Ferrocene tripeptide Gly-Pro-Arg conjugates: Synthesis and inhibitory effects on Alzheimer's Aβ1-42 fibrillogenesis and Aβ-induced cytotoxicity in vitro

Article abstract of DOI:10.1016/j.bmc.2012.11.030

Alzheimer's disease (AD) is the most common cause of dementia, and currently there is no clinical treatment to cure it or to halt its progression. Aggregation and fibril formation of β-amyloid peptides (Aβ) are central events in the pathogenesis of AD. Ma

Full text of DOI:10.1016/j.bmc.2012.11.030

Bioorganic & Medicinal Chemistry 21 (2013) 395–402
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Ferrocene tripeptide Gly-Pro-Arg conjugates: Synthesis and inhibitory effects
on Alzheimer’s Ab1–42 fibrillogenesis and Ab-induced cytotoxicity in vitro
a,b
a
a
a
a,b,
, Juan Li a,b,
⇑ ⇑
Binbin Zhou , Chun-Lan Li , Yuan-Qiang Hao , Muya Chabu Johnny , You-Nian Liu
a
College of Chemistry and Chemical Engineering, Central South University, Changsha, Hunan 410083, PR China
Key Laboratory of Resources Chemistry of Nonferrous Metals (Central South University), Ministry of Education, Changsha, Hunan 410083, PR China
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 26 July 2012
Revised 21 November 2012
Accepted 22 November 2012
Available online 2 December 2012
Alzheimer’s disease (AD) is the most common cause of dementia, and currently there is no clinical treat-
ment to cure it or to halt its progression. Aggregation and fibril formation of b-amyloid peptides (Ab) are
central events in the pathogenesis of AD. Many efforts have been spent on the development of effective
inhibitors to prevent Ab fibrillogenesis and cause disaggregation of preformed Ab fibrils. In this study, the
2
conjugates of ferrocene and Gly-Pro-Arg (GPR) tripeptide, Boc-Gly-Pro-Arg(NO )-Fca-OMe (4, GPR–Fca)
and Fc-Gly-Pro-Arg-OMe (7, Fc–GPR) (Fc: ferrocene; Fca: ferrocene amino acid) were synthesized by
HOBT/HBTU protocol in solution. These ferrocene GPR conjugates were employed to inhibit Ab1–42 fibril-
logenesis and to disaggregate preformed Ab fibrils. The inhibitory properties of ferrocene GPR conjugates
on Ab1–42 fibrillogenesis were evaluated by thioflavin T (ThT) fluorescence assay, and confirmed by
atomic force microscopy (AFM) analysis. The interaction between the ferrocene GPR conjugates and
Ab1–42 was monitored by electrochemical means. Our results showed that both GPR and GPR–Fca can sig-
nificantly inhibit the fibril formation of Ab1–42, and cause disaggregation of the preformed fibrils. As
expected, GPR–Fca shows stronger inhibitory effect on Ab1–42 fibrillogenesis than that of its parent pep-
tide GPR. In contrast, Fc–GPR shows no inhibitory effect on fibrillogenesis of Ab1–42. Furthermore, GPR–
Fca demonstrates significantly protection against Ab-induced cytotoxicity and exhibits high resistance to
proteolysis and good lipophilicity.
Keywords:
Ab1–42 fibrillogenesis
Ferrocene
GPR
Inhibitor
Cytotoxicity
Ó 2012 Elsevier Ltd. All rights reserved.
1
. Introduction
Alzheimer’s disease (AD) is a progressive and fatal neurodegen-
To date, lots of structurally different compounds have been
identified to prevent Ab aggregation and/or disaggregate pre-
formed fibrils. These compounds include small organic molecules
6
erative disorder in the elderly characterized by short-term memory
loss, continuous cognitive decline and behavioral disturbances.
AD affects approximately 26.6 million population worldwide,
and peptides or peptidomimetics. Small molecules such as the
1
9
0
0
10
antibiotic rifampicin, 4 -iodo-4 -deoxydoxorubicin,
tone derivatives
isaindigo-
have been
2
11
12
and scyllo-inositol derivatives
and the number of people with AD is increasing at a very high rate
that there could be about 106.2 million by 2050, resting an enor-
found to prevent Ab aggregation. Besides, peptides or peptidomi-
3
metic molecules, such as KLVFF and LPFFD show strong inhibition
7
,8,13
mous burden on the family, health care and society. In the past two
decades, a lot of effort has been spent to understand the pathogen-
esis of AD, in order to build a robust platform for effective pharma-
cological treatments. Although the precise mechanisms leading to
neurodegeneration in AD are not fully understood yet, several
pieces of evidence indicate that the conformational changes
accompanying the aggregation and fibril formation of b-amyloid
peptides (Ab), might play a pivotal role in the pathogenesis of
for Ab misfolding and aggregation.
Glyprolines (Gly-Pro-Arg,
GPRs) are the new family of regulatory peptides consisting of short
1
4
glycine and proline-containing peptides, which are produced in
the organism during degradation and synthesis of elastin, collagen,
1
5
and other connective tissue proteins. Recently, GPR was em-
1
4
ployed to inhibit the development of AD. For example, Uemura
and coworkers found that GPR is a small neuroprotective peptide
and can effectively prevent neuronal degeneration in vivo.16 Thus,
tripeptide GPR is considered as a potential lead compound for the
treatment of AD. However, natural peptides as drug compounds
are essentially limited by their poor membrane permeability and
instability in blood plasma.
