A pH-sensitive peptide-containing lasso molecular switch

Article abstract of DOI:10.3390/molecules180911553

The synthesis of a peptide-containing lasso molecular switch by a self-entanglement strategy is described. The interlocked [1] rotaxane molecular machine consists of a benzometaphenylene[25]crown-8 (BMP25C8) macrocycle surrounding a molecular axle. This molecular axle contains a tripeptidic sequence and two molecular stations: a N-benzyltriazolium and a pH-sensitive anilinium station. The tripeptide is located between the macrocycle and the triazolium station, so that its conformation can be tailored depending on the shuttling of the macrocycle from one station to the other. At acidic pH, the macrocycle resides around the anilinium moiety, whereas it shuttles around the triazolium station after deprotonation. This molecular machinery thus forces the lasso to adopt a tightened or a loosened conformation.

Full text of DOI:10.3390/molecules180911553

Molecules 2013, 18, 11553-11575; doi:10.3390/molecules180911553
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
A pH-Sensitive Peptide-Containing Lasso Molecular Switch
Caroline Clavel, Karine Fournel-Marotte and Frédéric Coutrot *
Supramolecular Machines and ARchitectures Team, Institut des Biomolécules Max Mousseron,
(IBMM) UMR 5247 CNRS-UM1-UM2, Université Montpellier 2, Place Eugène Bataillon,
Case Courrier 1706, F-34095 Montpellier Cedex 5, France
* Author to whom correspondence should be addressed; E-Mail: frederic.coutrot@univ-montp2.fr;
Tel.: +33-0-4-6714-3843; Fax: +33-0-4-6763-1046.
Received: 24 July 2013; in revised form: 9 September 2013 / Accepted: 11 September 2013 /
Published: 17 September 2013
Abstract: The synthesis of a peptide-containing lasso molecular switch by a self-
entanglement strategy is described. The interlocked [1] rotaxane molecular machine
consists of a benzometaphenylene[25]crown-8 (BMP25C8) macrocycle surrounding a
molecular axle. This molecular axle contains a tripeptidic sequence and two molecular
stations: a N-benzyltriazolium and a pH-sensitive anilinium station. The tripeptide is
located between the macrocycle and the triazolium station, so that its conformation can be
tailored depending on the shuttling of the macrocycle from one station to the other. At
acidic pH, the macrocycle resides around the anilinium moiety, whereas it shuttles around
the triazolium station after deprotonation. This molecular machinery thus forces the lasso
to adopt a tightened or a loosened conformation.
Keywords: [1]rotaxane; lasso; molecular switch; self-entanglement; peptide
1. Introduction
As part of our research concerning the synthesis of interlocked molecular machines, we were
interested in the synthesis of lasso compounds. In Nature, lasso molecules, more precisely lasso
peptides, can be secreted by bacteria. These compounds contain 16 to 21 amino acid residues which
define a stable lasso molecular shape (i.e., a peptide macrocycle covalently linked to a peptide axle
which threads the macrocycle) with a very constrained conformation, in comparison with their
unthreaded analogues. The macrocycle is threaded by the peptide tail, whose bulky side-chains act as

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molecular barriers to trap the macrocycle around the peptide axle in the lasso conformation. The
encircled peptide axle then adopts a looped conformation, conferring to the lasso a very compact
tridimensional structure, which is responsible for its biological activity. Beyond their remarkable
stability against proteolytic degradation, chemical and thermal denaturation [1–3], some lasso peptides
proved to inhibit HIV replication [4] or the Gram-negative RNA polymerase [5]. As a particular
example of lasso peptide, the ribosomally synthesized [6,7] antimicrobial peptide microcin J25
(MccJ25), which was isolated from E. coli in 1992, [8] has been extensively studied during the past
years [9–12]. The amino acid sequence was first proposed by Blond et al. in 1999, who proposed a
21-residue, head-to-tail cyclic structure [13]. Four years later, three teams (Montelione and Ebright
et al., Craik et al., and Darst et al.) demonstrated concomitantly that the initial proposed covalent and
three-dimensional structure was incorrect, and that the peptide was in fact a lasso compound [14–16].
In view of their structural-dependent properties, lasso peptides could be considered as potential
scaffold for therapeutic peptides [17]. With this aim, several biosyntheses and structure-activity
analysis of a wide range of lasso peptides were realized [18–20]. Recently, Marahiel et al. have
highlighted the interest of incorporating a RGD peptide sequence in the turn of the lasso MccJ25 [21].
They substituted the native Gly12Ile13Gly14 by the ArgGlyAsp sequence in the loop of the lasso, using
site-directed mutagenesis of the precursor protein McjA. Interestingly, they found that the use of a
lasso scaffold triggers a specific conformation of the incorporated bioactive peptide sequence, which is
responsible for specific and distinct physical, biological and chemical properties, with respect to their
linear shape. To date, no lasso peptide has been synthesized using chemical protocols, probably
because of the highly difficult synthetic challenge, especially due to the folding of peptides which
disturbs the necessary preorganization between the components to be assembled into an interlocked
lasso. However, other more or less related works concerning peptides and interlocked molecular
architectures such as peptido[2]rotaxanes have already been reported. Leigh et al. have been the first to
surround the dipeptide unit GlyGly with a tetramide macrocycle, using hydrogen bond interactions
between the peptide amide carbonyl groups of the thread and the NH of the macrocycle [22]. They
next extended their efficient five-components hydrogen bond directed clipping strategy to the synthesis
and the study of a wide range of dipeptides [23–26]. They also prepared many [2]rotaxane molecular
shuttles in which the encircled thread contains different peptide moieties as molecular stations [27–35].
Inversely, some [2]rotaxanes, in which the macrocycles consist of cyclic peptides were reported too [36].
More recently, Moretto et al. employed the Leigh’s strategy to yield an oligopeptide [2]rotaxane
shuttle, in which the longest part of the axle is a rigid helical peptide [37]. It is also noteworthy that
some biological applications of the peptide rotaxanes emerged. Indeed, Leigh et al. prepared a
pentapeptide [2]rotaxane derived from the Met-enkephalin in which the peptide core is protected
against peptidase-catalyzed hydrolysis, until it is released by the action of a galactosidase [38,39].
Anderson et al. reported an enzymatic synthesis, using α-chymotrypsin, of a peptide rotaxane based on
a cyclodextrin and a peptide thread containing a diazo moiety. They also described the resistance of the
obtained peptide rotaxane against enzyme-catalyzed hydrolysis depending on the photoisomerization
of the diazo moiety [40]. Meanwhile, Smithrud et al. designed rotaxanes as peptide carriers, using a
grafted crown ether and a calix[4]arene, cyclophane, or cleft as a blocking group [41]. It remains from
all these studies that protection of peptides against enzymatic degradation, on one hand, and the

