Chemo-enzymatic Total Syntheses of Jorunnamycin A, Saframycin A, and N-Fmoc Saframycin Y3

Article abstract of DOI:10.1021/jacs.8b07161

The antitumor tetrahydroisoquinoline (THIQ) alkaloids share a common pentacyclic scaffold that is biosynthesized by nonribosomal peptide synthetases involving unique enzymatic Pictet-Spengler cyclizations. Herein we report concise and divergent chemo-enzymatic total syntheses of THIQ alkaloids by merging precise chemical synthesis with in vitro engineered biosynthesis. A recombinant enzyme SfmC responsible for the biosynthesis of saframycin A was adapted for the assembly of these natural products and their derivatives, by optimizing designer substrates compatible with SfmC through chemical synthesis. The appropriately functionalized pentacyclic skeleton were efficiently synthesized by streamlining the linkage between SfmC-catalyzed multistep enzymatic conversions and chemical manipulations of the intermediates to install aminonitrile and N-methyl groups. This approach allowed rapid access to the elaborated pentacyclic skeleton in a single day starting from two simple synthetic substrates without isolation of the intermediates. Further functional group manipulations allowed operationally simple and expeditious syntheses of jorunnamycin A, saframycin A, and N-Fmoc saframycin Y3 that could be versatile and common precursors for the artificial production of other antitumor THIQ alkaloids and their variants.

Full text of DOI:10.1021/jacs.8b07161

Communication
pubs.acs.org/JACS
Cite This: J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
Chemo-enzymatic Total Syntheses of Jorunnamycin A, Saframycin
A, and N‑Fmoc Saframycin Y3
Ryo Tanifuji,† Kento Koketsu,§ Michiko Takakura,§ Ryutaro Asano,∥ Atsushi Minami,§
Hideaki Oikawa,*,§ and Hiroki Oguri*,†
†Department of Applied Chemistry, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16
Nakacho, Koganei, Tokyo 184-8588, Japan
§Division of Chemistry, Graduate School of Science, Hokkaido University, North 10 West 8, Sapporo 060-0810, Japan
∥Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology,
2-24-16 Nakacho, Koganei, Tokyo 184-8588, Japan
S
* Supporting Information
chemical manipulations to install an aminonitrile and a N-
ABSTRACT: The antitumor tetrahydroisoquinoline
(THIQ) alkaloids share a common pentacyclic scaffold
that is biosynthesized by nonribosomal peptide synthe-
tases involving unique enzymatic Pictet−Spengler cycliza-
tions. Herein we report concise and divergent chemo-
enzymatic total syntheses of THIQ alkaloids by merging
precise chemical synthesis with in vitro engineered
biosynthesis. A recombinant enzyme SfmC responsible
for the biosynthesis of saframycin A was adapted for the
assembly of these natural products and their derivatives,
by optimizing designer substrates compatible with SfmC
through chemical synthesis. The appropriately function-
alized pentacyclic skeleton were efficiently synthesized by
streamlining the linkage between SfmC-catalyzed multi-
step enzymatic conversions and chemical manipulations of
the intermediates to install aminonitrile and N-methyl
groups. This approach allowed rapid access to the
elaborated pentacyclic skeleton in a single day starting
from two simple synthetic substrates without isolation of
the intermediates. Further functional group manipulations
allowed operationally simple and expeditious syntheses of
jorunnamycin A, saframycin A, and N-Fmoc saframycin
Y3 that could be versatile and common precursors for the
artificial production of other antitumor THIQ alkaloids
and their variants.
methyl substituent to forge a pentacyclic core scaffold, and (iii)
subsequent two- or three-step chemical transformations to gain
rapid access to the natural products (1−3). These natural
products hold a promise as versatile intermediates for the
production of tetrahydroisoquinoline (THIQ) antitumor
antibiotics and their variants.
