Stereochemistry and Mechanism of Undecylprodigiosin Oxidative Carbocyclization to Streptorubin B by the Rieske Oxygenase RedG

Article abstract of DOI:10.1021/jacs.5b03994

The prodiginines are a group of specialized metabolites that share a 4-methoxypyrrolyldipyrromethene core structure. Streptorubin B is a structurally remarkable member of the prodiginine group produced by Streptomyces coelicolor A3(2) and other actinobacteria. It is biosynthesized from undecylprodigiosin by an oxidative carbocyclization catalyzed by the Rieske oxygenase-like enzyme RedG. Undecylprodigiosin derives from the RedH-catalyzed condensation of 2-undecylpyrrole and 4-methoxy-2, 2′-bipyrrole-5-carboxaldehyde (MBC). To probe the mechanism of the RedG-catalyzed reaction, we synthesized 2-(5-pentoxypentyl)-pyrrole, an analogue of 2-undecylpyrrole with an oxygen atom next to the site of C-C bond formation, and fed it, along with synthetic MBC, to Streptomyces albus expressing redH and redG. This resulted in the production of the 6′-oxa analogue of undecylprodigiosin. In addition, a small amount of a derivative of this analogue lacking the n-pentyl group was produced, consistent with a RedG catalytic mechanism involving hydrogen abstraction from the alkyl chain of undecylprodigiosin prior to pyrrole functionalization. To investigate the stereochemistry of the RedG-catalyzed oxidative carbocyclization, [7′-²H](7′R)-2-undecylpyrrole and [7′-²H](7′S)-2-undecylpyrrole were synthesized and fed separately, along with MBC, to S. albus expressing redH and redG. Analysis of the extent of deuterium incorporation into the streptorubin B produced in these experiments showed that the pro-R hydrogen atom is abstracted from C-7′ of undecylprodigiosin and that the reaction proceeds with inversion of configuration at C-7′. This contrasts sharply with oxidative heterocyclization reactions catalyzed by other nonheme iron-dependent oxygenase-like enzymes, such as isopenicillin N synthase and clavaminate synthase, which proceed with retention of configuration at the carbon center undergoing functionalization. (Chemical Equation Presented).

Full text of DOI:10.1021/jacs.5b03994

Article
pubs.acs.org/JACS
Stereochemistry and Mechanism of Undecylprodigiosin Oxidative
Carbocyclization to Streptorubin B by the Rieske Oxygenase RedG
David M. Withall, Stuart W. Haynes, and Gregory L. Challis*
Department of Chemistry, University of Warwick, Coventry CV4 7AL, United Kingdom
*
S Supporting Information
ABSTRACT: The prodiginines are a group of specialized metabolites that
share a 4-methoxypyrrolyldipyrromethene core structure. Streptorubin B is a
structurally remarkable member of the prodiginine group produced by
Streptomyces coelicolor A3(2) and other actinobacteria. It is biosynthesized
from undecylprodigiosin by an oxidative carbocyclization catalyzed by the
Rieske oxygenase-like enzyme RedG. Undecylprodigiosin derives from the
RedH-catalyzed condensation of 2-undecylpyrrole and 4-methoxy-2, 2′-
bipyrrole-5-carboxaldehyde (MBC). To probe the mechanism of the RedG-
catalyzed reaction, we synthesized 2-(5-pentoxypentyl)-pyrrole, an analogue
of 2-undecylpyrrole with an oxygen atom next to the site of C−C bond
formation, and fed it, along with synthetic MBC, to Streptomyces albus expressing redH and redG. This resulted in the production
of the 6′-oxa analogue of undecylprodigiosin. In addition, a small amount of a derivative of this analogue lacking the n-pentyl
group was produced, consistent with a RedG catalytic mechanism involving hydrogen abstraction from the alkyl chain of
undecylprodigiosin prior to pyrrole functionalization. To investigate the stereochemistry of the RedG-catalyzed oxidative
2
2
carbocyclization, [7′- H](7′R)-2-undecylpyrrole and [7′- H](7′S)-2-undecylpyrrole were synthesized and fed separately, along
with MBC, to S. albus expressing redH and redG. Analysis of the extent of deuterium incorporation into the streptorubin B
produced in these experiments showed that the pro-R hydrogen atom is abstracted from C-7′ of undecylprodigiosin and that the
reaction proceeds with inversion of configuration at C-7′. This contrasts sharply with oxidative heterocyclization reactions
catalyzed by other nonheme iron-dependent oxygenase-like enzymes, such as isopenicillin N synthase and clavaminate synthase,
which proceed with retention of configuration at the carbon center undergoing functionalization.
