Design of CID-cleavable protein cross-linkers: Identical mass modifications for simpler sequence analysis

Article abstract of DOI:10.1039/c5ob01410g

The cross-linking Mass Spectrometry (XL-MS) technique has enormous potential for studying the interactions between proteins, and it can provide detailed structural information about the interaction interfaces in large protein complexes. Such information h

Full text of DOI:10.1039/c5ob01410g

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Design of CID-cleavable protein cross-linkers:
identical mass modifications for simpler
sequence analysis†
Cite this: DOI: 10.1039/c5ob01410g
Wynne V. Kandur,a Athit Kao,b Danielle Vellucci,a Lan Huangb and
Scott D. Rychnovsky*a
The cross-linking Mass Spectrometry (XL-MS) technique has enormous potential for studying the inter-
actions between proteins, and it can provide detailed structural information about the interaction inter-
faces in large protein complexes. Such information has been difficult to obtain by conventional structural
methods. One of the primary impediments to the wider use of the XL-MS technique is the extreme chal-
lenge in sequencing cross-linked peptides because of their complex fragmentation patterns in MS. A
recent innovation is the development of MS-cleavable cross-linkers, which allows direct sequencing of
component peptides for facile identification. Sulfoxides are an intriguing class of thermally-cleavable
compounds that have been shown to fragment selectively during low-energy collisional induced dis-
sociation (CID) analysis. Current CID-cleavable cross-linkers create fragmentation patterns in MS2 of mul-
tiple peaks for each cross-linked peptide. Reducing the complexity of the fragmentation pattern in MS2
facilitates subsequent MS3 sequencing of the cross-linked peptides. The first authentic identical mass
linker (IML) has now been designed, prepared, and evaluated. Multistage tandem mass spectrometry (MSn)
analysis has demonstrated that the IML cross-linked peptides indeed yield one peak per peptide constitu-
ent in MS2 as predicted, thus allowing effective and sensitive MS3 analysis for unambiguous identification.
Selective fragmentation for IML cross-linked peptides from the 19S proteasome complex was observed,
providing a proof-of-concept demonstration for XL-MS studies on protein complexes.
Received 10th July 2015,
Accepted 4th August 2015
DOI: 10.1039/c5ob01410g
www.rsc.org/obc
Protein assemblies play a central role in the function of the cell.
One effective way to address the challenge of interpreting
Determining the structure of protein complexes is a daunting cross-linked sample data is to strategically design the linker to
challenge that often relies on a combination of methods to simplify MS sequencing of cross-linked peptides. Our group
construct multi-protein structural models.1 Recently, cross- has demonstrated such a simplification through the design of
linking Mass Spectrometry (XL-MS) analysis has been recognized CID-cleavable protein cross-linkers as outlined in Fig. 1. Other
as a valuable tool for the structural analysis of protein assem- groups have also reported CID-cleavable cross-linkers.4,5 DSSO
blies.2 Identification of cross-linked peptides provides amino (1) contains a sulfoxide group that cleaves in the MS instru-
acid level resolution information about protein physical contacts ment at lower collision energy than the peptide backbone.6,7
that can be used to assemble three-dimensional models of inter- This selective fragmentation separates the two linked peptide
acting proteins.2,3 The advantages of XL-MS studies include chains, allowing simplified sequencing in MS3 using conven-
small sample size, speed of sample preparation, and speed of tional database searching tools.3,7 In combination with new
data acquisition. One of the most significant challenges of cross- bioinformatics tools, DSSO has been proven as a very effective
linking studies is the difficulty in unambiguously sequencing protein cross-linker for elucidating structures of protein com-
low abundance cross-linked peptides in complex mixtures.
plexes because of its size, structural simplicity, and robust
cleavable bonds.3,7 In addition, two new derivatives of DSSO
have recently been developed for cross-linking studies, and
their successful applications in probing protein–protein
interactions further demonstrate the robustness of sulfoxide-
containing CID-cleavable reagents.8
The fragmentation pattern of DSSO interlinked peptides is
more complex than desired.9,10 Although the DSSO molecule
itself is symmetric, the fragmentation event is not, and this
aDepartment of Chemistry, University of California, Irvine, CA 92697, USA.
E-mail: srychnov@uci.edu
bDepartments of Physiology & Biophysics, University of California, Irvine, CA 92697,
USA
†Electronic supplementary information (ESI) available: General synthetic
methods, experimental data, and spectral data are presented in pdf format. See
DOI: 10.1039/c5ob01410g
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peaks, but it does not solve the complexity problem in MS2
and can compromise the peptide identification accuracy.
Therefore, it is desirable to develop a true equal-mass linker
that would generate identical (isobaric) fragments in the MS2,
and lead to only two predominant peaks in the MS2. Such an
identical mass linker (IML) was the goal of our investigation.
Herein we report the design and evaluation of new CID-cleav-
able identical mass cross-linkers based on the sulfoxide func-
tional group to facilitate MS2 and MS3 sequencing.
Results and discussion
Design and synthesis of a double-fragmentation cross-linker
Two approaches to solving the problem of four-peak MS2 were
explored: one double fragmentation event, where two identical
fragments would split from a central core, and one single frag-
mentation event, where two structurally different (yet isobaric),
fragments would result. Our efforts began with the double
fragmentation approach. It was hypothesized that a symmetri-
cal linker with two cleavable sites would allow for identical
mass modifications on both peptides in MS2. To this end, aryl
disulfoxide 2 was designed with the goal of inducing two sim-
ultaneous fragmentation events during MS2, resulting in pep-
tides with identical alkene modifications (Fig. 2).12 If the
desired fragmentation occurred, then MS2 would have two
peaks, corresponding to the two formerly interlinked peptides
(α and β represent different peptides in Fig. 2). Unfortunately,
when peptides interlinked by aryl disulfoxide 2 were exposed
to varying levels of CID energy in a Thermo Scientific LTQ
Orbitrap XL mass spectrometer, only one of the two sulfoxide
groups fragmented per CID event, resulting in a four-peak
pattern similar to DSSO (Fig. 2). The MS3 of the sulfenic acid
piece showed further fragmentation to the alkene, but did not
cleave the peptide backbone. Increasing the energy of the frag-
mentation event did not significantly alter the outcome.
Although the result is consistent with the physics of the CID
step, we were surprised that the selectivity for single bond clea-
vage was high enough to render the symmetric design of dis-
ulfoxide 2 ineffective.
Fig. 1 Representation of the detection of a DSSO (1) interlinked peptide
in MS1 and its fragmentation in MS2. The four-peak pattern in MS2
results from fragmentation on both sides of the sulfoxide. Subsequent
elimination of water is usually observed with the sulfenic acid
derivatives.
asymmetry leads each peptide chain in the cross-linked struc-
ture to produce two different daughter peaks in the MS2. A
representative four-peak MS2 pattern of a DSSO cross-linked
peptide (α–β) after CID-cleavage is illustrated in Fig. 1. Each
peptide (α and β) produces two MS2 peaks: an alkene fragment
and a sulfenic acid modified fragment.7 Although the four-
peak pattern is effective, it does require MS3 sequencing of at
least three of the four MS2 fragments to ensure unambiguous
identification of an inter-linked peptide in our current work-
flow. This increases MSn analysis time and leads to a fewer
peptides being sequenced in a given experiment. In addition,
the higher the number of MS2 fragment ions, the lower the
sensitivity observed for MS3 sequencing. These limitations that
are common to all published CID-cleavable linkers create a
need for new linker designs.4,5 An appropriate new linker
design would lead to one peak per peptide in MS2, facilitating
Design and synthesis of asymmetric single fragmentation
cross-linkers
targeted peak selection during MS3 analysis, improving sensi- The failure of the double fragmentation strategy stimulated a
tivity, and decreasing cycle time.
