U.S. patent application number 09/839622 was filed with the patent office on 2002-02-14 for water soluble tri-substituted 1,2-dioxetane compounds having increased storage stability, synthetic processes and intermediates.
Invention is credited to Akhavan-Tafti, Hashem, Arghavani, Zahra, DeSilva, Renuka, Thakur, Kumar.
Application Number | 20020019553 09/839622 |
Document ID | / |
Family ID | 27414317 |
Filed Date | 2002-02-14 |
United States Patent
Application |
20020019553 |
Kind Code |
A1 |
Akhavan-Tafti, Hashem ; et
al. |
February 14, 2002 |
Water soluble tri-substituted 1,2-dioxetane compounds having
increased storage stability, synthetic processes and
intermediates
Abstract
Stable, enzymatically triggered chemiluminescent 1,2-dioxetanes
with improved water solubility and storage stability are provided
as well as synthetic processes and intermediates used in their
preparation. Dioxetanes further substituted with two or more
water-solubilizing groups disposed on the dioxetane structure and
an additional fluorine atom or lower alkyl group provide superior
performance by eliminating the problem of reagent carryover when
used in assays performed on capsule chemistry analytical systems.
These dioxetanes display substantially improved stability on
storage. Compositions comprising these dioxetanes, a non-polymeric
cationic surfactant enhancer and optionally a fluorescer, for
providing enhanced chemiluminescence are also provided.
Inventors: |
Akhavan-Tafti, Hashem;
(Brighton, MI) ; DeSilva, Renuka; (Northville,
MI) ; Thakur, Kumar; (Southfield, MI) ;
Arghavani, Zahra; (Southfield, MI) |
Correspondence
Address: |
LUMIGEN, INC.
22900 W. EIGHT MILE ROAD
SOUTHFIELD
MI
48034
US
|
Family ID: |
27414317 |
Appl. No.: |
09/839622 |
Filed: |
April 20, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09839622 |
Apr 20, 2001 |
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09506263 |
Feb 17, 2000 |
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6245928 |
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09506263 |
Feb 17, 2000 |
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09101331 |
Jul 7, 1998 |
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6036892 |
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09101331 |
Jul 7, 1998 |
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PCT/US97/19618 |
Nov 7, 1997 |
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PCT/US97/19618 |
Nov 7, 1997 |
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08748107 |
Nov 8, 1996 |
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5721370 |
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08748107 |
Nov 8, 1996 |
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08509305 |
Jul 31, 1995 |
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5777135 |
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Current U.S.
Class: |
558/167 ;
549/510; 560/125; 560/126; 560/127; 560/128; 560/129 |
Current CPC
Class: |
C07C 69/734 20130101;
C07D 321/00 20130101; G01N 33/54326 20130101; G01N 33/5308
20130101; C07C 2603/74 20170501; C07F 9/12 20130101; C07F 9/65512
20130101; G01N 33/582 20130101 |
Class at
Publication: |
558/167 ;
549/510; 560/125; 560/126; 560/127; 560/128; 560/129 |
International
Class: |
C07C 057/02 |
Claims
What is claimed is:
1. A process for preparing a dioxetane salt compound of the
formula: 56having increased storage stability wherein R.sub.3 and
R.sub.4 are each selected from the group consisting of acyclic,
cyclic and polycyclic organic groups which can optionally be
substituted with heteroatoms and which can optionally be joined
together to form a cyclic or polycyclic ring group spiro-fused to
the dioxetane ring, wherein R.sub.2 is an aryl ring group selected
from the group consisting of phenyl and naphthyl groups which can
include additional substituents, wherein Z is selected from the
group consisting of halogen atoms and alkyl groups of 1-4
carbons,and M is selected from hydrogen, an alkali metal ion or a
quaternary ammonium or phosphonium ion comprising the steps of: a)
reacting a first alkene compound having the formula: 57wherein RG
is a removable group with a Z-substituted malonate ester and a base
to produce a malonate-substituted alkene compound having the
formula: 58wherein R' is an alkyl group of 1-4 carbons; b)
photooxygenating the malonate-substituted alkene compound by
irradiating a sensitizer in the presence of oxygen and the
malonate-substituted alkene compound to form a malonate-substituted
dioxetane having the formula: 59c) reacting the
malonate-substituted dioxetane with a phosphorylating reagent
having the formula WP(O)Y.sub.2 wherein W and Y are each selected
from halogen atoms, substituted or unsubstituted alkoxy, aryloxy,
aralkyloxy and trialkylsilyloxy groups to form a phosphorylated
dioxetane compound having the formula: 60d) hydrolyzing the
phosphorylated dioxetane in an aqueous solvent with a base of the
formula M--Q wherein Q is a basic anion to form the dioxetane salt
compound.
2. The process of claim 1 wherein the step of reacting the
malonate-substituted dioxetane compound with the phosphorylating
reagent comprises the steps of: a) reacting the
malonate-substituted dioxetane compound with a phosphorylating
reagent having the formula WP(O)Y'.sub.2 wherein Y' is a halogen
atom to form a dioxetane phosphoryl halide compound having the
formula 61b) reacting the dioxetane phosphoryl halide compound with
a hydroxyl compound of the formula Y--OH, wherein Y is selected
from substituted or unsubstituted alkyl groups to form the
phosphorylated dioxetane compound.
3. The process of claim 1 wherein the reagent WP(O)Y.sub.2 is
POCl.sub.3.
4. The process of claim 1 wherein R.sub.3 and R.sub.4 are combined
together to form a cyclic or polycyclic ring group R.sub.5
spiro-fused to the dioxetane ring and the dioxetane salt compound
has the formula: 62
5. The process of claim 4 wherein R.sub.5 is a substituted or
unsubstituted adamantyl group.
6. The process of claim 1 wherein R.sub.2 is a substituted or
unsubstituted meta-phenyl group.
7. The process of claim 1 wherein Z is selected from F and CH.sub.3
and M is Na.
8. The process of claim 5 wherein Z is selected from CH.sub.3 and
F, M is Na and R.sub.2 is a substituted or unsubstituted
meta-phenyl group.
9. The process of claim 5 wherein Z is CH.sub.3, M is Na, R.sub.2
is an unsubstituted meta-phenyl group, R.sub.5 is an unsubstituted
adamantyl group and the dioxetane salt compound has the formula:
63
10. The process of claim 5 wherein Z is F, M is Na, R.sub.2 is an
unsubstituted meta-phenyl group, R.sub.5 is an unsubstituted
adamantyl group and the dioxetane salt compound has the formula:
64
11. A process for preparing a dioxetane salt compound of the
formula: 65having increased storage stability wherein R.sub.3 and
R.sub.4 are each selected from the group consisting of acyclic,
cyclic and polycyclic organic groups which can optionally be
substituted with heteroatoms and which can optionally be joined
together to form a cyclic or polycyclic ring group spiro-fused to
the dioxetane ring, wherein R.sub.2 is an aryl ring group selected
from the group consisting of phenyl and naphthyl groups which can
include additional substituents, wherein Z is selected from the
group consisting of halogen atoms and alkyl groups of 1-4 carbons
and M is selected from hydrogen, an alkali metal ion, a quaternary
ammoniumion or a phosphonium ion comprising the steps of: a)
reacting a first alkene compound having the formula: 66wherein RG
is a removable group with a Z-substituted malonate ester and a base
to produce a malonate-substituted alkene compound having the
formula: 67wherein R' is an alkyl group of 1-4 carbons; b) reacting
the malonate-substituted alkene with a phosphorylating reagent
having the formula WP(O)Y.sub.2 wherein W and Y are each selected
from halogen atoms, to form a phosphorylated alkene compound having
the formula: 68c) reacting the phosphorylated alkene compound with
a hydroxyl compound of the formula Y'--OH, wherein Y' is selected
from substituted or unsubstituted alkyl groups to form a second
phosphorylated alkene compound having the formula: 69d) hydrolyzing
the second phosphorylated alkene compound in an aqueous solvent
with a base of the formula M--Q wherein Q is a basic anion to form
an alkene salt compound having the formula: 70e) photooxidizing the
alkene salt compound by irradiating a sensitizer in the presence of
oxygen and the alkene salt compound in aqueous solution to form the
dioxetane salt compound.
12. The process of claim 11 wherein R.sub.3 and R.sub.4 are
combined together to form a cyclic or polycyclic ring group R.sub.5
spiro-fused to the dioxetane ring and the dioxetane salt compound
has the formula: 71
13. The process of claim 12 wherein R.sub.5 is a substituted or
unsubstituted adamantyl group.
14. The process of claim 11 wherein R.sub.2 is a substituted or
unsubstituted meta-phenyl group.
15. The process of claim 11 wherein Z is selected from F and
CH.sub.3 and M is Na.
16. The process of claim 13 wherein wherein Z is selected from F
and CH.sub.3, M is Na, R.sub.2 is a substituted or unsubstituted
meta-phenyl group.
17. The process of claim 13 wherein Z is CH.sub.3, R.sub.2 is an
unsubstituted meta-phenyl group, R.sub.5is the unsubstituted
adamantyl group and the dioxetane salt compound has the formula:
72
18. The process of claim 13 wherein Z is F, R.sub.2 is an
unsubstituted meta-phenyl group, R.sub.5 is the unsubstituted
adamantyl group and the dioxetane salt compound has the formula:
73
19. An alkene compound of the formula: 74wherein Z is selected from
halogen atoms and alkyl groups of 1-4 carbons and each R' is an
alkyl group of 1-4 carbons.
20. The compound of claim 19 wherein Z is F and R' is ethyl.
21. The compound of claim 19 wherein Z if CH.sub.3 and R' is
ethyl.
22. An alkene compound of the formula: 75wherein Z is selected from
the group consisting of halogen atoms and alkyl groups of 1-4
carbons, each R' is an alkyl group of 1-4 carbons and each Y is
selected from halogen atoms, substituted or unsubstituted alkoxy,
aryloxy, aralkyloxy and trialkylsilyloxy groups.
23. The compound of claim 22 wherein Z is F and each Y is a
2-cyanoethyl group.
24. The compound of claim 22 wherein Z is CH.sub.3 each Y is a
2-cyanoethyl group.
25. An alkene compound of the formula: 76wherein Z is selected from
halogen atoms and alkyl groups of 1-4 carbons, each R' is an alkyl
group of 1-4 carbons and M is selected from hydrogen, an alkali
metal ion, a quaternary ammoniumion or a phosphonium ion.
26. The compound of claim 25 wherein Z is F and each M is a sodium
atom.
27. The compound of claim 25 wherein Z is CH.sub.3 and each M is a
sodium atom.
28. A dioxetane compound of the formula: 77wherein Z is selected
from halogen atoms and alkyl groups of 1-4 carbons and each R' is
an alkyl group of 1-4 carbons.
29. The compound of claim 28 wherein Z is F and R' is ethyl.
30. The compound of claim 28 wherein Z is CH.sub.3 and R' is
ethyl.
31. A dioxetane compound of the formula: 78wherein Z is selected
from halogen atoms and alkyl groups of 1-4 carbons, each R' is an
alkyl group of 1-4 carbons and each Y is selected from a Cl atom
and a 2-cyanoethyl group.
32. The compound of claim 31 wherein Z is F, Y is Cl and R' is
ethyl.
33. The compound of claim 31 wherein Z is CH.sub.3, Y is Cl and R'
is ethyl.
34. The compound of claim 31 wherein Z is F, Y is the 2-cyanoethyl
group and R' is ethyl.
