Enhancing the synthetic utility of aldolase antibody 38C2

Article abstract of DOI:10.1016/j.bmcl.2005.11.019

Three-phase partitioning (TPP) treated aldolase antibody 38C2 was evaluated for aldol reaction between p-nitrobenzaldehyde and acetone to give 4-(4′-nitrophenyl)-4-hydroxy-2-butanone. While TPP-treated 38C2 transformed 65% of p-nitrobenzaldehyde, the untr

Full text of DOI:10.1016/j.bmcl.2005.11.019

Bioorganic & Medicinal Chemistry Letters 16 (2006) 807–810
Enhancing the synthetic utility of aldolase antibody 38C2
Kalyani Mondal, Namakkal G. Ramesh, Ipsita Roy and Munishwar N. Gupta*
Chemistry Department, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi 110016, India
Received 15 June 2005; revised 25 October 2005; accepted 8 November 2005
Available online 29 November 2005
Abstract—Three-phase partitioning (TPP) treated aldolase antibody 38C2 was evaluated for aldol reaction between p-nitrobenzal-
0
dehyde and acetone to give 4-(4 -nitrophenyl)-4-hydroxy-2-butanone. While TPP-treated 38C2 transformed 65% of p-nitrobenzal-
dehyde, the untreated 38C2 gave only 24% transformation in 18 h at 25 ꢀC. However, since TPP-treated 38C2 also gave an
additional (unidentified) product, its synthetic utility was limited. Crosslinked aggregate of 38C2, however, gave the biocatalyst
which gave a single product and could be reused at 40 ꢀC five times without loss of activity.
ꢁ
2005 Elsevier Ltd. All rights reserved.
The aldolase antibody 38C2 is among the first antibody
preparation which is commercially available and has al-
1
ready proved its usefulness in catalyzing self aldol,
1
crossed aldol, retro aldol, Robinson annulation reac-
2
3
4
tions, and decarboxylation of b-ketoacids. Its conju-
gate with a water-soluble polymer has been used for
prodrug activation for selective chemotherapy for
cancer. Earlier, an organoinsulin molecule and etopside
derivative have been reported as prodrugs for diabetes
and cancer chemotherapy, respectively. The antibody
8C2 is reported to have 40 times less catalytic efficiency
as compared to the aldolase. It is also an expensive bio-
catalyst. The present work is aimed at enhancing its cat-
alytic efficiency and reusability.
0
Scheme 1. Synthesis of 4-(4 -nitrophenyl)-4-hydroxy-2-butanone by
aldol reaction between p-nitrobenzaldehyde and acetone.
5
6
,7
concentration, ammonium sulfate concentration, and
the volume of t-butanol added to 200 lL protein solution.
The optimized conditions are as described. Under these
3
8
16
conditions, 82% of the starting protein was found in the
interfacial layer in the precipitate form.
The aldol reaction used to monitor the catalytic
efficiency of 38C2 aldolase antibody was the reaction be-
The time course of the reaction with TPP-treated 38C2
(
Fig. 2, curve B) indicates that it shows a higher catalytic
conversion as compared to untreated 38C2 (Fig. 2, curve
A). Whereas untreated 38C2 gave 24% conversion in
1
tween p-nitrobenzaldehyde (1) and acetone (Scheme 1).
HPLC was used to estimate the disappearance of
1
,10
p-nitrobenzaldehyde.
18 h, the TPP-treated 38C2 gave 65% conversion in
the same time.
The first approach attempted was that of three-phase par-
titioning (TPP). It has been observed that when a pure en-
zyme is subjected to TPP, the treated enzyme shows
The nature of the product(s) formed is also different in
the case of TPP-treated 38C2 (Fig. 1). Apart from the
1
1–15
enhancement of the catalytic efficiency.
The condi-
17
expected aldol product (2, Scheme 1) with a retention
tions for subjecting 38C2 aldolase antibody were opti-
mized for obtaining the maximum amount of protein in
the interfacial layer. The parameters varied were protein
time of 6.62 min, the TPP-treated 38C2 also gave an
additional product (retention time, 7.74 min) (Fig. 1).
However, while the enhancement in catalytic efficiency
of 38C2 upon TPP-treatment is in agreement with earli-
1
4
er results with other enzymes such as a-chymotrypsin,
Keywords: Three-phase partitioning; Aldolase antibody 38C2; Cross-
linked aggregate; Reusability.
1
2
16
proteinase K, and lipase, the formation of an addi-
tional product indicated that TPP-treated biocatalysts
may function in a different way mechanistically. The
*
Corresponding author. Tel.: +91 11 2659 1503; fax: +91 11 2658
073; e-mail: munishwar48@yahoo.co.uk
1
0
960-894X/$ - see front matter ꢁ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.11.019

