Structure-Property Relationships of Polyethylene Glycol Modified Fluorophore as Near-Infrared Aβ Imaging Probes

Article abstract of DOI:10.1021/acs.analchem.8b01712

To optimize the lipophilicity and improve in vivo pharmacokinetics of near-infrared probes targeted Aβ plaques, we designed, synthesized, and evaluated a series of polyethylene glycol modified probes with hydroxyl and methoxyl terminals. The relationships between chemical structure and optical, biological properties were systemically elucidated. The results indicated that a desired Aβ probe should keep a balance among molecular rigidity, size, and lipophilicity. Probe 12d displayed improved properties including intense and selective response to Aβ_1-42 aggregates (K_d = 7.3 nM, 22-fold fluorescence enhancement and emission maxima at 715 nm upon interaction with Aβ_1-42 aggregates), sufficient blood-brain barrier penetration (3.04% ID/g), and fast wash out from the brain (brain_{2 min}/brain_{60 min} = 10.1). Clear fluorescence signals retention in transgenic mice than control mice in in vivo near-infrared imaging. Hence, polyethylene glycol modified probes retained favorable optical properties but displayed great improvement of biological properties for Aβ detection.

Full text of DOI:10.1021/acs.analchem.8b01712

Subscriber access provided by UNIVERSITY OF TOLEDO LIBRARIES
Article
Structure-Property Relationships of Polyethylene Glycol
Modified Fluorophore as Near-infrared A# Imaging Probes
Kaixiang Zhou, Yuying Li, Yi Peng, Xiaomei Cui, Jiapei Dai, and Mengchao Cui
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01712 • Publication Date (Web): 14 Jun 2018
Downloaded from http://pubs.acs.org on June 15, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 13
Analytical Chemistry
1
2
3
4
5
6
7
8
9
StructureꢀProperty Relationships of Polyethylene Glycol Modꢀ
ified Fluorophore as Nearꢀinfrared Aβ Imaging Probes
Kaixiang Zhoua, Yuying Lia, Yi Penga, Xiaomei Cuia, b, Jiapei Daic, Mengchao Cuia,*
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a Key Laboratory of Radiopharmaceuticals, Ministry of Education, College of Chemistry, Beijing Normal University, Beijing
100875, China
b Department of Chemistry and Environmental Sciences, Tibet University, Lhasa 850000, China
c Wuhan Institute for Neuroscience and Neuroengineering, SouthꢀCentral University for Nationalities, Wuhan 430074, China
ABSTRACT: To optimize the lipophilicity and improve in vivo pharmacokinetics of nearꢀinfrared probes targeted Aβ plaques, we
designed, synthesized and evaluated a series of polyethylene glycol modified probes with hydroxyl and methoxyl terminals. The
relationships between chemical structure and optical, biological properties were systemically elucidated. The results indicated that a
desired Aβ probe should keep a balance among molecular rigidity, size, and lipophilicity. Probe 12d displayed improved properties
including intense and selective response to Aβ1ꢀ42 aggregates (Kd = 7.3 nM, 22 fold fluorescence enhancement and emission maxiꢀ
ma at 715 nm upon interaction with Aβ1ꢀ42 aggregates), sufficient bloodꢀbrain barrier penetration (3.04 % ID/g), and fast wash out
from the brain (brain2min/brain60min = 10.1). Clear fluorescence signals retention in transgenic mice than control mice in in vivo nearꢀ
infrared imaging. Hence, polyethylene glycol modified probes retained favorable optical properties but displayed great improveꢀ
ment of biological properties for Aβ detection.
INTRODUCTION
radionuclide imaging, fluorescenceꢀbased optical imaging
gained incremental interest for detection of physiological and
biochemical process both in vitro and in vivo. However, fluoꢀ
rescence imaging is limited due to the poor light penetration of
the biological tissues, but it’s uplifting to remark that the light
in the nearꢀinfrared (NIR) region (650 ꢀ 900 nm) have better
tissue penetration and make it possible to realꢀtime imaging in
living organism10ꢀ14. With the assistant of NIR light, it’s possiꢀ
ble to achieve depth imaging, decrease the photodamage and
avoid autoflourescence from biological matters9. NIR optical
imaging using probes targeting to Aβ aggregates in the brain is
a strategy used to detect AD nonꢀinvasively in vivo. A number
of small NIR probes including charged oxazine dyes15 and
styrylꢀbased cyanine dyes16, as well as neutral probes such as
bithiophene derivatives17, difluoroboronate curcumin derivaꢀ
Alzheimer’s disease (AD) is a neurodegenerative disorder that
severely influences millions of people with the exacerbation of
the population ages, while there is no cure or stoppable theraꢀ
pies yet available1. To date, there’s still debate about the nosoꢀ
genesis of AD, the wellꢀestablished amyloid cascade hypotheꢀ
sis points out that the accumulation and deposition of the βꢀ
amyloid (Aβ) plaques is responsible for this disease. The proꢀ
teolysis pathway of the amyloid precursor protein produced
Aβ40 and Aβ42 fragments by βꢀ and γꢀsecretases, and these Aβ
monomers are easily aggregated to oligomers, fibers and finalꢀ
ly piled into Aβ plaques. The excess accumulation of Aβ
plaques lead to reduction of neurotransmitters, cognition deꢀ
fects and synaptic plasticity2ꢀ4. More recently, Moir and
coworkers revealed Aβ protein could act as antimicrobial pepꢀ
tides and exert positive physiological functions. The increased
Aβ generation may be the response of the received infection or
sterile inflammatory stimuli. Hence, Aβ protein plays a protecꢀ
tive role in innate immunity in the brain5, 6. In any case, the
aggregated Aβ protein is still believed to have close connecꢀ
tion with AD, trace or monitor the level of Aβ aggregates in
the brain may provide ample and embedded information to
better understand AD. In recent years, much effort have been
devoted to detect Aβ plaques nonꢀinvasively in the early stage
of AD, and various imaging techniques such as singleꢀphoton
emissionꢀcomputed tomography (SPECT)7, positron emission
computed tomography (PET)8 and optical imaging9 were emꢀ
ployed.
tives18ꢀ20, thiophene derivatives21, styrylpyran derivatives22ꢀ24
,
and boronꢀdipyrromethene probes25 were developed to label or
monitor Aβ species in the brain. These probes were commonly
designed on the basis of pullꢀpush structure with electron doꢀ
nor and acceptor bridged by conjugated π system (DꢀπꢀA).
Since 2014, according to the DꢀπꢀA structure, our group reꢀ
ported series of smart Aβ probes with different kinds of elecꢀ
tron acceptors, such as malononitrile, methyl cyanoacetate,
dimethyl malonate, meldrum’s acid, dimedone, and barbituric
27
acid26,
,
among
these
probes,
2ꢀ((2E,4E)ꢀ5ꢀ(4ꢀ
(dimethylamino)phen
yl)pentaꢀ2,4ꢀdienꢀ1ꢀ
ylidene)malononitrile (DANIR 2c)28 exhibited excellent ability
to image Aβ plaques in living mouse. Thereafter, (E)ꢀ2ꢀ(3ꢀ(6ꢀ
(dimethylamino)naphthalenꢀ2ꢀyl)allyli
(DANIR and
dene)malononitrile
2ꢀ((2E,4E)ꢀ5ꢀ(6ꢀ((2ꢀ
3b)29
PET and SPECT are powerful imaging tools and widely used
all over the world, however, their tracers must be labeled with
proper radionuclides, high cost, and exposure to the radioacꢀ
tivity impeded their extensive application. As an alternative to
hydroxyethyl)(methyl)amino)naphthalenꢀ2ꢀyl)pentaꢀ2,4ꢀdienꢀ
1ꢀylidene)malononitrile (DANIR 8c)30 with a naphthalene π
bridge were reported, compared with DANIR 2c, they disꢀ
ACS Paragon Plus Environment