35. The compound of claim 31 wherein Z is CH.sub.3, Y is the
2-cyanoethyl group and R' is ethyl.
36. A dioxetane of the formula: 79having increased storage
stability wherein R.sub.3 and R.sub.4 are each selected from the
group consisting of acyclic, cyclic and polycyclic organic groups
which can optionally be substituted with heteroatoms and which can
optionally be joined together to form a cyclic or polycyclic ring
group spiro-fused to the dioxetane ring, wherein R.sub.2 is an aryl
ring group selected from the group consisting of phenyl and
naphthyl groups which can include additional substituents, wherein
Z is selected from the group consisting of a fluorine atom and an
alkyl group of 1-4 carbons, M is selected from hydrogen, an alkali
metal ion or a quaternary ammonium or phosphonium ion and wherein X
is a protecting group which can be removed by an activating agent
to produce light.
37. A stable dioxetane of the formula: 80having increased storage
stability wherein R.sub.5 is selected from the group consisting of
cyclic and polycyclic alkyl groups which are spiro-fused to the
dioxetane ring and which contain 6 to 30 carbon atoms and which can
optionally include additional substituents, wherein R.sub.2 is an
aryl ring group selected from the group consisting of phenyl and
naphthyl groups which can optionally include additional
substituents, wherein Z is selected from the group consisting of a
fluorine atom and an alkyl group of 1-4 carbons, M is selected from
hydrogen, an alkali metal ion or a quaternary ammonium or
phosphonium ion and wherein X is a protecting group which can be
removed by an activating agent to produce light.
38. The dioxetane of claim 37 having the formula: 81
39. The dioxetane of any one of claims 36, 37 or 38 wherein the OX
group is selected from the group consisting of an O.sup.-M.sup.+
group wherein M is selected from the group consisting of hydrogen,
an alkali metal ion, a quaternary ammonium and a quaternary
phosphonium ion, an OOCR.sub.8 group wherein R.sub.8 is selected
from the group consisting of alkyl and aryl groups containing 2 to
8 carbon atoms and optionally containing heteroatoms,
OPO.sub.3.sup.-2 salt, OSO.sub.3.sup.- salt, .beta.-D-galactosidoxy
and .beta.-D-glucuronidyloxy groups.
40. A dioxetane of the formula: 82which has increased storage
stability.
41. A dioxetane of the formula: 83which has increased storage
stability.
42. A composition for producing light comprising in an aqueous
solution; (a) a stable dioxetane of the formula: 84having increased
storage stability wherein R.sub.3 and R.sub.4 are each selected
from the group consisting of acyclic, cyclic and polycyclic organic
groups which can optionally be substituted with heteroatoms and
which can optionally be joined together to form a cyclic or
polycyclic ring group spiro-fused to the dioxetane ring, wherein
R.sub.2 is an aryl ring group selected from the group consisting of
phenyl and naphthyl groups which can include additional
substituents, wherein Z is selected from the group consisting of a
fluorine atom and an alkyl group of 1-4 carbons, M is selected from
hydrogen, an alkali metal ion or a quaternary ammonium or
phosphonium ion and wherein X is a protecting group which can be
removed by an activating agent to produce the light; and (b) a
non-polymeric cationic enhancer substance which increases the
quantity of light produced by reacting the dioxetane with the
activating agent compared to the amount which is produced in the
absence of the enhancer.
43. The composition of claim 42 wherein the enhancer substance is a
dicationic surfactant of the formula: 85wherein each of A is
independently selected from the group consisting of P and N atoms,
wherein Link is an organic linking group containing at least two
carbon atoms selected from the group consisting of substituted and
unsubstituted aryl, alkyl, alkenyl and alkynyl groups and wherein
Link can optionally contain heteroatoms and wherein R is selected
from lower alkyl or aralkyl containing 1 to 20 carbon atoms and
wherein Y is an anion.
44. The composition of claim 45 wherein the enhancer substance is a
dicationic surfactant having the formula: 86and wherein link is
phenylene.
45. The composition of any one of claims 42, 43 or 44 wherein the
dioxetane has the formula: 87wherein R.sub.5 is selected from the
group consisting of cyclic and polycyclic alkyl groups which are
spiro-fused to the dioxetane ring and which contain 6 to 30 carbon
atoms and which can optionally include additional substituents.
46. The composition of claim 45 wherein R.sub.5 is selected from
the group consisting of an adamantyl group and a substituted
adamantyl group.
47. The composition of claim 45 wherein the dioxetane has the
formula: 88
48. The composition of claim 45 wherein the dioxetane has the
formula: 89
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation-in-part of applicants'
co-pending U.S. application Ser. No. 08/748,107 filed on Nov. 8,
1996 which is a continuation-in-part of applicants' co-pending U.S.
application Ser. No. 08/509,305 filed on Jul. 31, 1995.
BACKGROUND OF THE INVENTION
[0002] (1) Field of the Invention
[0003] The present invention relates generally to stable
1,2-dioxetanes and compositions which can be triggered by chemical
reagents, including enzymes, to generate chemiluminescence. The
dioxetanes contain more than one ionizable group which are part of
an alkoxy substituent. The dioxetanes further contain a fluorine
atom or lower alkyl group substituted for one of the hydrogen atoms
on the alkoxy substituent which improve the storage stability of
the dioxetane. The present invention, in particular, further
relates to methods of synthesis of such dioxetanes.
[0004] The dioxetanes which are prepared by the synthetic processes
of the present invention are useful in compositions containing the
dioxetane, a cationic surfactant and optionally a fluorescer which
enhance the amount of chemiluminescence which is produced.
Dioxetanes and enhanced compositions of the present invention are
useful in methods for generating light (chemiluminescence) and in
methods of analysis for detecting the presence or amount of an
analyte. Importantly, the ionizable groups afford a more water
soluble dioxetane and solve an unexpected chemical carryover
problem in capsule chemistry analytical systems, while the presence
of the fluorine atom or lower alkyl group improves the storage
stability of the dioxetane.
[0005] (2) Description of Related Art
[0006] a. Enzymatically Triggerable Dioxetanes. The first examples
of enzymatic triggering of dioxetanes are described in a U.S.
patent application (A. P. Schaap, U.S. patent application Ser. No.
887,139) and a series of papers (A. P. Schaap, R. S. Handley, and
B. P. Giri, Tetrahedron Lett., 935 (1987); A. P. Schaap, M. D.
Sandison, and R. S. Handley, Tetrahedron Lett., 1159 (1987) and A.
P. Schaap, Photochem. Photobiol., 47S, 50S (1988)). The highly
stable adamantyl-substituted dioxetanes bearing a protected
aryloxide substituent are triggered to decompose with emission of
light by the action of both an enzyme and aqueous buffer to give a
strongly electron-donating aryloxide anion which dramatically
increases the rate of decomposition of the dioxetane. As a result,
chemiluminescence is emitted at intensities several orders of
magnitude above that resulting from slow thermal decomposition of
the protected form of the dioxetane. U.S. Pat. No. 5,068,339 to
Schaap discloses enzymatically triggerable dioxetanes with
covalently linked fluorescer groups decomposition of which results
in enhanced chemiluminescence via energy transfer to the
fluorescer. U.S. Pat. Nos. 5,112,960 and 5,220,005 and a PCT
application (WO88/00695) to Bronstein disclose triggerable
dioxetanes bearing substituted adamantyl groups. U.S. Pat. No.
4,952,707 to Edwards discloses phosphate-substituted dioxetanes. A
PCT application (WO94/26726) to Bronstein discloses adamantyl
dioxetanes bearing a phenyl or naphthyl group substituted at a
non-conjugated position with an enzyme labile OX group and with an
additional group on the aryl ring.
[0007] Other triggerable dioxetanes are disclosed in a PCT
application (WO94/10258) to Wang. The dioxetanes disclosed in Wang
contain an alkoxy group which may be mono-substituted and a
substituted phenyl-OX group wherein one or more non-hydrogen groups
are present on the benzene ring substituent in addition to the
triggerable OX group.
[0008] Dioxetanes disclosed in all of the foregoing publications
generate a light-emitting carbonyl compound comprising an alkyl
ester of an aromatic carboxylic acid, typically the methyl ester of
a hydroxybenzoic or hydroxynaphthoic acid or else a hydroxyaryl
ketone.
[0009] Applicants' co-pending U.S. application Ser. No. 08/509,305
('305 application) filed on Jul. 31, 1995 discloses disubstituted
dioxetanes whose hydroxy dioxetane shows improved water solubility
and is fully incorporated herein by reference.
[0010] b. Surfactant Enhancement of Chemiluminescence from
Trigqerable Dioxetanes. Enhancement of chemiluminescence from the
enzyme-triggered decomposition of a stable 1,2-dioxetane in the
presence of water-soluble substances including an ammonium
surfactant and a fluorescer has been reported (A. P. Schaap, H.
Akhavan and L. J. Romano, Clin. Chem., 35(9), 1863 (1989)).
Fluorescent micelles consisting of cetyltrimethylammonium bromide
(CTAB) and 5-(N-tetradecanoyl)amino-fluorescein capture the
intermediate hydroxy-substituted dioxetane and lead to a 400-fold
increase in the chemiluminescence quantum yield by virtue of an
efficient transfer of energy from the anionic form of the excited
state ester to the fluorescein compound within the hydrophobic
environment of the micelle.
[0011] U.S. Pat. Nos. 4,959,182 and 5,004,565 to Schaap describe
additional examples of enhancement of chemiluminescence from
chemical and enzymatic triggering of stable dioxetanes in the
presence of micelles formed by the quaternary ammonium surfactant
CTAB. Fluorescent micelles also enhance light emission from the
base-triggered decomposition of hydroxy- and acetoxy-substituted
dioxetanes.
[0012] U.S. Pat. No. 5,145,772 to Voyta discloses enhancement of
enzymatically generated chemiluminescence from 1,2-dioxetanes in
the presence of polymers with pendant quaternary ammonium groups
alone or admixed with fluorescein. Other substances reported to
enhance chemiluminescence include globular proteins such as bovine
albumin and quaternary ammonium surfactants. Other cationic polymer
compounds were marginally effective as chemiluminescence enhancers;
nonionic polymeric compounds were generally ineffective and an
anionic polymer significantly decreased light emission. A PCT
application (WO 94/21821) to Bronstein describes the use of
mixtures of the aforementioned polymeric quaternary ammonium
surfactant enhancers with enhancement additives.
[0013] The enhancement and catalysis of a non-triggerable dioxetane
by pyranine in the presence of CTAB is described (Martin Josso,
Ph.D. Thesis,Wayne State University (1992), Diss. Abs. Int., Vol.
53, No. 12B, p. 6305).
[0014] U.S. Pat. No. 5,393,469 to Akhavan-Tafti discloses
enhancement of enzymatically generated chemiluminescence from
1,2-dioxetanes in the presence of polymeric quaternary phosphonium
salts optionally substituted with fluorescent energy acceptors.
[0015] European Patent Application Serial No. 94108100.2 discloses
enhancement of enzymatically generated chemiluminescence from
1,2-dioxetanes in the presence of dicationic phosphonium salts. No
documents disclose the combination of an anionic fluorescer and a
dicationic enhancer for enhancing chemiluminescence from a
triggerable dioxetane. No example of enhancement of substituted
dioxetanes of the type of the present invention has been
reported.
[0016] c. Triggerable Dioxetanes with Improved Water Solubility.
