Efficient low-cost preparation of trans-cyclooctenes using a simplified flow setup for photoisomerization

Article abstract of DOI:10.1007/s00706-016-1668-z

Bioorthogonal ligations have emerged as highly versatile chemical tools for biomedical research. The exceptionally fast reaction between 1,2,4,5-tetrazines and trans-cyclooctenes (TCOs), also known as tetrazine ligation, is frequently used in this regard. Growing numbers of applications for the tetrazine ligation led to an increased demand for TCO compounds, whose commercial availability is still very limited. Reported photochemical procedures for the preparation of TCOs using flow chemistry are straightforward and high yielding but require expensive equipment. Within this contribution, we present the construction and characterization of a low-cost flow photoreactor assembled from readily accessible components. Syntheses of all commonly used trans-cyclooctene derivatives were successfully carried out using the described system. We are convinced that the presented system for photoisomerization will promote access to bioorthogonally reactive TCO derivatives.

Full text of DOI:10.1007/s00706-016-1668-z

Monatsh Chem
DOI 10.1007/s00706-016-1668-z
ORIGINAL PAPER
Efficient low-cost preparation of trans-cyclooctenes using
a simplified flow setup for photoisomerization
Dennis Svatunek1,2 Christoph Denk1 Veronika Rosecker1 Barbara Sohr1
•
•
•
•
Christian Hametner1 Gu¨nter Allmaier2 Johannes Frohlich Hannes Mikula1
1
•
•
•
¨
Received: 15 November 2015 / Accepted: 24 December 2015
Ó The Author(s) 2016. This article is published with open access at Springerlink.com
Abstract Bioorthogonal ligations have emerged as
highly versatile chemical tools for biomedical research.
The exceptionally fast reaction between 1,2,4,5-tetrazines
and trans-cyclooctenes (TCOs), also known as tetrazine
ligation, is frequently used in this regard. Growing numbers
of applications for the tetrazine ligation led to an increased
demand for TCO compounds, whose commercial avail-
ability is still very limited. Reported photochemical
procedures for the preparation of TCOs using flow chem-
istry are straightforward and high yielding but require
expensive equipment. Within this contribution, we present
the construction and characterization of a low-cost flow
photoreactor assembled from readily accessible compo-
nents. Syntheses of all commonly used trans-cyclooctene
derivatives were successfully carried out using the descri-
bed system. We are convinced that the presented system
for photoisomerization will promote access to bioorthog-
onally reactive TCO derivatives.
Graphical abstract
Keywords Photochemistry Á Cycloadditions Á
Click chemistry Á Alkenes Á Isomerization Á
Bioorthogonal chemistry
Introduction
From a chemists perspective biological systems are
extraordinarily complex. Each individual cell combines a
broad range of functional groups, macromolecules,
enzymes, and organelles with different pH values as well as
sub-compartments with oxidizing or reducing conditions.
Stepping up from the single cell level to a higher organism
adds another magnitude of complexity to the ‘‘in vivo
chemical space’’, which are far off simple mixtures usually
present in a typical synthetic experiment.
Electronic supplementary material The online version of this
article (doi:10.1007/s00706-016-1668-z) contains supplementary
material, which is available to authorized users.
Bioorthogonal chemistry, a term first introduced by
Carolyn R. Bertozzi [1], is a tool that allows covalent bond
formation within highly complex environments, using
small molecule reaction partners. Those reactions (also
referred to as ‘‘in vivo click chemistry’’) meanwhile found
numerous applications in fluorescent imaging, drug deliv-
ery, PET and SPECT imaging, radionuclide therapy,
& Hannes Mikula
hannes.mikula@tuwien.ac.at
1
Institute of Applied Synthetic Chemistry, TU Wien, Vienna,
Austria
2
Institute of Chemical Technologies and Analytics, TU Wien,
Vienna, Austria
123

D. Svatunek et al.
radiochemistry or drug target identification among several
others [2–6]. High selectivity and fast ligation rates of the
involved bioorthogonal groups are as important as the
metabolic stability of reaction partners and ligation prod-
ucts, as well as their biocompatibility (also in regards of
toxicity). A prominent example is the reaction of organic
azides with cyclooctynes to form 1,2,3-triazoles, wherein
the strained conformation of the cyclooctyne eliminates the
need for (cytotoxic) copper(I)-catalysis thus also referred
to as the copper-free click (CFC) or strain-promoted alkyne
azide cycloaddition (SPAAC) [7]. SPAAC ligation rates
are highly dependent on substitution motifs on the
cyclooctyne moiety, whose synthesis is often cumbersome
and low-yielding. Several improved and more easily
accessible cyclooctyne derivatives have been reported, but
reaction rates of SPAAC ligations are still limited to
*5 M-1 s-1. Nevertheless, besides many in vitro appli-
cations SPAAC has been successfully used for in vivo
fluorescent imaging in developing zebrafish [8] as well as
for pretargeting in positron emission tomography (PET)
[9].
