A specific molecular beacon probe for the detection of human prostate cancer cells

Article abstract of DOI:10.1016/j.bmcl.2012.04.055

The small-molecule, water-soluble molecular beacon probe 1 is hydrolyzed by the lysate and living cells of human prostate cancer cell lines (LNCaP), resulting in strong green fluorescence. In contrast, probe 1 does not undergo significant hydrolysis in ei

Full text of DOI:10.1016/j.bmcl.2012.04.055

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3632–3638
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A specific molecular beacon probe for the detection of human prostate
cancer cells
Yu Lin Jiang a, , Christopher A. McGoldrick b, Deling Yin c, , Jing Zhao c, Vini Patel a, Marianne F. Brannon b,
⇑
⇑
Janet W. Lightner c, Koyamangalath Krishnan c, William L. Stone b,
⇑
a Department of Chemistry, College of Arts and Sciences, East Tennessee State University, Johnson City, TN 37614, United States
b Department of Pediatrics, College of Medicine, East Tennessee State University, Johnson City, TN 37614, United States
c Department of Internal Medicine, College of Medicine, East Tennessee State University, Johnson City, TN 37614, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 February 2012
Revised 3 April 2012
Accepted 10 April 2012
Available online 19 April 2012
The small-molecule, water-soluble molecular beacon probe 1 is hydrolyzed by the lysate and living cells
of human prostate cancer cell lines (LNCaP), resulting in strong green fluorescence. In contrast, probe 1
does not undergo significant hydrolysis in either the lysate or living cells of human nontumorigenic pros-
tate cells (RWPE-1). These results, corroborated by UV–Vis spectroscopy and fluorescent microscopy,
reveal that probe 1 is a sensitive and specific fluorogenic and chromogenic sensor for the detection of
human prostate cancer cells among nontumorigenic prostate cells and that carboxylesterase activity is
a specific biomarker for human prostate cancer cells.
Keywords:
Molecular beacon
Fluorescent probe
Fluorogenic
Ó 2012 Elsevier Ltd. All rights reserved.
Chromogenic
Detection
Prostate cancer
Carboxylesterase activity
Biomarker
Porcine liver esterase
Prostate cancer is the most common malignancy in American
men and the second leading cause of cancer mortality.1 There are
no symptoms during this cancer’s early stage; patients may die
due to lack of early detection and treatment.2 Therefore, there is
an urgent need to develop effective chemical methods for the
detection of prostate cancer in its early stages, potentially allowing
us to control the cancer from spreading and, ultimately, cure the
aggressive disease.2
At present, a chemical test measuring the level of prostate-
specific antigen (PSA) in serum is the most effective method for
the early detection of prostate cancer; its positive predictive value
is, however, only 35%.3 Therefore, the majority of patients will re-
ceive false-negative results, making them likely to lose the possi-
bility of early detection, prevention, and treatment. Another
method for the clinical diagnosis of human prostate cancer is
through biopsy, which checks the patient’s prostate tissues for can-
cerous cells.4 The diagnosis and prognosis of prostate cancer might
also be possible through analysis of the carboxylesterase activity in
the cells; some reports have described increased carboxylesterase
activity in the cells of other cancer types, such as human breast
cancer,5,6 relative to that in normal tissues. We are, however, una-
ware of any previous reports of increased carboxylesterase activity
in human prostate cancer cells for the diagnosis and prognosis of
prostate cancer cells.7
In a previous study, the carboxylesterase activity in breast can-
cer cells was determined through fluorescence monitoring, with
fluorescein diacetate used as the substrate for the enzyme.5 This
activated aryl acetate is, however, labile (high automatic hydroly-
sis rate), making it unstable in water or buffer.8 As a result, the
fluorescence background was high and the signal to noise ratio
was low.5 Fluorescein diacetate also possesses limited water solu-
bility.8 One approach toward overcoming the limitations of that
probe would be to develop a specific, water-soluble, ester-based
fluorescent probe, such as an alkyl aliphatic acid ester, with a
low rate of automatic hydrolysis.8
Recently, water-soluble 2-(20-phosphoryloxyphenyl)-4-(3H)-qui-
nazolinone (Q2-p) derivatives have been applied to detect the activity
of extra-cellular phosphatases around prostate cancer cells.9 In addi-
tion, a couple of large molecules and fluorescent probes containing
pyrenyl oligonucleotides have been employed in the detection of
intra-cellular carboxylesterase activity in HeLa cells.8 Notably, such
pyrenyl probes have not been used for the detection of human
⇑
Corresponding authors. Tel.: +1 423 439 6917; fax: +1 423 439 5835.
