INACTIVATION OF VIRUSES PRESENT IN BLOOD COMPONENTS USING CHEMICALLY-ACTIVATED COMPOUNDS

Abstract: A method of inactivating viral molecules present within a blood sample and compounds for use in same. The method involves adding to a virus-containing blood sample an effective quantity of a compound which both has an affinity for viral nucleic acid and which is activatable to an excited state in which the compound covalently binds viral nucleic acid. After permitting the compound to complex with viral nucleic acid, the compound is raised to its excited state by chemical activation. In a first preferred embodiment, psoralen, hypericin or a derivative of psoralen or hypericin is used as the activatable, viral-inactivating compound, and chemical activation of the compound is effected by the decomposition of a dioxetane proximate to the nucleic acid/compound complex. In a second preferred embodiment, the activatable, viral-inactivating compound is incorporated into a dioxetane molecule, and chemical activation of the compound is effected by decomposition of the dioxetane into a pair of carbonyl compounds.

Description: INACTIVATION OF VIRUSES PRESENT IN BLOOD COMPONENTS USING CHEMICALLY-ACTIVATED COMPOUNDS FIELD OF THE INVENTION The present invention relates generally to the inactivation of viruses present in blood components and more particularly to the inactivation of viruses present in blood components using chemically-activated compounds. BACKGROUND OF THE INVENTION Over the past several years, it has become widely recognized that AIDS (i. e., HIV) and other infectious diseases can be contracted from transfusions of contaminated blood and/or blood-derived products. In fact, it has been estimated that nearly 70% of all hemophiliacs have become HIV-positive due to their unsuspecting use of HIV-contaminated factor 8, factor 8 being a clotting factor derived from blood. Accordingly, in an effort to avert similar occurrences, many researchers have focused on various methods for destroying or inactivating viruses in blood. One approach that has been explored to inactivate viruses in blood is to treat the blood with psoralen or a derivative thereof. See e. g., Rai et al.,"Dramatic Improvements in Viral Inactivation with Brominated Psoralens, Naphthalenes and Anthracenes,"Photochemistry and Photobiology, Vol. 58, No. 1, pp. 59-65 (1993); Ronto et al., "Phage nucleoprotein-psoralen interaction: quantitative characterization of dark and photoreactions,"J. Photochem. Photobiol. B: BioL, Vol. 12, pp. 9-27 (1992); and U. S. Patent No. 4,169,204, inventors Hearst et al., which issued Sept. 25,1979, all of which are incorporated herein by reference. According to this approach, psoralen or a derivative thereof is added to a quantity of blood which contains viruses. Because of its relatively small size, the psoralen is readily taken up by the virus by diffusing through the viral capsule. Once inside the virus, the psoralen intercalates between the base pairs of the viral nucleic acids. The blood sample is then irradiated with light of a suitable wavelength to cause the psoralen to be raised to an excited state (e. g., 330 nm). Once in said excited or activated state, the psoralen covalently binds to viral nucleic acids, thereby impairing the operability of the bound nucleic acids. Unfortunately, the practical usefulness of the aforementioned psoralen technique has been limited by the fact that blood typically absorbs all wavelengths of light other than red light (i. e., 650-700 nm). One compound that may overcome the foregoing problem of light-absorption by blood is hypericin. Hypericin, which also has an affinity for nucleic acids and which also covalently binds to nucleic acids when raised to an excited state, is activatable by red light. In BioWorld Todav, Vol. 4, No. 229, pages 1 and 4 (Nov. 24,1993), auspicious results obtained from tests using photo-activated hypericin to inactivate viruses in blood are reported. As can be appreciated, it would be highly desirable to be able to activate in blood compounds like psoralen, hypericin and their derivatives without having to consider the light-absorption effects typically experienced in blood. Dioxetanes are compounds having a 4-membered ring wherein two of the four members are oxygen atoms bonded to one another and wherein the remaining two members are carbon atoms bonded to one another. A generalized structure of dioxetane is depicted below. EMI3.1 <tb> <SEP> o-o <tb> <SEP> i <tb> -c-c <tb> <SEP> I <SEP> I <tb> <SEP> dioxetane <tb> Examples of specific dioxetanes are disclosed in Schaap et al., Tetrahedron Letters, Vol. 28, No. 9, pp. 935-938 (1987), Schaap