A bioorthogonal quadricyclane ligation

Article abstract of DOI:10.1021/ja2072934

New additions to the bioorthogonal chemistry compendium can advance biological research by enabling multiplexed analysis of biomolecules in complex systems. Here we introduce the quadricyclane ligation, a new bioorthogonal reaction between the highly strained hydrocarbon quadricyclane and Ni bis(dithiolene) reagents. This reaction has a second-order rate constant of 0.25 M^-1 s^-1, on par with fast bioorthogonal reactions of azides, and proceeds readily in aqueous environments. Ni bis(dithiolene) probes selectively labeled quadricyclane-modified bovine serum albumin, even in the presence of cell lysate. We have demonstrated that the quadricyclane ligation is compatible with, and orthogonal to, strain-promoted azide-alkyne cycloaddition and oxime ligation chemistries by performing all three reactions in one pot on differentially functionalized protein substrates. The quadricyclane ligation joins a small but growing list of tools for the selective covalent modification of biomolecules.

Full text of DOI:10.1021/ja2072934

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A Bioorthogonal Quadricyclane Ligation
Ellen M. Sletten and Carolyn R. Bertozzi*
Departments of Chemistry and Molecular and Cell Biology and Howard Hughes Medical Institute, University of California, Berkeley,
California 94720, United States
S Supporting Information
b
ABSTRACT: New additions to the bioorthogonal chem-
istry compendium can advance biological research by enabl-
ing multiplexed analysis of biomolecules in complex sys-
tems. Here we introduce the quadricyclane ligation, a new
bioorthogonal reaction between the highly strained hydro-
carbon quadricyclane and Ni bis(dithiolene) reagents. This
reaction has a second-order rate constant of 0.25 Mꢀ1 sꢀ1
,
on par with fast bioorthogonal reactions of azides, and pro-
ceeds readily in aqueous environments. Ni bis(dithiolene)
probes selectively labeled quadricyclane-modified bovine
serum albumin, even in the presence of cell lysate. We have
demonstrated that the quadricyclane ligation is compatible
with, and orthogonal to, strain-promoted azideꢀalkyne
cycloaddition and oxime ligation chemistries by performing
all three reactions in one pot on differentially functionalized
protein substrates. The quadricyclane ligation joins a small
but growing list of tools for the selective covalent modifica-
tion of biomolecules.
Figure 1. (A) A generic [2 + 2 + 2] reaction between quadricyclane
and a π electrophile to yield a norbornene cycloaddition product. (B)
7-Acetoxyquadricyclane (1) reacts with bis(dithiobenzil)nickel(II) (2)
to form complex 3. Complex 3 undergoes photodegradation to 2 and
7-acetoxynorbornadiene (4) unless diethyldithiocarbamate (5) is present.
