (I) can be labelled using the "click" reaction to form fluorescent LNA oligonucleotides.
  • a method is therefore provided for synthesizing a fluorescent LNA oligonucleotide, said method comprising the step of reacting the
  • the fluorescent moiety FL is a cyanine, polyaromatic hydrocarbon (PAH, e.g .
  • linker atoms may be present between LF and N 3 .
  • the spectral properties of polyaromatic hydrocarbons can be remarkably improved by conjugation via the 1,2,3-triazole moiety.
  • the invention also relates to fluorescent LNA oligonucleotides per se. These comprise a fluorescent- LNA nucleotide monomer of formula (II) :
  • B is a purine or pyrimidine nucleobase, such as a pyrimidine nucleobase, e.g . thymine (T) .
  • FL constitutes a fluorescent moiety.
  • FL may comprise a cyanine, PAH (incl. perylene, pyrene) or xanthene fluorescent moiety.
  • Particular alkyne-LNA oligonucleotides are those comprising monomers M 2 -M 5 , in which B is thymine, and shown in Scheme 2. Further details of the synthesis and characterisation of alkyne-LNA
  • B is a purine or pyrimidine nucleobase
  • PI is H or a protecting group, e.g . DMT, MMT or Trityl (Tr)
  • P2 is H, a protecting group or a coupling group, e.g . a phosphoramidite, e.g. -P(N(Pr) 2 )OC 2 H 4 CN, a phosphodiester or phosphotriester
  • the reagent is of the formula 3 above.
  • Particular fluorescent LNA oligonucleotides of the invention comprise a sequence with SEQ ID No. 5-20.
  • SEQ ID No. 7, 8, 20 displayed the most promising sensing of DNA/RNA targets; SEQ ID No. 7 in complex with DNA showed high propensity in sensing autoimmune antibodies.
  • the present invention also relates to the duplex formed between fluorescent LNA
  • oligonucleotides of the invention and the complementary DNA/RNA strand.
  • the invention provides the use of the fluorescent LNA oligonucleotide according to the invention as a fluorescent LNA/DNA probe for the detection of complementary DNA and/or RNA. Further details of this are provided in the Examples and the discussion of Figure 1.
  • the present invention provides the use of the fluorescent LNA oligonucleotide according to the invention, or the duplex described above, for the detection of an
  • the probes disclosed herein demonstrate beneficial thermal stabilities of complementary complexes with DNA/RNA compared to the probes having 2'- uridine scaffold, as described in the above reference (stabilization by +1.5 - +13.0 °C vs. destabilization by -1.5 - 6.5 °C, respectively).
  • Fluorescence quantum yields for the probes described in this invention are higher than previously described in the above reference (OF 0.54-1.00 vs. OF 0.07-0.20, respectively), resulting in superior fluorescence brightnesses (FB up to 80) of the former probes and, hence, in remarkably low limit of target detection values.
  • Fluorescence light-up upon binding complementary DNA/RNA is similar for 2'-amino-LNA and 2'-uridina probes and is up to 7.8-fold. However in case of the former probes (in the above reference) the light-up is observed for more dyes attached, while in the latter a 7.7- fold increase of the dyes' fluorescence was an exceptional case of only one FRET pair.
  • the more consistent quenching of fluorescence in single-stranded LNA/DNA probes is caused by their increased propensity to form stable secondary, "duplex-like", structures (promoted by LNA monomers). Within the secondary structures, quenching of fluorescence of the attached hydrophobic dyes is more likely to occur.
  • POCs Peptide-Oligonucleotide Conjugates
  • Locked nucleic acids display excellent biomedical properties within synthetic oligonucleotides, such as improved target binding affinity, selectivity and enzymatic stability.
  • the present invention describes novel peptide-oligonucleotide conjugates (POCs) in which peptide chains are internally incorporated into oligonucleotides using the 2'-alkyne- 2'- amino-LNA scaffold (Scheme 3).
