Genetic incorporation of a 2-naphthol group into proteins for site-specific azo coupling

Article abstract of DOI:10.1021/bc400168u

The 2-naphthol analogue of tyrosine, 2-amino-3-(6-hydroxy-2-naphthyl) propanoic acid (NpOH), has been genetically introduced into proteins in Escherichia coli. This is achieved through the directed evolution of orthogonal aminoacyl-tRNA synthetase/tRNA pairs that selectively charge the target amino acid in response to the amber stop codon, UAG. Moreover, chemoselective azo coupling reactions have been revealed between the 2-naphthol group and diazotized aniline derivatives that are substituted with an electron donating moiety. The coupling reactions required a very mild condition (pH 7) with great reaction rate (less than 2 h at 0 C), high efficiency, and excellent selectivity.

Full text of DOI:10.1021/bc400168u

Communication
pubs.acs.org/bc
Genetic Incorporation of a 2‑Naphthol Group into Proteins for Site-
Specific Azo Coupling
Shuo Chen and Meng-Lin Tsao*
Chemistry and Chemical Biology Group, School of Natural Sciences, University of California, Merced, 5200 North Lake Road,
Merced, California 95343, United States
*
S Supporting Information
ABSTRACT: The 2-naphthol analogue of tyrosine, 2-amino-3-(6-hydroxy-2-naphthyl)propanoic acid (NpOH), has been
genetically introduced into proteins in Escherichia coli. This is achieved through the directed evolution of orthogonal aminoacyl-
tRNA synthetase/tRNA pairs that selectively charge the target amino acid in response to the amber stop codon, UAG. Moreover,
chemoselective azo coupling reactions have been revealed between the 2-naphthol group and diazotized aniline derivatives that
are substituted with an electron donating moiety. The coupling reactions required a very mild condition (pH 7) with great
reaction rate (less than 2 h at 0 °C), high efficiency, and excellent selectivity.
2
7,28
INTRODUCTION
tyrosine residues.
Despite the high reaction yield (>90%)
■
and fast reaction rate (2 h at 4 °C), the coupling reaction of
tyrosine was restricted to a basic environment (pH 9) and
required the presence of a strong electron withdrawing group at
the para-position of the diazotized aniline.
Genetic incorporation of noncanonical amino acids (NCAAs)
into proteins is a powerful method of introducing novel
chemistries into proteins at chosen sites,
1
,2
for which it
27−29
Moreover, there
enhances our ability to manipulate protein structures and
functions as well as to study post-translational modifications.
Several chemical transformations have been utilized at the sites
of NCAAs for selective bioconjugations. For instance, the
addition of an azido functionality into proteins provides a
perfect position for modifications through the Staudinger
ligation reaction, Cu(I) catalyzed azide−alkyne cycloaddition,
or the strain-promoted cycloaddition to conjugate with
triarylphosphines, terminal alkynes, or cyclooctynes, respec-
is no selectivity among all of the solvent-exposed tyrosine
residues. Thus, in order to utilize azo coupling for protein
modifications in a generally site specific manner, we envisioned
that diazotized aniline derivatives bearing an electron donating
group would selectively react with a genetically introduced 2-
naphthol group under neutral pH (Scheme 1). This hypothesis
is based on the coupling reaction’s electrophilic aromatic
substitution mechanism, for which electron-rich diazotized
anilines would have a reduced reactivity toward tyrosine due to
decreased electrophilicity; meanwhile, 2-naphthol would have a
faster reaction rate than the phenol site chain of tyrosine since
the former serves as a better nucleophile. Herein, we are
pleased to report our findings for the selective azo coupling
between the electron-rich diazonium compounds and a 2-
naphthol group that is genetically introduced into proteins.
3
−11
tively.
formation from a ketone or an aldehyde group,
cyanobenzothiazole condensation initiated with an 1,2-amino-
Other examples include the hydrazone or oxime
12−14
the
1
5
thiol, the photoinduced 1,3-dipolar cycloaddition between
1
6
tetrazoles and alkenes, the palladium catalyzed cross-coupling
17−20
with an iodophenyl or a boronophenyl group,
and the
2
1
cycloaddition of a norbornene moiety with tetrazines. These
chemical transformations, however, were sometimes hampered
by limitations such as speed, toxicity, and synthetic
RESULTS AND DISCUSSION
■
2
2−24
The first step of our study was to add the 2-naphthol
functionality into proteins. 2-Amino-3-(6-hydroxy-2-naphthyl)-
propanoic acid (NpOH in Scheme 2), the 2-naphthol analogue
accessibility.