assembly of functionalized ring systems.10 In practice, such rea-
ctions typically are metal-catalyzed as a means to overcome
an otherwise significant entropic barrier. However, the highly strai-
ned hydrocarbon quadricyclane directly undergoes [2 + 2 + 2]
cycloaddition with specific types of π systems (Figure 1A).11
Quadricyclane possesses many qualities that render it a
promising bioorthogonal reagent. First, it is abiotic, and we
envisioned that quadricyclane’s all sp3-hybridized carbon system
would be unreactive with native biomolecules.12 Second, the
molecule is relatively small13 and may therefore be amenable to
biosynthetic incorporation into biomolecules. But what intrigued
us most was quadricyclane’s reactivity profile. Its ∼80 kcal/mol
of strain14 promotes cycloaddition with diazodicarboxylates and
other electron-deficient π systems under mild conditions.11 By
contrast, quadricyclane does not react readily with simple alkenes,
alkynes, or even cyclooctynes. As well, previous work has shown
that the rates of these cycloaddition reactions are greatly enhanced
by the “on water” effect.15
To identify a bioorthogonal reaction partner for quadricy-
clane, we screened a variety of candidates for reactivity at room
temperature in aqueous or polar aprotic solvents [Table S1 in the
Supporting Information (SI)]. Diazodicarbonyl compounds
showed promising kinetics but were not sufficiently stable in
water for biological applications.16 α-Fluoroketones and fluori-
nated alkenes and alkynes reacted with quadricyclane too slowly
for practical utility, and some of these reagents were prone to side
ioorthogonal chemistry is an enabling tool for biological
B
research as well as a challenging frontier in synthetic metho-
dology.1 Bioorthogonal reaction development requires attention
to functional group reactivity and selectivity, as in any metho-
dology effort, but in addition one must consider issues of water
stability, biocompatibility, and reaction kinetics in physiological
settings. To invent reactions within the confines of such unusual
boundary conditions, chemists have been compelled to explore
manifolds of reactivity outside the mainstream of organic synth-
esis. Some notable success stories have come from adaptations of
classic azide chemistry, such as the Staudinger ligation2 and click
chemistry (both Cu-catalyzed3 and Cu-free versions4). These
bioorthogonal reactions have made it possible to probe a
particular biomolecule labeled with an azide using complemen-
tary reagents in vitro, in cells, and in live organisms.5 However,
multiplexed analysis of several biomolecules in a given system
requires parallel use of a collection of bioorthogonal reactions.
The toolkit has expanded considerably in recent years1 but is still
dwarfed by the compendium of conventional synthetic transfor-
mations, underscoring the need for new contributions.
In an effort to develop new bioorthogonal reactions that are
themselves orthogonal to the current cohort, we sought to exp-
lore unrepresented reactivity space. The published bioorthogonal
transformations represent four broad reaction types: 1,3-dipolar
cycloadditions,6 DielsꢀAlder reactions,7 metal-catalyzed couplings,3,8
and nucleophilic additions.2,9 Outside of this space lies the [2 +
2 + 2] cycloaddition reaction, a popular choice for the rapid
Received: August 3, 2011
Published: September 30, 2011
r
2011 American Chemical Society
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reactions. One reagent stood out as a promising lead: bis-
(dithiobenzil)nickel(II) (2), a Ni bis(dithiolene) complex that
reacted cleanly with 7-acetoxyquadricyclane (1) to yield adduct 3
(Figure 1B). Our results were consistent with a 1986 report by
Sugimori and co-workers highlighting the rapid reactivity of
2 with quadricyclane.17 However, transforming this finding into
a bioorthogonal reaction—a “quadricyclane ligation”—re-
quired that we address the central criteria of water stability,
biocompatibility, and reaction kinetics in physiological settings.
First, we assayed the stability of 1 in water and in the presence
of biological nucleophiles. We prepared 1 by the photochemical
[2 + 2] reaction of 4 as reported previously18 (Scheme S1 in the
SI). Compound 1 was found to be stable in phosphate-buffered
saline (PBS, pH 7.4), with no observed degradation after more
than 2 months at room temperature (Figure S1 in the SI). We
also found 1 to be unreactive with sugars, a variety of oxidants,
and free amino acids, most notably cysteine (Figure S2).
Furthermore, quadricyclane was stable in the presence of bovine
serum albumin (BSA) and cell culture media under conditions
emulating those necessary for metabolic labeling experiments.
The limited solubility of commercially acquired 2 in polar
solvents precluded a thorough stability study, but the compound
was found to be unreactive with a variety of nucleophiles in
dichloromethane.
Scheme 1. Water-Soluble Ni Bis(dithiolene) Complexes
We then turned our attention to the stability of norbornene 3,
the product formed by ligation of 1 and 2 (Figure 1B). There are
reports that quadricyclane’s adduct with 2 undergoes light-
induced reversion to norbornadiene (an isomer of quadricyclane)
and 2.19 Accordingly, we exposed 3 to ambient light and
monitored the formation of 2 and 4 (Figure 1B). The half-life
of the reversion was ∼35 h in CDCl3 (Figures S3 and S4), which
would be problematic for many biological labeling applications.
