Stereoselective synthesis of deuterium-labeled (2 s)-cyclohexenyl alanines, biosynthetic intermediates of cinnabaramide

Article abstract of DOI:10.1021/ol3029548

Dideuterated β-cyclohexenylalanines, proposed biosynthetic intermediates of the cinnabaramides, can be obtained from chiral alkynols via a sequence of Irland-Claisen rearrangement, ring closing metathesis, and radical decarboxylation. Feeding experiments indicate that both (2S)-β- cyclohexenylalanines can be incorporated into cinnabaramide, while the configuration at the cyclohexenyl ring does not restrict biosynthetic processing.

Full text of DOI:10.1021/ol3029548

ORGANIC
LETTERS
2012
Vol. 14, No. 23
6064–6067
Stereoselective Synthesis of
Deuterium-Labeled (2S)‑Cyclohexenyl
Alanines, Biosynthetic Intermediates
of Cinnabaramide
†
Philipp Barbie, Liujie Huo, Rolf Muller, and Uli Kazmaier*
‡
‡
,†
€
Saarland University, Institute of Organic Chemistry, Campus, Bldg. C4.2, and
Helmholtz Institute for Pharmaceutical Research Saarland, Helmholtz Centre for
Infection Research and Department of Pharmaceutical Biotechnology,
D-66123 Saarbruecken, Germany
u.kazmaier@mx.uni-saarland.de
Received October 26, 2012
ABSTRACT
Dideuterated β-cyclohexenylalanines, proposed biosynthetic intermediates of the cinnabaramides, can be obtained from chiral alkynols via a
sequence of IrlandÀClaisen rearrangement, ring closing metathesis, and radical decarboxylation. Feeding experiments indicate that both (2S)-β-
cyclohexenylalanines can be incorporated into cinnabaramide, while the configuration at the cyclohexenyl ring does not restrict biosynthetic
processing.
The proteasome is a key player in the regulation of a
wide range of cellularprocesses by degradation of proteins,
mediating processes such as amino acid recycling, cell
differentiation, and apoptosis.1 Therefore, proteasome
inhibitors are interesting candidates as antitumor drugs.2
Effective proteasome inhibitors are widespread in nature,
produced by a wide range of microorganisms. Many of
these inhibitors are peptide derived small molecules, such
as the group of γ-lactamÀβ-lactones.3
clasto-form omuralide (Figure 1).5 A closely related family
of compounds are the salinosporamides,6 isolated by Fenical
from the marine actinomycete Salinospora tropica.7 Almost
the same structural motif was found in the cinnabaramides,
a secondary metabolite of the terrestrial strain Streptomyces
JS 360.8
The cinnabaramides and salinosporamides only differ in
the side chain of the γ-lactam ring. Cinnabaramides and
salinosporamide A inhibit the 20S-proteasome in the low
nanomolar range.8,9 The strained β-lactone ring of these
In 1991, Omura et al. isolated a potent inhibitor from
Streptomyces lactacystineus, called lactacystin.4 But actu-
ally, the active compound was not lactacystin itself, but its
(5) Dick, L. R.; Cruikshank, A. A.; Grenier, L.; Melandri, F. D.;
Nunes, S. L.; Stein, R. L. J. Biol. Chem. 1996, 271, 7273–7275.
(6) Reed, K. A.; Manam, R. R.; Mitchell, S. S.; Xu, J.; Teisan, S.;
Chao, T. H.; Deyanat-Yazdi, G.; Neuteboom, S. T. C.; Lam, K. S.;
Potts, B. C. M. J. Nat. Prod. 2007, 70, 269–276.
(7) Feling, R. H.; Buchanan, G. O.; Minzer, T. J.; Kauffman, C. A.;
Jensen, P. R.; Fenical, W. Angew. Chem. 2003, 115, 369–371. Angew.
Chem., Int. Ed. 2003, 42, 355–357.
† Saarland University.
‡ Helmholtz Institute for Pharmaceutical Research Saarland.
(1) (a) Goldberg, A. L. Nature 2003, 426, 895–899. (b) Goldberg,
A. L. Biochem. Soc. Trans. 2007, 35, 12–17. (b) Murata, S.; Yashiroda,
H.; Tanaka, K. Nat. Rev. Mol. Cell Biol. 2009, 10, 104–115.
(2) Borissenko, L.; Groll, M. Chem. Rev. 2007, 107, 687–717.
