Biomimetic synthesis of a new class of bacterial signaling molecules

Article abstract of DOI:10.1021/ol9009893

The first synthesis of a newly discovered class of bacterial signaling molecules from Streptomyces coelicolor has been developed. These molecules, known as the methylenomycin furans (MMFs), trigger production of the antibiotic methylenomycin. The synthesis features a scandium triflatecatalyzed domino reaction of β-ketoesters and dihydroxyacetone yielding 2,3,4-substituted furans. The proposed reaction sequence (aldol reaction, cyclization, and dehydrative aromatization) may be reminiscent of the biosynthetic reaction in which dihydroxyacetone phosphate and a β-ketothioester are condensed by an enzyme.

Full text of DOI:10.1021/ol9009893

ORGANIC
LETTERS
2009
Vol. 11, No. 14
2984-2987
Biomimetic Synthesis of a New Class of
Bacterial Signaling Molecules
Jesse B. Davis, J. Daniel Bailey, and Jason K. Sello*
Department of Chemistry, Brown UniVersity, 324 Brook Street,
ProVidence, Rhode Island 02912
jason_sello@brown.edu
Received May 5, 2009
ABSTRACT
The first synthesis of a newly discovered class of bacterial signaling molecules from Streptomyces coelicolor has been developed. These molecules,
known as the methylenomycin furans (MMFs), trigger production of the antibiotic methylenomycin. The synthesis features a scandium triflate-
catalyzed domino reaction of ꢀ-ketoesters and dihydroxyacetone yielding 2,3,4-substituted furans. The proposed reaction sequence (aldol reaction,
cyclization, and dehydrative aromatization) may be reminiscent of the biosynthetic reaction in which dihydroxyacetone phosphate and a ꢀ-ketothioester
are condensed by an enzyme.
Bacteria commonly use low molecular weight signaling mol-
ecules to coordinate complex behaviors such as biofilm forma-
tion, differentiation, and antibiotic production.1 Streptomyces
bacteria are known to use signaling molecules to regulate
antibiotic production. These soil-dwelling bacteria are the
producers of two-thirds of the clinically used antibiotics.2
Recently, Challis and co-workers reported the discovery of a
furans (MMFs) as they induce the production of the antibiotic
methylenomycin and share a furan core structure (Figure 1).
These compounds are structurally distinct from the γ-butyro-
lactones, the major class of signaling molecules employed by
Scheme 1. Proposed Biosynthetic Pathways of γ-Butyrolactones
and MMFs
Figure 1. Methylenomycin furans (MMFs) from S. coelicolor.
new class of Streptomyces signaling molecules with a 2-alkyl-
4-hydroxymethylfuran-3-carboxylic acid core structure.3 Five
compounds differing in the identity of the alkyl group at C2
were isolated from S. coelicolor and termed the methylenomycin
the Streptomyces genus.4 The prototypical γ-butyrolactone is
A-Factor, an inducer of streptomycin biosynthesis in Strepto-
myces griseus (Scheme 1).
(1) Bassler, B. L.; Losick, R. Cell 2006, 125, 237–246.
(2) Hopwood, D. A. Streptomyces in Nature and Medicine: The
Antibiotic Makers; Oxford University Press: New York, 2007.
(3) Corre, C.; Song, J.; O’Rourke, S.; Chater, K. F.; Challis, G. L. Proc.
Natl. Acad. Sci. U.S.A. 2008, 105, 17510–17515.
(4) Takano, E. Curr. Opin. Microbiol. 2006, 9, 287–294.
10.1021/ol9009893 CCC: $40.75
Published on Web 06/22/2009
 2009 American Chemical Society

Interestingly, both the γ-butyrolactones and MMFs are
biosynthetically derived from the same starting materials,
dihydroxyacetone phosphate and a coenzyme A ꢀ-ke-
tothioester (Scheme 1). These substrates are condensed by
distinct enzymes belonging to the AfsA superfamily.3 Based
on the inherent reactivity of 1,3-dicarbonyl compounds and
ketones, we suspected that catalysts used in synthetic organic
chemistry could be to used to effect condensations reminis-
cent of the enzymatic reactions in the biosyntheses of the
signaling molecules. In model studies, we tested this
hypothesis using a ꢀ-ketoester (methyl 3-oxononanoate) in
place of the coenzyme A ꢀ-ketothioester and ketal-protected
dihydroxyacetone in place of dihydroxyacetone phosphate.
