Isotope-Coded Maleimide Affinity Tags for Proteomics Applications

Article abstract of DOI:10.1021/acs.bioconjchem.1c00206

Isotope-coded affinity tags (ICATs) are valuable tools for mass spectrometry-based quantitative proteomics, in particular, for comparison of protein (cysteine-residue) thiol oxidation state in normal, stressed, and diseased tissue. However, the iodoacetam

Full text of DOI:10.1021/acs.bioconjchem.1c00206

pubs.acs.org/bc
Article
Isotope-Coded Maleimide Affinity Tags for Proteomics Applications
#
#
Adam P. Wdowiak, Marisa N. Duong, Rohan D. Joyce, Amber E. Boyatzis, Mark C. Walkey,
Gareth L. Nealon, Peter G. Arthur,* and Matthew J. Piggott*
Cite This: https://doi.org/10.1021/acs.bioconjchem.1c00206
Read Online
ACCESS
Metrics & More
Article Recommendations
*
sı Supporting Information
ABSTRACT: Isotope-coded affinity tags (ICATs) are valuable tools
for mass spectrometry-based quantitative proteomics, in particular, for
comparison of protein (cysteine-residue) thiol oxidation state in
normal, stressed, and diseased tissue. However, the iodoacetamido
electrophile used in most commercial ICATs suffers from poor thiol-
selectivity and modest rates of adduct formation, which can lead to
spurious results. Hence, we designed and synthesized three ICATs
containing thiol-selective N-alkylmaleimide electrophiles (isotope-
coded maleimide affinity tags = ICMATs) and assessed these as mass
spectrometry probes for ratiometric analysis of lysozyme and muscle
proteomes. Two ICMAT pairs containing butylene/D -butylene
8
linkers were effective MS probes, but not ideal for typical proteomics
workflows, because peptides bearing these tags frequently did not coelute with HPLC. A switch to a phenylene/ C -phenylene
1
3
6
linker solved this issue without compromising the efficiency of adduct formation.
INTRODUCTION
convenient, higher throughput, and more accurate than gel-
■
10,11
based proteomics workflows.
The heavy ICAT typically incorporates a polydeuterated
Chemical probes for covalent protein labeling have proven
invaluable in quantitative proteomics. Isotope-coded affinity
tags (ICATs) were among the first probes used for high-
throughput quantitation workflows. While other techniques
and labels are now more commonly used for general
quantitative proteomics applications, probes that alkylate
cysteine residues (targets of cellular oxidation events) are
1
3
alkyl chain or C-enriched linker, while the corresponding
light ICAT contains the naturally abundant isotopes. A mass
difference of at least 5 Da is preferred, to enable tagged-peptide
ion resolution, which may otherwise be compromised by
1
3
10
naturally abundant C. In principle, the light/heavy ICAT
pair are chemically identical, meaning the reactivity is
unaffected on changing from a light probe to a heavy probe.
An iodoacetamide (IAM) moiety is most commonly used as
the thiol-reactive component in ICATs. Although this
1
−6
valuable for quantitative redox proteomics.
Typical ICATs
comprise three components: a reactive electrophile that
alkylates cysteine thiols, a biotin residue that allows enrich-
ment of tagged peptides, and an isotopically variable linker for
ratiometric analysis by mass spectrometry (MS). Additional
functionality can be introduced into the ICAT, such as a C or
fluorescent visualization motif and/or a cleavable linker that
simplifies, and enhances the sensitivity of, MS analysis.
A schematic illustrating the principle of ICAT methodology
is set out in Figure 1. Protein extracts from tissue samples or
cell culture are denatured and treated with the light tag (t).
Ideally, only polypeptides with free thiol groups are covalently
labeled in this first tagging step. The entire sample is then
reduced, converting oxidized cysteine residues to the free
thiols, which are then labeled with the heavy tag (T). The
mixture is then trypsinized, and tagged peptides are separated
from untagged peptides using (strept)avidin affinity chroma-
tography or pull down. Finally, LC−MS/MS analysis allows
protein identification, and ratiometric comparison of light:-
heavy tagged peptides reports on the ratios of the
reduced:oxidized cysteine residues present in the original
mixture. ICAT methodology is generally faster, more
2
1
electrophile was originally chosen to be thiol-specific, it has
1
4
since been found that that N-alkylation (e.g., of histidine
residues) is not just common, but it is the dominant
7
7
−9
12
conjugation mode for IAM using typical protocols. Indeed,
many undesired side-products have been observed in the
attempted labeling of cysteine thiol groups using IAM,
including alkylation of N- and C-terminal amino and
13
carboxylate groups, respectively. IAM also doubly alkylates
the ε-amino group of lysine residues, resulting in a tertiary
amine (2-acetamidoacetamide) adduct, which has been
Received: April 21, 2021
Revised: May 31, 2021
©
XXXX American Chemical Society
https://doi.org/10.1021/acs.bioconjchem.1c00206
A
Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
pubs.acs.org/bc
Article
Figure 1. Schematic example of ICAT methodology for the interrogation of protein−cysteine oxidation state (OxICAT). G = glutathione; SX = an
undefined oxidized cysteine sulfur; T = heavy tag, t = light tag.
misidentified as the diglycine modification characteristic of
protein ubiquitination, post-trypsin digestion.
use of biotinylated ICATs incorporating a maleimide
14
19−23
terminus;
however, to the best of our knowledge, no
Alkylation of thiols by soft Michael acceptor electrophiles is
considerably faster and more selective than with IAMs; N-
alkylmaleimides (NAMs) have been most widely studied. The
reaction of 2-nitro-5-thiobenzoic acid with N-ethylmaleimide
such chemical probes have been published in peer-reviewed
journals.
We recently reported the optimization of sample preparation
24
workflow using commercial biotin maleimide. Herein, we
describe the design, synthesis, and preliminary evaluation of
bespoke proteomics probes incorporating a maleimide moiety,
which we call ICMATs (isotope-coded maleimide affinity
tags).
15
(
NEM) was 20-fold faster than with IAM. In an analysis of
murine skeletal muscle tissue, 4 h of incubation with IAM was
necessary to alkylate all free thiols, but was complete in just 4
1
6
min with NEM. Indeed, alkylation of protein thiols by IAM
17
may be incomplete even after 6 h of incubation. For practical
purposes, the relatively slow reaction kinetics means that large
stoichiometric excesses of IAMs are required to complete
timely global thiol labeling. Much smaller excesses are required
for NAMs. In isolated myofibril preparations, complete
alkylation of thiols required a 1000-fold excess of IAM, but
RESULTS AND DISCUSSION
■
The key features required in the new thiol-reactive probes for
proteomic mass tagging are summarized in Figure 2. First, for
the reasons described above, a terminal maleimide moiety was
chosen as the thiol-selective trap. A biotin residue comprises
the other terminus of the ICMAT, to facilitate purification/
enrichment of tagged peptides, through (strept)avidin affinity
16
only a 125-fold excess of NEM. The larger excess required
for IAMs exacerbates their first major drawback, poor
chemoselectivity, leading to increased mislabeling of nucleo-
philic functional groups other than thiols, and consequent
misidentification of peptides. In addition, IAMs are photo-
2
,18
labile, and must be handled in the dark.
Overall, these
limitations of IAMs reduce the performance of the commercial
ICATs, especially when the amount of protein is unknown, as
using a molar excess can lead to indiscriminate “overlabeling”.
d5-NEM has been used alongside (light) NEM in
conjunction with gel-electrophoresis for MS-ratiometric
1
0
proteomics. There are also several patents that claim the
Figure 2. Generic structure and key features of ICMATs.
B
https://doi.org/10.1021/acs.bioconjchem.1c00206
Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
pubs.acs.org/bc
Article
a
Scheme 1. Synthesis of Light and Heavy* (d ) ICMAT-1 (7/7*) and ICMAT-2 (11/11*)
8
a
The red linker indicates the positions of perdeuteration. Yields for the light and heavy isotopologues are in black and red, respectively.
Figure 3. Left: Representative extracted-ion chromatogram for the ICMAT-1 (7/7*)-tagged peptide CELAAAMK derived from lysozyme. Right:
Representative standard curve based on the same tagged peptide. Ratios of light:heavy-tagged peptides were determined by extracted-ion
chromatogram peak integral.
chromatography/pull down. Finally, the linker connecting the
two components above must be appropriate for isotopic
labeling; that is, the heavy isotopologue must be synthetically
accessible from a reasonably priced, commercial starting
material. In addition, ideally the heavy and light linkers have
a mass difference of at least 5 Da to achieve clear resolution in
the mass spectra of peptide−probe adducts. These points were
considered in the design of the ICMATs described below.
outlined in Scheme 1. All steps were first conducted with
unlabeled (light) materials, followed by repetition with the d8
(heavy) isotopologues (denoted by *). Tetrahydrofuran
(THF, 1) is a convenient starting point, as d -THF is
8
reasonably inexpensive and readily available due to its use as
25
a solvent for NMR spectroscopy. Ring opening of THF (1)
provided 4-iodobutan-1-ol (2). Fischer esterification of D-
biotin (3) with this alcohol gave 4, from which the syntheses
1
2
26
Synthesis of H/ H-Labeled ICMATs. The syntheses of
the initially targeted ICMAT-1 (7) and ICMAT-2 (11) are
diverged. Alkylation of the protected maleimide 5 with
iodide 4 gave 6, which was subjected to a retro-Diels−Alder
C
https://doi.org/10.1021/acs.bioconjchem.1c00206
Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
pubs.acs.org/bc
Article
2
+
Figure 4. Extracted-ion chromatogram showing the different elution times of light- [TSGPDC(7)NFR] (m/z 696.2896, elution time 48.6 min)
2
+
and heavy-tagged [TSGPDC(7*)NFR] (m/z 700.3147, elution time 49.2 min) peptide derived from mouse muscle titin.
reaction to give ICMAT-1 (7) containing a simple butylene
linker. Alternatively, conversion of iodide 4 to the correspond-
ing azide 8, followed by copper-catalyzed cycloaddition with
terminal alkyne 9, gave triazole 10, which was thermally
unmasked to provide ICMAT-2 (11).
for example, using MALDI−TOF/TOF, or for use in
conjunction with gel-based fractionation, they are not ideal
for the most powerful and common protocols that incorporate
an HPLC fractionation step to improve resolution. Accord-
13
12
ingly, we designed a C/ C ICMAT pair, in which the
isotopologues were expected to have identical chromato-
Evaluation of ICMAT-1. The suitability of the ICMAT-1
pair 7/7* for ratiometric quantification of light- and heavy-
tagged proteins was assessed using a model protein, lysozyme,
which was chosen because it contains eight cysteine residues.
Briefly, commercial chicken egg lysozyme was reduced to
cleave all disulfide bonds, then exhaustively thiol-labeled by
treatment with ∼30 equiv of light 7 or heavy 7*. The light- and
heavy-tagged lysozyme samples were mixed in various ratios
and then subjected to a standard proteomic workflow for
bottom-up MS analysis (trypsin digestion, peptide cleanup,
graphic mobilities.2
13
13
Synthesis of 1 C/ C-Labeled ICMAT-3. C -THF is not
4
commercially available, and in any case would only provide an
M+4 heavy partner if applied to isotopologues of 7 and 11.
Instead, a p-phenylenediamine linker, as in ICMAT-3 (16)
Scheme 2. Synthesis of Light and Heavy ICMAT-3 pair 16/
a
1
6*
2
7
LC−MS/MS). A single tagged peptide was observed and, as
expected, automated MS/MS analysis (see Experimental
Procedures for details) confirmed this as being derived from
lysozyme. Encouragingly, the light- and heavy-tagged peptides
coeluted on HPLC (Figure 3, left). Relative quantification of
the light/heavy-tagged peptides by comparison of peak
integrals very closely matched the prepared ratios (Figure 3,
right).