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tailoring of the shape of peptides, on another hand, are two main problems to tackle for the conception
of new efficient peptide drugs.
Since the properties of a molecule highly rely on its topology, we were interested in combining a
lasso molecular architecture that contains a peptide sequence with the molecular machinery. Therefore,
we report, in this paper, a first synthetic approach of a lasso molecule containing, in its turn, the
simplest GlyGlyGly peptide as a model sequence (Figure 1).
Figure 1. Cartoon representation of the molecular lasso targets and of the synthetic
strategy used to prepare pH-sensitive peptide-containing lasso molecular switch.
Self-entanglement
stopper
Trapping with a
molecular barrier
Peptide in a bent
conformation
deprotonated pH-sensitive
protonated pH-sensitive
molecular station
Peptide
molecular station
Base
Acid
Tightened lasso
Loosened lasso
molecular station and
barrier at the same time
Affinity of the stations for the macrocycle
> >
The lasso has also been designed so that it could be possible to act as a pH-sensitive molecular
shuttle. Thus, the shuttling of the macrocycle along the threaded axle could trigger a variation in the
conformation of the lasso (more or less tightened), hence causing a more or less bent conformation of
the peptide. This concept should then be attractive for switching the properties of a peptide part of a
lasso after applying an external stimulus.
The molecular machinery described in this paper is based on interactions between a crown ether
macrocycle and our already reported system of molecular stations [42]. Indeed, the lasso compound is
composed of a benzometaphenylene[25]crown-8 (BMP25C8) [43–46] macrocycle which surrounds a
molecular axle containing two molecular stations for the BMP25C8. The anilinium moiety [47–49] is
the best molecular station and is used as the molecular template for the rotaxane formation. The
N-benzyltriazolium station is of poorer affinity, as long as the anilinium remains protonated. At the
protonated state, the lasso lies in a loosened conformation without many constraints for the conformation of
the peptide. However, after deprotonation, the BMP25C8 shuttles around the triazolium station, causing a
tightening of the lasso and forcing the peptide to adopt a more bent conformation.

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2. Results and Discussion
2.1. Synthetic Strategy to Synthesize the Lasso Compounds
The necessary driving force to yield our interlocked lasso molecules lies on the interactions between
a crown ether and an ammonium moiety [50,51]. Even though the binding affinities of the molecular
stations are lower for the larger macrocycle BMP25C8 than for the smaller DB24C8, the formation of
the [1]rotaxane was still possible. In the present paper, the strategy used to interlock the molecular
structure is based on a self-entanglement [52] of a “hermaphrodite” molecule (i.e., a molecule
containing both a macrocycle as host and an axle holding the anilinium template as guest) (Figure 1).
The size of the cavity of the macrocycle is crucial for the successful self-entanglement strategy,
especially because the end of the molecular axle is already capped with a bulky stopper, which stops
the axle from threading through the macrocycle by this extremity. However, the BMP25C8 macrocycle
appears large enough to allow for the rotation of the meta-substituted aromatic ring. This internal
rotational movement in the BMP25C8 leads to the threading of the covalently linked anilinium-
containing molecular axle, until the macrocycle surrounds the anilinium molecular template. At this
stage, the lasso compound remains in equilibrium with the initial un-interlocked hermaphrodite
molecule. We can then take advantage of the presence of the triazole moiety, which is located between
the macrocycle and the anilinium station, in order to create the second molecular station (i.e., triazolium)
on one hand, and especially on the other hand, to incorporate a bulky side-chain that acts as a kinetic
molecular barrier [53] and traps the interlocked architecture.
2.2. Preliminary Results Obtained on a Non-Peptidic Lasso Molecular Switch
We have recently reported the feasibility of the self-entanglement strategy on a non-peptidic
molecule [54] (Scheme 1, left side). The hermaphrodite unthreaded molecule 4u, consisting of the
BMP25C8 macrocycle linked to the anilinium molecular axle, was prepared in a two-step sequence
from the already synthesized alkyne 1 [55]. This alkyne compound possesses a carbamoylated
di-tert-butylaniline moiety, so that no template effect can occur at this time.
The first step was a copper(I)-catalyzed Huisgen [56–59] 1,3-dipolar cycloaddition, also called
“CuAAC click chemistry [60,61], between the alkyne 1 and the azido BMP25C8 2 in dichloromethane
in the presence of 2,6-lutidine and Cu(MeCN)4PF6. This step led very efficiently to the triazole
compound 3, which was then submitted to a decarbamoylation that revealed the anilinium template
moiety. A slow equilibrium, on the NMR time scale, between the interlocked lasso 4 and the
uncomplexed molecule 4u could then be observed. Since the di-tert-butylanilinium end of the
molecular axle is too bulky to thread into the macrocycle, the only way to assemble the lasso
architecture is through self-entanglement. It was demonstrated that the best conditions to obtain the
lasso 4 was the use of dichloromethane, as a good hydrogen bond promoting solvent, and at high
dilution. In these optimal conditions, a maximum ratio 4/4u of 45/55 was measured. Subsequent
trapping of the lasso structure was realized by taking advantage of the localization of the triazole
moiety between the macrocycle and the anilinium molecular station. The benzylation of the triazole
afforded the lasso 5 in a 27% yield. Here, no more equilibrium between interlocked compound 5 and
uncomplexed compound 5u was possible, due to the incorporation of the benzyl moiety on the triazole

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which acts as a steric molecular barrier for the BMP25C8. After deprotonation of the lasso 5, we found
that the BMP25C8 shuttles toward the triazolium molecular station, thus tightening the lasso structure.
This process could be reversed in acidic medium.
Scheme 1. Synthesis of the molecular lassos and molecular machinery.

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2.3. Extension to the Preparation of a pH-Sensitive Tripeptide-Containing Lasso Molecular Switch
2.3.1. Synthetic Part
We then aimed to incorporate a peptide sequence in the turn of the lasso molecular architecture. The
simplest GlyGlyGly tripeptidic sequence was chosen in order to fit with the structural prerequisites for
the self-entanglement synthetic strategy. Indeed, no such self-entanglement should be authorized with
bulky amino acid side-chains. With the aim to be able to tailor the conformation of the peptide
depending on the molecular machinery applied to the lasso, the peptide sequence has been localized
the closest to the macrocycle BMP25C8, that is to say in the loop of the lasso. Thus, the azido-
containing macrocycle 10 was first synthesized according to a three-step sequence from the
commercially available BocGlyGlyGlyOH. Activation of the C-terminal side of the tripeptide using
Castro’s reagent BOP [62] in the presence of triethylamine and 2-azidoethylamine [63] afforded the
azido tripeptide 8 in quantitative yield. The further decarbamoylation using trifluoroacetic acid
provided the ammonium intermediate 9, which was then submitted to a coupling reaction with the
carboxylic acid substituted BMP25C8 to yield the azido macrocycle 10. Due to the very poor solubility
of compound 10 in dichloromethane and in acetonitrile at room temperature, the copper(I)-catalyzed
Huisgen 1,3-dipolar cycloaddition was accomplished with alkyne 1 in acetonitrile and at a temperature
of 70 °C. In these experimental conditions, 90% of the unthreaded compound 11 was isolated.
Decarbamoylation of 11 unmasked the anilinium template and allowed for the self-entanglement of the
hermaphrodite molecule 12u. The equilibrium between the lasso 12 and its unthreaded analogue 12u
was studied in details (see Section 2.3.2.). The subsequent benzylation of the lasso 12 allowed for the
trapping of the lasso molecular architecture: it was carried out in dichloromethane at high dilution with
respect to 12 and using a very large excess of benzyl bromide. Although the conversion rate for the
benzylation of the non-peptidic lasso 4 was quasi quantitative (only 5% of the starting triazole
compounds 4/4u were recovered after chromatographic columns), the formation of the benzyl
triazolium appears much more tricky in the case of the peptide-containing lasso. Indeed, after 7 days,
33% of the triazole compounds 12/12u were still detected by NMR spectroscopy. Moreover, several
chromatographic columns on both silica gel and sephadex yielded to a mixture composed of 17% of
the unthreaded benzyltriazolium 13u and 17% of the desired triazolium lasso 13. The separation
between these compounds was found to be very tough and only 4% of 13 could be isolated. We were
curious about the possibility to realize the benzylation at a higher temperature in order to accelerate the
1
rate of the reaction. Unfortunately, VT H-NMR experiments in C2D2Cl4 on 12/12u showed that
increasing the temperature results in the dramatic decrease of the proportion of the lasso compound 12
(Table 1, entries 3–7). Despite the poorly efficient reaction of benzylation, the deprotonation of the
isolated loosened lasso 13 was carried out efficiently by adding the Hünig’s [64] base DIEA.
Deprotonation of the anilinium triggered the shuttling of the BMP25C8 towards the triazolium moiety,
hence causing the tightening of the lasso and inducing the more constraint conformation of the
tripeptide (see Section 2.3.3.).