tagged small molecules were used in vitro in combination
with fluorogenic tetrazines [23–26] to visualize sites of
drug accumulation at sub-cellular resolution. Tz ligations
were also applied for target identification studies [27] and
the synthesis of biocompatible polymers and hydrogels
[28, 29].
Ligation rates of IEDDA conjugations are strongly
dependent on steric and electronic influences on the tetra-
zine and the dienophile employed [30–32]. Norbornene and
terminal alkenes were used as dienophiles, but low rates
even with highly reactive Tz derivatives limit their use
[33–35]. Cyclopropenes were introduced as dienophiles
with less steric demand and higher reactivity compared to
norbornene [36–38]. However, the most reactive dieno-
philes are trans-cyclooctenes (TCOs) (Fig. 1b). The parent
compound trans-cyclooctene (TCO, 1) is useful as reagent
for ligation rate measurements using spectrophotometric
techniques, but lacks a site for further derivatization thus
making it unusable for bioconjugation. Consequently TCO
derivatives including a functional group for further modi-
fication have been developed, out of which trans-
cycloocten-5-ol (TCO-5-OH, 2), which exists in two
diastereomers, is the most frequently used. The diastere-
omer with axially configured hydroxyl group is formed in
smaller amounts using photoisomerization and is thus often
referred to as the ‘‘minor’’ TCO-5-OH (2a). 2a shows
moderately elevated reactivity compared to ‘‘major’’
(equatorial configuration) TCO-5-OH (2e). Nevertheless,
steric hindrance makes further modifications of 2a difficult,
which is why 2e is the most commonly used TCO in lit-
erature so far. TCO-5-OH has been proven to be reactive
in vivo for prolonged times ranging to several days, but the
mode of attachment and the microenvironment of the
functionality is reported to influence the half-life inside the
living system [39]. Annihilation of a cyclopropane ring to
the cyclooctene core structure led to the development of
(rel-1R,8S,9R,4E)-bicyclo[6.1.0]non-4-en-9-ylmethanol (s-
TCO, 4), which is one order of magnitude more reactive
than TCO-5-OH. This dienophile was reported in 2011 by
the Fox group [40], and is by far the fastest reacting TCO
derivative known so far. Unfortunately high reactivity
A significant improvement was achieved by the groups
of Joseph Fox [10] and Ralph Weissleder [11] in 2008,
when they independently introduced the inverse electron
demand Diels–Alder reaction (IEDDA) between 1,2,4,5-
tetrazines (Tz) and strained dienophiles as highly efficient
bioorthogonal reaction (Fig. 1a). When trans-cyclooctene
(TCO) derivatives react with electron deficient Tz com-
pounds second order rate constants of up to 3.3 9 106
M-1 s-1 (25 °C, H2O) [12] were observed, making the
IEDDA reaction several orders of magnitude faster than
SPAAC and the fastest bioorthogonal ligation discovered
so far. Hence, rapid ligation can be achieved even at very
high dilutions frequently encountered within living sys-
tems. IEDDA has successfully been applied in pretargeted
PET imaging [13, 14]. The recent development of tetrazi-
nes labeled with short-lived fluorine-18 [15–17] also allows
imaging of long-circulating nanomedicines with short-lived
isotopes as well as rapid radiolabeling of dienophile-tagged
(bio)molecules [18–22], avoiding further purification steps.
