Total Synthesis of the Proposed Structure of Mycobactin J

Article abstract of DOI:10.1021/acs.orglett.8b02832

The total synthesis of the proposed structure of mycobactin J (MJ), a metabolite of Mycobacterium tuberculosis, is presented. The highlights of the synthesis include a careful control of the Z-stereochemistry of the unsaturated long chain fatty acid, a biomimetic construction of the oxazoline building block and the carriage of an unprotected phenol throughout the synthesis.

Full text of DOI:10.1021/acs.orglett.8b02832

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Total Synthesis of the Proposed Structure of Mycobactin J
Chiranjit Ghosh, Sujit Pal, Akanksha Patel,† and Manmohan Kapur*
Department of Chemistry, Indian Institute of Science Education and Research Bhopal, Bhopal Bypass Road, Bhauri, Bhopal 462066,
Madhya Pradesh, India
S
* Supporting Information
ABSTRACT: The total synthesis of the proposed structure of mycobactin J
(MJ), a metabolite of Mycobacterium tuberculosis, is presented. The highlights
of the synthesis include a careful control of the Z-stereochemistry of the
unsaturated long chain fatty acid, a biomimetic construction of the oxazoline
building block and the carriage of an unprotected phenol throughout the
synthesis.
uberculosis (TB) is one of the world’s most predominant
diseases, with over one-fourth of the global population
proposed a revised structure for mycobactin J (1, Scheme 1).4
Almost all mycobacterial cell walls are lipid-rich and contain a
large amount of hydrocarbon content including fatty acids,
which produce an exceptionally tightly packed array, and this
limits the ability of many of drugs to penetrate inside the cell.5
By applying the “Trojan horse” approach, conjugate systems
with siderophores and other drugs have been developed, where
siderophores act as drug-delivery agents for mycobacterial
infections as well as killing of cancer cells.6 Miller and co-
workers prepared one such conjugate of mycobactin T with
artemisinin, and it displayed good activity against malaria as
well as tuberculosis.6a The mycobacterium produces side-
rophores such as mycobactins to harvest iron from the
environment, since the metal is a key nutrient and vital for
metabolic function as well as for cell division of the
bacterium.2d,7a,b Such iron-acquiring siderophores are also
routinely used for the treatment of iron-overloaded patients.7c
Groves and co-workers reported a mycobactin-mediated iron-
acquisition pathway within macrophages, which could
represent a new target for the control of mycobacterial
infection.8
T
being affected.1 Mycobacterium tuberculosis is the assassin
bacterium responsible for TB, and in recent years, considerable
research has been focused on identifying metabolites of
Mycobacterium sp. as potential drug-delivery candidates.2
Siderophores that contain hydroxamic acid residues are key
systems for the survival of the Mycobacterium sp. Siderophores
such as mycobactins (metabolites of Mycobacterium sp.),
formobactin, nocobactin, amamistatin (Figure 1),2f−k and
Depending upon their chemical structures, mycobactins
demonstrate either growth promoting or inhibitory activity
against MTB.9 Among all of the naturally occurring
mycobactins isolated so far, MJ relatively possesses the most
complex structure. To the best of our knowledge, there has
been no report of the total synthesis of MJ to date. The
challenges associated with the geometry and reactivity of the
olefin side chain, present as a (Z)-α,β-unsaturated hydroxamic
acid, and the assembly of the molecule overall drew us to
attempt its total synthesis. For this, we planned to investigate
the convergent/submonomer approach depicted in Scheme 1.
Two fragments 4 and 5 would be synthesized from
commercially available Cbz-lysine (6) via a common
Figure 1. General structure of naturally occurring mycobactins and
related compounds.
lasso peptides such as lariatins3 are some such metabolites
which also have the potential to be used in drug delivery
systems. These may possess good inhibitory potency against
Mycobacterium sp. as well as against other bacterial systems and
viruses.
