Enzymatic formation of an unnatural novel tetracyclic sesterterpene by β-amyrin synthase

Article abstract of DOI:10.1016/j.tetlet.2004.09.075

A C₂₅ oxidopolyprene was enzymatically converted to a novel unnatural tetracyclic sesterterpene by recombinant β-amyrin synthase from Pisum sativum, while a C₃₅ analogue did not afford any cyclization product. A convergent synthesis

Full text of DOI:10.1016/j.tetlet.2004.09.075

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 8299–8301
Enzymatic formation of an unnatural novel tetracyclic
sesterterpene by b-amyrin synthase
Hisashi Noma,a Hideya Tanaka,a Hiroshi Noguchi,a Masaaki Shibuya,b
Yutaka Ebizukab and Ikuro Abea,*
aSchool of Pharmaceutical Sciences and the 21st Century COE Program, University of Shizuoka, 52-1 Yada,
Shizuoka 422-8526, Japan
bGraduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
Received 5 August 2004; revised 9 September 2004; accepted 10 September 2004
Available online 28 September 2004
Abstract—A convergent synthesis provided a C25 and a C35 oxidopolyprene in which a farnesyl C15 unit is connected in a head-to-
head fashion to a geranyl C10 or a geranylgeranyl C20 unit. When incubated with recombinant b-amyrin synthase from Pisum
sativum the C25 oxidopolyprene was enzymatically converted to an unnatural novel tetracyclic sesterterpene, while the C35 analogue
did not afford any cyclization product.
ꢀ 2004 Elsevier Ltd. All rights reserved.
b-Amyrin synthase (bAS) (E.C. 5.4.99.-) catalyzes the
remarkable conversion of (3S)-2,3-oxidosqualene (1) to
b-amyrin (2), forming a 6/6/6/6/6-fused ring system with
a total of eight chiral centers in a single reaction.1 The
proton-initiated cyclization first produces the tetracyclic
dammarenyl C-20 cation, and the subsequent skeletal
rearrangements and 1,2-hydride shifts lead to formation
of the pentacyclic framework with the D12 double bond
(Scheme 1A). Interestingly, pentacyclic triterpene synth-
ases accept a variety of non-physiological substrate and
efficiently performs sequential ring-forming reactions to
produce a series of unnatural cyclic triterpenoids. We
have previously demonstrated that recombinant bAS
from Pisum sativum accepted 22,23-dihydro-2,3-oxido-
squalene (3) to yield a 4:1 mixture of euph-7-en-3b-ol
(4) and bacchar-12-en-3b-ol (5) (Scheme 1B).2 Further,
the enzyme efficiently converted 24,30-bisnor-2,3-oxido-
squalene into 29,30-bisnor-b-amyrin and its regioiso-
mers.3 On the other hand, it has been recently
reported that lupeol synthase from Arabidopsis thaliana
cyclized non-physiological 3-(x-oxidogeranylgeranyl)-
indole into petromindole, an indole diterpene with a 6/
6/6/6/6/5-fused ring system.4 Moreover, squalene:hopene
cyclase from a thermoacidophilic bacteria Alicyclobacil-
lus acidocaldarius accepted a C35 (geranylgeranyl-farne-
syl) polyprene hydrocarbon as a substrate to yield an
unnatural C35 hexacyclic polyprenoid with a 6/6/6/6/6/
5-fused ring system.5 Here in this paper, to further ex-
plorer the biosynthetic potential of the bAS enzyme,
we tested enzymatic conversion of newly synthesized
C25 and C35 oxidopolyprene in which a farnesyl C15 unit
is connected in a head-to-head fashion to a geranyl C10
or a geranylgeranyl C20 unit.