4
AD. Therefore, one of the most attractive therapeutic targets for
AD could rely on the use of the agents which could inhibit the fibril
formation of Ab or disaggregate the preformed Ab fibrils.5 A lot of
progress has recently been made in design and synthesis of inhib-
itors for Ab fibril formation.
–8
Ferrocene (Fc) and its derivatives exhibit a manifold of medici-
nal properties by serving as antimalarial, antibacterial and antitu-
mor agents due to their biological activity, low cytotoxicity, high
⇑
Corresponding authors.
E-mail addresses: liuyounian@csu.edu.cn (Y.-N. Liu), juanli@csu.edu.cn (J. Li).
1
7–21
lipophilicity and unique electrochemical behavior.
Especially,
0
968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.11.030

3
96
B. Zhou et al. / Bioorg. Med. Chem. 21 (2013) 395–402
when incorporated into a drug, ferrocene moiety could yield inter-
seen from Figure 1, when Ab1–42 was incubated in the presence
of GPR, the fluorescence intensity increases initially with incuba-
tion time, reaching its maximum at 4 days. However, a reduction
in fluorescence intensity shows that GPR is able to inhibit the fibril
formation of Ab1–42, in agreement with the neuroprotective behav-
ior of GPR in vivo. GPR–Fca exhibits similar ThT fluorescence pat-
tern as that of GPR. Both GPR and GPR–Fca reveal significant
inhibitory effects resulting in 40% and 50% reduction of the fibril
formation, respectively. In contrast to GPR and GPR–Fca, Fc–GPR
reveals negligible inhibitory effect on Ab1–42 fibrillogenesis. Our re-
sults point to robust inhibitory effects of GPR–Fca and GPR on Ab1–
42 fibrillogenesis.
2
2
esting results. For example, in the antimalarial compound oafer-
2
3
24
roquine and the antitumor agent ferrocifen, the introduction of
the ferrocenyl moieties improves the activity of the compounds
and alters their pharmacodynamic profiles. Recently, we conju-
gated ferrocene to pentapeptide KLVFF and found that Fc-KLVFF
shows improved lipophilicity and significant resistance towards
proteolytic degradation compared to its parent peptide, and high
1
6
25
inhibitory effect towards the fibrillogenesis of Ab1–42
.
Addition-
ally, ferrocene peptide conjugates, synthesized by the route either
from C-terminal or from N-terminal labelling, are recognized as
useful tools in bioorganometallic chemistry.26–28
The main objective of this work is to combine the lipophilic nat-
ure and enzymatic stability of ferrocene moiety and the neuropro-
tective nature of GPR peptide, by designing and synthesizing
several functional ferrocene GPR conjugates, and to investigate
the effect of the conjugates on Ab1–42 fibrillogenesis and on the
Ab-induced toxicity towards human neuroblastoma SH-SY5Y cells.
On the other hand, a control experiment was performed to test
the self-aggregation of GPR–Fca and GPR using ThT fluorescence
assay. A 25 lM GPR–Fca or GPR was incubated alone for 7 days
at 32 °C in 0.1 M PBS (pH 7.4), respectively. No obvious ThT fluores-
cence was observed in either solution (data no shown), which sug-
gests that neither GPR–Fca nor GPR undergoes self-aggregation in
solution.
2
2
. Results and discussion
2.3. Concentration-dependent effects of ferrocene GPR
conjugates on Ab1–42 fibrillogenesis
.1. Synthesis and characterization of GPR–Fca and Fc–GPR
GPR tripeptide, one of the members of the glyproline family was
To investigate the concentration responses of GPR and ferrocene
GPR conjugates on Ab1–42 fibrillogenesis, aliquots of Ab1–42 at a
found to possess the ability to protect and rescue cell death in-
concentration of 25 lM were incubated with ferrocene GPR conju-
1
6
duced by Ab. As mentioned in the Introduction, the aim of this
study is to design and synthesize of enzymatic resistance b-sheet
breakers which can inhibit the Ab1–42 fibril formation and attenu-
ate the Ab-induced cytotoxicity. Ferrocene, due to its biological
activity, low toxicity, and high lipophilicity may meet the require-
gates at 1:0.4, 1:1, 1:3, 1:5 and 1:10 (Ab1–42: GPR or ferrocene GPR
conjugates, molar ratio). The ThT fluorescence assay was also
exploited to monitor the formation of fibrils. As revealed by Fig-
ure 2, when Ab1–42 was incubated with different concentrations
of GPR or GPR–Fca, the inhibition of amyloid fibril formation was
apparently achieved and the inhibitory effect enhances rapidly
with increasing dosage of inhibitors. Moreover, our results indi-
cated that GPR–Fca is a more potent inhibitor of Ab1–42 fibrillogen-
esis than GPR. The IC50 values (the molar ratio of inhibitor to Ab1–42
fibrillogenesis with 50% maximum effect) for GPR and GPR–Fca are
3.28 ± 0.53 and 1.42 ± 0.31, respectively. We suggest that the dif-
ference in IC50 between GPR and GPR–Fca could be ascribed to
the hydrophobicity of ferrocene which makes the GPR–Fca more
easily to interact with Ab1–42. Furthermore, the introduction of
ferrocenoyl moiety could act as a good disrupting element for
Ab1–42 fibrillogenesis. In contrast, Fc–GPR shows no significant
inhibition of fibrillogenesis upon the Ab1–42 even at a molar ratio
of 1:10 and the IC50 values for Fc–GPR should be higher than 10.
