Facile synthesis of Fmoc-protected phosphonate pSer mimetic and its application in assembling a substrate peptide of 14-3-3 ζ

Article abstract of DOI:10.1016/j.tetlet.2017.05.037

Phosphatase-inert peptidomimetics containing phosphonate pSer analogue have been developed as valuable biological tools for probing and regulating pSer-dependent protein-protein interactions (PPIs) in cellular context. Herein, we report a facile and efficient synthesis route of Fmoc-protected phosphonate pSer mimetic and also present the application of this building block in the solid-phase synthesis of a phosphatase-resistant substrate peptide of 14-3-3 ζ, retaining 14-3-3 ζ binding efficacy similar to the parent pSer-containing peptide.

Full text of DOI:10.1016/j.tetlet.2017.05.037

Accepted Manuscript
Facile synthesis of Fmoc-protected phosphonate pSer mimetic and its applica-
tion in assembling a substrate peptide of 14-3-3 ζ
Jie Kang, Hong-Xue Chen, Si-Qi Huang, Yun-Lai Zhang, Rong Chang, Fang-
Yi Li, Yan-Mei Li, Yong-Xiang Chen
PII:
S0040-4039(17)30622-6
DOI:
http://dx.doi.org/10.1016/j.tetlet.2017.05.037
TETL 48930
Reference:
To appear in:
Tetrahedron Letters
Received Date:
Revised Date:
Accepted Date:
3 April 2017
9 May 2017
12 May 2017
Please cite this article as: Kang, J., Chen, H-X., Huang, S-Q., Zhang, Y-L., Chang, R., Li, F-Y., Li, Y-M., Chen,
Y-X., Facile synthesis of Fmoc-protected phosphonate pSer mimetic and its application in assembling a substrate
peptide of 14-3-3 ζ, Tetrahedron Letters (2017), doi: http://dx.doi.org/10.1016/j.tetlet.2017.05.037
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Graphical Abstract
To create your abstract, type over the instructions in the template box below.
Fonts or abstract dimensions should not be changed or altered.
Facile synthesis of Fmoc-protected phosphonate
pSer mimetic and its application in assembling a
substrate peptide of 14-3-3 ζ
Leave this area blank for abstract info.
‡
‡
Jie Kang , Hong-Xue Chen , Si-Qi Huang, Yun-Lai Zhang, Rong Chang, Fang-Yi Li, Yan-Mei Li, and Yong-Xiang Chen

