U.S. patent application number 14/901009 was filed with the patent office on 2017-11-09 for hydrophilic high quantum yield acridinium esters with improved stability and fast light emission.
The applicant listed for this patent is SIEMENS HEALTHCARE DIAGNOSTICS INC.. Invention is credited to Qingping Jiang, Anand Natrajan, David Sharpe, David Wen.
Application Number | 20170320830 14/901009 |
Document ID | / |
Family ID | 52280476 |
Filed Date | 2017-11-09 |
United States Patent
Application |
20170320830 |
Kind Code |
A1 |
Natrajan; Anand ; et
al. |
November 9, 2017 |
HYDROPHILIC HIGH QUANTUM YIELD ACRIDINIUM ESTERS WITH IMPROVED
STABILITY AND FAST LIGHT EMISSION
Abstract
Hydrophilic, high quantum yield, chemiluminescent acridinium
compounds with increased light output, improved stability, fast
light emission and decreased non specific binding are disclosed.
The chemiluminescent acridinium esters possess hydrophilic,
branched, electron-donating functional groups at the C2 and/or C7
positions of the acridinium nucleus.
Inventors: |
Natrajan; Anand;
(Manchester, NH) ; Sharpe; David; (Foxboro,
MA) ; Jiang; Qingping; (East Walpole, MA) ;
Wen; David; (Northborough, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SIEMENS HEALTHCARE DIAGNOSTICS INC. |
Tarrytown |
NY |
US |
|
|
Family ID: |
52280476 |
Appl. No.: |
14/901009 |
Filed: |
July 4, 2014 |
PCT Filed: |
July 4, 2014 |
PCT NO: |
PCT/US14/45505 |
371 Date: |
December 22, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61843528 |
Jul 8, 2013 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/582 20130101;
G01N 21/76 20130101; F21K 2/06 20130101; C07D 219/06 20130101; C09K
11/07 20130101 |
International
Class: |
C07D 219/06 20060101
C07D219/06; G01N 21/76 20060101 G01N021/76; F21K 2/06 20060101
F21K002/06; G01N 33/58 20060101 G01N033/58; C09K 11/07 20060101
C09K011/07 |
Claims
1. A hydrophilic, high quantum yield acridinium ester having the
following structure: ##STR00034## wherein, R.sub.1 is a methyl or
sulfopropyl group; G is a branched group independently selected at
each occurrence from: ##STR00035## where R.sub.2, R.sub.3, R.sub.4,
R.sub.5, R.sub.6 and R.sub.7 are independently at each occurrence a
methyl group or a group --(CH.sub.2CH.sub.2O).sub.nCH.sub.3, where
n is an integer from 1 to 5; and R.sub.12 is an electrophilic or
nucleophilic group for conjugating the acridinium compound to an
analyte, an analyte analog, or a binding molecule for an
analyte.
2. An acridinium ester according to claim 1 wherein G is, at one or
both occurrences, a group: ##STR00036## where R.sub.2 and R.sub.3
are independently at each occurrence a methyl group or a group
--(CH.sub.2CH.sub.2O).sub.nCH.sub.3, where n is an integer from 1
to 5.
3. An acridinium ester according to claim 2 wherein G is a group:
##STR00037## at one or both occurrences.
4. An acridinium ester according to claim 2 wherein G is a group:
##STR00038## at one or both occurrences.
5. An acridinium ester according to claim 2 wherein G is a group:
##STR00039## at one or both occurrences.
6. An acridinium ester according to claim 1 wherein G is, at one or
both occurrences, a group: ##STR00040## where R.sub.4, R.sub.5,
R.sub.6 and R.sub.7 are independently at each occurrence a methyl
group or a group --(CH.sub.2CH.sub.2O).sub.nCH.sub.3, where n is an
integer from 1 to 5.
7. An acridinium ester according to claim 6 wherein G is, at one or
both occurrences, a group: ##STR00041## at one or both
occurrences.
8. An acridinium ester according to claim 6 wherein G is a group:
##STR00042## at one or both occurrences.
9. An acridinium ester according to claim 1, where R.sub.12 is
selected from the group consisting of: (1) --OH; (2)
--O--N-succinimidyl; (3)
--NH--(CH.sub.2).sub.5--C(O)--O--N-succinimidyl; (4)
--NH--(CH.sub.2).sub.5--COOH; (5)
--NH--(C.sub.2H.sub.4O).sub.n--C.sub.2H.sub.4NH--C(O)--(CH.sub.2).sub.3---
C(O)--O--N-succinimidyl wherein n=1 to 5; (6)
--NH--(C.sub.2H.sub.4O).sub.n--C.sub.2H.sub.4NH--C(O)--(CH.sub.2).sub.3---
COOH, wherein n=1 to 5; (7)
--NH--(C.sub.2H.sub.4O).sub.n--C.sub.2H.sub.4NH.sub.2, wherein n=1
to 5; and (8) --NH--R--NHR, wherein R is independently hydrogen,
alkyl, alkenyl, alkynyl, or aralkyl; wherein R optionally comprises
up to 20 heteroatoms.
10. An acridinium ester according to claim 9, wherein R.sub.12 is
--OH.
11. An acridinium ester according to claim 9, wherein R.sub.12 is
--NH--(C.sub.2H.sub.4O).sub.n--C.sub.2H.sub.4NH.sub.2, wherein n=1
to 5.
12. An acridinium ester according to claim 9, wherein R.sub.12 is:
--NH--(C.sub.2H.sub.4O).sub.n--C.sub.2H.sub.4NH--C(O)--(CH.sub.2).sub.3---
C(O)--O--R'' wherein n=1 to 5; and where R'' is hydrogen or
--N-succinimidyl.
13. An acridinium ester according to claim 1, having the following
structure: ##STR00043## where R.sub.12 is an electrophilic or
nucleophilic group for conjugating the acridinium compound to an
analyte, an analyte analog, or a binding molecule for an
analyte.
14. An acridinium ester according to claim 1 having the following
structure: ##STR00044## where R.sub.12 is an electrophilic or
nucleophilic group for conjugating the acridinium compound to an
analyte, an analyte analog, or a binding molecule for an
analyte.
15. An acridinium ester according to claim 1 having the following
structure: ##STR00045## where R.sub.12 is an electrophilic or
nucleophilic group for conjugating the acridinium compound to an
analyte, an analyte analog, or a binding molecule for an
analyte.
16. An acridinium ester according to claim 1 having the following
structure: ##STR00046## where R.sub.12 is an electrophilic or
nucleophilic group for conjugating the acridinium compound to an
analyte, an analyte analog, or a binding molecule for an
analyte.
17. An acridinium ester according to claim 1 having the following
structure: ##STR00047## where R.sub.12 is an electrophilic or
nucleophilic group for conjugating the acridinium compound to an
analyte, an analyte analog, or a binding molecule for an
analyte.
18. The acridinium ester according to any of claims 14-18, wherein
R.sub.12 is --OH.
19. An assay for the detection or quantification of an analyte
comprising the steps of: (a) providing a conjugate comprising: (i)
a binding molecule specific for an analyte; and (ii) a hydrophilic,
high quantum yield and fast light emitting acridinium ester
according to claim 1; (b) providing a solid support having
immobilized thereon a second binding molecule specific for said
analyte; (c) mixing the conjugate, the solid phase and a sample
suspected of containing the analyte to form a binding complex; (d)
separating the binding complex captured on the solid support; (e)
triggering chemiluminescence of the binding complex from step (d)
by adding chemiluminescence triggering reagents; (f) measuring the
amount of light emission with a luminometer; and (g) detecting the
presence or calculating the concentration of the analyte by
comparing the amount of light emitted from the reaction mixture
with a standard dose response curve which relates the amount of
light emitted to a known concentration of the analyte.
20. An assay for the detection or quantification of an analyte
comprising the steps of: (a) providing a conjugate of an analyte
with a hydrophilic, high quantum yield and fast light emitting
acridinium ester according to claim 1; (b) providing a solid
support immobilized with a binding molecule specific for the
analyte; (c) mixing the conjugate, solid support and a sample
suspected of containing the analyte to form a binding complex; (d)
separating the binding complex captured on the solid support; (e)
triggering the chemiluminescence of the binding complex from step
(d) by adding chemiluminescence triggering reagents; (f) measuring
the amount of light with an luminometer; and (g) detecting the
presence or calculating the concentration of the analyte by
comparing the amount of light emitted from the reaction mixture
with a standard dose response curve which relates the amount of
light emitted to a known concentration of the analyte.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application Ser. No. 61/843,528 filed Jul. 8, 2013, which is
incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to hydrophilic, high quantum
yield, chemiluminescent acridinium compounds with increased light
output, improved stability, fast light emission and low
non-specific binding. These compounds because of their enhanced
quantum yield and hydrophilic nature, are useful in improving assay
sensitivity. The improved stability of these compounds is useful
for extending the shelf life of reagents using these compounds as
well as for minimizing variation in assay performance with time.
Their increased emission kinetics also permits faster light
measurements in assays especially in automated analyzers.
BACKGROUND OF THE INVENTION
[0003] Chemiluminescent acridinium esters (AEs) are extremely
useful labels that have been used extensively in immunoassays and
nucleic acid assays. A review by Pringle, M. J. Journal of Clinical
Ligand Assay vol. 22, pp. 105-122 (1999) summarizes past and
current developments in this class of chemiluminescent
compounds.
[0004] McCapra, F. et al. in Tetrahedron Lett. vol. 43,
pp.3167-3172 (1964) and Rahut et al. in J. Org. Chem vol. 301, pp.
3587-3592. (1965) disclosed that chemiluminescence from the esters
of acridinium salts can be triggered by alkaline peroxide. Since
these seminal studies, interest in acridinium compounds has
increased because of their utility as labels. The application of
the acridinium ester 9-carboxyphenyl-N-methylacridinium bromide in
an immunoassay was disclosed by Simpson, J. S. A. et al., Nature,
vol. 279, pp. 646-647 (1979). However, this acridinium ester is
quite unstable, thereby limiting its commercial utility. This
instability arises from hydrolysis of the 9-carboxyphenyl ester
linkage between the phenol and the acridinium ring.
[0005] Different strategies for increasing the stability of
acridinium compounds have been described. Law et al., Journal of
Bioluminescence and Chemiluminescence, vol. 4, pp. 88-89 (1989),
introduced two methyl groups to flank the acridinium ester moiety
to stabilize this linkage. The resulting sterically stabilized
acridinium ester, DMAE-NHS
[2',6'-dimethyl-4'-(N-succinimidyloxycarbonyl)phenyl
10-methylacridinium-9-carboxylate] was found to have the same light
output as an acridinium ester lacking the two methyl groups. The
stability of the former compound when conjugated to an
immunoglobulin was vastly superior and showed no loss of
chemiluminescent activity even after one week at 37.degree. C. at
pH 7. In contrast, the unsubstituted acridinium ester only retained
10% of its activity when subjected to the same treatment. U.S. Pat.
Nos. 4,918,192 and 5,110.932 describe DMAE and its
applications.
[0006] U.S. Pat. No. 5,656,426 to Law et al. discloses a
hydrophilic version of DMAE termed NSP-DMAE-NHS ester where the
N-methyl group has been replaced with an N-sulfopropyl (NSP) group.
The structures of these two compounds and the numbering system of
the acridinium ring are illustrated below.
