A polymeric and fluorescent gel for combinatorial screening of catalysts [23]

Full text of DOI:10.1021/ja0055763

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11270
J. Am. Chem. Soc. 2000, 122, 11270-11271
Scheme 1
A Polymeric and Fluorescent Gel for Combinatorial
Screening of Catalysts
Robert F. Harris, Andrew J. Nation, Gregory T. Copeland, and
Scott J. Miller*
Department of Chemistry, Merkert Chemistry Center
Boston College, Chestnut Hill, Massachusetts 02467-3860
ReceiVed September 5, 2000
Methods of increasing the pace of materials and catalyst
discovery have become a topic of intense interest in the field of
chemical synthesis.1 In particular, techniques that allow for the
parallel preparation and simultaneous screening of numerous
catalysts have gained particular attention as they promise to
accelerate what may be the rate-determining step in the catalyst
development process, the discovery of the catalyst itself.2 One
way to determine a catalyst’s activity is to monitor reaction
progress through the detection of a product with an appropriate
chemical sensor.3 In the present study, we report the design,
synthesis, and initial implementation of a sensor-functionalized
polymeric gel that allows for the pooled screening of certain types
of catalyst libraries.
Scheme 2
The method we describe involves deposition of catalysts that
have been immobilized on conventional resin-beads within a
polymeric matrix. The matrix is designed such that it possesses
sufficient permeability for diffusion of reagents to the individually
localized beads (Scheme 1).4 Simultaneously, the polymer
incorporates (by covalent attachment) a fluorescence-based sensor
that signals the presence of an appropriate reaction product by
an increase in fluorescence intensity.5 The method relies on the
slow diffusion of product out of the bead as it is formed into the
matrix, which affords a fluorescent zone around the site of an
active catalyst. Regions of the polymer surrounding beads that
are inactive remain dark as none of the product is present.
For an initial platform upon which to screen catalyst libraries,
we chose to synthesize a gel-based matrix in which we could
screen catalysts that mediate reactions that afford carboxylic acids
as products. Specifically, we chose to study catalysts for acylation
reactions of alcohols with acetic anhydride (eq 1). Work from
our laboratory has established that aminomethylanthracene 1 is a
suitable sensor for detection of the acidic products of this reaction6
and is an excellent probe for in situ monitoring of catalytic
activities.7 Earlier efforts focused on simultaneous attachment of
the fluorophore and catalyst to a unique bead; the present study
demonstrates that polymer-bound catalysts and polymer-bound
sensor may also be spatially segregated.
The matrix we set out to synthesize is based on the precedent
of Meldal who synthesized poly(ethylene glycol) dimethylacryl-
amide (PEGA) gels as peptide synthesis supports.8 These materials
have the advantage of swelling in both organic and aqueous
solvents. We speculated that the rate of diffusion of reagents
through these gels would be adequate to allow efficient mass
transport, but sufficiently slow such that real-time observation
of analyte migration would be possible. Therefore, we proposed
that sensor-functionalized gels, with a derivative of fluorophore
1 incorporated, would provide an ideal medium in which catalyst
activities might be monitored.9 The synthesis of such an ami-
nomethylanthracene-functionalized gel is outlined in Scheme 2.
A 3:1 molar ratio of N,N-dimethylacrylamide and acrylic acid is
copolymerized with AIBN as a radical initiator to afford pre-
polymer 2. Cross-linking is accomplished in a standard gel caster
using a solution of poly(ethylene glycol)-bridged diamine 3 that
contains a 0.1 M concentration of carboxylic acid-functionalized
fluorophore 4.10 This procedure affords a gel-like polymer with
the microstructure depicted by structure 5.
As in the case of the polymers reported by Meldal, this material
swells in a wide variety of solvents (e.g., DMF, i-PrOH, H2O).
