A trifunctional cyclooctyne for modifying azide-labeled biomolecules with photocrosslinking and affinity tags

Article abstract of DOI:10.1039/c5cc07536j

A bicyclo[6.1.0]nonyne (BCN)-based cyclooctyne reagent bearing a photocrosslinking diazirine (DAz) group and a biotin affinity handle, BCN-DAz-Biotin, is reported. BCN-DAz-Biotin is capable of simultaneously delivering photocrosslinking and affinity tags to azide-labeled biomolecules, enabling photoactivated capture and enrichment/detection of interacting species in native contexts.

Full text of DOI:10.1039/c5cc07536j

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A Trifunctional Cyclooctyne for Modifying Azide-Labeled
Biomolecules with Photocrosslinking and Affinity Tags
Jessica A. Stewart,a‡ Brent F. Piligian,a‡ Sarah R. Rundell,a and Benjamin M. Swartsa*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
A bicyclo[6.1.0]nonyne (BCN)-based cyclooctyne reagent bearing of being introduced into numerous biomolecules,8 the development
a photocrosslinking diazirine (DAz) group and a biotin affinity of new biomolecular labeling strategies and reagents is difficult and
handle, BCN-DAz-Biotin, is reported. BCN-DAz-Biotin is capable of sometimes unsuccessful. As well, other photocrosslinking groups,
simultaneously delivering photocrosslinking and affinity tags to such as aryl azides and benzophenones, are less amenable to
azide-labeled biomolecules, enabling photoactivated capture and metabolic incorporation due to their increased size.
enrichment/detection of interacting species in native contexts.
In some cases, it may be preferable or necessary to capitalize on
established chemical reporter strategies to first tag the biomolecule
of interest with an azide, then deliver a diazirine group (or other
photocrosslinker) to the biomolecule using an azide-specific reagent
(Figure 1A). Recently, the Jewett group reported the first such
reagent, an elegantly designed phosphine-based reagent called
PhosDAz (1, Figure 1B) that delivers diazirines to azides via a
“traceless” Staudinger ligation,9 which was inspired by chemistry
initially developed in the Bertozzi and Raines labs.10-12 PhosDAz is
readily synthesized from commercial materials and it selectively
converts azides into amide-linked diazirines without adding
significant bulk to the biomolecule, making it attractive for
photocrosslinking applications. A disadvantage of PhosDAz for some
applications is that it relies on relatively slow Staudinger ligation
chemistry (often requiring multi-hour incubations), which limits the
study of fast processes. For instance, our lab studies mycobacterial
species, some of which divide every 2 hours. In addition, PhosDAz
does not have a secondary handle for detection or enrichment of
crosslinked species, which limits its use in complex settings and
when relevant detection reagents (e.g., antibodies) are unavailable.
Here, we report a trifunctional cyclooctyne reagent, BCN-DAz-
Biotin (2, Figure 1B), which is designed to deliver both diazirine and
biotin tags to azide-labeled biomolecules via SPAAC, thus enabling
covalent photocrosslinking of interactors and subsequent detection
or enrichment of crosslinked species. BCN- DAz-Biotin is based on
the azide-reactive cyclooctyne bicyclo[6.1.0]nonyne (BCN), which
can be can be readily synthesized (or obtained commercially) and is
among the faster cyclooctynes for SPAAC.13, 14 The diazirine group
was chosen as the photocrosslinker, and biotin was chosen as the
affinity handle to allow either detection or enrichment using avidin
products.
Bioorthogonal chemistry has revolutionized the investigation of
biomolecules in living systems, proving to be particularly powerful
for studying cellular components that are not genetically encoded,
such as glycans and lipids.1 The bioorthogonal chemical reporter
strategy involves tagging a biomolecule of interest with a reactive
handle, which can undergo a selective bioorthogonal reaction to
deliver functional cargo to the biomolecule, thus enabling its
analysis in a native context.1, 2 The most commonly used chemical
reporter is the azide, which is small in size, inert to the biological
milieu, and capable of undergoing various bioorthogonal reactions,
including Cu-catalyzed and strain-promoted azide–alkyne
cycloadditions (CuAAC and SPAAC).3-5 Strategies have been
developed for incorporating azides into numerous types of
biomolecules—including glycans, lipids, proteins, and nucleic
acids—in various systems.2 The type of cargo delivered to azide-
labeled biomolecules via bioorthogonal reaction depends on the
application, but typically include fluorescent tags to enable
detection and affinity handles to enable enrichment.
