Esterification of an Unnatural Amino Acid Structurally Deviating from Canonical Amino Acids Promotes Its Uptake and Incorporation into Proteins in Mammalian Cells

Article abstract of DOI:10.1002/cbic.201000436

It's a cover up: Unnatural amino acids (UAAs) with noncanonical side chains can be efficiently transported into mammalian cells when the carboxyl group of the UAA is masked with selected ester groups. This greatly increases the UAA's uptake rate and intra

Full text of DOI:10.1002/cbic.201000436

DOI: 10.1002/cbic.201000436
Esterification of an Unnatural Amino Acid Structurally Deviating from
Canonical Amino Acids Promotes Its Uptake and Incorporation into
Proteins in Mammalian Cells
Jeffrey K. Takimoto, Zheng Xiang, Ji-Yong Kang, and Lei Wang*[a]
Genetically encoded unnatural amino acids (UAAs) enable
novel chemical and physical properties to be selectively intro-
duced into proteins directly in live cells; this provides great po-
tential for addressing biological questions at the molecular
and cellular level in native settings.[1–3] UAAs have been geneti-
cally incorporated into proteins in mammalian cells by using
orthogonal tRNA-codon–synthetase sets,[4–6] yet the current
low incorporation efficiency hinders their effective application.
Efforts to improve efficiency have focused on optimizing the
expression and activity of the orthogonal tRNA and synthe-
tase,[5–7] whereas the bioavailability of the UAA inside mamma-
lian cells, a prerequisite for incorporation, has not been ad-
dressed. In addition, there are many UAAs, such as glycosylat-
ed and phosphorylated amino acids, that have not been ge-
netically incorporated into proteins successfully. These amino
acids can be invaluable for studying the contribution of post-
translational modifications of protein function and the role of
a target protein in cellular signal transduction. Among many
reasons, the inability of the UAA to enter cells prevents the
evolution of a mutant synthetase specific for the UAA by using
cell-based selections or screens.[2] Here, we show that an UAA
that deviates structurally from the canonical amino acids in
side chain could not be efficiently transported into mammalian
cells, but that masking the carboxyl group of the UAA as an
ester greatly increased the rate of cellular uptake and intracel-
lular concentration of the UAA. This resulted in a significant in-
crease in the incorporation of this UAA into proteins in mam-
malian cells. Among three esters tested, acetoxymethyl ester
(AME) yielded the highest UAA incorporation efficiency, with a
concomitant reduction in the UAA required in the growth
medium.
pH, thereby making it difficult for them to cross the cell mem-
brane. Esters have been widely used in prodrugs to derivatize
carboxyl, hydroxyl, and thio functionalities so as to make the
parent drugs more membrane permeable.[10] In particular, Tsien
et al. pioneered the use of AME to enhance the cellular uptake
of various charged molecules, such as carboxylate-containing
Ca2+ chelators and phosphate-containing second messen-
gers.[11–12] We reasoned that masking the carboxyl group of the
UAA with an ester would convert the UAA into a protonated
weak base, which has a higher percentage of neutral form and
increased lipophilicity for crossing the membrane. Once inside
the cell, intracellular esterases can cleave the ester to regener-
ate the original UAA for incorporation.
We demonstrated this strategy with the UAA 2-amino-3-(5-
(dimethylamino)naphthalene-1-sulfonamide)propanoic
acid
(DanAla, 1), which does not resemble and is bulkier in size
than any canonical amino acid. Its fluorescence property also
makes it an attractive candidate to develop optical reporters
for imaging protein activities in live cells. We synthesized the
methyl, ethyl, and acetoxymethyl esters of DanAla by using
the methods shown in Scheme 1B. Compound 5 was synthe-
UAAs with side chains similar to canonical amino acids can
be transported into cells by endogenous amino acid transport-
ers,[3,8] which are relatively nonspecific for substrates.[9] Howev-
er, UAAs that deviate significantly from canonical amino acids
in side chain structure might not be recognized by these trans-
porters. Cell membranes are more permeable to neutral than
charged molecules. As zwitterions, UAAs have only a very
small proportion present in the neutral form at physiological
[a] J. K. Takimoto,+ Dr. Z. Xiang,+ J.-Y. Kang, Prof. L. Wang
The Jack H. Skirball Center for Chemical Biology and Proteomics
The Salk Institute for Biological Studies
10010 N. Torrey Pines Road, La Jolla, CA 92037 (USA)
Fax: (+1)858-597-0855
E-mail: lwang@salk.edu
Scheme 1. A) Structures of DanAla and DanAla esters. B) Synthetic approach
to DanAla esters. Reagents and conditions: a) MeI, DIPEA, DMF, 12 h, 87%
for 6; EtI, DIPEA, DMF, 12 h, 75% for 7; b) TFA, CH2Cl2, 100%; c) bromomethyl
acetate, DIPEA, CH3CN, 81%; d) BnBr, DIPEA, DMF, 08C to RT, 76%; e) Boc2O,
DMAP, Et3N, CH2Cl2, 97%; f) Pd/C, H2, MeOH; g) bromomethyl acetate, DIPEA,
CH3CN, 84% over two steps; h) TFA, CH2Cl2, 100%.