Analytical Chemistry
Page 2 of 13
played high quantum yield and more favorable in vivo properꢀ old, male; 71ꢀyearꢀold, female; 85ꢀyearꢀold, male) and healthy
1
2
3
4
5
6
7
8
ties. More recently, we expanded the scope of these DꢀπꢀA
probes and systemically elucidated the relationships between
πꢀconjugated system (different lengths of polyenic chains and
aromatic rings) and properties (optical and biological)31. Polyꢀ
ethylene glycol (PEG) chains are connected by repeated units
of ethylene glycol monomer and demonstrated favorable funcꢀ
tion on pharmacokinetic and pharmacodynamic in polypeptide
drugs32. Since 2005, Zhang et al. reported the conjugation of
short PEG chains to Aβ homing ligands with a terminal fluoꢀ
rineꢀ18 atom, and achieved simple adjustment of the ligand
lipophilicity and better in vivo pharmacokinetics33, 34. In this
study, to further investigate the relationship between structure
and properties including optical and biological, we applied
short PEG chains with hydroxyl and methoxyl terminals to the
electron acceptors of naphthalene ring based DꢀπꢀA probes.
humans (68ꢀyear old, female; 80ꢀyear old, female; 84ꢀyear old,
male; 89ꢀyear old, male; 74ꢀyear old, male) were obtained
from Chinese Brain Bank Center. All protocols requiring the
use of animals were approved by the animal care committee of
Beijing Normal University.
Chemistry. The PEG modified acceptors (1 - 6) and the final
NIR probes were outlined in Scheme 1 and S1 in Supporting
Information. Details of synthesis, original 1H NMR, 13C NMR,
MS, and HRMS spectra were also included in Supporting Inꢀ
formation.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Spectroscopic Determination. Ultraviolet absorption spectra,
fluorescence spectra and absolute quantum yields of these
probes in various solvents were tested using traditional methꢀ
ods. The fluorescence interaction of selected probes with
Aβ1−42 aggregates and bovine serum albumin (BSA) were deꢀ
termined according to our reported methods26, 28
.
EXPERIMENTAL SECTION
Biological Evaluations. In vitro fluorescence staining on
brain sections, in vitro saturation binding assay using Aβ1−42
aggregates, in vitro stability study, cytotoxicity study, brain
entry values and kinetics in normal mice, in vivo NIR imaging
and ex vivo histopathological staining in transgenic and ageꢀ
matched control mice were completed using our reported
methods26, 28, the detailed procedures were described in Supꢀ
porting Information.
General Information. Reagents and solvents were analyticalꢀ
grade and were purchased from OuheChem, TCI, and J&K
Chemicals and used without further purification. Reactions
were monitored by thin layer chromatography (TLC) plates
(aluminum sheets with silica gel 60 F254 plates, Merck). Colꢀ
umn chromatography was carried out with silica gel (45 ꢀ 75
ꢀm, Yantai Industry Research Institute). Synthetic Aβ1ꢀ42 pepꢀ
tides were purchased from Peptide Institute, Inc. (Japan), and
Aβ1ꢀ42 fibers for in vitro assays were aggregated using the proꢀ
RESULTS AND DISCUSSION
NIR Probes Synthesis
1
cedures reported previously35. H and 13C NMR spectra were
recorded on a Bruker Avance III (400 MHz or 100 MHz,
Germany) spectrometer in CDCl3, CF3COOD or DMSOꢀd6
solutions at room temperature (rt). Chemical shift (δ) is reportꢀ
ed in ppm downfield from tetramethylsilane (TMS) and couꢀ
pling constants (J) are reported in Hertz (Hz), and the multiꢀ
plicity is defined by s (singlet), d (doublet), t (triplet) or m
(multiplet). Mass spectrometry (MS) were acquired by the
Surveyor MSQ Plus (ESI) (Waltham, MA, USA) instrument.
High resolution mass spectra (HRMS) were acquired by
Thermo scientific QꢀExactive (ESI) (USA) mass spectrometer.
Fluorescence spectra were measured on
a
RFꢀ
5301PC spectrofluorophotometer (Shimadzu, Japan). The
absolute fluorescence quantum yields were acquired by the
Absolute PL Quantum Yield Spectrometer C11347 (Hamamaꢀ
tsu, Japan). UVꢀvisible spectra were carried out on the UVꢀ
3600 UVꢀVis spectrophotometer (Shimadzu, Japan). Highꢀ
performance liquid chromatography (HPLC) was conducted
on Primaide system (Hitachi, Japan). The sample was anaꢀ
lyzed on a Venusil MP C18 reverse column (5 ꢀm, 4.6
mm×250 mm, BonnaꢀAgela Technologies, China) eluted with
an isocratic system at a flow rate of 1.0 mL/min, mobile phase
was water and acetonitrile, respectively. Fluorescence obserꢀ
vation was performed on EVOS FL imaging system equipped
with GFP, RFP, Texas Red, and CY5 filter sets (Life, USA).
UV absorbance was read at 490 nm on Epoch microplate specꢀ
trophotometer (Biotek, USA). Male ICR mice (4 weeks, 18 ꢀ
22 g) used for brain uptake studies were purchased from Beiꢀ
jing Vital River Laboratories (China). Double transgenic mice
(C57BL6, APPsw/PSEN1, 19ꢀmonth old) used as AD model
and ageꢀmatched control mice (C57BL6, 19ꢀmonth old) were
purchased from Institute of Laboratory Animal Sciences, Chiꢀ
nese Academy of Medical Sciences. Postmortem brain tissues
of temporal lobe from autopsy confirmed AD patients (91ꢀyear
old, male; 68ꢀyear old, female; 64ꢀyear old, female; 76ꢀyear
Scheme 1. Reagents and conditions: (a) DCC, DMAP, anhyꢀ
drous CH2Cl2, rt, 5 h; (b) piperidine, MeOH or CH2Cl2, 60 °C,
reflux, 3 h.
Chemical synthesis was applied according to the methods
shown in Scheme 1. Naphthyl aldehydes with different lengths
of polyenic chains were obtained by iterative Wittig reaction
following the similar methods reported previously31. The PEG
modified acceptors (1 - 6) were produced by esterification
from 2ꢀcyanoacetate acid and short PEG chains (n = 1 ꢀ 3)
with hydroxyl and methoxyl terminals, dicyclohexylcarꢀ
bodiimide (DCC) and 4ꢀdimethylaminopyridine (DMAP) were
employed as dehydrant and catalyst, respectively. The final
NIR probes (7a-c to 13a-c) were readily synthesized using
Knoevengel condensation between appropriate naphthyl aldeꢀ
hydes and PEG modified cyanoacetate substrates catalyzed by
small amount of piperdine. All the probes were verified by 1H,
13C NMR, HRMS and proved to be greater than 95% purity
with HPLC. It should be noted that unlike our previously reꢀ
ported Knoevengel condensations using methanol or ethanol
as solvents, for these PEG modified cyanoacetate substrates,
transesterification is much more likely to happen in methanol
ACS Paragon Plus Environment

