Fluorodibenzocyclooctynes: A Trackable Click Reagent with Enhanced Reactivity

Article abstract of DOI:10.1002/chem.201902834

Bioorthogonal reactions have widespread applications in biological systems, and development of new bioorthogonal reactions has been of great interest over the past two decades. In this work, the design and synthesis of a family of fluorinated dibenzocyclooctynes (FDIBOs) are reported. The electron-deficient nature of fluorine atoms significantly accelerated the reaction of cyclooctynes in 1,3-dipolar cycloadditions, with either benzyl azide or ethyl diazoacetate, compared to conventional dibenzocyclooctyne (DIBO). In addition, FDIBOs showed unique trackable properties owing to the high NMR sensitivity of the naturally abundant ¹⁹F isotope. Biological molecules, including a monosaccharide, a peptide, and a protein, were tested with FDIBOs, and these reactions could be easily monitored by ¹⁹F NMR spectroscopy to evaluate the progress of the conjugation reactions. In addition, labeling of live cells was also demonstrated with metabolically modified bacteria to expand the possible applications of FDIBOs.

Full text of DOI:10.1002/chem.201902834

A Journal of
Accepted Article
Title: Fluorodibenzocyclooctynes: A Trackable Click Reagent with
Enhanced Reactivity
Authors: Wei Li, Juan Zou, Shiyu Zhu, Xianxian Mao, Hongyan Tian,
and Xiaojian Wang
This manuscript has been accepted after peer review and appears as an
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To be cited as: Chem. Eur. J. 10.1002/chem.201902834
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Fluorodibenzocyclooctynes: A Trackable Click Reagent with
Enhanced Reactivity
‡
‡
Wei Li, Juan Zou, Shiyu Zhu, Xianxian Mao, Hongyan Tian, and Xiaojian Wang*
Abstract: Bioorthogonal reactions have widespread applications in
biological systems, and development of new bioorthogonal reactions
has been of great interest over the past two decades. Herein we
to increase reactivity.[4b] Not long after its discovery, the DIBO
family has become one of the most widely used click reagents,
as it has been utilized and marketed by Invitrogen in its Click-IT®
products.
report the design and synthesis of
a family of fluorinated
dibenzocyclooctynes (FDIBOs). The electron-deficient nature of
fluorine atoms significantly accelerated the reaction of cyclooctynes
in 1,3-dipolar cycloadditions, with either benzyl azide or ethyl
diazoacetate, comparing to conventional dibenzocyclooctyne (DIBO).
In addition, FDIBOs showed unique trackable properties, because of
Despite the great success of these cycloalkynes in
bioconjugation applications, the moderate reaction rate has
been often an obstacle for certain applications, especially in
diluted conditions.[
9]
A more serious but often neglected
problem of current click reactions is the lack of tools to monitor
the reaction, especially in real time. To address these two
concerns, we designed and synthesized a family of fluorinated
dibenzocyclooctynes (FDIBOs, Figure 1A), based on DIBO. The
introduction of fluorine atoms into DIBO molecule benefited from
both the electron deficiency of fluorine, which should accelerate
the reaction rate, and the trackable nature of 19F, which is 100%
of naturally occurring isotope, highly sensitive to NMR
measurements, and has very low background in biological
systems.[
1
9
the high NMR sensitivity of naturally abundant
F isotope.
Biological molecules, including monosaccharide, peptide, and
protein were tested with FDIBOs, and these reactions could be
19
easily monitored by F NMR to evaluate the progress of conjugation
reactions. In addition, labeling of live cells was also demonstrated
with metabolically modified bacteria to expand the possible
applications of FDIBOs.
10]
The development of bioorthogonal reactions has drawn great
attention since its introduction in early 2000s.[1] Since then,
these reactions, often also known as “click reactions”, have been
widely used in the modification of biomolecules, including
proteins, oligonucleotides, lipids and carbohydrates.[1c,
Prominent among a variety of click reactions is the azide−alkyne
cycloaddition, first introduced as copper(I) catalyzed reactions.[3]
To overcome the limitations of copper catalyst, copper-free
reactions with cyclooctyanes have become one of the most
accepted reactions for biological labelings both in vitro and in
vivo.[4]
2]
Several families of different strained alkynes have been
reported, in the aim to enhance the reactivity. Bertozzi et al. first
reported the difluorocyclooctynes (DIFO) and then the more
reactive biarylazacyclooctynones (BARAC).[5] Debets et al.
reported dibenzo-aza-cyclooctyne (DIBAC), enjoying both the
rate enhancement from extra ring strain and increased
hydrophilicity from aniline moiety.[6] More recently, the same
group reported bicyclo[6.1.0]non-4-yne (BCN) as a favorable
reagent for the reaction with electron-deficient aryl azides, and
showed its unique selectivity in dual labeling experiments.[7]
Figure 1. (A) Structure of designed and synthesized FDIBOs. (*compound 6
was only computationally analyzed, but not synthesized, due to the difficulty of
its synthesis) (B) The trans conformation of FDIBOs used in DFT calculations
(FDIBO-1 shown as an example). Atoms: carbon-grey, oxygen-red, fluorine-
green, hydrogen-white.
