COMPOUNDS

BACKGROUND

Nucleotides and nucleosides such as sense and anti-sense oligonucleotides, dideoxynucleoside triphosphates. and genes have great potential as therapeutic compounds. Conjugates of such nucleotides and nucleosides with agents such as proteins, glycoproteins, antibodies, antibody fragments, hormones, saccharides or drugs are an effective way to direct the information of the nucleoside to the target.

A method of conjugating a nucleotide to an agent should yield conjugates which are stable and reversible. The method should also leave the nucleotide or polynucleotide intact so that it remains functional. In addition, the conjugates should be simple to synthesize and formed in relatively high yield. Several methods are currently being used to conjugate nucleotides to agents. These include photochemical reactions of azide groups and other light-activated crosslinkers; crosslinking by formation of nitrogen- phosphorous bonds through carbodiimide intermediate; crosslinking with thiol-containing alkyl-chains; electrostatic complexation with polycations such as polylysine or histones; and non-specific reactions of amino groups on nucleotide bases and protein side-chains with polyaldehydes such as paraformaldehyde or glutaraldehyde. However, none of these crosslinking methods completely fulfill all the criteria noted above. There exists a need for a method which meets the criteria and produces useful therapeutic and diagnostic agents.

SUMMARY OF THE INVENTION

This invention provides compounds which are comprised of an agent linked through a sulfur atom to a phosphorous atom of a nucleotide, polynucleotide, or analog thereof. A preferred embodiment is provided wherein a phosphorothioate-containing ester of a nucleotide, polynucleotide, or analog thereof, is attached to a maleimide group on a polypeptide, protein, hormone, or glycoprotein through a cyclic thioester linkage. Methods for producing the compounds and for introducing maleimide groups into polypeptides, proteins, hormones, or glycoproteins are also provided.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows a thin-layer chromatograph of phosphorothioate-DDI

Figure 2 shows a thin-layer chromatograph of thymidyl-phosphorothioate- thymidine. Figure 3 shows the fluorescence emission spectra of N-(l-pyrenyl) maleimide adducts of phosphorothioate nucleotides and 2-mercaptoethanol.

Figure 4 shows the fluorescence emission spectra of N-(l-pyrenyl) maleimide adducts of thymidyl-phosphorothioate-thymidine and 2-mercaptoethanol.

Figure 5 shows the absorbance spectra of the succinimidyl adduct of ^~ 5'- adenosine diphosphate beta-S (ADPS) and maleimide-modified bovine serum albumin (BSA- MAL) (BSA-SUC-ADPS; ■) and of blocked BSA-MAL (BLOCKED BSA-MAL; •) with control BSA-MAL absorbance subtracted from each.

DETAILED DESCRIPTION This invention pertains to compounds produced by and methods of coupling nucleotides, polynucleotides. or analogs thereof, to agents such as ligands, reporter groups, or solid supports while retaining their chemical activity. The method can be used to couple any phosphorothioate nucleotide (PN) to an agent which has a disulfide bond or a thiol or maleimide group. Phosphorothioate nucleotides can be synthesized by standard solution methods or by the procedure of Stec et al. (J. Am. Chem. Soc. 106: 6077-6089 (1984)) using an automated synthesizer. They can also be purchased from suppliers such as Oligos, Etc. (Guilford, CT). If an antibody or antibody fragment is used as the agent, the conjugate is a particularly useful targeting molecule allowing delivery of the PN to a specified cell type.

It has been known since 1968 (Neumann, Steinberg, Katchalski and Sela, i.

Am. Chem. Soc. 87:3841 -3848 (1 65)) that thiophosphoric acid can function as a thiol-like reducing agent. However, phosphorothioate nucleotides have not been used to conjugate proteins, glycoproteins, or saccharides to nucleotides, perhaps because of the belief that the decrease in the thiol character of the thiophosphoric ester by resonance stabilization of the sulfur would prevent such conjugation.

One aspect of the invention pertains to compounds comprised of a phosphorothioate-containing nucleotide, polynucleotide, or an analog thereof, conjugated to an agent. One or more atoms of the agent are bound to a sulfur atom which is bound to a phosphorous atom of the nucleotide, polynucleotide, or analog thereof.