Thus, this study aims to utilize the site
selective azo coupling as an alternative biocompatible method
for protein modifications.
Azo coupling, often used for producing azo dyes, has been
historically applied to the modification of proteins for immune
Received: April 4, 2013
Revised: August 22, 2013
2
5,26
tolerance studies.
The coupling reaction reportedly took
place very selectively at the phenol moieties of solvent-exposed
©
XXXX American Chemical Society
A
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Bioconjugate Chemistry
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Scheme 1. Azo Coupling at the Site of Phenol or 2-Naphthol
To test the ability of the evolved synthetases for selective
incorporation of NpOH into proteins, an amber stop codon
was substituted at a permissive site (Lys7) in the gene for a
34
mutant Z domain of protein A with a C-terminal 6 × His-tag.
Cells transformed with tRNACUA, NpOH-RS1, and this Z
domain gene were grown in the presence of 1 mM NpOH in
2
+
LB medium, and the mutant protein was purified by Ni
affinity column and subsequently analyzed by SDS-PAGE and
ESI-MS (Figure 1). The yield of the Z-domain mutant was
Scheme 2. Structure of NpOH
Figure 1. Incorporation of NpOH into a target protein. (a) SDS-
PAGE analysis of Lys7→TAG amber mutant of Z-domain protein
expressed under different conditions. Lane 1: molecular mass marker;
lane 2: expression with WT MjTyrRS; lane 3: expression with NpOH-
RS1 in the presence of NpOH; lane 4: expression with NpOH-RS1 in
the absence of NpOH. The SDS-PAGE gel was stained with
SimplyBlue SafeStain reagent. (b) ESI-MS deconvolution spectrum
of NpOH incorporated Z-domain protein: peak a is corresponding to
the Z domain protein without the first Met residue, peak b is the
acetylated product of peak a, and peak c is the full length protein.
of tyrosine, was chosen as the target amino acid and was
30
synthesized according to a published method. NpOH was
incorporated into proteins in Escherichia coli by means of a
unique pair of amber suppressor tRNACUA/aminoacyl tRNA
synthetase (MjTyrRS) derived from a Methanococcus jannaschii
Tyr
tRNA /TyrRS pair. The specificity of MjTyrRS was altered so
roughly 7 mg/L culture with the presence of NpOH, but was
insignificant without NpOH (Figure 1a), indicating a very high
fidelity in the incorporation of the unnatural amino acid.
Moreover, the ESI-MS spectrum showed peaks at m/z =
7847.0, 7889.1, and 7978.3 (Figure 1b), which match the
expected molecular weight for the full length NpOH-
incorporated Z-domain mutant (m/z = 7978.6) and that for
this mutant protein with the loss of its first methionine (m/z =
7847.4) and its subsequent post-translational acetylation
that the synthetase specifically charges with NpOH but none of
the endogenous amino acids. This was achieved through the
screening of a MjTyrRS library which was constructed based on
the crystal structure of a mutant MjTyrRS that selectively
3
1
charges with 3-(2-naphthyl)alanine. The mutations at
residues Pro1 , Ile , and Gln were preserved in this library,
58
159
162
3
2
65
160
and random mutations were introduced at Leu ,Leu , His
,
Tyr1 , and Ala to accommodate the hydroxyl group in
61
167
2
86
5
NpOH. In addition, an Arg mutation was introduced and
maintained in the library to substantially increase the amber
codon suppression rate. To evolve the synthetases, a one
product (m/z = 7889.5). Furthermore, under 335 nm
excitation, the purified Z-domain mutant displayed strong
blue fluorescence with an emission maximum at 423 nm, a new
feature acquired from the successful addition of NpOH (see
Figure S1 in Supporting Information). Similar results were
obtained when NpOH-RS1was replaced with NpOH-RS2.
