CN104513817A - Mono-substituted alkylation compound preparation method - Google Patents

Abstract

The invention relates to a mono-substituted alkylation compound preparation method, and specifically provides a method for preparing an alpha-amino acid asymmetric mono-substituted alkylation compound represented by the specific formula by using aldimine type Schiff base. According to the method of the present invention, in a media, in the presence of an optically-active quaternary ammonium salt phase transfer catalyst and an inorganic base, an aldimine type Schiff base alkylation step is started to be performed and quenching is performed before completing the stoichiometric reaction of the alkylation reaction so as to obtain the high optical-purity mono-substituted alkylation compound.

Description

A kind of method of mono-substituted alkylated compound

invention technology

The present invention relates to biomedicine field, particularly relate to a kind of method of mono-substituted alkylated compound.

background of invention

Mono-substituted alkylated compound, has become conventional clinical practice and assay laboratory in analyzing and testing that is biological and abiotic sample.These detection techniques can be divided into two large classes: (1) according to the interaction (technology such as, based on immunoassay) of part-acceptor and (2) based on nucleic acid hybridization (technology based on polynucleotide sequence).

Technology based on immunoassay, is characterized in that the step by a sequence comprises the antibody of Non-covalent binding, this antibody and antigen complementation.Detection technique based on polynucleotide sequence, it is characterized in that, comprised the complementary sequence of the polynucleotide sequence of the mark of Non-covalent binding or the assay of probe by the step of a sequence, under allowing the condition of the alkali of hybridization, match this hybridization check by Wo Sen-Ke Like.

Detection technique based on the polynucleotide sequence of the complementary sequence of the nucleic acid probe of the sequence of the mark of Non-covalent binding is main identification event.The nucleotide probe of the accurate molecular arrangement that this binding event is brought and interaction complementation and target.It is by the free energy of non-covalent bonding, such as, and hydrogen bond, the energy advantage of stacking free energy and release.

Summary of the invention

In order to adopt the Non-covalent binding for measuring the nucleic acid probe containing target sequence, it is necessary, so as can in conjunction with probe in detecting to target.This detection is the impact by signaling step or event.The generation of the main identification event that the mode that signaling step or event make some qualitative or quantitative detects.

The generation of the main identification event that various signal event also can be adopted to detect.The specific signal of the feature of adopted reporter molecules is depended in the selection of signal event.Although no matter labelled reagent itself, without the need to further process, may detect, time more, be that reporter molecule is covalently bound, or the Non-covalent binding combined is to labelled reagent.

Useful polynucleotide sequence is covalently bound as the labelled reagent of probe in nucleic acid detection system, also can adopt various reporter molecules.All it is required that reporter molecule provides may be detected a signal, namely by suitable mode, have the ability to be connected to labelled reagent with covalent linkage.

Molecule can be radioactivity or inactive.Radioactive signaling moiety, is characterized in that by one or more radioisotopic phosphorus, iodine, hydrogen, carbon, cobalt, nickel etc.Best radio isotope sends public testing.Gamma radiation, and there is the long transformation period.The detection of the radioactive reporter molecules detector of radiation normally by being caused by crystallization, or the stimulation photo emissions of the photographic fog of a photographic emulsion.

The advantage that inactive reporter molecules has is, their use can not cause and the danger being exposed to radiation, and does not require to use special treatment technology.In addition, they are normally more stable, and as a result, use more cheaply.Perhaps be the detection sensitivity of the inactive reporter molecules of equally high or higher specific activity reporter molecules.

It is made to be very suitable for being used in the application of various molecule and cytobiology with the ability of the DNA of inactive certification mark substance markers simply and reliably.The DNA of nonradioactive labeling or rna probe can be used for some and specifically apply, and comprise hybridization program (south, north, groove, or Dot blot, in situ hybridization), nucleic acid Position Research, DNA or RNA quantitatively and DNA enzymatic or RNA enzyme quantitative.