A further aspect of the invention pertains to compounds of the formula:

wherein B is a purine or a pyrimidine base; Z is H or -OH; and X and Y may be the same or different and each may be H. -OH, a nucleotide, a polynucleotide or an analog thereof provided that at least one of X and Y are of the formula:

O A — P =O

wherein Q is H, -OH, a nucleotide, nucleoside, a polynucleotide, or an analog thereof and A is -S-L wherein L is an agent. The compounds of this invention include those wherein A is of the formula:

In the above formula, B represents a purine or a pyrimidine base attached to the 1' carbon. Purine bases include adenine, inosine, guanine and any analog thereof. Pyrimidine bases include thymine, uracil, cytosine and any analog thereof.

The terms nucleotide and polynucleotide include any nucleotide, polynucleotide, or nucleoside monophosphate, diphosphate, or triphosphate as well as nucleotides attached to a polypeptide backbone. The term polynucleotide includes any chain of two or more nucleotides. The nucleotides may be linked through any type of group including phosphodiesters, methylphosphonates, and phosphorothioate diesters. In addition, phosphorothioate oligodeoxynucleotides which can be used as anti-sense molecules are also within the scope of this invention (See. Zon et al, WO 88/07544). Analogs of

polynucleotides and nucleotides are also within the scope of this invention, including ethylphosphonate nucleotides commonly used as antisense molecules.

The term agent encompasses any ligand, reporter group, bridging group, hormone, masking molecule, or solid support. A ligand is any molecule which would bond to another molecule. An example of a ligand is a molecule which is specific for a cellular receptor such as a glycoprotein like transferrin, allowing the conjugate to be targeted to a specific cell. Accordingly, the agent can be an antibody or an antibody fragment. Also, the agent can be a ligand such as a polypeptide. The term polypeptide includes proteins. A masking molecule is a molecule which would change or mask the characteristics of the nucleotide, polynucleotide, or analog thereof. This includes molecules which allow the nucleotide to permeate through the cell membrane or endosomal membrane such as fusigenic polypeptides. It also includes a molecule that would increase the molecular weight of the PN to allow for more efficient purification. A hormone includes substances that originate in a gland, organ, or part and is conveyed through the blood to another part of the body, stimulating it by chemical action to increase functional activity or to increase secretion of another hormone. Hormones within the scope of this invention include synthetic and naturally occurring hormones. A bridging molecule, such as SPDP (N-succinimidyl 3-(2- pyridyldithio)propionate), is one which would act as a bridge between the PN and another molecule. A bridging molecule can also act as a bridge between the PN and a protein. For example, SIAB (N-succinimidyl (4-iodoacetyI) aminobenzoate) can be reacted with a protein and then the SIAB-protein conjugate can be reacted with a PN to form a protein-PN complex bridged by the remnants of SIAB. A reporter group, such as N-(l-pyrenyl) maleimide, a probe for thiols, is a molecule which selectively bonds to certain molecules. Solid supports include materials to which a protein can be bound or nucleic acid can be attached. Solid supports can include beads, (e.g., magnetic beads (DYNABEADS™) and SEPHAROSE™ beads), filters, capillaries, plastic dipsticks (e.g., polystyrene strips) and microtiter wells.

In one embodiment of the method, a phosphorothioate-containing nucleotide, polynucleotide, or analog thereof, is reacted with a disulfide-, maleimide-, or thiol-containing agent to form a bond between the sulfur atom of the phosphorothioate nucleotide and the agent. For example, a nucleotide, polynucleotide, or analog thereof, possessing a free and unphosphorylated OH group, is reacted with thiophosphoryl chloride (PSCI3) in the cold. This reaction yields a precursor phosphorothioate nucleotide or nucleoside. The PN is purified by dialysis, or other treatment, so as to remove unreacted PSCI3 and thiophosphoric acid. The purified PN is then conjugated by reaction with a disulfide-containing or thiol- containing protein, glycoprotein, hormone, antibody, antibody-fragment, saccharide or drug.

The disulfide bond may be either naturally-occurring or artificially introduced prior to the conjugation reaction.

The sulfur of phosphorothioate diesters possesses considerable thiol character (Frey and Sammons, Science. 30: 541 -545 (1985)) and can react with thiols or disulfides to produce disulfide linkages between the agent and the nucleotide, polynucleotide, or analog thereof. In addition, the thiol character of the phosphorothioate moiety allows for reaction of the moiety with maleimide-containing agents to form cyclic thioesters of the formula:

wherein B is a purine or pyrimidine base; X, Y, and Q are H, -OH, a nucleotide, a polynucleotide, or an analog thereof; Z is H or -OH; and L is an agent. The agent may be a polypeptide, protein, glycoprotein, or saccharide. Further, the agent may be an antibody or antibody fragment.