The THIQ alkaloids represented by saframycins (1, 2),
jorunnamycins (3), renieramycins (4), and safracins (5)
constitute important families of antitumor antibiotics (Figure
1).7 These alkaloids share a densely functionalized common
ith the exception of the semisynthetic approach, the
Wtwo synthetic disciplines of chemical synthesis and
biological synthesis have been developed essentially independ-
ently in an effort to gain efficient access to structurally complex
and biologically intriguing natural products.1 The two
disciplines offer complementary strengths and weaknesses,
yet very few attempts have been made to explore the interface
of chemical and in vitro biological synthesis to achieve the total
synthesis of natural products and their analogs.2,3 Herein we
report the chemo-enzymatic divergent total syntheses of
saframycin A (1),4 N-protected derivative of saframycin Y3
(2),5 and jorunnamycin A (3)6 by streamlining the integration
of (i) chemical synthesis of designer substrates, (ii) an enzyme-
catalyzed cascade synthesis working in conjunction with
Figure 1. Structures of tetrahydroisoquinoline (THIQ) alkaloids.
pentacyclic scaffold capable of DNA alkylation through
generation of an electrophilic iminium species exploiting either
aminonitrile or hemiaminal group at C21. Ecteinascidin-743
(6) bearing an additional THIQ system with the formation of
a macrolactone exhibits exceptionally potent antitumor
activities and is clinically approved for the chemotherapy of
ovarian neoplasms and sarcoma.8 This anticancer agent 6
Received: July 7, 2018
© XXXX American Chemical Society
A
DOI: 10.1021/jacs.8b07161
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Communication
Figure 2. Proposed NRPS biosynthetic assembly line leading to saframycin A (1) and design of synthetic substrates (7, 8, and 14−19).
contains the common pentacyclic scaffold shared with other
THIQ alkaloids, and its semisynthetic production in 21
chemical steps from fermentation-derived cyanosafracin B
(5) has been developed to provide 6 for clinical applications.9
The promising therapeutic potentials of THIQ alkaloids
coupled with growing demand for artificial production of
these complex structures have made the common THIQ
scaffold an important target molecule for both chemical
synthesis and engineered biosynthesis.10,11
incorporate an additional stereocenter at C11 to produce
intermediate C bearing two THIQ substructures. Further
thioester reduction would furnish an aldehyde 10, and
spontaneous intramolecular cyclization would forge the
pentacyclic scaffold 11 having a hemiaminal moiety at C21.
The chain length of the fatty acid group was shown to be
critical for efficient enzymatic conversions. A series of
experiments revealed cryptic roles played by the long fatty
acyl chain and illustrated the elegant interplay of the four
domains of SfmC. Tyrosine derivative 7 as well as peptidyl
aldehydes such as 8 bearing the fatty acyl group are reported to
be commonly involved in the biosynthesis of saframycin
Mx1,12b quinocarcin,15 SF-1739/naphthyridinomycins,15,16
and ecteinscidins.17 Consequently, the related NRPS bio-
synthetic machinery can likely be adopted to very similar and
potentially universal strategies for the production of THIQ
alkaloids.
The engineered biosynthesis of functionalized tyrosine
derivative 7 has not been achieved yet, to our knowledge,
while the biosynthetic enzymes responsible for the conversion
of L-tyrosine into 7 have been suggested.17,18 To secure a
sufficient amount of 7, a catalytic asymmetric synthetic route
was developed.19 We previously investigated the SfmC-
catalyzed assembly of the tetracyclic scaffold (Scheme 1).
Employment of N-myristoylated peptidyl aldehyde 8, assumed
to be liberated from the upstream module SfmB, markedly
The pentacyclic scaffold of saframycin A (1), a representa-
tive member of the THIQ alkaloids, is biosynthesized from L-
alanine, glycine, and two molecules of the functionalized
tyrosine derivative 7 (Figure 2). Nonribosomal peptide
synthetases (NRPSs) composed of three modules (SfmA,
SfmB, and SfmC) are responsible for the assembly of the
pentacyclic scaffold.12 Unlike the canonical NRPS system
catalyzing the condensation of amino acid components,13 the
biosynthetic machinery of saframycins has two distinctive
features.14 First, SfmA contains an acyl-CoA ligase (AL)
domain for loading a fatty acid, whereas such a long acyl chain
is absent from the THIQ alkaloids. According to a collinearity
rule, two modules (SfmA and SfmB) catalyze formation of N-
acylated alanylglycinyl thioester A present on the peptidyl
carrier protein (PCP) in SfmB. The second feature is the
unique catalytic function of SfmC exploiting the Pictet−
Spengler (PS) and reduction (Red) domains. Instead of amide
bond formation being catalyzed by the canonical condensation
(C) domain, the Red domain could catalyze reduction of the
thioester substrate A on the upstream module (SfmB),
liberating peptidyl aldehyde 8. The Pictet−Spengler (PS)
domain could mediate imine formation between the resultant 8
and the tyrosine derivative 7 loaded on the PCP in SfmC.
