Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
  • C07D498/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
  • C07D498/04—Ortho-condensed systems
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
  • C07D498/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
• • 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
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
  • C12N9/1241—Nucleotidyltransferases (2.7.7)
  • C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/26—Preparation of nitrogen-containing carbohydrates
  • C12P19/28—N-glycosides
  • C12P19/30—Nucleotides
  • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6844—Nucleic acid amplification reactions
  • C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction

Definitions

• the present invention relates to reagents for the reversible protection of biological molecules. It relates in particular to compounds derived from aza-isatoic anhydride and their uses for the protection of biological molecules, including enzymes to block their activity. The invention also relates to the biological molecules thus protected and the methods for using these reagents.
• hot-start polymerases have been developed because they have many advantages over their native version and in particular give better sensitivity performance to diagnostic tests.
• the operating principle of a "hot-start" polymerase is to block its activity transiently by either an antibody, an aptamer or a chemical agent and then restore the activity as soon as the temperature rise during the PCR amplification .
• Blocking by a chemical agent is particularly advantageous because of its low cost.
• the inactivation of the polymerases by acylation of the amine functions of the lysine residues has been described in the state of the art (see for example: Enzyme and Microbial Technology 36 (2005) 947-952 "Thermally reversible inactivation of Taq polymerase in an organic solvent for application in hot start PCR "Ariel Louwrier, Anne van der Valk).
• the protecting groups are cleaved and the activity of the enzyme is restored.
• isatoic anhydride derivatives are capable of reacting with nucleophilic groups such as the amines present on the proteins.
• Moorman A.R. et al. have described the acylation of a protein, chymotripsin a, with isatoic anhydride (Moorman A.R., Abeles R.H. J. Am Chem Soc, 1982, 104, 6785-6786). This reaction inactivates the protein by generating stable anthranilic derivatives. More recently, Hooker et al.
• the patent application FR1257526 also describes acylating agents derived from isatoic anhydride for the functionahsation, labeling, capture or separation of ribonucleic acid (RNA) or chimeric nucleic acid (RNA / DNA).
• RNA ribonucleic acid
• RNA / DNA chimeric nucleic acid
• the acylating agents are more specifically described for the purpose of setting a group of interest on these biological molecules.
• the present invention proposes to provide novel reagents for the reversible protection of biological molecules, and in particular for the preparation of so-called "hot start" enzymes, that is to say which can be easily deprotected by a heat treatment, said new reagents preferably having one or more of the following advantages: - they are cheap,
• the reagents described in the present application are thus particularly suitable for transiently inactivating enzymes involved in the polymerization of nucleic acids such as Taq polymerase or certain reverse transcriptase, and more generally enzymes used in in vitro diagnostic techniques.
• X is a covalent bond or a C 1 -C 4 alkyl
• Y is a nucleophilic radical, preferably O, S, NR 4 , O-NR 4 , NH-O, or NH-NR 4
• R 4 is H or C 1 -C 4 alkyl, Z 1 , Z 2 , Z 3 each independently of one another, N or C, preferably Z 3 is C, more preferably Z 3 is C and R 3 is in the Z 3 position,
• R 1 is H, a substituted or unsubstituted C 1 -C 6 alkyl group, a substituted or unsubstituted alkenyl, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heterocycle, preferably R 1 is a methyl or ethyl group.
• R 2 is a thermolabile and / or acidolabile protecting group
• R 3 is H, substituted or unsubstituted C 1 -C 12 alkyl, for example, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl or 2,2-dimethylpropyl, substituted or unsubstituted aryl; , a substituted or unsubstituted heterocycle, an acyl group, a substituted or unsubstituted alkenyl group, a halogen (eg F, Cl, Br and I), or a cyano group.
• the compound according to the invention has the following formula (II):
• thermolabile and / or acidolabile group R 2 in formulas (I) and (II) above is selected from tert-butoxycarbonyl (BOC), substituted or unsubstituted phenoxyacetyl, trityl, methoxytrityl, dimethoxytrityl or citraconyl.
• R 1 is a methyl group.
• R 3 is iodine.
• Z 1 is N and Z 2 is C.
• the invention relates to one of the following structural compounds (VI)
• the invention also relates to a process for preparing a protected biological molecule, comprising contacting a compound according to the invention as described above with a biological molecule comprising one or more nucleophilic groups under conditions allowing acylation of one or more nucleophilic groups of said biological molecule to form a protected biological molecule.
• the biological molecule comprises amino functions.
• the biological molecule is a protein which comprises nucleophilic functions and in particular the amino functions of its lysine residues or of the terminal amino acid, the alcohol functions of the serines, and / or the thiol functions of the cysteines.