After NpOH was successfully introduced into proteins, our
next step was to uncover the proper conditions for selective azo
couplings. We started at the small molecule level by using p-
cresol and 2-naphthol as the model molecules to mimic the
reactions for tyrosine and NpOH, respectively. A series of
aniline derivatives were converted to their corresponding
diazonium salts after treatment with aqueous p-toluenesulfonic
acid and sodium nitrite. The diazotized aniline derivatives were
then mixed with p-cresol or 2-naphthol in a buffered solution at
pH 7 or pH 9 under an ice bath for 5 min. To avoid
complications due to the assumed instability of the diazonium
compounds, which were used in excess quantities, the coupling
reactions were subsequently stopped by adding sodium azide to
quench the remaining diazonium salts. The progress of the
reaction was easily observed from the appearance of the unique
bright color derived from the azo products. As depicted in
32
33
plasmid dual positive/negative selection system was applied,
for which the directed evolution is based on suppression of an
amber stop codon in the chloramphenicol acetyltransferase
gene in the presence of NpOH for positive selections, and
suppression of an amber codon for the production of uracil
phosphoribosyl-transferase in the presence of 5-fluoruracil but
the absence of NpOH for negative selections. After series
rounds of alternatively positive and negative selections, two
mutant MjTyrRS, namely, NpOH-RS1 and NpOH-RS2, were
identified. DNA sequencing revealed the following mutations in
the evolved synthetases compared to the wild-type MjTyrRS:
Y32E, L65T, D158S, I159A, H160P, Y161T, L162Q, A167W,
and D286R in NpOH-RS1; Y32E, L65 V, K90E, I159A,
H160W, Y161G, L162Q, A167I, and D286R in NpOH-RS2.
Interestingly, the D158S mutation in NpOH-RS1 and the K90E
mutation in NpOH-RS2 were not originally included in the
library and were possibly acquired by self-mutations during the
selections. Similar observations have been reported for the
3
3
selection system.
B
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Communication
Table 1, the azo coupling reaction selectively took place only
for 2-naphthol but not p-cresol at pH 7 when an electron
Table 1. Percent Yields for the Azo Coupling of p-Cresol and
a
2-Naphthol with Diazotized Aniline Derivatives
X
p-cresol (%)
2-naphthol (%)
NHCOCH3
0
0
0
76
76
77
CH3
H
−
b
b
SO3
High
High
CN
74
81
85
88
NO2
a
Based on the weights of the precipitated azo adducts produced from
2 μmol of p-cresol or 2-naphthol and 15 μmol of a diazonium salt.
No precipitation is formed but a high yield is reported here judged by
1
b
Figure 2. Chemoselective azo coupling for NpOH-incorporated
proteins. (a) NpOH incorporated Z-domain protein can be selectively
modified through the indicated azo coupling reactions. (b) ESI-MS
deconvolution spectrum for the azo coupling of NpOH incorporated
Z-domain (8 nmol) with diazotized 4′-aminoacetanilide (1.5 μmol).
Expected mass at 8008.6, 8050.6, and 8139.8 Da. (c) SDS-PAGE
analysis for the PEGylation of Z-domain proteins through azo
coupling. Lane 1: molecular mass marker; lane 2: NpOH Z-domain
mutant before the coupling reaction; lane 3: the azo coupling product
for NpOH Z-domain (6 nmol) coupled with the PEG5000 containing
reagent (2.5 μmol), the target protein band is emphasized with an
arrow sign; lane 4: the azo coupling between a control Z-domain
protein (6 nmol) and the PEG5000 containing reagent (2.5 μmol). The
SDS-PAGE gel was stained with SimplyBlue SafeStain reagent.
the appearance of the intense bright color from the azo adduct.
donating group was substituted at the para-position of the
diazonium reagents. Additionally, we also examined the azo
coupling of the electron-rich diazonium salts with phenyl-
alanine, histidine, tryptophan, or deoxyribonucleotide triphos-
phate mixtures (dNTPs). None of them displayed the color of
the azo product at pH 7. Unsurprisingly, the coupling reaction’s
preference toward 2-naphthol over p-cresol reduced drastically
when the diazonium salts were substituted with an electron
withdrawing group or when the reaction medium is kept at a
basic environment of pH 9 (see Figure S2 and S3 in Supporting
Information).
Ultimately, the selective azo coupling was proven to be
effective at the protein level as well. For instance, the NpOH
incorporated Z-domain was incubated with diazotized 4′-
but no NpOH. Results from the ESI-MS analysis of the control
reactions yielded only the mass peaks corresponding to the
unmodified protein, meaning that the azo adduct formation was
negligible for the control protein (see Figure S4 in Supporting
Information).