ICMAT-1 pair 7/7* was then used to interrogate the
oxidation status of cysteine residues in the proteome of mouse
muscle tissue, which was subjected to a typical OxICAT-type
2
8
workflow. Homogenized tissue was incubated with 7 (0.29
nmol per μg tissue). Tags were incorporated efficientlyno
untagged peptides were detected, as reported by Mascot search
engine from the MS/MS output filesand did not interfere
with the identification of the major muscle proteins (e.g., actin,
myosin, titin, tropomyosin, and troponin C). However, unlike
in the model protein lysozyme, the light and heavy
isotopologues in a subset of tagged peptides from muscle
tissue had slightly different HPLC retention times (e.g., Figure
4
). This effect, which is usually attributed to the slightly greater
hydrophobicity of protiated molecules relative to their
29
deteurated isotopologues, complicates ratiometric analysis,
7
,8
and has been noted previously with ICATs. Surprisingly, in
this case, the deuterated tagged peptide has a longer retention
time, indicating a stronger interaction with the C18 stationary
phase. Thus, while ICMAT-1 (7/7*) and, by analogy,
ICMAT-2 (11/11*) are suitable for direct proteome analysis,
aYields and positions of 13C labelling in the heavy series are in red.
Heavy isotopologues are denoted with * in the text and the
Experimental Procedures.
D
https://doi.org/10.1021/acs.bioconjchem.1c00206
Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
pubs.acs.org/bc
Article
3
0
Scheme 3. Synthetic Approach to Light ICMAT-3 16 by
Nitration of Biotin Anilide (18) and Coupling of p-
Nitroaniline (20)
microscopy, and as a linker for surface plasmon resonance
31
studies. By varying the order of steps in the previously
reported syntheses, we were able to improve on the overall
yield of 16 somewhat. As outlined in Scheme 2, ring-opening
of maleic anhydride with p-phenylenediamine (12) gave the
31
maleamic acid 14. Acylation with biotin chloride then
provided the anilide 15. Finally, cyclization with the peptide
coupling agent HCTU (1-[bis(dimethylamino)methylene]-5-
chloro-1H-benzotriazolium-3-oxide hexafluorophosphate) gave
the target 16. Alternatively, under the action of HCTU,
acylation of 14 with biotin was accompanied by cyclization of
the maleamic acid moiety to give maleimide 16 in good crude
yield. Despite substantial losses during chromatographic
purification of 16, this one-step procedure provided 16 in
slightly higher overall yield than the two separate reactions.
While providing the light isotopologue reasonably efficiently,
the route outlined in Scheme 2 is suboptimal for application to
1
3
the heavy congener, because C -p-phenylenediamine (12*) is
6
not (readily) commercially available, and its preparation from
C -aniline requires four steps, doubling the length of the
1
3
32
6
synthesis. Accordingly, other routes were investigated. The first
of these is depicted in Scheme 3. Aniline (17) was biotinylated,
either with biotin chloride or, more straightforwardly and in
better yield, with biotin and HCTU. Attempts to nitrate the
1
resulting anilide 18 were unsuccessful. While H NMR analysis
of the crude reaction products suggested the desired para
nitration of the anilide moiety had occurred, the signals due to
urea NH groups were absent. Although the identity of the
product was not confirmed, N,N′-dinitration of ureas is well-
33,34
precedented.
Efforts were then devoted to the coupling of p-nitroaniline
20) with biotin (Scheme 3). Unsurprisingly, this non-
(
nucleophilic aniline failed to react with biotin activated by
HCTU, despite extended heating. Even the reaction with
biotin chloride was sluggish. After extensive attempted
optimization, extended heating in pyridine was found to give
an acceptable yield of the p-nitroanilide 19, and these
conditions allowed the recovery of unreacted p-nitroaniline
(
Scheme 2), was selected, as the heavy version could be
13
synthesized from reasonably priced C -aniline.
6
The light ICMAT-3 (16) has been previously synthesized
for the purpose of fabricating protein-reactive heteropolytung-
state anions as labels for conventional transmission electron
Figure 5. Representative standard curves based on 16/16*−CELAAAMK. Ratios of light:heavy-tagged peptides are determined by integral of the
+
2+
[
16/16*−CELAAAMK] ions in MALDI mass spectra (left) or [16/16*−CELAAAMK] extracted-ion chromatogram peak integrals using LC−
MS/MS (right).
E
https://doi.org/10.1021/acs.bioconjchem.1c00206
Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
pubs.acs.org/bc
Article
Figure 6. MALDI MS of 16/16*−CELAAAMK (top) and an extracted ion chromatogram (ESI+) showing coelution of light- and heavy-tagged
peptides (bottom).
(
20), which would be important for the 13C -labeled version.
When ICMAT-3 pair 16/16* was used for the analysis of
dystrophic dog muscle using a typical proteomic workflow, all
light/heavy-tagged peptides were found to coelute. Represen-
tative examples of extracted-ion chromatograms of dystrophic
dog muscle-derived tagged peptides are shown in Figure 7.
6
1
The coupling conversion, as judged by H NMR spectroscopy,
was higher in refluxing THF/NEt (60%), but the reaction was
3
messier and these conditions did not allow the recovery of the
p-nitroaniline. Reduction of the nitro group to give aniline 21
proceeded smoothly with DMF as solvent. An initial attempted
reduction in methanol failed to go to completion, assumedly
due to the poor solubility of a partially reduced intermediate en
route to 21. Presumably, 21 could have been advanced to
ICMAT 16, but the failure of the nitration of 18 and poor yield
of the biotinylation of p-nitroaniline (20) made these routes
unattractive, so this line of investigation was abandoned.
CONCLUSION
■
Three novel isotope-coded affinity tags bearing pendant
maleimide groups, ICMATs-1−3 have been designed,
synthesized, characterized, and evaluated in typical proteomics
protocols. The electrophilic maleimide moiety has the
advantage of more rapid and selective thiol-reactivity than
the iodacetamide (IAM) residue in commercially available
ICAT tags. Thus, compared to ICATs, smaller excesses of
ICMATs are required, reducing non-cysteine conjugation and
associated protein misidentification.
1
3
Instead, C -p-phenylenediamine (12*) was prepared as
6
32
previously described and coupled with maleimide to give
4* (Scheme 2). Cyclization/biotinylation then provided the
target heavy ICMAT-3 (16*).
1
1
2
13
ICMAT-1, in which the heavy isotopologue 7* contains an
octadeuterobutylene linker, was shown to be effective for the
tagging, identification, and ratiometric analysis of the model
protein lysozyme and dystrophic dog muscle tissue proteome.
However, some light/heavy-tagged peptide pairs had slightly
different HPLC retention times, making this proto/deutero
pair nonideal for workflows incorporating a chromatographic
fractionation step. In contrast, lysozyme and mammalian
muscle-derived peptides tagged with ICMAT-3 (16*), bearing
Evaluation of C/ C-Labeled ICMAT-3. As with the
deuterated probe, the suitability of the ICMAT-3 pair 16/16*
for ratiometric protein quantification was initially assessed
using lysozyme. Once again, the ratios of light:heavy-tagged
peptide determined by MALDI mass spectrometry, or LC−
MS, matched the prepared ratios (Figure 5). A representative
MALDI spectrum and extracted-ion chromatogram are also
included (Figure 6). Importantly, in the LC−MS trace, perfect
coelution of light- and heavy-tagged peptides was observed.
1
3
a C -phenylenediamine linker, had identical chromatographic
6
F
https://doi.org/10.1021/acs.bioconjchem.1c00206
Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
pubs.acs.org/bc
Article
Figure 7. Representative extracted-ion chromatograms of ICMAT-16/16*-tagged peptides from dystrophic dog muscle. (A) Aconitate hydratase-
2
+
derived peptide [C(16/16*)KSQFTITPGSEQIR] with light/heavy m/z values of 847.93 and 850.94, respectively; (B) glycogen phosphorylase-
3
+
derived peptide [RWLVLC(7/7*)NPGLAEVIAER] with light/heavy m/z values of 647.02 and 649.03, respectively.
mobility to the light (16-tagged) isotopologues, and retained
all the benefits of the deuterated probe. ICMAT-3 has proven
useful for our proteomic studies comparing protein−cysteine
oxidation state in various normal, stressed, and diseased tissue
types.
raphy was performed using Davisil chromatographic silica
media LC60A 40−63 μm.
1
13
H and C NMR spectra were acquired using Bruker
1
13
Avance IIIHD (600 MHz for H and 150 MHz for C),
1
Bruker Avance IIIHD (500 MHz for H and 125 MHz for
C), Varian VNMRS (400 MHz for H and 100 MHz for
C), or Varian Inova (300 MHz for H and 75 MHz for C)
1
1
3
3
1
1
13
EXPERIMENTAL PROCEDURES
■
spectrometers and calibrated using residual monoprotiated
solvent as internal reference.
General. All solvents were distilled prior to use. Anhydrous
THF was obtained from a Pure Solv 5-Mid Solvent Purification
System (Innovative Technology Inc.). “Dry” DMF, DCM,
MeCN, toluene, and MeOH refers to solvents stored over
activated 3 Å molecular sieves for at least 24 h. Triethylamine
High-resolution mass spectra were recorded on a Waters
Liquid Chromatograph Premier mass spectrometer using ESI
or APCI in positive or negative mode, as indicated. IR spectra
were recorded on a PerkinElmer Spectrum One FT-IR
spectrometer with attenuated total reflectance (ATR) using
neat samples.
(
NEt ) and pyridine were dried over and distilled from CaH
3
2
onto KOH pellets under inert gas. All other reagents and
materials were purchased from commercial suppliers and used
as received.
Synthesis. The protected maleimide 5 was prepared as
35
described previously.
Denotes heavy isotopologues throughout. Skeleton
numbering is shown in the Supporting Information.
All reactions were conducted under an inert atmosphere,
unless otherwise specified. Where indicated, reaction temper-
atures refer to the temperature of the heating or cooling bath.
*
2
5
4
-Iodobutan-1-ol (2). BF •OEt (11.4 g, 80.0 mmol) was
All organic extracts were dried over MgSO , filtered, and
3
2
4
evaporated under reduced pressure at 40−50 °C. Trace
added dropwise over 5 min to a solution of NaI (12.0 g, 80.1
mmol) in anhydrous THF (2.88 g, 40.0 mmol) and dry MeCN
(80 mL). This solution was stirred overnight, whereupon it
residual solvent was removed under a stream of N or using
2
high vacuum.
Reaction progress was monitored by TLC using Merck
aluminum-backed TLC silica gel 60 F254 plates. Spots were
visualized directly (colored compounds), by UV light, or by
developed an orange/brown color. Saturated NaHCO (20
3
mL) was then added, and the reaction mixture was stirred for
30 min before extraction with Et O (3 × 30 mL). The extract
2
staining with KMnO or ninhydrin. Flash column chromatog-
was washed with water (2 × 20 mL), 1 M sodium thiosulfate
4
G
https://doi.org/10.1021/acs.bioconjchem.1c00206
Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
30 mL), H O (2 × 20 mL), and brine (20 mL), then dried
pubs.acs.org/bc
Article
(
and extracted into EtOAc (4 × 30 mL). The extract was
washed with H O (2 × 20 mL) and brine (20 mL), dried, and
evaporated. The brown residue was subjected to flash
chromatography. Elution with a gradient of DCM to 1:9
MeOH/DCM yielded 6 as a white powder (102 mg, 55%), mp
2
and evaporated to yield the iodide 1 as yellow oil (7.40 g,
2
1
9
3%), which was used without further purification. H NMR
(
2
2
CDCl , 400 MHz): δ 4.84 (br s, 1H, OH), 3.73 (t, J = 6.3 Hz,
3
H, OCH ), 3.23 (t, J = 6.9 Hz, 2H, CH I), 1.98−1.86 (m,
2
2
1
−1
H, CH ), 1.78−1.66 (m, 2H, CH ). The H NMR data
= 146−148 °C. IR (thin film) cm : 3320 (NH), 3212 (br,
2
2
36
match those reported.
NH), 1723 (OCO), 1703 (CO), 1683 (CO), 1657
37
1
1
,1,2,2,3,3,4,4-Octadeutero-4-iodobutan-1-ol (2*). Pre-
(CO). H NMR (CDCl , 300 MHz): δ 6.51 (s, H5‴/6‴),
3
pared in a similar fashion to 1, using d -THF (0.80 g, 10.0
5.48 (br s, 1H, NH), 5.27 (s, 2H, H4‴/7‴), 4.99 (br s, 1H,
NH), 4.53−4.49 (m, 1H, CH, H6a′), 4.34−4.30 (m, 1H, CH,
H3a′), 4.06 (m, 2H, H1″), 3.52 (m, 2H, H4″), 3.20−3.12 (m,
1H, H4′), 2.92 (dd, J = 5.1 Hz, 12.9 Hz, H6′), 2.86 (s, 2H,
H3a‴/7a‴), 2.72 (d, J = 13.2 Hz, 1H, H6′), 2.32 (t, J = 6.9 Hz,
8
mmol), BF •OEt (2.84 g, 20.0 mmol), and NaI (3.00 g, 20.0
3
2
mmol) in dry MeCN (20 mL). The iodide 2* was isolated as a
1
yellow oil (2.00 g, 96%). H NMR (CDCl , 300 MHz): δ 1.62
3
(
br s, 1H, OH + H O).