(3) (a) Gulder, T. A. M.; Moore, B. S. Angew. Chem. 2010, 122, 9534–
9556. Angew. Chem., Int. Ed. 2010, 49, 9346–9367. (b) Kale, A. J.;
McGlinchey, R. P.; Lechner, A.; Moore, B. S. ACS Chem. Biol. 2011, 6,
1257–1264.
€
(8) Stadler, M.; Bitzer, J.; Mayer-Bartschmid, A.; Muller, H.; Benet-
Buchholz, J.; Gantner, F.; Tichy, H.-V.; Reinemer, P.; Bacon, K. B.
J. Nat. Prod. 2007, 70, 246–252.
(9) (a) Fenteany, G.; Standaert, R. F.; Lane, W. S.; Choi, S.; Corey,
E. J.; Schreiber, S. L. Science 1995, 268, 726–731. (b) Groll, M.; Ditzel,
€
(4) Omura, S.; Fujimoto, T.; Matsuzaki, K.; Moriguchi, R.; Tanaka,
H.; Sasaki, Y. J. Antibiot. 1991, 44, 113–116.
L.; Lowe, J.; Stock, D.; Bochtler, M.; Bartunik, H. D.; Huber, R. Nature
1997, 386, 463–471.
r
10.1021/ol3029548
Published on Web 11/19/2012
2012 American Chemical Society

salinosporamides,3b,16 we are mainly interested in the
cinnabaramides.17
In principle, the molecule can be divided into two major
parts. In the case of the salinosporamides, the lower part of
the molecule is the product of a polyketide synthase (PKS)
leading to an activated β-ketothioester A (Scheme 1). The
upper part, the unusual amino acid β-hydroxy cyclohex-
enylalanine, is formed via a shunt in the phenylalanine
biosynthetic pathway, leading primarily to β-cyclohexeny-
lalanine B.16a After coupling to the peptidyl carrier protein
and oxidation by a cytochrome P450 hydroxylase (C), the
two building blocks are coupled on a nonribosomal pep-
tide synthetase (NRPS) to give D. Subsequent cyclization
gives rise to salinosporamide.16
Figure 1. Naturally occurring γ-lactamÀβ-lactones.
compounds interacts with a threonine in the active center
oftheproteasome, inactivatingthe enzymebyringopening
and covalent blocking of the active center.9 Salinosporamide
is found to trigger apoptosis and is in phase 1 of clinical trials
for the treatment of multiple myeloma.10 Not surprising,
these natural products arouse interest in the community of
synthetic organic chemists, and a wide range of interesting
syntheses have been developed in recent years for omuralide,11
cinnabaramide,12 and especially salinosporamide13 and
derivatives.14 The recent developments in this field are
nicely covered in reviews by Moore3a and Potts.15
Scheme 1. Biosynthesis of Salinosporamide A according to
Moore16a
Besides the development of straightforward protocols
for the synthesis of these compounds, much effort has
also been focused toward investigating their biosynthesis.
While Moore et al. are involved in the biosynthesis of the
(10) (a) Chauhan, D.; Catley, L.; Li, G.; Podar, K.; Hideshima, T.;
Velankar, M.; Mitsiades, C.; Mitsiades, N.; Yasui, H.; Letai, A.; Ovao,
H.; Berkers, C.; Nicholson, B.; Chao, T.-H.; Neuteboom, S. T. C.;
Richardson, P.; Palladino, M. A.; Anderson, K. C. Cancer Cell 2005, 8,
407–419. (b) Potts, B. C.; Albitar, M. X.; Anderson, K. C.; Baritaki, S.;
Berkers, C.; Bonavida, B.; Chandra, J.; Chauhan, D.; Cusack, J. C., Jr.;
Fenical, W.; et al. Curr. Cancer Drug Targets 2011, 11, 254–284.
(11) (a) Corey, E. J.; Li, W. D. Z. Chem. Pharm. Bull. 1999, 47, 1–10.
(b) Crane, S. N.; Corey, E. J. Org. Lett. 2001, 3, 1395–1397. (c) Gilley,
C. B.; Buller, M. J.; Kobayashi, Y. Org. Lett. 2007, 9, 3631–3634. (d)
Hayes, C. J.; Sherlock, A. E.; Green, M. P.; Wilson, C.; Blake, A. J.;
Selby, M. D.; Prodger, J. C. J. Org. Chem. 2008, 73, 2041–2051. (e) Gu,
W.; Silverman, R. B. J. Org. Chem. 2011, 76, 8287–8293.
(12) Ma, G.; Nguyen, H.; Romo, D. Org. Lett. 2007, 9, 2143–2146.
(13) (a) Reddy, L. R.; Saravanan, P.; Corey, E. J. J. Am. Chem. Soc.
