JPH0359914B2 - - Google Patents

Description

Claim 1 Formula: [wherein R' is hydrogen or hydroxy, B is a pyrimidine or purine base, and R is already bound to at least one reporter group or solid support that functions as a colored, fluorescent, luminescent or radioactive group or a ligand recognition group. or a linker arm capable of joining them] substantially pure consisting essentially of a preselected sequence of not more than 200 nucleotides having at least one nucleotide residue in a preselected position. single-stranded oligonucleotide. 2. The substantially pure single-stranded oligonucleotide of claim 1, wherein the linker arm is attached to a sterically acceptable site. 3 In the formula, B is a pyrimidine base, and its C5
2. The substantially pure single-stranded oligonucleotide according to claim 1, wherein said linker arm is attached to said position. 4. The substantially pure single-stranded oligonucleotide according to claim 1, wherein B is a purine base, and the linker arm is bonded to the C8 position. 5 The length of the preselected sequence is about 10 to about 40
A substantially pure single-stranded oligonucleotide according to claim 1, which is a nucleotide. 6 Formula: [wherein R' is hydrogen or hydroxy, B is a pyrimidine or purine base, and R is already associated with at least one reporter group or solid support that functions as a colored, fluorescent, luminescent or radioactive group or a ligand recognition group; a linker arm which is attached or capable of being attached to them] consisting essentially of a preselected sequence of not more than 200 nucleotides, having at least one nucleotide residue in a preselected position. A method for the chemical synthesis of pure single-stranded oligonucleotides having a preselected sequence,
consisting of the stepwise addition of selected reactive nucleotides to a substantially pure single-stranded oligonucleotide of less than 200 nucleotides, wherein the nucleotide or oligonucleotide has a reactive nucleotide attached to a 3' or 5' hydroxy group. containing a phosphorus-containing group, at least one of the reactive nucleotides having the formula: [wherein R', B and R have the same meanings as defined above] A method characterized in that the nucleotide is a nucleotide capable of forming a nucleotide residue represented by: TECHNICAL FIELD The present invention relates to the use of cell-based or cell-free systems.
A single-stranded oligonucleotide useful for immobilizing, positioning, and detecting complementary nucleic acid sequences of interest, the bases of which have substituents attached thereto that can function as one or more detectable reporter groups. single-stranded oligonucleotides of defined sequence, up to 200 base units in length, containing one or more nucleotide units, or having one or more detectable reporter groups attached thereto; Regarding. BACKGROUND OF THE INVENTION Nick translation method (P. Rigby et al., J.
Mol. Biol. 113:237-251, 1977) or gap-filling reactions (G. Bourguignon et al., J. Virol. 20: 290).
Methods for the enzymatic production of labeled double-stranded deoxypolynucleotides have been established using prior art techniques involving the insertion of radioactive isotopes into double-stranded DNA by the authors (B.-306, 1976). in particular,
A cut is made with DNase, and DNA polymerase is used to translate along the DNA strand. In this nitrogen translation process, the
DNA polymerase (Pol I) produces double strands in the presence of added deoxynucleoside triphosphates.
Nucleotides are condensed toward the 3' hydroxyl end of the single-stranded DNA break region. At the same time, the enzyme removes nucleotides from the 5' end of the break. If one or more of the triphosphates added are labeled, for example with ^32P phosphate, Pol
This label is introduced into the new strand by I. According to the gap-filling method, the concave end after cutting with a restriction enzyme is inserted into the Klenow fragment from Pol I or the Klenow fragment from Pol I.
It can be filled in using _T4 DNA polymerase. Both the nick translation and gap filling methods are used to
DNA is obtained. The length of this product is DNase
It depends on how much I is added to the reaction. Typically, 30% of the label is introduced and the chain length is 400-800 nucleotide units. The length of the product is
There is unpredictable heterogeneity within the 400-800 unit range. To conserve the enzyme as well as the labeled nucleotides, typically only 1 microgram of DNA is labeled in each reaction vessel. Double-stranded polynucleotides having a pyrimidine base modified with a carbon chain at the C-5 position have been enzymatically produced in a similar manner. This is based on J. Sagi et al. (Biochem. Biosphys. Acta. 606: 196-201, 1980
enzymatic extension of homopolymer primer templates as reported by P. Langer et al. (Proc. Nat. Acad. Sci. USA 78:6633-6637,
This was done by nick translation/gap filling with DNA polymerase using 2'-deoxyuridine 5'-triphosphate covalently linked to biotin as reported by (1981). This biotin can serve as a recognition site for avidin. Enzymatic methods described by Rigby et al., Bourguignon et al., and Langer et al. yield products with similar physical properties. The polynucleotide produced by this enzymatic method has a length of
400-800 units, requires double-stranded polynucleotides as starting material, produces double-stranded polynucleotides in all cases, and cannot label preselected sites. Furthermore, all enzymatic methods modify both strands of the polynucleotide and the resulting strands cannot be separated from each other. According to this method, the enzyme replaces all units with modified units or, if a mixture of modified and natural nucleoside triphosphates is added,
Insert modified units randomly. Furthermore, the enzymatic method of Langer et al. cannot produce polynucleotides into which fluorescent, luminescent, or antigenic reporter groups have been inserted. Using none of these techniques is it possible to produce oligonucleotides of defined length, defined sequence or single-stranded character with or without reporter groups. Furthermore, this prior art method also does not allow insertion of a modified base with a reporter group attached at a predetermined position in a polynucleotide. Double-stranded polynucleotides incorporating a modified adenine base at the C-8 position have also been produced by enzymatic methods. This is based on C. Vincent et al. (Nucl. Acids
Res. 10:6787-6796, 1982) by inserting 8-aminohexylamino-ATP (ribonucleotide) into the DNA fragment. However, this method is limited in scope and can only label the 3'-end with the triphosphate of the adenine ribonucleotide. Modified pyrimidine nucleosides cannot be inserted. Furthermore, like other enzymatic methods, 2
Both strands of a full-stranded polynucleotide are labeled, making it impossible to produce short (<100 units) oligonucleotides of defined sequence. The prior art enzymatic method described above requires chemical synthesis of the substituted nucleotide 5'-triphosphate, followed by enzymatic recognition and conversion of this non-natural substrate into nicked double-stranded DNA. Insertion is required. Such methods cannot produce polynucleotides of a preselected length or sequence, and the polymerases and
DNase is expensive. Furthermore, these methods are time-consuming, inefficient, require high enzymatic activity, and are limited to double-stranded DNA. Since only small amounts, ie a few micrograms, of unobtrusive polynucleotides, usually restriction fragments, are produced, they must be separated from their natural sources by complicated methods. Furthermore, the inability of DNA polymerases to recognize or insert potentially useful reporter groups such as fluorescent or dinitrophenyl severely limits the range of modifications that can be achieved in this polynucleotide product. For limited biological applications, a method for attaching a single fluorescent molecule to the 3' end of a long polyribonucleotide molecule (RNA) has been disclosed by JGJ Bauman et al. (J. Histochem.
Cytochem.29:238, 1981). This method also uses very small amounts (microgram quantities) of RNA, which is cumbersomely isolated from natural sources using enzymatic methods, and requires 2' and 3' hydroxyl groups. Therefore, it cannot be applied to DNA. Additionally, the chemically much greater instability of RNA compared to DNA limits the range of applications for polyribonucleotides produced by this method. A non-enzymatic method for the synthesis of defined sequences of oligonucleotides with natural nucleobases is available from SANarang.
(Meth.Enzymol 65:610, 1980), R.
Letsinger (J.Org.Chem・45:2715, 1980),
M. Matteucci et al. (J. Amer. Chem. Soc. 103:3185,
(1982) and G. Alvarado-Urbina et al. (Science 214:270, 1981),
or summarized. Such synthetic methods typically effect chain elongation by coupling activated nucleotide monomers with free hydroxyl-containing terminal units of the growing nucleotide chain. This coupling is accomplished via a phosphorus-containing group, similar to the phosphate triester method or one of the phosphite triester methods summarized by Narang et al. Of the latter, the methods of Letsinger et al. and Alvarado-Urbina et al. use phosphochloridite chemistry;
The method of Matt-eucci et al. uses phosphoramidite chemistry. In the chemical synthesis of oligonucleotides described above, only unmodified, i.e., natural, nucleobases are inserted, so the desired product is in short fragments and unmodified, i.e., natural, nucleobases.
Similar to RAN or DNA. It should be noted that the target product of such a synthesis does not insert either a label or a reporter group. Direct modification of homopolymeric polynucleotides has been reported with polyuridylic acid systems (Bigge et al.
et al., J. Carb., Nucleosides, Nucleotides 8:259,
(1981). However, the reported methods are limited in scope and cannot produce useful products. The treatment method causes significant polynucleotide cleavage and destruction, resulting in a product contaminated with metal ions that cannot be removed, and the method can only modify cytosine residues of DNA polynucleotides. . Furthermore, this method cannot produce oligonucleotides of specific length and defined sequence, cannot modify thymine or purine bases, and cannot modify preselected sites. As is clear from the above description, conventional polynucleotides with fixed sequences have the disadvantages mentioned above.
It has been produced only by enzymatic methods, which have drawbacks, particularly regarding time, cost, product length, sequence, and yield. Furthermore, such methods can only produce double-stranded products. Double-stranded polynucleotides can be denatured in solution with alkali or heat, allowing the strands to spontaneously separate for a short period of time. However, the individual single strands cannot be physically separated from each other, and once the denaturing conditions are removed, they rapidly revert to their natural double-stranded form. The conditions for hybridization are also the conditions for spontaneous reversion, so when a denatured polynucleotide is placed under hybridization conditions, it returns to its original double-stranded configuration. . Hybridization of either strand with the target polynucleotide is therefore limited by competition with the other strand. Modifications of nucleosides can include, for example, antiviral C
-Synthesis of 5-substituted pyrimidine nucleosides (Bergstrom et al., J.Amer.Chem.Soc.98:1587-
1589, 1976; Ruth et al., J.Org.Chem.43:2870−
2876, 1978; and Bergstrom et al., U.S. Pat.
4247544 and 4267171) or the synthesis of C-8 substituted adenine derivatives (Zappelli et al., U.S. Pat.
4336188 and 4199498). These nucleosides and those reported by Langer et al. and D. Ward (European Patent Application No. 0063879) are not useful in the method of the present invention. Such reported nucleosides have high reactivity at undesired sites, and their use in the synthesis of oligonucleotides by chemical methods may result in undesirable by-products or uncontrolled synthesis. The desired product may not be obtained. Furthermore, the above nucleosides do not have suitable sites in their substituents, cannot be modified by attaching reporter groups, and do not have masked reactive functional groups. None of these nucleosides are useful in the method of the invention. There is no prior art describing the chemical synthesis of oligonucleotides of defined sequence into which some modified bases have been introduced, with or without a reporter group. The rapid emergence of high-quality labeled single-stranded oligonucleotides of a certain sequence that does not contain dangerous and unstable radioactive isotopes is awaited, and meeting this need is the main objective of the present invention. It is. This objective has been achieved by chemical, ie non-enzymatic, methods which give predictable, superior product yields. More specifically, the method of the present invention includes:
The chemical incorporation of nucleotides modified with a wide variety of selected detectable reporter groups into oligonucleotides of defined sequence has been achieved, and such oligonucleotides have e.g. Useful for identifying, locating, separating and/or quantifying complementary sequences. Another object of the invention is to provide novel nucleotides useful for the chemical synthesis of labeled, single-stranded oligonucleotides of defined sequence. The method of the present invention is a novel chemical synthesis method for labeled oligonucleotides of a defined sequence,
It is superior to prior art enzymatic methods in various aspects.
