Cyclopropenones for Metabolic Targeting and Sequential Bioorthogonal Labeling

Article abstract of DOI:10.1021/jacs.7b03010

Cyclopropenones are attractive motifs for bioorthogonal chemistry, owing to their small size and unique modes of reactivity. Unfortunately, the fastest-reacting cyclopropenones are insufficiently stable for routine intracellular use. Here we report cyclop

Full text of DOI:10.1021/jacs.7b03010

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Article
Cyclopropenones for metabolic targeting
and sequential bioorthogonal labeling
R. David Row, Hui-Wen Shih, Austin T. Alexander, Ryan A. Mehl, and Jennifer A. Prescher
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Journal of the American Chemical Society
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Cyclopropenones for metabolic targeting and sequential bioorthogo-
nal labeling
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R. David Row,† Hui-Wen Shih,† Austin T. Alexander,# Ryan A. Mehl,# and Jennifer A.
Prescher*†,‡,§
†Departments of Chemistry, ‡Molecular Biology & Biochemistry, and §Pharmaceutical Science, University of Cali-
fornia, Irvine, California 92697, United States
#Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331, United States
ABSTRACT: Cyclopropenones are attractive motifs for bioorthogonal chemistry, owing to their small size and unique
modes of reactivity. Unfortunately, the fastest-reacting cyclopropenones are insufficiently stable for routine intracellular
use. Here we report cyclopropenones with improved stability that maintain robust reactivity with bioorthogonal phos-
phines. Functionalized cyclopropenones were synthesized and their lifetimes in aqueous media and cellular environments
were analyzed. The most robust cyclopropenones were further treated with a panel of phosphine probes, and reaction
rates were measured. Two of the phosphine scaffolds afforded ~100-fold rate enhancements compared to previously re-
ported reagents. Importantly, the stabilized cyclopropenones were suitable for recombinant protein production via genet-
ic code expansion. The products of the cyclopropenone ligation were also amenable to traceless Staudinger ligations, set-
ting the stage for tandem labeling experiments.
other,14-16 precluding multi-component imaging experi-
INTRODUCTION
ments. Thus, despite decades of work on bioorthogonal
reaction development, few reactions can be used relia-
The bioorthogonal chemical reporter strategy ranks
bly—and in combination—in living systems.
among the most powerful and popular methods for label-
ing biomolecules in their native environments (Figure 1).1,2
This strategy exploits cellular enzymes to incorporate
non-natural metabolites (endowed with unique chemical
functional groups or “reporters”) into target biomolecules.
In a second step, the reporters are selectively ligated with
Figure 1. The bioorthogonal chemical reporter strategy. First,
a non-natural precursor (small rectangle) bearing a chemical
reporter (circle) is metabolically introduced into a target
biomolecule (large rectangle). In a second step, the chemical
reporter can be covalently labeled with a complementary
chemical probe (arc) via a bioorthogonal reaction.
complementary functional groups bearing detectable
probes. Depending on the nature of the probe, this two-
step approach can be used to visualize and isolate a broad
range of biomolecules, including glycans,2-4 lipid metabo-
lites,5,6 and proteins.7 Recent applications have uncovered
new functional roles for post-translational modifica-
tions,8,9 mechanisms of cell wall assembly,10,11 and cell-
specific proteomes.12,13
To address these limitations, we are identifying more
stable and small reporters for cellular use. Included in this
group are cyclopropenes17,18 and triazines.19 These motifs
are small enough to target a variety of biomolecules in
cells, and can be selectively detected via biocompatible
cycloadditions. More recently, we turned our attention to
cyclopropenones. These motifs are also small and thus
appealing for general use. Cyclopropenones are found in
some natural products,20-22 underscoring their biocompat-
While the bioorthogonal chemical reporter strategy
continues to reveal new facets of biology, limitations re-
main. Many of the requisite reagents are not completely
inert to cysteine or other biological nucleophiles in cells
and tissues. Some scaffolds are also too big to traverse
cellular metabolic pathways. Another limitation is that
several bioorthogonal reagents cross-react with one an-
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Page 2 of 8
ibility and potential metabolic stability. They are also ro- expense of rapid reactivity, we also investigated a panel of
bustly reactive with triaryl phosphines,23,24 reagents that
have been used extensively for bioorthogonal Staudinger
ligation.4,25 The cyclopropenone-phosphine reaction pro-
ceeds through a ketene-ylide;23 this intermediate can be
trapped by a variety of nucleophiles (Figure 2). When the
nucleophile is attached to the phosphine itself, tethered
adducts are formed.
phosphine reagents to tune the kinetics of the ligation.
Cyclopropenones with improved stability were identified,
along with phosphines with markedly improved reaction
rates. These motifs are suitable for intracellular work and,
based on the unique products formed, can be used for
sequential labeling experiments.
