CA2214835A1 - Non-nucleosidic coumarin derivatives as polynucleotide-cross-linking agents - Google Patents

Abstract

Novel coumarin derivatives comprising a coumarin moiety linked to a nonnucleosidic backbone moiety are disclosed. The resulting molecules are typically used as photoactivate cross-linking groups when incorporated into polynucleotides as replacements for one or more of the complementary nucleoside bases present in probes used in procedures involving nucleic acid hybridization reactions.

Description

CA 0221483~ 1997-09-08 W 096128438 PCTrUS96/n313 NON-NUCLEOSIDIC CO~M~IN DERIVATIVES
AS POLYNUCLEOTID~CROSSL~K~G AGENTS

INTRODUCTION
s CROSS RE~ERENCE TO RELATED APPLICATIONS
This is a continn~tion-in-part of U.S. Patent Application 08/046,568 filed April 13, 1993. U.S. application 08/046,568 is herein incorporated by reference.
Technical Field This invention is related to photoactive nucleoside analogues that can be incorporated into synthetic oligonucleotides during automated DNA synthesis for use in crocclinking of complementary target nucleic acid sequences.

Back~round The use of crosclink~hle probes in nucleic acid hybridization assays to crosslink to target sequences is demonstrated in U.S. Patent 4,826,967 by K. Yabusaki et al.; compounds are based on furocoumarin (or psoralen) attached to existing polynucleotides (usually through adduct formation) and are satisfactory for many applications. However, the crocclinkin,, group/nucleoside adduct is difficult ,o ,y~tl~es.,c., ~al~icul~-li ~n l~,c, qu~ntities. In U.S. Patent 5,082,93cl, Saba et al. describe a photoactivatible nucleoside analogue comprising a coumarin moiety linked through its phenyl ring to the l-position of a ribose or deoxyribose sugar moiety in the absence of a~i intervening base moiety. The resulting nucleoside analogue is used as a photo-crosslinking group when inserted into a polynucleotide as a replacement for one or more of the complementary nucleoside bases present in a probe used in hybridization assays.
Nevertheless, new types of compounds that offer additional advantages, such as stability throughout probe synthesis and use, and conformational flexibility, continue to remain desirable.

CA 0221483~ 1997-09-08 W 096/28438 PCTrUS96/03134 2.
SUMMARY OF THE INVENTION
The current invention provides non-nucleosidic, stable, photoactive compounds that can be used as photo-cro~linking reagents in nucleic acid hybridization assays and theldl~euLic applications, as well as techniques and S interrne.li~tes that can be used to prepare the final products.
The compounds comprise coumarinyl derivatives prepared by linlcing the phenyl ring of a coumarin molecule or derivative to a hydroxy or polyhydroxy hydrocarbon molecule, such as one of the terminal hydroxy groups of a glycerol molecule. The (poly)hydroxy hydrocarbon moiety of the resulting compound is equivalent to the sugar of a nucleoside, while the coumarin moiety occupies the position of a base. Accordingly, the compounds can be inserted into growing polynucleotide chains using automated (or manual) techniques of polynucleotide synthesis. The double bond between the 3 and 4 positions of the coumarin ring system is a photoactive group that covalently crosslinks to nucleosides in the complementary strand when an oligonucleotide cont~ining this non-nucleoside analogue (the "probe") is used in a hybridization assay and/or therapeutic application.
For the most part, the photoactive compound has the formula ~n B X

~5 in which the substituents and linking groups are described below in more detail.The (poly)hydroxy hydrocarbon backbones give maximum flexibility and stability to the oligosaccharide structure in which they are located as well as good solubility in aqueous and organic media.~0 CA 0221483~ 1997-09-08 W O 96/28438 PCTrUS96/03134 3.
D E~CRU~YrIO N OF SPEC~'lC E~VnBO Dn~nEr~rS
The present invention provides cro.cclink~hle compounds that can be used as a photoactivatible non-nucleosidic crosslinker in oligonucleotide probes used inhybridi_ation assays and/or therapeutic applications. In hybridi7~ti~-n assays, the compounds of the inventions are typically used as part of synthetic DNA or RN~
oligonucleotides to determine the presence or absence of a specific DNA and RNA
base sequence in a sample. More specifically, this invention provides cou...a,ill derivatives ~tt~t'hP~ to a stable, flexible, (poly)hydroxy hydrocarbon backbone unit that act as photoactive crosclinking compounds in hybridi_ation assays.
Compounds of the invention have the general formula:

Backbone moiety - T inking moiety - Cro.cclinking moiety "Moiety" here and elsewhere in this speci~lcation indicates a part of a lS molecule that performs the indicated function. A given moiety is usually derived from another molecule by covalently linking together two or more molecules, withthe identifiable remnants of the original molecules being referred to as "moieties."
For example, if a pso~Llen molecule is attached to a glycerin molecule with a divalent linker, such as a methylene group, the resulting single molecule is referred to as being formed of glycerin, methylene, and psoralen moieties. It isnot necçcs~ry, however, that the three moieties actually arose from three separate molecules, as diccussed below. Thus "derived from" can refer to theoretical, as well as actual, precursors.
The crocslinking moiety will be derived from molecules having a fused be-~opyrone structure, such as the following: (1) coumarin and its simple derivatives; (2) psoralen and its derivatives, such as 8-methoxypsoralen or S-methoxypsoralen (at least 40 other naturally occurring psoralens have been described in the liLeldLIlle and are useful in practicing the present invention);
(3) cis-benzodipyrone and its derivatives; (4) trans-benzodipyrone; and s 30 (S) compounds cont~ining fused coumarin-cinnoline ring systems. All of these molecules contain the nP~ecc~ry crosclinking group (an activated double bond) located in the right orientation and at the right ~lict~n~e to crosslink with a -CA 0221483~ 1997-09-08 W096/28438 PCTrUS96/~3134 4.
nucleotide in the target strand. ALI of these molecules are coumarin derivatives, in that all contain the basic coulnalill (be-~upylone) ring system on which the remainder of the molecule is based.
The linking moiety will normally be formed from a precursor that contains from 1 to 100, preferably 1 to 25, more preferably 1 to 10, atoms with functional groups at two locations for attaching the other moieties to each other. After reaction of the precursor to form the linking moiety, the total number of atoms in the shortest linking chain of atoms between the coumarin ring system and the backbone moiety (sugar substitute) is generally from 1 to lS, preferably 1 to 7,more preferably 1 to 3. Otherwise this part of the structure can vary widely, asthis is essenti~lly just a flexible linkage from the cros~linkin~ moiety to the backbone moiety.
The linking moiety is most often a stable cyclic or acyclic moiety derived by reaction of a molecule bearing appropriate functional groups (usually at its termini) for linking the cr~!sClinkinE molecule at one end and the backbone molecule at the other end. However, if sufficient functional groups are present in the backbone and crncslinking moieties, a precursor to the linking moiety need not be used (i.e., the backbone and cros~linking moieties can be connPctP~ by a covalent bond).
It should be recognized that description of a particular part of the final molecule as belonging to a particular moiety of those identified above is somewhat arbitrary and does not necec~rily mean that there were three original molecules that reacted to form the final product. There are a number of coum~rin derivatives, for example, that have a functionalized methyl or methoxy group ~tt~ch~l to the coumarin ring that can react with a functional group on a backbone moiety precursor to form a product from only two starting materials. However, the re-sulting structure will generally appear to have three parts as indicated above:
the backbone molecule that is incorporated into the sugar backbone of a polynucleotide, the cros~linking moiety that occupies the space occupi~ by a base in a normal nucleoside, and the atoms (i.e., the linlcing moiety) that join the two principal parts together. For the sake of convenience, the linking moiety is considered to consist of atoms between the ring atom of the crosslinking moiety at CA 0221483~ 1997-09-08 W O 96/28438 PCTnUS96103134 5.
the point of attachment and the last carbon atom that clearly forms part of the backbone structure in the moiety that replaces the sugar molecule, which is usually the carbon atom bearing a hydroxyl group (or reaction product of a hydroxyl group) that is closest to the cro~Tinking moiety.
S The backbone moiety, so called because it nltim~tely functions in place of the ribose or deoxyribose portion of the backbone of a polynucleotide, will generally have 1 to 3 (sometimes more) hydroxyl groups (or similar functional groups, as discussed below) attached to dilre~ t sp3-hybridized carbon atoms.
The backbone moiety is generally uncharged so that it can function as a substitute for ribose or deoxyribose in the final modified nucleotide. Backbone moieties include but are not limited to the following: (1) linear hydrocarbon moieties such as a three-carbon propane unit or a longer hydrocarbon chain with appropriate functional groups, usually selected from the group con~i~tin~ of -OH, -NH2, -SH,-COOH, acid halides, and acid anhydrides, and (2) cyclic hydrocarbon moieties typically having a 5- to 7-membered carbon ring structure bearing one to three hydroxyl group or other functional groups as in (1) above. The functional groupsare shown in the prece ling sentence in unreacted form and will be present as derivatives of the in~ic~teA functional groups in many embodiments. The reactivefunctional groups mentioned above (other than -OH and -SH) are generally presentonly in intermediates; however, after reacting with other functional groups, they become stable groups or form covalent bonds to other parts of the molecule.
In addition to the basic structure described above, one or more coupling moieties can be ~tt~'ht-A to the backbone moiety to f?~cilit~te formation of bonds to existing or growing polynucleotide chains. The coupling moieties will typically comprise hydroxy coupling and/or protecting groups that are used in solution or solid-phase nucleic acid synthesis when the molecule in question is an interme~i~te being used in the preparation of a probe molecule. Typical coupling groups include phosphoramidite, phosphate, H-phosphonate, phosphorothioate, methyl phosphonate, trityl5 dimethoxytrityl, monomethoxytrityl, and pixyl groups. Non-phosphorous coupling groups include carbamates, amides, and linear and cyclic hydrocarbon groups, typically connecting to the rem~inder to the molecule with heLeloato.l, substit-~e-nts, such as -COCH3, -CH20H, -CF3, -NHCH3, and WO 96128438 PCTrUS96103134 6.
PO2CH2CH3. For a review of such cheihistry, see "Oligonucleotide Synthesis, A
Practical Approach," M.J. Gait, ed., IRL Press Ltd., Oxford, Great Britain, 1984, which is herein incorporated by reference.
Preferred compounds of the invention have the forrnul~
S

