General, Divergent Platform for Diastereoselective Synthesis of trans-Cyclooctenes with High Reactivity and Favorable Physiochemical Properties**

Article abstract of DOI:10.1002/anie.202101483

trans-Cyclooctenes (TCOs) are essential partners in the fastest known bioorthogonal reactions, but current synthetic methods are limited by poor diastereoselectivity. Especially hard to access are hydrophilic TCOs with favorable physicochemical properties for live cell or in vivo experiments. Described is a new class of TCOs, “a-TCOs”, prepared in high yield by stereocontrolled 1,2-additions of nucleophiles to trans-cyclooct-4-enone, which itself was prepared on a large scale in two steps from 1,5-cyclooctadiene. Computational transition-state models rationalize the diastereoselectivity of 1,2-additions to deliver a-TCO products, which were also shown to be more reactive than standard TCOs and less hydrophobic than even a trans-oxocene analogue. Illustrating the favorable physicochemical properties of a-TCOs, a fluorescent TAMRA derivative in live HeLa cells was shown to be cell-permeable through intracellular Diels–Alder chemistry and to wash out more rapidly than other TCOs.

Full text of DOI:10.1002/anie.202101483

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
www.angewandte.org
Accepted Article
Title: General, Divergent Platform for Diastereoselective Synthesis
of trans-Cyclooctenes with High Reactivity and Favorable
Physiochemical Properties
Authors: Joseph M. Fox, Jessica E Pigga, Julia E Rosenberger,
Andrew Jemas, Samantha J Boyd, Olga Dmitrenko, and Yixin
Xie
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202101483
Link to VoR: https://doi.org/10.1002/anie.202101483

10.1002/anie.202101483
Angewandte Chemie International Edition
RESEARCH ARTICLE
General, Divergent Platform for Diastereoselective Synthesis of
trans-Cyclooctenes with High Reactivity and Favorable
Physiochemical Properties
Jessica E. Pigga,[a] Julia E. Rosenberger,#[a] Andrew Jemas,#[a] Samantha J. Boyd,[a] Olga Dmitrenko,[a]
Yixin Xie,[a] Joseph M. Fox*[a]
[a]
J. E. Pigga, J. E. Rosenberger, A. Jemas, S. J. Boyd, O. Dmitrenko, Y. Xie, J. M. Fox
Department of Chemistry and Biochemistry
University of Delaware
163 The Green, Newark DE 19716
E-mail: jmfox@udel.edu
Supporting information for this article is given via a link at the end of the document.
Abstract: trans-Cyclooctenes (TCOs) are essential partners for the
fastest known bioorthogonal reactions, but current synthetic methods
are limited by poor diastereoselectivity. Especially hard to access are
hydrophilic TCOs with favorable physicochemical properties for live
cell or in vivo experiments. Described is a new class of TCOs, ‘a-
TCOs’, that is prepared in high yield via stereocontrolled 1,2-additions
of nucleophiles to trans-cyclooct-4-enone, which itself was prepared
on large scale in two steps from 1,5-cyclooctadiene. Computational
transition state models rationalize the diastereoselectivity of 1,2-
additions to deliver a-TCO products, which were also shown to be
more reactive than standard TCOs and less hydrophobic than even a
trans-oxocene analog. Illustrating the favorable physicochemical
properties of a-TCOs, a fluorescent TAMRA derivative in live HeLa
cells was shown to be cell-permeable through intracellular Diels-Alder
chemistry and to washout more rapidly than other TCOs.
For example, the most frequently utilized TCO derivatives are the
axial and equatorial diastereomers of 5-hydroxy-trans-
cyclooctene, which are produced through photoisomerization of
5-hydroxy-cis-cyclooctene in 72%.[6] Derivatives of the axial
diastereomer have emerged as especially useful, as they are an
order of magnitude
more reactive than
equatorial
diastereomers,[5,10] can promote fluorogenic effects for cell
imaging,[11] and have been employed in ‘click-to-release’
strategies for bioorthogonal uncaging.[3a,3b]
However,
photoisomerization produces the equatorial:axial isomers in 2.2:1
ratio, and thus produces ≤24% of the axial diastereomer.
