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Organic & Biomolecular Chemistry
Page 4 of 5
DOI: 10.1039/C8OB01076E
COMMUNICATION
Journal Name
Figure 4 Light-controlled bioorthogonal reactivity of 3-N scaffold will be amenable to control via additional wavelengths
spirocyclopropenes. ( ) Nvoc-protected cyclopropene 11 did of light through the application of the desired protecting
A
not display any ligation to tetrazine-modified BSA. Upon group. The kinetics of this reaction with 1,2,4,5-tetrazine is
exposure to 365 nm light, cyclopropene 11 generates 3N- sluggish relative to recent bioorthogonal ligations that have
deprotected cyclopropene 10b which ligates to the tetrazine- been optimized for speed, but this reagent provides control of
modified BSA protein. (
containing 3-N deprotected cyclopropene 10b and Nvoc application through the selection of an appropriate light-
protected 3-N cyclopropene 11. ( ) Chemiluminescence and cleavable protecting group. Additionally, kinetics for these
B) Molecular structures of biotin- reactivity in space and time that can be tuned to the particular
C
Ponceau analysis of ligation between the tetrazine-modified reactions are currently being optimized with analogs that
BSA and cyclopropenes 10b or 11. No signal was detected for lower the molecule’s pKa and decrease the strength of the
the Nvoc-caged 11 in the absence of light, whereas a strong rate-decelerating electron withdrawing groups on the spiro
signal was observed for the same compound after the sample ring system.
was exposed to 365 nm light. Each reaction contains 5.0 µg of
We thank Dr. Bela Ruzsicska and the Stony Brook
protein (BSA or tetrazine modified BSA) and 500 nM University Institute for Chemical Biology and Drug Discovery
cyclopropene (10b or 11) were either kept in dark or exposed for Mass Spectrometry resources and analyses, and Francis
to 365 nm light for 4 h in PBS (pH 7.4). The reactions were Picart for NMR analyses. We thank Prof. Nicole Sampson and
incubated at rt for 12–14 h and subjected to western-blot.
the Stony Brook Department of Biochemistry and Structural
Biology for access to instrumentation. This work was
S4, Supporting Information), which also allowed us to confirm supported by a grant to S.T.L. from the National Science
the formation of the expected ligation product (Compound Foundation 1451366.
nd
S11, Supporting Information). The observed 2 order rate
constant puts this spirocyclopropene in the same order of
magnitude as the original, Cu-free click reactions with
2
cyclooctyne . Additionally, the acceleration in rate with
1
Conflicts of interest
increasing pH suggests that next generation reagents with There are no conflicts to declare.
lowered pKa values are an avenue for future reaction rate
improvements.
Lastly, we sought to evaluate this ligation in biological Notes and references
contexts and to determine the success of the ligation after
1
D. M. Patterson, L. A. Nazarova and J. A. Prescher, ACS
light activation. Towards this end, we created a solution of
tetrazine-modified bovine serum albumin (BSA) and evaluated
its reactivity with the biotin-conjugated free-amine or Nvoc-
protected cyclopropenes 10b and 11 in a Western blot assay
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4
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6
7
protected cyclopropene 11 with tetrazine-BSA in the dark
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amine cyclopropene 10b displayed no reactivity with
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light at 365 nm we observed ligation to BSA-tetrazine,
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biology
with varying properties based on coumarins
(e.g., N-linked photocleavable protecting groups
2
3,25,26
27
, RuBi cage ,
, and thiochromone S,S-
dioxides ), we expect that this activatable cyclopropene
2
8–30
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nitroindonilyl
, 2-nitrobenzyl
3
2
4
| J. Name., 2012, 00, 1-3
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