Microwave-Accelerated SPOT-synthesis on cellulose supports

Article abstract of DOI:10.1021/ol049313f

Equation presented. We demonstrate that microwave irradiation can dramatically accelerate reaction rates for spatially addressable library synthesis on planar membrane supports. The development of a robust support/linker system, microwave-assisted synthesis of small molecule test libraries, and methods for solid-phase scale-up on cellulose are described.

Full text of DOI:10.1021/ol049313f

ORGANIC
LETTERS
2004
Vol. 6, No. 12
2019-2022
Microwave-Accelerated SPOT-Synthesis
on Cellulose Supports
Matthew D. Bowman, Ryan C. Jeske, and Helen E. Blackwell*
Department of Chemistry, UniVersity of WisconsinsMadison, 1101 UniVersity AVenue,
Madison, Wisconsin 53706-1322
blackwell@chem.wisc.edu
Received April 14, 2004
ABSTRACT
We demonstrate that microwave irradiation can dramatically accelerate reaction rates for spatially addressable library synthesis on planar
membrane supports. The development of a robust support/linker system, microwave-assisted synthesis of small molecule test libraries, and
methods for solid-phase scale-up on cellulose are described.
Combinatorial chemistry has become an important tool in
many areas of research, including chemical biology and drug
discovery.1 As the demand for libraries of small organic
molecules continues to grow, particularly due to the bur-
geoning fields of genomics and proteomics, the need to
develop new synthesis platforms for the rapid generation and
evaluation of “natural product-based” and “drug-like” com-
pounds becomes more urgent.2 Parallel and split-pool
synthesis on polymeric solid supports are currently the most
common strategies for the generation of combinatorial
libraries.3 While both of these approaches have enabled the
synthesis of large libraries, performing reactions on conven-
tional solid-phase resins can have significant drawbackss
the hydrophobic beads used are often expensive, difficult to
manipulate, fragile, and not amenable to many types of
biological assays. Furthermore, solid-phase reaction rates are
frequently 10- to 100-fold slower than their solution-phase
counterparts.4 This problem frequently outweighs the puri-
fication benefits permitted by solid supports and requires
significant time investment in reaction optimization. The
development of an inexpensive and robust library synthesis
platform that combines the purification benefits of solid-
phase resins with enhanced reaction rates would impact
significantly the role of combinatorial techniques in research
today. Here, we demonstrate that the strategic combination
of microwave-assisted organic reactions with SPOT-synthesis
on planar supports presents a platform that can address many
of these current limitations.5
SPOT-synthesis is a conceptually simple and flexible
approach for parallel library synthesis on planar cellulose
supports (Figure 1).6 This method involves spatially ad-
dressed, solid-phase synthesis on derivatized cellulose sheets
(readily prepared from inexpensive filter paper) to generate
arrays of unique molecules (1-10,000 SPOTs).7,8 In contrast
* To whom correspondence should be addressed.
(4) Li, W. B.; Yan, B. J. Org. Chem. 1998, 63, 4092-4097.
(5) For recent commentaries on microwave-assisted combinatorial chem-
istry, see: (a) Blackwell, H. E. Org. Biomol. Chem. 2003, 1, 1251-1255.
(b) Kappe, C. O. Curr. Opin. Chem. Biol. 2002, 6, 314-320.
(6) Frank, R. Tetrahedron 1992, 48, 9217-9232.
(1) (a) Ley, S. V.; Baxendale, I. R. Nat. ReV. Drug. DiscoV. 2002, 1,
573-586. (b) Lam, K. S.; Renil, M. Curr. Opin. Chem. Biol. 2002, 6, 353-
358. (c) Erhardt, P. W. Pure Appl. Chem. 2002, 74, 703-785.
(2) (a) Schreiber, S. L. Science 2000, 287, 1964-1969. (c) Seneci, P.;
Miertus, S. Mol. DiVers. 2000, 5, 75-89.
(3) (a) Boyle, N. A.; Janda, K. D. Curr. Opin. Chem. Biol. 2002, 6, 339-
346. (b) Tan, D. S.; Burbaum, J. J. Curr. Opin. Drug Discuss. DeVel. 2000,
3, 439-453.
(7) For a recent SPOT-synthesis review, see: Frank, R. J. Immunol.
Methods 2002, 267, 13-26.
