Copper-Free Huisgen Cycloaddition for the 14-3-3-Templated Synthesis of Fusicoccin-Peptide Conjugates

Article abstract of DOI:10.1002/asia.202000042

Mid-sized molecules have emerged as an attractive chemical space and potentially provide a robust basis for the development of synthetic agents to control intracellular protein interactions. However, the limited cell permeability and chemical tractability of such agents remain to be addressed. We envisioned that target-templated synthesis of such mid-sized molecules might provide a solution. Here, we exploited a copper-free Huisgen cycloaddition for template synthesis using a peptide fragment containing a 4,8-diazacyclononyne (DACN) moiety and an azide-containing fusicoccin derivative in the presence or absence of recombinant 14-3-3ζ protein in vitro. Time-course changes in the yield of products demonstrated that the reaction was accelerated in the presence of 14-3-3 and one of the regioisomers was generated predominantly, supporting the template effect.

Full text of DOI:10.1002/asia.202000042

CHEMISTRY
AN ASIAN JOURNAL
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Accepted Article
Title: Copper-free Huisgen Cycloaddition for the 14-3-3-Templated
Synthesis of Fusicoccin-Peptide Conjugates
Authors: Ryoma Masuda, Yuuya Kawasaki, Kazunobu Igawa,
Yoshiyuki Manabe, Hiroshi Fujii, Nobuo Kato, Katsuhiko
Tomooka, and Junko Ohkanda
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
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To be cited as: Chem. Asian J. 10.1002/asia.202000042
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Chemistry - An Asian Journal
10.1002/asia.202000042
COMMUNICATION
Copper-free Huisgen Cycloaddition for the 14-3-3-Templated
Synthesis of Fusicoccin-Peptide Conjugates
[
a]
[b]
[b]
[c]
[a]
Ryoma Masuda, Yuuya Kawasaki, Kazunobu Igawa, Yoshiyuki Manabe, Hiroshi Fujii, Nobuo
[
d]
[b]
[a]
Kato, Katsuhiko Tomooka, Junko Ohkanda*
Abstract: Mid-sized molecules have emerged as an attractive
chemical space and potentially provide a robust basis for the
development of synthetic agents to control intracellular protein
interactions. However, the limited cell permeability and chemical
tractability of such agents remain to be addressed. We envisioned that
target-templated synthesis of such mid-sized molecules might provide
a solution. Here, we exploited copper-free Huisgen cycloaddition for
template synthesis using a peptide fragment containing a 4,8-
diazacyclononyne (DACN) moiety and an azide-containing fusicoccin
derivative in the presence or absence of recombinant 14-3-3 protein
in vitro. Time-course changes in the yield of products demonstrated
that the reaction was accelerated in the presence of 14-3-3 and one
of the regioisomers was generated predominantly, supporting the
template effect.
Figure 1. Schematic representation of a protein-templated reaction for the in
situ generation of inhibitors. The protein of interest (POI) is 14-3-3 in this study.
Cell-permeable multivalent agents that are capable of recognizing
a large protein interface are highly desirable for the modulation of
intracellular signaling pathways. These dynamic and reversible
PPIs, but molecules of such size and structural complexity often
exhibit poor cell permeability and chemical tractability.
biological processes involve transient protein-protein interactions
To address these issues, we envisioned that target-templated
ligation would aid the generation of large conjugated molecules
(Figure 1). Protein-templated synthesis has been widely studied
s).[ The structural
1]
(PPIs) with short half-lives (0.1
-
1
transformation of PPIs is often regulated by reversible
posttranslational modifications of intrinsically disordered proteins
and/or regions (IDPs / IDRs).[2] Such PPI interfaces are generally
highly flexible and hydrophilic, making it challenging to develop
drug-like small molecules for directly binding to and controlling
these flexible proteins.