Subsequent regio- and stereocontrolled cyclization would
afford the THIQ intermediate B with formation of a sp3-
stereogenic center at C1. Then, SfmC composed of the four
domains (PS, A, PCP, and Red) could allow iterative assembly
of 7. Thioester reduction to generate aldehyde 9 followed by
reloading of 7 and subsequent Pictet−Spengler reaction could
Scheme 1. SfmC-Catalyzed Conversion of Synthetic
Substrates
B
DOI: 10.1021/jacs.8b07161
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Communication
Scheme 2. Chemo-enzymatic Total Syntheses of Jorunnamycin A (3) and Saframycin A (1)
accelerated (greater than 10-fold) the formation of 11, in
comparison to the corresponding CoA thioester 12. Upon
incubation of 8 and tyrosine derivative 7 with recombinant
holo-SfmC in the presence of ATP, Mg2+, Mn2+, and
NAD(P)H, we observed formation of the pentacyclic 11.
One-pot treatment of the resulting crude reaction mixture with
KCN and subsequent purification afforded aminonitrile 13 in
up to 22% overall yield based on 8.14a N-Myristoylated
peptidyl aldehyde 8 is highly compatible with SfmC as a
plausible biosynthetic substrate for the PS-domain, and thus
we synthesized several peptidyl aldehydes (14−16) with
various substitutions (Figure 2). Minor modifications of the
peptidyl moiety of 8, however, substantially decreased SfmC-
catalyzed formation of the corresponding pentacyclic scaffold
(SI-Figure S4−S6).
In addition to issues with the substrate tolerance, a major
obstacle for postenzymatic chemical conversions could be the
removal of the peptidyl side chain to access saframycins (1, 2)
and their congeners. While Tang and co-workers recently
revealed that a membrane-bound peptidase SfmE, hydrolyzes
the fatty acyl chain,20 we adopted a convenient chemical
approach employing artificial substrates compatible to SfmC.
Taking the relatively strict substrate specificity into account, we
minimized structural modification and thereby initially
designed the substrates, 17 and 18 bearing an ester linkage
in place of the amide bond in 8, with maintenance of an almost
identical chain length to that of 8 (Figure 2).
The SfmC-catalyzed conversion of 7 and 17 proceeded to
generate the corresponding pentacyclic products based on LC-
MS analysis (Scheme 2, SI-Figure S7). Chemical conversion of
the hemiaminal group through one-pot treatment with KCN
produced aminonitrile 20. The use of NADPH in place of
NADH provided a higher yield. However, despite the
promising LC-MS profiles indicating preferential formation
of the pentacyclic scaffolds (SI-Figure S7), the isolated yields
of 20 were less than 10%. We also struggled to reproducibly
attain optimum yield of 20. Even for the reported system
employing the plausible biosynthetic intermediate 8 of
SfmC,14a we also faced difficulties to consistently reproduce
the production of 13 in the range of 20% isolated yield
(Scheme 1, SI-Figures S2 and S3).
lated SfmC (holo-SfmC, SI-Figure S1). While these improve-
ments considerably shorten the preparation time for the holo-
SfmC, a substantial decrease of 20 was occasionally observed
upon handling and purification of the crude reaction mixture.
We thus assumed that the secondary amino group of 20 could
not only impair isolation efficiency but also entail unexpected
side reactions or oxidative degradations.21 According to this
working hypothesis, we next performed N-methylation of the
crude mixture containing secondary amine 20 by treatment
with excess amounts of formaldehyde and 2-picoline borane.22
Subsequent reductive amination to install the N-methyl group
proceeded cleanly, and more importantly, the corresponding
tertiary amine 21 was efficiently obtained in 18% overall yield
based on 17 in a reproducible manner (Scheme 2). This in situ
N-methylation provided significant improvements in the
chemo-enzymatic synthesis,23 which enabled facile access to
pentacyclic scaffold 21 incorporating the three stereocenters
(C1, C11, and C21) with accurate control of the chemo-,
regio- and stereoselectivities of the nine-step conversions.
