Targeting NF-κB p65 with a helenalin inspired bis-electrophile

Article abstract of DOI:10.1021/acschembio.6b00751

The canonical NF-κB signaling pathway is a mediator of the cellular inflammatory response and a target for developing therapeutics for multiple human diseases. The furthest downstream proteins in the pathway, the p50/p65 transcription factor heterodimer, have been recalcitrant toward small molecule inhibition despite the substantial number of compounds known to inhibit upstream proteins in the activation pathway. Given the roles of many of these upstream proteins in multiple biochemical pathways, targeting the p50/p65 heterodimer offers an opportunity for enhanced on-target specificity. Toward this end, the p65 protein presents two nondisulfide cysteines, Cys38 and Cys120, at its DNA-binding interface that are amenable to targeting by covalent molecules. The natural product helenalin, a sesquiterpene lactone, has been previously shown to target Cys38 on p65 and ablate its DNA-binding ability. Using helenalin as inspiration, simplified helenalin analogues were designed, synthesized, and shown to inhibit induced canonical NF-κB signaling in cell culture. Moreover, two simplified helenalin probes were proficient at forming covalent protein adducts, binding to Cys38 on recombinant p65, and targeting p65 in HeLa cells without engaging canonical NF-κB signaling proteins IκBα, p50, and IKKα/β. These studies further support that targeting the p65 transcription factor-DNA interface with covalent small molecule inhibitors is a viable approach toward regulating canonical NF-κB signaling.

Full text of DOI:10.1021/acschembio.6b00751

Articles
pubs.acs.org/acschemicalbiology
Targeting NF-κB p65 with a Helenalin Inspired Bis-electrophile
John C. Widen,† Aaron M. Kempema,† Peter W. Villalta,‡ and Daniel A. Harki*,†,‡
‡
†Department of Medicinal Chemistry and Masonic Cancer Center, University of Minnesota, 2231 Sixth Street SE, Minneapolis,
Minnesota 55455, United States
S
* Supporting Information
ABSTRACT: The canonical NF-κB signaling pathway is a
mediator of the cellular inflammatory response and a target for
developing therapeutics for multiple human diseases. The furthest
downstream proteins in the pathway, the p50/p65 transcription
factor heterodimer, have been recalcitrant toward small molecule
inhibition despite the substantial number of compounds known to
inhibit upstream proteins in the activation pathway. Given the
roles of many of these upstream proteins in multiple biochemical
pathways, targeting the p50/p65 heterodimer offers an oppor-
tunity for enhanced on-target specificity. Toward this end, the p65
protein presents two nondisulfide cysteines, Cys38 and Cys120, at
its DNA-binding interface that are amenable to targeting by
covalent molecules. The natural product helenalin, a sesquiterpene lactone, has been previously shown to target Cys38 on p65
and ablate its DNA-binding ability. Using helenalin as inspiration, simplified helenalin analogues were designed, synthesized, and
shown to inhibit induced canonical NF-κB signaling in cell culture. Moreover, two simplified helenalin probes were proficient at
forming covalent protein adducts, binding to Cys38 on recombinant p65, and targeting p65 in HeLa cells without engaging
canonical NF-κB signaling proteins IκBα, p50, and IKKα/β. These studies further support that targeting the p65 transcription
factor−DNA interface with covalent small molecule inhibitors is a viable approach toward regulating canonical NF-κB signaling.
berrant activation of canonical p50/p65 NF-κB tran-
pockets and nondiscrete protein tertiary structures, which result
Ascription factors and concomitant expression of their in weak binding by putative inhibitors.16,17 Irreversible covalent
binding by inhibitors to nucleophilic amino acids on tran-
scription factors may provide another strategy for transcrip-
tional regulation via direct protein binding provided the amino
acids that are covalently modified are intolerant of chemical
modification (e.g., chemical modification prevents DNA
binding and/or transcriptional activation).6
target genes has been implicated in a spectrum of human
diseases, including chronic inflammatory disease, atheroscle-
rosis, arthritis, and cancer.1−4 Consequently, interventional
strategies to regulate canonical NF-κB signaling may be broadly
useful for multiple therapeutic indications.5−8 Accordingly,
significant research efforts have been devoted toward the
discovery of inhibitors of canonical p50/p65 NF-κB signaling
with the targeting of upstream kinases that facilitate p50/p65
activation (via release from its repressor protein, IκBα) being
the prominent strategy.9,10 However, most of these enzymes
have degenerate activities with other cellular processes, and
therefore, their inhibition as a strategy to regulate canonical
NF-κB signaling results in off-target effects.9 Targeting the most
downstream proteins in the canonical NF-κB signaling pathway,
the p50/p65 transcription factor heterodimer itself would
ablate such specificity issues.
Chemical modulation of transcription factor−DNA interfaces
has shown promise as a strategy to regulate aberrant
transcription factor signaling. Pyrrole-imidazole polyamides
that target the DNA minor groove with sequence specificity
and disrupt transcription factor−DNA binding have demon-
strated promising utilities in both cell culture and animal
models, including modulation of canonical NF-κB signal-
ing.11−15 Conversely, targeting transcription factors with
protein-binding small molecules has proven more problematic,
which has been attributed to the typical shallow ligand binding
Many sesquiterpene lactone (SL) natural products are
known modulators of NF-κB signaling.18,19 In particular,
helenalin (Figure 1A), an SL isolable from some Arnica and
Helenium species,20−22 has provided inspiration for the rational
design of transcription-factor-targeting NF-κB inhibitors that
disrupt DNA binding. The medicinal properties of helenalin
have been known for over a century,23 which are structurally
endowed, in part, by its α-methylene-γ-butyrolactone; a moiety
found in scores of bioactive natural products.24 Exocyclic
methylene butyrolactones undergo hetero-Michael addition
with biological thiols to form covalent adducts. Helenalin also
contains an endocyclic α,β-unsaturated ketone (cyclopente-
none) that can also undergo hetero-Michael addition with
thiols. Removal of one of the two Michael acceptors of
helenalin, such as reduction of the cyclopenteone (yielding 2,3-
dihydrohelenalin) or α-methylene-γ-butyrolactone (yielding
Received: August 26, 2016
Accepted: November 7, 2016
© XXXX American Chemical Society
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approach.43 Our design would also enable the rapid synthesis of
analogues in comparison to helenalin, which required lengthy
total syntheses.44−47 Herein, we report the design, synthesis,
and characterization of simplified helenalin analogues and their
utilization in cell culture to target p65 of the canonical NF-κB
signaling pathway.
RESULTS AND DISCUSSION
■
Design and Synthesis of Simplified Helenalin Ana-
logues. The probe design was based on retaining both the
cyclopentenone and α-methylene-γ-butyrolactone of helenalin,
which are required for covalent reactivity at Cys38 and Cys120
of p65, but structurally simplifying the central 7-membered
ring, whereas the two above-mentioned ring systems would be
tethered in only one position (Figure 1A). In support of this
design, computational modeling of 6a and 6b predicted
distances between the electrophilic carbons (analogous to the
electrophilic carbons of helenalin denoted in Figure 1A) on
both simplified analogues to be 6.2 Å, which is comparable to
those distances measured from published X-ray crystal
structures of helenalin (6.2−6.4 Å; see Figure S1 for modeling
data).48,49 Additionally, the calculated distances between the
analogous electrophilic carbons on alkynylated probes 1a and
1b was similar (1a, 6.1 Å; 1b, 5.4 Å). An additional
consideration in our design was the utility of the
diastereoselective, Barbier-type, aldehyde allylation chemistry
developed by Hodgson and co-workers for synthesizing β-
substituted α-methylene-γ-butyrolactones.50 Accordingly, we
devised the five-step racemic synthesis of structurally simplified
helenalin probe 6a* shown in Scheme 1. The known, racemic
Figure 1. (A) Structure of the sesquiterpene lactone helenalin and the
design of helenalin mimics that contain both electrophiles found in the
parent natural product. The secondary alcohol is amenable to chemical
modifications to install reporter tags, such as alkynes. On the basis of
two reported crystal structures of helenalin, the distance between the
two electrophilic carbons (red asterisks) is 6.2−6.4 Å.48,49 (B) X-ray
crystal structure of the NF-κB p50−p65 heterodimer bound to DNA
(PDB 1VKX).33 Notably, p65 cysteine residues 38 and 120 are
adjacent in the DNA-binding interface (7.7 Å S−S distance, depicted
with dashed line).
11,13-dihydrohelenalin; plenolin), significantly diminishes
cytotoxicity in comparison to the parent natural product.25−28
Reduction of both Michael acceptors on helenalin ablates all
activity.26,27 The cyclopentenone and α-methylene-γ-butyro-
lactone of helenalin can engage biological thiols, such as
glutathione and cysteine, yielding covalent adducts.29,30
Previous studies by Merfort and colleagues have shown that
helenalin covalently targets Cys38 of NF-κB p65,31,32 which is
positioned at the DNA-binding interface upon heterodimeriza-
tion with p50 and DNA engagement (Figure 1B).33 Alkylation
of p65 by helenalin sterically prevents DNA binding of the
p50/p65 heterodimer and inhibits its transcriptional activa-
tion.34 Interestingly, molecular modeling of helenalin enabled
the hypothesis that it may engage in tandem hetero-Michael
additions with Cys38 and Cys120, which are 7.7 Å apart when
bound to DNA;35,36 however, the lack of sensitivity of helenalin
to a Cys120 → Ser mutation has drawn this cross-linking model
into question.18,34 Nonetheless, the nondisulfide, nucleophilic
cysteines, Cys38 and Cys120, located at the p65 DNA-binding
interface constitute unique structural features of p65 that lends
itself toward the development of covalent chemical modulators.