  • POCs novel peptide-oligonucleotide conjugates
  • Scheme 3 novel peptide-oligonucleotide conjugates
  • peptides have high potential to interact with oligonucleotide strands.
  • peptide chains can be incorporated into any position of oligonucleotide strand.
  • the distance and orientation of the peptide residues can be readily controlled from well-known nucleic acid structure parameters.
  • other peptides available as cell- penetrating and targeting specific cells can be covalently tethered to the 2'-alkyne.
  • the invention therefore provides a method for synthesizing a peptide-oligonucleotide conjugate (POC), said method comprising the step of reacting the oligonucleotide according to the invention with a peptide compound having the structure:
  • reaction is a copper (I)-catalyzed azide alkyne cycloaddition in which the alkyne moiety of the alkyne-LNA nucleotide of formula (I) reacts with the azide moiety of the peptide compound having the structure Q-N 3 so as to form a 1,2,3-triazole product.
  • one or two peptide moieties Q are reacted onto the alkylene-LNA oligonucleotide according to the invention.
  • the invention also relates to peptide-oligonucleotide conjugates (POCs) per se. These comprise a peptide-LNA oligonucleotide monomer of formula (III) :
  • B is a purine or pyrimidine nucleobase, such as a pyrimidine nucleobase, e.g. thymine (T).
  • Q constitutes a peptide moiety.
  • Q is a Met or Leu-enkephalin derivative containing additional lysine residues at the N-terminus.
  • the most preferred POCs are those having the structures set out in SEQ ID. Nos. 21-26.
  • one or two peptide moieties Q are joined to said peptide-oligonucleotide conjugate (POC).
  • the invention also provides a duplex formed between the peptide-oligonucleotide conjugate (POC) of this embodiment, and the complementary DNA/RNA, as well as the use of the peptide-oligonucleotide conjugate (POC) according to this embodiment as a LNA/DNA probe for the detection of complementary DNA and/or RNA.
  • POC peptide-oligonucleotide conjugate
  • the peptide-oligonucleotide conjugate (POC) according to this embodiment can be used in therapy, in particular antisense therapy.
  • click chemistry can also be used to attach other moieties to oligonucleotides by means of the alkyne-LNA nucleotides of the invention.
  • the oligonucleotide according to the invention can be reacted with a
  • COCs carbohydrate-oligonucleotide conjugates having the structure of Formula IV are provided :
  • B is a purine or pyrimidine nucleobase, such as a pyrimidine nucleobase, e.g . thymine (T) .
  • CHO constitutes a carbohydrate moiety.
  • Suitable carbohydrate moieties CHO include monosaccharides and polysaccharides.
  • the oligonucleotide according to the invention can be reacted with a lipid compound having the structure: in which L constitutes a lipid moiety and N 3 constitutes an azide moiety, wherein said reaction is a copper (I)-catalyzed azide alkyne cycloaddition in which the alkyne moiety of the alkyne- LNA nucleotide of formula (II) reacts with the azide moiety of the lipid compound having the structure L-N 3 so as to form a 1,2,3-triazole product.
  • Lipid-oligonucleotide conjugates having the structure of Formula V are provided :
  • B is a purine or pyrimidine nucleobase, such as a pyrimidine nucleobase, thymine (T) .
  • L constitutes a lipid moiety.
  • Suitable lipid moieties L include C 6 -C 2 4 fatty acid moieties.
  • oligonucleotide according to the invention can be reacted with a proteii compound having the structure:
  • Pro-N 3 in which Pro constitutes a protein moiety and N 3 constitutes an azide moiety, wherein said reaction is a copper (I)-catalyzed azide alkyne cycloaddition in which the alkyne moiety of the alkyne-LNA nucleotide of formula (I) reacts with the azide moiety of the protein compound having the structure Pro-N 3 so as to form a 1,2,3-triazole product.
