Chemoenzymatic synthesis of the polyketide macrolactone 10-deoxymethynolide

Article abstract of DOI:10.1021/ja0504340

The polyketide synthase-derived pikromycin thioesterase (Pik TE) is unique in its ability to catalyze the cyclization of 12- and 14-membered macrolactones. In this investigation, the total synthesis of the natural hexaketide chain elongation intermediate

Full text of DOI:10.1021/ja0504340

Published on Web 06/04/2005
Chemoenzymatic Synthesis of the Polyketide Macrolactone
10-Deoxymethynolide
Courtney C. Aldrich,†,‡ Lakshmanan Venkatraman,‡ David H. Sherman,*,† and Robert A. Fecik*,‡
UniVersity of Michigan Life Sciences Institute, Department of Medicinal Chemistry, 210 Washtenaw AVenue,
Ann Arbor, Michigan 48109-2216, and Department of Medicinal Chemistry, 308 HarVard St. S.E., 8-101 WDH,
UniVersity of Minnesota, Minneapolis, Minnesota 55455-0353
Received January 22, 2005; E-mail: fecik001@umn.edu; davidhs@umich.edu
Polyketides are a richly diverse class of natural products that
display a wide variety of clinically important biological activities.1
The enzymes that catalyze their formation, polyketide synthases
(PKSs), are complex multienzyme systems that are characterized
by a modular architecture that condense simple carboxylic acids
such as malonyl-CoA and methylmalonyl-CoA.
The pikromycin (Pik) PKS system of Streptomyces Venezuelae
ATCC 15439 is notable among modular PKS systems in that it
catalyzes the biosynthesis of both 12- and 14-membered macro-
lactones.2 Upon thioesterase (TE)-catalyzed cyclization of the linear
hexa- and heptaketide intermediates, 10-deoxymethynolide (1) and
narbonolide (2) undergo post-PKS oxidation and glycosylation to
methymycin and pikromycin, respectively (Figure 1). Our studies3
and those of others4 have focused on the ability of Pik monomodules
to catalyze chain extension and processing of natural and unnatural
substrates. To complement our work with Pik monomodules,3 we
Figure 1. Final steps in the biosynthesis of 10-deoxymethynolide and
narbonolide by the Pik PKS in S. Venezuelae.
initiated analysis of the Pik TE domain as an effective macrolac-
tonization catalyst.
Scheme 1 a
The ability of Pik TE to catalyze cyclization of both 12- and
14-membered ring macrolactones suggests that it has inherent
substrate tolerance, which is consistent with structural information
obtained by X-ray analysis.5 Cane and co-workers have reported
that relative to the 6-deoxyerythronolide B synthase (DEBS) TE,
Pik TE has enhanced specific activity and broader substrate
specificity.6 These conclusions were based upon the use of simple
diketide N-acetylcysteamine (SNAC) thioesters as substrates.
Recent work with TE domains from modular nonribosomal
peptide synthetase (NRPS) and PKS systems has demonstrated their
ability to accept natural and unnatural substrates for cyclization of
linear polypeptide and polyketide chains.7 For example, the TE
domain isolated from the epothilone PKS system has been shown
to catalyze macrocyclization of SNAC-seco-epothilone C obtained
from epothilone C.7e The biochemical analysis of modular PKSs
has been limited due in part to the challenge of obtaining suitable
quantities of natural substrates and substrate analogues, particularly
those involved in late stages of polyketide chain elongation. This
challenge can be met by total synthesis to obtain natural substrates
and substrate analogues in enantiomerically pure form.
a Key: (a) Me(OMe)NH•HCl, AlMe3, 84%; (b) NaH, PMBCl, NaI, 93%;
(c) DIBAL-H, 90%; (d) 7, CrCl2, NiCl2, 70%; (e) Dess-Martin, 95%; (f)
DDQ (see Supporting Information).
developed based on the potential to generate analogues for substrate
specificity studies. Synthesis of SNAC-seco-10-deoxymethynolide
began with the opening of lactone 39 to afford Weinreb amide 410
(Scheme 1). The primary alcohol in Weinreb amide 4 was protected
as PMB ether 5, which was reduced to aldehyde 6 (the mirror image
of 6 by different methodology has been reported11). Nozaki-
Hiyama-Kishi coupling of aldehyde 6 with vinyl iodide 712
provided allylic alcohol 8 as a 1:1 mixture of C-5 epimers, which
underwent Dess-Martin oxidation to ketone 9. Removal of the
PMB ether afforded alcohol 10 in equilibrium with its hemiketal,
which underwent SiO2-catalyzed dehydration to a dihydropyran
upon purification (see Supporting Information).
We report here the synthesis of SNAC-seco-10-deoxymethyno-
lide, a mimic of the natural hexaketide Pik TE chain elongation
intermediate, and its enzymatic macrolactonization by Pik TE to
complete the total synthesis of 10-deoxymethynolide. Further, we
have extended the study of the substrate specificity of Pik TE
through the use of unnatural hexaketide substrate analogues that
were obtained by total synthesis.
While the target hexaketide may be obtained by degradation of
10-deoxymethynolide (1),8 a more versatile total synthesis was
To avoid the problems associated with the instability of alcohol
10, allylic alcohol 8 was protected as TBS ether 11, followed by
debenzylation to give alcohol 12 (Scheme 2). Alcohol 12 was
† University of Michigan Life Sciences Institute, Department of Medicinal
Chemistry.
‡ Department of Medicinal Chemistry, University of Minnesota.
9
8910
J. AM. CHEM. SOC. 2005, 127, 8910-8911
10.1021/ja0504340 CCC: $30.25 © 2005 American Chemical Society