Furthermore, previous studies from other groups have
demonstrated covalent engagement of p65 Cys38 with diverse
small molecules,6,37−41 as well as covalent targeting of p65
Cys120.42
We hypothesized that chemical probes containing two
structurally similar, α,β-unsaturated carbonyls configured in
comparable chemical space to that of helenalin may recapitulate
the interesting biological activity of the parent natural product.
Consequently, we devised a strategy to develop structurally
simplified helenalin analogues that could be amenable to
structure−activity relationship studies, serve as suitable
chemical mimics of the parent natural product, and yield
powerful chemical biology tools for specificity studies and
target annotation by protein pulldown-mass spectrometry
analysis. Recently, bis-Michael acceptors have been developed
to regulate the Keap1/Nrf2/ARE pathway, which supports our
Scheme 1. Racemic Synthesis of Bifunctional Helenalin
a
Mimics and Their Alkyne Analogues
a
Reagents and conditions: (a) IBX, DMSO, 75 °C, 66%. (b) (i)
LiAlH4, Et2O 0 °C; (ii) PCC, CH2Cl2, RT, 40% (two steps). (c) Zn0,
NH4Cl (aq.), DMF, RT, 60%. (d) 4-Pentynoic acid, DCC, DMAP,
CH2Cl2, 40 °C, 80% (separable diastereomers). *Denotes 6a and 6b
prepared from racemic starting materials.
cyclopentanone rac-251−53 was oxidized to the α,β-unsaturated
ketone 3 using IBX in moderate yield.54 β-Keto ester 3 was
reduced to the diol with LiAlH4 and then subsequently oxidized
to the desired aldehyde 4 with PCC in a 40% yield over the two
steps. Notably, aldehyde 4 is volatile and unstable and must be
used immediately following careful purification and isolation.
The α-(bromo)methyl unsaturated furanone 5 was synthesized
in one step from α-methylene-γ-butyrolactone by a known
procedure.50,55 With building blocks 4 and 5 in hand, the
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diastereoselective Barbier coupling (vide supra) was performed,
which resulted in inseparable diastereomers 6a* and 6b*. Only
one diastereomer was observed in the Barbier coupling with
respect to β-substitution of the α-methylene-γ-butyrolactone
ring in accord with literature precedence of a six-membered
transition state. However, metal-catalyzed allylation of aldehyde
4, bearing a stereogenic center, occurs without facial selectivity,
yielding diastereomeric products 6a* and 6b* that are racemic.
Coupling the inseparable mixture of 6a* and 6b* with 4-
pentynoic acid and DCC yielded racemic 1a and 1b that were
separable on silica gel. The overall yield for the synthesis of 1a
and 1b was 13% (five steps).
A stereoselective synthesis of 6a was also developed to
deliver a simplified helenalin probe with the appropriate overall
stereochemistry as that found in helenalin and to overcome our
inability to separate diastereomers 6a* and 6b* from the
Barbier coupling in the racemic synthesis. Since our stereo-
selective synthesis would employ a different aldehyde coupling
partner, an opportunity to separate the diastereomers resulting
from the Barbier coupling would exist. Our synthesis began
with the stereoselective introduction of the quaternary methyl
group, yielding (S)-2, using an established protocol for the
stereoselective methylation of 1,3-ketoesters (Scheme 2).56−59
Diastereomers 10a and 10b were oxidized separately to their
corresponding cyclopentenones under catalytic Saegusa-Ito
oxidation conditions60,61 in DMSO under 1 atm of O2 in
46% yield (for 6a, 93:7 er) and 56% yield (for 6b, 91:9 er). It is
noteworthy that 10a, 10b, 6a, and 6b should not be dried
under high vacuum because of their propensity to decompose
(drying under low vacuum for a short period of time is
recommended). Enantiomerically pure 6a and 6b were
synthesized in six steps in 3% and 4% overall yields,
respectively.
Stereochemical Determination of Simplified Helen-
alin Probes. The Barbier coupling utilized for the synthesis of
our molecular probes resulted in a highly stereocontrolled β-
substitution of the butyrolactone ring. However, the lack of
facial selectivity for aldehyde allylation resulted in diaster-
eomers with respect to the quaternary center on the
cyclopentanone ring and secondary alcohol. To unambiguously
assign the stereochemistry of our probes by X-ray crystallog-
raphy, we esterified diastereomer 10b from the enantioselective
synthesis with 4-bromobenzoic acid, a moiety that bears a heavy
atom for determination of absolute configuration (Scheme 3).
a
Scheme 3. Synthesis and X-ray Crystal Structure of 11
a
Scheme 2. Enantioselective Synthesis of 6a and 6b
a
Reagents and conditions: (a) L-Valine tert-butyl ester, BF3·OEt, PhH,
reflux (Dean−Stark), 75%. (b) (i) LDA, toluene, THF (2 equiv), −78
°C; MeI; (ii) 3 M HCl (aq.), THF, RT, 53% (2 steps), 93:7 er. (c) (i)
LiHMDS, THF; TBSCl, −78 °C to RT; (ii) DIBAL-H, CH2Cl2, −78
°C, 27% (2 steps). (d) 5, Zn0, NH4Cl (aq.), DMF, RT, 64% (separable
diastereomers). (e) Pd(OAc)2, O2 (1 atm), DMSO, 46% (6a, 93:7 er),
56% (6b, 91:9 er).
a
Reagents and conditions: (a) 4-bromobenzoic acid, 4-DMAP, DCC,
DCM, 40 °C (99%). (b) TFA, DCM, RT (53%). Bottom: ORTEPII
plot of the X-ray diffraction data of 11.
Accordingly, 1,3-ketoester 7 was condensed with L-tert-
butylvaline to obtain enamine 8 in 75% yield. Enamine 8 was
deprotonated with LDA in toluene at −78 °C, and then two
equivalents of THF were added, followed by the addition of
excess CH3I at the same temperature. After formation of the
quaternary center, the crude product was isolated, and the
resulting Schiff base was hydrolyzed with aqueous HCl to
obtain (S)-2 in 53% yield and 93:7 er (85% ee) by chiral-GC
analysis. Cyclopentanone (S)-2 was then converted to the TBS-
protected enol ether, followed by two-electron reduction of the
ester to aldehyde 9 using DIBAL-H in 27% yield (two steps).
Notably, addition of the TBS protecting group resulted in a
nonvolatile and stable aldehyde 9 in comparison to 4. Aldehyde
9 was then subjected to the aforementioned Barbier conditions
with 5 to obtain diastereomers 10a and 10b in 1:1 dr (64%
yield) that were separable by silica gel chromatography.
Deprotection of the silyl enol ether to the ketone using TFA in
DCM afforded 11, which was crystallized and the X-ray
structure solved (Supporting Information). On the basis of this
information, diagnostic H NMR coupling constants, and the
known, absolute configuration of (S)-2, we unambiguously
assigned the structures of our chemical probes.
Simplified Helenalin Probes Inhibit NF-κB Signaling in
Cell Culture. Simplified helenalin analogues 6a and 6b and
alkynylated probes 1a and 1b were screened for inhibitory
activity toward canonical p50/p65 NF-κB signaling with a
cellular luciferase assay (Figure 2).62 Helenalin was utilized as a
benchmark and exhibited low micromolar inhibition of induced
NF-κB signaling (53.7 14.1% NF-κB activity at 2.5 μM). In
comparison, alkyne-functionalized probes 1a and 1b were
comparably active at 5 μM (54.4 16.7% and 52.9 7.1% NF-
1
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Figure 3. Proteome labeling of 1a and 1b in HeLa cells. Compounds
were dosed to HeLa cells at the concentrations shown. The cells were
lysed, and then TAMRA-N3 was conjugated to 1a and 1b via azide−
alkyne click chemistry. Proteins were separated by denaturing PAGE,
and gels were imaged for TAMRA fluorescence. Total proteins were
then visualized by staining with Oriole fluorescent protein stain
followed by gel imaging.
Figure 2. NF-κB-luciferase inhibition assay in A549 cells. Compounds
were dosed to A549 cells containing a stably transfected NF-κB
luciferase reporter construct and stimulated with TNF-α for 8 h
(except for noninduced control, Non-Ind). Luminescence was
normalized to the no compound induced (Ind) control and plotted
as % NF-κB luciferase activity. Mean standard deviation values are
shown (n ≥ 3 biological replicates). Compound-mediated toxicity to
A549 cells was measured concurrently (Figure S2).
given the designed probes are bis-electrophiles. However, the
distinct labeling patterns of 1a and 1b further reinforce the
relevance of the stereochemistry of our probes with respect to
protein targeting. Probe 1a (which contains the correct
stereochemistry with respect to helenalin) qualitatively labels
more proteins compared to 1b by visualization of the intense
bands seen at 50 and 10 μM. Additionally, pretreatment of cells
with helenalin prior to labeling studies with 1a competes away
protein binding properties by 1a, suggesting that our designed
probe mimics the proteome reactivity properties of the parent
natural product (Figure S3).
