Title of Invention

A COMPLEX COMPRISING A COMPOUND AND COMPOUNDS FROM FUNGI FOR THE DETECTION OF ORGANIC COMPOUNDS THROUGH FLUORESCENCE

Abstract The invention relates to the discovery of novel compounds from fungi that fluoresce upon interaction with organic or inorganic compounds. Such fluorophores can be used to detect and/or quantify compounds in a number of settings including (but not limited to) proteins in electrophoresis gels, in solution, on membranes or other surfaces.
Full Text A COMPLEX COMPRISING A COMPOUND AND COMPOUNDS
FROM FUNGI FOR THE DETECTION OF ORGANIC COMPOUNDS
THROUGH FLUORESCENCE
Technical Field
The present invention relates to a complex comprising a compound and
compounds from fungi for the detection of organic compounds through fluorescence which
can function as fluorescent dyes, methods of isloating such compounds from
microorganisms and uses of such compounds in scientific applications.
Background Art
Compounds that fluoresce have many uses and are known to be
particularly suitable for biological applications where fluorescence is
required for the detection of whole cells, cellular components, and cellular
functions. For example, many diagnostic and analytical techniques require
the samples to be fluorescently tagged so that they can be detected. This is
achieved by using fluorescent dyes or probes which interact with a wide
variety of materials such as cells, tissues, proteins, antibodies, enzymes,
drugs, hormones, lipids, nucleotides, nucleic acids, carbohydrates, or natural
or synthetic polymers to make fluorescent conjugates.
With synthetic fluorescent probes, ligands are frequency used to
confer a specificity for a biochemical reaction that is to be observed and the
fluorescent dye provides the means of detection or quantification of the
interaction. These applications include, among others, the detection of
proteins (for example in gels or aqueous solution), cell tracking, the detection
of proteins via fluorescently labelled antibodies, the assessment of enzymatic
activity, the staining of nucleic acids.
Long wavelength absorbance usually increases the utility of a
, fluorescent probe since it reduces the interference from cellular auto-
fluorescence and reduces the cytotoxic effect of the fluorophore in
combination with light. Although lasers are particularly useful as a
concentrated light source for the excitation of fluorescence and compounds
whose excitation spectra coincide with laser output are of high utility. The
argon laser is the most common light source for excitation of fluorescence,
and has principle outputs, light at 488 nm and 514 nm. Fluorescent
compounds that are excited by either of these wavelengths are therefore of
particular utility. Alternatively, excitation of fluorescence can be achieved
usin+g solid state light sources such as light emitting diodes. Fluorescent
compounds excited by the light emitted from these alternative sources are
also of particular utility.
Red fluorescent compounds are used extensively in many fields of
biological study. Many of these, including Texas red, Tetramethyl
rhodamine-isothiocyanate or red emitting BODEPY dyes require excitation at
green wavelengths such as 542 nm. This limits their use in many
applications, especially those where the argon-ion laser is used for excitation.
Compounds such as ethidium bromide, can be excited with light from the
argon-ion laser [520 nm band), but are not generally suitable for tagging of
organic molecules other than nucleic acids. Other compounds such as
phycoerythrin, can be excited using the argon-ion laser (488 nm) and does
emit in the orange/red wavelengths. Phycoerythrin, however, has poor
stability and a high molecular weight making it unsuitable for many
applications such as cell tracking, labelling of nucleic acids or staining
proteins.
For staining of proteins, there are a number of methods available.
These methods can utilise non-fluorescent compounds, or fluorescent
compounds. The most commonly used method utilises Coomassie blue
which is non-fluorescent, can require the use of large amounts of organic
solvents and is time consuming. Other fluorescence-based protein-detection
methods are available which are potentially more sensitive than non-
fluorescent methods. However, these methods are in general much more
expensive than non-fluorescent methods which limits their widespread use.
Therefore, compounds that combine useful spectral characteristics, and
relatively high sensitivity will be of particular utility.
There are several methods for the quantification of protein in solution.
These methods are based on a range of techniques, and include methods
where dyes bind to soluble proteins. These dyes can be either non-
fluorescent or fluorescent compounds. Fluorescent dye-based methods are
often more sensitive than the non-fluorescent dyes, and allow for the
determination of protein concentration over a wide range of concentrations.
Compounds that combine useful spectral characteristics with an ability to
bind proteins will be of particular utility.
In enzymatic studies, there is widespread use of fluorescent
compounds for the detection of particular enzymatic activities. For example,
fluorescein di-3-D-galactopyranoside (FDG) is a non-fluorescent compound
that is sequentially hydrolysed by the enzyme P-galactosidase first to
generate fluorescein monogalactoside and then to fluorescein which is highly
fluorescent. The cleavage of the FDG compound can be monitored by the
increase in fluorescence in the solution, and thus allows sensitive
quantification of enzymatic activity. At present, only a limited number of
fluorophores are suitable for this procedure. Therefore, novel fluorescent
compounds that can be conjugated to a variety of substrates will be of utility.
For dual colour staining, there is a very limited choice of low
molecular weight fluorophores. The predominant green fluorophore is
fluorescein, which strongly absorbs light from the 488 nm band of the argon
ion laser, and re-emits at 518 nm. At present there are few compatible red or
orange fluorophores that are of low molecular weight and are excited by the
488 nm or 514 nm bands of the argon ion laser. Therefore, low molecular
weight compounds that are excited by argon ion lasers and emit at
wavelengths greater than 600 nm will be of utility, particularly if there is
minimal spectral overlap with fluorescein.
The present inventors have isolated new compounds derived from a
fungus that are capable of combining readily with a range of organic .
molecules to produce fluorescent complexes.
Disclosure of Invention
In a first aspect of the invention, there is provided a compound
according to formula (I):
wherein:
each of R, R' and R" is a hydrogen atom, halogen atom or a straight or
branched chain C,_.,o aikyl, alkenyl or alkynyl group optionally substituted
with one or more halogen, hydroxy and/or oxo group;
rings A, B and C optionally include one or more double bonds;
rings B and C are optionally substituted with one or more halogen
atoms: and
the compound is capable of interacting with an organic compound and
when interacting with the organic compound, the compound emits
fluorescence after excitation at a broad range of wavelengths. Preferably, the
compound is excited at wavelengths in the range of 300-560 nm, more
preferably, 380-530 nm and even more preferably, UV wavelengths and/or
blue wavelengths. Preferably the compound emits in the wavelengths of 460
to 700 nm. More preferably its emission peak is centered around 530 nm
when interacting with an organic compound such as sodium dodecyl
sulphate and centered around 605 nm when interacting with a biomolecuie
such as a protein or cell.
Preferably, the first aspect of the invention provides a compound
according to formula (laj:
Preferably, the compound according to formula la has the
stereochemistry as depicted in formula (lb);
Preferably, R is a straight or branched chain C,..^o conjugated alkenyl
group optionally substituted with hydroxy and oxo groups; R' is a straight or
branched chain C^.^o alkyl group optionally substituted with a hydroxy group
and R" is a straight or branched chain C^.^o alkyl group.
More preferably, R=-C(OH]CHC(0)(CH)6CH3, K=-CHfiH and R"=Me
such that the present invention according to the first and second aspects
consists in the compound 5,6-dihydro-3-[(lZ, 4E, 6E, 8E]-l-hydroxy-3-oxo-
l,4,6,8-decatetraenyl]-6-hydroxymethyl-9a-methyl-2H-furo[3,2-
g][2]benzopyran-2-9(9aH]-dione according to formula (Ic):
In a second aspect, the present invention consists in a compound
according to formula (I), wherein each of R, R' and R" is a hydrogen atom,
halogen atom or a straight or branched chain C^.^^ alkyl, alkenyl or alkynyl
group optionally substituted with one or more halogen, hydroxy and/or oxo
groups;
ring A, B and C are optionally substituted with one or more halogen
atoms; and
rings A, B and C optionally include one or more double bonds;
with the proviso that:
(i) R*-C(OH)CHC(0](CH)eCH(Et)(Me) when R' = -CH20H, R" = Me,
ring A includes a double bond between C3 and C3a; ring B includes a
double bond between C4 and C4a; and ring C includes a double bond
between C8 and C8a;
(ii) R;^-C(0H)CHC(0](CH)6CH(Et)(Me) when R' = Me, R" = Me, rmg A
includes a double bond between C3 and C3a; ring B includes a double
bond between C4 and C4a; and ring C includes a double bond between
C8 and C8a;
(iii) R9i-C[0H)CHC(0)(CH)eCH(Et](Me) when R'=Me, R"=Me, ring A
includes a double bond between C3 and C3a; ring B includes a double
bond between C4 and C4a; and ring C includes a double bond between
C8 and C8a and C5 and C6;
(iv) R;t-C(0)(CH)4Me when R'=-77-propyI, R"=Me, ring A includes a
double bond between C3 and C3a; ring B includes a double bond
between C4 and C4a; and ring C includes a double bond between C8
and C8a and C5 and C6;
[v) R?t-C(0)(CH)eMe when R'=-i?-propyl, R"=Me, ring A includes a
double bond between C3 and C3a; ring B includes a double bond
between C4 and C4a; and ring C includes a double bond between C8
and C8a and C5 and C6;
(vi) R;t-C(0)(CH)sMe when R'^-lCHj^Me, R"=Me, ring A includes a
double bond between C3 and C3a; ring B includes a double bond
between C4 and C4a; and ring C includes a double bond between C8
and G8a and C5 and C6;
(vii) R;t-C(0)(CH)6Me when R'=-[Cl^JtAe, R"=Me, ring A includes a
double bond between C3 and C3a; ring B includes a double bond
between C4 and C4a; and ring C includes a double bond between C8
and C8a and C5 and C6;
(viii) R?i'(CH),C(Me)CHC(Me)(Et) when R'=-Ac, R"=Me, C4=C1, ring A
includes a double bond between C3 and C3a; ring B includes a double
bond between C4 and C4a; and ring C includes a double bond between
C8 and C8a and C5 and C6;
(ix) R;^-(CH]2C(Me)CHC(Me](Et) when R'=-Ac, R"=Me, ring A
includes a double bond between C3 and C3a; ring B includes a double
bond between C4 and C4a; and ring C includes a double bond between
C8 and C8a and C5 and C6;
(xj R?i-(CH)2C(Me)CH-;-propyl when R'=-Ac, R"=Me, ring A includes
a double bond between C3 and C3a; ring B includes a double bond
between C4 and C4a; and ring C includes a double bond between C8
and C8a and C5 and C6;
(xi) R;±-C(0](CH]>Ie when R'^-lCHj^Me. R" = Me, ring A includes a
double bond between C3 and C3a; ring B includes a double bond
between C4 and C4a; and ring C includes a double bond between C8
and C8a and C5 and C6:
(xii) R*-C(0)(CH),Me when R'=-/?-propyI. R"=Me, ring A does not
include a double bond; and rings B and C include double bonds