The preferred embodiment of the method involves reacting a PN with a maleimide-containing agent such as a polypeptide, protein, hormone, or glycoprotein. It is well known that alpha-beta unsaturated carbonyls are susceptible to nucleophilic addition by thiols (Gilman, H. (1943) Organic Chemistry. John Wiley and Sons Inc., New York pp. 835- 943). The ability to stabilize the anion intermediate through electron delocalization is a facilitating force for nucleophilic addition to alpha-beta unsaturated carbonyls (Morrison, R.T. et af (1987) Organic Chemistry. Fifth ed., Allyn and Bacon, Boston pp. 1086-1088) such as N-( 1 -pyrenyl) maleimide. Note that in N-(l-pyrenyl) maleimide, the alpha and beta carbons are equivalent due to the fact that there are two activating carboxyl groups symmetrically located about the olefinic double bond. The nucleophile can attack at either carbon center yielding identical main adducts.

Another aspect of the invention is a method of producing the above compound by first introducing maleimide groups into a polypeptide, protein, hormone, or glycoprotein and then reacting the resultant polypeptide, protein or glycoprotein with a phosphorothioate- containing nucleotide, polynucleotide, or analog thereof. For example, maleimide groups can be introduced into a polypeptide by reacting it with m-maleimidobenzoyl-N- hydroxysuccinimide ester (MBS)(Pierce Chemical, Rockford, IL). Conjugates are then formed by reacting the maleimide-containing polypeptide with a phosphorothioate-containing nucleotide, polynucleotide, or an analog thereof, to form a cyclic thioester linkage between the agent and the sulfur atom of the thiophosphoric ester.

One feature of the compounds of the invention is that the agent can remain attached to the polynucleotide while in the bloodstream to allow for targeted delivery of the polynucleotide to a cell and then the polynucleotide can be released once it has entered the cell through, for example, receptor-mediated endocytosis. It is believed that the phosphorothioate bond between the agent and the polynucleotide is not susceptible to nucleophilic attack at the pH of the bloodstream. However, at the considerably lower pH of an endosome that has absorbed the compound through receptor-mediated endocytosis, the phosphorothioate bond can be hydrolyzed and the polynucleotide released, thus, allowing the targeted delivery of the intact polynucleotide to a cell.

Polynucleotide probes that have been labeled through a phosphorothioate bond as described can be used to detect the presence of nucleic acids in a sample that contain nucleotide sequences complementary to the labeled polynucleotides. For example, an ohgonucleotide probe can be linked through a phosphorothioate diester bond to a protein such as an enzyme. The labeled ohgonucleotide probe can then be contacted with a sample of, for instance. DNA under conditions that allow for hybridization of the probe with a complementary nucleotide sequence in the DNA. The presence of nucleic acids that contain nucleotide sequences complementary to the ohgonucleotide probe and/or the degree of hybridization can be determined by the presence and/or amount of the protein (i.e., enzymatic activity) after a suitable washing step. Such a method can be used for diagnostic or identification purposes.

Linking PNs to antibodies or other agents through disulfide bonds and cyclic thioesters provides a chemical basis for synthesis of antibody-phosphorothioate-nucleotide conjugates that can be useful for immuno-targeted delivery of anti-AIDS phosphorothioate- dideoxynucleotides or phosphorothioate-ribozymes, of sense and anti-sense phosphorothioate-oligonucleotides to combat AIDS and other infections or to modulate gene

expression, and of phosphorothioate-DNA for gene therapy. In addition, conjugates prepared using the phosphorothioate-maleimide linking system should be stable in the blood yet cleavable at the P-S bond in the acidic endosome after internalization by the target cell.

Antisense technology has great potential for the regulation of gene expression in both natural and virally-induced genetic disorders. The technology takes advantage of the fact that a nucleotide made antisense to target mRNA according to Chargaff s base-pairing rule will bind to and prevent translation of the mRNA into protein products. Various groups have shown that the antisense nucleotide binds to its mRNA target and causes translation arrest (Cohen, J.S. (1991 ) Antiviral Res. 16:121-133; Ghosh, M.K. eJ aL (1992) Mol. Biol. 42:79-126), preventing target protein synthesis. Others have shown that phosphorothioate antisense oligonucleotides bound to target mRNA activate Rnase-H, thereby catalyzing the destruction of the mRNA target (Goodchild, J. (1989) Oligodeoxynucleotides. J.S. Cohen, ed., CRC Press, Boca Raton, FL, pp. 55-77). Phosphorothioate nucleotides have made an impact on the technology following the observation that they have higher biological half- lives due to their resistance to nuclease degradation. . The present invention can facilitate targeting these oligonucleotides to affected cells, thus providing an effective mode of delivery.

