Sequence-specific 2-cyanobenzothiazole ligation

Article abstract of DOI:10.1021/jacs.6b00982

The use of small, natural chemical reporters in conjunction with catalyst-free bioorthogonal reactions will greatly streamline protein labeling in a cellular environment with minimum perturbation to their function. Here we report the discovery of a 2-cyan

Full text of DOI:10.1021/jacs.6b00982

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Communication
Sequence-specific 2-cyanobenzothiazole ligation
Carlo P. Ramil, Peng An, Zhipeng Yu, and Qing Lin
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b00982 • Publication Date (Web): 15 Apr 2016
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Sequence-specific 2-cyanobenzothiazole ligation
Carlo P. Ramil, Peng An, Zhipeng Yu† and Qing Lin*
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Department of Chemistry, State University of New York at Buffalo, Buffalo, New York 14260, United States
Supporting Information Placeholder
ABSTRACT: The use of small, natural chemical reporters
in conjunction with catalystꢀfree bioorthogonal reactions will
greatly streamline protein labeling in cellular environment
with minimum perturbation to their function. Here we report
the discovery of a 2ꢀcyanobenzothiazole (CBT) reactive
peptide tag, CX10R7, from a cysteineꢀencoded peptide
phage library using the phageꢀassisted interrogation of
reactivity method. The fusion of CX10R7 with a protein of
interest allows siteꢀspecific labeling of the protein with CBT
both in vitro and on the surface of E. coli cells. Mutagenesis
studies indicated that the reactivity and specificity of
CX10R7 are attributed to the sequence environment in
which the residues surrounding cysteine help to stabilize the
ligation product.
Scheme 1. Cysteine―CBT ligation.
a) Nꢀterminal cysteine
1) TEV protease
2)
R
S
NC
N
b) Genetically encoded 1,2ꢀaminothiol
c) Cysteine tag (this work)
N
Q
S
I
P
M
V
T
C
C
E
N
Q
S
I
P
M
V
T
C
E
C
Bioorthogonal reactions typically require the use of a nonꢀ
natural chemical reporter, which is introduced into a
biomolecule of interest via natural or engineered
biosynthetic pathways.1 Since the overall bioorthogonal
labeling efficiency is the product of the bioorthogonal
reaction efficiency multiplying the incorporation efficiency, it
varies greatly depending on the specific enzymes and nonꢀ
natural chemical reporters used in the first step. To
circumvent this limitation, sequenceꢀspecific bioorthogonal
reactions involving only natural chemical reporters, e.g.,
natural peptides with unique chemical reactivity, would offer
selective conjugation with designed chemical probes such
as dimaleimide14 and perfluoroaromatics.15 Specifically, we
were intrigued by selective condensation reaction between
Nꢀterminal cysteine and 2ꢀcyanobenzothiazole (CBT)16 as
well as extensive medicinal chemistry literature showing
selective interactions of the enzyme active site cysteine with
the nitrileꢀcontaining ligands.17 Because Nꢀterminal cysteine
does not occur naturally, a TEV protease needs to be used
in order to generate requisite 1,2ꢀaminothiol prior to CBT
ligation (Scheme 1a). Alternatively, the 1,2ꢀaminothiol
moiety can be genetically encoded into proteins in the
thiaprolyl form, which is unmasked using methoxyamine
prior to CBT ligation (Scheme 1b).18 Inspired by these early
reports, we envisioned that a suitable peptide tag encoding
an internal cysteine could undergo the CBT ligation to form
a stabilized thioimidate adduct, affording efficient oneꢀstep
ligation while obviating the need for protease treatment or
protection/deprotection scheme (Scheme 1c). Here we
report the discovery of such a cysteine tag, CX10R7, using
phageꢀassisted interrogation of reactivity method we
reported recently.19 The mutagenesis and nitrile substrate
scope studies revealed that the CX10R7―CBT ligation
displays a balance of reactivity and selectivity. Moreover,
CX10R7 serves as a genetically encodable tag to direct
specific labeling of proteins in vitro as well as on the surface
of live E. coli cells.
a
streamlined approach toward biomolecular labeling.
