A versatile axle for the construction of disassemblage rotaxanes

Article abstract of DOI:10.3390/molecules21081043

Rotaxanes are unique mechanical devices that hold great promise as sensors. We report on two new rotaxanes that contain an acid or base sensitive trigger and readily disassemble in a wide range of environments. Disassemblage was observed under TLC and ¹H-NMR analysis. The axle is highly charged, which enhances solubility in aqueous environments, and can be readily derivatized with sensor components. The trigger was swapped in a one-pot method, which is promising for the rapid production of a series of sensors.

Full text of DOI:10.3390/molecules21081043

molecules
Article
A Versatile Axle for the Construction of
Disassemblage Rotaxanes
Lucas A. Powers and David B. Smithrud *
Department of Chemistry, University of Cincinnati, Cincinnati, OH 45221, USA; powersla@mail.uc.edu
* Correspondence: david.smithrud@uc.edu; Tel.: +1-513-556-9254
Academic Editor: Derek J. McPhee
Received: 30 June 2016; Accepted: 4 August 2016; Published: 10 August 2016
Abstract: Rotaxanes are unique mechanical devices that hold great promise as sensors. We report on
two new rotaxanes that contain an acid or base sensitive trigger and readily disassemble in a wide
range of environments. Disassemblage was observed under TLC and 1H-NMR analysis. The axle is
highly charged, which enhances solubility in aqueous environments, and can be readily derivatized
with sensor components. The trigger was swapped in a one-pot method, which is promising for the
rapid production of a series of sensors.
Keywords: rotaxanes; disassemble; switch; pH
1. Introduction
The detection of specific molecules or processes on the molecular scale has greatly benefited a
wide range of fields from medicine to environmental protection. This has spurred the rapid growth of
chemosensors [1,2]. Chemosensors undergo a physical or chemical change upon interaction with a
targeted molecule or environment to produce a signal that is observable to the eye or via instrumental
analysis. There are many challenges in creating such sensors. They must reach their target, which
requires sufficient stability and solubility, contain an efficient trigger mechanism, and produce a signal
intense enough to be sensed. If unique sensors had to be designed and built for every target, this
would be a daunting challenge.
The rotaxane architecture provides a promising universal framework for sensor development [3–7].
[2]Rotaxanes comprise a circular molecule (wheel) threaded over a linearly shaped molecule (axle)
with large groups (blocking groups) attached to the axle’s ends to keep the wheel threaded. Most
of the original rotaxane-sensors operated by the wheel switching positions on the axle after an
external stimulus is applied to give an observable signal [8–13]. While this class of sensors
continues to show great promise, we are constructing sensors that operate via the controlled
disassemblage of a rotaxane. Literature results show that an external stimulus can reduce the
stability of pseudorotaxane (a rotaxane with one blocking group)/rotaxanes, promoting disassemblage.
For example, Martinez-Cuezva et al promoted the dethreading of a [2]rotaxane using heat, flash
vacuum pyrolysis, or microwave radiation [14]. Leung showed that reducing the interaction strength
between axle and wheel via deprotonation or switch of solvent accelerated disassemblage [15]. Even the
simple protonation-deprotonation of a blocking group can provide control of rotaxane formation [16].
Recently, Huang showed that a polyrotaxane can be reversibly assembled and disassembled using
irradiation and heating steps [17].
Our goal is to construct rotaxane-sensors that disassemble when an external agent reduces the
size of a blocking group and operate in biological solutions. The first step is to develop an axle that can
be readily derivatized with sensor components. The axle cannot impede the release of the wheel and
be highly soluble in water. Finally, the wheel needs to efficiently thread onto an axle for high yields of
a pseudorotaxane prior to rotaxane synthesis using the end-capping method [18]. Thus, a potential
Molecules 2016, 21, 1043; doi:10.3390/molecules21081043
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axle needs to display the necessary charge state and solubility to enable wheel threading for rotaxane
formation while still being water soluble for sensor operation. Herein, we report on an optimal axle
that contains two alkylammonium ions for enhanced water solubility, forms pseudorotaxanes in good
yields, enabling rotaxane formation, and promotes rapid disassemblage once the blocking group is
reduced in size.
