Chemoenzymatic synthesis of cryptophycin anticancer agents by an ester bond-forming non-ribosomal peptide synthetase module

Article abstract of DOI:10.1021/ja204716f

Cryptophycins (Crp) are a group of cyanobacterial depsipeptides with activity against drug-resistant tumors. Although they have been shown to be promising, further efforts are required to return these highly potent compounds to the clinic through a new generation of analogues with improved medicinal properties. Herein, we report a chemosynthetic route relying on the multifunctional enzyme CrpD-M2 that incorporates a 2-hydroxy acid moiety (unit D) into Crp analogues. CrpD-M2 is a unique non-ribosomal peptide synthetase (NRPS) module comprised of condensation-adenylation-ketoreduction-thiolation (C-A-KR-T) domains. We interrogated A-domain 2-keto and 2-hydroxy acid activation and loading, and KR domain activity in the presence of NADPH and NADH. The resulting 2-hydroxy acid was elongated with three synthetic Crp chain elongation intermediate analogues through ester bond formation catalyzed by CrpD-M2 C domain. Finally, the enzyme-bound seco-Crp products were macrolactonized by the Crp thioesterase. Analysis of these sequential steps was enabled through LC-FTICR-MS of enzyme-bound intermediates and products. This novel chemoenzymatic synthesis of Crp involves four sequential catalytic steps leading to the incorporation of a 2-hydroxy acid moiety in the final chain elongation intermediate. The presented work constitutes the first example where a NRPS-embedded KR domain is employed for assembly of a fully elaborated natural product, and serves as a proof-of-principle for chemoenzymatic synthesis of new Crp analogues.

Full text of DOI:10.1021/ja204716f

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pubs.acs.org/JACS
Chemoenzymatic Synthesis of Cryptophycin Anticancer Agents by an
Ester Bond-Forming Non-ribosomal Peptide Synthetase Module
ꢀ
||
Yousong Ding,†,‡, ,# Christopher M. Rath,†,# Kyle L. Bolduc,†,‡ Kristina Hakansson,§ and
David H. Sherman*,†,‡,§,^
†Life Sciences Institute and Departments of ‡Medicinal Chemistry, §Chemistry, and ^Microbiology & Immunology,
University of Michigan, Ann Arbor, Michigan 48109, United States
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, United States
S Supporting Information
b
ABSTRACT: Cryptophycins (Crp) are a group of cyano-
bacterial depsipeptides with activity against drug-resistant
tumors. Although they have been shown to be promising,
further efforts are required to return these highly potent
compounds to the clinic through a new generation of
analogues with improved medicinal properties. Herein,
we report a chemosynthetic route relying on the multifunctional
enzyme CrpD-M2 that incorporates a 2-hydroxy acid moiety
(unit D) into Crp analogues. CrpD-M2 is a unique non-
ribosomal peptide synthetase (NRPS) module comprised
of condensationÀadenylationÀketoreductionÀthiolation
(C-A-KR-T) domains. We interrogated A-domain 2-keto
and 2-hydroxy acid activation and loading, and KR domain
activity in the presence of NADPH and NADH. The
resulting 2-hydroxy acid was elongated with three synthetic
Crp chain elongation intermediate analogues through ester
bond formation catalyzed by CrpD-M2 C domain. Finally, the
enzyme-bound seco-Crp products were macrolactonized by
the Crp thioesterase. Analysis of these sequential steps was
enabled through LC-FTICR-MS ofenzyme-boundintermedi-
ates and products. This novel chemoenzymatic synthesis of
Crp involves four sequential catalytic steps leading to the
incorporation of a 2-hydroxy acid moiety in the final chain
elongation intermediate. The presented work constitutes the
first example where a NRPS-embedded KR domain is em-
ployed for assembly of a fully elaborated natural product, and
serves as a proof-of-principle for chemoenzymatic synthesis of
new Crp analogues.
