Regulating the Rate of Molecular Self-Assembly for Targeting Cancer Cells

Article abstract of DOI:10.1002/anie.201600753

Besides tight and specific ligand-receptor interactions, the rate regulation of the formation of molecular assemblies is one of fundamental features of cells. But the latter receives little exploration for developing anticancer therapeutics. Here we show

Full text of DOI:10.1002/anie.201600753

Angewandte
Communications
Chemie
International Edition: DOI: 10.1002/anie.201600753
German Edition: DOI: 10.1002/ange.201600753
Anticancer Therapeutics
Regulating the Rate of Molecular Self-Assembly for Targeting Cancer
Cells
Jie Zhou, Xuewen Du, and Bing Xu*
Abstract: Besides tight and specific ligand–receptor interac-
tions, the rate regulation of the formation of molecular
assemblies is one of fundamental features of cells. But the
latter receives little exploration for developing anticancer
therapeutics. Here we show that a simple molecular design of
the substrates of phosphatases—tailoring the number of
phosphates on peptidic substrates—is able to regulate the rate
of molecular self-assembly of the enzyme reaction product.
Such a rate regulation allows selective inhibition of osteosar-
coma cells over hepatocytes, which promises to target cancer
cells in a specific organ. Moreover, our result reveals that the
direct measurement of the rate of the self-assembly in a cell-
based assay provides precise assessment of the cell targeting
capability of self-assembly. This work, as the first report
establishing rate regulation of a multiple-step process to inhibit
cells selectively, illustrates a fundamentally new approach for
controlling the fate of cells.
formation of nanofibrils of small peptidic hydrogelators in
pericellular space of cancer cells, thus achieved selective
[4c]
inhibition of cancer cells, including drug-resistant ones. This
approach turns out to be general since the use of a phosphory-
[
6]
[7]
lated carbohydrate or phosphorylated nanoparticles as the
substrates of ALP for EISA also selectively inhibit cancer
cells. These results, undoubtedly, establish that EISA is able
[
8]
to utilize “undruggable” enzymes, such as ALP, for gen-
erating molecular nanofibrils to inhibit cancer cells. Despite
the promises of the ALP-based EISA for potential cancer
therapy, the ubiquitous presence of ALPs in human body
presents a unique challenge. As revealed by the studies of
[
9]
mammalian ALPs, being a type of ectoenzymes and present
in all tissues throughout the entire body for important
biological functions, ALPs exist in four typical isozymes—
placental alkaline phosphatase (ALPP), germ cell alkaline
phosphatase (ALPP2), intestinal alkaline phosphatase
(ALPI), and tissue non-specific alkaline phosphatase
T
his study reports targeting cancer cells by the control of the
(ALPL). Although ALPP overexpresses only on certain
cancer cells and not on normal cells, ALPL expresses on
rate of the formation of supramolecular assemblies. Molec-
ular-targeted therapeutics, which mostly relies on tight
ligand–receptor interaction or enzyme inhibition, has been
a key strategy for developing cancer drugs. However, recent
advances in cancer biology have revealed the great complex-
ity of cancers, such as redundant signaling pathways, adaptive
drug resistance, genomic instability, intratumoral heteroge-
[10]
normal cells. While the apparent solution for selectivity is
to develop a substrate being dephosphorylated by ALPP only,
it would not be an easy task for two reasons. First, the inability
to develop a highly selective inhibitor for ALPP over ALPL
implies that ALPP and ALPL are indiscriminate to their
substrates. Second, there is little structural information of
ALPL, which makes the design of specific substrates difficult,
if not impossible. In addition, a single cell can co-express
different isozymes of ALP, albeit at different levels, further
complicating the situation. These facts demand a new strategy
for precisely targeting cancer cells in a desired organ or tissue,
particularly in the case of cancer cells overexpressing ALPL.
