Development of caged non-hydrolyzable phosphoamino acids and application to photo-control of binding affinity of phosphopeptide mimetic to phosphopeptide-recognizing protein

Article abstract of DOI:10.1016/j.bmc.2014.04.002

The design and synthesis of caged non-hydrolyzable phospho-serine, -threonine, and -tyrosine derivatives that generate parent non-hydrolyzable phosphoamino acids, containing a difluoromethylene unit instead of the oxygen of a phosphoester, after UV-irradiation are described. The caged non-hydrolyzable amino acids were incorporated into peptides by standard Fmoc solid-phase peptide synthesis, and the obtained peptides were successfully converted to the parent non-hydrolyzable phosphopeptides by UV-irradiation. Application of the caged non-hydrolyzable phosphoserine-containing peptide to photo-control the binding affinity of the peptide to 14-3-3β protein is also reported.

Full text of DOI:10.1016/j.bmc.2014.04.002

Bioorganic & Medicinal Chemistry 22 (2014) 2984–2991
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Development of caged non-hydrolyzable phosphoamino acids and
application to photo-control of binding affinity of phosphopeptide
mimetic to phosphopeptide-recognizing protein
⇑
⇑
Koji Ebisuno, Masaya Denda, Keiji Ogura, Tsubasa Inokuma, Akira Shigenaga , Akira Otaka
Institute of Health Biosciences and Graduate School of Pharmaceutical Sciences, The University of Tokushima, Shomachi, Tokushima 770-8505, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The design and synthesis of caged non-hydrolyzable phospho-serine, -threonine, and -tyrosine deriva-
tives that generate parent non-hydrolyzable phosphoamino acids, containing a difluoromethylene unit
instead of the oxygen of a phosphoester, after UV-irradiation are described. The caged non-hydrolyzable
amino acids were incorporated into peptides by standard Fmoc solid-phase peptide synthesis, and the
obtained peptides were successfully converted to the parent non-hydrolyzable phosphopeptides by
UV-irradiation. Application of the caged non-hydrolyzable phosphoserine-containing peptide to
photo-control the binding affinity of the peptide to 14-3-3b protein is also reported.
Received 13 March 2014
Revised 1 April 2014
Accepted 2 April 2014
Available online 13 April 2014
Keywords:
Caged peptide
Non-hydrolyzable
Phosphoamino acid
Phosphopeptide
UV-responsive
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
Protein phosphorylation/dephosphorylation is an indispensable
post-translational regulatory mechanism in a wide range of cellu-
lar processes.1 Numerous chemical tools have been developed for
controlling or monitoring phosphorylation and/or dephosphoryla-
tion events, enabling clarification of their biological significance.2
A caged phosphopeptide is a useful tool for spatiotemporal regula-
tion of phosphopeptide biological activity (Scheme 1).3 Caged
peptide 1 has a photo-removable protective group on a phosphate
moiety to mask its key functionality. Its biological activity derived
from interaction between the phosphate and a target biomolecule
is therefore turned off. On photo-irradiation followed by removal
of the protective group, the biologically active parent phosphopep-
tide 2 is generated. Although this technique is useful in the study of
Scheme 1. Caged phosphopeptide (PG: protecive group removable by photo-
irradiation).
phosphorylation/dephosphorylation events, there is
a risk of
conversion of the active phosphopeptide 2 to an inactive form by
hydrolysis of the phosphate by endogenous phosphatases. It is
therefore desirable to avoid removal of the phosphate of
phosphopeptides.
Scheme 2. Design of caged non-hydrolyzable phosphopeptide (PG: protective
group removable by photo-irradiation).
We and other groups have already developed non-hydrolyzable
phosphopeptides in which an oxygen of a phosphoester moiety has
been replaced by a difluoromethylene (CF2) unit to prevent
phosphate hydrolysis (Scheme 2).4–6 Because the physical
properties of difluoromethyl phosphonates are similar to those of
the corresponding phosphates (e.g., pKa2, charge under physiolog-
ical conditions, and bond angle of C–X–P in H2NCH2XPO3H2: 5.6,
⇑
Corresponding authors. Tel.: +81 88 633 9534; fax: +81 88 633 9505 (A.S.); tel.:
+81 88 633 7283; fax: +81 88 633 9505 (A.O.).
E-mail addresses: shigenaga.akira@tokushima-u.ac.jp (A. Shigenaga), aotaka@
tokushima-u.ac.jp (A. Otaka).
http://dx.doi.org/10.1016/j.bmc.2014.04.002
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

K. Ebisuno et al. / Bioorg. Med. Chem. 22 (2014) 2984–2991
2985
ꢀ2, and 117°, respectively, for CF2 derivative, X is CF2; 6.5, ꢀ2, and
119°, respectively, for the monoalkyl phosphate, X is oxygen),7
they can be successfully used to elucidate the biological functions
of phosphorylated peptides and proteins.8,9 We therefore designed
a caged non-hydrolyzable phosphopeptide that should work as a
caged phosphopeptide mimetic without enzymatic hydrolysis of
the phosphate.10 Although introduction of two photo-responsive
protective groups into the phosphonate is possible, monoprotec-
tion was used in this study because the bulkiness of the group
was expected to be enough to prevent binding of the caged peptide
to the phosphate-recognizing pockets of proteins. The mono-
protected phosphonate was presumed to be compatible with
solid-phase peptide synthesis (SPPS) because amino acid building
blocks containing the mono-protected phosphate or non-protected
phosphonate have been employed for preparation of phosphopep-
tides and their analogs.11 In this paper, the synthesis and photore-
actions of phosphopeptide mimetics with caged non-hydrolyzable
phospho-serine, -threonine, and -tyrosine are described. Applica-
tion of the serine derivative to photo-control of binding affinity
to 14-3-3b protein is also reported.
2. Results and discussion
2.1. Synthesis of caged non-hydrolyzable phosphoamino acid
derivatives and their incorporation into peptides
Fmoc-protected non-hydrolyzable phosphoserine 3a containing
a photo-removable protection was first synthesized as shown in
Scheme 3. In this study, an o-nitrobenzyl (oNB) group was chosen
as the photo-responsive protective group because of its wide-
spread use in a chemical biology field, simple structure without a
chiral center and commercial availability.3a,12 Carboxylic acid
4a4a was treated with allyl bromide in the presence of K2CO3 to
afford ester 5a. The Boc group of 5a was replaced by an Fmoc group
to give 6a. The ethyl groups of 6a were removed by treatment with
TMSBr in CH2Cl2 followed by aqueous MeCN. Monoesterification of
phosphonate 7a was then achieved by the use of a p-toluenesulfo-
nyl chloride (TsCl)-pyridine system. Compound 8a was used for a
subsequent reaction without hesitation because of its instability.