between C8a and C4a and C5 and C6;
(xiii) R^-CiO)iCH}Me when R'=-/7-propyl. R"=Me. ring A, B and C do
not include double bonds;
(xiv) R;*-C(0)(CH)eMe when R'^-CCHl^Me. R"=Me, ring A does not
include a double bond; and rings B and C include double bonds
between C8a and C4a and C5 and C6;
(xv) R7t-C(0)(CH),Me when R'=-C(CH2)(Me), R"=Me, ring A does not
include a double bond; and rings B and C include double bonds
between C8a and C4a and C5 and C6;
(xvi) R*-G(0)(CH),Me when R'=-(CH]2Me. R" = Me, ring A does not
include a double bond; and rings B and C include double bonds
between C8a and C4a and C5 and C6. including tautomers thereof.
In a third aspect, the present invention consists in a compound
according to formula (I), wherein each of R, R' and R" is a hydrogen atom,
halogen atom or a straight or branched chain C,.2o alkyl, alkenyl or alkynyl
group optionally substituted with one or more halogen, hydroxy and/or oxo
groups;
ring A, B and C are optionally substituted with one or more halogen
atoms; and
rings A, B and C optionally include one or more double bonds;
with the proviso that:
(i] R*-C(OH)CHC(0)(CH]eCH(Et)(Me) when R'=-CH20H. R"=Me,
ring A includes a double bond between C3 and C3a; ring B includes a
double bond between C4 and C4a; and ring C includes a double bond
between C8 and C8a;
(ii) R7i-C(OH)CHC(0]{CH)6CH(Et)(Me) when R'=Me, R" = Me, ring A
includes a double bond between C3 and C3a; ring B includes a double
bond between C4 and C4a; and ring C includes a double bond between
C8 and C8a;
(lii) R;=-C(0H)CHC(0)[CH)6CH(Et)(Me) when R' = Me. R" = Me, ring A
includes a double bond between C3 and C3a; ring B includes a double
bond between C4 and C4a: and ring C includes a double bond between
C8 and C8a and C5 and C6:
[iv) R;^-C{0]{CH^]JS'le when R'=-n-propyl, R"=Me, ring A includes a
double bond between C3 and G3a; ring B includes a double bond
between C4 and C4a; and ring C includes a double bond between C8
and C8a and C5 and C6;
(v) R?i-C(Oj(CHJeMe when R'=-i7-propyl, R"=Me, ring A includes a
double bond between C3 and C3a; ring B includes a double bond
between C4 and C4a; and ring C includes a double bond between C8
and C8a and C5 and C6;
(vi) R;t-C(0)(CH2}gMe when R'=-[CH)2Me, R"=Me, ring A includes a
double bond between C3 and C3a; ring B includes a double bond
between G4 and C4a; and ring C includes a double bond between C8
and C8a and C5 and C6;
(vii) R;t-C(0)(CH2)eMe when R'=-(CH)2Me, R"=Me, ring A includes a
double bond between C3 and C3a; ring B includes a double bond
between C4 and C4a; and ring C includes a double bond between C8
and C8a and C5 and C6;
(viii) R;=i-Ac when R'=-(CH]2C(Me]CHCH(iMe)(Et}, R"=Me, C4=C1, ring
A includes a double bond between C3 and C3a; ring B includes a
double bond between C4 and C4a; and ring C includes a double bond
between C8 and C8a and C5 and C6;
(ix) R;^-Ac when R'=-(CH)2C(Me)CHCH(Me)(Et), R"=Me, ring A
includes a double bond between C3 and C3a; ring B includes a double
bond between C4 and C4a; and ring C includes a double bond between
C8 and C8a and C5 and C6;
(x) R^i-Ac when R'=-(CH]2C(Me]CH-i-propyl, R"=Me, ring A includes
a double bond between C3 and C3a; ring B includes a double bond
between C4 and C4a; and ring C includes a double bond between C8
and C8a and C5 and C6;
(xi) R;^-C(0)(CH2},Me when R'=-{CH)2Me, R"=Me, ring A includes a
double bond between C3 and C3a; ring B includes a double bond
between C4 and C4a; and ring C includes a double bond between C8
and C8a and C5 and C6;
(xii] R5i-C(0](CH2),Me when R' = -/7-propyL R"=Me, ring A does not
include a double bond; and rings B and C include double bonds
between C8a and C4a and C5 and C6:
(xiii] R?^-C(0)(CH2]4Me when R'=-n-propyI, R"=Me, ring A, B and C do
not include double bonds;
(xiv) R5=-C(0)(CHJaMe when R'=-(CH)jMe, R"=Me, ring A does not
include a double bond; and rings B and C include double bonds
between C8a and C4a and C5 and C6;
(xv) R^-C(0)(CHj),Me when R'--C(CH2)(Me], R"-Me, ring A does not
include a double bond; and rings B and C include double bonds
between C8a and C4a and C5 and C6;
(xvi)R?^-C(0)(CH,),Me when W=-{CH]Me. R"=Me, ring A does not
include a double bond; and rings B and C include double bonds
between C8a and C4a and C5 and C6, including tautomers thereof,
[n a fourth aspect, the present invention consists in a compound
according to formula (I), wherein each of R, R' and R" is a hydrogen atom,
halogen atom or a straight or branchec chain C^.so alkyl, alkenyl or alkynyl
group optionally substituted with one or more halogen, hydroxy and/or oxo
groups;
ring A, B and C are opLioually substituted with one or more halogen
atoms; and
rings A, B and C optionally include one or more double bonds;
with the proviso that:
ii) R5:-C(0H)CHC(0)(CH]„CH(Et){Me) when R' = -CH20H, R"=Me,
ring A includes a double bond between C3 and C3a; ring B includes a
double bond between C4 and C4a; and ring C includes a double bond
between C8 and C8a;
iii) R?t-C(OH)CHC(0)[CH]«CH(Et)(Me) when R' = Me, R" = Me, ring A
includes a double bond between C3 and C3a; ring B includes a double
bond between C4 and C4a: and ring C includes a double bond between
CB and CSa;
iiii) R;±-C(0H)CHC(O)(CH)6CH(Et)(Me) when R' = Me. R" = Me, ring A
includes a double bond between C3 and C3a; nng B includes a double
bond between C4 and C4a: and ring C includes a double bond between
C8 and C8a and C5 and C6:
nv] R=-C(0)(CH),Me when R'=-n-propyl. R" = Me. nng A includes a
double bond between C3 and C2a; nng B includes a double bond
between C4 and C4a; and ring C includes a double bond between CB
and CSa and C5 and C6:
(v) R7t-C{0)(CH]eMe when R'=-n-propyl, R"=Me, ring A includes a
double bond between C3 and C3a; ring B includes a double bond
between C4 and C4a; and ring C includes a double bond between C8
and C8a and C5 and C6;
(vi) R;t-C(0)(CH]5Me when R'=-(CH)2Me, R"=Me, ring A includes a
double bond between C3 and C3a; ring B includes a double bond
between C4 and C4a; and ring C includes a double bond between C8
and C8a and C5 and C6;
(vii) R?i-C(0)(CH)eMe when R'=-(CH)2Me, R"=Me, ring A includes a
double bond between C3 and C3a; ring B includes a double bond
between C4 and C4a; and ring C includes a double bond between C8
and C8a and C5 and C6;
(viii)R^-(CH)2C(Me)CHC(Me)(Et) when R'=-Ac, R"=Me, C4=C1, ring A
includes a double bond between C3 and C3a; ring B includes a double
bond between C4 and C4a; and ring C includes a double bond between
C8 and C8a and C5 and C6;
(ix) R:^-(CH)2C(Me)CHC(Me)(Et) when R'=-Ac, R"=Me, ring A
includes a double bond between CS and C3a; ring B includes a double
bond between C4 and C4a; and ring C includes a double bond between
C8 and C8a and C5 and C6;
(x) R?i-(CH)2C(Me)CH-i-propyl when R'=-Ac, R"=Me, ring A includes
a double bond between C3 and C3a; ring B includes a double bond
between C4 and G4a; and ring C includes a double bond between C8
and C8a and C5 and C6;
(xi) R?t-C(0)(CH],Me when R'=-(CH)2Me, R"=Me, ring A includes a
double bond between C3 and C3a; ring B includes a double bond
between C4 and C4a; and ring C includes a double bond between C8
and CSa and C5 and CG;
(xii] R9t-C(0)(CH],Me when R'=-n-propyl, R"=Me, ring A does not
include a double bond; and rings B and C include double bonds
between C8a and C4a and C5 and C6;
(xiii) R?i-C(0)(CH),Me when R'=-n-propyl, R"=Me, ring A, B and C do
not include double bonds;
(xiv) R^-C(0)(CH)6Me when R'=-(CH]2Me, R" = Me, ring A does not
include a double bond; and rings B and C include double bonds
between CSa and C4a and C5 and C6;
(xv) R?i-C(0)(CH)4Me when R'=-C(CH2J(Me), R"=Me, ring A does not
include a double bond; and rings B and C include double bonds
between C8a and C4a and C5 and C6;
(xvi) R?t-C(0)(CH)4Me when R'=-(CH)2Me, R"=Me, ring A does not
include a double bond; and rings B and C include double bonds
between C8a and C4a and C5 and C6;
(xvii) R9t-C(0](CH2)4Me when R'=-ii-propyl, R"=Me, ring A includes a
double bond between C3 and C3a; ring B includes a double bond
between C4 and C4a; and ring C includes a double bond between C8
and C8a and C5 and C6;
(xviii) R;t-C(0)(CH2)6Me when R'=-n-propyl, R"=Me, ring A includes a
double bond between C3 and C3a; ring B includes a double bond
between C4 and C4a; and ring C includes a double bond between C8
and C8a and C5 and C6;
(xix) R^i-ClOKCHJgMe when R'=-{CH]2Me, R"=Me, ring A includes a
double bond between C3 and C3a; ring B includes a double bond
between C4 and C4a; and ring C includes a double bond between C8
and C8a and C5 and C6;
(xx) R7i-C(0)(CH2)9Me when R'=-(CH)2Me, R"=Me, ring A includes a
double bond between C3 and C3a; ring B includes a double bond
between C4 and C4a; and ring C includes a double bond between C8
and C8a and C5 and C6;
(xxi) R?^-Ac whenR'=-(CH)2C(Me)CHCH(Me)(Et), R"=Me, C4=C1, ring
A includes a double bond between C3 and C3a; ring B includes a
double bond between C4 and C4a; and ring C includes a double bond
between C8 and C8a and C5 and C6;
(xxii) R?i-Ac when R'=-(CH)2C(Me)CHCH(Me)(Et), R"=Me, ring A
includes a double bond between C3 and C3a; ring B includes a double
bond between C4 and C4a; and ring C includes a double bond between
C8 and C8a and C5 and C6;
(xxiii) R?t-Ac when R'=-(CH)2C(Me)CH-J-propyl, R"=Me, ring A
includes a double bond between C3 and C3a; ring B includes a double
bond between C4 and C4a; and ring C includes a double bond between
C8 and C8a and C5 and C6;
(xxiv) R^-C(0)(CH2]4Me when R' = -(CH)2Me, R" = Me, ring A includes a
double bond between C3 and C3a; ring B includes a double bond
between C4 and C4a; and ring C includes a double bond between C8
and C8a and C5 and C6;
(xxv) R;t-C(0)(CH2)4Me when R'=-n-propyl, R"=Me, ring A does not
include a double bond; and rings B and C include double bonds
between C8a and C4a and C5 and C6;
(xxvi) R;t.C[0){CH^]Me when R'=-/7-propyl, R" = Me, ring A, B and C do
not include double bonds;
(xxvii) R^-C(0){CH2]6Me when R'--(CH)2Me, R"=Me, ring A does not
include a double bond; and rings B and C include double bonds
between C8a and C4a and C5 and C6;
(xxviii) R7i-C(0)(CH2),Me when R'=-C[CH2)(Me), R"=Me, ring A does
not include a double bond; and rings B and C include double bonds
between C8a and C4a and C5 and C6;
(xxix)R?i-C(0)(CH2}4Me when R'=-(CH)2Me, R"-Me, ring A does not
include a double bond; and rings B and C include double bonds
betw^een C8a and C4a and C5 and C6, including tautomers thereof.
Preferably, the compound according to second, third and fourth aspect
of the invention is in accordance with formula (la) and more preferably, has
the stereochemistry as depicted in formula (lb).
Even more preferably, R--C(0H]CHC(0)(CH)gCH3, R'^-CH^OHand
R" = Me such that the present invention according to the second, third and
fourth aspect consists in the compound 5,6-dihydro-3-[(lZ, 4E, 6E, 8E]-1-
hydroxy-3-oxo-l,4,6,8-decatetraenyl]-6-hydroxymethyl-9a-methyl-2H-
furo[3,2-g][2]benzopyran-2-9(9aH)-dione acccording to formula (Ic).
It will be appreciated that the compound according to formula (I]-(Ic)
includes all corresponding tautomeric structures.
Chemically, the compounds of formula (I)-(Ic), such as 5,6-dihydro-3-
[(IZ, 4E, 6E, 8E]-l-hydroxy-3-oxo-l,4,6,8-decatetraenyl]-6-hydroxymethyl-9a-
methyl-2H-furo[3,2-g][2]benzopyran-2-9(gaH)-dione, are members of the
azaphilone family. The azaphilone compounds are produced by a variety of
fungi, and have been investigated for their antibiotic functions, their ability