Indeed, peptide tags comprised of 20 natural amino acids
have been reported, e.g., tetracysteine tag (CCPGCC) that
reacts selectively with the biarsenical fluorescent dyes such
as FlAsH2 and ReAsH3, tetraserine tag (SSPGSS) that
reacts selectively with rhodamineꢀderived bisboronic acid,4
hexahistidine tag that chelates with nickel nitriloacetate,5
and tetraaspartate tag that forms a stable Znꢀcoordination
complex.6 Separately, a large number of peptide tags that
undergo specific enzymeꢀmediated bioconjugation reactions
have also been reported.7 Despite these advances, short
peptide tags that show enzymeꢀfree, monovalent, selective
conjugation reactions with biocompatible chemical reagents
are still rare.8,9
In our search of reactivityꢀbased peptide tags, we turned
our attention to the cysteineꢀcontaining sequences because:
(i) cysteine is relatively infrequent in proteins (2.3%
occurrence in human10); (ii) the sulfhydryl group exhibits
exceptionally high nucleophiliꢀcity, which has been exploited
in the development of many useful cysteineꢀspecific
bioconjugation reactions;11ꢀ13 (iii) the cysteine reactivity can
be further tuned through its sequence environment for
To probe the sequence environment in an unbiased
manner, we designed a randomized 11ꢀmer peptide phage
library, X5CX5 (X = any natural amino acid), in which a
cysteine was placed in the middle and performed a series of
reactionꢀbased panning with biotinꢀPEG4ꢀCBT (1) (Figure
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labile on its own,20 we suspect that the surrounding polar
Table 1. Reactivity study of ubiquitin-CX10R7 mu-
1
2
3
4
5
6
7
8
residues might stabilize the thioimidate through some
noncovalent interactions such as hydrogen bonding. It
should be noted that the BLAST search of the bacterial and
human proteomes did not reveal protein sequences that are
similar to CX10R7 (Table S10).
tants a
S1). Representative clones that survived were subsequently
sequenced (Tables S1ꢀS3). Gratifyingly, two selections
converged to a single peptide sequence encoding 2
cysteines, VTNQECCSIPM, hereafter referred to as
CX10R7. Strikingly, several other selected clones also
encoded more than one cysteine (Table S3). Aside from the
convergence, the selected clones encode predominantly
polar and charged residues surrounding the central cysteine
(Tables S1ꢀS3), presumably stabilizing the cysteineꢀCBT
adducts. It is noteworthy that no significant enrichment was
observed in all three selections despite the sequence
convergence (Tables S4ꢀS6).
To verify the reactivity of the selected peptides, the
sequences that appeared at later rounds were individually
appended to the Cꢀterminus of ubiquitin (Ub)―a small
protein that does not contain cysteine. The resulting
ubiquitinꢀpeptide fusion proteins were expressed in E. coli
and purified by affinity chromatography, and their identities
were confirmed by mass spectrometry (Table S7). As a
negative control, we appended to the ubiquitin Cꢀterminus a
pentapeptide (CG4) encoding a cysteine flanked by two
glycines at each side on the basis that CG4 offers a
sterically unhindered cysteine for sequenceꢀindependent
ligation with CBT. Consistent with the literature reports,16,18
UbꢀCG4 did not form stable adduct with 6ꢀaminoꢀCBT
because Cys is located internally and not at the Nꢀterminus
(entry 1 in Table 1). In contrast, the selected peptide
sequences exhibited varying degree of reactivity towards 6ꢀ
aminoꢀCBT with CX10R7 giving the highest yield (Table S8
and Figure S2). The increased reactivity of CX10R7 is not a
result of additive effect due to the presence of two cysteines
as other diꢀ and triꢀcysteine containing sequences showed
significantly lower yields (Table S8). Interestingly, LCꢀMS
analysis revealed that the adduct masses correspond to a
mixture of one and two molecules of 6ꢀaminoꢀCBT added to
the ubiquitin, and consistent with the thioimidate formation
