WO1993000444A1

WO1993000444A1 - Biotinylated photoaffinity nucleotide analogues - synthesis and application

Info

Publication number: WO1993000444A1
Application number: PCT/US1991/004659
Authority: WO
Inventors: Shiuan Chen
Original assignee: City Of Hope
Priority date: 1991-06-28
Filing date: 1991-06-28
Publication date: 1993-01-07

Abstract

Nucleotide analogues having a photoreactive group and a biotinyl group which covalently attach to nucleotide dependent protein binding site are provided.

Description

BIOTINYLATED PHOTOAFFINITY NUCLEOTIDE ANALOGUES - SYNTHESIS AND APPLICATION

FIELD OF THE INVENTION

This invention relates to active site labelling useful in protein structure-function studies. More particularly the invention relates to nucleotide analogues having a photoreactive group and a biotinyl group which covalently attach to nucleotide dependent protein binding sites.

BACKGROUND OF THE INVENTION Protein structure-function studies are significantly facilitated by receptor binding site directed labelling. That protocol entails synthesis of binding site ligands containing a chemically reactive group which can be photo-generated. Such compounds covalently incorporate into the protein receptor binding site. Binding site identification is provided by the use of labelled derivatives.

In conventional practice the labelled peptide derivative is purified by reverse phase HPLC and characterized by microsequencing and mass spectral analyses. The purification and characterization procedures are tedious work. Frequently the binding site directed label is lost.

SUMMARY OF THE INVENTION This invention provides biotiπylated photoaffinity analogues useful in labelling nucleotide dependent protein binding sites. Upon photoirradiation these analogues covalently bond to the nucleotide binding site. The biotin radical is attached to the ribose moiety of the nucleotide through an ester linkage. Analogue modified proteins are detected by avidin-linked peroxidase or alkaline phosphatase methods. The modified protein can be purified by strepavidin-linked or avidin-linked affinity chromatography. After elution of the nonmodified protein, for example by using buffers at neutral pH, the modified proteins are released and collected from the avidin column by raising the pH to 8 or above to hydrolyze the ester linkage by which the biotin is bound to the ribose hydroxyl group. This invention thus combines the processes for the detection and purification of photoaffinity modified proteins and greatly simplifies the purification process.

GENERAL DESCRIPTION OF THE INVENTION This invention entails the use of biotinylated photoaffinity nucleotide analogues to characterize the structure of nucleotide dependent protein receptor binding sites. The invention has particular application to the characterization of the active sites of nucleotide dependent enzymes.

In the preferred practice of the invention a photoreactive group is initially introduced into the nucleotide of interest. The synthesis of photoaffinity analogues of nucleotides has been well documented. Several commonly used analogues are indicated in Table 1:

TABLE 1

Analogues References

Arylazido adenine nucleotides

(A)-arylazido nicotinamide adenine dinucleotides

(N)-arylazido nicotinamide adenine dinucleotides 3, 4

(A)-8-azido adenine nucleotides 5

(A)-2-azido adenine nucleotides 6

(A)-8-azido nicotinamide adenine dinucleotides

(A)-2-azido nicotinamide adenine dinucleotides

(N)-3-azido pyridine adenine dinucleotides

1. Jeng, S.J. and Guillory, R.J. J. Supramolecular Structure 3_:448-468 (1975)

2. Chen, S. and Guillory, R.J. J. Biol. Chem. 252:8990-9001 (1977)

3. Chen, S. and Guillory, R.J. J. Biol. Chem. 255:2445-2453 (1980)

4. Deng, P.S.K., Hatefi, Y. , and Chen, S. Biochemistry 2^:1094-1098 (1990)

5. Haley, B.E. Meth. Enzvmol £6:339-346 (1977)

6. Schaeffer, H.J. and Thomas, H.J. J. Am. Chem. Soc. 80:3738-3742 (1958)

7. Deng, P.S.K., Zhao, S.-H, lyanagi, T. , and Chen, S., Biochemistry in press (1991)

8. Hixson, S.S. and Hixson, S.S., Photochem. &. Photobiol. , 18: 135-138 (1973) photoreactive derivative of a nucleotide is first prepared in known manner. A biotin moiety is added to such derivatives through formation of an ester linkage between the biotin carboxyl group, and the hydroxyl group of a ribose moiety of the nucleotide. The biotinylated photoaffinity nucleotide analogues may be represented by the structural formula I in which X is a nucleotide including a ribose moiety y. A is a photoreactive group, and B is a biotin moiety:

I. o

II

A - X - y - O - C - B

A may be any photoreactive group. Preferred photoreactive groups are azido and diazo.

