Synthetic libraries of tyrosine-derived bacterial metabolites

Article abstract of DOI:10.1016/j.bmcl.2007.10.058

The preparation of a collection of 131 small molecules, reminiscent of families of long chain N-acyl tyrosines, enamides and enol esters that have been isolated from heterologous expression of environmental DNA (eDNA) in Escherichia coli, is reported. The synthetic libraries of N-acyl tyrosines and their 3-keto counterparts were prepared via solid-phase routes, whereas the enamides and enol esters were synthesized in solution-phase.

Full text of DOI:10.1016/j.bmcl.2007.10.058

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 3117–3121
Synthetic libraries of tyrosine-derived bacterial metabolites
Savvas N. Georgiades and Jon Clardy*
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School,
240 Longwood Avenue, Boston, MA 02115, USA
Received 19 September 2007; accepted 16 October 2007
Available online 22 October 2007
Abstract—The preparation of a collection of 131 small molecules, reminiscent of families of long chain N-acyl tyrosines, enamides
and enol esters that have been isolated from heterologous expression of environmental DNA (eDNA) in Escherichia coli, is reported.
The synthetic libraries of N-acyl tyrosines and their 3-keto counterparts were prepared via solid-phase routes, whereas the enamides
and enol esters were synthesized in solution-phase.
Ó 2007 Elsevier Ltd. All rights reserved.
Microbes, especially soil-dwelling bacteria, have made
enormous contributions to our stock of biologically ac-
tive small molecules.1 Discovering these molecules usu-
ally requires isolating the producing organism,
culturing it in the laboratory and assaying the culture
extracts for biological activity. The realization that only
a tiny and unrepresentative minority of soil and other
microorganisms can be cultured by currently described
techniques2 led many laboratories to develop alternative
strategies for accessing the small molecules produced by
the uncultured majority.3,4 Such processes typically in-
volve obtaining DNA—not the producing organism—
directly from the environment (thus termed environmen-
tal DNA or eDNA) and incorporating it into alternative
hosts to discover gene-host combinations with the
capacity to produce biologically active compounds. In
our laboratory we have employed this approach to ex-
press eDNA-encoded pathways in Escherichia coli, and
used an antibiotic assay to identify colonies producing
small molecule antibiotics. The most frequently identi-
fied metabolites have been long chain N-acyl amino
acids,3b,5 especially N-acyl tyrosines (NATs), and the
widespread occurrence of these compounds, which had
not been previously reported as microbial natural prod-
ucts, raised questions about their biosynthesis and bio-
logical function(s).
One pathway uncovered using this approach produced
NATs that were first converted to N-acyl (E)-enamides
through an oxidative decarboxylation, and finally to
N-acyl (E)-enol esters through an unusual N,O-exchan-
ge5a (Fig. 1a). Formation of the amide is catalyzed by an
N-acyl synthase that couples free tyrosine to a long
chain acid, delivered by an acyl carrier protein (ACP).6
The structure, mechanism and sequence alignment of
a
O
HO
HO
NATs
S
ACP
R1
O
NAT Synthase
HO
HO
N
R1
NH2
H
O
O
HO
Oxidative
HO
N,O-Transferase
O
O
Decarboxylase
O
R1
N
R1
H
(R1=Simple long chains, saturated or unsaturated)
O
AHLs
R2
b
HO
HO
O
O
N
S
ACP
S
H
R2
O
O
+
HO
OH
AHL Synthase
Adenine
O
NH2
Adenine
O
O
S
Keywords: Libraries; Bacterial metabolites; Antibiotics; Environmental
DNA; Solid-phase synthesis; Microwave-assisted reactions; Acyl Mel-
drum’s acids; Tyrosine; Long chain acids; N-Acyl amino acids; Ena-
mides; Enol esters.
(R2=Simple or 3-keto long chains, saturated or unsaturated)
Figure 1. (a) Proposed biosynthetic pathway for the production of
long chain N-acyl tyrosines and their conversion to (E)-enamides and
(E)-enol esters. (b) Formation of acyl homoserine lactones from
biosynthetic precursor S-adenosyl methionine (SAM).
