Thioesterase from Cereulide Biosynthesis Is Responsible for Oligomerization and Macrocyclization of a Linear Tetradepsipeptide

Article abstract of DOI:10.1021/acs.jnatprod.0c00333

Cereulide is a toxic cyclic depsidodecapeptide produced in Bacillus cereus by two nonribosomal peptide synthetases, CesA and CesB. While highly similar in structure to valinomycin and with a homologous biosynthetic gene cluster, recent work suggests that cereulide is produced via a different mechanism that relies on a noncanonical coupling of two didepsipeptide-peptidyl carrier protein (PCP) bound intermediates. Ultimately this alternative mechanism generates a tetradepsipeptide-PCP bound intermediate that differs from the tetradepsipeptide-PCP intermediate predicted from canonical activity of CesA and CesB. To differentiate between the mechanisms, both tetradepsipeptides were prepared as N-acetyl cysteamine thioesters (SNAC), and the ability of the purified recombinant terminal CesB thioesterase (CesB TE) to oligomerize and macrocyclize each substrate was probed. Only the canonical substrate is converted to cereulide, ruling out the alternative mechanism. It was demonstrated that CesB TE can use related tetradepsipeptide substrates, such as the valinomycin tetradespipetide and a hybrid cereulide-valinomycin tetradepsipetide in conjunction with its native substrate to generate chimeric natural products. This work clarifies the biosynthetic origins of cereulide and provides a powerful biocatalyst to access analogues of these ionophoric natural products.

Full text of DOI:10.1021/acs.jnatprod.0c00333

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Article
Thioesterase from Cereulide Biosynthesis Is Responsible for
Oligomerization and Macrocyclization of a Linear Tetradepsipeptide
Graham W. Heberlig and Christopher N. Boddy*
Cite This: J. Nat. Prod. 2020, 83, 1990−1997
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ABSTRACT: Cereulide is a toxic cyclic depsidodecapeptide
produced in Bacillus cereus by two nonribosomal peptide
synthetases, CesA and CesB. While highly similar in structure to
valinomycin and with a homologous biosynthetic gene cluster,
recent work suggests that cereulide is produced via a different
mechanism that relies on a noncanonical coupling of two
didepsipeptide-peptidyl carrier protein (PCP) bound intermedi-
ates. Ultimately this alternative mechanism generates a tetradep-
sipeptide-PCP bound intermediate that differs from the tetradep-
sipeptide-PCP intermediate predicted from canonical activity of
CesA and CesB. To differentiate between the mechanisms, both
tetradepsipeptides were prepared as N-acetyl cysteamine thioesters (SNAC), and the ability of the purified recombinant terminal
CesB thioesterase (CesB TE) to oligomerize and macrocyclize each substrate was probed. Only the canonical substrate is converted
to cereulide, ruling out the alternative mechanism. It was demonstrated that CesB TE can use related tetradepsipeptide substrates,
such as the valinomycin tetradespipetide and a hybrid cereulide−valinomycin tetradepsipetide in conjunction with its native
substrate to generate chimeric natural products. This work clarifies the biosynthetic origins of cereulide and provides a powerful
biocatalyst to access analogues of these ionophoric natural products.
ereulide, 1, is a cyclic depsidodecapeptide produced by
Bacillus cereus that was discovered in 1994. It is a
mechanism. It is proposed that a L-Hiv-L-Val-D-Hic-D-Ala PCP
bound intermediate is generated (Figure 1B) rather than the
1
,2
C
19
potassium ionophore, which rapidly depolarizes bacterial and
expected D-Hic-D-Ala-L-Hiv-L-Val intermediate (Figure 1A).
3
4,5
20−22
mitochondrial membranes, and as such it is a potent toxin.
Further it is suggested that some of the isocereulides
are
At lower doses it is an emetic toxin impacting the 5-HT3
produced by the unexpected coupling of a TE-bound
19
receptor, stimulating the vagus afferent nerve leading to
depsidipeptide with a PCP-bound tetradepsipeptide. This
study sets out to test if valinomycin and cereulide have
differing mechanisms of TE-mediated oligomerization and
macrocyclization by characterizing the ability of the CesTE to
oligomerize and macrocyclize tetradepsipeptide substrates.
2
vomiting. Due to the potential contamination of food
products with B. cereus and the high chemical and temperature
stability of cereulide, poisoning is a significant safety
6
−8
concern. The biosynthetic gene cluster responsible for
9−11
cereulide biosynthesis was identified in 2004.
Cereulide
is produced by two nonribosomal peptide synthetases, CesA
RESULTS AND DISCUSSION
12,13
■
and CesB, encoded on the pXO1 megaplasmids.
CesA uses
To test the TE-mediated oligomerization and macrocyclization
step from cereulide biosynthesis, both the proposed cereulide
α-ketoisocaproate, which is reduced in the pathway to D-α-
hydroxyisocaproate (D-Hic) and D-Ala to generate the peptidyl
19
14−16
TE substrate as an N-acetyl cysteamine (NAC) thioester (7,
carrier protein (PCP)-linked D-Hic-D-Ala depsipeptide.