  • protein-oligonucleotide conjugates having the structure of Formula VI are provided :
  • B is a purine or pyrimidine nucleobase, such as a pyrimidine nucleobase, e.g . thymine (T) .
  • Pro constitutes a protein moiety.
  • Phosphoramidite reagent 3 was prepared as described in Scheme 2. Fluorescent azides and TBTA for click chemistry were obtained from Lumiprobe LLC. 9,10-Diphenylantracene (DPA), perylene, crezyl violete perchlorate and oxazine 170 used in spectral studies were recrystallized. HPLC grade light petroleum ether, methanol, ethanol and DMF were distilled and stored over activated 4 A molecular sieves. DCM was always used freshly distilled over CaH 2 . Other reagents and solvents were used as received. Photochemical studies were performed using spectroquality methanol, ethanol and cyclohexane.
  • Oligonucleotide synthesis was carried out on a Perspective Biosystems Expedite 8909 instrument in 1 ⁇ scale using manufacturer's standard protocols. For incorporation of monomer M 1 a hand-coupling procedure was applied (20 min coupling). The coupling efficiencies of standard DNA phosphoramidites and reagent 1 based on the absorbance of the dimethoxytrityl cation released after each coupling varied between 95% and 100%. Cleavage from solid support and removal of nucleobase protecting groups was performed using 32% aqueous ammonia and methylamine 1 : 1, v/v, for 4 h at rt.
  • the resulting oligonucleotides were purified by DMT-ON RP-HPLC using the Waters System 600 equipped with Xterra MS C18-column (5 ⁇ , 150 mm x 7.8 mm). Elution was performed starting with an isocratic hold of A-buffer for 5 min followed by a linear gradient to 70% B-buffer over 40 min at a flow rate of 1.0 mL/min (A-buffer: 0.05 M triethyl ammonium acetate, pH 7.4; B-buffer: 25% buffer A, 75% CH 3 CN). RP-purification was followed by detritylation (80% aq.
  • oligonucleotides were then verified by MALDI-TOF mass spectrometry and IC-HPLC, respectively.
  • IC-HPLC was performed using the Merck Hitachi LaChrom instrument equipped with Dionex DNAPac Pa-100 column (250 mm x 4 mm).
  • nucleoside 1 (1.50 g, 2.62 mmol) in anhydrous DMF (12 mL) was added dropwise, and the reaction mixture was stirred at room temperature for a further 1 h.
  • the reaction mixture was diluted with ethyl acetate (150 mL) and washed with a saturated aqueous solution of NaHC0 3 (2 x 475 mL) and water (6 x 50 mL).
  • the aqueous phases were back-extracted in portions with ethyl acetate (160 mL in total).
  • the combined organic phases were dried over Na 2 S0 4 , filtered and the solvent removed in vacuo.
  • Lumiprobe LLC http://www.lumiprobe.com/
  • oligonucleotides were calculated using the following extinction coefficients (OD 250 / mol) : G, 10.5; A, 13.9; T, M 1 , 7.9; C, 6.6; M 2 , 11.8; M 3 , 5.7; M 4 , 4.3; 33.2; M 5 .
  • General method for CuAAC reactions (monomers M 2 -M 4 ).
  • Starting oligonucleotide ON1-ON4 (20 nmol) was dissolved in fresh MQ water (30 ⁇ _) in 1.5 mL plastic eppendorf.
  • DMSO 40 ⁇ _
  • 2 M triethylammonium acetate buffer pH 7.4; 10 ⁇ _
  • corresponding azide 3-6 (6 ⁇ .
  • the resulting mixture was deaerated, tightly closed, mixed on vortex and subjected to microwave conditions (microwave reactor, 60 °C, 15 minutes). The reaction was afterwards cooled to room temperature and filtrated through Illustra NAP-10 column (GE Healthcare) following manufacture's protocol. The resulting solution was evaporated followed by precipitation of the product conjugates from cold acetone (-18 °C, 12 h) and subsequent washing with acetone two times. The resulting conjugates ON17-ON20 were analyzed by MALDI TOF mass spectrometry and IE HPLC (Table S2). Final yields of products based on the absorbance at 260 nm : 81% (ON17), 72% (ON18), 76% (ON19), 65% (ON20).
  • the human monoclonal autoantibodies were purchased from Diarect AG (dsDNA-mAb32 and dsDNA-mAb33 correspond to clones 32. B9 and 33.H11, respectively).