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RESULTS AND DISCUSSION
Previous work
We observed that mono-substituted cycloprope-
nones, while robustly reactive with phosphines, are sus-
ceptible to background reactions with thiols in cellular
environments. To identify more stable cyclopropenones,
we synthesized a panel of di-substituted variants bearing
either alkyl or aryl appendages (Scheme 1). Many of these
scaffolds possessed higher LUMO energies (Figure S1) and
extra steric bulk to mitigate against thiol attack. As bulky
substituents would also likely impede phosphine addi-
tion, cyclopropenones with electron-withdrawing charac-
ter were also included to more precisely “tune” the reac-
tivity. Density functional theory (DFT) calculations (Fig-
ure S1) suggested that cyclopropenones bearing methox-
ymethyl or monofluoromethyl groups (as in compounds 5
or 7) might provide the appropriate balance between thiol
stability and phosphine reactivity. The LUMO energies
for these compounds fell between those of the dialkyl
(3a–b) and monoalkyl (8) scaffolds.
Nu
O
O
O
Nu
C
PPh2
H
Nu
H
PPh2
PPh2
H
cyclopropenone phosphine
carbonyl product
keteneꢀylide intermediate
This work
O
O
O
O
NH
CH3
2
Nu
R
PPh2
4
H2N
COOH
metabolic
reporter stability
R = Me, Ph, EWG
rate optimization
incorporation
Nu = OH, NH2, SH
Figure 2. Previous work in our lab demonstrated that triaryl
phosphines react efficiently with cyclopropenones to form
ketene-ylides. These intermediates can be trapped via teth-
ered nucleophiles to afford stable adducts. In this work, cy-
clopropenone stability, reactivity, and cellular use were in-
vestigated.
We previously showed that the unique features of
mono-substituted cyclopropenones could be exploited for
biomolecule labeling in aqueous conditions.23 The motifs
were appended to model proteins in vitro and selectively
targeted with phosphine-fluorophore conjugates.23 While
the mono-substituted cyclopropenones were observed to
react robustly with phosphines, they were susceptible to
reaction with cysteine and other biological thiols at pH >
7. Such side reactivity limits their utility for intracellular
imaging and other applications.
The desired cyclopropenones were prepared via a
common route involving difluorocarbene addition to al-
kyne precursors (Scheme 1).23 To access the water-soluble
cyclopropenones 3a and 3b (Scheme 1A), 5-hexynoic acid
and 6-heptynoic acid were first isomerized and amidated.
The resulting alkynes were then reacted with difluorocar-
bene (following the procedure of Olah et al.28) to form 2a–
b. These intermediates spontaneously hydrolyzed to the
corresponding cyclopropenones (3a–b) upon aqueous
workup. The yield of 3a was quite low (< 10%), as this
compound was unstable. By contrast, cyclopropenone 3b
was isolated in reasonable yield and was stable to concen-
tration. These observed differences may be attributed to
the shortened tether in compound 2a and undesirable
intramolecular reactivity (Figure S2).29
To optimize the cyclopropenones for intracellular
use, we aimed to identify scaffolds with improved stability
(Figure 2). We focused on di-substituted cyclopropenones
for this purpose, as previous observations by our lab and
others23,26,27 suggested that these reagents were less prone
to thiol attack. As improved stability often comes at the
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Scheme 1. Synthesis of model cyclopropenones.
1
2
3
4
5
6
O
A
1. KOtBu
DMSO, 75 °C
F
F
O
O
O
H2O
TMSCF3, NaI
THF, 23 °C
O
N
n
O
2. EDC, morpholine
CH2Cl2, 23 °C
OH
H3C
N
n
H3C
N
n
n
H3C
O
O
n = 2, 3
1a (n = 2) 61%
1b (n = 3) 66%
2a (n = 2)
2b (n = 3)
3a (n = 2)
3b (n = 3) 35%
7
8
9
O
B
TMSCF3, NaI
MeI, nꢀBuLi
CH3
MeO
MeO
CH3
CH3
THF, –78 °C
THF, 80 °C
then SiO2 (neat)
4
6
5
7% (2 steps)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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CH3
HO
O
1. MsCl, Et3N
THF, 0 °C
TMSCF3, NaI
CH3
F
F
2. CsF
i-PrOH, reflux
THF, 80 °C
then SiO2 (neat)
7 10% (3 steps)
were included for comparison. All compounds were dis-
solved in phosphate-buffered solutions (pH 7.4) and
analyzed via NMR spectroscopy (Table 1). After 1 week,
most of the cyclopropenones showed no signs of degra-
dation (Figures S3–S6), even at elevated temperatures.
One exception was diphenylcyclopropenone 10, which is
known to be photolytically unstable.23,30 We also exam-
ined cyclopropenone stabilities in the presence of thiols.
Table 1. Cyclopropenone stability.