Finally, the allyl group of 8a was removed in the presence of palla-
dium(0) to generate Fmoc-protected non-hydrolyzable phospho-
serine 3a. Purification of the product was highly complicated
because of its high polarity, so crude 3a without column chroma-
tography purification was used in subsequent reactions.13
To examine the photo-reactivity and photo-control of the bind-
ing affinity of the phosphoserine mimetic-containing peptide, ser-
ine derivative 3a was then incorporated into a model peptide. In
this study, a ligand analog of a 14-3-3b protein14 was synthesized
because a phosphate moiety on the ligand is critical for binding15,16
and the ligand sequence has been well studied.17 The caged phos-
phopeptide mimetic 9a, in which a phosphoserine of the original
sequence was replaced by the caged non-hydrolyzable derivative,
was prepared using standard Fmoc SPPS. For the coupling reaction,
O-(benzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium hexafluoro-
phosphate (HBTU) and N,N-diisopropylethylamine (DIEA) were
used before introduction of serine derivative 3a, whereas 2-(1H-
7-azabenzotriaol-1-yl)-1,1,3,3-tetramethyluronium
hexafluoro-
phosphate (HATU), 1-hydroxyl-7-azabenzotriazole (HOAt), and
DIEA were used for introduction of 3a. An HBTU/DIEA system
was used for further chain elongation.18 After introduction of an
8-amino-3,6-dioxaoctanoyl (miniPEG) linker, a 5-fluoresceinylam-
inothiocarbonyl (FTC) group was incorporated by treatment with
5-fluorescein isothiocyanate (FITC) in the presence of DIEA for
the subsequent fluorescence-based binding assay.
Fmoc-protected threonine derivative 3b and tyrosine derivative
3c were also prepared, starting from 4b5a,19 or 4c,6a similarly to the
serine derivative 3a except for the introduction of the oNB group
on 7b. When 7b was treated with TsCl and pyridine, the yield of
monoester 8b was not sufficient but a mass spectrometric analysis
of the crude reaction mixture gave a main peak identical to
[7bꢀH2OꢀH]ꢀ (data not shown). We thought that dehydrative
cyclization took place via electrophilic activation of the phospho-
nate by TsCl, therefore, o-NB bromide was employed to avoid the
cyclization induced by TsCl. Crude 3b and 3c, without column
chromatography purification, were successfully used in Fmoc SPPS
to prepare model peptide 9b and 9c, respectively.
Scheme 3. Synthesis of caged non-hydrolyzable phosphoamino acid derivatives
(oNB: o-nitrobenzyl). Reagents and conditions: (a) allyl bromide, K2CO3 for 5a and
5c or Na2CO3 for 5b, DMF, 97% (5a), 98% (5b), 85% (5c); (b) TFA; (c) N-(9H-fluoren-
2-ylmethoxycarbonyloxy)succinimide (FmocOSu), MeCN, 10% (w/v) Na2CO3 aq, 60%
(6a), 97% (6b), 77% (6c) (two steps); (d) TMSBr, CH2Cl2, then MeCN aq, 97% (7a), 81%
(7b), 51% (7c); (e) oNB alcohol, TsCl, pyridine, DMF for 8a and 8c, or oNB bromide,
DIEA, DMF, 50 °C for 8b, 68% (8a), 84% (8b), 54% (8c); (f) (Ph3P)4Pd, N-methylaniline,
THF; (g) Fmoc SPPS using HBTU/DIEA or HATU/HOAt/DIEA system (A: alanine; F:
phenylalanine; P: proline; R: arginine).
2.2. UV-induced generation of non-hydrolyzable
phosphopeptides
Next, UV-irradiation experiments were performed on caged
peptide 9a possessing the serine derivative (Fig. 1A and B). Caged
peptide 9a in sodium phosphate buffer (pH 7.6) was subjected to

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K. Ebisuno et al. / Bioorg. Med. Chem. 22 (2014) 2984–2991
Figure 1. Photo-irradiation reactions. (A) Reagents and conditions: method (a) UV-irradiation (>365 nm, 7 min) in sodium phosphate buffer (10 mM, pH 7.6); method (b) UV
irradiation (>365 nm, 7 min) in 0.1% (v/v) aqueous solution of TFA. (B–E) Analytical HPLC profiles of reaction of peptide 9a (B: method a; C: method b), 9b (D: method a), and
9c (E: method a), before and after UV irradiation. ⁄Non-peptidyl peaks. HPLC conditions: linear gradient of 0.1% (v/v) TFA/MeCN in 0.1% (v/v) TFA aq, 10–45% (B and C) or
5–45% (D and E) over 30 min.
UV irradiation (>365 nm). HPLC analysis showed that 9a com-
pletely disappeared within 7 min on UV irradiation. The observed
mass of the product, however, did not correspond to [10a+H]+ but
to [10a+Hꢀ16]+. The structure of the product was assumed to be
10a0 in which a sulfur atom of the thiocarbonyl moiety of 10a
was replaced by oxygen, presumably as a result of reaction with
singlet oxygen generated via photo-sensitization by the FTC
group.20 Although the conversion of 10a to 10a0 was not thought
to be problematic for biological applications,20c the uncaging
reaction was also examined under acidic conditions because the
absorption coefficient of a fluorescein under acidic conditions is
smaller than that under neutral conditions (0.1% (v/v) TFA in H2O,
pH 1.9; Fig. 1C).21 In this case, uncaged peptide 10a instead of
10a0 was obtained in high conversion yield. UV-irradiation experi-
ments were also performed on threonine derivative 9b and tyrosine
derivative 9c in phosphate buffer (pH 7.6), and high-purity uncaged
phosphopeptide mimetics 10b and 10c were generated (Fig. 1D and
E). These results encouraged us to examine whether photo-uncag-
ing of the caged non-hydrolyzable phosphopeptide can control
interactions with phosphopeptide-binding proteins.
D
mP values [
D
mP = (FP in the presence of 14-3-3b) ꢀ (FP in the
absence of 14-3-3b), where FP is the fluorescence polarization sig-
nal in mP (milli-polarization units)] before and after UV irradiation
were compared. If the peptide can bind to 14-3-3b protein, the FP
increases on addition of the protein. The
DmP value should there-
fore be positive. When 14-3-3b was added to a solution of caged
peptide 9a in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
(HEPES) buffer (pH 7.4),22
D
mP was close to zero, similar to that
for the negative control 11 (9a: ꢀ0.2 3.6 mP; 11: 5.2 0.7 mP).
In contrast, large
DmP values were observed in the case of UV-irra-
diated 9a (>365 nm, 7 min)23 and positive control 10a (UV-irradi-
ated 9a: 18.9 1.6 mP; 10a: 39.4 1.5 mP) compared with those
for 9a without UV irradiation and negative control 11, respectively.
Reason for a difference of
clear at present. However, in these experiments, the point is that
the mP value of 9a was increased by the UV irradiation. It was
therefore demonstrated that binding of the non-hydrolyzable
phosphopeptide to 14-3-3b was controlled by UV irradiation.
DmP values of 9a (UV+) and 10a is not
D
3. Conclusion
Caged non-hydrolyzable phosphoserine, phosphothreonine, and
phosphotyrosine derivatives were synthesized and incorporated
into peptides using standard Fmoc SPPS. UV-induced deprotection
of the caged peptides generated the parent non-hydrolyzable
phosphopeptide mimetics in high purity. The binding assay of
2.3. Photo-control of phosphopeptide mimetics-protein
interaction
Binding of the caged non-hydrolyzable phosphoserine deriva-
tive 9a to 14-3-3b protein was examined (Fig. 2). In this study,

K. Ebisuno et al. / Bioorg. Med. Chem. 22 (2014) 2984–2991
2987
(signals of difluoromethylene units sometimes were not detected).
For HPLC separations, a Cosmosil 5C18-AR-II analytical column
(Nacalai Tesque, 4.6 ꢁ 250 mm, flow rate 1.0 mL/min) or a Cosmo-
sil 5C18-AR-II preparative column (Nacalai Tesque, 20 ꢁ 250 mm,
flow rate 10.0 mL/min) was employed, and eluting products were
detected by UV at 220 nm. A solvent system consisting of 0.1%
(v/v) TFA in H2O (solvent A) and 0.1% (v/v) TFA in MeCN (solvent
B) was used for HPLC elution. UV-irradiation was performed using
a Moritex MUV-202U (3000 mW/cm2 HgeXe lamp) with the fil-
tered output (>365 nm) and the irradiation power was adjusted
to one third of the maximum.
