Oxidative Formation of Disulfide Bonds by a Chemiluminescent 1,2-Dioxetane under Mild Conditions

Article abstract of DOI:10.1021/acs.joc.0c00569

The oxidation of alkyl thiols to disulfides has been achieved under mild conditions using a chemiluminescent 1,2-dioxetane as a stoichiometric oxidant. Besides the mild and biocompatible reaction conditions, this approach offers the possibility to monitor the presence of thiols through oxidation and chemiluminescence of the remaining dioxetane.

Full text of DOI:10.1021/acs.joc.0c00569

Subscriber access provided by the University of Exeter
Note
Oxidative formation of disulfide bonds by a
chemiluminescent 1,2-dioxetane under mild conditions
Caroline Silvia Sauer, Johannes Köckenberger, and Markus Rolf Heinrich
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.0c00569 • Publication Date (Web): 12 Jun 2020
Downloaded from pubs.acs.org on June 18, 2020
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 9
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
Oxidative formation of disulfide bonds by a chemiluminescent 1,2-
dioxetane under mild conditions
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Caroline S. Sauer, Johannes Köckenberger and Markus R. Heinrich*
Department of Chemistry and Pharmacy, Pharmaceutical Chemistry, Friedrich-Alexander-Universität Erlangen-Nürnberg,
Nikolaus-Fiebiger-Straße 10, 91058 Erlangen, Germany.
Supporting Information Placeholder
whereat both strategies have been applied to determine choline
esterase activity.
ABSTRACT: The oxidation of alkyl thiols to disulfides has been
achieved under mild conditions using a chemiluminescent 1,2-
dioxetane as stoichiometric oxidant. Besides the mild and
biocompatible reaction conditions, this approach offers the
possibility to monitor the presence of thiols through oxidation and
chemiluminescence of the remaining dioxetane.
Scheme 1. Thiol oxidation in biology and synthetic
chemistry.
a) Enzymatical / biological and chemical oxidation
enzymes
2 R-SH
metallic or non-metal reagents
oxygen (co-oxidant)
R-S-S-R
In general, and with cysteine as the most prominent example,
thiols play an important role in nature ranging from the
formation of disulfide bridges to induce the folding of peptides
and proteins to the activation of signaling pathways to the
binding of metal ions. Moreover, cysteines and their related
thiolate anions may serve as nucleophiles in the active site of
enzymes or may be involved in redox catalytic processes such
as oxidations and reductions mediated by thioredoxins.1 While
the formation of disulfides from thiols in nature mainly occurs
through enzymatic oxidations,2, 3 a large and diverse number of
synthetic methods have been developed to achieve this highly
important transformation (Scheme 1a). Suitable oxidants
include metal-based reagents such as permanganate salts, ferric
chloride, cerium(IV) salts, copper(II) salts,4 chromates or
b) Oxidation by Ellman's reagent
NO2
CO2
O2C
2
O2N
S
O2C
O2N
S
h
S
2 H
R-S-S-R
2 R-SH
c) This work: oxidation by chemiluminescent dioxetane
O
O
MeO
OH
O
TBSO
TBSO
h
TBS -
cleavage
2 R-SH
R-S-S-R
In this work, we present a novel strategy for the mild formation
of disulfides using a chemiluminescent 1,2-dioxetane as
stoichiometric oxidant.31 The reaction can then be further
investigated by exploiting the light-emitting properties of the
1,2-dioxetane. Various derivatives of chemiluminescent
dioxetanes have so far been reported and been used for diverse
applications.32-34 For the present work, the tert-
butyldimethylsilyl (TBS) analog 1 was selected, from which
chemically initiated electron-exchange luminescence35
(CIEEL) can be easily triggered by the cleavage of the TBS
group by fluoride ions36, or by other conditions (Table 1).37 For
the synthesis of 1, we followed the McMurry cross-coupling
approach by Baader and co-workers38 using TBS-protected
methyl 3-hydroxybenzoate39 and 2-adamantanone as starting
materials.40 Photooxygenation of the resulting methyl vinyl
ether in the presence of methylene blue provided 1.41
7
dichromates,5 rhenium-sulfoxide complexes,6, as well as
diverse non-metal reagents comprising sodium perborate,
sodium chlorite, bromine on silica gel,8 N-bromosuccinimide,9
nitric oxide, sodium nitrite,4 hydrogen peroxide in
tetrafluoroethanol,10 hydantoin derivatives,11-13 sulfuryl
chloride,14 and peroxymonosulfate,15 among others. Further
methods involve oxygen as a co-oxidant combined with suitable
catalysts, as for example a FeCl3-NaI/air16 or a CsF-Celite/air
system.17 In this context, particularly mild and biocompatible
reaction conditions, such as oxidations under air4, 18, 19 or by
iodine,20 are highly desirable since they can be potentially
applied to complex biomolecules. In addition, the simultaneous
visualization of disulfide bond formation can be a useful
feature. So far, mainly mild oxidants such as Ellman’s reagent21
(5,5’-dithiobis-(2-nitrobenzoic acid), DTNB), solid-phase
versions thereof,22, 23 and related derivatives24-28 have been used
to determine the amount of free thiols groups along with
oxidation (Scheme 1b). With regard to visualization, structural
combinations of Ellman’s reagent with chemiluminescent
probes29 or with fluorescent dyes30 are particularly interesting,
Initial experiments showed that the reductive ring-opening of
the chemiluminescent 1,2-dioxetane 1 can be indeed coupled to
the oxidation of 2-mercaptoethanol 3a yielding disulfide 4a
along with the 2-hydroxy ketone 2, which is most likely formed
from 1 via an unstable α-hydroxy hemiacetal (Table 1). The
structure of the so far unknown ketone 2 was confirmed after
isolation using HRMS and NMR analysis, with a Cq signal at
205.1 ppm providing strong support.
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Except for the oxidation of DL-dithiothreitol (3b), the thiols
Page 2 of 9
1
2
3
4
5
6
7
8
3a,c,d were now converted with much lower deviations
regarding the formation of disulfide 4a,c,d and hydroxy ketone
2 (max. 7% for 3c→4c after 1 h). We currently assign this to an
at that time improved preparation of 1,2-dioxetane 1, which
provided the oxidant in higher purity than previously used for
the experiments in Table 1. Two-fold analysis of dioxetane 1,
Table 1. Influence of base on the 1,2-dioxetane-induced
oxidation of thiol 3a.
O
O
MeO
OH
O
TBSO
TBSO
1
2
1
as obtained by the improved procedure, by H NMR using
SH
S
OH
2
HO
HO
S
see table
for conditions
1,3,5-trimethoxybenzene as internal standard gave purities of
4a
3a
94% and 97%.43, 44
9
K2CO3
(eq.)g
4a
2
entry
solvent
CD3OD
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(%)h
(%)i
Table 2. Oxidation of thiols 3a-d.
O
O
MeO
OH
1a
2b
3b
4b
5b
6b
-
< 5
0
O
TBSO
TBSO
CD3OD
CD3OD
CD3OD
CD3OD
(CD3)2SO
0.1
0.2
0.4
1.0
1.0
33
60
72
76
63
35
61
80
88
66
1
2
2 R-SH
R-S-S-R
K2CO3, (CD3OD), r.t.