2
9,30
ment.
Moreover, the unique electrochemical behavior of ferro-
cene can be used to interrogate the interaction between b-sheet
breakers and Ab1–42. Since the introduced bulky groups into tripep-
tide GPR may render steric hindrance, resulting in affecting the
interaction with Ab1–42, ferrocene was attached to both N-terminal
and C-terminal of GPR to investigate the effect on Ab1–42 fibril
formation.
Both GPR–Fca and Fc–GPR were synthesized using HOBT/HBTU
protocol in solution. (see Scheme 1). GPR–Fca and Fc–GPR were
then characterized by FT-IR, 1H NMR and ESI-MS (data were pre-
sented in detail in experimental procedures section), which indi-
cating that ferrocene GPR conjugates were synthesized
successfully. In order to highlight the role of Fc in this kind of b-
sheet breaker, the tripeptide GPR was synthesized as a control.
2
.4. Kinetics of the interaction between Ab1–42 and ferrocene
2
.2. Kinetics of fibril formation of Ab1–42 in the presence of
GPR conjugates
ferrocene GPR conjugates
An attractive therapeutic strategy for AD is to block the aggre-
The binding of ThT to amyloid is specific, thus, ThT fluorescence
assay has been extensively used to monitor the fibril formation of
gation of Ab by inhibitors which contain a recognition element
3
2
and a disrupting element. During the past decades, KLVFF pep-
tide has been extensively used as a recognition element to devise
3
1
amyloid peptide. Herein, the kinetics of fibril formation of Ab1–
and the effects of ferrocene GPR conjugates on Ab1–42 fibrillogen-
8
,33
4
2
inhibitors for Ab fibrillation.
In our previous study, we devised
esis were examined by ThT fluorescence assay. Typically, ThT fluo-
rescence does not change when it binds to unstructured proteins.
However, when it binds to aggregated amyloid, a new excitation
peak at 450 nm and enhanced emission at 482 nm will be observed.
As shown in Figure 1, along with the incubation of Ab1–42 at a
an ferrocenoyl inhibitor for Ab fibrillation depending on KLVFF
and utilized the electrochemical method to study the interaction
between Ab and Fc–KLVFF according to the redox active properties
2
5
of ferrocenoyl conjugate. Here, the electrochemical properties of
GPR–Fca and Fc–GPR were also investigated by cyclic voltammetry
(CV). A pair of well-defined redox peaks, with the cathodic and
anodic peak potentials at 552 mV and 478 mV versus Ag/AgCl,
respectively, was observed for GPR–Fca, and the ratio of oxidative
to reductive peak currents is close to unity, indicating electro-
chemical reversibility of the electrode process. Similarly, Fc–GPR
also undergoes a reversible electron-transfer reaction (data not
shown). Due to the excellent intrinsic electrochemical signals of
the ferrocene GPR conjugates, differential pulse voltammetry
concentration of 25 lM alone at 32 °C, the ThT fluorescence in-
creases exponentially for a period and then remains almost con-
stant. Consistent with our previous studies, fibril formation of
Ab1–42 starts at very short beginning, and reaches its plateau at
the sixth day of incubation, after that, fluorescence intensity turns
2
5
to saturation. The equimolar composition of each inhibitor was
co-incubated with Ab1–42, GPR and GPR–Fca show inhibitory ef-
fects on fibrillogenesis of Ab1–42, although to different extents. As

B. Zhou et al. / Bioorg. Med. Chem. 21 (2013) 395–402
397
Scheme 1. Syntheses of ferrocene GPR conjugates.
Figure 2. Concentration-dependent effect of GPR and ferrocene GPR conjugates on
Ab1–42 fibrillogenesis. 25 M freshly prepared Ab1–42 was incubated at 32 °C in the
presence of GPR (s), Fc–GPR (j) or GPR–Fca (N) of difference concentrations in
.1 M PBS (pH 7.4) for 4 days. Reported data are expressed as a percentage of
maximum fluorescence intensity. Data represent the mean ThT fluorescence
measurement of three independent experiments (n = 3). Error bars represent the
standard deviation (SD) of the fluorescence measurement.
Figure 1. Inhibition of GPR peptide and ferrocene GPR conjugates on the kinetics of
Ab1–42 fibrillogenesis. Twenty five micrometer freshly prepared Ab1–42 was incu-
l
bated at 32 °C alone (s) or in the presence of 25
lM Fc–GPR (j), GPR (h), GPR–Fca
0
(d) for the indicated times. Fibril formation was quantitated by the ThT fluorometry
assay as described in methods. Data are expressed as a percentage of maximum
fibril formation (fluorescence intensity). Each experiment was repeated three times
(n = 3), and error bars represent the standard deviation (SD) of the fluorescence
measurement.
the solution containing the same concentration of GPR–Fca in the
absence of Ab1–42 was also monitored by the DPV, and the results
shows the current remained unchanged for 6 h, suggesting that
GPR–Fca does not undergo self-aggregation during this period.