1
Tetrahedron Letters
Facile synthesis of Fmoc-protected phosphonate pSer mimetic and its application in
assembling a substrate peptide of 14-3-3 ζ
‡
‡
Jie Kang , Hong-Xue Chen , Si-Qi Huang, Yun-Lai Zhang, Rong Chang, Fang-Yi Li, Yan-Mei Li, and
Yong-Xiang Chen
Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University, Beijing
1
00084, China
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Phosphatase-inert peptidomimetics containing phosphonate pSer analogue have been developed
as valuable biological tools for probing and regulating pSer-dependent protein-protein
interactions (PPIs) in cellular context. Herein, we report a facile and efficient synthesis route of
Fmoc-protected phosphonate pSer mimetic and also present the application of this building
block in the solid-phase synthesis of a phosphatase-resistant substrate peptide of 14-3-3 ζ,
retaining 14-3-3 ζ binding efficacy similar to the parent pSer-containing peptide.
Available online
Keywords:
2
017 Elsevier Ltd. All rights reserved.
Phosphonate
Phosphoserine mimetic
Peptide
1
4-3-3 ζ protein
Phosphoserine (pSer 1)-dependent protein-protein interactions
PPIs) are involved in important cellular signaling pathways,
Converting these natural phosphopeptides into phosphatases-inert
peptidomimetics by incorporating phosphonate pSer mimetic will
largely facilitate the regulation of 14-3-3 ζ involved protein-
protein interaction networks in cellular environment.
(
which regulate a variety of vital biological processes including
1
cell growth and proliferation. Many of such interactions are
2
relevant with cancer development and progression. Thus,
phosphopeptides derived from phosphoproteins, which can bind
tightly to pSer-binding proteins, have been developed as
3
competitive inhibitors of such PPIs. Because serine
phosphorylation is a reversible process respectively catalyzed by
kinases and phosphatases, the phosphate group is easily removed
4
from serine by phosphatases in cellular context. Thus,
nonhydrolyzable CH
Figure 1), in which CH
oxygen in pSer 1, has been incorporated into peptides and
2
-substituted phosphonate pSer mimetic 2
(
2
moiety replaces phosphoryl ester
2
Figure 1. Structures of pSer 1, CH -substituted phosphonate pSer
mimetic 2 and its Fmoc-protected forms suitable for solid-phase
peptide synthesis.
5
proteins to avoid cleavage by phosphatases.
These
phosphopeptide/phosphoprotein mimetics are useful tools for
biological studies in cellular context and provide starting points
Dating back to previous pioneering work, a few strategies have
been successfully developed to generate the nonhydrolyzable
phosphonate pSer mimetics. Walker group prepared racemic
phosphonate pSer mimetics through the addition of
vinylphosphonate diesters to potassium (N-diphenylmethylene)
5
for further design of therapeutics.
14-3-3 ζ, one isoform of highly conserved and ubiquitously
expressed 14-3-3 protein family, often binds to specific pSer-
containing motifs of interacting proteins in PPIs, which are
involved in some vital cellular signal transductions, cell cycle
8a
glycine esters. Weber group employed the Schoellkopf
bislactim ether asymmetric amino acid synthesis approach to
6a-d
control, and apoptosis.
Meanwhile, it was reported that 14-3-3
8b
ζ was upregulated in various types of carcinomas, such as
construct such chirality-pure building blocks, which had broad
6a,6d-f
8c
pancreatic adenocarcinoma and lung carcinoma.
Thus, 14-3-
applications. More recently, Mikołajczyk group reported an
3
ζ has the implication as prognostic and therapeutic target of
asymmetric synthetic route toward 2 by diastereoselective
6a,6d,6f
8d
some cancers.
The pSer-containing peptide sequences, such
addition of α-phosphonate carbanions to sulfinimines. Haufe
7
as RLYHpSLPA, have been developed as inhibitors of 14-3-3 ζ.
group described a straightforward synthesis route starting from
—
——
‡

Corresponding author. Tel.: +86-10-6277-2259; e-mail: chen-yx@mail.tsinghua.edu.cn; These authors contributed equally.

