Design, synthesis, and evaluation of cell permeable probes for protein kinases

Article abstract of DOI:10.1016/j.tet.2015.10.053

We have explored a novel molecular framework with a lysine core to prepare a probe library for protein kinases. Active site-directed probes, identified through screening of the library, displayed desirable labelling properties, including target specificity, good linear response to the corresponding enzymatic activity and cell permeability. Finally, a two-stage protocol utilizing the newly developed probe with an immunoprecipitation step was successfully established to monitor intracellular Src kinase activity in RK3E-v-Src cells.

Full text of DOI:10.1016/j.tet.2015.10.053

Tetrahedron 72 (2016) 58e68
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Design, synthesis, and evaluation of cell permeable probes
for protein kinases
Yu-Ling Hsu a,y, Chun-Chieh Yang , Tzyy-Chao Chou , Chih-Hsuan Tai ,
b
,
y
a
a
c
d
b
,
c
,
a,
*
*
Long-Yuan Chen , Shu-Ling Fu , Jing-Jer Lin
Department of Chemistry, National Taiwan University, Taipei, 106, Taiwan, ROC
Institute of Biopharmaceutical Sciences, National Yang-Ming University, Taipei, 112, Taiwan, ROC
Institute of Biochemistry and Molecular Biology, National Taiwan University College of Medicine, Taipei, 100, Taiwan, ROC
Institute of Traditional Medicine, National Yang-Ming University, Taipei, 112, Taiwan, ROC
, Lee-Chiang Lo
a
b
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 21 August 2015
Received in revised form 16 October 2015
Accepted 20 October 2015
Available online 22 October 2015
We have explored a novel molecular framework with a lysine core to prepare a probe library for protein
kinases. Active site-directed probes, identified through screening of the library, displayed desirable la-
belling properties, including target specificity, good linear response to the corresponding enzymatic
activity and cell permeability. Finally, a two-stage protocol utilizing the newly developed probe with an
immunoprecipitation step was successfully established to monitor intracellular Src kinase activity in
RK3E-v-Src cells.
Keywords:
Activity
Ó 2015 Elsevier Ltd. All rights reserved.
Synthesis
Click
ATP
Immunoprecipitation
1
. Introduction
intracellular protein kinases in light of their important roles in the
signalling pathways of protein phosphorylation and as drug
targets.
11e13
The development and applications of chemical probes have
contributed tremendously to the rapid progress in the research of
The most critical part in this attempt is the development of
probes that meet the following criteria; (i) cell permeability, (ii)
selective labelling to the catalytically active protein kinases, and
(iii) the labelled adduct recognizable by antibodies against the
target protein kinase. It should be noted that protein kinases in the
signalling pathways are often associated with and regulated by
other modulators through noncovalent interactions. Therefore
conducting the labelling event before the cells were ruptured
would be very helpful in reducing possible artifacts. This re-
quirement makes cell permeability an indispensable feature in the
probe design.
1
e4
modern chemical biology.
Despite their success in various ap-
plications,5 use of probes for directly measuring a designated
enzymatic activity in a proteomic sample is still a highly chal-
lenging task because the complex nature of the proteome fre-
,6
7,8
quently poses unforeseen obstacles. To overcome the difficulties,
we previously established an assay, which combines the use of
probes with target-specific antibody (Fig. 1). With such a two-stage
protocol, signals resulted from nonspecific reactions of the probes
in the first stage could be screened off by an immunocapture pro-
cess with a target-specific antibody. The assay platform was suc-
cessfully demonstrated on two model systems, which enabled
direct monitoring of src homology-2 (SH2) domain-containing
phosphatase 2 (SHP2) activity upon cisplatin treatment of cancer
cells and during erythroid differentiation.9 In continuation of our
efforts, we now focus on the development of an assay system for
To date, the molecular framework of probes targeting kinases
could generally be categorized into two major types; the one
derived from the known small-molecule inhibitors and the other
utilizing nucleoside or nucleotide derivatives. Probes in the first
,10
14e18
category usually work on selective subsets of kinases,
whereas probes in the second category works on a much
19,20
broader range of kinases.
It should also be noted that the
reporter group plays a crucial role in determining the cell per-
meability for both categories of probes. For example, probes
with a nucleotide acyl phosphate skeleton were estimated to
*
Corresponding authors. E-mail addresses: jingjerlin@ntu.edu.tw (J.-J. Lin), lclo@
14
ntu.edu.tw (L.-C. Lo).
y
Equal contribution.
http://dx.doi.org/10.1016/j.tet.2015.10.053
0
040-4020/Ó 2015 Elsevier Ltd. All rights reserved.

Y.-L. Hsu et al. / Tetrahedron 72 (2016) 58e68
59
Fig. 1. Schematic presentation for monitoring a designated enzymatic activity in proteomic samples by combining the use of a chemical probe with a target specific antibody.
cover more than 75% of the human protein kinases.19 However,
their highly negatively charged nature would prohibit them from
being used for the development of cell permeable probes.
0
5
’-p-Fluorosulfonylbenzoyl adenosine (5 -FSBA), on the other
hand, serves as a good starting point, as it is an uncharged ATP
analogue of which the triphosphate part was replaced with a p-
fluorosulfonylbenzoyl group (Fig. 2) and is a well-characterized
kinase inhibitor.2
1e23
Labelling studies with FSBA derivatives
revealed that the adenosine moiety could indeed facilitate binding
of the probe to the ATP binding pocket, and more importantly, the
electrophilic arenesulfonyl fluoride was able to react mainly with
2
2,24e26
a conserved lysine residue.
Recently, probes combining the
structural features of FSBA and PP1, a Src-family inhibitor, with
a terminal alknye tag attached to C-2 position of the ribose ring
were shown not only to be able to selectively label Src family
18
members, but also to possess cell permeability. In addition,
a later study demonstrated that altering the distance and orien-
tation of the reactive fluorosulfonyl moiety could help improve the
Fig. 3. The molecular framework of the active site-directed probe library (1aed, 2aed)
for protein kinases, which carries an electrophilic fluorosulfonylaryl group (p-/m-),
a nucleobase (T/A), and an azido reporter group (Z) on a lysine core.
2
7
labelling efficiency on kinases. Nevertheless, the rigid adenosine
skeleton limits the potential for further improvement. We herein
explored a novel molecular framework by attaching the three key
structural elements of a probe, including a fluorosulfonylaryl
warhead group for covalent bond formation, a nucleobase for
recognition, and an azido group to act as a cell penetrating re-
porter group,28 to a lysine core. Since there is no way to precisely
predict the outcome when a known binding moiety was struc-
turally connected to a warhead and a reporter group, the chances
of finding an optimal hit by betting on one specific combination
would be relatively slim. Therefore, in this study we performed
parallel synthesis to prepare a probe library (Fig. 3). The lysine
core is a trifunctionalized linker that not only places the nucleo-
base and the warhead units in proper distance and orientation to
resemble the corresponding pair in FSBA, but also allows for
possible fine tuning in the probe library construction. We envi-
sioned finding hits with better labelling performance could be
achieved by screening such a probe library.
thymine, were included as the recognition unit. The electrophilic
warhead was attached to the carboxyl group of the lysine core,
while the nucleobase (T/A) and the azido reporter (Z) components
occupied the two amino groups (
a
-/ 3 -). To expand the library, we
also exchanged the locations of the latter two components, T/A
versus Z, leading to probes 1aed where the nucleobase component
(T/A) was connected to the
a-amino group and to probes 2aed
where the nucleobase component was connected to the
3 -amino
group. A total of eight compounds were prepared and evaluated in
this study.
Prior to the library synthesis, building blocks 3e6 and a fluo-
rescent reagent 7 (Fig. 4) were prepared according to literature
28e31
procedures.
Building blocks 3e6 all carry a carboxyl func-
-/ -amino groups of the lysine
tionality that would react with the
a
3
core. It should be noted that acids 3e5 carry the nucleobase moiety,
whereas acid 6 carries the azido reporter group with an oligoe-
thyleneglycol linker. The rhodamine B-derived click reagent 7
(
l
ex¼567 nm,
l
em¼589 nm) would be used for visualization of the
2
. Results and discussion
labelled proteins after gel electrophoresis by Cu-catalyzed azide-
alkyne cycloaddition.
2
8,32
For the construction of the probe library (Fig. 3), two positional
isomers of arenesulfonyl fluorides (m- and p-) were used as the
electrophilic warhead and two kinds of nucleobase, adenine and
As shown in Scheme 1, the library synthesis begins with nitro-
benzenesulfonyl chlorides 8 (p-/m-). They were converted to sul-
fonyl fluorides 9 in good yields (94e95%) by treatment with KF in
the presence of 18-crown-6. The nitro group of 9 was subjected to
catalytic hydrogenation to give fluorosulfonyl anilines, which were
subsequently reacted with Fmoc-Lys(Boc)-OH in pyridine using
3
3
3
4
POCl
3
as the coupling reagent. The coupling reactions were car-
ꢁ
ried out at a low temperature (ꢀ16 C) to afford the key in-
termediates 10 in moderate yields (51e59%) over the two steps.
Intermediates 10 already carry the sulfonyl fluoride warhead at-
tached to the carboxyl group of the lysine core, and the two amino
groups where the nucleobase (T/A) and reporter (Z) moieties would
later be incorporated are temporarily protected by Fmoc (
a-) and
0
Boc ( -).
3
Fig. 2. The structures of ATP and 5 -FSBA.

60
Y.-L. Hsu et al. / Tetrahedron 72 (2016) 58e68
Fig. 4. Structures of the building blocks 3e6 and the fluorescent alkyne click reagent 7.
released -amino group was then coupled to the corresponding acid
3
building block 6/3/5 under standard EDC/HOBt method. In this step,
compounds 11aed led to probes 1aed whilst compounds 12a,b
provided probes 2a,b and the protected precursors 13a,b which
upon deprotection with TFA/CH
2 2
Cl afforded probes 2c,d, thus
completing the synthesis of the library.
