Synthesis of long-chain fatty acid enol esters isolated from an environmental DNA clone

Article abstract of DOI:10.1021/ol0267681

(Matrix presented) Long-chain fatty acid enol ester 1 is the major metabolite of a new family of small molecules isolated from the heterologous expression of environmentally derived DNA. A versatile synthesis of 1, in which an aromatic acetaldehyde is O-a

Full text of DOI:10.1021/ol0267681

ORGANIC
LETTERS
2003
Vol. 5, No. 2
121-124
Synthesis of Long-Chain Fatty Acid
Enol Esters Isolated from an
Environmental DNA Clone
Sean F. Brady and Jon Clardy*
Department of Chemistry and Chemical Biology, Cornell UniVersity,
Baker Laboratory, Ithaca, New York 14853-1301
jcc12@cornell.edu
Received August 22, 2002
ABSTRACT
Long-chain fatty acid enol ester 1 is the major metabolite of a new family of small molecules isolated from the heterologous expression of
environmentally derived DNA. A versatile synthesis of 1, in which an aromatic acetaldehyde is O-acylated with a long-chain acyl chloride
allowed for the rapid construction of both the isolated product (1) and a number of structural analogues (including 8, 17, and 18).
For the past 50 years natural products from cultured soil
microbes have played a central role in drug discovery.
Nucleic acid based studies now suggest that these cultured
bacteria represent only a small fraction of the bacterial
diversity present in most environmental samples.1 The
uncultured majority is a potentially rich source of structurally
unique natural products. Heterologous expression of DNA
extracted directly from environmental samples (environmen-
tal DNA, eDNA) in easily cultured hosts should provide
access to natural products produced by many previously
uncultured bacteria.2 The first new natural product biosyn-
thetic gene cluster characterized using this method, the fee
(fatty acid enol esters) gene cluster, produces long-chain fatty
acid enol esters that have not been previously characterized
from the extensive screening of cultured bacteria.3
synthesis of the major metabolite in this family was
undertaken to confirm the proposed structure 1 and provide
access to natural-product-like derivatives not found in the
culture broth of this heterologous expression system.
A
The fee gene cluster contains 13 opening reading frames
(feeA-feeM) and was cloned directly from soil collected
on the Cornell University campus. The N-acyl amino acid
synthase (NAS, FeeM) of the fee gene cluster together with
an acyl carrier protein (ACP) are thought to produce long-
(2) Brady, S. F.; Clardy, J. J. Am. Chem. Soc. 2000, 122, 12903-12904.
Brady, S. F.; Chao, C. J.; Handelsman, J.; Clardy, J. Org. Lett. 2001, 3,
1981-1984. Gillespie, D. E.; Brady, S. F.; Betterman, A. D.; Cianciotto,
N. P.; Liles, M. R.; Rondon, M. R.; Clardy, J.; Goodman, R. M.;
Handelsman, J. Appl. EnViron. Microbiol. 2002, 68, 4301-4306. Wang,
G.; Graziani, E.; Waters, B.; Pan, W.; Li, X.; McDermott, J.; Meurer, G.;
Saxena, G.; Anderson, R.; Davies, J. Org. Lett. 2000, 2, 2401-2404.
(3) Brady, S. F.; Chao, C. J.; Clardy, J. J. Am. Chem. Soc. 2002, 124,
9968-9969.
(1) Torsvik, V.; Sorheim, R.; Goksoyr, J. J. Ind. Microbiol. 1996, 17,
170-178. Torsvik, V.; Goksoyr, J.; Daae, F. L. Appl. EnViron. Microbiol.
1990, 56, 782-787. Hugenholtz, P.; Goebel, B. M.; Pace, N. R. J. Bacteriol.
1998, 180, 4765-4774. Stackebrandt, E.; Liesack, W.; Goebel, B. M.
FASEB J. 1993, 7, 232-236. Ward, D. M.; Weller, R.; Bateson, M. M.
Nature 1990, 345, 63-65.
10.1021/ol0267681 CCC: $25.00 © 2003 American Chemical Society
Published on Web 12/24/2002

