Thiol Probes to Detect Electrophilic Natural Products Based on Their Mechanism of Action

Article abstract of DOI:10.1021/acschembio.5b00924

New methods are urgently needed to find novel natural products as structural leads for the development of new drugs against emerging diseases such as cancer and multiresistant bacterial infections. Here we introduce a reactivity-guided drug discovery approach for electrophilic natural products, a therapeutically relevant class of natural products that covalently modify their cellular targets, in crude extracts. Using carefully designed halogenated aromatic reagents, the process furnishes derivatives that are UV-active and highly conspicuous via mass spectrometry by virtue of an isotopically unique bromine or chlorine tag. In addition to the identification of high-value metabolites, the process facilitates the difficult task of structure elucidation by providing derivatives that are primed for X-ray crystallographic analysis. We show that a cysteine probe efficiently and chemoselectively labels enone-, β-lactam-, and β-lactone-based electrophilic natural products (parthenolide, andrographolide, wortmannin, penicillin G, salinosporamide), while a thiophenol probe preferentially labels epoxide-based electrophilic natural products (triptolide, epoxomicin, eponemycin, cyclomarin, salinamide). Using the optimized method, we were able to detect and isolate the epoxide-bearing natural product tirandalydigin from Salinispora and thereby link an orphan gene cluster to its gene product.

Full text of DOI:10.1021/acschembio.5b00924

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Article
Thiol probes to detect electrophilic natural
products based on their mechanism of action
Gabriel Castro-Falcon, Dongyup Hahn, Daniela Reimer, and Chambers C. Hughes
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.5b00924 • Publication Date (Web): 13 Jun 2016
Downloaded from http://pubs.acs.org on June 15, 2016
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Thiol probes to detect electrophilic natural products based on their mechanism of action
Gabriel CastroꢀFalcón, Dongyup Hahn, Daniela Reimer, Chambers C. Hughes*
Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography,
University of California, San Diego, La Jolla, California, United States
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ABSTRACT
New methods are urgently needed to find novel natural products as structural leads for
the development of new drugs against emerging diseases like cancer and multiꢀresistant
bacterial infections. Here, we introduce a reactivityꢀguided drug discovery approach for
electrophilic natural products, a therapeuticallyꢀrelevant class of natural products that covalently
modifies their cellular targets, in crude extracts. Using carefully designed halogenated aromatic
reagents, the process furnishes derivatives that are UVꢀactive and highly conspicuous via mass
spectrometry by virtue of an isotopicallyꢀunique bromine or chlorine tag. In addition to the
identification of highꢀvalue metabolites, the process facilitates the difficult task of structure
elucidation by providing derivatives that are primed for Xꢀray crystallographic analysis. We show
that a cysteine probe efficiently and chemoselectively labels enoneꢀ, βꢀlactamꢀ, and βꢀlactoneꢀ
based electrophilic natural products (parthenolide, andrographolide, wortmannin, penicillin G,
salinosporamide), whilst a thiophenol probe preferentially labels epoxideꢀbased electrophilic
natural products (triptolide, epoxomicin, eponemycin, cyclomarin, salinamide). Using the
optimized method we were able to detect and isolate the epoxideꢀbearing natural product
tirandalydigin from Salinispora and thereby link an orphan gene cluster to its gene product.
Historically, natural products have had a tremendous influence on the development of
new drugs for cancer, antibacterial infections, immunosuppression, and immunostimulation.
Bioinformatic analyses of bacterial genomes clearly show the presence of many more
biosynthetic pathways encoding natural products than actual isolated compounds. This
disparity signals a need for the development of new methods for natural products research.
Some of these “orphan” gene clusters are “silent” and require a geneticsꢀbased solution for
production of a metabolite. However, the primary reason for the large quantity of orphan gene
clusters is that a natural product extract, in general, consist of miniscule amounts of the desired
metabolite in a hodgepodge of primary metabolites such as amino acids, sugars, lipids, natural
products and media components. Thus, new chemistryꢀbased methods to access the hidden
potential of bacterial genomes are needed.
