"Click-made" biaryl-linker improving efficiency in protein labelling for the membrane target protein of a bioactive compound

Article abstract of DOI:10.1039/c0ob00843e

We report on the design, synthesis and assessment of a novel biaryl-linked (BArL) molecular probe for the exploration of low-abundant target proteins for bioactive compounds based on the activity based protein profiling (ABPP) approach. Surprisingly, the

Full text of DOI:10.1039/c0ob00843e

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“Click-made” biaryl-linker improving efficiency in protein labelling for the
membrane target protein of a bioactive compound†
Yoko Nakamura,a Sho Inomata,a Makoto Ebine,a Yoshiyuki Manabe,a Izumi Iwakurab and Minoru Ueda*a
Received 6th October 2010, Accepted 28th October 2010
DOI: 10.1039/c0ob00843e
We report on the design, synthesis and assessment of a novel
biaryl-linked (BArL) molecular probe for the exploration of
low-abundant target proteins for bioactive compounds based
on the activity based protein profiling (ABPP) approach.
Surprisingly, the performance of the BArL probe was better
than that of the stepwise tagging approach that is considered
to be the most effective method used in ABPP study.
molecular probe for efficient tagging of low-abundance membrane
proteins. In this paper, we report the advantages of a biaryl-
linked (BArL) molecular probe which is of synthetic utility
using CuAAC9 “click” chemistry and provides remarkably high
performance in labelling of membrane target proteins.
Activity-based protein profiling (ABPP)1 has become a powerful
strategy for understanding biological processes in living cells and
organisms. Because most target proteins for bioactive natural
products are unknown, the prospect of using ABPP with a
bioactive natural product to induce a physiologically intriguing
phenomenon would launch a new field of chemical biology.
Chemical biology on jasmonate, an important plant hormone,
has evolved into one of the cutting-edge topics in plant science
since the identification of COI1-JAZs as a core signalling module.2
We identified b-D-glucopyranosyl 12-hydroxyjasmonic acid 1
as a bioactive metabolite which controls plant nyctinasty, a
rhythmic movement of plant leaves controlled by a biological
clock (Figure S1).3 Recently, our ABPP approach using probe
2, which is designed according to a previous FITC-probe used
in a fluorescence imaging study,4 identified a membrane target
protein for jasmonateglucoside 1 (MTJG).5 MTJG is expected to
be involved in shrinking of the motor cell which leads to nyctinastic
leaf-closure.6 The MTJG is completely different from COI1,7 a
cytosolic receptor of jasmonate. This result implies the existence
of an unknown mode of action on the jasmonates. However,
the limited availability of trace MTJG makes its identification
difficult. Certain classes of physiologically important proteins,
including membrane-associated and low-abundance proteins, are
difficult targets for chemical biology because of their low efficiency
in specific chemical tagging. For example, it is well known
that photolabelling efficiency for cross-link formation between a
photoaffinity probe and the target protein is always as low as a
few percent.8 Thus, it is essential to develop a high-performance
We employed the CuAAC reaction for a tractable probe
synthesis. CuAAC streamlines probe synthesis with concomitant
construction of a rigid biaryl linker moiety (Scheme 1). The use of
a “rigid” linker usually causes a difficulty in probe synthesis, such
as insolubility, difficult purification, and low yield.
CuAAC strategy enables these difficulties to be circumvented.
Alkylation of azide-containing 3 and subsequent CuAAC using
a biotin-containing ethynylaryl unit10 5 with triazole ligand11 10
gave BArL probe 6 without byproducts. In photoaffinity labelling
experiments against living motor cells of Samanea saman, BArL
probe 6 provided a distinct improvement of labelling efficiency
over triglycyl 25 (Fig. 1a). BArL probe 6 biotinylated MTJG
in a concentration-dependent manner between 1 and 100 mM,
and the efficiency in biotinylation was estimated to be twenty
times as much as that of 2. The specific binding was confirmed
by competitive inhibition using an excess amount of naturally
occurring 1 (Figure S2).
We found that this enhancement was due to the stronger affinity
of 6 with MTJG than that of 2. Thus, when shrinking rates of
motor cells, induced by structure recognition of 1 by MTJG,5 were
compared between 2 and 6, greater shrinking was observed for 6
(Figure S3). The high performance of the BArL probe may come
from the rigidity of its molecular shape due to the insertion of a
biaryl linker system which projects the pharmacophore away from
the molecular tag enough for the efficient binding with its target
protein.12 This model was supported by a comparison between the
most stable conformations of 2 and 6 obtained by MM and DFT
aDepartment of Chemistry, Tohoku University, 6-3 Aramaki-aza Aoba,
Aoba-ku, Sendai 980-8578, Japan. E-mail: ueda@m.tohoku.ac.jp; Fax: +81
22 795 6553; Tel: +81 22 795 6553
bInnovative Use of Light and Materials/Life, PREST, JST, 4-1-8 Honcho,