The incorporation of photocrosslinking groups into
biomolecules is
a powerful approach to identifying their
interactions in live cells, particularly when binding events are weak
or transient.6 For example, the Kohler lab has developed unnatural
monosaccharides bearing small, photoactivatable diazirine groups
that can be metabolically incorporated into glycoproteins and used
to identify glycosylation-dependent protein–protein interactions.7
Similar approaches have been developed to study various other
types of biomolecular interactions.8 Although diazirines are capable
For the synthesis of BCN-DAz-Biotin, shown in Scheme 1, we
used a modular strategy employing L-lysine as a trifunctional
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scaffold, similar to an approach previously used by Cravatt and co- workers
Journal Name
to
Figure 1. (A) Scheme for using bioorthogonal chemistry to probe biomolecular interactions. After labeling the biomolecule of interest using an established chemical
reporter strategy, a bioorthogonal reagent can be used to modify the biomolecule with a photocrosslinking group and, in this work, an affinity tag. Interacting species
can then be captured via photocrosslinking and subsequently enriched/detected. (B) Reagents for delivering photocrosslinking functionality to azide-labeled
biomolecules, including the previously reported PhosDAz and the reagent reported herein, BCN-DAz-Biotin.
access other trifunctional reagents.15 This strategy allows facile fluorescence were also observed when using BCN-DAz-Biotin
interchange of the type of cyclooctyne (or other bioorthogonal concentrations as low as 1 µM, allowing the reagent to be used
group), photocrosslinker, and detection tag, which provides sparingly if desired. If needed, enhanced sensitivity could be gained
flexibility to optimize all three components for specific applications. by exploiting the modular synthetic strategy to swap BCN for a
The synthesis began with mono-Boc-protected PEG diamine 3,16 more reactive cyclooctyne, such as BARAC.20
which was sequentially biotinylated at the free amino group, Boc-
Having confirmed the azide reactivity and detectability of BCN-
deprotected with 50% trifluoroacetic acid (TFA), and linked to Boc- DAz-Biotin in a cellular context, we wished to demonstrate its third
Lys(Fmoc)-OH (4). The resulting intermediate 5 was subjected to and key function, photocrosslinking. For this purpose, we adapted
TFA-mediated Boc removal followed by coupling with diazirine- the model system developed by Jewett and co-workers, in which
containing carboxylic acid 6.17 Removal of the Fmoc protecting the reagent is tested for photocrosslinking with purified bovine
group in 6 with 20% piperidine yielded compound 7, which carried serum albumin (BSA) in solution (Figure 3).9 As a control, we also
the biotin and diazirine groups, as well as a free amine to append evaluated commercial DBCO-Biotin, which contains a cyclooctyne
the cyclooctyne. Finally, 7 was coupled with exo-BCN para- and biotin but lacks a photocrosslinker, and would therefore not be
nitrophenyl carbonate (8)13 to provide the target compound, BCN- expected to bind to BSA in a UV light-dependent manner. First,
DAz-Biotin (2).
BCN-DAz-Biotin (or DBCO-Biotin) was reacted with an azide-
With BCN-DAz-Biotin in hand, we evaluated its ability to react modified 488 fluorophore (Az488) to simulate ligation to a
with azide-labeled biomolecules in living cells. Our group is focused biomolecule while also providing a detection tag for subsequent
on studying mycobacteria, including the pathogen Mycobacterium analysis. The ligation
tuberculosis. Therefore, as a model system for studying the azide-
specific reactivity of BCN-DAz-Biotin, we used an established
strategy employing azido trehalose (TreAz) analogues to label
trehalose glycolipids in live mycobacteria.18, 19 First, M. smegmatis
was treated with 6-TreAz, leading to efficient azide-labeling of
glycolipids. The cells were then reacted with varying concentrations
of BCN-DAz-Biotin (1–100 µM) for 30 minutes followed by
incubation with avidin–488. Analysis of cellular fluorescence
confirmed that BCN-DAz-Biotin was delivered to glycolipids in an
azide- and concentration-dependent manner (Figure 2). The optimal
concentration of BCN-DAz-Biotin was 50 µM, which provided a
signal-to-noise ratio (S/N) of 12. Significant increases in
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O
NH2
BocHN
O
3
1. Biotin, HBTU, DIEA, DMF
2. 50% TFA in CH2Cl2
3. 4, HBTU, DIEA, DMF
(66% over 3 steps)
FmocHN
NHBoc
O
HO
Boc-Lys(Fmoc)-OH (4)
FmocHN
NHBoc
HN
O
O
(CH2CH2O)2
HN
NH
H
H
HN
S
N
O
5
N
1. 50% TFA in CH2Cl2
HO
2. 6, HBTU, DIEA, DMF
3. 20% Piperidine in DMF
(48% over 3 steps)
O
DAz-CO2H (6)
N
N
H
N
H2N
O
HN
O
O
S
(CH2CH2O)2
HN
NH
Figure 2. BCN-DAz-Biotin reacts with azide-labeled biomolecules in living cells.