A
modified procedure using Ag(I) sulfonated silica gel slightly
improves the yield of the axial diastereomer to ≤27%.[12] Even for
the equatorial diastereomer, the need to separate diastereomers
represents a limitation for material throughput. For other TCO
derivatives, chromatographic separation of diastereomers can be
very difficult and not feasible by flash chromatography.[13] One
solution to the diastereomer issue has been to utilize s-TCO— a
highly strained dienophile prepared from the meso compound
precursor.[14] s-TCO is most reactive dienophile known, and is
suitable for applications as a probe molecule in bioorthogonal
chemistry,[3e,15] but due to alkene isomerization, s-TCO is often
unsuitable as a reporter where more prolonged cellular incubation
is required.[16] Currently, several TCO derivatives are available
for purchase, but they are very expensive. A general and
diastereoselective synthesis of TCO derivatives could greatly
increase the availability of these useful compounds for chemical
biology research.
In addition to stereoselectivity, hydrophobicity has been a
longstanding issue with TCOs where non-specific binding and
extensive wash-out protocols in live cell assays are undesirable
consequences.[17] Recently, heterocyclic trans-cyclooctenes with
backbone oxygen atoms have been shown to have higher
hydrophilicity and improved physiochemical properties for cellular
and in vivo imaging applications.[13,18] However, a drawback for
these oxo-TCOs are lengthy syntheses that produce
diastereomers that can be separated only with difficulty.
Introduction
Bioorthogonal reactions are a class of rapid, selective reactions
that can proceed efficiently and selectively in biological systems
without interfering with biological functional groups.[1]
Bioorthogonal chemistry has enabled a deeper understanding of
native biological processes and expanded the frontiers of
chemical biology through innovations in nuclear medicine,[2] drug
delivery,[3] and biomaterials.[4] The tetrazine ligation with trans-
cyclooctenes (TCOs) has been at the forefront of bioorthogonal
methodologies due to rapid reaction kinetics that generally
exceed k2 104 M–1s–1.[5]
TCO derivatives are synthesized by a photochemical flow-method
under singlet sensitized conditions, driving an otherwise
unfavorable isomeric ratio in favor of the trans-isomer via
selective metal complexation to Ag(I).[6] Flow photoisomerization
has been carried out on scales as large as 20 grams,[7] and
protocols have been developed where flow is mimicked by
periodically stopping irradiation and capturing TCO product by
filtering through AgNO3-silica and resubjecting the filtrate to
photoisomerization (254 nm).[8] While this method has been used
to synthesize a large number of different TCO derivatives,[9] the
production of diastereomers due to the planar chirality of the
alkene presents a bottleneck for the majority of TCO syntheses.
Described here is a diastereoselective method for synthesizing
TCOs with favorable physiochemical properties in high yield and
useful scale through the stereocontrolled additions of
nucleophiles to trans-cyclooct-4-enone 2 (Figure 1). As 2 lacks a
stereocenter, it therefore can be prepared on large scale using
1
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10.1002/anie.202101483
Angewandte Chemie International Edition
RESEARCH ARTICLE
Previously
A
• Poor diastereoselectivity
• Separation bottleneck
Pd(OAc)2
(5 mol %)
• single
diastereomer
• grams
hν
hν
flow
62%
1R
AcOH
benzoquinone
O
O
flow
1R
2
1R
1
3
44%
This work
• Single Diastereomer
• Improved hydrophilicity
• Divergent synthesis
• Facile purification
B
equatorial
face
R–
H
OH
hν
flow
LiAlH4
THF
R
O
OH
H
4a
4b
O
2
axial
face
O
OH
a-TCOs
1
2
C
1 step from 1,5-cyclooctadiene
Equatorial Face (favored)
ΔG‡ 14.09 kcal/mol
ΔH‡ 12.00 kcal/mol
νi = 477.2i cm-1
Figure 1. Previous approach to trans-cyclooctene syntheses compared to the
general, divergent, and diastereoselective platform described in this paper.