X may be any nucleotide. Preferably X is adenine, nicotinamide.

Modified proteins covalently bound to the biotinylated nucleotide analogues are detected by labelled avidin reagents such as avidin linked peroxidase or avidin linked alkaline phosphatase methods in known manner. See Kohanski, R.H., and Lane, M.D. (1990), Meth. Enz mol., 184, 194-200.

The modified labelled proteins are purified by avidin linked affinity chromatography. Only the modified proteins having a biotin moiety bind to the avidin in the column. The nonmodified proteins are eluted from the column using buffers such as phosphate buffer at substantially neutral pH. The pH of the elution medium e.g., 7 or 7.5 is then raised to at least eight to hydrolyze the ester linkage by which the biotin is bound to the nucleotide and thus release the modified proteins for collection and analysis.

EXEMPLIFICATION OF THE INVENTION This invention is exemplified by experiments to characterize the active site structure of nicotinamide nucleotide dependent enzymes by photoaffinity labelling techniques. The exemplification is for purposes of illustration only and does not limit the scope of the invention as claimed.

EXAMPLE I

This experiment describes a typical esterification reaction for attachment of a biotinyl group to a photoaffinity nucleotide analogue of the kind set forth in Table I.

Eleven mg of biotin (45.5 _mol) was dissolved in anhy. dimethylformamide (DMF) (182 μl) by heating the suspension to 80°C. l,l^,-Carbonyldiimidazole (8.5 mg; 50.7 μmol) was added to the biotin solution, and the mixture was kept at 80°C until evolution of CO2 had ceased. After the solution was cooled down to room temperature, a thick gel was formed.

An aqueous solution (910 μl) of 8-azidoadenosine 5-^,triphosphate (sodium salt) (5 mg; 9.1 μmol) was added to the reaction mixture described above. The suspension became a colorless clear solution after stirring at room temperature for 28 hours in the dark. At the end of 28 hour incubation, the solvent was removed by rotatory evaporation. The residue was washed with acetone (7 ml total) and then dissolved in 150 μl of water. The product, 3'-O-biotinyl 8_"-azidoadenosine 5'-triphosphate, was purified by preparative thin layer chromatography (Cellulose, 0.5 mm thick) . The solvent system is n-butanol:acetic acid:water (5/2/3) . The product has a Rf value of 0.45. It has a UV/VIS spectrum identical to 8-azidoadenine 5'-triphosphate. It is shown as a red spot upon spraying the TLC plate with a solution of 0.2% p-dimethylamino-cinnnamaldehyde in 2% sulfuric acid.

EXAMPLE II This example illustrates the use of the biotinylated photoaffinity analogue produced by methods described in Example I in active site directed labelling of a nicotinamide nucleotide dependent enzyme.

The interaction of four photoaffinity analogues of NAD+, (A)-2-azido NAD+, (A)-8-azido NAD+, (A)-arylazido-_/,-alanyl NAD+, and (N)-arylazido-ø-alanyl NAD+, with the rat liver NAD(P)H:quinone acceptor oxidoreductase was recently investigated in this laboratory (Deng et al., in press, Biochemistr , June 1991) . See Exhibit A incorporated herein by reference. The reduced analogues, (A)-2-azido NADH, (A)-8-azido NADH, (A)-arylazido-ø-alanyl NADH, and (N)-arylazido-,-alanyl NADH, were shown to be substrates of this quinone reductase. This enzyme was inhibited by (A)-2-azido NAD+ adn (A)-δ-azido NAD+ in a photodependent manner, and the inhibition of the enzyme could be prevented by the presence of nicotinamide nucleotide substrates during photolysis. On the other hand, no photodependent inhibition of this quinone reductase was found when the enzyme was photolyzed in the presence of either (A)-arylazido-_/,-alanyl NAD+ or (N)-arylazido-ø-alanyl NAD+. These results indicate that (A-2-azido NAD+ and (A)-δ-azido NAD+ are useful compounds to identify the nicotinamide nucleotide binding site of NAD(P)H:quinone acceptor oxidoreductase, and the presence of a bulky group linking to the ribose moiety of the nucleotide molecule, such as the arylazido-_/9-alanyl group in (A)-arylazido-ø-alanyl NAD+ and (N)-arylazido-ø-alanyl NAD+, would not affect the finding of the nicotinamide nucleotide derivatives to the active site of this enzyme.