Alternatively, cell-permeable multivalent mid-sized synthetic
molecules[3] (Mw: 600 ~ 2,000 kDa) are advantageous for
recognizing and regulating large protein interfaces. Examples
include the naturally occurring macrocycles cyclosporin A and
rapamycin, each of which forms a complex with a peptidyl proline
isomerase, thereby increasing their molecular size and enabling
binding to a flat surface on phosphatase calcineurin and kinase
mTOR, respectively.[4,5] These natural products highlight the
promise of mid-sized molecule-based strategies for controlling
for the generation of inhibitors[ and the synthesis of dynamic
11]
[12]
combinatorial libraries.
In both applications, complementary
reactive ligands are covalently linked in the presence of a protein
of interest.
14-3-3 proteins are a family of dimeric regulatory proteins
expressed in all eukaryotic cells and bind to diverse target
proteins containing phosphorylated serine or threonine-containing
consensus motifs.[ By binding to phosphorylated IDRs, 14-3-3
proteins play crucial roles in the regulation of enzyme activities, in
protein structural alterations, and in the cellular trafficking of
phosphorylated target proteins.[7] Studies have implicated the
dysregulation of 14-3-3 function in various diseases such as
cancer[8] and neurodegenerative diseases,[9] highlighting the
potential of new therapeutic strategies that disrupt the interaction
of 14-3-3 and phosphorylated IDPs.
6]
Several inhibitors of 14-3-3 have been reported,[10] including the
[
a]
R. Masuda, Prof. Dr. H. Fujii, Prof. Dr. J. Ohkanda
2
0-mer peptide R18, which binds to the groove of 14-3-3 with ~80
Academic Assembly, Institute of Agriculture, Shinshu University
nM affinity.[
10a]
Expression of R18 in cells induces apoptosis,
8304 Minami-Minowa, Kami-Ina, Nagano 399-4598, Japan
E-mail: johkanda@shinshu-u.ac.jp
validating the promise of 14-3-3 as a therapeutic target. However,
the long shallow amphiphilic binding groove of 14-3-3 makes the
development of cell-permeable inhibitors challenging.
To this end, the diterpene natural product fusicoccin A (FC,
Figure 2) attracted our attention because this fungal metabolite
binds to a hydrophobic cavity in the complex formed by plant 14-
b]
Dr. Y. Kawasaki, Dr. K. Igawa, Prof. Dr. K. Tomooka
Institute for Materials Chemistry and Engineering, Kyushu University
Kasuga-koen 6-1, Kasuga, Fukuoka 816-8580, Japan
Dr. Y. Manabe
Department of Chemistry, Graduate School of Science, Osaka
University
[
[
c]
1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan
3
-3 and PMA2 phosphopeptide (QSYpTV), which is the 14-3-3
d]
Prof. Dr. N. Kato
The Institute of Scientific Industrial Research, Osaka University
+
binding motif found in the C-terminal region of plant H -
ATPase.[13] Our previous study using an aldehyde-containing FC
8-1 Mihogaoka, Ibaraki, Osaka 567-0047, Japan
derivative
and
an
aminooxy-group-containing
peptide
Supporting information for this article is given via a link at the end of
the document.
demonstrated that oxime ligation between the two fragments was
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Figure 2. Design of azide-containing fusicoccin 1 and alkyne-containing
peptides 2 and 3.
Scheme 1. Synthesis of 1, 2, and 3. a) The protected peptide
DansGlyGln(Trt)Ser(tBu)Tyr(tBu)Asp(tBu)-OH was prepared by solid-phase
peptide synthesis (see S.I.).
significantly accelerated by 14-3-3 and generated the
corresponding conjugate in cells, resulting in significant
cytotoxicity.[ However, the FC-based molecule was unstable
due to the aldehyde group and potentially reacted nonspecifically
14]
group enhanced oxime ligation due to the proximity effect[14] and
thus we designed ortho-azide derivative 1 as a substrate for the
click reaction. The PMA2 peptide was used as the scaffold and
provided the alkyne counterpart following replacement of the
phosphothreonine residue with aspartic acid. Furthermore, dansyl
glycine was introduced at the N-terminus to improve cell
permeability and allow for fluorescence-based HPLC analysis.