et al., Tetrahedron Letters, Vol. 28, No. 11, pp. 1155-1158 (1987) and Schaap et al., Tetrahedron Letters, Vol. 28, No. 11, pp. 1159-1162 (1987), all of which are incorporated herein by reference. Dioxetanes typically decompose by cleavage of the O-O and C-C bonds of the 4-membered ring to release considerable energy and to form a pair of carbonyl compounds. Frequently, decomposition of dioxetanes is effected by providing a phenolic oxygen-X substituent on the 4-membered ring wherein X may be removed using a triggering agent. With the X group thus cleaved, the negatively charged phenolic oxygen group destabilizes the dioxetane, causing its decomposition. The energy released from cleavage of the O-O and C-C bonds of the 4membered ring typically causes a portion of one of the end-product carbonyl compounds adjacent to the 4-membered ring to be raised to an excited energy state. The excited portion of the compound then typically returns to its ground state by fluorescing. Examples of dioxetanes which decompose in the aforementioned manner are disclosed in European Patent Application No. 254,051, inventor Schaap, published January 27,1988 and PCT Patent Application No. PCT/US87/00554, inventor Bronstein, published January 28,1988, both of which are incorporated herein by reference. Instead of returning to the ground state by fluorescing, the excited portion of the carbonyl compound produced by dioxetane decomposition may return to the ground state by transferring its excitation energy to another portion of the carbonyl compound. An example of a dioxetane compound in which this form of energy transfer occurs is shown below. This compound is currently being sold under the trademark LUMIPHOS 530 by Lumigen Inc. (Southfield, MI) as a substrate for alkaline phosphatase. EMI4.1 When the above compound is exposed to alkaline phosphatase, the following sequence of events occurs: (1) the P032 group is cleaved from the phenol substituent on the 4-membered dioxetane ring; (2) the dioxetane compound then decomposes into the following pair of carbonyl compounds, with the phenol/ester portion of compound I being raised to an excited state: EMI4.2 and (3) next, energy transfer occurs from the phenol/ester portion of compound I to the fluorescein moiety of compound 1, raising the fluorescein moiety to an excited state. The fluorescein moiety then returns to its ground state by fluorescing. SUMMARY OF THE INVENTION It is an object of the present invention to provide a new method for inactivating viruses present in blood components. It is another object of the present invention to provide a method as described above which involves the use of activatable, viral-inactivating compounds. It is still another object of the present invention to provide a method as described above that does not require the use of light to activate the aforementioned activatable compounds. Additional objects, as well as features and advantages thereof, will be set forth in part in the description which follows, and in part will be obvious from the description or may be learned by practice of the invention. The objects of the invention also may be realized and attained by means of instrumentalities and combinations particularly pointed out in the appended claims. In furtherance of the objects broadly set forth above, a new method for inactivating viruses in blood components is provided, the method comprising the steps of: (a) adding to a virus-containing blood sample an effective quantity of a compound which both has an affinity for viral nucleic acid and is activatable to an excited state in which said compound covalently binds to the viral nucleic acid in such a way as to effectively disable the viral nucleic acid; and (b) chemically activating said compound to said excited state. Because the present method uses chemical activation, as opposed to photoactivation, to activate the viral-inactivating compound, the above-described problems of light absorption by blood are ameliorated. In a first preferred embodiment, psoralen, hypericin or a derivative of psoralen or hypericin is used as the activatable, viral-inactivating compound. The compound is added to a virus-containing blood sample and is allowed to complex with viral nucleic acids. Chemical activation of the compound is then effected by the decomposition of a dioxetane proximate to the nucleic acid/compound complex. In a second preferred embodiment, the activatable, viral-inactivating compound is itself incorporated into a dioxetane molecule. The dioxetane is preferably provided with means for controllably triggering its own decomposition, such as being provided with a cleavable group