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201000436.
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ChemBioChem 2010, 11, 2268 – 2272

sized from Boc-Dap-OH and dansyl chloride according to the
known procedure.[13] Alkylation of 5 with iodomethane and io-
doethane gave intermediates 6 and 7, which afforded DanAla-
OMe (2) and DanAla-OEt (3), respectively, after deprotection.
However, in the presence of bromomethyl acetate and N,N-dii-
sopropylethylamine (DIPEA), compound 5 was transformed to
the undesired cyclization product 8, instead of Boc-DanAla-
OAM. Therefore, the carboxyl group and the sulfonamide
group were protected with the benzyl group and the Boc
group,[14] respectively. After debenzylation, carboxylic acid 10
was transformed into acetoxymethyl ester 11,[15] which fur-
nished DanAla-OAM (4) after removal of the Boc groups.
To determine if the DanAla esters could enhance the incor-
poration of DanAla into proteins in mammalian cells, we used
an in cellulo fluorescence assay to evaluate the incorporation
efficiency of DanAla.[6–7] The incorporation of DanAla into
green fluorescent protein (GFP) was measured by using a
stable clonal HeLa cell line, into which a GFP gene containing
a premature UAG stop codon at a permissive site (Tyr182) was
integrated. The cells were transfected with the orthogonal
tRNA–synthetase pair specific for DanAla,[13] and the DanAla or
DanAla ester was added to the growth medium. The incorpo-
ration of DanAla at the UAG182 position results in full-length,
fluorescent GFP, whereas no incorporation results in a truncat-
ed, nonfluorescent protein. The total fluorescence intensity of
cells was measured by flow cytometry analysis. As shown in
Figure 1A, the cell fluorescence intensity increased when more
DanAla was added to the growth medium, but reached a pla-
teau from 0.25 to 1.00 mm. The plateau suggests that either
the concentration of DanAla inside the cells has saturated the
synthetase or that the cellular availability of DanAla is limiting.
When DanAla-OMe was used, the cell fluorescence intensity
doubled in comparison to that of cells incubated with the
same concentrations of DanAla; this indicates that the cellular
availability of DanAla was the limiting factor. Similar results
were also obtained for DanAla-OEt. There was no significant in-
crease in cell fluorescence intensity when the concentration of
DanAla-OMe or DanAla-OEt was increased from 0.10 to
0.25 mm. In contrast, DanAla-OAM doubled the fluorescence
intensity at 0.10 mm and quadrupled it at 0.25 mm. In compari-
son to 1.00 mm of DanAla, the amount often used in UAA in-
corporation, DanAla-OAM increased the cell fluorescence inten-
sity fourfold in addition to requiring 75% less compound in
the growth medium (0.25 mm). Western blot analysis of the
cell lysates confirmed the increase in the amount of GFP pro-
duced by the AME modification (Figure 1B). These results sug-
gest that all three ester modifications were able to increase
DanAla incorporation into proteins with a concomitant reduc-
tion in the extracellular supply of UAA. Furthermore, we identi-
fied DanAla-OAM as the most effective out of the three esters
tested.
To understand how DanAla-OAM increases DanAla incorpo-
ration, we first monitored the uptake of DanAla and DanAla-
OAM into cells by using fluorescence microscopy. We added
0.10 mm of DanAla or DanAla-OAM to the media of HEK293T
cells, and measured the cytosolic fluorescence intensity of cells
at sequential time points by using confocal microscopy (Fig-
ure 2A). For cells incubated with DanAla, the cytosolic fluores-
cence intensity was almost flat in the range of 620–750 AU. A
striking difference was observed in the DanAla-OAM incubated
sample. Within 5 min, the intracellular fluorescence intensity
rapidly rose to 1100 AU, a 48% increase from that of the
DanAla sample. The intensity increased for 3 h peaking at
1613 AU, which was twice the DanAla peak intensity. The in-
tensity of DanAla-OAM treated cells then gradually dropped,
possibly due to the depletion of the extracellular DanAla-OAM
in combination with the equilibration of converted DanAla in
and outside of cells. The AME modification thus significantly
accelerates the cellular uptake rate of the compound.