More specifically, double-stranded,
Unlike the heterogeneous and unpredictable production of 400 to 10,000 base units, the method of the present invention preferably produces 200
It allows the synthesis of subbase unit, homogeneous, predictably defined length, labeled, single stranded oligonucleotides of defined sequence. The product yields produced by the process of the invention are on the order of hundreds to tens of thousands of micrograms, as opposed to the 2-3 microgram yields obtained by prior art enzymatic methods. Furthermore, the oligonucleotide obtained by the method of the present invention is single-stranded, unlike the double-stranded oligonucleotide obtained by the enzymatic method. The single-stranded form of the oligonucleotide product avoids competition from complementary strands associated with rehybridization of double-stranded polynucleotides. DESCRIPTION OF THE INVENTION The present invention provides single-stranded oligonucleotides of defined sequence less than about 200 base units in length that can function as one or more detectable reporter groups or alternatively ,1
Substituents capable of binding the species or more detectable reporter groups are attached to a sterically tolerable position of the base (e.g. C-5 of a pyrimidine, C-8 of a purine). Single-stranded oligonucleotides containing at least one nucleotide unit are provided. More specifically, the present invention provides the formula [wherein R' is hydrogen or hydroxy, B is a pyrimidine or purine base, and R is already bound to at least one reporter group or solid support that functions as a colored, fluorescent, luminescent or radioactive group or a ligand recognition group. or a linker arm capable of joining them] substantially pure consisting essentially of a preselected sequence of not more than 200 nucleotides having at least one nucleotide residue in a preselected position. It concerns single-stranded oligonucleotides. The present invention also provides a method for chemically, i.e., non-enzymatically, synthesizing single-stranded oligonucleotides of defined sequence, the method comprising:
It consists of coupling an activated nucleotide monomer with a free hydroxyl terminal unit of a growing nucleotide chain, wherein at least one of the monomer and the terminal unit has a sterically tolerable base. A certain site is modified by attachment of a substituent that can function as one or more detectable reporter groups or bind to one or more detectable reporter groups. It is characterized by: The oligonucleotide produced by the method of the present invention contains one or more pyrimidine-based or purine-based units, and the unit may be a ribonucleotide or a deoxyribonucleotide. The reporter group may also be preselected prior to its synthesis, as well as the specific nucleotide unit to which it will bind. MOST PREFERRED EMBODIMENTS FOR CARRYING OUT THE INVENTION A preferred chemical method for synthesizing the defined sequence single-stranded oligonucleotides of the present invention is
The activated nucleotide monomer and at least one of the free hydroxyl terminal units of the growing nucleotide chain are detectable at one or more sterically tolerant sites of the base. can function as a reporter group,
Alternatively, it consists of coupling the terminal unit with the nucleotide monomer characterized in that it has been modified by attachment of a substituent capable of binding to one or more detectable reporter groups. It can be said that the substituent in the present invention that can bind to a reporter group generally exhibits nucleophilic properties. Examples of such substituents include those containing primary amines, aromatic amines, carboxylic acids, hydroxyl groups, and the like. The nucleotide monomers and bases of the terminal units are selected so as to obtain the desired predetermined sequence of nucleotide units in the desired product oligonucleotide. Such bases may take the form of the purines adenine (A), guanine (G), or hypoxanthine (H), or the pyrimidines uracil (U), cytosine (C), or thymine (T). ) can take the form. Such bases may also take the form of other bases that can be isolated from nature. A sterically tolerated site on a nucleotide unit is a position on the nucleobase of that unit that
The unit is modified by bonding a substituent at that position, and in this case, hybridization between the product oligonucleotide and the complementary nucleic acid component is not significantly hindered. , and can be defined as a position where the substituent is not sterically hindered from functioning as one or more reporter groups or from binding to one or more reporter groups. The sterically tolerated sites are the C-8 position of purines and the C-5 position of pyrimidines. Since the oligonucleotides of the present invention are particularly useful as probes for hybridization,
Modifications to attach substituents and/or reporter groups must not be made at sites on the pyrimidine or purine bases required for specific hybridization. Sites that should not be modified include adenine bases N ¹ and N ⁶ , guanine bases N ¹ , N ² and O ⁶ , and cytosine bases N ³ and
Includes ^N4 etc. Generally, a heteroatom (N
or O) should be avoided. Reporter groups may be aromatic and/or polycyclic groups having physical or chemical properties that are readily measurable or detectable by suitable physical or chemical detection systems or methods. can be defined as a chemical group. Reporter groups useful in oligonucleotides in the present invention can be easily detected. Easy detectability is provided by properties such as color change, luminescence, fluorescence or radioactivity. Alternatively, detectability is also provided by the ability of the reporter group to function as a ligand recognition site. Such groups are functionally referred to as colored, luminescent, fluorescent, radioactive or ligand recognition groups. Among such groups are those which are suitable for detection by conventional detection techniques, such as colorimetric, spectrophotometric, fluorescent or radioactive detection, or which do not require such conventional detection. These include those that may be involved in the formation of specific ligand-ligand complexes containing groups that can be detected by the method. As used herein, the reporter group is
Defined as a substituent that has physical, chemical, or other properties that can be readily measured or detected using appropriate measurement or detection methods. A reporter group as defined herein includes a reaction product or complex that ultimately has a physical, chemical or other property that can be easily measured or detected using an appropriate assay or detection method. Substituents capable of initiating or further interactions are also included. Examples of measurable or detectable properties possessed or caused by such groups, reaction products or complexes are color change, luminescence, fluorescence or radioactivity. Such properties can be measured or detected using conventional colorimeter, spectrophotometer, fluorometer or radioactivity sensitive meter. Interactions that can be initiated by reporter groups as defined herein include producing groups or complexes that can be easily detected, e.g. by colorimetric, spectrophotometric, fluorescent or radioactive detection methods. Appropriately specific and selective ligand-ligand interactions are involved. Such interactions may take the form of protein-ligand, enzyme-substrate, antigen-antibody, carbohydrate-lectin, protein-cofactor, protein-agonist, nucleic acid-nucleic acid or nucleic acid-ligand interactions, etc. . Examples of this ligand-ligand interaction include dinitrophenyl-dinitrophenyl antibody, biotin-avidin, oligonucleotide-complementary oligonucleotide, DNA-DNA, RNA-DNA, and
Examples include NADH-dehydrogenase.
Other useful interactions will occur to those skilled in the art. In the method of the present invention, the selected reporter group(s) may be attached to the nucleotide monomer before coupling to the terminal unit of the nucleotide chain, but it may also be attached after the oligonucleotide product is completed. can. The sequence of nucleotide units in the oligonucleotide product is preselected to yield an oligonucleotide with the correct specificity for its ultimate use. The oligonucleotides of the invention are useful in carrying out other projects involving recombinant DNA and nucleic acid rehybridization techniques. Such uses include identifying, positioning, separating and/or quantifying complementary sequences of interest in cell or cell-free systems. More specifically, such applications include all basic biological manipulations or diagnostic applications that involve hybridization of nucleic acid components or by modification of sterically tolerable sites. Purification (with or without subsequent detection) of complementary sequences by affinity chromatography is included when the oligonucleotide product is bound to a solid support. The nucleotide units in the oligonucleotide product are purine or pyrimidine based and may include units with natural bases mixed with units with modified bases.
Such units are ribonucleotides or deoxyribonucleotides. Preferably, the coupling step couples the monomer unit activated at the 3' position with the free 5' hydroxy of the terminal unit of the growing nucleotide chain. Alternatively, a monomer unit activated at the 5' position can be coupled with the free 3' hydroxy of the terminal unit of the nucleotide chain. This terminal unit is either the first or only unit of a growing nucleotide chain when the nucleotide monomers are coupled together, or one of the terminals of a group of nucleotide units. The method of the present invention produces oligonucleotides with a defined sequence represented by the following general formula. In the formula, n is 1 to about 199, preferably about 5 to about 60,
Most preferably from about 10 to about 40, R' is hydrogen or hydroxy and B is a naturally occurring purine or pyrimidine base such as adenine, guanine, cytosine, uracil, thymine or any other naturally occurring base; Each nucleotide unit with a base present is intermixed with one or more nucleotide units each with a modified base (B ^m ). Modified pyrimidine bases (Py ^m ) are those substituted at the C-5 position, typical examples of which are the uracil and cytosine bases of the following general formula: Modified purine bases ( ^Pum ) are substituted at the C-8 position, representative examples of which are modified adenine and guanine bases of the following general formula: The substituent R has the characteristic that it can function as one or more reporter groups or can bind to one or more reporter groups. In modified pyrimidine bases, the substituent R contains two or more carbon atoms, whereas in modified purine bases, R
contains one or more carbon atoms. In this regard, R preferably takes the _form of one of _the following functionalized carbon chains: R=-CH= _CR1R2 , _{-CH2CHR1R2 ,} -
_{CHR1R2 ,}

[expression] or

[Formula] In the above formula, R ₁ is hydrogen or alkyl, R ₂ is alkyl, alkenyl, aryl or a functionalized alkyl, alkenyl, aryl (where the functional group includes one or more amines, amides, nitriles, carboxylic acids, carboxylic esters, hydroxy, dinitrophenyl, aminobenzenesulfonates, etc.), Z is a polyvalent heteroatom such as nitrogen, oxygen or sulfur. Furthermore, R ₂ may be bound to a solid support or to one or more reporter groups that function as, for example, colored, fluorescent, luminescent, radioactive or ligand recognition groups. Functional fluorescent groups include fluorescein,
rhodamine, etc., and adducts thereof. Functional luminescent groups include luminol, acridine, luciferin, dioxetane, dioxamide, etc., and adducts thereof. Ligand recognition groups include vitamins (e.g. biotin, iminobiotin or desthiobiotin) that can be recognized by or can induce ligand-like interactions with proteins;
Antigens such as dinitrophenol, carbohydrates and other functional groups or adducts of such groups are included. Another oligonucleotide that can interact with nucleic acids is an example of a group that can induce ligand-like interactions. Ligand recognition groups also serve as functional colored reporter groups when recognition results in coloration. For example, when dinitrophenyl is used as a reporter group, a known detection system using an anti-dinitrophenyl antibody coupled to peroxidase that causes a color change can be used as the detection system. A functional radioactive group includes a radioactive element in a selected reporter group. In this specification, when the expression purine or pyrimidine base is used, analogs of such bases are also included. Such analogs include purine base analogs such as deazaadenosine (tubercidin, formicin, etc.), pyrimidine base analogs such as deazauracil, deazacytosine, azauracil, azacytosine, etc. Oligonucleotides of the formula are best prepared by chemical synthesis from monomeric nucleotide analog units of the formula: where _{R 6} = methyl, chlorophenyl; , B
and R′ are masked if appropriate;
is a phosphorus-containing group suitable for internucleotide bond formation during chain elongation in the synthesis of oligonucleotide products. Preferred phosphorus-containing groups suitable for internucleotide bond formation are alkylphosphomonochloridites or alkylphosphomonoamidites. Alternatively, phosphate triesters may be used for this purpose. The monomer may also have R ₃ attached to the 3'-hydroxy and R 3 attached to the 5'-hydroxy. In general, the term "masking group" or "blocking group" refers to a group that changes the chemical reactivity of an intact functional group and prevents undesired reactions from occurring.