new design principle for identical mass linkers. While the
Our group envisioned a new derivative of DSSO that would desired double fragmentation of cross-linker 2 did not occur,
result in equivalent mass modifications on all peptide frag- it was observed that the fragmentation occurred selectively on
ments in MS2, thereby decreasing the number of peaks per the aliphatic side of the sulfoxide group. This simple feature
cross-linked peptide in MS2 and increasing the signal-to-noise for controlling the direction of fragmentation of the sulfoxide
ratio for these peaks. The Ranish group previously reported was incorporated into the design of the new IML (3). The
the CID-cleavable BDRG cross-linker11 to address this design concept is outlined in Fig. 3. Unlike prior cross-linkers,
problem. It is structurally complex, and fragments under CID the new IML designs depend upon an asymmetric cross-linker
to produce two peaks (one for each peptide component) that and a selective sulfoxide cleavage. Cross-linking α- and β-pep-
are one Dalton apart, thus allowing the observation of a tides leads to two different structures, 4 and 4′. They should
pseudo two-fragment MS2 pattern in a low-resolution instru- have similar mobility in the LC and are likely to elute together.
ment. This predictable pattern makes it easier to process MS2 They have the same elemental composition, and will result in
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Fig. 3 The identical mass linker (IML) design includes a CID-cleavable
sulfoxide, lysine-reactive NHS esters, and identical formula modifi-
cations after fragmentation, allowing for one peak per peptide in MS2.
Initial investigations taught us that the thermal stability of
the sulfoxide group could not be taken for granted. An
effective sulfoxide bond must survive storage and crosslinking,
only cleaving during the low energy CID step in the LCMS. To
address this concern, control compounds 5 and 6 were pre-
pared to determine the stability of relevant sulfoxide structures
(Fig. 4). Both compounds 5 and 6 contain a secondary aro-
matic sulfoxide, but in one case it is benzylic and in the other
case it is aliphatic. The two thioether model systems were pre-
pared, oxidized with one equivalent of m-CPBA, and the result-
1
Fig. 2 Top: a proposed double-fragmentation event in CID would lead ing sulfoxides were monitored by H NMR spectroscopy. Alkyl
to only two peptide-containing peaks in the MS2. Bottom: aryl disulfox-
ide linker cleaves only one bond at a time in a CID step, creating a four-
peak pattern in MS2.
substituted compound 5 showed a small amount of elimin-
ation over the course of a week. However, aryl substituted com-
pound 6 completely eliminated over the course of 24 hours.
The latter outcome is surprising, and we note that stable sec-
ondary benzylic sulfoxides are known in the literature, but the
MS1 peaks of the same mass. The selective cleavage in the
CID step will produce an α-peptide alkene 4α and an
α-peptide sulfenic acid 4′α. Similarly, the β-peptide will be
represented by a sulfenic acid 4β and an alkene 4′β. If the two
sides of the cross-linker are carefully balanced, the sulfenic
acid and alkene will have identical formulas and thus con-
verge in MS2. Funneling both structures into MS3 will result
in a superimposable fragmentation pattern that could be
directly sequenced. At each step along the way the molecular
structures will be more complex than for DSSO, but the
resulting MS spectra will be simpler because the components
are isobaric.
Fig. 4 Elimination is slower for α-alkyl sulfoxides than for α-aryl
sulfoxides.
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addition of the ester group appears to accelerate the elimin-
ation reaction.13 This data demonstrated that benzylic sulf-
oxides should be avoided in the IML designs, but that aliphatic
sulfoxides are promising.
Over the course of this project several proposed IML struc-
tures were designed, synthesized, and evaluated. The three
IMLs structures are shown in Fig. 5 as compounds 7, 8, and 9.
In each design, a sulfur atom was incorporated as a stable thio-
phene moiety, to balance the sulfenic acid, and a methylene
group was added to avoid labile benzylic sulfoxides, vide supra.
In order to understand the CID-induced fragmentation of
these structures, model sulfoxides 10 and 11 were prepared.
They were designed to produce the same two molecular frag-
ments as IML 3 (9), but each of these isobaric molecular frag-
ments can be tracked in the MS separately. Sulfoxides 7–11
define the cross-linkers and model compounds that were eval-
uated in this investigation.
Scheme 1 Synthesis of thiol building block 15.
The common building block for these cross-linkers is thio- bromide. The ester was introduced using a Grubbs cross-meta-
phenol 15, the preparation of which is outlined in Scheme 1. thesis with methyl acrylate (Scheme 2).16,17 These sequences
The route closely follows literature precedent.14 3-Hydroxycin- provided enough material for compound characterization and
namic acid 12 was converted to the methyl ester with sulfuric
acid in methanol before reduction to the dihydrocinnamate
13. The resulting phenol was combined with N,N,-dimethyl-
carbamoyl chloride in the presence of DABCO, followed by
thermal rearrangement to the dimethyl thiocarbamate at
300 °C. Subsequent hydrolysis with KOH in methanol and
tetrahydrofuran yielded the free thiol carboxylic acid 15
(Scheme 1).15
The thiophene half (18) for IML 1 (7) was prepared by lithia-
tion of 2-methoxythiophene 16 and alkylation with allyl
Fig. 5 Three identical mass linkers (7, 8, & 9) and two control com-
pounds (10, 11) were prepared for these studies.
Scheme 2 Synthesis of IML 1 (7).
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MS evaluation; as a result, the yields for individual steps were
not optimized. Thiophene conjugate acceptor 18 was coupled
to thiophenol 15 with triethylamine in methanol to form the
diester. Subsequent hydrolysis with lithium hydroxide led to
the desired diacid 19. Preformed NHS·TFA was used to activate
the diacid to form the di-NHS ester.18,19 This procedure led to
fewer side products and higher yields than the traditional
EDCI coupling. Oxidation to sulfoxide 7 (IML 1) with m-CPBA
proceeded uneventfully.10 Subsequent cross-linking studies on
IML 1 showed that the compound decomposed (through loss
of water) to 21 prior to MS analysis (Scheme 2). This un-
expected instability stimulated the design of the two new linkers
IML 2 (8) and IML 3 (9), which feature α-branching in place of
β-branching to avoid the undesired rearrangement.
IML 2 (8) was comprised of the thiophenol fragments 15 and
a thiophene fragment with a benzylic methoxy substituent (24).
The desired thiophene was rapidly synthesized by a Baylis–
Hillman reaction of 2-thiophenecarboxaldehyde 22 and methyl
acrylate.20 Methylation of the resulting alcohol using silver oxide
and methyl iodide21 was followed by lithium hydroxide hydro-
lysis of the ester to deliver the carboxylic acid 24. Direct Michael
addition of thiols to acrylic acids with catalytic TBAF·3H2O has
been reported.22 We incorporated this step in the sequence to
avoid the hydrolysis step on the methyl ester since the hydrolysis
step led to large amounts of elimination side product in the syn-
thesis of IML 1 (7). This approach decreased the step count and
circumvented thiophenol elimination, but resulted in modest
yields. The TBAF·3H2O catalyzed reaction produced diacid 25,
Scheme 4 Synthesis of IML 3 (9).
which was further elaborated to the di-NHS ester and oxidized thiophene instead of on the benzylic position. Preparation of
to the sulfoxide 8 (IML 2, Scheme 3). IML 2 was stable enough IML 3 began with lithiation and carbonylation of 2-methoxy-
for cross-linking and MS evaluation.
thiophene (26) in Scheme 4.23 A Knoevenagel condensation
IML 3 differed from IML 2 only in the position of the methoxy with diethyl malonate introduced the required ester, and sub-
group. In this case, the methoxy resides on the C2 position of the sequent reduction with sodium borohydride selectively removed
the reactive alkene to give diester 28.24,25 The esters were hydro-
lyzed with potassium hydroxide. A decarboxylative Mannich
addition led to the desired acrylic acid 29.25 Thiophenol 15 and
acrylic acid 29 were combined using the TBAF·3H2O catalyzed
reaction22 to produce the diacid, and further elaboration to the
di-NHS ester was carried out using the NHS·TFA reagent.
