U.S. patent application number 13/037163 was filed with the patent office on 2011-09-01 for methods and systems for synthesis of a d-aminoluciferin precursor and related compounds.
Invention is credited to Amy L. GRYSHUK, Julie Perkins.
Application Number | 20110213124 13/037163 |
Document ID | / |
Family ID | 44505623 |
Filed Date | 2011-09-01 |
United States Patent
Application |
20110213124 |
Kind Code |
A1 |
GRYSHUK; Amy L. ; et
al. |
September 1, 2011 |
METHODS AND SYSTEMS FOR SYNTHESIS OF A D-AMINOLUCIFERIN PRECURSOR
AND RELATED COMPOUNDS
Abstract
Methods and systems to generate 6-amino-6-deoxy-D-luciferin
precursor, 2-cyano-6-aminobenzothiazole and related compounds and
derivatives
Inventors: |
GRYSHUK; Amy L.; (Livermore,
CA) ; Perkins; Julie; (Sunnyvale, CA) |
Family ID: |
44505623 |
Appl. No.: |
13/037163 |
Filed: |
February 28, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61308317 |
Feb 26, 2010 |
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61331072 |
May 4, 2010 |
|
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61331094 |
May 4, 2010 |
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Current U.S.
Class: |
530/329 ;
530/345 |
Current CPC
Class: |
C07K 1/13 20130101; A61K
49/0013 20130101 |
Class at
Publication: |
530/329 ;
530/345 |
International
Class: |
C07K 7/06 20060101
C07K007/06; C07K 1/113 20060101 C07K001/113 |
Goverment Interests
STATEMENT OF GOVERNMENT INTEREST
[0002] The United States Government has rights in this invention
pursuant to Contract No. DE-AC52-07NA27344 between the United
States Department of Energy and Lawrence Livermore National
Security, LLC for the operation of Lawrence Livermore National
Laboratory
Claims
1. A method to provide a peptide conjugated to
6-amino-6-deoxy-D-luciferin, the method comprising conjugating an
amino acid with a 2-cyano-6-aminobenzothiazole to provide a
2-cyano-6-amino-aminoacid-benzothiazole, the conjugating performed
to allow formation of a bond between the amino group of
6-amino-6-deoxy-D-luciferin and a carboxylic group of the amino
acid; conjugating the peptide with the
2-cyano-6-amino-aminoacid-benzothiazole to provide a
2-cyano-6-amino-peptide-benzothiazole; and reacting the
2-cyano-6-amino-peptide-benzothiazole with a D-cysteine to provide
a 6-amino-peptide-6-deoxy-D-luciferin, wherein the bond is a
peptide bond, carbamate bond or a urea/thiourea bond.
2. The method of claim 1, wherein the bond is a peptide bond.
3. The method of claim 1, wherein the amino acid is an amino acid
having a side chain hydrophobic, hydrophilic, acid or basic in
nature.
4. The method of claim 3, wherein the amino acid is Alanine,
Isoleucine, Leucine, Methionine, Phenylalanine, Tryptophan,
Tyrosine and Valine.
5. The method of claim 3, wherein the amino acid is Serine,
Threonine, Asparagine and Glutamine.
6. The method of claim 3, wherein the amino acid is Arginine,
histidine and Lysine, Aspartic acid and Glutamic acid.
7. The method of claim 1, wherein the peptide comprises at least 5
amino acid residues.
8. The method of claim 7, wherein the peptide comprises at least 6
or 7 amino acid residues.
9. The method of claim 1, wherein the peptide further comprises a
label.
10. A system to provide a peptide conjugated to a
6-amino-6-deoxy-D-luciferin, the system comprising one or more
peptides, amino acids, 2-cyano-6-aminobenzothiazole,
2-cyano-6-amino-aminoacid-benzothiazoles and/or
2-cyano-6-amino-peptide-benzothiazoles for simultaneous combined or
sequential use in the method to provide a peptide labeled with
6-amino-6-deoxy-D-luciferin according to claim 1.
11. An intermediate in the synthesis of an amino acid labeled with
6-amino-6-deoxy-D-luciferin, the intermediate being a
2-cyano-6-aminobenzothiazole of formula (III) ##STR00017## wherein
R1 is a single amino acid and R2 is a peptide attached to the
remainder of the compound of formula (IV) through a peptide bond a
carbamate bond or a urea/thiourea bond.
12. A labeled peptide obtainable by the method to provide a peptide
conjugated to a 6-amino-6-deoxy-D-luciferin according to claim 1.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is related and claims priority to U.S.
Provisional Application No. 61/308,317 entitled "Alternate
Syntheses of Precursor to D-Aminoluciferin" filed on Feb. 26, 2010,
docket IL-12088 to U.S. Provisional Application No. 61/331,072
entitled "Bioluminescent Protease Probe For The Detection Of
Disease" filed on, May 4, 2010 with docket number IL-12087, and to
U.S. Provisional Application No. 61/331,094 entitled
"Carboxylate-Modified Luciferin, Amino Acid/Peptide Probes For
Bioluminescent Protease Assays" filed on, May 4, 2010 with docket
number IL-11088, each of which is incorporated herein by reference
in its entirety.
FIELD
[0003] The present disclosure relates to bioluminescence and in
particular to methods and systems for the synthesis of
D-aminoluciferin precursor and related compounds.
BACKGROUND
[0004] Bioluminescence provides a useful read out for the detection
of reactions in a background of optically complex materials such as
cells and tissues and the signal-to-noise ratio can be very
high.
[0005] Luciferases are enzymes that generate visible light through
the oxidation of a specific substrate in the presence of oxygen and
usually a source of energy (such as Mg2.sup.+ and ATP). The
reaction between firefly luciferase and the substrate luciferin,
yields oxyluciferin, carbon dioxide and visible light with a
maximum around 560 nm and with other wavelengths of light output
identifiable by a skilled person.
[0006] For these reasons, bioluminescence and in particular
luciferin and related compounds are routinely used for in vivo
imaging applications and for additional techniques for in vitro
and/or in vivo detection of targets and/or reactions.
SUMMARY
[0007] Provided herein are methods and systems to synthesize a
precursor of D-aminoluciferin and related compounds and methods
that in several embodiments allow to minimize mixture of products
evident in the early steps and/or to conjugate D-aminoluciferin to
amino acids and peptide sequences to generate probes suitable for
bioluminescence assays.
[0008] According to a first aspect a method and system to provide
2-cyano 6-amino-benzothiazole from a monofunctional benzothiazole
is described. The method comprises providing a monofunctional
benzothiazole attaching in position C2 a functional group of
formula (I) (C(.dbd.X.sub.1)NH.sub.2) wherein X.sub.1 is O or S,
and converting the functional group of formula (I) to a cyanide
group, through elimination of H.sub.2X.sub.1 from the
monofunctional benzothiazole. In the method the monofunctional
benzothiazole either comprises an amino group in position C6 or is
modified to comprise an amino group in position C6. The system
comprises one or more monofunctional benzothiazoles attaching in
position C2 a functional group of formula (I), together suitable
reagents for simultaneous combined or sequential use in the method
to provide 2-cyano-6-aminobenzothiazole herein described
[0009] According to a second aspect a method and system to provide
2-cyano-6-aminobenzothiazole from a monofunctional benzothiazole is
described. The method comprises providing a monofunctional
benzothiazole attaching in position C2 a functional group of
formula (II) (C(.dbd.X.sub.1)X.sub.2R) wherein R is an alkyl group,
or a halogen atom; and X.sub.1 and X.sub.2 are independently O or
S. The method also comprises converting the functional group of
formula (II) into an amide of formula (I) (C(.dbd.X.sub.1)NH.sub.2)
and converting the functional group of formula (I) to a cyanide
group through elimination of a H.sub.2X.sub.1 from the
monofunctional benzothiazole. In the method the monofunctional
benzothiazole either comprises an amino group in position C6 or is
modified to comprise an amino group in position C6. The system
comprises at least two of one or more monofunctional benzothiazole
attaching in position C2 a functional group of formula (I), and/or
one or more monofunctional benzothiazole attaching in position C2 a
functional group of formula (II) together suitable reagents for
simultaneous combined or sequential use in the method to provide
2-cyano-6-aminobenzothiazole herein described.
[0010] According to a third aspect, a method and system to provide
an amino acid labeled with 6-amino-6-deoxy-D-luciferin and amino
acid obtainable thereby, are described. The method comprises
conjugating the amino acid with a 2-cyano-6-aminobenzothiazole to
provide an amino acid-conjugated 2-cyano-6-aminobenzothiazole or
2-cyano-6-aminobenzothiazole-[amino acid, the conjugating performed
to allow formation of a peptide bond, a carbamate bond or a
urea/thiourea bond between the amino group of
6-amino-6-deoxy-D-luciferin and a carboxylic group of the amino
acid. The method further comprises reacting the
2-cyano-6-aminobenzothiazole-[amino acid] with a D-cysteine to
provide an amino acid-conjugated 6-amino-6-deoxy-D-luciferin. In
the method, the amino acid is an amino acid having a side chain.
The system comprises at least two of one or more amino acids with
each amino acid having a side chain an amino acid-conjugated
2-cyano-6-aminobenzothiazole-[amino acid] and an amino
acid-conjugated 2-cyano-6-aminobenzothiazole-[amino acid] for
simultaneous combined or sequential use in the method to provide an
amino acid labeled with 6-amino-6-deoxy-D-luciferin herein
described.
[0011] According to a fourth aspect an intermediate in the
synthesis of an amino acid labeled with 6-amino-6-deoxy-D-luciferin
is described. The intermediate is a 2-cyano-6-aminobenzothiazole of
formula (III)
##STR00001##
wherein R1 is an amino acid attached to the remainder of the
compound of formula (III) through a peptide bond, a carbamate bond
or a urea/thiourea bond.
[0012] According to a fifth aspect, a method and system to provide
a peptide labeled with 6-amino-6-deoxy-D-luciferin is described.
The method comprises conjugating the peptide with an amino
acid-conjugated 2-cyano-6-aminobenzothiazole to provide a
peptide-conjugated 2-cyano-6-aminobenzothiazole, the conjugating
performed to allow formation of a peptide bond between the amino
group of the amino acid-conjugated 2-cyano-6-aminobenzothiazole and
a carboxylic acid group of the peptide. The method can further
comprise reacting the peptide-conjugated
2-cyano-6-aminobenzothiazole with D-cysteine to provide a
peptide-conjugated 6-amino-6-deoxy-D-luciferin. The system
comprises one or more peptides, amino acids,
2-cyano-6-aminobenzothiazole,
2-cyano-6-amino-aminoacid-benzothiazoles and/or
2-cyano-6-amino-peptide-benzothiazoles for simultaneous combined or
sequential use in the method to provide a peptide labeled with
6-amino-6-deoxy-D-luciferin herein described.
[0013] According to a sixth aspect an intermediate in the synthesis
of a peptide labeled with 6-amino-6-deoxy-D-luciferin is described.
The intermediate is a 2-cyano-6-aminobenzothiazole of formula
(IV)
##STR00002##
wherein R1 is a single amino acid and R2 is a peptide attached to
the remainder of the compound of formula (IV) through a peptide
bond a carbamate bond or a urea/thiourea bond.
[0014] According to a seventh aspect, a labeled peptide is
described. The labeled peptide is obtainable by the method to
provide a peptide conjugated to 6-amino-6-deoxy-D-luciferin herein
described.