In addition, the present invention can also be used to deliver the active dideoxynucleoside triphosphate drug into HIV-infected cells. Dideoxynucleoside triphosphates are the active antiviral drug forms in the treatment of HIV (Nakashima, H., et aj. (1986) Antimicrob. Agents Chemother. 30:933-937). Presently, the pro-drug dideoxynucleoside is administered since the unphosphorylated agents freely diffuse across the cell membrane. The therapeutic nucleosides are converted to the active triphosphate by cellular enzymes (Nakashima, H., et al (1986) Antimicrob. Agents Chemother. 30:933-937). The activity of these antivirals is therefore dependent on the rate at which they are intracellularly converted to their drug form. High doses must be administered for drug activity. Such high doses caused various side-effects such as peripheral neuropathy and pancreatitis (Yachoan, R. et al Ann. N.Y. Acad. Sci. 61:328-43). This invention would permit use of lower drug doses thereby decreasing the adverse side-effects.

The following nonlimiting examples will further explain and illustrate the methods and compounds of the invention.

EXEMPLIFICATION

Example 1 : Synthesis of phosphorothioate-dideoxyinosine (PS-DDI) and formation of mixed disulfide between PS-DDI and Ellman's reagent.

DD1 solution was prepared by dissolving 1 mg DDI (Sigma, St. Louis, MO) in 100 mcl of triethylphosphate (TEP) (Eastman Kodak, Rochester, NY). A 0.01m solution of Ellman's reagent (Sigma, St. Louis, MO) was prepared in 0.1 M Na2HPO4 (SPB). Ten test tubes were set up as follows:

Tube 1 10 mcl DDI solution + 10 mcl SPB;

Tube 2 10 mcl DDI solution + 10 mcl PSCI3 + 10 mcl SPB;

Tube 3 10 mcl Ellman's reagent + 10 mcl TEP;

Tube 4 10 mcl Ellman's reagent + 10 mcl PSCI3 + 10 mcl TEP; Tube 5 10 mcl Ellman's reagent + 10 mcl DDI solution;

Tube 6 10 mcl DDI solution + 10 mcl PSCI3 + 10 mcl Ellman's reagent;

Tube 7 10 mcl DDI solution + 10 mcl PSCI3 + 10 mcl Ellman's reagent + 20 mcl 2-mercaptoethanol ;

Tube 8 Thiophosphoric acid (PSCI3 + H2O); Tube 9 2-mercaptoethanol;

Tube 10 2-mercaptoethanol + thiophosphoric acid.

Thiophosphorylation was permitted to proceed for 1 hour in tubes 2, 4, 6 and 7. Following this, hydrolyzed PSCI3 was neutralized by adding concentrated NaOH and the solution was adjusted to pH 9-1 1. In tubes 6 and 7, Ellman's reagent was added after the thiophosphorylation reaction and pH adjustment. The 2-mercaptoethanol was added to tube 7 thirty minutes after the addition of Ellman's reagent.

Tubes 1 -10 were analyzed by thin-layer chromatography on silica gel (Eastman 6060 with fluorescent indicator) using a mobile phase containing isopropanol:ammonia:water (7:3:1). The thin-layer chromatogram was excited with a short- wavelength UV lamp (Fisher Scientific, Fairlawn, NJ) and photographed (Figure 1). The PS- DDI remained at the origin (Figure 1 , lane 2), in contrast with unreacted DDI, which had an rf value of 0.8 (Figure 1 , lane 1 ). The mixed disulfide between PS-DDI and Ellman's reagent is shown in Figure 1 , lanes 6 and 7.

Example 2: Formation of mixed disulfide between thymidyl-phosphorothioate-thymidine and Ellman's reagent.

66 meg thymidyl-PS-thymidine (T-PS-T; Oligos Etc., Guilford, CT) was dissolved in 100 mcl triethylphosphate. Six test tubes were set up as follows:

Tube 1 10 mcl T-PS-T solution + 10 mcl SPB;

Tube 2 10 mcl Ellman's reagent + 10 mcl TEP;

Tube 3 10 mcl T-PS-T solution + 10 mcl Ellman's reagent; Tube 4 10 mcl T-PS-T solution + 10 mcl Ellman's reagent + 20 mcl

2-mercaptoethanol; Tube 5 10 mcl Ellman's reagent + 10 mcl TEP + 10 mcl

2-mercaptoethanol ; Tube 6 10 mcl T-PS-T solution + 10 mcl SPB.