One promising approach that complements traditional bioactivityꢀguided isolation utilizes
the inherent reactivity of a metabolite to inform the detection and isolation process. One version
uses soluble reagents to label natural product functional groups or structural features in an
extract. Although the original metabolites are irreversibly modified, the resulting adducts often
have enhanced visibility using UVꢀdetected liquid chromatography/mass spectrometry (LC/MS)
instruments. The Marfey’s reaction is an early application of this method, in which amino acids
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are chemoselectively modified to give adducts with strongly absorbing chromophores. Another
method uses reactive resins to immobilize and selectively elute natural products based on
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chemical functionality.
including dehydroalanine,
alcohols, terminal alkynes,
In either case, only simple functional groups have been targeted,
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ketones and aldehydes, carboxylic acids, amines, thiols,
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and azides. Until recently, reactivityꢀguided isolation has not
been employed for the identification of natural product pharmacophores that would lead directly
to biologicallyꢀactive metabolites with a high potential for therapeutic development.
An important class of natural products owe their potent biological activity to an
electrophilic moiety that engages the cellular target of the molecule in a covalent linkage.
Due to the misconceived notion that they would be too toxic, the drug industry has historically
avoided the development of covalent drugs, despite the unparalleled success of aspirin and
penicillin. Recently, however, there has been a resurgence of interest in covalent drugs. In
addition to the newlyꢀapproved covalent proteasome inhibitor carfilzomib, whose structure is
based on the natural product epoxomicin, several covalent kinase inhibitors with striking
selectivity and potency are in clinical trials or have been approved by the FDA. Interestingly,
the electrophilic mechanism of action of all 39 FDAꢀapproved covalent drugs was discovered
serendipitously, only after they were selected and developed for their biological activity.
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RESULTS AND DISCUSSION
We set out to apply a reactivityꢀguided approach for the deliberate discovery of
electrophilic natural products. Given the reactivity of thiolates with naturallyꢀoccurring
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electrophilic moieties,
which has previously been exploited to classify certain electrophilic
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natural products, we wished to design and synthesize a thiolꢀbased probe that would tag these
compounds in a crude extract. These “pharmacophore probes” consist of three parts: 1. a highly
chemoselective reagent that reacts covalently with a specified pharmacophore, 2. an easily
identifiable UV/Vis or mass spectrometric tag, and 3. a linker that joins the reagent and tag. We
tested several different probe designs. Two probes proved superior to the others in terms of
reactivity, chemoselectivity, and stability (Figure 1). Cysteine probe 1 was synthesized in three
steps from Lꢀcystine, whilst thiophenol probe 2 was obtained commercially. These probes
possess each of the characteristics of an ideal pharmacophore probe. In addition to the thiol
functionality, both have a chromophore owing to the aromatic ring and a conspicuous MS
isotopic pattern ( Br: Br 1:1 and Cl: Cl 3:1). Though UVꢀactive halogenated metabolites are
not entirely uncommon, prior analysis of an extract can rapidly identify these endogenous
molecules and thereby discount them as labeled electrophilic natural products. The rationale for
different halogen atoms on probes 1 and 2 will be discussed later
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Both probes incorporate a halogenated aromatic ring. The introduction of this moiety,
especially bromobenzoyl substituents, is a longꢀestablished method for obtaining crystalline
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derivatives of natural products that are amenable to Xꢀray crystallographic analysis.
the probes not only react with the desired pharmacophore but produce an adduct that is
Thus,
inherently more crystalline. The cysteine probe, furthermore, contains a defined Rꢀstereocenter
that facilitates determinations of absolute stereochemistry.
Figure 1. Pharmacophore probes 1 and 2.
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We relied initially on four substrates to assess the coupling efficiency of our thiol probes
and optimize reaction conditions. Parthenolide (3) from feverfew (Tanacetum parthenium)
contains a reactive butenolide moiety that is known to interact covalently with Cys171 of Iκβ
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kinase and inhibit this enzyme (12) (Figure 2). Treatment of 3 with probe 1 in triethylamine and
N,Nꢀdimethylformamide (DMF) afforded 4 as a single isomer in 36% yield after HPLC
purification. The addition of triethylamine was essential for the reaction to proceed at an
appreciable rate, presumably via formation of the corresponding thiolate. Andrographolide (5)
from the plant Andrographis paniculata contains a substituted butenolide that reacts with Cys62
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of p50 and so inhibits NFꢀκB activation (13). The probe efficiently reacted with 5 as well to give
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as a mixture of diastereomers. The formation of diastereomeric products is not desired since it
complicates the analysis, but we were able to purify the major diastereomer using reversedꢀ
phase HPLC. Via reaction with Lys802, wortmannin (7) from the fungus Talaromyces wortmanii
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is a covalent inhibitor of phosphoinositide 3ꢀkinase (14).