H
H
Al
H
H
H
O
H
Li
flow photochemistry from cis-cyclooct-4-enone (1) without the
complication of diastereoselectivity in the photoisomerization step
encountered previously. The rigid structure of 2 distinguishes the
faces of the ketone, and computation was used to predict that
nucleophilic additions to 2 take place exclusively from the
equatorial-face of the ketone to produce axial products as single
diastereomers. By engaging a range of nucleophiles, ketone 2
ΔΔG‡ 2.8 kcal/mol
ΔΔH‡ 3.4 kcal/mol
Axial Face TS (disfavored)
O
ΔG‡ 16.85 kcal/mol
ΔH‡ 15.37 kcal/mol
νi = 370.6i cm-1
H
H
H
Li
Al
H
H
serves as
functionalized
a
general platform for preparing
axial-5-hydroxy-trans-cyclooctene
a
range of
(‘a-TCO’)
H
analogs. The method provides improved access to new
compounds as well as known TCO derivatives required for in vivo
radiochemistry,[10] click-to-release chemistry,[2a,3a,3e] and sulfenic
acid detection in live cells.[19] An a-TCO derivative was shown to
be more reactive than both axial and equatorial diastereomers of
Figure 2. (A) Synthesis of cis-cyclooct-4-enone 1 and trans-cyclooct-4-enone 2.
(B) Reaction of trans-cyclooct-4-enone 2 with LiAlH4. Nucleophilic addition can
occur to the equatorial or axial face of 2 producing diastereomers 4a and 4b,
respectively. (B) Transition state calculations predict nucleophilic addition to
equatorial face would be favored over the axial face.
5-hydroxy-trans-cyclooctene
as
well as oxo-TCO
in
cycloadditions with tetrazines. As a demonstration of the
favorable physicochemical properties of a-TCOs, a fluorescent
TAMRA derivative was shown to be cell-permeable by
demonstrating intracellular Diels-Alder reactions in live cells and
was shown to washout of HeLa cells more rapidly than even a
hydrophilic oxo-TCO analog.
barrier is significantly lower than that calculated for axial attack
(ΔΔG‡ 2.8 kcal/mol and ΔΔH‡ 3.4 kcal/mol). Encouraged by these
computational predictions, we experimentally investigated
additions of nucleophiles to TCO 2 (Scheme 1A). In agreement
with the computational data, nucleophilic addition of hydride
occurred exclusively to the equatorial face of 2 to produce 4a.
While LiAlH4 gave 4a in only 67% yield due partial alkene
isomerization, NaBH4 provided 4a as a single diastereomer in
90% yield with no isomerization. Derivatives of axial alcohol 4a
are especially useful for their rapid kinetics[10] and their utility in
click-to-release chemistry,[3e] but previous syntheses of 4a were
low yielding (≤27%) and required separation from major
diastereomer 4b.[6,12] The improved route described here is short,
selective and more scalable.
Nucleophilic additions with a diverse array of nucleophiles were
preformed to create a library of a-TCOs (Scheme 1). Compound
5 bearing a bioorthogonal alkyne tag was recently developed for
the capture of cellular protein sulfenic acids via transannular
thioetherification with subsequent proteomic analysis enabled by
a CuAAC-chemistry workflow.[19] We note that 5 (and other a-
TCOs) are still suitable for selective bioorthogonal chemistry as
they only modify sulfenic acids at relatively high TCO
concentration (generally ≥500 µM), but not under the conditions
typically used in intracellular bioorthogonal chemistry (generally
≤10 µM). In the previous study, 5 was synthesized by direct
photoisomerization in only 6% yield and required separation from
two isomers. As shown in Scheme 1A, compound 5 can be
Results and Discussion
Ketone 1 was prepared simply in a single step by the Wacker
oxidation (5 mol% Pd(OAc)2, AcOH, benzoquinone) of 1,5-
cyclooctadiene 3 on multigram scale (Figure 2A).[20] Alternately, 1
can be prepared in a single step by Dess-Martin oxidation of 5-
hydroxy-cis-cyclooctene.19 Photochemical isomerization of 1 was
conducted using the general photochemical flow method.[6,9]
Using a small cartridge and bed of capture silica, ketone 2 was
prepared in 62% yield and at a rate of ~150 mg/h, and in a typical
workflow, ketone 2 was prepared in 2.5 g batches.
