Synthesis and characterization of fluorinated conjugates of albumin

Article abstract of DOI:10.1016/j.jfluchem.2013.01.026

A small fluorocarbon dendron that contains nine chemically identical fluorine atoms was covalently conjugated to albumin via a flexible linker. Two versions were made, which differ by 10% in the linker length. Both versions display split ¹⁹F signal and much shorter ¹⁹F longitudinal relaxation time than their small molecule counterparts. 10% difference in the flexible linker length has negligible impact on the ¹⁹F signal.

Full text of DOI:10.1016/j.jfluchem.2013.01.026

Journal of Fluorine Chemistry 152 (2013) 173–181
Contents lists available at SciVerse ScienceDirect
Journal of Fluorine Chemistry
journal homepage: www.elsevier.com/locate/fluor
Synthesis and characterization of fluorinated conjugates of albumin
a
a
a,b,
Xuyi Yue , Yue Feng , Y. Bruce Yu
*
a
Department of Pharmaceutical Sciences, University of Maryland, Baltimore, MD 21201, United States
Fischell Department of Bioengineering, University of Maryland, College Park, MD 20742, United States
b
A R T I C L E I N F O
A B S T R A C T
Article history:
A small fluorocarbon dendron that contains nine chemically identical fluorine atoms was covalently
conjugated to albumin via a flexible linker. Two versions were made, which differ by 10% in the linker
Received 30 November 2012
Received in revised form 21 January 2013
Accepted 25 January 2013
Available online 4 February 2013
19
19
length. Both versions display split F signal and much shorter F longitudinal relaxation time than their
19
small molecule counterparts. 10% difference in the flexible linker length has negligible impact on the
signal.
F
ß 2013 Elsevier B.V. All rights reserved.
Keywords:
Albumin
Fluorination
Fluorocarbon dendron
Conjugation
NMR
Longitudinal relaxation time
1
. Introduction
albumin-bound gadolinium chelates, such as albumin-(Gd-
DTPA)30, dissociation of Gd-DTPA from albumin is prevented. As
a result, MRI-determined tumor microvascular parameters corre-
late much better with histopathological data [7]. However, the long
circulation time of covalently-bound Gd-DTPA increases the
possibility that the highly toxic Gd is released from the complex
into blood circulation. This risk factor limits albumin-(Gd-DTPA)30
and its analogs to preclinical studies [7].
Albumin is the most abundant protein in plasma with a
concentration around 35–50 g/L in human sera [1]. It is a
monomeric, multi-domain globular protein with a molecular
weight of 66.5 kDa. Human serum albumin has 585 amino acid
residues. Albumin has many medical [2,3] and pharmaceutical
applications [1,4], one of which is as macromolecular carrier of
imaging agents to prolong circulation time and to enhance tumor
accumulation. There are two classes of imaging agents that use
3
+
An alternative class of albumin-based imaging agents is
1
9
19
fluorinated albumins for F MRI. F is a stable isotope with a
99m
19
albumin as the carrier:
Tc-aggregated albumins for single-
spin quantum number of 1/2 and 100% natural abundance. F is
photon emission computed tomography (SPECT), such as Pulmo-
lite and Nanocoll ; and albumin-bound gadolinium chelates
the second most sensitive stable nucleus for MRI (83% as sensitive
1
1
1
1
19
as H). Compared with H MRI, F MRI is free of endogenous
1
9
for magnetic resonance imaging (MRI), such as gadofosveset
background signal. F imaging agents are directly MRI visible and
1
19
(
Ablavar ) [5].
the
F signal intensity is proportional to imaging agent
SPECT has lower spatial resolution than MRI and involves
concentration. In comparison, gadolinium-based contrast agents
99m
1
ionizing radiation. In fact, the use of
Tc-aggregated albumins in
are indirectly visible through the H signal whose intensity has
19
disease diagnosis has been in decline [6]. However, albumin-bound
gadolinium chelates have their own problems. For example, for
non-covalently albumin-bound gadolinium chelates, such as
gadofosveset, the release of gadofosveset from albumin diminishes
the correlation between MRI-determined tumor microvascular
parameters and histopathological data [7]. In the case of covalently
complex dependency on gadolinium concentration. Hence F MRI
1
is much more suited for quantitative imaging than H MRI.
1
9
To form albumin-based
F imaging agents, fluorocarbon
molecules need to bind to albumin, either non-covalently or
covalently, analogous to albumin-bound gadolinium chelates. Both
types have been explored before, but not in a site-specific manner.
Non-covalently fluorinated albumins can be formed simply by
mixing a fluorocarbon molecule (e.g., tetrafluorosuccinic acid)
with albumin [8]. However, non-covalently fluorinated albumins
would suffer the same problems of non-covalent albumin-
gadolinium chelates in that the imaging agent can dissociate
readily from albumin. This problem is avoided when fluorocarbon
*
Corresponding author at: Department of Pharmaceutical Sciences, University of
Maryland, Baltimore, MD 21201, United States. Tel.: +1 410 706 7514;
fax: +1 410 706 5017.
E-mail address: byu@rx.umaryland.edu (Y.B. Yu).
0022-1139/$ – see front matter ß 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jfluchem.2013.01.026

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X. Yue et al. / Journal of Fluorine Chemistry 152 (2013) 173–181
molecules are covalently linked to albumin. One prior example is
conjugating fluorinated anhydrides to the -amino group of lysine
9]. However, albumin has a large number of lysine residues (58 for
e
[
human serum albumin [10]). Therefore it is very difficult to obtain
a single product through the fluorination of lysine residues. As
imaging agents are pharmaceuticals that require regulatory
approval, it is highly desirable to have a single molecular entity
with well-defined chemical structure. The goal of this project is to
explore site-specific fluorination of albumin.
Fig. 1. Structure of fluorinated albumin. F represents the fluorocarbon dendron; TEG
refers to tetraethylene glycol; BSA is bovine serum albumin. The asterisk indicates a
chiral center generated during conjugation.
One approach to site-specific protein fluorination is to
introduce fluorinated amino acids such as
L-4-trifluorophenylala-
2.2. Synthesis of fluorinated albumins
nine through genetic engineering using expanded genetic codon
1
9
[
11]. However, the F signal from a single –CF
3
group in an amino
The synthesis of fluorinated albumins started with connecting
the fluorocarbon dendron to the flexible linker. As shown in
Scheme 1, protection of one of the hydroxyl groups in tetra-
19
acid is too weak for F MRI. Another approach to site-specific
protein fluorination is through chemical conjugation to the –SH
group of cysteine. Albumin has 35 cysteine residues, 34 of which
are paired to form 17 disulfide bonds [12]. This leaves a single
cysteine, Cys-34, for site-specific conjugation.