Burke et al. reported
cycloalkynes (SNO-OCTs), with tunable reactivity by the choice
of adjacent heteroatom.[8]
Ning et al. developed
dibenzocyclooctyne (DIBO), which utilized additional ring strain
a
series of sulfamate-containing
To estimate the reactivities of designed FDIBOs, DFT
calculations with M06-2X functional were first employed to study
the model reaction between FDIBOs and methyl azide (MeN
using DIBO/MeN as a reference, with ORCA programs (see
Supporting Information for calculation details).[
was used to simplify the calculation, since it was previously
reported that MeN predicted the reactivity similarly to benzyl
azide (BzN
) in such calculations.[5c] Only trans conformation
3
),
3
11]
Methyl azide
‡
‡
[
*]
W. Li, J. Zou, S. Zhu, X, Mao, H. Tian, Prof. Dr. X. Wang
Institute of Advanced Synthesis, School of Chemistry and
Molecular Engineering, Nanjing Tech University
Nanjing 211816, China
3
3
was analyzed (Figure 1B), because it was previously shown that
E-mail: ias_xjwang@njtech.edu.cn
‡] These authors contributed equally to this work.
different conformers of similar compounds followed same
[
[5c, 8]
reactivity trend in cycloadditions.
As shown in Table 1, the
Supporting information and the ORCID identification number for
the author of this article can be found online.
alkyne bond angles in all examined compounds were quite
similar, indicating similar strains. Distortion/interaction analysis,
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COMMUNICATION
a popular tool that is widely used for cycloaddition reaction
The reactivities of synthesized FDIBOs were all tested with
benzyl azide (BzN ) in methanol for quantification. As expected,
3
analysis,[ showed distinct differences of activation energies
12]
(
ΔE calc) between DIBO and FDIBO 1-4, and the ΔΔE‡calc was
‡
addition of fluorine atom(s) significantly enhanced reactivity of
cyclooctynes, comparing to conventional DIBO (Table 1, kexp).
Mono fluorination (FDIBO5) increased reaction rate by 67%, and
the difluorinated alcohol (FDIBO1) almost doubled rate. The
fastest one was tetrafluorinated FDIBO2, which had a rate 3.3
times faster. These results of reaction rates were in good
‡
between 0.3 to 1.7 kcal/mol. The activation free energy (ΔG calc
)
was similar among FDIBOs and DIBO, except tetrafluorinated
FDIBO-2, which had a ΔΔG‡calc of ~1 kcal/mol lower. Based on
previous study of BARACs, a ΔΔE‡calc of ~0.6 kcal/mol could
yield an increase of 75% in reaction rate,[5c] thus we were
encouraged by the DFT results and proceeded to synthesize
FDIBOs.
‡
accordance with the DFT calculated ΔE results, indicating the
successful design of FDIBOs.
To further evaluate the reactivities of FDIBOs, ethyl
diazoacetate (EDA) was used as an example of diazo
compounds, which were also often used in 1,3-dipolar
cycloaddition reactions with cycoloctynes.[13] FDIBO-1 was
tested with EDA in methanol, and DIBO was used as control.
The second-order reaction rate constants were determined to be
0.051 ± 0.001 M-1 s for FDIBO-1, and 0.019 ± 0.001 M s for
DIBO (see Supporting Information Figure S29 and S30). While
FDIBO1 has shown its superior reactivity over DIBO in both
reactions with azido and diazo compounds, its shelf life was still
good, as no significant degradation was observed after FDIBO1
was kept at room temperature for a period of two weeks (see
Supporting Information Figure S31). Another common concern
Table 1. Reactivity of synthesized FDIBOs.
angle
a (°)
angle
b (°)
ΔE‡calc
(kcal/mol)
ΔG‡calc
(kcal/mol)
k
exp
-1
molecule
(M-1 s )
[a,b]
-1
-1 -1
1
155.8
156.5
156.1
153.9
154.7
155.9
156.3
154.8
155.4
154.6
156.5
155.7
154.1
153.5
7.8
6.8
7.7
8.2
8.6
8.9
8.5
23.8
22.9
24.0
23.8
23.7
24.1
23.9
0.070 ± 0.001**
0.118 ± 0.005***
0.075 ± 0.006**
0.068 ± 0.006*
0.060 ± 0.001**
N/A
2
3
4
5
is the cross-reactivity with biologically available functional
6
groups, especially thiols.[
14]
A control experiment with β-
DIBO
0.036 ± 0.004
mercaptoethanol was performed and confirmed that no
significant reaction was observed (see Supporting Information
Figure S33).