E-mail addresses: jiangy@etsu.edu (Y.L. Jiang), yin@etsu.edu (D. Yin), stone@
etsu.edu (W.L. Stone).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.04.055

Y. L. Jiang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3632–3638
3633
O
N
N
N
O
H
O
O
NMe2
O
H
N
OH
cancer cell
cancer cell
O
O
OH
carboxyl-
esterase
cancer
lysate
1
O
N
N
N
HO
H
O
NMe2
OH
OH
2
cancer
lysate
O
O
H
N
O
O
3
OH
Scheme 1. Schematic representation of the operation of probe 1 for detection of human prostate cancer cells.
prostate cancer cells. Therefore, a small-molecule with better cell
permeabilityand a probe with specific fluorescenceappear necessary
for monitoring the carboxylesterase activity in human prostate
cancer cells.
quenching of the fluorescein moiety by the dabcyl group. Taken
together, we expected these features to make probe 1 a specific
substrate for carboxylesterases. After hydrolysis of 1 by a carboxy-
lesterase through acyl substitution, N-dabcylglycine (2) will be
expelled, causing the fluorescein unit to become fluorescent (i.e.,
no longer quenched). We synthesized the molecular beacon probe
1 from the dabcyl derivative 2 through Williamson ether synthesis15
and palladium-catalyzed Sonogashira coupling16,17 with compound
5 (Schemes 2 and S1–S3). We also prepared compounds 3 and 6 for
NMR spectroscopic studies (Fig. 1, Schemes S2 and S4).
We have designed the molecular beacon probe 1 as a substrate
for monitoring carboxylesterase activity (Scheme 1). This molecule
features one aliphatic ester group and two aromatic acid amide
groups; only the ester group will be hydrolyzed readily by carboxy-
lesterase.10 Aryl acid amide groups are typically less-reactive sub-
strates for most carboxylesterases, amidases and proteases.11,12 In
addition, we expected this probe to be resistant to hydrolyzes cata-
lyzed by intra- and extra-cellular phosphatases, nucleases, and
phosphodiesterases in prostate cancer cells due to probe 1 is non
phosphate salt/ester. Furthermore, probe 1 incorporates a fluoro-
phore—fluorescein, a water-soluble unit13 at physiological pH
(7.4)—and a typical fluorescence quencher (dabcyl group).14 In addi-
tion, the structure features an ortho-substituted phenyl group that
acts as a bridge to bring the fluorescein and dabcyl units together.
This design makes it possible to ensure effective fluorescence
To demonstrate whether the designed and synthesized 1 has a
hairpin like structure and whether it is a new molecular beacon
probe, and to determine whether fluorescence quenching was pos-
sible between the fluorescein and dabcyl units in 1, we investi-
gated their intramolecular hydrogen bonding and
p–p stacking
interactions using 1H NMR spectroscopy. We monitored the chem-
ical shifts of the protons of the probe 1, its synthetic precursor 6,
and its related compound 3 under the same conditions (in
acetone-d6, at a concentration of 38.0 mM, and at a temperature
I
O
OH
Br
O
HN
O
N
I
HN
N
Me2N
N
O
Cs2CO3, DMF
Me2N
N
O
2
49%
4
Me2N
OH
HO
O
O
HO
O
OH
O
N N
O
NH
O
O
5
O
NH O
NH
O
Pd(PPh3)4, NEt3, DMF
74%
O
1
Scheme 2. Synthesis of 1.