Page 3 of 13
Analytical Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
Figure 1. Fluorescent properties associated with the length of conjugated double bond and PEG chain. (A) Maximum emission of
methoxylꢀended probes (8a-d to 10a-d) measured in CH2Cl2 (white) and PBS (magenta). (B) Maximum emission of hydroxylꢀ
ended probes (11a-d to 13a-d) measured in CH2Cl2 (cyan) and PBS (magenta). (C) Absolute quantum yield of methoxylꢀended
probes (7a-d to 10a-d) determined in CH2Cl2.
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 1. Selected spectroscopic and biological data for the synthesized probes.
a
a
b
Probe
λem1
λex1
λem2
Φ/%
Folde
Kd (nM)f
Brain Entry (% ID gꢀ1)g
7a
7b
618
700
788
801
620
700
788
830
622
705
788
800
625
710
785
820
622
708
780
817
624
700
780
820
623
700
781
817
472
493
619
630
471
493
622
635
471
501
620
624
479
500
620
636
479
500
615
636
476
494
615
639
485
500
610
641
n.d.
615
660
700
n.d.
610
662
710
n.d.
613
662
712
n.d.
615
663
718
n.d.
614
664
720
n.d.
618
662
715
n.d.
620
665
720
16.6 c/0.8 d
58.2/0.7
39.2/0.2
11.8/0.1
18.1/0.9
57.9/1.0
39.5/0.1
10.9/0.1
18.5/0.9
56.8/1.2
39.4/0.2
10.8/0.1
78.2/0.9
57.3/1.3
36.1/0.2
29.4/0.2
19.4/0.8
52.1/1.0
32.9/0.3
9.8/0.1
n.d.
216.7
122.1
31.0
n.d.
359.13 ± 77.21
39.44 ± 12.32
10.34 ± 1.05
8.11 ± 0.65
n.d.
n.d.
7c
2.98 ± 0.43
1.42 ± 0.46
0.00 ± 0.00
0.40 ± 0.09
1.75 ± 0.25
1.00 ± 0.23
n.d.
7d
8a
164.5 ± 10.22
30.92 ± 6.38
30.97 ± 5.37
23.1 ± 10.43
415.17 ± 93.57
39.99 ± 12.84
24.59 ± 4.89
15.37 ± 3.4
8b
220.4
150.2
33.8
n.d.
8c
8d
9a
9b
186.1
159.3
20.2
n.d.
n.d.
9c
2.69 ± 0.25
1.07 ± 0.19
n.d.
9d
10a
10b
10c
10d
11a
11b
11c
11d
12a
12b
12c
12d
13a
13b
13c
13d
467.33 ± 136.5
32.93 ± 7.63
6.49 ± 1.06
180.5
163.1
10.8
n.d.
n.d.
1.74 ± 0.43
1.60 ± 0.59
0.85 ± 0.09
3.38 ± 0.26
3.87 ± 0.01
2.16 ± 0.15
n.d.
15.93 ± 3.81
144.77 ± 4.25
46.49 ± 6.1
161.0
125.9
27.4
n.d.
52.16 ± 9.6
8.24 ± 1.65
17.8/0.9
53.6/1.1
37.3/0.2
11.0/0.1
17.8/0.9
53.1/1.2
36.4/0.1
10.5/0.1
152.4 ± 1.31
62.43 ± 8.4
118.0
90.8
21.8
n.d.
n.d.
51.32 ± 11.29
7.28 ± 3.54
3.18 ± 0.66
3.04 ± 0.84
n.d.
259.53 ± 50.63
49.16 ± 12.6
44.57 ± 10.81
35.03 ± 12.1
99.0
80.2
23.5
n.d.
3.17 ± 0.40
2.56 ± 0.53
aEmission/Excitation maxima determined in PBS. bEmission maxima upon binding to Aβ1ꢀ42 aggregates. Absolute quantum yield measured
in CH2Cl2c and PBSd. eFluorescence enhancement upon binding to Aβ1ꢀ42 aggregates. fKd values of NIR probes were measured in triplicate
with results given as the mean ± SD. gInitial brain uptake after i.v. injection at 2 min, the values were shown as the mean ± SD (n = 3).
and only give the methyl cyanoacetate products. Thus for the
Optical Properties
synthesis of 8a-c to 13a-c, CH2Cl2 was selected as solvent.
ACS Paragon Plus Environment