Metabolite 7 reacted with thiol 1 in
a manner completely analogous to the natural product’s mechanism of action, producing a ringꢀ
opened structure. Importantly, this particular reaction was conducted in dichloromethane (DCM)
since undesired elimination reactions occurred in DMF. The most famous electrophilic natural
product, penicillin G (9), contains a βꢀlactam ring that is essential for its antibacterial activity.
The mechanism of action of 9 involves acylation of a key serine residue of the bacterial
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transpeptidase enzyme (15). Thiol 1 engaged in an efficient coupling reaction with 9 to cleanly
produce a pair of diastereomers. Purification and NMR analysis of the amino acid 10 proved
difficult so the labeling reaction was immediately followed by acetylation to give 11.
We then screened a number of strains from the tropical marine bacterial genus
Salinispora in order to demonstrate the method in crude extracts. Upon reaction of 1 with the
extract of S. tropica strain CNBꢀ392, we observed the formation of two new compounds with UV
absorption at 254 nm and brominated isotope patterns (Figure 3). Surprisingly, of the scores of
other metabolites in the crude extract, none appeared to react with the probe. The first adduct,
thioester 16, was purified and analyzed by NMR and then retrosynthetically disconnected to
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salinosporamide A (18), a wellꢀknown covalent proteasome inhibitor from Salinispora. The
second adduct, thioester 17, is highly reminiscent of the threonine adduct in the crystal structure
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of the enzymeꢀinhibitor complex (19). Thus, whether the electrophilic moiety is an α,βꢀ
unsaturated enone, an α,β,γ,δꢀunsaturated enone, a βꢀlactam or βꢀlactone, the thiol probe
simultaneously tags the compound and elucidates its mode of action by mimicking the key
nucleophilic residue on the cellular target responsible for covalent binding. This occurs whether
the key amino acid residue on the target is cysteine, lysine, serine or threonine (see Figure 2,
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2−15).
Notably, a saturated solution of thioester 17 in benzene produced crystals amenable to
Xꢀray analysis within 12 h (see Figure 3). A common characteristic of molecules containing a
heavy” bromine atom and a key feature of the method, a nearꢀzero Flack parameter of 0.068
was readily obtained and allowed for the unambiguous assignment of the absolute configuration
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of 17 and, hence, the natural product (18).
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Figure 2. (A) Reaction of probe 1 with enoneꢀ and βꢀlactamꢀbased electrophilic natural products
parthenolide (3), andrographolide (5), wortmannin (7), penicillin G (9). Et N = triethylamine,
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DMF = N,Nꢀdimethylformamide, DCM = dichloromethane, Ac O = acetic anhydride, pyr. =
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pyridine, rt = room temperature. (B) Natural products bound to the key nucleophilic amino acid
residue of their respective cellular targets.
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Figure 3. Reactivityꢀguided isolation of salinosporamide A (18). A) Reaction of probe 1 with the
crude extract of strain CNBꢀ392 produces brominated products 16 and 17, which can be
retrosynthetically traced back to salinosporamide A (18). (B) Salinosporamide A bound to the
proteasome. (C) Crystals of 17 formed via slow evaporation from benzene. (D) Xꢀray crystal
structure of 17. (E) LC/MS chromatogram (254 nm) of the crude extract before and after
treatment with 1, including the conspicuous MS isotope clusters of 16 and 17. The retention
time of 18 is indicated with an asterisk.
Although probe 1 is capable of engaging enoneꢀ, βꢀlactamꢀ, and βꢀlactoneꢀbased
electrophilic metabolites, its reactivity with epoxideꢀbased electrophilic natural products was
poor. Using triptolide (20), originally discovered from thunder god vine (Tripterygium wilfordii),
and the bacterial metabolite epoxomicin (22) as model substrates, we determined that
thiophenol probe 2 displayed remarkable reactivity toward epoxides (Figure 4). It has been
shown that triptolide (20) exerts its biological effects via inhibition and covalent modification of
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Cys342 of the transcription factor TFIIA (24).