##STR00001##
[0007] Natrajan et al. in U.S. Pat. No. 6,664,043 B2 disclosed
NSP-DMAE derivatives with hydrophilic modifiers attached to the
phenol. The structure of one such compound,
NSP-DMAE-HEG-Glutarate-NHS, (abbreviated as HEG-AE) is illustrated
in the above. In this compound a diamino hexa(ethylene) glycol
(diamino-HEG) moiety is attached to the phenol to increase the
aqueous solubility of the acridinium ester. A glutarate moiety was
appended to the end of HEG and was converted to the NHS ester to
enable labeling of various molecules.
[0008] A different class of stable chemiluminescent acridinium
compounds has been described by Kinkel et al., Journal of
Bioluminescence and Chemiluminescence vol. 4, pp. 136-139 (1989)
and Mattingly, Journal of Bioluminescence and Chemiluminescence
vol. 6, pp. 107-114 (1991) and U.S. Pat. No. 5,468,646. In this
class of compounds, the phenolic ester linkage is replaced by a
sulfonamide moiety, which is reported to impart hydrolytic
stability without compromising the light output. In acridinium
esters, the phenol is the `leaving group` whereas in acridinium
sulfonamides, the sulfonamide is the `leaving group` during the
chemiluminescent reaction with alkaline peroxide.
[0009] Light emission from acridinium compounds is normally
triggered by alkaline peroxide. The overall light output, which can
also be referred to as the chemiluminescence quantum yield, is a
combination of the efficiencies of the chemical reaction leading to
the formation of the excited-state acridone and the latter's
fluorescence quantum yield.
[0010] Recently, Natrajan et al. in U.S. Pat. No. 7,309,615 B2, the
disclosure of which is hereby incorporated by reference herein,
described hydrophilic, high quantum yield acridinium compounds
containing hydrophilic alkoxy groups (OR*) at C2 and/or C7 of the
acridinium ring, wherein R* is a group comprising a sulfopropyl
moiety or ethylene glycol moieties or a combination thereof. The
enhanced light output from such compounds and their hydrophilic
nature made them useful for improving the sensitivity of
immunoassays. The structure of one such compound,
NSP-2,7-(OMHEG).sub.2-DMAE-AC-NHS (abbreviated as HQYAE), is
illustrated below.
##STR00002##
SUMMARY OF INVENTION
[0011] It has surprisingly been found that hydrophilic, high
quantum yield, chemiluminescent acridinium esters possessing
electron-donating functional groups of the form --OG at C2 and/or
C7 of the acridinium ring, where G represents a branched
hydrophilic substituent, provide increased light output, improved
stability, fast light emission and/or low non-specific binding in
assays.
[0012] In one aspect of the invention, hydrophilic, high quantum
yield acridinium esters are provided having the structure of
formula (I):
##STR00003##
[0013] wherein, R.sub.1 is a methyl or sulfopropyl group; G is a
branched group independently selected at each occurrence from:
##STR00004##
[0014] where R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6 and
R.sub.7 are independently at each occurrence a methyl group or a
group --(CH.sub.2CH.sub.2O).sub.nCH.sub.3, where n is an integer
from 1 to 5; and R.sub.12 is an electrophilic or nucleophilic group
for conjugating the acridinium compound to an analyte, an analyte
analog, or a binding molecule for an analyte.
[0015] In some embodiments according to formula (I), G will be, at
one or both occurrences, a group:
##STR00005##
[0016] where R.sub.2 and R.sub.3 are independently at each
occurrence a methyl group or a group
--(CH.sub.2CH.sub.2O).sub.nCH.sub.3, where n is an integer from 1
to 5; and in particular, G may be a group:
##STR00006##
[0017] at one or both occurrences; or in another embodiment G may
be a group:
##STR00007##
[0018] at one or both occurrences. In a related embodiment, G is a
group:
##STR00008##
[0019] at one or both occurrences.
[0020] In other embodiments according to formula (I), G represents,
at one or both occurrences, a group:
##STR00009##
[0021] where R.sub.4, R.sub.5, R.sub.6 and R.sub.7 are
independently at each occurrence a methyl group or a group
--(CH.sub.2CH.sub.2O).sub.nCH.sub.3, where n is an integer from 1
to 5. In one variant according to this embodiment, R.sub.4-R.sub.7
may represent methyl groups, such that G is a group:
##STR00010##
[0022] at one or both occurrences.
[0023] In one embodiment according to formula (I), G is a
group:
##STR00011##
[0024] at one or both occurrences.
[0025] In the acridinium esters according to formula (I), R.sub.12
may be selected, for example, from the group consisting of: [0026]
(1) --OH; [0027] (2) --O--N-succinimidyl; [0028] (3)
--NH--(CH.sub.2).sub.5--C(O)--O--N-succinimidyl; [0029] (4)
--NH--(CH.sub.2).sub.5--COOH; [0030] (5)
--NH--(C.sub.2H.sub.4O).sub.n--C.sub.2H.sub.4NH--C(O)--(CH.sub.2).sub.3---
C(O)--O--N-succinimidyl wherein n=1 to 5; [0031] (6)
--NH--(C.sub.2H.sub.4O).sub.n--C.sub.2H.sub.4NH--C(O)--(CH.sub.2).sub.3---
COOH, wherein n=1 to 5; [0032] (7)
--NH--(C.sub.2H.sub.4O).sub.n--C.sub.2H.sub.4NH.sub.2, wherein n=1
to 5; and [0033] (8) --NH--R--NHR, wherein R is independently
hydrogen, alkyl, alkenyl, alkynyl, or aralkyl; wherein R optionally
comprises up to 20 heteroatoms.
[0034] In various illustrative embodiments, R.sub.12 will be --OH,
or R.sub.12 will be a group:
[0035] --NH--(C.sub.2H.sub.4O).sub.n--C.sub.2H.sub.4NH.sub.2,
wherein n=1 to 5,
[0036] or R.sub.12 will be a group:
[0037]
--NH--(C.sub.2H.sub.4O).sub.n--C.sub.2H.sub.4NH--C(O)--(CH.sub.2).s-
ub.3--C(O)--O--R'', where n=1 to 5; and where R'' is hydrogen or
--N-succinimidyl.
[0038] One acridinium ester according to formula (I) has the
following structure:
##STR00012##
[0039] where R.sub.12 is an electrophilic or nucleophilic group for
conjugating the acridinium compound to an analyte, an analyte
analog, or a binding molecule for an analyte.
[0040] Another acridinium ester according to formula (I) has the
following structure:
##STR00013##
[0041] where R.sub.12 is an electrophilic or nucleophilic group for
conjugating the acridinium compound to an analyte, an analyte
analog, or a binding molecule for an analyte.
[0042] Yet another acridinium ester according to formula (I) has
the following structure:
##STR00014##
[0043] where R.sub.12 is an electrophilic or nucleophilic group for
conjugating the acridinium compound to an analyte, an analyte
analog, or a binding molecule for an analyte.
[0044] Another acridinium ester according to formula (I) has the
following structure:
##STR00015##
[0045] where R.sub.12 is an electrophilic or nucleophilic group for
conjugating the acridinium compound to an analyte, an analyte
analog, or a binding molecule for an analyte.
[0046] Still another acridinium ester according to formula (I) has
the following structure:
##STR00016##
[0047] where R.sub.12 is an electrophilic or nucleophilic group for
conjugating the acridinium compound to an analyte, an analyte
analog, or a binding molecule for an analyte.
[0048] In one exemplary embodiment of the acridinium esters
according to formula (I), R.sub.12 represents --OH.
[0049] In another aspect of the invention, an assay for the
detection or quantification of an analyte is provided comprising
the steps of: (a) providing a conjugate comprising: (i) a binding
molecule specific for an analyte; and (ii) a hydrophilic, high
quantum yield and fast light emitting acridinium ester according to
formula (I); (b) providing a solid support having immobilized
thereon a second binding molecule specific for the analyte; (c)
mixing the conjugate, the solid phase and a sample suspected of
containing the analyte to form a binding complex; (d) separating
the binding complex captured on the solid support; (e) triggering
chemiluminescence of the binding complex from step (d) by adding
chemiluminescence triggering reagents; (f) measuring the amount of
light emission with a luminometer; and (g) detecting the presence
or calculating the concentration of the analyte by comparing the
amount of light emitted from the reaction mixture with a standard
dose response curve which relates the amount of light emitted to a
known concentration of the analyte.
[0050] In a related aspect, an assay for the detection or
quantification of an analyte is provided comprising the steps of:
(a) providing a conjugate of an analyte with a hydrophilic, high
quantum yield and fast light emitting acridinium ester according to
formula (I); (b) providing a solid support immobilized with a
binding molecule specific for the analyte; (c) mixing the
conjugate, solid support and a sample suspected of containing the
analyte to form a binding complex; (d) separating the binding
complex captured on the solid support; (e) triggering the
chemiluminescence of the binding complex from step (d) by adding
chemiluminescence triggering reagents; (f) measuring the amount of
light with an luminometer; and (g) detecting the presence or
calculating the concentration of the analyte by comparing the
amount of light emitted from the reaction mixture with a standard
dose response curve which relates the amount of light emitted to a
known concentration of the analyte.
[0051] These and other aspects of the invention may be more clearly
understood by reference to the following detailed description of
the invention and the appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0052] FIG. 1 illustrates structures of B-AEs with electrophilic
N-hydroxysuccinimidyl (NHS) functional groups suitable for
preparing conjugates of proteins or other molecules containing
nucleophilic functional groups.
[0053] FIG. 2 illustrates B-AE structures with nucleophilic,
hexaethylene glycol amine (HEG-NH.sub.2) functional groups useful
for conjugating the acridinium compound to molecules containing
electrophilic functional groups.
[0054] FIG. 3 shows the structures of estradiol conjugates
(abbreviated as B-AE-E2) prepared using the B-AEs of FIG. 2.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0055] The introduction of electron-donating functional groups such
as OR* at C2 and/or C7 of the acridinium ring increases the quantum
yield of the corresponding chemiluminescent acridinium compound.
When the R* group is hydrophilic, such as a sulfopropyl group or
methoxy poly(ethylene) glycol, the corresponding acridinium
compound not only exhibits increased light output but also shows
reduced non-specific binding in immunoassays. These two properties
in conjunction lead to an increase in the sensitivity of
immunoassays.
[0056] The main objectives of the current invention were to
identify structural features of acridinium compounds that result in
(a) faster light emission when compared to NSP-DMAE and derivatives
as well as HQYAE; (b) improved stability especially when compared
to HQYAE; (c) high light output that is comparable to HQYAE and (d)
low non-specific binding that is comparable to HQYAE.
[0057] The hydrophilic acridinium compounds according to the
present invention, abbreviated as B-AEs (Branched-Acridinium
Esters), not only show increased light output but also show
improved stability and faster light emission. By stability we refer
to the chemiluminescent activity of the acridinium compounds. An
increase in stability is thus manifested as increased retention of
chemiluminescent activity as a function of time. Increased
stability of acridinium compounds is useful because reagents
derived from such compounds are less likely to show a deterioration
of assay performance as a function of time and moreover, the shelf
life of regents derived from such compounds is likely to be
extended thereby leading to less waste. Typically, assay reagents
derived from acridinium compounds include conjugates of proteins or
small molecules. The second property of the acridinium compounds is
faster light emission by which is meant that these compounds emit
their total light in a significantly shorter period of time
compared to acridinium compounds lacking the unique structural
features of the acridinium compounds of the current invention.
Faster light emission enables faster measurements in assays and has
the potential to increase the throughput of automated analyzers.