2004, 126, 6230–6231. (b) Endo, A.; Danishefsky, S. J. J. Am. Chem. Soc.
2005, 127, 8298–8299. (c) Mulholland, N. P.; Pattenden, G.; Walters,
I. A. S. Org. Biomol. Chem. 2006, 4, 2845–2846. (d) Ling, T.; Macherla,
V. R.; Manam, R. R.; McArthur, K. A.; Potts, B. C. M. Org. Lett. 2007,
9, 2289–2292. (e) Mulholland, N. P.; Pattenden, G.; Walters, I. A. S.
Org. Biomol. Chem. 2008, 6, 2782–2789. (f) Takahashi, K.; Midori, M.;
Kawano, K.; Ishihara, J.; Hatakeyama, S. Angew. Chem. 2008, 120,
6340–6342. Angew. Chem., Int. Ed. 2008, 47, 6244–6246. (g) Fukuda, T.;
Sugiyama, K.; Arima, S.; Harigaya, Y.; Nagamitsu, T.; Omura, S. Org.
Lett. 2008, 10, 4239–4242. (h) Mosey, R. A.; Tepe, J. J. Tetrahedron
2009, 50, 295–297. (i) Nguyen, H.; Ma, G.; Romo, D. Chem. Commun.
2010, 46, 4803–4805. (k) Ling, M.; Potts, B. C.; Macherla, V. R. J. Org.
Chem. 2010, 75, 3882–3885. (l) Sato, Y.; Fukuda, H.; Tomizawa, M.;
Masaki, T.; Shibuya, M.; Kanoh, N.; Iwabuchi, Y. Heterocycles 2010,
81, 2239–2246. (m) Nguyer, H.; Ma, G.; Gladysheva, T.; Fremgen, T.;
Romo, D. J. Org. Chem. 2011, 76, 2–12. (n) Kaiya, Y.; Hasegawa, J.-L.;
Momose, T.; Sato, T.; Chida, N. Chem.;Asian J. 2011, 6, 209–219.
(o) Satoh, N.; Yokoshima, S.; Fukuyama, T. Org. Lett. 2011, 13, 3028–
3031.
In principle, one might expect a very similar biosynthetic
pathway also for the cinnabaramides, which should differ
mainly in the PKS-subunit. To study the cinnabaramide
biosynthesis, we were interested in obtaining deuterium-
labeled building blocks which can be used for feeding
experiments. Recently, we described the stereoselective
synthesis of dideuterated (2R,3S,4S)-β-cyclohexenyl-
serine,18 a postulated intermediate in an early biosynthetic
proposal.16a Later, the configuration of theamino acid was
determined to be (2S).16b Therefore, it was not so surpris-
ing that no incorporation of this amino acid itself, or in
the activated form, was observed in feeding experiments.
€
Recent biosynthetic studies by the Muller group indicate
that, in the case of the cinnabaramides, B is probably
coupled to the corresponding β-ketoester and that the
cyclochrome P-450 oxidation at the β-position proceeds
later on in the biosynthesis.19
To prove this proposal, we developed a stereoselective
synthesis of the two isomeric dideuterated (2S)-β-cyclo-
hexenylalanines. So far, the absolute configuration of the
cinnabaramide intermediates is not yet determined, but the
(2S) configuration seems reasonable, based on the analogy
to the salinosporamide biosynthesis. In the natural prod-
uct, the (4S)-configuration is found, and therefore the
(2S,4R)-β-cyclohexenylalanine 1 should be the correct
(14) Chen, Z. H.; Wang, B. C.; Kale, A. J.; Moore, B. S.; Wang,
R. W.; Qing, F. L. J. Fluorine Chem. 2012, 136, 12–19.
(15) Potts, B. C.; Lam, K. S. Mar. Drugs 2010, 8, 835–880.
(16) (a) Beer, L. L.; Moore, B. S. Org. Lett. 2007, 9, 845–848. (b)
ꢀ
McGlinchey, R. P.; Nett, M.; Eustaquio, A. S.; Asolkar, R. N.; Fenical,
€
W.; Moore, B. S. J. Am. Chem. Soc. 2008, 130, 7822–7823.
(17) Rachid, S.; Huo, L.; Hermann, J.; Stadler, M.; Kopcke, B.;
(18) Deska, J.; Hahn, S.; Kazmaier, U. Org. Lett. 2011, 13, 3210–
3213.
(19) Huo, L.; Muller, R., unpublished results.
€
€
€
Bitzer, J.; Muller, R. ChemBioChem 2011, 12, 922–931.
