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WO2021187748A1 - Procédé de modification sélective de nucléotides - Google Patents

Procédé de modification sélective de nucléotides Download PDF

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WO2021187748A1
WO2021187748A1 PCT/KR2021/001073 KR2021001073W WO2021187748A1 WO 2021187748 A1 WO2021187748 A1 WO 2021187748A1 KR 2021001073 W KR2021001073 W KR 2021001073W WO 2021187748 A1 WO2021187748 A1 WO 2021187748A1
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substituted
formula
otf
unsubstituted
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박철민
이양하
유은수
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UNIST Academy Industry Research Corp
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J23/00—Catalysts comprising metals or metal oxides or hydroxides, not provided for in group B01J21/00
    • B01J23/38—Catalysts comprising metals or metal oxides or hydroxides, not provided for in group B01J21/00 of noble metals
    • B01J23/40—Catalysts comprising metals or metal oxides or hydroxides, not provided for in group B01J21/00 of noble metals of the platinum group metals
    • B01J23/46—Ruthenium, rhodium, osmium or iridium
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C243/00—Compounds containing chains of nitrogen atoms singly-bound to each other, e.g. hydrazines, triazanes
    • C07C243/24—Hydrazines having nitrogen atoms of hydrazine groups acylated by carboxylic acids
    • C07C243/26—Hydrazines having nitrogen atoms of hydrazine groups acylated by carboxylic acids with acylating carboxyl groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C243/30—Hydrazines having nitrogen atoms of hydrazine groups acylated by carboxylic acids with acylating carboxyl groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of an unsaturated carbon skeleton
    • C07C243/32—Hydrazines having nitrogen atoms of hydrazine groups acylated by carboxylic acids with acylating carboxyl groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of an unsaturated carbon skeleton the carbon skeleton containing rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical

Definitions

  • It relates to a method for selective modification of nucleotides.
  • oligonucleotides The ability for site-selective functionalization of oligonucleotides is dependent on replication (Cell , 15 , 317-325 (1978)), repair ( ACS Chem. Biol. , 13 , 1721-1733 (2016)), transcription ( Nat. Rev. Mol). DNA or RNA and proteins, such as Cell Biol. , 7 , 557-567 (2006)), translation ( Cell , 4 , 11-20 (1975)), gene silencing ( Gen , 72, 51-58 (1988)) It provides a powerful tool for the exploration of fundamental biological processes, including the interactions between In addition, methods that enable the introduction of orthogonal functionalities include immobilization of proteins by DNA ( Chem. Soc. Rev.
  • One aspect is to provide a method for selectively modifying nucleotides, comprising reacting the nucleotides with a compound of Formula 1 under a rhodium catalyst:
  • R 1 and R 2 are each independently H, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted alkylaryl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycle, substituted or unsubstituted heteroaryl, substituted or unsubstituted acyl, substituted or unsubstituted aromatic, alcohol, alkoxy, amino , amido, nitro, ether, ester, halogen, ketone, cyano, carboxy, hydroxy, thiol, aldehyde, carbonyl, phosphorus, sulfur, phosphate, phosphat, phosphite, sulfate, disulfide, oxy, mer selected from the group consisting of
  • Another aspect comprises the steps of reacting a nucleotide with a compound of Formula 1 under a rhodium catalyst to obtain a first product;
  • a method for producing chemically linked nucleotides comprising reacting the first product with a compound of Formula 3,
  • the nucleotide forms a single-stranded nucleotide template and a duplex with the single-stranded nucleotide template, and consists of two or more single-stranded nucleotides having an overhanging guanosine, providing a manufacturing method :
  • R 1 and R 2 are each independently H, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted alkylaryl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycle, substituted or unsubstituted heteroaryl, substituted or unsubstituted acyl, substituted or unsubstituted aromatic, alcohol, alkoxy, amino , amido, nitro, ether, ester, halogen, ketone, cyano, carboxy, hydroxy, thiol, aldehyde, carbonyl, phosphorus, sulfur, phosphate, phosphat, phosphite, sulfate, disulfide, oxy, mer selected from the group consisting of
  • n 1 to 10
  • Another aspect is to provide a method for preparing photocaged nucleotides, comprising reacting the nucleotides with a compound of Formula 1 under a rhodium catalyst:
  • R 1 and R 2 are each independently H, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted alkylaryl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycle, substituted or unsubstituted heteroaryl, substituted or unsubstituted acyl, substituted or unsubstituted aromatic, alcohol, alkoxy, amino , amido, nitro, ether, ester, halogen, ketone, cyano, carboxy, hydroxy, thiol, aldehyde, carbonyl, phosphorus, sulfur, phosphate, phosphat, phosphite, sulfate, disulfide, oxy, mer selected from the group consisting of