  • BSA was obtained from Sigma-Aldrich and dissolved in a medium salt PBS at concentration 1.5 mg/mL.
  • a solution of corresponding nucleic acid complex prepared as described above in a plastic 1.5 mL tube 500 ⁇ _ 0.5 ⁇
  • 0.5 ⁇ 10 5 IU of the target autoantibody was added (53 ⁇ _, dsDNA-mAb32 9.4x l0 5 IU/mL at a protein concentration 0.43 mg/mL; 128 ⁇ , dsDNA-mAb33 3.9x l0 5 IU/mL at a protein concentration 0.70 mg/mL).
  • 33 ⁇ of the BSA stock solution was used in similar incubation reactions with the probes. Incubation was performed on Eppendorf Thermomixer Shaker (400 rpm) at 37 °C for 3 h. Upon cooling to ambident temperature over 1 h, the resulting solutions were analyzed by fluorescence spectroscopy using ⁇ 6 ⁇ 500 nm and monitoring A l 530 nm.
  • Limit of target detection (LOD) values were determined by series of incubations and subsequent analysis of dsDNA-mAb33 at concentrations ( ⁇ 5 ) : 1 IU/mL, 0.5 IU/mL, 0.25 IU/mL, 0.1 IU/mL, 0.05 IU/mL, 0.01 IU/mL, and ON7: DNA at concentration 0.5 ⁇ as described above. Resulting fluorescence intensities revealed LOD ⁇ 2.5 ⁇ 10 3 IU/mL, corresponding to the autoantibody concentration ⁇ 4.6 ⁇ g/mL.
  • oligonucleotides and duplexes containing monomer M 2 oligonucleotides and duplexes containing monomer M 2 .
  • ON7 DNA confirming no cross- reactivity for the prepared nucleic acid complex compared to single-stranded ON7, ON7: RNA and to triply modified ON8: DNA/RNA (Table S3, above).
  • Effective recognition of dsDNA-mAb33 is provided by steric and chemical complementarity of the unmodified internal region of ON7: DNA and heavy chain of the autoantibody, accompanied by effective hydrogen bonding.
  • limit of target detection (LOD) value for ON7 DNA was below 4.6 pg/mL of dsDNA- mAb33. This is comparable with currently applied enzyme-linked immunosorbent assay (ELISA), immunofluorescence tests (LOD approx. 1-2 pg/mL), and other fluorescent aptasensors.
  • ELISA enzyme-linked immunosorbent assay
  • LOD approx. 1-2 pg/mL immunofluorescence tests
  • POCs Peptide-Oligonucleotide Conjugates
  • oligonucleotides were calculated using the following extinction coefficients OD260/pmol) : G, 10.5; A, 13.9; T, Ml, 7.9; C, 6.6; M6, 8.1 ; M7, 9.2. Extinction coefficients at 260 nm of monomers M6-M7 were determined by summarizing extinction coefficient of monomer Ml and the corresponding azide 6-7. The latter values were measured at 19 °C in 5% DMSO-water, v/v.
  • the resulting solution was evaporated followed by precipitation of the product conjugates from cold acetone (-18 °C, 12 h) and subsequent washing with acetone two times.
  • M 6 and M 7 are long-chain enkefalins with K residues attached to LNA monomers, namely 5'- azidopentanoic acid-KKKYGGFM (M 6 ) and 5'-azidopentanoic acid-KKKYGGFL (M 7 ) .
  • Monomer M 5'-azidopentanoic acid-RQIKIWFQNRRMKWK-CONH 2
  • the invention relates to the following aspects:
  • Aspect 1 An oligonucleotide comprising an alkyne-LNA nucleotide unit of formula (I) :
  • Aspect 3 The oligonucleotide according to any one of the preceding aspects, wherein B is a pyrimidine nucleobase.
  • Aspect 4 The oligonucleotide according to any one of the preceding aspects, comprising a sequence with SEQ ID No. 1-4, preferably SEQ ID No. 2, 3 or 4.
  • a method for synthesizing a fluorescent LNA oligonucleotide comprising the step of reacting the oligonucleotide according to any one of aspects 1-4 with a fluorescent dye compound having the structure:
  • a fluorescent LNA oligonucleotide comprising a fluorescent-LNA nucleotide monomer of formula (II) :
  • B is a purine or pyrimidine nucleobase
  • Aspect 8 The fluorescent LNA oligonucleotide according to any one of aspects 6-7, wherein B is a pyrimidine nucleobase.
  • Aspect 9 The fluorescent LNA oligonucleotide according to any one of aspects 6-8, wherein FL comprises a cyanine, perylene, pyrene or other PAH, or a xanthene fluorescent moiety.
