U.S. patent application number 14/410296 was filed with the patent office on 2015-10-15 for 1,1 '-[[(substituted alkyl)imino]bis(alkylene)]bis-ferrocenes and their use in i electrochemical assays by labelling substrates of interest.
The applicant listed for this patent is Atlas Genetics Limited. Invention is credited to Christopher FROST, Barrie MARSH, David PEARCE.
Application Number | 20150293107 14/410296 |
Document ID | / |
Family ID | 46704099 |
Filed Date | 2015-10-15 |
United States Patent
Application |
20150293107 |
Kind Code |
A1 |
MARSH; Barrie ; et
al. |
October 15, 2015 |
1,1 '-[[(SUBSTITUTED ALKYL)IMINO]BIS(ALKYLENE)]BIS-FERROCENES AND
THEIR USE IN I ELECTROCHEMICAL ASSAYS BY LABELLING SUBSTRATES OF
INTEREST
Abstract
Compounds of general formula I wherein Fc and Fc' may be the
same or different and are substituted ferrocenyl moieties having at
least one ring substituent selected from sulfur-containing groups,
phosphorus-containing groups, iodo, chloro, silyl, fluoroalkyl
groups containing two or more fluorine atoms, heteroaryl,
substituted phenyl, and cyano, wherein if present as sole
substituent the cyano group is located on the proximal
cyclopentadienyl ring; X is a spacer, Y is a spacer, Z is a spacer;
and R is a linker group. Compound I may be used to make labelled
substrates, functionalised compounds for making labelled substrates
and may be used as labels in an electrochemical assay.
##STR00001##
Inventors: |
MARSH; Barrie; (Trowbridge,
GB) ; PEARCE; David; (Trowbridge, GB) ; FROST;
Christopher; (Bath and North East Somerset, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Atlas Genetics Limited |
Trowbridge |
|
GB |
|
|
Family ID: |
46704099 |
Appl. No.: |
14/410296 |
Filed: |
June 21, 2013 |
PCT Filed: |
June 21, 2013 |
PCT NO: |
PCT/GB2013/051643 |
371 Date: |
December 22, 2014 |
Current U.S.
Class: |
436/76 ;
536/23.1; 549/209; 556/144; 556/145 |
Current CPC
Class: |
C12Q 1/6816 20130101;
C07F 17/02 20130101; G01N 33/58 20130101; C12Q 1/6816 20130101;
C12Q 2563/113 20130101 |
International
Class: |
G01N 33/58 20060101
G01N033/58; C12Q 1/68 20060101 C12Q001/68; C07F 17/02 20060101
C07F017/02 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 22, 2012 |
GB |
1211157.1 |
Claims
1. A method of detecting a substrate in an electrochemical assay
comprising contact the substrate with a label comprising a compound
of general formula I: ##STR00064## in which: Fc is a substituted
ferrocenyl moiety having at least one ring substituent selected
from sulfur-containing groups, phosphorus-containing groups, iodo,
chloro, silyl, fluoroalkyl groups containing two or more fluorine
atoms, heteroaryl, substituted phenyl, and cyano, wherein if
present as sole substituent the cyano group is located on the
proximal cyclopentadienyl ring; Fc' is a substituted ferrocenyl
moiety having at least one ring substituent selected from
sulfur-containing groups, phosphorus-containing groups, iodo,
chloro, silyl, fluoroalkyl groups containing two or more fluorine
atoms, heteroaryl, substituted phenyl, and cyano, wherein if
present as sole substituent the cyano group is located on the
proximal cyclopentadienyl ring, and may be the same as or different
from Fc; X is a spacer; Y is a spacer; Z is a spacer; and R is a
linker group.
2. The method according to claim 1, wherein: X represents C1 to
C6-alkylene optionally interrupted by oxygen; Y represents C1 to
C6-alkylene optionally interrupted by oxygen, and Z represents C1
to C8 alkylene optionally interrupted by oxygen.
3. The method according to claim 1, wherein: X is
--(CH.sub.2).sub.x-- in which x is 1 or 2; Y is
--(CH.sub.2).sub.y-- in which y is 1 or 2; Z is (CH.sub.2).sub.z in
which z is from 1 to 8.
4. The method according to any claim 1, wherein Fc and Fc' are the
same and X and Y are the same.
5. (canceled)
6. The method according to claim 1, wherein the assay is for
detecting a biological or synthetic substrate selected from
nucleotides, nucleosides, oligonucleotides, and polynucleotides or
from amino acids, peptides, and proteins.
7. (canceled)
8. A method for manufacturing a functionalized labelling compound
comprising a label moiety for use in an electrochemical assay,
comprising reacting a compound of general formula I ##STR00065## in
which: Fc is a substituted ferrocenyl moiety having at least one
ring substituent selected from sulfur-containing groups,
phosphorus-containing groups, iodo, chloro, silyl, fluoroalkyl
groups containing two or more fluorine atoms, heteroaryl,
substituted phenyl, and cyano, wherein if present as sole
substituent the cyano group is located on the proximal
cyclopentadienyl ring; Fc' is a substituted ferrocenyl moiety
having at least one ring substituent selected from
sulfur-containing groups, phosphorus-containing groups, iodo,
chloro, silyl, fluoroalkyl groups containing two or more fluorine
atoms, heteroaryl, substituted phenyl, and cyano, wherein if
present as sole substituent the cyano group is located on the
proximal cyclopentadienyl ring, and may be the same as or different
from Fc; X is a spacer; Y is a spacer; Z is a spacer; and R is a
linker group, with a functionalising compound to obtain a
funtionalised labelling compound of general formula III: A-L-F III
in which A represents ##STR00066## wherein Fc, Fc', X, Y and Z are
as defined above, F represents a functionalising moiety; and L
represents a linker moiety.
9. A method for the manufacture of a labelled substrate, comprising
reacting a compound of general formula III: A-L-F III in which A, F
and L are as defined in claim 8; with a substrate to form a
labelled substrate, wherein the substrate is selected from amino
acids, nucleotides, nucleosides, sugars, peptides, proteins,
oligonucleotides, polynucleotides, carbohydrates, microparticles
and nanoparticles.
10. (canceled)
11. (canceled)
12. (canceled)
13. A functionalised labelling compound having the general formula
III: A-L-F III in which A represents a labelling moiety of general
formula Ia: ##STR00067## wherein: Fc is a substituted ferrocenyl
moiety having at least one ring substituent selected from
sulfur-containing groups, phosphorus-containing groups, iodo,
chloro, silyl, fluoroalkyl groups containing two or more fluorine
atoms, heteroaryl, substituted phenyl, and cyano, wherein if
present as sole substituent the cyano group is located on the
proximal cyclopentadienyl ring; Fc' is a substituted ferrocenyl
moiety having at least one ring substituent selected from
sulfur-containing groups, phosphorus-containing groups, iodo,
chloro, silyl, fluoroalkyl groups containing two or more fluorine
atoms, heteroaryl, substituted phenyl, and cyano, wherein if
present as sole substituent the cyano group is located on the
proximal cyclopentadienyl ring, and may be the same as or different
from Fc; X is a C1 to C6 alkylene chain which is optionally
interrupted by --O--, --S--, or --NR.sup.5 in which R.sup.5
represents hydrogen or C1 to C6 alkyl; Y is a C1 to C6 alkylene
chain which is optionally interrupted by --O-- S --, or
--NR.sup.5--, in which R.sup.5 represents hydrogen or C1 to C6
alkyl; Z is a C1 to C12 alkylene chain which may optionally be
substituted and/or may optionally be interrupted by --O--, --S--,
cycloalkyl, --CO--CONR.sup.1--, --NR.sup.1CO-- or --NR.sup.1 in
which R.sup.1 represents hydrogen or C1 to C4 alkyl; L represents a
linker moiety; and in which F represents a functionalising moiety
for reacting with a substrate for attachment of the labelling
moiety to the substrate.
14. The functionalised labelling compound according to claim 13,
wherein the substrate is a naturally occurring or synthetic
substrate selected from naturally occurring or synthetic amino
acids, nucleotides, nucleosides, sugars, peptides, proteins,
oligonucleotides, polynucleotides, carbohydrates, microparticles
and nanoparticles.
15. The functionalized labelling compound according to claim 13,
wherein F is selected from succinimidyl ester groups,
phosphoramidite groups, maleimide groups, biotin and azide
groups.
16. The functionalized labeling compound according to claim 13,
wherein F is or is derivable from a phosphoramidite moiety.
17. A labelled substrate for use in an electrochemical assay, the
labelled substrate being of general formula: A-L-F'--[S] IIIa in
which A represents ##STR00068## in which: Fc is a substituted
ferrocenyl moiety having at least one ring substituent selected
from sulfur-containing groups, phosphorus-containing groups, iodo,
chloro, silyl, fluoroalkyl groups containing two or more fluorine
atoms, heteroaryl, substituted phenyl, and cyano, wherein if
present as sole substituent the cyano group is located on the
proximal cyclopentadienyl ring; Fc' is a substituted ferrocenyl
moiety having at least one ring substituent selected from
sulfur-containing groups, phosphorus-containing groups, iodo,
chloro, silyl, fluoroalkyl groups containing two or more fluorine
atoms, heteroaryl, substituted phenyl, and cyano, wherein if
present as sole substituent the cyano group is located on the
proximal cyclopentadienyl ring, and may be the same as or different
from Fc; X is a spacer; Y is a spacer; Z is a spacer; L-F'
represents a linking moiety; and [S] represents a substrate.
18. (canceled)
19. A labelled substrate according to claim 17, wherein the
substrate is a microparticle, nanoparticle, biological molecule or
a synthetic analog selected from naturally occurring or synthetic
amino acids, nucleotides, nucleosides, sugars, peptides, proteins,
oligonucleotides, polynucleotides, and carbohydrates.
20. (canceled)
21. (canceled)
22. (canceled)
23. (canceled)
24. A compound according to general formula I ##STR00069## in
which: Fc is a substituted ferrocenyl moiety having at least one
ring substituent selected from sulfur-containing groups,
phosphorus-containing groups, iodo, chloro, silyl, fluoroalkyl
groups containing two or more fluorine atoms, heteroaryl,
substituted phenyl, and cyano, wherein if present as sole
substituent the cyano group is located on the proximal
cyclopentadienyl ring; Fc' is a substituted ferrocenyl moiety
having at least one substituent selected from the group consisting
of sulfonyl, having at least one ring substituent selected from
sulfur-containing groups, phosphorus-containing groups, iodo,
chloro, silyl, fluoroalkyl groups containing two or more fluorine
atoms, heteroaryl, substituted phenyl, and cyano, wherein if
present as sole substituent the cyano group is located on the
proximal cyclopentadienyl ring, and may be the same as or different
from Fc; X is a spacer; Y is a spacer; Z is a spacer; and R is a
linker group.
25. The compound according to claim 24, wherein either: (a) X
represents C1 to C6-alkylene optionally interrupted by oxygen; Y
represents C1 to C6-alkylene optionally interrupted by oxygen, and
Z represents C1 to C8 alkylene optionally interrupted by oxygen; or
(b) X represents --(CH2).sub.x-- in which x is from 1 to 6; and Y
represents --(CH2).sub.y-- in which y is from 1 to 6.
26. The compound according to claim 24, wherein Z represents C6 to
C8 alkylene optionally interrupted by oxygen.
27. The compound according to claim 24, in which the linker group R
comprises a group capable of reacting with a compatible group of a
functionalising moiety or of a substrate to attach the compound to
said functionalising moiety or said substrate.
28. (canceled)
29. The compound of claim 24, wherein Fc and Fc' are the same and
each comprise at least one substituent selected from the group
consisting of: sulfur-containing groups selected from sulfenyl,
sulfinyl and sulfonyl groups, phosphorus-containing groups selected
from phosphanyl and phosphinyl groups, iodo, chloro, fluoroalkyl
groups containing two or more fluorine atoms, heteroaryl, and
substituted phenyl.
30. The compound of claim 24, wherein Fc and Fc' are the same and
there is present as a said substituent a cyano group located on the
respective proximal cyclopentadienyl ring of each of said Fc and
Fc' moieties.
