Total synthesis of syringolin A and B

Article abstract of DOI:10.1021/ol100761z

Total syntheses of two recently discovered proteasome inhibitors, syringolin A and B, are reported. The key to our approach was creation of the α,β-unsaturated 12-membered lactam via intramolecular Horner-Wadsworth-Emmons reaction. Such reactions have been broadly used to prepared macrolactones, but this work presents a rarer example of its application to macrolactams. The final steps involved attachment of the bis(valinyl)urea side chain using peptide coupling procedures, including a method based on the unprotected valine N-carboxy anhydride. The additional alkene of syringolin A was created through cross-metathesis.

Full text of DOI:10.1021/ol100761z

ORGANIC
LETTERS
2010
Vol. 12, No. 10
2402-2405
Total Synthesis of Syringolin A and B
Michael C. Pirrung,* Goutam Biswas, and Tannya R. Ibarra-Rivera
Department of Chemistry, UniVersity of California, RiVerside, California 92521
michael.pirrung@ucr.edu
Received April 1, 2010
ABSTRACT
Total syntheses of two recently discovered proteasome inhibitors, syringolin A and B, are reported. The key to our approach was creation of
the r,ꢀ-unsaturated 12-membered lactam via intramolecular Horner-Wadsworth-Emmons reaction. Such reactions have been broadly used
to prepared macrolactones, but this work presents a rarer example of its application to macrolactams. The final steps involved attachment of
the bis(valinyl)urea side chain using peptide coupling procedures, including a method based on the unprotected valine N-carboxy anhydride.
The additional alkene of syringolin A was created through cross-metathesis.
Inactivation of the proteasome has recently been identified
as the mode of action of a variety of anticancer agents, both
natural products and synthetic compounds. These inhibitors
exploit the mechanism used by the proteasome, nucleophilic
catalysis by the N-terminal threonine,1 to trigger a reaction
with an electrophilic functional group in the inhibitor. The
types of electrophiles that are known to react with the
catalytic Thr1 of the proteasome include boronic acids,
ꢀ-lactones, epoxyketones,2 and cyclic thionocarbonates.3
In 2008, two macrolactams, syringolin A and B, were
shown to affect plant-pathogen interactions4 and to act
as virulence factors via inhibition of the proteasome.5 They
are members of a bioactive natural product family that
has been known for many years, includes the glidobactins/
cepafungins, and is now called the syrbactins. Crystal-
lography established that both syringolin A and B form a
covalent adduct with the proteasome via conjugate addition
of the N-terminal threonine hydroxyl group to their R,ꢀ-
unsaturated lactams. Given the known properties of
proteasome inhibitors, the action of syringolin A in cancer
cells was investigated. Treatment of a human neuroblas-
toma cell line with 25 µM syringolin A causes a dose-
dependent decrease in proteasome activity and a time-
dependent accumulation of ubiquitinated proteins via
irreversible inhibition of the proteasome.
These biological properties stimulated our interest in
the synthesis of the syringolins. Structures based on the
syrbactins would be valuable to access by total synthesis
in order to (1) develop novel anticancer therapeutics, (2)
better understand plant host-pathogen interactions, and
(3) elucidate fundamental questions concerning the struc-
ture and function of the proteasome in diverse eukaryotes,
including higher plants and even mycobacteria.3
(1) Marques, A. J.; Palanimurugan, R.; Matias, A. C.; Ramos, P. C.;
Dohmen, R. J. Chem. ReV. 2009, 109, 1509–1536.
(2) Fenteany, G.; Standaert, R. F.; Lane, W. S.; Choi, S.; Corey, E. J.;
Schreiber, S. L. Science 1995, 268, 726–731. Macherla, V. R.; Mitchell,
S. S.; Manam, R. R.; Reed, K. A.; Chao, T. H.; Nicholson, B.; Deyanat-
Yazdi, G.; Mai, B.; Jensen, P. R.; Fenical, W. F.; Neuteboom, S. T.; Lam,
K. S.; Palladino, M. A.; Potts, B. C. J. Med. Chem. 2005, 48, 3684–3687.
Meng, L.; Mohan, R.; Kwok, B. H.; Elofsson, M.; Sin, N.; Crews, C. M.
Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 10403–10408. Teicher, B. A.; Ara,
G.; Herbst, R.; Palombella, V. J.; Adams, J. Clin. Cancer Res. 1999, 5,
2638–2645.
Creation of the macrolactam poses the major challenge
for the synthesis of the syringolins. The Kaiser group
recently completed syntheses of both syringolin A and
B.6 Syringolin A was dissected into the bis(valinyl)urea
side chain and the 12-membered macrolactam core. The
(3) Lin, G.; Li, D.; de Carvalho, L. P.; Deng, H.; Tao, H.; Vogt, G.;
Wu, K.; Schneider, J.; Chidawanyika, T.; Warren, J. D.; Li, H.; Nathan, C.
Nature 2009, 461, 621–626.
(4) Wa¨spi, U.; Schweizer, P.; Dudler, R. Plant Cell 2001, 13, 153–61.
Michel, K.; Abderhalden, O.; Bruggmann, R.; Dudler, R. Plant Mol Biol.
2006, 62, 561–578.
(5) Groll, M.; Schellenberg, B.; Bachmann, A. S.; Archer, C. R.; Huber,
R.; Powell, T. K.; Lindow, S.; Kaiser, M.; Dudler, R. Nature 2008, 452,
755–758.
10.1021/ol100761z  2010 American Chemical Society
Published on Web 04/28/2010