Careful oxidation with m-CPBA produced the sulfoxide 9.
Two model sulfoxides were prepared, NHS esters 10 and 11,
to aid in understanding the MS behavior of IML 3. The syn-
thetic routes are presented in the Scheme 5. Thiophenol 15
was added to thiophene 29 under TBAF·H2O conditions to
produce adduct 31 in modest yield. NHS ester formation and
oxidation generated the target sulfoxide 10. Preparation of sulf-
oxide 11 began with thiol 15. Conjugate addition to ethyl acry-
late gave adduct 32 in modest yield. NHS ester formation used
the NHS·TFA reagent, and subsequent oxidation delivered sulf-
oxide 11. With the successful preparation of all three cross-
linkers and the two model sulfoxides, they were ready to be
evaluated in the peptide and protein LCMSn experiments.
MS evaluation of IML agents
The cross-linking behavior of IML 1 (7) was examined using
Scheme 3 Synthesis of IML 2 (8).
model protein cytochrome C.26 Analysis of cross-linked cyto-
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Fig. 6 SDS-PAGE analysis of cytochrome C (left) and lysozyme (right) with
DSSO, IML 2, and IML 3 shows comparable small protein cross-linking.
Scheme 5 Synthesis of sulfoxides 10 & 11.
chrome C by SDS-PAGE revealed that IML 1 exhibits compar-
able cross-linking efficiency to DSSO, but MS data showed no
sulfoxide fragmentation during CID. The unused cross-linker
was recovered and analyzed by ESI+ MS, showing loss of
water. 1H NMR showed the mixture of IML 1 diastereomers
had coalesced to a single compound. It is hypothesized that a
Pummerer-like rearrangement occurred, leading to an α,β-un-
saturated cross-linker 21 that is not CID-cleavable (Scheme 2).
The next two IML designs included α-branching instead of
β-branching to avoid the undesired rearrangement.
The cross-linking ability of IML 2 and IML 3 was compared
to that of DSSO. First, an SDS-PAGE analysis of model protein
(cytochrome C and lysozyme) cross-linking was explored. The
gel in Fig. 6 shows both IML linkers exhibit considerable
cross-linking activity. In the cytochrome C case, they were both
Fig. 7 IML 2 suffers from loss of the methoxy substituent at similar
energies to sulfoxide fragmentation. (DN = dead end).
comparable to DSSO; in the lysozyme case it appears they may the peak represents a mixture of both possible peptide
have induced higher order aggregate species. With proof of linkages. (Further detail of this experiment is presented in
protein cross-linking, IML cross-linked samples were subjected ESI.†) The complexity of the MS data for IML 2 arose from
to MS analysis.
competitive cleavage of the desired sulfoxide and the un-
The desired fragmentation (top) and example MS2 fragmen- desired benzylic methoxy group in 8. This problem led to
tation of IML 2 cross-linked sample (bottom) are shown in prioritization of IML 3 as the new target.
Fig. 7. Initial testing of IML 2 with synthetic peptide Ac-IR7
The challenges associated with interpreting the m/z 595.75
(Acetyl-IEAEKGR) in the MS instrument showed significant peak from IML 2 cross-linking experiments (Fig. 7 and ESI†)
loss of the methoxy group during CID (Fig. 7). The peak with led us to prepare model compounds to mimic the behavior
m/z 595.752+ represents a dead end-modified peptide10 that each side of the IML 3 cross-linker. Compound 10 mimics the
has lost the methoxy substituent during the first CID and has thiophene half, while compound 11 mimics the phenyl
not undergone sulfoxide elimination. This dead end linker portion. In each case, the CID sulfoxide elimination should
could be bonded to the peptide on either side, so it is likely generate one component of the full IML 3 CID process, and
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Fig. 9 MS2 and MS3 spectra of Ac-IR7 modified by sulfenic acid control
compound 11.
Fig. 8 MS2 and MS3 spectra of Ac-IR7 modified by alkene control com-
pound 10, i.e. 10βa.
these individual components can be directly analyzed in MS3
without complications from other peptidic components.
CID fragmentation of model compound 10 generated the
alkene-modified component of the fragmentation products
(Fig. 8). Compound 10-modified Ac-IR7 (β) appeared as a 1+ ion
in MS1 (m/z 1150.50) and MS2 (m/z 1024.48) (Fig. 8). The result-
ing 10βa peak was sequenced in the MS3 step without difficulty.
Model compound 11 was use to probe the sulfenic acid
fragmentation products. The 11-modified Ac-IR7 (β) appeared
as a 1+ ion in MS1 (m/z 1124.54) and MS2 (m/z 1024.48) (Fig. 9).
The MS2 peak representing the 1+ ion of thiophenol-modified
Ac-IR7 was successfully sequenced in MS3 (Fig. 9). As expected
the MS2 peaks generated from IR7-10 and IR7-11 had identical
m/z 1024.48, satisfying our expectations for the design of the
IML 3.
Testing of IML 3 began with peptide cross-linking of Ac-TR9
(Acetyl-TTSYKVTIR) and Ac-IR7 (Acetyl-IEAEKGR). As expected,
an IML 3 interlink showed identical mass modifications in
MS2, achieving proof of concept for the desired two-peak
pattern in MS2 (Fig. 10). As predicted, the MS2 of Ac-IR7 is
identical for IML 3 and for both control compounds 10 and 11
(Fig. 8–10). The extraordinarily clean MS2 for IML 3 cross-
linked peptides will dramatically simplify the XL-MS analysis
of more complex proteins.
IML 3 cross-linker was next evaluated in the analysis of a
protein complex. IML 3 was mixed with a purified sample of
the multi-subunit yeast 19S complex (∼1 MDa) in order to
demonstrate its utility as a tool for structural studies. Since the
19S has been thoroughly interrogated with DSSO in our
studies, we were able to compare to a previously identified
cross-link.3 In this case, IML 3 was used to successfully ident-
ify an Rpt4–Rpt5 interlink with clear MS2 and MS3 data
(Fig. 11). The MS2 shows two peaks representing two inter-
linked peptides (m/z 488.262+ and m/z 626.332+). Each of these
was selected for MS3 and, after undergoing CID, led to peptide
sequence data. It is worth noting that in an actual multi-
protein analysis, the MS2 spectrum contain a number of small
peaks from impurities in the peptide digest sample. The two
interlinked peptides were identified and automatically
sequenced in this sample because of the improved simplicity
in the MS2 spectrum.
Conclusion
Three different identical mass linkers (IML) for protein cross-
linking studies have been prepared and evaluated. The first
one, IML 1, was chemically unstable. IML 2 led to fragmenta-
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Fig. 11 MSn data of a representative IML 3 interlinked peptide of the
19S proteasome complex.
Experimental section
General information
Fig. 10 The simplified MS2 pattern with IML 3 cross-linked to two
different peptides is shown. Only two peaks are observed in the MS2.