In pharmaceutical and biomedical research dienophile-
Fig. 1 a Inverse electron
(a)
reaction between trans-
(b)
demand Diels–Alder (IEDDA)
cyclooctenes and 1,2,4,5-
tetrazines; b selected known
trans-cyclooctene derivatives
123

Efficient low-cost preparation of trans-cyclooctenes using a simplified flow setup for…
comes for the price of reduced stability, making s-TCO the
choice whenever rapid reaction is more relevant than pro-
longed stability. Another highly reactive dienophile
((2S,3aR,9aS,E)-3a,4,5,8,9,9a-hexahydrocycloocta[d] [1, 3]
dioxol-2-yl)methanol (d-TCO, 3) [12], contains a diox-
olane as structural motif enhancing its polarity and water
solubility, which is a clear benefit when working in bio-
logical media. It exhibits IEDDA reactivity in between the
rates for TCO-5-OH and s-TCO and is reported to display
high stability. Another compound, trans-cycloocten-3-ol
(TCO-3-OH, 5), which also exists in an equatorial (5e) and
axial configured (5a) isomer, deserves special attention due
to its ‘‘click-to-release’’ behavior. The cytotoxic drug
doxorubicin was conjugated to 5a using a carbamate
linkage on its 3-OH functionality leading to a pro-drug that
can be activated by bioorthogonal click reaction with
tetrazines [41]. This methodology gives rise to new ther-
apeutic strategies, and the full potential of release
chemistry is expected to unfold in years to come. The
choice of which dienophile is suited best has to be made for
every particular bioconjugation task depending on the
necessities for ligation rate, stability, steric impact, and
availability. Currently only the two isomers of TCO-5-OH,
2e and 2a are commercially available (2e also in activated
form for conjugation reactions) and quite cost intensive
when larger quantities are required.
SiO2) actively removes the TCO species from the equi-
librium, allowing excellent reaction yields of TCOs from
the respective cis-isomers in multigram quantities
(Fig. 2a). Despite being broadly applicable for TCO syn-
thesis the described setup uses a commercial UV reactor in
which a quartz flask containing the reaction mixture has to
be immersed (Fig. 2b). Both UV source and special
glassware are cost intensive, and differently sized quartz
flasks are necessary to maintain high flexibility regarding
the scale of the reaction. Systems including flow photore-
actors would eliminate this problem. Such reactors are
commercially available (e.g. UV-150 photochemical reac-
tor, Vapourtec Ltd.; Advanced-FlowTM G1 Photo Reactor,
CorningÒ), but cost intensive and often require even more
expensive flow chemistry equipment. We, therefore, aimed
to develop and characterize an effective, versatile, and low-
cost flow photoreactor using readily available components
for the preparation of various TCO derivatives.
Results and discussion
The most promising design featured a quartz glass tube (Ø
16 mm, length 790 mm) connected to tubes at both ends
and a total volume of 165 cm3 as a flow cell. Two 55 W
low-pressure mercury lamps (OSRAM, HNS 55 W OFR,
T8, 90 cm) with a dominant wavelength of 254 nm were
used for irradiation and placed alongside the quartz glass
tube (Fig. 3). For a picture of the used setup see Electronic
Supplementary Material (Fig. S1). This setup was mounted
onto a back plate, covered with aluminum foil to prevent
harmful radiation from exiting the system and aligned in
vertical orientation. Solutions were pumped through the
reactor upwards to allow for better filling of the reactor (see
Electronic Supplementary Material). The total cost of this
State of the art TCO synthesis was introduced by Fox
and coworkers in 2008 and made these useful small
molecules more accessible [42]. When cis-cyclooctenes are
irradiated with UV-C (254 nm) in the presence of a singlet
sensitizer an equilibrium between cis- and trans-isomers is
reached (photoisomerization) [43, 44]. The trans-isomers,
in contrast to the cis-compounds, form stable complexes
with silver cations. Hence, passing the reaction mixture
through a cartridge filled with AgNO3 on silica (AgNO3/
(a)
(b)
F
P
AgNO3
on
silica gel
silica gel
C
R
Fig. 2 a Photochemical isomerization of cis-cyclooctene with active removal of trans compound; b Reaction setup used by Fox et al.: C column,
P metering pump, F quartz glass flask filled with reaction solution, R Rayonet photochemical reactor
123

D. Svatunek et al.
Fig. 3 a 3D view of the flow
photoreactor; b dimensions in
mm
0.10
equation resulting in a predicted half-time of around
3.1 min (Fig. 4). The exact irradiation time for reaching
the equilibrium could not be determined due to the rising
temperature.