On the other hand, the current of Fc–GPR in the presence of Ab1–
42 was monitored at various incubation times, and we observed
that there were no significant changes in the peak currents. It is
(
DPV) method was conducted to calculate the kinetics of the inter-
action between Ab1–42 and the ferrocene GPR conjugates. As shown
in Figure 3, the DPV peak currents drop sharply when GPR–Fca was
incubated with Ab1–42 in a very short time, suggesting that GPR–
Fca is capable of interacting with Ab1–42 instantaneously. To
exclude the current drops from the self-aggregation of GPR–Fca,

3
98
B. Zhou et al. / Bioorg. Med. Chem. 21 (2013) 395–402
Figure 3. The kinetics of Ab1–42 fibrillogenesis in the present of ferrocene GPR
conjugates by using DPV method. Fifty micrometer Ab1–42 was incubated in the
Figure 4. Disaggregation effect of preformed amyloid fibrils by GPR and GPR–Fca.
Freshly prepared Ab1–42 at 25 M was incubated alone in 0.1 M PBS at 32 °C for
days. A solution of GPR or GPR–Fca at 25 M was then added to the preformed
Ab1–42 fibril solution and incubated at 32 °C. Fibril concentration was quantitated
by the ThT fluorescence assay, as described in Methods. Data are expressed as a
percentage of maximum fibril formation (fluorescence intensity). Each experiment
was repeated three times (n = 3), and error bars represent the standard deviation
l
presence of 50
lM Fc–GPR (d) or GPR–Fca (j) in PBS (pH 7.4) at 32 °C for the
6
l
indicated times. Additionally, 50
lM GPR–Fca (h) was incubated alone under the
same experimental conditions as a control experiment. Data are expressed as a
percentage of the maximum anodic peak current of ferrocene GPR conjugates alone.
The experiment of co-incubation of Ab1–42 with GPR–Fca was repeated three times
(n = 3), and error bars represent the standard deviation (SD) of the current.
(
SD) of the fluorescence measurement.
probably due to the attachment of ferrocene directly to the N-ter-
minal of GPR causes steric hindrance, thus may hinder the interac-
with GPR–Fca. To exclude the self-aggregates of GPR–Fca, we incu-
bated GPR–Fca solutions separately and used AFM to determine
insoluble species that might have formed at various incubation
times. There were no visible structures observed even after 7 days
by the AFM (data not shown). It demonstrated again GPR–Fca was
not self-aggregated and the amorphous aggregate was mainly con-
tributed to the strong interaction between GPR–Fca and the inter-
mediate aggregates of Ab1–42. The AFM results further confirmed
GPR–Fca can inhibit Ab1–42 fibrillogenesis, in agreement with the
ThT fluorescence results.
tions between GPR and Ab1–42
.
2
.5. Disaggregation effect of GPR and GPR–Fca on preformed
Ab1–42 fibrils
To determine whether GPR–Fca or GPR can cause the disaggre-
gation of mature Ab1–42 fibrils, we utilized GPR–Fca and GPR to
treat the preformed Ab1–42 fibrils. The mature Ab1–42 fibrils were
prepared by incubation of 25 lM Ab1-42 alone for 6 days at 32 °C,
then equimolar of the inhibitor was added to the preformed fibril
solution and co-incubated at 32 °C. As depicted in Figure 4, the
maximum level of fibril disaggregation with these inhibitors was
obtained after 5 days of incubation and remained unchanged
thereafter. The result is similar to that of reported by Soto and
coworkers. GPR–Fca can efficiently induce disaggregation of
Ab1–42 fibrils, achieving disaggregation of about 50% Ab1–42 fibril
reduction after incubated for 5 days. Disaggregation of mature fi-
brils by 61% by GPR was observed too. Together, these results show
GPR–Fca disaggregates mature fibrils in a more potent way than
GPR.
2.7. Effect of GPR–Fca and GPR on Ab-induced cytotoxicity
Aggregated Ab is revealed to be toxic to neurons and cause cog-
nitive decline in AD patients. In this study, both ThT assay and AFM
results showed that GPR–Fca and GPR inhibit the Ab1–42 fibril for-
mation successfully. Previous studies on the inhibition of the fibril
formation of Ab showed that most (but not all) Ab inhibitors have
3
4
3
5–37
the potency to prevent the Ab-induced cytotoxicity.
stance, El-Agnaf and co-workers found that though both
RGKLVFFGR and RGKLVFFGR-NH are able to effectively inhibit
Ab fibril formation, only RGKLVFFGR-NH can protect human neu-
For in-
2
2
3
8
2
.6. Effect of GPR–Fca on Ab1–42 fibril morphology
roblastoma SH-SY5Y cells from Ab toxicity. Thus, we determined
the effects of GPR and GPR–Fca on Ab-induced toxicity towards hu-
man neuroblastoma SH-SY5Y cells by MTT assay.