The throughput of automated analyzers is normally defined as the
number of tests the analyzer can perform in a given period of time.
The third and fourth properties of the acridinium compounds of the
current invention are their increased light output and low
non-specific binding, both extremely useful for improving assay
sensitivity.
[0058] It has unexpectedly been discovered that the placement of
branched functional groups derived from glycerol, of the type --OG,
where G is a branched functional group, at C2 and/or C7 of the
acridinium ring significantly increases the stability of the
corresponding acridinium compound and leads to faster light
emission. At the same time, the presence of these branched
functional groups increases the quantum yield and lowers the
non-specific binding of the corresponding acridinium compounds and
their conjugates. Non-specific binding in assays using solid phases
such as particles or microtiter plates are undesired binding
interactions of conjugates to these solid phases. These undesired
binding interactions typically increase the background of the assay
leading to a net lowering of the signal to background ratio in the
assay and thereby decreasing assay sensitivity.
[0059] The acridinium compounds of the current invention can be
represented by the general formula (I):
##STR00017##
[0060] where R.sub.1 is a methyl or sulfopropyl
(--CH.sub.2CH.sub.2CH.sub.2SO.sub.3.sup.-) group; G is defined
as
##STR00018##
[0061] where, R.sub.2 and R.sub.3 are the same or different and are
--(CH.sub.2CH.sub.2O).sub.nMe, where n=1-5; R.sub.4, R.sub.5,
R.sub.6 and R.sub.7 are the same or different and are either a
methyl group or --(CH.sub.2CH.sub.2O).sub.nMe, where n=1-5; and
where R.sub.12 is an electrophilic or nucleophilic group.
[0062] More specifically, the acridinium compounds of the present
invention can be represented by the following formula:
##STR00019##
[0063] where R.sub.2 and R.sub.3 are the same or different and are
--(CH.sub.2CH.sub.2O).sub.nMe groups, where n=1-3; and where
R.sub.12 is selected from the group consisting of: [0064] (1)
--O--N-succinimidyl; [0065] (2)
--NH--(CH.sub.2).sub.5--C(.dbd.O)--O--N-succinimidyl; and [0066]
(3)
--NH--(C.sub.2H.sub.4O).sub.n--C.sub.2H.sub.4NH--C(.dbd.O)--(CH.sub.2).su-
b.3--C(.dbd.O)--O--N-succinimidyl, wherein n=1 to 5; and [0067] (4)
--NH--(C.sub.2H.sub.4O).sub.n--C.sub.2H.sub.4NH.sub.2 where
n=1-5.
[0068] The acridinium compounds of the current invention can also
be represented by the following formula:
##STR00020##
[0069] where R.sub.4, R.sub.5, R.sub.6 and R.sub.7 are the same or
different and are either methyl or --(CH.sub.2CH.sub.2O).sub.nMe,
where n=1-3; and where R.sub.12 is selected from the group
consisting of: [0070] (1) --O--N-succinimidyl (NHS); [0071] (2)
--NH--(CH.sub.2).sub.5--C(.dbd.O)--O--N-succinimidyl; and [0072]
(3)
--NH--(C.sub.2H.sub.4O).sub.n--C.sub.2H.sub.4NH--C(.dbd.O)--(CH.sub.2).su-
b.3--C(.dbd.O)--O--N-succinimidyl, wherein n=1 to 5; and [0073] (4)
--NH--(C.sub.2H.sub.4O).sub.n--C.sub.2H.sub.4NH.sub.2 where
n=1-5.
[0074] Representative examples of the above general structures were
synthesized as discrete structures using traditional organic
chemistry techniques. The structures of these compounds along with
their abbreviated nomenclature are illustrated in FIGS. 1 and 2.
FIG. 1 illustrates structures of B-AEs with electrophilic
N-hydroxysuccinimidyl (NHS) functional groups whereas FIG. 2
illustrates B-AE structures with nucleophilic, hexaethylene glycol
amine (HEG-NH.sub.2) functional groups. The former compounds are
suitable for preparing conjugates of proteins or other molecules
containing nucleophilic functional groups. The latter compounds are
also useful for conjugating the acridinium compound to molecules
containing electrophilic functional groups. FIG. 3 shows the
structures of estradiol conjugates (abbreviated as B-AE-E2)
prepared using the B-AEs of FIG. 2. Estradiol is a steroidal
hormone that is commonly measured by immunoassay.
[0075] The B-AEs of FIG. 1 as well as NSP-DMAE,
NSP-DMAE-HEG-glutarate-NHS (abbreviated as HEG-AE) and the high
quantum yield acridinium ester NSP-2,7-(OMHEG)2-DMAE-AC-NHS
(abbreviated as HQYAE) were used to prepare conjugates of a murine,
monoclonal anti-TSH antibody (TSH=thyroid stimulating hormone) as
described in Example 9. Light emission from each conjugate was
triggered by the addition of two reagents. The first reagent
comprised 0.5% hydrogen peroxide in 100 mM nitric acid while the
second reagent contained a surfactant in 0.25 N sodium hydroxide.
Light was measured using a luminometer equipped with a
photo-multiplier tube as the detector. The amount of light emitted
by each acridinium compound conjugate was reported as Relative
Light Units (RLUs) by the luminometer. The total amount of light
emitted (100% RLUs) was measured for the various conjugates and
light emission at shorter measurement times, are represented as
fractions of this number and are also expressed as percentages in
Table 1. Other details pertaining to these measurements can be
found in the Examples section.
TABLE-US-00001 TABLE 1 % RLU as a function of measurement time of
acridinium compound-anti-TSH antibody conjugates. Entry Conjugate
0.5 s 1.0 s 2.0 s 5.0 s 1 HEG-AE 32 65 87 97 2 HQYAE 31 61 82 95 3
B1-AE 59 91 96 97 4 B2-AE 62 91 96 98 5 B3-AE 57 89 96 97 6 B04-AE
90 99 99 99 7 B4-AE 68 95 98 99
[0076] From Table 1, while HEG-AE and HQYAE emit only 65% and 61%
of their total light in one second, all the B-AE conjugates show
much faster light emission with .gtoreq.89% of the light emitted in
one second. The unique structural features in the B-AEs thus speed
up light emission from these compounds when conjugated to a
protein.
[0077] Similarly, the kinetics of light emission from the B-AE-E2
conjugates illustrated in FIG. 3 was compared with light emission
from the E2 conjugates of NSP-DMAE-E2 and HQYAE-E2. Both the latter
compounds incorporated the same HEG linkers. The results of these
measurements are tabulated in Table 2.
TABLE-US-00002 TABLE 2 % RLU as a function of measurement time of
E2 conjugates Entry Conjugate 0.5 s 1.0 s 2.0 s 5.0 s 1 NSP-DMAE 8
23 45 80 2 HQYAE 35 72 91 98 3 B1-AE 33 71 93 98 4 B2-AE 33 70 92
97 5 B4-AE 39 77 95 99
[0078] For the estradiol conjugates, all the B-AEs again show
faster light emission when compared to NSP-DMAE-HEG-E2
conjugate.
[0079] In addition to showing fast light emission, the acridinium
esters of the present invention also show good stability. By
"stability," is meant a minimal loss of chemiluminescent activity
as measured by the loss of RLUs when the compounds or conjugates
are stored in an aqueous solution typically, in the pH range of
7-8, which is within the physiological pH. From a mechanistic
viewpoint, hydrolysis of the phenolic ester is the main pathway by
which chemiluminescent acridinium esters become
non-chemiluminescent. Stable conjugates ensure long shelf life for
acridinium ester reagents and also ensure that assay performance
does not vary greatly over a given period of time. The stability of
various acridinium ester conjugates of the current invention are
listed in Tables 3 and 4. Aqueous solutions of the conjugates were
stored at 37.degree. C. in an aqueous buffer at pH 7.7 and RLUs
were recorded periodically using a luminometer. The RLUs that were
measured at the initial time point, also referred to as day 0, were
assigned a value of 100%. The RLUs that were measured at other time
points, are expressed as percentages of this number. Other details
pertaining to these measurements can be found in the Examples
section.
TABLE-US-00003 TABLE 3 Stability of anti-TSH antibody conjugates
expressed as % RLU Time HEG- (days) AE HQYAE B1-AE B2-AE B3-AE
B4-AE 0 100 100 100 100 100 100 7 94 86 97 91 92 96 16 89 77 91 85
87 93 23 82 68 84 78 81 85 33 82 68 87 79 83 86
TABLE-US-00004 TABLE 4 Stability of E2 conjugates expressed as %
RLU Time HEG- (days) AE HQYAE B1-AE B2-AE B4-AE 0 100 100 100 100
100 1 99 98 97 98 98 5 95 89 94 94 96 8 97 87 92 93 96 12 94 76 89
88 92 20 92 73 84 85 88 27 89 66 77 81 84 33 84 60 75 77 82
[0080] As is evident from Tables 3 and 4, the B-AE conjugates
retain a greater proportion of their chemiluminescent activity and
are more stable compared to the HQYAE conjugate. For example, the
anti-TSH antibody conjugate of HQYAE retains 68% of its
chemiluminescent activity after 33 days at 37.degree. C., the B-AE
conjugates retain .gtoreq.79% of their chemiluminescent activity in
the same period of time. A similar trend is noted for the estradiol
(E2) conjugates where the B-AE conjugates retain .gtoreq.75% of
their chemiluminescent activity after 33 days at 37.degree. C.,
whereas the HQYAE conjugate's chemiluminescent activity has dropped
to 60% in the same time period.
[0081] In addition to showing fast light emission, the B-AEs of the
present invention also show increased light output that is
comparable to or better than HQYAE. Table 5 summarizes the relative
quantum yields of the various B-AEs when conjugated to the anti-TSH
monoclonal antibody. In this table, the quantum yield of HEG-AE was
assigned a value of unity (1) and the quantum yields of all the
other conjugates are relative to this conjugate of this
compound.
TABLE-US-00005 TABLE 5 Relative quantum yields of AE conjugates of
anti-TSH antibody Relative quantum Entry Conjugate yield 1 HEG-AE
1.0 2 HQYAE 2.2 3 B1-AE 4.7 4 B2-AE 2.7 5 B3-AE 1.9 6 B04-AE 1.5 7
B4-AE 2.0
[0082] As can be noted from Table 5, all the B-AE conjugates show
greater light output (higher quantum yield) than the HEG-AE
conjugate and are either comparable or greater than the light
output of the HQYAE conjugate.
[0083] Finally, the B-AEs of the current invention also show low
non-specific binding to solid phases (Table 6). Non-specific
binding, as described earlier, in assays using solid phases such as
particles or microtiter plates are undesired binding interactions
of conjugates to these solid phases. These undesired binding
interactions typically increase the background of the assay leading
to a net lowering of the signal to background ratio in the assay
and thereby decreasing assay sensitivity. For the various
acridinium conjugates of the anti-TSH antibody listed in Table 6,
non-specific binding was measured on two different kinds of
particles; paramagnetic particles (PMP) and magnetic latex
particles (MLP). These two particles differ in their intrinsic
composition. PMPs are made mainly of iron oxide particles with a
silane coating containing amines. The amines are used to cross-link
proteins to the particle surface using reagents such as
glutaraldehyde. MLPs on the other hand are made of polystyrene. The
MLPs used in Table 6 contained a thin layer of magnetite to enable
magnetic separation and a polyacrylic acid coating for conjugating
proteins. The two types of particles were mixed with solutions of
the conjugates for a specific period of time and then the particles
were magnetically separated, washed once and then the
chemiluminescence associated with the particles was measured.
