EP0372002A1 - A covalent oligonucleotide-horseradish peroxidase conjugate - Google Patents

Abstract

Covalent conjugates of oligonucleotide chains and a horseradish peroxidase (HRP) are disclosed. The oligonucleotide chains are provided with one or more sulfhydryl moieties using selected sulfhydryl functionalization reagents, and are coupled through the sulfhydryl groups to a complex of HRP and an activated ester such as mal-sac-HNSA . Functionalization reagents are structured to introduce a spacer moiety that optimizes the distance between the oligonucleotide chain and the HRP tag.

Description

A COVALENT OLIGONUCLEOTIDE-HORSERADISH PEROXIDASE CONJUGATE

Description

Technical Field

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) .

Background Art • 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.

Methods of introducing a sulfhydryl group at the 5' terminus, of synthetic oligonucleotides are known. For example, Connolly, in Nuc. Acids Res. 13( 12) :4485-4502

(,1985) and in Nuc. Acids Res. 15(7 ):3131-3139 (1987), describes a method of incorporating a sulfhydryl inoiety. into synthetic DNA using S-trityl-O-methoxy-

^■ morpholinophosphite derivatives of 2-mercaptoethanol, 3- mercaptopropan-1-ol and 6-mercaptohexan-l-ol—i.e., re¬ agents given by the formula

where x is 2, 3 or 6. Connolly further describes deriva- tization of the sulfhydryl-containing oligonucleotides with thiol-specific probes.

However, this and other prior art_. methods suffer from one or more of the following disadvantages:

(1) 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;

(2) 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; (3) 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; ₍4) Conventionally used functionalizing reagents are frequently difficult to synthesize in high yield, necessitating complex, multi-step reactions;

₍5₎ Certain known reagents require treatment with multiple activating agents immediately prior to use;

₍6) Conventionally used functionalizing reagents do not allow for "tacking on" of multiple spacer chains to increase the distance between the terminal sulfhydryl, amino or hydroxyl moiety and the oligonucleotide chains, nor, generally, do they allow for multiple functionalization along an oligonucleotide chain;

(7) Conventionally used functionalizing reagents do not generally allow for functionalization at positions other than at the 5' hydroxyl terminus? and (8) Conventionally used functionalizing reagents sometimes require deprotection under harsh conditions, in such a way that, frequently, the deprotection reaction is not readily monitorable.

There is thus a need in the art for oligonucleotide functionalizing reagents and methods which address these considerations. 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. Also, Ruth and Jablonski, in Nucleosides and Nucleotides 6_,(1&2) :541-542 (1981), disclose conjugates of oligodeoxynucleotides and alkaline phosphatase having a 19-atom spacer chain between the oligomer and the enzyme. While these probes have been used successfully, it would nevertheless be desirable to provide probes which are more stable and which generate color faster, thus yielding a more effective and more readily monitorable means of detection.

Disclosure of the Invention

It is accordingly a primary object of the present invention to provide a stable, readily monitorable, "derivatized" oligonucleotide which comprises a sulfhydryl functionalized oligonucleotide covalently conjugated to HRP.

It is a further object of the present invention to provide a method of making such covalent conjugates. - It is another object of the invention to provide a method of using these conjugates in DNA probe-based ap¬ plications.

Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become appar¬ ent to those skilled in the art on examination of the- -fol¬ lowing, or may be learned by practice of the invention. The objects and advantages of the invention may be real¬ ized and attained by means of the- instrumentalities and combinations particularly^" pointed out in the appended claims.

In a preferred embodiment of the invention, oligonucleotide functionalizing reagents are used to functional!ze an oligonucleotide chain at a hydroxyl group contained therein to introduce a sulfhydryl group. The coύpling 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. After functionalization, the oligonucleotide is derivatized at the new sulfhydryl site with HRP as will be described.

Modes for Carrying Out the Invention

1. Definitions

"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. As noted above, the derivatizing species herein is the enzyme horseradish peroxidase.

An "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.

Use of the derivatized oligonucleotides in "probe-based" applications is intended to mean use of the labelled chain to detect or quantify oligonucleotide seg¬ ments or sequences in a specimen.

