Stabilized wittig olefination for bioconjugation

Article abstract of DOI:10.1039/c3cc45961f

Stabilized Wittig olefination holds great potential as a bioconjugation reaction. We demonstrate that the reaction of stabilized phosphorus ylides (or phosphonium salts) with aryl aldehydes is sufficiently robust to be used for live cell affinity isolation and fluorescence tagging of a protein, FKBP12.

Full text of DOI:10.1039/c3cc45961f

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Stabilized Wittig olefination for bioconjugation†
Kenneth M. Lum, Vanessa J. Xavier, Michelle J.-H. Ong, Charles W. Johannes and
Kok-Ping Chan*
Cite this: Chem. Commun., 2013,
49, 11188
Received 5th August 2013,
Accepted 11th October 2013
DOI: 10.1039/c3cc45961f
www.rsc.org/chemcomm
Stabilized Wittig olefination holds great potential as a bioconjugation byproduct of the reaction is triphenylphosphine oxide, which is also
reaction. We demonstrate that the reaction of stabilized phosphorus produced in the well-explored Staudinger ligation.4 Although these
ylides (or phosphonium salts) with aryl aldehydes is sufficiently robust coupling partners are not bioorthogonal in the strictest sense,11 we
to be used for live cell affinity isolation and fluorescence tagging of a show that they are sufficiently robust for protein affinity isolation
protein, FKBP12.
and fluorescence labeling in live cells.
From the outset, we recognized the inherent challenge of using
Bioconjugation reactions are indispensable for studying biology.1 an electrophile–nucleophile pair to do chemistry in biological
Current gold standards in the field have been developed from matrices. Aldehydes are increasingly being recognized as mild
decades-old name reactions: [3+2] azide–alkyne ‘‘Click’’ reaction ‘‘bioorthogonal’’ electrophiles,7a although they are well-known to
(Huisgen condensation),1b,2 tetrazine–cycloocetene [4+2] addition form reversible adducts with cellular nucleophiles.11,12 Likewise,
(Diels–Alder cycloaddition),3 and azide–ester Staudinger ligation stabilized phosphorus ylides are moderate nucleophiles, but little is
(Staudinger reaction, mediated by phosphine).4 Advances in these known about their reactivity with biologically-relevant electrophiles.
strategies such as the strain-promoted azide–alkyne condensation5 Collectively, these challenges have perhaps undermined the
have obviated harsh conditions (e.g. high temperatures, cytotoxic potential of Wittig olefination as a bioconjugation strategy.
metal catalysts) and enabled tremendous progress in understanding
Notwithstanding, we hoped to leverage on (1) the kinetic pre-
native cellular processes. Unsurprisingly, chemists are actively inves- ference of stabilized phosphoranylidenes for aldehydes, (2) the
tigating a variety of other reagent pairs,6 including aldehydes,7 as irreversibility of the Wittig reaction due to the formation of strong
chemical reporters in biological systems. These efforts underscore PQO and CQC bonds, and (3) the use of an aryl aldehyde coupling
the increasing demand for new strategies to do chemistry in vivo.
partner such that extensive p-conjugation in the final cinnamate
Despite such efforts, the Wittig reaction8 has not been adduct would provide an additional driving force for preferential
considered as a bioconjugation strategy. However, recent evidence conjugation with the phosphoranylidene (Scheme 1).
has shown that the reaction of stabilized phosphorus ylides with
Indeed, our preliminary studies (see ESI†) revealed that stabilized
aldehydes not only tolerates water but also benefits from a rate phosphorus ylides readily undergo Wittig olefination with aldehydes
enhancement in aqueous media.9 Therefore, we envisaged that we in common culture media and cell lysis buffers. The reaction of
might exploit stabilized Wittig olefination to modify aldehydes in methyl 2-triphenylphosphoranylideneacetate with benzaldehyde in
biological matrices. Partway into our investigation, we were grati- various aqueous buffers afforded the corresponding (E)-cinnamate
fied to learn that others too had begun to consider stabilized Wittig adduct stereoselectively (approximate E/Z = 9 : 1), with excellent
olefination, utilising it to modify proteins in aqueous–organic
mixtures;10 this report provided strong support for our efforts in
developing this methodology for bioconjugation.
