U.S. patent application number 14/801897 was filed with the patent office on 2015-11-12 for fluorescent molecular probes for use in assays that measure test compound competitive binding with sam-utilizing proteins.
The applicant listed for this patent is Cayman Chemical Company, Incorporated, The Regents of the University of Michigan. Invention is credited to Stephen Douglas Barrett, Levi Lynn Blazer, Daniel Austin Bochar, Fred Lawrence Ciske, Margaret Lynn Collins, Gregory William Endres, Jeffrey Keith Johnson, Gregory Scott Keyes, Ranjinder Singh Sidhu, Raymond C. Trievel.
Application Number | 20150322107 14/801897 |
Document ID | / |
Family ID | 47175189 |
Filed Date | 2015-11-12 |
United States Patent
Application |
20150322107 |
Kind Code |
A1 |
Barrett; Stephen Douglas ;
et al. |
November 12, 2015 |
FLUORESCENT MOLECULAR PROBES FOR USE IN ASSAYS THAT MEASURE TEST
COMPOUND COMPETITIVE BINDING WITH SAM-UTILIZING PROTEINS
Abstract
Assay methods may generally comprise forming homogeneous assay
mixtures comprising target SAM-utilizing protein, fluorescent
detection analyte, and test compound, incubating, and measuring FP
or TR-FRET signal emitted in order to determine a measure of test
compound-SAM-utilizing protein binding. Assay mixtures comprise a
SAM-utilizing protein, and a fluorescent detection analyte that
binds with the SAM-utilizing protein in the absence of test
compound. Assay mixtures may further comprise a test compound.
Assay mixture embodiments may generate FP or TR-FRET signal
properties that are a function of the inherent binding interactions
of both the test compound and the detection analyte with the
SAM-utilizing protein. Fluorescent detection analytes comprise a
fluorophore moiety, a covalent linker moiety, and a SAM-utilizing
protein ligand moiety and could be utilized in FP or TR-FRET assays
to measure test compound binding.
Inventors: |
Barrett; Stephen Douglas;
(Hartland, MI) ; Bochar; Daniel Austin; (Ann
Arbor, MI) ; Blazer; Levi Lynn; (Ann Arbor, MI)
; Ciske; Fred Lawrence; (Dexter, MI) ; Endres;
Gregory William; (Saline, MI) ; Johnson; Jeffrey
Keith; (Ann Arbor, MI) ; Keyes; Gregory Scott;
(Dexter, MI) ; Sidhu; Ranjinder Singh; (Ann Arbor,
MI) ; Trievel; Raymond C.; (Ypsilanti, MI) ;
Collins; Margaret Lynn; (Canton, MI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Cayman Chemical Company, Incorporated
The Regents of the University of Michigan |
Ann Arbor
Ann Arbor |
MI
MI |
US
US |
|
|
Family ID: |
47175189 |
Appl. No.: |
14/801897 |
Filed: |
July 17, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13475618 |
May 18, 2012 |
9120820 |
|
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14801897 |
|
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61487461 |
May 18, 2011 |
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Current U.S.
Class: |
536/27.6 |
Current CPC
Class: |
G01N 21/6428 20130101;
G01N 33/582 20130101; C07H 19/16 20130101; C07D 471/04 20130101;
G01N 2333/91011 20130101; G01N 2201/061 20130101; C07D 473/34
20130101; C07D 519/00 20130101 |
International
Class: |
C07H 19/16 20060101
C07H019/16; G01N 33/58 20060101 G01N033/58 |
Goverment Interests
GOVERNMENT CONTRACT
[0002] This invention was made with government support under
GM073839 awarded by the National Institute of Health. The
government has certain rights in this invention.
Claims
1-44. (canceled)
45. A fluorescent detection analyte having the structure of Formula
(I): ##STR00062## wherein the ----- between two atoms in either the
A ring or the B ring represents the bond involving the two atoms is
either a single or a double bond, and it may only represent a
double bond between the 4'-carbon and X.sup.9 when X.sup.9 is CH;
wherein X.sup.9 is O, NR.sup.7, S, CH (allowed when its bond to the
4'-carbon is a carbon-carbon double bond), or CH.sub.2 (allowed
when its bond to the 4'-carbon is a carbon-carbon single bond);
wherein R.sup.7 is hydrogen or methyl; wherein each of X.sup.5,
X.sup.6, X.sup.7, and X.sup.8 is independently a carbon or a
nitrogen; wherein no more than three of X.sup.5, X.sup.6, X.sup.7,
and X.sup.8 is nitrogen; wherein when any X.sup.5, X.sup.6,
X.sup.7, or X.sup.8 is nitrogen, the associated W.sup.5, W.sup.6,
W.sup.7, and W.sup.8, respectively, is not present; wherein when
X.sup.5 is carbon, W.sup.5 may be hydrogen, methyl, amino, or
chloro; wherein when X.sup.6 is carbon, W.sup.6 may be hydrogen,
methyl, amino, acetyl, carboxy, carboxamide, or hydroxy; wherein
when X.sup.7 or X.sup.8 is carbon, W.sup.7 or W.sup.8,
respectively, may independently be hydrogen, C.sub.1-6 alkyl,
C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.3-6 cycloalkyl,
(C.sub.3-6 cycloalkyl)methyl, phenyl, benzyl, five- or six-membered
heterocyclyl, five- or six-membered heteroaryl, cyano, amino,
acetyl, carboxy, hydroxy or CONR.sup.8R.sup.9; wherein each of
R.sup.8 and R.sup.9 is independently hydrogen, C.sub.1-6 alkyl,
C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.3-6 cycloalkyl,
(C.sub.3-6 cycloalkyl)methyl, phenyl, benzyl, five- or six-membered
heterocyclyl, five- or six-membered heteroaryl, or OR.sup.10, or
together with the nitrogen atom form a pyrrolidine, piperidine,
morpholine, or pyrazine ring; wherein R.sup.10 is hydrogen,
C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.3-6
cycloalkyl, (C.sub.3-6 cycloalkyl)methyl, phenyl, benzyl, five- or
six-membered heterocyclyl, or five- or six-membered heteroaryl; or
wherein W.sup.7 or W.sup.8 together form a five- or six-membered
aryl, carbocyclic, heterocyclic, or heteroaryl ring fused with the
B ring; wherein X.sup.2' is hydrogen, hydroxy, or OR.sup.2';
wherein X.sup.3' is hydrogen, hydroxy, or OR.sup.3'; wherein
X.sup.5' is C(.dbd.X.sup.4)X.sup.10R; wherein X.sup.4 is O or
H.sub.2; wherein X.sup.10 is C(H)NR.sup.11R.sup.12, NR.sup.1, or S;
wherein R.sup.11 and R.sup.12 each is independently hydrogen,
C.sub.1-4 alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl, C.sub.3-6
cycloalkyl, phenyl, benzyl, or acetyl, or together with the
nitrogen atom form an aziridine, azetidine, pyrrolidine,
piperidine, morpholine, or pyrazine ring; wherein R and R.sup.1
each is independently hydrogen, C.sub.1-8 alkyl, C.sub.2-6 alkenyl,
C.sub.2-6 alkynyl, C.sub.3-6 cycloalkyl, C.sub.6-10 aryl,
five-to-ten-membered heteroaryl, five- to ten-membered
heterocyclyl, C.sub.1-8 acyl, or [(S)-2-aminobutanoic acid]-4-yl,
wherein any alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl
or heteroaryl ring is optionally substituted with one or more of
fluoro, chloro, bromo, iodo, C.sub.1-6 alkyl, C.sub.2-6 alkenyl,
C.sub.2-6 alkynyl, methoxy, ethoxy, trifluoromethoxy,
trifluoromethyl, hydroxy, thiomethyl, cyano, NR.sup.8R.sup.9,
--N(H)C(.dbd.O)X.sup.11, acetyl, carboxy, carboxy(C.sub.1-4 alkyl),
or CONR.sup.8R.sup.9; wherein X.sup.11 is C.sub.1-6 alkyl,
C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.3-6 cycloalkyl,
(C.sub.3-6 cycloalkyl)methyl, phenyl, benzyl, OR.sup.10, or
NR.sup.8R.sup.9; wherein one of R.sup.2, R.sup.2' (if it exists),
and R.sup.3' (if it exists), comprises a linker component, wherein
the linker component comprises a linker moiety bonded to a
fluorophore moiety, and the other existing of R.sup.2, R.sup.2',
and R.sup.3' are hydrogen, and wherein R.sup.2, when not hydrogen,
substitutes a hydrogen atom of the B ring or a ring fused to the B
ring or a functional group covalently bound to the B ring or a ring
fused to the B ring; wherein the linker moiety comprises a
structure as illustrated in Formula (VI) or Formula (VII):
##STR00063## wherein when R.sup.2 comprises the linker component,
the linker moiety may alternatively comprise a structure as
illustrated in Formula (VIII): ##STR00064## wherein the
(CH.sub.2).sub.n group of Formula (IV) or the (CH.sub.2).sub.s
group of Formula (V) comprises the site of covalent attachment at
one of R.sup.2, R.sup.2', and R.sup.3' of Formulas (I), (II), and
(III); wherein X is CH.sub.2 or O; wherein X.sup.1 is N--H,
N--CH.sub.3, O, or S; wherein X.sup.2 is N--H, N--CH.sub.3, or,
when r is 0 and X.sup.1 is N--H or N-Me, X.sup.2 may alternatively
be O; wherein X.sup.3 is NH or O; wherein Z.sup.1 is a carbonyl,
thiocarbonyl, or sulfonyl group; wherein Y is a covalent bond that
binds the linker moiety to the fluorophore moiety, or
(CH.sub.2).sub.n wherein the last CH.sub.2 group in the chain (when
n is not 0) is farthest from Z.sup.1 is covalently bound to the
fluorophore moiety, or (CH.sub.2).sub.n--N(H)--Z.sup.2, or is of
the chemical structure: ##STR00065## wherein the oxygen atom end is
covalently bound to Z.sup.1; wherein each R.sup.5 and R.sup.6 is
independently H, methyl, or together are (CH.sub.2).sub.q; wherein
q is 1, 2, or 3; wherein p is 1 or 2; and wherein Z.sup.2 is a
carbonyl or thiocarbonyl group covalently bound by its carbon atom
to the fluorophore moiety, or a sulfonyl group covalently bound by
sulfur atom to the fluorophore moiety; wherein each n is
independently 0, 1, 2, 3, 4, or 5; wherein s is 1, 2, or 3; wherein
m is 1, 2, or 3; wherein each r is independently 0 or 1; wherein
the fluorophore moiety is a structure selected from the group of
chemical structures consisting of: ##STR00066## ##STR00067##
##STR00068## ##STR00069## wherein * represents the position at
which the linker moiety is covalently bound to the fluorophore
moiety; wherein A.sup.- is a PF.sub.6.sup.-, trifluoroacetate,
acetate, or halide anion; wherein B.sup.+ is a sodium, potassium,
cesium, ammonium, or .sup.+N(R.sup.4).sub.4 cation; and wherein
each R.sup.4 is independently H or C.sub.1-4 alkyl.
46. A fluorescent detection analyte according to claim 45, wherein
the fluorophore moiety comprises a mixture of chemical structure
(b) and (c), a mixture of chemical structure (b) and (n), a mixture
of chemical structure (d) and (e), a mixture of chemical structure
(f) and (g), a mixture of chemical structure (h) and (i), or a
mixture of chemical structure (j) and (k).
47-58. (canceled)
59. The fluorescent detection analyte of claim 45, wherein X.sup.9
is O.
60. The fluorescent detection analyte of claim 59, wherein X.sup.6
is N and wherein each of X.sup.5, X.sup.7 and X.sup.8 is C.
61. The fluorescent detection analyte of claim 45, wherein X.sup.9
is S.
62. The fluorescent detection analyte of claim 61, wherein X.sup.6
is N and wherein each of X.sup.5, X.sup.7 and X.sup.8 is C.
63. The fluorescent detection analyte of claim 45, wherein X.sup.9
is NR.sup.7.
64. The fluorescent detection analyte of claim 63, wherein X.sup.6
is N and wherein each of X.sup.5, X.sup.7 and X.sup.8 is C.
65. The fluorescent detection analyte of claim 45, wherein X.sup.6
is N and wherein each of X.sup.5, X.sup.7 and X.sup.8 is C.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority from U.S.
Provisional Application Ser. No. 61/487,461, filed May 18, 2011,
which is herein incorporated by reference.
FIELD OF THE INVENTION
[0003] The subject matter disclosed and claimed herein centers on
fluorescent molecular probes that may be used as detection analytes
in binding assay mixtures and the use of the assay mixtures for
measuring test compound binding to SAM-utilizing proteins.
BACKGROUND OF THE INVENTION
[0004] All references, including patents and patent applications,
are hereby incorporated by reference in their entireties.
[0005] S-Adenosylmethionine (SAM) is a ubiquitous metabolic
intermediate that is biosynthesized by the enzyme methionine
adenosyltransferase (SAM synthetase), which accelerates the
coupling of adenosine triphosphate (ATP) with methionine (Mato, J.
M., Pharmacology and Therapeutics, 1997, 73(3), 265-280). SAM plays
a key role in various biochemical processes such as enzymatic
reactions that involve transmethylation, transsulfuration, and the
polyamine-generating aminoalkylation pathway (Roje, S.,
Phytochemistry, 2006, 67(15), 1686-1698; Giulidori, P. et al., The
Journal of Biological Chemistry, 1984, 259(7), 4205-4211). Other
enzymatic reactions that involve interaction of proteins with SAM
or isostructural SAM analogs include transfer of methylene,
ribosyl, and 5'-deoxyadenosyl groups; formation of redox
intermediate 5'-deoxyadenosyl radical; and SAM decarboxylation.
SAM-nonenzymatic protein interactions also exist wherewith SAM acts
as a ligand affecting structural and functional modification in the
effector protein (Kozbial, P. Z. and Mushegian, A. R., BMC
Structural Biology, 2005, 5, 19-44).
##STR00001##
[0006] One of the most understood processes involving SAM-utilizing
proteins is biochemical transmethylation. The relatively unreactive
thioether methyl group of methionine is made very reactive toward
nitrogen, oxygen, sulfur, and carbon nucleophiles when coupled with
the adenosyl group to provide the chemically destabilizing
positively-charged sulfonium ion of SAM. SAM-utilizing
methyltransferases are enzymes that catalyze transfer of the
reactive methyl group from SAM to a substrate for covalent
modification, leaving the stabilized S-adenosylhomocysteine (SAH or
AdoHcy) by-product. Methyltransferases comprise a significant
percentage of the proteome and are found in all organisms
(Petrossian, T. C. and Clarke, S. G., Molecular and Cellular
Proteomics, 2011, 10(1)). A large and diverse set of SAM-utilizing
methyltransferase substrates is known. Broad substrate classes
include histone and non-histone proteins, nucleic acids,
polysaccharides, lipids, small organic molecules (e.g. catechol:
Mannisto, P. T. and Kaakkola, S., Pharmacological Reviews, 1999,
51(4), 593-628), and inorganic substrates (e.g. arsenic: Thomas, D.
J. et al., Experimental Biology and Medicine, 2007, 232(1), 3-13;
Hayakawa, T. et al., Archives of Toxicology, 2004, 79(4), 183-191);
and halides: (Ohsawa, N. et al., Bioscience, Biotechnology and
Biochemistry, 2001, 65, 2397-2404; Attieh, J. M. et al., 1995, 270,
9250-9257). SAM-utilizing methyltransferases play a role in
critical cellular processes including biosynthesis, signal
transduction, chromatin regulation, and gene silencing.
[0007] While the SAM-utilizing methyltransferases share a common
requirement for SAM, distinct differences exist in the SAM binding
structural fold and also in the SAM binding mode. These different
structural families can be grouped into at least seven classes,
five classes that are typically designated I through V (Schubert,
H. L. et al., Trends in Biochemical Sciences, 2003, 28(6),
329-335). Two other classes include the radical SAM enzymes (Frey,
P. A. et al., Critical Reviews in Biochemistry and Molecular
Biology, 2008, 43, 63-88), which catalyze diverse radical-based
reactions that include methylation, and the isoprenylcysteine
carboxy methyltransferases (ICMTs), which are integral membrane
proteins (Yang, J. et al., Molecular Cell, 2011, 44(6), 997-1004).
Amino-acid sequence homology within each class can be as low as
10%, showing that wide variations in molecular environment
mediating catalysis of methyl transfer from SAM to substrate are
allowed, which at least in part may be due to the favorable
energetics involved in the conversion of SAM to SAH.
Methyltransferases that bind and methylate protein substrates are
generally found in Class I (classical fold) or Class V (SET fold),
and methyltransferases that act on DNA substrates have been found
in Classes I or IV (Schubert, H. L. et al., Trends in Biochemical
Sciences, 2003, 28(6), 329-335). Non-catalytic domains outside the
core structure determine substrate recognition. SAM-utilizing
methyltransferases recruit SAM and a substrate to the SAM-dependent
methyltransferase fold, where methyl transfer occurs and modified
substrate and SAH are produced and released.
[0008] The involvement of methyltransferases in epigenetics is
currently an intense area of research (Copeland, R. A. et al.,
Nature Reviews Drug Discovery, 2009, 8, 724-732). The field of
epigenetics studies molecular changes such as DNA methylation and
post-translational histone modifications that influence phenotype
without alterations in the DNA sequence of the genome.
Methyltransferases play a major role in epigenetic regulation of
gene expression by catalyzing the modification of chromatin by
specific methylations of DNA, histones, or biomolecules associated
with chromatin (Kouzarides, T., Cell, 2007, 128, 693-705).
Chromatin remodeling methyltransferases can generally be divided
into two categories according to their substrates. DNA
methyltransferases (DNMTs) methylate the 5-position carbon atom of
cytosine in the CpG dinucleotide sites of the mammalian genome
(Cheng, X. and Blumenthal, R. M., Structure, 2008, 16, 341-350).
Protein methyltransferases (PMTs), which can generally be
subdivided into protein lysine methyltransferases (PKMTs) and
protein arginine methyltransferases (PRMTs), methylate protein
lysine or arginine residues, respectively. The histone targets of
PMTs are largely characterized while non-histone protein targets
such as the FOXO transcription factors continue to be discovered
(Yamagata, K. et al., Molecular Cell, 2008, 32, 221-231; Greer, E.