B-- X ~ ( wherein B represents (1) a linear, branched, or cyclic hydrocarbon group cont~ining from 2 to 15, preferably 3 to 10, more preferably 3 to 6, carbon atoms and, if cyclic, cont~ining a 5- or 6-membered ring or (2) a heterocyclic aromatic ring system comprising a 5- or 6-membered ring, both of B(l) and B(2) being substituted with 1, 2, or 3 groups of the formula OR,;
X represents (1) a linear, branched, or cyclic hydrocarbon group cont~ining I to 15, preferably 2 to 10, more preferably 3 to 6, carbon atoms or (2) such anX(l) group in which one to three (preferably one) carbon atom or atoms of the hydrocarbon group are replaced by an oxygen, sulfur, or nitrogen atom and in which the shortest linking chain of atoms in X between atoms in other parts of the formula attached tO X is 1 to 10 atoms, wherein X is optionally substituted with 1-3 subsfituent.c selected from the group con.ci.cting of hydroxy, halogen, amino,amido, azido, carboxy, carbonyl, nitro, thio, perfluoromethyl, and cyano functional groups;
nisO, 1,2,or3;
each W independently represents a hydroxy, halogen, amino, amido, azido, nitro, thio, carboxy, carbonyl, perfluoromethyl, or cyano functional group; an un.~ubstittlted hydrocarbyl group of 10 or fewer carbon atoms, preferably 6 or fewer, more preferably 3 or fewer; or such a hydrocarbyl group substituted with -CA 0221483~ 1997-09-08 W O 96/28438 PCTrUS96/03134 7.
1-3 of the functional groups or in which one carbon atom is replaced by an oxygen, sulfur, or nitrogen atom;
with the provisos that (1) when X or W is a substituted hydrocarbon, the total number of substituents in X or W is less than the total number of carbon S atoms in the X or W and no more than one substituent or heteroatom is ~tt~checl to a given carbon, unless the subctitl~ent~ are halogen atoms on the given carbon; (2) the total carbon atoms in all W substituents is 15 or fewer, preferably 10 or fewer, more preferably 6 or fewer; and (3) two W's together can form a ring when taken together with the rçm~in~l~r of the atoms to which they are attached (e.g., as in a psoralen);
Y and Z independently represent H, F or lower a~yl (usually 5 of fewer carbons, preferably 3 or fewer); and each Rl independently represent H, F or a hydroxy-protecting or hydroxy-coupling group capable of protecting or coupling a hydroxy group during synthesis of a polynucleotide or one or two (preferably two) R, represent a nucleotide or a polynucleotide conn~ctçd to the compound.
The oxygen atom or other non-C atom (if present) of a functional group (such as an ether or carboxylate) that bridges the B-X linkage often arises from a hydroxyl group in the precursor of B, but is considered part of the X linker (for ease of defining the various groups) in this and the following formulas, unless the contrary is clear from the context of the discussion.
Within general formula I above, certain compounds are preferred. The most important part of the molecule (at least in view of the difference between these compounds and what was previously known) is the B or backbone moiety.
Preferred B moieties belong to a group of a first sub-formula Q~

~1 a group of a second sub-formula R~ ~<

R~ R~

CA 0221483~ 1997-09-08 W 096/28438 PCTrUS96/03134 8.
or a group of a third sub-formula (C ~JL)~
C U (C~ )~ C IJ, ( C~ ) P C ( C ~L ) X ( C ~
2;"
wherein s is 2 or 3;
R~, Ry~ and Rz independently ~ rcsellt H or ORI;
m, n, p, q, and r independently represent 0 or 1;
one hydrogen of the sub-formula is replaced by a covalent bond to the X
group; and all other substituents and definitions of the formula of the compound are as previously defined for general formula I.
The hydrogen atom of the sub-formula that is replaced by a covalent bond to the X group is usually a hydrogen of a hydroxyl group (i.e, at least one OR, would represent a hydroxyl group in such a precursor molecule). However, this preference is for convenience of synthesis only, as the resulting B-X linkage can readily be prepared from (poly)hydroxy hydrocarbon precursors, many of which are commercially available. Other hydrogens can be replaced by the indicated covalent bond if desired. The actual molecules used in synthesis are often stillderived from a (poly)hydroxy compound in which one of the hydroxyl groups has been replaced by the functional group, often through a series of reactions. For example, a hydroxyl group can be replaced by a halogen atom or other leaving group, and the leaving group can participate in bond formation with an electron donating group in the precursor of the X group.
Compounds in which B is formed from a saturated hydrocarbon are ~lc;Çe.,ed, although unsaturated compounds (including cyclic aromatics) are permitted. In un~tur~ted compounds (including aromatics), the -OR, substituent preferably is not ~ft~chçcl directly to an sp2-hybridized carbon, but is ~tt~'h5CI to CA 0221483~ 1997-09-08 W 096128438 PCT~US96/03134 9.
an intervening sp3 carbon, as in -CZ2ORl in which each Z ~ lcs~lL~ H or an alkylgroup.
Compound of formula I in which B has the third sub-formula are ylc;rell~d among the three sub-formulas, especially those in which m + n + p + q + r =
0, 1, or 2. Even more preferably, these compounds of the third sub-formula represent an acycllc, saturated, di- or tri-hydroxy hydrocarbon, especially glycerol and 1,2- or 1,3-dihydroxyaLkanes of 3 to 5 carbons that are attached to the X
group at their terminal position furthest from the inrlic~tP~ hydroxyl groups, such as 4,5-dihydroxypentyl, 3,5-dihydroxypentyl, 2,4-dihydroxy-2-methylbutyl, 3-hydroxy-2-(hydroxymethyl)propyl, and 2,3-dihydroxypropyl.
Although such compounds are not preferred, as already inrlic~t~rl, aromatic ring systems can be present in the B moiety. These include both hydrocarbon and hetererocyclic aromatic ring systems. Of these compounds in which B comprises a benzene or naphth~lene ring system are preferred, especially 1,2-di(hydroxymethy)-substituted aromatics. The same substituents are preferred whenB comprises a heterocyclic ring system, such as a furan, pyran, pyrrole, pyrazole, imidazole, piperidine, pyridine, pyrazine, pyrimidine, pyrazidine, thiophene, acridine, indole, quinoline, isoquinoline, quinazoline, quinoxaline, x~nthene or 1,2-benzopyran ring systems.
Also not preferred but within the scope of the invention are compounds in which B comprises a bridged hydrocarbon ring system, such as bicyclo [3.1.0]
hexane or [2.2.1] heptane ring system. These molecules have configurations with reduced mobility so that various cis and trans substitution pattern can be easily prepared and m~int~in~ See, for example, Ferguson, "Organic Molecular Structure," Willard Grant, Boston, 1975, chapters 17-19, for a review of this chemistry and synthetic techniques. In a like manner, compounds in which B
comprises a spiro or dispiro hydrocarbon ring system are also within the scope of the invention.
As previously noted, the X linking group is not particularly restricted in - 30 structure, as it is not present in a part of the molecule that interacts either with the rem~inder of the backbone structure or with a complementary strand of a polynucleotide. However, there are preferred structures for this part of the CA 0221483~ 1997-09-08 WO 96/28438 PCT~US96J03134 10.
molecule, such as the following, which can represent X, in either of the two possible orientations:
O O
Il 11 -OCH2-, -SCH2-, - ~ CH2-, -CCH2-, -C-O-, O O
Il 11 -C-S-, -NH-C-, and -CL2(CH2) n ~, in which L
represents H, F, Cl, I, or Br and n = O, 1, or 2.