4.2. Preparation of caged non-hydrolyzable phosphoamino acid
derivatives
4.2.1. Allyl esters 5a–c
Typical procedure for preparation of 5a: Potassium carbonate
(121 mg, 0.880 mmol) and allyl bromide (76 lL, 0.880 mmol) were
added to a solution of carboxylic acid 4a4a (300 mg, 0.800 mmol) in
DMF (4.0 mL). The resulting mixture was stirred at room tempera-
ture overnight and was quenched by the addition of water. The
mixture was extracted with Et2O. The obtained organic layer was
washed with brine, dried over MgSO4, and concentrated in vacuo.
The resulting crude product was purified by column chromatogra-
phy (hexanes/EtOAc = 4:1 (v/v)) and 316 mg of allyl ester 5a
(0.760 mmol, 94%) was obtained as a pale yellow oil.
4.2.1.1. (S)-Allyl 2-{(tert-butoxycarbonyl)amino}-4-(diethoxy-
phosphoryl)-4,4-difluorobutanoate (5a).
Pale yellow oil;
19
316 mg; 94% yield; [a]
0.03 (c 1.0, CHCl3); 1H NMR (CDCl3,
D
400 MHz) d = 1.38 (6H, t, J = 7.2 Hz), 1.44 (9H, s), 2.53–2.80 (2H,
m), 4.27 (4H, q, J = 7.2 Hz), 4.58–4.63 (1H, br m), 4.65 (2H, d,
J = 6.0 Hz), 5.26 (1H, d, J = 10.4 Hz), 5.34 (1H, d, J = 16.4 Hz), 5.90
(1H, ddt, J = 16.4, 10.4 and 6.0 Hz); 13C NMR (CDCl3, 75 MHz)
d = 16.2 (d, J = 5.4 Hz), 28.2, 35.2 (td, J = 19.5 and 15.4 Hz), 48.4,
64.7 (d, J = 7.0 Hz), 66.3, 80.1, 118.8, 131.1, 154.9, 170.6; 31P NMR
(CDCl3, 162 MHz) d = ꢀ5.82 (t, J = 105.7 Hz); HRMS (ESI-TOF) m/z
calcd for C16H28F2NNaO7P ([M+Na]+) 438.1469, found 438.1479.
Figure 2. Binding assay based on FP. (A) Structures of caged non-hydrolyzable
peptide and reference peptides and (B) binding assays of peptides to 14-3-3b. The
peptide (20 nM, final) in HEPES buffer [10 mM HEPES, 150 mM NaCl, 0.05% (w/v)
Tween-20, 0.5 mM dithiothreitol, pH 7.4] was incubated at 20 °C in the absence or
presence of 14-3-3b (500 nM, final) for 1 h.22 The FP signals were recorded in mP
4.2.1.2. (2S,3R)-Allyl 2-{(tert-butoxycarbonyl)amino}-4-(dieth-
oxyphosphoryl)-4,4-difluoro-3-methylbutanoate
(5b).
Preparation of substrate 4b, see ref.5a Na2CO3 was used
(milli-polarization units), and the
D
mP values were calculated as follows.
27
instead of K2CO3; colorless oil; 11.3 mg; 98% yield; [
a]
17.5 (c
D
D
mP = (FP in the presence of 14-3-3b) ꢀ (FP in the absence of 14-3-3b). Averages
2.0, CHCl3); 1H NMR (CDCl3, 400 MHz) d = 1.30 (3H, d, J = 7.2 Hz),
1.37 (6H, t, J = 7.0 Hz), 1.45 (9H, s), 3.13 (1H, m), 4.26 (4H, m),
4.54 (1H, dd, J = 9.8 and 3.4 Hz), 4.63 (2H, ddd, J = 5.9, 1.5 and
1.5 Hz), 5.24 (1H, ddt, J = 11.7, 1.5 and 1.5 Hz), 5.34 (1H, ddt,
J = 17.2, 1.5 and 1.5 Hz), 5.44 (1H, d, J = 9.8 Hz), 5.90 (1H, ddt,
J = 17.2, 11.7 and 5.9 Hz); 13C NMR (CDCl3, 75 MHz) d = 12.0 (m),
16.3 (d, J = 1.2 Hz), 16.4 (d, J = 1.2 Hz), 28.3, 40.2 (m), 54.6 (m),
64.8 (m), 66.2, 80.1, 118.7, 131.6, 156.0, 170.6; 31P NMR (CDCl3,
162 MHz) d = 5.54 (dd, J = 109.6 and 104.1 Hz); HRMS (ESI-TOF)
m/z calcd for C17H30F2NNaO7P ([M+Na]+) 452.1626, found 452.1614.
of nine measurements are shown with the standard error of the mean. A statistical
analysis was performed using the t test (⁄p<0.005). 9a (UV+): UV (>365 nm) was
irradiated to 9a in the HEPES buffer for 7 min before the FP experiments. (C) HPLC
monitoring of preparation of 9a (UV+) for subsequent binding assay. HPLC charts
before and after UV irradiation to caged peptide 9a in HEPES buffer are depicted.
Reaction conditions are shown above and in the experimental section. Analytical
HPLC conditions: linear gradient of 0.1% (v/v) TFA/MeCN in 0.1% (v/v) TFA aq, 10–
45% over 30 min.
the non-hydrolyzable phosphoserine-containing peptide to 14-3-
3b protein was also examined, and the affinity was successfully
controlled by UV irradiation. Applications of the caged non-hydro-
lyzable phosphopeptide mimetics to tools for photo-control the
phosphopeptide-protein interaction events are being investigated.
4.2.1.3. (S)-Allyl 2-{(tert-butoxycarbonyl)amino}-3-[4-{(dieth-
oxyphosphoryl)difluoromethyl}-phenyl]propanoate
(5c).
Preparation of substrate 4c, see ref.;6a Colorless oil;
27
263 mg; 85% yield; [
a]
ꢀ5.06 (c 1.4, MeOH); 1H NMR (CDCl3,
D
4. Experimental section
4.1. General methods
400 MHz) d = 1.31 (6H, t, J = 7.2 Hz), 1.42 (9H, s), 3.10 (1H, dd,
J = 18.2 and 7.6 Hz), 3.19 (1H, dd, J = 18.2 and 7.2 Hz), 4.07–4.32
(4H, m), 4.59 (2H, d, J = 5.7 Hz), 4.62 (1H, m), 5.02 (1H, d,
J = 7.5 Hz), 5.25 (1H, d, J = 8.1 Hz), 5.30 (1H, d, J = 17.3 Hz), 5.85
(1H, ddt, J = 17.3, 8.1 and 5.7 Hz), 7.23 (2H, d, J = 8.1 Hz), 7.55
(2H, d, J = 8.1 Hz); 13C NMR (CDCl3, 75 MHz) d = 16.1 (d,
J = 5.0 Hz), 28.0, 37.8, 54.1, 64.5 (d, J = 6.2 Hz), 65.8, 79.7, 117.8
(td, J = 259.6 and 216.3 Hz), 118.8, 126.1, 129.3, 131.1 (m), 131.2,
All reactions of small molecules were carried out under a posi-
tive pressure of argon. For column chromatography, Silica Gel 60N
(spherical, neutral, Kanto Chemical Co., Inc.) was employed. NMR
spectra were recorded for 1H and 31P (proton decoupled) and 13C

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K. Ebisuno et al. / Bioorg. Med. Chem. 22 (2014) 2984–2991
139.1, 154.8, 171.0; 31P NMR (CDCl3, 162 MHz) d = ꢀ6.32 (t,
31P NMR (CDCl3, 162 MHz) d = ꢀ6.35 (t, J = 115.8 Hz); HRMS (ESI-
TOF) m/z calcd for C32H35F2NO7P ([M+H]+) 614.2119, found
614.2128.