The enzymatically triggerable dioxetanes are now undergoing
widespread use as substrates for marker enzymes in numerous
applications including immunoassays, gene expression studies,
Western blotting, Southern blotting, DNA sequencing and the
identification of nucleic acid segments in infectious agents.
Despite the growing use of these compounds, there are limitations
to there use in some assay methods. Triggerable dioxetanes whose
hydroxy dioxetane deprotected form are more water-soluble are
desirable. As shown in the structures below, it is especially
desirable that the hydroxy dioxetane formed by the
dephosphorylation of a phosphate dioxetane by alkaline phosphatase
be highly soluble in aqueous solutions and in compositions
containing chemiluminescence enhancing substances. Such dioxetanes
and compositions are of importance in certain solution assay
methods for detecting hydrolytic enzymes or conjugates of
hydrolytic enzymes. 1
[0017] As further background of the present invention and as more
fully explained in the examples below, it has been found that use
of conventional chemiluminescent dioxetane reagents in assays
performed on automated instrumentation based on the principles of
capsule chemistry analysis results in carryover of reagent from one
fluid segment to another, resulting in potentially inaccurate
measurements, erroneous results, and imprecision due to
non-reproducibility. Capsule chemistry analysis is described in
U.S. Pat. No. 5,399,497, which is fully incorporated by reference
herein. It has been postulated that, among other possible means for
overcoming the carryover problem, improved water solubility of the
hydroxy dioxetane, in particular, might eliminate or minimize
carryover of this luminescent reaction intermediate into adjacent
fluid segments of a capsule chemistry analysis system.
[0018] Dioxetane compounds in commercial use do not incorporate any
solubilizing groups which are appended to an alkoxy group. As such,
these dioxetanes are unsuitable for use in assay methods requiring
zero carryover. A suggestion of incorporating a solubilizing group
into a dioxetane has been made (U.S. Pat. No. 5,220,005). A
dioxetane with a carboxyl group substituted on an adamantyl
substituent is claimed, however, the preparation of such a
dioxetane is not described. Significantly, there is no disclosure
of what effect the addition of a carboxyl group had, if any, on
solubility and other properties of the dioxetane. There is no
teaching in the art of how many solubilizing groups are required or
what particular advantage might be conferred. Use of solubilizing
groups which interfere with the removal of the protecting group
which initiates light emission or which otherwise interfere with
light production would be of no value. Solubilizing groups which
would be removed during the luminescent reaction likewise would not
be useful.
[0019] In Applicant's co-pending '305 application it was
demonstrated that incorporation of one ionic solubilizing group was
insufficient to eliminate the carryover problem associated with the
hydroxy dioxetane produced by dephosphorylation of a phosphate
dioxetane. Phosphate dioxetanes whose hydroxy dioxetane product is
highly water soluble and enhanced compositions containing such
phosphate dioxetanes were provided to solve this problem. It was
subsequently discovered that dioxetanes which provided the solution
to the carryover problem, exhibited insufficient storage stability
at room temperature. Thus, no dioxetanes known in the art possessed
both high solubility of the hydroxy dioxetane and long term storage
stability.
[0020] Applicants' 08/748,107 application disclosed that
substitution of a hydrogen atom on the alkoxy group bearing two
ionic solubilizing groups with a fluorine atom or lower alkyl group
dramatically improves the storage stability of these dioxetanes.
Synthetic processes for preparing such dioxetanes were disclosed.
In the present application, improved processes are disclosed as
well as intermediates useful therein.
OBJECTS
[0021] It is an object of the present invention to provide
enzymatically triggered 1,2-dioxetanes with improved storage
stability whose hydroxy dioxetane product formed upon action of a
triggering enzyme is highly soluble in aqueous solution. It is a
second object of the present invention to provide 1,2-dioxetanes
substituted with two or more water-solubilizing ionic groups and
either a fluorine atom or lower alkyl group disposed on an alkoxy
substituent of the dioxetane structure which provide superior
storage stability. It is a further object of the present invention
to provide a composition comprising a fluorine or lower alkyl
group-substituted dioxetane with two or more ionic
water-solubilizing groups, a non-polymeric cationic enhancer and
optionally a fluorescer, for providing enhanced chemiluminescence.
It is a further object of the present invention to provide
dioxetanes and compositions which, when used in assays performed on
capsule chemistry analytical systems, eliminate the problem of
reagent carryover and have extended storage stability. It is yet
another object of the present invention to provide a synthetic
process and intermediates useful therein for the preparation of
1,2-dioxetanes substituted with two or more water-solubilizing
ionic groups and either a fluorine atom or lower alkyl group
disposed on an alkoxy substituent of the dioxetane structure.
IN THE DRAWINGS
[0022] FIG. 1 is a diagram of a capsule chemistry analysis system
in which carryover was determined to be a problem.
[0023] FIG. 2 is a profile of adjacent segments in the capsule
chemistry analysis system showing the observed luminescence
attributed to carryover as more fully described in the Examples
below.
[0024] FIG. 3 is a further profile of adjacent segments observed in
the experiments which are more fully described in the Examples
below and which established that the carryover was not optical in
nature.
[0025] FIG. 4 is a further profile of adjacent segments observed in
the experiments which are more fully described in the Examples
below and which established that the carryover was in fact chemical
in nature.
[0026] FIG. 5 is a graph depicting the relative rates of
decomposition at 25.degree. C. of a fluoro-substituted dioxetane, a
chloro-substituted dioxetane, a methyl-substituted dioxetane and a
reference dioxetane containing no halogen atoms.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0027] The present invention relates to dioxetanes with improved
storage stability and whose hydroxy dioxetane product formed upon
action of a triggering enzyme is highly soluble in aqueous solution
and which are triggerable by an enzyme to produce
chemiluminescence. Such triggerable dioxetanes eliminate or
minimize carryover of the luminescent hydroxy dioxetane into
adjacent segments in capsule chemistry analytical systems as
described in U.S. Pat. No. 5,399,497. Carryover can result from
solubilization, deposition or precipitation of light-emitting
material of low water solubility into the fluorocarbon oil which
serves as the isolating fluid in capsule chemistry systems. Reagent
carryover can lead to inaccurate measurements, erroneous results
and imprecision due to irreproducibility.
[0028] In the co-pending '305 application it was discovered that
dioxetane 1 below was particularly effective for the
chemiluminescent detection of alkaline phosphatase in aqueous
solution. 2
[0029] For comparison, dioxetane 2 which incorporates only one
ionizable group was prepared. This dioxetane did not eliminate the
carryover problem discussed above.
[0030] Use of dioxetane 1 in the test system described in U.S. Pat.
No. 5,399,497 led to complete elimination of the carryover problem.
However, it was subsequently discovered unexpectedly, that
solutions of dioxetane 1 in aqueous buffer displayed unsatisfactory
storage stability. Solutions containing 1 in alkaline buffer
displayed significant decomposition after storage at 25.degree. C.
for two weeks. Dioxetane 1, in fact, was found to be significantly
less stable than a related compound, Lumigen PPD, shown below which
has no ionic solubilizing groups on the alkoxy group. 3
[0031] As far as Applicants are aware, there is no teaching in the
art of dioxetane chemistry of the cause of the lower stability of
1. Means of structurally modifying 1 to improve its storage
stability while preserving its other beneficial properties were
disclosed in Applicants' co-pending application Ser. No. 08/748,107
which is fully incorporated herein by reference.
[0032] Definitions:
[0033] Storage stability is related to the rate of decomposition of
the dioxetane due to spontaneous reaction and is an intrinsic
property. Decomposition of triggerable dioxetanes can also be
induced by the presence of trace quantities of agents which
catalyze the removal of a protecting group and thus initiate the
decomposition. Storage stability of a dioxetane can be assessed by
measuring the quantity of dioxetane present in a known sample at
periodic intervals. The measurement can take any form known which
measures a property relatable to the quantity of dioxetane.
Techniques such as spectrophotometry, NMR spectrometry and the like
are exemplary. A convenient means is to measure the amount of light
produced by reacting a known quantity of dioxetane with a
triggering agent under a standard set of conditions. A decrease in
the amount or intensity of light emitted signals a loss of
dioxetane compound.
[0034] Storage stability refers to stability of the dioxetane in
both the pure form and as a solution or formulation in a buffer
solution. The formulation can also contain various additives for
increasing the amount of light produced or for improving the
activity of an enzymatic triggering agent. It is desirable that the
dioxetane in a formulation not undergo significant decomposition at
ambient temperature for a reasonable period of time. Compositions
to be used with automated analyzers should desirably be stable for
at least 1 week. Upon refrigeration at 0-5.degree. C., it is
desirable that no significant decomposition is observed for at
least 2-3 months. More desirably, compositions to be used with
automated analyzers should show not more than 2-3% change in the
observed indicator of storage stability in about 2-4 weeks.
[0035] The solution to the problem of storage stability was found
in dioxetanes having the formula I: 4
[0036] wherein Z is selected from the group consisting of a
fluorine atom and an alkyl group of 1-4 carbons and M is selected
from hydrogen, an alkali metal ion or a quaternary ammonium or
phosphonium ion, wherein R.sub.3 and R.sub.4 are each selected from
acyclic, cyclic and polycyclic organic groups which can optionally
be substituted with heteroatoms and which provide stability to the
dioxetane, wherein R.sub.2 is an aryl ring group selected from
phenyl and naphthyl groups which can include additional
substituents selected from halogens, alkyl, substituted alkyl,
alkoxy, substituted alkoxy, carbonyl, carboxyl, amino and
alkylamino groups and wherein X is a protecting group which can be
removed by an activating agent to form an oxyanion-substituted
dioxetane which decomposes and produces light and two
carbonyl-containing compounds, one of which is an
oxyanion-substituted ester compound containing two carboxylate
groups, as shown below. 5
[0037] When M is H it is recognized that the respective dioxetane
compound will preferably only be used under conditions of pH where
the carboxylic acid functions are ionized, i.e. pH.gtoreq.about 7.
Preferably M is an alkali metal ion, most preferably a sodium
ion.
[0038] The groups R.sub.3 and R.sub.4 in another embodiment are
combined together in a cyclic or polycyclic alkyl group R.sub.5
which is spiro-fused to the dioxetane ring, containing 6 to 30
carbon atoms which provides thermal stability and which can include
additional non-hydrogen substituents. 6
[0039] The group R.sub.5 is more preferably a polycyclic group,
preferably an adamantyl group or a substituted adamantyl group
having one or more substituent groups R.sub.6 selected from
halogens, alkyl, substituted alkyl, alkoxy, substituted alkoxy,
carbonyl, carboxyl, phenyl, substituted phenyl, amino and
alkylamino groups covalently bonded thereto. 7
[0040] In another preferred embodiment the group R.sub.2 is a
phenyl or naphthyl group. It is especially preferred that R.sub.2
is a phenyl group in which the OX group is oriented meta to the
dioxetane ring group as shown below. The phenyl ring may contain
additional ring substituents R.sub.7 independently selected from
halogens, alkyl, substituted alkyl, alkoxy, substituted alkoxy,
carbonyl, carboxyl, amino and alkylamino groups. Some exemplary
structures include by way of illustration: 8
[0041] Compounds of the latter two structural formulae in which
R.sub.6 is H or Cl and R.sub.7 is Cl as shown below are recognized
as further preferred compounds. 9
[0042] The nature of the OX group is dictated by the triggering
agent used in the assay for which it is to be used and may be
selected from hydroxyl, O.sup.-M.sup.+ wherein M is selected from
hydrogen, an alkali metal ion or a quaternary ammonium or
phosphonium ion, OOCR.sub.8 wherein R.sub.8 is selected from the
group consisting of alkyl and aryl groups containing 1 to 8 carbon
atoms and optionally containing heteroatoms, OPO.sub.3.sup.-2 salt,
OSO.sub.3.sup.- salt, .beta.-D-galactosidoxy and
.beta.-D-glucuronidyloxy groups. The OX group is preferably a
OPO.sub.3.sup.-2 salt group.
