Bioorganic & Medicinal Chemistry Letters 25 (2015) 44724476

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry Letters

journal homepage: www.elsevier.com/locate/bmcl

A pyrane based fluorescence probe for noninvasive prediction

of cerebral b-amyloid fibrils
Yan Cheng a,, Bi-yue Zhu a, Xue Li a, Guo-bo Li b, Sheng-yong Yang b, Zhi-rong Zhang a,
a
b

Key Laboratory of Drug Targeting and Drug Delivery Systems, West China School of Pharmacy, Sichuan University, Chengdu 610041, China
State Key Laboratory of Biotherapy/Collaborative Innovation Center of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Chengdu 610041, China

a r t i c l e

i n f o

a b s t r a c t
A potential fluorescence probe for in vivo detection of cerebral b-amyloid fibrils, (E)-2-(2-(2-(5-(dimethylamino)thiophen-2-yl)vinyl)-6-methyl-4H-pyran-4-ylidene)malononitrile (PT-1), was synthesized and
evaluated. In experiments in vitro, PT-1 exhibited clear labeling of b-amyloid fibrils and significant fluorescence changes upon binding to aggregated b-amyloid fibrils. It also showed favorite kinetics in the
brain, which is critical for cerebral imaging. In vivo fluorescence imaging with PT-1 and semi-quantitative
analysis of the images further confirmed noninvasive visualization of cerebral b-amyloid fibrils in vivo
and obvious distinction between APP/PS1 transgenic mice and wild-type controls. The results demonstrate the potential of PT-1 as a novel fluorescence probe for noninvasive prediction of cerebral b-amyloid
fibrils.
2015 Elsevier Ltd. All rights reserved.

Article history:
Received 15 July 2015
Revised 18 August 2015
Accepted 28 August 2015
Available online 29 August 2015
Keywords:
Neurodegenerative disease
b-Amyloid fibrils
Fluorescence probe

Neurodegenerative diseases, such as Alzheimers disease (AD),

Parkinsons disease (PD), and transmissible spongiform encephalopathies, are closely associated with the aggregation of amyloidogenic proteins in the brain. Although soluble amyloid precursor
proteins are distinct in amino acid sequence and native folding patterns, the aggregated amyloid fibrils are identified to share a characteristic cross-b structure by electron microscopy and X-ray
diffraction analysis.1,2 Approaches that allow in vivo visualization
of cerebral b-amyloid aggregation may provide beneficial insights
into a rational understanding of neuropathology, the monitoring
of efficacy of antiamyloid therapeutics, as well as the identification
of disease at risk and at an early stage.
Positron emission tomography (PET), which is capable of noninvasive human-scale neuroimaging, has been widely used for clinical applications. Over the past few years, numerous efforts have
been made in the field of in vivo amyloid detection utilizing
PET.311 Despite the remarkable progress in PET imaging of cerebral b-amyloid fibrils, its practical applications for testing the efficacy of candidate antiamyloid therapies in experimental animal
models may be hampered due to the complexity of the experimental procedure and data analysis, limited availability of radioisotopes, and radiation exposure.
On the contrary, optical imaging using fluorescence probes may
be a promising alternative approach for high throughout screening
Corresponding authors. Tel.: +86 28 85501566; fax: +86 28 85501615.
E-mail addresses:
(Z.-r. Zhang).

yancheng@scu.edu.cn

(Y.

http://dx.doi.org/10.1016/j.bmcl.2015.08.081
0960-894X/ 2015 Elsevier Ltd. All rights reserved.