31. The compound of claim 24, wherein Fc and Fc' are the same and
each comprise at least one ring substituent selected from
sulfur-containing groups and phosphorus-containing groups.
32. The compound of claim 24, wherein there is present as a said
sulfur-containing group at least one sulfonyl substituent selected
from groups of the formula R.sup.15S(0).sub.2-, wherein R.sup.15 is
selected from branched or straight-chain alkyl, haloalkyl, and
substituted or unsubstituted aryl, for example wherein R.sup.15
represents C1 to C4 alkyl; or C1 to C4 haloalkyl, for example C1 to
C4 fluoroalkyl, especially trifluoromethyl; or. R.sup.15 represents
(R.sup.16).sub.a--Ar--, or (R.sup.16).sub.a--HeAr-- in which Ar
represents aryl; HeAr represents heteroaryl; R.sup.16 is a
substituent selected from halo, alkyl, nitro, cyano, haloalkyl, and
alkoxy; and a is an integer in the range of from 0 to a number
equal to the maximum substitutable ring positions in the aryl or
heteroaryl ring.
33. (canceled)
34. (canceled)
35. The compound of claim 24, wherein Fc and Fc' are the same and
each comprise at least one substituent selected from groups of the
formula (R.sup.17).sub.2P(0)-, wherein R.sup.17 is independently
selected from branched or straight-chain alkyl, haloalkyl,
substituted or unsubstituted aryl and substituted or unsubstituted
heteroaryl, for example R.sup.17 represents C1 to C4 alkyl or
R.sup.17 represents unsubstituted aryl or unsubstituted heteroaryl
or represents (R.sup.18)b-Ar--, or (R.sup.18).sub.b--HeAr-- in
which Ar is aryl; HeAr is heteroaryl; R.sup.18 is a substituent
selected from halo, alkyl, nitro, cyano, haloalkyl, and alkoxy; and
b is an integer in the range of from 0 to a number equal to the
maximum substitutable ring positions in the aryl or heteroaryl
ring; for example wherein Ar represents phenyl and comprises one or
more substituents R.sup.18 (which may be the same or different)
selected from halo, alkyl, haloalkyl, nitro, cyano, and alkoxy, and
sulphur-containing groups.
36. (canceled)
37. (canceled)
38. (canceled)
39. The compound of claim 32, wherein R.sup.17 represents branched
C1 to C4 alkyl, for example t-butyl.
40. The compound of claim 24, wherein there is present as a said
substituent on each ferrocenyl at least one substituted phenyl
group in which the phenyl has at least one substituent selected
from halo, C1 to C4 alkyl, nitro, cyano, C1 to C4 haloalkyl, C1 to
C4 alkoxy and sulphur-containing groups, for example sulfinyl,
sulfenyl and sulfonyl.
41. The compound of claim 24, wherein there is present as a said
substituent on each ferrocenyl at least one heteroaryl group which
may be unsubstituted or substituted by at least one substituent
selected from halo, C1 to C4 alkyl, nitro, cyano, C1 to C4
haloalkyl and C1 to C4 alkoxy, for example wherein the heteroaryl
group is furanyl.
42. (canceled)
43. The compound of claim 24, wherein there is present as a said
substituent on each ferrocenyl at least one iodine or chlorine
atom.
44. The compound of claim 24, wherein Fc and Fc' are the same and
each comprise as a said substituent group at least one silyl
substituent, preferably a silyl group selected from alkyl silyl
groups, for example trimethylsilyl.
45. The compound of claim 24, wherein each ferrocenyl moiety is
further substituted by at least one additional substituent, for
example at least one additional substituent selected from bromo,
fluoro, C1 to C4 alkyl, haloalkyl, and C1 to C4 alkenyl.
46. (canceled)
47. The compound of claim 24, wherein Fc and Fc' each additionally
comprises at least one cyano group substituent on its distal
ring.
48. The compound of claim 24 wherein the compound is selected from
6-(bis((2-tert-butyl-sulfonylferrocenyl)1-methylferrocenyl)amino)hexan-1--
ol 6-(bis((2-cyanoferrocenyl) 1-methylferrocenyl)amino)hexan-1-ol
6-(bis((2-di-tert-butyl-phosphinyl-ferrocenyl)1-methylferrocenyl)amino)he-
xan-1-ol
6-(bis((1'-iodoferrocenyl)1-methylferrocenyl)amino)hexan-1-ol
6-(bis((1'-(4-nitrobenzyl)ferrocenyl)1-methylferrocenyl)amino)hexan-1-ol
6-(bis((1'-(1-furanyl)ferrocenyl)
1-methylferrocenyl)amino)hexan-1-ol 6-(bis((1'-chloroferrocenyl)
1-methylferrocenyl)amino)hexan-1-ol
6-(bis((2-tert-butyl-sulfinylferrocenyl)1-methylferrocenyl)amino)hexan-1--
ol
6-(bis((2-tert-butyl-sulfidylferrocenyl)1-methylferrocenyl)amino)hexan--
1-ol.
49. The compound of claim 24 wherein the compound is a
functionalised derivative of a compound according to claim 48.
50. The compound of claim 24, wherein R represents a linker group
comprising an oxygen atom, for example a linker group comprising a
hydroxyl group or a protected hydroxyl group.
51. A method of detecting a nucleic acid, comprising: (i)
contacting the nucleic acid with a complementary nucleic acid probe
under conditions to allow hybridization between the probe and
amplicon, (i) selectively degrading the either hybridized or
unhybridized probe, wherein said probe is labelled with compound
according to claim 24; and (iii) measuring the electrochemical
activity of the compound labelling the probe, wherein said
electrochemical activity is dependent either quantitatively or
qualitatively on the extent of degradation of the probe.
52. (canceled)
53. (canceled)
54. A nucleic acid labelled with a compound of claim 24.
Description
[0001] This application claims the benefit of United Kingdom patent
application 1211157.1 (filed 22 Jun. 2012), the complete contents
of which are hereby incorporated herein by reference for all
purposes.
FIELD OF THE INVENTION
[0002] The invention relates to electrochemical detection methods.
More especially, the invention relates to electrochemical assays,
to electrochemically active labels for use in electrochemical
detection methods, and to their use.
BACKGROUND OF THE INVENTION
[0003] The detection of certain biological molecules plays an
important part in many aspects of life. For example, in the medical
field, there is an ever-present need to detect bacterial or viral
pathogens, or biological molecules. Other fields in which sensitive
assays are essential include the food and beverage industries.
[0004] WO03/074731 discloses a method of probing for a nucleic
acid. A nucleic acid solution is contacted with an oligonucleotide
probe with an electrochemically active marker. The probe is caused
to at least partially hybridise with any complementary target
sequence which may be present in the nucleic acid solution.
Following enzymatic degradation of the nucleic acid probe,
information is electrochemically determined relating to the marker.
Compounds for use in the method are also disclosed.
[0005] WO2005/005657 discloses a method of detecting protease
activity in which a sample solution is contacted with a protease
substrate with an electrochemically active marker, providing
conditions under which any protease which may be present in the
sample may degrade the protease substrate and information relating
to the electrochemically active marker is electrochemically
determined. Certain novel compounds for use in the process were
also disclosed.
[0006] WO2012/085591 describes certain diferrocenyl compounds for
use as electrochemical labels.
[0007] There is a continuing need to develop labels that enable
detection of the presence in small concentrations of biological
substrates or indicators, for example, nucleic acids (in isolated
form or in the form of larger molecules, for example, natural or
synthetic oligonucleotides), or amino acids (in isolated form or in
the form of larger molecules, for example, natural or synthetic
peptides). In particular, there is a continuing need for new labels
with different oxidation potentials and/or with different chemical
or physical properties thereby widening the range of possible
assays available and increasing the scope for the development of
multiplex reactions.
SUMMARY OF THE INVENTION
[0008] The invention provides a compound according to general
formula I
##STR00002##
in which: [0009] Fc is a substituted ferrocenyl moiety having at
least one ring substituent selected from sulfur-containing groups,
phosphorus-containing groups, iodo, chloro, silyl, fluoroalkyl
groups containing two or more fluorine atoms, heteroaryl,
substituted phenyl, and cyano, wherein if present as sole
substituent the cyano group is located on the proximal
cyclopentadienyl ring; [0010] Fc' is a substituted ferrocenyl
moiety having at least one ring substituent selected from
sulfur-containing groups, phosphorus-containing groups, iodo,
chloro, silyl, fluoroalkyl groups containing two or more fluorine
atoms, heteroaryl, substituted phenyl, and cyano, wherein if
present as sole substituent the cyano group is located on the
proximal cyclopentadienyl ring, and may be the same as or different
from Fc; [0011] X is a spacer [0012] Y is a spacer [0013] Z is a
spacer; and [0014] R is a linker group.
[0015] The invention also provides use as a label in an
electrochemical assay of a compound of general formula I:
##STR00003##
in which: [0016] Fc is a substituted ferrocenyl moiety, having at
least one ring substituent selected from sulphur-containing groups,
phosphorus-containing groups, iodo, chloro, silyl, fluoroalkyl
groups containing two or more fluorine atoms, heteroaryl,
substituted phenyl, and cyano, wherein if present as sole
substituent the cyano group is located on the proximal
cyclopentadienyl ring; [0017] Fc' is a substituted ferrocenyl
moiety having at least one ring substituent selected from
sulphur-containing groups, phosphorus-containing groups, iodo,
chloro, silyl, fluoroalkyl groups containing two or more fluorine
atoms, heteroaryl, substituted phenyl, and cyano, wherein if
present as sole substituent the cyano group is located on the
proximal cyclopentadienyl ring, and may be the same as or different
from Fc; [0018] X is a spacer [0019] Y is a spacer [0020] Z is a
spacer; and [0021] R is a linker group.
[0022] The compounds used in accordance with the invention have
been found to be effective labels for use in electrochemical
assays. In particular, the compounds may be used to form labelled
substrates. Molecules of interest as substrates that may be
labelled include, but are not limited to: amino acids, nucleotides,
nucleosides, sugars, peptides, proteins, oligonucleotides,
polynucleotides, carbohydrates and derivatives or synthetic analogs
of any of those molecules. Other substrates that might be labelled
using the compounds of the invention include latex/paramagnetic
microparticles and nanoparticles. The labelling compounds of
general formula I and labelled molecules including labels derivable
from the labelling compounds are potentially useful in
electrochemical techniques in which their electrochemical
characteristics can be utilized to derive information about the
labels or their environment. For example, the compounds of the
invention may find use in a method as described in WO03/074731 or
in a method as described in WO2005/005657. The labelling compounds
of the invention and the labelled substrates derived therefrom
offer characteristics which make them useful complements to
previously known labelling compounds, permitting a wider spectrum
of applications, for example, offering additional opportunities for
avoidance of conditions under which measurement potential may be
compromised by interference with impurities that may be present
and/or offering differing electrochemical potential values and/or
allowing more greater flexibility in multiplex assays. A number of
the compounds and the corresponding labelled substrates have
relatively high electro potential values, as illustrated in
particular by Example 4 below. It is believed that, especially,
compounds of the invention having sulfur-containing or
phosphorus-containing substituents and their corresponding labelled
substrates will be useful in providing for assays in which the
measurement potential will be relatively high, for example, in
excess of 400 mV, for example in excess of 450 mV or even in excess
of 500 mV. Compounds having electrochemical potentials of at least
450 mV, for example 500 mV or more, will be particularly useful in
extending the range of available potential values and therefore,
for example, in potentially providing for more effective multiplex
assays. Other compounds of the invention having highly
electron-withdrawing substituents, for example, trifluoromethyl or
cyano, are believed to have similar advantages in terms of offering
high electrochemical potential values thereby extending the range
of useful labels and labelled substrates. Additionally, some
compounds of the invention and the corresponding labelled
substrates offer the advantage of having a narrower voltage peak,
which is advantageous in providing for the option of utilising a
greater number of labels in a multiplex assay, since the narrower
measurement peaks result in wider gaps between peaks, which may be
utilised if desired by incorporating additional labels with
potentials that will be within the gaps.
