Facile and highly efficient synthetic routes for the synthesis of (S)- and (R)-23-hydroxyundecylprodiginines ((23S)-2, and (23R)-2), 23-ketoundecylprodiginine (3), and deuterium-labeled 23-hydroxyundecylprodiginine ([23-d]-2) have been developed. We demonstrated a novel Rieske oxygenase MarG catalyzed stereoselective bicyclization of (23S)-2 to premarineosin A (4), a key step in the tailoring process of the biosynthesis of marineosins, using a marG heterologous expression system. The synthesis of various A-C-ring functionalized prodiginines 32-41 was achieved to investigate the substrate promiscuity of MarG. The two analogues 32 and 33 exhibit antimalarial and cytotoxic activities stronger than those of the marineosin intermediate 2, against Plasmodium falciparum strains (CQ(S)-D6, CQ(R)-Dd2, and 7G8) and hepatocellular HepG2 cancer cell line, respectively. Feeding of 34-36 to Streptomyces venezuelae expressing marG led to production of novel premarineosins, paving a way for the production of marineosin analogues via a combinatorial synthetic/biosynthetic approach. This study presents the first example of oxidative bicyclization mediated by a Rieske oxygenase.
Facile and highly efficient synthetic routes for the synthesis of <span class="Chemical">(S)-n> and <span class="Chemical">(R)-23-hydroxyundecylprodiginines (<span class="Chemical">(23S)-2, and (23R)-2), 23-ketoundecylprodiginine (3), and deuterium-labeled 23-hydroxyundecylprodiginine ([23-d]-2) have been developed. We demonstrated a novel Rieske oxygenase MarG catalyzed stereoselective bicyclization of (23S)-2 to premarineosin A (4), a key step in the tailoring process of the biosynthesis of marineosins, using a marG heterologous expression system. The synthesis of various A-C-ring functionalized prodiginines 32-41 was achieved to investigate the substrate promiscuity of MarG. The two analogues 32 and 33 exhibit antimalarial and cytotoxic activities stronger than those of the marineosin intermediate 2, against Plasmodium falciparum strains (CQ(S)-D6, CQ(R)-Dd2, and 7G8) and hepatocellular HepG2 cancer cell line, respectively. Feeding of 34-36 to Streptomyces venezuelae expressing marG led to production of novel premarineosins, paving a way for the production of marineosin analogues via a combinatorial synthetic/biosynthetic approach. This study presents the first example of oxidative bicyclization mediated by a Rieske oxygenase.
<span class="Chemical">Prodigininesn> (1–3, Figure 1)
are a family of red-pigmented natural products
characterized by a <span class="Chemical">4-methoxy bipyrrole moiety linked to a variety
of <span class="Chemical">alkyl-substituted pyrroles.[1] These compounds
have been extensively studied for their intriguing biological activities
(antimicrobial,[2] immunosuppressive,[3] antitumor,[2a,2b,4] anticancer,[5] and antimalarial[6,7]) and modes of action (transmembrane anion transport[5c−5e,8] and DNA intercalation[9]). Some of these compounds have shown clinical
potential, and the synthetic prodiginine analogue obatoclax-3 (GX15-070)
has completed phase II clinical trials for the treatment of small
cell lung cancer and is engaged in multiple clinical trials for the
treatment of other cancer conditions.[10,11]
Figure 1
Marineosin
biosynthetic pathway.
<span class="Chemical">Marineosinn>
biosynthetic pathway.
As a part of our ongoing interest in developing new ant<span class="Chemical">ipan>rasitic
agents, we recently reported the antimalarial activity of natural
and synthetic <span class="Chemical">prodiginines.[7] This work
showed that the terminal nonalkylated <span class="Chemical">pyrrole (ring A) and 3,5-dialkyl
substitutions on the other terminal pyrrole (ring C) of natural prodiginines
are crucial for potent antimalarial activity. In addition, we had
demonstrated in mice that prodiginines can be administered orally
with marked parasite clearance, including cures in some cases, without
evident weight loss and toxicity. Recently we also have developed
new methods for the synthesis of various 2,2′-bipyrrole-5-carboxaldehydes[12] and have subsequently generated a library of
middle ring (ring B) functionalized prodiginines and tambjamines for
their antimalarial activity and structure–activity relationship
(SAR) studies.
In 2008, Fencial and co-workers isolated <span class="Chemical">marineosinn>
A (6) and B (7) (Figure 1), a new
class of modified <span class="Chemical">prodiginines with an unusual <span class="Chemical">spiro-tetrahydropyran-aminal
and pyrrole-macrocyclic rings from the marine Streptomyces sp. CNQ-617.[13] Marineosins were shown
to have strong and selective anticancer activity, as well as antimalarial
activity.[13,14] The intriguing structure and biological
activities of marineosins has spurred efforts toward total synthesis
attempts. In recent years, several research groups have attempted
to accomplish the total synthesis but were successful only in the
synthesis of key fragments.[15] To date,
the total synthesis of marineosins is still incomplete.
Recently
we have demonstrated the final steps of the <span class="Chemical">marineosinn>
biosynthetic pathway through identification and characterization of
the corresponding mar gene cluster from marine Streptomyces sp. CNQ-617 (Figure 1).[14] Expression of the entire gene
cluster in a S. venezuelae host led
to production of marineosins, whereas gene replacement of marG, which encodes a Rieske nonheme iron-dependent oxygenase,
led to accumulation of 23-hydroxyundecylprodiginine (2) and 23-ketoundecylprodiginine (3). Replacement of marA, encoding a putative dehydrogenase, led to accumulation
of premarineosin A (4) and 16-ketopremarineosin A (5) (Figure 1). These observations did
not support either previous marineosin biosynthetic hypotheses, in
which the pathway either passes through an enone analogue of undecylprodiginine[13] or involves a hydroxylation of undecylprodiginine
by a RedG homologue (MarG).[15a] Rather,
they suggested a pathway which culminates in a MarG-catalyzed cyclization
of either 2 or 3 to form premarineosin A,
which is subsequently reduced by MarA to generate marineosins (Figure 1).
In the well-established <span class="Chemical">prodigininen> biosynthetic
pathway, it has
been shown that RedH condenses an <span class="Chemical">2-undecylpyrrole and a 4-methoxy-2,2′-bipyrrolyl-5-carboxaldehyde
(<span class="Disease">MBC, 8) to generate undecylprodiginine (1), which is then cyclized by the Rieske oxygenase RedG to form streptorubin
B (Figure 2).[16,17] An elegant
study on the biosynthesis of metacycloprodiginine by the same group
demonstrated an analogous oxidative cyclization catalyzed by another
RedG homologue, McpG.[17] These two Rieske
oxygenases, RedG and McpG, catalyze the regio- and stereoselective
cyclization of the same substrate, undecylprodiginine (1), to form 10- and 12-membered macrocyclic rings, respectively.[17] More interestingly, the absolute stereochemistry
of streptorubin B and metacycloprodiginine is varying at C-20 and
C-22, respectively, showing that the oxidative cyclization is stereospecific
(Figure 2).[17,18]
Figure 2
Roles of Rieske
oxygenases RedG, McpG, and MarG, along with other
tailoring enzymes, in the biosynthesis of the prodiginine natural
products streptorubin B, metacycloprodiginine and marineosin: (A)
late stages of the biosynthetic pathways leading to streptorubin B,
metacycloprodiginine, and marineosins; (B) biosynthetic gene cluster
of marineosins and the putative tailoring genes, with the Rieske oxygenase
genes redG, mcpG, and marG highlighted in red, genes involved in undecylprodiginine analogue
formation highlighted in blue, and the gene encoding premarineosin
reductase highlighted in green; (C) multiple amino acid alignment
of RedG, McpG, and MarG, with conserved sequences within CXH, CXXH
motifs that ligate to the [2Fe-2S] cluster (Rieske center) marked
with red asterisks and conserved sequences in the EXXHX4H motif that binds to ferrous ion (nonheme iron center) marked with
black asterisks.
Roles of Rieske
oxygenases RedG, McpG, and MarG, along with other
tailoring enzymes, in the biosynthesis of the <span class="Chemical">prodigininen> natural
products streptorubin B, metacycloprodiginine and marineosin: (A)
late stages of the biosynthetic pathways leading to streptorubin B,
metacycloprodiginine, and marineosins; (B) biosynthetic gene cluster
of marineosins and the putative tailoring genes, with the Rieske oxygenase
genes redG, mcpG, and marG highlighted in red, genes involved in undecylprodiginine analogue
formation highlighted in blue, and the gene encoding premarineosin
reductase highlighted in green; (C) multiple amino acid alignment
of RedG, McpG, and MarG, with conserved sequences within CXH, CXXH
motifs that ligate to the [2Fe-2S] cluster (Rieske center) marked
with red asterisks and conserved sequences in the EXXHX4H motif that binds to ferrous ion (nonheme iron center) marked with
black asterisks.
The product of the marH gene shares 80% similarity
with RedH, while the novel Rieske oxygenase MarG shares 80% similarity
with RedG and 70% similarity with McpG (Figure 2). It seems plausible that the putative <span class="Chemical">marineosinn> biosynthetic intermediate
(S)-23-hydroxyundecylprodiginine ((23S)-2) might be produced via a MarH condensation of 8 and (10′S)-hydroxyundecylpyrrole
((10′S)-9). Subsequently, the
unique bicyclization, including C–C bond formation, and the
intramolecular hydroalkoxylation are catalyzed by MarG to generate
premarineosins (Figure 2).
The proposed
final steps of <span class="Chemical">marineosinn> biosynthesis provide an
inspiration for a new approach for the elusive synthesis of <span class="Chemical">marineosins.