Cheng),

zrzzl@vip.sina.com

of cerebral b-amyloid fibrils. Notably, near-infrared fluorescence

imaging, which benefits from reduced autofluorescence and
acceptable penetration depth, is more amenable to in vivo detection of b-amyloid fibrils in the brain.12,13 To distinguish b-amyloid
fibrils from the background, in vivo fluorescence probes that are
specific for b-amyloid fibrils face more stringent design criteria
than in vitro reagents. Thioflavin T (ThT), which exhibits thousand-time brighter fluorescence upon binding, has been most
widely used for the histological staining of b-amyloid fibrils; however, short wavelength detection and limited bloodbrain barrier
permeability makes ThT not suitable for in vivo cerebral imaging.13,14 To obtain high target-to-background ratios, several empirical criteria are listed for an ideal amyloid imaging probe. First, the
probe should yield high local concentrations in the brain but contributes minimally to the background signal; second, the probe
shows specific labeling of b-amyloid fibrils in the brain; third,
the probe exhibits chemical or biological amplification upon binding to b-amyloid fibrils.12,13,15 At present, there are limited fluorescence probes for b-amyloid fibrils compared to PET probes.13,1625
Although most of the optical imaging systems are so far limited to
animal studies, several new technologies are poised to enter clinical trials. Thus, strategies for noninvasive optical imaging of cerebral b-amyloid fibrils with novel imaging agents are still worth
pursuing.
In the present study, we target cerebral b-amyloid fibrils of
transgenic mouse models by employing noninvasive fluorescence
imaging system in conjunction with a novel fluorescence probe
(PT-1, Fig. 1a). The design rationale of PT-1 was based on

Y. Cheng et al. / Bioorg. Med. Chem. Lett. 25 (2015) 44724476

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Figure 1. (a) Chemical structure of PT-1; (b) Emission spectra of PT-1 (1 lM) in the presence of Ab142 aggregates (dashed line) and without the aggregates (solid line).

the following findings. First, a compact structure with pushpull

architecture could provide long wavelength falling into near-infrared
range and good permeability of bloodbrain barrier. PAD-1 (CAS:
51325-91-8) is one of the typical fluorescent dyes with donor-pacceptor structure, exhibiting good affinity to b-amyloid fibrils.26
By substitution of phenyl group with thiophenyl group, emission
wavelength could be further redshifted.27 Second, N,N0 -dimethylamino group on the phenyl ring has proven to show relatively
higher affinity to b-amyloid than other substituents.17,24,28 Thus,
N,N0 -dimethylamino group on the thiophenyl ring may maintain
good binding affinity to b-amyloid fibrils. PT-1 was prepared by
the condensation of (2,6-dimethyl-4H-pyran-4-ylidene) malononitrile with 5-(dimethylamino)thiophene-2-carbaldehyde in the
presence of piperidine (yield: 22%, purity: P99%).
As expected, PT-1 displayed favorable fluorescence properties
(Table 1). PT-1 and PAD-1 fluoresces with emission maxima of
620 nm and 603 nm in methanol, respectively. The distinct bathochromic shift in emission appears to be a consequence of reduced
steric hindrance when phenyl ring is replaced by thiophenyl ring.
The thiophenyl ring and its excocyclic bond angle between its
2- and 5-substituents reduce the average torsion angle and lower
energy transitions. Emission maximum of PT-1 in PBS solution
further redshifted to 634 nm.27,29 Upon mixing with aggregated
b-amyloid fibrils (Ab142 fibrils), emission spectra of PT-1 exhibited
a blue-shift of 36 nm in wavelength and 25-fold increase in fluorescence intensity, suggesting an optical turn-on effect upon the
mixing (Fig. 1b). The main factor determining the blue-shift and
significant enhancement may be the steric restriction of rotation
of the molecule when incorporated in aggregated b-amyloid
fibrils.13,14
Next, saturation assays were carried out to quantify the binding
affinity of PT-1 to aggregated b-amyloid fibrils according to conventional methods.17,22,24,26 The amyloid plaques in AD brains are
mainly composed of aggregated Ab142 fibrils, so that Ab142 aggregates were used for in vitro binding assays. PT-1 binds to Ab142
aggregates with a binding affinity Kd of 54.3 nM, which is much
higher than that of the reported amyloid-specific fluorescence

Table 1
Fluorescence Properties of PT-1 and PAD-1 (1 lM)

PT-1
PAD-1b
a

kabs (nm)

kex (nm)

kem (nm)

e (cm1/M)

UF

528
467

530
480

620
603

42700
44936

0.61c
0.43

Data were obtained using methanol as solvent.