L-Hiv-L-Val-D-Hic-D-Ala-SNAC) and the expected NAC
substrate based on valinomycin homology (12, D-Hiv-D-Ala-
L-Hiv-L-Val-SNAC) were synthesized. For 7 (Scheme 1), a
Mitsunobu reaction was used to link Boc-L-Val to allyl L-α-
CesB uses α-ketoisovalerate, reduced in the pathway to L-α-
hydroxyisovalerate (L-Hiv) and L-Val and in conjunction with
14,15
CesA generates cereulide.
In the biosynthesis of the related
ionophore valinomycin, 2, Vlm1 generates a PCP-linked D-
Hiv-D-Val, which is condensed with L-lactate and then L-Val to
14,17
Received: March 26, 2020
Published: June 10, 2020
generate a PCP-linked D-Hiv-D-Val-L-Lac-L-Val.
Finally the
tetradepsipeptide is trimerized and macrocyclized by the C-
terminal thioesterase (TE) domain of Vlm2 to generate
1
8
valinomycin. Recent work suggests that despite the similarity
between the structures of cereulide and valinomycin and their
biosynthetic gene clusters, cereulide is produced via a different
©
2020 American Chemical Society and
American Society of Pharmacognosy
https://dx.doi.org/10.1021/acs.jnatprod.0c00333
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Figure 1. Two proposed pathways for the biosynthesis of cereulide by CesA and CesB. (A) This mechanism for cereulide formation is consistent
with the expected activity of all the catalytic domains in CesA and CesB, generating a D-Hic-D-Ala-L-Hiv-L-Val-PCP bound intermediate on CesB
that must be oligomerized and cyclized by the TE. (B) This proposed mechanism invokes a noncanonical use of the C* domain from CesA. Unlike
mechanism A, mechanism B leads to the formation of a L-Hiv-L-Val-D-Hic-D-Ala PCP bound intermediate on CesA that must be oligomerized and
19
cyclized by the TE. Note: Linker regions between domains are not to scale. The extended distance between the PCP and TE domains in CesB is
for clarity.
hydroxycaproate to generate the D-Hic-containing depsidipep-
tide. Deprotection of the allyl ester with Pd(PPh ) and
of 1, confirming its production is TE-dependent. This result
clearly demonstrates that the Ces TE is capable of
oligomerizing and macrocyclizing the expected tetradepsipep-
tide substrate derived from canonical NRPS activity, analogous
to valinomycin biosynthesis.
Kinetic characterization of Ces TE activity with 12 as
3
4
coupling with D-Ala-SNAC generated the depsitripeptide.
Finally Boc deprotection and coupling with L-Hiv furnished
the tetradepsipeptide substrate 7. Synthesis of 12 began with
coupling Boc-D-Ala and allyl L-α-hydroxyisovalerate to form
the ester linkage. From this intermediate the synthesis
mirrored the route to access 7, with allyl deprotection followed
by coupling to L-Val-SNAC, Boc deprotection, and coupling
with D-Hic to afford 12. The SNAC thioester serves to activate
the substrate for loading on the active site serine of the TE and
18
monitored by thiol release using Elman’s reagent provided an
−
1
−1
observed k /K = 0.28 ± 0.02 M s . This is comparable,
cat
M
though on the low end, to specificity constants seen for other
2
4
25
characterized TE domains, such as the epothilone and 6-
24
deoxyethythronolide B macrocyclizing TEs. The Ces TE
23
mimic the phosphopantetheine arm of the PCP domain.
could not be saturated even at 4 mM 12, preventing kcat and
The gene encoding the C-terminal TE domain from cesB
was synthesized and cloned into an expression vector under
the inducible T7 promoter. Ces TE was overexpressed in E. coli
BL21 and purified to homogeneity by metal affinity
chromatography. Treatment of Ces TE with 7 in 50 mM
Tris buffer pH 7.4 led to no detectable formation of cereulide
by LCMS (Figure 2). No oligomeric structures or hydrolysis of
KM from being independently determined. The K , which
M
based on our kinetic characterization is >4 mM, is significantly
higher than other TEs responsible for oligomerization and
macrocyclization of nonribosomal peptides. For example, the
tyrocidine TE (TycC TE) and the gramicidin TE (GrsB TE),
which both dimerize D-Phe-L-Pro-L-Val-L-Orn-L-Leu-SNAC
and cyclize the product to generate gramicidin S, show K ’s
M
26,27
7
was detected. This is consistent with Ces TE being unable to
in the range of 0.3−0.5 mM.
