Developing new chemical tools for DNA methyltransferase 1 (DNMT 1): A small-molecule activity-based probe and novel tetrazole-containing inhibitors

Article abstract of DOI:10.1016/j.bmc.2015.03.006

DNA methylation is an important epigenetic modification catalyzed by DNA methyltransferases (DNMTs). Abnormal expression of endogenous DNMTs in human causes alterations in the genome methylation patterns which subsequently lead to the development of cance

Full text of DOI:10.1016/j.bmc.2015.03.006

Accepted Manuscript
Developing new chemical tools for DNA methyltransferase 1 (DNMT 1): a
small-molecule activity-based probe and novel tetrazole-containing inhibitors
Biwei Zhu, Jingyan Ge, Shao Q. Yao
PII:
S0968-0896(15)00171-6
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.03.006
BMC 12130
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Bioorganic & Medicinal Chemistry
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21 January 2015
25 February 2015
2 March 2015
Please cite this article as: Zhu, B., Ge, J., Yao, S.Q., Developing new chemical tools for DNA methyltransferase 1
(DNMT 1): a small-molecule activity-based probe and novel tetrazole-containing inhibitors, Bioorganic &
Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.03.006
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small-molecule activity-based probe and novel
tetrazole-containing inhibitors
Biwei Zhu, Jingyan Ge and Shao Q. Yao*
Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543

Developing new chemical tools for DNA methyltransferase 1
(DNMT 1): a
small-molecule activity-based probe and novel tetrazole-
containing inhibitors
Biwei Zhu, Jingyan Ge and Shao Q. Yao*
Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543
*Corresponding author (SQY)
Prof Shao Q Yao
Department of Chemistry
National University of Singapore
3 Science Drive 3
Singapore 117543
Tel: (65) 65162925
Fax: (65) 67791691
Email: chmyaosq@nus.edu.sg

Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com
Developing new chemical tools for DNA methyltransferase 1 (DNMT 1): a small-
molecule activity-based probe and novel tetrazole-containing inhibitors
Biwei Zhu, Jingyan Ge and Shao Q. Yao*
aDepartment of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
DNA methylation is an important epigenetic modification catalyzed by DNA
methyltransferases (DNMTs). Abnormal expression of endogenous DNMTs in human
causes alterations in the genome methylation patterns which subsequently lead to the
development of cancers. Thus detection of endogenous DNMT activities and efficient
inhibition of DNMTs have important therapeutic significance. In this work, a small
molecule activity-based probe (ABP) of DNA methyltransferase 1 (DNMT1), T1, was
developed. The probe was a clickable analog of tryptophan and was able to covalently
label endogenous DNMT1 and inhibit its enzymatic activity more effectively than
previously known DNMT1 inhibitors (RG108 and its maleimide analogue 1149). In
addition, we also discovered a new type of small molecule DNMT inhibitors based on
tetrazole-containing compounds which were analogs of 1149. Amongst these
compounds, which we called Gn, one of them (G6) possessed reasonable inhibitory
Available online
Keywords:
DNA methylation
DNMT1 inhibitors
Activity-based probes
Tetrazoles
activity against DNMT1 in both in vitro enzymatic assays and
cell growth
proliferation experiments. Both T1 and G6 showed effective labeling of endogenous
DNMT1 from mammalian cells by using in vitro competitive pull-down and live-cell
bioimaging experiments.
2014 Elsevier Ltd. All rights reserved.
1. Introduction
hemi-methylated DNA and helps to maintain the methylation
patterns of DNA. DNMT3 is present in small quantities in
DNA methylation is an epigenetic modification that leads to the
addition of a methyl group at the C-5 position of cytosine
residues of DNA without interfering with usual base pairings.
The process prevents gene expression, stabilizes DNA-protein
complexes and promotes normal DNA expression.1-3 In
mammalian genes, the principal targets of DNA methylation are
CpG sequences, ~60-90% of which have been methylated.
However, there are some unmethylated regions called “CpG
islands” which occur predominantly in the promoters of
mammalian genes. Hypermethylation of CpG islands is regarded
as a common character of cancer cells because it will cause the
silencing of tumor suppressor genes. Failure in maintaining
normal DNA methylation patterns is associated with mis-
expression of certain proteins, which may lead to cancers or other
diseases. The abnormal DNA methylation has become an
interesting and promising cancer therapeutic target. 4-6
somatic cells and has no differential affinity between
unmethylated and hemi-methylated CpG sites, whose main role
is in de novo methylation. The catalytic mechanism of DNMTs
has been studied extensively (shown in Fig. 1A).12 During the
methylation processes, DNMTs first bind to the C-6 position of
cytosine residues of DNA, then transfer a methyl group from S-
adenosyl methionine (SAM; a methyl group donor) to the C-5 of
cytosine, followed by the concomitant release of S-adenosyl-L-
homocysteine (SAH). Subsequently, the proton at the C-5
position is removed and the free enzyme is released via -
elimination to generate the methylated cytosine.
Since the alteration in genome methylation and mis-DNA
methylation are caused by the mis-expression of DNMTs, based
on the catalytic mechanism of these enzymes, many small
molecule DNMT inhibitors have been developed, including
nucleoside analogues (5-Azacytidine and Zebularine), non-
nucleoside analogues (Procaine, Procainamide, Hydralazine,
Curcumin and RG108) and other SAH analogues.5,11,13 However,
their relatively poor inhibitory activities towards DNMTs
coupled with significant cytotoxicity make such compounds
DNA methylation is catalyzed by DNA methyltransferases
(DNMTs). This enzyme family mainly contains two categories,
including the maintain-type DNMT1 and the de novo-type
DNMT3.1,7-11 DNMT1 is the most abundant enzyme of human
DNMTs expressed in somatic cells, which has high affinity for

Figure. 1. A) Schematic showing DNA methyltransferases (DNMTs) catalyze DNA methylation. SAM acts as the methyl group donor; B)
Overall strategy of our probe (T1) and the inhibitor library (Gn) derived from known DNMT1 inhibitors 1149 and RG108; C) Workflow of
ABPP and the corresponding competitive ABPP technology used in the current study; D) Structures of the two different azide reporters used in
the present study.
unsuitable as potential therapeutic agents. New potent and highly
selective DNMT inhibitors are urgently needed.
bioactive compounds and their cellular mechanism-of-action can
now be investigated at the earliest stages of drug development,
thus potentially providing a better success rate in subsequent
clinical trials.15 These so-called “in situ drug profiling” strategies
have in recent years been successfully applied to a number of
biologically important drug candidates, including the FDA-
approved anti-obesity drug Orlistat,19 several anti-cancer small
molecule kinase inhibitors,20-24 and the newly developed
epigenetic protein-protein interaction (PPI) inhibitor JQ-1.25
Activity-based protein profiling (ABPP) technology has become
an important method for cell-based proteome profiling. Recently,
its corresponding probes, activity-based probes (ABPs) and
affinity-based probes (AfBPs) have also become promising
chemical tools that enable reliable and comprehensive
identification of potential “on” and “off” cellular targets of drug
candidates.14-18 With such probes, proteome-wide assessment of

Herein, we extend this approach to the study of DNMTs (Fig.
1B-D). Inspired by two known DNMT1 inhibitors 1149 and
RG108,26-28 two types of activity-based probes T1 and Gn were
designed and synthesized (Fig. 1B). To design these compounds
as potential covalent DNMT inhibitors, the maleic moiety in
1149 was strategically retained, which should result in covalent
interaction between the compound and the active-site cysteine
residue in DNMT1. In addition, a terminal alkyne and a tetrazole
moiety were introduced into T1 and Gn, respectively, which
would serve as click handles for reaction with azide- and alkene-
containing reporters under copper (I)-catalyzed azide-alkyne
[3+2] cycloaddition (CuAAC) conditions,29-32 and light-induced
tetrazole-alkene-1,3-dipolar cycloaddition (LITAC) conditions,
respectively (Fig. 1C & 1D).33-35 In the case of Gn, we envisaged
that the tetrazole moiety not only served as a well-known “photo-
click” handle, it could also be an effective aromatic mimetic to
introduce compound diversity (i.e. G1 to G12; see Table 1). The
tryptophan analogue probe T1 was designed via conversion of a
known DNMT1 inhibitor, 1149. The chemical structures of the
two previously reported, azide-containing reporters are shown in
Figure 1D (TER-PEG-N3 & TER-Biotin-N3), and would be used
to “click” to T1-labeled mammalian proteomes for subsequent
in-gel fluorescence scanning, pull-down (PD)/Western blotting
(WB) and bioimaging experiments.
Scheme 1. Chemical synthesis of A) T1 and B) Gn library