INTRODUCTION
natural material consists of the anti-(7′S), syn-(7′S), and anti-
■
5
,6
(
7′R) isomers in an approximately 88:7:5 ratio.
The prodiginines are a group of specialized metabolites with a
common 4-methoxypyrrolyldipyrromethene core produced by
a variety of bacteria, including Streptomyces and related
actinobacteria (Figure 1). Members of the prodiginine family
possess a range of interesting biological activities and have
recently been shown to be orally effective antimalarials in a
murine infection model.
Streptomyces coelicolor A3(2) produces undecylprodigiosin 1
and its carbocyclic derivative streptorubin B 2 (Figure 1).
Transannular interactions in the 10-membered carbocycle of
The biosynthesis of undecylprodigiosin 1 and streptorubin B
7
2
in S. coelicolor has been extensively studied. Hybrid
nonribosmomal peptide synthetase/polyketide synthase/α-oxo-
amine synthase assembly lines construct 4-methoxy-2,2′-
bipyrrole-5-carboxaldehyde (MBC) 3 and 2-undecylpyrrole 4
from L-proline, malonyl-CoA, L-serine, and S-adenosyl-L-
methionine, and one unit of acetyl-CoA, six units of malonyl-
CoA, and a unit of glycine, respectively (Figure 1) (CoA =
1
1
,2
3
,4
4,8
coenzyme A, Ad = adenosine). MBC 3 and 2-undecylpyrrole
4
are condensed to form undecylprodigiosin 1 by RedH
9
streptorubin B 2 restrict rotation around the C2−C1′ and C4−
(
Figure 1). The broad substrate tolerance of RedH has been
5
C7′ bonds, resulting in the formation of atropisomers. Nuclear
exploited to produce undecylprodigiosin analogues via a
9
Overhauser effect spectroscopy (NOESY) NMR experiments
showed that the hydrochloride salt of 2 exists as predominantly
mutasynthesis approach. Streptorubin B 2 is formed from
undecylprodigiosin 1 via an unusual oxidative carbocyclisation
5,6
10,11
the anti atropisomer in solution. The absolute configuration
of this atropisomer was assigned as 7′S by H and H NMR
spectroscopic analyses of stereoselectively deuterium labeled
reaction catalyzed by RedG,
a Rieske nonheme iron-
1
2
12
dependent oxygenase-like enzyme (Figure 1). Remarkably, in
Streptomyces longisporusruber DSM 40667 McpG, which is 75%
similar in sequence to RedG, catalyzes the regio- and
stereodivergent carbocylisation of undecylprodigiosin 1 to
5
streptorubin B.HCl produced via a mutasynthesis approach.
This stereochemical assignment was confirmed by enantiose6-
lective total synthesis and X-ray crystallographic analysis.
Chiral high pressure liquid chromatography (HPLC) compar-
isons of 2 isolated from S. coelicolor with chemically synthesized
10
form metacycloprodigiosin 5 (Figure 1).
Received: April 17, 2015
(
7′S)-, (7′R)-, and (7′RS)-streptorubin B showed that the
©
XXXX American Chemical Society
A
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Figure 1. Pathway for the biosynthesis of undecylprodigiosin 1 and streptorubin B 2 in S. coelicolor A3(2). RedH catalyzes the condensation of 2-
undecylpyrrole 4 and 4-methoxy-2,2′-bipyrrole-5-carboxaldehyde 3 to form 1, which undergoes RedG-catalyzed oxidative carbocyclization to form 2.
In Streptomyces longisporusruber DSM40667, McpG catalyzes the oxidative carbocyclisation of 1 to form metacycloprodigiosin 5.
Figure 2. Proposed mechanism for the RedG-catalyzed conversion of undecylprodigiosin 1 to streptorubin 2. Alternative Fe(V)O(OH) and C-7′/C-
5
cation intermediates are also possible.