Next, we quantified the intracellular concentrations of
DanAla and DanAla-OAM by using HPLC. HEK293T cells were
incubated with 0.10 mm of the compounds for 1 h, and cell
contents were extracted. Small molecules in the cell extracts
were separated by HPLC, and peaks corresponding to DanAla
Figure 1. A) Flow cytometric analysis of the incorporation efficiency of DanAla into GFP with different compounds added in the growth medium. Reporter
cells transfected with the orthogonal suppressor tRNA and the wild-type LeuRS were used as the positive control to normalize the total fluorescence intensity.
Error bars represent standard error of mean (SEM, n=3). B) Western blot analysis of the GFP protein with a GFP-specific antibody. The same number of cells
were used in lanes 2–5.
ChemBioChem 2010, 11, 2268 – 2272
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that all intracellular DanAla-OAM had been hydrolyzed into
DanAla. The peak area for DanAla was used to determine its in-
tracellular concentration. Cells incubated with DanAla and
DanAla-OAM had 0.28 and 8.9 mm intracellular concentration
of DanAla, respectively. The AME modification dramatically in-
creased the intracellular concentration of DanAla by 31-fold.
When the concentration of DanAla-OAM was further in-
creased to 0.50 mm, the cell fluorescence intensity decreased
unexpectedly (Figure 1A). We used propidium iodide (PI) stain-
ing to assess the health of cells incubated with DanAla or
DanAla-OAM (Figure 2C). For cells treated with 0.10 to
0.50 mm DanAla, the percentage of PI-positive cells was similar
to that of cells without UAA. A slight increase of PI-positive
cells was observed at 1.00 mm of DanAla. In contrast, cells
treated with DanAla-OAM showed a significantly higher per-
centage of PI-positive cells, especially at the concentration of
0.50 mm. DanAla-OAM hydrolysis releases formaldehyde, which
will negatively affect cell health at high concentration.[11] High
intracellular concentration of DanAla could be toxic to cells as
well. Therefore, an optimal concentration of the AME ester
should be used to achieve highest UAA incorporation efficien-
cy with minimal negative effect to cell health.
In summary, we have demonstrated that masking the car-
boxyl group of DanAla with AME increased the uptake rate
and intracellular concentration of the unnatural amino acid in
mammalian cells. This resulted in a higher DanAla incorpora-
tion efficiency accompanied by the added benefit of reducing
the amount of UAA in the growth medium. Efficient UAA in-
corporation could prove valuable for the effective application
of genetically encoded UAAs to studying various biological
processes in mammalian cells. Modification of UAAs with AME
could be generally applicable to other UAAs that have trouble
entering mammalian cells. To test this hypothesis, more UAAs
need to be modified, and their incorporation efficiency deter-
mined. However, there is currently no orthogonal tRNA–syn-
thetase available for the incorporation of these candidate
UAAs because a synthetase cannot be evolved for an UAA that
cannot readily enter the cell. We are applying the esterification
strategy to highly polar amino acids, such as glycosylated and
phosphorylated amino acids, so as to overcome this problem
with the final goal of genetically incorporating these biologi-
cally important amino acids into proteins.
Experimental Section
Synthesis of DanAla and DanAla esters: All reactions were carried
out under nitrogen with dry solvents in anhydrous conditions,
unless otherwise noted. Dry N,N-dimethylformamide (DMF), metha-
nol, methylene chloride, and acetonitrile were obtained by passing
commercially available predried, oxygen-free formulations through
activated alumina columns. Yields refer to chromatographically ho-
mogeneous materials. Reagents were purchased from Sigma–Al-
drich and used without further purification. Reactions were moni-
tored by TLC, and carried out on 0.25 mm Silicycle silica gel plates
(60F-254) by using UV light for visualization and an ethanolic solu-
tion of phosphomolybdic acid and cerium sulfate for developing
under heat. Silicycle silica gel (60, particle size 0.040–0.063 mm)
was used for flash column chromatography (FCC). NMR spectra
Figure 2. A) Cytosolic fluorescence intensity of cells incubated with 0.10 mm
DanAla or DanAla-OAM. Error bars represent standard deviation (SD).