on a phenolic oxygen substituent. The activatable compound may include a single moiety, such as a psoralen-or hypericin-containing moiety, which has an affinity for viral nucleic acids and which is capable, upon activation, of covalently binding to viral nucleic acids. Alternatively, the activatable compound may include a pair of moieties, one of which has an affinity for viral nucleic acids and the other of which is capable, upon activation, of covalently binding the nucleic acid. An activatable compound having two distinct moieties of the type described above could include, for example, a DNA or RNA probe as part of the first moiety and an aromatic azide or diazirine as part of the second moiety. In addition to being directed to a method of inactivating viruses in blood components, the present invention is also directed to the aforementioned class of dioxetanes which decompose to form activated, viral-inactivating compounds. DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS The present invention is directed to a new method for inactivating viruses in blood components. Broadly stated, this method involves chemically-activating, as opposed to optically-activating, a compound which has an affinity for a viral nucleic acid and which, upon being activated, covalently binds to the viral nucleic acid. In a first preferred embodiment, the activatable compound used to disable the viral nucleic acid is a compound such as psoralen, hypericin or a derivative of psoralen or hypericin which also possesses a strong affinity for viral nucleic acids and which covalently binds to viral nucleic acids when raised to an excited state. In accordance with the teachings of the present invention, an effective amount of such a compound is added to a quantity of a virus-containing blood sample. It is expected that an effective amount of psoralen, hypericin or a derivative thereof will typically be approximately the same amount of psoralen, hypericin or a derivative thereof used to inactivate viruses according to photoactivation methods. After waiting an appropriate period of time for the compound to be taken up by a virus and for the compound to associate with a viral nucleic acid, the compound is exposed to the decomposition of a dioxetane. This may be effected by adding to the blood sample a combination of hydrogen peroxide and a dioxetanedione precursor, such as an oxalate ester. These reagents react to form dioxetanedione, a highly unstable dioxetane. Dioxetanedione decomposes in the presence of the compound to raise the compound to an activated or excitedstate. This process is illustrated below for psoralen activation. EMI8.1 It is expected that at least the molar equivalent of the dioxetane to the compound will be required to chemically-activate the compound. Once activated, the activated compound returns to its ground state by covalently binding to its associated nucleic acid. As can readily be appreciated, removal of excess psoralen and/or dioxetanedione from a blood sample treated in the above manner may be desirable before administration of the blood sample to a patient. In a second preferred embodiment, the activatable, viral-inactivating compound is itself incorporated into a dioxetane. One such class of dioxetanes is represented below by formula III EMI8.2 wherein Tr is a cleavable blocking group used to trigger decomposition of the dioxetane, X is either H or a substituent used to control the pKa of the phenol group, and S, and S2 are atoms of psoralen, hypericin or a derivative of psoralen or hypericin joined directly to the four-membered dioxetane ring. Examples of suitable blocking groups Tr include (i) hydrogen; (ii) CF3CO2, acetate and other chemically-cleavable groups; and (iii) po3¯2, esters, sulfates and other enzymatically-cleavable groups. Preferably, blocking group Tr is cleavable by chemicals or enzymes endogenous to the blood sample (e. g, P03-2 by phosphatases). One advantage to using hydrogen as the blocking group Tr is that the rate of deprotonation of the phenolic oxygen and, hence the rate of dioxetane decomposition, can be adjusted simply by adjusting pH. To illustrate, at a pH above the pK, of the phenol group (e. g., a pH of 10), decomposition of the dioxetane is rapid with a half-life of about 5 seconds. In contrast, at a pH near 7, which is normal for blood, decomposition is much slower, perhaps up to 103 times slower. This slower rate at a pH of 7, however, may be beneficial since it affords the dioxetane more time to seek out and to associate with the nucleic acid before decomposing to the excited-state product. Also, by altering the PKa of the phenol group by, for