Based on the well-studied catalytic mechanism of naph-
streptorubin B 2. It was noted that an Fe(V)O(OH) complex, a
C-7′ carbocation and a 10-membered carbocycle bearing a
cation at C-5 are plausible alternative intermediates to 6, 7 and
9, respectively. One of the more remarkable facets of this
mechanistic proposal is that the hydrocarbon chain of
undecylprodigiosin 1 is oxidized by the Fe(III)OOH complex
in preference to the considerably more electron rich 4-
methoxypyrrolyldipyrromethene π-system.
thalene dioxygenase, we recently proposed a mechanism for the
RedG-catalyzed oxidative carbocyclization reaction involving
abstraction of a hydrogen atom from C-7′ of enzyme bound
undecylprodigiosin 1 by the active site Fe(III)OOH complex 6
12
to form a carbon-centered radical 7 and an Fe(IV)O(OH )
2
12
complex 8 (Figure 2). Addition of the radical to C-4 of the
proximal pyrrole leads to formation of the 10-membered
carbocycle 9 bearing a radical at C-5, which can be stabilized by
delocalization into the adjacent π-system. Hydrogen abstraction
Here we report the synthesis of 2-(5-pentoxypentyl)-pyrrole
10, a 2-undecylpyrrole analogue designed to probe the RedG
catalytic mechanism. The products resulting from feeding of
from C-4 of 9 by the Fe(IV)O(OH ) complex 8 yields
2
B
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Scheme 1. Synthesis of 2-(5-Pentoxypentyl)-pyrrole 10
Figure 3. Extracted ion chromatograms (EICs) at m/z = 396.26; 326.18 from LC-MS analyses of culture extracts of S. albus expressing redHG fed
with MBC 3 and 2-(5-pentoxypentyl)-pyrrole 10 (top panel), and S. albus expressing redH fed with MBC 3 and 2-(5-pentoxypentyl)-pyrrole 10
(
middle panel). The corresponding EIC from LC-MS analysis of a synthetic standard of 5-hydroxypentylprodigiosin 17 is shown in the bottom
panel. Peak doubling is due to geometrical isomerism, resulting from rotation around the bond between C-1″ and C-5 of ring C.
1
3
this 2-undecylpyrrole analogue, together with synthetic MBC 3,
to Streptomyces albus J1704 expressing redH and redG
demonstrate that hydrocarbon oxidation precedes functionali-
zation of the 4-methoxypyrrolyldipyrromethene moiety. We
butyldimethylsilyl (TBS)-protected 1,5-pentandiol 11 in six
steps (Scheme 1). Deprotonation of 11 with sodium hydride
and reaction of the resulting alkoxide with 1-bromopentane
afforded the corresponding ether 12. Deprotection of 12 with
tetra-n-butyl ammonium fluoride (TBAF) gave alcohol 13,
which was oxidized to carboxylic acid 14, via the aldehyde, by
sequential treatment with iodoxybenzoic acid (IBX) and
Coupling of 14 with pyrryl magnesium bromide,
followed by reduction of the resulting 2-acylpyrrole 15, were
both accomplished according to established procedures,
2
also report the synthesis of both enantiomers of [7′- H]-2-
undecylpyrrole. Feeding of these stereoselectively deuterium
labeled 2-undecylpyrroles, along with synthetic MBC 3, to S.
albus expressing redH and redG elucidated the stereochemical
course of the undecylprodigiosin to streptorubin B conversion,
allowing the remarkable chemoselectivity of RedG to be
rationalized.
14,15
oxone.
4
affording 2-(5-pentoxypentyl)-pyrrole 10.
Feeding of 2-(5-Pentoxypentyl)-pyrrole 10 and MBC 3
to S. albus Expressing redHG. We previously reported that
feeding of MBC 3 and 2-undecylpyrrole 4 to a strain of S. albus
expressing redH under the control of the constitutive ermE*
EXPERIMENTAL PROCEDURES
See the Supporting Information.
■
11
RESULTS AND DISCUSSION
promoter produces undecylprodigiosin 1. We also reported
that a similar experiment using a strain of S. albus expressing
redHG results in the production of streptorubin B 2, in addition
■
Synthesis of 2-(5-Pentoxypentyl)-pyrrole 10. We
reasoned that an analogue of undecylprodigiosin 1 with an
oxygen atom in place of the C-6′ methylene group would allow
us to probe the timing of hydrocarbon oxidation relative to
pyrrole functionalization in the RedG-catalyzed carbocycliza-
tion reaction, by stabilizing a radical/cationic intermediate at C-
11
to undecylprodigiosin 1.