to inhibit enzvmatic function, and their role as colouring agents in food.
The azaphilone nucleus has also been found in the pigments produced
bv Monascus spp. The pigments containing the common azaphilone core
include dihvdro-monascin and monascorubm. There is no record of their
utility as fluorescent dyes for the staining of biomolecules or organic
compounds.
None of the prior art known to the applicants teaches or suggests that a
compound of the structure described in formula (I)-[Ic) would be fluorescent,
nor suggests that it would be suitable for use in the fluorescent staining of
bio-molecules or organic compounds. There are accordingly unexpected and
advantageous properties associated with this compound including sensitive
detection of proteins in gels, cell tracking and dual colour staining.
The present inventors have unexpectedly found that compounds
according to formula (I)-(Ic) are not fluorescent in aqueous solution, but are
capable of interacting with organic compounds to produce an intensely
fluorescent stain. In a preferred embodiment, the compounds according to
formula (I)-(Ic) of the invention are used in the detection and tagging of
organic molecules.
Preferably, the compound according to formula (I)-(Ic), when bound to
an organic molecule emits fluorescence after excitation at blue wavelengths.
More preferably, the compounds according to formula (I)-(Ic] are excited by
light in the absorbance range 300-560 nm.
In a preferred embodiment, a compound according to formula [I)-(Ic)
interacts with proteins to produce a fluorescent complex that can be excited
by light generated by standard UV transilluminators (307 nm). Upon
excitation, the fluorescent complex emits light over a wide range of
wavelengths allowing the protein complexes to be detected. The excitation
wavelength of the protein/dye complex includes that of ethidium bromide
complexed with DNA (Absorption maximum 518 nm. Emission maxima 605
nm). This is of particular utility because it allows the same equipment to be
used to detect both DNA and protein in gels. The broad range of excitation
wavelengths allows the compound to be excited strongly by 488 nm. and 514
nm bands of the argon ion laser, as well as absorb light emitted from diode
light sources such as those the emit at around 400 nm
In another preferred embodiment, a fluorescent form of the compound
according to formula (I)-(Ic) is capable of strongly absorbing light from the
488 nm output of the argon-ion laser, and to re-emit the light at wavelengths
longer than 600 nm.
In another preferred embodiment, the compound according to formula
(I) is included in a composition, along with an analytically acceptable carrier,
14
and may be used in combination with a fluorochrome such as fluorescein in
dual staining appHcations. Such a fluorochrome may be included in the
composition or separately used.
The compound according to formula (I]-(Ic) may be produced by a
fungal species. Preferably, the fungal species is the strain deposited at the
Australian Government Analytical Laboratories (AGAL) on 15 January 1998
identified by Accession Number NM98/00507.
The methods for purification, isolation, as well of the structure of the
novel fluorophore 5,6-dihydro-3-[(lZ, 4E, 6E, 8E)-l-hydroxy-3-oxo-l,4,6,8-
decatetraenyl]-6-hydroxymethyl-9a-methyl-2H-furo[3,2-g][2]benzopyran-2-
9(9aH)-dione and related compounds have not previously been described.
The binding of the compound according to formula (I)-(Ic) to organic
molecules may involve direct chemical or physical binding or may be
achieved by the use of "linking molecules" known to the art. Organic
molecules which may be bound by the compound of the invention when
used as a fluorescent dye include, for example, proteins, peptides, sugars,
nucleic acids, antibodies, cell surface biomolecules, detergents and cells.
Due to the compound's fluorescent and organic compound-binding
characteristics, it will be appreciated that the compound will have use in any
application where detection of a fluorescent dye attached to an organic
compound is required.
In a fifth aspect, the present invention consists in a process of
producing a compound according to the first, second, third and fourth aspect
of the invention, the process comprising culturing a fungal species under
conditions such that the fungal species produces the compound; and
separating the compound from the culture.
The compound according to formula (I)-(Ic) can be used as a
fluorescent dye when in a crude culture extract or when purified by
extraction and separation techniques.
It has been found by the present inventors that after inoculation and
incubation of the strain identified by AGAL Acession Number NM98/00507
on the growth medium, a compound which is suitable as a fluorescent dye is
produced by the fungus.
It will be appreciated that it should be possible to produce compounds
according to formula (I)-(Ic) of the first, second, thu'd and fourth aspect of the
present mvention by using techniques other than from the supernatant of a
suitable fungal culture. For example, if the fungus produces a "pre-cursor",
then it will be possible to modify that pre-cursor to its fluorescent form by
chemical, physical or enzymatic means. The knowledge that such
compounds can be obtained from microorganisms should allow the discovery
and production of other compounds suitable for use as fluorescent dyes
belonging to the same family or quite distinct compounds with useful
characteristics. The compound according to formula (I) may also be
produced synthetically by direct chemical synthesis, or by modification of
intermediate(s) in the biosynthetic pathway used by the fungi.
The compound according to formula (I)-(Ic} of the present invention
may also be produced synthetically by chemical means. The knowledge that
a new fluorescent compound is produced by fungi may lead to other means
of producing the compound apart from culturing the fungi under the required
conditions.
The compound according to formula (I)-(Ic) of the present invention
has the distinct advantage that it binds to cells and other organic molecules
in its fluorescent form so can be used as a means to track the cells or the
other organic molecules when labelled with the compound.
In a sixth aspect, the present invention consists in use of the
compound according to the first and, second, third and fourth aspect of the
present invention as a fluorescent dye or marker, preferably in scientific
techniques for staining, labelling and/or detecting organic molecules.
Examples of the use of the compound according to the first, second,
third and fourth aspect of the present invention include but are not restricted
to cell tracking dyes for microscopy, membrane fluidity dyes, conjugation
with antibodies, conjugation to nucleic acids, cell surface ligand imaging
dyes, conjugation to sugars, cytometric analysis, and confocal microscopy. It
will be appreciated, however, that the compound would be suitable for any
use where fluorescence in the red wavelengths is required, including when
excited at 488 nm.
In a seventh aspect, the present invention consists in a method of
fluorescent-labelling an organic compound, the method comprising causing
the compound according to the first, second, third or fourth aspect of the
present invention to bind to an organic compound such that the organic
compound is fluorescently labelled with the compound.
Preferably, the fluorescently labelled organic compound is detected
when exposed to a wide range of wavelengths, preferably wavelengths in the
range of 300-560 nm.
The organic compound may include, but is not limited to, proteins,
peptides, sugars, nucleic acids, antibodies, cell surface biomolecules,
detergents and cells. The compound may bind directly to the organic
compound due to a chemical or physical association or may bind to the
organic compound via a linking molecule. If the compound is attached to a
ligand specific for the organic compound, for example an antibody or lectin,
then the binding of that ligand to the organic compound will cause the
organic compound to be fluorescently labelled.
In an eighth aspect, the present invention consists in a method of
detecting an organic compound in a sample including the organic compound,
the method comprising labelling the organic compound according to the
method of the seventh aspect of the present invention; and detecting the
organic compound in the sample by monitoring or detecting its fluorescence.
Preferably, the labelled organic compound is detected when the sample is
exposed to light from a wide range of wavelengths, preferably wavelengths in
the range of 300-560 nm, more preferably blue wavelengths.
The monitoring or detecting of the fluorescence of the labelled-organic
compound may be by any means known to the art. Such means include, but
are not limited to, transillumination, spectroscopy microscopy and
cytometry.
Throughout this specification the word "comprise", or variations such
as "comprises" or "comprising", will be understood to imply the inclusion of a
stated element, integer or step, or group of elements, integers or steps, but
not the exclusion of any other element, integer or step, or group of elements,
integers or steps.
Any discussion of documents, acts, materials, devices, articles or the
like which has been included in the present specification is solely for the
purpose of providing a context for the present invention. It is not to be taken
as an admission that any or all of these matters form part of the prior art base
or were common general knowledge in the field relevant to the present
invention as it existed in Australia before the priority date of each claim of
this application.
In order that the present invention may be more clearly understood a
preferred forms will be described with reference to the following examples
and accompanying drawings.
Brief Description of Drawings
Figure i shows an NMR spectrum of 5,6-dihydro-3-[(lZ, 4E, 6E, 8E)-1-
hydroxy-3-oxo-l,4,6,8-decatetraenyl]-6-hydroxyniethyl-9a-methyl-2H-
furo[3.2-g][2)ben2:opyran-2-9(9aH)-dione a fluorescent compound according
to the first, second, third and fourth aspects of the invention.
Figure 2 shows an example of an emission spectra of the compound
identified by Figure 1 when bound to bovine serum albumin (BSA) and
propidium iodide bound to DNA. The number of moles of the purified
compound used to bind to excess BSA, were equal to the number of moles of
propidium iodide used to bind to excess DNA. There are two excitation
bands resulting in emission at 605 nm. As seen, excitation by light at 390-
400 nm results in maximum emission of light at 605 nm.
Modes for Carrying Out the Invention
Example 1: Production of Extract A.
A biologically pure culture of the micro-organism having all of the
identifying characteristics of the strain identified by AGAL Accession
Number NlVl 98/00507 was obtained.
A growth medium for the fungus, AGAL Accession Number NIvI
98/00507, was prepared by adding 40 g Sucrose (GSR], 5 g Yeast Extract
(Difco), 10 g Peptone (Difco) and 10 g Agar (Difco) to 1 L of water. The
mixture was autoclaved for 15 min at 115°C to both sterilise the medium and
to dissolve the agar. The liquid was poured into culture dishes and allowed
to set at room temperature. After cooling, a culture of the fungus was
inoculated onto the surface of the medium, and incubated at 25°C for three
days. The culture was transferred to a refrigerator and incubated at 4°C until