A flow rate of 100 cm3/min was chosen for photoiso-
merization, which results in a rather short dwell time of
99 s. This is beneficial since the rate of isomerization is
highest within the first minutes and the increase of tem-
perature is only moderate.
ratio TCO/IS
temperature
35
30
25
20
0.05
0.00
0
2
4
6
8
t/min
For flow photoisomerization, the setup shown in
Fig. 5a was used, for a picture see Electronic Supple-
mentary Material (Fig. S2). An HPLC pump (P) was used
to pass the reaction mixture from a reservoir flask
(F) through a column (C) filled with silica and topped
with AgNO3-impregnated silica (AgNO3/SiO2) and cov-
ered with aluminum foil, then through the vertically
aligned flow reactor (R) back to the reservoir. The col-
umn was placed right after the pump and prior to the
photoreactor to prevent damage due to backpressure. The
use of the reservoir flask enables simple scaling of the
reaction without the use of expensive quartz glass flasks
in different dimensions, since containers of any size and
shape can be used for F. Implementation of cooling/
heating of the solution as well as reactions under inert gas
atmosphere can easily be realized in this setup. In this
case a 3-neck round bottom flask equipped with a con-
denser was used, which was cooled and put under inert
gas atmosphere.
Fig. 4 Development of TCO/IS ratio and temperature during
isomerization
device was below 300€ while the costs for commercially
available systems (photoreactor including lamps and quartz
glass flasks) are significantly higher (2000–5000€ or even
more).
To determine the required time for reaching the equi-
librium during photoisomerization a solution of cis-
cyclooctene (6), methyl benzoate as singlet sensitizer and
undecane as internal standard (IS, used for GC-FID mea-
surements) in diethyl ether/hexanes (1/1) was irradiated
without flow. Samples were taken after different irradiation
times and analyzed by GC-FID. The temperature in the
system reached 36 °C after about 6.5 min, which resulted
in boiling of the solvent. The TCO/IS ratio was calculated
over time and the data was fitted to a one phase association
123

Efficient low-cost preparation of trans-cyclooctenes using a simplified flow setup for…
(a)
(b)
inert gas
low pressure
Hg lamps
water cooling
P
AgNO3
on
R
silica gel
silica gel
C
F
quartz tube
Fig. 5 a Flow photoisomerization setup; b isomerization of compounds 2, 3, 4, and 5
(b)
(a)
5
4
3
2
1
0
1.0
0.5
0.0
6
7
8
9
10
0
50
100
150
200
250
6
7
8
9
10
t/min
cis-isomer
Fig. 6 a Decline of CCO derivatives during photoisomerization; b half-lifes of CCO compounds during photoisomerization
This setup was used for the synthesis of trans-cy-
clooctene (1) as a test substance followed by isomerization
of cis-cycloocten-5-ol (7), ((2S,3aR,9aS,Z)-3a,4,5,8,9,9a-
hexahydrocycloocta[d] [1, 3] dioxol-2-yl)methanol (8) and
(1R,8S,9R,4Z)-bicyclo[6.1.0]non-4-en-9-ylmethanol (9) as
well as cis-cycloocten-3-ol (10) to their corresponding
trans-isomers (Fig. 5b). Reactions were monitored by
measuring the remaining cis-configured starting material
by GC (Fig. 6a). Isomerizations of 6, 7, 8, and 9 were
performed at least in duplicates and revealed similar iso-
merization times and a mean half-life of 72 24 min for
all 9 reactions (Fig. 6b). The isomerization of 10 to TCO-
3-OH (mixture of 5a/5e) was significantly slower with a
half-life of 3.8 h, which is in good agreement with the
reported reaction time of 32 h [41] for this derivative in
comparison to 6-26 h reported for other TCOs [12, 40, 42].
Furthermore, isolated yields were compared with previ-
ously reported results (Table 1).
Conclusion
We were able to develop a low-cost and easy-to-build flow
photoreactor that can be used for efficient synthesis of trans-
cyclooctenes starting from respective cis-derivatives by
photoisomerization. The setup was tested and characterized
using different substituted and frequently used TCO deriva-
tives. Isomerization was possible in relatively short time for
all used substances with an average half-life of about 70 min
with exception of 10 that showed a half-life of 3.8 h.