J = 115.6 Hz); HRMS (ESI-TOF) m/z calcd for
C22H32F2KNO7P
([M+K]+) 530.1522, found 530.1526.
4.2.2. Fmoc derivatives 6a–c
4.2.3. Phosphonates 7a–c
Typical procedure for preparation of 6a: To allyl ester 5a
(313 mg, 0.760 mmol) was added TFA (6.0 mL) with stirring at
room temperature for 2 h. The mixture was concentrated in vacuo,
and the residue was dissolved in MeCN/10% (w/v) Na2CO3 aq (2:1
(v/v), 14 mL). To the solution was added FmocOSu (279 mg,
0.828 mmol) and the reaction mixture was stirred at room temper-
ature overnight. After addition of 5% (w/v) KHSO4 aq, the mixture
was extracted with EtOAc. The organic layer was washed with
brine, dried over MgSO4, and concentrated in vacuo. The obtained
crude material was purified by column chromatography (hexanes/
EtOAc = 4:1 (v/v)) and 311 mg of Fmoc derivative 6a (0.579 mmol,
77%) was obtained as a colorless oil.
Typical procedure for preparation of 7a: Trimethylsilyl bromide
(22 mL, 163 mmol) was added to a solution of diethyl ester 6a
(4.39 g, 8.17 mmol) in CH2Cl2 (162 mL), and the mixture was stir-
red at room temperature for 14 h. The reaction progress was mon-
itored using 31P NMR. After addition of H2O/MeCN, the mixture
was concentrated in vacuo. The crude material was purified by rep-
recipitation from hexanes and 3.39 g of phosphonate 7a
(7.04 mmol, 86%) was obtained as a white solid.
4.2.3.1. (S)-{3-([{(9H-Fluoren-9-yl)methoxy}carbonyl]amino)-4-
(allyloxy)-1,1-difluoro-4-oxobutyl}phosphonic
acid
ꢀ16.3 (c 0.90,
27
(7a).
White solid; 3.39 g; 86% yield; [
a]
D
MeOH); 1H NMR (methanol-d4, 400 MHz) d = 2.46–2.85 (2H, m),
4.23 (1H, t, J = 7.1 Hz), 4.31 (2H, d, J = 7.1 Hz), 4.64 (2H, d,
J = 5.6 Hz), 4.63–4.81 (1H, m), 5.20 (2H, dd, J = 10.5 and 1.3 Hz),
5.32 (2H, dd, J = 17.9 and 1.3 Hz), 5.93 (1H, ddt, J = 17.9, 10.5 and
5.6 Hz), 7.30 (2H, td, J = 7.3 and 1.2 Hz), 7.38 (2H, t, J = 7.3 Hz),
7.66 (2H, d, J = 7.3 Hz), 7.79 (2H, d, J = 7.3 Hz); 13C NMR (metha-
nol-d4, 75 MHz) d = 34.3 (m), 48.2, 48.8, 65.7, 66.7, 117.1, 119.4,
124.8, 126.7, 127.3, 131.6, 141.0, 143.7, 156.7, 170.9; 31P NMR
(methanol-d4, 162 MHz) d = 4.81 (t, J = 102.7 Hz); HRMS (ESI-TOF)
4.2.2.1. (S)-Allyl 2-([{(9H-fluoren-9-yl)methoxy}carbonyl]amino )-
4-(diethoxyphosphoryl)-4,4-difluorobutanoate (6a).
Color-
19
less oil; 311 mg; 77% yield; [a]
3.10 (c 0.86, CHCl3); 1H NMR
D
(CDCl3, 400 MHz) d = 1.38 (6H, td, J = 7.1 and 2.2 Hz), 2.58–2.82
(2H, m), 4.20–4.32 (5H, m), 4.37 (2H, d, J = 7.1 Hz), 4.67 (2H, d,
J = 5.4 Hz), 4.65–4.78 (1H, m), 5.26 (1H, dd, J = 10.5 and 1.2 Hz),
5.35 (1H, dd, J = 17.3 and 1.2 Hz), 5.76 (1H, d, J = 8.3 Hz), 5.91
(1H, ddt, J = 17.3, 10.5 and 5.4 Hz), 7.31 (2H, td, J = 7.3 and
1.2 Hz), 7.40 (2H, t, J = 7.3 Hz), 7.60 (2H, m), 7.76 (2H, d,
J = 7.3 Hz); 13C NMR (CDCl3, 75 MHz) d = 16.3 (d, J = 5.6 Hz), 35.5
(d, J = 16.2 Hz), 47.0, 48.9, 64.9 (dt, J = 6.2 and 3.7 Hz), 66.6, 67.4,
119.1, 119.9, 125.1, 127.1, 127.7, 131.3, 141.2, 143.7, 155.6,
170.3; 31P NMR (CDCl3, 162 MHz) d = –5.76 (t, J = 104.7 Hz); HRMS
(ESI-TOF) m/z calcd for C26H30F2NNaO7P ([M+Na]+) 560.1626,
found 560.1628.
m/z calcd for
C
22H22F2NNaO7P ([M+Na]+) 504.1000, found
504.0994.
4.2.3.2. {(2R,3S)-3-([{(9H-Fluoren-9-yl)methoxy}carbonyl]ami
no)-4-(allyloxy)-1,1-difluoro-2-methyl-4-oxobutyl}phosphonic
acid (7b).
For purification, column chromatography (CHCl3/
methanol = 99:1 (v/v)) was employed instead of reprecipitation;
22
colorless oil; 29 mg; 81% yield; [
a
]
10.1 (c 2.53, MeOH); 1H
D
4.2.2.2. (2S,3R)-Allyl 2-([{(9H-fluoren-9-yl)methoxy}carbonyl]
NMR (methanol-d4, 400 MHz) d = 1.12 (3H, d, J = 6.5 Hz), 2.93
(1H, m), 4.15 (1H, m), 4.24 (2H, m), 4.50 (1H, m), 4.54 (2H, d,
J = 5.6 Hz), 5.11 (1H, dd, J = 10.6 and 1.4 Hz), 5.25 (1H, dd, J = 17.3
and 1.4 Hz), 5.85 (1H, ddt, J = 17.3, 10.6 and 5.9 Hz), 7.21 (2H, t,
J = 7.4 Hz), 7.29 (2H, t, J = 7.4 Hz), 7.58 (1H, d, J = 7.4 Hz), 7.60
(1H, d, J = 7.4 Hz), 7.70 (2H, d, J = 7.4 Hz); 13C NMR (methanol-d4,
75 MHz) d = 12.4 (t, J = 8.6 Hz), 41.6 (m), 48.3, 56.0 (t, J = 12.4 Hz),
67.0, 68.3, 118.8, 120.9, 126.3, 126.3, 128.2, 128.8, 133.2, 142.5,
145.1, 145.2, 158.6, 171.9; 31P NMR (methanol-d4, 162 MHz)
d = 4.50 (d, J = 103.2 Hz); HRMS (ESI-TOF) m/z calcd for C23H23F2-
NO7P ([MꢀH]ꢀ) 494.1180, found 494.1180.
amino)-4-(diethoxyphosphoryl)-4,4-difluoro-3-methylbutano-
23
ate (6b).