2
4
-Iodobutyl 5-((4R)-2-oxohexahydro-1H-thieno[3,4-d]-
2H, H2), 1.80−1.60 (m, 8H, 4 × CH ), 1.50−1.38 (m, 2H,
2
1
3
imidazol-4-yl)pentanoate (4-iodobutyl D-biotinate) (4).
H4). C NMR (CDCl , 75 MHz): δ 176.6 (C1‴/3‴), 173.8
3
38
The method of Jung et al. was adapted. A dry round-bottom
flask was fitted with a Dean−Stark apparatus and charged with
D-(+)-biotin (3) (0.98 g, 3.9 mmol), TsOH (0.14 g, 0.80
mmol), and dry toluene (70 mL) under argon. The stirred
mixture was heated to 50 °C, and (1) (1.60 g, 8.00 mmol) was
added over 5 min. The solution was heated under reflux for 24
h, then the toluene was evaporated to give a brown residue,
which was subjected to flash chromatography. Elution with
DCM then 1:9 MeOH/DCM gave ester 4 as a yellow solid
(C1), 163.3 (C2′), 136.7 (C5‴/6‴), 81.1 (C4‴/7‴), 63.8
(C1″), 62.0 (C3a′), 60.2 (C6a′), 55.4 (C4′), 47.6 (C3a‴/
7a‴), 40.7 (C6′), 38.6, 34.0 (C2), 28.4, 26.0, 25.9, 24.9, 24.4.
+
+
HRMS (ESI+) m/z: Calcd for C H N NaO S [M + Na] ,
2
2
29
3
6
486.1669; found, 486.1674.
1,1,2,2,3,3,4,4-Octadeutero- (4-(4,7-epoxy-1,3-dioxo-3a,4-
dihydro-1H-isoindol-2(3H,7H,7aH)-yl)butyl D-biotinate (6*).
This compound was prepared as described for 6 using iodide
4* (300 mg, 0.70 mmol), 5 (165 mg, 1.00 mmol), K CO
(420 mg, 3.00 mmol), and anhydrous DMF (7 mL), yielding
3
5
2
3
−
1
(
0.75 g, 45%), mp = 94−96 °C. IR (thin film) cm : 3214 (br,
1
2
4
4
× NH), 1727 (OCO) 1697 (CO). H NMR (CDCl ,
6* as a white solid (148 mg, 48%), mp = 144−146 °C. IR
3
−
1
00 MHz): δ 5.37 (br s, 1H, NH), 5.03 (br s, 1H, NH), 4.54−
.49 (m, 1H, H6a′), 4.34−4.30 (m, 1H, H3a′), 4.10 (t, J = 6.4
(thin film) cm : 3322 (NH), 3219 (br, NH), 1702 (CO),
1
1680 (CO), 1658 (CO). H NMR (CDCl , 300 MHz): δ
3
Hz, 2H, H1″), 3.21 (t, J = 6.8 Hz, 2H, H4″), 3.20−3.14 (m,
6.51−6.52 (m, 2H, C5‴/6‴), 5.77 (br s, 1H, NH), 5.30 (br s,
1H, NH), 5.26 (s, 2H, H7‴, 4‴, 4.52−4.48 (m, 1H, CH,
H6a′), 4.33−4.29 (m, 1H, CH, H3a′), 3.16−3.12 (m, 1H,
H4′), 2.93 (dd, J = 4.8, 12.9 Hz, 1H, H6′), 2.86 (s, 2H, H3a‴/
7a‴), 2.72 (d, J = 12.9 Hz, 1H, H6′), 2.31 (t, J = 7.2 Hz, 2H,
1
1
(
1
H, H4′), 2.96 (dd, J = 4.8, 12.8 Hz, 1H, H6′), 2.74 (d, J =
2.8 Hz, 1H, H6′), 2.33 (t, J = 7.6 Hz, 2H, H2), 1.94−1.86
m, 2H, CH , H2″), 1.78−1.64 (m, 6H, 3 × CH , H3, 5, 3″),
2
2
13
.50−1.38 (m, 2H, CH , H4). C NMR (CDCl , 100 MHz):
2
3
1
3
δ 173.7 (C1), 163.3 (C2′), 63.4 (C1″), 62.1 (C3a′), 60.2
C6a′), 55.4 (C4′), 40.7 (C6′), 34.0 (C2), 30.2, 29.7, 28.5,
H2), 1.80−1.60 (m, 4H, H3, 5), 1.48−1.41 (m, 2H, H4). C
(
2
NMR (CDCl , 75 MHz): δ 176.6, (C1‴/3‴), 173.8 (C1),
3
8.4, 24.9, 6.1 (C4″). HRMS (ESI+) m/z: Calcd for
163.6 (C2′), 136.7 (C5‴/6‴), 81.1 (C4‴/7‴), 62.0 (C3a′),
60.2 (C6a′), 55.5 (C4′), 47.6 (C3a‴/7a‴), 40.7 (C6′), 34.0
(C2), 28.4, 28.3, 24.9. HRMS (ESI+) m/z: Calcd for
+
+
C H IN NaO S [M + Na] , 449.0366; found, 449.0362.
1
4
23
2
3
1
,1,2,2,3,3,4,4-Octadeutero-4-iodobutyl 5-((4R)-2-oxohex-
+
+
ahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoate
1,1,2,2,3,3,4,4-octadeutero-4-iodobutyl D-biotinate) (4*).
* was prepared as described for 4 from 2* (800 mg, 4.00
mmol), D-(+)-biotin (3) (488 mg, 2.00 mmol), and TsOH
C H D N NaO S [M + Na] , 494.2171; found, 494.2170.
22 21 8 3 6
(
4
4-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)butyl 5-((4R)-2-
oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoate
(7). Following a variation of the method of Reetz et al.,
39
6
(
(
(
(
70.0 mg 0.20 mmol), and yielding 4* as a yellow powder
(225 mg, 0.485 mmol) was dissolved in dry toluene (50 mL)
and heated under reflux for 24 h. The solution was evaporated,
and the white solid residue was subjected to flash
chromatography. Elution with DCM−1:9 MeOH:DCM
yielded 7 as a white powder (141 mg, 74%), mp = 90−92
−
1
0.588 g, 68%), mp = 94−96 °C. IR (thin film) cm : 3211
1
br, 2 × NH), 1726 (OCO), 1697 (CO). H NMR
CDCl , 400 MHz): δ 5.63 (br s, 1H, NH), 5.26 (br s, 1H,
3
NH), 4.52−4.49 (m, 1H, CH, H6a′), 4.33−4.30 (m, 1H, CH,
−
1
H3a′), 3.18−3.13 (m, 1H, H4′), 2.94 (dd, J = 5.1, 12.8 Hz,
°C. IR (thin film) cm : 3255 (br NH), 1698 (CO,
1
1
2
H, H6′), 2.74 (d, J = 12.8 Hz, 1H, H6′), 2.33 (t, J = 7.6 Hz,
unresolved). H NMR (CDCl , 300 MHz): δ 6.71 (s, 2H,
3
H, H2), 1.75−1.64 (m, 4H, H3, 5), 1.50−1.38 (m, 2H, CH ,
H4‴/5‴), 5.82 (br s, 1H, NH), 5.40 (br s, 1H, NH), 4.50−
4.48 (m, 1H, H6a′), 4.34−4.30 (m, 1H, H3a′), 4.07 (t, J = 6.4
Hz, 2H, H1″), 3.54 (t, J = 6.8 Hz, 2H, H4‴), 3.20−3.12 (m,
1H, H4′), 2.90 (dd, J = 4.8 Hz, J = 12.6 Hz, 1H, H6′), 2.72 (d,
J = 12.6 Hz, 1H, H6′), 2.31 (t, J = 7.6 Hz, 2H, H2), 1.78−1.64
2
1
3
H4). C NMR (CDCl , 100 MHz): δ 173.8 (C1), 163.6
3
(
(
C2′), 62.1 (C3a′), 60.2 (C6a′), 55.5 (C4′), 40.7 (C6′), 34.0
C2), 28.5, 28.4, 24.9. HRMS (ESI+) m/z: Calcd for
+
+
C H D IN NaO S [M + Na] , 457.0868; found, 457.0872.
1
4
15
8
2
3
1
3
(
4-(4,7-Epoxy-1,3-dioxo-3a,4-dihydro-1H-isoindol-2-
(m, 6H), 1.50−1.38 (m, 2H, H4). C NMR (CDCl , 100
3
(
3H,7H,7aH)-yl)butyl D-biotinate (6). This method was
MHz): δ 173.8 (C1), 171.0 (C1‴/3‴), 163.7 (C2′), 134.3
(C4‴/5‴), 63.8 (C1″), 62.1 (C3a′), 60.2 (C6a′), 55.5 (C4′),
40.7 (C6′), 37.5 (C4″), 34.0 (C2), 28.5, 28.3, 26.0, 25.3, 24.9.
39
adapted from that of Reetz et al. A solution of iodide 4
0.50 g, 1.2 mmol) in anhydrous DMF (5 mL) was added
(
3
5
+
+
dropwise over 2 min to a stirred suspension of 5 (0.231 g,
.40 mmol) and K CO (0.581 g, 4.20 mmol) in anhydrous
HRMS (ESI+) m/z: Calcd for C H N O SNa [M + Na] ,
18 25 3 5
1
418.1407; found, 418.1411.
2
3
DMF (10 mL) at 50 °C under N . The reaction mixture
1,1,2,2,3,3,4,4-Octadeutero-4-(2,5-dioxo-2,5-dihydro-1H-
pyrrol-1-yl)butyl 5-((4R)-2-oxohexahydro-1H-thieno[3,4-d]-
imidazol-4-yl)pentanoate (7*). Following the procedure for
2
turned yellow initially before developing a red/brown color.
After 4 h, the reaction mixture was diluted with H O (150 mL)
2
H
https://doi.org/10.1021/acs.bioconjchem.1c00206
Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
pubs.acs.org/bc
Article
the synthesis of 7, using 6* (225 mg, 0.477 mmol) gave 7* as a
white powder (167 mg, 84%), mp = 88−90 °C. IR (thin film)
evaporated, and the residue was subjected to flash chromatog-
raphy. Elution with 1:1 EtOAc/hexanes gave 12 as a white
−
1
1
1
cm : 3222 (br, NH), 1690 (CO unresolved). H NMR
solid (180 mg, 39%). H NMR (d -DMSO, 400 MHz): δ 6.53
6
(
CDCl , 300 MHz): δ 6.70 (s, 2H, H4‴, 5‴), 5.97 (br s, 1H,
(d, J = 0.9 Hz, 2H, HCCH), 5.31 (d, J = 0.6 Hz, 2H, HCO),
3
NH), 5.58 (br s, 1H, NH), 4.51−4.47 (m, 1H, CH, H6a′),
4.24 (d, J = 2.4 Hz, 2H, αCH), 2.91 (s, 2H, H CC), 2.20 (s,
2
1
39
4
.32−4.27 (m, 1H, CH, H3a′), 3.20−3.11 (m, 1H, CH, H4′),
.92 (dd, J = 4.8 Hz, J = 12.9 Hz, 1H, H6′), 2.72 (d, J = 12.9
1H, CH). The H NMR data match those reported.
