1
give 5 (53 mg, 70%) as a yellow solid. H NMR (500 MHz, CD3OD,
Mass spectrometric analysis of peptides (Figure S1): Starred
peaks from Figure 1C were manually collected and injected into an
Applied Biosystems 200 QTRAP mass spectrometer. The flow rate
was 3 mLminꢀ1, and mass spectra were recorded under the posi-
tive-enhanced multicharge mode.
258C): d=8.75 (s, 1H), 7.73 (d, J=8.7 Hz, 1H), 7.2–7.7 (m, 10H)
6.86 (dd, J=1.9, 8.6 Hz, 1H), 6.79 (s, 1H), 3.60 (s, 3H; CH3), 3.42 (m,
2H; CH2), 2.37 (t, 2H; CH2), 1.66 (m, 4H; CH2); 13C NMR (125 MHz,
CD3OD, 258C): d=174.2, 170.1, 162.2, 158.0, 155.8, 148.3, 130.7,
130.5, 130.1, 129.8, 129.7, 128.8, 119.2, 116.6, 114.7, 108.1, 51.9,
39.7, 34.0, 30.2, 22.8; HR ESI-MS calcd: 483.1914 [M+H]+; obs.:
483.1932.
Mammalian cell culture: Human embryonic kidney (HEK) cells
were cultured in Dulbecco’s modified Eagle medium (DMEM; Cell-
gro, Manassas, VA, USA) supplemented with 10% v/v fetal bovine
serum (PAA Laboratories, Etobicoke, Ontario, Canada). For imaging,
cells were plated as a monolayer on glass coverslips. Adherence of
HEK cells was promoted by precoating the coverslip with fibronec-
tin (50 mgmLꢀ1, Millipore). All cells were maintained at 378C under
5% CO2.
Synthesis of 7-aminocoumarin 6: HCl (0.5 mL, 1m) was added to a
stirring solution of 5 (10 mg, 21 mmol) in THF/water (1:1, 10 mL).
The mixture was stirred at 258C for 48 h, then concentrated in
vacuo. The yellow residue was purified by column chromatography
(silica gel, EtOAc/MeOH/NH4OH 94:5:1) to afford 6 as a light yellow
1
solid (5 mg, 76%). H NMR (500 MHz, D2O, 258C): d=8.30 (s, 1H),
PRIME cell-surface labeling (Figure 2): HEK cells were transfected
at ~70% confluency with expression plasmids for LAP4.2[16]-neurex-
in-1b (400 ng for a 0.95 cm2 dish) and H2B-YFP (100 ng) using Lipo-
fectamine 2000 (Invitrogen). 18 h after transfection, cells were
treated with W37VLplA enzyme (10 mm), coumarin probe (200 mm),
ATP (1 mm), and Mg(OAc)2 (5 mm) in cell growth medium for
20 min at room temperature. After excess labeling reagents had
been removed by replacing the medium 2–3 times, cells were im-
mediately imaged or incubated at 378C for 20 min to allow cell-
surface protein turnover.
7.36 (d, J=8.3 Hz, 1H), 6.66 (d, J=8.6 Hz, 1H), 6.40 (s, 1H), 3.36 (m,
2H; CH2), 2.29 (t, 2H; CH2), 1.66 (m, 4H; CH2); 13C NMR (125 MHz,
CD3OD, 258C): d=181.8, 164.6, 163.3, 158.4, 148.9, 132.2, 113.5,
109.7, 109.5, 98.4, 39.8, 38.1, 29.8, 24.5; UV/Vis (phosphate buffer
pH 7): lmax (e)=380 nm (18400mꢀ1 cmꢀ1); HR ESI-MS calcd:
303.0986 [MꢀH]ꢀ, obs.: 303.0973.
Synthesis of 7-aminocoumarin-AM: Silver(I) oxide (6 mg, 30 mmol)
followed by acetoxymethyl bromide (1.5 mL, 15 mmol) was added
to a stirring solution of 7-aminocoumarin 6 (3 mg, 9 mmol) in anhy-
drous acetonitrile (1 mL). The mixture was stirred at 258C for 12 h,
then concentrated in vacuo. The yellow residue was purified by
column chromatography (silica gel, EtOAc/hexane 8:1) to afford 7-
aminocoumarin-AM as a light yellow solid (3 mg, 81% yield).
1H NMR (300 MHz, CDCl3, 258C): d=8.39 (s, 1H), 7.28 (d, J=8.7 Hz,
1H), 6.70 (dd, J=8.6, 2.4 Hz, 1H), 6.45 (d, J=2.4 Hz, 1H), 5.72 (s,
2H), 3.34 (m, 2H; CH2), 2.31 (t, 2H; CH2), 2.09 (s, 3H; CH3), 1.70 (m,
4H; CH2); HR ESI-MS calcd: 377.1343 [M+H]+, obs.: 377.1348.
PRIME intracellular labeling (Figure 3): HEK or HeLa cells were
transfected with expression plasmids for W37VLplA (20 ng) and LAP
substrate (LAP2-YFP, LAP2-YFP-NLS, or LAP2-b-actin; 400 ng) by
using Lipofectamine 2000. 18 h after transfection, cells were treat-
ed with 7-aminocoumarin-AM (20 mm) in serum-free DMEM for
10 min at 378C. Excess coumarin probe was removed by washing
the cells with cell-growth medium (4ꢁ15 min). Cells were then
imaged live.
7-Aminocoumarin and 7-hydroxycoumarin pH profiles (Fig-
ure 1B): Fluorescence emission was recorded for 150 mm solutions
by using a TECAN Safire Microplate Reader and a plastic transpar-
ent-bottomed 384-well plate (Greiner). pH 3–6 buffers were pre-
pared by mixing different ratios of acetic acid (0.1m) and sodium
acetate-trihydrate (0.1m) solutions. pH 7–10 buffers were prepared
by mixing different ratios of Na2HPO4 (0.1m) and either HCl (0.1m;
for pH 7–9 buffers) or NaOH (0.1m; for pH 10 buffer). Final pH
adjustments in all buffer solutions were made by adding small
amounts of HCl (1m) or NaOH (1m).
Fluorescence imaging: Cells were imaged in Dulbecco’s phos-
phate-buffered saline (DPBS) in confocal mode. We used a Zeiss
Axiovert 200M inverted microscope with a 40ꢁ oil-immersion ob-
jective. The microscope was equipped with a Yokogawa spinning-
disk confocal head, a Quad-band notch dichroic mirror (405/488/
568/647), and 405 (diode), 491 (DPSS), and 561 nm (DPSS) lasers
(all 50 mW). 7-Aminocoumarin (405 laser excitation, 445/40 emis-
sion), YFP (491 laser excitation, 528/38 emission), and DIC images
were collected by using Slidebook software. Fluorescence images
in each experiment were normalized to the same intensity ranges.
Acquisition times ranged from 10–1000 ms.
In vitro 7-aminocoumarin ligation reactions (Figures 1C and S2):
For Figure 1C, reactions were assembled as follows: LplA enzyme
(2 mm), LAP2 synthetic peptide (150 mm, sequence: GFEIDKVWYDL-
DA[16]), 7-aminocoumarin 6 probe (500 mm), ATP (5 mm), and
Mg(OAc)2 (5 mm) in Na2HPO4 (25 mm, pH 7.2). The reaction mixture
was incubated at 308C for 2 h and quenched with EDTA (final con-
centration 100 mm). The mixture was analyzed on a Varian Prostar
HPLC by using a reversed-phase C18 Microsorb–MV 100 column
(250ꢁ4.6 mm). Chromatograms were recorded at 210 nm. We used
a 10-min gradient of 30–60% acetonitrile in water with 0.1% tri-
fluoroacetic acid at a flow rate of 1 mLminꢀ1. LAP2 had a retention
time of 7 min; after ligation to 7-aminocoumarin, tR increased to
9 min.
Acknowledgements
Funding was provided by the NIH (R01 GM072670) and MIT. X.J.
was supported by John Reed (MIT Class of 1961) and Paul E.
Gray (MIT Class of 1954) Funds, administered by the MIT Under-
graduate Research Opportunity Program. We thank Jennifer Yao
for LplA enzymes, Daniel S. Liu and Katharine A. White for plas-
mids, David Surry (MIT) for synthetic advice, and Peng Zou for
helpful feedback on the manuscript.
For Figure S2, W37VLplA (2 mm) and coumarin probe (500 mm) were
used in one case. Aliquots from the reaction were collected and
quenched with EDTA over 55 min. For the other case, W37VLplA
(1 mm) and coumarin probe (100 mm) were used, and aliquots were
collected and quenched over 70 min. After HPLC analysis, percent
product conversions were calculated by dividing the product peak
area by the sum of (product+starting material) peak areas.
Keywords: cell imaging
engineering · fluorescent probes · palladium
·
cross-coupling
·
enzyme
[1] C. Uttamapinant, K. A. White, H. Baruah, S. Thompson, M. Fernandez-
ChemBioChem 2011, 12, 65 – 70
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
69