core was prepared with a protected diol as a synthon for
the trans-R,ꢀ-unsaturated amide, and the formation of the
macrolactam was achieved by ring-closing metathesis in
49% yield. After side chain attachment, the unsaturated
amide was revealed in the penultimate step. This synthesis
entails 16 linear steps (not including preparation of the
side chain) and proceeds in an excellent 9% overall yield.
The syringolin B synthesis used lysine and valine as
starting materials, assembled a polypeptide using standard
coupling methods, and then used a peptide coupling agent
at high dilution to close the macrolactam, which proceeded
in 30% yield. Glidobactin A, a natural product with a core
ring closely related to the syringolins, has also been
synthesized.7 Here, macrolactamization via a pentafluo-
rophenyl active ester gave the 12-membered ring in only
20% yield.
Our aim was a modular and general synthetic approach
to the syringolins with the potential for straightforward
preparation of structural variants simply by the substitution
of structurally variant modules (diversity-oriented syn-
thesis).8 The R,ꢀ-unsaturated amide of the syringolins
suggested an intramolecular Horner-Wadsworth-Emmons
condensation for the preparation of the 12-membered ring.
Such reactions have found utility in the high-yield
synthesis of macrolactones9 but have been applied to only
a few macrolactams.10
The total synthesis of syringolin B was a proving ground
for the macrocyclization. The C2-symmetric bis(valinyl)
urea side chain was prepared from two commercial
compounds: L-valine-derived isocyanate 1 and L-valine
tert-butyl ester hydrochloride, using a route developed by
Clerc et al.6 The monoacid was activated as the nicely
crystalline NHS active ester 2. Boc-L-Lysine was acylated
with the NHS active ester of diethylphosphonoacetic acid
(3) using a protocol developed for other acids,11 and then
a conventional peptide coupling with L-valinol was
executed. The two-step yield of 4 is 75%. Its Dess-Martin
oxidation sets up the key cylization reaction. The very
mild Horner-Wadsworth-Emmons protocol developed
by Helquist for the formation of acrylamides with high
selectivity for (E) stereochemistry was uniquely success-
ful.12 This contrasted with the outcome using several other
common methods. Helquist’s method was applied using
2 equiv of Zn2+, 1 equiv of TMEDA, and 4 equiv of Et3N
for 15 h at 5 mM concentration; the cyclization product
5 was obtained in 75% yield. No stereoisomers (NMR
and HPLC data) or dimers (MS data) were detected in
this product. The removal of the Boc group was performed
with HBr in acetic acid to give the amine salt in 100%
yield, and the free base was obtained by treatment with
the ion-exchange resin MP-carbonate in methanol. Cou-
pling of the amine with 2, a route similar to that used by
Clerc et al. for syringolin A,6 was disappointing, giving