L. and Brunet, A., Oncogene, 2005, 24, 7410-7425).
[0009] Aberrant DNA and histone methylation due to abnormal
methyltransferase expression levels or mutations is associated with
the onset and progression of a variety of cancers and other
diseases and conditions (Egger, G., Nature, 2004, 429, 457-463;
Esteller, M., New England Journal of Medicine, 2008, 358,
1148-1159).
[0010] Selective, small-molecule inhibitors of epigenetic targets,
such as histone deacetylases (HDACs) and DNMTs, have been
successfully designed and deployed as therapeutic agents for the
treatment of myelomas, lymphomas, and other cancers and have been
further investigated for use against inflammatory and autoimmune
disorders (Copeland, R. A. et al., Nature Reviews Drug Discovery,
2009, 8, 724-732; Szyf, M., Clinical Reviews in Allergy and
Immunology, 2010, 39(1), 62-77; Kaiser, J., Science, 2010,
330(6004), 576-578). PKMTs and PRMTs are favorable drug targets
amenable to small-molecule inhibition (Copeland, R. A. et al.,
Current Opinion in Chemical Biology, 2010, 4, 505-510). Each
methyltransferase is structurally unique and has a distinct
functional profile (Dillon, S. C. et al., Genome Biology, 2005, 6,
227; Cheng, X. et al., Annual Reviews in Biophysical and
Biomolecular Structure, 2005, 34, 267-294). Small molecule
inhibitors are currently sought after for a variety of
methyltransferases in the search for new drug therapies (Shaaban,
S. A. and Bedford, M. A., Chemistry and Biology, 2007, 14(3),
242-244), and the potential pharmaceutical utility in areas such as
antibiotics and treatment of Parkinson's Disease (e.g.
catechol-O-methyltransferase inhibitors) and beyond is vast; thus,
an efficient means for rapidly screening large compound collections
against an ever-growing number of known SAM-utilizing
methyltransferases is needed.
[0011] Various SAM-utilizing methyltransferase screening assays
have been developed and used for identifying compound inhibitors.
One such assay reported is a universal competitive fluorescence
polarization (FP) methyltransferase activity immunoassay that
measures formation of SAH (Graves, T. L. et al., Analytical
Biochemistry, 2008, 373, 296-306). The assay employs an anti-AdoHcy
antibody and fluorescence-labeled AdoHcy conjugate tracer to
measure AdoHcy generated from the methyltransferase activity.
Another SAM-utilizing methyltransferase assay reported is an
enzyme-coupled continuous spectrophotometric screen (Dorgan, K. M.
et al., Analytical Biochemistry, 2006, 350, 249-255). In this assay
SAH generated from demethylation of SAM is hydrolyzed to
S-ribosylhomocysteine and adenine by recombinant
S-adenosylhomocysteine/5'-methylthioadenosine nucleosidase. Adenine
is subsequently hydrolyzed to hypoxanthine and ammonia by
recombinant adenine deaminase, a process which is monitored
continuously by measuring absorbance at a wavelength of 256 nm.
Another enzyme coupled assay used for measuring SAM-utilizing
methyltransferase activity involves the conjugation of homocysteine
(Hcy), which is generated from cleavage of SAH by SAH hydrolase
(SAHH), to a thiol-reactive fluorophore (Collazo, E. et al.,
Analytical Biochemistry, 2005, 342, 86-92). A commercial
radiometric histone methyltransferase assay is also known and has
been adapted for high throughput screening (Horiuchi, K. Y. et al.,
FASEB J, 2010, 24, lb61). All of these assays gauge SAM-utilizing
methyltransferase activity by measuring the generation of products
formed as a consequence of the signature methyl-transfer reaction
from SAM to the substrate but do not directly provide information
as to the specific binding interactions of the test compound
without subsequent enzymological study. In addition, target
screening using a coupled enzyme assay method suffers from the
potential generation of false positive leads due to inhibition of
the coupling enzymes. The radiometric assay is a robust binding
assay but possesses the inherent liability of generating
radioactive waste. The assays developed here overcome these
shortfalls and will also provide specific binding information by
directly measuring binding affinities and dissociation constants of
the test compounds.
[0012] A fluorescence polarization or TR-FRET assay could measure
test compound binding to a SAM-utilizing protein by measuring
displacement of a fluorescence-labeled ligand ("detection analyte"
or "probe") from the protein. The universal cofactor SAM provides a
structural template with which to design a versatile detection
analyte. SAM itself is a chemically reactive methyl donor and thus
is not a suitable compound for incorporation into a stable
detection analyte. A robust, chemically stable SAM mimic possessing
steric and electronic characteristics similar to those of SAM is
therefore desirable. Such an analyte design would provide a
SAM-utilizing protein ligand moiety seeking to take advantage of
inherent pan-methyltransferase recruitment of SAM and could thus
allow the analyte to be utilized across the SAM-dependent
methyltransferase enzyme family. Herein are disclosed fluorescent
detection analytes, assays that employ them, and their uses for
assessing binding of test compounds.
[0013] Sinefungin, a SAM- and SAH-analog nucleoside isolated from
Streptomyces griseolus and Streptomyces incarnatus, exhibits an
array of antimicrobial effects due primarily to its inhibition of
SAM-utilizing methyltransferases (Malina, H. et al., Journal of
Antibiotics, 1985, 38(9), 1204-1210; Berry, D. R. and Abbott, B.
J., Journal of Antibiotics, 1978, 31(3), 185-191; Vedel M. et el.,
Biochemical and Biophysical Research Communications, 1978, 85(1),
371-376). The sinefungin molecular structure may be divided into
three subunits: a central ribose ring, an adenine ring connected by
its 9-nitrogen position to the 1'-ribose ring carbon atom, and an
ornithine side chain connected by its amino acid .delta.-carbon to
the 5'-carbon atom of the ribose ring. Recent reports disclose
structures of sinefungin bound in the SET domain of histone PKMTs
SETT/9, LSMT, SmyD1 and SmyD3 (Subramanian, K. et al., Molecular
Cell, 2008, 30(3), 336-47; Couture et al., The Journal of
Biological Chemistry, 2006, 281(28), 19280-19287; Sirinupong, N. et
al., The Journal of Biological Chemistry, 2010, 285(52),
40635-40644; Sirinupong, N. et al., Journal of Molecular Biology,
2010). Earlier published sinefungin-nucleic acid methyltransferase
complex structures wherein sinefungin is shown to bind in the SAM
binding site show only minor structural changes in the complex
versus the cases in which SAM or SAH are shown bound (Zheng, S. et
al., The Journal of Biological Chemistry, 2006, 281(47),
35904-35913; Thomas, C. B. et al., The Journal of Biological
Chemistry, 2003, 278(28), 26094-26101).
##STR00002##
[0014] SAM-like affinity for a wide range of methyltransferases and
relative chemical stability make sinefungin a reasonable detection
analyte SAM-utilizing protein methyltransferase ligand moiety. For
example, sinefungin binds to human SETT/9 with only a six-fold
lower affinity than SAM, and sinefungin binds to Arabidopsis LSMT
with only a fourteen-fold lower affinity than SAM (Couture et al.,
The Journal of Biological Chemistry, 2006, 281(28), 19280-19287;
Horowitz et al., The Journal of Biological Chemistry, 2011, Epub
jbc.M111.232876). Variations in spatial requirements among
different methyltransferase catalytic domains may require that an
assortment of sinefungin-based detection analytes be utilized to
screen test compounds against a broad panel of these enzymes.
Sinefungin-based detection analyte alternatives may vary based on
different linker-fluorophore attachment positions on sinefungin.
The linker moiety, for example, may be covalently bound to
sinefungin at a nitrogen or carboxy oxygen position on the
ornithine residue, one of the two ribose hydroxyl oxygen atoms, or
to an open carbon or the free amino position on the C-6 carbon atom
of the adenine base ring. Sinefungin-based probes may be designed
that are selective to SET domain-containing lysine
methyltransferases or other classes of methyltransferases, which
can use different binding modes to recognize SAM. These differences
may necessitate that the linker moieties tethering sinefungin or
sinefungin analogs to the fluorophore be attached to sinefungin at
different positions to accommodate the varying binding modes.
[0015] Fluorescence polarization and TR-FRET assays generally
provide advantages in the study of protein-ligand binding over
other conventional assay types. These assay formats allow rapid
real-time measurements, avoid the use of radioactive materials, are
homogeneous requiring minimal additions and no washing steps, and
may possess sub-nanomolar detection limits. FP and TR-FRET assays
are currently used in drug discovery and are routinely converted to
high-throughput screening (HTS) formats (Burke, T. J. et al.,
Combinatorial Chemistry and High Throughput Screening, 2003, 6(3),
183-194). The uses, advantages, and photophysical principles
associated with FP and TR-FRET assays have been described and are
well known to those ordinarily skilled in the art (Lakowicz, J. R.,
Principles of Fluorescence Spectroscopy, Springer, New York, USA,
1999; Owicki, J. C., Journal of Biomolecular Screening, 2000, 5(5),
297-306; Nasir, M. S., Jolley, M. E., Combinatorial Chemistry &
High Throughput Screening, 1999, 2, 177-190; Klostermeier, D. and
Millar, D. P., Biopolymers (Nucleic Acid Sciences), 2002, 61,
159-179).
SUMMARY OF THE INVENTION
[0016] Assay methods of the exemplary embodiments may generally
comprise forming homogeneous assay mixtures comprising target
SAM-utilizing protein, fluorescent detection analyte, and test
compound, incubating, and measuring FP or TR-FRET signal emitted in
order to determine a measure of test compound-SAM-utilizing protein
binding.
[0017] Assay mixtures of the exemplary embodiments comprise a
SAM-utilizing protein, and a fluorescent detection analyte that
binds with the SAM-utilizing protein in the absence of test
compound. Assay mixtures of the exemplary embodiments may further
comprise a test compound. Exemplary assay mixture embodiments may
generate FP or TR-FRET fluorescence emissions, such as but not
limited to signal intensity, polarization (for FP), or ratio of
donor/acceptor emissions (for TR-FRET), that gauge test compound
and detection analyte binding with the SAM-utilizing protein.
[0018] Fluorescent detection analytes of the exemplary embodiments
comprise a fluorophore moiety, a SAM-utilizing protein ligand
moiety, and a linker moiety that covalently links the fluorophore
moiety with the SAM-utilizing protein ligand moiety, and could be
utilized in FP or TR-FRET assays to measure test compound
binding.
[0019] Other exemplary embodiments of the invention will become
apparent from the detailed description provided hereinafter. It
should be understood that the detailed description and specific
examples, while disclosing exemplary embodiments of the invention,
are intended for purposes of illustration only and are not intended
to limit the scope of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 illustrates a synthetic pathway for preparing the
fluorescent detection analytes Compound 1A (Sinefungin Probe 1A)
and Compound 2A (Sinefungin Probe 2A).
[0021] FIG. 2 is a plot illustrating saturation binding of
Sinefungin Probe 1A with each of five different methyltransferase
enzymes.
[0022] FIG. 3 is a plot illustrating concentration-response curves
for SAM, SAH, and unlabeled sinefungin (SF) competing for binding
to SET7/9 (30 nM) with Sinefungin Probe 1A (10 nM).
[0023] FIG. 4 shows the Z'-factor analysis of the SET7/9 FP assay
utilizing Sinefungin Probe 1A.
[0024] FIG. 5 illustrates a general synthetic pathway for preparing
thioadenosine fluorescent detection analytes.
[0025] FIG. 6 illustrates an alternative general synthetic pathway
for preparing thioadenosine fluorescent detection analytes.
[0026] FIG. 7 illustrates a general synthetic pathway for preparing
aza-adenosine fluorescent detection analytes.
[0027] FIG. 8 illustrates an alternative general synthetic pathway
for preparing aza-adenosine fluorescent detection analytes.
[0028] FIG. 9 illustrates a general synthetic pathway for preparing
sinefungin fluorescent detection analytes and sinefungin analog
fluorescent detection analytes wherein the linker moiety is
attached to the adenine ring or adenine ring replacement
portion.
[0029] FIG. 9A illustrates general synthetic pathways for obtaining
various sinefungin base ring portions covalently bound to linker
moieties or linker moiety precursors.
[0030] FIG. 10 is a plot illustrating binding affinity of
Sinefungin Probe 1A in high-throughput fluorescence
polarization-based assays for MLL and SET7/9.
[0031] FIG. 11 is a plot illustrating the binding affinity of
Sinefungin Probe 1A and Sinefungin Probe 1B to SET7/9.
[0032] FIG. 12 is a plot illustrating the binding affinities of
Sinefungin Probe 3 and Sinefungin Probe 1B to SET7/9.
[0033] FIG. 13 plots profiling data for Probe synthetic precursors
Compounds 16A, 20A-iv, and 20A-iii against seven
methyltransferases.
[0034] FIG. 14 is a plot illustrating the binding affinity of
Thioadenosine Probe 1 to PRMT1.
DETAILED DESCRIPTION OF THE INVENTION
[0035] The exemplary embodiments may be directed to FP or TR-FRET
assays, assay mixtures, and fluorescent detection analytes for
identifying compounds that bind to SAM-utilizing proteins. Certain
embodiments may be directed to FP or TR-FRET assays and assay
mixtures wherein the SAM-utilizing proteins are methyltransferases.
Certain other embodiments may be directed to FP or TR-FRET assays
and assay mixtures wherein the methyltransferases are DNA
methyltransferases, histone methyltransferases, methyltransferases
for transcription factors and chromatin-associated proteins, or
methyltransferases for cellular molecules.
[0036] FP signal is typically generated by measuring the
polarization of the fluorescence emitted from a detection analyte.
As the detection analyte freely tumbles in solution, the
polarization signal is low. This signal increases when the analyte
is bound by a molecule (e.g. a methyltransferase) that is
relatively large as compared to the analyte. Thus, any test
compound that displaces the analyte from the methyltransferase will
induce a measurable decrease in the FP signal.
[0037] TR-FRET signal is typically a function of measured emission
of an acceptor fluorophore following excitation of the donor
fluorophore. The acceptor fluorophore emission is only generated if
the donor fluorophore is in close proximity (generally less than
100 angstroms) to the acceptor fluorophore and energy transfer from
donor to acceptor fluorophore occurs. Thus, any test compound that
dissociates the acceptor fluorophore from the close proximity of
the donor fluorophore-bearing SAM-utilizing protein will induce a
measurable decrease in the TR-FRET signal.
[0038] Exemplary methods employed for measuring FP and TR-FRET
signals are described herein, and are generally known to those
skilled in the art. The compounds identified as binding ligands may
provide novel therapies for the treatment of diseases or conditions
mediated by the SAM-utilizing protein employed in the assay.
[0039] As used herein below with respect to the exemplary
embodiments and claims, the term "fluorescence signal or property"
is meant to describe a property or properties associated with
fluorescence emitted by the assay mixture. Such properties comprise
those typically found in the art, including but not limited to
polarization signal, intensity, or signal decay. Such property or
properties can be used to measure and quantify test compound
binding to SAM-utilizing proteins as described previously or in the
exemplary embodiments or claims.
[0040] As used herein, the properties of the FP signal may be
described in terms of the measured polarization signal.
[0041] As used herein, the properties of the TR-FRET signal may be
described in terms of the intensity of the measured fluorescence
emission spectrum.
[0042] The exemplary embodiments herein may provide a homogeneous,
rapid, and consistent assay for high-throughput screening of
compounds or agents for binding to a SAM-utilizing protein.
[0043] A moiety, as defined herein with respect to the exemplary
embodiments and claims, generally refers to a functional group or a
particular portion of a molecule. A moiety may be bound to other
portions of the molecule, or moieties, by a covalent chemical bond
or bonds.
[0044] A nucleoside moiety is a portion of a molecule comprising a
nucleoside. A nucleoside, as defined herein with respect to the
exemplary embodiments and claims, generally refers to a
glycosylamine comprising a nucleobase (base) bound to a ribose or
deoxyribose (sugar) by way of a .beta.-glycosidic linkage. One
nucleoside known to those ordinarily skilled in the art is
adenosine, which consists of the base, adenine, bound by a
.beta.-glycosidic linkage to the sugar, ribose. Other nucleosides
known to those ordinarily skilled in the art include cytidine,
guanosine, inosine, thymidine, and uridine.
[0045] A nucleoside-type moiety, as defined herein with respect to
the exemplary embodiments and claims, is a portion of a molecule
comprising a nucleoside or a nucleoside having at least one
structural modification. An exemplary structural modification may
include, but is not limited to, a stereochemical variation from a
naturally-derived molecule or moiety. Another exemplary structural
modification may include, but is not limited to, a variation in the
position of an atom or functional group, such as is the case for a
structural isomer or regioisomer of a naturally-derived molecule or
moiety. Another exemplary structural modification may include, but
is not limited to, the substitution of an atom or functional group
of a naturally-derived molecule or moiety with an alternative atom
or functional group. An exemplary nucleoside-type moiety may
possess one or more of the same type of exemplary structural
modifications described above, or a combination of more than one
variety of exemplary structural modifications described above.
[0046] An exemplary nucleoside-type moiety that may be utilized is
an adenosine moiety. Another exemplary nucleoside-type moiety that
may be utilized is a deaza-adenosine moiety, such as a
3-deaza-adenosine moiety, having one adenine-ring nitrogen atom
replaced with a carbon atom bearing a hydrogen atom, as illustrated
below:
##STR00003##
[0047] Other exemplary nucleoside-type moieties that may be
utilized may include, but are not limited to, an adenosine moiety
or deaza-adenosine moiety substituted with a functional group (FG)
at one of the base ring positions as illustrated below:
##STR00004##
[0048] Further exemplary nucleoside-type moieties that may be
utilized include, but are not limited to, a sinefungin moiety, an
S-substituted-5'-thioadenosine (also referred to herein as a
"sulfur-based," a "thioadenosine," or a "SAM-like") moiety, and a
5'-aza-adenosine (also referred to herein as a "nitrogen-based," an
"aza-adenosine," or an "aza-SAM-like") moiety, as illustrated
below:
##STR00005##
R and R.sup.1 are further described in various embodiments
below.
