EP0372002A1 - Ein kovalentes oligonucleotid-meerrettich-peroxidase-konjugat - Google Patents

Ein kovalentes oligonucleotid-meerrettich-peroxidase-konjugat

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Publication number
EP0372002A1
EP0372002A1 EP88908882A EP88908882A EP0372002A1 EP 0372002 A1 EP0372002 A1 EP 0372002A1 EP 88908882 A EP88908882 A EP 88908882A EP 88908882 A EP88908882 A EP 88908882A EP 0372002 A1 EP0372002 A1 EP 0372002A1
Authority
EP
European Patent Office
Prior art keywords
oligonucleotide
mmole
sulfhydryl
conjugate
hrp
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP88908882A
Other languages
English (en)
French (fr)
Inventor
Corey Levenson
Chu-An Chang
Fred T. Oakes
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cetus Oncology Corp
Original Assignee
Cetus Corp
Cetus Oncology Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Cetus Corp, Cetus Oncology Corp filed Critical Cetus Corp
Publication of EP0372002A1 publication Critical patent/EP0372002A1/de
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids

Definitions

  • the present invention relates generally to DNA hybridization probes, and more particularly relates to a stable,- covalent conjugate of an oligonucleotide and horseradish peroxidase (HRP) .
  • HRP horseradish peroxidase
  • Non-isotopically labelled synthetic DNA frag ⁇ ments have found broad application in molecular biology— e.g., in the areas of DNA sequencing, DNA probe-based diagnostics, and the like.
  • the conjugate disclosed herein is prepared using reagents which facilitate the labeling of oligonucleotides with specific groups by incorporating one or more modifiable sulfhydryl groups at one or more hydroxyl sites within the oligonucleotide.
  • a short spacer chain linking the 5' terminus of the oligonucleotide to the sulfhydryl, a ino or hydroxyl group results in destabilization of the derivatized structure—i.e., proximity of a solid support or a bulky labeling species to the oligonucleotide chain causes s.teric interference.and thus hinders use of the derivatized oligonucleotide in- robe-based applications;
  • a hydrophobic spacer chain linking the 5' terminus of the oligonucleotide to the sulfhydryl, amino or hydroxyl group provides problems with solubility in the aqueous solvents commonly used in DNA probe-based methods;
  • Conventionally used functionalizing reagents are often incompatible with commonly used DNA synthesis methodology, primarily because the functionalizing re ⁇ agents are incompatible with the reagents and solvents typically used therewith;
  • Conventionally used functionalizing reagents are frequently difficult to synthesize in high yield, necessitating complex, multi-step reactions;
  • the present invention involves certain novel functionalizing reagents which overcome the aforementioned problems. More specifically, the invention is directed to. a method of "derivatizing" sulfhydryl-functionalized oligonucleotides which can be prepared using novel oligonucleotide functionalizing reagents as will be described.
  • Covalent conjugates of oligonucleotides and labelling enzymes have been described in the literature. For example, Jablonski et. al., in Nuc. Acids Res. 14.(15) :6115-6128 (1986), describe covalent conjugates of alkaline phosphatase and oligonucleotides prepared using the homobifunctional reagent- disuccini idyl suberate. Renz and Kurz, in Nuc. Acids Res. 12(8);3435-3445 (1981), describe a covalent complex of HRP and oligonucleotides using a polyethyleneimine spacer chain having a molecular weight of about 1400.
  • oligonucleotide functionalizing reagents are used to functional!ze an oligonucleotide chain at a hydroxyl group contained therein to introduce a sulfhydryl group.
  • the co ⁇ pling reaction is effected using standard techniques for coupling a phosphoramidite to a hydroxyl group of an oligonucleotide, as described, inter alia, by Beaucage and Caruthers, Tetrahedron Lett. (1981) 22_:1859-1862.
  • the oligonucleotide is derivatized at the new sulfhydryl site with HRP as will be described.
  • “Sulfhydryl functionalizing” or simply “functionalizing” as used herein means incorporating a protected or unprotected sulfhydryl moiety into an oligonucleotide chain.
  • the sulfhydryl group introduced by functionalization is spaced apart from the oligonucleotide chain by a spacer chain as will be described herein.
  • Derivatizing as used herein means reacting a functionalized oligonucleotide at the added sulfhydryl group with a detectable species, i.e., one that serves as a label in probe-based * applications.
  • a "derivatized” oligonucleotide is thus one- that is detectable by virtue of the "derivatizing” species.
  • the derivatizing species herein is the enzyme horseradish peroxidase.
