Synthesis of fluorinated rhodamines and application for confocal laser scanning microscopy

Article abstract of DOI:10.1016/j.jfluchem.2016.07.025

Four different fluorinated fluorescence dyes were prepared by attaching perfluoroalkyl ponytails (including CH₂or C₂H₄spacer) to each of the two amine groups of rhodamine (Rh) and characterized with respect to their fluorescence properties in 2,2,2-trifluoroethanol (TFE), N,N-dimethylformamide (DMF), tetrahydrofuran (THF), and toluene. They showed an excellent quantum yield in TFE. Double staining with Rh-C₂H₄-C₁₀F₂₁and Rh-DPPE was employed to visualize the distribution of perfluoropalmitic acid in mixed giant unilamellar vesicles with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) observed by confocal laser scanning microscopy.

Full text of DOI:10.1016/j.jfluchem.2016.07.025

Journal of Fluorine Chemistry 189 (2016) 70–78
Contents lists available at ScienceDirect
Journal of Fluorine Chemistry
journal homepage: www.elsevier.com/locate/fluor
Synthesis of fluorinated rhodamines and application for confocal laser
scanning microscopy
*
Mark Jbeily, Regina Schöps, Jörg Kressler
Department of Chemistry, Martin Luther University Halle-Wittenberg, D-06099 Halle (Saale), Germany
A R T I C L E I N F O
A B S T R A C T
Article history:
Four different fluorinated fluorescence dyes were prepared by attaching perfluoroalkyl ponytails
(including CH2 or C2H4 spacer) to each of the two amine groups of rhodamine (Rh) and characterized with
respect to their fluorescence properties in 2,2,2-trifluoroethanol (TFE), N,N-dimethylformamide (DMF),
tetrahydrofuran (THF), and toluene. They showed an excellent quantum yield in TFE. Double staining with
Rh-C2H4-C10F21 and Rh-DPPE was employed to visualize the distribution of perfluoropalmitic acid in
mixed giant unilamellar vesicles with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) observed by
confocal laser scanning microscopy.
Received 23 June 2016
Received in revised form 28 July 2016
Accepted 31 July 2016
Available online 1 August 2016
Chemical compounds studied in this article:
Heptafluorobutyric anhydride (PubChem
CID: 67643)
Pentadecafluorooctanoyl chloride
(PubChem CID: 78978)
ã 2016 Elsevier B.V. All rights reserved.
1-iodo-1H,1H,2H,2H-perfluorodecane
(PubChem CID: 74885)
1-iodo-1H,1H,2H,2H-perfluorododecane
(PubChem CID: 74886)
2,2,2-trifluoroethanol (PubChem CID: 6409)
DPPC (PubChem CID: 452110)
Perfluoropalmitic acid (PubChem
CID:106027)
Keywords:
Fluorinated rhodamines
Giant unilamellar vesicles
Confocal laser scanning microscopy
Fluorophilicity
1. Introduction
of fluorine-rich domains by fluorescence techniques since more
and more pharmaceutical active ingredients contain some per-
Rhodamine (Rh) based fluorescence dyes have been employed
frequently for all kinds of fluorescence microscopy [1]. In life
sciences, Rh-based fluorescence dyes with one or two fatty acid
chains are used since they incorporate selectively into the
hydrophobic part of the double layer of cell or model membranes
[2]. Fluorinated Rh dyes are synthesized since they have high
quantum yields, and good photostability [3,4]. Additionally, it has
been demonstrated that they exhibit some kind of fluorophilicity
when functionalized with fluorous ponytails [5] since they tend to
adsorb on fluorous silica gel in flash columns (F-SPE) when eluted
with a fluorophobic solvent as e.g. aqueous methanol as reported
by Kölmel et al. [6]. There seems to be some need for the detection
fluoroalkyl groups [7,8]. Furthermore, designer proteins employ
highly fluorinated non-native amino acids [9], polar hydrophobic
fluorinated sugars are introduced [10,11], fluorinated amphiphiles
are synthesized for drug delivery [12], fluorinated compounds are
used in medicinal chemistry [13,14], and fluorinated block
copolymers are designed for specific membrane interactions
[15,16].
Here, we describe the synthesis of four fluorinated rhodamines
Rh-C,nH2n-CmF2m+1 (F-rhodamines with the m,n combinations of
(1,3), (1,7), (2,8), and (2,10)). One F-ponytail (-CnH2n-CmF2m+1) is
attached to each of the two amine groups of rhodamine. Then, UV–
vis and fluorescence spectra are measured and the quantum yield
is determined. Finally, the dye with the longest F-ponytails Rh-
C2H4-C10F21 is employed together with Rh-DPPE to stain selectively
fluorine-rich domains in mixed giant unilamellar vesicles (GUVs)
of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and
* Corresponding author.
E-mail address: joerg.kressler@chemie.uni-halle.de (J. Kressler).
http://dx.doi.org/10.1016/j.jfluchem.2016.07.025
0022-1139/ã 2016 Elsevier B.V. All rights reserved.

M. Jbeily et al. / Journal of Fluorine Chemistry 189 (2016) 70–78
71
perfluoropalmitic acid (PFPA) as observed by confocal laser
scanning microscopy (CLSM).
bearing C8F17 and C10F21 groups with two methylene spacers in
1-methyl-2-pyrrolidone (NMP) with ethyldiisopropylamine
(DIPEA) as an organic base at 100 ꢀC for 24 h to obtain the C8F17
-
2. Results and discussion
C2H4-secondary amino phenol intermediates (i) and C10F21C2H4-
secondary amino phenol (j), respectively. The last synthesis part
yielding fluorinated fluorescence dyes Rh-CH2-C7F15, Rh-C2H4-
C8F17, and Rh-C2H4-C10F21 was a Friedel-Crafts condensation step
[18] similar to the one used to synthesize Rh-CH2-C3F7 (d). As an
example, the characteristic 1H and 19F NMR spectra are given for
Rh-C2H4-C10F21 (l) in Figs.1 and 2, respectively. The NMR spectra of
the other three F-rhodamines and their electrospray ionization
time of flight (ESI-TOF) spectra are shown in the Supplementary
data (Fig. S1–S10).