The convergent synthesis of the C25 (6a)6 and the C35
(6b)7 oxidopropyrene (both in racemic form) involved
the coupling of oxidofarnesyl phenylsulfone with gera-
nyl or geranylgeranyl bromide, which was followed by
dephenylsulfonation by LiHBEt3 in the presence of
catalytic amount of PdCl2 [1,3-bis(diphenylphos-
phino)propane] as described before (Scheme 2).5
When enzymatic conversion of the C35 oxidopolyprene
(6b) was attempted with the recombinant P. sativum
bAS,8 no cyclization product could be detected by
TLC or GC. Thus, the C35 oxidopolyprene was not
accepted as a substrate, suggesting that the catalytic site
of the bAS does not have enough space to accept the
larger substrate analogue with an additional C5 isoprene
unit. In contrast, enzyme reaction with the C25 oxido-
polyprene (6a) resulted in isolation of a single cycliza-
tion product (0.3mg, 0.3% yield).8 Spectroscopic data
Keywords: b-Amyrin synthase; Oxidosqualene cyclase; Triterpene
synthase; Unnatural sesterterpene.
*
Corresponding author. Tel./fax: +81 54 264 5662; e-mail:
abei@ys7.u-shizuoka-ken.ac.jp
13
(1H and C NMR, HMQC, HMBC, and MS)9 of the
0040-4039/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2004.09.075

8300
H. Noma et al. / Tetrahedron Letters 45 (2004) 8299–8301
(A)
chair
-chair
-chair
-boat
H
12
H
12
13
- H-12
H
HO
HO
HO
HO
HO
O
Dammarenyl cation
Lupanyl cation
1
Baccharenyl cation
Oleanyl cation
2
(B)
22
chair
-chair
-chair
-boat
23
12
12
- H-12
HO
HO
HO
O
5
H
3
H
- H-7
HO
13
17
20
16
7
HO
H
7
H
4
(C)
chair
12
-chair
-chair
-boat
HO
HO
O
21
22
18
12
20
17
6a
11
9
16
15
19
H
25
19
13
14
- H-7
H
1
4
24
13
17
21
17
2
3
20
10
8
HO
5
22
20
16
HO
25
7
H
7
7
HO
23
18
6
H
24
23
7
Scheme 1. Proposed mechanism for the conversion of (A) 2,3-oxidosqualene (1) to b-amyrin (2); (B) 22,23-dihydro-2,3-oxidosqualene (3) to a 4:1
mixture of euph-7-en-3b-ol (4) and bacchar-12-en-3b-ol (5); (C) C25 oxidopropyrene (6a) to compound 7 by recombinant P. sativum b-amyrin
synthase.
product were characteristic of those of tetracyclic hydro-
carbon alcohols, and showed good agreement with
euph-7-en-3b-ol (4)2 except the signals due to the alkyl
side chain, suggesting the structure of a novel tetracyclic
OH
sesterterpene alcohol (7) (Scheme 1C). The 6/6/6/5 ring
system was uniquely consistent with both biogenetic
reasoning and the heteronuclear correlation NMR
spectroscopy (HMQC and HMBC). Finally, the stereo-
chemistry of C-17 was confirmed by NOEs observed
between Me-19/Me-24 and Me-19/Me-25, but absent
between Me-18/Me-25 and Me-25/Me-21,22.
a, b
c, d
SO2Ph
SO2Ph
The enzymatic conversion of the C25 oxidopolyprene
(6a) (0.3% yield) by the recombinant bAS was much less
efficient than that of 22,23-dihydro-2,3-oxidosqualene
(3%)2 and 24,30-bisnor-2,3-oxidosqualene (30%).3 The
absence of the terminal C5 isoprene unit of 2,3-oxid-
osqaulene thus resulted in the poor yield of the cycliza-
tion reaction. As in the case of the enzymatic formation
of euph-7-en-3b-ol (4) from 22,23-dihydro-2,3-oxido-
squalene (3), a substrate analogue lacking the terminal
double bond of 2,3-oxidosqualene (Scheme 1B), bAS
initiated cyclization of 6a from a chair–chair–chair–boat
conformation to generate the Markovnikov damma-
rene-type tertiary cation with the 17b-isopropyl group.
