JPH0630574B2 - Oligonucleotide-horseradish peroxidase covalent conjugate - Google Patents

Description

TECHNICAL FIELD The present invention relates generally to DNA hybridization probes, and more particularly to covalent conjugates of oligonucleotides and horseradish peroxidase (HRP).

BACKGROUND OF THE INVENTION Non-isotopic labeled synthetic DNA fragments have wide application in molecular biology, for example in the fields of DNA sequencing, DNA probe based diagnostics and the like. The conjugates disclosed herein are prepared using reagents that facilitate the labeling of the oligonucleotide with a particular group by introducing one or more sulfhydryl groups at one or more hydroxyl sites in the oligonucleotide. To be done.

Sulfhydryl group at 5'-end of synthetic oligonucleotide
How to introduce is known. Connolly,Nuc.Acids R
es.13(12): 4485-4502 (1985) andNuc. Acids Res.15
(7): 3131-3139 (1987) is 2-mercaptoethanol,
3-mercaptopropan-1-ol and 6-mercap
Toxan-1-ol S-trityl-0-methoxy
A morpholinophosphine derivative, ie of the formula:(Wherein x is 2, 3 or 6) is used to synthesize a synthetic DNA with sulfhydryl.
It describes how to introduce minutes.

However, this and other prior art methods have one or more of the following drawbacks.

(1) Instability of the structure in which a short spacer chain connecting the 5'-end of an oligonucleotide to a sulfhydryl, amino or hydroxy group is derivatized, ie, a solid support for the oligonucleotide or bulky The proximity of the labeled species causes structural hindrance and thus prevents the use of derivatized oligonucleotides in probe-based applications; (2) the 5'-end of the oligonucleotide to a sulfhydryl, amino or hydroxy group. The linking hydrophobic spacer strands cause solubility problems in aqueous solvents commonly used in DNA probe-based methods; (3) commonly used functionalizing reagents are often commonly used DNA synthesis. The main reason for not complying with the law is
This is because the functionalizing reagents are incompatible with the reagents and the solvents typically used with them; (4) commonly used functionalizing reagents are often difficult to synthesize in high yields and are complex and complex. It is a stepwise reaction; (5) certain known reagents need to be treated with multiple activators immediately before use; (6) commonly used functionalizing reagents are terminal sulfhydryl, amino or hydroxy moieties and oligos. Not only does it not allow "sticking" of multiple spacer chains to increase the distance to the nucleotide chain, but generally they do not allow multiple functionalization along the oligonucleotide chain; (7) Commonly used Functionalization reagents generally do not allow functionalizations at positions other than the 5'-hydroxyl terminus; and (8) conventional functionalization reagents and sometimes deprotection under vigorous conditions. Principal cities, the deprotection reaction can not often easily detected.

Therefore, there is a need in the art for oligonucleotide functionalizing agents that takes into account the above points. The present invention relates to some novel functionalizing reagents that overcome the above problems. More specifically, the present invention is directed to a method of "derivatizing" sulfhydryl-functionalized oligonucleotides that can be prepared using the novel oligonucleotide-functionalizing reagents as described.

The covalent bond conjugate between the oligonucleotide and the labeling enzyme
It is described in the literature. For example, Jablonski et al.Nuc.Aci
ds Res.14(15): 6115-6128 (1986) is a homobifunctional reagent disa.
Oligonuku produced using cuccinimidyl suberate
Covalent conjugation of reotide with alkaline phosphatase
It describes the body. Renz and Kurz,Nuc. Acids Res.12
(8): 3435-3445 (1981) is a polymer having a molecular weight of about 1400.
Oligonucleotides using a thyleneimine spacer
A covalent complex with HRP is described. In addition, Ru
th and Jablonski,Nucleosides and Nucleotides 6(1
And 2): 541-542 (1981) between the oligomer and the enzyme.
Oligodeoxynucleotidation with a 19-atom spacer chain at
Describing a conjugate of Otide and alkaline phosphatase
There is. Although these probes have been used successfully,
However, it is more stable and develops faster,
More effective and faster monitoring means
It is desired to provide.

DISCLOSURE OF THE INVENTION Accordingly, a first object of the invention is to provide stable, easily monitorable "derivatized" oligonucleotides comprising sulfhydryl-functionalized oligonucleotides covalently conjugated to HRP. is there.

Another object of the present invention is to provide a method for producing such a covalent bond conjugate.

Another object of the invention is to provide a method of using these conjugates in DNA probe based applications.

Other objects, advantages and novel features of the present invention will be set forth in part in the description that follows, and in part will be known to those skilled in the art based on subsequent testing or by practice of the invention. . The objects and advantages of the invention will be realized and attained by means of the instruments and combinations particularly pointed out in the appended claims.