AFM was used to image the morphologies of insoluble species
produced in Ab1–42 solution as well as in the mixture of Ab1–42
/
Consideration that ferrocene is innocuous to many cell lines,39
and consequently the ferrocene tag would not compromise the
ability of GPR–Fca for alleviating the cytotoxicity inflicted by
Ab1–42 aggregates. Thus, the human neuroblastoma SH-SY5Y cells
were employed to investigate this consideration. As shown in
Figure 6, the SH-SY5Y cells viability which was exposed to the
pre-aggregated Ab1–42 exhibits only as 55%. At the molar ratio of
1:1 (GPR: Ab1–42), the mixture solution was pre-incubated under
the same conditions, the treated cell viability was rescued to 80%
which is consistent with the result in Uemura’s work, showing
GPR prevents and rescues Ab-induced neuronal apoptosis. When
GPR–Fca was incubated with Ab1–42, the cell viability was rescued
to 85% which is higher than the one treated with the GPR/Ab1–42
mixture, in good agreement with our ThT results (Figs. 1 and 2).
GPR–Fca. As can be seen from Figure 5A, in the absence of GPR–
Fca, Ab1–42 formed typical amyloid-like, 8-10 nm in length
unbranched fine fibrils after 2 days incubation at 32 °C. The fibrils
become more clearly visible after 6 days incubation (Fig. 5B), sim-
ilar to our previous studies.25 In contrast, in the presence of GPR–
Fca, a lot of insoluble globular aggregates were observed after
2
days incubation, and a few larger aggregates (as shown in
Fig. 5C, the size is about sub-micrometer) are exclusively of the
amorphous morphology. As revealed by Figure 5D, the amount of
the amorphous aggregates became greater after 6 days of incuba-
tion, and the protofibrils as well as any kind of fibrillar materials
were absent completely. The images show dramatic difference be-
tween inhibitor-free Ab1–42 control sample and samples incubated
1
6

B. Zhou et al. / Bioorg. Med. Chem. 21 (2013) 395–402
399
Figure 5. AFM observation of fibril formation. Aliquots of 25
lM Ab1–42 were incubated alone (top) or in the presence of 25
l
M GPR–Fca (bottom) at 32 °C for 2 days (A, C)
and 6 days (B, D), respectively.
pass.40 One of our aims to modify the GPR peptide with ferrocene
moiety is to improve the proteolytic stability of GPR in blood
stream. The proteolytic stability of GPR and ferrocene GPR conju-
gates were studied by HPLC method. As shown in Figure 7, after
2
4 h incubation at 25 °C, only 20% of intact GPR was detected,
while, 45% and 50% of Fc–GPR and GPR–Fca remain intact, respec-
tively. The results are consistent with the experimental hypothesis,
that is, the ferrocene tethered GPRs exhibit significantly resistance
to proteolytic degradation in comparison with their parent peptide
GPR.
It is worth noting that lipophilicity is the most important factor
in determining the permeability of drug candidates and plays a
4
1
dominant role in toxicity predictions. It is well known that ferro-
cene possess a high lipophilicity and our previous studies demon-
strated that the conjugation of ferrocene group to KLVFF peptide
results in an increase in the lipophilic value of Fc–KLVFF.25 In this
study, the octanol/water partition coefficient (log P value) was
used to describe the lipophilicity of the inhibitors. To determine
w
the lipophilicity of GPR–Fca and GPR, log k values of all com-
pounds were determined by RP–HPLC and then converted to log P
4
2
Figure 6. SH-SY5Y cell viabilities in the presence of different solutions. All the
added solutions were pre-incubated for 48 h at 32 °C. The cell viability was
determined at 24 h after the addition of pre-incubated solutions. The error bars
correspond to relative standard deviation values determined from measurements
conducted on three different days.
value by comparison to reference compounds. As listed in Table
1, the attachment of ferrocene to GPR leads to an increase in the
lipophilicity of GPR–Fca compared to parent GPR. Metzler-Nolte
and co-workers found that the conjugation of ferrocene moiety
to a hydrophilic peptide lead to an increase in lipophilicity thus
enhancing the permeation coefficient of peptide across the
4
3
blood–brain barrier (BBB). Thus, We are inclined to infer that
the membrane permeability of GPR would be improved by the con-
jugation of ferrocene.
2
.8. Enzymatic stability and lipophilicity of GPR and ferrocene
GPR conjugates
A major drawback with the use of peptides as drugs in central
nervous system diseases is their rapid degradation by proteolytic
enzymes. For example, Molineaux and coworkers showed that
the use of cholyl–LVFFA as a therapeutic agent is restricted by its
propensity to be cleared up completely after the hepatic first
3. Conclusions
The ferrocene GPR conjugates were synthesized successfully.
The synthesized GPR–Fca shows high inhibitory effect upon Ab1–

4
00
B. Zhou et al. / Bioorg. Med. Chem. 21 (2013) 395–402
Boer Chemical Reagents Co. (Shanghai, China). Dichloromethane
CH Cl ) obtained from Hengxing (Tianjin, China) was dried and
distilled over CaH before use. SH-SY5Y cell (human neuroblas-
toma) was purchased from American Type Culture Collection Inc.
Manassas, VA). Fetal bovine serum (FBS), Dulbecco’s modified Ea-
(
2
2
2
(
gle medium (DMEM), Ham’s F12 medium were obtained from Hy-
Clone (Logan, UT), and the mixture of penicillin and streptomycin
was from Sigma (St. Louis, MO). All chemicals were of analytical
grade and used without further purification.
4
.2. Characterization of compounds
FT-IR spectra of all compounds prepared in KBr pellets were re-
1
corded with AVATAR360 Fourier transform spectrophotometer. H
NMR spectra were recorded on a Bruker AMX-500 Spectrometer
4
with Me Si (TMS) as internal standard. Chemical shifts were re-
1
ported in ppm relative to TMS for H NMR spectra. Electrospray
ionization mass spectrometry (ESI-MS) was conducted on a Ther-
mal Fisher LTQ linear ion-trap mass spectrometer (San Jose, CA).