Computation was used to predict if nucleophilic addition to TCO 2
would be diastereoselective (Figure 2B). It was reasoned that 1,3-
diaxial interactions in the lowest energy ‘crown’ conformation of
the parent TCO would favor addition to the equatorial face by
nucleophiles, akin to the facial selectivity of conformationally
biased cyclohexanones.[21] The barriers for the reaction of LiAlH4
with 2 were calculated at the M06L/6-311+G(d,p) level using a
SCRF solvent model for THF (Figure 2C). The calculated barriers
relative to the pre-reaction complex for the equatorial attack by
hydride are ΔG‡ 14.09 kcal mol-1 and ΔH‡ 12.00 kcal mol–1. The
2
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10.1002/anie.202101483
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RESEARCH ARTICLE
Universal Platform
A
axial
alcohol
oxime
conjugates
RO
H
OH
4a
N
OH
CuAAC
MgBr
ZnBr
18: R = H, 81%
19: R = OBn, 83%
5
RO–NH2
NaBH4
90%
2 86%
Clickable tags
LiHMDS
MeCN
Br
98%
N
85%
Zn
OH
10
2
O
OH
bis-dienophile
(Tetrazine)
LiHMDS
MeOAc
6
Conjugation
precursors
Ph
TMEDA
n-BuLi
92%
PhLi
98%
86%
MeO
O
OH
9
7
Raman tag
OH
8
OH
O
B
Functional Derivatives
+
Me2N
Acid/aldehyde reactive
NMe2
Amine reactive
Fluorescent
H
N
O
O
N
TAMRA-aTCO
(12)
H2N
–
CO2
O
OH
O
13
OH
11
79%, 1 step from 11
O
quant.
1 step from 11
89%, 2 steps from 9
H
N
(
)
4
O
N
H
O
OH
O
O
N
Alkylation Precursor
Clickable tag (CuAAC)
Acid reactive
Thiol reactive
HN
HO
H2N
O
O
OH
17
OH
89%, 1 step from 9
OH
93%, 1 step from 10
OH
14
15
16
96%, 1 step from 14
86%, 1 step from 11
Scheme 1. (A) Nucleophilic addition reactions of trans-cyclooct-4-enone (2) serves as a universal platform for the diastereoselective synthesis of a-
TCOs as well as oxime conjugates. (B) Functional derivatives readily available from conjugation precursors 9 and 10
prepared in 86% yield as a single diastereomer by adding
propargyl magnesium bromide to ketone 2. Similarly, compound
6 bearing a simple alkene tag can be constructed in 85% yield
and as a single diastereomer by the addition of allyl zinc bromide
to ketone 2. Like trans-cyclooctenes, simple α-olefins can
function as dienophiles in tetrazine ligation but with much slower
kinetics, providing handles for potential sequential bioorthogonal
chemistry applications.[22] Organolithium nucleophiles generated
in-situ were used to synthesize TCOs 7-8. Illustrating the ability
to introduce a tag that may be useful for Raman spectroscopy and
trimethyltin hydroxide[24] followed by DIC-mediated coupling with
N-hydroxysuccinimide gave NHS ester 11 in 89% yield (Scheme
1B). Alternately, using LiOH gave 11 in 46% yield after DIC
coupling. Through amide coupling, 11 gave the fluorescent
TAMRA-conjugate 12 with favorable physiochemical properties,
and 11 was also combined with hydrazine monohydrate to give
hydrazide 13 with a potential handle for aldehyde conjugation.[25]
TCO 9 also was combined with LiAlH4 to provide diol 14 in 89%
yield which was next used in a Williamson ether synthesis with
NaH/propargyl bromide to give 96% yield of propargyl ether 15.