It is a functional expression of chemically modifying or "blocking" a functional group by attaching a second moiety to that functional group. Such modification is reversible and can be converted to the original functional group by appropriate treatment. In many cases, such masking is an interconversion of structure to functionality in form. For example, a primary amine can be masked by acetylation to a substituted amide, which can then be converted back to the primary amine by appropriate hydrolysis. The compounds of formula also include their acceptable conjugate salts. Conjugate acids that can be used to prepare such salts are those containing non-reactive cations, such as nitrogen-containing bases such as ammonium salts, mono-,
such as g-, tri- or tetra-substituted amine salts,
Alternatively, it is a suitable metal salt, such as a sodium or potassium salt. The manufacturing process of the present invention will be outlined below and illustrated schematically. After that, the present invention is illustrated more specifically,
Give a detailed explanation. Since the present invention relates to an oligonucleotide containing both a pyrimidine-based nucleotide unit and a purine-based nucleotide unit, examples are given of oligonucleotides that use both pyrimidine- and purine-based compounds in the synthesis process. do. The individual pyrimidine and purine-based compounds exemplified are only examples of the pyrimidine and purine groups, respectively, and they may be incorporated into the respective groups in their manufacturing processes and oligonucleotide products, if appropriate or desired. It should be understood that others may be substituted. Although for the most part deoxyribonucleotides are shown, it should be understood that the invention also contemplates ribonucleotides, and if ribonucleotide compounds are required in the oligonucleotide product. , deoxyribonucleotide compounds can be replaced by ribonucleotide compounds. A more important point of the present invention is to provide a new group of nucleotides that are essential as intermediates in a new method of synthesizing oligonucleotides. All such nucleotides have a base modified by a functionalized carbon chain and one or more amide substituents, the amide nitrogen being a sterically tolerable base. In the part where you get it,
Bonded through carbon chains. In the case of pyrimidine-based nucleosides, the carbon chain is attached at the C-5 position; in the case of purine-based nucleosides, the carbon chain is linked via a polyvalent heteroatom, e.g. nitrogen, oxygen or sulfur. It is bonded at the C-8 position. Additionally, such nucleosides are chemically blocked at the 5' (or 3') position with a group such as dimethoxytrityl, which is suitable for chemical synthesis of oligonucleotides. In this new group of nucleosides, the substituents are -CH ₂ CHR ₁ C _o H _2o Y, -CH=CR ₁ C _o H _2o Y,

[expression] or

Selected from [formula]. Here, R ₁ is hydrogen or C _1-6 lower alkyl, and Y is one or more amide, substituted amide, substituted amino or substituted aminoalkylphenyl group. More specifically, Y is one or more

[Formula] (X is hydrogen, fluorine or chlorine) may be included. Synthesis of these nucleotides and their masked versions is described in Examples,
, , , , , XII and. Preferred nucleosides include a substituent at C-5 of the pyrimidine nucleoside.

[Formula] (n=3-12, Y teeth

[Formula]). Most preferred are nucleosides where the pyrimidine base is uracil. The method of the invention can begin with the production of a selected nucleoside. Generally, the most preferred nucleosides are best prepared as follows. Bergs-trom and Ruth's method [J. Amer.
Chem.Soc.96:1587 (1976)], 5-(methyl-3-acrylyl)-
Produce 2'-deoxyuridine. The nucleoside is then treated with 1.05 equivalents of dimethoxytrityl chloride in pyridine to protect the 5' hydroxy with dimethoxytrityl (DMT). 0 in chloroform containing 2% triethylamine
The product obtained is purified by chromatography on silica, eluting with a gradient of 10% methanol. Purified 5'-DMT-5-(methyl 3-acrylyl)-2'-deoxyuridine is treated with 1N KOH at ambient temperature for 24 hours to hydrolyze the methyl ester. The obtained 5′-DMT-5-(3
-acrylyl)-2'-deoxyuridine is treated with excess dicyclohexylcarbodiimide and hydroxybenztriazole in pyridine. After 4 hours, a 2-5 fold excess of 1,7-diaminoheptane is added and the reaction is stirred overnight. 12～
After 20 hours, a 10-20 fold excess of trifluoroacetic anhydride is added and the reaction is stirred at room temperature for 4 hours. 2%
The product was purified by chromatography on silica eluting with a gradient of 0-10% methanol in chloroform containing 1% triethylamine, followed by exclusion chromatography using a Sephadex LH-20 eluting with methanol containing 1% triethylamine. refine. Collecting the appropriate fractions yields 5′DMT-5-[N-(7-trifluoroacetylaminoheptyl)-1-acrylamide].
-2'-deoxyuridine is obtained. This product is suitable for oligonucleotide synthesis by the phosphochloridite method as described in Examples and . Alternatively, this compound can be prepared by combining the methods described in the Examples and Sections. Diaminoheptane in this method can be used with other diaminoalkanes (e.g. diaminopropane, diaminopropane,
diaminohexane, diaminododecane), n is 3, 6 or 12, and R is

Other compounds with substituent lengths of the formula are obtained. Two such nucleosides, one based on pyrimidine (uracil) and the other on purine (adenine)
is shown at the beginning of the scheme below illustrating this method. The reactive site on the base of the nucleoside is then masked, for example by attaching a benzoyl group (Bz) to the 6-position amine of the adenine-based nucleoside, as shown in Reaction 1. For information on this type of masking, please refer to "Synthetic Procedures in Nucleic Acid".
"Chemistry", Volume 1, W. Zorbach and R.
Tipson (ed.), Wiley-Int-erscience, NY,
Generally described in 1968. As shown in reaction 1, for example, the unprotected amine on the substituent is masked by attaching a trifluoroacetyl group (Ac). Selected 3' or 5' hydroxys of the nucleoside are then masked by attaching dimethoxytrityl (DMT) groups. In reaction 2 shown later, 5'-hydroxy is masked,
The 3' hydroxy is left free, ie, available for reaction. Alternatively, mask the 3′ hydroxy,
The 5' hydroxy may be left free. This nucleoside is then preferably
The 3' hydroxy is converted to an activated nucleotide monomer by attaching a phosphorus-containing group containing an activating moiety. If you want to block this modified nucleoside appropriately,
Modifications of the methods described by Letsinger et al., Matteucci et al., or summarized by Narang can be used for oligonucleotide synthesis. A method using phosphochloridite chemistry as described by Letsinger et al. is detailed in the Examples. To use phosphoramidite chemistry, similar to the modified method of Dorper et al. [Nucleic Acids Res. 11:2575 (1983)],
The protected, modified nucleoside is phosphitylated with methylchloro(N,N-diisopropyl)phosphoramidate or methylchloro-phosphomorpholinate using a modification of the method of Matteucci et al. Alternatively, the protected, modified nucleoside can be phosphorylated with 1.2 equivalents of chlorophenyl dichlorophosphate in trimethyl phosphate at room temperature and then placed in water to form the 3'-chlorophenyl phosphate adduct of the modified nucleoside. Obtainable. Such adducts are useful in modifying the phosphotriester approach, as exemplarily summarized by Narang et al. The scheme for Reaction 3 shows the synthesis of an activated monomeric nucleotide unit of the formula by attaching a phosphomonochloridite group to the 3' hydroxy of the nucleoside, with chlorine serving as the activating moiety. Coupling or condensation of a selected activated nucleoside monomer, i.e., a uracil-based monomer or an adenine-based monomer, to the terminal unit of a growing nucleotide chain.
This is shown in reaction 4 of the scheme. The nucleotide chain is
At its right-hand end, a naturally occurring group and its
It is depicted as containing a masking group R ₄ attached to the 3' hydroxy or a nucleoside unit with a solid support. The illustrated chain also includes one or more (n') nucleotide units with a naturally occurring base attached to the 5' hydroxy of the nucleoside unit and at the terminal end of the nucleotide unit. One has a free hydroxyl at the 5' position. In this coupling reaction, the chlorine of the monomer is removed by reacting with the water of the free hydroxyl of the terminal unit, and the oxygen of the terminal unit is coupled to the phosphorus of the monomer as shown in the figure, and this monomer is attached to the nucleotide chain. becomes a new terminal unit. The DMT 5' blocking group is then removed and further activated nucleotide monomer units are coupled in sequence to allow elongation of the nucleotide chain. The nucleotide units added to the chain can be preselected and are either naturally occurring bases or modified bases. Further chain extension by adding one or more (n″) nucleotide units with naturally occurring bases is shown in diagrammatic reaction 4a. Synthesis of oligonucleotides of selected length and sequence Once done, the DMT group is removed from its terminal unit and the masked reactive groups are unmasked. Examples of modified uracil and adenine bases with unmasked reactive groups are shown schematically in Reaction 5. If the first nucleotide unit is attached to a solid support _R4 , the chain is detached from the solid support. The appropriate order of unmasking can be preselected. If the nucleotide product cannot function as a reporter group in the intended use, a suitable reporter group R ₅ can be attached to such a substituent, as exemplified in Reaction 6. ,
Each base is shown with a reporter group attached to each substituent of the base. In the above formula, for example, B ^m in the oligonucleotide product is: A variety of useful reporter groups (R ₅ ) can be attached to the oligonucleotide product. for example: Oligonucleotide Products with Reporter Groups (R ₅ =Biotin or Fluorescein) Having generally described and illustrated the process of the invention, each of the described reactions will be described in more detail below. For reaction 1, N ⁴ of cytosine, adenine
Masking of chemically reactive amines such as N ⁶ , N ² of guanine, and alkyl or aryl amines of modified bases with suitable masking groups can be applied to alcohols, pyridines, lutidines, chloroform, etc. The nucleoside is mixed with an excess amount of the appropriate acid anhydride in a suitable solvent such as 0°C and 110°C.
The reaction can be suitably carried out by reacting at a temperature in the range of 20°C to 80°C, usually for about 1 to 24 hours. Suitable acid anhydrides include acetic anhydride, trifluoroacetic anhydride, benzoic anhydride, anisic anhydride, and the like. Preferred are acetic anhydride, trifluoroacetic anhydride, benzoic anhydride and isobutyric anhydride. Masking of the 5'-hydroxy in reaction 2 is achieved by treating the nucleoside with a slight excess of a suitable acid-labile masking agent, such as trityl chloride,
This can be conveniently carried out by reacting with monomethoxytrityl chloride, dimethoxytrityl chloride (DMTCl), trimethoxytrityl chloride, or the like. Preferred is dimethoxytrityl chloride. A typical reaction is
For example, the reaction is carried out in a suitable solvent such as pyridines, lutidines, trialkylamines, etc. at a temperature in the range of -20°C to 120°C, usually 20°C to 100°C, for about 1 to 48 hours. A preferred reaction uses 1.1 equivalents of DMTCl in pyridine and is reacted for about 2 hours at room temperature. It is generally preferred to separate and/or isolate the respective product of each of the above reactions before using it as a starting material for the next reaction. Separation and isolation can be carried out by suitable purification methods such as evaporation, filtration, crystallization, column chromatography, thin layer chromatography, and the like. For specific examples of typical separation and isolation methods, reference may be made to the appropriate Examples below. However, other equivalent separation methods can of course also be employed. It is also noted that although typical reaction conditions (e.g. temperature, molar ratio, reaction time) are given, conditions above or below these ranges may also be used, although usually less suitable. must be understood. Activation to phosphite shown in reaction 3 is as follows:
The nucleoside compound is heated to −90°C in an appropriate solvent.