In 1970, Snow and co-workers reported the existence of a
variety of naturally occurring mycobactins.2a In 1982,
mycobactin J (MJ) was isolated and characterized by Merkal
and co-workers, and in the year 2001, De Voss and co-workers
Received: September 5, 2018
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.8b02832
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Scheme 1. Retrosynthetic Analysis
intermediate, with the oxazoline synthon 7 being obtained
from salicylic acid and threonine by following a biomimetic
cyclization approach in which the β-C of the threonine would
retain its configuration.10 This is one of the critical chiral
centers present in this mycobactin, which cannot be obtained
via regular acid- or DAST-mediated cyclization.11 The
submonomer 2, the chiral 3-hydroxypentanoic acid derivative,
can be accessed easily via the Evans aldol reaction,12 and the
crucial (Z)-pentadec-2-enoic acid 3 can be derived via the
Corey−Fuchs reaction13 and subsequent Lindlar reduction.
Our synthesis started with the construction of the (Z)-long
chain fatty acid 3. The aldehyde synthesized from commer-
cially available 1-tetradecanol (10) was subjected under
Corey−Fuchs reaction conditions,13 followed by quenching
with ethyl chloroformate and hydrolysis, which delivered the
alkyne acid 11 in good overall yield (Scheme 2). Reduction of
11 using Lindlar catalyst yielded 3 in almost quantitative yield.
heating 12 with hydroxylamine hydrochloride in MeOH at
reflux followed by a quick HATU-promoted cyclization and
TBDPS protection to afford the cyclolysine component 18. We
then took up the construction of the eastern building block,
cobactin 21 (Scheme 3). Hydrogenolysis of 18 followed by
HATU/EDC.HCl-mediated coupling with the chiral pentanoic
acid 2 led to the silyl-protected cobactin 20. The unprotected
cobactin 21 was obtained in good yield when 20 was treated
with Amberlyst-15. Attempts to obtain 21 directly via coupling
of 19 with the hydroxy acid 2 led to failure.
For the construction of the western building block
mycobactic acid 27b, salicylic acid was coupled with the HCl
salt of L-threonine methyl ester 22 to afford the amide 23
(Scheme 4). Catalytic, ammonium molybdate-mediated
dehydrative biomimetic cyclization with retention of config-
uration at the β-position of the threonine led to oxazoline ester
24, which was then saponified to obtain the oxazoline acid 7.10
This was used as such for the next step without any
purification. The amine 16 which was obtained by Pd/C-
mediated hydrogenolysis of 15 (Scheme 3) was coupled with
acid 7 using EDC·HCl to achieve 25a in good yield (Scheme
4). Controlled di-Boc cleavage using TFA followed by HATU/
DIPEA-mediated coupling with acid 3 led to the largest
building block, the diacylated product 26a (Scheme 4), with
trace quantities of the monoacylated product 27a. The control
of pH in the reaction to obtain 26a via the coupling reaction
was found to be very critical, with several of our attempts
resulting in scrambling of the olefin geometry as well as some
conjugate addition. After considerable optimization, DIPEA (7
equiv) and the unsaturated acid (2.7 equiv) were found to
prevent the olefin isomerization and provide an improved yield
of 26a. DIPEA-mediated transesterification followed by
saponification delivered mycobactic acid 27b.
Scheme 2. Synthesis of Long-Chain Acid
The nitrone 12 precursor was synthesized via a protocol
developed by Miller and co-workers (Scheme 3).9a Treatment
of this nitrone with hydroxyl amine hydrochloride and SOCl2-
mediated esterification delivered the desired ester 14.
