Chemistry Reference
In-Depth Information
of dibenzocyclooctynes (dIbo) to detect azide, and they exhibited comparable reactivity to dIFo. Without electron withdrawing
groups, the dibenzo system achieved similar kinetics as dIFo, probably through increased strain energy. Researchers from the
netherlands have reported a bicyclo[6.1.0]nonyne (bCn) as a novel ring-strained alkyne [151]. The synthesis of bCn conjugates
was highly straightforward from cyclopropanation of 1,5-cyclooctadiene, and the bCn probes showed around twofold better
kinetics compared with dIbo in the cycloaddition reaction with azides. It has been used in the labelling of proteins and glycans,
and in the three-dimensional visualisation of living melanoma [151]. The so-called bARAC, biarylazacyclooctynone, introduced
amide in the cyclooctyne ring adding even more strain energy through its resonant structure (Figure 2.5) [152]. The easily syn-
thesised (seven-step) bRACo, in fact, was around threefold more sensitive to the azido group than dIFo, making it promising
for
in vivo
labelling. Interestingly, a fluorogenic cyclooctyne was prepared based on the parent structure of bARAC and gave
10-fold enhancement in fluorescence quantum yield upon triazole formation with organic azides. This may find its use in
real-time imaging of azide-labelled biomolecules [153]. To overcome the possible bioavailability problem caused by the hydro-
phobic nature of dIFo, hydrophilic azacyclooctyne, therein termed dIMAC, was synthesised in nine steps from glucose
(Figure 2.5) [154]. The reactivity of dIMAC was similar to that of the nonfluorinated cyclooctynes. examples using monofluo-
rinated cyclooctyne (MoFo) [142] and dibenzoazacyclooctyne (dIbAC) [155] were also examined. besides reactivity, lipophi-
licity of these compounds is another parameter for selection of proper reagents (Figure 2.5), because lipophilic compounds have
poor solubility in water, and may have hydrophobic interactions with proteins in the reaction mixture such as that in cells [149].
Along with the frequently used Alexa Fluor dyes and FITC in bertozzi's and other groups, bodIPy-cyclooctyne conjugates
[156] and luminescent quantum dot conjugates [157] were also applied for labelling in live cells.
besides
in vivo
labelling, the highly efficient 'copper-free' azide-alkyne cycloaddition can be used simply as a biocon-
jugation strategy
ex vivo
, such as
in situ
cross-linking of photodegradable polymeric networks using bifunctional, fluori-
nated cyclooctynes [158], cross-linking of communication-mediating (CoM) domains of nonribosomal peptide synthetase
(nRPS) proteins to study their protein-protein interactions [159], as well as dendrimer synthesis [88, 160].
Monofluorocyclooctyne functionalisation of radionuclide chelators, such as doTA and noTA, has been used to introduce
radiolabels such as In-111 to azide-modified peptides via this copper-free click chemistry [161, 162]. F-18 radiolabels were
also introduced to bombesin, a 14-amino acid neuropeptide that binds to gastrin-releasing peptide receptor, for diagnosis
and imaging of cancer [163].
Similar to the cyclooctyne-azide click chemistry, strain-promoted cycloadditions of cyclic nitrones with cyclooctynes are also
very efficient, or even faster in some cases than the reactions of azides [164, 165] and can also be useful tools in bioconjugation.
2.2.3
through reactions with alkenes
2.2.3.1 Diels-Alder Reaction with Tetrazine
1,2,4,5-Tetrazine or s-tetrazine can react with electron rich dienophiles at
room temperature via inverse electron-demand diels-Alder reactions, followed by a retro-[4
+
2] cycloaddition to release n
2
as the only byproduct [166, 167]. Later, Sauer studied the kinetics of inverse electron-demand diels-Alder reactions between
electron-deficient tetrazines with various linear and cyclic dienophiles, among which the highly strained
trans
-cyclooctene
was the fastest (seven orders of magnitude faster than the
cis
-cyclooctene) (Scheme 2.8) [168]. In protic solvents, the 4,5-dihy-
dropyridizine rapidly rearranges to its isomer.
However, the tetrazines in Sauer's work underwent rapid hydrolysis in water. Fox and co-workers replaced the strongly
electron-withdrawing ester or trifluoromethyl groups with aromatic groups, achieving more stable tetrazine derivatives in
aqueous media, and made this extremely efficient reaction promising for bioconjugation (Scheme 2.8) [169]. The reactions
are orthogonal to a variety of functionalities and proceed in most organic solvents, water, cell media, or cell lysates in high
yields. For instance, in methanol/water mixture, the reaction between 3,6-di-(2-pyridyl)-s-tetrazine and
trans
-cyclooctene
was extremely fast at a second-order rate constant of 2000 M
-1
s
-1
with quantitative yield, while only micromolar reagents
were needed. Recently Fox et al. reported a group of even more reactive
trans
-cyclooctenes [170]. use of the
R=R
′
=CO
2
Me or CF
3
(In Sauer's work)
R
′
R
′
H
N
2
N
R
′
N
Rearr.
Water
N
NH
N
Diels-Alder
solvent
N
R=R
′
= Ph or
(In Fox's work)
N
Retro-[4+2]
N
H
R
N
N
R
R
NH
2
R
R
′
R=H, R
′
=
(In Weissleder's work)
NN
scheme 2.8
Strain-promoted diels-Alder reaction between tetrazine and
trans
-cyclooctene (TCo).