2.1. Synthesis and characterization
Four different rhodamines having partially fluorinated pony-
tails as shown in Scheme 1 d,h,k,l were synthesized similarly to
procedures applied by Belov and Hell et al. [3,17] and Kölmel et al.
[6]. Compound h has been reported by Kölmel et al. synthesized in
a different way. For the synthesis of Rh-CH2-C3F7 heptafluorobu-
tyric anhydride was reacted with m-anisidine in dichloromethane
(DCM) in the presence of triethylamine (TEA) as an organic base for
12 h to obtain the C3F7-secondary amide intermediate (a). It was
further reduced in the presence of lithium aluminum hydride
(LiAlH4) in THF under reflux for 12 h to obtain the C3F7-CH2-
secondary amine intermediate (b). It was demethylated by reacting
with boron tribromide in DCM under reflux for 12 h to obtain the
C3F7-CH2-secondary amino phenol intermediate (c). It was then
reacted with phthalic anhydride in the presence of p-toluenesul-
fonic acid monohydrate and an excess of propionic acid at 160 ꢀC
for 24 h to obtain the Rh-CH2-C3F7 fluorescence dye (d) in a yield of
24%. Rh-CH2-C7F15 (h) was synthesized in a similar way to Rh-CH2-
C3F7 (d) with a main difference regarding the first synthesis step of
the C7F15-secondary amide intermediate (e) in which pentadeca-
fluorooctanoyl chloride was used to functionalize m-anisidine with
a C7F15-alkyl group in the presence of pyridine. C7F15-CH2-
secondary amine intermediate (f) was synthesized similarly to
(b) and the C7F15-CH2-secondary amino phenol (g) was prepared in
a similar way to (c). Compounds (i) and (j) were synthesized by
reacting 3-aminophenol with the corresponding alkyl iodide
19F NMR peaks were assigned according to data given by W. R.
Dolbier [19].
2.2. UV–vis and fluorescence spectroscopy
The absorbance and emission of the four dyes was measured in
2,2,2-trifluoroethanol (TFE), N,N-dimethylformamide (DMF), tet-
rahydrofuran (THF), and toluene as a function of concentration in
the dilute regime where the Lambert-Beer law is obeyed
(absorbance less than 0.1, Supplementary data, Fig. S11–S22).
Thus, all UV–vis and fluorescence measurements were done at
concentrations less than 2 mM (equivalent to an absorption of less
than 0.1 in TFE) to be safely within the linear regime. Quantum
yields of the F-rhodamines Fx in TFE, DMF, THF, and toluene were
calculated relative to a standard (fluorescein in 0.1 M NaOH
aqueous solution with a quantum yield of Fst = 0.89) as shown in
Table 1 based on a protocol published by Würth et al. [20].
Absorbance f and emission flux F were measured at 490 and
491 nm for Rh-CH2-C3F7 and Rh-CH2-C7F15, respectively, in TFE,
Scheme 1. Synthesis of F-rhodamines Rh-CnH2n-CmF2m+1. i) DCM, TEA, 0 ꢀC to RT,12 h, ii) DCM, pyridine, 0 ꢀC to RT,12 h; iii) THF, LiAlH4, reflux,12 h; iv) DCM, BBr3, reflux,12 h;
v) NMP, DIPEA, 100 ꢀC, 24 h; vi) propionic acid, phthalic anhydride, p-toluenesulfonic acid monohydrate, 160 ꢀC, 24 h. The following F-rhodamines (n,m) were synthesized d
(1,3), h(1,7), k(2,8), and l(2,10).

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M. Jbeily et al. / Journal of Fluorine Chemistry 189 (2016) 70–78
Fig. 1. 1H NMR spectrum (500 MHz, THF-d8) of Rh-C2H4-C10F21 (l) measured at 27 ꢀC.
Fig. 2. 19F NMR spectrum (470 MHz, THF-d8) of Rh-C2H4-C10F21 (l) measured at 27 ꢀC.
Table 1
Relative quantum yields of F-rhodamines in TFE, DMF, THF, and toluene measured against a fluorescein standard in 0.1 M NaOH as solvent at 20 ꢀC.
Quantum Yield
F
Rh-CH2-C3F7
Rh-CH2-C7F15
Rh-C2H4-C8F17
Rh-C2H4-C10F21
TFE
DMF
THF
0.91a
0.14a
0.06a
0.26a
0.93b
0.10b
0.08b
0.12b
0.88c
0.19d
0.22d
0.18d
0.87c
0.26d
0.20d
0.21d
Toluene
aEmission spectra were recorded at 490 nm, b491 nm, c496 nm, and d504 nm excitation.

M. Jbeily et al. / Journal of Fluorine Chemistry 189 (2016) 70–78
73
DMF, THF, and toluene. The quantum yields of Rh-C2H4-C8F17 and
2.3. Confocal laser scanning microscopy
Rh-C2H4-C10F21 were determined at 496 nm excitation in TFE and
at 504 nm excitation in DMF, THF, and toluene. Eq. (1) was used to
calculate the relative quantum yields of the synthesized F-
rhodamines
The fluorophilic character of the synthesized fluorescence dye
bearing the longest fluorinated ponytails (Rh-C2H4-C10F21) was
tested in GUVs formed by the hydration of a DPPC/PFPA 10:1 (mol
%) mixture including 0.1 mol% Rh-C2H4-C10F21 and 0.1 mol% Rh-
DPPE on agarose [22]. The chemical structures of both dyes are
given on top of Fig. 5. It should be noted that Rh-C2H4-C10F21 is not
able to incorporate into pure DPPC GUVs. An equatorial image of
the obtained GUVs (Fig. 5A) shows that the two dyes in use are
incorporated into the mixed lipid bilayer but not homogeneously.