Analytical Chemistry
Page 4 of 13
In this study, our probes are composed of dimethylamino doꢀ shift of probes upon binding to Aβ aggregates usually bring
1
2
3
4
5
6
7
8
nors, naphthalene and polyene based π bridge, and PEG modiꢀ
fied cyanoacetate acceptors, which possess typical DꢀπꢀA arꢀ
chitecture, and they could form an internal charge transfer
(ICT) state. In general, ICT probes exhibited great solvatoꢀ
chromism due to the diverse stabilization of the dipole moꢀ
ment between ground and excited states in solvents with difꢀ
ferent polarity9,36. Hence, the solvatochromism is a good
pathway to perform specific signal for the fluorescence probes
upon responded target. As shown in Figure 1A, B, S1ꢀ7 and
Table 1, S2ꢀ3, our probes displayed highly sensitive to solvent
polarity (in five slovents), and give bathochromic shifts
around 40 to 100 nm from tetrahydrofuran to phosphate buffer
saline (PBS) (concentration of 0.01 M). The narrowed
HOMOꢀLUMO gap contributed to the bathochromic shift of
the absorption and emission wavelength, and extend the conꢀ
negative effect，which may push the emission band out of
NIR region, fortunately, the emission maxima of the NIR
probes with three and four conjugated double bonds were in
excess of 650 nm upon interaction with Aβ aggregates. These
results suggested that our probes meet the fluorescent prereqꢀ
uisites as NIR sensor.
Histopathological Staining
In vitro fluorescence staining on formalinꢀfixed brain slices
from transgenic (Tg) mice model (APPswe/PSEN1) of AD,
autopsyꢀconfirmed AD patients and patients with cerebral
amyloid angiopathy (CAA) was selected as the easiest method
to screen the binding ability of these NIR probes to Aβ
plaques. As shown in Figure S23ꢀ26, all probes could specifiꢀ
cally label Aβ deposits on brain tissues from Tg mice and AD
patients. Taking probe 12d as an example, we select brain
tissues from four AD and two CAA patients to further validate
it’s binding ability to Aβ deposits. Various Aβ forms such as
diffuse plaques (Fig 2C, D), denseꢀcore plaques (Fig 2A, F)
and vascular plaques (Fig 2B, E) could be stained by probe
12d, which suggest that 12d possessed hypersensitive to the βꢀ
sheet structure of Aβ deposits. Negative staining was observed
on the brain slices from five healthy humans and a wildꢀtype
(WT) mouse, indicate low nonꢀspecific binding (Figure S27).
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
jugatedꢀπ system is the most effective way to achieve a
38
HOMOꢀLUMO gap reduction37,
.
As expected,
for each additional trans double bond added to these probes,
the λem increased about 80 nm in CH2Cl2, and finally we
pushed the maxima emission wavelength to the NIR region,
for probes with three or four conjugated double bonds, their
λem were greater than 750 nm in PBS (0.01 M) solutions. In
addition, these probes displayed high quantum yield (10% ꢀ
58%) in CH2Cl2 solution with a trend of rise first then decrease
by expanding the conjugatedꢀπ system (Figure 1C), and this
regular pattern is completely in accordance with our previous
studies31. It is also found that the pegylated acceptors (n = 0 ꢀ
3, with hydroxyl and methoxyl terminals) has minor effect on
optical properties. Compared with the probes with malonoꢀ
nitrile acceptors29, the probes with PEG modified cyanoacetate
acceptors reach a same convergence of absorption maxima,
emission maxima, Stokes shift and quantum yield. In general,
these properties were mainly dominated by the conjugatꢀ
ed system area, moreover, we could conclude that malonoꢀ
nitrile and cyanoacetate has similar electron withdrawing abilꢀ
ity, and we can improve their biological properties by simply
modify the cyanoacetate acceptors without affecting optical
properties.
The fluorescence switch on based biosensor provides a simple
and quick analysis tool for the detection of specific proteins12.
As shown in Table 1 and Fig S15ꢀ22, our probe achieved “turn
on” phenomenon reposed on the sensitivity of DꢀπꢀA structure
to the polarity of surrounding environmental, the quantum
yield of probe in CH2Cl2 is greatly increased compared to PBS
(0.01 M) with more polarity. This phenomenon could be corꢀ
roborated by the results of intense fluorescence enhancement
(20.2 ꢀ 220.4 fold) accompanied with significant hypsoꢀ
chromic shift (101 ꢀ 141 nm) of these probes when bound to
Aβ1ꢀ42 aggregates. The “turn on” mechanism could
be considered that our probes bind to the hydrophobic area of
Aβ aggregates. Similar to our previous studies31, sigꢀ
nal enhancement was gradually reduced when increase the
length of conjugatedꢀπ system while the length of PEG chain
has little effect on this. The fluorescence enhancement of the
hydroxylꢀended probes (11a-d to 13a-d) was slight lower than
methoxylꢀended probes (7a-d to 10a-d), which means that the
hydrogen bond formed by hydroxyl group may reduce the
hydrophobic interaction between probes and Aβ aggregates. In
addition, very weak interaction between our probes with boꢀ
vine serum albumin and monomeric Aβ peptide (for 12d) indiꢀ
cated low nonꢀspecific binding. In general, the hypsochromic
Figure 2. In vitro histopathological fluorescence staining of probe
12d on brain slices from patients of AD and CAA. (A) AD tissue,
64ꢀyear old, female, temporal lobe, 20X; (B) CAA tissue, 68ꢀyear
old, female, temporal lobe, 10X; (C) AD tissue, 71ꢀyear old, feꢀ
male, temporal lobe, 20X; (D) AD tissue, 85ꢀyear old, male, temꢀ
poral lobe, 20X; (E) CAA tissue, 76ꢀyear old, male, temporal
lobe, 20X; (F) AD tissue, 91ꢀyear old, male, temporal lobe, 20X.
Saturation Binding Assay
Thanks to the fluorescence enhancement effect of these probes
upon binding to Aβ1ꢀ42 aggregates, we can measure quantitaꢀ
tive binding affinity of these NIR probes to Aβ1ꢀ42 aggregates
by saturation binding assays. As shown in Figure 3B, S28ꢀ31
and Table 1, they displayed enhancive binding affinity toꢀ
wards prolonged polyenic chains, while the length of PEG
chains with hydroxyl or methoxyl terminals didn’t express
ACS Paragon Plus Environment

