Storable palladacycles for selective functionalization of alkyne-containing proteins

Article abstract of DOI:10.1039/c3cc43479f

We report the facile preparation of palladacycles as storable arylpalladium(ii) reagents from acetanilides via cyclopalladation. The palladacycles exhibit good stability in PBS buffer and are capable of functionalizing a metabolically encoded HPG-containing protein, thus providing a new type of biocompatible organometallic reagent for selectively functionalizing the alkyne-encoded proteins.

Full text of DOI:10.1039/c3cc43479f

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Storable palladacycles for selective functionalization of
alkyne-containing proteins†‡
Cite this: Chem. Commun., 2013,
49, 6809
Gang Cheng, Reyna K. V. Lim, Nan Li and Qing Lin*
Received 10th May 2013,
Accepted 3rd June 2013
DOI: 10.1039/c3cc43479f
www.rsc.org/chemcomm
We report the facile preparation of palladacycles as storable in PBS buffer, and their use in selective functionalization of an
arylpalladium(II) reagents from acetanilides via cyclopalladation. alkyne-encoded protein in PBS under mild conditions.
The palladacycles exhibit good stability in PBS buffer and are
We first prepared a series of palladacycles with varying
capable of functionalizing a metabolically encoded HPG-containing substituents on the aromatic ring from the acetanilide derivatives
protein, thus providing a new type of biocompatible organometallic by following Yu’s cyclopalladation protocol.14 We found that
reagent for selectively functionalizing the alkyne-encoded proteins. substrates with methoxy substituents (except at the ortho position
which is probably due to the ortho-effect14) provided the desired
Bioorthogonal reactions have provided chemical tools to study palladacycles in excellent yields (Table 1, entries 2 and 12) whereas
biomolecular dynamics and function in living systems.1–4 Similar to substrates with methyl substituents afforded modest yields15
azides, terminal alkynes have become attractive bioorthogonal (entries 8 and 9) similar to 2a without any substituents.14,16,17
chemical reporters due to their small size, excellent biocompatibility, Interestingly, fluorine substitution gave rise to high yields
and ease of incorporation into proteins. Importantly, the alkynes (entries 6 and 10) compared to chlorine which gave much lower
readily react with the azide probes via Cu-catalyzed azide–alkyne yields (entries 5 and 7). BODIPY substrate 1k served as a poor
cycloaddition5–8 or strain-promoted cycloaddition reaction.9 substrate for the cyclopalladation reaction, giving only 35%
Recently, we developed a new protein bioconjugation strategy yield (entry 11). Notably, high regioselectivity was observed in
through a palladium-mediated cross-coupling reaction to label an the formation of palladacycle 2d from 1-acetamidonaphthalene
alkyne-encoded protein in vitro and in E. coli.10 In this strategy, we 1d (see ESI‡). Using Pd(O2CCF3) as a palladium source,18 we
employed a two-step procedure in which we generate the ‘preacti- can also readily generate palladacycles containing a fluorescein
vated’ arylpalladium(II) complex first followed by the cross-coupling or a PEG (MW E 5 kDa) group (entries 14 and 15).
reaction with the alkyne-encoded protein. The reaction was clean
With the palladacycles in hand, we examined their stability in
and efficient, however, the in situ generated arylpalladium(II) PBS buffer. Gratifyingly, most palladacycles exhibited high tolerance
complex gradually loses its reactivity in phosphate buffered saline to the PBS buffer–DMSO (19 : 1) mixture as monitored by 1H NMR
(PBS) over time. Hence, excess amount of reagent is typically within 24 h (see Table S1 in ESI‡). Next, we assessed whether
required in order to achieve high conversion. At about the same palladacycle 2a can be employed to modify a metabolically encoded
time, Myers and co-workers reported the preparation of storable homopropargylglycine (HPG)-containing ubiquitin (HPG-Ub)10 via a
arylpalladium(II) reagents11 through the decarboxylative palladation cross-coupling reaction process (Scheme 1). Treating 2.5 mM of
and showed that these reagents were capable of labeling the olefinic HPG-Ub with 4 equiv. of palladacycle 2a in PBS buffer at 37 1C for
substrates via stoichiometric Heck-type reaction in aqueous media. 30 min afforded the ligation product 3a in 70% yield, based on
Inspired by this report, we envisioned that by introducing a weak- LC-MS analysis. Prolonging the reaction time to 4 h increased the
coordination directing group12,13 in our palladium complex, we may yield to 93%. Likewise, addition of a large excess of palladacycle 2a
achieve an optimum balance between reactivity and stability. (25 equiv.) increased the yield to 92% (Scheme 1). This encouraging
Herein, we describe the preparation of the storable palladacycles result led us to investigate the rest of the palladacycles (2b–2o) in the