Our expectation was that these starting materials would
undergo a Knoevenagel-type reaction to give an R,ꢀ-
unsaturated dicarbonyl compound that would subsequently
lactonize upon ketal deprotection, yielding the butenolide
intermediate (1) in γ-butyrolactone biosynthesis (Scheme 1).
In initial condensations, we examined the catalytic activity
of piperidine, ammonium acetate, and diethylamine, which
are known to catalyze carbon-carbon bond-forming reac-
unsaturated dicarbonyl compound product was not observed;
however, a methyl 2-alkyl-4-hydroxymethylfuran-3-carboxylate
product was isolated in good yield. This product has the identical
skeleton observed in the S. coelicolor MMF signaling mol-
ecules. In subsequent experiments, we found that this reaction
is also catalyzed by ytterbium triflate but not significantly by
bismuth triflate or zinc chloride (Table 1). In the absence of
scandium or ytterbium triflate, no reaction of the substrates was
observed. Apparently, Sc(OTf)3 and Yb(OTf)3 catalyze the
condensation and dehydrative aromatization of dihydroxyac-
etone and ꢀ-ketoesters.
Given the mechanistic curiosity of the metal-catalyzed
condensation and its potential use in the chemical synthesis
of the MMFs, we studied the reaction in more detail. The
structure of the product indicated that in situ deprotection
of the dihydroxyacetone ketal took place prior to furan
formation. Indeed, we observed rapid (<1 min) deprotection
of the dihydroxyacetone by scandium triflate in methanol
by thin-layer chromatography.8 The rate of deprotection is
clearly important because condensations in THF, where
deprotection is markedly slower, yield much less product
than those performed in methanol (42% vs 72% yield). Next,
we examined the importance of the order of substrate and
catalyst addition because both dihydroxyacetone and ꢀ-ke-
toesters are prone to self-condensation. We found that
premixing scandium triflate with either of the substrates
followed by addition of the other substrate led to an increase
in side reactions and a ∼30% decrease in furan yield. The
optimal reaction conditions involved premixing of the
substrates followed by addition of the catalyst. Then, we
investigated the importance of reactant stoichiometry given
the propensity of the ꢀ-ketoester and dihydroxyacetone to
self-condense. It was found that a 1:1 stoichiometry was
optimal. Finally, catalyst loadings were screened and the
reaction was found to be highest yielding when 10 mol %
of scandium triflate was used. Any more than 10 mol %
catalyst resulted in side reactions that lowered the yield, while
5 mol % led to a slower reaction rate. With these optimized
reaction conditions, we obtained methyl 2-hexyl-4-hy-
droxymethylfuran-3-carboxylate from ketal-protected dihy-
droxyacetone and methyl 3-oxononanoate in 72% yield.
Furan formation likely involves an aldol reaction of the
ꢀ-ketoester and dihydroxyacetone. To examine the substrate
scope of this reaction, we reacted a ꢀ-ketoester with two
different substrates containing R-hydroxy groups, hydroxy-
acetone, and glycolaldehyde (Table 2). These substrates
reacted to form the expected furans in 51 and 56% yields,
respectively. In contrast, only starting materials were recov-
ered from reactions with ketones lacking an R-hydroxy group
(cyclohexanone, cyclopentanone, or 3-pentanone). The ab-
sence of aldol products in those reactions suggested that the
R-hydroxy group is crucial for irreversible carbon-carbon
bond formation. Thus, we propose that under these conditions
ꢀ-ketoesters and R-hydroxycarbonyl compounds undergo a
domino reaction.5b,9 They first react in a reversible aldol
Table 1. Condensation Catalyst Screeninga
catalyst
loading (mol %)
% yield
Sc(OTf)3
Yb(OTf)3
Bi(OTf)3
ZnCl2
10
10
10
10
72
56
trace
trace
a Reactions were performed at room temperature for 15 h with equimolar
substrates.