  • oligonucleotide as used herein is a single- stranded or double-stranded, typically a single-stranded, chain of nucleotide, typically deoxyribonucleotide, monomer units. While the reagents and methods of the present invention may be used in conjunction with a single nucleotide monomer or with a full-length DNA strand, the "oligonucleotides” herein are typically single-stranded and of from about 2 to about 400 monomer units, and, more typically for most probe-based applications, from about 2 to about 100 monomer units. Optimal length for use as an allele-specific oligonucleotide (or "ASO") is about 135-21 base pairs.
  • a free sulfhydryl group that is "protected” is one that has been reacted with a protecting moiety such that the resulting protected group will not be susceptible to any- sort of chemical reaction during the synthetic step or steps during which the protecting group is present.
  • stability of the functionalized or derivatized oligonucleotide chain is meant substantial absence of steric interference as well as chemical stabil- ity under the conditions of most probe-based applications.
  • lower alkyl and lower alkoxy are meant alkyl and alkoxy substituents, respectively, having from about 1 to 6, more typically from about 1 to 3, carbon atoms.
  • the sulfhydryl functionalizing reagents used to prepare the probes of the present invention i.e., the covalent oligonucle ⁇ tide-HRP conjugates— re substantially linear reagents having a phosphoramidite moiety at one end linked through a hydrophilic spacer chain to an opposing end provided with a protected or unprotected sulfhydryl moiety.
  • These functionalizing reagents are given by the
  • R is a protected or unprotected sulfhydryl moiety
  • R is a hydrogen, -CH-OH, or a substituent hav- ing the formula
  • X 1 , X 2 , X 3 , X 4 , X 5 and X 6 may be the same or dif ⁇ ferent and are selected from the group consisting of hydrogen, lower alkyl and lower alkoxy;
  • R 1 and R2 are independently selected from the group consisting of hydrogen and lower alkyl
  • R is £-cyanoethyl or methyl; the Q moieties are selected .from the group consisting of
  • n', n'' and n' ' ' are integers in the range of 2 and 10 inclusive; and n is an integer in the range of 2 and 30 inclusive.
  • Formula (4) represents one examples of a particularly preferred embodiment
  • hydrophilic spacer chain in such a case is a polyether linkage, e.g., as shown, formed from polyethylene glycol.
  • the spacer chain may also be formed from polypropylene glycol or the like, or from poly(oxyalkyleneamines) such as the Jeffamines sold by
  • the R moiety is a protected sulfhydryl moiety.
  • the protecting group is selected so that the sulfhydryl moiety remains intact during the phosphoramidite coupling .
  • ste i.e., in which the phosphoramidite group of the reagent reacts with, the hydroxyl moiety on the oligonucleotide chain.
  • R 1 and R2 are either hydrogen or lower alkyl, and may be the same or different; in a particularly preferred embodiment, both R 1 and R2 are isopropyl .
  • R3 is either methyl or ?-cyanoethyl; in a particularly preferred embodiment, R is /3-cy ' anoethyl.
  • the spacer chain (9) is the spacer chain (9)
  • n, n', n' ' and n' ' ' are integers having values as set forth above.
  • the spacer chain is the polyether moiety
  • n is typically 2-30, more typically 2-20 (in some, cases, however, n ' may ' be larger than 30, i.e., where increased distance is desired between the derivatizing moiety and the oligonucleotide chain) .
  • Optimal values for n provide the spacer chain with a total of at least about 8 carbon atoms along its length. The length of the spacer chain is quite relevant to the effectiveness of the re- agents, " as.
  • sulfhydryl group and the oligonucleotide chain (1) facilitates coupling of the reagent to DNA; (2) avoids steric interference which would hinder hybridization and destabilize the functionalized or derivatized oligonucleotide chain; (3) simulates a "solution" type environment in that freedom of movement of the derivatized sulfhydryl moiety is enhanced; and (4) avoids interference with the .activity of the derivatizing species, in this case the enzymatic activity of horseradish peroxidase.
  • the hydrophilicity of the spacer chain also enhances the solubility of the functionalized or derivatized oligonucleotide chains in aqueous media.
  • R is either hydrogen, hydroxyl, or the aromatic
  • R is (3), it is selected so that the chromogenic cation
  • DMT i.e., : is -CH 2 -0-DMT.
  • R is bonded to the carbon atom adjacent to the phosphoramidite group
  • R may be bonded to one or more other carbon atoms along the spacer chain, as illustrated by formula (2).
  • the coupling reaction between the functionalizing reagents and a hydroxyl-containing compound may be represented by the following scheme:
  • X is typically an oligonucleotide chain.