[0043] Dioxetanes of the present invention having the formula:
10
[0044] wherein R.sub.2, R.sub.3, R.sub.4, M and Z are as described
above can be prepared using methods described in Applicants'
co-pending application Ser. No. 08/748,107 and other methods known
in the art of dioxetane chemistry. For example, a ketone and ester
having the formulas below wherein RG is a replaceable atom or group
and X' is a replaceable atom or group such as a hydrogen or an
alkyl group or a trialkylsilyl group can be coupled by a low-valent
titanium reagent to form an intermediate vinyl ether. Removable
groups include leaving groups such as halogen atoms selected from
Cl, Br and I, sulfates, sulfonates such as tosylate, mesylate and
triflate, quaternary ammonium groups, and azide. 11
[0045] The intermediate vinyl ether is converted in a process of
one or more steps to a precursor vinyl ether phosphate salt. It may
be desired for synthetic convenience to replace one removable group
with another removable group. The group RG is replaced by a
CZ(COOM).sub.2 fragment by reaction with a Z-substituted malonate
ester and later saponification of the ester groups. The group X' is
converted to the group X in the case where X and X' are not
identical by removing X' and reacting with a reagent which adds the
X group or a protected form of the X group. For example when X' is
H and X is PO.sub.3Na.sub.2, treatment with base to deprotonate
followed by reaction with a phosphorylating agent produces a
phosphate triester-protected vinyl ether which is converted to the
phosphate salt by hydrolysis of the triester to the disodium salt.
In this multi-step process, two or more operations may occur in the
same process step, for example hydrolysis of carboxylic esters and
phosphate esters can be effected in the same step. 12
[0046] The precursor vinyl ether phosphate salt is directly
converted to the dioxetane by known reactions including, for
example, addition of singlet oxygen generated by dye sensitization.
13
[0047] Each of these processes is exemplified by way of
illustration in the specific examples below. In particular, Scheme
1 depicts schematically a synthetic pathway used to prepare
dioxetanes 3-5 according to the steps described above as disclosed
and embodied in the aforementioned 748,107 application. 14
[0048] A preferred embodiment of the present invention concerns a
process for preparing a dioxetane salt compound of the formula IV:
15
[0049] having increased storage stability wherein R.sub.3 and
R.sub.4 are each selected from the group consisting of acyclic,
cyclic and polycyclic organic groups which can optionally be
substituted with heteroatoms and which can optionally be joined
together to form a cyclic or polycyclic ring group spiro-fused to
the dioxetane ring, wherein R.sub.2 is an aryl ring group selected
from the group consisting of phenyl and naphthyl groups which can
include additional substituents, wherein Z is selected from the
group consisting of halogen atoms and alkyl groups of 1-4 carbons
and M is selected from hydrogen, an alkali metal ion or a
quaternary ammonium or phosphonium ion comprising the steps of:
[0050] a) reacting a first alkene compound having the formula:
16
[0051] wherein RG is a removable group with a Z-substituted
malonate ester and a base to produce a malonate-substituted alkene
compound having the formula: 17
[0052] wherein R' is an alkyl group of 1-4 carbons;
[0053] b) reacting the malonate-substituted alkene with a
phosphorylating reagent having the formula WP(O)Y.sub.2 wherein W
and Y are each halogen atoms, to form a phosphorylated alkene
compound having the formula: 18
[0054] c) reacting the phosphorylated alkene compound with a
hydroxyl compound of the formula Y'--OH, wherein Y' is selected
from substituted or unsubstituted alkyl groups to form a second
phosphorylated alkene compound having the formula: 19
[0055] and
[0056] d) hydrolyzing the second phosphorylated alkene compound in
an aqueous solvent with a base of the formula M--Q wherein Q is a
basic anion to form an alkene salt compound having the formula:
20
[0057] and
[0058] e) photooxidizing the alkene salt compound by irradiating a
sensitizer in the presence of oxygen and the alkene salt compound
in aqueous solution to form the dioxetane salt compound.
[0059] It is more preferred that this process is used to prepare a
dioxetane in which R.sub.3 and R.sub.4 are combined together to
form a cyclic or polycyclic ring group R.sub.5 spiro-fused to the
dioxetane ring and the dioxetane salt compound has the formula:
21
[0060] In other preferred processes, the group R.sub.2 is a
meta-phenyl group, Z is a halogen or an alkyl group having 1-4
carbons atoms, more preferably Z is F or CH.sub.3, and M is an
alkali metal ion, more preferably M is Na.
[0061] It has now been discovered that compounds of formula IV
22
[0062] having increased storage stability wherein R.sub.3 and
R.sub.4 are each selected from the group consisting of acyclic,
cyclic and polycyclic organic groups which can optionally be
substituted with heteroatoms and which can optionally be joined
together to form a cyclic or polycyclic ring group spiro-fused to
the dioxetane ring, wherein R.sub.2 is an aryl ring group selected
from the group consisting of phenyl and naphthyl groups which can
include additional substituents, wherein Z is selected from the
group consisting of halogen atoms and alkyl groups of 1-4
carbons,and M is selected from hydrogen, an alkali metal ion or a
quaternary ammonium or phosphonium ion can be advantageously
prepared by an improved process comprising the steps of:
[0063] a) reacting a first alkene compound having the formula:
23
[0064] wherein RG is a removable group with a Z-substituted
malonate ester and a base to produce a malonate-substituted alkene
compound having the formula: 24
[0065] wherein R' is an alkyl group of 1-4 carbons;
[0066] b) photooxygenating the malonate-substituted alkene compound
by irradiating a sensitizer in the presence of oxygen and the
malonate-substituted alkene compound to form a malonate-substituted
dioxetane having the formula: 25
[0067] c) reacting the malonate-substituted dioxetane with a
phosphorylating reagent having the formula WP(O)Y.sub.2 wherein W
is selected from halogens and Y is selected from halogen atoms,
substituted or unsubstituted alkoxy, aryloxy, aralkyloxy and
trialkylsilyloxy groups to form a phosphorylated dioxetane compound
having the formula: 26
[0068] d) hydrolyzing the phosphorylated dioxetane in an aqueous
solvent with a base of the formula M--Q wherein Q is a basic anion
to form the dioxetane salt compound.
[0069] It is more preferred that this process is used to prepare a
dioxetane in which R.sub.3 and R.sub.4 are combined together to
form a cyclic or polycyclic ring group R.sub.5 spiro-fused to the
dioxetane ring and which can contain additional substituents and
the dioxetane salt compound has the formula: 27
[0070] In other preferred embodiments, the process is used to
prepare a dioxetane in which the group R.sub.2 is a meta-phenyl
group which can contain additional substituents, Z is a halogen or
an alkyl group having 1-4 carbons atoms, more preferably Z is F or
CH.sub.3, and M is an alkali metali ion, more preferably M is
Na.
[0071] The step of reacting the first alkene compound with the
Z-substituted malonate ester CHZ(COOR').sub.2 and a base to produce
a malonate-substituted alkene compound is generally performed in a
polar aprotic solvent such as DMSO, DMF, N,N-dimethylacetamide,
N-methylpyrollidone using a poorly nucleophilic base, preferably
sodium or potassium hydride. The reaction is preferably performed
at an elevated temperature to decrease reaction time, generally
between 50 and 150.degree. C., more usually between 80 and
120.degree. C. Removable groups include leaving groups such as
halogen atoms selected from Cl, Br and I, sulfates, sulfonates such
as tosylate, mesylate and triflate, quaternary ammonium groups, and
azide.
[0072] In the improved process described herein, the
photooxygneation step is performed on the intermediate
malonate-substituted alkene instead of photooxygenating a phosphate
alkene as the final step of the overall process as described in the
748,107 application. In this step, the malonate-substituted alkene
compound bearing a phenol group is dissolved in an organic solvent
and irradiated in the presence of a sensitizer and oxygen to form a
malonate-substituted dioxetane. Irradiation of a sensitizer and
oxygen with light, usually visible light, generates singlet oxygen
which reacts with the vinyl ether-type double bond of the
malonate-substituted alkene. The sensitizer is can be dissolved in
the solvent or, preferably, immobilized on a polymeric particle as
is commonly known in the art. Sensitizers useful for generating
singlet oxygen include, without limitation, Rose Bengal, methylene
blue, eosin, tetraphenylporphyrin (TPP) metal complexes of TPP,
especially zinc and manganese and C.sub.60. Preferred organic
solvents include halocarbons such as CH.sub.2 Cl.sub.2, CHCl.sub.3
and CCl.sub.4, deuterated halocarbons, low molecular weight ketones
and their deuterated analogs, aliphatic and aromatic hydrocarbons
and their deuterated analogs. Most preferred is CH.sub.2Cl.sub.2.
Conducting the photooxygenation in an organic solvent
advantageously provides a reaction medium in which the lifetime of
singlet oxygen is maximized. This has the effect of significantly
decreasing reaction times and permitting the photooxygenation to
proceed more readily to completion. Product isolation is
facilitated as well, in most cases requiring only a simple
filtration of sensitizer and evaporation of solvent.
[0073] The step of reacting the malonate-substituted dioxetane with
a phosphorylating reagent having the formula WP(O)Y.sub.2 wherein W
is selected from halogens and Y is selected from halogen atoms,
substituted or unsubstituted alkyloxy groups and trialkylsilyloxy
groups to form a phosphorylated dioxetane compound is performed in
an organic solvent, preferably a halocarbon such as
CH.sub.2Cl.sub.2 or CHCl.sub.3 or an ether such as diethyl ether or
tetrahydrofuran (THF) in the presence of an amine base. Useful
amine bases include, without limitation, pyridine and
triethylamine. When Y is a substituted or unsubstituted alkyloxy
group, an aryloxy, aralkyloxy or trialkylsilyloxy group,
representative Y groups include, by way of example, alkoxy such as
OCH.sub.3, OCH.sub.2CH.sub.3, and the like, substituted alkoxy such
as cyanoethoxy (OCH.sub.2CH.sub.2CN) or trimethylsilylethoxy
(OCH.sub.2CH.sub.2Si(CH.sub- .3).sub.3), phenoxy, substituted
phenoxy, benzyloxy, trimethylsilyloxy and others as are generally
known to the skilled organic chemist. The two groups Y can also be
combined together as a single group such as ethylenedioxy as occurs
in the reagent 28
[0074] Preferred groups Y are cyanoethoxy groups. In a more
preferred embodiment, Y is a halogen, preferably Y and W are both
Cl.
[0075] The phosphorylation step is performed in solution at a
temperature in the range of about -78.degree. C. to about
25.degree. C. A temperature of about 0-5.degree. C. is particularly
convenient. The phosphorylating agent WP(O)Y.sub.2 is added in a
controlled fashion so as not to cause the reaction solution to
become hot. The phosphorylating reagent is preferably accompanied
by an amine base during the addition, preferably pyridine.