The bacterium M. smegmatis was cultured in the presence or absence of the
chemical reporter 6-TreAz, then reacted with BCN-DAz-Biotin and stained with
avidin-488. Flow cytometry (A) and fluorescence microscopy (B) analysis of
azide-labeled cells reacted with BCN-DAz-Biotin at the indicated concentration
H
H
HN
and stained with avidin-488. Scale bars, 5 µm.
O
7
detection of the photocrosslinked product via its biotin handle.
These experiments set the stage for performing in vivo crosslinking
experiments with BCN-DAz-Biotin.
H
H
O
O-pNP
8, Et3N, DMF (50%)
O
exo-BCN-O-pNP (8)
BCN-DAz-Biotin (2)
Scheme 1. Synthesis of BCN-DAz-Biotin. BCN, bicyclo[6.1.0]nonyne; Boc, tert-
Conclusions
In summary, we developed the trifunctional cyclooctyne BCN-
DAz-Biotin, which is the first reagent capable of modifying
azide-labeled biomolecules with functional tags that allow
both photocrosslinking and subsequent detection/enrichment
of interacting species. We showed that BCN-DAz-Biotin, which
was accessed via an efficient and modular synthetic strategy,
possesses both of these functions by carrying out a proof-of-
principle photocrosslinking experiment with BSA. Furthermore,
the reagent successfully modified cell surface azides in the
bacterium M. smegmatis, demonstrating its ability to be
deployed in living systems. We envision that BCN-DAz-Biotin
will facilitate the study of biomolecular interactions,
particularly in cases where strategies already exist for
incorporating azides into the biomolecule of interest. For
instance, in relation to the cell labeling work presented herein,
BCN-DAz-Biotin could be used to investigate host–pathogen
interactions mediated by trehalose glycolipids during M.
tuberculosis infection. Reagents such as PhosDAz and BCN-
DAz-Biotin should be valuable tools given the large and
growing number of strategies for incorporating azides into
butoxycarbonyl;
DIEA,
N,N-diisopropylethylamine;
DMF,
N,N-
dimethylformamide; Fmoc, 9-fluorenylmethoxycarbonyl; HBTU, N,N,N′,N′-
tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate; pNP, para-
nitrophenyl; TFA, trifluoroacetic acid.
product was then incubated with BSA and irradiated at 365 nm for
20 min (or left non-irradiated). SDS-PAGE followed by in-gel
fluorescence analysis confirmed that photocrosslinking occurred
between BCN-DAz-Biotin and BSA only when exposed to UV light.
Finally, to demonstrate that the photocrosslinked product could be
detected via its biotin tag, Western blot analysis using avidin–HRP
was performed. As expected, signal was only observed in the UV-
irradiated BCN-DAz-Biotin sample. No photocrosslinking between
the control compound DBCO-Biotin and BSA was observed by in-gel
fluorescence or Western blot. Notably, we did not observe
background signal in these experiments, which stood in contrast to
the significant background observed when PhosDAz (1) was used in
photocrosslinking experiments with BSA. Overall, the BCN-DAz-
Biotin reagent functioned as designed in a model system,
sequentially undergoing: (i) Efficient SPAAC reaction with an organic
azide; (ii) photocrosslinking with a proximal biomolecule; and (iii)
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various types of biomolecules. PhosDAz and BCN-DAz-Biotin This work was supported by CMU and by the Research
have complementary attributes
Corporation for Science Advancement (22525). We thank Dr. R.
J. Hood for assistance with NMR and MS. Flow cytometry
instrumentation was supported by the NSF (1337647). We thank
K. K. Palaniappan for helpful discussions.
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Figure 3. BCN-DAz-Biotin enables UV light-dependent photocrosslinking between
an organic azide and a model protein, as well as subsequent detection of the
photocrosslinked product. (A) Experimental workflow. BCN-DAz-Biotin (or
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fluorophore (Az488), then incubated with BSA and irradiated with 365 nm UV
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SDS-PAGE with in-gel fluorescence to visualize 488-modified species (top),
avidin-HRP Western blot to visualize biotinylated species (middle), and Ponceau
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that dictate their appropriateness for a given application:
PhosDAz delivers diazirines to azides tracelessly and with
minimal steric bulk, but does so with slower chemistry and
lacks detection/enrichment capability; on the other hand,
BCN-DAz-Biotin uses SPAAC to rapidly deliver diazirines to
azides complete with detection/enrichment capability, but the
size of the resulting conjugate is substantial. Future iterations
of such reagents should combine fast and selective azide
reactivity with the delivery of both photocrosslinking and
affinity capabilities in the smallest form possible. Additionally,
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involving
the
development
and
applications
of
photocrosslinking reagents for mycobacteria research is
ongoing in our lab.
Notes and references
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