Then, a backbone rearrangement (H-17a ! 20a, H-
13b ! 17b, CH3-14a ! 13a, CH3-8b ! 14b) with elim-
ination of H-7a proton yielded the unnatural tetracyclic
+
Br
O
n
n = 1 or 3
e, f
O
n
6a: n = 1
6b: n = 3
Scheme 2. Reagents and conditions: (a) PBr3, hexane, ꢀ5ꢁC; (b)
PhSO2, DMF, 83%; (c) NBS, 20% THF/H2O, 28%; (d) K2CO3,
MeOH, 88%; (e) n-BuLi (1equiv), THF, ꢀ78ꢁC, 72%; (f)
[PdCl2(dppp)] (10mol%), LiBEt3H (2equiv), 0ꢁC, 58%.

H. Noma et al. / Tetrahedron Letters 45 (2004) 8299–8301
8301
3. Abe, I.; Sakano, Y.; Sodeyama, M.; Tanaka, H.; Noguchi,
H.; Shibuya, M.; Ebizuka, Y. J. Am. Chem. Soc. 2004, 126,
6880–6881.
4. Xiong, Q.; Zhu, X.; Wilson, W. K.; Ganesan, A.; Matsuda,
S. P. T. J. Am. Chem. Soc. 2003, 125, 9002–9003.
product 7 with a Markovnikov five-membered D-ring
(Scheme 1C). It was remarkable that the stereochemistry
of the non-physiological product, whose structure is
apparently different from b-amyrin, was strictly control-
led by the enzyme, even in the absence of the terminal C5
isoprene unit. Interestingly, no evidence was obtained
for the formation of baccharene-type product with the
anti-Markovnikov six-membered D-ring in the reaction
mixture. Further analyses of the catalytic potential of
functionally divergent triterpene synthases promise to
reveal intimate structural details of the enzyme catalyzed
processes and suggest strategies for manipulating sub-
strate and product specificities of the polyene cyclization
reactions.
5. Abe, I.; Tanaka, H.; Noguchi, H. J. Am. Chem. Soc. 2002,
124, 14514–14515.
6. C25 oxidopolyprene 6a: 1H NMR (400MHz, CDCl3): d 5.13
(m, 4H), 2.70 (t, 1H, J = 6.2Hz), 2.09–1.98 (m, 16H), 1.68
(s, 3H), 1.60 (s, 9H), 1.55 (s, 3H), 1.30 (s, 3H), 1.26 (s, 3H);
13C NMR (100MHz, CDCl3): d 135.1, 134.9, 134.0, 131.2,
124.9, 124.4 (·2), 124.3, 64.2, 58.3, 39.7, 39.7, 36.3, 28.2
(·2), 27.5, 26.7, 26.7, 25.7, 24.9 (·2), 18.7, 17.7, 16.0, 16.0.
HRMS (EI): found for [C25H42O] 358.3240; calcd 358.3236.
7. C35 oxidopolyprene 6b: 1H NMR (400MHz, CDCl3): d 5.13
(m, 6H), 2.70 (t, 1H, J = 5.6Hz), 2.20–1.96 (m, 24H), 1.68
(s, 3H), 1.60 (s, 15H), 1.55 (s, 3H), 1.30 (s, 3H), 1.26 (s, 3H);
13C NMR (100MHz, CDCl3): d 135.1, 134.9 (·2), 134.9,
134.0, 131.2, 124.9, 124.8, 124.4 (·2), 124.3 (·2), 64.2, 58.3,
39.8, 39.7 (·3), 36.3, 28.3 (·2), 27.5, 26.8, 26.7 (·3), 25.7,
24.9, 18.7, 17.7, 16.0 (·3), 16.0 (·2). HRMS (EI): found for
[C35H58O] 494.4472; calcd 494.4488.