tions of 1,3-dicarbonyls.5 No reaction was observed with any
of the amine catalysts. These results can be ascribed to the
lower reactivity of ketones relative to that of aldehydes in
Knoevenagel condensations.6 Next, we tested Lewis acidic
metals as condensation catalysts because they have been
shown to effect additions of 1,3-dicarbonyl compounds to
aldehydes or ketones.7 For practical and mechanistic consid-
erations, we focused our efforts on air- and moisture-insensitive
Lewis acid catalysts (Table 1). First, we reacted methyl
3-oxononanoate and ketal-protected dihydroxyacetone in metha-
nol using scandium triflate as a catalyst. The expected R,ꢀ-
(5) (a) Tietze, L. F.; Beifuss, U. The Knoevenagel Reaction. In
ComprehensiVe Organic Syntheses; Trost, B. M., Fleming, I., Eds.;
Pergamon: Oxford, 1991; Vol. 2, pp 341-394. (b) Tietze, L. F.; Rackelmann
N. The Domino Knoevenagel-Hetero-Diels-Alder Reaction and Related
Transformations. In Multicomponent Reactions; Zhu J., Bie, H., Eds.; Wiley-
VCH: Weinheim, 2005; Vol. 12, pp 1-168.
(6) Smith, M., March, J., Eds. March’s AdVanced Organic Chemistry:
Reactions, Mechanisms, and Structure, 5th ed.; John Wiley & Sons, Inc.:
New York, 2001.
(7) (a) Rao, P. S.; Venkataratnam, R. V. Tetrahedron Lett. 1991, 32,
5821–5822. (b) Narsaiah, A. V.; Nagaiah, K. Synth. Commun. 2003, 33,
3825–3832.
(8) The deprotection of acetals by scandium triflate is known. Oriyama,
T.; Watahiki, T.; Kobayashi, Y.; Hirano, H.; Suzuki, T. Synth. Commun.
2001, 31, 2305–2311.
Org. Lett., Vol. 11, No. 14, 2009
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NMR spectra and the masses of the compounds were
identical to those reported for the natural MMFs (see the
Supporting Information).3
Table 2. Condensation Substrate Scopea
The dihydroxyacetone and ꢀ-ketoesters used in our chemi-
cal synthesis are structurally reminiscent of the enzymatic
Scheme 3. Condensation with a ꢀ-Ketothioester Substratea
a The reaction was performed at room temperature for 18 h with
equimolar substrates.
substrates, dihydroxyacetone phosphate and coenzyme A
ꢀ-ketothioesters. To directly test whether these structural
similarities translate into similar reactivities, we carried out a
scandium-catalyzed condensation using ketal-protected dihy-
droxyacetone and a coenzyme A thioester mimic, the N-
acetylcysteamine ꢀ-ketothioester (SNAC).10 This reaction gave
the expected furan thioester, 3, in 42% yield (Scheme 3).11
Our proposed mechanism for furan formation has interest-
ing implications for the biosyntheses of both the γ-butyro-
lactones and MMF signaling molecules in Streptomyces
bacteria. Both classes of signaling molecules are derived from
dihydroxyacetone phosphate and a coenzyme A ꢀ-ke-
tothioester via the action of homologous enzymes. It is not
clear how homologous enzymes generate different molecular
skeletons from the same substrates. In contrast to the
proposed biosyntheses of γ-butyrolactones and MMFs,3,12
we suspect that an intermediate, 4, resulting from the aldol
reaction of a coenzyme A ꢀ-ketothioester and dihydroxyac-
etone phosphate substrates is common to the biosynthesis
of both classes of signaling molecules (Scheme 4). Although
only the MMF-like furan product is observed in the labora-
tory, it is conceivable that an enzyme could generate either
the furan (2) or butenolide (1) product via different chemose-
lective ring-closing reactions (Scheme 4). Reaction of the
primary alcohol with the thioester will yield the butenolide
intermediate of γ-butyrolactone biosynthesis. Reaction of the
primary alcohol with the ketone will yield a furan product
leading to formation of the MMFs. The true test of this
hypothesis awaits biochemical characterization of the en-
zymes involved in MMF biosynthesis.
a Reactions were performed at room temperature for 18 h with equimolar
substrates.
reaction, followed by addition of the R-hydroxy group to
the ketone yielding a ketal intermediate that undergoes dehy-
drative aromatization to give the furan. The equilibrium most
likely favors starting materials, but product formation is driven
by irreversible aromatization. Curiously, the nucleophilic pri-
mary alcohol of the aldol product does not react with the
electrophilic carbonyl of the methyl ester; a butenolide product,
which would result from such a reaction, was not isolated.