This can be most suitably carried out by treatment with a suitable phosphitylating agent at a temperature of 60 DEG C. for 1 minute to 2 hours. Suitable phosphitylating agents include methylphosphodichloridite, o-chlorophenylphosphodichloridite, p-chlorophenylphosphodichloridite, methylphospho(dialkylamino)monochloridite, and the like. Suitable solvents include 0-20% of a suitable base (usually 1-20%).
5% by volume), for example, pyridine containing lutidines, collidines, trialkylamines, etc.
Examples include lutidines, acetonitrile, tetrahydrofuran, dioxane, and chloroform. Preferred phosphitylating agents are methylphosphodichloridite, o-chlorophenylphosphodichloridite, and methylphospho(di-isopropylamino)-monochloridite. Preferred phosphitylation conditions are 0.9 equivalents of methylphosphodichloridite in acetonitrile or pyridine containing 5% 2,6-lutidine for 5 to 10 minutes at room temperature or below. An example of chemically inserting modified nucleotide monomers into a growing nucleotide chain to create a defined nucleotide sequence is shown in Reactions 4 and 4a. Typical conditions are in a suitable solvent at -20°C to 50°C, preferably at ambient temperature, for about 1 to 60 minutes. Suitable solvent mixtures include 0-20% of a suitable base (usually 1-5% by volume);
Examples include pyridine, lutidine, acetonitrile, tetrahydrofuran, dioxane, and chloroform containing lutidines, collidines, and trialkylamines. The growing chain may be soluble, insoluble, or attached to a suitable solid support by suitable chemical methods known in the art. Preferably, it is bound to a solid carrier. Furthermore, the growing chain may or may not already contain one or more modified nucleosides. After condensation of the activated monomer onto the growing chain in reaction 4, the initial product is treated with appropriate reagents to oxidize the intermediate phosphite triester and remove any unreacted oligonucleotide. Capping is performed to block the 5'-hydroxy of the 5'-DMT group and remove the 5'-DMT group. Oxidation of the phosphite triester can be carried out by treatment with 0.1 to 5% w/v iodine in a suitable solvent, for example a tetrahydrofuran/water/lutidine mixture. Chemical capping of unreacted 5'-hydroxy can be accomplished by acetylation or acylation using, for example, acetic anhydride and 4-dimethylaminopyridine in a tetrahydrofuran/lutidine mixture. Removal of the 5'-blocking group, usually DMT, is most conveniently carried out by treatment with a mild organic acid in an aprotic solvent, such as chloroformacetic acid or 1-5% by volume dichloroacetic acid or trichloroacetic acid in dichloromethane. I can do it well. DMT
The growing nucleotide chain that has been removed is
It becomes an acceptor for further elongation in the next reaction by the activated monomer, resulting in an oligonucleotide of the desired length and sequence, as shown in reaction 4a. Once the oligonucleotide of the desired sequence has been produced, reaction 5 is performed to obtain the oligonucleotide product. For this purpose, thiophenol treatment removes the methyl masking group from the phosphate triester and appropriate aqueous alkali or ammonia treatment removes the benzoyl from the protected amine.
Acetyl, isobutyl, trifluoroacetyl or other groups are removed and/or the product is desorbed from the solid support. Removal of DMT from the oligonucleotide product is carried out by appropriate treatment with a mild acid such as aqueous acetic acid for 10-60 minutes at ambient temperature to 40°C. Such reactions can be carried out at or before the final purification step. Final purification is carried out by suitable methods, such as polyacrylamide gel electrophoresis, high pressure liquid chromatography (HPLC), reverse phase or anion exchange on DEAE-cellulose, or a combination of these methods. The oligonucleotide synthesis method described here is
Modified deoxyribonucleoside (R′ is H)
Alternatively, modified ribonucleosides (R' is hydroxy) can also be used. When ribonucleosides are used, the 2'-hydroxy is masked by a suitable masking group, such as that provided by a silyl ether. Other ribose forms, including arabinose and 3'-deoxyribose, can also be used in the method of producing the desired oligonucleotides. Substituents that modify nucleotide bases may themselves function as one or more reporter groups. An example thereof is a substituent containing a dinitrophenyl group. If a substituent cannot function as a reporter group, it must be capable of binding one or more reporter groups before or after the chain extension coupling reaction. Selected oligonucleotide products are reacted with appropriate reagents to bind such reporter groups. For example, if a modified base is inserted into an oligonucleotide and the substituent R ₂ contains one or more primary amines, the amine-
Coupling with reactive groups such as isocyanates, isothiocyanates, active carboxylic acid conjugates, expoxides or active aromatic compounds to form amide, urea, thiourea, amine or aromatic amine bonds. For example, as shown in the scheme for Reaction 5, oligonucleotides containing uracil or adenine bases modified with substituents bearing primary amines can be reacted with a suitable reagent such as fluorescein isothiocyanate. , as shown in reaction 6, a reporter group R ₅ (fluorescein) attached to a substituent can be obtained. Other reporter groups that can be similarly attached include a wide variety of organic moieties such as fluoresceins, rhodamines, acridinium salts, dinitrophenyls, benzenesulfonyls, reminols, luciferins, biotins, vitamins, Contains carbohydrates.
Suitable active reporter groups can be commercially available, such as “Bioluminescence and
M. DeLuca and W. McElroy
ed., Acad.Press, New York (1981)], D.
The method of Roswell et al. or H. Schroeder et al. [ Meth .
Enzymol. LX ₁₁ , 1978] and the literature cited therein. Typically, addition of the reporter group involves adding a substituent of the modified base, where _R2 is C _{o H2o NH2} , with an excess of the selected reporter group, preferably in an aqueous solvent, at about -20 °C. ~50℃ (preferably 20
This is conveniently achieved by reacting for 1 to 24 hours at a temperature in the range of 1 to 40°C. Suitable solvents are aqueous buffers and 0-50% organic solvents such as lower alcohols, tetrahydrofuran, dimethylformamide, pyridine, and the like. Preferred reporter group reactants include fluorescein isothiocyanates, dinitrophenyl isothiocyanates, fluorodinitrobenzene, N-hydroxysuccinimidylbiotin,
active esters of carboxyfluorescein, such as N-hydroxysuccinimidyl dinitrobenzoate, isothiocyanates, such as aminobutylethylisoluminol isothiocyanate;
rhodamine, biotin adducts, dioxetanes,
Contains dioxamides, carboxyacridines, carbohydrates, etc. Additionally, if the oligonucleotide product contains a modified base where R includes one or more carboxylic acids, it may be mildly condensed with, for example, primary alkylamines to form an amide linkage. obtain. Typically, this involves combining the oligonucleotide with an excess of a reporter group containing a primary amine, preferably in an aqueous solvent, in the presence of a suitable condensing agent such as a water-soluble carbodiimide.
It is convenient to carry out the reaction at a temperature in the range of about -20°C to 50°C (preferably 20°C to 40°C) for a period of 6 to 72 hours. Anti-preferred reporter groups of this type include (aminoalkyl)-amino-naphthalene-1,2-dicarboxylic acid hydrazides, amino-
These include fluoresceins, aminorhodamins, aminoalkyluminols, aminoalkylaminobenzenesulfonyl adducts, amino sugars, and the like. Additionally, chemical synthesis of the initial oligonucleotide product may be performed using modified nucleotide monomers whose substituents include such reporter groups. If such groups need to be masked, they are appropriately masked during the coupling reaction. On the other hand, some reporter groups do not require masking. For example, an unmasked nitrophenyl adduct can be present on a modified nucleotide monomer during the coupling reaction without adverse effects. Reporter groups used in the inventive method typically include aromatic, polyaromatic, cyclic, and polycyclic organic moieties, which can also include heteroatoms such as nitrogen, oxygen, and sulfur. It is functionalized by containing. The oligonucleotide product can contain one or more types of modifications, or one or more modified bases. An example of this type of oligonucleotide is shown in the following structural formula: In the above formula, C ^m is 5-(3-aminopropyl)cytosine, U ^m is 5-[N-(4-aminobutyl)-1
-acrylamide]uracil, A ^m is 8-[6-
2,4-dinitrophenyl]-aminohexyl]
It is aminoadenine. This product is further modified by reaction with fluorescein isothiocyanate to provide fluorescein reporter groups on C ^m and U ^m . Such oligonucleotide products demonstrate the diversity of choices of modified and unmodified nucleotide units in oligonucleotide products made possible by the method of the present invention. More specifically, such oligonucleotides are exemplified by the use of one or more nucleotide units whose bases have been modified with substituents that provide the same or a different type of reporter group function. ing. Also exemplified are units whose base is modified by a substituent to which a reporter group is attached, namely C ^m and U ^m , while A ^m is a unit whose base is itself modified with a dinitrophenyl group. is an example of a unit modified with a substituent that can function as a reporter group because it contains It is further understood that such an oligonucleotide may contain more than one nucleotide unit of the same type, and that it may contain units with unmodified bases mixed with units with modified bases. It shows. As shown in Reaction 6, instead of attaching the reporter group to the primary amine of the substituent, such an amine or other group may alternatively be attached to a suitably activated solid support. be able to. This results in certain oligonucleotide sequences being covalently linked to such carriers via modified bases. Such solid supports are useful for detecting and isolating complementary nucleic acid components. Alternatively, the modified nucleotide monomers may be coupled to a solid support prior to the chain extension coupling reaction 4, thereby providing a solid support for such monomers during the coupling reaction. Specific examples are provided below to enable those skilled in the art to practice the invention. This example should not be construed as limiting the scope of the invention, but merely as illustrative and representative of the invention. To clarify the structure, reference may be made to the process diagrams described above. EXAMPLE This example describes the synthesis of a modified nucleoside precursor, 5-(3-trifluoroacetylaminopropenyl)-2'-deoxyuridine. 5-chloromercury-2'-deoxyuridine (3.6 g, 7.8 mmol) is suspended in 200 ml of methanol. N-allyl trifluoroacetamide (6.8 ml, 55 mmol) was added followed by 0.2 N lithium tetrachloropalladate in methanol.
Add ml. After stirring at room temperature for 18 hours, the reaction was gravity filtered to remove the black solid palladium, and the yellow methanolic liquid was treated with five 200 mg portions of sodium borohydride and then concentrated in vacuo to a solid. Obtain mold residue. The residue was subjected to flash column chromatography on silica gel.
