Antibody-catalyzed hydrolysis of oligomeric esters: A model for the degradation of polymeric materials

Article abstract of DOI:10.1039/b008065i

A catalytic antibody has been discovered that degrades oligomeric ester substrates.

Full text of DOI:10.1039/b008065i

Antibody-catalyzed hydrolysis of oligomeric esters: a model for the
degradation of polymeric materials†
Oliver Brümmer, Timothy Z. Hoffman, Da-Wei Chen and Kim D. Janda*
Department of Chemistry, The Scripps Research Institute and The Skaggs Institute for Chemical Biology, 10550
North Torrey Pines Road, La Jolla, CA 92037, USA. E-mail: kdjanda@scripps.edu
Received (in Cambridge, UK) 5th October 2000, Accepted 15th November 2000
First published as an Advance Article on the web 11th December 2000
A catalytic antibody has been discovered that degrades
oligomeric ester substrates.
ity and workable solubility parameters for the kinetic analysis of
the appearance and disappearance of distinct oligomeric
subunits. Based on these considerations (vide supra), and the
need for maximal antibody substrate recognition, 4-hydroxy-
phenylacetic acid 3 was chosen as our monomer for oligomeric
synthesis.
Twenty-five monoclonal antibodies (mAbs) were screened
against tetramer 4, where the term ‘tetramer’ refers to the total
number of aromatic residues, that contained distinct terminal
units (Fig. 1). Though 4 was only soluble up to 250 mM in
aqueous buffer (50 mM PIPES, 50 mM NaCl, pH 6.7, 5%
DMSO cosolvent), we were still able to conduct the assay at 500
mM of 4 under heterogeneous conditions using an orbital shaker.
Three mAbs were found that cleaved the tetramer by cutting at
A topic of current interest to both the chemical and global
community alike concerns the development of readily degrad-
able polymers and benign agents necessary for polymer
breakdown.1,2 From an environmental perspective, microbial/
enzymatic approaches for polymer breakdown are very attrac-
tive, and while a number of commercially used polymers are
sensitive to biodegradation, a large variety of commonly used
materials are essentially resistant.3–5 Furthermore, our current
understanding is not sufficient to fully predict a degradability
profile or to specifically target an enzyme/microorganism that
could degrade a man-made, synthetic polymer.6
Antibodies bind biological macromolecules or haptens with
high affinities and specificities.7 We and others have reported
antibodies that bind phosphorus transition-state analogues can
catalyze the hydrolysis of carbonate, ester, carbamate and amide
substrates.8 Yet, to date there have been no reports of antibody
catalysts that accept oligomeric versions of these simple
substrate classes. Herein we report the first example of a
catalytic antibody capable of degrading synthetic oligomeric
esters.
Several tactics could be envisioned in a hapten design for an
antibody-catalyzed hydrolysis of an oligomer. Among the ones
we considered include: (1) a transition-state analogue used as a
repeating unit, and (2) a pseudo-symmetric transition-state
analog. Both approaches have merit, but to date no method-
ology for antibody-catalyzed oligomer degradation has been
disclosed.8 We recently synthesized the new transition-state
analogue phosphorodithioate hapten 1 and used this molecule
attached to keyhole limpet hemocyanin (KLH) to obtain
antibodies with excellent catalytic activity for the hydrolysis of
carbonate 2a (kcat 2.7 min21) and good activity to ester 2b (kcat
0.27 min21).9 Based on the carbonate and esterolytic activities
seen from antibodies derived from 1 and its pseudo-symmetric
structure we reexamined this panel of antibodies for possession
of ‘deoligomerase’ activity. Our objective was thus to see if an
antibody could perform either a stepwise degradation from an
oligomer’s terminus or mediate nonselective endo-cleavage on
a starting oligomer and resulting subunit structures.
Before antibody screening could be engaged a set of
oligomers that could be recognized based on 1’s haptenic
structure needed to be prepared. While aromatic oligocarbonate
structures were deemed to be the most highly congruent
substrates for antibody library recognition we found such
molecules to be unstable for screening. As a second choice,
consideration was given to oligoesters. Polyesters are notori-
ously insoluble in both aqueous and organic solvents.2 How-
ever, we found that smaller molecular weight oligoesters could
be synthesized in a controlled stepwise and highly pure
manner.10 Furthermore, these oligomers displayed good stabil-
† Electronic supplementary information (ESI) available: experimental
procedures are provided including kinetic assays, and full characterization
(1H and 13C NMR, HRMS, HPLC traces) of the compounds under
discussion (4, 6–8, 10–12). See http://www.rsc.org/suppdata/cc/b0/
b008065i/
Fig. 1 Structures of hapten 1, inhibitor 9, carbonate 2a, oligoesters 4, 6,
10–12, simple esters and products derived from hydrolysis, 2b, 3, 5, 7 and
8.
DOI: 10.1039/b008065i
Chem. Commun., 2001, 19–20
This journal is © The Royal Society of Chemistry 2001
19