Our group has developed fluorinated dendrimers as F imaging
agents [13,14]. Due to the spherical symmetry of the dendritic
structure, F signals from multiple fluorine atoms coalesce into a
ethylene glycol
compound 2 [16]. The resulting tosylate 2 was subjected to S
substitution with sodium azide to give compound [16].
1 with 4-toluenesulfonyl chloride afforded
N
2
3
19
Conversion of the remaining hydroxyl group in compound 3 to
tosylate afforded precursor 4 [17], which then reacted with the
small fluorocarbon dendron, sodium perfluoro-tert-butanoate, to
give the azide compound 5. Reduction of the azide group in 5 with
19
19
single
F signal for imaging. We have made a family of
fluorocarbon dendrons with each dendron containing 9–243
chemically identical fluorine atoms [15].
To explore the chemistry of site-specific labeling of albumin
with these fluorocarbon dendrons, we successfully conjugated the
smallest dendron of this family, a perfluoro-tert-butanol deriva-
tive, to Cys-34 of bovine serum albumin (BSA) in this work.
3
Ph P followed by hydrolysis gave free amine 6. The coupling
between amine 6 and Fmoc-Lys(Boc)-OH proceeded smoothly to
give compound 7 with good yield. It should be noted that the
reaction temperature and the molar ratio of the reactants are both
important; the coupling reaction was conducted at 0 8C and Fmoc-
Lys(Boc)-OH was slightly in excess of 6 at a molar ratio of 1.2:1.
Removal of the Fmoc group in compound 7 with piperidine in DMF
afforded the secondary amine 8 with high yield.
2
. Results and discussion
Next we turned our attention to introducing the prosthetic
group maleimide to the fluorocarbon dendron via the flexible
linker (Scheme 2). Reaction of the azide compound 3 with bromo
tert-butyl acetate by a modified procedure afforded a new azide
compound 9 [18]. This reaction extends the flexible linker by two
carbon atoms and at the same time introduces a protected
carboxylic group for further condensation with the amino group in
compound 8. Reduction of the azide group in compound 9 with
2
.1. Design of fluorinated albumins
Our conjugation of the small fluorocarbon dendron to albumin
utilizes the prosthetic group maleimide, which is commonly used
for albumin conjugation through Cys-34 [1]. Fig. 1 shows the
structure outline of the fluorinated albumin. Between the
fluorocarbon dendron and the prosthetic group is a 3-segment
flexible linker, made of one lysine flanked by two tetraethylene
glycol units. This 3-segment linker serves several purposes: to
avoid steric clash between the fluorocarbon dendron and albumin;
to provide motion freedom to the F signal emitter to prevent
signal broadening; and to facilitate albumin conjugation through
electrostatic attraction because lysine and albumin are oppositely
charged at neutral pH.
3
Ph P afforded the free amine compound 10 [18]. Reaction of 10
with N-methoxycarbonyl-maleimide 12, which was prepared from
maleimide 11 according to literature procedure [19], afforded
compound 13, an ester. Slight excess of compound 12 (the 12:10
molar ratio was 1.25:1) and moderate reaction time (1 h) at 0 8C
were required to achieve good yield for this reaction. Deprotection
of the tert-butyl group in compound 13 with TFA, followed by
coupling with amine 8, afforded compound 14 at 61% yield.
19
Fig. 2 shows detailed structures of two fluorinated albumins.
The two versions differ in the way lysine is incorporated into the
flexible linker. As a result, one linker is four methylene units longer
than the other. The intention is to explore whether the length of the
Exposing 14 to TFA removed the Boc protection group of the e-
amino group of lysine, resulting in compound 15, which was
purified by preparative HPLC with high yield. Compound 15 has
excellent aqueous solubility at neutral pH.
19
flexible linker has any effect on the resulting F signal.
Fig. 2. Fluorinated albumins with shorter (top) and longer (bottom) flexible linker between the fluorocarbon dendron and albumin.

X. Yue et al. / Journal of Fluorine Chemistry 152 (2013) 173–181
175
Scheme 1. Linking fluorocarbon dendron to flexible linker.
Fluorinated albumin 16 was synthesized by reacting the
maleimide-functionalized compound 15 with BSA at a 2:1 molar
ratio. The reaction was conducted at pH 8 in 40 mM ammonium
bicarbonate aqueous solution (Scheme 3). The reaction proceeded
smoothly under ambient temperature. Conjugation to Cys-34 was
demonstrated using Ellman’s test, which indicates presence of the
instead of Fmoc-Lys(Boc)-OH, reacted with amine 6 to form
compound 17. Note that compounds 7 and 17 are isomers; they
differ in the location of the Boc and Fmoc protection groups.
Deprotection of the Fmoc group in 17 with piperidine in DMF gave
compound 18. In compound 18 the
e
-amine of lysine is exposed for
-amine of
further condensation with 13 while in compound 8 the
a
–
SH group before conjugation and absence of the –SH group after
lysine is exposed for further condensation with 13. This difference
extends the flexible linker by four methylene units. After this
condensation, the prosthetic group maleimide is now connected to
the fluorocarbon dendron via a longer flexible linker to give
compound 19. Removal of the Boc-group in 19 led to compound 20,
which was functionalized with a maleimide group for reaction
with the –SH group of Cys-34 in BSA. Conjugation of 20 to BSA
proceeded smoothly at ambient temperature in 40 mM ammoni-
um bicarbonate aqueous solution at pH 8.0 to give the fluorinated
albumin 21. Again, conjugation to Cys-34 was verified using
conjugation. Judged by the disappearance of the –SH group,
conjugation took less than 30 min to complete. Excess 15 was
removed via exhaustive dialysis against phosphate-buffered saline
(PBS) at pH 7.4 at room temperature using a dialysis membrane
with a molecular weight cut-off (MWCO) of 25,000 Da.
With fluorinated albumin 16 in hand, we embarked on the
synthesis of a second version of fluorinated albumins, which
contains a longer flexible linker. This is achieved by switching the
orientation of lysine in the linker. To this end, Boc-Lys(Fmoc)-OH,
Scheme 2. Connecting the prosthetic group maleimide to the fluorocarbon dendron.

1
76
X. Yue et al. / Journal of Fluorine Chemistry 152 (2013) 173–181
Scheme 3. Conjugation of 15 to BSA. The asterisk indicates a new chiral center generated during conjugation.
Ellman’s test. After the reaction completed, excess 20 was removed
through extensive dialysis against PBS, again using a dialysis
membrane with MWCO of 25,000 Da.
albumin. In contrast, the two diastereomers in each of the control
compounds 22 and 23 experience identical chemical environment
due to their small size.