o
2. Results and discussion
pyridine and cooled in an ice/NaCl bath to -10 ~ -20 C for 20
min, then used to react with the above-described, freshly
prepared diazonium salt (Reaction A). The resulting mixtures
were allowed to warm to room temperature, thus affording the
corresponding tetrazole compounds 2dn in acceptable yields (24-
89%). Subsequently, the Cbz group in each compound was
removed by H2/Pd-C treatment to afford compound 2en. Finally,
further reaction of the primary amine in each compound with
maleic anhydride in acetic anhydride/sodium acetate under
refluxing conditions delivered the desired products Gn in
acceptable yields (9-52% over two steps). The 12 analogues of
Gn (n= 1-12) were so chosen based on their structural diversity
and the commercial availability of their aldehyde precursors, thus
delivering a variety of diverse aromatic groups linked to the core
inhibitor structure via the centrally located tetrazole moiety (see
summary Table in Scheme 1B).
2.1. Chemistry
2.1.1 Synthesis of activity-based probe (T1)
Probe T1 was a derivative of 1149, containing an additional
small alkyne handle at the 5-position of the tryptophan indole
ring. The probe was synthesized in five steps from the
commercially available L-5-hydroxytryptophan as shown in
Scheme 1A. Briefly, both the carboxylic acid and the primary
amine group in L-5-hydroxytryptophan were first protected with
a methyl and a tert-butyloxycarbonyl group, respectively. The
resulted compound 1a was reacted with propargyl bromide under
basic conditions to generate compound 1b (99.0% yield).
Deprotection of the Boc group by TFA gave the free primary
amine which was further reacted with maleic anhydride under
refluxing conditions to afford the final probe T1 in 31.0% yield
(two steps).
2.2. Biological evaluation
With all compounds in hand, we next confirmed the biological
properties of these two types of probes by side-by-side
comparison with known DNMT inhibitors. As earlier mentioned,
for the 12 Gn compounds, they were initially designed as
covalent modifiers of DNMT1 (via the maleimide moiety). Upon
covalent reaction with endogenous DNMT1, we had planned to
subsequently carry out LITAC reaction with a suitable reporter
tag to detect the probe-bound DNMT1 by taking advantage of the
presumably bioorthogonal nature of the photo-induced tetrazole-
alkene ligation reaction. To our great surprise, we found this
reaction was not as “bioorthogonal” as expected (Fig. S1);33-35 by
taking G6 as a model substrate, and UV-irradiated it for 2 min
following published conditions,36 we found the compound rapidly
reacted with a variety of biological nucleophiles including acids,
amines, thiols and others, yielding highly fluorescent products
which were distinctly different from the nitrile imine
2.1.2 Synthesis of tetrazole-based library (Gn)
The 12 tetrazole-based compounds Gn (n = 1-12) were
synthesized in several steps as shown in Scheme 1B. Starting
from the commercially available 4-nitro-3-phenyl-L-alanine, the
carboxylic acid moiety was protected by a methyl group,
followed by amine protection to give the corresponding Cbz-
protected methyl ester, 2a. Subsequently, the nitro group in 2a
was reduced by tin (II) chloride under acidic conditions to give
2b in 81.3% yield. Next, the diazonium salt was prepared by
slowly adding a cold NaNO2 solution into a cold solution of 2b
(dissolved in 1:1 ethanol/water). Independently, 12 commercially
available common aldehydes, N1-12, were each individually mixed
with benzenesulfonyl hydrazide in ethanol and stirred at room
temperature. Upon solvent evaporation, the corresponding 2cn (n
= 1-12) were obtained. Subsequently, they were re-dissolved in
Figure. 2. A) Left: dose-dependent inhibition assay of T1, Gn as well as the positive controls (RG108 and 1149) against recombinant
DNMT1; Right: Inhibition assay of different inhibitors at 2 M; B) GI50 plot of several inhibitors against MCF-7 cells; C) Fluorescence-
activated cell sorting (FACS) results of MCF-7 cells treated with 10 M probes (T1, G6, 1149 and RG108); D) Protein profiling results of
MCF-7 cells treated with T1 (10 M)/competitors (G6, 1149 & RG108; 50, 100 & 500 M); minus sign (-): cells treated with DMSO
(negative control); E) competitive PD/WB results of T1 against DNMT1. The size of DNMT1 is around 183 kDa. MCF-7 cells were treated
with T1 (10 M) and competitors including G6, 1149 & RG108 (50, 100 & 500 M); the minus sign (-) means cells were treated with DMSO
(negative control).

of cancer cells by 50%. In this study, GI50 values of selected
compounds, together with those of the two known inhibitors
(RG108 and 1149) were subsequently determined and
summarized in Table 1. According to the results, it was obvious
that T1 showed better inhibition than both 1149 and RG108. The
results once again confirmed that the tetrazole-based compounds
had reasonable GI50 against MCF-7. G6, the smallest-size
tetrazole amongst the 12 compounds, showed the best inhibition
efficiency which was consistent with the earlier in vitro DNMT1
enzyme inhibition results. The good inhibition potency of T1 and
G6 against MCF-7 was further determined by Fluorescence-
Activated Cell Sorting (FACS) experiments (Fig. 2C); by using a
BD Pharmingen TM FITC Annexin V Apoptosis Detection Kit I to
stain the inhibitor-treated cells with FITC Annexin V (detecting
apoptosis) and propidium iodide (PI; detecting cell death), the
fluorescence signals were quantified. Based on the data
histogram, it was observed that after treating live MCF-7 cells
with inhibitors at a concentration of 10 μM, both T1 and G6
showed a noticeable increase in the number of Annexin V
labelled cells (Fig. 2C). From the statistics values, T1 caused
61~62% cell apoptosis and death; the value was higher than that
of the known DNMT1 inhibitor RG108. These results thus
indicate that both T1 and G6 indeed were relatively potent and
novel DNMT1 inhibitors in causing mammalian cancer cell death
and apoptosis.
Table 1. GI values of all the probes/inhibitors
50
GI /M
GI /M
Probe
T1
Probe
G6
G7
G8
G9
10
50
50
0.29 ± 0.07
7.31 ± 0.06
127.90 ± 0.07
0.38 ± 0.06
1.16 ± 0.07
0.48 ± 0.06
0.87 ± 0.06
1.08 ± 0.06
0.30 ± 0.06
1.10 ± 0.06
6.15 ± 0.07
1.86 ± 0.05
0.73 ± 0.07
1.77 ± 0.06
0.78 ± 0.05
-
1149
RG108
G1
G2
G3
G11
G12
-
G4
G5
intermediate initially released from UV irradiation. Upon further
checking in the literature, we found several reports had
previously documented that the nitrile imine generated from a
UV-irradiated tetrazole indeed readily react with different
nucleophiles.37,38 Thus, Gn were not employed as activity-based
probes in our subsequent experiments. They were nevertheless
found to act as novel covalent inhibitors of DNMT1 from our
present study (Table 1, Fig. 2 & Fig. 3). Overall, we assessed T1
and Gn’s ability to inhibit recombinantly purified DNMT1 in an
in vitro enzyme inhibition assay. We subsequently evaluated
their effect in inhibition of mammalian cell growth in an XTT
anti-proliferation assay. Finally, we carried out in vitro cell-based
proteome profiling experiments with T1 and competitive ABPP
with Gn followed by cellular bioimaging.
2.2.3 Target profiling with T1 and competitors
T1 showed better potency against DNMT1 than both RG108
1149, and as a covalent modifier of the enzyme, its terminal
alkyne could serve as a clickable tag for subsequent cell-based
proteome profiling and target identification.15,19 To do so, we
first obtained the optimized T1-proteome labeling conditions:
100 g of freshly prepared MCF-7 cell lysates were incubated
with 10 M of T1 for 1 h at room temperature, followed by 2 h
of CuAAC click chemistry with previously reported protocols
(using TER-PEG-N3 or TER-Biotin-N3).19 Subsequently, the
same conditions were used for dose-dependent, competitive
ABPP by using lysates of mammalian cells pretreated with
either G6, 1149 or RG108 (Fig. 2D); in-gel fluorescence
scanning results showed some distinctly fluorescent bands at <
70 kDa, but not at ~183 kDa, where the labeled endogenous
DNMT1 was expected to appear. This indicates T1 was not
able to directly profile the presence of endogenous DNMT1
under such experimental conditions, presumably due to the low
endogenous expression level of the enzyme in mammalian cells
and insufficient detection sensitivity of the probe. We therefore
carried out profiling experiments by using an alternative
strategy - the competitive ABPP followed by click chemistry
with TER-Biotin-N3, then protein enrichment by pull-down (PD)
followed by DNMT1 visualization with Western blotting (WB)
analysis (Fig. 2E); successful detection of the endogenous
DNMT1 (~183 KDa) in T1-labeled proteomes, but not in the
unlabeled proteome clearly showed that T1 indeed was a novel
activity-based probe of DNMT1 capable of covalent labeling of
endogenous DNMT1 from complex mammalian protein lysates,
albeit with a low sensitivity (thus rendering the labeled target
detected only upon enrichment and signal amplification). When
the same labeling/PD/WB experiments were carried out on
lysates of mammalian cells pre-treated with increasing
concentrations of G6, 1149 or RG108, we observed
concomitant decreases in the signals of the labeled DNMT1,
indicating all three inhibitors effectively competed for binding
to the active site of DNMT1 even under complex cellular
2.2.1 Inhibition against DNMT1
First, T1 and Gn library were evaluated for their inhibition
efficiency against pure DNMT1 using DNMT Activity/Inhibition
Assay Ultra Kit (Fluorometric, EpiQuikTM). Generally, with an
increase in the inhibitor concentrations, a concomitant decrease
in the enzyme activity was observed until a saturation point was
reached (Fig. 2A, left). For T1, when compared to RG108 or
1149, it appeared that introduction of the terminal alkyne in 5-
hydroxytrptophan did not have significant impact toward the
inhibition of DNMT1. Interestingly, all tetrazole-based
inhibitors, Gn (n = 1-12), appeared to possess reasonable
inhibitory property against DNMT1 (Fig. 2A, right); among
them, G6 possessed the highest inhibitory activity against
DNMT1 and inhibited the enzyme even more effectively than the
two known DNMT1 inhibitors, RG108 and 1149. This thus
provides the first direct evidence that the tetrazole moiety in
these compounds indeed served as a good mimic to the indole
ring of tryptophan and could effectively bind to the DNMT1
active site via non-covalent interactions. Encouraged by these
preliminary results and due to high cost of the DNMT1
enzymatic assay, we subsequently carried out cell-based assays,
ABPP and cell-based imaging experiments to further assess
selected compounds.
2.2.2 Cell proliferation
Inhibition of DNMT1 induces cell death and apoptosis of human
cancer cells that harbor abnormal active DNMTs, such as MCF-7
breast cancer cell line.1 In the XTT cell proliferation assay (Fig.
2B, S2 & Table 1), we observed the corresponding dose-
dependent inhibition of T1 and Gn towards MCF-7. GI50 value is
the concentration of the anti-cancer drug that inhibits the growth
environments.
The success of competitive ABPP also
confirmed that the covalent labeling of DNMT1 by T1 was