[0023] In all aspects of the invention, the following spacers are
preferred: X is a C1 to C6 alkylene chain which is optionally
interrupted by --O--, --S--, or --NR.sup.5--, in which R.sup.5
represents hydrogen or C1 to C6 alkyl; Y is a C1 to C6 alkylene
chain which is optionally interrupted by --O--, --S--, or
--NR.sup.5--, in which R.sup.5 represents hydrogen or C1 to C6
alkyl; and Z is a C1 to C12 alkylene chain which may optionally be
substituted and/or may optionally be interrupted by --O--, --S--,
cycloalkyl, --CO--, --CONR.sup.1--, --NR.sup.1CO-- or --NR.sup.1--
in which R.sup.1 represents hydrogen or C1 to C4 alkyl.
[0024] In the compounds used according to the invention it is
preferred that X represents C1- to C6-alkylene optionally
interrupted by oxygen; Y represents C1 to C6-alkylene optionally
interrupted by oxygen; and Z represents C1 to C8 alkylene
optionally interrupted by oxygen. Thus in these embodiments X, Y
and Z can be represented by the formula
(CH.sub.2).sub.a--O--(CH.sub.2).sub.b wherein a.gtoreq.0 and
b.gtoreq.0. For X and Y, a+b=1-6. For Z, a+b=1-8. Ideally
a.gtoreq.1 and b.gtoreq.1.
[0025] X is preferably --(CH.sub.2).sub.x-- in which x is from 1 to
6, preferably 1 to 4, especially 1 or 2; or C1 to C6-alkylene
interrupted by oxygen, for example
--(CH.sub.2).sub.3--O--CH.sub.2--, --(CH.sub.2).sub.2--O--
(CH.sub.2).sub.2--, or --CH.sub.2--O-- (CH.sub.2).sub.3--.
[0026] Y is preferably --(CH.sub.2).sub.y-- in which y is from 1 to
6, preferably 1 to 4, especially 1 or 2; or C1 to C6-alkylene
interrupted by oxygen, for example --(CH.sub.2).sub.3--O--
CH.sub.2--, --(CH.sub.2).sub.2--O-- (CH.sub.2).sub.2-- or
--CH.sub.2--O--(CH.sub.2).sub.3--.
[0027] Preferably X and Y are the same. Preferably Fc and Fc' are
the same and X and Y are the same.
[0028] In an embodiment, Z is a C1 to C12 alkylene chain which may
optionally be substituted and/or may optionally be interrupted by
--O--, --S-- or --NR.sup.1-- in which R.sup.1 represents hydrogen
or C1 to C4 alkyl. Preferably Z is --(CH.sub.2).sub.z-- in which z
is from 1 to 8, with z preferably representing from 1 to 6,
especially from 2 to 6; or is C1 to C8 alkylene interrupted by
oxygen, for example --(CH.sub.2).sub.2--O--(CH.sub.2).sub.3-- or
--(CH.sub.2).sub.3--O--(CH.sub.2).sub.2--. In one preferred
embodiment: X is --(CH.sub.2).sub.x-- in which x is 1 or 2; Y is
--(CH.sub.2).sub.y-- in which y is 1 or 2; and Z is
--(CH.sub.2).sub.z-- in which z is from 1 to 8. Where X and Y
represent an alkylene chain interrupted by --NR.sup.5--, R.sup.5
preferably represents hydrogen or C1 to C4 alkyl, more preferably
hydrogen.
[0029] In one preferred embodiment, the invention provides use, as
an electrochemical label, of a compound of the general formula
II:
##STR00004##
in which
[0030] Fc is a substituted ferrocenyl moiety as defined above with
reference to general formula I,
[0031] Fc' is a substituted ferrocenyl moiety as defined above with
reference to general formula I, and may be the same as or different
from Fc; [0032] x is 1 or 2; [0033] y is 1 or 2; [0034] z is from 1
to 8; [0035] and R is a linker group.
[0036] Preferably, x and y are each equal to 1.
[0037] It is preferred that the ferrocenyl moieties are the same,
and it is therefore preferred that Fc and Fc' carry the same
substituents in the same positions.
[0038] Except where the contrary is apparent from the context, the
term "substrate" is used throughout the remainder of this document
to include both naturally occurring substrates and synthetic
substrates, and references herein to amino acids, nucleotides,
nucleosides, sugars, peptides, proteins, oligonucleotides,
polynucleotides, or carbohydrates, are to be understood as
referring to naturally occurring or synthetic amino acids,
nucleotides, nucleosides, sugars, peptides, proteins,
oligonucleotides, polynucleotides, or carbohydrates. Substrates can
also be polypeptides. Synthetic substrates include synthetic
analogues of naturally occurring substrates. Substrates include
single nucleotides and single amino acids. In the case of an assay
relying upon cleavage of a substrate, for example by an enzyme, a
single amino acid may be regarded as a substrate because, although
it lacks an internal bond capable of being cleaved by a protease
enzyme, such a bond may be formed through the attachment of a
marker. Where derivatives of naturally occurring substrates are
referred to herein, those derivatives may be naturally occurring
derivatives or synthetic derivatives of the substrate.
[0039] The invention provides a method of detecting a chemical
entity using a compound according to the invention. Use in an
electrochemical assay according to the invention may be for example
in an assay for detecting an electrochemically labelled substrate.
The electrochemical assay may for example be an assay for
determination of the amount of an electrochemically labelled
substrate. The assay may advantageously be for detecting or
determining the amount of a labelled substrate wherein the labelled
substrate is selected from amino acids, nucleotides, nucleosides,
sugars, peptides, proteins, oligonucleotides, polynucleotides,
carbohydrates, microparticles and nanoparticles. In certain
preferred embodiments, the assay is for detecting or determining
the amount of a labelled substrate in which the labelled substrate
is selected from nucleotides, nucleosides, oligonucleotides, and
polynucleotides. In another advantageous embodiment, the assay is
for detecting or determining the amount of a labelled substrate in
which the labelled substrate is selected from amino acids,
peptides, and proteins.
[0040] For the purpose of attachment to substrates, the label may
be functionalised by addition of a functionalising group. Thus, the
invention further provides functionalised derivatives comprising a
moiety derivable from the compounds of the invention attached to a
functionalising group suitable for enhancing attachment to a
substrate.
[0041] The invention also provides a method for manufacturing a
functionalized labelling compound comprising a label moiety for use
in an electrochemical assay, comprising reacting a compound of
general formula I:
##STR00005##
in which: [0042] Fc is a substituted ferrocenyl moiety having at
least one ring substituent selected from sulphur-containing groups,
phosphorus-containing groups, iodo, chloro, silyl, fluoroalkyl
groups containing two or more fluorine atoms, heteroaryl,
substituted phenyl, and cyano, wherein if present as sole
substituent the cyano group is located on the proximal
cyclopentadienyl ring, [0043] Fc' is a substituted ferrocenyl
moiety having at least one ring substituent selected from
sulphur-containing groups, phosphorus-containing groups, iodo,
chloro, silyl, fluoroalkyl groups containing two or more fluorine
atoms, heteroaryl, substituted phenyl, and cyano, wherein if
present as sole substituent the cyano group is located on the
proximal cyclopentadienyl ring, and may be the same as or different
from Fc; [0044] X is a spacer [0045] Y is a spacer [0046] Z is a
spacer; and [0047] R is a linker group, with a functionalising
compound to obtain a funtionalised labelling compound of general
formula III:
[0047] A-L-F III
in which A represents
##STR00006##
wherein [0048] Fc, Fc', X, Y and Z are as defined above with
reference to general formula I; [0049] F represents a
functionalising moiety, especially a functionalising moiety for
reacting with a substrate for attachment of the labelling moiety to
the substrate; and [0050] L represents a linker moiety.
[0051] The linker moiety L will generally be a linker moiety
derivable from the linker group R. For example where R is or
contains an OH group L will usually represent or comprise
--O--.
[0052] Furthermore the invention provides a method for the
manufacture of a labelled substrate, comprising reacting a compound
of general formula III:
A-L-F III [0053] in which A, L and F are as defined above; with a
substrate to form a labelled substrate.
[0054] The invention moreover provides a functionalised labelling
compound for use in the manufacture of a labelled substrate, the
functionalised labelling compound having the general formula
III:
A-L-F III
in which A, L and F are as defined above.
[0055] The invention also provides a labelled substrate for use in
an electrochemical assay, the labelled substrate being of general
formula IIIa:
A-L-F'-[S] IIIa
in which A represents
##STR00007##
in which: [0056] Fc is a substituted ferrocenyl moiety having at
least one ring substituent selected from sulphur-containing groups,
phosphorus-containing groups, iodo, chloro, silyl, fluoroalkyl
groups containing two or more fluorine atoms, heteroaryl,
substituted phenyl, and cyano, wherein if present as sole
substituent the cyano group is located on the proximal
cyclopentadienyl ring, [0057] Fc' is a substituted ferrocenyl
moiety having at least one ring substituent selected from
sulphur-containing groups, phosphorus-containing groups, iodo,
chloro, silyl, fluoroalkyl groups containing two or more fluorine
atoms, heteroaryl, substituted phenyl, and cyano, wherein if
present as sole substituent the cyano group is located on the
proximal cyclopentadienyl ring, and may be the same as or different
from Fc; [0058] X is a spacer [0059] Y is a spacer [0060] Z is a
spacer; [0061] L-F' represents a linking moiety; and [0062] [S]
represents a substrate.
[0063] The linking moiety -L-F'-- is in general a moiety derivable
from the moiety -L-F according to general formula III or a moiety
derivable from the moiety --R according to general formula I. In an
embodiment the linking moiety -L-F'-- is a moiety derivable from
the moiety -L-F according to general formula III.
[0064] By analogy, the invention also provides a labelled substrate
for use in an electrochemical assay, the labelled substrate being
of general formula IIIb:
A-L-R'-[S] IIIb
where R' is a residue of R formed when reacting a compound of the
invention with a substrate.
[0065] The invention further provides assays comprising substrates
according to the invention.
DETAILED DESCRIPTION
[0066] Except where it is clear that the contrary is intended,
references herein to "alkyl" are to straight- or branched-chain
alkyl groups preferably having from 1 to 6 carbon atoms, more
preferably from 1 to 4 carbon atoms, optionally interrupted by a
heteroatom selected from O, S and N and/or optionally having one or
more substituents; or to cycloalkyl groups. Illustrative alkyl
groups include, for example, methyl, ethyl, n-propyl, i-propyl,
n-butyl, t-butyl.
[0067] References herein to "cycloalkyl" are to cycloalkyl groups
having up to eight, preferably up to six, ring atoms, optionally
including one or more heteroatoms. Illustrative cycloalkyl groups
include, for example, cyclohexyl and heterocyclic groups such as
piperidinyl and morpholinyl.
[0068] References herein to "alkenyl" are to straight- or
branched-chain alkenyl groups preferably having from 1 to 6 carbon
atoms, more preferably from 1 to 4 carbon atoms, optionally having
one or more substituents. Illustrative alkenyl groups include, for
example, ethenyl, propenyl, butenyl.
[0069] The term "haloalkyl" is used herein, except where the
contrary is indicated, to refer to alkyl groups having one or more
halogen atoms present as substituents, said one or more halogen
atoms being selected from fluorine, chlorine, bromine and
iodine.
[0070] Unless the contrary is indicated, "sulfur-containing group"
will be understood as including, without limitation, substituent
groups including an --S(O).sub.2-- moiety (referred to herein as
"sulfonyl"), an --S(O)-- moiety (referred to herein as "sulfinyl")
or an --S-- moiety (referred to herein as "sulfenyl"). Preferred
sulfur-containing groups that may be present as substituents on the
ferrocenyl rings in accordance with the invention are those in
which the sulfur atom is directly bonded to a ring carbon.
[0071] Unless the contrary is indicated, "phosphorus-containing
group" will be understood as including, without limitation,
substituent groups including those based on phosphines or phosphine
oxides, more particularly phosphanyl (>P--) and phosphinyl
(>P(O)--) groups. Preferred phosphorus-containing groups that
may be present as substituents on the ferrocenyl rings in
accordance with the invention are those in which the phosphorus
atom is directly bonded to a ring carbon.