1
9
Up to this point, we have synthesized two fluorinated albumins,
Another possible cause of the split F signal is that more than
one copy of the fluorinated tag, 15 or 20, is conjugated to BSA.
However, this possibility is ruled out by MALDI-TOF mass
spectrometry analysis of the two fluorinated albumins, 16 and
21, which showed that only one copy of the fluorinated tag was
conjugated to BSA (Supporting Information). Based on the results
from mass spectrometry, which show a single fluorinated tag is
conjugated to BSA, and Ellman’s test, which show that the free –SH
in BSA disappeared after conjugation, we conclude that the site-
specific conjugation has been achieved.
1
6 and 21. In 16, the number of bonds between fluorine and sulfur
is 36 while in 21 this number is 40, a 10% difference.
Two control compounds, 22 and 23, were also made, as shown
in Scheme 5. 22 and 23 are respectively small molecule analogs of
fluorinated albumins 16 and 21. In 22 and 23, the maleimide group
reacted b-mercaptoethanol instead of albumin. All the conjugation
reactions between maleimide and the –SH group created a new
chiral center as shown in Schemes 3–5. The resulting products, 16,
2
1, 22 and 23, each is a mixture of two diastereomers, as indicated
by an asterisk in Schemes 3–5.
The fluorinated albumins, 16 and 21, were dialyzed against PBS
using a dialysis membrane with 25,000 Da MWCO. After extensive
dialysisat room temperature and pH 7.4, the F NMR signalremains
1
9
2.3. Characterization of fluorinated albumins
unchanged. In contrast, when 22 and 23 were mixed with albumin,
the F signal disappears afterextensive dialysis. Note that 22and 23
19
19
Fig. 3 shows the F NMR spectra of the two fluorinated
albumins (16 and 21) and their respective small molecule
counterparts (22 and 23). The two fluorinated albumins each give
cannot form covalent bond with BSA because their maleimide group
has been blocked. As a result, 22 and 23 can only bind to albumin
non-covalently and thereby can be removed by dialysis. This control
experiment proves that in 16 and 21 the fluorocarbon dendron has
indeed been covalently conjugated to albumin.
19
split F signals in aqueous solutions while the two small molecule
19
controls each give a single F signal in both aqueous and organic
19
solutions. One plausible explanation of the F signal splitting is
that the two diastereomers in each fluorinated albumin experience
slightly different chemical environment due to the large size of
1
9
1
The F longitudinal relaxation time, T , of fluorinated albumins
and their controls were measured using the inversion-recovery
Scheme 4. Synthesis of the longer version of fluorinated albumin. The asterisk indicates a new chiral center generated during conjugation.

X. Yue et al. / Journal of Fluorine Chemistry 152 (2013) 173–181
177
Scheme 5. Syntheses of control compounds 22 and 23. The asterisk indicates a new chiral center generated during conjugation.
1
9
Fig. 3. F NMR spectra of 16 (a), 21 (b), 22 (c), 23 (d) in PBS buffer (pH 7.4, to provide deuterium lock, D
CDCl at 25 8C.
2
O was added to 20% of the total volume) at 25 8C, and 22 (e), 23 (f) in
3
pulse sequence [20]. Attempts to measure the transverse relaxa-
tion time T and the diffusion coefficient were unsuccessful due to
the very short T relaxation time and poor signal to noise ratio. The
reason that T can be measured but not T is because T
measurement involves only signal decay while T measurement
that 10% difference in the length of the flexible linker has little
1
9
2
1
effect on F T relaxation both in fluorinated albumins (16 and 21)
and in the small molecules (22 and 23).
2
1
2
2
1
3. Conclusions
involves both signal decay and signal recovery and hence produces
more data for analysis. The fluorinated albumins have much
A small fluorocarbon dendron that contains nine chemically
identical fluorine atoms was covalently conjugated to albumin via
a flexible linker. Conjugation exploits the reaction between
maleimide and Cys-34 of albumin. This reaction creates a new
chiral center and each fluorinated albumin is a mixture of two
19
1
shorter F T relaxation time, consistent with the formation of
fluorinated macromolecules [21]. Also can be seen from Table 1 is
Table 1
19
1
9
diastereoisomers. The fluorinated albumin displays split F signal,
probably because the fluorocarbon dendron in the two diaster-
eoisomers have non-identical chemical environment due to the
large size of albumin. 10% difference in the length of the flexible
linker between the fluorocabron dendron and albumin made little
1
F longitudinal relaxation time T of fluorinated albumins and their controls.
Fluorinated
albumins
Control
compounds
Shorter version (Compounds 16 and 22)
Longer version (Compounds 21 and 23)
T
T
1
1
(16) = 0.63 s
(21) = 0.68 s
T
T
1
1
(22) = 1.52 s
(23) = 1.47 s
1
9
difference in F signal shape and longitudinal relaxation time.

1
78
X. Yue et al. / Journal of Fluorine Chemistry 152 (2013) 173–181
4
. Outlook
Based on the results presented here, future development of
5.3. Procedures and characterization for new compounds
5.3.1. Synthesis of 14-Azido-1,1,1-trifluoro-2,2-bis(trifluoromethyl)-
3,6,9,12-tetraoxa-tetradecane, 5
19
fluorinated conjugates of albumin for F MRI applications should
19
take the following issues into account. Firstly, F signal intensity,
which would require conjugating fluorocarbons with more
fluorine atoms to albumin. In this regard, we have already made
fluorocarbon dendrons containing 243 chemically identical
fluorine atoms [15]. Secondly, F relaxation times. This work
shows that coupling a fluorocarbon dendron to albumin leads to
shorter T
the concern is that such coupling might lead to overly short T
result of the slow rotational motions of albumin. Overly short T
broadens the F signal and thereby is detrimental to MRI. Longer
linker is probably needed to fully uncouple the rotational motions
of the fluorocarbon dendron from the rotational motions of
albumin. Thirdly, F signal splitting, which was likely caused by
the new chiral center generated in the conjugation process. To
avoid forming
alternative prosthetic group other than maleimide should be
used. One such possibility is bromide, which can react quickly with
To the azide compound 4 (22.4 g, 60 mmol) in dry DMF
(300 mL) was added sodium perfluoro-tert-butanate (24.8 g,
96 mmol). The reaction mixture was stirred at 60 8C overnight.
TLC showed that the starting material was consumed completely.
