A new visual screening assay for catalytic antibodies with retro-aldol retro-Michael activity

Article abstract of DOI:10.1016/j.bmcl.2006.12.057

Fast and convenient methods are required for the detection of novel catalysts. We have developed a new assay to allow direct visualization of retro-aldol retro-Michael catalytic activity and have demonstrated it with catalytic antibody 38C2. The assay is

Full text of DOI:10.1016/j.bmcl.2006.12.057

Bioorganic & Medicinal Chemistry Letters 17 (2007) 1172–1175
A new visual screening assay for catalytic antibodies with
retro-aldol retro-Michael activity
Marina Shamis,a Carlos F. Barbas, IIIb and Doron Shabata,*
aDepartment of Organic Chemistry, School of Chemistry, Raymond and Beverly Sackler Faculty of Exact Sciences,
Tel-Aviv University, Tel Aviv 69978, Israel
bDepartment of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
Received 8 November 2006; revised 7 December 2006; accepted 8 December 2006
Available online 22 December 2006
Abstract—Fast and convenient methods are required for the detection of novel catalysts. We have developed a new assay to allow
direct visualization of retro-aldol retro-Michael catalytic activity and have demonstrated it with catalytic antibody 38C2. The assay
is based on a catalytic cleavage of a physiologically stable substrate to release 3,4-cyclohexeneoesculetin. The latter then reacts with
iron(III) to generate a non-soluble complex that precipitates in the form of a black dye. This assay may be used for screening new
catalysts for retro-aldol retro-Michael activity with improved efficiency for specific prodrug activation.
Ó 2006 Elsevier Ltd. All rights reserved.
The tandem retro-aldol retro-Michael reaction catalyzed
by antibody 38C2 is an efficient cleavage reaction that
has potential for prodrug activation.1–3 This reaction
is not known to be catalyzed by natural enzymes and,
therefore, non-specific prodrug activation by endoge-
nous enzymes should not occur. The unique activity of
this antibody is derived from an e-amino-lysine residue
with a significantly perturbed pKa (5.8) buried deeply
in the antigen binding pocket. This lysine is capable of
reacting with a ketone functionality at physiological
pH and consequently generates a highly reactive enam-
ine species.4 Several variations of fluorogenic assays
example of such a substrate (Fig. 1). Antibody 38C2 cat-
alyzes the retro-aldol retro-Michael cleavage reaction to
generate an amine intermediate that is cyclized sponta-
neously to release 3,4-cyclohexenoesculetin. In the pres-
ence of iron(III) ion, 3,4-cyclohexenoesculetin forms
complex 3, a black precipitate that is constructed of
three molecules of 3,4-cyclohexenoesculetin per ion of
iron III (Fig. 2). This assay has been recently used for
staining cells with cloned DNA that contains the se-
quence for b-galactosidase.9 We sought to replace the
b-galactosidase substrate with a retro-aldol retro-Mi-
chael substrate in order to detect the aldolase activity
of catalytic antibody 38C2.
have been developed to monitor aldolase activity5,6
,
including a direct visual detection assay7 and one
amperometric assay.8 Here, we report on a new visual
detection assay for the screening of retro-aldol retro-
Michael catalytic activity.
Substrate 1 was synthesized as outlined in Figure 3.10
3,4-Cyclohexenoesculetin 2 was selectively protected as
methoxymethyl-ether 4 and then reacted with 4-nitro-
phenyl-chloroformate to give the 4-nitrophenyl-carbon-
ate 5. Reaction of the retro-aldol retro-Michael linker 6a
(prepared as previously described1) with carbonate 5
afforded compound 6, which was deprotected with
TFA to give substrate 1.
Simple, sensitive in vitro detection of a catalyst can be
achieved if the desired reaction converts a non-visible
substrate into a new product that can react with an addi-
tional reagent to generate a colorful precipitate. Com-
pound 1, generated from 3,4-cyclohexenoesculetin 2
and a retro-aldol retro-Michael linker, is a promising
To determine whether compound 1 is stable under phys-
iological conditions, it was incubated in phosphate-buf-
fered saline, pH 7.4 (PBS) at 37 °C for 72 h. No
decomposition was observed. The reaction of substrate
1 upon incubation with catalytic antibody 38C2 was
monitored by reverse-phase HPLC. We found that the
antibody indeed catalyzed the retro-aldol retro-Michael
Keywords: Catalytic antibodies; Prodrug activation; Aldol reaction;
Assay.
*
Corresponding author. Tel.: +972 3 640 8340; fax: +972 3 640
9293; e-mail: chdoron@post.tau.ac.il
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.12.057