This is even more evident in Fig. 5B (z-stacking). The red, middle
channel of Fig. 5B shows the DPPC-rich continuous phase stained
by Rh-DPPE. This is not surprising since the fluorescence dye and
the phospholipid have identical acyl chain residues. But the yellow
color of the overlay image indicates that also the F-rhodamine dye
incorporates into the continuous phase. The dark spots in the
middle, red image represent regions where Rh-DPPE is excluded
but the green channel, left image and the overlay, right image
strongly indicate an enrichment of the fluorophilic dye Rh-C2H4-
Fx fst n2x ðsodium lineÞ
Fx
¼
Fstꢁ ꢁ ꢁ
ð1Þ
n2stðsodium lineÞ
Fst fx
where the subscript x refers to the values of the F-rhodamines and
st to the values of the fluorescein standard. For the F-rhodamine
solutions the refractive index of their corresponding solvents were
used and the refractive index of 0.1 M NaOH aqueous solution was
used for the fluorescein standard. The relative quantum yields are
summarized in Table 1.
As observed from Table 1, the F-rhodamines display a high
quantum yield in TFE in contrast to DMF, THF, and toluene where
the quantum yields were significantly lower. Simultaneously, the
UV–vis absorption and emission spectra differ drastically between
TFE solutions and the other organic solvents used.
The absorption peaks in the 400–500 nm range visible in the
TFE solutions (Fig. 3a) disappear when the F-rhodamines are
dissolved in THF (Fig. 4a) and a strong absorption at 285 nm is
observed due to the shift in equilibrium towards the non-
fluorescent spirolactone “closed form” [3]. The “open form”
exhibits strong fluorescence and the spirolactone is non-fluores-
cent as illustrated in Scheme 2. The strong emission observed for
the F-rhodamines in TFE (Fig. 3b) is dramatically reduced in THF
C10-F21. The exclusion of Rh-DPPE from the ordered gel phase of
DPPC is due to the very large head group of the dye compared with
the head group of the lipid [2,23]. Obviously, PFPA is also located in
the dark domains indicated by the fluorescence of the F-
rhodamine. Thus the double staining is a suitable tool to analyze
the internal structure of the hydrophobic part of the double layer of
the GUVs.
(Fig. 4b) and almost vanished for Rh-CH2-C3F7 and Rh-CH2-C7F15
The appearance of the elastic scattering Rayleigh peaks observed at
.
3. Conclusion
the excitation wavelengths indicates the very weak fluorescence in
1 mM THF solutions (Fig. 4b). The situation is similar for DMF and
toluene where the fluorescence is practically switched off as seen
in Fig. S23–26 in comparison with the emission peak when TFE is
used as a solvent.
The synthesized dyes have similar absorption and fluorescence
properties in TFE as shown in Fig. 3a and b. A significant red shift of
about 13 nm in the absorption and emission spectra is observed
when the length of the spacer increases from one to two methylene
groups. This is mainly caused by a decrease of the electron
withdrawing effect of the fluorinated alkyl group on the nitrogen of
the amine group with increasing spacer length [21]. The 22 nm
Stokes shift, i.e. the difference between the maximum absorption
wavelength (lmax,abs) and the maximum emission wavelength
We synthesized four fluorinated rhodamine fluorescence dyes
which were characterized by their UV–vis and fluorescence spectra
and showed excellent quantum yields in TFE. As an example Rh-
C2H4-C10F21 was used as CLSM fluorescence dye and proved to be
stable and resistant to photobleaching after repetitive laser scans.
Rh-C2H4-C10F21 was successfully used to stain fluorinated moieties
partitioned into DPPC/PFPA mixed GUVs (obtained by hydration on
agarose). This is a proof of principle for the possibility of using
highly fluorinated fluorescence dyes to selectively stain fluorine-
rich domains in model membranes.
4. Experimental
4.1. Materials
(lmax,em) remains constant for all four synthesized F-rhodamines.
Thus, it depends neither on the length of the spacer nor on the
length of the fluorinated ponytail. The quantum yields measured in
TFE were similar to other rhodamines measured in other alcohols.
F-rhodamines have a high resistance to photo bleaching and can be
used as laser dyes such as in confocal laser scanning microscopy
(CLSM) [3,4,18].
m-Anisidine, heptafluorobutyric anhydride, perfluoropalmitic
acid (PFPA), and phthalic anhydride were purchased from Alfa
Aesar. Triethylamine (TEA), pyridine, anhydrous dichloromethane
(DCM), anhydrous THF, anhydrous DMF, toluene, agarose super LM,
tert-butyl methyl ether (MTBE), diethyl ether, n-hexane, ethanol,
Fig. 3. Normalized absorption (a) and emission (b) spectra of 1 m
M F-rhodamines in TFE at 20 ꢀC.

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M. Jbeily et al. / Journal of Fluorine Chemistry 189 (2016) 70–78
Fig. 4. Normalized absorption (a) and emission (b) spectra (Rh-CH2-C3F7 and Rh-CH2-C7F15 were excited at 490 and 491 nm respectively; Rh-C2H4-C8F17 and Rh-C2H4-C10F21
were both excited at 504 nm) of 1
M F-rhodamines in THF at 20 ꢀC.
m
Scheme 2. Equilibrium between the fluorescent (open) and the non-fluorescent (closed) spirolactone.
and chloroform were purchased from Carl Roth. Lithium aluminum
hydride [2 M] THF solution, boron tribromide [1 M] dichloro-
methane solution, propionic acid, p-toluenesulfonic acid mono-
hydrate, pentadecafluorooctanoyl chloride, 3-aminophenol, 1-
iodo-1H,1H,2H,2H-perfluorodecane,1-iodo-1H,1H,2H,2H-perfluor-
ododecane, anhydrous 1-methyl-2-pyrrolidone (NMP), ethyldiiso-
propylamine (DIPEA), and nonafluorobutyl methyl ether (HFE-
7100) were purchased from Sigma-Aldrich. 1,2-dipalmitoyl-sn-
glycero-3-phosphocholine (DPPC) was purchased from Avanti
Polar Lipids. Rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phos-
phoethanolamine (Rh-DPPE) was purchased from Thermo Fisher
Scientific. Deuterated solvents for solution NMR spectroscopy
(CDCl3, CD3OD, DMSO-d6, and THF-d8) were purchased from Armar
Chemicals. All chemicals were used without further purification.