We predicted that the photochemical reversion could be inhib-
ited by removal of Ni from the product.20 After screening a
variety of metal chelators (Table S2), we found that diethyl-
dithiocarbamate (5; Figure 1B) entirely prevented the photo-
degradation of 3 (Figure 1B and Figure S5).
With these promising results, we pursued the synthesis of a Ni
bis(dithiolene) reagent that is both water-soluble and function-
alized with a probe to detect biomolecule labeling. Compound
15 bearing two sulfonate groups and two biotin moieties was
designed for this purpose (Scheme 1). We prepared 15 and a
model compound, bis(isopropyl amide) 14, from dithiol-2-one
ligand precursors.21 Briefly, alkyne 6 was combined with xantho-
gen disulfide 7 in the presence of the radical initiator 1,10-
azobis(cyclohexanecarbonitrile) to yield dithiocarbonate 8.22
Treatment of 8 with fuming sulfuric acid installed a single
sulfonate group and also hydrolyzed the methyl ester, producing
9 in good yield. Standard amide bond-coupling conditions were
used to conjugate either isopropylamine or an amine-functionalized
biotin derivative to 9, affording 10 and 11, respectively. These
intermediates were then converted to anionic Ni bis(dithiolene)
species 12 and 13 in situ by treatment with tetramethylammonium
hydroxide and NiCl2. Immediate oxidation with 0.5 equiv of iodine
afforded the desired neutral complexes 14 and 15.23 Gratifyingly, we
found that 14 and 15 are soluble in PBS at concentrations up to 5
and 10 mM, respectively.
that is not present in the adduct with quadricyclane, allowing for
pseudo-first-order kinetic measurements by absorption spectros-
copy. We found that the second-order rate constant for the
reaction of 14 and 1 in a 1:1 PBS/EtOH mixture24 was 0.25 (
0.05 Mꢀ1 sꢀ1 at room temperature (Figures S6 and S7). This rate
constant is comparable to those for cyclooctyneꢀazide cycload-
ditions currently used for biological labeling applica-
tions25 and should allow for the use of mild reaction conditions.
The stability of 14 was also monitored using the NIR absorp-
tion band, which is dependent on both the oxidation state of the
complex (i.e., 14 vs 12) and the connectivity of the dithiolene
ligands.26 The absorption at 850 nm remained essentially un-
altered when 14 was incubated in PBS for 20 h (Figure S8). As
well, exposure to amino acids had either no effect or a minimal
effect on the absorption intensity (Figure S9). A marked excep-
tion was cysteine, which caused an immediate decrease in the
intensity of the 850 nm absorption band and the concomitant
appearance of a new band at ∼900 nm (Figure S10). This
transformation is consistent with reduction of 14 to 12,26b and
treatment of 14 with other reducing agents yielded similar results
(Figure S11). The neutral complex could be regenerated by
addition of K3Fe(CN)6, as judged by the reappearance of the
absorption band at 850 nm and restoration of the reactivity with
1 (Figures S12 and S13). Concerned that 14 would undergo
redox reactions with free thiol groups of protein-associated
cysteine residues, we monitored the integrity of the Ni bis-
(dithiolene) complex in the presence of BSA, which possesses a
solvent-exposed cysteine side chain.27 Incubation with BSA led
to reduction of 14 but at a lower rate than observed with free
cysteine (Figure S14). Therefore, protein-mediated reduction
should not undermine the much faster quadricyclane ligation.
Notably, 14 was stable toward oxidized insulin over a 5 h period
(Figure S15).