Data from Ref. 26.
c
Quantum yield was measured in dichloromethane with rhodamine B as a
reference.
b

probe AOI987 (Kd = 220 nM), and is close to that of CRANAD-2

(Kd = 38.9 nM) and NIR-2c (Kd = 26.9 nM).16,17,24 Notably, PT-1
maintained high binding affinity after substitution of phenyl group
of PAD-1 (Kd = 58.9 nM) with thiophenyl group. The result indicated that PT-1 may share the same binding site(s) or binding pattern as PAD-1.
APP/PS1 transgenic mice are a type of double transgenic mice
that express a mutant human presenilin 1 (PS1) and chimeric
human/mouse amyloid precursor protein (APPswe) in cerebral
neurons, which have been widely used in studies of neurological
diseases related to amyloid formation in the brain.30 As shown in
Figure 2, b-amyloid plaques were clearly observed by staining
APP/PS1 brain sections with PT-1. The fluorescent spots were
consistent with the presence of b-amyloid plaques on the adjacent
section stained by thioflavin S, a pathological dye widely used for
b-amyloid fibrils. Autofluorescence was not observed under the
same imaging condition. The results suggested that PT-1 could
image natural formed b-amyloid fibrils with good specificity and
negligible autofluorescence.
To further investigate the possible interaction between PT-1
and b-amyloid fibrils, molecular dock simulation was carried out
on two-fold Ab140 fibril structure (PDB ID: 2LMO).25,3133 Ab142
and Ab140 are two major isoforms of Ab. The only difference
between Ab142 and Ab140 is that Ab142 has two extra residues
at the C-terminus. Both Ab142 and Ab140 have been shown to form
fibrils with parallel b-sheet structures.34 As presented in Figure 3,
the 16-KLVFF-20 and 30-AII-32 hydrophobic clefts of Ab140 fibril
structure were found to be the major binding site of PT-1 with
the GoldScore value of 69.9. The simulation showed the binding
between PT-1 and b-amyloid fibrils with a preference for nonpolar
16-KLVFF-20 and 30-AII-32 residues, and PT-1 forms a hydrogenbonding interaction with the ILE31 of b-amyloid fibrils. It is plausible that the nearly planar structure of PT-1 inserts into the
hydrophobic cleft of Ab140 fibril structure with minor stereo
hindrance and interacts with the side chains of nonpolar residues
16-KLVFF-20 through strong hydrophobic interaction. Thus, it is
likely that the optical turn-on and emission shift behavior of
PT-1 in the presence of b-amyloid fibrils come from a lower
dielectric constant microenvironment around PT-1.25
Efficient permeability of the bloodbrain barrier is critical for
in vivo cerebral imaging. Brain uptakes of PT-1 were analyzed by
ex vivo imaging studies using normal mice (Fig. 4). PT-1 penetrated
the bloodbrain barrier efficiently with high initial concentration
in the brain, and washed out rapidly from the brain with time.
The relative fluorescence intensity of PT-1 decreased to 15% at
120 min postinjection, while signals from PAD-1 remained above
20% in normal mice.26 The comparison suggests minimal
background signals from PT-1 during in vivo imaging of cerebral

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Y. Cheng et al. / Bioorg. Med. Chem. Lett. 25 (2015) 44724476

Figure 2. Fluorescence staining of b-amyloid fibrils from an aged APP/PS1 brain section with PT-1 (A). b-Amyloid plaques were confirmed by thioflavin S staining of the
adjacent brain section (B). Autofluorescence was not observed under the same imaging condition (C).

Figure 3. The binding site of PT-1 on two-fold Ab140 fibril structure based on molecular dock simulation using GOLD program.

Figure 4. Ex vivo analysis of relative fluorescence intensities of PT-1 in normal

brains at each timepoint postinjection (2.0 mg/kg, n = 5).

amyloid fibrils. The kinetics of PT-1 in the brain may be mainly

caused by passive diffusion mechanism with calculated Log P value
of 2.24 (ChemDraw Ultra 8.0).15

In vivo imaging of cerebral amyloid fibrils was performed using

fluorescence reflectance imaging modality, which is commonly
used for fast real-time imaging. Aged APP/PS1 transgenic mice
were selected as amyloid positive models and age-matched
C57BL/6 mice were used as wild type controls. A solution of PT-1
(2.0 mg/kg) was intravenously injected into the tail of the mice.
Real-time images of the mice were recorded immediately after
PT-1 injection. As shown in Figure 5, both of APP/PS1 and control
group showed high fluorescence signals from brain areas within
initial 5 min, and the signals diminished with time. To overcome
individual difference in brain images, the measured fluorescence
intensity was normalized to that at an initial timepoint. Semiquantitative analysis of the images verified the slower clearance
of fluorescence signals around brain areas from APP/PS1 group
compared to that of the control group (Fig. 6). The fluorescence signal of APP/PS1 group was higher than that of the wild type group
after 5 min postinjection, and the decay of fluorescence signal
was slower in APP/PS1 group compared to the wild type group,
which allowed a gradual discrimination between APP/PS1 and wild
type group after the administration of PT-1. The retention of fluorescence signals around brain area from APP/PS1 group may result
from the binding of PT-1 to cerebral amyloid fibrils. To confirm the

Y. Cheng et al. / Bioorg. Med. Chem. Lett. 25 (2015) 44724476

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Figure 5. Representative images around brain areas of APP/PS1 and age-matched wild-type controls preinjection and postinjection of PT-1 (2.0 mg/kg).