spectra, labs,max was used to excite the corresponding F-rhod-
amines. Rh-CH2-C3F7 and Rh-CH2-C7F15 were excited at 490 and
491 nm, respectively, whereas Rh-C2H4-C8F17 and Rh-C2H4-C10F21
were excited at 504 nm. The calibration of the FluoroMax 2
spectrometer was checked with the water Raman peak at 397 nm
before and after the measurement of each sample. All fluorescence
measurements were done at 20 ꢀC. Emission spectra were recorded
with 0.5 nm increments, 0.1 s integration time, and 2 nm excitation
and emission slits. The emission spectra were recorded as counts
per second (cps) as a function of the wavelength (nm) using S/R
(signal/reference) detection mode to compensate the aging effect
of the xenon lamp.
50
Rh-DPPE were mixed with 50
diethyl ether/nonafluorobutyl methyl ether 1:1 (v/v) containing
0.1 mol% Rh-C2H4-C10F21. 5 l of the homogeneous organic solution
m
l of a 20 mM DPPC chloroform solution containing 0.1 mol%
m
l of a 2 mM PFPA acid solution of
4.2. Methods
m
were applied as a thin layer on an agarose coated cover glass, dried
for 10 min at RT, 1 min at 50 ꢀC, followed by gentle hydration in
deionized water for 2 h at 45 ꢀC [23]. Double staining was realized
with Rh-DPPE and Rh-C2H4-C10F21. A Leica TCS SP2 DM IRE2
confocal microscope equipped with an HCX PL APO lbd.BL
63 ꢂ1.2 W CORR (water immersion) objective (Leica Microsystems,
Wetzlar, Germany) was used [24]. Commercially available Rh-DPPE
(0.1 mol%) was used to stain the GUVs, excited with the 543 nm
laser and a detection range of 580–620 nm (red channel). The F-
rhodamine Rh-C2H4-C10F21 (0.1 mol%) was excited with the 514 nm
laser and a detection range of 525–540 nm (green channel).
Imaging of the obtained GUVs was done at room temperature (RT).
Z-stacking was performed from bottom to top.
Solution NMR spectra were recorded at 27 ꢀC by Agilent
Technologies 400 MHz 1H VNMRS spectrometer or Agilent
Technologies 500 MHz 1H DD2 spectrometer. Samples were
dissolved in deuterated solvents (CDCl3, CD3OD, DMSO-d6, or
THF-d8). 1H and 13C NMR spectra were reported relative to TMS, 19
F
spectra relative to CFCl3, and 13C NMR spectra were proton
decoupled.
ESI-TOF measurements were performed on a Focus microToF by
Bruker Daltonics. The samples were dissolved in 2,2,2-trifluor-
oethanol to a final concentration of 30
(180.00 L/h, positive mode).
mM and directly infused
m
Varian-Cary 4000 double beam UV–vis spectrometer was used
for UV–vis measurements. BRAND1 UV micro single-use cuvettes
(mfr. no. BRAND1 7592 20) with center height 15 mm having a
4.3. Synthesis of fluorescence dyes
1 cm path length were filled with 500 mL 2,2,2-trifluoroethanol
(TFE) solutions. UV–vis measurements were done with baseline
correction (subtraction) at a temperature of 20 ꢀC. Fluorescence
was measured by a FluoroMax 2 spectrometer (Jobin-Yvon).
200
(105.250-QS) with center height 15 mm. For fluorescence emission
4.3.1. Rh-CH2-C3F7 synthesis (a–d)
Secondary amide intermediate (a): 5 g (40.6 mmol) m-anisi-
dine, 19.98 g (48.72 mmol) heptafluorobutyric anhydride, and
0.493 g (4.872 mmol) triethylamine were added to a Schlenk flask
equipped with a magnetic stirrer, containing 50 mL anhydrous
m
L solutions were placed in a Hellma SUPRASIL1 cuvette

M. Jbeily et al. / Journal of Fluorine Chemistry 189 (2016) 70–78
75
Fig. 5. The left chemical structure gives the Rh-C2H4-C10F21 employed for CLSM and the right chemical structure represents Rh-DPPE. CLSM equatorial image (A) and z-
stacking image (B) of GUVs made from DPPC/PFPA 10:1 (mol%) binary mixture. The Rh-C2H4-C10F21 dye (0.1 mol%) is observed through the green (left) channel, the Rh-DPPE
dye (0.1 mol%) is observed through the red (middle) channel, and the right images show the overlay of both channels. (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.)
DCM maintained at 0 ꢀC in an ice-water bath. The reaction was left
under continuous stirring for 1 h in the ice-water bath then
removed and left under constant stirring at RT overnight. The
reaction was opened to the atmosphere, and the organic phase was
further diluted with 100 mL DCM. The DCM phase was washed
three times with 5% NaHCO3 aqueous solution, one time with
water, three times with 0.01 M HCl aqueous solution, and three
times with sodium chloride brine. The DCM phase was dried over
sodium sulfate, filtered and concentrated at 30 ꢀC using a rotary
evaporator under reduced pressure. 150 mL n-hexane were added
to the round bottom flask and heated to boiling. The clear warm n-
hexane phase was decanted into a beaker, and the product was left
to crystallize at 4 ꢀC overnight. The product was filtered with a
Büchner funnel, washed with portions of cold n-hexane and dried
under vacuum at RT. Recrystallization from n-hexane was repeated
a second time to give compound (a) as a white solid with 54% yield.
aluminum hydride in THF solution. The reaction was refluxed for
12 h, left to cool down slowly to RT, and quenched with 1 mL water.
