Development of a Ratiometric Fluorescent Probe with Two Reactive Sulfoxides for Monitoring the Activity of Methionine Sulfoxide Reductase A

Article abstract of DOI:10.1055/s-0036-1591888

Biological oxidation of methionine side chains in proteins is a process that affects the functions of many proteins. One of the key regulators of this signaling is the enzyme methionine sulfoxide reductase A (MsrA). MsrA is implicated in a number of disea

Full text of DOI:10.1055/s-0036-1591888

SYNTHESIS
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Georg Thieme Verlag Stuttgart · New York
2018, 50, A–F
paper
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Synthesis
N. Makukhin et al.
Paper
Development of a Ratiometric Fluorescent Probe with Two
Reactive Sulfoxides for Monitoring the Activity of Methionine
Sulfoxide Reductase A
Nikolai Makukhin
Vladimír Nosek
Jiří Míšek*
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Department of Organic Chemistry, Faculty of Science, Charles
University, Hlavova 2030/8, 12843 Prague 2, Czech Republic
misek@natur.cuni.cz
Published as part of the Bürgenstock Special Section 2017 Future
Stars in Organic Chemistry
Received: 05.12.2017
Accepted after revision: 15.12.2017
Published online: 18.01.2018
level of MsrA is inversely proportional to the aggressiveness
of breast cancer. However, despite the obvious importance
of these enzymes, there is a lack of detailed studies on their
mechanisms in vivo, which in turn stems from the lack of
suitable tools for monitoring the in vivo activity of the
enzyme in real time.
DOI: 10.1055/s-0036-1591888; Art ID: ss-2017-z0786-op
Abstract Biological oxidation of methionine side chains in proteins is a
process that affects the functions of many proteins. One of the key reg-
ulators of this signaling is the enzyme methionine sulfoxide reductase A
We have previously reported the first ratiometric fluo-
rescent probe named (S)-Sulfox-1 for the monitoring of the
(
MsrA). MsrA is implicated in a number of diseases, but detailed under-
standing of its function is hindered by the lack of tools for monitoring
the enzyme’s activity. We have designed and synthesized a probe
named (S,S)-Sulfox-2 that is based on a BODIPY fluorophore and is
equipped with two chiral sulfoxide units of defined stereochemistry.
17
activity of MsrA in real time in cells. Recently, other
small-molecule- and protein-based fluorescent probes for
Msr activity monitoring have emerged.1
8,19
(S,S)-Sulfox-2 is shown to be highly responsive to MsrA and allows trac-
The design of our original probe, (S)-Sulfox-1, relies on
the fact that the absorption/emission characteristics of the
ing of the MsrA activity by a significant change in the fluorescence pro-
file.
BODIPY fluorophore can be altered by the substituents at
Keywords chirality, fluorescent probes, oxidoreductases, Suzuki–
Miyaura coupling, redox chemistry
20–22
positions 3 and 5 (Figure 1).
Aryl substituents in these
positions cause a bathochromic shift as the conjugated
system is being extended. Furthermore, substituents on the
Organismal oxidation of proteins by reactive oxygen
species is historically associated with cysteine residues;1
our previous work
this study
R
however, the methionine side chain can be oxidized as
readily as that of cysteine.2–4 The product of methionine ox-
8
idation is the corresponding methionine sulfoxide that can
occur in two stereoisomeric forms: S and R. Nature has
evolved the enzyme methionine sulfoxide reductase (Msr)
that can reduce free or protein-bound methionine sulfoxide
back to methionine. Interestingly, two distinct classes of
Msrs exist with complementary stereoselectivity, MsrA re-
ducing the (S)-epimer and MsrB reducing the (R)-epimer
N
N
N
N
3
5
3
5
B
B
R
R
R
R
S+
S+
S+
O–
OMe
O–
–O
R =
O
3
5–7
(Scheme 1). It is well established that oxidation of pro-
(
S)-Sulfox-1
(S,S)-Sulfox-2
tein-bound methionine residues can alter the function of a
large number of proteins, such that Msrs act as a central
Figure 1 Chemical structures of the fluorescent probe (S)-Sulfox-1
(that has been previously reported for monitoring the activity of
methionine sulfoxide reductase A) and the probe (S,S)-Sulfox-2
described in this article
8–16
hub for this redox regulation and signaling.