2. Results and Discussions
2.1. Synthesis of the [2]Rotaxanes
[2]Rotaxanes FMOC-R
route for rotaxane synthesis, and test the switches. Since many biological environments display
unique pH values, such as the acidic environment of cancerous sites [19 21], changes in pH were
used to reduce the size of the blocking group. Amine bases found in the body, such as serotonin, are
attractive targets for monitoring health [22 23]. The FMOC group of FMOC-R is cleaved under basic
1 and Trityl-R2 were constructed (Figure 1) to develop the axle, find a
–
,
1
conditions, whereas, the trityl group of Trityl-R2 is cleaved under acidic conditions. A Boc-protected,
dibenzyl-24-crown-8 ether was used as the wheel, and a 1,3-dimethyl benzenoid ring was used as a
model blocking group. Future [2]rotaxanes will contain various signal groups along with appropriate
triggers, depending on the need of a sensor. Biological solutions also have a variety of polarities,
therefore we measured disassemblage in chloroform, DMSO, and aqueous solutions.
Figure 1. FMOC-R1 and Trityl-R2 were constructed and investigated as disassemblage rotaxanes.
The [2]rotaxanes were readily synthesized in a few steps in an overall 20% yield (Scheme 1).
The short, highly polar axle
3 was chosen to enhance the solubility of the rotaxanes in aqueous
solutions with an eye towards constructing biologically active sensors. Having an amino and carboxy
termini enables a wide range of signal and trigger groups to be coupled either through nucleophilic
or electrophilic atoms. Axle 3 was synthesized through amide coupling of 4-bromobutyric acid with
1,4-xylene diamine. It was purified by simple extraction into water, which demonstrates its high
polarity, followed by recrystallization. After a protecting group was added to its benzylic amine
(we have used FMOC, BOC, and trityl), the carboxylic acid was activated with CDI, followed by the
addition of blocking group
5
. Once the protecting group was removed, the alkylammonium ions
were converted to PF6´ salts to promote pseudorotaxane
8
formation. Blocking group-axle , as the
7
PF6´ salt, and wheel were combined in a minimal amount of chloroform, followed by the addition
of Trityl-Cl or FMOC-Cl. The [2]rotaxanes were obtained in good yields of 50% after purification via
column chromatography. The wheel and blocking group-axle 7 were also recovered and reused.
Adding the trigger in the last step is convenient for synthesizing a set of sensors for different
targets. The trigger can be switched readily in a one-pot method. For example, FMOC-R
1 was
readily converted to Trityl-R . FMOC-R was exposed to piperidine in a minimal amount of DMSO.
2
1

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After removing DMSO via evaporation, a minimal amount of dried chloroform was added to the
pseudorotaxane
30% yield.
8 followed by the addition of Trityl-Cl. Trityl-R2 was produced in a non-optimized,
Scheme 1. Synthesis of the [2]rotaxanes.
2.2. Measuring the Stability of Pseudorotaxane 8
To operate as a sensor, the pseudorotaxane needs to disassemble once the trigger is activated.
The stability of pseudorotaxane
(Figure 2). These solvents were chosen to represent environments a sensor may operate in in a
biological system. Furthermore, blocked-axle was investigated as the chloride salt, which will
likely exist in biological environments. In CDCl3, the strong ion pairs greatly weakens pseudorotaxane
(KA = 17
0.6 M´1) when compared to the typical association observed in the millimolar range for
the complex of DB24C8 with R2NH2+ PF6 salts [24
(KA = 250
10 M´1). This value is consistent with literature values [27
8 was determined in chloroform, DMSO, and a DMSO/water mixture
7
8
˘
´
–26]. Tighter association was observed in DMSO-d6
˘
–
29]. Association was not
observed in a 70/30 (v/v) mixture of DMSO-d6/D2O up to a 50 mM solution of both components.
This finding is not surprising considering that water forms strong hydrogen bonds that out-compete
the favorable charge-induced dipole interactions between the alkylammonium ions of blocked-axle
7
and the wheel. These results show that the pseudorotaxanes will disassemble in the high millimolar
concentration range for most environments in biological systems. For future sensors, additional potential
favorable interactions between the wheel and sensor components would have to be considered.
Figure 2. Changes in the chemical shift of an amide proton of pseudorotaxane
8 held at a constant
concentration in CDCl3 and DMSO-d6 (10 mM and 30 mM, respectively) caused by the increasing
concentration of the wheel.

