Design and synthesis of new mass tags for matrix-free laser desorption ionization mass spectrometry (LDI-MS) based on 6,11-dihydrothiochromeno[4,3-b]indole

Article abstract of DOI:10.1016/j.tet.2016.07.052

We have rationally designed and synthesized new mass tags that are heterolytically cleavable upon UV-irradiation; these tags are based on a 6,11-dihydrothiochromeno[4,3-b]indole skeleton. Unless exposed to UV light, the dithioacetal group maintained its stability under normal conditions. After chemical conjugation of the mass tags with biomolecules of interest, such as proteins, the resulting conjugates efficiently and selectively generated the corresponding mass-tag fragment ions without the aid of a matrix under laser desorption/ionization (LDI) conditions. We envision that these new dithioacetal-based tags would provide a new platform of the so-called matrix-free laser desorption ionization mass spectrometry (LDI-MS), that would allow multiple detection of biomarkers with high sensitivity and selectivity. The limit of detection (LOD) of these tags was measured to be 5?fmol in the case of non-conjugated mass tag themselves and 2.8?fmol in the case of mass-tag-conjugated myoglobin.

Full text of DOI:10.1016/j.tet.2016.07.052

Tetrahedron 72 (2016) 5612e5619
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Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Design and synthesis of new mass tags for matrix-free laser
desorption ionization mass spectrometry (LDI-MS) based on
6,11-dihydrothiochromeno[4,3-b]indole
*
Nana Kang, Jung-Min Lee, Aeran Jeon, Han Bin Oh, Bongjin Moon
Department of Chemistry, Sogang University, Seoul 04107, South Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 June 2016
Received in revised form 16 July 2016
Accepted 19 July 2016
Available online 20 July 2016
We have rationally designed and synthesized new mass tags that are heterolytically cleavable upon UV-
irradiation; these tags are based on a 6,11-dihydrothiochromeno[4,3-b]indole skeleton. Unless exposed
to UV light, the dithioacetal group maintained its stability under normal conditions. After chemical
conjugation of the mass tags with biomolecules of interest, such as proteins, the resulting conjugates
efficiently and selectively generated the corresponding mass-tag fragment ions without the aid of
a matrix under laser desorption/ionization (LDI) conditions. We envision that these new dithioacetal-
based tags would provide a new platform of the so-called matrix-free laser desorption ionization
mass spectrometry (LDI-MS), that would allow multiple detection of biomarkers with high sensitivity
and selectivity. The limit of detection (LOD) of these tags was measured to be 5 fmol in the case of non-
conjugated mass tag themselves and 2.8 fmol in the case of mass-tag-conjugated myoglobin.
Ó 2016 Elsevier Ltd. All rights reserved.
Keywords:
Matrix-free LDI-MS
Photocleavable mass tag
Thiochromene
Pummerer rearrangement
MALDI-TOF-MS
1. Introduction
employed under matrix-free conditions (see Fig. 1b). In 1999,
Shchepinov and co-workers have firstly reported the use of trityl
Matrix-assisted laser desorption ionization mass spectrometry
(MALDI-MS) is a powerful tool for analyzing large biomolecules1
and has been used in various clinical diagnostic applications.2 Al-
though the MALDI method enables direct ionization of high-
molecular-weight biomolecules and their subsequent identifica-
tion by mass spectrometry, the technique suffers from the inherent
limitation arising from the required use of a matrix. Typically, the
matrix consists of small molecules, such as 3,5-dimethoxy-4-
group-based LDI mass tags for encoding in combinatorial oligo-
nucleotide synthesis.6 Since then, trityl type LDI tags have been
employed in many applications such as mass spectrometry imag-
ing,7 mass tagging in solid phase synthesis of oligonucleotides,8 the
hydroxycinnamic
acid
(sinapinic
acid),3
a-cyano-4-
hydroxycinnamic acid (CHCA),4 and 2,5-dihydroxybenzoic acid
(DHB),5 that assist the ionization of analytes upon UV laser irradi-
ation. The matrix can thereby contaminate the mass signals in the
low-molecular-weight region. Furthermore, because the use of the
matrix ensures that all heavy biomolecules in the sample are ion-
ized, the mass spectrum not only shows the mass information of
the specific biomolecules of interest but also may contain those of
other heavy contaminants unless the sample is highly pure (see
Fig.1a). To circumvent this problem, a new technique called matrix-
free laser desorption ionization mass spectrometry (LDI-MS) was
suggested, where heterolytically photocleavable mass tags were
Fig. 1. Difference between the detection principles of (a) conventional MALDI-MS and
of (b) matrix-free LDI-MS. (a) In MALDI-MS, background contamination from the
matrix is the inherent limitation; (b) by contrast, in LDI-MS, background contamina-
tion is avoided because no matrix is used.
* Corresponding author. Fax: þ82 2 701 0967; e-mail address: bjmoon@sogang.
ac.kr (B. Moon).
http://dx.doi.org/10.1016/j.tet.2016.07.052
0040-4020/Ó 2016 Elsevier Ltd. All rights reserved.

N. Kang et al. / Tetrahedron 72 (2016) 5612e5619
5613
detection of single nucleotide polymorphism,9 calibration of mass
2. Results and discussion
spectrometers,10 signal amplification in MALDI-MS,11 and immu-
noassays for detecting intact protein antigens using a MALDI-MS
instrument.12 Recently, Topolyan and co-workers reported a new
mass tag based on another stable carbocation species, triphe-
nylcyclopropenylium ion, and demonstrated its high sensitivity in
both in ESI and LDI mass spectrometry.13
Another type of photocleavable mass tag for LDI-MS was de-
veloped by Maki and co-workers in 2007, where p-alkoxybenzoate
was employed as the core group. Chemical conjugation of this tag to
an analyte allowed the generation of the negative ion bearing the
analyte and enabled detection of the analyte by matrix-free LDI-
MS.14 It is noteworthy that while trityl group-based mass tags
generate positive ions, p-alkoxybenzoate group-based tags gener-
ate negative ions.
Ortho-nitrobenzyl (ONB) group is a well-known photo-cleavable
group and has been introduced to several mass tags for MALDI-
MS.15 However, the role of ONB group in these tags was simply
homolytic chemical bond cleavage upon UV-irradiation. The use of
matrix was still required to promote the ionization of the photo-
chemically cleaved tags.
One of the most promising future applications of LDI-MS mass
tags represents multiple detection of biomarkers via immunoas-
says. With respect to the preparation of ideal mass tags for multi-
plex analysis to detect many different biomarkers, preparing
a library of mass tags with a ‘mass-variation group’ should be easy.
Fig. 2 describes the working principle of the mass-tagged antibody-
based multiple detection of biomarkers using the matrix-free LDI-
MS technique. The overall procedure is similar to that of conven-
tional antibody-based biomarker detection, where a surface-
immobilized capture antibody and a signaling detection antibody
are used to detect a target antigen (biomarker). If the detection
antibodies are conjugated with the photocleavable mass tags with
specific molecular masses, the matrix-free LDI should immediately
generate a set of detectable ions and the TOF MS of the ions should
specify the identity and amount of each detection antibody. When
a larger number of mass tags are employed, an increased number of
biomarkers can be detected simultaneously. Therefore, further ef-
forts to design improved tag molecules for multiplexing are cer-
tainly desired.