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.

By "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. By "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.

2. Structure of the Functionalizing Reagents

The sulfhydryl functionalizing reagents used to prepare the probes of the present invention—i.e., the covalent oligonucleαtide-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

- structure

wherein :

R is a protected or unprotected sulfhydryl moiety;

* R is a hydrogen, -CH-OH, or a substituent hav- ing the formula

in which X¹, X², X³, X⁴, X⁵ and X⁶ 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;

3 R is £-cyanoethyl or methyl; the Q moieties are selected .from the group consisting of

-0-, -NH-, -S-, -NH- -NH- and may be the same or different; 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

-CH₂-CH₂{θ-CH₂-CH₂)-_nO-CH (4)

where R, R *, R1, R2, R3 and n are as given above. The hydrophilic spacer chain in such a case is a polyether linkage, e.g., as shown, formed from polyethylene glycol.

(In other embodiments encompassed by general, structure

(2), the spacer chain may also be formed from polypropylene glycol or the like, or from poly(oxyalkyleneamines) such as the Jeffamines sold by

Texaco Chemical Co.) When it is desired to couple the functionalizing reagent to an oligonucleotide chain, at any position, generally, that a nucleoside phosphoramidite could be coupled to the chain, 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. The condi¬ tions for this reaction are those used in the conventional method of synthesizing DNA via the so-called "phosphoramidite" route, described, for example, in Beaucag and Garuthers, Tetrahedron Lett. 22;1859-1862 (1981). Examples of particularly preferred sulfhydryl protecting groups 'are given by R=

It is to be understood that the aforementioned exemplary protecting groups are illustrative only, and that any number of sulfhydryl protecting groups may be used so long as the above-described "protecting" criteria are met.

The opposing, second end of the functionalizing reagent defined by the phosphoramidite group

is selected so as to couple, typically, to the terminal 5' hydroxyl of a growing or completed oligonucleotide chain. As noted above, 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. Use of the phosphoramidite group as a coupling means is well known in. the art of DNA synthesis, and reference may be had to Beaucage and Caruthers (1981), supra, for further descrip¬ tion on point.

The spacer chain (9)

is a hydrophilic chain wherein n, n', n' ' and n' ' ' are integers having values as set forth above.

In the preferred embodiment represented by formula (4), the spacer chain is the polyether moiety

-CH₂-CH₂{θ-CH₂-CH₂}_nO-CH₂-CH- (10)

wherein 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. providing greater distance between the 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

* substituent given by (3). Where R is (3), it is selected so that the chromogenic cation

is monitorable upon release. That is, after coupling of the functionalizing reagent to DNA, deprotection will yield cation (11) in solution. An example of a particularly, preferred substituent is dimethoxytrityl

(DMT)—i.e., : is -CH₂-0-DMT.

While in a preferred embodiment, as illustrated

* by structures (2) and (4), R is bonded to the carbon atom adjacent to the phosphoramidite group, it is also possible

* that R may be bonded to one or more other carbon atoms along the spacer chain, as illustrated by formula (2).

■3. ^■ Use of_. the^' Novel Reagents to Functionalize Oliqo- -nucleotide Chains

In general, the coupling reaction between the functionalizing reagents and a hydroxyl-containing compound may be represented by the following scheme:

X-OH^{' ♦} Reagent (2^

(Scheme I

(12) In Scheme I, 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.

Multiple functionalization of an oligonucleotide is possible by making use of multiple R sites where R is -CH-OH or given by formula (3). After .acid deprotection, further functionalization by reaction at the deprotected hydroxyl site is enabled. Thus, in the case of functionalized oligonucleotide (13), for example,

R-CH₂-CH₂{θ-CH₂-CH₂}_nO-CH₂-CH-O ( 3 )

R^*

deprotection of R and further functionalization at the -CH₂OH-OH moiety so provided, using a standard phosphoramidite coupling procedure, gives the compound of formula (14) :

Multiple functionalization at a plurality of hydroxyl groups along an oligonucleotide chain is also possible using the same chemistry.