This simple design of a flow photoisomerization appa-
ratus, which can easily be set up in any lab, enables the
123

D. Svatunek et al.
Table 1 Comparison of yields for photoisomerization of 2, 3, 4, and 5 reported in literature and those reached in this work
TCO
2 (2e/2a)
3
4
5 (5e/5a)
Literature
This work
71 % [42] (47/24)
47 % (32/15)
59 % [12]
58 %
74 % [40]
79 %
43 % [41] (25/18)
75 % (36/39)
efficient and cheap production of TCO derivatives up to
multigram scale.
the starting material in the reservoir flask using either
methyl benzoate or n-undecane as internal standard. The
reservoir flask was cooled with ice-water during isomer-
ization. After the reaction was completed, as indicated by
consumption of the starting material, the lamps were turned
off. The column was flushed with generous amounts of the
solvent as used during isomerization ([60 cm3/mmol of
cis-configured starting material) and then dried with com-
pressed air. Silica and AgNO3/SiO2 were taken out of the
column, stirred in a mixture of concentrated NH4OH
(37 %) and dichloromethane (each 10 cm3/g AgNO3/SiO2)
and filtrated. The filter cake was washed twice with con-
Experimental
Unless otherwise noted, all reagents were purchased from
commercial suppliers and used without further purification.
Cis-isomers 7 [42], 8 [12], 9 [40], and 10 [45] as well as
AgNO3-impregnated silica gel (AgNO3/SiO2) [42] were
prepared following known procedures. GC-FID measure-
ments were performed on
a
TraceTM 1310 GC
chromatograph from Thermo Scientific equipped with TR-
5MS columns (length 15 m, I.D. 0.25 mm, film 1.0 lm).
1H and 13C NMR spectra were recorded on a Bruker AC
200 or Bruker Avance UltraShield 400 spectrometer at
20 °C.
centrated
NH4OH
(5 cm3/g
AgNO3/SiO2)
and
dichloromethane (5 cm3/g AgNO3/SiO2). The organic
layer was separated, washed twice with water, dried over
Na2SO4, and concentrated. Column chromatography was
used for further purification if needed.
trans-Cyclooctene (1)
Kinetics of photoisomerization
Following the general procedure using 9.50 g 6 (86 mmol),
12.30 g methyl benzoate (91 mmol), 130 g AgNO3/SiO2
(10 %), 100 g SiO2, and 3500 cm3 Et2O/hexanes (1:99);
5.50 g (50 mmol, 58 %) of pure 1 were obtained without
further purification. Spectroscopic data matched those
reported in the literature [42].
The flow photoreactor as illustrated in Fig. 3 was closed at
the bottom and filled with a 10 mM solution of 187 mg cis-
cyclooctene
(1.7 mmol),
231 mg
methylbenzoate
(1.7 mmol), and 265 mg n-undecane (1.7 mmol) in
170 cm3 hexanes/Et2O (1:1). A resistance thermometer
was placed inside the tube to follow the temperature of the
solvent. The lamps were turned on and samples were taken
at different time points and analyzed by GC-FID.
TCO-5-OH, rel-(1R,4E,pR)-cyclooct-4-enol (2e)
and rel-(1R,4E,pS)-cyclooct-4-enol (2a)
Following the general procedure using 2.03 g
7
(11.9 mmol), 2.00 g methyl benzoate (14.7 mmol), 27 g
AgNO3/SiO2 (10 %), 18 g SiO2, 700 cm3 Et2O/hexanes
(9:1), 1.00 g n-undecane (6.4 mmol); the crude product
was purified by column chromatography (90 g silica,
Photoisomerization and synthesis of TCO
compounds: general procedure
hexanes/ethyl
acetate,
4:1)
to
yield
658 mg
The setup shown in Fig. 5a was used. The column was
filled with silica gel, topped with AgNO3/SiO2 (10 %) and
flushed with solvent. Then the reservoir was filled with
solvent and put under argon atmosphere. The pump was
turned on and set to a flow rate of 100 cm3/min. After the
solvent had filled the whole system cis-cyclooctene (final
concentration between 5 and 20 mM), methyl benzoate
and, if applicable, n-undecane was slowly added to the
reservoir over the course of several minutes to ensure
homogeneous distribution of all reactants. Afterwards the
solution was pumped through the system for another
20 min. The lamps were turned on and the reaction pro-
gress was monitored by GC-FID following the decline of
(32 %, 5.2 mmol) of equatorial product (2e) and 313 mg
(15 %, 2.5 mmol) of axial product (2a). Spectroscopic data
matched that reported in the literature [42].
d-TCO, ((2 s,3aR,9aS,E)-3a,4,5,8,9,9a-hexahydrocy
cloocta[d][1, 3]dioxol-2-yl)methanol (3)
Following the general procedure using 1.50 g
8
(8.2 mmol), 2.00 g methyl benzoate (14.7 mmol), 19 g
AgNO3/SiO2 (10 %), 20 g SiO2, 500 cm3 Et2O/hexanes
(1:1), 1.00 g n-undecane (6.4 mmol); column chromatog-
raphy (Et2O/hexanes, 2:1) was performed to yield 875 mg
(58 %, 4.8 mmol) of desired product 3. Spectroscopic data
matched that reported in the literature [12].