2.1. Photocleavable LDI-MS mass tag design
The design principle of the mass tag is shown in Fig. 3. Most
importantly, the tag should have a photocleavable part that favor-
ably cleaves in heterolytic fashion so that the resulting ions can be
detected by MS. To promote this phenomenon, the tag should
contain an appropriate chromophore that can absorb UV light and
direct its energy toward bond cleavage. A mass variation group
should also be present in the ion for detection, and the other ionic
side should bear a reactive group16 that allows the conjugation of
the tag with biomolecules. With these design principles in mind,
we chose the 2-(alkylthio)-2H-thiocromene skeleton (1) as the UV
chromophore (see Fig. 3). We envision that UV absorption by the
2H-thiochromene core could induce heterolytic cleavage of CeS
bonds, resulting in thiochromenium ion (2) and alkyl thiolate (RSꢀ),
because this process is facilitated by the aromatization of 2H-thi-
ochromene into thiochromenium ion. Furthermore, we conjectured
that the UV absorbance of 1 could be tuned by modifying the fused
aromatic group (Ar). Notably, thiochromene dithioacetal 1 should
be sufficiently stable under physiological conditions so that it is not
ionized until it is subjected to the LDI conditions.
Fig. 3. Design principle of a heterolytically photocleavable LDI mass tag reagent.
Initially, two thiochromene dithioacetals, 3 and 4 (Fig. 4), were
prepared via modified versions of procedures reported in the lit-
erature;17 these dithioacetals were subsequently subjected to LDI-
MS. Whereas 4 exhibited a weak mass signal under LDI condi-
tions, 3 did not exhibit any signal under the same conditions (see
Fig. S1 and S2 in Supplementary data). The para-OMe group on the
aryl ring appeared to enhance the stability of the thiochromenium
ion. On the basis of this observation, we attempted to stabilize the
thiochromenium ion by introducing an indole ring, as shown in 5a
(Fig. 4). By altering the benzene ring with an indole ring, we an-
ticipated that both the UV absorption at 355 nm, which is the
typical UV laser wavelength used in a MALDI instrument, and the
stability of the resulting thiochromenium ion should be enhanced.
Indeed, the absorbance of 5a at 355 nm was 8-fold greater than that
of 4, whereas those of 3 and 4 were negligible, as shown in the
UVevis spectra (see Fig. 5). To our delight, 5a exhibited 75-fold
higher photocleavage efficiency than 4 under matrix-free LDI-MS
conditions (vide infra).
Fig. 2. Schematic of the multiplex detection strategy using three different photo-
cleavable mass tags by the matrix-free LDI-MS method.
In this article, we wish to report new mass tags for LDI-MS based
on a new chemical scaffold; 6,11-dihydrothio-chromeno[4,3-b]in-
dole. The tags were rationally designed and synthesized consider-
ing synthetic flexibility, photochemical property, and improved
solubility under bio-conjugation conditions. The sensitivity of the
tags under the LDI-MS conditions was satisfactory enough so that
they can be used for biomarker detection in the future.

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N. Kang et al. / Tetrahedron 72 (2016) 5612e5619
substrate because of their thiophilic ‘soft’ character.20 For example,
oxidation and the concomitant rearrangement of 10c to 12c could
be achieved in a single step in high yield by treating with
PhI(OTFA)2. The impurities in the crude thiochromenium salts were
removed by washing with diethyl ether, and the resulting residues
were sufficiently pure for the next reaction. Thiochromenium salts
12aed were then trapped with 3-mercaptopropionic acid in ace-
tonitrile in the presence of Na2CO3. The use of a base such as
Na2CO3 was critical in this case. Otherwise, the reaction did not
proceed to completion even when excess thiol was employed. We
suspect that the reaction appears to be reversible under acidic
conditions in which dithioacetals are re-protonated by the residual
trifluoroacetic acid (TFA) and subsequent CeS bond re-cleavage
occurs. The resulting mixture was simply purified by being
washed with H2O and aq NH4Cl; these products were then sub-
jected to the next step without further purification. Finally, the
carboxylic acid groups were transformed into N-hydrox-
ysuccinimide active esters (NHS ester) 5aed by the Steglich ester-
Fig. 4. Structures of mass tags 3, 4, and 5a in which fused aryl groups are varied.
ification21
carbodiimide hydrochloride (EDCI).
using
N-(3-dimethyl
aminopropyl)-N’-ethyl-
Fig. 5. UVevis spectra of compounds 3, 4, and 5a in CH2Cl2 solution (5.00ꢁ10ꢀ5 M).
Molar extinction coefficients ( 3 ) at 355 nm are; 149 Mꢀ1 cmꢀ1 (3), 1200 Mꢀ1 cmꢀ1 (4),
and 9430 Mꢀ1 cmꢀ1 (5a), respectively.
2.2. Synthesis of photocleavable LDI-MS mass tags
The syntheses of 5a and its mass-varied derivatives 5bed are
described in Scheme 1.18 The indole moiety was conveniently in-
troduced via the Fischer indole synthesis reaction between ketone
6 and phenyl hydrazine (7).18b In this step, trimethylsilyl chloride
(TMSCl) was added to the equimolar mixture of 6 and 7 in EtOH to
generate anhydrous HCl in situ, and the mixture was heated at
reflux to give 6,11-dihydro-thiochromeno[4,3-b]indole (8) in good
yield (96%). m-CPBA oxidation of 8 afforded sulfoxide 9 in 78% yield.
In contrast to our expectation, the subsequent N-alkylation attempt
of 9 provided undesired
a-alkylation products in addition to the
desired N-alkylation products, likely because of the
a-proton of the
sulfoxide becoming more acidic than that of the sulfide. Therefore,
N-alkylation was performed prior to S-oxidation. N-Alkylation of
indole 8 was carried out with various alkyl halides using sodium
hydride as a base to give N-alkylated thiochromenoindole de-
rivatives 10aed in excellent yields (70e98%). In this case, we chose
four alkyl groups with lower masses, i.e., methyl, ethyl, propyl, and
butyl groups, to minimize the increase in hydrophobicity. Because
a large number of alkyl halides are commercially available and are
relatively inexpensive, mass variation of the mass tags can be easily
extended to a wide range of molecules. Sulfides 10aed could be
easily oxidized to sulfoxides 11aed with m-chloroperbenzoic acid
(m-CPBA) in good yields. In the next step, conversion of 11-alkyl-
6,11-dihydro-thiochromeno[4,3-b]indole 5-oxide (11) into thio-
chromenium salt 12 by the Pummerer rearrangement19 was carried
out using trifluoroacetic anhydride (TFAA). Treatment of sulfoxides
11aed with TFAA (3 equiv) in acetonitrile provided the thio-
chromenium ion pair 12aed in 5 min. In this case, at least 3 equiv of
TFAA was required to complete the reaction. After excess TFAA and
solvent were removed under reduced pressure, anhydrous diethyl
ether was added to the mixture to obtain thiochromenium salts
12aed as deep-yellow solids; these products were subjected to the
next reaction without further purification. Some hypervalent iodine
reagents, such as PhI(OTFA)2 and PhI(CN)OTf, are known to pro-
mote the Pummerer rearrangement starting from the sulfide
Scheme 1. Synthetic routes for new photocleavable mass tags 5aed.

