Rapid Cu-free click chemistry with readily synthesized biarylazacyclooctynones

Article abstract of DOI:10.1021/ja100014q

Chemical equation presented Bioorthogonal chemical reactions, those that do not interact or interfere with biology, have allowed for exploration of numerous biological processes that were previously difficult to study. The reaction of azides with strained alkynes, such as cyclooctynes, readily forms a triazole product without the need for a toxic catalyst. Here we describe a iarylazacyclooctynone (BARAC) that has exceptional reaction kinetics and whose synthesis is designed to be both modular and scalable. We employed BARAC for live cell fluorescence imaging of azide-labeled glycans. The high signal-to-background ratio obtained using nanomolar concentrations of BARAC obviated the need for washing steps. Thus, BARAC is a promising reagent for in vivo imaging.

Full text of DOI:10.1021/ja100014q

Published on Web 02/26/2010
Rapid Cu-Free Click Chemistry with Readily Synthesized
Biarylazacyclooctynones
John C. Jewett, 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
Received January 8, 2010; E-mail: crb@berkeley.edu
Bioorthogonal chemical reactions, those that do not interact or
interfere with biology, have allowed for exploration of numerous
difluorinated cyclooctynes (DIFO, 2) have been employed in
applications ranging from synthesis of star dendrimers7 to labeling
live zebrafish embryos.8 Recently, Boons and co-workers reported
on the usage of dibenzocyclooctynes (DIBO, 3a,b) as detection
reagents for the azide.9 These compounds were remarkably reactive,
with rate constants approaching those of the difluorinated cyclooc-
tynes. While the fluorine atoms affected the rate by altering the
electronics of the alkyne,10 the dibenzo system accomplished a
similar rate enhancement through increased strain energy.11 Ad-
dition of one more sp2-like center to the dibenzocyclooctyne ring
can have a further rate-enhancing effect, as reported by Debets et
al. in their study of an aza-dibenzocyclooctyne bearing an exocyclic
amide linkage.12
The dibenzocyclooctynes are stable, but one extra degree of
unsaturation across the ring renders an ene-yne (4) that is highly
reactive but also unstable (Figure 1B).13 In designing new cyclooc-
tyne probes we sought to brush against the line between stability
and reactivity without crossing it. In looking for functional groups
that have varying degrees of double-bond character we arrived at
the amide. Amides have a significant resonance structure wherein
the lone pair of electrons on the nitrogen atom is delocalized
between the nitrogen and carbonyl. There are no reported cyclooc-
tynes that have an amide within the ring, as in BARAC; thus their
balance of stability and reactivity is an intriguing unanswered
question. Further, BARAC derivatives may have solubility and
pharmacokinetic properties that are superior to their parent all-
carbon cyclooctynes.14,15
In planning a synthesis of BARAC, we designed a modular route
that would allow for future modifications to examine the relationship
of structure and activity within biological systems. When synthe-
sizing cyclooctynes, it is essential to build strain after ring
formation; accordingly the amide, functionalized with a linking
moiety, was not an ideal bond disconnection to pursue. The
reactions previously employed to form the cyclooctyne’s strained
triple bond have undermined efficient syntheses by imposing long
reaction times and low yields.6,14,16 To overcome this hurdle, we
sought to install the alkyne from R-trimethylsilyl ketone 5 (Scheme
1). Although unprecedented in the synthesis of cyclooctynes,
biological processes that were previously difficult to study.1
A
widely used bioorthogonal functional group is the azide, which can
be incorporated into myriad biological molecules by feeding cells
or organisms azide-functionalized metabolic substrates.2 The
abundance, location, and dynamics of the azide-labeled bio-
molecules can be monitored by chemical ligation with probes
bearing complementary functionality.3 The copper-catalyzed click
reaction between azides and terminal alkynes is ideal for many
applications, but copper(I) has the undesirable side effect of being
cytotoxic at low concentrations.4 The reaction of azides with
strained alkynes, such as cyclooctynes, relieves that burden by
readily forming a triazole product without a toxic catalyst (Figure
1A). Such reactions, in addition to other select cycloadditions, are
now being referred to as Cu-free click chemistries.5
In our ongoing work toward the design of cyclooctyne probes
for live cell and animal imaging, we have focused on two central
attributes: kinetic parameters and synthetic facility. Herein we
describe a biarylazacyclooctynone (BARAC, 1) that has exceptional
reaction kinetics and whose synthesis is designed to be both modular
and scalable (Figure 1B). We employed BARAC for live cell
fluorescence imaging of azide-labeled glycans. The high signal-to-
background ratio obtained using nanomolar concentrations of
BARAC obviated the need for washing steps. Thus, BARAC is a
promising reagent for in vivo imaging.