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2.3.2. Studies of the Equilibrium between the Unthreaded Compound 12u and the Lasso 12
The equilibrium between the unthreaded compound 12u and the lasso 12 highly depends on the
polarity of the solvent (Figure 2), on the concentration (Figure 3), and on the temperature.
1
Figure 2. H-NMR spectra (600 MHz, 298 K) at 5.10−4 M of a mixture of compounds
12/12u in: (a) CD3OD. (b) CD3CN. (c) CD2Cl2. The lettering and numbering correspond to
the proton assignments indicated in scheme 1. The orange color corresponds to the
unthreaded molecule 12u, whereas the green color corresponds to lasso compound 12. The
black assignments correspond to both compounds.
Table 1 summarizes the 12/12u ratios measured in the different solvents from the best hydrogen
bond promoting solvents dichloromethane and tetrachloroethane to the more polar solvent methanol
and upon variation of the concentration and temperature. As the equilibrium between 12 and 12u
1
proved to be slow on the NMR time scale, two sets of H-NMR signals were observed for each
1
unthreaded and lasso compounds. The ratio values reported in Table 1 were obtained by H- NMR
spectroscopy at 600 MHz by integrating the signal of hydrogens H15 or H12.

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Figure 3. 1H-NMR spectra (600 MHz, 298 K) of a mixture of compound 12/12u in CD2Cl2
at: (a) 5.10−2 M. (b) 5.10−3 M. (c) 5.10−4 M. The lettering and numbering correspond to the
proton assignments indicated in scheme 1. The orange color corresponds to the unthreaded
molecule 12u, whereas the green color corresponds to lasso compound 12.
Table 1. Ratio between lasso compound 12 and unthreaded molecule 12u depending on
concentration, solvent and temperature.
Entries C (12/12u) [M]
T (K)
298
298
298
318
328
338
348
CD3OD
CD3CN
CD2Cl2
C2D2Cl4
-
1
2
3
4
5
6
7
5.10−2
5.10−3
5.10−4
5.10−4
5.10−4
5.10−4
5.10−4
-
-
35/65
-
-
44/56
-
0/100
25/75
47/53
55/45
38/61
29/71
14/86
8/92
-
-
-
-
-
-
-
-
-
-
-
-
Obviously, it can be seen from the data reported in Table 1 that the nature of the solvent has a big
influence on the 12/12u ratio. At 298 K, the best self-entanglement was noticed in the less polar
dichloromethane and tetrachloroethane and at the highest dilution (Table 1, entry 3). At a similar
concentration, the proportion of the lasso 12 dramatically declines in the more polar acetonitrile and no
presence of the lasso was detected in methanol (Figure 2 and Table 1, entry 3). Eventually, increasing
the concentration of the sample in dichloromethane tends to be unfavorable to the interlocked lasso
architecture (Figure 3 and Table 1, entries 1–3).

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Besides, the equilibrium between 12u and 12 could be highlighted using drift tube ion mobility
mass spectrometry (DT IM-MS). By this analytical method, it was possible to separate compounds 12
and 12u, because they have different velocities which are due to their distinct sizes related to their
unlike conformational structures. A similar analytical study was utilized by Leigh et al. to distinguish a
trefoil knot from its unknotted-macrocycle analogue [65]. Figure 4 shows the DT IM-MS results of
various samples of a mixture of compounds 12/12u at the same concentration (5 × 10−4 M) upon
variation of the ratio of a solvent mixture of dichloromethane/acetonitrile. Interestingly, and whatever
the polarity of the solvent mixture used for the injection, the lasso compound 12, whose conformation
is smaller and more compact, have a lower arrival time distribution (63 scans). On the contrary, the
unthreaded molecule 12u consists of a larger ion with less conformational restrictions. As a
consequence, it takes more time for this ion to travel through the drift cell (arrival time distribution of
67 scans). Moreover, the same trend in the 12/12u ratio could be detected upon variation of the solvent
polarity by this method as that observed by NMR. Indeed, a 12/12u ratio of 18/82 in favor of 12u was
measured in the more polar acetonitrile, whereas the ratio was inverted in pure dichloromethane. The
consistency of this trend observed from the dichloromethane to the acetonitrile, with respect to the
NMR study, suggests that the equilibrium between 12 and 12u is slow on the ion mobility mass
spectrometry time scale. The width at half-height of each pick was also measured in pure acetonitrile
and dichloromethane: the peak related to the lasso 12 has a slightly narrowest distribution, which
reveals a more persistent size and shape, in accordance with the more restraint lasso structure. On the
contrary, the slightly broadest distribution observed for the peak of the unthreaded molecule 12u
exhibits its larger degree of flexibility.
2.3.3. 1H-NMR Investigation of the Molecular Machinery between Lasso Compounds 13 and 14
1
The comparison between the H-NMR spectra of the protonated and deprotonated unthreaded
molecules 13u, 14u with their respective protonated and deprotonated molecular lassos 13 and 14
allowed to demonstrate the lasso interlocked architecture and the localization of the BMP25C8 around
the molecular axle (Figure 5).
In the 1H-NMR spectrum of the protonated molecular lasso 13, signals for the hydrogen atoms HA-B
and HC-F of the BMP25C8 appear split because they are facing the two non-symmetrical ends of the
molecular axle of the interlocked lasso architecture. Moreover, the localization of the macrocycle
1
around the anilinium station in 13 can be deduced from the direct comparison between the H-NMR
spectra of 13 and 13u [Figure 5(a) and (b)]. Indeed, in the lasso 13, the signals for the hydrogen atoms
H21 appear at very high chemical shift (δ = 8.60 ppm). Likewise, hydrogens H20 belonging to the anilinium
station are shifted downfield with respect to the protonated unthreaded molecule 13u (Δδ = 0.15 ppm),
because they interact by hydrogen bonds with the oxygen atoms of the BMP25C8. Concerning the
hydrogen HI of the BMP25C8, it is dramatically shifted upfield in 13 (Δδ = −0.67 ppm) because it
experiences the shielding effect of the anilinium aromatic ring. The same trend is observed for
hydrogen atoms HE-F of the BMP25C8. Eventually, the hydrogen atoms H13 and H15–19 are all more or
less shielded (respectively Δδ = −0.17, −0.15, −0.40, −0.50, −0.63 and −0.30 ppm) because they all
undergo the shielding effect of the aromatic rings of the BMP25C8. Among them, it is noteworthy that
hydrogen atoms H16-17-18 are the more concerned by this effect.