N. Kang et al. / Tetrahedron 72 (2016) 5612e5619
5615
2.3. LDI-MS study of the photocleavable mass tags
difference can be sufficiently resolved by TOF MS analysis in this
mass region, multiplex analyses should be achievable. The sensi-
tivity of the mass tags was also tested, and the limit of detection
(LOD) of mass tag 5d, which has the highest efficiency, was mea-
sured to be 5 fmol (see Fig. S3 in Supplementary data). In order to
compare the relative sensitivity of our tag to that of trityl group
based LDI-MS tags, we have prepared an analog of 5 (Thc-Tag) in
which the alkylthio group is replaced with a phenylthio group and
subjected it to LDI-MS with equimolar amount of tris(4-
methoxyphenyl)methyl phenyl thioether (Trit-Tag). In this exper-
iment, Thc-Tag exhibited w2.2 fold higher signal intensity than
Trit-Tag (see Fig. S4 in Supplementary data).
After obtaining the mass tags 5aed, we tested their photo-
cleavage efficiencies under matrix-free LDI-MS conditions (See
Fig. 6). To our delight, the LDI-MS signals exhibited single m/z peaks
that correspond to the masses of thiochromenium ions 5aDedD
with high signal to noise (S/N) ratios at 100 pmol loading. To check
the feasibility of multiple detection with these mass tags, we also
subjected an equimolar mixture of 5aed to LDI-MS (see Fig. 7).
Interestingly, heavier mass tags were observed to exhibit greater
LDI-MS abundance. In repeated experiments, this propensity was
reproducibly observed; however, the reason for this greater abun-
dance is not yet fully understood. Nevertheless, we believe that the
unequal LDI-MS sensitivity of the mass tags could be compensated
through the use of isotope-labeled internal standards that will be
introduced in the future applications. Because a 14 Da mass
2.4. Demonstration of protein detection by LDI-MS
We next examined the use of mass tags 5aed for bioconjugation
of proteins and subsequent detection by LDI-MS. Myoglobin from
equine skeletal muscle (17.6 kDa) was chosen as a test substrate. To
a TEAB buffer solution (triethylammonium bicarbonate 50 mM,
70
m
L, pH¼8.0) were added a solution of myoglobin (142
mM, 20 mL,
2.8 nmol) in TEAB buffer and a solution of mass tag 5d (20 mM in
DMSO, 10 mL, 202 nmol). The resulting mixture was incubated for
1 h at 25 ꢂC. The insoluble precipitates were removed by centrifu-
gation. To scavenge the residual unreacted tags, a commercially
available polymer-supported amine [tris(2-aminoethyl)amine,
polymer-bound, 4.0e5.0 mmol/g N loading, 30 mg, 105e150 mmol]
was added to the supernatant. After removing the resin by a simple
centrifugation, the supernatant was diluted 10-fold with the mix-
ture of TEAB buffer solution (50 mM):DMSO¼9:1 and 1
mL of the
solution [2.8 pmol (w50 ng) of myoglobin] was loaded onto
a MALDI plate. Subjection of the sample to LDI-MS resulted in
a mass spectrum with a single peak at 278 Da; this peak corre-
sponds to the thiochromenium ions cleaved from the tagged
myoglobin without any interference peaks, as shown in Fig. 8. To
verify that the peaks originated only from the tags bound to myo-
globin and not from the residual non-reacted mass tags, we
Fig. 6. LDI-MS spectra of mass tags 5aed (100 pmol loading).
Fig. 8. Sensitivity test of 5d-mass-tagged myoglobin under LDI-MS conditions; (a)
2.8 pmol, (b) 280 fmol, (c) 28 fmol, and (d) 2.8 fmol loading; (e) LDI-MS spectrum of
a negative control experiment performed in the absence of myoglobin to ensure that
all of the excess mass tags were removed by the purification method.
Fig. 7. LDI-MS spectrum of an equimolar mixture of 5aed.

5616
N. Kang et al. / Tetrahedron 72 (2016) 5612e5619
conducted a negative control experiment. When the experiment
was performed in the same manner except but without myoglobin,
no peaks were observed in the spectrum, as shown in Fig. 8e. In this
protein conjugation and LDI-MS detection experiment, the LOD for
myoglobin was determined to be lower than 2.8 fmol (w50 pg).
obtained using Autoflex Speed series of Bruker Daltonics (Leipzig,
Germany) equipped with 355 nm laser pulse. An MTP 384 ground
steel plate was used for sample loading. A flexControl software was
used as a data acquisition system to transfer mass spectra to
hpZ4000 computer.
3. Conclusion
4.2. Synthesis
In conclusion, we have successfully designed and synthesized
new mass tags based on dithioacetal derivatives of 6,11-
dihydrothiochromeno[4,3-b]indole; these tags are easily ionisable
upon UV irradiation in a MALDI-MS instrument, without the use of
a matrix. The tags were readily synthesized in five or six steps and
in reasonably high overall yields (16e27%) starting from commer-
cially available starting materials. The dithioacetal structure was
selectively photocleavable upon UV irradiation while maintaining
its stability under normal dark conditions. The introduction of the
indole moiety to the tag was critical to induce greater photo-
ionization efficiency by increasing both the absorbance of UV
light and the stability of the resulting thiochromenium ion. Addi-
tionally, easy mass variation of the tags was also possible through
simple N-alkylation of the indole moiety, which is critical for the
application of these tags to the future multiple detection of bio-
markers. The protein tagging and purification procedures were
optimized and demonstrated using myoglobin as a model protein.
The detection limit of the mass tags under normal LDI conditions
was estimated to be w5 fmol level, whereas that of tagged myo-
globin was w50 pg (2.8 fmol), comparable to the lower limits for
other conventional bioassay techniques.22 Currently, we are
attempting to apply these tags to multiplex detection of a set of
biomarkers by employing antibody-based assays; the results will
be reported in due course.
4.2.1. 6,11-Dihydrothiochromeno[4,3-b]indole (8). Thiochro-man-4-
one (0.700 g, 4.26 mmol) and phenyl hydrazine (0.461 g,
4.26 mmol) were diluted with EtOH (7 mL) and to the solution was
added trimethylsilyl chloride (0.463 g, 4.26 mmol) in one portion.