[0049] A sinefungin moiety, as defined herein, refers to a moiety
wherein the molecular structure of that moiety is that of
sinefungin, a stereoisomer of sinefungin, or a related analog to
sinefungin as described herein. Exemplary sinefungin moieties that
may be utilized include, but are not limited to, sinefungin, an N-
or O-protected or an N-- or O--C.sub.1-4 alkylated derivative
(including alkyl esters of the sinefungin carboxylic acid) of
sinefungin, a carboxamide analog (wherein the sinefungin carboxylic
acid is instead a carboxamide or N-alkylcarboxamide or
N,N-dialkylcarboxamide) of sinefungin, a deaza-adenine analog of
sinefungin, a deoxyribose analog of sinefungin, a sinefungin analog
wherein one of the sinefungin-unsubstituted adenine carbon atoms is
substituted with a halogen, amino, alkylamino, trifluoromethyl,
methoxy, ethoxy, trifluoromethoxy, thiomethyl, cyano, C.sub.1-4
alkyl, C.sub.2-4 alkenyl, or C.sub.2-4 alkynyl, a sinefungin analog
wherein the adenine ring is replaced with another heterocyclic
ring, or any sinefungin stereoisomer, sinefungin regioisomer,
sinefungin analog stereoisomer, or sinefungin analog regioisomer
thereof.
[0050] Exemplary nucleoside-type moieties may be covalently bonded
to linker moieties as described elsewhere herein. Exemplary
structural positions at which a nucleoside-type moiety may be
covalently bonded to a linker moiety may include, but are not
limited to, a base-ring carbon atom position, a base ring
functional group that possesses a suitable site for covalent
linkage to a linker moiety, a ribose-2'-hydroxyl oxygen atom, or a
ribose-3'-hydroxyl oxygen atom.
[0051] Exemplary detection analytes may be prepared by methods
generally and specifically described herein.
[0052] One exemplary embodiment may be directed to a method for
identifying compounds that bind to SAM-utilizing proteins
comprising: [0053] (a) forming an assay mixture comprising: [0054]
(1) a fluorescent detection analyte comprising: [0055] (i) a
fluorophore moiety; [0056] (ii) a SAM-utilizing protein ligand
moiety; and [0057] (iii) a linker moiety that covalently links the
fluorophore moiety with the SAM-utilizing protein ligand moiety;
[0058] (2) a SAM-utilizing protein or a SAM-utilizing protein
labeled with a donor fluorophore or an acceptor fluorophore; and
[0059] (3) a test compound; [0060] (b) irradiating the assay
mixture at a particular excitation wavelength to generate a
fluorescence signal or property; [0061] (c) measuring the
fluorescence signal or property generated by the assay mixture; and
[0062] (d) determining the level of binding of the test compound to
the SAM-utilizing protein or to the SAM-utilizing protein labeled
with the donor fluorophore or the acceptor fluorophore from the
measured fluorescence signal or property.
[0063] In certain of these embodiments, the SAM-utilizing protein
ligand moiety (ii) comprises a nucleoside-type moiety. Exemplary
nucleoside-type moieties that may be utilized include those
described above, such as, for example, a sinefungin moiety, a
sulfur-based moiety, or a nitrogen-based moiety.
[0064] In certain other of these embodiments, wherein the
SAM-utilizing protein ligand moiety (ii) comprises a sinefungin
moiety, the linker moiety that covalently links the fluorophore
moiety with the sinefungin moiety (iii) comprises a linker moiety
that covalently links the fluorophore moiety with the sinefungin
moiety through an atom on the sinefungin moiety selected from the
group consisting of a ribose 2'-hydroxy oxygen atom and a ribose
3'-hydroxy oxygen atom.
[0065] In certain other of these embodiments, wherein the
SAM-utilizing protein ligand moiety (ii) comprises a sinefungin
moiety, the linker moiety that covalently links the fluorophore
moiety with the sinefungin or sinefungin analog moiety (iii)
comprises a linker moiety that covalently links the fluorophore
moiety with the sinefungin moiety through an atom on the base ring
or base ring replacement portion of the sinefungin moiety.
[0066] Another exemplary embodiment may be directed to a method for
identifying compounds that bind to SET domain-containing lysine
methyltransferase enzymes comprising: [0067] (a) forming an assay
mixture comprising: [0068] (1) a fluorescent detection analyte
comprising: [0069] (i) a fluorophore moiety; [0070] (ii) a
sinefungin moiety; and [0071] (iii) a linker moiety that covalently
links the fluorophore moiety with the sinefungin moiety through an
atom on the sinefungin moiety selected from the group consisting of
the ribose 2'-hydroxy oxygen atom, the ribose 3'-hydroxy oxygen
atom, and the adenine 2-carbon atom; [0072] (2) a SET
domain-containing lysine methyltransferase enzyme or a SET
domain-containing lysine methyltransferase enzyme labeled with a
donor fluorophore; and [0073] (3) a test compound; [0074] (b)
irradiating the assay mixture at a particular excitation wavelength
to generate a fluorescence signal or property; [0075] (c) measuring
the fluorescence signal or property emitted by the assay mixture;
and [0076] (d) determining the level of binding of the test
compound to the SET domain-containing lysine methyltransferase
enzyme or to the SET domain-containing lysine methyltransferase
enzyme labeled with the donor fluorophore or the acceptor
fluorophore from the measured fluorescence signal or property.
[0077] In certain of these embodiments, the SET domain-containing
lysine methyltransferase enzyme (2) is selected from the group
consisting of SET7/9, GLP, MLL, and G9a.
[0078] In certain of these embodiments, wherein the SET
domain-containing lysine methyltransferase enzyme (2) is selected
from the group consisting of SET7/9, GLP, MLL, and G9a, the
detection analyte (1) is selected from the group consisting of
Sinefungin Probe 1A, Sinefungin Probe 1B, Sinefungin Probe 2A,
Sinefungin Probe 3 and Sinefungin Probe 4. Sinefungin Probe 1A,
Sinefungin Probe 1B, Sinefungin Probe 2A, Sinefungin Probe 3 and
Sinefungin Probe 4 are each illustrated and described in the
Examples below.
[0079] Another exemplary embodiment may be directed to a method for
identifying compounds that bind to lysine methyltransferase enzyme
SET7/9 comprising: [0080] (a) measuring a fluorescence signal or
property of an irradiated assay mixture, wherein the assay mixture
comprises: [0081] (1) a fluorescent detection analyte comprising:
[0082] (i) a fluorophore moiety; [0083] (ii) a sinefungin moiety;
and [0084] (iii) a linker moiety that covalently links the
fluorophore moiety with the sinefungin moiety through an atom on
the sinefungin moiety selected from the group consisting of a
ribose 2'-hydroxy oxygen atom and a ribose 3'-hydroxy oxygen atom;
[0085] (2) a lysine methyltransferase enzyme SET7/9 or a lysine
methyltransferase enzyme SET7/9 labeled with a donor fluorophore or
an acceptor fluorophore; and [0086] (3) a test compound; [0087] (b)
irradiating the assay mixture at a particular excitation wavelength
to generate a fluorescence signal or property; [0088] (c) measuring
the fluorescence signal or property generated by the assay mixture;
and [0089] (d) determining the level of binding of the test
compound to the lysine methyltransferase enzyme SET7/9 or to the
lysine methyltransferase enzyme SET7/9 labeled with the donor
fluorophore or the acceptor fluorophore from the measured
fluorescence signal or property.
[0090] In certain of these embodiments, the fluorescent detection
analyte (1) is selected from the group consisting of Sinefungin
Probe 1A, Sinefungin Probe 1B, Sinefungin Probe 2A, Sinefungin
Probe 3 and Sinefungin Probe 4.
[0091] Another exemplary embodiment may be directed to a method for
identifying compounds that bind to lysine methyltransferase enzyme
G9a comprising: [0092] (a) measuring a fluorescence signal or
property of an irradiated assay mixture, wherein the assay mixture
comprises: [0093] (1) a fluorescent detection analyte comprising:
[0094] (i) a fluorophore moiety; [0095] (ii) a sinefungin moiety;
and [0096] (iii) a linker moiety that covalently links the
fluorophore moiety with the sinefungin moiety through an atom on
the sinefungin moiety selected from the group consisting of a
ribose 2'-hydroxy oxygen atom and a ribose 3'-hydroxy oxygen atom;
[0097] (2) a lysine methyltransferase enzyme G9a or a lysine
methyltransferase enzyme G9a labeled with a donor fluorophore or an
acceptor fluorophore; and [0098] (3) a test compound; [0099] (b)
irradiating the assay mixture at a particular excitation wavelength
to generate a fluorescence signal or property; [0100] (c) measuring
the fluorescence signal or property generated by the assay mixture;
and [0101] (d) determining the level of binding of the test
compound to the lysine methyltransferase enzyme G9a or to the
lysine methyltransferase enzyme G9a labeled with the donor
fluorophore or the acceptor fluorophore from the measured
fluorescence signal or property.
[0102] In certain exemplary embodiments, the fluorescent detection
analyte (1) is selected from the group consisting of Sinefungin
Probe 1A, Sinefungin Probe 1B, Sinefungin Probe 2A, Sinefungin
Probe 3 and Sinefungin Probe 4.
[0103] Another exemplary embodiment may be directed to a method for
identifying compounds that bind to lysine methyltransferase enzyme
GLP comprising: [0104] (a) measuring a fluorescence signal or
property of an irradiated assay mixture, wherein the assay mixture
comprises: [0105] (1) a fluorescent detection analyte comprising:
[0106] (i) a fluorophore moiety; [0107] (ii) a sinefungin moiety;
and [0108] (iii) a linker moiety that covalently links the
fluorophore moiety with the sinefungin moiety through an atom on
the sinefungin moiety selected from the group consisting of a
ribose 2'-hydroxy oxygen atom and a ribose 3'-hydroxy oxygen atom;
[0109] (2) a lysine methyltransferase enzyme GLP or a lysine
methyltransferase enzyme GLP labeled with a donor fluorophore or an
acceptor fluorophore; and [0110] (3) a test compound; [0111] (b)
irradiating the assay mixture at a particular excitation wavelength
to generate a fluorescence signal or property; [0112] (c) measuring
the fluorescence signal or property generated by the assay mixture;
and [0113] (d) determining the level of binding of the test
compound to the lysine methyltransferase enzyme GLP or to the
lysine methyltransferase enzyme GLP labeled with the donor
fluorophore or the acceptor fluorophore from the measured
fluorescence signal or property.
[0114] In certain exemplary embodiments, the fluorescent detection
analyte (1) is selected from the group consisting of Sinefungin
Probe 1A, Sinefungin Probe 1B, Sinefungin Probe 2A, Sinefungin
Probe 3 and Sinefungin Probe 4.
[0115] Another exemplary embodiment may be directed to a method for
identifying compounds that bind to lysine methyltransferase enzyme
MLL comprising: [0116] (a) measuring a fluorescence signal or
property of an irradiated assay mixture, wherein the assay mixture
comprises: [0117] (1) a fluorescent detection analyte comprising:
[0118] (i) a fluorophore moiety; [0119] (ii) a sinefungin moiety;
and [0120] (iii) a linker moiety that covalently links the
fluorophore moiety with the sinefungin moiety through an atom on
the sinefungin moiety selected from the group consisting of a
ribose 2'-hydroxy oxygen atom and a ribose 3'-hydroxy oxygen atom;
[0121] (2) a lysine methyltransferase enzyme MLL or a lysine
methyltransferase enzyme MLL labeled with a donor fluorophore or an
acceptor fluorophore; and [0122] (3) a test compound; [0123] (b)
irradiating the assay mixture at a particular excitation wavelength
to generate a fluorescence signal or property; [0124] (c) measuring
the fluorescence signal or property generated by the assay mixture;
and [0125] (d) determining the level of binding of the test
compound to the lysine methyltransferase enzyme MLL or to the
lysine methyltransferase enzyme MLL labeled with the donor
fluorophore or the acceptor fluorophore from the measured
fluorescence signal or property.
[0126] In certain exemplary embodiments, the fluorescent detection
analyte (1) is selected from the group consisting of Sinefungin
Probe 1A, Sinefungin Probe 1B, Sinefungin Probe 2A, Sinefungin
Probe 3 and Sinefungin Probe 4.
[0127] Another exemplary embodiment may be directed to a method for
identifying compounds that bind to SET domain-containing lysine
methyltransferase enzymes comprising: [0128] (a) forming an assay
mixture comprising: [0129] (1) a fluorescent detection analyte
comprising [0130] (i) a fluorophore moiety; [0131] (ii) an
S-substituted-5'-thioadenosine moiety; and [0132] (iii) a linker
moiety that covalently links the fluorophore moiety with the
S-substituted-5'-thioadenosine moiety; [0133] (2) a SET
domain-containing lysine methyltransferase enzyme or a SET
domain-containing lysine methyltransferase enzyme labeled with a
donor fluorophore; and [0134] (3) a test compound; [0135] (b)
irradiating the assay mixture at a particular excitation wavelength
to generate a fluorescence signal or property; [0136] (c) measuring
the fluorescence signal or property generated by the assay mixture;
and [0137] (d) determining the level of binding of the test
compound to the SET domain-containing lysine methyltransferase
enzyme or to the SET domain-containing lysine methyltransferase
enzyme labeled with the donor fluorophore or the acceptor
fluorophore from the measured fluorescence signal or property.
[0138] Another exemplary embodiment may be directed to a method for
identifying compounds that bind to the SET domain-containing lysine
methyltransferase enzyme PRDM9 comprising: [0139] (a) forming an
assay mixture comprising: [0140] (1) a fluorescent detection
analyte comprising [0141] (i) a fluorophore moiety; [0142] (ii) an
S-substituted-5'-thioadenosine moiety; and [0143] (iii) a linker
moiety that covalently links the fluorophore moiety with the
S-substituted-5'-thioadenosine moiety; [0144] (2) the SET
domain-containing lysine methyltransferase enzyme PRDM9 or PRDM9
labeled with a donor fluorophore; and [0145] (3) a test compound;
[0146] (b) irradiating the assay mixture at a particular excitation
wavelength to generate a fluorescence signal or property; [0147]
(c) measuring the fluorescence signal or property generated by the
assay mixture; and [0148] (d) determining the level of binding of
the test compound to PRDM9 or to the PRDM9 labeled with the donor
fluorophore or the acceptor fluorophore from the measured
fluorescence signal or property.
[0149] In certain of these embodiments, the fluorescent detection
analyte (1) comprises Thioadenosine Probe 1, which is described and
illustrated below in the Examples.
[0150] Another exemplary embodiment may be directed to a method for
identifying compounds that bind to arginine methyltransferase
enzymes comprising: [0151] (a) forming an assay mixture comprising:
[0152] (1) a fluorescent detection analyte comprising [0153] (i) a
fluorophore moiety; [0154] (ii) an S-substituted-5'-thioadenosine
moiety; and [0155] (iii) a linker moiety that covalently links the
fluorophore moiety with the S-substituted-5'-thioadenosine moiety;
[0156] (2) a protein arginine methyltransferase (PRMT) enzyme or
PRMT enzyme labeled with a donor fluorophore; and [0157] (3) a test
compound; [0158] (b) irradiating the assay mixture at a particular
excitation wavelength to generate a fluorescence signal or
property; [0159] (c) measuring the fluorescence signal or property
generated by the assay mixture; and [0160] (d) determining the
level of binding of the test compound to the PRMT enzyme or to the
PRMT enzyme labeled with the donor fluorophore or the acceptor
fluorophore from the measured fluorescence signal or property.
[0161] In certain of these embodiments, the PRMT enzyme (2) is
selected from the group consisting of PRMT1 and PRMT4 (CARM1).
[0162] In certain of these embodiments, the fluorescent detection
analyte (1) comprises Thioadenosine Probe 1.
[0163] Another exemplary embodiment may be directed to an assay
mixture for identifying compounds that bind to a SAM-utilizing
protein comprising: [0164] (a) a detection analyte comprising:
[0165] (1) a fluorophore moiety; [0166] (2) a SAM-utilizing protein
ligand moiety; and [0167] (3) a linker moiety that covalently links
the fluorophore moiety with the SAM-utilizing protein ligand
moiety; [0168] (b) a SAM-utilizing protein or a SAM-utilizing
protein labeled with a donor fluorophore or an acceptor
fluorophore; and [0169] (c) a test compound.
[0170] In certain of these embodiments, the SAM-utilizing protein
ligand moiety used in the assay mixture comprises a nucleoside-type
moiety. Exemplary nucleoside-type moieties that may be utilized
include those described above, such as, for example, a sinefungin
moiety, a sulfur-based moiety, or a nitrogen-based moiety.
[0171] In certain other of these embodiments, wherein the
SAM-utilizing protein ligand moiety (ii) used in the assay mixture
comprises a sinefungin moiety, the linker moiety that covalently
links the fluorophore moiety with the sinefungin moiety (iii)
comprises a linker moiety that covalently links the fluorophore
moiety with the sinefungin moiety through an atom on the sinefungin
moiety selected from the group consisting of a ribose 2'-hydroxy
oxygen atom and a ribose 3'-hydroxy oxygen atom.
[0172] In certain other of these embodiments, wherein the
SAM-utilizing protein ligand moiety (ii) used in the assay mixture
comprises a sinefungin moiety, the linker moiety that covalently
links the fluorophore moiety with the sinefungin or sinefungin
analog moiety (iii) comprises a linker moiety that covalently links
the fluorophore moiety with the sinefungin moiety through an atom
on the base ring or base ring replacement portion of the sinefungin
moiety.
[0173] Another exemplary embodiment may be directed to an assay
mixture for identifying compounds that bind to a SAM-utilizing
methyltransferase enzyme comprising: [0174] (a) a detection analyte
comprising: [0175] (1) a fluorophore moiety; [0176] (2) a
sinefungin moiety; and [0177] (3) a linker moiety that covalently
links the fluorophore moiety with the sinefungin moiety; [0178] (b)
a SAM-utilizing methyltransferase enzyme or a SAM-utilizing
methyltransferase enzyme labeled with a donor fluorophore or an
acceptor fluorophore; and [0179] (c) a test compound.
[0180] Another exemplary embodiment may be directed to an assay
mixture for identifying compounds that bind to a SAM-utilizing
methyltransferase enzyme comprising: [0181] (a) a detection analyte
comprising: [0182] (1) a fluorophore moiety; [0183] (2) a
sinefungin moiety; and [0184] (3) a linker moiety that covalently
links the fluorophore moiety with the sinefungin moiety through an
atom on the sinefungin moiety selected from the group consisting of
the ribose 2'-hydroxy oxygen atom, the ribose 3'-hydroxy oxygen
atom, and the adenine 2-carbon atom; [0185] (b) a SAM-utilizing
methyltransferase enzyme or a SAM-utilizing methyltransferase
enzyme labeled with a donor fluorophore or an acceptor fluorophore;
and [0186] (c) a test compound.