To test if the newly designed probes could label kinases, we
compared their reactivity on two kinases, focal adhesion kinase
(Fak) and lymphocyte-specific protein tyrosine kinase (Lck). After
Scheme 1. Synthesis of the intermediates 10. Reagents and conditions: (a) KF, 18-
crown-6, CH
Lys(Boc)-OH, POCl
3
CN, 24 h, 94e95%; (b) (i) H
2
, Pd/C, EtOAc/AcOH (1/1), 3 h, (ii) Fmoc-
labelling, the reaction mixtures were subjected to click reaction
with fluorescent reagent 7 in order to generate fluorescent readout
signals for the labelled proteins following the separation with SDS-
polyacrylamide gel electrophoresis. As shown in Fig. 5A, Coomassie
blue staining shows the relative amount of each protein in the
mixture. On the right are the fluorescent signals from which dif-
ferential labelling activities were clearly observed. As a general
trend, probes carrying a thymine unit (1a, 2a, 1b, and 2b) showed
higher labelling efficiency toward both protein kinases than those
with an adenine unit (1c, 2c, 1d, and 2d). When examined more
closely, we found neither the attachment site of the thymine unit
ꢁ
3
, py, -16 C, 2.5 h, 51e59%.
Next, intermediates 10 were treated with TBAF to remove the
Fmoc protecting group to release the -amino group which was
then subjected to amide bond formation reaction with each acid
building block (3/5/6) under standard EDC/HOBt method to give
intermediates 11aed and 12a,b in moderate yields (48e73%)
a
(Scheme 2). It should be pointed out that in our initial attempts
using building block 4 to incorporate the A moiety to the lysine
core, we encountered extremely low yields. Fortunately, this diffi-
culty could be overcome by replacing it with the di-Boc protected
building block 5.30 We thus used building block 5 when the A
moiety was needed in the synthesis. Accordingly, through amide
on the lysine core (
a
-/ 3 -) nor the positional isomer of the sulfonyl
fluoride warhead (p-/m-) seem to greatly affect the labelling out-
come. In contrast, isomeric position of the warhead played a more
significant role, with m-isomers giving a better labelling, for probes
carrying an adenine unit (1d/2d versus 1c/2c). These observations
indicated that screening a probe library is an appropriate approach
to identify a promising combination of warhead and nucleobase.
Since probes 1b and 2b apparently showed better labelling activity
bond formation reaction at the a-amino group we obtained com-
pounds 11aed when nucleobase blocks 3/5 were used, and com-
pounds 12a,b when the azido acid 6 was used. To set the stage for
the second amide bond formation reaction, the Boc group of these
2 2
compounds was removed by treatment with TFA in CH Cl . The
Scheme 2. Synthesis of probes 1aed and 2a-d. Reagents and conditions: (a) (i) 1 M TBAF in THF, 30 min, (ii) corresponding acid (3/5/6), EDC, HOBt, DIEA, CH
i) TFA, CH Cl , 30 min, (ii) corresponding acid (6/3/5), EDC, HOBt, DIEA, DMF/CH Cl (1/1), 16 h, 51e95%. (c) TFA/CH Cl , 30 min, 80e82%.
2 2
Cl , 16 h, 48e73%; (b)
(
2
2
2
2
2
2

Y.-L. Hsu et al. / Tetrahedron 72 (2016) 58e68
61
and could be synthesized in fewer steps, they were chosen for
further studies. To demonstrate that the probes showed selective
labelling toward kinases, we compared their reactivity on other
proteins including carbonic anhydrase, glucosidase, phosphorylase,
PTP1B, g-globulin, RNase A, and lysozyme. As shown in Fig. 5B, no
labelling signals were detected for the non-kinase proteins. We
have further tested the labelling selectivity in a protein mixture
containing these proteins and found only Lck was labelled. Thus,
our newly designed probes showed specific labelling toward pro-
tein kinases.
The sensitivity of the labelling reactions by 1b and 2b was next
determined. Under saturated probe concentration (0.5 mM), both
1b and 2b were capable of detecting as little as 6.25 ng of Lck,
which is about 4-fold more sensitive than silver stain (Figure S1A).
Moreover, there is a good linear correlation between the extent of
labelling and the protein level (Figure S1B), suggesting that the
extent of 1b and 2b labelling can be used as a measurement of
protein activity. We next characterized the labelling properties of
probes 1b and 2b. To test if the labelling requires native confor-
mations of the proteins, labelling with heat-denatured Lck was
first examined. As shown in Fig. 6A, heat-treated Lck failed to be
labelled by both 1b and 2b, indicating that an active protein is
required for the labelling. We then tested if active-site binding of
1b and 2b is required for the labelling reaction. As shown in
Fig. 6B, the labelling signal was diminished with FSBA-treated Lck,
suggesting that the labelling events of 1b and 2b occurred in the
2
2,24e26
ATP binding pocket of Lck.
Since these two probes behaved
similarly, we chose probe 2b for further analysis. The labelling
reactions were also tested in the presence of ATP. We reasoned
that if the probes bind to the active site of the kinase, ATP should
be able to compete away the labelling reaction. As shown in
Fig. 6C, ATP effectively inhibited the labelling of 2b to Lck in a dose
dependent manner.
Fig. 5. Screening, reactivity, and specificity of probes toward kinase. (A) One mg
each of the catalytic domains of kinases Fak or Lck proteins were incubated with 25
m
M
ꢁ
probes at 25 C for 1 h. The reaction products were coupled with 7 using click
chemistry, and then separated with a 13.5% SDS-polyacrylamide gel. The proteins were
visualized by Coomassie blue staining and fluorescence scanning analysis using Ty-
phoon 9400D. The excitation wavelength is 532 nm and the emission wavelength is
Src is a non-receptor protein tyrosine kinase protein that me-
3
5
diates important signalling pathways for tumorigenesis. An
elevated level of Src kinase activity was suggestively linked
to cancer progression. Inhibitors targeting Src kinase have been
considered promising agents for anti-cancer chemotherap-
580 nm. (B) As above, one mg each of the indicated protein or mixture of them (Mix)
were labelled with probe 1b or 2b and analyzed.
Fig. 6. Active site-directed labelling of Lck with 1b and 2b. One
m
g each of heat-denatured (A) and FSBA-blocked (B) Lck was labelled with 1b and 2b, respectively, and analyzed as
described in Fig. 5. (C) One
, were separated by a 13.5% SDS-polyacrylamide gel, and subjected to Coomassie blue staining and fluorescence scanning analysis using Typhoon 9400D (upper panel). The
fluorescence intensity was quantified, normalized, and then plotted against Log[ATP] (lower panel).
mg each of Lck was incubated with 0.5 mM of 2b in the presence of varying amounts of ATP. The reaction products, undergoing click reaction with reagent
7

62
Y.-L. Hsu et al. / Tetrahedron 72 (2016) 58e68
eutics.36 To evaluate if probe 2b could be applied in the assay
platform (Fig. 1) for an intracellular kinase assay, we examined the
detection of Src kinase using a cell-based model system. Here,
a rat kidney epithelial cell RK3E was transfected with v-Src to
immunoprecipitation step would be of great help in the success
of the assay.
3. Conclusion
3
7
generate a stable line, RK3E-v-Src that expressed v-Src. The
RK3E-v-Src cells were treated with probe 2b (100 M), and the
m
We have explored a novel molecular framework with a lysine
protein extracts were collected, clicked, and analyzed. Under the
labelling condition, no apparent cytotoxic effect was observed
core to prepare a probe library for protein kinases. The active site-
directed probes, identified through screening of the library, dis-
played desirable labelling properties, including target specificity,
good linear response to the kinase activity and cell permeability.
More importantly, the labelled kinase remained recognizable by the
corresponding antibody. These features make the probes extremely
(
Figure S2). As shown in Fig. 7A, cells showed some background
fluorescence even without probe treatment. Probe 2b labelled
many additional protein bands in RK3E-v-Src cells. Among the
additional bands, a band corresponding to the size of v-Src was
3
8
9
apparent. Addition of PP2, a specific Src-family kinase inhibitor,
decreased the labelling of this band, suggesting that it is likely to
be v-Src. More significantly, the data showed that probe 2b pos-
sesses cell permeable property.
valuable in the two-stage protocol for the assay of intracellular ki-
nase activities. In addition, the novel molecular framework de-
scribed in this paper would allow the expansion of the probe library
by incorporating various heterocyclic arylamines other than thy-
Fig. 7. Detection of intracellular Src kinase with probe 2b. (A) RK3E-v-Src cells were incubated with probe 2b (100 mM) for 1 h. Cell extracts were prepared from the treated cells,
coupled to 7 by click chemistry, and separated by a 10% SDS-polyacrylamide gel. The gel was then visualized by Coomassie blue staining (left panel) and fluorescence scanning
analysis (middle panel) and immunoblotting analysis using anti src antibody (right). Cell extracts prepared from the treated cells were immunoprecipitated with anti-Src antibody,
and then coupled to 7 using click chemistry. The immunoprecipitated proteins were then separated by a 10% SDS-polyacrylamide gel and then subjected to fluorescence scanning
analysis (right panel). Immunoblotting analyses were also conducted to detect the Src protein levels (bottom panels). (B) Quantification of the relative Src labelling values in the
immunoprecipitates presented in Fig. 7A, right panel.
Although Src kinase could be detected by 2b, it is difficult to
give a reliable quantitative result under such a circumstance,
which also represents one of the major limitations of using probe
alone to determine the designated enzymatic activity in a pro-
teomic sample. Nevertheless, the difficulty can be overcome by
our protocol. A clear signal appeared after the complicated la-
belled proteins had been subjected to an immunoprecipitation
mine and adenine to the lysine core, making it a useful strategy for
further development and improvement of the probes.
4
4
. Experimental
.1. Synthesis of intermediates 10
All of the chemical reagents (except where indicated otherwise)
(
Fig. 7A). Thus, combination of our probe and an immunopre-
cipitation step enables the detection of a specific kinase within
a complex proteome. Consistent with the in vitro labelling ex-
periments, the 2b labelling was inhibited by PP2 (Fig. 7A and B).