The reaction mixture was heated at reflux for 4 h and cooled to
room temperature. The solution was basified with saturated
NaHCO3 aqueous solution and diluted with EtOAc (10 mL). The
organic layer was separated and the aqueous layer was extracted
with ethyl acetate three times (20 mLꢁ3). The combined organic
layers were washed with brine, dried over anhydrous Na2SO4, fil-
tered, and concentrated in vacuo. The resulting solid was dispersed
in EtOAc:Et2O¼1:8 (v/v) solution, filtered, and dried under reduced
pressure to give 9 as an ivory solid (0.961 g, 4.08 mmol, 96%):
mp¼162e165 ꢂC; Rf¼0.33 (EtOAc:hexane¼1:4); 1H NMR (400 MHz,
DMSO-d6):
d
11.58 (s, 1H), 7.71 (d, J¼7.2 Hz, 1H), 7.53 (d, J¼8.0 Hz,
1H), 7.39 (d, J¼8.0 Hz, 1H), 7.34 (d, J¼7.6 Hz, 1H), 7.24 (dd, J¼8.0,
7.2 Hz, 1H), 7.13e7.17 (m, 2H), 7.04 (dd, J¼7.2, 7.0 Hz, 1H), 4.28 (s,
2H); 13C NMR (100 MHz, DMSO-d6):
d 136.7, 132.4, 131.8, 127.5,
127.4, 127.2, 125.84, 125.78, 122.9, 122.4, 119.4, 118.5, 111.4, 105.9,
22.7; IR (ZnSe-ATR) 3336 (w), 1957 (w), 1915 (w), 1871 (w), 1785
(w), 1453 (w), 1440 (w), 1416 (w), 1312 (w), 1276 (w), 1177 (w), 1006
(w), 1036 (w), 918 (w), 864 (w), 761 (m), 733 (vs), 670 (w) cmꢀ1
;
Anal. Calcd for C15H11NS: C, 75.92; H, 4.67; N, 5.90; S, 13.51. Found:
C, 75.92; H, 4.62; N, 5.91; S, 13.63.
4. Experimental section
4.1. General methods
4.2.2. 6,11-Dihydrothiochromeno[4,3-b]indole 5-oxide (9). 6,11-
Dihydrothiochromeno[4,3-b]indol (8, 0.480 g, 2.02 mmol,
1.0 equiv) was dissolved in distilled CH2Cl2 (15 mL) and to the so-
lution was added 3-chloroperbenzoic acid (69%, 0.556 g,
2.23 mmol) at 0 ꢂC. After 1 h, to the solution was added 20% sodium
thiosulfate aqueous solution (4 mL) to quench the reaction. The
organic layer was separated and the aqueous layer was extracted
with ethyl acetate three times (30 mLꢁ3). The combined organic
layers were sequentially washed with saturated Na2CO3 aqueous
solution and brine, dried over anhydrous Na2SO4, filtered, and
concentrated in vacuo. The crude was dispersed in cold CH2Cl2 and
then insoluble solid was filtered and washed with cold CH2Cl2 to
give 10 as a light green solid in 78% yield; mp¼223e226 ꢂC dec;
Rf¼0.41 (EtOAc:hexane¼1:1þMeOH 10%); 1H NMR (400 MHz,
All commercially obtained solvents and reagents were used
without further purification unless noted below. All reactions were
performed in oven dried glassware. Anhydrous diethyl ether (Et2O)
and tetrahydrofuran (THF) were purified by refluxing with, and
distilling from sodium under N2 atmosphere. Anhydrous CH2Cl2
were purified by the same techniques from CaH2. N,N-dime-
thylformamide (DMF) was distilled over anhydrous MgSO4 under
N2 atmosphere. Triethylammonium bicarbonate buffer (1.0 M,
pH¼8.0, TEAB buffer), myoglobin from equine skeletal, and tris(2-
aminoethyl)amine; polymer-bound (4.0e5.0 mmol/g N loading)
were purchased from SigmaeAldrich. Reactions were monitored by
thin-layer chromatography (TLC) carried out on 0.25 nm Merck
silica gel plates (60F254). The spots were visualized by UV light
irradiation and ceric ammonium molybdate staining. Flash chro-
matography was performed by using hand-packed columns of
Merck silica gel (230e400 mesh). 1H NMR and 13C NMR spectra
were obtained using a Varian 400-Mercury INOVA 400 (400 MHz
for 1H spectrometer and 100 MHz for 13C spectrometer). Chemical
DMSO-d6):
d
11.98 (s, 1H), 7.89 (d, J¼7.6 Hz, 1H), 7.84 (d, J¼7.6 Hz,
1H), 7.70 (dd, J¼8.0, 7.6 Hz, 1H), 7.66 (d, J¼8.0 Hz, 1H), 7.51 (dd,
J¼8.0, 7.6 Hz, 1H), 7.47 (d, J¼8.0 Hz, 1H), 7.20 (dd, J¼7.6, 7.2 Hz, 1H),
7.09 (dd, J¼7.6, 7.2 Hz, 1H), 4.66 (d, J¼15.6 Hz, 1H), 4.40 (d,
J¼15.6 Hz, 1H); 13C NMR (100 MHz, DMSO-d6):
d 137.2, 137.1, 132.3,
130.9,128.9,128.2,127.4, 126.7, 123.2,123.0, 119.8, 118.9, 111.9, 101.0,
43.8; IR (ZnSe-ATR) 3140 (w br), 1592 (w), 1444 (w), 1349 (w), 1321
(w), 1282 (w), 1211 (w), 1112 (w), 1037 (w), 1009 (m), 951 (w), 824
(w), 788 (w), 758 (m), 742 (vs), 731 (s), 656 (w). cmꢀ1; HRMS (ESI)
m/z calcd for C15H11NNaOS ([MþNa]þ) 276.0454, found 276.0456.
shifts were reported relative to tetramethylsilane (
d 0.00) and
DMSO-d6 peak (
d
2.50) for 1H NMR spectra and DMSO-d6 peak (
d
39.52) for 13C NMR spectra. IR spectra were obtained using
a Thermo-Nicholet Avartar-330 IR spectrometer with a single-
bounce ATR mode using a ZnSe crystal (Smart MIRacle). HRMS
were obtained using a Varian Ion Trap Mass Spectrometer 500 3D-
ION. UVevis spectra were obtained using a JASCO V-660 UVevis
spectrophotometer. Elemental analyses were performed at the
Organic Chemistry Research Center (OCRC) using a Thermo Flash
EA 1112 elemental analyzer. All matrix-free assisted laser de-
sorption ionization time-of-flight mass (LDI-TOF MS) spectra were
4.2.3. General procedure for preparation of 11-alkyl-6,11-
dihydrothiochromeno[4,3-b]indoles (10aed). To the mixture of
6,11-dihydrothiochromeno[4,3-b]indol (8, 1.50 g, 6.32 mmol,
1.0 equiv) and NaH (60%, dispersion in mineral oil, 0.556 g,
12.6 mmol, 2.0 equiv) was added anhydrous DMF (10 mL) at 0 ꢂC
under N2 atmosphere. The reaction mixture was stirred at 0 ꢂC.