Previously we reported that the addition of electronegative
fluorine atoms at the propargylic position of cyclooctyne dramat-
ically increases its rate of reaction with azides (Figure 1B).6 These
Scheme 1. Retrosynthesis of BARAC (1)
Figure 1. Bioorthogonal reaction of cyclooctyne probes with azide-labeled
biomolecules allows their interrogation in cell-based systems. (A) Cells are
treated with azide-functionalized metabolic substrates. The azides are then
detected with a cyclooctyne-functionalized probe. (B) Cyclooctynes designed
for fast Cu-free click chemistry (1-3) and reactivity studies (4). The R-group
denotes the location for linkage to a probe moiety.
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C O M M U N I C A T I O N S
Scheme 2. Synthesis of BARAC (15)
is amenable to large-scale preparation with only one purification
step at the end of the sequence. The TMS group was then installed
in a straightforward manner to form 10.
Oxidation of indole 10 with excess m-CPBA cracked open the
central rings to unveil cyclic keto-amide 11, which was unstable
to silica-gel purification. Interestingly, these conditions left the
terminal alkene untouched. Treatment of the potassium enolate of
ketone 11 with trifluoromethanesulfonyl anhydride gave 12. Selec-
tive reaction of the terminal alkene with a nitrile oxide generated
in situ from chlorooxime 13 installed a linker for conjugation to a
probe molecule.23 Reaction of 14 with CsF introduced the strained
alkyne in excellent yield in under 30 min at rt. In summary, the
synthesis of BARAC (15) from commercial intermediate 9 was
accomplished in 6 steps in 18% overall yield. Importantly, BARAC
was stable to traditional chromatography and on the benchtop,
unlike its prototype 4.
We studied the reactivity of BARAC derivative 15 using benzyl
azide as a model substrate. Gratifyingly, the second-order rate
constant in acetonitrile at rt was 0.96 M-1 s-1 (Figures S1 and S2,
Supporting Information), over 12-fold higher than the rate constant
for DIFO under identical conditions and over 450 times higher than
that for an unactivated cyclooctyne.11,24
formation of the enol triflate from this ketone could allow for
strained alkyne formation using mild conditions and short reaction
times.17 However, such R-silyl ketones are synthetically challenging.
Selective carbon-silicon bond formation under enolate-forming
conditions is problematic due to the oxophilic nature of silicon
coupled with the electronegativity of oxygen.18
We envisioned introducing the silyl group prior to the carbonyl,
and toward this end, we harnessed the susceptibility of indoles to
oxidative C3-C4 bond cleavage.19 Silylation of the C8-position
of indole 6 should be facile due to its relatively acidic protons.20
The indole, formed from a Fisher indole synthesis between phenyl
hydrazine (7) and 1-indanone (8), was an ideal early precursor
because of the wide variety of aryl hydrazines and indanones that
are either commercially available or readily synthesized in large
quantities.
We next sought to evaluate BARAC’s performance in live cell
labeling experiments. Using a carbamate linkage, we conjugated
BARAC to biotin, affording BARAC-biotin (16), or to fluorescein,
affording BARAC-Fluor (17, Figure 2A) (Scheme S1 and Figure
S7, Supporting Information). Azides were introduced into cell
surface glycans using a strategy we employ routinely.25 We treated
cultured Jurkat cells with peracetylated N-azidoacetyl mannosamine
(Ac4ManNAz) to introduce the corresponding azido sialic acids
(SiaNAz) into their cell-surface glycoproteins and lipids. Then, the
cells were labeled with BARAC-biotin at a concentration of 1 µM
for 1 h. Staining with fluorescein isothiocyanate (FITC-avidin) and
flow cytometry analysis produced the data shown in Figure 2B.