5 4. Synthesis of the Functionalizing Reagents

The inventors herein have developed various routes to the novel reagents. For the purpose of simplic¬ ity, syntheses of the functionalizing reagents will be discussed in terms of exemplary structure (6) rather than

10 general structure (4). It is to be understood, however, that the synthetic methods described apply, in general, substantially identically to compounds represented by (4). In a first embodiment, where the functionalizing reagent to be synthesized is an amine functionalizing reagent, l^rι Scheme II may be followed:

HO-CH₂-CH₂{ -CH₂-CH₂}_nO-CH₂-CH₂-OH ^♦ (15)

20

Step (l) ø,P R-H

5

0

(18)

5 Scheme II 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.

An alternative method of synthesizing the amine functionalizing reagents herein, and one which may also be used to give the sulfhydryl functionalizing reagents, is given by Scheme III:

(continued next page)

R-CH₂-CH₂{o-CH₂-CH₂}_n -CH₂-CH₂-O-CH₂-CH-CH

(23) - OH OH

R'

NR¹R² (4)

R-CH₂-CH₂{o-CH₂-CH₂}_nCH₂-CH-OP ^₃ ^J

In Scheme III, steps la-lc and la'-lb' represent alternative routes to intermediate (21). In steps la-lc, the protected diol (21) is formed by: reaction of the polyethylene glycol with allyl bromide (reaction carried

• out at room temperature for at least about a few hours, preferably overnight) to give (19); reaction of (19) with osmium tetroxide to give diol (20) under conventional, known conditions; and protection of the diol by reaction with 2,2-dimethoxypropane. 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)

* (and may thus be omitted where R is hydrogen) and Step 4-

2 introduces the phosphoramidite. "X" in both Steps 4-1 and 4-2 is a halogen leaving group, preferably chlorine. A third synthetic method, specific for the production of sulfhydryl functionalizing reagents, is given by Scheme IV.

HO-CH₂-CH₂{θ-CH₂-CH₂}-_nO-CH₂-CH₂-OH ( 8)

_/NR¹R^:

X-R Step (2)

NDR³

Scheme IV

In Scheme IV, 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. Here, "R" of structure (4) is shown as -S-CO3 (0=phenyl throughout) but may in fact be any number of protected sulfhydryl moieties.

5. Derivatization with HRP 5 The functionalized oligonucleotide chains prepared using the above-described reagents are primarily useful in probe-based applications. That is, the primary purpose of introducing a sulfhydryl group into an oligonucleotide chain is to enable derivatization at that

10 site with a labeled species. The present application is directed to derivatization with the enzyme horseradish peroxidase.

The derivatized oligonucleotides of the- ^* present invention are conjugates comprising an oligonucleotide

15 chain covalently coupled to HRP, the conjugates given by the structure (26)

)

•25 wherein

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

30 typically in the range of about 2 and 100 monomer units. Where the conjugate is to be used as an ASO, as noted earlier, the number of monomer units in the chain is preferably about 13-21.

35 In an exemplary embodiment, the conjugates of the invention may be represented by the structure (27)

0-X

(27)

where R , X and n are as given above. The conjugates of formula (27) result from coupling of exemplary sulfhydryl functionalizing reagent (4) to oligomer X.

The covalent conjugates represented by Formula (26) are prepared by the procedure illustrated in Scheme V:

mal-sac HRP complex ( 28 ) ^'• ( 29 )

( 26 )

Scheme V Preparation of mal-sac-HNSA, i.e., the (N- maleimido-6-aminocaproyl [mal-sac] derivative of 4- hydroxyl-3-nitrobenzene sulfonic acid sodium salt [HNSA] ) and the corresponding mal-sac-HNSA HRP complex (28) is described in Examples 6 and 7 below.

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.

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.

For use in hybridization, 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).. The general procedure followed is well known in the art, and typically involves:

(1) providing a covalent conjugate according to the inven- tion, which 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.

Generally, the covalent conjugate hybridizes to an analyte that is attached to a solid support- and is then detected thereon.

