CA1293939C - Photochemical nucleic acid-labeling reagent having a polyalkylamine spacer - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
A photochemical nucleic acid-labeling reagent of the formula wherein Q is a photoreactive residue of a nucleic acid-binding ligand; L is a detectable label residue; R
is hydrogen, C1 to C7-alkyl, aryl, hydroxy, or C1 to C7-alkoxy; x is an integer from 2 through 7; and y is an integer from 3 through 10; wherein R and x, respectively, can be the same or different each time they appear in the formula. The reagent is useful in the highly efficient labeling of nucleic acids for the purpose of detection in hybridization assays.

Description

3~

BACKGROUND OF TI~E INVENTION
Field of the Invention This application relates to thc provision of labeled nucleic acids suitable for hybridi~ation assays.

e~
In Canadian patent number 1,222,705, issued June 9, 1987, there is described a photochemical method of labelling nucleic acids for detection'purposes in hybridization assays for the determination of specific polynucleotide sequences. The assays are of a known type.

The'labeIed probes' of patent number 1,222,705 comprise (a) a nucleic acid component, (b) a nucleic acid-binding ligand photochemically linked to the nucleic acid component, and (c) a label chemically linked to lb). ~hese probes generally perform quite satisfactorily, but in some instanccs it is desirable to improve their performance.

SUMM~RY OF THE INVENTION
It is an object of the present invention to provide a labeled nucleic acid of improved solubility, to increase the efficiency of preparing such molecules which is important when the test sample is beinq labeled, and to improve the sensitivity of the probe in an assay.
These and other objects and advantages are realized in accordance with the present invention :
, ., i'`' ''~

~2~3~3~

pursuant to which there ls provided a photochemical nucleic-acid labeling reayent o~ the forrnula R ¦ ~1 Q - -N - ~ CH2- ~ N~ - L
Y' wherein Q is a photoreactive residue of a nucleic acid-binding ligand; I. is a detectable label residue; R
is hydrogen, Cl to C7-alkyl, aryl (e.g., phenyl, naphthyl and anthracyl), hydroxy, or Cl to C7- alkoxy; x is an integer from 2 to 7; y is an inteqer from 3 to 10;
wherein R and x, respectively, can be the same or different each time they appear in the formula.
The present invention also concerns a labeled nucleic acid comprising (a) a nucleic acid component, (b) a nucleic acid-binding ligand photochemically linked to the nucleic acid component, (c) a label and (d) a polyalkylamine spacer chemically linking (b) and (c), such as a residue of spermine.
, BRIEF DESCRIPTION OF THE DR~W_ G
! The Eiqure is a photograph of immobilized cell lysates that have not been photochemically labeled fox comparison purposes with compounds of the present invention and compounds of the prior art.

DETAILED DESCRIPTION OF THE INV~NTION
The spacer of the present invention is a polyalkylamine residu~ of the formula r I I
-N - ~ (CH2)x- N~
, y, 3~

wherein R is hydrogen, Cl to C7-alkyl, aryl (e.g., phenyl, naphthyl and anthracyl), hydroxy, or Cl to C7-alkoxy; x is an in~eger ~rom 2 throuyh 7; and y is an integer from 3 through 10. ~ and x, respectively, can be the same or different each t.ime they appear in the spacer residue, that is, the R groups that appear along the spacer residue do not have to be identical, e.g., one or more `can be hydrogen, one or more can be alkyl, one or more can be hydroxyl, and so forth, and the alkylene yroups -(CH2)- do not have to be of the same length along the spacer residue, e.g., the first may be propylene, the second butylene, the third propylene, and so forth.
Generally, the R and alkylene groups can vary independently of their position on the spacer residue.
Preferably, the R groups are independently selected from hydrogen and C1 to C4-alkyl, e.g., methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl; x is, independently, an integer from 2 through 5; and y is an integer from 3 through 6.
particularly useful spacer residue is the residue of N-4,N-9-dimethylspermine having the formula CH3 ClH3 H
-N -~CH2- ~ N --(CH2 ~ -N - CH2 ~ N- .

The spacer can be incorporated at any stage of the process of making the labeled nucleic acid a-b-d-c defined hereinabove. Thus the sequence can be any oE the following:
a-~b+d+c, b+d-~c~a, d+c+b-~a, b+d+a+c, etc.

i The conditions for the individual steps are well known in chemistry.
As set forth in Canadian Patent L, 227,705, supra, the nucleic acid is joined to the ligand photochemically, employing a photoreactive nucleic acid-binding ligand, e.g., an intercalator compound such as a furocoumarin, a phenanthridine, an anthracycline, a phenazine, an acridine, a phenothiazine, a quinoline, or a non-intercalator compound such as netropsin, distamycin, Hoechst 33258* or bis-benzimidazole to link the nucleic acid to a la~el which can be "read" or assayed in a conventional manner, including fluorescence detection.
The end product is thus a labeled nucleic acid comprising (a) a nucleic acid component, (b) an intercalator or other nucleic acid-binding ligand photochemically linked to the nucleic acid component, and (c) a label chemically linked to (b) through the spacer residue of the present invention.
The nucleic acid can be DNA, RN~, an oligonucleotide or a probe such as described in Canadian Patent 1,227,705, described supra, from higher eukaryotes such as humans and other animals, procaryotes such as plants, fungi, bacteria, viruses, and the like, yeasts, etc. The nucleic acid can be of known composition or can be unknown, as in a sample to be assayed, e.g., a known unlabeled probe is immobilized and thereafter subjected to hybridization conditions with an unknown nucleic acid sample labeled in accordance with the invention.
The novel photochemical method provides more favorable reaction conditions than the usual chemical coupling method for biochemically sensitive substances.
By using proper wavelen~ths for irradiation, DNA, RNA and oligonucleotides can be modified without affecting the native structure of the polymers. The nucleic *Trade Mark ~2~3~g acid-bindin~ ligalld, hereillafter exemplified by an intercalator, and lahel can first be coupled and then photoreacted witll the nucleic acid or the nuclei~ ac;d can first be photoreac-ted with the intercalator and then coupled to the label. A general scheme for coupling a nucleic acid, exemplified by double-stranded DNA, to a label such as a hapten or enzyme is as follows:

Label . +
Photoreactive Intercalator Labeled Nucleic Acid, e.g., double-stranded DNA
Photoreactive Intercalator ~ _ _ ~ _ Photoreactive Intercalator h~-- ~ ~
Chemically - Functionalized nucleic / acid, e.g., DNA
/~
Label Labeled nucleic acid, e.g., DNA
Where the hybridizable portion of the probe is in a douple stranded ~orm, such portion is then denatured to yield a hybridizable single stranded portion.
Alternatively, where the labeled nucleic acid, e.g., DNA, comprises the hybridizable portion already in single stranded~form, such denaturization can be avoided if desired. Alternatively, nucleic acid, e.g., double stranded DNA, can be labeled by the approach o~ the present invention after hybridization has occurred using a hybridization format which genera-tes nucleic acid, e.g., double stranded DNA, only in the presence of the sequence to be detected.
To produce specific and efficient photochemical products, it is desirable that the nucleic acid component ~293939 and the photoreactive intercalator compound be allowed to react in tll~ dark in a spccific manl-er.
For couplincJ ~o nucleic acid, e.y., DN~, aminomethyl psoralen, amlnomethyl angelicin and amino alkyl ethidium or methidium azides are particularly useful compounds. They bind to double-stranded DNA and only the complex produces a photoadduct. In the case where`labeled double~stranded DNA must be denatured in order to yield a hybridizable single stranded region, conditions are employed so that simultaneous interaction of two strands of DNA with a single photoadduct is prevented. It is necessary that the frequency of modification along a hybridiz.able single stranded portion of the;probe not be so great as to substantially prevent hybridi~ation, and accordingly there preferably will be ~ot more than one site of modification per 25, more usually 50, and preferably 100, nucleotide bases.
Suitable angelicin derivatives have the following formula ~ L ~ 1 J ~ ,~
~, .. /
j.~, in which R1, R2 and R3 each independently is hydroqen or Cl-C7-alkyl; and R4 is hydrogen, C1-C7-alkyl or lower alkyl substituted with hydroxy, C1~C7-alkoxy, amino, halo, or ~3939 ~ N-Particularly preferred angelicin derivatives have the following moieties for Rl, R2, R3 and ~4:

H H H H

3 ~ N-CH2 O

Many other compounds with different R's can be synthesized following published procedures.
Suitable psoralen derivatives have the formula i~z in which Rl, R3 and R6 each independently is hydrogen or lower aIkyl, .

~3~3~

is hydrogen, C1-C7 alkyl Gr Cl-C7-alkyl substitutecl by hydroxy C1-C7~ oxy, ~-nino, h~lo or ~\/
, and R2 and R5 each independently is hydrogen, hydroxy, carboxy, carbo-CI-C7-alkoxy or C1-C7-alkoxy.

Angelicin derivatives are superior to psoralen compounds for monoadduct formation. If a single-stranded probe is covalently attached to some extra double-stranded nucleic acid, e.g., DNA, use of phenanthridium and psoralen compounds is desirable since these compounds interact specifically to double-stranded nucleic acid, e.g., DNA, in the dark. The chemistry for the synthesis of the coupled reayents to modify nucleic acids for labeling, described more fully hereinbelow, is similar for all cases.
! The nucleic acid component can be singly or doubly stranded DNA or RNA or fragments thereof such as are produced by restriction en7ymes or even relatively short oligcmers.
The nucleic acid-binding ligands of the present invention used to link the nucleic acid component to the label can be any suitable photoreactive form of known nucleic acid-binding ligands. Particularly preferred nucleic acid-binding ligands are intercalator compounds such as the furocoumarins, e.g., angelicin (isopsoralen) or psoralen or derivatives thereof which photochemically will react with nucleic aclds, e.g., 4'-aminomethyl-4,5'-;

3~i39 dimethyl angelicin, ~'-am.inomethyl-trioxsalen ~4'-amino-methyl-4,5',8-trimethyl-psoralen), 3-carboxy-5-amino-psoralen, 3-carboxy-8-aminopsc)r~lcn, 3-c;lrbo~y-~-h~d~o~y-psoralen, and 9,5'-dime.llyl-~-methoxy psoralen, as well as the phenanthridines, e.g., the mono~ or bis-azido amino-alkyl methidium or ethidium compounds.
Photoreac~ive forms of a variety of other intercalating agen~s can generally be used as exemplified in the following table:
Intercalator Classes and Representative Compounds Literature References A. Acridine dyes Lerman, J. Mol. Biol.
3:18(196I); Bloomfield et al, "Physcial Chemistry - of Nucleic Acids", Chapter 7, pp. 429-476, ~!arper and Rowe, NY(1974) proflavin, acridine Miller et al, Bio-orange, quinacrine, polymers 19:2091~1.980) acriflavine B. Phenanthriditles Bloomfield et al, supra Miller et al, supra ethidium coralyne Wilson et al, J. Med.
Chem. l9:i261(1976) ellipticine, ellipticine Fety et al, FEBS
cation and derivatives Letters 17:321(1971);
Kohn et al, Cancer Res.
35:71(197G); LePecq et al, PNAS (VSA)71:
5078(1974); Pelaprat et al, J. Med. Chem.
23:1330(1980) C. Phenazines Bloomfield et al, supra 5-methylphenazine cation D. Phenothiazines ibid chlopromazine E. Quinolines ibid chloroquine quinine F. Aflatoxin ibid ~ ~ 293~3~

G. Polycyclic hydrocarbons ibid and their oxirane derivatives 3,4-benzpyL-erle benzopyrene diol Yange et al, Biochem.
epoxidie, l-pyrenyl- Biophys. Res. Comm.
oxlrane 82:929(1978~
benzankhracene-5,6-oxide Amea et al, Science ~ 176:47(197~) H. Actinomycins Bloomfield et al, supra actinomycin D
I. Anthracyclinones ibid beta-rhodomycin A
daunamycin J. Thiaxanthenones ibid miracil D
X. Anthramycin ibid L. Mitomycin Ogawa et al, Nucl.
Acids Res., Spec. Publ.
3:79(1977) Akhtar et al, Can. J. Chem.
53:2891(1975) M. Platinium Complexes Lippard, Accts. Chem.
Res. 11:211(1978) N. Poly~ntercalators echinomycin Waring et al, Nature 252:653(197~);
--Wakelin t Biochem. J.
157:721(1976) quinomycin Lee et al, Biochem. J.
triostin 173:115(1978): Huang et BBM928A al, Biochem. 19:
tandem 5537(1980): Viswamitra et al, Nature 289:
gl7(1981) diacridines LePecq et al, PNAS
(USA)72:2915(1975):
Carrellakis et al, Biochim. Biophys. Acta 418:277(1976);; Wakelin et al, Biochem 17:5057(1978); Wakelin et 3~9 al , FEBS Lett.
104:261~1979); Capelle et al, Biochem. 18:335~
(1979); Wright et al, ~iochem; 19:5825(1980);
Berllier et al, Biochem.
J. 199:479 (1981); King et al, 8iochem. 21:4982 ~19~2) ethidium dimer Gau~ain et al, Biochem.
17:5078(1978); ~uhlman et al, Nucl. Acids Res.
5:2629(1978); Marlcovits et al, Anal. Biochem.
94:259(1979): Dervan et al, JACS 100:1968(1978);
ibid 101:3664(1979).
ellipticene dimers Debarre et al, Compt.
and analogs Rend. Ser. D. 284:
81(1977); ~elaprat et al, J. Med. Chem.
~3:1336(1980) heterodimers Cain et al, J. Med.
Chem. 21:658(1978);
Gaugain et al, Biochem.
17:5078(1978) trimers Hansen et al, JCS
Chem. Comm. 162(1983);
Atnell et al, JACS
! 105:2913tl983) O. Norphillin A Loun et al, JACS 104:
3213(1982) - --.
P. Fluorenes and fluorenones Bloomfleld et al, supra fluorenodiamines Witkowski et al, Wiss. Beitr.-Martin-Luther-Univ. Halle Wittenberg, 11(1981) Q. Furocoumarins angelicin Venema et al, MGG, Mol. Gen. Genet.
179;1 (1980) 4,5'-dimethylangelicin Vedaldi et al, Chem.~
Biol. Interact. 36:
275(1981) .