• the invention relates in particular to a protected biological molecule, and represented by the following formula (III):
• W is a nucleophilic radical of the biological molecule, preferably NH, S or O;
• X is a covalent bond or a C 1 -C 4 alkyl
• Y is a nucleophilic radical, preferably O, S, NR 4 , O-NR 4 , NH-O, or NH-
• NR 4 , and R 4 is H or C 1 -C 4 alkyl
• Z 1 , Z 2 , Z 3 are each independently of one another, N or C, preferably Z 3 is C, more preferably Z 3 is C and R 3 is in position
• R 1 is H, substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted aryl or substituted or unsubstituted heterocycle, preferably R 1 is methyl or ethyl,
• R 2 is H or a thermolabile and / or acidolabile protecting group
• R 3 is H, a substituted or unsubstituted C 1 -C 12 alkyl group, for example an iso-propyl, isobutyl, sec-butyl, tert-butyl, isopentyl or 2,2-dimethylpropyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heterocycle, an acyl group, a substituted or unsubstituted alkenyl group, a halogen (eg F, Cl, Br and I), or a cyano group.
• a substituted or unsubstituted C 1 -C 12 alkyl group for example an iso-propyl, isobutyl, sec-butyl, tert-butyl, isopentyl or 2,2-dimethylpropyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heterocycle, an acyl group
• Biomol is selected from among proteins and especially enzymes.
• Biomol is an enzyme for use in a nucleic acid polymerization reaction, for example a DNA polymerase.
• the invention also relates to the uses of a compound according to the invention for the reversible inactivation of an enzyme.
• the compound according to the invention is used for the reversible inactivation of an enzyme for use in a nucleic acid polymerization reaction, for example a DNA polymerase.
• the invention also provides a method of amplifying a nucleic acid comprising the implementation of an inactivated polymerase enzyme by the compounds according to the invention, and at least one heat treatment step at a temperature allowing the cleavage of the or R 2 thermolabile groups and deprotection of the nucleophilic groups, said heat treatment step preceding said nucleic acid amplification step.
• substituted or unsubstituted means that one or more hydrogen (s) present in a group may be substituted by a functional group, for example selected from amine, imine, nitrile, cyano, amide, imide, hydroxyl, alkoxyl, carbonyl, carboxyl, ester, thiol, thioether, thioester and halide.
• a functional group for example selected from amine, imine, nitrile, cyano, amide, imide, hydroxyl, alkoxyl, carbonyl, carboxyl, ester, thiol, thioether, thioester and halide.
• C1-C4 alkyl refers to a straight or branched alkyl chain, or cycloalkyl, having x to y carbon atoms.
• a linear alkyl chain mention may be made of: methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl and n-decyl.
• branched alkyl chain mention may be made of: iso-propyl, isobutyl, sec-butyl and tert-butyl, isopentyl, 2,2-dimethylpropyl, isooctyl, iso-nonyl and iso-decyl.
• cycloalkyl there may be mentioned cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.
• aryl refers to aromatic cyclic hydrocarbons which do not include hetero atoms in the ring.
• the aryl group may comprise from 6 to 14 carbon atoms in the aromatic ring moiety.
• aryl groups include phenyl, biphenyl, phenanthrenyl, pyrenyl, chrysenyl, anthracenyl and naphthyl.
• heterocycle denotes a group comprising at least one saturated or unsaturated ring, at least one of the ring atoms of which is a heteroatom, for example N, O or S.
• a heterocycle may be composed of several fused rings.
• the heterocycle may comprise between 6 and 14 atoms in the cyclic portion.
• acyl refers to a group comprising a carbonyl group, the acyl group being bonded to the molecule via the carbonyl carbon atom. This carbon atom is also bonded to another carbon atom belonging to a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group or a substituted or unsubstituted heterocycle.
• the acyl group comprises between 2 and 12 carbon atoms.
• alkenyl denotes a linear or branched alkyl chain, or a cycloalkyl, comprising at least one carbon-carbon double bond.
• the alkenyl group comprises between 2 and 12 carbon atoms.
• nucleophile refers to a group capable of forming a covalent bond with a reactive (electrophilic) site by giving the two electrons necessary for the creation of the bond, under appropriate reaction conditions.
• nucleophilic groups may be naturally present on a biological molecule, for example the amine group of a lysine, the terminal amine of a polypeptide chain, the alcohol of a serine or the thiol group of a cysteine present on a protein or an enzyme.
• protecting group refers to a functional group introduced into a molecule from a chemical function to mask all or part of its reactivity.
• the masking (protection) of a chemical function on the molecule thus improves the selectivity of the following reactions.
• the term “protection” or “protected” thus refers to the state of the molecule after reaction with a protecting group.
• a “protected biological molecule” is a biological molecule that has one or more chemical functions protected by protective groups. In the case of an enzyme, a protected enzyme can then be inactive. Deprotection refers to the step of release of all or part of the protective groups of a protected molecule and, preferably, obtaining the molecule in its original state, before its protection by the compounds according to the invention.
• thermolabile protecting group refers to a protecting group which is stable at room temperature, for example between 15 ° C and 25 ° C, and which is cleaved, dissociated or released from a molecule to which it is bound by a treatment.
• thermal heating step
• Thermolabile protecting groups include in particular those described by Koukhareva et al., Anal. Chem., 2009, 81, 4955-4962; or Trinlink Biotechnologies (WO2012 / 09434 and US8,133,669).