[0072] References herein to "heteroaryl" are to be understood as
including any single or fused aromatic moiety including one or more
heteroatoms, the heteroatoms preferably being selected from oxygen,
sulphur and nitrogen. Where more than one heteroatom is present the
heteroatoms may be the same or different, each advantageously being
independently selected from oxygen, sulphur and nitrogen.
Illustrative heteroaryl groups include without limitation furanyl,
imidazolyl, thiazolyl.
[0073] References herein to "aryl" are to be understood as
including any single or fused aromatic ring system and include both
hetero and other ring systems.
[0074] The expression "substituted phenyl" as used herein includes
any phenyl group having one or more substituents attached to the
phenyl ring, at any ring carbon atom of the ring. Where there is
more than one substituent on the phenyl ring those substituents may
be the same or may be different from one another.
[0075] The application of electrochemical detection has a number of
advantages over fluorescent detection. Electrochemical detection
has the potential for very high levels of sensitivity and exhibits
a wider linear dynamic range than fluorescence. There is no
requirement for samples to be optically clear. There is also less
interference from background contaminants (many biological samples
auto-fluoresce).
[0076] Electrochemical detection is based on the observation that
an electrochemically active marker exhibits different
electrochemical characteristics depending on whether or not it is
attached to a substrate and on the nature of the substrate. For
example, in the case of an electrochemical label attached to an
amino acid, the exhibited characteristics will depend not only on
the identity of the amino acid but also on whether or not that
amino acid residue is incorporated into a peptide or protein, and
on the length of any such peptide or protein. Under appropriate
circumstances, the electrochemical activity of a marker attached to
an amino acid residue can change by a detectable degree following
loss of attachment of a single or very few amino acid residues.
[0077] The size and characteristics of a molecule to which an
electrochemically active marker is attached influence the
observable characteristics of the electrochemical marker. That may
occur, for example, by influencing the rate of migration of the
marker by diffusion or its rate of migration in response to an
electric field.
[0078] Electrochemical activity of a marker may also be influenced
by steric effects resulting from the presence of the molecule to
which it is linked. For example, steric hindrance may prevent the
marker from approaching an electrode and accepting or donating
electrons.
[0079] If the marker is attached to a peptide then the secondary
structure of the peptide (as largely determined by the primary
sequence) may influence the physical properties of the marker. For
example, if the marker is attached to an amino acid residue in a
peptide such that the structure of the peptide sterically hinders
the electrochemically active marker then the signals observable by
voltammetry may be reduced. Digestion of the peptide may destroy or
release secondary structure elements and thus reduce or abolish the
influence of the peptide structure on the marker. Accordingly,
digestion of the peptide results in a change, usually an increase,
in the electrochemical signal produced by the marker moiety. In a
differential pulse voltammetry experiment, the Faradaic current
response at a particular applied voltage may increase upon
digestion of the peptide.
[0080] Analogously, if a marker is attached to a nucleotide, the
electrochemical characteristics will be influenced by whether or
not the nucleotide is incorporated into an oligonucleotide, upon
the length of that oligonucleotide, and upon the sequence of the
oligonucleotide especially in the vicinity of the point of
attachment.
[0081] The information relating to the electrochemically active
marker can be obtained by voltammetry or by an amperometric method.
Differential pulse voltammetry is particularly suitable. If
desired, the electrochemical detection step may be carried out
using one or more electrodes covered by a membrane which is able
selectively to exclude molecules based on one or more
characteristics, for example, size, charge or hydrophobicity. That
may assist in eliminating background noise current arising from,
for example, charged species in the solution.
[0082] In the compounds (including labelling compounds,
functionalised labelling compounds and labelled substrates) used in
accordance with the invention, including the compounds according to
general formulae I, II and III, the labelled substrate of general
formula IIIa and the label moiety of general formula Ia, the two
ferrocenyl groups Fc and Fc' are each independently selected from
substituted ferrocenyl groups having one or more substituents as
defined above with reference to general formula I. One or both
pentadienyl rings of one or each of the ferrocenyl moieties may be
substituted by one or more substituents, the nature and location of
which are selected so as to influence in a desired manner the redox
characteristics of the ferrocene moiety. In addition to the
substituents defined above, the pentadienyl rings of the ferrocenyl
moiety may further be substituted by any further ring
substituent(s) that do not materially reduce the electrochemical
sensitivity of the label, or by any further ring substituent(s)
that will enhance the electrochemical or other characteristics of
the label in any respect.
[0083] In a preferred embodiment, Fc and Fc' are the same and each
comprise at least one substituent selected from the group
consisting of: [0084] sulfur-containing groups selected from
sulfenyl, sulfinyl and sulfonyl groups, phosphorus-containing
groups selected from phosphanyl and phosphinyl groups, [0085] iodo,
[0086] chloro, [0087] fluoroalkyl groups containing two or more
fluorine atoms, especially trifluoroalkyl, heteroaryl, and [0088]
substituted phenyl.
[0089] In another embodiment, Fc and Fc' are the same and there is
as substituent a cyano group located on the respective proximal
cyclopentadienyl ring of each of said Fc and Fc' moieties.
[0090] In one preferred embodiment of the invention, Fc and Fc' are
the same and each comprise at least one ring substituent selected
from sulfur-containing groups and phosphorus-containing groups. In
one illustrative Example below from the group consisting of
sulfur-containing groups and phosphorus-containing groups, there is
disclosed a compound with an electrochemical potential value of in
excess of 500 mV. It is believed that compounds of the invention
that include sulfur-containing groups or phosphorus-containing
groups, especially those in which the sulfur or phosphorus atom is
directly boded to a ring carbon of the ferrocenyl, will be
advantageous in extending the range of available potential values
of such labels, making them useful as labels in electrochemical
assays. In particular, those compounds offer the possibility of use
in assays in which the high electrochemical potential value may be
valuable, for example in multiplex assays where a range of
different labels with differentiable electrochemical potentials are
used.
[0091] In certain preferred compounds there is present as a said
sulfur-containing group at least one sulfonyl substituent selected
from groups of the formula R.sup.15S(O).sub.2--, wherein R.sup.15
is selected from branched- or straight-chain alkyl, haloalkyl, and
substituted or unsubstituted aryl. Illustrative alkyl groups
R.sup.15 include, for example, methyl, ethyl, propyl, or butyl,
especially t-butyl. Preferred haloalkyl groups R.sup.15 include,
for example, fluoroalkyl groups with one of more fluoro
substituents, especially trifluoromethyl. In certain preferred
embodiments, R.sup.15 represents unsubstituted C1 to C4 alkyl; or
C1 to C4-haloalkyl, for example C1 to C4-fluoroalkyl, especially
trifluoromethyl. Illustrative aryl groups R.sup.15 include,
especially, phenyl, which may be substituted or unsubstituted, with
preferred substituents including, for example, halo, unsubstituted
alkyl (preferably C1 to C4 alkyl), substituted alkyl (for example
haloalkyl), nitro, cyano, alkoxy (for example, C1 to C4 alkoxy,
preferably methoxy) and sulfur-containing groups, for example
sulfonyl. Other illustrative aryl substituents R.sup.15 include
heteroaryl groups containing at least one heteroatom selected from
oxygen, sulphur and nitrogen. Illustrative of preferred aryl groups
R.sup.15 are those of general formula (R.sup.16).sub.a--Ar--, or
(R.sup.16).sub.a--HeAr-- in which Ar represents aryl; HeAr
represents heteroaryl; R.sup.16 is a substituent selected from
halo, alkyl, nitro, cyano, haloalkyl, alkoxy, and
sulphur-containing groups, for example sulfonyl; and a is an
integer in the range of from 0 to a number equal to the maximum
substitutable ring positions in the aryl, or heteroaryl ring. For
example, R.sup.15 may represent phenyl substituted by F; Cl; Br; I;
unsubstituted C1 to C4 alkyl; C1 to C4 haloalkyl, for example
trifluoromethyl; nitro; cyano; methoxy; or sulfur-containing
groups, for example sulfonyl.
[0092] In further embodiments, there is present as a said
phosphorus-containing substituent a group of the general formula
(R.sup.17).sub.2P(O)--, wherein each R.sup.17 is independently
selected from branched- or straight-chain alkyl, haloalkyl,
substituted or unsubstituted aryl and substituted or unsubstituted
heteroaryl. Advantageously, R.sup.17 represents C1 to C4 alkyl,
which is preferably unsubstituted, for example, methyl, ethyl,
propyl, or butyl, especially t-butyl.
[0093] Illustrative aryl substituents R.sup.17 include phenyl and
heteroaryl groups containing at least one heteroatom selected from
oxygen, sulphur and nitrogen, each of which may be unsubstituted or
substituted. Preferred aryl groups R.sup.17 include those of
general formula (R.sup.18).sub.b--Ar--, or (R.sup.18).sub.b--HeAr--
in which Ar is aryl; HeAr is heteroaryl; R.sup.18 is a substituent
selected from halo, alkyl, nitro, cyano, haloalkyl, alkoxy and
sulphur-containing groups, for example sulfonyl; and b is an
integer in the range of from 0 to a number equal to the maximum
substitutable ring positions in the aryl or heteroaryl ring. For
example, R.sup.17 may represent phenyl substituted by F, Cl, Br, I,
unsubstituted C1 to C4 alkyl, C1 to C4 haloalkyl, for example
trifluoromethyl, nitro, cyano, or methoxy. Preferably, Ar
represents phenyl and comprises one or more substituents R.sup.18
(which may be the same or different) selected from halo, alkyl,
haloalkyl, nitro, cyano, and alkoxy.
[0094] More preferably, each R.sup.17 is the same and represents
phenyl with at least one substituent, especially phenyl with one
subsistent in the 4-position. In one preferred embodiment, R.sup.7
represents branched C1 to C4 alkyl, for example t-butyl.
[0095] In another advantageous embodiment the ferrocenyl groups are
the same and there is present as a said substituent on each
ferrocenyl at least one substituted phenyl group in which the
phenyl has at least one substituent selected from halo, C1 to C4
alkyl, nitro, cyano, C1 to C4 haloalkyl, C1 to C4 alkoxy, and
sulfur-containing radicals, for example, sulfonyl. Illustrative of
such substituents on the phenyl are, for example, fluorine,
chlorine, bromine, iodine atoms, nitro, cyano, trifluoromethyl, and
methoxy. In certain embodiments, each phenyl has one substituent
which may be located in the 4-position, for example, 4-nitrophenyl
(wherein the ferrocenyl group is attached to the phenyl group at
the 1-position).
[0096] In a further embodiment there is present as a said
substituent on each ferrocenyl at least one heteroaryl group which
may be unsubstituted or substituted by at least one substituent
selected from halo, C1 to C4 alkyl, nitro, cyano, C1 to C4
haloalkyl and C1 to C4 alkoxy. For example, there may be present as
a heteroaryl group furanyl.
[0097] In yet further embodiments, there may be present as a said
substituent on each ferrocenyl at least one iodine or chlorine
atom. Other possible substituents include at least one silyl
substituent, preferably a silyl group selected from alkyl silyl
groups, for example trialkylsilyl, especially trimethylsilyl.
[0098] In addition to at least one substituent as defined with
reference to general formula I herein, each ferrocenyl moiety may
optionally be further substituted by at least one additional
substituent, for example, by at least one additional substituent
selected from bromo, fluoro, C1 to C4-alkyl, haloalkyl, and C1 to
C4 alkenyl. In accordance with a further embodiment, Fc and Fc' may
each additionally comprise at least one cyano group substituent on
its distal ring.
[0099] It is preferred that the ferrocenyl moieties are identical.
That is thought to give a stronger signal.
[0100] The moiety Z may be unsubstituted or substituted.
