A R T I C L E S
Amstutz et al.
MOZ-ampicillin (513 mg, 1.00 mmol) was dissolved in 3.6 mL of
cold H2O, and the pH was adjusted to 7.0. KMnO4 (190 mg, 1.2 mmol)
was dissolved in 5 mL H2O and treated with 65 µL 10% H3PO4. The
KMnO4 solution was added dropwise to the first solution on ice, while
maintaining the pH between 6.8 and 7.3 with 10% H3PO4 and 10%
NaOH. After 40 min, the reaction mixture was brought to pH 8.0 and
filtered over Celite, and the filtrate was acidified to pH 3.0 with 10%
H3PO4.50 The product was extracted with EtOAc (5×, 100 mL, dried
with NaSO4, and concentrated in vacuo to afford 495 mg (91% yield)
of MOZ-ampicillin sulfone as an egg white powder: IR (CHCl3 [cm-1])
3410, 2960, 1810, 1730, 1700, 1612, 1515, 1495, 1328, 1118; 1H NMR
(DMSO): δ 1.33, 1.46, 3.74 (3 × 3H, 3s), 4.36 (1H, s), 4.97 (2H, s),
5.31 (1H, d, J ) 4.5), 5.51 (1H, d, J ) 8.3), 5.90 (1H, dd, J ) 4.4,
8.8), 6.90 (2H, d, J ) 8.6), 7.28-7.31 (5H, m), 7.35-7.50 (2H, m),
7.97 (1H, br d, J ) 8.5), 8.65 (1H, br d, J ) 8.8); 13C NMR (DMSO):
δ 17.4, 19.8, 55.1 (3q), 56.8, 59.1, 63.7 (3d), 64.6 (s), 65.4 (d), 67.3
(t), 113.8, 127.0 (2× 2C, 2d), 128.4 (s), 128.7 (d), 129.1, 129.9 (2×
2C, 2d), 136.0, 156.0, 159.5, 168.9, 170.6, 173.6 (6s); MS: 546.2 m/z.
A solution of MOZ-ampicillin sulfone (220 mg, 0.40 mmol) in 2
mL of CH2Cl2 and 0.2 mL of TFA was stirred at room temperature for
30 min.49 The solvent was removed in vacuo. The product was triturated
with ether that was removed as the supernatant after centrifugation.
This procedure was repeated twice, and the product was dried in vacuo.
Ampicillin sulfone (150 mg, 0.39 mmol, 97% yield) was isolated as a
pale yellow powder: IR (KBr [cm-1]): 3390, 2980, 1802, 1674, 1525,
sulfone on ice. Aliquots were taken after various time points and excess
inhibitor was removed by gel filtration (NAP-500 column, Pharmacia,
according to the manufacturer’s instructions). Alternatively, one aliquot
was incubated at 25 °C for 2 h while another was kept on ice at a pH
8 for 2 h. For inhibition studies, one aliquot was preincubated with 1
mM ampicillin sulfone for 1 h at room temperature prior to incubation
with biotinylated ampicillin sulfone (as described above). The proteins
were separated by SDS-PAGE (12%) and blotted onto a membrane
(Immunoblot, Millipore). The membrane was blocked with milk, and
the presence of the biotinylated suicide inhibitor was detected with an
avidin-alkaline phosphatase conjugate (Pierce).
DNA Constructs for the Model System. The â-lactamase gene
encoding the double cysteine to alanine mutant was PCR amplified
51
from the plasmid pAP5•2C•2A
with the primers SDA-BLA
5′-(AGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAA-
CTTTAAGAAGGAGATA TATCCATGGACTACAAAACCAGCCA-
GAAACGCTGGTGAAAGT), which codes for the Shine Dalgarno box
and the beginning of the â-lactamase gene, and BLArev 5′-(ACA-
CAGGCCCCCGAGGCCCAATGCTTAATCAGTGA), annealing at
the end of the â-lactamase gene and introducing a unique SfiI restriction
site, while removing the stop codon. To create an inactive â-lactamase
to use as a negative control for activity, the active site serine was
mutated to alanine (S70A) by PCR mutagenesis. A unique MscI
restriction site was added at the site of the mutation to allow for
discrimination of the genes encoding active and inactive enzyme. In
parallel, a 171 amino acid portion of the tolA gene (residues 131 to
302) was amplified by PCR from chromosomal E. coli DNA in order
to serve as a C-terminal tether for the displayed â-lactamase. The
primers used were tolAforSFI 5′-(TATATGGCCTCGGGGGCCGAAT-
TCCAGAAGCAAGCTGAAG), introducing an SfiI-site, and tolAmrev
5′-(CCGCACACCAGTAAGGTGTGCGGTTAGCTCACCGAAAA-
TATCATC), which introduced a RNA-stabilizing 3′-loop. The inactive
S70A mutant and the active â-lactamase were fused to the tolA spacer
by SfiI digestion-ligation. The resulting constructs were further
amplified by PCR with the primers tolAmrev and T7B 5′-(ATAC-
GAAATTAATACGACTCACTATAGGGAGACCACAACGG), which
introduced an RNA stabilizing 5′-loop and the T7 promoter for in vitro
transcription. Both constructs were verified by DNA sequencing.