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2.3. Investigating the Rate of Disassemblage
Sensors need to disassemble quickly after a trigger is activated to localize the target of interest.
FMOC-R and Trityl-R were tested for the rate of wheel slippage. Thin layer chromatography
analysis was used initially. The three components of FMOC-R and Trityl-R (wheel, axle, and the
1
2
1
2
FMOC or trityl byproducts) are clearly differentiated from the starting rotaxanes via TLC. The rate of
trityl clippage was sluggish, requiring a very high percentage of acid to be observed within 30 min
(for example, 50% TFA in CHCl3). On the other hand, exposing FMOC-R1 to ethanolamine or
piperidine from a 5% (v/v) solution down to 2 equivalence of amine to rotaxane in DMSO resulted in
rapid disassemblage of the rotaxane within two minutes. FMOC cleavage via amines is sluggish in
chloroform so t-butoxide dissolved in a minimal amount of DMSO (5% v/v) was used instead. Once
again, FMOC-R1 disassembled within five minutes after exposing it to solutions containing 2 eq. of
t-butoxide to FMOC-R1 in CHCl3.
1H-NMR analysis confirmed rapid disassemblage. We needed to demonstrate that pseudorotaxane
8
disassembled quickly. FMOC-R
percentage of base. For these studies, the concentration of FMOC-R
that only an insignificant amount of pseudorotaxane would exist after FMOC clippage. Under all
conditions (2 eq. t-butoxide or 1% piperidine in 95/5 CDCl3/DMSO-d6, 1% ethanolamine in DMSO-d6,
and 1% ethanolamine in 70/30 DMSO-d6/D2O), FMOC-R was disassembled within 5 minutes, which
is the minimum time needed to run an 1H-NMR experiment. Figure 3 highlights the shifts in 1H-NMR
spectra for FMOC-R in CDCl3 caused by the addition of piperidine (see Supplementary Materials for
1
was chosen since the FMOC group is rapidly removed in a small
1
was kept low (2 mM) to ensure
8
1
1
results obtained in DMSO-d6). These results are consistent with the slippage rates of axles containing
benzylammonium ions, which are in the millisecond range [30]. These findings suggest that sensors
built from Axle
3 should rapidly disassemble and accurately identify the location of a desired target in
a variety of environments, including aqueous environments.
Figure 3. Example of the disassemblage of FMOC-R
) FMOC-R (2 mM) in CDCl3/DMSO-d6 (95%/5% v/v); (
of piperidine (1% v/v) after approximately 5 min. Disassemblage is evident with the chemical shifts
of the wheel and blocked-axle more closely matching the chemical shifts of the pure wheel and
blocked-axle 7 (c,d), respectively.
1
caused by the presence of piperidine.
(a
1
b
) the same solution as ( ) with the addition
a
7

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3. Materials and Methods
3.1. General Synthetic Protocol
Solvents, reagents, and starting materials for synthesis were purchased from Sigma-Aldrich
(St. Louis, MO, USA). Moisture sensitive reactions were carried out under positive argon pressure.
1
All organic solvents were freshly distilled over a suitable drying agent. H- and 13C-NMR spectra were
obtained using a Bruker AMX400 spectrometer (The Woodlands, TX, USA) operating at 400.13 MHz for
proton and 100.61 MHz for carbon nuclei. Chemical shifts are in ppm and referenced using an internal
TMS standard for 1H-NMR or deuterated solvent for 13C-NMR. ESI mass spectra were obtained using
Micromass Q-TOF-2 spectrometer. Samples were injected from a MeOH/formic acid solution.
3.2. Synthetic Procedures
Axle 3: 1,4-Xylene diamine (2.0 g, 15 mmol) was dissolved in 20 mL of dry DMF. Sodium
carbonate was added (3.1 g, 29 mmol) to the solution. In a separate flask, 4-bromobutyric acid (2.5 g,
15 mmol) was dissolved in 1 mL of dry DMF and deprotonated with 1.5 equivalences of triethylamine.