κB activity, respectively). Notably, 1a elicits more cellular
cytotoxicity (64.5 3.0% cell viability) during this 8 h assay
compared to 1b (97.8
15.1% cell viability) at 10 μM
treatment. Cellular cytotoxicity for 1a at 20 μM treatment was
comparable to that observed at 10 μM, whereas cellular viability
was >80% for all other doses of all compounds shown in Figure
2 (refer to Figure S2 for cellular viability data). Intriguingly,
enantiomerically pure 6b, the diastereomer with the “incorrect”
stereochemistry with respect to helenalin, has approximately 6-
fold more NF-κB inhibitory activity at 25 μM compared to 6a,
the enantiomerically pure “correct” diastereomer from the
Barbier coupling (6b, 14.5 7.4% NF-κB activity; 6a, 88.8
4.9% NF-κB activity). Furthermore, 6b achieves the same NF-
κB inhibitory activity as helenalin at only a 4-fold higher dose
(6b, 51.6 16.6% NF-κB activity at 10 μM; helenalin, 53.7
14.1% NF-κB activity at 2.5 μM). These data suggest the
stereochemistries of our helenalin analogues are important with
respect to cellular potency. The observation that 1a and 1b are
equivalently potent in this assay hints at the possibility that 6a
may have poor uptake properties or may be poorly retained in
cells; however, no data currently exist to support this proposal.
Simplified Helenalin Probes Covalently Bind Proteins
in Cell Culture. To qualitatively assess the protein-binding
properties of our probes in cell culture, we performed protein
labeling studies in HeLa cells. Alkyne-functionalized 1a and 1b
were dosed separately to HeLa cells for 1 h, cells were then
lysed and the lysate cleared, and then protein-probe adducts
were labeled with tetramethylrhodamine-azide (TAMRA-N3)
via a copper mediated [3 + 2] Huisgen reaction (click
chemistry).63 Protein-1a/1b-TAMRA adducts were separated
by denaturing PAGE and fluorescence visualization of TAMRA
(Figure 3). To demonstrate uniform protein labeling, total
proteins were also imaged. Both 1a and 1b labeled multiple
proteins in a concentration dependent manner (Figure 3).
Gratifyingly, protein bands at approximately 65 kDa were
observed for both 1a and 1b, which would be expected for
targeting NF-κB p65. Off-target binding by 1a and 1b, which is
evident by the multiple protein bands observed in our
qualitative analysis, was observed and was certainly anticipated
Helenalin and Simplified Helenalin Probes Covalently
Label Cys38 of Recombinant p65. Previous studies with
wild-type p65 and Cys → Ser mutant p65 proteins have
demonstrated that helenalin covalently targets Cys38.34 To
evaluate if helenalin mimics 1a and 1b also target Cys38,
recombinant human p65 was incubated with 1a and 1b,
proteins were digested, and LC-MS/MS was performed to
identify adducted thiols (Figure 4). The predicted trypsin
digestion of p65 proximal to Cys38 yields C38EGR41 or
Y36KCEGR41 if the cleavage site closest to Cys38 is missed.
Searching for the exact masses of the probes as a cysteine
modification found the expected probe-peptide adducts for
helenalin (Y36KCEGR41 adduct), 1a (C38EGR41 adduct) and 1b
(C38EGR41 adduct). Analysis of the MS2 data for studies with
1a and 1b and recombinant p65 consistently revealed a
fragment that was cleaved at the secondary hydroxyl group,
which demonstrates that the α-methylene-γ-butyrolactone was
reacting with Cys38 (Figure 4 and Figures S4−S5). On the
other hand, no fragments were found to support reactivity at
the endocyclic enones of 1a and 1b, although such adducts
might be reversible and unstable to digestion and MS analysis.
Our reactivity data are consistent with previous reports
demonstrating that α-methylene-γ-butyrolactones form irrever-
sible hetero-Michael addition adducts with cysteines.30,31
Interestingly, attempts to identify Cys38 adducts for 6a and
6b were unsuccessful, although adducts to a surface cysteine on
p65, Cys105, was observed (Figure S7). Cys105 adducts were
not found in experiments with 1a, 1b, or helenalin. The
biological significance of labeling Cys105 on p65 remains
unclear.
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Figure 4. Annotation of compound binding sites on p65. Helenalin, 1a, and 1b were incubated separately with recombinant human p65 in 1 × PBS
buffer for 1 h. Proteins were digested with trypsin and peptides analyzed by LC-MS/MS. Peptide-probe adducts were identified by extracting the
theoretical mass from the total ion chromatogram and then analysis of the MS2 fragmentation pattern. Helenalin, 1a, and 1b all bound Cys38 as
expected. The MS2 Fragment 1 (shown on the bottom left) was detected after dosing 1a and 1b, indicating that covalent adducts are forming at the
exocyclic methylene butyrolactone.
Simplified Helenalin Probes Covalently Target p65 in
Cell Culture. After determining that simplified helenalin
probes 1a and 1b can inhibit canonical NF-κB signaling in cell
culture, covalently label cellular proteins, and target Cys38 of
recombinant p65, we turned our attention to evaluating
whether 1a and 1b can directly bind p65 in cell culture and
avoid other well-established canonical NF-κB signaling
enzymes. Accordingly, 1a and 1b were dosed to HeLa cells at
50 μM and incubated for 30 min. Cells were then harvested,
washed extensively to remove unincorporated probe, and then
lysed by sonication. Protein-1a/1b adducts were then labeled
with biotin-azide (biotin-N3) via a copper mediated [3 + 2]
Huisgen reaction (click chemistry).63 Protein-1a/1b-biotin
adducts were enriched with a monomeric avidin column and
separated by denaturing PAGE. Protein-1a/1b-biotin adducts
were transferred to a membrane and then incubated with
primary antibodies for p65 (primary target), IκBα (p50/p65
repressor protein; for specificity assessment), p50 (forms
transcription factor heterodimer with p65; for specificity
assessment), and IKKα/β (upstream kinases that contribute
to activation of the NF-κB signaling pathway; for specificity
Figure 5. Immunodetection of NF-κB signaling pathway proteins for
assessment; antibody recognizes both proteins). To our delight,
covalent binding by 1a and 1b. Compounds were dosed to HeLa cells
simplified helenalin probes 1a and 1b successfully pulled down
p65 from live HeLa cells but did not target IκBα, p50, or
at the concentrations shown, the cells were lysed, and then Biotin-N3
was conjugated to 1a and 1b via azide−alkyne click chemistry. Protein-
IKKα/β (Figure 5). The negative control demonstrates no
proteins are pulled down in the absence of probes, and the
input lysates show that all proteins being evaluated are present
in the input lysate (prior to monomeric avidin isolation of
protein-1a/1b-biotin adducts). Furthermore, a head-to-head
comparison of the ability of 1a and 1b to target p65 reveals
comparable efficiencies (Figure 5C).
We also wanted to determine if 6a and 6b could compete
away binding of alkyne probes 1a and 1b in live cells; especially
since p65 adducts were not observed with 6a and 6b by MS
analysis. To this end, 6a and 6b were dosed to HeLa cells at 50
μM for 20 min prior to dosing 1a or 1b. Interestingly, 6b was
able to completely block labeling and pulldown by both 1a and
1b, whereas 6a exhibited only partial activity at the same
concentration (Figure 5). These data suggest that 6a and 6b
1a/1b adducts were isolated on monomeric avidin resin and identified
by Western blotting with the antibodies shown. Notably, IKKα/β is
recognized with the same antibody. (A, B) 1a (for A) and 1b (for B)
label p65 but not other NF-κB pathway proteins, as shown in
pulldown experiments. Competition experiments were performed by
pretreating cells with 6a and 6b 20 min before the addition of 1a (for
A) or 1b (for B). (C) Head-to-head comparison of labeling efficiency
of 1a and 1b. Input lysate is the crude protein product before
monomeric avidin enrichment.
target Cys38 on p65. Additionally, the difference in efficiencies
between the two compounds is consistent with the cellular
reporter data (Figure 2), in which 6a was dramatically less
potent than 6b for inhibition of canonical NF-κB signaling.
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min) over 18.1 min. The mass spectrometer electron ionization source
temperature was set to 250 °C for detection. Area under the peak for
each enantiomer was used to determine the enantiopurity, reported as
enantiomeric ratio (er). The enantiopurity of other compounds was
determined using normal phase chiral-HPLC or 19F analysis after
derivatization with (S)-(−)-α-methoxy-α-(trifluoromethyl)-
phenylacetic acid ((S)-MTPA). Information regarding these two
methods can be found in the Supporting Information.
Conclusions. We have developed simplified helenalin
probes that target Cys38 of NF-κB p65 by a covalent
mechanism of action. Our chemical probes are easily accessed
in six synthetic steps (to probes 1a and 1b) and contain an
alkyne handle for imaging and protein pulldown applications.
Simplified helenalin probes 1a and 1b inhibit induced,
canonical NF-κB signaling in a cellular reporter assay with
low micromolar efficiencies and selectively engage p65 over
other well-established proteins whose targeting by small
molecules affects canonical NF-κB signaling, namely, IκBα,
p50, and IKKα/β. The strategy of covalent targeting of solvent
accessible, nucleophilic amino acids at key transcription factor
interfaces, such as the DNA-binding cleft for p65 in this work,
represents a viable strategy to rationally design small molecule
transcription factor binders with biochemical utilities for
transcriptional control of aberrant gene expression in human
diseases. Current efforts are focused on improving the
specificities and potencies of the first-generation helenalin-
based probes reported in this work, with a particular focus on
the size and rigidity of the moieties that mimic the central
seven-membered ring of helenalin.