example, adding certain substituents to the phenolic aromatic ring, various rates of decomposition at a given pH may be obtainable. Examples of substituents X which may be used to lower the pKa of the phenol group are electron-withdrawing groups, such as chloro, cyano, and carboxy groups. Examples of substituents X which may be used to raise the pKa of the phenol group are electron-donating groups, such as methoxy and dialkylamino groups. For activation of the dioxetane in extracorporeal blood, it is believed that the best triggering mechanism might simply be deprotonation of the phenol by the blood's buffering capacity. Although, as described above, the free phenolic dioxetane decomposes slowly at a pH of 7 in solution, the dioxetane may be stable for longer periods of time as a solid material. In this manner, the dioxetane could possibly be stored for long periods of time in a blood collection bag and then activated, when needed, by its dissolution in blood. If necessary, acidic buffer salts could be used in the formulation to increase the stability of the free phenolic dioxetane. If greater stability than the free phenol is needed, the phenol could be blocked with a group that is more slowly removed at a pH near 7, such as trifluoroacetyl ester or a phosphate group. An example of a specific dioxetane falling within the class defined by formula III is represented below as compound IV. As can be seen, compound IV includes a psoralen-derivative joined directly to the 4-membered dioxetane ring. EMI10.1 The synthesis of compound IV is represented below. (The synthesis of precursor A is detailed by Carlassare et al. in Journal of Photochemistry and Photobiology, B : Biology, Vol. 5, pp. 25-39 (1990), which is incorporated herein by reference.) EMI10.2 EMI11.1 <tb> <SEP> H, <SEP> C <SEP> HjC <tb> <SEP> OH <tb> <SEP> O <tb> <SEP> H3f <SEP> H3C <tb> <SEP> O <tb> <SEP> C <SEP> fi <SEP> 5 <SEP> C <tb> OH'OPO <tb> <SEP> OP029 <tb> <SEP> H <SEP> 3C <tb> <SEP> CHEZ <tb> <SEP> --,.. <tb> <SEP> v <tb> <SEP> CH3 <tb> Compounds of the type represented by formula III are typically rather unstable (due to the attachment of the psoralen or hypericin moiety directly to the 4-membered dioxetane ring) and may require storage at very low temperatures until immediately before use to prevent their premature decomposition. Another class of dioxetanes into which an activatable, viral-inactivating compound is incorporated is represented below by formula V EMI11.2 wherein Tr is a cleavable blocking group used to trigger decomposition of the dioxetane; S, and S2 are either independent groups that stabilize the blocked dioxetane or are part of a single group that stabilizes the blocked dioxetane; L is a linking group; Y is a group that includes either (a) a moiety which has nucleic acid affinity but no moiety which, upon decomposition of the dioxetane, is raised to an excited state which covalently binds nucleic acid, (b) a moiety which, upon decomposition of the dioxetane, is raised to an excited state which covalently binds nucleic acid but no moiety which has nucleic acid affinity, (c) a single moiety which has nucleic acid affinity and which, upon decomposition of the dioxetane, is raised to an excited state which covalently binds nucleic acid, or (d) a first moiety which has nucleic acid affinity and a second moiety, distinct from the first moiety which, upon decomposition of the dioxetane, is raised to an excited state which covalently binds nucleic acid; and X is either (a) hydrogen provided that Y is either a single moiety or a pair of moieties which cumulatively possess both nucleic acid affinity and a capacity to be raised, upon decomposition of the dioxetane, to an excited state which covalently binds nucleic acid, (b) a substituent group which alters the pKa of the phenolic oxygen provided that Y is either a single moiety or a pair of moieties which cumulatively possess both nucleic acid affinity and a capacity to be raised, upon decomposition of the dioxetane, to an excited state which covalently binds nucleic acid, (c) a moiety which has nucleic acid affinity provided that Y includes a moiety which, upon decomposition of the dioxetane, is raised to an excited state which covalently binds nucleic acid, or (d) a moiety which, upon decomposition of the dioxetane, is raised to an excited state which covalently binds nucleic acid provided that Y includes a moiety which possesses nucleic acid affinity. The cleavable blocking group Tr may be hydrogen or any of the other substituents discussed above in connection with the class of dioxetanes represented by formula Ill. S, and S2 are preferably atoms of spiro-adamantyl, spiro-9bicyclo {3.3.1} nonane or similar compounds that impede stretching of the O-O bond of the 4-membered dioxetane ring. L is preferably oxygen since oxygen facilitates in the preparation of dioxetanes during the photo-oxygenation process typically used to make dioxetanes. Other possible but less desirable linking groups are other electron-rich atoms, such as sulfur and nitrogen. Examples of substituents suitable for use as the aforementioned moiety having nucleic acid affinity include psoralen, hypericin, derivatives of psoralen and hypericin, and various nucleic acid probes. As can readily be appreciated, one advantage to using nucleic acid probes, as opposed to psoralen, hypericin and their derivatives, is that nucleic acid probes can be used to target specific viral nucleic acid sequences (e. g., sequences essential for the survival of the virus) whereas psoralen, hypericin and their derivatives possess a more non-specific affinity for nucleic acids. For purposes of the present specification and claims, the term "nucleic acid probes"is broadly defined to include stabilized oligonucleotides. Examples of substituents suitable for use as the aforementioned moiety which, upon decomposition of the dioxetane, is raised to an excited state which covalently binds the nucleic acid include aromatic azides and diazirines, hypericin and those derivatives of psoralen (non-derivatized psoralen will not work) which have an absorption spectrum which is sufficiently compatible with the emission spectrum of the phenolic group to permit radiationiess energy transfer to occur from the phenolic oxygen to the psoralen derivative. As can readily be appreciated, hypericin and those psoralen derivatives meeting the above-described energy-transfer criterion can serve both as the nucleic acid affinity moiety and the binding moiety. Information pertaining to the synthesis of compounds of formula V may be obtained, for example, from U. S. Patent No. 5,068,339, inventors Schaap et al., which issued November 26,1991, and is incorporated herein by reference. Three examples of dioxetanes (compounds VI-VIII) falling within the class defined broadly by formula V and proposed syntheses therefor are represented below. The nucleic acid sequence (SEQ ID NO: 1) which forms a part of compounds VI and Vil is a nucleic acid probe for the HIV protease gene. (The synthesis of precursor B used to make compounds VI-VIII is detailed in U. S. Pat. No. 5,068,339.) EMI14.1 EMI15.1 EMI15.2 <tb> <SEP> N3 <tb> <SEP> ptotooxygcnation <tb> <SEP> N <SEP> 02 <tb> 0 <SEP> (CH2) <SEP> nOP020-SEC) <SEP> kt) <SEP> VAO'L <tb> <SEP> OP03-2 <tb> EMI15.3 EMI16.1 EMI16.2 <tb> <SEP> N3 <tb> <SEP> N=N <tb> <SEP> 02 <SEP> pliotooxygenation <tb> <SEP> t <SEP> 2 <SEP> pilotOOX4 <SEP> <SEP> genatiOtl0-----N <SEP> (CH2) <SEP> nOH-. <SEP> o <tb> <SEP> OP032 <SEP> + <SEP> XPO2o-SEQ <SEP> 10 <SEP> NO.: <SEP> L <tb> <SEP> (0rt. <SEP> on <SEP> o <tb> EMI17.1 EMI18.1 <SEP> OH <SEP> 0 <SEP> OH <tb> <SEP> CH,,OI- <SEP> ? <SEP> photooaygenation <tb> <SEP> OI <tb> Oh <tb> <SEP> H <SEP> OIl <tb> EMI18.2 Additional dioxetanes (compounds IX through XI) falling within the class defined broadly by formula V are represented below. EMI18.3 EMI19.1 wherein Li is a linking group, such as amide, ester, urea, thiourea or the like, m is an integer from 2 to 10 (preferably 2 to 6), n is an integer from 2 to 10 (preferably 2 to 6), and R is H, OCH, or N (CH3) 2. The embodiments of the present invention described above are intended to be merely exemplary and those skilled in the art shall be able to make numerous variations and modifications to it without departing from the spirit of the present invention. All such variations and modifications are intended to be within the scope of the present invention as defined in the appended claims. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Zepp, Charles M. Heefner, Donald L. (ii) TITLE OF INVENTION: Inactivation of Viruses Present in Blood Components Using Chemically-Activated Compounds (iii) NUMBER OF SEQUENCES: 1 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Kriegsman & Kriegsman (B) STREET: 883 Edgell Road (C) CITY: Framingham (D) STATE: Massachusetts (E) COUNTRY: USA (F) ZIP: 01701 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Diskette, 3.50 inch, 1.44 Mb storage (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: MS-DOS 6.2 (D) SOFTWARE: WordPerfect 5.1 (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US08179437 (B) FILING DATE: 10-JAN-1994 (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Kriegsman, Edward M. (B) REGISTRATION NUMBER: 33,529 (C) REFERENCE/DOCKET NUMBER: 80822PCT (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (508) 877-8588 (B) TELEFAX: (508) 877-3797 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESSS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: CAT CCC ACA CCC AAA AAA 18 His Pro Thr Pro Lys Lys 1 5