808
K. Mondal et al. / Bioorg. Med. Chem. Lett. 16 (2006) 807–810
1
00
80
0.20
0.10
0.00
A
B
6
4
2
0
0
0
0
0
5
10
15
Time (h)
20
25
Figure 2. Time course of TPP-treated antibody reaction. The aldol
reaction between p-nitrobenzaldehyde and acetone was carried out
using untreated antibody (d) and TPP-treated antibody (s) as
1
6
described. Samples were withdrawn at different time intervals and
the amount of p-nitrobenzaldehyde (substrate) used in the reaction was
monitored through HPLC at 254 nm. The experiment was done in
duplicate and the error bars show the variation in the readings. The
observed standard deviation in each set of readings was less than 0.2%.
sulfate (80% w/v) precipitated 95% protein which re-
tained 94% aldolase activity. Under optimum condi-
tions, 40 mM glutaraldehyde formed CLEA which
showed 120% activity. Marginal increase (in this
range) in the activity after CLEA formation has been
2
0
1
9
reported by earlier workers as well. CLEA gave only
one product and that was the expected aldol product
1
1
20
00
80
14.70
0.00
Retention time (min)
6
4
2
0
0
0
0
Figure 1. HPLC chromatogram of aldol condensation between p-nitro
benzaldehyde (t = 9.71 min) and acetone (t = 2.12 min) with TPP-
treated aldolase antibody 38C2 resulting in the formation of the usual
aldol product (t = 6.21 min) and a new product (t = 7.74 min).
R
R
R
R
above catalytic efficiency was based upon an assay
which measures depletion of the substrate. The forma-
tion of second product in significant amount implies
that product yield of desirable aldol would be consider-
ably lower (see Fig. 2). In that respect, the TPP did not
yield the desired biocatalyst with better synthetic utility.
A recent biocatalyst design is crosslinked enzyme aggre-
gates (CLEA) which are based upon crosslinking of a
protein which is initially precipitated by adding an
0
5
10
15
Time (h)
Figure 3. Thermal stability of antibody 38C2 and CLEA of 38C2 at
5
0 ꢀC. Catalytic antibody 38C2 (100 lg/100 lL) samples (d) were
incubated at 50 ꢀC for different periods of time. Thereafter, the sample
was cooled to 25 ꢀC and assayed for its activity with p-nitrobenzal-
dehyde (ref). The experiment was repeated with CLEA of 38C2 (s).
Residual activity was calculated by taking the activity of 38C2 at 0
time to be 100%. The experiment was done in duplicate and the error
bars show the variation in the readings. The observed standard
deviation in each set of readings was less than 0.1%.
1
8,19
organic solvent or a salt.
After screening some pre-
cipitating agents (data not shown), precipitation with
ammonium sulfate was found to work best. Ammonium