3634
Y. L. Jiang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3632–3638
Me2N
Hd16.870
7.889
6.638
OHf
Hf1
Hd2
N N
9.124
HO
O
Hd37.880
6.644
Hf2
O
O
Hf3
O
6.702
8.062
Hd4
N
H
8.216
a1
NH
O
O
6
HO
O
3
Me2N
Hd16.854
7.828
6.616
Hf1
Hf3
Hd2
N N
HO
O
O
OHf9.079
Hd37.868
6.622
Hf2
O
8.040
Hd4
N
6.671
H
8.499
a1
O
O
O
NH
O
1
Figure 1. Partial 1H NMR spectra of compounds 1, 3, and 6 at 38.0 mM in acetone-d6, with assignments of the chemical shifts (ppm) of selected protons of OH, NH, and
aromatic groups.
of 25 °C (Fig. 1). Relative to its position in the NMR spectrum of 6,
the signal for the amide proton Hal of 1 had shifted downfield by
0.283 ppm, suggesting that this hydrogen atom was involved in
significant intramolecular hydrogen bonding ( Fig. 2A). Further-
more, the signals of the OH proton and aromatic protons Hdl-4
and Hf1-3 of 1 were located upfield—by 0.040, 0.016, 0.061, 0.012,
0.022, 0.022, 0.022, and 0.031 ppm, respectively—relative to the
corresponding protons of 6 and 3, indicating their stacking
p–p
interactions occurred between the fluorescein and dabcyl
groups.18,19
To investigate the intramolecular interactions suggested by the
NMR spectra further, we performed a structural calculation for 1 at

Y. L. Jiang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3632–3638
3635
(A)
(B)
O
N
O
N
N
O
H
O
NMe2
O
H
N
OH
O
O
OH
Figure 2. (A) Chemical structure of the intramolecular hydrogen bond NG-HꢀꢀꢀOF (G, glycine; F, 6-carboxamide fluorescein). (B) Calculated structure of probe 1. Dotted line:
NG-HꢀꢀꢀOF hydrogen bond.
Figure 3. (A) Fluorescence spectra of probe 1 (10.0
lM) after 5 h at room temperature in 0.10 M tris–maleate buffer at pH 8 in the presence (green) and absence (purple) of
porcine liver carboxylesterase (PLC, 1.10 M). (B) Green fluorescence (right) generated from the probe 1 upon hydrolysis with PLC. In the control reaction (left), no or little
l
green fluorescence was observed. The picture was taken under irradiation with UV light (350 nm). All the fluorescence measurements were recorded on a fluorometer by an
excitation at 490 nm.
the RM1 level. Figure 2B reveals the presence of an NG-HꢀꢀꢀOF intra-
molecular hydrogen bond having a hydrogen bond distance of
2.159 Å and angle of 166.4°. These results are consistent with sig-
nificant hydrogen bonding occurring within the molecule.18,19 The
intramolecular hydrogen bond of probe 1 would promote the for-
mation of a hairpin-like structure, bringing the xanthenyl group
of the fluorescein moiety toward the dabcyl group to ensure effec-
fluorescence intensity from the control, the fluorescence intensity
of the sample from the reaction catalyzed by the lysate of human
prostate cancer cells was 30.8-fold greater than that catalyzed by
the lysate of human nontumorigenic prostate cells (Fig. 4C), indicat-
ing that probe 1 is a selective and specific sensor for the detection of
carboxylesterase activityin the lysate of humanprostate cancer cells
and that carboxylesterase activity is a reliable biomarker for human
prostate cancer. Interestingly, the carboxylesterase activity of the
lysate from human prostate cancer cells lasted for a long time (up
to 10 days at room temperature), suggesting that 1 can detect the
carboxylesterase activity of samples after long periods of storage
(Fig 4D), making it a potentially more useful probe.
