2,2,2-Trifluoroethyl 6-thio-β-d-glucopyranoside as a selective tag for cysteines in proteins

Article abstract of DOI:10.1016/j.carres.2012.08.010

A synthetic route to a trifluoromethyl and thiol containing glucose derivative (2,2,2-trifluoroethyl 6-thio-β-d-glucopyranoside) is presented, which is based on microwave-assisted Fischer glycosylation under increased pressure. This water-soluble, neutral thiol-compound can be used to selectively introduce a fluorine probe for ¹⁹F NMR spectroscopy on cysteines in proteins. It can be attached under mild conditions in an aqueous environment without the risk of denaturing the protein. This tag has been applied to determine the redox-state of two cysteine residues in a bacterial transcription activator. Qualitative information about the solvent accessibility can be obtained from F-19 solvent PREs.

Full text of DOI:10.1016/j.carres.2012.08.010

Carbohydrate Research 361 (2012) 100–104
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
2,2,2-Trifluoroethyl 6-thio-b-D-glucopyranoside as a selective tag for cysteines in
proteins
Richard F. G. Fröhlich a, Evelyne Schrank, Klaus Zangger
⇑
Institute of Chemistry/Organic and Bioorganic Chemistry, University of Graz, Heinrichstrasse 28, A-8010 Graz, Austria
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 June 2012
Received in revised form 16 August 2012
Accepted 17 August 2012
Available online 25 August 2012
A synthetic route to a trifluoromethyl and thiol containing glucose derivative (2,2,2-trifluoroethyl 6-thio-
b-D-glucopyranoside) is presented, which is based on microwave-assisted Fischer glycosylation under
increased pressure. This water-soluble, neutral thiol-compound can be used to selectively introduce a
fluorine probe for 19F NMR spectroscopy on cysteines in proteins. It can be attached under mild condi-
tions in an aqueous environment without the risk of denaturing the protein. This tag has been applied
to determine the redox-state of two cysteine residues in a bacterial transcription activator. Qualitative
information about the solvent accessibility can be obtained from F-19 solvent PREs.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Fluorinated glucose
NMR spectroscopy
Fischer glycosylation
Microwave irradiation
Cysteine redox state
1. Introduction
conditions in aqueous environment to avoid protein denaturation.
We used this tag to determine the redox state of two functionally
The only naturally occurring fluorine isotope 19F constitutes one
of the most versatile NMR detectable nuclei.1 Due to its high sensi-
tivity (second most NMR sensitive element after hydrogen), virtual
absence in natural organic and biological samples, and its very large
chemical shift range, fluorine is often used to provide a unique per-
spective on structural and dynamical molecular features by solu-
tion1,2 and solid-state NMR spectroscopy.3 In particular, 19F NMR
has been used for biomolecules to answer questions related to,
for example, protein–protein and protein–ligand interactions,4,5
protein unfolding,6 enzymatic reactions,7 or membrane-binding.8
In order to incorporate the fluorine nucleus into the protein of inter-
est it can be inserted biosynthetically by using fluorinated amino
acid analogs or chemically attached, typically via protein –SH, –
NH, or –OH groups.1 Fluorine tags in use are often rather hydropho-
bic and show low water solubility. Here we describe the synthesis
of a fluorine tag based on glucose, containing a trifluoromethyl-
and a thiol group. Glucose was the carrier substance of choice for
being a water soluble, neutral, nontoxic, and cheap compound.
The thiol can be used to selectively attach the compound to cysteine
SH groups. The polarity due to the large number of OH groups
makes it water soluble and allows the attachment to any solvent
accessible, reduced cysteine in a protein. As a prerequisite to be
useful for biomolecules, the tag can be attached under mild reaction
important cysteine residues of a bacterial transcription activator
protein. To get information about the solvent accessibility, 19F
relaxation enhancements in a paramagnetic environment (solvent
PREs)9,10 can be used. So far, solvent PREs have been used for 1H
and 13C only.9 However, the large gyromagnetic ratio makes fluo-
rine a very convenient nucleus for PREs. Their size is comparable
to proton solvent PREs. For higher 19F NMR sensitivity a trifluoro
group on the tag is advantageous.