We speculate that in order to
load 7. In contrast, incubation of 12 with Ces TE generated
cereulide, 1, as well as dimeric, trimeric, and tetrameric SNAC
products, 13, 14, and 15, respectively (Figure 3). Both the no
enzyme and the boiled enzyme controls showed no production
effectively catalyze oligomerization and macrocyclization, these
TEs likely possess enhanced affinity for oligomerized substrates
over monomeric substrates as the oligomers have higher
structural homology to the macrocyclization substrates and
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Scheme 1. Synthesis of SNAC Substrates 7 and 12
Figure 2. LCMS traces of incubation of synthetic tetradesipeptide N-acetyl cysteamine substrates with CesB TE. (A) Negative control. Boiled CesB
TE (15 μM) with 1 mM 7 in 50 mM Tris pH 7.4, 14 h 22 °C. (B) CesB TE (15 μM) with 1 mM 7 in 50 mM Tris pH 7.4 for 14 h at 22 °C. No
cereulide or oligomeric product is formed. (C) Negative control. Boiled CesB TE (15 μM) with 1 mM 12 in 50 mM Tris pH 7.4, 14 h 22 °C. (D)
CesB TE (15 μM) with 1 mM 12 in 50 mM Tris pH 7.4 for 14 h at 22 °C. Cereulide (1) as well as dimeric, trimeric, and tetrameric N-acetyl
cysteamine thioester products (13, 14, and 15, Figure 3) are formed. All traces are ion extraction for m/z = 504, 888, 1272, 1658, and 1170, which
+
+
+
correspond to the [M + H] for 7/12, 13, 14, and 15, and the [M + NH ] for 1, respectively. *12 hydrolysis product, m/z = 403 [M + H] .
4
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Figure 3. Structures of cereulide oligomers and additional SNAC substrates.
Figure 4. LCMS traces of incubation of two different synthetic tetradesipeptide N-acetyl cysteamine substrates with CesB TE. (A) CesB TE (15
μM) with 2 mM 12 and 1 mM 16 in in 50 mM Tris pH 7.4, 14 h at 22 °C. The expected natural products 1 and 2 were formed as well as two
hybrid natural products comprising two cereulide monomers and one valinomycin monomer (cyclo-Cer -Vlm) and the other of two valinomycin
2
monomers and one cereulide monomer (cyclco-Cer-Vlm ). A boiled enzyme control showed no product formation. (B) CesB TE (15 μM) with 2
2
mM 12 and 1 mM 17 in in 50 mM Tris pH 7.4, 14 h at 22 °C. Cereulide and a complex mixture of oligomeric-SNAC compounds were generated.
+
A boiled enzyme control generated no oligomeric or cyclic products. Traces are ion extraction for [M + Na] ions with m/z = 526 (12), 512 (16),
+
and 498 (17), [M + H] ions with m/z = 889 (13), 875 (Cer-Vlm-SNAC), 861 (Cer-Hyb-SNAC), 1273 (14), 1259 (Cer -Vlm-SNAC), 1245
2
+
(
(
Cer-Vlm -SNAC and Cer -Hyb-SNAC), 1217 (Cer-Hyb -SNAC), and 1658 (15), and [M + NH ] ions with m/z = 1170 (1), 1128 (2), 1156
cyclo-Cer -Vlm), 1132 (Cyclo-Cer-Vlm ), 1142 (cyclo-Cer -Hyb), and 1114 (cyclo-Cer-Hyb ).
2
2
2
4
2
2
2
2
thus can be stabilized by more of the enzyme−substrate
interactions involved in macrocyclization activity. In the case of
Ces TE, we expect higher affinity for the more mature
trimerized intermediate over the dimerized intermediate and
monomeric substrate. This is consistent with the observation
that the dimeric and trimeric SNAC intermediates do not
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Article
accumulate to appreciable levels in the biochemical assays but
are rather processed to products preferentially over the SNAC-
activated monomer. Unlike the GrsB TE, which catalyzes
dimerization and macrocyclization, Ces TE must trimerize and
misincorporation of α-ketoisocaproate in place of α-ketoiso-
valerate by A1 of CesB, α-ketoisovalerate in place of α-
ketoisocaproate by A1 of CesA, D-Ser in place of D-Ala by A2
of CesA, and L-Ala in place of L-Val by A2 of CesB,
respectively.
macrocyclize. This is also consistent with a higher K for the
M
monomeric substrate of Ces TE than the monomeric substrate
of GrsB TE.
The unexpected linear tetra-, hexa-, octa-, deca-, and
dodecadepsipeptide esters isolated from alcoholic extracts of
cereulide-producing strains were initially proposed to be due to
alcoholic hydrolysis of PCP- and TE-linked biosynthetic
Treatment of the Ces TE with the related tetradepsipeptide
substrate from the valinomycin biosynthetic pathway (16, D-
Hiv-D-Val-L-Lac-L-Val-SNAC) resulted in very little processing
of the substrate (Figure S3D). While trace formation of
valinomycin, 2, and the trimeric intermediate is observable, the
main product is the dimeric intermediate. Dimer formation
shows that 16 is loaded onto the active site Ser of Ces TE.