We also used UV spectroscopy to monitor the hydrolysis of
probe 1 in the presence of the lysates of human prostate cancer
and nontumorigenic prostate cells (Fig. 5). In the presence of the
lysates of human prostate cancer and nontumorigenic prostate
cells and in their absence, the wavelengths of maximum absor-
bance for the solutions were 494, 497, and 497 nm, respectively,
indicating that the hydrolysis catalyzed by the cancer cell lysate re-
sulted in a blue shift of the UV absorbance by 3 nm (Fig. 5A). The
UV absorbance for a mixture of 2 and 3 also appeared at 494 nm
(Fig. 5B), the same wavelength as that for the hydrolysis sample
catalyzed by the lysate of human prostate cancer cells, suggesting
that the hydrolysis of probe 1 by the lysate of the cancer cells led to
the production of compounds 2 and 3 (Scheme 1). In addition, the
relative intensities at 494 nm in the spectra recorded in the pres-
ence of the lysates of human prostate cancer and nontumorigenic
prostate cells and in their absence were 1.29, 1.10, and 1.00,
respectively. The increase of absorbance of the sample catalyzed
by the lysate of human prostate cancer cells was 29%, which is
2.9-fold of the increase of absorbance (10%) of the sample
tive p–p stacking interactions (Fig. 2B).
To test whether probe 1 would be an effective sensor for
carboxylesterase activity, we applied it as a substrate for hydrolysis
catalyzed by porcine liver carboxylesterase (PLC) at room temper-
ature in 0.10 M tris–maleate buffer at pH 8.0. For the control exper-
iment, we repeated this reaction, but with the absence of PLC. After
5 h, we measured the fluorescence intensity at 525 nm for each
sample upon excitation at 490 nm. The spectra revealed a 23-fold
increase in fluorescence for the sample in the presence of the
esterase, indicating that 1 was sensitive probe for carboxylesterase
activity (Fig. 3). The hydrolysis produced compounds 2 and 3, as
detected by mass spectrometry, indicating that the reaction was
the hydrolysis of the ester group in probe 1 (Figs. S1 and S2,
Scheme 1).
Next, we used probe 1 to detect carboxylesterase activity in the
total cellular lysate of human prostate cancer cells under the same
conditions as those described above for porcine liver carboxylester-
ase. For comparison, we also determined the carboxylesterase activ-
ity of the total cellular lysate of human nontumorigenic prostate
cells and performed a control experiment without any lysates. After
26 h, the fluorescence intensity at 525 nm of the sample from the
reaction catalyzed by the lysate of human prostate cancer cells
was 3.8-fold greater than that catalyzed by the lysate of nontumor-
igenic prostate cells ( Fig. 4A and B). After subtraction of the

3636
Y. L. Jiang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3632–3638
Figure 4. (A) Relative fluorescence intensities upon hydrolysis of probe 1 (10.0
the total cellular lysates (0.60 g/ L) of (a) prostate cancer cells (LNCaP) and (b) nontumorigenic prostate cells (RWPE-1) and (c) in the absence of cell lysates. (B) (a) Green
fluorescence generated from probe 1 upon hydrolysis with the lysate of prostate cancer cells for 26 h. (b) Weak fluorescence observed upon hydrolysis with the lysate of
nontumorigenic prostate cells (0.60 g/ L). (c) In the control reaction, no or little green fluorescence was observed. The picture was taken under irradiation with UV light
(350 nm). (C) Relative increase in fluorescence intensity at 525 nm upon hydrolysis of probe 1 catalyzed by the lysates of nontumorigenic prostate and prostate cancer cells
for 26 h. (D) Incremental increase in fluorescence at 525 nm upon hydrolysis of probe 1 (10.0 M) for 0 (indigo), 1 (dark yellow), 2 (purple), 4 (blue), 6 (turquoise), 8 (red), and
L) at room temperature in 0.10 M tris–maleate buffer at pH 8.0. All the fluorescence
lM) for 26 h at room temperature in 0.10 M tris–maleate buffer at pH 8.0 in the presence of
l
l
l
l
l
10 (green) days, catalyzed by the lysate of human prostate cancer cells (0.60
measurements were recorded on a fluorometer by an excitation at 490 nm.