The mixture was poured into water and extracted with EtOAc. The
pooled organic extracts were washed with water and brine, the
combined organic phase was concentrated under reduced
pressure through rotary evaporation, the residue was purified
by silica gel flash chromatography using hexane/EtOAc (4/1) as
19
1
relaxation time, which is beneficial for MRI. However,
2
as a
2
19
the eluent to afford compound 5 (21 g, 48.1 mmol, 80% yield) as a
1
clear liquid. H NMR (500 MHz, CDCl
3
)
d
4.13 (s, 2H), 3.72–3.70
1
9
(m, 2H), 3.64 (s, 10H), 3.35 (t, J = 5.0 Hz, 2H); F NMR (470 MHz,
19
13
CDCl À73.47 (s); C NMR (126 MHz, CDCl
J = 292.4 Hz), 80.2–79.5 (m), 71.1, 71.0, 70.7, 70.1, 69.39, 69.36,
69.34, 50.7; MS (ESI) m/z Calcd. for C12 , 437.0997, found
3
)
d
3
) d 120.4 (q,
a
new chiral center during conjugation, an
16 9 3 4
H F N O
+
4
455 (M + NH ) .
–
SH but generates no new chiral center. Work is under way to take
5.3.2. Synthesis of 14,14,14-trifluoro-13,13-bis(trifluoromethyl)-
3,6,9,12-tetraoxatet-radecan-1-amine, 6
these factors into account and will be reported in due time.
3
To the azide compound 5 in dry THF (180 mL) was added Ph P
(
10.8 g, 41.2 mmol). The reaction mixture was monitored by TLC.
After the starting material has been completely consumed, water
4 mL) was added and the reaction mixture was stirred for 40 h at
5
. Experimental procedures
(
5.1. Materials
room temperature. After removal of the solvent in vacuum
through rotary evaporation, the residue was purified by silica gel
Unless otherwise stated, all chemicals were obtained from
column chromatography first using CH
2 2
Cl /MeOH (8/1) then
commercial sources and used without further purification.
Analytical thin layer chromatography (TLC) was performed on
Merck precoated silica gel 60 F254 plates with visualization by
MeOH as the eluent to give compound 6 (7.8 g, 19.0 mmol, 83%
1
yield) as yellow oil. H NMR (500 MHz, CDCl
3
) d4.16 (t, J = 5.0 Hz,
2H), 3.73 (t, J = 5.0 Hz, 2H), 3.67–3.62 (m, 8H), 3.51 (t, J = 5.5 Hz,
1
9
ultraviolet (UV) irradiation at
l
= 254 nm or staining with KMnO
4
.
2H), 2.87 (t, J = 5.5 Hz, 2H), 1.43 (br, 2H); F NMR (470 MHz,
1
3
Purifications by silica gel chromatography were performed using
Angela silica gel. BSA was purchased from Sigma.
CDCl
3
)
d
À73.50 (s); C NMR (126 MHz, CDCl
3
) d 120.0 (q,
J = 292.4), 79.8–79.1 (m), 72.9, 70.7, 70.3, 70.2, 69.9, 69.0, 41.4;
MS (ESI) m/z Calcd. for C12
(M + H) .
H
18
F
9
NO
4
, 411.1092, found 412
+
5.2. General information
Compounds 1, 11 are commercially available; compounds 2, 3,
, 9, 10, 12 are known compounds synthesized in this work; the
syntheses and characterization for these compounds, along with
MALDI-TOF mass spectrometry results for fluorinated albumins 16
and 21, are provided in Supporting Information; compounds 5, 6, 7,
5.3.3. Synthesis of (S)-(9H-fluoren-9-yl)methyl tert-butyl (1,1,1-
trifluoro-16-oxo-2,2-bis(trifluorometh-yl)-3,6,9,12-tetraoxa-15-
azahenicosane-17,21-diyl)dicarbamate, 7
To a solution of amine 6 (3 g, 7.3 mmol) in 70 mL of anhydrous
DMF at 0 8C was added DIEPA (1.4 g, 11 mmol), Fmoc-Lys(Boc)-
4
˚
8
, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 are new compounds
OH (4.1 g, 8.8 mmol), 4 A molecular sieve (5 g), Cl-HOBt (1.9 g,
synthesized in this work.
11 mmol), HBTU (4.2 g, 11 mmol) successively. The mixture was
stirred at 0 8C for 2 h and at room temperature overnight. TLC
showed that the starting material was consumed completely.
The mixture was quenched with ice water and extracted with
EtOAc. The pooled organic extracts were washed with water and
brine. After drying off the organic layer and evaporation of the
solvent under reduced pressure through rotary evaporation, the
residue was purified by silica gel column chromatography using
1
19
13
H, F and C NMR spectra were carried out on a Varian
INOVA 500 MHz NMR spectrometer, equipped with
a
1
19 13 15
1
19
H/ F{ C/ N} 5-mm triple resonance probe. The H, F and
1
3
C NMR spectra were recorded at 500 MHz, 470 MHz and
1
26 MHz, respectively. Unless otherwise specified, all NMR
1
spectra were recorded in CDCl
) are reported in parts per million (ppm) relative to a residual
proton peak of the solvent, = 7.24 for CDCl , and = 3.31 for
CD OD. Multiplicities are reported as follows: s (singlet), d
3
solution. H NMR chemical shifts
(
d
d
3
d
CH
2 2
Cl /MeOH (16/1) as the eluent to afford compound 7 (5.3 g,
1
3
6.2 mmol, 84% yield) as a yellow oil. H NMR (500 MHz, CDCl ) d
3
(
(
doublet), t (triplet), q (quartet), dd (doublet of doublets), or m
multiplet). Broad peaks are indicated by the addition of br.
7.80 (d, J = 7.0 Hz, 2H), 7.64 (d, J = 6.5 Hz, 2H), 7.44 (d, J = 7.0 Hz,
2H), 7.36–7.32 (m, 2H), 6.70 (br, 1H), 5.75 (br, 1H), 4.78 (br, 1H),
4.43 (s, 2H), 4.25 (t, J = 6.5 Hz, 1H), 4.18 (s, 3H), 3.74 (s, 2H), 3.68
(s, 2H), 3.63 (s, 6H), 3.59 (s, 2H), 3.50 (s, 2H), 3.15 (br, 2H), 1.88
(br, 1H), 1.72 (br, 1H), 1.53 (br, 2H), 1.48 (s, 9H), 1.42–1.41 (m,
Coupling constants are reported as J value in hertz (Hz). The
number of protons (n) for a given resonance is indicated as nH and
is based on spectral integration values. C NMR chemical shifts
(
(
1
3
1
9
13
d
d
) are reported in ppm relative to CD
= 77.2). For F NMR, hexafluorobenzene was used as the
3
OD (
d
= 49.5) or CDCl
3
2H); F NMR (470 MHz, CDCl
3
)
d
À73.47 (s); C NMR (126 MHz,
1
9
CDCl ) d 171.9, 156.3, 143.99, 143.94, 141.4, 127.9, 127.2,
3
standard with
d
= À164.9 ppm.