So far sugars bearing the 2,2,2-trifluoroethyl moiety as aglycone
were accessible on preparative scale under Mitsunobo reaction
conditions from 2,3,4,6-tetra-O-benzyl-
excessive Lewis-acid promoted glycosylation on peracetylated
glucose12,13 or 1-O-acetyl-2,3,4,6-tetra-O-benzyl-b-
-glucopyrano-
side,14 or via transglycosylation employing an
-galactosidase on
4-nitrophenyl
-galactopyranoside.15 Under classic Fischer gly-
D
-glucopyranose,11 via
D-
D
a
-D
a
-D
cosylation conditions—catalytic amounts of in situ formed HCl, re-
flux in the respective alcohol serving as reaction solvent at ambient
pressure—we were unsuccessful in our efforts to react 2,2,2-trifluo-
roethanol (TFE) with D-glucose. This is presumably due to the fact
that the sugar is insoluble in TFE, and the latter’s low nucleophilic-
ity and boiling point. Therefore, the presented glycosylation was
performed under increased pressure and microwave irradiation16
yielding the desired product.
2. Results and discussion
⇑
Corresponding author. Tel.: +43 316 380 8673; fax: +43 316 380 9840.
E-mail address: klaus.zangger@uni-graz.at (K. Zangger).
Current address: piCHEM Forschungs- und Entwicklungs GmbH, Kahngasse 25, A
a
The reaction rate acceleration by means of microwave irradia-
tion is known for the Fischer glycosylation.17 Therefore, we
8045 Graz, Austria.
0008-6215/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2012.08.010

R. F. G. Fröhlich et al. / Carbohydrate Research 361 (2012) 100–104
101
performed the synthesis in a sealed pressurized tube in a micro-
wave reactor at temperatures above the melting point of -glucose
D
and obtained a conversion to the expected mixture of four trifluo-
roethyl glucosides (Scheme 1) with the anomeric pair of pyrano-
sides 1 being the predominant product (ꢀ75%). Although 20,20,20-
trifluoroethyl
a-D-glucopyranoside a-1 was the major product
(60%) the only isomer that could be isolated with satisfactory pur-
ity was found to be the b-anomer b-1. The other isomers could not
be separated by silica-gel flash chromatography. The stereochem-
istry of
a-1, b-1 and its subsequent derivatives was supported by
HMBC and NOESY NMR experiments. The primary hydroxy group
in b-1 was selectively converted with 2,4,6-trisisopropyl-benzene-
sulfonic chloride into sulfonate 3 in 73% yield. Although a success-
ful substitution of the leaving group on carbon-6 with potassium
thioacetate has been published by Ramström and co-workers for
methyl 6-tosyl-a-D
-mannopyranoside,18 applying this method to
the 6-O-tosyl analog of 3 led to the same difficulty, which was also
Figure 1. Fluorine-19 NMR spectra of compound 6 stored under aerobic conditions
and treated with H2O2 or with DTT.