The cells showed robust azide-specific labeling with no significant
background labeling compared to cells treated with FITC-avidin
alone (Figures S3 and S4, Supporting Information). Even at a
concentration of 50 nM, BARAC-biotin still showed significant cell
labeling in 1 h (Figures S3 and S4, Supporting Information).
As shown in Scheme 2, Fisher indole synthesis gave com-
mercially available indole 9.21 The indole nitrogen was alkylated
with allyl bromide using phase transfer conditions,22 thereby
protecting this sensitive functionality and also introducing a
functional handle for future probe attachment. This initial sequence
Figure 2. BARAC-probe conjugates label live cells with superior sensitivity compared to DIFO and DIBO reagents. (A) Structures of BARAC-biotin (16)
and BARAC-Fluor (17). (B-C) Flow cytometry plots of live cell labeling with BARAC-biotin. Jurkat cells were incubated with (+Az) or without (-Az)
25 µM Ac4ManNAz for 3 days. The cells were labeled with 1 µM cyclooctyne-biotin for various times and then treated with FITC-avidin. Cyclooctyne-
biotin probes used were DIBO-biotin, BARAC-biotin, or DIFO-biotin. The degree of labeling was quantified by flow cytometry. The level of fluorescence
is reported in mean fluorescence intensity (MFI, arbitrary unit). Error bars represent the standard deviation of three replicate experiments. (B) Comparison
of the efficiencies of labeling of different cyclooctyne reagents after 1 h. (C) Time-dependent labeling of cyclooctyne-biotin probes. MFI reported as difference
between signal of cells +Az and signal of cells -Az.
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For comparative purposes, we performed similar experiments
using DIFO-biotin6 and DIBO-biotin16c (Figure 2B). BARAC-biotin
showed the best labeling after 1 h. To quantify the differences in
reaction kinetics among the reagents in the context of cell surface
labeling, we measured cell surface fluorescence at various time
points during a 30-min incubation with the cyclooctynes (Figure
2C). After 1 min, BARAC-biotin gave a 10-fold higher signal than
either DIFO-biotin or DIBO-biotin, consistent with BARAC’s ∼12-
fold higher rate constant. The high level of reactivity of BARAC-
biotin was not accompanied by any cytotoxicity compared to cells
treated with no cyclooctyne reagent (Figures S5 and S6, Supporting
Information).
reagent for real-time and in vivo imaging of azide-labeled
biomolecules.
Acknowledgment. J.C.J. was supported by a postdoctoral
fellowship from the American Cancer Society, and E.M.S. was
supported by a predoctoral fellowship from the Organic Division
of the American Chemical Society (Genentech). This work was
supported by a grant to C.R.B. from the National Institutes of Health
(GM58867).
Supporting Information Available: Experimental procedures,
spectral data, and assay data. This material is available free of charge
via the Internet at http://pubs.acs.org.
We next evaluated BARAC-Fluor (17) as a reagent for direct
fluorescence imaging of live Chinese hamster ovary (CHO) cells.
CHO cells grown in either the presence or absence of 50 µM
Ac4ManNAz were treated with 5 µM BARAC-Fluor (11) for 5 min
at rt, washed, and then imaged (Figure 3A-H). The azide-labeled
CHO cells showed robust cell surface fluorescence.
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In some situations, washing away unbound fluorescent labeling
reagents is difficult or impossible. Such cases include whole animal
imaging experiments, real-time imaging of cultured cells, and
situations in which the biomolecular targets are intracellular. We
reasoned that BARAC’s superior sensitivity, the consequence of
its kinetics and low nonspecific background reactivity, would allow
for use of low labeling concentrations that eliminate the need for
washing steps. Cells were cultured as before but labeled with 250
nM BARAC-Fluor for 30 min at rt. Without washing away excess
reagent we imaged the cells (Figure 3I-P) and observed clear
labeling above background. BARAC is therefore a promising
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