Ethyl 1-Methyl-2-oxocyclopent-3-enecarboxylate (3).51−53 To a
stirred solution of rac-2 (1.00 g, 5.88 mmol) in DMSO (20 mL) was
added 4.11 g (14.7 mmol) of IBX,64 and the solution was heated to 85
°C for 18 h. The reaction was cooled to 0 °C, and aqueous NaHCO3
(saturated, 40 mL) was added slowly. The reaction was filtered to
remove IBX decomposition products. The solution was extracted with
Et2O (30 mL, 3×), and the organic layer was dried over Na2SO4 and
concentrated in vacuo. The crude mixture was SiO2 purified with
EtOAc (10−30%) in hexanes to afford 3 (0.52 g, 53% yield) as a
slightly tinted yellow oil. 1H NMR (CDCl3): 7.76−7.71 (m, 1H),
6.19−6.15 (m, 1 H), 4.14 (q, J = 7.2 Hz, 2 H), 3.25 (d, J = 19.1 Hz, 1
H), 2.53 (d, J = 19.2 Hz, 1 H), 1.39 (s, 3H), 1.21 (t, J = 7.2 Hz, 3H)
ppm. 13C NMR (CDCl3): 206.9, 171.7, 163.2, 131.8, 61.7, 53.5, 42.9,
20.8, 14.2 ppm. HRMS-ESI+ (m/z) calcd [M + H]+ for C9H12O3:
169.0859. Found: 169.0855.
5-(Hydroxymethyl)-5-methylcyclopent-2-enol. To a stirred sus-
pension of LiAlH4 (0.66 g, 17 mmol) in EtO2 (25 mL) at 0 °C was
added 3 (0.75 g, 4.4 mmol) in EtO2 (6 mL) dropwise. The reaction
was stirred at 0 °C for 2 h. The reaction was carefully quenched with
H2O (1 mL), then aqueous NaOH (10%, 0.5 mL), and finally H2O (2
mL) and stirred overnight. The reaction was then filtered through
Celite, and the filtrate was concentrated in vacuo. The crude mixture
was SiO2 purified with EtOAc (30−100%) in hexanes to afford a clear
MATERIALS AND METHODS
■
Materials and Synthetic Methods. Unless otherwise noted,
reactions were performed in flame-dried glassware under a nitrogen or
argon atmosphere and stirred with a Teflon-coated magnetic stir bar.
Liquid reagents and solvents were transferred via syringe and cannula
using standard techniques. Reaction solvents dichloromethane
(DCM), N,N-dimethylformamide (DMF), tetrahydrofuran (THF),
and diethyl ether (Et2O) were dried by passage over a column of
activated alumina using a solvent purification system (MBraun). All
other chemicals were used as received unless otherwise noted.
Helenalin was purchased from Enzo Life Sciences and purified by SiO2
flash column chromatography before use in biological assays. The
molarities of n-butyllithium solutions were determined by titration
against diphenylacetic acid as an indicator (average of three
determinations). Reaction temperatures above 23 °C refer to oil
bath temperature, which was controlled by a temperature modulator.
Reaction progress was monitored by thin layer chromatography using
EMD Chemicals Silica Gel 60 F254 glass plates (250 μm thickness) and
visualized by UV irradiation (at 254 nm) and/or KMnO4 stain. Silica
gel chromatography was performed on a Teledyne-Isco Combiflash
Rf-200 instrument utilizing Redisep Rf High Performance silica gel
columns (Teledyne-Isco), or flash column chromatography was
performed using SiliCycle silica gel (32−63 μm particle size, 60 Å
1
oil (0.46 g, 80% yield). H NMR analysis was consistent with that
reported previously.65−67
1-Methyl-2-oxocyclopent-3-enecarbaldehyde (4). The diol prod-
uct (0.31 g, 2.3 mmol) from above was dissolved in DCM (20 mL) at
RT and stirred. PCC (1.51 g, 7.03 mmol) was added in 3 equiv
portions over 4 h while stirring. This reaction was not kept under an
inert atmosphere. The reaction was vacuum filtered through Celite.
The resulting solution was carefully concentrated on a rotary
evaporator (no water bath; 150 Torr vacuum) until only a small
amount of solution remained. This solution was introduced onto a
silica column (1 in. diameter, 4 in. length) and quickly purified (10%
Et2O in pentanes). The fractions containing the desired compound
were collected and concentrated carefully (as described above) to
afford a volatile, clear oil 4 (0.16 g, 55% yield). Note: Concentration
until no residual solvent is left will result in loss of product and decreased
yields. Do not use a high vacuum to dry product. After isolation, the
product was used immediately in the next reaction. If stored at −20 °C in
1
pore size). H NMR (500 MHz), 13C NMR (125 MHz), and 19F
1
DCM, the compound degrades in the span of 24 h. H NMR (CDCl3):
NMR (470 MHz) spectra were recorded on a Bruker Avance NMR
9.43 (s, 1H), 7.82−7.77 (m, 1H), 6.16−6.12 (m, 1H), 3.37 (dt, J =
19.4, 2.5 Hz, 1H), 2.41 (dt, J = 19.4, 2.3 Hz, 1H), 1.45 (s, 3H) ppm.
13C NMR (CDCl3): 206.0, 198.1, 164.4, 132.0, 60.6, 37.4, 18.2 ppm.
1
spectrometer. H and 13C chemical shifts (δ) are reported relative to
the solvent signal, CHCl3 (δ = 7.26 for 1H NMR and δ = 77.00 for 13
C
HRMS-ESI+ (m/z) calcd [M + H]+ for C7H8O2: 125.0597. Found:
125.0594.
NMR). Some spectra contain TMS (0.05% v/v). All NMR spectra
were obtained at RT unless otherwise specified. High resolution mass
spectral data were obtained at the Analytical Biochemistry Core
Facility of the University of Minnesota Masonic Cancer Center on an
LTQ Orbitrap Velos Mass Spectrometer (Thermo Fisher).
The purities of all compounds tested in biological assays were
checked via analytical HPLC analysis on an Agilent 1200 series
instrument equipped with a diode array detector (wavelength
monitored = 215 nm) and a Zorbax SBC18 column (4.6 × 150
mm, 5.0 μm, Agilent Technologies). All compounds tested in
biological assays were >95% pure by HPLC. Information regarding
the HPLC method and purity traces can be found in the Supporting
Information.
Rac-(R)-4-((S)-Hydroxy((R)-1-methyl-2-oxocyclopent-3-en-1-yl)-
methyl)-3-methylenedihydrofuran-2(3H)-one (6a*) and rac-(S)-4-
((R)-Hydroxy((R)-1-methyl-2-oxocyclopent-3-en-1-yl)methyl)-3-
methylenedihydrofuran-2(3H)-one (6b*). Compounds 4 (69 mg,
0.56 mmol) and 550,55 (55 mg, 0.84 mmol) were combined in a round-
bottom flask containing DMF (3 mL) and a stir bar. Activated Zn0
(109 mg, 1.68 mmol) was added to the stirring solution. Zn0 was
freshly activated before each reaction by stirring in aqueous HCl (4 M)
for 15 min and then filtered; washed with H2O (200 mL, 3×), MeOH
(200 mL), EtOAc (200 mL), and Et2O (100 mL); and dried under a
high vacuum for at least 1 h. Aqueous NH4Cl (saturated, 1 drop) was
added to the solution. The reaction was degassed and backfilled with
Ar(g) 3× and then allowed to stir at RT for 16 h. The reaction was
quenched with H2O (15 mL) and extracted with Et2O (20 mL, 3×),
then dried over Na2SO4 and concentrated in vacuo to a crude oil. The
crude oil was SiO2 purified with EtOAc (0 to 70%) in hexanes to give
The enantiopurity of (S)-2 was determined by chiral-GC/MS using
an Agilent 7200B GC/Q-TOF with an Agilent J&W CycloSil-B GC
column (30 m × 0.25 mm, 0.25 μm film). The injector port was set at
250 °C and the column flow (helium gas) at 1.0 mL/min. The
temperature method began at 35 °C and increased to 180 °C (8 °C/
F
DOI: 10.1021/acschembio.6b00751
ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology
Articles
74 mg (60% yield) of a clear oil containing 6a* and 6b* that was an
inseparable mixture of diastereomers. The NMR characterization data
for the separated, enantiomerically enriched compounds are below.
See the Supporting Information for methodology used for
enantiopurity analysis of synthesized compounds. For 6a*, a modest
chiral induction was observed in the Barbier coupling reaction in a
32:68 dr for the resulting esterified, (S)-MTPA analogues by 19F
NMR. For 6b*, no chiral induction was observed, as evidenced by the
1:1 er by chiral-HPLC analysis.