3a-d
4a-d
time
(h)
4a-d
(%)c
2
entry
1a
thiol 3a-d
(%)d
1
3
1
3
1
3
1
3
54
57
87
57
74
51
84
53
82
CD3CN, D2O,
CD3OD
7c
1.0
75
84
SH
3a
3b
3c
3d
HO
82
78
100
44
80
53
79
8d,e
9d,f
-
-
< 5
23
0
OH
SH
CD3OD, Tris
buffer
2b
HS
< 15
OH
a Standard conditions (NMR tube):
1
(0.01 mmol), 2-
SH
3a
1
mercaptoethanol (3a) (0.02 mmol) in solvent (700 µL). H NMR
spectrum recorded after 3 hours at r.t. b CD3OD or (CD3)2SO
previously incubated with K2CO3 (15 min). c 10% CD3CN, 10%
D2O, only CD3OD incubated with K2CO3. d 10% Tris buffer (D2O,
pH = 8.6) in CD3OD. e 1H NMR spectrum recorded after 1.5 hours.
f 1H NMR spectrum recorded after 18 hours. g Equivalents refer to
3a.h Yield of 4a derived from ratio of 3a and 4a. i Yield of 2 derived
from ratio of 1 and 2.
OMe
O
O
4a,e
N
SH
a Standard conditions (NMR tube): 1 (0.01 mmol) and thiol 3
(0.02 mmol) in CD3OD (700 µL), previously incubated with
1
K2CO3 (0.02 mmol, 15 min). H NMR spectrum recorded after 1
hour and 3 hours at r.t. b Thiol 3b (0.01 mmol). c The yield of 4a-d
derived from ratio of 3a-d and 4a-d. d Yield of 2 derived from ratio
of 1 and 2. e Transesterification (-OCH3 → -OCD3) observed for 3d
to 4d.
The reactions conducted for optimization were monitored by 1H
NMR spectroscopy in CD3OD, whereas the emerging triplet
signals of disulfide 4a at 2.84 ppm and 3.79 ppm turned out as
reliable indicators. Since major side products could not be
detected, the yields were calculated from ratios of 1:2 and
3a:4a. The experiments summarized in Table 1 show that the
use of CD3OD saturated with potassium carbonate (approx. 11
mM) (entry 5) provides the highest yield of disulfide 4a (76%).
While lower amounts of base (entries 1-4) and the change to
(CD3)2SO slowed down the reaction (entry 6), the mixture of
CD3OD, D2O, and CD3CN also provided disulfide 4a in a good
yield of 75% (entry 7). Finally, an attempt with alkaline Tris
buffer42 (entries 8 and 9) revealed that the desired oxidation
proceeds much more readily in methanol (entry 5). In aqueous
systems (entries 7-9), changes in the additives can lead to strong
deviations, whereat the limited solubility of dioxetane 1 may
also play a role. This assumption is in agreement with earlier
results by Adam,31 who achieved the oxidation of thiols with a
structurally far more simple and more polar, but non-
chemiluminescent 1,2-dioxetane in pure water.
In the oxidation of 3b, the formation of the cyclic disulfide 4b
occurred faster than the formation of 2, with deviations of 21%
after 1 h and 26% after 3 h. This is most likely due to the
intramolecular reaction course, thereby opening an additional
mechanistic pathway. As control reactions ruled out a
dioxetane-independent oxidation as background reaction,45 an
alternative dioxetane-dependent oxidative mechanism appears
plausible for thiol 3b, which, however, does not follow the
typical 1:2 stoichiometry of dioxetane to thiol.
To investigate whether a disulfide 4 reacts further with
dioxetane 1, a control reaction between trans-4,5-dihydroxy-
1,2-dithiane (4b) and 1 in basic methanol under standard
1
reaction conditions was monitored by H NMR spectroscopy.
Under the conditions of Table 2, and over 24 hours, the cyclic
disulfide 4b was found to be stable. To demonstrate the
synthetic applicability and reliability of the novel oxidation
method, three disulfides were prepared on a larger than
0.01 mmol scale, followed by isolation and purification. These
experiments gave 4a, 4c, and 4d in yields of 81%, 83%, and
83%, respectively, which are well comparable to the NMR
yields reported in Table 2 (4a: 82%, 4c: 80%, 4d: 79%).
While an equable, ideally 1:1-formation of 2 and 4a would be
advantageous regarding quantification of the oxidation by
chemiluminescence, increased reaction times - on the other
hand - might give rise to side reactions. Against this
background, the dioxetane-mediated oxidation of thiol 3a was
evaluated in comparison with three further thiols 3b-d (Table
2).
When moving to N-acetyl L-cysteine methyl ester (3e) and N-
acetyl L-cysteine amide (3f), we found that these oxidations
surprisingly proceeded faster than those summarized in Table
ACS Paragon Plus Environment

Page 3 of 9
The Journal of Organic Chemistry
2
1 (1 eq.)
S
AcHN
O
S
2, so that the reaction temperature had to be lowered to 0°C. For
SH
S
S
AcO
1
2
3
4
3e
(2 eq.)
+
+
+ 4e
+ 4e
comparison, the oxidation of 2-mercaptoethanol 3a to disulfide
4a, which was repeated at 0 °C, was added as a reference
reaction to Figure 1.
3h
(2 eq.)
OAc
K2CO3, (CD3OD), 0 °C
AcO
OMe
4eh
OAc
4h
2
1 (1 eq.)
S
AcHN
O
S
SH
S
S
AcHN
3e
(2 eq.)
Figure 1. Oxidation of cysteine derivatives 3e,f,g, and thiol
3a at 0 °C.
3i
(2 eq.)
AcHN
OMe
4ei
NHAc
K2CO3, (CD3OD), 0 °C
NHAc
5
4i
6
7
8
9
O
O
MeO
SH
OH
O
In the first competition experiment (Figure 2, top), it was
surprising to see that the formation of the cysteine disulfide 4e
was now even slower than that of 4a, whereas the mixed
disulfide 4ae arose as major product leading to a final product
ratio of 4a:4e:4ae = 28:16:56. The summarized yields of the
disulfides 4a (12.5%), 4e (7%) and 4ae (25%), when multiplied
by a factor of 2 (89%), nicely correspond to the amount of
hydroxy ketone 2 (87%), thereby further supporting the
suitability of 1,2-dioxetane 1 as a clean 1:2 oxidant for thiols
(see Table S2).
TBSO
TBSO
X
O
1
2
RHN
S
RHN
O
NHR
S
K2CO3, (CD3OD), 0 °C
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
X
X
4e,f,g
3e: R = Ac, X = OMe
3f: R = Ac, X = NH2
3g: R = H, X = OMe
When 2-mercaptoethanol (3a) was replaced by its O-acetylated
derivative 3h, and competition was studied with cysteine 3e, a
final product ratio of 4h:4e:4eh = 27:19:54 was obtained
(Figure 2, bottom). This ratio is only slightly different from the
product distribution resulting from the competition of 3a and
3e, as shown above. N-Acetyl cysteamine (3i), however, when
competing with 3e, led to an altered ratio of 4i:4e:4ei =
42:16:42. In comparison to the thiols 3a, 3e, and 3h, N-acetyl
cysteamine (3i) thus shows the highest reactivity to form
symmetric disulfides.