*
Corresponding author. Tel.: +1 617 432 2845; fax: +1 617 738
3702; e-mail: jon_clardy@hms.harvard.edu
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.10.058

3118
S. N. Georgiades, J. Clardy / Bioorg. Med. Chem. Lett. 18 (2008) 3117–3121
this N-acyl synthase5b,6 suggest a relationship to the syn-
thases that produce the acyl homoserine lactones
(AHLs, Fig. 1b), quorum sensing mediators in many
Gram-negative bacteria.7 In both the AHLs and NATs,
a common head group—either homoserine lactone or
tyrosine—is attached to a variety of long chain acids,
and biological activity requires the correct acid frag-
ment. In the E. coli heterologous expression system we
use, ACP-bound long chain acids from E. coli pathways
can replace those of the original producer, and conse-
quently the identities of the biologically relevant prod-
ucts of NAT pathways are not known with certainty.5a
In order to explore the relationship between acid struc-
ture and biological activity in the amide, enamide and
enol ester pathway, we developed an efficient chemical
synthesis of plausible library members, which is de-
scribed herein.
to carry out this reaction in THF using diethyl phos-
phocyanidate (DEPC) as the activating agent in the
presence of TEA, for 24 h at room temperature. Excess
of acid, DEPC and TEA was required. Under these con-
ditions, 30 simple acids with various degrees of unsatu-
ration (5–34) were combined with the resin-bound
nucleophiles in parallel to furnish 60 coupling products
(5aL–34aL and 5aD–34aD). All couplings were quanti-
tative and free of impurities, as indicated by LC–MS.
Parallel removal of the allyl protecting group from these
intermediates was achieved with 5 mol % Pd(PPh3)4 in
THF and excess morpholine in 6–8 h. The mild depro-
tection conditions were compatible with the presence
of unsaturated long chains. This sensitive reaction
required absence of light and initial degassing of the
reaction container, to avoid catalyst degradation.
Cleavage of the NATs from the beads was carried out
using a 70/30 (v/v) HF/pyridine mixture in THF, buf-
fered with additional pyridine. The reaction was typi-
cally quenched with TMSOMe, to provide >95% pure
products (5cL–34cL and 5cD–34cD) after the superna-
tant was separated from the beads and the volatile
byproducts were evaporated.
Long chain NATs were prepared via a solid-phase route
(Scheme 1). Polystyrene macrobeads of 500–600 lm
diameter, functionalized with a trialkylaryl silicon linker
(1), were chosen as the solid support.8 First, the resin
was activated with excess trifluoromethanesulfonic acid
in CH2Cl2, followed by quenching with 2,6-lutidine.
The activated beads were then treated for 24 h with a
saturated solution of a bis-protected tyrosine substrate,
the Fmoc carbamate/allyl ester of either L- or D-tyrosine
(2L and 2D, respectively),9 to afford the corresponding
resin-bound species (3L and 3D).10 Complete removal
of the Fmoc group with 20% (v/v) piperidine in DMF
led to the resin-bound allyl esters 4L and 4D. After
screening various amide coupling conditions, we elected
To augment the library with long chains reminiscent of
AHLs, solid-phase synthesis of NATs bearing a 3-keto
functionality was carried out using a modification of
the above procedure (Scheme 2). Long chain 5-acyl-
2,2-dimethyl-1,3-dioxane-4,6-diones (5-acyl Meldrum’s
acids),11 a masked form of the labile 3-oxo carboxylic
acids, were used as building blocks in the amide cou-
pling step. An alternative to conventional heating in
O
O
O
NH2
O
d
a-c
NHFmoc
Si
Si
O
Si
O
OCH3
3L
3D
4L
4D
1
O
R
O
O
O
R
OH
O
OH
O
HN
g-h
f
e
O
HN
HO
HN
Si
Si
O
O
R
60 Examples
5aL-34aL
5aD-34aD
60 Examples
5bL-34bL
5bD-34bD
60 Examples
5cL-34cL
5cD-34cD
RCO2H
R=
RCO2H
R=
5
6
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
n-C3H7
n-C18H37
n-C19H39
O
n-C4H9
7
n-C5H11
n-C6H13
n-C7H15
n-C8H17
n-C9H19
n-C10H21
O
8
9
NHFmoc
HO
HO
10
11
12
13
14
15
16
17
18
19
2L
O
n-C11 23
n-C12 25
n-C13 27
n-C14 29
n-C15 31
n-C16 33
n-C17 35
H
O
H
NHFmoc
H
H
2D
H
H
H
Scheme 1. Solid-phase synthesis of long chain N-acyl tyrosines. Reagents and conditions: (a) CH2Cl2/TfOH/rt/40 min; (b) CH2Cl2/2,6-lutidine/rt/5–
10 min; (c) CH2Cl2/2L or 2D/rt/24 h; (d) DMF/piperidine/rt/4 h; (e) RCO2H (5–34)/THF/DEPC/TEA/rt/24 h; (f) THF/Pd(PPh3)4/morpholine/rt/8–
12 h; (g) THF/HF/pyridine/rt/2.5 h; (h) TMSOMe/rt/15 min.