To meet the above need, we choose to use the rate of
molecular self-assembly to amplify the difference of the
expression level of ALPs in cancer and normal cells for
targeting cancer cells. As a demonstration of concept, we
select two cell lines (i.e., HepG2 (liver hepatocellular
carcinoma) and Saos-2 (osteosarcoma)) known to express
ALPL and design two kinds of substrates (i.e., monophos-
phorylated and diphosphorylated peptides, Figure 1) of
ALPL for regulating the rate of self-assembly. We choose
HepG2 and Saos-2 because the former acts as the model cell
[
1]
neity, and tumor microenvironment. These conceptual
advances not only elucidate that the major root of drug
resistance in current cancer therapy is the reliance on specific,
tight ligand–receptor binding of only one or two molecular
targets (e.g., enzymes, receptors, or transcription factors), but
also underscore an urgent need of new approaches for cancer
therapy. Recognizing that cancer immunotherapy essentially
is a form of spatiotemporal controlled apoptosis in human
body and enzyme-instructed self-assembly (EISA) is an
[
2]
inherent feature of apoptosis, we have departed from the
dogma of specific ligand–receptor binding and are focusing on
integrating enzyme transformation (ET) and self-assembly
[
3]
(
SA) to generate the fibrils of small molecules as potential
[
4]
anticancer therapeutics.
Based on the development of enzymatic self-assembly and
[5]
hydrogelation/aggregation of small molecules, we have
employed alkaline phosphatases (ALP) to catalyse the
[11]
of hepatocytes
and the latter is known to overexpress
[6]
ALPL on membrane. Our antibody staining reveals that
HepG2 cells, indeed, express less ALPLs than Saos-2 does.
Tailoring the rate of self-assembly is able to amplify such
a subtle difference in the expression of ALPL. As illustrated
in Scheme 1, the rate for generating the self-assembling
peptide (i.e., TPD) should be slower from the diphosphory-
lated substrates (i.e., TPD-2p) than from the monophos-
[
*] J. Zhou, X. W. Du, Prof. Dr. B. Xu
Department of Chemistry, Brandeis University
4
15 South St, Waltham, MA 02454 (USA)
E-mail: bxu@brandeis.edu
Supporting information for this article can be found under:
http://dx.doi.org/10.1002/anie.201600753.
5
770
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2016, 55, 5770 –5775

Angewandte
Communications
Chemie
[4e]
tyrosine promotes self-assembly, we design a d-tetrapep-
tide derivative (TPD-1p, Figure 1) by inserting a tyrosine
between two phenylalanine residues of that d-tripeptide
derivative. Besides potentially retaining or even enhancing
the self-assembly ability of the d-tetrapeptide, the additional
tyrosine residue provides another site for phosphorylation,
which affords a diphosphorylated substrate (TPD-2p) for
ALP. We expect the ALP-catalysed formation of TPD would
be slower from TPD-2p than from TPD-1P. To directly
visualize the self-assembly behaviour of TPD generated from
these two types of precursors, we design fTPD-2p and fTPD-
1
p (Figure 1) as the fluorescent analogues of TPD-2p and
Figure 1. Molecular structures of the tetrapeptide derivatives/precur-
sors with two or one phosphotyrosines (TPD-2p and TPD-1p) and their
fluorescent analogs (fTPD-2p and fTPD-1p). These precursors turn into
the self-assembling d-peptidic hydrogelators (i.e., TPD and fTPD) after
enzymatic dephosphorylation by alkaline phosphatase (ALP).
TPD-1p by replacing the Nap motif with nitrobenzoxadiazole
(NBD), a fluorophore known to fluoresce brightly in assem-
[
4e]
bled state.