2
Tetrahedron Letters
D-methionine to produce Boc-protected phosphonate D-pSer
The key step in constructing phosphonate pSer mimetic is the
8e
11
mimetic.
formation of P-C bond, which was achieved by using
Herein, we report a facile and efficient synthesis approach
toward nonhydrolyzable phosphonate pSer mimetic 3 in a form
suitable for Fmoc solid-phase peptide synthesis (SPPS) using
commercially available N-Boc protected L-aspartic α-tert-butyl
ester as starting material. Furthermore, we present the application
of this easily accessible building block in the solid-phase
synthesis of a substrate peptide of 14-3-3 ζ, leading to a
phosphatase-resistant inhibitor (Figure 2).
Michaelis-Becker reaction via nucleophilic attack of bromide 10
8e
by dibenzyl phosphite according to a literature procedure. The
resultant compound 11 underwent a deprotection by treatment
with a solution of TFA (50%, v), followed by re-protection using
Fmoc-OSu to give the final crude product containing mono-
benzyl and bis-benzyl phosphonate derivatives 3 and 4. After
HPLC purification, the major component 3 was obtained in a
yield of 43% over two steps. Due to the convenient removal of
benzyl groups on phosphonate in the peptide deprotection step,
the final crude product actually can be directly used for peptide
synthesis.
The obtained Fmoc-protected phosphonate pSer mimetics were
applied in the solid-phase synthesis of a phosphopeptide mimetic
(Peptide-CP 6) derived from the 14-3-3
ζ
substrate
(RLYHpSLPA) (Figure 2). Rink amide resin was chosen for
anchoring the peptide leading to an amide C-terminus. The
peptide was elongated following the standard Fmoc-based SPPS
procedures. Considering further evaluation of the binding affinity
between peptide target and 14-3-3 ζ, the 5(6)-carboxyfluorescein
(FAM) was introduced into the N-terminus, while a PEG linker
was inserted between FAM and peptide sequence to suppress the
effect of fluorophore on the peptide-protein interaction. Finally,
the peptide was released from the resin by treatment with a high
concentration of TFA (82.5%, v) while all of the protecting
groups including benzyl groups on phosphonate were removed
simultaneously. After HPLC purification, the desired Peptide-CP
6
was characterized by analytical HPLC and ESI-MS (Figure 3).
Meanwhile, the corresponding parent pSer-containing Peptide-
OP 5 and nonphosphorylated Peptide-OH 12 were prepared as
control following the similar synthetic procedures of Peptide-CP
Figure 2. Structures of the parent pSer-containing Peptide-OP 5 and
phosphatases-resistant Peptide-CP 6 containing phosphonate pSer
mimetic as substrates of 14-3-3 ζ.
6
.
Following this concise synthesis route of phosphonate pSer
mimetic (Scheme 1), the commercially available N-Boc protected
L-aspartic α-tert-butyl ester 7 was first treated with N-
hydroxysuccinimide and dicyclohexylcarbodiimide (DCC) to
give a succinimide ester intermediate 8, followed by reduction
9
4
using NaBH according to previously reported procedures,
leading to an L-homoserine derivative 9. Next, the hydroxyl
group in compound 9 was substituted with bromo group through
a derived Appel reaction using triphenylphosphine and N-
10
bromosuccinimide to afford the desired bromide 10.
Figure 3. Characterization of Peptide-CP 6. a) Analytical HPLC
profile of Peptide-CP 6. b) ESI-MS spectrum of Peptide-CP 6, Mcalcd
=1593.7 Da.
To evaluate the activities of these peptides to inhibit 14-3-3 ζ,
we applied fluorescence polarization technique to respectively
measure the binding affinities of Peptide-CP 6 and Peptide-OP 5
with 14-3-3 ζ. The nonphosphorylated Peptide-OH 12 was used
as a negative control. 100 nM FAM-containing peptides were
titrated with increasing amount of 14-3-3 ζ until the polarization
signals of Peptide-CP 6 and Peptide-OP 5 reached saturation,
while the signal of Peptide-OH 12 displays no obvious increase.
Scheme 1. Synthesis of Fmoc-protected phosphonate pSer mimetics.