Rac-(S)-((R)-1-Methyl-2-oxocyclopent-3-en-1-yl)((R)-4-methyl-
ene-5-oxotetrahydrofuran-3-yl)methyl pent-4-ynoate (1a) and rac-
(R)-((R)-1-Methyl-2-oxocyclopent-3-en-1-yl)((S)-4-methylene-5-oxo-
tetrahydrofuran-3-yl)methyl pent-4-ynoate (1b). The previously
isolated mixture of 6a* and 6b* (14 mg, 0.06 mmol) were dissolved in
DCM (5 mL); then 4-pentynoic acid (12 mg, 0.13 mmol) was added
to this solution. 4-DMAP (31 mg, 0.25 mmol) was then added
followed by DCC (39 mg, 0.19 mmol). The reaction was heated to 40
°C for 4 h. The reaction was quenched with H2O (5 mL) and
extracted with DCM (10 mL, 3×). The organic layer was dried over
Na2SO4, filtered, and concentrated in vacuo to afford a crude oil. The
crude product was SiO2 purified with EtOAc (0−70%) in hexanes to
afford two white solids with an overall yield of 15 mg (80% yield, 1:1
ratio of 1a/1b).
and the reaction was stirred at −78 °C for 16 h. The reaction was then
quenched with aqueous NH4Cl (saturated, 50 mL) and extracted with
Et2O (30 mL, 3×), washed with brine (15 mL, 1×), dried over
Na2SO4, and concentrated in vacuo. The crude products were then
dissolved in THF (50 mL), and aqueous HCl (3M, 50 mL) was added
and stirred at RT for 6 h. The reaction was extracted with Et2O (50
mL, 3×), washed with water (10 mL, 1×) and then brine (15 mL, 1×),
and dried over Na2SO4. The solvent was concentrated in vacuo. The
crude mixture was SiO2 purified with EtOAc (0%−20%) in hexanes to
afford (S)-2 as a colorless oil (0.54 g, 53%, 93:7 er by chiral-GCMS).
NMR characterization was consistent with previously reported data for
this compound.51
Ethyl (S)-2-((tert-Butyldimethylsilyl)oxy)-1-methylcyclopent-2-
ene-1-carboxylate. LiHMDS solution in THF (1 M solution, 4.20
mL, 4.19 mmol) was added to anhydrous THF (30 mL) at −78 °C.
Next, a solution of 9 (0.59 g, 3.5 mmol) in THF (5 mL) was added
dropwise and stirred for 1 h at −78 °C. A solution of TBSCl (1.05 g,
6.98 mmol) in THF (10 mL) was then added dropwise at −78 °C, and
then the reaction was allowed to slowly come to RT and stirred for 16
h. The reaction mixture was quenched with aqueous NH4Cl
(saturated, 30 mL) and extracted with Et2O (30 mL, 3×). The
organic layer was washed with brine (15 mL, 1×), dried over Na2SO4,
and concentrated in vacuo. The crude material was SiO2 purified with
EtOAc (0 to 10%) in hexanes, resulting in a clear oil (0.78 g, 79%
yield). 1H NMR (500 MHz): 4.61 (t, J = 2.2 Hz, 1H), 4.12 (q, J = 7.2
Hz, 2H), 2.40−2.29 (m, 2H), 2.29−2.19 (m, 1H), 1.77−1.67 (m, 1H),
1.72 (m, 1H), 1.30 (s, 3H), 1.24 (t, J = 7.1 Hz, 3H), 0.90 (s, 9H), 0.16
(s, 3H), 0.15 (s, 3H). 13C NMR (125 MHz): 176.1, 156.2, 101.2, 60.5,
54.3, 35.3, 26.2, 25.5, 21.3, 18.0, 14.2, −5.0, −5.2 ppm. HRMS-ESI+
(m/z) calcd [M + H]+ for C15H28O3Si: 285.1881. Found: 285.1883.
(S)-2-((tert-Butyldimethylsilyl)oxy)-1-methylcyclopent-2-enecar-
baldehyde (9). To a solution of anhydrous DCM (20 mL) was added
the silyl enol ether from the above reaction (0.91 g, 3.19 mmol), and
the resulting solution was cooled to −78 °C. A solution of DIBAL-H
in DCM (1M, 3.19 mL, 3.19 mmol) was added dropwise to the
reaction mixture and stirred for 4 h at −78 °C. The reaction was then
carefully quenched with H2O (1 mL); aqueous NaOH (0.5M, ∼ 0.1
mL) was added and then an additional aliquot of H2O (1 mL). The
reaction was gravity filtered through qualitative filter paper, washed
with brine (10 mL, 1×), dried over Na2SO4, and concentrated in vacuo.
The crude product was SiO2 purified with EtOAc (0−5%) in hexanes
to afford a clear oil (0.29 g, 38% yield). 1H NMR (500 MHz): 9.53 (s,
1H), 4.75 (t, J = 2.3 Hz, 1H), 2.34−2.25 (m, 3H), 1.72−1.60 (m, 1H),
1.20 (s, 3H), 0.90 (s, 9H), 0.17 (s, 3H), 0.15 (s, 3H) ppm. 13C NMR
(125 MHz): 202.5, 154.2, 103.2, 59.3, 30.7, 26.0, 25.5, 18.0, 17.6, −4.9,
−5.0; impurities at 25.7, −3.6 ppm. HRMS-ESI+ (m/z) calcd [M +
H]+ for C13H24O2Si: 241.1618. Found: 241.1616.
1a. 1H NMR (CDCl3): 7.73−7.68 (m, 1H), 6.38 (s, 1H), 6.19−6.14
(m, 1H), 5.72 (s, 1H), 5.17 (d, J = 4.9 Hz, 1H), 4.36 (dd, J = 9.5, 7.6
1H), 4.27 (dd, J = 9.6, 2.8 Hz, 1H), 3.60−3.52 (m, 1H), 2.97 (dt, J =
19.0, 2.4 Hz, 1H), 2.49−2.33 (m, 5H), 1.95 (t, J = 2.3 Hz, 1H), 1.20
(s, 3H) ppm. 13C NMR (CDCl3): 210.1, 170.4, 169.8, 163.2, 134.5,
132.0, 125.4, 82.0, 77.0, 69.9, 69.3, 49.8, 40.8, 40.2, 33.2, 20.3, 14.1
ppm. HRMS-ESI+ (m/z) calcd [M + H]+ for C18H20O3: 303.1227.
Found: 303.1222.
1
1b. H NMR (CDCl3): 7.76−7.72 (m, 1H), 6.28 (s, 1H), 6.26−
6.20 (m, 1H), 5.56 (s, 1H), 5.10 (d, J = 6.3 Hz, 1H), 4.30−4.27 (m,
2H), 3.56−3.50 (m, 1H), 2.92 (dt, J = 19.8, 2.4 Hz, 1H), 2.54−2.42
(m, 5H), 1.98 (t, J = 2.4 Hz, 1H), 1.19 (s, 3H) ppm. 13C NMR
(CDCl3): 209.8, 169.9, 168.8, 162.4, 133.5, 132.4, 124.3, 81.0, 76.8,
69.0, 68.5, 48.4, 40.2, 38.5, 32.4, 21.6, 13.2 ppm. HRMS-ESI+ (m/z)
calcd [M + H]+ for C18H20O3: 303.1227. Found: 303.1222.
(S)-2-((1-(tert-Butoxy)-3-methyl-1-oxobutan-2-yl)amino)-
cyclopent-1-ene-1-carboxylate (8). The synthetic procedure was
adapted from that previously reported.56−59 To remove the salt of L-
valine tert-butyl ester hydrochloride, the material was dissolved in
EtOAc (100 mL), and aqueous NaOH (0.5 M, 100 mL) was added
and stirred for 10 min. The material was extracted with EtOAc (100
mL, 3×), washed with brine (15 mL, 1×), dried over Na2SO4,
concentrated in vacuo to a clear oil, and then immediately used. To a
stirred solution of 7 (2.99 g, 19.1 mmol) and L-valine tert-butyl ester
(4.31 g, 24.9 mmol) in benzene (50 mL) was added BF3·OEt2 (1.18
mL, 9.55 mmol). The reaction mixture was refluxed with a Dean−
Stark trap for 24 h. The reaction was allowed to cool to RT and
quenched with aqueous NaHCO3 (saturated, 50 mL) and then
extracted with Et2O (50 mL, 3×), washed with brine (10 mL, 1×), and
dried over Na2SO4. The organic layer was concentrated in vacuo. The
crude product was SiO2 purified with EtOAc (0−5%) in hexanes to
afford 8 (4.46 g, 75%) as a white solid. 1H NMR (500 MHz): 7.63 (bs,
1H), 4.22−2.11 (m, 2H), 3.64 (dd, J = 10.0, 5.5 Hz, 1H), 2.52 (t, J =
7.1 Hz, 2H), 2.47 (t, J = 7.6 Hz, 2H), 2.17−2.06 (m, 1H), 1.81 (p, J =
7.4 Hz, 2H), 1.46 (s, 9H), 1.27 (t, J = 7.1 Hz, 3H), 0.98 (app t, 6H)
ppm. 13C NMR (125 MHz): 171.3, 168.2, 163.1, 94.7, 81.6, 63.9, 58.6,
32.3, 31.8, 29.3, 28.0, 20.9, 19.2, 17.8, 14.8. HRMS-ESI+ (m/z) calcd
[M + H]+ for C17H29NO4: 312.2169. Found: 312.2163.