4-Bromobutyrate was added dropwise to the xylene diamine solution over 1 hour. The solution was
˝
then stirred at 70 C for 16 hours. Solid Na2CO3 was removed via filtration, and the solvent was
removed under vacuum. The resulting solid was extracted CH2Cl2/water (3 ). The aqueous phase
ˆ
was kept, and water was removed under vacuum. The residue was recrystallized in 10% MeOH/90%
CH2Cl2 (v/v). Solid 1, 4-Xylene diamine was removed via filtration, leaving axle 3 as an orange oil in
1
a 72% yield (2.35 g, 10.6 mmol). H-NMR: (D2O):
δ 1.66 (2H, m), 2.17 (2H, t), 3.38 (2H, t), 4.21–4.28
(4H, m), 7.27 (4H, S). 13C-NMR:
138.98, 176.14. Mass spectral analysis for C12H18N2O2-H+ (cal. 223.14 found 223.14).
Trityl-Axle 4: Axle (2.0 g, 9.0 mmol) was dissolved in 20 mL of dry DMF. Sodium carbonate was
δ 27.76, 32.32, 38.75, 42.47, 60.76, 127.77, 129.11, 131.54, 133.35, 136.89,
3
added (950 mg, 9.0 mmol) to the solution, followed by the addition of trityl chloride (2.5 g, 9.0 mmol).
˝
The solution was stirred at 70 C for 16 h. The solution was filtered to remove Na2CO3, and the
DMF was removed under vacuum. The crude product was extracted with CH2Cl2/pH = 7 water
(3 ). The organic layers were collected and concentrated. The crude product was purified via column
ˆ
chromatography (100% CH2Cl2, then 5% to 50% methanol). Trityl-Axle
4
was obtained as an orange oil
1
in 80% yield (3.4 g, 7.0 mmol). H-NMR (DMSO-d6):
δ
1.45 (2H, m), 2.00 (2H, t), 3.22 (2H, t), 3.95–4.05
(4H, m), 6.80–7.00 (16H, m), 7.20 (3H, d). 13C-NMR:
δ 27.54, 32.12, 38.67, 42.39, 60.83, 80.25, 125.35,
127.23, 127.58, 128.78, 129.21, 129.35, 129.78, 130.56, 131.62, 133.22, 136.95, 139.07, 176.28. Mass spectral
analysis for C31H32N2O2-H+ (cal. 465.25 found 465.20).
Blocking Group 5: A solution containing 3,5-dimethyl benzoic acid (2.0 g, 13 mmol) in 20 mL
of dry DMF was cooled to 0 ˝C. CDI was added (2.2 g, 15 mmol) to the reaction mixture, which was
stirred under Ar. After two hours, the solution was transferred to a dropping funnel. It was added
dropwise over two hours to a solution containing ethylene diamine (1.6 g, 26.6 mmol) in 5 mL of dry
DMF. The reaction mixture was stirred at room temperature for 16 h. DMF was evaporated, and the
crude product was purified by column chromatography (5% to 20% methanol). Blocking group
5 was
1
obtained as a yellow oil in 85% yield (2.2 g, 11 mmol). H-NMR (DMSO-d6):
δ 2.30 (6H, s), 2.89 (2H, t),
3.46 (2H, m), 7.08 (1H, s), 7.42 (2H, s). 13C-NMR:
137.41, 138.42, 170.21. Mass spectral analysis for C11H16N2O-Na+, (cal. 215.12 found 215.18).
Blocked-Trityl-Axle 6: A solution of trityl-axle (1 g, 2.1mmol) in 10 mL of dry DMF was cooled
to 0 C, and CDI (350 mg, 2.3 mmol) was added. After two hours, blocking group (413 mg, 2.1 mmol)
δ 21.23, 43.51, 61.47, 129.58, 130.32, 130.96, 136.28,
4
˝
5
was added to the reaction mixture. The reaction mixture was warmed to room temperature and stirred
for 16 hours. DMF was removed via evaporation under vacuum, and the crude material was extracted
in CH2Cl2/1N NaOH. The organic layer was concentrated, and the material was purified via column
chromatography (100% CH2Cl2, then 5% to 20% methanol). Blocked-trityl-axle
6
was obtained as a
1
yellow oil in 73% yield (1.0 g, 1.6 mmol). H-NMR (DMSO-d6):
δ
1.65 (2H, m), 2.33 (6H, s), 2.38 (2H,
21.26,
m), 3.37 (2H, m), 3.49 (2H, m), 3.64 (2H, t), 4.38–4.44 (4H, m), 7.00–7.50 (22H, m). 13C-NMR:
δ

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27.59, 32.18, 38.72, 42.35, 43.56, 60.75, 61.50, 80.28, 125.32, 127.31, 127.63, 128,72, 129.26, 129.42, 129.51,
129.82, 130.39, 130.53, 130.91, 131.57, 133.27, 136.25, 136.91, 137.43, 138.45, 139.02, 170.27, 176.31. Mass
spectral analysis for C42H46N4O2-H+, NaCl (cal. 697.33 found 697.30).