Figure 1. CrpD-M2 biosynthetic scheme. (A) Enzymes used in this
study, CrpD-M2 and Crp TE. Domains within the CrpD-M2 polypep-
tide are noted with squares, and the phosphopantetheinyl arm is denoted
by the linked SH group. (B) Reactions catalyzed by CrpD-M2- and Crp
TE-mediated cyclization. (C) Utilized SNAC-ABC analogues (3À5).8
(D) Cyclic Crp (6À8)11À13 and linear seco-Crp (9À11) generated by
CrpD-M2 and Crp TE:8 units A (red), B (dark red), C (green), and D
(blue).
approaches.6 The gene cluster is comprised of two type I polyketide
synthase (PKS) genes, crpA and crpB, two non-ribosomal peptide
synthetase (NRPS) genes, crpC and crpD, and four tailoring enzyme
genes, including a key P450 epoxidase (crpE). Previous studies from
this laboratory have demonstrated the feasibility and efficiency of
biocatalysts from this metabolic pathway to properly macrocyclize
and regio- and stereospecifically epoxidize Crp intermediates to
generate these natural products and novel analogues.6À8
CrpD is a bimodular NRPS involved in late-stage assembly of
the Crp chain elongation intermediate. Bioinformatic analysis
and precursor incorporation studies revealed that the substrate of
its first module is methyl-β-alanine, converted from L-aspartic acid
by CrpG, a β-methylaspartate-R-decarboxylase.6,9 These studies
also suggested that 2-ketoisocaproic acid (2KIC, 1) instead of
atural products have been widely applied to fight disease
N
and offer chemical scaffolds for development of new
analogues with improved/modified functions, achieved through
semisynthesis, total synthesis, or chemoenzymatic synthesis.1À3
Cryptophycins are potent anticancer agents at picomolar con-
centrations and exert their cytotoxic effects in both vinca
alkaloid- and taxol-resistant cancer cells that contribute to the
proliferation of drug-resistant tumors.4 Their clinical potential
and the synthetic challenges they pose have stimulated the
development of alternative strategies to provide suitable amounts
of material and new analogues with improved physiochemical
properties for clinical studies.5 The cryptophycin gene cluster
was recently elucidated and offers unique opportunities for
assembly of the drug and new analogues using chemoenzymatic
Received: May 23, 2011
Published: August 08, 2011
r
2011 American Chemical Society
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L-2-hydroxyisocaproic acid (L-2HIC, 2) was the substrate of
CrpD module 2 (CrpD-M2), which was incorporated into Crp
asunit D (Figure1).6 Severalnatural Crp analogues contain unit D
variations, including 3-methyl-2-hydroxyvalerate, 2-hydroxyvale-
rate, and 3-methyl-2-hydroxybutyrate (Figure S1).6 Altered
bioactivity of these analogues suggests the importance of the
unit D in anticancer activity, but limited efforts have been made
to query analogues carrying unnatural unit D structures.10
Four distinct steps are catalyzed by CrpD-M2 in Crp
biosynthesis (Figure 1). In step I, the free-acid extender unit
(e.g., 1) is activated by the CrpD-M2 adenylation (A) domain
to form the corresponding acyl-AMP. This intermediate is then
loaded onto the thiolation (T) domain active-site-bound phos-
phopantetheine arm through a transthioesterification reaction to
form the acyl enzyme intermediate in step II. In the presence of
reducing cofactors, the 2KIC enzyme intermediate is converted
stereoselectively to L-2HIC in step III by the unique ketoreduction
(2-KR) domain, based upon analysis of known products. The
L-2-hydroxy group in unit D is condensed with Crp unit ABC
biosynthetic intermediate, transferred from CrpD module 1 T
domain through formation of an atypical (in an NRPS module)
ester linkage by the condensation (C) domain (Figure 1, step IV).
In this report, four sequential steps were assessed to investigate the
unique incorporation of a 2-hydroxy acid subunit into Crp natural
products, and to probe the intrinsic substrate flexibility and synthetic
potential of CrpD-M2. Chemoenzymatic synthesis of Crps 3,11 24,12
and 5113 (6À8) with CrpD-M2 and Crp thioesterase (TE)
(Figure 1, step V) serves as a proof-of-principle for efforts to generate
Crp analogues with unnatural structures of units C and D through
synthetic and biochemical methods.
A CrpD-M2 expression construct was generated by amplifying
a DNA fragment consisting of C, A, KR, and T domains by PCR,
and cloning into the BamHI and XhoI sites of pET28a. This
construct was overexpressed in Escherichia coli BAP1 strain for
production of phosphopantetheinylated proteins.14 The N-terminal
His-tagged protein was purified with Ni-NTA resin to ∼80% purity
(Figure S2). The integrity of the purified protein was verified by
peptide map fingerprinting and FTICR-MS (Figure S3A, Table S1).