the culture turned an intense red colour and dye production was high
(usually 3 to 5 days).
Once the culture produced sufficient dye for harvesting, the culture
medium, including the fungal biomass was transferred mto ethanoi at a ratio
of one volume of culture medium to two volumes of ethanoi. The dye was
extracted mto the ethanolic phase by mcubation at 4'^C and shaking for 16
hours. The liquid phase was decanted from the residual culture medium,
and centrifuged at 3000 rpm foi 10 min at 4°C.
The clarified extract (Extract A) was either stored for use, or purified
further according to one of the procedures described under examples 2 and 3.
Example 2: Purification of Extract A to produce Extract B.
The crude ethanolic extract of Extract A produced according to the
method described in example 1 was reduced in volume under high vacuum,
chromatographed on cellulose powder using methanol as a solvent and
applied to a sephadex LH-20 column, also eluted with methanol. Fractions
were collected over 48 hours and the purple band eluting near the end was
collected. This fraction was freeze dried and resuspended in a minimum
volume of (0.1% acetic acid) methanol and stored.
Example 3: Purification of 5,6-dihydro-3-[(lZ, 4E, 6E, 8E]-l-hydroxy-3-
oxo-l,4,6,8-decatetraenyl]-6-hydroxymetliyl-9a-methyl-2H-furo[3,2-
g][2]benzopyran-2-9(gaH)-dione.
Extract B produced according to the method described in example 2
was subjected to HPLC purification (Supelco Cl6-amide column, resolved in
75% methanol/water, 0.05% acetic acid, then 50% acetonitrile/water, 0.05%
acetic acid). The final fraction was frozen (-20°C) to remove water and the
remaining acetonitrile was evaporated under a stream of nitrogen.
This procedure produced analytically pure, 6-dihydro-3-{(lZ, 4E, 6E,
8E)-l-hydroxy-3-oxo-l,4,6,8-decatetraenyl]-6-hydroxymethyl-9a-methyl-2H-
furo[3,2-g][2]benzopyran-2-9(9aH)-dione which was an orange solid.
Example 4: Structure determination of ,6-dihydro-3-[(lZ, 4E, 6E, 8E)-1-
hydroxy-3-oxo-l,4,6,8-decatetraenyI]-6-hydroxymethyl-9a-methyI-2H-
furo[3,2-g][2]benzopyran-2-9(9aH)-dione.
The compound produced according to example 3 was analysed using a
combination of NMR spectroscopy and high resolution mass spectrometry to
determine the structure of, 6-dihydro-3-[(lZ, 4E, 6E, 8E)-l-hydroxy-3-oxo-
l,4.6,8-decatetraenyl]-6-hydroxymethyl-9a-methyl-2H-furo[3.2-
g][2]benzopyran-2-9(9aH)-dione.
Nuclear magnetic resonance spectroscopy.
The jSTN'IR sample was prepared by dissolving the ITPLC purified
compound obtained from Example 3 (~5 mg) in CDCl^ (0.5 aiL: 99.96 atom%.
Aldrich) and filtering it into an NMR tube (PP527, VVilmald). The sample
was degassed and equilibrated under an atmosphere of nitrogen.
The NiVlR data were acquired on a Bruker DRX600 (600 MPIz) NIvIR
spectrometer at 27°C and processed using xwinNlvIR (version 2.6; Bruker).
All 2D N^LR experiments were run with quadrature detection with an *H
spectral width of 6009 Hz and a recycle delay of 2 s. Chemical shifts were
referenced to the residual CHCI3 {5H 7.26 ppm; 5C 77.01 ppm). High power
^H n:/2 pulses were determined to be 9.5 ms and low power [for MLEV spin
lock] at 25.15 ms. "C high power 7c/2 pulse was 10.5 ms and a low power
pulse of 65 ms was used for GARP decoupling. Gradient pulses were
delivered along the z-axis using a 100 step sine program.
Data for ID experiments were acquired using 32 K real points and zero
filled to 64 K and then Gaussian multiplied for resolution enhancement.
Carbon - hydrogen correlation (HSQC) was achieved via a sensitivity
enhanced double INEPT transfer using echo/antiecho-TPPI gradient selection
(Palmer, A. G., Ill, Cavanagh, J., and Wright, P. (1991] /. Magn. Reson. 93,
151-70; Schleucher, J., Schwendinger, M., Sattler, M., and Schmidt, P. (1994]
/. Biomol. NMR 4, 301-6; Kay, L. E., Keifer, P., and Saarinen, T. (1992) /. Am.
Chem. Soc. 114(26], 10663-5]. 2K data points were collected in t2 (128 scans
per increment] with a 1.3 s recycle delay with decoupling during acquisition.
In tl, 512 increments were used (10-170 ppm] and the INEPT sequence was
optimized for a X-H coupling of 145 Hz. A gradient ratio of 80:20.1 was used
to select echo/antiecho-TPPI phase sensitivity.
One dimensional ROESY spectra were measured using a selective
Gaussian pulse on the proton of interest (Kessler, H., H. Oschkinat, C.
Griesinger & W. Bermel (1986]/. Magn. Reson. 70, 106; Stonehouse, J., P.
Adell, }. Keeler & A.J. Shaka, (1994]/. Am. Chem. Soc. 116, 6037; Stott, K., J.
Stonehouse, J. Keeler, T.L. Hwang & A.J. Shaka, (1995] / Am. Chem. Soc. 117,
4199 4200]. A 1000 step Gaussian program (60 ms, 64.6 dB] was used to
achieve a ti/2 pulse. A mixing time of 100 ms (13 dB] was used for a
continues wave spin lock. Gradient selection was achieved with a 15%
gradient along the z-axis. lOK transients were accumulated over 6009 Hz.
ROE enhancements were measured as a percentage of the irradiated peak and
not compensated for offset from the carrier frequency.
Two dimensional homonuclear Hartman-Hahn transfer spectra
(TOCSY) were measured using the MLEV17 (Bax. A., & Davis. D. G. (1985) /
Magn. Reson. 63(1), 207-13] pulse sequence flanked with 2 ms low power
trim pulses. Sine bell shifted (90°) apodisation was used in the processing of
both dimensions.
2D 'H-'^C correlation via heteronuclear zero and double quantum
coherence optimized for long range couplings (E[IvD3C) with low-pass J-filter
(145Hz) to suppress one-bond correlations (Bax, A., & M.F. Summers, (1986)
/. Am. Chem. Soc. 108, 2093 - 2094] was acquired with no decoupling during
acquisition time and using gradient pulses (50:30:40.1) for selection. The
delay for evolution of long range couplings was optimized for couplings of
20, 10, 5 and 2Hz in separate experiments. A spectral width of 210ppm was
used in Fl and the final spectrum magnitude calculated to destroy phase
information.
'H NMR (600MHz, CDCIJ 5 1.75, s, C9a-Me; 1.84, dd, J 6.7, 1.1 Hz, C9'-
Me; 2.80, dd, J 17.2, 3.6 Hz, H5a; 2.89, ddd, J 17.1, 11.5, 1.9 Hz, H5p; 3.85,
dd, J 12.2, 5.5 Hz, CeCH^OH; 3.97, dd, J 12.2, 3.4 Hz, C6CH2OH; 4.39, m, H6;
5.97, m, H9'; 6.10, d, J 15.1 Hz, H4', 6.19, ddd, J 15.1, 10.6, 1.6 Hz, H8'; 6.25,
dd, J 14.6, 11.2 Hz, H6'; 6.58, dd, J 14.5, 10.5 Hz, H7'; 6.79, s. H2"; 7.16, bs,
H4, 7.32, dd, J 15.2, 11.2 Hz, H5'; 7.85, s, H8.
"C NIVIR (125MHz, CDCI3) 5 18.8, C9'-Me; 28.0, C9a-Me; 29.3, C5; 63.4,
C6-CH2OH; 79.0, C6; 86.2, C9a; 101.0, C2'-, 112.2, C8a; 113.4, G4; 115.3, C3;
126.5, C4'; 128.6, C6'; 131.7, C8'; 136.0, C9'; 140.9, C4a; 142.0, C7'; 142.6, C5';
159.0, C8; 168.2, C2/C3a; 177.7, Cl-, 185.0, C3'; 190.0, C9.
Heteronuclear single quantum coherence (HSQC) was used to correlate
and assign all protons to protonated carbons. In addition, each spin system
was characterised by running a series of total correlation spectroscopy
(TOCSY) experiments with mixing times of 8 msec, 20 msec, 100 msec. The
shortest mixing time gave correlations to only the directly coupled protons
whereas the longest mixing time identified the entire spin system and gave
information about very small long range couplings which were valuable for
assigning the positions of the many (apparently) uncoupled protons.
Thorough space connectivities were achieved by a series of one dimensional
selective rotating frame correlation spectroscopy (ROESY) experiments.
Selective excitation was achieved by a lo-w power Gaussian 90 degree pulse
and using gradient section to observe only ROE's free of TOCSY transfers. A
mixing time of 100 msec was found to be optimal. Long range heteronuclear
multiple quantum coherence (HJvIBC) spectra with mixing tunes of 25, 50.
100 and 250 msec was found to be essential to assign all non-protonated
carbons. Even after this. C2 was found not to correlate with any protons and
was coincident with C3a at 168.2 ppm.
From the spectral data, several tricyclic skeletons were theoretically
possible but one (6-dihydro-3-[(lZ, 4E, 6E, 8E)-l-hydroxy-3-oxo-l,4,6,8-
decatetraenyl]-6-hydroxymethyl-9a-methyl-2H-furo[3,2-g][2]benzopyran-2-
9(9aH)-dione), fitted the available data exactly.
Mass Spectrometry
The compound produced according to Example 3 was subjected to high
resolution mass spectrometry to determine that the molecular formula of the
compound was C23H22O7.
Mass spectrum (ESI, positive ion) m/e 411 (M+H"", 55%), 317 (5), 249
(12), 163 (10), 121 (100), 93 (4). Negative ion ESI-FTICR Found: M-H"",
409.1304, C23H21O7 requires 409.1293; found: 247.0617, CuH^jOj requires
247.0685.
UV-Visible and Infrared Spectrometry
X^^ (MeOH) 432, 555 nm, e 10000, 4000. X^^ (alkaline MeOH) 432 nm.
s 16000. k^^ (acidic MeOH) 390, 560 nm, s 6000, 10000.
v„„ (neat) 3400 (br), 2925, 2854, 1744, 1712, 1589, 1259, 1010 cm-Example 5: Protein gel staining using Extract A.
A protein gel was prepared according to any one of a number of
standard protocols. For example, an SDS page gel was prepared according to
the protocol of Laemmli (U.K. 1970, Nature, 227:680-685). After
electrophoresis was completed, the gel was removed from the electrophoresis
apparatus. The gel was fixed in a container containing 100 mL of a staining
solution composed of 90 mL water, 10 mL glycerol and 5 mL of Extract A.
The gel and staining solution were incubated with gentle shaking for 90
minutes. After the 90 minute staining procedure, the solution was removed
and the gel briefly washed three times in water. After these brief washes, the
gel was placed in 100 mL of 10% glycerol in water and incubated with
shaking for a further 30 min.
To visualise the proteins in the gel, the gel was transferred to a UV
transilluminator (307 nm), and photographed using polaroid 667 black and
white film and a filter set designed for the detection of ethidium bromide gels
(eg. Molecular probes E-7591).
Example 6: Epifluoroescence differentiation of sporulated and vegetative
cells.
A micro-organism is cultured under conditions to promote sporulation
and Extract B is used to facilitate the identification of sporulated ceils within
a predominantly vegetative cell culture. For example, Saccharomyces
cerevisiae was grown in a water based broth containing 10 g/L Glucose, 5 g/1
yeast extract, and 10 g/L peptone. After a 16 hour incubation at 30°C, the
cells were harvested by centrifugation and resuspended in 1 mL of water.
The 5 IJ.L aliquots of the culture were spotted onto a semi-solid medium
composed of 5% Potassium acetate, 2% Agar and water. The cultures were
incubated at 22°C for 4 days to allow sporulation of the yeast cells. The
sporulated culture was resuspended in water to a density of 1 x 10' cells per
mL. 5 \iL of Extract B was added to 1 mL of the sporulated culture. To
counterstain, 5 p-L of a 10 mA'I solution (in DMSO] of 5(6)-carboxyfluorecein
diacetate (CFDA), was added to the sporulated culture. After 5 minutes, the
sporulated culture was harvested by centrifugation and resuspended in 1 ml
of water. Kxamination under an epifluorescence microscope (excitation 488
nm) allowed for rapid differentiation between sporulated and vegetative
cells.
It -will be appreciated by persons skilled in the art that numerous
variations and/or modifications may be made to the invention as shown in
the specific embodiments without departing from the spirit or scope of the
invention as broadly described. The present embodiments are, therefore, to
be considered in all respects as illustrative and not restrictive.
CLAIMS
1. A complex comprising a compound according to formula (I):