Importantly,
decreased levels of MsrA have been implicated in a number
of diseases. Thus, for instance, a decrease of the expression
©
Georg Thieme Verlag Stuttgart · New York — Synthesis 2018, 50, A–F

B
Synthesis
N. Makukhin et al.
Paper
Scheme 1 Representation of the reversible oxidation of methionine residues in proteins
phenyl ring can affect the emission wavelength, with elec-
tron-donating substituents increasing the red shift. Reduc-
tion of the sulfoxide substituent of (S)-Sulfox-1 to a sulfide
by MsrA resulted in red-shifting of the emission by 25 nm.
This difference in emission characteristics is the basis for
the ratiometric sensing. In general, ratiometric probes are
preferable to intensity probes for biological imaging. Inten-
sity probes might suffer from artifacts caused by variation
in concentrations that are due to external and environmen-
stituent at position 5 of (S)-Sulfox-1 would further increase
the change in the emission wavelength upon reduction
with MsrA.
The synthesis commenced with the preparation of
dibromo BODIPY 2 in an acceptable yield (35%), which was
utilized for further steps (Scheme 2). Dibromo substitution
was selected over dichloro as bromides have higher relative
reactivity in Suzuki–Miyaura coupling.25 Further, the
fluorine atoms in 2 were substituted for triethyleneglycol
arms to enhance the solubility and brightness in aqueous
media, which provided key precursor 3 for the synthesis of
Sulfox-2. The coupling partner 417 for the final Suzuki–Mi-
yaura coupling was synthesized by a racemic or asymmetric
oxidation/borylation sequence. It should be noted that Pd-
catalyzed borylation leading to chiral sulfoxide (S)-4 re-
quired strictly oxygen-free conditions (see the Supporting
Information). Dioxane was freshly distilled from
sodium/benzophenone right before the reaction and water
was thoroughly degassed by three freeze/thaw cycles.
Keeping the freshly distilled dioxane for just one day under
argon in a fume hood or deliberate injection of air into the
reaction led to partial racemization of the chiral sulfoxide
(S)-4 (from >99% ee to ~80% ee, as observed by chiral HPLC).
23,24
tal factors.
These factors are inherently canceled out for
ratiometric probes.
Our goal was to prepare a ratiometric fluorescent probe
for MsrA with enhanced difference in the emission wave-
length of the oxidized and reduced forms. This would allow
the probe to be used with more standard fluorescent filter
sets and thus further promote its applicability.
Herein, we report on the development of the ratio-
metric fluorescent probe (S,S)-Sulfox-2 with two reactive
sulfoxide moieties and demonstrate its superiority over
(
S)-Sulfox-1 in monitoring the activity of MsrA.
Substitution at positions 3 and 5 of the BODIPY fluoro-
phore has been previously shown to have an additive effect
20,21
on the wavelength of emission.
Therefore, we hypothe-
sized that addition of a second methylsulfinylphenyl sub-
No racemization was observed without a palladium
OMe
OMe
OMe
1. NBS, THF, –78 °C
1. AlCl3, CH2Cl2, rt
2
3
. DDQ, THF, rt
. Et3N, BF3·OEt2, rt
35%
2. RH, CH2Cl2, rt
32%
N
N
N
N
B
NH HN
B
Br
F
F
Br
Br
R
R
Br
1
2
OMe
R =
O
3
3
Scheme 2 Synthesis of the intermediate 3
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Georg Thieme Verlag Stuttgart · New York — Synthesis 2018, 50, A–F

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Synthesis
N. Makukhin et al.
Paper
catalyst. Since racemization of sulfoxides through radical
species has been demonstrated previously,26–28 we hypoth-
esized that a combination of palladium catalyst and oxygen
generates a radical intermediate that is capable of partial
racemization of sulfoxide (S)-4. Hence, this behavior had to
be considered during the final Suzuki–Miyaura coupling
leading to (S,S)-Sulfox-2.