Based on these results, it is important to note that the probe
could have greater potential in the development of assay system
for an array of intracellular kinases, given the ready availability of
the corresponding antibodies from various commercial sources. It
is also worth mentioning that the quality of the antibodies is an
important factor in a practical application of the assay platform.
Therefore, a reliable source of high quality antibodies for the
were purchased from Acros, Aldrich, and Merck and were used
without further purification. N-9-Fluorenylmethoxycarbonyl
(
Fmoc) amino acids and 1-hydroxybenzotriazole (HOBt) were
from Advanced ChemTech. Analytical thin-layer chromatography
TLC) plates (silica gel, 60F-54, Merck) were visualized under UV
(
light and/or phosphomolybdic acidꢀethanol stain. Column chro-
matography was performed with Kiesegel 60 (70e230 mesh) silica
gel (Merck). IR spectra were recorded on a Varian 640 FT-IR spec-
1
13
trometer. H and C NMR spectra were recorded using a Bruker
Avance 400 spectrometer. The proton and carbon chemical shifts

Y.-L. Hsu et al. / Tetrahedron 72 (2016) 58e68
63
(
d
) are given in ppm using CDCl
and 49.00), DMSOed 2.50 and 39.52), acetoneed
9.84), and D O ( 4.70) as internal standards, and coupling con-
3
(
d
H
7.24 and 77.0), CD
3
OD (
d
H
3.31
31.1 (CH
2
), 29.2 (CH
2
), 28.4 (CH
3
), 22.2 (CH
2
); 19F NMR (376 MHz,
6
(d
H
6
(d
H
2.05 and
CDCl ): d 65.83. IR (KBr): 3283, 2940, 1685, 1588, 1516, 1404, 1250,
3
ꢀ1
SNa (MþNa)þ
2
2
d
H
1209, 1178 cm . HRMS calcd for C32
648.2156, found 648.2136.
3 7
H36FN O
stants are in hertz (Hz). IR spectra were recorded using Varian 640
FTeIR spectrometer. The high resolution mass spectra (HRMS) were
recorded on an LCT Premier XE mass spectrometer. Melting points
are uncorrected.
4.1.4. (S)-(9H-Fluoren-9-yl)methyl tert-butyl (6-((3-(fluorosulfonyl)
p h e n y l ) a m i n o ) - 6 - o x o h e x a n e - 1, 5 - d i y l ) d i c a r b a m a t e
(10b). Compound 10b was prepared with the same procedure as
4
.1.1. 4-Nitrobenzene-1-sulfonyl fluoride (9a). To a solution of 4-
nitrobenzene-1-sulfonyl chloride (20.0 g, 90.2 mmol) in 500 mL
CH CN was added KF (26.0 g, 451 mmol) and 18-crown-6 (1.2 g,
.5 mmol). After stirring for 24 h, the reaction mixture was evap-
orated to dryness and EtOAc (600 mL) was added. It was washed
with H
dried over Na
described for compound 10a, except compound 9b was used as the
starting material. Compound 10b was obtained in 59% yield as
ꢁ
3
a white solid, with mp 143e144 C. R
f
¼0.43 (hexane/EtOAc¼1/1).
1
4
3
H NMR (400 MHz, CDCl ): d 9.26 (s, 1H, NH), 8.27 (s, 1H, aromatic),
7.97 (d, J¼6.4 Hz, 1H, aromatic), 7.73 (d, J¼6.8 Hz, 2H, aromatic),
7.68 (d, J¼7.7 Hz,1H, aromatic), 7.56 (d, J¼6.3 Hz, 2H, aromatic), 7.50
(m, 1H, aromatic), 7.36 (m, 2H, aromatic), 7.25 (m, 2H, aromatic),
2
O (300 mLꢂ3) and brine (300 mL). The organic layer was
2
SO
4
, filtered, and evaporated under vacuum to pro-
ꢁ
vide 9a (17.4 g, 94%) as a yellow solid, mp 76e77 C. R
f
¼0.63
5.67 (d, 1H, J¼6.5 Hz, NH), 4.41 (d, J¼6.7 Hz, 2H), 4.32 (m, 1H,
aeH),
1
(
2
hexane/EtOAc¼6/4). H NMR (400 MHz, CDCl
3
):
d
8.47 (d, J¼8.7 Hz,
4.19 (t, J¼6.7 Hz, 1H), 3.18 (m, 1H), 3.07 (m, 1H), 1.98 (m, 1H), 1.72
1
3
13
H, aromatic), 8.25 (d, J¼8.7 Hz, 2H, aromatic); C NMR (100 MHz,
(m, 1H), 1.53 (m, 2H), 1.41 (m, 2H), 1.41 (s, 9H); C NMR (100 MHz,
1
9
CDCl
3
):
d
151.8 (C),138.2 (d, J¼26.7 Hz, C),130.0 (CH),124.9 (CH);
F
3
CDCl ): d 171.3 (C), 156.9 (C), 156.5 (C), 143.5 (C), 141.2 (C), 139.4 (C),
NMR (376 MHz, CDCl
3
): 65.1. IR (KBr): 3105, 2872,1951,1818,1613,
d
133.3 (d, J¼24.2 Hz, C), 130.1 (CH), 127.7 (CH), 127.0 (CH), 126.1 (CH),
ꢀ1
1540, 1420, 1354, 1314, 1215 cm . HRMS calcd for C
6
H
5
4
FNO S
124.9 (CH), 123.3 (CH), 120.0 (CH), 118.9 (CH), 79.5 (C), 67.3 (CH
55.5 (CH), 46.9 (CH), 39.3 (CH ), 31.4 (CH ), 29.2 (CH ), 28.3 (CH
); F NMR (376 MHz, CDCl ):
2
),
),
þ
(
MþH) 205.9923, found 205.9918.
2
2
2
3
19
22.3 (CH
2
3
d
64.60. IR (KBr): 3436,1680,
ꢀ
1
4.1.2. 3-Nitrobenzene-1-sulfonyl fluoride (9b). Compound 9b was
1534, 1516, 1409, 1213, 1209, 1170 cm . HRMS calcd for
prepared with the same procedure as described for compound 9a,
except 3-nitrobenzene-1-sulfonyl chloride was used as the starting
C
32
H36FN
3
O
7
SNa (MþNa)þ 648.2150, found 648.2102.
material instead of 4-nitrobenzene-1-sulfonyl chloride. Compound
4.2. Synthesis of 11aed, 12a, and 12b: general procedure
ꢁ
9
b was obtained in 95% yield as a brown solid, mp 43e45 C (lit. mp
ꢁ
39
1
4
CDCl
5e46 C).
):
R
f
¼0.55 (hexane/EtOAc¼6/4). H NMR (400 MHz,
Compound 10a/10b (159.0 mg, 0.2542 mmol) was dissolved in
1.1 mLTBAF/THF (1 M,1.1 mmol), and the reaction mixture wasstirred
3
d
8.57 (s, 1H, aromatic), 8.38 (d, J¼8.0 Hz, 1H, aromatic), 8.16
1
3
(
d, J¼7.9 Hz, 1H, aromatic), 7.85 (dd, J¼8.0, 7.9 Hz, 1H, aromatic);
NMR (100 MHz, CDCl ):
148.4 (C),134.4 (d, J¼27.2 Hz, C),133.9 (CH),
31.7 (CH),130.2 (CH),123.7 (CH); F NMR (376 MHz, CDCl
IR (KBr): 3451, 3097, 2109, 1607, 1538, 1417, 1353, 1216, 1079 cm
C
at rt for 30 min CH
0.0763 mmol), DIEA (84 m
2
Cl
2
(2.5 mL), 3/5/6 (0.2542 mmol), HOBt (10.3 mg,
L, 0.51 mmol), and EDC (73.1 mg,
3
d
19
1
3
):
d
65.15.
0.381 mmol)were added sequentially, and themixturewasstirred for
another 16 h. The reaction was quenched with 5% aqueous citric acid
ꢀ
1
.
(
1 mL) and was concentrated to dryness under vacuum. The residue
was dissolved in 50 mL EtOAc, which was washed with 5% aqueous
citric acid (10 mLꢂ2), 5% aqueous NaHCO (10 mLꢂ2), H O (10 mLꢂ2)
and brine (10 mL). The organic layer was dried over Na SO , filtered,
and concentrated. The desired product was then purified with silica
gel column chromatography eluted with CH Cl /MeOH (95/5).