125.24, 125.20, 120.5 (q, J = 294.0 Hz), 79.2, 71.2, 70.7, 70.6, 70.4,
For high performance liquid chromatography (HPLC) experi-
ments, eluent and gradient conditions are indicated along with
each compound purified by HPLC chromatography.
70.0, 69.5, 67.1, 55.0, 47.3, 40.2, 39.4, 32.7, 29.8, 28.5,22.7; MS
+
(ESI) m/z Calcd. for C38
884 (M + Na) .
H
48
F
9
N
3
O
9
, 861.3247, found 862 (M + H) ,
+

X. Yue et al. / Journal of Fluorine Chemistry 152 (2013) 173–181
179
5
2
.3.4. Synthesis of (S)-tert-butyl-17-amino-1,1,1-trifluoro-16-oxo-
,2-bis(trifluoro-methyl)-3,6,9,12-tetraoxa-15-azahenicosan-21-
120.4 (q, J = 293.0 Hz), 78.9, 71.2, 71.1, 70.7, 70.64, 70.59, 70.5,
70.44, 70.37, 70.3, 70.1, 69.6, 69.5, 67.9, 52.6, 40.3, 39.3, 37.2, 32.2,
ylcarbamate, 8
57 9 4
29.6, 28.5, 22.9; MS (ESI) m/z Calcd. for C37H F N O14, 952.3728,
+
+
+
To a solution of compound 7 (1.4 g, 1.6 mmol) in DMF (15 mL)
was added piperidine (3 mL). The solution was stirred at room
temperature overnight. TLC showed that the starting material was
consumed completely. After the solvent was removed in vacuum
through rotary evaporation, the residue was purified by silica gel
found 953 (M + H) , 975 (M + Na) , 991 (M + K) .
5.3.7. Synthesis of (S)-N-(21-amino-1,1,1-trifluoro-16-oxo-2,2-
bis(trifluoromethyl)-3, 6,9,12-tetraoxa-15-azahenicosan-17-yl)-14-
(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12-
tetraoxatetradecan-1-amide, 15
column chromatography first using CH
2 2
Cl /MeOH (8/1) then
MeOH as the eluent to give compound 8 (0.96 g, 1.5 mmol, 92%
To
a solution of Boc-protected compound 14 (240 mg,
1
yield) as a yellow viscous oil. H NMR (500 MHz, CDCl
3
)
d
7.60 (br,
0.25 mmol) in 6 mL of DCM was added 1 mL of TFA. The reaction
mixture was stirred at room temperature for 3 h, TLC showed that
the starting material was consumed completely. Afterwards, the
solvent was evaporated under vacuum through rotary evaporation.
The residue was dissolved in water and purified by preparative
HPLC (Eclipse XDB-C18, 9.4 mm  250 mm, 5.0 mL/min, solvent A,
0.1% HCl in water, solvent B, 0.1% HCl in methanol, using a 0–100%
solvent B in 0–100 min gradient, detection wavelength 210 nm) to
give compound 15 (205 mg, 0.24 mmol, 95% yield) as a yellow oil.
Analytical HPLC (Eclipse XDB-C18, 4.6 mm  150 mm, 1.0 mL/min,
solvent A, 0.1% TFA in water, solvent B, 0.1% TFA in methanol, using
1
3
1
H), 4.64 (br, 1H), 4.17 (t, J = 3.5 Hz, 2H), 3.75 (t, J = 4.5 Hz, 2H),
.69–3.64 (m, 8H), 3.59–3.56 (m, 2H), 3.48–3.45 (m, 2H), 3.40 (br,
H), 3.13–3.12 (m, 2H), 2.00 (br, 2H), 1.86–1.82 (m, 1H), 1.58–1.48
19
3
(m, 3H), 1.44 (s, 9H), 1.42–1.39 (m, 2H); F NMR (470 MHz, CDCl )
13
d
3
–73.52 (s); C NMR (126 MHz, CDCl ) d 174.7, 156.3, 120.5 (q,
J = 293.0 Hz), 79.2, 71.3, 70.74, 70.69, 70.4, 70.0, 69.6, 69.5, 55.1,
4
C
0.3, 39.0, 34.6, 30.0, 28.5, 23.0; MS (ESI) m/z Calcd. for
+
23
38 9 3 7
H F N O , 639.2566, found 640 (M + H) .
5
.3.5. Synthesis of tert-butyl-14-(2,5-dioxo-2,5-dihydro-1H-pyrrol-
-yl)-3,6,9,12-tetr-aoxatetradecan-1-oate, 13
1
a 0–100%–100% solvent B in 0–20–24 min gradient, detection
wavelength 210 nm): t
1
Amine 10 (692 mg, 2.3 mmol) was dissolved in a saturated
R
= 16.88 min. H NMR (500 MHz, CDCl
3
)
d
aqueous solution of sodium bicarbonate (6 mL). The solution was
cooled to 0 8C and stirred for 10 min before adding N-(methox-
ycarbonyl)maleimide 12 (453 mg, 2.9 mmol). The resulting solu-
tion was stirred at 0 8C for 20 min and at room temperature for an
additional 10 min. TLC showed that the starting material was
consumed completely. The mixture was directly subjected to silica
7.75 (br, 1H), 7.62 (d, J = 7.0 Hz, 1H), 6.72 (s, 2H), 4.57 (s, 1H), 4.14
(s, 2H), 4.10–3.99 (m, 2H), 3.73–3.69 (m, 8H), 3.66–3.63 (m, 14H),
3.59 (s, 6H), 3.42 (d, J = 14.5 Hz, 2H), 3.02 (s, 2H), 1.84 (br, 3H), 1.70
1
9
13
(br, 1H), 1.48 (br, 2H); F NMR (470 MHz, CDCl
3
)
d
À73.47 (s);
C
NMR (126 MHz, CDCl 172.4, 171.4, 170.7, 134.2, 120.2 (q,
3
) d
J = 293.8 Hz), 70.9, 70.4, 70.3, 70.2, 70.1, 69.94, 69.90, 69.8, 69.3,
gel flash chromatography using CH
2
Cl
2
/MeOH (15/1) as the eluent
69.0, 67.8, 52.5, 39.6, 39.5, 37.1, 31.8, 26.3, 22.3; MS (ESI) m/z Calcd.
+
to afford compound 13 (700 mg, 1.8 mmol, 80% yield) as a light
for C32
H
49
F
N
9 4
O
12, 852.3203, found 853 (M + H) .
