Enzymatic formation of pyrrole-containing novel cyclic polyprenoids by bacterial squalene:hopene cyclase

Article abstract of DOI:10.1016/j.tetlet.2006.02.151

A convergent synthesis provided two pyrrole-containing squalene analogues, in which a C₂₀ isoprene unit is connected to pyrrole, 2-(geranylgeranyl)pyrrole and 2-(farnesyldimethylallyl)pyrrole. When incubated with recombinant squalene:hopene cyclase from Alicyclobacillus acidocaldarius, 2-(farnesyldimethylallyl)pyrrole was enzymatically converted to a 10:1 mixture of a tricyclic and a bicyclic unnatural novel polyprenoids, whereas 2-(geranylgeranyl)pyrrole was not a substrate for the enzyme.

Full text of DOI:10.1016/j.tetlet.2006.02.151

Tetrahedron Letters 47 (2006) 3085–3089
Enzymatic formation of pyrrole-containing novel cyclic
polyprenoids by bacterial squalene:hopene cyclase
Hideya Tanaka,a Hisashi Noma,a Hiroshi Noguchia and Ikuro Abea,b,
*
aSchool of Pharmaceutical Sciences and the 21st Century COE Program, University of Shizuoka, 52-1 Yada,
Shizuoka 422-8526, Japan
bPRESTO, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012, Japan
Received 6 February 2006; revised 20 February 2006; accepted 27 February 2006
Abstract—A convergent synthesis provided two pyrrole-containing squalene analogues, in which a C20 isoprene unit is connected to
pyrrole, 2-(geranylgeranyl)pyrrole and 2-(farnesyldimethylallyl)pyrrole. When incubated with recombinant squalene:hopene cyclase
from Alicyclobacillus acidocaldarius, 2-(farnesyldimethylallyl)pyrrole was enzymatically converted to a 10:1 mixture of a tricyclic
and a bicyclic unnatural novel polyprenoids, whereas 2-(geranylgeranyl)pyrrole was not a substrate for the enzyme.
ꢀ 2006 Elsevier Ltd. All rights reserved.
The broad substrate tolerance and catalytic potential of
squalene:hopene cyclase (SHC) (E.C. 5.4.99.7) from a
thermoacidophilic bacteria Alicyclobacillus acidocalda-
rius is remarkable.1–3 The enzyme normally catalyzing
cyclization of squalene (1) into a 5:1 mixture of hop-
22(29)-ene (2) and hopan-22-ol (3) (Scheme 1A) accepts
a variety of substrate analogues (C15–C35) and efficiently
performs sequential ring-forming reactions to produce a
series of unnatural cyclic polyprenoids. One of the most
impressive examples is the cyclization of a C35 squalene
analogue (4) into a ‘supra-natural’ hexacyclic polypre-
noid (5) with a 6.6.6.6.6.5-fused ring system (Scheme
1B).3a Further, we have recently reported formation of
indole-containing novel cyclic polyprenoids (7 and 8)
from 3-(farnesyldimethylallyl)indole (6) (Scheme 1C).3b
Here we now describe synthesis and enzymatic cycliza-
tion of two new substrate analogues in which a C20 iso-
prene unit is connected to pyrrole. It was anticipated
that incorporation of the less bulky pyrrole ring, instead
of indole, would not significantly disturb the cyclization
reaction and thus lead to the formation of novel unnat-
ural cyclic polyprenoids with highly fused ring systems.
In addition, interesting biological activities of the cycli-
zation products were also expected due to the presence
of the heteroaromatic ring moiety.
The convergent synthesis of the substrate analogues, 2-
(geranylgeranyl)pyrrole (9)4 and 2-(farnesyldimethyl-
allyl)pyrrole (10),5 involved regioselective alkylation of
pyrrole at C-2 by geranylgeranyl bromide or farnesyl-
dimethylallyl bromide, which was mediated by zinc tri-
flate as described before (Scheme 2).3b When incubated
with purified recombinant A. acidocaldarius SHC,6 2-
(geranylgeranyl)pyrrole (9) was completely inactive
and did not afford any reaction products (Scheme 3A),
which was confirmed by HPLC and GLC analysis. This
was consistent with the previous observation that
3-(geranylgeranyl)indole was not a substrate for A. aci-
docaldarius SHC,3b suggesting that the enzyme is parti-
cularly sensitive to structural changes on the pro-C14b
face and thus fails to bind the substrate analogue
(Scheme 3A). Presumably, a-orientation of the pro-
C14 methyl group is crucial for the correct folding of
the substrate as in the case of the folding of squalene
(Scheme 1A).3a–d The alternative b-orientation of the
pro-C14 methyl group may interact repulsively with
the pro-C10 methyl or a nearest neighbor from the sub-
strate binding pocket of the enzyme; possibly with the
residue I261 on the basis of the crystal structure of A.
acidocaldarius SHC1,2 (Fig. 1).
In contrast, 2-(farnesyldimethylallyl)pyrrole (10), in
which a farnesyl C15 unit is connected in a head-to-head
fashion to a dimethylallyl C5 unit, was converted to a
10:1 mixture of the two novel tri- and bicyclic products
11 and 12 (Scheme 3B), whose structures were
Keywords: Squalene cyclase; Triterpene synthase; Unnatural poly-
prenoids; Pyrrole.
*
Corresponding author. Tel./fax: +81 54 264 5662; e-mail: abei@
ys7.u-shizuoka-ken.ac.jp
0040-4039/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2006.02.151

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H. Tanaka et al. / Tetrahedron Letters 47 (2006) 3085–3089
Scheme 1. Proposed mechanism for the conversion of (A) squalene (1) to hop-22(29)-ene (2) and hopan-22-ol (3); (B) a C35 squalene analogue (4)
into a supra-natural hexacyclic polyprenoid (5); (C) 3-(farnesyldimethylallyl)indole (6) to indole-containing novel cyclic polyprenoids 7 and 8 by
recombinant Alicyclobacillus acidocaldarius SHC.
Scheme 2. Synthesis of the pyrrole-containing substrate analogues.

H. Tanaka et al. / Tetrahedron Letters 47 (2006) 3085–3089
3087
Scheme 3. Proposed mechanism for the enzymatic conversion of the pyrrole-containing substrate analogues.
Figure 1. Proposed interactions between the active-site amino acid residues of Alicyclobacillus acidocaldarius SHC and 3-(farnesyldimethyl-
allyl)indole (6) (adapted from Ref. 1c).
determined by NMR (1H and 13C NMR, HMQC,
HMBC, NOESY, and differential NOE) and MS spec-
tween Me-19 and H-20. Interestingly, the core structure
of 11 with the D14(20) double bond was different from
that of the previously reported indole-containing
6.6.5-tricyclic product 7 with the D9(11) double bond
(Scheme 1C).3b On the other hand, the 1H NMR of
the minor product 12 (0.1 mg, 0.1% yield) indicated
the presence of one methyl doublet (d 0.85), three
methyl singlets (d 0.61, 1.02, and 1.06), one vinylic
methyl (d 1.56), and one vinylic proton (d 5.95), which
was in good agreement with those of the indole-contain-
ing minor product 8 with a 6.6-fused ring system
(Scheme 1C).3b Indeed, NMR data (HMQC, HMBC,
and NOE) unambiguously established the bicyclic struc-
ture with the D5 double bond. This was the first demon-
1
troscopy.7,8 Thus, the H NMR spectrum of the major
product 11 (1.0 mg, 1.0% yield) revealed the presence of
four methyl singlets (d 0.64, 0.76, 0.77, and 0.80) and
two vinylic protons (d 4.71 and 4.82), in addition to
the signals due to the pyrrole ring. A structure with
the 6.6.5-fused tricyclic ring system was uniquely consis-
tent with both biogenetic reasoning and the hetero-
nuclear correlation spectroscopy (HMQC and HMBC).
Further, the ring junctions and the stereochemistry of
ring substituents were established by NOE experiments.
The stereochemistry of C-13 was confirmed by NOEs
observed between Me-18 and Me-19, but absent be-

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H. Tanaka et al. / Tetrahedron Letters 47 (2006) 3085–3089
stration of the enzymatic formation of pyrrole-contain-
ing cyclic polyprenoids.
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) Hoshino, T.; Sato, T. Chem. Commun.
2002, 291–301.
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.
As in the case of 3-(farnesyldimethylallyl)indole (6),3b
the enzymatic cyclization of 2-(farnesyldimethylallyl)-
pyrrole (10), folded in all pre-chair conformation,
was interrupted at the bicyclic or tricyclic intermediate
cation (Scheme 3B). From the 6.6.5-tricyclic tertiary cat-
ion, proton elimination at H-20 yielded 11 as the major
product (Scheme 3B), whereas, in the case of the indole-
containing analogue 6, an alternative backbone rear-
rangement (H-13b!14, CH3-8b!13b, H-9a!8a) with
elimination of H-11b afforded 7 with the D9(11) double
bond (Scheme 1C).3b Presumably, the presence of the
bulky indole moiety caused perturbation in the folding
conformation of the intermediate cation, which resulted
in the irregular rearrangement reaction and the proton
abstraction at the different position. On the other hand,
from the 6.6-bicyclic tertiary intermediate cation, a
backbone rearrangement (H-9a!8a, CH3-10b!9b, H-
5a!10a) with elimination of H-6b yielded the minor
product 12 (Scheme 3B). In both cases, it is remarkable
that the stereochemistry of the cyclization reactions of
the unnatural substrates was strictly controlled by the
enzyme in a regio- and stereo-specific manner.
3. For enzymatic conversion of squalene analogs 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. 2005, 7, 5873–5876; (c)
Tanaka, H.; Noguchi, H.; Abe, I. Org. Lett. 2004, 6, 803–
806; (d) Tanaka, H.; Noguchi, H.; Abe, I. Tetrahedron Lett.
2004, 45, 3093–3096; (e) Rohmer, M.; Anding, C.; Ouris-
son, G. Eur. J. Biochem. 1980, 112, 541–547; (f) Bouvier, P.;
Berger, Y.; Rohmer, M.; Ourisson, G. Eur. J. Biochem.
1980, 112, 549–556; (g) Rohmer, M.; Bouvier, P.; Ourisson,
G. Eur. J. Biochem. 1980, 112, 557–560; (h) Renoux, J.-M.;
Rohmer, M. Eur. J. Biochem. 1986, 155, 125–132; (i) Abe,
I.; Rohmer, M. J. Chem. Soc., Perkin Trans. 1 1994, 783–
791; (j) 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; (k) Robustell, B.; Abe, I.; Prest-
wich, G. D. Tetrahedron Lett. 1998, 39, 957–960; (l)
Robustell, B.; Abe, I.; Prestwich, G. D. Tetrahedron Lett.
1998, 39, 9385–9388; (m) Zheng, Y. F.; Abe, I.; Prestwich,
G. D. J. Org. Chem. 1998, 63, 4872–4873; (n) Sato, T.; Abe,
T.; Hoshino, T. Chem. Commun. 1998, 2617–2618; (o)
Hoshino, T.; Kondo, T. Chem. Commun. 1999, 731–732; (p)
Hoshino, T.; Ohashi, S. Org. Lett. 2002, 4, 2553–2556; (q)
Hoshino, T.; Nakano, S.; Kondo, T.; Sato, T.; Miyoshi, A.
Org. Biomol. Chem. 2004, 2, 1456–1470; (r) Nakano, S.;
Ohashi, S.; Hoshino, T. Org. Biomol. Chem. 2004, 2, 2012–
2022; (s) Hoshino, T.; Kumai, Y.; Kudo, I.; Nakano, S.;
Ohashi, S. Org. Biomol. Chem. 2004, 2, 2650–2657.
Here it should be noted that the less bulky pyrrole-con-
taining analogue 10 was not as a good substrate as the
indole-containing analogue 6. The yield of the pyrrole
product 11 was five times less than that of the indole
product 7. This may be due to the stereoelectronic effect
of the heteroaromatic ring. Thus, the p-electron rich in-
dole ring moiety fits better into the active-site of the en-
zyme because of more efficient p–p interactions with the
active-site aromatic residues; possibly with W169 and/or
F605 lining the active-site cavity of A. acidocaldarius
SHC1,2 (Fig. 1). Manipulation of the enzyme reaction
by combination of newly designed substrate analogues
and structure-based modulation of the active-site geo-
metry of the enzyme would lead to further production
of structurally distinct ‘supra-natural steroids’, which
is now in progress in our laboratories.
4. 2-((2E,6E,10E)-3,7,11,15-Tetramethylhexadeca-2,6,10,14-
1
tetraenyl)-pyrrole (9): H NMR (400 MHz, CDCl3): d 7.95
(br s, 1H), 6.65 (m, 1H), 6.14 (m, 1H), 5.92 (m, 1H), 5.36
(m, 1H), 5.12 (m, 3H), 3.36 (d, 2H, J = 7.3 Hz), 2.05 (m,
12H), 1.70 (s, 3H), 1.62 (s, 3H), 1.61 (s, 9H). 13C NMR
(100 MHz, CDCl3): d 137.4, 135.4, 135.0, 131.2, 124.4,
124.1, 124.1, 120.9, 116.1, 108.5, 104.9, 39.7, 39.6, 26.7,
26.6, 26.6, 26.4, 26.3, 25.7, 21.0, 17.7, 16.1, 16.0, 16.0.
HRMS (FAB): found for [C24H37N]+ 339.2898; calcd.
339.2926.
Acknowledgements
5. 3-((2E,6E,10E)-2,7,11,15-Tetramethylhexadeca-2,6,10,14-
tetraenyl)-pyrrole (10): 1H NMR (400 MHz, CDCl3): d 7.90
(br s, 1H), 6.66 (m, 1H), 6.13 (m, 1H), 5.94 (m, 1H), 5.29
(m, 1H), 5.13 (m, 3H), 3.28 (s, 2H), 2.00 (m, 12H), 1.69 (s,
3H), 1.63 (s, 3H), 1.61 (s, 9H). 13C NMR (100 MHz,
CDCl3): d 135.5, 135.0, 135.0, 133.6, 131.2, 126.5, 124.4,
124.2, 124.1, 116.4, 108.3, 106.1, 39.7, 39.7, 38.3, 28.2, 28.1,
26.8, 26.6, 25.7, 17.7, 16.1, 16.0, 15.8. HRMS (FAB): found
for [C24H37N]+ 339.2938; calcd 339.2926.
This work was in part supported by the COE21 Pro-
gram, and Grant-in-Aid for Scientific Research from
the Ministry of Education, Culture, Sports, Science
and Technology, Japan, and by Grant-in-Aid from
The Sankyo Foundation of Life Science, Japan. H.T.
is a recipient of the JSPS Fellowship for Young Scientist
(No. 175479).
6. The recombinant A. acidocaldarius SHC was prepared as
described in the previous papers.3a–d Reaction mixture
contained the substrate analog 9 (or 10) (20 mg) and
purified recombinant SHC (120 mg) in 400 ml of 50 mM Na
citrate, pH 6.0, 0.1% Triton X-100, was incubated at 60 ꢁC
for 16 h. The incubations were stopped by freezing and
lyophilization, followed by extraction with hexane
(3 · 200 ml). The combined extracts were evaporated to
dryness, separated on SiO2 TLC (20% EtOAc in hexane)
and reverse-phase HPLC (Phenomex Gemini 5m ODS
column, 250 · 4.6 mm, 5% THF in MeCN, 0.5 ml/min) to
Supplementary data
Supplementary data (Complete set of spectroscopic data
(1H and 13C NMR, HMQC, HMBC, and NOE) of the
substrate analogues (9 and 10) and enzyme reaction
products (11 and 12) (nine pages)) associated with this
article can be found, in the online version, at
doi:10.1016/j.tetlet.2006.02.151.

H. Tanaka et al. / Tetrahedron Letters 47 (2006) 3085–3089
3089
give 11 (0.2 mg) and 12 (0.02 mg) (from 10). In order to get
enough amount of products for NMR analysis, the
experiments were repeated five times.
191, 231, 340 (M+). HRMS (FAB+): found for [C24H37N]
25
339.2923; calcd 339.2926. ½aꢀD ꢁ21.7 (c 0.10 in CHCl3).
1
8. Minor product (12): H NMR (400 MHz, CD3OD): d 6.57
1
7. Major product (11): H NMR (500 MHz, CD3OD): d 6.48
(m, 1H, H-50), 5.95 (m, 1H, H-40), 5.77 (m, 1H, H-30), 5.47
(m, 1H, H-6), 5.21 (m, 1H, H-13), 1.56 (s, 3H, Me-20), 1.06
(s, 3H, Me-17), 1.02 (s, 3H, Me-16), 0.85 (d, 3H, J = 6.8 Hz,
Me-19), 0.61 (s, 3H, Me-18). 13C NMR (100 MHz,
CD3OD): d 149.2 (C-5), 134.5 (C-14), 117.3 (C0-5), 127.1
(C-13), 117.4, 117.3, 108.2, 106.5, 42.2 (C-3), 41.2 (C-10),
39.1, 37.3, 34.7 (C-4), 32.7 (C-8), 31.2, 30.3 (C-17), 29.5 (C-
16), 29.2, 28.7, 23.3, 22.4, 22.1, 19.8, 16.5 (C-18), 15.5 (C-
19). The NMR assignments were performed according to
data from H–H COSY, HMQC, HMBC, differential NOE
experiments. LRMS (FAB+): m/z 69, 80, 119, 134, 176, 191,
340 (M+). HRMS (FAB+): found for [C24H37N] 339.2925;
(m, 1H, H-50), 5.87 (m, 1H, H-40), 5.66 (m, 1H, H-30), 4.82
(s, 1H, H-20), 4.71 (s, 1H, H-20), 3.25 (s, 2H, H-15), 1.99 (t,
1H, J = 6.8 Hz, H-13), 0.80 (s, 3H, Me-18), 0.77 (s, 3H, Me-
16), 0.76 (s, 3H, Me-17), 0.64 (s, 3H, Me-19). 13C NMR
(125 MHz, CD3OD): d 149.3 (C-14), 130.0 (C0-2), 117.3
(C0-5), 112.5 (C-20), 108.2 (C0-4), 107.0 (C0-3), 64.7 (C-9),
58.8 (C-5), 57.3 (C-13), 43.7 (C-3), 41.3 (C-1), 37.9 (C-15),
34.0 (C-16), 34.0 (C-10), 30.8 (C-7), 26.7 (C-12), 21.8 (C-
17), 20.7 (C-11), 20.5 (C-2), 19.4 (C-6), 15.9 (C-18), 15.8 (C-
19). The NMR assignments were performed according to
data from H–H COSY, HMQC, HMBC, NOESY, differ-
ential NOE experiments. LRMS (FAB+): m/z 69, 95, 134,
25
calcd 339.2926. ½aꢀD 16.9 (c 0.10 in CHCl3).