S. N. Georgiades, J. Clardy / Bioorg. Med. Chem. Lett. 18 (2008) 3117–3121
3119
O
O
O
OH
O
O
O
O
HN
HN
b
NH2
a
Si
Si
Si
O
O
O
O
O
4L
4D
38 Examples
35bL-53bL
35bD-53bD
38 Examples
35aL-53aL
35aD-53aD
R
R
c-d
O
5-Acyl M.A.
R=
5-Acyl M.A.
R=
35
36
37
38
39
40
41
42
43
44
CH3
45
46
47
48
49
50
51
52
53
n-C11H23
n-C12H25
O
O
R
O
C2H5
H
O
OH
O
n-C3H7
n-C4H9
n-C5H11
n-C6H13
n-C7H15
n-C8H17
n-C9H19
n-C10H21
n-C13 27
H
O
n-C14 29
n-C15 31
n-C16 33
n-C17 35
H
HN
HO
H
O
H
38 Examples
35cL-53cL
35cD-53cD
H
R
5-Acyl M.A.
Scheme 2. Solid-phase synthesis of long chain 3-oxo-N-acyl tyrosines. Reagents and conditions: (a) NMP/5-acyl Meldrum’s acid (35–53)/microwave/
200 °C/10 min; (b) THF/Pd(PPh3)4/morpholine/rt/8–12 h; (c) THF/HF/pyridine/rt/2.5 h; (d) TMSOMe/rt/15 min.
O
OH
O
OH
the presence of an external base, which is the usual pro-
tocol of choice for a solution-phase reaction involving
nucleophilic attack on a 5-acyl Meldrum’s acid, was nec-
essary in order to avoid bead degradation. Thus a
microwave-assisted method was developed. Optimal
reaction conditions required the use of N-methyl pyrrol-
idone (NMP) as the solvent, which has both excellent
absorbance characteristics and ideal interaction with
this solid-phase, excess of the 5-acyl Meldrum’s acid
building block and no external base, at 200 °C with con-
trolled microwave irradiation in a commercial reactor
for 10 min. Parallel coupling of 19 different saturated
and unsaturated building blocks (35–53) to resin-bound
substrates 4L and 4D led to full consumption of the sub-
strate in all cases, and formation of 70–80% of the de-
sired products (35aL–53aL and 35aD–53aD). The 38
intermediates were successfully carried through the allyl
deprotection and cleavage steps as described above, to
yield 38 final 3-oxo-N-acyl tyrosines (35cL–53cL and
35cD–53cD).
Br
a
b
c
54
56
55
OTBDMS
OH
HN
OTBDMS
O
O
R
HN
R
RCONH2
R=
57
58
59
60
61
62
63
n-C7H15
n-C9H19
n-C10H21
n-C11H23
n-C13H27
n-C14 29
n-C15H31
d
OTBDMS
OH
H
7 Examples
57aE 63aE
7 Examples
57bE-63bE
-
Scheme 3. Solution-phase synthesis of long chain N-acyl enamides.