Org. Lett., Vol. 14, No. 23, 2012
6065

intermediate. Interestingly, the biosynthetic enzyme re-
sponsible for the activation of the β-cyclohexenylalanine
seems to have a rather broad substrate spectrum, allowing
also the incorporation of other amino acids.16b,20 There-
fore, we were interested to see if the configuration of the
4-position does play any role.
dideuterated allyl alcohol (R)-4. Coupling to semipro-
tected aspartate22 using the Steglich protocol provided
the required allylester 5, the substrate for the subsequent
Claisen rearrangement. Our group was investigating
Claisen rearrangements of amino acids for a long time.23
While the best results are obtained for R-amino acid esters
with chelated enolates,24 this protocol is not really suitable
for β-amino acid esters, such as 5. Here the IrelandÀClaisen
rearrangement25 is the method of choice,26 providing 6 in
high yield and diastereoselectivity. While the stereogenic
center at C-2 remains unchanged, the configuration at
C-4 is determined by the stereogenic center of the allyl
alcohol (chirality transfer almost perfect). The configura-
tion at C-3 is the result of the enolate formation in the
deprotonation step. This center is formed as a 9:1 mixture,
according to NMR. But this center is negligible, because
it is removed later on. The diastereomeric rearrangement
products could be separated by flash chromatography.
Diastereomerically pure 6 was subjected to ring closing
metathesis providing cyclohexenyl aspartate 7 in high
yield. In case the diastereomeric mixture was subjected
to RCM also at this stage, a separation of the isomers was
possible.
Our retrosynthetic plan toward the (2S,4R)-1 is shown
in Scheme 2. D2-labeled β-cyclohexenylalanine 1 should be
accessible via radical decarboxylation of the corresponding
aspartate derivative E, while the cyclohexenyl ring should
be the result of a ring closing metathesis (RCM). The key
step of the synthesis is a stereoselective Claisen rearrange-
ment providing the diene F from the allyl ester G. In this
step the stereogenic center at C-4 is incorporated. Depend-
ing on the configuration of the allyl alcohol used, both
isomers can be obtained stereoselectively. The configura-
tion at C-3 of F does not play any role, because this
stereogenic center is removed later on. The required di-
deuterated trans allyl alcohol should be available from the
corresponding propargyl alcohol (R)-2 via reduction using
LiAlD4.
The next step, the radical decarboxylation, was found to
be the most critical one of the whole sequence. We decided
to apply the Barton protocol using the corresponding
N-hydroxythiopyridone esters.27 Unfortunately, this ester
was found to be very sensitive, undergoing fast decom-
position. Attempts to isolate and purify it were unsuccess-
ful. Therefore, we had to carry out the activation and
decarboxylation in a one-pot protocol. We investigated
a wide range of coupling reagents for the initial step, the
formation of the active ester. No ester formation was
observed by using chloroformates, carbonyldiimidazole,
or T3P as coupling reagents. DCC at À20 °C provided the
required ester, but in impure form, and therefore, the
decarboxylated product was very difficult to separate from
the byproducts formed. The best results were obtained
using PhPOCl2/NEt3 in the activation step, and the very
mild BEt3/O2 protocol28 for the radical formation. t-BuSH
was used as a H-source. Under these optimized conditions,
the required product 8 could be obtained in 49% yield. The
final steps toward 1 proceeded quantitatively. The isomeric
(2S,4S)-derivative was obtained from (S)-2 in an analo-
gous fashion and comparable yield.
Scheme 2. Retrosynthesis of (2S,4R)-Dideuterated Cyclohexe-
nylalanine 1
We began our synthesis with the coupling of 5-bromo-
pentene with butyn-3-ol (Scheme 3). Although this reac-
tion is described in the literature,21 we were able toimprove
the yield of 2 significantly by using LiNH2 as a base
(instead of BuLi). The racemic alkynol 2 was subsequently
subjected to an enzymatic kinetic resolution using Novozym
435. The reaction stops after 50% conversion, and both
enantiomeric compounds were obtained with high yield
and ee. Unfortunately, we were not able to determine the
ee-value of the unreacted alcohol directly by GC (no
separation) and therefore, for analytical purposes, the
remaining alcohol (S)-2 was also converted into the acetate
(S)-3. Upon saponification, both enantiomers of alkynol 2
were obtained.
(22) Sato, S.; Tetsuhashi, M.; Sekine, K.; Miyachi, H.; Naito, M.;
Hashimoto, Y.; Aoyama, H. Bioorg. Med. Chem. 2008, 16, 4685–4698.