Attack by a second equivalent of 16, which is required for
formation of dimer and ultimately other higher order
structures including 2, occurs; however, the low processivity
of Ces TE with this substrate suggests that attack by the
second equivalent of substrate may be slow. Treatment of Ces
TE with a mixture of 12 and 16 (Figure 4A) results in the
formation of cereulide (1) and valinomycin (2) as well as
hybrid cereulide−valinomycin macrocycles. Based on our data
both the cereulide and valinomycin SNAC monomers can be
loaded onto the active site Ser. We propose that a second
equivalent of the cereulide monomer 12 is more effective than
the valinomycin monomer 16 at acting as a substrate to effect
off-loading for formation of dimeric and trimeric products.
Following this mechanism, the hybrid cereulide−valinomycin
product is generated from valinomycin monomer 16 loading
onto Ces TE followed by a cereulide monomer 12 effecting
off-loading via transesterification to make the activated
valinomycin−cereulide dimer (D-Hiv-D-Val-L-Lac-L-Val-D-Hiv-
D-Ala-L-Hiv-L-Val-SNAC). This substrate is then loaded back
on the Ces TE and transesterified with the cereulide monomer
19
intermediates. To rationalize hexa- and decadepsipeptide
formation, it was proposed that noncanonical C domain
activity could be occurring to couple dipeptide products
1
9
produced by CesA and CesB. On the basis of our
biochemical characterization of Ces TE, we suggest an
alternative hypothesis fully consistent with the data and the
canonical activity of C domains. In biochemical treatment of
Ces TE with the native SNAC-activated substrate 12, we
observe formation of linear SNAC-activated tetramer (hexde-
cadepsipeptide). This occurs when the TE-O-trimer inter-
mediate undergoes attack by another equivalent of monomer
12 before macrocyclization can occur. In our assays the linear
activated tetramer is never observed to undergo macro-
cyclization. Thus, in the producing organism if the TE
elongates the substrate beyond trimer, macrocyclization cannot
occur and the tetramer will be either further elongated or
hydrolyzed off. Unlike cereulide, these extra-long linear
oligomers should be sensitive to alcoholic hydrolysis and
readily generate the esters observed. Significantly, this
mechanism provides a route to produce multiple equivalents
of the linear esters, rather than being limited to between one
and three equivalents relative to the amounts of CesA and
CesB, as would be required if the esters were formed by
alcoholic hydrolysis of the PCP- and TE-linked intermediates.
Thus, in summary this work demonstrates that cereulide is
generated highly analogously to valinomycin where the TE
mediates trimerization and macrocyclization of the expected
tetradepsipeptide substrate based on canonical activity of the
NRPS domains present in CesA and CesB. In addition the Ces
TE is selective, in our biochemical assays, for the nucleophile
that hydrolyzes the depsipeptidyl-TE intermediates. This can
be used to access hybrid cereulide macrocycles, with a single
non-native monomer incorporated into the final macrocycle.
Lastly we propose a mechanism consistent with all data to
account for the formation of an unexpected linear ester from
cereulide-producing strains, which eliminates the need to
resort to noncanonical activity of the CesB C domain.
12, generating the linear hybrid trimer with two cereulide
monomers and one valinomycin monomer (Cer -Vlm-SNAC).
2
Subsequent Ces TE loading and macrocyclization then gives
the hybrid product. Because the valinomycin monomer 16 is
not as effective a substrate to effect off-loading, misincorpora-
tion is limited, as clearly seen in the ratio of trimeric Cer3-
SNAC versus Cer -Vlm-SNAC products (Figure 4A).
2
On the basis of this working model, we investigated a hybrid
cereulide−valinomycin hybrid monomer with the expectation
that this substrate would load on the Ces TE but not be able to
act as an off-loading substrate to make higher order oligomers.
The hybrid tetradepsipeptide substrate, D-Hic-D-Ala-L-Lac-L-
Val-SNAC, 17 (Scheme S1), was synthesized. Treatment of
Ces TE with 17 led to minimal dimer formation and no
macrocycle formation, consistent with our working model
EXPERIMENTAL SECTION
■
General Experimental Procedures. All solvents were purchased
from Fisher Scientific. All reagents were purchased at the highest
available purity and used without further purification. EDC was
purchased from Oakwood Chemical (Estill, SC, USA), HOBt was
purchased from GL Biochem (Shanghai, PRC), and all other
chemicals were purchased from Sigma-Aldrich (Oakville, ON,
Canada). All reactions were carried out using dry solvents under a
nitrogen atmosphere unless otherwise noted. NMR spectroscopy was
performed with either a Bruker Avance II operating at 400 MHz for
(Figure S3B). Treatment of Ces TE with a combination of 12
and 17 generated both cereulide and the hybrid product
containing two cereulide monomers and one hybrid monomer
from 17, also fully consistent with our model (Figure 4B).