(Experimental details can be found in Example 11.) The ratio of
this chemiluminescence value in comparison to the total
chemiluminescence input is referred to fraction non-specific
binding (fNSB). Conjugates with low non-specific binding will have
low fNSB values. In examining Table 6, it is evident that all the
B-AE conjugates have lower non-specific binding than HEG-AE on both
types of particles. The fNSB values of the B-AE conjugates were
also found to be comparable to the previously described hydrophilic
HQYAE.
TABLE-US-00006 TABLE 6 Fractional Nonspecific Binding (fNSB) of
anti-TSH antibody-acridinium conjugates to particles. Particle PMP
MLP Acridinium Ester Conjugate Fractional Nonspecific Binding
(fNSB) HEG-AE 4.1E-05 1.2E-05 HQYAE 6.0E-06 7.5E-07 B1-AE 4.5E-06
1.8E-06 B2-AE 5.9E-06 2.8E-06 B3-AE 3.9E-06 1.2E-06 B4-AE 5.8E-06
1.9E-06 B04-AE 5.9E-06 7.1E-06
[0084] The hydrolytically stable, fast light emitting, hydrophilic,
high quantum yield acridinium compounds of the invention are useful
as labels in assays for the determination or quantitation of
analytes. Analytes that are typically measured in such assays are
often substances of some clinical relevance and can span a wide
range of molecules from large macromolecules such as proteins,
nucleic acids, viruses bacteria, etc. to small molecules such as
ethanol, vitamins, steroids, hormones, therapeutic drugs, etc. A
`sandwich` immunoassay typically involves the detection of a large
molecule, also referred to as macromolecular analyte, using two
binding molecules such as antibodies. One antibody is immobilized
or attached to a solid phase such as a particle, bead, membrane,
microtiter plate or any other solid surface. Methods for the
attachment of binding molecules such as antibodies to solid phases
are well known in the art. For example, an antibody can be
covalently attached to a particle containing amines on its surface
by using a cross-linking molecule such as glutaraldehyde. The
attachment may also be non-covalent and may involve simple
adsorption of the binding molecule to the surface of the solid
phase, such as polystyrene beads and microtiter plate. The second
antibody is often covalently attached with a chemiluminescent or
fluorescent molecule often referred to as a label. Labeling of
binding molecules such as antibodies and other binding proteins are
also well known in the art and are commonly called conjugation
reactions and the labeled antibody is often called a conjugate.
Typically, an amine-reactive moiety on the label reacts with an
amine on the antibody to form an amide linkage. Other linkages such
as thioether, ester, carbamate, and the like, between the antibody
and the label are also well known. In the assay, the two antibodies
bind to different regions of the macromolecular analyte. The
macromolecular analyte can be, for example, proteins, nucleic
acids, oligosaccharides, antibodies, antibody fragments, cells,
viruses, receptors, or synthetic polymers. The binding molecules
can be antibodies, antibody fragments, nucleic acids, peptides,
binding proteins or synthetic binding polymers. For example the
folate binding protein ("FBP") binds the analyte folate. Synthetic
binding molecules that can bind a variety of analytes have also
been disclosed by Mossbach et al. Biotechnology vol. 14, pp.
163-170 (1995).
[0085] When the solid phase with the immobilized antibody and the
labeled antibody is mixed with a sample containing the analyte, a
binding complex is formed between the analyte and the two
antibodies. This type of assay is often called a heterogenous assay
because of the involvement of a solid phase. The chemiluminescent
or fluorescent signal associated with the binding complex can then
be measured and the presence or absence of the analyte can be
inferred. Usually, the binding complex is separated from the rest
of the binding reaction components such as excess, labeled antibody
prior to signal generation. For example if the binding complex is
associated with a magnetic bead, a magnet can be used to separate
the binding complex associated with the bead from bulk solution. By
using a series of `standards`, that is, known concentrations of the
analyte, a `dose-response` curve can be generated using the two
antibodies. Thus, the dose-response curve correlates a certain
amount of measured signal with a specific concentration of analyte.
In a sandwich assay, as the concentration of the analyte increases,
the amount of signal also increases. The concentration of the
analyte in an unknown sample can then be calculated by comparing
the signal generated by an unknown sample containing the
macromolecular analyte, with the dose-response curve.
[0086] In a similar vein, the two binding components can also be
nucleic acids that bind or hybridize to different regions of a
nucleic acid analyte. The concentration of the nucleic acid analyte
can then be deduced in a similar manner.
[0087] Another class of immunoassays for small molecule analytes
such as steroids, vitamins, hormones, therapeutic drugs or small
peptides employs an assay format that is commonly referred to as a
competitive assay. Typically, in a competitive assay, a conjugate
is made of the analyte of interest and a chemiluminescent or
fluorescent label by covalently linking the two molecules. The
small molecule analyte can be used as such or its structure can be
altered prior to conjugation to the label. The analyte with the
altered structure is called an analog. It is often necessary to use
a structural analog of the analyte to permit the chemistry for
linking the label with the analyte. Sometimes a structural analog
of an analyte is used to attenuate or enhance its binding to a
binding molecule such an antibody. Such techniques are well known
in the art. The antibody or a binding protein to the analyte of
interest is often immobilized on a solid phase either directly or
through a secondary binding interaction such as the biotin-avidin
system.
[0088] The concentration of the analyte in a sample can be deduced
in a competitive assay by allowing the analyte-containing sample
and the analyte-label conjugate to compete for a limited amount of
solid phase-immobilized binding molecule. As the concentration of
analyte in a sample increases, the amount of analyte-label
conjugate captured by the binding molecule on the solid phase
decreases. By employing a series of `standards`, that is, known
concentrations of the analyte, a dose-response curve can be
constructed where the signal from the analyte-label conjugate
captured by the binding molecule on the solid phase is inversely
correlated with the concentration of analyte. Once a dose-response
curve has been devised in this manner, the concentration of the
same analyte in an unknown sample can be deduced by comparing the
signal obtained from the unknown sample with the signal in the
dose-response curve.
[0089] Another format of the competitive assay for small molecules
analytes involves the use of a solid phase that is immobilized with
the analyte of interest or an analyte analog and an antibody or a
binding protein specific for the analyte that is conjugated with a
chemiluminescent or fluorescent label. In this format, the
antibody-label conjugate is captured onto the solid phase through
the binding interaction with the analyte or the analyte analog on
the solid phase. The analyte of interest present in a sample then
"competitively" binds to the antibody-label conjugate and thus
inhibits or replaces the interaction of the antibody-label
conjugate with the solid phase. In this fashion, the amount of
signal generated from the antibody-label conjugate captured on the
solid phase is correlated to the amount of the analyte in
sample.
[0090] In accordance with the foregoing, an assay for the detection
or quantification of an analyte comprises, according to one
embodiment of the invention, the following steps:
[0091] (a) providing a conjugate comprising: (i) a binding molecule
specific for an analyte; and (ii) any of the inventive hydrophilic,
high quantum yield and fast light emitting acridinium esters
according to the invention;
[0092] (b) providing a solid support having immobilized thereon a
second binding molecule specific for said analyte;
[0093] (c) mixing the conjugate, the solid phase and a sample
suspected of containing the analyte to form a binding complex;
[0094] (d) separating the binding complex captured on the solid
support;
[0095] (e) triggering chemiluminescence of the binding complex from
step (d) by adding chemiluminescence triggering reagents;
[0096] (f) measuring the amount of light emission with a
luminometer; and
[0097] (g) detecting the presence or calculating the concentration
of the analyte by comparing the amount of light emitted from the
reaction mixture with a standard dose response curve which relates
the amount of light emitted to a known concentration of the
analyte.
[0098] In another embodiment, an assay for the detection or
quantification of an analyte is provided comprising the steps
of:
[0099] (a) providing a conjugate of an analyte with any of the any
of the inventive hydrophilic, high quantum yield and fast light
emitting acridinium esters (b) providing a solid support
immobilized with a binding molecule specific for the analyte;
[0100] (c) mixing the conjugate, solid support and a sample
suspected of containing the analyte to form a binding complex;
[0101] (d) separating the binding complex captured on the solid
support;
[0102] (e) triggering the chemiluminescence of the binding complex
from step (d) by adding chemiluminescence triggering reagents;
[0103] (f) measuring the amount of light with an luminometer;
and
[0104] (g) detecting the presence or calculating the concentration
of the analyte by comparing the amount of light emitted from the
reaction mixture with a standard dose response curve which relates
the amount of light emitted to a known concentration of the
analyte.
[0105] Macromolecular analytes can be proteins, nucleic acids,
oligosaccharides, antibodies, antibody fragments, cells, viruses,
synthetic polymers, and the like. Small molecule analytes can be
steroids, vitamins, hormones, therapeutic drugs, small peptides,
and the like. The binding molecules in the assays can be an
antibody, an antibody fragment, a binding protein, a nucleic acid,
a peptide, a receptor or a synthetic binding molecule.
EXAMPLE 1
Synthesis of B1-AE-NHS ester, 1i
[0106] a) 1,3-Bis(methoxyethoxy)-2-propyl toluenesulfonate, 1b. The
compound 1a, 1,3-bis(methoxyethoxy)-2-propanol was synthesized as
described by Cormier and Gregg in Chem. Mater. 1998, 10, 1309-1319.
A solution of 1a (2 g, 9.6 mmol) in anhydrous pyridine (10 mL) was
treated with 4-dimethylaminopyridine (0.234 g, 1.92 mmol) followed
by p-toluenesulfonyl chloride (3.67 g, 19.25 mmol). The reaction
was stirred at room temperature under a nitrogen atmosphere for 3
days. The solvent was then removed under reduced pressure and the
residue was partitioned between ethyl acetate (75 mL) and 10% HCl
(100 mL). The ethyl acetate layer was washed with brine, dried over
anhydrous magnesium sulfate and concentrated under reduced
pressure. The crude product (4.05 g) was purified by flash
chromatography on silica gel using 1:1, ethyl acetate/hexanes as
eluent. The product was recovered as a light yellow oil. Yield=2.42
g (70%).
[0107] b) Compound 1d. A mixture of 2,7-dihydroxy acridine methyl
ester, 1c (0.2 g, 0.48 mmol), (U.S. Pat. No. 7,309,615), compound
1b (0.868 g, 2.39 mmol) and cesium carbonate (0.39 g, 1.2 mmol) in
anhydrous DMF (10 mL) was heated at 100.degree. C. under a nitrogen
atmosphere for 4-5 hours. A small portion of the reaction mixture
was then analyzed by HPLC using a Phenomenex, C.sub.18 4.6
mm.times.25 cm column and a 30 minute gradient of 10.fwdarw.100% B
(A=water with 0.05% TFA, B=MeCN with 0.05% TFA) at a flow rate of
1.0 mL/minute and UV detection at 260 nm. Product was observed
eluting at Rt=25 minutes and was the major component. The reaction
was then cooled to room temperature and concentrated under reduced
pressure. The residue was partitioned between ethyl acetate (50 mL)
and water (50 mL). The ethyl acetate layer was separated, dried
over anhydrous magnesium sulfate and concentrated under reduced
pressure. The crude product (0.48 g) was purified by flash
chromatography on silica gel using ethyl acetate as eluent.
Yield=0.134 g (34%); MALDI-TOF MS 797.8 observed.