Colorless oil; 137 mg; 97% yield; [
a]
12.0 (c 2.91,
D
CHCl3); 1H NMR (CDCl3, 400 MHz) d = 1.35 (3H, d, J = 7.4 Hz), 1.42
(6H, td, J = 7.1 and 2.7 Hz), 3.21 (1H, m), 4.23–4.60 (7H, m), 4.61–
4.74 (3H, m), 5.28 (1H, dd, J = 10.4 and 1.2 Hz), 5.38 (1H, dd,
J = 17.2 and 1.2 Hz), 5.94 (1H, ddt, J = 17.2, 10.4 and 5.8 Hz), 6.01
(1H, d, J = 9.8 Hz), 7.33 (1H, t, J = 7.5 Hz), 7.34 (1H, t, J = 7.5 Hz),
7.42 (2H, t, J = 7.5 Hz), 7.64 (1H, d, J = 7.5 Hz), 7.66 (1H, d,
J = 7.5 Hz), 7.79 (2H, d, J = 7.5 Hz); 13C NMR (CDCl3, 75 MHz)
d = 12.1 (t, J = 9.8 Hz), 16.3, 16.4, 40.5 (m), 47.1, 55.0 (m), 64.8 (d,
J = 7.5 Hz), 65.0 (d, J = 6.8 Hz), 66.3, 67.4, 118.9, 119.9, 125.2,
125.2, 127.0, 127.1, 127.7, 127.7, 131.5, 141.3, 143.7, 143.9,
156.6, 170.2; 31P NMR (CDCl3, 162 MHz) d = 5.51 (dd, J = 109.2
and 103.5 Hz); HRMS (ESI-TOF) m/z calcd for C27H32F2NNaO7P
([M+Na]+) 574.1782, found 574.1786.
4.2.3.3. (S)-([4-{2-([{(9H-Fluoren-9-yl)methoxy}carbonyl]amino )-
3-(allyloxy)-3-oxopropyl}-phenyl]difluoromethyl)phosphonic
27
acid (7c).
White powder; 465 mg; 52% yield; [
a]
ꢀ4.30 (c
D
1.6, MeOH); 1H NMR (methanol-d4, 400 MHz) d = 3.02 (1H, dd,
J = 13.7 and 9.3 Hz), 3.21 (1H, dd, J = 13.7 and 5.0 Hz), 4.14 (1H, t,
J = 6.5 Hz), 4.29 (2H, t, J = 6.5 Hz), 4.49 (1H, dd, J = 9.3 and
5.0 Hz), 4.60 (2H, d, J = 5.5 Hz), 5.20 (1H, d, J =10.7 Hz), 5.29 (1H,
d, J = 17.3 Hz), 5.88 (1H, ddt, J = 17.3, 10.7 and 5.5 Hz), 7.25–7.40
(6H, m), 7.57 (2H, t, J = 8.2 Hz), 7.59 (2H, d, J = 7.5 Hz), 7.76 (2H,
d, J = 7.5 Hz); 13C NMR (methanol-d4, 75 MHz) d = 38.1, 48.2, 56.7,
66.8, 67.9, 118.8, 119.8 (td, J = 260.3 and 211.8 Hz), 120.9, 126.1,
127.5 (t, J = 6.2 Hz), 128.1, 128.7, 130.2, 133.0, 133.6 (td, J = 22.4
and 13.1 Hz), 140.1, 142.5, 1450, 145.0, 158.2, 172.8; 31P NMR
(methanol-d4, 162 MHz) d = ꢀ4.9 (t, J = 113.7 Hz); HRMS (ESI-
TOF) m/z calcd for C28H26F2KNO7P ([M+K]+) 596.1052, found
596.1051.
4.2.2.3. (S)-Allyl 2-([{(9H-fluoren-9-yl)methoxy}carbonyl]amino )-
3-[4-{(diethoxyphosphoryl)-difluoromethyl}phenyl]propano-
27
ate (6c).
Colorless oil; 518 mg; 55% yield; [
a]
D ꢀ7.58 (c 1.0,
MeOH); 1H NMR (CDCl3, 400 MHz) d = 1.30 (6H, t, J = 7.1 Hz), 3.15
(1H, dd, J = 14.0 and 5.8 Hz), 3.20 (1H, dd, J = 14.0 and 5.8 Hz),
4.10–4.25 (5H, m), 4.38 (1H, dd, J = 10.5 and 7.1 Hz), 4.45 (1H,
dd, J = 10.5 and 7.1 Hz), 4.64 (2H, d, J = 5.9 Hz), 4.70 (1H, dt,
J = 8.0 and 5.8 Hz), 5.27 (1H, d, J = 11.2 Hz), 5.83 (1H, d,
J = 17.6 Hz), 5.85 (1H, ddt, J = 17.6, 11.2 and 5.9 Hz), 7.20 (2H, d,
J = 8.1 Hz), 7.32 (2H, t, J = 7.4 Hz), 7.41 (2H, t, J = 7.4 Hz), 7.55
(2H, d, J = 8.1 Hz), 7.57 (2H, d, J = 7.4 Hz), 7.72 (2H, d, J = 7.4 Hz);
13C NMR (CDCl3, 75 MHz) d = 16.3 (d, J = 6.9 Hz), 37.9, 47.1, 54.7,
64.8 (d, J = 6.9 Hz), 66.2, 66.9, 119.3, 120.0, 125.0 (d, J = 4.4 Hz),
126.5 (td, J = 22.0 and 14.4 Hz), 127.1, 127.8, 129.5, 1310, 131.4
(td, J = 21.8 and 13.7 Hz), 139.0, 141.3, 143.7, 143.8, 155.6, 170.1;
4.2.4. o-Nitrobenzyl esters 8a and 8c
Typical procedure for preparation of 8a: To a solution of phos-
phonate 7a (50.0 mg, 104 lmmol) in DMF (520 lL), pyridine

K. Ebisuno et al. / Bioorg. Med. Chem. 22 (2014) 2984–2991
2989
(20
added. After stirring at room temperature for 15 min followed by
addition of o-nitrobenzyl alcohol (79.5 mg, 519 mol), the reaction
mixture was stirred at room temperature for additional 26 h. The
crude material was concentrated in vacuo, and the obtained mix-
ture was purified by column chromatography (CHCl3/MeOH = 49:1
l
L) and p-toluenesulfonyl chloride (99.2 mg, 520
l
mol) were
dependent decomposition of 8b was observed, it was used without
hesitation for a subsequent reaction after measurement of 1H and
31P NMR followed by HRMS and optical rotation. In this
experiment, time-consuming 13C NMR was not recorded.
l
4.2.6. Non-hydrolyzable amino acid derivatives 3a–c
(v/v)). o-Nitrobenzyl ester 8a (43.7 mg, 70.9 lmol, 68%) was
obtained as a colorless oil.