Purify by eluting with a 15% by volume mixture of methanol in chloroform. Fractions in which the product has been appropriately purified are combined and concentrated under reduced pressure to obtain 2.4 g of crystals of 5-(3-trifluoroacetylaminopropenyl)-2'-deoxyuridine. UVλ _nax 291nm
(ε7800), λ _nio 266nm (ε(4400); TLC (Silica,
Elute with a 15% volume solution of methanol in chloroform)
R _f =0.4. EXAMPLE This example describes the synthesis of the modified nucleoside precursor 5-[N-(trifluoroacetylaminoheptyl)-1-acrylamide]-2'-deoxyuridine. 5-chloromercury-2'-deoxyuridine (3.6 g, 7.8 mmol) is suspended in 200 ml of methanol. Add N-(7-trifluoroacetylaminoheptyl)-acrylamide (55 mmol),
Then 41 ml of 0.2N lithium tetrachloropalladate in methanol are added. After stirring at room temperature for 18 hours, the reaction is filtered by gravity to remove the black solid palladium. Yellow methanolic liquid
treated with 200 mg of sodium borohydride five times,
It is then concentrated under reduced pressure to obtain a solid residue. The residue is purified by flash column chromatography on silica gel, eluting with a 10% by volume mixture of methanol in chloroform. The fractions in which the product has been appropriately purified are combined and concentrated under reduced pressure, and 5-
2.8 g of crystals of [N-(7-trifluoroacetylaminoheptyl)-1-acrylamide]-2'-deoxyuridine are obtained. UVλ _nax 302nm (ε18000),
λ _nio 230 nm, 280 nm; TLC (silica, eluted with 15% methanol by volume in chloroform) R _f =
0.3. EXAMPLE This example describes the masking of 5'-hydroxy for the preparation of 5'-dimethoxytrityl-5-(3-trifluoroacetylaminopropenyl)-2'-deoxyuridine as shown in the reaction. do. 5-(3-trifluoroacetylaminopropenyl)-2'-deoxyuridine (2.4 g) twice;
Completely evaporate from pyridine, then pyridine 40
Stir in ml. Dimethoxytrityl (DMT)
Chloride (2.3 g, 6.6 mmol) is added and the mixture is stirred at room temperature for 4 hours. Thin layer chromatography (on silica, methanol in chloroform)
After confirming the completion of the reaction by elution with a 10% by volume mixture, the reaction is concentrated to give a residual solid. The residue is subjected to column chromatography on silica, all fast-flowing impurities have been eluted with chloroform, and the product is then eluted with a 5% by volume mixture of methanol in chloroform. The residue was then concentrated to give 5'-dimethoxytrityl-5-(3-trifluoroacetylaminopropen-1-yl)-2'-deoxyuridine as a white fluffy solid (4 g).
get as. This product decomposes when heated;
UV λ _nax 291 nm, λ _nio 266 nm; (TLC (silica, eluted with 10% methanol by volume in chloroform): R _f 0.6. Example In this example 5'-dimethoxytrityl-5-
(3-trifluoroacetylaminopropyl)-
Hydrogenation of the exocyclic double bond and masking of 5'-hydroxy to obtain 2'-deoxyuridine will be explained. Repeat the synthesis of nucleoside precursors and 5'-hydroxy masking of Example and [However, before addition of DMT chloride, purification 5
-(3-trifluoroacetylaminopropenyl)
5'-dimethoxytrityl-5-(3-trifluoroacetylamino Propyl)-2'-deoxyuridine is produced. From the examples, the synthesis of other modified uracil nucleosides, followed by reaction 2.
The masking of hydroxy shown in will be explained. Example N-allyl trifluoroacetamide was replaced with the following compound group (numbers 1 to 8 ), and the steps and operations were repeated to synthesize a nucleoside precursor and mask 5' hydroxy,
In each case, the following groups of compounds (numbered 1' to 8' ) are obtained: i.e. replaced by the following compounds: 1 N-(3-butenyl)trichloroacetamide 2 N-(5-hexenyl) trifluoroacetylamide 3 N -(2-methyl-2-propenyl)trifluoroacetamide 4 N-(4-ethenyl phenylmethyl) trifluoroacetamide 5 N-(1-methyl-3-butenyl) trifluoroacetamide 6 N-(12-trichloroamino dodecyl)acrylamide 7 (pertrifluoroacetylpolylysyl)acrylamide 8 N-(3-trifluoroacetylamidopropyl)acrylamide, and the following compound is obtained: 1'5'-dimethoxytrityl-5-(4-trichloroacetylaminobuten-1-yl)-2'-
Deoxyuridine 2'5'-dimethoxytrityl-5-(6-trifluoroacetylaminohexen-1-yl)-
2'-deoxyuridine3'5'-dimethoxytrityl-5-(3-trifluoroacetylamino-2-methylpropene-
1-yl)-2'-deoxyuridine4'5'-dimethoxytrityl-5-[2-(4-trifluoroacetylaminomethylphenyl)ethen-1-yl]-2'-deoxyuridine5'5' -dimethoxytrityl-5-(4-trifluoroacetylamino-4-methylbutene-1
-yl)-2'-deoxyuridine 6'5'-dimethoxytrityl-5-[N-(12-trichloroacetylaminododecyl)-1-acrylamide]-2'-deoxyuridine7'5'-dimethoxytrityl-5-[N-(pertrifluoroacetylpolylysyl)-1-acrylamide]-2'-deoxyuridine8'5'-dimethoxytrityl-5-[N-(3-trichloroacetylaminopropyl)-acrylamide]-2' -Deoxyuridine Example 5-(3-Trifluoroacetylaminopropenyl)-2'-deoxyuridine was replaced with the following 5-substituted-2'-deoxyuridines (numbers 9 to 18 ) to obtain 5' of Example. By repeating the hydroxy masking operation, the following product group ( 9′ to 18′ )
Manufacture. That is, replacing with the following compounds: 9 5-(propen-1-yl)-2'-deoxyuridine 10 5-(carbutoxyethyl)-2'-deoxyuridine 11 5-(3-carbumethoxylpropane-1-
yl)-2'-deoxyuridine 12 5-(4-carbumethoxy-2-methylbuten-1-yl)-2'-deoxyuridine 13 5-(3-cyanopropen-1-yl)-2'-
Deoxyuridine 14 5-(4-cyano-2-methylbutene-1-
yl)-2'-deoxyuridine15 5-[2-(4-carbumethoxyphenyl)ethen-1-yl]-2'-deoxyuridine16 5-(4-acetoxybuten-1-yl)-
2'-deoxyuridine17 5-(4-acetoxybutan-1-yl)-
2'-deoxyuridine, and the following 5'-dimethoxytrityl-5-alkyl-2'-deoxyuridines are obtained: 9'5'-dimethoxytrityl-5-(propene-
1-yl)-2'-deoxyuridine10'5'-dimethoxytrityl-5-(2-carbumethoxyethyl)-2'-deoxyuridine11'5'-dimethoxytrityl-5-(3-carbumethoxypropane-1-yl)-2'-deoxyuridine12'5'-dimethoxytrityl-5-(4-carbumethoxy-2-methylbuten-1-yl)-
2'-deoxyuridine13'5'-dimethoxytrityl-5-(3-cyanopropen-1-yl)-2'-deoxyuridine14'5'-dimethoxytrityl-5-(4-cyano-2-methylbutene-1-yl)-2'-deoxyuridine15'5'-dimethoxytrityl-5-[2-(4-
Carbumethoxyphenyl)ethen-1-yl]
-2'-deoxyuridine 16'5'-dimethoxytrityl-5-(4-acetoxybuten-1-yl)-2'-deoxyuridine17'5'-dimethoxytrityl-5-(4-acetoxybutane-1-yl)yl)-2'-deoxyuridine18'5'-dimethoxytrityl-5-[4-(2,
4-dinitrophenyl)butyl]-2'-deoxyuridine Example Synthesis of the nucleoside precursor of Example by replacing 5-chloromerkyry-2'-deoxyuridine with 5-chloromerkyryuridine and synthesis of the 5'-hydroxy nucleoside precursor. By repeating the masking method, the corresponding 5'-dimethoxytrityl-5-substituted uridines are prepared. Example-XI illustrates the synthesis of modified cytosine nucleosides. Cytosine nucleosides, like adenosine nucleosides and unlike uracil nucleosides, have reactive groups on the base moiety, so such reactive groups can be masked to protect against undesired reactions. . In these examples, in reaction 2
Similar to the masking of 5'-hydroxy, masking of the reactive group in the base portion of cytosine shown in reaction 1 will be explained. Example 5-(3-Trifluoroacetylaminopropenyl) ^-N4 -benzoyl-2'-deoxyuridine 5-chloromerkyry-2'-deoxyuridine was replaced with 5-chloromerkyry-2'-deoxycytidine, The nucleoside precursor synthesis method of the example is repeated to produce 5-(3-trifluoroacetylaminopropenyl)-2'-deoxycytidine (UVλ _nax 287 nm). Purification 5-
(3-trifluoroacetylaminopropenyl)-
2'-Deoxycytidine (1.3 g, 4.6 mmol) is stirred in 80 ml of absolute ethanol, anhydrous benzoyl (1.5 g, 7 mmol) is added and the reaction is brought to reflux. Additionally, 1.5 g of anhydrous benzoyl is added 5 times every hour. Thin layer chromatography [silica plate, n-butanol/methanol/conc.
After determining the completion of the reaction (during 6-10 hours) by elution with NH ₄ OH/water (60:20:1:20).