• thermolabile protecting groups generally include amides, ethers, esters, acetals, carbonates, thioethers, thioesters, thioacetals, thiocarbonates and especially those described in Koukhareva et al., Anal. Chem., 2009, 81, 4955-4962, or monomethoxy trityl (MMT), dimethoxy trityl (DMT), and / or phenoxyacetate, substituted or not.
• MMT monomethoxy trityl
• DMT dimethoxy trityl
• phenoxyacetate substituted or not.
• acidolabile protecting group means a protecting group which is stable under neutral or basic conditions and which is cleaved, dissociated or salted out, under acidic conditions at ambient temperature, for example by treatment at a pH below 6.0, or less than 5.0.
• acid-labile protecting groups include, in particular, citraconyl, tert-butoxycarbonyl (BOC), benzyloxycarbonyl, trityl (Trt), methoxytrityl, dimethoxytrityl, benzyloxymethyl (Bom) or t-butoxymethyl (Bum).
• biological molecule is broadly understood to include all macro molecules synthesizable by biological organisms.
• nucleic acid polymers including DNA or AR, polysaccharides, peptides, polypeptides and proteins, including enzymes.
• polypeptide refers to amino acid polymers or oligomers.
• the amino acids of such a polymer are linked together by a peptide bond between a carboxyl group and an amino group of two amino acids.
• a protein may comprise several polypeptides linked together by non-covalent bonds and / or disulfide bridges.
• amino acids includes natural or unnatural amino acids or their substituted derivatives.
• the compounds according to the invention or reagents for the protection of biological molecules are derivatives of isatoic anhydride useful for transiently protecting ("reversible" protection) the nucleophilic groups, for example the amines, of a biological molecule, for example a protein.
• the compounds according to the invention react with the nucleophilic group of a biological molecule and in particular a protein.
• they can react with the amine ⁇ of a lysine of a protein (or enzyme) or the N-terminal amine of a protein (or enzyme), the alcohol of a serine of a protein (or enzyme) or the thiol of a cysteine of a protein to form respectively an amide, ester, or thioester bond.
• nucleophilic functions of a protein or an enzyme preferably at least one contributes substantially to maintaining the conformation of the protein and / or the enzymatic function.
• the protection of said essential nucleophilic function thus causes the inactivation of the protein or the enzyme.
• the amide, ester and / or thioester bond thus obtained between the biological molecules and the compounds according to the invention is particularly stable at low temperature or ambient temperature.
• Biological molecules can be rendered inactive with the compounds according to the invention, for example under storage or transport conditions at room temperature.
• it is possible to master the starting point of an enzymatic reaction by inducing the conditions for the deprotection of an enzyme, according to the principle below:
• the compounds according to the invention are reacted by reacting the nucleophilic groups of an enzyme with the compound and obtaining a protected enzyme.
• a nucleophilic function of the compound according to the invention is deprotected by acidic and / or thermal treatment by cleavage of a thermolabile and / or acidolabile protective group present on the compound according to the invention and protecting said nucleophilic function of the compound. according to the invention.
• the cyclization is obtained between the nucleotide deprotected function of the compound according to the invention and the carbonyl of the amide, the ester or thioester obtained by the acylation of the enzyme, which concomitantly allows the reversion of acylation, deprotection of the enzyme and restoration of enzymatic activity.
• the expression “compounds according to the invention” refers to the compounds of formula (I) below and to all their sub-formulas specifically described (and in particular the formulas (II) to (VI), the salts of these compounds, their stereoisomers (including diastereoisomers and enantiomers), tautomers and isotopically labeled compounds (including deuterium substitutions and radioactive isotopes).
• nucleophilic function Y protected by a thermolabile and / or acidolabile group R 2 and once released, the cleavage of the amide bond generated by the acylation
• a bulky group R 3 allowing steric hindrance around the nucleophile and the acceleration of the intramolecular cyclization kinetics.
• X is a covalent bond or a C 1 -C 4 alkyl
• Y is a nucleophilic radical
• Zi, Z 2i Z 3 are each independently of one another, N or C, preferably Z 3 is C, more preferably Z is C 3 and R 3 is in position Z 3, R 1 is H, a substituted or unsubstituted C 1 -C 6 alkyl group, a substituted or unsubstituted alkenyl, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heterocycle, preferably R 1 is a methyl or ethyl group.
• R 2 is a thermolabile and / or acidolabile protecting group
• R 3 is H, a substituted or unsubstituted C 1 -C 12 alkyl group, for example an isopropyl, isobutyl, sec-butyl, tert-butyl or isopentyl group or 2 , 2-dimethylpropyl, a substituted or unsubstituted aryl group, a substituted or unsubstituted heterocycle, an acyl group, a substituted or unsubstituted alkenyl group, a halogen (eg F, Cl, Br and I), or a cyano group.
• a halogen eg F, Cl, Br and I
• Z 1 , Z 2 and Z 3 are N.
• X is unsubstituted C 1 -C 3 alkyl, for example methyl.