M. Shamis et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1172–1175
1173
O
O
HO
Antibody 38C2
OH
OH
O
N
O
N
O
O
O
O
O
OH
2
1
O
O
O
N
N
CO
2
Figure 1. Antibody 38C2 catalyzed retro-aldol retro-Michael cleavage reaction of substrate 1, followed by spontaneous cyclization to release
3,4-cyclohexenoesculetin 2.
O
O
H
O
H
O
OH
OH
O
Fe+3
Fe
O
O
O
O
O
O
O H
3
2
O
O
Figure 2. Three molecules of 3,4-cyclohexenoesculetin react with iron(III) ion to generate a complex that forms a black precipitate.
O
NO
2
4-nitrophenyl-
chloroformate
O
OH
CH OCH Cl
3
2
2
O
O
O
O
O
OCH OCH
OCH OCH
3
2
3
2
5
4
6aO
N
O
HO
O
O
HO
N
O
BOC
O
N
TFA/MeOH
1
O
N
1. TFA
2. Et N
O
3
O
O
6
OCH OCH
2
3
Figure 3. Chemical synthesis of substrate 1.
cleavage reactions to generate 3,4-cyclohexenoesculetin.
As shown in Figure 4, compound 1 was gradually con-
verted to the product 3,4-cyclohexenoesculetin; no
amine intermediate was observed. Lack of detection of
the intermediate can be explained by the fast cyclization
step that occurs spontaneously after the retro-aldol
retro-Michael cleavage. Since substrate 1 was synthe-
sized in its racemic form, one enantiomer was cleaved
much faster by the catalytic antibody than the other.
This results in a slower reaction rate after 50%
conversion of the substrate to product.
70
60
50
40
30
20
10
0
0
200 400 600 800 1000 1200 1400 1600 1800
To evaluate the utility of the substrate in a visual assay,
we incubated substrate 1 and iron(III) chloride with a
catalytic amount of antibody 38C2 in PBS (pH 7.4). A
control reaction included substrate 1 and iron(III) chlo-
ride in PBS (pH 7.4). A generation of a black precipitate
was clearly observed in the tube with catalytic antibody
Time [min]
Figure 4. Conversion of substrate 1 to 3,4-cyclohexenoesculetin versus
time. Substrate 1 [500 lM] in PBS (pH 7.4) with catalytic antibody
38C2 (50 lM) at 37 °C (blue). Substrate 1 in the absence of the
antibody (red).

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M. Shamis et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1172–1175
In summary, we have developed a new screening assay
for retro-aldol retro-Michael catalytic activity that can
be clearly visualized. The assay is based on a catalytic
cleavage of a physiologically stable substrate to release
3,4-cyclohexenoesculetin. The substrate is cleaved by
catalytic antibody 38C2. 3,4-Cyclohexenoesculetin
reacts with iron(III) ion to generate a non-soluble
complex that precipitates as a black dye. The black
dye was clearly observed in the solution in the presence
of the antibody, whereas the control solution remained
clear. This assay may be used in a search for new cata-
lysts with retro-aldol retro-Michael activity that can
have improved efficiency for specific prodrug activation.
Acknowledgment
D.S. thanks the Israel Science Foundation, the Israel
Ministry of Science ‘Tashtiot’ program, and the Israel
Cancer Association for financial support.
References and notes
1. Shabat, D.; Lode, H. N.; Pertl, U.; Reisfeld, R. A.; Rader,
C.; Lerner, R. A.; Barbas, C. F., 3rd. Proc. Natl. Acad.
Sci. U.S.A. 2001, 98, 7528.
2. Shabat, D.; Rader, C.; List, B.; Lerner, R. A.; Barbas, C.
F., III Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 6925.
3. Satchi-Fainaro, R.; Wrasidlo, W.; Lode, H. N.; Shabat,
D. Bioorg. Med. Chem. 2002, 10, 3023.
4. Wagner, J.; Lerner, R. A.; Barbas, C. F., III Science 1995,
270, 1797.
5. List, B.; Barbas, C. F., 3rd; Lerner, R. A. Proc. Natl.
Acad. Sci. U.S.A. 1998, 95, 15351.
6. (a) Carlon, R. P.; Jourdain, N.; Reymond, J.-L. Chem.
Eur. J. 2000, 6, 4154; (b) Jourdain, N.; Perez Carlon, R.;
Reymond, J.-L. Tet. Lett. 1998, 39, 9415.
Figure 5. (A) Photograph of a tube containing substrate 1 (500 lM),
iron(III) chloride (200 lM), and catalytic antibody 38C2 (50 lM) (on
the left), and a tube with substrate 1 and iron(III) chloride (on the
right). (B) Photograph of the tubes described in (A) under 340 nm UV
light.
7. Tanaka, F.; Kerwin, L.; Kubitz, D.; Lerner, R. A.;
Barbas, C. F., 3rd. Bioorg. Med. Chem. Lett. 2001, 11,
2983.
8. Sagi, A.; Rishpon, J.; Shabat, D. Anal. Chem. 2006, 78,
1459.
38C2, while the control reaction remained completely
clear (Fig. 5A). 3,4-Cyclohexenoesculetin and derivative
1 are both fluorescent. When the control reaction was
exposed to 340 nm UV light, fluorescence was clearly
observed; no fluorescence was observed in the tube
containing antibody (Fig. 5B). This phenomenon is
explained by the reaction of the 3,4-cyclohexenoescule-
tin with iron(III) ion, resulting in metal complex 3
that quenches the fluorescence generated by the free
3,4-cyclohexenoesculetin. We evaluated the sensitivity
of the assay by analyzing a series of reactions prepared
with a range of antibody concentrations. The black
precipitate and quenched UV signal could be detected
down to 1 lM of catalytic antibody 38C2.
9. Heuermann, K.; Cosgrove, J. BioTechniques 2001, 30,
1142, 1146.
10. Compound 4. The commercially available 3,4-cyclohexe-
noesculetin 2 (100 mg, 0.431 mmol) and tert-butyl potas-
sium hydroxide (48.36 mg, 0.431 mmol) were dissolved in
2 mL DMF and cooled to 0 °C. Chloromethyl-methyl-
ether (33 lL, 0.431 mmol) was added dropwise to the
stirred solution. The reaction mixture was stirred for 2 h at
room temperature and monitored by TLC (EtOAc/He,
3:1). After completion, the mixture was diluted with
EtOAc, washed with satd solution of NH4Cl, dried over
sodium sulfate, and the solvent was removed under
reduced pressure. The product was purified by column
chromatography on silica gel (EtOAc/Hex, 2:3) to give
One important advantage of this assay is that it may be
applied for selection of proteins expressed from cloned
DNA in Escherichia coli colonies. If the expressed
protein has a retro-aldol retro-Michael catalytic activity,
it will form the black dye/iron complex that will
precipitate. Since the black dye is not water-soluble, it
will gradually accumulate in the cell and form a visual
stain that will indicate the colony that expresses a
protein with the desired catalytic activity.
1
compound 4 (67 mg, 56%). H NMR (200 MHz, CDCl3):
d = 7.09 ppm (2H, s); 3.53 (2H, s); 2.72 (2H, m); 1.86–1.81
(4H, m); 1.58 (3H, s).
Compound 5. Compound 4 (67 mg, 0.24 mmol) was
dissolved in dried 2 mL THF. Triethylamine (30 lL) was
added. The reaction mixture was cooled to 0 °C and 4-
nitrophenylchloroformate (48.7 mg, 0.24 mmol) dissolved
in 2 mL THF was added dropwise. The reaction mixture
was stirred at room temperature for 1 h and monitored by