123

Efficient low-cost preparation of trans-cyclooctenes using a simplified flow setup for…
14. Nichols B, Qin Z, Yang J, Vera DR, Devaraj NK (2014) Chem
Commun 50:5215
s-TCO, (rel-1R,8S,9R,4E)-bicyclo[6.1.0]non-4-en-
9-ylmethanol (4)
Following the general procedure using 1.50 g
¨
15. Denk C, Svatunek D, Filip T, Wanek T, Lumpi D, Frohlich J,
Kuntner C, Mikula H (2014) Angew Chemie Int Ed 53:9655
9
(9.9 mmol), 1.30 g methyl benzoate (9.5 mmol), 33 g
AgNO3/SiO2 (10 %), 50 g SiO2, 1000 cm3 Et2O/hexanes
(1:1), 1.00 g n-undecane (6.4 mmol); 1.19 g (79 %,
7.8 mmol) of pure 4 were obtained without chromato-
graphic purification. Spectroscopic data matched that
reported in the literature [40].
¨
¨
16. Zhu J, Li S, Wangler C, Wangler B, Lennox RB, Schirrmacher R
(2015) Chem Commun 51:12415
17. Rashidian M, Keliher EJ, Dougan M, Juras PK, Cavallari M,
Wojtkiewicz GR, Jacobsen JT, Edens JG, Tas JMJ, Victora G,
Weissleder R, Ploegh H (2015) ACS Cent Sci 1:142
18. Wu Z, Liu S, Hassink M, Nair I, Park R, Li L, Todorov I, Fox JM,
Li Z, Shively JE, Conti PS, Kandeel F (2013) J Nucl Med 54:244
19. Selvaraj R, Liu S, Hassink M, Huang C-W, Yap L-P, Park R, Fox
JM, Li Z, Conti PS (2011) Bioorg Med Chem Lett 21:5011
20. Liu S, Hassink M, Selvaraj R, Yap L, Park R, Wang H, Chen X,
Fox JM, Li Z, Conti PS (2013) Mol Imaging 12:121
21. Selvaraj R, Giglio BC, Liu S, Wang H, Wang M, Yuan H,
Chintala SR, Yap L, Conti PS, Fox JM, Li Z (2015) Bioconjugate
Chem 26:435
TCO-3-OH, (1RS,2RS)-trans-cyclooct-2-enol (5e)
and (1SR,2RS)-trans-cyclooct-2-enol (5a)
Following the general procedure using 2.00 g 10
(15.8 mmol), 2.15 g methyl benzoate (15.8 mmol), 35 g
AgNO3/SiO2 (10 %), 40 g SiO2, 800 cm3 Et2O/hexanes
(1:1), 2.50 g n-undecane (15.8 mmol); the crude product
was purified by column chromatography (90 g silica gel,
10-50 % ethyl acetate in hexanes) to yield 694 mg (35 %,
5.5 mmol) of axial isomer 5a, 669 mg (33 %, 5.3 mmol) of
equatorial isomer 5e and 146 mg of a mixture (5a/
5e = 1.3:1, 7 %, 1.2 mmol). NMR spectra matched those
reported in the literature [41].
22. Li Z, Cai H, Hassink M, Blackman ML, Brown RCD, Conti PS,
Fox JM (2010) Chem Commun 46:8043
23. Meimetis LG, Carlson JCT, Giedt RJ, Kohler RH, Weissleder R
(2014) Angew Chem Int Ed 29:7531
ˇ
24. Yang J, Seckute J, Cole CM, Devaraj NK (2012) Angew Chem
ˇ
Int Ed 51:7476
_
25. Devaraj NK, Hilderbrand S, Upadhyay R, Mazitschek R, Weis-
sleder R (2010) Angew Chem Int Ed 49:2869
26. Carlson JCT, Meimetis LG, Hilderbrand SA, Weissleder R
(2013) Angew Chem Int Ed 52:6917
Acknowledgments We thank Rudolf Svatunek for the design
drawings and the doctoral program ‘Molecular and Elemental Imag-
ing in Biosciences (MEIBio)’ of TU Wien for funding.
