Storable arylpalladium(II) reagents for alkene labeling in aqueous media

Article abstract of DOI:10.1021/ja206339s

We show that arylpalladium(II) reagents linked to biotin and indocyanine dye residues can be prepared by decarboxylative palladation of appropriately substituted electron-rich benzoic acid derivatives. When prepared under the conditions described, these organometallic intermediates are tolerant of air and water, can be stored for several months in solution in dimethyl sulfoxide, and permit biotin- and indocyanine dye-labeling of functionally complex olefinic substrates in water by Heck-type coupling reactions.

Full text of DOI:10.1021/ja206339s

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Storable Arylpalladium(II) Reagents for Alkene Labeling in
Aqueous Media
Rebecca L. Simmons, Robert T. Yu, and Andrew G. Myers*
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, United States
S Supporting Information
b
ABSTRACT: We show that arylpalladium(II) reagents
linked to biotin and indocyanine dye residues can be
prepared by decarboxylative palladation of appropriately
substituted electron-rich benzoic acid derivatives. When
prepared under the conditions described, these organo-
metallic intermediates are tolerant of air and water, can be
stored for several months in solution in dimethyl sulfoxide,
and permit biotin- and indocyanine dye-labeling of func-
tionally complex olefinic substrates in water by Heck-type
coupling reactions.
he value of selective bond-forming reactions between reci-
procally reactive functional groups in water at ambient
T
temperature has been noted by Sharpless and co-workers and
was strikingly illustrated by them with the disclosure of the
copper-catalyzed [3 + 2] dipolar cycloaddition reaction of azides
and alkynes.1 Bertozzi and co-workers as well as others have
developed strain-promoted cycloaddition reactions that proceed
efficiently in water without the use of catalysts.2 In this work, we
report preliminary steps toward the development of a different
type of noncatalyzed (carbonÀcarbon) bond-forming reaction
in water: a stoichiometric Heck-type coupling of discrete, stor-
able arylpalladium(II) reagents with olefinic reactants. We
describe the synthesis of three specific arylpalladium(II) reagents
bearing biotin and indocyanine dye labels and demonstrate that
these can be coupled with functionally complex olefinic reactants
in aqueous media at ambient temperature.
Figure 1. Decarboxylative palladation of biotin-linked benzoic acid
derivative 1.
(Figure 1c; also see the SI).5 A marked upfield shift of the
biotin carbonyl carbon resonance in the 13C NMR spectrum
of the product suggests that the cyclic urea was complexed
with palladium. While we depict the complex as N-bound in
structure 2, we do not rule out η3- or O-bound complexes or
the possibility of dinuclear complexes (see the SI for examples
of like-shifted amido- and imidopalladium complexes). Stock
solutions of reagent 2 in DMSO-d6 are stable to handling in air
at ambient temperature, and when stored frozen at À20 °C and
then thawed just prior to use in coupling reactions (see below),
they can be used reliably for several months.
Decarboxylative palladation of the indocyanine dye-linked
benzoic acid derivative 3 (Figure 2a), also synthesized in seven
steps (see the SI), was studied next. The Cy5 indocyanine dye
residue of substrate 3 proved to be incompatible with the
conditions developed for decarboxylative palladation, as heat-
ing of substrate 3 with 1À3 equiv of palladium(II) trifluor-
oacetate at 80 °C for 25 min in DMSO-d6 led to byproducts
Decarboxylative palladation of the biotin-linked benzoic acid
derivative 1 (Figure 1), synthesized in seven steps from 2,
5-dimethoxybenzaldehyde and D-biotin [see the Supporting
Information (SI)], was studied first. Substrate 1 was chosen
because electron-rich benzoic acids (and their sodium salts) with
the 2,4,5-substitution pattern had previously been shown to be
particularly good substrates for decarboxylative palladation.3,4
When substrate 1 (monosodium salt) was heated with palladium-
(II) trifluoroacetate (1À3 equiv) in DMSO-d6 at 85 °C for
15 min, we observed that the expected arylpalladium(II) inter-
mediate had formed but was contaminated with the product of
protonolysis of the arylpalladium(II) intermediate. When instead
substrate 1 was first deprotonated with excess sodium hydroxide
in DMSO-d6 (1H NMR analysis suggested that a dianion was
formed; see Figure 1b), decarboxylative palladation in the
presence of 3.0 equiv of palladium(II) trifluoroacetate was
remarkably clean and efficient, forming the stable arylpalladium-
(II) intermediate 2, as evident from 1H and 13C NMR analysis
Received: July 13, 2011
Published: September 04, 2011
r
2011 American Chemical Society
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Figure 2. (a) Cy5-linked benzoic acid derivative 3 can be palladated at two sites. (b) Structures of indocyanine dyes 4 and 5.
Figure 3. Decarboxylative palladation of Cy5-linked benzoic acid derivatives 6 and 8.
arising from palladation of the central methine carbon of the dye
(Figure 2) as well as the expected decarboxylative palladation
product, which was the major component of the mixture.
Scheme 1. Heck-Type Coupling Reaction of 2 and 10 in
Aqueous Media
This complication was circumvented by substitution of the
central carbon of the dye. The unsymmetrically and symme-
trically substituted Cy5 dyes 4 and 5,6 respectively (Figure 2b),
were synthesized in two to four steps from commercially avail-
able materials (see the SI). These were linked to the 2,4,5-
substituted benzoic acid derivative as before. When each of the
Cy5 derivatives 6 and 8 was heated with 3.0 equiv of palladium-
(II) trifluoroacetate and 2.0 equiv of sodium carbonate in
DMSO-d6 at 80 °C for 25 min, decarboxylative pallada-
tion to form a stable arylpalladium(II) intermediate (7 or 9,
respectively) was both clean and efficient, as evident from 1H and
13C NMR analysis (Figure 3 and the SI).7 As with reagent 2,
stock solutions of reagents 7 and 9 in DMSO-d6 were stable to
handling in air at ambient temperature but were stored frozen at
À20 °C and then thawed just prior to use in coupling reactions.
We initiated our studies of Heck-type coupling reactions with
biotin-linked arylpalladium(II) reagent 2 using DMSO as the
solvent and 6-vinylquinaldic acid derivative 10 as the substrate
(Scheme 1), chosen because it served as a model for a projected
mechanistic study of saframycins8 and because the coupling
product was anticipated to be fluorescent.9 When a stock
solution of 2 in DMSO (18 mM, 1.4 equiv) was combined with
10 (1 equiv) in air at 23 °C (20 h), the (fluorescent) trans
coupling product 11 was obtained in 68% yield [HPLC iso-
lation]. We next evaluated the same coupling reaction in water,
limiting the total concentration of DMSO to 5%, in the presence
of varying buffer compositions and using equimolar amounts of
each substrate but at much higher dilution (0.1 mM). The
coupling product 11 was formed in yields of 35À70% at 23 °C
within 4À5 h (see the SI). Optimum results (70% yield) were
obtained using RIPA buffer, which contains 1% Triton X-100 and
0.1% SDS (Scheme 1). Lipshutz and co-workers extensively
studied organometallic coupling reactions in water, including
conventional Heck reactions of aryl halides using exogenous
palladium catalysts, and noted that surfactants were critical for
achieving successful coupling in the systems they examined.10
We too have observed beneficial effects of surfactants in the
(noncatalytic) transformations we have examined, including
Heck-type coupling of 2 and 10, but in the latter case, surfactants
were not absolutely required.11 Lastly, we found that while the
efficiency of Heck-type coupling of 2 and 10 was greatly
attenuated in the presence of tris(2-carboxyethyl)phosphine,
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Scheme 2. Heck-Type Coupling Reaction of Styryl-Modified Taxol Derivative 12 and Reagent 7 in Aqueous Media
Figure 4. (a) Styryl residues were introduced by the reaction of lysine residues with NHS-ester 14. Subsequent treatment with PdÀArÀCy5 reagent 7
led to labeling of the modified protein by the dye. (b) SDS-PAGE with visualization by fluorescence imaging (bottom) and Coomassie staining (top).
ascorbic acid, or DTT, amines or pyridine-based ligands were
well-tolerated.
determined by LCÀMS analysis (see the SI).12 When this protein
mixture (39 μM) was incubated with Cy5-linked reagent 7 (115
μM; abbreviated as PdÀArÀCy5 in Figure 4) in pH 8.0 Tris buffer
containing 5% DMSO at 23 °C or, separately, at 37 °C for 4 h, a
major fluorescent band with MW ≈ 15 kDa was observed by
denaturing SDS-PAGE analysis along with a minor fluorescent
band of higher molecular weight (∼16 kDa, believed to be the
doubly modified Heck-type coupling product; see Figure 3b, lanes
3 and 5). High-resolution LCÀMS analysis of the product mixture
after purification by size-exclusion chromatography showed a major
product peak at 15 273 Da, corresponding to the singly modified
Heck-type coupling product (est. conversion 75%). Control
experiments conducted with the styryl-modified lysozyme sub-
strate and a nonpalladated Cy5 dye reactant [protiodepalladation
product HÀArÀCy5 (17)] or with unmodified lysozyme and
Cy5-linked reagent 7 did not produce any significant fluorescent
bands (lanes 2 or 4 and 6, respectively; Figure 4b).
We next conducted an affinity-enrichment experiment using
biotin-conjugated arylpalladium(II) reagent 2 and styryl-mod-
ified FK-506 probe 18 (Figure 5a; see the SI for its synthesis and
biological evaluation using an NFAT reporter gene assay).13,14
Fresh cell lysate from healthy Chinese hamster ovary (CHO)
cells was diluted to a total protein concentration of 1.0 mg/mL
with Tris-buffered saline containing Triton X-100 (pH 8.0), and
the diluted protein solution was treated with 18 (0.02 mM) for
4 h at 4 °C. This sample was then incubated with 2 (0.05 M) for
14 h at 23 °C. DTT was added, followed by an amine-based
palladium scavenger resin. Proteins complexed to the biotin-
conjugated probe molecule were then sequestered onto
In a different test of the feasibility of Heck-type coupling,
unsymmetrically substituted Cy5 arylpalladium(II) reagent 7
(14 mM, 1 equiv) in DMSO was mixed with taxol-derived
substrate 12 (1.5 equiv) in air at 23 °C for 2 h. The trans
Heck-type coupling product 13 was obtained as a blue solid in
70% yield after isolation by flash-column chromatography.
Remarkably, when substrate 12 (25 μM, a >500-fold dilution,
1 equiv) was combined with reagent 7 (50 μM, 2 equiv) in
aqueous potassium phosphate buffer solution (pH 8.0, 0.1%
Triton X-100) containing 5% DMSO at 23 °C, 13 was formed in
67% yield after 1 h (Scheme 2 and the SI). Variation of the pH
(5.8À8.0) in the latter system was found not to influence the
yield of the coupling product (63À68% yield of 13, 1 h).
Coupling was slower but equally efficient when Tris buffer was
used in lieu of phosphate buffer (23% yield after 3 h and 70%
yield after 17 h at 23 °C). In both cases, we found that Triton
X-100 was an essential additive (<5% coupling in its absence),
consistent with the prior observations of Lipshutz and
co-workers,10 although we found that lower loadings of surfactant
were required in this particular system (0.1% vs 5À15%).
We next evaluated the feasibility of labeling a protein substrate
that had been covalently modified to present an alkene function.
Acylation of lysozyme (MW = 14 307 Da) with styryl succini-
mide (14) at pH 8.5 for 1.5 h (Figure 4a) gave rise to a mixture
comprising ∼34% unmodified lysozyme, 40% singly modified
lysozyme (15, MW = 14 451 Da), and lesser amounts of doubly
and triply modified lysozyme (20 and 6%, respectively), as
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gratefully acknowledges Eli Lilly for an Organic Chemistry
Fellowship. This research was supported by NSF Grant CHE-
0749566 and NIH Grant CA-047148.
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Figure 5. (a) Structures of FK506 and FK506-based affinity probes and
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storable arylpalladium(II) reagents bearingbiotinand indocyanine
dye residues. We have also demonstrated that these reagents
function effectively in Heck-type coupling reactions with styrene-
containing substrates in aqueous media at 23 °C, including a stryl-
modified protein substrate and a styryl-modified FK-506 analogue
in the presence of a cell lysate containing its protein receptors.
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reagents, subsequent coupling reactions do not rely upon any
catalytic cycle, which we believe to be advantageous. In addition to
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less stable, undergoing slow protiodepalladation over the course of
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’ ASSOCIATED CONTENT
S
Supporting Information. Complete experimental proce-
b
dures and spectral data. This material is available free of charge
via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
myers@chemistry.harvard.edu
’ ACKNOWLEDGMENT
(15) Preliminary experiments have suggested that labeling of ribo-
nucleic acids can also be achieved by the present methodology.
We thank David Liu, Tobias Ritter, Alan Saghatelian, Stuart
Schreiber, and Joshua Vaughan for helpful discussions. R.L.S.
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