reported by the aforementioned group, where the conversion of
methyl b-D
-galactopyranoside had failed.19 As described, this prob-
lem was circumvented by acetylation of the remaining three hy-
droxy groups prior to the substitution reaction with potassium
thioacetate. Acetylation of 3 in pyridine and acetic anhydride gave
4 in quantitative yield. The displacement with thioacetate afforded
5 in very good yield (92%). Finally, the global deprotection with so-
dium methoxide in methanol yielded thiol compound 6 as a white
crystalline powder in 71% yield. When stored at air for several
days, the 19F NMR spectrum of 6 contains two signals (see Fig. 1)
where the larger one at ꢁ74.12 ppm corresponds to the tag in
the reduced state and the smaller peak at ꢁ74.04 ppm to the oxi-
dized tag. This was confirmed by reaction with DTT or H2O2 which
results in spectra containing only the reduced or oxidized tag,
respectively (Fig. 1). Compound 6 was attached to free thiol groups
of reduced cysteines of the Vibrio cholerae transcription activator
ToxR. Due to very inefficient direct reaction of 6 with free thiol
groups, the latter were first activated with Ellman’s reagent 5,50-
dithiobis(2-nitrobenzoic acid) (DTNB).20,21 The better leaving
group 5-thio-2-nitrobenzoic acid allows much higher yields by
disulfide interchange. Without activation, tagging with 6 gave less
than 5% yield on the model compound glutathione (not shown),
while after activation with DTNB the reaction gave 90–95% of the
desired tagged compound. This procedure was applied to investi-
gate the redox state of ToxR. It has been speculated that ToxR
forms dimers through disulfide bond formation in its initial activa-
tion step, which finally leads to cholera toxin production.22 How-
ever, recently a preformed intramolecular disulfide bond has
been postulated.23 To determine the redox state and solvent acces-
sibility of the two cysteines of ToxR the protein was incubated with
DTNB and then 6 both with and without prior treatment with D,L
-
dithiothreitol (DTT) to reduce any cysteine disulfide bonds. After
chromatographic separation from the majority of excess tag 6,
19F NMR spectra were recorded. The fluorine spectrum of ToxR
without prior DTT treatment (Fig. 2) only shows signals from the
free tag and oxidized free tag which is always present and obvi-
ously not separated completely by the short gel filtration chroma-
tography step. After reduction by DTT two additional broad signals
appear downfield (Fig. 2) which belong to 6 attached to the two
cysteine residues at positions 236 and 293 in ToxR. The additional
two fluorine signals in the 19F NMR spectrum of ToxR previously
treated with DTT are much broader than the one from the free
tag and no scalar coupling to the neighboring CH2-group can be
HO
O
HO
HO
HO
HO
OH
O
a)
D-Glucose
O
CF3
O
CF3
α-1
OH
HO
HO
HO
OH
O
2
O
CF3
OH
β-1
b)
O2S
RS
RO
RO
O
d)
O
O
RO
O
CF3
O
CF3
RO
OR
5 R = Ac
6 R = H
OR
3 R = H
e)
c)
4 R = Ac
Scheme 1. Reagents and conditions: (a) TFE, H+ cat., 160 °C mw, 10 min, 51%; (b) N2, 2,4,6-trisisopropylbenzene-sulfonyl chloride, pyridine, 71%; (c) Ac2O, pyridine, quant.;
(d) N2, KSAc, DMF, 78 °C, 20 h, 92%; (e) N2, NaOMe, MeOH, 71%.

102
R. F. G. Fröhlich et al. / Carbohydrate Research 361 (2012) 100–104
the addition of the water soluble inert paramagnetic molecule gad-
olinium-diethylenetriamine pentaacetic acid-bismethylamide
(Gd(DTPA-BMA)). The resulting solvent PREs are larger for solvent
exposed atoms and smaller the more protected the fluorine atom
is. This effect has been used on proteins,9,24 membrane-bound
peptides,25–28 and also small organic molecules bound to large
molecular assemblies.29 Addition of Gd(DTPA-BMA) to the fluo-
rine-tagged ToxR sample even at relatively low concentration
(2 mM) of the paramagnetic agent led to significant broadening
of the signals of free and oxidized tag, but almost no influence on
the signals from the protein (Fig. 3). Because of its size the free
tag is accessible to the paramagnetic solvent from each side, while
the fluorine tag on the protein is more protected. Only at a concen-
tration of 9 mM Gd(DTPA-BMA) more significant changes can be
seen on the protein fluorine signals. When compared to solvent
PREs found on proteins,9 this indicates that the tags on ToxR are
somewhat protected from the solvent but are certainly not deeply
buried within the protein. Solvent PREs are proportional to the ob-
served nucleus gyromagnetic moment. Since fluorine and protons
have very similar values their solvent PREs should be comparable.