O
t1/2 (h)
R
R2
R
1
1
Cyclopropenone
Conditions
– Cys
N.R.*
+ Cys
< 0.2
O
R1 = H
R2
dꢀPBS
pH 7.4, 37 °C
8
N
=
3
O
The compounds were incubated with L-cysteine or glu-
R1 = CH2OCH3
R2 = hexyl
20% CD3CN / dꢀPBS
tathione and monitored at room temperature or at 37 °C
(Figures S7–S15). While the methoxymethyl (5) and
monofluoromethyl (7) cyclopropenones were stable in
aqueous solution, they were found to react rapidly with
cysteine (Figures S7-S8), making their use in cellular
contexts impractical. The half-lives of most cycloprope-
none scaffolds, in general, were < 12 h. However, cyclo-
propenone 3b remained inert to cysteine over a 24-hour
period, even at elevated pH (Figures S12–S14) and tem-
perature. Similar trends were observed with glutathione
(Figure S15). These data suggested that the dialkyl-
substituted scaffold was an excellent candidate for in
cellulo work.
5
7
9
N.R.
N.R.
N.R.
3 d*
~1.5
< 0.2
~12.5
< 0.2
pH 7.4, 37 °C
R1 = CH2F
R2 = hexyl
20% CD3CN / dꢀPBS
pH 7.4, 37 °C
R1 = CH3
R2 = Ph
20% CD3CN / dꢀPBS
pH 7.4, 23 °C
R1 = Ph
20% CD3CN / dꢀPBS
10
R
2 = Ph
pH 7.4, 23 °C
O
R1 = CH3
dꢀPBS
pH 7.4–8.4, 37 °C
3b
N
N.R.
N.R.
3
R2
=
O
*As reported in ref. 23
The remaining cyclopropenones were prepared in a
similar fashion (Scheme 1B). In brief, methoxymethyl
cyclopropenone 5 was accessed by methylating 2-nonyn-
1-ol prior to forming the difluorocyclopropene. Interest-
ingly, this intermediate did not hydrolyze to the corre-
sponding cyclopropenone upon aqueous workup. Com-
plete conversion was only realized upon prolonged ex-
posure to silica gel. To access the fluorinated cyclopro-
penone 7, 2-nonyn-1-ol was first mesylated. Subsequent
SN2 displacement provided scaffold 6. This alkyne was
ultimately converted to the cyclopropenone 7 as previ-
ously described.
Encouraged by the enhanced stability of 3b, we pro-
ceeded to investigate its reactivity with a panel of phos-
phines (Figure 3). The panel included triaryl phosphines
bearing tethered nucleophiles, such as alcohols (11),
amines (12, 13), and thiols (14), along with alkyl phos-
phine variants (15, 16). Some of these probes (11–13) were
previously shown to react with cyclopropenones and
efficiently trap ketene-ylide intermediates.23 These
phosphines also had the potential to afford a variety of
covalent linkages (e.g., esters, amides, and thioesters)
for biomolecule derivatization. We hypothesized that
scaffolds with more electron-donating substituents
would boost phosphine nucleophilicity and thus in-
crease reaction rates. Similarly, alkyl phosphine probes
were expected to provide enhanced reactivity.
With the desired cyclopropenones in hand, we
monitored their stabilities under physiological condi-
tions. Phenyl-substituted cyclopropenones 9 and 10
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A
O
O
O
O
O
O
Ph2P
Nu
1
2
3
benzeneꢀd6
23 o
N
Nu
Nu
N
R
N
C
O
PPh2
PPh2
O
O
R
R
3b, 8
11–18
4
5
6
7
8
B
NO2
NO2
Ph2P
HO
Ph2P
H2N
Ph2P
H3CHN
Ph2P
HS
Ph2P
HO
Ph2P
H2N
Ph2P
HO
Ph2P
H2N
k2
(Mꢀ1sꢀ1)
(±)
11
12
13
14
≤ 10–5
9.3 ± 0.4 x 10–2
15
16
17
18
9
3b
1.9 ± 0.2 x 10–2 2.3 ± 0.2 x 10–4 1.6 ± 0.1 x 10–4
*
**
10
11
12
13
14
15
16
17
18
19
20
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23
24
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0.34 ± 0.06
≥ 20*
2.2 ± 0.3 x 10–2
≥ 20*
2.1 ± 0.3 x 10–2
≥ 20*
–
(R = CH3)
8
4.2 ± 0.6 x 10–2
0.31 ± 0.07
0.18 ± 0.05
≥ 20*
(R = H)
Figure 3. Kinetic experiments to analyze cyclopropenone and phosphine reactivity. A) Reactions were performed in benzene-d6
and monitored via NMR. Two products were formed, corresponding to phosphine addition at either side of the cyclopropenone
ring. B) Second order rate constants for reactions between mono- and di-substituted cyclopropenones and a panel of phosphine
probes. Errors are given as the standard deviation for n = 3 experiments. For 12, 16, and 18, multiple addition products were ob-
served late in the reactions (see Figure S18). *Reactions were too fast or too slow to measure via NMR. **Upon mixing 3b and 18,
an unidentified precipitate formed, preventing accurate rate measurements.