Table 1 Optimization of the Conditions for the Suzuki Coupling
OMe
rac-4
OMe
or
(S)-4
O–
S+
B(pin)
N
N
B
The first attempt to install racemic phenylsulfoxide
moieties followed the protocol developed for the synthesis
of Sulfox-1, but it turned out to be unsuccessful. By employ-
ing rac-4 and intermediate 3 under the standard Suzuki–
Miyaura conditions with Pd(PPh ) , formation of only a
[Pd], Na2CO3, solvent, 85 °C
R
R
N
N
B
Br
R
R
Br
S+
S+
O–
–O
3
4
OMe
R =
O
trace amount of the monosubstituted product was ob-
served by HPLC-MS (Table 1), along with an intractable
mixture of other products. Judged from the molecular
masses and the isotopic patterns of these side products,
possible side-reaction pathways include loss of the tri-
ethyleneglycol arms, BODIPY deborylation, and substitution
of bromides. Consistent with our experiments on Sulfox-1,
water-free conditions or toluene as a solvent did not pro-
vide even traces of the monosubstituted product. We rea-
soned that speeding up the Suzuki–Miyaura coupling by a
careful choice of ligands could outcompete the undesired
side reactions. A number of ligands have been developed
that accelerate the Suzuki–Miyaura coupling, from the ini-
3
(S,S)-Sulfox-2
3
Entry
Solvent
dioxane
[Pd]
Yielda
1
2
3
4
5
6
7
Pd(PPh
3
)
4
nd
dioxane + 5% H O
2
Pd(PPh3)4
ndb
nd
toluene
Pd(PPh
Pd(PPh
3
)
)
4
4
toluene + 5% H
2
O
3
nd
dioxane
Pd(OAc)
Pd(OAc)
Pd(OAc)
2
2
2
/CyJohnPhos
/CyJohnPhos
/CyJohnPhos
ndb
dioxane + 5% H
dioxane + 10% H
nd = not detected.
2
O
24% (30%)c
nd
2
O
a
b
c
29
Only the monocoupled product was observed.
The yield in parentheses is that of the coupling with racemic sulfoxide rac-4.
tial MAP-type phosphines to the most recent examples,
30
such as Buchwald’s CyJohnPhos.
Delightfully, with
Pd(OAc) and CyJohnPhos, the desired bis-coupled product
2
was isolated in 30% yield (entry 6). The presence of 5% (v/v)
of water was also crucial for the success of the reaction. It is
believed that water helps to hydrolyze the boronic ester
fluoroethanol (TFAE) was employed to determine the stereo-
chemical purity of (S,S)-Sulfox-2. Addition of 10 equivalents
of TFAE to racemic Sulfox-2 caused a significant shift and
split of the proton signals of the methyl sulfoxide moiety in
the 1H NMR spectrum (see the Supporting Information).
Three new peaks were observed in a 2:1:1 ratio, which is in
agreement with the presence of two enantiomeric forms
(S,S- and R,R-) and a meso compound (R,S- and S,R-). The
same analysis was carried out with (S,S)-Sulfox-2 and only
one signal corresponding to one enantiomeric form was ob-
served. These experiments confirmed that the absolute
stereochemistry of the sulfoxide moieties was retained in
(S,S)-Sulfox-2, as was the stereochemical integrity.
Next, the spectroscopic characteristics of (S,S)-Sulfox-2
were measured and compared with those of (S)-Sulfox-1.
As expected, a bathochromic shift in the absorption maxi-
mum occurred for Sulfox-2 since the conjugation system is
extended (Sulfox-1, λmax = 509 nm; Sulfox-2, λmax = 548
nm). A similar trend was also seen in the emission spectra;
when excited at 548 nm, (S,S)-Sulfox-2 exhibited a major
emission peak centered at 589 nm. This gives a red shift in
emission of 69 nm compared to (S)-Sulfox-1, the emission
maximum of which is at 520 nm. The quantum yield in the
case of (S,S)-Sulfox-2 was lower than that of (S)-Sulfox-1
(0.14 vs 0.60), presumably due to the presence of additional
into a boronic acid, which is more reactive in the Suzuki–
Miyaura coupling.3
1,32
Indeed, under strictly anhydrous
conditions, only the monosubstituted product was detect-
ed. On the other hand, raising the water content to 10%
(v/v) did not provide any of the desired product (entry 7)
and decomposition side reactions began to dominate.