A very rough estimation of the insertion depth can be given com-
paring the influence of Gd(DTPA-BMA) on the observed fluorine
signals to proton immersion depths on proteins and micelle-bound
peptides.9,10,28 The distance from the paramagnetically accessible
surface of the fluorine tags has to be between 3 and 15 Å. However,
it should be mentioned that so far the theory of solvent PREs has
not been developed for 19F and any quantitative interpretation
should be treated with caution. While the oxidation state of cyste-
ines can be determined by UV/Vis or MS measurements of proteins
reacted with DTNB,20,30 the use of fluorine tagging and 19F NMR not
only allows the determination of surface accessibility by solvent
PREs, but could for example also be used to monitor protein–
protein or protein–ligand interactions, structural changes of the
protein upon changing environmental conditions, or follow chem-
ical reactions. In contrast to the traditional UV/Vis based determi-
nation of cysteine redox states by DTNB, the exact protein
concentration does not need to be known for the presented NMR
approach and it also works if the reaction with DTNB would not
proceed to completion for whatever reason.
Figure 2. Schematic description of protein fluor-tagging of reduced cysteines and
19F NMR spectra of ToxR tagged with 2,2,2-trifluoroethyl 6-thio-
a-D-glucopyran-
oside after activation with DTNB and previous DTT treatment (a) and without DTT
treatment (b).
seen. This is a result of the slow tumbling of the protein and there-
fore faster T2 relaxation. In contrast, the signals of free tag and oxi-
dized tag are much sharper and the scalar coupling to the
neighboring CH2-group of 6 leads to triplets in the 19F NMR spec-
tra. To assign the two broad protein-bound fluorine signals, single
cysteine mutants (C236S and C293S) were constructed. Both mu-
tants were much less soluble than the wild-type protein and only
C293S was stable at a concentration that allowed the acquisition of
a
19F spectrum. The 19F NMR spectrum of this tagged mutant is
shown in the Supplementary data and contains beside free tag only
the low-field signal at ꢁ73.80 ppm. Therefore, the peak at
ꢁ73.95 ppm has to belong to cysteine 236. Treatment of ToxR with
DTT is necessary to enable the attachment of DTNB and subse-
quently the fluorine tag. Therefore, both cysteines exist in the oxi-
dized state in native ToxR, which is in accordance with the recently
proposed intramolecular disulfide bond of ToxR23 and in contradic-
tion with the previously hypothesized disulfide bond formation
after interaction with ToxS.22 Considering that the two cysteines
of ToxR are located in the oxidative environment of the periplasm,
the formation of a disulfide seems even more realistic. To check if
oxidation by oxygen during protein expression and/or purification
could have produced the cysteine disulfide in ToxR, we artificially
reduced ToxR by DTT and let it stand at air. Even after several
weeks only very minor amounts of oxidized ToxR were found.
To the best of our knowledge this study is the first example of
using a fluorine tag to determine the oxidation state of cysteines
in proteins. The large fluorine chemical shift range and its superb
sensitivity to small changes in the chemical environment allow
the separation of fluorine signals due to changes in the protein
environment of the individual cysteines and thus simply determin-
ing the number of reduced cysteines by counting the peaks in the
19F NMR spectrum. Information about the solvent accessibility or
distance to the molecular surface of a fluorine nucleus can be ob-
tained by monitoring 19F NMR relaxation times or line widths upon
Figure 3. Fluorine-19 NMR spectra of tagged ToxR before (blue) and after the
addition of 2 mM (red) and 9 mM (green) Gd(DTPA-BMA).

R. F. G. Fröhlich et al. / Carbohydrate Research 361 (2012) 100–104
103
In conclusion, we have synthesized a water-soluble glucose
based fluorine-tag, which can easily be attached selectively to cys-
teines in proteins. This compound can be covalently bound under
very mild conditions at neutral pH values, which makes it a favor-
able tag for biomolecules. As an example it has been attached to a
bacterial transcription activator to determine the redox state of its
two cysteines by 19F NMR spectroscopy. Qualitative information
about the solvent accessibility can be obtained from solvent PREs
on the fluorine NMR signals.
ture was incubated at room temperature for approximately 45 min
prior to being applied to a PD MidiTrap G-25 Column. All subse-
quent steps were then carried out in the exact same way as de-
scribed above for the untreated ToxR sample. For the NMR
measurements D2O (10% v/v) was added for field/frequency
locking.