wherein:
.each of R, R' and R" is a hydrogen atom, halogen atom or a straight or branched
chain C1-20 alkyl, alkenyl or alkynyl group optionally substituted with one to ten halogen,
hydroxy and/or 0x0 group;
rings A, B and C optionally comprising one to five double bonds;
rings B and C are optionally substituted with one to five halogen atoms, and
an organic molecule,
wherein the organic molecule is a nitrogen containing compound, and wherein the
compound of formula (I) interacts with the organic molecule so as to fluoresce after
excitation.
2. A complex as claimed in claim 1 comprising a compound of formula (la):

3. A complex as claimed in claim 2 comprising a compound having the
stereochemistry as depicted in formula (lb):
(lb)
4. A complex as claimed in any one of claims 1 to 3 comprising a compoimd wherein
R is a straight or branched chain C1-20 conjuga.ted alkenyl group optionally substituted
with hydroxy and 0x0 groups; R' is a straight or branched chain C1-20 alkyl group
optionally substituted with a hydroxy group and R" is a straight or branched chain C1-20
alkyl group.
5. ¦ A complex as claimed in any one of claims 1 to 4, wherein the nitrogen containing
compound is selected from the group consisting of proteins, amino acids, peptides,
nucleic acids and aliphatic or aromatic amines.
6. A complex as claimed in any one of claims 1 to 5, wherein the compound of
formula (I) interacts with the organic molecule in the presence of a surfactant
(soap/detergent) so as to fluoresce after excitation.
7. A complex as claimed in claim 6, wherein the surfactant is selected from the group
consisting of sahs of long chain (C4 - C40) fatty acids or sulfonic acids.
8. A compound according to formula (I):