  • Another aspect is to obtain a second product by reacting a double-stranded nucleotide in which one strand is guanosine bulged (G-bulged) with a compound of Formula 1 under a rhodium catalyst;
  • R 1 and R 2 are each independently H, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted alkylaryl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycle, substituted or unsubstituted heteroaryl, substituted or unsubstituted acyl, substituted or unsubstituted aromatic, alcohol, alkoxy, amino , amido, nitro, ether, ester, halogen, ketone, cyano, carboxy, hydroxy, thiol, aldehyde, carbonyl, phosphorus, sulfur, phosphate, phosphat, phosphite, sulfate, disulfide, oxy, mer selected from the group consisting of
  • X is a fluorescent material
  • Another aspect is to provide a method for preparing a derivative of a modified nucleotide comprising the step of reacting a derivative of the nucleotide with a compound of Formula 1 under a rhodium catalyst:
  • R 1 and R 2 are each independently H, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted alkylaryl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycle, substituted or unsubstituted heteroaryl, substituted or unsubstituted acyl, substituted or unsubstituted aromatic, alcohol, alkoxy, amino , amido, nitro, ether, ester, halogen, ketone, cyano, carboxy, hydroxy, thiol, aldehyde, carbonyl, phosphorus, sulfur, phosphate, phosphat, phosphite, sulfate, disulfide, oxy, mer selected from the group consisting of
  • One aspect relates to a method for selective modification of nucleotides comprising reacting the nucleotides with a compound of Formula 1 under a rhodium catalyst:
  • R 1 and R 2 are each independently H, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted alkylaryl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycle, substituted or unsubstituted heteroaryl, substituted or unsubstituted acyl, substituted or unsubstituted aromatic, alcohol, alkoxy, amino , amido, nitro, ether, ester, halogen, ketone, cyano, carboxy, hydroxy, thiol, aldehyde, carbonyl, phosphorus, sulfur, phosphate, phosphat, phosphite, sulfate, disulfide, oxy, mer selected from the group consisting of
  • the nucleotide it is possible to deform the O 6 position of guanine with selective of the four bases of the nucleotide, it is possible to deform the O 6 position of guanine Specifically, unless paired (unpaired), More specifically, the modification may be that a carbonyl group is bonded to the O 6 position of an unpaired guanine.
  • the reaction is possible with a small amount of catalyst and a low reaction concentration, and it is kinetically fast, and the reaction is possible in air.
  • the rhodium catalyst is [Rh(COD)Cl] 2 , [Rh(OH)(COD)] 2 , Rh(COD)(acac), [Rh(COD)(MeCN) 2 ]BF 4 , [Rh(COD) 2 ]OTf, Rh(C 8 H 14 ) 2 (acac), Rh(C 2 H 4 ) 2 (acac), Rh(nbd)(acac), Rh(nbd) 2 BF 4 , Rh(nbd) 2 ( OTf), [Rh(nbd)Cl] 2 , Rh(COD) 2 SbF 6 , (CAAC-Cy)Rh(COD)Cl, [Rh(COD) 2 ]BARF, [Rh(COE) 2 Cl] 2 , [RhCl(C 6 H 10 )] 2 , (1,5-Cyclooctadiene)(8-quinolinolato)rhodium(I
  • the [Rh(COD)Cl] 2 may be specifically a compound of Formula 8:
  • the rhodium catalyst When the rhodium catalyst is a Rh(II) catalyst, it may not be able to cause nucleotide modification due to poor reactivity, and when the rhodium catalyst is a Rh(I) catalyst, a reaction may occur.
  • the rhodium catalyst is [Rh(OH)(COD)] 2 or Rh(COD)(acac)
  • the yield of modified nucleotides may be high, the reaction time may be small, and the rhodium catalyst is [Rh(COD)] )Cl] 2 , the yield of the modified nucleotide is highest and the reaction time may be short.
  • the amount of the rhodium catalyst is 0.01 to 1000 mol%, 0.01 to 500 mol%, 0.01 to 100 mol%, 0.1 to 1000 mol%, 0.1 to 500 mol%, relative to the total number of moles of the nucleotide, the compound of Formula 1 and the rhodium catalyst , 0.1 to 100 mol%, 1 to 1000 mol%, 1 to 500 mol% or 1 to 100 mol%, specifically 0.1 to 500 mol%, 0.1 to 100 mol%, %, 1 to 500 mol% or It may be 1 to 100 mol%, more specifically 1 to 100 mol%.
  • the reaction ratio of the rhodium catalyst is less than 0.01 mol% relative to the total number of moles of the nucleotides, the compound of Formula 1 and the rhodium catalyst, the reaction may not occur or the reaction time may be prolonged, and if it exceeds 1000 mol%, the yield may be poor have.
  • the reaction ratio of the rhodium catalyst is 0.01 to 1000 mol% relative to the total number of moles of the nucleotides, the compound of Formula 1 and the rhodium catalyst, the yield may be better or the reaction time may be reduced, and when it is 1 to 100 mol%, the most The yield is good, and the reaction time can be reduced.
  • the nucleotide may include a nucleotide having a guanine (G) base or a derivative of a nucleotide having a guanine (G) base, wherein the nucleotide is selected from the group consisting of single nucleotides, oligonucleotides and polynucleotides at least one nucleotide.