3.3. 2,2,2-Trifluoroethyl b-D-glucopyranoside (b-1)
In a 30 ml microwave reactor vessel fine powdered
(1.35 g, 7.5 mmol) was suspended in 2,2,2-trifluoroethanol
(15 ml). Acetic chloride (10 L, 0.14 mmol) was added, the tube
D-glucose
3. Experimental
l
was sealed by a Teflon lid and the suspension was stirred
(600 rpm) at 160 °C for 10 min. After cooling to rt the resulting yel-
3.1. General methods
lowish solution was quenched with triethylamine (100 lL) and
All chemicals were purchased from Sigma Aldrich (St. Louis,
MO, USA). A microwave synthesis reactor monowave 300 by Anton
Paar (Graz, Austria) was used. Analytical TLC was performed on
precoated aluminum plates Silica Gel 60 F254 (Merck), detected
with UV light (254 nm), dipped either in p-anisaldehyde (6 vol %
p-anisaldehyde and 1 vol % H2SO4 conc. in EtOH), or ceric ammo-
nium molybdate (100 g ammonium molybdate tetrahydrate and
4 g ceric sulfate dihydrate in 1 L H2SO4 10%) and developed by a
heatgun. For flash chromatography Silica Gel 60 220–440 mesh
(Merck) was used. All 1H, 13C, and most 19F NMR spectra were re-
corded on a Bruker Avance III 300 MHz spectrometer at 298 K.
They were referenced to the residual non-deuterated solvent peak.
Qualitative 19F solvent PREs were acquired at 300 K on a Bruker
Avance III 500 MHz NMR spectrometer, using a 5 mm SEF probe
operating at a F-19 frequency of 470 MHz. They were obtained
by addition of a 60 mM stock solution of Gd(DTPA-BMA) to final
concentrations of 2 and 9 mM. Gd(DTPA-BMA) was purified from
the commercially available contrast agent OmniscanÒ as described
previously.10 Optical rotations were measured on a Perkin Elmer
341 polarimeter at 589 nm and a path length of 10 cm. High reso-
lution mass spectra were recorded on a Waters GCT Premier
equipped with Electron impact (EI, 70 eV) and direct insertion.
concentrated under reduced pressure to give a tan colored foam.
The residue was purified by flash chromatography (200 g SiO2,
eluted with 5% EtOH in EtOAc, 1.5 L, then 10% EtOH in EtOAc,
500 ml) which gave b-
amorphous white solid in addition to a mixture of other isomers
(835 mg, 43%). Fractions containing mainly -1 were purified twice
D-glucopyranoside b-1 (160 mg, 8%) as
a
again to give the material sufficiently clean for NMR and optical
rotation.
