A Cyclopropenethione-Phosphine Ligation for Rapid Biomolecule Labeling

Article abstract of DOI:10.1021/acs.orglett.8b02296

Cyclopropenethiones are reported as new bioorthogonal reagents. These motifs react readily with substituted phosphines to provide thiocarbonyl adducts. In some cases, the ligations are >300-fold faster than analogous reactions with bioorthogonal cyclopropenones. Dialkyl cyclopropenethiones are also stable in aqueous buffers and can be used for biomolecule labeling in vitro and in cell lysate. The rapid reactivity and biocompatibility of cyclopropenethiones suggest that they will be useful probes for cellular studies.

Full text of DOI:10.1021/acs.orglett.8b02296

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
A Cyclopropenethione-Phosphine Ligation for Rapid Biomolecule
Labeling
R. David Row† and Jennifer A. Prescher*,†,‡,§
‡
§
†Departments of Chemistry, Molecular Biology & Biochemistry, and Pharmaceutical Sciences, University of California, Irvine,
California 92697, United States
S
* Supporting Information
ABSTRACT: Cyclopropenethiones are reported as new
bioorthogonal reagents. These motifs react readily with
substituted phosphines to provide thiocarbonyl adducts. In
some cases, the ligations are >300-fold faster than analogous
reactions with bioorthogonal cyclopropenones. Dialkyl cyclo-
propenethiones are also stable in aqueous buffers and can be
used for biomolecule labeling in vitro and in cell lysate. The
rapid reactivity and biocompatibility of cyclopropenethiones suggest that they will be useful probes for cellular studies.
ioorthogonal chemistries have enabled a broad range of
B
applications in living organisms,1,2 including biomolecule
imaging,3 metabolic profiling,4−6 and targeted drug delivery.7,8
Despite their ubiquity in numerous fields, these reactions are
not without limitation. Only a handful of bioorthogonal
reagents are reliable in the most demanding environments,
including inside cells. Additionally, many bioorthogonal probes
cross-react with one another, precluding dual imaging and
other multicomponent studies.9 These and other applications
demand new reagents and new reactions.
Our laboratory has focused on expanding the scope of
bioorthogonal chemistry by developing probes that are small,
stable, and tunable.10 We recently reported one such class
of reagents, cyclopropenones.11,12 These motifs are stable in
biological media and react robustly with ortho-substituted
phosphines. The ligation involves initial formation of a reactive
ketene-ylide, followed by intramolecular trapping (Figure 1A).
The resulting products are stable in cellular environments. The
unique mechanism of the cyclopropenone-phosphine ligation
renders it compatible with many classic bioorthogonal
reagents.12 Cyclopropenones are also small and innocuous to
cellular enzymes and metabolic pathways.12 Dialkyl-substituted
scaffolds, in particular, are well suited for time intensive
studies. We used these probes in long-term cultures for site-
specific protein modification.12 The most stable cyclo-
propenones, though, required lengthy reaction times with
bioorthogonal phosphines. Faster rates could be achieved with
monosubstituted scaffolds, but these probes were more
susceptible to side reactions with biological nucleophiles.
We hypothesized that cyclopropenone heteroanalogs could
strike the right balance between kinetic stability and rapid
reactivity. We were particularly drawn to cyclopropenethione
(CpS) scaffolds. CpS differs from cyclopropenone (CpO) by
only a single atom and would thus likely be small enough to
avoid perturbing target biomolecules or pathways. CpS is also
known to participate in a variety of transformations, including
photocleavage reactions13,14 and ring-opening cycloaddi-
Figure 1. Bioorthogonal ligations of cyclopropenones and cyclo-
propenethiones. (A) Cyclopropenones (CpO, X = O) react with
phosphines to form ketene-ylides. These intermediates can be trapped
with pendant nucleophiles to afford stable adducts. Cyclopropene-
thiones (CpS, X = S) were hypothesized to react similarly with
phosphine probes. (B) CpS scaffolds harbor lower LUMO energies
than analogous CpO probes. Density functional theory (DFT)
calculations were performed with Spartan, using the B3LYP level of
theory and basis set 6-31G*.
tions.15,16 Previous work by Yoneda and Matsumura further
revealed that triarylphosphines can react with diphenyl CpS via
nucleophilic addition to the alkene.17,18 Based on this
precedent, we surmised that CpS derivatives could react with
substituted phosphines (similar to CpO probes), ultimately
providing thiocarbonyl products (Figure 1A). Thiocarbonyls
are widely used in fluorescence quenching and other
Received: July 21, 2018
© XXXX American Chemical Society
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DOI: 10.1021/acs.orglett.8b02296
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Organic Letters
Letter
biophysical experiments with proteins,19,20 suggesting that they
too are stable and biocompatible.21,22 Thioamides, in
particular, are known to exhibit improved proteolytic stabilities
and can increase peptide half-lives in biological media.23
To evaluate the proposed phosphine ligation, we first
examined the frontier molecular orbitals of CpS derivatives.