At 0 °C, very low deviations between the formation of
disulfides 4a,e,f,g and the hydroxy ketone 2 were found in each
of the four experiments (see Table S1). Furthermore, a control
experiment with cysteine 3f ruled out a dioxetane-independent
formation of 4f. The clean formation of disulfide 4g from the
N-unprotected cysteine 3g demonstrated that free amino groups
do not interfere with the oxidation. Dioxetane 1 thus appears to
be more stable towards nucleophiles than the sterically less
hindered dioxetanes studied by Adam.31, 46
Although these competition experiments give interesting
insights into the relative reactivity of particular thiols in
combination with dioxetane 1 as mild oxidant, it is yet difficult
to draw mechanistic conclusions.47 In general, radical31 as well
as ionic mechanisms46 have been described for the oxidation of
thiols by 1,2-dioxetanes, whereat seminal and pioneering work
on the reaction of 1,2-dioxetanes with thiols has been conducted
by the Adam research group.31
The faster oxidation of the cysteines 3e and 3f - compared to
thiol 3a - then turned our interest towards the question of
whether the dioxetane 1 might be able to oxidize particular
thiols selectively, which would be a unique feature (Figure 2).
Finally, we turned to evaluate whether the chemiluminescent
properties of dioxetane 1 can be employed to monitor the thiol
oxidation process. In a series of preliminary experiments (see
Table S3 and Figure S1), it turned out that the dioxetane 1
remaining in the reaction mixture needs to be separated from
the newly formed disulfide, as chemiluminescence is otherwise
significantly quenched. Moreover, we found that a mixture of
basic methanol and DMSO was well suited to cleave the TBS
group from 2 and to trigger light emission. On this basis, a
simple and practical general procedure could be developed.
Accordingly, and as exemplified for the oxidation of cysteine
3e (Scheme 2), remaining dioxetane 1 and hydroxy ketone 2
were extracted with MTBE from the reaction mixture after
oxidation. Alternatively, samples may be taken from an
Figure 2. Competitive oxidation of thiols 3a, h or i and
cysteine derivative 3e.
2
1 (1 eq.)
S
AcHN
S
SH
S
S
HO
3e
(2 eq.)
+
+ 4e
3a
(2 eq.)
OH
K2CO3, (CD3OD), 0 °C
HO
O
OMe
4ae
OH
4a
ongoing
oxidation.
After
washing
with
water,
chemiluminescence was triggered by the addition of DMSO and
basic CH3OH.
Scheme 2. Procedure for the oxidation of cysteine 3e
combined with chemiluminescence read-out.
ACS Paragon Plus Environment

The Journal of Organic Chemistry
decay curve (as shown in Figure 3) is not necessary, which adds
Page 4 of 9
O
O
OH
MeO
3e
O
TBSO
O
TBSO
1
2
3
4
5
6
7
8
to the attractiveness of the overall method.
1
K2CO3,
(CH3OH), 0 °C,
75 min
In conclusion, we have shown that 1,2-dioxetane 1 can serve as
a particularly mild oxidant for thiols in methanol to give
disulfides. In single reactions and competition experiments,
insights into the relative reactivities of the thiols were obtained.
Furthermore, a first procedure has been developed, which
allows linking the thiol oxidation to the highly sensitive
chemiluminescent properties of 1,2-dioxetane 1, so that the
course of thiol oxidation or the thiol content of a sample can be
monitored. Regarding the importance of thiols in chemistry and
biology, these results represent a valuable starting point for
future investigations. Experiments towards more polar and thus
fully water-soluble chemiluminescent derivatives of dioxetane
1 are currently underway in our laboratory.
1
2
MeO
1. extraction of reaction mixture
(or sample) with MTBE
2. washing with water
3. TBS-cleavage by addition of
DMSO and basic CH3OH
4. chemiluminescence
measurement
S
AcHN
O
NHAc
S
4e
OMe
no h
h
The chemiluminescence read-out curves depicted in Figure 3
were obtained according to the overall procedure described in
Scheme 3. In the left part of Figure 3, the decay curves from
three calibration experiments (A)-(C) are summarized. In the
right part, the results of two dioxetane-mediated oxidations of
cysteine 3e with subsequent optical read-out are shown
((E),(F)). The experiment (D), which was performed in the
absence of thiol 3e, gives an impression on the good
reproducibility, as it corresponds to the calibration experiment
(A).
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
EXPERIMENTAL SECTION
In the oxidation experiments, the ratio of 3e to 1 of 2:1 led to a
full consumption of 1 and basically no light emission (F).
Starting with a ratio of 3e to 1 of 4:3 (E), left one-fourth of
dioxetane 1 unchanged, which should result in approximately
25% of the maximal light emission. As the read-out of
experiment (E) is well comparable with calibration experiment
(C), where 25% of the full amount of 1 was used, this underlines
the applicability of the method.
General experimental. All reactions were carried out following
common organic chemistry laboratory procedures. Chemicals and
solvents were obtained from commercial sources and were used as
received. Reactions with air- or moist-sensitive reagents were
carried out in glass equipment, previously heated under vacuum
and subsequently purged with nitrogen or argon gas. Air or water-
sensitive reactions were performed in anhydrous solvents
(indicated in each case). Potassium carbonate (anhydrous, free-
flowing, Redi-Dri™, ACS reagent, ≥ 99%) was purchased from
Sigma-Aldrich (product number: 791776). NMR spectra were
recorded on a Bruker Avance III HD 400 (1H-NMR: 400 MHz;
DEPTQ: 101 MHz) and a Bruker Avance III HD 600 (1H-NMR:
600 MHz; DEPTQ: 151 MHz) spectrometers. Chemical shifts (δ)
are reported in parts per million (ppm). For 1H NMR, CDCl3,
CD3OD or (CD3)2SO were used as solvents referenced to the
internal standard tetramethylsilane (TMS, δ = 0.00 ppm) or the
residue proton signal of the used solvents CDCl3 (δ = 7.26 ppm),
CD3OD (δ = 3.31 ppm) or (CD3)2SO (δ = 2.50 ppm). The DEPTQ
spectra were calibrated to the characteristic signals of the solvents:
CDCl3 (δ = 77.0 ppm), CD3OD (δ = 49.0 ppm) or (CD3)2SO
(δ = 39.4 ppm). The following abbreviations were used for the
signal multiplicity: s (singlet), d (doublet), dd (doublet doublet), t
(triplet), dt (doublet triplet), q (quadruplet), m (multiplet), br
(broad). Coupling constants J were indicated in Hertz (Hz). The
spectra were evaluated with the program MestReNova.
Figure 3. Oxidation of varying amounts of cysteine 3e by
dioxetane 1 combined with chemiluminescence read-out.a
a Calibration measurements (A)-(C): (A): 100% of 1, (B): 50% of
1, (C) 25% of 1. Ratios of reactants in the experiments (D)-(F):
(D): only 1 (no thiol added), (E): 1:3e (3:4), (F): 1:3e (1:2).
Thin-layer chromatography (TLC) analyses were performed on
Merck 60 F254 aluminum sheets (0.25 mm silica gel) and
visualized by fluorescent detection using UV light (254 nm and
In addition to the monitoring of completed oxidation reactions,
which allows determining the thiol content (Scheme 2 and
Figure 3), the optical read-out can also be applied to follow the
reaction course. For this purpose, the oxidation of cysteine 3e
to 4e at 0°C was quenched after three different intervals. The
amounts of remaining dioxetane 1 were determined by
chemiluminescence and the values were compared to those
measured by 1H NMR (Figure 1 and Table S1). After reaction
times of 22.5, 43.5, and 64.5 minutes, calculation of the amount
of 1 from the chemiluminescence read-out gave values of 50%,
33%, and 23%, respectively (see Figure S2). Within typical
error ranges, these values correspond well to those measured by
1H NMR (47%, 29%, and 18%) after identical reaction times.