Blocked-Axle 7: Blocked-trityl-axle
6 (600 mg, 0.94 mmol) was exposed to 200 µL of 1N HCl
in 10 mL of methanol. The reaction mixture was stirred at room temperature for 4 hours. Sodium
bicarbonate was added to neutralize the solution. Solid Na2CO3 was removed via filtration, and the
methanol was evaporated under vacuum. The crude product was washed with ether (3
the trityl by-products. Blocked-axle was obtained as a yellow solid in 95% yield (350 mg, 0.89 mmol).
1H-NMR:
1.68 (2H, m), 2.20 (6H, s), 2.30 (2H, m), 3.15 (2H, m), 3.32 (2H, m), 3.40 (2H, t), 4.22–4.29
(4H, m), 7.14 (1H, s), 7.18–7.23 (4H, m), 7.49 (2H, s). 13C-NMR:
21.24, 27.53, 32.16, 38.70, 42.31, 43.59,
ˆ) to remove
7
δ
δ
60.73, 61.55, 125.35, 127.33, 129.47, 129.80, 130.87, 131.61, 133.31, 136.22, 136.94, 137.40, 138.41, 138.96,
170.22, 176.27. Mass spectral analysis for C23H32N4O2-H+, MeOH (cal. 429.29 found 429.22).
FMOC-R1: Blocked-axle
7 (600 mg, 1.5 mmol) was suspended in a diethyl ether/ethyl acetate
(50/50 (v/v)) solution. Sodium hexafluorophosphate (300 mg 1.8 mmol) was added, and the mixture
was extracted with pH = 3 water. Organic solvents were evaporated under vacuum, and the residue
was dissolved in 0.5 mL of freshly distilled chloroform. The wheel (400 mg, 0.6 mmol) was added,
and the solution was stirred under Ar. After 30 min, FMOC-Cl (390 mg, 1.5mmol) was added to the
solution. The reaction mixture was stirred at room temperature for 12 h. Excess Na2CO3 was added to
the reaction mixture, which was stirred for an additional 12 h. The solution was filtered to remove
Na2CO3, and CHCl3 was evaporated under vacuum. The crude material was purified via column
chromatography (100% CH2Cl2, then 3% to 5% methanol). FMOC-R
1
was isolated as a red foam in a
1
52% yield (400 mg, 0.3 mmol). H-NMR (CDCl3):
δ
1.50 (18H, s), 2.07 (2H, t), 2.27–2.31 (8H, m), 3.41
21.19, 28.38, 31.46, 35.42, 36.60, 40.81, 41.11,
(2H, t), 3.50–4.38 (35H, m), 6.78–7.80 (21H, m). 13C-NMR:
δ
46.57, 46.92, 52.17, 53.59, 66.74, 67.03, 67.36, 68.05, 69.60, 70.79, 77.36, 80.50, 106.66, 113.11, 113.59, 120.33,
124.91, 127.24, 127.39, 127.76, 128.42, 133.05, 133.93, 134.00, 134.59, 135.02, 138.10, 158.12, 162.74, 168.88,
169.12. Mass spectral analysis for C72H92N6O16-H+, HCl, HCO2H, NaPF6 (cal 1547.60 found 1547.64).
Trityl-R2: The same procedure was followed for FMOC-R
1
synthesis with Trityl-Cl replacing
1
FMOC-Cl. Trityl-R
2
was isolated as a yellow oil in a 51% yield (400 mg, 0.3 mmol). H-NMR (CDCl3):
δ
1.50 (18H, s), 2.27 (2H, t), 2.32 (6H, s), 2.38 (2H, m), 3.41 (2H, m), 3.54 (2H, m), 3.65–4.48 (28H, m), 6.82
(6H, m), 6.96–7.8 (14H, m). 13C-NMR:
δ
21.16, 28.12, 28.37, 30.93, 33.89, 40.97, 43.41, 47.57, 52.51, 69.03,
69.54, 70.34, 77.31, 80.37, 106.05, 111.88, 115.76, 124.81, 126.42, 127.29, 127.83, 127.91, 128.06, 128.11,
128.19 128.60, 133.10, 134.13, 138.20, 145.96, 149.19, 153.10, 158.15, 168.60. Mass spectral analysis for
C76H96N6O14-H+, MeOH, H2O, NaCl (cal 1425.74 found 1425.71).