B) HPLC analysis of cell extracts from cells incubated with 0.10 mm DanAla
or DanAla-OAM. Arrows indicate the peak positions for DanAla and DanAla-
OAM, as determined by using pure compounds. C) PI staining analysis of cell
health at indicated conditions. Error bars represent SEM, n=3.
and DanAla-OAM were verified by using pure compounds and
mass spectrometry. Surprisingly, after only 1 h of incubation,
cells incubated with DanAla-OAM showed no peak for DanAla-
OAM but a large peak for DanAla (Figure 2B); this indicates
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were recorded on a Varian Oxford AS500 instrument and calibrated
by using residual undeuterated solvent or tetramethylsilane as an
internal reference. LCMS experiments were performed on an Agi-
lent 1100 Series LC/MSD instrument with a Phenomenex Synergi
4u Fusion-RP 80 A column (150ꢁ4.6 mm). DanAla was synthesized
as described.[13]
0.885 mmol) and 4-dimethylaminopyridine (DMAP; 21.6 mg,
0.177 mmol) were dissolved in CH2Cl2 (4 mL), and the mixture was
cooled to 08C. Et3N (0.136 mL, 0.974 mmol) and Boc2O (289.8 mg,
1.328 mmol) in CH2Cl2 (2 mL) were added dropwise to this stirring
mixture. The reaction mixture was allowed to warm to room tem-
perature and stirred for 4 h. Saturated aqueous NH4Cl solution was
added to the mixture. The aqueous phase was separated and
washed with CH2Cl2 (3ꢁ10 mL). The combined organic phase was
washed with brine, dried with sodium sulfate, and concentrated
under reduced pressure. The residue was purified by FCC (ethyl
acetate/hexanes 1:3, v/v) to give compound 9 (538.6 mg, 97%).
Rf =0.34 (SiO2, ethyl acetate/hexanes 1:2, v/v).
NMR (1H and 13C) and HRMS data for compounds are given in the
Supporting Information.
Compound 5: Boc-Dap-OH (2.0 g, 9.8 mmol) was added in one
portion to a stirring solution of dansyl chloride (2.4 g, 8.9 mmol)
and Et3N (2.6 mL) in CH2Cl2 (50 mL) at 08C. The reaction mixture
was allowed to warm to room temperature. After being stirred for
12 h, the reaction mixture was concentrated under reduced pres-
sure. The residue was purified by FCC (MeOH/CH2Cl2 7:93, v/v) to
give compound 5 (3.675 g, 94%) as a yellow solid. Rf =0.22 (SiO2,
MeOH/CHCl3 1:8, v/v).
Compound 10: Pd/C (87.3 mg) was added to a solution of com-
pound 9 (515.0 mg, 0.82 mmol) in methanol. The reaction mixture
was stirred under hydrogen for 2 h at room temperature. The reac-
tion mixture was passed through a short plug of celite and eluted
with ethyl acetate. The filtrate was concentrated to give com-
pound 10 (467.3 mg) as
MeOH/CHCl3 1:10, v/v).
a yellow-green solid. Rf =0.22 (SiO2,
Compound 6: Compound 5 (218.8 mg, 0.50 mmol) was dissolved
in DMF (2 mL), and the mixture was cooled to 08C. DIPEA
(0.096 mL, 0.55 mmol) and MeI (0.062 mL, 1.00 mmol) were added
dropwise to the solution. The reaction mixture was allowed to
warm to room temperature and stirred for 12 h. Water (20 mL) was
added, and the solution was extracted with ethyl acetate (3ꢁ
20 mL). The organic phase was washed with brine, dried with
sodium sulfate, and concentrated under reduced pressure. The res-
idue was purified by FCC (ethyl acetate/hexanes 3:7, v/v) to give
compound 6 (197.4 mg, 87%) as a yellow solid.
Compound 11: Compound 10 (434.9 mg, 0.809 mmol) was dis-
solved in CH3CN, and the mixture was cooled to 08C. DIPEA
(0.56 mL, 3.236 mmol) and bromomethyl acetate (0.24 mL,
2.427 mmol) were added dropwise to the solution. The reaction
mixture was allowed to warm to room temperature and stirred for
12 h, then concentrated under reduced pressure. The residue was
purified by FCC (ethyl acetate/hexanes 1:2, v/v) to give com-
pound 11 (389.5 mg, 84% over 2 steps) as yellow-green solid. Rf =
0.31 (SiO2, ethyl acetate/hexanes 1:2, v/v).