Kawaguchi, Saitama 332-0012, Japan
† Electronic supplementary information (ESI) available: Fig. S1–S8,
materials and methods, experimental procedures and NMR spectra. See
DOI: 10.1039/c0ob00843e
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Fig. 1 (a) Photoaffinity labelling and chemiluminescence detection of MTJG by 2 (100 mM) or 6 (1–100 mM).(b) Stereostructures of 2 and 6 (CP:
cyclopentanone; BP: benzophenonyl; BT: biotinyl). The plausible conformer was obtained by MM and DFT(B3LYP/6-31G*) calculations. Each atom
is represented by colors as follows: carbon (black), oxygen (red), nitrogen (blue), sulfur (green), fluorine (purple). (c) Important ROE correlations in 2
and 6(See also Figures S4 and S5).
calculations13 (Fig. 1b) coupled with ROESY experiments (Fig.
1c, S4 and S5). Probe 2 has a globular conformation in which
the benzophenone group, molecular tag, and pharmacophore are
folded in close proximity. On the other hand, probe 6 has a
unique T-shaped conformation in which the benzophenone group,
molecular tag, and pharmacophore are spatially separate from
each other. This is because rigid biaryl linker moiety prevents
the formation of folded conformation. In addition to the ease of
synthesis, the BArL probe had a marked improvement in protein
labelling efficiency because of its unique T-shaped conformation.
The stepwise tagging approach using on-the-cell CuAAC14 is
also known as a powerful method for tagging of a membrane
protein (Figure S6). Protein labelling by the sterically minimal
azide probe and subsequent introduction of tag using CuAAC was
expected to improve its affinity to the target protein and enhance
labelling efficiency.15 Surprisingly, the labelling performance of
BArL probe was better than that of the stepwise tagging approach
in protein labelling. BP-containing azide4 was used for affinity-
based introduction of an azide handle to MTJG on a living cell.
Successive CuAAC with alkyne5 was carried out at 4 ◦C to provide
specific tagging of MTJG. The reaction conditions of CuAAC
were determined on the basis of in vitro examination using 4 and
5 (Figure S7). Considering the toxicity of copper to the living
cell,16 CuAAC conditions that require 500 mM copper(I) were
employed.14,17 Copper ion provokes a decrease in the number
of living cells during CuAAC reactions. The proteins released
from the dead cells easily form insoluble copper aggregates during
CuAAC. Thus, it is important to maintain the number of living
cells during the reaction. The copper ion will increase the loss of
the target protein because of formation of insoluble aggregates. A
remarkable decrease in cell viability was observed for CuAAC at
a higher temperature (30 ◦C) (data not shown). We compared the
labelling performance with that of the BArL probe approach using
6. Unexpectedly, the BArL probe 6 turned out to give labelling
efficiency twice as good as stepwise tagging using 4 with 5 (Fig.
2a). Thus, it was concluded that the BArL probe exhibited the best
performance in the labelling of the membrane target among three
different approaches using 2, 4 and 6.
Next, we synthesized four biotin-bearing BArL probes with
different reactive functionalities, such as benzophenone (BP: 6),
trifluoromethyldiazirine (TFMD: 11), iodoacetyl (IA: 12), and to-
syloxyacetyl (Ts: 13) and compared MTJG-labelling performances
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is considered to be better for living cells. However, in this case,
TFMD probe 11 gave the best result among these four probes (Fig.
2b). In this case, the labeling efficiency of TFMD functionality may
be superior to those of BP and IA because there would be little
difference among the affinity of three photoaffinity probes (11–13)
with MTJG. The use of the BArL probe and screening for the best
reactive functionality realized as much as ca. 70-fold enhancement
in labelling efficiency compared to 2.
Conclusions
In summary, we have found important advantages of a “click”-
made BArL probe, which has remarkably high performance in
protein labelling, attributed to its unique T-shaped conformation.
The efficiency surpassed that of the stepwise tagging approach for
the MTJG protein. “Click”-made BArL probes promise to make
ABPP-based exploration of trace membrane targets for bioactive
compounds a powerful method.
Notes and references
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Scheme 1 CuAAC synthesis of BArL probes: a = benzophenone; b =
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Fig. 2 (a) Photoaffinity labelling and chemiluminescence detection of
MTJG by a stepwise approach using 4 with 5 or the BArL approach using
6. (b) Chemiluminescence detection of labeled MTJG using 6, 11, 12 and
13.
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probes (Scheme 1). All the CuAAC couplings proceeded in good
yields (ca. 70–90%).
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◦
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specific binding with 13 and 27 kDa proteins (Figure S8). Ts 13
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This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 83–85 | 85
©