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Figure 4. DT IM-MS of the mixture 12/12u in: (a) CH3CN. (b) CH3CN/CH2Cl2 30/70.
(c) CH3CN/CH2Cl2 20/80. (d) CH3CN/CH2Cl2 10/90. (e) CH2Cl2. Data shows: the average
time distribution (in scan units) at a drift voltage of 40V and the peak area (in %) for the
detected molecular ion [M−PF6]+.

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1
Figure 5. H-NMR spectra (600 MHz, CD2Cl2, 298 K) of: (a) the protonated unthreaded
compound 13u. (b) the protonated lasso-based compound 13. (c) the deprotonated lasso-
based compound 14. (d) the deprotonated unthreaded compound 14u. The coloring,
lettering and numbering correspond to the proton assignments indicated in scheme 1.
Deprotonation of the loosened lasso 13 led to the tightened lasso 14. The direct comparison of the
1H-NMR spectra between the two lasso [1]rotaxanes 13 and 14 reveals the shuttling of the BMP25C8
1
[Figure 5(b) and (c)]. Unsurprisingly, H-NMR chemical shifts of hydrogens H20, H23 and H25, which
belong to the anilinium moiety, are shifted upfield in the deprotonated lasso 14, due to the
deprotonation of the anilinium unit. At the same time, in 14, HI is shifted downfield (Δδ = 0.31 ppm)
because it does not experience anymore the strong shielding effect of the aniline extremity. However,
this signal is still at a lower chemical shift than those observed in the unthreaded protonated and
deprotonated molecules 13u and 14u (respectively Δδ = −0.36 and −0.34 ppm) [Figure 5(c) and (d)].
This result can be explained by the new localization of HI which now experiences the shielding effect
of the benzyl triazolium moiety, this latter effect being weaker than those of the anilinium aromatic
ring. This observation is corroborated by the upfield shift underwent by the other hydrogen atoms of
the aromatic rings of the crown ether HJ, HA and HB (respectively Δδ = −0.18, −0.04 and −0.16 ppm),
which experience the same shielding effect from the triazolium ring. Moreover, the displacement of

Molecules 2013, 18
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the BMP25C8 can account for the upfield shift observed in 14 for the hydrogen atoms of the benzyl
molecular barrier H28, H30 (respectively Δδ = −0.23, −0.24 ppm) and in a lesser extent H32 (with
respect to 13), due to their localization in the shielding cavity of the aromatic rings of the BMP25C8.
Similarly, the hydrogen atoms of the molecular axle located on the other side of the molecular barrier
H11 and H12 are both shielded by the BMP25C8: actually, this latter lies on the triazolium moiety and its
aromatic rings can reach the other side of the molecular barrier (respectively Δδ = −0.23, −0.41 ppm).
On the contrary, the hydrogen atoms located between the aniline and the triazolium unit H16, H17, H18
and H19 and in a much lesser extent H13, are shifted downfield in 14 (respectively Δδ = 0.62, 0.73, 0.82,
0.22 and 0.13 ppm) because they do not experience anymore the shielding effect of the BMP25C8. The
1
direct comparison between the H-NMR spectra of the tightened deprotonated lasso 14 and the
deprotonated unthreaded analogue 14u corroborates the localization of the BMP25C8 [Figure 5(c) and (d)].
1
Figure 6. H-NMR ROESY experiment (600 MHz, CD2Cl2, 298 K) of the protonated
loosened molecular lasso 13. The coloring, lettering and numbering correspond to the
proton assignments indicated in Scheme 1.

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1
The ROESY H-NMR experiments provided complementary and consistent insights into the
1
structure of the lasso molecular switch 13/14 (Figures 6 and 7). The ROESY H-NMR experiment
carried out on the protonated lasso (Figure 6) provided the evidences of the lasso architecture with a
localization of the BMP25C8 around the anilinium station (relevant correlation peaks highlighted in
cyan). It is particularly true for the correlation peaks between respectively H23 and H27 of the anilinium
site with HC-H of the BMP25C8, or between HI of the BMP25C8 with H20, H23 and H27. Interestingly,
no correlation peak between the BMP25C8 and the triazolium site was observed, which is consistent
with the localization of the macrocycle around the anilinium moiety.
1
Figure 7. H-NMR ROESY experiment (600 MHz, CD2Cl2, 298 K) of the deprotonated
tightened molecular lasso 14. The coloring, lettering and numbering correspond to the
proton assignments indicated in Scheme 1.

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After deprotonation (Figure 7), the relevant highlighted cross peaks relative to the loosened lasso
disappear, whereas new correlation peaks demonstrate the new main localization of the macrocycle
around the triazolium. In particular, new correlation peaks are observed for the hydrogens HC-H
belonging to the BMP25C8 with respectively the triazolium hydrogen H13, the benzyl barrier H28 and
H30, and the next hydrogen H15. At the same time, hydrogen atoms HE-F correlate with H31-32 of the
aromatic benzyl molecular barrier. The aromatic hydrogen HI of the BMP25C8 is now correlated with
the hydrogen atoms H15, H16 and H17, whereas the other aromatic hydrogens HA and HB of the
BMP25C8 are correlated with the benzyl barrier H28. In a consistent way, the aromatic hydrogen HJ of
the BMP25C8 is correlated with H12, whereas the last aromatic hydrogen of the BMP25C8 HA is now
correlated with H11. The whole NMR studies are consistent with the two main localizations of the
BMP25C8 around either the anilinium or the triazolium station upon variation in pH.
Concerning the peptide part GlyGlyGly of the lasso molecule, the overlapping in ROESY spectra
between methylene hydrogens H2, H5, H8 and the very split methylene hydrogens HC-H of the
BMP25C8 unfortunately prevents us from giving dependable assumptions concerning an eventual
conformational change of the tripeptide sequence. However, slight changes in chemical shifts could be
1
observed when comparing the 1D H-NMR spectra of the protonated loosened lasso 13 with the
1
deprotonated tightened lasso 14. The H-NMR chemical shift of methylene hydrogens H2 remained
almost unchanged in 14 (Δδ = +0.02 ppm), whereas H5 and H8 are more or less shielded in 14
(Δδ = −0.05 and −0.18 ppm). This latter observation can be assigned to the shielding effect of the
aromatic of the BMP25C8, corroborating the new localization of the BMP25C8.
3. Experimental
3.1. General
All reactions were achieved under an atmosphere of argon unless otherwise indicated.
Dichloromethane was distilled over P2O5 and was degassed by bubbling Ar for 20 min. Analytical
thin-layer chromatography (TLC) was performed on Merck silicagel 60 F254 plates. Compounds were
1
visualized by dipping the plates in an ethanolic solution of ninhydrine, followed by heating. H-NMR
13
and C-NMR spectra were obtained from a Bruker Avance III spectrometers (respectively at 400.13
MHz or 600.13 MHz and 100.62 MHz or 150.95 MHz). Chemical shifts of 1H-NMR and 13C-NMR are
given in ppm by using CHD2OH or CHDCl2 as references (3.31 ppm and 5.32 ppm respectively for 1H
13
spectrum and 49 ppm and 54 ppm respectively for C spectrum). Coupling constants (J) are reported
in hertz (Hz). Standard abbreviations indicating multiplicity were used as follows: s (singlet), br
(broad), d (doublet), t (triplet), q (quartet), m (multiplet). High-resolution mass spectra (HRMS) and
mass spectra were recorded on a Q-TOF Micro (water) apparatus.
3.2. Synthesis and Characterizations of [1]Rotaxanes 13 and 14
Boc-GlyGlyGly-NH-CH2CH2-N3 (8). 2-Azidoethylamine was first prepared by adding sodium azide
(800 mg, 12.2 mmol, 2.3 equiv) to a solution of 2-bromoethylamine hydrobromide (1.08 g, 5.3 mmol,
1 equiv) in H2O (20 mL). The resulting solution was then stirred overnight at 65 °C. After cooling down
to RT, NaOH (232 mg, 5.8 mmol, 1.1 equiv) was added to the reaction mixture. The resulting aqueous