1
yellow oil. H NMR (500 MHz, CDCl
3
)
d
6.64 (s, 2H), 3.94 (s, 2H),
3
4
8
.64–3.62 (m, 4H), 3.60–3.59 (m, 2H), 3.57–3.55 (m, 6H), 3.52 (s,
5.3.8. Conjugation of 15 to BSA to form shorter fluorinated BSA 16
BSA (332 mg, 5 mol, 1 equiv.) was dissolved in 10 mmol
13
H), 1.39 (s, 9H); C NMR (126 MHz, CDCl
3
)
d
170.8, 169.8, 134.3,
m
1.6, 70.8, 70.69, 70.67, 70.63, 70.2, 69.1, 67.9, 37.2, 28.2; MS (ESI)
ammonium bicaronate aqueous solution (8 mL, pH 8.0) equipped
with a magnetic stirring bar. The solution was purged with
nitrogen for 15 min, then compound 15 (7 mg, 0.02 mmol, 4
equiv.) was added and the solution was stirred using a shaker at
25 8C for 0.5 h. Ellman’s test showed that no free sulfhydryl group
was left. Subsequently, the reaction mixture was extensively
dialyzed against 10 mmol ammonium bicarbonate (pH 6.0) and
deionized water using a dialysis membrane of 25,000 Da MWCO to
remove low molecular weight impurities, including excess
compound 15. The solution was then lyophilized to yield
+
8 4
m/z Calcd. for C18H29NO , 387.1893, found 405 (M + NH ) , 410
+
(M + Na) .
5.3.6. Synthesis of (S)-tert-butyl-17-(14-(2,5-dioxo-2,5-dihydro-1H-
pyrrol-1-yl)-3,6,9, 12-tetraoxatetradecanamido)-1,1,1-trifluoro-16-
oxo-2,2-bis(trifluoromethyl)-3,6,9,12-tetraoxa-15-azahenicosan-21-
ylcarbamate, 14
Compound 13 (400 mg, 1.03 mmol) was dissolved in methylene
chloride (4 mL), and TFA (2 mL) was added. The reaction was
monitored by TLC. After the reaction is complete, the solvent was
removed through rotary evaporate and the residue, an acid, was
used for the next step without further purification. To a solution of
amine 8 (550 mg, 0.86 mmol) in 6 mL of anhydrous DMF at 0 8C
fluorinated BSA 16 as a white solid (246 mg, 3.7
mmol, 73% yield).
Analytical HPLC (Eclipse XDB-C18, 4.6 mm  150 mm, 1.0 mL/min,
solvent A, 0.1% TFA in water, solvent B, 0.1% TFA in methanol, using
a 0–100%–100% solvent B in 0–20–24 min gradient, detection
1
9
was added DIEPA (166 mg, 1.29 mmol), the acid as prepared above,
˚
wavelength 210 nm): t
in PBS, pH 7.4)
R 2
= 17.06 min. F NMR (470 MHz, 20% D O
4
A molecular sieve (2 g), Cl-HOBt (219 mg, 1.29 mmol), HBTU
d
À70.09 (s),À70.17. MS (MALDI-TOF) m/z Calcd. for
(489 mg, 1.29 mmol) successively. The mixture was stirred at 0 8C
16, 67,283, found 67,508, 0.3% of error between calcd. and found
MS. Note: the calculated value is obtained as 66,431 (found MS of
reference BSA) + 852 (Calcd. MS of 15).
for 2 h and at room temperature overnight. TLC showed that the
starting material was consumed completely. The mixture was
quenched with water at 0 8C and extracted with EtOAc. The pooled
organic extracts were washed with water and brine. After drying
off the organic layer and evaporation of the solvent under reduced
pressure through rotary evaporation, the residue was purified by
5.3.9. Ellman’s assay
5,5-dithio-bis-(2-nitrobenzoic acid) (Ellman’s reagent, 20 mg,
0.05 mmol) was dissolved in 0.1 sodium phosphate (5 mL, pH 8.0,
containing 1 mM EDTA) to prepare the Ellman’s reagent solution
(10 mM). 0.12 mL of free BSA (2 mM) or fluorinated BSA
silica gel column chromatography using CH
the eluent to afford compound 14 (500 mg, 0.53 mmol, 61% yield)
2 2
Cl /MeOH (15/1) as
1
as a light yellow oil. H NMR (500 MHz, CDCl
3
)
d
7.26 (d, J = 8.0 Hz,
H), 6.75 (br, 1H), 6.62 (s, 2H), 4.86 (s, 1H), 4.34 (dd, J = 15.0 Hz,
.5 Hz, 1H), 4.05 (s, 2H), 3.91 (s, 2H), 3.64–3.49 (m, 26H), 3.44 (t,
conjugation solution (2 mM), 50 mL of Ellman’s reagent and
1
2
2.50 mL of sodium phosphate buffer was incubated for 15 min
at room temperature. UV absorption spectra were acquired using a
1-cm cuvette without dilution. The molar extinction coefficient of
2-nitro-5-thiobenzoic acid under our experimental conditions is
J = 5.0 Hz, 2H), 3.39–3.35 (m, 1H), 3.29–3.25 (m, 1H), 2.98–2.95 (m,
H), 1.76–1.74 (m, 1H), 1.58–1.54 (m, 1H), 1.43–1.37 (m, 2H), 1.32
2
1
9
À1
À1
(
s, 9H), 1.29–1.24 (m, 2H); F NMR (470 MHz, CDCl
3
)
d
À73.52 (s);
14,150 M cm at 412 nm. Before conjugation, the absorbance of
1
3
C NMR (126 MHz, CDCl
3
)
d
171.4, 170.7, 170.1, 156.1, 134.3,
free BSA at 412 nm was 0.512, giving ꢀ0.4-SH per BSA

1
80
X. Yue et al. / Journal of Fluorine Chemistry 152 (2013) 173–181
(
commercially available BSA has part of Cys-34 blocked); after
3.68–3.67 (m, 5H), 3.63–3.59 (m, 16H), 3.46–3.42 (m, 2H), 3.31–
3.27 (m, 6H), 1.98–1.95 (m, 2H), 1.59–1.55 (m, 2H), 1.51–1.48 (m,
conjugation, the absorbance at 412 nm was 0.022, giving ꢀ0.017-
SH per BSA, indicating ꢀ96% blockage of free-SH in BSA.
1
9
13
2H); F NMR (470 MHz, CDCl
CDCl 170.9, 169.2, 134.4, 120.4 (q, J = 294.5 Hz), 71.2, 71.0, 70.6,
70.5, 70.4, 70.3, 70.2, 70.1, 69.5, 68.0, 53.6, 39.4, 38.3, 37.3, 30.9,
3
) d–73.50 (s); C NMR (126 MHz,
3
) d
5
.3.10. Synthesis of (S)-(9H-fluoren-9-yl)methyl tert-butyl-(1,1,1-
trifluoro-16-oxo-2,2-bis(trifluoromethyl)-3,6,9,12-tetraoxa-15-
azahenicosane-17,21-diyl)dicarbamate, 17
28.9, 22.0; MS (ESI) m/z Calcd. for C32
853 (M + H) .