[0076] The hydrolysis or deprotection step is accomplished by
hydrolyzing the phosphorylated dioxetane in an aqueous solvent with
a base of the formula M--Q wherein Q is a basic anion in a quantity
sufficient to cause removal of the protecting groups Y and R' to
form the dioxetane salt compound. The solvent can comprise water,
an aqueous buffer or a mixture of water and one or more organic
solvents. Preferred orgnaic solvents are water-miscible solvents
such as methanol, ethanol, acetone and THF. Four equivalents of the
base are typically required, however for convenience, an excess can
be employed. Removal of the protecting groups can be performed
sequentially or simultaneously. Depending on the particular groups
Y and R' and the base it may or may not be possible to isolate
partially hydrolyzed intermediates.
[0077] The choice of the basic deprotecting agent will be
determined, in part, by the nature of the groups Y and R' to be
removed. The deprotecting agent must also not cause undesired side
reactions such as hydrolysis of the vinyl ether group in the
process where the vinyl ether phosphate salt is first prepared or
decomposition of the dioxetane ring group in the process where the
protected dioxetane is prepared. Preferred deprotecting agents
include organic and inorganic bases such as sodium hydroxide,
potassium hydroxide, potassium carbonate, sodium methoxide, sodium
ethoxide, potassium t-butoxide, ammonia, ammonium hydroxide and the
like. Other preferred deprotecting agents include nucleophilic
agents such as cyanide ion, fluoride ion.
[0078] In another embodiment, the step of reacting the
malonate-substituted dioxetane compound with the phosphorylating
reagent comprises the steps of:
[0079] a) reacting the malonate-substituted dioxetane compound with
a phosphorylating reagent having the formula WP(O)Y'.sub.2 wherein
W and Y' are each halogen atoms to form a dioxetane phosphoryl
halide compound having the formula 29
[0080] b) reacting the dioxetane phosphoryl halide compound with a
hydroxyl compound of the formula Y--OH, wherein Y is selected from
substituted or unsubstituted alkyl groups to form the
phosphorylated dioxetane compound.
[0081] The dioxetane phosphoryl halide compound is converted to the
phosphorylated dioxetane compound by reaction with at least two
equivalents of a hydroxyl compound Y--OH and preferably with an
excess. Exemplary compounds which can serve as the hydroxyl
compound Y--OH include, without limitation, lower alcohols such as
methanol and ethanol, substituted lower alcohols such as
3-hydroxypropionitrile (HOCH.sub.2CH.sub.2CN) and
2-trimethylsilylethanol, phenol, substituted phenols, benzyl
alcohol and others as are generally known.
[0082] In another aspect, the present invention relates to
synthetic intermediates used in the processs for preparing the
present dioxetanes. In particular the following novel alkene
intermediate compounds are useful. 30
[0083] Additionally the following novel dioxetane compounds are
useful as synthetic intermediates in the preparation of the present
dioxetane compounds. 31
[0084] An exemplary synthesis of a dioxetane of the present
invention by this improved process is shown in Scheme 2. 32
[0085] The starting material (precursor alkene) in the above
described synthetic processes having the formula: 33
[0086] wherein RG is a removable group can be prepared by methods
known in the art. In one method, Lhe vinyl ether function is
prepared by Ti-mediated coupling of a ketone R.sub.3R.sub.4C.dbd.O
and an ester HOR.sub.2COOCH.sub.2CH.sub.2--G as described in U.S.
Pat. Nos. 4,983,779 and 4,982,192 wherein G is a group which may be
identical with RG or may be a group which can be replaced by RG or
converted into RG. An exemplary synthetic process in which RG is an
iodine atom and G is a chlorine atom is presented hereinbelow. It
is further recognized that, for convenience, the ester component of
the coupling reaction may be used in protected form in which the
hydroxyl group is present in a masked form such as a silyl ether or
an alkyl ether. After the coupling reaction, the free hydroxyl
group is then liberated using standard synthetic means.
[0087] It has further been discovered by Applicants that these
precuror alkenes can also be prepared by a new process not
previously reported for the preparation of this type of vinyl
ether. While the foregoing Ti-mediated process requires the
preparation of individual ester compounds bearing the G or RG
group, adding additional complexity and cost, the new process
utilizes a common vinyl ether intermediate which can be prepared
from commercially available starting materials.
[0088] An example of a reaction for preparing the precursor alkene
by the new process is depicted below. A lower alkyl vinyl ether
compound, wherein lower alkyl, R.sub.9, here indicates a
C.sub.1-C.sub.4 straight or branched alkyl group, is reacted with a
catalytic amount of a mercury salt in the presence of at least one
mole equivalent of another alcohol R.sub.10--OH, e.g. one having
the formula HOCH.sub.2CH.sub.2G, to produce the desired precursor
alkene. 34
[0089] The conversion of unsubstituted vinyl ethers having the
formula CH.sub.2.dbd.CHOR.sub.a to other unsubstituted vinyl ethers
having the formula CH.sub.2.dbd.CHOR.sub.b is known and described,
e.g. in W. H. Watanabe and L. E. Conlon, J. Am. Chem.Soc. 79, 2828
(1957), the preparation by a mercury salt-catalyzed reaction of
trisubstituted alkenes used in the present processes has not been
reported to the best of Applicants' knowledge.
[0090] In this reaction process, R.sub.3 and R.sub.4 are each
selected from acyclic, cyclic and polycyclic organic groups which
can optionally be substituted with heteroatoms and which can
optionally be joined together to form a cyclic or polycyclic ring
group R.sub.5 spiro-fused to the dioxetane ring, R.sub.2 is an aryl
ring group selected from phenyl and naphthyl groups which can
include additional substituents. An example of the use of this
mercury-catalyzed reaction for the preparation of an alkene
precursor to a dioxetane of the invention is 35
[0091] wherein G is a chlorine atom. The mercury salt is any Hg(II)
salt which functions to catalyze the vinyl ether groups and is
preferably a salt of a weak acid such as acetate or
trifluoroacetate. The mercury salt is used in catalytic quantitity,
typically from 0.01 to 0.5 moles per mole of alkene, more typically
from 0.05 to 0.25. The alcohol component R.sub.10--OH can be any
alkanol, substituted alkanol, benzyl alcohol, unsaturated alcohol,
such as allyl alcohol. The alcohol is used in excess, at least two
moles per mole of alkene and preferably at least 5 moles per mole
of alkene. In a preferred process, the alcohol is used as the
reaction solvent. The reaction is typically but not necessarily
conducted above ambient temperature up to the boiling point of the
solvent. Preferable reaction temperatures are in the range of about
70-120.degree. C. Additional solvents for purposes of improving the
solubility of reactants or altering polarity or boiling point can
be used.
[0092] It is recognized that while the mercury-catalyzed vinyl
ether exchange reaction described above will find particular use in
the preparation of intermediates used for the further elaboration
to water soluble tri-substituted dioxetane of the present
invention, it is more generally applicable to the preparation of a
wide variety of alkene or vinyl ether compounds.
Specific Embodiments
[0093] A fluoro-substituted analog of dioxetane 1, identified as 3,
a chloro-substituted analog 4 and a methyl-substituted analog 5
have been prepared and their storage stability evaluated over
several weeks. Storage stability of a solution of 1 was measured
for comparison. All solutions were prepared with the same
composition, differing only in the identity of the dioxetane.
Stability was evaluated by chemiluminescent enzyme assay with a
fixed volume of test solution and fixed limiting amount of alkaline
phosphatase and measuring the plateau light intensity at 25.degree.
C. Unexpectedly, aqueous solutions containing dioxetanes 3 and 5
were substantially more stable than 1, while dioxetane 4 was not.
Solutions of dioxetanes 3 or 5 underwent essentially no
decomposition after four weeks at 25.degree. C. Surprisingly, the
storage stability of dioxetane 4 was actually worse than that of 1.
36
[0094] The reasons for this difference in the properties of these
four dioxetanes are not presently understood. It is particularly
significant that dioxetanes 3 and 4 should show such marked
difference in storage stability when they differ structurally only
by having different halogen substituents. Applicants are aware of
no teachings in the art of dioxetane chemistry to explain or
predict these results.
[0095] Furthermore, tests on dioxetane 3, showed that, like
dioxetane 1, it caused no carryover in the capsule chemistry assay
system. Dioxetanes such as 3 and 5 bearing a substituent containing
two carboxylate groups and either a fluorine atom or a lower alkyl
group and compositions containing such dioxetanes are therefore
superior to other known dioxetanes and compositions for use in
capsule chemistry analysis systems.
[0096] In another aspect of the invention, compositions providing
enhanced chemiluminescence are provided. Enhanced compositions are
advantageous in assays requiring the highest analytical
sensitivity. Increasing the chemiluminescence efficiency of the
dioxetane decomposition reaction while maintaining or reducing
extraneous light emission from spontaneous dioxetane decomposition
is one manner in which sensitivity can be enhanced or improved.
[0097] The present invention, therefore, also relates to
compositions comprising a cationic enhancer and a stable
1,2-dioxetane as described above having increased storage stability
which can be triggered to generate chemiluminescence. Such
compositions for providing enhanced chemiluminescence comprise a
dioxetane as described above in an aqueous solution, and a
non-polymeric cationic enhancer substance which increases the
quantity of light produced by reacting the dioxetane with the
activating agent compared to the amount which is produced in the
absence of the enhancer. It is preferred that the enhancer
substance is a dicationic surfactant of the formula: 37
[0098] wherein each of A is independently selected from P and N
atoms and wherein Link is an organic linking group containing at
least two carbon atoms selected from the group consisting of
substituted and unsubstituted aryl, alkyl, alkenyl and alkynyl
groups and wherein Link may contain heteroatoms and wherein R is
selected from lower alkyl or aralkyl containing 1 to 20 carbon
atoms and wherein Y is an anion. It is especially preferred that
the enhancer substance is a dicationic surfactant having the
formula: 38
[0099] and wherein link is phenylene.
[0100] Compositions of the present invention for providing enhanced
chemiluminescence may optionally contain at least one fluorescer as
a supplementary enhancer. Fluorescers useful are those compounds
which are capable of increasing the quantity of light produced
through energy transfer. Anionic fluorescers are particularly
effective it is believed due to favorable electrostatic
interactions with the cationic enhancer. Particularly preferred
fluorescers are anionic compounds and include, without limitation,
pyranine and fluorescein.
[0101] In order to more fully describe the various aspects of the
present invention, the following non-limiting examples describing
particular embodiments are presented for purposes of illustration
of the invention.
EXAMPLES
Example 1
Preparation of Dioxetane 1
[0102] The dioxetane
[4-(3,3-biscarboxy)propoxy)-4-(3-phosphoryloxyphenyl)-
]spiro[1,2-dioxetane-3,2'-tricyclo[3.3.1.1.sup.3,7]decane],
tetrasodium salt was prepared by the sequence of reactions
described in Applicants' U.S. Pat. No. 5,631,167. The synthesis up
to the intermediate alkene
[(3-hydroxyphenyl)-(2-iodoethoxy)-methylene]tricyclo[3.3.1.1.sup.3,7
]decane was conducted essentially as described in U.S. Pat. Nos.
5,013,827 and 5,068,339. 39
Example 2
Preparation of Dioxetane 2
[0103] The dioxetane
[4-(3-carboxypropoxy)-4-(3-phosphoryloxyphenyl)]spiro-
[1,2-dioxetane-3,2'-tricyclo[3.3.1.1.sup.3,7 ]-decane] (2) was
prepared by the sequence of reactions described in Applicants' U.S.
Pat. No. 5,631,167. The synthesis up to the intermediate alkene
[(3-carboxypropoxy)-(3-hydroxyphenyl)methylene]-tricyclo[3.3.1.1.sup.3,7]-
decane was conducted essentially as described. in U.S. Pat. Nos.