The peptidic scaffold was then used to produce either 2 (in which
the C-terminal valine residue was replaced by O-propargylic
valinol to provide a propargyl group) or 3 (in which the valine was
replaced by a DACN moiety).
[
14]
with biomolecules.
We therefore decided to apply a more
bioorthogonally selective reaction to improve the specificity of
target-guided synthesis.
Huisgen cycloaddition is a type of click reaction[15] that has been
widely used in biological applications and drug discovery.[16]
Cyclooctynes have in particular been utilized for the copper-free
Huisgen reaction due to their high reactivity resulting from their
strained alkyne, and numerous applications of cyclooctynes in
[
17]
biological studies have been reported. However, the instability
of cyclooctynes[
18]
and their non-specific reactions with cysteine
and glutathione[19] must be addressed prior to their use in protein-
Compounds 1-3 were readily synthesized by straightforward
chemical modification of fusicoccin J, a biosynthetic intermediate
for FC, or by standard peptide couplings (Scheme 1; for details
see S.I.). Acetonide-protected fusicoccin J[14] was treated with
ortho-azido benzyl bromide and then deprotected to afford 1 in
moderate yield. Compounds 2 and 3 were synthesized by
coupling the protected pentapeptide derivative either with O-
propargylic valinol derivative, which was prepared by the Nicholas
templated reactions. In addition, although their fast reaction rate
is largely favorable for bio-probing applications,[20] it can be
disadvantageous for templated reactions if the ligand binding rate
is slower than the reaction rate. A slower but more selective
copper-free reaction is thus desirable.
To this end, some of the authors previously reported a series of
heteroatom-embedded cycloalkynes with different-sized ring
systems, and demonstrated that their reactivities can be tuned by
altering the size of the ring and incorporating heteroatoms that
affect the distortion angle of the alkyne moiety.[21] In particular,
reaction with N-protected valinol,[
22]
or with NMs-DACN,
[21c]
followed by deprotection.
The cycloaddition of 1 and 2 was first examined in the presence
of copper sulfate in DMSO. The reaction was complete after 2 h
at r.t., producing the corresponding conjugate product (see S.I.).
The reaction was then performed in a buffer solution in the
presence of recombinant 14-3-3 protein without the copper
catalyst at 37 °C. However, no product was detected after 20 h,
indicating that the cycloaddition of 1 and 2 requires a catalyst and
that the proximity effect does not drive the reaction (data not
shown).
4
,8-diazacyclononyne (DACN) was attractive as its reaction rate
is faster than that of simple cyclooctyne (OCT) but slower than
that of the conventional difluorinated cyclooctyne, DIFO. In
addition, two nitrogen atoms in the ring system make it a useful
building block as they allow for further chemical modification.
These chemical properties prompted us to incorporate DACN into
our module design and test its utility in a 14-3-3-templated
conjugate reaction. In this study, we designed and synthesized
FC- and peptide-based fragments containing an azide and an
alkyne functional group, respectively, and evaluated their utility for
the 14-3-3-templated copper-free click reaction in vitro.
On the other hand, although the copper-free reaction of 1 and
3
in DMSO at 37 °C produced only trace amounts of product after
8
0 h, the reaction accelerated significantly as the temperature
Benzylated FC derivative (1) was designed as the azide-
containing fragment (Figure 2). Our previous study demonstrated
that a formyl group introduced at the ortho position of the benzyl
was raised to 70 °C, producing a mixture of two products in an
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Figure 3. (a) HPLC profile of product formation after heating 1 (300 M) and 3
(300 M) in DMSO at 70 °C for 4 h (solid line) and 37 °C for 80 h (dashed line).