[
3
a] rate constants for the reaction with benzyl azide in CD OD at 25 °C,
reported as mean ± standard deviation. [b] The statistical significances were
determined using a two-sample Student’s t-test (*p ≤ 0.05, **p ≤ 0.01, ***p ≤
While the tetrafluorinated FDIBO2 showed the best reactivity
with azides, the difluorinated FDIBO1 was used for the following
biological applications, because of its ease of further
functionalization from the hydroxyl group. As shown in Figure
3
0.001), comparing to the rate of DIBO/BzN .
3A, FDIBO1 was activated by 4-nitrophenyl chloroformate to
form compound 13, which can then react with molecule
containing amino groups, such as amine containing fluorophores,
peptides, proteins, synthesized amino oligonucleotides, etc.
A
highly water-soluble fluorescent dye 14 (termed as AENDS,
Amino Ethoxy Naphthalene Di-Sulfonic acid， see Supporting
Information Figure S34 for absorption and emission spectra)
was synthesized and conjugated to 13, which was used as an
example of trackable cyclooctyne-containing fluorophore 15
(
termed as AENDS-FDIBO1). The coupling of 15 with azide-
Figure 2. Synthesis of difluorodibenzocyclooctynol 1. a. TMSCHN
2
, BF
SiCl, THF, -40 C to rt, 1.5 h; c.
Selectfluor, DMF, rt, 30 min, 83% over two steps; d. KHMDS, TBSCl, THF, -40
3 2
·OEt ,
o
o
3
modified mannose (Man-N ) was achieved in water, and can be
monitored by 19F NMR for the reaction process (Figure 3B). As
the reaction proceeded, the F peaks between -85 to -90 ppm,
corresponding to the unreacted 15, shrank, while the peaks
between -65 to -75 ppm, corresponding to the products,
increased. To further test the stability and reaction selectivity of
FDIBO compounds, AENDS-FDIBO1 was mixed with fetal
bovine serum (FBS) and incubated for 24 h, and no significant
reaction or degradation was observed (see Supporting
Information Figure S32).
DCM, -10 C, 3 h, 70%; b. LiHMDS, Me
3
o
19
C to rt, 2 h; e. Selectfluor, DMF, rt, 30 min, 94% over two steps; f. NaBH
4
,
THF, rt, 7 h, 95%; g. TBSCl, DBU, DCM, rt, overnight, 90%; h. Br
overnight, 74%; i. LDA, THF, rt, 2 h, 75%.
2
, DCM, rt,
A facile synthetic route was developed from commercially
available dibenzosuberenone (7) and good yields of targeted
compounds were achieved over multi-step synthesis. The
synthesis of difluorodibenzocyclooctynol (FDIBO1) was shown in
Figure 2 as a representative and the synthesis of FDIBOs 2-5
were included in Supporting Information (see Supporting
Information Figure S1 – S12), while the synthesis of FDIBO6
failed due to unstable intermediate.
Similarly, a widely used αvβ3 integrin inhibitor peptide, cyclo-
RGDfK (cRGDfK),[15] was examined for the 19F signal tracking.
The cRGDfK was first labeled with compound 13, and the
product (cRGDfK-FDIBO1) was then reacted with azido-modified
fluorescein (FITC-Az, see Supporting Information Figure S16) in
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DMSO. The labeling with 13 went smoothly, and the completion
of the reaction with FITC-Az was confirmed by 19F NMR, as the
peaks between -85 to -95 ppm corresponding to unreacted
corresponding to the starting FITC-FDIOB1 (Figure 4C).
However, the peaks corresponding to the product were not
19
observed on F NMR, even though the covalent linkage
between BSA-N and FITC-FDIBO1 was confirmed by SDS-
cRGDfK-FDIBO1 completely diminished (Figure 3C).
The
3
successful conjugation was later confirmed by MS spectroscopy,
as the peaks corresponding to the products of both reaction
were observed (see Supporting Information for details).