Other (but lesser) preferred compounds are those in which X comprises a cyclic structure with a 5- or 6-membered carbon or heterocyclic ring (the lattercont~ining one O, S, or N atom), such as cyclopentane, cyclohexene, dihydrofuran, pyrrole, or pyridine.
In the crosclinking moiety, Y and Z generally have 5 or fewer carbons, preferably 3 or fewer, and are most preferably methyl if they are aL'cyl groups.Compounds in which W, Y, and Z are all hydrogen are preferred, as are compounds in which W is a pyrone or furan ring fused to the phenyl ring of the formula. These later compounds are preferably compounds in which all of the formula to the right of X in forrnula I represents coumarin, psoralen, cis-benzodipyrone, or trans-benzodipyrone or a derivative thereof within the formula.
The compounds of formula I in which a nucleotide or polynucleotide is connected to the compound are usually (but not always) connected via a phosphorous-cont~ining linl~irlg group. Preferred phocFhorouc-containing linkinggroups, as well as other linking groups, are discussed elsewhere. Such compoundsare ~ierel,ed compounds of the invention, as they can be used directly in the assays and crosciinking processes that are the principal end use of this invention.
These compounds have the formula (Nm,Q4N",2)m3 in which ml and m2 are integers (usually less than 200, preferably less than 100; one of ml and m2 is usually at least 14, preferably at least 17, most preferably at least 20); m3 is an integer from 1 to 10, preferably 1 to 5 (m3 is generally less than (ml +m2)/10);each N independently represents a nucleotide of a desired polynucleotide sequence;
Q l~lc;sents the nucleotide-replacing molecule of the invention incorporated into CA 0221483~ 1997-09-08 WO 96/28438 PCTnUS96/03134 11.
the normal polynucleotide sequence; and m4 is 1-5, preferably 1-3. It is also possible to have two or more Q moieties sep~r~t~d from each other by a few (usually one or two) normal bases in a polynucleotide sequence as long as there is an nninterrupted sequence of nucleotides to make the hybrid stable. Such S sequences are considered to be equivalent to ul~illlell.lpted Q sequences. Preferred lengths of In~ ellùpled normal nucleotide sequences are as set out above for ml and m2.
Q can be present either in the interior of the polynucleotide or at one of its terminal positions. In an interior position, at least two R, groups must be present in order to allow the Q molecule to connect to ends of two separate strands; if Q
is inserted at a terminal position, only one Rl is required, although others may be present in both cases.
In these formulas it should be recognized that each Nm,Q4Nm2 can differ from each other in a polynucleotide sequence in which m3 is greater than 1; i.e., multiple Q moieties can be present randomly along the length of a molecule, provided that the rem~ining parameters described above are complied with.
One group of preferred polynucleotides has a long sequence of uninterrupted normal bases with 1-5 Q moieties present at either or both ends ofthe molecule (preferably 1-3 Q moieties). As noted, the Q moieties can be eitherconsecutive or can be interrupted with a few normal nucleotides. Plural Q
moieties (either consecutive or not) in the middle of a probe also represents a preferred embodiment, with relatively long uninterrupted sequences to either side of the cros~linking Q units.
In all ~ elled embodiments, there is at least one unhllellupted sequence of nucleotides that is complementary to the corresponding target nucleotides. This unint~llul,t~d sequence provides stability during the hybridization process so that proper recognition of the target will occur. The factors that lead to stability and selectivity are the same in the present process as in any other hybridization process. Unilllellupted sequences of complementary nucleotides followed by Q
~ 30 moieties are no difrel~-lt in this regard from uninterrupted sequences of target nucleotides followed by a non-complementary normal base. Thus, the stability of -CA 0221483~ 1997-09-08 W 096/28438 PCT~US96/03134 12.
polynucleotides cont~ining the croc~linking moiety of the invention can readily be predicted from standard considerations of nucleic acid hybridization.
Also ~.~fel-~d are compounds in which two R1 groups are present in the B
moiety and both represent a dirrel~lll hydroxyl-coupling or hydroxyl-protecting group, as such compounds are ready for use in the synthesis of a cross-lin~able polynucleotide. These protecting and activating groups are also discussed elsewhere in this speci~lcation.
Another particularly preferred group of compounds of the invention have the formula II below, many of which are within and a preferred embodiment of compounds of the scope of formula I:

~/n Y

C ~ CC~ ) 3 C ~ (C~ ~ X -(C~ o 15 ~~

where n, is 0 to 10 (preferably 0 to 5, more preferably I to 3);
n2 is O to S (preferably 0 to 2, more preferably 0 or 1);
n3 is 0 to 5 (preferably 0 to 2, more prefe~bly 0 or 1);
each W is independently a small stable substituent cont~ining Up to 15 atoms (especially a lower hydrocarbyl group; a halogen, nitro, thio, cyano, carbonyl, carboxy, hydroxy, amino, amido, or polyfluoroaL~cyl group; or a hydrocarbyl substituent cont~ining one or more hetero atoms (i.e., an atom otherthan carbon or hydrogen that forms a stable covalent bond with carbon at 25~C inwater));
Y and Z independently l~;pl~se"l H, F or a lower aL~cyl group;
X is an organic group containing (a) 1 to 10 carbon atoms and (b) 0 to 10, preferably 0 to 2, hetero atom selecte~l from the group consisting of O, S and N, and wherein X comprises a shortest linking chain of 1 to 10 atoms between the other atoms of the formula to which it is attached;
R2 is H or ORI; and CA 0221483~ 1997-09-08 W O 96/2843X PCT~US96/03134 13.
Rl is H or a group capable of coupling with or protecting (the former preferably being located only on a terminal hydroxyl of the backbone moiety) a hydroxyl group during automated polynucleotide synthesis. Alternatively R
represents a nucleotide or polynucleotide linked to the compound by a phosphodiester linkage or other typical group used to couple sugars in polynucleotides. Preferred coupling groups include phosphorous cont~inin~ groupssuch as phosphite, phospohramidite, phosphate, H-phosphonate, phosphorothioate, phosphorodithioate, and methyl phosphonate. Non-phosphorous coupling groups include carb~m~tes and amides. Lower hydrocarbon groups include Cl-C6 alkenyl and alkenyl group as well as C3-C6 cyclic groups, and preferably include Cl-C4 aLkyl and a~cenyl groups, especially methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl, and tert-butyl. Typical hydrocarbyl groups with hetero atom substituents include -COCH3, -CH20H, -CF3, -NHCH3, -CO2CH2CH3, and-CON(CH3)2.
Compounds of the invention are useful either as intermediates in the preparation of or as components of photoactivate polynucleotides used for example as probes in hybridization assays. Since the intention is that one or more of these molecules eventually form part of a polynucleotide, the backbone moiety that forrns part of the molecules is derived either from glycerin or a different polyhydroxyl hydrocarbon molecule in most cases. The glyceryl or other polyhydroxyl hydrocarbon molecule is incorporated at any position into the backbone of a nucleic acid typically by phosphodiester type linkage with the 3' and/or S' hydroxyl groups of the ~ cellt nucleotides in the molecule, with the crosclinking moiety normally being ~ ched to the backbone moiety prior to such incorporation.
The crosslinking moiety portion of the compound of the invention can be derived from coumarin itself or any number of substituted coumarins. An organic functional group at the position in the crosslinkin~ moiety precursor where glycerin or another backbone moiety will be att~h~cl is typically used to join the crosclinking moiety to the backbone moiety in the final product. Since final products can be often prepared by alternative synthetic routes, any given final product will likely have several possible precursors. The linlcing moiety can arise =
CA 0221483~ 1997-09-08 WO 96/28438 PCTrUS96tO3134 14.
from a separate molecule or be formed by reaction portions of the cro~linking moiety precursor and the backbone moiety precursor.
At locations other than the linking position, the coumarin (or other) ring system can be either unsubstituted or substitntecl Typical sub~ llelll~ on the phenyl ring are small, stable sub~it~llell~c normally found on aromatic rings inorganic compounds. Substit It~nt~ can be selected as desired to change the excitation wavelength of the coumarin. Substit--tents at the 3- and 4- positions are typically non-polar and are most often hydrocarbon substih-tentc, with methyl substitlltent~ being most common. Although the location of coumarin sub~ r."C
can vary, substitutents are most often found at the 4-,5-,6-7, and 8-positions.
In certain preferred embodiment the coumarin moiety precursor, prior to reaction with the backbone moiety precursor, will have the formula:

~

in which Y, Z, n2, M and W have the meanings previously defined; and X, is a precursor of all or part of the X linking moiety. Xl will react with an organic function group on the precursor of the linker moiety to form a covalent bond. Typical reactive functional groups include hydroxy, amine, halogen, thio, carbonyl, carboxy ester, carboxy amide, silyl and vinyl groups. These precursorscan be synthesi7e~1 by standard methods of organic synthesis from coumarin itself or from the many commercially available coumarin derivatives.

CA 0221483~ 1997-09-08 W O 96/28438 PCT~US96/03134 15.
In certain ~ rt;lled embo-limentc the glycerol backbone moiety precursor has the formula:

~ 1 L ) 1~ 3 in which Rl, R2, and n" and n3 have the me~ning previously defined and X2 is a precursor of all or part of the X linking group.
X2 will react with an organic functional group on the coumarin moiety to form a covalent bond in the final linking X moiety. X2 typically will be selected from reactive functional groups and nucleophilic and electrophilic groups that are capable of undergoing nucleophilic or electrophilic substitution or addition.
Examples of specific functional groups include hydroxy, amino, halogen, thio, carbonyl, carboxy ester, carboxy amide, vinyl, and silicon derivatives. This precursor can be synthesi7PA by standard methods of organic synthesis from (poly)hydroxy hydrocarbons such as glycerin, commercial available 1,2- or 1,3-dihydroxy a~cane derivatives, or such compounds with a protected hydroxyl group at the location of the indicated hydroxyl groups. See Misiura, K., Durrant, I., Evans, M.R., and Gait, M.J., Nucleic Acids Res. (1990) 18, 4345-4354, which is herein incorporated by reference, for a ~iiccllCcion of attaching moieties having structures similar to those of the present backbone moieties to bases used in polynucleotide synthesis.
Compounds of the invention can be ~ d by standard techniques of synthetic organic chPmictry, using the guidelines outlined in this specification.
For example, a typical synthesis based on commercially available starting materials is set forth in the following reaction scheme.