Claims: WHAT IS CLAIMED IS: 1. A method of inactivating a virus present in a blood sample comprising the steps of: (a) adding to the blood sample an effective quantity of a compound which both has a nucleic acid affinity and is activatable to an excited state in which said compound covalently binds to a nucleic acid in such a way as to effectively disable the nucleic acid; and (b) chemically activating said compound to said excited state. 2. The method as claimed in claim 1 wherein said compound is selected from the group consisting of psoralen, hypericin and derivatives of psoralen and hypericin, and wherein said chemically activating step comprises bringing said compound into proximity with a decomposing dioxetane whereby the decomposition of said decomposing dioxetane causes said compound to be raised to said excited state. 3. The method as claimed in claim 1 wherein said compound is incorporated into a dioxetane molecule and wherein said chemically activating step comprises decomposing said dioxetane molecule so as to form said compound in said excited state. 4. The method as claimed in claim 3 wherein said compound includes a moiety which both possesses a nucleic acid affinity and is activated to said excited state upon the decomposition of said dioxetane molecule. 5. The method as claimed in claim 4 wherein said moiety is a psoralen, psoralen-derivative, hypericin or hypericin-derivative moiety. 6. The method as claimed in claim 3 wherein said compound includes a first moiety which possesses a nucleic acid affinity and a second moiety, distinct from the first moiety, which is activated to said excited state upon decomposition of the dioxetane molecule. 7. The method as claimed in claim 6 wherein said first moiety is a nucleic acid probe and wherein said second moiety is an aromatic azide or diazirine moiety. 8. The method as claimed in claim 3 wherein said compound is incorporated into a dioxetane molecule of the formula EMI23.1 wherein Tr is a cleavable blocking group used to trigger decomposition of the dioxetane molecule, X is either H or a substituent used to control the pKa of the phenol group of the dioxetane molecule, and S, and S2 are atoms of psoralen, hypericin or a derivative of psoralen or hypericin joined directly to the fourmembered dioxetane ring of the dioxetane molecule. 9. The method as claimed in claim 3 wherein said compound is incorporated into a dioxetane molecule of the formula EMI23.2 wherein Tr is a cleavable blocking group used to trigger decomposition of the dioxetane; S, and S2 are either independent groups that stabilize the blocked dioxetane or are part of a single group that stabilizes the blocked dioxetane; L is a linking group; Y is a group that includes either (a) a moiety which has nucleic acid affinity but no moiety which, upon decomposition of the dioxetane, is raised to an excited state which covalently binds nucleic acid, (b) a moiety which, upon decomposition of the dioxetane, is raised to an excited state which covalently binds nucleic acid but no moiety which has nucleic acid affinity, (c) a single moiety which has nucleic acid affinity and which, upon decomposition of the dioxetane, is raised to an excited state which covalently binds nucleic acid, or (d) a first moiety which has nucleic acid affinity and a second moiety, distinct from the first moiety which, upon decomposition of the dioxetane, is raised to an excited state which covalently binds nucleic acid; and X is either (a) hydrogen provided that Y is either a single moiety or a pair of moieties which cumulatively possess both nucleic acid affinity and a capacity to be raised, upon decomposition of the dioxetane, to an excited state which covalently binds nucleic acid, (b) a substituent group which alters the pKa of the phenolic oxygen provided that Y is either a single moiety or a pair of moieties which cumulatively possess both nucleic acid affinity and a capacity to be raised, upon decomposition of the dioxetane, to an excited state which covalently binds nucleic acid, (c) a moiety which has nucleic acid affinity provided that Y includes a moiety which, upon decomposition of the dioxetane, is raised to an excited state which covalently binds nucleic acid, or (d) a moiety which, upon decomposition of the dioxetane, is raised to an excited state which covalently binds nucleic acid provided that Y includes a moiety which possesses nucleic acid affinity. 