lg/l
Figure 5. (A) Relative UV absorbance intensities upon hydrolysis of probe 1 (10
l
M) for 43 h at room temperature in 0.10 M tris–maleate buffer at pH 8.0 in the presence of
M) and 3
the lysates of (a) human prostate cancer cells and (b) nontumorigenic prostate cells and (c) in the absence of any lysates. (B) UV spectrum of a mixture of 2 (10
(10 M) in the same buffer.
l
l
catalyzed by the lysate of nontumorigenic prostate cells, suggest-
ing that increased UV absorbance occurred significantly upon the
cleavage of probe 1 by the carboxylesterase in the lysate of human
prostate cancer cells (Fig. 5A). These results are consistent with
significant hydrolysis of probe 1 occurring in the presence of the
lysate of human prostate cancer cells; the hydrolysis was relatively
insignificant when catalyzed by the lysate of human nontumori-
genic prostate cells. Thus, the carboxylesterase activity is higher

Y. L. Jiang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3632–3638
3637
Figure 6. Microscopy images of live human prostate (A) nontumorigenic prostate cells (RWPE-1) and (B) prostate cancer cells (LNCaP) after treatment with probe 1 (10.0
for 30 min in PBS buffer. The fluorescence image was obtained by excitation with 490 nm and detection from 500–560 nm with 200ꢁ magnification.
lM)
in the lysate of human prostate cancer cells than it is in that of non-
tumorigenic prostate cells. This experiment confirms that carboxy-
lesterase activity is a biomarker for human prostate cancer cells
and that 1 is an effective probe for the detection of the cancer cells.
To determine the effectiveness of probe 1 for the detection of
live human prostate cancer cells, we performed experiments using
LNCaP cancer cells and RWPE-1 nontumorigenic prostate cells with
fluorescent microscopy (Fig. 6). No green fluorescence appeared
within the human prostate nontumorigenic prostate cells
(Fig. 6A), but green fluorescence appeared within human prostate
cancer cells (Fig. 6B), suggesting that 1 is a sensitive probe for
the detection of human prostate cancer cells. Furthermore, the
green fluorescence appears clear and bright in the image 6B, indi-
cating that 1 is an effective probe for highlighting human prostate
cancer cells. The treatment time was only 30 min, consistent with
the high reactivity of probe 1 during analysis. Furthermore, the
green fluorescence was present within the human prostate cancer
cells, suggesting that probe 1 is cell-permeable to human prostate
cancer cells. Therefore, probe 1 is specific for the detection of
carboxylesterase activity within human prostate cancer cells.
Prostate cancer is one of the leading causes of death in
American men; early detection of this deadly disease is necessary
for effective treatment. We suggest that unusual carboxylesterase
activity in human prostate cancer cells should be listed as another
important biomarker, in addition to PSA, for this particular disease.
The synthesis of probe 1 is crucial for the measurement of the in-
crease in carboxylesterase activity in human prostate cancer cells.
This probe aids the identification of a human prostate cancer bio-
marker. It is also likely that probe 1 would find applications in clin-
ical trials for the identification of human prostate cancer cells,
because human prostate cancer is not diagnosed by a single test,
but rather through many tests (including testing for PSA and
biopsy). During biopsies, the probe should be useful for testing
the carboxylesterase activity, thereby judging the progression
and severity of the human prostate cancer. Probe 1 might also be
useful for human prostate cancer prognosis, monitoring either
the deterioration of the cancer or the patient’s recovery. In addi-
tion, it is possible that probe 1 might also be used for the detection
of other human cancers, including breast cancer. Therefore, further
evaluation and application of probe 1 has the potential to improve
human health.