(R)-4-((S)-((R)-2-((tert-Butyldimethylsilyl)oxy)-1-methylcyclopent-
2-en-1-yl)(hydroxy)methyl)-3-methylenedihydrofuran-2(3H)-one
(10a) and (S)-4-((R)-((R)-2-((tert-Butyldimethylsilyl)oxy)-1-methylcy-
clopent-2-en-1-yl)(hydroxy)methyl)-3-methylenedihydrofuran-
2(3H)-one (10b). To a solution of DMF (3 mL), 9 (0.29 g, 1.2 mmol),
and 550,55 (0.32 g, 1.8 mmol) was added powdered Zn0 (0.32 g, 4.8
mmol, activated as described for 6a*/6b*). Aqueous NH4Cl
(saturated, one drop) was added, and the reaction was degassed and
backfilled with Ar(g) (3×). The reaction was then stirred at RT for 16
h. The reaction mixture was quenched with H2O (15 mL) and
extracted with Et2O (15 mL, 3×). The organic layer was washed with
H2O (10 mL, 1×) and then brine (10 mL, 1×). The subsequent
organic layer was dried over Na2SO4 and concentrated in vacuo. The
crude mixture was SiO2 purified with an isocratic eluent system EtOAc
(20%) in hexanes to afford the separated diastereomers 10a and 10b as
white solids (0.41 g, 64% yield, 1:1 dr). Note: 10a/10b will polymerize
when concentrated, and/or the silyl enol ether will be deprotected if allowed
to sit at RT as a solid for an extended period of time. To avoid this, do not
dry under a high vacuum, and after concentration with a low vacuum
pump immediately take 10a and 10b on to the next reaction.
Ethyl (S)-1-Methyl-2-oxocyclopentane-1-carboxylate ((S)-2). Ster-
eoselective methylation was adapted from that previously re-
ported.56−59 n-BuLi in hexanes (2.0 M solution, 3.56 mL, 7.13
mmol) was added to a solution of DIPA (1.00 mL, 7.13 mmol) at −78
°C in anhydrous toluene (30 mL), and the reaction was warmed to 0
°C and stirred for 30 min. The reaction mixture was cooled to −78 °C,
and 8 (1.85 g, 5.94 mmol) in anhydrous toluene (10 mL) was added
dropwise and stirred for 1 h. THF (0.96 mL, 11 mmol) was added to
the reaction and stirred for 2 h. MeI (1.85 mL, 29.7 mmol) was added,
10a. 1H NMR (500 MHz): 6.42 (d, J = 1.6 Hz, 1H), 5.91 (d, J = 1.0
Hz, 1H), 4.66 (t, J = 2.4 Hz, 1H), 4.22 (dd, J = 9.2, 4.1 Hz, 1H), 4.22
(dd, J = 9.2, 4.1 Hz, 1H), 3.62 (dd, J = 6.6, 4.1 Hz, 1H), 3.29−3.22 (m,
G
DOI: 10.1021/acschembio.6b00751
ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology
Articles
1H), 2.27−2.19 (m, 2H), 2.17−2.08 (m, 1H), 2.0 (d, J = 4.2 Hz, 1H),
1.62−1.51 (m, 1H), 1.10 (s, 3H), 0.93 (s, 9H), 0.20 (s, 3H), 0.17 (s,
3H) ppm. 13C NMR (125 MHz): 170.8, 156.9, 135.0, 126.2, 102.0,
76.1, 70.3, 51.9, 41.3, 31.1, 25.7, 25.4, 21.8, 18.0, −4.6, −5.3 ppm.
HRMS-ESI+ (m/z) calcd [M + H]+ for C18H30O4Si: 339.1986. Found:
339.1982.
Rac-(R)-((R)-1-Methyl-2-oxocyclopentyl)((S)-4-methylene-5-oxo-
tetrahydrofuran-3-yl)methyl 4-Bromobenzoate (11). The 4-bromo
benzoate silyl enol ether (0.180 g, 0.035 mmol) from the previous
reaction was dissolved in DCM (2 mL), and TFA (200 μL) was added
to the reaction at RT. The reaction was stirred for 1 h, then quenched
with aqueous NaHCO3 (saturated, 10 mL) and extracted with DCM
(10 mL, 3×). The organic layer was dried over Na2SO4 and
concentrated in vacuo. The crude product was SiO2 purified with
EtOAc (0−40%) in hexanes to afford a white solid (0.100 g, 53%
1
10b. H NMR (500 MHz): 6.40 (d, J = 1.1 Hz, 1H), 6.01 (s, 1H),
4.62 (t, J = 2.3 Hz, 1H), 4.24 (dd, J = 9.4, 3.1 Hz, 1H), 4.24 (dd, J =
9.4, 5.3 Hz, 1H), 3.64 (dd, J = 8.6, 3.7 Hz, 1H), 3.23−3.16 (m, 1H),
2.51 (d, J = 3.7 Hz, 1H), 2.30−2.12 (m, 2H), 2.07−1.99 (m, 1H),
1.63−1.55 (m, 1H), 1.20 (s, 3H), 0.94 (s, 9H), 0.22 (s, 3H), 0.18 (s,
3H) ppm. 13C NMR: 170.9, 157.4, 135.7, 126.0, 101.9, 77.4, 68.9, 51.3,
42.4, 30.7, 25.8, 25.6, 22.7, 18.0, −4.4, −5.5 (125 MHz). HRMS-ESI+
(m/z) calcd [M + H]+ for C18H30O4Si: 339.1986. Found: 339.1978.
(R)-4-((S)-Hydroxy((R)-1-methyl-2-oxocyclopent-3-en-1-yl)-
methyl)-3-methylenedihydrofuran-2(3H)-one (6a). Compound 10a
(22 mg, 0.074 mmol) was dissolved in DMSO (2 mL), and Pd(OAc)2
(7.0 mg, 0.013 mmol) was added. The reaction was placed under 1
atm of O2 (g) and stirred at RT for 24 h. The reaction mixture was
quenched with H2O (10 mL) and extracted with Et2O (15 mL, 3×).
The organic layer was washed with H2O (10 mL, 1×) and then brine
(10 mL, 1×), dried over Na2SO4, and concentrated in vacuo. The
crude mixture was SiO2 purified with EtOAc (0−80%) in hexanes to
1
yield). H NMR (500 MHz): 7.83−74 (m, 2H), 7.62−7.54 (m, 2H),
6.14 (d, J = 2.4 Hz, 1H), 5.53 (d, J = 1.7 Hz, 1H), 5.19 (d, J = 6.7 Hz,
1H), 4.47−4.35 (m, 2H), 3.87−3.81 (m, 1H), 2.52−2.42 (m, 1H),
2.33−2.21 (m, 1H), 2.15−2.07 (m, 1H), 2.05−1.88 (m, 3H), 1.17 (s,
3H) ppm. 13C NMR (125 MHz): 220.0, 169.7, 164.8, 134.2, 132.1,
131.0, 128.9, 128.1, 126.0, 78.5, 69.8, 51.4, 39.9, 38.8, 34.2, 20.2, 18.6
ppm. HRMS-ESI+ (m/z) calcd [M + H]+ for C19H19BrO5: 407.0489.
Found: 407.0471.
Cell Culture. All cell lines were maintained in a humidified 5% CO2
environment at 37 °C in tissue culture flasks (Corning) under
normoxic conditions. Adherent cells were dissociated using Trypsin-
EDTA solution (0.25%, Gibco). A549-NF-κB luciferase cells were
cultured as described previously.62 HeLa were cultured in MEM media
(Cellgro) supplemented with 10% FBS (Gibco), penicillin (100 IU/
mL, ATCC), and streptomycin (100 μg/mL, ATCC).
1
afford 6a (10 mg, 46% yield, 93:7 er), as a clear oil. H NMR (500
A549-Luciferase NF-κB reporter assay. The NF-κB-luciferase assay
in stably transfected A549 cells was conducted according to a
previously described protocol.62
MHz): 7.78−7.72 (m, 1H), 6.46 (d, J = 2.1 Hz, 1H), 6.20−6.16 (m,
1H), 5.81 (d, J = 1.9 Hz, 1H), 4.43 (dd, J = 9.4, 7.7 Hz, 1H), 4.24 (dd,
J = 9.4, 3.1 Hz, 1H), 3.97 (d, J = 5.7 Hz, 1H), 3.35−3.29 (m, 1H), 3.10
(app dt, J = 18.9, 2.5 Hz, 1H), 2.35 (app dt, J = 18.9, 2.8 Hz, 1H), 1.14
(s, 3H) ppm. 13C NMR (125 MHz): 213.0, 170.3, 164.3, 134.6, 132.0,
126.3, 75.9, 69.9, 51.4, 41.4, 39.5, 21.1 ppm. HRMS-ESI+ (m/z) calcd
[M + H]+ for C12H14O4: 223.0965. Found: 223.0963.
Labeling in HeLa Cell Culture. HeLa cells were grown to 90%
confluency in a 75 cm2 culture flask. The culture flasks were dosed
with the respective probe concentrations or a DMSO control and
incubated for 1 h at 37 °C under normoxic conditions. The cells were
detached with nonenzymatic cell dissociation solution (Life Tech-
nologies) and washed with cold 1× PBS buffer (10 mL, 3×). The cells
were pelleted after each suspension for 5 min at 1000 rpm. After the
last wash, the cells were suspended in cold 1× PBS buffer (1 mL)
containing Complete EDTA-Free Protease Cocktail (Promega). The
cells were lysed via sonication with a Vibra Cell VCX 750 (750 W, 20
kHz, 120 V) at 40% power for 30 s, while on ice. The lysates were
stored at −80 °C until further use.