The reaction mixture is cooled and concentrated under reduced pressure to obtain a semi-solid. Knead this solid with ether three times,
Decant and dry. The crude product is recrystallized from water to give chromatographically pure ^N4 -benzoyl-5-(3-trifluoroacetylaminopropenyl)-2'-deoxycytidine as a white solid. This product decomposes above 120°C; UV _nax = 311nm. Example 5'-dimethoxytrityl-5-(3-
trifluoroacetylaminopropenyl)-N ⁴
-Benzoyl-2'-deoxycytidine 5-(3-trifluoroacetylaminopropenyl)-2'-deoxyuridine is replaced with 5-(3-trifluoroacetylaminopropenyl) ^-N4 -benzoyl-2'-deoxycytidine , 5'-dimethoxytrityl-5-(3-trifluoroacetylaminopropenyl) ^-N4 -benzoyl-2'-deoxycytidine is produced by repeating the 5'-hydroxy masking operation in Example. Example By replacing N-allyl trifluoroacetamide with the N-alkyl trifluoroacetamides of Examples and repeating the nucleoside precursor synthesis and 5' hydroxy masking operations of Examples, the corresponding 5'-dimethoxytrityl -5-(trifluoroacetyl)aminoalkyl) ^-N4 -benzoyl-2'-deoxycytidines are produced. That is, the following compound groups. 5'-dimethoxytrityl-5-(4-trifluoroacetylaminobuten-1-yl) ^-N4 -benzoyl-2'-deoxycytidine 5'-dimethoxytrityl-5-(6-trifluoroacetylaminohexene-1 -il) -N ⁴ -
Benzoyl-2'-deoxycytidine 5'-dimethoxytrityl-5-(3-trifluoroacetylamino-2-methylpropene-1-
yl) ^-N4 -benzoyl-2'-deoxycytidine 5'-dimethoxytrityl-5-[2-(4-trifluoroacetylaminomethylphenyl)ethen-1-yl] ^-N4 -benzoyl-2'- Deoxycytidine 5'-dimethoxytrityl-5-(4-trifluoroacetylamino-4-methylbuten-1-yl) ^-N4 -benzoyl-2'-deoxycytidine 5'-dimethoxytrityl-5-[N-(12 -trifluoroacetylaminododecyl)-1-acrylamide]-N ⁴ -benzoyl-2'-deoxycytidine 5'-dimethoxytrityl-5-[N-(pertrifluoroacetylpolylysyl)-1-acrylamide]-N ⁴ -Benzoyl-2'-deoxycytidine Example XI 5'-dimethoxytrityl- ^N4 -benzoyl-5-(2-carbumethoxyethenyl)-
Synthesis of 2'-deoxycytidine 5-(2-carbumethoxyethenyl)-2'-deoxycytidine (0.82 g, 2.6 mmol) is stirred in 50 ml of absolute ethanol. Benzoic anhydride (500mg,
2.2 mmol) is added and the reaction is heated to reflux. Additionally, 500 mg of benzoic anhydride is added every hour for 5 times. After the reaction is determined to be complete by thin layer chromatography (usually 6-8 hours), the reaction is cooled and vacuum distilled to yield a yellow semi-solid material. Chromatography on silica gel, eluting with a mixture of methanol/chloroform in linear proportions ranging from 1:19 to 1:3, combining and evaporating the appropriate fractions to remove ^N4-
Benzoyl-5-(2-carbumethoxyethenyl)
-2'-deoxycytidine is obtained as an amorphous white solid. UV λ _nax 296nm, λ _nio 270nm. Dry this solid completely and dissolve it in 20 ml of pyridine. Dimethoxytrityl chloride (1.1 equivalent)
is added and the reaction is stirred at ambient temperature for 6 hours. Concentration gave a solid, which was then subjected to column chromatography on silica gel, eluting with a 10% mixture of methanol in chloroform to give 5'-dimethoxytrityl- ^N4 -benzoyl-5-(2-carbumethoxyethyl). (tenyl)-2'-deoxycytidine is obtained as an off-white fluffy solid. Example XII By replacing 5-(2-carbumethoxyethenyl)-2'-deoxycytidine with the following compound group (numbers 19 to 27 ) and repeating the method for synthesizing the nucleoside precursor of Example XI. , the following corresponding compound groups (numbers 19' to 27' ) are obtained. That is, the following compounds are substituted: 19 5-(2-carbumethoxyethyl)-2'-deoxycytidine20 5-(3-carbumethoxypropan-1-yl)-2'-deoxycytidine21 5-(4 -carbumethoxy-2-methylbuten-1-yl)-2'-deoxycytidine22 5-(3-cyanopropen-1-yl)-2'-
Deoxycytidine 23 5-(4-cyano-2-methylbutene-1-
yl)-2'-deoxycytidine24 5-[2-(4-carbumethoxyphenyl)ethen-1-yl)-2'-deoxycytidine25 5-(4-acetoxybuten-1-yl)-
2'-deoxycytidine26 5-(4-acetoxybutan-1-yl)-
2'-deoxycytidine27 5-[4-(2,4-dinitrophenyl)butyl]-2'-deoxycytidine and the following 5'-dimethoxytrityl- ^N4 -benzoyl-5-alkyl-2'-deoxycytidines obtain: 19'5'-DMT-N ⁴ -benzoyl-5-(2-carbumethoxyethen-1-yl))-2'-deoxycytidine 20'5'-DMT-N ⁴ -benzoyl-5-( 3-carbumethoxypropan-1-yl)-2'-deoxycytidine 21'5'-DMT-N ⁴ -benzoyl-5-(4-carbumethoxy-2-methylbuten-1-yl)
-2'-deoxycytidine 22'5'-DMT-N ⁴ -benzoyl-5-(3-cyanopropen-1-yl)-2'-deoxycytidine 23'5'-DMT-N ⁴ -benzoyl-5- (4-cyano-2-methylbuten-1-yl)-2'-deoxycytidine 24'5'-DMT-N ⁴ -benzoyl-5-[2(4-
Carbumethoxyphenyl)ethen-1-yl]
-2'-deoxycytidine 25'5'-DMT-N ⁴ -benzoyl-5-(4-acetoxybuten-1-yl)-2'-deoxycytidine 26'5'-DMT-N ⁴ -benzoyl-5- (4-acetoxybutan-1-yl)-2'-deoxycytidine 27'5'-DMT-N ⁴ -benzoyl-5-[4-
(2,4-dinitrophenyl)butyl]-2'-deoxycytidine Similarly, other acid anhydrides such as acetic anhydride,
By using anisoyl anhydride or tolyl anhydride, in Examples and XI, ^N4 -acyl or ^N4 -acetyl-5-alkyl-2'-deoxycytidine in which benzoyl is replaced by acetyl or acyl is prepared. , respectively, are manufactured. Example 5-chloromer-kyry-2'-deoxycytidine was replaced with 5-chloromer-kyry-cytidine,
By repeating the nucleoside precursor synthesis method and 5'-hydroxy masking method from Examples, the corresponding 5'-dimethoxytrityl- ^N4
-Preparing benzoyl-5-substituted cytidines. EXAMPLE This example illustrates the masking of the reactive base moiety of the adenine nucleoside and the masking of the 5' hydroxy. N ⁶ -benzoyl-8-(6-aminohexyl)
Amino-2'-deoxycyadenosine (4 mmol) is stirred in 60 ml of absolute ethanol. Trifluoroacetic anhydride (6 mmol) is added and the reaction is stirred at room temperature. Two additional portions of trifluoroacetic anhydride are added every hour. After 4 hours, the reaction is concentrated to give a solid residue, which is lyophilized overnight.
The ^N6 -benzoyl-8-(6-trifluoroacetylaminohexyl)amino-2'-deoxyadenosine crude product is thoroughly dried and concentrated twice in pyridine to a solid residue. This solid material is placed in 40 ml of pyridine under stirring and dimethoxytrityl chloride (6.5 mmol) is added. 4
After an hour, the reaction is concentrated to obtain a solid residue. This was subjected to column chromatography on silica gel, and the methanol content in chloroform was reduced from 0 to 0.
A multi-step gradient elution between 15% and 5'-dimethoxytrityl- ^N6 -benzoyl-8-(6-trifluoroacetylaminohexyl)amino-2'-deoxyadenosine is obtained as an off-white solid. From the example, as in reaction 3 in the diagram,
Activation of 5-substituted-nucleosides and naturally occurring nucleosides masked at the 5' position to the respective phosphomonochloridites. Example Preparation of 3'-phosphomonochloridite of 5'-DMT-5-(3-trifluoroacetylaminopropan-1-yl)-2'-deoxyuridine. Fluoroacetylaminopropan-1-yl)-2'-deoxyuridine (1.54 g, 2.2 mmol) was dissolved in 20 ml of benzene three times over 12 hours each to remove residual water and solvent. Freeze-dry from.
The resulting very fluffy white powder is transferred under reduced pressure into a nitrogen atmosphere and dissolved in anhydrous acetonitrile containing 5% by volume of 2,6-lutidine to a final concentration of 30mM. A large pill of methylphosphodichloridite (1.0 eq.) is quickly added via syringe under a nitrogen atmosphere and with vigorous stirring. This reactant was mixed under a nitrogen atmosphere.
Swirl for about 1 minute. The resulting reaction solution of crude 5'-DMT-5-(3-trifluoroacetylaminopropan-1-yl)-2'-deoxyuridine 3'-methylphosphomonochloridite was then purified without further purification. , used as is in the synthesis of deoxyoligonucleotides (Example). The ^31P -NMR (CH ₃ CN/CDCl ₃ ) of this product is:
Typically, 40-70 mol% represents the desired product (167.5 ppm). The rest are bis-3′,
3′-[5′DMT-5-(3-trifluoroacetylaminopropan-1-yl)-2′-deoxyuridylyl]methylphosphite (140 ppm),
5'-DMT-5-(3-trifluoroacetylaminopropan-1-yl)-2'-deoxyuridine
Composed of 3′-methylphosphonate (9.5ppm),
This latter compound is produced in an amount that reflects the presence of water in the reaction system. Example Production of 3'-phosphomonochloridites of naturally occurring 2'-deoxynucleoside 5'-DMT-5-(3-trifluoroacetylaminopropan-1-yl)-2'-deoxyuridine, 5 '-DMT-2'-deoxythymidine 5'-DMT-N ⁴ -benzoyl-2'-deoxycytidine 5'-DMT-N ⁶ -benzoyl-2'-deoxyadenosine 5'-DMT-N ² -isobutyryl-2 By substituting with '-deoxyguanosine and repeating the procedure of the example, the following corresponding phosphomonochloridites are prepared. That is, 5'-DMT-2'-deoxythymidine 3'-methylphosphomonochloridite 5' ^- DMT-N 4 -benzoyl-2'-deoxycytidine 3'-methylphosphomonochloridite 5'-DMT-N ⁶ -benzoyl-2'-deoxyadenosine 3'-methylphosphomonochloridite 5'-DMT-N 6 -benzoyl-2'-deoxyadenosine 3'-methylphosphomonochloridite 5'-DMT- ^{N 2 -isobutyryl} -2 '-Deoxyguanosine 3'-methylphosphomonochloridite Example By replacing methylphosphodichloridite with o -chlorophenylphosphodichloridite and repeating the synthesis method of phosphomonochloridite in Example and 5'-DMT-nucleoside 3'-phosphomonochloridites, namely 5'-DMT-5-(3-trifluoroacetylaminopropyl)-2'-deoxyuridine 3'- o -
Chlorophenylphosphomonochloridite 5'-DMT-2'-deoxythymidine 3'- o -chlorophenylphosphomonochloridite, 5'-DMT-N ⁴ -benzoyl-2'-deoxycytidine 3'- o -chlorophenyl Phosphomonochloridite 5'-DMT-N ⁶ -benzoyl-2'-deoxyadenosine-3'- o -chlorophenylphosphomonochloridite 5'-DMT-N ² -isobutyryl-2'-deoxyguanosine 3'- o - Obtain chlorophenylphosphomonochloridite. Similarly, by using p -chlorophenylphosphodichloridite, a 3'- p -chlorophenylphosphomonochloridite adduct of a similar material is prepared. [ ^32P ]NMR of o- chlorophenylphosphomonochloridite product ( _{CH3CNCDCl3 )}
are 160.7, 160.5ppm (diastereomer). Example - Reactions 4 and 5 of the diagram
This exemplifies the chemical synthesis of oligonucleotides incorporating modified bases, as shown in FIG. Example 5 Synthesis of deoxyoligonucleotides containing -(3-aminopropyl)-uracil and naturally occurring nucleotide units Immediately before the synthesis of deoxyoligonucleotides, the phosphomonochloridite synthesis procedure of Example was carried out. The product was directly dissolved in anhydrous acetonitrile/5% by volume 2,6-lutidine.