Develop the on-radiation certification mark that the direct tag protocol of two kinds of enzymes mediation is additional, as the DNA of the fluorescein of fluorescent chemicals and rhodamine (and other).Although these marking methods allow the method for on-radiation detection system or exceed radiometry method still there is the shortcoming shown in sensitivity, the nonradioactive labeling's system of each developed so far.1) reagent of enzymatic DNA Mk system needs, comprises some nucleotide precursors that are unmarked and mark, primer, and/or the enzyme promoting DNA synthesis.Labeling effciency is not easy to control and modal two kinds of labeled reactants (nick translation, random primer) be the label probe that impossible create are identical sizes as initiate dna.2) directly marking method also has obvious limitation, and the susceptibility caused comprising a label reduces, and the label protocol of effort multistep, severe reaction conditions, makes a variation from reaction, and the reaction efficiency of instability is lower.

Under normal circumstances, must first be that size classification is separated according to the polynucleotide (RNA, DNA) analyzed, transfer on solid support, then by a series of step process, to guarantee the probe only having specific binding.Detect hybridization product usual Qu Jue Yu Unit, the derivative or antibody complexing of heavy metal.The method of hybridization is the fundamental research of staple food always, and also serve as commercial reagents box now, for diagnosing heredity, the quantity of pernicious transmissible disease constantly increases.

Desirable marking method identifies in conjunction with the simplicity of a step and high-level efficiency, complete in maintenance, result that the is stable and marked product of the initial DNA of same size.In order to develop and these features, the ability that we have with mustargen, with the DNA alkylation DNA marker reagent of the adducts containing covalent linkage connection be obtained by reacting in one step.

Between the step of mark result in the compound of formation of chemical bond and Polynucleotide and/or protein.Labeling process comprises hatching with described compound at water-based or non-aqueous solution, is secondly the polynucleotide be separated with the Polynucleotide of the mark of unreacted labelled reagent.Degree on label can be controlled by the relative quantity of aignment mark reagent and Polynucleotide, by the length that adjustment is cultivated, controlled by the temperature of incubation, by controlling the absolute concentration of Polynucleotide and labelled reagent, and the solution by controlling, use water or organic solvent, pH value, ionic strength and buffer composition.

specific implementation method:

The Polynucleotide of mark can be used for multiple use:

1) technology detects the Polynucleotide of particular sequence, rely on nucleic acid or the albumen of target, comprise Dot blot, slot blot, Southern trace, Northern blot hybridization, the west and south, the binding affinity of the Polynucleotide of FISH(fluorescence in situ hybridization or mark, hybridization), the RNA of in situ hybridization and DNA sequence dna, " chip " in new development or the mobile equipment of porous or multi-slot wherein be the combination technique of Polynucleotide.

2) mark, be delivered to the Polynucleotide of cell in vitro or in body with the position of the ubcellular and tissue of determining them.

3) oligonucleotide marked is used as primer as pcr amplification technology (polymerase chain reaction).

4) polynucleic acid is quantitative.

5) quantitative nucleic acid (rnase and DNA enzymatic), fluorescence polarization or fluorescence go quencher.

6) polynucleic acid order-checking.

7) direct mutagenesis detects.

8) reactive group is covalently bound, uses the application program of antisense.

Also describe the digoxin that a kind of method is functionalized.The saccharide residue of digoxin functional group first in terminal forms the function of digoxin dialdehyde oxide diol, standard reductive alkylation reaction sodium cyanoborohydride subsequently and connection compound.Linker comprises and the organic molecule of primary amine and shielded primary amine, after by the derivative of digoxin and the removal of blocking group with primary amino.The reactive group that primary amine can be converted into aldehydes or ketones as the thiol-reactive group of thiosemicarbazide or hydrazide group, as Haloacetamide, maleimide, disulfide, or pyridyl two sulphur; ; The reactive group (as succinimide ester) of amine, lsothiocyanates, or SULPHURYL CHLORIDE, or the carboxylic acid of reactive group as diazomethane or haloacetyl.The result of this method, can be used for the protein of label, peptide, polysaccharide, lipid, and digoxin molecule in other interested molecule.Also be applicable to the synthesis utility routine of straight ahead from the precursor of cheapness, with the method for this uniqueness, provide Gaoxin label.

Provide the method for the covalently bound nucleic acid of single step label, comprise, forming covalent linkage for alkylating nucleic acid has dismountable labelled reagent.Then, in conjunction with covalent linkage have the mixture of dismountable labelled reagent, the nucleic acid wherein comprised, is characterized in that, under the condition of described labelled reagent, thus forms the reactivity of covalent linkage with nucleic acid.

The compound provided is tagged molecule, and it contains a covalent linkage has dismountable for alkylation, and chemical at the labelled reagent of an one-step reaction, thus provides the chemistry with detectable reporter molecule.