We describe herein a strategy for tagging proteins that is based
on Wittig olefination of a functionalized phosphoranylidene or a
phosphonium salt probe with an aryl aldehyde bait. The only
Division of Organic Chemistry, Institute of Chemical and Engineering Sciences,
Agency for Science Technology and Research Singapore, 11 Biopolis Way,
Helios Block #03-08, Singapore. E-mail: chan_kok_ping@ices.a-star.edu.sg;
Fax: +65 6874 5870; Tel: +65 6799 8599
Scheme 1 Mechanistic rationale behind the development of stabilized Wittig
† Electronic supplementary information (ESI) available: Full experimental details
and compound characterization. See DOI: 10.1039/c3cc45961f
olefination for bioconjugation.
c
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Table 1 Wittig-mediated bioconjugation of aldehydes in Tris buffer
Phosphor-
anylidene
probe
Yield
(%)
Aldehyde
Product
3
3
78
94
3
4
73
76
7
Scheme 2 Synthesis of Wittig probes. (a) BrCH2COBr, Et3N, CH2Cl2. (b) PPh3, PhMe.
(c) 2 M NaOH–CH2Cl2 (1 : 1). (d) (D)-biotin, K2CO3, DMF, 60 1C. (e) 1 M HCl. (f) N-dansyl-
6-aminohexanoic acid, K2CO3, DMF, 60 1C. (g) (D)-biotin, K2CO3, DMSO, 50 1C, then 1 M
HCl. (h) N-dansyl-3-aminopropanoic acid, K2CO3, DMF, 60 1C, then 1 M HCl.
FK506 is a small-molecule macrolide with high affinity for FKBP12. The
aryl aldehyde analog 7 is designed to demonstrate Wittig olefination in
biological systems (see ESI for respective structures).
chemoselectivity towards aldehydes and no observable reaction
with simple ketones. In rate studies of stabilized Wittig olefina-
tion in Tris buffer, we found that the second order rate constant
at 4 1C was comparable to that of the mechanistically-related
Staudinger ligation (see ESI†).
Scheme 3 Synthesis of dansyl probe 17 bearing an ethylenediamine linker.
(a) BrCH2COBr, Et3N, CH2Cl2. (b) PPh3, PhMe.
Encouraged by these results, we developed a rapid synthesis to
access probes bearing a stabilized phosphoranylidene joined to a
functional tag by a nonyl linker (Scheme 2A). These phosphorany-
lidene probes underwent Wittig olefination smoothly with aldehydes
of varying structural complexity, in Tris buffer at 4 1C (Table 1).
Leveraging on this synthetic approach, we developed a second
generation of probes linked by tetraethylene glycol (Scheme 2B),
which we hoped would improve probe uptake for cellular bioconju-
gation.13 A similar synthetic route starting from mono-tosylated
tetraethylene glycol allowed quick access to biotin probe 13 and
dansyl probe 14, analogues of 3a and 4 respectively (Scheme 3).
Having established the effectiveness of our probes in buffered
media, we turned our attention to demonstrating proof of con-
cept in biological matrices. We conducted streptavidin-mediated
affinity isolation on cell lysates treated with bait molecule 7
followed by phosphoranylidene probe 3. Western blot analysis
revealed bands at 12 kDa indicating successful isolation of
FKBP12 through Wittig conjugation of 3 and 7 in vitro. The
intensity of the FKBP12 band increased in a dose dependent
manner with probe concentration at fixed bait concentration
(Fig. 1A). Likewise, band intensity also increased with bait
concentration when probe concentration was fixed, appearing
to saturate at bait concentrations exceeding 0.5 mM (Fig. 1B).