[0015] According to an eighth aspect a carboxylate-modified
luciferin amino acid/peptide probe, is described and related method
for synthesis and/or use of said carboxylate modified probe. The
carboxylate-modified D-luciferin, amino acid/peptide probe
comprises a D-luciferin molecule conjugated to an amino acid or
peptide wherein conjugation between the D-luciferin and the amino
acid or peptide is performed with peptide bond between the
amino-terminus of the amino acid or peptide and a carboxyl group of
the said D-luciferin.
[0016] The methods and systems described allow in several
embodiments facilitating the generation of amounts of pure
6-amino-6-deoxy-D-luciferin (D-aminoluciferin) precursor.
[0017] The methods and systems described allow in several
embodiments facilitating the coupling of
6-amino-6-deoxy-D-luciferin (D-aminoluciferin) precursor to peptide
sequences and/or to single amino acids in solution phase.
[0018] The methods and systems described allow in several
embodiments generation of pure 6-amino-6-deoxy-D-luciferin
(D-aminoluciferin) precursor and/or subsequent coupling to peptide
sequences and/or to single amino acids in a solution phase.
[0019] The methods and systems described herein can be used in
connection with applications wherein production of D-aminoluciferin
and/or related uses and/or derivatives is desired, including but
not limited to medical application, biological analysis and
diagnostics including but not limited to clinical applications.
[0020] The details of one or more embodiments of the disclosure are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages will be apparent from the
description and drawings, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] The accompanying drawings, which are incorporated into and
constitute a part of this specification, illustrate one or more
embodiments of the present disclosure and, together with the
detailed description and the examples, serve to explain the
principles and implementations of the disclosure.
[0022] FIG. 1 shows a schematic representation of a bioluminescent
assay based on release of D-aminoluciferin from labeled compounds
according to an embodiment herein described.
[0023] FIG. 2 shows a schematic representation of a method to
provide 6-amino-2-cyanobenzothiazole from a monofunctional
benzothiazole according to an embodiment herein described. In
particular, the schematic of FIG. 2 illustrates a synthetic route
of a 4 step process to generate D-aminoluciferin precursor,
2-cyano-6-aminobenzothiazole beginning with a monofunctional
benzothiazole(6-nitrobenzothiazole).
[0024] FIG. 3 shows a schematic representation of the four steps
depicted in FIG. 2 according to an embodiment herein described. In
particular, the schematic of FIG. 3 shows a specific route where a
carboxyethyl functional group is introduced in the position C2 of
6-nitrobenzothiazole and then converted to a carboxamide group and
on to a cyano group. The nitro group in position C6 of the
resulting 2-cyano-6-nitrobenzothiazole is then reduced to an amino
group.
[0025] FIG. 4 shows nuclear magnetic resonance spectra for
compounds of methods and systems of the disclosure, according to an
embodiment herein described. FIG. 4A shows a nuclear magnetic
resonance (.sup.1H-NMR) spectra of ethyl
6-nitrobenzothiazole-2-carboxylate 2 obtained on a Bruker 500 MHz
machine in d.sub.6-DMSO. FIG. 4B shows nuclear magnetic resonance
(.sup.13C-NMR) spectra of ethyl 6-nitrobenzothiazole-2-carboxylate
2 obtained on a Bruker 500 MHz machine in d.sub.6-DMSO. FIG. 4C
shows a nuclear magnetic resonance (.sup.1H-NMR) spectra of
6-nitrobenzothiazole-2-carboxamide 3 obtained on a Bruker 500 MHz
machine in d.sub.6-DMSO. FIG. 4D shows Nuclear magnetic resonance
(.sup.13C-NMR) spectra of 6-nitrobenzothiazole-2-carboxamide 3
obtained on a Bruker 500 MHz machine in d.sub.6-DMSO. FIG. 4E shows
a nuclear magnetic resonance (.sup.1H-NMR) spectra of
2-cyano-6-nitrobenzothiazole 4 obtained on a Bruker 500 MHz machine
in d.sub.6-DMSO. FIG. 4F shows a nuclear magnetic resonance
(.sup.13C-NMR) spectra of 2-cyano-6-nitrobenzothiazole 4 obtained
on a Bruker 500 MHz machine in d.sub.6-DMSO. FIG. 4G shows a
nuclear magnetic resonance (.sup.1H-NMR) spectra of
2-cyano-6-aminobenzothiazole 5 obtained on a Bruker 500 MHz machine
in d.sub.6-DMSO. FIG. 4H shows a nuclear magnetic resonance
(.sup.13C-NMR) spectra of 2-cyano-6-aminobenzothiazole 5 obtained
on a Bruker 500 MHz machine in d.sub.6-DMSO. FIG. 4I shows Mass
spectra were acquired on a Micromass Quattro Micro API mass
spectrometer operating in positive ion mode. The samples were
dissolved in MeCN/H.sub.2O (1:1), 0.1% formic acid for mass
spectrometry analysis.
[0026] FIG. 5 shows schematic representation of a method to provide
2-cyano-6-aminobenzothiazole from a monofunctional benzothiazole
according to an embodiment herein described. In particular, the
illustration of FIG. 5 shows a general scheme for syntheses of
2-cyano-6-aminobenzothizaole, starting with a ethyl
benzothiazole-2-carboxylate according to an embodiment herein
described.
[0027] FIG. 6 shows a schematic representation of the four steps
depicted in FIG. 5 according to an embodiment herein described. In
particular, in the illustration of FIG. 6A, the ethyl
benzothiazole-2-carboxylate is first nitrated to provide a ethyl
6-nitrobenzothiazole-2-carboxylate; the ester group in position C2
of the resulting ethyl 6-nitrobenzothiazole-2-carboxylate is then
expected to be reduced to a carboxamide group and then to a cyano
group. In the illustration of FIG. 6B nitration of C6 of the
benzothiazole is performed after the reducing the carboxylate group
in C2 to a carboxamide and before the expected conversion of the
carboxamide group to a cyano group. In the illustration of FIG. 6C
nitration of C6 of the benzothiazole is performed after reducing
the carboxylate group in C2 to a carboxamide and before the
expected conversion of the carboxamide to a cyano group, followed
by a reduction of the C6 position to an amino group.
[0028] FIG. 7 shows schematic representation of a method to provide
2-cyano-6-aminobenzothiazole from a monofunctional benzothiazole
according to an embodiment herein described. In particular, the
illustration of FIG. 7 shows synthetic routes consisting of a
series of nitration reactions expected to generate the intermediate
derivatives to the D-aminoluciferin precursor,
2-cyano-6-aminobenzothiazole according to embodiments herein
described.
[0029] FIG. 8 shows a nuclear magnetic resonance spectra for a
compound herein described. In particular FIG. 8 shows a nuclear
magnetic resonance (.sup.13C-NMR) spectra of ethyl
6-nitrobenzothiazole-2-carboxylate 7 obtained on a Bruker 500 MHz
machine in d.sub.6-DMSO.
[0030] FIG. 9 shows a schematic representation of a method to
provide an amino acid or a peptide labeled with
6-amino-6-deoxy-D-luciferin according to an embodiment herein
described. In particular, the illustration of FIG. 9, schematically
shows a 3-step process of the synthesis of
2-cyano-6-aminobenzothiazole, the precursor to
6-amino-6-deoxy-D-luciferin, conjugated to single amino acids
(R1=one of the 20 amino acids) followed by subsequent conjugation
to R.sub.2 (R.sub.2=either another amino acid or a peptide
sequence) and then cyclization of the ring structure to generate
bioluminescent probes according to embodiments herein
described.
[0031] FIG. 10 shows a schematic representation of the two steps
depicted in FIG. 9 according to an embodiment herein described. In
particular, FIG. 10A shows a schematic representation of a
conjugation of single amino acids to 2-cyano-6-aminobenzothiazole,
the precursor to 6-amino-6-deoxy-D-luciferin (D-aminoluciferin) and
subsequent schematic representation of a cyclization of ring
structure to form amino acid conjugated 6-amino-D-luciferin. FIG.
10B shows a schematic representation of a method to provide an
amino acid or a peptide labeled with 6-amino-6-deoxy-D-luciferin
from a 2-cyano-6-aminobenzothiazole according to an embodiment
herein described. In particular, the schematics of FIG. 10B show a
generalization of a step-wise process for preparation of the
precursor to D-aminoluciferin comprising first conjugating a single
amino acid (R1), followed by subsequent conjugation with a longer
peptide sequence (R2), and then formation of the peptide-conjugated
D-aminoluciferin substrate via cyclization.
[0032] FIG. 11 shows a schematic representation of a method to
provide an amino acid or a peptide labeled with
6-amino-6-deoxy-D-luciferin from a monofunctionalized benzothiazole
according to an embodiment herein described
[0033] FIG. 12 shows a schematic representation of a method to
provide an amino acid or a peptide labeled with
6-amino-6-deoxy-D-luciferin from a 2-cyano-6-aminobenzothiazole
according to an embodiment herein described. In particular, the
schematics of FIG. 13 show a specific example of the method
schematically illustrated in FIG. 11 where R.sub.1 is a tyrosine
amino acid and R.sub.2 is a specifically designed biotin-labeled
peptide sequence that can be recognized by the serine protease,
prostate specific antigen. The biotin functional group on the end
of the peptide sequence facilitates coupling to a streptavidin
compound wherein this final compound could be utilized in an assay
system.
[0034] FIG. 13 shows a schematic representation of a method to
provide an amino acid or a peptide labeled with
6-amino-6-deoxy-D-luciferin from a monofunctionalized benzothiazole
according to an embodiment herein described.
[0035] FIG. 14 shows a schematic representation of a method to
provide a carboxylate modified luciferin amino acid/peptide probes
according to an embodiment herein described.
[0036] FIG. 15 shows a schematic representation of a method using a
carboxylate modified luciferin amino acid/peptide probes according
to an embodiment herein described.
[0037] FIG. 16 shows liquid chromatography/mass spectrometry
spectra for compounds of methods and systems of the disclosure,
according to an embodiment herein described.
[0038] FIG. 17 shows liquid chromatography/mass spectrometry (FIG.
17A) and nuclear magnetic resonance spectra (FIG. 17B) for a
compound according to an embodiment herein described.
[0039] FIG. 18 shows liquid chromatography/mass spectrometry
spectra for a compound according to an embodiment herein
described.
[0040] FIG. 19 shows liquid chromatography/mass spectrometry (FIG.
19A) and nuclear magnetic resonance spectra (FIG. 19B) for a
compound according to an embodiment herein described.
[0041] FIG. 20 shows liquid chromatography/mass spectrometry
spectra for a compound according to an embodiment herein
described.
[0042] FIG. 21 shows liquid chromatography/mass spectrometry
spectra for a compound according to an embodiment herein
described.
DETAILED DESCRIPTION
[0043] Provided herein are methods and systems to synthesize a
precursor of 6-amino-6-deoxy-D-luciferin, (herein also
D-aminoluciferin), and derivatives thereof, which can be used in
connection with various applications that are based on the reaction
between luciferase enzyme of the North American firefly (Photinus
pyralis) and substrate D-luciferin.