indeed activity-based, making it the first known ABP of
DNMT1 to our knowledge.
that T1 was able to bind to endogenous DNMT1 in live
mammalian cells.
Figure. 3. Competitive fluorescence imaging of MCF-7 cells with T1. A total of four imaging channels were employed, including bright-field
(BF), probe-treated channel (upon click chemistry with TER-PEG-N3), immunofluorescence (IF) and Hoechst-stained channel (NU). Panels 1-
6: cells were treated with 5 µΜ of T1; Panels 7-12: cells were pretreated with G6 (100 µΜ); Panels 13-18: cells were pretreated with 1149 (100
µΜ); Panels 19-24: cells were pretreated with RG108 (100 µΜ). Scale Bar: 10 µm.
2.2.4 Bioimaging
A competitive imaging experiment was developed to test
whether our newly developed, tetrazole-based DNMT1
inhibitors, e.g. G6, were indeed able to bind to the same
DNMT1 active site in live mammalian cells as T1, thus directly
competing with T1 for binding to the enzyme. Live MCF-7
mammalian cells were pre-treated with either G6, RG108 or
1149 (100 M) for 1 h prior to incubation with T1 as earlier
described, then imaged (Fig. 3; panels 7-24); a significant
decrease in the fluorescence signals was detected in T1-labeled
cells pre-treated with G6. Interestingly, neither RG108 nor
1149 were as effective as G6 in competitively decreasing the
fluorescence signals of labeled cells, once again indicating G6
was a better DNMT1 inhibitor than both RG108 and 1148
under native cellular environments.
Bioimaging is another interesting way to report endogenous
enzyme-drug interactions, including drug uptake amount and
target localizations.39-48 In recent years, small molecule-based
activity-based probes have been used to successfully image
endogenous cellular targets.24,25,44,45-48 Since T1 appeared to
possess better cellular activities than both RG108 and 1149,
and it was also a more potent DNMT1 inhibitor, we reasoned
that it might serve as a potential DNMT1 imaging probe as well.
To confirm this hypothesis, we carried out cellular imaging
experiments (Fig. 3). MCF-7 cells were first treated with an
optimized amount of T1 (5 M) for 30 min in a cell culture
incubator. Subsequently, cells were fixed with 3.7%
formaldehyde, permeabilized by 0.1% Triton X-100, and
clicked with TER-PEG-N3 by following previously optimized
protocols.19,24 The same cells were further treated with anti-
DNMT1 antibodies by following standard immunofluorescence
(IF) protocols, then stained with Hoechst to visualize their
nuclei. Finally, cells were imaged by fluorescence microscopy
under four different imaging channels (Fig. 3); no fluorescence
was detected in cells treated with DMSO alone (Fig S6; panels
5-8). Strong T1 fluorescence signals were detected throughout
the nuclei of the cells (green), which co-localized reasonably
well with signals obtained from the immunofluorescence with
anit-DNMT1 antibodies (IF; red; panels 1-6), clearly indicating
3. Conclusion
We have successfully developed two types of novel chemical
tools for the study of DNMT1 – an small molecule, activity-
based probe (T1) and twelve tetrazole-containing small molecule
covalent inhibitors (Gn; n = 1-12). We found T1 could serve as
an effective activity-based profile for cell-based profiling of
endogenous DNMT1 expressed in mammalian cells, albeit with
relatively low detection sensitivity. Further improvement will
clearly be needed on this probe in order to make such profiling
experiments a routine endeavor for future chemical and cellular
studies of DNMT1 biology. As tetrazole-derived tryptophan