  • Aspect 10 The fluorescent LNA oligonucleotide according to any one of aspects 6-9, comprising a sequence with SEQ ID No. 5-20, preferably SEQ ID No. 7, 8, 20, most preferably SEQ ID No. 7.
  • Aspect 11 A duplex formed between the fluorescent LNA oligonucleotide according to any one of aspects 6-10, and the complementary DNA/RNA.
  • Aspect 12 Use of the fluorescent LNA oligonucleotide according to any one of aspects 6-10 as a fluorescent LNA/DNA probe for the detection of complementary DNA and/or RNA.
  • Aspect 13 Use of the fluorescent LNA oligonucleotide according to any one of aspects 6-10, or the duplex according to aspect 11, for the detection of an autoimmune antibody.
  • a peptide-oligonucleotide conjugate (POC) comprising a peptide-LNA
  • B is a purine or pyrimidine nucleobase
  • Aspect 17 The peptide-oligonucleotide conjugate (POC) according to any one of aspects 15-
  • B is a pyrimidine nucleobase
  • Aspect 18 The peptide-oligonucleotide conjugate (POC) according to any one of aspects 15-
  • Q is a Met or Leu- enkephalin derivative containing additional lysine residues at the N-terminus.
  • Aspect 19 A duplex formed between the peptide-oligonucleotide conjugate (POC) according to any one of aspects 15-18, and the complementary DNA/RNA.
  • Aspect 20 Use of the peptide-oligonucleotide conjugate (POC) according to any one of aspects 15-18 as a LNA/DNA probe for the detection of complementary DNA and/or RNA.
  • POC peptide-oligonucleotide conjugate
  • Aspect 21 The peptide-oligonucleotide conjugate (POC) according to any one of aspects 15- 18 for use in therapy, in particular antisense therapy.
  • COC carbohydrate-oligonucleotide conjugate
  • a carbohydrate-oligonucleotide conjugate (COC) comprising a carbohydrate-LNA oligonucleotide monomer of formula (IV) :
  • B is a purine or pyrimidine nucleobase
  • a method for synthesizing a lipid-oligonucleotide conjugate comprising the step of reacting the oligonucleotide according to any one of aspects 1-4 with a lipid compound having the structure: in which L constitutes a lipid moiety and N 3 constitutes an azide moiety, wherein said reaction is a copper (I)-catalyzed azide alkyne cycloaddition in which the alkyne moiety of the alkyne- LNA nucleotide of formula (I) reacts with the azide moiety of the lipid compound having the structure L-N 3 so as to form a 1,2,3-triazole product.
  • LOC lipid-oligonucleotide conjugate
  • a lipid-oligonucleotide conjugate comprising a lipid-LNA oligonucleotide monomer of formula (V) :
  • B is a purine or pyrimidine nucleobase
  • a method for synthesizing a protein-oligonucleotide conjugate comprising the step of reacting the oligonucleotide according to any one of aspect 1-4 with a protein compound having the structure:
  • Pro-N 3 in which Pro constitutes a protein moiety and N 3 constitutes an azide moiety, wherein said reaction is a copper (I)-catalyzed azide alkyne cycloaddition in which the alkyne moiety of the alkyne-LNA nucleotide of formula (I) reacts with the azide moiety of the protein compound having the structure Pro-N 3 so as to form a 1,2,3-triazole product.
  • a protein-oligonucleotide conjugate comprising a protein-LNA oligonucleotide monomer of formula (VI) :
  • B is a purine or pyrimidine nucleobase
  • Aspect 29 An alkyne-LNA nucleoside of formula (X) according to aspect 28, wherein PI is protecting group, e.g. DMT, MMT or Trityl (Tr).
  • PI is protecting group, e.g. DMT, MMT or Trityl (Tr).
  • Aspect 30 An alkyne-LNA nucleoside of formula (X) according to aspect 28-29, wherein P2 is coupling group, e.g. a phosphoramidite, e.g. -P(N(Pr) 2 )OC 2 H 4 CN, a phosphodiester or phosphotriester.
  • P2 is coupling group, e.g. a phosphoramidite, e.g. -P(N(Pr) 2 )OC 2 H 4 CN, a phosphodiester or phosphotriester.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Saccharide Compounds (AREA)