CA 022l4835 l997-09-08 W 096/28438 PCTrUS96/03134 16.
REACTION SCHEME FOR TYPICAL SYNTHESIS

~~~ ~~~o ~ C~ (b) O~<o (c) (~) ~"~ ~o~
r 0~ 0 0 o o~ , o ~ ~
0~1 l'r 25Reagents (a) sodium hydride (NaH)/CH3-O-CH2CH2-O-CH3 (b) 7-bromomethyl coumarin (c) HCl (aq.), l~F (tetrahydrofuran) (d) DMTrCl (4~4~ methoxytritylchloride), pyridine (e) ClPN(ipr)20CH2CH2CN, CH3CH2N(ipr)2, CH2C12 -CA 0221483~ 1997-09-08 W O 96/28438 PCTrUS96/03131 17.

7-Coumarinyl methyl soL~cetal To 120 g ethylene glycol dimethyl ether was added soL~cetal (2.64 g, 19.0 mmole) and sodium hydride (0.88 g, 22.0 mmole, 60% in mineral oil). To the resulting suspension was added 7-bromomethylcoumarin (4.8 g., 19.0 mmole) in small portions over a period of 7 minutes. After lO min. 1.5 ml of glacial acetic acid was added to stop the reaction. The solid was then sep~r~tPd from the suspension solution by centrifugation. The solution was then conce~ dt~d to a solid. The solid was then purified by silica gel chromatography using chloroform/ethyl acetate 97:3 as the eluant. The fractions cont~inin,, product were identified by TLC and were combined and concentrated to a white solid in vacuo.
Yield was 630 mg; the melting point was 75-80~C. Rf2=0.55 in CHCl3/ethyl~eet~te 9: 1.

1 -0-(4.4'-dimethoxytrityl)-3-0-(7-coumarinvl methyl) glycerol 7-Coumarinyl methyl so~cetal (800 mg, 2.74 mmole) was dissolved in a solution of tetrahydrofuran (12 ml) and in hydrochloric acid (6 ml) for 20 min~ltçs The solution was then dried by co-evaporation with absolute ethanol (2 x 5 ml) to give an oil. The res--lting solution was washed with 25 ml of saturated sodium caFbonate solution and then extracted with 3 x ''5 rnl of diethyl ether. The solution was concentrated to an oil in vacuo. The oil was dried by co-evaporation with pyridine (2 x 5 ml) to give a dry product. To the liquid was added pyridine(30 ml), 4-dimethylaminopyridine (25 mg) and triethylamine (200 ~41). To the res~ ing solution was added 4,4'-dimethoxy trityl chloride (905 mg, 2.95 mmole).The reaction mixture was stirred for two hours. 37.5 ml of water was added to - stop the reaction, and the resnlting solution was extracted with 2 x 180 ml of diethyl ether. The combined ether extracts were concentrated in vacuo, dissolvedin 15 ml methylene chloride, and purified by silica gel chromatography using acetone/hexane 4:6 as the elution solvent. Fractions with Rf=0.5 were collected and evaporated to dryness to yield the product (770 mg, 55% yield).

CA 0221483~ 1997-09-08 WO 96/28438 PCTrUS96103134 18.
EX~nPLE 3 1 -0-(4~4 '-Dimethoxytrityl)-3-0-(7-coumarinylmethyl)-2-0-r(N.N-diisopropyl)(2-cyanoethyl) phosphoramidite)l-glvcerol 1-0-(4,4'-Dimethoxytrityl)-3-0-(7-coumarinyl methyl)glycerol (1.20 g, 2.18 mmole) was co-evaporated with 2 x 6.5 ml mixed solution (5 ml pyridine and l.S methylene chloride) two times. To the dry reactant was added methylene chloride (4.6 ml) and diisopropylethylamine (1.87 ml, 8.59 mmole). The suspension was stirred until it became a clear solution. Then, 2-cyanoethyl N,N-diisopropyl chlorophosphoramidite (0.62 ml, 3.24 mmole) was added to the solution. The rt-snlting solution was stirred for 65 min. The reaction mixture was then diluted with 45 ml of ethyl acetate and 2.2 ml triethylamine, extracted with 10% aqueous sodium carbonate (2 x 30 ml), and with saturated sodium carbonate (2 x 30 ml), and with saturated sodium chloride (2 x 30 ml). The organic phase was concentrated in vacuo. The resnlting product was purified by silica gel lS chromatography with a solvent system (methylene chloride/diethyl ether/triethylamine 90:7.5:1). Fractions with R~=0.73 were collected. The yield was concentrated in vacuo to a solid. Yield was 1.06 g (1.41 mmole, 64%).

Preparation of Oli~odeoxynucleotides Containin~ a Non-Nucleosidic Coumarin Functionality Using tne reagent prepared in Example 3, above, an uli~onucieotide was prepared via the ,l~-cyanoethylphosphoramidite method of DNA synthesis that was identit~l to a segment of human papilloma virus type 16, comprising nucleotides 397 to 417 of the E6 gene in which the 20th base (~de.nine) was replaced by 3-(7-coumarinylmethyl)glycerol .
After assembly, the oligonucleotides were cleaved from the solid support with 3 ml 30% NH40H for 1.5 h at room temperature. The ammonia solution was then heated at 55~C for 1.5 h. After cooling, the NH40H was removed in vacuo. The crude oligonucleotide was purified to homogeneity by reversed phase high perforrnance liquid chromatography.

CA 0221483~ 1997-09-08 W096/28438 PCTrUS96/0313 19.
The oligonucleotide was hybridized in 0.75 M NaCl buffer (20,uL) with a complementary 5'-32P-labeled target oligonucleotide (molar ratio of probe/target = 10:1) for 1 hour at 40~C. At this time the solution was irradiated with 302 nm wavelength light for 10 minlltes. Denaturing polyacrylamide gel electrophoresis analysis of the irradiated mixture indicated that the level of photochPmic~l crosslinking achieved with respect to the radiolabeled target was 80%. Control experiments with analogous oligonucleotides cont~ining one of the nucleosidic coumarin derivative described in Saba et al., U.S. Patent No.
5,082,934, were carried out in parallel. The optimal crocslinking efficiencies obtained with these reagents were 60%. Accordingly, the compound of the invention underwent photochPmic~l crosslinking with 20% more efficiency (1/3 greater relative efficiency).

EXAMPLE S
By following a similar reaction shown in the previous examples 1, 2, and 3, a product with the following structure could be synthesized as well.

o-or1r_ o~ O~ P~
1.

C~

This compound would be also useful for the preparation of oligodeoxynucleotides cont~ining non-nucleotide psoralen derivatives.

CA 0221483~ 1997-09-08 W O 96/28438 PCTrUS96/03134 20.

Using the reagent prepared in Example 3, oligonucleotides were prepared via the ,B-cyanoethylphosphoramidite method of DNA synthesis that were i~l~nti~lto segments of the genome of human papilloma virus type 16. The oligonucleotides were complementary to nucleotides 89-108 and 283-302 of the E6 gene, respectively (the sequence of which is herein incorporated by reference). In each molecule, the 5' terrninal nucleotide of the natural sequence (adenosine) was replaced by 3-(7-coumarinylmethyl) glycerol. The 3' end terminated with a biolinmoiety.
In parallel, two additional DNA molecules were synth~ ec1. These oligonucleotides had complementary sequences to either nucleotides 89-108 or 283-302 of the E6 gene; however, in these modifled oligonucleotides 3-(7-coumarinylmethyl) glycol was replaced by the nucleosidic coumarin derivative described in Saba et al., U.S. Patent No. 5,082,934, by using the 3'-0-(N,N-diisopropyl phosphoramidite) 5'-~ (4,4'-dimethoxytrityl) derivative at the 5' position of the 2'-deoxyribonucleotide, herein referred to as the "Saba compound."
After assembly, the four oligonucleotides were cleaved from the solid support with 1 ml 30% NH40H for 1.5 hours at room temperature. The ammonia solution was then heated at 55~C for a further 1.5 hours. After cooling, the NH40H was removed in vacuo. The crude oligonucleotides were purifled to homogeneity by high performance liquid chromatography.

W 096/28438 PCTnUS96/03134 21.
The oligonucleotides were hybridi~ed in 0.75 M NaC1 buffer (20,u1) with complementary 5'-32P-labeled oligonucleotides (molar ratio of unlabelled:labelled oligonucletides = 100: 1) for 1 hour at 40~C. At this time the solutions were irradiated with W-B wavelength light (XL 1500 W cross-linker, Spectronics, S Inc.) for 15 minl-tes. The extent of crocslinking (with respect to the radiolabeled targets) was determined by denaturing polyacrylamide gel electrophoresis followed by scintillation counting of the excised bands. The results are set forth in thefollowing table:

10E6 Gene Cros.clinkingCro.c.clinking Sequence Cros.clink~r Used inRe~ction Efficiency Position Oligonucleotide Site S' ~3' %
89-108 3-(7-Coumarinylmethyl) glycerol Tl-r 64%
89-108 Saba compound Tl l 54%

15283-302 3-(7-Coumarinylmethyl) glycerol TTT 76%
283-302 Saba compound TTT 68%

The results indicate that the compounds of the current invention undergo photochemical crosclinking more efficiently than the compound of U.S. Patent 5,082,934 ( > 10 % greater relative efficiency).

W O 96/28438 PCTrUS96/03134 22.
EX~MPLE 7 1-0-(4 4'-Dimethoxytrityl)-3-0-(7-coumarinyl)-2-0-(2-cyanoethyl-N.N-diisopropyl phosphoramidite) ~Iycerol Another embodiment of the invention was synthesi7Y1 using 7-hydroxycoumarin instead of 7-bromomethylcoumarin as in Example 1. The reaction scheme for the synthesis of 1-0-(4,4'-dimethoxytrityl)-3-0-(7-coumarinyl)-2-0-(2-cyanoethyl-N,N-diisopropyl phosphoramidite) glycerol is as follows:

C~-- C~l - c~, - I;y . ~ .'~ C~ ~ c~, -c~-C~,--o '~0 o . ~lo ~o ~ ~'c ,,~ \0/

~- S , ~ . - C :1 -C.~ - O ~ '~ + ~ ~ ~ C ~, 15 i~ o '? 'a Y; C ~ ~ C ~ z _ C .L7 _ c ~ c) -r C~