In sum, the advantages of the novel HRP conjugates in probe-based applications are many. 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. Finally, in contrast to other enzyme/oligomer conjugates, e.g., alkaline phosphatase systems, ease of detection is enhanced by the rapid generation of color.

It is to be understood that while the invention has been described in conjunction with the preferred ^■specific embodiments thereof, that the foregoing descrip¬ tion as well as. the examples which follow are intended to illustrate and not limit 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 to which the invention pertains. Exampie 1 (a) Reaction of tetraethylene glycol with phthalimide (see Step(l), Scheme II): Tetraethylene glycol (38.85 g, 200 mmole) and triphenyl phosphine (52.46 g, 200 mmole) were dissolved in 200 L of dry THF, and phthalimide (29.43 g, 200 mmole) added. A solution of diethylazo dicarboxylate (DEAD) (34.83 g; 200 mmole) in 100 mL of dry THF was added dropwise to the reaction mixture, with cooling and stirring. The reaction mixture was stirred overnight at room temperature. Solvent was then removed under reduced pressure, and the residue partitioned between 250 mL of H~0 and 250 ' L of diethyl ether. The aqueous layer was washed five times with 200 mL of diethyl ether and concentrated under vacuum. The residue was dried by azeotropic distillation of toluene (3 x^* 100 mL) and weighed. 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.

The structure of the product obtained in (a) was confirmed by H NMR asJ

(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;

21.67 mmole) 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 an SiO_ column using a mixture of ethyl acetate and hexane (70:30) as eluant. Fractions containing the desired product were pooled and concentrated to a syrup weighing 2.84 g (7.82 mmole; 54.3%). Elemental analysis was as follows. Calc: C, 62.80; H, 6.93; N, 3.85. Found: C, 62.49; H, 6.99; N, 3.82.

Proposed structure of the product obtained:

(c) Synthesis of the corresponding diol (see Step lb, Scheme III) : To a solution of the allyl ether prepared in step (b) (2.84 g; 7.82 mmole)^' and N-methyl morpholine N-oxide (1.83 g; 15.63 mmole) in 180 L of DMF/ H-0 (8:1) was added osmium tetroxide (8.13 mL of a solu¬ tion 25 mg/mL in t-butanol; 800 t mole) The resulting amber solution was stirred at room temperature. After 48 hours, .a solution of sodium hydrosulfite (2.13 g) in water (10 mL)' was added to the reaction mixture. A black precipitate formed and the suspension was stirred for 1^' hour. The mixture was filtered and concentrated under reduced pressure. The residue was purified on; an SiO~ column using a mixture of methylene chloride- and methanol as the eluant. Elemental analysis was as follows. Calc: C, 62.80; H, 6.93; N, 3.85. Found: C, 62.49; H, 6.99; N, 3.82. Proposed structure of the product:

(d) Labelling with DMT: The diol obtained in part (c) (1.0 g; 2.50 mmole) was coevaporated with anhydrous pyridine (2 x 15 mL) . The dry residue was then dissolved in 25 L of the same. DMT-C1 (0.92 g; 2.75 mmole) was added to the solution. The reaction was car¬ ried out at room temperature and monitored by TLC (CH-«Cl:MeOH approximately 97:3) until appearance of the product.

After one hour, 10 mL of methanol was added and the reaction mixture was stirred for ten additional minutes. Next, the reaction was quenched with 10 L of ice water and extracted with ethyl acetate (2 x 75 L) .

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

This residue was chromatographed using the above solvent system. The final product was used without further purification in step (e) . Yield: 86.3% of theoretical (1.51 g actual / 1.75 g theoretical).

Proposed structure of the product obtained in this 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^' [(CH₃)₂~CH]₂-N-Et -was added. Then, the phosphoramidite

(0.66 g; 2.8 mmole) was added with a syringe over a two- minute period. The reaction was carried out at room temperature and under argon. After one hour, the mixture was transferred with 50 L of ethyl acetate in a 250 L separatory funnel and extracted with saturated NaCl solu¬ tion four times. The organic layer was dried over Na-SO. and evaporated down to an oily residue under vacuum-. This residue was chromatographed with 1% Et-.N in ethyl acetate. Yield.: 4.8.4% . of theoretical (0.610 g actual / 1.26 g theoretical) .