with an ortho-directing group, the characterization of their stability series using 4 equiv. at three time points, namely, 30 min, 2 h, and
4 h, and the results are summarized in Table 2. In the palladacycle
Department of Chemistry, State University of New York at Buffalo, Buffalo,
series, 2d with the naphthyl ring showed the highest ligation
efficiency, reaching essentially quantitative yield within 30 min.
Palladacycles with methyl, methoxy, or fluorine substituents on the
New York 14260, USA. E-mail: qinglin@buffalo.edu; Fax: +1 716 6456963;
Tel: +1 716 645 4254
† This manuscript is dedicated to Professor Andrew Hamilton on the occasion of
aromatic ring (Table 2, entries 2, 3, 6, 8, 9, 10, and 12) also gave high
his 60th birthday.
‡ Electronic supplementary information (ESI) available. See DOI: 10.1039/c3cc43479f yields. However, the reaction with chloro-substituted palladacycles
c
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Table 1 Synthesis of palladacyclesa
Table 1 (continued)
Entry Aryl acetanilide
Palladacycles
Yieldb (%)
85c
15
a
Reactions were carried out with 0.1 mmol acetanilide, 1 equiv. of
Yieldb (%)
75
Pd(OAc)2 and 1 equiv. of p-TsOHÁH2O in 1 mL of dioxane at room
Entry
1
Aryl acetanilide
Palladacycles
b
c
temperature for 1–10 h. Isolated yields. Estimated based on 1H NMR.
2
3
4
93
68
87
Scheme 1 Optimization of reaction conditions.
Table 2 Reactions of palladacycles with HPG-Uba
5
6
54
95
Yieldb (%)
Entry
1
Palladacycles
0.5 h
2 h
96
4 h
93
70
7
67
70
71
73
2
3
65
49
84
70
93
80
8
4
5
6
96
20
68
97
33
85
99
42
89
9
10
11
12
13
35
92
72
7
45
56
61
8
9
82
83
90
88
98
92
14
60
c
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Table 2 (continued)
Yieldb (%)
0.5 h
Entry
10
Palladacycles
2 h
70
4 h
80
59
11
30c
60
ND
76
ND
85
Scheme 2 Proposed mechanism for the generation of a styrene product.
this discrepancy, we propose that palladacycle 2 would undergo
carbopalladation with alkyne (alkyne insertion) to afford a vinyl-
palladium(II) intermediate A (Scheme 2), which could be stabilized
by coordination with nearby residues such as Arg in the protein
substrate, thus preventing a path to indole formation via inter-
mediate B or other side reactions such as multi-alkyne-insertion.
Upon treatment with a reducing reagent such as 3-mercapto-
propanoic acid or dithiothreitol, intermediate A would undergo
reductive depalladation to generate the styrene product.
In conclusion, we have synthesized stable palladacycles that
are suitable for protein labelling in aqueous medium. These
palladacycles were used to modify a terminal alkyne-encoded
protein in PBS buffer at 37 1C to form the styrene adducts with
moderate to high yields. Because of their superior stability and
reactivity, these palladacycles may be useful for functionalizing
the terminal alkyne-encoded proteins in cellular systems.
We gratefully acknowledge the National Institutes of Health
(GM 085092) for financial support.
12
13
14c
60c
ND
ND
ND
ND
14
a
Reactions were carried out using 2.5 mM of HPG-Ub and 4 equiv. of
b
palladacycle at 37 1C. Yields were determined based on LC-MS analysis:
yield% = Iproduct/(IHPG-Ub + Iproduct), where Iproduct and IHPG-Ub represent
the ion counts of the ligated product and HPG-Ub, respectively.
c
25 equiv. of palladacycle was used. ND, not determined.
2e and 2g proceeded sluggishly, giving lower yields (Table 2,
entries 5 and 7). In addition, palladacycles 2k and 2m carrying a
BODIPY group gave only 30% and 14% yields, respectively, when
25 equiv. of palladacycles were employed (Table 2, entries 11 and
13). The lower reactivity can be attributed to their poor solubility in
PBS buffer. Palladacycle 2n with a fluorescein group gave a relatively
higher yield (60%) under the same conditions (Table 2, entry 14).
To elucidate the structure of the ligation product, the adduct
of HPG-Ub with palladacycle 2d was digested with trypsin and
subsequently analysed by LC-MS. The mass of the naphthyl
acetanilide-modified C-terminal pentapeptide is consistent with a
structure in which the linkage between naphthalene and the peptide
fragment is a double bond (Fig. S1, Tables S2 and S3, ESI‡).
To verify the selectivity of this type of ligation, we analysed the
reaction mixture of HPG-Ub with the BODIPY-functionalized palla-
dacycle 2k by SDS-PAGE. In-gel fluorescence analysis revealed that
only HPG-Ub was fluorescently labelled, while no fluorescent band
was detected with wild-type ubiquitin under identical conditions
(Fig. S2, ESI‡). Similarly, the reaction of HPG-Ub with PEG-
containing palladacycle 2o led to the concentration-dependent
formation of a distinct higher molecular weight band by
SDS-PAGE, indicating the selective formation of the PEGylated
HPG-Ub adduct (Fig. S3, ESI‡).
Notes and references
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c
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Chem. Commun., 2013, 49, 6809--6811 6811