  • G guanine
  • G guanine
  • the method for selectively modifying a nucleotide selectively modifies the O 6 position of guanine in a nucleotide having a guanine base, and the nucleotide may be a single nucleotide, an oligonucleotide, or a polynucleotide, and the length of the nucleotide is not limited.
  • the nucleotide may include at least one structure selected from the group consisting of a hairpin structure, a duplex structure, a single-stranded structure, a double-stranded structure, and a guanosine bulged structure.
  • the guanosine bulged (G-bulged) structure refers to a structure that is good to participate in the reaction by protruding only the guanosine portion because guanosine is not paired among the linked nucleotides.
  • hairpin structure duplex structure, single-stranded structure, double-stranded structure, and guanosine bulged (G-bulged) structure, and may cause modification of unpaired guanosine in the structure.
  • the nucleotide derivative may be at least one compound selected from the group consisting of a compound of Formula 9, a compound of Formula 10, and a compound of Formula 12:
  • the compound of Formula 1 may be at least one compound selected from the group consisting of diazoacetone (DAX) and a compound of Formula 2 below, and specifically diazoacetone (DAX) may be:
  • the yield of the modified nucleotide may be higher and the reaction rate may be faster, and the compound of Formula 1 is diazoacetone In the case of (diazoacetone, DAX), the yield may be higher and the reaction rate may be faster.
  • the diazoacetone (DAX) may be a compound of Formula 14:
  • the reaction ratio of the compound of Formula 1 is 0.1 to 100 equivalents (equiv.), 0.1 to 50 equivalents, 0.1 to 30 equivalents, 0.5 to 100 equivalents, 0.5 to 50 equivalents, 0.5 to 30 equivalents, 1 to 100 equivalents relative to the nucleotides. , 1 to 50 equivalents or 1 to 30 equivalents, specifically 0.5 to 50 equivalents, 0.5 to 30 equivalents, 1 to 50 equivalents, or 1 to 30 equivalents, and more specifically 1 to 30 equivalents.
  • reaction ratio of the compound of Formula 1 When the reaction ratio of the compound of Formula 1 is less than 0.1 equivalents (equiv.) relative to the nucleotide, the reaction may not occur or the yield may be poor, and if it exceeds 100 equivalents, the reaction time may be prolonged or the yield may be poor, and the When the reaction ratio of the compound of Formula 1 is 0.1 to 100 equivalents, the yield may be better or the reaction time may be shortened. When the reaction ratio of the compound of Formula 1 is 1 to 30 equivalents, the yield is the best and the reaction time is short. can
  • the solvent of the reaction may be a cosolvent or water in which at least one compound selected from the group consisting of tetrahydrofuran (THF) and dimethyl sulfoxide (DMSO) and water are mixed, specifically, the reaction
  • the solvent may be a cosolvent in which tetrahydrofuran and water are mixed or water.
  • the solvent of the reaction is a cosolvent or water in which at least one compound selected from the group consisting of tetrahydrofuran (THF) and dimethyl sulfoxide (DMSO) and water are mixed
  • nucleotides may be selectively modified, and the reaction may not occur when a cosolvent containing other compounds or a solvent not containing water is used.
  • the concentration in the cosolvent of the at least one compound selected from the group consisting of tetrahydrofuran and dimethyl sulfoxide is 0.1 to 70% by volume, 0.1 to 60% by volume, 0.1 to 55% by volume, 1 to 70% by volume, 1 to 60% by volume, 1 to 55% by volume, 5 to 70% by volume, 5 to 60% by volume, or 5 to 55% by volume, specifically 1 to 60% by volume , 1 to 55% by volume, 5 to 60% by volume or 5 to 55% by volume, more specifically 5 to 55% by volume.
  • the solvent of the reaction is a cosolvent
  • concentration in the cosolvent of at least one compound selected from the group consisting of tetrohydrofuran and dimethyl sulfoxide exceeds 70% by volume
  • the reaction may not occur.
  • the yield of the modified nucleotides may be good or the reaction time may be shortened, and if it is 55 vol% or less, the yield may be better or the reaction time may be shorter.
  • the reaction is pH 3 to 10, pH 3 to 9, pH 3 to 8, pH 4 to 10, pH 4 to 9, pH 4 to 8, pH 5 to 10, pH 5 to 9 or It may be made under the conditions of pH 5 to 8, specifically, it may be made under the conditions of pH 4 to 9, pH 4 to 8, pH 5 to 9, or pH 5 to 8, and more specifically, conditions of pH 5 to 8 may be made in
  • the reaction proceeds under the conditions of less than 3 or more than 10, the reaction does not proceed, the yield of modified nucleotides is not good, the reaction time may be too long, and the reaction is carried out under the conditions of pH 4 to 9
  • the yield of the modified nucleotide may be better, the reaction time may be shortened, and when the reaction is carried out at pH 5 to 8, the yield of the modified nucleotide may be the best, and the reaction time may be shortened the most .