Figure 6. Relative fluorescence intensity around brain areas of APP/PS1 transgenic

mice and wild-type controls after intravenous injection of 2.0 mg/kg of PT-1 (n = 3).
The fluorescence signal of APP/PS1 group was higher than that of the wild type
group after 5 min postinjection, and the decay of fluorescence signal was slower in
APP/PS1 group compared to the wild type group.

specific labeling of cerebral b-amyloid fibrils in vivo, fluorescence

observation of ex vivo brain sections was performed using aged
APP/PS1 transgenic mice and age-matched controls. As shown in

Figure 7, fluorescent spots were clearly observed in APP/PS1 brains,

which were confirmed to be b-amyloid plaques by thioflavin S
staining. In contrast, no significant fluorescence was observed in
the brains of wild type controls. The results further confirmed that
the imaging probe (PT-1) crossed the bloodbrain barrier and
bound to b-amyloid plaques selectively in the brain.
To achieve clearer imaging of cerebral b-amyloid fibrils in vivo,
targeted fluorescence probes with emission wavelengths in nearinfrared range are preferred. Our previously reported pyrane-based
probe (PAD-1) with N,N0 -dimethylaminophenyl group showed
good binding to b-amyloid fibrils with relatively short wavelength
of emission (603 nm). Consequently, PT-1 with N,N0 -dimethylaminothiophenyl group was designed to further lengthen the
emission wavelength (634 nm). Compared to PAD-1, PT-1 displayed better spectral properties and kinetics in normal brains,
which is favorable for enhancing specific binding and minimizing
background signals during in vivo imaging. Although the emission
wavelength decreased upon amyloid binding, PT-1 showed longer
excitation and emission wavelengths than those of the most common biological autofluorescencing molecules, such as NADPH, flavins, and extracellular matrix (collagens).35 In vitro fluorescent
staining and in vivo imaging experiments further confirmed that
the autofluorescence from biological matter was negligible under
the imaging condition. Despite of the substitution of phenyl group
with thiophenyl group, PT-1 showed specific labeling of cerebral
b-amyloid fibrils in vivo, with high binding affinity similar to that

Figure 7. Fluorescence observation of ex vivo brain sections from APP/PS1 transgenic mice and wild-type controls. Brains were removed at 60 min postinjection of PT-1
(2.0 mg/kg). b-Amyloid plaques in brain tissue from APP/PS1 transgenic mouse was clearly visualized (A) while no labeling was observed by this probe in wild-type brains (C).
b-Amyloid plaques were confirmed by staining the adjacent brain section with thioflavin S (B).

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Y. Cheng et al. / Bioorg. Med. Chem. Lett. 25 (2015) 44724476