property for biological studies of human prostate cancer cells
in vitro and in vivo.24 Due to complexities associated with the
metastasis of cancer and the possibility that blood might also be
involved, other fluorescence wavelengths might also be useful.25
For this purpose, we propose that tetramethylrhodamine
(TMR)26–28 and naphthofluorescein29,30 units be used instead of
the fluorescein unit in probe 1; these two fluorophores provide
bright yellow and deep-red fluorescence, respectively. Indeed, the
synthesis of probe 1 is the first step in the development of a series
of sensors for carboxylesterase activity in human prostate cancer
and other cancers.
In summary, we have synthesized and evaluated a sensitive and
specific probe for the detection of human prostate cancer cells
among nontumorigenic prostate cells through the analysis of carb-
oxylesterase activity in cell lysates and living cells. We have also
determined that carboxylesterase activity is a specific biomarker
for human prostate cancer cells. The probe 1 is the first small-
molecule, water-soluble molecular beacon for the detection of
human prostate cancer cells.
Acknowledgments
This research is supported by the Office of Research and Spon-
sored Programs (RD09010, RD 0E82016 and RS0026 for Y.L.J. and
RD583 for W.L.S.) and SFRA Grants for V.P. and Y.L.J. from the Hon-
ors College at East Tennessee State University. This work was also
supported in part by NIH grant NIDA020120 to D.Y. We acknowl-
edge funds from the Dishner Chair of Excellence Endowment Funds
(KK).
Supplementary data
Supplementary data (1H and 13C NMR spectra of new
compounds) associated with this article can be found, in the online
version, at http://dx.doi.org/10.1016/j.bmcl.2012.04.055.
References and notes
1. Hassan, H. M.; Elnagar, A. Y.; Khanfar, M. A.; Sallam, A. A.; Mohammed, R.;
Shaala, L. A.; Youssef, D. T. A.; Hifnawy, M. S.; El Sayed, K. A. Eur. J. Med. Chem.
2011, 46, 1122.
2. Ghosh, S. K.; Kim, P.; Zhang, X.; Yun, S.-H.; Moore, A.; Lippard, S. J.; Medarova, Z.
Cancer Res. 2010, 70, 6119.
3. Andriole, G. L. et al N. Eng. J. Med. 2009, 360, 1310.
4. Debes, J. D.; Sebo, T. J.; Heemers, H. V.; Kipp, B. R.; Haugen, D. A. L.; Lohse, C. M.;
Tindall, D. J. Cancer Res. 2005, 65, 708.
5. Afrimzon, E.; Deutsch, A.; Shafran, Y.; Zurgil, N.; Sandbank, J.; Pappo, I.;
Deutsch, M. Clin. Exp. Metastasis 2008, 25, 213.
6. Katz, J.; Levitz, M.; Kadner, S. S.; Finlay, T. H. J. Steroid Biochem. Mol. Biol. 1991,
38, 17.
7. (a) LeRoy, B. E.; Nadella, M. V. P.; Toribio, R. E.; Leav, I.; Rosol, T. J. Vet. Pathol.
2004, 41, 131; (b) Xu, G.; Zhang, W.; Ma, M. K.; McLeod, H. L. Clin. Cancer Res.
2002, 8, 2605.
This Letter presents the first small-molecule molecular beacon
that is useful as a probe for detecting human cancer cells. To
reduce the fluorescence background during analysis, we suspect
that the quenching dabcyl unit could be substituted by a BHQ-1
unit, which has its maximum UV absorbance near 521 nm and
quenches most of the fluorescence of fluorescein at 525 nm; this
new quencher might also provide superior quenching effi-
ciency.20–23 Furthermore, the hydrolysis of the molecular beacon
probe 1 results in impressive green fluorescence, a desirable

3638
Y. L. Jiang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3632–3638
8. Dai, N.; Teo, Y. N.; Kool, E. T. Chem. Commun. 2010, 46, 1221.
9. Pospisil, P.; Wang, K.; Aowad, A. F.; Iyer, L. K.; Adelstein, S. J.; Kassis, A. I. Cancer
Res. 2007, 67, 2197.