  • the reaction conditions are the same as those used in the phosphoramidite route to DNA synthesis, as noted earlier and as described, inter alia, by Beaucage and Caruthers (1981) , supra.
  • Compound (12) is deprotected as follows. Where R is given by formula (3), conversion to an unprotected hydroxyl group is carried out by treatment with acid.
  • the protected sulfhydryl moiety at R may be deprotected with, e.g., silver nitrate.
  • Step (1) represents the Mitsunobu reaction as is well known in the art. Briefly, the reaction involves admixture of compounds (15), (16), (17) and (18) in a polar, organic solvent for a least several hours, prefer ⁇ ably overnight (see Example 1).
  • Compound (19) is isolated and coupled to the phosphoramidite (wherein X represents a halogen, preferably chlorine) as follows. A molar excess of the phosphoramidite is added to compound (19) in a suitable solvent, again, one that is preferably a polar, organic solvent, under an inert atmosphere.
  • Compound (20) is isolated—e.g., by column chromatography.
  • steps la-lc and la'-lb' represent alternative routes to intermediate (21).
  • the protected diol (21) is formed by: reaction of the polyethylene glycol with allyl bromide (reaction carried
  • Steps la'-lb' give (21) via reaction of the tosylated glycol with the solketal anion.
  • Step 2 represents the Mitsunobu reaction as shown in ⁇ cheme II, " where R is as defined earlier, while the acid treatment of Step 3 deprotects the diol.
  • Step 4-1 introduces a chromogenic moiety where R is given by (5)
  • Step 1 is carried out at a low temperature, ' preferably about 0 C or less, and the triphenylphosphine, diisopropylazodicarboxylate and S-tritylmercaptan are al ⁇ lowed to react overnight.
  • the phosphoramidite is added in Step 2, and (25) is obtained in good yield.
  • the present application is directed to derivatization with the enzyme horseradish peroxidase.
  • the derivatized oligonucleotides of the- * present invention are conjugates comprising an oligonucleotide
  • R r Q r n, n', n" and n'" are as defined above for compound (2), and X is an oligonucleotide chain.
  • the length of the oligonucleotide chain is
  • the number of monomer units in the chain is preferably about 13-21.
  • the conjugates of the invention may be represented by the structure (27)
  • Thiolated oligonucleotide (29) is prepared as described in Example 8. Typically, the tritylthio oligonucleotides are detritylated just prior to use in the reaction of Scheme V.
  • the mal-sac HRP complex (28) is coupled to thiolated oligonucleotide (29) by simple admixture, preferably at room temperature or lower.
  • the reaction mixture is allowed .to remain at low temperature—e.g., about 0 C—at least overnight and preferably at least about several days, at which point the covalent HRP conjugate (26) is isolated and purified, preferably chromatographically.
  • the conjugates Prior to use in probe-based applications, the conjugates are stored in a phosphate buffer (added salts optional) maintained at a pH of from about 5.5 to about 7.5, preferably about 6.0, at a temperature of from about -10 C to about 30°C (with the proviso that the solution not be frozen), optimally about 4°C.
  • a phosphate buffer added salts optional maintained at a pH of from about 5.5 to about 7.5, preferably about 6.0, at a temperature of from about -10 C to about 30°C (with the proviso that the solution not be frozen), optimally about 4°C.
  • the conjugate s.olu- tions are normally diluted (the final concentration varying depending on use) with hybridization buffer and used according to standard hybridization techniques (see, e.g., Maniatis, et al. , Molecular Cloning, New York: Cold Spring Harbor Laboratory, 1982).
  • standard hybridization techniques see, e.g., Maniatis, et al. , Molecular Cloning, New York: Cold Spring Harbor Laboratory, 1982.
  • the general procedure followed is well known in the art, and typically involves:
  • conjugate includes an oligomer having a nucleotide sequence substantially complementary to that of an analyte of interest, i.e., sufficiently complementary to enable hybridization; (2) contacting, in solution, the analyte of interest with the covalent conjugate; and (3) detecting the presence of nucleic acid complexes which form by assaying for HRP activity.
  • the covalent conjugate hybridizes to an analyte that is attached to a solid support- and is then detected thereon.
  • a primary advantage is the relatively long, hydrophilic spacer chain which provides an optimum distance between the HRP and the oligonucleotide, ensuring that full bio ⁇ logical activity of the HRP is retained and enhancing the effectiveness of hybridization.
  • the novel conjugates by virtue of the "R " moiety, also allow multiple derivatization of one oligonucleotide, i.e., attachment of two or more "spacer-HRP" chains either linked end-to-end,, bound at various points within an oligonucleotide chain, or both.