Molecules 2013, 18
11567
solution was saturated with NaCl and extracted with CHCl3 (3 × 20 mL). The organic layer was dried
over anhydrous MgSO4 and used directly for the next step without being concentrated. The solution
containing the 2-azidoethylamine (5.3 mmol, 1.5 equiv) was diluted with CHCl3 (total volume: 150
mL) before adding successively Boc-glycylglycylglycine (1 g, 3.5 mmol, 1 equiv) and triethylamine
(1.43 mL, 10.6 mmol, 3 equiv). The mixture was then stirred until complete dissolution of the
tripeptide (15 min). BOP (1.88 g, 4.6 mmol, 1.3 equiv) was added and the mixture was stirred at RT
for 18 h. The resulting white precipitate 8 was filtered, washed with CH2Cl2 and dried under vacuum.
It was used in the next step without further purification (1.25g, quantitative). Rf: 0.40 (CH2Cl2/MeOH
1
9/1). H-NMR (600 MHz, CD3OD, 298K): δ ppm = 3.90 & 3.86 & 3.75 (3s, 3x2H, H2 H5 H8),
3.45–3.34 (m, 4H, H11 H12), 1.45 (s, 9H, HCH3-Boc). 13C-NMR (150 MHz, CD3OD, 298K): δ ppm = 173.6
& 172.2 & 171.9 (C3 C6 C9), 158.8 (CO-Boc), 81.0 (C(CH3)3-Boc), 51.3 (C12), 44.9 & 43.8 & 43.4 (C2 C5
C8), 39.9 (C11), 28.7 (CH3-Boc). MS (ESI): [M + H]+ calcd for C13H24N7O5: 358.18, found: 358.19.
TFA,H-GlyGlyGly-NH-CH2CH2-N3 (9). TFA (5 mL) was added to a suspension of the
Boc-GlyGlyGly-NHCH2CH2N3 8 (1.25g) in CH2Cl2 (20 mL), leading to complete dissolution of this
compound. After stirring for 15 min at RT, the reaction mixture was concentrated and co-evaporated 5
times with CH2Cl2 (20 mL) in order to remove the excess of TFA. The resulting white solid 9 (1.15 g,
1
quantitative) could be used without further purification. Rf: 0.09 (CH2Cl2/MeOH 9/1). H-NMR (600
MHz, CD3OD, 298K): δ ppm = 3.98 & 3.88 & 3.78 (3s, 3 × 2H, H2 H5 H8), 3.39 (s, 4H, H11 H12).
13C-NMR (150 MHz, CD3OD, 298K): δ ppm = 171.9 & 171.8 & 168.4 (C3 C6 C9), 161.0 (q,
2JC-F = 38.3 Hz, CO2-TFA), 117.1 (q, 1JC-F = 287.6 Hz, CF3-TFA), 51.4 (C12), 43.6 & 43.4 & 41.6 (C2 C5
C8), 39.9 (C11). MS (ESI): [M−TFA]+ calcd for C8H16N7O5: 258.13, found: 258.13.
Azido macrocycle (10). The preliminary synthesized carboxylic acid macrocycle [8] (894 mg,
1.82 mmol, 1 equiv) and compound 9 (707 mg, 2 mmol, 1.1 equiv) were dissolved in DMF (20 mL).
Then, triethylamine (755 µL, 5.45 mmol, 3 equiv) and BOP (973 mg, 2.36 mmol, 1.3 equiv) were
added and the mixture was stirred at RT for 17h. DMF was evaporated under vacuum and the resulting
yellow oil was diluted with CH2Cl2 (100 mL), before being successively washed with an aqueous
solution of HCl 1M (100 mL), a saturated aqueous solution of NaHCO3 (100 mL) and brine (100 mL).
The organic layer was then dried over MgSO4 and concentrated to yield a beige precipitate, which was
then triturated, washed with Et2O (200 mL) and dried under vacuum. The compound 10 was pure
enough to be used without further purification (1.33g, quantitative). Rf: 0.63(CH2Cl2/MeOH 9/1).
1H-NMR (600 MHz, CD3OD, 298K): δ ppm = 7.04 (d, 2H, 4JHJ-HI = 2.1 Hz, HJ), 6.98–6.92 (m, 2H, HA),
6.91–6.85 (m, 3H, HI HB), 4.22–4.16 (m, 4H, HH), 4.14–4.08 (m, 4H, HC), 4.04 (s, 2H, H2), 3.91 &
3.89 (2s, 2 × 2H, H5 H8), 3.84–3.77 (m, 8H, HD HG), 3.69 (s, 8H, HE HF), 3.35–3.32 (2m, 2x2H, H11
13
H12). C-NMR (150 MHz, CD3OD, 298K): δ ppm = 172.9 & 172.2 & 171.9 & 170.4 (CK C3 C6 C9),
161.6 & 150.5 & 136.6 (CIV arom BMP25C8), 123.0 (CB), 116.8 (CA), 108.0 (CJ), 107.5 (CI), 72.0 & 71.9
(CE CF), 71.0 (2s, CD CG), 70.3 (CC), 69.4 (CH), 51.3 (C12), 44.5 (C2), 44.0 & 43.4 (C5 C8), 39.9 (C11).
MS (ESI): [M + H]+ calcd for C33H46N7O12: 732.32, found: 732.32.
Compound (11). To a solution of the azido macrocycle 10 (165 mg, 0.225 mmol, 1 equiv) and the
preliminary synthesized alkyne 1 [9] (103 mg, 0.25 mmol, 1.1 equiv) in dry CH3CN (8 mL) at 70 °C,