After preparing the non-commercial starting
materials (e.g., Fmoc-phosphotyrosine and NBD-COOH
(
Figure S1 in the Supporting Information, SI)) prior to solid
[12]
phase peptide synthesis (SPPS),
we used SPPS for the
phorylated substrates (i.e., TPD-1p) because the former
requires dephosphorylation twice. Cell viability assays indi-
cate that, while TPD-2p and TPD-1p exhibit similar inhib-
itory activities against Saos-2 cells, TPD-2p is more cell
compatible than TPD-1p towards HepG2 cells. Moreover, the
fluorescent analogues of TPD-2p and TPD-1p (i.e., fTPD-2p
and fTPD-1p) confirm that the diphosphorylated substrate
results in little molecular assemblies on HepG2 cells, but
monophosphorylated substrate affords significant amount of
molecular assemblies on HepG2 cells. These results establish
that it is feasible to achieve excellent targeting selectivity by
using the rate of molecular self-assembly to amplify the
difference of ectoenzyme expressions on different cells/
tissues. Besides being the first report of using rate regulation
of a multiple-step process to inhibit cells selectively, this work
illustrates a novel approach for developing therapeutics that
may pass by liver and target the tumor site (e.g., sites of
osteosarcoma) for selectively killing cancer cells.
synthesis and high-performance liquid chromatography
(HPLC) for the purification and obtained the molecules
shown in Figure 1 in good yields (60–80%). LC-MS and
H NMR confirm the purity and structures of these designed
molecules (SI)
1
Both TPD-2p and TPD-1p dissolve well in water to make
transparent solutions at physiological pH 7.4 at the concen-
tration of 500 mm (the highest concentration used in cell
experiment) (Figure S2A). The solubility of TPD-2p is better
than that of TPD-1p due to an additional phosphate. Trans-
mission electron microscopy (TEM) images of their solutions
after water evaporation show that both TPD-2p and TPD-1p
form few amorphous aggregates (Figure 2A), suggesting that
the majorities are still oligomers or monomers that cannot be
visualized by TEM. Having the same peptide backbone,
either TPD-2p or TPD-1p results in the same self-assembling
molecule TPD after its complete dephosphorylation cata-
lyzed by ALP. This feature explains that treating the above
À1
We have validated that a small d-tripeptide derivative,
solutions with ALP (1 UmL ) leads to the same viscous
Nap-d-Phe-d-Phe-d- Tyr, with the tyrosine phosphorylated,
solutions (Figure S2A) with same nanofibrils (d = 5 Æ 2 nm)
in them (Figure 2A). TPD-2p takes longer time to be
completely dephosphorylated due to the second phosphate,
as evidenced by that TPD-2p takes longer time (Figure S2B)
p
selectively inhibits cancer cells due to the different expression
of ALPs between cancer (e.g., HeLa) and normal (e.g., HS-5)
[
4c,d,10b]
cells.
Based on this result and that aromatic ring of
Scheme 1. Illustration of the use of the rate of molecular self-assembly (controlled by numbers of enzymatic site) to amplify the difference of the
expression level of ALPs in different cell lines.
Angew. Chem. Int. Ed. 2016, 55, 5770 –5775
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5771

Angewandte
Communications
Chemie
Figure 3. A) Confocal microscopy images of HepG2 and Saos-2 cells
after ALPL antibody staining. Nuclei are stained by Hoechst 33342.
B) Fluorescence quantification of ALPL antibody staining of HepG2
and Saos-2. The values are normalized so that of Saos-2 equals to 1.
high level of ALPL, as the whole cell membrane emits bright
green fluorescence visualized by confocal fluorescence mi-
croscopy. Fluorescence quantification of the antibody staining
indicates at least 5 times more ALPL on Saos-2 cell
membrane than HepG2 cells. The expression level of
ALPLs on HepG2 cell surface is moderate, compared with
that of Saos-2 cells. This result justifies our choices of HepG2
and Saos-2 cells for proving the concept outlined in Scheme 1.