Reagents and conditions: (a) TBDMSCl/imidazole/DMF/rt/12 h/85%;
(b) NBS/LiOAc/CH3CN/H2O/60 °C/1 h/83%; (c) RCONH2 (57–63)/
(CH3NHCH2)2/CuI/Rb2CO3/toluene/80 °C/24 h/65–70%; (d) HF/pyri-
dine/THF/rt/6 h/85–90%.
Since our devised synthetic routes leading to the
enamide and enol ester families involved steps that were
either heterogeneous or incompatible with the solid-
phase, both were realized in solution-phase. The (E)-
enamides were prepared as shown in Scheme 3, starting
with protection of 4-hydroxycinnamic acid (54) as a silyl
ether (55, 85% yield) with TBDMSCl in DMF, in the
presence of imidazole. Intermediate 55 was stereoselec-
tively converted to the corresponding (E)-vinyl bromide
(56, 83% yield) via a decarboxylative bromination with
N-bromosuccinimide.12 This LiOAc-catalyzed Huns-
diecker transformation was carried out in CH3CN/
H2O with mild heating. A modification of Buchwald’s
conditions for copper-catalyzed amidation13 was em-
ployed for coupling of the vinyl bromide to a series of
saturated long chain carboxamides (57–63),14 generating
the long chain (E)-enamides 57aE–63aE at 65–70% yield
with retention of the trans geometry. The amidation was
carried out in toluene, using CuI as the copper source,
N,N0-dimethylethylenediamine as the bidentate ligand
and Rb2CO3 as the base,13 and required strictly anhy-
drous conditions and heating at 70 °C for 24 h. Depro-
tection of the intermediates was achieved with 70/30
(v/v) HF/pyridine in THF/pyridine and afforded 85–
90% of the final (E)-enamides (57bE–63bE).
The last class of eDNA-associated compounds, the enol
esters, were derived from methyl 4-hydroxyphenyl ace-
tate (64, Scheme 4), which was quantitatively protected
under conditions similar to the protection of 54 above
to afford TBDMS-silyl ether 65. Intermediate 65 was
converted to an aldehyde in two steps: reduction to an
alcohol (66) with LiAlH4 in THF at ꢀ78 °C (94% yield),
followed by alcohol oxidation to the aldehyde (67) with
2-iodoxybenzoic acid (IBX)15 in DMSO (93% yield). By
means of KHMDS in THF/toluene, 67 was converted to
a mixture of trans and cis potassium enolates, in accord

3120
S. N. Georgiades, J. Clardy / Bioorg. Med. Chem. Lett. 18 (2008) 3117–3121
OH
OCH3
O
helpful discussions and preliminary biological studies
on the library, Dr. Li Lai (formerly HMS) for assistance
with solid-phase handling, and Dr. Ralph Mazitschek
(Broad Institute of MIT and Harvard) and Dr. Jared
Shaw (formerly Broad Institute) for useful suggestions.
OCH3
O
RCO2Suc
R=
68
69
70
71
72
73
74
75
76
77
78
79
80
n-C5H11
n-C6H13
n-C7H15
n-C8H17
n-C9H19
n-C10H21
c
a
b
66
65
64
OTBDMS
O
OTBDMS
O
OH
n-C11 23
n-C12 25
n-C13 27
n-C14 29
n-C15 31
n-C16 33
n-C17 35
H
H
H
O
O
R
O
R
H
Supplementary data
H
H
H
f
d-e
H
Detailed experimental procedures and spectroscopic
data associated with this article can be found in the on-
line version, at doi:10.1016/j.bmcl.2007.10.058.
67
O
OTBDMS
OTBDMS
OH
O
26 Examples
68aE-80aE
68aZ-80aZ
26 Examples
68bE-80bE
68bZ-80bZ
N
R
O
O
RCO2Suc
References and notes
(E)/(Z)= ~2/1
1. (a) Clardy, J.; Fischbach, M. A.; Walsh, C. T. Nat.