[0187] Another exemplary embodiment may be directed to an assay
mixture for identifying compounds that bind to a SET
domain-containing methyltransferase enzyme comprising: [0188] (a) a
detection analyte comprising: [0189] (1) a fluorophore moiety;
[0190] (2) a sinefungin moiety; and [0191] (3) a linker moiety that
covalently links the fluorophore moiety with the sinefungin moiety
through an atom on the sinefungin moiety selected from the group
consisting of the ribose 2'-hydroxy oxygen atom, the ribose
3'-hydroxy oxygen atom, and the adenine 2-carbon atom; [0192] (b) a
SET domain-containing lysine methyltransferase enzyme or a SET
domain-containing lysine methyltransferase enzyme labeled with a
donor fluorophore or an acceptor fluorophore; and [0193] (c) a test
compound.
[0194] In certain of these embodiments, the SET domain-containing
lysine methyltransferase enzyme (2) of the assay mixture is
selected from the group consisting of SET7/9, GLP, MLL, and
G9a.
[0195] In certain of these embodiments, wherein the SET
domain-containing lysine methyltransferase enzyme (2) of the assay
mixture is selected from the group consisting of SET7/9, GLP, MLL,
and G9a, the detection analyte (1) is selected from the group
consisting of Sinefungin Probe 1A, Sinefungin Probe 1B, Sinefungin
Probe 2A, Sinefungin Probe 3 and Sinefungin Probe 4.
[0196] Another exemplary embodiment may be directed to an assay
mixture for identifying compounds that bind to a SET
domain-containing methyltransferase enzyme comprising: [0197] (a) a
detection analyte selected from the group consisting of Sinefungin
Probe 1A, Sinefungin Probe 1B, Sinefungin Probe 2A, Sinefungin
Probe 3, and Sinefungin Probe 4; [0198] (b) a SET domain-containing
lysine methyltransferase enzyme or a SET domain-containing lysine
methyltransferase enzyme labeled with a donor fluorophore or an
acceptor fluorophore, the SET domain-containing lysine
methyltransferase enzyme selected from the group consisting of
SET7/9, GLP, MLL, and G9a; and [0199] (c) a test compound.
[0200] Another exemplary embodiment may be directed to an assay
mixture for identifying compounds that bind to lysine
methyltransferase enzyme SET7/9 comprising: [0201] (a) a detection
analyte selected from the group consisting of Sinefungin Probe 1A,
Sinefungin Probe 1B, Sinefungin Probe 2A, Sinefungin Probe 3, and
Sinefungin Probe 4; [0202] (b) SET7/9 or SET7/9 labeled with a
donor fluorophore or an acceptor fluorophore; and [0203] (c) a test
compound.
[0204] Another exemplary embodiment may be directed to an assay
mixture for identifying compounds that bind to lysine
methyltransferase enzyme G9a comprising: [0205] (a) a detection
analyte selected from the group consisting of Sinefungin Probe 1A,
Sinefungin Probe 1B, Sinefungin Probe 2A, Sinefungin Probe 3, and
Sinefungin Probe 4; [0206] (b) G9a or G9a labeled with a donor
fluorophore or an acceptor fluorophore; and [0207] (c) a test
compound.
[0208] Another exemplary embodiment may be directed to an assay
mixture for identifying compounds that bind to lysine
methyltransferase enzyme GLP comprising: [0209] (a) a detection
analyte selected from the group consisting of Sinefungin Probe 1A,
Sinefungin Probe 1B, Sinefungin Probe 2A, Sinefungin Probe 3, and
Sinefungin Probe 4; [0210] (b) GLP or GLP labeled with a donor
fluorophore or an acceptor fluorophore; and [0211] (c) a test
compound.
[0212] Another exemplary embodiment may be directed to an assay
mixture for identifying compounds that bind to lysine
methyltransferase enzyme MLL comprising: [0213] (a) a detection
analyte selected from the group consisting of Sinefungin Probe 1A,
Sinefungin Probe 1B, Sinefungin Probe 2A, Sinefungin Probe 3, and
Sinefungin Probe 4; [0214] (b) MLL or MLL labeled with a donor
fluorophore or an acceptor fluorophore; and [0215] (c) a test
compound.
[0216] Another exemplary embodiment may be directed to an assay
mixture for identifying compounds that bind to SET
domain-containing lysine methyltransferase enzymes comprising:
[0217] (a) a detection analyte comprising: [0218] (1) a fluorophore
moiety; [0219] (2) an S-substituted-5'-thioadenosine moiety; and
[0220] (3) a linker moiety that covalently links the fluorophore
moiety with the S-substituted-5'-thioadenosine moiety; [0221] (b) a
SET domain-containing lysine methyltransferase enzyme or a SET
domain-containing lysine methyltransferase enzyme labeled with a
donor fluorophore; and [0222] (c) a test compound.
[0223] Another exemplary embodiment may be directed to an assay
mixture for identifying compounds that bind to SET
domain-containing lysine methyltransferase enzyme PRDM9 comprising:
[0224] (a) a detection analyte comprising: [0225] (1) a fluorophore
moiety; [0226] (2) an S-substituted-5'-thioadenosine moiety; and
[0227] (3) a linker moiety that covalently links the fluorophore
moiety with the S-substituted-5'-thioadenosine moiety; [0228] (b)
the SET domain-containing lysine methyltransferase enzyme PRDM9 or
PRDM9 labeled with a donor fluorophore; and [0229] (c) a test
compound.
[0230] In certain of these embodiments, the detection analyte (a)
of the assay mixture comprises the fluorescent detection analyte
Thioadenosine Probe 1.
[0231] Another exemplary embodiment may be directed to an assay
mixture for identifying compounds that bind to arginine
methyltransferase enzymes comprising: [0232] (a) a detection
analyte comprising: [0233] (1) a fluorophore moiety; [0234] (2) an
S-substituted-5'-thioadenosine moiety; and [0235] (3) a linker
moiety that covalently links the fluorophore moiety with the
S-substituted-5'-thioadenosine moiety; [0236] (b) an arginine
methyltransferase enzyme or an arginine methyltransferase enzyme
labeled with a donor fluorophore; and [0237] (c) a test
compound.
[0238] In certain of these embodiments, the arginine
methyltransferase enzyme of the assay mixture is selected from the
group consisting of PRMT1 and PRMT4 (CARM1).
[0239] In certain of these embodiments, the detection analyte (a)
of the assay mixture comprises the fluorescent detection analyte
Thioadenosine Probe 1.
[0240] Another exemplary embodiment may be directed to a
fluorescent detection analyte having the structure of Formula
(I):
##STR00006## [0241] wherein the ----- between two atoms in either
the A ring or the B ring represents the bond involving the two
atoms is either a single or a double bond, and it may only
represent a double bond between the 4'-carbon and X.sup.9 when
X.sup.9 is CH; [0242] wherein X.sup.9 is O, NR.sup.7, S, CH
(allowed when its bond to the 4'-carbon is a carbon-carbon double
bond), or CH.sub.2 (allowed when its bond to the 4'-carbon is a
carbon-carbon single bond); [0243] wherein R.sup.7 is hydrogen or
methyl; [0244] wherein each of X.sup.5, X.sup.6, X.sup.7, and
X.sup.8 is independently a carbon or a nitrogen; [0245] wherein no
more than three of X.sup.5, X.sup.6, X.sup.7, and X.sup.8 is
nitrogen; [0246] wherein when any X.sup.5, X.sup.6, X.sup.7, or
X.sup.8 is nitrogen, the associated W.sup.5, W.sup.6, W.sup.7, and
W.sup.8, respectively, is not present; [0247] wherein when X.sup.5
is carbon, W.sup.5 may be hydrogen, methyl, amino, or chloro;
[0248] wherein when X.sup.6 is carbon, W.sup.6 may be hydrogen,
methyl, amino, acetyl, carboxy, carboxamide, or hydroxy; [0249]
wherein when X.sup.7 or X.sup.8 is carbon, W.sup.7 or W.sup.8,
respectively, may independently be hydrogen, C.sub.1-6 alkyl,
C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.3-6 cycloalkyl,
(C.sub.3-6 cycloalkyl)methyl, phenyl, benzyl, five- or six-membered
heterocyclyl, five- or six-membered heteroaryl, cyano, amino,
acetyl, carboxy, hydroxy or CONR.sup.8R.sup.9; [0250] wherein each
of R.sup.8 and R.sup.9 is independently hydrogen, C.sub.1-6 alkyl,
C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.3-6 cycloalkyl,
(C.sub.3-6 cycloalkyl)methyl, phenyl, benzyl, five- or six-membered
heterocyclyl, five- or six-membered heteroaryl, or OR.sup.10, or
together with the nitrogen atom form a pyrrolidine, piperidine,
morpholine, or pyrazine ring; [0251] wherein R.sup.10 is hydrogen,
C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.3-6
cycloalkyl, (C.sub.3-6 cycloalkyl)methyl, phenyl, benzyl, five- or
six-membered heterocyclyl, or five- or six-membered heteroaryl;
[0252] or wherein W.sup.7 or W.sup.8 together form a five- or
six-membered aryl, carbocyclic, heterocyclic, or heteroaryl ring
fused with the B ring; [0253] wherein X.sup.2' is hydrogen,
hydroxy, or OR.sup.2'; [0254] wherein X.sup.3' is hydrogen,
hydroxy, or OR.sup.3'; [0255] wherein X.sup.5 is
C(.dbd.X.sup.4)X.sup.10R; [0256] wherein X.sup.4 is O or H.sub.2;
[0257] wherein X.sup.10 is C(H)NR.sup.11R.sup.12, NR.sup.1, or S;
[0258] wherein R.sup.11 and R.sup.12 each is independently
hydrogen, C.sub.1-4 alkyl, C.sub.2-C.sub.2-3 alkenyl, C.sub.2-3
alkynyl, C.sub.3-6 cycloalkyl, phenyl, benzyl, or acetyl, or
together with the nitrogen atom form an aziridine, azetidine,
pyrrolidine, piperidine, morpholine, [0259] or pyrazine ring;
[0260] wherein R and R.sup.1 each is independently hydrogen,
C.sub.1-8 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.3-6
cycloalkyl, C.sub.6-10 aryl, five-to-ten-membered heteroaryl, five-
to ten-membered heterocyclyl, C.sub.1-8 acyl, or
[(S)-2-aminobutanoic acid]-4-yl, [0261] wherein any alkyl, alkenyl,
alkynyl, cycloalkyl, heterocyclyl, aryl or heteroaryl ring is
optionally substituted with one or more of fluoro, chloro, bromo,
iodo, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl,
methoxy, ethoxy, trifluoromethoxy, trifluoromethyl, hydroxy,
thiomethyl, cyano, NR.sup.8R.sup.9, --N(H)C(.dbd.O)X.sup.11,
acetyl, carboxy, carboxy(C.sub.1-4 alkyl), or CONR.sup.8R.sup.9;
[0262] wherein X.sup.11 is C.sub.1-6 alkyl, C.sub.2-6 alkenyl,
C.sub.2-6 alkynyl, C.sub.3-6 cycloalkyl, (C.sub.3-6
cycloalkyl)methyl, phenyl, benzyl, OR.sup.10, or NR.sup.8R.sup.9;
and [0263] wherein one of R.sup.2, R.sup.2' (if it exists), and
R.sup.3' (if it exists), comprises a linker component, wherein the
linker component comprises a linker moiety bonded to a fluorophore
moiety, and the other existing of R.sup.2, R.sup.2', and R.sup.3'
are hydrogen, and wherein R.sup.2, when not hydrogen, substitutes a
hydrogen atom of the B ring or a ring fused to the B ring or a
functional group covalently bound to the B ring or a ring fused to
the B ring.
[0264] Another exemplary embodiment may be directed to a
fluorescent detection analyte having the structure of Formula
(II):
##STR00007## [0265] wherein any two of R.sup.2, R.sup.2', and
R.sup.3' comprises hydrogen and the third comprises a linker
component, wherein the linker component comprises a linker moiety
bonded to a fluorophore moiety.
[0266] Another exemplary embodiment may be directed to a
fluorescent detection analyte having the structure of Formula
(III):
##STR00008## [0267] wherein any two of R.sup.2, R.sup.2', and
R.sup.3' comprises hydrogen and the third comprises a linker
component, wherein the linker component comprises a linker moiety
bonded to a fluorophore moiety.
[0268] Another exemplary embodiment may be directed to a
fluorescent detection analyte having the structure of Formula
(IV):
##STR00009## [0269] wherein any two of R.sup.2, R.sup.2', and
R.sup.3' comprises hydrogen and the third comprises a linker
component, wherein the linker component comprises a linker moiety
bonded to a fluorophore moiety and wherein R is hydrogen, C.sub.1-8
alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.3-6 cycloalkyl,
C.sub.6-10 aryl, five-to-ten-membered heteroaryl, five- to
ten-membered heterocyclyl, C.sub.1-8 acyl, or [(S)-2-aminobutanoic
acid]-4-yl.
[0270] Another exemplary embodiment may be directed to a
fluorescent detection analyte having the structure of Formula
(V):
##STR00010## [0271] wherein any two of R.sup.2, R.sup.2', and
R.sup.3' comprises hydrogen and the third comprises a linker
component, wherein the linker component comprises a linker moiety
bonded to a fluorophore moiety; [0272] wherein R and R.sup.1 are
each independently hydrogen, C.sub.1-8 alkyl, C.sub.2-6 alkenyl,
C.sub.2-6 alkynyl, C.sub.3-6 cycloalkyl, C.sub.6-10 aryl,
five-to-ten-membered heteroaryl, five- to ten-membered
heterocyclyl, C.sub.1-8 acyl, or [(S)-2-aminobutanoic acid]-4-yl;
and [0273] wherein X.sup.4 is O or H.sub.2.
[0274] As noted above, each of the detection analytes according to
Formulas (I)-(V) includes a linker moiety. In certain embodiments
associated with Formulas (I)-(V), the linker moiety comprises a
structure as illustrated in Formula (VI) or Formula (VII):
##STR00011## [0275] wherein when R.sup.2 comprises the linker
component, the linker moiety may alternatively comprise a structure
as illustrated in Formula (VIII):
[0275] ##STR00012## [0276] wherein the (CH.sub.2).sub.n group of
Formula (IV) or the (CH.sub.2), group of Formula (V) comprises the
site of covalent attachment at one of R.sup.2, R.sup.2', and
R.sup.3' of Formulas (I), (II), and (III); [0277] wherein X is
CH.sub.2 or O; [0278] wherein X.sup.1 is N--H, N--CH.sub.3, O, or
S; [0279] wherein X.sup.2 is N--H, N--CH.sub.3, or, when r is 0 and
X.sup.1 is N--H or N-Me, X.sup.2 may alternatively be O; [0280]
wherein X.sup.3 is NH or O; [0281] wherein Z.sup.1 is a carbonyl,
thiocarbonyl, or sulfonyl group; [0282] wherein Y is a covalent
bond that binds the linker moiety to the fluorophore moiety, or
(CH.sub.2).sub.n wherein the last CH.sub.2 group in the chain (when
n is not 0) is farthest from Z.sup.1 is covalently bound to the
fluorophore moiety, or [0283] (CH.sub.2).sub.n--N(H)--Z.sup.2, or
is of the chemical structure:
[0283] ##STR00013## [0284] wherein the oxygen atom end is
covalently bound to Z.sup.1; [0285] wherein each R.sup.5 and
R.sup.6 is independently H, methyl, or together are
(CH.sub.2).sub.q; [0286] wherein q is 1, 2, or 3; [0287] wherein p
is 1 or 2; and [0288] wherein Z.sup.2 is a carbonyl or thiocarbonyl
group covalently bound by its carbon atom to the fluorophore
moiety, or a sulfonyl group covalently bound by sulfur atom to the
fluorophore moiety; [0289] wherein each n is independently 0, 1, 2,
3, 4, or 5; [0290] wherein s is 1, 2, or 3; [0291] wherein m is 1,
2, or 3; [0292] wherein each r is independently 0 or 1; [0293]
wherein the fluorophore moiety is a structure selected from the
group of chemical structures consisting of:
[0293] ##STR00014## ##STR00015## ##STR00016## ##STR00017## [0294]
wherein * represents the position at which the linker moiety is
covalently bound to the fluorophore moiety; [0295] wherein A.sup.-
is a PF6.sup.-, trifluoroacetate, acetate, or halide anion; [0296]
wherein B.sup.+ is a sodium, potassium, cesium, ammonium, or
.sup.+N(R.sup.4).sub.4 cation; and [0297] wherein each R.sup.4 is
independently H or C.sub.1-4 alkyl.
[0298] An exemplary detection analyte embodiment of any of Formulas
(I)-(V) comprises a fluorophore moiety mixture that is a
fluorophore regioisomer pair, such as one of the pairs (b) and (c);
(b) and (n); (d) and (e); (f) and (g); (h) and (i); and (j) and
(k).
[0299] An exemplary detection analyte embodiment of any of Formulas
(I)-(V) comprises a fluorophore moiety that is a salt mixture
having more than one kind of anion A.sup.- or cation B.sup.+; for
example, an exemplary detection analyte embodiment may comprise a
fluorophore moiety (a) salt mixture wherein A.sup.- is a PF6.sup.-
and trifluoroacetate anion mixture.
[0300] Another exemplary embodiment may be directed to a
fluorescent detection analyte selected from the group consisting
of: [0301] Sinefungin Probe 1A, Sinefungin Probe 1B, Sinefungin
Probe 2A,
[0301] ##STR00018## ##STR00019## ##STR00020## ##STR00021##
##STR00022## [0302] wherein any A.sup.- is PF6.sup.-, halide,
acetate, or trifluoroacetate, or a mixture thereof.
[0303] Another exemplary embodiment may be directed to a
fluorescent detection analyte selected from the group consisting
of:
Thioadenosine Probe 1 and
##STR00023##
[0305] For all exemplary embodiments herein, the drawings that
include "4.fwdarw." and "2.fwdarw." elements represent a
regioisomeric mixture wherein each of the two groups with undefined
covalent attachment to the phenyl ring may be covalently bound at
either the "2-" or "4-" position as indicated, with the other of
the two groups is covalently bound at the other of the "2-" or "4-"
positions of the phenyl ring. The regioisomers may or may not be
present in equal amounts in the mixture.