Compound 15 serves a more stable alternative to alkyne-tagged
TCO 6, which is found to polymerize if not stored in solution.
Amine 16 was synthesized by LiAlH4 reduction of TCO nitrile 10
in 93% yield to introduce an acid reactive conjugation handle.
Maleimide 17 was prepared though amide coupling of TCO 11
with N-(2-aminoethyl)maleimide in 86% yield. The yield of 17 is
significantly higher than what was observed in previous
imaging,[23] TCO
7 bearing a phenylacetylene group was
synthesized by the addition of lithium phenyl acetylene to 2 in 92%
yield. TCO 8 was synthesized similarly by the addition of phenyl
lithium to 2 in 98% yield and serves as a model reaction for
nucleophilic addition of aryl groups to TCO 2.
The diastereoselective additions of 2 with methyl α-lithioacetate
or lithioacetonitrile provided a straightforward path to introduce
handles for conjugation via amide bond or ether formation. The
reaction of ketone 2 with LiHMDS/methyl acetate produced ester
9 in 86% yield. Similarly, the combination of 2 with LiHMDS and
acetonitrile produced nitrile 10 in 98% yield. Hydrolysis of 9 with
preparations
of
TCO-maleimides
from
activated
nitrophenylcarbonates.[14] Furthermore, oxime conjugates 18 and
19 could be prepared by the direct condensation of the
corresponding hydroxylamines in the presence of pyridine
3
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10.1002/anie.202101483
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RESEARCH ARTICLE
A
kinetics
compare:
a-TCO derivatives also display rapid kinetics in Diels-Alder
reactions compared to most other TCOs. The kinetics for the
reaction of a-TCO derivative 14 toward a 3,6-dipyridyl-s- tetrazinyl
succinamic acid derivative 20 were measured by stopped flow
kinetics at 25 °C in 95:5 PBS:MeOH (Figure 3A). With a second-
order rate constant of 150,000 ± 8000 M–1s–1, a- TCO is more than
twice as reactive toward 20 as the axial isomer of 5-hydroxy-trans-
cyclooctene (4a, 70,000 ± 1800 M–1s–1), and nearly 7-times more
reactive than equatorial isomer 4b (22,400 ± 40 M–1s–1).[5] The
faster kinetics are likely due an increase in olefinic strain for a-
TCO due to steric effect of geminal substitution in the 8-
membered ring backbone. Likely for the same reason, similar rate
accelerations have been observed for more sterically
encumbered derivatives of 4a.[10] a-TCO 14 is also more reactive
than oxo-TCO,[13] and the conformationally strained, bicyclic d-
TCO is only 2.2-times more reactive than 14.[5] As shown in
Figure 3B, a-TCO derivatives are also calculated to have
improved physiochemical properties relative to other TCO
derivatives. While both oxo-TCO and d-TCO were previously
introduced as less hydrophobic bioorthogonal reagents, the
methylamine conjugate of a-TCO is calculated to have even a
lower cLogP value.
4bd:
HO
a-TCOa: 150,000 M-1s-1
4aa,b
:
O
HO
HO2C
0.1 mM
NH
0.20
0.15
0.10
0.05
0.00
axial
equatorial
2.6 mM
HO
N
22,400 M-1s-1
70,000 M-1s-1
20
HO
N
N
N
d-TCOd
oxoTCOc
N
OH
OH
a-TCO (14)
2-pyr
O
O
O
stopped flow
kinetics
0.00
0.05
0.10
94,600 M-1s-1 366,000 M-1s-1
NHMe
Time (s)
B
O
LogP calculations
NHMe
O
NHMe
NHMe
O
O
O
O
O
HO
O
O
O
a-TCO
cLogP: 1.11
TCO
oxoTCO
cLogP: 1.33
d-TCO
cLogP: 1.76
cLogP: 1.95
C
cell permeability and microscopy
fusion proteins localized
to nucleus (HaloTag-H2B-
GFP) or cytoplasm
The stability of a-TCO 14 is very similar to that of axial-5-hydroxy-
trans-cyclooctene 4a, which is used broadly for applications in
bioorthgonal chemistry. In MeOD, 35 mM solutions of both 14
and 4a are >99% stable after 1 week at room temperature. After
24 h at room temperature in D2O-PBS (pD 7.4), 33 mM solutions
of 14 and 4a display 90% and 85% stability, respectively. In D2O-
PBS containing 25 mM mercaptoethanol, 49% of both 14 and 4
remained after 20 h.