In a preferred embodiment of the invention, an oligonucleotide functionalizing reagent is used to functionalize the oligonucleotide chain at the hydroxyl groups contained therein in order to introduce sulfhydryl groups. The coupling reaction is
Beaucage and Caruthers, Tetrahedron Lett . (1981) 2
2 : 1859-1862, using standard techniques for coupling phosphoramidites to the hydroxyl groups of oligonucleotides. After functionalization, the oligonucleotide was labeled with HRP as described.
Is derivatized at the new sulfhydryl site.

Aspects of Carrying Out the Invention "Sulfhydryl functionalization" or simply "functionalization" as used herein refers to the introduction of a protected or unprotected sulfhydryl moiety into an oligonucleotide chain. means. The sulfhydryl group introduced by functionalization is separated from the oligonucleotide chain by a spacer chain as described herein.

"Derivatization," as used herein, refers to a species in which a functionalized oligonucleotide is a detectable species in an added sulfhydryl, amino, or hydroxyl moiety, that is, a species that serves as a label in probe-based applications. Means to react with. Thus, a "derivatized" oligonucleotide is one that is detectable by a "derivatized" species. As mentioned above, the derivatized species in the present invention is the enzyme horseradish peroxidase.

An "oligonucleotide" as used herein is a nucleotide, typically a single or double stranded deoxyribonucleotide monomer unit, typically one.
It is a main chain. Although the reagents and methods of the present invention can be used in combination with single nucleotide monomers or full length DNA strands, "oligonucleotides in the present invention are typically about 2 to about 400 monomer units, and more typically Is a single strand of about 2 to about 100 monomer units for most probe-based applications.Allele-specific oligonucleotides (or "AS").
The optimal length for use as "O") is about 135-21 base pairs.

The use of derivatized oligonucleotides in "probe-based" applications is intended to mean the use of labeled strands to detect or quantify oligonucleotide segments or sequences in an analyte.

A "protected" free sulfhydryl group is such that the resulting protected group is not susceptible to any type of chemical reaction during the synthetic step or steps in which the protecting group is present.
It is a group that reacts with the protective component.

By "stability" of a functionalized or derivatized oligonucleotide chain is meant the substantial absence of steric hindrance and chemical stability in most probe-based applications.

"Lower alkyl" or "lower alkoxy" means about 1-6
Respectively, more typically about 1 to 3 carbon atoms and alkyl and alkoxy substituents, respectively.

2. Structure of Functionalizing Reagent The sulfhydryl functionalizing reagent used to produce the probe of the invention, i.e. the covalently bound oligonucleotide-HRP conjugate, has a phosphoramidite moiety at one end, which moiety is linked via a hydrophilic spacer. And a substantially linear reagent linked to the other end with a protected or unprotected sulfhydryl moiety. These functionalizing reagents generally have the following structural formula: Wherein R is a protected or unprotected amino, sulfhydryl or hydroxy moiety; R ^* is hydrogen, -CH ₂ OH, or the following formula: Wherein X ¹ , X ² , X ³ , X ⁴ , X ⁵ and X ⁶ may be the same or different and are selected from the group consisting of hydrogen, lower alkyl and lower alkoxy. R ¹ and R ² are independently selected from the group consisting of hydrogen and lower alkyl groups; R ³ is β-cyanoethyl or methyl; the Q component is of the formula: —O—, —NH -, -S-, as well as Selected from the group consisting of and may be the same or different; n ', n "and n are in the range 2-10 (inclusive of 2 and 10)
And n is an integer in the range of 2-30 (inclusive of 2 and 30)].

Structural formula (4): (Wherein R, R ^* , R ¹ , R ² , R ³ and n are as described above) is an example of a particularly preferred embodiment. The hydrophilic spacer chain is, in the case shown above, a polyether linkage formed, for example, from polyethylene glycol.
[In another embodiment included in the general structural formula (4), the spacer chain is polyethylene glycol or the like, or Texaco.
It can also be formed from poly (oxyaralkyleneamine) such as JEFFANINES ™ sold by Texaco Chemical Co.

If it is desired to couple the functionalizing reagent to the oligonucleotide chain at any position, generally a nucleoside phosphoramidite can be coupled to the chain and the R component is a protected sulfhydryl component. The protecting group is chosen such that the sulfhydryl moiety is intact during the sulfolamidite coupling step, i.e. while the reagent phosphoramidite group reacts with the hydroxyl moiety on the oligonucleotide chain. Conditions for this reaction include, for example, Beaucage and Caruthers, Tetrahedro.
n Lett . 22 : 1859-1862 (1981) and is used in a conventional method for synthesizing DNA via the so-called "phosphoramidite" route.

Examples of particularly preferred sulfhydryl protecting groups are the following R: Or It is represented by.

It should be understood that the protecting groups exemplified above are merely examples, and any sulfhydryl protecting group can be used as long as it meets the above-mentioned "protection" criteria.