[0306] Another exemplary embodiment may be directed to a
fluorescent detection analyte selected from the group consisting
of:
Aza-adenosine Probe 1, Aza-adenosine Probe 3,
##STR00024## ##STR00025##
[0308] Another exemplary embodiment may be directed to a
fluorescent detection analyte selected from the group consisting
of:
(Aza-adenosine Probe 2), (Aza-adenosine Probe 4),
##STR00026## ##STR00027## ##STR00028##
[0310] Detection analytes of the exemplary embodiments comprising a
sinefungin moiety covalently bound to the linker moiety at its 2'-
or 3'-hydroxy oxygen were prepared generally according to the
synthetic scheme described in FIG. 1, wherein the N-protection
reaction of Step 1, the O-derivatization reaction of Step 2, the
deprotection reaction of Step 3, and the fluorophore moiety
attachment reaction of Step 4 are carried out by methods known in
the art to provide a fluorescent detection analyte product mixture
comprising at least one compound selected from the group consisting
of a Compound 1A, a Compound Regioisomer Mixture 1B, and a Compound
2. The N-protection reactions of Step 1 include but are not limited
to tert-butoxycarbonyl (Boc) protections that are carried out by
contacting sinefungin (Compound 3) with suitable amounts of
di-tert-butyl dicarbonate (Boc.sub.2O) and a suitable base such as
sodium hydroxide in a solvent or solvent mixture such as
water-dioxane. The O-derivatization reactions of Step 2 may take
place by contacting an intermediate Compound 4 with an appropriate
acetylene electrophile and a suitable base in a suitable solvent or
solvent mixture to provide an intermediate comprising at least one
compound selected from the group consisting of a Compound 5 and a
Compound 6. The deprotection reactions of Step 3 may be carried out
with known conditions suitable for removal of the
nitrogen-protecting groups to provide an intermediate comprising at
least one compound selected from the group consisting of a Compound
7 and a Compound 8. For example, Boc protecting groups may be
removed by contacting an appropriately-protected intermediate
prepared by Step 2 with a suitable amount of an acid such as
trifluoroacetic acid (TFA). The fluorophore moiety attachment
reactions of Step 4 may subsequently be carried out by contacting
an intermediate prepared by Step 3 with a reagent comprising an
appropriate fluorophore covalently linked to an azide group in a
reaction mixture further comprising suitable amounts of copper(II)
sulfate, sodium ascorbate, and a suitable solvent or solvent system
such as water-DMF to provide a fluorescent detection analyte
product comprising at least one compound selected from the group
consisting of a Compound 1A, a Compound Regioisomer Mixture 1B, and
a Compound 2.
[0311] Detection analytes of the exemplary embodiments comprising a
sinefungin moiety covalently bound to the linker moiety at its base
ring are prepared generally according to the synthetic scheme
illustrated in FIG. 9, wherein the syntheses proceed through
Compound 25. The synthesis of Compound 25 is described by Rapoport
et al in the Journal of Organic Chemistry, 1990, 55, 948-955.
Preparation of various base ring portions of the sinefungin moiety
wherein various linker moieties or a linker moiety precursors are
covalently bound to the base ring portion are generally illustrated
in FIG. 9A.
[0312] Detection analytes of the exemplary embodiments comprising a
sinefungin moiety are alternatively prepared using other methods
known in the art, including those that comprise the assembly of
sinefungin probes comprising linker moieties having the general
structures represented by Formulas (VI), (VII), and (VIII).
[0313] Detection analytes of the exemplary embodiments comprising a
thioadenosine moiety were prepared generally according to the
synthetic scheme illustrated in FIG. 5. Commercially-available
2-chloroadenosine may be subjected to chlorination conditions to
provide the 2,5'-dichloro-5'-deoxy-adenosine (Compound 14), which
may be subsequently reacted with a thiol or thiolate anion to
provide the corresponding S-substituted-2-chloro-5'-thioadenosine
intermediate (Compound 15). The
S-substituted-2-chloro-5'-thioadenosine intermediate was treated
with a reagent comprising a linker moiety or a linker moiety
precursor of Formula (VIII), the nucleophilic end (X.sup.1--) of
which reacts with the S-substituted-2-chloro-5'-thioadenosine
intermediate at the adenine-2-carbon position of the adenine
portion of the nucleoside moiety, displacing the 2-chloro group,
forming the Boc-protected intermediate of the general structure
represented as Compound 16. Deprotection and subsequent addition of
the fluorophore reagent under appropriate conditions afforded
thioadenosine detection analytes of the general structure
represented as Compound 17.
[0314] Detection analytes of the exemplary embodiments comprising a
thioadenosine moiety are alternatively prepared generally according
to the synthetic scheme illustrated in FIG. 6. 2-Chloroadenosine is
treated with a reagent comprising a linker moiety or a linker
moiety precursor of Formula (VIII), the nucleophilic end
(X.sup.1--) of which reacts with 2-chloroadenosine at the
adenine-2-carbon position, displacing the 2-chloro group, forming
the Boc-protected intermediate of the general structure represented
as Compound 18. Subjection of intermediates of the general
structure represented as Compound 18 to chlorination conditions
afford the 5'-chloro intermediates of the general structure
represented as Compound 19. Addition of the appropriate thiol or
thiolate provides intermediates of the general structure
represented as Compound 16, which may be carried forward to the
corresponding detection analytes according to the general route
described above.
[0315] Detection analytes of the exemplary embodiments comprising a
thioadenosine moiety are alternatively prepared using other methods
known in the art, including those that comprise the assembly of
thioadenosine probes comprising linker moieties having the general
structures represented by Formulas (VI), (VII), and (VIII).
[0316] Detection analytes of the exemplary embodiments comprising
an aza-adenosine moiety are prepared generally according to the
synthetic schemes illustrated in FIGS. 7 and 8.
[0317] Detection analytes of the exemplary embodiments comprising
an aza-adenosine moiety are alternatively prepared using other
methods known in the art, including those that comprise the
assembly of aza-adenosine probes comprising linker moieties having
the general structures represented by Formulas (VI), (VII), and
(VIII).
[0318] Unless otherwise defined herein, scientific and technical
terms used in connection with the exemplary embodiments shall have
the meanings that are commonly understood by those of ordinary
skill in the art.
[0319] Further, unless otherwise required by context, singular
terms shall include pluralities and plural terms shall include the
singular. Generally, nomenclature used in connection with, and
techniques of chemistry and molecular biology described herein are
those well known and commonly used in the art.
[0320] While specific embodiments of the invention have been
described in detail, it will be appreciated by those skilled in the
art that various modifications and alternatives to those details
could be developed in light of the overall teachings of the
disclosure. Accordingly, the particular arrangements disclosed are
meant to be illustrative only and not limiting as to the scope of
the invention which is to be given the full breadth of the claims
appended and any and all equivalents thereof.
EXAMPLES
[0321] Liquid chromatography-mass spectra (LC/MS) were obtained
using an Agilent LC/MSD G1946D or an Agilent 1100 Series LC/MSD
Trap G1311A. Quantifications were obtained on a Cary 50 Bio
UV-visible spectrophotometer.
[0322] Nuclear magnetic resonance (NMR) spectra were obtained using
a Varian INOVA (400 MHz) nuclear magnetic resonance
spectrometer.
[0323] High performance liquid chromatography (HPLC) analytical
separations were performed on an Agilent 1200 HPLC and followed by
an Agilent Technologies G1315B Diode Array Detector with UV.sub.max
@ 260 nm or 585 nm.
[0324] High performance liquid chromatography (HPLC) preparatory
separations were performed on an Agilent 1100 HPLC G1361A and
followed by an Agilent Technologies G1315B Diode Array Detector
with UV.sub.max @ 260 nm or 585 nm.
Example 1
Preparation of Sinefungin Probe 1A, Sinefungin Probe 2A, and
Sinefungin Probe 1B
##STR00029## ##STR00030##
[0326] The synthetic route including the divergence at Step 3 upon
separation of the regioisomers isolated in Step 2 is illustrated in
FIG. 1.
Step 1: Preparation of
(2S,5S)-6-((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydr-
ofuran-2-yl)-2,5-bis((tert-butoxycarbonyl)amino)hexanoic acid
(Compound 4)
##STR00031##
[0328] The title intermediate Compound 4 was prepared by reacting
sinefungin (Compound 3, 5 mg, 0.013 mmol) in 1M NaOH (32 .mu.L,
0.032 mmol) and water (268 .mu.L) with di-tert-butyl dicarbonate (7
mg, 0.032 mmol) in 1,4-dioxane (300 .mu.L). The reaction was
stirred at room temperature overnight. LC/MS (Gemini C18, 3.mu.,
2.0.times.50 mm, 400 .mu.L/min, A: 90/10/0.01 H.sub.2O/MeOH/HOAc, B
90/10/0.01 MeOH/H.sub.2O/HOAc 0-6 min 0-100% B, 6-9 hold at 100% B,
9.1-15 min re-equilibrate at 0% B) showed mostly desired
intermediate (retention time 7.7 min, ESI m/z 582, ESI.sup.- m/z
580) with a small amount of mono-protected intermediate (retention
time 4.4 min, ESI.sup.+ m/z 482, ESE m/z 480). All solvent was
removed, water was added and the pH was found to be about 8. 1M HCl
was added to bring the pH to about 7. An extraction was done with
ethyl acetate to remove impurities. Water was removed to give the
title intermediate 4 (6 mg, 79% yield) as a white solid.
Step 2: Preparation and separation of a product mixture comprising
(2S,5S)-6-((2R,3R,4R,5R)-5-(6-amino-9H-purin-9-yl)-3-hydroxy-4-(prop-2-yn-
-1-yloxy)tetrahydrofuran-2-yl)-2,5-bis((tert-butoxycarbonyl)amino)hexanoic
acid (Compound 5) and
(2S,5S)-6-((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-4-hydroxy-3-(prop-2-yn-
-1-yloxy)tetrahydrofuran-2-yl)-2,5-bis((tert-butoxycarbonyl)amino)hexanoic
acid (Compound 6)
##STR00032##
[0330] To a mixture consisting of
(2S,5S)-6-((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydr-
ofuran-2-yl)-2,5-bis((tert-butoxycarbonyl)amino)hexanoic acid
(Compound 4, 6 mg, 0.010 mmol) in 0.6 M KOH (83 .mu.L, 0.050 mmol)
was added 18-crown-6 (7 mg, 0.026 mmol) in 1,4-dioxane (83 .mu.L)
followed by propargyl bromide (80% in toluene, 2.2 .mu.L, 0.020
mmol). The reaction mixture was stirred at room temperature
overnight. HPLC (Gemini C18, 110 .ANG., 5.mu., 250.times.4.6 mm, 1
mL/min, 5/95/0.05.fwdarw.50/50/0.05 ACN/H.sub.2O/TFA) showed a
mixture of mostly starting material (20.2 min, 35%), and two
products possessing the mass of the desired mono-propargylated
reaction product (21.9 min, 29%; 22.6 min, 6%) and undesired
di-propargyl product (25.0 min, 14%). LC/MS (Gemini C18, 3.mu.,
2.0.times.50 mm, 400 .mu.L/min, A: 90/10/0.01 H.sub.2O/MeOH/HOAc, B
90/10/0.01 MeOH/H.sub.2O/HOAc 0-6 min 0-100% B, 6-9 hold at 100% B,
9.1-15 min re-equilibrate at 0% B) confirmed the peak identities of
starting Compound 4 (7.4 min, ESI.sup.+ m/z 582), Compound 5/6
mixture (7.6 min, ESI.sup.+ m/z 620), di-propargyl (7.9 min,
ESI.sup.+ m/z 658). Prep HPLC (Gemini C18, 110 .ANG., 10.mu.,
250.times.21.2 mm, 20 mL/min, 15/85/0.05.fwdarw.90/10/0.05
ACN/H.sub.2O/TFA) was done to isolate the 4 aforementioned
materials (starting material and three products). The major
mono-propargyl product solution was concentrated to remove the ACN
and then lyophilized to afford its purified isomer (Compound 5, 2
mg, 32% yield) as a white powder; LC/MS (same conditions as above)
7.5 min, ESI.sup.+ m/z 620. The minor mono-propargyl product
solution was concentrated to remove the ACN and then lyophilized to
afford its purified isomer (Compound 6, <1 mg, <16% yield) as
a white powder; LC/MS (same conditions as above) 7.0 min, ESI.sup.+
m/z 620, ESI.sup.- m/z 618.
Step 3a: Preparation of
(2S,5S)-2,5-diamino-6-((2R,3R,4R,5R)-5-(6-amino-9H-purin-9-yl)-3-hydroxy--
4-(prop-2-yn-1-yloxy)tetrahydrofuran-2-yl)hexanoic acid (Compound
7)
##STR00033##
[0332] Compound 5 (1.0 mg, 0.0016 mmol) was deprotected using
trifluoroacetic acid (TFA, 500 .mu.L, 6.5 mmol) by stirring at room
temperature for 30 min. The TFA was removed and the deprotected
product was carried onto the next reaction step without further
purification or characterization.
Step 3b: Preparation of
(2S,5S)-2,5-diamino-6-((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-4-hydroxy--
3-(prop-2-yn-1-yloxy)tetrahydrofuran-2-yl)hexanoic acid (Compound
8)
##STR00034##
[0334] Compound 6 (<1.0 mg, <0.0016 mmol) was deprotected
using trifluoroacetic acid (TFA, 500 .mu.L, 6.5 mmol) by stifling
at room temperature for 30 min. The TFA was removed and the
deprotected product was carried onto the next reaction step without
further purification or characterization.
Step 4a(i): Preparation of Sinefungin Probe 1A (Compound 1A)
[0335] To a mixture consisting of Compound 7 (.about.671 .mu.g,
0.0016 mmol) in water (100 .mu.L) with sodium ascorbate (1 mg,
0.005 mmol) in water (200 .mu.L) was added copper(II) sulfate (500
.mu.g, 0.0031 mmol) in water (100 .mu.L) and CAL Fluor.RTM. Red 610
azide (Biosearch Technologies, Inc. Lot #MVR1716, 1.4 mg, 0.0016
mmol) in DMF (400 .mu.L). The reaction mixture was stirred at room
temperature overnight. HPLC (Gemini C18, 110 .ANG., 5.mu.,
250.times.4.6 mm, 1 mL/min, 5/95/0.05.fwdarw.90/10/0.05
ACN/H.sub.2O/TFA) showed mainly one product (13.2 min, 21%) and
starting fluorophore azide (19.6 min, 60%). Prep HPLC (Gemini C18,
110 .ANG., 10.mu., 250.times.21.2 mm, 20 mL/min,
15/85/0.05.fwdarw.90/10/0.05 ACN/H.sub.2O/TFA) was done to purify
the product which eluted from the column at around 9 min. The
product solution was concentrated to remove the ACN and then
lyophilized to afford the title detection analyte compound (248
.mu.g as quantified by UV (MeOH, extinction coefficient 91,000),
13% yield) as a dark purple solid; LC/MS (Gemini C18, 3.mu.,
2.0.times.50 mm, 400 .mu.L/min, A: 90/10/0.01 H.sub.2O/MeOH/HOAc, B
90/10/0.01 MeOH/H.sub.2O/HOAc 0-6 min 0-100% B, 6-9 hold at 100% B,
9.1-15 min re-equilibrate at 0% B) consistent with that expected
for pure title detection analyte compound (5.2 min, ESI.sup.+
m/z=1162/2=581).
Step 4a(ii): Preparation of Sinefungin Probe 1B (Compound
Regioisomer Mixture 1B)
[0336] To a mixture consisting of Compound 7 (.about.1 mg, 0.0016
mmol) in water (100 .mu.L) with sodium ascorbate (1 mg, 0.005 mmol)
in water (200 .mu.L) was added copper(II) sulfate (500 .mu.g,
0.0031 mmol) in water (100 .mu.L) and Azide-Fluor585 (Click
Chemistry Tools, Catalog # AZ110, 1.3 mg, 0.0016 mmol) in DMF (600
.mu.L). The reaction mixture was stirred at room temperature
overnight. HPLC (Gemini C18, 110 .ANG., 5.mu., 250.times.4.6 mm, 1
mL/min, 5/95/0.05.fwdarw.90/10/0.05 ACN/H.sub.2O/TFA) showed mainly
one product mixture (12.3 min and 13 min, 40%) and starting
fluorophore azide mixture (18.7 min and 20.5 min, 50%). The
reaction mixture was allowed to continue stifling a second night.
Prep HPLC (Gemini C18, 110 .ANG., 10.mu., 250.times.21.2 mm, 20
mL/min, 15/85/0.05.fwdarw.90/10/0.05 ACN/H.sub.2O/TFA) was done to
purify the product which eluted from the column at around 8-9 min.
The product solution was concentrated to remove the ACN and then
lyophilized to afford the title detection analyte compound, which
was taken up in methanol (500 .mu.L) and quantified by UV (895
.mu.g/mL, or 448 rig); LC/MS (Gemini C18, 3.mu., 2.0.times.50 mm,
400 .mu.L/min, A: 90/10/0.01 H.sub.2O/MeOH/HOAc, B 90/10/0.01
MeOH/H.sub.2O/HOAc 0-6 min 0-100% B, 6-9 hold at 100% B, 9.1-15 min
re-equilibrate at 0% B) consistent with that expected for pure
title detection analyte compound regioisomer mixture (5.4 min,
ESI+m/z=1226/2=613).