H
49
F
9
N
4
O
12, 852.3203, found
+
Compound 17 was prepared using a procedure identical to the
preparation of compound 7. 6.1 g, 7.1 mmol, 95% yield, light yellow
5.3.14. Conjugation of 20 to BSA to form longer fluorinated BSA 21
The procedure is the same as the preparation of compound 16.
1
oil. H NMR (500 MHz, CD
3
OD)
d
7.74 (d, J = 8.0 Hz, 2H), 7.60 (d,
J = 8.0 Hz, 2H), 7.35 (t, J = 7.0 Hz, 2H), 7.27 (t, J = 7.0 Hz, 2H), 4.31 (d,
J = 6.5 Hz, 2H), 4.14 (m, 3H), 4.00 (s, 1H), 3.66 (t, J = 3.5 Hz, 2H),
Product 21 (160 mg, 2.4 mmol, 48% yield) was obtained as a white
solid. Analytical HPLC (Eclipse XDB-C18, 4.6 mm  150 mm,
3
3
.58–3.53 (m, 6H), 3.48 (d, J = 4.5 Hz, 2H), 3.39 (d, J = 5.5 Hz, 2H),
.37–3.31 (m, 2H), 3.09 (t, J = 6.0 Hz, 2H), 1.76–1.71 (m, 1H), 1.59–
1.0 mL/min, solvent A, 0.1% TFA in water, solvent B, 0.1% TFA in
methanol, using a 0–100%–100% solvent B in 0–20–24 min
1
9
19
1
.53 (m, 1H), 1.48–1.45 (m, 2H), 1.41 (s, 9H), 1.36–1.35 (m, 2H);
F
d
gradient, detection wavelength 210 nm):
NMR (470 MHz, 20% D O in PBS, pH 7.4)
t
R
= 17.05 min.
F
13
NMR (470 MHz, CDCl
3
)
d
À70.12 (s); C NMR (126 MHz, CD
3
OD)
2
d
À73.05 (s), À73.12
1
1
7
74.4, 158.1, 157.0, 144.70, 144.67, 141.9, 128.1, 127.5, 125.5,
(s). MS (MALDI-TOF) m/z Calcd. for 21, 67,283, found 67,341, 0.09%
of error between calcd. and found MS. Note: the calculated value is
obtained as 66,431 (found MS of reference BSA) + 852 (Calcd.
MS of 20).
21.1 (q, J = 292.8 Hz), 120.3, 79.9, 71.2, 70.9, 70.8, 70.5, 70.37,
0.36, 69.8, 66.9, 55.4, 47.8, 40.7, 39.7, 32.5, 29.8, 28.1, 23.4; MS
+
(ESI) m/z Calcd. for C38
H
48
F
9
N
3
O
9
, 861.3247, found, 862 (M + H) .
5
2
.3.11. Synthesis of (S)-tert-butyl-21-amino-1,1,1-trifluoro-16-oxo-
,2-bis(trifluoro-methyl)-3,6,9,12-tetraoxa-15-azahenicosan-17-
5.3.15. Synthesis of N-((S)-21-amino-1,1,1-trifluoro-16-oxo-2,2-
bis(trifluoromethyl)-3,6,9,12-tetraoxa-15-azahenicosan-17-yl)-14-
(3-(2-hydroxyethylthio)-2,5-dioxopyrrolidin-1-yl)-3,6,9,12-
tetraoxatetradecan-1-amide, 22
ylcarbamate, 18
Compound 18 was prepared using a procedure identical to the
preparation of compound 8. 1.3 g, 2.0 mmol, 88% yield as a yellow
2-Mercaptoethanol (22
of maleimide 15 (90 mg, 0.11 mmol) and Et
in anhydrous CH Cl (6 mL). The mixture was stirred at room
m
L, 0.32 mmol) was added to a solution
1
oil. H NMR (500 MHz, CDCl
3
)
d
6.82 (d, J = 3.5 Hz, 1H), 5.22–5.20
3
N (28 L, 0.22 mmol)
m
(br, 1H), 4.16 (t, J = 4.0 Hz, 2H), 4.09 (d, J = 6.0 Hz, 1H), 3.74 (t,
2
2
J = 5.0 Hz, 2H), 3.71–3.69 (m, 2H), 3.66–3.61 (m, 6H), 3.56 (t,
J = 4.5 Hz, 2H), 3.46 (d, J = 4.0 Hz, 2H), 2.73–2.70 (m, 2H), 1.93 (br,
temperature overnight, then the solvent was evaporated under
vacuum through rotary evaporation. The residue was dissolved in
water and purified by preparative HPLC (Eclipse XDB-C18,
9.4 mm  250 mm, 5.0 mL/min, solvent A, 0.1% HCl in water,
solvent B, 0.1% HCl in methanol, using a 0–100% solvent B in 0–
100 min gradient, detection wavelength 210 nm) to give com-
pound 22 (47 mg, 0.05 mmol, 48% yield) as a yellow oil. Analytical
HPLC (Zorbax 300SB-C8, 4.6 mm  150 mm, 1.0 mL/min, solvent A,
2
H), 1.84–1.80 (m, 1H), 1.62–1.57 (m, 1H), 1.51–1.45 (m, 2H), 1.44
19
(
s, 9H), 1.41–1.38 (m, 2H); F NMR (470 MHz, CDCl
3
)
d
À73.52 (s);
177.0, 155.5, 120.3 (q, J = 291.7 Hz),
9.8, 71.1, 70.55, 70.47, 70.2, 69.7, 69.4, 69.3, 54.3, 41.6, 39.2, 32.8,
1
3
3
C NMR (126 MHz, CDCl ) d
7
2
6
8.3, 22.6; MS (ESI) Calcd. for C23
40 (M + H) .