360 nm) or the following TLC stain: KMnO4
[3.0 g KMnO4,
20 g K2CO3, 5 mL NaOH (5% w/w) in 300 mL H2O]. Flash
chromatography was carried out using silica gel 60 (40 – 63 μm,
230 – 400 mesh ASTM) as stationary phase and under pressure of
1.0 – 1.5 bar. The used solvent mixtures are listed at the respective
procedures. Liquid chromatography – mass spectrometry (LC-MS)
was carried out on a Waters e2695 HPLC system with a photodiode
array detector for detection at λ = 220 and 240 nm and a quantum
diode array detector for mass analysis. A binary solvent gradient of
acetonitrile and Milli-Q® water was applied on a C18 column
(2.1 × 50 mm × 2.6 µm). High resolution mass spectrometry
(HRMS) was carried out on a timsTOF Pro from the company
Brucker with ESI source. A warm-white LED lamp (PAR38,
2800K, 18 W) was used for light irradiation. Measurements of the
luminescence intensities were carried out with a Victor3 V 1420
Multilabel Plate Counter from the company Perkin Elmer in
triplicates. The operation “Chemolumineszenz” was applied and
the delay between repeats was 2 seconds. 96-well white clear-
From the experiments combined with chemiluminescence
measurements, it became obvious that the initial values
recorded during the chemiluminescence read-out (performed in
triplicates) are well suited for analysis and calculation of the
dioxetane concentration. As a result, a recording of the whole
ACS Paragon Plus Environment

Page 5 of 9
The Journal of Organic Chemistry
bottom microtest plates were used for the measurement of the
probes.
[(3-tert-Butyldimethylsilyloxy)phenyl]-2-hydroxyadamantan-2-
yl]methanone (2): To confirm the structure of hydroxy ketone 2, an
experiment was performed on a larger scale to isolate the product 2.
CH3OH (3.36 mL) was previously incubated with K2CO3 (12.7 mg,
0.92 mmol) for 15 minutes at room temperature. The supernatant
solution was transferred to a tube and used to dissolve dioxetane 1
(20.0 mg, 0.05 mmol) and 2-mercaptoethanol (3a) (7.00 µL,
0.10 mmol). After stirring for 3 hours at room temperature, the
solvent was removed under reduced pressure. The crude product
was purified by column chromatography (iso-hexane / ethyl
acetate = 20:1) to yield 2 as a clear oil. Rf value: 0.2 (iso-
1
2
3
4
5
6
7
8
Among the starting materials 1, 3a-i, the compounds 3a-c,e,g,h and
3i were commercially available. Dioxetane 139-41 and thiols 3d48
and 3f49 were prepared according to known methods. Among the
disulfides 4a-i, 4a,c,e,g were commercially available and were
used as authentic samples for comparison. Compounds 4b,d,f,h,i
were prepared according to known methods to be available for
comparison.20, 50, 51
Preparation and characterization of compounds.
9
hexane / ethyl acetate
=
20:1) [UV]. 1H-NMR (400 MHz,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Preparation of 1 was carried out according to known methods.39-41
4-[(3-tert-Butyldimethylsilyloxy)phenyl]-4-methoxyspiro[1,2-
dioxetane-3,2’-adamantane] (1): 1H NMR (400 MHz, CDCl3):
δ (ppm) = 0.19 (s, 6 H), 0.98 (s, 9 H), 1.23 – 1.90 (m, 12 H), 2.24
(br s, 1 H), 3.02 (br s, 1 H), 3.24 (s, 3 H), 6.93 (dd, J = 2.4 Hz,
J = 9.1 Hz, 1 H), 7.38 (t, J = 7.6 Hz, 1 H). 2 signals are missing.
1H-NMR (600 MHz, CD3OD): δ (ppm) = 0.21 (s, 6 H), 1.00 (s,
9 H), 1.08 (dt, J = 2.7 Hz, J = 13.1 Hz, 1 H), 1.26 (dd, J = 2.9 Hz,
J = 13.1 Hz, 1 H), 1.47 – 1.93 (m, 10 H), 2.17 (br s, 1 H), 2.97 (br
s, 1 H), 3.20 (s, 3 H), 6.93 (ddd, J = 1.0 Hz, J = 2.5 Hz, J = 8.1 Hz,
CD3OD): δ (ppm) = 0.22 (s, 6 H), 1.00 (s, 9 H), 1.55 – 1.82 (m,
10 H), 2.29 (br s, 2 H), 2.38 (d, J = 12.6 Hz, 2 H), 6.99 (ddd,
J = 1.0 Hz, J = 2.5 Hz, J = 8.1 Hz, 1 H), 7.28 (t, J = 7.7 Hz, 1 H),
7.55 (ddd, J = 1.1 Hz, J = 1.6 Hz, J = 7.7 Hz, 1 H), 7.56 – 7.62 (m,
1 H). DEPTQ (151 MHz, CD3OD): δ (ppm) = −4.3 (2 × CH3), 19.1
(Cq), 26.2 (3 × CH3), 28.5 (CH), 28.6 (CH), 33.4 (CH2), 35.6 (CH2),
35.9 (2 × CH), 38.6 (CH2), 39.3 (2 × CH2), 81.0 (Cq), 121.8 (CH),
123.3 (CH), 124.3 (CH), 130.1 (CH), 140.0 (Cq), 156.4 (Cq), 205.1
(Cq). HRMS (ESI) m/z: [M + H]+ calcd. for C23H35O3Si 387.2350;
Found: 387.2349.
1 H), 7.35 (t, J = 7.8 Hz, 1 H).
2 signals are missing.
DEPTQ (151 MHz, CD3OD): δ (ppm) = -4.2 (2 × CH3), 19.1 (Cq),
26.1 (3 × CH3), 27.4 (CH), 27.6 (CH), 32.6 (CH2), 33.0 (CH), 33.3
(CH2), 34.0 (CH2), 34.7 (CH), 35.7 (CH2), 37.4 (CH2), 50.1 (CH3),
96.5 (Cq), 113.1 (Cq), 122.6 (br, CH), 130.6 (br, CH), 137.6 (Cq),
157.2 (Cq). 2 signals are missing. The analytical data are in
agreement with those reported in literature.38
2-Hydroxyethyl disulfide (4a): 1H NMR (400 MHz, CD3OD):
δ (ppm) = 2.84 (t, J = 6.5 Hz, 4 H), 3.79 (t, J = 6.5 Hz, 4 H). This
data is in agreement with those obtained from an authentic sample
under identical conditions.
General Procedure for Table 2.
CD3OD (700 µL) was previously incubated with K2CO3
(0.02 mmol) for 15 minutes at room temperature. The supernatant
solution was transferred to a NMR tube and used to dissolve
dioxetane 1 (5.00 mg, 0.01 mmol) and the respective thiol 3a-d
(0.01 or 0.02 mmol). After incubation for 1 hour and 3 hours at
room temperature, a 1H NMR spectrum was recorded at room
temperature, respectively.