In a manner that predicted the particular
epoxide that is modified by the enzyme, the probe reacted with the 12,13ꢀepoxide of 20 to yield
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1. Likewise, epoxomicin (22), a covalent proteasome inhibitor with an epoxy ketone
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functionality, gave 23 under the same conditions. Unlike adducts formed from 1 and enoneꢀ, βꢀ
lactamꢀ, and βꢀlactoneꢀbased electrophilic natural products, the structure of 23 does not mimic
the morpholino adduct observed in the coꢀcrystal structure of 22 and the proteasome, which like
other covalent proteasome inhibitors involves reaction with the Nꢀterminal threonine residue
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Figure 4. (A) Reaction of probe 2 with epoxideꢀbased electrophilic natural products triptolide
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20) and epoxomicin (22). Et N = triethylamine, TCEP HCl = tris(2ꢀcarboxyethyl)phosphine
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hydrochloride, DMF = N,Nꢀdimethylformamide, rt = room temperature. (B) Natural products
bound to the key nucleophilic amino acid residue of their respective cellular targets.
In order to demonstrate the effectiveness of probe 2 in crude extracts, we first treated
extract from Streptomyces hygroscopicus strain ATCC 53709 with probe 2. This gave a major
chlorinated adduct (26), which was retrosynthetically disconnected to the proteasome inhibitor
eponemycin (27), in addition to several minor chlorinated compounds from eponemycin
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analogues (Figure 5).
Like epoxomicin (22) the epoxide moiety in 27 is essential for the
molecule’s potent irreversible inhibition of the proteasome. When probe 2 was treated with
extract from Streptomyces sp. strain CNBꢀ382, a suite of chlorinated compounds was produced.
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The major adduct (28) can be traced back to cyclomarin A (29). The other chlorinated
compounds were derived from new cyclomarin analogues. In terms of its purported activity
against Mycobacterium tuberculosis, which involves inhibition of the ClpC1 subunit of the
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caseinolytic protease,
and its activity against malaria, the epoxide is dispensable.
Treatment of extract from Streptomyces sp. strain CNBꢀ091 with probe 2 yielded a single
chlorinated product (30), which is derived from salinamide A (31), in addition to the chlorideꢀ
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containing analogue salinamide B.
bacterial RNA polymerase.
Again, the epoxide ring is not required for inhibition of
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Thus, reactivityꢀguided isolation of epoxideꢀcontaining natural
products with probe 2 leads to the discovery of epoxideꢀbased electrophilic natural products, like
eponemycin, as well as natural products that contain epoxides that do not appear involved in
the mechanism of action, like cyclomarin and salinamide.
Probe 1 reacts much more readily with βꢀlactamꢀ and βꢀlactoneꢀbased electrophilic
natural products and probe 2 reacts much faster with epoxideꢀcontaining natural products. This
orthogonal reactivity allows them to be employed in extracts simultaneously. Since probe 1 is
brominated and probe 2 is chlorinated, the isotopic patterns of labelled natural products point
toward their structural origin. Competition experiments using probes 1 and 2 indicated that
brominated probe 1 reacted exclusively with the βꢀlactam in penicillin G (9) and the βꢀlactone in
salinosporamide A (18) whilst chlorinated probe 2 alone reacted with the epoxide in salinamide
A (31) (See Supporting Information). Interestingly, some chlorinated products formed from
reaction of salinosporamide A (18) and probe 2 via a substitution reaction with the alkyl chloride
functionality in 18. This result was validated using salinosporamide B, which lacks an alkyl
chloride and shows no reactivity with probe 2. Probe reactivity was not exclusive with
parthenolide (3), which bears a highlyꢀreactive exocyclic enone, as a ~1:1 mixture of brominated
and chlorinated parthenolide adducts was produced.
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Figure 5. Reactivityꢀguided isolation of eponemycin (27), cyclomarin (29), and salinamide (31).
Reaction of probe 2 with the crude extract of (A) strain ATCC 53709 produces chlorinated
product 26, (C) strain CNBꢀ382 gives chlorinated product 28, and (E) strain CNBꢀ091 gives
chlorinated product 30. (B,D,F) LC/MS chromatograms (254 nm) of the crude extracts before
and after treatment with 2, including the conspicuous MS isotope clusters of 26, 28, and 30. The
retention times of 27, 29, and 31 are indicated with an asterisk.