C O M M U N I C A T I O N S
Scheme 2 a
Figure 2. HPLC trace of SNAC-seco-10-deoxymethynolide (18, red)
overlaid with trace following enzymatic reaction with Pik TE showing
exclusive conversion to 10-deoxymethynolide (1, black).
a Key: (a) i-Pr2NEt, TBSOTf, 97%; (b) DDQ, 87%; (c) TPAP, NMO,
81%; (d) R-4-benzyl-3-propionyl-2-oxazolidinone, Bu2BOTf, i-Pr2NEt, 75%;
(e) LiOH, H2O2, 85%; (f) HSNAC, EDC, DMAP, 62%; (g) HF•pyr., pyr.,
70%; (h) MnO2, 35%.
on the reactivity of Pik TE with its native substrates will be the
subject of future investigations.
As shown here, de novo synthesis of the natural pikromycin
hexaketide chain elongation intermediate (SNAC-seco-10-deoxy-
methynolide) and its analysis as a substrate for Pik TE has revealed
the exquisite selectivity and efficiency of this enzyme. Positional
variation of functional groups and stereochemistry will enable
investigation of the overall tolerance of Pik TE and suggest
strategies for its optimization as a versatile biocatalyst toward
diverse hydroxyl acyl-SNAC esters.
oxidized to aldehyde 13, which underwent a syn-aldol reaction to
afford alcohol 14. Removal of the chiral auxiliary gave acid 15,
and coupling with N-acetylcysteamine (HSNAC) afforded thioester
16 which was deprotected to triol 17. For subsequent enzymatic
reactions, the C-7 epimeric mixture of triol 17 was separated by
reverse-phase HPLC. Chemoselective allylic oxidation of triol 17
with MnO2 afforded SNAC-seco-10-deoxymethynolide 18 in 35%
yield (based upon 60% conversion). Thioester 18 slowly decom-
poses at 23 °C, thus precluding longer reaction times to achieve
Acknowledgment. This research was supported by grant FRD
01-13 from the Academic Health Center, University of Minnesota
(to R.A.F. and D.H.S), and NSF 0118926 (to D.H.S.).
1
100% conversion. Upon the basis of H NMR analysis, thioester
1
Supporting Information Available: Experimental details and H
18 exists in equilibrium with hemiketal 19 as an approximately
4:5:1 ratio of 18:19â:19R.
and 13C NMR spectra for compounds 4-6, 8, 9, and 11-18, scheme
for decomposition of compound 10, enzymatic reaction conditions, and
steady-state kinetic analysis details. This material is available free of
charge via the Internet at http://pubs.acs.org.
Incubation of SNAC-seco-10-deoxymethynolide (18, as a mixture
with hemiketal 19) with the purified Pik TE6 (10 µM) at pH 7.0
afforded 10-deoxymethynolide (1) as the exclusive product as
monitored by HPLC and mass spectrometry (Figure 2). Steady-
state kinetic analysis was used to determine the kinetic parameters.
The specificity constant kcat/KM for this reaction is 1.67 ( 0.027
mM-1 min-1, which is approximately 4-fold greater than that
obtained for the cyclization of SNAC-seco-epothilone C chain
elongation intermediate with its cognate TE.7e As observed in the
enzymatic cyclization of SNAC-seco-epothilone C,7e the low