Feeding of 2-(5-pentoxypentyl)-pyrrole 10 and synthetic
8
MBC 3 to S. albus expressing redH resulted in the formation of
a red pigment that gave rise to ions with m/z = 396.26 in liquid
7
′, which may lead to the formation of shunt products. Thus,
chromatography-mass spectrometry (LC-MS) analyses, corre-
sponding to the [M + H] ion for the 6′-oxa undecylprodi-
+
we synthesized 2-(5-pentoxypentyl)-pyrrole 10 from t-
C
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Scheme 2. Synthesis of 5-Hydroxypentylprodigiosin 17
2
a
Scheme 3. Synthesis of [7′- H](7′R)-2-Undecylpyrrole 22
a
18
2
Compound 26 is the TBS ether of (R)-glycidol, which was prepared according to a literature procedure. [7′- H](7′S)-2-undecylpyrrole 23 was
synthesized via the same route using the TBS ether of (S)-glycidol.
giosin analogue 16 (Figure 3). No production of 16 was
observed when 10 and 3 were fed to wild type S. albus. When 2-
pentoxypentyl)-pyrrole 10 and MBC 3 are fed to S. albus
expressing redHG has the same retention time and
fragmentation pattern as the authentic standard of 17 (Figure
3 and Supporting Information).
(
5-pentoxypentyl)-pyrrole 10 and synthetic MBC 3 were fed to
S. albus expressing redHG, the 6′-oxa undecylprodigiosin
analogue 16 was also produced (Figure 3), but none of the
corresponding streptorubin B analogue could be detected.
Instead ultrahigh resolution LC-MS analyses showed that a
small amount of a compound with a molecular formula
corresponding to 5-hydroxypentylprodigiosin 17 (calculated for
Synthesis of 2-Undecylpyrrole Stereoselectively Deu-
terium Labeled at C-7′. To probe the stereoselectivity of
hydrogen abstraction from C-7′ and the stereochemical course
of the subsequent carbocyclization reaction, we synthesized
2
2
[7′- H](7′R)-2-undecylpyrrole 22 and [7′- H](7′S)-2-undecyl-
pyrrole 23 from commercially available R- and S-glycidol,
respectively, (Scheme 3). 4-Pentyn-1-ol 24 was protected as its
+
2
C H N O , 326.1863; found, 326.1858), a derivative of 16
19
24
3
lacking the n-pentyl group, was produced (Figure 3).
Compound 17 was not produced when 10 and 3 were fed to
S. albus expressing redH alone (Figure 3).
18
4-methoxybenzyl ether 25. TBS-protected R-glycidol 26 was
reacted in the presence of BF ·OEt with the acetylide derived
from 25 by deprotonation with n-BuLi to yield homopropar-
3
2
To confirm the production of 5-hydroxypentylprodigiosin 17
when 2-(5-pentoxypentyl)-pyrrole 10 and MBC 3 are fed to S.
albus expressing redHG, an authentic synthetic standard was
gyllic alcohol 27. Reduction with H over 10% Pd−C converted
2
27 to the saturated diol 28. Selective protection of the primary
alcohol in 28 with pivaloyl choride in pyridine gave ester 29,
which was converted to the corresponding tosylate 30 by
treatment with 4-toluenesulfonyl chloride (TsCl), 4-dimethy-
13
prepared in six steps from TBS-protected 1,5-pentandiol 11
(
Scheme 2). Swern oxidation of 11 yielded the corresponding
1
6
aldehyde, which was further oxidized to the acid 18 using
Pinnick’s reagents. Coupling of acid 18 with pyrryl
1
7
laminopyridine (DMAP) and Et N. Reaction of 30 with TBAF
3
magnesium bromide and reduction of the resulting 2-
acylpyrrole 19 to the corresponding alkylpyrrole 20, was
followed by deprotection with TBAF to give 2-(5-hydrox-
yielded epoxide 31, which was converted to the alcohol 32
4
19
20
using n-PrLi, CuCN, and BF ·OEt . The enantiopurity of
3 2
32 was assessed by converting a small portion to the
21
1
ypentyl)-pyrrole 21. Acid-promoted condensation of 21 with
corresponding Mosher’s esters, which were analyzed by H
NMR spectroscopy (see the Supporting Information). While a
precise enantiometric excess for the esters could not be
6
BOC-protected MBC afforded 5-hydroxypentylprodigiosin 17
as its HCl salt. LC-MS/MS analyses confirmed that the
dealkylated undecylprodigiosin analogue produced when 2-(5-
1
determined due to signal overlap, the H NMR data confirmed
D
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2
Figure 4. Mass spectra of streptorubin B 2 from LC-MS analyses of mycelial extracts of S. albus expressing redHG fed with MBC 3 and [7′- H](7′S)-
2
2
-undecylpyrrole 23 (top spectrum) or [7′- H](7′R)-2-undecylpyrrole 22 (bottom spectrum). When 23 is fed, 92% of the label is incorporated into
streptorubin B, whereas when 22 is fed, 77% of the label is calculated to be lost (after subtraction from the m/z = 393.2750 signal in the bottom
panel of the component due to unlabeled 13C streptorubin B isotopomer).