Our data and working model for Ces TE oligomerizing and
macrocyclizing activity are consistent with the mechanism of
formation for the closely related ionophore natural product
1
8
valinomycin and provide a mechanism for the biosynthesis of
the isocereulides, which are produced along with cereulide in
1
13
H and 100 MHz for C spectra or a Bruker Avance III with a
1
13
2
0−22
cryoprobe operating at 600 MHz for H and 150 MHz for
C
trace amounts.
The ability of Ces TE to cyclize the two
spectra. LCMS analysis was carried out using a Shimadzu LC20A
HPLC coupled with a Shimadzu LCMS2020 single quad mass
spectrometer with an ESI source. High-resolution mass spectroscopy
(HRMS) was conducted on a Micromass Q-TOF I (John L. Holmes
Mass Spectroscopy Facility, Ottawa, ON, Canada).
non-native cereulide−valinomycin hybrids above clearly
indicates that some degree of non-native side chain
composition can be accommodated by the TE. Thus,
isocereulides A, B, C, and D are generated by the single
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Boc-L-Val-D-Hic Allyl Ester (5). In a round-bottom flask, 453 mg
of Boc-L-Val (2.08 mmol, 1.2 equiv) 172 mg of L-Hic allyl ester (1.74
6.6 Hz, 1H), 3.39−3.32 (m, 1H), 3.17 (dt, J = 13.4, 6.1 Hz, 1H),
2.16−2.07 (m, 2H), 1.88−1.70 (m, 3H), 1.48 (d, J = 7.3 Hz, 3H),
1.08 (d, J = 6.7 Hz, 3H), 1.04 (d, J = 3.1 Hz, 3H), 1.03 (d, J = 2.8 Hz,
3H), 0.97 (d, J = 6.3 Hz, 3H), 0.94 (d, J = 6.2 Hz, 3H), 0.90 (d, J =
mmol, 1 equiv), and 915 mg of triphenylphosphine (PPh , 3.49 mmol,
3
2
equiv) were dissolved in 18 mL of dry THF. The stirring solution
1
3
was cooled to 0 °C on an ice bath for 10 min. To this was added 685
μL of diisopropyl azodicarboxylate (DIAD, 705 mg, 3.49 mmol, 2
equiv) dropwise, and the reaction was stirred on ice for an additional
hour, then allowed to warm to ambient temperature and stirred
overnight. At completion the reaction was concentrated and the title
6.9 Hz, 3H); C NMR (150 MHz, CDCl ) δ 201.79, 175.88, 172.10,
3
171.54, 171.13, 76.11, 72.94, 59.13, 55.02, 40.98, 40.29, 38.90, 31.82,
29.57, 28.58, 24.48, 23.33, 23.04, 20.86, 19.06, 19.03, 18.95, 17.00,
16.09; HRMS (ESI+) exact mass calculated for C H N NaO S
2
3
41
3
7
526.2563, found 526.2551.
compound (550 mg, 85%) was purified by silica column
Boc-D-Ala-L-Hiv Allyl Ester (10). In a round-bottom flask, 200
mg of L-2-hydroxyisovalerate allyl ester (1.26 mmol, 1 equiv) and 264
mg of Boc-D-Ala (1.39 mmol, 1.1 equiv) were dissolved in 6.5 mL of
CH Cl . To this solution were added 155 mg of 4-dimethylaminopyr-
1
chromatography (gradient 10% to 15% EtOAc in hexanes).
H
NMR (400 MHz, CDCl ) δ 5.87 (ddt, J = 17.2, 10.4, 5.8 Hz, 1H),
3
.30 (dd, J = 17.2, 1.4 Hz, 1H), 5.23 (dd, J = 10.4, 1.1 Hz, 1H), 5.05
2
2
idine (DMAP, 1.26 mmol, 1 equiv) and 365 mg of 1-ethyl-3-(3-
(dimethylamino)propyl)carbodiimide hydrochloride (EDC, 1.9
mmol, 1.5 equiv) at ambient temperature. The resulting solution
was stirred for 14 h at ambient temperature. The reaction was
quenched with saturated NH Cl(aq), extracted 3× with CH Cl ,
0
.9 Hz, 2H), 4.29 (dd, J = 9.0, 4.6 Hz, 1H), 2.18 (dt, J = 12.5, 6.4 Hz,
H), 1.82 (tt, J = 15.5, 5.1 Hz, 1H), 1.78−1.67 (m, 1H), 1.68−1.59
1
(m, 1H), 1.42 (s, 9H), 0.97 (d, J = 6.8 Hz, 3H), 0.93 (d, J = 6.5 Hz,
1
3
3
1
3
H), 0.92−0.87 (m, 6H); C NMR (100 MHz, CDCl ) δ 171.83,
3
4
2
2
69.96, 155.51, 131.47, 118.84, 79.72, 71.57, 65.88, 58.64, 39.72,
1.25, 28.30, 24.53, 23.07, 21.28, 19.08, 17.38; HRMS (ESI+) exact
washed with NaHCO (aq), washed with brine, dried over Na SO ,
3
2
4
and concentrated. The title compound (368 mg, 88%) was purified by
1
mass calculated for C H NNaO 394.2200, found 394.2192.