When cyclopropenones 3b and 8 were treated with
the phosphine panel, the expected trends in reactivity
were observed. Reactions with the di-substituted scaf-
fold (3b) proceeded more slowly than reactions with the
mono-substituted analog 8 (Figures 3, S16, and S17). In-
creased reaction rates were achieved with cyclohexyl
phosphines 15–16 compared to their triaryl counterparts
11-14. It should be noted that the enhanced nucleophilic-
ity of 15 and 16 also renders these phosphines more sus-
ceptible to oxidation. However, only minimal levels of
oxidation were observed in ligations performed with the
cyclohexyl variants (<5% observed by NMR). The unu-
sual stability of phosphine 16 has been previously re-
ported,31 and related scaffolds are known to be stable,32
suggesting their feasibility in biological experiments.
explanation for the rate differential is that phosphines 11
and 15 activate the cyclopropenone for attack via an in-
termolecular hydrogen bond (Figure 4A). Dialkylcyclo-
propenones are known to readily form hydrogen bonds
with phenol.33 The pKa values (in DMSO) for phenol
(pKa = 18)34 and aniline (pKa = 31)35 further suggest that
11 should be a better hydrogen bond donor than 12–13.
Indeed, when 11 was reacted with 3b, the rate increased
with additional equivalents of exogenous phenol (Fig-
ures 4B and S19). We further measured rates with phos-
phines 17 and 18. These scaffolds comprise hydrogen
bond donors and para-nitro groups. We hypothesized
that these phosphines would provide increased reaction
rates as a result of stronger hydrogen bonding. Howev-
er, the rate of the reaction between 17 and 3b was nearly
identical to that of 11 and 3b. One potential explanation
is that the rate increase achieved by lowering the pKa of
17 is offset by a decrease in phosphine nucleophilicity
(due to the electron-withdrawing nitro group). For ani-
line phosphine 18, a noticeable decrease in rate was ob-
served. Phosphine 18 does not likely provide for a strong
hydrogen bond. The pKa values for phenol and p-
nitroaniline (18.0 and 20.9, respectively)34,35 support this
hypothesis.
0.12
A
B
0.1
0.08
0.06
0.04
0.02
0
O
H
O
PPh2
R
R²2=
0.9.99866
= 0 98
R
R
R
1
hydrogen bond
activation
0
0.2
0.4
0.6
0.8
1
1.2
Equiv. phenol
Figure 4. Hydrogen bonding enhances cyclopropenone
reactivity. A) Hydrogen bonding can position the phospho-
rus atom near the vinyl carbon. B) Reaction rates between
3b and 11 increase with additional equivalents of phenol.
To showcase the stability and reactivity of the dial-
kyl cyclopropenone scaffold, we investigated its use for
intracellular biomolecule labeling. We aimed to site-
specifically target proteins with cyclopropenones via
genetic code expansion. Toward this end, we synthe-
sized a non-canonical amino acid for in cellulo experi-
ments (Scheme 2). Briefly, 3-pentyn-1-ol was protected
with tetrahydropyran (THP) to afford 19. The alkyne was
A surprising observation was made upon treating
cyclopropenone 3b with phosphine phenol 11. These
reactions were ~100 times faster than reactions with the
corresponding anilines (12, 13). Similar trends were ob-
served with cyclohexyl phosphines 15–16. One possible
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hibited significant toxicity in bacteria, likely due to cel-
then subjected to the aforementioned carbene insertion
and hydrolysis sequence to provide 20. The acid-labile
THP group was removed to give alcohol 21. This inter-
lular interference from the TFA counterion.36 Subse-
quent deprotections were carried out using hydrochloric
acid in dioxane. The chloride salt (24b) was compatible
with cells (data not shown). Importantly, the cyclopro-
penone amino acids were also stable in physiological
buffers and in the presence of cysteine (Figures S20-S21).
1
2
3
4
5
6
7
mediate was then appended to Boc-L-lysine. To remove
the Boc group and liberate the desired amino acid,
compound 23 was initially subjected to trifluoroacetic
acid (TFA). The isolated trifluoroacetate salt (24a) ex-
8
9
Scheme 2. Synthesis of cyclopropenone-functionalized amino acid.