3
The increasing of fluorescence polarization signal reflects the
binding of 14-3-3 ζ to the fluorescent peptides. The polarization
signal changes were respectively fitted to the saturation titration
Peptide-OP 5 and Peptide-CP 6 was 100 nM. The concentration of
14-3-3 ζ was 5 μM.
12
Equation 1 (Supporting Information, SI), generating the K
values. As shown in Figure 4a, Peptide-CP 6 displays a K value
of 0.86 μM, which is close to the K value of Peptide-OP 5 (0.46
D
Acknowledgments
D
D
This work was supported by grants from the Major State Basic
Research Development Program of China (2013CB910700) and
the National Natural Science Foundation of China (21372140).
μM) binding to 14-3-3 ζ. The results indicate that the substitution
of pSer by phosphonate pSer mimetic slightly reduces the
binding affinity of peptide substrate (RLYHpSLPA) to 14-3-3 ζ,
proving that Peptide-CP 6 can be used as an inhibitor of 14-3-3 ζ.
We deduced that the slightly reduced binding affinity might be
References and notes
caused by the less electronegativity of CH
phosphonate pSer mimetic compared to that of pSer.
2
-substituted
1.
(a) Hunter, T. Cell. 2000, 100, 113-127.
(b) McCubrey, J. A.; May, W. S.; Duronio, V.; Mufson, A. Leukemia.
2
000, 14, 9-21.
In addition, we detected the association between these peptides
and 14-3-3 ζ in the presence or absence of alkaline phosphatase
by using fluorescence polarization assay. As mentioned above,
the strength of fluorescence polarization signal reflects the degree
of 14-3-3 ζ binding to these fluorescent peptides. As shown in
Figure 4b, the addition of alkaline phosphatase clearly disrupted
the association of Peptide-OP 5 with 14-3-3 ζ due to the removal
of phosphate group from serine, while the binding of Peptide-CP
(
(
(
c) Cohen, P. Nat Cell Biol. 2002, 4, 127-130.
d) Hunter, T. Cell. 1995, 80, 225-236.
e) Seet, B. T.; Dikic, I.; Zhou, M.-M.; Pawson, T. Nat Rev Mol Cell
Biol. 2006, 7, 473-483.
2. Watanabe, N.; Osada, H. Curr Drug Targets. 2012, 13, 1654-1658.
3
.
Na, Z.; Pan, S.; Uttamchandani, M.; Yao, S. Q. Angew Chem Int Ed.
014, 53, 8421-8426.
Ma, M.-R.; Hu, Z.-W.; Zhao, Y.-F.; Chen, Y.-X.; Li, Y.-M. Sci Rep.
016, 6, 37130.
2
4
.
2
6
to 14-3-3 ζ kept intact in the presence of alkaline phosphatase.
5.
(a) Arrendale, A.; Kim, K.; Choi, J. Y.; Li, W.; Geahlen, R. L.; Borch,
These results demonstrate that Peptide-CP containing
6
R. F. Chem Biol. 2012, 19, 764-771.
(b) Tarrant, M. K.; Rho, H. S.; Xie, Z.; Jiang, Y. L.; Gross, C.; Culhane,
phosphonate pSer analogue can be developed as useful tools for
probing and regulating 14-3-3 ζ involved PPI networks in cellular
context.
J. C.; Yan, G.; Qian, J.; Ichikawa, Y.; Matsuoka, T.; Zachara, N.;
Etzkorn, F. A.; Hart, G. W.; Jeong, J. S.; Blackshaw, S.; Zhu, H.; Cole,
P. A. Nat Chem Biol. 2012, 8, 262-269.
In conclusion, we have developed a facile and efficient
synthesis route for producing Fmoc-protected phosphonate pSer
analogue. This synthetic approach has the potential to be used for
generating pSer mimetics bearing biorthogonal protecting groups
on phosphonate. In addition, the prepared phosphonate pSer
analogue has been successfully applied in the solid-phase
synthesis of a phosphatase-resistant substrate peptide of 14-3-3 ζ,
