Siloxyl ether functionalized resins for chemoselective enrichment of carboxylic acids

Article abstract of DOI:10.1021/ol202376m

Although the carboxylic acid moiety is prevalent in many biologically produced molecules, including natural products and proteins, methods to chemoselectively target this functional group have remained elusive. Generally, strategies that utilize carboxylate nucleophilicity also promote reactions with other nucleophiles such as amines and hydroxyls. A reagent was sought to facilitate the selective isolation of carboxylic acid containing compounds from complex mixtures. Here, the development of siloxyl ether functionalized solid supports is described.

Full text of DOI:10.1021/ol202376m

ORGANIC
LETTERS
2
011
Vol. 13, No. 20
652–5655
Siloxyl Ether Functionalized Resins for
Chemoselective Enrichment of Carboxylic
Acids
5
†
Darci J. Trader and Erin E. Carlson*
,†,‡
Departments of Chemistry and Molecular and Cellular Biochemistry, Indiana
University, 212 South Hawthorne Drive, Bloomington, Indiana 47405, United States
carlsone@indiana.edu
Received September 1, 2011
ABSTRACT
Although the carboxylic acid moiety is prevalent in many biologically produced molecules, including natural products and proteins, methods to
chemoselectively target this functional group have remained elusive. Generally, strategies that utilize carboxylate nucleophilicity also promote
reactions with other nucleophiles such as amines and hydroxyls. A reagent was sought to facilitate the selective isolation of carboxylic acid
containing compounds from complex mixtures. Here, the development of siloxyl ether functionalized solid supports is described.
The search for molecules possessing the features
required to modulate biological processes is a long-
standing scientific goal. Natural products have been a
continual source of inspiration, yielding many ther-
apeutic agents and targets for (bio)synthetic studies.
Natural products and their derivatives account for
nearly half of the drugs currently on the market and
2
have been used extensively as chemical probes. Despite
3
recent advances, the isolation of novel natural pro-
Current purification methods, such as high perfor-
mance liquid chromatography (HPLC) or size exclusion
chromatography, separate molecules by their physico-
chemical properties including polarity, charge, and size.
New technologies are needed that isolate molecules
based upon alternative characteristics than those pre-
sently used for natural product discovery, which are
biased toward abundant molecules. We have reported
the development of chemoselective isolation strategies
to facilitate enrichment of a subclass of biological mo-
1
ducts remains challenging as the use of traditional
isolation methods often results in rediscovery of known
5
lecules including metabolites and alcohol-containing
6
natural products. For natural products discovery, we
4
compounds and/or the loss of bioactivity.
employ a controllably reversible reaction to immobilize
the targeted functional group class onto a solid support.
Following an extensive wash protocol, the enriched
subpool is tracelessly released to enable immediate
characterization of the natural products of interest.
†
Department of Chemistry
Department of Molecular and Cellular Biochemistry
‡
(
1) Newman, D. J.; Cragg, G. M. J. Nat. Prod. 2007, 70, 461.
(2) (a) Carlson, E. E. ACS Chem. Biol 2010, 5, 639. (b) Bottcher, T.;
Pitscheider, M.; Sieber, S. A. Angew. Chem., Int. Ed. 2010, 49, 2680.
3) (a) Mansson, M.; Phipps, R. K.; Gram, L.; Munro, M. H.;
(
Larsen, T. O.; Nielsen, K. F. J. Nat. Prod. 2010, 73, 1126. (b) Araya,
J. J.; Montenegro, G.; Mitscher, L. A.; Timmermann, B. N. J. Nat. Prod.
(5) (a) Carlson, E. E.; Cravatt, B. F. Nat. Methods 2007, 4, 429. (b)
Carlson, E. E.; Cravatt, B. F. J. Am. Chem. Soc. 2007, 129, 15780.
(6) Odendaal, A. Y.; Trader, D. J.; Carlson, E. E. Chem. Sci. 2011, 2,
760.
2
010, 73, 1568.
4) Watve, M. G.; Tickoo, R.; Jog, M. M.; Bhole, B. D. Arch.
Microbiol. 2001, 176, 386.
(
1
0.1021/ol202376m r 2011 American Chemical Society
Published on Web 09/29/2011