(8) We and others use Whatman 1Chr chromatography paper for SPOT-
synthesis (thickness ) 0.34 mm and cost ca. 0.5¢/cm2).
10.1021/ol049313f CCC: $27.50 © 2004 American Chemical Society
Published on Web 05/18/2004

We initiated our investigations through the development
of a robust, linker-functionalized planar cellulose support
(Scheme 1). Previous cellulose derivatization methods for
Scheme 1. Support Modification and Linker Installation
Figure 1. Schematic of the SPOT-synthesis process.
to conventional polystyrene resins, the hydrophilic, mechani-
cally robust membrane sheets are easy to manipulate during
synthesis and washing steps. Furthermore, cellulose mem-
branes are compatible with various on-support biological
screening methods, including protein-binding assays, enzyme-
linked immunosorbent assays (ELISA), and agar overlay.7
Despite the advantages outlined above, the application of
the SPOT-synthesis technique has been largely restricted to
the construction of simple peptide libraries.7 Examples of
nonpeptide libraries generated via SPOT-synthesis are scarce,
and most have been based on simple acylation chemistry.9
More challenging chemistry has not been pursued, in part,
because (1) SPOT-synthesis suffers from slow reaction rates
similar to conventional solid-phase synthesis and (2) general,
reproducible methods for heating spatially addressed reac-
tions are not available. We hypothesized that microwave
(MW) assistance could broaden the reaction scope of SPOT-
synthesis and facilitate the generation of complex small
molecule libraries. The use of MW irradiation as a non-
conventional heating method for organic synthesis has
increased over the past decade, primarily due to marked
reductions in reaction times and enhancements in conversion
and purity.10 To date, however, the generality and utility of
MW-assisted reactions on planar polymeric supports have
not been explored.5a,11
SPOT-synthesis have relied on reaction of cellulose with
epibromohydrin.7,11 We chose to avoid this toxic, expensive
reagent and found direct tosylation of support 18 to be an
effective method to activate cellulose prior to introduction
of diamine 2 as a “spacer” element. By controlling the
concentration of TsCl and reaction time, the loading level
of the support could be varied from 50 nmol/cm2 to 10 µmol/
cm2.17 Diamine 2 was installed as a “spacer” since this unit
has been shown to improve the accessibility of support-bound
molecules for subsequent reactions and on-support assays.11
MW irradiation of tosyl cellulose in neat diamine 2 (300
W) significantly expedited spacer incorporation; support 3
was generated in 15 min as opposed to the 6 h required for
similar incorporation using traditional methods (100 °C, in
a drying oven). We found MW heating of membranes in
shallow glass vessels to be straightforward and reproducible
using a commercial multimodal MW reactor.18 In contrast,
a household MW oven (Kenmore model 721; 300 W) gave
irreproducible results for heating planar support reactions and
underscores the importance of using dedicated, commercial
MW reactors for synthesis.5,10
We chose to examine the scope and limitations of MW-
assisted SPOT-synthesis through the preparation of libraries
of chalcone-derived molecules, as chalcone synthesis via
Claisen-Schmidt condensation has been performed with
success in solution under MW conditions previously.10,12
Chalcones can be derivatized easily to generate diverse
second-generation libraries.13,14 Further, chalcones present
a proven molecular scaffold for biological evaluation, as
certain derivatives exhibit potent and specific activities, e.g.,
activation of Sirtuin proteins15 and broad anticancer activity.16
We next introduced a versatile acid-cleavable, Wang-type
linker system onto amino-cellulose support 3 (Scheme 1).19
(13) Katritzky, A. R.; Serdyuk, L.; Chassaing, C.; Toader, D.; Wang,
X.; Forood, B.; Flatt, B.; Sun, C.; Vo, K. J. Comb. Chem. 2000, 2, 182-
185 and references therein.
(14) Powers, D. G.; Casebier, D. S.; Fokas, D.; Ryan, W. J.; Troth, J.
R.; Coffen, D. L. Tetrahedron 1998, 54, 4085-4096.
(15) Howitz, K. T.; Bitterman, K. J.; Cohen, H. Y.; Lamming, D. W.;
Lavu, S.; Wood, J. G.; Zipkin, R. E.; Chung, P.; Kisielewski, A.; Zhang,
L. L.; Scherer, B.; Sinclair, D. A. Nature 2003, 425, 191-196.
(16) Lawrence, N. J.; Rennison, D.; McGown, A. T.; Ducki, S.; Gul, L.