analogs, G6 was found to be a novel small molecule inhibitor of
DNMT1, with better inhibitory activities than well-known
DNMT1 inhibitors such as RG108 and 1148 under both in vitro
and cellular settings. G6 was initially designed to serve not only
as a covalent inhibitor, but also a potential activity-based probe
of DNMT1. Unfortunately, throughout our preliminary studies,
we found the so-called “photo-click” reaction involving tetrzoles
was not bioorthogonal as previously reported,33-36 and the UV-
irradiated tetrozole intermediate (nitrile imine) appeared to react
with many common biological nucleophiles.37,38 More extensive
studies are underway to better understand such reactions and
results will be reported in due course. We caution however, the
bioorthogonality of “photo-click” chemistry needs to be carefully
scrutinized before it is used in other chemical biological
applications. Nevertheless, based on our in vitro DNMT1
inhibition assay, XTT anti-cell proliferation assay, FACS, in situ
competitive APBB as well as cellular imaging results, we could
confirmed that G6 was indeed a reasonable DNMT1 inhibitor
and may be further developed into a useful chemical tool for the
study of DNMT1 biology.
the reaction was completed, the solution was evaporated to
remove the solvent and excessive SOCl2 under reduced pressure.
The dark green residue was dissolved in a minimal amount of hot
methanol. Et2O was added to obtain the dark green product
precipitation as a solid. The crude product was then re-dissolved
in dioxane-H2O cosolvent (110 mL, dioxane:H2O = 1:1). To the
solution, a NaHCO3 solution (sat.) was added at 0 oC to adjust the
pH of the solution to 8. Then Boc2O (0.99 g, 4.53 mmol) was
added to the above solution at 0 oC, followed by stirring
overnight at room temperature. The reaction mixture was
extracted with EtOAc for three times, the organic layer was
combined, washed with brine and dried over Na2SO4. Flash
column chromatography (Hexane:EtOAc = 3:1) was used to
afford the protected unnatural amino acid 1a as a pale white solid
(1.25 g, 83.0% in two steps); 1H NMR (300 MHz, MeOD) δ 7.15
(d, J = 8.6 Hz, 1H), 7.00 (s, 1H), 6.90 (d, J = 2.2 Hz, 1H), 6.67
(dd, J = 8.6, 2.1 Hz, 1H), 4.39 (d, J = 6.7 Hz, 1H), 3.65 (s, 3H),
3.22-2.92 (m, 2H), 1.31 (d, J = 48.1 Hz, 9H); 13C NMR (75 MHz,
MeOD) δ 174.76, 157.51, 151.34, 132.95, 129.40, 125.19, 112.73,
112.51, 109.97, 103.41, 80.67, 55.98, 52.58, 28.65. MS (ESI):
[M+H+] calcd for C17H22N2O5 335.15, found 335.20.
4.1.2. (S)-methyl 2-((tert-butoxycarbonyl)amino)-3-(5-(prop-
2-yn-1-yloxy)-1H-indol-3-yl)propanoate (1b)
4. Experimental
All chemicals were purchased from commercial vendors and
used without further purification, unless otherwise noted. N,N-
Dimethylformamide (DMF) and Dichloromethane (CH2Cl2,
DCM) were distilled over CaH2. All non-aqueous reactions
were carried out under nitrogen atmosphere in oven-dried
glassware. Reaction progress was monitored by TLC on pre-
coated silica plates (Merck 60 F254, 250 μm thicknesses) and
spots were visualized by ceric ammonium molybdate,
ninhydrin, basic KMnO4, UV light or iodine. Flash column
chromatography was carried out using Merck silica gel (0.040-
Compound 1a (200 mg, 0.598 mmol) was dissolved in anhydrous
DMF (2 mL). Then anhydrous potassium carbonate (124 mg,
0.897 mmol) was added to the solution. The obtained mixture
was stirred at room temperature for 30 min. Propargyl bromide
(80% in toluene, 0.1 mL, 0.897 mmol) was added to the reaction
mixture, which was subsequently stirred at room temperature
overnight. After the completion of the reaction, water was added
to dilute the reaction solution. The crude product was extracted
with EtOAc for three times, the organic layer was combined,
washed with water and brine, dried over Na2SO4 and
concentrated. Flash column chromatography (Hexane:EtOAc =
2:1) was used to generate the titled product 1b as a yellow oil
(220.35 mg, 99.0%); 1H NMR (300 MHz, CDCl3) δ 8.28 (s, 1H),
7.23 (d, J = 8.8 Hz, 1H), 7.10 (d, J = 1.5 Hz, 1H), 6.99-6.87 (m,
2H), 5.28 (s, 1H), 4.72 (d, J = 2.2 Hz, 2H), 3.67 (s, 3H), 3.24 (s,
1H), 3.22 (s, 1H), 2.53 (s, 1H), 1.42 (s, 9H); 13C NMR (75 MHz,
CDCl3) δ 172.78, 155.12, 152.05, 131.90, 127.86, 123.81, 112.93,
111.93, 109.81, 102.93, 79.94, 79.18, 75.19, 57.05, 54.17, 52.22,
28.25. MS (ESI): [M+H+] calcd for C20H24N2O5 373.17, found
373.14.
1
0.063). H NMR and 13C NMR spectra were recorded on a
Bruker model Advance 300 MHz or DPX-500 MHz NMR
spectrometer. Chemical shifts are reported in parts per million
relative to internal standard tetramethylsilane (Si(CH3)4 = 0.00
ppm) or residual solvent peaks (CDCl3 = 7.26 ppm, MeOD =
3.31 ppm). 1H NMR coupling constants (J) are reported in
Hertz (Hz) and multiplicity is indicated as follows: s (singlet), d
(doublet), t (triplet), m (multiplet), dd (doublet of doublet), dt
(doublet of triplet). MS spectra were recorded on a Finnigan
LCQ mass spectrometer, a Shimadzu LC-IT-TOF spectrometer
or a Shimadzu LC-ESI spectrometer. Analytical HPLC was
carried out on Shimadzu LC-IT-TOF or LC-ESI systems
equipped with an autosampler, using reverse-phase
Phenomenex Luna 5 μm C18 100 Å 50 × 3.0 mm columns.
Preparative HPLC was carried out on Gilson preparative HPLC
system using Trilution software and reverse-phase Phenomenex
Luna 5 μm C18 (2) 100 Å 50 × 30.00 mm column. 0.1%
TFA/H2O and 0.1% TFA/acetonitrile were used as eluents for
all HPLC experiments. The flow rate was 0.6 mL/min for
analytical HPLC and 8 mL/min for preparative HPLC. UV-vis
absorption and fluorescence spectra were measured by using a
Shimadzu UV-vis spectrophotometer and a Perkin Elmer LS50
spectrofluorometer, respectively. All the measurements were
performed at room temperature.
4.1.3. (S)-methyl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-
(5-(prop-2-yn-1-yloxy)-1H-indol-3-yl) propanoate (T1)
Compound 1b (200 mg, 0.54 mmol) was dissolved in HPLC-
grade DCM (2 mL). To the solution, TFA (2 mL) was added
dropwise. The reaction mixture was stirred at room temperature
for 1 h. After the reaction was completed, the volatile was
removed under reduced pressure. The residue was diluted with
water. After adjusting the pH value of the solution to 7~8 with
saturated NaHCO3 solution, then the product was extracted with
EtOAc. The organic phase was combined, washed with brine,
dried over Na2SO4 and concentrated. The product 1b-amine can
be directly used in the next step without further purification.
Maleic anhydride (19.8 mg, 0.202 mmol) was dissolved in glacial
acetic acid (0.5 mL) at room temperature. To the solution, a
solution of compound 1b-amine (50 mg, 0.184 mmol) in glacial
acetic acid (0.5 mL) was added dropwise. The reaction mixture
was refluxed for 4 h. After the reaction completion, the reaction
was cooled to room temperature and water was added to quench
the reaction. Saturated sodium bicarbonate solution was
4.1. Synthesis of activity-based probe T1
4.1.1.
(S)-methyl
2-((tert-butoxycarbonyl)amino)-3-(5-
hydroxy-1H-indol-3-yl) Propanoate (1a)
Thionyl Chloride (2.25 mL) was added dropwise to L-5-
Hydroxytryptophan (1 g, 4.54 mmol) in a methanol (22.5 mL)