all three ester linkages. Interestingly, none of these antibodies
were noted previously to be catalysts for the hydrolysis of the
simple carbonate 2a, hence they had not been previously
examined as catalytic antibodies for monoester hydrolysis.9 The
most impressive mAb, OB2-48F8, was studied in greater detail
since it degraded 4 to the products 3, 5, 6, 7 and 8 (Fig. 1) with
the best overall rate estimated semi-quantitatively from the sum
of product concentrations measured by HPLC. Thereafter, the
antibody was freshly prepared and extensively purified by
protein G affinity chromatography followed by MonoQ ion
exchange chromatography. Notably, the antibody specific
activity increased upon purification, as required. Also, the Fab
fragment of OB2-48F8 was prepared, purified, and displayed a
similar activity compared to the whole IgG. Hence, we ruled out
contamination by esterases as a source of the deoligomerization
reaction.
Due to the solubility limitations of 4, the trimer 6 was used as
a substrate to quantitatively obtain an assessment of antibody
activity. The upper limit of solubility of 6 in the above buffer–
cosolvent system was 750 mM and allowed a full kinetic
analysis of the formation of 3 and 7 by OB2-48F8 (kcat = 2.2 3
1022 min21, Km = 580 mM, kcat/kuncat = 1.5 3 103). The
phosphorodithioate 9 was a competitive inhibitor (Ki = 6.2 mM)
for this route of trimer degradation.11,12 Concurrently, the
antibody also catalyzed the cleavage of 6 to subunits 5 and 8
(kcat = 1.5 3 1023 min21, Km = 520 mM, kcat/kuncat = 1.2 3
102). The observed activity was interesting in the light of the
haptenic structure for which conventional wisdom would
predict a preferred hydrolysis to yield 5 and 8. We also tested
the antibody-catalyzed hydrolysis of 2b,9 the ester most
congruent to 1 and 9, as well as the cleavage of the two primary
ester products 7 and 8. OB2-48F8 catalyzed the hydrolysis of 2b
(kcat = 1.0 3 1022 min21, Km = 289 mM, kcat/kuncat = 3.3 3
102) and was competitively inhibited by 9 (Ki = 16.5 mM). The
Fig. 2 Reaction profiles showing products from the antibody-catalyzed
hydrolysis of oligoester 10. Reactions were carried out in 50 mM PIPES,
50 mM NaCl, pH 6.7, 5% DMSO at 21 °C in the presence of 500 mM 10 and
20 mM OB2-48F8. The uncatalyzed reactions were carried out under the
same conditions in the absence of antibody and the rates subtracted.
We thank Dr Peter Wirsching for reviewing the manuscript.
Financial support was provided by The National Institute of
Health (GM 43858) and The Skaggs Institute of Chemical
Biology.
antibody also cleaved 7 (kcat = 1.7 3 1023 min21, Km
=
695 mM, kcat/kuncat = 1.1 3 102) and 8 (kcat = 2.0 3 1023
min21, Km = 940 mM, kcat/kuncat = 90). Thus, mAb OB2-48F8
was capable of completely degrading 6 to its component
monomeric building blocks. Unexpectedly, the trimer 6 was a
better substrate than ester 2b for reasons that remain unclear.
Additional studies employing the novel phosphorodithioate
moiety might reveal unusual features with regard to the
correlation between hapten and substrate structures.
Notes and references
1 A. C. Albertsson and S. Karlsson, Acta Polym., 1995, 46, 114.
2 M. P. Stevens, Polymer Chemistry, 2nd edition, Carl-Hanser Verlag,
Munich, 1990.
3 U. Witt, M. Yamamoto, U. Seeliger, R.-J. Muller and V. Warzelhan,
Angew. Chem., Int. Ed., 1999, 38, 1438; S. Kobayashi, H. Uyama and T.
Takamoto, Biomacromolecules, 2000, 1, 3.
Finally, to investigate the capabilities of OB2-48F8 for