Phosphoramidite group: The other end of the functionalizing reagent as defined by is most often selected to couple to the terminal 5'-hydroxyl group of the growing or completed oligonucleotide chain. As mentioned above, R ¹ and R ² are hydrogen or lower alkyl and may be the same or different, and in a particularly preferred embodiment both R ¹ and R ² are isopropyl. R ³ is methyl or β-cyanoethyl,
In a particularly preferred embodiment R ³ is β-cyanoethyl. The use of phosphoramidites as coupling means is well known in the field of DNA synthesis and for a more detailed description Beaucage and Caruthers (1981).
We will have to mention (above).

Spacer chain (9): Is a hydrophilic chain, where n, n ', n "and n are integers having the above values.

In a preferred embodiment represented by formula (4), the spacer chain comprises a polyether component: Where n is typically 2-30, more typically 2-20, but in some cases, ie where increased distance between the derivatizing component and the oligonucleotide chain is desired. Can be greater than 30.

The optimum value for n provides a spacer chain having about 8 or more total carbon atoms along the length of the spacer chain. The length of the spacer chain is highly related to the effectiveness of the reagents of the invention, the larger the distance between the sulfhydryl and the oligonucleotide chain, (1) facilitating the coupling of the test to DNA, (2) avoid steric hindrance that would interfere with hybridization and destabilize the functionalized or derivatized oligonucleotide chain, and (3)
It stimulates a "solution" type environment to enhance the freedom of movement of the derivatized sulfhydryl components. The fact that the spacer strand is hydrophilic also enhances the solubility of the functionalized or derivatized oligonucleotide strand in aqueous medium.

R ^* is hydrogen, an aromatic substituent represented -CH ₂ OH, or by (3). If R ^* is (3), this is the chromogen cation: Are selected so that they can be monitored after release. That is, after coupling of the functionalizing reagent to the DNA, deprotection will result in the cation (11) in solution. Examples of particularly preferred substituents include dimethoxytrityl (DMT), that is, R ^* is -CH ₂ -O-DMT.

In a preferred embodiment, R ^* is attached to the carbon atom adjacent to the phosphoramidite group, as shown by structural formulas (2) and (4), but as shown in formula (2), R ^* is It may be attached to one or more other carbon atoms along the spacer chain.

3. Use of Novel Reagents to Functionalize Oligonucleotide Strands In general, the coupling reaction between novel functionalizing reagents and hydroxy group-containing compounds is described by the following scheme: Scheme I Is represented by In Scheme I, X is typically an oligonucleotide chain. The reaction conditions are as previously described, and in particular Beaucage and Caruthers (1981).
The same as that used in the phosphoramidite route to DNA synthesis, as described by (supra).

The compound (12) is deprotected as follows. When R ^* is represented by formula (3), conversion to unprotected hydroxy is carried out by treatment with acid. The protected sulfhydryl substituent on "R" is accomplished by treatment with, for example, silver nitrate.

Polyfunctionalized oligonucleotides plurality of R ^* sites [R ^* is expressed in -CH ₂ OH, or Formula (3)] is possible by using the. After acid deprotection, further functionalization can be achieved by reaction at the deprotected hydroxyl site. That is, the functionalized oligonucleotide (13): , For example, by deprotection of R ^* and deprotection using standard phosphoramidite coupling methods-
Further functionalization in the CH ₂ OH, -OH component is described by the following formula (1
Four): A compound represented by

Multiple functionalization at multiple hydroxyl groups along the oligonucleotide chain is also possible using the same chemistry.

4. Synthesis of Functionalized Reagents We have developed various routes to new reagents. For simplicity, let us consider the synthesis of functionalizing reagents by the exemplary structural formula (6) rather than by the general structural formula (4). However, it should be understood that the synthetic methods described generally apply to compounds represented by (4) substantially similarly. In the first embodiment, where the functionalizing reagent to be synthesized is an amine functionalizing reagent, Scheme II is as follows.

Stage (1) is Mitsun well known in the art.
Shows obu reaction. In summary, this reaction is compound (15),
The mixture of (16), (17) and (18) is used in a polar organic solvent for at least several hours, preferably overnight (Example 1).
checking). Compound (19) is isolated and coupled to a phosphoramidite where X is a halogen, preferably chlorine, as follows. A molar excess of phosphoramidite is added to compound (19) in a suitable solvent, also preferably a polar organic solvent, under an inert atmosphere. Compound (20) is isolated, for example, by column chromatography.

Another method for synthesizing an amine functionalizing reagent, comprising:
What can also be used to obtain the sulfhydryl functionalizing reagent is represented by Scheme III below.