Step 4b: Preparation of Sinefungin Probe 2A (Compound 2A)
[0337] To a mixture consisting of Compound 8 (<671 .mu.g,
<0.0016 mmol) in water (100 .mu.L) with sodium ascorbate (1 mg,
0.005 mmol) in water (200 .mu.L) was added copper(II) sulfate (500
.mu.g, 0.0031 mmol) in water (100 .mu.L) and CAL Fluor.RTM. Red 610
azide (Biosearch Technologies, Inc. Lot #MVR1716, 1.4 mg, 0.0016
mmol) in DMF (400 .mu.L). The reaction mixture was stirred at room
temperature overnight. HPLC (Gemini C18, 110 .ANG., 5.mu.,
250.times.4.6 mm, 1 mL/min, 5/95/0.05.fwdarw.90/10/0.05
ACN/H.sub.2O/TFA) showed mainly one product (13.1 min, 3%) and
starting fluorophore azide (19.4 min, 75%). Prep HPLC (Gemini C18,
110 .ANG., 10.mu., 250.times.21.2 mm, 20 mL/min,
15/85/0.05.fwdarw.90/10/0.05 ACN/H.sub.2O/TFA) was done to purify
the product which eluted from the column at around 8.6 min. The
product solution was concentrated to remove the ACN and then
lyophilized to afford the title detection analyte compound (12
.mu.g as quantified by UV (MeOH, extinction coefficient 91,000),
.about.0.6% yield) as a dark purple solid; LC/MS (Gemini C18,
3.mu., 2.0.times.50 mm, 400 .mu.L/min, A: 90/10/0.01
H.sub.2O/MeOH/HOAc, B 90/10/0.01 MeOH/H.sub.2O/HOAc 0-6 min 0-100%
B, 6-9 hold at 100% B, 9.1-15 min re-equilibrate at 0% B)
consistent with that expected for pure title detection analyte
compound (5.1 min, ESI.sup.+ m/z=1162/2=581).
Example 2
Preparation of Sinefungin Probe 3
##STR00035##
[0338] Step 1: Preparation of
(2S,5S)-6-((2R,3R,4R,5R)-4-(allyloxy)-5-(6-amino-9H-purin-9-yl)-3-hydroxy-
tetrahydrofuran-2-yl)-2,5-bis((tert-butoxycarbonyl)amino)hexanoic
acid (Compound 10)
##STR00036##
[0340] To a mixture consisting of
(2S,5S)-6-((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydr-
ofuran-2-yl)-2,5-bis((tert-butoxycarbonyl)amino)hexanoic acid
(Compound 4, 5 mg, 0.0086 mmol) in 0.6 M KOH (72 .mu.L, 0.043 mmol)
was added 18-crown-6 (9 mg, 0.034 mmol) in 1,4-dioxane (72 .mu.L)
followed by allyl bromide (1.7 .mu.L, 0.020 mmol). The reaction
mixture was stirred at room temperature over the weekend. HPLC
(Gemini C18, 110 .ANG., 5.mu., 250.times.4.6 mm, 1 mL/min,
5/95/0.05.fwdarw.50/50/0.05 ACN/H.sub.2O/TFA) showed a mixture of
mostly starting material (19.7 min, 45%), and two products
possessing the mass of the desired mono-allylated reaction product
(21.9 min, 34%; 22.6 min, 9%) and undesired di-allyl product (25.6
min, 7%). The desired (major) mono-allyl ether product was isolated
by prep HPLC (Gemini C18, 110 .ANG., 10.mu., 250.times.21.2 mm, 20
mL/min, 5/95/0.05.fwdarw.50/50/0.05 ACN/H.sub.2O/TFA)--peak
collected at about 13.5 min. The major mono-allyl ether product
solution was concentrated to afford the title intermediate
(Compound 10, 2.3 mg, 43% yield) as a colorless solid; LC/MS
(Gemini C18, 3.mu., 2.0.times.50 mm, 400 .mu.L/min, A: 90/10/0.01
H.sub.2O/MeOH/HOAc, B 90/10/0.01 MeOH/H.sub.2O/HOAc 0-6 min 0-100%
B, 6-9 hold at 100% B, 9.1-15 min re-equilibrate at 0% B) 6.2 min,
ESI m/z 622, ESI.sup.- m/z 620.
Step 2: Preparation of
(2S,5S)-6-((2R,3R,4R,5R)-5-(6-amino-9H-purin-9-yl)-3-hydroxy-4-(2-oxoetho-
xy)tetrahydrofuran-2-yl)-2,5-bis((tert-butoxycarbonyl)amino)hexanoic
acid (Compound 11)
##STR00037##
[0342] To a 7-mL scintillation vial containing Compound 10 (2.0 mg,
0.0032 mmol) was added sequentially tetrahydrofuran (200 .mu.L),
water (132 .mu.L), sodium periodate (3.3 mg, 0.015 mmol), and a
mixture consisting of potassium osmium tetroxide dihydrate (68
.mu.g, 0.00018 mmol) and water (68 .mu.L). The reaction mixture was
stirred overnight in the dark at room temperature. HPLC (Gemini
C18, 110 .ANG., 5.mu., 250.times.4.6 mm, 1 mL/min,
5/95/0.05.fwdarw.50/50/0.05 ACN/H.sub.2O/TFA) showed complete
disappearance of starting material. The reaction mixture was placed
in the freezer overnight and the desired product isolated the
following day by prep HPLC (Gemini C18, 110 .ANG., 10.mu.,
250.times.21.2 mm, 20 mL/min, 5/95/0.05.fwdarw.50/50/0.05
ACN/H.sub.2O/TFA)--split peak collected at about 20.5 min. The
relevant fractions were combined and concentrated to afford the
title intermediate (1 mg, 50% yield) as a clear solid; LC/MS
ESI.sup.+ m/z 624, ESI.sup.- m/z 622.
Step 3: Preparation of bis-BOC-protected
sinefungin-2'-O-(amine-PEG.sub.3-aminosulfonyl linker)-Fluor585
(Compound Regioisomer Mixture 12)
##STR00038##
[0344] To a 7-mL scintillation vial containing Compound 11 (500
.mu.g, 0.00080 mmol) was added a mixture consisting of
Sulforhodamine 101-PEG.sub.3-amine (Biotium, 675 .mu.g, 0.00084
mmol) and methanol (135 .mu.L). The reaction mixture was stirred in
the dark at room temperature for 4.5 hours. HPLC (Gemini C18, 110
.ANG., 5.mu., 250.times.4.6 mm, 1 mL/min,
5/95/0.05.fwdarw.90/10/0.05 ACN/H.sub.2O/TFA) showed only starting
materials present. The reaction mixture was subsequently treated
with a mixture consisting of sodium cyanoborohydride (1 mg, 0.016
mmol) in pH 5.5 sodium acetate buffer (50 .mu.L) followed by
additional buffer rinse (50 .mu.L). The reaction mixture was
stirred in the dark at room temperature overnight. Analysis by HPLC
(same method as above) showed a product peak. Prep HPLC (Gemini
C18, 110 .ANG., 10.mu., 250.times.21.2 mm, 20 mL/min,
30/70/0.05.fwdarw.90/10/0.05 ACN/H.sub.2O/TFA) resulted the
collection of two products, one eluting at about 8 minutes, and
another at about 9 minutes. The combined products were concentrated
and lyophilized to afford the title intermediate (<1 mg); LC/MS
ESI.sup.+ m/z=1416/2=708).
Step 4: Preparation of Sinefungin Probe 3 (Compound Regioisomer
Mixture 9)
[0345] To an ice-chilled 2-mL amber vial containing Compound
Regioisomer Mixture 12 (<1 mg) was added a mixture consisting of
1:1 v/v trifluoroacetic acid-dichloromethane (50 .mu.L). The
reaction mixture was allowed to stand cold for five minutes and was
subsequently concentrated by blowing off the solvent mixture with a
nitrogen stream. The concentrate was taken up into methanol and was
purified by prep HPLC (Gemini C18, 1101, 10.mu., 250.times.21.2 mm,
20 mL/min, 5/95/0.05.fwdarw.90/10/0.05 ACN/H.sub.2O/TFA over 20
min). The two major (product regioisomers--LC/MS ESI.sup.+ m/z 608
for both peaks) peaks were collected, concentrated, and lyophilized
to afford the title compound (138 .mu.g--quantified by UV in
methanol using 80,000 extinction coefficient) as a dark purple
solid.
Example 3
Preparation of Sinefungin Probe 4 (Compound 13)
##STR00039##
[0347] The title detection analyte compound was prepared from
Compound 7 (Example 1, Step 3a) by coupling with
6-carboxyfluorescein-PEG.sub.3 azide (Berry & Associates, CAS
#412319-45-0, Catalog No. FF6110) using the click chemistry methods
reported herein; LC/MS (Gemini C18, 3.mu., 2.0.times.50 mm, 400
.mu.L/min, A: 90/10/0.01 H.sub.2O/MeOH/HOAc, B 90/10/0.01
MeOH/H.sub.2O/HOAc 0-6 min 0-100% B, 6-9 hold at 100% B, 9.1-15 min
re-equilibrate at 0% B) 4.3 min (100% purity), ESI.sup.+ m/z 498
(z=2; expected molecular mass: 996).
Example 4
Preparation of Thioadenosine Probe 1
##STR00040##
[0348] Route I
Step 1: Preparation of
(2R,3R,4S,5S)-2-(6-amino-2-chloro-9H-purin-9-yl)-5-(chloromethyl)tetrahyd-
rofuran-3,4-diol, also called
5'-deoxy-5'-(chloro)-2-chloroadenosine Compound 14
##STR00041##
[0350] To a flask containing hexamethylphosphoramide (10 mL) was
added thionyl chloride (1.5 mL, 2.4 g, 21 mmol) at 0.degree. C.
with stifling. The solution was allowed to reach room temperature
before 2-chloroadenosine (Toronto Research Chemicals, 1.0 g, 3.3
mmol) was added portionwise. The orange suspension was stirred
under nitrogen at room temperature overnight. The mixture was
poured into water (90 mL) and the resulting mixture was
subsequently filtered. The yellow solids were washed with water
then stirred in 1N NH.sub.4OH (50 mL) for two hours. The mixture
was filtered then washed with cold water. The white solid was dried
under high vacuum to obtain the title intermediate (0.80 g, 76%
yield); .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 3.83 (dd, 1H),
3.91 (dd, 1H), 4.07 (dd, 1H), 4.16 (dd, 1H), 4.65 (dd, 1H), 5.46
(d, 1H), 5.60 (d, 1H), 5.84 (d, 1H), 7.84 (br s, 2H), 8.35 (s, 1H);
MS (ESI.sup.+): m/z 291, 293 (M+1).
Step 2: Preparation of
(2R,3R,4S,5S)-2-(6-amino-2-chloro-9H-purin-9-yl)-5-((methylthio)methyl)te-
trahydrofuran-3,4-diol, also called
5'-deoxy-5'-(methylthio)-2-chloroadenosine (Compound 15A)
##STR00042##
[0352] Compound 14 (320 mg, 1.0 mmol) was stirred under nitrogen in
DMF (3 mL) at 0.degree. C. Sodium methanethiolate (77 mg, 1.1 mmol)
was added portionwise and the mixture allowed to reach room
temperature with stifling overnight. The mixture was poured into
saturated aqueous NaCl (30 mL). The pH of the solution was adjusted
to .about.7 with 6N HCl. The solution was diluted with more
saturated NaCl to precipitate a gummy solid. The aqueous portion
was decanted and the remaining solid purified by silica
chromatography (2 to 10% methanol in chloroform). Appropriate
fractions were collected, solvent removed and the title
intermediate obtained as a white solid (0.80 g, 36% yield); MS
(ESI.sup.+): m/z 332.0, 334.0 (M+1) and (ESI.sup.-): m/z 330.0
(M-1).
Step 3: Preparation of tert-butyl
(2-((6-amino-9-((2R,3R,4S,5S)-3,4-dihydroxy-5-((methylthio)methyl)tetrahy-
drofuran-2-yl)-9H-purin-2-yl)amino)ethyl)carbamate, also called
5'-deoxy-5'-(methylthio)-2-(ethylamino-tert-butylcarbamate)-adenosine
(Compound 16A)
##STR00043##
[0354] To a microwave tube containing compound 15A (120 mg, 0.36
mmol) was added tert-butyl N-(2-aminoethyl)carbamate (580 mg, 3.62
mmol). The mixture was heated in a microwave for 20 min. at
120.degree. C. under nitrogen. After cooling to room temperature
the mixture was dissolved in dichloromethane and the crude reaction
mixture was loaded on a 12 g Silicycle column. The residue was then
purified by silica chromatography (0 to 11% 2 to 10% methanol in
dichloromethane). Appropriate fractions were collected, solvent
removed and the title intermediate obtained as a white solid (0.017
g, 11% yield); MS (ESI.sup.+): m/z 456.1 (M+1), 478.0 (M+Na).
Step 4: Preparation of Thioadenosine Probe 1 (Compound 17A)
Deprotection of Compound 16a to Produce Boc-Deprotected Compound
16a Trifluoroacetate Salt
[0355] Compound 16A (11 mg, 0.024 mmol) was treated with an excess
of trifluoroacetic acid (500 .mu.L, 6.5 mmol) at room temperature
in a 7-mL scintillation vial. The resulting cloudy mixture was
clarified partially by the addition of a few drops of methanol
followed by a few drops of dichloromethane, and the reaction was
allowed to carry on for 30 minutes. The solvent was subsequently
blown off and the
5'-deoxy-5'-(methylthio)-2-(1,2-diaminoethyl-1-yl)-adenosine
residue taken up into dichloromethane with a slight methanol
additive to aid in dissolution, and the mixture analyzed by thin
layer chromatography (TLC) and LCMS. TLC: 10:90:0.5 v/v
methanol-dichloromethane-conc. NH.sub.4OH, no starting material
present, product spot R.sub.f=0; LCMS (ESI+) m/z 356 (M+1).
Analysis by HPLC (gradient: 5:95:0.05 to 90:10:0.05
acetonitrile-water-TFA) showed the product comprised two major
entities: 5.8 min (21%), 6.1 min (70%).
[0356] Conjugation of Boc-deprotected Compound 16A trifluoroacetate
salt with Texas Red-X, succinimidyl ester (Life Technologies
Catalog # T-6134) to produce Thioadenosine Probe 1: The
Boc-deprotected Compound 16A trifluoroacetate salt (500 .mu.g,
0.00086 mmol) was dissolved in DMF in a 2-mL amber vial, to which
diisopropylethylamine (50 .mu.L, 0.24 mmol) was added followed by
the Texas Red-X, succinimidyl ester (Invitrogen) solution (1.5 mg
in 100 .mu.L DMF) and 2.times.50 .mu.L DMF rinses. The reaction
mixture was stirred overnight. The crude product was purified by
prep HPLC (5:95:0.05 to 90:10:0.05 acetonitrile-water-TFA afforded
separation of the desired material as shown by a major peak elution
at 12.5 minutes. The purest fraction was concentrated and the
residue taken up in methanol for quantification after the usual
manner. The methanol solution (0.78 mL) concentration was measured
and calculated to be 184 .mu.g/mL, affording the purified
Thioadenosine Probe 1 (free form) with 15.8% yield; HPLC-UV purity:
96.9%; LCMS (ESI.sup.+) m/z 1057.
Route 2
Step 1: Preparation of tert-butyl
(2-((6-amino-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofu-
ran-2-yl)-9H-purin-2-yl)amino)ethyl)carbamate Compound 18
##STR00044##
[0358] A NEAT mixture of 2-chloroadenosine (1.15 g, 8.81 mmol) and
tert-butyl N-(2-aminoethyl)carbamate (3.2 g, 20 mmol) under a
nitrogen stream in a 250-mL round bottom flask was heated to
150.degree. C. for two hours, over which time turned the mixture to
a red oil. The mixture was subsequently cooled to room temperature
and was treated with 9:1 v/v dichloromethane-methanol (about 60 mL)
with stifling, generating an orange precipitate. The precipitate
was collected by filtration and dried to afford the title
intermediate (1.48 g, 91.4% yield) as a white solid; TLC R.sub.f
(9:1 v/v dichloromethane-methanol) 0.26; LC/MS (ESI+) m/z 426.
Step 2: Preparation of tert-butyl
(2-((6-amino-9-((2R,3R,4S,5S)-5-(chloromethyl)-3,4-dihydroxytetrahydrofur-
an-2-yl)-9H-purin-2-yl)amino)ethyl)carbamate Compound 19
##STR00045##
[0360] To a stifling mixture consisting of Compound 18 (1.03 g,
2.42 mmol) and acetonitrile (80 mL) under a nitrogen atmosphere and
cooled over an ice-acetone bath was added thionyl chloride (0.53
mL, 7.3 mmol). The reaction mixture continued stirring cold for 15
minutes and was subsequently treated with pyridine (0.469 g, 4.84
mmol) with dropwise addition. An immediate color change was
observed, and the mixture was stirred cold for another 45 minutes.
The cold bath was removed and the mixture was allowed to stir
overnight at room temperature. The reaction mixture was
concentrated under reduced pressure, and the residue was dissolved
in methanol. The ice bath-cooled mixture under a nitrogen
atmosphere was treated with ammonium hydroxide solution to pH 10.
The resulting mixture was stirred for 1.5 hours, as it was allowed
to warm to room temperature. The mixture was concentrated under
reduced pressure, and the residue was purified by silica column (80
g) chromatography. Elution with a gradient (100% dichloromethane to
10% methanol in dichloromethane), combination of fractions
containing separated desired product, and concentration under
reduced pressure afforded the title intermediate (0.175 g, 16.4%
yield) as a white solid; .sup.1H NMR (400 MHz, DMSO-d.sub.6)
.delta. 7.87 (s, 1H), 6.81 (t, 1H, J=5.5 Hz), 6.73 (s, 2H), 6.26
(t, 1H, J=5.5 Hz), 5.73 (d, 1H, J=5.5 Hz), 5.47 (d, 1H, J=5.9 Hz),
5.36 (d, 1H, J=5.1 Hz), 4.75 (broad m, 1H), 4.20 (m, 1H), 4.01 (m,
1H), 3.90 (dd, 1H, 11.4, 5.2 Hz), 3.79 (m, 1H), 3.23 (m, 2H), 3.08
(m, 2H), 2.06 (s, 2H), 1.34 (s, 9H); LC/MS (ESI+) m/z 444.
Step 3: Preparation of Compound 16A
[0361] Compound 19 (limiting reagent) is stirred under nitrogen in
DMF at 0.degree. C. Sodium methanethiolate (1.1 molar equivalent)
is added portionwise and the mixture allowed to reach room
temperature with stirring overnight. The reaction mixture is worked
up and the product purified and isolated in a manner similar to
that described for Compound 15A, Route 1, Step 2 above.
Step 4: Preparation of Thioadenosine Probe 1 (Compound 17A)
[0362] The title detection analyte is prepared from Compound 16A
according to the manner described in Route 1, Step 4 above.
