Cell Surface Presentation of Azides
A R T I C L E S
of TfN3 in CH2Cl2 was subsequently added slowly to a solution of
Boc-Dap-OH (1.81 g, 8.86 mmol), K2CO3 (1.84 g, 13.3 mmol), and
CuSO4‚5H2O (22 mg, 0.088 mmol) in H2O (30 mL) and MeOH (45
mL). The resulting mixture was stirred overnight, and the organic
solvents were evaporated under reduced pressure. The water layer was
acidified to pH 6 with concd HCl, diluted with 0.25 M, pH 6.2
phosphate buffer (50 mL), and extracted with CH2Cl2 (4 × 100 mL).
The combined organic layers were washed with brine (50 mL), dried
over Na2SO4, and concentrated in vacuo. The residue was dissolved in
5 mL concd HCl (5 mL), and after being stirred for 24 h, the product
was purified using cationic Dowex exchange resin to afford the product
vated biotin-PEO ester with excess neat propargylamine as described
elsewhere.12
Plasmids and Expression Hosts. The plasmid pQE-60/OmpC
encodes a mutant form of outer membrane protein C that contains six
additional methionine residues in its exposed loops.12 pQE-60/OmpC
was linearized by digestion with Nhe I. The plasmid pQE-15 MRS,
which encodes a methionyl-tRNA synthetase expression cassette,15 was
digested with Nhe I, and a 2.5 kb fragment corresponding to the MetRS
cassette was isolated by agarose gel electrophoresis. This fragment was
ligated to the linearized pQE-60/OmpC to generate the plasmid pAJL-
20. This plasmid was transformed into the E. coli methionine auxotroph
M15MA12 to generate the expression host M15MA[pAJL-20]. The
expression host M15MA[pQE-60/OmpC] has been previously de-
scribed.12
1
as an off-white powder (696 mg, 60%). H NMR (300 MHz, D2O):
3.80-3.63 (m, 3H). 13C NMR (75 MHz, D2O): 171.8, 53.9, 50.8.
HRMS (FAB) calcd for C3H7N4O2 (MH+), 131.0569; found, 131.0569.
Expression of OmpC Containing Non-Canonical Amino Acids.
An overnight culture of the expression host was diluted 50-fold in M9
minimal medium containing all 20 natural amino acids (40 mg/L each)
as well as the antibiotics ampicillin (200 mg/L) and kanamycin (35
mg/L). Upon reaching an OD600 of 0.9-1, the culture was centrifuged
at 6000g for 7 min and resuspended in M9 medium containing 19 amino
acids (no methionine). The cells were shaken at 37 °C for 10 minutes
to deplete the intracellular pool of methionine, centrifuged again, and
resuspended in M9 medium containing 19 amino acids (no methionine).
To these cells was added either methionine, the noncanonical amino
acid, or no amino acid. Methionine (7) and azidohomoalanine (2) were
added to a final concentration of 40 mg/L while 1, 3, and 4 were added
to a final concentration of 750 mg/L. Protein expression was induced
for 3 h at 37 °C by the addition of isopropyl-â-D-thiogalactoside (IPTG)
to a final concentration of 1 mM.
2-Amino-5-azidopentanoic acid (azidonorvaline, 3): Boc-Ornithine-Z
(Boc-Orn(Z)-OH, 2.00 g, 5.46 mmol, Novabiochem) and 10% Pd/C
(581 mg, 0.819 mmol Pd) in EtOAc (50 mL) were subjected to a
hydrogen atmosphere of 40 psi for 16 h. The mixture was filtered over
Celite, the filter was washed with water, and the solvents were
evaporated in vacuo. The resulting product (1.7 g) was used in the
next reaction without further purification. Tf2O (1.84 mL, 10.9 mmol)
was added dropwise to a vigorously stirred mixture of NaN3 (3.55 g,
54.6 mmol) in H2O (9 mL) and CH2Cl2 (15 mL) at 0 °C. The resulting
mixture was allowed to warm to room temperature and was stirred for
2 h. The water layer was extracted with CH2Cl2 (2 × 5 mL), and the
combined organic layers were washed with saturated aqueous Na2CO3
(25 mL). The resulting solution of TfN3 in CH2Cl2 was subsequently
added slowly to a solution of Boc-ornithine, K2CO3 (1.13 g, 8.18 mmol),
and CuSO4‚5H2O (14 mg, 0.056 mmol) in H2O (17 mL) and MeOH
(34 mL). The mixture was stirred overnight, and the organic solvents
were evaporated under reduced pressure. The water layer was acidified
to pH 6 with concd HCl, diluted with 0.25 M, pH 6.2 phosphate buffer
(50 mL), and extracted with CH2Cl2 (4 × 100 mL). The combined
organic layers were washed with brine (50 mL), dried over Na2SO4,
and concentrated in vacuo. The residue was dissolved in concd HCl (5
mL), and after being stirred for 24 h, the product was purified using
cationic Dowex exchange resin to afford the product as an off-white
Copper-Catalyzed Triazole Formation on the Cell Surface. An
aliquot of cells expressing recombinant OmpC (1 mL) was centrifuged
at 4 °C and washed once in 1 mL of PBS (pH 7.4). The cells were
centrifuged and resuspended in 1 mL of PBS. Triazole ligand 5 was
added to a final concentration of 200 µM, and biotin-PEO-propargy-
lamide 6 was added to a final concentration of 50 µM. Addition of the
active copper species was accomplished in two different ways. For in
situ generation of Cu(I), 100 µM CuSO4 and 200 µM of tris-
(carboxyethyl)phosphine (TCEP) were added to the cells. Alternatively,
the Cu(I) ion was added directly to the cells in the form of an aqueous
suspension of CuBr. Briefly, 10 µL of a 10 mM suspension of CuBr
(99.999% purity, Aldrich) was thoroughly agitated and added to the
cells. As discussed in the Results section, the quality of the CuBr is
critical for the success of the experiment. All labeling reactions were
allowed to continue for 16 h at 4 °C and were stopped by washing the
cells with PBS.