Procedures used for optimization (Table 1).
The equivalents of K2CO3 refer to 2-mercaptoethanol (3a).
Entry 1: Dioxetane
1
(5.00 mg, 0.01 mmol) and 2-
mercaptoethanol (3a) (1.40 µL, 0.02 mmol) were dissolved in
CD3OD (700 µL) in a NMR tube. After 3 hours at room
temperature, a 1H NMR spectrum was recorded at room
temperature.
Entries 2-6: CD3OD or (CD3)2SO (700 µL) was previously
incubated with K2CO3 (0.1 – 1.0 eq.) for 15 minutes at room
temperature. Subsequently, dioxetane 1 (5.00 mg, 0.01 mmol) and
2-mercaptoethanol (3a) (1.40 µL, 0.02 mmol) were dissolved in the
CD3OD supernatant in a NMR tube. After incubation of 3 hours at
room temperature, a 1H NMR spectrum was recorded at room
temperature.
Entry 7: CD3OD (700 µL) was previously incubated with K2CO3
(1.0 eq.) for 15 minutes at room temperature. Subsequently,
dioxetane 1 (5.00 mg, 0.01 mmol) and 2-mercaptoethanol (3a)
(1.40 µL, 0.02 mmol) were dissolved in a solvent mixture,
comprising of 80% CD3OD supernatant (560µL), 10% D2O
(70 µL) and 10% CD3CN (70 µL) in a NMR tube. After incubation
of 3 hours at room temperature, a 1H NMR spectrum was recorded
at room temperature.
Entries 8 and 9: Dioxetane 1 (5.00 mg, 0.01 mmol) and 2-
mercaptoethanol (3a) (1.40 µL, 0.02 mmol) were dissolved in a
solvent mixture, comprising of CD3OD (600 µL) and Tris buffer in
D2O (60 µL, 100 mM, pH = 8.6) in a NMR tube. After incubation
of 1.5 and 18 hours at room temperature, a 1H NMR spectrum was
recorded at room temperature, respectively.
Trans-4,5-dihydroxy-1,2-dithiane (4b): 1H NMR (400 MHz,
CD3OD): δ (ppm) = 2.87 (dd, J = 9.9 Hz, J = 13.4 Hz, 2 H), 3.03
(d, J = 13.3 Hz, 2 H), 3.46 – 3.54 (m, 2 H). This data is in
agreement with those obtained from an authentic sample under
identical conditions.
Dibenzyl disulfide (4c): 1H NMR (600 MHz, CD3OD):
δ (ppm) = 3.61 (s, 4 H), 7.21 – 7.33 (m, 10 H). This data is in
agreement with those obtained from an authentic sample under
identical conditions.
Captopril methyl ester disulfide (4d): Rf value: 0.2
(dichloromethane / methanol = 40:1)
[KMnO4].
1H-
NMR (400 MHz, CD3OD): δ (ppm) = 1.21 (d, J = 6.9 Hz, 6 H),
1.91 – 2.09 (m, 6 H), 2.19 – 2.31 (m, 2 H), 2.72 (dd, J = 5.1 Hz,
J = 13.3 Hz, 2 H)., 2.93 (dd, J = 9.0 Hz, J = 13.3 Hz, 2 H), 3.11 –
3.20 (m, 2 H), 3.70 (s, 6 H), 3.72 – 3.79 (m, 4 H), 4.54 (dd,
J = 4.1 Hz, J = 8.7 Hz, 2 H). DEPTQ (151 MHz, CD3OD):
δ (ppm) = 17.1 (2 × CH3), 25.7 (2 × CH2), 30.1 (2 × CH2), 39.0
(2 × CH), 42.1 (2 × CH2), 48.5 (2 × CH2), 52.7 (2 × CH3), 60.3
(2 × CH), 174.2 (2 × Cq), 175.9 (2 × Cq). MS (ESI) m/z: [M + H]+
461.23. HRMS (ESI) m/z: [M + H]+ calcd. for C20H33N2O6S2
461.1775; Found: 461.1776.
Determination of the amount of dissolved K2CO3 in CD3OD.
K2CO3 (37.9 mg, 0.27 mmol) was incubated in CH3OH (10.0 mL)
for 15 minutes at room temperature. The solvent of the CH3OH
supernatant was removed under reduced pressure to yield the
K2CO3 (15.2 mg). Referring to the NMR experiment (Table 1,
Entry 5), this means that in 700 µL CD3OH 1.03 mg K2CO3 is
dissolved. This corresponds to a molarity of 10.6 mM.
Procedures for large scale reactions to disulfides 4a,c,d.
Preparation of disulfide 4a: CH3OH (4.20 mL) was previously
incubated with K2CO3 (15.9 mg, 0.12 mmol.) for 15 minutes at
room temperature. The supernatant solution was transferred to a
tube and used to dissolve dioxetane 1 (25.0 mg, 0.06 mmol) and 2-
mercaptoethanol (3a) (8.40 µL, 0.12 mmol). After gentle stirring
(250 rpm) of 3 hours at room temperature, the solvent was removed
under reduced pressure under nitrogen atmosphere. The crude
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 6 of 9
product was purified by column chromatography (100%
J = 6.5 Hz, 4 H). This data is in agreement with those obtained
1
2
3
4
5
6
7
8
dichloromethane  dichloromethane / methanol = 40:130:1) to
give 4a (7.50 mg, 48.6 µmol, 81%) as a clear oil.
from an authentic sample under identical conditions.
N,N’-Diacetylcystamine (4i): 1H NMR (600 MHz, CD3OD):
δ (ppm) = 1.94 (s, 6 H), 2.82 (t, J = 6.7 Hz, 4 H), 3.47 (t,
J = 6.7 Hz, 4 H). This data is in agreement with those obtained
from an authentic sample under identical conditions.
Preparation of disulfide 4c: CH3OH l (16.8 mL) was previously
incubated with K2CO3 (66.6 mg, 0.48 mmol) for 15 minutes at
room temperature. The supernatant solution was transferred to a
tube and used to dissolve dioxetane 1 (100 mg, 0.24 mmol) and
phenylmethanethiol (3c) (56.4 µL, 0.48 mmol). After gentle
stirring (250 rpm) of 3 hours at room temperature, the solvent was
removed under reduced pressure under nitrogen atmosphere. The
crude product was purified by column chromatography (100% iso-
hexane  iso-hexane / ethyl acetate = 40:1) to give 4c (49.2 mg,
0.20 mmol, 83%) as a white solid.
Chemiluminescence measurement with 1 in the absence of
thiols (reference reaction).