of PAD-1. Therefore, further structural modifications of pyrane-based

compounds, such as prolonged double bonds, may lead to more
near-infrared probes with specific amyloid binding. Currently, fluorescence reflectance imaging is most commonly used for the evaluation of fluorescence probes for cerebral b-amyloid fibrils. Due to
several methodological limitations, the spatial resolution and
quantitative ability of fluorescence reflectance imaging is relatively
lower than PET. Thus, fluorescence reflectance imaging could only
obtain semi-quantitative analysis of the images. To improve image
visualization and enable quantitative analysis, tomographic
fluorescence imaging system, which enables reconstruction of
three-dimensional maps, may further be applied.12,17 As PT-1
showed excellent labeling ability of cerebral b-amyloid fibrils
in vivo, quantitative analysis would be possible with the application of three-dimensional tomographic fluorescence imaging.
In conclusion, optical imaging approaches are potentially useful
for in vivo visualization of cerebral b-amyloid fibrils of neurodegenerative disorders. In the present study, we demonstrated noninvasive detection of cerebral b-amyloid fibrils using in vivo
fluorescence imaging system with a novel probe PT-1. PT-1 exhibited optical turn-on effect upon binding to aggregated b-amyloid
fibrils. Obvious distinction was observed between APP/PS1 transgenic mice and wild-type controls by real-time image analysis
after intravenous administration of PT-1. It is expected that this
fluorescence imaging strategy with PT-1 be applied to detect cerebral b-amyloid fibrils, facilitating noninvasive evaluation of the
efficacy of antiamyloid therapies as well as the identification of
stages of amyloid pathology.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (Grant No. 81402891), China Postdoctoral Science
Foundation (Grant No. 2014M560723), Doctoral Programs of
Higher Education of China (Grant No. 20130181120114), and
Scientific Research Foundation for Youth Scholars from Sichuan
University (No. 2012SCU11091). The authors would like to thank
Dr. Mengchao Cui (College of Chemistry, Beijing Normal University) for help with the preparation of APP/PS1 transgenic mouse
brain sections.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.08.
081.
References and notes
1. Knowles, T. P.; Vendruscolo, M.; Dobson, C. M. Nat. Rev. Mol. Cell. Biol. 2014, 15,
384.