5,013,827 and 5,068,339. 40
Example 3
Preparation of Dioxetane 3
[0104] This dioxetane was prepared by the sequence of reactions
described below. The synthesis up to the intermediate alkene
[(3-hydroxyphenyl)-(2-iodoethoxy)methylene]tricyclo[3.3.1.1.sup.3,7]decan-
e was conducted as described in Example 1. 41
[0105] (a) Synthesis of
[((3,3-biscarboethoxy)-3-fluoropropoxy)-(3-hydroxy-
phenyl)methylenetricyclo[3.3.1.1.sup.3,7]decane. Sodium hydride (75
mg of a 60% dispersion in oil) was washed free of oil with hexane,
dried under vacuum and added to 4 mL of anhydrous DMSO. Diethyl
fluoromalonate (0.3 g) was added and the suspension stirred under
Ar for 15 min. A solution of the iodoethoxy alkene (0.5 g) in 5 mL
of anhydrous DMSO was added to the reaction mixture. The reaction
was heated to 100.degree. C. and stirred for 2 h. After cooling,
the mixture was diluted with 30 mL of ethyl acetate. The ethyl
acetate solution was extracted 3-4 times with water, dried and
evaporated. The crude material was chromatographed using 5-20%
ethyl acetate in hexane. The desired compound (0.25 g) was obtained
in 45% yield: .sup.1H NMR (CDCl.sub.3) .delta. 1.28 (t, 6H),
1.66-1.95 (m, 12H), 2.45 (t, 1H), 2.52 (t, 1H), 2.67(br s, 1H),
3.20 (br s, 1H), 3.52(t, 2H), 4.23-4.30 (q, 4H), 6.74-7.22 (m, 4H).
42
[0106] (b) Synthesis of
[((3,3-biscarboethoxy)-3-fluoropropoxy-(3-(bis-(2--
cyanoethyl)phosphoryloxy)phenyl)methylene]tricyclo[3.3.1.1.sup.3,7]decane.
A flask containing 10 mL of CH.sub.2Cl.sub.2 under a layer of argon
was cooled in an ice bath. Pyridine (1.71 mL) was added followed by
slow addition of PoCl.sub.3 (0.61 mL) and stirring continued for 15
min. A solution of the alkene (0.972 g) from step (a) in 10 mL of
CH.sub.2Cl.sub.2 was added dropwise. The ice bath was removed and
the solution stirred for 2.5 h. To this solution was added 1.71 mL
of pyridine and 1.44 mL of 2-cyanoethanol. The reaction mixture was
stirred for 12-15 h resulting in formation of a white precipitate.
The mixture was diluted with CH.sub.2Cl.sub.2 and washed with
4.times.50 mL of water. The CH.sub.2Cl.sub.2 extract was dried and
evaporated. The crude product was purified by chromatography using
75% ethyl acetate in hexane. A total of 1.2 g of an oil (88%) was
obtained: .sup.1H NMR (CDCl.sub.3) .delta. 1.29 (s, 6H), 1.79-1.97
(m, 12H), 2.46-2.53 (2t, 2H), 2.63 (br s, 1H), 2.83 (t, 4H), 3.20
(br s, 1H), 3.50 (t, 2H), 4.24-4.31 (q, 4H), 4.35-4.51 (m, 4H),
7.13-7.36 (m, 4H); .sup.31P NMR (CDCl.sub.3) .delta.-9.49 (p).
43
[0107] (c) Synthesis of
[(3,3-biscarboxy-3-fluoropropoxy)-(3-phosphoryloxy-
phenyl)methylene]tricyclo[3.3.1.1.sup.3,7]-decane, tetrasodium
salt. The alkene (1.2 g) from step (b) was dissolved in 20 mL of
acetone. A solution of 297 mg of sodium hydroxide in 4 mL of water
was added. The solution was stirred over night during which time a
precipitate formed. The liquid was decanted and the solid washed
with 10.times.5 mL of acetone. After drying under vacuum, a white
solid (1.0 g) was obtained: .sup.1H NMR (D.sub.2O) .delta.
1.75-1.89 (m, 12H), 2.29 (t, 2H), 2.37 (t, 2H), 2.57 (br s, 1H),
3.12 (br s, 1H), 3.56 (t, 2H), 6.99-7.30 (m, 4H) .sup.31P NMR
(D.sub.2O) .delta.0.69 (s). 44
[0108] (d) Synthesis of
[4-(3,3-biscarboxy)-3-fluoropropoxy)-4-(3-phosphor-
yloxyphenyl)]spiro[1,2-dioxetane-3,2'-tricyclo[3.3.1.1.sup.3,7]decane],
tetrasodium salt (3). The alkene (348.6 mg) from step (c) was
dissolved in 10 mL of D.sub.2O. Polymer-bound Rose Bengal (500 mg)
was suspended in 10 mL of p-dioxane and added to the water
solution. The reaction mixture was cooled to 5-8.degree. C., oxygen
bubbling was started and the mixture irradiated with a sodium lamp
through a 5 mil KAPTON filter. After a total of 2.5 h, the polymer
beads were filtered off and the solution was evaporated to dryness
producing a white solid (3). .sup.1H NMR (D.sub.2O) .delta.
0.93-1.79 (m, 12H), 2.19 (br s, 1H), 2.41-2.49 (m, 2H), 2.97 (br s,
1H), 3.40-3.49 (m, 2H), 7.19-7.42 (m, 4H); .sup.31P NMR (D.sub.2O)
.delta. 0.575 (s).
Example 4
Preparation of Dioxetane 4
[0109] This dioxetane was prepared by the sequence of reactions
described below. The synthesis up to the intermediate alkene
[(3-hydroxyphenyl)-(3,3-biscarboethoxy)propoxymethylene]tricyclo[3.3.1.1.-
sup.3,7]decane was conducted as described in Example 1. 45
[0110] (a) Synthesis of
[((3,3-biscarboethoxy)-3-chloropropoxy)-(3-hydroxy-
phenyl)methylenetricyclo[3.3.1.1.sup.3,7]decane. A solution of
(3,3-biscarboethoxypropoxy)-(3-hydroxyphenyl)methylenetricyclo[3.3.1.1.su-
p.3,7]decane (1.2 g) in 10 mL of dry THF was added to a 2.4 eq. of
LDA in 25-30 mL of dry THF at -78.degree. C. under argon. The
reaction was stirred for 30 min at -78.degree. C. and treated with
a solution of N-chlorosuccinimide (0.58 g) in 15 mL of dry THF. The
reaction was allowed to warm to room temperature over an hour and
stirred for an additional hour. The THF was removed in vacuo and
the residue dissolved in 100 mL of ethyl acetate. The organic
solution was washed with water, dried and evaporated. The crude
material was separated by column chromatography. .sup.1H NMR
(CDCl.sub.3) .delta. 1.23 (t, 6H), 1.7-2.00 (m, 12H), 2.57 (t, 2H),
2.65 (br s, 1H), 3.2 (br s, 1H), 3.56 (t, 2H), 4.22 (q, 4H),
6.65-7.25 (m, 4H). 46
[0111] (b) Synthesis of [((3,3-biscarboethoxy)-3-chloropropoxy-(3-(
bis-(2-cyanoethyl)phosphoryloxy)phenyl)methylene]tricyclo[3.3.1.1.sup.3,7-
]decane. A flask containing 25 mL of CH.sub.2Cl.sub.2 under a layer
of argon was cooled in an ice bath. Pyridine (1.5 g) was added
followed by slow addition of POCl.sub.3 (1.82 g) and stirring
continued for 15 min. A solution of the alkene (1.5 g) from step
(a) and 1.5 g of pyridine in 25 mL of CH.sub.2Cl.sub.2 was added
dropwise. The ice bath was then removed and the solution stirred
for 1 h. The solution was again cooled with an ice bath and treated
sequentially with 3.0 g of pyridine and 2.8 g of 2-cyanoethanol.
The reaction mixture was stirred for 12-15 h resulting in formation
of a white precipitate. The mixture was diluted with
CH.sub.2Cl.sub.2 and washed with water. The CH.sub.2Cl.sub.2
extract was dried and evaporated. The crude product was purified by
chromatography using 50% ethyl acetate in hexane. A total of 1.4 g
of product was obtained an oil: .sup.1H NMR (CDCl.sub.3) .delta.
1.278 (t, 6H), 1.80-1.97 (m, 12H), 2.565 (t, 2H), 2.63 (br s, 1H),
2.826 (t, 4H), 3.20 (br s, 1H), 3.556 (t, 2H), 4.271 (q, 4H),
4.40-4.47 (m, 4H), 7.15-7.36 (m, 4H). 47
[0112] (c) Synthesis of
[(3,3-biscarboxy-3-chloropropoxy)-(3-phosphoryloxy-
phenyl)methylene]tricyclo[3.3.1.1.sup.3,7]decane, tetrasodium salt.
The alkene (0.9 g) from step (b) was dissolved in 25 mL of acetone.
A solution of 0.22 g of sodium hydroxide in 3 mL of water was
added. The solution was stirred over night during which time a
precipitate formed. The liquid was decanted and the solid
triturated with acetone. The white solid was filtered, washed
further with acetone and dried under vacuum: .sup.1H NMR (D.sub.2O)
.delta. 1.77-1.92 (m, 12H), 2.422 (t, 2H), 2.59 (br s, 1H), 3.15
(br s, 1H), 3.635 (t, 2H), 7.02-7.33 (m, 4H). 48
[0113] (d) Synthesis of
[4-(3,3-biscarboxy)-3-chloropropoxy)-4-(3-phosphor-
yloxyphenyl)]spiro[1,2-dioxetane-3,2'-tricyclo[3.3.1.1.sup.3,7]decane],
tetrasodium salt (4). The alkene (35 mg) from step (c) was
dissolved in 1.0 mL of D.sub.2O. Polymer-bound Rose Bengal (500 mg)
was soaked in 1.0 mL of p-dioxane-d.sub.8 for 5 min and then added
to the water solution. The reaction mixture was cooled to 0.degree.
C., oxygen bubbling was started and the mixture irradiated with a
sodium lamp through a 5 mil KAPTON filter for 45 min to produce 4
as determined by NMR. The mixture was filtered and the
solution-diluted in buffer for enzyme assay: .sup.1H NMR (D.sub.2O)
.delta. 1.05-1.96 (m, 12H), 2.19 (br s, 1H), 2.60-2.62 (m, 2H),
3.07 (br s, 1H), 3.56-3.58 (m, 2H), 7.25-7.44 (m, 4H).
Example 5
Preparation of Dioxetane 5
[0114] This dioxetane was prepared by the sequence of reactions
described below. The synthesis up to the intermediate alkene
[(3-hydroxyphenyl)-(2-iodoethoxy)methylene]tricyclo[3.3.1.1.sup.3,7]decan-
e was conducted as described in Example 1. 49
[0115] (a) Synthesis of
[((3,3-biscarboethoxybutoxy)-(3-hydroxyphenyl)meth-
ylenetricyclo[3.3.1.1.sup.3,7]decane. Sodium hydride (0.866 g of a
60% dispersion in oil) was washed free of oil with hexane, dried
under vacuum and added to 15 mL of anhydrous DMSO. Diethyl
methylmalonate (2.4 g) was added and the suspension stirred under
Ar for 15 min. A solution of the iodoethoxy alkene (2.8 g) in 15 mL
of anhydrous DMSO was added to the reaction mixture. The reaction
was heated to 100.degree. C. and stirred for 2 h. After cooling,
the mixture was diluted with 30 mL of ethyl acetate. The ethyl
acetate solution was extracted 3-4 times with water, dried and
evaporated. The crude material was chromatographed using 5-20%
ethyl acetate in hexane. The desired compound (0.80 g) was obtained
in 25% yield: .sup.1H NMR (CDCl.sub.3) .delta. 1.208 (t, 6H), 1.347
(s, 3H), 1.76-1.96 (m, 12H), 2.20 (t, 2H), 2.66 (br s, 1H), 3.20
(br s, 1H), 3.41 (t, 2H), 4.09-4.17 (q, 4H), 6.78-7.26 (m, 4H).