Lysate was allowed to thaw and kept on ice. The protein
concentration was measured via BCA analysis (Pierce BCA Protein
Assay Kit, Thermo Scientific), and all lysates were normalized to the
sample with the lowest concentration. Click reagents were added to
each sample (1 μL CuSO4, 100 mM stock in ddH2O; 1 μL TBTA, 20
mM stock in DMSO; 0.5 μL TAMRA-N3,68 40 mM stock in DMSO; 2
μL TCEP, 100 mM in ddH2O) and allowed to react for 3 h at RT.
LDS 4× sample buffer (8 μL, NuPAGE) and 10× sample reducing
agent (2 μL, NuPAGE) were then added to each sample and heated to
90 °C for 5 min before being pipetted into a 15-well NuPAGE Novex
4−12% polyacrylamide bis-tris gel and separated with electrophoresis
(180 V, 54 min) in NuPAGE MES SDS running buffer (1×). Gels
were imaged using a TyphoonFLA7000 gel imager (General Electric).
Images were analyzed using ImageQuant TL v7.0 software.
Pulldown Experiments. HeLa cells were allowed to grow to 90%
confluency in a 150 cm2 flask under normoxic conditions at 37 °C in a
humidified CO2 incubator. The medium was replaced with 20 mL of
fresh medium, and the competition compounds 6a and 6b or a DMSO
control was dosed to achieve the final concentration (50 μM, DMSO
concentration <0.05%) in each flask and incubated for 20 min. Alkyne
probes 1a or 1b were dosed at 50 μM and incubated for an additional
30 min. After incubation, the medium was removed, and the cells were
washed with cold 1× PBS (10 mL). The cells were then dissociated
from the flask using nonenzymatic dissociation media (4 mL, Life
Technologies). The cells were collected in 1× PBS (8 mL) and
centrifuged (1000 rpm, 5 min, RT) in a conical tube. The cells were
washed with cold 1× PBS (10 mL) and centrifuged again. The cells
were then taken up in 1× PBS (2.5 mL) containing protease inhibitor
(Complete EDTA-free protease inhibitor cocktail, Life Technologies).
The cells were lysed via sonication with a Vibra Cell VCX 750 (750 W,
(S)-4-((R)-Hydroxy((R)-1-methyl-2-oxocyclopent-3-en-1-yl)-
methyl)-3-methylenedihydrofuran-2(3H)-one (6b). Compound 10b
(12 mg, 0.04 mmol) was dissolved in DMSO (2 mL), and Pd(OAc)2
(1.00 mg, 0.004 mmol) was added. The reaction was placed under 1
atm of O2(g) and stirred at RT for 24 h. The reaction mixture was
quenched with H2O (10 mL) and extracted with Et2O (15 mL, 3×).
The organic layer was washed with H2O (10 mL, 1×) and then brine
(10 mL, 1×). The subsequent organic layer was dried over Na2SO4
and concentrated in vacuo. The crude mixture was SiO2 purified with
EtOAc (0−80%) in hexanes to afford 6b (4.0 mg, 56% yield, 91:9 er)
1
as a clear oil. H NMR (500 MHz): 7.77−7.73 (m, 1H), 6.39 (d, J =
2.7 Hz, 1H), 6.24−6.16 (m, 1H), 5.98 (d, J = 2.3 Hz, 1H), 4.32−4.23
(m, 1H), 4.15 (dd, J = 9.4, 5.2 Hz, 1H), 3.77 (dd, J = 7.9, 2.6 Hz, 1H),
3.45 (d, J = 2.8 Hz, 1H), 3.41−3.32 (m, 1H), 2.87 (dt, J = 19.5, 2.5 Hz,
1H), 2.44 (dt, J = 19.4, 2.4 Hz, 1H), 1.26 (s, 3H). 13C NMR (125
MHz): 213.8, 170.4, 163.8, 134.9, 132.7, 126.4, 75.4, 68.5, 49.9, 42.0,
41.1, 20.8 ppm. HRMS-ESI+ (m/z) calcd [M + H]+ for C12H14O4:
223.0965. Found: 223.0963.
Rac-(R)-((R)-2-((tert-Butyldimethylsilyl)oxy)-1-methylcyclopent-2-
en-1-yl)((S)-4-methylene-5-oxotetrahydrofuran-3-yl)methyl 4-Bro-
mobenzoate. Compound 10b (0.150 g, 0.443 mmol) was dissolved
in DCM (4 mL). Next, 4-DMAP (0.325 g, 2.66 mmol) and 4-
bromobenzoic acid (0.445 g, 2.22 mmol) were added, followed by
DCC (0.457 g, 2.22 mmol). The reaction was heated to 40 °C for 16
h. The reaction was allowed to cool to RT and quenched with H2O (5
mL). The mixture was extracted with DCM (20 mL, 3×). The organic
layer was dried over Na2SO4 and concentrated in vacuo. The crude
reaction product was SiO2 purified with EtOAc (0−20%) in hexanes.
1
The reaction afforded 0.180 g (99% yield) of a white solid. H NMR
(500 MHz): 7.89−7.82 (m, 2H), 7.63−7.56 (m, 2H), 6.12 (d, J = 2.5
Hz, 1H), 5.51 (d, J = 2.1 Hz, 1H), 5.11 (d, J = 7.5 Hz, 1H), 4.71 (t, J =
2.3 Hz, 1H), 4.46 (dd, J = 9.5, 3.8 Hz, 1H), 4.34 (dd, J = 9.5, 7.9 Hz,
1H), 3.50−3.41 (m, 1H), 2.38−2.21 (m, 3H), 1.84−1.74 (m, 1H),
1.14 (s, 3H), 0.96 (s, 9H), 0.23 (s, 3H), 0.19 (s, 3H) ppm. 13C NMR
(125 MHz): 170.1, 165.1, 155.8, 134.9, 132.0, 131.0, 128.7, 128.5,
125.2, 102.8, 79.5, 69.7, 51.5, 40.3, 30.5, 26.1, 25.7, 24.3, 18.1, 4.4, 5.4,
impurity at 53.4 ppm. HRMS-ESI+ (m/z) calcd [M + H]+ for
C25H33BrO5Si: 521.1353. Found: 521.1331.
H
DOI: 10.1021/acschembio.6b00751
ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology
Articles
20 kHz, 120 V) at 40% power for 30 s, while on ice. The lysates were
stored at −80 °C until further use.
equipped with a Dionex Ultimate UHPLC pump (Thermo Scientific),
a nanospray source, and Xcalibur 3.0.63 software for instrument
control. Peptide mixtures were directly injected onto a nanoHPLC
column (75 μm i.d., 10 cm packed bed, 15 μm orifice) created by hand
packing a commercially purchased fused-silica emitter (New
Objective) with Zorbax SB-C18 5 μm separation media (Agilent).
The gradient program started from 0 to 17 min at 2% MeCN/H2O
(1% formic acid) with a flow rate of 3 μL/min, followed by a linear
increase to 30% MeCN/H2O (1% formic acid) from 17 to 80 min,
followed by a linear increase to 80% MeCN/H2O (1% formic acid)
from 80 to 91 min. Finally, the column was equilibrated with 2%
MeCN/H2O (1% formic acid) from 91 to 99 min with a flow rate of 9
μL/min. Liquid chromatography was carried out at an ambient
temperature. The mass spectrometer was calibrated prior to each
analysis, and the spray voltage was adjusted to ensure a stable spray.
The MS tune parameters were as follows: spray voltage of 2.460 kV,
capillary temperature of 300 °C, and an S-lens RF level of 60%. MS/
MS spectra were collected using simultaneous data-dependent
scanning and target mass analysis, in which one full scan mass
spectrum is acquired in the Orbitrap detector (R = 120 000, scan range
320−2000 m/z), followed by a target list analyzing masses
corresponding to expected theoretical probe-peptide adducts
(343.6443, 489.2235, 640.8083, 770.8567, 229.4320, 326.4848,
427.5413, 514.2402, 923.7783, 693.0855, 569.7730, 383.6574,
529.2365, 353.1601, 680.8214, 454.2167 m/z), followed by 12 data-
dependent MS/MS spectra acquired with the Orbitrap detector (R =
15 000) with charge states 2−7, dynamic exclusion after one detection
for 20 s, an intensity threshold of 5.0 × 104, and a mass tolerance of
10.00 ppm. The method uses an isolation width of 1.6 m/z, maximum
injection time of 150 ms, 40% HCD collision energy, and 1 AGC
microscan. Spectral data were analyzed using the Proteomic
Discoverer software package (v1.4.0.288, ThermoFisher). Data were
processed using the SEQUEST v.27 algorithm.71 Peptide spectra were
searched against the UniProt Human Protein Database. Helenalin
(+262.1205 Da), 6a and 6b (+222.0892), 1a and 1b (+302.3260 Da),
and/or cysteine carboxamidomethylation (+57.0215 Da) were set as a
dynamic modification. Precursor mass tolerance was set to 10 ppm
within the calculated mass, and fragment ion mass tolerance was set to
10 mmu of their monoisotopic mass. Probe-peptide adducts found
using the Proteome Discoverer software were further scrutinized by
manually extracting the mass using the Xcalibur software from the total
ion chromatogram. The MS2 fragmentation data were analyzed
manually to confirm the identity of probe-peptide adducts.