1
that 32 is predominantly the 7S enantiomer. Alcohol 32 was
Comparison of chromatograms resulting from monitoring of
absorbance at 533 nm (corresponding to the λmax for the
protonated 4-methoxypyrrolyldipyrromethene chromophore of
1 and 2) showed that the amount of streptorubin B 2 produced
relative to undecylprodigiosin 1 was much lower in the
converted to the tosylate 33, which was reacted with LiAlD to
4
22
afford (7R)-7-deuterioundecan-1-ol 34. Swern oxidation of 34
gave the corresponding aldehyde, which was further oxidized
to the acid 35 using oxone. Coupling of acid 35 with pyrryl
16
15
2
magnesium bromide and reduction of the resulting 2-
experiment utilizing [7′- H](7′R)-2-undecylpyrrole 22 than in
2
4
2
acylpyrrole 36 yielded [7′- H](7′R)-2-undecylpyrrole 22.
that using [7′- H](7′S)-2-undecylpyrrole 23 (see the Support-
2
Using TBS-protected S-glycidol in place of 26, [7′- H](7′S)-
-undecylpyrrole 23 was synthesized via an analogous sequence
ing Information). This is consistent with a primary kinetic
2
isotope effect for the abstraction of the deuterium atom from
2
of transformations.
[7′- H](7′R)-undecylprodigiosin, which may account for the
Feeding of Deuterium-Labeled Pyrroles 22 and 23,
and MBC 3, to S. albus Expressing redHG. LC-MS analysis
of mycelial extracts of S. albus expressing redHG that had been
discrepancy in the extent of deuterium retention in streptorubin
2
B 2 when [7′- H](7′S)-2-undecylpyrrole 23 was used
compared with the amount of deuterium loss from streptorubin
2
2
fed with [7′- H](7′S)-2-undecylpyrrole 23 and MBC 3 showed
B 2 when [7′- H](7′R)-2-undecylpyrrole 22 was employed.
that 92% of the resulting streptorubin B 2 is deuterium labeled
Mechanistic Implications. The observed production of 5-
hydroxypentylprodigiosin 17 when 2-(5-pentoxypentyl)-pyr-
role 10 and MBC 3 are fed to S. albus expressing redHG is
consistent with a RedG catalytic mechanism involving oxidation
of C-7′ prior to pyrrole functionalization. Presumably, 17 arises
via abstraction of a hydrogen atom at C-7′ of 10 by the
Fe(III)OOH complex 6 to form a radical (or cation) that is
(
Figure 4). In contrast, 77% of the deuterium label is lost in
2
streptorubin B 2 resulting from the feeding of [7′- H](7′R)-2-
undecylpyrrole 22 and MBC 3 to S. albus expressing redHG
(
Figure 4). These data show that the pro-R hydrogen atom is
preferentially abstracted from C-7′ in the RedG-catalyzed
conversion of undecylprodigiosin 1 to streptorubin B 2.