1
powder (382 mg, 41%). H NMR (600 MHz, D2O): 3.70 (t, J ) 6.1
Hz, 1H), 3.34 (t, J ) 6.6 Hz, 2H), 1.92-1.84 (m, 2H), 1.69-1.56 (m,
2H). 13C NMR (75 MHz, D2O): 174.9, 54.6, 50.7, 28.1, 24.2. HRMS
(EI) calculated for C5H10N4O2, 158.0804; found, 158.0170.
2-Amino-6-azidohexanoic acid (azidonorleucine, 4): Tf2O (2.7 mL,
16 mmol) was added dropwise to a vigorously stirred mixture of NaN3
(5.27 g, 81.1 mmol) in H2O (13 mL) and CH2Cl2 (22 mL) at 0 °C. The
resulting mixture was allowed to warm to room temperature and was
stirred for 2 h. The water layer was extracted with CH2Cl2 (2 × 8
mL), and the combined organic layers were washed with saturated
aqueous Na2CO3 (25 mL). The resulting solution of TfN3 in CH2Cl2
was subsequently added slowly to a solution of N-R-Boc-lysine (2.0 g,
8.1 mmol, Bachem), K2CO3 (1.68 g, 12.2 mmol), and CuSO4‚5H2O
(20 mg, 0.080 mmol) in H2O (26 mL) and MeOH (52 mL). The mixture
was stirred overnight, and the organic solvents were evaporated under
reduced pressure. The water layer was acidified to pH 6 with concd
HCl, diluted with 0.25 M, pH 6.2 phosphate buffer (50 mL), and
extracted with CH2Cl2 (4 × 100 mL). The combined organic layers
were washed with brine (50 mL), dried over Na2SO4, and concentrated
in vacuo. The residue was dissolved in concd HCl (5 mL), and after
stirring for 24 h, the product was purified using cationic Dowex
exchange resin to afford the product as a white powder (244 mg, 18%).
1H NMR (600 MHz, D2O): 3.69-3.67 (m, 1H), 3.30 (t, J ) 6.6 Hz,
Outer Membrane Fraction Isolation and Western Blotting. The
outer membrane protein fraction of cells labeled via the azide-alkyne
cycloaddition was isolated as described.16 The outer membrane protein
fractions from equal numbers of cells were electrophoresed (12% tris-
tricine gel, 165 V, 45 min) and subsequently transferred to a
nitrocellulose membrane (30 V, 1 h at 4 °C). The membrane was
blocked for 1 h with 5% milk in PBS/Tween. After being washed with
PBS, the membrane was probed with an avidin-HRP conjugate
(Amersham Biosciences). Blots were visualized using appropriate
detection reagents (ECL Western Blotting Analysis System, Amersham
Biosciences).
Flow Cytometry. After OmpC expression and biotinylation, 1 mL
of cells was washed twice with 1 mL of PBS (pH 7.4). The cells were
stained with 5 µL of a 1 mg/mL solution of an avidin-Alexa Fluor
488 conjugate (Molecular Probes, Eugene, OR) for 2-3 h at 4 °C.
The cells were washed three more times with PBS, diluted 40-fold,
2H), 1.86-1.78 (m, 2H), 1.62-1.57 (m, 2H), 1.44-1.35 (m, 2H). 13
C
NMR (75 MHz, D2O): 177.6, 57.4, 53.4, 32.7, 30.4, 24.4. HRMS
(14) Mangold, J. B.; Mischke, M. R.; Lavelle, J. M. Mutat. Res. 1989, 216,
27-33.
(FAB) calculated for C6H13N4O2 (MH+), 173.1039; found, 173.1040.
(15) Kiick, K. L.; van Hest, J. C. M.; Tirrell, D. A. Angew. Chem., Int. Ed.
2000, 39, 2148-2152.
Azidohomoalanine (2) was prepared as previously described.14
Biotin-PEO-propargylamide (6) was synthesized by reacting an acti-
(16) Xu, Z. H.; Lee, S. Y. Appl. EnViron. Microbiol. 1999, 65, 5142-5147.
9
J. AM. CHEM. SOC. VOL. 126, NO. 34, 2004 10599