[0044] The reaction between the luciferase enzyme and substrate
D-luciferin is widely used as an optical reporter for in vitro and
in vivo assays. Broad ranging applications of the luciferase enzyme
as optical reporter comprise reporting gene expression.sup.1,
proliferation of cancerous cells,.sup.2 efficacy of cancer
therapies.sup.3, bacterial infection.sup.4 and enzymatic
action..sup.5, 6 Under various reaction conditions identifiable by
a skilled person, photons are generated during the enzymatic
conversion of D-luciferin to oxyluciferin in the presence of
compounds such as ATP, O.sub.2 and Mg.sup.2+..sup.7-9 In several
cases bioluminescence can be detected with a low intrinsic
background in cells and tissues, sometimes in contrast to
fluorescent-based detection, where autofluorescence emits
significant background noise. Studies to modify the natural
substrate D-luciferin in numerous positions have generated
substrates with novel properties. For example, masking the carboxyl
group and/or methylating the 6-position phenolic group of
D-luciferin inhibits reaction with the luciferase enzyme. Enzymatic
or chemical hydrolysis of the carboxylic acid mask and
demethylation of the methoxy group releases the free substrate for
subsequent reaction with the luciferase enzyme..sup.6 In various
cases 6-amino-6-deoxy-D-luciferin, D-aminoluciferin, with an amino
group in the 6-position on the benzothiazole ring,.sup.10 allows
conjugation of the optical reporter substrate to peptide sequences
to generate protease assay probes..sup.5,10,11-13
[0045] FIG. 1 shows a cartoon scheme depicting an exemplary
application of bioluminescent assay performed with labeled probes
obtainable with the methods and systems herein described. In
particular, in the illustration of FIG. 1, a complex biological
sample (ex. blood sample) when interacting with a specifically
designed protease probe has the capability to release the
D-aminoluciferin, which ultimately provides light output in the
presence of Mg.sup.2+ and ATP when proteases (e.g. indicative of
disease) are present in the biological sample. In some embodiments,
this assay can serve as a marker for disease.
[0046] Methods and systems are herein described to provide a
D-aminoluciferin precursor formed by 2-cyano-6-aminobenzothiazole
(herein also D-aminoluciferin precursor or simply precursor) and/or
probes labeled with D-aminoluciferin provided starting from the
precursor.
[0047] In some embodiments, methods and systems are described to
provide the precursor with method starting from a monofunctional
benzothiazole. The term "benzothiazole" as used herein indicates an
organosulfur compound of formula (V)
##STR00003##
which can be substituted or unsubstituted in the various positions.
Benzothiazole is typically colorless, and takes the form of a
slightly viscous liquid while most of its derivatives are solid.
Benzothiazoles are commercially available and can be prepared using
methods such as treatment of 2-aminobenzenethiol with acid
chlorides according to the reaction
C.sub.6H.sub.4(NH.sub.2)SH+RC(O)Cl.fwdarw.C.sub.6H.sub.4(NH)SCR+-
HCl+H.sub.2O and to additional procedures identifiable by a skilled
person.
[0048] The term "monofunctional benzothiazole" indicates a
benzothiazole including one functional group in the C2 position
wherein the term "functional group" as used herein indicates a
specific group of atoms within a molecular structure that are
responsible for the characteristic chemical interactions or
reactions of that structure. In general, exemplary functional
groups include hydrocarbons, groups containing halogen, groups
containing oxygen, groups containing nitrogen and groups containing
sulfur all identifiable by a skilled person. In particular,
exemplary functional groups in the sense of the present disclosure
comprise alkoxy or alkyl groups, secondary amines, amides and
additional functional groups can be identified by a skilled person
upon reading of the present disclosure. In some embodiments, the
monofunctional benzothiazole can be substituted in any positions,
wherein the terms "substituted" or "substitution" herein indicates
replacement of one or more hydrogens with chemical groups that do
not substantially interfere with the characteristic chemical
interactions or reactions of the functional group as described
herein.
[0049] In some embodiments, methods and systems are described to
provide the precursor with methods starting from a monofunctional
benzothiazole attaching in position C2 a functional group of
formula (I) (C(.dbd.X.sub.1)NH.sub.2) wherein X.sub.1 is O or S,
wherein the amide in the functional group of formula (I) is
converted to a cyanide group by elimination of a H.sub.2X.sub.1
compound.
[0050] The term "amide" as used herein indicates an organic
compound that contains the carbonyl group (R.sub.4--C.dbd.O )
linked to a nitrogen atom (N) (herein also carboxamide) or a
thiamide group R.sub.4--CS--NR'R.sub.5, where R.sub.4, R', and
R.sub.5 are same or different and are organic groups with R' and
R.sub.5 possibly formed by H. The term refers both to a class of
compounds and a functional group within those compounds.
[0051] In particular, the term "carboxamide" as used herein
indicates an organic compound that contains a carbonyl group
(R4-C.dbd.O) linked to a nitrogen atom (N). Typically, carboxamides
of the general structure R6-CO--NR7R2R8 can be synthesized by
replacement of the hydroxyl group (OH) of a carboxylic acid by an
amino group (NR1R2), where R6, R7, and R8 are same or different and
are organic substituents with R7 and R8 possibly formed by H.
Exemplary compounds containing carboxamide groups comprise
asparagine and glutamine.
[0052] The term thioamides indicates sulfur analogues to amides,
where the oxygen atom (O) in amide is replaced by a sulfur atom
(S). The general structure of thioamide is R.sub.4--CS--NR'R.sub.5,
where R.sub.4, R', and R.sub.5 are same or different and are
organic groups with R' and R.sub.5 possibly formed by H. Typically
thioamides are analogous to carboxamides but they exhibit greater
multiple bond character along the C--N bond, resulting in a larger
rotational barrier. Thioamides are typically prepared by treating
amides with phosphorus sulfides such as phosphorus pentasulfide
and, in more specialized applications, Lawesson's reagent. An
alternative route entails the reaction of nitriles with hydrogen
sulfide. The Willgerodt-Kindler reaction also affords
benzylthioamides.
[0053] The terms "eliminate" and "elimination reaction" as used
herein with reference to a reaction indicate an elimination
reaction. An elimination reaction is a type of organic reaction in
which two substituents are removed from a molecule in either a one
or two-step mechanism (E2 and E1 mechanisms, respectively). Either
the unsaturation of the molecule increases or the valence of an
atom in the molecule decreases by two. E1 mechanism: generally
follows the following form: the first step requires the loss of the
leaving group, forming a carbocation intermediate. A nucleophilic
species, usually a base, then attacks a neighboring hydrogen
forming the double bond. E2 mechanism generally follows the
following form: a nucleophilic species or base attacks a hydrogen
neighboring the leaving group, pushing the electrons into the
double bond as the leaving group leaves. In elimination reactions
herein described does not necessarily result in an actual
production of an H.sub.2X.sub.1 product, as long as the
H.sub.2X.sub.1 compound is removed from the starting compound. In
this connection for example, in embodiments where X.sub.1 is oxygen
and a POCl.sub.3 reagent is used the oxygen atom is eventually
attached to phosphorus. Exemplary elimination reactions comprise
dehydration and dehydrosulfurization. A dehydration reaction is
usually defined as a chemical reaction that involves the loss of
water from the reacting molecule. Dehydration reactions are a
subset of elimination reactions where the leaving group is water
(H.sub.2O). Dehydrosulfurization reactions or H.sub.2S elimination
reactions are another subset of elimination reaction where the
leaving group is (H.sub.2S).
[0054] In some embodiments, in the functional group of formula (I)
X.sub.1 is S, the conversion from thioamide to CN group can be
performed by dehydrosulfurization directed to elimination the
H.sub.2S compound. The dehydrosulfurization can be performed with
suitable reagents (e.g. tellurium chloride) and under suitable
conditions, such as the ones described for example in ref. 17. A
skilled person will be able to identify additional reactions and
related procedures that are functional to the dehydrosulfurization
of the monofunctional benzothiazole herein described.
[0055] In some embodiments, in the functional group of formula (I)
X.sub.1 is O the reduction can be performed by dehydration of the
corresponding ester group with production of H.sub.2O according to
procedures such as the ones exemplified in the Examples section and
additional procedures identifiable by a skilled person.
[0056] In some embodiments, the benzothiazole presenting the
functional group of formula (I) in position C2 and/or the
benzothiazole presenting the functional group CN comprises an amino
group in position C6. In some embodiments, the benzothiazole
presenting the functional group of formula (I) in position C2 is
modified to comprise an amino group in position C6. In particular
in some of those embodiments, a nitrile group is first introduced
in position C6 before or after converting the functional group of
formula (I) to a CN group. The nitrile group in C6 of the
benzothiazole is then converted to an amino group. A skilled person
will be able to identify suitable methods to introduce an NO2 group
in monofunctional benzothiazoles herein described at any stage of
the procedures. For example, typical nitrations use nitric acid and
sulfuric acid to produces the nitronium ion (NO.sub.2), which is
the active species of nitration reactions and attacks the
electron-rich reactant, such as a benzene ring, to initiate
electrophilic substitution reaction.
[0057] In some embodiments, an NH2 is introduced directly into the
C6 position of the benzothiazole ring. A skilled reader will be
able to identify suitable reaction for introducing an NH2 group
directly in monofunctional benzothiazoles herein described at any
stage of the procedures herein described.
[0058] In some embodiments, the functional group of formula (I) can
be provided in the monofunctional benzothiazole herein described
starting from a monofunctional benzothiazole attaching in position
C2 a functional group of formula (II) (C(.dbd.X.sub.1)X.sub.2R)
wherein R is an alkyl group, or a halogen atom and X.sub.1 and
X.sub.2 are independently O or S.
[0059] The term "alkyl" as used herein refers to a linear,
branched, or cyclic saturated hydrocarbon group typically although
not necessarily containing 1 to about 10 carbon atoms, preferably 1
to about 6 carbon atoms, such as methyl, ethyl, n-propyl,
isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl, and the like,
as well as cycloalkyl groups such as cyclopentyl, cyclohexyl and
the like. Generally, although again not necessarily, alkyl groups
herein contain 1 to about 6 carbon atoms. The term "cycloalkyl"
intends a cyclic alkyl group, typically having 4 to 8, preferably 5
to 7, carbon atoms. The term "substituted alkyl" refers to alkyl
substituted with one or more substituent groups, and the terms
"heteroatom-containing alkyl" and "heteroalkyl" refer to alkyl in
which at least one carbon atom is replaced with a heteroatom. If
not otherwise indicated, the terms "alkyl" and "lower alkyl"
include linear, branched, cyclic, unsubstituted, and/or
substituted. In some embodiments, in the functional group of
formula (I), R can be a substituted alkyl which can comprise
heteroatoms such as N, O, S, P, Si, F, Cl, Br, and I, as well as
additional groups and heteroatoms identifiable by a skilled
person.
[0060] In embodiments where X.sub.2 is O, the resulting X.sub.2--R
group can be an alkoxy group or an acid halide. The term "alkoxy"
as used herein intends an alkyl group bound through a single,
terminal ether linkage; that is, an "alkoxy" group may be
represented as --O-alkyl where alkyl is as defined above. A "lower
alkoxy" group intends an alkoxy group containing 1 to 6 carbon
atoms. Exemplary alkoxy group comprised in the functional group of
formula (I) comprise methoxy, ethoxy, propoxy and additional alkoxy
group comprising a lower alkyl. The term "acid halide" indicates a
chemical compound that can be typically derived from an oxoacid by
replacing a hydroxyl group with a halide group (i.e. a binary
compound, of which one part is a halogen atom and the other part is
an element or radical that is less electronegative (or more
electropositive) than the halogen). Exemplary acid halides comprise
(C(.dbd.O)Cl) and additional halides identifiable by a skilled
person. In some embodiments, the acyl halide can be treated with
ammonia, and the reaction is expected to proceed faster than the
reaction performed with the ester group.