The CrpD-M2 T domain active site was also identified, and proper
post-translational modification of the T domain active-site Ser was
verified by MS/MS (Figure S3B, Table S2).15
Bioinformatic analysis can reliably predict NRPS A domain
specificity on the basis of binding pocket residue motifs.16 The
conserved Asp235 involved in ionic interaction with the amino
group of the substrate amino acid is replaced by Val235 in CrpD-M2
A domain (Table S3). Similar to unit D of Crp, a 2-hydroxy acid
moiety is also involved in at least nine other natural products:
bacillaene,17 barbamide,18 bassianolide,19 beauvericin,20 cereulide,21
enniatin,22 hectochlorin,23 kutzneride,24 and valinomycin.21 A similar
replacement of Asp235 is conserved across all A domains responsible
for incorporation of 2-hydroxy acid into these natural products
(Table S3), predicting that the CrpD-M2 A domain prefers
2-hydroxy and/or 2-keto acids.
Figure 2. Examination of CrpD-M2 A domain substrate specificity
using the radio-PPi exchange assay. (A) Relative activity of the A domain
normalized to 2. (B) Extender units investigated in this assay.
selectivity for the natural substrate 2-oxovalerate (15) was observed.
CrpD-M2 specificity to two other natural unit D fragments,
3-methyl-2-oxovalerate (13) and 3-methyl-2-oxobutyrate (16),
was decreased ∼50% and 90%, respectively. This result, along
with the observed weak activation of unnatural substrates
2-oxobutyrate (17) and phenyl pyruvate (19), suggests that
the size and bulk of substrate side chains are important in
CrpD-M2 A domain recognition.
In an effort to assess further the flexibility of the CrpD-M2 A
domain, we tested 2-keto-γ-(methylthio)butyrate (AKGB, 12)
as a substrate. Its effective activation (Figure 2) demonstrates the
potential of native CrpD-M2 in producing novel Crp analogues
with an altered unit D moiety. Moreover, the weak activation of
4-methylvalerate (18) by CrpD-M2 A domain reveals the
importance of the R-position functional group in enzyme
recognition. Therefore, CrpD-M2 A domain has relatively re-
laxed substrate specificity and exhibits a similar selectivity toward
2-keto and 2-hydroxy acids (Figure 1, step I). ATP-PPi exchange
assays have been applied to examine substrate preference of A
domains in the biosynthesis of bacillaene,17 barbamide,18 cereulide,21
enniatin,22 hectochlorin,23 and kutzneride.24 Only HctEIVA from the
hectochlorin pathway displayed a similar selectivity to 2-keto and
2-hydroxy acids compared to the CrpD-M2 A domain.23
We next monitored substrate loading directly on the T-do-
main of CrpD-M2 (Figure 1, step II).25À28 Enzyme reactions
were terminated by proteolysis with trypsin, and the active-site
peptides bound with extender units were separated and analyzed
by LC-FTICR-MS and LC-IT-MS/MS (Tables S4 and S5,
Figures S26ÀS35). As shown in Figure S28A,C, T domain
active-site peptides bound with L-2HIC and 2-KIC showed
masses of 4116.21 and 4114.15, respectively, at a charge state
of 4+, matching theoretical values within (30 ppm (Table S4).
D-2HIC (20) was also loaded on the CrpD-M2 T domain active
site, as shown by the observed mass of 4116.18 (Figure S28B).
Since only L-2HIC-containing Crp analogues have been iso-
lated and characterized from the cyanobacteriumNostoc sp. ATCC
53789, we suspect that factors other than A domain selectivity,
such as substrate availability and/or downstream processing,
determine the final outcome. It is well-known that 2-keto
acids are indispensible intermediates in amino acid biosynthesis,
such as 1, 13, and 16 in the biosynthesis of leucine, isoleucine,
and valine, respectively. The availability of free 2-hydroxy acid may
be ascribed to a pathway-specific enzyme. For example, A
domains for the biosynthesis of bassianolide,19 beauvericin,20
and enniatin22 are specific to D-2-hydroxy isovalerate (D-2HIV),
and a pathway-specific NADPH-dependent reductase is essential
The well-established ATP-PPi exchange assay was employed to
biochemically determine the substrate specificity of the CrpD-M2
A domain with 10 acyl acid substrates (Figure 2). CrpD-M2
activated 2-KIC (1) about 20 times more efficiently than its
cognate amino acid L-leucine (14), consistent with bioinformatic
prediction and previous feeding experiments.6 Interestingly,
L-2HIC (2), which was not incorporated into the Crp final
structure during an in vivo precursor incorporation study,6 was
among the best substrates in the assay. A similar high level of
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for stereospecific reduction of 2-keto isovalerate.19,20,29 Since a
corresponding reducing enzyme is not encoded in the Crp gene
cluster,6 it is possible that 2KIC is the native substrate of CrpD-M2
A domain.