Phosphonium probe 3a was likewise effective (Fig. 1C, lane 2).
Neither 3 nor 3a would effect a significant level of pulldown with
unmodified FK506 that lacked aldehyde functionality (Fig. 1C,
lanes 3 and 4), confirming that the probes react preferentially
with aldehydes over ketones.
With positive results from the affinity isolation of FKBP12 in
cell lysates, we investigated if the reaction would tolerate
cellular matrices. HeLa cells were incubated with bait 7 for
8 hours, followed by probe 3 or 13 in fresh media for 15 hours.
We were delighted to find that both probes 3 and 13 were able
to isolate FKBP12, indicating successful Wittig olefination
within the cells (Fig. 1D). The relative amount of FKBP12
isolated with 13 (C log P = 4.7 of ylide) was higher than that of
3 (C log P = 8.2),‡ consistent with the notion that the linkers
alter solubility and membrane permeability. Interestingly, the
relative amount of FKBP12 pulled down with either probe was
less than that obtained with pre-conjugated adduct 8, suggesting
that further optimization is required to improve the bioconjugation
yields in live cells (see ESI†). Nevertheless, successful pulldown of
FKBP12 from the complex environment of a living cell indicates
that stabilized Wittig olefination is indeed a viable bioconjugation
strategy.
Concurrently, we were interested to show that stabilized
Wittig olefination might be used for fluorescently labeling
proteins in live cells. To this end, we conducted imaging
experiments with bait 7 and dansyl probes 4 and 14 to explore
if we could fluorescently label FKBP12 in live cells. HeLa cells
were dosed with either bait 7 or DMSO as a control, followed by
c
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Given the various genetic7a,14 and chemical methods for
incorporating aldehyde reporters into cells, we anticipate that
stabilized Wittig olefination will find increased use in chemical
biology, both on its own and possibly in tandem with other
bioorthogonal reactions. Towards this goal, we have demon-
strated that stabilized Wittig olefination possesses desirable
attributes for bioconjugation: (1) the reacting functionalities
are sufficiently stable and chemoselective in complex environ-
ments, and (2) when installed on appropriately tuned scaffolds,
the resulting reagents can be used to biotinylate or fluores-
cently-label target proteins in live cells.
This research was supported by A*STAR ICES (Project code
ICES/11-240B08). We thank Michelle Su (Singapore BioImaging
Corsortium) for her assistance in the cell biology experiments
and Graham Wright (Institute of Medical Biology, Microscopy
Unit) for his invaluable microscopy advice.
Fig. 1 Affinity isolation of FKBP12. Titration experiments with (A) increasing
probe concentration and (B) increasing bait concentration. The intensity of the
FKBP12 band increased in a dose-dependent manner in these experiments.
(C) Comparison of phosphoranylidene probe 3 and phosphonium probe 3a.
Both probes react with bait 7, but not unmodified FK506. (D) Pulldown of
FKBP12 from live HeLa cells using probes 3 and 13.
Notes and references
‡ C log P values were calculated using ChemBioDraw Ultra.
the respective dansyl probe in a similar manner to the affinity
isolation assays. We hypothesized that cells treated with both
bait 7 and a dansyl probe would be more fluorescent than cells
treated with a probe alone: the irreversible reaction of 7 with
the probe traps the fluorophore in the cell as the dansylated-
FK506–FKBP12 complex, consequently shifting the equilibrium
to favor the diffusion of more probe into the cell.
Unfortunately, dansyl probes 4 and 14 were unsuitable for
this purpose, since cells treated with bait 7 and these probes
displayed only weak fluorescence (see Fig. 2A and B). These
findings prompted us to further synthesize dansyl probe 17
(Scheme 3), which bears an ethylenediamine linker, in the hope
that (1) a shorter linker would decrease non-radiative modes of
relaxation and lead to a stronger fluorescence signal, and
(2) the solubility properties associated with this linker would
further improve membrane permeability of the probe.
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