K. Mondal et al. / Bioorg. Med. Chem. Lett. 16 (2006) 807–810
809
1
00
References and notes
1
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. Fainaro, R. S.; Wrasidlo, W.; Lode, H. N.; Shabat, D.
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3514.
Cycles
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7528.
Figure 4. Reusability of CLEA of 38C2. The aldol reaction with
CLEA was carried out at 40 ꢀC for 10 h. Thereafter the reaction
mixture was removed, CLEA washed with 1 mL buffer and fresh
reactants added. After the 5th cycle, loss of protein (5 lg) was detected.
After the 10th cycle, the washings were pooled and the amount of
protein that leached out was found to be 15 lg (as estimated by the
8. Tanaka, F.; Barbas, C. F., III J. Immunol. Methods 2002,
269, 67.
9. Bradford, M. M. Anal. Biochem. 1976, 72, 248.
10. Murine aldolase antibody 38C2 was purchased from
Sigma Chemicals Co. (Cat. No. 479950). p-Nitro benzal-
dehyde was recrystallized from an ethanol–water mixture
9
Bradford method ). Residual activity of CLEA of 38C2 was calculated
by taking the activity of 38C2 in the first cycle as 100%.
(
drous CaSO , distilled, and further dried over activated
1:1, v/v) for further use. Acetone was dried over anhy-
4
molecular sieves before use. Diisopropylamine was dis-
tilled over sodium hydride just before use. All other
solvents were freshly distilled before use. All antibody
catalyzed reactions were carried out in buffered saline
(
as checked by the HPLC assay). Investigating thermal
stability of 38C2 and CLEA of 38C2 showed that CLEA
had a higher thermal stability (Fig. 3). At 50 ꢀC, CLEA
retained 100% activity, while 38C2 had only 75% activ-
ity after 5 h of thermal exposure. However, CLEA also
lost 20% activity after 10 h. At 40 ꢀC, both 38C2 and its
CLEA retained complete activity even after 12 h. While
at 25 ꢀC, free 38C2 gave 10% conversion and CLEA
gave 13% conversion in 10 h, at 40 ꢀC the corresponding
conversion figures were 33% and 38%, respectively (con-
version in the range of 30% has been reported during
(
0.01 M phosphate, 0.15 M NaCl, pH 7.4) and monitored
by high performance liquid chromatography (HPLC)
(Beckmann System Gold HPLC) using a reverse-phase
C18 column (150 mm · 4.6 mm). The eluent was 25%
acetonitrile in water containing trifluoroacetic acid (0.1%)
with a flow rate of 1.0 mL/min. The peaks were detected at
1
54 nm .
2
1. Singh, R. K.; Gourinath, S.; Sharma, S.; Roy, I.; Gupta,
1
1
M. N.; Betzel, C.; Srinivasan, A.; Singh, T. P. Protein Eng.
001, 14, 307.
2
2
1
synthetic application of 38C2). Hence, 40 ꢀC was
chosen to evaluate reusability of CLEA. Figure 4 shows
the real advantage of working with CLEA. CLEA of
2. Roy, I.; Sharma, A.; Gupta, M. N. Bioorg. Med. Chem.
Lett. 2004, 18, 887.
13. Roy, I.; Gupta, M. N. Biocatal. Biotransform. 2004, 22,
261.
3
8C2 could be used up to 5 cycles without losing any
aldolase activity. Even after 10 cycles, it lost only 20%
activity. It may be mentioned that very few immobilized
catalytic antibodies have been described in the
14. Singh, N.; Jabeen, T.; Sharma, S.; Roy, I.; Gupta, M. N.;
Bilgrami, S.; Somvanshi, R. K.; Dey, S.; Perbandt, M.;
Betzel, C.; Singh, T. P. FEBS J. 2005, 272, 562.
15. Roy, I.; Gupta, M. N. Enzyme Microb. Technol. 2005, 36,
2
2,23
literature.
catalytic antibody has also been reported during immo-
Significant loss in biological activity of
8
96.
1
6. Catalytic antibody 38C2 solutions (200 lL, 10 mg/mL in
buffered saline) were mixed with 30% saturated ammo-
nium sulfate (w/v). This was followed by the addition of
t-butanol (200 lL). The solution was gently vortexed
and incubated at 25 ꢀC for 30 min. Three phases (i.e.,
upper layer of t-butanol, interfacial precipitate of
protein, and lower aqueous layer) were formed. The
mixture was then centrifuged at 1800g for 5 min. This
2
3
bilization. Considering the expensive nature of 38C2,
CLEA of 38C2 offers an efficient approach for the
synthetic application of this versatile biocatalyst.
Acknowledgments
low-speed centrifugation gave
a compact interfacial
This work was supported by funds from Astrazeneca
Research Foundation, Bangalore, India. The authors
thank Dr. S. Anand Kumar, Director, Astrazeneca
Research Foundation, for initiating their interest in
catalytic antibody 38C2. The authors also thank Ms
Parul Jain for preparing the aldol product by a known
synthetic route for HPLC analysis. The financial
support by IIT to KM in the form of Senior Research
Fellowship is also acknowledged.
precipitate and clearly separated the lower aqueous
and upper organic layers. The aqueous layer was
pipetted out using a Pasteur pipette, after piercing the
precipitate layer. The t-butanol layer was similarly
removed. The precipitate was then dissolved in phos-
phate-buffered saline so that the total volume was
200 lL. The dissolved precipitates were desalted on a
prewashed (with buffered saline) PD10 column (pre-
packed Sephadex G25) to remove ammonium sulfate