It is used containing 30mM crude 3'-methylphosphomonochloridite. Solid support (5-DMT-N ⁶ -benzoyl-
2'-deoxyadenosine 3'-succinamidopropyl silica (250 mg, 20 μeq) is placed in a suitable reaction flow vessel (glass or Teflon column or funnel). This solid carrier was prepared in advance with acetonitrile/5% lutidine, tetrahydrofuran/water/lutidine containing 2 w/v% iodine for 2 minutes,
Conditions were adjusted in advance by sequentially treating with chloroform, 4% dichloroacetic acid in chloroform for 2.5 minutes, and acetonitrile/5% lutidine. In this case, each treatment may be carried out in a total volume of 5-15 ml in two or three portions or in constant flow, as desired. Deoxyoligonucleotides follow reaction 4,
Desired activated 5'-DMT-nucleotide
The terminal unit of the growing nucleotide chain (which is initially the only unit of this chain; i.e., the deoxyadenosine containing solid support) is added sequentially to the 3' methylphosphomonochloridite monomer. The free 5′- of
It is synthesized by bonding this to a hydroxyl group. Additions are selected from examples and
This is done by reacting 10 ml of 30 mM crude monochloridite with the freshly deprotected 5' hydroxy on the chain in 2-3 portions or by constant flow and reaction for 2-6 minutes. The first phosphomonochloridite addition, followed by one complete reagent cycle, consists of the following sequential treatments: -5'-DMT-5-(3-trifluoroacetylaminopropyl)-2'-deoxy Uridine 3'-methylphosphomonochloridite, - acetonitrile/lutidine wash, - capping for 5 minutes with 0.3M 4-dimethylaminopyridine in acetic anhydride/lutidine/tetrahydrofuran (1:3:2), - acetonitrile/5% lutidine wash. , -tetrahydrofuran/water/lutidine (6:
oxidize for 2 minutes with 2% iodine in 2:1), - acetonitrile/5% lutidine wash, - chloroform wash, - 2.5% by volume dichloroacetic acid in chloroform.
- Chloroform wash, - Acetonitrile/lutidine wash. Repeat the above cycle with 5'-DMT-5
-(3-trifluoroacetylaminopropyl)-
The 2'-deoxyuridine 3'-methylphosphomonochloridite is replaced with a different one of the 3'-methylphosphomonochloridites listed below and the procedure is repeated 13 times. However, the dichloroacetic acid treatment is excluded in the final reagent cycle: 5'-DMT-2'-deoxythymidine 3'-methylphosphomonochloridite 5'-DMT-5-(3-trifluoroacetylaminopropyl)-2'- Deoxyuridine 3'-methylphosphomonochloridite 5'-DMT-N ⁶ -benzoyl-2'-deoxyadenosine 3'-methylphosphomonochloridite 5'-DMT-N ⁴ -benzoyl-2'-deoxycytidine 3' -Methylphosphomonochloridite 5'-DMT-N ² -isobutyryl-2'-deoxyguanosine 3'-methylphosphomonochloridite 5'-DMT-5-(3-trifluoroacetylaminopropyl)-2'-deoxy Uridine 3'-methylphosphomonochloridite 5'-DMT-2'-deoxythymidine 3'-methylphosphomonochloridite 5'-DMT-5-(3-trifluoroacetylaminopropyl)-2'-deoxyuridine 3 '-Methylphosphomonochloridite 5'-DMT-deoxythymidine 3'-methylphosphomonochloridite 5'-DMT-5-(3-trifluoroacetylaminopropyl)-2'-deoxyuridine 3'-methylphosphomonochloridite Lydite 5'-DTM-N ² -isobutyryl-2'-deoxyguanosine 3'-methylphosphomonochloridite 5'-DMT-N ⁶ -benzoyl-2'-deoxyadenosine 3'-methylphosphomonochloridite 5' -DMT- ^N4 -benzoyl-2'-deoxycytidine 3'-methylphosphomonochloridite The support is transferred and treated with 2 ml of concentrated ammonium hydroxide for 4 hours at ambient temperature to release the product from the support. The supernatant is removed and the solid is washed three times with 0.5 ml of concentrated ammonium hydroxide, the supernatants are combined, sealed and heated at 50°C overnight. Freeze-dry the clear yellow supernatant completely. The primary purification is
On a RP-8 (C-8) column, the ratio of acetonitrile in 25mM ammonium acetate at pH 9.8 was 0-30.
Gradient elution as volume% (60 minutes)
Performed using reversed-phase high performance liquid chromatography (HPLC). Collect the 5'-DMT-terminated products that elute as a sharp peak after about 40 minutes; those with shorter chains, both capped and uncapped, elute before 25 minutes. Resulting in. The collected product was distilled to give a solid residue, which was diluted with 80% acetic acid at ambient temperature for 20 min.
After a minute treatment (to remove DMT), lyophilization yields a solid residue, which is dissolved in a small amount of aqueous buffer. Products are typically 90% after HPLC
There is more homogeneity. This is then electrophoresed on a regular 20% polyacrylamide gel (1-6 mm thick) to produce the appropriate product band (the product is usually smaller than unmodified deoxyoligonucleotides of similar length). It can be further purified by cutting out and extracting the molecule (which migrates). The purified 5-aminopropyl-uracil-containing pentadecadeoxy oligonucleotide product thus produced is shown diagrammatically below [in the figure, U ^m =5-(3-aminopropyl)uracil]. Note that conventional deprotection of the oligonucleotide with ammonia also removed the masking trifluoroacetyl group on the substituent. The length and sequence of this oligonucleotide is then determined using an appropriate protocol (e.g., determining the length and sequence of oligonucleotides belonging to the prior art, in which the bases contained in the nucleotide unit are unmodified). This can be carried out by the ³² P-kinase method and sequencing using protocols already used for 32 P-kinase analysis, etc.). Similarly, other 5-(modified) uracil- Containing deoxyoligonucleotides are produced. In addition, in order to replace the nucleoside 3'-methylphosphomonochloridite adduct of Examples and with the 3'- o- or p -chlorophenylphosphomonochloridite adduct of the corresponding Example, and to remove the chlorophenyl protecting group. The same deoxyoligonucleotide product is obtained by treating the deoxyoligonucleotide with pyridinium oximate (at the end of deoxyoligonucleotide synthesis and before treatment with concentrated ammonium hydroxide). Example 5'-DMT-5-(3-trifluoroacetylaminopropyl)-2'-deoxyuridine was prepared as follows.
Substituted with 5'-DMT-5-alkyl-2'-deoxyuridines ( numbers 28-36 ), Example-
By repeating the phosphomonochloridite and deoxyoligonucleotide synthesis methods described above, the following oligonucleotides (numbers 28' to 36' ) having the corresponding uracil base U ^m are produced. That is, when replaced with the following compound: 28 5'-dimethoxytrityl-5-(3-trifluoroacetylaminopropen-1-yl)-
Deoxyuridine29 5'-dimethoxytrityl-5-(4-trifluoroacetylaminobutan-1-yl)-
2'-deoxyuridine30 5'-dimethoxytrityl-5-(4-trifluoroacetylaminobuten-1-yl)-
2'-deoxyuridine31 5'-dimethoxytrityl-5-(6-trifluoroacetylaminohexan-1-yl)-
2'-deoxyuridine32 5'-dimethoxytrityl-5-(6-trifluoroacetylaminohexen-1-yl)-
2'-deoxyuridine33 5'-dimethoxytrityl-5-(2-trifluoroacetylaminopropan-2-yl)-
2'-deoxyuridine34 5'-dimethoxytrityl-5-(3-trifluoroacetylamino-2-methyl-propen-1-yl)-2'-deoxyuridine35 5'-dimethoxytrityl-5-(3- Trifluoroacetylamino-2-methyl-propan-1-yl)-2'-deoxyuridine36 5'-dimethoxytrityl-5-[2-(4-trichloroacetylaminomethylphenyl)ethen-1-yl] -2'-deoxyuridine 37 5'-dimethoxytrityl-5-[N-(pertrifluoroacetylpolylysyl)-1-acrylamide]-2'-deoxyuridine 38 5'-DMT-5-[N-(7 -trifluoroacetylaminoheptyl)-1-acrylamide]
-2'-Deoxyuridine, deoxynucleotides are prepared corresponding to the products of the examples in which U ^m is: 28'5-(3-aminopropen-1-yl)uracil29'5-(4-aminobutan-1-yl)laucil30'5-(4-aminobuten-1-yl)laucil31' 5-(6- Aminohexan-1-yl) laucyl 32' 5-(6-aminohexen-1-yl) laucyl 33' 5-(3-aminopropan-2-yl) laucyl 34' 5-(3-ami-2-methyl Propene-1-
yl) laucyl 35' 5-(3-amino-2-methylpropane-1
-yl) laucyl 36' 5-[2-(4-aminoethylphenyl)ethen-1-yl] laucyl 37' 5-[N-(polylysyl)-1-acrylamide] laucyl 38' 5-[N-( 7-aminoheptyl)-1-acrylamide]laucil. Similarly, similar deoxyoligonucleotides are prepared using other 5'-DMT-5(acylaminoalkyl)-2'-deoxyuridines. Example 5'-DMT-5-(3-acetylaminopropyl)-2'-deoxyuridine was converted into the following 5-substituted-
By repeating the synthesis method of phosphomonochloridite and deoxyoligonucleotide in Examples ~ by substituting 2'-deoxyuridines (numbers 37 to 46 ), the following corresponding U ^m
Oligonucleotides with uracil bases (numbers 37' - 46' are prepared, i.e., substituted with the following compounds: 37'5'-DMT-5-(propen-1-yl)-
2'-Deoxyuridine385'-DMT-5-(2-carbumethoxyethyl)-2'-deoxyuridine395'-DMT-5-(3-carbumethoxypropan-1-yl)-2'-deoxy Uridine 40 5'-DMT-5-(4-carbumethoxy-2-
methylbuten-1-yl)-2'-deoxyuridine41 5'-DMT-5-(3-cyanopropene-1-
yl)-2'-deoxyuridine42 5'-DMT-5-(4-cyano-2-methylbuten-1-yl)-2'-deoxyuridine43 5'-DMT-5-[2-(4-carb methoxyphenyl)ethen-1-yl)-2'-deoxyuridine44 5'-DMT-5-(4-acetoxybutene-1
-yl)-2'-deoxyuridine45 5'-DMT-5-(4-acetoxybutane-1
-yl)-2'-deoxyuridine 46 5'-DMT-5-[4-(2,4-dinitrophenyl)butyl]-2'-deoxyuridine. and obtain a product in which U ^m is: 37' 5-(propen-1-yl) uracil 38' 5-(2-carboxyethyl) uracil 39' 5-(3-carboxypropan-1-yl)
Uracil 40' 5-(4-carboxy-2-methylbutene-
1-yl)uracil 41' 5-(3-cyanopropen-1-yl)uracil 42' 5-(4-cyano-2-methylbutene-1-