Generation of a chemoselective isolation reagent toolkit
is significant because it will provide a means for discovery
of natural products that are unlikely to be identified by
traditional strategies due to low isolation yields, poor
compound resolution, and/or the presence of interfering
compounds. Furthermore, a functional group targeted
method can offer insight into the structural content of
compounds prior to characterization efforts.
compounds. We anticipated that the steric environment
about the silicon atom would be critical to ester stability.
Resin 3 displays relatively hindered isopropyl groups.
Thus, we wished to examine resin variants with altered
steric properties. The more hindered derivative, a di-tert-
butyl resin, could not be synthesized. We expect that this is
a result of the bulky nature of the intended product. Both
the dimethyl- and diethyl-substituted resins, S1 and S2,
respectively, favored enrichment of alcohol-containing
compounds with yields of ∼70% and exhibited dramati-
cally lower yields of carboxylic acids (∼20%; Table S1).
These data suggest that the nature of the alkyl substituents
on the silicon atom is critical to preferential functional
group capture.
Our first reversible enrichment tag was for the hydroxyl
functional group, which was targeted by formation of a
silyl ether bond using a chlorodiethylsiloxane polystyrene
6
resin. The isolated compound pool was readily released
from the resin by treatment with a fluoride source. Here,
we describe a chemoselective strategy for the carboxylic
acid. This functional group is present in approximately
7
1
5% of natural products and 25% of drugs, making it an
important group to target with our reversible enrichment
method. A previousstudyreportsuse of ananionexchange
resin to separate a subpool of natural products, including
carboxylic acids. However, other acidic groups, such as
7
phenols, were readily isolated using the reported strategy.
Scheme 1. Synthesis of the Diisopropyl Siloxyl Chloride Resin
3
b
Additionally, the noncovalent interaction that this method
is dependent upon is not strong enough to tolerate exten-
sive washing protocols resulting in substantial carryover
between fractions. Clearly, development of a covalent and
chemoselective enrichment strategy is warranted.
Few transformations that selectively target carboxylic
8
acids have been reported. During the development of our
hydroxyl group enrichment tag, we identified a silicon-
functionalized resin architecture (3) that displayed signifi-
cant preference for reaction with carboxylic acid contain-
ing molecules. This resin utilizes a diisopropylsiloxane
capture moiety, which forms the corresponding siloxyl
These results are consistent with what is known about
silyl and siloxyl ester stability in that bulkier esters are
9
ester bond with carboxylic acids (4; Scheme 1). Following
capture, unaltered compounds are readily released from
resin using a fluoride source. This result was surprising
1
0
significantly more stable. However, silylating reagents
are generally not chemoselective for carboxylic acids often
1
1
forming more stable bonds with the hydroxyl group.
Recently, a bulky reagent, di-tert-butylisobutylsilyl tri-
flate, was reported to selectively protect carboxylic acids
1
0
given the general instability of silyl esters; however,
carboxylic acids were enriched using resin 3 with yields
of ∼90% (Table S1). This resin also promoted isolation of
alcohol containing compounds (yields 6À18%) suggesting
that optimization would be required to achieve chemos-
electivity. Amine- and thiol-containing compounds were
not captured to an appreciable extent by this resin (0À3%;
Table S1).
1
2
over alcohols in some substrates. This protecting group
also readily forms stable conjugates with amines. Intrigu-
ingly, we found that the presence of the diisopropyl sub-
stituents on the silicon was not enough to afford a selective
reagent. The oxygen linker between the resin and the
silicon atom is also important for selectivity, as a diisopro-
pylsilyl-functionalized resin facilitated the isolation of
alcohols, carboxylic acids, thiols, and amines (S3, Table
S1 and ref 6). Thus, our studies indicate that privileged
capture of carboxylic acids is best accomplished with the
diisopropyl siloxyl scaffold.
With the ultimate goal of generating a chemoselective
capture resin, we first sought to identify the chemical
characteristics that resulted in formation of a stable ester
species while also affording selectivity toward carboxylic
acids. A series of resin derivatives were synthesized and
assessed for their ability to capture a standard set of
Efforts to achieve the desired chemoselectivity by pre-
ferentially cleaving either the captured alcohols or acids
from resin 3 were unsuccessful (data not shown). We
reasoned that tuning of the leaving group would enable
selective capture of the more nucleophilic carboxylate.
Accordingly, a series of dialkyldisiloxane resins was
(
7) Henkel, T.; Brunne, R. M.; M u€ ller, H.; Reichel, F. Angew. Chem.,
Int. Ed. 1999, 38, 643.
8) Hermanson, G. T. Bioconjugate Techniques, 2nd ed.; Elsevier Inc.:
Rockford, IL, 2008.
9) Meloni, M. M.; White, P. D.; Armour, D.; Brown, R. C. D.
Tetrahedron 2007, 63, 299.
10) (a) Weinberg, J. M.; Gitto, S. P.; Wooley, K. L. Macromolecules
998, 31, 15. (b) Wang, M.; Weinberg, J. M.; Wooley, K. L. Macro-
(
(
(
1
(11) Ojima, Y.; Yamaguchi, K.; Mizuno, N. Adv. Synth. Catal. 2009,
351, 1405.
molecules 1998, 31, 7606. (c) Kocienski, P. J. Protecting Groups, 3rd ed.;
Thieme: Stuttgart, 2005.
(12) Liang, H.; Hu, L.; Corey, E. J. Org. Lett. 2011, 13, 4120.
Org. Lett., Vol. 13, No. 20, 2011
5653