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.

(a) Synthesis of the allyl derivative of pentaethylene. glycol: To a solution of pentaethylene glycol (5,65 g; 20 mmole) in 100 mL of dry THF was added the potassium salt of t-butanol (2.24 g; 20 mmole). . The mixture was stirred for 30 minutes and 18-crown-6 (53 mg; 0.2 mmole) was added. The mixture was stirred for an ad¬ ditional 30 minutes and then allyl bromide (2.42 g; 1.73 mL; 20 mmole) was added. A white precipitate, presumably potassium bromide, was noted to form and stirring was continued overnight. The 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:

(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. After 45 minutes of stirring, 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_ using a mixture of methylene chloride, methanol, and acetic acid (80:20:5) [?] as eluant. The fractions containing product were pooled and concentrated to yield 3.3 g (9.26 mmole; 69.7% yield) product.

Proposed structure of the product:

(c) The triol prepared in step (b) (3-3 g; 9.26 mmole) was taken up in 60 mL acetone and cupric sulfate

(45 g; 28.20 mmole) was added. To the resulting bluish suspension was added 60 mL H2SO4, at which point the solu- tion turned yellow. The flask was stoppered and stirred over a weekend. The suspension was then filtered through a Whatman GFB filter and the filtrate treated in 2.5 g Ca(OH)_ for one hour. The suspension was filtered again and the filtrate concentrated and purified on an Si°2 column. The column was run in 97:3 chloroform: methanol and then again using 8:1 chloroform: methanol. The column fractions were pooled, yielding 3.03 g (7.64 mmole; 82.5%) product. Proposed structure of the product:

Example 3

Synthesis of

was carried out as follows.

(a) Hexaethylene glycol (10.0 g; 35.40 mmole) was coevaporated with anhydrous pyridine (3 x 25 mL) and then dissolved in 100 mL of the same. DMT-C1 (13.17 g; 38.94. mmole) was added to the solution. The reaction was carried out at room temperature and monitored by TLC (CHCl,:MeOH approximately 8:1).^' until the appearance of product. After two hours, 25 mL of methanol was added and the reaction mixture was stirred for 15 additional minutes. Next, the reaction was quenched with 50 mL ice water and extracted with ethyl acetate (3 x 150 mL) . The organic layer was washed with 5% NaHCO., (2 x 100 mL) , saturated NaCl (2 x 100 mL) , dried over Na₂S0₄ and then evaporated down to an oily residue (yellowish color) . This oily residue was chromatographed on a silica gel column (400 g) . The column was eluted first with CHCl₃:Me^'0H (approximately 97:3), then with CHCl₃:MeOH (ap¬ proximately 90:10). The fractions were combined and evaporated to dryness to give an oily residue. The material obtained was presumed to be of the structure

HO -o- O-OMT

and was used without further purification in the synthesis of the corresponding phosphoramidite.

(b) The procedure of Example 1(e) was followed using 2.0 g (3.40 mmole) of the compound obtained in (a), 1.6 g (6.80 mmole) of the phosphoramidite

.^' CH(CH₃)₂

and 1.76 g (13.60 mmole) of [ (CH₃)₂-CH]-N-Et. Elemental analysis of the product was as expected for C₄₂H_βlN_O_ιnP xH₂0. Calc.r C, 63.49; H, 7.93; N, 3.52. Found: C, 63.36; H, 7.95; N, 4.11. Yield: 85.4% of theoretical (2.28 g / 2.67 g) . ^■ Example 4 (a) Synthesis of

(compound (26); see Step 1, Scheme IV) was carried out as follows. To a 0°C solution of triphenylphosphine (7.87 g;

10 30 mmole) in 75 mL dry THF was added the azodicarboxylate(NCOOCH(CH₃) )₂ (6.07 g; 30 mmole) with stirring. After one hour, a solution of tetraethylene glycol (5.83 g; 30 mmole) in 10 mL dry THF was added. All material dissolved to give a pale yellow solution. After