1H NMR and 13C NMR spectra were recorded at ambient temp-
erature at 500 MHz and 125 MHz, respectively, on a Bruker
DRX500 NMR instrument. 1H and 13C NMR data is reported as
follows: chemical shifts are reported in ppm on a δ scale and
referenced to internal tetramethylsilane or residual solvent
(TMS: δ 0.00; CHCl3: δ 7.27(1H), 77.23 (13C)), multiplicity (s =
singlet, d = doublet, t = triplet, q = quartet, qu = quintet, m =
multiplet), coupling constants (Hz), and integration. Infrared
(IR) spectra were obtained using a FT-IR spectrometer. Accu-
rate mass spectra were acquired on a Waters LCT Premier
quadrupole time-of-flight spectrometer and were obtained by
peak matching. Gas chromatography/mass spectrometry
(GC/MS) was performed with a Thermo-Finnigan Trace Mass
Spectrometer Plus quadrupole system with a fused silica
capillary column (30 m × 0.32 mm × 0.25 mm) wall-coated
with DB-5 (J & W Scientific) using electron ionization (70 eV)
or a Waters GCT Premier orthogonal acceleration time-of-
flight spectrometer using chemical ionization. Melting points
tion patterns complicated by the loss of a labile methoxy
group. The third linker, IML 3, showed both good chemical
stability and clean fragmentation patterns in LCMSn. IML 3 led
to much simpler MS2 patterns upon CID, facilitating the selec-
tion of meaningful peaks in the MS3 analysis. These proof-of-
concept experiments have elucidated important design criteria
for CID cleavable sulfoxide cross-linkers. Foremost among
these criteria is the use of primary sulfoxides for their balance
of thermal stability and CID lability. The IML strategy, where
the cross-linker is designed to produce identical mass and
formula components upon CID, is a promising approach to
improve the analysis of protein–protein interactions by cross-
linking and LCMSn methods.
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are uncorrected and were obtained using a Büchi 510 melting Experimental procedures
point apparatus. Analytical thin layer chromatography was
performed on EMD Chemicals Inc. silica gel 60 F254 plates.
Liquid chromatography was performed using forced flow
(flash chromatography) of the indicated solvent system on
Sorbent Technologies silica gel (SiO2) 60 (230–400 mesh).
Unless otherwise noted, all reactions were carried out
Methyl 3-(3-hydroxyphenyl)propanoate (13). Acetyl chloride
under an atmosphere of argon in flame-dried glassware. Sol- (4.39 mL, 61.5 mmol), m-hydroxycinnamic acid (12) (10.0 g,
vents were distilled from CaH2 or filtered through alumina 60.9 mmol), and methanol (300 mL) were combined in a
before use.27
round bottom flask, fitted with a cold water condenser, and
heated to 70 °C for 4.5 h. The reaction mixture was cooled to
room temperature and the solvent was removed and flushed
General procedure 1: ester hydrolysis
Starting ester was dissolved in 2 : 1 or 4 : 1 THF : H2O, depend- with CH2Cl2. The crude methyl ester was dissolved in ethanol
ing on solubility. The mixture was cooled to 0 °C and lithium (206 mL) and acetic acid (11.0 mL) before addition of Pd/C
hydroxide (98%, 5 equiv.) dissolved in minimal H2O was (5% by wt, 6.50 g, 3.05 mmol). The headspace was evacuated
added slowly. The reaction was monitored by thin layer and a balloon of H2 gas was attached to the flask. The reaction
chromatography, while stirring vigorously. Upon completion, stirred for two days and there was no change in Rf. The reac-
the mixture was diluted with diethyl ether and water before the tion mixture was filtered through Celite and solvent was
layers were separated. Diethyl ether was added to the resulting removed in vacuo. The crude product was purified by flash
aqueous layer, followed by addition of HCl to pH 1. The layers column chromatography (30% EtOAc/hexanes) to yield desired
were separated and the aqueous layer was extracted with methyl ester 13 (8.82 g, 80%). Spectra were identical to pre-
diethyl ether (2×). Organics were combined, washed with viously reported data.28
brine, dried over Na2SO4, filtered and evaporated in vacuo.
General procedure 2: TBAF-promoted Michael additions
Acrylic acid (0.28 mmol), thiophenol (0.51 mmol), and THF
(1 mL) were combined in a round bottom flask to which
TBAF·3H2O (0.06 mmol) was added at rt. The flask was fitted
Methyl 3-(3-((dimethylcarbamothioyl)oxy)phenyl)propanoate
with a cold-water condenser and the mixture was heated to
(14). Methyl ester 13 (5.00 g, 27.7 mmol) was combined with
50 °C for 16 h. The mixture was cooled to rt, evaporated
dimethylthiocarbamoyl chloride (10.3 g, 83.3 mmol) and
in vacuo, diluted with EtOAc and washed with 1 N HCl (2×).
DABCO (9.3 g, 82.9 mmol) in DMF (14 mL) in a round bottom
The organic layer was washed with brine, dried over Na2SO4,
flask. The mixture was heated to 80 °C for 18 h before the
filtered, and evaporated in vacuo to yield the crude product. In
addition of EtOAc, H2O, and brine. The layers were separated
some cases, this reaction was run neat.22
and the organic layer was washed with brine. The aqueous
layer was extracted with EtOAc and the organic layer was
washed with brine. The organic layers were combined, dried
over Na2SO4, and the solvent was removed in vacuo. The crude
material was purified by flash column chromatography (20%
EtOAc/hexanes) to afford the desired product (4.60 g, 62%).
Spectra were comparable to previously reported data.28
1H NMR (500 MHz, CDCl3) δ 7.31 (t, J = 7.7, 1H), 7.10 (d, J =
7.5, 1H), 6.93 (overlapping d, J = 7.5, 2H), 3.68 (s, 3H),
3.46 (s, 3H), 3.35 (s, 3H), 2.98 (t, J = 7.7, 2H), 2.66 (t, J = 7.7,
2H); 13C NMR (125 MHz, CDCl3) δ 30.9, 35.6, 38.9, 43.5,
51.9, 120.9, 122.9, 126.1, 129.4, 142.1, 154.3, 173.4, 188.0;
IR (thin film) 3404, 2948, 1736, 1532, 1286 cm−1; Accurate
Mass ES+ m/z calcd for C13H17NO3SNa (M + Na)+ 290.0827,
found 290.0835.
General procedure 3: NHS ester preparation
Crude diacid (0.08 mmol) was dissolved in dry dichloro-
methane (0.62 mL) in a flame dried round bottom flask under
argon. NHS·TFA (0.41 mmol) was added before a slow addition
of triethylamine (0.49 mmol) at 0 °C. The mixture was left to
warm slowly overnight while stirring under argon. After 16 h,
the mixture was diluted with dichloromethane and washed
with water (2×). The organic layer was dried with Na2SO4, fil-
tered, and evaporated in vacuo.
General procedure 4: m-CPBA-oxidation to sulfoxide
Di-NHS ester (0.026 mmol) was dissolved in CDCl3 (1 mL) and
m-CPBA (77% w/w, 0.026 mmol) was added slowly while
monitoring by LRMS ESI+ and 1H NMR. When this reaction
was run on larger scale than 30 mg starting material, the
solution was cooled in an ice bath prior to m-CPBA addition.
Once the reaction was complete, the solution was diluted
with dichloromethane (2 mL) and washed with 10% sodium
bicarbonate solution (3 × 2 mL). The organic layer was dried
over Na2SO4, filtered, and evaporated in vacuo to afford the
product.
Methyl
3-(3-((dimethylcarbamoyl)thio)phenyl)propanoate
(34). Starting protected alcohol 14 (0.2 g, 0.748 mmol) was
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placed in a crimp-top vial and sealed under argon. The neat oil (125 MHz, CDCl3) δ 164.9, 136.8, 129.3, 121.7, 116.3, 103.4,
was placed in a 310 °C sand bath. After 20 min, the reaction 60.4, 34.9; IR (thin film) 3008, 2831, 1562, 1512, 1215 cm−1
;
was complete by TLC and the vial was allowed to cool to rt. Accurate Mass (GCMS CI+) m/z calcd for C8H11OS (M + H)+
The crude product was purified by flash column chromato- 155.0531, found 155.0528.
graphy (20% EtOAc/hexanes) to yield dark yellow oil product
34 (91 mg, 45%). 1H NMR (500 MHz, CDCl3) δ 7.35–7.29
(m, 3H), 7.22 (d, J = 7.4, 1H), 3.08 (br s, 3H), 3.03 (br s, 3H),
2.96 (t, J = 8.0, 2H), 2.64 (t, J = 8.0, 2H); 13C NMR (125 MHz,
CDCl3) δ 173.4, 167.1, 141.5, 135.7, 133.9, 129.4, 129.2, 129.0,
51.9, 37.1, 36.0, 30.9; IR (thin film) 2951, 2360, 1736, 1670,
(E)-Methyl 4-(5-methoxythiophen-2-yl)but-2-enoate (18).