Molecules 2013, 18
11568
were added successively Cu(CH3CN)4PF6 (84 mg, 0.225 mmol, 1 equiv) and 2,6-lutidine (3 μL, 0.03
mmol, 0.1 equiv). The mixture was stirred for 24 h at 70 °C. After filtration, the solvent was
evaporated under vacuum. The crude was then purified by chromatography on a silicagel column
(solvent gradient elution CH2Cl2 to CH2Cl2/MeOH 80/20) to give compound 11 (234 mg, 90%) as a
beige powder. Rf 0.65 (CH2Cl2/MeOH 9/1). 1H-NMR (600 MHz, CD2Cl2, 298K): δ (ppm) = 7.99 (br t,
1H, H1), 7.70 & 7.46 (2 br t, 2 × 1H, H4 H7), 7.36 (s, 1H, H13), 7.26 (s, 1H, H25), 7.11 (br t, 1H, H10),
7.01–6.95 (m, 4H, H23 HJ), 6.92–6.86 (m, 4H, HA HB), 6.83 (s, 1H, HI), 4.32 (t, 2H, 3JH12-H11 = 5.3 Hz, H12),
4.18–4.13 (m, 4H, HH), 4.12–4.07 (m, 4H, HC), 4.00 (d, 2H, 3JH2-H1 = 5.1 Hz, H2), 3.86–3.74 (m, 12H,
3
H5 H8 HD HG), 3.67 (s, 8H, HE HF), 3.59–3.51 (m, 4H, H11 H20), 2.61 (t, 2H, JH15-H16 = 7.4 Hz, H15),
1.64–1.56 (m, 2H, H16), 1.56–1.48 (m, 2H, H19), 1.39 (s, 9H, HCH3-Boc), 1.31 (s, 18H, H27), 1.36–1.25
13
(m, 4H, H17 H18). C-NMR (150 MHz, CD2Cl2, 298K): δ (ppm) = 171.8 & 170.6 & 170.4 & 168.7
(CK C3 C6 C9), 160.7 & 149.4 & 135.8 (CIV arom BMP25C8), 155.6 (CO-Boc), 151.7 (C24), 142.7 (C22), 122.3
(C13), 122.2 (CB), 121.9 (C23), 120.3 (C25), 115.4 (CA), 107.5 (CJ), 106.5 (CI), 80.0 (C(CH3)3-Boc), 71.3
(2s, CE CF), 70.3 & 70.2 (CD CG), 69.3 (CC), 68.9 (CH), 50.7 (C20), 49.7 (C12), 44.7 (C2), 43.9 & 43.5
(C5 C8), 39.8 (C11), 35.3 (C26), 31.7 (C27), 29.9 (C16), 28.9 (C19), 28.7 (CH3-Boc), 29.5 & 27.1 (C17 C18),
26.0 (C15). MS (ESI): [M + H]+ calcd for C60H89N8O14: 1145.65, found: 1145.65.
Compounds (12/12u). TFA (2.5 mL) was added to a solution of compound 11 (156 mg, 0.14 mmol,
1 equiv.) in CH2Cl2 (10 mL). After stirring for 30 min at RT, the reaction mixture was concentrated
and co-evaporated 5 times with CH2Cl2 (20 mL) in order to remove the excess of TFA. The residue
was then diluted in CH2Cl2 (10 mL). To this solution was added NH4PF6 (114 mg, 0.7 mmol, 5 equiv)
and H2O milliQ (5 mL): the biphasic solution was stirred vigorously for 30 minutes. The aqueous layer
was then extracted with CH2Cl2 (3 × 5 mL) and the combined organic layers were dried over MgSO4 and
concentrated to give compounds 12/12u (162 mg, quantitative) as a beige powder. Rf 0.52
(CH2Cl2/MeOH 9/1). HRMS (ESI): [M+H−PF6]2+ calcd for C55H82N8O12: 523.3026, found: 523.3007.
1
Unthreaded Compound (12u). H-NMR (600 MHz, 5.10−2 M in CD2Cl2, 298K): δ (ppm) = 7.98 (br t,
1H, H1), 7.81 (br t, 1H, H4 or H7), 7.69–7.62 (m, 1H, H4 or H7), 7.60 (s, 1H, H13), 7.50 (t, 1H,
4
4JH25-H23 = 0.9 Hz, H25), 7.31–7.26 (m, 1H, H10), 7.29 (d, 2H, JH23-H25 = 0.9 Hz, H23), 6.97 (d, 2H,
4JHJ-HI = 1.7 Hz, HJ), 6.83 (s, 4H, HA HB), 6.84 (br t, 1H, HI), 4.40 (br t, 2H, H12), 4.14–4.10 (m, 4H, HH),
4.10–4.07 (m, 4H, HC), 4.05–4.02 (m, 2H, H2), 3.85–3.74 (m, 12H, H5 H8 HD HG), 3.65 (s, 8H, HE HF),
3
3
3.62–3.56 (m, 2H, H11), 3.36 (t, 2H, JH20-H19 = 7.3 Hz, H20), 2.62 (t, 2H, JH15-H16 = 6.9 Hz, H15),
1.76–1.69 (m, 2H, H19), 1.63–1.54 (m, 2H, H16), 1.41–1.32 (m, 2H, H18), 1.35–1.22 (m, 2H, H17), 1.29
(s, 18H, H27). 13C-NMR (150 MHz, 5.10−2 M in CD2Cl2, 298K): δ (ppm) = 172.0 & 170.8 & 169.2 (4s,
CK C3 C6 C9), 160.8 & 149.1 & 135.5 (CIV arom BMP25C8), 154.3 (C24), 147.5 (C22), 124.3 (C25), 124.0
(C13), 122.3 (CA), 117.3 (C23), 115.3 (CB), 107.5 (CJ), 107.1 (CI), 71.1 (CE CF), 70.4 & 70.0 (CD CG),
69.2 (CC), 68.9 (CH), 53.9 (C20), 50.3 (C12), 44.6 (C2), 43.9 & 43.4 (C5 C8), 39.9 (C11), 35.6 (C26), 31.5
(C27), 28.7 & 28.0 (C16 C17), 25.9 (C18), 25.8 (C19), 24.9 (C15).
Pseudo[1]rotaxane (12). 1H-NMR (600 MHz, 5.10−2 M in CD2Cl2, 298K): δ (ppm) = 8.63 (br s, 2H, H21),
8.19 (br t, 1H, H1), 7.69–7.62 (m, 2H, H4 H7), 7.56 (s, 1H, H13), 7.52 (br t, 1H, H25), 7.39 (d, 2H,
4JH23-H25= 1.2 Hz, H23), 7.19 (t, 1H, 3JH10-H11 = 5.8 Hz, H10), 7.03 (d, 2H, 4JHJ-HI = 1.7 Hz, HJ), 6.96–6.92