In a preferred embodiment, the compound provided has following structure, comprising:

In formula, D is selected from fluorescence radiation compound, radioactive compound, haptens, immunogenic molecules, chemiluminescent luminophor, and in the group of protein composition, the molecule in the group that B is selected has the electrostatic interaction of the nucleic acid composition of affinity, minor groove binding, major groove combines, interlayer; Select the group formed from alkylating agent mustard and 3 membered ring derivatives of A.

In a preferred embodiment, have the compound of formula, it is characterized in that, one has the container of dismountable labelled reagent, for alkylation in an one-step reaction containing covalent linkage.Also with the operation instruction that test kit provides.The operation instruction of term used, refers to the concrete manifestation of the method for measurement of concentration of at least one in the reagent suited, the parameter of mixing, as the relative quantity of reagent and sample, and the maintenance time cycle of reagent/specimen admixture, temperature, buffer conditions etc.

People can determine whether a specific compound be suitable for of the present invention by comparing the compound shown in candidate compound and successful embodiment.A suitable alkylating target molecule of alkylated compound, at an one-step reaction.The compound of this example demonstration is the suitable method of the alkylated reaction of successful target molecule and preparation thereof.If candidate compound carries out the efficiency of at least 80%, compound described in an embodiment, this compound is the alkylated compound of a suitable step.

According to the nucleotide sequence of the nucleic acid molecule of the present invention of a preferred embodiment, this may be complementary target sequence, is converted into by covalently bound reporter molecules tagged compound.It is useful reporter molecules that several compound describes, for by nucleic acid and protein.In a preferred embodiment, what be suitable for that compound of the present invention comprises in following three parts is one or more:

A-alkylation group---the chemical functional of electricity, allows them become the compound of bearing nucleophilic group covalently bound.Alkylating reagent comprises yperite (mustargen and sulfur mustard), triatomic ring (aziridine, oxyethane, cyclopranes, the cyclopropane of activation, episulfide).

B-pad---the connection between alkylation group and reporter molecule-spacer groups, comprise alkane, alkene, ester, ether, glycerol, acid amides, sugar, polysaccharide, and heteroatoms is as oxygen, and sulphur or nitrogen, and/or under the physiological condition of molecular cleavage, as a disulphide bridges or the responsive group of enzyme.Distance piece can with clean positive charge, or following any one: minor groove binding, major groove bonding agent, insertion group, or the protein that combines of other DNA or peptide, and as transcription factor, thus the compound provided has the Polynucleotide increasing avidity.Distance piece is arranged, to alleviate the interference of the reporter molecule of the nucleic acid after the alkylated compound of possible molecular separation and alkylated reaction.

C-reporter's molecule---the additional chemical part being detected as the compound of object.Reporter's molecule can be fluorescence, as the derivative of a rhodamine or flourescein.Reporter's molecule can be haptens, as digoxin, or is attached to other molecule as vitamin H, the molecule of the oligosaccharides combination of the Sugar receptors of avidin and avidin streptavidin or combination, this molecule.Reporter's molecule can be that protein or enzyme are as alkaline phosphatase.Reporter's molecule also may comprise radioactive atom as H.sup.3, C.sup.14, P.sup.32, P.sup.33 S.sup.35 I.sup.125 I.sup.131 Tc.sup.99, and other radioelement.

Some compounds are in another preferred embodiment useful crosslinked polynucleic acid and other compound of protein, comprise reporter molecules, protein, lipid, polynucleotide.In a preferred embodiment, what be suitable for that compound of the present invention comprises in following three parts is one or more:

A-alkylation group---the chemical functional of electricity, allows them become the compound of bearing nucleophilic group covalently bound.Alkylating reagent comprises yperite (mustargen and sulfur mustard), triatomic ring (aziridine, oxyethane, cyclopranes, the cyclopropane of activation, episulfide).

B-pad---the connection between alkylation group and reporter molecule-spacer groups, comprise alkane, alkene, ester, ether, glycerol, acid amides, sugar, polysaccharide, and heteroatoms is as oxygen, and sulphur or nitrogen, and/or under the physiological condition of molecular cleavage, as a disulphide bridges or the responsive group of enzyme.Distance piece can with clean positive charge, or following any one: minor groove binding, major groove bonding agent, insertion group, or the protein that combines of other DNA or peptide, and as transcription factor, thus the compound provided has the Polynucleotide increasing avidity.