~3~3~

psoralen Marciaili et al, Z.
Naturforsch B 27(2):
196(]972) 8-methoxyp~;oralen Belo~nzov Qt al, Mutat.
Res. 84:11(1981);
Scott et al, Photochem.
Pho-tobiol. 34:6~(1981) 5-aminomethyl-~- Hanserl et al, Tet. Lett.
methoxypsoralen 22:1847(1981) 4,5,8-trimethylpsoralen Ben-Hur et al, sioche~. Biophys.
Acta 331:181(1973) 4'-aminomethyl-4,5,8- Issacs et al, Biochem.
trimethylpsoralen 16:1058(1977) xanthoto:cin Hradecma et al, Acta Virol. (Engl. Ed.) 26:
305(1982) khellin Beaumont et al, Biochim. 8iophys.
Acta 608:1829(1980) R. Benzodipyrones Murx et al, J. Het.
Chem. 12:417(1975);
- Horter et al, Photo-chem. Photobiol. 20:
407(1974) S. Monostral Fast Blue Juarranz et al, Acta Histochem. 70:130 (1982) Particularly useful photoreactive for~s of such inteL-calating agents are the azidointercalators. Their reactive nitrenes are readily generated at long wavelength ultraviolet or visib]e light and the nitrenes of arylazides prefer insertion reactions over their rearrangement products [see White et al, Methods in Enzymol., 46, 644j (1977)~. Representative azidointercalators are 3-azidoacridine, 9-azidoacridine, ethidium monoazide, ethidium diazide, ethidium dimer azide ~Mitchell et al, JACS, 104, 4265, (1982)], 4-azido-7-chloroquinoline, and 2-azidofluorene. Other 3~3~

useful photoreactable intercalators are the furocoumarins which form [2+2] cycloadducts with pyrimidine residues.
Alkylating ag~nt.s can also be use~ such as bis-chloroethylamines and epoxides or aziridines, e.g., aflatoxins, polycyclic hydrocarbon epoxides, mitomycin, and noxphillin A.
The label which is linked to the nucleic acid compoilent according to the present invention can be any chemical group or residue having a detectable physical or chemical property. Tlle label will bear a functional chemical group to enable it to be chemically linked to the intercalator compound. Such labeling materials have been well developed in the field of immunoassays and in general most any label useful in such methods can be -applied to the present invention. Particularly useful are enzymatically active groups, such as enzymes ~see Clin. Chem., ~197~), 22, 1243), enzyme substrates (see sritish Pat. Spec. 1,548,741), coenzymes (see U.S.
Patents 4,230,797 and 4,238,565), and enzyme inhibitors (see U.S. Patent 4,134,792; fluorescers (see Clin. Chem., (1979), 25, 353) and chromophores including phycobili-proteins; luminescers such as chemiluminescers and bioluminescers (see Clin Chem., (1979), 25, 512, and ibid, 1531); specifically bindable ligands; and residues comprising-radioisot~pes such as 3H, 35S, 32p, 125I, and 4C. Such labels are detected on the basis of their own physical properties (e.g., fluorescers, chromophores and radioisotopes) or their re~ctive or binding properties (e.g., enzymes, substrates, coenzymes and inhibitors).
For example, a cofactor-labeled nucleic acid can be detected by addinq the enzyme for which the label is a cofactor and a substrate for the enzyme. A hapten or ligand (e.g., biotin) labeled nucleic acid can be detected by adding an antibody or an antibody fragment to the hapten or a protein (e.g., avidin) which binds the .

12~3~

ligand, tag~ed with a detectable molecule. Such detectable m~lecule can be some molecule with a measurable physic~l property (e.g., fluorescence or absorbance) or a participant in an enz~me reaction (e.g., see above list). For exaMple, one can-use an enzyme which acts upon a substrate to generate a product with a measurable physical property. Examples of the la-tter include, but are not limited to, beta-galactosidase, alkaline phosphatase, papain, and peroxidase. For ln situ hybridization studies, ideally the final product is water insoluble. Other labels will be evident to one of the ordinary skill in the art.
The particular sequence in making the labeled nucleic acid can be varied. Thus, for example, an amino-substituted psoralen can first be photometrically coupled with a nucleic acid, the product having pendant amino groups by which it can be coupled to the label.
Alternatively, the psoralen can first be coupled to a label such as an enzyMe and then to the nucleic acid The spacer chain length between the nucleic acid-binding ligand and the label can be extended via hydrocarbon or peptide. A typical example involves extending an 8-hydroxy psoralen derivative with an alkyl halide,according to the method described by J. L. DeCout and J. Lhomme, Photochelllistry Photob~ y, 37, 155-161 (1983). The haloalkylated derivative is then reacted either with thiol or amines to produce the reactive residue, as has been described by W. A. Saffran et al., Proc. Natl. Acad. Sci., U.S.A., 79, q594 ~1982).
If the label is an enzyme, for example, the product will ultimately be plàced on a suitable medium and the extent of catalysis will be determined. Thus, if the enzyme is a phosphatase the medium could contain nitrophenyl phosphate and one would monitor the amount of nitrophenol generated by observing the color. If the ~3~3~

enzyme is a beta-~alactosidase the mcdium can contain o-nitrophenyl-D-galac~o~pyranoslde which also will liberate nitrophe~llol.
The la~eled nucleic acid of the present invention is applicable to all conventional hybridization assay formats, and in general to any format that is possible based on formation of a hybridization product or aggregate comprising the labeled nucleic acid. In particular, the unique labeled probe of the present invention can be used in solution and solid-phase hybridization formats, including, in the latter case, formats involving immobilization of either sample or probe nucleic acids and sandwich formats.
- The labeled nucleic acid probe will comprise at least one single stranded base sequence substantially complementary to or homologous with the sequence to be detected. However, such base sequence need not be a single continuous polynucleotide segment, but can be comprised of two or more individual seqments interrupt~d by nonhomologous sequences. These nonhomologous sequences can be linear or they can be self-complementary and form hairpin loops. In addition, the homologous region o~ the probe can be flanked at the 3' - and 5' -terminii by nonhomologous sequences, such as those comprising the DNA or RNA of a vector into which the homologous sequence had been inserted for propagation.
In either instance, the probe as presented as an analytical reagent will exhibit detectable hybridization at one or more points with sample nucleic acids of interest. Linear or circular single stranded polynucleotides can be used as the probe element, witl major or minor portions being duplexed with a complementary polynucleoti.de strand or strands, provided that the critical homologous segment or segments are in single stranded form and available for hybridization with ~2~ 3~
sample DNA or RN~. Useful probes include linear or circular pro~cs wherein the hon~ologous probe sequcnce is in essentially onlysingle stranded fo~m ISee particularly, HU ~nd Messing, Gene, 17, 271, (1~82)].
The labeled probe of the present invention can be used in any conventional hybridization technlque. As improvements are made and as conceptually new forrnats are developed, such can be readily applied to the present labeled probe. Conventional hybridization formats which are particularly useful include those wherein the sample nucleic acids or the polynucleotide probe is immobilized on a solid support (solid-phase hybridiza~ion) and those wherein the polynucleotide species are all in solution (solution hybridization).
In solid-phase hybridization formats, one of the polynucleotide species participating in hybridization is fixed in an appropriate manner in its single stranded form to a solid support. Useful solid supports are well known in the art and include those which bind nucleic acids either covalently or non-covalently. Noncovalent supports which are generally understood to involve hydrophobic bonding include naturally occurring and synthetic polymeric materials, such as nitrocellulose, derivatized n~lon, and fluorinated polyhydrocarbons, in a variety of forms such as flters or solid sheets.
Covalent binding supports are also useful and comprise materials having chemically reactive groups or groups, such as dichlorotriazine, diazobenzyloxymethyl, and the like, which can be activated for binding to polynucleotides.
A typical solid-phase hybridization technique begins with immobilization of sample nucleic acids onto the support in single stranded form. This initial step essentially preverlts reannealing of complementary strands from the sample and can be used as a means for ~3~39 concentratitlg samp~e m~teri~l Ol- the support for enhanced detectability. The polynucleotide probc is then contacted with the ~upport ~nd hybridiza~ion detected by measurement of ~he label as described here~in. The solid support provides a convenient means for separating labeled probe which has hybridized to the sequence to be detected from that which has not hybridized.
~ nother method of interest is ~he s~ndwich hybridi2ation technique wherein one o~ two mutually exclusive fraqmen~s of the homologous sequence of the probe is immobilized and the other is la~elIed. The presence of the polynucleotide sequence of interest results in dual hybridization to the immobilized and labèled probe segments. See Methods in Enzymology 65,468, tl980) and Gene 21, 77-85, (1983) for further details.
The present invention will now be described with reference to the following non-limiting examples.