[0061] In embodiments where X.sub.2 is S the resulting X.sub.2--R
group is an alkyl group and in particular a lower alkyl group, or a
halogen such as Cl. Exemplary functional groups of formula (II)
comprise (C(.dbd.S)OR), which can be made for example by treating
the ester with P.sub.2S.sub.5 as described in ref. 19;
(C(.dbd.O)SR) which can be made for example by reacting the ester
with a mercaptan under basic conditions as identifiable by a
skilled person; and (C(.dbd.S)SR), which can be made for example by
treating the chloromethylbenzothiazole with sulfur and methyl
iodide, according to the procedure described in ref. 20. A skilled
reader will be able to identify additional functional groups of
formula (II) and suitable procedure to provide monofunctional
benzothiazole comprising those functional groups.
[0062] In some embodiments, wherein the functional group of formula
(I) and (II) X.sub.1 is S, the conversion from ester to thioamide
can be performed using reagents and procedures identifiable by a
skilled person. For the conversion from ester to thioamide can be
performed by using NH.sub.3, then P.sub.2S.sub.5 as described in
ref 18. Additional reactions will be identifiable by a skilled
person upon reading of the present disclosure.
[0063] In some embodiments, wherein the functional group of formula
(I) and (II) X.sub.1 is O, the conversion from ester to carboxamide
can be performed by using amines alone or in presence of reagents
(such as dicyclohexylcarbodiimide (DCC)) which are suitable to
convert the hydroxyl oxygen in a more efficient leaving group.
Examples of those conversions are reported in the Example section
of the present disclosure. A skilled reader will be able to
identify additional reactions using amines or other reagents that
are suitable to convert an ester according to formula (I) wherein
X.sub.1 is oxygen into a carboxamide of formula (II) wherein
X.sub.1 is oxygen.
[0064] In some embodiments of methods and systems wherein the
D-aminoluciferin precursor is provided from a monofunctional
benzothiazole, the monofunctional benzothiazole is
6-nitrobenzothiazole and the precursor can be synthesized according
to a method schematically illustrated in FIG. 2. In particular, in
the illustration of FIG. 2 production of the precursor can be
performed in four steps. In particular a thio or carboxy ester
functional group of formula (II) can be introduced in C2 with
suitable reactions. The thio or carboxy ester can then be converted
into a corresponding amide which can then be converted into a
nitrile. The NO2 group in C6 can then be reduced to NH2 using
procedure identifiable by a skilled person. Possible variations of
this reaction scheme comprise a three steps procedure performed
starting from a monofunctional benzothiazole attaching an ester of
formula (II), three steps procedure performed starting from a
monofunctional benzothiazole attaching an amide of formula (I) and
other variations wherein the reduction of the NO2 group in C6 is
performed before the final reduction of the amide of formula (I) to
a CN group.
[0065] In an embodiment, wherein X.sub.1 is O the method can be
performed as schematically illustrated in FIG. 3. In particular a
free radical method can be used to generate
2-carboxyethylester-6-nitrobenzothiazole from commercially
available 6-nitrobenzothiazole according to procedures exemplified
in Example 1.
[0066] In the exemplary illustration of FIG. 3, the process further
comprises converting the 2-carboxyethylester-6-nitrobenzothiazole
into a carboxamide which in some embodiments can be carried out
using suitable reagents to introduce nitrogen in the functional
group of formula (II) which are identifiable by a skilled person
upon reading of the present disclosure. Exemplary procedures using
reagents such as potassium amide or tritylamine and use a diverse
range of catalysts such as various forms of palladium. In an
exemplary procedure, nitrogen is introduced by adding ammonia gas
in methanol with 100% conversion (TLC and analytical HPLC) (see
Example 2.
[0067] In the exemplary illustration of FIG. 3, the
6-nitrobenzothiazole-2-carboxamide is then converted into a
2-cyano-6-nitrobenzothiazole. In some embodiments, this conversion
can be performed by dehydration of the carboxamide to a nitrile
which can be carried out for example using POCl.sub.3 in pyridine,
again in quantitative yield (TLC and analytical HPLC). The
corresponding nitrile can then be purified using gravity column
silica chromatography before the final step and starting material
6-nitrobenzothiazole can be recovered (see Example 3). Additional
procedures to perform dehydration comprise procedures which use
silanes, zirconium catalysts, palladium catalysts, phosphines
according to techniques identifiable by a skilled person.
Additional procedures and techniques which are also suitable for
the conversion of carboxamide to a nitrile are also identifiable by
a skilled person.
[0068] In the exemplary illustration of FIG. 3, the reduction of
the NO2 group in C6 to a NH2 is performed following conversion of
the C2 into a nitrile group as a final reaction to generate the
6-amino-6-deoxy-D-luciferin, D-aminoluciferin, precursor. The
reduction of NO2 in C6 can be performed using methods identifiable
by a skilled person, such as, procedures performed using stannous
chloride in ethanol exemplified in the Example section. In those
reactions, a sufficient number of equivalents of the reductant
(e.g. about 2.5 to about 5 eq) can be used to reduce the nitro
group in a predetermined amount of time (e.g. about 2 h). Silica
chromatography purification can also be used to yield
D-aminoluciferin precursor with high purity for subsequent coupling
reactions (e.g. to amino acids and peptides) (see Example 4).
[0069] In some embodiments according to the illustration of FIG. 3,
reactions are selected so that subsequent conversions of ethyl
6-nitrobenzothiazole-2-carboxylate are not impeded by a mixture of
ethyl 6-nitrobenzothiazole-2-carboxylate and 6-nitrobenzothiazole,
and therefore, the mixture can be carried forward without
purification. In some of those embodiments, the synthesis is
particularly suitable for actual production of material for use in
assays, kits, and additional applications identifiable by a skilled
person since purification is not necessary.
[0070] A skilled person will be able to identify variants of the
procedure schematically illustrated in FIG. 3 with other functional
groups of formula (I) and (II) and with reduction of the NO.sub.2
in C6 position at different stages before the conversion of the
functional group in C2 upon reading of the present disclosure as
also exemplified by the Examples section and figures.
[0071] For example additional exemplary reaction schemes starting
from a different monofunctional benzothiazole herein described, are
schematically illustrated in FIG. 5 which shows a specific
functional group of formula (II) wherein X.sub.1 and X.sub.2 are
oxygen and R is an ethyl group. In the exemplary illustration of
FIG. 5, the starting monofunctional benzothiazole is ethyl
benzothiazole-2-carboxylate and the nitration of C6, and conversion
of the ethyl ester C2 group into a carboxamide and then to a
nitrile group can be performed according to any of the procedure
herein described.
[0072] Some specific routes are further illustrated in FIGS. 6 and
7. In particular, FIGS. 6 and 7, show specific schemes for
alternative synthesis of 2-cyano-6-aminobenzothiazole starting with
a commercially available 2-carboxyethyl ester. As shown in the
exemplary illustration of FIG. 6, it is expected that nitration can
be performed at any point and the possibility to nitrate either the
ester (FIG. 6A) or the carboxamide (FIG. 6B) has been
demonstrated.
[0073] In the exemplary illustration of FIG. 6A, the monofunctional
benzothiazole is ethyl benzothiazole-2-carboxylate and the
2-cyano-6-aminobenzothiazole can be provided following nitration
and subsequent reduction of the benzothiazole. Nitration of the
commercially available, ethyl benzothiazole-2-carboxylate was also
carried out using concentrated H.sub.2SO.sub.4 and KNO.sub.3
resulting in a mixture of 2-carboxyethylester-6-nitrobenzothiazole
with starting material (TLC). Gravity silica column chromatography
of 2-carboxyethylester-6-nitrobenzothiazole proved challenging to
separate from starting material 2-carboxytheylester-benzothiazole
(10% yield). (see Example 5).
[0074] In the exemplary illustration of FIGS. 6B and 6C, converting
the ethyl benzothiazole-2-carboxylate into a
2-carboxamide-benzothiazole is carried out with ammonia (e.g. using
ammonia gas in methanol with 100% conversion (TLC, analytical HPLC)
see Example 6). Without purification 2-carboxamide-benzothiazole
can be reacted (e.g. with conc. H.sub.2SO.sub.4 and HNO.sub.3) to
yield a mixture of 6-nitrobenzothiazole-2-carboxamide and
2-carboxamide-nitrobenzothiazole (.about.50% by TLC) (see Example
7). Suitable techniques can be used to separate the mixture (for
example gravity silica column chromatography of
6-nitrobenzothiazole-2-carboxamide (.about.38% yield) see Example
7).
[0075] In some embodiments, converting the
2-carboxamide-benzothiazole to a corresponding nitrile in C2
position can be performed elimination reactions and additional
reactions identifiable by a skilled person. In the methods herein
described for example conversion of the C2 carboxamide can be
performed by dehydration to the nitrile using suitable reagents
such as phosphoryl chloride in pyridine, proceeding with
quantitative conversion. In some of those embodiments,
benzothiazole-2-carboxamide can be converted to the nitrile to
yield 2-cyano-benzothiazole with 100% conversion (analytical HPLC,
mass spect) (see Example 8). Without purification, the nitrile was
reacted with H.sub.2SO.sub.4/HNO.sub.3 and the reaction was
monitored over time (over a period of .about.2 h) via analytical
HPLC. (see Example 11).
[0076] A skilled person will be able to identify alternative
reactions schemes according to the illustration of FIGS. 5 to 7,
wherein in the functional group (II) of the monofunctional
benzothiazoles, X.sub.1 and/or X.sub.2 are S and/or R is provided
by a different alkyl group (in particular a lower alkyl group) or a
halogen (e.g. Cl), upon reading of the present disclosure. In any
of those embodiments wherein the monofunctional benzothiazole used
in methods and systems herein described does not initially comprise
a nitro or amino group in position C6, the amino group is
introduced at various stages as will be understood by a skilled
person. For example in some embodiment, it is expected that the
NO.sub.2 group can be introduced using concentrated nitric and
sulfuric acids, giving regio selectivity for the C6 position over
the C4,C5, or C7 positions of the benzothiazole ring presenting a
carboxamide, before converting the carboxamide to a cyanide group.
In some embodiments, the NH2 group can be introduced at any stage
with other reactions which do not require nitration of the C6
position that will be identifiable by a skilled person upon reading
of the present disclosure.
[0077] In an embodiment of methods and systems provide the
D-aminoluciferin precursor the monofunctional benzothiazole is
6-nitrobenzothiazole. In some of those embodiments cyanation of the
2-position of 6-nitrobenzothiazole can be performed by forming a
lithium salt of benzothiazole with butyllithium then reacting this
with a soluble cyanide followed by quenching of excess butyl
lithium with water.sup.15. Starting material and only a small
amount of 6-nitrobenzothiazole-2-carbonitrile resulted. Direct
cyanation of the C2 position of other monofunctional benzothiazoles
herein described can be performed according to techniques and
procedures identifiable by a skilled person upon reading of the
present disclosure.
[0078] In some embodiments, the 6-amino-6-deoxy-D-luciferin,
D-aminoluciferin, precursor obtained with any of the methods and/or
systems herein described or provided using other methods can be
coupled to an amino acid or peptide labeled to provide an amino
acid-conjugated or a peptide
conjugated-2-cyano-6-aminobenzothiazole.
[0079] As used herein the term "amino acid", "amino acid monomer",
or "amino acid residue" refers to any of the twenty naturally
occurring amino acids including synthetic amino acids with
unnatural side chains and including both D and L optical isomers.
The term "polypeptide" or "peptide" as used herein indicates an
organic polymer composed of two or more amino acid monomers and/or
analogs thereof. The term "polypeptide" includes amino acid
polymers of any length including full length proteins and peptides,
as well as analogs and fragments thereof. A polypeptide of three or
more amino acids is also called a protein oligomer or oligopeptide.
The term "amino acid analog" refers to an amino acid in which one
or more individual atoms have been replaced, either with a
different atom, isotope, or with a different functional group but
is otherwise identical to its natural amino acid analog.