The material is also reversibly fluorescent. Treatment of gel 5
with acetic acid (HOAc) induces the gel to fluoresce (excitation
λ, 390 nm; emission λ, 420 nm); washing with base followed by
rinsing with DMF results in a gel that is nonfluorescent. Of
(1) Jandeleit, B.; Schaefer, D. J.; Powers, T. S.; Turner, H. W.; Weinberg,
W. H. Angew. Chem., Int. Ed. 1999, 38, 2494.
(5) (a) Reddington, E.; Sapienza, A.; Gurau, B.; Viswanathan, R.; Saran-
gapani, S.; Smotkin, E. S.; Mallouk, T. E. Science 1998, 280, 1735. (b)
Shaughnessy, K. H.; Kim, P.; Hartwig, J. F. J. Am. Chem. Soc. 1999, 121,
2123. (c) Yeung, E. S.; Su, H. J. Am. Chem. Soc. 2000, 122, 7422.
(6) For pioneering studies on the use of aminomethylanthracenes as pH
and metal-ion sensors, see ref 3a.
(2) For several recent reviews of combinatorial catalysis, see: (a) Crabtree,
R. H. Chem. Commun. 1999, 17, 1611. (b) Kuntz, K. W.; Snapper, M. L.;
Hoveyda, A. H. Curr. Opin. Chem. Biol. 1999, 3, 313. (c) Francis, M. B.;
Jamison, T. F.; Jacobsen, E. N. Curr. Opin. Chem. Biol. 1998, 2, 422.
(3) For comprehensive reviews of the field, see: (a) Fluorescent Chemosen-
sors for Ion And Molecule Recognition; Czarnik, A. W., Ed.; American
Chemical Society: Washington, DC, 1993. (b) de Silva, A. P.; Gunaratne, H.
Q. N.; Gunnlaugsson, T.; Huxley, A. J. M.; McCoy, C. P.; Rademacher, J.
T.; Rice, T. E. Chem. ReV. 1997, 97, 1515.
(7) Copeland, G. T.; Miller, S. J. J. Am. Chem. Soc. 1999, 121, 4306.
(8) Meldal, M. Tetrahedron Lett. 1992, 33, 3077.
(9) Fluorophore-tagged poly(acrylates) have been synthesized previously
for the purpose of studying photophysical and photochemical events in rigid
networks. For example, see: Clements, J. H.; Webber, S. E. J. Phys. Chem.
A 1999, 103, 2513.
(4) For a radiographic binding assay with functionalized beads dispersed
in a photographic emulsion, see: Nestler, H. P.; Wennemers, H.; Sherlock,
R.; Dong, D. L.-Y. Bioorg. Med. Chem. Lett. 1996, 6, 1327.
(10) For details on the synthesis of 5, see the Supporting Information.
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Figure 1. Gel reponse to HOAc. (a) Injection of 10 µL down the side
of a well (diameter ) 1 cm). (b) Spotting of six samples, v/v (HOAc/i-
PrOH): (i) 30:70; (ii) 40:60; (iii) 50:50; (iv) 60:40; (v) 70:30; (vi) 80:
20.
Figure 2. Fluorescence micrographs of catalyst-functionalized beads
immobilized in gel 5. (a) Micrograph of active bead (6) after introduction
of reagents (t ) 5 min). (b) Micrograph of catalytically inactive bead (7)
after introduction of reagents (t ) 5 min).
Figure 3. Screen of peptide library deposited in sensor-functionalized
gel. (a) Library composition. Each member is terminated in either Pmh
or Phe. (b) Excerpt of fluorescence micrograph of gel containing library
upon exposure to reaction conditions. (c) Sequences of other “hit” and
“miss” beads obtained by single-bead Edman degradation.
particular note is that the rate of diffusion of HOAc within the
polymer is slow, a fact that suggests that localization of a
fluorescence response in a spatially arrayed region of the polymer
should be possible. In Figure 1a, we show a fluorescence
micrograph of a portion of the polymer cast in a microwell
(diameter ) 3 mm). At t ) 0 min, no fluorescence is evident in
the gel. After one drop (∼10 µL) of HOAc is injected down the
side of the well, an increase in fluorescence intensity is apparent,
but the rate of diffusion is greatly decreased relative to the rate
in free solution. The fluorescence intensity is also proportional
to the concentration of the HOAc that is present. Figure 1b shows
a fluorescence micrograph of six spots placed upon the gel,
increasingly more concentrated in HOAc.