Based on the results described above, the following experiments were designed to identify and charactize the nicotinamide nucleotide binding site of rat liver NAD(P)H;quinone acceptor oxidoreductase using the biotinylated photoaffinity nucleotide analogues of this invention.

By methods described in Example l, the following two compounds, 3'-0-biotinyl-(A)-2-azido NAD+ and 3'-0-biotinyl-(A)-8-azido NAD+ were synthesized. Proper kinetic experiments to show that these compounds indeed bind to the active site of this quinone reductase and to determine the best codition for our photoaffinity labeling experiments are described in Deng et al. , 1990, Biochemistry 29, 1094-1098 [or FASEB J. 4 (7) Abstract No. 848]; and Deng et al, 1991, supra. The actual labeling experiment is performed using radioactive derivatives of the above described compounds. The modified enzyme (1 nmol) is digested by proper proteases and an aliquot of digested protein mixtures is applied to reverse phase HPLC. The modified peptide(s) are identified by the radioactivity associated with it. The modified peptide(s) can also be identified in the following manner. The resolved peptides will be spotted on a piece of nitrocellulose or PVDF membrane. This membrane is processed as for a Western blotting experiment. See Brakel, C.E. , et al (1990) Meth. Enzy ol 184, 437-441. The identification of modified protein(s) will be done using by ELISA Amplification System (GIBCO/BRL, Gaithersburg, MD) . The results from the different identification methods are compared. The rest of digested protein mixture is incubated with 5 ml Strepavidin- or Avidin-agarose (Sigma Co.) in 50 mM phosphate buffer, pH 7.0. After 3 hour incubation, the Avidin-agaraose is then incubated with one bed volume of 50 mM ammonium bicarbonate buffer, pH 8.5 for one hour and packed into a small column. The column is washed using the same buffer. The eluent will be concentrated, and the protein(s) present in the ammonium bicarbonate buffer is analyzed by protein microsequencing techniques and mass spectrometry. If needed, the protein is purified by passing through reverse phase HPLC before characterization. The invention includes application to other types of nucleotide dependent enzymes by synthesizing biotinylated photoaffinity analogues of adenine, quanine, and other nucleotides. These analogues are also incorporated into RNA or DNA probes by chemical or molecular biological methods. These DNA or RNA derivatives may be useful for the detection and removal of undesired DNA or RNA from cells, e.g., AIDS viral RNA. For example, under proper photoirradiation condition, biotinylated photoaffinity derivatives of DNA or RNA probes, containing sequences complementary to important regions of AIDS viral RNA, would form a nondissociable complex with AIDS viral RNA. The detection of these RNA can be accomplished by methods described above, i.e., membrane hybridization or ELISA amplification method. The viral RNA can be removed by avidin affinity chromatography.

Claims

CLAIMS :

1. A biotinylated photoaffinity nucleotide analogue having the formula

o

II

A - X - Y - O - C - B

in which A is a photoactive group X is a nucleotide; Y is a ribose moiety of X; and B is a biotin moiety.

2. A nucleotide analogue, as defined by Claim 1, in which the photoactive group A is azido or a diazo group.

3. A nucleotide analogue, as defined by Claim 1, in which X is an adenine nucleotide or a nicotinamide adenine dinucleotide.

4. In a process for labelling of nucleotide dependent enzyme receptor binding site which comprises covalent bonding of a labelled nucleotide substrate or inhibitor of said enzyme to said binding site the improvement of which comprises

(i) bonding a biotinylated photoactive nucleotide analogue to said binding site to produce modified enzyme molecules, said biotin being bound to said analogue by an ester linkage;

(ii) determining the presence or absence of said modified enzyme molecules by reaction with an avidin linked label.

5. A process as defined by claim 4 in which said nucleotide analogue is an addenine nucleotide, a nicotinamide adenine dinucleotide or a pyridine adenine dinucleotide.

6. A process which comprises

(i) bonding a biotinylated photoactive nucleotide analogue to a nucleotide dependent protein receptor binding site on a protein to produce a biotinylated protein derivative;

(ii) labelling said biotinylated derivative by reaction with labelled aviden;

(iii) subjecting the reaction products of step iii to aviden linked affinity chromatography under conditions effective to bind said labelled derivative to said column and provide an eluate substantially free of said labelled derivative;

(iv) releasing and collecting the bound modified protein by adjusting the eluate pH to a level appropriate to hydrolyze the ester linkage by which said biotin is bound to said nucleotide

7. A process as defined by claim 6 in which said nucleotide analogue is an adenine nucleotide, a nicotinamide adenine dinucleotide.