silica column chromatography (20% EtOAc in hexanes). H NMR
2
9
33
6
Boc-L-Val-D-Hic-D-Ala-SNAC (6). In a round-bottom flask, 100
(400 MHz, CDCl ) δ 5.90 (ddt, J = 17.2, 10.4, 5.8 Hz, 1H), 5.33 (dq,
3
mg of 5 (0.27 mmol, 1 equiv) was dissolved in 2 mL of CH Cl under
J = 17.2, 1.5 Hz, 1H), 5.25 (ddd, J = 10.4, 2.4, 1.2 Hz, 1H), 5.05 (d, J
= 7.6 Hz, 1H), 4.68−4.59 (m, 2H), 4.49−4.36 (m, 1H), 2.36−2.21
(m, 1H), 1.44 (d, J = 6.0 Hz, 12H), 1.01 (d, J = 6.9 Hz, 3H), 0.98 (d,
2
2
a nitrogen atmosphere. To this solution were added 31 μL of
morpholine (30 mg, 0.35 mmol, 1.3 equiv) and 16 mg of Pd(PPh3)4
in a single portion. The reaction was stirred at ambient temperature
and monitored by TLC. At completion the reaction was quenched by
the addition of 10% aqueous HCl, the organic layer was removed, and
the remaining aqueous fraction was extracted 3× with CH Cl . The
1
3
J = 6.9 Hz, 3H); C NMR (100 MHz, CDCl ) δ 172.79, 168.91,
3
131.50, 118.97, 84.84, 65.80, 56.04, 30.15, 28.33, 27.92, 18.74, 18.66,
17.10; HRMS (ESI+) exact mass calculated for C H NNaO
6
1
6
27
352.1736, found 352.1736.
2
2
combined organic fractions were washed with brine, dried over
Boc-D-Ala-L-Hiv-L-Val-SNAC (11). In a round-bottom flask, 124
mg of 10 (0.37 mmol, 1 equiv) was dissolved in 2 mL of CH Cl
2
Na SO , and concentrated in a dry round-bottom flask. This
2
4
2
intermediate was used immediately in the subsequent reaction. To
under a nitrogen atmosphere. To this solution were added 43 μL of
morpholine (43 mg, 0.49 mmol, 1.3 equiv) and 30 mg of Pd(PPh3)4
in a single portion. The reaction was stirred at ambient temperature
and monitored by TLC. At completion the reaction was quenched by
the addition of 10% aqueous HCl, the organic layer was removed, and
the remaining aqueous fraction was extracted 3× with CH Cl . The
the flask were added 2 mL of CH Cl and 61 mg of D-Ala-SNAC (as a
2
2
HCl salt, 0.27 mmol, 1 equiv). To the resulting solution were added
40 μL of Hunig’s base (104 mg, 0.81 mmol, 3 equiv), 55 mg of
1
̈
HOBt (0.4 mmol, 1.5 equiv), and 78 mg of EDC (0.4 mmol, 1.5
equiv). The reaction was stirred under argon at ambient temperature
2
2
for 20 h. The reaction was quenched with saturated NH Cl(aq),
combined organic fractions were washed with brine, dried over
4
extracted 3× with CH Cl , washed with NaHCO (aq), then with
Na SO , and concentrated in a dry round-bottom flask. This
intermediate was used immediately in the subsequent reaction. To
2
2
3
2
4
brine, dried over Na SO , and concentrated. The title compound (124
2
4
mg, 91%) was partially purified by silica column chromatography
the flask were added 4 mL of CH Cl and 120 mg of L-Val-SNAC (as
2
2
1
(40% acetone in hexanes) and taken into the subsequent reaction. H
HCl salt, 0.37 mmol, 1 equiv). To the resulting solution were added
197 μL of Hunig’s base (146 mg, 1.12 mmol, 3 equiv), 77 mg of
NMR (400 MHz, CDCl ) δ 6.09 (s, 1H), 5.32 (t, J = 6.7 Hz, 1H),
̈
3
5
3
2
1
3
.06 (d, J = 6.2 Hz, 1H), 4.57−4.45 (m, 1H), 3.92 (t, J = 6.7 Hz, 1H),
.44−3.26 (m, 3H), 2.99 (td, J = 6.3, 3.6 Hz, 1H), 2.94 (t, J = 6.0 Hz,
H), 2.02 (tt, J = 13.5, 6.7 Hz, 1H), 1.92 (s, J = 2.4 Hz, 4H), 1.80−
.75 (m, 2H), 1.67 (dt, J = 20.2, 6.6 Hz, 1H), 1.42 (d, J = 2.7 Hz,
HOBt (0.56 mmol, 1.5 equiv), and 109 mg of EDC (0.56 mmol, 1.5
equiv). The reaction was stirred under argon at ambient temperature
for 20 h. The reaction was quenched with saturated NH Cl(aq),
4
extracted 3× with CH Cl , washed with NaHCO (aq), then with
2
2
3
1
3
H), 1.37 (s, 10H), 1.02−0.96 (m, 6H), 0.93−0.88 (m, 6H);
C
brine, dried over Na SO , and concentrated. The title compound (192
2 4
NMR (100 MHz, CDCl ) δ 201.33, 172.17, 170.77, 170.45, 156.23,
mg, 94%) was partially purified by silica column chromatography
3
1
8
2
0.67, 72.66, 60.17, 55.59, 40.19, 39.20, 30.02, 28.48, 28.32, 24.41,
3.29, 23.12, 21.17, 19.08, 18.66, 16.99.