To that end, the proposed <span class="Chemical">marineosin biosynthetic pathway intermediates 8 and (10′S)-9 would
be synthesized and then condensed to form (23S)-2. MarG would then provide enzymatic access to premarineosin
A (4), and thus marineosins (6 and 7), after a final chemical reduction step. In this work we
report the successful synthesis of (S)- and (R)-23-hydroxyundecylprodiginines ((23S)-2 and (23R)-2), 23-ketoundecylprodiginine
(3), and deuterium-labeled 23-hydroxyundecylprodiginine
([23-d]-2). In vivo feeding experiments
using a marG heterologous expression system unequivocally
demonstrated the role played by MarG to catalyze stereoselective bicyclization
of (23S)-2 to premarineosin A (4). In addition, we also report the elucidation of the substrate
promiscuity of MarG using a series of chemically synthesized new analogues
of 2. Finally, we report on the potent antimalarial activity
of two derivatives of 2, 23-alkoxyundecylprodiginines 32 and 33.
Results and Discussion
Synthesis
of (S)- and (R)-23-Hydroxyundecylprodiginines
(23S)-2 and (23R)-2
Our initial efforts focused on the synthesis of
the proposed biosynthetic intermediates 8, (10′S)-9, and (10′R)-9 from the key precursors 10–13 (Scheme 1). By use of literature methodologies,
4-methoxy-2,2′-bi<span class="Chemical">pyrrolen>-5-carboxaldehyde (<span class="Disease">MBC, 8) was prepared from the commercially available <span class="Chemical">4-methoxy-3-pyrrolin-2-one
(10) in two steps.[19]
Scheme 1
Retrosynthesis
of (S)- and (R)-23-(Hydroxy/keto)undecylprodiginines
(23S)-2, (23R)-2, and 3
The synthesis of (10′S)-hydroxyundecyl<span class="Chemical">pyrrolen>
((10′S)-9) and (10′R)-hydroxyundecyl<span class="Chemical">pyrrole ((10′R)-9) is outlined in Schemes 2 and 3. <span class="Chemical">1-(Phenylsulfonyl)-pyrrole (14) was
treated with dichloromethyl methyl ether to give 1-(phenylsulfonyl)-2-pyrrolecarboxaldehyde
(15) in 82% yield,[20] which
was further treated with 3-butenylmagnesium bromide in THF at 0 °C
to give compound 16 in 85% yield. To avoid a two-step
sequence (oxidation of alcohol group, and deoxygenation, and deprotection
by NaBH4/reflux)[15b,15c] for the conversion
of compound 16 to 2-(4-pentenyl)pyrrole (11), we successfully developed a one-pot cascade reaction by using
LiAlH4 in THF at 0 °C to reflux conditions (see the Experimental Section), and this proceeded with excellent
yields (Scheme 2). Conversely, (S)-propylene oxide (17) was treated with 4-pentenylmagnesium
bromide (18) in the presence of CuI to give the (S)-2-hydroxy-7-octene (12) in 92% yield.[21] Subsequently, a standard cross-metathesis reaction
between 11 and 12 by Grubbs II catalyst
and further hydrogenation of the olefin bond of 19 with
Pd/C (10%) under H2 gas at room temperature led to the
desired compound (10′S)-9 (Scheme 2). We employed a similar reaction sequence to furnish
the anti isomer (10′R)-9 from
(R)-propylene oxide (20) via the appropriate
intermediates (R)-2-hydroxy-7-octene (13), 11, and 21 as outlined in Scheme 3.
Scheme 2
Synthesis of (10′S)-Hydroxyundecylpyrrole
((10′S)-9)
Scheme 3
Synthesis of (10′R)-Hydroxyundecylpyrrole
((10′R)-9)
With the proposed biosynthetic intermediates
(10′S)-9 and (10′R)-9 in hand, we then sought and successfully
synthesized the
desired <span class="Chemical">(S)-23-hydroxyundecylprodigininen> ((23S)-2) and (R)-23-hydroxyundecylprodiginine
((23R)-2) using an acid-mediated (methanolicHCl) condensation with 4-methoxy-2,2′-bipyrrole-5-carboxaldehyde
(8) in good yields as outlined in Scheme 4.[7] Compounds (23S)-2 and (23R)-2 were fully
characterized by NMR and MS analysis and compared with the TLC and
HPLC profiles of natural product 2 (Figure S1, Supporting Information).
Scheme 4
Synthesis of (S)- and (R)-23-Hydroxyundecylprodiginines
((23S)-2 and (23R)-2)
Stereochemical Analysis
and Conversion of (23S)-2 and (23R)-2 to Premarineosin
A (4) by a MarG-Catalyzed Bicyclization
Sequence
analysis suggests that MarG is a Rieske nonheme <span class="Chemical">ironn>-dependent oxygenase,
containing the universally conserved N-terminal CXH and CXXH motifs
that ligate to the [2Fe-2S] cluster in the Rieske center and conserved
EXXHX4H motif that binds to ferrous ion in the nonheme
<span class="Chemical">iron center. It was initially proposed that MarG is involved in the
<span class="Chemical">hydroxylation and subsequent spiroaminal ring formation of marineosin
from an undecylprodiginine intermediate.[15a] Our previous work suggests that MarG is not responsible for hydroxylation
but catalyzes spiroaminal formation from an isomer of 2 (Figure 1).[14] Taking
into consideration the difficulties associated with in vitro protein
reconstitution of this class of enzymes, we sought the overexpression
of MarG in S. venezuelae ATCC 15439
(S. venezuelae MarG, see the Experimental Section and Tables S1 and S2 (Supporting Information)) in order to study its
role in the biosynthesis of marineosin in vivo. Feeding of synthesized
(23S)-2 to S. venezuelae MarG resulted in the production of both premarineosin A (4) and 16-ketopremarineosin A (5, a presumptive shunt
product) (Scheme 5). The production of 4 and 5 was confirmed by LC-MS analysis using
side by side comparison with standard premarineosins (Figure 3 and Figure S2 (Supporting Information)). Surprisingly, feeding of the opposite isomer, (23R)-2, to S. venezuelae MarG also provided both premarineosin A (4) and 16-ketopremarineosin
A (5), as shown in Scheme 5 (Figure 3 and Figure S2). Unfortunately,
the conversion of prodiginines to premarineosins was very poor, and
the resulting products were not isolated. However, they were confirmed
by LC-MS analysis using side by side comparison with standard premarineosins.
The conversion of both isomers (23S)-2 and (23R)-2 into premarinesosin A,
which has the S stereocenter at C-23, by the S. venezuelae MarG strain presented an enigma. The
diastereomer of 4, premarineosin B,[14] was not observed in detectable levels. As premarineosins
A and B differ only in the stereochemistry at the spiroaminal carbon
(C-8), it is most likely that premarineosin B arises from an inversion
of the aminal nitrogen of 4. The absence of detectable
levels of premarineosin B indicates that premarineosin A (4) is more thermodynamically stable under pathway conditions. It is
noteworthy that feeding (23R)-2 to S. venezuelae MarG resulted in the production of
23-ketoundecylprodiginine (3) as detected by LC-MS analysis
(Figure 3). Intriguingly, 3 was
not observed when the experiment was carried out with (23S)-2 (Figure 3). The production
of 3 suggests that (23R)-2 is initially converted into 3, and the latter may also
be participating in the MarG-catalyzed cyclization to provide premarineosins.
To test this hypothesis and determine the actual intermediate for
MarG, we sought the synthesis of 3.
Scheme 5
Conversion of (23S)-2, (23R)-2, 3, and [23-d]-2 to Premarineosins
by MarG
Figure 3
LC-MS (EIC) profiles
of feeding (23S)-2 and (23R)-2 to S. venezuelae MarG:
(A) EIC for m/z 408.26–408.27,
corresponding to [M + H]+ for 4, from an LC-MS
analysis of standard 4; (B) EIC for m/z 410.28–410.29, corresponding to [M + H]+ for (23S)-2, from an LC-MS
analysis of synthesized (23S)-2; (C)
EIC for m/z 408.26–408.27,
corresponding to [M + H]+ for 4, from an LC-MS
analysis of extracts of S. venezuelae MarG fed with (23S)-2; (D) EIC for m/z 408.26–408.27, corresponding
to [M + H]+ for 3, from an LC-MS analysis
of synthesized 3; (E) EIC for m/z 408.26–408.27, corresponding to [M + H]+ for 4 and [M + H]+ for 3, from
an LC-MS analysis of extracts of S. venezuelae MarG fed with (23R)-2.
LC-MS (EIC) profiles
of feeding <span class="Chemical">(23S)-2n> and (23R)-2 to <span class="Species">S. venezuelae MarG:
(A) EIC for m/z 408.26–408.27,
corresponding to [M + H]+ for 4, from an LC-MS
analysis of standard 4; (B) EIC for m/z 410.28–410.29, corresponding to [M + H]+ for <span class="Chemical">(23S)-2, from an LC-MS
analysis of synthesized (23S)-2; (C)
EIC for m/z 408.26–408.27,
corresponding to [M + H]+ for 4, from an LC-MS
analysis of extracts of S. venezuelae MarG fed with (23S)-2; (D) EIC for m/z 408.26–408.27, corresponding
to [M + H]+ for 3, from an LC-MS analysis
of synthesized 3; (E) EIC for m/z 408.26–408.27, corresponding to [M + H]+ for 4 and [M + H]+ for 3, from
an LC-MS analysis of extracts of S. venezuelae MarG fed with (23R)-2.