In Scheme III, steps 1a-1c and 1a'-1
b'denotes an alternative route to intermediate (21). Stage 1
In a-1c, the protected diol (21) is reacted with polyethylene glycol (15) and allyl bromide to form (19) by reaction at room temperature for at least about several hours, preferably overnight. Diols by reaction of (19) with osmium tetroxide under commonly known conditions
Formation of (20); and protection of the diol by reaction with 2,2-dimethoxypropane. Stage 1
a'-1b 'provides (21) via the reaction of tosylated glycol (21') with the solketal anion. Stage 2
Represents the Mitsunobu reaction shown in Scheme II, where R
Is as previously defined, while the acid treatment of Step 3 deprotects the diol. Step 4-1 is chromogen component (11)
(Where Y is represented by (11)) and step 4-2 introduces phosphoramidate. Both stages 4-
"X" in 1 and 4-2 is a halogen leaving group, preferably chlorine.

A third synthetic method specific for the preparation of sulfhydryl functional reagents is illustrated by Scheme IV.

Scheme IV In Scheme IV, step 1 is carried out at low temperature, preferably below 0 ° C. and the triphenylphosphine, diisopropylazodicarboxylate and S-trityl mercaptan are reacted overnight. Intermediate (24) is purified and phosphoramidate is added in step (2) and (25) is obtained in high yield. Here, “R” in Structural Formula (4) is shown as —S—Cφ ₃ (always φ = phenyl), but can actually be any protected sulfhydryl moiety.

5. Derivatization with HRP Functionalized oligonucleotide chains produced using the reagents described above are primarily useful in probe-based applications. Thus, the main purpose of introducing sulfhydryls into oligonucleotides is to allow derivatization at the site with a labeled species. The present invention is directed to derivatization with horseradish peroxidase enzyme.

The derivatized oligonucleotide of the invention is an anti-coalescence comprising an oligonucleotide chain covalently attached to HRP, the anti-coalescence having the following structural formula (26): Where R ^* , Q, 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 typically in the range of 2-100 monomer units. When the conjugate is used as an allele-specific oligonucleotide (ASO). The number of monomer units in the chain is preferably about 13-21.

In one embodiment, such a conjugate has the structural formula (2
7): (Wherein R ^* , X and n are as described above). The conjugate of formula (27) results from the coupling of sulfhydryl functionalizing reagent (4) to oligomer X.

The covalent bond conjugate (28) represented by the formula (28) has the scheme V
Manufactured by the method exemplified by.

Scheme V mal-sac-HNSA, an N-maleimido-6-aminocaproyl [mal-sac] derivative of 4-hydroxy-3-nitrobenzenesulfonic acid sodium salt [HNSA], and the corresponding mal-sac-HNSA HRP complex ( 28) is described later in Examples 6 and 7.

The thiolated oligonucleotide (29) is prepared as described in Example 8. Typically, the trityllithio oligonucleotide is derivatized just prior to use in the reaction of Scheme V.

The mal-sac HRP complex (28) is coupled to the thiolated oligonucleotide (29) by simple mixing, preferably below room temperature. The reaction mixture is kept at room temperature, for example at about 0 ° C., for at least overnight, and preferably for at least several days, at which point the covalent HRP conjugate.
(26) is isolated and preferably purified by chromatography.

Prior to use in probe-based applications, the conjugate is treated with phosphate buffer (optionally acid added) maintained at a pH of about 5.5 to about 7.5, preferably about 6.0, at about −10. ℃ ~ about 30 ℃ (but do not freeze the solution),
Optimally, hold at a temperature of about 4 ° C.

For use in hybridization, the conjugate solution is usually diluted with hybridization buffer (final concentration depends on the application) and standard hybridization techniques (eg Maniatis et al., Molecu.
lar Cloning , New York, Cold Spring Harbor Labor
atory, 1982). General methods are well known in the art, and typically (1)
Providing a covalent conjugate that is substantially complementary to the nucleotide sequence of interest of interest, ie, sufficiently complementary to allow hybridization;
(2) contacting the analyte of interest in solution with the covalent conjugate; and (3) the presence of the resulting nucleic acid complex
Detecting by measuring RP activity.

Generally, the covalent complex will hybridize to the analyte bound to the solid support and then be detected thereon.

In summary, there are many transition points for the novel HRP conjugates of the invention in probe-based applications. The main advantage is the relatively long hydrophilic spacer chain, which provides the optimum distance between the HRP and the oligonucleotide,
It ensures that the full biological activity of P is retained and enhances the efficiency of hybridization. The novel conjugates are also multiderivatized of one oligonucleotide due to the "R ^* " component, ie, two or more linked end-to-end or joined at various points within the oligonucleotide chain. To allow the binding of "spacer-HRP". Finally, unlike other enzyme / oligomer conjugates such as the alkaline phosphatase system, rapid color development enhances ease of detection.

While this invention has been described in connection with certain preferred embodiments thereof, it is understood that the foregoing description and examples are illustrative and not limiting of the scope of the invention. Other aspects, advantages, and modifications within the scope of the invention will be apparent to those skilled in the art.