After 30 min, to the solution was added alkyl halide (12.6 mmol,
2.0 equiv) under N2 atmosphere and the reaction mixture was

N. Kang et al. / Tetrahedron 72 (2016) 5612e5619
5617
stirred at room temperature. After 2 h, H2O (10 mL) and EtOAc
(30 mL) were sequentially added to the solution. The organic layer
was separated and the aqueous layer was extracted with ethyl ac-
etate three times (40 mLꢁ3). The combined organic layers were
washed with brine, dried over anhydrous Na2SO4, filtered, and
concentrated in vacuo. The crude was purified by flash column
chromatography on silica gel (EtOAc:hexane¼1:15) to give the
desired product 10aed as an ivory solid.
dihydrothiochromeno[4,3-b]indole (9ae9d, 1.0 equiv) was dis-
solved in distilled CH2Cl2 and to the solution was added 3-
chloroperbenzoic acid (69%, 1.1 equiv) at 0 ꢂC. After 1 h, to the so-
lution was added 20% sodium thiosulfate aqueous solution to
quench the reaction. The organic layer was separated and the
aqueous layer was extracted with ethyl acetate three times. The
combined organic layers were sequentially washed with saturated
Na2CO3 aqueous solution and brine, dried over anhydrous Na2SO4,
filtered, and concentrated in vacuo. The crude was purified using
the method specified below.
4.2.3.1. 11-Methyl-6,11-dihydrothiochromeno[4,3-b]indole
(10a). Yield: 90%; mp¼94e97 ꢂC; Rf¼0.32 (EtOAc:hexane¼1:15);
1H NMR (400 MHz, DMSO-d6):
d
7.83 (d, J¼7.6 Hz, 1H), 7.62 (d,
4.2.4.1. 11-Methyl-6,11-dihydrothiochromeno[4,3-b]indole 5-
oxide (11a). The crude was purified by filtration to give 11a as
J¼8.0 Hz, 1H), 7.50e7.54 (m, 2H), 7.33 (dd, J¼7.6, 6.8 Hz, 1H),
7.21e7.25 (m, 2H), 7.11 (d, J¼7.6, 7.2 Hz, 1H), 4.15 (s, 2H), 3.95 (s,
a
yellow solid in 76% yield; mp¼180e183 ꢂC; Rf¼0.29
3H); 13C NMR (100 MHz, DMSO-d6):
d
138.5, 134.5, 133.7, 128.6,
(EtOAc:hexane¼1:1); 1H NMR (400 MHz, DMSO-d6):
d 7.89 (d,
127.8, 127.1, 126.2, 125.0, 124.1, 122.5, 119.7, 118.7, 110.3, 109.7, 32.8,
22.8; IR (ZnSe-ATR) 2887 (w), 1908 (w), 1875 (w), 1834 (w), 1470
(w), 1426 (w), 1360 (w), 1275 (w), 1235 (w), 1219 (w), 1121 (w), 1164
(w), 1082 (w), 1046 (w), 943 (w), 821 (w), 737 (vs), 714 (m), 675 (w)
cmꢀ1; Anal. Calcd for C16H13NS: C, 76.46; H, 5.21; N, 5.57; S, 12.76.
Found: C, 76.49; H, 5.22; N, 5.43; S, 12.64.
J¼7.6 Hz, 1H), 7.83 (d, J¼7.2 Hz, 1H), 7.68e7.71 (m, 2H), 7.58e7.61
(m, 2H), 7.27 (dd, J¼7.6, 7.2 Hz, 1H), 7.15 (dd, J¼7.6, 7.2 Hz, 1H),
4.55 (d, J¼14.2 Hz, 1H), 4.47 (d, J¼14.2 Hz, 1H), 4.03 (s, 3H); 13C
NMR (100 MHz, DMSO-d6):
d 140.3, 138.6, 133.1, 131.5, 128.4, 127.0,
126.4, 125.9, 125.1, 123.2, 120.2, 119.0, 110.6, 100.7, 44.2, 32.3; IR
(ZnSe-ATR) 2962 (w), 1581 (w), 1524 (w), 1468 (w), 1423 (w), 1356
(w), 1367 (w), 1261 (w), 1227 (w), 1123 (w), 1070 (m), 1048 (s),
4.2.3.2. 11-Ethyl-6,11-dihydrothiochromeno[4,3-b]indole
1035 (s), 1025 (m), 1016 (m), 817 (w), 759 (vs), 667 (w) cmꢀ1
;
(10b). Yield: 70%; mp¼122e126 ꢂC; Rf¼0.36 (EtOAc:hexane¼1:15);
HRMS (ESI) m/z calcd for C16H13NNaOS ([MþNa]þ) 290.0610,
1H NMR (400 MHz, DMSO-d6):
d
7.68 (d, J¼7.6 Hz, 1H), 7.63 (d,
found 290.0610.
J¼8.4 Hz, 1H), 7.51e7.56 (m, 2H), 7.35 (dd, J¼7.6, 7.2 Hz, 1H),
7.21e7.26 (m, 2H), 7.12 (dd, J¼7.6, 7.2 Hz, 1H), 4.40 (q, J¼7.0 Hz, 2H),
4.13 (s, 2H), 1.39 (t, J¼7.0 Hz, 3H); 13C NMR (100 MHz, DMSO-d6):
4.2.4.2. 11-Ethyl-6,11-dihydrothiochromeno[4,3-b]indole 5-oxide
(11b). The crude was purified by filtration to give 11b as a yellow
solid in 79% yield; mp¼210e213 ꢂC; Rf¼0.33 (EtOAc:hexane¼1:1);
d
137.7, 133.8, 133.7, 128.7, 127.7, 127.0, 126.4, 124.4, 124.3, 122.6,
119.8, 118.8, 118.7, 110.2, 22.7, 15.5, 15.4; IR (ZnSe-ATR) 2970 (w),
1936 (w), 1847 (w), 1477 (w), 1457 (w), 1363 (w), 1340 (w), 1284
(w), 1208 (w), 1162 (w), 1131 (w), 1086 (w), 1040 (w), 1102 (w), 784
(w), 746 (vs), 712 (w), 670 (w) cmꢀ1; Anal. Calcd for C17H15NS: C,
76.94; H, 5.70; N, 5.28; S, 12.08. Found: C, 76.88; H, 5.71; N, 5.29; S,
12.07.
1H NMR (400 MHz, DMSO-d6):
d
7.83 (d, J¼7.6 Hz, 1H), 7.70e7.75
(m, 3H), 7.59e7.62 (m, 2H), 7.27 (dd, J¼7.6, 7.2 Hz, 1H), 7.15 (dd,
J¼7.6, 7.2 Hz, 1H), 4.43e4.57 (m, 4H), 1.41 (t, J¼7.0 Hz, 3H); 13C NMR
(100 MHz, DMSO-d6): d 140.6, 137.7, 132.3, 131.7, 128.5, 127.0, 126.4,
126.2, 124.4, 123.2, 120.3, 119.2, 110.6, 101.1, 44.1, 15.32; IR (ZnSe-
ATR) 2974 (w), 1585 (w), 1459 (w), 1339 (w), 1159 (w), 1133 (w),
1075 (w),1024 (m), 831 (w), 768 (w), 750 (vs), 737 (m), 700 (w), 668
(w) cmꢀ1; HRMS (ESI) m/z calcd for C17H15NNaOS ([MþNa]þ)
304.0767, found 304.0768.