Figure 7. Resistance to proteolytic degradation of GPR and ferrocene GPR conju-
–1
gates. Aliquots of GPR, GPR–Fca and Fc–GPR at a concentration of 0.76 mg mL in
–
1
4.3. Synthesis of GPR, ferrocene monocarboxylic acid (Fc-COOH)
and Fca
0
.1 M PBS were incubated overnight with 0.1 mg mL of chymotrypsin at 25 °C.
The percentage of the compounds resistant to proteolysis was determined by
comparing to the original intact peptide by HPLC.
Tripeptide GPR was synthesized following our previous proce-
dures by standard peptide synthesis procedure in solution using
Table 1
4
4
Octanol/water partition coefficients of GPR–Fca and GPRa
HOBt and HBTU as coupling reagents. Fc-COOH and Fca were
2
6,27
synthesized according to the literature procedure.
Compound
Logk
w
LogP
GPR–Fca
GPR
2.97
1.79
3.15
2.01
4.4. Synthesis of GPR–Fca (4)
a
was determined by standard method.43,44
LogP and logk
w
GPR–Fca was synthesized according to the literature proce-
2
7,45
dure,
and the synthetic procedure was shown in Scheme 1.
Cl
Briefly, to a solution of Boc-Gly-OH (5 mmol, 1.25 g) in dry CH
2
2
4
2
fibril formation and can lead to the disaggregation of existing
(DCM) at 0 °C, HOBt (5.5 mmol, 0.79 g) and HBTU (5.5 mmol, 2.1 g)
were added. After stirring for 1 h at 0 °C, H-Pro-OMe (5 mmol,
Ab1–42 mature fibrils. Moreover, the MTT cytotoxicity results indi-
cate that GPR–Fca can effectively inhibit Ab-induced toxicity to-
wards human neuroblastoma SH-SY5Y cells in vitro. The
attachment of ferrocene moiety onto GPR tripeptide allows us to
investigate the kinetics of the process that disrupts the Ab1–42
aggregation pathway. And our results suggest that the inhibitory
effect is realized through a strong interaction between GPR–Fca
and Ab1–42 at the very beginning of incubation. In particular, the
good lipophilicity and high resistance to proteolytic degradation
of GPR–Fca makes it possible to resist Ab1–42 fibril formation and
prevent the Ab-induced cytotoxicity in vivo. Our results suggest
that GPR–Fca is a potential candidate for therapeutic treatment
of AD. To develop these inhibitors for the practical treatment of
AD in the near future, we are trying to evaluate their pharmacolog-
ical and therapeutic properties on in vivo animal models, and to
improve their efficacy across BBB by modifying their chemical
structure or apply new techniques such as nanoparticulate drug
delivery systems if necessary.
0.84 g) by treatment with Et
mixture was then stirred overnight, and washed with saturated
aqueous solutions of NaHCO , citric acid (10%), and water, then
dried over Na SO , and evaporated to dryness under reduced pres-
3
N in DCM was added. The reaction
3
2
4
sure. The crude product was purified by flash column chromatog-
raphy (EtOAc/hexanes = 1:1). Then Boc-Gly-Pro-OMe was treated
by NaOH aqueous solution and the free acid Boc-Gly-Pro-OH was
given. Similarly to the synthetic procedure of Boc-Gly-Pro-OMe,
Boc-Arg(NO
2
)-Fc-OMe was synthesized. Then Boc-Arg(NO
2
)-Fc-
)-Fc-
OMe was treated by 50% TFA with DCM and NH
2
-Arg(NO
2
OMe was given.
To a solution of Boc-Gly-Pro-OH (4.0 mmol, 1.09 g) in dry DCM
at 0 °C, Et N (4.2 mmol, 0.59 mL), HOBt (4.2 mmol, 0.568 g) and
HBTU (4.2 mmol, 1.68 g) were added, reacted 1 h at 0 °C, then
1.7 g H-Arg(NO )-Fc-OMe by treatment with Et N in THF/DMF
3
2
3
was added. The reaction mixture was then stirred overnight, and
evaporated to dryness under reduced pressure. The crude product
was purified by flash column chromatography (DCM/EtOAc/
MeOH = 90:30:1) to give Boc-Gly-Pro-Arg(NO
2
)-Fca-OMe (GPR–
4
4
. Materials and methods
ꢀ
1
ꢀ1
Fca) (yield 30.6%). IR (KBr, cm ): 3116, 1149, 825 cm (í(Fc)),
ꢀ
ꢀ
ꢀ
1
1
ꢀ1
ꢀ1
ꢀ1
ꢀ1
1
1
1
712 cm
(í(C@O)), 1544 cm
(í(C–N)), 3376 cm
(s, N–H),
.1. Materials
246 cm (s, C–C), 1284 cm (s, C–N), 1069 cm (s, C–O–C),
1
617 cm (í(C@N, Arg)).