Example 1. (A) Reaction of tetraethylene glycol with phthalimide [see scheme II, step (1)] Tetraethylene glycol (38.85g, 200mmole) and triphenylphosphine (52.46g, 200mmole) were dissolved in 200m dry THF. And then phthalimide (29.43g, 200mmole)
Was added. A solution of diethyl azodicarboxylate (DEAD) in 100 m of dry THF was added dropwise to the reaction mixture with freezing and stirring. The reaction mixture was stirred at room temperature overnight. Then the solvent was removed under reduced pressure and the residue was removed to 250 m.
Partitioned between water and 250 m diethyl ether. The aqueous layer was washed 5 times with 200m diethyl ether and dried under vacuum. The residue was dried by azeotropic distillation of toluene (3 x 100 m). Then got 25.89
g Purified on SiO ₂ column with ethyl acetate as eluent. The product fractions were collected and syrupy (11.75g; 3
Concentrate to 6.3 mmole; 18.2%) and crystallize it overnight.

The structure of the product obtained in (a) was determined by ¹ H-NMR to be Was confirmed.

(B) Synthesis of allyl derivative [Scheme III, step (1
See b)] To a solution of the alcohol (4.67 g, 14.4 mmole) obtained in step (a) in 100 m dry THF was added NaH (520 mg, 21.67 mmole). The mixture was stirred for 1 hour and then allyl bromide (1.9m, 2.61g, 21.67mmole) was added. The suspension was stirred overnight, at which point it was filtered and the solvent removed under reduced pressure. The residue was purified on a SiO ₂ column with a mixture of ethyl acetate and hexane (70:30) as eluent. Fractions containing the desired product were pooled and concentrated to 2.84 g (7.82 mmole, 54.3%) syrup.
The elemental analysis was as follows.

Calculated: C 62.80; H 6.93; N 3.85 Found: C 62.49; H 6.99; N 3.82.

The proposed structure of the resulting product is Met.

(c) Synthesis of the corresponding diol [Scheme III, step (1
See b)] Allyl ether (2.84 g, 7.82 mmol) and N-methylmorpholine N-oxide (1.83 g, 15.63 mmole) obtained in step (b) in 180 m DMF / water (8: 1). Osmium tetroxide (25 mg / m solution in t-butanol 8.13
m; 800 μmole) was added. The resulting amber solution was stirred at room temperature. After 48 hours, a solution of sodium hydrosulfite (2.13g) in water (10m) was added to the reaction mixture. The black precipitate and suspension that formed was stirred for 1 hour. The mixture was filtered and concentrated under reduced pressure. The residue was purified on a SiO ₂ column with a mixture of methylene chloride and methanol as eluent. The elemental analysis was as follows.

The proposed structure of the product is Met.

(D) Labeling with DMT The diol obtained in (c) above (1 g, 2.25 mmol
e) was coevaporated with anhydrous pyridine (2 × 15 m).
The dry residue was then dissolved in 25m anhydrous pyridine. DMT-Cl (0.92 g, 2.75 mmole) was added to this solution. The reaction is carried out at room temperature and until TLC (CH ₃ Cl: Me) appears.
It was monitored by OH about 97: 3).

After 1 hour, 10m of methanol was added and the reaction mixture was stirred for a further 10 minutes. Then quench the reaction with 10m of ice water and ethyl acetate (2 x 75m).
It was extracted with.

The organic layer was washed once with 5% NaHCO ₃ (50 m) and twice with saturated NaCl solution and dried over Na ₂ SO ₄ . The product was evaporated under reduced pressure to an oily residue.

The residue was chromatographed using the solvent system described above.
The final product was used in step (e) without further purification. Yield: 86.3% of theoretical amount (actual amount 1.51 g / theoretical amount 1.75 g). The proposed structure obtained at this stage is Met.

(E) Preparation of phosphoramidite 1 product of the product (1.0 g, 1.4 mmole) obtained in step (d) was added.
Dissolved in 0 m acid free chloroform and refreshed with dry argon. Place in a 250 m round bottom flask. To this solution, 0.72 g (5.6 mmole) of ((CH ₃ ) _2-
CH] _2- N-Et was added. Then the phosphoramidite: 0.66g (2.8mmole) was added using a syringe over 2 minutes. The reaction was carried out at room temperature and under argon.
After 1 hour, the mixture was 250m with 50m ethyl acetate.
To a separatory funnel and extracted 4 times with saturated NaCl solution. The organic layer was dried over Na ₂ SO ₄ and evaporated under vacuum to an oily residue. This residue is 1% Et ₃ N in ethyl acetate.
Chromatographed by. Yield: 48.4% of theory
(Actual amount 0.61 g / theoretical amount 1.26 g).

Example 2. Following essentially the same method as described in Example 1,
However, in this case the tetraethylene glycol starting material was not first reacted with phthalimide.