[0108] c) Compound 1f. A mixture of compound 1d (60 mg, 75.3
umoles), distilled 1,3-propane sultone (1 g, 8.2 mmol) and sodium
bicarbonate (65 mg, 0.77 mmol) was heated at 150.degree. C. under a
nitrogen atmosphere for 1 hour. A portion of the reaction mixture
was withdrawn, diluted with methanol and analyzed by HPLC as
described in section (b). The acridinium ester 1e was observed
eluting at Rt=19 minutes. The reaction was cooled to room
temperature and 20 mL of 1:1, ethyl acetate/hexanes was added. The
mixture was sonicated briefly to disperse the gummy solid and the
solvent was then decanted. The crude product was dried under
reduced pressure. This crude product was suspended in 1 N HCl (10
mL) and was refluxed under a nitrogen atmosphere for 2 hours. HPLC
analysis of the crude reaction mixture showed complete hydrolysis
of the reaction mixture with product eluting at Rt=16 minutes. The
product was purified by preparative HPLC using an YMC, C.sub.18
30.times.300 mm column and the same gradient at described in
section (b) at a solvent flow rate of 20 mL/minute and UV detection
at 260 nm. The HPLC fractions containing product were combined and
concentrated under reduced pressure. Yield=55 mg (81%); MALDI-TOF
MS 904.7 observed.
[0109] d) Compound 1g. A solution of compound 1f (53 mg, 58.2
umoles) in anhydrous DMF (2 mL) was treated with
diisopropylethylamine (15.2 uL, 87.3 umoles) and TSTU (20 mg, 64
umoles). The reaction was stirred at room temperature. After 30
minutes, HPLC analysis of the reaction mixture as described in
section (b) indicated complete conversion to the NHS ester eluting
at Rt=18 minutes. This reaction was added dropwise to a stirred
solution of 2,2'-(ethylenedioxy)bis(ethylamine) (86 ul, 0.582 mmol)
in anhydrous DMF (1.0 mL). After 30 minutes, HPLC analysis of the
reaction mixture, as described in section (b) showed complete
conversion to the product 1g eluting at Rt=13.6 minutes. The
product was purified by preparative HPLC as described in section
(c). Yield=50 mg (83%); MALDI-TOF MS 1035.6 observed.
[0110] e) B1-AE-NHS, compound 1i. A solution of compound 1g (47.5
mg, 46 umoles) in anhydrous methanol (3 mL) was treated with
diisopropylethylamine (40 uL, 0.23 mmol) and glutaric anhydride (26
mg, 0.23 mmol). The reaction was stirred at room temperature. After
30 minutes, HPLC analysis, as described in section (b), showed
complete conversion to the glutarate derivative 1h eluting at
Rt=14.8 minutes. The reaction mixture was diluted with anhydrous
toluene (3 mL) and concentrated under reduced pressure. The crude
product was dissolved in anhydrous DMF (3 mL) and treated with
diisopropylethylamine (80 uL, 0.46 mmol) and TSTU (138 mg, 0.46
mmol). After stirring for 30 minutes, HPLC analysis, as described
in section (b), showed >80% conversion to the product 1i eluting
at Rt=16 minutes. The product was purified by preparative HPLC as
described in section (c). The HPLC fractions containing product
were frozen at -80.degree. C. and lyophilized to dryness. The
lyophilized product was dissolved in anhydrous MeCN and transferred
to a tared round bottom flask and concentrated under reduced
pressure. Yield=32 mg (56%); MALDI-TOF MS 1247.1 observed.
[0111] The following reactions describe the synthesis of B1-AE-NHS,
compound 1i.
##STR00021## ##STR00022##
EXAMPLE 2
Synthesis of B2-AE-NHS ester, 2h
[0112] a). 1,3-Bis(3,6-dioxaheptanyl)glycerol-2-toluenesulfonate,
2b. The compound 1,3-Bis(3,6-dioxaheptanyl)glycerol, 2a, was
synthesized as described by Vacus and Simon in Adv. Mater. 1995, 7,
797-800. Crude 2a (16 g, 0.054 mol) was dissolved in anhydrous
pyridine (50 mL) and treated with 4-dimethylaminopyridine (1.32 g,
0.011 mol) followed by p-toluenesulfonyl chloride (12.4 g, 0.065
mol). The reaction was stirred under a nitrogen atmosphere for 16
hours. The solvent was then removed under reduced pressure and the
residue was partitioned between ethyl acetate (100 mL) and 2N HCl
(100 mL). The ethyl acetate layer was washed with saturated sodium
bicarbonate solution followed by brine. It was then dried over
magnesium sulfate and concentrated under reduced pressure. The
crude product (14 g) was purified by flash chromatography on silica
gel using 1:4, hexanes/ethyl acetate as eluent. Yield=6.3 g, light
yellow oil. MALDI-TOF MS 473.4 observed, (M+Na.sup.+).
[0113] b) Compound 2c. A mixture of 1c (0.2 g, 0.48 mmol), 2b (1.08
g, 2.4 mmol) and cesium carbonate (0.39 g, 0.12 mmol) in anhydrous
DMF (10 mL) was heated in an oil bath at 100.degree. C. under a
nitrogen atmosphere. After 5 hours, the reaction was cooled to room
temperature and concentrated under reduced pressure. The residue
was partitioned between ethyl acetate (75 mL) and water (75 mL).
The ethyl acetate layer was washed with brine and dried over
anhydrous magnesium sulfate. The solvent was then removed under
reduced pressure to afford 0.974 g of crude product which was
purified by flash chromatography on silca gel using 3% methanol in
ethyl acetate as eluent. Yield=99.4 mg (21%); MALDI-TOF MS 974.4
observed.
[0114] c) Compound 2e. A mixture of compound 2c (58 mg, 60 umoles),
distilled 1,3-propane sultone (0.75 g, 6.15 mmol) and sodium
bicarbonate (50 mg, 0.59 mmol) was heated at 150.degree. C. under a
nitrogen atmosphere. After 1 hour, a small portion was withdrawn,
diluted with methanol and analyzed by HPLC using a Phenomenex,
C.sub.18 4.6 mm.times.25 cm column and a 30 minute gradient of
10.fwdarw.100% B (A=water with 0.05% TFA, B=MeCN with 0.05% TFA) at
a flow rate of 1.0 mL/minute and UV detection at 260 nm. Product
was observed eluting at Rt=18.5 minutes (>80% conversion,
startimg material Rt=23.5 minutes). The reaction was cooled to room
temperature and 20 mL of 1:1, ethyl acetate/hexanes was added.
After brief sonication to disperse the gummy product, the solvent
was decanted and the product 2d was dried under vacuum.
[0115] The crude acridinium ester 2d was suspended in I N HCl (10
mL) and refluxed under a nitrogen atmosphere for 2 hours. HPLC
analysis, as described above, indicated complete conversion to
product 2e eluting at 16 minutes. The product was purified by
preparative HPLC using an YMC, C.sub.18 30.times.300 mm column and
the same gradient described above at a solvent flow rate of 20
mL/minute and UV detection at 260 nm. The HPLC fractions containing
product were combined and concentrated under reduced pressure.
Yield=42 mg (65%); MALDI-TOF MS 1083.3 observed.
[0116] d) Compound 2f. A solution of compound 2e (42 mg, 39 umoles)
in anhydrous DMF (2 mL) was treated with diisopropylethylamine (10
uL, 59 umoles) and TSTU (14 mg, 46.5 umoles). The reaction was
stirred at room temperature. After 15 minutes, HPLC analysis, as
described in section (c) showed complete conversion to the NHS
ester eluting at Rt=17.7 minutes. This reaction was added dropwise
to a stirred solution of 2,2'-(ethylenedioxy)bis(ethylamine) (58
ul, 0.39 mmol) in anhydrous DMF (1.0 mL). After 30 minutes, HPLC
analysis of the reaction mixture, as described in section (b)
showed complete conversion to the product 2f eluting at Rt=13.6
minutes. The product was purified by preparative HPLC as described
in section (c). Yield=37 mg (79%); MALDI-TOF MS 1217.9
observed.
[0117] e) B2-AE-NHS, compound 2h. A solution of compound 2f (37 mg,
30 umoles) in anhydrous methanol (3 mL) was treated with
diisopropylethylamine (26 uL, 0.15 mmol) and glutaric anhydride (17
mg, 0.15 mmol). The reaction was stirred at room temperature. After
30 minutes, HPLC analysis, as described in section (c), showed
complete conversion to the glutarate derivative 2g eluting at Rt=15
minutes. The reaction mixture was diluted with anhydrous toluene (3
mL) and concentrated under reduced pressure. The crude product was
dissolved in anhydrous DMF (2 mL) and treated with
diisopropylethylamine (52 uL, 0.3 mmol) and TSTU (89 mg, 0.3 mmol).
After stirring for 30 minutes, HPLC analysis, as described in
section (c), showed >80% conversion to the product 2h eluting at
Rt=16 minutes. The product was purified by preparative HPLC as
described in section (c). The HPLC fractions containing product
were frozen at -80.degree. C. and lyophilized to dryness. The
lyophilized product was dissolved in anhydrous MeCN and transferred
to a tared round bottom flask and concentrated under reduced
pressure. Yield=39 mg (91%); MALDI-TOF MS 1425.4 observed.
[0118] The following reactions describe the synthesis of B2-AE-NHS,
2h.
##STR00023## ##STR00024##
EXAMPLE 3
B3-AE-NHS ester 3h
[0119] a) 1,3-Bis(3,6,9-dioxadecanyl)glycerol-2-toluenesulfonate,
3b. The compound 1,3-Bis(3,6,9-dioxadecanyl)glycerol, 3a, was
synthesized as described by Lauter et al. in Macromol. Chem. Phys.
1998, 199, 2129-2140. The alcohol (7 g, 0.0182 mol) was dissolved
in anhydrous pyridine (30 mL) and treated with
4-dimethylaminopyridine (0.444 g, 3.6 mmol) and p-toluenesulfonyl
chloride (3.85 g, 0.02 mol). The reaction was stirred under a
nitrogen atmosphere for 3 days. The solvent was then removed under
reduced pressure and the residue was partitioned between ethyl
acetate (100 mL) and 10% HCl (100 mL). The ethyl acetate layer was
washed with saturated sodium bicarbonate solution and brine. It was
then dried over anhydrous magnesium sulfate and concentrated under
reduced pressure. The crude product was purified by flash
chromatography on silica gel using 5:4.5:0.5, hexanes:ethyl
acetate:methanol. Yield=4.47 g (45%); light yellow oil.
[0120] b) Compound 3c. A mixture of 1c (0.2 g, 0.48 mmol), 3b (1.3
g, 2.4 mmol) and cesium carbonate (0.35 g, 0.11 mmol) in anhydrous
DMF (10 mL) was heated in an oil bath at 100.degree. C. under a
nitrogen atmosphere. After 6 hours, the reaction was cooled to room
temperature and concentrated under reduced pressure. The residue
was partitioned between ethyl acetate (75 mL) and water (75 mL).
The ethyl acetate layer was washed with brine and dried over
anhydrous magnesium sulfate. The solvent was then removed under
reduced pressure to afford 1.3 g of crude product which was
purified by flash chromatography on silca gel using 5% methanol in
ethyl acetate as eluent. Yield=134 mg (22%); MALDI-TOF MS 1148.9
observed.