27. Yang KS, Budin G, Tassa C, Kister O, Weissleder R (2013)
Angew Chem Int Ed 52:10593
28. Zhang H, Dicker KT, Xu X, Jia X, Fox JM (2014) ACS Macro
Lett 3:727
Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License (http://
creativecommons.org/licenses/by/4.0/), which permits unrestricted
use, distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a
link to the Creative Commons license, and indicate if changes were
made.
29. Liu S, Zhang H, Remy RA, Deng F, Mackay ME, Fox JM, Jia X
(2015) Adv Mater 27:2783
30. Liu F, Liang Y, Houk KN (2014) J Am Chem Soc 136:11483
31. Liu F, Paton RS, Kim S, Liang Y, Houk KN (2013) J Am Chem
Soc 135:15642
32. Karver M, Weissleder R, Hilderbrand SA (2011) Bioconjugate
Chem 22:2263
33. Lang K, Davis L, Torres-Kolbus J, Chou C, Deiters A, Chin JW
(2012) Nat Chem 4:298
34. Knall A-C, Hollauf M, Slugovc C (2014) Tetrahedron Lett
55:4763
References
35. Beckmann HSG, Niederwieser A, Wiessler M, Wittmann V
(2012) Chem Eur J 18:6548
36. Patterson DM, Nazarova LA, Xie B, Kamber DN, Prescher JA
(2012) J Am Chem Soc 134:18638
37. Devaraj N (2012) Synlett 23:2147
1. Sletten EM, Bertozzi CR (2009) Angew Chem Int Ed 48:6974
2. Devaraj NK, Weissleder R (2011) Acc Chem Res 44:816
3. Knall A-C, Slugovc C (2013) Chem Soc Rev 42:5131
4. Lang K, Chin JW (2014) ACS Chem Biol 9:16
5. Willems LI, van der Linden WA, Li N, Li K, Liu N, Hoogen-
doorn S, van der Marel GA, Florea BI, Overkleeft HS (2011) Acc
Chem Res 44:718
ˇ
ˇ
38. Yang J, Liang Y, Seckute J, Houk KN, Devaraj NK (2014)
Chemistry 20:3365
_
39. Rossin R, van den Bosch SM, Ten Hoeve W, Carvelli M, Ver-
steegen RM, Lub J, Robillard MS (2013) Bioconjugate Chem
24:1210
6. Knight JC, Cornelissen B (2014) Am J Nucl Med Mol Imaging
4:96
40. Taylor MT, Blackman ML, Dmitrenko O, Fox JM (2011) J Am
Chem Soc 133:9646
41. Versteegen RM, Rossin R, ten Hoeve W, Janssen HM, Robillard
MS (2013) Angew Chem Int Ed 52:14112
42. Royzen M, Yap GPA, Fox JM (2008) J Am Chem Soc 130:3760
43. Inoue Y, Kunitomi Y, Takamuku S, Sakurai H (1978) J Chem
Soc Chem Commun 1024
7. Jewett JC, Bertozzi CR (2010) Chem Soc Rev 39:1272
8. Laughlin ST, Baskin JM, Amacher SL, Bertozzi CR (2008)
Science 320:664
9. Lee SB, Kim HL, Jeong H-J, Lim ST, Sohn M-H, Kim DW
(2013) Angew Chem Int Ed 52:10549
10. Blackman ML, Royzen M, Fox JM (2008) J Am Chem Soc
130:13518
44. Inoue Y, Takamuku S, Kunitomi Y, Sakurai H (1980) J Chem
Soc Perkin Trans 2:1672
11. Devaraj NK, Weissleder R, Hilderbrand SA (2008) Bioconjugate
Chem 19:2297
45. Zhang J, Matta ME, Martinez H, Hillmyer MA (2013) Macro-
molecules 46:2535
12. Darko A, Wallace S, Dmitrenko O, Machovina MM, Mehl RA,
Chin JW, Fox JM (2014) Chem Sci 5:3770
¨
13. Rossin R, van Duijnhoven SMJ, Lappchen T, van den Bosch SM,
Robillard MS (2014) Mol Pharm 11:3090
123