  • the reaction is a 2-[N-morpholino]ethanesulfonic acid (2-[N-morpholino]ethansulfonic acid, MES) buffered aqueous solution and 3-[N-morpholino]propanesulfonic acid (3-morpholinopropane -1-sulfonic acid, MOPS) may be formed from at least one buffered aqueous solution selected from the group consisting of buffered aqueous solutions.
  • the nucleotide modification yield may be poorer or the reaction may not proceed at all.
  • the reaction is sodium chloride (NaCl), magnesium chloride (MgCl 2 ), potassium chloride (KCl), sodium acetate (sodium acetate, NaOAc), magnesium acetate (magnesium aceate, Mg (OAc) 2 ) and potassium acetate (Potassium acetate, KOAc) may be formed by adding at least one additive selected from the group consisting of.
  • the cation of the additive masks the negative charge of the phosphate of the nucleotide to help form a secondary structure.
  • Another aspect comprises the steps of reacting a nucleotide with a compound of Formula 1 under a rhodium catalyst to obtain a first product;
  • the nucleotides form a single-stranded nucleotide template and a duplex with the single-stranded nucleotide template, and are composed of two or more single-stranded nucleotides with overhangs guanosine.
  • R 1 and R 2 are each independently H, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted alkylaryl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycle, substituted or unsubstituted heteroaryl, substituted or unsubstituted acyl, substituted or unsubstituted aromatic, alcohol, alkoxy, amino , amido, nitro, ether, ester, halogen, ketone, cyano, carboxy, hydroxy, thiol, aldehyde, carbonyl, phosphorus, sulfur, phosphate, phosphat, phosphite, sulfate, disulfide, oxy, mer selected from the group consisting of
  • n 1 to 10
  • Chemically ligated nucleotides can be easily and quickly prepared by using the nucleotide modification method according to an aspect.
  • a method for producing a chemically ligated nucleotide according to an aspect includes the step of reacting the nucleotide with the compound of Formula 1 under a rhodium catalyst to obtain a first product, some of the steps of obtaining the first product are as described above It may be within the range, and the solvent, aqueous buffer solution, pH conditions, additives, etc. may also be within the above-mentioned range.
  • the compound of Formula 1 may be at least one compound selected from the group consisting of diazoacetone (DAX) and a compound of Formula 2 below:
  • DAX diazoacetone
  • the rhodium catalyst is [Rh(COD)Cl] 2 , [Rh(OH)(COD)] 2 , Rh(COD)(acac), [Rh(COD)(MeCN) 2 ]BF 4 , [Rh( COD) 2 ]OTf, Rh(C 8 H 14 ) 2 (acac), Rh(C 2 H 4 ) 2 (acac), Rh(nbd)(acac), Rh(nbd) 2 BF 4 , Rh(nbd) 2 (OTf), [Rh(nbd)Cl] 2 , Rh(COD) 2 SbF 6 , (CAAC-Cy)Rh(COD)Cl, [Rh(COD) 2 ]BARF, [Rh(COE) 2 Cl] 2 , [RhCl(C 6 H 10 )] 2 , (1,5-Cyclooctadiene)(8-quinolinolato)rhodium(
  • the [Rh(COD)Cl] 2 may be specifically a compound of Formula 8:
  • the first product forms a duplex with the single-stranded nucleotide template
  • the O 6 position of the guanine of guanosine of two or more single-stranded nucleotides having overhanged guanosine is modified may be one, the O 6 position of the unpaired guanine may be modified, and more specifically, the carbonyl group may be bonded to the O 6 position of the unpaired guanine.
  • the number of single-stranded nucleotides having the overhanging guanosine may be two.
  • the prepared nucleotide may be a total of three nucleotides with two chemically linked single-stranded nucleotides and a nucleotide template.
  • a method for preparing chemically ligated nucleotides according to an aspect includes reacting the first product with the compound of Formula 3;
  • the step of reacting the first product with the compound of Formula 3 forms a duplex with the single-stranded nucleotide template, and two or more single-stranded nucleotides having overhanging guanosine guanosine O of guanine
  • the carbonyl group present at the 6- position may react with the compound of Formula 3 as a linker to be connected in the form of oxime ether.
  • n may be 1 to 5, and more specifically, 2 to 3. When n is 0, there is no linker to be chemically connected, and when n is more than 10, too large a ring is formed and may be unstable.
  • Another aspect relates to a method for preparing photocaged nucleotides comprising reacting the nucleotides with a compound of Formula 1 under a rhodium catalyst:
  • R 1 and R 2 are each independently H, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted alkylaryl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycle, substituted or unsubstituted heteroaryl, substituted or unsubstituted acyl, substituted or unsubstituted aromatic, alcohol, alkoxy, amino , amido, nitro, ether, ester, halogen, ketone, cyano, carboxy, hydroxy, thiol, aldehyde, carbonyl, phosphorus, sulfur, phosphate, phosphat, phosphite, sulfate, disulfide, oxy, mer selected from the group consisting of
  • nucleotide modification method it is possible to make photocaged nucleotides that have to undergo a complex synthesis process in a single step, thereby reducing the reaction time, excellent yield, and not wasting the compound for production.