Density functional theory (DFT) calculations revealed that the
LUMO values for CpS scaffolds were consistently lower than
those for analogous CpO molecules (Figure 1B). The reduced
LUMO values suggested the potential for increased reactivity
with phosphines. Whether or not CpS molecules would also be
susceptible to rapid reactions with thiols or other biological
nucleophiles remained to be determined.
We synthesized a panel of CpS compounds to examine their
suitability as bioorthogonal reagents. A symmetric dialkyl CpS
(3a) was included for ease of product characterization. We also
prepared scaffolds bearing aryl substituents (3b−c) and water-
solubilizing groups (3d). All of the desired cyclopropene-
thiones were prepared via the corresponding cyclopropenones
(2a−d, Scheme 1). Alkynes 1a−d were first treated with
Figure 2. Cyclopropenethiones are stable to L-glutathione (GSH) at
physiological pH. Cyclopropenethione 3d (5 mM) was incubated
with GSH (5 mM) in d-PBS (pH 7.4) and monitored by H NMR
1
spectroscopy.
Scheme 2. Cyclopropenethiones Exhibit Faster Ligation
Rates than Analogous Cyclopropenones
Scheme 1. Synthesis of Model Cyclopropenethiones
contrast, CpS 3d reacted sluggishly with thiol-substituted
phosphine 5. The reduced rate may be attributed to increased
steric congestion at the reactive center or decreased phosphine
nucleophilicity.12,26 Dithioester products were also initially
detected in the reaction of 3d with 5 (via NMR spectroscopy),
but complex mixtures ultimately formed. It is likely that the
dithioesters reacted further with CpS itself.
difluorocarbene to generate 3,3-difluorocyclopropenes.24
These intermediates were then hydrolyzed to provide 2a−d.
The desired cyclopropenethiones were ultimately obtained by
treating 2a−d with Lawesson’s reagent. We also attempted to
prepare monosubstituted CpS probes. However, these scaffolds
were not stable to concentration (data not shown), and were
likely subject to intermolecular attack.
To form more stable thioamides, we evaluated CpS
reactivity with amine-substituted phosphine 6. This probe
did not react with 3d, though, even at elevated concentrations.
Similarly poor reactivity was observed with the analogous CpO
probe.12 Rapid CpO-phosphine ligations (in organic solvents)
require hydrogen-bond activation (Figure S8). Thus, we
hypothesized that stronger hydrogen-bond donors could
boost reactivity with CpS probes. Indeed, when phosphine 7
(bearing a nitro group para to the amine) was incubated with
3d, ligation products were observed within a few hours. The
lowered pKa of the pendant amine promotes reactivity, despite
deactivating the phosphine via inductive effects. More
nucleophilic alkylphosphine probes also enabled efficient
production of thioamides and thionoesters (Table 1 and
Figure S9). Surprisingly, 3d reacted slower with phosphine 8
than phosphine 4. The opposite trend was observed with CpO
analogs.12 It is possible that the CpS ligation is more
dependent on initial hydrogen-bond activation. These data
further imply that CpO and CpS, despite their structural
similarity, might be useful probes for orthogonal labeling
applications.
With the CpS scaffolds in hand, we first evaluated their
stabilities in aqueous solution. Compounds 3b−d were
1
incubated in d-PBS (pH 7.4) and monitored by H NMR
spectroscopy (Figures S1−S3). Probes 3b and 3d were stable
for >1 week at 37 °C. The diaryl scaffold 3c degraded, resulting
in an insoluble and unidentified precipitate. CpS 3c may be
susceptible to dimerization or intermolecular reactivity over
prolonged time periods.13,25 The stabilities of 3b and 3d
toward biological nucleophiles were further examined. The
molecules were incubated with L-glutathione (GSH) in d-PBS
(pH 7.4) and monitored via 1H NMR spectroscopy (Figures 2
and S4−S5). No reactivity was observed over 24 h at 37 °C,
indicating that both scaffolds were stable to thiols.
Encouraged by the biocompatibility of dialkyl CpS scaffolds,
we investigated their reactivities with a panel of phosphines.
Symmetric analog 3a was initially used to determine the
structure of the ligated adduct. When 3a was incubated with
phosphine 4, thionoester S5 was formed in high yield (Scheme
S1). The expected thionoester was also observed upon treating
3d with phosphine 4. Importantly, this latter reaction
proceeded ∼300-fold faster than the analogous CpO-
phosphine ligation (Scheme 2 and Figure S6). Such rapid
reactions are desirable for live cell and tissue studies, where
only small amounts of reagents are typically tolerated. By
The reactivity and stability profiles of CpS probes suggested
that dialkyl variants would be suitable for biomolecule labeling.
To assess the efficiency of the CpS-phosphine ligation in this
context, we performed reactions on a model protein. Hen egg-
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Table 1. Cyclopropenethione Reactivity with Phosphine
Probes
a
b
Products were not stable to isolation conditions. Multiple addition
products were observed (see Figure S7).
white lysozyme was nonspecifically functionalized with dialkyl
CpS motifs. Mass spectrometry revealed 1−5 CpS units were
appended to the protein surface (Figures 3 and S10). The
derivatized protein (Lys-CpS) was then treated with
phosphine-biotin probe 9. This reagent was previously used
to tag CpO-protein conjugates.12 Based on reactivity data from
above , we anticipated that 9 could also efficiently ligate CpS-
modified proteins to provide stable thioamide products.