To demonstrate that sensor-functionalized polymer 5 is a
suitable medium in which to screen catalysts, we prepared samples
of 5 in which gel cross-linking was accomplished around samples
of resin beads that are functionalized with potential catalyst
candidates (Figure 2). As controls, we prepared two sets of beads.
In one case, we made beads that contained the dipeptide sequence
p(NO2)Phe-π(Me)His-BOC (6). This sequence contains the
N-alkylimidazole heterocycle as part of the π(methyl)histidine
(Pmh) residue; this moiety is known to be highly active for acyl-
transfer reactions involving alcohols and anhydrides.11,12 A second
set of beads was made that contained the sequence p(NO2)Phe-
Phe-BOC (7), which we have shown is catalytically inactive under
these acyl transfer conditions. Each set contained p(NO2)Phe to
ensure that all beads are uniformly dark throughout the experiment
as a result of efficient fluorescence quenching.13 When a sample
of the gel containing the active beads was exposed to the reaction
conditions for 5 min, an increase in fluorescence intensity was
noted in zones of the gel where beads were localized (Figure 2a).
In contrast, when the same experiment was performed with a gel
sample containing the inactive beads (Figure 2b), the gel remained
dark.
We then screened an actual library of catalyst candidates that
had been deposited in sensor-functionalized gel 5 (Figure 3). We
synthesized a 50-member tetrapeptide library using the split-and-
pool method.14 Each member of the library was initiated with
the p(NO2)Phe residue (Figure 3a). The second and third residues
were composed of five different amino acids introduced in two
“split” steps.15 The final residue of the library was either the
catalytically competent residue Pmh or the catalytically inactive
residue Phe. Sensor-functionalized gel 5 was then cross-linked
around the beads containing the library, and the gel was exposed
to the reaction conditions. Figure 3b shows a fluorescence
micrograph of a portion of the gel. Four beads surrounded by
halos of fluorescence were extracted from the gel, washed, and
subjected to single-bead Edman degradation16 to determine the
sequences of the peptides immobilized on the beads. In addition,
four beads that produced no increase in fluorescence intensity
were extracted and subjected to the same analysis. In each of the
four “hits” selected, the peptides were found to contain the
catalytically active Pmh residue. In each of the four “miss” cases,
the peptides were found to be terminated with Phe (Figure 3c).
These results validate the concept of using sensor-functionalized
gels as a screening tool for reactions that produce appropriate
analytes. Future studies from our laboratory will address the
incorporation of additional sensors for other products, and on
applications.
Acknowledgment. This research is supported by the NSF (CHE-
9874963). We also thank the NIH, DuPont, Eli Lilly, Glaxo-Wellcome,
and Merck for research support. S.J.M. is a Fellow of the Alfred P. Sloan
Foundation, a Cottrell Scholar of Research Corporation, and a Camille
Dreyfus Teacher-Scholar.
Supporting Information Available: Experimental details for the
preparation of the gel, catalyst library and for screening (PDF). This
material is available free of charge via the Internet at http://pubs.acs.org.
JA0055763
(11) Ho¨fle, G.; Steglich, W.; Vorbru¨ggen, H. Angew. Chem., Int. Ed. Engl.
1978, 17, 569.
(14) (a) Furka, A.; Sebestyen, F.; Asgedom, M.; Dibo, G.; Int. J. Pept.
Protein Res. 1991, 37, 487. (b) Lam, K. S.; Salmon, S. E.; Hersh, E. M.;
Hruby, V. J.; Kazmierski, W. M.; Knapp, R. J. Nature 1991, 354, 82.
(15) Residues two and three: Ala, Gly, Leu, Phe, and Val.
(16) Single-bead Edman degradation analyses were performed at Midwest
Analytical, Inc., St Louis, MO 63123.
(12) For peptide-based analogues, see: Jarvo, E. R.; Copeland, G. T.;
Papaioannou, N.; Bonitatebus, P. J., Jr.; Miller, S. J. J. Am. Chem. Soc. 1999,
121, 11638 and references therein.
(13) Garcia-Echeverria, C.; Kofron, J. L.; Kuzmic, P.; Kishore, V.; Rich,
D. H. J. Am. Chem. Soc. 1992, 114, 2758.