cJ ~ c ;~ _ c ;~ ~
''~
/~'c~
C ~ ~--c~ _c~f2 _ c~-cl~ I
p _ l? ~
c"

This synthetic route requires less time to complete than the reaction sequence using 7-bromomethylcoumarin and provides a cost savings of about 50 percent compared to the 7-bromomethylcoumarin synthetic sequence. The 7~
hydroxycoumarin derivatives can be introduced into oligonucleotides and are morestable during deprotection of the oligonucleotides (exposure to conrçnt~ted NH3 at == =~
CA 022l483~ l997-09-08 W 096/28438 PCTnUS96/03134 23.
room temperature) than compaunds of U.S. Patent 5,082,934. The 7-hydroxycoumarin derivatives exhibit a dilre~ l absorption spectrum (A maxirnum of 325 nm) col"~al~d to the 7-bromomethylcoumarin derivatives (~ maximum of 310 nm). The 7-hydroxycoumarin derivatives are red shffled relative to the 7-bromomethylcoumarin derivatives, which reduces the effect of quenchers, such as nucleic acids. The spectral shift also allows for more selective excitation of the 7-hydroxycoumarin derivatives.
Synthesis of 7-Glycidyl Coumarin The intermediate 7-glycidyl coumarin was prepared in a reaction flask equipped with a reflux condenser cont~ining 16.2 g of 7-hydroxycoumarin, 15.8 g of epibromohydrin, 13.8 g of potassium carbonate and 270 ml of acetone ("reaction solution"). The reaction solution was boiled and refluxed overnight, cooled, treated with 100 ml of 5 % NaOH aqueous solution, and extracted three times with 80 ml of methylene chloride. After evaporating the solvent a crude yellow solid was obtained. The crude solid (1.5g) was dissolved in a solution of30 ml hexane and 20 ml acetone at 50~C. The hexane/acetone solution was then cooled at 0~C for 2 to 3 hours. White crystals formed and were collected by filtering and dried to a white powder. 290 mg of white powder was obtained. The melting point of this new compound (7-glycidyl coumarin) was 110- 112 ~C . Thin layer chromatography (TLC) was done in 8% (v/v ethyl acetate/CHCI3; the Rf value of the 7-glycidyl coumarin was 0.6.
Hydrolvsis of Glycidyl Coumarin 7-Glycidyl coumarin (2.0g) was dissolved in a solution of 80 ml acetone and 50 ml of 1.8 M aqueous H2SO4. The acetone/acid solution was heated to a boil for 20 min~ltes. The solution was cooled and neutralized with a 1.6 M
NF,f4OH aqueous solution until a pH 7-8 was reached. The neutralized solution was extracted with 50 ml ethyl acetate three times. After evaporating the solvent, the produc~, 7-(1-O-glyceryloxy)coumarin, was obtained with a melting point of 118-120~C. Synthesis of 1-0-(4.4'-Dimethoxytrityl)-3-0-(7-coumarinyl) glycerol Coumarinyl glycerol (1.37g) was coevaporated with 11 ml of purified pyridine by rotary evaporation three times. The coevaporated coumarinyl glycerolwas added to 44 mg 4-dimethylaminopyridine, 330 ~I triethylamine, 45 ml CA 0221483~ 1997-09-08 W 096/28438 PCTrUS96/03134 24.
pyridine and 1.78 g of dimethaxytrityl chloride. The solution was stirred at room temperature for 3 hours. The reaction was stopped by adding 66 ml of deionized water. The reaction solution was then extracted three times with 35 ml of methyiene chloride. The organic phase was dried over sodium sulfate. The crude product obtained by evaporating the solvent was purified by chromatography usinga silica gel column and eluting with a solution of 70% hexane, 28% acetone and 2% triethylamine. 2.6 g of purified product (1-0-(4,4' dimethoxytrityl)-3-0-(7-coumarinyl)glycerol) gave a single TLC spot with an Rf of 0.43 using the same solvent system.
Synthesis of 1-0-(4 4'-dimethoxytrityl)-3-0-(7-coumarinyl)-2-0-(2-cyanoethyl-N~N-diisopropyl phosphoramidite) ~Iycerol 1-0-4,4'-Dimethoxytrityl-3-0-(7-coumarinyl)glycerol (2.5g) was coevaporated with 12 ml of 75% pyridine and 25% methylene chloride two times.
A solution of 5 ml methylene chloride and 5 ml pyridine was added to the dry viscous liquid (methylene chloride/coumarin solution). The methylene chloride/coumarin solution was added under argon to a 50 ml flask contz~inin~ a solution of 3 ml of diisopropylethylamine, 10 ml of methylene chloride and 1.8 gof 2-cyanoethyl ~V,N-diisopropyl chlorophosphoramidite. The solution was stirredfor 90 minutes. The reaction mixture was diluted with a solution of 60 ml ethyl acetate and 3 ml triethylamine. The reaction mixture was extracted two times with 50 ml of saturated sodium chloride aqueous solution. The organic phase was then dried over sodium sulfate. The crude product was purified by a silica gel chromatography column. 2.6 g of purified product gave a single spot by TLC
with an Rf of 0.2 using 80% hexane and 20% acetone eluant.

Using the reagent prepared in Example 7, oligonucleotides were prepared via the ,B-cyanoethylphosphoramidite method of DNA synthesis that were identicalto segments of the cryptic plasmid of Chlamydia trachomatis. The oligonucleotides were complementary to nucleotides 876-900, 6857-6878, 7118-7140, and 6725-6752 of the cryptic plasmid (the sequence of which is herein incorporated by reference), the first two oligonucleotides cont~ining one CA 022l483~ l997-09-08 W096/28438 PCT~US96/03134 25.
crocclinking compound per oligonucleotide and the latter two oligonucleotides cont~ining two crosslinking compounds per oligonucleotide.
After automated synthesis, the oligonucleotides were cleaved from the solid support and deprotected with 3 ml 30% NH40H for 2 h at room temperature. The S NH40H was removed in vacuo, and the crude oligonucleotide was purified to homogeneity by de~ g polyacrylamide gel electrophoresis.
The oligonucleotides were hybridized in 0.75 M NaCl buffer (195 ~41) with complementary 5'-32P-labeled oligonucleotides (molar ratio of unlabeled:labeled oligonucleotides = 100:1) for 20 minutes at 40~C, at which time the solutions were irradiated with W-A wavelength light (8 W lamp) for 20 minutt-s. The extent of crosslinking (with respect to the radiolabeled oligonucleotide) was determined by denaturing polyacrylamide gel electrophoresis followed by scintillation counting of the exicsed bands. The results are set forth in the following table:

Cryptic Plasmid ofNumber of CrosslinkersCr-clinkin~Cro~linkin~
Chlan~ydia trachomatisin OligonucleotideReaction Efficiency, Site %
~' ~ 3' 6857-6878 1 Tl-r 86 207118-7140 2 TIT, TAT 99 6725-6752 2 TAC, Tl-r 98 The results indicate that the compounds of the current invention undergo photoch~mic~l crosslinlcing more efficiently than the compound of U.S. Patent 5,082,934.

Coumarin derivatives can be synthesi7eci cont~inin, various side chains, including, (1) short side chains, such as glycerol, (2) long side chains, such as poly(ethylene glycols), (3) aromatic rings, and (4) aliphatic cyclic rings, such as ethylene-dioxy rings. Such coumarin derivatives can be synthe~i7~ from the WO 96/28438 PCTnUS96103134 26.
a~lo~liate coumarin starting materials, such as, 7-methyl coumarin, 7-hydroxy coumarin, esculetin (6,7-dihydroxycoumarin) or 7-glycidyl coumarin. Attached to each coumarin starting material is the desired side chain Cont~ining active functional groups.
s W 096/28438 PCT~US96/03134 27.
REACTION SCHEME FOR CO~JMARIN
CONTAINING AN ALIPHATIC ~lHKOcycLIc RING DERIVATIVE
l-O-r2-cyanoethyl-N.N-diispropyl phosphoramiditel-2.3-0-(6.7-coumarinyl)-~Iycerol .
This compound is not itself a compound within the general formulas described above, but is an intermediate that can be used to prepare such compounds via reaction of X and/or B unit precursors with the hydroxyl group that is activated by formation of a phosphoramidite in the last step of the reaction shown.

o~3 A ~ ) ~~(~~~
A ~L,(b) A ~i~cc) ~ ~ ~

c ,~/
Reagent (a) potassium carbonate/acetone (b) potassium hydroxide (c) 2-cyanoethyl N,l\r-diisopropyl chlorophosphor~mi~lit~ldiisopropylethylamine/pyridine * mixture of ~"~ 0"~o~

W 096/28438 PCTrUS96/03134 28.
Reaction at step (a) with 2,3-dibromo-1,4-dihydroxybutane instead of epibromohydrin gives the similar compound 2,3-0-(6,7-coumarinyl)-1.2.3.4-tetrahydroxybutane; which then can be converted to 1-0-0-(4,4'-dimethoxytrityl)-4-0-(~B-cyanoethyl-l~T,N-diisopropyl phosphoramidite)-2,3-0,0-(6,7-coumarinyl~-1 ,2,3,4-tetrahydroxybutane as shown beiow.

~~,~

NC----/ ~p~
I

~l~N~I~

CA 0221483~ 1997-09-08 W 096/28438 PCTrUS96/03134 29.
Preparation Of 6 7-(Hydroxymethylethylenedioxy)coumarin (Steps AI & AII) Esculetin (0.9Og) was stirred with a solution of potassium carbonate (1.40g) and 200 ml of anhydrous acetone for 1 hour at room temperature.
Epibromohydrin (1.05g) was added to the solution. The yellow suspension solution was then refluxed overnight. Potassium hydroxide (0.70g) was then added and refluxed for one hour. The solution was then separated from the solidsby centrifugation. The solution was then evaporated by a water aspirator. The resulting product was then dissolved in 50 ml of water. The aqueous solution wasthen extracted three times with 35 ml of methylene chloride. The organic solution was extracted twice with 50 ml of 2M sodium hydroxide. The resulting organic phase was dried over sodium sulfate. After evaporating the solvent, 200 mg of white solid was obtained. TLC in 50% acetone/hexane shows Rf 0.42. The yield was 26% by weight.