810
K. Mondal et al. / Bioorg. Med. Chem. Lett. 16 (2006) 807–810
and t-butanol. The absorbance of the fractions collected
1 mL each) was read at 280 nm. The fractions contain-
18. Cao, L.; van Langen, L.; Sheldon, R. A. Curr. Opin.
Biotechnol. 2003, 14, 387.
(
ing the protein were pooled and concentrated (against
sucrose) to 1 mL. 82% of the starting protein was found
to partition to the interfacial layer during TPP as
19. Schoevaart, R.; Wolbers, M. W.; Golubovic, M.; Ottens,
M.; Kieboom, A. P. G.; van Rantwijk, F.; van der Wielen,
L. A. M.; Sheldon, R. A. Biotechnol. Bioeng. 2004, 87, 754.
20. Procedure for preparation of CLEA of 38C2: Catalytic
antibody 38C2 solution (100 lg in 100 lL of 0.01 M
phosphate buffer, pH 7.4) was added to 2 mg bovine
serum albumin (BSA) and the volume made up to
200 lL. Ammonium sulfate (80%, w/v) was then added to
precipitate the protein and incubated at 4 ꢀC for 30 min
for complete precipitation. Glutaraldehyde (40 mM)
solution was then added to the precipitated protein
solution and the reaction mixture was incubated at 25 ꢀC
for 3 h. The aggregates formed were washed thrice with
1 mL PBS to remove unreacted glutaraldehyde. The
CLEA of antibody 38C2 thus formed were used for
further reactions.
9
estimated by BradfordÕs method. To this solution of
3
1
8C2,
p-nitrobenzaldehyde
(final
concentration
.04 mM) and acetone (final concentration 0.12 M) were
added and kept in an incubator-shaker at 25 ꢀC for
different time periods. Thereafter, the reaction was
monitored by HPLC at 254 nm. A similar reaction
was carried out with untreated-catalytic antibody. The
amount of product formed was calculated by taking the
substrate concentration at 0 h as 100% and subtracting
the amount of substrate that was used up at the given
time.
1
7. Compound 2. This compound (aldol product) was chem-
1
ically synthesized as per the method described. HPLC
showed the same retention time (6.62 min) as the aldol
product obtained in the antibody reaction. Also, upon
coinjection of the reaction mixture with an aliquot of the
chemically synthesized aldol, the peak for the aldol
product was found to be overlapped, thus proving that
the chemically synthesized product was the same as in the
aldol mixture.
21. Shulman, A.; Keinan, E.; Shabat, D.; Barbas, C. F., III J.
Chem. Edu. 1999, 76, 977.
22. Janda, K. D.; Ashley, J. A.; Jones, T. M.; McLeod, D. A.;
Schloeder, D. M.; Weinhouse, M. I. J. Am. Chem. Soc.
1990, 112, 8886.
23. Spitznagel, T. M.; Jacobs, J. W.; Clark, D. S. Enzyme
Microb. Technol. 1993, 15, 916.