Substituents, when present, may be for example one or more
substituents selected from hydroxy, halo, cyano, amino, and
unsubstituted or substituted C1-C4 alkyl, C1-C4 alkenyl, or aryl;
wherein in each case optional substituents include without
limitation hydroxy, halo, cyano, oxo, amino, ester or amido. The
moiety Z may, if desired, be interrupted by one, or optionally more
than one, atom or moiety selected from --O--, --S--, cycloalkyl,
including heterocycloalkyl, --CO--, --CONH --, --NHCO-- and --NH--
and --NR.sup.1-- in which R.sup.1 is C1 to C4 alkyl. Illustrative
of cycloalkyl moieties that may be included as interruptions within
the moiety Z are cycloalkyl rings with from 5 to 7 ring atoms,
especially 6 ring atoms, for example cyclohexyl, piperidinyl,
morpholinyl.
[0101] The moieties X and Y, which are preferably the same,
advantageously have a chain length of from 1 to 6, preferably from
1 to 4 carbon atoms, especially one or two carbon atoms, and more
especially one carbon atom. The moieties X and Y may each represent
an alkylene chain, optionally interrupted by --O--, --S-- or
--NR.sup.5-- for example --NH--. Preferred moieties X and Y
include, for example, --CH.sub.2--, --CH.sub.2-- CH.sub.2--,
--(CH.sub.2).sub.3--O-- CH.sub.2--, --CH.sub.2--O--
(CH.sub.2).sub.3--, --(CH.sub.2).sub.3--O-- (CH.sub.2).sub.2--, and
--(CH.sub.2).sub.2--O-- (CH.sub.2).sub.3--.
[0102] In some embodiments, labels according to the present
invention may be prepared by reacting two equivalents of a suitable
substituted ferrocene carboxaldehyde in a suitable solvent in the
presence of a reducing agent. The structure of the desired label,
including the structure of moiety Z, may be determined by selection
of suitable starting materials and/or routine modification of the
synthesis method. In one illustrative method, for example a
ferrocene derivative such as 1'-iodo ferrocene carboxaldehyde,
1'-chloro ferrocene carboxaldehyde, 1'-furanyl ferrocene
carboxaldehyde or 2-tert-butyl sulphonyl ferrocene carboxaldehyde
may be reacted with a suitable amine (for example,
6-aminohexan-1-ol, glycine or (aminoethoxy)ethanol)) in a suitable
solvent, for example THF, in the presence of a reducing agent, for
example sodium triacetoxyborohydride. When glycine is used as the
amine, the resulting di-ferrocenyl glycine derivative may be
further modified to generate a desired structure. For example, it
may be reacted with oxalyl chloride in dichloromethane then treated
with 4-(hydroxymnethyl)piperidine to generate a
ferrocene-substituted derivative of
2-((di-ferrocenylmethyl)amino)-1-(4-(hydroxymethyl)piperidin-1-yl)ethanon-
e. In another embodiment, when glycine is used as the amine, the
resulting di-ferrocenyl glycine derivative may be further reacted
with oxalyl chloride in dichloromethane then treated with
6-aminohexan-1-ol to generate a ferrocene-substituted derivative of
N,N-2-(diferrocenylmethylamino)acetyl-6-aminohexanol (also named
N-(6-hydroxylhexyl)-2-((diferrocenylmethyl)amino)-acetamide).
Suitable methods for synthesis of other compounds according to the
invention will be apparent to those skilled in the art in the light
of the disclosure herein.
[0103] Linkage to the substrate can be by any suitable linkage,
typically by linkage to a substrate side chain. The linker group R
in the compounds of general formula I may be any group suitable for
effecting linkage to the substrate either directly or via a
functionalising group as described herein. R is advantageously,
although not necessarily, a linker group comprising an oxygen atom.
R is preferably a hydroxyl group or a protected hydroxyl group or a
group containing a hydroxyl group or a protected hydroxyl group. It
will be appreciated, however, that any other suitable linker group
R may be selected having regard to the substrate to which, in use,
the compound is to be attached. Various synthetic methods have been
developed for the derivatisation of protein, peptide or amino acid
side chains or protein, peptide or amino acid terminal moieties.
For example, lysine residues in a protein may be derivatised by
reaction with a succinimidyl ester. For derivatisation at other
amino acid residues, other known synthetic methods may be used. For
example, a maleimide reagent may be used to derivatise cysteine
residues. An N-hydroxy succinimide ester may be used to derivatise
the amino terminus or side chain amino group of a protein or
peptide, or an amino moiety of an amino acid.
[0104] Suitable derivatisation methods for nucleotides are also
well-known, for example, using a phosphoramidite moiety.
[0105] The above derivatisation methods are illustrative of the
methods that may be used to link the compounds of the invention to
a substrate, although other methods may be used.
[0106] Labelled substrates according to the invention may be
prepared by reaction of a compound according to the invention,
optionally after functionalisation to obtain a functionalised
labelling compound, with a substrate, for example, with a substrate
selected from amino acids, nucleotides (for example oligo
deoxyribonucleotides or oligo ribonucleotides), nucleosides,
sugars, peptides, proteins, oligonucleotides, polynucleotides,
carbohydrates and derivatives of any of those molecules.
[0107] In a preferred embodiment, the substrate is a nucleotide or
an oligonucleotide. The nucleotide may be selected from adenosine,
thymidine, guanosine, cytidine or uridine nucleotides. Preferably
the nucleotide, or a nucleotide of the oligonucleotide, is attached
to the label through a group attached to the ribose or deoxyribose
group of the nucleotide, for example in the 2',3' or 5' position,
for example through an oxygen or nitrogen atom. Most preferably,
the nucleotide is attached at the 3' or 5' position, for example at
the 5' position. Linking at other positions is also possible.
[0108] In the case of nucleotides, one advantageous way of
attaching labels of the invention is by functionalization with
phosphoramidite. The linking of phosphoramidite groups to
oligonucleotides is widely practised in oligonucleotide synthesis
and thus methods and conditions for attachment to an
oligonucleotide of labels functionalised with phosphoramidite will
be well-known and a routine matter to those skilled in the art.
Further, it advantageously permits the use of standard oligo
manufacturing methods.
[0109] Oligonucleotides for use in an assay in accordance with the
invention are advantageously nucleotides having from 2 to 50
nucleotides, more preferably from 2 to 40 nucleotides especially
from 15 to 35 nucleotides, with from 18 to 30 nucleotides being
especially preferred. For some applications, shorter
oligonucleotides may be useful, for example oligonucleotides with
from 2 to 14 nucleotides, more preferably from 2 to 10
nucleotides.
[0110] Attachment to proteins, for example via cysteine or lysine,
may be accomplished in some cases by incubation of the protein and
ferrocenyl label together at room temperature in an appropriate
buffer solution. Where the label is advantageously to be linked to
cysteine or lysine but the substrate sequence does not contain
cysteine or lysine at a suitable position the sequence may if
desired be mutated to add one or more cysteine or lysine residue
either as an additional residue or as a substitution for another
residue. An alternative method for attachment to proteins may
include biotinylation of the labels and use of commercial
streptavidinated proteins (or vice versa). By way of example, the
substrate may be biotinylated by any standard technique for example
by use of a commercially available biotinylation kit. Biotinylated
substrate will bind to strepavidin or avidin conjugated compounds
such as antibodies (which are commercially and widely
available).
[0111] It will however be apparent to the skilled person that
similar labels may be attached to a substrate at a selected one of
a number of locations by use of an appropriate labelling functional
group.
[0112] In functionalised labelling compounds of the general formula
III:
A-L-F III
A-L is preferably a moiety derived from a compound according to
general formula I and F is a functionalising group. Preferred
functionalised labelling compounds of the general formula III
include compounds of the general formula IIIb:
A-O--F IIIb
wherein A-O is a moiety derived from a compound according to
general formula I, preferably by loss of a hydroxy hydrogen atom or
protecting group when the linker group R of general formula I is
hydroxyl or a hydroxyl-containing group or is a protected hydroxyl
group, and F is a functionalising group.
[0113] Suitable functionalising groups that may be usable with
labels of the invention, including as functionalising group F in
general formula III and general formula IIIb, may include, without
limitation, succinimidyl ester groups, phosphoramidite groups,
maleimide groups, biotin and azide groups. It will be appreciated,
however, that there may be used any functionalising group that
facilitates attachment of the labelling compound to the substrate
to be labelled.
[0114] The invention also provides a method of detecting a nucleic
acid (for example RNA or DNA) in a sample comprising the optional
step of amplifying the nucleic acid (for example by PCR or another
nucleic acid amplification technique) followed by the step of
contacting the amplicon (or the nucleic acid) with a complementary
nucleic acid probe under conditions to allow hybridization between
the probe and amplicon (or the nucleic acid), followed by the step
of selectively degrading either hybridized or unhybridized probe
(for example by use of single or double strand specific nucleases),
wherein said probe is labelled with an electrochemically active
compound of the invention and wherein the method provides the step
of measuring the electrochemical activity of the compound labelling
the probe of wherein said electrochemical activity is dependent
either quantitatively or qualitatively on the extent of degradation
of the probe.
[0115] The invention also provides a method of detecting an
antibody or derivative (which may for example be bound to target
antigen in an assay) with an electrochemically active compound of
the invention comprising the step of measuring the electrochemical
activity of the compound. This method can be performed
quantitatively or qualitatively.
[0116] The invention also provides methods of diagnosing or
monitoring a disease in a subject comprising using a method of the
invention in the detection of a protease or a protease inhibitor
associated with said disease in a tissue or body fluid of the
subject. A substrate for the protease can be labelled according to
the invention.
[0117] The invention also provides methods of diagnosing or
maintaining a disease in a subject comprising using a method of the
invention to detect a peptide or protein associated with said
disease in a tissue or body fluid of the subject.
[0118] The invention also provides methods of diagnosing or
monitoring a disease in a subject comprising using a method of the
invention in the detection of a nuclease or a nuclease inhibitor
associated with said disease in a tissue or body fluid of the
subject.
[0119] Furthermore, the invention provides use of a method of the
invention for detecting a disease in a subject.
[0120] The invention also provides methods of detecting a
microorganism (in particular, a pathogen or other undesirable
organism, for example a food spoilage organism), comprising using a
method of the invention. A substrate from the microorganism (or
derived from the pathogen e.g. a nucleic acid amplicon produced
using a target nucleic acid sequence in the pathogen) can be
labelled according to the invention. Detection of the labelled
substrate can be used to indicate detection of the
microorganism.
[0121] The invention also provides an assay comprising a step which
uses a labelled substrate of the invention, optionally in
combination with other assay components for example a sample
vessel, a container comprising electrodes for electrochemical
detection, enzymes for use in the assay or standards and controls.
Said assay may use more than one different labelled substrate of
the invention. If that is the case the presence of different
labelled substrates may be differentially detected by labelling
them with electrochemical labels of the invention having different
electrochemical characteristics (for example different oxidation
potentials) thereby permitting the assay to be a multiplex (for
example a duplex) assay in which different substrates may be
discriminated when present in the same sample vessel. Simplex
assays are also encompassed by the invention.
[0122] Table 1a below sets out certain illustrative formulae of
compounds according to the invention which may be used as labels in
electrochemical assays in accordance with the invention, and which
may be used to make functionalised labelling compounds and labelled
substrates according to the invention. Table 1a also sets out in
the second column illustrative corresponding functionalised
labelling compounds according to the invention. Tables 1b, 2, 3 and
4 set out general formulae of further illustrative compounds of the
invention. Whilst functionalised compounds corresponding to the
compounds identified in Tables 1b, 2, 3 and 4 are not shown, it
will be appreciated that the compounds shown may be functionalised
by addition of any suitable functionalising moiety. In the formulae
in Tables 1a, 1b, and 2 to 4, except where considerations of steric
hindrance mitigate against it, each ferrocenyl may have more than
one substituent, which may be the same or different, and in any
ring position. Both ferrocenyl groups preferably have the same
substituent(s) in the same positions, i.e. both ferrocenyl groups
are the same.