(b) Time-course change in the HPLC profile of product formation when a
mixture of 1 (300 M) and 3 (300 M) was reacted in the presence of 14-3-3
300 M) in HEPES buffer (10 mM HEPES, pH 7.3, 150 mM NaCl, 0.05% (v/v)
(
Tween 20), 37 °C. HPLC analysis was conducted at 0, 5, 28, 48, 72, and 80 h
after initiation of the reaction. Data obtained after 80 h are shown as a solid line.
(c) Time-course changes in the total yield of 4-1 and 4-2 in the absence (dashed
line, white circles) and presence (solid line, red circles) of 14-3-3 (300 M) in
HEPES buffer (10 mM HEPES, pH 7.3, 150 mM NaCl, 0.05% (v/v) Tween 20),
37 °C. Commercially available R18 was added to the reaction mixture in the
presence of 14-3-3 (300 M) (dashed line, white triangles).
We obtained insights into the significant regiospecificity of the
reaction by conducting molecular dynamics (MD) simulations
using a model of the ternary complex of 14-3-3 bound to 1 and
3, followed by probability analysis that the positioning of 1 and 3
in the complex resembles the transition state (TS) yielding 4-1 or
Scheme 2. 14-3-3-templated Huisgen cycloaddition between azide-containing
1
and DACN-containing peptide 3. a) [1] = [3] = 1 mM in DMSO, b) [1] = [3] =
300 µM, in HEPES buffer (10 mM HEPES pH 7.3, 150 mM NaCl, 0.05% (v/v)
4
-2. To this end, we first docked 1 and 3 into the crystal structure
Tween 20) in the presence or absence of 14-3-3 (300 µM) at 37 °C.
of 14-3-3 and obtained six structures which gave the top G-
scores. We then performed an MD simulation using each initial
structure for 100 ns, and each initial structure produced 2,000
poses. The resulting total 12,000 poses were analyzed to
examine how often the azide and alkyne moiety in 1 and 3,
respectively, were positioned in a conformation resembling the TS
approximately 1:1 ratio after 4 h (Figure 3a). The products had
HPLC retention times of 11.3 and 11.6 min; following their
isolation, high resolution H NMR analysis and mass spectrometry
1
confirmed these products to be the regioisomeric conjugates 4-1
or 4-2 (see S.I.). Further attempts to determine their
for 4-1 or 4-2. These TS models were obtained by DFT calculation
using a simplified model of the reaction.[
24]
We judged the
1
regiochemistry by H NMR were unsuccessful due to the
presence of a number of rotamors. [23]
similarity of a pose to either of the TS models by defining the
pseudo” TS structures 4-1TS’ and 4-2TS’, where the distances
“
Intriguingly, the reaction of 1 and 3 in buffer was significantly
accelerated at 37 °C by the addition of 14-3-3. The ratio of the
two products after 80 h was approximately 1:18, clearly indicating
that the reaction proceeded regioselectively (Figure 3b and 3c).
As seen in Figure 3c, comparison of the time-course changes of
the total yield of 4-1 and 4-2 in the presence (red circles) and
absence (white circles) of 14-3-3 clearly shows that the protein
significantly accelerates the reaction, and generates the products
efficiently in approximately 68% yield after 80 h.
We confirmed the effect of 14-3-3 by using R18 peptide, a
general 14-3-3 inhibitor, to examine whether the peptide
suppressed the reaction. As expected, the reaction rate was
significantly reduced, supporting the template effect of 14-3-3
between the azide nitrogen atoms (N1 and N3) and the alkyne
carbon atoms (C8 and C9), and the two distortion angles (N4-
C10-C9-N1 and N5-C13-C8-N3), were set as the criteria (Figure
4
a). A typical example of a pose judged as 4-1TS’ and as 4-2TS’
(Figure 4b and 4c) possesses a geometry similar to the
corresponding TS model, validating the criteria. As a result, 24
and 111 poses were judged as 4-1TS’ and 4-2TS’, respectively
(Figure 4d, see also S.I.), suggesting that 4-2 could be a preferred
product of the reaction.