[0080] The term "couple" or "conjugate" as used herein indicates
formation of a covalent bond between two compounds. Typically, in
some embodiments, coupling or conjugation of the amino acid or to
the amino group in 2-cyano-6-aminobenzothiazole can be performed
with an efficient peptide coupling method such as DCC, or N-methyl
morpholine and isobutylchloroformate, since the aniline amino group
is relatively unreactive. In other embodiments, coupling or
conjugation can be performed reactions suitable to form a
urea/thiourea (NH(C.dbd.X)NH) or a carbamate/thiocarbamate
(X(C.dbd.X)NH) linkage, where X.dbd.O or S.
[0081] The term "carbamate" as used herein indicates organic
compounds derived from carbamic acid (NH2COOH). Specifically, the
term refers to any salt or ester of carbamic acid with the general
structure R8O--CO--NR9R10, where R8, R9, and R10 are same or
different and are organic substituents with one or two possibly
formed by H. The term "thiocarbamate" as used herein indicates
sulfur analogues of carbamates, where one of the oxygen atoms (O)
in a carbamate is replaced by a sulfur atom (S). Specifically,
there are two structurally isomeric types of thiocarbamates:
O-thiocarbamates, ROC(.dbd.S)NR2, where the carbonyl group
(C.dbd.O) is replaced with a thiocarbonyl group (C.dbd.S) and
S-thiocarbamates, RSC(.dbd.O)NR2, where the R--O--group is replaced
with an R--S-- group. Further, O- and S-thiocarbamates can
interconvert, for example in the Newman-Kwart rearrangement.
[0082] In particular, in some embodiments coupling of the peptide
with the precursor can be performed according to a two step process
schematically illustrated in FIGS. 9 and 10 wherein a process is
illustrated that comprises conjugating an amino acid and/or a
peptide to the precursor. In particular, in some embodiments the
conjugation between amino acid and/or peptide can be performed in a
two step reaction wherein the amino acid or peptide is first
conjugated with the peptide and then the resulting amino
acid-conjugated or peptide-conjugated precursor is reacted with
cysteine to provide an amino acid-conjugated D-aminoluciferin or a
peptide-conjugated D-aminoluciferin (FIG. 10A). In other
embodiments, a three step reaction is performed wherein the an
amino acid-conjugation with the precursor is performed, followed by
conjugation with the specific peptide sequence, and then reacting
with D-cysteine to provide the peptide-conjugated D-aminoluciferin
(FIG. 10B)
[0083] In some embodiments, conjugation of the amino group of the
2-cyano-6-aminobenzothiazole to single amino acids or peptides can
be achieved using isobutylchloroformate activation of the free acid
of the amino acid in the presence of N-methyl morphiline in THF as
previously reported..sup.11, 13 (see Example 10). In some
embodiments, conjugation of the amino group of the
2-cyano-6-aminobenzothiazole to single amino acids can be achieved
using carbodiimide activation. In particular carbodiimide
activation involves attack of the carboxylate oxygen on the central
carbodiimide carbon, creating a leaving group with that oxygen atom
according to reactions identifiable by a skilled person. The amine
then attacks the carboxyl carbonyl, displacing the now urea leaving
group. The insolubility of some ureas can in some cases help drive
the reaction.
[0084] In some embodiments, suitable amino acids to be conjugated
with the D-aminoluciferin precursor comprise the 20 naturally
occurring amino acid amino acids, including Isoleucine, Alanine,
Leucine, Asparagine, Lysine, Aspartic Acid, Methionine, Cysteine,
Phenylalanine, Glutamic Acid , Threonine, Glutamine, Tryptophan
Glycine, Valine, Proline, Serine, Tyrosine, Arginine, Histidine,
and additional amino acids such as Selenocysteine Ornithine, and
Taurine. In particular, in some embodiments, the amino acids
comprise in particular Alanine, Valine, Isoleucine, Leucine,
Tyrosine, Methionine, Threonine. In some embodiments, suitable
amino acids to be conjugated with the D-aminoluciferin precursor
comprise in particular Phenylalanine, Histidine and Glutamic Acid.
In some embodiments, amino acids comprising a side chain
(hydrophobic, hydrophilic, acid or basic in nature) can be coupled
with the precursor and then cyclized with D-cysteine to yield the
amino acid-conjugated 6-amino-6-deoxy-D-luciferin, which it is
expected to contribute to varying pharmacokinetics/pharmacodynamics
behaviors as measured by bioluminescence.
[0085] In some embodiments, the amino acids comprise a side chain,
which have been surprisingly found to be labelable with the
precursor and then converted in to functional amino acid labeled
with D-luciferin despite the expected modifications in
biodistribution (pharmacokinetics/pharmacodynamics) as measured by
bioluminescence In particular in some embodiments, the amino acid
is an amino acid having a hydrophobic side chain (including for
example Alanine, Isoleucine, Leucine, Methionine, Phenylalanine,
Tryptophan, Tyrosine and Valine). In some embodiments, the amino
acid is an amino acid having a non-polar uncharged side chain
(including for example Serine, Threonine, Asparagine and
Glutamine). In some embodiments, the amino acid is an amino acid
having an electrically charged side chain (including for example
Arginine, histidine and Lysine, Aspartic acid and Glutamic
acid).
[0086] In some embodiments, an intermediate in the synthesis of
peptide labeled at the carboxy terminus with
6-amino-6-deoxy-D-luciferin formed by a substituted
2-cyano-6-aminobenzothiazole can be provided.
[0087] In some embodiments, methods and systems to provide a
peptide labeled at the carboxy terminus with
6-amino-6-deoxy-D-luciferin from a monofunctional benzothiazole. In
some embodiments, the peptide can be of at least 6 or 7 amino acid
resides in length and in some of those embodiments the system can
comprise a further label such a biotin label or a linker possibly
attached and in particular conjugated to the peptide at the
terminus opposite to the terminus where the D-amino luciferin is
conjugated directly or indirectly through conjugation to an amino
acid-conjugated D-aminoluciferin precursor. Without any intention
of being limiting and for guidance purpose only it is expected that
use of the three step process schematically illustrated in FIG. 10B
will be particularly useful for synthesis of a peptide with a
sequence of 5 amino acid residues or longer and/or to a peptide
further labeled with biotin or other labels. In particular, in some
of those embodiments, the D-aminoluciferin precursor is then first
reacted with an amino acid and then redirectly with a long peptide
chain thus allowing conjugation when the amino group on the
precursor is not reactive enough to directly conjugate the peptide
chain in an efficient manner according to an experimental
design.
[0088] In an embodiment, a method and system to provide a peptide
labeled with 6-amino-6-deoxy-D-luciferin is described. The method
comprises conjugating the peptide with an amino acid-conjugated
2-cyano-6-aminobenzothiazole to provide a peptide-conjugated
2-cyano-6-aminobenzothiazole, the conjugating performed to allow
formation of a peptide bond between the amino group of the amino
acid-conjugated 2-cyano-6-aminobenzothiazole and a carboxylic group
of the peptide. The method further comprises reacting the
peptide-conjugated 2-cyano-6-aminobenzothiazole with D-cysteine to
provide a peptide-conjugated 6-amino-6-deoxy-D-luciferin. The
system comprises one or more peptides, peptide-conjugated
6-amino-2-cyanobenzothiazole and/or amino acid-conjugated
2-cyano-6-aminobenzothiazole for simultaneous combined or
sequential use in the method to provide a peptide labeled with
6-amino-6-deoxy-D-luciferin herein described.
[0089] In some embodiments, modification of D-luciferin,
6-hydroxy-D-luciferin, to D-aminoluciferin,
6-amino-6-deoxy-D-luciferin, is expected to result in substrates
that generate novel bioluminescent properties and offers the
opportunity to expand the range of bioluminescence-based assays.
D-aminoluciferin, with an amino group in the C6-position of the
benzothiazole ring, allows conjugation of the optical reporter
substrate to single amino acids and/or peptide sequences to
generate novel assay probes with the peptide sequences targeting
proteases.
[0090] In some embodiments, methods and systems herein described
provide an alternate route for the synthesis of the precursor
2-cyano-6-aminobenzothiazole to D-aminoluciferin has been
identified and used to generate material for subsequent conjugation
to amino acids and peptide sequences for thorough investigation of
the utility of bioluminescent protease probes.
[0091] In some embodiments, methods and systems herein described
provide convenient synthetic route to D-aminoluciferin derivatives
of peptides. In particular in some embodiments aminoluciferin
derivatives herein described can be used in conjunction with
firefly luciferase to report on cellular events in vivo as well as
in bioluminescent assays. In particular, in some embodiments, the
synthetic route begins with a monofunctional benzothiazole (either
6-nitrobenzothiazole or ethyl benzothiazole-2-carboxylate, proceeds
through 2-cyano-6-aminobenzothiazole, and results in an amino acid
or peptide conjugated to 6-amino-6-deoxy-D-luciferin.
[0092] In some embodiments a carboxylate-modified Luciferin, amino
acid or peptide probe, is described, that comprises a D-luciferin
molecule conjugated with the amino-terminus of an amino acid or
peptide at the carboxyl group of said D-luciferin. In some of those
embodiments, the carboxyl-terminus of the amino acid residue or
peptide is blocked with a blocking group, such as an amide.
[0093] The carboxylate-modified luciferin, amino acid or peptide
probe can be provided using methods and systems based on reactions
performed on a resin base. In particular the amino acid or peptide
can be provided on a resin base (e.g. by synthesizing the peptide
on the resin base). A D-luciferin molecule can then be conjugated
at the amino terminus of the peptide or amino acid on the resin
bases via formation of a peptide bond. The resulting conjugate can
be cleaved from the resin to produce a produce free
carboxylate-modified luciferin, amino acid/peptide probe that can
then be optionally purified if desired. In some embodiments the
resin base is a chlorotrityl chloride (CTC) resin. In some
embodiments synthesizing the peptide can be performed using
standard Fmoc amino acid synthesis. Purification can be performed
with techniques identifiable by a skilled person, for examples
purification can be using reverse phase high performance liquid
chromatography. Following purification the carboxyl-terminus of the
said synthesized probe can be blocked using a blocking agent such
as an amide.
[0094] The carboxylate-modified Luciferin, amino acid or peptide
probe can be used to perform various assays and procedure aimed at
detection of targets and/or reactions. For example in some
embodiments the carboxylate modified luciferin amino acid or
peptide can be used in connection with luciferase for releasing the
D-luciferin molecule in the assay.
[0095] In particular, in some embodiments, carboxylate-modified
luciferin probes are provided, where D-luciferin is modified at the
carboxylic acid with amino acids/peptides to inhibit the reaction
with the luciferase enzyme. In some of those embodiments, upon
carboxypeptidase digestion of the amino acids conjugated to the
D-luciferin, the free bioluminescent substrate is released for
subsequent reaction with the luciferase enzyme. Further, in some
embodiment, if the carboxyl-terminus of a D-luciferin-peptide
sequence is blocked as a carboxamide or other suitable blocking
agent, only upon proteolytic action is a free carboxylic acid
generated for carboxypeptidase to begin digestion to release the
D-luciferin. Combined together, these concepts are used to generate
carboxylate-modified luciferin, amino acid/peptide probes and
assays using such probes or any derivative thereof to monitor the
action of proteases such as endoproteases or exopeptidase of
interest.