without subsequent loss of NH3 to form the thiazoline ring
(Figure S2 and Table S9). Since the thioimidate is generally
To probe the effect of sequence environment on the CBT
ligation efficiency, we prepared a series of UbꢀCX10R7
mutants (Table S7) and determined their ligation yields with
6ꢀaminoꢀCBT (Table 1 and Figure S3). When only one
cysteine is present, the yield dropped by more than 4ꢀfold
(compare entries 3 and 4 to 2 in Table 1). The removal of
both cysteines abolished the product formation, indicating
that the CBT reactivity is specific toward cysteine (entry 5 in
Table 1). Since the 7ꢀmer retains the majority of the
reactivity (entry 6 in Table 1), we replaced the surrounding
polar residue (Asn3, Gln4, Glu5, and Ser8) that may serve
as potential Hꢀbond donors or acceptors with alanine, and
found these mutations reduced the reactivity by 3∼10ꢀfold
(entries 7ꢀ10 in Table 1). The Q4A mutant showed the
lowest yield, indicating that Gln4 may play a major role in
stabilizing the thioimidate product. To probe which Cys
adduct is stabilized by Gln4, double alanine mutants were
prepared and their reactivities were examined. Both
mutants produced minimal adducts (entries 11 and 12 in
Table 1), suggesting Gln4 is important for both cysteines
(compare entries 11 to 3; 12 to 4). Since the amide side
chain in Gln4 can serve as either a Hꢀbond donor or
acceptor, we generated the Q4E mutant in which the
carboxylate side chain can only serve as a Hꢀbond acceptor
at pH 8.5, and found that this mutation completely abolished
the CX10R7 reactivity (entry 13 in Table 1), indicating that
Gln4 likely serves as a Hꢀbond donor in stabilizing the
thioimidate product and that the presence of the negatively
charged carboxylate also destabilizes the adduct (compare
entry 13 to 7). Since deamination from the thioimidate to the
thiazoline was not observed, we further tested whether this
could occur when a proximal –NH2 group reminiscent of the
Nꢀterminal cysteine is present. Thus, a lysine scan of
CX10R7 was carried out by replacing three proximal
residues on either side of cysteine with lysine. Upon
incubation with 6ꢀaminoꢀCBT, all lysine mutants showed
reduced reactivity (entries 14ꢀ19 in Table 1). Pairwise
comparisons between the lysine mutants and the alanine
mutants (compare entries 14ꢀ17 to 7ꢀ10) indicate that the
lysine mutants afforded higher yields, consistent with the
notion that the polar side chains help to stabilize the
thioimidate adduct.
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a A mixture of 5 ꢁM ubiquitinꢀpeptide fusion protein, 500 ꢁM
6ꢀaminoꢀCBT, and 1 mM TCEP was incubated in NH4bHCO3
buffer/ acetonitrile (1:1), pH 8.5, at 37 °C for 1 h.
The
conversion was determined by LCꢀMS based on the ion counts
and calculated using the following equation: conversion % =
Iproducts/(IUbꢀpeptide + Iproducts), where Iproducts and IUbꢀpeptide represent
the ion counts of the CBT adducts (both monoꢀ and diꢀCBT
adducts) and Ubꢀpeptide, respectively. Data shown are
averages of two independent measurements
± standard
deviation. See Table S9 and Figure S3 in the Supporting
Information for details.
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We then tested the reactivity of other aryl nitriles toward
UbꢀCX10R7. As expected, the reactivity of nitriles correlates
1
with their
electrophili
city21
(Table 2).
To
2
3
4
5
6
7
8
9
a
mixture of 5 ꢁM ubiquitinꢀpeptide fusion protein, 500 ꢁM
aryl nitrile, and 1 mM TCEP was incubated in NH4HCO3
b
evaluate
buffer/acetonitrile (1:1), pH 8.5, at 37 °C for 1 h.
The
conversion was determined by LCꢀMS as described previously.
Data shown are averages of two independent measurements ±
standard deviation. See Table S11 and Figure S4 in
Supporting Information for details. ND, not determined.
entry
peptide name
peptide
% conꢀ
version
sequence
b
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1
2
CG4
GGCGG
0
The initial aryl nitrile scope studies suggest that the CBT