1365 cm−1; Accurate Mass ES+ m/z calcd for C13H17NO3SNa
2-Allyl-5-methoxythiophene 17 (0.256 g, 1.66 mmol) was com-
(M + Na)+ 290.0827, found 290.0830.
bined with methyl acrylate (4.15 mL) and Grubbs 2 catalyst
(0.035 g, 0.0415 mmol) in a sealed vial under argon. The
reaction was heated at 90 °C for eight hours. The reaction
mixture was cooled to rt, evaporated, and placed on the high
vacuum. The crude product was purified by flash column
chromatography (20–40% CH2Cl2/pentane) to yield desired
product (0.100 g, 28%). 1H NMR (500 MHz, CDCl3) δ 7.06–7.02
3-(3-Mercaptophenyl)propanoic acid 15. Dimethyl thio-
(dt, 1H, J = 6.6, 15.5), 6.42 (d, J = 3.7, 1H), 6.01 (d, J = 3.7, 1H),
carbamate 34 (0.20 g, 0.75 mmol) was combined with KOH
5.88 (d, J = 15.5, 1H), 3.85 (s, 3H), 3.73 (s, 3H), 3.54 (d, J = 6.6,
(0.17 g) in methanol (5 mL) and THF (5 mL) in a sealed vial
under argon. The vial was heated to 70 °C for five h. The vial
was allowed to cool and CH2Cl2 and 5% HCl were added. The
layers were separated before the aqueous layer was extracted
three times with CH2Cl2. The organic layer was dried with
Na2SO4, filtered, and the solvent was removed in vacuo. The
2H); 13C NMR (125 MHz, CDCl3) δ 167.0, 165.4, 146.7,
126.0, 123.1, 122.3, 103.6, 60.5, 51.8, 33.0; IR (liquid)
2951, 2831, 1728, 1658, 1512 cm−1; Accurate Mass (GCMS
CI+) m/z calcd for C10H12O3S (M + H)+ 213.0585, found
213.0589.
crude product was purified by flash column chromatography
(40% EtOAc/hexanes) to yield the product (86 mg, 63%).
m.p. 62–65 °C; 1H NMR (500 MHz, CDCl3) δ 7.18–7.10 (m, 3H),
6.98 (d, J = 7.2, 1H), 3.43 (s, 1H), 2.89 (t, J = 7.8, 2H), 2.66 (t, J =
7.8, 2H); 13C NMR (125 MHz, CDCl3) δ 179.4, 141.4, 131.2,
129.5, 129.4, 127.6, 35.5, 30.4; IR (thin film) 3055, 2931, 2862,
2557, 1693 cm−1; Accurate Mass ES− m/z calcd for C9H9O2S
(M − H)− 181.0323, found 181.0323.
3-(3-((4-Methoxy-1-(5-methoxythiophen-2-yl)-4-oxobutan-2-yl)-
thio)phenyl)propanoic acid (35). Dimethyl thiocarbamate 34
(0.10 g, 0.38 mmol) was combined with KOH (0.10 g,
1.8 mmol) in methanol (5.0 mL) and THF (5.0 mL) in a sealed
vial under argon. The vial was heated to 70 °C for five hours.
2-Allyl-5-methoxythiophene (17). 2-Methoxythiophene (16) The vial was allowed to cool and 1 M HCl (0.50 mL) was
(1.1 g, 1.0 mL, 9.9 mmol) was dissolved in dry diethyl ether added. Methyl acrylate 18 (0.027 g, 0.13 mmol) and triethyl-
(28 mL) in a flame dried round bottom flask. sec-Butyllithium amine (0.038 mL, 0.38 mmol) were added to the solution and
(13.7 mL of [0.72] solution, 9.9 mmol) was added to the the reaction mixture was left to stir at rt overnight. The reac-
mixture dropwise at 0 °C. After 90 min, the reaction mixture tion was extracted with CH2Cl2 (3×) and the organics were com-
was cooled to −78 °C allyl bromide (0.86 mL, 9.9 mmol) was bined, dried over Na2SO4, filtered, and evaporated. The crude
added. The reaction was allowed to slowly warm and stir product was purified by flash column chromatography (20%
overnight. The reaction was cooled to 0 °C before addition of EtOAc/hexanes) to yield desired product (0.017 g, 18%).
saturated ammonium chloride solution (14 mL). The layers 1H NMR (500 MHz, CDCl3) δ 7.31 (app d, J = 8.7, 2H), 7.23
were separated before the aqueous layer was extracted with (app d, J = 7.5, 2H), 7.11 (d, J = 7.6, 1H), 6.43 (d, J = 3.7, 1H),
diethyl ether (2×). The organic layers were combined, washed 5.99 (d, J = 3.7, 1H), 3.84 (s, 3H), 3.66 (s, 3H), 3.67–3.64 (over-
with brine, and dried over MgSO4 before being filtered and lapping m, 1H), 3.01 (dd, J = 15.0, 5.0, 1H), 2.98–2.86 (m, 3H),
evaporated. The crude product was purified by flash column 2.71–2.60 (m, 3H), 2.53 (dd, J = 15.0, 5.0, 1H); 13C NMR
chromatography (pentane) to yield the product as a green oil (125 MHz, CDCl3) δ 177.7, 172.1, 165.4, 141.3, 133.8, 133.1,
(0.70 g, 46%). 1H NMR (500 MHz, CDCl3) δ 6.39 (d, J = 3.7, 131.3, 129.4, 127.9, 126.6, 124.0, 103.2, 60.4, 52.1, 46.3, 38.9,
1H), 6.00 (d, J = 3.7, 1H), 5.97–5.92 (m, 1H), 5.13 (d, J = 15.5, 35.7, 35.5, 30.6; IR (thin film) 2951, 1736, 1709, 1435,
1H), 5.07 (dt, J = 8.7, 1H), 3.40 (d, J = 5.5, 2H); 13C NMR 1211 cm−1; Accurate Mass ES+ m/z calcd for C19H22O5S2Na
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(M + Na)+ 417.0806, found 417.0807.
1207 cm−1; Accurate Mass ES+ m/z calcd for C26H26N2O9S2
(M + Na)+ 597.0977, found 597.0980.
3-((3-(2-Carboxyethyl)phenyl)thio)-4-(5-methoxythiophen-
2-yl)butanoic acid (19). Monocarboxylic acid 35 (0.017 g,
0.043 mmol) was dissolved in 4 : 1 THF/H2O (0.40 mL) and
LiOH·H2O was added and the mixture was stirred vigorously
for 2.5 h. H2O and diethyl ether were added and the aqueous
layer was washed twice more with diethyl ether. 1 M HCl was
added until pH 1 was reached, and the aqueous layer was
extracted with diethyl ether (3×). The combined organic layers
were washed with brine and evaporated. The crude product
was redissolved in CH2Cl2, filtered through cotton and evapo-
rated down to yield an orange oil (0.0060 g, 34%). 1H NMR
(500 MHz, CDCl3) δ 7.35 (s, 1H), 7.30 (d, J = 7.7, 1H), 7.22 (t, J =
7.6, 1H), 7.11 (d, J = 7.5, 1H), 6.45 (d, J = 3.6, 1H), 6.00
(d, J = 3.7, 1H), 3.84 (s, 3H), 3.61 (quintet, J = 7.0, 1H), 3.03
(dd, J = 15.2, 6.5, 1H), 3.00–2.90 (m, 3H), 2.68 (t, J = 7.2, 2H),
2.64 (app dd, J = 11.3, 5.9, 1H), 2.53 (app dd, J = 16.1, 8.2, 1H);
13C NMR (125 MHz, CDCl3) δ 178.9, 177.6, 165.4, 141.3, 133.7,
133.6, 131.8, 129.4, 128.2, 126.5, 124.1, 103.3, 60.4, 46.7, 39.4,
36.0, 35.6, 30.6; IR (thin film) 3086, 2924, 2357, 1709,
1211 cm−1; Accurate Mass ES− m/z calcd for C18H19O5S2 (M − H)−
379.0674, found 379.0669.