Abstract

La présente invention concerne des nucléotides alcyne-LNA, pouvant être efficacement dérivatisés par chimie par cycloaddition azide-alcyne catalysée par le cuivre(I) (Cu AAC ou "click"). Dans un mode de réalisation, ceci permet de préparer des sondes fluorescentes. Dans un autre mode de réalisation, la chime "click" permet de préparer des conjugués peptide-oligonucléotide (POC).
PCT/EP2014/052694 2013-02-12 2014-02-12 Oligonucléotides alcyne-lna pouvant être traités par "chimie click" Ceased WO2014124952A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US201361763576P 2013-02-12 2013-02-12
DKPA201370074 2013-02-12
US61/763,576 2013-02-12
DKPA201370074 2013-02-12

Publications (1)

Publication Number Publication Date
WO2014124952A1 true WO2014124952A1 (fr) 2014-08-21

Family

ID=51353487

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2014/052694 Ceased WO2014124952A1 (fr) 2013-02-12 2014-02-12 Oligonucléotides alcyne-lna pouvant être traités par "chimie click"

Country Status (1)

Country Link
WO (1) WO2014124952A1 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017047816A1 (fr) * 2015-09-18 2017-03-23 田辺三菱製薬株式会社 Acide nucléique réticulé guna, procédé de production de ce dernier, et composé intermédiaire
WO2020100826A1 (fr) 2018-11-12 2020-05-22 田辺三菱製薬株式会社 Acide nucléique artificiel réticulé alna
JP2020537654A (ja) * 2017-10-17 2020-12-24 サレプタ セラピューティクス， インコーポレイテッド アンチセンス送達のための細胞透過性ペプチド
JP2021518361A (ja) * 2018-03-16 2021-08-02 サレプタ セラピューティクス， インコーポレイテッド アンチセンス送達用キメラペプチド
CN114199846A (zh) * 2021-12-15 2022-03-18 上海理工大学 一种基于肽-寡核苷酸共轭识别的新型铅(ii)荧光生物传感器及其应用
TWI816703B (zh) * 2017-10-17 2023-10-01 美商薩羅塔治療公司 雙環肽寡核苷酸結合物
RU2820226C2 (ru) * 2018-11-12 2024-05-31 Мицубиси Танабе Фарма Корпорейшн Сшитая искусственная нуклеиновая кислота alna

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013019794A1 (fr) * 2011-08-01 2013-02-07 The General Hospital Corporation Banques de protéines et de peptides

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013019794A1 (fr) * 2011-08-01 2013-02-07 The General Hospital Corporation Banques de protéines et de peptides

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ANNA S. JØRGENSEN ET AL: "The synthesis of double-headed nucleosides by the CuAAC reaction and their effect in secondary nucleic acid structures", ORGANIC & BIOMOLECULAR CHEMISTRY, vol. 9, no. 5, 1 January 2011 (2011-01-01), pages 1381, XP055107469, ISSN: 1477-0520, DOI: 10.1039/c0ob00438c *
HEERA KRISHNA ET AL: "Alkynyl Phosphonate DNA: A Versatile "Click"able Backbone for DNA-Based Biological Applications", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 134, no. 28, 18 July 2012 (2012-07-18), pages 11618 - 11631, XP055107467, ISSN: 0002-7863, DOI: 10.1021/ja3026714 *
I. KIRA ASTAKHOVA ET AL: "Interfacing Click Chemistry with Automated Oligonucleotide Synthesis for the Preparation of Fluorescent DNA Probes Containing Internal Xanthene and Cyanine Dyes", CHEMISTRY - A EUROPEAN JOURNAL, vol. 19, no. 3, 23 November 2012 (2012-11-23), pages 1112 - 1122, XP055107465, ISSN: 0947-6539, DOI: 10.1002/chem.201202621 *
SINA BERNDL ET AL: "Comparison of a Nucleosidic vs Non-Nucleosidic Postsynthetic "Click" Modification of DNA with Base-Labile Fluorescent Probes", BIOCONJUGATE CHEMISTRY, vol. 20, no. 3, 18 March 2009 (2009-03-18), pages 558 - 564, XP055084883, ISSN: 1043-1802, DOI: 10.1021/bc8004864 *