TABLE-US-00001 TABLE 1a Illustrative embodiments with
cyclopentadienyl ring substituents in accordance with the invention
General formula of compound 1 ##STR00008## 2 ##STR00009## 3
##STR00010## 4 ##STR00011## 5 ##STR00012## General formula -
illustrative functionalised label 1 ##STR00013## 2 ##STR00014## 3
##STR00015## 4 ##STR00016## 5 ##STR00017## Identification of
symbols 1 R.sup.10 represents a radical selected from S-containing
groups, P-containing groups, I, Cl, trialkylsilyl, CF.sub.3,
heteroaryl, substituted phenyl; q represents from 1 to 5, for
example 1; and W represents (CH.sub.2).sub.n where n is from 0 to
6, O, S or NR.sup.20 where R.sup.20 is alkyl, for example C1 to C4
alkyl 2 R.sup.11 represents a radical selected from S-containing
groups, P-containing groups, I, Cl, trialkylsilyl, CF.sub.3,
heteroaryl, substituted phenyl and cyano; r represents from 1 to 5,
for example 1; and W represents (CH.sub.2).sub.n where n is from 0
to 6, O, S or NR.sup.20 where R.sup.20 is alkyl, for example C1 to
C4 alkyl 3 R.sup.12 represents a radical selected from S-containing
groups, P-containing groups, I, Cl, trialkylsilyl, CF.sub.3,
heteroaryl, substituted phenyl; q represents represents from 1 to
5, for example 1; and W represents (CH.sub.2).sub.n where n is from
0 to 6, O, S or NR.sup.20 where R.sup.20 is alkyl, for example C1
to C4 alkyl 4 R.sup.13 represents a radical selected from
S-containing groups, P-containing groups, I, Cl, trialkylsilyl,
CF.sub.3, heteroaryl, substituted phenyl; q represents from 1 to 5,
for example 1; and W represents (CH.sub.2).sub.n where n is from 0
to 6, O, S or NR.sup.20 where R.sup.20 is alkyl, for example C1 to
C4 alkyl 5 R.sup.14 represents a radical selected from S-containing
groups, P-containing groups, I, Cl, trialkylsilyl, CF.sub.3,
heteroaryl, substituted phenyl; q represents from1 to 5, for
example 1; W represents (CH.sub.2).sub.n where n is from 0 to 6, O,
S or NR.sup.20 where R.sup.20 is alkyl, for example C1 to C4 alkyl;
and V represents (CH.sub.2).sub.m where m represents from 2 to 6,
optionally interrupted by an O atom
TABLE-US-00002 TABLE 1b Further illustrative embodiments with
cyclopentadienyl ring substituents in accordance with the invention
General formula of compound Identification of symbols 6
##STR00018## R.sup.12 represents a radical selected from S-
containing groups, P-containing groups, I, Cl, trialkylsilyl,
CF.sub.3, heteroaryl, substituted phenyl, cyano; q represents from
1 to 4, for example 1; and W represents (CH.sub.2).sub.n where n is
from 0 to 6, O, S or NR.sup.20 where R.sup.20 is alkyl, for example
C1 to C4 alkyl 7 ##STR00019## R.sup.13 represents a radical
selected from S- containing groups, P-containing groups, I, Cl,
trialkylsilyl, CF.sub.3, heteroaryl, substituted phenyl, cyano; q
represents from 1 to 4, for example 1; and W represents
(CH.sub.2).sub.n where n is from 0 to 6, O, S or NR.sup.20 where
R.sup.20 is alkyl, for example C1 to C4 alkyl 8 ##STR00020##
R.sup.14 represents a radical selected from S- containing groups,
P-containing groups, I, Cl, trialkylsilyl, CF.sub.3, heteroaryl,
substituted phenyl, cyano; q represents from 1 to 5, for example 1;
W represents (CH.sub.2).sub.n where n is from 0 to 6, O, S or
NR.sup.20 where R.sup.20 is alkyl, for example C1 to C4 alkyl; and
V represents (CH.sub.2).sub.m where m represents from 2 to 6
optionally interrupted by an O atom
[0123] The compounds 6 to 8 above may be functionalised by any
suitable method, for example by phosphoramidation analogously to
the compounds 1 to 5 shown in Table 1 above.
[0124] In Table 2 below there are shown general formulae describing
certain preferred embodiments of the invention in which each
ferrocenyl group is substituted by a sulfonyl group. Table 3 below
shows general formulae describing certain preferred embodiments of
the invention in which each ferrocenyl group is substituted by a
phosphinyl group. The compounds in Tables 2 and 3 may each be
functionalised by any suitable method, for example
phosphoramidation. The present invention encompasses the
functionalised analogs of the compounds defined in the Tables 1a,
1b, 2 and 3 as well as labelled substrates derived therefrom.
TABLE-US-00003 TABLE 2 Illustrative embodiments with sulfonyl
radicals as cyclopentadienyl ring substituents 9. ##STR00021## 10.
##STR00022## 11. ##STR00023## 12. ##STR00024## 13. ##STR00025## 14.
##STR00026## 15. ##STR00027## 16. ##STR00028## 17. ##STR00029## 18.
##STR00030## 19. ##STR00031## 20. ##STR00032##
[0125] Where present in the formulae 9 to 20 above: [0126] R.sup.15
represents alkyl, for example t-butyl, haloalkyl, especially
fluoroalkyl, for example CF.sub.3, phenyl or substituted phenyl;
[0127] W represents (CH.sub.2).sub.n where n is from 0 to 6, O, S
or NR.sup.20 where R.sup.20 is alkyl, for example C1 to C4 alkyl;
and [0128] V, where present, represents (CH.sub.2).sub.m where m
represents from 2 to 6, optionally interrupted by an O atom; and
[0129] R.sup.16, where present, represents one or more radicals
selected from F, Cl, Br, I, alkyl, NO.sub.2, cyano, CF.sub.3 and
methoxy.
TABLE-US-00004 [0129] TABLE 3 Illustrative embodiments with
phosphinyl radicals as cyclopentadienyl ring substituents 21.
##STR00033## 22. ##STR00034## 23. ##STR00035## 24. ##STR00036## 25.
##STR00037## 26. ##STR00038## 27. ##STR00039## 28. ##STR00040## 29.
##STR00041## 30. ##STR00042## 31. ##STR00043## 32. ##STR00044##
[0130] Where present in the respective formulae 21 to 32 above:
[0131] R.sup.17 represents alkyl, for example t-butyl, phenyl or
substituted phenyl; [0132] W represents (CH.sub.2).sub.n where n is
from 0 to 6, O, S or NR.sup.20 where R.sup.20 is alkyl, for example
C1 to C4 alkyl; [0133] V represents (CH.sub.2).sub.m where m
represents from 2 to 6, optionally interrupted by an O atom; and
[0134] R.sup.18 represents one or more radicals selected from F,
Cl, Br, I, alkyl, NO.sub.2, cyano, CF.sub.3 and methoxy; in an
especially preferred embodiment, R.sup.8 represents one of said
radicals and is located in the 4-position.
[0135] In the general formulae and their functionalised
counterparts in Tables 1a, 1b, 2 and 3 when one or more ring
substituents is present on the proximal pentadienyl ring of each
ferrocenyl, that is, the ring that is directly bonded to the rest
of the molecule, there is preferably a said ring substituent at an
adjacent ring position to that bond. When more than one ring
substituent is present on each proximal pentadienyl ring, those
substituents may be in any position relative to one another. When
more than one ring substituent is present on each distal
pentadienyl ring of each ferrocenyl, that is, the ring remote from
the bond linking the ferrocenyl to the rest of the molecule, those
substituents may be in any position relative to one another. Whilst
in the compounds shown in the above Tables there are shown ring
substituents on either the proximal or the distal ring, it is also
possible for both pentadienyl rings of each ferrocenyl to carry one
or more substituents.
[0136] As illustrated in the Examples herein, incorporation of one
or more substituents on each of the ferrocenyl groups (the
substituents on each ferrocenyl being the same) can be used to
obtain compounds with modified electrochemical characteristics,
providing through appropriate substituent selection a suite of
compounds from which two or more may be selected for the purpose of
multiplex reactions.
[0137] Certain illustrative compounds according to the invention
which have been found to have good electrochemical properties are
set out in Table 4 below. The invention includes in addition to the
compounds in Table 4 functionalised labelling compounds and
labelled substrates which are derivable from those compounds in
accordance with the invention.
TABLE-US-00005 TABLE 4 Illustrative labelling compounds according
to the invention ##STR00045##
6-(bis((2-tert-butyl-sulfonylferrocenyl)1-
methylferrocenyl)amino)hexan-1-ol ##STR00046##
6-(bis((2-cyanoferrocenyl)1- methylferrocenyl)amino)hexan-1-ol
##STR00047## 6-(bis((2-di-tert-butyl-phosphinyl-
ferrocenyl)1-methylferrocenyl)amino)hexan-1-ol ##STR00048##
6-(bis((1'-iodoferrocenyl)1- methylferrocenyl)amino)hexan-1-ol
##STR00049## 6-(bis((1'-(4-nitrobenzyl)ferrocenyl)1-
methylferrocenyl)amino)hexan-1-ol ##STR00050##
6-(bis((1'-(1-furanyl)ferrocenyl)1-
methylferrocenyl)amino)hexan-1-ol ##STR00051##
6-(bis((1'-chloroferrocenyl)1- methylferrocenyl)amino)hexan-1-ol
##STR00052## 6-(bis((2-tert-butyl-sulfidylferrocenyl)1-
methylferrocenyl)amino)hexan-1-ol ##STR00053##
6-(bis((2-tert-butyl-sulfinylferrocenyl)1-
methylferrocenyl)amino)hexan-1-ol
DETAILED DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS
[0138] The following examples illustrate compounds of the invention
wherein Fc and Fc' are the same:
Materials and Methods
[0139] 1'-Iodo ferrocene carboxaldehyde was synthesised from
iodoferrocene using the method described in Organometallics, 2011,
30, 3504-3511. [0140] 1'-Furanyl ferrocene carboxaldehyde was
synthesised from 1'-iodoferrocene carboxaldehyde, using method
adapted from Angewandte Chemie, 2006, 45, 1282-1284. [0141]
1-[(Dimethylamino)methyl]-2-(di-tert-butyl phosphinyl)-ferrocene
was synthesised from dimethylaminomethyl ferrocene using method
adapted from Organometallics, 1985, 7 1297-1302. [0142] 1'-Chloro
ferrocene carboxaldehyde was prepared from chloroferrocene using
the procedure from Coll. Chechoslovak. Chemm. Commun., 1987, 52,
174-181. [0143] 2-tert-Butyl sulphonyl ferrocene carboxaldehyde was
obtained from synthesised from dimethylaminomethyl ferrocene using
method adapted from Organometallics, 1985, 7, 1297-1302. [0144]
2-Cyanoethyldiisopropylchlorophosphoramidite was obtained from
Sigma-Aldrich. [0145] 6-(Bis((2-formyl)
1-methylferrocenyl)amino)hexan-1-ol was synthesised from
(+/-)-4-(methoxymethyl)-2-ferrocenyl-1,3-dioxane using a method
adapted from Journal of Organic Chemistry, 1997, 62, 6733-6745.
[0146] Iodoferrocene was synthesised from ferrocene, from an
adaptation of the method described in Journal of Organometallic
Chemistry, 2011, 696, 1536-1540, utilising iodine as a suitable
electrophile. [0147] Chloroferrocene prepared from ferrocene using
a modified procedure from J. Organomet. Chem., 1996, 512, 219-224,
using hexachloroethane as a chlorinating reagent. [0148]
6-Aminohexanol, ferrocene, dimethylaminomethyl ferrocene and
4-nitrobenzene boronic acid were obtained from Sigma-Aldrich.
[0149] 1-[(Dimethylamino)methyl]-2-(t-butylthio)-ferrocene was
prepared using the procedure from Organomet., 1988, 7, 1297-1302.
[0150] 3-tert-butylsulfinyl ferrocene carboxaldehyde was prepared
according to the procedure described in Chem. Commun., 2004,
598-599.