  • the step of reacting the nucleotide with the compound of Formula 1 under the rhodium catalyst may be within the aforementioned range, and a solvent, aqueous buffer solution, pH conditions, additives, etc. may also be within the aforementioned range.
  • the compound of Formula 1 may be at least one compound selected from the group consisting of diazoacetone (DAX) and a compound of Formula 2 below:
  • DAX diazoacetone
  • the rhodium catalyst is [Rh(COD)Cl] 2 , [Rh(OH)(COD)] 2 , Rh(COD)(acac), [Rh(COD)(MeCN) 2 ]BF 4 , [Rh( COD) 2 ]OTf, Rh(C 8 H 14 ) 2 (acac), Rh(C 2 H 4 ) 2 (acac), Rh(nbd)(acac), Rh(nbd) 2 BF 4 , Rh(nbd) 2 (OTf), [Rh(nbd)Cl] 2 , Rh(COD) 2 SbF 6 , (CAAC-Cy)Rh(COD)Cl, [Rh(COD) 2 ]BARF, [Rh(COE) 2 Cl] 2 , [RhCl(C 6 H 10 )] 2 , (1,5-Cyclooctadiene)(8-quinolinolato)rhodium(
  • the [Rh(COD)Cl] 2 may be specifically a compound of Formula 8:
  • the nucleotide may be a nucleotide having a guanine (G) base, and the nucleotide may be at least one nucleotide selected from the group consisting of a single nucleotide, an oligonucleotide, and a polynucleotide.
  • G guanine
  • the method for producing a photocaged nucleotide according to an aspect may be to selectively modify the O 6 position of guanine in a nucleotide having a guanine base, specifically, the O 6 position of an unpaired guanine is modified It may be one, and more specifically, it may be to bond a carbonyl group to the O 6 position of an unpaired guanine.
  • the nucleotide may be a single nucleotide, an oligonucleotide, or a polynucleotide, and the length of the nucleotide is not limited.
  • Another aspect is to obtain a second product by reacting a double-stranded nucleotide in which one strand is guanosine bulged (G-bulged) with a compound of Formula 1 under a rhodium catalyst;
  • a method for preparing a conjugate of a fluorescent substance-labeled nucleotide and a protein comprising subjecting the sixth product to a reductive amination reaction with a protein having a lysine residue under a reducing agent:
  • R 1 and R 2 are each independently H, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted alkylaryl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycle, substituted or unsubstituted heteroaryl, substituted or unsubstituted acyl, substituted or unsubstituted aromatic, alcohol, alkoxy, amino , amido, nitro, ether, ester, halogen, ketone, cyano, carboxy, hydroxy, thiol, aldehyde, carbonyl, phosphorus, sulfur, phosphate, phosphat, phosphite, sulfate, disulfide, oxy, mer selected from the group consisting of
  • X is a fluorescent material
  • a conjugate of a fluorescent substance-labeled nucleotide and a protein can be easily and quickly prepared.
  • Some of the steps of obtaining the second product and obtaining the sixth product may be within the above-described ranges, and solvents, aqueous buffer solutions, pH conditions, additives, and the like may also be within the above-described ranges.
  • the compound of Formula 1 may be at least one compound selected from the group consisting of diazoacetone (DAX) and a compound of Formula 2 below:
  • DAX diazoacetone
  • the rhodium catalyst is [Rh(COD)Cl] 2 , [Rh(OH)(COD)] 2 , Rh(COD)(acac), [Rh(COD)(MeCN) 2 ]BF 4 , [Rh( COD) 2 ]OTf, Rh(C 8 H 14 ) 2 (acac), Rh(C 2 H 4 ) 2 (acac), Rh(nbd)(acac), Rh(nbd) 2 BF 4 , Rh(nbd) 2 (OTf), [Rh(nbd)Cl] 2 , Rh(COD) 2 SbF 6 , (CAAC-Cy)Rh(COD)Cl, [Rh(COD) 2 ]BARF, [Rh(COE) 2 Cl] 2 , [RhCl(C 6 H 10 )] 2 , (1,5-Cyclooctadiene)(8-quinolinolato)rhodium(
  • the [Rh(COD)Cl] 2 may be specifically a compound of Formula 8:
  • the step of obtaining the second product may be one in which the O 6 position of the guanine of bulged guanosine in the guanosine bulged strand among the double-stranded nucleotides is selectively modified, specifically, the O 6 position of unpaired guanine.
  • the 6- position may be modified, and more specifically, a carbonyl group may be bonded to the O 6 position of an unpaired guanine.
  • the second product may be one in which the O 6 position of the guanine of the bulged guanosine is modified.
  • a method for preparing a conjugate of a fluorescent substance-labeled nucleotide and a protein includes reacting the second product with the compound of Formula 4 to obtain a third product.
  • the second product may be bound by reaction with an amine group of the compound of Formula 4, for example, the bound form may be an oxime form.