The solubilities of compounds 14 and 15 enabled us to assess
the reaction kinetics with quadricyclane in aqueous/organic
solvent mixtures as well as to probe in more detail the stability
of these Ni bis(dithiolene) complexes in the presence of biomo-
lecules. Complex 14 has a large NIR absorption band at 850 nm
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Figure 2. Ni bis(dithiolene) reagents selectively label quadricyclane-modified BSA. (A) Modification of BSA’s solvent-exposed lysine residues with
compound 16 and subsequent labeling with biotinylated Ni bis(dithiolene) reagent 15. (B, C) Ni bis(dithiolene) 15 displays (B) time- and (C) dose-
dependent labeling of QC-BSA. BSA (ꢀ) or QC-BSA (+) (5 μg) was treated with (B) 50 μM 15 for various amounts of time or (C) various
concentrations of 15 for 30 min. The reactions were quenched with 1 and 5, and the presence of product was detected by Western blot using α-biotin-
HRP. (D) Reaction of 15 with QC-BSA can be prevented by pretreatment with tetrasulfonated Ni bis(dithiolene) 17. BSA (ꢀ) or QC-BSA (+) (5 μg)
was treated with various amounts of 17 for 30 min followed by 50 μM 15 for 30 min. The reactions were quenched with 1 and 5, and the samples were
analyzed by Western blot probing with α-biotin-HRP. (E) QC-BSA can be selectively labeled in a mixture of proteins. To 25 μg of lysate in the presence
of 1 mM K3Fe(CN)6 was added no BSA or reagent (lane 1), BSA (1.5 μg) and 50 μM 15 (lane 2), and QC-BSA (1.5μg) and 50 μM 15 (lane 3). After 30 min,
the reactions were quenched with 1 and 5 and analyzed by Western blot. The BSA monomer and dimer bands are visible in the QC-BSA-treated sample. The
bands denoted with asterisks represent a biotinylated E. coli protein. In (BꢀE), equal protein loading was verified by Ponceau stain.
The quadricyclane ligation was then subjected to the first
critical test of bioorthogonality: selective protein labeling. We
modified lysine residues on BSA with quadricyclane p-nitrophenyl
carbonate 16 (Figure 2A).28 We then treated either quadricyclane-
modified BSA (QC-BSA) or native BSA with 50 μM 15 for
various amounts of time and, after quenching with excess 5 and 1,
assayed the products by Western blot probing with an α-biotin
antibody conjugated to horseradish peroxidase (α-biotin HRP).
We were excited to see that 15 selectively labeled QC-BSA in
a time-dependent manner with very little background labeling
of unmodified BSA, even upon prolonged exposure of the
Western blot (Figure 2B). Similarly, when the reaction time was
held constant (30 min) and the concentration of 15 was varied,
dose-dependent labeling was observed, again with minimal non-
specific reactivity (Figure 2C). As well, pretreatment of QC-
BSA with tetrasulfonated Ni bis(dithiolene) 1729 quenched the
protein-bound quadricyclane moiety, as demonstrated by reduced
labeling with 15 (Figure 2D). To determine whether the quad-
ricyclane ligation possessed the heightened selectivity required for
labeling of target biomolecules within more complex samples, we
combined 1.5 μg of QC-BSA (or unmodified BSA) with 25 μg of
Figure 3. The quadricyclane ligation is orthogonal to Cu-free click
E. coli lysate. This mixture was treated with 50 μM 15 for 30 min,
chemistry and the oxime ligation. (A) A mixture of QC-BSA, Az-DHFR,
quenched as described above, and analyzed by Western blot.
and CHO-MBP (8 μg each) was treated with 15 (150 μM), DIMAC-
fluor (250 μM), and H2NO-FLAG (1 mM) for 3 h at 37 °C, pH 4.5. This
mixture was basified with 850 mM tris buffer and quenched with excess
Selective labeling of QC-BSA was observed, but the signal was
weak (Figure S16). We hypothesized that the diminished signal was
due to reduction of 15 by a species present in the lysate. Gratifyingly,
when we added K3Fe(CN)6 (1 mM) to the reaction mixture, robust
and selective labeling of QC-BSA was observed (Figure 2E).