(A) Synthesis of allyl derivative of pentaethylene glycol Pentaethylene glycol (5.65
g, 20mmole) in a solution of t-butanol potassium salt (2.24
g, 20 mmole) was added. The mixture was stirred for 30 minutes.
Then 18-crown-6 (53 mg, 0.2 mmole) was added.
The mixture was stirred for a further 30 minutes and then allyl bromide (2.42g, 1.73m, 20mmole) was added. A white precipitate, believed to be potassium bromide, was observed and stirring was continued overnight. The reaction mixture was filtered through a Whatman GFB filter, adsorbed onto 8 g of SiO ₂ and fractionated on a SiO ₂ column using a mixture of methylene chloride and acetone (1: 1) as solvent. 4.28g pooled fraction
This gave (13.28mmole, 66.4%) product. The elemental analysis was as follows.

Calculated: C 55.88; H 9.83 Found: C 55.56; H 9.76.

The proposed structure of the product is Met.

(B) Synthesis of the corresponding diol Mixture of acetone and water (8: 1) 270 m Middle allyl ether prepared in step (a) (4.28 g, 13.28 mm
ole) in N-methylmorpholine (3.11 g, 4.6 m, 2
6.55mmole, 2eq) followed by osmium tetroxide (25mg / m in t-butanol; 338mg; 13.5m; 1.33
mmole (0.1 equiv.) was added. The reaction mixture was stirred overnight. The next morning, a solution of 15m sodium hydrosulfite (3.62g) in water was added. After stirring for 45 minutes, the suspension was filtered through a Whatman GFB filter.
The solvent was evaporated, the residue taken up in methanol and the suspension filtered. The filtrate was concentrated to an amber syrup, which was then purified on SiO ₂ using a mixture of methylene chloride, methanol and acetic acid (80: 20: 5) as eluent. Fractions containing product were concentrated to 3.3 g
The product (9.26mmole, yield 69.7%) was obtained.

The proposed structure of this product is Met.

(C) Triol prepared in step (b) (3.3 g, 9.
26mmole) in 60m of acetone, and cupric sulfate
(45 g, 28.20 mmole) was added. It was added 60 m H ₂ SO ₄ in the bluish tinged resulting suspension, a solution has changed to yellow at this point. The flask was stoppered and stirred over the weekend. The suspension was then filtered through a Whatman GFB filter and the filtrate treated in 2.5 g Ca (OH) ₂ for 1 hour. The suspension was filtered again and the filtrate was concentrated and purified on a SiO ₂ column. Chloroform /
Methanol (97: 3) was passed through and then again chloroform / methanol (8: 1). Column fractions were pooled to give 3.03g (7.64mmole, 82.5%) of product.

The proposed structure of the product is Met.

Example 3. Was synthesized as follows.

(a) Hexaethylene glycol (10.0 g, 35.40 mmole) was coevaporated with anhydrous pyridine (3 x 25 m) and then dissolved in 100 m of it. DMT-Cl (13.1
7g, 38.94mmole) was added. The reaction was carried out at room temperature and TLC (CHCl ₃ : MeOH ca. 8: 1) until product appeared.
Monitored by. After 2 hours, 25m of methanol was added and the reaction mixture was stirred for a further 15 minutes. The reaction was then quenched with 50m ice water and extracted with ethyl acetate (3 x 150m). The organic layer was washed with 5% NaHCO ₃ (2 × 100 m) and saturated NaCl (2 × 100 m).
), Dried over Na ₂ SO ₄ and then evaporated to an oily residue (yellowish). The oily residue was chromatographed on a silica gel column (400g). The column was eluted first with CHCl ₃ / MeOH (ca 97: 3) and then with CHCl ₃ / MeOH (ca 90:10). The fractions were combined and evaporated to dryness to give an oily residue. The resulting material has the following structure: And was used in the synthesis of the corresponding phosphoramidites without further purification.

(B) 2.0 g (3.40 mmole) of the compound obtained in (a), 1.6 g (6.80 mmole) phosphoramidite: And it was repeated method of Example 1 (e) using [(CH _{3) 2} -CH] -N-Et of 1.76g (13.60mmole). Elemental analysis of the product was as expected for C ₄₂ H ₆₁ N ₂ O ₁₀ PxH ₂ O.

Calculated: C 63.49; H 7.93; N 3.52. Found: C 63.36; H 7.95; N 4.11.

Yield: 85.4% of theory (2.28g / 2.67g).