CH3OH (700 µL) was previously incubated with K2CO3
(0.02 mmol) for 15 minutes at room temperature. The supernatant
solution was transferred to a NMR tube and used to dissolve
dioxetane 1 (5.00 mg, 0.01 mmol). The solution was transferred
into a centrifuge tube and methyl tert-butyl ether (1.40 mL), water
(3.00 mL), and aqueous barium chloride solution (0.5 M, 20 drops)
were added. The organic phase (200 µL) was diluted with
methyl tert-butyl ether (1/10) and was subsequently washed with
water (2 mL). The chemiluminescent decay of 1 was triggered by
the addition of the MTBE phase (7.16 µL) to a solution of dimethyl
sulfoxide (183 µL) and potassium carbonate saturated methanolic
solution (10 µL) in a well. Light emission was measured as
described above. The basic methanolic solution was prepared as
mentioned above and the final concentration of 1 was 25 µM per
well.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Preparation of disulfide 4d: CH3OH (4.20 mL) was previously
incubated with K2CO3 (15.9 mg, 0.12 mmol.) for 15 minutes at
room temperature. The supernatant solution was transferred to a
tube and used to dissolve dioxetane 1 (25.0 mg, 0.06 mmol) and
captopril methyl ester (3d) (27.8 mg, 0.12 mmol). After gentle
stirring (250 rpm) of 3 hours at room temperature, the solvent was
removed under reduced pressure under nitrogen atmosphere. The
crude product was purified by column chromatography (iso-
hexane / ethyl acetate = 1:11:2 100% ethyl acetate) to give 4d
(23.2 mg, 0.05 mmol, 83%) as a clear oil.
Chemiluminescence measurement with dioxetane
disulfide 4e (verification of extraction procedure).
1 and
General Procedure for Figure 1.
Cold CD3OD (700 µL) was previously incubated with K2CO3
(0.02 mmol) for 15 minutes. The supernatant solution was
transferred to a NMR tube and used to dissolve dioxetane 1
(5.00 mg, 0.01 mmol) and the respective thiol 3a,e,f,g
(0.02 mmol). The 1H NMR spectra were recorded continuously at
0 °C.
CH3OH (700 µL) was previously incubated with K2CO3
(0.02 mmol) for 15 minutes at room temperature. The supernatant
solution was transferred to a NMR tube and used to dissolve
dioxetane 1 (5.00 mg, 0.01 mmol) and N,N’-diacetyl-L-cystine
dimethyl ester (4e) (0.01 mmol). The solution was transferred into
a centrifuge tube and methyl tert-butyl ether (1.40 mL), water
(3.00 mL), and aqueous barium chloride solution (0.5 M, 20 drops)
were added. The organic phase (200 µL) was diluted with
methyl tert-butyl ether (1/10) and was subsequently washed with
water (2 mL). The chemiluminescent decay of 1 was triggered by
the addition of the MTBE phase (7.16 µL) to a solution of dimethyl
sulfoxide (183 µL) and potassium carbonate saturated methanolic
solution (10 µL) in a well. Light emission was measured as
described above. The basic methanolic solution was prepared as
mentioned above.
N,N‘-Diacetyl-L-cystine dimethyl ester (4e):1H NMR (400 MHz,
CD3OD): δ (ppm): 2.00 (s, 6 H), 2.97 (dd, J = 8.7 Hz, J = 14.0 Hz,
2 H), 3.21 (dd, J = 5.0 Hz, J = 14.0 Hz, 2 H), 3.75 (s, 3 H), 4.74
(dd, J = 5.0 Hz, J = 8.6 Hz, 2 H). This data is in agreement with
those obtained from an authentic sample under identical conditions.
N,N‘-Diacetyl-L-cystine bisamide (4f): 1H NMR (600 MHz,
CD3OD): δ (ppm): 2.01 (s, 6 H), 2.91 (dd, J = 9.2 Hz, J = 13.9 Hz,
2 H), 3.22 (dd, J = 4.9 Hz, J = 13.9 Hz, 2 H), 4.71 (dd, J = 4.9 Hz,
J = 9.2 Hz, 2 H). This data is in agreement with those obtained
from an authentic sample under identical conditions.
General procedure for Figure 3.
The chemiluminescence measurement with 1 in the absence of
thiols was carried out for three different concentrations (reference
reactions used for calibration).
1
L-cystine dimethyl ester (4g): H NMR (400 MHz, CD3OD):
δ (ppm): 2.97 (dd, J = 6.8 Hz, J = 13.8 Hz, 2 H), 3.11 (dd,
J = 5.4 Hz, J = 13.8 Hz, 2 H), 3.75 (s, 3 H), 3.79 (dd, J = 5.5 Hz,
J = 6.8 Hz, 2 H). This data is in agreement with those obtained
from an authentic sample under identical conditions.
CH3OH (700 µL) was previously incubated with K2CO3
(0.02 mmol) for 15 minutes at room temperature. The supernatant
solution was transferred to a NMR tube and used to dissolve
dioxetane 1 (5.00 mg, 0.01 mmol) The solution was transferred into
a centrifuge tube and methyl tert-butyl ether (1.40 mL), water
(3.00 mL) and aqueous barium chloride solution (0.5 M, 20 drops)
were added. The organic phase (200 µL) was diluted with
methyl tert-butyl ether (1/10) and was subsequently washed with
water (2 mL). The organic phase was used unchanged or was partly
further diluted (1/1 and 1/4) to give the three desired
concentrations. The chemiluminescent decay of 1 was triggered by
the addition of the MTBE phase (7.16 µL) to a solution of dimethyl
sulfoxide (183 µL) and potassium carbonate saturated methanolic
solution (10 µL) in a well. Light emission was measured as
described above. The basic methanolic solution was prepared as
mentioned above.
General Procedure for competition experiments (Figure 2).
Cold CD3OD (700 µL) was previously incubated with K2CO3
(0.02 mmol) for 15 minutes. The supernatant solution was
transferred to a NMR tube and used to dissolve dioxetane 1
(5.00 mg, 0.01 mmol), thiol 3a, and cysteine 3e (each 0.02 mmol).
Additional K2CO3 (1.03 mg, 7.45 µmol) was added to the solution
1
in the tube. The H NMR spectra were recorded continuously at
0 °C. Analysis of the product distribution is based on 1H NMR data
of previously prepared 4a and 4e. The same reaction conditions
were used for the competition experiment with thiol 3h and 3i,
respectively.
Bis(2-acetoxyethyl) disulfide (4h):1H NMR (600 MHz, CD3OD):
δ (ppm) = 2.05 (s, 6 H), 2.96 (t, J = 6.5 Hz, 4 H), 4.32 (t,
Chemiluminescence measurement with 1 and 3e at ratios of 3:4
and 1:2.
ACS Paragon Plus Environment

Page 7 of 9
The Journal of Organic Chemistry
Cold CH3OH (700 µL) was previously incubated with K2CO3
1
2
3
4
5
6
7
8
(0.02 mmol) for 15 minutes. The supernatant solution was
transferred to a NMR tube and used to dissolve dioxetane 1
(5.00 mg, 0.01 mmol) and N-acetyl-L-cysteine methyl ester (3e)
(0.015 mmol or 0.02 mmol). The solution was incubated for 75
minutes at 0 °C. The solution was transferred into a centrifuge tube
and methyl tert-butyl ether (1.40 mL), water (3.00 mL), and
aqueous barium chloride solution (0.5 M, 20 drops) were added.
The organic phase (200 µL) was diluted with methyl tert-butyl
ether (1/10) and was subsequently washed with water (2 mL). The
chemiluminescent decay of 1 was triggered by the addition of the
MTBE phase (7.16 µL) to a solution of dimethyl sulfoxide
(183 µL) and potassium carbonate saturated methanolic solution
(10 µL) in a standard 96-well plate. Light emission was measured
as described above. The basic methanolic solution was prepared as
mentioned above.
REFERENCES
(1) Pace, N. J.; Weerapana, E. Diverse functional roles of reactive
cysteines. ACS Chem. Biol. 2012, 8, 283-296.
