NON-NUCLEOSIDIC COUMARIN DERIVATIVES AS POLYNUCLEOTIDE-CROSS-LINKING AGENTS

DERIVES NON NUCLEOSIDIQUES DE COUMARINE UTILISES COMME AGENTS DE RETICULATION DE POLYNUCLEOTIDES

English Abstract

Novel coumarin derivatives comprising a coumarin moiety linked to a non-
nucleosidic 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.

French Abstract

Cette invention concerne de nouveaux dérivés de coumarine comportant une fraction de coumarine liée à une fraction squelette non nucléosidique. Les molécules résultantes servent typiquement de groupements de réticulation photosensibles lorsqu'elles sont incorporées à des polynucléotides à la place d'une ou de plusieurs bases nucléotidiques complémentaires présentes à l'intérieur de sondes utilisées dans des procédures faisant appel à des réactions d'hybridation moléculaire d'acides nucléiques.

Claims

35.
WHAT IS CLAIMED IS:
1. A compound having the formula:
<IMG>
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-, <IMG> , <IMG> ,
<IMG>, <IMG>, 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
<IMG>

37.
a group of a second sub-formula
<IMG>
or a group of a third sub-formula
<IMG>
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
<IMG>

40.
27. A compound having the formula:
<IMG>
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
<IMG>
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-, <IMG> , <IMG> ,
<IMG>, <IMG>, 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
<IMG>
a group of a second sub-formula
<IMG>
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.

Description

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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.

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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
0
~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

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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

-
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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

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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

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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~

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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
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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

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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

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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

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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

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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

=
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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.

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In certain ~ rt;lled embo-limentc the glycerol backbone moiety precursor
has the formula:
~ 1 L ) 1~ 3
0
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.

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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

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17.
EXAMPLE 1
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.
EXAMPLE 2
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).

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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%).
EXAMPLE 4
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.

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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.

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20.
EXAMPLE 6
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.

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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).

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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

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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

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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.
EXAMPLE 8
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

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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'
876-900 1 TAA 88
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.
EXAMPLE 9
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

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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

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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~

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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~

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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).

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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 .

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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.

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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~.

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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

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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.