HaloTag-transfected
HeLa cells
(HaloTag-GAP43-GFP)
H
N
2)
1)
O
TAMRA
O
N
O
N
(
)
5
O
OH
N
Me
N
Cl
MeTz-Halo (21)
TAMRA-a-TCO (12)
The fluorescent conjugate TAMRA-a-TCO 12 was shown to be
cell permeable through selective bioorthogonal reaction inside
live cells using the HaloTag self-labeling platform (Figure 3C).
Here, cells are transfected with a GFP-HaloTag construct fused
to a protein that controls subcellular localization, and then labeled
by a tetrazine-HaloTag ligand 21. Upon subsequent reaction with
fluorescent TAMRA-a-TCO, conjugation is expected only in those
cells that express the HaloTag fusion protein, and co-localization
of GFP and TAMRA fluorescence is expected. As shown in
Figure 3C, HeLa cells were transfected with either HaloTag-H2B-
GFP (nucleus) or HaloTag-GAP43-GFP (cytoplasm), labeled with
MeTz-Halo 21 (10 µM), washed and then treated with TAMRA-a-
TCO (1 µM) for 30 min, at which point the TCO reagent was
chased by a non-fluorescent tetrazine, and the cells were fixed
and imaged. As shown in Figure 3C, for both nuclear and
cytoplasmic targets, selective colocalization of the TAMRA signal
with GFP was observed in both cases.
The advantage of the increased hydrophilicity for a-TCO
conjugates was demonstrated by comparing the washout times
for fluorescent derivatives in mammalian cells. While TCO-
derivatives offer rapid labeling kinetics, a consideration for
labeling by fluorophore-tagged TCOs has been the background
fluorescence due to non-covalent cellular binding that can be
ameliorated only by extended washout times. More hydrophilic
oxo-[13] and dioxo-trans-cyclooctenes[18a] with backbone oxygens
offer improvements, but are difficult to synthesize and, for dioxo-
TCO, display reduced Diels-Alder kinetics. As shown in Figure 4,
we prepared the fluorescent conjugates TAMRA-TCO, TAMRA-
oxo-TCO and TAMRA-a-TCO and compared their cellular
washout times to unconjugated TAMRA. Thus, HeLa cells were
incubated for 30 min with TAMRA-dyes, and cells were initially
GAP43 (cytoplasm)
H2B (nucleus)
GFP
DAPI
DAPI
GFP
merge
TAMRA
merge
TAMRA
ak2 measured in 95:5 water:MeOH. bThe reaction of 4a with a PEGylated
amide of 20 in 100% H2O was previously measured as k2 80,200 M–1s–1
.
[5]
ck2 previously measured with 20 in 100% H2O.[13] dk2 previously measured
with PEGylated amide of 20 in 100% H2O.[5]
Figure 3. (A) Stopped flow kinetics under pseudo-first order conditions were
used to determine second order rate constants for the reactions of tetrazine 20
with 14, allowing comparison to less reactive 4a and 4b. (B) cLogP calculations
for a series of analogs of TCO, oxoTCO, d-TCO, and a-TCO illustrate the
improved hydrophilicity of a-TCO conjugates. (C) Cell permeability was
demonstrated by incorporation of MeTz-Halo (21) into HeLa cells transfected
with either H2B-HaloTag-GFP (nuclear) or GAP43-HaloTag-GFP (cytoplasmic),
followed by labeling with TAMRA-a-TCO (1 µM). Confocal microscopy images
of transfected cells labeled with TAMRA-a-TCO show subcellular colocalization
of GFP and TAMRA fluorescence, consistent with selective intracellular labeling.