4.2.3.3. 11-Propyl-6,11-dihydrothiochromeno[4,3-b]indole
(10c). Yield: 94%; mp¼75e80 ꢂC; Rf¼0.40 (EtOAc:hexane¼1:15);
1H NMR (400 MHz, DMSO-d6):
d
7.69 (d, J¼8.0 Hz, 1H), 7.62 (d,
J¼7.6 Hz, 1H), 7.56 (d, J¼8.4 Hz, 1H), 7.51 (d, J¼7.6 Hz, 1H), 7.35 (dd,
J¼7.6, 7.2 Hz,1H), 7.20e7.25 (m, 2H), 7.11 (dd, J¼8.0, 7.2 Hz,1H), 4.34
(t, J¼7.2 Hz, 2H), 4.13 (s, 2H), 1.73 (tq, J¼7.2, 7.2 Hz, 2H), 0.82 (t,
4.2.4.3. 11-Propyl-6,11-dihydrothiochromeno[4,3-b]indole 5-
oxide (11c). The crude was purified by flash column chromatogra-
phy on silica gel (EtOAc:hexane¼1:1) to give 12c as a yellow solid in
80% yield; mp¼128e132 ꢂC; Rf¼0.27 (EtOAc:hexane¼1:1); 1H NMR
J¼7.2 Hz, 3H); 13C NMR (100 MHz, DMSO-d6):
d 138.2, 133.9, 133.8,
128.8, 128.0, 127.0, 126.4, 124.30, 124.25, 122.6, 119.8, 118.8, 110.6,
110.4, 46.1, 23.3, 22.7, 11.0; IR (ZnSe-ATR) 3056 (w), 2970 (w), 1475
(w), 1460 (w), 1414 (w), 1348 (w), 1204 (w), 1158 (w), 1039 (w), 1012
(w), 754 (m), 738 (vs), 669 (w) cmꢀ1; Anal. Calcd for C18H17NS: C,
77.38; H, 6.13; N, 5.01; S, 11.47. Found: C, 77.38; H, 6.17; N, 4.95; S,
11.35.
(400 MHz, DMSO-d6):
d
7.82 (d, J¼8.0 Hz, 1H), 7.69e7.77 (m, 3H),
7.58e7.64 (m, 2H), 7.26 (dd, J¼7.6, 7.2 Hz,1H), 7.15 (dd, J¼7.6, 7.2 Hz,
1H), 4.56 (d, J¼14.0 Hz, 1H), 4.42e4.46 (m, 3H), 1.77 (tq, J¼7.4,
7.0 Hz, 2H), 0.84 (t, J¼7.4 Hz, 3H); 13C NMR (100 MHz, DMSO-d6):
d
140.6, 138.3, 132.6, 131.6, 128.4, 126.8, 126.6, 126.1, 124.4, 123.2,
120.3, 119.2, 110.9, 101.2, 45.8, 44.1, 23.2, 11.0; IR (ZnSe-ATR) 2958
(w),1581 (w),1481 (w),1455 (w),1410 (w),1362 (w),1209 (w),1077
(m), 1056 (m), 1036 (m), 1024 (m), 902 (w), 763 (s), 753 (vs), 730
(m) cmꢀ1; HRMS (ESI) m/z calcd for C18H17NNaOS ([MþNa]þ)
318.0923, found 318.0925.
4.2.3.4. 11-Butyl-6,11-dihydrothiochromeno[4,3-b]indole
(10d). Yield: 98%; mp¼86e90 ꢂC; Rf¼0.47 (EtOAc:hexane¼1:15);
1H NMR (400 MHz, DMSO-d6):
d
7.71 (d, J¼7.6 Hz, 1H), 7.62 (d,
J¼8.0 Hz, 1H), 7.55 (d, J¼8.4 Hz, 2H), 7.52 (d, J¼8.0 Hz, 1H), 7.34 (dd,
J¼7.6, 7.2 Hz,1H), 7.20e7.25 (m, 2H), 7.11 (dd, J¼7.6, 7.2 Hz,1H), 4.38
(t, J¼7.6 Hz, 2H), 4.12 (s, 2H), 1.68 (tt, J¼7.6, 7.6 Hz, 2H), 1.25 (tq,
J¼7.6, 7.2 Hz, 2H), 0.85 (t, J¼7.2 Hz, 3H); 13C NMR (100 MHz, DMSO-
4.2.4.4. 11-Butyl-6,11-dihydrothiochromeno[4,3-b]indole 5-oxide
(11d). The crude was purified by flash column chromatography
on silica gel (EtOAc:hexane¼1:1) to give 12d as a yellow solid in
65% yield; mp¼64e68 ꢂC; Rf¼0.33 (EtOAc:hexane¼1:1); 1H NMR
d6): d 138.1,134.0,133.8,128.7,127.9, 127.0, 126.3,124.4,124.3,122.5,
119.8, 118.8, 110.6, 110.5, 44.3, 31.9, 22.7, 19.4, 13.5; IR (ZnSe-ATR)
2962 (w), 2921 (w), 1469 (w), 1454 (w), 1360 (w), 1347 (w), 1196
(400 MHz, DMSO-d6):
d
7.82 (d, J¼7.2 Hz, 1H), 7.77 (d, J¼7.6 Hz, 1H),
7.69e7.71 (m, 2H), 7.58e7.62 (m, 2H), 7.26 (dd, J¼7.8, 7.6 Hz, 1H),
7.15 (dd, J¼7.8, 7.6 Hz, 1H), 4.41e4.57 (m, 4H), 1.72 (tt, J¼7.2, 7.2 Hz,
2H), 1.27 (tq, J¼7.2, 7.2 Hz, 2H), 0.85 (t, J¼7.2 Hz, 3H); 13C NMR
(w), 1120 (w), 1042 (w), 819 (w), 757 (m), 743 (vs), 681 (w) cmꢀ1
;
Anal. Calcd for C19H19NS: C, 77.77; H, 6.53; N, 4.77; S, 10.93: Found:
C, 77.74; H, 6.55; N, 4.81; S, 10.98.
(100 MHz, DMSO-d6): d 140.7, 138.2, 132.6, 131.6, 128.5, 126.8, 126.6,
126.1, 124.3, 123.2, 120.3, 119.2, 110.9, 101.2, 44.2, 44.1, 31.8, 19.4,
13.5; IR (ZnSe-ATR) 2950 (w), 1585 (w), 1482 (w), 1456 (w), 1432
(w), 1349 (w), 1266 (w), 1131 (w), 1079 (m), 1052 (m), 1027 (m), 737
4.2.4. General procedure for preparation of 11-alkyl-6,11-
dihydrothiochromeno[4,3-b]indole 5-oxide (11aed). 11-Alkyl-6,11-

5618
N. Kang et al. / Tetrahedron 72 (2016) 5612e5619
(s), 668 (w) cmꢀ1; HRMS (ESI) m/z calcd for C19H19NNaOS
([MþNa]þ) 332.1080, found 332.1080.