yields of 7 of ca. 50%.
More reactive and sterically smaller acylating agents
for the macrolactam amine were therefore sought, and
amino acid N-carboxy anhydrides were considered. While
such reagents were popularized for solid-phase peptide
synthesis as the urethane derivatives,13 several examples
are known of peptide bond formation using free amino
acid N-carboxy anhydrides, which should be among the
most sterically undemanding of amino acid acylating
reagents. Although they can undergo ring-opening po-
lymerization, the use of organic solvents and low tem-
peratures has enabled them to be used for monoacylation
reactions.14 The appeal of this strategy is enhanced by
the commercial availability of many amino acid N-carboxy
anhydrides, as well as excellent procedures to prepare
them from Boc-amino acids.15 In the event, the free base
derived from 5 was treated with the commercially avail-
able valine N-carboxy anhydride 6 in DMF/dichlo-
romethane for 4 h at -78 °C and then 2 h at rt. Without
purification, the acylation mixture was subjected to urea
formation with 1, and the ester 7 was obtained in 55%
yield. As this route obviates the advanced preparation of
reagents like 2, it considerably streamlines side chain
preparation. It also makes the synthesis more modular,
enabling the simple preparation of side chain variants by
subsitution of the isocyanate and N-carboxy anhydride
components. Finally, it may be considered biomimetic,
as recent studies on the biosynthesis of the syringolins
have invoked N-carboxy anhydride 6 as one possible
intermediate in the formation of this unusual amino acid
urea.16 The saponification of 7 gave syringolin B, which
exhibited spectroscopic properties (1H NMR, 13C NMR)
matching those reported for the natural product.
(6) Clerc, J.; Groll, M.; Illich, D. J.; Bachmann, A. S.; Huber, R.;
Schellenberg, B.; Dudler, R.; Kaiser, M. Proc. Natl. Acad. Sci. U.S.A. 2009,
106, 6507–6512.
With the phosphonate macrocyclization proven in the
syringolin B synthesis, we moved on to syringolin A
(Scheme 2). We required a method to prepare 3,4-
dehydrolysine intermediates, and the strategy that emerged
to address this need made for an even more modular
synthesis. Modules used for the macrolactam core include
(7) Schmidt, U.; Kleefeldt, A.; Mangold, R. J. Chem. Soc., Chem.
Commun. 1992, 1687–1689.
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J. Am. Chem. Soc. 1981, 103, 1222–1224. Tatsuta, K.; Masuda, N. J.
Antibiot. 1998, 51, 602–606. Harvey, J. E.; Raw, S. A.; Taylor, R. J. K.
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Chem. Soc. 2006, 128, 5340–5341. Haidle, A. M.; Myers, A. G. Proc. Natl.
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Mitsos, C. A. Angew. Chem., Int. Ed. 2005, 44, 3714–3717.
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Org. Lett., Vol. 12, No. 10, 2010
2403