degrading larger oligomers approaching ‘real’ polymers, we
synthesized polyesters 10–12 that contained additional repeat-
ing units (Fig. 1). The pentamer 10 (MW 729) was sparingly
soluble (less than 30 mM) in our aqueous buffer conditions,
however using a 500 mM heterogeneous mixture of 10
degradation still occurred into subunit structures (Fig. 2). As
detailed in Fig. 2, there was an initial increase in the dimers 7
and 8 followed by a decrease as monomers 3 and 5 increased.
This was rationalized based on the above cleavage preferences
and consecutive-reaction kinetics. Over a one month period
near neutral pH at ambient temperature in the presence of
antibody, approximately 200 mM of 3 was formed from
degraded 10. Without antibody, less than 3% occurred. In a
similar fashion, OB2-48F8 also broke down polyesters 11 (MW
863) and 12 (MW 997) that were virtually insoluble under our
assay conditions, yet cleavage occurred at multiple sites to yield
suboligomers as primary products (data not shown). Sig-
nificantly, the antibody was stable for more than a month under
heterogeneous conditions while the progress of these reactions
was being monitored.13 All the observations and data confirmed
that the antibody performed oligomer degradations by ‘multi-
mer’ processing using nonregioselective, kinetically biased
endo-cleavage, rather than a stepwise deoligomerization
through cleavage of monomers from a terminus.
4 E. C. King, A. J. Blacker and T. D. H. Bugg, Biomacromolecules, 2000,
1, 75; K. Tabata, H. Abe and Y. Doi, Biomacromolecules, 2000, 1,
157.
5 J. J Jesudason, R. H. Marchessault and T. Saito, J. Environ. Polym.
Degrad., 1993, 1, 89.
6 E. Rantze, I. Kleeberg, U. Witt, R.-J. Mueller and W.-D. Deckwer,
Macromol. Symp., 1998, 130, 319; U. Witt, R.-J. Mueller and W.-D.
Deckwer, Macromol. Chem. Phys., 1996, 197, 1525.
7 H. N. Eisen, General Immunology, 3rd ed., J. B. Lippincott Company,
Philadelphia, 1990.
8 K. D. Janda, C. G. Shelvin and C.-H. L. Lo, in Comprehensive
Supramolecular Chemistry, Vol. 4: Supramolecular Reactivity and
Transport: Bioorganic Systems, ed. J. L. Atwood, J. E. D. Davies, D. D.
MacNicol, F. Vögtle, J.-L. Lehn, Y. Murakami, Elsevier, New York,
1996, p. 43; P. Wentworth and K. D. Janda, Curr. Opin. Chem. Biol.,
1998, 2, 138.
9 O. Brümmer, P. Wentworth, D. P. Weiner and K. D. Janda, Tetrahedron
Lett., 1999, 40, 7307.
10 The synthesis of the compounds under discussion (4, 6–8 and 10–12)
can be found in the supplementary information.
11 Phosphorodithioate 9 is a tight-binding inhibitor of OB2-48F8. Ki
determinations were therefore not made using the classical Lineweaver–
Burk analysis over a range of inhibitor concentrations, since this is
known to be subject to error for tight-binding inhibitors. Rather the
method of Copeland et al. (R. A. Copeland, D. Lombardo, J. Glannaras
and C. P. Decicco, Bioorg. Med. Chem. Lett., 1995, 5, 1947) was
employed, which allows for the accurate determination of the Ki values
of tight-binding inhibitors.
In conclusion, a catalytic antibody has been discovered that
degrades oligomeric esters. Our findings are of fundamental
importance as now catalytic antibodies share another trait
thought only to be associated with enzymes, the biodegradation
of oligo and polymeric materials.1
12 Symmetrical phosphorodithioate 9 was prepared starting from 4-acet-
amidophenol.9
13 Aliquots of sample were removed periodically and checked by ELISA
for hapten binding.
20
Chem. Commun., 2001, 19–20