Page 5 of 13
Analytical Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
Figure 3. (A) The clogP value of the NIRF probes 7a - 10d (methoxylꢀended probes) (white), 11a - 13d (hydroxylꢀended probes) (cyan),
calculated by the online ALOGPS 2.1 program. (B) Kd value for 7a - 10d (white), 11a - 13d (cyan). (C) Initial brain uptake of the 8a - 8d
(black curve) and 11a -11d (red curve) after i.v. injection at 2 min.
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
regular or negative impact on binding affinity. In previous
study, the malononitrile acceptor conjugated well to the π
bridge to form a coplanar structure, which is favourable for Aβ
binding. However, the probes with unconjugated PEG chains
have more torsion may block their ready access to Aβ fibrils.
Compared to the probes with malononitrile acceptor (DANIR
3aꢀc)29, the binding affinity of PEG modified probes is slightly
decreased, but still good enough to bind to Aβ aggregates. In
summary, all these results indicate that conjugated π bridge is
the essential factor in binding affinity and PEG modification is
a straightforward method to adjust the hydrophilicity without
passive effect on binding affinity.
than DANIR 3b with two double bonds (brain2min/brain60min :
3.7 vs 21.7)29. However, the PEG modified probe 12d with
four conjugated double bonds still possess satisfactory washꢀ
out rate (brain2min/brain60min = 10.1), which verified the incorꢀ
porate of PEG chains is benefit for pharmacokinetic. All these
results demonstrated that lipophilicity of probe has
strong relativity to their initial brain uptake and kinetics, the
PEG modification is a simple way to adjust and improve these
properties of NIR probes.
In addition, 12d displayed high stability in PBS or in the presꢀ
ence of reducing agents including glutathione and cysteine
(Figure S32). Furthermore, from the results of MTT assay,
12d displayed poor cytotoxicity to human neuronal cell line
(SHꢀSY5Y) (Figure S33).
Brain Penetration and Clearance Study
Optimal lipophilicity is one of the most important factors conꢀ
tributed to the bloodꢀbrain barrier (BBB) penetration ability of
small molecule through passive diffusion39,40. As expected,
lipophilicity of the probes (clogP) increases with the exꢀ
tend of conjugated π system, meanwhile, the increase of PEG
chains has little effect on clogP value, mainly due to the relaꢀ
tive short PEG units (n = 1 ꢀ 3). In addition, after adding hyꢀ
droxyl group, the probe lipophilicity has reduced obviously
(Figure 3A). Compared with DANIR 3aꢀc, methoxylꢀended
probes (7a-d to 10a-d) exhibited increased lipophilicity with
the same length of polyenic chains, while hydroxylꢀended
probes (11a-d to 13a-d) decreased. Then the brain uptakes
were measured by a HPLC based quantification method, conꢀ
centrations of the probes in the brain homogenate were anaꢀ
lyzed using HPLC after intravenous (i.v.) injection to ICR
mice. As shown in Table 1, under the same length of polyenic
chain, hydroxylꢀended probes (11a-d to 13a-d) displayed
higher initial brain uptake than methoxylꢀended probes (7a-d
to 10a-d), while the number of PEG units has less impact
on the brain uptake. In order to study the effects of polyenic
chain on brain uptake, we selected probe 8a-d and 11a-d with
same PEG unit (n = 1) but different length of polyenic chain
(m = 0 ꢀ 3). In accordance with our previous results, the initial
brain uptake of these probes increase first and then decrease;
by extending the number of polyenic unit (Figure 3C). Howꢀ
ever, compared with DANIR 3a ꢀ c, the PEG modified probes
in this study displayed lower brain uptake, which indicate that
molecular weight and size have negative effect on the ability
of brain penetration. In addition, according to our previous
studies, the brain kinetics of probes expressed as clearance rate
decreased sharply with expanding polyenic chains probably
due to the increase of lipophilicity, e.g. DANIR 3c with three
conjugated double bonds displayed much lower clearance rate
Figure 4. (A) In vivo NIR brain images from Tg and WT control
mouse on representative time points before and after i.v. injection
of probe 12d. (B) Quantitative analysis of the relative fluoresꢀ
cence signals to show the brain kinetic curves of probe 12d at
selected time points.
In vivo NIR Imaging
Finally, to investigate the in vivo imaging capacity of the NIR
probes, probe 12d with favorable optical and biological propꢀ
ACS Paragon Plus Environment