C-reaction group---the function of a kind of chemical substance of further chemical reaction can be carried out.Reactive group includes, but are not limited to alkylation group, amine, alcohol; sulfydryl, lsothiocyanates, isocyanic ester, acyl azide; N-hydroxysuccinimides, sufonyl chlorine, aldehyde, epoxide; carbonic ether, imido-ester, carboxylate salt, alkylposphates arylhalides(is as difluorodinitrobenzene); iodoacetamides, maleimide, the acryl muriate flourobenzes of aziridine, disulphide; succinic diamide, carboxylic acid, the hydroxy-acid group of activation.

In another preferred embodiment, for identifying the method using the Polynucleotide being applicable to using the one or more compounds that the present invention includes in following three parts:

A-alkylation group---the chemical functional of electricity, allows them become the compound of bearing nucleophilic group covalently bound.Alkylating reagent comprises yperite (mustargen and sulfur mustard), triatomic ring (aziridine, is also referred to as epoxide, cyclopranes, activates cyclopropane, oxyethane and episulfide).

B-pad---the connection between alkylation group and reporter molecule-spacer groups, comprise alkane, alkene, ester, ether, glycerol, acid amides, sugar, polysaccharide, and heteroatoms is as oxygen, and sulphur or nitrogen, and/or under the physiological condition of molecular cleavage, as a disulphide bridges or the responsive group of enzyme.Distance piece can with clean positive charge, or following any one: minor groove binding, major groove bonding agent, insertion group, or the protein that combines of other DNA or peptide, and as transcription factor, thus the compound provided has the Polynucleotide increasing avidity.

C-reporter's molecule-reporter molecules---the additional chemical part being detected as the compound of object.Reporter's molecule can be fluorescence, as the derivative of a rhodamine or flourescein.Reporter's molecule can be haptens, as digoxin, or is attached to other molecule as vitamin H, the molecule of the oligosaccharides combination of the Sugar receptors of avidin and avidin streptavidin or combination, this molecule.Reporter's molecule can be that protein or enzyme are as alkaline phosphatase.Reporter's molecule also may comprise radioactive atom as H.sup.3, C.sup.14, P.sup.32, P.sup.33 S.sup.35 I.sup.125 I.sup.113 Tc.sup.99, and other radioelement.

Any one in a large amount of nucleotide sequences, also can adopt according to the present invention for detecting target molecule as probe.Comprise, such as, at the target sequence of RNA and DNA, and various virus, viroid, fungi, parasite or bacteriological infection, the feature of the polynucleotide sequence of the sequence of heredopathia or other desirable detection target molecules.Probe can be synthesis, semisynthetic or natural origin.Probe molecule, comprises polyribonucleotide and deoxidation.

Although about 30 of the nucleic acid used in the preferred embodiment of the invention bases or longer times, also shorter nucleic acid can be used, as long as can particularly, the target sequence of stably hybridizing.The nucleic acid hybridization that can be designed, no matter the DNA double chain of justice or antisense strand.But when target is messenger RNA(mRNA), the sequence of probe should be supplementing of it.

Claims (7)

1. a method for mono-substituted alkylated compound, comprising: a) forming a covalent linkage has dismountable labelled reagent, is selected from mustard, and aziridine, for alkylating nucleic acid;

B) covalent linkage there is the mixture of dismountable labelled reagent, the nucleic acid wherein comprised, is characterized in that, under the condition of described labelled reagent, there is nonspecific sequence and maximum one hour of the time of nucleic acid within for some time, thus form the reactivity of covalent linkage.

2. method according to claim 1, wherein, described covalent linkage has dismountable labelled reagent to comprise the alkylated compound with reporter molecule.

3. method according to claim 2, is characterized in that, described alkylating agent is selected from the group of mustard and aziridine composition.

4. method according to claim 3, is characterized in that, mustard is selected from mustargen and sulfur mustard.

5. method according to claim 2, is characterized in that, described reporter molecule comprises from fluorescence radiation molecule, containing hapten molecule, and protein, the molecule selected in the group of Radiochemicals composition.

6. method according to claim 2, is characterized in that, described reporter molecule to be connected to alkylating compound and spacerarm.

7. method according to claim 7, is characterized in that, connecting arm is positively charged ion.