Compound b-1: Rf = 0.59 (20% EtOH in EtOAc), ½a D20
ꢁ20 (c 1.4,
ꢂ
MeOH), 1H NMR (CD3OD) d 4.40 (d, J = 7.7 Hz, 1H, H-1), 4.29 (dq,
3JH,F = 9.1 Hz, 2JH,H = 12.3 Hz, 1H), 4.11 (dq, 3JH,F = 8.9 Hz,
2JH,H = 12.3 Hz, 1H), 3.90 (dd, J = 1.1, 11.9 Hz, 1H), 3.74–3.63 (m,
1H), 3.43–3.28 (m, 3H), 3.24 (dd, J = 7.8, 8.8 Hz, 1H, H-2); 13C
1
NMR (CD3OD) d 125.4 (q, JC,F = 277 Hz, C-20), 104.1 (C-1), 78.1,
2
77.8, 74.7, 71.4, 66.6 (q, JC,F = 35 Hz, C-10), 62.6 (C-6); 19F NMR
3
(CD3OD) d ꢁ75.6 (t, JH,F = 9.0 Hz);
Compound
a
-1: Rf = 0.52 (20% EtOH in EtOAc), ½a D20
ꢂ
+120 (c
4.95, MeOH), 1H NMR (CD3OD) d 4.90 (d, J = 3.7 Hz, 1H, H-1),
3
2
4.13 (dq, JH,F = 9.1 Hz, JH,H = 12.4 Hz, 1H, H-10), 4.03 (dq,
3JH,F = 8.9 Hz, JH,H = 12.4 Hz, 1H, H-10), 3.82 (dd, J = 2.2, 11.8 Hz,
2
1H, H-6), 3.72–3.61 (m, 2H), 3.58 (ddd, J = 2.2, 5.5, 9.8 Hz, 1H, H-
5), 3.44 (dd, J = 3.7, 9.8 Hz, 1H, H-2), 3.35–3.26 (m, 1H, H-4); 13C
1
NMR (CD3OD) d 125.5 (q, JC,F = 278 Hz, C-20), 100.8 (C-1), 74.5
3.2. Cysteine tagging via disulfide bond formation
2
(C-3), 74.3 (C-5), 73.1 (C-2), 71.4 (C-4), 65.5 (q, JC,F = 35 Hz, C-10),
3
62.4 (C-6); 19F NMR (CD3OD) d ꢁ75.4 (t, JH,F = 9.0 Hz).
The attachment of 6 to reduced cysteines in proteins was tested
on the Vibrio cholerae transcription activator ToxR. The purified
protein was dialyzed extensively against 50 mM Tris buffer (pH
7.6) before applying 1 ml of the protein solution (2 mg/ml) to a
PD MidiTrap G-25 Column (Life Technologies) equilibrated with
the same buffer. The protein was eluted from the column with
1.5 ml of the Tris buffer. The fractions containing protein were
pooled and DTNB (dissolved in Tris buffer and adjusted to approx-
imately pH 7 with 1 M Tris buffer pH 8.0) was added to a final con-
centration of 10 mM. The resulting light yellow mixture was
incubated at room temperature for 30 min prior to being applied
to the newly equilibrated column. Again the protein solution was
eluted with 1.5 ml of the Tris buffer, the protein fractions united
and once more applied to the equilibrated column. This step was
repeated twice to remove the excess DTNB, which could be moni-
tored easily due to the yellowish color of the DTNB. In a next step, 6
dissolved in the same Tris buffer (colorless) was added to the pro-
tein solution in a final concentration of 5 mM and the resulting
light yellow mixture was incubated at room temperature for an-
other 30 min before being applied to the equilibrated column.
Again the protein solution was eluted with 1.5 ml of the Tris buffer
and the combined protein fractions once more applied to the col-
umn. This step was repeated twice. In parallel another ToxR sample
of the same concentration but pre-treated with DTT was tagged.