The THF phase was filtered, diluted with 200 mL DCM, and filtered
a second time. The organic phase was washed two times with
water, twice with 5% NaHCO3 aqueous solution and twice with
NaCl brine. The organic phase was dried over sodium sulfate,
filtered and evaporated at 30 ꢀC using a rotary evaporator under
reduced pressure. The product was purified by silica gel column
chromatography with mobile phase composition cyclohexane/
ethyl acetate 4:1 (v/v) to obtain compound (b) as a slightly yellow
viscous liquid with 61% yield. 1H NMR (400 MHz, CDCl3)
d 7.12
(t, J = 8.1 Hz, 1H), 6.37 (dd, J = 8.2, 2.3 Hz, 1H), 6.30 (dd, J = 8.1, 2.1 Hz,
1H), 6.24 (t, J = 2.3 Hz, 1H), 3.92–3.79 (m, 3H), 3.78 (s, 3H). 13C NMR
(101 MHz, CDCl3)
104.12 (s), 99.58 (s), 55.13 (s), 44.18 (t, J = 23.4 Hz). 19F NMR
(376 MHz, CDCl3)
d 160.83 (s), 147.76 (s), 130.19 (s), 106.08 (s),
d
ꢃ80.74 (t, J = 9.4 Hz, 3F), ꢃ118.89–119.22 (m,
1H NMR (400 MHz, CDCl3)
7.01 (m, 1H), 6.83–6.78 (m, 1H), 3.83 (s, 3H). 13C NMR (101 MHz,
CDCl3) 160.34 (s), 136.17 (s), 130.10 (s), 112.46 (s), 112.36 (s),
106.20 (s), 55.42 (s). 19F NMR (376 MHz, CDCl3)
d
7.86 (s, 1H), 7.33–7.27 (m, 2H), 7.07–
2F), ꢃ127.54–127.73 (m, 2F).
d
4.3.3. Secondary amino phenol intermediate (c)
d
ꢃ80.51 (t,
1 g (3.277 mmol) C3F7-CH2-sec-amine (b) was transferred to a
two-neck round-bottom flask equipped with a magnetic stirrer,
reflux condenser and a drying tube filled with calcium chloride.
20 mL DCM were added followed by 4.92 mL (4.92 mmol) of 1 M
boron tribromide solution in DCM. The reaction was refluxed for
12 h, left to cool down slowly to RT, and quenched with 1 mL water.
The DCM phase was added to 100 mL 5% NaHCO3 aqueous solution
and the pH was checked with pH paper to be around 8. The product
J = 8.8 Hz, 3F), ꢃ120.06–120.50 (m, 2F), ꢃ126.54–126.87 (m, 2F).
4.3.2. Secondary amine intermediate (b)
2 g (6.27 mmol) C3F7-sec-amide (a) were transferred into a two-
neck round-bottom flask equipped with a magnetic stirrer, reflux
condenser and a drying tube filled with calcium chloride. 40 mL
THF were added followed by 7.84 mL (15.68 mmol) of a 2 M lithium

76
M. Jbeily et al. / Journal of Fluorine Chemistry 189 (2016) 70–78
was extracted with portions of DCM, and the organic portions were
combined and washed three times with NaCl brine, dried over
sodium sulfate and filtered. DCM was removed on a rotary
evaporator at 30 ꢀC under reduced pressure. The product was
purified by silica gel column chromatography with mobile phase
composition cyclohexane/ethyl acetate 3:1 (v/v) to obtain com-
pound (c) as a brownish waxy solid with 58% yield. 1H NMR
4.3.6. Secondary amine intermediate (f)
1 g (1.926 mmol) C7F15-secondary amide (e) was transferred
into a two-neck round-bottom flask equipped with a magnetic
stirrer, reflux condenser, and a drying tube filled with calcium
chloride. 30 mL THF were added followed by 2.4 mL (4.8 mmol) of a
2 M lithium aluminum hydride solution in THF. The reaction was
refluxed for 12 h, left to cool down slowly to RT and quenched with
0.5 mL water. The THF phase was filtered to remove insoluble salts,
diluted with 200 mL diethyl ether, and filtered. The organic phase
was washed one time with water, twice with 5% NaHCO3 aqueous
solution and twice with sodium chloride brine. The organic phase
was dried over sodium sulfate and filtered. The organic solvents
were evaporated at 30 ꢀC using a rotary evaporator under reduced
pressure to obtain compound (f) as a yellowish viscous liquid with
68% yield. No further purification steps were needed. 1H NMR
(400 MHz, DMSO-d6)
2H), 6.09–6.05 (m, 1H), 6.05–5.98 (m, 1H), 3.96–3.81 (m, 2H). 13
NMR (101 MHz, DMSO-d6) 158.60 (s),149.46 (s),129.99 (s),105.16
(s), 104.21 (s), 100.07 (s), 43.28 (t, J = 22.5 Hz). 19F NMR (376 MHz,
DMSO-d6)
d 9.03 (s,1H), 6.93–6.82 (m,1H), 6.19–6.11 (m,
C
d
d
ꢃ80.29 (t, J = 9.4 Hz, 3F), ꢃ117.46–117.76 (m, 2F),
ꢃ127.05–127.40 (m, 2F).
4.3.4. Rh-CH2-C3F7 (d)
246.6 mg (0.8469 mmol) C3F7-CH2-amino-phenol (c), 100.4 mg
(0.6778 mmol) phthalic anhydride, 564.6 mg (7.621 mmol) pro-
pionic acid, and 12.08 mg (0.0635 mmol) p-toluenesulfonic acid
monohydrate were added to a Schlenk flask equipped with a
(500 MHz, CDCl3)
6.29 (m, 1H), 6.25 (t, J = 2.3 Hz, 1H), 3.97–3.80 (m, 3H), 3.78 (s, 3H).