3.3. Measuring the Association Constants
Complex formation between pseudorotaxane
solutions (CDCl3, DMSO-d6, and 70/30 (v/v) DMSO-d6 to D2O) at 25.0 C. For the assay performed in
8
and the wheel ˝were investigated in various
CDCl3, a constant concentration of pseudorotaxane
8 (10 mM) in 0.5 mL CDCl3 was exposed to an
increasing amount of wheel. The wheel was added to the NMR tube via small volume additions from
a stock solution of wheel in CDCl3 (0.15 M). The change in volume of 11% after the final addition of
wheel was not considered in the determination of the association constant. A similar procedure was
followed for the assays performed in the DMSO-d6, except that the concentration of the wheel-stock
solution was higher (25 mM). For the assay performed in 70/30 (v/v) DMSO-d6 to D2O, the wheel
and pseudorotaxane
concentration, precipitation was observed. To obtain association constants, shifts in the chemical shift
of an amide proton of pseudorotaxane , caused by changes in a wheel’s concentration, were plotted
and fitted, using non-linear least-square fitting procedure to derive the association constants [31].
The following binding equation was used to calculate the association constants, ∆δ
8 were mixed to give a final 50 mM solutions of both components. At a higher
8
=
∆δobs ´ ∆δ0
= KA[T] ∆δmax / (1 + KA[T]), where the difference in the chemical shift (∆δ) of an axle proton in the
presence of wheel (∆δobs) and in its absence (∆δ0) depends on the concentration of the wheel (T),

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the chemical shift of the proton when it is completely bound to the wheel (∆δmax), and the association
constant of this complex (KA).
3.4. Observing Disassemblage
A series of 1 dram vials containing a dilute solution of a rotaxane (0.5 mM) was exposed to
various bases at different concentrations. After addition of a base, the samples were run at various
time points on a TLC plate and the amount of rotaxane was estimated via eye. For the 1H-NMR assays,
a 1H-NMR spectrum was taken of a 1 mM solution of FMOC-R
1 in CDCl3 or DMSO-d6 with or without
a cosolvent. A base was added to this solution to give a final solution containing: 2 eq. t-butoxide
or 1% piperidine in 95/5 CDCl3/DMSO-d6, 1% ethanolamine in DMSO-d6, or 1% ethanolamine in
70/30 DMSO-d6/D2O. A second 1H-NMR spectrum was taken immediately after adding a base.
4. Conclusions
A new class of disassemblage [2]rotaxanes were successfully constructed and shown to
disassemble readily in a variety of solvents once the trigger was activated. The [2]rotaxanes were
synthesized in a few steps, resulting in a good, overall yield from starting materials of 20%. The
-
pseudorotaxane is stable enough as the PF6 salt to form [2]rotaxanes in good yields, while as with
the chloride salt, the wheel is only weakly threaded, resulting in dethreading in the millimolar
concentration range in CDCl3, DMSO-d6, and in DMSO-d6/D2O mixtures. Disassemblage is also rapid,
which is important for future sensors to locate a target of interest. The axle is very versatile. It can be
readily derivatized with sensor components and is highly polar once the trigger is activated. High
polarity is crucial for developing sensors that operate in biological solutions. We also demonstrated
that the trigger can be swapped efficiently in a one pot, synthetic procedure.
Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/21/
8/1043/s1.
Acknowledgments: Research reported in this publication was supported by the National Institute of Biomedical
Imaging and Bioengineering of the National Institutes of Health under Award Number R21EB012122.
Author Contributions: D.S. conceived and designed the experiments, performed the experiments; analyzed the
data, and wrote the paper. L.P. performed the experiments.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Disassemblage of FMOC-R1 in DMSO-d6 are available from authors.
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