Given the racemic nature of the sulfoxide rac-4, the
product was obtained as a mixture of diastereoisomers.
Next, enantiopure sulfoxide (S)-4 was subjected to the opti-
mized conditions and the desired product was isolated in a
comparable yield (24%) (Table 1, entry 6). It should be noted
that the same synthetic protocol was used for the prepara-
tion of the reduced form 5 of Sulfox-2 with two sulfide
units. However, the resulting product was prone to oxi-
dation under ambient conditions and was never isolated as
a pure compound. However, this fact should not be an issue
for sensing purposes as both in vitro and in vivo experi-
ments are carried out under reducing environments.
In order to confirm that the chirality of the sulfoxide
moieties in (S,S)-Sulfox-2 was retained, analysis by chiral
HPLC was attempted. However, partial decomposition of
(
S,S)-Sulfox-2 on the chiral columns (OD series) negated
this approach. Thus, the chiral shift reagent (S)-anthryltri-
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Georg Thieme Verlag Stuttgart · New York — Synthesis 2018, 50, A–F

D
Synthesis
N. Makukhin et al.
Paper
phenyl rings that can further enhance relaxation of the ex-
cited state through non-radiative molecular motion pro-
cesses.
In summary, we have developed an asymmetric syn-
thetic protocol for the preparation of a new fluorescent
probe, (S,S)-Sulfox-2. The synthesis was enabled by thor-
ough optimization of the final Suzuki–Miyaura coupling
procedure. The resulting chiral probe (S,S)-Sulfox-2 is readi-
ly reduced by MsrA. The observed reduction of both sulfox-
ide groups of (S,S)-Sulfox-2 by MsrA is associated with a
marked red shift of the emission wavelength. The difference
between emission maxima for the oxidized and reduced
forms increased from 25 nm for (S)-Sulfox-1 to 40 nm for
(S,S)-Sulfox-2. This feature can be utilized for convenient
monitoring of the reactivity of MsrA in a ratiometric fash-
ion.
33
The activity of MsrA was tested against (S,S)-Sulfox-2.
Although (S,S)-Sulfox-2 has a second methylsulfinylphenyl
arm that makes both sulfoxide groups rather crowded,
MsrA (E. coli) was found to efficiently reduce both sulfoxide
units as confirmed by HPLC–MS analysis (see the Support-
ing Information). Finally, the activity of MsrA was moni-
tored by fluorescence spectroscopy using (S,S)-Sulfox-2 as a
surrogate substrate. The course of the reduction of (S,S)-
Sulfox-2 can be easily followed from the emission spectra
(Scheme 3). The emission maximum of the reduced form 5
is red-shifted by 40 nm compared to (S,S)-Sulfox-2, giving
an emission maximum at 629 nm. The control experiment
without MsrA did not show any significant change in the
emission profile (see the Supporting Information). As the
original probe (S)-Sulfox-1 and its reduced form provides a
difference in the emission maxima of 25 nm, this result
confirms that the addition of a second methylsulfinylphe-
nyl unit increases the red shift, which is beneficial for sens-
ing purposes. The observed emission ratio at 589 nm and
All starting reagents were commercially available and of analytical
purity, and were used without further treatment. Solvents were dried
according to standard methods. Aryldipyrromethane 1 was prepared
34
according to the literature procedure. (S)-boronate 4 was obtained
17
as described earlier. Analytical TLC was performed using Merck pre-
coated silica gel 60 Å F254 plates (0.2 mm thickness) and samples were
visualized with UV light at 254 nm. Preparative column chromatogra-
phy was carried out using Across Organies silica gel 60 Å (particle size
0.063–0.200 mm). Purifications by HPLC were performed under the
following conditions: Agilent ZORBAX SB-C18 column (5 μL, 9.4 × 150
mm); UV/Vis detection at λobs = 254 and 548 nm; flow rate: 4
629 nm (F589/F629) changed upon reaction from 5.1 to 0.2,
giving a 26-fold difference [whereas (S)-Sulfox-1 gave a 13-
fold difference].