We sought a flexible synthesis of 1 that could be used
to create a library of natural-product-like compounds for
future biological screening. It seemed likely that the most
versatile route to 1 would exploit the O-acylation of an
aldehyde (3) with an acid chloride (4) (Scheme 1). Because
Scheme 1
numerous long-chain acid chlorides and aromatic aldehydes
are commercially available the head, tail and configura-
tion of the double bond could be easily varied with this
approach.
Phenylacetaldehyde (5) was used to construct a family of
long-chain fatty acid enol esters, 6-8, as a model for the
formation of long-chain enol esters from an aromatic
acetaldehyde and a long-chain acyl chloride (Scheme 1,
Figure 2). These compounds are of particular interest because
Figure 1. (a) Long-chain enol esters 1 and 2 are derived from
tyrosine in successive steps catalyzed by an N-acyl amino acid
synthase (NAS),4 a decarboxylase, and an N-O transferase. (b)
When the fee gene cluster is expressed in E. coli the fatty acids
found in these natural products are likely specified by the E. coli
fatty acid loading machinery and not the fatty acid loading system
specific to the fee gene cluster.
chain N-acyltyrosines4 that are oxidatively decarboxylated
(FeeG) and converted from the eneamide to the enol ester
(FeeH) to give 1 and 2 (Figure 1). Transposon mutagenesis
studies suggest that the ACP could either be the fee-specific
ACP (FeeL) or the endogenous E. coli ACP. In each case it
appears that the native E. coli fatty acid loading machinery
and not the pathway-specific fatty acid loading system is
likely used to charge the ACP (Figure 1).3 The biological
properties of many natural products that contain long-chain
fatty acids (i.e. homoserine lactones, Nod factors, insect
pheromones, and lipid A) are highly dependent on the
specific long-chain fatty acids found within these molecules.
Because the E. coli fatty acid loading machinery is being
used in the biosynthesis of these molecules, the specific fatty
acid(s) encoded by the fatty acid loading system of the fee
gene cluster may not be incorporated into 1 and 2. Thus the
isolated metabolites 1 and 2 may not be the compounds
produced by the fee gene cluster in the organism from which
the eDNA was derived. The synthesis of compounds related
to 1 was undertaken to gain access to a pool of molecules
resembling metabolites that might be produced by the fee
gene cluster in the wild.
Figure 2. Long-chain enol esters prepared from phenylacetalde-
hyde as examples of synthetic phenylalanine derivatives of the
natural tyrosine based enol esters 1 and 2.
(4) The proposed biosynthesis of long-chain N-acyl amino acids is
inferred from published studies on the biosynthesis of long-chain homoserine
lactones. More, M. I.; Finger, L. D.; Stryker, J. L.; Fuqua, C.; Eberhard,
A.; Winans, S. C. Science 1996, 272, 1655-1658. Watson, W. T.; Minogue,
T. D.; Val, D. L.; Bec von Bodman, S.; Churchill, M. E. A. Mol. Cell
2002, 9, 685-694.
they represent the products of a fee-like biosynthetic path-
waythat uses phenylalanine in place of tyrosine as the amino
acid precursor.
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Org. Lett., Vol. 5, No. 2, 2003