(2) Bindoli, A.; Fukuto, J. M.; Forman, H. J. Thiol chemistry in
peroxidase catalysis and redox signaling. Antioxid. Redox Signal.
2008, 10, 1549-1564.
(3) Faccio, G.; Nivala, O.; Kruus, K.; Buchert, J.; Saloheimo, M.
Sulfhydryl oxidases: sources, properties, production and
applications. Appl. Microbiol. Biotechnol. 2011, 91, 957-966.
(4) Ruano, J. L. G.; Parra, A.; Alemán, J. Efficient synthesis of
disulfides by air oxidation of thiols under sonication. Green Chem.
2008, 10, 706-711.
(5) Witt, D. Recent developments in disulfide bond formation.
Synthesis 2008, 2491-2509.
(6) Arterburn, J. B.; Perry, M. C.; Nelson, S. L.; Dible, B. R.;
Holguin, M. S. Rhenium-catalyzed oxidation of thiols and
disulfides with sulfoxides. J. Am. Chem. Soc. 1997, 119, 9309-
9310.
(7) Abu-Omar, M. M.; Khan, S. I. Molecular rhenium (V)
oxotransferases: oxidation of thiols to disulfides with sulfoxides.
The case of substrate-inhibited catalysis. Inorg. Chem. 1998, 37,
4979-4985.
(8) Ali, M. H.; McDermott, M. Oxidation of thiols to disulfides with
molecular bromine on hydrated silica gel support. Tetrahedron
Lett. 2002, 43, 6271-6273.
(9) Ghafuri, H.; Hashemi, M. M. A simple, economical, and catalyst-
free oxidation of thiols to disulfides. J. Sulfur Chem. 2009, 30, 578-
580.
(10) Kesavan, V.; Bonnet-Delpon, D.; Bégué, J.-P. Oxidation in fluoro
alcohols: mild and efficient preparation of disulfides from thiols.
Synthesis 2000, 223-225.
(11) Khazaei, A.; Zolfigol, M. A.; Rostami, A. 1, 3-dibromo-5, 5-
dimethylhydantoin [DBDMH] as an efficient and selective agent
for the oxidation of thiols to disulfides in solution or under solvent-
free conditions. Synthesis 2004, 2959-2961.
(12) Alam, A.; Takaguchi, Y.; Tsuboi, S. Simple, extremely fast, and
high‐yielding oxidation of thiols to disulfides. Synth. Commun.
2005, 35, 1329-1333.
(13) Akdag, A.; Webb, T.; Worley, S. D. Oxidation of thiols to
disulfides with monochloro poly (styrenehydantoin) beads.
Tetrahedron Lett. 2006, 47, 3509-3510.
(14) Leino, R.; Lönnqvist, J.-E. A very simple method for the
preparation of symmetrical disulfides. Tetrahedron Lett. 2004, 45,
8489-8491.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Monitoring of the reaction course: comparison of NMR yields
and chemiluminescence read-out after quenching.
Cold CH3OH (700 µL) was previously incubated with K2CO3
(0.02 mmol) for 15 minutes. The supernatant solution was
transferred to a NMR tube and used to dissolve dioxetane 1
(5.00 mg, 0.01 mmol) and N-acetyl-L-cysteine methyl ester (3e)
(0.02 mmol). The solution was incubated for 22.5, 43.5 or 64.5
minutes at 0 °C. After incubation for the time indicated, the
solution was transferred into a centrifuge tube and methyl tert-butyl
ether (1.40 mL), water (3.00 mL), and aqueous barium chloride
solution (0.5 M, 20 drops) were added. The organic phase (200 µL)
was diluted with methyl tert-butyl ether (1/10) and was
subsequently washed with water (2 mL). The chemiluminescent
decay of 1 was triggered by the addition of the MTBE phase
(7.16 µL) to a solution of dimethyl sulfoxide (183 µL) and
potassium carbonate saturated methanolic solution (10 µL) in a
standard 96-well plate. Light emission was measured as described
above. The basic methanolic solution was prepared as mentioned
above.
ASSOCIATED CONTENT
Supporting Information
Preliminary results and NMR spectra. The Supporting Information
is available free of charge on the ACS Publications website.
(15) Hajipour, A. R.; Mallakpour, S. E.; Adibi, H. Selective and
efficient
oxidation
of
sulfides
and
thiols
with
benzyltriphenylphosphonium peroxymonosulfate in aprotic
solvent. J. Org. Chem. 2002, 67, 8666-8668.
AUTHOR INFORMATION
(16) Iranpoor, N.; Zeynizadeh, B. Air oxidative coupling of thiols to
disulfides catalyzed by Fe (III)/NaI. Synthesis 1999, 49-50.
(17) Shah, S. T. A.; Khan, K. M.; Fecker, M.; Voelter, W. A novel
method for the syntheses of symmetrical disulfides using CsF–
Celite as a solid base. Tetrahedron Lett. 2003, 44, 6789-6791.
(18) Liu, Y.; Wang, H.; Wang, C.; Wan, J.-P.; Wen, C. Bio-based
green solvent mediated disulfide synthesis via thiol couplings free
of catalyst and additive. RSC Adv. 2013, 3, 21369-21372.
(19) Sridhar, M.; Vadivel, S. K.; Bhalerao, U. T. Novel method for
preparation of symmetric disulfides from thiols using enzyme
catalysis. Synth. Commun. 1998, 28, 1499-1502.
(20) Bettanin, L.; Saba, S.; Galetto, F. Z.; Mike, G. A.; Rafique, J.;
Braga, A. L. Solvent-and metal-free selective oxidation of thiols to
disulfides using I2/DMSO catalytic system. Tetrahedron Lett.
2017, 58, 4713-4716.
(21) Ellman, G. L. Tissue sulfhydryl groups. Arch. Biochem. Biophys.
1959, 82, 70-77.
Corresponding Author
*E-mail: markus.heinrich@fau.de; Phone: +49-9131-85-65670
ORCID
Caroline S. Sauer: 0000-0003-0401-6803
Johannes Köckenberger: 0000-0001-8707-9054
Markus R. Heinrich: 0000-0001-7113-2025
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
The authors would like to acknowledge the financial support of
Emerging Fields Initiative (EFI) by the FAU Erlangen-Nürnberg.
We are also grateful for the support of the Graduate School
Molecular Science (GSMS) and for experimental assistance by Ms.
Anja Hubert (Pharmaceutical Chemistry, FAU Erlangen-
Nürnberg).
(22) Annis, I.; Chen, L.; Barany, G. Novel solid-phase reagents for
facile formation of intramolecular disulfide bridges in peptides
under mild conditions. J. Am. Chem. Soc. 1998, 120, 7226-7238.
(23) Hargittai, B.; Annis, I.; Barany, G. Application of solid-phase
Ellman's reagent for preparation of disulfide-paired isomers of α-
conotoxin SI. Lett. Pept. Sci. 2000, 7, 47-52.
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 8 of 9
(24) Zhu, J.; Dhimitruka, I.; Pei, D. 5-(2-Aminoethyl) dithio-2-
(46) Adam, W.; Heil, M. Reaction of 1, 2-dioxetanes with heteroatom
nucleophiles: adduct formation by nucleophilic attack at the
peroxide bond. J. Am. Chem. Soc. 1992, 114, 5591-5598.