A plausible application for site-specific PEGylation via azo
coupling is demonstrated with a diazonium reagent bearing a
poly(ethylene glycol) moiety (Figure 2a, R = PEG5000). As
depicted in Figure 2c, the PEGylated product was obtained
efficiently for the conjugation of the NpOH incorporated
protein (lane 3), while applying the same condition to the
control protein did not yield any adduct (lane 4). This indicates
again that the coupling reaction is highly selective and also that
it can be utilized to modify proteins with not only small
molecules but also macromolecules.
aminoacetanilide (Figure 2a, R = CH ) for 90 min at 0 °C and
3
neutral pH. The product was analyzed by ESI-MS spectroscopy
and the result is shown in Figure 2b. Compared to the MS
peaks from the protein measured before the coupling reaction
(
peaks a, b, and c in Figure 1b), a mass increment of +161.2 Da
for each of them was expected at 8008.6, 8050.6, and 8139.8 Da
due to the addition of the azo moiety. Indeed, the major peaks
showed in Figure 2b at 8008.3 and 8050.0 Da can be attributed
unambiguously to these assignments. Examining the MS
spectra further, the mass peaks for the unmodified Z-domain
mutant at 7847 and 7889 Da were insignificant in Figure 2b.
This suggests that the coupling reaction is very efficient and has
a near quantitative conversion yield (>96%, estimated from the
heights of the MS peaks). In addition to the mass spectroscopic
evidence, the modified protein displayed a distinct pink color
with a broad UV−visible absorption band at 520 nm (see
Figure S5 in Supporting Information). This strongly supports
that the azo coupling took place at the NpOH sites. We further
examined the kinetics of the reaction by monitoring at this
unique visible band. A pseudo first-order kinetics was obtained
CONCLUSION
■
In summary, we have evolved MjTyrRS synthetase mutants to
genetically encode 2-amino-3-(6-hydroxy-2-naphthyl)-
propanoic acid (NpOH) in Escherichia coli with high fidelity
and efficiency. We also demonstrated that the 2-naphthol side
chain in NpOH can serve as the target for site specific protein
modifications through the selective azo coupling reaction under
neutral pH using diazonium reagents with an electron donating
substituent. Our findings provide an alternative way for
biocompatible chemical transformation of proteins. The
selective azo coupling reported here could be potentially
applied to reactions other than protein modifications and could
extend to other aromatic compounds that undergo a faster
electrophilic substitution than 2-naphthol does. Moreover, the
−3
−1
with a rate constant of (8.4 ± 0.4) × 10
conditions of 140 μM protein and 30 mM diazonium salt at pH
in a 4 °C cold room (see Figure S6 in Supporting
s
under the
7
Information). To further demonstrate that the coupling
reaction is triggered by the presence of NpOH, azo coupling
reactions were tested for a Z-domain control protein (Figure
1
a, lane 2), in which the NpOH residue was replaced with a
tyrosine so that the control protein contains tyrosine residues
C
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Bioconjugate Chemistry
Communication
chemoselective azo coupling could potentially bring forth a
photoswitchable functionality into proteins, where the photo-
switch can be activated by nonultraviolet light for which the
excitation wavelength can be tuned in accordance with the
diazonium compounds. Ongoing directions include the kinetic
study of the azo coupling reactions, the transformation of
several established procedures for selective tyrosine modifica-
of blomolecules in living systems. J. Am. Chem. Soc. 126, 15046−
1
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ASSOCIATED CONTENT
Supporting Information
Experimental details about the directed evolution, protein
expression and purification, and the azo coupling reactions with
small molecules and proteins. This material is available free of
charge via the Internet at http://pubs.acs.org.
■
*
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(
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AUTHOR INFORMATION
Corresponding Author
E-mail: mtsao@ucmerced.edu.
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*
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Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
The authors are grateful for financial support from University of
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ABBREVIATIONS
selective Suzuki-Miyaura protein modification at genetically encoded
■
aryl halides. Chem. Commun. 47, 1698−1700.
NpOH, 2-amino-3-(6-hydroxy-2-naphthyl)propanoic acid; Tyr,
tyrosine; RS, aminoacyl tRNA synthetase; PEG, poly(ethylene
glycol)
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