A.; Hadfield, J. A.; Khan, N. J. Comb. Chem. 2001, 3, 421-426.
(17) Functionalization levels were determined by quantitation of Fmoc-
derivatized amino-support 3 using UV spectroscopy (at 296 nm).
(18) All MW-assisted reactions on cellulose supports were performed
in a Milestone Ethos Microsynth multimodal reactor using power (wattage)
control. Temperature control was not possible due to the low solvent
volumes used. Supports were washed with various solvents and dried
routinely between each synthesis step; see the Supporting Information.
(9) For selected examples, see: (a) Scharn, D.; Germeroth, L.; Schneider-
Mergener, J.; Wenschuh, H. J. Org. Chem. 2001, 66, 507-513. (b) Jobron,
L.; Hummel, G. Angew. Chem., Int. Ed. 2000, 39, 1621-1624. (c) Ast, T.;
Heine, N.; Germeroth, L.; Schneider-Mergener, J.; Wenschuh, H. Tetra-
hedron Lett. 1999, 40, 4317-4318.
(10) For a recent MW review, see: Lidstrom, P.; Tierney, J.; Wathey,
B.; Westman, J. Tetrahedron 2001, 57, 9225-9283.
(11) Scharn, D.; Wenschuh, H.; Reineke, U.; Schneider-Mergener, J.;
Germeroth, L. J. Comb. Chem. 2000, 2, 361-369.
(12) Babu, G.; Perumal, P. T. Synth. Commun. 1997, 27, 3677-3682.
2020
Org. Lett., Vol. 6, No. 12, 2004

First, 4-formylphenoxyacetic acid (4) was coupled to support
3 via a standard carbodiimide coupling. Aldehyde support 5
was then subjected to NaBH4 to yield benzyl alcohol-derived
support 6. Notably, this four-step reaction sequence did not
impact the integrity of the cellulose membrane; we routinely
generate support 6 on large scale (ca. 300 cm2 sheets) and
find this support/linker system to be highly robust for SPOT-
synthesis.
Table 1. Chalcone Test Library Synthesized on Support 6
We evaluated the feasibility of MW-assisted synthesis on
support 6 through the generation of select chalcones. First,
we attached various hydroxyacetophenones 7 to support 6
via MW-assisted nucleophilic substitution (Scheme 2).
entrya
R1 (7)
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
3-OH
3-OH
3-OH
3-OH
3-OH
3-OH
3-OH
R2 (9)
purityb (%)
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
10l
10m
10n
10o
10p
10q
10r
3-OH
3-Br
4-Cl
92
89
99
93
70
69
79
75
90
>99
92
86
84
81
91
90
63
67
90
74
43
62
68
66
95
90
92
96
87
81
84
87
66
65
51
59
40
52
3-OH, 4-NO2
4-OH
4-OH, 3-Br
4-OH, 3-OMe
5-I, 4-OH, 3-OMe
3-OH
Scheme 2. Initial Building Block Loading
3-Br
4-Cl
3-OH, 4-NO2
4-OH
4-OH, 3-Br
4-OH, 3-OMe
5-I, 4-OH, 3-OMe
3-OH
Support 6 was activated immediately prior to use by
treatment with TsCl in DMF. Solutions of acetophenones 7
and base (KO-t-Bu, in DMSO) were then spotted onto
activated support 6 in a spatially addressable manner (SPOT
size ) 0.28 cm2) and subjected to MW irradiation at 500 W
for 10 min.18 Reproducible acetophenone (7) loadings ranging
from 100 to 200 nmol/cm2 were obtained via this protocol
(as determined after physically punching-out the SPOT and
TFA-mediated cleavage).20 This short reaction time contrasts
with the 5-10 h required for analogous solid-phase nucleo-
philic displacements using traditional heating methods (50
°C).13 Detailed optimization work revealed that high-boiling
polar solvents, such as DMSO, and concentrated building
block solutions (ca. 2.0 M 7) were required for high loadings
(data not shown). These conditions allow for sustained MW
heating5,10 and were used for all subsequent MW-assisted
SPOT-syntheses.