10. Liang, Y.; Sharon, A.; Grier, J. P.; Rapp, K. L.; Schinazi, R. F.; Chu, C. K. Bioorg.
Med. Chem. 2009, 17, 1404.
11. Soong, C.-L.; Ogawa, J.; Shimizu, S. Appl. Environ. Microbiol. 1947, 2000, 66.
12. Fournand, D.; Bigey, F.; Arnaud, A. Appl. Environ. Microbiol. 1998, 64, 2844.
13. Jiang, Y. L.; Patel, P.; Klein, M. M. Bioorg. Med. Chem. 2010, 18, 7034.
14. Wang, H.; Kim, Y.; Liu, H.; Zhu, Z.; Bamrungsap, S.; Tan, W. J. Am. Chem. Soc.
2009, 131, 8221.
21. Johansson, M. K.; Fidder, H.; Dick, D.; Cook, R. M. J. Am. Chem. Soc. 2002, 124,
6950.
22. Peng, X.; Chen, H.; Draney, D. R.; Volcheck, W. M. Anal. Biochem. 2009, 388, 220.
23. Lovell, J. F.; Chen, J.; Jarvi, M. T.; Cao, W. G.; Allen, A. D.; Liu, Y.; Tidwell, T. T.;
Wilson, B. C.; Zheng, G. J. Phys. Chem. B 2009, 113, 3203.
24. Kobayashi, T.; Urano, Y.; Kamiya, M.; Ueno, T.; Kojima, H.; Nagano, T. J. Am.
Chem. Soc. 2007, 129, 6696.
25. Ho, N.-H.; Weissleder, R.; Tung, C.-H. Bioorg. Med. Chem. Lett. 2006, 16, 2599.
26. Welser, K.; Perera, M. D. A.; Aylott, J. W.; Chan, W. C. Chem. Commun. 2009,
6601.
15. Poeylaut-Palena, A. A.; Tomatis, P. E.; Karsisiotis, A. I.; Damblon, C.; Mata, E. G.;
Vila, A. J. Bioorg. Med. Chem. Lett. 2007, 17, 5171.
16. Crisp, G. T.; Jiang, Y. L. Tetrahedron 1999, 55, 549.
17. Crisp, G. T.; Jiang, Y. L. Tetrahedron Lett. 2002, 43, 3157.
18. Jiang, Y. L.; Gao, X.; Zhou, G.; Patel, A.; Javer, A. J. Org. Chem. 2010, 75, 324.
19. Lamale, B.; Henry, W. P.; Daniels, L. M.; Zhang, C.; Klein, S. M.; Jiang, Y. L.
Tetrahedron 2009, 65, 62.
20. Kadirvel, M.; Rattray, N. J. W.; Rajendran, R.; Zalloum, W. A.; Gbaj, A.;
Demonacos, C.; Bichenkova, E. V.; Freeman, S. New J. Chem. 2011, 35, 701.
27. Clark, P. M.; Dweck, J. F.; Mason, D. E.; Hart, C. R.; Buck, S. B.; Peters, E. C.;
Agnew, B. J.; Hsieh-Wilson, L. C. J. Am. Chem. Soc. 2008, 130, 11576.
28. Ngo, J. T.; Champion, J. A.; Mahdavi, A.; Tanrikulu, I. C.; Beatty, K. E.; Connor, R.
E.; Yoo, T. H.; Dieterich, D. C.; Schuman, E. M.; Tirrell, D. A. Nat. Chem. Biol.
2009, 5, 715.
29. Yao, S. Q.; Yeo, S. D. U.S. 7,449,558. Chem. Abst. 2005, 143, 128801.
30. Sun, W.-C.; Gee, K. R.; Klaubert, D. H.; Haugland, R. P. J. Org. Chem. 1997, 62,
6469.