For this purpose DTT dissolved in the same Tris buffer was added
to the protein solution to a final concentration of 10 mM. This mix-
3.4. 20,20,20-Trifluoroethyl 6-O-(2,4,6-
trisisopropybenzenesulfonyl)-b-D-glucopyranoside (3)
Under nitrogen protection b-1 (285 mg, 1.1 mmol) was dis-
solved in dry pyridine (4 ml) and cooled to 0 °C. 2,4,6-Tris-
isopropylbenzenesulfonyl chloride (97%, 440 mg, 1.4 mmol) was
added, the ice bath was removed 50 min later and the clear slightly
orange reaction mixture was stirred for 3 d at rt after which time
the reaction was quenched with water (50 ml) and extracted with
EtOAc (3 ꢃ 25 ml). The combined organic layers were washed with
HCl (1 M, 2 ꢃ 30 ml) and saturated NaHCO3 (30 ml), dried (MgSO4),
filtered, and concentrated under reduced pressure. The residue was
purified by flash chromatography (30 g SiO2, eluted with 30%
cyclohexane in EtOAc, 350 ml) which gave compound 3 as white
solid (417 mg, 71%). Rf = 0.22 (30% cyclohexane in EtOAc), ½a D20
ꢂ
ꢁ23.8 (c 4.29, CHCl3); 1H NMR (CDCl3) d 7.19 (s, 2H), 4.79 (br s,
3H, D2O-exchangeable), 4.44 (d, J = 7.5 Hz, 1H, H-1), 4.35 (dd,
J = 4.0, 10.3 Hz, 1H, H-6), 4.23 (d, J = 10.3 Hz, 1H, H-6), 4.17–3.82
(m, 4H), 3.70–3.50 (m, 3H, H-3, H-4, H-5), 3.44 (dd, J = 8.2,
8.4 Hz, 1H, H-2), 2.91 (hept, J = 6.8 Hz, 1H), 1.34–1.14 (m, 18H);
13C NMR (CDCl3)
d 154.0, 151.0, 129.0, 123.9, 123.6 (q,
1JC,F = 278 Hz, C-20), 102.3 (C-1), 75.9 (C-3), 73.7 (C-5), 73.1 (C-2),
2
69.4 (C-4), 68.0 (C-6), 65.8 (q, JC,F = 35 Hz, C-10), 34.3, 29.8, 24.8,
24.6, 23.6; 19F NMR (CDCl3) d ꢁ74.0 (t, JH,F = 8.6 Hz); HRMS
3

104
R. F. G. Fröhlich et al. / Carbohydrate Research 361 (2012) 100–104
(EI TOF): [M–CF3CH2OH]+ calcd for C21H32F3O7S: 428.1869, found:
428.1881.
MeOH); 1H NMR (D2O, referenced with acetone as internal stan-
dard at 2.22 ppm) d 4.60 (d, J = 7.9 Hz, 1H, H-1), 4.41–4.15 (m,
2H, H-10), 3.56–3.36 (m, 3H), 3.33 (t, J = 8.6 Hz, 1H, H-2), 3.01 (br
d, J = 14.2 Hz, 1H, H-6), 2.74 (dd, J = 7.1, 14.2 Hz, 1H, H-6); 13C
NMR (D2O, referenced with acetone as internal standard at
3.5. 20,20,20-Trifluoroethyl 2,3,4-tri-O-acetyl-6-O-(2,4,6-
trisisopropybenzenesulfonyl)-b-D-glucopyranoside (4)
1
30.89 ppm) d 124.3 (q, JH,F = 278 Hz, C-20), 103.3 (C-1), 76.7,
2
Under nitrogen protection 3 (387 mg, 0.73 mmol) was dissolved
in dry pyridine (5 ml, 62 mmol) and acetic anhydride (2 ml,
21 mmol) was added. The resulting solution was stirred overnight
at rt. The reaction was quenched with water (40 ml) and extracted
with EtOAc (2 ꢃ 25 ml), washed with HCl (1 M, 2 ꢃ 30 ml) and sat-
urated NaHCO3 (35 ml), dried (MgSO4), filtered, and concentrated
under reduced pressure to give compound 4 as a colorless glass
(479 mg, 100%), which was used without further purification in
75.8, 73.6, 72.3, 66.9 (q, JC,F = 34 Hz, C-10), 25.7 (C-6); 19F NMR
3
(D2O) d ꢁ74.2 (t, JH,F = 8.8 Hz); HRMS (EI TOF): [M–SH]+ calcd
for C8H12F3O5: 245.0637, found: 245.0666, [M–H2O]+ calcd for
C8H11F3O4S: 260.0330, found: 260.0350.