M. Shamis et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1172–1175
1175
TLC (EtOAc/He, 3:1). After completion of reaction, the
precipitate was recovered by filtration and the remaining
solvent was removed under reduced pressure. The product
was purified by column chromatography on silica gel
(EtOAc/Hex, 2:3) to give compound 5 in the form of white
powder (69.5 mg, 65%). 1H NMR (200 MHz, CDCl3):
d = 8.3 ppm (2H, d, J=7); 7.5 (2H, d, J=7); 7.4 (1H, s);
7.23 (1H, s); 3.5 (2H, s); 2.72 (2H, m); 2.58 (2H, m); 1.84
(4H, m) 1.56 (3H, s).
tion, the DMF was removed under reduced pressure and
the crude product was purified by flash chromatography
(MeOH/EtOAc, 2:98) to give pure compound 6 in the
form of white powder (47 mg, 54%). 1H NMR (200 MHz,
CDCl3): d = 7.14 (2H, s); 4.24(2H, m); 3.51–3.48 (4H, m);
3.13 (2H, s); 3.04 (2H, s); 2.99–2.98 (3H, m); 2.72 (2H, m);
2.65 (2H, d, J=4); 2.58 (2H, m); 2.17 (3H, s); 1.88 (2H, m);
1.84 (4H, m); 1.23 (3H, s). MS(FAB): C28H38N2 O10
[M+Na]+ 585.1
Compound 6. Retro-aldol–retro-Michael linker 6a
(57.1 mg, 0.156 mmol) was deprotected from the Boc
with 1 mL TFA for 2 min at 0 °C. The excess of the acid
was removed under reduced pressure and the amine salt
was dissolved in 2 mL DMF. Compound 5 (69.5 mg,
0.156 mmol) was added in with 0.5 mL of triethylamine
and the solution was stirred for 10 min. The reaction was
monitored by TLC (EtOAc/MeOH, 9:1). After comple-
Compound 1. Compound 6 (47 mg, 0.08 mmol) was
dissolved in 1 mL DCM and 1 mL TFA at 0 °C. The
solution was stirred for10 min and reaction was monitored
by TLC (EtOAc/MeOH, 9:1). After completion, the
solvent was removed under reduced pressure and purified
by column chromatography on silica gel (EtOAc, 100%) to
give desired compound
6 (30 mg, 75%). MS(FAB):
C26H34N2 O9 [M+H]+ 519.1.