Synthesis of 23-Ketoundecylprodiginine (3) and
Subsequent Conversion to Premarineosins
We antic<span class="Chemical">ipan>ted that
the <span class="Chemical">prodiginine 3 could be obtained directly from <span class="Chemical">(23S)-2 and/or (23R)-2 by an oxidation of the hydroxyl group at C-23, as shown in Scheme 6. Unfortunately, we were unable to convert compound
(23S)-2 and/or (23R)-2 to the desired product 3 under a variety
of oxidation reagents/conditions, which resulted in either formation
of several products or extensive decomposition. We therefore took
an alternative route that exclusively leads to the final product 3 (Scheme 6). To achieve this, we took
either the (10′S)-9 and/or (10′R)-9 as a starting material and subjected them
to several oxidizing agents. A Ley–Griffith oxidation provided
the desired 10′-ketoundecylpyrrole (22) in 51%
isolated yield (Scheme 6). It is noteworthy
that the Ley–Griffith oxidation of (10′S)-9 and/or (10′R)-9 also provided the 1,5-dihydropyrrol-2-one 23 as a side
product in considerable amount (15% isolated yield). The individual
compound 23 was fully characterized by extensive NMR
and MS analysis (Supporting Information). This kind of controlled oxidation of N-protected pyrroles has
been previously reported by using Dess–Martin periodinane,[22] but to our knowledge, Ley–Griffith oxidation
conditions (TPAP/NMO) have never been exploited to unprotected pyrroles.
To demonstrate the generality of this controlled oxidation, the reaction
was carried out under the same reaction conditions on 2-ethylpyrrole
(24a), 2,4-dimethylpyrrole (24b), and 3-ethyl-2,4-dimethylpyrrole
(24c) and successfully led to the desired 5-alkylene-1,5-dihydropyrrol-2-ones 25a–c (Table 1).
Conversely, the same reaction conditions with N-protected 2-ethylpyrroles 24d–f failed to provide the corresponding
oxidative products in detectable levels. The N-methylpyrrole 24g was completely converted into the corresponding oxidized
product 25d in excellent yield (Table 1).
Scheme 6
Synthesis of 23-Ketoundecylprodiginine (3)
Table 1
Synthesis of 5-Alkylene-1,5-dihydropyrrol-2-ones
(25a–c) and 25d
nr = no reaction.
nr = no reaction.These <span class="Chemical">5-alkylene-1,5-dihydropyrrol-2-onesn> 23 and 25a–c are highly functionalized and therefore
are excellent building blocks for the synthesis of natural and synthetic
products of biological importance: for example, <span class="Chemical">pulchellalactam is
a potent CD45 protein tyrosine phosphatase (PTP) inhibitor, which
was isolated from the marine <span class="Disease">fungus Corollospora pulchella.[23] Finally, the acid-catalyzed condensation
of the key intermediate 22 with 8 provided
the desired 23-ketoundecylprodiginine (3) in 50% isolated
yield (Scheme 6), and it was fully characterized
by NMR and MS analysis and compared with the TLC and HPLC profiles
of natural product 3 (Figure S1, Supporting Information).
Feeding of <span class="Chemical">23-ketoundecylprodigininen>
(3) to the <span class="Species">S. venezuelae MarG expression strain clearly provided
both <span class="Chemical">premarineosin A (4) and 16-ketopremarineosin A (5), as shown in Scheme 5 (Figure 4 and Figures S2 and S3 (Supporting
Information)). The conversion of hydroxy- and ketoprodiginines(23S)-2, (23R)-2, and 3 to premarinesosin A by MarG expression
strain did not resolve the question of whether the keto or hydroxyl
derivative is the correct intermediate. We hypothesized that hydroxy-
and ketoprodiginines are most likely interconverting via a redox reaction
in the MarG expression host, S. venezuelae (Figure 1 and Scheme 5). Therefore, we sought the synthesis of [23-d]-23-hydroxyundecylprodiginine
([23-d]-2) to further study the intermediates
involved in marineosin biosynthesis.
Figure 4
LC-MS (EIC) profiles of 4 and [23-d]-4 from feeding of 3 and [23-d]-2 to S. venezuelae MarG:
(A) EIC for m/z 408.26–408.27,
corresponding to [M + H]+ for 4, from an LC-MS
analysis of standard 4; (B) EIC for m/z 408.26–408.27, corresponding to [M + H]+ for 3, from an LC-MS analysis of synthesized 3; (C) EIC for m/z 411.28–411.29,
corresponding to [M + H]+ for [23-d]-2, from an LC-MS analysis of synthesized [23-d]-2; (D) EIC for m/z 408.26–408.27, corresponding to [M + H]+ for 4, from an LC-MS analysis of extracts of S.
venezuelae MarG fed with 3; (E) EIC for m/z 408.26–408.27, corresponding
to [M + H]+ for 4, from an LC-MS analysis
of extracts of S. venezuelae MarG fed
with [23-d]-2; (F) EIC for m/z 409.27–409.28, corresponding to [M + H]+ for [23-d]-4, from LC-MS analysis
of extracts of S. venezuelae MarG fed
with [23-d]-2.
LC-MS (EIC) profiles of 4 and [23-d]-4 from feeding of 3 and <span class="Chemical">[23-d]-2n> to <span class="Species">S. venezuelae MarG:
(A) EIC for m/z 408.26–408.27,
corresponding to [M + H]+ for 4, from an LC-MS
analysis of standard 4; (B) EIC for m/z 408.26–408.27, corresponding to [M + H]+ for 3, from an LC-MS analysis of synthesized 3; (C) EIC for m/z 411.28–411.29,
corresponding to [M + H]+ for <span class="Chemical">[23-d]-2, from an LC-MS analysis of synthesized [23-d]-2; (D) EIC for m/z 408.26–408.27, corresponding to [M + H]+ for 4, from an LC-MS analysis of extracts of S.
venezuelae MarG fed with 3; (E) EIC for m/z 408.26–408.27, corresponding
to [M + H]+ for 4, from an LC-MS analysis
of extracts of S. venezuelae MarG fed
with [23-d]-2; (F) EIC for m/z 409.27–409.28, corresponding to [M + H]+ for [23-d]-4, from LC-MS analysis
of extracts of S. venezuelae MarG fed
with [23-d]-2.
Synthesis of [23-d]-23-Hydroxyundecylprodiginine
([23-d]-2) and Subsequent Conversion
into Deuterium-Labeled Premarineosin A ([23-d]-4) via MarG-Catalyzed Cyclization
Adopting an approach
developed by Li and Herzon in our syntheses,[24] <span class="Chemical">10-undecynoic acidn> (26) was treated with Au(IPr)Cl
(0.02 equiv), AgO2CCF3 (0.02 equiv), and Shvo’s
catalyst (0.02 equiv) in a mixture of water and isopropyl alcohol
(14% v/v), to give the desired 10-hydroxyundecanoic acid (27) in 82% yield (Scheme 7). 10-Oxoundecanoic
acid (28) was synthesized by oxidation of the hydroxyl
group of 27 using Dess–Martin periodinane conditions,
which upon treatment with sodium borodeuteride (NaBD4)
gave the [10-d]-10-hydroxyundecanoic acid ([10-d]-27). Attempts to convert the acid group
of [10-d]-27 to acid chloride using
either oxalyl chloride (COCl)2 or thionyl chloride (SOCl2) in dichloromethane/toluene, with a subsequent Friedel–Crafts
acylation on pyrrole in the presence of Zn/toluene[25] led to the formation of several products. Therefore, the
hydroxyl group at C-10 needed to be protected. The efficiency of this
reaction sequence (formation of acid chloride and Friedel–Crafts
acylation of pyrrole) toward the 2-acylpyrrole [10′-d]-30 was improved by the protection of the
hydroxyl group (C10–OH) of [10-d]-27 with Ac2O/pyridine. The reaction of [10-d]-10-acetoxyundecanoic acid ([10-d]-29) with SOCl2/DMF (catalytic)/toluene with a subsequent
acylation of pyrrole in the presence of Zn/toluene provided the desired
2-acylpyrrole [10′-d]-30 as a
major product along with the 3-acylpyrrole [10′-d]-31 as a minor product. This ratio of products is in
contrast with the standard regioselective acylation method, in which
2-acylpyrrole is the single product from pyrrole and acid chloride.[25] The two isomers [10′-d]-30 (57%) and [10′-d]-31 (8%) were isolated by silica gel column chromatography
and characterized by NMR and MS analysis. Then the 2-acylpyrrole [10′-d]-30 was smoothly converted to [10′-d]-9 in 67% isolated yield by using an excess
(12 equiv) of NaBH4 in isopropyl alcohol (IPA) under reflux
(Scheme 7).[7,26] The acid-catalyzed
condensation of [10′-d]-9 with 8 provided the desired racemic [23-d]-23-hydroxyundecylprodiginine
([23-d]-2) in 70% isolated yield (Scheme 7).
Scheme 7
Synthesis of Racemic [23-d]-23-Hydroxyundecylprodiginine
([23-d]-2)
Feeding of ∼90% <span class="Chemical">deuteriumn>-labeled racemic compound
<span class="Chemical">[23-d]-2 to the <span class="Species">S. venezuelae MarG expression host led to production of [23-d]-4 and 4 in ∼1.2:1 ratio, along
with the shunt products [23-d]-5 and 5, as shown in Scheme 5 (Figure 4 and Figures S2–S4 (Supporting
Information)). Production of deuterium-labeled premarineosin
A ([23-d]-4) is consistent with a pathway
that directly proceeds from the isomer [23S-23-d]-2 without oxidation to 3 (Scheme 5). The detection of ∼45% unlabeled product
premarineosin A (4) via MS analysis (Figure S4) from ∼90% labeled [23-d]-2 is consistent with a pathway in which the hydroxyl
group at C-23 of [23R-23-d]-2 is oxidized to 3 (as observed with (23R)-2 feeding experiment), the deuterium label
is lost, and then the compound is reduced to the unlabeled (23S)-2, which is then converted by MarG to premarineosin
A (4) (Scheme 5). Our findings
are thus all consistent with a role of MarG in catalyzing formation
of premarineosins exclusively from (S)-23-hydroxyundecylprodiginine
((23S)-2).