core is crucial for the ligation and that its moderate
CX10R7
VTNQECCSIPM
VTNQEACSIPM
VTNQECASIPM
VTNQEAASIPM
NQECCSI
54 ± 1
14 ± 4
12 ± 1
0
% conversion b
3
CX10R7ꢀC6A
CX10R7ꢀC7A
CX10R7ꢀC6AꢀC7A
CX10R7ꢀ7mer
CX10R7ꢀN3A
CX10R7ꢀQ4A
CX10R7ꢀE5A
CX10R7ꢀS8A
CX10R7ꢀQ4AꢀC6A
CX10R7ꢀQ4AꢀC7A
CX10R7ꢀQ4E
CX10R7ꢀN3K
CX10R7ꢀQ4K
CX10R7ꢀE5K
CX10R7ꢀS8K
CX10R7ꢀI9K
entry
nitrile
4
UbꢀCX10R7
UbꢀCG4
0
5
1
54 ± 1
6
41 ± 2
13 ± 2
6 ± 1
17 ± 1
21 ± 1
3 ± 1
5 ± 1
0
7
VTAQECCSIPM
VTNAECCSIPM
VTNQACCSIPM
VTNQECCAIPM
VTNAEACSIPM
VTNAECASIPM
VTNEECCSIPM
VTKQECCSIPM
VTNKECCSIPM
VTNQKCCSIPM
VTNQECCKIPM
VTNQECCSKPM
VTNQECCSIKM
2
3
0
0
ND
ND
8
9
4
5
0
ND
10
11
12
13
14
15
16
17
18
19
64 ± 1
4 ± 1
6
7
8
0
0
ND
ND
0
20 ± 2
29 ± 2
25 ± 3
35 ± 8
35 ± 2
42 ± 6
12 ± 1
9
71 ± 1
27 ± 4
CX10R7ꢀP10K
10
11
0
ND
selectivity of the reactive aryl nitriles, we also determined
their ligation yield with UbꢀCG4, a negative control for
sequenceꢀindependent reactivity. Simple nitrileꢀcontaining
heterocycles that are less electrophilic than 6ꢀaminoꢀCBT
including 2ꢀ and 4ꢀcyanopyridine, 8ꢀcyanoꢀquinoline, 2ꢀ
cyanopyrrole and 2ꢀcyanothiophene showed no reactivity
(entries 2ꢀ4, 6 and 7 in Table 2). A more electrophilic nitrile,
2ꢀcyanopyrimidine, showed increased reactivity (entry 5 in
Table 2); however, up to four molecules can be added to
UbꢀCX10R7 based on LCꢀMS analysis (Figure S4a),
suggesting selectivity was lost by reacting with other
nucleophiles, probably the two lysines on the surface of
ubiquitin. 2ꢀCyanothiazole (half of CBT) reacted with Ubꢀ
CX10R7 at a much lower yield (entry 8 in Table 2),
indicating that the benzene ring in the CBT structure is
37 ± 1
20 ± 1
12
13
2 ± 1
0
61 ± 7
22 ± 9
14
15
83 ± 2
52 ± 2
84 ± 3
22 ± 4
important.
Among
three
CBT
bioisosteres,
7ꢀ
methylthiazolo[5,4ꢀb]pyridineꢀ2ꢀcarbonitrile
(2)
showed
higher reactivity but suffered poor selectivity, 2ꢀ
cyanobenzimidazole (3) showed no reactivity, and 2ꢀ
cyanobenzoxazole (4) showed modest yield along with poor
selectivity (entries 9ꢀ11 in Table 2).
electrophilicity ensures exquisite selectivity towards Ubꢀ
CX10R7. To fineꢀtune the CBT reactivity and selectivity, we
placed various electronꢀdonating and electronꢀwithdrawing
groups on the benzene ring. Compound 5 containing two
electronꢀdonating methoxy groups, thus decreasing the
electrophilicity of the nitrile group, showed essentially no
reactivity (entry 12 in Table 2). Substitution of the electronꢀ
withdrawing bromo and nitro groups resulted in higher
Table 2. Reactivity and selectivity of aryl nitriles a
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reactivity toward UbꢀCX10R7; however, selectivity was lost
CX10R7 peptide tag did not yield any ligation product
despite the presence of two free cysteines (Cys48, Cys70)22
on its surface (Figure 1b). Furthermore, streptavidin blot
confirmed that the biotin conjugate was stable and
detectable after denaturing electrophoresis and protein
blotting (Figures 1c and S10).
1
2
3
4
5
6
7
8
(entries 13 and 14 in Table 2). Taken together, the brief
survey of the various aryl nitriles and substituted CBTs
indicate that 6ꢀaminoꢀCBT displays a delicate balance
between reactivity and selectivity. Further testing of the
various reaction parameters for the ligation reaction
between UbꢀCX10R7 and biotinꢀPEG4ꢀCBT (Figure S5)
revealed that the optimal reaction conditions involved the
use of PBS buffer, pH 7.4, at 37 °C for 1 h. The secondꢀ
order rate constant, k2, for this ligation was measured to be
17 Mꢀ1 sꢀ1 (Figure S6), about twice as fast as the
condensation between cysteine and CBT.16 The adducts
appear to be stable at room temperature after removing the
excess biotinꢀPEG4ꢀCBT (Figure S7). Notably, replacing
biotin in the biotinꢀPEG4ꢀCBT with a fluorophore (Figure S8)
abolished the reaction, indicating that the remote functional
groups present on the CBT structure may participate in
secondary interactions with the CX10R7.