Finally, we evaluated the cytotoxicity of the compounds against
human colon cancer HCT116 cells; the results are shown in
Figure 5. Treatment of the cells with high concentrations of 1
resulted in moderate activity whereas 3 was inactive, indicating
that the benzylated FC derivative exhibits some toxicity,
(dashed line, white triangles in Figure 3c). We attempted to
estimate the K values of 4-1 and 4-2 for 14-3-3 by isothermal
d
[
14]
consistent with previous results. Treating HCT116 cells with a
titration calorimetry (ITC) but were unsuccessful, presumably in
part due to the low solubilities of the compounds.
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4
Figure 5. Cell proliferation assay results for HCT116 cells. Cells (5 × 10 cells
/
2
well) were treated with 1, 3, 4 (1:1 mixture of chemically prepared 4-1 and 4-
), an equimolar mixture of 1 and 3, or DMSO and incubated at 37 °C, 5% CO
2
for 3 days prior to WST assay. n=12.
1:1 mixture of 4-1 and 4-2 had no effect, suggesting that these
large conjugates lack cell permeability. In contrast, an equimolar
mixture of 1 and 3 showed apparent cytotoxicity. The pattern of
the concentration response of activity was sharper in the range
2
0 – 40 M than that observed for 1 (n=12), indicating that the
activity of the mixture of 1 and 3 did not stem from 1 alone. We
also examined the cell lysate and found that it was fluorescently
active (see S.I.). These results suggest that 1 and 3 penetrated
into the cells and reacted to intracellularly generate 4. This is the
first example of 14-3-3-templated regiospecific Huisgen
cycloaddition, suggesting that this highly bioorthogonal reaction
might provide a useful tool for controlling endogenous 14-3-3
proteins in cells.
In conclusion, we designed FC-based 1 and peptide 3
containing a DACN moiety and evaluated their utility for copper-
free Huisgen cycloaddition in the presence of 14-3-3. The results
demonstrated that the addition of 14-3-3 significantly accelerates
the reaction and promotes regiospecificity, supporting the
template effect of 14-3-3. Although we have not completed full
structural assignments of 4-1 and 4-2, our theoretical models
suggest that 4-2 might be the dominant product. A limitation of the
current system is its moderate cellular activity, and this needs to
be addressed for further development of practical biological tools.
Improving the reaction rate by changing the heteroatoms in the
cyclononyne ring system[21] and the solubility of the resulting
conjugates by modifying the chemical structures may help
address these issues. Work toward these goals is currently
underway in our laboratory.
Acknowledgements
Figure 4. a) Geometrical models defined for the “pseudo” transition state for 4-
This work was supported by grants from the Japan Society for the
1
and 4-2 (4-1TS’ and 4-2TS’, respectively). A typical example of a pose that
satisfied the criteria for b) 4-1TS’ (the pose highlighted with a black asterisk in Promotion of Science (19K15567 to Y.K.,18H02106 and
the raw data for #6 in Fig. 4d), and for c) 4-2TS’ (the pose highlighted with a 16K13098 to J.O.) and the Ministry of Education, Culture, Sports,
red asterisk in the raw data for #1 in Fig. 4d). The dansyl moiety in 3 was
excluded for clarity. d) The C-N distances for each pose obtained from MD
simulation of 6 initial structures (#1 - #6). The poses which satisfied the criteria
Science and Technology (18H04419 to K. T., 18H04394 to J.O.),
and by the Nagase Science and Technology Foundation, Astellas
for 4-1TS’ and 4-2TS’ are indicated in black and red bars, respectively at the Foundation for Research on Metabolic Disorders, Naito
bottom of each diagram (Glide in Schrödinger Suites 2015-2).
Foundation, Koyanagi Foundation, and Uehara Foundation (J.O.).