10. The method as claimed in claim 9 wherein S, and S2 are atoms of a spiro-adamantyl or spiro-9-bicyclo {3.3.1} nonane substituent and L is oxygen. 11. The method as claimed in claim 10 wherein Y includes a nucleic acid probe substituent and an aromatic azide or diazirine substituent. 12. The method as claimed in claim 10 wherein Y includes a suitable psoralen or hypericin substituent. 13. A method of chemically modifying a viral nucleic acid comprising the steps of: (a) exposing the viral nucleic acid to a compound which both has a nucleic acid affinity and is activatable to an excited state in which said compound covalently binds to the viral nucleic acid; and (b) chemically activating said compound to said excited state. 14. A dioxetane compound of the formula EMI24.1 wherein Tr is a cleavable blocking group used to trigger decomposition of the dioxetane compound, X is either H or a substituent used to control the pKa of the phenol group of the dioxetane compound, and S, and S2 are atoms of psoralen, hypericin or a derivative of psoralen or hypericin joined directly to the four-membered dioxetane ring of the dioxetane compound. 15. The dioxetane compound of claim 14 wherein S, and S2 are atoms of psoralen or a derivative thereof. 16. A dioxetane compound of the formula EMI25.1 wherein Tr is a cleavable blocking group used to trigger decomposition of the dioxetane; S, and S2 are either independent groups that stabilize the blocked dioxetane or are part of a single group that stabilizes the blocked dioxetane; L is a linking group; Y is a group that includes either (a) a moiety which has nucleic acid affinity but no moiety which, upon decomposition of the dioxetane, is raised to an excited state which covalently binds nucleic acid, (b) a moiety which, upon decomposition of the dioxetane, is raised to an excited state which covalently binds nucleic acid but no moiety which has nucleic acid affinity, (c) a single moiety which has nucleic acid affinity and which, upon decomposition of the dioxetane, is raised to an excited state which covalently binds nucleic acid, or (d) a first moiety which has nucleic acid affinity and a second moiety, distinct from the first moiety which, upon decomposition of the dioxetane, is raised to an excited state which covalently binds nucleic acid; and X is either (a) hydrogen provided that Y is either a single moiety or a pair of moieties which cumulatively possess both nucleic acid affinity and a capacity to be raised, upon decomposition of the dioxetane, to an excited state which covalently binds nucleic acid, (b) a substituent group which alters the pKa of the phenolic oxygen provided that Y is either a single moiety or a pair of moieties which cumulatively possess both nucleic acid affinity and a capacity to be raised, upon decomposition of the dioxetane, to an excited state which covalently binds nucleic acid, (¯) a moiety which has nucleic acid affinity provided that Y includes a moiety which, upon decomposition of the dioxetane, is raised to an excited state which covalently binds nucleic acid, or (d) a moiety which, upon decomposition of the dioxetane, is raised to an excited state which covalently binds nucleic acid provided that Y includes a moiety which possesses nucleic acid affinity. 17. The dioxetane compound of claim 16 wherein S, and S2 are atoms of a spiro-adamantyl or spiro-9-bicyclo {3.3.1} nonane substituent. 18. The dioxetane compound of claim 16 wherein one of X or Y includes a nucleic acid probe and the other of X or Y includes an aromatic azide or diazirine. 19. The dioxetane compound of claim 16 wherein at least one of X and Y includes a psoralen or hypericin substituent having both nucleic acid affinity and the capacity to be raised to said excited state upon decomposition of said dioxetane compound. 20. A method of inactivating a virus comprising the steps of: (a) treating the virus with an effective quantity of a compound which has an affinity for viral nucleic acid and which is activatable to an excited state in which said compound covalently binds to viral nulceic acid in such a way as to effectively disable the bound viral nucleic acid; and (b) chemically activating said compound to said excited state. 21. The method as claimed in claim 20 wherein said compound includes a nucleic acid probe specific for a viral nucleic acid sequence essential to survival of the virus.

Inventors: HEEFNER DONALD L, ZEPP CHARLES M, HEEFNER, DONALD, L., ZEPP, CHARLES, M.
Applicants: HEEFNER DONALD L, HEMASURE INC, ZEPP CHARLES M, HEEFNER, DONALD, L., HEMASURE INC., ZEPP, CHARLES, M.
Application year: 1995
Main classification: A01N1
IPC codes: A61L2, C07F9