Example 4. (a) Compound: [Compound (26), see scheme IV, step (1)] was synthesized as follows. 75m dry THF
Azodicarboxylate (NCOOCH (C) was added to a solution of medium triphenylphosphine (7.87g, 30mmole) at 0 ° C with stirring.
_{H 3)) 2 (6.07g,} 30mmole) was added. After 1 hour, tetraethylene glycol (5.83g, 30mmole) in 10m dry THF
Solution was added. All material dissolved gave a pale yellow solution. After 1 hour, a solution of mercaptan φ ₃ C-SH in 20 m of dry THF was added dropwise with cooling and stirring.
The reaction mixture was stirred overnight and the solvent removed under reduced pressure. The residue was applied to a SiO ₂ column and methylene chloride and then a mixture of methylene chloride and CH ₃ CN (2:
It fractionated using 1). CH2 as the eluent on SiO ₂
Rechromatographed with ₃ CN and the product removed from φP = 0 by taking a small (about 15 m) fraction. Fractions were pooled, 5.22 g (11.53 mmole, 38.
4% overall yield, 77% of theory) was obtained. The results of elemental analysis were as follows.

Calculated: C 71.65; H 7.12; S 7.08 Measured: C 71.32; H 7.21; S 7.15.

(B) Next, the corresponding phosphoramidite: The reaction product of step (a) (4.22 g, 9.30 mmole), phosphoramidite, according to the method described in Example 1 (e): (4.40g, 18.60mmole), and ((CH ₃ ) ₂ CH) ₂ N-Et (4.81g, 3
7.20 mmole). Yield: 75.3% of theoretical amount (4.5%
7g / 6.07g).

Example 5. (a) Compound: Was synthesized as follows. 75m of dry THF triphenylphosphine (7.87 g, 30 mmole) in a solution of 0 ℃ of stirring diisopropyl azodicarboxylate (NCOOCH (CH ₃₎₎ with ₂ (6.07 g, 30 mmole) was added. After 1 hour, tetraethylene glycol in 10m dry THF
A solution of (5.83g, 30mmole) was added. All material dissolved gave a pale yellow solution. After 1 hour, 20m dry TH
A solution of mercaptan φ ₃ C-SH in F was added dropwise with cooling and stirring. The reaction mixture was stirred overnight and the solvent was removed under reduced pressure. The residue was applied to a SiO ₂ column and fractionated with methylene chloride followed by a mixture of methylene chloride and CH ₃ CN (2: 1). The material was rechromatographed on SiO ₂ with CH ₃ CN as the eluent, and the product was removed from φ ₃ P = 0 by taking a small (about 15 m) fraction. Fractions were pooled to 5.22g (11.53m
mole; overall yield 38.4%; 77% of theory). The elemental analysis was as follows.

Calculated: C 71.65; H 7.12; S 7.08 Measured: C 71.32; H 7.21; S 7.15.

(b) Phosphoramidite: The product (4.22 g, 9.30 mmole) obtained in step (a) of Example 1 was dissolved in 10 m of acid-free chloroform and placed in a 250 m round bottom flask refreshed with argon. 0.72 g (5.6 mmole) of [(CH ₃ ) ₂ CH] ₂ N-Et was added to this solution. Then the phosphoramidite: (0.66g, 2.8mmole) was added with a syringe over 2 minutes. The reaction was carried out at room temperature under argon. After 1 hour, the mixture was transferred to a 250 m separatory funnel with 50 m ethyl acetate and extracted 4 times with saturated NaCl solution.
The organic layer was dried over Na ₂ SO ₄ and evaporated under vacuum to an oily residue. The residue was chromatographed with 1% Et ₃ N in ethyl acetate.

Example 6. Preparation of mal-sac-HNSA ester 1 molar equivalent (2.24 g) of 4-hydroxy-3-nitrobenzenesulfonic acid sodium salt (HNSA) was added at 1 molar equivalent (2.06 g).
g) dicyclohexylcarbodiimide and 1 molar equivalent
(2.10 g) N-maleimido-6-aminocaproic acid and 25
m in dimethylformamide at room temperature. A white precipitate of dicyclohexylurea formed. The precipitate was filtered off and 300 ml of diethyl ether were added to the mother liquor. A rubbery solid precipitated from the mother liquor after about 10 minutes to 4 hours. This solid was found to contain 58% active HNSA ester and 42% free HNSA.

Analysis was performed by dissolving a small amount of the precipitate in 10 mM phosphate buffer (pH 7.0) and measuring the absorbance at 406 nm.
This reading gives the amount of unreacted free HNSA, which is the crude HNSA.
It is a contaminant in the ester. Addition of a very small amount of strong base (5N NaOH) hydrolyzed the ester.
I took the second reading. The difference between the second reading and the first reading gave the amount of ester in the original material. For production purposes, the solid was dissolved in DMF, placed on a LH20 Sephadex column, and the ester separated from the entrapped free HNSA by elution with DMF. The progress of purification was monitored by thin layer chromatography using chloroform, acetone and acetic acid (6: 3: 1 = v: v: v) as the eluting solvent. The product was positively identified as mal-sac HNSA by its activity of reacting with amines. The amount of crude ester produced was calculated to be about 30% of theory and the material produced consisted of 99% ester.

The ester thus obtained was found to be fully soluble in water and stable in water for several hours without the addition of nucleophile. The purified ester was found to be stable for long periods when stored in the dry state.