Typical procedure for preparation of 3a: N-methylaniline
(241 mg, 2.24 mmol) and (Ph3P)4Pd (25.9 mg, 22.4
added to a solution of allyl ester 8a (138 mg, 224
l
mol) were
l
mol) in THF
4.2.4.1. (2S)-Allyl 2-([{(9H-fluoren-9-yl)methoxy}carbonyl]amin
o)-4,4-difluoro-4-[hydroxy-{(2-nitrobenzyl)oxy}phosphoryl] but-
(4.48 mL), and the resulting mixture was stirred at room tempera-
ture for 4 h. After addition of 1 M HCl aq, the mixture was
extracted with CH2Cl2. The organic layer was washed with water
followed by brine, dried over Na2SO4, and concentrated in vacuo.
The obtained crude material was used to a subsequent reaction
without further purification.
27
anoate (8a).
Colorless oil; 43.7 mg; 68% yield; [
a]
D ꢀ7.43 (c
0.92, MeOH); 1H NMR (methanol-d4, 400 MHz) d = 2.35–2.77 (2H,
m), 4.10 (1H, t, J = 7.1 Hz), 4.18–4.21 (2H, m), 4.50–4.59 (3H, m),
5.14 (1H, dt, J = 10.5 and 1.4 Hz), 5.23 (1H, dt, J = 17.2 and
1.4 Hz), 5.37 (2H, d, J = 7.0 Hz), 5.82 (1H, ddt, J = 17.2, 10.5 and
5.6 Hz), 7.20 (2H, t, J = 7.5 Hz), 7.28 (2H, t, J = 7.5 Hz), 7.38 (1H, t,
J = 7.8 Hz), 7.57 (2H, d, J = 7.5 Hz), 7.61 (1H, dd, J = 7.8 and
7.5 Hz), 7.68 (2H, d, J = 7.5 Hz), 7.89 (1H, d, J = 7.8 Hz), 8.00 (1H,
d, J = 7.5 Hz); 31P NMR (methanol-d4, 162 MHz) d = 3.58 (t,
4.2.6.1. (2S)-2-([{(9H-Fluoren-9-yl)methoxy}carbonyl]amino)-
4,4-difluoro-4-[hydroxy{(2-nitrobenzyl)oxy}phosphoryl]buta-
noic acid (3a).
White powder; 123 mg; HRMS (ESI-TOF) m/z
calcd for C26H23F2N2NaO9P ([M+Na]+) 599.1007, found 599.0990.
J = 90.7 Hz); HRMS (ESI-TOF) m/z calcd for
C29H26F2N2O9P
([MꢀH]ꢀ) 615.1344, found 615.1346. Because time dependent
decomposition of 8a was observed, it was used without hesitation
for a subsequent reaction after measurement of 1H and 31P NMR
followed by HRMS and optical rotation. In this experiment, time-
consuming 13C NMR was not recorded.
4.2.6.2.
(2S,3R)-2-([{(9H-Fluoren-9-yl)methoxy}carbonyl]ami
no)-4,4-difluoro-4-[hydroxy{(2-nitrobenzyl)oxy}phosphoryl]-
3-methylbutanoic acid (3b).
White powder; 26.2 mg; HRMS
(ESI-TOF) m/z calcd for C27H24F2N2O9P ([MꢀH]ꢀ) 589.1188, found
589.1184.
4.2.4.2.
(2S)-Allyl
2-([{(9H-fluoren-9-yl)methoxy}carbonyl]
4.2.6.3. (2S)-2-([{(9H-Fluoren-9-yl) methoxy}carbonyl]amino)
3-{4-(difluoro[hydroxy{(2-nitro-benzyl)oxy}phosphoryl] methyl)-
amino)-3-{4-(difluoro[hydroxy{(2-nitrobenzyl)oxy}phosphoryl]
methyl)phenyl}propanoate (8c).
54% yield; [
White powder; 42.0 mg;
phenyl} propanoic acid (3c).
White powder; 53.0 mg; HRMS
27
a]
ꢀ6.10 (c 0.76, MeOH); 1H NMR (methanol-d4,
D
(ESI-TOF) m/z calcd for C32H28F2N2O9P ([M+H]+) 653.1501, found
400 MHz) d = 3.01 (1H, dd, J = 14.0 and 9.2 Hz), 3.20 (1H, dd,
J = 14.0 and 5.1 Hz), 4.15 (1H, t, J = 6.9 Hz), 4.29 (2H, d,
J = 6.9 Hz), 4.49 (1H, dd, J = 9.2 and 5.1 Hz), 4.62 (2H, d,
J = 5.6 Hz), 5.21 (1H, ddt, J = 10.5, 1.4 and 1.1 Hz), 5.28–5.37 (3H,
m), 5.91 (1H, ddt, J = 16.9, 10.5 and 5.6 Hz), 7.22–7.34 (4H, m),
7.47 (1H, t, J = 7.9 Hz), 7.54–7.64 (4H, m), 7.68 (1H, t, J = 7.7 Hz),
7.78 (2H, d, J = 7.6 Hz), 7.96 (1H, d, J = 7.7 Hz), 8.06 (1H, d,
J = 7.9 Hz); 13C NMR (pyridine-d5, 75 MHz) d = 37.4, 47.3, 55.9,
65.3 (d, J = 9.1 Hz), 65.4, 66.3, 118.0, 120.0, 124.4, 125.2, 125.2,
126.8 (br m), 127.1, 127.7, 127.8, 128.7, 129.0, 132.1, 133.7,
139.1, 141.3, 114.0, 144.1, 146.7, 156.7, 171.7; 31P NMR (pyri-
dine-d5, 162 MHz) d = 4.44 (t, J = 98.1 Hz); HRMS (ESI-TOF) m/z
calcd for C35H30F2N2O9P ([MꢀH]ꢀ) 691.1657, found 691.1656.
653.1487.
4.3. Preparation of caged peptides 9a–c
Typical procedure for preparation of 9a: On NovaSynÒ TGR resin
(loading: 0.22 mmol/g, 30 mg, 6.6 lmol) were coupled Fmoc pro-
tected amino acids (3.0 equiv) in the presence of O-(benzotriazol-
1-yl)-N,N,N0,N0-tetramethyluronium hexafluorophosphate (HBTU,
2.9 equiv) and N,N-diisopropylethyamine (DIEA, 2.9 equiv) in
DMF at room temperature for 0.5 h before coupling of the caged
amino acid. Incorporation of crude 3a (7.6 mg, <2.0 equiv) was
achieved by the use of 2-(1H-7-azabenzotriaol-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate (HATU, 1.95 equiv) in
the presence of DIEA (4.0 equiv) and 1-hydroxyl-7-azabenzotria-
zole (HOAt, 2.2 equiv) in DMF at room temperature for 4 h. After
acetyl capping (10 equiv of acetic anhydride and 10 equiv of pyri-
dine in DMF, 0.5 h at room temperature), following couplings were
performed for elongation of a peptide using an Fmoc protected
amino acid (5.0 equiv), HBTU (4.5 equiv) and DIEA (10 equiv) in
DMF at room temperature for 1 h. Then Fmoc-8-amino-3,6-dioxa-
octanoic acid (Fmoc-miniPEG-OH, 3.0 equiv) was incorporated
using HBTU (2.9 equiv) and DIEA (2.9 equiv) in DMF for 1 h at room
temperature. After removal of N-terminal Fmoc group of the mini-
PEG, the resin was reacted with 5-fluorescein isothiocyanate (FITC,
2.5 equiv) and DIEA (2.5 equiv) in DMF for 1 h twice. The resulted
complete resin was treated with TFA/m-cresol/thioanisole/triethyl-
silane/H2O (80:5:5:5:5 (v/v)) at room temperature for 2 h. After the
resin was filtered off, cooled diethyl ether was added to the filtrate,
and the resulting precipitate was collected by centrifugation. The
obtained precipitate was washed with diethyl ether and purified
by preparative HPLC to give caged peptide 9a (4.6 mg, 44%) as a
yellowish lyophilized powder. Analytical HPLC conditions: a linear
gradient of solvent B in solvent A over 30 min. Preparative HPLC
conditions: a linear gradient of solvent B in solvent A over 30 min.