To verify that TPD-2p amplifies the difference of the ALP
expression levels in different cell lines for targeting cancer
cells, we use TPD-2p to treat HepG2 and Saos-2 cells, and use
TPD-1p as a control. After treating HepG2 and Saos-2 cells
with TPD-2p or TPD-1p at different concentrations, the cell
viabilities at different time (i.e., 24, 48, and 72 hr) (Figure 4
and Figure S3) show that TPD-2p and TPD-1p exhibit similar
cytotoxicity on Saos-2 cell, with the 48hr-IC50 values of 130,
1
1
50 mm, respectively, but TPD-2p is less cytotoxic than TPD-
p on HepG2 cells (with the 48hr-IC50 value of 300, 180 mm,
respectively). More specifically, as shown in Figure 4A, by
adding a second phosphate on the molecule of TPD-1p to
obtain TPD-2p, we successfully enhance the selectivity for
targeting Saos-2 cells over HepG2 cells, from 30% cell
viability difference to 48% cell viability difference, 1.6-fold
enhancement, at the concentration of 200 mm, and 1.56, 1.82,
1.90 fold enhancement at the concentration of 300, 400,
500 mm, respectively.
Figure 2. A) TEM images and B) static light scattering signals of the
solutions of different precursors (TPD-2p, TPD-1p and fTPD-2p, fTPD-
1
p) before and after ALP treatment. [precursor]=500 mm, pH 7.4,
À1
[
ALP]=1 UmL , in PBS buffer. The scale bar is 100 nm.
to form a solid hydrogel (1.5 h) than TPD-1p does (0.5 h)
0.5 wt%). fTPD-2p (or fTPD-1p) exhibits similar behavior
(
as TPD-2p (or TPD-1p) (Figure 2A), except that replacing
the Nap motif by NBD group slightly decreases the self-
assembly ability. TEM images reveal similar nanofibrils (d =
The above results agree with the significantly higher
ALPL expression on Saos-2 cells (Figure 3) and support the
concept illustrated in Scheme 1. That is, the dephosphoryla-
tion of TPD-1p generates TPD for immediate self-assembly,
but the dephosphorylation of TPD-2p first results in inter-
mediates (TPD-1p and TPD-1p’, SI), which are more soluble
and diffuse more freely than TPD, before the second
dephosphorylation to form TPD. High ALPL level on Saos-
2 cells warrants rapidly dephosphorylate TPD-2p to TPD
before the intermediates diffuse away. So TPD-2p and TDP-
1p exhibit almost same inhibitory activities to Saos-2 cells.
However, in the case of less ALPL on HepG2, dephosphor-
ylation of TPD-1p forms TPD that self-assembles immedi-
ately to result in the nanofibrils on cell surface to cause cell
death, but dephosphorylation the TPD-2p firstly generates
the intermediates (TPD-1p and TPD-1p’) (Figure S4), which
can drift away due to their good solubility before continuously
4
Æ 2 nm) in their solutions after ALP treatment. Static light
scattering (SLS) signals of the solutions of different precur-
sors before and after the addition of ALP confirm that all the
four small molecules hardly self-assemble before enzymatic
dephosphorylation, but form aggregates/nanofibrils after
ALP treatment, because the signals of the solutions of
precursors are almost the same with that of PBS buffer and
dramatically increase after the addition of ALP.
[13]
Despite that Western blot
indicates a significantly
higher ALPL expression in Saos-2, it represents the enzyme
amount in cell lysate, mainly from the cytosol. We thus use
antibody staining to confirm the different ALPL levels on the
membranes of HepG2 and Saos-2 cells. According to the
results in Figure 3, Saos-2 cells, indeed, express extremely
5
772
www.angewandte.org
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2016, 55, 5770 –5775

Angewandte
Communications
Chemie
The replacement of N-terminal-capped motifs results in
reduced self-assembly ability and reduced cytotoxicity (Fig-
ure S5), but still allows their corresponding self-assembling
molecules fTPD to self-assemble and form nanofibrils in
pericellular space of cells. According to Figure 5, treating
Figure 5. Confocal microscopy images and the corresponding fluores-
cence quantification of A) HepG2 and B) Saos-2 cells treated with
fTPD-2p and fTPD-1p at the concentration of 500 mm for 12 hours.
Nuclei are stained by Hoechst 33342.
HepG2 cells with fTPD-2p (500 mm) for 12 hours only leads to
faint yellow fluorescence in the cells, likely due to endocy-
tosis, while the addition of fTPD-1p into HepG2 cell culture
results in strong fluorescence on the surface of the cells.