Biotechnol. 2006, 24, 1541; (b) Clardy, J.; Walsh, C.
Nature 2004, 432, 829; (c) Koehn, F. E.; Carter, G. T. Nat.
Rev. Drug Discov. 2005, 4, 206; (d) Butler, M. S. J. Nat.
Prod. 2004, 67, 2141; (e) Leeds, J. A.; Schmitt, E. K.;
Krastel, P. Expert Opin. Investig. Drugs 2006, 15, 211; (f)
Pelaez, F. Biochem. Pharmacol. 2006, 71, 981.
Scheme 4. Solution-phase synthesis of long chain N-acyl enol esters.
Reagents and conditions: (a) TBDMSCl/imidazole/DMF/rt/12 h/
100%; (b) LiAlH4/THF/ꢀ78 °C/1 h/94%; (c) IBX/DMSO/rt/4 h/93%;
(d) KHMDS/toluene/THF/0 °C/5 min; (e) THF/RCO2Suc (68–80)/rt/
30 min/30–40%; (f) HF/pyridine/rt/6 h/85–90%.
2. (a) Torsvik, V.; Salte, K.; Sørheim, R.; Goksøyr, J. Appl.
Environ. Microbiol. 1990, 56, 776; (b) Torsvik, V.;
Goksøyr, J.; Daae, F. L. Appl. Environ. Microbiol. 1990,
56, 782; (c) Ward, D. M.; Weller, R.; Bateson, M. M.
Nature 1990, 345, 63; (d) Stackebrandt, E.; Liesack, W.;
Goebel, B. M. FASEB J. 1993, 7, 232; (e) Amann, R. I.;
Ludwig, W.; Schleifer, K. H. Microbiol. Rev. 1995, 59,
143; (f) Hugenholtz, P.; Goebel, B. M.; Pace, N. R.
J. Bacteriol. 1998, 180, 4765.
3. (a) Wang, G.-Y.-S.; Graziani, E.; Waters, B.; Pan, W.; Li,
X.; McDermott, J.; Meurer, G.; Saxena, G.; Andersen, R.
J.; Davies, J. Org. Lett. 2000, 2, 2401; (b) Brady, S. F.;
Clardy, J. J. Am. Chem. Soc. 2000, 122, 12903; (c)
MacNeil, I. A.; Tiong, C. L.; Minor, C.; August, P. R.;
Grossman, T. H.; Loiacono, K. A.; Lynch, B. A.; Phillips,
T.; Narula, S.; Sundaramoorthi, R.; Tyler, A.; Aldredge,
T.; Long, H.; Gilman, M.; Holt, D.; Osburne, M. S. J.
Mol. Microbiol. Biotechnol. 2001, 3, 301.
4. A similar approach has been used for enzyme catalyst
discovery: DeSantis, G.; Zhu, Z.; Greenberg, W. A.;
Wong, K.; Chaplin, J.; Hanson, S. R.; Farwell, B.;
Nicholson, L. W.; Rand, C. L.; Weiner, D. P.; Robertson,
D. E.; Burk, M. J. J. Am. Chem. Soc. 2002, 124, 9024.
5. (a) Brady, S. F.; Chao, C. J.; Clardy, J. J. Am. Chem. Soc.
2002, 124, 9968; (b) Brady, S. F.; Chao, C. J.; Clardy, J.
J. Appl. Environ. Microbiol. 2004, 70, 6865; (c) Brady, S.
F.; Clardy, J. J. Nat. Prod. 2004, 67, 1283; (d) Brady, S. F.;
Clardy, J. Org. Lett. 2005, 7, 3613.
with a previous report from our laboratory for a similar
system.16 Under the specific reaction conditions em-
ployed here, moderate selectivity for the trans enolate
(precursor to the only isomeric product present in the
natural extract from the eDNA clone) was observed.