Examples All preparations involving either azidophenyl or furocoumarin derivati~es were handled in a darkroom under sa~elight conditions.
Infrared (IR) spectra were obtained with a Perkin-Elmer*Model 710B or 237 infrared spectrophotometer, or on a Nicolet* SDBXB FT IR
spectrometer, unless o~herwise noted. A 1602 cm 1 band of polystyrene film was used as an external calibration standard. Signals were reported as cm 1.
Proton magnetic resonance (lH NMR) spectra were obtained at 89.55 MHz using a JEOL FX-90~ spectrometer or at 60 MHz using a Vari-an T-60*spectrometer; spectra were obtained in CDC13 solution, unless othe~ise noted.
Chemical shifts were reported in parts per million downfield from the internal standard tetramethylsilane.

* Trade Mark -3~3~9 Mass spectra (~S) were obtained on a llewlett-Packard 59B5~ spectrom~te~ operating in either an electron impact (EI). chemical ioniza~i.on (CI), or ~a~t atom bombardment lF~) mode.
Organic rea~ents were obtained Erom Alclrich Chemical Company and were used without purification, unless otherwise noted. Inorganic reagents were ACS
reagant grade from Fisher Scientific Company or any other major vendor. R~action solvents were ACS reagent grade;
tetrahydrofuran lTHF) was HPLC grade from J.T. ~aker Chemical Company. Brine refers to a saturated aqueous sodium chloride solution.
Thin layer chromatograph ~TCL) was performed using silica gel 60F-254 plates from E. Merck. Flash column chromatography was performed using E. Merck or American Scientific Products Silica Gel 60 (230-400 mesh). All melting points and boilinq points reported were uncorrected.

Example 1: .
1-Amino-17-N-(Biotinylamido?-3,6,9,12,15-Pentaoxaheptade-cane ! A solution containing 7.2 g of 1,17-diamino-3,6,9,12,15-pentaoxaheptadecane, prepared according to Kern et al, Makromol. Chem., 150 ~10):2539, (1979), (25 mmol) in 75 mL of DMF under an argorl atmosphere was treated with 3.41 g of N-oxysuccinimidyl biotin (10 mmol) added in portions over 1.0 hour. The resulting solution was stirred for 9 hours at ambient temperature. TLC
(Sio2, 70:10:1 CHC13-CH30H- concentrated NH40H) visualized by dimethylaminocinnamaldehyde spray reagent showed excellent conversion to a new product (Rf=0.18).
The reaction mixture was divided in half and each half was absorbed onto 5io2 and flash-chromatographed on S00 g of SiO2-60 (230-400 mesh) using a 70:10:1 * Trade Mark ~3~:3~

CHC13-CH3OH-c:oncelltrat~d NH4OH solvenl: mixture.
Fractions containi.ll~ the product were poolcicl and concentrated to cJi.ve 2~42 ~ of a cJel.atino~;, waxy 30Ii.cl.
The product was preci.pitatecl as a soll.d from isopropanol-ether, washed with hexane, and dried at 55C
(0.1 mm~ to give 1.761 g of a white powder ~35~ yield).

Anal~sis: Calculated for C22H42N4O7S-3/2 H2O: C, 49.51; H, 8.50;
N, 10.49 Found: C, 49.59; H, 8.13;
N, 10.39 H NMR (90 MHz, dmso-d6)~: 1.1-1.7 (m, 6H~; 2.05 (t, J=7Hz, 2H); 2.62 (t, J=4l~z, lEI); 2.74 (t, J=4Hz, lH); 3.0-3.4 (m, 14H); 3.50 (s, 14H);
4.14 (m, lH); 4.30 (m, lH); 6.35 (d, J=qHz, lH); 7.80 (m, lH).

13C NMR (22.5 MHz, dmso-d6)~: 25.2, 28.0, 28.2, 35.1, 40.6, 55.3, 59.2, 61.1, 69.6, 69.8, 71.3, 162.7, 172.1.
i: .
IR (KBr) cm 1 2900, 2850, 1690, 1640, 1580, 1540, 1450, 1100.

Mass Spectrum (FAB) m/e: 507.3 (M+l, 56%).
!

Example 2:
4'-(Biotinyl-PEG)-4,5'-Dimethylangelicin ~Biotinyl-Peq-Angelicin) Example 2A:
Small Scale Procedure A solution of 203 mg of 1-amino-17-N-(biotinylamido)-3,6,9,12,15-pentao~aheptadecane (0.4 mmol) in 1 mL of DMF under an argon atmosphere was treated with 78 mg of N, N-carbonyldimidazole (0.48 mmol). The resulting mixture was stirred for 4 hours and :~2~3~3~

was then trea~ed wi~h 55 mg of 4'-aminomethyl-4,5'-dimethylangelicin h~drochloride (Dall'Acqua et al, J
Med. Chem., 24, l78, ~19~1)) (0.2 mmol), l40 ~L of diisopropylethylamine and 100 ~L of DMF. The resulting mixture was stirred overnight at 50C. The mixture was then evaporated onto SiO2 ln vacuo and the impregnated solid flash chromatographed on 6() g of SiO2 (230-400 mesh) eluted with 1. 5 L of 7% CH3-CHC13 fcllowed by 1 L
of 10% CH30H-CHC13. Fractions containing the product were pooled and concentrated to give 72 mg of a glassy solid (47% yield).