Synthesis of Hydroxyundecylprodiginine
Analogues To Probe MarG
Substrate Promiscuity
Having determined the intermediates
involved in the biosynthesis of <span class="Chemical">marineosin an>nd the role of MarG, we
wanted to study the potential of MarG-generated <span class="Chemical">marineosin analogues.
Using the general approach for synthesis of <span class="Chemical">(23S)-2, (23R)-2, 3,
and [23-d]-2 and various 2,2′-bipyrrole-5-carboxaldehydes
(52a–d),[7,12] we
synthesized various A- and B-ring functionalized (hydroxy/alkoxy)undecylprodiginines 2 and 32–41, as shown in
Schemes 8 and 9. These
compounds and undecylprodiginine (1) were fed to the
MarG expression strain.
Scheme 8
Synthesis of A- and B-Ring Functionalized
(Hydroxy/alkoxy)undecylprodiginines 2 and 32–38
Scheme 9
Synthesis of Hydroxyundecylprodiginines 39–41 Containing a Terminal Hydroxyl Group at the Alkyl Chain
Neither <n class="Chemical">span class="Chemical">premarineosin A (4) nor C9–C21 cyclized
<span>n class="Chemical">prodiginine was observed at a detectable level after feeding undecylprodiginine
(1), which has no hydroxyl group at C-23 of the alkyl
chain (Figure 1). This observation clearly
indicates that the hydroxyl group of 2 is required for
the MarG-catalyzed process and must be introduced early in the pathway.
One option is the hydroxylation of the 2-UP subunit by an unidentified
oxidase (route 1, Figure 1) or recruitment
of 2-hydroxybutyric acid starter unit by MarP (route 2, Figure 1). The gene product(s) required for introduction
of the hydroxyl group remains unknown. We also did not observe any
C9–C21 cyclized prodiginine related products after feeding
of 23-alkoxyundecylprodiginines 32 and 33 (Scheme 8), further demonstrating that the
hydroxyl group at C-23 is required for the formation of the spiro-tetrahydropyran-aminal
ring and that hydroxyundecylprodiginines are the preferred substrates
for MarG. Prodiginines 34–36 with
shorter alkyl chain length (octyl) on ring C and C-alkyl substitutions on rings A and B, containing the key secondary
alcohol substituent, were synthesized (Scheme 8) and fed to S. venezuelae MarG mutant.
LC-MS analyses clearly demonstrated the production of both the corresponding
premarineosin A and 16-ketopremarineosin analogues 58–63. The compounds were not isolated and characterized
due to poor conversion of hydroxyundecylprodiginines to premarineosins;
however, their corresponding chemical structures were proposed on
the basis of the above feeding results of S. venezuelae MarG mutant, MS/MS, and LC-MS analyses (Figure 5 and Figures S5–S8 (Supporting
Information)). Conversely, cyclic products of prodiginines
with N-methylpyrrole and furan in the place of terminal
pyrrole (ring A) (37 and 38, Scheme 8), were not detected via LC-MS after feeding their
respective substrates to the S. venezuelae MarG expression strain. Interestingly, prodiginines 39–41 (Scheme 9), with a
terminal hydroxyl group at the alkyl chain (primary alcohols), were
converted to their corresponding 16-ketoprodiginines 64–66 (Figure 5), but their
corresponding cyclized products were not detected via LC-MS. These
products 64–66 also were not isolated
and characterized, but their chemical structures were proposed on
the basis of the above feeding results of S. venezuelae MarG mutant and MS analysis (Figure S9 (Supporting
Information)). The above data demonstrate that MarG catalyzed
bicyclization requires the presence of a free secondary hydroxyl group
on an alkyl chain on ring C but can tolerate C-alkyl
substitutions on rings A and B (notably replacement of the methoxy
group). Substitution of terminal pyrrole (ring A) with other heterocycles
does not lead to cyclized products.
Figure 5
Proposed structures of expected products
from feeding of 34–36 and 39–41 to S. venezuelae MarG.
Proposed structures of expected products
from feeding of 34–36 and 39–41 to <span class="Species">S. venezuelaen> MarG.
Antimalarial Activity and
Cytotoxicity of Prodiginines
All synthesized natural <span class="Chemical">prodigininesn> 1–3, and their analogues 32–41, were evaluated for their antimalarial activity
against the <span class="Chemical">chloroquine-sensitive
(CQS) D6 and the <span class="Chemical">chloroquine-resistant (CQR)
Dd2 and 7G8 strains of Plasmodium falciparum with chloroquine (CQ) as a reference drug,[27] and the results are shown in Table 2. In
parallel, the cytotoxicity of all tested compounds was also tested
against hepatocellular HepG2 cancer cell line using mefloquine (MQ)
as a control drug.[27d] The synthesized prodiginines 2, (23R)-2, (23S)-2, and 3, containing an extra hydroxyl
and/or keto group at C-23 of the alkyl chain, have decreased antimalarial
activity and selectivity in comparison to undecylprodiginine (1), which has no substitutions on the alkyl chain (Table 2 and Figure 1). It is noteworthy
that the isomer (23S)-2 is only slightly
more potent than the isomer (23R)-2 and
racemic compound 2 (Table 2),
which demonstrates that the stereochemistry at C-23 does not alter
the antimalarial activity. More significantly, 23-alkoxy analogues 32 and 33 have much improved antimalarial activity
and selectivity in comparison to 2 and 3. Indeed, 32 and 33 have antimalarial activity
comparable to that of 1, with slightly reduced toxicity
(as measured by activity against HepG2cancer cells), and thus offer
increased selectivity. In contrast, retention of the free hydroxyl
group on the alkyl chain, with a reduction in the alkyl chain length,
led to a 3-fold decrease in the antimalarial potency (34 versus 2). Similarly, a loss of antimalarial activity
was observed with replacement of the OMe group of ring B by methyl
groups (35 versus 2), demonstrating the
importance of the methoxy group on ring B for potency. Surprisingly,
prodiginine 36, which contains an extra alkyl residue
(ethyl) on ring A, showed a substantially higher potency with an IC50 of <24 nM against all strains of P. falciparum (36 versus 2), and it had reduced selectivity.
Replacement of the terminal nonalkylated pyrrole ring (ring A) of
the core moiety by N-methylpyrrole and furan moieties
(compounds 37 and 38) resulted in a large
decrease in antimalarial activity (37 and 38 versus 2). These results, indicating that the pyrrole
NH of the prodiginines is required for potent antimalarial activity,
support our previous findings.[7] Our previous
studies also demonstrated that undecylprodiginine, with a terminal
amine group at the alkyl chain, showed poor antimalarial activity
(IC50 = 1700 nM).[7] However,
prodiginines 39–41, with a terminal
hydroxyl group at the alkyl chain (primary alcohols), exhibited better
activity (IC50 < 110 nM). Notably, in these cases, longer
alkyl chains led to an increase in the antimalarial activity (IC50 of 39 (C10) > 40 (C11) > 41 (C12)). These results thus demonstrate that the presence
of hydroxyl and/or keto substituents on the alkyl chain, replacement
of methoxy group of ring B by alkyl substituents, and replacement
of the terminal nonalkylated pyrrole ring (ring A) by N-methylpyrrole and/or other heterocycles have an adverse effect on
the antimalarial activities. Conversely, the hydroxyl group of 2 masked with methyl and benzyl groups, as in 32 and 33, respectively, dramatically increased the potency
and selectivity (Table 2).
Table 2
Antimalarial Activity and Cytotoxicity
of Prodiginines
antimalarial
activity (IC50 in nM)a
compd
D6
Dd2
7G8
cytotoxicity (IC50 in nM)a HepG2
SIb (D6)
1
7.2
7.5
7.0
1713
238
2
110
98
118
7358
67
(23R)-2
92
51
114
7961
86
(23S)-2
66
46
100
9152
139
3
191
121
199
14667
77
32
7.1
10.2
11.3
2682
378
33
5.7
3.7
2.3
2770
486
34
383
293
368
12050
31
35
1084
1188
627
9385
9
36
17
15
24
1757
103
37
>2500
>2500
>2500
>100000
>40
38
2328
>2500
1001
>100000
>43
39
108
80
168
11416
106
40
59
36
93
11363
193
41
38
23
58
6184
163
CQ
10
102
63
NDc
ND
MQ
ND
ND
ND
10970
ND
IC50 values are represented
as averages of triplicate measurements (SD ± 10%).
SI (selectivity index) = IC50 (cytotoxicity)/IC50 (D6).
ND: not determined.
IC50 values are represented
as averages of triplicate measurements (SD ± 10%).SI (selectivity index) = IC50 (<span class="Disease">cytotoxicityn>)/IC50 (D6).
ND: not determined.
Conclusions
In summary, we have accomplished the first synthesis
of <span class="Chemical">(S)-23-hydroxyundecylprodigininen> (<span class="Chemical">(23S)-2), <span class="Chemical">(R)-23-hydroxyundecylprodiginine
((23R)-2)), 23-ketoundecylprodiginine
(3), [23-d]-23-hydroxyundecylprodiginine
([23-d]-2), and their synthetic analogues 32–41 from commercially available starting
materials in straightforward approaches. These feasible synthetic
routes can be carried out on a large scale and are suited for the
generation of a library of novel prodiginines for their advanced biological
activities and SAR studies. Formation of the critical unusual spiro-tetrahydropyran-aminal
ring of marineosins and conversion of (23S)-2 to premarineosin A (4) were accomplished by
MarG expressed in S. venezuelae. Conversion
of [23-d]-2 to the deuterium-labeled
premarineosin A ([23-d]-4) unambiguously
demonstrates that the pathway directly proceeds stereospecifically
from (23S)-2. The synthesis of 23-alkoxyundecylprodiginines
(32 and 33), 20-hydroxyoctylprodiginine
(34), and A- and B-ring functionalized prodiginines (35–41) was used to investigate the substrate
promiscuity of MarG. As such, this one-step enzymatic (C–C
bond formation and intramolecular hydroalkoxylation) work represents
a complementary approach to the widespread synthetic approaches directed
toward this class of complex natural products. Investigations to improve
the conversion of prodiginines to premarineosins by MarG and subsequent
isolation and chemical reduction step to the final marineosins and
their analogues are underway in our laboratory. Significantly, alkoxy
analogues 32 and 33 exhibited potent antimalarial
activity with better selectivity than natural prodiginines 1–3. In the course of this work, we also have
discovered a simple and convenient method for the synthesis of 5-alkylene-1,5-dihydropyrrol-2-ones
from C-alkyl-substituted pyrroles by using TPAP/NMO.