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Eyers, S. J. Gaskell, R. Liu, H. Kohn, J. Am. Chem. Soc. 2011, 133,
We thank the Comprehensive Analysis Center of ISIR, Osaka
University, and A. Oda of the Instrumental Center of Shinshu
University for spectral measurements. Y. Hosoya and Y. Nagaoka
performed part of the organic synthesis.
11320−11330.
[
11] a) X. Hu, J. Sun, H.-G. Wang, H. R. Manetsch, J. Am. Chem. Soc. 2008,
130, 13820-13821.; b) J. F. A. Chase, P. K. Tubbs, Biochem. J. 1969,
111, 225–235. c) E. Queis, C. Sabot, P.-Y. Renard, Chem. Commun.
2015, 51, 12158-12169, and references therein.
Keywords: fusicoccin • 14-3-3 • protein-templated reaction •
Huisgen cycloaddition • heteroatom-embedded cycloalkyne
[12] a) I. Huc, J. –M. Lehn, Proc. Natl. Acad. Sci. U. S. A. 1997, 94, 2106–
110. b) D. A. Erlanson, A. C. Braisted, D. R. Raphael, M. Randal, R. M.
Stroud, E. M. Gordon, J. A. Wells, Proc. Natl. Acad. Sci. U. S. A. 2000,
7, 9367–9372.
13] M. Wꢀrtele, C. J. Ottmann, A. Wittinghofer, C. Oecking, EMBO J. 2003,
2, 987-994.
2
9
[
[
1]
2]
J. R. Perkins, I. Diboun, B. H. Dessailly, J. G. Lees, C. Orengo, Structure
010, 13, 1233-1243.
[
[
[
[
[
[
[
[
2
2
a) J. Sohn, G. Buhrman, J. Rudolph, Biochemistry 2007, 46, 807-818; b)
C. Oldfield, A. K. Dunker, Ann. Rev. Biochem. 2014, 83, 553-584. c) J.
D. A. Bah, R. Vernon, Z. Siddiqui, M. Krzeminski, R. Muhandiram, C.
Zhao, N. Sonenberg, L.E. Kay, J.D. Forman-Kay, Nature 2015, 519, 106-
14] P. Parvatkar, N. Kato, M. Uesugi, S. Sato, J. Ohkanda, J. Am. Chem.
Soc. 2015, 137, 15624-15627.
15] H. C. Kolb, M. G. Finn, K. B. Sharpless, Angew. Chem. Int. Ed. 2001, 40,
2
004-2021.
16] P. Thirumurugan, D. Matosiuk, K. Jozwiak, Chem. Rev. 2013, 113, 4905-
979, and references therein.
N. J. Agard, J. A. Prescher, C. R. Bertozzi, J. Am. Chem. Soc. 2004,
26, 15046-15047.
109.
[
[
3]
4]
J. Ohkanda, Chem. Rec. 2013, 13, 561-575.
4
J. Liu, J. D. Farmer, Jr., W. S. Lane, J. Friedman, I. Weissman, S. L.
Schreiber, Cell 1991, 66, 807-815.
17]
1
[
[
[
5]
6]
7]
D. M. Sabatini, H. Erdijument-Bromage, M. Lui, P. Tempst, S. H. Snyder,
Cell 1994, 78, 35-43.
18] G. de Almeida, E. M. Sletten, H. Nakamura, K. K. Palaniappan, C. R.
Bertozzi, Angew. Chem. Int. Ed. 2012, 51, 2443-2447.
M. B. Yaffe, K. Rittinger, S. Voilnia, P. R. Caron, A. Aitken, H. Leffers, S.
J. Gamblin, S. J. Smerdon, L. C. Cantley, Cell 1997, 91, 961-971.
a) Y. Kondo, J. Ognjenovic, S. Banerjee, D. Karandur, A. Merk, K.
Kulhanek, K. Wong, J. P. Roose, S. Subramaniam, J. Kuriyan, Science
19] R. van Geel, G. J. M. Pruijin, F. L. van Delft, W. C. Boelens, Bioconjugate
Chem. 2012, 23, 392-398.