[0096] The term "derivative" as used herein with reference to a
first compound (e.g., D-aminoluciferin), indicates a second
compound that is structurally related to the first compound and is
derivable from the first compound by a modification that introduces
a feature that is not present in the first compound while retaining
functional properties of the first compound. Accordingly, a
derivative of D-aminoluciferin, usually differs from the original
D-aminoluciferin by modification of the substituents of the
D-aminoluciferin that might or might not be associated with an
additional function not present in the original D-aminoluciferin.
The additional function, and the properties associated with
D-aminoluciferin can be verified with methods and systems such as
the ones described in the Examples section in connection with
exemplary processes and derivatives herein described.
[0097] In some embodiments, derivatives of D-aminoluciferin herein
described can be used in connection with several assays directed to
in vitro or in vivo detection of targets and/or reactions.
[0098] The term "detect" or "detection" as used herein indicates
the determination of the existence, presence or fact of a target or
signal in a limited portion of space, including but not limited to
a sample, a reaction mixture, a molecular complex and a substrate
including a platform and an array. In particular, D-aminoluciferin
typically provides bioluminescence which is an optically detectable
signal.
[0099] In some embodiments, the D-aminoluciferin derivatives herein
described can be used in assays where bioluminescent detection has
extremely high sensitivity because the low background interference
is not affected by cellular auto-fluorescence and other fluorescent
contaminants and does not require extreme operation conditions. An
example is provided by protease assays based on bioluminescence in
connection with in vitro and in vivo applications which are
identifiable by a skilled person. Proteases can be specific or
non-specific in their action and therefore, a wide range of
protease substrates may be required to characterize protease
proteomics in different complex backgrounds (e.g. in vivo or
serum). A wide variety of proteases are present in all living
organisms and in protease activity levels are often a part of the
host response to infection or disease..sup.16 Therefore, monitoring
protease activity in both in vitro and in vivo experiments is
potentially an early warning system for a particular diseased state
before clinical symptoms occur.
[0100] Exemplary suitable assays are provided by in vitro assays
for certain protease developed with different reporting techniques.
The most common techniques are the proteolytic release of p-nitro
aniline for UV detection, the proteolytic release of fluorescence
dyes for fluorescence detection and mass spectrometry detection of
proteolytic cleavage products. However, there are various problems
associated with the detection techniques currently available when
developing an assay for detecting specific reactions in a complex
background (e.g.in vivo--the low sensitivity of UV absorption,
auto-fluorescence of cells or fluorescent interference from
chemical and natural products, and the vacuum and other expensive
and cumbersome parts required for mass detection).
[0101] In methods and systems herein described, any of the above
compounds can be synthesized or added according to techniques
identifiable by a skilled person.
[0102] As disclosed herein, the compounds herein described can be
provided as a part of systems to detect targets according to any of
the methods described herein. The systems can be provided in the
form of kits of parts.
[0103] In a kit of parts, the monofunctionalized benzothiazoles,
reducing agents, and other reagents to perform the methods can be
comprised in the kit independently. One or more compounds and
reagents can be included in one or more compositions alone or in
mixtures identifiable by a skilled person. Each of the one or more
of compounds and reagents can be in a composition together with a
suitable vehicle.
[0104] Additional reagents can include salts (such as Mg.sup.2+)
and reagents (e.g. ATP) and/or molecules suitable to enhance or
favor the reaction according to any embodiments herein described
and/or molecules, standards and/or equipment to allow detection of
pressure temperature and possibly other suitable conditions. For
example luciferin/luciferase as control reagents can be comprised
in a kit to serve as the "parent compounds" that will provide the
baseline for comparison to the modified bioluminescence
substrates.
[0105] In particular, the components of the kit can be provided,
with suitable instructions and other necessary reagents, in order
to perform the methods here described. The kit will normally
contain the compositions in separate containers. Instructions, for
example written or audio instructions, on paper or electronic
support such as tapes or CD-ROMs, for carrying out the assay, will
usually be included in the kit. The kit can also contain, depending
on the particular method used, other packaged reagents and
materials (i.e. wash buffers and the like).
[0106] Further advantages and characteristics of the present
disclosure will become more apparent hereinafter from the following
detailed disclosure by way of illustration only with reference to
an experimental section.
EXAMPLES
[0107] The methods system herein described and related
intermediates and derivatives are further illustrated in the
following examples, which are provided by way of illustration and
are not intended to be limiting.
[0108] In particular, the following examples illustrate exemplary
synthesis and uses of 2-cyano-6-aminobenzothiazole starting from
6-nitrobenzothiazole, ethyl 6-nitrobenzothiazole-2-carboxylate, and
other compounds performed according to the reaction schemes
summarized in FIGS. 1-13. A person skilled in the art will
appreciate the applicability of the features described in detail
for 2-cyano-6-aminobenzothiazole for additional compounds (e.g.
additional monofunctional benzothiazole) having same or different
chemical characteristics according to the present disclosure, and
to related derivatives. The following examples also illustrated
exemplary synthesis and uses of a carboxylate modified luciferin
amino acid probe for bioluminescent protease assay according to
reactions schemes summarized in FIGS. 14 and 15. A skilled person
will appreciate the applicability of the features described in
detail for the specific phenylalanine probe shown in the examples
to other probes according to the present disclosure.
[0109] The following experimental procedures and characterization
data were used for all compounds and their precursors exemplified
herein.
[0110] General. Commercially available reagents and solvents were
used as received without further purification. Anhydrous pyridine
and phosphoryl chloride were purchased from Aldrich.
6-nitrobenzothiazole was purchased from Alfa Aesar. Anhydrous
ammonia was purchased from Mattheson Tri-Gas, ethyl pyruvate from
Fluka, ferrous sulfate from Mallinckrodt Chemicals, sodium
bicarbonate from EMD Biosciences, and tin(II) chloride from Sigma.
The amino acid compounds were purchased from NovaBioChem and the
biotin-conjugated peptides were specifically designed and purchased
from Peptides International.
[0111] Characterization/Instrumentation. Analytical thin layer
chromatography (TLC) was carried out using aluminum sheets coated
with silica gel 60 F.sub.254. Reaction conversions were followed by
analytical HPLC at 1 mL/min on an Agilent 1100 machine (Waters
Symmetry C18, 5 .mu.m, 4.2.times.150 mm column, diode array
detector) with a linear gradient from 95% H.sub.2O (0.1% TFA) to
80% MeCN (0.1% TFA) over 15 mi. For analytical characterization,
small portions were purified by semi-preparative HPLC at 10 mL/min
on a Waters preparative machine (Waters Symmetry prep C18, 7 .mu.m,
19.times.300 mm column, photodiode array detection) with a linear
gradient from 90-95% H.sub.2O (0.1% TFA) to 50-90% MeCN (0.1% TFA)
for 30-35 min. Semi-preparative HPLC fractions were collected and
lyophilized using Kinetics Flexi-Dry freeze-dryer. Nuclear magnetic
resonance (NMR) spectra were obtained on a Bruker 500 MHz machine
in d.sub.6-DMSO. Splitting patterns are denoted s, singlet; d,
doublet; dd, doublet of doublets; t, triplet; m, multiplet; br s,
broad singlet. Mass spectra were acquired on a Micromass Quattro
Micro API mass spectrometer operating in positive ion mode. The
samples were dissolved in MeCN/H.sub.2O (1:1), 0.1% formic acid for
mass spectrometry analysis.
EXAMPLE 1
Synthesis of ethyl 6-nitrobenzothiazole-2-carboxylate (2)
[0112] Ethyl 6-nitrobenzothiazole-2-carboxylate was synthesized
from 6-nitrobenzothiazole according to the following reaction
scheme.
##STR00004##
[0113] In particular, 6-nitrobenzothiazole 1 (16.7 mmol, 3.0 g) was
suspended in 6.6 ml deionized H.sub.2O. 2.7 ml conc.
H.sub.2SO.sub.4 was added dropwise to the reaction flask.
Separately, 30% H.sub.2O.sub.2 (167.6 mmol, 5.7 g) was added
dropwise to a solution of ethyl pyruvate (75.6 mmol, 8.4 ml) at
0.degree. C. The resultant oxyhydroperoxide solution and a solution
of FeSO.sub.4.7H.sub.2O (48.9 mmol, 13.6 g) in 13.2 ml deionized
H.sub.2O were simultaneously added dropwise to the
6-nitrobenzothiazole reaction flask at 0.degree. C. After 30 min,
the reaction was poured onto ice and basified with NaHCO.sub.3 (pH
from 2 to .about.pH 6). The organic product 2 was washed with a
saturated NaCl solution, extracted with EtOAc, dried over
Na.sub.2SO.sub.4, and concentrated in vacuo. The material was
additionally filtered with 200 proof EtOH yielding a pale yellow
solid (.about.61% yield crude material). Agilent HPLC displayed
partial conversion to the crude product at 10.4 min with a peak at
8.7 min corresponding to the starting material.
[0114] Compound 2 was used in the subsequent step without
purification. For analytical characterization, a small portion was
purified by semi-preparative HPLC at 10 mL/min on a Waters
preparative machine (Waters Symmetry prep C18, 7 .mu.m,
19.times.300 mm column, photodiode array detection) with a linear
gradient from 95% H.sub.2O (0.1% TFA) to 90% MeCN (0.1% TFA) for 35
min yielding a large product peak at 27 min. The fractions were
collected and lyophilized using Kinetics Flexi-Dry freeze-dryer.
ESI-MS: m/z calcd for C.sub.10H.sub.8N.sub.2O.sub.4S (M+H).sup.+
253.26, found 252.85 (100%). .sup.1H (d.sub.6-DMSO) .delta.
(ppm)=1.39 (t, J=7.0 Hz, 3H), 4.48 (q, J=7.0 Hz, 2H), 8.44 (m, 2H),
9.32 (s, 1H). .sup.13C NMR (d.sub.6-DMSO) .delta. (ppm)=13.8, 63.0,
120.1, 122.1, 125.5, 136.5, 145.9, 155.9, 159.3, 164.1.
[0115] Bernadi et al reported almost quantatative conversion of
benzothiazole to ethyl 6-nitrobenzothiazole-2-carboxylate..sup.14
In this case, the low solubility of starting material 1 in aqueous
H.sub.2SO.sub.4 limited the conversion to the desired product
(.about.40% by TLC). The product 2 was also impossible to separate
from the starting material 1 either by flash or gravity silica
column chromatography.
EXAMPLE 2
Synthesis of 6-nitrobenzothiazole-2-carboxamide (3)
[0116] 6-Nitrobenzothiazole-2-carboxamide was synthesized from
ethyl 6-nitrobenzothiazole-2-carboxylate according to the following
reaction scheme.
##STR00005##
[0117] In particular, crude ethyl
6-nitrobenzothiazole-2-carboxylate 2 (7.9 mmol, 2.0 g) was
dissolved in 140 ml MeOH and purged with NH.sub.3 gas. Conversion
to the product was monitored by analytical TLC (3 Hexane: 2 EtOAc).
Following complete conversion to the product (20 min) the solvent
was removed in vacuo. The crude material was filtered with
chloroform (30 ml) to yield a pale tan-colored solid (80% yield
crude material). Agilent HPLC displayed partial conversion to the
crude product 3 at 8.1 min. The compound 3 was used in the
subsequent step without purification.
[0118] For analytical characterization, a small portion was
purified by semi-preparative HPLC at 10 mL/min on a Waters
preparative machine (Waters Symmetry prep C18, 7 .mu.m,
19.times.300 mm column, photodiode array detection) with a linear
gradient from 90% H.sub.2O (0.1% TFA) to 50% MeCN (0.1% TFA) for 30
min yielding a large product peak at 22.5 minutes. The fractions
were collected and lyophilized using Kinetics Flexi-Dry
freeze-dryer. ESI-MS: m/z calcd for C.sub.8H.sub.5N.sub.3O.sub.3S
(M+H).sup.+ 224.22, found 223.88 (100%). .sup.1H (d.sub.6-DMSO)
.delta. (ppm)=8.30 (br s, NH), 8.31 (dd, J=9.0 Hz, J=0.5 Hz), 8.41
(dd, J=9.0 Hz, J=2.5 Hz), 8.65 (br s, NH), 9.28 (d, J=2.0 Hz).