(23) (a) Kazmaier, U. Amino Acids 1996, 11, 283–299. (b) Kazmaier,
U.; Maier, S. Org. Lett. 1999, 1, 1763–1766. (c) Kazmaier, U.; Mues, H.;
Krebs, A. Chem.;Eur. J. 2002, 8, 1850–1855.
(24) (a) Kazmaier, U.; Maier, S. Tetrahedron 1996, 52, 941–954. (b)
Kazmaier, U.; Schneider, C. Synthesis 1998, 1321–1326. (c) Kazmaier,
U.; Schneider, C. Tetrahedron Lett. 1998, 39, 817–818. (d) Kazmaier, U.;
Maier, S. J. Org. Chem. 1999, 64, 4574–4575.
For the synthesis of the deuterated (2S,4R)-cyclohex-
enylalanine 1, (R)-2 was reduced with LiAlD4 (Scheme 3).
The in situ formed vinylalumoxane was quenched by
the addition of D2O giving rise to the trans-configured
(25) (a) Ireland, R. E.; Mueller, R. H. J. Am. Chem. Soc. 1972, 94,
5897–5898. (b) Ireland, R. E.; Mueller, R. H.; Willard, A. K. J. Am.
Chem. Soc. 1976, 98, 2868–2877.
(26) Becker, D.; Kazmaier, U. J. Org. Chem., in press (DOI: 10.1021/
jo301693d).
(27) Barton, D. H. R.; Crich, D.; Motherwell, W. B. Tetrahedron
1985, 41, 3901–3924.
(20) Nett, M.; Gulder, T. A. M.; Kale, A. J.; Hughes, C. C.; Moore,
B. S. J. Med. Chem. 2009, 52, 6163–6167.
(21) Roush, W. R.; Spada, A. P. Tetrahedron Lett. 1982, 23, 3773–
3776.
(28) Ollivier, C.; Renaud, P. Chem. Rev. 2001, 101, 3415–3434.
6066
Org. Lett., Vol. 14, No. 23, 2012

Based on this efficient incorporation we can conclude that
(2S,4R)-β-cyclohexenylalanine 1 most likely serves as the
native substrate of the cinnabaramide peptide synthetase.
Interestingly, while the salinosporamide pathway shows
a broad substrate tolerance allowing for the incorporation
of alternative amino acids, no cinnabaramides derived
from incorporation of proteinogenic amino acids have
been detected in extracts of the wild type producer. But
interestingly, the diastereomeric (2S,4S)-cyclohexenylala-
nine wasincorporatedaswell. Thismay indicate thateither
(1) the free amino acids cannot be activated for further
incorporation or (2) in vivo insufficient quantities of amino
acids are available for secondary metabolite biosynthesis.
As the latter scenario is unlikely we assume that it should
be possible to incorporate alternative amino acids in the
respective position of cinnabaramides. Ongoing work in
our laboratories is addressing this question. After con-
ducting a bioactivity screening, we expect to discover more
cytotoxic novel cinnabaramides.
Scheme 3. Synthesis of d2-(2S,4R)-1
In conclusion, we have shown that the IrlandÀClaisen
rearrangement of β-amino acid allylic esters is a highly
suitable tool for the stereoselective synthesis of β-substi-
tuted aspartates. Subsequent ring closing metatheses and
decarboxylations give rise to deuterated β-cyclohexenyla-
lanine derivatives. Feeding experiments indicate that (2S)-
β-cyclohexenylalanine is an intermediate in the biosynthesis
of cinnabaramide. The configuration at the cyclohexenyl
ring doesnot play any role, asobviouslybothstereoisomers
are accepted by the peptidyl carrier protein. Further bio-
synthetic studies are currently under investigation.
Acknowledgment. Financial support from the Deutsche
Forschungsgemeinschaft was gratefully acknowledged.
Subsequent feeding experiments were conducted using
compound 1, which was stepwise administrated to the
culture broth of Streptomyces sp. JS360 (see Supporting
Information). Organic extracts of the culture supernatants
were analyzed using HPLC-MS, which unambiguously
demonstrated incorporation of 1 into cinnabaramide re-
sulting in the production of dideuterated cinnabaramides
in a yield comparable to that of native cinnabaramides.
Supporting Information Available. Experimental pro-
cedures as well as analytical and spectroscopic data of all
new compounds, details of the feeding experiments, and
copies of NMR spectra. This material is available free of
charge via the Internet at http://pubs.acs.org.
The authors declare no competing financial interest.
Org. Lett., Vol. 14, No. 23, 2012
6067