wherein each of R, R' and R" is a hydrogen atom, halogen atom or a straight or branched
chain C1-20 alkyl, alkenyl or alkynyl group optionally substituted with one to ten
halogen, hydroxy and/or 0x0 groups;
rings A, B and C are optionally substituted with one to five halogen atoms; and
rings A, B and C optionally comprise one to five double bonds;
with the proviso that:
(i) R^-C(0H)CHC(0)(CH)6CH(Et)(Me) when R'=-CH20H, R"=Me, ring A
comprising a double bond between C3 and C3a; ring B comprising a double bond
between C4 and C4a; and ring C comprising a double bond between C8 and C8a;
(ii) R9i-C(OH)CHC(0)(CH)6CH(Et)(Me) when R'=Me, R"=Me, ring A
comprising a double bond between C3 and C3a; ring B comprising a double bond
between C4 and C4a; and ring C comprising a double bond between C8 and C8a;
(iii) R?t-C(0H)CHC(0)(CH)6CH(Et)(Me) when R'=Me, R"=Me, ring A
comprising a double bond between C3 and C3a; ring B comprising a double bond
between C4 and C4a; and ring C comprising a double bond between C8 and C8a
and C5 and C6;
(iv) R9i:-C(0)(CH)4Me when R'=n-propyl, R"=Me, ring A comprising a double
bond between C3 and C3a; ring B comprising a double bond between C4 and C4a;
and ring C comprising a double bond between C8 and C8a and C5 and C6;
(v) R9!:-C(0)(CH)6Me when R'=n-propyl, R"=Me, ring A comprising a double
bond between C3 and C3a; ring B comprising a double bond between C4 and C4a;
and ring C comprising a double bond between C8 and C8a and C5 and C6;
(vi) R9!:-C(0)(CH)5Me when R'=-(CH)2Me, R"=Me, ring A comprising a
double bond between C3 and C3a; ring B comprising a double bond between C4
and C4a; and ring C comprising a double bond between C8 and C8a and C5 and
C6;
(vii) R^-C(0)(CH)6Me when R'=-(CH)2Me, R"=Me, ring A comprising a
double bond between C3 and C3a; ring B comprising a double bond between C4
and C4a; and ring C comprising a double bond between C8 and C8a and C5 and
C6;
(viii) R9i-(CH)2C(Me)CHC(Me)(Et), when R'=-Ac, R"=Me, C4=C 1, ring A
comprising a double bond between C3 and C3a; ring B comprising a double bond