IML 1 (7). Di-NHS-ester 20 (0.006 g, 0.01 mmol) was sub-
jected to general procedure 4 to yield desired product as a
mixture of diastereomers contaminated with grease (0.006 g
crude after workup). 1H NMR (500 MHz, CDCl3) δ 7.51–7.30
(m, 4H), 6.45 (d, J = 3.8, 1H), 6.04 (d, J = 3.8, 1H), 3.88–3.83 (m,
2H), 3.63 (d, J = 6.5, 2H), 3.13–2.97 (m, 3H), 2.94–2.87 (m, 2H),
2.85 (s, 8H), 2.62 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 169.4,
169.3, 168.9, 153.0, 140.6, 140.5, 135.4, 134.1, 131.8, 130.6,
129.8, 129.7, 129.4, 127.42, 127.37, 126.1, 124.3, 124.2, 123.9,
123.0, 116.7, 103.8, 60.5, 41.2, 33.4, 32.6, 32.5, 30.5, 30.4, 30.2,
29.9, 26.1, 25.82, 25.76; IR (thin film) 2924, 2256, 1817, 1782,
1739 cm−1; Accurate Mass ES+ m/z calcd for C26H26N2O10S2Na
(M + Na)+ 613.0927, found 613.0920.
Methyl 2-(hydroxy(thiophen-2-yl)methyl)acrylate 23. Thio-
phene carboxaldehyde 22 (5 mL, 0.054 mmol) and methyl acry-
late (6.24 mL, 0.070 mmol) were combined with DABCO (3.9 g,
0.035 mmol) in a round bottom flask and the mixture was
sonicated for 24–48 h. The resulting mixture was loaded onto a
silica gel column and the product was isolated by flash chromato-
graphy as yellow oil (7.14 g, 66%). Spectra were identical to
previously reported data.20
Di-NHS-ester 20. Diacid 19 (0.005 g, 0.013 mmol) was com-
bined with CH2Cl2 (0.10 mL), triethylamine (0.011 mL,
0.080 mmol) and NHS·TFA (0.017 g, 0.080 mmol) at rt under
argon. After 3 h stirring at rt, TLC showed the reaction was
complete. CH2Cl2 and H2O were added before the organic
layer was washed with H2O (2×) and brine before it was filtered
Methyl 2-(methoxy(thiophen-2-yl)methyl)acrylate (36). Benzylic
through cotton and evaporated. The crude product was puri- alcohol 23 (0.50 g, 2.5 mmol) was combined with Ag2O (1.2 g,
fied by flash column chromatography (50% EtOAc/hexanes) to 5.0 mmol) and methyl iodide (0.47 mL, 7.6 mmol) in CH2Cl2
yield desired product (0.0060 g, 80%). 1H NMR (500 MHz, (10 mL). The mixture was sealed in a capped vial and heated to
CDCl3) δ 7.45 (s, 1H), 7.39 (dd, J = 7.7, 12.8, 1H), 7.28 (t, J = 7.6, 50 °C over 72 h.21 The reaction mixture was cooled, filtered
1H), 7.18 (d, J = 7.5, 1H), 6.50 (d, J = 3.7, 1H), 6.01 (d, J = 3.7, through a silica plug with CH2Cl2 rinses, and evaporated
1H), 3.86 (s, 3H), 3.62 (quintet, J = 6.8, 1H), 3.08 (dd, 1H), in vacuo to afford the product as a clear colorless oil (0.374 g,
1
3.08–3.00 (m, 4H), 2.93 (dd, J = 8.0, 7.3, 2H), 2.93–2.75 70%) H NMR (500 MHz, CDCl3) δ 7.27 (d, J = 5.5, 1H), 7.01
(m, 9H); 13C NMR (125 MHz, CDCl3) δ 169.3, 169.2, 168.9, (d, J = 3.1, 1H), 6.95 (dd, J = 3.6, 4.8, 1H), 6.37 (s, 1H), 6.04
168.0, 166.8, 165.6, 140.4, 134.3, 132.9, 132.6, 129.6, 128.6, (s, 1H), 5.40 (s, 1H), 3.74 (s, 3H), 3.38 (s, 3H); 13C NMR
126.1, 125.8, 124.5, 103.4, 60.4, 46.1, 35.4, 35.2, 32.6, 30.4, (125 MHz, CDCl3) δ 166.3, 143.6, 140.8, 126.8, 126.5, 125.8,
26.1, 25.8, 25.8, 25.7; IR (thin film) 2927, 1813, 1786, 1739, 125.4, 57.3, 52.2; IR (thin film) 2993, 2951, 2827, 1724,
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1631 cm−1; Accurate Mass ES+ m/z calcd for C10H12O3SNa 79.2, 58.1, 51.5, 32.7, 32.6, 30.5, 30.0, 25.8; IR (thin film) 2940,
(M + Na)+ 235.0405, found 235.0406.
2255, 1813, 1784, 1739 cm−1; Accurate Mass ES+ m/z calcd for
C26H26N2O9S2Na (M + Na)+ 597.0977, found 597.0980.
2-(Methoxy(thiophen-2-yl)methyl)acrylic acid (24). Acid 24
was prepared from methyl ether 36 (0.374 g, 1.76 mmol) by
general procedure 1. Crude product was purified by flash
chromatography (30% EtOAc/hexanes) (0.1 g, 0.471 mmol)
(300 mg, 86%) 1H NMR (500 MHz, CDCl3) δ 7.29 (d, J = 5.0,
1H), 7.02 (d, J = 3.3, 1H), 6.96 (t, J = 3.5, 1H), 6.53 (s, 1H), 6.15
(s, 1H), 5.37 (s, 1H), 3.38 (s, 3H); 13C NMR (125 MHz, CDCl3)
δ 171.1, 143.1, 140.2, 128.0, 126.8, 126.7, 126.0, 76.3, 57.3;
IR (thin film) 2934, 2664, 1697, 1629, 1435 cm−1; Accurate
Mass ES+ m/z calcd for C9H10O3SNa (M + Na)+ 221.0248, found
221.0240.
IML 2 (8). Di-NHS ester 37 (0.015 g, 0.026 mmol) was sub-
jected to general procedure 4 to yield desired product (0.013 g,
84%). 1H NMR (500 MHz, CDCl3) δ 7.53–7.29 (m, 4H), 7.03
(s, 1H), 7.01–6.96 (m, 1H), 5.17 (d, J = 4.3, 0.5H), 5.12 (d, J =
4.5, 0.5H), 3.60–3.22 (m, 6H), 3.20–3.00 (m, 1H), 3.12 (over-
lapping t, J = 7.6, 2H), 2.95 (t, J = 7.2, 2H), 2.83 (br s, 8H);
13C NMR (125 MHz, CDCl3) δ 169.27, 169.26, 169.23, 169.20,
169.1, 168.7, 167.8, 166.9, 166.6, 140.94, 140.93, 140.88, 140.6,
136.8, 135.5, 134.1, 131.8, 131.4, 130.6, 130.1, 130.0, 129.9,
129.8, 129.5, 127.6, 127.4, 127.0, 126.8, 126.42, 126.38, 126.30,
126.28, 126.18, 126.15, 124.33, 124.25, 123.9, 123.0, 122.9,
122.7, 79.1, 79.0, 76.4, 58.4, 58.3, 57.5, 54.8, 53.1, 46.1, 45.1,
32.6, 32.50, 32.47, 32.45, 30.5, 30.44, 30.38, 25.8; IR (thin film)
2939, 2251, 1812, 1781, 1738 cm−1. Accurate Mass ES+ m/z
calcd for C26H26O10N2S2Na (M
613.0925.