H NMR (90 MHz, ~nso-d6)S: 1.1-1.8 (m, 6H); 2.04 (bt, J=7Hz,-2H); 2.5 (s, 6H); 2.56 (m, lH); 2.74 (bd, J=4Hz, lH); 2.8-3.4 (m, 14H); 3.40 (m, 14H); 4.14 (m, lH); 4.25 (m, lH); 4.40 (bd, J-6HZ, 2H); 6.15 (m, lH~;
6.35 ~s, lH); 7.02 (s, lH), 7.45 (d, J-8HZ, lH); 7.62 (d~ J=8HZ, lH), 7.80 (m, lH) .

13C M~R (22.5 MHz, dmso-d6)~: 11.9, 18.9, 25.3, 28.2, 28.3, 33.4, 35.2, 55.4, 59.2, 61.0, 69.2, 69.6, 69.8, 70.0, 89.0, 107.8, 112.0, 113.1, 114.3, 120.6, 121.6, 153.6, 154.4, 155.6, 157.9, 159.5, 162.7, 172.1.

Example 2B:
Large Scale Preparation A solution containing 760 mg of amino-Peg-biotin (1. 5 mmol) and 2 mL of DMF under an argon atmosphere was treated with 2g2 mg of N,N-carbonyldiimid azol~ (1. 8 mmol). The resulting solution was stirrecl for 4 hours at room -temperature.
Analytical TLC (siO2 r 4: 1 CHC13-CH30H) indicated a complete conversion of starting material (Rf=0.1) to imidazourea (Rf=0. 5) . The solution was then txeated with 243 mg of q'-aminomethyl-4,5'-dimethylangelicin (lmmol) and 1. 39 mL of diisopropylethylmine (1. 03 g, 8 mmol).
The resulting mixture was then stirred at 50 C under an ,.

3~:3~
arqon dtmosphere overnight~ The solvents were rcmoved in vacuo. Th~ residue was then dissolvecl in CI~3OII and adsorbed on to Sio2, which was thcrl pl~cc~ atop a 100 g of SiO2-60 column ~230~400 mesh) which had becn packed and equilibrated with a 9:1 C~IC13-CH3OH solvent: mixture.
The column was then flash eluted with this solvent mixture with 25 mI. fractions collected. Fractions contàining the product were then pooled and concentrated to give 0.63 g of an oil. The product was dissolved in acetone containing a trace of methanol and precipitated with ether. Centrifuga~cion was used to isolate the flocculant white precipitate. The product was dried at 53C, 0.1 mm, to give 450 mg of a white, taclcy solid (52~6 yield). mp 80-83C.

Analysis calculated for C37H53N5011S: C, 57-11; ~, 6-91; N, 9-06 Found: C, 56.61; I~, 7.04; N, 9.18 IR ~XBr) cm 1 2910, 2860, 1700, 1635, 1550, 1440, 1375, 1275, 1240, Iloo.

The lH ~MR spectrum agreed with that o~ 1 he previously prepared sample.

Example 3:
N,N'-Dicyanoethyl-N,N'-1,4-Diaminobutane A solution of 18.6~I g of N,N'-dimethyl-1,4-diaminobutane (0.161 mole) was cooled to 0C and treated with 23 mL of ~reshly distilled acrylonitrile (18.54 g, 0.35 mole) added dropwise over O.S hours. A mild exotherm was noted during this addition. The resulting mixture was allowed to cool to ambient temperature over a 1.5 hour period. The mix'cure was tIlen concentrated in vacuo to remove acrylonitrile at 12 mn~ and was then distilled at 0.1 mm. Fractions distilling at 207-222C

were pooled to yield 30 5S g o~ product as a colorless oil (85.6% yield).

A~alysis: Calculated for C12~122N4: C, 6~.82; H, 9.98; N, 25.20 Fou~d: C, 64.55; H, 10.12; N, 24.84 H NMR (60 MHz, CDC13)~: 1.45 (m, 4H); 1.92 (s, 6H, -N-CH3); 2.4-3 ~m, 12H) C NMR (22.5 MHz, COC13)~: 15.9, 24.7, 41.4, 52.5, 56.6, 118.8 ppm.-- IR (CHC13) cm 1 29S0, 2850, 2810, 2250, 1510.
~ . . . .
Mass Spectrum: (EI) m/e: 222.2 (~ , 11.9~);
223.3 (M~l, 2.6%).

Example 4:
N-4 ,N-9-Dimethylspermine A solution containing 6.2 g of N, N ' -Dicyano-ethyl-N,N'-dimethyl-1,4'-diaminobutane (28 mmol) in 200 mL of CH30H was saturated with gaseous ammonia. The solution was then treated with several spatulas full of freshly ~ashed (]OX with H2O) Raney Nickel. The mixture was then hydrogenated at 50-70C and 50 psig of H2 in a Paar shak2r apparatus. The theoretical amount of H2 was taken-up in 2 hours, but hydrogenation was continued overnight. The mixture was then filtered through Celite and the precipitate washed with CH3OH and H2O. The filtrate was concentrated ln vacuo to give 6.48 g of an oily residue. Evaporative distillation at 100-llO~C, 0.05 mm, gave 6.272 g of the product as a colorless oil (97%).

Analysis: Calculate~ for C12H30N4: C, 62.56; H, 13.13; N, 24.32 Found: C, 62.43; H, 13.20; N, 24.03 .

~5a3~i3~

H ~R (90 MHz, CDC13) S 1.l (s, 4H, Nli~); 1.3 (m, 9H, C6-, C7-CH2)j~
1.4 tq~untet, ~lli, J=71-!~., C2-, C11 C112); 2.0 ~s, 611, N~~, N9-C113); 2.15 (m, 8ll, C3-, C5-, C~3-, C1o-,CII;~); 2.65 (~:, J=711%, ~11, Cl, C12~1~).

IR ~C}ICl3) cm 1 3500_3050, 3380, 2920, 2840, 2780, 2500, 1580, 1470.

Upon standing at room temperature several days, a white preci~pitate resulted which was determined to be the carbonate salt on the basis of the following data:
nalysis: Calculated for Cl2H30N4-HC02: C, 59.73; H, 11.95, N, 21.43.
Found: C, 59.84; H, 12.00, N, 22.12.

C ~IR (CI~C13)~: 163.2 ppn.

Example 5:
N-l-(Biotinylamido)-N~4,N-9-Dimethylspermine A solution containin~ 2. 907 g of N-4, N-9-dimPthylspermine (12. 62 mmol) in 25 mL of DMAC
(dimethylacetamide) under argon was treated with 1. 722 g of N-oxysuccinimidyl biotin (5 . 05 mmol) added in portions over a 3 hour period. A precipitate was noted one hour after addition. The resulting solution was stirred overnight at ambient temperature. Analytical TLC (SiO2, 15:5:1 CHCl3-CH30H-concentrated NH40H, ninhydrin or dimethylaminocinnamaldehyde visualization) indicated the formation of monoacylation product (R~-0.57). The mixture was filtered and the precipitate washed with DMAC
and dried at 55C, 0.1 mm. This product was identified as the bis-N-oxysuccinimidyl salt of N-4,N-9-dimethyl-spermine on the basis of the following data. mp:
decomposes slowly 96.5-105C.

Analysis: Calculated for C20H40N606: C, 52.16; H, 8.75; N, 18~24 Fo ~ : C, 52.14; H, 8.33; N, 17.69 ~3~ 9 H ~ (90~1z, ~qO)~: 1.55 (m, 4~1); 1.86 (q, J=8llz, 4~1); 2.36 (s, 611);2.6 (m, 6~i); 2.64 (s, 8il); 2.92 (t, J-8~-lz, 4ll).