  • X of the compound of Formula 4 may be a fluorescent material, and the fluorescent material is fluorescein, BODIPY, Tetramethylrhodamine, Alexa, Cyanine, Allo. It may be at least one fluorescent material selected from the group consisting of phycocyanin, rhodamine B, and derivatives thereof, and specifically may be a derivative of rhodamine B represented by the compound of Formula 5 below. have:
  • the method for preparing a fluorescent substance-labeled nucleotide and protein conjugate includes the step of isolating the third product to obtain a fluorescent substance-labeled single-stranded fourth product.
  • the third product may be a modified double-stranded nucleotide in the form of a fluorescent substance and an oxime, and separation of the third product corresponds to denaturation as separating the double-stranded into a single-stranded. After that, only a single strand labeled with a fluorescent substance can be isolated and obtained.
  • the denaturation may be used without limitation as long as it is a known method, for example, it may be by applying heat.
  • the separation of the fourth product which is a single-stranded single-stranded fluorescent substance, can be used without limitation as long as it is a known separation method.
  • it may be separation using chromatography, specifically, separation using HPLC can
  • one strand is guanosine bulged (G-bulged) by binding a single-stranded nucleotide complementary to the fourth product to the fourth product. obtaining a fifth product of the strand.
  • Single-stranded nucleotides complementary to the fourth product may be prepared by designing from the base sequence of the fourth product.
  • the fifth product of the guanosine bulged double strand is not limited, as the guanosine bulged strand may be a fluorescent material-labeled strand or a complementary strand thereof.
  • the method for preparing a conjugate of a fluorescent substance-labeled nucleotide and a protein comprises the step of reacting the fifth product of the double strand with a compound of Formula 1 under a rhodium catalyst to obtain a sixth product, the specific method is It may be within the above-mentioned range.
  • the guanosine bulged double-stranded fifth product may be reacted with Formula 1 to selectively modify the O 6 position of the guanine of bulged guanosine to obtain a sixth product, specifically unpaired
  • the O 6 position of guanine may be modified, and more specifically, a carbonyl group may be bonded to the O 6 position of unpaired guanine.
  • the method for preparing a conjugate of a fluorescent substance-labeled nucleotide and a protein comprises the step of subjecting the sixth product to a reductive amination reaction with a protein having a lysine residue under a reducing agent.
  • the reducing agent may be used without limitation as long as it is a reducing agent capable of causing reductive amination, for example, NaBH 3 CN, NaBH 4 , LiBH 3 CN and NaBH(OCOCH 3 ) 3 Selected from the group consisting of It may be at least one reducing agent.
  • the sixth product may be a double-stranded nucleotide labeled with a fluorescent substance in which the O 6 position of the guanine of bulged guanosine is selectively modified, and the bulged guanine position is combined with a protein through a reductive amination reaction to fluoresce
  • a conjugate of a substance-labeled nucleotide and a protein may be prepared.
  • the protein must have lysine as an amino acid residue, and through this, the reductive amination reaction occurs well, so that a conjugate of a fluorescent substance-labeled nucleotide and a protein can be easily and quickly prepared.
  • Another aspect relates to a method for preparing a derivative of a modified nucleotide comprising the step of reacting the derivative of the nucleotide with a compound of Formula 1 under a rhodium catalyst:
  • R 1 and R 2 are each independently H, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted alkylaryl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycle, substituted or unsubstituted heteroaryl, substituted or unsubstituted acyl, substituted or unsubstituted aromatic, alcohol, alkoxy, amino , amido, nitro, ether, ester, halogen, ketone, cyano, carboxy, hydroxy, thiol, aldehyde, carbonyl, phosphorus, sulfur, phosphate, phosphat, phosphite, sulfate, disulfide, oxy, mer selected from the group consisting of
  • nucleotide in the method for preparing a derivative of a modified nucleotide according to an aspect, “nucleotide”, “solvent”, “aqueous buffer solution”, “pH condition”, “additive”, etc. may be within the above-described range.
  • the nucleotide derivative may include a nucleotide derivative having a guanine (G) base, and specifically, the nucleotide derivative is a compound of Formula 9, a compound of Formula 10, and Formula It may be at least one compound selected from the group consisting of 12 compounds:
  • the derivative of the modified nucleotide may be one in which a guanine base portion in the structure of a derivative of a nucleotide having a guanine base is modified, and specifically, the derivative of the modified nucleotide is a compound of Formula 10 below, It may be at least one compound selected from the group consisting of a compound of Formula 11 and a compound of Formula 13 below:
  • the rhodium catalyst is [Rh(COD)Cl] 2 , [Rh(OH)(COD)] 2 , Rh(COD)(acac), [Rh(COD)(MeCN) 2 ]BF 4 , [Rh(COD) 2 ]OTf, Rh(C 8 H 14 ) 2 (acac), Rh(C 2 H 4 ) 2 (acac), Rh(nbd)(acac), Rh(nbd) 2 BF 4 , Rh (nbd) 2 (OTf), [Rh(nbd)Cl] 2 , Rh(COD) 2 SbF 6 , (CAAC-Cy)Rh(COD)Cl, [Rh(COD) 2 ]BARF, [Rh(COE) 2 Cl] 2 , [RhCl(C 6 H 10 )] 2 , (1,5-Cyclooctadiene)(8-quinolinolato)rhodium
  • the [Rh(COD)Cl] 2 may be specifically a compound of Formula 8:
  • a nucleotide modification method includes chemical ligation, preparation of photocaged nucleotides, preparation of a fluorescent substance-labeled nucleotide and protein conjugate (interaction between protein and nucleic acid), sequencing, It can be used in various fields such as DNA nanotechnology, DNA damage and repair, and development of a molecular beacon probe, and the application fields are the above-described chemical ligation, photo It is not limited to the preparation of the caged (photocaged) nucleotide and the preparation of the fluorescent substance-labeled nucleotide and the protein conjugate.