solubility of the substrate prevented determination of individual
kcat and KM parameters, but the KM is greater than 1 mM. In contrast
to the reaction of SNAC-seco-epothilone C with its cognate TE,7e
enzymatic hydrolysis of SNAC-seco-10-deoxymethynolide to seco-
10-deoxymethynolide was not detected. The quantitative conversion
and lack of observed partitioning to the seco-acid demonstrates the
pronounced selectivity and efficiency of this biocatalytic process.
We also used non-native substrates to conduct a preliminary
substrate specificity study of Pik TE. The reaction of the individual
C-7 epimers of triol 17 with Pik TE at pH 7.0 was monitored,
however, cyclization was not observed and the exclusive products
were due to hydrolysis (see Supporting Information). These results
show that Pik TE is highly optimized for its native substrate, as
minor structural changes at C-7 are not tolerated. One potential
explanation for this is the pH (7.0) at which these enzymatic
reactions were conducted. Crystal structures of Pik TE show that
the size of the substrate channel increases at higher pH,5 suggesting
that pH can influence the reactivity of Pik TE.6 The effect of pH
References
(1) Katz, L. Chem. ReV. 1997, 97, 2557-2575.
(2) (a) Xue, Y.; Zhao, L.; Liu, H.-W.; Sherman, D. H. Proc. Natl. Acad. Sci.
U.S.A. 1998, 95, 12111-12116. (b) Xue, Y.; Sherman, D. H. Met. Eng.
2001, 3, 15-26.
(3) (a) Beck, B. J.; Aldrich, C. C.; Fecik, R. A.; Reynolds, K. A.; Sherman,
D. H. J. Am. Chem. Soc. 2003, 125, 4683-4684. (b) Beck, B. J.; Aldrich,
C. C.; Fecik, R. A.; Reynolds, K. A.; Sherman, D. H. J. Am. Chem. Soc.
2003, 125, 12551-12557.
(4) Yin, Y.; Lu, H.; Khosla, C.; Cane, D. E. J. Am. Chem. Soc. 2003, 125,
5671-5676.
(5) Tsai, S.-C.; Lu, H.; Cane, D. E.; Khosla, C.; Stroud, R. M. Biochemistry
2002, 41, 12598-12606.
(6) Lu, H.; Tsai, S.-C.; Khosla, C.; Cane, D. E. Biochemistry 2002, 41, 12590-
12597.
(7) (a) Kohli, R. M.; Takagi, J.; Walsh, C. T. Proc. Natl. Acad. Sci. U.S.A.
2002, 99, 1247-1252. (b) Kohli, R. M.; Walsh, C. T.; Burkart, M. D.
Nature 2002, 418, 658-661. (c) Grunewald, J.; Sieber, S. A.; Marahiel,
M. A. Biochemistry 2004, 43, 2915-2925. (d) Sieber, S. A.; Walsh, C.
T.; Marahiel, M. A. J. Am. Chem. Soc. 2003, 125, 10862-10866. (e)
Boddy, C. N.; Schneider, T. L.; Hotta, K.; Walsh, C. T.; Khosla, C. J.
Am. Chem. Soc. 2003, 125, 3428-3429.
(8) Aldrich, C. C.; Beck, B. J.; Fecik, R. A. Sherman, D. H. J. Am. Chem.
Soc. 2005, 127, in press.
(9) Hoffman, R. W.; Zeiss, H.-J.; Ladner, W.; Tabche, S. Chem. Ber. 1982,
115, 2357-2369.
(10) Nicolaou, K. C.; Pihko, P. M.; Diedrichs, N.; Zou, N.; Bernal, F. Angew.
Chem., Int. Ed. 2001, 40, 1262-1265.
(11) Meyer, S. D.; Miwa, T.; Nakatsuka, M.; Schreiber, S. L. J. Org. Chem.
1992, 57, 5058-5060.
(12) Pilli, R. A.; de Andrade, C. K. Z.; Souto, C. R. O.; de Meijere, A. J. Org.
Chem. 1998, 63, 7811-7819.
JA0504340
9
J. AM. CHEM. SOC. VOL. 127, NO. 25, 2005 8911