(40% acetone in hexanes) and taken into the subsequent reaction. H
NMR (600 MHz, CDCl ) δ 7.15 (d, J = 8.6 Hz, 1H), 6.50 (d, J = 29.8
3
HO-L-Hiv-L-Val-D-Hic-D-Ala-SNAC (7). To a round-bottom flask
Hz, 1H), 5.47 (dd, J = 41.1, 5.9 Hz, 1H), 5.13 (t, J = 12.6 Hz, 1H),
4.42 (dd, J = 17.2, 9.8 Hz, 1H), 4.30−4.16 (m, 1H), 3.36−3.27 (m,
2H), 3.00−2.88 (m, 3H), 2.27 (td, J = 13.3, 6.6 Hz, 1H), 1.90 (d, J =
1.6 Hz, 4H), 1.40 (d, J = 7.2 Hz, 3H), 1.35 (s, 8H), 0.95−0.88 (m,
was added 100 mg of 6 (0.2 mmol, 1 equiv) in a minimal amount of
EtOAc and cooled to 0 °C. To this was added 2 mL of 4 M HCl in
dioxane (Sigma), and the reaction allowed to warm to ambient
temperature. The reaction was monitored by TLC, and at completion
all solvent was removed by rotary evaporation. The unpurified
intermediate was used immediately and was dissolved in 1 mL of
1
3
14H); C NMR (150 MHz, CDCl ) δ 199.98, 172.91, 170.67,
3
170.01, 155.58, 80.13, 78.28, 64.55, 49.77, 39.05, 30.14, 30.03, 28.41,
28.24, 23.00, 19.38, 18.79, 17.84, 17.43, 16.54.
CH Cl , and to this were added 105 μL of Hu
mmol, 3 equiv), 26 mg of L-α-hydroxyisovaleric acid (0.21 mmol, 1.1
̈
nig’s base (78 mg, 0.6
HO-D-Hic-D-Ala-L-Hiv-L-Val-SNAC (12). To a round-bottom flask
was added 55 mg of 11 (0.11 mmol, 1 equiv) in a minimal amount of
EtOAc and cooled to 0 °C. To this was added 1 mL of 4 M HCl in
dioxane (Sigma), and the reaction allowed to warm to ambient
temperature. The reaction was monitored by TLC, and at completion
all solvent was removed by rotary evaporation. The unpurified
intermediate was used immediately and was dissolved in 1 mL of
2
2
equiv), 40 mg of HOBt (0.3 mmol, 1.5 equiv), and 60 mg of EDC
0.3 mmol, 1.5 equiv). The reaction was stirred at ambient
temperature overnight and at completion was quenched with
saturated NH Cl(aq), extracted 5× with CH Cl , washed with
NaHCO (aq), then with brine, dried over Na SO , and concentrated.
(
4
2
2
3
2
4
The title compound (18 mg, 18%) was purified by silica column
chromatography (50% acetone in hexanes). H NMR (600 MHz,
CH Cl , and to this was added 60 μL of Hunig’s base (44 mg, 0.33
̈
2 2
1
mmol, 3 equiv), 17 mg of D-α-hydroxyisocaproic acid (0.12 mmol, 1.1
equiv), 19 mg of HOBt (0.16 mmol, 1.5 equiv), and 33 mg of EDC
(0.16 mmol, 1.5 equiv). The reaction was stirred at ambient
temperature overnight and at completion was quenched with
CDCl ) δ 7.77 (d, J = 8.1 Hz, 1H), 7.35 (d, J = 6.8 Hz, 1H), 6.16 (s,
3
1
H), 5.28 (dd, J = 10.6, 3.0 Hz, 1H), 4.62 (p, J = 7.4 Hz, 1H), 4.14
(dd, J = 9.0, 6.8 Hz, 1H), 3.92 (d, J = 3.3 Hz, 1H), 3.52 (td, J = 13.8,
1
995
https://dx.doi.org/10.1021/acs.jnatprod.0c00333
J. Nat. Prod. 2020, 83, 1990−1997

Journal of Natural Products
pubs.acs.org/jnp
Article
saturated NH Cl(aq), extracted 5× with CH Cl , washed with
Biology, University of Ottawa, Ottawa, ON K1N 6N5,
Canada; orcid.org/0000-0002-3259-3184; Phone: 613-
62-8900; Email: cboddy@uottawa.ca
4
2
2
NaHCO (aq), then with brine, dried over Na SO , and concentrated.