Analytical Chemistry
Page 6 of 13
erties was selected for nonꢀinvasive detection of Aβ plaques in of conjugated π system and PEG chains, we can obtain new
1
2
3
4
5
6
7
8
Tg mice (C57BL6, APPsw/PSEN1, 19ꢀmonth old, male), and
ageꢀmatched WT mice as control. After i.v. injection of 12d,
one hour dynamic imaging was acquired by an IVIS Lumina
III system with time intervals of 2 minutes. Quantitative analꢀ
ysis of region of interesting (ROI) in the brain area indicated
probe 12d sufficiently penetrate the BBB and displayed conꢀ
siderable differences between Tg and WT at later time points.
The fluorescence signal discrepancies between Tg and WT
[F(Tg)/F(WT) ranging from 1.5 to 3.4] of 12d were signifiꢀ
cantly higher than that DANIR 3b [F(Tg)/F(WT) ranging from
1.0 to 2.1] and DANIR 3c [F(Tg)/F(WT) ranging from 1.0 to
1.5] under the same dose and imaging parameters (Figure 4).
NIR probes with better Aβ detection capability.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website.
Details of synthesis, additional figures, tables, NMR and MS
spectra (PDF).
9
AUTHOR INFORMATION
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Corresponding Author
*Phone/Fax: +86ꢀ10ꢀ58808891. Eꢀmail: cmc@bnu.edu.cn.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
This work was funded by the National Natural Science Foundaꢀ
tion of China (No. 21571022), and the National Science and
Technology Major Projects for Major New Drugs Innovation and
Development (No. 2014ZX09507007ꢀ002).
REFERENCES
(1) Brookmeyer, R.; Johnson, E.; ZieglerꢀGraham, K.; Arrighi, H.
M. Alzheimer's Dementia 2007, 3, 186ꢀ191.
(2) Hardy, J.; Higgins, G. Science 1992, 256, 184ꢀ185.
(3) Hardy, J.; Selkoe, D. J. Science 2002, 297, 353ꢀ356.
(4) Hardy, J. J. Alzheimer's Dis. 2006, 9, 151ꢀ153.
(5) J Soscia, S.; E Kirby, J.; Washicosky, K.; Tucker, S.; Ingelsson,
M.; Hyman, B.; Burton, M.; Goldstein, L.; Duong, S.; E Tanzi, R.;
Moir, R. Plos One, 2010; Vol. 5, p e9505.
(6) Kumar, D. K. V.; Choi, S. H.; Washicosky, K. J.; Eimer, W. A.;
Tucker, S.; Ghofrani, J.; Lefkowitz, A.; McColl, G.; Goldstein, L. E.;
Tanzi, R. E.; Moir, R. D. Sci. Transl. Med. 2016, 8, 340ra372ꢀ
340ra372.
(7) Ono, M.; Saji, H. Int. J. Mol. Imaging 2011, 2011, 543267.
(8) Rowe, C. C.; Villemagne, V. L. J Nucl Med 2011, 52, 1733ꢀ
1740.
(9) Staderini, M.; Martin, M. A.; Bolognesi, M. L.; Menendez, J. C.
Chem. Soc. Rev. 2015, 44, 1807ꢀ1819.
(10) Weissleder, R.; Pittet, M. J. Nature 2008, 452, 580ꢀ589.
(11) Gibson, E. A.; Masihzadeh, O.; Lei, T. C.; Ammar, D. A.;
Kahook, M. Y. J. Ophthalmol. 2011, 2011, 870879.
(12) Ntziachristos, V. Annu. Rev. Biomed. Eng. 2006, 8, 1ꢀ33.
(13) Ntziachristos, V. Nat. Methods 2010, 7, 603.
(14) Stuker, F.; Ripoll, J.; Rudin, M. Pharmaceutics 2011, 3, 229ꢀ
274.
(15) Hintersteiner, M.; Enz, A.; Frey, P.; Jaton, A.ꢀL.; Kinzy, W.;
Kneuer, R.; Neumann, U.; Rudin, M.; Staufenbiel, M.; Stoeckli, M.
Nat. Biotechnol. 2005, 23, 577ꢀ583.
(16) Li, Y.; Xu, D.; Ho, S.ꢀL.; Li, H. W.; Yang, R.; Wong, M. S.
Biomaterials 2016, 94, 84ꢀ92.
(17) Nesterov, E. E.; Skoch, J.; Hyman, B. T.; Klunk, W. E.;
Bacskai, B. J.; Swager, T. M. Angew. Chem. Int. Ed. 2005, 44,
5452ꢀ5456.
Figure 5. Ex vivo histopathological staining results of brain slices
(cortex and hippocampus region) from a Tg mouse (C57BL6,
APPsw/PSEN1, 19ꢀmonth old, male) (A, Texas Red channel, 4X)
and an ageꢀmatched WT mouse (C57BL6, 19ꢀmonth old, male)
(D, Texas Red channel, 4X) after dosing with 12d. The homoloꢀ
gous staining results were confirmed by ThS (B, E, GFP chanꢀ
nel,). The merged images were shown in C and F, respectively.
Furthermore, after NIR imaging, subsequent ex vivo experiꢀ
ment was performed on Tg and WT mice to confirm the bindꢀ
ing
of
12d
to
Aβ
deposits.
Fluoresꢀ
cence microscopy observation of the frozen brain sections in
Figure 5 indicate that high signalꢀtoꢀnoise fluorescence spots
were turnedꢀon and mostly concentrated in the cortex and hipꢀ
pocampus regions of Tg mouse, while no specific signal was
found in WT mouse brain. The same sections were further
stained by ThioflavinꢀS (ThS) in GFP channel, and the spots
merged well with the Texas Red channel of 12d (Figure 5C).
These results demonstrated probe 12d could label Aβ plaques
in vivo and effectively distinguish Tg and WT mouse.
CONCLUSIONS
In conclusion, we designed; synthesized and evaluated a series
of DꢀπꢀA based Aβ probes with PEG modified acceptor. From
in vitro studies, the PEG modified acceptors displayed very
weak impact on emission wavelength, quantum yield, fluoresꢀ
cence enhancement upon binding to Aβ aggregates and bindꢀ
ing affinity. The length of π bridge is still to be the dominant
factor in above properties. Besides, the introduction of PEG
chains caused the lipophilicity change as well as increased the
molecule weight and size, and eventually led to decreased
brain uptakes. In addition, Probe 12d held a good capacity of
crossing the BBB and favorable clearance rate from mice
brain with improved brain kinetics compared with previously
reported probes. Hence, PEG modification of the DꢀπꢀA
probes is a direct and convenient way to adjust and improve
their biological properties without changing much on optical
properties. We believe that by carefully balancing the length
(18) 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,
15257ꢀ15261.
(19) Zhang, X.; Tian, Y.; Li, Z.; Tian, X.; Sun, H.; Liu, H.; Moore,
A.; Ran, C. J. Am. Chem. Soc. 2013, 135, 16397ꢀ16409.
(20) Zhang, X.; Tian, Y.; Zhang, C.; Tian, X.; Ross, A. W.; Moir,
R. D.; Sun, H.; Tanzi, R. E.; Moore, A.; Ran, C. P. Natl. Acad. Sci.
USA 2015, 112, 9734ꢀ9739.
(21) Watanabe, H.; Ono, M.; Saji, H. Chem. Comm. 2015, 51,
17124ꢀ17127.
ACS Paragon Plus Environment