Because we can complete the overall manipulations (7 + 17 →
21) within a single day, this chemo-enzymatic approach, which
does not require the isolation of any intermediates, greatly
reduces the time and labor involved in assembling the highly
elaborated pentacyclic THIQ scaffold.
Having established the sequential process, we secured
sufficient amount of 21 as the appropriately functionalized
intermediates to achieve chemical conversions into 3 (Scheme
2). Hydrolysis of the ester linkage of 21 smoothly proceeded to
furnish 22 in 83% yield. Oxidation of the phenols in the
presence of a catalytic amount of salcomine gave rise to 3 (2.9
mg).24 We thus developed the chemo-enzymatic synthetic
process of jorunnamycin A (3) in just four-pot starting from
the tyrosine derivative 7. While elegant total syntheses of 3 by
Zhu and Chen have been reported,6b,c our approach described
herein could provide an alternative synthetic route to 3.
This approach employing a designer peptidyl aldehyde can
be readily applied to saframycin A (1) (Figure 2 and Scheme
2). The change of peptidyl aldehyde substrate 8 into 18 was
demonstrated to be well tolerated to SfmC, and 4.6 mg of 24
was obtained in 13% yield based on 18 (SI-Figure S8).
Hydrolysis of 24 (12.2 mg) gave secondary alcohol 25 in 91%
yield. Salcomine-mediated oxidation of phenols followed by
Swern oxidation of the alcohol allowed chemo-enzymatic total
synthesis of 1 (5.0 mg).
In efforts to improve the enzymatic conversion, we
optimized purification methods of the labile SfmC and also
conducted direct in vivo preparation of phosphopantetheiny-
C
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To expand the scope of this approach, we further designed
substrate 19 having an allyl carbamate linkage to access
saframycin Y3 (3) bearing a N-terminal alanine unit (Figure 2
and Scheme 3). SfmC-catalyzed skeleton forming reaction
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.8b07161.
■
S
Detailed experimental procedures, spectroscopic data,
LC-MS chromatograms, and copies of NMR spectra
(PDF)
Scheme 3. Chemo-enzymatic Synthesis of N-Fmoc
Saframycin Y3 (29)
AUTHOR INFORMATION
■
Corresponding Authors
*h_oguri@cc.tuat.ac.jp
*hoik@sci.hokudai.ac.jp
ORCID
Hideaki Oikawa: 0000-0002-6947-3397
Hiroki Oguri: 0000-0001-8007-1631
Notes
The authors declare the following competing financial
interest(s): We have patent applications on the development
of the chemo-enzymatic synthetic process to generate
tetrahydroisoquinoline scaffolds.
ACKNOWLEDGMENTS
■
We thank Dr. Yasushi Ogasawara (Hokkaido Univ.) for the
generous gift of the plasmid pACYC-sfp. We are grateful for
financial supports from the Japan Science and Technology
Agency (JST) “Precursory Research for Embryonic Science
and Technology (PRESTO)” for a project of “Molecular
technology and creation of new functions”, JSPS KAKENHI
[Grant No. JP15H01835 (H. Oikawa), JP17H05433 (H.
Oguri), JP16H06446 (A. Minami)], Grant-in-Aid for JSPS
Research Fellow [Grant No. JP17J00931 (R. Tanifuji)],
Daiichi Sankyo Foundation of Life Science and Nagase Science
Technology Foundation. The authors dedicate this paper to
Prof. Masahiro Hirama on the occasion of his 70th birthday.
employing 19 with 7 also proceeded efficiently to furnish the
corresponding 27 in 20% overall yield based on 19 (SI-Figure
S9). Notably, 7.3 mg of 27 was successfully obtained in a day.
Treatment of 27 (12.8 mg) with Pd(PPh3)4 in CH2Cl2
employing PhSiH3 as a hydride source allowed rapid
transformation to liberate the corresponding primary
amine,25 which was isolated after protection with Fmoc
group to give 28 in 80% yield (2 steps). Salcomine-mediated
oxidation of the phenols provided 6.9 mg of N-Fmoc
saframycin Y3 (29) in 64% yield. Although removal of the
N-Fmoc group in 29 to liberate saframycin Y3 (2) was feasible
based on ESI-MS analysis, it was quite difficult to isolate 2
having both the primary amine and the bisquinone groups in a
practical yield. Eventually, we thus report the synthesis of the
N-Fmoc protected derivative of 2.
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E
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