Subsequent di-Boc protection provided the linear component
15 in good yield. The synthesis of 13 was accomplished by
Scheme 3. Synthesis of Lysine-Based Fragment and Cobactin
B
DOI: 10.1021/acs.orglett.8b02832
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Scheme 4. Synthesis of Mycobactic Acid and Related Acids
We then attempted the final esterification to reach the target
molecule. Inspired by the synthesis of different mycobactin
analogues developed by Miller and co-workers,9a,14 the final
esterification was attempted by EDC·HCl-mediated coupling
of mycobactic acid 27b with the unprotected cobactin 21
(Scheme 5A). A careful NMR analysis of the product
suggested that instead of the desired esterification, this
coupling occurred at the eastern N(OH) of cobactin 21 to
yield 28, which was further confirmed by formation of methyl
ester of mycobactic acid 27a via a DIPEA-mediated trans-
esterification reaction of 28. From the above information, we
realized that protection of the N-OH would be necessary, and
accordingly, we prepared m-nitrobenzoate and acetate-
protected cobactins 29 and 30 (Scheme 5B). However,
attempts at esterification of these two cobactin derivatives
with mycobactic acid 27b as well as acid 26b (to form the O-
acyl-protected MJ) failed.15 Coupling of 29 and 30 with acid
25b to form the ester and di-Boc-protected mycobactin
analogues resulted in failure even when a variety of peptide
coupling agents as well as Yamaguchi esterification were tried
(Scheme 5B).15 When these esterification approaches failed to
produce the desired result, we envisaged an approach for
coupling TBDPS-cobactin 20 and mycobactic acid 27b.15
EDC·HCl-mediated coupling of 27b and 20 gave the best yield
of TBDPS protected-MJ 31 along with the undesired product
28 and some intramolecular-cyclization product of acid 27b
(Scheme 5C). The yield of the product 31 was improved when
the TBDPS-protected cobactin 20 was prepared in situ.
Further optimization to improve the yield of the final
esterification, including the coupling between acid 26b with
TBDPS-protected cobactin 20, were attempted.15 AOP-
mediated coupling between 26b and 20 delivered 31 in a
trace amount, instead of the TBDPS-protected diacyl-MJ.
During purification of 31, we faced considerable issues in the
separation of the unreacted 20 from 31. We therefore decided
to go ahead with removing the TBDPS group. Based on our
earlier experiences of double-bond isomerization in the
synthesis of 26a and 28,16 we avoided the use of TFA or
AcOH. TBAF worked the best, delivering MJ (1) along with
some unidentified side products.15 1H NMR indicated that a
considerable amount of the tetrabutylammonium ion pair of
MJ 3217 along with MJ 1 was present (Scheme 5C). Attempts
to exchange the ions using Amberlyst-15 or Dowex-50 resins17
were unfortunately unsuccessful. To overcome this problem,
we synthesized the TBS-protected cobactin 34 and coupled it
with acid 27b, but no TBS-protected MJ was detected; only
the undesired product 28 was formed (Scheme 5D).18
Scheme 5. Coupling of Mycobactic Acid as Well as Related
Acids with Cobactin and Elaboration of the Synthesis of MJ
At this stage, we then altered our synthetic strategy (Scheme
6) and planned the deprotection of the silyl protecting group
and the two Boc groups from 36 simultaneously in a single
step. Installation of the long-chain acid 3 at the final stage to
get 35 and deprotection of the acyl group by trans-
esterification (for mono- or di-N,O-acyl group) would yield
MJ.
Toward this end, TBDPS/TBS-protected cobactin was
employed for the union with acid 25b under several
C
DOI: 10.1021/acs.orglett.8b02832
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Scheme 6. Modified Retrosynthesis
Scheme 7. Synthesis of Mycobactin J
esterification conditions, but unfortunately for us, none of
these attempts yielded the desired product.15
observed at δ 2.6 ppm, corresponding to the proton of β-C of
the chiral acid part; this was observed as a broad peak in
synthetic MJ. One more deviation was observed for the
doublet peak at δ 1.2 ppm present in the authentic compound;
in the synthetic sample, it was observed as a set of rotamer
peaks. Such rotamer peaks were also observed for the advanced
stage building blocks acid 26b, ester 27a, and mycobactic acid
27b. It is also observed that the α-protons of amino acids
(which usually are not isomerized under standard coupling
reactions) showed deviations from the authentic one. At this
stage, the exact structure of MJ remains elusive to us, and
detailed studies to prove the actual structure of 1 are currently
To circumvent these unforeseen difficulties, we detoured
from the esterification reaction of the cobactin and mycobactic
acid (Scheme 7). We then explored EDC·HCl/HATU-
mediated coupling of mycobactic acid 27b with the chiral
acid 2. The resulting acid 37 was freed from the unreacted
pentanoic acid 2 by simple workup (which indirectly also
solved the problem of separation of 20 from 31, faced at the
earlier stage). EDC·HCl/HATU-mediated coupling of 37 with
18 furnished 31. At this deprotection stage, treatment of 31
with TFA yielded MJ-TFA salt in good yield with no
isomerization of the double bond. The silyl trifluoroacetate
was washed out with pentane was followed by an aqueous
NaHCO3 wash to yield 1 as a free base in 50% yield. Further
attempts at purification over silica gel led to considerable
decomposition of the compound, thereby decreasing the yield
of the product. This product contained trace amounts of other
impurities which could not be separated. At this stage, when
in progress via the synthesis of other diastereomers.