• Ri is H, alkyl to C 6, an alkenyl, an aryl group or a heterocycle, said alkyl, alkenyl, aryl, or heterocycle being optionally substituted with one or more selected functional groups among nitrile, cyano, amide, imide, alkoxyl, carbonyl, carboxyl, ester, thioether, thioester and halide.
• R 1 is C 1 -C 6 alkyl, optionally substituted with one or more functional groups selected from nitrile, cyano, amide, imide, alkoxyl, carbonyl, carboxyl, ester, thioether, thioester and halide.
• R 2 is a protective group selected from amides, ethers, esters, acetals, carbonates, thioethers, thioesters, thioacetals, thiocarbonates, monomethoxy trityl (MMT), dimethoxy trityl (DMT), substituted or unsubstituted phenoxyacetate, citraconyl, tert-butoxycarbonyl (BOC), benzyloxycarbonyl, trityl (Trt), methoxytrityl, dimethoxytrityl, benzyloxymethyl (Bom) or t-Butoxymethyl (Bum) .
• MMT monomethoxy trityl
• DMT dimethoxy trityl
• BOC tert-butoxycarbonyl
• Trt benzyloxycarbonyl
• Trt methoxytrityl
• dimethoxytrityl benzyloxymethyl
• R 3 is H, C 1 -C 12 alkyl, aryl, heterocycle, acyl, alkenyl, halogen (eg F, Cl, Br and I), or cyano group, said alkyl, aryl, heterocycle or alkenyl groups being optionally substituted by one or more functional groups chosen from nitrile, cyano, amide, imide, alkoxyl, carbonyl, carboxyl, ester, thioether, thioester and halide groups.
• halogen eg F, Cl, Br and I
• R 3 is a C 1 -C 12 alkyl group or a halogen (eg F, Cl, Br and I), said alkyl group being optionally substituted by one or more functional groups selected from the groups nitrile, cyano amide, imide, alkoxyl, carbonyl, carboxyl, ester, thioether, thioester and halide.
• a halogen eg F, Cl, Br and I
• R 3 is placed so as to promote the kinetics of cyclization.
• R 3 is preferably in the 6-position.
• R 3 is a tert-butyl group, an isopropyl group, a cyano group, or an iodine atom.
• Y is different from SS.
• the divalent nucleophilic radical Y is chosen from O, S, NR 4 , O-NR 4 , NH-O, NH-NR 4, C (O) -O-NR 4, C (O) - NH-O, C (O) -NH-NR4, O-C (O) -NH-NR4, NH-C (O) -NH-NR4, O-C (O) -NH-O, NH-C ( 0) -NH-O, O-C (O) -O-NR 4, NH-C (O) -O-NR 4, C (O) -S and R 4 is H or a C 1 -C 4 alkyl group.
• the number of linear X + Y atom is greater than or equal to 2, for example between 2 and 4.
• Y is chosen from O, S and NH.
• X is not a link.
• Y is chosen from O, S and NR 4 , and X is not a bond.
• the compound according to the invention is of formula (II) below :
• thermolabile or acidolabile group R 2 in formulas (I) and (II) above is selected from tert-butoxycarbonyl (BOC), substituted or unsubstituted phenoxyacetyl, trityl, methoxytrityl, dimethoxytrityl or citraconyle.
• the protective group R 2 is the thermolabile phenoxyacetyl protecting group, substituted or unsubstituted, it is preferably a phenoxyacetyl substituted by a halogen, for example fluorophenoxyacetyl.
• thermolabile and / or acidolabile particularly suited to the desired function as described for example in WO2012 / 094343, US8,133,669, or in the following books:
• R 1 is a methyl group.
• R 3 is a halogen, for example, iodine.
• N and Z 2 is C.
• the invention relates to one of the following structural compounds, also described as examples:
• the compounds according to the invention can be easily synthesized according to the methods described in examples or other synthetic methods known in the state of the art.
• some of the compounds according to the invention of formula (II) can be synthesized from the precursor hydroxyl compound 6 whose synthesis is described in example 1.
• the precursor compound 6 can then be modified by reaction of a thermolabile group or acidolabile R 2 on the hydroxyl function of the precursor. It is also possible to react on these precursor compounds or their derivatives the Ri and / or R 3 groups by conventional methods of chemistry.
• the compound according to the invention of formula (II) can be finally obtained by nucleophilic substitution cyclization, as described for example in step (i) of scheme 3 in Example 1 below to give the compounds according to the invention.
• the compounds according to the invention are useful for reversibly protecting, modifying or inactivating enzymes, proteins and more generally biological molecules comprising one or more nucleophilic groups, for example biological molecules comprising one or more amines.
• the invention relates in particular to a process for preparing a biological molecule comprising one or more protected nucleophilic groups, comprising contacting a compound according to the invention as described above with a biological molecule under conditions allowing acylation of one or more nucleophilic groups of said biological molecule to form a biological molecule comprising one or more protected nucleophilic groups (hereinafter also referred to as "protected biological molecule").