13C NMR (126 MHz, CDCl3)
160.84 (s), 147.76 (s), 130.18 (s), 106.06
(s), 104.11 (s), 99.56 (s), 55.11 (s), 44.40 (t, J = 23.2 Hz). 19F NMR
(470 MHz, CDCl3)
d 7.12 (t, J = 8.1 Hz, 1H), 6.39–6.36 (m, 1H), 6.32–
d
magnetic stirrer. The reaction was carried out in
a
160 ꢀC
d
ꢃ80.79 (tt, J = 10.1, 2.4 Hz, 3F), ꢃ117.90–118.37
thermostated oil bath for 24 h under a nitrogen atmosphere and
continuous stirring. The reaction was left to cool down, and the
Schlenk flask was opened to the atmosphere. The products were
dissolved in chloroform/ethanol 10:1 (v/v) mixture, filtered and
washed six times with 0.1 M HCl aqueous solution. The organic
phase was dried over sodium sulfate and filtered. The product was
purified by silica gel column chromatography with a chloroform/
methanol 3:1 (v/v) mobile phase and later precipitated from a
concentrated acetone solution into excess cold n-hexane. The
product was filtered using a Büchner funnel and washed with
portions of cold n-hexane then put under vacuum at RT to yield Rh-
CH2-C3F7 (d) as an orange solid with 24% yield. 1H NMR (400 MHz,
(m, 2F), ꢃ121.63–121.90 (m, 2F), ꢃ121.91–122.21 (m, 2F), ꢃ122.56–
122.87 (m, 2F), ꢃ123.11–123.53 (m, 2F), ꢃ125.90–126.34 (m, 2F).
4.3.7. Secondary amino phenol intermediate (g)
0.5 g (0.99 mmol) C7F15-CH2-secondary amine (f) was trans-
ferred into a two-neck round-bottom flask equipped with a
magnetic stirrer, reflux condenser and a drying tube filled with
calcium chloride. 20 mL DCM were added followed by 1.5 mL
(1.5 mmol) 1 M boron tribromide solution in DCM and refluxed for
12 h. The reaction was left to cool down slowly to RT and quenched
with 0.25 mL water. The DCM phase was added to 100 mL 5%
NaHCO3 aqueous solution and the pH was checked with pH paper
to be around 8. The product was extracted with portions of DCM,
and the organic portions were combined and washed three times
with NaCl brine, dried over sodium sulfate and filtered. DCM was
removed on a rotary evaporator at 30 ꢀC under reduced pressure to
obtain compound (g) as a brownish waxy solid with 73% yield. No
further purification steps were needed. 1H NMR (500 MHz, CDCl3)
CD3OD)
J = 7.5, 1.0 Hz, 1H), 7.32 (d, J = 7.3 Hz, 1H), 6.94–6.86 (m, 4H), 6.79–
6.75 (m, 2H), 4.18 (t, J = 15.7 Hz, 4H). 19F NMR (376 MHz, CD3OD)
d 8.16 (d, J = 7.5 Hz, 1H), 7.79 (td, J = 7.4, 1.2 Hz, 1H), 7.74 (td,
d
ꢃ82.33 (t, J = 9.7 Hz, 6F), ꢃ119.19–119.47 (m, 4F), ꢃ128.45–129.00
(m, 4F). ESI-TOF, m/z: calculated for C28H17F14N2O3 695.101 [M]+;
found 695.0288.
d
7.06 (t, J = 8.1 Hz,1H), 6.28 (d, J = 2.3 Hz,1H), 6.27 (d, J = 2.3 Hz,1H),
6.20 (t, J = 2.3 Hz, 1H), 4.78 (s, 1H), 3.96–3.76 (m, 3H). 13C NMR
(126 MHz, CDCl3) 156.67 (s), 148.03 (s), 130.40 (s), 106.11 (s),
106.07 (s), 100.23 (s), 44.33 (t, J = 23.3 Hz). 19F NMR (470 MHz,
CDCl3)
4.3.5. Rh-CH2-C7F15 synthesis (e–h)
d
Secondary amide intermediate (e): 5 g (11.56 mmol) pentade-
cafluorooctanoyl chloride, 1.186 g (9.631 mmol) m-anisidine, and
0.9525 g (12.04 mmol) pyridine were added to a Schlenk flask
equipped with a magnetic stirrer, containing 50 mL DCM main-
tained at 0 ꢀC in an ice water bath. The reaction was left under
continuous stirring for one hour in the ice-water bath then at RT
overnight. The reaction was opened to the atmosphere, and the
organic phase was further diluted with 100 mL DCM and filtered to
remove part of the precipitated pyridinium chloride. The DCM
phase was washed three times with 0.01 M HCl aqueous solution,
one time with water, three times with 5% NaHCO3, three times with
NaCl brine, dried over sodium sulfate, and filtered. The DCM phase
was concentrated with a rotary evaporator at 30 ꢀC under reduced
pressure until the onset of turbidity. Approximately 250 mL n-
hexane were added to the DCM concentrate and the solution was
heated to boiling. The upper clear organic phase was decanted into
a beaker and allowed to crystallize at RT overnight to yield the
product C7F15-secondary amide (e) as a white solid with 59% yield.
d
ꢃ80.80 (tt, J = 10.1, 2.4 Hz, 3F), ꢃ117.84–118.42 (m, 2F),
ꢃ121.57–121.90 (m, 2F), ꢃ121.91–122.21 (m, 2F), ꢃ122.57–122.93
(m, 2F), ꢃ123.17–123.47 (m, 2F), ꢃ125.96–126.29 (m, 2F).