Example 5
Preparation of Aza-adenosine Probes 1 and 2
##STR00046##
[0363] A. Preparation of Aza-Adenosine Probe 1
Step 1: Preparation of 2-chloroadenosine-5'-N-ethylcarboxamide
(Compound 20A-iii)
##STR00047##
[0365] To a mixture consisting of
2-chloro-2',3'-O-isopropylideneadenosine-5'-N-ethylcarboxamide (CAS
#120225-75-4, Toronto Research Chemicals, Catalog # C367370, 2 mg,
0.0052 mmol) in methanol (250 .mu.L) was added 0.1 M HCl (250
.mu.L). The reaction was stirred at room temperature overnight. TLC
(5:95 MeOH:CH.sub.2Cl.sub.2) still showed starting material. The
reaction was heated to 50.degree. C. and allowed to react
overnight. TLC showed the starting material was gone. Upon cooling
to room temperature, white solid precipitated. Water was added to
rinse away excess acid followed by a second rinse checking to make
sure the water was neutral. The product was a white solid (1.2 mg,
60% yield as the HCl salt). HPLC (Gemini C18, 110 .ANG., 5.mu.,
250.times.4.6 mm, 1 mL/min, 5/95/0.05.fwdarw.50/50/0.05
ACN/H.sub.2O/TFA) showed the purity to be .gtoreq.99%. LC/MS
(Gemini C18, 3.mu., 2.0.times.50 mm, 400 .mu.L/min, A: 90/10/0.01
H.sub.2O/MeOH/HOAc, B 90/10/0.01 MeOH/H.sub.2O/HOAc 0-6 min 0-100%
B, 6-9 hold at 100% B, 9.1-15 min re-equilibrate at 0% B) confirmed
the peak identity as product (6.3 min, ESL.sup.+ m/z 343, ESE m/z
342).
Step 2: Preparation of tert-butyl
(2-((6-amino-9-42R,3R,4S,5S)-5-(ethylcarbamoyl)-3,4-dihydroxytetrahydrofu-
ran-2-yl)-9H-purin-2-yl)amino)ethyl)carbamate (Compound
21A-iii)
##STR00048##
[0367] To a microwave tube containing Compound 20A-iii is added
tert-butyl N-(2-aminoethyl)carbamate (10-fold molar excess). The
mixture is heated in a microwave for 20 min. at 120.degree. C.
under nitrogen. Workup of the reaction mixture, purification, and
isolation of the title intermediate is carried out in a manner
similar to that of Example 4, ROUTE 1, Step 3 used for Compound
16A.
Step 3: Preparation of Aza-Adenosine Probe 1 (Compound 22A-iii)
[0368] Boc-deprotection of Compound 21A-iii and subsequent
conjugation with Texas Red-X, succinimidyl ester is carried out in
a manner similar to that of Example 4, ROUTE 1, Step 4 for Compound
17A (Thioadenosine Probe 1).
B. Preparation of Aza-Adenosine Probe 2
[0369] Step 1: Preparation of
2-chloro-2',3'-O-isopropylideneadenosine-5'-N-ethylamine (Compound
20A-ii)
##STR00049##
[0370] To a mixture consisting of
2-chloro-2',3'-O-isopropylideneadenosine-5'-N-ethylcarboxamide (2
mg, 0.0052 mmol) in THF (400 .mu.L) cooled to 0.degree. C. was
added borane dimethyl sulfide complex (2 M in THF, 50 .mu.L, 0.1
mmol). The reaction was allowed to slowly warm to room temperature
overnight. The reaction was worked up by cooling it in an ice bath
and adding methanol (1 mL) and stirred for one hour. The reaction
mixture was concentrated, cooled to 0.degree. C. followed by
addition of methanolic HCl (8 .mu.L acetyl chloride, 2 mL MeOH).
The reaction was stirred and allowed to come to room temperature
over two hours. The reaction mixture was concentrated and then
triturated with ether to give an off-white solid (2.3 mg,
quantitative crude yield). HPLC (Gemini C18, 1101, 5.mu.,
250.times.4.6 mm, 1 mL/min, 5/95/0.05.fwdarw.50/50/0.05
ACN/H.sub.2O/TFA) showed starting material was mostly gone and 3
new peaks (6 min, 34%; 8.1 min, 12%; 9 min, 21%). LC/MS (Gemini
C18, 3.mu., 2.0.times.50 mm, 400 .mu.L/min, A: 90/10/0.01
H.sub.2O/MeOH/HOAc, B 90/10/0.01 MeOH/H.sub.2O/HOAc 0-6 min 0-100%
B, 6-9 hold at 100% B, 9.1-15 min re-equilibrate at 0% B) confirmed
the presence of product (0.5 min, 4.2 min ESI.sup.+ m/z 369).
Step 2: Preparation of 2-chloroadenosine-5'-N-ethylamine (Compound
20A-iv)
##STR00050##
[0372] To a mixture consisting of Compound 20A-ii (400 .mu.g,
0.0011 mmol) in methanol (200 .mu.L) was added 0.1 M HCl (200
.mu.L). The reaction was heated to 50.degree. C. and allowed to
proceed overnight. TLC showed the starting material was gone. HPLC
(Gemini C18, 110 .ANG., 5.mu., 250.times.4.6 mm, 1 mL/min,
5/95/0.05.fwdarw.90/10/0.05 ACN/H.sub.2O/TFA) showed mostly one
peak (6.1 min, 72%). Prep HPLC (Gemini C18, 110 .ANG., 10.mu.,
250.times.21.2 mm, 20 mL/min, 5/95/0.05.fwdarw.50/50/0.05
ACN/H.sub.2O/TFA) was done to isolate the product peak. Evaporation
of solvents yielded the product as a white solid (400 .mu.g, 66%
yield as the TFA salt). LC/MS (Gemini C18, 3.mu., 2.0.times.50 mm,
400 .mu.L/min, A: 90/10/0.01 H.sub.2O/MeOH/HOAc, B 90/10/0.01
MeOH/H.sub.2O/HOAc 0-6 min 0-100% B, 6-9 hold at 100% B, 9.1-15 min
re-equilibrate at 0% B) confirmed the peak identity as product
(ESI.sup.+ m/z 329).
[0373] Aza-adenosine Probe 2 is prepared from Compound 20A-iv in a
manner similar to that described in Steps 2 and 3 used for
preparation of Aza-adenine Probe 1.
Example 6
Preparation of Aza-adenosine Probes 3 and 4
##STR00051##
[0374] A. Preparation of Aza-Adenosine Probe 3
Step 1: Preparation of
2-chloro-2',3'-O-isopropylideneadenosine-5'-N-Boc-ornithineamide
t-butyl ester (Compound 20B-i)
##STR00052##
[0376] 2-Chloroadenosine-5'-carboxy-2',3'-O-isopropylidene (CAS
#72209-19-9, 10 mg, 0.028 mmol), Boc-L-Dab-OtBu hydrochloride (17
mg, 0.055 mmol), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide
hydrochloride (11 mg, 0.057 mmol) and N,N-diisopropylethylamine (10
.mu.L, 0.057 mmol) were combined in DMF (300 .mu.L). The reaction
was allowed to proceed overnight at room temperature. HPLC (Gemini
C18, 110 .ANG., 5.mu., 250.times.4.6 mm, 1 mL/min,
5/95/0.05.fwdarw.90/10/0.05 ACN/H.sub.2O/TFA) showed 3 new peaks
(9.9 min, 24%; 10.1 min, 16%; 17.3, 52%). Prep HPLC (Gemini C18,
110 .ANG., 10.mu., 250.times.21.2 mm, 20 mL/min,
5/95/0.05.fwdarw.50/50/0.05 ACN/H.sub.2O/TFA) was done to isolate
the 2 pks close to 10 min together and the 17.3 min pk. LC/MS
(Gemini C18, 3.mu., 2.0.times.50 mm, 400 .mu.L/min, A: 90/10/0.01
H.sub.2O/MeOH/HOAc, B 90/10/0.01 MeOH/H.sub.2O/HOAc 0-6 min 0-100%
B, 6-9 hold at 100% B, 9.1-15 min re-equilibrate at 0% B) showed
the 2 earlier pks were EDC adducts of the starting materials and
confirmed the latter pk as product (ESI.sup.+ m/z 612). The product
solution was concentrated to give the title intermediate (10 mg,
59% yield) as a white solid.
Step 2: Preparation of 2-chloroadenosine-5'-N-ornithineamide
(Compound 20C-iii)
##STR00053##
[0378] To Compound 20B-i (2 mg, 0.0033 mmol) was added 3 M HCl (500
.mu.L, 1.5 mmol) and a few drops methanol for solubility. The
reaction mixture was stirred at room temperature overnight. TLC
(5:95 MeOH:CH.sub.2Cl.sub.2) showed that starting material was gone
and that a new baseline spot had appeared. HPLC (Gemini C18, 110
.ANG., 5.mu., 250.times.4.6 mm, 1 mL/min,
5/95/0.05.fwdarw.90/10/0.05 ACN/H.sub.2O/TFA) showed mostly one new
peak (6.5 min, 78%). LC/MS (Gemini C18, 3.mu., 2.0.times.50 mm, 400
.mu.L/min, A: 90/10/0.01 H.sub.2O/MeOH/HOAc, B 90/10/0.01
MeOH/H.sub.2O/HOAc 0-6 min 0-100% B, 6-9 hold at 100% B, 9.1-15 min
re-equilibrate at 0% B) confirmed the peak identity as product
(ESI.sup.+ m/z 416).
[0379] Aza-adenosine Probe 3 is prepared from Compound 20A-iv in a
manner similar to that described in Steps 2 and 3 used for
preparation of Aza-adenine Probe 1.
B. Preparation of Aza-Adenosine Probe 4
Step 1: Preparation of
2-chloro-2',3'-O-isopropylideneadenosine-5'-N-Boc-ornithineamine
t-butyl ester (Compound 20B-ii)
##STR00054##
[0381] Following a procedure similar to that described in Example 5
(Aza-adenosine Probe 2, Step 1), borane dimethyl sulfide complex (2
M in THF, 30 .mu.L, 0.1 mmol) was added to a 0.degree. C. solution
of Compound 20B-i (2 mg, 0.0033 mmol) in THF (400 .mu.L). The
reaction mixture was allowed to slowly warm to room temperature
overnight. The same work up was done to afford the crude title
intermediate (1.6 mg, 80% yield) as a white solid. HPLC (Gemini
C18, 1101, 5.mu., 250.times.4.6 mm, 1 mL/min,
5/95/0.05.fwdarw.90/10/0.05 ACN/H.sub.2O/TFA) showed some starting
material and other peaks. LC/MS (Gemini C18, 3.mu., 2.0.times.50
mm, 400 .mu.L/min, A: 90/10/0.01 H.sub.2O/MeOH/HOAc, B 90/10/0.01
MeOH/H.sub.2O/HOAc 0-6 min 0-100% B, 6-9 hold at 100% B, 9.1-15 min
re-equilibrate at 0% B) confirmed the presence of product
(ESI.sup.+ m/z 598).
Step 2: Preparation of 2-chloroadenosine-5'-N-ornithineamine
(Compound 20C-iv)
##STR00055##
[0383] A mixture consisting of Compound 20B-ii (1.6 mg, 0.0027
mmol) and methanol (100 .mu.L) was treated with 3 M HCl (300
.mu.L). The reaction mixture was stirred at room temperature
overnight. HPLC (Gemini C18, 110 .ANG., 5.mu., 250.times.4.6 mm, 1
mL/min, 5/95/0.05.fwdarw.90/10/0.05 ACN/H.sub.2O/TFA) showed mostly
2 peaks--deprotected starting material (6.5 min, 31%) and a new
peak (4.3 min, 23%). LC/MS (Gemini C18, 3.mu., 2.0.times.50 mm, 400
.mu.L/min, A: 90/10/0.01 H.sub.2O/MeOH/HOAc, B 90/10/0.01
MeOH/H.sub.2O/HOAc 0-6 min 0-100% B, 6-9 hold at 100% B, 9.1-15 min
re-equilibrate at 0% B) confirmed the presence of product
(ESI.sup.+ m/z 402).
[0384] Aza-adenosine Probe 4 is prepared from Compound 20C-iv in a
manner similar to that described in Steps 2 and 3 used for
preparation of Aza-adenine Probe 1.
Example 7
Preparation of Sinefungin Probe 5
##STR00056##
[0385] Step 1: Preparation of tert-butyl
(2-((6-amino-9H-purin-2-yl)amino)ethyl)carbamate (Compound 27A)
##STR00057##
[0387] To a microwave tube containing 2-chloroadenosine (Aldrich
Chemical) is added tert-butyl N-(2-aminoethyl)carbamate (AK
Scientific, 10-fold molar excess). The mixture is heated in a
microwave for 20 minutes at 120.degree. C. under nitrogen. Workup
of the reaction mixture, purification, and isolation of the title
intermediate is carried out in a manner similar to that of Example
4, ROUTE 1, Step 3 used for Compound 16A.
Step 2: Preparation of
(2R,3R,4R,5R)-2-(6-amino-2-((2-((tert-butoxycarbonyl)amino)ethyl)amino)-9-
H-purin-9-yl)-5-((2S,5S)-2-azido-6-(tert-butoxy)-5-(4-methylphenylsulfonam-
ido)-6-oxohexyl)tetrahydrofuran-3,4-diyl diacetate (Compound
28A)
##STR00058##
[0389] To a mixture consisting of Compound 27A (10 molar
equivalents) in an appropriate solvent is added SnCl.sub.4
(equimolar) in dichloroethane. A mixture consisting of
(2S,3R,4R,5R)-5-((2S,5S)-2-azido-6-(tert-butoxy)-5-(4-methylphenylsulfona-
mido)-6-oxohexyl)tetrahydrofuran-2,3,4-triyl triacetate (limiting
reagent, prepared in the manner of Rapoport et. al. as 25.beta.) is
added and the mixture stirred at room temperature for 16 hours. The
mixture is partitioned between ethyl acetate and 0.5N
Na.sub.2HPO.sub.4 and the organics washed with brine, dried and
evaporated. The residue is chromatographed on silica gel to obtain
the title intermediate.
Step 3: Preparation of (2S,5S)-tert-butyl
6-((2R,3S,4R,5R)-5-(6-amino-2-((2-((tert-butoxycarbonyl)amino)ethyl)amino-
)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)-5-azido-2-(4-methylphe-
nylsulfonamido)hexanoate (Compound 29A)
##STR00059##
[0391] To a mixture consisting of Compound 28A in methanol is added
potassium carbonate (10 molar equivalents) and the mixture stirred
for 30 minutes at room temperature. Acetic acid is added to neutral
pH and the solution is stirred 30 minutes. Solvent is evaporated
and the mixture is partitioned between ethyl acetate and water. The
organics are washed with brine, dried and evaporated. The residue
is chromatographed on silica gel to obtain the title
intermediate.
Step 4: Preparation of
(2S,5S)-2,5-diamino-6-((2R,3S,4R,5R)-5-(6-amino-2-((2-aminoethyl)amino)-9-
H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)hexanoic acid
(Compound 30A)
##STR00060##
[0393] To a mixture consisting of Compound 29A in methanol is added
Pd(OH).sub.2 on carbon. The mixture is shaken under hydrogen for
two days and is subsequently diluted with methanol and ammonium
hydroxide. The mixture is filtered through Celite and solvent
evaporated to give intermediate amine. The material is dissolved
into trifluoroacetic acid/water (9/1) and stirred for one hour.
Solvent is evaporated and the residue is stirred in liquid ammonia
at -78.degree. C. Sodium is added (10 molar equivalents) and the
solution is stirred for about one minute. Excess solid ammonium
chloride is added and the ammonia is evaporated to give crude
Compound 30A. This material is either used without further
purification or is purified by preparative reverse phase HPLC.
Step 5: Preparation of Sinefungin Probe 5 (Compound 31A)
Conjugation of Compound 30A with Texas Red-X, Succinimidyl Ester
(Life Technologies Catalog # T-6134)
[0394] Compound 30A and Texas Red-X, succinimidyl ester are
dissolved in a suitable solvent with a slight excess of a suitable
base. The desired product, Sinefungin Probe 5, is obtained through
workup and purification procedures disclosed elsewhere herein for
other detection analytes.
Example 8
Saturation Binding: Sinefungin Probe 1A
[0395] Methods:
[0396] The experiment was performed once, with two replicate wells
for all methyltransferases per condition. The sole exception to
this is NSD2 (see details below). The assay was performed in a 1/2
area, black, 96-well plate (Corning #3686) in a final volume of 30
.mu.L. Methyltransferases (Table 1) were serially diluted two-fold
in FP buffer (Cayman Catalog #600028) and 15 .mu.L of a given
dilution were added per well to the assay plate. Fifteen
microliters of sinefungin probe 1A (10 nM) were added to each well,
giving a final sinefungin probe 1A concentration of 5 nM. This
concentration of sinefungin probe 1A was chosen because it had at
least a 10-fold increase in fluorescence (excitation max 575
nm/emission max 615 nm) over background (buffer). The mixture was
incubated in the dark at room temperature for 30 minutes and its
fluorescence polarization was read using a BioTek Synergy 4.
[0397] For NSD2, the assay was performed essentially as above, but
with the following changes. The assay was performed in a 20 .mu.L
total volume (10 .mu.L NSD2/10 .mu.L, sinefungin probe 1) in
low-volume, black, 384-well plates (Nunc #264705). The
concentrations of sinefungin probe 1A and methyltransferase were
identical to that of the 96-well format. The assay was performed
once with three replicate wells per condition.
TABLE-US-00001 TABLE 1 Cayman Methyltransferase Product Item #
SET7/9 10320 PRMT1 10350 SET8 10319 G9a 49010353 NSD2 10758
[0398] Results and Interpretation:
[0399] Of the 5 methyltransferases tested, only SET7/9 and G9a
displayed measurable affinity for sinefungin probe 1A (FIG. 2).
SET7/9 bound sinefungin probe 1A with high affinity (Kd: 17 nM),
while G9a has a lower affinity (Kd: .about.700 nM) for the probe.
These data suggest that sinefungin probe 1A is not a general
S-adenosyl methionine (SAM)-binding site probe, but is selective
for those binding sites that have structural characteristics which
allow for the linker-fluorophore moiety of sinefungin probe 1A to
be covalently attached at the sinefungin moiety 2'-ribose hydroxyl
oxygen atom.