4.2.5.3. 2,5-Dioxopyrrolidin-1-yl 3-((11-propyl-6,11-dihydrothio
chromeno[4,3-b]indol-6-yl)thio)propanoate
(5c). Yield:
61%;
mp¼77e82 ꢂC dec; Rf¼0.36 (EtOAc:hexane¼1:1); 1H NMR
4.2.5. General procedure for preparation of 2,5-dioxopyrrolidin-1-yl
3-((11-alkyl-6,11-dihydrothiochromeno [4,3-b]indol-6-yl)thio)prop-
anoate (5aed) from 11aed. 11-Alkyl-6,11-dihydrothiochromeno
[4,3-b]indole 5-oxide (11aed, 1.0 equiv) was dispersed in CH3CN
and to the suspension was added trifluoroacetic anhydride
(3.0 equiv) at 0 ꢂC under N2 atmosphere. After 5 min, the deep
yellow solution was concentrated in vacuo and the residue was
precipitated in anhydrous Et2O at 0 ꢂC. The resulting yellow solid
was filtered, washed with cold anhydrous Et2O, and dried under
reduced pressure to give the thionium salt (12aed) as a deep yel-
low solid, which was used without further purification.
(400 MHz, DMSO-d6):
d
7.83 (d, J¼8.0 Hz, 1H), 7.70 (d, J¼8.0 Hz, 1H),
7.59 (d, J¼7.6 Hz, 2H), 7.43 (dd, J¼7.6, 7.6 Hz, 1H), 7.32 (dd, J¼7.6,
7.6 Hz, 1H), 7.26 (dd, J¼8.0, 7.2 Hz, 1H), 7.15 (dd, J¼8.0, 7.2 Hz, 1H),
6.21 (s, 1H), 4.31e4.46 (m, 2H), 3.08e3.21 (m, 2H), 2.96e3.03 (m,
1H), 2.75e2.82 (m, 5H),1.67e1.82 (m, 2H), 0.83 (t, J¼7.2 Hz, 3H); 13
C
NMR (100 MHz, DMSO-d6):
d 170.1, 167.9, 138.0, 134.1, 130.5, 129.8,
127.5, 126.9, 124.3, 123.0, 122.9, 120.23, 120.20, 118.8, 111.5, 110.7,
46.2, 42.7, 31.4, 25.7, 25.5, 23.1, 10.9; IR (ZnSe-ATR) 2961 (w), 1812
(w), 1783 (w), 1733 (s), 1460 (w), 1424 (w), 1348 (m), 1201 (m), 1065
(m), 1045 (m), 908 (w), 810 (w), 744 (vs) cmꢀ1; Anal. Calcd for
C
25H24N2O4S2: C, 62.48; H, 5.03; N, 5.83; S, 13.34. Found: C, 62.42;
The freshly prepared thionium salt (12aed, 1.0 equiv) was
dissolved in CH3CN and to the solution was added 3-
mercaptopropionic acid (1.0 equiv), followed by Na2CO3
(1.0 equiv). The reaction mixture was stirred at room temperature
until it turns to colorless and diluted with EtOAc. H2O was added to
the mixture until all the solid had dissolved. The organic layer was
separated and the aqueous layer was extracted with EtOAc three
times. The combined organic layers were wash with saturated
NH4Cl aqueous solution, followed by brine, dried over anhydrous
Na2SO4, filtered, and concentrated in vacuo. The resulting solid
was used without further purification. The crude carboxylic acid
and N-hydroxysuccinimide (1.1 equiv) were dissolved in
anhydrous CH2Cl2 and to the solution was added N-(3-
H, 5.03; N, 5.82 S, 13.40.
4.2.5.4. 2,5-Dioxopyrrolidin-1-yl 3-((11-butyl-6,11-dihydrothio
chromeno[4,3-b]indol-6-yl)thio)propanoate
(5d). Yield:
51%;
mp¼76e81 ꢂC dec; Rf¼0.35 (EtOAc:hexane¼1:1); 1H NMR
(400 MHz, DMSO-d6):
d
7.85 (d, J¼8.0 Hz, 1H), 7.70 (d, J¼8.0 Hz, 1H),
7.58e7.60 (m, 2H), 7.43 (dd, J¼8.0, 8.0 Hz, 1H), 7.32 (dd, J¼8.0,
7.2 Hz, 1H), 7.26 (dd, J¼7.6, 7.2 Hz, 1H), 7.15 (dd, J¼7.6, 7.2 Hz, 1H),
6.21 (s, 1H), 4.40e4.46 (m, 2H), 3.08e3.21 (m, 2H), 2.96e3.03 (m,
1H), 2.77e2.82 (m, 5H), 1.65e1.75 (m, 2H), 1.21e1.29 (m, 2H), 0.86
(t, J¼7.0 Hz, 3H); 13C NMR (100 MHz, DMSO-d6):
d 170.1,167.9,137.9,
134.2, 130.5, 129.9, 127.6, 126.9, 126.8, 124.3, 123.02, 122.96, 120.2,
118.8, 111.5, 110.6, 44.5, 42.8, 31.8, 31.5, 25.8, 25.5, 19.3, 13.5; IR
(ZnSe-ATR) 2954 (w), 2925 (w), 1811 (w), 1783 (w), 1735 (s), 1459
(w), 1422 (w), 1359 (w), 1200 (m), 1066 (m), 1042 (w), 810 (w), 745
(s), 669 (m) cmꢀ1; Anal. Calcd for C26H26N2O4S2: C, 63.14; H, 5.30;
N, 5.66; S, 12.96. Found: C, 63.14; H, 5.30; N, 5.69 S, 12.90.
dimethylaminopropyl)-N0-ethylcarbodiimide
hydrochloride
(3.0 equiv) in CH2Cl2 via cannula at 0 ꢂC under N2 atmosphere. The
reaction mixture was stirred at room temperature for 4 h and di-
luted with CH2Cl2. The resulting solution was washed twice with
H2O, dried over anhydrous Na2SO4, filtered, and concentrated in
vacuo. The crude was purified by flash column chromatography on
silica gel (EtOAc:hexane¼1:1) to give the desired product as
a white solid.
4.2.6. General procedure for preparation of 11-alkyl-11H-thio-
chromeno[4,3-b]indol-5-ium (12c) from 9c. To the mixture of 11-
propyl-6,11-dihydrothiochromeno[4,3-b]indole (9c, 0.200 g,
0.716 mmol) and bis(trifluoroacetoxy)iodobenzene (0.369 g,
0.859 mmol) was added anhydrous CH3CN (1 mL) at 0 ꢂC under N2
atmosphere. After 5 min, the deep yellow solution was concen-
trated in vacuo and the residue was precipitated in anhydrous Et2O
(4 mL) at 0 ꢂC. The resulting yellow solid was filtered, washed with
cold anhydrous Et2O, and dried under reduced pressure to give the
thionium salt 12c (277 mg, 0.709 mmol) as a deep yellow solid,
which was used without further purification.
