Novel fluorogenic substrates for imaging β-lactamase gene expression

Article abstract of DOI:10.1021/ja036126o

A new class of small nonfluorescent fluorogenic substrates becomes brightly fluorescent after β-lactamase hydrolysis with up to 153-fold enhancement in the fluorescence intensity. Less than 500 fM of β-lactamase in cell lysates can be readily detected, and β-lactamase expression in living cells can be imaged with a red fluorescence derivative. These new fluorogenic substrates should find uses in clinical diagnostics and facilitate the applications of β-lactamase as a biosensor. Copyright

Full text of DOI:10.1021/ja036126o

Published on Web 08/19/2003
Novel Fluorogenic Substrates for Imaging â-Lactamase Gene Expression
Wenzhong Gao,† Bengang Xing,† Roger Y. Tsien,‡ and Jianghong Rao*,†
Department of Molecular and Medical Pharmacology, Crump Institute for Molecular Imaging,
UniVersity of California, Los Angeles, California 90095-1770, and Howard Hughes Medical Institute,
Departments of Pharmacology and Chemistry & Biochemistry, UniVersity of California San Diego,
La Jolla, California 92093-0647
Received May 13, 2003; E-mail: jhrao@mednet.ucla.edu
Scheme 1. Synthesis of CC1 and CC2a
â-Lactamases are a family of bacterial enzymes that cleave
penicillins and cephalosporins with high catalytic efficiency and
render these bacteria resistant to â-lactam antibiotics.1 Rapid and
sensitive detection of â-lactamase activity in biological samples is
thus of large clinical importance. While â-lactamases are the
biochemical markers for identification of â-lactam antibiotics-
resistant bacterial pathogens, the â-lactamase activity can also serve
as a “reporter” or “sensor” for monitoring biological processes and
interactions of interest. TEM-1 â-lactamase (Bla), the 29 kDa
isoform product of the ampicillin resistance gene (ampr), is
especially useful because it is relatively small and monomeric, does
not exist in eukaryotes, but can be easily expressed in eukaryotic
a (a) BrCH2CH(OCH3)2, DMF, K2CO3, 115 °C, 12 h, 82%; (b)
concentrated HCl/CHCl3, 0 °C, 30 min, 63%; (c) NaI, acetone, 1 h; (d)
PPh3, EtOAc, o/n, 81% in two steps; (e) CH2Cl2, 1 M NaOH, room
temperature, o/n, 28%; (f) m-CPBA, CH2Cl2, 20 min, 93%; (g) TFA,
cells without any noticeable toxicity.2,3 TEM-1 Bla has been
developed as a reporter for examining the promoter/regulatory
elements activity in living mammalian tissue culture cells.4-6 Protein
CH2Cl2, anisole, 1 h, 0 °C, 71% (for n ) 0) and 81% (for n ) 1).
fragment complementation assays based on Bla have been suc-
cessfully applied to monitor constitutive and inducible protein
interactions both in vitro and in vivo.7-9 Incorporation of Bla into
HIV-1 virions allows convenient detection of HIV-1 virion fusion
in primary T lymphocytes in complex cell populations.10
Substrates that detect Bla activity with high sensitivity are critical
for the successful implementations of Bla assays. Fluorogenic
substrates are superior to chromogenic substrates (such as well-
known nitrocefin and PADAC)11-13 in detecting enzyme activity
because of the high sensitivity of fluorescence detection. The first
reported fluorogenic Bla substrate shown to work with cells was
CCF2, which consists of a donor 7-hydroxycoumarin linked via a
cephalosporin to an acceptor fluorescein.4 CCF2 fluoresces green
because of fluorescence resonance energy transfer from the cou-
marin donor to the fluorescein acceptor. Hydrolysis of the cepha-
losporin by Bla splits off the fluorescein, disrupts energy transfer,
and shifts the emission to blue.4 As the only fluorogenic Bla
substrate currently available, CCF2 has many successful applications
in tissue culture,4-10 but its high molecular weight and low aqueous
solubility have prevented applications in intact mammalian tissues
or in cells with thick walls such as in yeast or plants. More
fluorogenic substrates would expand the usefulness of Bla as a
biosensor. Here, we report a new class of small fluorogenic
substrates that work by releasing a phenolic dye from a vinylogous
cephalosporin.
Figure 1. (A) Hydrolysis of CC1 by â-lactamase releases the fluorophore
umbelliferone. (B) Emission of CC1 (10 nM in PBS) before (dash dot line)
and after (solid line) treatment of â-lactamase (excitation at 400 nm).
the cephalosporin is linked to the 7-hydroxy group of umbelliferone
alkylated through an allylic ether bond (Scheme 1).15 With the
7-hydroxy group of umbelliferone alkylated, CC1 should be
essentially nonfluorescent, and if Bla hydrolysis leads to spontane-
ous release of umbelliferone, in this case through an extended
conjugation system, fluorescent signals will be produced (Figure
1A). The complete synthesis of CC1 is illustrated in Scheme 1.
The Wittig coupling reaction exclusively afforded the Z-isomer,
1
as shown by its H NMR spectrum.
We first examined the emission spectrum of CC1 before and
after the Bla treatment and observed a 153-fold increase in the
intensity at the wavelength of 460 nm upon complete hydrolysis
of CC1 (Figure 1B). This result validates that Bla cleaves CC1
and releases the fluorophore umbelliferone. The increase in the
fluorescent emission at 460 nm is considerably larger than the 20-
fold enhancement of CCF2 at similar wavelengths and affords a
convenient means to measure the Bla activity. In the phosphate-
buffered saline (PBS) at pH 7.1, CC1 is hydrolyzed by TEM-1
Bla with a catalytic constant (kcat) of 52 ( 1 s-1 and a Michaelis
constant (Km) of 70 ( 7 µM (values were obtained from weighted
least-squares fit of a double-reciprocal plot of the hydrolysis rate
Cleavage of the â-lactam ring of a cephalosporin creates a free
amino group, which triggers spontaneous elimination of any leaving
group previously attached to the 3′ position.14 Umbelliferone is a
widely used fluorophore with maximal excitation at 360 nm and
emission at 460 nm. When its 7-hydroxy group is alkylated, the
compound becomes essentially nonfluorescent. We thus designed
and synthesized a â-lactam called CC1 in which the 3′ position of
† University of California, Los Angeles.
‡ University of California, San Diego.
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J. AM. CHEM. SOC. 2003, 125, 11146-11147
10.1021/ja036126o CCC: $25.00 © 2003 American Chemical Society