The carbon skeleton of the condensation product was the
same as in the proposed structures of the S. coelicolor
Scheme 2. Synthesis of the S. coelicolor MMFs
MMFs.3 To validate those structures, we synthesized all five
MMFs using the appropriate ꢀ-ketoester substrates and ketal-
protected dihydroxyacetone. The furan methyl esters were
formed in good yields (60-72%) by the scandium-catalyzed
condensation of the ꢀ-ketoester and ketal-protected dihy-
droxyacetone in methanol at room temperature within 18 h.
We obtained the S. coelicolor MMFs 1-5 in 71-92% yield
by saponification of the corresponding furan methyl esters
using 2.5 equiv of LiOH in 1:1 THF/water (Scheme 2). The
The scandium-catalyzed condensation and dehydrative
aromatization of ꢀ-ketoesters and R-hydroxy ketones under
mild conditions is a remarkable domino reaction. This
reaction is a variant of the Garcia Gonzalez reaction, which
is a metal-catalyzed condensation of ring-opened sugars and
(10) (a) Cane, D. E.; Yang, C. J. Am. Chem. Soc. 1987, 109, 1255–
1257. (b) Gilbert, I. H.; Ginty, M.; O’Neill, J. A.; Simpson, T. J.; Staunton,
J.; Willis, C. L Bioorg. Med. Chem. Lett. 1995, 5, 1587–1590.
(11) All reactions with the SNAC substrate were perfomed using THF
as a solvent as the thioester was not sufficiently stable in methanol.
(12) Kato, J.; Funa, N.; Watanabe, H.; Ohnishi, Y.; Horinouchi, S. Proc.
(9) Tietze, L. F. Chem. ReV. 1996, 96, 115–136.
Natl. Acad. Sci. U. S. A. 2007, 104, 2378–2383
.
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Org. Lett., Vol. 11, No. 14, 2009

dicarbonyl compounds.13,14 In contrast to the Garcia Gonza-
lez reaction, the key reaction reported in the MMF synthesis
uses an R-hydroxy ketone as a substrate with only 10 mol
% catalyst, and it proceeds at room temperature in good yield.
Our findings are of further interest because a metal catalyst
appears to mimic a reaction catalyzed by an enzyme. In any
case, this work highlights the potential for using synthetic
organic chemistry to provide mechanistic insight into com-
plex enzymatic reactions.15
Scheme 4. Proposed Biosynthetic Pathways of the
γ-Butyrolactones and MMFs
Acknowledgment. Brown University and a Frontiers in
Chemistry grant from the Department of Chemistry at Brown
are gratefully acknowledged for financial support. Dr. Russell
Hopson and Dr. Tun-Li Shen from the Department of
Chemistry at Brown University are gratefully acknowledged
for technical support in NMR and MS analysis.
Supporting Information Available: Procedures and
spectroscopic data for all compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
OL9009893
(13) (a) Rodrigues, F.; Canac, Y.; Lubineau, A. Chem. Commun. 2000,
2049–2050. (b) Bartoli, G.; Fernandez-Bolaos, J. G.; Di Antonio, G.; Foglia,
G.; Giuli, S.; Gunella, R.; Mancinelli, M.; Marcantoni, E.; Paoletti, M. J.
Org. Chem. 2007, 72, 6029–6036
(14) A furan methyl ester product was isolated from a zinc chloride-
catalyzed reaction of hydroxyacetone and ethyl acetoacetate. Garcia
.
Gonzalez, F. AdV. Carbohydr. Chem. 1956, 11, 97–143
(15) Kim, J.; Ashenhurst, J. A.; Movassaghi, M. Science 2009, 324,
238–241.
.
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