1. TMSCF3, NaI
THF, 23 °C
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
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58
59
60
O
O
OH
O
O
Amberlyst–15
MeOH, 23 °C
DHP, TsOH
H3C
MeOH, 23 °C
H3C
2. H2O
H3C
O
O
O
H3C
OH
19 80%
20 68%
21 96%
O
O
OH
1. Boc–
L
–Lys–OH
NO2
O
O
pyridine, DMF, 23 °C
p–nitrochloroformate
R
NH3
H3C
O
N
H3C
O
O
2. TFA : CH2Cl2 (1:1), 23 °C
or
4 N HCl in dioxane, 23 °C
pyridine, CH2Cl2, 23 °C
H
22 77%
24a, (R = trifluoroacetate)
24b, (R = chloride)
32% (2 steps)
33% (2 steps)
O
A
O
R
H
N
H
N
N
H
HN
O
CH3
O
O
PPh2
25
4
4
O
O
siteꢀspecific
O
genetic
bioorthogonal
reaction
O
incorporation
N
R
H2N
COOH
24b
GFP-Cpo
GFP-Cpo-Phos
PPh2
H3C
CH3
H
O
O
O
N
H
O
R =
N
H
O
O
N
H
NH
H
S
s
it
e
ꢀs
p
e
c
if
i
GFP-Cpo (µg)
Lysate (30 µg)
25 (100 µM)
c
2
–
+
2
–
–
–
+
–
–
+
+
1
+
+
2
+
+
1000
B
D
E
[24b] 0 mM
[24b] 0 mM
[24b] 1 mM
GFP-Lys
5.04E+07
100
800
600
400
200
0
[24b] 1 mM
27979.0 ± 1 Da
GFP-Cpo
–Met
s
i
te
ꢀ
s
p
e
c
i
f
GFP-Cpo
i
c
0.00E+00
0
27500
28000
28500
29000
A1
D1
E1
F1
G1
1.39E+06
100
kDa
28762.3 ± 1 Da
GFP-Cpo-Phos
pBAD vector
Amino acid
+TAG +TAG +TAG WT
C
75
37
25
PCK 24b
–
–
37 kDa
s
i
te
ꢀ
s
p
e
c
i
f
GFP-Cpo
i
c
0.00E+00
0
27500
28000
28500
29000
15
10
25 kDa
lane
27,500
28,000
28,500
29,000
1
2
3
4
5
Mass (Da)
Figure 5. Cyclopropenones can be used for recombinant protein production. A) Overall strategy for incorporating non-canonical
amino acid 24b into GFP via amber codon suppression. GFP-Cpo was targeted with phosphine probes. B) A library of PylRS mu-
tants was screened to identify synthetases compatible with 24b. C) GFP-Cpo was expressed in the presence of 24b. Minimal GFP
expression was observed in the absence of 24b. D) ESI-Q-TOF mass spectrometry analysis of the genetic incorporation and reac-
tivity of cyclopropenone 24b. Reactions between GFP-Cpo (30 µM) and phosphine 25 (500 µM) were performed in PBS (pH 7)
at 37 °C. Quantitative conversion was observed. E) The cyclopropenone-phosphine ligation enables sensitive imaging in com-
plex mixtures. Bacterial cell lysate containing GFP-Cpo (1-2 µg) was treated with phosphine-biotin 25 (100 µM) for 1 h at 37 °C.
Covalent adducts were detected via Western blot with avidin staining (above). Protein loading was assessed with Coomassie
staining (below).
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With the amino acid in hand, we needed to identify We further identified conditions to ligate cyclopro-
1
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an appropriate non-canonical aminoacyl-tRNA synthe-
tase (ncAA-RS)/tRNA pair for genetic incorporation.
The ncAA-RS/tRNA pair is responsible for installing the
unnatural amino acid in response to a single codon (in
this case, TAG, the amber stop codon). Frequently, a
compatible ncAA-RS can be identified via screening of
existing synthetase libraries that have been previously
selected for structurally similar ncAAs (Figure S22).
When successful, this strategy of using permissive
ncAA-RSs avoids the need for evolving a new mutant
from wild-type AARSs. We used such existing libraries
to identify ncAA-RS mutants that were permissive to the
cyclopropenone amino acid 24b (Figure S23).
penone-modified proteins with phosphine probes. GFP-
Cpo was incubated with various concentrations of
phosphine-biotin 25 in PBS (pH 7) at 37 °C. Dose- and
time-dependent labeling was observed via Western blot
(Figure S29), and reactions appeared complete within 1
h (using 50 µM phosphine-biotin). The identity of the
ligated product was confirmed using mass spectrometry
(expected 28763.4 Da; observed 28762.3 ± 1 Da, Figure
5D). Successful ligation of GFP-Cpo was also achieved
using triaryl phosphine probe S13 (Figure S28).
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As a testament to the exquisite selectivity of the cy-
clopropenone-phosphine reaction, GFP-Cpo was suc-
cessfully ligated in complex mixtures. Micromolar con-
centrations of phosphine-biotin 25 and short labeling
times (1 h) were sufficient to detect GFP-Cpo in cell
lysate (Figure 5E), with no background labeling ob-
served (Figure 5E). Overall, these data suggest that cy-
clopropenone non-canonical amino acids will be suita-
ble for genetic code expansion and other applications in
the most complex biological settings.