E
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Figure 5. Mechanistic interpretation of the results from feeding of 2-(5-pentoxypentyl)-pyrrole 10 and stereoselectively deuterium labeled 2-
undecylpyrroles 22 and 23, along with MBC 3, to S. albus expressing redHG. The C-7′ radical formed by abstraction of the pro-R hydrogen atom in
undecylprodigiosin 1 by the RedG ferric hydroperoxide complex 6 undergoes inversion prior to adding to C-4 of the proximal pyrrole, resulting in S-
configured streptorubin B 2. The reduced reactivity of the radical formed via abstraction of a hydrogen atom from C-7′ of 2-(5-pentoxypentyl)-
pyrrole 10 by ferric hydroperoxide complex 6 prevents it from adding to C-4 of the proximal pyrrole. Instead, the radical “rebounds” onto the ferryl
complex 8 to form the hemiketal complex 38, which undergoes protonation-triggered collapse to liberate pentanal and form 5-
hydroxypentylprodigiosin 17.
unable to add to C-4 of the proximal pyrrole, either because the
reactivity of the radical (cation) toward the pyrrole is reduced
by the neighboring oxygen lone pair, or because of repulsion
between the lone pairs of the C-6′ oxygen atom and the pyrrole
from 4-methoxypyrrolyldipyrromethene moiety, which could
prevent its electron rich π-system from being directly oxidized
by the Fe(III)OOH complex 6. However, this arrangement
would probably place the Fe(IV)O(OH ) complex 8 too far
2
π-face (or both). The Fe(IV)O(OH ) complex 8 formed by
away from the hydrogen atom that needs to be removed from
intermediate 9 to produce streptorubin B 2 for this to occur via
direct abstraction. This hydrogen atom could be extracted
directly if positioned near one of the oxygen atoms of the
hydroperoxide ligand in the Fe(III)OOH complex 6, but this
would require a change in the conformation of the alkyl chain
after abstraction of the pro-R hydrogen atom from C-7′ to
permit C−C bond formation. Alternatively, the orientation of
intermediate 9 in the active site could change to allow direct
2
hydrogen abstraction from 10 by 6 reacts with the C-7′ radical
instead, forming the hemiketal complex 37, which upon
protonation can collapse with loss of n-pentanal to give 5-
hydroxypentylprodigiosin 17 (Figure 5). Similar dealkylation
reactions are known to be catalyzed by other members of the
Rieske nonheme iron-dependent oxygenase family, such as
Dicamba monoxygenase, which demethylates Dicamba (3, 6-
dichloro-2-methoxybenzoate) via hydroxylation to form a
23
hemiacetal that collapses with elimination of formaldehyde.
hydrogen abstraction by the Fe(IV)O(OH ) complex 8, or
2
Analysis of deuterium incorporation into streptorubin B 2
resulting from feeding of stereoselectively deuterium labeled 2-
undecylpyrroles 22 and 23, together with MBC 3, to S. albus
expressing redHG shows that RedG abstracts predominantly
the pro-R hydrogen atom from C-7′ of undecylprodigiosin 1.
Together with the previous observation that streptorubin B 2
produced by S. coelicolor has predominantly the 7′S
indirect hydrogen abstraction from intermediate 9 by complex
8 could occur via an appropriately placed active site water
molecule. It is even conceivable that an electron is transferred
from intermediate 9 to complex 8. The resulting C-5 cation
intermediate could then be deprotonated by a basic residue
within the active site, obviating the need for the nonheme iron
center to be located near H-4 of the pyrrole. This C-5 cation
intermediate could also be formed via hydride abstraction from
C-7′ by the Fe(III)OOH complex 6 to form a transient alkyl
cation that reacts with C-4 of the pyrrole. Alternatively, the
transient alkyl cation could result from reaction of intermediate
5
configuration, this result shows that the RedG-catalyzed C−
C bond forming reaction proceeds with inversion of
configuration at C-7′ (Figure 5). In contrast, carbon-
heteroatom bond formation in oxidative cyclizations catalyzed
by other nonheme iron-dependent enzymes, such as
isopenicillin N synthase and clavaminate synthase proceeds
with retention of configuration at the carbon atom undergoing
7
with complex 8, installing a hydroxyl group that undergoes
subsequent protonation to promote its departure. Further
experiments will be required to discriminate between these
various possibilities.
2
4,25
functionalization.
This stereochemical outcome is observed
because the heteroatom participating in oxidative cyclization is
CONCLUSIONS
ligated cis to the oxygen binding site of the nonheme iron
■
2
6,27
center in these enzymes.