Example 7. Preparation of conjugate of mal-sac HNSA ester and horseradish peroxidase (HRP) An amide of mal-sac HNSA ester and HRS was prepared as follows.

40 mg (1.0 μmole) total HRP (Sigma Chemical Co.)
Was dissolved in 0.5m 0.1M phosphate buffer (pH 7.0),
The total amine concentration was 3.7 x 10 ^-3 M. Next, Example 6A
5 mg (1.1 × 10 ⁻⁵ mole) of the mal-sac HNSA ester of Example 5A calculated from the data of Example 1 was dissolved in 0.5 m of HRP solution. The mixture is stirred at room temperature and HRP
Fractions (2.8 m) were collected on a Pharmacia G-25 column and used as a solvent was 0.1 M phosphate buffer (pH 6.0).

Example 8. Manufacture of HRP-oligonucleotide conjugates The following 19-synthesized with Biosearch 8630 DNA synthesizer
Thiol-functionalized oligomers were prepared using mer: d (TGTTTGCCTGTTCTCAGAC).

The sulfhydryl functionalizing reagent obtained in Example 1 (b) was mixed with a solution of oligomers and standard phosphoramidite coupling conditions [eg Beaucage and Caruthers.
(1981), supra] and coupled to it below.

Trityl thio-oligomer, PRP-column for preparative,
And the following solvent system [where solvent "A" is CH ₃ CN,
And “B” is 5% CH ₃ CN in 0.1M TEAA (pH 7.2)]: (1) A: 10% → 40%, 15 minutes; (2) A: 40%
→ 100%, 15 minutes; and (3) A: 100%, 5 minutes. The tritylthio oligomer elutes after about 20 minutes.

The purified tritylthio oligomer thus obtained was mixed with silver nitrate and dithiothreitol [0.1M TEAA (pH6.5)].
Detritylation with 0.1 M and 0.15 M respectively. The ditritylated oligomer was then passed through a G-25 (NAP-10) column, concentrated in vacuo to about 100μ and used immediately in the next conjugation reaction.

The mal-sac HRP complex prepared in Example 7 (700 μm
Was partitioned into thio-oligomers to give a final volume of 800μ. The individual reaction vessels were held at room temperature for about 1 hour and then at about 4 ° C. for about 2 days, at which point the four conjugates were removed and the following solvent gradient (““ “B” is 20 mM Na ₂ PO ₄ (pH 6); “C” is 20 mM
Means Na ₂ PO ₄ + 1M NaCl (pH 6)]: (1)
B: 0 → 100%, 30 minutes; (2) C: 100%, 10 minutes; and (3) C: 100% → 0%, 5 minutes. The remaining unconjugated HRP and oligomer are each about 2
After about 15 minutes and about 15-40 minutes (depending on the size of the oligomer), the conjugate was similarly eluted after about 15-40 minutes (also depending on the size of the oligomer).
The identity of the product was confirmed by UV spectroscopy by monitoring the peak absorbance of the oligomer (260 nm) and HRP heme group (402 nm).

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Orches, Fred Tee. New York, USA 14617, Rochester, Stanton Lane 216

Claims (8)

[Claims] 1. The following structural formula: (Wherein R ^* is hydrogen or —CH ₂ OH; n ′, n ″ and n are integers in the range of 2 to 10 (inclusive of 2 and 10); n is 2 to 30 (2 and 30) A covalent conjugate of an oligonucleotide chain with horseradish peroxidase, wherein X is an integer in the range; and X is an oligonucleotide chain). 2. The conjugate according to claim 1, wherein R ^* is hydrogen. 3. Useful as an allele-specific oligonucleotide, and wherein said oligonucleotide chain has about 13 to about
The conjugate of claim 1 having a length of about 21 monomer units. 4. The following structural formula: Wherein R ^* is hydrogen or --CH ₂ OH; n is an integer in the range 2 to 30 (inclusive of 2 and 30; and X is an oligonucleotide chain) A covalent conjugate of the chain with horseradish peroxidase. 5. The conjugate according to claim 4, wherein R ^* is hydrogen. 6. Useful as an allele-specific oligonucleotide, and wherein said oligonucleotide chain comprises from about 13 to
The conjugate of claim 4 having a length of about 21 monomer units. 7. The following structural formula: (Wherein R ^* is hydrogen or —CH ₂ OH; n ′, n ″ and n are integers in the range of 2 to 10 (inclusive of 2 and 10); n is 2 to 30 (2 and 30) And X is an oligonucleotide chain), wherein X is an oligonucleotide chain), and X has the following structure: Reacting a functionalized oligonucleotide having ## STR1 ## with a complex of mal-sac-HNSA ester and HRP under coupling conditions; 8. The functionalized oligonucleotide chain has the following structural formula: 8. The method of claim 7, wherein the method is represented by: