Enzymatic formation of indole-containing unnatural cyclic polyprenoids by bacterial squalene:hopene cyclase

Article abstract of DOI:10.1021/ol052507q

(Chemical Equation Presented) Two indole-containing substrate analogues, in which a C₂₀ isoprene unit is connected to indole (3-(geranylgeranyl)indole and 3-(farnesyldimethylallyl)indole), were synthesized and tested for enzymatic cyclization by squalene:hopene cyclase from Alicyclobacillus acidocaldarius. Interestingly, 3-(geranylgeranyl)indole was not a substrate for the bacterial squalene cyclase, while 3-(farnesyldimethylallyl) indole was efficiently converted to a 2:1 mixture of unnatural novel products.

Full text of DOI:10.1021/ol052507q

ORGANIC
LETTERS
2005
Vol. 7, No. 26
5873-5876
Enzymatic Formation of
Indole-Containing Unnatural Cyclic
Polyprenoids by Bacterial
Squalene:Hopene Cyclase
Hideya Tanaka,† Hiroshi Noguchi,† and Ikuro Abe*,†,¶
School of Pharmaceutical Sciences and the COE 21 Program, UniVersity of Shizuoka,
Shizuoka 422-8526, Japan, and PRESTO, Japan Science and Technology Agency,
Kawaguchi, Saitama 332-0012, Japan
abei@ys7.u-shizuoka-ken.ac.jp
Received October 17, 2005
ABSTRACT
Two indole-containing substrate analogues, in which a C20 isoprene unit is connected to indole (3-(geranylgeranyl)indole and 3-(farnesyldim-
ethylallyl)indole), were synthesized and tested for enzymatic cyclization by squalene:hopene cyclase from Alicyclobacillus acidocaldarius.
Interestingly, 3-(geranylgeranyl)indole was not a substrate for the bacterial squalene cyclase, while 3-(farnesyldimethylallyl)indole was efficiently
converted to a 2:1 mixture of unnatural novel products.
Squalene:hopene cyclase (SHC) (E.C. 5.4.99.7) from a
thermoacidophilic bacteria, Alicyclobacillus acidocaldarius,
Scheme 1. Enzymatic Cyclization of Squalene by SHC
catalyzes remarkable cyclization of squalene (1) into a 5:1
mixture of hop-22(29)-ene (2) and hopan-22-ol (3) (Scheme
1). The enzyme binds the substrate in an all-chair conforma-
tion and mediates sequential C-C bond formation reactions
through a progression of rigidly held carbocationic interme-
diates. Recent crystallographic and structure-based mutagen-
esis studies as well as utilization of active-site probes have
revealed intimate structural details of the enzyme-templated
polyene cyclization reaction.1,2
Interestingly, the bacterial SHC exhibits remarkably broad
substrate tolerance. The enzyme accepts a wide variety of
† University of Shizuoka.
¶ PRESTO.
nonphysiological substrate analogues (C15-C35) and ef-
ficiently performs sequential ring-forming reactions to
produce a series of unnatural cyclic polyprenoids.3 On the
basis of crystal structure of the enzyme, the hydrophobic
(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) Hoshino, T.;
Sato, T. Chem. Commun. 2002, 291-301.
10.1021/ol052507q CCC: $30.25
© 2005 American Chemical Society
Published on Web 11/23/2005

active-site cavity lined with aromatic amino acid residues
appears to have enough space to accept larger substrate
analogues.2 One of the most impressive examples is the
enzymatic formation of a supranatural hexacyclic polyprenoid
with a 6/6/6/6/6/5-fused ring system from a C35 analogue in
which a farnesyl C15 unit is connected in a head-to-head
fashion to a geranylgeranyl C20 unit.3a It is remarkable that
even for the unnatural substrate with an additional isoprene
unit stereochemistry of the cyclization reaction was strictly
controlled by the enzyme, leading to formation of 11 chiral
centers in a regio- and stereospecific manner. Manipulation
of the enzyme reaction by substrate analogues would thus
lead to further production of chemically and structurally
disparate unnatural polycyclic polyprenoids.
cyclase from Arabidopsis thaliana has been recently reported
by Matsuda and co-workers.6a Substrate 5 has a farnesyl C15
unit as in the case of the natural substrate, squalene (C15
unit × 2). Our previous studies have suggested that the
presence of the farnesyl C15 unit in the substrate is important
for enzymatic formation of polycyclic products by A.
acidocaldarius SHC.3a,c Further, the π-π interactions of the
indole ring with the active-site aromatic residues of the
enzyme were also anticipated for both substrate analogues.
The convergent synthesis of 4 involved regioselective
alkylation of indole at C3 by geranylgeranyl C20 bromide
mediated by zinc triflate according to the literature (Scheme
2A).6 On the other hand, 5, in which a farnesyl C15 unit is
We report herein the synthesis and enzymatic cyclization
of two indole-containing substrate analogues in which a C20
isoprene unit is connected to indole, 3-(geranylgeranyl)indole
(4)4 and 3-(farnesyldimethylallyl)indole (5).5 Substrate 4 is
a putative precursor for natural indole diterpenes,6a,7 and en-
zymatic cyclization of 3-(ω-oxidogeranylgeranyl)indole into
a hexacyclic petromindole by a plant oxidosqualene:lupeol
Scheme 2. Synthesis of Indole-Containing Substrate
Analogues
(2) For crystal structure of A. acidocaldarius SHC, see: (a) Wendt, K.
U.; Poralla, K.; Schulz, G. E. Science 1997, 277, 1811-1815. (b) Wendt,
K. U.; Lenhart, A.; Schulz, G. E. J. Mol. Biol. 1999, 286, 175-187.
(3) For enzymatic conversion of squalene analogues (C15-C35) by
bacterial squalene cyclase, see: (a) Abe, I.; Tanaka, H.; Noguchi, H. J.
Am. Chem. Soc. 2002, 124, 14514-14515. (b) Tanaka, H.; Noguchi, H.;
Abe, I. Org Lett. 2004, 6, 803-806. (c) Tanaka, H.; Noguchi, H.; Abe, I.
Tetrahedron Lett. 2004, 45, 3093-3096. (d) Rohmer, M.; Anding, C.;
Ourisson, G. Eur. J. Biochem. 1980, 112, 541-547. (e) Bouvier, P.; Berger,
Y.; Rohmer, M.; Ourisson, G. Eur. J. Biochem. 1980, 112, 549-556. (f)
Rohmer, M.; Bouvier, P.; Ourisson, G. Eur. J. Biochem. 1980, 112, 557-
560. (g) Renoux, J.-M.; Rohmer, M. Eur. J. Biochem. 1986, 155, 125-
132. (h) Abe, I.; Rohmer, M. J. Chem. Soc., Perkin Trans. 1 1994, 783-
791. (i) Abe, I.; Dang, T.; Zheng, Y. F.; Madden, B. A.; Feil, C.; Poralla,
K.; Prestwich, G. D. J. Am. Chem. Soc. 1997, 119, 11333-11334. (j)
Robustell, B.; Abe, I.; Prestwich, G. D. Tetrahedron Lett. 1998, 39, 957-
960. (k) Robustell, B.; Abe, I.; Prestwich, G. D. Tetrahedron Lett. 1998,
39, 9385-9388. (l) Zheng, Y. F.; Abe, I.; Prestwich, G. D. J. Org. Chem.
1998, 63, 4872-4873. (m) Sato, T.; Abe, T.; Hoshino, T. Chem. Commun.
1998, 2617-2618. (n) Hoshino, T.; Kondo, T. Chem. Commun. 1999, 731-
732. (o) Hoshino, T.; Ohashi, S. Org. Lett. 2002, 4, 2553-2556. (p)
Hoshino, T.; Nakano, S.; Kondo, T.; Sato, T.; Miyoshi, A. Org. Biomol.
Chem. 2004, 2, 1456-1470. (q) Nakano, S.; Ohashi, S.; Hoshino, T. Org.
Biomol. Chem. 2004, 2, 2012-2022. (r) Hoshino, T.; Kumai, Y.; Kudo, I.;
Nakano, S.; Ohashi, S. Org. Biomol. Chem. 2004, 2, 2650-2657.
(4) 3-((2E,6E,10E)-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraenyl)-
indole (4): 1H NMR (400 MHz, CDCl3): δ 7.87 (brs, 1H), 7.61 (dtd, 1H,
J ) 7.8, 1.1, 0.9 Hz), 7.35 (dt, 1H, J ) 8.3, 0.9 Hz), 7.20 (ddd, 1H, J )
8.2, 7.0, 1.1 Hz), 7.13 (ddd, 1H, J ) 7.9, 7.0, 1.1 Hz), 6.95 (dt, 1H, J )
2.3, 1.0 Hz), 5.48 (dd, 1H, J ) 7.8, 6.8 Hz), 5.28 (m, 3H), 3.48 (d, 2H, J
) 6.8 Hz), 2.09 (m, 12H), 1.78 (s, 3H, Me-17), 1.70 (s, 3H, Me-18), 1.62
(s, 6H, Me-19, 20), 1.61 (s, 3H, Me-21). 13C NMR (100 MHz, CDCl3): δ
136.5, 135.6, 135.0, 134.9, 131.2, 127.5, 124.4, 124.3, 123.6, 122.9, 121.9,
121.1, 119.1, 119.0, 116.1, 111.0, 39.7, 26.8, 26.7, 26.6, 25.7, 24.0, 17.7,
16.1, 16.0, 16.0. HRMS (FAB): found for [C28H39N]+ 389.3059; calcd
389.3082.
(5) 3-((2E,6E,10E)-2,7,11,15-tetramethylhexadeca-2,6,10,14-tetraenyl)-
indole (5): 1H NMR (400 MHz, CDCl3): δ 7.91 (brs, 1H), 7.62 (ddt, 1H,
J ) 7.8, 1.1, 0.9 Hz), 7.34 (dt, 1H, J ) 8.3, 0.9 Hz), 7.18 (ddd, 1H, J )
8.2, 7.0, 1.2 Hz), 7.10 (ddd, 1H, J ) 7.9, 7.0, 1.1 Hz), 6.97 (dt, 1H, J )
2.3, 1.1 Hz), 5.39 (m, 1H), 5.14 (m, 3H), 3.46 (s, 2H), 2.04 (m, 12H), 1.70
(s, 3H, Me-17), 1.63 (s, 3H, Me-18), 1.62 (s, 9H, Me-19, 20, 21). 13C NMR
(100 MHz, CDCl3): δ 136.4, 135.1, 134.9, 134.2, 131.2, 127.9, 125.4, 124.4,
124.3, 122.0, 121.8, 119.4, 119.1, 114.8, 110.9, 39.8, 39.7, 35.8, 28.4, 28.2,
26.8, 26.7, 25.7, 17.7, 16.1, 16.0. HRMS (FAB): found for [C28H39N]+
389.3094; calcd 389.3082.
(6) (a) Xiong, Q.; Zhu, X.; Wilson, W. K.; Ganesan, A.; Matsuda, S. P.
T. J. Am. Chem. Soc. 2003, 125, 9002-9003. (b) Zhu, X.; Ganesan, A. J.
Org. Chem. 2002, 67, 2705-2708.
(7) Fueki, S.; Tokiwano, T.; Toshima, H.; Oikawa, H. Org. Lett. 2004,
6, 2697-2700.
connected in a head-to-head fashion to a dimethylallyl C5
unit, was synthesized starting from farnesol,8 and finally
coupled with indole in the same manner (Scheme 2B).
When incubated with purified recombinant A. acidocal-
darius SHC,9 3-(geranylgeranyl)indole (4) did not afford any
cyclization product (Scheme 3A), which was confirmed by
TLC and GLC analysis. This is in sharp contrast with the
above-mentioned enzymatic cyclization of 3-(ω-oxidogera-
nylgeranyl)indole by the plant oxidosqualene cyclase.6a This
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Org. Lett., Vol. 7, No. 26, 2005

C14 methyl group is crucial to the correct folding and binding
of the substrate, as in the case of the cyclization of squalene
into hopene.3a,c Presumably, the alternative â-orientation of
the pro-C14 methyl group may interact repulsively with the
pro-C8 methyl or a nearest neighbor from the substrate
binding pocket of the enzyme.3a
Scheme 3. Enzymatic Conversion of Indole-Containing
Analogues
In contrast, 3-(farnesyldimethylallyl)indole (5) was a
competent substrate for the enzyme and efficiently converted
to a 2:1 mixture of a tetracyclic and a pentacyclic product,
6 and 7, which were completely separated by reverse-phase
HPLC (Scheme 3B).9 The structures of the cyclization
products were determined by NMR (1H and 13C NMR,
HMQC, HMBC, NOESY, and differential NOE) and MS
spectroscopy.10,11 The 1H NMR spectrum of the major
product 6 (1.0 mg, 5.0% yield) revealed the presence of four
methyl singlets (δ 0.88, 0.88, 0.92, and 1.10), one methyl
doublet (δ 0.78, J ) 6.9 Hz), and one vinylic proton (δ 5.19),
in addition to the signals due to the indole ring.10
A structure with a 6/6/5-fused tricyclic ring moiety was
uniquely consistent with both biogenetic reasoning and the
heteronuclear correlation spectroscopy (HMQC and HMBC).
Further, the ring junctions and the stereochemistry of ring
substituents were established by NOE experiments. Interest-
ingly, formation of a similar 6/6/5-fused tricyclic ring moiety
with the ∆9(11) double bond has been recently reported for
one of the enzymatic cyclization products of (3S)-2,3-
(8) (a) Coates, R. M.; Ley, D. A.; Cavender, P. L. J. Org. Chem. 1978,
43, 4915-4922. (b) Zheng, Y. F.; Oehlschlager, A. C. J. Org. Chem. 1994,
59, 5803-5809.
(9) The recombinant A. acidocaldarius SHC was prepared as described
in the previous papers.3a-c The reaction mixture contained the substrate
analogue 5 (or 4) (20 mg) and purified recombinant SHC (120 mg) in 400
mL of 50 mM Na citrate, pH 6.0, 0.1% Triton X-100, and was incubated
at 60 °C for 16 h. The incubations were stopped by freezing and
lyophilization, followed by extraction with 200 mL of hexane (×3). The
combined extracts were evaporated to dryness, separated on SiO2 TLC (20%
EtOAc in hexane) and by reverse-phase HPLC (Phenomex Gemini 5m ODS
column, 250 × 4.6 mm, 5% THF in MeCN, 0.5 mL/min) to give 1.0 mg
of 6 (Rt ) 21.9 min) and 0.5 mg of 7 (Rt ) 20.3 min) (from 5).
(10) Major product (6): 1H NMR (500 MHz, CDCl3): δ 7.92 (brs, 1H,
NH), 7.64 (dt, 1H, J ) 7.9, 1.1 Hz), 7.36 (dt, 1H, J ) 8.0, 1.0 Hz), 7.19
(ddd, 1H, J ) 8.0, 7.0, 1.1 Hz), 7.12 (ddd, 1H, J ) 8.0, 7.0, 1.0 Hz), 6.98
(d, 1H, J ) 1.7 Hz), 5.19 (m, 1H), 3.07 (dd, 1H, J ) 14.0, 2.6 Hz), 2.77
(brd, 1H), 2.36 (1H), 2.36 (1H), 1.10 (s, 3H, Me-18), 0.92 (s, 3H, Me-17),
0.88 (s, 3H, Me-16), 0.88 (s, 3H, Me-19), 0.78 (d, 3H, J ) 6.9 Hz, Me-
20). 13C NMR (125 MHz, CDCl3): δ 158.4 (C-9), 136.4 (C-8′), 127.8 (C-
9′), 121.7 (C-2′), 121.7 (C-6′), 119.1 (C-5′), 119.0 (C-4′), 116.4 (C-3′),
115.5 (C-11), 111.0 (C-7′), 50.26 (C-8), 48.2 (C-13), 47.0 (C-12), 45.7 (C-
5), 44.6 (C-14), 42.6 (C-3), 40.1 (C-1), 36.4 (C-10), 33.4 (C-4), 33.0 (C-
16), 28.4 (C-15), 25.1 (C-18), 22.1 (C-7), 21.5 (C-17), 19.4 (C-5), 19.2
(C-2), 17.0 (C-19), 15.7 (C-20). The NMR assignments were performed
according to data from H-H COSY, HMQC, HMBC, NOESY, and
differential NOE experiments. LRMS (FAB+, %): m/z 130 (98), 158 (67),
231 (100), 390 (M+, 15). HRMS (FAB+): found for [C28H39N] 389.3059;
calcd 389.3082. [R]25 ) -6.7° (c ) 0.10 in CHCl3).
D
(11) Minor product (7): 1H NMR (500 MHz, CDCl3): δ 7.93 (brs, 1H,
NH), 7.62 (dt, 1H, J ) 8.0, 1.1 Hz), 7.35 (dt, 1H, J ) 8.2, 1.0 Hz), 7.17
(ddd, 1H, J ) 8.2, 7.0, 1.2 Hz), 7.09 (ddd, 3H, J ) 8.0, 7.0, 1.2 Hz), 6.97
(t, 1H, J ) 1.1 Hz), 5.43 (m, 1H), 5.34 (m, 1H), 3.44 (s, 2H, Me-30), 1.95
(m, 2H), 1.63 (s, 3H, Me-34), 1.55 (s, 3H, Me-34), 1.06 (s, 3H, Me-17),
1.00 (s, 3H, Me-16), 0.83 (d, 3H, J ) 6.8 Hz, Me-19), 0.61 (s, 3H, Me-
18). 13C NMR (125 MHz, CDCl3): δ 146.3 (C-5), 133.6 (C-14), 133.6
(C-8′), 126.1 (C-13), 126.0 (C-9′), 122.1 (C-9), 122.1 (C-6′), 121.8 (C-2′),
119.4 (C-4′), 119.1 (C-5′), 116.1 (C-6), 114.9 (C-3′), 110.9 (C-7′), 41.0
(C-3), 39.9 (C-10), 39.9 (C-10), 37.1 (C-15), 36.7 (C-11), 35.8 (C-4), 35.8
(C-9), 35.8 (C-15), 33.5 (C-8), 31.7 (C-7), 29.8 (C-17), 29.0 (C-16), 27.6
(C-1), 22.3 (C-2), 21.5 (C-12), 16.1 (C-18), 15.9 (C-20), 15.2 (C-19). The
NMR assignments were performed according to data from H-H COSY,
HMQC, HMBC, and differential NOE experiments. LRMS (FAB+, %):
m/z 130 (100), 184 (48), 390 (M+, 30). HRMS (FAB+): found for [C28H39N]
suggested that the bacterial SHC is particularly sensitive to
the structural changes on the pro-C14â face (numbering in
hopene) (Scheme 3A) and thus fails to bind the substrate 4.
As we have previously proposed, R-orientation of the pro-
389.3059; calcd 389.3082. [R]25 ) 0.1° (c ) 0.10 in CHCl3).
D
Org. Lett., Vol. 7, No. 26, 2005
5875

oxidosqualene by a Gly600 deletion mutant of A. acidocal-
darius SHC.12a
On the other hand, the H NMR spectrum of the minor
the unnatural novel pentacyclic 6 as the major product, while
from the bicyclic tertiary cation with a 6/6-fused ring system,
a backbone rearrangement (H-9Rf8R, CH3-10âf9â,
H-5Rf10R) with elimination of H-6â produced the unnatural
novel tetracyclic 7 as the minor product (Scheme 3B).
Remarkably, the enzyme strictly controlled the stereochem-
istry of the cyclization and the rather unusual rearrangement
reactions even for the indole-containing substrate analogue.
Further, it is also interesting that triterpenes with the similar
tricyclic and bicyclic skeletons have been reported for the
products of A. acidocaldarius SHC mutants as mentioned
above.12
1
product 7 (0.5 mg, 2.5% yield) indicated the presence of
doublet (δ 0.83, J ) 6.8 Hz), one vinylic methyl (δ 1.63),
three methyl singlets (δ 0.61, 1.00, and 1.06), one methyl,
and one vinylic proton (δ 5.43),11 which is in good agreement
with those of a bicyclic product from squalene by a F365A
mutant of A. acidocaldarius SHC.12b Indeed, NMR spectro-
scopic data (HMQC, HMBC, and NOE) unambiguously
established the structure with a 6/6-fused bicyclic ring moiety
with the ∆5 double bond.
Enzymatic formation of a petromindole-like hexacyclic
product 8 from 3-(farnesyldimethylallyl)indole (5) would
apparently involve the formation of a tricyclic secondary
cationic intermediate with a 6/6/6-fused ring system and a
subsequent ring closure to indole at C4′ (Scheme 3C).
However, the cyclization reaction of 5 by A. acidocaldarius
SHC, initiated in a chair-chair-chair conformation, was
interrupted at the bicyclic or tricyclic cationic stage probably
because of the presence of the bulky indole ring. In addition,
the π-π interactions of the indole ring with the active-site
aromatic residues (e.g., Trp169 and Phe605 on the basis of
the crystal structure of A. acidocaldarius SHC2) would also
be anticipated, which may affect the folding conformation
of the substrate at the active site of the enzyme as well as
the subsequent cyclization and rearrangement reactions. As
a result, from the tricyclic Markovnikov cation with a 6/6/
5-fused ring system, a backbone rearrangement (H-13âf14,
CH3-8âf13â, H-9Rf8R) with elimination of H-11â yielded
For further production of structurally divergent unnatural
natural products, manipulation of the enzyme reaction by
substrate analogues with different isoprene chain length and/
or with other heteroaromatic ring systems is now in progress
in our laboratories.
Acknowledgment. This work was in part supported by
the COE21 Program, Grant-in-Aid for Scientific Research
(Nos. 16510164, 17310130, and 17035069), and Cooperation
of Innovative Technology and Advanced Research in Evo-
lutional Area (CITY AREA) from the Ministry of Education,
Culture, Sports, Science and Technology, Japan. H.T. is a
recipient of the JSPS Fellowship for Young Scientist (No.
175479).
Supporting Information Available: Complete set of
spectroscopic data (1H and 13C NMR, HMQC, HMBC, and
NOE) of the substrate analogues (4, 5) and enzyme reaction
products (6, 7) (15 pages). This material is available free of
charge via the Internet at http://pubs.acs.org.
(12) (a) Hoshino, T.; Shimizu, K.; Sato, T. Angew. Chem., Int. Ed. 2004,
43, 6700-6703. (b) Hoshino, T.; Sato, T. Chem. Commun. 1999, 2005-
2006.
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