o
solution at 0 C. The brown solution was refluxed for 4 h. When

employed to neutralize the reaction solution. The crude product
was extracted with EtOAc for three times, the organic layer was
combined, washed with water and brine, dried over Na2SO4 and
concentrated. Flash column chromatography (DCM:MeOH =
95:5) was used to afford the titled product T1 as a yellowish solid
(20 mg, 31.0% in two steps): 1H NMR (300 MHz, CDCl3) δ 7.22
(d, J = 8.8 Hz, 1H), 7.08 (d, J = 2.3 Hz, 1H), 6.98 (d, J = 2.3 Hz,
1H), 6.89 (dd, J = 8.8, 2.4 Hz, 1H), 6.57 (s, 2H), 5.04 (dd, J =
9.2, 7.0 Hz, 1H), 4.73 (d, J = 2.4 Hz, 2H), 3.80 (s, 3H), 3.64-3.54
(m, 2H), 2.54 (t, J = 2.4 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ
170.04, 169.65, 152.26, 134.18, 131.89, 127.63, 123.64, 113.39,
112.06, 111.01, 102.63, 79.34, 75.35, 57.14, 53.00, 52.63, 24.79.
HRMS: [M+Na+] calcd for C19H16N2O5 375.0951, found
375.0965.
filtrate was collected and extracted with EtOAc. The organic
phase was washed with water and brine, dried over Na2SO4 and
concentrated. The crude product was further purified by flash
column chromatography (Hexane:EtOAc = 3:1) to obtain the
titled product 2b as a golden yellow oil (2.28 g, 81.3%): 1H NMR
(500 MHz, CDCl3) δ 7.40-7.28 (m, 5H), 6.88 (d, J = 8.3 Hz, 2H),
6.62 (d, J = 8.2 Hz, 2H), 5.22 (d, J = 7.3 Hz, 1H), 5.09 (d, J = 2.1
Hz, 2H), 4.59 (dd, J = 13.9, 5.8 Hz, 1H), 3.71 (s, 3H), 3.06-2.94
(m, 2H); 13C NMR (75 MHz, CDCl3) δ 172.30, 155.76, 145.46,
136.28, 130.17, 128.57, 128.14, 128.07, 125.36, 115.42, 66.97,
55.06, 52.31, 37.40. MS (ESI): [M+H+] calcd for C18H20N2O4
329.14, found 329.12;
4.2.3. General procedure
4.2.3.1. Synthesis of tetrazole compound (2dn)
4.2. Synthesis of tetrazole-based compounds (Gn)
Aldehyde N and PhSO2NHNH2 (1:1.2 equiv.) were dissolved in
ethanol (6.8 mL/mmol) and the resulting solution was stirred at
room temperature for 3 h. After the completion of the reaction,
EtOH was removed under reduced pressure to give compound
2cn. The crude product was used in the next step directly without
further purification.36
4.2.1. (S)-methyl 2-amino-3-(4-nitrophenyl)propanoate (2a)
Thionyl chloride (12 mL) was added dropwise to a solution of 4-
nitro-3-phenyl-L-alanine (10 g, 47.58 mmol) in HPLC-grade
methanol (150 mL) at 0 oC. The mixture was refluxed for 2 h.
When the reaction was completed, the reaction mixture was
cooled to room temperature and removed the solvent under
reduced pressure. The residue was re-dissolved in water and
NaHCO3 was added to adjust the pH of the solution to 7~8. The
mixture was extracted with DCM for three times. The organic
phase was combined, washed with water and brine. Finally the
organic phase was dried with Na2SO4 and concentrated in vacuo
to afford the methylated amino acid as yellow oil (10.46 g,
98.1%). The product was used in the next step without further
purification.
Compound 2cn (0.85 equiv.) was re-dissolved in pyridine (10.4
o
mL/mmol) and cooled to -10 ~ -20 C in an ice/NaCl bath to
derive solution A. Separately, to a solution of compound 2b (1
equiv.) in mixed EtOH-H2O (10.4 mL/mmol, EtOH:H2O = 1:1)
was added concentrated hydrochloric acid (0.48 mL/mmol), and
the mixture was cooled to -5 oC with an ice/NaCl bath.
Subsequently, a cold solution of NaNO2 (1.5 equiv.) in water
(1.74 mL/mmol) was added dropwise into to derive solution B (it
o
is very important to keep the temperature below 0 C during the
The methylated amino acid (1.933 g, 8.62 mmol) was dissolved
in a dioxane/H2O co-solvent (50 mL, dioxane:H2O = 1:1). To the
solution, a NaHCO3 solution (sat.) was added at 0 oC to adjust the
pH of the solution to around 8. To this solution, a solution of
benzyl chloroformate (1.765 g, 10.34 mmol) in dioxane was
entire addition process!).
Solution B was added to solution A slowly and kept the
o
temperature below -5 C at all the time. Upon the completion of
addition, the reaction mixture was allowed to warm to room
temperature. The solution was extracted with EtOAc/H2O,
washed with diluted HCl solution and brine, then dried with
Na2SO4 and concentrated. The crude product was further purified
via column chromatography (1% EtOAc in DCM) to afford the
titled compound 2dn.
o
added dropwise at 0 C. After addition, the reaction mixture was
allowed to warm to room temperature, followed by stirring
overnight. After the reaction was completed, EtOAc was used to
extract the crude product for three times, the organic layer was
collected and combined, washed with diluted HCl solution,
saturated NaHCO3 solution, water and brine, dried over Na2SO4
and concentrated. The crude product was purified by flash
column chromatography (Hexane:EtOAc = 3.5:1) to yield the
protected unnatural amino acid 2a as a white solid (3.06 g,
4.2.3.2. Synthesis of the target probe Gn (avoiding light
during all the process)
To a solution of tetrazole amino acid derivatives 2dn (100 mg) in
HPLC MeOH was added 10% Pd/C (20 mg). The reaction was
sealed and air was removed by inletting nitrogen gas into the
system. While air was totally removed, the reaction mixture was
hydrogenated at room temperature (1 atm) overnight. And the
catalyst was removed by filtration through a short pad of celite
and washing with EtOAc. The solvent was removed under
reduced pressure and the obtained product 2en can be directly
used in the next step.
1
99.2%): H NMR (500 MHz, CDCl3) δ 8.07 (d, J = 8.3 Hz, 2H),
7.37-7.27 (m, 5H), 7.24 (s, 2H), 5.51 (s, 1H), 5.06 (dd, J = 30.8,
12.2 Hz, 2H), 4.68 (dd, J = 13.3, 6.6 Hz, 1H), 3.72 (s, 3H), 3.27
(dd, J = 13.8, 5.4 Hz, 1H), 3.10 (dd, J = 13.8, 6.8 Hz, 1H); 13C
NMR (125 MHz, CDCl3) δ 171.38, 155.58, 147.08, 143.85,
136.09, 130.19, 128.51, 128.30, 128.09, 123.62, 67.06, 54.52,
52.60, 38.06. MS (ESI): [M+H+] calcd for C18H18N2O6 359.12,
found 359.10.
To a solution of maleic anhydride in HPLC-grade CHCl3 (0.8
mL/mmol), a solution of compound 2en (1 equiv.) in HPLC
CHCl3 (0.2 mL/mmol) was added dropwise. The resulting
mixture was stirred at room temperature for 2 h. After starting
materials were completely reacted, the solvent was removed
under reduced pressure. The residue was re-dissolved in acetic
anhydride (1 mL/mmol) and sodium acetate (20 mg/mmol) was
added. The mixture was refluxed for 2 h, cooled and quenched
with water. The aqueous solution was extracted with EtOAc,
washed with saturated NaHCO3 solution, water and brine, dried
over Na2SO4 and concentrated. The crude product Gn was
purified by flash column chromatography.
4.2.2. (S)-methyl 3-(4-aminophenyl)-2 (((benzyloxy)carbonyl)
amino)propanoate (2b)
Compound 2a (3.06 g, 8.54 mmol) was dissolved in a mixed
solution of ethanol/H2O (125 mL, EtOH:H2O = 3:2). After that,
7.9 mL concentrated hydrochloric acid (37%) was added to the
solution. Anhydrous tin (II) chloride (14.34 g, 75.62 mmol) was
added and the resulting solution was refluxed at 70 ~ 80 oC for 50
min. When the reaction was completed, the reaction mixture was
basified with NaHCO3 to adjust the pH around 8 immediately.
The precipitated Sn(OH)2 was removed by filtration through a
short pad of celite and washed with EtOAc for three times. The