  • Selective deformation of the nucleotide it is possible to deform the O 6 position of guanine with selective of the four bases of the nucleotide, more specifically, (unpaired) that are paired, and can deform the O 6 position of guanine,
  • the reaction is possible with a small amount of catalyst and a low reaction concentration, and it is kinetically fast, and the reaction is possible in air.
  • chemically ligated nucleotides can be easily and quickly prepared, and photocaged nucleotides, which have to undergo a complex synthesis process, can be produced in a single step, and fluorescent material-labeled nucleotides and proteins can be prepared, and even derivatives of nucleotides can be modified, so it can be applied to various techniques using nucleotides with modified guanine.
  • FIG. 1 is a diagram illustrating a selective modification method according to an aspect of an oligonucleotide.
  • FIG. 2 is a view showing a selective modification method according to an aspect of guanosine bulged (G-bulged) nucleotides.
  • FIG. 3 is a diagram illustrating a method of chemically ligating ODN by applying a selective modification method according to an aspect.
  • FIG. 4 is a diagram showing denaturing PAGE analysis of ligation visualized using SYBR gold (Lanes; 1: before acetonylation, 2: after acetonylation, 3: after ligation, 4: 5a of FIG. 3, 5: 5b of FIG. 3) , 6: 5c of FIG. 3)
  • FIG. 5A is a diagram illustrating a photocaged oligonucleotide
  • FIG. 5B is a diagram illustrating a synthesis step according to an existing method of a photocaged oligonucleotide.
  • FIG. 6 is a diagram illustrating the preparation of a photocaged oligonucleotide using a selective modification method according to an aspect.
  • FIG. 7 is a diagram illustrating modification of various oligonucleotides by a selective modification method according to an aspect.
  • FIG. 8 is a schematic diagram illustrating cross-linking between modified DNA and a protein using a method for selectively modifying nucleotides according to an aspect.
  • FIG. 9 is a diagram schematically illustrating a process of fluorescent labeling and crosslinking with proteins using a method for selectively modifying nucleotides according to an aspect in whole cell lysate of E. coli.
  • FIG. 11 is a schematic diagram illustrating a method for selective modification of nucleotides according to an aspect.
  • FIG. 12 is a diagram showing the process and results of selective modification of nucleotides according to solvents.
  • reaction vessel was dried in an oven overnight and then cooled to room temperature before use.
  • Thin layer chromatography was performed using a plate coated with Kieselgel 60F254 (Merck), and for flash column chromatography, P60 silica gel (230 - 400 mesh) was used.
  • Nuclear magnetic resonance spectra ( 1 H and 13 C NMR) were recorded on an Agilent 400-MR DD2 Fourier-transform NMR spectrometer, and chemical shifts are described in parts per million (ppm). Resonance patterns are represented together by s (singlet), d (doublet), t (triplet), q (quartet), q (quintet), sext (sextet) and m (multiplet), and br represents a broad signal. used The coupling constant (J) is expressed in hertz (Hz).
  • IR spectra were recorded on a Perkin Elmer Spectrum Two infrared spectrophotometer and expressed in cm ⁇ 1 .
  • HRMS High-resolution mass spectra
  • HPLC-MS High performance liquid chromatography-mass spectrometry
  • a metal carbene complex including Cu(I, II), Fe(III), Ir(I), and Rh(I, II) is formed.
  • Metal catalysts (5 mol%) were screened for base-selectivity with DAX and four nucleobases in 50% THF-H 2 O as model substrates (Table 1, entry 1 to 7).
  • nucleoside 5 mM
  • catalyst 10 mol%
  • diazo compound 8 equiv.
  • 10% THF-H 2 O MES (20 mM)
  • pH 6.0 room temperature
  • the catalyst suitable for the reaction of the present invention should have a) the ability to form a metal carbene complex that balances the reactivity and selectivity to the target and the stability in the aqueous buffer solution, b) a high turnover number for a small amount of catalyst used did.
  • DAX diazoacetone
  • Example 3 as in the reaction shown in Figure 1, various types of oligonucleotides (ssODNs (single-stranded oligodeoxynucleotides), ssORNs (single-stranded oligoribonucleotides), hairpins and duplexes) are also guano with DAX and Rh(I) catalysts.