yl) uracil 43' 5-[2-(4-carboxyphenyl)ethen-1-yl]uracil 44' 5-(4-hydroxybuten-1-yl)uracil 45' 5-(4-hydroxybutane-1 -yl)uracil 46' 5-[4-(2,4-dinitrophenyl)butyl]uracil Note: 46' directly functions as a reporter group, ie can be used as a ligand for anti-dinitrophenyl antibodies. Similarly, other suitable 5′-DMT
Similar deoxyoligonucleotides are prepared using -5-alkyl-2'-deoxyuridines. Example XI 5′-DMT-5-(3-trifluoroacetylaminopropyl)-2′-deoxyuridine
By substituting DMT-N ⁴ -benzoyl-5-(3-trichloroacetylaminopropyl)-2'-deoxycytidine and repeating the procedure in Example, the same procedure as in Example (but U ^m [5-
(3-aminopropyl)-uracil] is 5-(3-
aminopropyl) substituted with cytosines),
Deoxyoligonucleotides are produced. For example, the formula: (wherein, C ^m represents 5-(3-aminopropyl)cytosine). Example XII Deoxyribonucleotide synthesis of Example XI by replacing 5'-DMT- ^N4 -benzoyl-5- ( 3-trichloroacetylaminopropyl)-2'-deoxycytidine with the following compound group (numbers 47-57 ) By repeating the method, each of the following C ^m
Corresponding oligonucleotides with cytosine bases (numbers 47' - 57' ) are prepared. That is,
Replace with the following compound. 47 5-DMT- ^N4 -benzoyl-5-(3-trifluoroacetylaminopropen-1-yl)-2'-deoxycytidine48 5'-DMT- ^N4 -benzoyl-5-(4-trifluoroacetyl aminobutan-1-yl)
-2'-deoxycytidine 49 5'-DMT-N ⁴ -benzoyl-5-(4-trifluoroacetylaminobuten-1-yl)
-2'-deoxycytidine 50 5'-DMT-N ⁴ -benzoyl-5-(6-trifluoroacetylaminohexan-1-yl)-2'-deoxycytidine 51 5'-DMT-N ⁴ -benzoyl-5 -(6-Trifluoroacetylaminohexen-1-yl)-2'-deoxycytidine52 5'-DMT-N4 -benzoyl-5-( ³ -trifluoroacetylaminopropan-2-yl)-2'- Deoxycytidine 53 5'-DMT-N ⁴ -benzoyl-5-(3-trifluoroacetylamino-2-methylpropen-1-yl)-2'-deoxycytidine 54 5'-DMT-N ⁴ -benzoyl-5 -(3-Trifluoroacetylamino-2-methylpropan-1-yl)-2'-deoxycytidine 55 5'-DMT-N ⁴ -benzoyl-5-[2-(4
-trifluoroacetylaminomethylphenyl)ethen- ¹ -yl]-2'-deoxycytidine56 5'-DMT-N4 -benzoyl-5-[N-(pertrifluoroacetylpolylysyl)-1-acrylamide] -2'-deoxycytidine 57 5'-DMT- ^N4 -benzoyl-5-[N-(trifluoroacetylaminoheptyl)-acrylamide]-2'-deoxycytidine. and obtain a product in which C ^m is: 47' 5-(3-aminopropen-1-yl)cytosine 48' 5-(4-aminobutan-1-yl)cytosine 49' 5-(4-aminobuten-1-yl)cytosine 50' 5-(6- aminohexan-1-yl)cytosine 51' 5-(6-aminohexen-1-yl)cytosine 52' 5-(3-aminopropan-2-yl)cytosine 53' 5-(3-amino-2-methyl Propen-1
-yl)cytosine 54' 5-(3-amino-2-methylpropane-1
-yl)cytosine 55' 5-[2-(4-aminomethylphenyl)ten-1-yl)cytosine 56' 5-[N-(polylysyl)-1-acrylamide]cytosine 57' 5-[N-( 7-aminoheptyl)-1-acrylamido]cytosine Similarly, similar deoxyoligonucleotides are prepared by using other ^N4 -acyl-5-(acylaminoalkyl)-2'-deoxycytidines. . Example 5'-DMT- ^N4 -benzoyl-5-(3-trifluoroacetylaminopropyl)-2'-deoxycytidine was replaced with the following compound group (numbers 58 to 68 ) to synthesize the deoxyoligonucleotide of Example XI. By repeating the method, each of the following C ^m
Corresponding oligonucleotides with cytosine bases (numbers 58'- 68') are prepared. That is,
Replace with the following compound. 58 5'-DMT-N ⁴ -benzoyl-5-(propen-1-yl)-2'-deoxycytidine 59 5'-DMT-N ⁴ -benzoyl-5-(2-carbumethoxyethyl)-2'- Deoxycytidine 60 5'-DMT-N ⁴ -benzoyl-5-(2-carbumethoxyethen-1-yl)-2'-deoxycytidine 61 5'-DMT-N ⁴ -benzoyl-5-(3-carbumethoxy propan-1-yl)-2'-deoxycytidine62 5'-DMT-N ⁴ -benzoyl-5-(4-carbumethoxy-2-methylbuten-1-yl)
-2'-deoxycytidine63 5'-DMT-N4-benzoyl-5-(3-cyanopropen-1- ^yl )-2'-deoxycytidine64 5'-DMT- ^N4 -benzoyl-5-(4 -cyano-2-methylbuten-1-yl)-2'-deoxycytidine 65 5'-DMT-N ⁴ -benzoyl-5-[2-(4
-carbumethoxyphenyl)ethen-1-yl]-2'-deoxycytidine66 5'-DMT-N4 -benzoyl-5-( ⁴ -acetoxybuten-1-yl)-2'-deoxycytidine67 5' -DMT-N ⁴ -benzoyl-5-(4-acetoxybutan-1-yl)-2'-deoxycytidine68 5'-DMT-N ⁴ -benzoyl-5-[4-
(2,4-dinitrophenyl)butyl]-2-deoxycytidine. We then obtain a product in which C ^m is: 58' 5-(propen-1-yl)cytosine 59' 5-(2-carboxyethyl)cytosine 60' 5-(2-carboxyethen-1-yl)cytosine 61' 5-(3-carboxypropane-1- )
Cytosine 62' 5-(4-carboxy-2-methylbutene-
1-yl)cytosine 63' 5-(3-cyanopropen-1-yl)cytosine 64' 5-(4-cyano-2-methylbutene-1-
yl)cytosine 65' 5-[2-(4-carboxyphenyl)ethen-1-yl]cytosine 66' 5-(4-hydroxybuten-1-yl)cytosine 67' 5-(4-hydroxybutane-1 -yl)cytosine 68' 5-[4-(2,4-dinitrophenyl)butyl]cytosine as well as other suitable 5'-DMT- ^N4 -acyl-5
Similar deoxyoligonucleotides are prepared by using -alkyl-2'-deoxycytidines. Example XI 5′-DMT-5-(3-trifluoroacetylaminopropyl)-2′-deoxyuridine
By repeating the phosphomonochloridite and deoxyoligonucleotide synthesis method of Example by substituting DMT- ^N6 -benzoyl-8-(4-trifluoroacetylaminohexyl)amino-2'-deoxyadenosine, Similarly, deoxyoligonucleotides (however, U ^m is replaced with A ^m , and this A ^m is 8-
(6-aminohexyl)amino-2'-deoxyadenosine] is produced. Example 6 illustrates the attachment of a reporter group to oligonucleotides having appropriately modified bases, as shown in Reaction 6. Examples Fluoresceinated deoxyoligonucleotide Formula: [In the formula, U ^m is 5-[N-(7-aminoheptyl)
The purified pentadeca-nucleotide (obtained from the example) represented by -1-acrylamide [represents uracil] was added to a 300 mM aqueous sodium borate solution or sodium carbonate buffer at pH 9.5 containing 30 mM sodium chloride in 25A260. Dissolve at the rate of unit/ml. Solid fluorescein isothiocyanate (0.5 mg/ml) is added and the mixture is sealed and shaken gently at 4°C to 25°C overnight. This reaction product was directly mixed with G-50 Sephadex.
Column to separate unbound-fluorescein additive (which is retained); fluoresceinated deoxyoligonucleotide adduct elutes near empty volume. The initial fractions containing significant A260 units are combined and lyophilized to obtain a solid product with a structure similar to the starting pentadecaoxyoligonucleotide. However, in this case, U ^m
is the formula: or unreacted 5-[N-(7-aminoheptyl)-1-acrylamido]uracil. λ _nax (H ₂ O) 262 nm, 498 nm If this operation is carried out using the compound groups described in Examples, XI, XII, and the like, the corresponding fluoresceinated or polyfluoresceinated deoxyoligo Nucleotides are produced. Examples Attachment of a reporter group other than fluorescein can be achieved by replacing fluorescein isothiocyanate with, for example, the compound below and repeating the procedure of the example. These compound groups include, for example, 2,4-dinitrophenyl isothiocyanate 1-fluoro-2,4-dinitrobenzene aminoethyl isoluminol isothiocyanate aminoethylaminonaphthalene-1,2-carboxylic hydrazide isothiocyanate N ,N'-bis(alkylsulfonyl)-N-aryl-N'-isothiocyanateAryl-dioxamide m -sulfonyl aniline isothiocyanate 9-(N-hydroxysuccinimidyl)biotin 9-(N-hydroxysuccinimidyl) - carboxy)-N-methylacridine or cyanogen bromide-activated Sepharose, with which the corresponding adducts with groups other than fluorescene attached can be prepared. Illustrative Example Binding of isoluminol and free primary amine-containing reporter group Formula obtained from the example: [In the formula, U ^m is 5-(2-carboxyethenyl)
[representing uracil]] in water.
Dissolve at the rate of 30A260 units/ml and dilute with 1 volume of pyridine. Aminobutylethylisoluminol is added to a final concentration of 1 mg/ml, followed by a 5-fold molar excess of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. Seal the reaction and shake gently in the dark for 12-48 hours. The reaction mixture was concentrated under reduced pressure to obtain a solid residue, which was directly transferred to G-50 Sephadex.
Chromatograph on a column. Isoluminol
The deoxyoligonucleotide conjugate elutes near empty volume. The initial fractions containing significant A260 units are combined and lyophilized to obtain a solid product with a structure similar to the starting deoxyoligonucleotide. However, in this case U ^m has the formula: Or unreacted 5-(2-carboxyethenyl)
Either uracil. If this operation is carried out using examples and compounds in which R ₂ contains carboxy, the corresponding deoxyoligonucleotide isoluminol is similarly produced. Alternatively, aminobutylisoluminol could be replaced with other free primary amine-containing reporter groups;
By repeating this operation, a corresponding deoxyoligonucleotide-reporter adduct is produced in the same manner. Examples Binding of Dinitrophenyl Reporter Group Formula: [In the formula, A ^m represents 8-(6-aminohexyl)aminoadenine] The purified nonanucleotide represented by
20A260 units/ml in 250mM sodium carbonate buffer
Dissolve in the ratio of 1-fluoro-2,4-dinitrobenzene. The reaction solution is shaken overnight at ambient temperature and then chromatographed directly onto a Sephadex G-50 column. The initial fractions containing significant A260 units are combined and concentrated to yield an oligonucleotide product with a similar structure to the starting decanucleotide. However, in this case,
A ^m is the formula: or unreacted 8-(6-aminohexyl)aminoadenine. Repeating this procedure by replacing 8-(6-aminohexyl)aminoadenine with other modified bases containing free primary amines also yields the corresponding dinitrophenylated oligonucleotide adducts. Manufactured.