4.2.4. (S)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(4-(5-(4-
isopropylphenyl)-2H-tetrazol-2-yl) phenyl) propanoate (2d1)
A yellowish solid (79.2 mg, 56.9%): 1H NMR (500 MHz, CDCl3)
δ 8.25 (dd, J = 7.8, 1.7 Hz, 2H), 8.10 (d, J = 8.4 Hz, 2H), 7.52
(dd, J = 5.7, 3.8 Hz, 3H), 7.38-7.27 (m, 7H), 5.31 (d, J = 7.7 Hz,
1H), 5.09 (t, J = 12.4 Hz, 2H), 4.73 (dd, J = 13.8, 6.0 Hz, 1H),
3.76 (s, 3H), 3.27 (dd, J = 13.9, 5.7 Hz, 1H), 3.16 (dd, J = 13.9,
6.2 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 171.60, 165.22,
155.58, 137.77, 135.94, 130.60, 130.57, 128.98, 128.56, 128.31,
128.17, 127.14, 127.07, 120.00, 67.14, 54.71, 52.56, 38.03. MS
(IT-TOF): [M+H+] calcd for C25H23N5O4 458.18, found 458.17.
A yellowish solid (130 mg, 85.5%): 1H NMR (500 MHz, CDCl3)
δ 8.17 (d, J = 8.2 Hz, 2H), 8.09 (d, J = 8.3 Hz, 2H), 7.41-7.27 (m,
9H), 5.31 (d, J = 7.7 Hz, 1H), 5.09 (t, J = 12.7 Hz, 2H), 4.75-4.69
(m, 1H), 3.75 (s, 3H), 3.27 (dd, J = 13.9, 5.6 Hz, 1H), 3.15 (dd, J
= 13.9, 6.1 Hz, 1H), 3.03-2.95 (m, 1H), 1.31 (d, J = 6.9 Hz, 6H);
13C NMR (125 MHz, CDCl3) δ 171.61, 165.29, 155.56, 151.72,
137.64, 136.13, 135.98, 130.53, 128.56, 128.30, 128.17, 127.11,
127.08, 124.68, 119.98, 67.13, 54.71, 52.55, 38.02, 34.18, 23.84.
MS (IT-TOF): [M+H+] calcd for C28H29N5O4 500.22, found
500.20.
4.2.10. (S)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(4-(5-
(3,5-difluorophenyl)-2H-tetrazol-2-yl) phenyl) propanoate
(2d7)
4.2.5. (S)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(4-(5-(4-
fluorophenyl)-2H-tetrazol-2-yl)phenyl) propanoate (2d2)
A yellowish solid (111.6 mg, 74.3%): 1H NMR (500 MHz,
CDCl3) δ 8.08 (d, J = 8.3 Hz, 2H), 7.84-7.73 (m, 2H), 7.33 (dd, J
= 16.8, 7.3 Hz, 7H), 6.95 (tt, J = 8.8, 2.3 Hz, 1H), 5.32 (d, J = 6.4
Hz, 1H), 5.09 (t, J = 11.8 Hz, 2H), 4.73 (dd, J = 13.5, 6.1 Hz,
1H), 3.76 (s, 3H), 3.28 (dd, J = 13.9, 5.6 Hz, 1H), 3.16 (dd, J =
13.9, 6.2 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 171.56, 164.37
(d, J = 12.8 Hz), 163.45, 162.44, 155.54, 138.24, 135.69, 130.67,
128.57, 128.32, 128.18, 120.02, 110.20 (d, J = 7.0 Hz), 110.03
(d, J = 6.8 Hz), 105.95, 67.15, 54.69, 52.58, 38.04. MS (IT-
TOF): [M+H+] calcd for C25H21F2N5O4 494.16, found 494.14.
A yellowish solid (128 mg, 88.4%): 1H NMR (500 MHz, CDCl3)
δ 8.27-8.19 (m, 2H), 8.09 (d, J = 8.2 Hz, 2H), 7.36-7.27 (m, 6H),
7.25-7.18 (m, 3H), 5.29 (s, 1H), 5.09 (t, J = 11.8 Hz, 2H), 4.72 (s,
1H), 3.75 (s, 3H), 3.25 (s, 1H), 3.15 (d, J = 7.4 Hz, 1H); 13C
NMR (125 MHz, CDCl3) δ 171.57, 165.23, 163.83 (d, J = 148.6
Hz), 155.53, 137.85, 136.11, 135.86, 130.58, 129.10 (d, J = 8.6
Hz), 128.55, 128.29, 128.16, 123.39, 119.95, 116.13 (d, J = 22.0
Hz). MS (IT-TOF): [M+H+] calcd for C25H22FN5O4 476.17,
found 476.16.
4.2.11. (S)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(4-(5-(4-
(hexyloxy)phenyl)-2H-tetrazol-2-yl) phenyl) propanoate (2d8)
4.2.6. (S)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(4-(5-
(naphthalen-1-yl)-2H-tetrazol-2-yl)phenyl) propanoate (2d3)
A yellowish solid (88.3 mg, 68.8%): 1H NMR (500 MHz, CDCl3)
δ 8.17 (d, J = 8.8 Hz, 2H), 8.08 (d, J = 8.4 Hz, 2H), 7.38-7.26 (m,
7H), 7.03 (d, J = 8.9 Hz, 2H), 5.29 (d, J = 7.9 Hz, 1H), 5.11 (q, J
= 12.2 Hz, 2H), 4.72 (d, J = 7.7 Hz, 1H), 4.04 (t, J = 6.6 Hz, 2H),
3.75 (s, 3H), 3.26 (dd, J = 14.0, 5.7 Hz, 1H), 3.15 (dd, J = 13.9,
6.0 Hz, 1H), 1.86-1.78 (m, 2H), 1.53-1.25 (m, 6H), 0.92 (t, J =
7.1 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 171.58, 165.16,
161.12, 155.34, 136.00, 130.50, 128.55, 128.28, 128.15, 119.92,
119.43, 114.90, 68.19, 67.11, 54.70, 52.52, 37.88, 31.57, 29.17,
25.70, 22.59, 14.01. MS (IT-TOF): [M+H+] calcd for C31H35N5O5
558.26, found 558.23.
A yellowish solid (208.1 mg, 83.7%): 1H NMR (500 MHz,
CDCl3) δ 9.02 (d, J = 8.5 Hz, 1H), 8.38 (d, J = 7.2 Hz, 1H), 8.18
(d, J = 8.0 Hz, 2H), 8.03 (d, J = 8.1 Hz, 1H), 7.96 (d, J = 8.0 Hz,
1H), 7.68-7.56 (m, 3H), 7.34 (d, J = 9.5 Hz, 7H), 5.33 (d, J = 7.8
Hz, 1H), 5.10 (t, J = 13.2 Hz, 2H), 4.74 (d, J = 6.9 Hz, 1H), 3.77
(s, 3H), 3.30 (dd, J = 13.6, 5.6 Hz, 1H), 3.18 (dd, J = 14.1, 6.0
Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 171.61, 165.35, 155.56,
137.84, 136.13, 135.97, 134.01, 131.28, 130.64, 128.69, 128.67,
128.57, 128.32, 128.19, 127.44, 126.33, 125.78, 125.25, 123.92,
120.06, 67.14, 54.72, 52.58, 38.04. MS (IT-TOF): [M+H+] calcd
for C29H25N5O4 508.19, found 508.18.
4.2.12. (S)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(4-(5-
(pyridin-4-yl)-2H-tetrazol-2-yl)phenyl)propanoate (2d9)
4.2.7. (S)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(4-(5-(4-
methoxyphenyl)-2H-tetrazol-2-yl) phenyl) propanoate (2d4)
A yellowish solid (39 mg, 32.7%): 1H NMR (500 MHz, CDCl3) δ
8.83 (s, 2H), 8.12 (dd, J = 21.4, 6.5 Hz, 4H), 7.34 (dd, J = 7.5,
4.9 Hz, 7H), 5.33 (d, J = 7.8 Hz, 1H), 5.17-5.03 (m, 2H), 4.73
(dd, J = 13.7, 6.1 Hz, 1H), 3.76 (s, 3H), 3.29 (dd, J = 13.9, 5.6
Hz, 1H), 3.16 (dd, J = 13.9, 6.1 Hz, 1H); 13C NMR (125 MHz,
CDCl3) δ 171.55, 163.12, 155.57, 150.41, 138.44, 136.13,
135.65, 130.72, 128.57, 128.33, 128.19, 121.10, 120.09, 67.16,
54.69, 52.60, 38.06. MS (IT-TOF): [M+H+] calcd for C24H22N6O4
459.17, found 459.14.
A yellowish solid (55.4 mg, 37.3%): 1H NMR (500 MHz, CDCl3)
δ 8.18 (d, J = 8.8 Hz, 2H), 8.08 (d, J = 8.4 Hz, 2H), 7.40-7.26 (m,
7H), 7.04 (d, J = 8.8 Hz, 2H), 5.30 (d, J = 8.2 Hz, 1H), 5.10 (q, J
= 12.2 Hz, 2H), 4.72 (d, J = 7.6 Hz, 1H), 3.89 (s, 3H), 3.75 (s,
3H), 3.26 (dd, J = 13.7, 5.7 Hz, 1H), 3.15 (dd, J = 14.0, 6.1 Hz,
1H); 13C NMR (125 MHz, CDCl3) δ 171.60, 165.10, 161.51,
155.55, 137.58, 136.14, 135.99, 130.53, 128.60, 128.56, 128.30,
128.17, 119.93, 119.72, 114.39, 67.13, 55.41, 54.71, 52.54,
38.02. MS (IT-TOF): [M+H+] calcd for C26H25N5O5 488.19,
found 488.18.
4.2.13. (S)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(4-(5-(4-
(difluoromethoxy)phenyl)-2H-tetrazol-2yl)phenyl)propanoate
(2d10)
4.2.8. (S)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(4-(5-(4-
(dimethylamino)phenyl)-2H-tetrazol-2-yl)phenyl) propanoate
(2d5)
A yellowish solid (35.8 mg, 23.5%): 1H NMR (500 MHz, CDCl3)
δ 8.13-8.07 (m, 2H), 7.35-7.26 (m, 9H), 6.85 (s, 2H), 5.29 (d, J =
7.6 Hz, 1H), 5.11 (q, J = 12.2 Hz, 2H), 4.72 (d, J = 7.7 Hz, 1H),
3.75 (s, 3H), 3.28-3.23 (m, 1H), 3.18-3.13 (m, 1H), 3.07 (s, 6H).
MS (IT-TOF): [M+H+] calcd for C27H28N6O4 501.22, found
501.19.
A yellowish solid (34 mg, 26.5%): 1H NMR (500 MHz, CDCl3) δ
8.26 (d, J = 8.7 Hz, 2H), 8.09 (d, J = 8.3 Hz, 2H), 7.38-7.26 (m,
9H), 6.60 (t, J = 73.4 Hz, 1H), 5.31 (d, J = 8.0 Hz, 1H), 5.14-5.07
(m, 2H), 4.72 (d, J = 7.5 Hz, 1H), 3.76 (s, 3H), 3.27 (dd, J = 13.9,
5.6 Hz, 1H), 3.15 (dd, J = 13.9, 6.2 Hz, 1H); 13C NMR (125
MHz, CDCl3) δ 171.58, 164.34, 155.55, 152.87, 137.92, 136.12,
135.86, 130.60, 128.76, 128.56, 128.31, 128.17, 124.37, 119.99,
119.80, 115.64, 67.14, 54.70, 52.55, 38.04. MS (IT-TOF):
[M+H+] calcd for C26H23F2N5O5 524.17, found 524.15.
4.2.9. (S)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(4-(5-
phenyl-2H-tetrazol-2-yl)phenyl)propanoate (2d6)
4.2.14. (S)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(4-(5-(4-
(trifluoromethyl)phenyl)-2H-tetrazol-2-yl)phenyl)propanoate
(2d11)