Sodium hydrogen phosphate, sodium dihydrogen phosphate,
1
3
H NMR for GPR–Fca (500 MHz, CDCl ): d 8.405–8.426 (d, 1H,
thioflavine T (ThT) and hexafluoroisopropanol (HFIP) were pur-
chased from Sigma (St Louis, MO). o-(Benzotriazol-1-yl)-
NH, Pro) 8.01 (m, 1H, á-NH, Arg), 7.776–7.795 (t, 1H, NH, Gly),
7.54–7.60 (m, 1H, â–NH, Arg), 7.42–7.46 (m, H, C@NH Arg), 5.74
0
0
N,N,N ,N -tetramethyluronium (HBTU) and 1-hydroxybenzotrizole
HOBt) were acquired from HighFine Biotech Co. (Shuzhou, China).
Amino acids, Boc-Gly-OH, H-Pro-OMe, Boc-Arg(NO )-OH, and H-
Arg(NO )-OMe were purchased from GL Biochem (Shanghai, Chi-
na). Triethylamine (Et N) and ferrocene (Fc) were obtained from
0
0
(
s, 1H, NH Fc), 4.83 (m, 1H, CH, Pro), 4.81 (s, 4H, 2,2 H, 4,4 H Cp),
.49 (s, 2H, CH2, Gly), 4.41–4.42 (m, 2H, CH Pro and Arg), 4.12
m, 4H, 3,3 H, 5,5 H Cp), 3.81 (s, 3H, OCH3, Fc), 2.78 (m, 2H, Arg
â–CH ), 2.55 (m, 2H, Arg ã-CH ), 1.59 (9H, s, Boc).
(
4
(
,
2
0
0
2
2
2
3

B. Zhou et al. / Bioorg. Med. Chem. 21 (2013) 395–402
401
ESI-MS for GPR–Fca: m/z calcd for C30
59.1; found: 359.8.
H
44
N
8
O
9
Fe [M+2H]2+
:
(diameter 3 mm), platinum wire was the counter electrode, and
Ag/AgCl/3.0 M KCl was used as the reference electrode. Ferrocene
GPR conjugates were diluted with 0.1 M PBS (pH 7.4) to a final con-
3
4
.5. Synthesis of Fc–GPR (7)
centration of 50 lM. For differential pulse voltammetric (DPV)
determinations, the following conditions were used: sample width
17 ms, pulse amplitude 50 mV, pulse width 50 ms and pulse period
200 ms.
The synthetic procedure of the Fc–GPR was shown in Scheme 1
following our previous work.44 Briefly, to a solution of Fc-COOH
5.65 mmol, 1.3 g) in dry DCM at 0 °C, Et N (6.19 mmol, 0.87 mL),
(
3
HOBt (6.19 mmol, 0.84 g) and HBTU (6.19 mmol, 2.4 g) were
added, reacted 1 h at 0 °C, then 1 g H-Gly-OMe by treatment with
4
.9. Atomic force microscopy
Et
red overnight at room temperature, washed with saturated aque-
ous solutions of NaHCO , citric acid (10%), and water, and
evaporated to dryness under reduced pressure at 40 °C. The crude
product (Fc-Gly-OMe) was purified by flash column chromatogra-
phy (DCM/EtOAc = 2:1). Then Fc-Gly-OMe was treated by NaOH
aqueous solution and the free acid Fc-Gly-OH was given. Similarly
3
N in DCM (5 mL) was added. The reaction mixture was then stir-
The morphology of Ab1–42 aggregates was obtained on a Pico-
Scan SPM microscope (Molecular Imaging, Phoenix, AZ, USA) using
a MAC Mode. For imaging, aliquots of Ab1–42 or Ab1–42/GPR–Fca
3
(
10
cast onto these mica sheets that had been pretreated with Ni(II)
in 10 mM NiCl for 15 min. Afterwards, the slides were gently
rinsed with water to remove salt, and then dried with N
lL) were taken out at a predetermined incubation time and
2
2
.
to the synthesis of Fc-Gly-OMe, Pro-Arg(NO
1
1
2
)-OMe (3.4 mmol,
.45 g) was added to the solution of Fc-Gly-OH (3.61 mmol,
.04 g) in DCM. The mixture was then stirred for 24 h at room tem-
4
.10. Cytotoxicity assay
perature. The crude product was purified by flash column chroma-
tography (DCM/EtOAc/MeOH = 90:30:5) to give Fc-Gly-Pro-
SH-SY5Y cells were maintained in a medium of DMEM, Ham’s
F12, FBS, and the mixture of penicillin and streptomycin (V/V/V/
V = 44.5%:44.5%:10%:1%) at 37 °C under a humidified atmosphere
2
of 5% CO . The cultured cells were then transferred to a 96-well
plate with approximately 10,000 cells per well. After overnight
incubation at 37 °C the medium was replaced with a DMEM/F12
medium containing 5% FBS and Ab1-42 sample or Ab1–42/GPR,
Ab1–42/GPR–Fca mixture which were pre-incubated at 32 °C for
ꢀ1
ꢀ1
ꢀ1
Arg(NO
2
)-OMe (Fc–GPR) (yield 48.5%). IR (cm ): 1105, 824 cm
ꢀ1
ꢀ1
(
(
(
m
(Fc)), 1740 cm
(m
(C@O)), 1438 cm
(m(C–N)), 3307 cm
ꢀ1
ꢀ1
1
m
(N–H)), 1539 cm (d(N–H)), 1629 cm
(m(C@N, Arg)). H NMR
3
500 MHz, CDCl ,): ä 8.206(s, 1H, á–NH, Arg),7.38(s, 1H, NH, Gly),
7
2
.021(s, 1H, C@NH, Arg), 4.627(m, 1H, CH, Arg), 4.398(s, 2H,
0
0
,2 H Cp), 4.351(s, 2H, 3,3 H, Cp), 4.215(s, 2H, Gly), 4.141(s, 5H,
, Arg), 3.55 (m, 2H, CH2, Pro), 3.38 (m, 2H,
Cp), 3.792 (s, 3H, OCH
CH
, Pro), 2.97ꢁ2.76 (m, 4H, Arg â–CH
ESI-MS for Fc–GPR: m/z calcd for C25
3
4
8 h to form the different aggregates, then the SH-SY5Y cells were
incubated with the resultant media at 37 °C in an atmosphere of 5%
CO for 24 h. Viability of the cells exposed to each solution was
2
2 2
, ã–CH ).