Reaction of T-PS-T with Ellman's reagent was carried out for 45 minutes at 25° C. At the end of that time, concentrated NaOH was added and the solution was adjusted to pH 9- 1 1. Tube 3 had a more intense yellow color than tube 2, demonstrating formation of a mixed disulfide between T-PS-T and Ellman's reagent. Thin layer chromatography was performed as described in Example 1 (Figure 2). The mixed disulfide between T-PS-T and Ellman's reagent is shown in Figure 2, lane 3. The mixed disulfide between T-PS-T and Ellman's reagent was reduced by 2 -mercaptoethanol (Figure 2, lane 4). The data shown in Figure 2 demonstrate that the internucleotide thiophosphate forms a mixed disulfide with Ellman's reagent, and. thus, forms the chemical basis of crosslinking PS-nucleotides to antibodies and other proteins.

That PN forms mixed disulfide compounds with Ellman's reagent a disulfide- containing molecule is unexpected in view of the resonance stabilization of the sulfur of PN. These results show that thiophosphoric esters can in fact be linked to disulfide or to thiol- containing groups.

Example 3: Formation of cyclic thioesters.

Cyclic thioesters were formed between N-(l -pyrenyl) maleimide (Weltman gt al, J. Biol. Chem. 248: 3173-3177 ( 1973)) and the following phosphorothioates thiophosphoric acid, thymidyl-PS-thymidine, and 2'-deoxycytosine 5'-O-(l- thiotriphosphate)(CTP-alphaS).

3 mg of N-(l -pyrenyl) maleimide (PM) (Sigma, St. Louis, MO) were dissolved in 10 ml acetone (HPLC grade; Aldrich, Milwaukee, WI) to achieve a 1 mM concentration of PM. Test solutions were prepared in the following manner:

(1 ) thiophosphoric acid was prepared by reacting 10 mcl PSCI3 (Aldrich, Milwaukee, WI) with 100 ml of 0.1 M Na ₂ HPO4.

(2) 1 mM solution of 2-mercaptoethanol was prepared in 0.1 M Na2HPO4.

(3) 20 mcl of CTP-alphaS (US Biochemical Corp., Cleveland, OH) were added to 2 ml Na2HPO4 to achieve a final concentration of 0.1 mM CTP-alphaS.

(4) 70 meg T-PS-T were added to 2 ml Na2HPO4 to achieve a final concentration of 0.06 mM T-PS-T.

1 10 mcl of PM solution in acetone were added to 1.0 ml of each of the four test solutions. Control solutions were prepared by adding 110 mcl 0.1 M Na2HPO4 to 1 ml of each of the four test solutions. Blanks consisted of 0.1 M Na2HPO4 and of 110 mcl of PM in 1 ml of Na2HPO4 . Final concentration of PM was 0.1 mM.

Reactions were permitted to progress, and reaction products were observed visually with a long- wavelength UV lamp (UVP, Inc., San Gabriel, CA, Model UVL-56, λ

366 nm). After, about one hour, blue fluorescence was observed in the thiophosphoric acid, 2-mercaptoethanol, CTP-alphaS, and T-PS-T test solutions. No fluorescence was observed in the corresponding controls and blanks. Spectrofluorometry was performed with an Aminco

Bowman spectrofluorometer (Urbana, IL). The wavelength of maximum excitation was 350 nm, and of maximum emission was 390-392 nm. Fluorescence of each sample at 400 nm, excited at 330 nm, is given in the following table (meter multiplier=0.03):

TABLE 1

SAMPLE . FLUORESCENCE

Buffer 00.5 ± 00.7

Buffer + PM 01.5 ± 00.7

Thiophosphoric acid 00.5 ± 00.7

Thiophosphoric acid + PM 67.0 ± 11.3

T-PS-T 01.0 ± 00.0 T-PS-T + PM 19.5 ± 06.4

CTP-alphaS 00.0 ± 00.0

CTP-alphaS + PM 08.5 ± 05.0

Mercaptoethanol 00.0 ± 00.0

Mercaptoethanol + PM 15.5 ± 00.7

The data given in Table I shows that PM formed fluorescent adducts not only with 2-mercaptoethanol, but also with thiophosphoric acid, and the thiophosphoric esters CTP-alphaS and T-PS-T. Maleimide groups can be introduced into antibodies, and other proteins, using N-hydroxysuccinimide esters such as MBS. The data in Table I, therefore, demonstrate the feasibility of coupling phosphorothioate nucleotides to antibodies by the formation of cyclic thioester crosslinks.