2
4-(4-((4,7-Epoxy-1,3-dioxo-3a,4-dihydro-1H-isoindol-2-
(3H,7H,7aH)-yl)methyl)-1H-1,2,3-triazol-1-yl)butyl D-bioti-
nate (10). This was adapted from the method of Mukai et
Hz, 1H, H6′), 2.30 (t, J = 7.2 Hz, 2H, H2), 1.78−1.60 (m,
13
4
H), 1.50−1.38 (m, 2H, CH , H4). C NMR (CDCl , 100
2
3
41
MHz): δ 173.7 (C1), 171.0 (C1‴/3‴), 163.1 (C2′), 134.3
C4‴/5‴), 62.0 (C3a′), 60.2 (C6a′), 55.3 (C4′), 40.7 (C6′),
4.0 (C2), 28.4, 28.4, 24.9. HRMS (ESI+) m/z: Calcd for
al. The masked N-propargylmaleimide 9 (142 mg, 0.699
mmol) was added to a stirred solution of CuSO ·5H O (15
mg, 0.060 mmol) and sodium ascorbate (24 mg, 0.12 mmol)
(
3
4
2
+
+
C H D N O SNa [M + Na] , 426.1909; found, 426.1927.
in 1:1 DMF:H O (5 mL). After 5 min, 8 (171 mg, 0.501
1
8
17
8
3
5
2
4
-Azidobutyl 5-((4R)-2-Oxohexahydro-1H-thieno[3,4-d]-
mmol) was added, whereupon a deep red color developed.
imidazol-4-yl)pentanoate (8). Following a method adapted
from Bates et al., 4 (213 mg, 0.500 mmol) was dissolved in
anhydrous DMF (5 mL) at 0 °C. NaN (100 mg, 1.50 mmol)
was added portion-wise, and the resultant suspension was
allowed to warm to room temperature and stirring was
continued for 24 h. The solution was diluted with H O (150
mL) and extracted with CHCl (4 × 30 mL). The extract was
After 24 h, the reaction mixture was diluted with H O (200
2
40
mL) and extracted with EtOAc (6 × 20 mL). The extract was
washed with water (20 mL) and brine (3 × 20 mL), dried, and
evaporated, and the residue was subjected to flash chromatog-
raphy. Elution with DCM then 1:9 MeOH:DCM gave 13 as a
white solid (70 mg, 28%), mp = 150−152 °C. IR (thin film)
3
2
−
1
1
cm : 3245 (br, NH), 1695 (CO unresolved). H NMR
3
washed with water (2 × 20 mL) and brine (20 mL), dried, and
evaporated, and the residue was subjected to flash chromatog-
raphy. Elution with DCM−1:9 MeOH/DCM yielded 8 as a
yellow waxy solid (160 mg, 94%), mp = 86 °C. IR (thin film)
(CDCl , 300 MHz): δ 7.50 (s, 1H, H5‴), 6.53 (s, 2H, H5′‴/
3
6′‴), 5.34 (br s, 1H, NH), 5.29 (s, 2H, H4′‴/7′‴), 4.94 (br s,
1H, NH), 4.79 (s, 2H, Hα4‴), 4.54−4.50 (m, 1H, CH, H6a′),
4.34 (t, J = 6.9 Hz, 2H, H4″), 4.34 (m, 1H, H3a′) 4.08 (t, J =
6.3 Hz, 2H, H1″), 3.20−3.12 (m, 1H, H4′), 2.92 (dd, J = 5.1
Hz, J = 12.6 Hz, 1H, H6′), 2.91 (s, 2H, H3a′‴/7a′‴), 2.72 (d,
J = 12.9 Hz, 1H, H6′), 2.32 (t, J = 7.2 Hz, 2H, H2), 2.00−1.90
−
1
cm : 3216 (NH), 2095 (N ), 1727 (CO), 1697 (CO).
3
1
H NMR (CDCl , 400 MHz): δ 5.77 (br s, 1H, NH), 5.39 (br
3
s, 1H, NH), 4.53−4.48 (m, 1H, CH, H6a′), 4.33−4.29 (m,
1
H, CH, H3a′), 4.09 (t, J = 6.4 Hz, 2H, 1H″), 3.32 (t, J = 6.8
(m, 2H), 1.80−1.60 (m, 6H, CH
2
), 1.50−1.38 (m, 2H, H4).
1
3
Hz, 2H, H4″), 3.20−3.12 (m, 1H, H4′), 2.90 (dd, J = 4.8 Hz, J
C NMR (CDCl3, 75 MHz): δ 175.9 (C1′‴/3′‴), 173.7
=
=
6
12.8 Hz, 1H, H6′), 2.73 (d, J = 12.8 Hz, 1H, H6′), 2.33 (t, J
(C1), 163.2 (C2′), 142.4, 136.7 (C5′‴/6′‴), 122.6, 81.2
(C4′‴/7′‴), 63.5 (C1″), 62.1 (C3a′), 60.2 (C6a′), 55.4 (C4′),
50.0 47.7 (C3a′‴/7a′‴), 40.7, 34.3, 34.0, 28.55, 28.4, 27.2,
7.6 Hz, 2H, H2), 2.00−1.86 (m, 2H, CH ), 1.76−1.62 (m,
2
13
H), 1.50−1.38 (m, 2H, CH , H4). C NMR (CDCl , 100
2
3
+
MHz): δ 173.9 (C1), 163.7 (C2′), 63.8 (C1″), 62.1 (C3a″),
25.8, 24.9. HRMS (ESI+) m/z: Calcd for C H N O SK [M
2
5
32
6
6
+
6
2
0.2 (C6a′), 55.5 (C4′), 51.1, 40.7, 34.0, 28.5, 28.4, 26.0, 25.7,
+K] , 583.1736; found, 583.1724.
+
4.9. HRMS (ESI+) m/z: Calcd for C H N O SNa [M +
4-(4-((1,3-Dioxo-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoin-
dol-2(3H)-yl)methyl-1H-1,2,3-tirazol-1-ylbutyl biotinate
(10*). The procedure above was applied to 8* (191 mg,
0.547 mmol), 9 (176 mg, 0.866 mmol), CuSO ·5H O (13 mg,
14
23
5
3
+
Na] , 364.1414; found, 364.1420.
,1,2,2,3,3,4,4-Octadeutero-4-azidobutyl 5-((4R)-2-oxo-
1
hexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoate (8*).
A procedure similar to that above was applied to 4* (294
mg, 0.677 mmol) and NaN3 (137 mg, 2.10 mmol) in
anhydrous DMF (5 mL) to afford 8* as a yellow waxy solid
4
2
0.052 mmol) and sodium ascorbate (20 mg, 0.10 mmol) in 1:1
DMF:H O (5 mL). Purification as above gave 10* as a white
2
powder (129 mg, 47%), mp = 148−150 °C. IR (thin film)
−
1
−1
1
(
214 mg, 90%), mp = 84−86 °C. IR (thin film) cm : 3215,
cm : 3312 (br, NH), 1742 (CO), 1698 (CO). H NMR
1
2
4
4
3
096 (N ), 1728 (CO), 1699 (CO). H NMR (CDCl ,
(CDCl , 300 MHz): δ 7.49 (s, 1H, H5‴), 6.50 (s, 2H, H5′‴/
3
3
3
00 MHz): δ 5.65 (br s, 1H, NH), 5.28 (br s, 1H, NH), 4.52−
.49 (m, 1H, CH, H6a′), 4.32−4.29 (m, 1H, CH, H3a′),
.18−3.13 (m, 1H, H4′), 2.91 (dd, J = 4.8 Hz, J = 12.8 Hz, 1H,
6′‴), 5.99 (br s, 1H, NH), 5.58 (br s, 1H, NH), 5.26 (s, 2H,
H4′‴/7′‴), 4.75 (s, 2H, CH , Hα4‴), 4.50−4.46 (m, 1H, CH,
2
H6a′), 4.30−4.26 (m, 1H, CH, H3a′), 3.14−3.12 (m, 1H,
H4′), 2.89 (dd, J = 7.5 Hz, J = 19.5 Hz, 1H, H6′), 2.89 (s, 2H,
H3a′‴/7a′‴), 2.70 (d, J = 12.9 Hz, 1H, H6′), 2.29 (t, J = 7.5
H6′), 2.73 (d, J = 12.8 Hz, 1H, H6′), 2.33 (t, J = 7.6 Hz, 2H,
1
3
H2), 1.76−1.63 (m, 4H), 1.50−1.38 (m, 2H, CH , H4).
C
2
NMR (CDCl , 100 MHz): δ 173.8 (C1), 163.6 (C2′), 62.1
Hz, 2H, H2), 1.80−1.60 (m, 4H, CH ), 1.46−1.38 (m, 2H,
3
2
1
3
(
2
[
C3a′), 60.2 (C6a′), 55.5 (C4′), 40.7 (C6′), 34.0 (C2), 28.5,
CH , H4). C NMR (CDCl , 75 MHz): δ 175.8, (C1′‴/3′‴),
2
3
+
8.4, 24.9. HRMS (ESI+) m/z: Calcd for C H D N O SNa
173.8 (C1), 163.8 (C2′), 142.3, 136.7 (C5′‴/6′‴), 122.6, 81.1
(C4′‴/7′‴), 62.0 (C3a′), 60.2 (C6a′), 55.6 (C4′), 47.6
(C3a′‴/7a′‴), 40.6, 34.3, 34.0, 28.56, 28.3, 24.9. HRMS (ESI
14
15
8
5
3
+
M + Na] , 372.1916; found, 372.1924.
-(2-Propynyl)-3a,4,7,7a-tetrahydro-4,7-epoxy-1H-isoin-
dole-1,3(2H)-dione (9). This method was adapted from Reetz
2
+
+
+) m/z: Calcd for C H D N O SNa [M + Na] , 575.2498;
2
5
24
8
6
6
3
9
et al. A solution of propargyl bromide (0.55 g, 4.6 mmol) in
anhydrous DMF (10 mL) was added dropwise to a suspension
of the protected maleimide (5) (0.39 g, 2.3 mmol) and
K CO (1.60 g, 11.5 mmol) in anhydrous DMF (10 mL)
under argon. The reaction mixture was heated to 50 °C and
stirred for 6 h. It was then diluted with H O (400 mL) and
found, 575.2510.
4-(4-((2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)-1H-
1,2,3-triazol-1-yl)butyl biotinate (11). A solution of 10 (70
mg, 0.15 mmol) in dry toluene (20 mL) was heated under
reflux for 24 h. The volatiles were evaporated, and the residue
was subjected to flash chromatography. Elution with DCM−
1:9 MeOH:DCM gave 11 as a white powder (49 mg, 81%),
35
2
3
2
extracted with EtOAc (4 × 30 mL). The extract was washed
with water (2 × 20 mL) and brine (20 mL), dried, and
−
1
mp = 144 °C. IR (thin film) cm : 3227 (br, NH), 1700 (C
I
https://doi.org/10.1021/acs.bioconjchem.1c00206
Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
pubs.acs.org/bc
Article
1
O unresolved), 1646 (CO). H NMR (CDCl , 400 MHz): δ
by rapid silica filtration (1:19−1:10 MeOH/DCM) gave 14*
3
1
7
.56 (s, 1H, H5‴), 6.74 (s, 2H, H4′‴/5′‴), 5.11 (br s, 1H,
as an off-white powder (55 mg, 58%). H NMR (d -DMSO,
6
NH), 4.82 (s, 2H, Hα4″), 4.79 (br s, 1H, NH), 4.54−4.50 (m,
500 MHz): δ 10.64 (s, 1H, NH), 7.30 (dm, J = 158.3 Hz, 2H,
H2′/6′), 6.52 (dm, J = 154.8 Hz, 2H, H3′/5′), 6.45 (d, J =
1
1
3
H, CH, H6a′), 4.36 (t, J = 7.2 Hz, 2H, H4″), 4.35−4.32 (m,
H, CH, H3a′), 4.09 (t, J = 6.4 Hz, 2H, CH2, H1″), 3.20−
.15 (m, 1H, H4′), 2.95 (dd, J = 4.8 Hz, J = 12.8 Hz, 1H,
12.4 Hz, 1H, H2), 6.25 (d, J = 12.4 Hz, 1H, H3), NH and
2
1
3
COOH not observed. C NMR (d -DMSO, 100 MHz): δ
6
H6′), 2.73 (d, J = 12.8 Hz, 1H, H6′), 2.33 (t, J = 7.6 Hz, 2H,
166.2 (C4), 162.5 (C1), 146.0 (td, J = 60.5 Hz, J = 8.9 Hz,
1
2
H2), 2.01−1.94 (m, 2H), 1.74−1.59 (m, 6H, CH ), 1.50−1.42
C1′), 132.3 (C2), 131.8 (d, J = 3.7 Hz, C3), 126.8 (td, J =
2
1
13
(
(
m, 2H, CH , H4). C NMR (CDCl , 75 MHz): δ 173.6
63.9 Hz, J = 8.8 Hz, C4′), 121.4 (td, J = 61.8 Hz, J = 5.4 Hz,
2
3
2
1
2
C1′‴/3′‴), 170.3 (C1), 163.0 (C2′), 142.8, 134.5 (C4′‴/
′‴), 122.8, 63.58 (C1″), 62.0 (C3a′), 60.2 (C6a′), 55.4
C4′), 50.0, 40.7, 33.9, 33.0, 28.4, 28.4, 27.2, 25.8, 24.9.