These pyrrol-2-ones are excellent building blocks for the synthesis
of natural and synthetic products of biological importance, and further
investigations to improve the yields and expand the substrate scope
of the oxidation with this reagent as well as mechanistic studies
are underway in our laboratory.
Experimental
Section
General Considerations
NMR spectra were recorded on
a spectrometer at 400 MHz (n class="Chemical">1H) and 100 MHz (<span class="Chemical">13C). Experiments were recorded in <span class="Chemical">CDCl3 and acetone-d6 at 25 °C. Chemical shifts are given in
parts per million (ppm) downfield from internal standard Me4Si (TMS). HRMS (ESI) were recorded on a high-resolution (30000) LTQ-Orbitrap
mass spectrometer. Unless otherwise stated, all reagents and solvents
were purchased from commercial suppliers and used without further
purification. Reactions which required the use of anhydrous, inert
atmosphere techniques were carried out under an atmosphere of argon/nitrogen.
Chromatography was executed using silica gel (230–400 mesh)
and/or neutral alumina as the stationary phase and mixtures of ethyl
acetate and hexane as eluents. Analytical HPLC analyses were performed
using a C18 column (4.6 × 150 mm) with a linear elution gradient
ranging from CH3OH/CH3CN/H2O (40%/10%/50%)
to CH3OH (100%) acidified with 0.1% trifluoroacetic acid
at a flow rate of 0.3 mL/min.
Synthesis of 1-(1-(Phenylsulfonyl)-pyrrol-2-yl)pent-4-en-1-ol
(16)
To a stirred solution of 15 (4.0 g, 17.02 mmol) in 200 mL of <span class="Chemical">THFn> was added 3-butenylmagnesium
bromide solution (0.5 M in <span class="Chemical">THF) (68 mL, 34.04 mmol) at 0 °C,
and the reaction mixture was stirred at 0 °C for 2 h. Then the
reaction was quenched by addition of <span class="Chemical">2 N HCl, with the temperature
maintained at <10 °C, and extracted with diethyl ether (3
× 100 mL). The combined organic extracts were washed with water
and brine and dried over anhydrous Na2SO4. The
solvent was evaporated under reduced pressure, and the crude product
was chromatographed on silica gel to afford the title compound 16 (4.21 g, 85%) as a syrup: Rf = 0.45 (20% EtOAc/hexanes); 1H NMR (CDCl3,
400 MHz) δ 7.78 (d, J = 8.1 Hz, 2H), 7.59 (m,
1H), 7.50 (m, 2H), 7.31 (dd, J = 1.7, 3.3 Hz, 1H),
6.29 (m, 2H), 5.72 (m, 1H), 4.93 (m, 2H), 4.85 (m, 1H), 2.92 (br s,
1H), 2.12 (m, 2H), 1.90 (m, 2H); 13C NMR (CDCl3, 100 MHz) δ 139.1, 138.1, 137.8, 134.0, 129.5 (2C), 126.6
(2C), 123.5, 115.1, 112.5, 111.8, 64.5, 34.2, 30.1; HRMS (ESI) calcd
for C15H17NaNO3S (M + Na)+ 314.0821, found 314.0815; IR (KBr) νmax 3365, 2956,
2846, 1678, 1425 cm–1.
Synthesis of 2-(4-Pentenyl)pyrrole
(11)
To a stirred suspension of <span class="Chemical">LiAlH4n> (2.7 g, 72.16 mmol)
in dry <span class="Chemical">THF (200 mL) was added dropwise 16 (3.0 g, 10.30
mmol) in <span class="Chemical">THF (50 mL) at 0 °C, the reaction mixture was stirred
for 3 h at 0 °C, and it was warmed to room temperature. Then
the resulting solution was heated to reflux for 12 h. The reaction
was quenched with a saturated solution of sodium sulfate. The insoluble
solid was filtrated off and washed with DCM (200 mL). Then the combined
organic solution was concentrated under reduced pressure to give the
crude product, which was further chromatographed on silica gel, with
ethyl acetate/hexanes as eluent, to afford 11 (1.14 g,
82%) as a colorless syrup: Rf = 0.80 (20%
EtOAc/hexanes); 1H NMR (CDCl3, 400 MHz) δ
7.95 (br s, 1H), 6.72 (m, 1H), 6.22 (dd, J = 2.8,
5.8 Hz, 1H), 6.01 (m, 1H), 5.88 (m, 1H), 5.09 (m, 2H), 2.68 (t, J = 7.2 Hz, 2H), 2.18 (m, 2H), 1.80 (m, 2H); 13C NMR (CDCl3, 100 MHz) δ 138.5, 132.4, 116.2, 115.0,
108.3, 105.1, 33.3, 28.9, 27.1; HRMS (ESI) calcd for C9H14N (M + H)+ 136.1121, found 136.1126; IR
(KBr) νmax 2931, 2857, 1681, 1439 cm–1.
Representative Procedure for the Synthesis of (S)-2-Hydroxy-7-octene (12)
To a stirred solution
of <span class="Chemical">(S)-propylene oxiden> (17; 1.0 g, 17.24
mmol) and copper iodide (327 mg, 1.72 mmol) in 50 mL of <span class="Chemical">THF was added
<span class="Chemical">4-pentenylmagnesium bromide solution (18; 0.5 M in THF)
(52 mL, 25.86 mmol) at 0 °C, and the reaction mixture was stirred
at 0 °C for 2 h. Then the reaction mixture was quenched by addition
of 2 N HCl, with the temperature maintained at <10 °C, and
extracted with diethyl ether (3 × 100 mL). Then the combined
organic extracts were washed with 10% sodium thiosulfate solution,
water, and brine and dried over anhydrous Na2SO4. The solvent was evaporated under reduced pressure, and the crude
product was chromatographed on silica gel to afford the title compound 12 (2.03 g, 92%) as a colorless liquid: Rf = 0.50 (50% EtOAc/hexanes); 1H NMR (CDCl3, 400 MHz) δ 5.79 (m, 1H), 4.98–4.92 (m, 2H),
3.80 (m, 1H), 2.04 (m, 2H), 1.40 (m, 6H), 1.18 (d, J = 6.2 Hz, 3H); 13C NMR (CDCl3, 100 MHz) δ
138.9, 114.4, 68.1, 39.2, 33.7, 28.9, 25.2, 23.5; HRMS (ESI) calcd
for C8H16NaO (M + Na)+ 151.1093,
found 151.1079; IR (KBr) νmax 3361, 2972, 2930, 2857,
1640 cm–1. Compound 13 has the same
spectral data.
Representative Procedure for the Synthesis
of (S)-11-(Pyrrol-2-yl)undec-7-en-2-ol (19)
Grubbs’
second-generation catalyst (Grubbs’ II; 160 mg, 0.19 mmol)
was added to a stirred solution of 11 (500 mg, 3.70 mmol),
and 12 (948 mg, 7.40 mmol) in <span class="Chemical">dichloromethanen> (25 mL)
at room temperature. Then the solution was stirred for 6 h at 40 °C.
After completion of the reaction, the solvent was removed under reduced
pressure and the product was chromatographed on <span class="Chemical">silica gel, with ethyl
acetate/<span class="Chemical">hexanes as eluent, to afford the desired product 19 (390 mg, 45%) as a syrup: Rf = 0.45
(30% EtOAc/hexanes); 1H NMR (CDCl3, 400 MHz)
δ 8.15 (br s, 1H), 6.65 (dd, J = 2.5, 6.6 Hz,
1H), 6.13 (dd, J = 2.5, 5.7 Hz, 1H), 5.92 (m, 1H),
5.42 (m, 2H), 3.81 (m, 1H), 2.60 (t, J = 7.6 Hz,
2H), 2.01 (m, 4H), 1.66 (m, 2H), 1.35 (m, 6H), 1.18 (d, J = 6.2 Hz, 3H); 13C NMR (CDCl3, 100 MHz) δ
132.5, 130.8, 129.9, 116.0, 108.1, 104.8, 68.1, 39.1, 32.4, 32.0,
29.4 (2C), 27.0, 25.1, 23.4; HRMS (ESI) calcd for C15H25NaNO (M + Na)+ 258.1828, found 258.1819; IR (KBr)
νmax 3381, 2928, 2856, 1679, 1437 cm–1. Compound 21 has the same spectral data.
Representative
Procedure for the Synthesis of (10′S)-Hydroxyundecylpyrrole
((10′S)-9)
To a degassed solution of 19 (200 mg, 0.85 mmol) in
<span class="Chemical">methanoln> (10 mL) was added a catalytic amount of 10% Pd/C. The reaction
mixture was stirred at room temperature for 12 h under <span class="Chemical">hydrogen gas.