Cited By (32)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017047816A1 (fr) * 2015-09-18 2017-03-23 田辺三菱製薬株式会社 Acide nucléique réticulé guna, procédé de production de ce dernier, et composé intermédiaire
CN108137638A (zh) * 2015-09-18 2018-06-08 田边三菱制药株式会社 桥连型核酸GuNA、其制造方法及中间体化合物
JPWO2017047816A1 (ja) * 2015-09-18 2018-07-05 田辺三菱製薬株式会社 架橋型核酸ＧｕＮＡ、その製造方法および中間体化合物
CN108137638B (zh) * 2015-09-18 2022-05-03 田边三菱制药株式会社 桥连型核酸GuNA、其制造方法及中间体化合物
JP2023075302A (ja) * 2015-09-18 2023-05-30 田辺三菱製薬株式会社 架橋型核酸ＧｕＮＡ、その製造方法および中間体化合物
US10961269B2 (en) 2015-09-18 2021-03-30 Mitsubishi Tanabe Pharma Corporation Bridged nucleic acid GuNA, method for producing same, and intermediate compound
JP2022033869A (ja) * 2015-09-18 2022-03-02 田辺三菱製薬株式会社 架橋型核酸ＧｕＮＡ、その製造方法および中間体化合物
JP6994197B2 (ja) 2015-09-18 2022-01-14 田辺三菱製薬株式会社 架橋型核酸ＧｕＮＡ、その製造方法および中間体化合物
JP2020537654A (ja) * 2017-10-17 2020-12-24 サレプタ セラピューティクス， インコーポレイテッド アンチセンス送達のための細胞透過性ペプチド
EP3697422A4 (fr) * 2017-10-17 2021-08-11 Sarepta Therapeutics, Inc. Peptides de pénétration cellulaire pour administration antisens
TWI837102B (zh) * 2017-10-17 2024-04-01 美商薩羅塔治療公司 用於反義遞送之細胞穿透肽
US12138312B2 (en) 2017-10-17 2024-11-12 Sarepta Therapeutics, Inc. Cell-penetrating peptides for antisense delivery
TWI816703B (zh) * 2017-10-17 2023-10-01 美商薩羅塔治療公司 雙環肽寡核苷酸結合物
JP7702444B2 (ja) 2017-10-17 2025-07-03 サレプタ セラピューティクス， インコーポレイテッド アンチセンス送達のための細胞透過性ペプチド
JP7320500B2 (ja) 2017-10-17 2023-08-03 サレプタ セラピューティクス， インコーポレイテッド アンチセンス送達のための細胞透過性ペプチド
US12502432B2 (en) 2017-10-17 2025-12-23 Sarepta Therapeutics, Inc. Bicyclic peptide oligonucleotide conjugates
JP2023083557A (ja) * 2017-10-17 2023-06-15 サレプタ セラピューティクス， インコーポレイテッド アンチセンス送達のための細胞透過性ペプチド
JP2021518361A (ja) * 2018-03-16 2021-08-02 サレプタ セラピューティクス， インコーポレイテッド アンチセンス送達用キメラペプチド
JP7596148B2 (ja) 2018-03-16 2024-12-09 サレプタ セラピューティクス， インコーポレイテッド アンチセンス送達用キメラペプチド
US12370261B2 (en) 2018-03-16 2025-07-29 Sarepta Therapeutics, Inc. Chimeric peptides for antisense delivery
EP3765030A4 (fr) * 2018-03-16 2022-01-26 Sarepta Therapeutics, Inc. Peptides chimères pour administration d'antisens
JP7356448B2 (ja) 2018-11-12 2023-10-04 田辺三菱製薬株式会社 架橋型人工核酸ａｌｎａ
EP4275689A2 (fr) 2018-11-12 2023-11-15 Mitsubishi Tanabe Pharma Corporation Acide nucléique artificiel réticulé alna
EP4275690A2 (fr) 2018-11-12 2023-11-15 Mitsubishi Tanabe Pharma Corporation Acide nucléique artificiel réticulé alna
JPWO2020100826A1 (ja) * 2018-11-12 2021-09-30 田辺三菱製薬株式会社 架橋型人工核酸ａｌｎａ
RU2820226C2 (ru) * 2018-11-12 2024-05-31 Мицубиси Танабе Фарма Корпорейшн Сшитая искусственная нуклеиновая кислота alna
KR20210090659A (ko) 2018-11-12 2021-07-20 미쓰비시 타나베 파마 코퍼레이션 가교형 인공 핵산 alna
US12338265B2 (en) 2018-11-12 2025-06-24 Mitsubishi Tanabe Pharma Corporation Crosslinked artificial nucleic acid ALNA
CN112996522A (zh) * 2018-11-12 2021-06-18 田边三菱制药株式会社 桥连型人工核酸alna
WO2020100826A1 (fr) 2018-11-12 2020-05-22 田辺三菱製薬株式会社 Acide nucléique artificiel réticulé alna
CN114199846B (zh) * 2021-12-15 2023-05-23 上海理工大学 一种基于肽-寡核苷酸共轭识别的铅（ii）荧光生物传感器及其应用
CN114199846A (zh) * 2021-12-15 2022-03-18 上海理工大学 一种基于肽-寡核苷酸共轭识别的新型铅(ii)荧光生物传感器及其应用