The loaded 2KIC is proposed to be reduced to L-2HIC by the
R-KR domain of CrpD-M2 (Figure 1, step III). This type of KR
domain is also embedded in NRPS modules of cereulide,21
hectochlorin,23 kutzneride,24 and valinomycin,21 and its R-keto
reduction activity and stereospecificity were biochemically con-
firmed in the cereulide system.21 Bioinformatic analysis indicates
that CrpD-M2 KR domain is grouped with these NRPS KR
domains and is phylogenetically distinct from the more thor-
oughly studied PKS β-KR domains (Figure S4A). The stereo-
chemical outcome of PKS β-KR domains can be predicted on the
basis of conserved amino acid sequence motifs.30À32 A similar
analysis was performed to predict 2-hydroxy chirality introduced
by KR domains from CrpD-M2, CesA, and CesB (Figure S4B).
The corresponding KR domain in CesA and CesB produces
L-2HIC and D-2HIV, respectively. However, none of these enzymes
contain the conserved motifs expected for either type A or type B
β-KR polypetides. Given the phylogenetic distance and posi-
tional difference of the keto group in their respective substrates
between PKS β-KR domains and NRPS R-KR domains, this
sequence divergence was not unexpected. The first KR domain in
the hybrid PKS/NRPS PksJ involved in bacillaene biosynthesis
catalyzes both β- and R-ketone reduction, with 10-fold preference
toward the former reaction.17 Based on structural analysis, the
product of β-ketone reduction was consistent with the KR A-type
sequence motif. However, as with NRPS R-KR domains, the PksJ
KR cannot be grouped as type A or B (Figure S4B). Therefore, it is
not possible to classify NRPS R-KR domains using current PKS β-
KR domain bioinformatic tools,30À32 and product chirality must be
determined experimentally (Figure S5). In the CrpD-M2 KR
reaction, the S-configuration (e.g., L-2HIC) is expected since all
natural Crp contain this stereochemistry.
After loading of 2KIC (Figure S28C), addition of reducing
cofactors (e.g., NAD(P)H) to the CrpD-M2 reaction resulted in
a mass shift observed by FTICR-MS (Figure S28D,E). The
increase of 0.5 m/z unit (a 2-Da shift in the deconvoluted mass)
is consistent with 2KIC conversion to 2HIC as the product of the
R-KR reaction. Both NADH and NADPH operated within a
similar (1À2-fold) efficiency as hydride donors, based on peak
abundance (Figure S28D,E). Future structural analysis of the
CrpD-M2 R-KR domain may contribute to understanding
stereochemical control in this enzyme subclass.
Next, the ability of CrpD-M2 to form the seco-Crp intermedi-
ate was investigated using synthetic SNAC-ABC chain elonga-
tion intermediates 3À5 as the starting point (Figure 1B). The
synthetic scheme followed our previously established route,33
and the SNAC-ABC intermediates were confirmed using
NMR and high-resolution mass spectrometry (Supporting In-
formation). The intermediate with the monomethylated unit C
(3-amino-2(R)-methylpropionyl, 3) was then combined with
CrpD-M2 and 2. The C domain of CrpD-M2 is proposed to
catalyze formation of an ester bond with the 2-hydroxy group of
unit D as the nucleophile (Figure 1, step IV). Formation of the ester
bond was confirmed by detecting the reaction products released
from CrpD-M2 T domain following addition of the excised Crp TE
(Figure 1, step V; Figure 3A,B). Both cyclic 6 (Figure 3A) and linear
9 (Figure 3B) were observed in the extracted ion chromatograms
(EICs). Previously, a didomain NRPS (T-C), Fum14p, was shown
to form a CÀO bond in the biosynthesis of the fungal mycotoxin
Figure 3. FTICR-MS analysis of Crp products from the reaction of unit
C monomethyl chain elongation intermediate 3 with L/D-2HIC, ATP,
CrpD-M2, and Crp TE. EICs are presented at (15 ppm as time versus
absolute signal. Inset mass spectra are time averaged over the 1 min
elution window corresponding to the asterisk in the EIC. Inset mass
spectra are presented as m/z versus absolute signal. Monoisotopic mass
MH+ and the experimental mass error in ppm are reported. Reactions
with L-2HIC and D-2HIC monitor the formation of cyclic (A,C) and
linear products (B,D). No enzyme control reactions for cyclic (E) and
linear product formation (F) are provided.
fumonisin.34 The only other C domain shown to catalyze
CÀO bond formation is a discrete enzyme, SgcC5, in C-1027
biosynthesis.35 In both cases, donor substrates are tethered to T
domains, while the nucleophile (ÀOH) is from a small-molecule
extender unit. This study represents the first example of a C domain in
a complete NRPS module that catalyzes the incorporation of non-
amino acid moieties as an ester synthase.