• the acylation is done by nucleophilic substitution on one of the carbonyl functions of the anhydride with addition of the nucleophilic group of the biological molecule and elimination of CO 2 .
• the biological molecule comprises amino functions.
• the biological molecule is a protein which comprises the ⁇ -amine functions of lysine or amino residues of the terminal amino acid.
• the acylation of the ⁇ -amine functions of lysine residues or of the terminal amine makes it possible to form very stable amide bonds, in particular under long-term storage conditions (for example over 24 hours). or even days at room temperature).
• the amount of reagents used can be adjusted according to the desired degree of modification (protection).
• the desired degree of modification corresponds to the modification threshold beyond which the enzyme is inactivated. This threshold can be determined empirically by simple tests.
• the buffer used for the protection reaction is generally a buffer with a pH of between 6 and 9, preferably between 7 and 9, more preferably between 7 and 8.
• buffers include phosphate buffers, and non-phosphate buffers. nucleophiles operating in this pH zone, which can contain up to 50% of DMSO.
• Such buffers are known to those skilled in the art as indicated in http://www.sigmaaldrich.com/life-science/core-bioreagents/biological-buffers.html
• the acylation reaction, especially acylation of a enzyme with at least one compound of formula (I) or (II) is preferably obtained in a phosphate buffer, for example at pH 7.4.
• the protection process is preferably carried out at a temperature between 4 ° C and 40 ° C, for example a temperature between 4 ° C and 25 ° C.
• the biological molecules comprising one or more protected nucleophilic groups that can be obtained by the above process are also part of the present invention.
• an enzyme may comprise several protected amino groups because of the presence of multiple lysines in its polypeptide sequence, or OH or SH groups because of the presence of serine or cysteine respectively.
• the invention therefore relates to a protected biological molecule, and represented by the following formula (III):
• Biomol is a biological molecule
• W is a nucleophilic group of the biological molecule, preferably NH, S or O, X is a covalent bond or an alkyl to C 4,
• Y is a nucleophilic radical, preferably O, S, NR 4 , O-NR 4 , NH-O, or NH-NR 4
• R 4 is H or C 1 -C 4 alkyl
• Z 1 , Z 2 , Z 3 are each independently of one another, N or C, preferably Z 3 is C, more preferably Z 3 is C and R 3 is in position
• R 1 is H, substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted aryl or substituted or unsubstituted heterocycle, preferably R 1 is methyl or ethyl,
• R 2 is H or a thermolabile and / or acidolabile protecting group
• R 3 is H, a substituted or unsubstituted C 1 -C 12 alkyl group, for example an iso-propyl, isobutyl, sec-butyl, tert-butyl, isopentyl or 2,2-dimethylpropyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heterocycle, an acyl group, a substituted or unsubstituted alkenyl group, a halogen (eg F, Cl, Br and I), or a cyano group.
• a substituted or unsubstituted C 1 -C 12 alkyl group for example an iso-propyl, isobutyl, sec-butyl, tert-butyl, isopentyl or 2,2-dimethylpropyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heterocycle, an acyl group
• the biological molecule comprises several nucleophilic groups, for example a protein comprising several lysines
• the same biological molecule may comprise all or part of these nucleophilic groups thus protected by acylation with the reagent according to the invention.
• the reagent of formula (II) is reacted with a biological molecule comprising one or more nucleophilic groups, a protected biological molecule of formula (VII) is obtained:
• the biological molecule is selected from enzymes.
• the process is applicable to any type of enzyme.
• the enzymes of interest mention will be made more particularly of the enzymes used in molecular biology techniques, for the genetic engineering or the polymerization of nucleic acids, and more particularly the enzymes used in in vitro diagnostic techniques.
• These enzymes include, by way of illustration, lipases, proteases, glycolases or nucleases.
• the enzymes of interest include restriction enzymes, ligases, RNA polymerases, DNA polymerases, such as DNA polymerase I, II or III, or DNA polymerase ⁇ , ⁇ , or ⁇ , the terminal deoxynucleotidyl transferase (TdT) or telomerase, the DNA dependent RNA polymerase, the primase, or the RNA dependent DNA polymerase (reverse transcriptase).
• restriction enzymes such as DNA polymerase I, II or III, or DNA polymerase ⁇ , ⁇ , or ⁇
• DNA polymerase ⁇ , ⁇ , or ⁇ the terminal deoxynucleotidyl transferase (TdT) or telomerase
• TdT terminal deoxynucleotidyl transferase
• telomerase the DNA dependent RNA polymerase
• reverse transcriptase reverse transcriptase
• the biological molecule according to the invention is an enzyme intended to be used in a nucleic acid polymerization reaction, for example a DNA polymerase.
• a nucleic acid polymerization reaction for example a DNA polymerase.
• the polymerases that can be used for the polymerization of nucleic acids are well known to those skilled in the art.
• the polymerization reaction is in particular a polymerization reaction in the context of polymerase chain reaction (PCR) amplification.