C2′/6′), 113.7 (td, J = 60.2 Hz, J = 5.8 Hz, C3′/5′).
1
2
5
(Z)-4-Oxo-4-((4-(((5-((3aR,4R,6aS)-2-oxohexahydro-1H-
thieno[3,4-d]imidazol-4-yl)pentanoyl)oxy)amino)phenyl)-
amino)but-2-enoic acid (N-4-(D-biotinylaminophenyl)-
maleamic acid) (15). D-(+)-Biotin (3) (245 mg, 1.00
mmol) was stirred with SOCl (1.5 mL) under a CaCl
(
+
+
HRMS (ESI+) m/z: Calcd for C H N O SNa [M + Na] ,
21
28
6
5
4
99.1734; found, 499.1746.
,1,2,2,3,3,4,4-Octadeutero-4-(4-((2,5-dioxo-2,5-dihydro-
H-pyrrol-1-yl)methyl)-1H-1,2,3-triazol-1-yl)butyl biotinate
1
2
2
1
guard tube for 2 h, after which the SOCl was evaporated
2
(
0
1
11*). The procedure above was applied to 10* (110 mg,
under a stream of N . The residue was dissolved in THF (5
mL) and DMF (3 drops). Aniline 14 (206 mg, 1.00 mmol)
2
.20 mmol) in dry toluene (10 mL). Purification as above gave
1* as a white powder (83 mg, 87%), mp = 150−152 °C. IR
and NEt (0.28 mL, 2.0 mmol) were dissolved in anhydrous
3
−
1
(
thin film) cm : 3228 (br, NH), 1700 (CO), 1684 (C
DMF (2.5 mL) and cooled to 0 °C. The biotin chloride
solution was added dropwise to the aniline solution over 40
min. The solvent was evaporated, and the residue was diluted
1
O), 1646 (CO). H NMR (CDCl , 400 MHz): δ 7.55 (s,
3
1
H, CH, H5‴), 6.74 (s, 2H, H4′‴/5′‴), 5.19 (br s, 1H, NH),
.86 (br s, 1H, NH), 4.82 (s, 2H, Hα4′), 4.54−4.51 (m, 1H,
4
with H O (20 mL) added. The resulting precipitate was
2
CH, H6a′), 4.34−4.31 (m, 1H, CH, H3a′), 3.20−3.14 (m, 1H,
H4′), 2.93 (dd, J = 5.2 Hz, J = 12.8 Hz, 1H, H6′), 2.73 (d, J =
collected and triturated with boiling EtOH, affording a
1
yellow−green powder (246 mg, 57%). H NMR (d -DMSO,
6
1
2.8 Hz, 1H, H6′), 2.33 (t, J = 7.2 Hz, 2H, H2), 1.75−1.59
400 MHz): δ 11.17 (br s, 1H, NH), 9.85 (s, 1H, NH), 7.53 (s,
4H, ArH), 6.43 (s, 1H, NH), 6.21−6.36 (m, 3H, NH + H2/3),
4.28−4.32 (m, 1H, CH, H6a′), 4.12−4.16 (m, 1H, CH, H3a′),
3.10−3.14 (m, 1H, CH, H4′), 2.82 (dd, J = 5.1 Hz, J = 12.4
1
3
(
(
(
(
(
5
m, 4H, CH ), 1.50−1.42 (m, 2H, CH , H4). C NMR
2
2
CDCl , 75 MHz): δ 173.7 (C1′‴/3′‴), 170.3 (C1), 163.1
3
C2′), 142.8, 134.5 (C4′‴/5′‴), 122.8, 62.1 (C3a′), 60.2
1
2
C6a′), 55.4 (C4′), 40.7, 33.9, 33.0, 28.4, 28.4, 24.9. HRMS
Hz, 1H, H6′), 2.58 (d, J = 12.4 Hz, 1H, H6′), 2.33 (t, J = 8.0
Hz, 2H, CH , H2″), 1.33−1.69 (m, 6H, 3 × CH , H3″/4′′/
+
+
ESI+) m/z: Calcd for C H D N O SNa [M + Na] ,
21
20
8
6
5
2
2
07.2236; found, 507.2242.
5′′).
1
3
C -p-Phenylenediamine (12*). A suspension of 10% Pd/
1-(4-(((5-((3aR,4R,6aS)-2-Oxohexahydro-1H-thieno[3,4-
d]imidazol-4-yl)pentanoyl)oxy)amino)phenyl)-1H-pyrrole-
2,5-dione (N-4-(D-biotinylaminophenyl)maleimide) (16).
6
13
42
C (72 mg) in a solution of C -p-nitroaniline (20*) (75 mg,
6
0
.52 mmol) in dry MeOH (2 mL) was stirred under a balloon
31
of H for 5 h, after which time TLC (EtOAc) indicated the
Method A (Adapted from Quideau et al. ): HCTU (63 mg,
0.15 mmol) was added to a stirred solution of the maleamic
acid 15 (42 mg, 0.098 mmol) and Hunig’s base (26 mg, 0.20
2
reaction was complete. The mixture was diluted with DCM
(
10 mL) and filtered through Celite. The solvent was
evaporated to afford 12* as pale pink crystals (51 mg, 86%).
mmol) in anhydrous DMF (5 mL) at 0 °C, under N . The
2
1
H NMR (CDCl , 500 MHz) δ 6.33−6.85 (m, 4H, ArH), 3.35
solution was allowed to warm to room temperature. After 4.5
h, the reaction was poured into water and extracted with
EtOAc (3 × 20 mL). The combined organic phase was washed
with H O (20 mL), dilute HCl (20 mL), NaHCO (20 mL),
3
3
1
(
(
s, 4H, NH ). C NMR (CDCl , 125 MHz) δ 138.1−139.1
2
3
m, CH), 116.4−116.9 (m, CNH ). HRMS (ESI+) m/z:
2
1
3
+
+
calcd. for C H N [M + H] 115.0962, found 115.0960.
6
9
2
2
3
(
Z)-3-((4-Aminophenyl)carbamoyl)prop-2-enoic acid (N-
and brine (20 mL). The solution was dried and evaporated,
and the residue was subjected to flash chromatography. Elution
3
1
4
-aminophenylmaleamic acid) (14). A solution of maleic
anhydride (0.98 g, 10 mmol) in anhydrous THF (10 mL) was
added dropwise to a stirred solution of p-phenylenediamine
with 1:19 MeOH/DCM gave the maleimide 16 as a pale
1
yellow powder (18 mg, 43%). H NMR (d -DMSO, 500
6
(
12) (1.08 g, 10.0 mmol) in anhydrous THF (20 mL) over 30
MHz) δ 10.03 (s, 1H, NH), 7.67 (d, J = 8.9 Hz, 2H, H2‴/6‴),
7.23 (d, J = 8.9 Hz, 2H, H3‴/5‴), 7.16 (s, 2H, H3/4) 6.43 (s,
1H, NH), 6.35 (s, 1H, NH), 4.28−4.32 (m, 1H, CH, H6a′),
4.12−4.16 (m, 1H, CH, H3a′), 3.10−3.14 (m, 1H, CH, H4′),
2.82 (dd, J = 5.1 Hz, J = 12.4 Hz, 1H, H6′), 2.58 (d, J = 12.4
min, whereupon a precipitate formed. After 24 h, the
suspension was vacuum filtered and the precipitate washed
with DCM (30 mL). The olive-green powder was triturated
with boiling MeOH (50 mL) and collected by vacuum
filtration to give the maleamic acid 14 as a gray powder (1.85
g, 90%). H NMR (d -DMSO, 400 MHz): δ 10.53 (s, 1H,
1
2
Hz, 1H, H6′), 2.33 (t, J = 8.0 Hz, 2H, CH , H2″), 1.33−1.69
2
1
1
(m, 6H, 3 × CH , H3″/4″/5″). The H NMR data match
6
2
31
NH), 7.29 (d, J = 8.8 Hz, 2H, H2′/6′), 6.53 (d, J = 8.8 Hz, 2H,
those reported.
H3′/5′), 6.46 (d, J = 12.4 Hz, 1H, H2), 6.28 (d, J = 12.4 Hz,
Method B: Maleamic acid 14 (202 mg, 0.980 mmol) was
added to a stirred, dark yellow solution of D-(+)-biotin (3)
(250 mg, 1.02 mmol), HCTU (1.28 g, 3.09 mmol), and
1
1
H, H3), NH and COOH not observed. The H NMR data
2
31
match those reported.
1
3
(
Z)-3-((4-Amino(1,2,3,4,5,6- C )phenyl)carbamoyl)prop-
Hunig’s base (0.7 mL, 4 mmol) in anhydrous DMF (7 mL),
̈
6
1
3
2
-enoic acid (N-4-amino(1,2,3,4,5,6- C )phenylmaleamic
upon which the solution turned dark red. After stirring
overnight, the reaction mixture was diluted with water (100
mL) to afford a gray precipitate, which was collected by
vacuum filtration, then dissolved in 1:9 MeOH:DCM (100
6
acid) (14*). The procedure above was applied to solutions
of 12* (51 mg, 0.45 mmol) in EtOAc (3 mL) and maleic
anhydride (44 mg, 0.45 mmol) in EtOAc (1 mL). Purification
J
https://doi.org/10.1021/acs.bioconjchem.1c00206
Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
pubs.acs.org/bc
Article
3
1
mL). The solution was washed with 5% NaHCO (3 × 100
al. D-(+)-Biotin (3) (500 mg, 2.05 mmol) was suspended in
3
mL) and water (100 mL), dried, and evaporated. Purification
by rapid silica filtration (1:19 MeOH:DCM) afforded
maleimide 16 as a pale yellow powder (108 mg, 27%),
identical to the material described above.
SOCl (10 mL, 140 mmol) under moisture guard and stirred
2
until complete dissolution to give an orange solution. The
excess thionyl chloride was evaporated under a stream of N2,
after which p-nitroaniline (20) (190 mg, 1.37 mmol) and
pyridine (3.0 mL, 37 mmol) were added to form a brown
suspension. Anhydrous DMF (1 mL) was added, and the
resulting brown solution was stirred at room temperature for 4
days. The solvent was evaporated, and the residue was
dissolved in 1:9 MeOH:DCM (5 mL) and stirred with solid
1
-(4-(((5-((3aR,4R,6aS)-2-Oxohexahydro-1H-thieno[3,4-
d]imidazol-4-yl)pentanoyl)oxy)amino)phenyl-
1
3
1
(
,2,3,4,5,6- C )-1H-pyrrole-2,5-dione (N-4-(D-biotinylamino-
6
1
3
1,2,3,4,5,6- C )phenyl)maleimide) (16*). Method B was
6
applied to 14* (50 mg, 0.24 mmol), D-(+)-biotin (3) (60
mg, 0.25 mmol), TCTU (252 mg, 0.710 mmol), and Hunig’s
̈
NaHCO (1.17 g, 14.0 mmol) for 30 min. The resulting
3
base (0.16 mL, 0.71 mmol) in anhydrous DMF (2 mL).