4.2.5.1. 2,5-Dioxopyrrolidin-1-yl 3-((11-methyl-6,11-dihydrothio
chromeno[4,3-b]indol-6-yl)thio)propanoate
(5a). Yield:
49%;
mp¼90e96 ꢂC dec; Rf¼0.52 (EtOAc:hexane¼1:1); 1H NMR
(400 MHz, DMSO-d6):
d
7.98 (d, J¼8.0 Hz, 1H), 7.71 (d, J¼8.0 Hz, 1H),
7.59 (d, J¼7.6 Hz, 1H), 7.56 (d, J¼8.4 Hz, 1H), 7.42 (dd, J¼8.0, 7.2 Hz,
1H), 7.33 (dd, J¼7.6, 7.2 Hz, 1H), 7.27 (dd, J¼8.4, 7.2 Hz, 1H), 7.15 (dd,
J¼7.6, 7.2 Hz, 1H), 6.23 (s, 1H), 4.00 (s, 3H), 3.10e3.24 (m, 2H),
2.96e3.03 (m, 1H), 2.75e2.82 (m, 5H); 13C NMR (100 MHz, DMSO-
d6):
d
170.2, 168.0, 138.2, 134.7, 130.5, 129.7, 127.7, 126.8, 126.7, 125.1,
4.3. General procedure for the bioconjugation of mass tags to
myoglobin
123.0, 122.7, 120.2, 118.7, 110.7, 110.4, 42.9, 33.0, 31.5, 25.7, 25.5; IR
(ZnSe-ATR) 1811 (w), 1782 (w), 1732 (s), 1470 (w), 1426 (w), 1361
(w), 1201 (m), 1064 (m), 824 (w), 743 (s), 668 (w) cmꢀ1; Anal. Calcd
for C23H20N2O4S2: C, 61.04; H, 4.45; N, 6.19; S, 14.17. Found: C, 61.14;
H, 4.48; N, 6.10 S, 14.04.
In the conjugation procedure, in order to ensure that mass tags
are not photo-degraded, the glass-tubes containing mass tags or
conjugated myoglobin were wrapped with aluminum foil. The
mass tag (5d, 2,5-dioxopyrrolidin-1-yl 3-((11-butyl-6,11-
dihydrothiochromeno[4,3-b]indol-6-yl)thio)propanoate, 0.50 mg,
4.2.5.2. 2,5-Dioxopyrrolidin-1-yl 3-((11-ethyl-6,11-dihydrothio
chromeno[4,3-b]indol-6-yl)thio)propanoate
(5b). Yield:
40%;
1.0
prepare the stock solution at 20 mM. A stock solution of myo-
globin (142 M in TEAB buffer), which was used as a representa-
tive protein for conjugation, was prepared by dissolving
myoglobin (17.6 kDa, 2.5 mg, 140 nmol) in TEAB buffer solution
(pH¼8.0, 50 mM, 1 mL). For conjugation of mass tag with myo-
mmol) was dissolved in DMSO (dimethylsulfoxide, 50 mL) to
mp¼82e87 ꢂC dec; Rf¼0.32 (EtOAc:hexane¼1:1); 1H NMR
(400 MHz, DMSO-d6):
d
7.81 (d, J¼8.0 Hz, 1H), 7.71 (d, J¼8.0 Hz, 1H),
m
7.60 (d, J¼7.2 Hz, 1H), 7.58 (d, J¼8.0 Hz, 1H), 7.44 (dd, J¼7.6, 7.6 Hz,
1H), 7.33 (dd, J¼7.6, 7.6 Hz, 1H), 7.27 (dd, J¼7.6, 7.6 Hz, 1H), 7.16 (dd,
J¼7.6, 7.2 Hz, 1H), 6.21 (s, 1H), 4.44 (q, J¼7.0 Hz, 2H), 3.08e3.23 (m,
2H), 2.96e3.03 (m, 1H), 2.75e2.82 (m, 5H), 1.42 (t, J¼7.0 Hz, 3H);
globin, the myoglobin stock solution (142
20 mL, 2.8 nmol) was diluted with TEAB buffer (70 m
m
M in TEAB buffer,
L, 50 mM),
and then the solution was reacted with the stock solution of mass
tag (20 mM in DMSO, 10 L, 202 nmol) (The resulting concen-
tration of myoglobin was 28 M). In the case of a negative control
sample (without myoglobin), TEAB buffer (90 L, 50 mM) was
mixed with the stock solution of mass tag (10 L, 202 nmol). The
reaction mixture was vortexed for 1 h at 25 ꢂC. Subsequently, the
13C NMR (100 MHz, DMSO-d6):
d
170.1, 167.9, 137.5, 133.9, 130.6,
129.8, 127.6, 126.9,126.7, 124.4, 123.1, 123.0, 120.3, 118.8, 111.2, 110.4,
42.83, 42.76, 31.5, 25.7, 25.5, 15.4; IR (ZnSe-ATR) 1810 (w), 1781 (w),
1733 (s), 1459 (w),1426 (w),1345 (w),1203 (m),1066 (m),1044 (m),
991 (w), 813 (w), 745 (vs) cmꢀ1; Anal. Calcd for C24H22N2O4S2: C,
61.78; H, 4.75; N, 6.00; S, 13.74. Found: C, 61.83; H, 4.79; N, 5.97 S,
13.91.
m
m
m
m

N. Kang et al. / Tetrahedron 72 (2016) 5612e5619
5619
Banoub, J. Appl. Spectrosc. Rev. 2009, 44, 210e230; (c) Fenselau, C. Anal. Chem.
1997, 69, 661Ae665A.
2. (a) Sparbier, K.; Wenzel, T.; Dihazi, H.; Blaschke, S.; Muller, G.; Deelder, A.; Flad,
mixtures were subjected to centrifugation (7000 rpm, 5 min, rt).
To the supernatant (90 L) was added tris(2-aminoethyl)amine,
polymer-bound (Sigma Aldrich, 4.0e5.0 mmol/g loading,
30 mg, 105e150 mol) to scavenge the residual unreacted tags.
m
€
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This research was supported by a grant from the Emerging In-
dustrial Technology Development Program funded by the Ministry
of Trade, Industry& Energy of Korea (10044352).
Supplementary data
Supplementary data (The LDI-MS spectra of compounds 3 and 4.
The sensitivity test of mass tags (5d) and photocleavage efficiency
comparison between Thc-Tag and Trit-Tag in LDI-MS. The 1H and
13C NMR spectra of the compounds 5aed, 8, 9, 10aed, and 11aed)
associated with this article can be found in the online version, at
http://dx.doi.org/10.1016/j.tet.2016.07.052.
References and notes
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