New additions to the bioorthogonal reaction compendium are
most powerful when they can be used in conjunction with other
bioorthogonal chemistries. We therefore aimed to determine
whether the quadricyclane ligation can be performed simulta-
neously with two established bioorthogonal reactions, Cu-free click
chemistry and oxime formation, which are already known to be
mutually compatible.30 A mixture containing equal protein loads
1, 5, and 2-azidoethanol. (B) The mixture was separated into three por-
tions, and each portion was analyzed by Western blot probing with a
different antibody: α-biotin-HRP (quadricyclane ligation), α-fluores-
cein-HRP (Cu-free click chemistry), or α-FLAG-HRP (oxime ligation).
The Ponceau stain indicates that all three proteins were present. Oligomer
bands were observed for BSA and DHFR.34
of QC-BSA (MW ∼66 kDa), azidohomoalanine-containing
dihydrofolate reductase (Az-DHFR, MW ∼23 kDa),31 and alde-
hyde-taggedmaltosebindingprotein(CHO-MBP, MW ∼42kDa)32
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was treated with nickel complex 15, an azacyclooctyne conjugated to
fluorescein (DIMAC-fluor),33 and an aminooxy-functionalized
FLAG peptide (H2NO-FLAG)32 (Figure 3A). After incubation
for 3 h, the mixture was separated into three portions, each of which
was analyzed by Western blot probing with one of the following
antibodies: α-biotin-HRP, α-fluorescein-HRP, or α-FLAG-HRP
(Figure 3B). As shown in Figure 3B, each labeling reagent, including
15, reacted only with its complementary bioorthogonal partner.34
Like the cyclooctyne and aminooxy probes, compound 15 showed
no significant labeling of proteins lacking its partner (quadricyclane),
nor did it interfere with the other bioorthogonal reactions.
In summary, the quadricyclane ligation is a promising bio-
orthogonal reaction that can be used for selective protein labeling
alongside other popular bioorthogonal chemistries. The two
reaction partners reliably form their covalent adduct in environs
as complex as cell lysates. The next challenge for this chemistry
will be applications to cell labeling.35 However, the quadricyclane
ligation in its present form is best suited for in vitro labeling
experiments; improvements will be important for its use in living
systems, where stabilizing additives such as K3Fe(CN)6 and diethyl-
dithiocarbamate are not ideal.36 More detailed studies into the balance
of Ni bis(dithiolene) reactivity and redox stability are warranted, as
well as investigations of the adduct’s photochemistry. Nonetheless,
our results herein suggest that reactions of quadricyclane constitute a
fertile sector of reactivity space for bioorthogonal chemistry.
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’ ASSOCIATED CONTENT
S
Supporting Information. Experimental procedures and
b
supporting figures, schemes, and tables. This material is available
free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
(24) Compound 1’s hydrophobicity mandated the use of ethanol.
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Corresponding Author
crb@berkeley.edu
’ ACKNOWLEDGMENT
Wethank N. Agard and J. Hudak for Az-DHFR andCHO-MBP
samples and R. Bergman, C. Gordon, J. Jewett, and K. Palaniappan
for helpful discussions. This work was funded by a grant to C.R.B.
from the NIH (GM058867). E.M.S. was supported by a predoc-
toral fellowship from the Organic Division of the ACS.
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(28) For the synthesis of 16, see Scheme S1.
(29) For the synthesis of 17, see Scheme S2.
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(33) For the synthesis of DIMAC-fluorescein, see Scheme S3.
(34) Controls for Figure 3B can be found in Figure S17.
(35) Notably, compound 17 showed no significant toxicity to
cultured mammalian cells at concentrations up to 500 μM after 1 h of
treatment. By contrast, Cu(I) displays significant toxicity under these
conditions. See Figures S18 and S19.
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