suspension was filtered through a plug of silica gel. The filtrate
was evaporated, and the residue was subjected to rapid silica
filtration. Elution with DCM afforded p-nitroaniline 20 (63
mg, 33% recovery). Further elution with 1:19 MeOH:DCM
afforded amide 19 as an orange powder (145 mg, 29%). IR
Purification as above gave 16* as a pale yellow powder (25 mg,
1
2
7
7
7
4%). H NMR (500 MHz, d -DMSO): δ 10.03 (s, 1H, NH),
6
.80−7.87 and 7.47−7.54 (dm, J = 165 Hz, 2H, H2‴/6‴),
CH
.34−7.42 and 7.03−7.11 (dm, J = 155 Hz, 2H, H3‴/5‴),
CH
−
1
1
.16 (s, 2H, H3/4), 6.43 (s, 1H, NH), 6.35 (s, 1H, NH), 4.30
(ATR) cm : 3220 (NH), 1696 (CO unresolved). H NMR
(
1
=
dd, J = 7.5 Hz, J = 5.3 Hz, 1H, CH, H6a′), 4.12−4.16 (m,
(d -DMSO, 400 MHz) δ 10.50 (s, 1H, NH), 8.21 (d, J = 9.2
1
2
6
H, CH, H3a′), 3.10−3.15 (m, 1H, CH, H4′), 2.83 (dd, 1H, J
Hz, 2H, ArH, H3″/5″), 7.28 (d, J = 9.2 Hz, ArH, H2″/6″),
6.43 (s, 1H, NH), 6.36 (s, 1H, NH), 4.28−4.32 (m, 1H, CH,
H6a), 4.12−4.16 (m, 1H, CH, H3a), 3.10−3.14 (m, 1H, CH,
H4), 2.82 (m, 1H, H6), 2.58 (d, J = 12.4 Hz, 1H, H6), 2.39 (t,
J = 7.5 Hz, 2H, CH , H2′), 1.33−1.69 (m, 6H, 3 × CH , H3′/
1
12.5 Hz, J = 5.1 Hz, H6′), 2.58 (d, 1H, J = 12.5 Hz, H6′),
2
2
.33 (t, 2H, J = 7.5 Hz, CH , H2″), 1.30−1.70 (m, 6H, 3 ×
2
13
CH , H3″/4″/5″). C NMR (150 MHz, d -DMSO): δ 171.4
2
6
(
9
C1″), 170.1 (C2/5), 162.7 (C2′), 138.8 (dt, J = 62.5 Hz, J =
1
2
2
2
1
3
.6 Hz, C1′), 134.6 (C3/4), 126.8−127.9 (m, C3‴/5‴),
25.5−126.5 (m, C4‴), 118.6−119.6 (m, C2‴/6‴), 61.1
4′/5′). C NMR (d -DMSO, 100 MHz) δ 172.7 (C1′), 163.2
6
1
(C2), 146.0 (C4″), 142.5 (C3′′/5′′), 125.5 (C1′′), 119.1
(C2′′/6′′), 61.5 (C6a), 59.7 (C3a), 55.9 (C4), 36.9 (C6),
28.66 (C4′), 28.58 (C5′), 25.3 (C3′). HRMS (ESI+) m/z:
(
C6a′), 59.2 (C3a′), 55.4 (C4′), 36.2 (C2″), 28.2 (C4″), 28.1
C5″), 25.1 (C3″). HRMS (ESI+) m/z: calcd. for
(
C
1
3
+
+
+
+
C H N O SNa [M + Na] 443.1455, found 443.1465.
calcd. for C H N O S [M + H] 365.1284; found 365.1295.
16 21 4 4
1
4
6
22
4
4
NMR assignments were made with the assistance of COSY,
HSQC, and HMBC experiments.
NMR assignments were made with the assistance of COSY,
HSQC, and HMBC experiments.
13
5
-((3aS,4S,6aR)-2-Oxohexahydro-1H-thieno[3,4-d]-
Acet- C -anilide. The procedure was adapted from Law-
6
3
2
imidazol-4-yl)pentanamide (18) (D-Biotinanilide). The pro-
rie. Ac O (0.40 mL, 4.2 mmol) was added to a solution of
C -aniline (250 mg, 2.52 mmol) in dry DCM (15 mL), and
2
43
13
cedure for (18) was adapted from Feng et al. A suspension of
D-(+)-biotin (3) (54 mg, 0.22 mmol) and HCTU (87.5 mg,
6
stirred for 1 h. The solution was washed with NaHCO (3 ×
3
0
.212 mmol) in anhydrous DMF (1 mL) was stirred for 20
10 mL), and the aqueous layer was back-extracted with EtOAc
min, followed by addition of NEt (0.20 mL, 1.4 mmol),
(2 × 10 mL). The combined organic phase was dried and
3
forming a yellow solution. Aniline (17) (0.020 mL, 0.14
mmol) was added, and the solution was stirred at 80 °C
overnight. The solvent was evaporated, and the brown residue
was dissolved in 1:9 MeOH/DCM (5 mL). Solid NaHCO3
evaporated to afford the title compound as beige crystals (299
1
mg, 84%). H NMR (CDCl , 500 MHz): δ 7.49 (m [app. dq],
3
JCH = 160 Hz, 2H, H2/6), 7.31 (m [app. dquint.] JCH = 160
Hz, 2H, H3/5), 7.09 (m [app. dquint.], J = 160 Hz, 1H,
CH
1
3
(120 mg, 1.43 mmol) was added, and the suspension was
H4), 2.18 (s, 3H, CH ), NH obscured. C NMR (CDCl , 125
3
3
1
3
stirred for 30 min, then filtered through a plug of silica. The
filtrate was evaporated and the residue was purified by rapid
silica filtration. Elution with 1:19 MeOH/DCM afforded
benzamide 18 as a pale-beige powder (35 mg, 79%). IR (ATR)
MHz): δ 168.6 (CO), 138.2 (dt, J = 63.5 Hz, J = 9.9 Hz,
1
1
3
C1′), 129.3 (ddd [app. dt], J = J = 55.9 Hz, J = 7.5 Hz,
1
2
1
2
3
C2′/6′), 125.0 (ttd, J = 55.8 Hz, J = 2.5 Hz, J = 9.9 Hz,
C4′), 120.1 (m, C3′/5′), 25.0 (d, J = 2.5 Hz, CH ). This
3
−
1
1
32
cm : 3300 (NH), 1696 (CO), 1659 (CO). H NMR
compound has been synthesized previously, but no analytical
(
d -DMSO, 400 MHz) δ 9.87 (s, 1H, NH), 7.58 (d, J = 7.6 Hz,
6
data were reported.
13
32
2
H, ArH, H3″/5″), 7.28 (dd [app. t], J = J = 7.5 Hz, 2H,
p-Nitroacet- C -anilide. Adapted from Lawrie. NaNO3
1
2
6
ArH, H2″/6″), 7.01 (t, J = 7.4 Hz, H4″), 6.43 (s, 1H, NH),
.35 (s, 1H, NH), 4.28−4.32 (m, 1H, CH, H6a), 4.12−4.16
m, 1H, CH, H3a), 3.10−3.14 (m, 1H, CH , H4), 2.82 (dd, J
(260 mg, 3.08 mmol) was added to a stirred solution of
1
3
6
(
=
acet- C -anilide (290 mg, 2.05 mmol) in conc. H SO (5 mL)
6
2
4
under a moisture guard at 0 °C. The reaction was stirred for 4
h, during which time a pale orange color developed. The
solution was poured out onto ice/water (200 mL), and the
precipitate was filtered and air-dried to afford the title
2
1
5.1 Hz, J = 12.4 Hz, 1H, H6), 2.58 (d, J = 12.4 Hz, 1H,
2
H6), 2.30 (t, J = 7.5 Hz, 2H, CH , H2′), 1.33−1.69 (m, 6H, 3
2
13
×
CH , H3′/4′/5′). C NMR (d -DMSO, 100 MHz): δ 171.6
2
6
1
(
C1′), 163.1 (C2), 139.8 (C1″), 129.1 (C3″/5″), 123.4
C4″), 119.5 (C2″/6″), 61.5 (C3a), 59.7 (C6a), 55.8 (C4),
compounds as a pale yellow powder (222 mg, 59%). H
(
3
(
NMR (500 MHz, d -DMSO) δ 10.55 (s, 1H, NH), 7.62−8.40
6
1
3
6.7 (C6), 28.67 (C4′), 28.53 (C5′), 25.6 (C3′). HRMS
(m, 4H, ArH), 2.11 (1H, CH , s). C NMR (125 MHz, d -
DMSO) δ 168.6 (s, CO), 145.9 (dt, J = 61.2 Hz, J = 9.0 Hz,
3
6
−
−
1
3
ESI−) m/z: calcd. for C H N O S [M−H] 318.1276;
16
20
3
2
1
3
found 318.1281. NMR assignments were made with the
assistance of COSY, HSQC, and HMBC experiments.
C4′), 142.4 (dt, J = 67.0 Hz, J = 9.0 Hz, C3′/5′), 125.4 (m,
C2′/6′), 119.0 (m, C1′), 24.7 (d, J = 2.9 Hz, CH ). HRMS
3
1
2
13
+
+
N-(4-Nitrophenyl)-5-((3aS,4S,6aR)-2-oxohexahydro-1H-
thieno[3,4-d]imidazol-4-yl)pentanamide (19) (N-biotinyl-4-
nitroaniline). The procedure was adapted from Quideau et
(ESI+) m/z: calcd. for C C H N O [M + H] 187.0809,
2 6 9 2 3
found 187.0814. This compound has been synthesized
32
previously, but no analytical data were reported.
K
https://doi.org/10.1021/acs.bioconjchem.1c00206
Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
pubs.acs.org/bc
Article
1
3
C -p-Nitroaniline (20*). The procedure for the synthesis
sterile microtubes, frozen in liquid nitrogen, and subsequently
stored at −80 °C until analysis.
6
32
of 20* was adapted from Lawrie. A stirred suspension of p-
nitroacet- C -anilide (212 mg, 1.17 mmol) in 35% aq. NaOH
10 mL) was heated at 50 °C for 12 h. After cooling, the
aqueous solution was extracted with EtOAc (3 × 10 mL). The
organic extract was dried and evaporated to afford 20* as
yellow crystals (160 mg, 97%). H NMR (500 MHz, CDCl ):
δ 7.87−8.26 (m, 2H, H3/5), 6.43−6.81 (m, 2H, H2/6), 4.31
s, 2H, NH ). C NMR (125 MHz, CDCl ): δ 152.6 (td, J =
0.8 Hz, J = 8.5 Hz, C1), 139.3 (br td, J = 67.9 Hz, J = 8.7
Hz, C4), 125.9−127.0 (m, C3/5), 113.0−114.0 (m, C2/6).
HRMS (ESI+) m/z: calcd. for C C H N O [M+H
MeOH] 177.0966, found 177.0966. This compound has
been synthesized previously, but no analytical data were
reported.
1
3
Labeling of Lysozyme Standards. A 2 mg/mL solution
of purified lysozyme was prepared in SDS/Tris buffer (0.5%
SDS, 0.5 M Tris, pH 7.0). A lysozyme aliquot (200 μg) was
reduced by adding 50 mM tris(2-carboxyethyl)phosphine
(TCEP; pH 7.0) (4.8 μL). The sample was vortexed and
incubated for 2 h at room temperature. More SDS/Tris buffer
was added to dilute the reduced lysozyme solution to 100 μg/
mL. DMSO solutions of ICMATs (5 mM) (7, 7*, 16, and
6
(
1
3
1
3
1
(
6
2
3
3
1
3
1
6*) were stored at −20 °C. Aliquots of reduced lysozyme
12
13
+
solution (400 μL, containing 40 μg lysozyme) were alkylated
by the addition of 16 μL of 5 mM ICMAT solution (i.e., each
ICMAT was added to a separate aliquot of reduced lysozyme
at a ratio of 2 nmol ICMAT/μg lysozyme), followed by
vigorous vortexing at room temperature for 1 h. Standards of
different proportions of 7:7* or 16:16* labeled lysozyme
6
10
2
3
+
+
32
N-(4-Aminophenyl)-5-((3aS,4S,6aR)-2-oxohexahydro-1H-
thieno[3,4-d]imidazol-4-yl)pentanamide (21). A suspension
of 10% Pd/C (145 mg) in a solution of 19 (140 mg, 0.384
mmol) in anhydrous DMF (6 mL) was stirred under a balloon
(
0:100, 20:80, 35:65, 50:50, 65:35, 80:20, and 100:0 ratios)
were prepared such that each standard contained 8 μg of
lysozyme. To remove excess tags and SDS, 1 mL of acetone,
precooled to −20 °C, was added to each standard, briefly
vortexed and incubated at −20 °C overnight. The samples
were then centrifuged at 10 000 g for 5 min at 4 °C, and the
supernatant was removed. The protein pellets were redissolved
in of 100 mM pH 7.0 Tris (200 μL) and were digested at 37
of H overnight, after which time TLC (1:9 MeOH:DCM)
2
indicated the reaction was complete. The mixture was diluted
with MeOH (20 mL) and filtered through Celite. The solvent
was evaporated to afford the slightly impure product 21 as an
−
1
orange powder (115 mg, 90%). IR (ATR) cm : 3243 (NH),
1
1
690 (CO), 1649 (CO). H NMR (d -DMSO, 400
6
°
C for 16 h with trypsin (Promega), at 1:25 protease:protein
MHz): δ 9.41 (s, 1H, NH), 7.19 (d, J = 8.7 Hz, ArH, H2″/
(
w/w) ratio. Each sample was then split into 4 μg protein
6
6
(
2
′′), 6.47 (d, J = 8.7 Hz, 2H, ArH, H3″/5′′), 6.43 (s, 1H, NH),
.36 (s, 1H, NH), 4.28−4.32 (m, 1H, CH, H6a), 4.12−4.16
aliquots and desiccated by vacuum centrifugation.