Determination of Electrochemical Potential
[0151] The electrochemical potential values mentioned hereafter
were measured using an electrochemical cell including as background
electrolyte an aqueous 100 mM solution of sodium chloride, using a
printed carbon working electrode, a printed carbon counter
electrode and a silver/silver chloride reference electrode, all
with silver connectors. The electrodes were ink based and were
screen printed on to a polymer substrate (for example Mylar)
followed by heat curing. By way of illustration, the sample may be
prepared as follows: Ferrocenyl label precursor (2 ng) is dissolved
in DMSO (1 mL). An aliquot of 10 .mu.L is taken of this solution
and is then further diluted in the buffer (500 .mu.L). Then an
aliquot (20 .mu.L) is applied to the screen printed electrode to
run the electrochemical scan. An illustrative form of suitable cell
is described and shown schematically in WO2012/085591.
Example 1
Synthesis of
6-(bis((1'-iodoferrocenyl)1-methylferrocenyl)amino)hexan-1-ol
##STR00054##
[0153] 1'-Iodo ferrocene carboxaldehyde (259 mg, 0.76 mmol) was
dissolved in dry THF (7 cm.sup.3) and treated with
6-aminohexan-1-ol (44 mg, 0.38 mmol) and sodium
trisacetoxyborohydride (313 mg, 1.91 mmol) successively. The
solution was allowed to stir at room temperature overnight. After
this time the reaction was quenched by addition of NaHCO.sub.3
(sat) (10 cm.sup.3). The organic layer was separated, then the
aqueous layer back extracted with EtOAc (3.times.10 cm.sup.3).
Combined organic extracts were dried over MgSO.sub.4, filtered then
concentrated in vacuo to give an orange oil. Product was purified
by silica chromatography, eluting with 1:1 (EtOAc:Hexane)+1%
NH.sub.3OH. To give the desired product as an orange oil 123 mg,
42%. .sup.1H NMR (300 MHz; d.sub.6-benzene) .delta..sub.H: 4.28
(4H, t, J 1.8, F.sub.cH), 4.14 (4H, t, J 1.5, F.sub.cH), 4.11 (4H,
t, J 1.5, F.sub.cH), 4.06 (4H, t, J 1.8, F.sub.cH), 3.60 (2H, t, J
6.6, CH.sub.2O), 3.41 (4H, s, F.sub.cCH.sub.2N), 2.27 (2H, t, J7.4,
NCH.sub.2), 1.55-1.23 (8H, m, CH.sub.2); .sup.13C NMR (75 Mhz;
d.sub.6-benzene) .delta..sub.C: 85.2, 75.1, 73.3, 70.9, 69.4,
62.98, 52.02, 51.8, 40.5, 32.7, 27.1, 27.0, 25.5; HRMS (ESI) m/z
calcd for C.sub.28H.sub.33NOFe.sub.2I.sub.2 765.9428, m/z. found
765.9460.
[0154] The electrochemical potential was measured and found to be
442 mV.
Example 2
Synthesis of
6-(bis((1'-(4-nitrophenyl)ferrocenyl)1-methylferrocenyl)amino)hexan-1-ol
##STR00055##
[0156] To a Schlenk tube was added 4-nitrobenzene boronic acid
(39.6 mg, 0.23 mmol), trisdibenzylideneacetone palladium
(Pd.sub.2(dba).sub.3-1.9 mg, 2 mol %), tricyclohexylphosphine (1.4
mg, 4.8 mol %). The flask was sealed and evacuated and back filled
with argon four times. The
6-(bis((1'-iodoferrocenyl)1-methylferrocenyl)amino)hexan-1-ol (83
mg, 0.11 mmol) in 1,4-dioxane (2 cm.sup.3) was then added to the
flask. This was then followed by 1.27 M K.sub.3PO.sub.4 (aq) (141
.mu.l, 0.18 mmol). The flask was then heated at 100.degree. C.
overnight. After this time the reaction was allowed to cool to room
temperature then diluted with EtOAc (5 cm.sup.3) and H.sub.2O (5
cm.sup.3). The organic layer was separated and the aqueous layer
back extracted with EtOAc (3.times.5 cm.sup.3). The combined
organics were then washed with brine (sat) (10 cm.sup.3), dried
over Na.sub.2SO.sub.4, filtered and concentrated in vacuo to give a
purple solid. Product was purified by silica chromatography,
eluting with EtOAc+1% NH.sub.3OH to give the desired product as an
amorphous solid 11 mg, 13%. .sup.1H NMR (300 Mhz; d.sub.6-benzene)
.delta..sub.H: 8.05 (4H, app d, J 8.9, ArH), 7.08 (4H, app t, J
8.9, ArH), 4.39 (4H, t, J1.8, F.sub.cH), 4.24 (4H, t, J1.8,
F.sub.cH), 3.96 (4H, t, J1.8, F.sub.cH), 3.88 (4H, t, J1.8,
F.sub.cH), 3.52 (2H, t, J5.8, CH.sub.2OH), 3.01 (4H, s,
CH.sub.2F.sub.c), 2.28 (2H, t, J6.9, NCH.sub.2), 1.50-1.26 (8H, m,
CH.sub.2).
[0157] The electrochemical potential was measured and found to be
437 mV.
Example 3
Synthesis of
6-(bis((1'-(1-furanyl)ferrocenyl)1-methylferrocenyl)amino)hexan-1-ol
##STR00056##
[0159] 1'-Furanyl ferrocene carboxaldehyde (34 mg, 0.12 mmol) was
dissolved in dry THF (1 cm.sup.3) and treated with
6-aminohexan-1-ol (7 mg, 0.06 mmol) and sodium
trisacetoxyborohydride (49 mg, 0.3 mmol) successively. The solution
was allowed to stir at room temperature overnight. After this time
the reaction was quenched by addition of NaHCO.sub.3 (sat) (5
cm.sup.3). The organic layer was separated, then the aqueous layer
back extracted with EtOAc (3.times.5 cm.sup.3). Combined organic
extracts were dried over MgSO.sub.4, filtered then concentrated in
vacuo to give an orange oil. Product was purified by silica
chromatography, eluting with 1:1 (EtOAc:Hexane)+1% NH.sub.3OH to
give the desired product as an orange oil 15 mg, 39%. .sup.1H NMR
(300 Mhz; d.sub.6-benzene) .delta..sub.H: 7.26 (2H m, ArH), 6.25
(2H, dd, J6.5, 1.8, ArH), 6.20 (2H, dd, J6.5, 0.7, ArH), 4.61 (4H,
t, J 1.9, F.sub.cH), 4.21 (4H, t, J1.9, F.sub.cH), 4.12 (4H, t,
J1.9, F.sub.cH), 4.05 (4H, t, J1.9, F.sub.cH), 3.44 (2H, t, J 6.3,
CH.sub.2OH), 3.35 (4H, s, CH.sub.2F.sub.c), 2.40 (2H, t, J7.2,
CH.sub.2N), 1.56-1.27 (8H, m, CH.sub.2), .sup.13C NMR (75 Mhz;
d.sub.6-benzene) .delta..sub.C: 154.2, 141.6, 111.9, 104.4, 86.1,
77.8, 72.2, 69.6, 66.5, 63.03, 52.4, 33.5, 27.9, 27.63, 26.3, HRMS
(ESI) m/z calcd for C.sub.36H.sub.39Fe.sub.2NO.sub.3 668.15264 m/z
found 668.1558.
[0160] The electrochemical potential was measured and determined to
be 339 mV.
Example 4
Synthesis of
6-(bis((2-di-tert-butyl-phosphinyl-ferrocenyl)1-methylferrocenyl)amino)he-
xan-1-ol
[0161] 6-(bis((2-di-tert-butyl-phosphinyl-ferrocenyl)
1-methylferrocenyl)amino)hexan-1-ol was synthesised as shown in the
scheme below.
##STR00057##
[0162] To a solution of 1-[(dimethylamino)methyl]-2-(di-tert-butyl
phosphinyl)-ferrocene (145 mg, 0.3 mmol) in diethyl ether (5
cm.sup.3) was added methyl iodide (111 .mu.l, 1.7 mmol). The orange
solution was allowed to stir at room temperature under N.sub.2 for
1 hour. The orange suspension that was formed was then concentrated
in vacuo to give a bright orange solid. The bright orange solid was
then taken up in dry acetonitrile (3 cm.sup.3) and then treated
with 6-amino-hexan-1-ol (17.5 mg, 0.15 mmol). The flask was then
sealed and heated at reflux for 18 hours. After this time the dark
orange solution was allowed to cool to room temperature. The
reaction was partitioned between CH.sub.2Cl.sub.2 (5 cm.sup.3) and
NaHCO.sub.3 (sat) (5 cm.sup.3), the organic layer was separated and
then dried over Na.sub.2SO.sub.4, filtered and then concentrated
under reduced pressure to give a brown oil. Purification by basic
alumina chromatography eluting with 2% MeOH:CH.sub.2Cl.sub.2 gave
the desired product as an orange oil (8 mg, 7% yield)
[0163] .sup.1H NMR (250 MHz; d.sub.6-benzene) .delta..sub.H 4.63
(2H, app s, F.sub.cH), 4.14 (12H, s, F.sub.cH), 3.85 (6H, br s,
F.sub.cH+F.sub.cNCH.sub.2), 3.55 (2H, app s, OCH.sub.2), 2.45 (2H,
app s, CH.sub.2N), 1.47-0.95 (44H, m, tBu+CH.sub.2), .sup.31P NMR
(121 MHz; d.sub.6-benzene) .delta..sub.P 61.3, HRMS (ESI .mu.TOF)
m/z calcd for C.sub.44H.sub.69NO.sub.3Fe.sub.2P.sub.2 834.3529 m/z.
found 834.3603.
[0164] The electrochemical potential (DPV) was measured and found
to be 512 mV.
Example 5
6-(bis((1'-chloroferrocenyl)1-methylferrocenyl)amino)hexan-1-ol
[0165] 6-(bis((1'-chloroferrocenyl)
1-methylferrocenyl)amino)hexan-1-ol was synthesised as shown in the
scheme below.
##STR00058##
1'-chloro ferrocene carboxaldehyde (1.35 g, 5.44 mmol) was
dissolved in dry THF (50 cm.sup.3) and treated with
6-aminohexan-1-ol (318 mg, 2.72 mmol) and
sodiumtrisacetoxyborohydride (1.11 g, 6.80 mmol) successively. The
solution was allowed to stir at room temperature overnight. After
this time the reaction was quenched by addition of NaHCO.sub.3
(sat) (30 cm.sup.3). The organic layer was separated, then the
aqueous layer back extracted with EtOAc (3.times.30 cm.sup.3).
Combined organic extracts were dried over MgSO.sub.4, filtered then
concentrated in vacuo to give an orange oil. Product was purified
by silica chromatography, eluting with 1:1 (EtOAc:Hexane)+1%
NH.sub.3OH to give the desired product as an orange oil 892 mg,
56%.
[0166] .sup.1H NMR (300 MHz; d.sub.6-benzene) .delta..sub.H 4.18
(4H, t, J 1.6, F.sub.cH), 4.14 (4H, t, J 1.8, F.sub.cH), 3.98 (4H,
t, J 1.6, F.sub.cH), 3.65 (4H, t, J 1.8, F.sub.cH), 3.65 (4H, s,
NCH.sub.2, F.sub.cCH.sub.2N), 3.34 (2H, t, J6.3, OCH.sub.2), 2.41
(2H, t, J7.0, NCH.sub.2), 1.55-1.23 (8H, m, CH.sub.2), .sup.13C NMR
(75 MHz; d.sub.6-benzene) .delta..sub.C 93.4, 86.6, 72.9, 70.6,
69.1, 68.1, 67.3, 63.0, 52.8, 33.6, 28.1, 27.7, 26.3, HRMS (ESI
.mu.TOF) m/z calcd for C.sub.28H.sub.33NOFe.sub.2Cl.sub.2 582.0716
m/z. found 582.0722.
[0167] The electrochemical potential (DPV) was measured and found
to be 452 mV.