+
Na)+ 613.0927, found
2-(((3-(2-Carboxyethyl)phenyl)thio)methyl)-3-methoxy-3-
(thiophen-2-yl)propanoic acid (25). Thiophenol 15 (0.054 g,
0.30 mmol), carboxylic acid 24 (0.039 g, 0.20 mmol), and THF
(enough to dissolve) were combined with TBAF·H2O (0.012 g,
0.039 mmol) in a capped vial. The vial was evacuated and
backfilled with argon before heating to 50 °C overnight. The
solvent was removed in vacuo before the crude mixture was
purified by flash column chromatography (60% EtOAc/
hexanes) to yield desired product (0.047 g, 63%). The material
was not clean; it was taken on to the next step without further
2-Methoxythiophene carboxaldehyde 26. 2-Methoxythio-
phene (1.13 g, 9.89 mmol) was combined with diethyl ether
(27.5 mL) in a flame-dried round bottom flask under argon.
The reaction mixture was cooled to 0 °C before addition of
s-butyllithium (13.7 mL, [0.72]) over 20 min. After one hour,
the reaction mixture was cooled to −78 °C before slow addition
of N,N-dimethylformamide (1.38 mL, 17.8 mmol). Once the
addition was complete, the reaction mixture was left to warm
slowly to rt and stir overnight. Crude product was purified by
flash chromatography to yield 1.1 g (78%) of the desired
product. Spectra were identical to previously reported data.23
purification. IR (thin film) 2930, 2566, 1708, 1594, 1420 cm−1
;
Accurate Mass ES+ m/z calcd for C18H20O5S2Na (M + Na)+
403.0650, found 403.0656.
Di-NHS ester 37. Diacid 25 (0.031 g, 0.081 mmol) was sub-
jected to general procedure 3. The crude product was purified
Diethyl 2-((5-methoxythiophen-2-yl)methyl)malonate (28).
by flash column chromatography (70% EtOAc/pentane) to 2-Methoxythiophene-carboxaldehyde 26 (3.15 g, 22.2 mmol),
yield desired product (0.031 g, 67%). 1H NMR (500 MHz, cyclohexane (111 mL), diethyl malonate (3.48 mL, 22.8 mmol),
CDCl3) δ 7.38 (d, J = 4.7, 1H), 7.21–7.16 (m, 1H), 7.13 (d, J = piperidine (0.330 mL, 0.890 mmol), and benzoic acid (0.108 g,
8.3, 1H), 7.07–7.01 (m, 4H), 5.01 (d, J = 5.3, 1H), 3.44 (dd, J = 0.890 mmol) were combined in a round bottom flask fitted
13.9, 3.2, 1H), 3.38 (s, 3H), 3.32 (dd, J = 13.9, 9.9, 1H), with a Dean Stark trap, cold water condenser, and fitted with a
3.27–3.21 (m, 1H), 2.98–2.93 (m, 2H), 2.90–2.76 (m, 10H); calcium carbonate drying tube. The reaction mixture was
13C NMR (125 MHz, CDCl3) δ 169.3, 168.9, 168.0, 167.1, 141.5, heated in a 105 °C oil bath for 20 h. The reaction flask was
140.2, 135.5, 129.5, 129.1, 128.4, 127.3, 126.7, 126.6, 126.1, allowed to cool to rt before the solvent was removed in vacuo.
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Diethyl ether was added to redissolve the mixture and the hours at room temperature, the mixture was heated to 100 °C
organic solution was washed with 10% hydrochloric acid solu- for 2 h. The mixture was cooled to rt, chloroform was added,
tion (3×), saturated sodium bicarbonate solution (3×), and and the organic layer was extracted with saturated bicarbonate
brine (1×) before it was filtered and the solvent was removed solution (3 × 50 mL). Chloroform was added to the aqueous
in vacuo. The product 27 (6.20 g, 98%) was not purified.24 The layer and concentrated HCl was added slowly, with stirring, to
resulting crude product was dissolved in ethanol and cooled pH 1. The resulting mixture was extracted with chloroform
to 0 °C. Sodium borohydride (0.429 g, 11.3 mmol) was added (3×), dried over Na2SO4, filtered, and evaporated in vacuo. The
to the solution and gas evolved. After 45 min, the ice bath product was isolated crude as a light brown solid (0.77 g,
was removed and the solution was heated to 40 °C in an oil 47%),29 m.p. 59 °C–62 °C; 1H NMR (500 MHz, CDCl3) δ 6.45
bath. Reaction progress was monitored by TLC (20% EtOAc/ (d, J = 3.7, 1H), 6.38 (s, 1H), 6.01 (d, J = 3.7, 1H), 5.74 (d, J =
hexanes). Once the reaction was complete, water was added 1.0, 1H), 3.85 (s, 3H), 3.66 (s, 2H); 13C NMR (125 MHz, CDCl3)
before adding acetic acid to pH 4. Solids were filtered off δ 171.5, 165.1, 138.9, 128.7, 126.9, 123.1, 103.3, 60.2, 32.2; IR
and diethyl ether and water were added to the filtrate. The (thin film) 2995, 1696, 1560, 1512, 1203 cm−1; Accurate Mass
layers were separated, the organics washed with water and ES− m/z calcd for C9H9O3S (M − H)− 197.0272, found
brine before drying with Na2SO4, filtering, and evaporating 197.0262.
in vacuo. The crude product 28 was isolated as a yellow oil
(5.26 g, 85%)24,25 1H NMR (500 MHz, CDCl3) δ 6.44 (d, J =
3.7, 1H), 5.96 (d, J = 3.8, 1H), 4.19 (qd, J = 7.2, 2.1, 4H), 3.83
(s, 3H), 3.59 (t, J = 7.6, 1H), 3.26 (d, J = 7.6, 2H), 1.25 (t, J =
7.1, 6H); 13C NMR (125 MHz, CDCl3) δ 168.7, 165.2, 126.2,
123.5, 103.3, 61.8, 60.4, 54.3, 29.7, 14.3; IR (thin film) 2939,
1731, 1561, 1506, 1208 cm−1; Accurate Mass ES+ m/z calcd for
C13H18O5SNa (M + Na)+ 309.0773, found 309.0770.
3-((3-(2-Carboxyethyl)phenyl)thio)-2-((5-methoxythiophen-
2-yl)methyl)propanoic acid (30). Acid 29 (0.77 g, 3.9 mmol),
thiophenol 15 (0.76 g, 4.2 mmol), and THF (7 mL) were com-
bined in a round bottom flask to which TBAF trihydrate
(0.25 g, 0.78 mmol) was added at rt. The flask was fitted with a
cold-water condenser and the mixture was heated to 50 °C for
2-((5-Methoxythiophen-2-yl)methyl)malonic acid (38). Diethyl
16 h. The mixture was cooled to rt, evaporated in vacuo, and
ester 28 (5.26 g, 18.4 mmol) was dissolved in ethanol (47 mL)
diluted with EtOAc and washed with 1 N HCl (2×). The organic
at rt. A solution of KOH (2.1 g, 36.7 mmol) in ethanol (20 mL)
layer was washed with brine, dried over Na2SO4, filtered, and
was added slowly to the mixture by addition funnel. After
150 min, the mixture was cooled to 0 °C and diethyl ether was
added to induce further crystallization. The solids were filtered
off and rinsed with heptane. The isolated solids were dissolved
evaporated in vacuo to yield dark red oil. The product was a
mixture of thiophene starting material 29 and desired product
30 (crude: 1.48 g, quantitative). A small amount of desired
product was purified for characterization by column chromato-
in saturated bicarbonate solution and EtOAc was added. Con-
graphy (50% EtOAc/pentane). Pure product appeared as a
centrated HCl was added to pH 1 and the aqueous layer was
1
white crystalline solid m.p. = 110–115 °C. H NMR (500 MHz,
extracted (3 × 75 mL). The organic layer was washed with
CDCl3) δ 7.21–7.18 (m, 3H), 7.07–7.02 (m, 1H), 6.41 (d, J = 3.7,
brine, dried over Na2SO4, filtered, and evaporated in vacuo.