mL/min; gradient elution method: H O/MeCN from 50:50 to 0:100,
2
15 min). Optical rotations were measured on an Autopol III automatic
polarimeter. Infrared spectra were recorded on a Thermo Nicolet
1
13
Avatar 370 FT-IR spectrophotometer. H and C NMR spectra were
recorded on Bruker Avance-400 instruments; C spectra were 1
13
H
decoupled. Chemical shifts (δ) are reported in ppm relative to the
solvent (CDCl : δ = 77.0; CD OD: δ = 49.0) or the residual solvent
3
C
3
C
peak (CHCl : δH = 7.26; CD OH: δH = 3.31; H O in CD OH: δ = 4.87,
3
3
2
3
H
C D H: δ = 7.16) as internal standards. Accurate mass measurements
6
5
H
(HRMS) were obtained by ESI on an Agilent 6530 Q-TOF MS spectro-
meter. UV/Vis absorption spectra were recorded on a BioDrop Touch
spectrophotometer. Fluorescence measurements were performed on
an AMINCO-Bowman Series 2 spectrofluorometer.
3,5-Dibromo-4,4-difluoro-8-(4-methoxyphenyl)-BODIPY (2)
5-(4-Methoxyphenyl)dipyrromethane (1) (530 mg, 2.1 mmol) was
dissolved in anhyd THF (50 mL) and cooled to –78 °C. N-Bromosuccin-
imide (750 mg, 4.2 mmol) was added to the mixture in two portions
over a period of 1 h (375 mg every 30 min). The mixture was stirred
for a further 2 h at –78 °C. A solution of DDQ (477 mg, 2.1 mmol) in
THF (10 mL) was added to the mixture dropwise over 10 min. The re-
action mixture was warmed to r.t. and the solvent was removed un-
der reduced pressure. The crude product was subjected to flash col-
umn chromatography using CH Cl (R = 0.95). The red solid (534 mg)
2
2
f
thus obtained was dissolved in anhyd CH Cl (50 mL) and treated with
2
2
Et N (1 mL), followed by the addition of BF ·Et O (1 mL). After stirring
3
3
2
at r.t. for 1 h, the reaction mixture was washed with 0.1 M NaOH
2 × 50 mL), H O (2 × 50 mL) and brine, then dried over MgSO4 and
(
Scheme 3 (a) A sensing mechanism of (S,S)-Sulfox-2; DTT = dithio-
threitol. (b) Changes in the emission profile of (S,S)-Sulfox-2 (5 μM)
upon incubation with MsrA (5 μM) for 30 min at 20 °C (excitation at 548
nm). The inset shows the changes in the emission ratio F589/F629 as a
function of time.
2
concentrated by rotary evaporation under reduced pressure. The resi-
due was purified by column chromatography on silica gel (PhMe/CH
Cl = 4:1) to afford 2 in 35% yield (340 mg) as an amorphous red solid.
-
2
2
35
The NMR spectra were in agreement with the published data.
R = 0.45 (PhMe/CH Cl = 4:1).
f
2
2
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E
Synthesis
N. Makukhin et al.
Paper
1
1
H NMR (400 MHz, CDCl ): δ = 3.89 (s, 3 H, OCH ), 6.52 (d, J = 4.4 Hz, 2
The enantiomeric excess of (S,S)-Sulfox-2 was determined by H NMR
3
3
H, 2 pyrrole-H), 6.82 (d, J = 4.4 Hz, 2 H, 2 pyrrole-H), 7.02 (d, J = 8.8 Hz,
spectroscopy as follows. Sulfox-2 (2 mg) and the chiral shift reagent
2
H, ArH), 7.44 (d, J = 8.8 Hz, 2 H, ArH).
(S)-(+)-1-(9-anthryl)-2,2,2-trifluoroethanol (TFAE) (6.4 mg, 10 equiv)
1
were dissolved in C D (0.6 mL) in an NMR tube and the H NMR spec-
6
6
trum was recorded.