A yellowish solid (48 mg, 35.1%): 1H NMR (500 MHz, CDCl3) δ
8.38 (d, J = 8.2 Hz, 2H), 8.10 (d, J = 8.3 Hz, 2H), 7.80 (d, J = 8.3
Hz, 2H), 7.31 (dd, J = 24.4, 16.4 Hz, 7H), 5.31 (d, J = 7.6 Hz,
1H), 5.09 (dd, J = 15.8, 7.2 Hz, 2H), 4.73 (d, J = 6.0 Hz, 1H),
3.76 (s, 3H), 3.31-3.22 (m, 1H), 3.16 (dd, J = 13.8, 6.1 Hz, 1H);
13C NMR (125 MHz, CDCl3) δ 171.54, 164.02, 155.55, 138.12,
136.11, 135.77, 132.50, 132.24, 130.66, 128.57, 128.32, 128.18,
127.35, 126.01 (d, J = 3.9 Hz), 120.05, 67.15, 54.70, 52.58,
38.05. MS (IT-TOF): [M+H+] calcd for C26H22F3N5O4 526.16,
found 526.16.
4.2.19. (S)-methyl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-
(4-(5-(4-methoxyphenyl)-2H-tetrazol-2-yl)phenyl) propanoate
(G4)
1
A yellowish solid (21.5mg, 35.1%): H NMR (500 MHz, CDCl3)
δ 8.19-8.14 (m, 2H), 8.07 (d, J = 8.6 Hz, 2H), 7.33 (d, J = 8.5 Hz,
2H), 7.06-7.01 (m, 2H), 6.64 (s, 2H), 5.01 (dd, J = 11.5, 5.3 Hz,
1H), 3.89 (s, 3H), 3.81 (s, 3H), 3.61-3.53 (m, 2H); 13C NMR (125
MHz, CDCl3) δ 169.73, 168.85, 165.07, 161.49, 130.08, 128.57,
120.03, 119.66, 114.37, 55.40, 53.06, 52.83, 34.23. HRMS:
[M+Na+] calcd for C22H19N5O5 456.1278, found 456.1284.
4.2.15. (S)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(4-(5-
(3,5-dimethylphenyl)-2H-tetrazol-2-yl) phenyl) propanoate
(2d12)
4.2.20. (S)-methyl 3-(4-(5-(4-(dimethylamino)phenyl)-2H-
tetrazol-2-yl)phenyl)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-
yl) propanoate (G5)
A yellowish solid (97 mg, 76.8%): 1H NMR (500 MHz, CDCl3) δ
8.10 (d, J = 8.3 Hz, 2H), 7.87 (s, 2H), 7.40-7.26 (m, 7H), 7.14 (s,
1H), 5.30 (d, J = 7.9 Hz, 1H), 5.11 (q, J = 12.2 Hz, 2H), 4.72 (dd,
J = 13.5, 6.0 Hz, 1H), 3.75 (s, 3H), 3.27 (dd, J = 13.9, 5.6 Hz,
1H), 3.15 (dd, J = 13.9, 6.1 Hz, 1H), 2.42 (s, 6H); 13C NMR (125
MHz, CDCl3) δ 171.60, 165.44, 155.31, 138.66, 137.68, 136.12,
135.98, 132.29, 130.54, 128.56, 128.30, 128.16, 126.86, 124.81,
119.99, 67.13, 54.71, 52.53, 38.03, 21.30 (d, J = 1.7 Hz). MS
(IT-TOF): [M+H+] calcd for C27H27N5O4 486.21, found 486.19.
1
A yellowish solid (1 mg, 9.4%): H NMR (500 MHz, CDCl3) δ
8.15-8.02 (m, 4H), 7.32 (d, J = 8.5 Hz, 2H), 6.80 (d, J = 8.9 Hz,
2H), 6.63 (s, 2H), 5.01 (dd, J = 11.5, 5.4 Hz, 1H), 3.80 (s, 3H),
3.57 (dd, J = 16.5, 8.4 Hz, 2H), 3.05 (s, 6H); 13C NMR (125
MHz, CDCl3) δ 169.74, 168.74, 165.65, 151.59, 137.98, 134.16,
130.01, 128.17, 119.98, 111.94, 53.05, 52.86, 40.24, 34.23.
HRMS: [M+Na+] calcd for C23H22N6O4 469.1595, found
469.1611.
4.2.21. (S)-methyl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-
(4-(5-phenyl-2H-tetrazol-2-yl)phenyl)propanoate (G6)
4.2.16. (S)-methyl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-
(4-(5-(4-isopropylphenyl)-2H-tetrazol-2-yl)phenyl)propanoate
(G1)
A yellowish solid (27.5 mg, 51.4%): 1H NMR (500 MHz, CDCl3)
δ 8.23 (dd, J = 7.7, 1.8 Hz, 1H), 8.09 (d, J = 8.5 Hz, 1H), 7.56-
7.45 (m, 2H), 7.35 (d, J = 8.5 Hz, 1H), 6.64 (s, 1H), 5.01 (dd, J =
11.5, 5.3 Hz, 1H), 3.81 (s, 2H), 3.58 (ddd, J = 25.9, 14.4, 8.5 Hz,
1H), 13C NMR (125 MHz, CDCl3) δ 169.88, 168.98, 165.33,
138.62, 135.96, 134.31, 130.73, 130.26, 129.10, 127.23, 127.18,
53.21, 52.97, 34.39. HRMS: [M+Na+] calcd for C21H17N5O4
426.1173, found 426.1181.
A yellowish solid (21 mg, 34.5%): 1H NMR (300 MHz, CDCl3) δ
8.11 (dd, J = 19.3, 8.5 Hz, 4H), 7.36 (dd, J = 11.7, 8.4 Hz, 4H),
6.64 (s, 2H), 5.01 (dd, J = 11.1, 5.7 Hz, 1H), 3.80 (s, 3H), 3.57 (t,
J = 8.3 Hz, 2H), 2.98 (dt, J = 13.8, 6.8 Hz, 1H), 1.30 (d, J = 6.9
Hz, 6H); 13C NMR (125 MHz, CDCl3) δ 169.75, 168.86, 165.27,
151.71, 138.36, 135.87, 134.18, 130.10, 127.09, 127.07, 124.63,
120.08, 77.29, 77.04, 76.78, 53.08, 52.84, 34.25, 34.18, 29.66,
23.84. HRMS: [M+Na+] calcd for C24H23N5O4 468.1642, found
468.1655.
4.2.22. (S)-methyl 3-(4-(5-(3,5-difluorophenyl)-2H-tetrazol-2-
yl)phenyl)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)
propanoate (G7)
4.2.17. (S)-methyl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-
(4-(5-(4-fluorophenyl)-2H-tetrazol-2-yl)phenyl) propanoate
(G2)
1
A white solid (16.1 mg, 43.9%): H NMR (500 MHz, CDCl3) δ
8.07 (d, J = 8.4 Hz, 2H), 7.76 (d, J = 6.0 Hz, 2H), 7.36 (d, J = 8.4
Hz, 2H), 6.99-6.90 (m, 1H), 6.64 (s, 2H), 5.01 (dd, J = 11.5, 5.3
Hz, 1H), 3.80 (s, 3H), 3.58 (ddd, J = 25.9, 14.4, 8.5 Hz, 2H); 13C
NMR (125 MHz, CDCl3) δ 169.86, 168.93, 164.49 (d, J = 12.6
Hz), 163.57, 162.56, 139.08, 135.72, 134.32, 130.37, 120.27,
110.23 (dd, J = 20.8, 6.9 Hz), 106.07 (t, J = 25.4 Hz), 53.23,
52.94, 34.42. HRMS: [M+Na+] calcd for C21H15F2N5O4
462.0984, found 462.0975.
A yellowish solid (18.6mg, 42.8%): 1H NMR (300 MHz, CDCl3)
δ 8.23 (dd, J = 8.8, 5.4 Hz, 2H), 8.07 (d, J = 8.5 Hz, 2H), 7.35 (d,
J = 8.5 Hz, 2H), 7.22 (dd, J = 16.4, 7.7 Hz, 3H), 6.64 (s, 2H),
5.01 (dd, J = 11.1, 5.7 Hz, 1H), 3.81 (s, 3H), 3.65-3.52 (m, 2H).
13C NMR (125 MHz, CDCl3) δ 169.88, 168.97, 165.36, 163.96
(d, J = 146.3 Hz), 138.71, 135.88, 134.31, 130.29, 129.26,
123.49, 120.20, 116.28 (d, J = 22.5 Hz), 53.23, 52.96, 34.39.
HRMS: [M+Na+] calcd for C21H16FN5O4 444.1079, found
444.1090.
4.2.23. (S)-methyl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-
(4-(5-(4-(hexyloxy)phenyl)-2H-tetrazol-2-yl)phenyl)
propanoate (G8)
4.2.18. (S)-methyl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-
(4-(5-(naphthalen-1-yl)-2H-tetrazol-2-yl)phenyl) propanoate
(G3)
1
A white solid (13.2 mg, 17.3%): H NMR (500 MHz, CDCl3) δ
8.14 (d, J = 8.8 Hz, 2H), 8.07 (d, J = 8.5 Hz, 2H), 7.33 (d, J = 8.5
Hz, 2H), 7.02 (d, J = 8.8 Hz, 2H), 6.63 (s, 2H), 5.01 (dd, J =
11.5, 5.3 Hz, 1H), 4.03 (t, J = 6.6 Hz, 2H), 3.80 (s, 3H), 3.57
(ddd, J = 25.9, 14.4, 8.5 Hz, 2H), 1.86-1.76 (m, 2H), 1.53-1.45
(m, 2H), 1.36 (d, J = 3.7 Hz, 4H), 0.92 (t, J = 7.0 Hz, 3H); 13C
NMR (125 MHz, CDCl3) δ 169.71, 168.83, 165.13, 161.10,
138.24, 135.86, 134.14, 130.06, 128.51, 120.00, 119.37, 114.87,
68.16, 53.03, 52.82, 34.22, 31.56, 29.15, 25.69, 22.58, 14.01.
HRMS: [M+Na+] calcd for C27H29N5O5 526.2061, found
526.2080.
A yellowish solid (29.2mg, 48.1%): 1H NMR (500 MHz, CDCl3)
δ 8.99 (d, J = 8.5 Hz, 1H), 8.36 (d, J = 7.2 Hz, 1H), 8.17 (d, J =
8.4 Hz, 2H), 8.02 (d, J = 8.2 Hz, 1H), 7.95 (d, J = 8.0 Hz, 1H),
7.67-7.56 (m, 3H), 7.38 (d, J = 8.4 Hz, 2H), 6.65 (s, 2H), 5.03
(dd, J = 11.5, 5.3 Hz, 1H), 3.81 (s, 3H), 3.67-3.51 (m, 2H).13C
NMR (125 MHz, CDCl3) δ 169.90, 168.99, 165.46, 138.69,
135.98, 134.33, 134.11, 131.41, 130.72, 130.33, 128.82, 128.79,
127.58, 126.46, 125.87, 125.37, 124.00, 120.29, 53.23, 52.98,
34.42. HRMS: [M+Na+] calcd for C25H19N5O4 476.1329, found
476.1335.
4.2.24. (S)-methyl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-
(4-(5-(pyridin-4-yl)-2H-tetrazol-2-yl)phenyl) propanoate (G9)

