2
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W. Li et al. / Tetrahedron Letters 57 (2016) 2539–2543
and explored their conformation-bioactivity interconnectivity. It
was found that the synergetic combination of the distinct proper-
ties of azobenzene and bile acid moieties, the stable and control-
lable tweezer conformation with tunable hydrophilic and
hydrophobic channels can be obtained conveniently. Interestingly,
at this tweezer-like conformation the antibacterial activity of the
synthesized bile acid conjugates for both Gram-positive and
Gram-negative bacteria increased apparently. However, when the
molecules conformation changed back to the extended state, the
antibacterial activity decreased simultaneously. For azobenzene-
bridged deoxycholic acid dimers, the antibacterial activity was
even switched off for Gram-positive bacteria. More importantly,
this bioactivity conversion can be carried out reversibly upon the
molecule conformation changes from the extended state to the
tweezer-like form.
Scheme 1. Chemical structure of the azobenzene linked bile acid dimers (CA-azo,
DA-azo and LA-azo) and the model compound (P-azo).
However, the further investigation proves that the rates of the
conformation change of these three bile acid-azobenzene conju-
gates are also varied and strongly affected by the subtle variation
of the pendant groups, especially in the relative concentrated solu-
Scheme 1 shows the chemical structures of synthesized azoben-
zene linked dimeric cholic acid (CA-azo), deoxycholic acid (DA-
azo), and lithocholic acid (LA-azo). Attempts to directly condense
1
0
the bile acid with 4,4 -azodianiline in the presence of DCC proved
tion (ꢀ10 mg/mL). For this concentration, H NMR measurement
to be rather sluggish and afforded a complex mixture of products.
In an alternative way (Scheme S1), the hydroxyl groups of bile acid
were first protected by formylation with anhydrous formic acid in
almost quantitative yield. Then coupling of the acid chloride with
diaminoazobenzene in the presence of triethylamine gave the bis-
can be performed to calculate the ratio of different configurations
accurately by the integral of the corresponding signals. As shown
in Figure S1, for CA-azo the fraction of the tweezer-like conforma-
tion is much lower than that of LA-azo under the same intensity of
light irritation. The conformation change rate of DA-azo, which
possesses two hydroxyl groups at the steroid nucleus, lies right
in between the values of CA-azo and LA-azo. It is clear that the
only thing different between the three bile acid derivatives is the
number of the hydroxyl groups on the skeleton, so the different
rate of isomerization could no longer be simply explained by the
steric hindrance due to size of the pendant groups. In the concen-
trated solution (Scheme S2), it was assumed that the hydrophobic
face of bile acid tends to aggregate together in the polar media,
leaving the hydrophilic groups pointing toward the solvents and
reducing the interfacial energy. The hydroxyl groups are expected
to form hydrogen bonds with the solvent molecules. The more
hydroxyl groups the compounds present, the stronger hydrogen
bond will be formed between the pendant groups and solvent
molecules. Therefore, not only the size of the pendant group, but
also the properties of the substituents around the azobenzene core
determine the rate and activation energy barrier for the conforma-
tion change process.
Bridged by azobenzene groups, these bile acid derivatives with
tweezer-like configuration tend to revert to the extended state
thermally once the optical irritation was absence. This behavior
may interfere with our bioactivity test and leave unpredictable
result for the tweezer-like configuration, so it is necessary to char-
acterize the stability of the tweezer-like state before the antimicro-
bial screen. In order to obtain a useful reference for the conversion
rate during the bioactivity test, the tweezer-like state of both CA-
azo and P-azo in solution was placed in dark at 30 °C, which is
the temperature that the bacteria were cultured. Then the kinetics
of the thermal-induced conformation reversion was measured by
following the changes in absorbance at 371 nm. As displayed in
2
coupled products. Hydrolysis of the diamide with LiOH in THF–H O
led to the desired bile acid dimers in about 80% yield. The three
kinds of molecules are differentiated only by the number of hydro-
xyl groups. As the model compound for comparison, azobenzene
with two butane end groups (P-azo) was also synthesized.
It was well recognized that azobenzene can undergo photoiso-
merization from a full-conjugated trans configuration to a cis iso-
mer under illumination by 365 nm light.12 Thermal cis to trans
relaxation in the dark leads to 100% trans isomer reversibly. As
1
demonstrated by H NMR spectra of azobenzene-bridged bile
acid dimers (Figs. S15, S17, S19), the peaks at about 7.80 ppm
are ascribed to the aromatic protons of trans isomers and no
peaks belonging to the cis isomers were observed,13 revealing a
pure trans isomers before irradiating by 365 nm light. The
extended trans conformation can change to a tweezer-like state
in a reversible manner. And the tweezer-like conformation is
expected to be adaptable to the polarity of the surroundings
and led to the tunable hydrophilic and hydrophobic channels
(Fig. 1a). Due to the facial amphiphilic feature of the pendant bile
acid moieties, the tweezer-like conformation can be more pre-
dictable and stable, thereby facilitating our effort to evaluate
the bioactivity of these two different conformation states and
further probe the conformation-bioactivity relationship of these
bile acid derivatives.
The conformation switchable behavior of these azobenzene-
bridged bile acid dimers and the model compound P-azo was first
3
investigated. When irradiating the CA-azo solution (1 mM in CH -
OH) with 365 nm light, the absorption band at 371 nm which is
⁄
attributed to the
p
–p
transition of the trans-azobenzene
decreases, along with an increase of the band around 455 nm,
Figure S2, the thermal half-lives (
which was found to be extraordinarily extended as compared to
P-azo ( 1/2 < 1.5 h) in CH OH. The result indicated that once the
s1/2) of CA-azo are about 3.5 h,
⁄
which is attributed to the n–
p
absorption (Fig. 1b). This result
indicates that the CA-azo undergoes the expected conformation
change and the Uv–vis spectra further confirmed the presence of
pure trans isomers before irradiation by 365 nm light. The isomer-
ization of CA-azo proceeds relatively slowly compared to the
model compound P-azo (Fig. 1c), which only need less than
s
3
molecule conformation changed to tweezer-like state, CA-azo pre-
sented a higher thermal-energy barrier to convert back to the
extended form compared with that of P-azo. Besides the above
mentioned steric hindrance effect, this phenomenon could also
be contributed to the formation of tightly folded conformation by
hydrophobic and hydrophilic interactions in solvents. In other
words, the tweezer-like configuration of bile acid dimers is more
stable than other typical azo derivatives.
3
min to complete isomerization. This phenomenon can be easily
understood by the fact that the azo unit of CA-azo was placed at
core of the molecule which was surrounded by two large and rigid
steroid skeletons, so that its conformation state change encounters
more significant resistance than P-azo with only two short and
flexible end groups.
As demonstrated by the conformation change of bile acid
derivatives, the 100% tweezer-like isomers is not expected to be