We screened a panel of 96 previously selected pyr-
rolysine Methanosarcina barkeri amino acyl-tRNA syn-
thetases/(Mb)tRNACUA pairs (ncAA-PylRS/tRNA) for
permissivity toward 24b, while maintaining their fidelity
against native amino acids.18,37 Two PylRS/tRNA pairs
that had been previously selected for photoprotected
lysine (PPK) and photoprotected ornithine (PPO) deriv-
atives were able to incorporate 24b (Figures 5B and S22).
It is important to note that the two PylRS/tRNA mu-
tants (D1 and G1) harbor Cys at the active site, highlight-
ing again the biocompatibility of the cyclopropenone
motif.
Scheme 3. Sequential cyclopropenone-phosphine
and traceless Staudinger ligations.
O
O
Ph2P
HO
H3C
N
benzene, 23 °C
3
O
O
While the efficiency of incorporation was low com-
pared to other ncAA-PylRS/tRNA pairs, G1-RS was capa-
ble of incorporating 24b in response to an amber codon-
disrupted GFP gene (GFP-150-TAG), resulting in expres-
sion of GFP-Cpo (Figure 5C, lane 3, and Figures S24–
S25). We also found that only a single mutation (Y306T)
in G1-RS was necessary to facilitate 24b incorporation
and GFP-Cpo production (Figure S26). In all cases,
minimal GFP was translated when 24b was withheld
(Figure 5C, lane 4 and Figures S24-S26). To verify that
24b was stable in complex media and could be incorpo-
rated into recombinant proteins, we compared the
masses of GFP-Cpo to GFP-WT and GFP-PPK using
ESI-Q-TOF mass analysis. Native GFP (GFP-WT) exhib-
ited the expected mass of 27828.9 ± 1 Da and GFP-Cpo
showed the expected mass increase to 27979.0 ± 1 Da,
verifying that 24b is installed at a single site (Figure 5D).
Lysine incorporation was also detected in the GFP-Cpo
sample, perhaps due to carbamate cleavage (Figure S27)
or reduced synthetase fidelity (i.e., canonical lysine was
incorporated in response to the amber codon). Lysine
incorporation was also observed when G1-RS was used
to install PPK or PPO into GFP-150-TAG, (Figures S24
and S28).
3b
11
O
O
O
N
N
N3
Ph
O
Ph
N
3
O
3
O
H
DMF (5% H2O)
23 °C
PPh2
26a + regioisomer 26b 68%
27a + regioisomer 27b 91%
As noted earlier, the unique mechanism of the cy-
clopropenone-phosphine ligation makes it a good can-
didate for labeling applications in conjunction with oth-
er bioorthogonal chemistries, including inverse elec-
tron-demand Diels-Alder cycloadditions,17,38,39 dipolar
cycloadditions, and several azide-alkyne reactions.1,2,14
The latter class of transformations is particularly note-
worthy, as the reagents – like the cyclopropenones re-
ported here – are robust enough for intracellular work.
It also did not escape our attention that the products of
the cyclopropenone-phosphine ligation are analogous to
classic traceless Staudinger reagents,40,41 making them
potential bioorthogonal probes for azides. To demon-
strate this reactivity, we treated regioisomers 26a-b with
a model azide (benzyl azide) in acetonitrile (Scheme 3).
Only trace amounts of the desired amides were ob-
served, although the starting materials were fully con-
sumed. Mass spectrometry and NMR analyses indicated
the formation of the stabilized aza-ylide (data not
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Journal of the American Chemical Society
shown).42-44 More polar solvents (5% H2O/DMF) facili-
This work was supported by the UC Irvine School of Physi-
tated clean conversion to the expected amides 27a-b.44
Collectively, these data demonstrate that the cyclopro-
penone ligation will be useful for sequential labeling
applications with azides and other bioorthogonal rea-
gents. Future studies will examine the sequential liga-
tions in cellular environments.
1
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3
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7
8
cal Sciences and the Alfred P. Sloan Foundation (J.A.P.),
along with the NSF (MCB-1518265 to R.A.M.). J.A.P. is a
Camille Dreyfus Teacher-Scholar and Cottrell Scholar.
R.D.R. is supported by an NSF Graduate Research Fellow-
ship. We thank the Heyduk, Nowick, and Chamberlin la-
boratories for providing reagents and equipment, Philip
Dennison for assistance with NMR experiments, and mem-
bers of the Prescher lab for manuscript edits and helpful
discussions.