Similar Documents

Publication Publication Date Title
JP5364380B2 (ja) ポリヌクレオチド標識試薬
WO2014124952A1 (fr) Oligonucléotides alcyne-lna pouvant être traités par "chimie click"
JPH11513388A (ja) 選択的結合性相補的オリゴヌクレオチド
JP4499141B2 (ja) 修飾核酸合成用アミダイド及び修飾核酸合成方法
EP2526113A1 (fr) Modification chimique post-synthétique d'arn à la position 2' du cycle ribose par chimie « click »
JP2013520438A (ja) 逆方向合成ｒｎａのためのホスホルアミダイト
WO2010034024A2 (fr) Monomères protégés et procédés de déprotection pour la synthèse de l’arn
JP2009190983A (ja) オリゴヌクレオチド誘導体
AU1027897A (en) Linker arm for solid support oligonucleotide synthesis and process for production thereof
JP5195757B2 (ja) 核酸合成用ダイマーアミダイド及び核酸合成方法
CN118852286A (zh) 化合物、缀合物及其用途
JP5750209B2 (ja) 機能性分子、機能性分子合成用アミダイド、及び標的物質解析方法
JP2016130232A (ja) オリゴヌクレオチド
JP5975524B2 (ja) 化合物、核酸、核酸の製造方法および核酸を製造するためのキット
US20080167459A1 (en) Amidite for nucleic acid synthesis and nucleic acid synthesizing method
Winkler et al. A novel concept for ligand attachment to oligonucleotides via a 2′‐succinyl linker
US9353142B2 (en) Protecting group for indole group, nucleic acid-synthesizing amidite and nucleic acid-synthesizing method
AU2013361605A1 (en) Post-synthetic orthogonal amidation plus metal catalyzed azide-alkyne cycloaddition click chemistry on siRNA
CN119019354B (zh) 化合物及其制备方法和用途
US20080009612A1 (en) Novel Polynucleotide Synthesis Labeling Chemistry
JPWO2011105610A1 (ja) インスレーター及びその利用
Kolganova et al. Simple and Stereoselective Preparation of an 4‐(Aminomethyl)‐1, 2, 3‐triazolyl Nucleoside Phosphoramidite
WO2021081881A1 (fr) Molécule de glucide fonctionnel à base d'un échafaudage moléculaire à tdg et procédé de préparation s'y rapportant
WO2011090968A1 (fr) Modification chimique post-synthétique d'arn à la position 2' du cycle ribose par chimie « click »
CN120398985A (zh) 一种核糖偶联脂肪链核苷酸单体的制备和应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 14703869

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 14703869

Country of ref document: EP

Kind code of ref document: A1