4
.1.3. (S)-(9H-Fluoren-9-yl)methyl tert-butyl (6-((4-(fluorosulfonyl)
phenyl)amino)-6-oxohexane-1,5-diyl)dicarbamate (10a). To a solu-
tion of compound 9a (200.0 mg, 0.975 mmol) in 10 mL EtOAc/AcOH
3
2
2
4
(
1:1) was added 20% Pd/C catalyst (40.0 mg). The reaction mixture
was stirred under H atmosphere for 3 h. The reaction mixture was
2
2
2
filtered through Celite and the filtrate was evaporated to dryness
under vacuum. The residue was dissolved in EtOAc (40 mL) and
4.2.1. (S)-tert-Butyl (6-((4-(fluorosulfonyl)phenyl)amino)-5-(2-(5-
washed with 5% NaHCO
3
(10 mLꢂ3), H
2
O (10 mLꢂ2) and brine
methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamido)-6-
oxohexyl)carbamate (11a). Compound 11a was obtained in 49%
(
10 mL). The organic layer was dried over Na
2
SO , filtered, and
4
ꢁ
evaporated under vacuum to afford the crude aniline derivative,
which was used without further purification. To a solution of the
above crude aniline derivative and Fmoc-Lys(Boc)-OH (435.5 mg,
yield as a white solid, mp 140e143 C. R
f
¼0.33 (Chloroform/
1
MeOH¼9/1). H NMR (400 MHz, acetone-d
6
): d 10.35 (s, 1H, NH),
9.83 (s, 1H, NH), 8.09 (m, 1H, NH), 8.09 (d, J¼8.8 Hz, 2H, aromatic),
7.98 (d, J¼8.8 Hz, 2H, aromatic), 7.45 (s, 1H), 6.00 (m, 1H, NH), 4.58
1
.023 mmol) in 3 mL anhydrous pyridine cooled in an ice/salt bath
ꢁ
(ꢀ16 C) under argon, was added freshly distilled POCl
3
(90.6
mL,
(m, 1H, aeH), 4.55 (s, 2H), 3.05 (m, 2H), 1.97 (m, 1H), 1.82 (s, 3H),
13
0
.9747 mmol). The reaction mixture was stirred for 2.5 h, and then
1.78 (m, 1H), 1.48 (m, 2H), 1.43 (m, 2H), 1.38 (s, 9H); C NMR
(100 MHz, acetone-d ): 172.2 (C), 168.8 (C), 165.0 (C), 156.9 (C),
152.7 (C), 146.8 (C), 142.9 (CH), 130.7 (CH), 126.8 (d, J¼24.4 Hz, C),
120.7 (CH), 110.4 (C), 79.3 (C), 78.6 (C), 55.4 (CH), 51.8 (CH ), 40.8
diluted with EtOAc (40 mL). The EtOAc solution was washed with
% citric acid (10 mLꢂ3), water (10 mLꢂ2), and brine (10 mL). The
organic layer was dried over Na SO , filtered, and evaporated. The
6
d
5
2
4
2
19
desired product was purified with silica gel column chromatogra-
(CH
2 2 3 2 3
), 32.1 (CH ), 28.7 (CH ), 23.8 (CH ), 12.4 (CH ); F NMR
phy eluted with hexane/EtOAc (65/35). Compound 10a was ob-
(376 MHz, acetone-d
1598, 1537, 1470, 1404, 1245, 1204, 1168 cm . HRMS calcd for
6
):
d
65.8. IR (KBr): 3293, 3068, 2930, 1680,
ꢁ
ꢀ1
tained as a white solid (311 mg, 51%), mp 113e114 C. R
f
¼0.5
1
ꢀ
(
hexane/EtOAc¼1/1). H NMR (400 MHz, CDCl
.87 (d, J¼8.4 Hz, 2H, aromatic), 7.77 (d, J¼8.8 Hz, 2H, aromatic),
.74 (d, J¼7.6 Hz, 2H, aromatic), 7.55 (d, J¼7.1 Hz, 2H, aromatic), 7.37
m, 2H, aromatic), 7.26 (m, 2H, aromatic), 5.69 (s, 1H, NH), 4.72 (s,
3
):
d
9.32 (s, 1H, NH),
C
24
H
31FN
5
O
8
S (MeH) 568.1877, found 568.1880.
7
7
(
4.2.2. (S)-tert-Butyl (6-((3-(fluorosulfonyl)phenyl)amino)-5-(2-(5-
methyl-2,4-dioxo-3,4- dihydropyrimidin-1(2H)-yl)acetamido)-6-
1
1
2
H, NH), 4.41 (d, J¼6.8 Hz, 2H), 4.30 (m, 1H,
a
eH), 4.19 (t, J¼6.8 Hz,
oxohexyl)carbamate (11b). Compound 11b was obtained in 48%
ꢁ
H), 3.17 (m, 1H), 3.05 (m, 1H), 1.96 (m, 1H), 1.72 (m, 1H), 1.52 (m,
yield as a white solid, mp 136e138 C. R
f
¼0.43 (Chloroform/
13
1
H), 1.44 (m, 2H), 1.42 (s, 9H); C NMR (100 MHz, CDCl ): 171.4
3
d
MeOH¼85/15). H NMR (400 MHz, acetone-d
6
): 10.24 (s, 1H, NH),
d
(
(
(
C), 156.9 (C), 156.6 (C), 144.6 (C), 143.5 (C), 143.4 (C), 141.2(C), 129.7
9.77 (s, 1H, NH), 8.70 (s, 1H, aromatic), 8.03 (d, J¼8.3 Hz, 1H, aro-
matic), 8.01 (m, 1H, NH), 7.76 (d, J¼8.0 Hz, 1H, aromatic), 7.69 (dd,
J¼8.3, 8.0 Hz, 1H, aromatic), 7.44 (d, J¼1.0 Hz, 1H), 5.99 (m, 1H, NH),
CH), 127.8 (CH), 127.1 (CH), 126.8 (d, J¼24.3 Hz, C), 124.9 (CH), 120.0
2 2
CH), 119.5 (CH), 79.6 (C), 67.4 (CH ), 55.6(CH), 46.9 (CH), 39.1 (CH ),

64
Y.-L. Hsu et al. / Tetrahedron 72 (2016) 58e68
4
.57 (m, 1H,
a
eH), 4.55 (m, 2H), 3.05 (m, 2H), 1.96 (m, 1H), 1.81 (d,
(12b). Compound 12b was obtained in 73% yield as colourless oil,
1
J¼1.0 Hz, 3H), 1.77 (m, 1H), 1.49 (m, 2H), 1.42 (m, 2H), 1.38 (s, 9H);
R
f
¼0.5 (chloroform/MeOH¼9/1). H NMR (400 MHz, CDCl
3
): d 9.66
1
3
C NMR (100 MHz, acetone-d
56.9 (C), 152.5 (C), 143.0 (CH), 141.2 (C), 133.7 (d, J¼24.1 Hz, C),
31.5 (CH), 127.3 (CH), 123.8 (CH), 119.1 (CH), 110.3 (C), 78.6 (C) 55.3
CH), 51.4 (CH ), 40.7 (CH ), 32.2 (CH ), 30.4 (CH ), 28.7 (CH ), 23.7
CH ), 12.3 (CH ); F NMR (376 MHz, acetone-d ): 63.5; IR (KBr):
6
):
d
172.0 (C), 168.9 (C), 165.2 (C),
(s, 1H, NH), 8.24 (s, 1H, aromatic), 7.99 (d, J¼8.2 Hz, 1H, aromatic),
7.66 (d, J¼8.0 Hz, 1H, aromatic), 7.60 (d, J¼8.3 Hz, 1H, NH), 7.49 (dd,
1
1
(
(
J¼8.2, 8.0 Hz, 1H, aromatic), 4.77 (s, 1H, NH), 4.66 (m, 1H,
aeH), 4.11
2
2
2
2
3
(d, J¼15.9 Hz, 1H), 4.05 (d, J¼15.9 Hz, 1H), 3.73e3.63 (m, 10H), 3.36
19
2
3
6
d
(dd, J¼5.1 Hz, 4.9 Hz, 2H), 3.13 (m, 1H), 3.07 (m, 1H), 2.01 (m, 1H),
ꢀ
1
13
3
288, 3063, 2930, 1680, 1537, 1470, 1409, 1250, 1204, 1163 cm
.
1.76 (m, 1H), 1.53 (m, 2H), 1.43 (m, 2H), 1.40 (s, 9H); C NMR
ꢀ
HRMS calcd for C27
H36FN
8
O
10S (MeH) 568.1877, found 568.1873.
3
(100 MHz, CDCl ): d 170.8 (C), 170.3 (C), 156.0 (C), 139.6 (C), 133.0 (d,
J¼24.4 Hz, C),130.0 (CH), 125.8 (CH), 122.9 (CH), 118.6 (CH), 78.9 (C),
4
.2.3. (S)-tert-Butyl
ycarbonyl)amino)-1-((4- (fluorosulfonyl)phenyl)amino)-1-oxohexan-
- y l ) a m i n o ) - 2 - o x o e t h y l ) - 9 H - p u r i n - 6 - y l ) c a r b a m a t e
11c). Compound 11c was obtained in 56% yield as a white solid mp
tert-butoxycarbonyl(9-(2-((6-((tert-butox-
71.0 (CH
2
), 70.4 (CH
2
), 70.3 (CH
), 39.8 (CH
); F NMR (376 MHz, CDCl
2
), 70.0 (CH
), 32.0 (CH
):
2
), 69.9 (CH ), 69.7 (CH
2
), 29.3 (CH ), 28.2 (CH
2
2
),
),
53.3 (CH), 50.4 (CH
2
2
2
3
19
2
(
22.6 (CH
2
3
d
64.7. IR (neat): 3329, 3278,
ꢀ
1
2919, 2858, 2105, 1711, 1583, 1532, 1404, 1250, 1209, 1178 (cm );
HRMS calcd for
641.2359.
ꢁ
1
SNa (MþNa)þ 641.2381, found
1
(
(
15e117
400 MHz, CDCl
d, J¼8.7 Hz, 2H, aromatic), 7.76 (d, J¼7.2 Hz, 1H, NH), 7.68 (d,
C.
R
f
¼0.44 (Chloroform/MeOH¼85/15).
H
NMR
25 6 9
C H39FN O
3
): 9.68 (s, 1H, NH), 8.76 (s, 1H), 8.24 (s, 1H), 7.80
d
J¼8.7 Hz, 2H, aromatic), 5.04 (d, J¼16.8 Hz, 1H), 5.04 (d, J¼16.8 Hz,
H), 4.96 (m, 1H, eH), 4.49 (m, 1H), 3.08 (m, 1H), 2.99 (m, 1H), 1.84
m, 1H), 1.66 (m, 1H), 1.55e1.39 (m, 31H). C NMR (100 MHz,
CDCl ): 170.7 (C), 166.6 (C), 156.7 (C), 153.4 (CH), 152.1 (CH), 150.6
C), 150.2 (C), 145.9 (CH), 144.6 (C), 129.7 (CH), 128.1 (C), 126.9 (d,
J¼24.7 Hz, C), 119.7 (C), 84.2 (C), 79.6 (C), 54.5 (CH), 45.9 (CH ), 39.3
CH ), 30.9 (CH ), 29.1 (CH ), 28.4 (CH ), 27.7 (CH ), 22.2 (CH
NMR (376 MHz, CDCl ):
4.3. Synthesis of 1aed, 13a, 13b, 2a, and 2b: general procedure
1
(
a
13
To a solution of 11aed/12a/12b (0.10 mmol) in 0.8 mL CH
2 2
Cl
3
d
was added 200 L TFA. The reaction mixture was stirred at rt for
m
(
30 min. Toluene (1 mL) was added and the mixture was evaporated
to dryness under vacuum. The residue was dissolved in 1 mL DMF/
2
19
(
2
2
2
3
3
2
).