The results reported herein not only illuminate the mechanism
of the RedG-catalyzed oxidative carbocyclization of undecyl-
prodigiosin 1 to streptorubin B 2, but also provide a starting
point for understanding and investigating the mechanism and
stereochemistry of similar oxidative carbocyclization reactions
catalyzed by RedG homologues in the biosynthesis of other
prodiginine alkaloids. For example, McpG has been shown to
The inversion of configuration at C-7′ in the RedG-catalyzed
reaction suggests that the alkyl chain of the substrate may be
juxtaposed between its 4-methoxypyrrolyldipyrromethene
moiety and the nonheme iron center (Figure 5), preventing
the former from coordinating to the latter. Such an arrange-
ment uses the alkyl chain to “insulate” the nonheme iron center
F
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Figure 6. Proposed catalytic mechanism for MarG. The key difference from the RedG catalytic mechanism is that the intermediate 41 resulting from
addition of the alkyl radical to the 4-methoxypyrrolyldipyrromethene π-system is hypothesized to react with the Fe(IV)O(OH ) complex 8 to
2
produce the hydroxylated product 42.
Figure 7. Proposed mechanism for formation of the premarineosins 39 from intermediate 42. The premarineosins are reduced to form the
marineosins 40 by MarA.
catalyze the conversion of undecylprodigiosin 1 to metacyclo-
prodigiosin 5 with opposite absolute stereocontrol, resulting in
attacks C-1″. The resulting delocalized radical 41 can be
quenched by “rebounding” onto the Fe(IV)O(OH ) complex 8
2
10
the 9′R absolute configuration (Figure 1). It seems reasonable
to propose that this also proceeds with inversion of
configuration, for the reasons discussed above, but involves
abstraction of the pro-S hydrogen atom from C-9′. This
hypothesis could be tested by synthesizing both enantiomers of
(rather than by hydrogen atom abstraction as in the RedG
proposed catalytic mechanism) to give a product 42 that has
undergone both oxidative carbocyclisation and hydroxylation
(Figure 6). Elimination of water from 42, followed by addition
of the C-10′ hydroxyl group to the π-system would yield the
premarineosins 39 (Figure 7). Addition of the C-10′ hydroxyl
group to the alternative delocalized cation intermediate would
yield the premarineosins 39 directly.
2
-undecylpyrrole stereoselectively deuterium-labeled at C-9′
and analyzing whether the deuterium atom is retained when
each is fed, along with MBC, to strains of S. albus expressing
mcpH and mcpG.
The gene cluster believed to direct the biosynthesis of
deschlororoseophilin 43 and prodigiosin R1 44 in Streptomyces
griseoviridis contains four RedG homologues (RphG, RphG2,
Recently, Reynolds and co-workers reported that the RedG
homologue MarG catalyzes the conversion of (10′S)-10′-
hydroxyundecylprodigiosin 38 to the premarineosins 39
30
RphG3 and RphG4). One of these (RphG3) is likely
nonfunctional because it lacks the conserved Cys and His
residues that ligate the [2Fe-2S] cluster in the N-terminal
Rieske domain. Another presumably catalyzes C−C bond
formation between C-4 and C-9′ in 11′-dimethyl-undecylpro-
digiosin 45 to form prodigiosin R1 44 via a very similar process
(
Figures 6 and 7), which undergo MarA-catalyzed reduction
28,29
to marineosins A and B 40 (Figure 7).
We hypothesize that
the MarG catalytic cycle involves abstraction of a hydrogen
atom from C-8′ of 38 by the Fe(III)OOH complex 6 (or an
Fe(V)O(OH) complex) to yield a radical (or cation) that
G
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Figure 8. Proposed mechanism for the biosynthesis of deschlororoseophilin 43 and prodigiosin R1 44 in Streptomyces griseoviridis.
to the McpG-catalyzed conversion of undecylprodigiosin 1 to
(3) Tsao, S.-W.; Rudd, B. A. M.; He, X.-G.; Chang, C.-J.; Floss, H. G.
J. Antibiot. 1985, 38, 128−131.
4) Mo, S.; Sydor, P. K.; Corre, C.; Alhamadsheh, M. M.; Stanley, A.
E.; Haynes, S. W.; Song, L.; Reynolds, K. A.; Challis, G. L. Chem. Biol.
008, 15, 137−148.
5) Haynes, S. W.; Sydor, P. K.; Corre, C.; Song, L.; Challis, G. L. J.
Am. Chem. Soc. 2011, 133, 1793−1798.