Contrasting to the case of HepG2 cells, the addition of fTPD-
2
p or fTPD-1p in Saos-2 cell culture leads to the similar
result—significant fluorescence on cell surface (Figure 5),
which is more fluorescent than that on HepG2 cell treated by
fTPD-1p. Because NBD-modified peptides fluoresce
Figure 4. A) 48-hour cytotoxicity of TPD-2p and TPD-1p on HepG2 and
Saos-2 cells at different concentration. The viability differences
between the two cell lines are labelled as well. B) 48-hour cell viability
of HepG2 and Saos-2 cells incubated with TPD-2p and TPD-1p
[4e]
intensely in self-assembled nanofibrils, the yellow fluores-
cence reflects the amount of nanofibrils formed by EISA of
fTPD-2p or fTPD-1p. Quantification of the yellow fluores-
cence in pericellular space of HepG2 and Saos-2 cells treated
by fTPD-1p and fTPD-2p is highly consistent with cell
viability results, which is also supported by antibody staining
of ALPL. Moreover, treating the Saos-2 and HepG2 cells with
fTPD-2p or fTPD-1p together with ALPL inhibitor DQB
(10 mm) for 4 hours prior to imaging, we observe obvious
difference between the cells with and without DQB even by
naked eyes (Figure S6). Confocal fluorescence images further
confirm the inhibitory effect (Figure S7). These results
indicate that ALPL catalysed self-assembly to generate the
strong fluorescence, agreeing with the cell viability data
discussed above.
The direct measurement of the formation rate of the self-
assembling molecules in a cell-based assay (SI) provides more
accurate assessment of the cell targeting capability of self-
assembly. Specifically, we treat the solution of TPD-2p and
TPD-1p (500 mm in PBS buffer, pH 7.4) with the cell lysate of
HepG2 or Saos-2 ( ꢀ 500 mg total protein) and examine the
amount (%) of each component at different time points by
(
300 mm) with or without ALPL inhibitors (DQB, 2 mm) for 48 h. The
4
initial cell number is 1”10 cells/well.
interacting with ALPL to lose another phosphate and then
self-assemble. So TPD-2p is more cell compatible than TDP-
1
p.
To further confirm the ALPL-catalyzed dephosphoryla-
tion is responsible for the observed cell inhibition, we
incubate HepG2 and Saos-2 cell by TPD-2p and TPD-1p
[14]
together with ALPL inhibitor (2,5-dimethoxy-N-(quinolin-
-yl)benzenesulfonamide, denoted as DQB and at 2 mm). As
3
shown in Figure 4B and Figure S3, the presence of ALPL
inhibitor, DQB, significantly reduces the inhibition of Saos-2
and HepG2 cells. Obviously, DQB rescues more Saos-2 cells
than HepG2 cells (e.g., 50% vs. 20%) for both TPD-2p and
TPD-1p. These results not only validate the critical role of
ALPLs for instructing the self-assembly of small d-peptide
derivatives to inhibit cancer cells, but also agree with the
different ALPL expression levels on Saos2 and HepG2 cells.
Angew. Chem. Int. Ed. 2016, 55, 5770 –5775
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5773

Angewandte
Communications
Chemie
Acknowledgements
This work was partially supported by NIH (R01CA142746)
and NSF (MRSEC 1420382). We thank Brandeis EM and
Optical Imaging facilities for TEM. J.Z. is a HHMI interna-
tional student fellow.
Keywords: cancer · enzymes · rate regulation ·
selective inhibition · self-assembly
How to cite: Angew. Chem. Int. Ed. 2016, 55, 5770–5775
Angew. Chem. 2016, 128, 5864–5869
[
1] a) D. Hanahan, R. A. Weinberg, Cell 2000, 100, 57 – 70; b) D.
Hanahan, R. A. Weinberg, Cell 2011, 144, 646 – 674.
[
[
2] J. Zhou, B. Xu, Bioconjugate Chem. 2015, 26, 987 – 999.