4.2.5. o-Nitrobenzyl esters 8b
To a solution of phosphonate 7b (24.8 mg, 50.1
(500 L) were added o-nitrobenzyl bromide (43.0 mg, 200
and DIEA (19.1 L, 110 mol). After stirring at 50 °C for 3 h, the
lmmol) in DMF
l
l
mol)
l
l
mixture was concentrated in vacuo. Following to purification of
the obtained crude material by column chromatography (CHCl3/
MeOH = 49:1 (v/v)), o-nitrobenzyl ester 8b (26.6 mg, 42.2 lmol,
84%) was obtained as a white powder.
4.2.5.1. (2S,3R)-Allyl 2-([{(9H-fluoren-9-yl)methoxy}carbonyl]
amino)-4,4- difluoro-4-[hydroxy{(2-nitro-benzyl)oxy}phosphoryl]-
16
3-methylbutanoate (8b).
[a]
4.22 (c 1.2, MeOH); 1H NMR
D
(methanol-d4, 400 MHz) d = 1.24 (3H, d, J = 7.2 Hz), 4.08–4.18
(1H, m), 4.19–4.23 (2H, m), 4.44 (1H, m), 4.53 (2H, m), 5.09 (1H,
ddt, J = 10.6, 1.4 and 1.4 Hz), 5.24 (1H, ddt, J = 17.2, 1.4 and
1.4 Hz), 5.37 (2H, d, J = 7.1 Hz), 5.83 (1H, ddt, J = 17.2, 10.6 and
5.8 Hz), 7.20 (2H, t, J = 7.6 Hz), 7.28 (2H, t, J = 7.6 Hz), 7.40 (1H, t,
J = 7.7 Hz), 7.56–7.66 (3H, m), 7.69 (2H, d, J = 7.6 Hz), 7.92 (1H, d,
J = 7.7 Hz), 8.02 (1H, d, J = 8.1 Hz); 31P NMR (methanol-d4,
162 MHz) d = 2.91 (t, J = 91.5 Hz); HRMS (ESI-TOF) m/z calcd for
C
30H28F2N2O9P ([MꢀH]ꢀ) 629.1501, found 629.1491. Because time

2990
K. Ebisuno et al. / Bioorg. Med. Chem. 22 (2014) 2984–2991
Figure 3. Structure of phosphoserine mimic 14.
Scheme 4. Preparation of synthetic intermediate 13. Reagents and conditions: (a)
tetrabutylammonium azide, MeCN, 0 °C, 51%.
4.3.1. Peptide 9a
4.5.4. Peptide 10c
Yellow lyophilized powder; 4.60 mg; 44% yield; Analytical HPLC
conditions: 10–45% over 30 min. Retention time = 27.2 min; Pre-
parative HPLC conditions: 10–35% over 50 min; LRMS (ESI-TOF)
m/z calcd for [M+H]+ 1586.6, found; 1586.7.
Analytical HPLC conditions: 5–45% over 30 min. Retention
time = 17.8 min; LRMS (ESI-TOF) m/z calcd for [M+H]+ 993.5,
found; 993.2.
4.6. Preparation of control peptides 10a and 11
4.3.2. Peptide 9b
White lyophilized powder; 0.21 mg; 3% yield; Analytical HPLC
conditions: 5–45% over 30 min. Retention time = 21.8 min; Pre-
parative HPLC conditions: 15–35% over 30 min; LRMS (ESI-TOF)
m/z calcd for [M+H]+ 1066.5, found; 1066.1.
Peptides 10a and 11 were prepared as similar to the caged pep-
tides. For construction of peptide 11 and a synthetic intermediate
of 10a before introduction of a non-hydrolyzable phosphoserine
unit, N,N0-diisopropylcarbodiimide (DIC)/HOBt system was
employed (3.0 equiv amino acid, 3.2 equiv DIC, 3.0 equiv HOBtꢂH2O
in DMF, 2 h at room temperature). Coupling of phosphoserine
mimic 144a,16b was achieved using HATU/HOAt/DIEA system
(3 equiv 14, 2.9 equiv HATU, 2.9 equiv HOAt, 8.7 equiv DIEA in
DMF, 1 h at room temperature). Elongation of following amino
acids was performed using the DIC/HOBt and/or the HATU/HOAt/
DIEA system (Fig. 3).
4.3.3. Peptide 9c
white lyophilized powder; 1.12 mg; 15% yield; Analytical HPLC
conditions: 5–45% over 30 min. Retention time = 24.2 min; Pre-
parative HPLC conditions: 15–35% over 30 min; LRMS (ESI-TOF)
m/z calcd for [M+H]+ 1128.5, found; 1128.1.
4.4. Modified preparation of threonine derivative 4b
To a solution of substrate 125a (230 mg, 0.51 mmol) in acetoni-
trile (1.7 mL) was added tetrabutylammonium azide (440 mg,
1.6 mmol), and the reaction mixture was stirred at 0 °C for 12 h.
After addition of water, the mixture was extracted with Et2O. The
obtained organic layer was washed with brine, dried over MgSO4,
and concentrated in vacuo. The resulting crude product was puri-
fied by column chromatography (hexanes/EtOAc = 4:1 (v/v)) and
110 mg of azide 13 (0.26 mmol, 51%) was obtained as a pale yellow
oil. 1H NMR spectrum was identical with that reported
previously.5a
4.6.1. Peptide 10a
Yellow lyophilized powder; 2.39 mg; 14% yield; Analytical HPLC
conditions: 20–40% over 30 min. Retention time = 18.9 min; Pre-
parative HPLC conditions: 10–35% over 45 min; LRMS (ESI-TOF)
m/z calcd for [M+2H]2+ 726.3, found; 726.2.
4.6.2. Peptide 11
Yellow lyophilized powder; 2.20 mg; 8% yield; Analytical HPLC
conditions: 20–40% over 30 min. Retention time = 18.6 min; Pre-
parative HPLC conditions: 10–35% over 45 min; LRMS (ESI-TOF)
m/z calcd for [M+H]+ 1337.6, found; 1336.9.
4.5. UV-irradiation experiment of peptide 9a–c
4.7. Binding assay using FP
Typical procedure for caged peptide 9a: Peptide 9a (50
0.032 mol) in sodium phosphate buffer (10 mM phosphate, pH
7.6, 500 L) was irradiated by UV (>365 nm; irradiation power:
one third of the maximum) for 7 min. Progress of the reaction
was monitored by analytical HPLC and uncaged product 10a0 was
characterized using ESI-MS. Analytical HPLC conditions: a linear
gradient of solvent B in solvent A over 30 min.
lg,
Typical procedure: caged peptide 9a (20 nM final) in HEPES buf-
l
fer [10 mM HEPES, 150 mM NaCl, 0.05% (w/v) Tween-20, 0.5 mM
l
dithiothreitol, pH 7.4, 50 l
L]22 was incubated at 20 °C in the
absence or presence of 14-3-3b protein (500 nM, final) for 1 h.17
After recording the FP response in mP (milli-polarization units),
mP values were calculated as follows: DmP = (FP in the presence
D
of 14-3-3b) ꢀ (FP in the absence of 14-3-3b). Averages of nine mea-
surements with the standard error of the mean are shown in
Figure 2. For the photo-irradiation experiments, peptide 9a in
HEPES buffer was subjected to UV irradiation (>365 nm; irradiation
power: one third of the maximum) for 7 min before the FP exper-
iments. Completion of the uncaging reaction was confirmed by
HPLC monitoring.