After replacement of air by <span class="Chemical">nitrogen, Pd/C was filtered off and methanol
was evaporated under reduced pressure. The crude product was chromatographed
on silica gel to afford the title compound (10′S)-9 (190 mg, 95%) as a white solid: mp 43–45
°C; Rf = 0.56 (20% EtOAc/hexanes); 1H NMR (CDCl3, 400 MHz) δ 8.01 (br s, 1H),
6.66 (m, 1H), 6.13 (dd, J = 2.8, 5.7 Hz, 1H), 5.91
(m, 1H), 3.80 (m, 1H), 2.59 (t, J = 7.4 Hz, 2H),
1.65–1.58 (m, 3H), 1.47–1.29 (m, 13H), 1.18 (d, J = 6.2 Hz, 3H); 13C NMR (CDCl3, 100
MHz) δ 132.9, 116.0, 108.2, 104.8, 68.2, 39.4, 29.7, 29.6 (2C),
29.5, 29.4 (2C), 27.7, 25.8, 23.5; HRMS (ESI) calcd for C15H28NO (M + H)+ 238.2165, found 238.2159; IR
(KBr) νmax 3511, 3243, 2923, 2849, 1574, 1470 cm–1. Compound (10′R)-9 has the same spectral data.
Representative Procedure
for the Synthesis of (S)-23-Hydroxyundecylprodiginines
((23S)-2)
To a stirred suspension
of <span class="Disease">MBCn> (8; 75 mg,
0.39 mmol) and (10′S)-9 (187
mg, 0.78 mmol) in anhydrous <span class="Chemical">methanol (10 mL) was added <span class="Chemical">methanolic
2 N HCl (catalytic amount) at room temperature. The resulting brightly
colored solution was stirred for 5 h at same temperature. The methanol
was removed under reduced pressure, and the crude material was dissolved
in ethyl acetate (75 mL) and washed with saturated NaHCO3 solution (2 × 25 mL). The organic layer was dried over anhydrous
Na2SO4, solvent was removed under reduced pressure,
and the crude product was chromatographed on neutral alumina as the
stationary phase and hexane/ethyl acetate as the mobile phase to afford
the desired (23S)-2 (102 mg, 63%): mp
93–95 °C; Rf = 0.30 (70% EtOAc/hexanes); 1H NMR (CDCl3, 400 MHz) δ 6.86 (s, 1H), 6.67
(dd, J = 1.1, 3.6 Hz, 1H), 6.61 (dd, J = 1.1, 2.7 Hz, 1H), 6.45 (d, J = 3.6 Hz, 1H), 6.11
(dd, J = 2.7, 3.6 Hz, 1H), 6.08 (s, 1H), 5.82 (d, J = 3.6 Hz, 1H), 3.97 (s, 3H), 3.79 (m, 1H), 2.05 (t, J = 7.6 Hz, 2H), 1.46–1.12 (m, 16H), 1.19 (d, J = 6.2 Hz, 3H); 13C NMR (CDCl3, 100
MHz) δ 169.1, 160.2, 144.7, 138.5, 128.4, 128.3, 123.0, 121.1,
116.0, 112.9, 110.0, 108.6, 95.7, 68.2, 58.4, 39.4, 29.7, 29.6 (3C),
29.4, 29.2, 27.3, 25.8, 23.5; HRMS (ESI) calcd for C25H36N3O2 (M + H)+ 410.2802,
found 410.2791; IR (KBr) νmax 3368, 3227, 2925, 2851,
1606, 1574, 1192 cm–1. Compound (23R)-2 has the same spectral data.
Synthesis of 10′-Ketoundecylpyrrole
(22)
Tetrapropylammonium perruthenate (<span class="Chemical">TPAPn>;
44 mg, 0.12 mmol) was added
to a stirred solution of (10′S)-9 (200 mg, 0.84 mmol), NMO (197 mg, 1.68 mmol), and 4 Å molecular
sieves (1.0 g) in <span class="Chemical">dichloromethane (10 mL) at 0 °C. The solution
was warmed to room temperature and stirred for 1.5 h. The reaction
mixture was filtered through Celite and washed with <span class="Chemical">dichloromethane
(100 mL). The filtrate was concentrated under reduced pressure to
give a crude residue, which was further chromatographed on silica
gel, with ethyl acetate/hexanes as eluent, to afford 22 (101 mg, 51%), and 23 (31 mg, 15%).
<span class="Chemical">Au(IPr)Cln> (136 mg, 0.22 mmol), <span class="Chemical">AgO2CCF3 (49
mg, 0.22 mmol), Shvo’s catalyst (239 mg, 0.22 mmol), 10-undecynoic
acid (26; 2.0 g, 10.98 mmol), <span class="Chemical">H2O (7 mL, 373.6
mmol), and isopropyl alcohol (IPA) (43 mL) were placed in a sealed
tube with a Teflon-lined cap under an argon atmosphere at room temperature.
The reaction mixture was stirred and heated for 48 h at 70 °C.
The mixture was filtered through Celite and washed with dichloromethane
(200 mL). The filtrate was concentrated under reduced pressure to
give a crude orange residue, which was further chromatographed on
silica gel, with ethyl acetate/hexanes as eluent, to afford the pure
10-hydroxyundecanoic acid (27) as a white solid (1.82
g, 82%): mp 46–48 °C; Rf =
0.35 (70% EtOAc/hexanes); 1H NMR (CDCl3, 400
MHz) δ 3.79 (m, 1H), 2.32 (t, J = 7.4 Hz, 2H),
1.59 (m, 2H), 1.44–1.24 (m, 12H), 1.17 (d, J = 6.2 Hz, 3H); 13C NMR (CDCl3, 100 MHz) δ
179.4, 68.3, 39.2, 34.0, 29.5, 29.3, 29.2, 29.0, 25.6, 24.7, 23.3;
HRMS (ESI) calcd for C11H22NaO3 (M
+ Na)+ 225.1461, found 225.1463; IR (KBr) νmax 3450, 2934, 2853, 1728, 1220 cm–1.
Synthesis
of 10-Oxoundecanoic Acid (28)
To a stirred solution
of 27 (1.0 g, 4.95 mmol) in anhydrous
<span class="Chemical">CH2Cl2n> (50 mL) was added <span class="Chemical">Dess–Martin
<span class="Chemical">periodinane (DMP; 3.14 g, 7.42 mmol) at 0 °C. The mixture was
stirred at room temperature for 10 h, and then the solvent was evaporated,
and diethyl ether was added to the crude product, and the reaction
mixture was stirred until a white solid was formed. The reaction mixture
was filtered through Celite and washed with diethyl ether (200 mL).
The filtrate was concentrated under reduced pressure to give a crude
residue, which was further chromatographed on silica gel, with ethyl
acetate/hexanes as eluent, to afford the pure product 28 as a white solid (881 mg, 89%): mp 51–53 °C; Rf = 0.45 (70% EtOAc/hexanes); 1H
NMR (CDCl3, 400 MHz) δ 2.40 (t, J = 7.4 Hz, 2H), 2.32 (t, J = 7.4 Hz, 2H), 2.11 (m,
3H), 1.64–1.50 (m, 4H), 1.27 (m, 8H); 13C NMR (CDCl3, 100 MHz) δ 209.6, 180.0, 43.7, 34.0, 29.8, 29.1, 29.0
(2C), 28.9, 24.6, 23.8; HRMS (ESI) calcd for C11H21O3 (M + H)+. 201.1485, found 201.1486; IR (KBr)
νmax 2921, 2845, 1726, 1701, 1235 cm–1.
Synthesis of [10-d]-10-Hydroxyundecanoic Acid
([10-d]-27)
To a stirred solution
of 28 (850 mg, 4.25 mmol) in anhydrous <span class="Chemical">methanoln> (15 mL)
was added slowly <span class="Chemical">NaBD4 (209 mg, 5.10 mmol) at 0 °C,
and the reaction mixture was stirred at the same temperature for 2
h. The resulting solution was quenched with 1 N <span class="Chemical">HCl and extracted
with ethyl acetate (3 × 50 mL), and the combined extracts were
washed with water and brine, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The crude residue
was further chromatographed on silica gel, with ethyl acetate/hexanes
as eluent, to afford the pure product [10-d]-27 as a white solid (690 mg, 80%): mp 45–47 °C; Rf = 0.35 (70% EtOAc/hexanes); 1H
NMR (CDCl3, 400 MHz) δ 2.32 (t, J = 7.4 Hz, 2H), 1.59 (m, 2H), 1.44–1.24 (m, 12H), 1.17 (s,
3H); 13C NMR (CDCl3, 100 MHz) δ 179.4,
67.8 (t, C-23), 39.0, 34.1, 29.5, 29.3, 29.1, 29.0, 25.6, 24.7, 23.2;
HRMS (ESI) calcd for C11H21DNaO3 (M
+ Na)+ 226.1522, found 226.1523; IR (KBr) νmax 3450, 2934, 2853, 1728, 1220 cm–1.
Representative
Procedure for the Synthesis of [10-d]-10-Acetoxyundecanoic
Acid ([10-d]-29)
<span class="Chemical">Acetic anhydriden>
(<span class="Chemical">Ac2O; 1.88 g, 18.47 mmol)
was added to a stirred solution of [10-d]-27 (1.5 g, 7.39 mmol) in <span class="Chemical">pyridine (10 mL), and the reaction mixture
was stirred for 12 h at room temperature. The reaction mixture was
diluted with ethyl acetate (200 mL), the solution was washed with
2 N HCl (3 × 50 mL) and brine, dried over anhydrous Na2SO4, and filtered, and the solvent was removed by rotary
evaporation to furnish the crude product. The crude compound was chromatographed
on silica gel using hexane/ethyl acetate as the mobile phase to afford
[10-d]-29 (1.70 g, 94%) as a syrup: Rf = 0.50 (40% EtOAc/hexanes); 1H
NMR (CDCl3, 400 MHz) δ 2.42 (m, 2H), 2.00 (s, 3H),
1.65–1.26 (m, 14H), 1.17 (s, 3H); HRMS (ESI) calcd for C13H24DO4 (M + H)+ 246.1810,
found 246.1811; IR (KBr) νmax 2931, 2857, 1742, 1710,
1264 cm–1.