We next investigated the feasibility of MW-assisted
Claisen-Schmidt condensations in a SPOT format (Table
1). In preliminary experiments, spotting of support 8 with
various benzaldehydes and base in polar solvents (e.g., KOH
in H2O) followed by MW irradiation at 400 W for 20 min
generated chalcone species 10 (after TFA cleavage) with
good to excellent conversions (60-98%).18,21 This represents
a marked reduction in reaction time and a concomitant
enhancement in yield; these condensation reactions typically
require ca. 2-48 h for lower conversions using conventional
solid-support synthesis methods.13,22
3-OH
4-OH, 2-Me
4-OH, 2-Me
4-OH, 2-Me
4-OH, 2-Me
4-OH, 2-Me
4-OH, 2-Me
4-OH, 2-Me
4-OH, 2-Me
4-OH, 3-OMe
4-OH, 3-OMe
4-OH, 3-OMe
4-OH, 3-OMe
4-OH, 3-OMe
4-OH, 3-OMe
4-OH, 3-OMe
4-OH, 3-OMe
4-OH, 3-NO2
4-OH, 3-NO2
4-OH, 3-NO2
4-OH, 3-NO2
4-OH, 2-OH
4-OH, 2-OH
3-Br
4-Cl
10s
10t
3-OH, 4-NO2
4-OH
4-OH, 3-Br
4-OH, 3-OMe
5-I, 4-OH, 3-OMe
3-OH
10u
10v
10w
10x
10y
10z
10a ′
10b′
10c′
10d ′
10e′
10f ′
10g′
10h ′
10i′
10j′
10k ′
10l′
3-Br
4-Cl
3-OH, 4-NO2
4-OH
4-OH, 3-Br
4-OH, 3-OMe
5-I, 4-OH, 3-OMe
3-OH
3-Br
4-Cl
3-OH, 4-NO2
3-OH
3-Br
a MW reaction conditions, solvent volumes, and base concentration vary
for certain substrates. See text and Supporting Information. b Determined
by integration of the HPLC trace at 254 nm.
nones 7 and eight benzaldehydes 9 were selected as library
building blocks, based on their solubility in polar solvents
and the presence of functional group handles, i.e., hydroxyl
and/or halogen, for the incorporation of additional library
diversity elements.2b The building blocks were combined to
yield a parallel array of 40 unique chalcones (10a-10l′) with
good to excellent conversions in almost all cases (as deter-
mined after TFA cleavage).20,21 Different basic reaction con-
ditions were required depending on the benzaldehyde species
(9). More reactive benzaldehydes, such as 3-hydroxy-
benzaldehyde, underwent efficient condensation at 1.0 M in
With these two reactions optimized, we synthesized a test
library of chalcones on support 6 (Table 1). Six acetophe-
(19) Wang, S. S. J. Am. Chem. Soc. 1973, 95, 1328-1333.
(20) Compounds were cleaved quantitatively from the Wang linker by
treatment with TFA vapor (in a vacuum desiccator, 25 °C, 60 min). See
refs 11 and 19.
(21) Both cis and trans chalcone isomers were observed in certain cases;
see the Supporting Information.
(22) Control SPOT reactions (30 min, 25 °C) generated chalcones in
low yields (ca. 10%); see the Supporting Information.