13C ~ (22.5 ~l~'l)2)~ 22.6, 2~.1, 2~.7, 37.6, 39.~, 53.1, 55.6, 179.0 IR (KBr) cm : 3060, 2840, 1760, 1670, 1250, 1100.

The filtrate which contained the desired product was evaporated in vacuo and flash chromatographed on 500 g of Sio2-60 (230-400 mesh) using a 15:5:1 CHC13-CH30H-concentrated NH40H solvent mixture. Fractions of 25 rnL
were collected. Fractions containing the product (136-245i were pooled and concentrated to give 1.127 g of a colorless foam, which was precipi~ated as a white powder from isopropanol-ether and dried at 55C, 0.1 ~.
The yield of product was 958 mg (42%).
Analysis: Calculated for C22H44N602S 2H20: C, 53.63; H, 9.82; N, 17.06 Found: C, 54.08; H, 9.46; N, 17.25 H NMR (90 MHz, D2O)$: 1.2-1.9 (m, 14H, C6, C7, Cll, C2-H2, Bio-CB , c3, C~-H2), 2.25 (d, 6H, CH3-N-); 2.3-3.0 (m, 12H); 3.2 (t, J=7Hz, 2H); 3.35 (m, lH, Bio-C2H), 4.4 (m, lH, Bio-C3H); 4.6 (m, lH, Bio-C4H).

3C NMR (22.5 MHz, D2O)d: 25.8, 27.3, 27.5, 28.9, 29.8, 30.0, 37-7 (Bio ~) 39.6 (N4,Ng-CH3); 40.7 (Bio-C5); 41.8 (C12); 43.0 (Cl); 55.9 (Bio-C2); 57.5 (C3,C10); 58.3 (C5, C8); 62.3 (Bio-C4); 64.2 (Bio-C3);
107.3 (Bio-C2?; 178.6.

IR (XBr) an : 3300, 3100, 2950, 2870, 2810, 1700, ]640, 1550, 1460.

Mass Spectrum: ~FAB) m/e: 457 (M+l, 10.4~) 458 1M~2, 3.3~) 459 (M+3, 1.4%) 3~3~

Example 6:
Biotinyl-Spermine;~ -Anclelicin 1~ solut:icn containin-J 29U n~cJ of N-1 (rllotinyl-amido)-N-4~N-9-cIimethylspermiIle (0.5 mmol~ in 3 mL of DMAC under argon was treated with 97 mg of N,N-carbonyl-diimidazole (0.6 mmol). Complete conversion to imidazolide was noted after 4 hours by analytical TLC
(SiO2, 15:5:1 CHCl3-CH30H-concentrated NH40H, Rf=0.5).
The reaction mixture was then treated wi-th 80 mg of 4'-aminomethyl-4,5'-dimethylangelicin (0.33 mmol) and 436 jUL of diisopropyle-thylamine (2.6 mmol). The resulting solution was then stirred at 40C overnight. ~ new product was then noted by analytical TLC (SiO2, 50:10:1 CHCl3-CH30H-concentrated NH40}1, I~f=0.1). The mixture was then concentrated in vacuo and flash chromatographed on 60 g of SiO2 eluted with a 50:lO:1 CHCl3-CH30H-concentrated NH40H solvent mixture. Fractions of 25 mL
were collected. Fractions containing the product were pooled and concentrated to a solid, which was dissolved in 5N HCl and evaporated to an oil, which as azeotropically evaporated t~"ice with toluene. The product was then precipitat'ed as a white powder from ethanol-ether and dried at 55C, 0.1 mm. The yield of product was 209 mg (76~6).
, Analysis: Calculated for C37H55N706S SH20: C, 50.00; H, 7.37; N, 11.03 Found: C, 50.15; H, 7.65; N, 10. 46 H N~ (90 MHz, D20)~: 1.1-1.2 (m, 16H); 2.23 (s, 3H, N-CH3); 2.41 (s, 3H, N-CH3); 2.75 (m, 2H); 2-80 (s, 3H, Ang C4-CH3); 2.86 (s, 3H, Ang C5'-CH3); 2.9-3.4 (m, 13H); 4.16 (m, 2H, Ang C4'-H2); 4.35 (m, lH, Bio C3-H); 4.6 (m, lH, Bio-C4-H); 6.06 (m, lH, Ang C3-H); 7.12 (AB quartet, J=8Hz, 2H, Ang C5-H, C6-il). ' 3~9 Cl~ll~ (2~.5 ~Iz, D20)S: 13.3, 20 6, 22.8, 25.8, 2fi.3, 27.1, 29.8, 30.1, 36.0, 37.4,:~.2, 38.7, ~ , 55.6, 57.3, 57,4, 62.4, 6~1.2, 110.2, 112.'1, 113.1, 1l6.1, 117.6, 122.1, 1~7.7, 15fi.6, 157.'1, 158.(J, lh2.0, 165.0, 167.2.

I~ (KBr) om 1 3400, 3260, 2940, 2625, 2500, 1730-1640, 1615, 1560, 1450, 1390, 1285, 1250, 1080.

Example 7:
4,5'-Dimethyl-4'-Hydroxymethylangelicin A suspension containing 750 mg o 4'-chloro-methyl-4,5'-dimethylangelicin (2.86 mmol) in 500 mL of H2O was refluxed for 4 hours and then cooled. The precipitate was filtered to give 600 mg of crude product mixture which was flash chromato~raphed on a 100 g column of SiO2-60 eluted with a 1% CH30H-CHC13 solvent mixture.
Fractions of 25 mL were collected. Fractions containing the product were pooled and concentrated to give a white solid which was recrystallized from ehtyl acetate and dried at 55C, 0.1 mm.

Yield = 343 mg (49~). mp 201.5-203C (lit mp 201C).

Analysis: Calculated for C14H12O4: C, 68.84; H, 4-95 Found: C, 68.98; H, 4.82 H NMR (90 MHz, dmso-d6)S: 2.40 (s, 3Hj; 2.45 (s, 3H); 4.72 (d, J=4Hz, 2H,-CH2-); 4.9 ~t, J=4Hz, OH); 6.3 (m, lH); 7.5 (AB quartet, J=9Hz, C5-H, C6-H).

IR (KBr) cm : 3430, 3080, 2940, 2900, 1700, 1635, 1620, 1450, 1400, 1380, 1300, 1270, 1090.

Mass Spectrum: ~I (m/e): 244.1 (~l, 72.6%);
245.1 (M+1, 12.1%);
198.1 (M -46), 100~).