3
2
4
The title compound (32 mg, 56%) was purified by silica column
7
1
chromatography (50% acetone in hexanes). H NMR (400 MHz,
CDCl ) δ 7.28−7.25 (m, 1H), 7.22 (d, J = 9.1 Hz, 1H), 6.12 (s, 1H),
3
Author
5
4
3
1
1
.11−5.04 (m, 1H), 4.65 (s, 1H), 4.55 (dd, J = 13.9, 7.0 Hz, 1H),
.51−4.45 (m, 1H), 4.14 (d, J = 5.9 Hz, 1H), 3.49−3.35 (m, 2H),
.35−3.20 (m, 1H), 3.14−3.04 (m, 1H), 2.84 (dt, J = 7.1, 6.5 Hz,
H), 2.41−2.23 (m, 3H), 1.96 (s, 3H), 1.89−1.79 (m, 1H), 1.60−
Graham W. Heberlig − Department of Chemistry and
Biomolecular Sciences Centre for Chemical and Synthetic
Biology, University of Ottawa, Ottawa, ON K1N 6N5,
Canada; orcid.org/0000-0001-8439-274X
1
3
.46 (m, 3H), 1.44 (t, J = 6.6 Hz, 3H), 1.03−0.86 (m, 26H);
C
NMR (100 MHz, CDCl ) δ 199.88, 176.11, 172.67, 171.29, 169.99,
3
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jnatprod.0c00333
7
2
8.99, 70.45, 64.46, 48.18, 43.55, 38.72, 30.26, 29.94, 28.58, 24.50,
3.37, 23.09, 21.45, 19.48, 18.84, 18.05, 17.19, 16.74; HRMS (ESI+)
exact mass calculated for C H N NaO S 526.2563, found 526.2558.
Protein Expression and Purification. The CesB TE gene
2
3
41
3
7
Notes
(
UNIPROT Q20CI8 residues 2420-2681) was generated syntheti-
The authors declare no competing financial interest.
cally and cloned into pET21 (pGWH43). Expression conditions are
15
modified from Alonzo et al. Briefly, the CesB TE was expressed in E.
coli BL21 Rosetta grown in LB media supplemented with 100 μg/mL
ampicillin and 34 μg/mL chloramphenicol. Cells were induced at an
OD600 = 0.45 with 0.1 mM IPTG and further incubated at 16 °C for
ACKNOWLEDGMENTS
■
This work was funded by the Natural Sciences and Engineering
Research Council of Canada (NSERC).
1
6 h. Cells were harvested by centrifugation and resuspended in buffer
A (50 mM Tris pH 8.0, 300 mM NaCl, 10% v/v glycerol) and lysed
by sonication. The lysate was centrifuged at 10 000 rpm for 45 min,
and the clarified supernatant was incubated with 0.75 mL of Ni-NTA
superflow resin (QIAGEN, Valenica, CA, USA) at 4 °C for 1 h. The
resin was loaded into a column and first washed with buffer A
supplemented with 20 mM imidazole; then CesB TE was eluted with
buffer A supplemented with 150 mM imidazole. The purified protein
was exchanged into dialysis buffer (25 mM HEPES, 100 mM NaCl,
pH 8) and concentrated by centrifugation (Amicon 10 000 MWCO).
The concentrated protein was flash frozen and stored at −78 °C. Of
note, the original Alonzo et al. protocol calls for the addition of
TCEP; this was omitted from our preparation to facilitate the use of a
colormetric assay to monitor substrate loading. We observed more
variability in activity and lower stability of the Ces TE in the absence
of TCEP, indicating the enzyme may be redox sensitive. Protein
concentration was determined by the Bradford assay (Biorad).
Approximately 8 mg of purified protein was obtained per L of cell
culture.
REFERENCES
■
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(
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5
4
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ASSOCIATED CONTENT
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jnatprod.0c00333.
■
(
2
(
̈
*
M.; Nguyen, D. P.; Dornan, M. H.; Boddy, C. N.; Schmeing, T. M.;
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Additional synthetic procedures, 1H and 13C NMR
spectra, and kinetic characterization (PDF)
DOI: 10.1038/srep10637.
AUTHOR INFORMATION
Corresponding Author
Christopher N. Boddy − Department of Chemistry and
Biomolecular Sciences Centre for Chemical and Synthetic
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