Org. Lett., Vol. 6, No. 12, 2004
2021

a 6 N aq KOH solution, while less reactive substrates, such
as vanillin, required a more concentrated solution of both
aldehyde (2.0 M) and base (12 N aq KOH) to achieve good
conversion. Preparation of the library in a spatially addressed
format permitted the application of different reaction condi-
tions in parallel and expedited library synthesis. Indeed, this
library was synthesized in only 60 min (for three reaction
steps starting from support 6). These results underscore the
utility of MW-assisted reactions in library synthesis.5
To further investigate the scope of MW-assisted synthesis
on support 6, we examined several heterocycle-generating
reactions on support-bound chalcones.13 We discovered that
the condensation of chalcones 11 with either acetamidine or
benzamidine hydrochloride (12) yielded dihydropyrimidines
13 with good to excellent conversions under MW-assisted
conditions (Table 2).23 We then prepared a small library of
Figure 2. Overlaid LC traces for crude 4-hydroxyacetophenone,
chalcone 10b, and dihydropyrimidine 13a cleaved from individual
SPOTs of support 6. UV detection at 254 nm. The smaller peak
(*) is the cis isomer of 10b.21
(nanomolar to micromolar scale) that are sufficient for
characterization and biological evaluation. The ability to
translate SPOT-synthesis protocols directly from membrane
supports to another form of cellulose that is amenable to
larger scale synthesis would expand the utility of this
methodology further. We examined MW-assisted synthesis
on cellulose powder, fibers, and Perloza beads24,25 as potential
scale-up synthesis methods. We found inexpensive Perloza
beads (100 µm diameter) to be mechanically robust and
readily functionalized with our Wang-type linker/spacer
system using chemistry analogous to that outlined in Scheme
1 for support 6 (ca. 10-80 µmol/g loadings). In initial
studies, this alternate support system was compatible with
MW-assisted chalcone synthesis,18 yielding chalcone 10e,
for example, in good purity (74%) at a loading of 4.3 µmol/
g. We note that large-scale synthesis on Perloza (10-100
g) will require different MW reaction conditions; current
work involves evaluation of continuous flow MW methods.5
In summary, we have demonstrated that MW-assisted
SPOT-synthesis is a viable approach for the generation of
parallel combinatorial libraries. We have developed a robust
support/linker system for SPOT-synthesis (6), established its
compatibility with a range of MW-assisted organic reactions,
and extended this methodology to another cellulose support
type for larger scale reactions. This combination of tech-
niques presents an accessible and versatile platform for
combinatorial discovery and should allow parallel libraries
of moderate size to be prepared and screened on the order
of days. Ongoing work is directed at the synthesis and
biological evaluation of larger, second-generation chalcone
libraries.
Table 2. Dihydropyrimidines Synthesized on Support 6
entry
R1
R2
R3
puritya (%)
13a
13b
13c
13d
13e
13f
13g
13h
13i
13j
13k
13l
4-OH
4-OH
4-OH
4-OH
3-OH
3-OH
3-OH
3-OH
3-OMe, 4-OH
3-OMe, 4-OH
3-OMe, 4-OH
3-OMe, 4-OH
3-Br
3-Br
4-Cl
4-Cl
3-Br
3-Br
4-Cl
4-Cl
3-Br
3-Br
4-Cl
4-Cl
Me
Ph
Me
Ph
Me
Ph
Me
Ph
Me
Ph
Me
Ph
88
80
83
69
86
86
73
84
79
76
82
83
a Determined by integration of the HPLC trace at 254 nm.
12 dihydropyrimidines (13a-l) using MW-assisted SPOT-
synthesis. Interestingly, we observed the formation of only
trace amounts of the related pyrimidine derivatives under
these reaction conditions (30 min, MW 400 W, air atmo-
sphere).18 This is in contrast to previous pyrimidine syntheses
on solid-phase (ca. 20 h, 100 °C in oil bath)13 where no
dihydropyrimidine intermediates were isolated. These data
demonstrate how MW-assisted SPOT methods may comple-
ment current solid-phase techniques.
Acknowledgment. This work was supported by generous
start-up funds from UWsMadison and CEM Corp. We thank
Prof. Laura Kiessling for helpful discussions and CEM Corp.
and Milestone, Inc. for technical assistance with MW
synthesis instrumentation.
Representative LC traces for a three-step sequence of
single SPOT reactions (ca. 100 nmol/SPOT) are shown in
Figure 2 and reveal the efficient conversions achievable using
MW-assisted SPOT-synthesis on support 6.
One advantage of the SPOT-synthesis technique is that it
provides rapid access to compounds in small quantities
Supporting Information Available: Full experimental
details for support derivatization and MW-assisted synthesis.
This material is available free of charge via the Internet at
http://pubs.acs.org.
OL049313F
(23) The absolute tautomeric form of 13 remains unknown. Dihydro-
pyrimidines can exist as 1,4 or 1,6 annular tautomers, see: Weis, A. L.
Tetrahedron Lett. 1982, 23, 449-452.
(24) Perloza MT-100 is commercially available from Iontosorb Co.
(25) De Luca, L.; Giacomelli, G.; Porcheddu, A.; Salaris, M.; Taddei,
M. J. Comb. Chem. 2003, 5, 465-471.
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Org. Lett., Vol. 6, No. 12, 2004