1
1458, 1433, 1376, 1323, 1116; H NMR (DMSO): δ 1.32, 1.42 (2×
3H, 2s); 4.33 (1 H, s); 5.31-5.29 (2H, m); 5.95 (1 H, dd, J ) 8.4,
4.5,); 7.30-7.54 (5H, m); 9.12 (1H, d like m, J ) 8.5); 13C NMR
(DMSO): δ 17.0, 19.1 (2q); 54.7, 55.6, 63.1 (3d); 63.7 (s), 64.6 (d),
127.4, 128.7 (2× 2C, 2d), 129.1 (d), 132.9 167.7, 168.0, 173.1 (4s),
MS: 382.2 m/z.
EZ-LinkSulfo-NHS-LC-LC-Biotin (7.6 mg, 1.14 × 10-2 mmol,
Pierce) was dissolved in 7 mL borate buffer pH 7.8. Ampicillin sulfone
(7.1 mg, 2.3 × 10-2 mmol) was added. This solution was sonicated
and stirred for 1 h at room temperature. Tris(hydroxymethyl)ami-
nomethane base (2 mg, 1.7 × 10-2 mmol) was added, stirred for an
additional 10 min, and lyophilized to afford 71.5 mg of a white powder
(as a complex with borate) from which 30 mg was dissolved in MeOH
and applied to a silica gel column (1 g, in a Pasteur pipet). The product
was eluted with 10 mL of MeOH, which was removed in vacuo. The
product was obtained quantitatively as a white powder from ampicillin
sulfone. MS: 834.4 m/z.
Inhibition Studies. â-Lactamase (from E. cloacae (Fluka) or E. coli
R-TEM â-lactamase from in vitro translation) in concentrations from
10-9 to 10-10 M (as determined by activity measurements,51 and
assuming that the specific activity of the in vitro translated â-lactamase
is similar to that of the bacterially expressed enzyme) was incubated
at room temperature in 50 mM potassium phosphate buffer (pH 6.0,
5% DMSO), with or without 1 mM ampicillin sulfone. DMSO is
required to dissolve ampicillin sulfone. Aliquots of 500 µL were taken
after 10, 20, 30, 45, and 90 min and were mixed with 500 µL of 0.4
mM nitrocefin (Becton Dickinson) in 50 mM potassium phosphate
buffer (pH 6.0, 1% DMSO). Reaction kinetics were followed at OD486
during 2 min at room temperature with a Perkin-Elmer Lambda 20
spectrophotometer.51 To show the covalent nature of the inhibition, a
sample (500 µL) of inhibited and, as a control, not inhibited enzyme
were dialyzed against 500 mL potassium phosphate buffer (50 mM,
pH 7) in “Slide-A-Lycer 10K” devices (molecular weight cut-off, 10
kD, 0.5-3 mL sample volume) (Pierce) for 2 h before activity was
measured.
Separation of Ternary Complexes from Free â-Lactamase by
Gel Filtration. For ribosome display, the in vitro transcription and in
vitro translation of the constructs were carried out essentially as
previously described.44 Following a 10-min translation in 110 µL, the
reaction was stopped by 4-fold dilution in ice-cold wash buffer (WB;
50 mM potassium phosphate buffer, pH 6.0; 150 mM NaCl; 50 mM
MgCl2). A 250-µL aliquot was treated with 2.5 µL RNase A (Qiagen,
10 mg/mL in WB) for 180 min at 4 °C to release ribosome-bound
â-lactamase, while another aliquot was treated with WB only. An
aliquot (200 µL) of each solution was applied to a 1-mL gel filtration
column (CL-4B, Pharmacia) that had been equilibrated with 10-20
mL of ice-cold WB. Fractions of 100 µL were collected, and the enzyme
activity was assayed in a nitrocefin assay (200 µM nitrocefin in WB
buffer, pH 6.0, 1% DMSO). In addition, an aliquot of diluted translation
mix was treated with RNaseA for 1 h at room temperature without
subsequent gel filtration. The â-lactamase activity of this sample was
determined in order to quantify the total activity, as free enzyme,
resulting from translation.
Enrichment for Catalytic Activity with Ribosome Display. RNA
coding for active as well as inactive S70A mutant â-lactamase was
mixed in a ratio of 1 to 10 (10 µg total of RNA in 10 µL water) and
translated in 110 µL for 10 min.44 Translation was stopped by 4-fold
dilution into ice-cold WB with 1% BSA (bovine serum albumin,
Sigma). Biotinylated ampicillin sulfone (stock solution, 0.01 M in
DMSO) was added to a final concentration of 0.01 mM. A control
sample contained the same final concentration of DMSO but no
biotinylated ampicillin sulfone. After 30 min at 4 °C, the reaction was
stopped by removal of the excess biotinylated ampicillin sulfone by
Western Blot Analysis. E. coli S30-extract (as used for ribosome
display 44) was diluted 2-fold in 50 mM potassium phosphate buffer
(pH 6.0, 1% DMSO) and incubated with 0.1 mM biotinylated ampicillin
(50) Johnson, D. A.; Panetta, C. A.; Cooper, D. E. J. Antibiot. 1963, 28, 1927-
1928.
(51) Laminet, A. A.; Plu¨ckthun, A. EMBO J. 1989, 8, 1469-1477.
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9402 J. AM. CHEM. SOC. VOL. 124, NO. 32, 2002