  • ease of detection is enhanced by the rapid generation of color.
  • the 25.89 g obtained was then purified on an SiO- column using ethyl acetate as an eluant.
  • the product fractions were collected and " concentrated to a syrup (11. ' 75 g; 36.3 mmole; 18.2%) which was allowed to crystallize overnight.
  • step (b) Synthesis of the allyl derivative (see Step lb, Scheme III) : To a solution of the alcohol obtained in step (a) (4.67 g; 14.4 mmole) in 100 mL of dry THF was added NaH (520 mg; 21.67 mmole). The mixture was stirred for one hour, and then allyl bromide (1.9 mL; 2.61 g;
  • step (e) Preparation of the phosphoramidite: The product obtained in step (d) (1.0 g; 1.4 mmole) was dis ⁇ solved in 10 mL of acid-free chloroform and placed in a 250 L round bottom flask preflushed with dry argon. To this solution (.72 g, 5.6 mmole) of ' [(CH 3 ) 2 ⁇ CH] 2 -N-Et -was added. Then, the phosphoramidite
  • Example 2 Essentially the same procedure was followed as set forth in Example • 1, but the tetraethylene glycol starting material was not in this case ' initially reacted with phthalimide.
  • reaction mixture was filtered through a Whatman GFB filter, adsorbed onto 8 g of SiO-, and fractionated on an SiO- column using a mixture of methylene chloride and acetone (1:1) as eluant * .
  • the pooled fractions yielded 4.28 g (13.28 mmole; 66.4%) product. Elemental analysis was as follows Calc, C, 55.88; H, 9.38. Found: C, 55.56; H, 9.76. Proposed structure of the product:
  • step (b) Synthesis of the corresponding diol: To a solution of the allyl ether prepared in step (a) (4.28g; 13.28 mmole) in 270 L of a mixture of acetone and water (8:1) was. added N-methyl morpholine (3.11 g; 4.6 L; 26.55 mmole; 2 eq. ) followed by osmium tetroxide (25 mg/mL in t- butanol; 338 mg; 13.5 mL; 1.33 mmole [0.1 eq.]). The re- action mixture was stirred overnight. The next morning, a solution of sodium hydrosulfite (3.62 g) in 15 mL water was added.
  • N-methyl morpholine 3.11 g; 4.6 L; 26.55 mmole; 2 eq.
  • osmium tetroxide 25 mg/mL in t- butanol; 338 mg; 13.5 mL;
  • step (c) The triol prepared in step (b) (3-3 g; 9.26 mmole) was taken up in 60 mL acetone and cupric sulfate
  • step (a) The product obtained in step (a) (4.22 g; 9.30 mmole) was dissolved in 10 L of acid-free chloroform and placed in a 250 mL round bottom flask preflushed with dry argon. To this solution (.72 g, 5.6 mmole) of [ (CH.,) 2 -CH] 2 ⁇ N-Et was added. Then, the phosphoramidite
  • HNSA -nitrobenzene sulfonic acid sodium salt
  • the analysis consisted of dissolving a small amount of the precipitate in 10 mM phosphate buffer at pH 7.0 and measuring absorbance at 406 nm; this reading provides the amount of unreacted free HNSA which is the contaminating material in the crude HNSA ester. Addition of very small amounts of concentrated strong base (5N NaOH) hydrolyzed the ester. A second reading was . taken. Subtraction of the first reading from the second yielded the amount of ester in the original material. For purification purposes, the solid was dissolved in DMF, placed on a LH20 Sephadex column and eluted with DMF so that the ester was separated from the contaminating free HNSA.
  • HNSA Ester and Horseradish Peroxidase (HRP) HNSA Ester and Horseradish Peroxidase
  • a thiol-functionalized oligomer was prepared using the following 19-mer which had been synthesized on a Biosearch 8630 DNA Synthesizer: d(TGTTTGCCTGTTCTCAGAC) .
  • the sulfhydryl functionalizing reagent obtained 0 in Example 1(b) was mixed with a solution of the oligomer
  • the tritylthio oligomer was purified by a 5 standard chromatographic technique ' using a preparative PRP-1 column and the following solvent gradient (wherein solvent “A” designates CH.XN and "B” designates 5% CH 3 C in 0.1M TEAA, pH 7.3): (1) A, 10% —> 40%, 15 in.; (2) A, 40% —> 100%, 15 min.; and (3) A, 100%, 5 min. The tritylthio oligomers eluted after about 20 minutes.
  • the purified tritylthio oligomer so obtained was detritylated using silver nitrate and dithiothreitol (0.1 M and 0.15 M, respectively, in 0.1 M TEAA, pH 6.5).
  • the ditritylated oligomer was then passed through a G-25 (NAP-10) column, concentrated under vacuum to ap ⁇ proximately 100 ⁇ l r and used right away in the following conjugation reaction.
  • the mal-sac HRP complex prepared in Example 7 was aliquoted into the thiooligomer to give a final volume of 800 ul.
  • the individual reaction vessels were allowed to remain at room temperature for ap ⁇ proximately one hour, and then at about 4 C for two days, at which point the four conjugates were removed and puri ⁇ fie on a DEAE Nucleogen column using the following solvent gradient ("B” designates 20 mM Na 2 P0., pH 6; "C” designates 20 mM Na 2 P0 4 + 1M NaCl, pH 6): (1) B, 0 —> 100%, 30 min.; (2) C, 100%, 10 min.; and (3) C, 100 —> 0%, 5 min..