Molecules 2013, 18
11569
(m, 2H, HA), 6.90–6.86 (m, 2H, HB), 6.20 (br t, 1H, HI), 4.43 (br t, 2H, H12), 4.24–3.99 (m, 8H, HH
HC), 3.94–3.91 (m, 2H, H2), 3.96–3.67 (m, 12H, H5 H8 HD HG), 3.73–3.46 (m, 8H, HE HF), 3.67–3.56
3
(m, 2H, H11), 3.56-3.48 (m, 2H, H20), 2.41 (t, 2H, JH15-H16 = 8 Hz, H15), 1.62–1.52 (m, 2H, H19),
1.38–1.29 (m, 2H, H16), 1.27 (s, 18H, H27), 1.01–0.93 (m, 2H, H17), 0.87–0.80 (m, 2H, H18). 13C-NMR
(150 MHz, 5.10−2 M in CD2Cl2, 298K): δ (ppm) = 172.4 & 171.2 & 169.1 (4s, CK C3 C6 C9), 160.1 &
149.1 & 135.7 (CIV arom BMP25C8), 154.2 (C24), 147.8 (C22), 124.9 (C25), 123.9 (C13), 122.8 (CA), 117.3
(C23), 113.2 (CB), 108.2 (CI), 106.8 (CJ), 71.9 & 71.3 (CE CF), 70.7 & 70.6 (CD CG), 69.1 & 68.6 (CC
CH), 52.3 (C20), 50.2 (C12), 45.3 (C2), 44.2 & 44.0 (C5 C8), 39.9 (C11), 35.7 (C26), 31.6 (C27), 30.2 (C16),
29.4 (C17), 29.3 (C19), 26.2 (C18), 25.4 (C15).
[1]rotaxane (13) A solution of compound 12 (162 mg, 0.14 mmol, 1 equiv) dissolved in CH2Cl2 (10
mL) was added dropwise over 24 h to a solution of benzyl bromide (55 mL) and CH2Cl2 (218 mL).
The mixture was stirred for further 6d at RT. CH2Cl2 was then evaporated and the excess of benzyl
bromide was removed by filtration on a silicagel column (solvent gradient elution CH2Cl2 to
CH2Cl2/MeOH 80/20). The remaining solid (224 mg) was then diluted in CH2Cl2 (16 mL). To this
solution was added NH4PF6 (133 mg, 0.82 mmol, 5 equiv) and H2O milliQ (8 mL): the biphasic
solution was stirred vigorously for 30 minutes. The aqueous layer was extracted with CH2Cl2 (3 × 20 mL)
and the combined organic layers were dried over MgSO4 and concentrated under vacuum. The crude
was purified twice by chromatography on a silicagel column (CH2Cl2 to CH2Cl2/MeOH 80/20) to give
several mixed fractions and the isolated [1]rotaxane 13 (7 mg, 4%) as a colourless oil. Rf 0.86
(CH2Cl2/MeOH 9/1). 1H-NMR (600 MHz, CD2Cl2, 298K): δ (ppm) = 8.60 (br s, 2H, H21), 8.16 (s, 1H,
H13), 7.56 (br t, 1H, H1), 7.52 (s, 1H, H25), 7.41 (s, 2H, H23), 7.41–7.33 (m, 3H, H32 H31), 7.27 (d, 2H,
3JH30-H31 = 7 Hz, H30), 7.27–7.19 (m, 2H, H4 H7), 7.03 (br t, 1H, H10), 7.01 (s, 2H, HJ), 6.94–6.88 (m,
2H, HA), 6.88–6.83 (m, 2H, HB), 6.20 (s, 1H, HI), 5.64 (s, 2H, H28), 4.70–4.63 (m, 2H, H12), 4.24–4.18
& 4.18–4.12 & 4.09–4.03 (m, 8H, HH HC), 4.01–3.95 (m, 2H, H2), 3.92–3.73 (m, 12H, H5 H8 HD HG),
3.78–3.71 (m, 2H, H11), 3.73–3.41 (m, 8H, HE HF), 3.51–3.41 (m, 2H, H20), 2.51(t, 2H, 3JH15-H16 = 7.5
Hz, H15), 1.48–1.41 (m, 2H, H19), 1.31–1.21 (m, 2H, H16), 1.29 (s, 18H, H27), 0.88–0.80 (m, 2H, H17),
0.75–0.67 (m, 2H, H18). HSQC 13C-NMR (150 MHz, CD2Cl2, 298K): δ (ppm) = 129.9 (C31 C32), 129.6
(C13), 128.5 (C30), 124.7 (C25), 122.5 (CA), 117.3 (C23), 113.1 (CB), 108.8 (CI), 106.4 (CJ), 72.0 & 71.2
(CE CF), 70.6 & 69.7 (CD CG), 68.8 & 68.5 (CH CC), 55.2 (C28), 53.8 (C12), 52.3 (C20), 44.9 (C2), 43.5
(C5 C8), 39.3 (C11), 31.8 (C27), 28.8 (C17), 27.6 (C19), 27.1 (C16), 25.8 (C18), 23.8 (C15). HRMS (ESI):
[M-PF6]+ calcd for C62H88F6N8O12P: 1281.6164, found: 1281.6177.
[1]Rotaxane (14). To a solution of the [1]rotaxane 13 (3 mg, 2.1 µmol, 1 equiv) in CD2Cl2 (0.6 mL)
was added 74 µL of a 1% solution of DIEA in CD2Cl2 (4.2 µmol, 2 equiv.). Rf 0.73(CH2Cl2/MeOH
1
9/1). H-NMR (600 MHz, CD2Cl2, 298K): δ (ppm) = 8.29 (s, 1H, H13), 7.33–7.28 (m, 1H, H32),
7.28–7.24 (m, 2H, H31), 7.20 (br t, 1H, H1), 7.12 & 7.08 (2 br t, 2H, H4 H7), 7.03 (d, 2H, 3JH30-H31 = 7.4 Hz,
H30), 6.93 (br t, 1H, H10), 6.88–6.84 (m, 2H, HA), 6.83 (s, 2H, HJ), 6.76 (s, 1H, H25), 6.73–6.68 (m, 2H,
3
HB), 6.51 (s, 1H, HI), 6.48 (m, 2H, H23), 5.41 (s, 2H, H28), 4.26 (t, 2H, JH12-H11 = 6.3 Hz, H12),
3
4.19–4.14 & 4.10–4.04 (m, 4H, HH), 4.03–3.97 & 3.94–3.89 (m, 4H, HC), 4.00 (d, 2H, JH2-H1 = 5.3
Hz, H2), 3.86–3.66 (m, 12H, H5 H8 HD HG), 3.71–3.55 (m, 8H, HE HF), 3.55–3.50 (m, 2H, H11), 3.15 (t,
2H, 3JH20-H19 = 6.5 Hz, H20), 3.07-3.01 (m, 2H, H15), 1.95–1.82 (m, 2H, H16), 1.69–1.62 (m, 2H, H19),