• an enzyme of interest used in the protection method according to the invention is chosen from the following polymerases: T7 DNA polymerase, Kornberg DNA polymerase, Klenow DNA polymerase, Taq DNA polymerase, Micrococcal DNA polymerase, DNA polymerase alpha, Pfu DNA polymerase, AMV reverse transcriptase, MMLV reverse transcriptase, E.coli RNA polymerase, SP6 RNA polymerase, T3 or T7.
• thermostable DNA polymerases and / or having exonuclease activity are used in the method according to the invention to prepare protected polymerases.
• polymerases thus protected are more particularly useful for Hot Start applications.
• Polymerase protection prevents the non-specific amplification of DNA at low temperatures and thus promotes a better efficiency of the amplification reaction, the enzyme being deprotected only at high temperature, by cleavage of the thermolabile group.
• thermostable polymerases include those which are not subject to denaturation of their structure and / or inactivation of their enzymatic activity between 50 ° C and 100 ° C, those that work and are active once they are deprotected, between 50 ° C and 100 ° C. More particularly, the polymerases: TAQ Polymerase and KLen TAQ polymerase.
• thermostable enzymes may be derived from the following biological organisms: Thermus antranikiiding, Thermus aquaticus, Thermus caldophilus, Thermus chliarophilus, Thermus fyliformis, Thermus flavus, Thermus igniterrae, Thermus lacteus, Thermus oshimai, Thermus ruber, Thermus rubens, Thermus scotoductus, Thermus silvanus, Thermus species Z05, Thermus species sps 17, Thermus thermophilus, Thermotoga maritima, Thermotoga neapolitana, Thermosipho africanus, Anaerocellum thermophilum, Bacillus caldotenax, Bacillus stearothermophilus, etc. Methods of use of the compounds according to the invention
• the biological molecules protected according to the present invention may advantageously be deprotected by a simple thermal and / or acid treatment according to the chosen R 2 group.
• the deprotection step can be carried out by heat treatment under pH conditions of between 6.5 and 9.5, preferably between 8 and 9.0, more preferably between 8.5 and 9.0.
• methods of deprotecting the nucleophilic groups of a biological molecule protected by the compounds according to the invention are also described, said method comprising a step of cleavage of the thermolabile and / or acidolabilizable group or groups R 2 by heat treatment and / or or acid respectively, and concomitant deprotection of the nucleophilic groups.
• One of the advantages of the process described above is that it makes it possible to inactivate enzymes. By “inactivation” it should be understood that the catalytic activity of an enzyme protected by the process of the invention is greatly reduced or even in relation to its catalytic activity prior to protection under optimal conditions of activity.
• the deprotection method described above then makes it possible to restore the enzymatic activity of the protected enzymes.
• the invention also relates to the uses of a compound according to the invention for the reversible inactivation of an enzyme.
• the compound according to the invention is used for the reversible inactivation of an enzyme for use in a nucleic acid amplification reaction, for example a polymerase.
• the invention is particularly suitable for hot start applications of enzymes, for example polymerases used in a nucleic acid amplification reaction (so-called "PCR" reaction).
• the invention also provides a method for amplifying a nucleic acid comprising (i) implementing a protected polymerase enzyme or inactivated by the compounds according to the invention, (ii) at least one step for the deprotection of the polymerase, for example by a heat treatment at a temperature allowing the cleavage of the thermolabile group or groups R 2 , (iii) a step of amplification of nucleic acid using the deprotected polymerase in step (ii).
• the amplification process is carried out using a DNA polymerase, such as Taq polymerase, Pfu polymerase, T7 polymerase, Klenow fragment of E DNA polymerase. . coli and / or reverse transcriptase, or any other polymerase as described above.
• the amplification method is a polymerase chain reaction (PCR) reaction, well known to those skilled in the art.
• the PCR protocol comprises, for example, 20 to 40 cycles, each cycle comprising at least (i) a denaturation phase of the DNA to be amplified at a temperature generally of between 90 ° C. and 95 ° C., (ii) a phase of hybridization of the primers with the DNA to be amplified at a temperature generally between 55 ° C and 65 ° C and (iii) an extension phase at a temperature generally between 68 ° C and 75 ° C.
• the protected polymerase according to the invention is in this case preferably a protected thermostable polymerase, for example a protected Taq (Thermus aquaticus) polymerase, a protected Pfu (Pyrococcus furiosus) polymerase, protected Wind or Ti (Thermococcus litoralis) polymerase or their variants. , especially recombinant.
• a sufficient number of amines of said polymerase are protected so that the protected polymerase is inactive or substantially inactive at room temperature and can be deprotected at a temperature equivalent to or greater than the primer hybridization temperature (primer) used for PCR amplification.
• nested PCR nested PCR
• quantitative or qPCR
• semi-quantitative or real-time PCRs error-prone PCRs
• RT-PCR reverse transcription PCR
• the invention also relates to kits or implementation kits comprising the protected enzymes according to the invention, and optionally reagents, buffers, controls and / or an instruction leaflet for implementation.