(47) Denes, F.; Pichowicz, M.; Povie, G.; Renaud, P. Thiyl radicals in
organic synthesis. Chem. Rev. 2014, 114, 2587-2693.
(48) Moss, G. P.; Gullick, D. R.; Cox, P. A.; Alexander, C.; Ingram,
M. J.; Smart, J. D.; Pugh, W. J. Design, synthesis and
characterization of captopril prodrugs for enhanced percutaneous
absorption. J. Pharm. Pharmacol. 2006, 58, 167-177.
nitrobenzoate as a more base-stable alternative to Ellman's reagent.
Org. Lett. 2004, 6, 3809-3812.
(25) Yang, X.; Gelfanov, V.; Liu, F.; DiMarchi, R. Synthetic route to
human relaxin-2 via iodine-free sequential disulfide bond
formation. Org. Lett. 2016, 18, 5516-5519.
(26) Riener, C. K.; Kada, G.; Gruber, H. J. Quick measurement of
protein sulfhydryls with Ellman's reagent and with 4, 4′-
dithiodipyridine. Anal. Bioanal. Chem. 2002, 373, 266-276.
(27) Grassetti, D. R.; Murray Jr, J. F. Determination of sulfhydryl
groups with 2, 2′-or 4, 4′-dithiodipyridine. Arch. Biochem.
Biophys. 1967, 119, 41-49.
1
2
3
4
5
6
7
8
(49) Warner, J. C.; Cheruku, S.; Thota, S.; Lee, J. W. Method for the
preparation of N-acetyl cysteine amide. WO2015148880A1,
October 01, 2015.
9
(50) Nakae, Y.; Kusaki, I.; Sato, T. Lithium perchlorate catalyzed
acetylation of alcohols under mild reaction conditions. Synlett
2001, 1584-1586.
(51) Van den Broek, L. A. G. M.; Lazaro, E.; Zylicz, Z.; Fennis, P. J.;
Missler, F. A. N.; Lelieveld, P.; Garzotto, M.; Wagener, D. J. T.;
Ballesta, J. P. G.; Ottenheijm, H. C. J. Lipophilic analogs of
sparsomycin as strong inhibitors of protein synthesis and tumor
growth: a structure-activity relationship study. J. Med. Chem.
1989, 32, 2002-2015.
(28) Winther, J. R.; Thorpe, C. Quantification of thiols and disulfides.
Biochim. Biophys. Acta 2014, 1840, 838-846.
(29) Sabelle, S.; Renard, P.-Y.; Pecorella, K.; de Suzzoni-Dézard, S.;
Créminon, C.; Grassi, J.; Mioskowski, C. Design and synthesis of
chemiluminescent probes for the detection of cholinesterase
activity. J. Am. Chem. Soc. 2002, 124, 4874-4880.
(30) Maeda, H.; Matsuno, H.; Ushida, M.; Katayama, K.; Saeki, K.;
Itoh, N. 2, 4‐Dinitrobenzenesulfonyl fluoresceins as fluorescent
alternatives to Ellman's reagent in thiol‐quantification enzyme
assays. Angew. Chem. Int. Ed. 2005, 44, 2922-2925.
(31) Adam, W.; Epe, B.; Schiffmann, D.; Vargas, F.; Wild, D. Facile
reduction of 1, 2‐dioxetanes by thiols as potential protective
measure against photochemical damage of cellular DNA. Angew.
Chem. Int. Ed. 1988, 27, 429-431.
(32) Schaap, A. P.; Handley, R. S.; Giri, B. P. Chemical and enzymatic
triggering of 1, 2-dioxetanes. 1: aryl esterase-catalyzed
chemiluminescence from a naphthyl acetate-substituted dioxetane.
Tetrahedron Lett. 1987, 28, 935-938.
(33) Schaap, A. P.; Sandison, M. D.; Handley, R. S. Chemical and
enzymatic triggering of 1, 2-dioxetanes. 3: alkaline phosphatase-
catalyzed chemiluminescence from an aryl phosphate-substituted
dioxetane. Tetrahedron Lett. 1987, 28, 1159-1162.
(34) Hananya, N.; Shabat, D. A Glowing Trajectory between Bio‐and
Chemiluminescence: From Luciferin‐Based Probes to Triggerable
Dioxetanes. Angew. Chem. Int. Ed. 2017, 56, 16454-16463.
(35) Koo, J.-Y.; Schuster, G. B. Chemically initiated electron exchange
luminescence. A new chemiluminescent reaction path for organic
peroxides. J. Am. Chem. Soc. 1977, 99, 6107-6109.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(36) Schaap, A. P.; Chen, T.-S.; Handley, R. S.; DeSilva, R.; Giri, B.
P. Chemical and enzymatic triggering of 1, 2-dioxetanes. 2:
fluoride-induced
chemiluminescence
from
tert-
butyldimethylsilyloxy-substituted dioxetanes. Tetrahedron Lett.
1987, 28, 1155-1158.
(37) Wuts, P. G. M.; Greene, T. W., Greene's Protective Groups in
Organic Synthesis. 4th ed.; John Wiley & Sons: Hoboken, NJ,
2006.
(38) Bastos, E. L.; Ciscato, L. F. M. L.; Weiss, D.; Beckert, R.; Baader,
W. J. Comparison of convenient alternative synthetic approaches
to 4-[(3-tert-butyldimethylsilyloxy) phenyl]-4-methoxyspiro [1, 2-
dioxetane-3, 2′-adamantane]. Synthesis 2006, 11, 1781-1786.
(39) Kwon, S.; Myers, A. G. Synthesis of (−)-quinocarcin by directed
condensation of α-amino aldehydes. J. Am. Chem. Soc. 2005, 127,
16796-16797.
(40) Juo, R.-R.; Edwards, B.; Gee, M.; Wang, Z.; Skaare, K.
Chemiluminescent compositions, methods, assays and kits for
oxidative enzymes. WO2010101839A2, March 01, 2010.
(41) Andronico, L. A.; Quintavalla, A.; Lombardo, M.; Mirasoli, M.;
Guardigli, M.; Trombini, C.; Roda, A. Synthesis of 1, 2‐dioxetanes
as thermochemiluminescent labels for ultrasensitive bioassays:
rational prediction of olefin photooxygenation outcome by using a
chemometric approach. Chem. Eur. J. 2016, 22, 18156-18168.
(42) Annis, I.; Hargittai, B.; Barany, G. [10] Disulfide bond formation
in peptides. Methods Enzymol. 1997, 289, 198-221.
(43) Adam, W.; Baader, W. J. Effects of methylation on the thermal
stability and chemiluminescence properties of 1, 2-dioxetanes. J.
Am. Chem. Soc. 1985, 107, 410-416.
(44) Wilson, T.; Schaap, A. P. Chemiluminescence from cis-diethoxy-
1, 2-dioxetane. Unexpected effect of oxygen. J. Am. Chem. Soc.
1971, 93, 4126-4136.
(45) Misra, H. P. Generation of superoxide free radical during the
autoxidation of thiols. J. Biol. Chem. 1974, 249, 2151-2155.
ACS Paragon Plus Environment

Page 9 of 9
The Journal of Organic Chemistry
1
2
3
4
5
6
no h
O

O
MeO
OH
O
TBSO
TBSO
chemi-
luminescence
7
8
9
2 R-SH
R-S-S-R
clean oxidation
mild conditions
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment