Catch and release of alkyne-tagged molecules in water by a polymer-supported cobalt complex

Article abstract of DOI:10.1039/c1ob06123b

A cobalt-phosphine complex supported on PS-PEG beads was found to react with a propargyl carbamate tag, and the tagged molecules immobilized on the beads could be released by acidic treatment through the Nicholas reaction pathway. These reactions work in

Full text of DOI:10.1039/c1ob06123b

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Catch and release of alkyne-tagged molecules in water by a
polymer-supported cobalt complex†
Hiromichi Egami,a,b Shinji Kamisuki,a,b Kosuke Dodo,a,b Miwako Asanuma,a,b Yoshitaka Hamashimab and
Mikiko Sodeoka*a,b
Received 8th July 2011, Accepted 18th August 2011
DOI: 10.1039/c1ob06123b
A cobalt–phosphine complex supported on PS-PEG beads
was found to react with a propargyl carbamate tag, and the
tagged molecules immobilized on the beads could be released
by acidic treatment through the Nicholas reaction pathway.
◦
These reactions work in aqueous media at 4 C, so that this
catch and release procedure is compatible with conditions
generally used in biochemical experiments.
Enrichment of a specific molecule from complex mixtures, such
as cell lysates, is indispensable for chemical biology studies, e.g.
to identify a binding protein or the protein binding site of
a low-molecular-weight bioactive compound. The development
of methods for the enrichment of various target molecules is
consequently of great importance, and the topic has attracted
the attention of many researchers.1 Affinity purification using a
combination of a biotin tag and immobilized avidin is widely
used nowadays (Scheme 1a).2,3 However, two issues have been
found with this technique: 1) harsh conditions are required to
release tagged molecules from avidin beads, due to the extremely
high affinity between biotin and avidin;4 and 2) the bioactivity
and membrane permeability of bioactive compounds are often
decreased by the introduction of biotin with a large linker.
The former problem has been addressed by the introduction of
a cleavable linker, such as disulfide,5 peptide,6 diazobenzene,7
hydrazone8 or acid-cleavable linker9 (Scheme 1b). However, the
efficiency and selectivity of these reactions are not necessarily
adequate for chemical biology studies. The latter problem has
been overcome by the introduction of click reactions, such as the
Sharpless–Meldal reaction, but these are indirect and multistep
procedures (Scheme 1c).10
Scheme 1 Outline of catch and release reactions.
alkyne-modified phospholipid with Co2(CO)8 in organic solvents
and subsequent purification of the resulting alkyne cobalt complex
using phosphine-functionalized silica gel.11 Organic solvents and
elevated temperature were required for the formation of the
alkyne–cobalt complex followed by its reaction with phosphine,
and an oxidant was required for release of the phospholipid.
Independently we have been working on the development of
click- and biotin-free methods for the detection and enrichment of
alkyne-tagged molecules useful for chemical biology research. We
have already reported a direct detection method of alkyne-tagged
molecules in living cells by using Raman microscopy.12 Herein,
we disclose a new catch and release method for the enrichment
of alkyne-tagged molecules using a polymer-supported cobalt
complex in water.
For the application to the enrichment of alkyne-tagged
biomolecules, e.g. peptides and proteins, catch and release reac-
tions that are highly selective to the alkyne are required. The
reactions should also be carried out in aqueous media in the
presence of various functional groups under mild conditions.
Thus, we decided to use an alkyne–cobalt carbonyl complex
as a key intermediate to meet the above criteria. It is well
known that Co2(CO)8 and its phosphine complex readily and
selectively react with alkyne, and the resulting complexes are
usually stable at room temperature under air.13 First, we tested
reaction sequences similar to the ones described in Brown’s report.
Namely, the reactions of alkyne with Co2(CO)8 in an aqueous
buffer were examined. But only a small amount of the desired
Alkyne is now widely used as a bioorthogonal tag in chemical
biology. Various functionalities such as fluorophore and biotin can
easily be installed by means of a Cu-mediated click reaction,9 and
can be used for the detection and enrichment of tagged molecules.
Recently, Brown and co-workers reported the reaction of an
aSodeoka Live Cell Chemistry Project, ERATO, Japan Science and Technol-
ogy, 2-1 Hirosawa, Wako-shi, Saitama, 351-0198, Japan. E-mail: sodeoka@
riken.jp; Fax: 81 48 462 4666
bRIKEN Advanced Science Institute, 2-1 Hirosawa, Wako-shi, Saitama,
351-0198, Japan
† Electronic supplementary information (ESI) available: Experimental
details and spectroscopic data. See DOI: 10.1039/c1ob06123b
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Co2(CO)6–alkyne complex was generated, and the reproducibility
was low, probably because Co2(CO)8 is not soluble in water and
is unstable under aerobic aqueous conditions.11 In addition, the
oxidative conditions required for decomplexation of the cobalt
complex may cause damage to some of the amino acid residues
in proteins. Therefore, we decided to use the polymer-supported
cobalt complex and the propargyl carbamate tag, anticipating that
it would form the polymer-supported alkyne–cobalt complex A
(Scheme 1d). The bound molecules would be released as an amine
compound through the Nicholas reaction in the presence of a
Brønsted acid.14,15 In this paper, we present proof-of-concept of
this direct catch and release procedure for alkyne-tagged molecules
in water, using the cobalt complex supported on PS-PEG. These
mild conditions should be applicable to biomolecules.