Representative Procedure
for the Synthesis of 11-Oxo-11-(pyrrol-2-yl)undecan-2-yl-2-d Acetate ([10′-d]-30) and 11-Oxo-11-(pyrrol-3-yl)undecan-2-yl-2-d Acetate
([10′-d]-31)
To a stirred
solution of [10-d]-29 (1.5 g, 6.12 mmol)
and <span class="Chemical">dimethylformamiden> (<span class="Chemical">DMF; catalytic amount) in <span class="Chemical">toluene (10 mL) was
added thionyl chloride (SOCl2; 1.32 mL, 18.36 mmol). The
resulting solution was stirred at 80 °C for 2 h. After all solvents
were removed in vacuo, the residue was dissolved in toluene (50 mL),
and this solution was transferred into a stirred solution of pyrrole
(250 mg, 3.73 mmol) and Zn powder (485 mg, 7.46 mmol) in toluene (100
mL). After completion of the reaction (monitored by TLC, 2 h), the
reaction mixture was filtered through Celite and washed with dichloromethane
(200 mL). The filtrate was concentrated under reduced pressure to
give a crude residue, which was further chromatographed on silica
gel, with ethyl acetate/hexanes as eluent, to afford the pure [10′-d]-30 (625 mg, 57%), and [10′-d]-31 (88 mg, 8%).
Representative Procedure for the Synthesis
of [10′-d]-Hydroxyundecylpyrrole ([10′-d]-9)
To a stirred solution of [10′-d]-30 (500 mg, 1.70 mmol) in 100 mL of isopropyl
<span class="Chemical">alcoholn> (<span class="Chemical">IPA) at 25 °C was added slowly <span class="Chemical">sodium borohydride (NaBH4) (755 mg, 20.40 mmol), and the reaction mixture was heated
at reflux for 12 h. The hot reaction mixture was poured into 100 mL
of ice water, and the solution was acidified with 2 N HCl. The suspension
was extracted with dichloromethane (3 × 50 mL), and the combined
organic extracts were washed with water and brine and dried over anhydrous
Na2SO4. The solvent was evaporated under reduced
pressure, and the crude product was chromatographed on silica gel
to afford the title compound ([10′-d]-9 (271 mg, 67%) as a white solid: mp 46–48 °C; Rf = 0.55 (20% EtOAc/hexanes); 1H
NMR (CDCl3, 400 MHz) δ 7.99 (br s, 1H), 6.66 (dd, J = 2.7, 4.0 Hz, 1H), 6.13 (dd, J = 2.7,
5.7 Hz, 1H), 5.91 (m, 1H), 2.59 (t, J = 7.4 Hz, 2H),
1.64–1.58 (m, 2H), 1.47–1.27 (m, 14H), 1.18 (s, 3H); 13C NMR (CDCl3, 100 MHz) δ 132.9, 116.0, 108.2,
104.8, 67.7 (t, C-23), 39.2, 29.7, 29.6 (2C), 29.5, 29.4 (2C), 27.7,
25.7, 23.4; HRMS (ESI) calcd for C15H27DNO (M
+ H)+ 239.2228, found 239.2227; IR (KBr) νmax 3511, 3243, 2923, 2849, 1574, 1470 cm–1.
Synthesis
of [23-d]-23-Hydroxyundecylprodiginine
([23-d]-2)
Compound 42 (1.62 g,
80%) was synthesized from 7-oxooctanoic
acid by the same procedure as described for [10-d]-27 using <span class="Chemical">n class="Chemical">NaBH4: syrup; Rf = 0.25 (60% EtOAc/<span>n class="Chemical">hexanes); 1H NMR (CDCl3, 400 MHz) δ 3.77 (m, 1H), 2.31 (t, J = 7.4 Hz, 2H), 1.61 (m, 2H), 1.43–1.30 (m, 6H), 1.16 (t, J = 6.2 Hz, 3H); 13C NMR (CDCl3, 100
MHz) δ 179.1, 68.1, 38.8, 34.0, 29.0, 25.3, 24.6, 23.2; HRMS
(ESI) calcd for C8H16NaO3 (M + Na)+ 183.0992, found 183.0987; IR (KBr) νmax 3417,
2935, 2861, 1731, 1713 cm–1.
Synthesis
of 10-Acetoxyundecanoic Acid (29)
Compound 29 (1.72 g, 95%) was synthesized by the same
procedure as described for [10-d]-29: syrup; Rf = 0.50 (40% EtOAc/<span class="Chemical">hexanesn>); 1H NMR (CDCl3, 400 MHz) δ 4.85 (m, 1H), 2.31
(t, J = 7.5 Hz, 2H), 2.00 (s, 3H), 1.64–1.53
(m, 3H), 1.44 (m, 1H), 1.33–1.22 (m, 10H), 1.16 (d, J = 6.2 Hz, 3H); 13C NMR (CDCl3, 100
MHz) δ 180.0, 170.9, 71.1, 35.9, 34.0, 29.3, 29.2, 29.1, 29.0,
25.3, 24.6, 21.4, 19.9; HRMS (ESI) calcd for C13H25O4 (M + H)+ 245.1747, found 245.1740; IR (KBr)
νmax 2931, 2857, 1743, 1730, 1245 cm–1.
Synthesis of 10-Methoxyundecanoic Acid (43)
To a stirred solution of 27 (1.0 g, 4.95 mmol) in <span class="Chemical">DMFn>
(20 mL) was added gradually <span class="Chemical">NaH (237 mg, 9.90 mmol) at 0 °C.
The resulting suspension was stirred for 30 min, and <span class="Chemical">methyl iodide
(CH3I) (1.05 g, 7.42 mmol) was added over 10 min at the
same temperature. Then the reaction mixture was warmed to room temperature
and stirring was continued for 12 h. After completion of the reaction,
the reaction mixture was gradually poured into ice cold water and
extracted with ethyl acetate (3 × 100 mL). The combined organic
layers were washed with water and brine. The solvent was evaporated
under reduced pressure to give the crude product, which was dissolved
in THF (20 mL), and aqueous 5 N NaOH (10 mL) was added. The resulting
solution was stirred at 60 °C for 3 h, and then it was acidified
(pH ∼6) by 2 N HCl and extracted with ethyl acetate (3 ×
100 mL). The combined organic extracts were washed with water and
brine and dried over anhydrous Na2SO4. The solvent
was evaporated under reduced pressure to afford the pure product 43 (866 mg, 81%) as a syrup: Rf = 0.35 (30% EtOAc/hexanes); 1H NMR (CDCl3,
400 MHz) δ 3.32 (s, 3H), 3.30 (m, 1H), 2.33 (t, J = 7.5 Hz, 2H), 1.64–1.61 (m, 3H), 1.34–1.18 (m, 11H),
1.12 (d, J = 6.2 Hz, 3H); 13C NMR (CDCl3, 100 MHz) δ 179.7, 77.0, 55.8, 36.2, 34.1, 29.6, 29.4,
29.2, 29.0, 25.4, 24.7, 18.9; HRMS (ESI) calcd for C12H24NaO3 (M + Na)+ 239.1618, found 239.1619;
IR (KBr) νmax 2930, 2855, 1738, 1710, 1463 cm–1.
Synthesis of 10-(Benzyloxy)undecanoic Acid
(44)
A stirred solution of 2-benzyloxy-1-methylpyridinium
triflate (Dudley
reagent;[28] 3.45 g, 9.90 mmol), <span class="Chemical">MgOn> (396
mg, 9.90 mmol), and compound 27 (1.0 g, 4.95 mmol) in
<span class="Chemical">toluene (75 mL) was heated at 85 °C for 24 h. The reaction mixture
was cooled to room temperature, filtered through Celite, and washed
with <span class="Chemical">dichloromethane (100 mL). The filtrate was concentrated under
vacuum, the obtained crude product was dissolved in THF (20 mL), and
aqueous 5 N NaOH (10 mL) was added. The resulting solution was stirred
at 60 °C for 3 h and then acidified (pH ∼6) by 2 N HCl
and extracted with ethyl acetate (3 × 100 mL). The combined organic
extracts were washed with water and brine and dried over anhydrous
Na2SO4. The solvent was evaporated under reduced
pressure to afford the pure product 44 (1.09 g, 76%)
as a syrup: Rf = 0.30 (30% EtOAc/hexanes); 1H NMR (CDCl3, 400 MHz) δ 7.36–2.28
(m, 5H), 4.59 (d, J = 11.8 Hz, 1H), 4.49 (d, J = 11.8 Hz, 1H), 3.52 (m, 1H), 2.36 (t, J = 7.4 Hz, 2H), 1.67–1.61 (m, 3H), 1.45–1.35 (m, 11H),
1.18 (d, J = 6.2 Hz, 3H); 13C NMR (CDCl3, 100 MHz) δ 180.0, 139.1, 128.3 (2C), 127.7 (2C), 127.4,
74.9, 70.3, 36.6, 34.1, 29.6, 29.4, 29.2, 29.1, 25.5, 24.7, 19.6;
HRMS (ESI) calcd for C18H28NaO3 (M
+ Na)+ 315.1931, found 315.1930; IR (KBr) νmax 3030, 2929, 2855, 1731, 1454 cm–1.
Synthesis
of 7-Acetoxyoctanoic Acid (45)
Compound 45 (1.78 g, 94%) was synthesized by the same
procedure as described for [10-d]-29: syrup; Rf = 0.55 (60% EtOAc/<span class="Chemical">hexanesn>); 1H NMR (CDCl3, 400 MHz) δ 4.86 (m, 1H), 2.31
(t, J = 7.4 Hz, 2H), 2.00 (s, 3H), 1.62–1.28
(m, 8H), 1.16 (d, J = 6.2 Hz, 3H); 13C
NMR (CDCl3, 100 MHz) δ 179.6, 171.0, 71.0, 35.6,
33.9, 28.8, 25.0, 24.5, 21.3, 19.9; HRMS (ESI) calcd for C10H18NaO4 (M + Na)+ 225.1097, found
225.1088; IR (KBr) νmax 2977, 2939, 2863, 1738, 1710,
1374, 1246 cm–1.