~ 2~3~

Example 8:
4,5'-Dimethyl-4'-Formy:lallgelicln __ ~ solu~ion containllg 175 mCJ o~ 4,5'-dimetl1yl-4'-hydro~ymethyl angelicin (0.71 ~ol) in 10 mL of CH2Cl2 was treated with 306 mg of pyridinium chlorochromate (PCC), ~0.42 mmol). After 2 hours, an addi-tional 75 mg of PCC was added and the mixture stirred for 1 hour. The mixtu`re was filtered and the precipitate was washed thrice with S mL aliquots of CH2Cl~. The combined organic layers were concentrated and the residue flash chromatographed on 60 g of SiO2-60 (230-400 mesh) el~lted with a 0.5% CH30H-CHC13 mixture. Fractions of 20 mL were collected. Fractions containing the product were pooled and concentrated to a white solid which was recrystallized from toluene and dried at 55C, 0.1 mm.
The yield was 79 mg (46%). A second crop of 24 mg (14%) was obtained from concentration of the mother liquor to half volume, precipitation from ether and drying at 55C, 0.1 mm was conducted. The combined yield was 103 mg (60~). mp 219-220C.

Analysis: Calculated for C14H1004: C, 69.42; H, 4-16 ! Found: C, 69.44; H, 4.47 H NMR (90 ~Z' ~ImS~l6)$ ~ 51 (s~ C4-CH3); 2.90 (s, C5'-cH3); 6.3 (m, C3-H); 7.5 (d, J=8Hz, C5-H); 7.66 (d, J=8Hz, C6-H); 10.4 (~O).

3C NMR (22.5 ~Iz, clmso-d6)~S 18.2, 22.8, 112.2, 117.0, 119.9, 120.4, 126.2, 131.8, 158.0, 159.5, 160.9, 163.1, 168.2, 189.8.

IR (KBr) cm 1 3060, 2970, 2860, 1730, 1690, 1615, 1570, 1390, 1070.

Mass Spectrum: EI (m/e): 242.1 (M , 100%);
243.1 (M+l, 14.9%).

2~

3~3~

Example 9:
Biotinyl-Sp~rllline -Any~licln -A suspension oE 58 mcJ of 9'-~ormy]-~,5~-dimethylangelicill (0.24 nunol) and 137 m~ oE
N-l-~biotinylamido)-N-4,N-9-dimethylspermine (0.3 mmol) in 3 mL of CH30H was treated with 25 mg of NaBH3CN (0.4 mmol). The resulting suspension was warmed briefly to dissolve the contentsj and was then stirred two hours at ambient temperature. Analytical TLC tsio2, 30:10:1 CHC13-CH30H-concentrated NE140H) indicated complete conversion of aldehyde to a mixture containing a major product ~Rr=0.3). The mixture was treated with 50 ~L of 0.5 N HCl and stirred for 5 minutes and was then neutralized with 50juL of 0.5 N NaOH. The solvents were then evaporated _ vacuo and the residue flash chromatographed on 60 g of SiO2-60 (230-400 mesh) eluted with a 40:10:1 CHC13-CH30H-concentrated NH40H solvent mixture. Fractions of 25 mL were collected. Fractions containing the product were pooled and concentrated to an oil which was dissolved in 3 mL of 4N HCl and evaporated to a solid which was azeotropically distilled twice with toluene. The white solid was then precipitated from ethanol-ether and dried at 53C, 0.1 mm. The yield was 96 mg (51~). mp: effervesced slowly above 147C, decomposed at 202-220C.

Analysis: Calculated for C36H57C13N605S 6~120:
C, 47.92;1l, 7.71; N, 9.31 Found: C, 47.91; H, 7.04; N, 9.97 H NMR (90 ~Iz, D20)~: 1.1-2.0 (m, 12H), 2.18 (t, J=7Hz, 4H); 2.35 (s, 3H, N-CH3), 2.48 (s, 3H, N-CH3); 2.8 (m, 2H); 2.79 (s, 3H, Ang C4-CH3);
2-84 (s, 3H, Ang C5-CH3); 2.9-3.5 (m, 13H)i 4.3 (s, 2H, Ang-C4'-CH2);

4.2-4.4 (m, 211, Bio C3-H, C~-H); 6.2 (s, 1~-1, Anq C3-ll); 7.36 (d, J=~lz, lH, Ang C5-H); 7.52 (d, J=8llz, 1~ g C6-~l).

13c NMR 122-5~nlz, D2o)s: 13.6, 20.a, 22.~, 25.~, 27.1, ~s.a, 30.0, 37.5, 38.0, ~1.8, ~.2, ~fi~l, Sq.~, 55.7, 57.1, 62.3, 6~.1, 106.~, 110.6, 113.3, 116.7, 117.1, 123.8, 148.1, 158.1, 159.5, 160.6, 165.0, 167.2, 179.2.

IR (KBr) cm 1 3400, 3250, 3050, 2940, 2650, 1720, 1690, 1620, 1450, 1080.

Mass Spectl~m: (FAB) m/e: 683 (M+1, 3.5~) 684 (M~2, 1.2~) 685 (M+3, 5.1%) Example 10:
Labeling Efficiencies of Three Angelicin Linker Biotin Analogues_for DNA Labell1ng Each of the biotin analogues (products of Examples 2, 6 and 9) were diluted to 10 ~M into 0.1 M
sodium phosphate buffer, pH 7.0, 0.15 M NaCl containing 33 pg/mL (50 luM) calf thymus dsDNA. A sample of biotinylrpEG-angelicin was also diluted into 0.1 mM EDTA, pH 8, with the same amount of DNA. Samples of 50 juL were exposed to light of 340 + 30 nm for one hour. The samples, along with 50 ~L samples not irradiated, were diluted with 450 /uL H2O (0.1 M Tris-HCl, p~l 8.0, for the samples not containing salt) and extracted 2x with 0.5 mL
2-butanol, diluted 10% v/v with 3 M sodium acetate, and the DNA precipitated with two volumes of 100% ethanol.
The centrifuged pellets were dried, then dissolved in 250 uL 10 mM Tris-HCl, pH 7.4, 1 mM EDTA. The DNA and biotin concentrations were determined by the method as described in the previous example.

~33~3~

The following results clearly show that the spermine linkers ~re superior to PEG linker:

P~uct of nase Pairsr~t~iotin Non-I~radiat~d _rad~
2 Biotinyl-PEG-~elicin 0 154 -~ (No Salt~ 5100 80 6 Biotinyl-spermine -angelicin 0 15 9 Biotinyl-spermine -an~licin 625 32 Example 11:
Efficiency of Whole Cell Lysate Labelinq of Different Compounds For each labelling reaction appro~imately 108 E. Coli cells were taken in 1 ml borate buffer (10 mM pH
8.2). To the suspension 0.1 ml 1 (N) sodium hydroxide solution was added. The mixture was then heated in a boiling water bath to lyse the cells for 10 minutes. The lysed cells were then neutralized by HCl solution in ice. "
After the neutralization, 50 ~g of labelling reagent dissolved in water (approximately 1 mg/mll was added.
The mixture was then irradiated for 60 minutes using a long wavelength setting of UVL 56* (UV Products, Inc., San ~abriel, California, U.S.A.) hand held lamp (for furocoumarines) or a "~ODAK"*projector lamp (ELH 120v 300w~ (for a~ido photobiotin or Forster's compound). For two step labelling, an overnight incubation with N-hydroxysuccinimidobiotin was done after the photoreaction. No purification was carried ou~ after any of these reactions. A two step process for whole cell lysate produces non-specific biotinylated products, e.g., proteins.
The labelled cells were then spotted onto nitrocellulose paper in equivalent amounts of 105, 104, * Trade Mark , 3~3 10 , 10 , 10 , and 0 cell number; 5, 4, 3, 2, 1 and 0 are respective amoullts on the filter. In the ~igure, the letters f, Si, bp, b~ and ~ re~pective1y repr~sent Forster's photobiotirl (Nucleic Acids ~es., 13(3), 745, (1985)) spermine linked angelicin (Example 9) PEG linked angelicin ~Example 2) two step labelling and blank as the photolabelling reagents.
The chemiluminescent detection of label was done as follows:
The nitrocellulose strip containing the biotinylated cell lysate was incubated in a solution tapproximately 3 ml) containing 250 Jug/ml avidin linked horseradish peroxidase 0.1 M tris (pH 7.5); 0.1 M NaCl; 2 mM MgC12, 0.05% Triton X*100 and 20lug/ml calf thymus DNA
for 30 minutes at room temperature. The strip was then washed twice with the same buffer without the avidin linked horseradish peroxidase and calf thymus DNA. Then it was placed in a petri dish containing 5 ml 60 ~uM
iodophenol in 40 mM tris and 40 mM ammonium acetate (p~
8.2) mixture. Then l:l v/v (10 ml) luminol: H2O2 (1 mM: , 2.5 mM final) was added and the emitted light was recorded by exposing a "POLAROIDn*film (type 57) in a dark room. The results clearly indicate that spermine linked angelicin is superior to PEG linked angelicin in its labelling efficiency.
Conclusion: With pure DNA or with cell lysate nucleic acids polyamine linkers show superior labelling property than the corresponding PEG linkers.
It will be appreciated that the instant specification and cl~ims are set forth by way of illustration and not limitation, and that various modifications and changes may be made without departure from the spirit and scope of the present invention.

* Trade Mark

Claims (29)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY OR
PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A photochemical nucleic acid-labeling reagent of the formula wherein Q is a photoreactive residue of a nucleic acid-binding ligand; L is a detectable label residue; R
is hydrogen, C1 to C7-alkyl, aryl, hydroxy, or C1 to C7-alkoxy; x is an integer from 2 through 7; and y is an integer from 3 through 10; wherein R and x, respectively, can be the same or different each time they appear in the formula.

2. A reagent according to claim 1, wherein R
is hydrogen or C1 to C4-alkyl, x is an integer from 2 through 5, and y is an integer from 3 through 6.

3. A reagent according to claim 1 of the formula

4. A reagent according to claim 1, wherein the photoreactive residue of a nucleic acid-binding ligand is a photoreactive residue of an intercalator compound.

5. A reagent according to claim 4, wherein the intercalator compound is selected from the group consisting of acridine dyes, phenanthridines, phenazines, furocoumarins, phenothiazines, quinolines and antracyclines.

6. A reagent according to claim 4, wherein the intercalator compound is selected from the group consisting of furocoumarins and phenanthridines.

7. A reagent according to claim 4, wherein the intercalator compound is of the formula in which R1, R2 and R3 each independently is hydrogen or C1-C7-alkyl; and R4 is hydrogen, C1-C7-alkyl or C1-C7-alkyl substituted with hydroxy, C1-C7-alkoxy, amino, halo, or is of the formula .

8. A reagent according to claim 4, wherein the intercalator compound is 4'-aminomethyl-4,5'-dimethylangelicin.

9. A reagent according to claim 4, wherein the intercalator compound is of the formula in which R1, R3 and R6 each independently is hydrogen or C1-C7-alkyl; R4 is hydrogen, C1-C7-alkyl or C1-C7-alkyl substituted with hydroxy, C1-C7-alkoxy, amino, halo, or is of the formula ; and R2 and R5 each independently is hydrogen, hydroxy, carboxy, carbo-C1-C7-alkoxy or C1-C7-alkoxy.
!

10. A reagent according to claim 4, wherein the intercalator compound is 4'-aminomethyl-4,5',8-trimethyl-psoralen.

11. A reagent according to claim 1, wherein the detectable label residue is a residue of a moiety selected from the group consisting of biotin, a hapten, an enzyme, a fluorescent molecule and a luminescent molecule.

12. A reagent according to claim 1, wherein the detectable label residue is selected from the group consisting of a biotin and a hapten residue.

13. A labeled nucleic acid comprising (a) a nucleic acid component, (b) a nucleic acid-binding ligand photochemically linked to the nucleic acid component, (c) a label, and (d) a spacer chemically linking (b) and (c) and being of the formula wherein R is hydrogen, C1 to C7 alkyl, aryl, hydroxy, or C1 to C7 alkoxy; x is an integer from 2 through 7; and y is an integer from 3 through 10; wherein R and x, respectively, can be the same or different each time they appear in the formula.

14. A labeled nucleic acid according to claim 13, wherein R is hydrogen or C1 to C4 alkyl, x is an integer from 2 through 5, and y is an integer from 3 through 6.

15. A labeled nucleic acid according to claim 13, wherein the spacer is of the formula .

16. A labeled nucleic acid according to claim 13, wherein the nucleic acid-binding ligand is an intercalator compound.

17. A labeled nucleic acid according to claim 16, wherein the intercalator compound is selected from the group consisting of acridine dyes, phenanthridines, phenazines, furocoumarins, phenothiazines, quinolines and anthracyclines.

18. A labeled nucleic acid according to claim 16, wherein the intercalator compound is selected from the group consisting of furocoumarins and phenanthridines.

19. A labeled nucleic acid according to claim 16, wherein the intercalator compound is of the formula in which R1, R2 and R3 each independently is hydrogen or lower alkyl; and R4 is hydrogen, C1-C7-alkyl or C1-C7-alkyl substituted with hydroxy, C1-C7-alkoxy, amino, halo, or is of the formula

20. A labeled nucleic acid according to claim 16, wherein the intercalator compound is 4'-aminomethyl-4,5'-dimethylangelicin.

21. A labeled nucleic acid according to claim 16, wherein the intercalator compound is of the formula in which R1, R3 and R6 each independently is hydrogen or C1-C7-alkyl; R4 is hydrogen, C1-C7-alkyl or C1-C7-alkyl substituted with hydroxy, C1-C7-alkoxy, amino, halo, or is of the formula ; and R2 and R5 each independently is hydrogen, hydroxy, carboxy, carbo-C1-C7-alkoxy or C1-C7-alkoxy.

22. A labeled nucleic acid according to claim 16, wherein the intercalator compound is 4'-aminomethyl-4,5'-8-trimethylpsoralen.

23. A labeled nucleic acid according to claim 13, wherein the label is selected from the group consisting of biotin, a hapten, an enzyme, a fluorescent molecule and a luminescent molecule.

29. A labeled nucleic acid according to claim 13, wherein the label is selected from the group consisting of biotin and a hapten.

25. A labeled nucleic acid according to claim 13, wherein the nucleic acid component is DNA.

26. A labeled nucleic acid according to claim 13, wherein the nucleic acid component is RNA.

27. A labeled nucleic acid according to claim 13, wherein the nucleic acid component is an oligonueleotide.

28. A labeled nucleic acid according to claim 13, wherein the nucleic acid component is a probe.

29. A labeled nucleic acid according to claim 13, wherein the nucleic acid component is an unknown.