Although cobalt carbonyl complexes have been intensively stud-
ied, there is little information about the reactivity and properties
of cobalt carbonyl complexes in water. Thus, we first examined the
stability and reactivity of the alkyne–cobalt–phosphine complex in
water. We prepared a dansyl derivative 1 with the triethylene glycol-
linked propargyl carbamate tag as a model substrate (Scheme 2).
The propargyl carbamate tag was introduced into amine 2 by
using the activated carbonate 3. Subsequently the alkyne–cobalt–
phosphine complex 4 was prepared by the reaction of alkyne
1 with Co2(CO)7PPh3 complex in THF at room temperature.
The stability and reactivity of cobalt complex 4 in water were
Fig. 1 Stability and reactivity of the cobalt complex 4.
procedure16b and cobalt–phosphine complex 7 was prepared by
mixing 6 and Co2(CO)8 in THF. The reaction of cobalt complex
7 supported on PS-PEG with alkyne 1 was examined in an
aqueous Hepes buffer (pH 7.0),17 which is often employed in
biochemical experiments (Scheme 3). As expected, the reaction
proceeded to give alkyne–cobalt–phosphine complex 8 at ambient
temperature. Formation of cobalt complex 7 and its alkyne
complex 8 was characterized by IR spectroscopy. Absorptions of
the carbonyl groups of polymer-supported complexes 718 and 8 are
in good agreement with those of Co2(CO)7PPh3, the corresponding
non-polymer-supported complex 4, and similar known cobalt
complexes19 (Fig. 2).
The above results prompted us to investigate the catch and
release reaction of alkyne 1 on cobalt beads 7. Since the reaction
would generally be operated at low concentration and at low tem-
perature in biological experiments, the reaction of alkyne 1 with
Scheme 2 Synthesis of model substrate.
traced by HPLC analysis with detection at 280 nm to minimize
background peaks (Fig. 1). In the presence of 5% trifluoroacetic
acid (TFA), the reaction proceeded smoothly even at 4 ◦C to give
the corresponding amine 2 in 98% yield, together with a small
amount (2%) of 1 (Fig. 1a, b). Complex 4 was shown to be
stable under neutral aqueous conditions (Fig. 1b). To examine
whether 2 was formed directly from 4 via the Nicholas reaction,
or by stepwise decomplexation and hydrolysis of 1, 1 was treated
with 5% TFA. No reaction was observed, indicating that amine
2 was obtained via the Nicholas reaction pathway from complex
4. The Nicholas reaction is expected to selectively cleave the C–O
bond at the a-position of the alkyne–cobalt–phosphine complex.
Furthermore, under the same conditions, we observed formation
and gradual decomposition of the cobalt complex 5, which is also
a Nicholas reaction product, by means of LC–MS analysis.
Encouraged by these results, we next attempted to prepare
the polymer-supported cobalt complex. We employed PS-PEG,
because it has been utilized as a polymer support for catalysts in
aqueous media and is also tolerant to organic solvents.16 PS-PEG-
supported phosphine 6 was synthesized according to a reported
Scheme 3 Synthesis of the cobalt-alkyne complex supported on PS-PEG
in water.
Scheme 4 Catch and release of 1 at high dilution.
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Scheme 5 Enrichment of the alkyne-tagged molecule.
Further optimization is under way in our laboratory prior to
application of this new method to the enrichment of biomolecules,
such as peptide and proteins.
Notes and references
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3 His-tag technology is an another reliable method for purification of
proteins bearing the tag introduced by genetic engineering, see ref. 1.
However, this tag is too large to utilize for a low-molecular-weight
bioactive compound.
4 J.-N. Rybak, S. B. Scheurer, D. Neri and G. Elia, Proteomics, 2004, 4,
2296–2299.
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Fig. 2 IR spectra of cobalt complexes.
cobalt beads 7 was carried out at 4 ◦C on a 50 nmol scale (50 mM).
The catch reaction was monitored by fluorescence analysis of the
supernatant after removal of the beads by centrifugation, and the
release reaction was separately monitored by HPLC analysis to
detect the released product (Scheme 4). To our delight, alkyne 1
reacted with cobalt complex 7 even under low concentration and
low temperature conditions, and 57% of the alkyne was held on
to the resin after it had been washed ten times with Hepes buffer.
Finally, amine 2 was obtained in 59% yield after treatment with
TFA.
Furthermore, in order to examine the selectivity of the catch
and release reactions, a mixture of equal amounts of alkyne-tagged
molecule 1 and its benzyl derivative 9 was treated with cobalt resin
7 (Scheme 5). Again, 57% of alkyne 1 was caught by the cobalt
resin, while 93% of 9 and 43% of 1 were recovered in the residual
and wash solutions. Subsequently, treatment of the resin with TFA
afforded the Nicholas product 2 in 43% yield together with 4% of
starting alkyne 1. It is noteworthy that only a negligible amount
of benzyl derivative 9 was detected. Hence, great enrichment of
alkyne-tagged molecule 1 has been achieved.
In summary, we have demonstrated direct and selective catch
and release reactions of alkyne-tagged molecules utilizing a
polymer-supported cobalt complex under mild conditions. To our
knowledge, this is the first example of direct catch and release
reactions of alkyne-tagged molecules in water. These proof-of-
concept experiments strongly indicate that this cobalt chemistry
would be in principle applicable to chemical biology studies.
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17 The buffer contained 150 mM NaCl, 10 mM Hepes-Na (pH 7.0) and
0.5% (w/w) Triton X-100 in water.
18 The peak of 1886 cm-1 indicates that ionic cobalt complex
[Co(CO)3L2]+[Co(CO)4]- was located on the polymer, see ref. 19.
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