Scheme 1. Total Synthesis of Syringolin B
Scheme 2. Total Synthesis of Syringolin A
last nitrogen. Staudinger reduction of the azide and then
coupling with the phosphonoacetic acid active ester 3 give
12 (72% for two steps). The oxidation and cyclization of
this material (2 equiv Zn2+, 1 equiv TMEDA, 8 equiv
TEA, rt, 2 mM, 12 h) is nearly as successful as in the
first synthesis (55%). When the aldehyde is added to the
reaction mixture over 8 h, the yield increases to an
outstanding 81%. While the presence of two alkenes in
the 12-membered ring is expected to increase the strain
relative to syringolin B (calculated by MMFF to be 13
kcal/mol), this does not appear to be a major impediment
to the cyclization reaction. Again, no stereoisomers are
detected in product 13. This synthesis was completed by
removal of the Cbz group with HBr in acetic acid and
acylation with 2. This process was enhanced by neutral-
izing the amine salt with the ion-exchange resin MP-
carbonate in DMF and performing the acylation in the
same pot. The known ester 14 is obtained in 73% yield.
The final deprotection used a high-yielding procedure
reported by Clerc et al.6 Synthetic syringolin A gave
spectroscopic and physicochemical properties matching
those reported for the natural product and was identical
by comparison to an authentic sample (1H NMR, 13C
NMR, HPLC).
valinol and 3 from the first synthesis, vinyl glycine
derivative 8, and 1-bromo-3-butene. Compound 8 is
available in three steps from commercial Z-Met-OMe.17
Peptide coupling of vinyl glycine derivatives is known
using conventional mixed anhydride protocols as well as
the more exotic reagent 2-isobutoxy-1-isobutoxycarbonyl-
1,2-dihydroquin-oline (IIDQ).18 These studies prepared
vinyl glycine amides for use in following metathesis
reactions, as performed here. The coupling of L-valinol
with 8 gives 9 efficiently, though slowly. This product
can be crystallized in stereochemically homogeneous form.
This coupling is significantly enhanced by microwave
irradiation (80 °C internal temperature, CEM Discover),
wherein it can be completed in 10 min. We take advantage
of the commercial availability of 1-bromo-3-butene to use
a 10-fold excess in cross-metathesis19 with 9 using the
Grubbs second-generation catalyst.20 This gives a product
10 that has exclusively the (E) stereochemistry. Nucleo-
philic substitution of 10 with sodium azide introduces the
(17) Afzali-Ardakani, A.; Rapoport, H. J. Org. Chem. 1980, 45, 4817–
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56, 10113–10125. Duggan, H. M. E.; Hitchcock, P. B.; Young, D. W. Org.
Biomol. Chem. 2005, 3, 2287–2295. Hoffmann, T.; Waibel, R.; Gmeiner,
P. J. Org. Chem. 2003, 68, 62–69.
These syntheses proceed in 7 steps and 27% overall
yield and 10 steps and 22% overall yield. The number of
functional group interconversions and redox steps was
(19) Huwe, C. M.; Blechert, S. A. Tetrahedron Lett. 1995, 36, 1621–
1624. Crowe, W. E.; Goldberg, D. R.; Zhang, Z. J. Tetrahedron Lett. 1996,
37, 2117–2120. Campagne, J.-M.; Ghosez, L. Tetrahedron Lett. 1998, 39,
6175–6178. Hoffmann, T.; Waibel, R.; Gmeiner, P. J. Org. Chem. 2003,
68, 62–69. Nolen, E. G.; Kurish, A. J.; Wong, K. A.; Orlando, M. D.
Tetrahedron Lett. 2003, 44, 2449–2453.
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Ed. 2001, 40, 1277–1279.
2404
Org. Lett., Vol. 12, No. 10, 2010

minimized by the use of commercial and amino acid
derived building blocks. These syntheses efficiently con-
struct the large lactam rings via the Horner-Wadsworth-
Emmons reaction and in particular use mild reaction
conditions developed by Helquist that were uniquely
effective in these cyclizations. Valine N-carboxy anhydride
proved a suitably active reagent to efficiently acylate the
somewhat hindered and electronically deactivated syrin-
golin B macrolactam amine. These syntheses are quite
modular and should permit the preparation of many
structural variants that could improve on the biological
properties of the parent natural products.
Acknowledgment. Fellowship support from UC-MEXUS
(TRI-R) is appreciated. Mass spectra were obtained on an
instrument purchased with NSF grant CHE0541848. We are
grateful to Paul Xu and Amber Scroggs (UC-Riverside) for
preparing starting materials and to Prof. Dudler (ETH) for
providing a comparison sample of syringolin A.
Supporting Information Available: NMR spectra for
new compounds and other experimentals and characterization
data. This material is available free of charge via the Internet
at http://pubs.acs.org.
OL100761Z
Org. Lett., Vol. 12, No. 10, 2010
2405