4.5.1. Peptide 10a0
Analytical HPLC conditions: 10–45% over 30 min. Retention
time = 21.8 min; LRMS (ESI-TOF) m/z calcd for [M+H]+ 1435.6,
found; 1434.8.
4.5.2. Peptide 10a
Analytical HPLC conditions: 10–45% over 30 min. Retention
time = 23.3 min; LRMS (ESI-TOF) m/z calcd for [M+2H]2+ 726.3,
found; 726.1. As the reaction solvent, 1% (v/v) TFA in water was
used instead of the phosphate buffer.
Acknowledgments
We thank Prof. H. Kosako (The University of Tokushima) for
valuable discussion. This research was supported in part by a
Grant-in-Aid for Scientific Research (KAKENHI) including Innova-
tive Areas ‘Fusion Materials’ (No. 2206). The Takeda Pharmaceuti-
cal Company is also acknowledged. M.D. is grateful for JSPS
fellowship.
4.5.3. Peptide 10b
Analytical HPLC conditions: 5–45% over 30 min. Retention
time = 14.5 min; LRMS (ESI-TOF) m/z calcd for [M+H]+ 931.4,
found; 931.2.

K. Ebisuno et al. / Bioorg. Med. Chem. 22 (2014) 2984–2991
2991
13. Serine derivative 3a could be purified by the use of normal phase silica gel
column chromatography (CHCl3 then CHCl3/MeOH = 99:1 (v/v)) for removal of
less polar compounds including triphenylphosphine followed by reverse phase
preparative HPLC (0.1% (v/v) TFA/MeCN in 0.1% (v/v) TFA aq, 38–48% over
30 min). However, this purification protocol is not useful because the weight of
3a obtained after HPLC purification at once was insufficient. The protocol
without chromatographic purification was therefore developed for practical
reasons. Reverse phase HPLC analyses for estimation of purity of the crude 3a–
c were not examined because the crude product included a less polar and
easily crystallizable triphenylphosphine. Instead, HPLC charts of the crude
peptides containing 3a–c are shown in Figure S2 in the Supporting Information
14. Mhawech, P. Cell Res. 2005, 15, 228–236. and references therein.
15. Application of caged phosphopeptides to elucidate a role of 14-3-3b protein:
Nguyen, A.; Rothman, D. M.; Stehn, J.; Imperiali, B.; Yaffe, M. B. Nat. Biotechnol.
2004, 22, 993.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.04.002.
References and notes
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28; (b) Gonzalez-Vera, J. A. Chem. Soc. Rev. 2012, 41, 1652.
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Chem., Int. Ed. 2006, 45, 4900; (c) Rothman, D. M.; Shults, M. D.; Imperiali, B.
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Kubota, H.; Tamamura, H.; Fujii, N. Tetrahedron Lett. 1995, 36, 927; (b)
Berkowitz, D. B.; Shen, Q.; Maeng, J.-H. Tetrahedron Lett. 1994, 35, 6445.
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551; (b) Burke, T. R., Jr.; Smyth, M. S.; Otaka, A.; Roller, P. P. Tetrahedron Lett.
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16. The CF2-substituted phosphoserine derivatives as
a ligand of 14-3-3 (a)
Arrendale, A.; Kim, K.; Choi, J. Y.; Li, W.; Geahlen, R. L.; Borch, R. F. Chem. Biol.
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18. For incorporation of phosphoamino acid building blocks followed by
elongation of the peptides, uronium-based coupling reagents are known to
be effective. See: Perich, J. W.; Ede, N. J.; Eagle, S.; Bray, A. M. Lett. Pept. Sci.
1999, 6, 91.
19. In
a
previously report of the preparation of 4b, N,N,N0,N0-
tetramethylguanidinium azide was used to introduce an azide unit.5a
However, it is no longer commercially available for us. In this study,
therefore, tetrabutylammonium azide purchased from
a major chemical
reagent supplier was employed for the azidation. Details are shown in an
experimental section of ‘Modified preparation of threonine derivative 4b’ and
Scheme 4.
7. (a) Otaka, A.; Mitsuyama, E.; Watanabe, J.; Watanabe, H.; Fujii, N. Biopolymers
(Pept. Sci.) 2004, 76, 140; (b) Chambers, R. D.; O’Hagan, D.; Lamont, R. B.; Jain, S.
C. J. Chem. Soc., Chem. Commun. 1990, 1053; (c) O’Hagan, D.; Rzepa, H. S. Chem.
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Panigrahi, K.; Eggen, M.; Maeng, J.-H.; Shen, Q.; Berkowitz, D. B. Chem. Biol.
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9. The non-hydrolyzable phosphotyrosine derivative for Fmoc solid phase peptide
synthesis has already been commercially available.
20. Generation of singlet oxygen by FTC sensitization: (a) Beck, S.; Sakurai, T.;
Eustace, B. K.; Beste, G.; Schier, R.; Rudert, F.; Jay, D. G. Proteomics 2002, 2, 247;
Reaction of singlet oxygen with
a thiocarbonyl group: (b) Ramnath, N.;
Ramesh, V.; Ramamurthy, V. J. Org. Chem. 1983, 48, 214; Example of photo-
induced replacement of a sulfur atom of an FTC group to an oxygen: (c)
Shigenaga, A.; Tsuji, D.; Nishioka, N.; Tsuda, S.; Itoh, K.; Otaka, A. ChemBioChem
1929, 2007, 8.
21. pH Dependency of absorption coefficient of a fluorescein: Sjoback, R.; Nygren,
J.; Kubista, M. Spectrochim. Acta, Part A 1995, 51, L7.
10. Other hydrolysis-resistant caged phosphoamino acid derivatives based on O-
phosphorothioyl amino acids had also been reported and successfully applied
to photo-control of a biological event: (a) Aemissegger, A.; Carrigan, C. N.;
Imperiali, B. Tetrahedron 2007, 63, 6185; (b) Goguen, B. N.; Hoffman, B. D.;
Sellers, J. R.; Schwartz, M. A.; Imperiali, B. Angew. Chem., Int. Ed. 2011, 50, 5667.
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phosphonate derivatives, see (a) Wakamiya, T.; Saruta, K.; Yasuoka, J.-I.;
Kusumoto, S. Chem. Lett. 1994, 23, 1099; (b) Gordeev, M. F.; Patel, D. V.; Barker,
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23. Completion of removal of the o-nitrobenzyl group was confirmed by HPLC
monitoring (Fig. 2C, peak areas are shown in Fig. S2 in the Supporting
information). In this case, sulfur-replaced peptide 10a0 was not detected
presumably due to quenching of singlet oxygen by an amine moiety of HEPES.
Reaction of singlet oxygen with amines, see: (a) Clennan, E. L.; Pace, A.
Tetrahedron 2005, 61, 6665. and references therein; (b) Smith, W. F., Jr. J. Am.
Chem. Soc. 1972, 94, 186; (c) Ogryzlo, E. A.; Tang, C. W. J. Am. Chem. Soc. 1970,
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2010, 39, 2835.