between C4 and C4a; and ring C comprising a double bond between C8 and C8a
and C5 and C6;
(ix) R?i-(CH)2C(Me)CHC(Me)(Et), when R'=-Ac R"=Me, ring A comprising a
double bond between C3 and C3a; ring B comprising a double bond between C4
and C4a; and ring C comprising a double bond between C8 and C8a and C5 and
C6;
(x) R9!:-(CH)2C(Me)CH-i-propyl when R'=-Ac, R"=Me, ring A comprising a
double bond between C3 and C3a; ring B comprising a double bond between C4
and C4a; and ring C comprising a double bond between C8 and C8a and C5 and
C6;
(xi) R?i-C(0)(CH)4Me when R'=-(CH)2Me, R"=Me, ring A comprising a
double bond between C3 and C3a; ring B comprising a double bond between C4
and C4a; and ring C comprising a double bond between C8 and C8a and C5 and
C6;
(xii) R?i-C(0)(CH)4Me when R'=-n-propyl, R"=Me, ring A does not comprise a
double bond; and rings B and C comprising double bonds between C8a and C4a
and C5 and C6;
(xiii) R9i-C(0)(CH)4Me when R'=-n-propyl, R"=Me, rings A, B and C do not
comprise double bonds.
(xiv) R?t-C(0)(CH)6Me when R'=-C(CH)2Me, R"=Me, ring A does not
comprise a double bond; and rings B and C comprising double bonds between C8a
and C4a and C5 and C6;
(XV) R^-C(0)(CH)4Me when R'=-C(CH)2(Me), R"=Me, ring A does not
comprise a double bond; and rings B and C comprising double bonds between C8
and C4a and C5 and C6;
(xvi) R9i-C(0)(CH)4Me when R'=-(CH)2Me, R"=Me, ring A does not comprise
a double bond; and rings B and C comprising double bonds between C8a and C4a
and C5 and C6;
(xvii) R7i:-C(0)(CH2)4Me when R'=-n-propyl, R"=Me, ring A comprising a
double bond between C3 and C3a; ring B comprising a double bond between C4
and C4a; and ring C comprising a double bond between C8 and C8a and C5 and
C6;

(xviii) R7t-C(0)(CH2)6Me when R'=-n-propyl, R"=Me, ring A comprising a
double bond between C3 and C3a; ring B comprising a double bond between C4
and C4a; and ring C comprising a double bond between C8 and C8a and C5 and
C6;
(xix) R?i:-C(0)(CH2)5Me when R'=-(CH)2Me, R"=Me, ring A comprising a
double bond between C3 and C3a; ring B comprising a double bond between C4
and C4a; and ring C comprising a double bond between C8 and C8a and C5 and
C6;
(xx) R9t-C(0)(CH2)6Me when R'=-(CH)2Me, R"=Me, ring A comprising a
double bond between C3 and C3a; ring B comprising a double bond between C4
and C4a; and ring C comprising a double bond between C8 and C8a and C5 and
C6;
(xxi) R^i-Ac when R'=-(CH)2C(Me)CHCH(Me)(Et), R"=Me, C4=C 1, ring A
comprising a double bond between C3 and C3a; ring B comprising a double bond
between C4 and C4a; and ring C comprising a double bond between C8 and C8a
and C5 and C6;
(xxii) Rit-Ac when R'=-(CH)2C(Me)CHCH(Me)(Et), R"=Me, ring A
comprising a double bond between C3 and C3a; ring B comprising a double bond
between C4 and C4a; and ring C comprising a double bond between C8 and C8a
and C5 and C6;
(xxiii) R^-Ac when R'=-(CH)2C(Me)CH-/-propyl, R"=Me, ring A comprising a
double bond between C3 and C3a; ring B comprising a double bond between C4
and C4a; and ring C comprising a double bond between C8 and C8a and C5 and
C6;
(xxiv) R?i-C(0)(CH2)4Me when R'=-(CH)2Me, R"=Me, ring A comprising a
double bond between C3 and C3a; ring B comprising a double bond between C4
and C4a; and ring C comprising a double bond between C8 and C8a and C5 and
C6;
(xxv) R9t-C(0)(CH2)4Me when R'=-n-propyl, R"=Me, ring A does not comprise
a double bond; and rings B and C comprising double bonds between C8a and C4a
and C5 and C6;

(xxvi) R9t-C(0)(CH2)4Me when R'=-n-propyl, R"=Me, rings A, B and C do not
comprise double bonds.
(xxvii) R^-C(0)(CH2)6Me when R'=-(CH)2Me, R"=Me, ring A does not
comprise a double bond; and rings B and C comprising double bonds between C8a
and C4a and C5 and C6;
(xxviii) R7t-C(0)(CH2)4Me when R'=-C(CH2)(Me), R"=Me, ring A does not
comprise a double bond; and rings B and C comprising double bonds between CSa
and C4a and C5 and C6;
(xxix) R?t-C(0)(CH2)4Me when R'=-(CH)2Me, R"=Me, ring A does not
comprise a double bond; and rings B and C comprising double bonds between CSa
and C4a and C5 and C6, comprising tautomers thereof.
9. A compound as claimed in claim 8 according to the formula (la):

10. A compound as claimed in claim 9 having the stereochemistry as depicted in
formula (lb):

11. A compound according to formula (Ic) wherein R is -
C(OH)CHC(0)(CH)6CH3, R' is -CH2OH and R" is Me being the compound 5,6-
dihydro-3-[(lZ, 4E, 6E, 8E)-l-hydroxy-3-oxo-l,4,6,8-decatetraenyl]-6-hydroxymethyl-
9a-methyl-2H-furo [3,2-g][2]benzopyran-2-9(9aH)-dione according to formula (Ic),
comprising tautomers thereof:
(Ic)
12. A compound according to any one of claims 8 to 11, wherein the compound is
capable of interacting with a nitrogen containing organic molecule so as to fluoresce
after excitation.
13. A complex according to any one of claims 1 to 7 or a compound as claimed in any
one of claims 8 to 12, wherein the compound when interacting with a nitrogen
containing organic molecule, emits fluorescence after excitation at ultraviolet to blue
wavelength.
14. A complex as claimed in any one of claims 1 to 7 or a compound as claimed in any
one of claims 8 to 12, wherein the compound when interacting with a nitrogen
containing organic molecule, emits fluorescence when excited by light in the range 300-
560nm.
15. A complex or a compound as claimed in claim 14, wherein the organic molecule is
a protein and the compound when interacting with the protein emits fluorescence after
excitation at 307 nm.
16. A complex or a compound as claimed in claim 14, wherein compound emits
fluorescence when excited by light at about 400 run, about 488 nm and about 514 nm.
17. A complex or a compound as claimed in claim 16, wherein the compound emits
fluorescence at about 600 nm after excitation at 488 nm.
18. A composition including a complex or a compound as claimed in any one of
claims 1 to 17, an analytically acceptable carrier and optionally, a flurochrome.
19. A process for the preparation of a compound as claimed in any one of claims 8 to
12 comprising culturing a fungal species under conditions such that the fungal species
produces the compound, and separating the compound from the culture.
20. A method for fluorescently labelling a nitrogen containing organic molecule,
comprising causing a compound according to formula (I):

wherein:
each of R, R' and R" is a hydrogen atom, halogen atom or a straight or branched
chain C1-20 alkyl, alkenyl or alkynyl group optionally substituted with one to ten
halogen, hydroxy and/or 0x0 group;
rings A, B and C optionally having one to five double bonds;
rings B and C are optionally substituted with one to five halogen atoms,
comprising tautomers of formula (I);
or a compound of formula (la), comprising tautomers thereof:

or a compound having the stereochemistry as depicted in formula (lb), comprising
tautomers thereof:


or a compound of formula (I), (la) or (lb) wherein R is a straight or branched chain C1-20
conjugated alkenyl group optionally substituted with hydroxy and 0x0 groups; R' is a
straight or branched chain Ci-io alkyl group optionally substituted with a hydroxy group
and R" is a straight or branched chain C1-20 alkyl group, comprising tautomers thereof,
or a compound according to any one of claims 8 to 12, which bind to the nitrogen
containing organic molecule in a manner such that the organic molecule is fluorescently
labelled with the compound.
21. A complex, compound, method, or composition as claimed in any one of claims 1
to 8, 13 to 15 and 19, wherein the organic molecule is a protein, peptide, sugar, nucleic
acid, cell surface molecule or detergent, or is included in an antibody or a cell.
22. A complex, compound, composition, or method as claimed in claim 24 wherein
the compound is bound to the organic molecule through a linking moiety.
23. A method of detecting a nitrogen containing organic molecule in a sample
comprising the organic molecule, the method comprising labelling the organic molecule
according to the method of claim 20, and detecting the organic molecule in the sample
by monitoring or detecting its fluorescence.
24. A method as claimed in claim 23, wherein the labelled organic molecule is
detected when the sample is exposed to light from a wide range of wavelengths,
preferably wavelengths in the range of 300-560 nm, more preferably ultraviolet to blue
wavelengths.
25. A method as claimed in claim 23 or claim 24, wherein the monitoring or detecting
of the fluorescence of the labelled-organic molecule is by transillumination,
spectroscopy, microscopy or cytometry.
26. A composition including:
(i) a compound according to formula (I)
(I)
wherein:
each of R, R' and R" is a hydrogen atom, halogen atom or a straight or branched
chain Ci-20 alkyl, alkenyl or alkynyl group optionally substituted with one to ten
halogen, hydroxy and/or 0x0 group;
rings A, B and C optionally comprising one to five double bonds;
rings B and C are optionally substituted with one to five halogen atoms,
comprising tautomers of formula (I), and
(ii) an organic molecule
wherein the organic molecule is a nitrogen containing molecule,
wherein the compound interacts with the organic molecule so as to fluoresce after
excitation.
27. A complex comprising a compound according to formula (I):

wherein:
each of R, R' and R" is a hydrogen atom, halogen atom or a straight or branched
chain C1-20 alkyl, alkenyl or alkynyl group optionally substituted with one to ten
halogen, hydroxy and/or 0x0 group;
rings A, B and C optionally comprising one to five double bonds;
rings B and C are optionally substituted with one to five halogen atoms,
comprising tautomers of formula (I), and
an organic molecule.

wherein the organic molecule is a nitrogen containing molecule, and
wherein the compound is capable of interacting with the organic molecule so as to
fluoresce after excitation.
28. A complex as claimed in claim 29, comprising a compound according to formula
(la), comprising tautomers thereof:
(la)
29. A complex as claimed in claim 28, wherein the compound has the stereochemistry
as depicted in formula (lb), comprising tautomers thereof:
(lb)
30. A complex as claimed in any one of claims 27 to 29 wherein R is a straight or
branched chain C1-20 conjugated alkenyl group optionally substituted with hydroxy and
0x0 groups; R' is a straight or branched chain C1-20 alkyl group optionally substituted
with a hydroxy group and R" is a straight or branched chain C1-20 alkyl group,
comprising tautomers thereof.
31. A complex as claimed in claim 29 or 30 comprising a compound wherein R is -
C(OH)CHC(0)(CH)6CH3, R' is -CH2OH and R" is Me being the compound 5,6-
dihydro-3-[(lZ, 4E, 6E, 8E)-l-hydroxy-3-oxo-l,4,6,8-decatetraenyl]-6-hydroxymethyl-
9a-methyl-2H-furo [3,2-g][2]benzopyran-2-9(9aH)-dione according to formula (Ic),
comprising tautomers thereof:
32. A complex as claimed in any one of claims 27 to 31 wherein the nitrogen
containing molecule is selected from the group consisting of ammonia, hydrazine and
hydroxylamine, ammonia containing compounds, hydrazine containing compounds and
hydroxylamine containing compounds.
33. A complex as claimed in any one of claims 27 to 32, wherein the compound
interacts with the inorganic molecule in the presence of a surfactant so as to fluoresce
after excitation at a broad range of wavelengths.
34. A complex as claimed in claim 33, wherein the surfactant is selected from the
group consisting of salts of long chain (C4 - C40) fatty acids or sulfonic acids.
35. A complex as claimed in any one of claims 27 to 34, wherein the compound when
interacting with an inorganic molecule, emits fluorescence after excitation at ultraviolet
to blue wavelengths.
36. A complex as claimed in any one of claims 27 to 34, wherein the compound emits
fluorescence when excited by light in the range of 300-560 nm.
37. A complex as claimed in claim 36, wherein the compound emits fluorescence
when excited by light at about 400 nm, 488 nm or 514 nm.
38. A complex as claimed in claim 37, wherein compound emits fluorescence at
visible wavelengths after excitation at 488 nm.
39. A composition including a complex as claimed in any one of claims 27 to 38, an
analytically acceptable carrier and optionally, another fluorochrome.
40. A method of detecting biomolecules by fluorescence, the method comprising
interacting a compound according to formula (I)
(I)
with the biomolecule;
wherein:
each of R, R' and R" is a hydrogen atom, halogen atom or a straight or branched
chain C1-20 alkyl, alkenyl or alkynyl group optionally substituted with one to ten
halogen, hydroxy and/or 0x0 group;
rings A, B and C optionally comprising one to five double bonds;
rings B and C are optionally substituted with one to five halogen atoms,
comprising tautomers of formula (I).
41. A method as claimed in claim 40, wherein the biomolecule is detected when the
sample is exposed to light from a wide range of wavelengths, preferably wavelengths in
the range of 300-560 nm, more preferably ultraviolet to blue wavelengths.
42. A method as claimed in claim 40 or claim 41 wherein the monitoring or detecting
of the fluorescence of the biomolecule is by transillumination, spectroscopy, microscopy
or cytometry.
43. A composition including:
(i) a compound according to formula (I)
(I)
wherein:
each of R, R' and R" is a hydrogen atom, halogen atom or a straight or branched
chain C1-20 alkyl, alkenyl or alkynyl group optionally substituted with one to ten
halogen, hydroxy and/or 0x0 group;

rings A, B and C optionally comprising one to five double bonds;
rings B and C are optionally substituted with one to five halogen atoms,
comprising tautomers of formula (I), and
(ii) an inorganic molecule,
wherein the inorganic molecule is a nitrogen containing molecule;
wherein the compound is capable of interacting with an inorganic molecule so as
to fluoresce after excitation.

The invention relates to the discovery of novel compounds from fungi that
fluoresce upon interaction with organic or inorganic compounds. Such
fluorophores can be used to detect and/or quantify compounds in a number of
settings including (but not limited to) proteins in electrophoresis gels, in solution,
on membranes or other surfaces.

Documents:

in-pct-2002-1294-kol-abstract.pdf

in-pct-2002-1294-kol-assignment.pdf

in-pct-2002-1294-kol-claims.pdf

in-pct-2002-1294-kol-correspondence.pdf

in-pct-2002-1294-kol-description (complete).pdf

in-pct-2002-1294-kol-drawings.pdf

in-pct-2002-1294-kol-examination report.pdf

in-pct-2002-1294-kol-form 1.pdf

in-pct-2002-1294-kol-form 18.pdf

in-pct-2002-1294-kol-form 3.pdf

in-pct-2002-1294-kol-form 5.pdf

in-pct-2002-1294-kol-gpa.pdf

in-pct-2002-1294-kol-granted-abstract.pdf

in-pct-2002-1294-kol-granted-claims.pdf

in-pct-2002-1294-kol-granted-drawings.pdf

in-pct-2002-1294-kol-granted-form 1.pdf

in-pct-2002-1294-kol-granted-specification.pdf

in-pct-2002-1294-kol-others.pdf

in-pct-2002-1294-kol-reply to examination report.pdf

in-pct-2002-1294-kol-specification.pdf


Patent Number 244889
Indian Patent Application Number IN/PCT/2002/1294/KOL
PG Journal Number 52/2010
Publication Date 24-Dec-2010
Grant Date 23-Dec-2010
Date of Filing 17-Oct-2002
Name of Patentee FLUOROTECHNICS PTY LTD.
Applicant Address R557 BUILDING E8C, MACQUARIE UNIVERSITY, NORTH RYDE NSW
Inventors:
# Inventor's Name Inventor's Address
1 BELL PHILIP JOHN LIVINGSTON 18 APPS AVENUE, TURRAMURRA NSW 2074
2 KERUSO PETER 1/108 CAMMERAY ROAD, NSW 2062
PCT International Classification Number C07D 493/04
PCT International Application Number PCT/AU2001/00472
PCT International Filing date 2001-04-26
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 PQ 7117 2000-04-26 Australia