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  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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EP88908882A 1987-10-02 1988-09-19 Ein kovalentes oligonucleotid-meerrettich-peroxidase-konjugat Withdrawn EP0372002A1 (de)

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US103978 1987-10-02

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CA (1) CA1300532C (de)
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WO1990010718A1 (en) * 1989-03-10 1990-09-20 Millipore Corporation Sequencing nucleic acids using chemiluminescent detection
US5272077A (en) * 1989-09-12 1993-12-21 Eastman Kodak Company Method for preparing a covalent conjugate of an oligonucleotide and an enzyme
US5082780A (en) * 1989-09-12 1992-01-21 Eastman Kodak Company Oligonucleotide-enzyme conjugate that can be used as a probe in hybridization assays and polymerase chain reaction procedures
CA2032331A1 (en) * 1990-01-22 1991-07-23 Annie L. Wu Method and kits for detecting human leukocyte antigen dna
WO1992000989A1 (en) * 1990-07-10 1992-01-23 Imperial Chemical Industries Plc Non-isotopic nucleic acid labelling method
US5633230A (en) * 1990-10-24 1997-05-27 Allelix Biopharmaceuticals, Inc. Treatment of cytomegalovirus infection
JPH06502766A (ja) * 1990-11-14 1994-03-31 アクゾ・ノベル・ナムローゼ・フェンノートシャップ ポリスチレン支持体ベース−サンドイッチハイブリダイゼーションアッセイ法を用いる核酸の非同位体検出およびそれに用いる組成物
US5495006A (en) * 1991-09-27 1996-02-27 Allelix Biopharmaceuticals, Inc. Antiviral polynucleotide conjugates
WO2017097973A1 (en) * 2015-12-09 2017-06-15 Universitaet Konstanz Modified nucleosides

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GB8306426D0 (en) * 1983-03-09 1983-04-13 Malcolm A D B Detecting polynucleotide sequence
US4626501A (en) * 1983-09-02 1986-12-02 Integrated Genetics, Inc. Labeled DNA
US4657853A (en) * 1984-09-14 1987-04-14 E. I. Du Pont De Nemours And Company Immunoassays utilizing covalent conjugates of polymerized enzyme and antibody
GB8509880D0 (en) * 1985-04-17 1985-05-22 Ici Plc Testing device

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Title
See references of WO8902932A1 *

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CA1300532C (en) 1992-05-12
WO1989002932A1 (en) 1989-04-06
IL87880A0 (en) 1989-03-31
IL87880A (en) 1993-08-18
JPH0630574B2 (ja) 1994-04-27

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