FeNa [M+Na]+:
33 7 7
H N O
2
6
22.2; found 622.6.
determined based on the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphe-
nyltetrazolium bromide (MTT) assay. Briefly, media with the Ab1–
4
.6. Preparation of Ab1–42 solutions
42
sample or inhibitor/Ab1-42 mixture in the wells were replaced
–
1
with 100
solutions were incubated for 4 h. After removal of the MTT-con-
taining media, 150 L of DMSO was added into each well to dis-
lL media containing 0.5 mg mL . MTT and the resultant
Ab1–42 purchased from American Peptide Company Inc. (Sunny-
vale, CA) was prepared as follows. Briefly, HFIP (250 L) was added
l
l
to 0.25 mg of lyophilized Ab1–42 and the solution was kept at room
temperature for 2 h. This was followed by sonication for 30 min
and then centrifugation at 14,000 rpm for 30 min. The supernatant
was pipetted out and freeze-dried. The as-treated Ab1–42 sample
was then dissolved in 20 mM NaOH and sonicated for 1 min, which
was followed by centrifugation at 14,000 rpm for 30 min. The
supernatant was again pipetted out and used to prepare the stock
solution of Ab1–42. Ferrocene GPR conjugates were dissolved in
DMSO, followed by dilution with water to prepare the stock solu-
tion of 1 mM ferrocene GPR conjugates. All aqueous solutions were
solve the formazan precipitate. UV–Vis absorption was then
recorded at 570 nm using a microplate reader (Tecan, San Jose,
CA). MTT assay in five separate wells was conducted, and the re-
ported viability was the average value obtained on three different
days.
4
.11. Protease-resistance assay
Aliquots of GPR, GPR–Fca and Fc–GPR at a concentration of
ꢀ
1
0
.76 mg mL in 0.1 M phosphate buffered saline (PBS) were incu-
prepared using Millipore water (18 M
purification system (Millipore Co., Billerica, MA).
X cm) treated by a water
ꢀ
1
bated overnight with 0.1 mg mL of chymotrypsin (Bio Basic Inc.,
NY, USA) at 25 °C. Thereafter, the samples were injected to reverse-
phase HPLC (Shimadzu 6AD, Columbia, MO) on a C18 column
4
.7. Fluorometric quantitation of fibrillogenesis
(
dimension of 250 ꢂ 4.6 mm i.d.) from Phenomenex (Torrance,
CA) with a linear gradient of 10–50% acetonitrile containing 0.1%
trifluoroacetic acid in 20 min. The percentage of the compounds
resistant to proteolysis was measured by comparing the peak area
Formation of fibrils was monitored using ThT fluorescence as-
say. Briefly, an aliquot of the aggregated Ab1–42 from the stock
was collected at indicated time intervals and treated with 10
lM
(
UV absorbance at 215 nm) corresponding to the intact compound
ThT in 50 mM glycine–NaOH (pH 9.2) solution immediately before
the measurement. ThT fluorescence was measured at 482 nm with
excitation at 450 nm and slits of 5 nm for both excitation and emis-
sion using an F-2500 FL Spectrophotometer (Hitachi) in a 1.0 cm
quartz cuvette.
which was incubated in PBS.
4
.12. Determination of the octanol/water partition coefficient
(
logP value) by RP-HPLC
LogP values were determined by RP-HPLC according to a litera-
4
.8. Electrochemical measurements
4
2,43
ture procedure.
Water and methanol each containing 0.1% (v/
v) TFA were used as eluents. Four different standards with known
logP (naphthalene, phenol, acetophenone and nitrophenol) were
used for calibrating the system and to establish a correlation be-
Electrochemical measurement was carried out with a model
CHI660B Electrochemical Workstation (CH Instruments, Austin
TX) at room temperature. Glassy carbon was the working electrode

4
02
B. Zhou et al. / Bioorg. Med. Chem. 21 (2013) 395–402
18. Bincoletto, C.; Tersariol, I. L. S.; Oliveira, C. R.; Dreher, S.; Fausto, D. M.; Soufen,
w
tween logP and logk . These compounds were dissolved in meth-
anol and uracil was exploited to determine the system’s dead time.
M. A.; Nascimento, F. D.; Caires, A. C. F. Bioorg. Med. Chem. 2005, 13, 3047.
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1
2
Acknowledgments
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Partial support of this work by the Natural Science Foundation
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