Labeling of Muscle Samples. The labeling technique
m, 1H, H3a), 3.10−3.14 (m, 1H, H4), 2.82 (m, 1H, H6),
used was adapted from the fluorescent method for quantifying
.58 (d, J = 12.4 Hz, 1H, H6), 2.21 (t, J = 7.5 Hz, 2H, H2′),
44,45
protein thiol oxidation,
involving the sequential labeling of
13
1
.33−1.69 (m, 6H, H3′/4′/5′). C NMR (d -DMSO, 100
6
reduced and oxidized protein thiol groups, using ICMATs
either the 7/7* pair or the 16/16* pair). Frozen muscle (∼10
MHz): δ 170.6 (C1′), 163.2 (C2), 145.0 (C1″), 129.0 (C4′′),
(
1
5
21.4 (C2′′/6′′), 114.2 (C3′′/5′′), 61.5 (C6a), 59.7 (C3a),
mg) was crushed under liquid nitrogen, and suspended in ice-
cold 20% (w/v) trichloroacetic acid (TCA)/acetone (500 μL),
then homogenized (Ultra-Turrax T25; Rose Scientific) on
maximum for 15 s to produce an even suspension, which was
incubated at −20 °C for at least 1 h. An aliquot of the
suspension (50 μL) was diluted with acetone precooled to −20
5.9 (C4), 36.9 (C6), 28.74 (C4′), 28.56 (C5′), 25.8 (C3′).
−
−
HRMS (ESI−) m/z: calcd. for C H N O S [M−H]
1
6
21
4
2
3
33.1385; found 333.1377.
Animal Procedures. Mouse muscle samples were obtained
from either adult male dystrophic mdx (C57Bl/10ScSnmdx/
mdx) or non-dystrophic control C57Bl/10ScSn (C57) mice
°
C (1 mL), then centrifuged at 10,000 g for 5 min at 4 °C. The
(
the parental strain for mdx). All mice were obtained from the
supernatant was discarded and the protein pellet was dissolved
in 0.5% pH 7.3 SDS/Tris buffer (300 μL). Native protein
thiols were labeled with either ICMAT-1 (7) or ICMAT-3
Animal Resource Centre, Murdoch, Western Australia. They
were maintained at the University of Western Australia on a 12
h light/dark cycle, under standard conditions, with free access
to food and drinking water. All animal experiments were
conducted in strict accordance with the guidelines of the
National Health and Medical Research Council Code of
practice for the care and use of animals for scientific purposes
2004), and the Animal Welfare act of Western Australia
2002), and were approved by the Animal Ethics committee at
the University of Western Australia.
As part of a broader study on oxidative stress in muscular
dystrophy, dog muscle samples were obtained from dystrophic
male golden retriever dogs or healthy controls, aged
approximately 8 months. These dogs were handled and
housed in the Boisbonne Center for Gene Therapy (ONIRIS,
Atlantic Gene Therapies, Nantes, France). The Institutional
Animal Care and Use Committee of the Region des Pays de la
Loire (University of Angers, France) approved all the
experimental protocols. Skeletal muscle samples (biceps
femoris) were obtained after the dogs were sacrificed,
performed by intravenous injection of pentobarbital sodium
(
16) by adding a 5 mM DMSO solution (30 μL; final
concentration, 0.46 mM) followed by incubation for 30 min at
room temperature. Excess 7 or 16 ICMAT was consumed by
the addition of 50 mM aqueous cysteine (6.5 μL; final
concentration ∼1 mM cysteine). Oxidized thiols were reduced
with 3.7 mM TCEP in 0.5% pH 7.3 SDS/Tris buffer, then
labeled with 5 mM 7* or 16* in DMSO (respectively,
depending on which ICMAT pair was being used in the
experiment) to a final ICMAT concentration of 0.046 mM.
Labeled protein was recovered by acetone precipitation (6:1 v/
v) and centrifugation at 3000 g for 15 min. Protein was
digested by addition of a solution of trypsin (20 μg) in 100
mM TRIS pH 7.0 (520 μL) and incubation at 37 °C for 16 h.
Digested proteins (peptides) were purified using the cation
exchange and affinity purification columns provided with the
SCIEX Cleavable ICAT kit, as per manufacturers specifications
(Sciex). Purified peptide fractions were desiccated using
vacuum centrifugation.
(
(
StrataX Polymeric Reversed Phase Desalting. Each
(
Dolethal, Vetoquinol). Muscle samples were placed into
StrataX column was preconditioned with MeOH (2 mL) then
L
https://doi.org/10.1021/acs.bioconjchem.1c00206
Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
pubs.acs.org/bc
Article
MeCN (2 mL), followed by 80% MeCN/H O/0.1% formic
oxidation of methionine residues, and alkylation of cysteine
residues (by the relevant ICMAT). Other search criteria
included a MS tolerance of 0.2 Da nd MS/MS tolerance of 0.2
Da. Protein identification was determined on the basis of two
or more identified peptides with ion scores exceeding the
significance threshold.
2
acid (0.5 mL) then water (1 mL). Peptide sample were
reconstituted in water (200 μL), then loaded onto the
preconditioned StrataX column, washed with water (2 mL),
and eluted with 80% MeCN/H O/0.1% formic acid (0.5 mL).
2
All samples were then desiccated by vacuum centrifugation and
stored at −80 °C until analysis by mass spectrometry.
Matrix-Assisted Laser Desorption/Ionization (MALDI-
TOF/TOF) Mass Spectrometry. Labeled peptide samples
derived from lysozyme were reconstituted in 80% MeCN/
ASSOCIATED CONTENT
sı Supporting Information
■
*
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.bioconjchem.1c00206.
H O/0.05% TFA (5 μL). Peptide solutions (0.6 μL) were
2
combined with matrix solution (0.6 μL 5 mg/mL α-cyano-4-
hydroxysuccinamic acid, 10 mM ammonium citrate, 80%
1H and 13C NMR spectra of all new compounds (PDF)
MeCN/H O/0.1% TFA) on a MALDI-TOF/TOF plate, and
2
allowed to air-dry. Analysis was performed with a 5800
MALDI-TOF/TOF Mass Spectrometer (AB Sciex). Parent
mass peaks (mass range m/z 800−4000 from combined MS
and MS/MS spectra) were submitted to the MASCOT
database for confirmation of labeled lysozyme peptides, using
the following search conditions: Swissprot database, all
mammalian species, trypsin digest with allowance for up to
one missed cleavage per peptide, no fixed modifications,
variable modification of oxidation on methionine residues and
alkylation of cysteine residues with ICMATs, MS tolerance of
AUTHOR INFORMATION
Corresponding Authors
■
Peter G. Arthur − School of Molecular Sciences, University of
Western Australia, Perth 6009, Australia;
Email: peter.arthur@uwa.edu.au
Matthew J. Piggott − School of Molecular Sciences, University
of Western Australia, Perth 6009, Australia; orcid.org/
0000-0002-5857-7051; Email: matthew.piggott@
uwa.edu.au
0
.4 Da, MS/MS tolerance of 0.4 Da. Areas under the peaks
Authors
corresponding to the labeled lysozyme peptide CELAAAMK
were obtained from the TOF/TOF Explorer Series software (v
4
Adam P. Wdowiak − School of Molecular Sciences, University
of Western Australia, Perth 6009, Australia
Marisa N. Duong − School of Molecular Sciences, University of
Western Australia, Perth 6009, Australia; Proteomics
International, Perth 6009, Australia
Rohan D. Joyce − School of Molecular Sciences, University of
Western Australia, Perth 6009, Australia
Amber E. Boyatzis − School of Molecular Sciences and School
of Biomedical Sciences, University of Western Australia, Perth
6009, Australia; orcid.org/0000-0002-5940-4531
Mark C. Walkey − School of Molecular Sciences, University of
Western Australia, Perth 6009, Australia
Gareth L. Nealon − School of Molecular Sciences and Centre
for Microscopy, University of Western Australia, Perth 6009,
Australia
.1.0, AB Sciex) to quantify the experimental ratio of the
isotopic ICMATS.
HPLC−MS/MS. Each labeled peptide sample was recon-
stituted in solvent A (2% MeCN/0.1% formic acid) at
approximately 0.1 μg/μL, and a volume of 20 μL was injected
into a Shimadzu Prominence nano HPLC system coupled to a
5
600 TripleTOF (Sciex) mass spectrometer. Separation was
achieved on an Agilent Zorbax 300SB-C18, 3.5 μm column
eluted with a linear gradient of H O/MeCN/0.1% formic acid
2
(
v/v) at 40 °C. The HPLC mobile phase was prepared by
combining 0.1% formic acid and 2% MeCN in H O (A) and
2
0
.1% formic acid and 2% H O in MeCN (B). Gradient elution
2
was performed with 2% B for 3 min, increased to 40% B at 15
min, then ramped to 98% B by 16 min and held for 2 min
before returning to 2% B within 1 min. MS spectra were
acquired using electrospray ionization in mode, by informa-
tion-dependent acquisition (IDA), whereby only the most
intense 20 MS peaks between 400 and 1250 m/z were selected
for MS/MS scans. Mass tolerance was 50 mDa. A calibration
with trypsin-digested bovine serum albumen (BSA) was
conducted before a batch of samples was run.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.bioconjchem.1c00206
Author Contributions
#
A.P.W. and M.N.D. contributed equally.
Notes
The authors declare no competing financial interest.
For labeled lysozyme standards, areas under the peaks of the
extracted ion chromatograms corresponding to the labeled
lysozyme peptide CELAAAMK were obtained from the
Skyline software (AB Sciex, v 4.1.0) to quantify the
experimental ratio of the isotopic ICMATs. Sample prepara-
tion variation was accounted for with a correction factor
calculated from the 50:50 light:heavy sample. Quantification of
muscle-derived peptides was performed in the same manner,
but without the correction factor.
MS/MS Proteomic Analysis. MS/MS data were imported
into the database search engine Mascot (v 2.4.1, www.
matrixscience.com) for identification of peptides and proteins,
using the following search conditions: Swissprot database, all
mammalian species, trypsin digest with allowance for up to one
missed cleavage per peptide, no fixed modifications, variable
ACKNOWLEDGMENTS
■
A.P.W. was an undergraduate student on exchange from
University of Bristol at the time. M.N.D. was the recipient of a
Forrest Research Foundation Scholarship and Australian
Government Research Training Program Scholarship at
UWA. A.E.B. was the recipient of an Australian Postgraduate
Award. We thank Proteomics International for access to mass
spectrometers; the UWA Centre for Microcopy, Character-
isation and Analysis for access to NMR and mass
spectrometers and technical support; and the National Health
and Medical Research Council (NHMCR) for funding. The
TOC graphic contains an image derived from the crystal
structure of hen egg lysozyme from PDB, DOI: 10.2210/
pdb3LYZ/pdb.
M
https://doi.org/10.1021/acs.bioconjchem.1c00206
Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
pubs.acs.org/bc
Article
spectrometric anal. of proteins. WO2002-EP121062003040093,
0021030, 2003.
20) Lerchen, H.-G., Siegmund, H.-U., Immler, D., Schumacher, A.,
and Auriel, D. Preparation of isotopically-coded affinity markers for
mass spectrometric analysis of proteins. WO2002-EP12105
REFERENCES
■
2
(
(
1) Gygi, S. P., Rist, B., Gerber, S. A., Turecek, F., Gelb, M. H., and
Aebersold, R. (1999) Quantitative analysis of complex protein
mixtures using isotope-coded affinity tags. Nat. Biotechnol. 17, 994−
999.