The enolates were trapped with saturated long chain
N-hydroxysuccinimide esters (68–80).17 LC–MS analysis
of an aliquot from each reaction revealed in all cases a
mixture of products with the same mass, that included
(E)- and (Z)-enol esters as well as what appeared to be
carbon acylation products. Partial purification of the
crude products afforded inseparable mixtures of (E)-
and (Z)-enol esters (68aE–80aE and 68aZ–80aZ), for
which 1H NMR indicated a ratio of approximately
2:1. The ratio was independent of acid chain length
and reflects the thermodynamic preference for the trans
enolate. The (E)/(Z) mixtures were resolved after HF re-
moval of the TBDMS protecting group in pyridine,
which proceeded at 85–90% yield. Thirteen deprotected
(E)-enol esters (68bE–80bE) and 13 (Z)-enol esters
(68bZ–80bZ) were obtained in pure form.
In summary, solid- and solution-phase methods are de-
scribed for the preparation of synthetic libraries of tyro-
sine-derived bacterial metabolites (131 compounds have
been delivered), resembling small molecules isolated
from heterologous expression of eDNA in E. coli. Preli-
minary biological studies on library members have
shown antibiotic activity against Bacillus subtilis and
moderate inhibitory potential of Pseudomonas aerugin-
osa biofilm formation. Additional assays are planned
and will be reported elsewhere in due course.
6. (a) Van Wagoner, R. M.; Clardy, J. Structure 2006, 14,
1425; Commentary by (b) Churchill, M. E. A. Structure
2006, 14, 1342.
7. For recent reviews on AHLs and bacterial signalling, see: (a)
Waters, C. M.; Bassler, B. L. Annu. Rev. Cell Dev. Biol. 2005,
21, 319; (b) Bassler, B. L.; Losick, R. Cell 2006, 125, 237.
8. Preparation of this resin is described in: Tallarico, J. A.;
Depew, K. M.; Pelish, H. E.; Westwood, N. J.; Lindsley,
C. W.; Shair, M. D.; Schreiber, S. L.; Foley, M. A.
J. Comb. Chem. 2001, 3, 312.
9. Substrates 2L and 2D were prepared from commercial
Fmoc-tyrosine following the method of: Jensen, K. J.;
Meldal, M.; Bock, K. J. Chem. Soc. Perkin Trans. 1 1993,
17, 2119.
10. The loading in this step was typically 1.3–1.4 mmol of
substrate per gram of resin, approximately equal to the
Acknowledgments
We gratefully acknowledge the NIH for funding of this
project (CA24487 to J.C.). We thank Dr. Sean Brady
(formerly HMS) and Dr. Lauren Junker (HMS) for

S. N. Georgiades, J. Clardy / Bioorg. Med. Chem. Lett. 18 (2008) 3117–3121
3121
empirical maximum for this type and batch of resin. It was
determined by UV analysis as explained in the supple-
mentary data.
13. Jiang, L.; Job, G. E.; Klapars, A.; Buchwald, S. L. Org.
Lett. 2003, 5, 3667.
14. The carboxamide building blocks were prepared from
fatty acids and NH3 with DEPC in 1,4-dioxane/DMF, as
described in the supplementary data.
11. The long chain 5-acyl Meldrum’s acids were prepared
from carboxylic acids and Meldrum’s acid, as detailed in
the supplementary data, using
a
modification of
a
15. For an efficient preparation of IBX from 2-iodobenzoic
acid, see: Frigerio, M.; Santagostino, M.; Sputore, S.
J. Org. Chem. 1999, 64, 4537.
16. Brady, S. F.; Clardy, J. Org. Lett. 2003, 5, 121.
17. The N-hydroxysuccinimide ester building blocks were
prepared from fatty acids and N-hydroxysuccinimide with
DCC in THF, as described in the supplementary data.
published method: Raillard, S. P.; Chen, W.; Sullivan,
E.; Bajjalieh, W.; Bhandari, A.; Baer, T. A. J. Comb.
Chem. 2002, 4, 470.
12. (a) Kuang, C.; Yang, Q.; Senboku, H.; Tokuda, M.
Synthesis 2005, 8, 1319; (b) Kuang, C.; Senboku, H.;
Tokuda, M. Synlett 2000, 10, 1439.