Scheme 2. Generation of Dialkyldisiloxyl Resin Derivatives
Useful for Chemoselective Capture of Carboxylic Acids
Table 1. Enrichment Yields of Model Carboxylic Acid, Alcohol,
Amine, and Thiol Containing Molecules with Derivatives of 5
a
a
Upon cleavage, the resin can be regenerated (Table S4).
generated by alcoholysis of the silyl chloride (5, Scheme 2).
First, we examined the loading capacity and enrichment
yields of three resins, methoxy- (6), isopropoxy- (7), and
tert-butoxy-substituted (8), with a model set of car-
boxylic acids (Tables 1, S2, and S3). The best combina-
tion of loading capacity and breadth of carboxylic acid
capture was obtained with the isopropoxy derivative (7).
The tert-butoxy derivative (8) was effective for capture
of less hindered substrates but was too bulky to enable
efficient enrichment of sterically encumbered com-
pounds, while the methoxy derivative (6) showed mod-
erate to low yields likely because of its relatively poor
leaving group potential. Importantly, all three deriva-
tives displayed complete carboxylic acid chemoselectiv-
ity (Table 1). Further studies will be required to
determine if chemoselectivity is due exclusively to the
nucleophilicity of the carboxylate, or to other character-
istics such as an increased ability of the carboxylate to
form a pentavalent silicon complex.
To identify a resin with increased loading capacity and
consistently high enrichment yields, we explored alkoxyl
moieties with electron-withdrawing groups (EWG) or
electron-donating groups (EDG). A variety of alcohols,
We next sought to show the utility of this resin for the
isolation of carboxylic acids from complex mixtures. Mon-
ensin (13) is a polyether ionphore antibiotic secreted by
2
2 in all, were tested to see which properties (i.e., sterics,
EWG, or EDG) yielded resins with the highest loading
capacity with three carboxylic acids (Table S2). The high-
est loading values were obtained by activation with secon-
dary alcohols. Efficient isolation of tertiary carboxylic
acids was only seen with resins derived from electron-poor
alcohols verifying the influence of the leaving group on
capture potential (e.g., 9, 10, S23, S24).
From this resin set, 9 and 10 were selected for further
characterization based upon both loading capacity and
cost of the reagents. These resins were subjected to
coupling with a large model set of carboxylic acids
ranging in steric hindrance and molecular complexity
13
Streptomyces cinnamonensis. The growth media of this
bacterium was collected to yield a crude natural product
extract. Prior to enrichment, the extract was doped with
four model carboxylic acids and two alcohols, two amines,
and a thiol (17À20, S9) to ensure chemoselectivity in a
biological setting. We analyzed the crude extract by LC-
MS, yielding trace A (Figure 1).
The extract was subjected to enrichment with resin 9.
The resulting material was analyzed by LC-MS using the
same gradient (trace B, Figure 1). Monensin (13) was
recovered in a 64% yield along with 3-oxo-1-indancar-
boxylic acid (S32), 3,5-dimethyl-4-methoxybenzoic acid
(S38), probenecid (16), and abietic acid (15) recovered in a
96%, 87%, 75%, and 57% yield, respectively. None of the
chemoselective model compounds were detected, confirm-
ing that 9 remains specific for carboxylic acid containing
(
20 compounds; Tables 1 and S3). Amine-, thiol- and
alcohol-containing compounds were included to assess
chemoselectivity (Table 1). High and consistent enrich-
ment yields of a wide array of carboxylic acids were
observed with resin 9, which was activated with a 1,
3
-dichloro-2-propoxyl group. This resin was also found
to be completely chemoselective and was chosen as our
carboxylic acid enrichment tag.
(
13) Huczynski, A.; Stefanska, J.; Przybylski, P.; Brzezinski, B.;
Bartl, F. Bioorg. Med. Chem. Lett. 2008, 18, 2585.
5
654
Org. Lett., Vol. 13, No. 20, 2011