  • ssODNs single-stranded oligodeoxynucleotides
  • ssORNs single-stranded oligoribonucleotides
  • hairpins and duplexes are also guano with DAX and Rh(I) catalysts.
  • Example 4 as in the reaction shown in FIG. 2, it was attempted to check whether the selective acetonylation reaction of guanosine with DAX and Rh(I) catalysts is possible even in bulge G duplexes and hairpins.
  • FIG. 3 it was attempted to chemically link the ODN by the method of the present invention. Specifically, as shown in FIG. 3, it forms a duplex with the oligonucleotide template (5a (SEQ ID NO: 45) in FIG. 3), and two single-stranded oligonucleotides having an overhang guanosine (in FIG. 3) 5b (SEQ ID NO: 46) and 5c (SEQ ID NO: 47)) in the system, using a catalyst of [Rh(COD)Cl] 2 and diazoacetone (DAX) 5a, 6b (SEQ ID NO: 48) and 6c (SEQ ID NO: 48) and 6c (sequences No. 49) was formed, and a ligated oligodeoxyribonucleotide (ODN) was obtained by reacting a compound of Formula 6 (dialkoxyamine) as a linker.
  • DAX diazoacetone
  • FIG. 5A The basic synthesis of photocaged oligonucleotides starts from the synthesis of phosphoramidite monomolecules with photocleavable groups (FIG. 5A), which mainly involves a long synthesis process.
  • Woodson group reported a temporally controlled Hfq-promoting annealing study using photocaged oligoribonucleotides (ORNs).
  • ORNs photocaged oligoribonucleotides
  • oligonucleotides (7a (SEQ ID NO: 51), 7c (SEQ ID NO: 53) and 7e (SEQ ID NO: 55)) were mixed with [Rh(COD)Cl] 2 with a catalyst of Formula 2 Oligonucleotides (7b (SEQ ID NO: 52), 7d (SEQ ID NO: 54) and 7f (SEQ ID NO: 56) with external, internal and hairpin overhang) modified using the compound of formed.
  • Crosslinking was performed through reductive amination, and 14a of FIG. 9 was incubated with NaCNBH 3 in E. coli lysate mixed with T7 RNAP (lane 1 in Table 5).
  • Control experiments were also performed as follows: a) without T7 RNAP (lane 2 of Table 5), b) with only lysate (lane 3 of Table 5), c) without NaCNBH 3 (lane 4 of Table 5) ), d) without lysate (lane 5 of Table 5), e) with only T7 RNAP (lane 6 of Table 5), f) with only 14a of FIG. 9 (lane 7 of Table 5).
  • Example 1 a cosolvent of THF-H 2 O was used for nucleotide modification, but in Example 8, THF was not included and only H 2 O was used as a solvent to test whether nucleotide modification could occur.
  • the experimental conditions were the same as in FIG. 12A, and the oligonucleotide of a hairpin structure (SEQ ID NO: 57, compound 7b) was reacted with diazoacetone (DAX) under 10 mol% [Rh(COD)Cl] 2 catalyst to selectively modify guanine.
  • DAX diazoacetone
  • the solvent was reacted by comparison with H 2 O or 10% THF-H 2 O.
  • an oligonucleotide (SEQ ID NO: 59, compound 8b-OX) having oxime bound to guanine was formed through the subsequent oxime formation reaction, and it was confirmed whether the compounds 8b and 8b-OX were well formed.
  • Example 9 it was attempted to check whether the selective acetonylation of guanosine with DAX and Rh(I) catalysts is possible with nucleotide derivatives in addition to various types of nucleotides.
  • Cyclic diguanylate (c-di-GMP, cyclic diguanylate, CAS: 61093-23-0), which is a nucleotide derivative, was also subjected to an experiment as shown in Scheme 3 to see if selective modification was possible:

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Abstract

Selon un aspect, l'invention concerne un procédé de modification sélective de nucléotides comprenant une étape de réaction de nucléotides et d'un composé de formule 1 en présence d'un catalyseur au rhodium, la position O6 d'une guanine parmi les quatre bases de nucléotides pouvant être sélectivement modifiée. En particulier, la position O6 d'une guanine non appariée peut être modifiée. La réaction peut être effectuée à une faible concentration de réaction en présence d'une petite quantité de catalyseur, est cinétiquement rapide et peut même avoir lieu dans l'air. De plus, tous les nucléotides, y compris les mononucléotides, les oligonucléotides et les polynucléotides, peuvent être modifiés de manière sélective. En outre, à l'aide du procédé, des nucléotides ligaturés chimiquement peuvent être produits facilement et rapidement, des nucléotides photocagés, qui doivent habituellement passer par un procédé de synthèse compliqué, peuvent être produits en une seule étape, un conjugué d'une protéine et d'un nucléotide marqué par un matériau fluorescent peut être produit et même des dérivés de nucléotides peuvent être modifiés et ainsi, le procédé peut être appliqué à diverses techniques utilisant des nucléotides contenant une guanine modifiée.
PCT/KR2021/001073 2020-03-16 2021-01-27 Procédé de modification sélective de nucléotides Ceased WO2021187748A1 (fr)

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