3,5-Dibromo-4,4-{2-[2-(2-methoxyethoxy)ethoxy]ethoxy}-8-(4-
methoxyphenyl)-BODIPY (3)
Preparation and Purification of E. coli Methionine Sulfoxide Re-
ductase A (MsrA)
Compound 2 (340 mg, 0.68 mmol) and aluminum chloride (273.5 mg,
2
.0 mmol) were dissolved in anhyd CH Cl (20 mL) under an argon at-
2 2
5
mosphere. The resulting mixture was refluxed for 5 min. After cooling
to r.t., 2-[2-(2-methoxyethoxy)ethoxy]ethanol (1 mL, 6.8 mmol) was
added and the mixture was stirred at r.t. for 1 h. The reaction mixture
MsrA gene in pGEX vector was transformed into chemically compe-
tent E. coli BL21(DE3) cells, and an aliquot was plated on LB agar with
–1
ampicillin (100 mg L ) and grown overnight at 37 °C. A single colony
was used for the preculture of which 0.5 mL was inoculated in 500 mL
was washed with 2 M NaOH (2 × 50 mL), H O (5 × 50 mL) and brine,
2
–1
then dried over MgSO and concentrated by rotary evaporation under
LB medium with ampicillin (100 mg L ). The bacterial culture was in-
cubated at 37 °C, 220 rpm until an OD600 of 0.6 was reached. Next,
production of the protein was induced with IPTG (0.5 mM) and the
culture was incubated at 37 °C, 220 rpm for an additional 3 h. The
cells were harvested by centrifugation (3000 × g for 30 min at 4 °C).
The cell pellet was resuspended in PBS buffer [Na HPO (10 mM),
4
reduced pressure. The crude product was purified by column chroma-
tography using silica gel (hexane/EtOAc/MeOH/Et N = 3:2:0.5:0.1) to
3
give 215 mg of product. The final product was additionally purified
by HPLC (t = 7 min) to afford 3 in 32% yield (180 mg) as an orange oil.
R
R = 0.52 (hexane/EtOAc/MeOH/Et N = 3:2:0.5:0.1).
2
4
f
3
KH PO (1.8 mM), KCl (2.7 mM), NaCl (140 mM), pH 7.3] and lysed by
2
4
IR (KBr): 2866, 1571, 1539, 1380, 1245, 1177, 1114, 1084 cm–1
.
sonication. Cell debris was removed by centrifugation (10000 × g for
30 min at 4 °C), and the soluble fraction was loaded onto a glutathi-
one agarose column (Protino Glutathione Agarose 4B, Macherey-Na-
gel) and the resulting suspension gently agitated for 30 min at r.t. The
column was washed with 3 × 10 bed volumes of PBS and finally the
protein was eluted with 3 × 1 bed volumes of elution buffer [Tris-HCl
1
H NMR (400 MHz, CD OD): δ = 3.27–3.31 (m, 4 H, 2 OCH ), 3.36 (s, 6
3
2
H, 2 OCH ), 3.41–3.61 (m, 20 H, 5 OCH ), 3.94 (s, 3 H, OCH ), 6.68 (d,
3
2
3
J = 4.4 Hz, 2 H, 2 pyrrole-H), 6.97 (d, J = 4.4 Hz, 2 H, 2 pyrrole-H), 7.17
(d, J = 8.8 Hz, 2 H, ArH), 7.58 (d, J = 8.8 Hz, 2 H, ArH).
13
C NMR (101 MHz, CD OD): δ = 56.1, 59.1, 62.2, 71.3, 71.4, 71.5, 72.8,
3
(50 mM), glutathione (10 mM), pH 8]. The elution buffer was ex-
72.9, 115.2, 123.7, 126.2, 132.3, 133.0, 133.7, 137.8, 144.7, 163.6.
changed with storage buffer [Tris-HCl buffer (20 mM), DTT (1 mM),
glycerol (10%), NaCl (0.1 M), pH 8.0] with an Amicon Ultra-4 centrifu-
gal filter (3000 MWCO). This procedure typically yielded ~20 mg of
protein per liter of culture. The protein concentration was deter-
mined from the absorbance at 280 nm.