1
A yellow solid (4.8 mg, 22.0%): H NMR (500 MHz, CDCl3) δ
6. Nadinet, M.; Michel, B. Y.; Bertrand, P. and Benhida, R., Med. Chem.
Common., 2012, 3, 263.
8.82 (s, 2H), 8.16 (d, J = 4.7 Hz, 2H), 8.10 (d, J = 8.4 Hz, 2H),
7.38 (d, J = 8.4 Hz, 2H), 6.65 (s, 2H), 5.01 (dd, J = 11.4, 5.3 Hz,
1H), 3.81 (s, 3H), 3.59 (dt, J = 14.2, 5.9 Hz, 2H); 13C NMR (125
MHz, CDCl3) δ 169.71, 168.74, 162.93, 149.89, 139.21, 135.52,
135.30, 134.18, 130.29, 121.21, 120.21, 53.08, 52.78, 34.29.
HRMS: [M+Na+] calcd for C20H16N6O4 427.1125, found
427.1130.
7. Allis, C. D.; Jenuwein, T.; Reinberg, D. and Caparros, M. L.,
Epigenetics, 2007.
8. Fatemi, M.; Fatemi, M.; Pao, M. M.; Jeong, S.; Gal-Yam, E. N.; Egger,
G.; Weisenberger, D. J. and Jones, P. A., Nucleic Acids Res., 2005, 33,
e176.
9. Saxonov, S.; Berg, P. and Brutlag, D. L., Proc. Natl. Acad. Sci. U.S.A.,
2006, 103, 1412.
4.2.25. (S)-methyl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-
(4-(5-(4-(trifluoromethyl)phenyl)-2H-tetrazol-2-yl)phenyl)
propanoate (G10)
10. Gros, C; Fahy, J; Halby, L; Dufau, I; Erdmann, A; Gregoire, J-M;
Ausseil, F; Vispé, S; Arimondo, P. B., Biochimie, 2012, 94, 2280.
11. Yu, N. and Wang, M., Curr Med Chem, 2008, 15, 1350.
12. Song, J.; Rechkoblit, O.; Bestor, T. H. and Patel, D. J., Science, 2011,
331, 1036.
1
A yellowish solid (2.6 mg, 16.6%): H NMR (500 MHz, CDCl3)
δ 8.24 (d, J = 8.8 Hz, 2H), 8.08 (d, J = 8.5 Hz, 2H), 7.35 (d, J =
8.5 Hz, 2H), 7.27 (d, J = 6.9 Hz, 2H), 6.78-6.40 (m, 3H), 5.01
(dd, J = 11.5, 5.3 Hz, 1H), 3.81 (s, 3H), 3.63-3.53 (m, 2H); 13C
NMR (125 MHz, CDCl3) δ 169.72, 168.80, 164.31, 152.85,
138.62, 135.73, 134.16, 130.15, 128.73, 124.32, 120.08, 119.76,
115.62. HRMS: [M+Na+] calcd for C22H17F2N5O5 492.1090,
found 492.1084.
13. Suzuki, T.; Tanaka, R.; Hamada, S.; Nakagawa, H. and Miyata, N.,
Bioorg. Med. Chem. Lett., 2010, 20, 1124.
14. Evans, M. J. and Cravatt, B. F., Chem. Rev., 2006, 106, 3279.
15. Su, Y.; Ge, J.; Zhu, B.; Zheng, Y-G; Zhu, Q. and Yao, S. Q., Curr. Opin.
Chem. Biol., 2013, 17, 768.
16. Lee, J. and Bogyo, M., Curr. Opin. Chem. Biol., 2013, 17, 118.
17. Park, J.; Koh, M. and Park, S. B., Mol. BioSyst., 2013, 9, 544.
18. Gersch, M.; Kreuzer, J. and Sieber, S. A., Nat. Prod. Rep., 2012, 29, 659.
19. Yang, P.-Y.; Liu, K.; Ngai, M. H.; Lear, M. J.; Wenk, M. R. and Yao, S.
Q., J. Am. Chem. Soc., 2010, 132, 656.
4.2.26. (S)-methyl 3-(4-(5-(4-(difluoromethoxy)phenyl)-2H-
tetrazol-2-yl)phenyl)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-
yl) propanoate (G11)
1
A yellowish solid (7.9 mg, 28.5%): H NMR (500 MHz, CDCl3)
20. Shi, H.; Zhang, .C-J; Chen, G. Y. J. and Yao, S. Q, J. Am. Chem. Soc.,
2012, 134, 3001.
δ 8.36 (d, J = 8.1 Hz, 2H), 8.10 (d, J = 8.6 Hz, 2H), 7.79 (d, J =
8.2 Hz, 2H), 7.36 (d, J = 8.5 Hz, 2H), 6.64 (s, 2H), 5.01 (dd, J =
11.5, 5.3 Hz, 1H), 3.81 (s, 3H), 3.62-3.54 (m, 2H); 13C NMR
(125 MHz, CDCl3) δ 169.72, 168.78, 164.00, 138.87, 135.66,
134.17, 130.21, 127.32, 126.00, 125.97, 120.15, 53.07, 52.81,
34.28. HRMS: [M+Na+] calcd for C22H16F3N5O4 494.1047, found
494.1033.
21. Li, Z.; Hao, P.; Li, L.; Tan, C. Y. J., Cheng, X.; Chen, G. Y. J.; Sze, S.
K.; Shen, H.-M and Yao, S. Q, Angew. Chem. Int. Ed., 2013, 52, 8551.
22. Lannine, B. R.; Whitby, L. R.; Dix, M. M.; Douhan, J.; Gilbert, A. M.;
Hett, E. C.; Johnson, T.; Joslynl, C.; Kath, J. C.; Niessen, S.; Roberts, L.
R.; Schnute, M. E.; Wang, C.; Hulce, J. J.; Wei, B. X.; Whiteley, L. O.;
Hayward, M. M. and Cravatt, B. F., Nat. Chem. Biol., 2014, 10, 760.
23. Krishnamurty, R.; Brigham, J. L.; Leonard, S. E.; Ranjitkar, P.; Larson,
E. T.; Dale, E. J.; Merritt, E. A. and Maly, D. J., Nat. Chem. Biol., 2013,
9, 43.
4.2.27. (S)-methyl 3-(4-(5-(3,5-dimethylphenyl)-2H-tetrazol-2-
yl)phenyl)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)
propanoate (G12)
1
A white solid (11 mg, 17.9%): H NMR (500 MHz, CDCl3) δ
24. Su, Y.; S. Pan.; Li, Z.; Li, L.; Wu, X.; Hao, P.; Sze, S. K.; Yao, S. Q.,
Sci. Rep., 2015, 5, 7724.
8.08 (d, J = 8.0 Hz, 2H), 7.85 (s, 2H), 7.34 (d, J = 8.1 Hz, 2H),
7.13 (s, 1H), 6.64 (s, 2H), 5.01 (dd, J = 11.4, 5.2 Hz, 1H), 3.80 (s,
3H), 3.65-3.48 (m, 2H), 2.41 (s, 6H); 13C NMR (125 MHz,
CDCl3) δ 169.71, 168.83, 165.41, 138.63, 138.38, 135.84,
134.15, 132.26, 130.08, 126.80, 124.77, 120.08, 53.03, 52.82,
34.23, 21.26. HRMS: [M+Na+] calcd for C23H21N5O4 454.1486
[M+H+], found 454.1477.
25. Li, Z.; Wang, D.; Li, L.; Pan, S.; Na, Z.; Tan, C. Y. J. and Yao, S. Q, J.
Am. Chem. Soc., 2014, 162, 9990.
26. Brueckner, B.; Boy, R. G.; Siedlecki, P.; Musch, T.; Kliem, H. C.;
Zielenkiewicz, P.; Suhai, S.; Wiessler, M. and Lyko, F., Cancer Res.,
2005, 65, 6305.
27. Schirrmacher, E.; Beck, C.; Brueckner, B.; Schmitges, F.; Siedlecki, P.;
Bartenstein, P.; Lyko, F. and Schirrmacher, R., Bioconj. Chem., 2006, 17,
261.
Acknowledgments
28. Kuck, D.; Singh, N.; Lyko, F. and Medina-Franco, J. L., Bioorg. Med.
Chem., 2010, 18, 822.
Funding was provided by the National Medical Research Council
(CBRG/0038/2013) and Ministry of Education (MOE2012-T2-1-
116/MOE2012-T2-2-051/MOE2013-T2-1-048) of Singapore. We
also acknowledge the financial support from the Singapore-
29. Kolb, H. C. and Sharpless, K. B., Drug Discovery Today, 2003, 8, 1128.
30. Meldal, M. and Tornøe, C. W., Chem. Rev., 2008, 108, 2952.
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6974.
Peking-Oxford
Research
Enterprise
(COY-15-EWI-
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8, 1749.
`RCFSA/N197-1).
33. Song, W.; Wang, Y.; Qu, J.; Madden, M. M. and Lin, Q., Angew. Chem.
Int. Ed. 2008, 47, 2832.
34. Song, W.; Wang, Y.; Qu, J. and Lin, Q., J. Am. Chem. Soc. 2008, 130,
9654.
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