Indeed, rapid and quantitative conversion to the expected
adducts was observed (Figures 3B and S11). The resulting
conjugates were stable for >1 week at 37 °C (Figure S12).
Additionally, minimal phosphine oxidation was observed upon
product formation, consistent with previous reports on CpO
reactivity.11,12
Reactions with CpS scaffolds were also faster than those
carried out with analogous cyclopropenones. Lysozyme
samples functionalized with CpO or CpS (Lys-CpO or Lys-
CpS, respectively) were prepared as above. Mass spectrometry
confirmed that both conjugates were modified to a similar
extent (Figure S10). The proteins were then treated with
phosphine 9 and analyzed via Western blot. As shown in
Figure 3C, shorter reaction times and lower phosphine
concentrations were required to achieve robust labeling with
Lys-CpS. Adducts were observed within minutes, and
reactions were complete by 30 min. By contrast, reactions
with Lys-CpO required ∼1 h to observe initial covalent
adducts.
We further examined the CpS-phosphine ligation in a more
complex setting. Probes compatible with cells and intracellular
environments are often in high demand.1,27 To mimic the
cellular milieu, Lys-CpS was incubated with freshly prepared
cell lysate (Figure 3D). The samples were then treated with
phosphine 9 and analyzed via Western blot as described above.
Figure 3. Cyclopropenethione conjugates are readily ligated in vitro
and in cell lysate. (A) Lys-CpS samples were treated with a
phosphine-biotin probe. (B) Lys-CpS (20 μM, 1−5 modifications)
was incubated with phosphine-biotin probe 9 (500 μM), and the
reaction was monitored by ESI mass spectrometry. Deconvoluted
mass spectra are shown. Full conversion to the expected products
(Lys-Phos, 1−5 modifications) was observed. (C) Lys-CpS and Lys-
CpO (20 μM) were reacted with phosphine 9 (50 μM) for 0−60 min
at 37 °C. Covalent adducts were detected via Western blot with
streptavidin staining (top). Coomassie staining was used to assess
protein loading (bottom). (D) Lys-CpS (20 μM) was incubated with
9 (1 mM) in bacterial lysate for 1 h at 37 °C. Covalent adducts were
detected via Western blot with streptavidin staining (top). Coomassie
staining was used to assess protein loading (bottom). *Protein
impurities present in commercial lysozyme stock.
Rapid and selective labeling was observed after just 1 h. Larger
concentrations of 9 were required to achieve robust ligation,
likely due to competing phosphine oxidation pathways. The
lack of background reactivity observed, though, demonstrates
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In summary, we found that substituted cyclopropenethiones
are viable bioorthogonal reagents. These scaffolds are stable in
aqueous buffers and in the presence of biological nucleophiles.
Cyclopropenethiones differ from cyclopropenones by a single
atom, but react ∼300-fold faster with some phosphines.
Biocompatible probes with improved reaction rates are
welcome additions to the bioorthogonal toolkit. CpS-
phosphine ligations also proceed readily in biological buffers
and cell lysate. Future experiments will examine compatibilities
with live cells. Cyclopropenethiones are subject to react with
strong electrophiles and may require further tuning for
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thiones also exhibit distinct reactivity profiles compared to
other bioorthogonal reagents, including cyclopropenones,
suggesting opportunities for multicomponent studies. An
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ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.8b02296.
Experimental details, spectroscopic data for new
compounds, and additional images (PDF)
(23) Chen, X.; Mietlicki-Baase, E. G.; Barrett, T. M.; McGrath, L. E.;
Koch-Laskowski, K.; Ferrie, J. J.; Hayes, M. R.; Petersson, E. J. J. Am.
Chem. Soc. 2017, 139, 16688.
(24) Wang, F.; Luo, T.; Hu, J.; Wang, Y.; Krishnan, H. S.; Jog, P. V.;
Ganesh, S. K.; Prakash, G. K. S.; Olah, G. A. Angew. Chem., Int. Ed.
2011, 50, 7153.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: jpresche@uci.edu.
ORCID
̈
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(26) Soellner, M. B.; Nilsson, B. L.; Raines, R. T. J. Am. Chem. Soc.
2006, 128, 8820.
Jennifer A. Prescher: 0000-0002-9250-4702
Notes
(27) Shih, H.-W.; Kamber, D. N.; Prescher, J. A. Curr. Opin. Chem.
Biol. 2014, 21, 103.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the U.S. National Institutes of
Health (R01 GM126226 to J.A.P.) and the Alfred P. Sloan
Foundation (J.A.P.). R.D.R. was supported by an NSF
Graduate Research Fellowship. We thank the Heyduk, Nowick,
and Chamberlin laboratories for providing reagents and
equipment. We also acknowledge Philip Dennison (UCI) for
assistance with NMR experiments, Benjamin Katz (UCI) and
Felix Grun (UCI) for assistance with mass spectrometry
̈
experiments, and members of the Prescher laboratory for
manuscript edits and helpful discussions.
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