retaining 14-3-3 ζ binding efficacy similar to the parent pSer-
containing peptide.
(c) Klingberg, R.; Jost, J. O.; Schumann, M.; Gelato, K. A.; Fischle, W.;
Krause, E.; Schwarzer, D. ACS Chem Biol. 2015, 10 , 138-145.
(
d) Rogerson, D. T.; Sachdeva, A.; Wang, K.; Haq, T.; Kazlauskaite,
A.; Hancock, S. M.; Huguenin-Dezot, N.; Muqit, M. M.; Fry, A. M.;
Bayliss, R.; Chin, J. W. Nat Chem Biol. 2015, 11, 496-503.
(a) Aghazadeh, Y.; Papadopoulos, V. Drug Discov Today. 2016, 21,
6
.
2
78-287.
(b) Aitken, A. Semin Cancer Biol. 2006, 16, 162-172.
(
(
c) Hermeking, H. Nat Rev Cancer. 2003, 3, 931-943.
d) Neal, C. L.; Yu, D. Expert Opin Ther Tar. 2010, 14, 1343-1354.
(
e) Niemantsverdriet, M.; Wagner, K.; Visser, M.; Backendorf, C.
Oncogene. 2008, 27, 1315-1319.
(
f) Fan, T.; Li, R.; Todd, N. W.; Qiu, Q.; Fang, H. B.; Wang, H.; Shen,
J.; Zhao, R. Y.; Caraway, N. P.; Katz, R. L.; Stass, S. A.; Jiang, F.
Cancer Res. 2007, 67, 7901-7906.
(a) Rittinger, K.; Budman, J.; Xu, J.; Volinia, S.; Cantley, L. C.;
Smerdon, S. J.; Gamblin, S. J.; Yaffe, M. B. Mol Cell. 1999, 4, 153-166.
7
8
.
.
(
b) Vazquez, M. E.; Nitz, M.; Stehn, J.; Yaffe, M. B.; Imperiali, B. J Am
Chem Soc. 2003, 125, 10150-10151.
(a) Hamilton, R.; Shute, R. E.; Travers, J.; Walker, B.; Walker, B. J.
Tetrahedron Lett. 1994, 35, 3597-3600.
(
b) Shapiro, G.; Buechler, D.; Ojea, V.; Pombovillar, E.; Ruiz, M.;
Weber, H. P. Tetrahedron Lett. 1993, 34, 6255-6258.
c) Rigger, L.; Schmidt, R. L.; Holman, K. M.; Simonovic, M.; Micura,
R. Chem Eur J. 2013, 19, 15872-15878.
(
(
(
d) Łyżwa, P.; Mikołajczyk, M. Heteroatom Chem 2011, 22, 594-598.
e) Prasad, V. P.; Wagner, S.; Keul, P.; Hermann, S.; Levkau, B.;
Schafers, M.; Haufe, G. Bioorg Med Chem. 2014, 22, 5168-5181.
Jackson, R. F. W.; Moore, R. J.; Dexter, C. S.; Elliott, J.; Mowbray, C.
E. J Org Chem. 1998, 63, 7875-7884.
9
1
.
0. (a) Bhat, R. G.; Porhiel, E.; Saravanan, V.; Chandrasekaran, S.
Tetrahedron Lett. 2003, 44, 5251-5253.
(
5
b) Howarth, N. M.; Wakelin, L. P. G. J Org Chem. 1997, 62, 5441-
450.
1
1
1. Fields, S. C., Tetrahedron. 1999, 55, 12237-12273.
2. (a) Chen, Y.-X.; Koch, S.; Uhlenbrock, K.; Weise, K.; Das, D.;
Gremer, L.; Brunsveld, L.; Wittinghofer, A.; Winter, R.; Triola, G.;
Waldmann, H. Angew. Chem. Int. Ed. 2010, 49, 6090-6095.
(
b) Chu, T.-T.; Gao, N.; Li, Q.-Q.; Chen, P.-G.; Yang, X.-F.; Chen, Y.-
X.; Zhao, Y.-F.; Li, Y.-M. Cell Chem Biol. 2016, 23, 453-461.
Figure 4. a) Fluorescence polarization measurements of Peptide-OP
5
, Peptide-CP 6 and Peptide-OH 12 respectively upon addition of
different concentration of 14-3-3 ζ. The fluorescence polarization
changes of Peptide-OP 5 and Peptide-CP 6 were fitted to Equation 1
(
D
SI) to yield K values. b) Fluorescence polarization measurements
of Peptide-OP 5 and Peptide-CP 6 upon addition of 14-3-3 ζ in the
presence or absence of alkaline phosphatases. The concentration of

4
Tetrahedron Letters
Supplementary Material
The experimental description and characterization data of
compounds in this article can be found XXXX, in the online
version.
Highlights:



Developing a facile synthesis method
to CH -substituted phosphonate pSer
2
mimetic.
Applying phosphonate pSer mimetic
in SPPS synthesis of 14-3-3 ζ
substrate.
The phosphatase-resistant substrate
peptide retains 14-3-3 ζ binding
efficacy.