Acknowledgements
This work was in part supported by the 21st Century
COE Program and Grant-in-Aid for Scientific Research
(Nos. 16510164, 1531053, and 15101007) from the Min-
istry of Education, Culture, Sports, Science and Tech-
nology, Japan, and by Grant-in-Aid from The
Mochida Memorial Foundation for Medical and Pharma-
ceutical Research, and the Tokyo Biochemical Research
Foundation, Japan.
8. P. sativum bAS was expressed in the yeast mutant strain
GIL77 (10L of culture) as described before.2,3 The reaction
mixture containing 100mg of C25 oxidopolyprene 6a (or
C35 oxidopolyprene 6b), 0.45M sucrose, 1mM EDTA,
1mM DTT, and 0.1% Triton X-100 in 200mL of 0.1M
KPB, pH7.4, was incubated at 30ꢁC for 18h. The incuba-
tions were stopped by adding equal volume of 20% KOH in
50% aq EtOH, saponified at 30ꢁC for 24h, and extracted
with 400mL of hexane (·3). The combined extracts were
evaporated to dryness, separated on SiO2 column (15%
EtOAc/hexane), and finally purified by HPLC (YMC-Pack
PRO C4; 5% THF/CH3CN; 0.3mL/min) to give 0.3mg of
compound 7, along with 3.5mg of b-amyrin derived from
2,3-oxidosqualene accumulated in the mutant yeast cells.
9. Compound 7: 1H NMR (400MHz, CDCl3): d 5.26 (dt, 1H,
J = 4.0, 2.8Hz, H-7), 3.24 (dd, 1H, J = 11.0, 4.2Hz, H-3),
0.97 (s, 6H, Me-23, Me-25), 0.88 (d, 6H, J = 6.4Hz, Me-21,
Me-22), 0.86 (s, 3H, Me-24), 0.81 (s, 3H, Me-18), 0.75 (s,
3H, Me-19); 13C NMR (100MHz, CDCl3): d 146.9 (C-8),
117.8 (C-7), 79.2 (C-3), 54.9 (C-17), 51.2 (C-14), 50.6 (C-5),
48.9 (C-9), 43.5 (C-13), 38.9 (C-4), 37.2 (C-1), 35.0 (C-10),
34.0 (C-15), 33.5 (C-12), 29.7 (C-20), 28.3 (C-16), 27.7 (C-
23), 27.6 (C-25), 24.0 (C-6), 22.2 (C-21), 22.2 (C-22), 21.9
(C-18), 18.0 (C-11), 14.7 (C-24), 13.1 (C-19). The NMR
assignments were performed according to data from
HMQC, HMBC experiments, and by comparison with
the data of euph-7-en-3b-ol. LRMS (EI): m/z 358 [M]+, 343,
325, 203, 175, 119, 95, 69, 43. HRMS (EI): found for
[C25H42O] 358.3241; calcd 358.3236.
Supplementary data
Complete set of spectroscopic data (1H and 13C NMR,
HMQC, HMBC, NOE, and MS) of compound 7 (7
pages). Supplementary data associated with this article
can be found, in the online version, at doi:10.1016/
j.tetlet.2004.09.075.
References and notes
1. For reviews, see: (a) Abe, I.; Rohmer, M.; Prestwich, G. D.
Chem. Rev. 1993, 93, 2189–2206; (b) Wendt, K. U.; Schulz,
G. E.; Corey, E. J.; Liu, D. R. Angew. Chem., Int. Ed. 2000,
39, 2812–2833; (c) Xu, R.; Fazio, G. C.; Matsuda, S. P. T.
Phytochemistry 2004, 65, 261–291.
2. Abe, I.; Sakano, Y.; Tanaka, H.; Lou, W.; Noguchi, H.;
Shibuya, M.; Ebizuka, Y. J. Am. Chem. Soc. 2004, 126,
3462–3463.