• the invention relates to a hot-start nucleic acid amplification kit, comprising i) a thermostable DNA polymerase protected according to the invention, for example of formula (III), (VII ), (VIII), (IX) or (X) wherein Biomol is a thermostable DNA polymerase,
• nucleic acid detection reagents for example a fluorescent reagent
• kits are particularly useful for the implementation of amplification methods as described above.
• the invention will be better understood using the examples detailed below and with reference to the appended figures.
• Figure 1 HPLC monitoring of the cleavage of the aza-anthranylate phenoxyacetate derivative and the release of phenylethylamine.
• Figure 2 HPLC monitoring of the cleavage of the derivative aza-anthranylate fluoro-phenoxyacetate 14 and the release of phenylethylamine.
• Figure 3 HPLC monitoring of the cleavage of the N-Boc aza-anthranylate derivative 19 and the release of phenylethylamine.
• FIG. 4 Acylation of hemoglobin by compound 13 used at different concentrations.
• Figure 5 Reversion of the acylation of hemoglobin following a heat treatment.
• Figure 6 Inactivation of Taq polymerase following its acylation with compound 13 and restoration of its activity after heat treatment at 95 ° C. for 15 min under PCR conditions (experiments carried out in duplicate, the average is represented). Dashed clear left: without activation; dotted gray right: after 15 minutes of activation at 95 ° C.
• MilliQ water ultrapure water (Millipore, Molsheim, France),
• the LC-MS analyzes were performed with a WATERS Alliance 2795 HPLC chain equipped with a PDA 996 diode array detector (Waters), a ZQ 2000 mass spectrometry detector (Waters), an Empower version software 2 and a WATERS XTerra MS C18 column (4.6 x 2.5 ⁇ ) used with a flow rate of 1 ml / min at 30 ° C (detection at 260 nm or max plot).
• the ZQ 2000 mass spectrometer has an Electrospray ionization source. Ionizations were performed in positive mode with a cone voltage of 20V and a voltage at the level of the capillary of 3.5kV.
• the conditions used for HPLC analyzes are as follows:
• EXAMPLE 1 Synthesis of an Aza-Isatoic Anhydride Molecule According to the Invention Containing a Thermolabile O-Phenoxyacetate Protecting Group (9) (Corresponding to Compound IV)
• a first hydroxyl precursor compound 6 is first synthesized in 6 steps. It can serve as a starting compound for the synthesis of other compounds according to the invention.
• Compound 6 is then phenoxyacetylated to give compound 7, then iodinated to give compound 8 and finally cyclized in order to obtain the expected 9-aza-isatoic compound capable of reacting with a protein or any other biological molecule to protect it transiently. .
• the final product is obtained in the form of a white powder with a yield of 69% (4.88 g, 26.30 mmol).
• the mixture is filtered on celite, evaporated and purified on a column of silica gel using an eluent gradient (cyclohexane / AcOEt 95/5 with cyclohexane / AcOEt 90/10).
• the final product is obtained in the form of a white powder with a yield of 67% (3.07 g, 12.70 mmol).
• the final product is obtained in the form of a white powder with a yield of 97% (2.94 g, 12.06 mmol).
• CITHMCINOSS® M 357.95 g / mole
• 2.25 g of 3 (9.23 mmol, 1 eq) 30 ml of DCM, 1.88 g of imidazole (27.70 mmol, 3 eq) are introduced and 2.78 g of TBDMSCI (18.47 mmol, 2 eq).
• the reaction mixture is stirred for 4 hours at room temperature.
• 50 mL of water is then added and the solution is extracted with DCM (3x75 mL).
• the organic phases are then combined, washed with saturated NaCl solution (2 x 50 mL), dried over MgSO 4 and evaporated.
• reaction crude is then purified on a silica gel column using an eluent gradient (EP to EP / Et 2 0 95/5).
• the final product is obtained in the form of a colorless oil with a yield of 95% (3.15 g, 8.80 mmol).
• the final product is obtained in the form of a white powder with a yield of 70% (385 mg, 1.03 mmol).
• the final product is obtained in the form of a yellow powder with a yield of 91% (340 mg, 0.68 mmol).
• the reaction mixture was evaporated to dryness and directly purified on a Cl 8 grafted silica gel column, using an eluent gradient (H 2 O / ACN 95/5 to H 2 O / ACN 5/95).
• the final product is obtained in the form of a white powder with a yield of 90% (255 mg, 0.54 mmol).
• Precursor compound 6 is fluoro-phenoxyacetylated (a group which may be more heat labile) to give compound 11, then iodinated to give compound 12 and finally cyclized to obtain the 13-aza-isatoic compound.
• the final product is obtained in the form of a yellowish powder with a yield of 57% (190 mg, 0.49 mmol).
• Ci 7 H 12 END 2 0 6 M 486.19 g / mole
• the reaction mixture is evaporated to dryness and directly purified on a C18 grafted silica gel column, using an eluent gradient (H 2 O / ACN 95/5 to H 2 O / ACN 5/95).
• the final product is obtained in the form of a white powder with a yield of 77% (115 mg, 0.24 mmol).