Acknowledgements
Financial support to K.Z. by the Austrian Science Foundation
(FWF) (project numbers P20020 and P22630) is gratefully
acknowledged. E.S. thanks the Austrian Academy of Sciences for a
DOCfFORTE fellowship.
the following step. Rf = 0.61 (50% EtOAc in cyclohexane), ½a D20
ꢂ
+0.38 (c 2.9, CHCl3); 1H NMR (CDCl3) d 7.19 (s, 2H), 5,21 (t,
J = 9.5 Hz, 1H, H-3), 4.97 (dd, J = 7.9, 9.6 Hz, 1H, H-2), 4.92 (dd,
J = 9.5, 9.8 Hz, 1H, H-4), 4.61 (d, J = 7.9 Hz, 1H, H-1), 4.17–3.80
(m, 7H), 2.91 (hept, J = 6.9 Hz, 1H), 2.03 (s, 3H), 2.00 (s, 3H), 1.99
(s, 3H), 1.28–1.22 (m, 18H); 13C NMR (CDCl3) d 170.2, 169.5,
Supplementary data
1
169.4, 154.3, 151.0, 129.1, 124.0, 123.4 (q, JC,F = 278 Hz, C-20),
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.carres.2012.08.
010.
100.7 (C-1), 72.5 (C-5), 72.2 (C-3), 70.7 (C-2), 68.6 (C-4), 66.8 (C-
2
6), 65.9 (q, JC,F = 35 Hz, C-10), 34.4, 29.8, 24.7, 23.6, 20.7, 20.7,
3
20.6, 20.6, 20.5, 20.5; 19F NMR (CDCl3) d ꢁ74.5 (t, JH,F = 8.5 Hz);
HRMS (EI TOF) [M–H]+ calcd for C29H40F3O11S: 653.2244, found:
653.2266.
References
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glucopyranoside (5)
Under nitrogen protection 4 (448 mg, 0.68 mmol) was dissolved
in anhydrous DMF (4.5 ml) and potassium thioacetate (98%,
400 mg, 3.43 mmol, 5 equiv) was added. The clear yellow solution
was heated to 78 °C for 20 h. The reaction mixture was diluted
with water (50 ml) and extracted with EtOAc (3 ꢃ 20 ml). The com-
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EtOAc in cyclohexane), ½a D20
ꢂ
ꢁ18.1 (c 3.31, CHCl3); 1H NMR (CDCl3)
d 5.14 (dd, J = 9.3, 9.5 Hz, 1H, H-3), 5.00–4.86 (m, 2H), 4.56 (d,
J = 7.9 Hz, 1H, H-1), 4.05 (dq, J = 8.8, 12.4 Hz, 1H, H-20), 3.89 (dq,
J = 8.3, 12.4 Hz, 1H, H-20), 3.60 (m, 1H, H-5), 3.21 (dd, J = 1.5,
14.4 Hz, 1H, H-6), 3.02 (dd, J = 6.6, 14.4 Hz, 1H, H-6), 2.29 (s, 3H,
SAc), 2.02 (s, 3H), 1.98 (s, 3H), 1.94 (s, 3H); 13C NMR (CDCl3) d
1
194.5 (SAc), 170.1, 169.7, 169.3, 123.4 (q, JC,F = 279 Hz, C-20),
2
100.5 (C-1), 73.3 (C-5), 72.3 (C-3), 70.8, 70.3, 65.7 (q, JC,F = 35 Hz,
C-10), 30.4, 30.4, 29.9 (C-6), 20.7, 20.6, 20.6, 20.5, 20.4, 20.4; 19F
3
NMR (CDCl3) d ꢁ74.5 (t, JH,F = 8.5 Hz); HRMS (EI TOF): [M–H]+
calcd for C16H20F3O9S: 445.0780, found: 445.0810.
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submitted for publication.
3.7. 20,20,20-Trifluoroethyl 6-thio-b-
D-glucopyranoside (6)
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MeOH, 2 ml, 0.67 mmol) was added. The resulting homogeneous
mixture was stirred for 2.5 h at rt. Silica (2 g) was added and the
solvent was removed under reduced pressure. The dried residue
was loaded onto a flash chromatography column (28 g SiO2, eluted
with EtOAc, 250 ml) which gave title compound 6 as a hygroscopic
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ˇ ´
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2408–2416.
white gum (120 mg, 71%). Rf = 0.32 (in EtOAc), ½a D20
ꢁ17 (c 0.56,
ꢂ