The potassium enolate of phenylacetaldehyde (5) was
prepared using KH in ethylene glycol diethyl ether (EGDE)
at -5 °C.5 Upon the addition of a long-chain acid chloride
(2 equiv) to the potassium enolate, long-chain enol esters
6-8 were obtained in 64-84% yield. In general we have
observed that our yields decrease as the length of the fatty
acid increases. After purification by normal phase flash
chromatography (hexanes/dichloromethane) the cis and trans
isomers of each long-chain enol ester were obtained as
approximately 1:1 mixtures. The initial six members of the
phenylalanine-based library are cis and trans isomers of long-
chain fatty acid enol esters containing palmitoyl (6),6
myristoyl (7), and lauryl (8) groups (Figure 2).
19 were obtained in 65-85% yield. Each of the TBDMS-
protected enol esters were then deprotected using 1.1 equiv
of TBAF in THF (56-85% yield).11 With the appropriate
mobile phase (hexanes/dichloromethane) the cis and trans
isomers of the deprotected products could be separated by
normal phase flash chromatography.
The large coupling constant (J ) 13 Hz) observed in the
natural sample of 1 suggested that the enolate contained a
trans double bond. The cis and trans isomers of 1 were
separated cleanly by silica gel flash chromatography (90:
10, hexanes/dichloromethane) and the synthetic trans isomer
1a (1a trans, J ) 13 Hz, 1b cis, J ) 7.5 Hz) was found to
be spectroscopically identical to the isolated compound. In
addition to 1, cis and trans isomers of three analogues that
differ in the fatty acid attached to the tyrosine derived
headgroup were constructed. These derivatives include
analogues containing linoleic (17), arachidonic (18), and
capric (19) fatty acids (Figure 3).
The acetaldehyde needed for the synthesis of long-chain
enol esters based on tyrosine was obtained from the oxidation
of tert-butyldimethylsilyl (TBS)-protected 4-hydroxyphen-
ethyl alcohol (9). 4-Hydroxyphenethyl alcohol (9) was
protected as the bis TBS silyl ether7 (10) followed by
selective deprotection of the secondary silyl ether with I2 in
methanol8 to afford 11 in 87% yield over two steps. A Swern
oxidation of the TBS protected 4-hydroxyphenethyl alcohol
(11) provided aldehyde 12 in 67% yield.9
The synthesis of long-chain enol esters from 12 using KH
gave only poor yields in our hands. Even when acetyl
chloride was used as a model reactant in place of the less
soluble longer-chain acid chlorides, a large amount of the
starting aldehyde (12) was recovered from the reaction.
Under these conditions it is appears that KH is not able to
efficiently form the K enolate of the para-substituted acetal-
dehyde 12. We found that replacing KH with potassium bis-
(trimethylsilane)amide (KHMDS) under essentially the same
reaction conditions greatly increased the yield of long-chain
enol esters.10 Using KHMDS to trap the potassium enolate
of 12, the TBS-protected long-chain enol esters of 1 and 17-
(5) The procedure for the formation of the enol ester from a potassium
enolate was modified slightly from: Ladjama, D.; Riehl, J. J. Synthesis
1979, 7, 504-507. KH (1.1 equiv) was cleaned of mineral oil using three
pentane washes and then resuspended in EGDE at 1 mmol/500 µL; 0.5
mmol of the aldehyde (5) in 200 µL of EGDE was added to 1.1 equiv of
KH stirring in EGDE at -5 °C. Two equivalents of acid chloride (chilled
in 400 µL of EGDE) was added to the K enolate after 10 min and then
stirred at room temperature for 15 min. After the addition of 1 mL of water
the reaction was extracted five times with pentane. The dried organic extracts
were then chromatographed using normal phase flash chromatography with
hexanes/diethyl ether and hexanes/dichloromethane.
(6) NMR data for representative long-chain enol esters with phenyl-