9
CONCLUSIONS
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In conclusion, improved dialkyl cyclopropenones
for bioorthogonal labeling were generated. These motifs
are highly stable in aqueous environments and in the
presence of biological nucleophiles. Through kinetic
analyses, we further identified phosphines that react
with dialkyl cyclopropenones at rates suitable for bio-
logical application. Interestingly, our data suggest that
hydrogen bonding can accelerate the reactions.
REFERENCES
(1)
Patterson, D. M.; Nazarova, L. A.; Prescher, J. A. ACS
Chem. Biol. 2014, 9, 592.
(2)
1, 13.
(3)
Prescher, J. A.; Bertozzi, C. R. Nat. Chem. Biol. 2005,
Chang, P. V.; Prescher, J. A.; Hangauer, M. J.;
Bertozzi, C. R. J. Am. Chem. Soc. 2007, 129, 8400.
(4) Prescher, J. A.; Dube, D. H.; Bertozzi, C. R. Nature
2004, 430, 873.
(5)
Kho, Y.; Kim, S. C.; Jiang, C.; Barma, D.; Kwon, S. W.;
Cheng, J.; Jaunbergs, J.; Weinbaum, C.; Tamanoi, F.; Falck, J.;
Zhao, Y. Proc. Natl. Acad. Sci. USA 2004, 101, 12479.
We also synthesized a non-canonical amino acid
comprising a cyclopropenone motif, and identified
compatible mutant synthetases for genetic incorpora-
tion. These tools can be employed for recombinant pro-
tein production and site-specific tagging experiments.
Downstream cellular applications will benefit from im-
proved incorporation efficiencies with the PylRS/tRNA
pairs, and work along these lines is underway.
(6)
Charron, G.; Zhang, M. M.; Yount, J. S.; Wilson, J.;
Raghavan, A. S.; Shamir, E.; Hang, H. C. J. Am. Chem. Soc.
2009, 131, 4967.
(7)
(8)
Lang, K.; Chin, J. W. Chem. Rev. 2014, 114, 4764.
Charron, G.; Li, M. M. H.; MacDonald, M. R.; Hang,
H. C. Proc. Natl. Acad. Sci. USA 2013, 110, 11085.
(9) Zaro, B. W.; Yang, Y. Y.; Hang, H. C.; Pratt, M. R.
Proc. Natl. Acad. Sci. USA 2011, 108, 8146.
(10) Siegrist, M. S.; Whiteside, S.; Jewett, J. C.; Aditham,
A.; Cava, F.; Bertozzi, C. R. ACS Chem. Biol. 2013, 8, 500.
(11) Liechti, G. W.; Kuru, E.; Hall, E.; Kalinda, A.; Brun, Y.
V.; VanNieuwenhze, M.; Maurelli, A. T. Nature 2014, 5065, 507.
(12) Elliott, T. S.; Townsley, F. M.; Bianco, A.; Ernst, R. J.;
More broadly, the stabilized cyclopropenones iden-
tified here will provide new opportunities for biological
application. The cyclopropenones are suitable for use in
intracellular and other stringent environments, and by
virtue of their unique reactivities, these scaffolds are
likely compatible with a variety of other bioorthogonal
functionality. The products of the cyclopropenone-
phosphine ligation are also reactive with azides, sug-
gesting utility in tandem labeling experiments.
Sachdeva, A.; Elsässer, S. J. E. A.; Davis, L.; Lang, K.; Pisa, R.;
Greiss, S.; Lilley, K. S.; Chin, J. W. Nat. Biotechnol. 2014, 32,
465.
(13)
Yuet, K. P.; Doma, M. K.; Ngo, J. T.; Sweredoski, M. J.;
Graham, R. L. J.; Moradian, A.; Hess, S.; Schuman, E. M.;
Sternberg, P. W.; Tirrell, D. A. Proc. Natl. Acad. Sci. USA 2015,
112, 2705.
(14)
Patterson, D. M.; Prescher, J. A. Curr. Opin. Chem.
Biol. 2015, 28, 141.
ASSOCIATED CONTENT
Supporting Information
(15)
Karver, M. R.; Weissleder, R.; Hilderbrand, S. A.
Angew. Chem. Int. Ed. 2012, 51, 920.
(16)
Lang, K.; Davis, L.; Wallace, S.; Mahesh, M.; Cox, D.
The Supporting Information is available free of charge on
the ACS Publications website. Experimental details, spec-
troscopic data for new compounds, kinetic plots, and addi-
tional images are included.
J.; Blackman, M. L.; Fox, J. M.; Chin, J. W. J. Am. Chem. Soc.
2012, 134, 10317.
(17)
N.; Prescher, J. A. J. Am. Chem. Soc. 2012, 134, 18638.
(18) Kamber, D. N.; Nazarova, L. A.; Liang, Y.; Lopez, S.
Patterson, D. M.; Nazarova, L. A.; Xie, B.; Kamber, D.
AUTHOR INFORMATION
A.; Patterson, D. M.; Shih, H.-W.; Houk, K. N.; Prescher, J. A. J.
Am. Chem. Soc. 2013, 135, 13680.
Corresponding Author
(19)
Kamber, D. N.; Liang, Y.; Blizzard, R. J.; Liu, F.; Mehl,
* jpresche@uci.edu
R. A.; Houk, K. N.; Prescher, J. A. J. Am. Chem. Soc. 2015, 137,
8388.
(20)
Kogen, H.; Kiho, T.; Tago, K.; Miyamoto, S.; Fujioka,
Author Contributions
T.; Otsuka, N.; Suzuki-Konagai, K.; Ogita, T. J. Am. Chem. Soc.
2000, 122, 1842.
The manuscript was written through contributions of all
authors. All authors have given approval to the final version
of the manuscript.
(21)
Okuda, T.; Yokose, K.; Furumai, T.; Maruyama, H. B.
J. Antibiot. 1984, 37, 718.
(22)
Bohlmann, F.; Jakupovic, J.; Müller, L.; Schusrer, A.
ACKNOWLEDGMENTS
Angew. Chem. Int. Ed. 1981, 3, 292.
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 8 of 8
(23)
Shih, H.-W.; Prescher, J. A. J. Am. Chem. Soc. 2015,
(34)
Bordwell, F. G.; McCallum, R. J.; Olmstead, W. N. J.
1
2
3
4
5
6
7
8
137, 10036.
(24)
Org. Chem. 1984, 49, 1424.
Hamada, A.; Takizawa, T. Tetrahedron Lett. 1972, 13,
(35)
Bordwell, F. G.; Algrim, D. J. J. Am. Chem. Soc. 1988,
1849.
(25)
110, 2964.
(36)
Saxon, E.; Bertozzi, C. R. Science 2000, 287, 2007.
Pini, A.; Lozzi, L.; Bernini, A.; Brunetti, J.; Falciani,
(26)
Breslow, R.; Altman, L. J.; Krebs, A.; Mohacsi, E.;
C.; Scali, S.; Bindi, S.; Di Maggio, T.; Rossolini, G. M.; Niccolai,
N.; Bracci, L. Amino Acids 2012, 43, 467.
Murata, I.; Peterson, R. A.; Posner, J. J. Am. Chem. Soc. 1965, 87,
1326.
(37)
Cooley, R. B.; Karplus, P. A.; Mehl, R. A.
(27)
(28)
Potts, K. T.; Baum, J. S. Chem. Rev. 1974, 74, 189.
Wang, F.; Luo, T.; Hu, J.; Wang, Y.; Krishnan, H. S.;
ChemBioChem 2014, 15, 1810.
(38)
Blizzard, R. J.; Backus, D. R.; Brown, W.; Bazewicz, C.
9
Jog, P. V.; Ganesh, S. K.; Prakash, G. K. S.; Olah, G. A. Angew.
Chem. Int. Ed. 2011, 50, 7153.
G.; Li, Y.; Mehl, R. A. J. Am. Chem. Soc. 2015, 137, 10044.
(39)
Blackman, M. L.; Royzen, M.; Fox, J. M. J. Am. Chem.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(29)
1219.
(30)
Cheng, Z. L.; Chen, Q. Y. Chin. J. Chem. 2006, 24,
Soc. 2008, 130, 13518.
(40)
Saxon, E.; Armstrong, J. I.; Bertozzi, C. R. Org. Lett.
Wilkerson, M. G.; Henkin, J.; Wilkin, J. K. J. Am.
2000, 2, 2141.
Acad. Dermatol. 2015, 11, 802.
(31)
2597.
(32)
129, 5096.
(33)
(41)
Soellner, M. B.; Nilsson, B. L.; Raines, R. T. J. Am.
Caiazzo, A.; Dalili, S.; Yudin, A. K. Org. Lett. 2002, 4,
Chem. Soc. 2006, 128, 8820.
(42)
Bianchi, A.; Russo, A.; Bernardi, A. Tetrahedron:
Barder, T. E.; Buchwald, S. L. J. Am. Chem. Soc. 2007,
Asymmetry 2005, 16, 381.
(43)
(44)
Bianchi, A.; Bernardi, A. J. Org. Chem. 2006, 71, 4565.
Bernardes, G. J. L.; Linderoth, L.; Doores, K. J.;
Bostwick, D.; Henneike, H. F.; Hopkins, H. P. J. Am.
Chem. Soc. 1975, 97, 1505.
Boutureira, O.; Davis, B. G. ChemBioChem 2011, 12, 1383.
TOC Graphic
H
N
H
N
O
O
N
H
4
4
PPh2
O
O
O
bioorthogonal
reaction
O
N
tagging via genetic
code expansion
PPh2
H3C
CH3
optimized Cpo
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