6) Hu, D. X.; Clift, M. D.; Lazarski, K. E.; Thomson, R. J. J. Am.
Chem. Soc. 2011, 133, 1798−1804.
7) For a recent review, see: Challis, G. L. J. Ind. Microbiol. Biotechnol.
014, 41, 219−232.
8) Stanley, A. E.; Walton, L. J.; Kourdi-Zerikly, M.; Corre, C.;
Challis, G. L. Chem. Commun. 2006, 3981−3983.
9) Haynes, S. W.; Sydor, P. K.; Stanley, A. E.; Song, L.; Challis, G. L.
10
metacycloprodigiosin 5 (Figure 8). The remaining two RedG
homologues are hypothesized to be responsible for the
conversion of 45 to deschlororoseophilin 43 (Figure 8). One
is proposed to catalyze C−C bond formation between C-10′
and C-4 in 45 to give 46 (Figure 8). The other is proposed to
catalyze the conversion of 46 to 47 via an analogous process to
the MarG-catalyzed conversion of (10′S)-10′-hydroxyundecyl-
prodigiosin 38 to 42 (Figures 6 and 8). Rearrangement of
intermediate 47 via ring opening, tautomerization, ring closure
and elimination of ammonia would give deschloroseophilin 43
(
2
(
(
(
2
(
(
Figure 8).
(
ASSOCIATED CONTENT
■
Chem. Commun. 2008, 1865−1867.
*
S
Supporting Information
(10) Sydor, P. K.; Barry, S. M.; Odulate, O. M.; Barona-Gomez, F.;
Haynes, S. W.; Corre, C.; Song, L.; Challis, G. L. Nat. Chem. 2011, 3,
388−392.
1
Experimental procedures and H NMR spectra of the Mosher’s
esters of alcohol 32 and its enantiomer. The Supporting
Information is available free of charge on the ACS Publications
website at DOI: 10.1021/jacs.5b03994.
(
11) Sydor, P. K.; Challis, G. L. Methods Enzymol. 2012, 516, 195−
18.
12) Barry, S. M.; Challis, G. L. ACS Catal. 2013, 3, 2362−2370.
13) McDougal, P. G.; Rico, J. G.; Oh, Y.-I.; Condon, B. D. J. Org.
Chem. 1986, 51, 3388−3390.
14) Travis, B. R.; Sivakumar, M.; Hollist, G. O.; Borhan, B. Org. Lett.
2
(
(
AUTHOR INFORMATION
Corresponding Author
g.l.challis@warwick.ac.uk
■
(
*
2
003, 5, 1031−1034.
Notes
(15) More, J. D.; Finney, N. S. Org. Lett. 2002, 4, 3001−3003.
The authors declare no competing financial interest.
(16) Omura, K.; Swern, D. Tetrahdedron 1978, 34, 1651−1660.
(17) Bal, B. S.; Childers, W. E.; Pinnick, H. W. Tetrahedron 1981, 37,
ACKNOWLEDGMENTS
This work was funded by BBSRC through a doctoral training
grant to the University of Warwick, the RSC Briggs Scholarship
2091−2096.
18) Ichimaru, N.; Murai, M.; Kakutani, N.; Kako, J.; Ishihara, A.;
Nakagawa, Y.; Nishioka, T.; Yagi, T.; Miyoshi, H. Biochemistry 2008,
7, 10816−10826.
19) Freeman, P. K.; Hutchinson, L. L. J. Org. Chem. 1980, 45,
924−1930.
20) Lipshutz, B. H.; Wilhelm, R. S.; Kozlowski, J. A.; Parker, D. J.
Org. Chem. 1984, 49, 3928−3938.
■
(
4
(
1
(
(
to D.M.W.) and the European Commission (Framework 6
integrated project, contract no. 005224). The assistance of Dr.
Ivan Prokes and Dr. Lijiang Song with acquisition of NMR and
mass spectra, respectively, is gratefully acknowledged. Regan
Thomson and Dennis Hu (Northwestern University) are
thanked for helpful discussions.
(
21) Dale, J. A.; Mosher, H. S. J. Am. Chem. Soc. 1973, 95, 512−519.
(22) The displacement of homochiral secondary tosylates with
LiAlD is known to proceed with clean inversion. See: Ashby, E. C.;
4
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DOI: 10.1021/jacs.5b03994
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