Example 9
Sinefungin Probe 1A Competition with SAM, SAH, and Unlabeled
Sinefungin
[0400] Methods:
[0401] Carboxymethyl cellulose-purified SAM, SAH (Cayman Catalog
#700145), and sinefungin were diluted in a semi-Log manner in FP
buffer (Cayman Catalog #600028). Five microliters of these
dilutions were added to the wells of a low-volume, black, 384-well
plate (Nunc #264705). Ten microliters of SET7/9 (60 nM) was added
to the wells, followed by the addition of 40 nM sinefungin probe 1A
(5 .mu.L), providing final concentrations of 30 nM SET7/9 and 10 nM
sinefungin probe 1. The assay was allowed to equilibrate at room
temperature for 30 minutes in the dark. Fluorescence polarization
was measured as above. The experiment was performed two independent
times, each in duplicate.
[0402] Results and Interpretation:
[0403] Sinefungin Probe 1A was displaced in a
concentration-dependent manner by SAM and unlabeled sinefungin with
IC.sub.50 values of 130 nM and 220 nM, respectively (FIG. 3). SAH
did not compete with probe binding at concentrations below 1 .mu.M.
The potent inhibition of probe binding by SAM suggests that the
compound is indeed binding at the SAM binding site. The inability
of SAH to compete with sinefungin binding is likely explained by
the low affinity of SAH for SET7/9. These data therefore suggest
that Sinefungin Probe 1A is binding to SET7/9 in the SAM binding
site.
Example 10
High-Throughput Screening of SET7/9 Using Sinefungin Probe 1A and
Adaptation for Screening of GLP and MLL ("SAM-Screener" Assay)
[0404] Methods:
[0405] A collection of 14,400 compounds from the Maybridge
HitFinder collection of compounds was assayed for the displacement
of Sinefungin Probe 1A from SET7/9 in a high-throughput manner
using the SET7/9 SAM-Screener FP assay Cayman Catalog Item
#600490). Thirty microliters of assay buffer (50 mM Tris pH 8.0, 50
mM KCl, 5 mM CHAPS, 2 mM DTT) was added to every well of a black
384-well polystyrene microtiter plate (Thermo-Fisher 364576). To
every well, excluding control wells, was added one microliter of a
DMSO solution containing a test compound. Control wells received
either 1 .mu.l DMSO or 1 .mu.l of a sinefungin solution. The final
concentration of sinefungin control or test compounds in the assay
was 50 .mu.M or 10 .mu.M, respectively. To every well of the plate
was added 10 .mu.l of a SET7/9 solution to give a final assay
concentration of 30 nM. After a 15-minute incubation at ambient
temperature, 10 .mu.l of Sinefungin Probe 1A solution was added to
every well to give a final assay concentration of 10 nM in a total
assay volume of 50 .mu.l Samples were analyzed using a Biotek
Synergy H4 plate reader in fluorescence polarization mode after a
30-minute incubation at ambient temperature in the dark. Data were
analyzed using GeneData (v. 9.01).
[0406] Adaptation for GLP and MLL:
[0407] Assays for probe binding to two other methyltransferases
(GLP and MLL) were also adapted to high throughput format. The
assay conditions were identical to the SET7/9 assay, however the
protein concentration was increased to raise the signal:noise ratio
for the assay. For GLP, the protein concentration was raised to 500
nM, while the concentration of MLL was raised to 1 .mu.M. Data were
analyzed in GraphPad Prism (v. 5.04) and/or GeneData (v. 9.01).
[0408] Concentration-Response Experiments:
[0409] Compounds that displayed a Z-score <-3 from the mean of
the test compounds wells were chosen for concentration-response
follow-up experiments. In total, 47 compounds were selected for
concentration-response follow up and were tested in 8-point serial
concentration response curves in duplicate using the assay
conditions and analysis methodology as described above.
[0410] Profiling of a pre-production version of the Cayman Chemical
Epigenetics Screening Library (Cayman Catalog Item #11076) against
SET7/9 and GLP was performed in a similar manner using Sinefungin
Probe 1B. For GLP, the enzyme concentration was raised to 500 nM to
produce a robust signal:noise ratio. Data were analyzed in GraphPad
Prism (v. 5.04) and/or GeneData (v. 9.01).
[0411] Results:
[0412] High-throughput adaptation of the SET7/9 SAM-Screener assay
produced an assay capable of analyzing >20,000 data points per
day with robust Z' scores (>0.6). High-throughput screening of
the Maybridge HitFinder collection was performed in one day with an
overall Z' score across the entire assay of 0.68 (Table 1). From
this assay, 47 compounds were identified that had a Z score <-3
from the mean of the test compound wells. These compounds were
subjected to confirmation concentration-response experiments, from
which two compounds were determined to be active with an
IC50<250 .mu.M. These compounds, KMN-10305 and KMN-10719 are
currently being evaluated for their ability to inhibit
methyltransferase activity.
TABLE-US-00002 TABLE 1 High-throughput screening data from an
experiment that tested 14,400 compounds for Sinefungin Probe
1A-displacement activity from SET7/9. Confirmation Compounds Tested
"Actives" Rate (% total) Primary Screen 14400 47 0.3 CRC* 2.degree.
Assay 47 2 0.01 *CRC = concentration response curve
[0413] A pre-production version of the Cayman Epigenetics Screening
Library was profiled in an 8-point concentration-response format
against the methyltransferases SET7/9 and GLP. For both sets of
experiments the Z' values were >0.6 and from each compound. Of
the 44 compounds tested in this experiment, only sinefungin showed
appreciable inhibition against SET7/9. Similar results were
observed for GLP, however, UNC0638 also inhibited probe binding
with an IC.sub.50 value of 32 nM.
[0414] This assay was also adapted for high throughput format
against the methyltransferase MLL (Cayman Catalog Item #10658).
Relevant data are illustrated in FIG. 10: The fluorescence
polarization (FP) Sinefungin Probe 1A binding assay was adapted to
a high-throughput inhibitor screening format for both A) MLL and B)
SET7/9. The probe polarization is increased by >120 mp by
binding to SET7/9 (30 nM) and by >145 mp by binding to MLL (1
.mu.M). As controls, sinefungin (50 .mu.M for SET7/9, 300 .mu.M for
MLL) or SAM fully inhibits probe binding. Sixty-four replicates
were tested for each condition for SET7/9, sixteen replicates were
tested for MLL. Z' factor scores were >0.5 for all conditions
tested (SET7/9:sinefungin: 0.71; SAM: 0.72; no SET7/9: 0.69;
MLL:sinefungin: 0.72).
Example 11
Sinefungin Probe SAR Studies
[0415] Sinefungin Probe 1A was tested for the ability to bind to 16
proteins, including several non-methlytransferase protein controls
(Table 2). This probe binds to 4 methyltransferases (SET7/9, G9a,
GLP, MLL) with Kd values <2 .mu.M.
TABLE-US-00003 TABLE 2 Sinefungin Probe 1A affinity for each of the
proteins tested. All proteins were tested in a saturation-binding
format and data are presented as the mean .+-. SEM from at least
two independent experiments performed in duplicate. Enzymatic
activities of the proteins were confirmed using an orthogonal assay
as listed in the table. Confirmation Protein Sinefungin Probe 1A
Affinity (.mu.M) Assay Type SET7/9 0.027 .+-. 0.0015 Colorimetric
G9a 1.8 .+-. 1.0 Colorimetric KMT5b 6.1 .+-. 0.8 Radiometric PRDM9
2.6 .+-. 0.6 Radiometric GLP 0.58 .+-. 0.05 Colorimetric MLL 0.75
.+-. 0.065 Radiometric PRMT1 >1.2 Colorimetric NSD2 >2
Radiometric SET8 >2 Colorimetric KMT5c >2 Radiometric DNMT3b
>2 N/A PRMT4 >2 Radiometric ASH2L >10 N/A Lysozyme >10
N/A Trypsin >10 N/A Sumo >10 N/A
[0416] An analog of Sinefungin Probe 1A, Sinefungin Probe 1B,
wherein the hexyl portion of the linker moiety is replaced with
PEG.sub.3 and the Cal Fluor.RTM. Red 610 fluorophore moiety is
replaced with the "Fluor585" (Texas Red)-derived fluorophore
moiety, was tested for the ability to bind several
methyltransferases, including SET7/9. These two probes bind to
SET7/9 with essentially identical affinities and are displaced by
sinefungin in a similar manner (FIG. 11: A) Sinefungin Probe 1A
(comprising Cal Fluor.RTM. fluorophore) and Sinefungin Probe 1B
(comprising Fluor585 fluorophore) bind to SET7/9 with similar
affinities. Similar data have been observed for GLP and G9a. B)
Sinefungin is able to displace Sinefungin Probe 1B binding to
SET7/9 with a K.sub.i value of 120 nM, which corresponds well with
the 168 nM K.sub.i values calculated for displacement of Sinefungin
Probe 1A).
[0417] Similar experiments were performed using Sinefungin Probe 3,
wherein the "click" triazole ring portion of the linker moiety of
Sinefungin Probe 1B is replaced with the "non-click" tether-portion
of the linker moiety as shown in Example 2 above.
[0418] Sinefungin Probe 3 bound to SET7/9 with similar affinity to
that of the click-linker based probes Sinefungin Probes 1A and 1B
(FIG. 12). This probe was tested in parallel with Sinefungin Probe
1B to determine whether it possessed increased affinity for any
methyltransferases. None of the 7 methyltransferases tested
(SET7/9, PRDM9, G9a, NSD2, MLL, KMT1b, SETS) showed significantly
greater affinity for the non-click probe than the click-based
probe.
Example 12
Radiometric Profiling of Various Thioadenosine and Aza-Adenosine
Probe Synthetic Precursors
[0419] Overview:
[0420] Three adenosine-type probe synthetic precursors were tested
against a panel of 7 methyltransferases using a radiometric
methyltransferase assay. In general, Thioadenosine Probe 1
synthetic precursor Compound 16A displayed inhibitory activity
against 4 of the 7 enzymes tested, while two Aza-adenosine probe
precursors, Compounds 20A-iv and 20A-iii, showed substantially less
potency against the panel of methyltransferases.
[0421] Reagents:
[0422] Compound 16A was prepared by the method described in Example
4 and illustrated in FIG. 5. The Compounds 20A-iv and 20A-iii were
prepared according to methods described in Example 6 and
illustrated in FIG. 7. Enzymes were obtained from the Cayman
Catalog with the exception of PRMT4 and PRMT6, which were produced
in baculovirus and purified to near homogeneity using Ni-affinity
chromatography. Human core histones were purified from HeLa nuclear
pellet per the method of Cote et al. S-adenosylmethionine (SAM) was
obtained from Sigma-Aldrich (Cat # A7007) and S-[methyl-3H]-SAM was
obtained from Perkin-Elmer (Cat# NET155H001MC).
##STR00061##
Structures of the compounds that were tested in this profiling
experiment.
[0423] Methods: Compounds or vehicle control were diluted in assay
buffer (50 mM Tris pH 8.0, 150 mM NaCl, 3 mM MgCl2, 5% glycerol, 1
mM dithiothreitol) to a final assay concentration of 10 .mu.M, 1
.mu.M, or 0.1 .mu.M and were plated into V-bottom polypropylene
96-well plates. To this was added enzyme diluted to provide a final
assay concentration (GLP: 10 nM; PRMT1: 100 nM; SET7/9: 120 nM;
PRDM9: 120 nM; PRMT4: 150 nM; PRMT6: 120 nM; G9a: 200 nM) and the
compound was allowed to preincubate for 10 minutes at room
temperature. Reactions were initiated by adding core histones, SAM,
and S-[methyl-3H]-SAM to final assay concentrations of 2.5 .mu.M,
700 nM and 300 nM, respectively. Reactions were allowed to progress
at room temperature for 30 minutes, at which time they were
quenched with bovine serum albumin and SAM to final reaction
concentrations of 0.33% and 100 .mu.M, respectively. Samples were
mixed immediately after quenching and transferred to a 96-well GFC
Uni-Filter microtiter filter plate (Perkin Elmer, Cat#6005174) that
contained 50 .mu.l of 20% TCA. Samples were allowed to precipitate
for at least 5 minutes at room temperature. The samples were
filtered onto the GFC membrane and washed four times with 100 .mu.l
of 10% TCA and once with 100 .mu.l of 95% EtOH. To each well was
added 25 .mu.l of MicroScint-20 (Perkin-Elmer Cat#6013621) and
samples were analyzed using a TopCount NXT microplate scintillation
counter. The experiment was performed one time in duplicate for all
compound concentrations tested and in triplicate for the vehicle
and no enzyme controls. All data was analyzed in GraphPad Prism (v.
5.04).
[0424] Results:
[0425] Compound 16A displayed the greatest inhibitory activity
against the broadest panel of enzymes (FIG. 13). This compound
inhibits PRMT1 and PRMT4, PRDM9, and G9a, while not dramatically
altering the activity of GLP, PRMT6 and SET7/9. Compounds 20A-iv
and 20A-iii did not show appreciable inhibition against the
enzymatic activity of any of the enzymes tested, with the exception
of PRMT6, for which Compound 20A-iii displayed some modest
inhibition.
[0426] Thioadenosine Probe 1 displayed about a 5 .mu.M binding
affinity (Kd) with PRMT1 (FIG. 14).
Example 13
TR-FRET Assay Procedure
Procedure:
Materials Required:
[0427] Buffer: FP buffer (e.g. Cayman Catalog #600028) Receptor:
Lanthanide chelate-labeled SAM-utilizing protein (direct chemical
labeling or secondary labeling via labeled antibody or
streptavidin; label example: LanthaScreen Tb chelate, Invitrogen)
Assay Plate: low volume, 384 well black plate (e.g. Nunc #264705)
Dilution plate: 1/2 area 96-well plate (e.g. Corning 3695) Suitable
multimode plate reader (e.g. BioTek Synergy H4)
[0428] The assay described here is an isothermal saturation binding
experiment designed to measure the affinity (Kd) of Sinefungin
Probe 1A, Sinefungin Probe 2A, or a similar
sinefungin-linker-fluorophore probe (probe), as exemplified in the
embodiments described herein, with a donor fluorophore (a
lanthanide chelate)-labeled SAM-utilizing protein (receptor).
Simple adaptation would allow this assay to be used for competition
binding experiments including high throughput screening. The assay
is described in a 384-well format, but can be scaled to desired
volume or plate format as needed.
[0429] The assay is performed in a total volume of twenty
microliters, with ten microliters comprising a solution of the
receptor and ten microliters of the probe. Final concentrations of
the reagents are as such: receptor: 0 nM or 10 nM; probe or general
probe: 1.4 nM.fwdarw.3 .mu.M in a series of twelve two-fold
dilutions. [0430] 1) The probe is serially diluted two-fold in a 96
well plate across one of the rows, generating 12 dilutions of the
probe or general probe. [0431] a. The probe is diluted to 6 .mu.M
in buffer and is subsequently serially diluted across plate by
adding 100 .mu.L of 6 .mu.M stock to well A1 and 50 .mu.L buffer to
wells A2-A12. Serially dilute by transferring 50 .mu.L in a
stepwise manner across the row, mixing thoroughly in each well.
[0432] 2) Transfer 10 .mu.L of the diluted probe to two sets of
duplicate wells (four wells total per concentration of probe) in a
low-volume 384 well black plate. [0433] 3) To one set of duplicate
wells, add 10 .mu.L of 20 nM receptor. To the other set of
duplicate wells, add 10 .mu.L buffer. [0434] 4) Allow the reaction
to incubate for 30 minutes at room temperature, protected from
light. [0435] 5) Analyze the reaction using a BioTek Synergy H4 (or
similar) multimode plate reader with TR-FRET capability. [0436] a.
Instrument sensitivity is adjusted by normalizing the fluorescence
values to a buffer-filled (20 .mu.L) well. [0437] b. Samples are
measured in a time resolved manner using a 200 .mu.s delay before
initiating a 500 .mu.s data collection window. [0438] c. Data are
collected using a xenon flash bulb and appropriate excitation and
emission filter sets for the FRET pair: [0439] i. E.g. Tb
chelate/fluorescein FRET pair: Excitation 360 nm/40 nm bandpass;
Emission 460 nm/40 nm bandpass and 528 nm/20 nm bandpass. [0440] 6)
Data analysis and Expected results: [0441] a. Data are transformed
as the ratio of the 528 nm emission/460 nm emission (TR-FRET ratio)
and are plotted on an XY scatter graph as TR-FRET ratio vs. [probe]
(nM). [0442] i. For the probe dilution series that contains only
buffer and probe, it is expected that the TR-FRET ratio will
increase in a linear fashion with a shallow slope. This is
background signal from the assay. [0443] ii. For the probe dilution
series that contains both probe and receptor, the TR-FRET ratio
will increase in the shape of a rectangular hyperbola summed with a
background linear component. [0444] b. For each concentration of
probe, subtract the assay background (probe+buffer wells) from the
saturation binding wells (probe+receptor wells) to obtain the
receptor-specific TR-FRET signal. [0445] c. Calculate Kd using
nonlinear regression to fit the background-corrected data to the
model:
[0445] Y=B.sub.max*X/(Kd+X)
Adaptations:
[0446] The assay can be adapted for competition analysis of
unlabeled competitors by testing increasing concentrations of the
unlabeled competitor against a fixed concentration of probe and
receptor. Ideally, the probe concentration in this format should be
approximately 80% of the maximal TR-FRET signal and receptor
concentration should be less than twice the expected IC.sub.50
value of the competitor. For this experiment, plot the data as
TR-FRET Ratio vs. Log.sub.10[competitor] (M). Calculate IC.sub.50
using nonlinear regression to fit the data to the model:
Y=Bottom+(Top-Bottom)/(1+10 ((Log IC.sub.50-X)*HillSlope))
Where:
[0447] Bottom=lower bound of TR-FRET signal (assay background, e.g.
vast excess of competitor) Top=upper bound of TR-FRET signal (no
competitor present) HillSlope=Hill coefficient; the slope of the
curve around the IC.sub.50 value
REFERENCES
[0448] The standard fitting models described here were obtained
from GraphPad Prism version 5.04.
[0449] It should be understood that the detailed description and
specific examples, while disclosing exemplary embodiments of the
invention, are intended for purposes of illustration only and are
not intended to limit the scope of the invention.
* * * * *