Page 7 of 13
Analytical Chemistry
(22) Zhu, B. Y.; Cheng, Y.; Li, G. B.; Yang, S. Y.; Zhang, Z. R.
Bioorg. Med. Chem.. 2016, 24, 827ꢀ834.
(32) Harris, J. M.; Chess, R. B. Nat. Rev. Drug Discovery 2003, 2,
214.
(33) Zhang, W.; Oya, S.; Kung, M.ꢀP.; Hou, C.; Maier, D. L.;
1
2
3
4
5
6
7
8
(23) Yang, H. Y.; Zhang, J. J.; Zang, Y.; Zhang, H. Y.; Li, J.; Chen,
G. R.; He, X. P. Dyes Pigm. 2017, 136, 224ꢀ228.
(24) Zhu, J. Y.; Zhou, L. Y.; Li, Y. K.; Chen, S. B.; Yan, J. W.;
Zhang, L. Anal. Chim. Acta. 2017, 961, 112ꢀ118.
(25) Ren, W.; Xu, M.; Liang, S. H.; Xiang, H.; Tang, L.; Zhang,
M.; Ding, D.; Li, X.; Zhang, H.; Hu, Y. Biosens. Bioelectron. 2016,
75, 136ꢀ141.
Kung, H. F. Nucl. Med. Biol. 2005, 32, 799ꢀ809.
(34) Stephenson, K. A.; Chandra, R.; Zhuang, Z.ꢀP.; Hou, C.; Oya,
S.; Kung, M.ꢀP.; Kung, H. F. Bioconjugate Chem. 2007, 18, 238ꢀ246.
(35) Li, Z.; Cui, M.; Dai, J.; Wang, X.; Yu, P.; Yang, Y.; Jia, J.; Fu,
H.; Ono, M.; Jia, H. J. Med. Chem.. 2013, 56, 471ꢀ482.
(36) de Silva, A. P.; Gunaratne, H. Q. N.; Gunnlaugsson, T.;
Huxley, A. J. M.; McCoy, C. P.; Rademacher, J. T.; Rice, T. E. Chem.
Rev. 1997, 97, 1515ꢀ1566.
(26) Fu, H.; Cui, M.; Tu, P.; Pan, Z.; Liu, B. Chem. Comm. 2014,
50, 11875ꢀ11878.
9
(27) Zhou, K. X.; Fu, H. L.; Feng, L.; Cui, M. C.; Dai, J. P.; Liu, B.
L. Chem. Comm. 2015, 51, 11665ꢀ11668.
(37) Peccati, F.; Wisniewska, M.; SolansꢀMonfort, X.; Sodupe, M.
Phys. Chem. Chem. Phys. 2016, 18, 11634ꢀ11643.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(28) Cui, M.; Ono, M.; Watanabe, H.; Kimura, H.; Liu, B.; Saji, H.
J. Am. Chem. Soc.2014, 136, 3388ꢀ3394.
(29) Fu, H. L.; Cui, M. C.; Zhao, L.; Tu, P. Y.; Zhou, K. X.; Dai, J.
P.; Liu, B. L. J. Med. Chem. 2015, 58, 6972ꢀ6983.
(30) Fu, H. L.; Tu, P. Y.; Zhao, L.; Dai, J. P.; Liu, B. L.; Cui, M. C.
Ana.l Chem. 2016, 88, 1944ꢀ1950.
(38) Yao, L.; Zhang, S.; Wang, R.; Li, W.; Shen, F.; Yang, B.; Ma,
Y. Angew. Chem. Int .Ed. Engl.2014, 53, 2119ꢀ2123.
(39) Mathis, C. A.; Wang, Y.; Holt, D. P.; Huang, G.ꢀF.; Debnath,
M. L.; Klunk, W. E. J. Med. Chem. 2003, 46, 2740ꢀ2754.
(40) Waterhouse, R. N. Mol. Imaging Biol. 2003, 5, 376ꢀ389.
(31) Zhou, K.; Bai, H.; Feng, L.; Dai, J.; Cui, M. Anal. Chem.
2017, 89, 9432ꢀ9437.
Insert Table of Contents artwork here
ACS Paragon Plus Environment