2
(
003040288, 20021030, 2003.
21) Lockhoff, O., Lehmann, T., Lerchen, H.-G., Immler, D., and
(
2) Mendoza, V. L., and Vachet, R. W. (2009) Probing protein
structure by amino acid-specific covalent labeling and mass
spectrometry. Mass Spectrom. Rev. 28, 785−815.
3) El-Shafey, A. F., Armstrong, A. E., Terrill, J. R., Grounds, M. D.,
and Arthur, P. G. (2011) Screening for increased protein thiol
oxidation in oxidatively stressed muscle tissue. Free Radical Res. 45,
Siegmund, H.-U. Preparation of isotope-coded affinity tags for use in
mass-spectrometric anal. of proteins. WO2002-EP12006 2003040287,
(
2
(
0021028, 2003.
22) Lockhoff, O., Schumacher, A., Lerchen, H.-G., and Immler, D.
Synthesis of isotope-coded affinity tags containing a carbohydrate
linker group and their use for protein analysis. DE2003−
9
(
91−999.
4) Murray, C. I., and Van Eyk, J. E. (2012) Chasing cysteine
1
032207710322077, 20030515, 2004.
oxidative modifications: proteomic tools for characterizing cysteine
redox status. Circ.: Cardiovasc. Genet. 5, 591.
5) Liu, P., Zhang, H., Wang, H., and Xia, Y. (2014) Identification of
redox-sensitive cysteines in the Arabidopsis proteome using
(
23) Olejnik, J., and Rothschild, K. J. Photocleavable isotope-coded
affinity tags. US2003−68729720050085630, 20031016, 2005.
24) Boyatzis, A. E., Bringans, S. D., Piggott, M. J., Duong, M. N.,
(
(
Lipscombe, R. J., and Arthur, P. G. (2017) Limiting the Hydrolysis
and Oxidation of Maleimide-Peptide Adducts Improves Detection of
Protein Thiol Oxidation. J. Proteome Res. 16, 2004−2015.
OxiTRAQ, a quantitative redox proteomics method. Proteomics 14,
7
(
50−762.
6) Loescher, C. M., Breitkreuz, M., Li, Y., Nickel, A., Unger, A.,
Dietl, A., Schmidt, A., Mohamed, B. A., Kotter, S., Schmitt, J. P., et al.
2020) Regulation of titin-based cardiac stiffness by unfolded domain
oxidation (UnDOx). Proc. Natl. Acad. Sci. U. S. A. 117, 24545−24556.
7) Bottari, P., Aebersold, R., Turecek, F., and Gelb, M. H. (2004)
(25) Cao, Y., Yang, X., Du, D., Xu, X., Song, F., and Xu, L. (2011)
New system of deprotection step for the hydroxide radicals: boron
trifluoride etherate/sodium iodide. Int. J. Chem. (Toronto, ON, Can.)
(
3
(
, 113−117.
26) Gooden, D. M. (2009) 6-(4-(2-{2-[2-(2-Hydroxy-ethoxy)-
ethoxy]-ethoxy}-ethyl)-10-oxa-4-aza-tricyclo[5.2.1.02,6]dec-8-ene-
,5-dione). Molbank 2009, M638.
27) Li, C., Johnson, R. P., and Porco, J. A., Jr. (2003) Total
(
Design and Synthesis of Visible Isotope-Coded Affinity Tags for the
Absolute Quantification of Specific Proteins in Complex Mixtures.
Bioconjugate Chem. 15, 380−388.
3
(
(
8) Yi, E. C., Li, X.-j., Cooke, K., Lee, H., Raught, B., Page, A.,
Synthesis of the Quinone Epoxide Dimer (+)-Torreyanic Acid:
Application of a Biomimetic Oxidation/Electrocyclization/Diels-
Alder Dimerization Cascade. J. Am. Chem. Soc. 125, 5095−5106.
Aneliunas, V., Hieter, P., Goodlett, D. R., and Aebersold, R. (2005)
Increased quantitative proteome coverage with 13C/12C-based, acid-
cleavable isotope-coded affinity tag reagent and modified data
acquisition scheme. Proteomics 5, 380−387.
9) Fauq, A. H., Kache, R., Khan, M. A., and Vega, I. E. (2006)
Synthesis of Acid-Cleavable Light Isotope-Coded Affinity Tags
ICAT-L) for Potential Use in Proteomic Expression Profiling
Analysis. Bioconjugate Chem. 17, 248−254.
10) Kurono, S., Kurono, T., Komori, N., Niwayama, S., and
(28) Leichert, L. I., Gehrke, F., Gudiseva, H. V., Blackwell, T., Ilbert,
M., Walker, A. K., Strahler, J. R., Andrews, P. C., and Jakob, U. (2008)
Quantifying changes in the thiol redox proteome upon oxidative stress
in vivo. Proc. Natl. Acad. Sci. U. S. A. 105, 8197−8202.
(
(
(29) Turowski, M., Yamakawa, N., Meller, J., Kimata, K., Ikegami,
T., Hosoya, K., Tanaka, N., and Thornton, E. R. (2003) Deuterium
isotope effects on hydrophobic interactions: the importance of
dispersion interactions in the hydrophobic phase. J. Am. Chem. Soc.
(
Matsumoto, H. (2006) Quantitative proteome analysis using D-
labeled N-ethylmaleimide and 13C-labeled iodoacetanilide by matrix-
assisted laser desorption/ionization time-of-flight mass spectrometry.
Bioorg. Med. Chem. 14, 8197−8209.
1
(
(
25, 13836−13849.
30) Keana, J. F. W., Ogan, M. D., Lu, Y., Beer, M., and Varkey, J.
1986) Functionalized Keggin- and Dawson-type cyclopentadienylti-
(
11) Guaragna, A., Amoresano, A., Pinto, V., Monti, G.,
tanium heteropolytungstate anions: small, individually distinguishable
labels for conventional transmission electron microscopy. 2.
Reactions. J. Am. Chem. Soc. 108, 7957−63.
Mastrobuoni, G., Marino, G., and Palumbo, G. (2008) Synthesis
and Proteomic Activity Evaluation of a new Isotope-Coded Affinity
Tagging (ICAT) Reagent. Bioconjugate Chem. 19, 1095−1104.
(31) Quideau, S., Douat-Casassus, C., Delannoy Lopez, D. M., Di
(
12) Boja, E. S., and Fales, H. M. (2001) Overalkylation of a protein
Primo, C., Chassaing, S., Jacquet, R., Saltel, F., and Genot, E. (2011)
Binding of Filamentous Actin and Winding into Fibrillar Aggregates
by the Polyphenolic C-Glucosidic Ellagitannin Vescalagin. Angew.
Chem., Int. Ed. 50, 5099−5104.
digest with iodoacetamide. Anal. Chem. 73, 3576−3582.
(13) Yang, Z., and Attygalle, A. B. (2007) LC/MS characterization
of undesired products formed during iodoacetamide derivatization of
sulfhydryl groups of peptides. J. Mass Spectrom. 42, 233−243.
(32) Lawrie, K. W. M. (2007) The synthesis of (13C6)N-(3,4-
(14) Nielsen, M. L., Vermeulen, M., Bonaldi, T., Cox, J., Moroder,
L., and Mann, M. (2008) Iodoacetamide-induced artifact mimics
dichlorophenyl)-2,2-dimethylpropanamide. J. Labelled Compd. Radio-
pharm. 50, 504.
ubiquitination in mass spectrometry. Nat. Methods 5, 459−460.
(15) Hansen, R. E., and Winther, J. R. (2009) An introduction to
(33) Mondet, J. C., and Roussat, G. (1987) Some properties of
N,N’-dinitro-N,N’-dimethylurea. Propellants, Explos., Pyrotech. 12, 29.
(34) Suri, S. C., and Chapman, R. D. (1988) A convenient method
for N-nitration using ammonium nitrate-trifluoroacetic anhydride.
Synthesis 1988, 743−5.
(35) Zhu, J., Waengler, C., Lennox, R. B., and Schirrmacher, R.
(2012) Preparation of Water-Soluble Maleimide-Functionalized 3 nm
Gold Nanoparticles: A New Bioconjugation Template. Langmuir 28,
5508−5512.
(36) Buijs, W., Elburg, P. v., and van der Gen, A. (1983) The
Conversion of α,ω-Diols into Mono Iodo Alcohols. Synth. Commun.
13, 387−392.
methods for analyzing thiols and disulfides: Reactions, reagents, and
practical considerations. Anal. Biochem. 394, 147−158.
(16) Rogers, L. K., Leinweber, B. L., and Smith, C. V. (2006)
Detection of reversible protein thiol modifications in tissues. Anal.
Biochem. 358, 171−184.
(
17) Galvani, M., Hamdan, M., Herbert, B., and Righetti, P. G.
(
2001) Alkylation kinetics of proteins in preparation for two-
dimensional maps: a matrix assisted laser desorption/ionization-
mass spectrometry investigation. Electrophoresis 22, 2058−2065.
(18) Mueller, T., and Winter, D. (2017) Systematic Evaluation of
Protein Reduction and Alkylation Reveals Massive Unspecific Side
Effects by Iodine-containing Reagents. Mol. Cell. Proteomics 16, 1173−
(37) Sheepwash, E. E., Rowntree, P. A., and Schwan, A. L. (2008)
The preparation of three new partially deuterated hexadecanethiols
for applications in surface chemistry. J. Labelled Compd. Radiopharm.
51, 391−398.
1
(
187.
19) Lerchen, H.-G., Lockhoff, O., Immler, D., and Siegmund, H.-U.
Preparation of isotopically coded affinity markers for mass
N
https://doi.org/10.1021/acs.bioconjchem.1c00206
Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
pubs.acs.org/bc
Article
(38) Jung, H. J., Shim, J. S., Park, J., Ha, H.-J., Kim, J.-H., Kim, J.-G.,
Kim, N. D., Yoon, J. H., and Kwon, H. J. (2009) Identification and
validation of calmodulin as a binding protein of an anti-proliferative
small molecule 3,4-dihydroisoquinolinium salt. Proteomics: Clin. Appl.
3
, 423−432.
39) Reetz, M. T., Rentzsch, M., Pletsch, A., Taglieber, A.,
Hollmann, F., Mondiere, R. J. G., Dickmann, N., Höcker, B.,
(
̀
Cerrone, S., Haeger, M. C., et al. (2008) A Robust Protein Host for
Anchoring Chelating Ligands and Organocatalysts. ChemBioChem 9,
5
(
52−564.
40) Bates, R. W., and Dewey, M. R. (2009) A Formal Synthesis of
Swainsonine by Gold-Catalyzed Allene Cyclization. Org. Lett. 11,
706−3708.
41) Mukai, S., Flematti, G., Byrne, L., Besant, P., Attwood, P., and
3
(
Piggott, M. (2012) Stable triazolylphosphonate analogues of
phosphohistidine. Amino Acids 43, 857−874.
(42) Lawrie, K. W. M. (2007) The synthesis of [13C6]N-(3,4-
dichlorophenyl)-2,2-dimethylpropanamide. J. Labelled Compd. Radio-
pharm. 50, 504−504.
(43) Feng, S., Li, S., Kang, X., and Liu, K. (2010) One step synthesis
of biotinylated amino acids. J. Chem. Res. 34, 348−350.
44) Armstrong, A. E., Zerbes, R., Fournier, P. A., and Arthur, P. G.
2011) A fluorescent dual labeling technique for the quantitative
(
(
measurement of reduced and oxidized protein thiols in tissue samples.
Free Radical Biol. Med. 50, 510−517.
(45) Terrill, J. R., Duong, M. N., Turner, R., Le Guiner, C., Boyatzis,
A., Kettle, A. J., Grounds, M. D., and Arthur, P. G. (2016) Levels of
inflammation and oxidative stress, and a role for taurine in
dystropathology of the Golden Retriever Muscular Dystrophy dog
model for Duchenne Muscular Dystrophy. Redox Biol. 9, 276−286.
O
https://doi.org/10.1021/acs.bioconjchem.1c00206
Bioconjugate Chem. XXXX, XXX, XXX−XXX