C O M M U N I C A T I O N S
Figure 2. Linear dependence of the CC1 hydrolysis rate on the â-lactamase
concentration. Wild-type and CMV-bla Jurkat cells were mixed at different
ratios (with the percentage of CMV-bla Jurkat cells from 0, 0.5, 1, 5, 10,
25, 50, and 75 to 100%), lysed, and diluted for CC1 assay with excitation
at 365/42 nm and emission at 465/35 nm.
Figure 3. (A) Structures of CR2 and CR2/AM. (B) Fluorescence images
of wild-type (left) and Bla-stably transfected (right) C6 glioma cells loaded
with CR2/AM in Hank’s Balanced Salts Solution for 25 min at room
temperature. Excitation filter, 540/25; emission filter, 635/55; and 40×
magnification.
versus CC1 concentration); its catalytic efficiency (kcat /Km) is 7.4
× 105 M-1 s-1. The spontaneous hydrolysis rate constant of CC1
in the PBS is ∼1.3 × 10-6 s-1, and the enzymatic acceleration is
∼4 × 107 fold.
with â-lactamase as a biosensor. The design reported here is not
limited to umbelliferone and resorufin and could be extended to
other molecules containing phenolic leaving groups. It may serve
as a general strategy to create a wide variety of fluorogenic,
chromogenic, and lumogenic substrates for â-lactamase.
As compared to CCF2 (kcat ) 29 s-1 and Km ) 23 µM),4 CC1
has a 2-fold lower affinity for Bla, but its kcat is nearly twice as
fast. Because CC1 itself is nonfluorescent, a higher concentration
can be used to obtain a faster hydrolysis rate. The stability of CC1
in the absence of Bla can be further improved by oxidation of the
sulfide in the six-membered ring to sulfoxide CC2, which resulted
in a 4-fold decrease in the spontaneous hydrolysis rate (∼2.6 ×
10-7 s-1). CC2 remains as a substrate with slightly less catalytic
efficiency (kcat ) 10 s-1 and Km ) 0.35 mM). CC2 may be preferred
for experiments where a longer incubation is needed.
Acknowledgment. This work was supported in part by a Damon
Runyon Cancer Research Foundation fellowship and Burroughs
Wellcome Fund (to J.R.), UCLA start-up funds (to J.R.), and the
NIH NS27177 and DOE DE-FG03-01ER63276 (to R.Y.T.). We
are grateful to Dr. Q. Li at NewBiotics, Inc., for the generous gift
of purified TEM-1 â-lactamase. We thank Dr. Hasegawa for his
assistance with Jurkat lymphocyte experiments.
We tested the ability of CC1 in detecting Bla activity in
measuring the percentage of Bla-expressed cells in a cell mixture.
Wild-type Jurkat cells do not express any Bla, and a clonal Jurkat
cell line constitutively transfected with Bla gene under the cyto-
megalovirus (CMV) promoter control (CMV-bla Jurkat cells)
expresses approximately 1.5 × 104 Bla/cell.5 Two cell lines were
mixed at different ratios, and the cell lysates were analyzed with
CC1 for the Bla activity. A plot of the apparent hydrolysis rate
versus the percentage of CMV-bla Jurkat cells reveals a linear
relationship (Figure 2). This assay can reliably detect 0.5% CMV-
bla Jurkat cells in the background of wild-type cells, which
corresponds to approximately 500 fM of Bla.
The importance of the inserted double bond is further exemplified
in making a red fluorescent substrate, which is preferred due to a
longer excitation and emission wavelength. Resorufin fluoresces
maximally at 585 nm when excited at 550 nm, and a structure with
resorufin directly linked to the 3′-position of cephalosporin has been
made but found to spontaneously hydrolyze rapidly in water.16
However, an analogue of CC2 with umbelliferone replaced by
resorufin (CR2) is stable in PBS buffer with a half-life of ∼182 h.
CR2 displays a 42-fold increase in the fluorescence intensity upon
the hydrolysis by Bla with the catalytic parameters kcat ) 17 ( 3
s-1 and Km ) 114 ( 12 µM. A membrane-permeable acetoxymethyl
ester of CR2 (CR2/AM) was able to image the Bla activity in Bla-
stably transfected C6 glioma cells (Figure 3).
Supporting Information Available: Synthesis of CC1, CC2, and
CR2, and procedures for the measurements of kinetic parameters and
Bla activity in cell lysates (PDF). This material is available free of
charge via the Internet at http://pubs.acs.org.
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This report describes a design of a new class of fluorogenic
substrates of â-lactamase and characterization of their enzymatic
kinetics, and it demonstrates their applicability in detecting â-lac-
tamase activity in biological samples. These new fluorogenic
substrates are easy to make, simple to use, have high sensitivity
for detecting â-lactamase activity, and should facilitate applications
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