.sup.13C NMR (d.sub.6-DMSO) .delta. (ppm)=120.3, 122.1, 124.8,
137.1, 145.6, 156.6, 160.8, 171.1.
EXAMPLE 3
Synthesis of 2-cyano-6-nitrobenzothiazole (4)
[0119] 2-Cyano-6-nitrobenzothiazole was synthesized from
6-nitrobenzothiazole-2-carboxamide according to the following
reaction scheme.
##STR00006##
[0120] In particular, crude 6-nitrobenzothiazole-2-carboxamide 3
(4.5 mmol, 1.0 g), was dissolved in anhydrous pyridine (0.74 mol,
60 ml) and stirred at RT under N.sub.2. The temperature of the
reaction was dropped to 0.degree. C. and POCl.sub.3 (0.14 mol, 12.5
ml) was added dropwise to the reaction flask. After 20 min, the
acetone/ice bath was removed and the reaction was stirred at RT for
an additional 2 h. The contents were then transferred to a larger
reaction flask containing 150 ml EtOAc at 0.degree. C. While
stifling, the reaction was quenched with the dropwise addition of
water (150 ml).
[0121] The organic layer was separated, dried over
Na.sub.2SO.sub.4, and concentrated in vacuo yielding an
orange/brown solid material. TLC analysis (3 Hexane: 7
CH.sub.2Cl.sub.2) displayed 100% conversion to the product 4.
Agilent HPLC displayed conversion to the crude product at 10.1 min.
The product 4 was purified by gravity silica column chromatography
(3 Hexane: 7 CH.sub.2Cl.sub.2) to yield a pale white solid
(.about.24% pure product). ESI-MS: m/z calcd for
C.sub.8H.sub.3N.sub.3O.sub.2S (M+H).sup.+ 206.20, found 205.97
(100%). .sup.1H (d.sub.6-DMSO): .delta. (ppm)=8.47 (s, 2H, 1H),
9.39 (s, 1H). .sup.13C NMR (d.sub.6-DMSO): .delta. (ppm)=112.7,
120.3, 122.7, 125.3, 136.0, 143.2, 146.4, 154.6.
EXAMPLE 4
Synthesis of 2-cyano-6-aminobenzothiazole (5)
[0122] 2-Cyano-6-aminobenzothiazole was synthesized from
2-cyano-6-nitrobenzothiazole according to the following reaction
scheme.
##STR00007##
[0123] In particular, purified 2-cyano-6-nitrobenzothiazole 4 (0.49
mmol, 100 mg) was dissolved in 5 ml EtOH (200 Proof). 2.5
equivalents of SnCl.sub.2 (1.3 mmol, 322 mg) was added to the
flask. The reaction was heated to 60.degree. C. under N.sub.2, and
stirred for 2 h. Over time the mixture changed from a bright
fluorescent yellow to an orange/yellow coloring. After cooling to
room temperature (RT), the reaction was poured into ice water
(.about.5 ml) and the pH was adjusted using NaHCO.sub.3 (pH 7).
[0124] The product 5 was extracted using EtOAc, dried over
MgSO.sub.4 and removed in vacuo (.about.65% yield). Agilent HPLC
displayed conversion to the crude product at 7.4 min. The product 5
was purified by gravity silica column chromatography (3 Hexane: 2
EtOAc) to yield an orange/yellow solid. ESI-MS: m/z calcd for
C.sub.8H.sub.5N.sub.3S (M +H).sup.+ 176.22, found 175.85 (100%).
.sup.1H NMR (d.sub.6-DMSO, 500 MHz) .delta. (ppm)=6.15 and 6.55 (br
s, NH.sub.2), 6.97 (dd, J=9.0 Hz, J=2.0 Hz), 7.15 (d, J=2.5 Hz),
7.86 (d, J=9.0 Hz). .sup.13C NMR (d.sub.6-DMSO) .delta.
(ppm)=102.2, 114.1, 117.3, 125.0, 127.7, 138.2, 143.2, 150.4.
EXAMPLE 5
Synthesis of ethyl 6-nitrobenzothiazole-2-carboxylate (7)
[0125] Ethyl 6-nitrobenzothiazole-2-carboxylate was synthesized
from ethyl benzothiazole-2-carboxylate according to the following
reaction scheme.
##STR00008##
[0126] In particular, commercially available ethyl
benzothiazole-2-carboxylate 6 (24.1 mmol, 5.0 g) was suspended in
22 ml conc. H.sub.2SO.sub.4 at 0.degree. C. At 10.degree. C.
KNO.sub.3 (26.4 mmol, 26 g) was added portionwise over 30 min to
the stifling solution, not to exceed 15.degree. C. Color of
reaction changed from bright green/yellow to yellow over time. The
reaction warmed from 25.degree. C. to 40.degree. C. for an
additional 30 min. When the temperature of the reaction was
decreased, the solution was poured over ice/water). The precipitate
forming in solution was collected via glass frit vacuum filtration
to yield a light yellow solid 7.
[0127] Resultant solid 7 was additionally washed with water,
extracted into EtOAc, and concentrated in vacuo. Agilent HPLC
displayed partial conversion to the crude product at .about.10.0
min with a peak at .about.9.9 min corresponding to the starting
material. Attempts to purify the product were made using gravity
silica column chromatography (4 Hexane: 1 EtOAc) to yield a yellow
solid (.about.10% yield). Compound 7 was identified with LC/MS
spectroscopic analysis as illustrated in FIG. 16.
EXAMPLE 6
Synthesis of benzothiazole-2-carboxamide (8)
[0128] Benzothiazole-2-carboxamide was synthesized from ethyl
benzothiazole-2-carboxylate according to the following reaction
scheme.
##STR00009##
[0129] In particular, commercially available ethyl
benzothiazole-2-carboxylate 6 (2.4 mmol, 0.5 g) was dissolved in 34
mL MeOH and purged with NH.sub.3 gas. Conversion to the product was
monitored by TLC (3 Hexane: 2 EtOAc). Following complete conversion
to the product 8 (.about.30 min), the solvent was removed in vacuo.
Agilent HPLC displayed 100% conversion to the desired product 8
with a single peak at .about.8.0 min. The solid white compound was
used in the subsequent step without purification. ESI-MS: m/z calcd
for C.sub.8H.sub.6N.sub.2OS 179.02 (M+H).sup.+, 179.88 found (100%)
(see LC/MS spectra of FIG. 17A and NMR spectra of FIG. 17B).
EXAMPLE 7
Synthesis of 6-nitrobenzothiazole-2-carboxamide (9)
[0130] 6-nitrobenzothiazole-2-carboxamide was synthesized from
benzothiazole-2-carboxamide according to the following reaction
scheme.
##STR00010##
[0131] In particular, 2-carboxamide-benzothiazole 8 (0.88 mmol,
0.156 g) was suspended in 0.655 ml conc. H.sub.2SO.sub.4 at
0.degree. C. 1.3 equivalents of concentrated HNO.sub.3 (1.1 mmol,
0.069 g) was added portionwise over 10 min to the stirring
solution; not to exceed 0.degree. C. The reaction remained at
0.degree. C. for .about.6 h and continued overnight; warming to
25.degree. C. overnight. The following day the reaction was poured
over ice/water forming a white precipitate in solution. The
reaction was washed with water, extracted into EtOAc, and
concentrated in vacuo to yield crude product. Attempts to purify
the product were made using gravity silica column chromatography
(Initially 3 Hexane: 7 CH.sub.2Cl.sub.2 followed by 3 Hexane: 2
EtOAc) to yield a pure white solid (.about.30% yield). (see LC/MS
spectra of FIG. 18). Further NMR can be performed to detect the
monofunctional benzothiazole comprising the N.sub.O2 at position C6
versus thiazole substituted in the C4, C5, or C7 positions.
EXAMPLE 8
Synthesis of 2-cyanobenzothiazole (10)
[0132] 2-Cyanobenzothiazole was synthesized from
benzothiazole-2-carboxamide according to the following reaction
scheme.
##STR00011##
[0133] Benzothiazole-2-carboxamide 8 (0.61 mmol, 0.1 g) was
dissolved in anhydrous pyridine (0.080 mol, 6.5 ml) and stirred
under N.sub.2 at 0.degree. C. The temperature of the reaction was
dropped to 0.degree. C. during dropwise addition of POCl.sub.3
(0.015 mol, 1.4 ml). The solution immediately changed from clear to
a pale pink coloring with addition of POCl.sub.3. After 20 min, the
acetone/ice bath was removed and the reaction was stirred at RT for
2 h further. The tan/brown-colored reaction was then transferred to
a larger reaction flask containing 10 ml EtOAc at 0.degree. C. The
reaction was quenched by the dropwise addition of water. The
organic product 10 was washed with water, extracted with EtOAc, and
concentrated in vacuo to yield a light yellow/orange product. TLC
analysis (3 Hexane: 2 EtOAc) and Agilent HPLC displayed 100%
conversion to the desired product (single peak at 10.5 min). The
compound was used in the subsequent step without purification (95%
yield). ESI-MS: m/z calcd for C.sub.8H.sub.4N.sub.2S 161.01 (M
+H).sup.+, 160.86 found (100%). (see LC/MS spectra of FIG. 19A and
NMR spectra of FIG. 19B).
EXAMPLE 9
Synthesis of 2-cyano-6-nitrobenzothiazole (11)
[0134] 2-Cyano-6-nitrobenzothiazole was synthesized from
2-cyanobenzothiazole according to the following reaction
scheme.
##STR00012##
[0135] In particular 2-cyano-benzothiazole 10 (0.84 mmol, 0.134 g)
was suspended in 0.563 ml conc. H.sub.2SO.sub.4 at 0.degree. C. 1.3
equivalents of concentrated HNO.sub.3 (1.1 mmol, 0.069 g) was added
portionwise over 10 min to the stirring solution; not to exceed
0.degree. C. over a total of .about.45 min. The reaction proceeded
further for 2 h and at 5 h the product was evident. The reaction
was then poured over ice/water, extracted into EtOAc, and
concentrated in vacuo to yield a crude yellow residue 11. For
analytical characterization, a small portion was purified by
semi-preparative HPLC at 10 mL/min on a Waters preparative machine
(Waters Symmetry prep C18, 7 .mu.m, 19.times.300 mm column,
photodiode array detection) with a linear gradient from 90%
H.sub.2O (0.1% TFA) to 50% MeCN (0.1% TFA) for 30 min yielding a
large product peak at .about.27 minutes. The fractions were
collected and lyophilized using Kinetics Flexi-Dry
freeze-dryer.
[0136] Further NMR can be performed to detect the monofunctional
benzothiazole comprising the NO.sub.2 at position C6 versus
thiazole substituted in the C4, C5, or C7 positions.
EXAMPLE 10
Synthesis of Tyrosine-Aminocyano Derivative of
2-cyano-6-aminobenzothiazole
[0137] The tyrosine-aminocyano derivative of
2-cyano-6-aminobenzothiazole was synthesized according to the
following reaction scheme.