2. Rochet, J. C.; Lansbury, P. T., Jr. Curr. Opin. Struct. Biol. 2000, 10, 60.
3. Nordberg, A. Lancet. Neurol. 2004, 3, 519.
4. Ono, M.; Wilson, A.; Nobrega, J.; Westaway, D.; Verhoeff, P.; Zhuang, Z. P.;
Kung, M. P.; Kung, H. F. Nucl. Med. Biol. 2003, 30, 565.
5. Mathis, C. A.; Wang, Y.; Holt, D. P.; Huang, G. F.; Debnath, M. L.; Klunk, W. E. J.
Med. Chem. 2003, 46, 2740.
6. Klunk, W. E.; Engler, H.; Nordberg, A.; Wang, Y.; Blomqvist, G.; Holt, D. P.;
Bergstrom, M.; Savitcheva, I.; Huang, G. F.; Estrada, S.; Ausen, B.; Debnath, M.
L.; Barletta, J.; Price, J. C.; Sandell, J.; Lopresti, B. J.; Wall, A.; Koivisto, P.; Antoni,
G.; Mathis, C. A.; Langstrom, B. Ann. Neurol. 2004, 55, 306.
7. Koole, M.; Lewis, D. M.; Buckley, C.; Nelissen, N.; Vandenbulcke, M.; Brooks, D.
J.; Vandenberghe, R.; Van Laere, K. J. Nucl. Med. 2009, 50, 818.
8. Villemagne, V. L.; Ong, K.; Mulligan, R. S.; Holl, G.; Pejoska, S.; Jones, G.; OKeefe,
G.; Ackerman, U.; Tochon-Danguy, H.; Chan, J. G.; Reininger, C. B.; Fels, L.; Putz,
B.; Rohde, B.; Masters, C. L.; Rowe, C. C. J. Nucl. Med. 2011, 52, 1210.
9. Choi, S. R.; Golding, G.; Zhuang, Z. P.; Zhang, W.; Lim, N.; Hefti, F.; Benedum, T.
E.; Kilbourn, M. R.; Skovronsky, D.; Kung, H. F. J. Nucl. Med. 1887, 2009, 50.
10. Clark, C. M.; Schneider, J. A.; Bedell, B. J.; Beach, T. G.; Bilker, W. B.; Mintun, M.
A.; Pontecorvo, M. J.; Hefti, F.; Carpenter, A. P.; Flitter, M. L.; Krautkramer, M. J.;
Kung, H. F.; Coleman, R. E.; Doraiswamy, P. M.; Fleisher, A. S.; Sabbagh, M. N.;
Sadowsky, C. H.; Reiman, E. P.; Zehntner, S. P.; Skovronsky, D. M. JAMA 2011,
305, 275.
11. FDA approves 18F-florbetapir PET agent. J. Nucl. Med. 2012, 53, 15N.
12. Weissleder, R.; Pittet, M. J. Nature 2008, 452, 580.
13. Nesterov, E. E.; Skoch, J.; Hyman, B. T.; Klunk, W. E.; Bacskai, B. J.; Swager, T. M.
Angew. Chem., Int. Ed. 2005, 44, 5452.
14. Voropai, E. S.; Samtsov, M. P.; Kaplevskii, K. N.; Maskevich, A. A.; Stepuro, V. I.;
Povarova, O. I.; Kuznetsova, I. M.; Turoverov, K. K.; Fink, A. L.; Uverskii, V. N. J.
Appl. Spectrosc. 2003, 70, 868.
15. Mathis, C. A.; Wang, Y.; Klunk, W. E. Curr. Pharm. Des. 2004, 10, 1469.
16. Hintersteiner, M.; Enz, A.; Frey, P.; Jaton, A. L.; Kinzy, W.; Kneuer, R.; Neumann,
U.; Rudin, M.; Staufenbiel, M.; Stoeckli, M.; Wiederhold, K. H.; Gremlich, H. U.
Nat. Biotechnol. 2005, 23, 577.
17. Ran, C.; Xu, X.; Raymond, S. B.; Ferrara, B. J.; Neal, K.; Bacskai, B. J.; Medarova,
Z.; Moore, A. J. Am. Chem. Soc. 2009, 131, 15257.
18. Okamura, N.; Mori, M.; Furumoto, S.; Yoshikawa, T.; Harada, R.; Ito, S.;
Fujikawa, Y.; Arai, H.; Yanai, K.; Kudo, Y. J. Alzheimers Dis. 2011, 23, 37.
19. Chang, W. M.; Dakanali, M.; Capule, C. C.; Sigurdson, C. J.; Yang, J.; Theodorakis,
E. A. ACS Chem. Neurosci. 2011, 2, 249.
20. Liu, K.; Guo, T. L.; Chojnacki, J.; Lee, H. G.; Wang, X.; Siedlak, S. L.; Rao, W.; Zhu,
X.; Zhang, S. ACS Chem. Neurosci. 2012, 3, 141.
21. Ono, M.; Ishikawa, M.; Kimura, H.; Hayashi, S.; Matsumura, K.; Watanabe, H.;
Shimizu, Y.; Cheng, Y.; Cui, M.; Kawashima, H.; Saji, H. Bioorg. Med. Chem. Lett.
2010, 20, 3885.
22. Ono, M.; Watanabe, H.; Kimura, H.; Saji, H. ACS Chem. Neurosci. 2012, 3, 319.
23. Watanabe, H.; Ono, M.; Matsumura, K.; Yoshimura, M.; Kimura, H.; Saji, H. Mol.
Imaging 2013, 12, 338.
24. Cui, M.; Ono, M.; Watanabe, H.; Kimura, H.; Liu, B.; Saji, H. J. Am. Chem. Soc.
2014, 136, 3388.
25. Fu, H.; Cui, M.; Tu, P.; Pan, Z.; Liu, B. Chem. Commun. 2014, 11875.
26. Cheng, Y.; Zhu, B.; Deng, Y.; Zhang, Z. Anal. Chem. 2015, 87, 4781.
27. Dingemans, T. J.; Bacher, A.; Thelakkat, M.; Pedersen, L. G.; Samulski, E. T.;
Schmidt, H. W. Synth. Metals 1999, 105, 171.
28. Cheng, Y.; Ono, M.; Kimura, H.; Kagawa, S.; Nishii, R.; Kawashima, H.; Saji, H.
ACS Med. Chem. Lett. 2010, 1, 321.
29. Park, K. C.; Dodd, L. R.; Levon, K.; Kwei, T. K. Macromolecules 1996, 29, 7149.
30. Bilkei-Gorzo, A. Pharmacol. Ther. 2014, 142, 244.
31. Petkova, A. T.; Ishii, Y.; Balbach, J. J.; Antzutkin, O. N.; Leapman, R. D.; Delaglio,
F.; Tycko, R. Proc. Natl. Acad. Sci. 2002, 99, 16742.
32. Cook, N. P.; Ozbil, M.; Katsampes, C.; Prabhakar, R.; Mart, A. A. J. Am. Chem. Soc.
2013, 135, 10810.
33. Yang, Y.; Cui, M.; Zhan, g. X.; Dai, J.; Zhang, Z.; Lin, C.; Guo, Y.; Liu, B. J. Med.
Chem. 2014, 57, 6030.
34. Gu, L.; Guo, Z. J. Neurochem. 2013, 126, 305.
35. Monici, M. Biotechnol. Annu. Rev. 2005, 1, 227.