Though genome mining techniques that can predict natural product structures have
progressed considerably, detecting and isolating gene products produced in minute quantities
still remains a challenge. As reactivityꢀguided isolation directly probes molecular structure, it is
an ideal method for enabling genome mining efforts. For instance, the genome of Salinospora
pacifica strain CNTꢀ003 contained an orphan gene cluster (Table S1) that was predicted to
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encode a member of the tirandamycin family of natural products (Figure 6). While screening
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Salinispora extracts with the probes, we noticed the production of a distinct chlorinated adduct
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32) upon reaction of 2 with the extract of strain CNTꢀ003. We suspected that we had labelled
the gene product of the tirandamycin cluster, as many of these metabolites contain epoxides. In
this case, the probeꢀnatural product adduct was not isolated. Instead, the molecular formula of
the original metabolite was calculated to be C H NO , and the compound was then directly
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isolated from the crude extract using its calculated mass and predicted UV properties. The H
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NMR spectrum clearly matched tirandalydigin (33), a compound closely related biosynthetically
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and structurally to the tirandamycins.
Like cyclomarin A (28) and salinamide A (31), the
epoxide in 33 does not appear essential to its activity as a bacterial RNA polymerase inhibitor.
Given the central role that the electrophilic centers play in the natural products’ biological
activity, we examined the relative cytotoxicity of the natural products and their corresponding
thiol adducts (Table 1). Against the HCTꢀ116 colon cancer cell line, enones 3, 5, and 7, as well
as their corresponding thiol adducts 4, 6, and 8, showed only moderate cytotoxicity (11−32 ꢁM).
Salinosporamide (18) (IC = 41 nM) was converted into thioester 16, which maintained
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remarkable activity (IC = 14 nM), and thioester 17, which showed little cytotoxicity (IC = 14
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M). Like lactacystin and other salinosporamideꢀbased thioesters,
16 is capable of reforming
the βꢀlactone ring that is crucial to proteasome inhibition, while 17 cannot undergo this
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transformation.
The cytotoxicity of the epoxideꢀbased electrophilic natural products 20, 22,
and 27 is greatly diminished 100−1000 fold upon ringꢀopening thioether formation, highlighting
the importance of this reactive pharmacophore. Metabolites 9, 29, and 31, which have
demonstrated antibacterial activity and prokaryotic targets, and their derivatives were not tested
in the HCTꢀ116 bioassay.
Figure 6. Using reactivityꢀguided isolation to validate genome mining efforts: A) A partial NRPSꢀ
PKS hybrid gene cluster found in Salinispora pacifica strain CNTꢀ003 and analyzed
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bioinformatically to code for a natural product in the tirandamycin family (Tdy). The gene cluster
is located on two contigs and is missing 6 PKS modules; B) Reaction of probe 2 with crude
CNTꢀ003 extract gave a single labelled compound (32). The molecular formula culated for the
original natural product was C H NO , which matched tirandalydigin (33). Epoxide 33 was then
detected and isolated from the extract using its calculated mass. NRPS (nonribosomal peptide
synthetase), PKS (polyketide synthase), KS (ketosynthase domain), AT (acyltransferase
domain), ACP (acyl carrier protein), C (condensation domain), A (adenylation domain), PCP
(peptidyl carrier protein)
2
2
27
6
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 1. Cytotoxicity of selected
compounds against the human colon
cancer line HCTꢀ116
natural cytotoxicity
product (IC , ꢂM)
thiol
cytotoxicity
adduct (IC , ꢂM)
50
50
1
1
6
7
0.014
14
18
0.041
2
0
2
0.0080
0.0054
0.17
21
23
26
62
2
85
27
24
METHODS
o
Preparation of cysteine thiol probe (1). To cooled methanol (150 mL) at 0 C was
slowly added thionyl chloride (7.60 mL, 105 mmol) followed by Lꢀcystine (10.0 g, 41.6 mmol),
and the reaction mixture was heated at reflux for 12 h. The solution was concentrated until the
volume was reduced to 15 mL. Diethyl ether (30 mL) was added, and 12.4 g of cystine dimethyl
ester precipitated out of solution. To a white heterogeneous solution of the dimethyl ester (3.64
g, 13.6 mmol) in acetonitrile (150 mL), 4ꢀbromobenzoyl chloride (6.22 g, 28.7 mmol) and
triethylamine (7.50 mL, 54.3 mmol) were added. The reaction mixture left stirring for 2 h. A
saturated ammonium chloride solution (300 mL) was added, and the resulting solution was
extracted with ethyl acetate (2 x 200 mL). The combined organic extracts were washed with a
saturated sodium bicarbonate solution (400 mL), brine (200 mL), dried over Na SO , and
concentrated. A saturated solution of the product in dichloromethane (50 mL) was prepared.