Preparation of Aliphatic Heterocyclic Rin~ Derivative (Step Am) 6,7-(Hydroxymethylethylenedioxy)coumarin (200 mg) was coevaporated with 1 ml of dry pyridine, twice. To the dry r~ t~nt, 0.9 ml of dichloromethane and 0.9 ml of pyridine was added. 2-Cyanoethyl-N,N-diisopropyl chlorophosphoramidite (280 mg), was dissolved in a solution of 0.2 ml of diisopropylethylamine and 0.9 ml of dichloromethane. The phosphoramidite solution was added to the coumarin solution. The rçs~ ing solution was stirred at room temperature for 2 hours. The reaction mixture was then diluted with a solution of 10 ml of ethyl acetate and 0.5 ml of triethylamine. The solution wasextracted three times with 6 ml of saturated sodium chloride solution. After evaporation of the solvent, the resulting product was purified by a silica gel column with the following eluant: acetone/hexane/triethylamine = 36160/4. The purified product (100 mg) was obtained with an Rf = 0.57 (in TLC using the same eluant).

W 096/28438 PCTrUS96/03134 30.
REACTION SCHEME FOR COUMARIN CONNECTED TO AN AROMATIC
SIDE
CHAIN
3-0-(7-Coumarinylmethoxy)- 1 -0-r2-cyanoethyl-N,N-diisoprophyl S phosphoramidite~ 3-dihydroxybenzene ~3 + ~1 13S , 1 0 /,L~o c~3 C~(~ o~'C~ o 'o~

B ~ o~ C~ ( B

~,~C~ ~ V/~~~ ~C ~

~ \~

Reagents (a) 2-cyanoethyl-N,N-diisopropyl chlorophosphoramidite, diisopropylethylamine/pyridine/dichloromethane .

-CA 022l483~ l997-09-08 W 096/28438 PCTrUS96/0313 31.
Preparation of 7-Bromomethyl Coumarin (Step BI) 7-Methylcoumarin (28g), 70% benzoylperoxide (1.68g), and N-bromosuccinimde (30.8g) was added to 140 ml of chloroform in a one liter flaskand the suspension was refluxed overnight. The solution was diluted with 100 ml of chloroform. The resulting crude product was recryst;llli7e-~ from 750 ml of acetone. A white solid (21g) was obtained with a melting point of 172-176~C was obtained.
Preparation of 3-0-(7-Coumarinylmethyl)-1.3-dihydroxybenzene (Step BII) 7-Bromomethylcoumarin (0.70g) was added to a suspension of resorcinol (2.25g), pot~sil-m carbonate (1.75g) and acetone (200 ml). The solution was heated and stirred for 4 hours. The solution was then separated from the solid.
After evaporating the solvent, the crude product was dissolved in 40 ml of dichloromethane. The organic solution was then extracted three times with 40 ml of water. TLC using 20% (v/v) ethyl acetate/chloroform gave R, = 0.32. After evaporating the solvent, the product was recrystallized from CH2CI2/ethyl acetate solution; 300 mg of purified product was obtained.
Preparation Of 3-0-(7-Coumarinylmethoxy)-l-O-r2-cyanoethyl-N.N-diisopropyl phosphoramiditel-1~3-dihvdroxybenzene (Step Bm) 2-cyanoethyl N,N-diisopropyl chlorophosphoramidite was reacted with the product of Step BII in a fashion similar to that of Step AIII.

WO 96/28438 PCTrUS96103134 32.
REACTION SCH~E FOR COllMARIN CONTAINING A LONG SIDE
CHAIN
3-0-(7-Coumarinyl)-2-0-f2-cyanoethyl-N.N-diisoprophyl phosphoramidite)-1-0-(2-r2-(4~4'-dimethoxytrityloxy)ethoxylethyl)glycerol ~'o~ Cr~ ~3 C I ~ 3 o t ~to~ 2c~- o-Cr~ -o~ C ~ ~3 b) o' 'o ~ CH ~-C~(~C~2ci~0c~!qC~
C

> ~--o ~J~C;f-C~= OC~hch~:-o-c;~ cr~ D ~1 C)~

C~ o~e~~O~C~ Cr~ cr~L-OC~CH2-o--DL~IT
~,,P A/--<
S J' C~

25Reagents (a) potassium carbonate/acetone (b) sodium hydroxide/ethylene glycol dimethyl ether (c) 4, 4'-~iimethoxytritylchloride/pyridine (d) 2-cyanoethyl N,N-diisopropyl chlorophosphoramidite, diisopropyl ethylamine/dichlorometh~n~.

CA 0221483~ 1997-09-08 W 096/28438 PCTrUS96/03134 33.
Preparation Of 7-Glycidyl Coumarin (Step CI) This compound was prepared as described in Example 6.
Preparation Of 3-0-(7-Coumarinyl)-1-0-(2-r2-hvdroxyethoxy~ethyl)~lycerol (Step CII) 7-Glycidylcoumarin (lg) was dissolved in a solution of 10 mg of sodium hydroxide, 2.65g of diethylene glycol and 5 ml of ethylene glycol dimethyl ether.
The solution was heated to reflux for 6 hours. The reaction mixture was diluted with 10 ml of de-ionized water and was extracted three times with 10 ml of dichloromethane. The organic phase was then dried over sodium sulfate. After evaporating the solvent, the crude product was then purifled by a silica gel column with 50% (v/v) acetone/hexane. A major product with Rf 0.09 (260 mg) and a minor product with Rf 0.34 (50 mg) (TLC solvent 50% (v/v) hexane/acetone) were obtained.
Preparation Of 3-0-(7-Coumarinyl)-1-0-(2-r2-(4~4'-dimethoxytrityloxy)ethoxy~ethyl)~lycerol (Step Cm) The dihydroxy coumarin derivative (230 mg) obtained as the product of Step CII was coevaporated with dry pyridine. Dimethoxytritylchloride (320 mg), 60 ml of triethylamine and 10 mg of 4-dimethylaminopyridine were added to the coumarin derivative. The solution was stirred at room temperature for 16 hours.
The solution was diluted with water and extracted with dichloromethane, then dried with sodium sulfate. After evaporating the solvent, the crude product was purified by a silica gel column with 40% (v/v) acetone/hexane as eluant.
Preparation Of 3-0-(7-Coumarinyl)-2-0-(2-cyanoethyl-N.N-diisopropyl phosphoramidite)-1-0-(2-r2-(4~4-dimethoxytrityloxy)ethoxylethyl)~lycerol (Step CIV) 2-Cyanoethyl-N,N-diisopropyl chlorophosphoramidite is reacted with the product of Step cm as described in Step Am.
Preparation Of Phosphoramidite Used In Steps Am, Bm AND CIV
The general procedure for preparing the phosphoramidite is as follows.
Under an inert atmosphere 1.2 eq of 2-cyanoethyl-N,N-diisopropyl chlorophosphor~mi~litP and 2.4 eq of N,N-diisopropylethylamine are dissolved in 0.9 ml of dichloromethane in a glass container capped with a septum. The -CA 022l483~ l997-09-08 W 096/28438 PCTnUS96/0313 34.
coumarin precursor (1.0 eq) (such as the product of steps AII, BII or Cm) was dissolved 9.9 ml of pyridine and 0.9 ml of dichloromethane. While the chlorophosphoramidite solution is stirred, the coumarin solution is added. Stirring is continued for 2 hours. Ethylacetate is added, and the organic solution is washed 4 S with NaCI (aqueous) three times and dried with Na2SO4. After removing the solvent, the crude product is purified by column chromatography using acetone/hexane/triethylamine (36:60:4). Al~plu~liate fractions are collected andconcentrated under vacuum.
The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.
All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and indi-~!idually indicated to be incorporated by reference.

Claims (42)

35.
WHAT IS CLAIMED IS:

1. A compound having the formula:

wherein B represents (1) a linear, branched, or cyclic hydrocarbon group containing from 2 to 10 carbon atoms and, if cyclic, containing a 5- or 6-membered ring or (2) a heterocyclic aromatic ring system containing a 5- or 6-membered ring, saidB(1) or B(2) being substituted with 1, 2, or 3 groups of the formula OR1, wherein R1 independently represent H or a hydroxy-protecting or hydroxy-coupling group capable of protecting or coupling a hydroxy group during synthesis of a polynucleotide, or OR1 represent a nucleotide or a polynucleotide connected to the remainder of said formula, and wherein one to three carbon atoms of the hydrocarbon group can be replaced by an oxygen, nitrogen, or sulfur atom;
X represents (1) a bond, (2) a linear, branched, or cyclic hydrocarbon group containing 1 to 10 carbon atoms or (3) the X(2) group in which one to three carbon atoms of the hydrocarbon group are replaced by an oxygen, sulfur, or nitrogen atom and wherein the shortest linking chain of atoms in X between atomsin other parts of said formula attached to X is 1 to 10 atoms, wherein X is optionally substituted with 1-3 substituents selected from the group consisting of hydroxy, halogen, amino, amido, azido, carboxy, carbonyl, perfluoromethyl, and cyano functional groups; and wherein X is attached to the phenyl ring of said formula directly or through W;
n is 0, 1, 2, or 3;
each W independently represents a hydroxy, halogen, amino, amido, azido, nitro, thio, carboxy, carbonyl, perfluoromethyl, or cyano functional group; an unsubstituted hydrocarbyl group of 10 or fewer carbon atoms; or said hydrocarbylgroup substituted with 1-3 of said functional groups or in which one carbon atoms 36.
replaced by an oxygen, sulfur, or nitrogen atom and wherein two Ws together can represent a ring fused to the phenyl ring of said formula;
with the provisos that (1) when X or W is a substituted hydrocarbon, the total number of substituents in X or W is less than the total number of carbon atoms in said X or W and no more than one substituent or heteroatom is attached to a given carbon, unless said substituents are halogen atoms on said given carbon, and (2) the total carbon atoms in all W substituents is 15 or fewer; and Y and Z independently represent H or lower alkyl or F;