alanine-derived headgroups 6a,b (cis:trans): 1H NMR (500 MHz,
CD2Cl2) δ 7.89 (d, J ) 13, 1H), 7.62 (m, 2H), 7.30-7.38 (m, 7H), 7.22-
7.28 (m, 2H), 6.41 (d, J ) 13, 1H), 5.72 (d, J ) 7, 1H), 2.54 (t, J ) 7.5,
2H), 2.45 (t, J ) 7.5, 2H), 1.66-1.75 (m, 4H), 1.24-1.42 (m, 48H), 0.91
(t, J ) 7.5, 6H); 13C NMR (100 MHz, CD2Cl2) δ 171.3, 170.8, 137.0,
134.9, 134.8, 134.6, 129.7, 129.3, 129.0, 127.9, 127.8, 126.7, 115.4, 112.0,
34.7, 34.5, 32.6, 30.3, 30.3, 30.2, 30.1, 30.0, 29.9, 29.7, 25.3, 25.3, 23.3,
14.5.
Figure 3. The isolated long-chain enol ester 1a and analogues
constructed as initial members of a library of compounds based on
1 and 2.
The general strategy presented here (Scheme 2) is ap-
plicable to the rapid synthesis of a modest library of
(11) NMR data for representative long-chain enol esters with tyrosine-
derived headgroups 1a (trans): 1H NMR (500 MHz, CD2Cl2) δ 7.74 (d, J
) 13, 1H), 7.22, (d, J ) 9, 2H), 6.78 (d, J ) 9, 2H), 6.33 (d, J ) 13, 1H),
2.42 (t, J ) 7.5, 2H), 1.66 (m, 2H), 1.2-1.4 (m, 24H), 0.88 (t, J ) 6.5,
3H); 13C NMR (100 MHz, CD2Cl2) δ 171.5, 155.8, 135.6, 128.1, 127.4,
116.1, 115.0, 34.5, 32.5, 30.3, 30.2, 30.2, 30.0, 29.9, 29.8, 29.6, 25.3, 23.3,
14.5. 1b (cis): 1H NMR (500 MHz, CD2Cl2) δ 7.49 (d, J ) 9, 2H), 7.20
(d, J ) 7.5, 1H), 6.81 (d, J ) 9, 2H), 5.63 (d, J ) 7.5, 1H), 2.52 (t, J )
7, 2H), 1.71 (m, 2H), 1.2-1.4 (m, 24H), 0.88 (t, J ) 6.5, 3H); 13CNMR
(100 MHz, CD2Cl2) δ 170.9, 155.4, 133.1, 131.1, 127.6, 115.8, 111.5, 34.7,
32.5, 30.3, 30.2, 30.2, 30.0, 29.9, 29.8, 29.6, 25.3, 23.3, 14.4. 19a,b (cis
and trans): 1H NMR (500 MHz, CD2Cl2) δ 7.73 (d, J ) 13), 7.49 (d, J )
8.5), 7.20-7.23 (m), 6.81 (d, J ) 8.5), 6.78 (d, J ) 8.5), 6.33 (d, J ) 13),
5.63 (d, J ) 7), 2.52 (t, J ) 8), 1.6-1.8 (m), 1.2-1.4 (m), 0.88 (t, J )
6.5); 13CNMR (100 MHz, CD2Cl2) δ 171.5, 171.0, 155.8, 155.5, 135.5,
133.1, 131.1, 128.1, 127.6, 127.4, 116.1, 115.8, 115.1, 111.6, 34.7, 34.5,
32.4, 30.0, 29.8, 29.6, 25.3, 25.3, 23.2, 14.4.
(7) Corey, E. J.; Venkateswarlu, A. J. Am. Chem. Soc. 1972, 94, 6190-
6191.
(8) Lipshutz, B. H.; Keith, J. Tetrahedron Lett. 1998, 39, 2495-2498.
(9) Mancuso, A. J.; Huang, S.-L.; Swern, D. J. Org. Chem. 1978, 43,
2480-2482.
(10) KHMDS (1.1 equiv, as a 0.5 M solution in THF) was added to 0.5
mmol of 12 in 100 µL of EGDE. After 1 min at -5 °C a chilled solution
of acid chloride (2 equiv) dissolved in EGDE (160 µL) was added to the K
enolate and allowed to stir at room temperature for 15 min. After the addition
of 1 mL of water the reactions were extracted five times with pentane. The
dried organic extracts were then chromatographed using normal phase flash
chromatography with hexanes/ethyl acetate.
Org. Lett., Vol. 5, No. 2, 2003
123

produced 12 analogues to examine the overall applicability
of this strategy. A larger library is currently being made and
will be screened in a number of bioassays in an attempt to
define the role of this new family of long-chain enol esters.
Using organic synthesis to understand the roles that biosyn-
thetic small molecules play among uncultured soil bacteria
is a unique contribution of chemistry to illuminating the
unknown world of uncultured microorganisms.
Scheme 2
Acknowledgment. We thank Professor David B. Collum
for his prophetic and helpful suggestions. This work was
supported by a grant from the Ellison Medical Foundation
and CA24487.
Supporting Information Available: 1H and 13C NMR
spectra for representative long-chain enol esters based on
phenylalanine (6a,b) and tyrosine (1a,b and 19a,b). This
material is available free of charge via the Internet at
http://pubs.acs.org.
compounds similar to 1. Using this approach we both
confirmed the structure of the isolated product (1) and
OL0267681
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Org. Lett., Vol. 5, No. 2, 2003