Assuming both cyclic and linear products share similar ionization
efficiency in the positive ion mode, we conclude that cyclic 6 was
formed as the predominant species over linear 9(Figure3A,B). This
result demonstrates chemoenzymatic synthesis of 6 through five
catalytic steps (Figure 1, steps IÀV). Formation of 6 was con-
firmed by MS/MS (Figure S6A) and LC co-elution with an authentic
standard (Figure S7). Observed isotope patterns of cyclic product
andMS2 fragmentswerealsoconsistentwith the +237Clshiftfrom
unit B.
Similarly, the synthetic chain elongation intermediate with
desmethyl (3-amino-propionyl, 4) or gem-dimethyl (3-amino-
dimethylpropionyl, 5) unit C and L-2HIC (2) were used as
substrates in the CrpD-M2 reaction, along with Crp TE for off-
loading/cyclization. Both Crp 24 (7) and Crp 51 (8) were
generated by this chemoenzymatic route, indicating the versati-
lity of the CrpD-M2 C domain and Crp TE (Figure S8A,E). The
corresponding hydrolyzed linear products (10, 11) were also
observed (Figure S8B,F). Assuming similar ionization efficiency
between linear and cyclic products, linear 10 was the predomi-
nant species compared to cyclic 7 (Figure S8A,B), and cyclic 8
was present in similar abundance compared to its linear counter-
part, 11 (Figure S8E,F). Using SNAC-ABCD analogues as native
substrate mimics, Beck et al. investigated the impact of unit C
methylation on Crp TE-mediated macrocylization.8 The ana-
logue bearing the 3-amino-dimethylpropionyl group was found
to produce more cyclic product (6:1) than the one with a
3-amino-propionyl moiety (5:1) but less than the one with a
3-amino-2(R)-methylpropionyl group (10:1). We found a similar
order of unit C reactivity when the native T domain-bound substrates
generated by CrpD-M2 were supplied to CrpD TE.
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In an effort to further probe the flexibility of CrpD-M2 to
substrate stereochemistry, D-2HIC (20) was substituted for
2 in the chemoenzymatic reaction. Following loading of chain
elongation intermediate 3, no cyclic depsipeptide product was
detected (Figure 3C), but a small amount of linear product was
formed (Figure 3D), suggesting that the C domain of CrpD-M2
was able to recognize the stereoisomer of its acceptor substrate
and catalyze ester bond formation with its donor ABC chain
elongation intermediate, albeit at a lower level. It also indicated
that the natural outcome of the unit D KR reaction was L-2HIC,
followed by esterification and macrocyclization. The failure to
macrocyclize ABC-D-2HIC substrates suggests that the “gate-
keepers” for unit D stereochemical selection are the CrpD-M2 R-
KR domain and Crp TE rather than its C or A domain.
In summary, non-amino acid extender subunits selected and
processed by NRPS enzymes have been found in a handful of
natural products isolated from bacteria and fungi. However, a
complete mechanism for understanding their incorporation has
not been established. In this study, CrpD-M2 coupled with Crp
TE was used to further explore unit D selection, loading, reduction,
elongation through an ester bond, and final product formation.
FTICR-MS was employed to assess these five sequential biochemical
reactions that occurred through a complete NRPS module including
A, C, KR, T, and TE domains. This is also the first study in which a
NRPS bearing an embedded KR domain was used to directly
generate bioactive compounds from fully elaborated natural and
unnatural chain elongation intermediates to provide cyclic crypto-
phycins 3, 24, and 51. Thus, CrpD-M2 as a chemoenzymatic reagent
or as part of a microbial-derived production strategy has the potential
to generate novel Crp analogues. Future efforts will focus on new
analogues with improved physicochemical properties that may be
beneficial as clinical agents for treatment of malignant diseases.
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’ ASSOCIATED CONTENT
S
Supporting Information. Methods, figures, and tables.
b
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’ AUTHOR INFORMATION
Corresponding Author
davidhs@umich.edu
Author Contributions
#These authors contributed equally to this work.
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Y.D. was supported by a Rackham Predoctoral Fellowship.
This work was supported by NIH grant CA108874, GM076477
and the HansW. VahlteichProfessorship(toD.H.S.).WorkinK.H.’s
laboratory is supported by an NSF Career Award (CHE-05-47699).
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