PAGE (Figure 4D). We speculate that the absence of 19F signal
for the conjugate was due to the peak broadening, because of
the loss of rotational freedom and the increase of viscosity after
conjugation, as reported before.[16]
Figure 4. (A) Modification of BSA with Az-TEG-PNP. (B) MALDI-TOF MS
1
9
spectrum of BSA (black) and BSA-N
3
(red). (C) F NMR spectra of the
reaction mixture of BSA-N with FITC-FDIBO1 before and after the reaction,
3
with trifluoroethanol as the internal standard (76.5 ppm). Numbers within the
spectra were corresponding the integral of nearby peaks. (D) SDS-PAGE
visualized under UV light (top) and after Coomassie blue stain (bottom). Well
from left to right: protein ladder (each line: 80 kDa, 60 kDa, 50 kDa, and 40
3
kDa from top to bottom), BSA, BSA-N , BSA treated with FITC-FDIBO, and
BSA-N treated with FITC-FDIBO.
3
Bacterial peptidoglycan (PG) has emerged to be an
important target to study, because it is a critical component of
Figure 3. (A) Synthesis of AENDS-FDIBO1 (15). a. Et
7
3
N, DMF, rt, overnight,
5%. (B) F NMR spectra of the reaction mixture of Man-N with 15 at
different reaction time, with trifluoroethanol as the internal standard (76.5 ppm).
1
9
3
bacteria and its synthesis pathway has been a key target for
1
9
Inset: the structure of Man-N
3
. (C) F NMR spectra of the reaction mixture of
[17]
antibiotics.
Thus we metabolically labeled Corynebacterium
cRGDfK-FDIBO1 with FITC-Az before and after the reaction, with
trifluoroacetic acid as the internal standard (76.5 ppm). Inset: the structure of
cRGDfK-FDIBO1.
glutamicum’s (C. glutamicum) PG with azido-D-norvaline (ADN) ,
following previously reported protocol.[
18]
The azido-modified C.
glutamicum was then treated with 20 M FITC-FDIBO1 for 1 h,
and imaged under fluorescent microscopy after washing with
To further study the feasibility of FDIBO and 19F NMR for
bioconjugation monitoring with larger biological molecules,
bovine serum albumin (BSA) was tested as an example of large
proteins. The native BSA was first modified with activated
PBS and fixation with cold methanol.
As expected, C.
glutamicum that were treated with ADN displayed a strong
fluorescence, while minimal fluorescence was observed in the
control group, with untreated C. glutamicum (Figure 5).
azido-tetraethylene glycol (Az-TEG-PNP), which yielded BSA-N
3
In summary, we have successfully designed and
synthesized a series of fluorinated dibenzocyclooctynes, termed
as FDIBOs. Favorable reaction kinetics of FDIBOs with either
azido- or diazo- compounds were observed, comparing to the
conventional DIBO, and the reactivity of FDIBOs was positively
correlated with the amount of fluorine atoms within the molecule.
Addition of fluorine atoms further provided a trackable moiety,
with an average of eight azido groups per BSA molecule based
on MALDI-TOF MS results (Figure 4B). A fluorescein derivative
containing FDIBO1 (FITC-FDIBO1, see Supporting Information
Figure S17) was synthesized, and used for the study with BSA-
N . The conjugation between BSA-N and FITC-FDIBO1 was
3 3
done in a solution of DMSO/water 3:1 mixture, and as the
reaction proceeded, the 19F NMR showed a decrease of peaks
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which provided strong 19F NMR signal with negligible
background from most biological molecules. Monosaccharide,
small peptide and protein were tested with FDIBO molecules,
and utilization of 19F NMR showed great advantage for real-time
Keywords: click chemistry • 19F NMR spectroscopy•
cyclooctynes • bioconjugation
monitoring of the reaction progress.
In vitro labeling of
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Acknowledgements
[
[
This work was supported by grants from the National Natural
Science Foundation of China (21708019), Natural Science
Foundation of Jiangsu (BK20170987), Fund of the State Key
Laboratory of Analytical Chemistry for Life Science of Nanjing
University (SKLACLS1701), Jiangsu province “Double Plan” and
start-up fund at Nanjing Tech University (39837135). We thank
Dr. Jin Huang and Linlin Su for help with fluorescent microscopy,
Dr. Lin Ding and Siqiao Li (Nanjing University) for help with
MALDI-TOF MS, Dr. Chin H. Lin (New York University) for help
with 19F NMR analysis, and we are grateful to the High
Performance Computing Center of Nanjing Tech University for
supporting the computational resources.
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COMMUNICATION
‡
‡
Wei Li, Juan Zou, Shiyu Zhu,
Xianxian Mao, Hongyan Tian, and
Xiaojian Wang*
Page No. – Page No.
Fluorodibenzocyclooctynes: A
Trackable Click Reagent with
Enhanced Reactivity
Design and synthesis of fluorinated dibenzocyclooctynes (FDIBOs) were reported.
These compounds showed better reactivity than conventional DIBO and the
conjugation process with different biological molecules were evaluated by 19F NMR
in real time. Live bacteria were also labeled and imaged with FDIBO-fluorophore
conjugate.
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