wherein said formula contains one photoactive bond and said bond is located between the carbons to which Y and Z are attached in said formula.

2. The compound of claim 1, wherein X, in either orientation, is -OCH2-, -SCH2-, -NHCH2-, , , , , or -CL2(CH2)n-, in which L = H, F, Cl, I , or Br and n = 0, 1 , or 2.

3. The compound of Claim 1, wherein X is a cyclic structure with a 5- or 6-membered ring or a 5- or 6-membered heteroring containing one O, S, or Natom.

4. The compound of Claim 1, wherein all of said formula to the right of X represents coumarin or a derivative thereof within said formula.

5. The compound of Claim 1, wherein X is covalently connected to the 7 position of a coumarin moiety.

6. The compound of Claim 1, wherein B represents:
a group of a first sub-formula 37.

a group of a second sub-formula or a group of a third sub-formula wherein Rx, Ry, and Rz independently represent H or OR1;
m, n, p, q, and r independently represent 0 or 1;
one hydrogen of said sub-formula is replaced by a covalent bond to said X
group; and all other substituents and definitions of said formula of said compound are otherwise as previously defined.

7. The compound of Claim 6, wherein B is saturated.

8. The compound of Claim 6, wherein B has said third sub-formula.

9. The compound of Claim 6, wherein m + n + p + q + r = 0, 1, or 2.

10. The compound of Claim 6, wherein said third sub-formula represents an acyclic, saturated, di- or tri-hydroxyhydrocarbon.

38.

11. The compound of Claim 6, wherein said third sub-formula represents tri-O-substituted glycerin.

12. The compound of Claim 1, wherein said nucleotide or polynucleotide is connected to said compound via a phosphorous-containing linking group.

13. The compound of Claim 12, wherein said phosphorous-containing group is a phosphate group.

14. The compound of Claim 1, wherein B contains a benzene or naphthalene ring.

15. The compound of Claim 1, wherein B contains a bridged hydrocarbon ring.

16. The compound of Claim 1, wherein B contains a bicyclo [3.1.0] or hexane or [2.2.1] heptane ring.

17. The compound of Claim 1, wherein B contains a spiro or dispiro hydrocarbon ring.

18. The compound of Claim 1, wherein at least one R1 represents a trityl, pixyl, dimethoxytrityl, monomethoxytrityl, phosphite, phosphoramidite, phosphate, H-phosphonate, phosphorothioate, methylphosphonate, phosphodithioate or phosphotriester group.

19. The compound of Claim 1, wherein B contains a heterocyclic ring selected from the group consisting of furan, pyran, pyrrole, pyrazole, imidazole, piperidine, pyridine, pyrazine, pyrimidine, pyridazine, thiophene, acridine, indole, quinoline, isoquinoline, quinazoline, quinoxaline, xanthene, and 1,2-benzopyran.

39.

20. A compound of Claim 1, wherein said compound is a polynucleotide of the formula (Nm1Qm4Nm2)m3 in which each N represents a nucleotide of a desired polynucleotide sequence, Q represents the nucleotide-replacing molecule in the formula of claim 1, and m1, m2, and m3 are integers, and m3 is from 1 to 10.

21. A compound of Claim 20 wherein m1 and m2 are less than 100.

22. A compound of Claim 21 wherein at least one Q is at a terminal position and at least one Q is at an interior position of said polynucleotide sequence.

23. A compound of Claim 21 wherein at least one Q is at a interior position of said polynucleotide sequence.

24. A compound of Claim 21 wherein Q is at a terminal position of said polynucleotide sequence.

25. A compound of Claim 1 wherein B-X- is R1-O-CH2-CH2-O-CH2-CH2-O-CH2-CH(OR1)-CH2-O-.

26. A compound of Claim 1 wherein X is -CH2-O- and B is 40.

27. A compound having the formula:

wherein B represents (1) a branched or cyclic hydrocarbon group containing from 3 to 10 carbon atoms and, if cyclic, containing a 5- or 6-membered ring or (2) a heterocyclic aromatic ring system containing a 5- or 6-membered ring, said B(1) or B(2) being substituted with 1, 2, or 3 groups of the formula OR1, wherein R1 represent H or a hydroxy-protecting or hydroxy-coupling group capable of protecting or coupling a hydroxy group during synthesis of a polynucleotide, or 2 groups when OR1 represents a nucleotide or a polynucleotide connected to the remainder of said formula, and wherein one to three carbon atoms of the hydrocarbon group may be replaced by an oxygen, nitrogen, or sulfur atom; or B
represents a group of a third sub-formula wherein Rx, Ry, and Rz independently represent OR1;
m, n, p, q, and r independently represent 0 or 1; and one hydrogen of said sub-formula is replaced by a covalent bond to said X group;
X represents (1) a bond, (2) a linear, branched, or cyclic hydrocarbon group containing 1 to 10 carbon atoms or (3) said X(2) group in which one to three carbon atoms of the hydrocarbon group are replaced by an oxygen, sulfur, or nitrogen atom, and wherein the shortest linking chain of atoms in X between atoms in other parts of said formula attached to X is 1 to 10 atoms, wherein X is optionally substituted with 1-3 substituents selected from the group consisting of 41.
hydroxy, halogen, amino, amido, azido, carboxy, carbonyl, perfluoromethyl, and cyano functional groups; and wherein X is attached to the phenyl ring of said formula directly or through W;
n is 1, 2, or 3;
each W independently represents a hydroxy, halogen, amino, amido, azido, nitro, thio, carboxy, carbonyl, perfluoromethyl, or cyano functional group; an unsubstituted hydrocarbyl group of 10 or fewer carbon atoms; or said hydrocarbylgroup substituted with 1-3 of said functional groups or in which one carbon atoms replaced by an oxygen, sulfur, or nitrogen atom and wherein two Ws together can represent a ring fused to the phenyl ring of said formula;
with the provisos that (1) when X or W is a substituted hydrocarbon, the total number of substituents in X or W is less than the total number of carbon atoms in said X or W and no more than one substituent or heteroatom is attached to a given carbon, unless said substituents are halogen atoms on said given carbon, and (2) the total carbon atoms in all W substituents is 15 or fewer; and Y and Z independently represent H or lower alkyl.

28. The compound of claim 27, wherein X, in either orientation, is -OCH2-, -SCH2-, -NHCH2-, , , , , or -CL2(CH2)n-, in which L = H, F, Cl, I, or Br and n = 0, 1, or 2.

29. The compound of Claim 27, wherein X is a cyclic structure with a 5- or 6-membered ring or a 5- or 6-membered heteroring containing one O, S, or Natom.

30. The compound of Claim 27, wherein W is a pyrone or furan fing fused to the phenyl ring of said formula.

42.

31. The compound of Claim 27, wherein all of said formula to the right of X represents psoralen, cis-benzodipyrone, or trans-benzodipyrone or a derivative thereof within said formula.

32. The compound of Claim 27, wherein X is covalently connected to the 4 position of a furan ring of a psoralen moiety.

33. The compound of Claim 27, wherein B represents:
a group of a first sub-formula a group of a second sub-formula and all other substituents and definitions of said formula of said compound are otherwise as previously defined.

34. The compound of Claim 27, wherein B is saturated.

35. The compound of Claim 27, wherein B has said third sub-formula.

36. The compound of Claim 35, wherein m + n + p + q + r = 0, 1, or 2.

37. The compound of Claim 36, wherein B represents an acyclic, saturated, tri-hydroxyhydrocarbon.

43.

38. The compound of Claim 36, wherein B represents tri-O-substituted glycerin.

39. The compound of Claim 27, wherein at least one R1 represents a trityl, pixyl, dimethoxytrityl, monomethoxytrityl, phosphite, phosphoramidite, phosphate, H-phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate or phosphotriester group.

40. The compound of Claim 27, wherein B contains a heterocyclic ring selected from the group consisting of furan, pyran, pyrrole, pyrazole, imidazole, piperidine, pyridine, pyrazine, pyrimidine, pyridazine, thiophene, acridine, indole, quinoline, isoquinoline, quinazoline, quinoxaline, xanthene, and 1,2-benzopyran.

41. A compound of Claim 27, wherein said compound is a polynucleotide of the formula (Nm1Qm4Nm2)m3 in which each N represents a nucleotide of a desired polynucleotide sequence, Q represents the nucleotide-replacing molecule in the formula of claim 1, and m1, m2, and m3 are integers, and m3 is from 1 to 10.

42. The compounds: 1-O-(4,4'-Dimethoxytrityl)-4-O-(.beta.-cyanoethyl-N,N-diisopropyl phosphoramidite)-2,3-O,O-(6,7-coumarinyl)-1,2,3,4-tetrahydroxybutane; 2,3-O,O(6,7-coumarinyl)-1,2,3,4-tetrahydroxybutane; 1-O-.beta.-cyanoethyl-N,N-diisopropyl phosphoramidite]-2,3-O,O-(6,7-coumarinyl)-glycerol;
or 2,3-O-(6,7-coumarinyl)-glycerol.