3] a) M. Yamanaka, N. Haraya, S. Yamamichi, Chem. Asian J. 2011,
6, 1022 – 1025; b) L. A. Estroff, A. D. Hamilton, Chem. Rev.
2
004, 104, 1201 – 1217; c) T. Noguchi, B. Roy, D. Yoshihara, Y.
Tsuchiya, T. Yamamoto, S. Shinkai, Chem. Sci. 2015, 6, 3863 –
867.
4] a) Z. M. Yang, K. M. Xu, Z. F. Guo, Z. H. Guo, B. Xu, Adv.
Mater. 2007, 19, 3152 – 3156; b) J. Li, Y. Kuang, J. Shi, J. Zhou,
J. E. Medina, R. Zhou, D. Yuan, C. Yang, H. Wang, Z. Yang, J.
Liu, D. M. Dinulescu, B. Xu, Angew. Chem. Int. Ed. 2015, 54,
3
Figure 6. The time-dependent curves for dephosphorylation process of
TPD-2p and TPD-1p (500 mm) after incubation with the cell lysate of
HepG2 and Saos-2 at 378C in PBS buffer.
[
1
3307 – 13311; Angew. Chem. 2015, 127, 13505 – 13509; c) Y.
analytical HPLC. As shown in Figure 6 and Figure S8, in the
first hour, the lysates of Saos-2 and HepG2, respectively,
dephosphorylate more than 40% and less than 5% of TPD-
Kuang, J. Shi, J. Li, D. Yuan, K. A. Alberti, Q. Xu, B. Xu, Angew.
Chem. Int. Ed. 2014, 53, 8104 – 8107; Angew. Chem. 2014, 126,
8
242 – 8245; d) J. Shi, X. Du, D. Yuan, J. Zhou, N. Zhou, Y.
Huang, B. Xu, Biomacromolecules 2014, 15, 3559 – 3568; e) J.
Zhou, X. Du, J. Li, N. Yamagata, B. Xu, J. Am. Chem. Soc. 2015,
1
p. The conversion of TPD-2p follows the similar trend as that
of TPD-1p. Moreover, Saos-2 lysate dephosphorylates 50%
of TPD-2p or TPD-1p within 2 hours, while HepG2 lysate
takes more than 15 hours to consume half of TPD-2p and
more than 6 hours to dephosphorylate half of TPD-1p. This
significant difference, agreeing with the antibody staining,
further supports the cell viability data and fluorescence
imaging. Besides the cell-based assay, we examined the
preference of ALP to the two tyrosine phosphates of TPD-
1
37, 10040 – 10043.
[
5] a) Z. Yang, H. Gu, D. Fu, P. Gao, J. K. Lam, B. Xu, Adv. Mater.
2004, 16, 1440 – 1444; b) S. Toledano, R. J. Williams, V. Jaya-
warna, R. V. Ulijn, J. Am. Chem. Soc. 2006, 128, 1070 – 1071;
c) Z. Yang, G. Liang, B. Xu, Acc. Chem. Res. 2008, 41, 315 – 326;
d) X. Miao, W. Cao, W. Zheng, J. Wang, X. Zhang, J. Gao, C.
Yang, D. Kong, H. Xu, L. Wang, Z. Yang, Angew. Chem. Int. Ed.
2
013, 52, 7781 – 7785; Angew. Chem. 2013, 125, 7935 – 7939;
e) M. Zelzer, S. J. Todd, A. R. Hirst, T. O. McDonald, R. V.
Ulijn, Biomater. Sci. 2013, 1, 11 – 39; f) A. G. Cheetham, D.
Keith, P. Zhang, R. Lin, H. Su, H. Cui, Curr. Cancer Drug
Targets, 2015, 16, 1 – 20; g) L. Cui, D. Tokarz, R. Cisek, K. K. Ng,
F. Wang, J. Chen, V. Barzda, G. Zheng, Angew. Chem. Int. Ed.
2
p in PBS buffer with commercially available ALP, and found
that ALP exhibits little preference to the terminal or mid
tyrosine phosphates in TPD-2p (Figure S9).