4.3.8. Rh-CH2-C7F15 (h)
300 mg (0.6107 mmol) C7F15-CH2-phenol (g), 72.39 mg
(0.4887 mmol) phthalic anhydride, 407.3 mg (5.498 mmol) pro-
pionic acid, and 8.716 mg (0.04582 mmol) p-toluenesulfonic acid
monohydrate were added to a Schlenk flask equipped with a
magnetic stirrer. The reaction was carried out in
a
160 ꢀC
thermostated oil bath for 24 h under a nitrogen atmosphere and
continuous stirring. The reaction was left to cool down, and the
Schlenk flask was opened to the atmosphere. The products were
dissolved in chloroform/ethanol 10:1 (v/v) mixture, filtered and
washed six times with 0.1 M HCl aqueous solution. The organic
phase was dried over sodium sulfate and filtered. The product was
purified by silica gel column chromatography with a chloroform/
methanol 5:1 (v/v) mobile phase and later precipitated from a
concentrated acetone solution into excess cold n-hexane. The
product was filtrated using a Büchner funnel and washed with
portions of cold n-hexane then put under vacuum at RT to obtain
Rh-CH2-C7F15 (h) as an orange solid with 19% yield. 1H NMR
1H NMR (500 MHz, CDCl3)
(ddd, J = 8.1, 2.0, 0.6 Hz, 1H), 6.80 (ddd, J = 8.4, 2.4, 0.7 Hz, 1H), 3.82
(s, 3H).13C NMR (126 MHz, CDCl3)
160.35 (s),136.19 (s),130.09 (s),
112.47 (s),112.35 (s),106.20 (s), 55.41 (s).19F NMR (470 MHz, CDCl3)
d 7.87 (s, 1H), 7.32–7.27 (m, 2H), 7.04
d
d
ꢃ80.77 (tt, J = 10.1, 2.4 Hz, 3F), ꢃ119.21–119.34 (m, 2F), ꢃ121.31–
121.55 (m, 2F), ꢃ121.80–122.04 (m, 2F), ꢃ122.15–122.32 (m, 2F),
(500 MHz, CD3OD)
d 8.02 (d, J = 7.4 Hz, 1H), 7.74 (td, J = 7.5, 1.2 Hz,
ꢃ122.56–122.81 (m, 2F), ꢃ126.02–126.15 (m, 2F).
1H), 7.68 (td, J = 7.5,1.0 Hz,1H), 7.24 (d, J = 7.5 Hz,1H), 6.72–6.68 (m,

M. Jbeily et al. / Journal of Fluorine Chemistry 189 (2016) 70–78
77
4H), 6.61–6.57 (m, 2H), 4.08 (t, J = 15.8 Hz, 4H). 19F NMR (470 MHz,
CD3OD)
ꢃ122.59–122.88 (m, 4F), ꢃ122.89–123.21 (m, 4F), ꢃ123.59–123.92
(m, 4F), ꢃ124.12–124.43 (m, 4F), ꢃ127.14–127.53 (m, 4F). ESI-TOF,
m/z: calculated for C36H17F30N2O3 1095.0755 [M]+; found
1094.9658.
The reaction mixture was bubbled with nitrogen for 30 min then
transferred to a 100 ꢀC thermostated oil bath and kept under
stirring for 24 h. The Schlenk flask was removed from the oil bath
and left to cool down to RT then opened to the atmosphere. The
reaction was diluted with 200 mL MTBE and filtered to remove the
precipitated DIPEA.HI salt. The organic phase was washed six times
with a 10% NaCl aqueous solution (w/w), dried over sodium sulfate
d
ꢃ82.40 (tt, J = 10.1, 2.4 Hz, 6F), ꢃ118.41–118.87 (m, 4F),
4.3.9. Rh-C2H4-C8F17 synthesis (i,k)
Secondary amino phenol intermediate (i): 861.2 mg
(7.892 mmol) 3-aminophenol, 10 g (17.42 mmol) 1-Iodo-
and filtered. The MTBE phase was evaporated on a rotary
evaporator at 40 ꢀC under reduced pressure, and the product
was crystallized twice from a clear boiling n-hexane solution at RT
to yield C10F21-C2H4-secondary amino phenol (j) as an off-white
1H,1H,2H,2H-perfluorodecane,
20 mL
NMP,
and
2.559 g
(19.80 mmol) DIPEA were transferred into a Schlenk flask equipped
with a magnetic stirrer. The reaction mixture was bubbled with
nitrogen for 30 min then transferred to a 100 ꢀC thermostated oil
bath and kept under stirring for 24 h. The Schlenk flask was
removed from the oil bath and left to cool down to RT then opened
to the atmosphere. The reaction was diluted with 300 mL MTBE
and filtered to remove the precipitated DIPEA.HI salt. The organic
phase was washed ten times with a 10% NaCl aqueous solution (w/
w), dried over sodium sulfate, and filtered. The MTBE phase was
evaporated on a rotary evaporator at 40 ꢀC under reduced pressure,
and the product was purified by silica gel column chromatography
solid with 70% yield. 1H NMR (500 MHz, CDCl3)
d 7.06 (t, J = 8.0 Hz,
1H), 6.25 (ddd, J = 8.0, 2.3, 0.7 Hz, 1H), 6.21 (ddd, J = 8.1, 2.2, 0.7 Hz,
1H), 6.14 (t, J = 2.3 Hz,1H), 3.80 (s,1H), 3.57–3.46 (m, 2H), 2.49–2.32
(m, 2H). 19F NMR (470 MHz, CDCl3)
ꢃ113.65–113.98 (m, 2F), ꢃ121.23–122.11 (m, 10F), ꢃ122.49–122.86
(m, 2F), ꢃ123.20–123.73 (m, 2F), ꢃ125.82–126.34 (m, 2F).