concentrations. An average recovery yield of ∼80% was
observed across ratios of natural product background to
targeted acid ranging from 2:1 to 800:1 (8 μmolÀ15 nmol;
Table S7). This provides strong evidence that our enrich-
ment strategy will enable detection of low abundance
species.
Finally, we confirmed the utility of resin 9 for isola-
tion of carboxylic acids following solution phase synthe-
sis. We converted the methyl ester derivative of serine to
the corresponding carboxylic acid and subjected the crude
reaction to 9, resulting in isolation of the product in good
yield and high purity (Figure S5). Thus, our resin can be
used to purify synthetically derived carboxylic acids,
mostly notably, polar compounds that are difficult to
isolate using standard chromatography methods.
We have synthesized a chemoselective enrichment tag
for carboxylic acid containing molecules and applied it to a
variety of systems. We determined that a diisopropyl
siloxyl architechure is required for carboxylic acid prefer-
ence and that an alkoxyl leaving group is essential to
achieve complete chemoselectivity. After determining the
loading capacities of 22 diisopropyldisiloxane resins, we
established that resin 9 had all of the desired properties to
serve as our enrichment reagent. This is the first example of
a chemoselective method for solid-phase enrichment of
carboxylic acids. In addition, to the best of our knowledge,
Figure 1. Enrichment from the extract of Streptomyces cinna-
monensis. (A) TIC of the crude natural product extract with
monensin and four doped model carboxylic acids, which are
highlighted. (B) TIC of the carboxylic acid containing molecules
enriched from the extract. (C) Two alcohols (17, 18), two amines
formation of silyl or siloxyl esters via the siloxyl ether has
10,14
not been previously reported.
We expect that this
carboxylic acid enrichment tag, along with our previously
developed tag for hydroxyl containing compounds, will
find utility in many applications, including the discovery of
natural products.
(
19, S9), and a thiol (20) were spiked into the extract. Red trace is
the extracted ion chromatogram of the compounds prior to
enrichment. Blue trace indicates that none of the compounds are
present following capture and release of the extract.
compounds in a biological background (Figures 1C and S3).
Itisevidentfromthechromatogramsthatmanyofthehighly
abundant compounds were eliminated and that fewer
compounds are present after carboxylic acid capture en-
abling better resolution of the remaining components.
Additionally, some features represent a greater proportion
of the carboxylic acid fraction than of the crude extract
demonstrating enrichment. We quantified the extent of
carboxylic acid enrichment in comparison to compounds
containing other functional groups that were spiked into
the sample. Carboxylic acids were enriched by an average
of ∼300-fold over other functional groups (Table S5).
To show that enrichment occurs equivalently indepen-
dent of the proportion of the sample that the targeted
compounds comprise, we spiked three carboxylic acids
into the Streptomyces cinnamonensis extract at varying
Acknowledgment. This work was supported by NIH
R00 GM82983 and a Pew Biomedical Scholar Award
(E.E.C.). We thank L. Brown and M. K. Brown for their
thorough reading of the manuscript.
Supporting Information Available. Methods for resin
synthesis, compound capture, and cleavage; methods for
determination of loading capacities and enrichment
yields; Streptomyces cinnamonensis enrichment data;
characterization of resins and mass spectrometry data
for enrichment experiments. This material is available
free of charge via the Internet at http://pubs.acs.org.
(
14) (a) Mbah, G. C.; Speier, J. L. J. Organomet. Chem. 1984, 271, 77.
b) Chauhan, M.; Chauhan, B. P. S.; Boudjouk, P. Org. Lett. 2000, 2,
027. (c) Huang, X.; Craita, C.; Awad, L.; Vogel, P. Chem. Commun.
2005, 1297.
(
1
Org. Lett., Vol. 13, No. 20, 2011
5655