Example 4: Comparison of Relative Reactions of thymidyl-PS-thymidine and 2-mercaptoethanol with N-( 1 -pyrenyl) maleimide.

2 ml 6x10" ⁵ M T-PS-T (Oligos Etc., Guilford, CT) and lxlO" ⁴ M CTP-alphaS

(U.S. Biochemical Corp., Cleveland, OH) solutions prepared in sodium phosphate dibasic buffer (pH 8.8) were each reacted with 200 mcl of lxlO'^M N-(l-pyrenyl) maleimide (Sigma, St. Louis, MO) solution prepared in acetone (HPLC grade; Aldrich, Milwaukee, WI). A solution containing N-(l -pyrenyl) maleimide in acetone was added to 2-mercaptoethanol as a standard thiol-PM reaction control. The resulting mixtures were left to react for seven days at ambient temperature in the dark after which the solutions were analyzed by fluorescence spectroscopy using a Gilford spectrofluorometer (Oberlin, OH). Emission intensities were recorded from 450-350 nm upon excitation at 330 nm. Background N-(l -pyrenyl) maleimide fluorescence was subtracted from the emission spectra. (Fig. 3)

2 ml lxlO'-^M T-PS-T solution prepared in sodium phosphate monobasic/dibasic buffer (pH 7.1 ) was reacted with 20 microliters of lxlO'^M N-(l -pyrenyl) maleimide solution prepared in acetone such that the final concentrations of T-PS-T and N-(l -pyrenyl) maleimide in the reaction mixture were lxlO'^M and lxlO'^M, respectively. An analogous reaction was prepared with the standard thiol-2-mercaptoethanol and N-(l-pyrenyl) maleimide with the final concentrations of 2-mercaptoethanol and N-(l -pyrenyl) maleimide being lxlO'^M and lxl O'^M, respectively. The reactions were allowed to go for 48 hours and the emission spectra of the resulting solutions were recorded from 450-350 nm upon excitation at 330 nm. Background emission of pyrene maleimide was subtracted from the emission spectra (Fig. 4). Figure 3 shows the fluorescence emission spectra of T-PS-T-PM, CTP-alphaS-PM and 2-mercaptoethanol-PM from 450-350 nm upon excitation at 330 nm. The background fluorescence of N-(l -pyrenyl) maleimide was subtracted. The fluorescence of the phosphorothioate-PM solutions above background indicated the formation of the

maleimide adducts. Figure 4 shows the emission spectra of T-PS-T-PM and 2-mercaptoethanol-PM solutions 48 hours after reaction was initiated. Equal concentrations of 2-mercaptoethanol and T-PS-T were each reacted at lOOx excess of N-(l -pyrenyl) maleimide-adduct yield than T-PS-T as is indicated by higher fluorescence intensity of the 2- mercaptoethanol-PM solution in comparison to T-PS-T-PM solution.

Example 5

5'-adenosine diphosphate beta-S (ADPS) was reacted with maleimide-modified bovine serum albumin (BSA-MAL, 20 maleimides/BSA) purchased from Pierce Chemical Co. (Rockford, IL). BSA-MAL (0.035mM) was reacted with ADPS (0.03M) for 17 hours at room temperature, at pH 7.0 in 0.1 M sodium phosphate buffer to produce the succinimidyl adduct of ADPS and BSA-MAL (BSA-SUC-ADPS). Controls for the (BSA-MAL)- ADPS reaction were untreated BSA-MAL and BSA-MAL in which maleimide was inactivated (BLOCKED BSA-MAL) by 2-mercaptoethanol (0.1M) prior to the addition of ADPS. All sample were dialyzed exhaustively against sodium phosphate buffer (0.1M, pH 7.0), diluted appropriately and absorbance of the diluted solutions was measured from 250-400nm with a recording spectrophotometer.

The absorbance spectrum of BSA-SUC-ADPS (Figure 5), with a peak at 260nm indicates the formation of protein-nucleotide conjugates. ADPS added to 20% of the maleimide groups in BSA-MAL, thereby crosslinking approximately four ADPS molecules to each BSA molecule.

Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the following claims.