In conclusion, this work demonstrates a simple strategy to
regulate the rate of molecular self-assembly of the enzyme
reaction product by tailoring the number of phosphates on
peptidic substrates. Our results verify the feasibility to
achieve better target selectivity by using the self-assembling
rate to amplify the difference in enzyme expressions on
different cells/tissues and using molecular engineering to
reduce side effects. For example, comparing to TPD-1p, TDP-
2
015, 54, 13928 – 13932; Angew. Chem. 2015, 127, 14134 – 14138.
[
6] R. A. Pires, Y. M. Abul-Haija, D. S. Costa, R. Novoa-Carballal,
R. L. Reis, R. V. Ulijn, I. Pashkuleva, J. Am. Chem. Soc. 2015,
1
37, 576 – 579.
[7] X. Du, J. Zhou, L. Wu, S. Sun, B. Xu, Bioconjugate Chem. 2014,
25, 2129 – 2133.
8] M. A. T. Blaskovich, Curr. Med. Chem. 2009, 16, 2095 – 2176.
9] J. L. Millan, Mammalian Alkaline Phosphatases: From Biology
to Applications in Medicine and Biotechnology, Wiley-VCH,
Weinheim, 2006.
[
[
2
p would be a better choice for targeting osteosarcoma (e.g.,
Saos-2) by intravenous injection because TPD-2p would be
more likely than TPD-1p to pass by liver (i.e. hepatocytes like
HepG2), which expresses ALPL, to finally reach the tumor
site (e.g., Saos-2) (Scheme 1). Although this work focuses on
the same isoenzyme, the principle established would be
applicable to different isoenzymes that exhibit different
reactivity, which promises a new way for controlling dosage
in precision medicine.
[
10] a) W. H. Fishman, N. R. Inglis, S. Green, C. L. Anstiss, N. K.
Gosh, A. E. Reif, R. Rustigian, M. J. Krant, L. L. Stolbach,
Nature 1968, 219, 697 – 699; b) M. Uhlen, L. Fagerberg, B. M.
Hallstroem, C. Lindskog, P. Oksvold, A. Mardinoglu, A.
Sivertsson, C. Kampf, E. Sjoestedt, A. Asplund, I. Olsson, K.
Edlund, E. Lundberg, S. Navani, C. A.-K. Szigyarto, J. Odeberg,
D. Djureinovic, J. O. Takanen, S. Hober, T. Alm, P. -H. Edqvist,
H. Berling, H. Tegel, J. Mulder, J. Rockberg, P. Nilsson, J. M.
Schwenk, M. Hamsten, K. von Feilitzen, M. Forsberg, L.
Persson, F. Johansson, M. Zwahlen, G. von Heijne, J. Nielsen,
F. Ponten, Science 2015, 347, 1260419.
5
774
www.angewandte.org
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2016, 55, 5770 –5775

Angewandte
Communications
Chemie
[
[
[
11] E. G. Schuetz, J. D. Schuetz, S. C. Strom, M. T. Thompson, R. A.
Fisher, D. T. Molowa, D. Li, P. S. Guzelian, Hepatology 1993, 18,
254 – 1262.
12] Fmoc Solid Phase Peptide Synthesis: A Practical Approach
Eds.: W. C. Chan, P. D. White), Oxford Univ. Press, Oxford,
000.
[14] R. Dahl, E. A. Sergienko, Y. Su, Y. S. Mostofi, L. Yang, A. M.
Simao, S. Narisawa, B. Brown, A. Mangravita-Novo, M.
Vicchiarelli, L. H. Smith, W. C. OꢀNeill, J. L. Millan, N. D. P.
Cosford, J. Med. Chem. 2009, 52, 6919 – 6925.
1
(
2
Received: January 22, 2016
Revised: March 11, 2016
Published online: April 6, 2016
13] http://www.abcam.com/Alkaline-Phosphatase-Tissue-Non-
Specific-antibody-EPR4477-ab108337-references.html.
Angew. Chem. Int. Ed. 2016, 55, 5770 –5775
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5775