Example 6
6-(bis((2-cyano)1-methylferrocenyl)amino) hexan-1-ol
##STR00059##
[0169] 6-(bis((2-formyl)1-methylferrocenyl)amino)hexan-1-ol (1 eq)
is dissolved in ethanol and treated with hydroxylamine
hydrochloride (5 eq) and sodium acetate (5 eq). The resulting
suspension is then heated at reflux for 18 hrs. After this time the
reaction is allowed to cool to room temperature and concentrated in
vacuo. The solid residue is then partitioned between chloroform and
NaHCO.sub.3 (sat). The organic layer is separated and dried over
Na.sub.2SO.sub.4, filtered and concentrated in vacuo to give the
corresponding oxime. The oxime is then taken up in dry THF and
treated with (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium
hexafluorophosphate (BOP-2 eq) and stirred for 5 mins. Then
1,8-diazabicyclo[5.4.0]undec-7-ene (2.3 eq) is added. The solution
is then stirred for 90 mins. The reaction is then diluted with
EtOAc and washed with water and brine (sat). The organic phase is
dried over MgSO.sub.4, filtered and then concentrated in vacuo.
Example 7
6-(bis((2-tert-butyl-sulfonylferrocenyl)1-methylferrocenyl)amino)hexan-1-o-
l
##STR00060##
[0171] 2-tert-Butyl sulphonyl ferrocene carboxaldehyde (1 eq) is
dissolved in dry THF and treated with 6-aminohexan-1-ol (0.5 eq)
and sodium trisacetoxyborohydride (2.5 eq) successively. The
solution is allowed to stir at room temperature overnight. After
this time the reaction is quenched by addition of NaHCO.sub.3
(sat). The organic layer is separated, then the aqueous layer back
extracted with EtOAc. Combined organic extracts are dried over
MgSO.sub.4, filtered then concentrated in vacuo.
Example 8
6-(bis((2-tert-butyl-sulfidylferrocenyl)1-methylferrocenyl)amino)hexan-1-o-
l
[0172]
6-(bis((2-tert-butyl-sulfidylferrocenyl)1-methylferrocenyl)amino)he-
xan-1-ol was synthesised as shown in the scheme below.
##STR00061##
[0173] 1-[(Dimethylamino)methyl]-2-(t-butylthio)-ferrocene (1.21 g,
3.49 mmol) was dissolved in acetic anhydride (10 cm.sup.3). The
brown solution was then refluxed for 1 hour, TLC at this indicated
full consumption of the starting material. The solution was allowed
to cool to room temperature, the solution was then concentrated in
vacuo to approximately 90% of original volume. The resulting brown
oil was then taken up in EtOAc (25 cm.sup.3) and washed with
NaHCO.sub.3 (sat) (20 cm.sup.3) and brine (sat) (20 cm.sup.3). The
brown solution was then dried over MgSO.sub.4, filtered and
concentrated in vacuo to give the desired acetoxy ester as a
orange/brown oil (1.12 g, 93%) without need for further
purification.
[0174] .sup.1H NMR (250 MHz, C.sub.6D.sub.6) .delta. 5.37 (2H, d,
J=1.43 Hz), 4.51 (1H, dd, J=2.6, 1.4), 4.44 (1H, dd, J=2.6, 1.4),
4.07 (1H, t, J 2.6), 4.07 (5H, s) 1.82 (s, 3H), 1.33 (9H, s).
[0175] To a suspension of lithium aluminium hydride (369 mg, 9.71
mmol) in Et.sub.2O (15 cm.sup.3) at 0.degree. C. was added the
acetoxy ester (1.12 g, 3.23 mmol) dropwise via syringe. Once
addition was complete the slurry was allowed to warm to room
temperature and stir for 30 mins. After this time the flask was
cooled to 0.degree. C. and then quenched by sequential addition of
H.sub.2O (369 .mu.l), followed by 15% NaOH (aq) (369 .mu.l) and
H.sub.2O (1.1 cm.sup.3) the suspension was then allowed to warm to
room temperature stirred for 10 minutes, filtered and concentrated
in vacuo to the desired product as an orange solid (790 mg, 80%)
without the need for further purification.
[0176] .sup.1H NMR (250 MHz, C.sub.6D.sub.6) .delta. 4.64 (2H, s),
4.41 (1H, dd, J=2.4, 1.5 Hz), 4.32 (1H, dd, J=2.4, 1.5 Hz), 4.17
(5H, s), 4.08 (1H, t, J=2.6 Hz), 1.28 (9H, s).
[0177] 1-tert-butyl sulfidyl-2-hydroxymethyl ferrocene (304 mg, 1
mmol) and barium manganate (1.02 g, 4 mmol) was placed in a schelnk
tube, the flask sealed, then evacuated and back filled with argon
four times and finally left over an Argon atmosphere. The flask was
then charged with benzene (15 cm.sup.3) and the slurry was allowed
to stir at room temperature overnight. The slurry was then filtered
through celite, and washed with Et.sub.2O until the washing ran
clear. The orange solution was concentrated in vacuo to give the
desired product as a red oil (239 mg, 79%) without the need for
further purification.
[0178] .sup.1H NMR (250 MHz, C.sub.6D.sub.6) .delta. 10.69 (1H, s),
5.08 (1H, dd, J=2.5, 1.3 Hz), 4.47 (1H, dd, J=2.5, 1.3 Hz), 4.27
(1H, t, J=2.5 Hz), 4.13 (5H, s), 1.17 (9H, s).
[0179] The 2-tert-butylsulfidyl-ferrocene carboxaldehyde (237 mg,
0.78 mmol) was placed in a round bottomed flask with
6-amino-hexanol (46 mg, 0.39 mmol) and dissolved in dry THF (5
cm.sup.3). The suspension was then treated with sodium
trisacetoxyborohydride (322 mg, 1.96 mmol). The flask was equipped
with a condenser and refluxed overnight. After this time the flask
was allowed to cool to room temperature. The reaction was quenched
by addition of NaHCO.sub.3(sat) (10 cm.sup.3). Organics were
separated and the aqueous layer back extracted with EtOAc
(3.times.5 cm.sup.3). Combined organics were washed with brine
(sat) (10 cm.sup.3), dried over MgSO.sub.4, filtered and
concentrated in vacuo to give a brown oil. Purification by silica
chromatography eluting with 50% EtOAc:nHex+2% TEA gave the desired
product as an orange oil (18 mg, 7%)
[0180] 1H NMR (250 MHz, C.sub.6D.sub.6) .delta. 4.52 (2H, app s),
4.19-4.15 (14H, m), 3.91 (4H, s), 3.54 (2H, t, J=7.3), 2.64 (2H, t,
J=7.3), 1.69-1.43 (26H, m); m/z (ESI .mu.TOF, M+H) calcd for
C.sub.36H.sub.52NOS.sub.2Fe.sub.2 m/z 690.2143. found 690.2158.
Electrochemical potential (DPV) 369 mV.
Example 9
6-(bis((2-tert-butyl-sulfinylferrocenyl)1-methylferrocenyl)amino)hexan-1-o-
l
[0181] 6-(bis((2-tert-butyl-sulfinylferrocenyl)
1-methylferrocenyl)amino)hexan-1-ol was synthesised as shown in the
scheme below.
##STR00062##
[0182] 3-tert-Butylsulfinyl ferrocene carboxaldehyde (26.5 mg,
0.083 mmol) was dissolved in dry THF (1 cm.sup.3), treated with
6-amino-hexan-1-ol (4.8 mg, 0.041 mmol). The brown solution was
stirred for 10 mins before sodium trixacetoxyborohydride (34 mg,
0.2 mmol) was added in one portion. The brown suspension was then
stirrer at room temperature overnight. After this time the material
was concentrated in vacuo to give a brown solid. Purification by
silica chromatography eluting with 2.5% MeOH:CH.sub.2Cl.sub.2+2%
NH.sub.3OH to give the desired product as a yellow oil (4 mg,
14%)
[0183] .sup.1H NMR (250 MHz, C.sub.6D.sub.6) .delta. 5.12 (2H, app
s), 4.92 (2H, app s), 4.39 (14H, m), 4.22 (4H, s), 3.69 (2H, t,
J=7.1 Hz), 2.45 (2H, t, J=7.1 Hz), 1.50-1.18 (26H, m), m/z (ESI
.mu.TOF, M+H) calcd for C.sub.36H.sub.52NO.sub.3S.sub.2Fe.sub.2 m/z
722.2087. found 722.2089. m/z; Electrochemical potential (DPV) 532
mV.
Example 10
General Synthetic Procedure for Attaching Phosphoramidite
Functional Group
##STR00063##
[0185] The ferrocenyl derivative shown as a starting material in
the above reaction scheme is illustrative, and may be replaced by a
molar equivalent of any of the compounds made in Examples 1 to 9
above.
[0186] N,N-diisopropylethylamine (0.4 mL, 8.4 mmol) was added to a
stirred solution of the ferrocene derivative (2.1 mmol) in dry THF
(25 mL) under a nitrogen atmosphere.
2-cyanoethyldiisopropylchlorophosphoramidite (0.2 ml, 3.15 mmol)
was added dropwise and the resulting mixture was stirred for 15
mins. MilliQ filtered water (200 mL) was added and the solution was
stirred for a further 30 mins. Ethyl Acetate-Triethylamine (1:1, 25
mL) was added, a precipitate formed. The mixture was washed with
saturated NaCHCO.sub.3 (25 mL) and MilliQ filtered water (25 mL).
The organic fraction was dried over MgSO.sub.4 and the solvent was
removed under vacuo. The crude product was then purified by silica
gel chromatography (petroleum ether:ethyl acetate 9:1).
Example 11
Binding of Labels to Protein
[0187] The labels of the invention are attached to a peptide by
attachment of the label to a free amine of, for example, a lysine
residue in the peptide. Attachment may be accomplished conventional
techniques including functionalisation of the labelling compound to
form an active NHS ester and reaction of the functionalised ester
with the free amine group of the peptide.
Example 12
Binding of Labels to Microparticles
[0188] A biotin molecule is coupled to a label, for example a label
as made in any of Examples 1 to 9. The biotinylation can be carried
out in an automated oligonucleotide synthesiser or using standard
laboratory conditions by reaction of ferrocenyl phosphoramidite
label with N-hydroxysuccinimide (NHS) esters of biotin.
[0189] Paramagnetic treptavidin particles are washed .times.3
(phosphate buffer) and mixed with biotinylated label, followed by
incubation for 1 hour at room temperature with mixing. The
particles are washed .times.2 (phosphate buffer) and washed
.times.1 (PCR buffer). They are resuspended in final buffer (PCR
buffer). Following each wash step the supernatants are tested for
electrochemical signal, and if necessary washing is repeated until
the supernatants show no indication of free electrochemical
label.
[0190] These particles are assayed at a range of concentrations to
validate that the observed electrochemical signal is attributable
to the label coupled to the magnetic particles, using magnetic
capture of the particles and resuspension in a range of buffer
volumes.
[0191] In Table 5 below, the electrode potentials of the compounds
made in Examples 1 to 5, 8 and 9 are listed, together with the
comparison value for N,N-diferrocenylmethyl-6-aminohexanol, a
compound in which the ferrocenyl groups are unsubstituted. A method
for synthesis of N,N-diferrocenylmethyl-6-aminohexanol is disclosed
in WO2012/085591.
TABLE-US-00006 TABLE 5 Effect on electrode potential of
substituents on ferrocenyl moieties Example Fc substituent
Electrode potential A None 275 mV 1 1'-Iodo 442 mV 2
1'-(4-Nitrophenyl) 437 mV 3 1'-Furanyl 339 mV 4
2-di-t-butylphosphinyl 512 mV 5 1'-Chloro 452 mV 8
2-tert-butyl-sulfidyl 369 mV 9 2-tert-butyl-sulfinyl 532 mV
[0192] The data in the above table shows that the compounds of
Examples 1 to 5, 8 and 9 provide useful electrochemically active
labels. The labels may be used to provide an electrochemical signal
within a desired range of values. They may be useful as alternative
labels to other labelling compounds with similar potential values,
for example, where those other labelling compounds have
disadvantageous properties in the assay in question, for example,
incompatibility with impurities or other components present in the
assay or incompatibility with the measurement conditions, any of
which could affect measurement sensitivity. As well, or instead,
they may be used with one or more other labels in a multiplex assay
in which more than one label is present to provide two or more
determinations in a single sample, the use of two or more labels
with different electrochemical properties in those circumstances
permitting effective distinction between measurements relating to
the respective species to be determined.
* * * * *