Molecules 2013, 18
11570
1.60–1.54 (m, 2H, H17), 1.56–1.49 (m, 2H, H18), 1.28 (s, 18H, H27). HSQC 13C-NMR (150 MHz,
CD2Cl2, 298K): δ (ppm) = 129.4 (C31 C32), 129.3 (C13), 129.1 (C30), 121.9 (CA), 113.8 (CB), 112.1
(C25), 108.3 (CI), 107.8 (C23), 105.1 (CJ), 71.8 & 71.5 & 69.9 (CE CF), 70.9 & 70.8 & 68.4 (CD CG),
69.8 & 69.7(CC), 68.6 & 68.5 (CH), 54.3 (C28), 52.3 (C12), 44.7 (C20), 44.4 (C2), 44.6 & 43.4 (C5 C8),
38.5 (C11), 31.1 (C27), 30.5 (C19), 29.9 (C17), 27.8 (C18), 26.6 (C16), 23.3 (C15). HRMS (ESI): [M+H]+
calcd for C62H88F6N8O12P: 1281.6164, found: 1281.6144.
3.3. Synthesis and Characterizations of the Uncomplexed Threads 13u and 14u
Protonated Unthreaded Compound (13u): Compound 11 (110 mg, 0.096 mmol, 1 equiv) was
dissolved in benzyl bromide (3.4 mL, 300 equiv) and the mixture was stirred for 6d at RT before being
directly filtered on a silicagel column to remove the excess of benzyl bromide (solvent gradient elution
CH2Cl2 to CH2Cl2/MeOH 85/15). To a solution of the residue in CH2Cl2 (4 mL) was then added TFA
(1 mL). The mixture was stirred for 30 minutes before being concentrated and co-evaporated 5 times
with 20 mL of CH2Cl2 in order to remove the excess of TFA. The residue was then diluted in CH2Cl2
(20 mL). To this solution was added NH4PF6 (78 mg, 0.48 mmol, 5 equiv) and MilliQ H2O (10 mL):
the biphasic solution was stirred vigorously for 30 minutes. The aqueous layer was extracted with
CH2Cl2 (3 × 5 mL) and the combined organic layers were dried over MgSO4 and concentrated to give
1
compound 13u (106 mg, 80%) as a white foam. Rf 0.86 (CH2Cl2/MeOH 9/1). H-NMR (600 MHz,
CD2Cl2, 298K): δ (ppm) = 8.33 (s, 1H, H13), 7.67 (br t, 1H, H1), 7.48 (br t, 1H, H25), 7.43 & 7.28 (2 br
t, 2H, H4 H7), 7.42–7.39 (m, 3H, H31 H32), 7.30–7.26 (m, 2H, H30), 7.25 (d, 2H, 4JH23-H25= 1.4 Hz, H23),
7.04 (br t, 1H, H10), 6.99 (d, 2H, 4JHJ-HI= 2.1 Hz, HJ), 6.90–6.86 (m, 5H, HA HB HI), 5.64 (s, 2H, H28),
4.67 (t, 2H, 3JH12-H11 = 5 Hz, H12), 4.19–4.14 (m, 4H, HH), 4.11–4.06 (m, 4H, HC), 4.04 (d, 2H, 3JH2-H1
3
= 5.7 Hz, H2), 3.88 (d, 2H, 3J = 5.7 Hz, H5 or H8), 3.83 (d, 2H, J = 5.6 Hz, H5 or H8), 3.81–3.75 (m,
8H, HD HG), 3.76–3.73 (m, 2H, H11), 3.67 (s, 8H, HE HF), 3.31 (t, 2H, 3JH20-H19 = 7.8 Hz 2H, H20), 2.66
(t, 2H, 3JH15-H16 = 7.4 Hz, H15), 1.78–1.70 (m, 2H, H19), 1.69–1.62 (m, 2H, H16), 1.38–1.30 (m, 4H, H17
13
H18), 1.28 (s, 18H, H27). C-NMR (150 MHz, CD2Cl2, 298K): δ (ppm) = 171.9 & 171.2 & 171.0 &
169.4 (CK C3 C6 C9), 160.8 & 149.2 & 135.7 & 135.5 (CIV arom BMP25C8), 154.3 (C24), 145.1 (C22), 131.8
(C29), 130.0 (C13 C31 C32), 128.8 (C30), 124.3 (C25), 122.4 (CB), 117.2 (C23), 115.4 (CA), 107.4 (CJ),
107.3 (CI), 71.1 (CE CF), 70.3 & 70.0 (CD CG), 69.2 (CC), 68.9 (CH), 55.4 (C28), 54.0 (C20), 53.9 (C12),
44.5 (C2), 43.8 & 43.4 (C5 C8), 39.4 (C11), 35.6 (C26), 31.4 (C27), 26.3 (C16), 28.1 & 25.9 (C17 C18 C19),
23.6 (C15). HRMS (ESI): [M−PF6]+ calcd for C62H88F6N8O12P: 1281.6164, found: 1281.6173.
Deprotonated Unthreaded Compound (14u): A solution of the thread 13u (48 mg, 0.034 mmol, 1
equiv) in CH2Cl2 (10 mL) was washed with an aqueous solution of 1 M NaOH (5 mL). After
separation and further extraction of the remaining aqueous layer with CH2Cl2 (10 mL), the combined
organic layer were dried over MgSO4 and then evaporated to obtain the deprotonated unthreaded
1
compound 14u (43 mg, 99%) as a colourless oil. Rf 0.73 (CH2Cl2/MeOH 9/1). H-NMR (600 MHz,
3
CD2Cl2, 298K): δ (ppm) = 8.25 (s, 1H, H13), 7.47 (t, 1H, JH1-H2 = 6 Hz, H1), 7.44–7.40 (m, 3H, H31
H32), 7.42 (br t , 1H, H4 or H7), 7.22 (t, 1H, 3J= 5.4 Hz, H4 or H7), 7.30-7.26 (m, 2H, H30), 7.11 (t, 1H,
3JH10-H11 = 5.9 Hz, H10), 6.98 (d, 2H, 4JHJ-HI = 2 Hz, HJ), 6.91–6.87 (m, 4H, HA HB), 6.85 (br t, 1H, HI),
6.75 (br t, 1H, H25), 6.43 (d, 2H, 4JH23-H25 = 1 Hz, H23), 5.64 (s, 2H, H28), 4.63 (t, 2H, 3JH12-H11 = 5 Hz,

Molecules 2013, 18
11571
H12), 4.20–4.16 (m, 4H, HH), 4.11–4.07 (m, 4H, HC), 4.00 (d, 2H, 3JH2-H1 = 5.5 Hz, H2), 3.83–3.74 (m,
3
12H, H5 H8 HD HG), 3.71–3.65 (m, 2H, H11), 3.69 (s, 8H, HE HF), 3.06 (t, 2H, JH20-H19 = 7 Hz, H20),
3
2.71 (t, 2H, JH15-H16 = 7.8 Hz, H15), 1.64–1.54 (m, 2H, H16), 1.58–1.49 (m, 2H, H19), 1.40–1.28 (m,
13
4H, H17 H18), 1.29 (s, 18H, H27). C-NMR (150 MHz, CD2Cl2, 298K): δ (ppm) = 172.1 & 171.0 &
170.8 & 168.9 (CK C3 C6 C9), 160.8 & 149.4 & 135.7 (CIV arom BMP25C8), 152.2 (C24), 148.8 (C14), 145.2
(C22), 131.8 (C29), 130.2 & 130.1 & 130.0 (C13 C31 C32), 128.6 (C30), 122.2 (CB), 115.4 (CA), 112.2
(C25), 107.9 (C23), 107.3 (CJ), 107.0 (CI), 71.3 (2s, CE CF), 70.3 & 70.2 (CD CG), 69.3 (CC), 68.9 (CH),
55.5 (C28), 54.1 (C12), 44.8 (C2), 44.5 (C20), 44.4 & 43.6 (C5 C8), 39.2 (C11), 35.2 (C26), 31.8 (C27),
29.9 (C19), 27.4 (C16), 29.2 & 27.1 (C17 C18), 23.9 (C15). HRMS (ESI): [M + H]+ calcd for
C62H88F6N8O12P: 1281.6164, found: 1281.6168.
4. Conclusions
We have reported the synthesis of a new lasso molecular switch containing a peptide part, using a
two-step sequence strategy: (1) the self-entanglement of a hermaphrodite molecule, (2) the trapping of
the interlocked pseudo lasso structure with a molecular barrier. 1D and 2D 1H-NMR spectroscopies, as
well as DT IM-MS studies provided information about the self-interlocking of the molecular lasso and
about the pH-dependent molecular machinery. In the protonated state, the lasso remains loosened
because the BMP25C8 resides around the best anilinium molecular site. However, deprotonation of the
anilinium triggers the shuttling of the BMP25C8 towards the triazolium station, resulting in a more
tightened conformation. It appears consistent to say that the peptide part of the lasso, which is located
between the macrocycle and the triazolium unit (that is to say in the loop of the lasso), does not play
any role in the molecular machinery: however, it undergoes a conformational change from a more
extended and flexible shape at acidic pH to a more constraint bent conformation at basic pH. To the
best of our knowledge, this is the first example of a pH-sensitive lasso molecular switch incorporating
a peptide backbone. Further investigations towards the structure-activity relationship of such a
peptide-containing molecular machine are in progress.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/18/9/11553/s1.
Conflicts of Interest
The authors declare no conflict of interest.
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