H
38
F
9
N
3
O
7
, 639.2566, found m/z
+
0.1% TFA in water, solvent B, 0.1% TFA in methanol, using a 0–
5
.3.12. Synthesis of (S)-tert-butyl-36-(2,5-dioxo-2,5-dihydro-1H-
pyrrol-1-yl)-1,1,1-trifluoro-16,23-dioxo-2,2-bis(trifluoromethyl)-
,6,9,12,25,28,31,34-octaoxa-15,22-diazahexatriacontan-17-
100%–100% solvent B in 0–20–24 min gradient, detection wave-
1
R 3
length 210 nm): t = 15.56 min. H NMR (500 MHz, CDCl ) d 8.03
3
(s, 1H), 7.97 (S, 2H), 7.93 (d, J = 8.0 Hz, 1H), 5.20 (s, 3H), 4.62 (d,
J = 5.0 Hz, 1H), 4.19–4.11 (m, 3H), 4.02 (d, J = 5.5 Hz, 1H), 3.85 (t,
J = 5.5 Hz, 2H), 3.76–3.72 (m, 7H), 3.68–3.60 (m, 19H), 3.48–3.45
(m, 3H), 3.29–3.23 (m, 1H), 3.08–3.06 (m, 3H), 2.91–2.85 (m, 1H),
2.64 (dd, J = 23.5 Hz, 7.5 Hz, 1H), 1.85 (t, J = 24.5 Hz, 4H), 1.53 (d,
ylcarbamate, 19
Compound 19 was prepared using a procedure identical to the
preparation of compound 14. 380 mg, 0.4 mmol, 61% yield, clear
oil. H NMR (500 MHz, CDCl
1
3
)
d
7.04 (br, 1H), 6.69 (br, 1H), 6.65 (s,
1
9
13
2
(
3
H), 5.29 (br, 1H), 4.08 (s, 2H), 3.97 (s, 1H), 3.90 (s, 2H), 3.66–3.63
m, 4H), 3.59–3.52 (m, 22H), 3.47–3.46 (m, 2H), 3.37–3.36 (m, 2H),
.20 (t, J = 6.0 Hz, 2H), 1.77–1.74 (m, 1H), 1.57–1.44 (m, 3H), 1.37
J = 6.0 Hz, 2H); F NMR (470 MHz, CDCl
3
)
d
À73.49 (s); C NMR
(126 MHz, CDCl ) d 177.8, 175.4, 172.6, 171.5, 120.5 (q,
3
J = 293.7 Hz), 71.2, 71.1, 70.7, 70.6, 70.5, 70.2, 70.1, 69.6, 69.4,
1
9
(
s, 9H), 1.32–1.30 (m, 2H); F NMR (470 MHz, CDCl
3
)
d
À73.40 (s);
67.3, 61.7, 52.8, 39.9, 39.7, 38.6, 36.8, 34.5, 32.0, 26.7, 22.6; MS (ESI)
1
3
+
C NMR (126 MHz, CDCl
3
)
d
172.2, 170.6, 170.1, 155.7, 134.2,
m/z Calcd. for C34
H
55
F
9
N
4
O
13S, 930.3343, found 931 (M + H) .
1
20.3 (q, J = 295.6 Hz), 79.7, 71.1, 71.0, 70.6, 70.53, 70.47, 70.4,
7
2
0.3, 70.2, 70.1, 69.7, 69.4, 67.8, 54.5, 39.2, 38.3, 37.1, 32.1, 29.6,
5.3.16. Synthesis of N-((S)-17-amino-1,1,1-trifluoro-16-oxo-2,2-
bis(trifluoromethyl)-3,6,9,12-tetraoxa-15-azahenicosan-21-yl)-14-
(3-(2-hydroxyethylthio)-2,5-dioxopyrrolidin-1-yl)-3,6,9,12-
tetraoxatetradecan-1-amide, 23
57 9 4
9.2, 28.3, 22.7; MS (ESI) m/z Calcd. for C37H F N O14, 952.3728,
+
+
4
found 970 (M + NH ) , 975 (M + Na) .
5
.3.13. Synthesis of (S)-N-(17-amino-1,1,1-trifluoro-16-oxo-2,2-
bis(trifluoromethyl)-3,6,9,12-tetraoxa-15-azahenicosan-21-yl)-14-
2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12-
Compound 23 was prepared using a procedure identical to the
preparation of compound 22. 58 mg, 0.062 mmol, 46% yield as a
clear oil. Analytical HPLC (Zorbax 300SB-C8, 4.6 mm  150 mm,
1.0 mL/min, solvent A, 0.1% TFA in water, solvent B, 0.1% TFA in
(
tetraoxatetradecan-1-amide, 20
Compound 20 was prepared using a procedure identical to the
preparation of compound 15. 240 mg, 0.25 mmol, 71% yield as a
yellow oil. Analytical HPLC (Eclipse XDB-C18, 4.6 mm  150 mm,
methanol, using a 0–100%–100% solvent B in 0–20–24 min
1
gradient, detection wavelength 210 nm): t
(500 MHz, CDCl
R
= 15.64 min. H NMR
8.43 (s, 1H), 8.17 (s, 3H), 7.88 (s, 1H), 4.71 (s,
3
)
d
1
.0 mL/min, solvent A, 0.1% TFA in water, solvent B, 0.1% TFA in
3H), 4.16 (s, 2H), 4.11 (s, 2H), 4.04 (br, 1H), 3.85–3.84 (m, 2H),
3.74–3.62 (m, 27H), 3.46 (br, 2H), 3.31–3.24 (m, 3H), 3.08–3.05 (m,
1H), 2.88–2.85 (m, 1H), 2.64–2.60 (m, 1H), 1.97 (br, 1H), 1.61 (br,
methanol, using a 0–100%–100% solvent B in 0–20–24 min
1
gradient, detection wavelength 210 nm): t
500 MHz, CDCl 8.48 (s, 1H), 8.22 (s, 3H), 7.53 (t, J = 6.0 Hz, 1H),
.74 (s, 2H), 4.18–4.15 (m, 2H), 4.02 (s, 2H), 3.75–3.71 (m, 4H),
R
= 16.89 min. H NMR
1
9
13
(
3
)
d
2H), 1.51 (br, 2H); F NMR (470 MHz, CDCl
3
)
d
À73.46 (s);
C
6
NMR (126 MHz, CDCl 177.5, 175.1, 171.1, 169.1, 120.3
3
) d

X. Yue et al. / Journal of Fluorine Chemistry 152 (2013) 173–181
181
[
[
3] N. Soni, M. Margarson, Curr. Anaes. Crit. Care 15 (2004) 61–68.
4] A.O. Elzoghby, W.M. Samy, N.A. Elgindy, J. Controlled Release 157 (2012) 168–
1
(
6
2
q, J = 293.6 Hz), 71.0, 70.7, 70.4, 70.3, 70.2, 70.1, 70.0, 69.9, 69.3,
9.2, 67.1, 61.4, 53.4, 39.6, 39.3, 38.4, 38.2, 36.5, 34.3, 30.8, 28.6,
1.9; MS (ESI) m/z Calcd. for C34 13S, 930.3343, found 931
82.
[5] B. Elsadek, F. Kratz, J. Controlled Release 157 (2012) 4–28.
55 9 4
H F N O
+
+
[
[
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