50
[0116] (b) Synthesis of
[((3,3-biscarboethoxybutoxy-3-(bis-(2-cyanoethyl)p-
hosphoryloxy)phenyl)methylene]tricyclo[3.3.1.1.sup.3,7]decane. A
flask containing 15 mL of CH.sub.2Cl.sub.2 under a layer of argon
was cooled in an ice bath. Pyridine (1.38 g) was added followed by
slow addition of POCl.sub.3 (0.8 g) and stirring continued for 15
min. A solution of the alkene (0.8 g) from step (a) in 15 mL of
CH.sub.2Cl.sub.2 was added dropwise. The ice bath was removed and
the solution stirred for 1 h. To this solution was added 1.38 g of
pyridine and 1.24 g of 2-cyanoethanol. The reaction mixture was
stirred for 12-15 h resulting in formation of a white precipitate.
The mixture was diluted with CH.sub.2Cl.sub.2 and washed with
4.times.50 mL of water. The CH.sub.2Cl.sub.2 extract was dried and
evaporated. The crude product was purified by chromatography using
75% ethyl acetate in hexane. A total of 0.55 g of an oil (50%) was
obtained: .sup.1H NMR (CDCl.sub.3) .delta. 1.208 (t, 6H), 1.34 (s,
3H), 1.78-1.97 (m, 12H), 2.18 (t, 2H), 2.61 (br s, 1H), 2.81 (t,
4H), 3.21 (br s, 1H), 3.41 (t, 2H), 4.09-4.16 (q, 4H), 4.37-4.46
(m, 4H), 7.14-7.34 (m, 4H). 51
[0117] (c) Synthesis of
[(3,3-biscarboxybutoxy)-(3-phosphoryloxyphenyl)met-
hylene]tricyclo[3.3.1.1.sup.3,7]decane, tetrasodium salt. The
alkene (0.47 g) from step (b) was dissolved in 14 mL of acetone. A
solution of 0.117 g of NaOH in 1.5 mL of water was added. The
solution was stirred over night during which time a precipitate
formed. The liquid was decanted and the solid washed with
10.times.5 mL of acetone. After drying under vacuum, a white solid
(0.383 g, 92%) was obtained: .sup.1H NMR (D.sub.2O) .delta. 1.09
(s, 3H), 1.75-1.90 (m, 12H)), 2.00 (t, 2H), 2.57 (br s, 1H), 3.13
(br s, 1H), 3.47 (t, 2H), 7.01-7.29 (m, 4H). 52
[0118] (d) Synthesis of [4-(3,3-biscarboxybutoxy)-4-
(3-phosphoryloxyphenyl)]spiro[1,2-dioxetane-3,2'-tricyclo[3.3.1.1.sup.3,7-
]decane], tetrasodium salt (5). The alkene (65 mg) from step (c)
;was dissolved in 3 mL of D.sub.2O. Polymer-bound Rose Bengal (35
mg) was suspended in 3 mL of p-dioxane and added to the water
solution. The reaction mixture was cooled to 5-8.degree. C., oxygen
bubbling was started and the mixture irradiated with a sodium lamp
through a 5 mul KAPTON filter for 1 h to produce (5). The polymer
heads were filtered off and the solution used for preparing stock
solutions for testing. .sup.1 H NMR (D.sub.2O) .delta. 0.92-1.33
(m, 5H), 1.38-2.21 (m, 13H), 2.92 (br s, 1H), 3.19-3.32 (m, 2H),
7.14-7.73 (m, 4H).
Example 6
Alternative Preparation of Dioxetane 3
[0119] The dioxetane was prepared by the sequence of reactions
described below using
[(3-hydroxyphenyl)methoxymethylenetricyclo[3.3.1.1.sup.3,7]de- cane
as starting material. This compound can be prepared as described in
U.S. Pat. No. 4,983,779.
[0120] (a) The alkene
[(3-hydroxyphenyl)methoxymethylenetricyclo[3.3.1.1.s- up.3,7]decane
(12 g) was added to 100 mL of 2-chloroethanol and stirred. A
catalytic amount of Hg(OAc).sub.2 (2.8 g) was then added to the
mixture under an argon atmosphere. The reaction was stirred for 5 h
at 110.degree. C. After cooling to room temperature, the
chloroethanol was remove under vacuum. The solid was dissolved in
EtOAc and washed with water. The EtOAc layer was dried over
Na.sub.2SO.sub.4 and evaporated to produce
[(3-hydroxyphenyl)-(2-chloroethoxy)methylene]tricyclo[3.3.1.1.sup-
.3,7]decane.
[0121] (b) Replacement of the chlorine atom in the above compound
with an iodine atom was conducted essentially as described in U.S.
Pat. Nos. 5,013,827 and 5,068,339.
[0122] (c) Synthesis of
[(3-hydroxyphenyl)-(3,3-biscarboethoxy)-3-fluoropr-
opoxymethylene]tricyclo[3.3.1.1.sup.3,7]decane from
[(3-hydroxyphenyl)-(2-iodoethoxy)methylene]tricyclo[3.3.1.1.sup.3,7]decan-
e is described in Example 3 above. 53
[0123] (d) The fluoromalonate alkene from step (c) (0.375 g) was
photooxygenated with ca. 1 mg of methylene blue in 15 mL of
CH.sub.2Cl.sub.2. After cooling the solution to -78.degree. C. with
O.sub.2 bubbling, the solution was irradiated with a sodium lamp
through a 5 mil KAPTON filter for 45 min and then allowed to warm
to room temperature. The CH.sub.2Cl.sub.2 was evaporated and the
residue chromatographed using from 0-5% EtAc in CH.sub.2Cl.sub.2 as
eluent to produce
[4-(3,3-biscarboethoxy-3-fluoropropoxy)-4-(3-hydroxyphenyl)]spiro-
[1,2-dioxetane-3,2'-tricyclo[3.3.1.1.sup.3,7]-decane]: .sup.1H NMR
(CDCl.sub.3) .delta. 0.97-1.02 (m, 1H), 1.21-1.33 (m, 7H),
1.45-1.91 (m, 10H), 2.23 (br s, 1H), 2.48-2.80 (m, 2H), 2.96 (br s,
1H), 3.35-3.44 (m, 1H), 3.65-3.75 (m, 1H), 4.21-4.40 (m, 4H),
6.85-7.40 (m, 4H). 54
[0124] (e) The dioxetane from the previous step was phosphorylated
by the following process. A solution of 2 mL of anhydrous pyridine
and 10 mL of CH.sub.2Cl.sub.2 under argon was cooled to 0.degree.
C. and a solution of 0.424 g of POCl.sub.3 in 10 mL of
CH.sub.2Cl.sub.2 was added dropwise. After 15 min, a solution of
0.424 g of the dioxetane in 10 mL of CH.sub.2Cl.sub.2 was added
dropwise. The solution was allowed to warm to room temperature and
stirred for 4 h. The solution was again cooled to 0.degree. C. and
a solution of 0.75 g of cyanoethanol in 10 mL of CH.sub.2Cl.sub.2
was added dropwise. This solution was allowed to warm to room
temperature as it was stirred for 2.5 h. After evaporating to
dryness, the residue was chromatographed using from 50-100% ethyl
acetate in hexanes as eluent. The solvents were then removed in
vacuo yielding a colorless oil. The dioxetane was then dissolved in
100 mL of CH.sub.2Cl.sub.2 and washed three times with type I
water. The organic layer was then dried over Na.sub.2SO.sub.4,
filtered, and evaporated to produce the phosphorylated dioxetane:
.sup.1H NMR (CDCl.sub.3) .delta. 0.90-0.95 (m, 1H), 1.24-1.33 (m,
7H), 1.46-2.20 (m, 11H), 2.50-2.86 (m, 6H), 2.96 (br s, 1H),
3.32-3.41 (m, 1H), 3.62-3.73 (m, 1H), 4.20-4.48 (m, 8H), 7.30-7.70
(m, 4H); .sup.31P (CDCl.sub.3) -9.53 (p). 55
[0125] (f) the alkyl groups were removed by reacting the dioxetane
from the previous step with 47.2 mg of NaOH in 1 mL of type I water
and 10 mL of acetone under argon over night. Solvent was decanted
from the oily residue which had formed. The oil was then washed
twice with 2 mL of acetone and then triturated with another 10 mL
of acetone to produce a powdery white solid. Solid dioxetane 3 was
collected by suction filtration and washed with another 20 mL of
acetone.
[0126] In an alternative procedure, the dioxetane product of step
(d) can be directly converted to dioxetane 3 by by the following
process. A solution of 2 mL of anhydrous pyridine and 10 mL of
CH.sub.2Cl.sub.2 under argon is cooled to 0.degree. C. and a
solution of 0.424 g of POCl.sub.3 in 10 mL of CH.sub.2Cl.sub.2 is
added dropwise. After 15 min, a solution of 0.424 g of the
dioxetane in 10 mL of CH.sub.2Cl.sub.2 is added dropwise. The
solution is allowed to warm to room temperature and stirred for 4
h. The phosphate salt is formed and the ester groups are hydrolyzed
by reacting the resulting dichlorophosphate dioxetane with 47.2 mg
of NaOH in 1 mL of type I water and 10 mL of acetone under argon
over night. The solvent is removed from the residue containing the
product. The product is then washed with acetone and, if needed,
triturated with acetone to produce a powdery white solid. Dioxetane
3 is collected by suction filtration.
Example 7
Discovery of Reagent Carryover Problem in Capsule Chemistry
Analysis System
[0127] The experiments described below were performed on a
prototype capsule chemistry analysis system essentially as
described by Kumar et al in U.S. Pat. No. 5,399,497, with the
detection system configured to measure light emission
(luminescence). The method and apparatus comprises feeding a stream
of fluid segments through a Teflon tube, where the tube has an
isolating layer of fluorocarbon oil on the inner surface. Sample
and reagents are aspirated into this tube, and the resulting liquid
segments are moved through the tube. Separation steps and washing
steps which are required by heterogeneous immunoassay methods were
facilitated by means of magnets, which transferred magnetic
particles from one aqueous segment to another. The detection system
was comprised of a photon counter and a fiber optic read head, in
which the fibers were radially arranged around the Teflon tube to
maximize the efficiency of light collection.
[0128] The TECHNICON IMMUNO 1.RTM. TSH method (Bayer Corporation,
Tarrytown, N.Y., USA) was used as a representative immunoassay
method for the testing of luminogenic reagents. The method
principle involved incubation of a specimen containing the antigen
TSH with a first reagent (R1), which contained a
fluorescein-labeled antibody, and simultaneously with a second
reagent (R2), which contained an antibody-alkaline phosphatase
(ALP) conjugate. Each antibody was specific for a different epitope
on the TSH antigen, so that formation of a "sandwich" was promoted
between these two antibodies and the TSH antigen. Magnetic
particles containing bound anti-fluorescein were used to capture
the sandwich, and the particles were subsequently washed to remove
unbound reagents. The particles were then exposed to the
luminogenic reagent, which contained a substrate for ALP, and
luminescence was measured.