F
CH
2
Cl
2
(1/1), followed by adding azido acid 6 (22.4 mg, 0.10 mmol),
L, 0.40 mmol), and EDC
3
d
63.8. IR (KBr): 3391, 2980, 1786, 1694,
HOBt (4.1 mg, 0.03 mmol), DIEA (66 m
ꢀ
1
10FS (MþH)þ
1537, 1410, 1212, 1113 cm . HRMS calcd for C34
H
48
N
8
O
(23.1 mg, 0.120 mmol). The reaction mixture was stirred for 24 h. It
was then quenched with 5% aqueous citric acid (1 mL) and evap-
orated to dryness under vacuum. The residue was dissolved in
EtOAc (50 mL) and washed with 5% aqueous citric acid (10 mLꢂ2),
7
79.3198, Found: 779.3203.
.2.4. (S)-tert-Butyl
4
tert-butoxycarbonyl(9-(2-((6-((tert-butox-
ycarbonyl)amino)-1-((3- (fluorosulfonyl)phenyl)amino)-1-oxohexan-
- y l ) a m i n o ) - 2 - o x o e t h y l ) - 9 H - p u r i n - 6 - y l ) c a r b a m a t e
5% aqueous NaHCO
The organic layer was dried over Na
3
(10 mLꢂ2), H
2
O (10 mLꢂ2) and brine (10 mL).
2
2
SO , filtered, and evaporated.
4
(
11d). Compound 11d was obtained in 45% yield as a white solid,
The desired product was purified with silica gel column chroma-
tography eluted with chloroform/MeOH (95/5).
ꢁ
1
mp 102e104 C. R
400 MHz, CD OD):
f
¼0.50 (chloroform/MeOH¼85/15). H NMR
(
3
d
8.83 (s, 1H), 8.53 (s, 1H), 8.48 (m, 1H, aro-
matic), 7.88 (d, J¼8.7 Hz, 1H, aromatic), 7.73 (d, J¼7.9 Hz, 1H, aro-
4. 3.1. (S)-4-(1-Azido-17-(2-(5-methyl-2, 4-dioxo-3, 4-
dihydropyrimidin-1(2H)-yl)acetamido)-11-oxo-3,6,9-trioxa-12-
azaoctadecanamido)benzene-1-sulfonyl fluoride (1a). Compound 1a
matic), 7.63 (m, 1H, aromatic), 5.25 (d, J¼16.8 Hz, 1H), 5.20 (d,
J¼16.8 Hz, 1H), 4.49 (dd, J¼8.4, 5.5 Hz, 1H,
aeH), 3.06 (m, 2H), 1.91
13
(
(
(
m,1H),1.83 (m,1H),1.52 (m, 4H),1.41 (s, 9H),1.36 (s,18H); C NMR
100 MHz, CD OD): 173.0 (C), 168.5 (C), 158.6 (C), 155.3 (C), 153.1
CH), 151.6 (C), 150.9 (C), 149.3(CH), 141.3 (C), 134.6 (d, J¼24.5 Hz, C),
31.8 (CH), 129.8 (C), 127.6 (CH), 124.5 (CH), 120.0 (CH), 85.4 (C),
0.0 (C), 56.0 (CH), 48.5 (CH ), 41.1 (CH ), 32.9 (CH ), 30.7 (CH ),
8.9 (CH ), 28.1 (CH ), 24.2 (CH ); F NMR (376 MHz, CD OD):
64.9. IR (neat): 3304, 2976, 2930, 1778, 1675, 1603, 1547, 1404,
was obtained in 95% yield as colourless oil, R
f
¼0.45 (chloroform/
1
3
d
MeOH¼85/15). H NMR (400 MHz, acetoneed
6
): 10.36 (s, 1H, NH),
9.96 (s, 1H, NH), 8.16 (d, J¼7.1 Hz, 1H, NH), 8.11 (d, J¼8.8 Hz, 2H,
aromatic), 7.99 (d, J¼8.8 Hz, 2H, aromatic), 7.53 (m, 1H, NH), 7.46 (s,
d
1
8
2
d
2
2
2
2
1H), 4.55 (s, 2H), 4.55 (m, 1H, aeH), 3.96 (s, 2H), 3.69e3.65 (m,
19
3
3
2
3
10H), 3.40 (t, J¼4.8 Hz, 2H), 3.27 (m, 2H), 1.99 (m, 1H), 1.84 (m, 1H),
13
1.81 (s, 3H), 1.55 (m, 2H), 1.44 (m, 2H); C NMR (100 MHz,
acetoneed ): 172.3 (C), 171.1 (C), 168.8 (C), 165.0 (C), 152.6 (C),
ꢀ1
1373, 1281, 1250, 1143, 1107 cm . HRMS calcd for C34
H
48
N
8
O10FS
6
d
þ
(
MþH) 779.3198, found 779.3209.
146.8 (C), 142.9 (CH), 130.6 (CH), 126.7 (d, J¼24.6 Hz, C), 120.7 (CH),
1
10.3 (C), 71.7 (CH
70.6 (CH ), 55.4 (CH), 51.6 (CH
29.9 (CH ), 23.5 (CH ), 12.3 (CH
2
), 71.2 (CH
2
), 71.1 (CH
2
), 71.0 (CH
), 38.6 (CH
); F NMR (376 MHz, acetoneed
2
), 70.9 (CH
2
),
),
):
4.2.5. (S)-tert-Butyl (1-azido-13-((4-(fluorosulfonyl)phenyl)carba-
2
2
), 51.4 (CH
2
2
), 31.7 (CH
2
1
9
moyl)-11-oxo-3,6,9-
trioxa-12-azaheptadecan-17-yl)carbamate
2
2
3
6
(
R
1
12a). Compound 12a was obtained in 63% yield as colourless oil.
d
64.8. IR (neat): 3304, 2925, 2868, 2110, 1665, 1593, 1542, 1409,
1
ꢀ1
NaO10S (MþNa)þ 707.2235,
f
¼0.40 (hexane/EtOAc¼1/1). H NMR (400 MHz, CDCl
3
):
d
9.69 (s,
1214 cm . HRMS calcd for C27
found 707.2218.
H37FN
8
H, NH), 7.87 (d, J¼8.9 Hz, 2H, aromatic), 7.79 (d, J¼8.9 Hz, 2H,
aromatic), 7.55 (d, J¼8.1 Hz, 1H, NH), 4.72 (m, 1H, NH), 4.60 (m, 1H,
a
1
eH), 4.08 (d, J¼15.9 Hz, 1H), 4.04 (d, J¼15.9 Hz, 1H), 3.72e3.62 (m,
4. 3. 2. (S)-3-(1-Azido-17-(2-(5-methyl-2, 4-dioxo-3, 4-
dihydropyrimidin-1(2H)-yl)acetamido)-11- oxo-3,6,9-trioxa-12-
azaoctadecanamido)benzene-1-sulfonyl fluoride (1b). Compound
0H), 3.36 (m, 2H), 3.15 (m, 1H), 3.06 (m, 1H), 1.98 (m, 1H), 1.75 (m,
13
1
H), 1.53 (m, 2H), 1.43 (m, 2H), 1.40 (s, 9H); C NMR (100 MHz,
): 172.2 (C), 171.6 (C), 157.2 (C), 145.7 (C), 130.5 (C), 127.4 (d,
J¼24.7 Hz, C), 120.2 (CH), 79.8 (C), 71.5 (CH
0.7 (CH ), 70.5 (CH ), 70.3 (CH ), 54.0 (CH), 50.9 (CH
1.3 (CH ), 29.5 (CH ), 28.5 (CH
CDCl ): 65.7. IR (neat): 3324, 3283, 2925, 2863, 2105, 1711, 1593,
CDCl
3
d
1b was obtained in 74% yield as colourless oil, R
MeOH¼85/15). H NMR (400 MHz, acetoneed
f
¼0.46 (chloroform/
1
2
), 70.9 (CH
2
), 70.9 (CH
), 40.0 (CH
2
2
),
),
6
): 10.28 (s, 1H, NH),
d
7
3
2
2
2
2
9.82 (s, 1H, NH), 8.71 (s, 1H, aromatic), 8.06 (d, J¼8.2 Hz, 1H, NH),
8.03 (d, J¼7.9 Hz, 1H), 7.76 (d, J¼8.0 Hz, 1H, aromatic), 7.69 (dd,
J¼8.0, 7.9 Hz,1H, aromatic), 7.49 (m, 1H, NH), 7.45 (s, 1H), 4.56e4.50
(m, 3H), 3.96 (s, 2H), 3.69e3.65 (m, 10H), 3.40 (t, J¼4.8 Hz, 2H), 3.28
(m, 2H), 1.97 (m, 1H), 1.81 (s, 3H), 1.79 (m, 1H), 1.56 (m, 2H), 1.50 (m,
19
2
2
3 2
), 22.8 (CH ); F NMR (376 MHz,
3
d
ꢀ
1
1
C
527, 1404, 1368, 1306, 1250, 1209 cm . HRMS calcd for
25
H
39FN
6
O
9
SNa (MþNa)þ 641.2381, found 641.2378.
13
2
6
H); C NMR (100 MHz, acetoneed ): d 171.2 (C), 170.4 (C), 168.1
4
.2.6. (S)-tert-Butyl (1-azido-13-((3-(fluorosulfonyl)phenyl)carba-
(C), 164.5 (C), 151.7 (C), 142.2 (CH), 140.4 (C), 132.8 (d, J¼24.1 Hz, C),
moyl)-11-oxo-3,6,9-trioxa-12-azaheptadecan-17-yl)carbamate
130.7 (CH), 126.5 (CH), 123.0 (CH), 118.2 (CH), 109.4 (C), 78.4 (CH),

Y.-L. Hsu et al. / Tetrahedron 72 (2016) 58e68
65
7
0.8 (CH
2
), 70.3 (CH
2
), 70.2 (CH
), 37.9 (CH
); F NMR (376 MHz, acetoneed
2
), 70.2 (CH
2
), 70.0 (CH
), 29.1 (CH
):
2
), 69.7 (CH
), 22.7 (CH
2
2
),
),
acetoneed
6
):
d
10.14 (s, 1H, NH), 9.98 (s, 1H, NH), 8.62 (m, 1H, ar-
5
4.5 (CH), 50.5 (CH
2
2
), 31.1 (CH
2
2
omatic), 8.01 (d, J¼8.1 Hz, 1H, aromatic), 7.78 (d, J¼8.1 Hz, 1H, ar-
19
11.5 (CH
3
6
d
64.5. IR (neat): 3217,
omatic), 7.71 (dd, J¼8.1, 8.1 Hz, 1H, aromatic), 7.66 (d, J¼8.0 Hz, 1H,
ꢀ
1
3
058, 2930, 2858, 2105, 1685, 1532, 1414, 1245, 1204, 1122 cm
.
NH), 7.52 (m, 1H, NH), 7.37 (d, J¼1.2 Hz, 1H), 4.62 (m, 1H,
aeH), 4.40
þ
HRMS calcd for C27
H
36FN
8
O
10S (MeH) 683.2259, found 683.2250.
(s, 2H), 4.07 (d, J¼15.8 Hz, 1H), 4.02 (d, J¼15.8 Hz, 1H), 3.78e3.68
m, 10H), 3.38 (m, 2H), 3.27 (m, 1H), 3.21 (m, 1H), 1.97 (m, 1H),
(
13
4.3.3. (S)-4-(17-(2-(6-Amino-9H-purin-9-yl)acetamido)-1-azido-11-
1.85e1.76 (m, 4H), 1.55 (m, 2H), 1.48 (m, 2H); C NMR (100 MHz,
DMSOed ): 171.9 (C), 170.0 (C), 167.1 (C), 164.9 (C), 151.5 (C), 142.8
(CH), 140.8 (C), 132.4 (d, J¼23.1 Hz, C), 131.6 (CH), 126.9 (CH), 123.3
(CH), 118.2 (CH), 108.4 (C), 70.8 (CH ), 70.3 (CH ), 70.1 (CH ), 70.1
), 53.4 (CH), 50.4 (CH ), 49.8 (CH ),38.9
), 29.1 (CH ), 23.3 (CH ), 12.3 (CH
): 63.5; IR (KBr): 3263, 3058, 2925, 2105,
oxo-3,6,9-trioxa-12-azaocta- decanamido)benzene-1-sulfonyl fluo-
ride (1c). Compound 1c was obtained in 70% yield as a white solid,
6
d
ꢁ
1
mp 118e119 C.
400 MHz, CD OD):
R
d
f
¼0.53 (chloroform/MeOH¼8/2).
H
NMR
2
2
2
(
3
8.19 (s, 1H), 8.12 (s, 1H), 7.98 (d, J¼9.0 Hz, 2H,
(CH
(CH
2
), 70.1 (CH
2
), 69.7 (CH
2
2
2
1
9
aromatic), 7.92 (d, J¼9.0 Hz, 2H, aromatic), 5.09 (d, J¼16.9 Hz, 1H),
2
), 31.9 (CH
2
2
2
3
); F NMR
5
.04 (d, J¼16.9 Hz, 1H), 4.58 (dd, J¼8.5, 5.4 Hz, 1H,
a
eH), 3.96 (2H),
(376 MHz, acetoneed
6
d
ꢀ
1
3
.65e3.63 (m, 10H), 3.35 (dd, J¼5.1, 4.7 Hz, 2H), 3.28 (m, 2H), 1.92
1680, 1537, 1424, 1245, 1204, 1117 cm
. HRMS calcd for
13
ꢀ
(
m, 1H), 1.81 (m, 1H), 1.59 (m, 2H), 1.48 (m, 2H); C NMR (100 MHz,
27
C H36FN
8
O
10S (MeH) 683.2259, found 683.2271.
CD OD): 173.2 (C), 172.9 (C), 157.4 (C), 154.0 (CH), 151.2 (C),
3
d
1
46.9(C), 143.8 (CH), 131.0 (CH), 128.1 (d, J¼25.0 Hz, C), 121.2 (CH),
4.3.7. (S)-tert-Butyl tert-butoxycarbonyl (9-(1-azido-13-((4-(fluo-
rosulfonyl)phenyl)carbamoyl)-11,19-dioxo-3,6,9-trioxa-12,18-
diazaicosan-20-yl)-9H-purin-6-yl)carbamate (13a). Compound 13a
1
7
3
19.8 (C), 72.2 (CH
1.4 (CH ), 71.2 (CH
), 30.3 (CH
2
), 72.0 (CH
2
), 71.7 (CH
2
), 71.6 (CH
), 46.5 (CH
2
), 71.6 (CH
), 39.6 (CH
2
),
),
2
2
), 56.0 (CH), 51.9 (CH
2
2
2
19
ꢁ
2.8 (CH
2
2
), 24.2 (CH
2
); F NMR (376 MHz, CD
3
OD):
was obtained in 85% yield as a white solid, mp 181ꢀ182 C. R
f
¼0.50
1
d
63.8. IR (KBr): 3314, 2925, 2858, 2100, 1665, 1593, 1532, 1424,
(chloroform/MeOH¼85/15). H NMR (400 MHz, CDCl
3
):
d
9.85 (s,
ꢀ
1
FS (MþH)þ
1245, 1204, 1107 cm . HRMS calcd for C27
H
37
N
11
O
8
1H, NH), 8.77 (s, 1H), 8.26 (s, 1H), 7.81 (d, J¼8.7 Hz, 2H, aromatic),
7.74 (d, J¼8.7 Hz, 2H, aromatic), 7.59 (d, J¼8.1 Hz, 1H, NH), 7.13 (t,
6
94.2531, found 694.2534.
J¼5.2 Hz,1H, NH), 4.88 (s, 2H), 4.62 (m,1H, eH), 4.07 (d, J¼15.8 Hz,
a
4.3.4. (S)-3-(17-(2-(6-Amino-9H-purin-9-yl)acetamido)-1-azido-11-
1H), 4.01 (d, J¼15.8 Hz, 1H), 3.67e3.58 (m, 10H), 3.32 (t, J¼4.8 Hz,
oxo-3,6,9-trioxa-12-azaocta- decanamido)benzene-1-sulfonyl fluo-
ride (1d). Compound 1d was obtained in 51% yield as colourless oil,
R
d
2H), 3.20 (m, 1H), 3.12 (m, 1H), 1.89 (m, 1H), 1.70 (m, 1H), 1.46 (m,
13
3
2H), 1.38 (m, 2H), 1.38 (s, 18H); C NMR (100 MHz, CD OD): d 171.0
1
f
¼0.35 (chloroform/MeOH¼85/15). H NMR (400 MHz, CD
3
OD):
(C), 170.6 (C), 165.8 (C), 153.4 (C), 152.0 (CH), 150.6 (C), 150.0 (C),
8.45 (s,1H, aromatic), 8.19 (s,1H), 8.13 (s,1H), 7.86 (d, J¼8.1 Hz,1H,
146.0 (CH), 144.8 (C), 129.6 (CH), 128.2 (C), 126.6 (d, J¼24.5 Hz, C),
aromatic), 7.71 (d, J¼7.8 Hz,1H, aromatic), 7.60 (dd, J¼8.1, 7.8 Hz,1H,
aromatic), 5.11 (d, J¼17.0 Hz, 1H), 5.05 (d, J¼17.0 Hz, 1H), 4.48 (dd,
119.6 (CH), 84.1 (C), 71.1 (CH
2
), 70.4 (CH
2
), 70.4 (CH ), 70.2 (CH
2
), 45.9 (CH ), 39.2 (CH
2
2
),
),
70.1 (CH
31.4 (CH
CDCl ):
2
), 69.8 (CH
), 28.3 (CH
63.9; IR (KBr): 3304, 2925, 2100, 1783, 1665, 1583, 1409,
2
), 53.3 (CH), 50.5 (CH
2
2
19
1
H,
a
eH), 3.96 (s, 2H), 3.63e3.61 (m, 10H), 3.34 (m, 2H), 3.26 (m,
2
2
), 27.6 (CH ), 22.6 (CH
3
2
); F NMR (376 MHz,
13
2
H), 1.91 (m, 1H), 1.81 (m, 1H), 1.56 (m, 2H), 1.46 (m, 2H); C NMR
OD): 173.0 (C), 172.8 (C), 169.1 (C), 157.3 (C), 153.9
CH), 151.1(C), 143.7 (CH), 141.3 (C), 134.6 (d, J¼24.4 Hz, C), 131.7
CH), 127.8 (CH), 124.5 (CH), 120.1 (CH), 119.7 (C), 72.0 (CH ), 71.6
), 55.9 (CH), 51.8
3
d
ꢀ
1
ꢀ
(
(
(
(
(
100 MHz, CD
3
d
1250, 1209, 1112 cm . HRMS calcd for C37
892.3423, found 892.3428.
51 11
H N O12FS (MeH)
2
CH
CH
2
2
), 71.5 (CH
), 46.5 (CH
2
), 71.5 (CH ), 71.3 (CH
), 39.6 (CH ), 32.7 (CH
OD): d 62.6. IR (neat): 3401, 3078, 2925, 2868,
2
2
), 71.1 (CH
2
4.3.8. (S)-tert-Butyl tert-butoxycarbonyl (9-(1-azido-13-((3-(fluo-
rosulfonyl)phenyl)carbamoyl)- 11,19-dioxo-3,6,9-trioxa-12,18-
diazaicosan-20-yl)-9H-purin-6-yl)carbamate (13b). Compound 13b
19
2
2
2
), 30.2 (CH
2
), 24.1 (CH
2
);
F
NMR (376 MHz, CD
3
ꢀ1
2110, 1685, 1598, 1542, 1475, 1424, 1204, 1127, 1091 cm . HRMS
was obtained in 73% yield as colourless oil, R
f
¼0.36 (chloroform/
9.75 (s, 1H, NH), 8.64
s, 1H), 8.35 (s, 1H), 8.29 (s, 1H, aromatic), 8.25 (s, 1H), 7.92 (d,
FS (MþH)þ 694.2531, found 694.2532.
MeOH¼85/15). H NMR (400 MHz, CDCl
1
calcd for C27
H
37
N
11
O
8
3
): d
(
4
.3.5. (S)-4-(14-Azido-2-(4-(2-(5-methyl-2,4-dioxo-3,4-
J¼8.3 Hz, 1H, aromatic), 7.64 (d, J¼6.8 Hz, 1H, aromatic), 7.48 (dd,
dihydropyrimidin-1(2H)-yl)acetamido) butyl)-4-oxo-6,9,12-trioxa-3-
azatetradecanamido)benzene-1-sulfonyl fluoride (2a). Compound
2
R
DMSOed
1
J¼8.3, 6.8 Hz, 1H, aromatic), 7.00 (t, J¼5.6 Hz, 1H, NH), 4.89 (s, 2H),
4.67 (m, 1H,
a
eH), 4.11 (d, J¼15.9 Hz, 1H), 4.04 (d, J¼15.9 Hz, 1H),
ꢁ
a was obtained in 80% yield as a white solid, mp 177e178 C.
3.70e3.61 (m, 10H), 3.34 (t, J¼4.9 Hz, 2H), 3.18 (m, 2H), 1.93 (m, 1H),
1
13
f
¼0.36 (chloroform/MeOH¼85/15).
):
11.25 (s, 1H, NH), 10.86 (s, 1H, NH), 8.15 (t, J¼5.3 Hz,
H, NH), 8.07 (d, J¼8.9 Hz, 2H, aromatic), 7.98 (d, J¼8.9 Hz, 2H,
eH),
.25 (s, 2H), 3.97 (s, 2H), 3.66e3.57 (m, 10H), 3.37 (dd, J¼4.9, 4.7 Hz,
H), 3.05 (m, 2H), 1.74 (s, 3H), 1.74 (m, 2H), 1.43 (m, 2H), 1.34 (m,
H
NMR (400 MHz,
1.72 (m, 1H), 1.51 (m, 2H), 1.42 (m, 2H), 1.42 (s, 18H); C NMR
6
d
3
(100 MHz, CDCl ): d 170.9 (C), 170.3 (C), 165.8 (C), 153.4 (C), 151.9
(CH), 150.5 (C), 149.9 (C), 146.0 (CH), 139.6 (C), 133.1 (d, J¼24.2 Hz,
aromatic), 7.89 (d, J¼7.8 Hz, 1H, NH), 7.40 (s, 1H), 4.50 (m, 1H,
a
C), 130.1 (CH), 128.1 (CH), 126.0 (CH), 123.1 (CH), 118.8 (C), 83.9 (C),
4
2
2
71.0 (CH
53.1 (CH), 50.5 (CH
27.6 (CH ), 22.6 (CH
2
), 70.4 (CH
2
), 70.4 (CH
2
), 70.4 (CH
), 39.2 (CH
); F NMR (376 MHz, CDCl
2
), 70.1 (CH
), 31.8 (CH
):
2
), 69.8 (CH
), 28.3 (CH
64.5; IR (neat):
2
),
),
2
), 45.8 (CH
2
2
2
2
13
19
H); C NMR (100 MHz, DMSOed
64.6 (C), 151.1 (C), 146.0 (C), 142.5 (CH), 130.1 (CH), 124.7 (d,
), 69.9 (CH ), 69.7
), 53.2 (CH), 50.1 (CH ), 49.4
), 22.9 (CH ), 12.0(CH
66.51. IR (KBr): 3273, 3063, 2935,
6
):
d
171.9 (C), 169.7 (C), 166.8 (C),
3
2
3
d
1
3268, 2930, 2100, 1783, 1670, 1588, 1527, 1404, 1281, 1250, 1204,
ꢀ
1
þ
J¼23.4 Hz, C), 119.7 (CH), 108.0 (C), 70.4 (CH
2
2
1178, 1112 cm . HRMS calcd for C37
found 894.3581.
H
53
11
N O
12FS (MþH) 894.3580,
(
CH
2
), 69.7 (CH
), 38.6 (CH
2
), 69.7 (CH
2
), 69.3 (CH
), 28.7 (CH
):
100, 1690, 1537, 1475, 1409, 1245, 1204, 1096 cm . HRMS calcd for
2
2
19
(
CH
2
2
), 31.5 (CH
2
2
2
3
);
F
NMR (376 MHz, DMSOed
2
6
d
4.3.9. (S)-4-(2-(4-(2-(6-Amino-9H-purin-9-yl)acetamido)butyl)-14-
azido-4-oxo-6,9,12-trioxa- 3-azatetradecanamido)benzene-1-
sulfonyl fluoride (2c). To a solution of compound 13a (147.0 mg,
ꢀ
1
ꢀ
C
27
H
36
N
8
O
10FS (MeH) 683.2259, found 683.2250.
2 2
0.164 mmol) in 1.1 mL CH Cl was added TFA (0.5 mL). The reaction
4
.3.6. (S)-3-(14-Azido-2-(4-(2-(5-methyl-2,4-dioxo-3,4-
mixture was stirred at rt for 30 min. Toluene (1 mL) was added and
the mixture was evaporated to dryness under vacuum to yield
dihydropyrimidin-1(2H)-yl)acetamido) butyl)-4-oxo-6,9,12-trioxa-3-
azatetradecanamido)benzene-1-sulfonyl fluoride (2b). Compound
compound 2c (93.3 mg, 82%) as colourless oil. R
f
¼0.30 (chloroform/
8.20 (s, 1H), 8.11 (s,
1H), 7.99 (d, J¼9.0 Hz, 2H, aromatic), 7.93 (d, J¼9.0 Hz, 2H,
ꢁ
1
2
b was obtained in 82% yield as a white solid, mp 147ꢀ148 C.
MeOH¼85/15). H NMR (400 MHz, CD
3
OD): d
1
R
f
¼0.40 (chloroform/MeOH¼85/15).
H
NMR (400 MHz,

66
Y.-L. Hsu et al. / Tetrahedron 72 (2016) 58e68
aromatic), 4.94 (s, 2H), 4.58 (dd, J¼8.8, 5.2 Hz, 1H,
a
eH), 4.07 (s,
(50 mL, ꢂ3), 5% NaHCO
(50 mL, ꢂ2). The organic phase was dried over anhydrous Na
filtered and concentrated. Purification by silica gel column chro-
matography (eluent: 100e90% CHCl /MeOH) provided the desired
product 15 (2.4 g, 76%) as a pale yellow oil. R
¼0.44 (CHCl
6.44 (br, 1H, NH), 5.12 (br,
3
(50 mL, ꢂ3), H
2
O (50 mL, ꢂ2), and brine
2
H), 3.76e3.64 (m, 10H), 3.35 (dd, J¼5.0, 4.8 Hz, 2H), 3.25 (m, 2H),
2
SO ,
4
13
1.94 (m, 1H), 1.81 (m, 1H), 1.60 (m, 2H), 1.49 (m, 2H); C NMR
(
(
100 MHz, CD OD): 172.9 (C), 172.9 (C), 168.6 (C), 157.3 (C), 153.9
3
d
3
CH), 151.0 (C), 146.8 (C), 143.6 (CH), 130.9 (CH), 127.9 (d, J¼24.9 Hz,
f
3
/
1
C),121.0 (CH),119.7 (C), 72.1 (CH
1.1 (CH ), 71.1 (CH ), 54.9 (CH), 51.7 (CH
), 29.8 (CH
2
), 71.6 (CH
), 46.5 (CH
); F NMR (376 MHz, CD
2
), 71.4 (CH
2
), 71.4 (CH
2
),
),
MeOH¼9/1). H NMR (400 MHz, CDCl
3
): d
7
3
2
2
2
2
), 40.3 (CH
2
1H, NH), 3.52e3.34 (m, 8H), 3.25 (q, J¼5.4 Hz, 2H), 3.12 (d, J¼4.9 Hz,
19
2.8 (CH
2
2
), 24.1 (CH
2
3
OD):
2H), 2.08e1.96 (m, 4H), 1.81 (t, J¼2.5 Hz, 1H), 1.62e1.50 (m, 2H),
1
3
d
64.8. IR (neat): 3314, 3073, 2930, 2110, 1690, 1598, 1537, 1470,
1.44e1.30 (m, 2H), 1.26 (s, 9H); C NMR (100 MHz, CDCl
3
):
), 69.8 (CH ), 69.8 (CH
2
), 35.4 (CH ), 28.0 (CH
d 172.6
ꢀ1
FS (MþH)þ
1
424, 1204, 1132 cm . HRMS calcd for C27
H
37
N
11
O
8
(C), 155.7 (C), 83.7 (C), 78.8 (C), 69.8 (CH
69.5 (CH ), 68.4 (CH), 39.9 (CH ), 38.8 (CH
7.6 (CH ), 24.4 (CH ), 17.8 (CH
2
2
2
),
),
6
94.2531, found 694.2534.
2
2
2
3
2
2
2
2
). IR (neat): 3301, 3083, 2933, 2869,
ꢀ
1
4
.3.10. (S)-3-(2-(4-(2-(6-Amino-9H-purin-9-yl)acetamido)butyl)-
2116, 1695, 1651, 1455, 1392, 1366, 1279, 1251, 1171, 863, 638 cm .
14-azido-4-oxo-6,9,12-trioxa-3-azatetradecanamido)benzene-1-
ESI-MS calcd for C18
33
H N
2
O
5
(MþH)þ 357.2389, found 357.2390.
sulfonyl fluoride (2d). Compound 2d was prepared with the same
procedure as described for compound 2c, except compound 13b
was used as the starting material instead of 13a. Compound 2d was
4.4.2. N-(6-(Diethylamino)-9-(2-(4-(4,15-dioxo-8,11-dioxa-5,14-
diazahenicos-20-yn-1-oyl) piperazine-1-carbonyl)phenyl)-3H-
obtained in 80% yield as colourless oil, R
f
¼0.33 (chloroform/
): 10.38 (s, 1H, NH),
.64 (d, J¼1.5 Hz, 1H), 8.35e8.25 (m, 3H), 8.00 (m, 1H, aromatic),
.90 (s, 2H, NH), 7.75 (d, J¼7.5 Hz, 1H, aromatic), 7.69 (m, 1H), 5.04
s, 2H), 4.70 (m, 1H, eH), 4.08 (s, 2H), 3.73e3.65 (m, 10H), 3.39 (t,
J¼4.9 Hz, 2H), 3.25 (m, 2H), 1.97 (m, 1H), 1.81 (m, 1H), 1.56 (m, 2H),
.48 (m, 2H); 13C NMR (100 MHz, CD
OD): 172.9 (C), 172.9 (C),
68.1 (C), 153.5 (C), 150.8 (C), 148.1(CH), 145.6 (CH), 141.3 (C), 134.5
d, J¼24.3 Hz, C), 131.7 (CH), 127.7 (CH), 124.5 (CH), 120.0 (CH), 119.5
C), 72.1 (CH ), 71.6 (CH ), 71.4 (CH ), 71.4 (CH ), 71.1 (CH ), 71.0
), 51.7 (CH ), 46.9 (CH ), 40.4 (CH ), 32.9 (CH ), 29.9 (CH ), 24.2
); F NMR (376 MHz, CD OD): 62.7. IR (neat): 3309, 2919,
xanthen-3-ylidene)-N-ethylethanaminium chloride (7). The reaction
mixture containing compound 15 (125.9 mg, 0.35 mmol) in 3 mL of
20% TFA/CH Cl was stirred at rt for 1 h. It was then concentrated to
2 2
1
MeOH¼85/15). H NMR (400 MHz, acetoneed
6
d
8
7
dryness under reduced pressure and kept under high vacuum for
3 h to remove the residual TFA. The residue was dissolved in DMF
(
a
41
(1 mL) and the solution was added compound 16 (180 mg,
0.29 mmol), HOBt (19.9 mg, 0.15 mmol) and TEA (331.3 L,
1
3
d
m
1
2.32 mmol). The mixture was cooled in ice bath for 10 min, fol-
lowed by addition of a solution of DCC (72.9 mg, 0.35 mmol) in DMF
(2 mL). The reaction mixture was stirred at rt for 22 h under Ar
atmosphere. The organic solvent was removed, and the residue
obtained was purified with size-exclusion chromatography over
Sephadex LH-20 (eluent: MeOH). The desired product 7 (220.0 mg)
(
(
(
(
2
2
2
2
2
CH
CH
2
2
2
2
2
2
2
19
3
d
ꢀ1
2
858, 2095, 1670, 1598, 1542, 1419, 1245, 1209, 1107, cm . HRMS
calcd for C27
H
37
N
11
O
8
FS (MþH)þ 694.2531, found 694.2526.
was obtained as a purple red oil in 71% yield over two steps, R
f
¼0.51
7.88e7.72 (m,
H), 7.62e7.54 (m, 1H), 7.33 (d, J¼9.5 Hz, 2H), 7.12 (dd, J¼9.5, 1.9 Hz,
H), 7.01 (d, J¼2.4 Hz, 2H), 3.73 (q, J¼7.0 Hz, 8H), 3.65 (s, 4H),
1
(
CHCl
3
/MeOH¼8/2). H NMR (400 MHz, CDCl
3
): d
3
2
4
.4. Synthesis of the click reagent 7
4
.4.1. tert-Butyl (2-(2-(2-(hept-6-ynamido)ethoxy)ethoxy)ethyl)car-
3.62e3.50 (m, 4H), 3.50e3.36 (m, 12H), 2.66 (t, J¼6.6 Hz, 2H), 2.52
(t, J¼6.6 Hz, 2H), 2.30e2.16 (m, 5H), 1.80e1.68 (m, 2H), 1.62e1.48
4
0
bamate (15). To an ice-cooled solution of compound 14 (2.2 g,
8
3
slowly added a solution of DCC (2.0 g, 9.51 mmol) in DMF (20 mL).
The reaction mixture was stirred at rt for 22 h. It was then con-
centrated to dryness under vacuum. The residue was dissolved in
13
.72 mmol), 6-heptynoic acid (1.0 g, 7.93 mmol), HOBt (535.4 mg,
.96 mmol) and TEA (8.9 mL, 63.44 mmol) in DMF (60 mL) was
(m, 2H), 1.35 (t, J¼7.0 Hz, 12H); C NMR (100 MHz, CDCl
3
): d 175.9
(C), 175.9 (C), 174.7 (C), 172.7 (C), 169.5 (C), 159.3 (C), 157.2 (C), 157.0
(C),136.5 (C),133.2 (CH),132.2 (C),131.8 (CH),131.3 (CH),128.9 (CH),
115.4 (CH), 114.8 (C), 97.3 (CH), 84.7 (C), 71.3 (CH
2
), 71.3 (CH
), 43.0 (CH
2
), 70.6
), 42.2
(CH
(CH
2
), 70.6 (CH
), 40.3 (CH
2
), 69.9 (CH), 46.9 (CH
), 36.4 (CH ), 31.5 (CH
2
), 46.6 (CH
), 29.2 (CH
2
2
CH
2
Cl
2
(200 mL), and the solution was washed with 5% citric acid
2
2
2
2
2
), 29.0 (CH
2
), 26.1

Y.-L. Hsu et al. / Tetrahedron 72 (2016) 58e68
67
(
CH
2
), 18.8 (CH
2
), 12.8 (CH
3
). IR (neat): 3417, 3297, 2971, 2920, 1633,
4.9. Labelling and detection of intracellular kinase
1
6
8
588, 1468, 1414, 1339, 1180, 1274, 1247, 1132, 1075, 1007, 922, 822,
ꢀ1
(MþH)þ 849.4915, found
83 cm . ESI-MS calcd for C49
49.4917.
H
65
N
6
O
7
Probe 2b (100
m
M) was added directly to the RK3E and RK3E-v-
Src cells and incubated with for 1 h. The cells were then washed
with PBS and then lysed in RIPA lysis buffer (50 mM TriseHCl pH
7
.5, 1% NP-40, 150 mM NaCl, 1 mM PMSF, and 1ꢂprotease inhibitors
cocktail (Calbiochem)) to prepare total cell extract. Total cell ex-
tracts, 500 g, were then coupled to compound 7 by click chemistry,
4
.5. Cell lines
m
Rat kidney epithelial cell RK3E expressing v-Src, RK3E-v-Src,
and separated by a 10% SDS-polyacrylamide gel. The gel was then
subjected to fluorescence scanning analysis followed by Coomassie
blue staining of the gel.
were used in this study. This cell was maintained in Dulbecco’s
modified Eagle’s medium containing 10% foetal bovine serum, 100
units/mL penicillin, and 100 mg/mL streptomycin in a 5% CO hu-
2
ꢁ
42
midified incubator at 37 C.
4.10. Immuno-detection of probe-labelled protein
4
.6. Cloning, expression, and purification of the catalytic do-
As above, the probe-treated RK3E-v-Src cells were lysed to
mains of Fak and Lck
prepare extracts and then immunoprecipitated with anti-Src anti-
body (EC10, mouse monoclonal antibody, purchased from Merck
Millipore). The precipitated proteins were coupled to compound 7
using click chemistry, separated by a 10% SDS-polyacrylamide gel
and then analyzed by fluorescence scanning analysis and Coo-
massie blue staining of the gel.
The cDNA encoding the catalytic domains of human Fak (resi-
dues 411e686), Lck (residues 225e509), and Src (residues
53e535) were amplified from cDNA clone and then expressed in E.
coli BL21(DE3) as 6-His tagged recombinant proteins. To isolate
recombinant proteins, culture of E. coli harbouring the expression
2
plasmid was grown in LB2 medium with 50 mg/mL ampicillin at
ꢁ
2
5 C to OD600¼0.6. At this point, IPTG was added to a final con-
4.11. Src kinase activity measurement
ꢁ
centration of 1 mM and the cells were allowed to grow at 25 C for
an additional 3 h before harvesting by centrifugation. Cells were
resuspended in guanidineeHCl buffer (6 M guanidineeHCl, 0.1 M
One
m
g purified Src protein was incubated with varying amounts
ꢁ
of PP2 (Sigma, P0042) at 25 C for 30 min. The kinase activities were
then analyzed using an ADP-GloÔ kinase activity assay kit
(Promega).
2 4
NaH PO , 0.01 M Tris, pH 8.0) and followed by gentle shaking for
ꢁ
1
1
h at 4 C. The suspension was then centrifuged at 13,000 g for
ꢁ
5 min at 4 C. The clear supernatant was collected and applied to
a Ni SepharoseÔ column (GE-Amersham Biosciences Ltd) pre-
viously equilibrated with guanidineeHCl buffer. The column was
washed and the bound protein was eluted by guanidineeHCl buffer
containing 200 mM imidazole. To renature the purified protein,
protein eluted from column was diluted to 50 mg/mL using gua-
nidineeHCl buffer and dialyzed against renaturation buffer using
tubing with molecular weight cutoff 12,000 (Sigma) (100 mM Tris,
Acknowledgements
This research was supported by National Science Council Taiwan
and National Health Research Institutes grants (NSC 100-2311-B-
002-016, 101-2311-B-002-020-MY3, NHRI-EX101-10050SI to J.-J.L.
and, NSC 101-2113-M-002-007, NSC 102-2113-M-002-004-MY3 to
L.-C.L.).
pH 8.0, 2 mM EDTA, 3 mM DTT, 0.4 M
L
-arginine, 50% glycerol) at
ꢁ
4
C for 12e16 h. Purified protein was dialyzed against storage
Supplementary data
buffer (50 mM Tris pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM DTT, 50%
glycerol), aliquoted, and frozen by dry ice-ethanol bath. The kinase
activities of these renatured proteins were analyzed using an ADP-
GloÔ kinase activity assay kit (Promega).
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.tet.2015.10.053. These data in-
clude MOL files and InChiKeys of the most important compounds
described in this article.
4
.7. Other recombinant proteins
References and notes
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g
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