a)
CX10R7
extracelluar
periplasm
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b)
overlay
Alexa Fluor 568
DIC
a)
OmpXꢀA7
C70
C48
OmpXꢀ
CX10R7
b)
CX10R7-sfGFP
+2
biotinꢀ
CX10R7
ꢀsfGFP
PEG4ꢀ
CBT
+1
biotinꢀ
PEG4ꢀ
CBT
OmpXꢀ
CX10R7
(3X)
sfGFP
Figure 2. Selective labeling of the CX10R7ꢀtagged membrane
protein OmpX on E. coli surface with biotinꢀPEG4ꢀCBT. (a)
Labeling scheme. (b) Micrographs of E. coli expressing either
OmpXꢀA7 or OmpXꢀCX10R7. The cells were treated with 500
ꢁM biotinꢀ PEG4ꢀCBT for 1 h before PBS wash and subsequent
staining with streptavidinꢀAlexa Fluor 568 for 45 min. The last
row shows the 3× digitally zoomedꢀin images. Scale bar = 2
ꢁm.
To probe whether CX10R7 can direct selective protein
labeling on live cells, we inserted the sequence onto the
outer loop of the bacterial membrane protein OmpX to
obtain OmpXꢀCX10R7. A string of 7 alanines were also
placed in the same location to obtain OmpXꢀA7 as a
negative control. The E. coli cells expressing either OmpXꢀ
CX10R7 or OmpXꢀA7 were treated with 500 ꢁM of biotinꢀ
PEG4ꢀCBT. The biotinylation of OmpX on cell surface was
probed with streptavidinꢀAlexa Fluor 568 and visualized
using confocal microscopy. In Alexa Fluor channel (ex 580
nm, em 585ꢀ712 nm), only cells expressing OmpXꢀCX10R7
showed red fluorescence (Figure 2b). The zoomedꢀin
singleꢀcell image showed that the fluorescent labeled
OmpXꢀCX10R7 accumulated preferentially at the two poles
of E. coli cell (last row in Figure 2b), similar to the previous
observation.23
sfGFP
sfGFP
− Met
c)
Streptavidin
Coomassie
Figure 1. Selective labeling of the CX10R7ꢀtagged sfGFP by
biotinꢀPEG4ꢀCBT. (a) Reaction scheme. The reaction was set
up by incubating 5 ꢀM of CX10R7ꢀsfGFP (or sfGFP), 500 ꢁM
of biotinꢀPEG4ꢀCBT, and 1 mM of TCEP in PBS, pH 7.4, at 37
°C for 1 h. (b) Deconvoluted masses of the product mixtures.
See Table S12 in the Supporting Information for mass
calculations. (c) Streptavidin blot to probe selective
biotinylation of CX10R7ꢀsfGFP by biotinꢀPEG4ꢀCBT. Equal
protein loading was verified by Coomassie blue staining.
In summary, we have identified a unique cysteineꢀ
containing peptide tag, CX10R7, which reacts selectively
with aminoꢀCBT to form stable adducts without the need of
the 1,2ꢀaminothiol group. The CX10R7 tag represents a
new natural chemical reporter with which the CBT ligation
reaction can be performed both in vitro and on live cells.
Mutagenesis and aryl nitrile scope studies indicate that both
the surrounding environment and the requisite CBT
structure are important to the reactivity and selectivity of this
To evaluate whether CX10R7 can serve as a general
peptide tag for selective protein labeling, we fused CX10R7
to the Nꢀterminus of superfolder green fluorescent protein to
obtain CX10R7ꢀsfGFP (Figures 1a and S9). After incubating
with biotinꢀPEG4ꢀCBT in PBS buffer, pH 7.4, at 37 °C for 1
h, CX10R7ꢀsfGFP gave the desired ligation products in 65%
yield (Figure 1b). As a control, wildꢀtype sfGFP without the
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ASSOCIATED CONTENT
Supporting Information
Supplemental figures and tables, synthetic schemes,
experimental procedures, characterization of all new
compounds and proteins. This material is available free of
charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
Qing Lin (qinglin@buffalo.edu).
Present Addresses
†Sichuan University College of Chemistry, China.
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
We gratefully acknowledge the NSF (CHEꢀ1305826) and
NIH (GM 85092) for financial support. We thank Prof.
Wenshe Liu at Texas A&M University for generously
sharing the pBADꢀsfGFPꢀ134TAG and pETDuetꢀOmpXꢀA7
plasmids.
(20) MacFaul, P. A.; Morley, A. D.; Crawford, J. J. Bioorg Med
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S.; Crane, S.; Berthelette, C. Bioorg. Med. Chem. Lett. 2007, 17,
998ꢀ1002.
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Wooley, K. L.; Liu, W. R. ACS Chem. Biol. 2013, 8, 1664ꢀ1670.
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