• Example 3 Synthesis of a Type of aza-isatoic anhydride Molecule According to the Invention Provided with an N-Boc Acid / Thermolabile Protecting Group 18 (Corresponding to Compound VI)
• the synthesis of another example of compound according to the invention (according to scheme 5).
• the precursor compound 6 is first converted to the precursor azido compound of an amino compound on which the amino function will be coupled to the protecting group BOC to access the compound 16.
• An iodination reaction gives the compound 17 and finally cyclization allows to access the compound 18 aza-isatoic.
• reaction crude is then purified on a column of silica gel using an eluent gradient (EP / AcOEt 9/1 to EP / AcOEt 8/2).
• the final product is obtained in the form of a yellow oil with a yield of 59% (135 mg, 0.40 mmol).
• the final product is obtained in the form of a yellow powder with a yield of 67% (121 mg, 0.26 mmol).
• the final product is obtained in the form of a yellowish powder with a yield of 71% (50 mg, 0.09 mmol).
• the azaisatoic anhydride 18 is dissolved in 1 ml of DCM. 27.6 g of phenylethylamine (0.22 mmol, 1 eq) are then added and the reaction medium is left stirring at room temperature for 1h. 10 mL of water is then added and the solution is extracted with Et 2 (4x20 mL). The organic phases are then combined, dried over MgSO 4 and evaporated. The raw reaction is then purified on a silica gel column using an eluent gradient (EP / AcOEt 7/3 to EP / AcOEt 4/6).
• a solution of amide (10, 14 or 19) at 2.5 mM in DMSO and 180 of a buffer conventionally used in a reaction of amplification of genetic material 60 mM Tris pH 9, 50 mM KO, 1 mM MgCl 2 .
• the mixture is then stirred at 95 ° C. in a thermomixer.
• the deprotection process was also carried out starting from the fluorophenoxyacetate derivative 14. After 15 minutes at 95 ° C., the amide population is very low. In particular, it is lower compared to the result observed with the derivative 10 (see FIG. 2). The presence of the fluorine atom accelerates the cleavage of the group phenoxy acetate. Thus, after 30 minutes, this population of amide has almost completely disappeared in favor of alcohol and the lactone very majority indicating the release of phenylethylamine.
• the AL602 CTRLE 0.25X chromatogram shows the control hemoglobin that has not reacted with the compound 13.
• the theme and the 2 alpha and beta subunits of the protein part are visualized therein.
• the following 3 chromatograms (AL 600 0.25 X-1X) show the disappearance of the protein part in favor of a right-shifted solid corresponding to alpha and beta subunits acylated by the aza-isatoic compound 13. The widening of the peak corresponds to the random acylation of the reactive sites of the protein .
• reaction media were taken up after reaction in 8M GuHCl in order to solubilize the protein part which would have precipitated during the heat treatment.
• This example demonstrates the reversible acylation of a model protein by compound 13 as described in the invention.
• Example 7 Demonstration of the reversible acylation of TAQ polymerase by the aza-isatoic compound 13 as described in the invention
• the aza-isatoic derivative 13 with the fluoro-phenoxy thermolabile group is capable of reacting with a thermostable polymerase (Taq) under mild conditions and in an aqueous medium to form a Taq-aza anthranylate fluorophenoxy derivative. Elimination of this protecting group following a heat treatment at 95 ° C. under PCR conditions makes it possible to restore the activity of the polymerase, which is demonstrated by the assay of its activity.
• the concept of using aza-isatoic anhydrides suitably modified to transiently mask the activity of a polymerase and then restore it after a heat treatment is thus demonstrated.
• the polymerization activity of the TAQ polymerase is carried out using a 45-base oligonucleotide probe terminated by a "hairpin" structure. This is characterized by the presence of a quencher of fluorescence at the beginning of the structure and by a fluorophore at the end of the sequence of the probe, so that the quencher is found spatially close to the fluorophore and that no signal of fluorescence can not be measured in this configuration. Under the action of the polymerase activity, this probe is elongated with a 19 base oligonucleotide complementary to the beginning of the previous probe.
• the hairpin structure of the probe unfolds and the fluorophore can emit and a fluorescence is then measurable.
• This fluorescence measurement is carried out at a temperature of 60 ° C. for 20 minutes in the presence of the reagents necessary for the activity of the enzyme, that is to say, dNTPs, MgCl 2 and a basic buffer at pH 9.5.
• the increase in fluorescence is linear at the beginning of the measurement and makes it possible to calculate an initial speed corresponding to the amount of fluorescence emitted per minute of elongation.
• Chemical modification of the polymerase to render it inactive is measured by this method. By measuring the level of residual activity after modification of the enzyme, it is possible to determine the effectiveness of the protection put in place. A polymerization completely inactivated by chemical modification must no longer generate activity without thermal activation at temperatures above 90 ° C.
• FIG. 6 therefore demonstrates that, depending on the concentration chosen for the acylating agent, the Taq polymerase activator can be completely inactivated and restored after treatment at 95 ° C. for 15 min under "hot-start” conditions.
• PCR see column corresponding to AL 604 0.375X).

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