After thawing, the samples were centrifuged at 4000 rpm for 20 min
at 0 °C to clear the lysate. The samples were transferred to clean
conical tubes, and 200 μL of 10 w/v% SDS in ddH2O were added and
heated to 65 °C for 10 min. The protein concentration of each sample
was measured via BCA analysis (Pierce BCA Protein Assay Kit,
Thermo Scientific), and all lysates were normalized to the sample with
the lowest concentration (between 1.0 to 1.6 mg mL−1). Click
reagents were added (10 μL CuSO4, 100 mM stock in ddH2O; 20 μL
TBTA, 20 mM stock in DMSO; 20 μL Biotin-N3 [Sigma-Aldrich
762024; CAS: 875770−34−6], 20 mM stock in DMSO; 10 μL TCEP,
100 mM in ddH2O) and allowed to react for 3 h at RT. After
incubation, 15 μL of each sample was collected and saved for the input
lysate control.
The samples were then separated on a monomeric avidin column
according to the manufacturer’s instructions (Pierce) at 4 °C. The
biotinylated samples were eluted using the regeneration buffer (0.1 M
HCl glycine buffer, pH 2.8). Note: biotinylated samples did not elute
using the elution biotin buffer. After the samples were collected, they
were concentrated using a 10 kDa molecular weight cutoff filter
(Amicon), ddH2O (20 mL, 2×) was added, centrifuged, and finally the
samples were concentrated to ∼500 μL. The samples were collected
and concentrated to dryness in a SpeedVac for 10 h at RT. These
samples were then dissolved in ddH2O (20 μL), 10× reducing agent
(2 μL, NuPAGE), and 4× sample buffer (10 μL, NuPAGE) and
vortexed. Sample buffer and reducing agent were added to the input
lysates in the same fashion, and all samples were heated to 90 °C for 5
min before pipetting 15 μL of each sample into a 15-well NuPAGE
Novex 4−12% polyacrylamide bis-tris gel and separated with
electrophoresis (180 V, 54 min) in NuPAGE MES SDS running
buffer (1×). The samples were separated and transferred (30 V, 1 h,
RT) in 1× TBE buffer to a PVDF membrane (Immobilon-FL) for
Western blot analysis. Membranes were incubated with the respective
primary antibodies (p65, Santa Cruz, sc-372; p50, Santa Cruz sc-8414;
IκBα, Santa Cruz sc-371; IKKα/β, Santa Cruz sc-7607) with a 1:1000
dilution in 0.5% nonfat milk (BioRad) in 1× PBS (10 mL) overnight
at 4 °C. Secondary HRP-conjugated antibodies (antirabbit poly-HRP,
Pierce cat # 32260; secondary antimouse, Novex HRP cat # A16072)
were added to 0.5% nonfat milk (BioRad) in 1× PBS (10 mL) at a
1:5000 dilution for 1 h at RT. Membranes were washed in ddH2O
between each incubation (30 mL for 1 min, 5×). Super Signal West
Dura Extended Duration Luminol/Enhancer Solution (1 mL) and
Stable Peroxide Buffer (1 mL) were added to the top of the membrane
and imaged with a Li-COR Odyssey Fc imaging system. After each
antibody was detected, the membrane was stripped with Restore PLUS
Western Blot Stripping Buffer (Thermo), washed with ddH2O for 30
min, and blocked overnight at 4 °C in 0.5% w/v nonfat dry milk
(BioRad) in 1× PBS before incubating with the next antibody.
Labeling Recombinant Human p65. Recombinant human p65 in
buffer (this clone has five point mutations compared to the p65
sequence listed under accession no. AAA36408: L159V, P180S, F309S,
A439V and V462M; Active Motif) was placed in an Eppendorf tube (3
μL, 100 ng/μL) with ddH2O (6 μL) and incubated with 100 μM of
helenalin, 6a, 6b, 1a, or 1b for 1 h at RT. LDS 4× sample buffer (4 μL,
NuPAGE) and 10× sample reducing agent (1 μL, NuPAGE) were
then added to each sample and heated to 90 °C for 5 min before being
pipetted into a 15-well NuPAGE Novex 4−12% polyacrylamide bis-tris
gel and run into the top of the gel with electrophoresis (180 V, 5 min)
in NuPAGE MES SDS running buffer (1×). The top of each well
where the protein was located was excised and placed into an
Eppendorf tube.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acschem-
bio.6b00751.
NMR and analytical HPLC characterization of synthe-
sized compounds, determination of enantiomeric ratio of
6a and 6b, molecular modeling of probe compounds,
peptide-probe MS2 fragmentation data, and crystallo-
graphic information (PDF)
Crystallographic information for 11 (CIF)
AUTHOR INFORMATION
■
The gel pieces were then processed by in-gel trypsin digestion
according to a previously reported protocol.69,70 The peptides were
desalted using a P10 C18 Zip-Tip (Millipore). Samples were dried with
a SpeedVac for 3 h at RT, and the dried peptides were dissolved in
95:5 H2O/MeCN 0.1% formic acid solution (12 μL) for HPLC- ESI+-
MS/MS analysis.
Corresponding Author
*E-mail: daharki@umn.edu.
ORCID
Daniel A. Harki: 0000-0001-5950-931X
HPLC-ESI+-MS/MS analyses of tryptic peptides were conducted
using an Orbitrap Fusion mass spectrometer (Thermo Scientific)
Notes
The authors declare no competing financial interest.
I
DOI: 10.1021/acschembio.6b00751
ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology
Articles
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ACKNOWLEDGMENTS
■
The work was generously supported by the University of
Minnesota (Academic Health Center Seed Grant #2010.01),
The V Foundation for Cancer Research (V Scholar Award to D.
A. Harki), Hyundai Hope on Wheels (Hope Grant), and the
NIH (R21-CA194661). J. C. Widen acknowledges the
University of Minnesota, College of Pharmacy for a Bighley
Graduate Fellowship. Mass spectrometry was performed at the
Analytical Biochemistry Core Facility of the Masonic Cancer
Center, which is supported by the NIH (P30-CA77598 and S10
RR-024618). We gratefully acknowledge V. G. Young, Jr.
(University of Minnesota, Department of Chemistry, X-ray
Crystallographic Laboratory) for solving the structure of 11 and
funding from the NSF/MRI (#1229400) and the University of
Minnesota for the purchase of the Bruker-AXS D8 Venture
diffractometer. The Minnesota Supercomputing Institute
(MSI) at the University of Minnesota is acknowledged for
molecular modeling resources.
(15) Yang, F., Nickols, N. G., Li, B. C., Szablowski, J. O., Hamilton, S.
R., Meier, J. L., Wang, C. M., and Dervan, P. B. (2013) Animal toxicity
of hairpin pyrrole-imidazole polyamides varies with the turn unit. J.
Med. Chem. 56, 7449−7457.
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transcription factors with small molecules. Curr. Opin. Chem. Biol. 14,
331−340.
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organic molecules. Curr. Opin. Chem. Biol. 12, 464−471.
(18) Merfort, I. (2011) Perspectives on sesquiterpene lactones in
inflammation and cancer. Curr. Drug Targets 12, 1560−1573.
(19) Siedle, B., Garcia-Pineres, A. J., Murillo, R., Schulte-Monting, J.,
Castro, V., Rungeler, P., Klaas, C. A., Da Costa, F. B., Kisiel, W., and
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sesquiterpene lactones as inhibitors of the transcription factor NF-
κB. J. Med. Chem. 47, 6042−6054.
ABREVIATIONS
■
Cys, cysteine; IBX, 2-Iodoxybenzoic acid; DMSO, dimethyl
sulfoxide; PCC, pyridinium chlorochromate; DCC, N,N′-
dicyclohexylcarbodiimide; RT, room temperature; PhH,
benzene; LDA, lithium diisopropylamine; LiHMDS, Lithium
bis(trimethylsilyl)amide; 4-DMAP, 4-dimethylaminopyridine;
TBSCl, tert-butyldimethylsilyl chloride; atm, atmosphere; sec,
seconds; min, minutes; dr, diastereomeric ratio; er, enantio-
meric ratio; sat, saturated; aq, aqueous; GC, gas chromatog-
raphy; TFA, trifluoroacetic acid; LC-MS, liquid chromatog-
raphy−mass spectrometry; SDS-PAGE, sodium dodecyl sulfate-
polyacrylamide gel electrophoresis; (S)-MTPA, (S)-α-Me-
thoxy-α-trifluoromethylphenylacetic acid; TMS, tetramethylsi-
lane; EtOAc, ethyl acetate; EDTA, Ethylenediaminetetraacetic
acid; ESI, electrospray ionization; HPLC, high-performance
liquid chromatography; UHPLC, ultra high-performance liquid
chromatography; TBE, tris boric acid EDTA buffer; ddH2O,
distilled and deionized water; HRMS-ESI, high resolution mass
spectrometry-electrospray ionization; PVDF, polyvinylidene
difluoride
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Johnston, L. D. (1974) Antineoplastic Agents. 34. Helenium-
Autumnale-L. J. Med. Chem. 17, 1013−1016.
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