Then, hexanes (30 mL) was added to the solution, and 6.96 g (81%) of dibromobenzoyl
disulfide precipitated out of solution. To a solution of the disulfide (6.0 g, 9.4 mmol) in a 2:1
dichloromethaneꢀmethanol mixture, tris(2ꢀchloroethyl)phosphine hydrochloride salt (4.0 g, 14
mmol) and triethylamine (5.0 mL, 38 mmol) were added. The reaction was left for 3 h. Flash
chromatography of the crude material on silica gel (hexanesꢀethyl acetate, 4:6) yielded 5.4 g
(90%) of cysteine thiol probe (1) as a white solid. UV/Vis: λ = 241 nm; [α] = +3.3 (c 0.020,
2
4
max
D
‒
1 1
CHCl ); IR (KBr) ṽ =3323, 1742, 1635, 1524, 1219 cm ; H NMR 7.71 (d, 8.6 Hz, 2H), 7.61 (d,
3
8
.6 Hz, 2H), 7.01 (br d, 1H), 5.07 (ddd, 7.1, 4.0, 4.0 Hz, 1H), 3.84 (s, 3H), 3.14 (dd, 9.1, 4.0 Hz,
13
2H), 1.38 (t, 9.1 Hz, 1H); C NMR 170.3, 165.8, 132.1, 131.7, 128.5, 126.6, 53.7, 52.8, 26.7;
+
79
HRESIꢀQꢀTOFꢀMS: m/z (M+H) 317.9800 calcd for C H BrNO S, found 317.9784.
1
1
13
3
Generation of crude extracts. Streptomyces hygroscopicus ATCCꢀ53709 was grown in
‒1
‒1
ISP1 medium (5 g L of tryptone and 3 g L of yeast extract in deionized water) while
Streptomyces spp. CNBꢀ091 and CNBꢀ382 and Salinispora spp. CNBꢀ392 and CNTꢀ003 were
‒1
‒1
‒1
grown in A1 seawater medium (10 g L of starch, 4 g L of yeast extract and 2 g L of peptone
in 75% seawater and 25% deionized water). From cryovials, these strains were first grown in
starter cultures (25 mL) and after 7 days the whole volume was used to inoculate preꢀcultures (1
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L). After 3−4 days, 25 mL aliquots of the preꢀcultures were used to inoculate producing cultures
o
(
1 L). The cultures were incubated at 27 C with rotatory shaking at 150 rpm for 3−7 days, after
‒1
which Amberlite XADꢀ16N resin (~20 g L ) was added and left shaking for 2 h. The resin was
filtered through cheesecloth, washed with deionized water, and extracted with acetone. The
acetone was removed under reduced pressure, and the resulting aqueous layer was extracted
with ethyl acetate. The combined organic extracts were concentrated.
0
1
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9
0
1
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0
Labeling reaction. To pure natural product or crude extract, probe 1 or 2 (1.2−1.5
equiv), and triethylamine (2.0−4.2 equiv) was added dry N,Nꢀdimethylformamide which had
been previously sparged with gaseous nitrogen for 10 min. The reaction vessel was kept under
inert atmosphere using a nitrogen gas source and bubbler. The reaction was conducted with or
without added tris(2ꢀchloroethyl)phosphine hydrochloride salt (1.2−1.4 equiv) to prevent
unwanted dimerization of the probes. The progress of the reaction was monitored on an
‒1
analytical Agilent 1100 Series HP system (1.0 mL min ) with UV (210 and 254 nm) and ELS
detection, and also on an analytical Agilent 1260 Infinity Series LC system coupled to a 6530
Series QꢀTOF mass spectrometer, both using a C18(2) Phenomenex Luna column (5 ꢁm, 100 x
4.6 mm) with a 10 minute solvent gradient from 10 % to 100 % acetonitrile + 0.1% formic acid in
water. When HPLC analysis indicated complete conversion (1.5−24 h), the mixture was
concentrated. The adducts were then purified by isocratic reversedꢀphase HPLC (acetonitrile
and water + 0.1% trifluoroacetic acid) using a C8(2) Phenomenex Luna column (5 ꢁm, 250 x 10
mm) with UV detection (254 nm). Preparative reversedꢀphase HPLC purifications were
performed with a C18(2) Phenomenex Luna column (10 ꢁm, 250 x 21.2 mm) with UV detection
(
254 nm).