Synthesis of 30, 31, 46a,b, 47a,b, and 48a,b
Compounds 30 (600 mg, 55%), 31 (76 mg, 7%), 46a (450 mg, 65%), 46b (62.2 mg, 9%), 47a (560
mg, 63%), 47b (71.2
mg, 8%), 48a (496 mg, 53%), and 48b (94
mg, 10%) were synthesized by the same procedure as described for [10′-d]-30 and [10′-d]-31.
Compounds 55a (614 mg, 59%), 55b (601 mg, 55%), 55c (641
mg, 56%), 56a (73 mg, 7%), 56b (87 mg, 8%),
and 56c (114 mg, 10%) were synthesized by the same procedure
as described for [10′-d]-30 and
[10′-d]-31.
Compounds 39 (83 mg, 80%), 40 (85 g, 79%), and 41 (83 g, 75%) were synthesized
by
the same procedure as described for <span class="Chemical">(23S)-2n>.
S.
venezuelae ATCC 15439 was used as a heterologous
host for marG overexpression.[14]<span class="Species">Escherichia colin> One Shot
Top10 (Invitrogen) was used as a host strain for the construction
of recombinant plasmids. <span class="Species">E. coli <span class="Chemical">ET12567
(pUZ8002) was used as donor strain in conjugation experiments.[29] pCR8-GW-TOPO (Invitrogen) was used as cloning
vector. pSE34 is a E. coli-Streptomycesshuttle vector, which contains pIJ101
derivative replication element and the constitutive strong ermE* promoter. pIJ778 is a plasmid containing an OriT transfer element required for conjugation and the spectinomycin-resistant
gene aadA. All bacterial strains, plasmids, and cosmid
used in this study are given in Table S1 (Supporting
Information).
General DNA Manipulations
8A7, a
SuperCosl cosmid carrying
the entire mar gene cluster, was isolated by the
standard protocol.[14,30] DNA fragments were recovered
from an <n class="Chemical">span class="Chemical">agarose gel by using the QIAquick Gel Extraction Kit (QIAGEN).
Restriction endonucleases were purchased from New England Biolab.
Preparation of plasmid DNA was done by using a QIAprep Spin Miniprep
Kit (QIAGEN). All other DNA manipulations were performed according
to standard protocols.[30,31] PCR was performed in 35 cycles
by using a GeneAmp PCR system 2700 (Applied Biosystems). Platinum Taq DNA polymerase high fidelity (Invitrogen) was used for
amplification of marG gene. <span>n class="Chemical">Oligodeoxyribonucleotides
for PCR primers were synthesized by Integrated DNA Technologies, and
their sequences are shown in Table S2 (Supporting
Information). The nucleotide sequences of the gene fragment
were determined at the MMI DNA analysis core facility, Oregon Health
and Sciences University (Portland, OR, USA).
Generation of marG Overexpression
Strain
The marG ORF was amplified using
forward primer marG-15bF, introducing a unique NdeI
site at the 5′-end of the
gene, and the reverse primer marG-15bR, introducing
a unique BamHI site downstream to the TGA translational
stop codon. Cosmid Dn class="Chemical">NA 8A7 was used as a template. The PCR fragment
was first cloned into the pCR8-GW-TOPO vector. The 1.4-kbp NdeI-BamHI insert was further subcloned into pET15b to generate
pMarG-15b. After sequencing to confirm the inset, this plasmid was
digested with XbaI and HindIII. The fragment containing
the marG gene, along with the ribosome binding site
(<span class="Disease">RBS), was cloned into the corresponding sites of pSE34 to generate
pMarG-34. The ampicillin resistance marker bla on
the pMarG-34 plasmid was replaced by aadA-oriT cassette generated by PCR from pIJ778 with primers Amp-SpF/Amp-SpR using the standard method of PCR targeting.[32] The resulting pMarG-34S was
introduced into S. venezuelae strain
by conjugation following the established protocol, except mannitol-soy
flour agar was replaced with AS1 media containing 10 mM MgCl2.[33] Thiostrepton-resistant exconjugants
representing S. venezuelae strains
host with marG overexpression plasmids. The new strain
was named S. venezuelae MarG.
Media,
Culture Techniques, and Feeding Experiments
Recombinant <span class="Species">S. venezuelaen> MarG was
grown on sporulation <span class="Chemical">agar (SPA; 0.1% <span class="Species">yeast extract, 0.1% beef extract,
0.2% tryptose, 0.01% ferrous sulfate, 1.0% glucose, and 2.0% Difco
bacto agar) containing 50 μg/mL of spectinomycin at 30 °C
for 7 days. Fresh spores were used to inoculate 10 mL of SCM seed
cultures (1.5% soluble starch, 2.0% soytone, 0.01% CaCl2, 0.15% yeast extract and 1.0% MOPS, pH 7.2) containing 10 μg/mL
of spectinomycin and were incubated at 30 °C for 3 days. Production
cultures were prepared with the same medium (150 mL each flask) and
inoculated with seed cultures (5.0% v/v). The production cultures
were grown in Baffled Erlenmeyer flasks at 30 °C with vigorous
shaking (220 rpm). After incubation for 3 days, the cultures were
grouped into four groups; the first group was fed with (23S)-2 (2.0 mg in 200 μL of DMSO), the
second group was fed with [23-d]-2 (2.0
mg in 200 μL of DMSO), the third group was fed with 3 (2.0 mg in 200 μL of DMSO), and the fourth group (control
group) was fed with 200 μL of DMSO. After 4 days of incubation,
the cultures were harvested by centrifugation at 5000 rpm, 4 °C
for 20 min. The harvested cells were disrupted by the addition of
20 mL of methanol solution containing 1.0% 2 N HCl followed by vortex
for 1 min. After filtration using cotton wool, the methanol extract
was dried under reduced pressure and stored at −20 °C
until further analysis. The same procedures were used for other analogues.
Premarineosin Production Analysis
The crude <span class="Chemical">methanolicn>
extract was dissolved in <span class="Chemical">methanol, centrifuged, and filtered through
a 0.22 μm filter to give a clear solution, and this was used
for our analysis. Analytical HPLC and LC-MS analyses were performed
on a C18 column (2.1 mm × 250 mm, 5 μm) with a linear elution
gradient as specified in Table S4 (Supporting
Information). The molecular weight of each compound was determined
by electrospray mass spectrometry. Extracted ion chromatograms and
tandem MS analyses were performed to analyze the consumption of feeding
substrates and the production of pre<span class="Chemical">marineosins.
Authors: Mai Nguyen; Richard C Marcellus; Anne Roulston; Mark Watson; Lucile Serfass; S R Murthy Madiraju; Daniel Goulet; Jean Viallet; Laurent Bélec; Xavier Billot; Stephane Acoca; Enrico Purisima; Adrian Wiegmans; Leonie Cluse; Ricky W Johnstone; Pierre Beauparlant; Gordon C Shore Journal: Proc Natl Acad Sci U S A Date: 2007-11-26 Impact factor: 11.205
Authors: Kancharla Papireddy; Martin Smilkstein; Jane Xu Kelly; Shaimaa M Salem; Mamoun Alhamadsheh; Stuart W Haynes; Gregory L Challis; Kevin A Reynolds Journal: J Med Chem Date: 2011-07-08 Impact factor: 7.446
Authors: Deborah A Smithen; A Michael Forrester; Dale P Corkery; Graham Dellaire; Julie Colpitts; Sherri A McFarland; Jason N Berman; Alison Thompson Journal: Org Biomol Chem Date: 2013-01-07 Impact factor: 3.876
Authors: Estelle Marchal; Deborah A Smithen; Md Imam Uddin; Andrew W Robertson; David L Jakeman; Vanessa Mollard; Christopher D Goodman; Kristopher S MacDougall; Sherri A McFarland; Geoffrey I McFadden; Alison Thompson Journal: Org Biomol Chem Date: 2014-06-28 Impact factor: 3.876
Authors: Jeffery T Davis; Philip A Gale; Oluyomi A Okunola; Pilar Prados; Jose Carlos Iglesias-Sánchez; Tomás Torroba; Roberto Quesada Journal: Nat Chem Date: 2009-04-19 Impact factor: 24.427
Authors: Papireddy Kancharla; Yuexin Li; Monish Yeluguri; Rozalia A Dodean; Kevin A Reynolds; Jane X Kelly Journal: J Med Chem Date: 2021-06-10 Impact factor: 7.446
Authors: Tristan de Rond; Parker Stow; Ian Eigl; Rebecca E Johnson; Leanne Jade G Chan; Garima Goyal; Edward E K Baidoo; Nathan J Hillson; Christopher J Petzold; Richmond Sarpong; Jay D Keasling Journal: Nat Chem Biol Date: 2017-09-11 Impact factor: 15.040
Authors: Ramsés A Gamboa-Suasnavart; Norma A Valdez-Cruz; Gerardo Gaytan-Ortega; Greta I Reynoso-Cereceda; Daniel Cabrera-Santos; Lorena López-Griego; Wolf Klöckner; Jochen Büchs; Mauricio A Trujillo-Roldán Journal: Microb Cell Fact Date: 2018-11-28 Impact factor: 5.328
Figure 1
Figure 2
Scheme 1
Scheme 2
Scheme 3
Scheme 4
Scheme 5
Figure 3
Scheme 6
Figure 4
Scheme 7
Scheme 8
Scheme 9
Figure 5