Analytical Chemistry
Page 8 of 13
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 1. Fluorescent properties associated with the length of conjugated double bond and PEG chain. (A)
Maximum emis-sion of methoxyl-ended probes (8a-d to 10a-d) measured in CH2Cl2 (white) and PBS
(magenta). (B) Maximum emission of hydroxyl-ended probes (11a-d to 13a-d) measured in CH2Cl2 (cyan)
and PBS (magenta). (C) Absolute quantum yield of methoxyl-ended probes (7a-d to 10a-d) determined in
CH2Cl2.
30x9mm (300 x 300 DPI)
ACS Paragon Plus Environment

Page 9 of 13
Analytical Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2. In vitro histopathological fluorescence staining of probe 12d on brain slices from patients of AD and
CAA. (A) AD tissue, 64-year old, female, temporal lobe, 20X; (B) CAA tissue, 68-year old, female, temporal
lobe, 10X; (C) AD tissue, 71-year old, female, temporal lobe, 20X; (D) AD tissue, 85-year old, male,
temporal lobe, 20X; (E) CAA tissue, 76-year old, male, temporal lobe, 20X; (F) AD tissue, 91-year old,
male, temporal lobe, 20X.
75x86mm (300 x 300 DPI)
ACS Paragon Plus Environment

Analytical Chemistry
Page 10 of 13
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 3. (A) The clogP value of the NIRF probes 7a - 10d (methoxyl-ended probes) (white), 11a - 13d
(hydroxyl-ended probes) (cyan), calculated by the online ALOGPS 2.1 program. (B) Kd value for 7a - 10d
(white), 11a - 13d (cyan). (C) Initial brain uptake of the 8a - 8d (black curve) and 11a -11d (red curve)
after i.v. injection at 2 min.
29x8mm (300 x 300 DPI)
ACS Paragon Plus Environment

Page 11 of 13
Analytical Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 4. (A) In vivo NIR brain images from Tg and WT control mouse on representative time points before
and after i.v. injection of probe 12d. (B) Quantitative analysis of the relative fluorescence signals to show
the brain kinetic curves of probe 12d at selected time points.
60x67mm (300 x 300 DPI)
ACS Paragon Plus Environment

Analytical Chemistry
Page 12 of 13
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 5. Ex vivo histopathological staining results of brain slices (cortex and hippocampus region) from a Tg
mouse (C57BL6, APPsw/PSEN1, 19-month old, male) (A, Texas Red channel, 4X) and an age-matched WT
mouse (C57BL6, 19-month old, male) (D, Texas Red channel, 4X) after dosing with 12d. The homologous
staining results were confirmed by ThS (B, E, GFP channel,). The merged images were shown in C and F,
respectively.
62x38mm (300 x 300 DPI)
ACS Paragon Plus Environment

Page 13 of 13
Analytical Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
182x110mm (300 x 300 DPI)
ACS Paragon Plus Environment