HRMS (ESI): m/z [M + NH ] calcd for C30H45B79Br81BrN O : 762.1598;
found: 762.1587.
+
4
3
9
3
,5-[(S)-4-(Methylsulfinyl)phenyl]-4,4-{2-[2-(2-methoxye-
thoxy)ethoxy]ethoxy}-8-(4-methoxyphenyl)-BODIPY [(S,S)-Sulf-
ox-2]
Real-Time Monitoring of MsrA Activity
A mixture of 3 (15 mg, 0.02 mmol), (S)-4 (16 mg, 0.06 mmol),
Pd(OAc)2 (0.9 mg, 0.004 mmol), CyJohnPhos (2.8 mg, 0.008 mmol),
Na CO (6 mg, 0.06 mmol) in dioxane (2 mL, freshly distilled over so-
A solution of (S,S)-Sulfox-2 (5 μM, 1.6 mL) in the buffer [phosphate
buffer (50 mM), NaCl (50 mM), pH 7.5 + 10% MeCN] containing DTT
2
3
(20 mM) was prepared in a 1 cm quartz cuvette and MsrA (E.coli) was
dium and benzophenone) and H O (0.1 mL, degassed by
3
2
added to a final concentration of 5 μM. The solution was mixed gen-
tly. The emission spectra (λexc = 548 nm) were registered every 1 min
up to 30 min at ambient temperature in air (25 °C).
freeze/thaw cycles) was stirred at 85 °C for 30 min (TLC monitoring)
under an argon atmosphere. The solvent was removed by rotary
evaporation under reduced pressure and the crude product was puri-
fied by column chromatography using silica gel (hexane/EtO-
Ac/MeOH/Et N = 3:2:0.5:0.1) to give 6 mg of the product. The final
3
Funding Information
product was additionally purified by HPLC (t = 5 min) to afford (S,S)-
R
Sulfox-2 in 23% yield (4 mg, >95% ee) as a pink oil.
This research was supported by the Czech Science Foundation (Grant-
R
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Y)
(
c = 0.1, MeOH).
IR (KBr): 2926, 2848, 1272, 1257, 1120, 1090, 1048, 952 cm–1
.
Supporting Information
1
H NMR (400 MHz, CD OD): δ = 2.83 (s, 6 H, 2 SCH ), 2.97–3.01 (m,
3
3
4
3
2
H, 2 OCH ), 3.06–3.11 (m, 4 H, 2 OCH ), 3.24–3.28 (m, 4 H, 2 OCH ),
2
2
2
Supporting information for this article is available online at
.28 (s, 6 H, 2 OCH ), 3.37–3.45 (m, 8 H, 4 OCH ), 3.45–3.49 (m, 4 H,
3
2
https://doi.org/10.1055/s-0036-1591888.
S
u
p
p
o
nrtogI
i
f
rm oaitn
S
u
p
p
ortioIgnfmr oaitn
OCH ), 3.93 (s, 3 H, OCH ), 6.79 (d, J = 4.4 Hz, 2 H, 2 pyrrole-H), 7.05
2
3
(
=
d, J = 4.4 Hz, 2 H, 2 pyrrole-H), 7.17 (d, J = 8.6 Hz, 2 H, ArH), 7.63 (d, J
8.6 Hz, 2 H, ArH), 7.73 (d, J = 8.3 Hz, 4 H, ArH), 8.27 (d, J = 8.3 Hz, 4 H,
ArH).
References
1
3
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C NMR (101 MHz, CD OD): δ = 43.7, 56.1, 59.0, 61.7, 71.2, 71.3, 71.4,
3
7
1
2.5, 72.9, 115.1, 122.4, 124.3, 127.9, 131.9, 132.4, 133.7, 137.8, 139.3,
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HRMS (ESI): m/z [M + NH4]+ calcd for C44H59BN O11S2: 880.3686;
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= 548 nm
max
–1
–1
(16188 ε/M cm ).
©
Georg Thieme Verlag Stuttgart · New York — Synthesis 2018, 50, A–F

F
Synthesis
N. Makukhin et al.
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©
Georg Thieme Verlag Stuttgart · New York — Synthesis 2018, 50, A–F