##STR00013##
[0138] Tyrosine protected amino acid (0.32 mmoles, 0.11 g) was
dissolved in 4 mL of anhydrous THF and stirred under N.sub.2 at
0.degree. C. in the dark. N-methylmorpholine (2 eq, 0.64 mmol,
0.070 mL) and isobutyl chloroformate (1.3 eq, 0.42 mmol, 0.055 mL)
were added dropwise to the reaction at 0.degree. C. and stirred for
30 min in the dark. Separately, purified
2-cyano-6-aminobenzothiazole 5 (0.32 mmol, 0.056 g) was dissolved
in 1 mL of anhydrous THF and then added dropwise to the reaction
flask over a period of 30 min. The reaction was stirred under
N.sub.2 in the dark at 0.degree. C. for 2 h, followed by an
overnight stir at room temperature in the dark. 24 h later the
anhydrous THF was removed in vacuo. The product 12 was dissolved in
EtOAc and washed with saturated NaHCO.sub.3 and NaCl to quench any
remaining isobutyl chloroformate. The organic layer was collected,
dried with MgSO.sub.4, and evaporated to dryness. The protected
group on the tyrosine portion of the product was removed using 20%
TFA in anhydrous CH.sub.2Cl.sub.2; stirred at room temperature in
the dark for 2 h. Following deprotection, the product 12 was
evaporated to dryness and placed on vacuum.
[0139] The residue was redissolved in 50/50 0.1% TFA in MeCN &
H.sub.2O (+drops of DMF to solubilize) and any solid residue
removed by filtration through a 0.45 .mu.m filter. Analytical HPLC
with a linear gradient from 95% H.sub.2O (0.1% TFA) to 80% MeCN
(0.1% TFA) over 15 min yielded a peak at 7.3 min. The material was
purified using the semi-preparative HPLC at 10 mL/min on a Waters
preparative machine (Waters Symmetry prep C18, 7 .mu.m,
19.times.300 mm column, photodiode array detection) with a linear
gradient from 90% H.sub.2O (0.1% TFA) to 50% MeCN (0.1% TFA) for 30
min. Semi-preparative HPLC fractions at 17 min were collected and
lyophilized using Kinetics Flexi-Dry freeze-dryer. Compound 12 was
identified with LC/MS spectroscopic analysis as illustrated in FIG.
20.
[0140] This same procedure can be applied for various other amino
acids (e.g. the 20 amino acids) as a means to develop new
conjugates as bioluminescence probes that may vary in their kinetic
activity (pharmacodynamics/pharmacokinetics) in vitro and in
vivo
EXAMPLE 11
Synthesis of tyrosine-D-aminoluciferin from Amino Acid-Conjugated
2-cyano-6-aminobenzothiazole Intermediate
[0141] The tyrosine-d-aminoluciferin compound was synthesized
according to the following reaction scheme
##STR00014##
[0142] The deprotected and purified amino acid-conjugated
2-cyano-6-aminobenzothiazole (e.g. tyrosine conjugated; 0.048
mmoles, 0.024 g) was dissolved in anhydrous THF (0.5 mL), followed
by dropwise addition of D-cysteine (1.2 eq, 0.06 mmoles, 0.011 g,
in 0.056 mL; adjusted to pH 8) while stifling the reaction at RT in
the dark for 2 h. After 2 h, the reaction was evaporated to dryness
and placed on vacuum. The residue was re-dissolved in a mixture of
anhy. THF and any solid residue removed by filtration through a
0.45 .mu.m filter. The solvents were removed in vacuo.
EXAMPLE 12
Synthesis of Peptide Conjugated
2-cyano-6-aminoacid-aminobenzothiazole from the Amino
Acid-Conjugated 2-cyano-6-aminobenzothiazole Intermediate
[0143] The peptide-conjugated
2-cyano-6-aminoacid-aminobenzothiazole was synthesized according to
the following reaction scheme.
##STR00015##
[0144] The peptide sequence was designed to be recognized by a
specific protease in the blood that is indicative of disease (e.g.
prostate specific antigen (PSA), which correlates to prostate
cancer; a wide variety of proteases are also present and these
levels may change in response to biological insults conferred by
infection, malignant growth and autoimmune responses.sup.16. The
protected peptide sequence (0.033 mmoles, 0.067 g) was dissolved in
2.3 mL of anhydrous THF (+small amount of anhydrous DMF),
sonicated, and stirred under N.sub.2 at 0.degree. C. in the dark
(material in suspension). N-methylmorpholine (2 eq, 0.07 mmol,
0.007 mL) and isobutyl chloroformate (1.3 eq, 0.04 mmol, 0.006 mL)
were added drop-wise to the reaction at 0.degree. C. and stirred
for 30 min in the dark. Separately, purified amino acid-conjugated
2-cyano-6-aminobenzothiazole (e.g. tyrosine conjugated) 12 (0.03
mmol, 0.011 g) was dissolved in 0.2 mL of anhydrous THF and then
added dropwise to the reaction flask over a period of 30 min. The
reaction was stirred under N.sub.2 in the dark at 0.degree. C. for
2 h, followed by a 72 h stir at room temperature in the dark. After
72 h, the anhydrous THF/DMF was removed in vacuo. The product 14
was dissolved in EtOAc and washed with saturated NaHCO.sub.3 to
quench any remaining isobutyl chloroformate. The organic layer was
collected and evaporated to dryness to yield a bright yellow to
golden orange/yellow residue. The protected group on the peptide
portion of the product was removed using 50% TFA in anhydrous
CH.sub.2Cl.sub.2; stirred at RT in the dark for 3 h. Following
deprotection, the product 14 was evaporated to dryness and placed
on vacuum. Compound 14 was identified with LC/MS spectroscopic
analysis as illustrated in FIG. 21.
[0145] This same procedure can be applied for various other peptide
sequences as a means to develop new conjugates as bioluminescence
probes for detection of various other proteases that link to
disease.
EXAMPLE 13
Synthesis of Peptide Conjugated tyrosine-D-aminoluciferin from the
Peptide Conjugated 2-cyano-6-aminobenzothiazole-tyrosine-Conjugated
Intermediate
[0146] The peptide-conjugated tyrosine-D-aminoluciferin was
synthesized according to the following reaction scheme.
##STR00016##
[0147] The deprotected and purified peptide-conjugated
2-cyano-6-aminobenzothiazole-[amino acid] (0.006 mmoles, 0.008 g)
was dissolved in anhydrous THF (0.01 mL), followed by drop-wise
addition of D-cysteine (1.2 eq, 0.007 mmoles, 0.001 g, 0.001 mL)
while stifling the reaction at RT in the dark for 2 h. After 2 h,
the reaction was evaporated to dryness and placed on vacuum. The
residue was re-dissolved in a mixture of anhy. THF and any solid
residue removed by filtration through a 0.45 .mu.m filter. The THF
was removed in vacuo
EXAMPLE 14
Synthesis of a Carboxylate-Modified Luciferin Amino Acid/Peptide
Probe
[0148] Chlorotrityl chloride (CTC) resin can be used to synthesize
amino acid/peptide derivatives of D-Luciferin as outlined below. An
example for phenylalanine is shown below however chlorotryl
chloride resin is commonly used to synthesize peptides, A peptide
of any length can be synthesized using standard Fmoc amino add
synthesis the D-luciferin can be conjugated as a last step and the
conjugate cleaved from the resin. Purification is using reversed
phase high performance liquid chromatography (HPLC)
[0149] Reference is made to the reaction Scheme of FIG. 14 showing
a schematic illustration of an exemplary reaction scheme.
EXAMPLE 14
Methods Using a Carboxylate-Modified Luciferin Amino Acid/Peptide
Probe
[0150] Carboxypeptidase Y was used but other carboxypeptidases are
expected to be suitable depending on the nature of the amino acids
attached to the D-luciferin as will be understood by a skilled
person. Carboxypeptidase Y (sold supported or free in solution) can
be used to remove C-termini amino acids from a peptide. In the case
shown here, it will remove phenylalanine from the conjugate,
releasing the D-luciferin for reaction with the enzyme.
Carboxypeptidase Y is expected to be confirmed as functioning
optimally using hippuryl-L-phenylalanine, a commercially available
UV-active substrate for carboxypeptidases. Reference is made to the
reaction
[0151] The scheme of FIG. 15A showing a schematic illustration of
an exemplary reaction scheme.
[0152] A blocking carboxypeptidase activity using a blocking agent
(e.g. CONH2) on the C. terminus of the conjugates was investigated
as outlined in FIG. 15B.
[0153] These determinations are expected to confirm the utility of
D-luciferin amino acid I peptide conjugates as extremely sensitive
probes for proteolytic action for in vitro and in vivo assays as
outlined below. This example shows the C terminus of the substrate
blocked as a carboxamide. Only upon proteolytic cleavage of the
probe will free carboxy terminus be revealed for the
carboxypeptidase to digest and release the D-luciferin for
subsequent reaction with luciferase. In this connection reference
is made to the schematic illustration of FIG. 15C.
[0154] The examples set forth above are provided to give those of
ordinary skill in the art a complete disclosure and description of
how to make and use the embodiments of the compounds, compositions,
systems and methods of the disclosure, and are not intended to
limit the scope of what the inventors regard as their disclosure.
All patents and publications mentioned in the specification are
indicative of the levels of skill of those skilled in the art to
which the disclosure pertains.
[0155] The entire disclosure of each document cited (including
patents, patent applications, journal articles, abstracts,
laboratory manuals, books, or other disclosures) in the Background,
Summary, Detailed Description, and Examples is hereby incorporated
herein by reference. All references cited in this disclosure are
incorporated by reference to the same extent as if each reference
had been incorporated by reference in its entirety
individually.
[0156] It is to be understood that the disclosures are not limited
to particular compositions or biological systems, which can, of
course, vary. It is also to be understood that the terminology used
herein is for the purpose of describing particular embodiments
only, and is not intended to be limiting. As used in this
specification and the appended claims, the singular forms "a,"
"an," and "the" include plural referents unless the content clearly
dictates otherwise. The term "plurality" includes two or more
referents unless the content clearly dictates otherwise. Unless
defined otherwise, all technical and scientific terms used herein
have the same meaning as commonly understood by one of ordinary
skill in the art to which the disclosure pertains.
[0157] When a Markush group or other grouping is used herein, all
individual members of the group and all combinations and possible
subcombinations of the group are intended to be individually
included in the disclosure. Every combination of components or
materials described or exemplified herein can be used to practice
the disclosure, unless otherwise stated. One of ordinary skill in
the art will appreciate that methods, device elements, and
materials other than those specifically exemplified can be employed
in the practice of the disclosure without resort to undue
experimentation. All art-known functional equivalents, of any such
methods, device elements, and materials are intended to be included
in this disclosure. Whenever a range is given in the specification,
for example, a temperature range, a frequency range, a time range,
or a composition range, all intermediate ranges and all subranges,
as well as, all individual values included in the ranges given are
intended to be included in the disclosure. Any one or more
individual members of a range or group disclosed herein can be
excluded from a claim of this disclosure. The disclosure
illustratively described herein suitably can be practiced in the
absence of any element or elements, limitation or limitations which
is not specifically disclosed herein.
[0158] A number of embodiments of the disclosure have been
described. The specific embodiments provided herein are examples of
useful embodiments of the disclosure and it will be apparent to one
skilled in the art that the disclosure can be carried out using a
large number of variations of the monofunctional benzothiazoles,
compositions, methods steps, and systems set forth in the present
description. As will be obvious to one of skill in the art, methods
and devices useful for the present methods can include a large
number of optional composition and processing elements and
steps.
[0159] It will be understood that various modifications may be made
without departing from the spirit and scope of the present
disclosure. Accordingly, other embodiments are within the scope of
the following claims.
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