15 one hour, a solution of the mercaptan jZfx-SH in 20 mL 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 an Si0₂ column and fractionated using methylene

20 chloride followed by a mixture of mixture of methylene chloride and CH_,CN (2:1).- The material was rechromatographed on SiO- using CH^CN as eluant, and the product was removed from j2f"₃P=0 by taking small (ap¬ proximately 15 mL) fractions. The fractions were pooled,

25. yielding 5;22 ,g (11.53 mmole; 38.4% overall; 77% of theoretical). Elemental analysis was as follows. Calc:

. C, 71.65; H, 7.12; S, 7.08. Found: C, 71.32; H, 7.21; S,

7.15.

30

35 (b) Synthesis of the corresponding phosphoramidite

was then carried out according to the method described in Example 1(e), using the reaction product of step (a) (4.22 g; 9.30 mmole), the phosphoramidite

CH(CH₃)

(4.40 g; 18.60 mmole) and [ (CH.,)₂-CH]₂-N-Et (4.8.1 g; 37.20 mmole). Yield: 75.3% of theoretical (4.57 g / 6.07 g) .

Example 5 (a) Synthesis of

was carried out as follows. To a 0 C solution of triphenylphosphine (7.87 g; 30 mmole) in 75 mL dry THF was added the diisopropyl azodicarboxylate (NC00CH(CH₃) )₂ (6.0.7 g; 30 mmole) with stirring. After one hour, a solu¬ tion of tetraethylene glycol (5-83 g; 30 mmole) in 10 mL dry THF was added. All material dissolved to give a pale yellow solution. After one hour, a solution of the mercaptan in 20 mL 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 an SiO- column and fractionated using methylene chloride followed by a mixture of mixture of methylene chloride and CH,CN (2:1). The material was rechromatographed on SiO- using CHXN as eluant, and the product was removed from _/0'₃P=O by taking small (ap¬ proximately 15 L) fractions. The fractions were pooled, yielding 5.22 g (11.53 mmole; 38.4% overall; 77% of theoretical). Elemental analysis was as follows. Calc: C, 71.65; H, 7.12; S, 7.08. Found: C, 71.32; H, 7.21; S, 7.15.

(b) Preparation of the phosphoramidite:

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

CH(CH₃)₂

(0.66 g; 2.8 mmole) was added with a syringe over a two- minute period. The reaction was carried out at room temperature and under argon. After one hour, the mixture was transferred with 50 L of ethyl acetate in a 250 ml separatory funnel and extracted with saturated NaCl solu¬ tion four times. The organic layer was dried over Na2Sθ4 and evaporated down to an oily residue under vacuum. This residue was chromatographed with 1% Et-.N in ethyl acetate.

Example 6 Preparation of mal-sac-HNSA Ester

One molar equivalent (2.24 g) of 4-hydroxy-3-

-nitrobenzene sulfonic acid sodium salt (HNSA) was mixed together with one molar equivalent (2.06 g) of dicyclohexylcarbodiimide and one molar equivalent (2.10 g) of N-maleimido-6-aminocaproic acid in 25 mL of dimethylformamide (DMF) at room temperature overnight. A white precipitate of dicyclohexylurea was formed. The precipitate was filtered and 300 mL diethyl ether was added to the mother liquor. After about 10 minutes to 4 hours a gummy solid precipitated from the mother liquor. This solid was found to contain 58% of active HNSA ester and 42% of free HNSA.

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. The progress of purification was monitored by thin layer chromatography using chloroform, acetone and acetic acid (6:3:1 v:v:v) as eluting solvent. The product was positively identified as mal-sac HNSA ester by its re¬ activity with amines. The yield of crude ester produced was estimated to be approximately 30% of theoretical; the purified material consisted of 99% ester.

The ester thus obtained was found to dissolve fully in water and was found to be stable in water for

5 several hours, provided no nucleophiles were added. The purified ester was found to be stable for extended periods when stored desiccated.