20] J. M. Baskin, J. A. Prescher, S. T. Laughlin, N. J. Agard, P. V. Chang, I.
A. Miller, A. Lo, J. A. Codelli, C. R. Bertozzi, Proc. Natl. Acad. Sci. 2007,
2
019, 366, 109-115. b) H. Hermeking, A. Benzinger, Semin. Cancer Biol.
006, 16, 183–192.
104, 16793-16797.
2
[
21] a) R. Ni, N. Mitsuda, T. Kashiwagi, K. Igawa, K. Tomooka, Angew. Chem.
Int. Ed. 2015, 54, 1190-1194. b) K. Igawa, S. Aoyama, Y. Kawasaki, T.
Kashiwagi, Y. Seto, R. Ni, N. Mitsuda, K. Tomooka, Synlett 2017, 28,
[
[
[
8]
9]
C. L. Neal, J. Xu, P. Li, S. Mori, J. Yang, N. N. Neal, X. Zhou, S. L.
Wyszomierski, D. Yu, Oncogene 2012, 31, 897-906.
P. Steinacker, A. Aitken, M. Otto, Semin. Cell Dev. Biol. 2011, 22, 696-
2110-2114. c) Y. Kawasaki, Y. Yamanaka, Y. Seto, K. Igawa, K.
704.
Tomooka, Chem. Lett. 2019, 48, 495-497.
10] a) B. Wang, H. Yang, Y. C. Liu, T. Jelinek, L. Zhang, E. Ruoslahti, H. Fu,
Biochemistry 1999, 38, 12499-12504.; (a) H. Wu, J. Ge, S. Q. Yao,
Angew. Chem., Int. Ed. 2010, 49, 6528-6532. (b) A. Arrendale, K. Kim,
J. Y. Choi, R. L. Geahlen, R. Borch, Chem. Biol. 2012, 19, 764-771. (c)
J. Zhao, Y. Du, J. R. Horton, A. K. Upadhyay, B. Lou, Y. Bai, X. Zhang,
L. Du, M. Li, B. Wang, L. Zhang, J. T. Barbieri, F. R. Khuri, X. Cheng, H.
Fu, Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 16212-16216. (d) V. Corradi,
M. Mancini, F. Manetti, S. Petta, M. A. Santucci, M. Botta, Bioorg. Med.
Chem. Lett. 2010, 20, 6133-6137. e) K. D. Park, D. Kim,O. Reamtong, C.
[
[
22] S. M. Wells, J. C. Winden, D. A. Harki, K. M. Brummond, Org. Lett. 2016,
18, 4566-4569.
23] These products were determined as either isomers by 1H NMR and
HRMS. NOE measurement under heating condition did not detect
apparent signals, and at present the regiochemistry is not yet determined.
24] DFT calculations for cycloaddition of NMs,N'Ac-DACN with phenyl azide
were performed at B3LYP/6-311G(d,p) level of theory as a model
reaction to set the criteria for 4-1TS' and 4-2TS' (see S.I.).
[
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Entry for the Table of Contents
COMMUNICATION
Copper-free Huisgen cycloaddition
of an azide-containing fusicoccin
derivative and a peptide fragment
containing a nitrogen-embedded
cyclic alkyne moiety (DACN) in the
presence of 14-3-3 protein was
evaluated. The results demonstrated
that the reaction was accelerated in
the presence of 14-3-3 and
Ryoma Masuda, Yuuya Kawasaki,
Kazunobu Igawa, Yoshiyuki Manabe,
Hiroshi Fujii, Nobuo Kato, Katsuhiko
Tomooka, Junko Ohkanda*
Page No. – Page No.
Copper-free Huisgen Cycloaddition
for the 14-3-3-Templated Synthesis
of Fusicoccin-peptide Conjugates
generated one of the regioisomers
predominantly, supporting the
template effect of 14-3-3.
For internal use, please do not delete. Submitted_Manuscript
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