d
ꢃ80.76 (t, J = 10.0 Hz, 3F),
4.3.12. Rh-C2H4-C10F21 (l)
458 mg (0.699 mmol) C10F21-C2H4-secondary amino phenol (j),
82.83 mg (0.5592 mmol) phthalic anhydride, 466 mg (6.290 mmol)
propionic acid and 9.97 mg (52.4
monohydrate were added to a Schlenk flask equipped with a
magnetic stirrer. The reaction was carried out in
160 ꢀC
mmol) p-toluenesulfonic acid
with an n-hexane/MTBE 1:1 (v/v) mobile phase yielding C8F17
C2H4-phenol (i) as an off-white solid with 61% yield. 1H NMR
(400 MHz, CDCl3) 7.05 (t, J = 8.0 Hz, 1H), 6.25–6.17 (m, 2H), 6.14–
6.08 (m, 1H), 4.59 (s, 1H), 3.79 (s, 1H), 3.52 (t, J = 7.1 Hz, 2H), 2.53–
2.26 (m, 2H). 19F NMR (376 MHz, CDCl3)
-
a
d
thermostated oil bath for 24 h under nitrogen atmosphere and
continuous stirring. The reaction was left to cool down, and the
Schlenk flask was opened to the atmosphere. The products were
dissolved in HFE-7100/THF/ethanol 10:2:1 (v/v/v) mixture, filtered
and washed six times with 0.1 M HCl aqueous solution. The organic
phase was dried over sodium sulfate and filtered. Organic solvents
were evaporated on a rotary evaporator at 45 ꢀC under reduced
pressure. Silica gel column chromatography with gradient elution
starting from MTBE/THF 10:1 (v/v) and slowly decreasing the MTBE
portion reaching pure THF was used to obtain Rh-C2H4-C10F21 (l) as
a red solid. The product was dissolved in a small quantity of
acetone and precipitated into excess cold n-hexane, filtered using a
Büchner funnel and dried under vacuum at RT to obtain Rh-C2H4-
d
ꢃ80.76 (t, J = 10.0 Hz, 3F),
ꢃ113.49–114.13 (m, 2F), ꢃ121.24–122.21 (m, 6F), ꢃ122.44–122.92
(m, 2F), ꢃ123.17–123.70 (m, 2F), ꢃ125.75–126.36 (m, 2F).
4.3.10. Rh-C2H4-C8F17 (k)
0.6 g
(0.8648 mmol) phthalic anhydride, 0.721 g (9.73 mmol) propionic
acid, and 15.42 mg (81.06 mol) p-toluenesulfonic acid monohy-
(1.081 mmol) C8F17-C2H4-phenol
(i),
128.1 mg
m
drate were added to a Schlenk flask equipped with a magnetic
stirrer. The reaction was carried out in a 160 ꢀC thermostated oil
bath for 24 h under nitrogen atmosphere and continuous stirring.
The reaction was left to cool down, and the Schlenk flask was
opened to the atmosphere. The products were dissolved in
chloroform/THF/ethanol 10:1:1 (v/v/v) mixture, filtered and
washed six times with 0.1 M HCl aqueous solution. The organic
phase was dried over sodium sulfate and filtered. Organic solvents
were evaporated on a rotary evaporator at 45 ꢀC under reduced
pressure and silica gel column chromatography with gradient
elution starting from diethyl ether/THF 25:1 (v/v) and slowly
C
10F21 (l) as a red solid with 20% yield. 1H NMR (500 MHz, THF-d8)
d
7.90 (d, J = 7.6 Hz, 1H), 7.64 (td, J = 7.4, 1.1 Hz, 1H), 7.58 (td, J = 7.4,
0.7 Hz, 1H), 7.15 (d, J = 7.6 Hz, 1H), 6.48 (d, J = 8.6 Hz, 2H), 6.42 (d,
J = 2.3 Hz, 2H), 6.29 (dd, J = 8.7, 2.3 Hz, 2H), 5.54 (t, J = 6.1 Hz, 2H),
3.56–3.49 (m, 4H), 2.64–2.35 (m, 4H). 19F NMR (470 MHz, THF-d8)
d
ꢃ79.95 (t, J = 10.0 Hz, 6F), ꢃ112.54–112.89 (m, 4F), ꢃ120.09–120.90
(m, 20F), ꢃ121.23–121.62 (m, 4F), ꢃ121.94–122.35 (m, 4F),
ꢃ124.78–125.13 (m, 4F). ESI-TOF, m/z: calculated for
increasing the THF portion reaching
a
final mobile phase
C
44H21F42N2O3 1423.0876 [M]+; found 1422.9367.
composition of diethyl ether/THF 1:1 (v/v) was used to obtain
the Rh-C2H4-C8F17 (k) as a red solid. The product was dissolved in a
small quantity of acetone and precipitated into excess cold n-
hexane, filtered using a Büchner funnel and dried under vacuum at
RT to obtain Rh-C2H4-C8F17 (k) as a red solid with 23% yield. 1H
Acknowledgments
The authors thank Deutsche Forschungsgemeinschaft (DFG),
Forschergruppe FOR 1145, TP3.
NMR (500 MHz, THF-d8)
d 7.91 (d, J = 7.6 Hz, 1H), 7.65 (td, J = 7.5,
1.1 Hz,1H), 7.59 (td, J = 7.5, 0.7 Hz,1H), 7.16 (d, J = 7.6 Hz,1H), 6.48 (d,
J = 8.6 Hz, 2H), 6.42 (d, J = 2.3 Hz, 2H), 6.30 (dd, J = 8.7, 2.3 Hz, 2H),
5.55 (t, J = 6.1 Hz, 2H), 3.56–3.48 (m, 4H), 2.58–2.45 (m, 4H). 19F
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
NMR (376 MHz, THF-d8)
d
ꢃ81.81 (ꢃ81.81 (t, J = 10.1 Hz, 6F),
the
online
version,
at
http://dx.doi.org/10.1016/j.
ꢃ114.37–114.78 (m, 4F), ꢃ121.84–122.92 (m, 12F), ꢃ123.06–123.58
(m, 4F), ꢃ123.69–124.35 (m, 4F), ꢃ126.55–127.18 (m, 4F). ESI-TOF,
m/z: calculated for C40H21F34N2O3 1223.1004 [M]+; found
1222.9859.
jfluchem.2016.07.025.
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