To conclude, the first total synthesis of the proposed
structure of mycobactin J is described. Our synthetic approach
should allow explorations of other members of the mycobactin
family. The highlight of the work is the design of the pathway
to overcome the repeated scrambling of the critical Z-olefin
geometry as well as problems associated with conjugate
addition to the Z-olefin and the choice of the crucial silyl
protecting group.
1
the H NMR of the synthetic sample was compared with a
commercial sample in CDCl3 and as well as MeOH-d4, it was
found that peaks at certain regions did not concur with each
other (see the SI for details). First, peaks for the proton of the
ε-C of the two-lysine parts between δ 3.5−4.0 ppm were found
to be broader in synthetic MJ as compared to the authentic
sample. For the authentic sample, a well-resolved peak was
D
DOI: 10.1021/acs.orglett.8b02832
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.8b02832.
■
S
Experimental details and spectral characterization of all
new compounds (PDF)
(8) Luo, M.; Fadeev, E. A.; Groves, J. T. Nat. Chem. Biol. 2005, 1,
149.
̈
(9) (a) Walz, A. J.; Mollmann, U.; Miller, M. J. Org. Biomol. Chem.
́
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2007, 5, 1621. (b) Juarez-Hernandez, R. E.; Franzblau, S. G.; Miller,
M. J. Org. Biomol. Chem. 2012, 10, 7584.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: mk@iiserb.ac.in.
ORCID
(10) (a) Sakakura, A.; Kondo, R.; Ishihara, K. Org. Lett. 2005, 7,
1971. (b) Sakakura, A.; Umemura, S.; Kondo, R.; Ishihara, K. Adv.
Synth. Catal. 2007, 349, 551.
(11) (a) Phillips, A. J.; Uto, Y.; Wipf, P.; Reno, M. J.; Williams, D. R.
Org. Lett. 2000, 2, 1165. (b) Reddy, L. R.; Saravanan, P.; Corey, E. J.
J. Am. Chem. Soc. 2004, 126, 6230.
(12) Sharma, K. K.; Boddy, C. N. Bioorg. Med. Chem. Lett. 2007, 17,
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(13) (a) Sahu, B.; Muruganantham, R.; Namboothiri, I. N. N. Eur. J.
Org. Chem. 2007, 2007, 2477. (b) Shingala, S. D.; Reddy, G. V.;
Kumar, R. S. C.; Yadav, P. A.; Babu, K. S. J. Asian Nat. Prod. Res.
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Manmohan Kapur: 0000-0003-2592-6726
Present Address
†(A.P.) The State University of New York at Buffalo, Buffalo,
NY.
Notes
The authors declare no competing financial interest.
(14) Xu, Y.; Miller, M. J. J. Org. Chem. 1998, 63, 4314.
(15) See the Supporting Information for details.
(16) Use of 1 equiv of DIPEA in the coupling led to E-product 28.
(17) Kaburagi, Y.; Kishi, Y. Org. Lett. 2007, 9, 723.
(18) Cobactin 34 was confirmed by HRMS and by crude H NMR
(not given in the Supporting Information); purification on column led
to deprotected cobactin 20.
ACKNOWLEDGMENTS
■
Funding from CSIR-India and SERB-India (EMR/2016/
004298/OC) is gratefully acknowledged. C.G. thanks UGC−
India for a research fellowship. S.P. and A.P. thank IISERB for
fellowships. We thank CIF, IISERB, for the analytical data and
the Director, IISERB, for funding and the infrastructure
facilities.
1
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