The crude product was isolated as a white crystalline solid
1H), 5.97 (d, J = 3.7, 1H), 3.19 (dd, J = 7.5, 13.8, 1H), 3.11 (dd,
(5.63 g, 48%). m.p. 266–279 °C; 1H NMR (500 MHz, CD3OD)
J = 6.9, 14.8, 1H), 3.07 (dd, J = 6.0, 13.8, 1H), 3.02 (dd, J = 7.0,
15.2, 1H), 2.91 (t, J = 7.4, 2H), 2.86 (quintet, J = 6.7, 1H), 2.65
δ 6.50 (d, 1H, J = 3.8), 5.99 (d, 1H, J = 3.9), 3.85 (s, 3H), 3.73
(app t, J = 7.1, 2H); 13C NMR (125 MHz, CDCl3) δ 179.5, 179.0,
165.2, 141.2, 135.4, 130.6, 129.5, 128.9, 127.5, 127.1, 126.1,
123.8, 103.4, 60.4, 47.3, 35.7, 34.9, 31.4, 30.7; IR (thin film)
2942, 1702, 1556, 1506, 1432 cm−1; Accurate Mass ES− m/z
calcd for C18H19O5S2 (M − H)− 379.0674, found 379.0675.
(t, 1H, J = 7.3), 3.33 (app d, 2H, J = 7.3); 13C NMR (125 MHz,
D2O) δ 178.4, 163.6, 129.9, 122.2, 103.9, 70.9, 60.5, 31.0;
IR (thin film) 3354, 1576, 1512, 1332, 1201 cm−1; Accurate
Mass ES− m/z calcd for C9H9O5S (M − H)− 229.0171, found
229.0174.
2-((5-Methoxythiophen-2-yl)methyl)acrylic acid (29). Diacid
38 (1.89 g, 8.21 mmol) was combined with 37% formalin
(5.96 mL) and diethylamine (1.44 mL) under argon. After three
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Di-NHS ester 39. Crude diacid 30 (1.48 g) was dissolved in on-line with an Easy-nLC 1000 (Thermo Fisher, San Jose, CA)
dry dichloromethane (30 mL) in a flame dried round bottom as previously described.3,7 Each MSn experiment consists of
flask under argon. NHS·TFA (4.12 g, 19.5 mmol) was added one MS scan in FT mode (350–1400 m/z, resolution of 60 000
before a slow addition of triethylamine (3.24 mL, 23.4 mmol) at m/z 400) followed by two data-dependent MS2 scans in FT
at 0 °C. The mixture was left to warm slowly overnight while mode (resolution of 7500) with normalized collision energy at
stirring under argon. After 16 h, the mixture was diluted with 20% on the top two MS peaks with charges at 3+ or up, and
dichloromethane and washed with water (2×). The organic three MS3 scans in the LTQ with normalized collision energy
layer was dried with Na2SO4, filtered, and evaporated in vacuo. at 35% on the top three peaks from each MS2.
The products were separated using flash column chromato-
graphy (50% EtOAc/pentane–70% EtOAc/pentane), yielding di-
1
NHS ester 39 (0.952 g, 43%). H NMR (500 MHz, CDCl3) δ 7.29
Abbreviations
(s, 1H), 7.24 (overlapping d, 2H), 7.10 (d, 1H), 6.49 (d, J = 3.8,
1H), 6.00 (d, J = 3.8, 1H), 3.83 (s, 3H), 3.28 (dd, J = 7.4, 13.7, IML
1H), 3.24 (dd, J = 6.5, 15.2, 1H), 3.17–3.04 (m, 3H), 2.92 (d, J = CID
8.3, 3H), 2.83 (s, 8H); 13C NMR (125 MHz, CDCl3) δ 169.3, TR9
169.2, 169.1, 168.0, 165.5, 140.4, 134.9, 130.7, 129.7, 129.4, Ac-IR7
127.4, 124.8, 124.5, 103.5, 60.4, 44.9, 34.5, 32.6, 31.5, 30.5, XLMS
Identical mass linker
Collision-induced dissociation
Acetyl-TTSYKVTIR
Acetyl-IEAEKGR
Cross-linker mass spectrometry
Liquid chromatography mass spectrometry
Disuccinimidyl sulfoxide
25.8, 25.8; IR (thin film) 2945, 1811, 1781, 1738, 1206 cm−1
Accurate Mass ES+ m/z calcd for C26H26N2O9S2Na (M + Na)+ DSSO
;
LCMS
597.0977, found 597.0967.
m-CPBA
DABCO
KOH
meta-Chloroperoxybenzoic acid
1,4-Diazabicyclo[2.2.2]octane
Potassium hydroxide
NHS·TFA
N-Hydroxysuccinimide trifluoroacetic acid complex
TBAF·3H2O Tetra-N-butylammonium fluoride trihydrate
SDS-PAGE
Sodium dodecyl sulfate polyacrylamide gel
electrophoresis
ESI
Electrospray ionization
Rpt4–Rpt5 Two interlinked proteasome regulatory subunits
IML 3 (9). Sulfoxide 9 was prepared from di-NHS ester 39
(0.030 g, 0.052 mmol) using general procedure 4. The isolated
product appeared as an orange oil (27 mg, 87%). The product
was characterized as a 1 : 0.7 mixture of diastereomers, with a
very small amount of starting sulfide that was not consumed
in the reaction. 1H NMR (500 MHz, CDCl3) δ 7.58–7.46
(m, 3H), 7.41–7.36 (m, 1H), 6.60 (d, J = 3.8, 0.6H), 6.50 (d, J =
3.8, 0.4H), 6.03 (d, J = 3.8, 0.6H), 5.99 (d, J = 3.8, 0.4H), 3.86
(s, 1.8H), 3.84 (s, 1.2H), 3.55–3.47 (m, 0.6H), 3.37–3.25
(m, 3.8H), 3.15 (t, J = 7.6, 2H), 3.08–3.01 (m, 0.6H), 2.97 (t, J =
7.6, 2H), 2.83 (br s, 8H); 13C NMR (125 MHz, CDCl3) δ 169.29,
169.25, 169.2, 168.9, 168.8, 168.5, 167.82, 167.80, 165.8, 165.7,
144.0, 143.5, 141.03, 140.96, 140.4, 131.9, 131.6, 131.1, 130.1,
129.9, 129.6, 127.5, 127.4, 126.0, 125.4, 124.9, 124.2, 124.1,
123.9, 123.8, 123.5, 122.9, 122.7, 103.7, 103.6, 60.39, 60.37,
60.3, 57.7, 56.5, 39.7, 39.2, 32.6, 32.5, 32.40, 32.39, 31.4, 30.5,
30.4, 29.9, 25.8, 25.8; IR (thin film) 1811, 1781, 1734, 1505,
1205 cm−1; Accurate Mass ES+ m/z calcd for C26H26N2O10S2Na
(M + Na)+ 613.0927, found 613.0908.
Acknowledgements
This work was supported by National Institutes of Health
grants RO1GM074830-06A1 and R21CA161807 to L.H., and
Biomedical Informatics Training predoctoral fellowship to A.K.
(5T15LM007443-10 to Pierre Baldi). The authors acknowledge
Dr John Greaves for his support of this project.
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