Cytotoxicity assays. The human colorectal cancer cell line HCTꢀ116 (ATCC CCLꢀ247)
was cultured in McCoy's 5A medium (Life Technologies) supplemented with 10% fetal bovine
‒1
serum (FBS, Invitrogen), 30 mM HEPES (Fisher Scientific) and 100 ꢂg mL
penicillin/streptomycin (Invitrogen) and incubated in tissue culture flasks with a surface of 75
cm² at 37 °C in a 5% CO atmosphere. Routine passaging of cells was conducted every 3 to 4
2
days. The cell layer was briefly rinsed in 10 mL Dulbecco's phosphateꢀbuffered saline (DPBS)
2+
2+
with Ca and Mg (Life Technologies) and detached from the culture flask using 2 mL of 0.25%
w/v) trypsinꢀEDTA (Life Technologies) at 37 °C. For the subcultivation 8 mL of complete
(
McCoy's 5A medium was added to the detached cells, cells were aspirated by gently pipetting
and then transferred into a new culture flask (1 mL into 20 mL fresh medium). Cytotoxicity in half
maximal inhibitory concentrations (IC ) of the natural products (3, 5, 7, 18, 20, 22, 27) and their
50
adducts (4, 6, 8, 16, 17, 21, 23, 26) were tested using the colorimetric 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀ
yl)ꢀ5ꢀ(3ꢀcarboxymethoxyphenyl)ꢀ2ꢀ(4ꢀsulfophenyl)ꢀ2Hꢀtetrazolium (MTS) bioassay. HCTꢀ116
4
‒1
cells (2.5 x 10 cells mL ) were incubated for 24 h at 37 °C in a 5% CO atmosphere in flatꢀ
2
‒1
bottom 96ꢀwell plates. Compounds in DMSO were added in a concentration of 10 mg mL , 1
mg mL or 0.1 mg mL based on their activity, serially diluted, and incubated for additional 72
h. As reference, the standard anticancer drug etoposide VPꢀ16 (Sigma Aldrich) with an IC of
.49−4.9 ꢁM in a concentration of 4 mg mL in DMSO and DMSO (ATCC) were tested as
positive and negative controls, respectively. IC values were determined after a 3 h incubation
‒1
‒1
50
‒1
0
50
‒1
of the colorimetric indicator using MTS (1.9 mg mL in DPBS, Promega) in the presence of
phenazine methosulfate (PMS, 0.044 mg mL in DPBS, Sigma Aldrich). MTS is reduced by
‒1
living cells into a formazan product with an absorbance maximum at 490 nm in phosphateꢀ
buffered saline. Quantification of the formazan product at 490 nm representing the proportional
Max
cell survival rates and the calculation of the IC values were determined by an E microplate
50
reader (Molecular Devices) using the analysis software SoftMax Pro.
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CONCLUSIONS
The tremendous potential for the discovery of natural products that inspire new drugs
has not been realized because there is a shortage in the number of new methods. Reactivityꢀ
guided isolation, a hybrid of traditional natural products chemistry and synthetic organic
chemistry, was invented to help access the high value chemical space of natural product
extracts. This approach, which can be applied to bacterial, fungal, and plant extracts alike, could
transform the field of natural products.
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI:
XXXXXXX. Bioinformatic analysis of the tirandalydigin biosynthetic gene cluster, detailed
experimental procedures, chromatograms of reaction mixtures, UV/vis and MS spectra, and
1
1
1
tabulated spectroscopic data including original H, Hꢀ H COSY, HSQC, and HMBC spectra for
, 4, 6, 8, 16, 17, 21, 23, 26, 28, 30, 33 (PDF).
1
AUTHOR INFORMATION
Corresponding Author
*Eꢀmail: chughes@ucsd.edu
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENTS
This work was primarily supported by startꢀup funds from the Scripps Institution of
Oceanography. C.C.H. acknowledges the UCSD Academic Senate for a Research Award
(
RN143SꢀHUGHES) and the Hellman Foundation for a generous fellowship. G.C.ꢀF. thanks SIO
for a San Diego Fellowship (2T32GM067550ꢀ10) as part of The Training Program in Marine
Biotechnology (NIH GM067550). D.R. thanks the Deutsche Forschungsgemeinschaft (RE
3704/1ꢀ1) for postdoctoral funding. We acknowledge the NIH (S10 OD0106400) for an HRLCꢀ
MS instrument.
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