[0121] c) Compound 3e. A mixture of compound 3c (45 mg, 39 umoles),
distilled 1,3-propane sultone (0.5 g, 4.1 mmol) and sodium
bicarbonate (33 mg, 0.39 mmol) was heated at 150.degree. C. under a
nitrogen atmosphere. After 2 hours, a small portion was withdrawn,
diluted with methanol and analyzed by HPLC using a Phenomenex,
C.sub.18 4.6 mm.times.25 cm column and a 30 minute gradient of
10.fwdarw.100% B (A=water with 0.05% TFA, B=MeCN with 0.05% TFA) at
a flow rate of 1.0 mL/minute and UV detection at 260 nm. Product
was observed eluting at Rt=18.3 minutes (>80% conversion,
startimg material Rt=22.5 minutes). The reaction was cooled to room
temperature and 20 mL of 1:1, ethyl acetate/hexanes was added.
After brief sonication to disperse the gummy product, the solvent
was decanted and the product 3d was dried under vacuum.
[0122] The crude acridinium ester 3d was suspended in I N HCl (10
mL) and refluxed under a nitrogen atmosphere for 2 hours. HPLC
analysis, as described above, indicated complete conversion to
product 3e eluting at 16.3 minutes. The product was purified by
preparative HPLC using an YMC, C.sub.18 30.times.300 mm column and
the same gradient at described above at a solvent flow rate of 20
mL/minute and UV detection at 260 nm. The HPLC fractions containing
product were combined and concentrated under reduced pressure.
Yield=28 mg (57%); MALDI-TOF MS 1255.9 observed.
[0123] d) Compound 3f A solution of compound 3e (28 mg, 22.3
umoles) in anhydrous DMF (2 mL) was treated with
diisopropylethylamine (6.4 uL, 33.5 umoles) and TSTU (8 mg, 26.6
umoles). The reaction was stirred at room temperature. After 30
minutes, HPLC analysis, as described in section (c) showed complete
conversion to the NHS ester eluting at Rt=17.7 minutes. This
reaction was added dropwise to a stirred solution of
2,2'-(ethylenedioxy)bis(ethylamine) (32 ul, 0.22 mmol) in anhydrous
DMF (1.0 mL). After one hour, HPLC analysis of the reaction
mixture, as described in section (c) showed complete conversion to
the product 3f eluting at Rt=14 minutes. The product was purified
by preparative HPLC as described in section (c). Yield=28 mg (90%);
MALDI-TOF MS 1388.6 observed.
[0124] e) B3-AE-NHS, compound 3h. A solution of compound 3f (28 mg,
20 umoles) in anhydrous methanol (2 mL) was treated with
diisopropylethylamine (17.6 uL, 0.1 mmol) and glutaric anhydride
(11.5 mg, 0.1 mmol). The reaction was stirred at room temperature.
After 30 minutes, HPLC analysis, as described in section (c),
showed complete conversion to the glutarate derivative 3g eluting
at Rt=15.2 minutes. The reaction mixture was diluted with anhydrous
toluene (3 mL) and concentrated under reduced pressure. The crude
product was dissolved in anhydrous DMF (2 mL) and treated with
diisopropylethylamine (35 uL, 0.2 mmol) and TSTU (60 mg, 0.2 mmol).
After stirring for 30 minutes, HPLC analysis, as described in
section (c), showed >70% conversion to the product 3h eluting at
Rt=16.2 minutes. The product was purified by preparative HPLC as
described in section (c). The HPLC fractions containing product
were frozen at -80.degree. C. and lyophilized to dryness. The
lyophilized product was dissolved in anhydrous MeCN and transferred
to a tared round bottom flask and concentrated under reduced
pressure. Yield=17.6 mg (55%); MALDI-TOF MS 1598 observed.
[0125] The following reactions describe the synthesis of B3-AE-NHS,
3h.
##STR00025## ##STR00026##
EXAMPLE 4
B4-AE-NHS ester, 4h
[0126] a) Compound 4a. 1,3-Bis(methoxyethoxy)-2-propanol, 1a, (12
g, 0.058 mol) and potassium hydroxide (2.43 g, 0.043 mol) were
stirred vigorously and epichlorohydrin (1.334 g, 0.0144 mol) was
added dropwise. The reaction was heated at 80.degree. C. for 24
hours. It was then cooled to room temperature and water (50 mL) was
added. The solution was extracted with dichloromethane (3.times.50
mL). The combined dichloromethane extracts were dried over
anhydrous magnesium sulfate and concentrated under reduced
pressure. The crude product (10.85 g) was sued as such for the next
reaction.
[0127] b) Compound 4b. A solution of 4a (10.5 g crude, 0.023 mol)
in anhydrous pyridine (25 mL) was treated with
4-dimethylaminopyridine (0.56 g, 4.6 mmol) and p-toluenesulfonyl
chloride (0.046 mol, 8.8 g). The reaction was stirred at room
temperature under a nitrogen atmosphere for 16 hours. The solvent
was then removed under reduced pressure and the residue was
partitioned between ethyl acetate (100 mL) and 2 N HCl (100 mL).
The ethyl acetate layer was washed with saturated sodium
bicarbonate and brine. It was then dried over anhydrous magnesium
sulfate and concentrated under reduced pressure. The crude product
(16.4 g) was purified by flash chromatography on silica gel using
1:1 ethyl acetate:hexanes to elute 1,3-bis(methoxyethoxy)-2-propyl
toluenesulfonate followed by ethyl acetate to elute product.
Yield=2.82 g (32%); MALDI-TOF MS 648.6 (M+Na.sup.+).
[0128] c) Compound 4c. A mixture of 2,7-dihydroxy acridine methyl
ester, 1c (0.2 g, 0.48 mmol) compound 4b (1.5 g, 2.4 mmol) and
cesium carbonate (0.39 g, 1.2 mmol) in anhydrous DMF (10 mL) was
heated at 100.degree. C. under a nitrogen atmosphere for 4-5 hours.
A small portion of the reaction mixture was then analyzed by HPLC
using a Phenomenex, C.sub.18 4.6 mm.times.25 cm column and a 30
minute gradient of 10.fwdarw.100% B (A=water with 0.05% TFA, B=MeCN
with 0.05% TFA) at a flow rate of 1.0 mL/minute and UV detection at
260 nm. Product was observed eluting at Rt=23.5 minutes and was the
major component. The reaction was then cooled to room temperature
and concentrated under reduced pressure. The residue was
partitioned between ethyl acetate (50 mL) and water (50 mL). The
ethyl acetate layer was separated, dried over anhydrous magnesium
sulfate and concentrated under reduced pressure. The crude product
(1.36 g) was purified by flash chromatography on silica gel using
5% methanol in ethyl acetate as eluent. Yield=0.156 g (25%);
MALDI-TOF MS 1325 observed.
[0129] d) Compound 4e. A mixture of compound 4c (60 mg, 45.3
umoles), distilled 1,3-propane sultone (1.0 g, 8.2 mmol) and sodium
bicarbonate (76 mg, 0.9 mmol) was heated at 150.degree. C. under a
nitrogen atmosphere. After 2 hours, a small portion was withdrawn,
diluted with methanol and analyzed by HPLC using a Phenomenex,
C.sub.18 4.6 mm.times.25 cm column and a 30 minute gradient of
10.fwdarw.100% B (A=water with 0.05% TFA, B=MeCN with 0.05% TFA) at
a flow rate of 1.0 mL/minute and UV detection at 260 nm. Product
was observed eluting at Rt=17.8 minutes (>80% conversion,
startimg material Rt=23.5 minutes). The reaction was cooled to room
temperature and 20 mL of 1:1, ethyl acetate/hexanes was added.
After brief sonication to disperse the gummy product, the solvent
was decanted and the product 4d was dried under vacuum.
[0130] The crude acridinium ester 4d was suspended in I N HCl (10
mL) and refluxed under a nitrogen atmosphere for 2 hours. HPLC
analysis, as described above, indicated complete conversion to
product 4e eluting at 17 minutes. The product was purified by
preparative HPLC using an YMC, C.sub.18 30.times.300 mm column and
the same gradient at described above at a solvent flow rate of 20
mL/minute and UV detection at 260 nm. The HPLC fractions containing
product were combined and concentrated under reduced pressure.
Yield=33.5 mg (52%); MALDI-TOF MS 1433.1 observed.
[0131] e) Compound 4f A solution of compound 4e (33.5 mg, 23.4
umoles) in anhydrous DMF (2 mL) was treated with
diisopropylethylamine (6.1 uL, 35 umoles) and TSTU (8.5 mg, 28.2
umoles). The reaction was stirred at room temperature. After 30
minutes, HPLC analysis, as described in section (c) showed complete
conversion to the NHS ester eluting at Rt=18.7 minutes. This
reaction was added dropwise to a stirred solution of
2,2'-(ethylenedioxy)bis(ethylamine) (35 ul, 0.24 mmol) in anhydrous
DMF (1.0 mL). After one hour, HPLC analysis of the reaction
mixture, as described in section (c) showed complete conversion to
the product 4f eluting at Rt=14.5 minutes. The product was purified
by preparative HPLC as described in section (d). Yield=22 mg (59%);
MALDI-TOF MS 1565.8 observed.
[0132] B4-AE-NHS, compound 4h. A solution of compound 4f (22 mg, 14
umoles) in anhydrous methanol (2 mL) was treated with
diisopropylethylamine (12.3 uL, 70 umoles) and glutaric anhydride
(8 mg, 70 mmoles). The reaction was stirred at room temperature.
After 30 minutes, HPLC analysis, as described in section (d),
showed complete conversion to the glutarate derivative 4g eluting
at Rt=15.9 minutes. The reaction mixture was diluted with anhydrous
toluene (3 mL) and concentrated under reduced pressure.
[0133] The crude product was dissolved in anhydrous DMF (2 mL) and
treated with diisopropylethylamine (24.6 uL, 0.14 mmol) and TSTU
(42 mg, 0.14 mmol). After stirring for 30 minutes, HPLC analysis,
as described in section (c), showed >70% conversion to the
product 4h eluting at Rt=16.9 minutes. The product was purified by
preparative HPLC as described in section (d). The HPLC fractions
containing product were frozen at -80.degree. C. and lyophilized to
dryness. The lyophilized product was dissolved in anhydrous MeCN
and transferred to a tared round bottom flask and concentrated
under reduced pressure. Yield=18.8 mg (75%); MALDI-TOF MS 1776.5
observed.
[0134] The following reactions describe the synthesis of B4-AE-NHS,
4h.
##STR00027## ##STR00028##
EXAMPLE 5
B04-AE-NHS, 5i
[0135] a) Compound 5b. 1,3-Dimethoxy-2-propanol, 5a, was
synthesized as described by Kang et al. in Bull. Korean Chem. Soc.
2006, 27, 1364-1370. Crude 1,3-dimethoxy-2-propanol (10.66 g, 0.089
mol) and potassium hydroxide (3 g, 0.00534 mol) was stirred at
80.degree. C. under a nitrogen atmosphere until all the potassium
hydroxide dissolved. Epichlorohydrin (1.65 g, 0.00178 mol) was then
added dropwise and the reaction was heated at 100.degree. C. under
a nitrogen atmosphere for 24 hours. The reaction was then cooled to
room temperature and partitioned between ethyl acetate (75 mL) and
saturated ammonium chloride solution (75 mL). The ethyl acetate
layer was separated and the aqueous layer was extracted once more
with ethyl acetate (50 mL). The combined ethyl acetate extracts
were dried over anhydrous magnesium sulfate and concentrated under
reduced pressure. The recovered light brown oil (3.7 g) was used a
such in the next reaction.
[0136] b) Compound 5c. Compound 5b (3.7 g, 0.0125 mol) was
dissolved an anhydrous pyridine (15 mL) and treated with
4-dimethylpyridine (0.381 g, 3.1 mmol) and p-toluenesulfonyl
chloride (4.8 g, 0.0025 mol). The reaction was stirred at room
temperature under a nitrogen atmosphere for 3 days. The solvent was
then removed under reduced pressure and the residue was partitioned
between ethyl acetate (75 mL) and 1N HCl (50 mL). The ethyl acetate
layer was separated and washed with 2% sodium hydroxide solution
(50 mL) and saturated ammonium chloride solution (50 mL). It was
then dried over anhydrous magnesium sulfate and concentrated under
reduced pressure. The crude product (6.6 g) was purified by flash
chromatography on silica gel using 75:24:1; hexanes:ethyl acetate:
methanol as eluent. Yield=1.73 g, light yellow oil.
[0137] c) Compound 5d. A mixture of 2,7-dihydroxy acridine methyl
ester, 1c (0.1 g, 0.24 mmol) compound 5c (0.54 g, 1.2 mmol) and
cesium carbonate (0.2 g, 0.06 mmol) in anhydrous DMF (5 mL) was
heated at 100.degree. C. under a nitrogen atmosphere for 4-5 hours.
A small portion of the reaction mixture was then analyzed by HPLC
using a Phenomenex, C.sub.18 4.6 mm.times.25 cm column and a 30
minute gradient of 10.fwdarw.100% B (A=water with 0.05% TFA, B=MeCN
with 0.05% TFA) at a flow rate of 1.0 mL/minute and UV detection at
260 nm. Product was observed eluting at Rt=26.2 minutes and was the
major component. The reaction was then cooled to room temperature
and concentrated under reduced pressure. The residue was
partitioned between ethyl acetate (75 mL) and water (50 mL). The
ethyl acetate layer was separated, dried over anhydrous magnesium
sulfate and concentrated under reduced pressure. The crude product
(0.45 g) was purified by preparative TLC on silica gel using 1%
methanol in ethyl acetate as eluent. Yield=64 g (28%); MALDI-TOF MS
973.8 observed.
[0138] d) Compound 5f A mixture of compound 5d (64 mg, 65.7
umoles), distilled 1,3-propane sultone (1.6 g, 13.1 mmol) and
sodium bicarbonate (110 mg, 1.3 mmol) was heated at 150.degree. C.
under a nitrogen atmosphere. After 2 hours, a small portion was
withdrawn, diluted with methanol and analyzed by HPLC using a
Phenomenex, C.sub.18 4.6 mm.times.25 cm column and a 30 minute
gradient of 10.fwdarw.100% B (A=water with 0.05% TFA, B=MeCN with
0.05% TFA) at a flow rate of 1.0 mL/minute and UV detection at 260
nm. Product was observed eluting at Rt=20.5 minutes (>60%
conversion). The reaction was cooled to room temperature and 20 mL
of 1:1, ethyl acetate/hexanes was added. After brief sonication to
disperse the gummy product, the solvent was decanted and the
product 5e was dried under vacuum.
[0139] The crude acridinium ester 5e was suspended in IN HCl (10
mL) and refluxed under a nitrogen atmosphere for 2 hours. HPLC
analysis, as described above, indicated complete conversion to
product 5f eluting at 17.5 minutes. The product was purified by
preparative HPLC using an YMC, C.sub.18 30.times.300 mm column and
the same gradient at described above at a solvent flow rate of 20
mL/minute and UV detection at 260 nm. The HPLC fractions containing
product were combined and concentrated under reduced pressure.
Yield=12 mg (17%); MALDI-TOF MS 1082.4 observed.
[0140] e) Compound 5g. A solution of compound 5f (12 mg, 11.1
umoles) in anhydrous DMF (1 mL) was treated with
diisopropylethylamine (4.0 uL, 22 umoles) and TSTU (5 mg, 16.7
umoles). The reaction was stirred at room temperature. After 30
minutes, HPLC analysis, as described in section (c) showed complete
conversion to the NHS ester eluting at Rt=19.5 minutes. This
reaction was added dropwise to a stirred solution of
2,2'-(ethylenedioxy)bis(ethylamine) (16 ul, 0.11mmol) in anhydrous
DMF (1.0 mL). After one hour, HPLC analysis of the reaction
mixture, as described in section (c) showed complete conversion to
the product 5g eluting at Rt=14.7 minutes. The product was purified
by preparative HPLC as described in section (d). Yield=15.4 mg
(quantitative); MALDI-TOF MS 1212.9 observed.
[0141] B04-AE-NHS, compound 5i. A solution of compound 5g (15.4 mg,
12.7 umoles) in anhydrous methanol (2 mL) was treated with
diisopropylethylamine (11 uL, 63.5 umoles) and glutaric anhydride
(7.2 mg, 63.5 umoles). The reaction was stirred at room
temperature. After 30 minutes, HPLC analysis, as described in
section (c), showed complete conversion to the glutarate derivative
5h eluting at Rt=16.2 minutes. The reaction mixture was diluted
with anhydrous toluene (3 mL) and concentrated under reduced
pressure. The crude product was dissolved in anhydrous DMF (2 mL)
and treated with diisopropylethylamine (22 uL, 0.126 mmol) and TSTU
(38 mg, 0.126 mmol). After stirring for 15 minutes, HPLC analysis,
as described in section (c), showed >80% conversion to the
product 5i eluting at Rt=17.2 minutes. The product was purified by
preparative HPLC as described in section (d). The HPLC fractions
containing product were frozen at -80.degree. C. and lyophilized to
dryness. The lyophilized product was dissolved in anhydrous MeCN
and transferred to a tared round bottom flask and concentrated
under reduced pressure. Yield=12.3 mg (68%); MALDI-TOF MS 1423.8
observed.
[0142] The following reactions describe the synthesis of
B04-AE-NHS, compound 5i
##STR00029## ##STR00030##
EXAMPLE 6
B1-AE-E2, 6b
[0143] a) Compound 6a. A solution of compound 1f (26 mg, 28.7
umoles) in anhydrous DMF (2 mL) was treated with
diisopropylethylamine (7.5 uL, 43 umoles) and TSTU (10.4 mg, 35
umoles). The reaction was stirred at room temperature. After 30
minutes, HPLC analysis of the reaction mixture using a Phenomenex,
C.sub.18 4.6 mm.times.25 cm column and a 30 minute gradient of
10.fwdarw.100% B (A=water with 0.05% TFA, B=MeCN with 0.05% TFA) at
a flow rate of 1.0 mL/minute and UV detection at 260 nm, indicated
complete conversion to the NHS ester eluting at Rt=18.2 minutes.
This reaction was added dropwise to a stirred solution of diamino
hexa(ethylene) glycol (U.S. Pat. No. 6,664,043), (40 mg, 0.142
mmol) in anhydrous DMF (2.0 mL). After 30 minutes, HPLC analysis of
the reaction mixture showed complete conversion to the product 6a
eluting at Rt=14.1 minutes. The product was purified by preparative
HPLC using an YMC, C.sub.18 30.times.300 mm column and the same
gradient as described above at a solvent flow rate of 20 mL/minute
and UV detection at 260 nm. The HPLC fractions containing product
were combined and concentrated under reduced pressure. Yield=26 mg
(78%); MALDI-TOF MS 1168.6 observed.
[0144] b) B1-AE-E2, 6b. Estradiol-6-carboxymethyloxime (1 mg, 2.78
umoles) in DMF (0.1 mL) was combined with compound 6a (3.25 mg,
2.78 umoles) and treated with diisopropylethylamine (1 uL, 5.56
umoles) followed by BOP reagent (1.84 mg, 4.17 umoles) added as a
solution in DMF (0.184 mL of a 10 mg/mL solution). The reaction was
stirred at room temperature 2 h. HPLC analysis, as described in
section (a), indicated >80% conversion to the product eluting at
Rt=18.2 minutes. The product was purified by preparative HPLC using
an YMC, C.sub.18 20.times.250 mm column and a 30 minute of
10.fwdarw.70% B (A=water with 0.05% TFA, B=MeCN with 0.05% TFA)
gradient of at a solvent flow rate of 16 mL/minute and UV detection
at 260 nm. The HPLC fractions containing product were combined,
frozen at -80oC and lyophilized to dryness. Yield=2.8 mg (67%);
MALDI-TOF MS 1511.2 observed.
[0145] The following reactions describe the synthesis of B1-AE-E2,
6b.
##STR00031##
EXAMPLE 7
B2-AE-E2, 7b
[0146] a) Compound 7a. A solution of compound 2e (30 mg, 28 umoles)
in anhydrous DMF (2 mL) was treated with diisopropylethylamine (7.2
uL, 42 umoles) and TSTU (10 mg, 34 umoles). The reaction was
stirred at room temperature. After 30 minutes, HPLC analysis of the
reaction mixture using a Phenomenex, C.sub.18 4.6 mm.times.25 cm
column and a 30 minute gradient of 10.fwdarw.100% B (A=water with
0.05% TFA, B=MeCN with 0.05% TFA) at a flow rate of 1.0 mL/minute
and UV detection at 260 nm, indicated complete conversion to the
NHS ester eluting at Rt=17.7 minutes. This reaction was added
dropwise to a stirred solution of diamino hexa(ethylene) glycol
(U.S. Pat. No. 6,664,043), (40 mg, 0.142 mmol) in anhydrous DMF
(1.0 mL). After 30 minutes, HPLC analysis of the reaction mixture
showed complete conversion to the product 7a eluting at Rt=14
minutes. The product was purified by preparative HPLC using an YMC,
C.sub.18 30.times.300 mm column and the same gradient as described
above at a solvent flow rate of 20 mL/minute and UV detection at
260 nm. The HPLC fractions containing product were combined and
concentrated under reduced pressure. Yield=28.3 mg (76%); MALDI-TOF
MS 1345.4 observed.
[0147] b) B2-AE-E2, 7b. Estradiol-6-carboxymethyloxime (1 mg, 2.78
umoles) in DMF (0.1 mL) was combined with compound 7a (3.74 mg,
2.78 umoles) and treated with diisopropylethylamine (1 uL, 5.56
umoles) followed by BOP reagent (1.84 mg, 4.17 umoles) added as a
solution in DMF (0.184 mL of a 10 mg/mL solution). The reaction was
stirred at room temperature 2 h. HPLC analysis, as described in
section (a), indicated >80% conversion to the product eluting at
Rt=18.1 minutes. The product was purified by preparative HPLC using
an YMC, C.sub.18 20.times.250 mm column and a 30 minute of
10.fwdarw.70% B (A=water with 0.05% TFA, B=MeCN with 0.05% TFA)
gradient of at a solvent flow rate of 16 mL/minute and UV detection
at 260 nm. The HPLC fractions containing product were combined,
frozen at -80oC and lyophilized to dryness. Yield=4.6 mg (98%);
MALDI-TOF MS 1688.4 observed.
[0148] The following reactions describe the synthesis of B2-AE-E2,
7b.
##STR00032##
EXAMPLE 8
B4-AE-E2, 8b
[0149] a) Compound 8a. A solution of compound 4e (26 mg, 18 umoles)
in anhydrous DMF (2 mL) was treated with diisopropylethylamine (4.0
uL, 27 umoles) and TSTU (6.6 mg, 22 umoles). The reaction was
stirred at room temperature. After 30 minutes, HPLC analysis of the
reaction mixture using a Phenomenex, C.sub.18 4.6 mm.times.25 cm
column and a 30 minute gradient of 10.fwdarw.100% B (A=water with
0.05% TFA, B=MeCN with 0.05% TFA) at a flow rate of 1.0 mL/minute
and UV detection at 260 nm, indicated complete conversion to the
NHS ester eluting at Rt=18.7 minutes. This reaction was added
dropwise to a stirred solution of diamino hexa(ethylene) glycol
(U.S. Pat. No. 6,664,043), (25 mg, 0.089 mmol) in anhydrous DMF
(2.0 mL). After 30 minutes, HPLC analysis of the reaction mixture
showed complete conversion to the product 8a eluting at Rt=15.1
minutes. The product was purified by preparative HPLC using an YMC,
C.sub.18 30.times.300 mm column and the same gradient as described
above at a solvent flow rate of 20 mL/minute and UV detection at
260 nm. The HPLC fractions containing product were combined and
concentrated under reduced pressure. Yield=22.5 mg (73%); MALDI-TOF
MS 1698.6 observed.
[0150] b) B4-AE-E2, 8b. Estradiol-6-carboxymethyloxime (1 mg, 2.78
umoles) in DMF (0.1 mL) was combined with compound 8a (4.72 mg,
2.78 umoles) and treated with diisopropylethylamine (1 uL, 5.56
umoles) followed by BOP reagent (1.84 mg, 4.17 umoles) added as a
solution in DMF (0.184 mL of a 10 mg/mL solution). The reaction was
stirred at room temperature 2 h. HPLC analysis, as described in
section (a), indicated >80% conversion to the product eluting at
Rt=18.9 minutes. The product was purified by preparative HPLC using
an YMC, C.sub.18 20.times.250 mm column and a 30 minute of
10.fwdarw.70% B (A=water with 0.05% TFA, B=MeCN with 0.05% TFA)
gradient of at a solvent flow rate of 16 mL/minute and UV detection
at 260 nm. The HPLC fractions containing product were combined,
frozen at -80oC and lyophilized to dryness. Yield=4.0 mg (70%);
MALDI-TOF MS 2040.9 observed.
[0151] The following reactions describe the synthesis of B4-AE-E2,
8b.
##STR00033##
EXAMPLE 9
[0152] General procedure for labeling anti-TSH Mab with acridinium
ester. A stock solution of the antibody (5 mg/mL, 50 uL, 0.5 mg,
3.4 nmoles) was diluted with either 0.1 M phosphate buffer pH 8
(150 uL) or 0.1 M sodium carbonate pH 9 (150 uL) to give a 2.5
mg/mL solution. To this solution was added 20 equivalents of the
acridinium NHS ester as a DMF solution. For example, using
B1-AE-NHS, this entailed the addition of 83 ug added as 8.3 uL of a
10 mg/mL DMF solution of the acridinium ester.
[0153] The labeling reactions were stirred gently at room
temperature for 3-4 hours and were then diluted with de-ionized
water (1.8 mL). These diluted solutions were then transferred to 2
mL Centricon filters (MW 30,000 cutoff) and centrifuged at 4500 G
to reduce the volume to .about.0.2 mL. This process was repeated
three more times. The filtered conjugates were finally diluted into
a total volume of 200 uL de-ionized water for mass spectral
analysis and RLU measurements.
[0154] Mass spectra were recorded on a Voyager DE MALDI-TOF mass
spectrometer and the unlabeled antibody was used as the reference.
Approximately 2 uL of the conjugate solution was mixed with 2 uL of
sinnapinic acid matrix solution (HP) and the spotted on a MALDI
plate. After complete drying, mass spectra were recorded. From the
difference in mass values for the unlabeled antibody and the
conjugates, the extent of AE incorporation could be measured.
Typically, under these labeling conditions, 3-6 AE labels were
incorporated in the antibody.
EXAMPLE 10
[0155] Measurement of Stability. Maximization of stability of
acridinium esters is one parameter by which assay precision is
enhanced. Chemiluminescence stability of several acridinium esters
covalently attached to anti-TSH antibody were analyzed for the
correlation of molecular structure of the acridinium ester to the
stabilty of chemiluminescent under both a nominal storage
temperature of 4.degree. C. and an elevated storage temperature of
37.degree. C. Equivalent amounts of acridinium ester-labeled
antiTSH (thyroid stimulating hormone) antibody each conjugated to a
different acridinium ester were diluted to a concentration of 0.2
nanomolar in Siemens Healthcare Diagnostics TSH3 (thyroid
stimulating hormone) Lite Reagent buffer consisting of 0.1 M sodium
N-(2-hydroxyethyl) piperazine-N'-2-ethanesulfonate (HEPES), 0.15 M
sodium chloride, 7.7 mM sodium azide, 1.0 mM tetrasodium
ethylenediaminetetraacetate, (EDTA), 12 mM
t-octylphenoxypolyethoxyethanol (Triton X-100), 76 uM bovine serum
albumin (BSA), 7 uM mouse immunoglobin (IgG), pH 7.7. Each
acridinium ester solution was partitioned into two sets of storage
vessels. One set of storage vessels was kept at 4.degree. C. and
the other at 37.degree. C. Starting from the day of initial
dilution the chemiluminescence from 10 microliters of each
acridinium ester-antibody solution was determined under standard
conditions on a Berthold Technolgies Autolumat LB953 luminometer
with sequeuntial addition of 300 microliters each of Siemens
Healthcare Diagnostics Flash Reagent 1 (0.1 M nitric acid and 0.5%
hydrogenperoxide) and Siemens Healthcare Diagnostics Flash Reagent
2 (0.25 M sodium hydroxide and 0.05% cetyltrimethylammonium
chloride).
EXAMPLE 11
[0156] Measurement of Fractional Non-specific Binding. Minimization
of fractional nonspecific binding (fNSB) of acridinium esters to a
solid phase is one parameter by which assay sensitivity is
enhanced. The fractional nonspecific bindings of several acridinium
esters covalently attached to antiTSH antibody were analyzed for
correlation to the molecular structure of the acridinium ester.
Equivalent amounts of acridinium ester-labeled antiTSH (thyroid
stimulating hormone) antibody each conjugated to a different
acridinium ester were diluted to a concentration of 2 nanomolar in
Siemens Healthcare Diagnostics TSH3 (thyroid stimulating hormone)
Lite Reagent buffer consisting of 0.1 M sodium N-(2-hydroxyethyl)
piperazine-N'-2-ethanesulfonate (HEPES), 0.15 M sodium chloride,
7.7 mM sodium azide, 1.0 mM tetrasodium
ethylenediaminetetraacetate, (EDTA), 12 mM
t-octylphenoxypolyethoxyethanol (Triton X-100), 76 uM bovine serum
albumin (BSA), 7 uM mouse immunoglobin (IgG), pH 7.7. Following
dilution 100 microliters of the acridinium ester containing
solutions were each with 200 microliters of horse serum (Siemens
Healthcare Diagnostics Multi-diluent 1) and 200 microliters of
either of two solid phases. The first solid phase was 200
microliters of Siemens Healthcare Diagnostics ACS PTH (parathyroid
hormone) Solid Phase containing 50 micrograms of magnetic latex
microparticles (MLP) derivatized with antiPTH antibody. The second
solid phase was 200 microliters of Siemens Healthcare Diagnostics
ACS TSH3 (thyroid stimulating hormone) Solid Phase containing 60
micrograms of paramagnetic microparticles (PMP) derivatized with
antiTSH antibody. The particles were magnetically collected and
washed twice with water after an incubation of 10 minutes to allow
interaction between the acridinium ester labeled antbodies and the
solid phases. The chemiluminescence of acridinium ester associated
with the particles was meaured under standard conditions on a
Berthold Technolgies Autolumat LB953 luminometer with sequeuntial
addition of 300 microliters each of Siemens Healthcare Diagnostics
Flash Reagent 1 (0.1 M nitric acid and 0.5% hydrogenperoxide) and
Siemens Healthcare Diagnostics Flash Reagent 2 (0.25 M sodium
hydroxide and 0.05% cetyltrimethylammonium chloride).
Chemiluminescence was measured for 5.0 seconds. Fractional
nonspecific binding (fNSB) is calculated as the ratio of
particle-bound chemiluminescence to total chemiluminescence input.
In general hydrophobicity of an acridinium ester elevates fNSB and
is undesirable when distinguishing small amounts of specific
signal, conversely hydrophilicity of an acridinium ester lowers
fNSB and is desirable when distinguishing small amounts of specific
signal.
EXAMPLE 12
[0157] Measurement of Chemiluminescence Kinetics. Hastening of
acridinium ester chemiluminescence rates is one parameter by which
assay throughput rates can be increased. Chemiluminescence kinetics
of several acridinium esters covalently attached to anti-TSH
antibody were analyzed for the correlation of molecular structure
of the acridinium ester to its rate of chemiluminescence light
emission. Each acridinium ester labeled antibody was diluted to a
concentration of 0.2 nanomolar in a buffer consisting of 0.1 M
sodium phosphate, 0.15 M sodium chloride, 6 mM sodium azide and 1
g/L bovine serum albumin (BSA). The chemiluminescence kinetics for
10 microliters of each acridinium ester-antibody conjugate tested
was integrated in 0.1 second intervals for 20 seconds under
standard conditions on a Berthold Technolgies Autolumat LB953
luminometer with sequeuntial addition of 300 microliters each of
Siemens Healthcare Diagnostics Flash Reagent 1 (0.1 M nitric acid
and 0.5% hydrogenperoxide) and Siemens Healthcare Diagnostics Flash
Reagent 2 (0.25 M sodium hydroxide and 0.05% cetyltrimethylammonium
chloride). The chemiluminescence kinetics of the tested acridinium
esters were compared for relative rate of light emission.
EXAMPLE 13
[0158] Measurement of Quantum Yield. Increasing acridinium ester
chemiluminescence quantum yield is one parameter by which assay
sensitivity can be increased. Chemiluminescence quantum yields of
several acridinium esters covalently attached to antiTSH antibody
were tested for the correlation of molecular structure of the
acridinium esters to the magnitude of their chemiluminescence light
output. Each acridinium ester labeled antibody was diluted to a
concentration of 0.2 nanomolar in a buffer consisting of 0.1 M
sodium phosphate, 0.15 M sodium chloride, 6 mM sodium azide and 1
g/L bovine serum albumin (BSA). The chemiluminescence kinetics for
10 microliters of each acridinium ester-antibody conjugate tested
was measured for 10 seconds under standard conditions on a Berthold
Technolgies Autolumat LB953 luminometer with sequeuntial addition
of 300 microliters each of Siemens Healthcare Diagnostics Flash
Reagent 1 (0.1 M nitric acid and 0.5% hydrogenperoxide) and Siemens
Healthcare Diagnostics Flash Reagent 2 (0.25 M sodium hydroxide and
0.05% cetyltrimethylammonium chloride). The chemiluminescence
quantum yield was calculated as the ratio of the chemiluminescence
to the amount of acridinium ester tested.
[0159] All patent and non-patent literature referenced in this
specification is hereby incorporated by reference.
[0160] The invention having been described by the foregoing
description of the preferred embodiments, it will be understood
that the skilled artisan may make modifications and variations of
these embodiments without departing from the spirit or scope of the
invention as set forth in the following claims.
* * * * *