Example 7 10 Preparation of Conjugate of mal-sac

HNSA Ester and Horseradish Peroxidase (HRP)

An amide of mal-sac HNSA ester and HRP was prepared as follows:

A total of 40 mg (1.0 v oles) of HRP (Sigma 1.5 Chemical Co.) was dissolved in 0.5 mL of 0.1 M phosphate buffer at pH 7.0 to yield a total amine- concentration of 3.7 L 10^"3 M. Then, 5 mg (1.1 x 10^"5 moles) of the mal- sac HNSA ester of Example 5A, calculated from the data in Example 6A, was dissolved in 0.5 L of the HRP solution. 20 The mixture was stirred at room temperature, and the HRP fraction (2.8 mL) was collected on a Pharmacia G-25 column using 0.1 M phosphate buffer, pH 6.0, as eluant.

Example 8 5 ^' Preparation of HRP-Oligonucleotide Conjugates

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

, and ^• coupled thereto under standard phosphoramidite

^■coupling conditions (see, e.g., Beaucage and Caruthers

(1981) , supra) .

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% CH₃C 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 υ l _r and used right away in the following conjugation reaction.

The mal-sac HRP complex prepared in Example 7 (700 ιi) 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 Na₂P0., pH 6; "C" designates 20 mM Na₂P0₄ + 1M NaCl, pH 6): (1) B, 0 —> 100%, 30 min.; (2) C, 100%, 10 min.; and (3) C, 100 —> 0%, 5 min.. Remaining unconjugated HRP and oligomer eluted after about 2 and about 15-40 min (depending on the size of the oligomer) , respectively, while the conjugate eluted after about 15-40 min as, .well (also depending on the size of the oligomer) . The identity of the product was confirmed by ultraviolet spectroscopy, monitoring peak absorbances of the oligomer (at 260 n ) and of the heme group of HRP (at 402 nm) .

Claims

1. A covalent conjugate of an oligonucleotide chain and horseradish peroxidase given by the structure

wherein

* R is hydrogen or -CH₂OH; the Q moieties, are selected from the group consisting of 0 0 0

II II II

-0-, -NH-, -S-, -NH-C-, -NH-C-0-, and -NH-C-NH- and may be the same or different; n', n'.' and n' ' ' are integers in the range of 2 and 10 inclusive; n is -.an integer in the range of 2 and ^'30 inclusive; and

X is an oligonucleotide chain.

* 2. The conjugate of claim 1, wherein R is hydrogen.

3. The conjugate of claim 1 which is useful as an allele-specific oligonucleotide and wherein the oligonucleotide chain is from about 13 to about 21 monomer units in length. 4. A covalent conjugate of an oligonucleotide chain and horseradish peroxidase given by the structure

wherein

R is hydrogen or -CH-OH; the Q moieties are selected from the group consisting of 0 0 0 u n I.

-0-, -NH-, -S-, -NH-C-, -NH-C-0-, and -NH-C-NH- and may be the same or different; n' , n'' and n' ' ' are integers in the range of 2 and 10 inclusive; n is an integer in the range of 2 and 30 inclusive; and

X is an oligonucleotide chain.

* The conjugate of claim 4, wherein R ^■ is hydrogen..

6. The conjugate of claim 4 which is useful as an allele-specific oligonucleotide and wherein oligonucleotide chain is from about 13 to about 21 monomer units in length. '7, A method of preparing a covalent conjugate of an oligonucleotide chain and horseradish peroxidase, the conjugate given by the structure

wherein

R is hydrogen or -CH₂0H; the Q moieties are selected from the group consisting of - 0 0 0

-0-, -NH-, -S-, -NH-C-, -.NH-C-0-, and -NH-C-NH- and may be the same^' or different; n', n' ' and n''' are integers in the range of 2 and 10 inclusive; and • X is an oligonucleotide chain wherein the method comprises the steps of: reacting a functionalized oligonucleotide chain having the structure

with a complex of mal-sac-HNSA ester and HRP under coupling conditions. 8. The method of claim 10, wherein the functionalized oligonucleotide chain is given by the structure