[0129] The luminogenic R3 reagent was comprised of 0.2 mM CSPD
(disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo
[3.3.1.1.sup.3,7]decan}-4-yl)phenyl phosphate, (Tropix, Inc.,
Bedford, Mass., USA), 3 mM pyranine (hydroxypyrenesulfonic acid), 1
mM MgCl.sub.2, 1 M diethanolamine buffer (pH 10.0), 0.1% Triton
X-100 and 0.1% NaN.sub.3. The sequence of events on the capsule
chemistry analysis system is depicted in FIG. 1 of the drawings.
The fluid capsule or test package was comprised of six liquid
segments, each of which had a volume of 28 .mu.l. Magnetic
particles (1.4 .mu.l of the magnetic particle reagent used in the
TECHNICON IMMUNO 1 system were aspirated into the first segment
(MP), with the remainder of fluid being particle wash buffer (25 mM
Tris, pH 7.5, containing 0.2 M NaCl, 0.1% Triton X-100 and
preservative). R1 (10.4 .mu.l of serum-based solution containing
fluorescein-labeled antibody to TSH), R2 (10.4 .mu.l of serum-based
solution containing antibody to TSH conjugated with ALP) and S (7.2
.mu.l of serum sample) were aspirated into the second segment. The
next two segments (W1 and W2) were comprised of the same wash
buffer used above in the MP segment. The fifth segment was R3, of
the composition described above, with the key elements being the
luminogenic substrate and the luminescence enhancer. The sixth
segment was an inter-test buffer (same as the particle buffer
described above), which was used to isolate adjacent tests.
Magnetic transfers are depicted by the arrows in the FIG. 1. These
transfers were facilitated by one of two magnetic transfer
assemblies (M1 or M2). After an incubation of 13 minutes, during
which sandwich formation occurred, M1 transferred the magnetic
particles into the R1+R2+S segment to initiate capture. After an
additional period of 6 minutes, M2 transferred the particles into
the first wash segment. After an additional period of 12 seconds,
M2 transferred the particles into the second wash segment. After
another period of 12 seconds, M2 transferred the particles into the
R3 segment, and light emission from this segment was detected as
the stream of aqueous segments passed back and forth through the
luminometer readhead.
[0130] Since the Teflon tube is transparent to light, a problem
with light piping (or "optical carryover") was expected.
Specifically, some of the photons emitted from the R3 segment of an
adjacent test could enter the Teflon material, propagate down the
length of the tube and be scattered into the detector during the
measurement of the signal of the test of interest. However, while a
signal was detected in the adjacent tests, it did not occur in the
expected manner. Instead of declining rapidly with distance from
test N, peaks of light output were observed centered around the R3
segments of the adjacent test packages, as shown in FIG. 2 of the
drawings. In FIG. 2, test N produced a high level of luminescence,
approximately 7.5 million counts per seconds (cps). Tests N-1 and
N-2 were aspirated into the tube before test N and preceded this
test through the luminometer, and tests N+1 and N+2 followed after
test N. The analysis system recorded photons counted for each
individual air and liquid segment in the stream. The profile in
FIG. 2 represents the average of 10 replicate panels of 5 tests
each corrected for background luminescence signal produced in the
absence of ALP. The reagent blank values subtracted from each data
point were an average obtained from 10 replicate panels of 5 tests
each. The magnitude of the carryover signal was computed by
dividing the peak cps in each adjacent test by the peak cps in test
N, expressed in parts per million (ppm).
[0131] Another possible explanation for this behavior was physical
carryover of ALP from test N into the neighboring tests in an
unintended manner. This could happen, for example, if the tube
contained particulate materials deposited on the walls, which could
disrupt the smooth motion of the liquid segments through the tube.
However, placement of 10 mM inorganic phosphate in the R3 segments
of the adjacent tests had no effect on the magnitude of the signals
in the adjacent tests. Since this amount of phosphate would have
inhibited ALP by at least 90% under these test conditions, the
possibility of physical carryover was ruled out.
[0132] To further rule out optical carryover, the fluorescent
enhancer pyranine was omitted from test N only, but present in the
adjacent tests. As a result, the magnitude of the signal in test N
was lower by a factor of approximately 10. However, as shown in
FIG. 3 of the drawings, the height of the peaks in the adjacent
tests did not change significantly. The fact that the carryover
signal did not change in the adjacent tests proportionately clearly
demonstrated that this carryover was not optical.
[0133] An additional and unexpected type of carryover was the cause
of the carryover problem. It was found that the hydroxy dioxetane
intermediate was sufficiently soluble in the fluorocarbon oil used
to coat the inner wall of the Teflon tube, such that the carryover
was due to transfer of dissolved hydroxy dioxetane intermediate via
the oil into the R3 segments of the neighboring tests. This process
was tested by changing the buffer of the R3 segments in the
adjacent tests from 1 M DEA at pH 10 to 1 M Tris at pH 7. At pH 7,
dissolved hydroxy dioxetane intermediate in these R3 segments is
stable and does not emit light. As shown in FIG. 4 of the drawings,
this change in pH resulted in the complete elimination of the side
bands of luminescence. The residual minor carryover in the N+1 and
N-1 tests was due to the anticipated optical carryover. These
results verified that the source of light emission in the peaks in
the neighboring tests was "chemical carryover" of the hydroxy
dioxetane derived from CSPD into the R3 segments of adjacent
tests.
Example 8
Elimination of Observed Chemical Carryover with Dicarboxylic
Acid-Substituted Dioxetane 1
[0134] Table 1 shows the effect of using three other dioxetanes on
the chemical carryover of the reaction intermediate. LUMIGEN PPD
[4-(methoxy)-4-(3-phosphoryloxyphenyl)]spiro-[1,2-dioxetane-3,2'-tricyclo-
[3.3.1.1.sup.3,7]-decane], (Lumigen, Inc., Southfield, Mich., USA),
dioxetane 2, a monocarboxylic acid derivative and dioxetane 1, a
dicarboxylic acid derivative were each used in test formulations at
the same concentration. The ppm column is the signal for the N+1
test, which represents worst case behavior. The carryover of the
unmodified parent compound, PPD, was found to be more than twice as
high as that observed with CSPD. Surprisingly, the monocarboxylic
acid derivative, dioxetane 3, showed a reduction of only 84% in the
magnitude of the chemical carryover. This indicated that a single
charged group was insufficient to completely prevent solubilization
of the reaction intermediate in the fluorocarbon oil. However, the
dicarboxylic acid derivative was 100% effective, indicating that
two charged groups were fully adequate to achieve the desired
behavior.
1TABLE 1 Reduction of Chemical Carryover Compound ppm % Reduction
LUMIGEN PPD 1640 Dioxetane 2 260 84 Dioxetane 1 0 100
Example 9
The Role of Enhancers
[0135] As part of the optimization of a reagent based on dioxetane
1, a number of enhancer materials was examined. At pH 9.6, Enhancer
A (1-trioctylphosphoniummethyl-4-tributylphosphoniummethylbenzene
dichloride) increased the luminescent signal by a factor of 6.2,
and Enhancer B (poly(vinylbenzyltributylphosphonium chloride))
increased the signal by a factor of 19.7. At pH 10.0, Enhancer A
increased the signal by a factor of 4.8, and Enhancer B increased
the signal by a factor of 18.9.
[0136] Despite the fact that Enhancer B achieved higher light
intensities, Enhancer A was preferred for use on the analysis
system since it is a low molecular weight monomeric compound.
Polymeric compounds, especially if they are polycationic, interact
with serum components, causing precipitation, which would pose
significant problems for the operation of the analysis system.
[0137] Both fluorescein and pyranine were found to be effective as
supplementary fluorescers in combination with Enhancer A. Alone,
these fluorescers must be used at relatively high concentrations (3
mM) in order to achieve an enhancement of about ten-fold. However,
in combination with Enhancer A, a synergistic effect was observed,
in which a comparable enhancement resulted at 100-fold lower
concentrations of fluorescer than needed in the absence of the
enhancer. Tables 2 and 3 show the extent of enhancement by pyranine
and fluorescein, respectively, in the presence of 1 mg/mL of
Enhancer A.
2TABLE 2 Enhancement by Pyranine with Enhancer A [Pyranine] (mM)
Enhancement Factor 0.01 3.7 0.02 7.3 0.03 9.8 0.04 12.2 0.05
13.7
[0138]
3TABLE 3 Enhancement by Fluorescein with Enhancer A [Fluorescein]
(mM) Enhancement Factor 0.01 2.6 0.02 4.0 0.05 7.1 0.10 8.7
Example 10
Optimized Formulation for Capsule Chemistry Analysis System
[0139] The above described observations have led to the development
of an optimized formulation for the capsule chemistry analysis
system. This formulation is comprised of 0.1-1 mM dioxetane 1,
0-0.05 mM pyranine, 0.1-5 mg/mL Enhancer A, 0-1 mM Mg.sup.+2 0.1-1
M 2-amino-2-methyl-1-propa- nol (pH 10.0) and 0.01-1% Triton X-100.
Use of this formulation results in complete elimination of the
chemical carryover problem and enhanced performance.
Example 11
Stability of 1, 3, 4 and 5 Measured by Enzyme Assay.
[0140] Formulations comprising 0.1 mg/mL Enhancer A, 0.88 mM
Mg.sup.+2 0.2 M 2-amino-2-methyl-1-propanol, pH 10, 0.1% Triton
X-100 and 0.5 mM dioxetane 1, 3, 4 and 5, respectively, were
prepared and stored in opaque polyethylene bottles at 4.degree. C.,
25.degree. C. and 40.degree. C. Twenty four 100 .mu.L aliquots from
each bottle were pipetted into the wells of a 96 well plate and the
solutions incubated at 37.degree. C. Into each well 10 .mu.L
solutions containing 8.times.10.sup.-17 moles of AP were injected
and light intensity integrated over five hours. Data are the
average of all 24 wells. The experiment was repeated at the
indicated time intervals for each dioxetane. The results in FIG. 5
show the comparative stability of the three formulations at
25.degree. C. As shown in FIG. 5, fluoro-substituted dioxetane 3
was found to exhibit substantially better storage stability than
chloro-substituted dioxetane 4 and non-halo-substituted dioxetane
1. Dioxetanes 3 and 5 were also substantially more stable than 1 or
4 at 40.degree. C.
4TABLE 4 Storage Stability of Formulations % of Dioxetane Remaining
Time (wks) 1 3 4 5 0 100 100 100 100 1 94.8 100 2 91.1 77.0 99.8 3
87.5 99.1 66.0 4 84.1 65.6 99.4 5 81.8 6 80.7 9 76.5 96.9 10 57.5
12 96.7 14 93.8 21 93.6
Example 12
Performance of 3
[0141] A detection reagent incorporating dioxetane 3 was evaluated
in a test system as described in Example 7. The test material was a
fluorescein-labeled alkaline phosphatase conjugate which was
captured onto the magnetic particles. Assays for AP using the
reagent containing 3 produced results with sensitivity, dynamic
range and precision comparable to the results using dioxetane
1.
[0142] The foregoing examples are illustrative only and not
intended to be restrictive. The scope of the invention is indicated
only by the appended claims and equivalents.
* * * * *