Scale bars for H2B and GAP43 labeling are 5 µM and 10 µM, respectively.
(Scheme 1A). In summary, ketone 2 can serve as a readily
prepared, central intermediate for the diastereoselective
preparation of a range of hydrophilic a-TCO conjugates.
4
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10.1002/anie.202101483
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RESEARCH ARTICLE
additions to deliver a-TCO products. The strategy can be applied
to the synthesis of a range of usefully functionalized a-TCOs with
high yield, selectivity. a-TCOs were also shown to be more
reactive than standard TCOs and less hydrophobic than even
hydrophilic oxo-TCO analogs. As a demonstration of the
favorable physicochemical properties of a-TCOs, a fluorescent
TAMRA derivative was shown to be cell-permeable by
demonstrating intracellular Diels-Alder chemistry in live cells and
to washout of HeLa cells more rapidly and completely than TCO
and oxo-TCO analogs.
Acknowledgements
This work was supported by NIH GM132460 and Pfizer.
Instrumentation was supported by NIH awards P20GM104316,
P30GM110758, S10RR026962, and S10OD016267 and NSF
awards CHE-0840401, CHE-1229234, and CHE-1048367. J. R.
and A. J. were supported as CBI fellows through NIH T32-
GM1333395.
# =equal contributions
Keywords: trans-cyclooctene • tetrazine • diastereoselective •
hydrophilic • bioorthogonal
Figure 4. (A) Structures of TAMRA and conjugates with TCO, oxo-TCO and a-
TCO. (B,C) HeLa cells were incubated for 30 min with TAMRA-dyes, and cells
were initially washed three times with PBS, and then cell media was exchanged
after 10, 40 and 120 minutes. After each wash, cells were imaged live by
fluorescence microscopy with illumination at 531 nm and with fixed-intensity
across all samples. (B) Widefield images cells after 3 washes. (C) Comparison
of background fluorescence across all experiments, quantified by dividing total
fluorescence by the number of cells in each image.
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washed three times with DPBS, and then cell media was
exchanged after 10, 40 and 120 minutes. After each wash, cells
were imaged live by fluorescence microscopy with illumination at
531 nm and fixed-intensity across all samples. Widefield images
of the cells after 3 washes are shown in Figure 4B; images after
the earlier and later washings are shown in Figure S10.
Background fluorescence was quantified by dividing total
fluorescence by the number of cells in each image (Figure 4C).
For TAMRA-TCO, cells are markedly fluorescent after 3 washings,
and still display significant background after washing for 2 hours.
The background is improved with TAMRA-oxo-TCO and
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fluorescence even after 2 hours.
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Conclusion
In summary, a-TCOs are a class of trans-cyclooctenes with
favorable physiochemical properties that can be prepared in high
yield through the stereocontrolled additions of nucleophiles to
trans-cyclooct-4-enone (2), a trans-cyclooctene that can be
prepared on large scale in two steps from 1,5-cyclooctadiene.
Computation was used to rationalize diastereoselectivity of 1,2-
5
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RESEARCH ARTICLE
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6
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RESEARCH ARTICLE
Entry for the Table of Contents
equatorial face
• Divergent synthesis
• Diastereoselective
• Improved hydrophilicity
• Rapid kinetics
R–
R
O
OH
a-TCOs
axial
face
Bottleneck removed! Readily accessible trans-cyclooct-4-enone
undergoes diastereoselective additions with a broad range of
nucleophiles to give access to a-TCOs, a family of trans-
cyclooctenes with high reactivity and improved physiochemical
properties.
Institute and/or researcher Twitter usernames: @FoxGroupUD,
@ChemistryUD
7
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