Compound 7: Compound 5 (218.8 mg, 0.50 mmol) was dissolved
in DMF (2 mL), and the mixture was cooled to 08C. DIPEA
(0.096 mL, 0.55 mmol) and EtI (0.080 mL, 1.00 mmol) were added
dropwise to the solution. The reaction mixture was allowed to
warm to room temperature and stirred for 12 h. Water (20 mL) was
added, and the solution was extracted with ethyl acetate (3ꢁ
20 mL). The organic phase was washed with brine, dried with
sodium sulfate, and concentrated under reduced pressure. The res-
idue was purified by FCC (ethyl acetate/hexanes 3:7, v/v) to give
compound 7 (174.1 mg, 75%) as a yellow solid.
Compound 4: Compound 11 (61.0 mg, 0.10 mmol) was dissolved
in CH2Cl2 (0.9 mL), and the mixture was cooled to 08C. TFA (0.3 mL)
was added dropwise to the solution. The reaction mixture was al-
lowed to warm to room temperature and stirred for 6 h, then con-
centrated under reduced pressure. After being under vacuum for
48 h, the residue was dissolved in DMSO (1.0 mL) for use.
Cell lines and culture conditions: HEK293T cells were used for de-
termining the UAA intracellular concentration and imaging experi-
ments. A stable clonal HeLa cell line containing the GFP gene with
Tyr182UAG mutation was previously established in this lab and
was used here for assaying UAA incorporation efficiency.[6]
HEK293T and HeLa-GFP(Tyr182UAG) cells were cultured in Dulbec-
co’s modified Eagle’s medium (DMEM, Mediatech) supplemented
with 10% fetal bovine serum (FBS).
Compound 2: Compound 6 (45.2 mg, 0.10 mmol) was dissolved in
CH2Cl2 (0.9 mL), and the mixture was cooled to 08C. Trifluoroacetic
acid (TFA; 0.3 mL) was added dropwise to the solution. The reac-
tion mixture was allowed to warm to room temperature and
stirred for 4 h, then concentrated under reduced pressure. After
being under vacuum for 48 h, the residue was dissolved in dimeth-
yl sulfoxide (DMSO; 1.0 mL) for use.
Fluorescence imaging: HEK293T cells (3ꢁ105) were seeded into an
imaging dish supplemented with DMEM with 10% FBS, but with-
out phenol red. After 18–24 h, DanAla or DanAla-OAM was added
to the medium to a final concentration of 0.10 mm. Cells were im-
mediately imaged with an Olympus IX81 spinning disc microscope
with a Hamamatsu EM-CCD under the same conditions, lex =360Æ
30 nm, lem =535Æ20 nm. After each time point, cells were placed
back into the incubator. Images were analyzed in Slidebook 4.2 by
using the masking tool and average intensity function. Cells not
treated with any compound were used as control to determine
background fluorescence, which was subtracted from the mea-
sured intensities of samples to yield the final net intensities.
Compound 3: Compound 7 (46.6 mg, 0.10 mmol) was dissolved in
CH2Cl2 (0.9 mL), and the mixture was cooled to 08C. TFA (0.3 mL)
was added dropwise to the solution. The reaction mixture was al-
lowed to warm to room temperature and stirred for 4 h, then con-
centrated under reduced pressure. After being under vacuum for
48 h, the residue was dissolved in DMSO (1.0 mL) for use.
Compound 9: Compound 5 (569.4 mg, 1.30 mmol) was dissolved
in DMF (10 mL), and the mixture was cooled to 08C. DIPEA
(0.25 mL, 1.43 mmol) and benzyl bromide (0.31 mL, 2.60 mmol)
were added dropwise to the solution. The reaction mixture was al-
lowed to warm to room temperature and stirred for 24 h. Water
(70 mL) was added and the solution was extracted with ethyl ace-
tate (50 mL). The organic phase was washed with brine, dried with
sodium sulfate, and concentrated under reduced pressure. The res-
idue was purified by FCC (ethyl acetate/hexanes 1:3–1:2, v/v) to
give Boc-DanAla-OBn (522.1 mg, 76%) as a yellow solid. Rf =0.25
(SiO2, ethyl acetate/hexanes 1:2, v/v). Boc-DanAla-OBn (466.7 mg,
HPLC analysis of intracellular concentration: HEK293T cells (8.5ꢁ
106) were seeded into a 10 cm tissue culture dish supplemented
with DMEM containing 10% FBS. After 18–24 h, DanAla or DanAla-
OAM was added to the medium to a final concentration of 0.1 mm.
The compounds were incubated with cells for 1 h. Cells were then
washed with phosphate-buffered saline (PBS; 1 mL) several times.
ChemBioChem 2010, 11, 2268 – 2272
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After trypsinization with trypsin (1 mL, 0.05%; Mediatech), cells
were centrifuged, and the medium was removed. Cells were
washed again with water (1 mL) and centrifuged. The supernatant
was removed, and cells were resuspended in water (250 mL). Tolu-
ene (50 mL) was added, and the cells were placed in a shaking incu-
bator at 378C for 30 min. Cells were centrifuged at 20000 relative
centrifugal force (RCF) for 10 min. The aqueous layer was placed
into a Microcon Ultracel YM10 column (Millipore) and spun for
30 min at 12000 RCF. The flow-through was analyzed by HPLC-MS
(Agilent 1100 Series LC/MSD instrument): and aliquot (5 mL) of the
aqueous layer was separated on a Phenomenex Synergi 4u Fusion-
RP 80 A column (150ꢁ4.6 mm) with a gradient of aqueous acetoni-
trile/0.1% formic acid (75:25 to 25:75) over 10 min. DanAla and
DanAla-OAM were identified by extracting their MW (+1) from the
total ion mass spectrum. Pure DanAla and DanAla-OAM were also
added into the flow-though prepared from cells not treated with
compounds so as to verify the peak positions of DanAla (3.3 min)
and DanAla-OAM (3.7 min). Calculation of DanAla concentrations in
each sample was based on the extracted ion area of DanAla and a
standard curve. The standard curve was made by extracting the
area of DanAla in solutions with different concentrations (0.10,
0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, and 1.00 mm). Cells not
treated with any compound were used to measure the back-
ground area at 3.3 and 3.7 min. The intracellular concentration was
given by the total molar amount of DanAla divided by the total
volume of cells, by using the volume of a single cell as in ref. [16].
sonication. The lysis procedure was repeated three times to ensure
complete lysis. Loading buffer was added, and the samples were
boiled. Prepared samples were loaded onto a 12% SDS-PAGE gel.
For the pELcua-LeuRS sample, 104 cells were loaded, while 7.5ꢁ104
cells were loaded for all other samples. The primary antibody (Anti-
GFP Monoclonal Antibody 7G9, BioPioneer, San Diego, USA) and
horseradish peroxidase (HRP) conjugated secondary antibody
(Goat Anti-Mouse IgG-HRP Conjugate, Santa Cruz Biotechnology)
were used to detect GFP proteins.
Acknowledgements
We thank Dr. Michael Burkart for help with the HRMS. This work
was supported by the Ray Thomas Edwards Foundation, Beck-
man Young Investigator Program, March of Dimes Foundation
(5-FY08-110), California Institute for Regenerative Medicine (RN1-
00577-1, and National Institutes of Health (1DP2OD004744-01).
Keywords: amino acids
· cellular uptake · enzymes ·
fluorescence · genetic code expansion
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Flow cytometry: Stable HeLa–GFP(Tyr182TAG) clonal cells were
seeded in a 3.5 cm culture dish and transfected with pELcua-LeuRS
or pELcua-DanAlaRS (5 mg) plasmid DNA by using Lipofectamine
2000.[6] HeLa–GFP(Tyr182TAG) cells that were not transfected with
a tRNA–synthetase pair were used as a negative control. DanAla
and DanAla-OAM were added 24 h post transfection, and flow cy-
tometry was carried out 24 h after the addition of the compounds.
Cells were trypsinized and washed with PBS twice. Samples were
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Western blot: HeLa–GFP(Tyr182TAG) clonal cells were transfected
and incubated with the appropriate DanAla or DanAla ester, as
previously described for flow cytometry analysis. Cells were trypsi-
nized and washed with PBS, and cell number was counted by
using a hemocytometer. Samples were centrifuged and resuspend-
ed with PBS (20 mL) containing ethylendiaminetetraacetate-free
protease inhibitor cocktail (Roche) and DNase I (Roche). These sam-
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Received: July 30, 2010
Published online on September 24, 2010
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