Chemoenzymatic syntheses of prenylated aromatic small molecules using Streptomyces prenyltransferases with relaxed substrate specificities

Article abstract of DOI:10.1016/j.bmc.2008.07.052

NphB is a soluble prenyltransferase from Streptomyces sp. strain CL190 that attaches a geranyl group to a 1,3,6,8-tetrahydroxynaphthalene-derived polyketide during the biosynthesis of anti-oxidant naphterpin. Here we report multiple chemoenzymatic syntheses of various prenylated compounds from aromatic substrates including flavonoids using two prenyltransferases NphB and SCO7190, a NphB homolog from Streptomyces coelicolor A3(2), as biocatalysts. NphB catalyzes carbon-carbon-based and carbon-oxygen-based geranylation of a diverse collection of hydroxyl-containing aromatic acceptors. Thus, this simple method using the prenyltransferases can be used to explore novel prenylated aromatic compounds with biological activities. Kinetic studies with NphB reveal that the prenylation reaction follows a sequential ordered mechanism.

Full text of DOI:10.1016/j.bmc.2008.07.052

Bioorganic & Medicinal Chemistry 16 (2008) 8117–8126
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Chemoenzymatic syntheses of prenylated aromatic small molecules using
Streptomyces prenyltransferases with relaxed substrate specificities
Takuto Kumano a, Stéphane B. Richard b, Joseph P. Noel b, Makoto Nishiyama a, Tomohisa Kuzuyama a,
*
a Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
b Howard Hughes Medical Institute, Jack H. Skirball Center for Chemical Biology and Proteomics, The Salk Institute for Biological Studies,
10010 N. Torrey Pines Road, La Jolla, CA 92037, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
NphB is a soluble prenyltransferase from Streptomyces sp. strain CL190 that attaches a geranyl group to a
1,3,6,8-tetrahydroxynaphthalene-derived polyketide during the biosynthesis of anti-oxidant naphterpin.
Here we report multiple chemoenzymatic syntheses of various prenylated compounds from aromatic
substrates including flavonoids using two prenyltransferases NphB and SCO7190, a NphB homolog from
Streptomyces coelicolor A3(2), as biocatalysts. NphB catalyzes carbon–carbon-based and carbon–oxygen-
based geranylation of a diverse collection of hydroxyl-containing aromatic acceptors. Thus, this simple
method using the prenyltransferases can be used to explore novel prenylated aromatic compounds with
biological activities. Kinetic studies with NphB reveal that the prenylation reaction follows a sequential
ordered mechanism.
Received 13 June 2008
Revised 18 July 2008
Accepted 18 July 2008
Available online 24 July 2008
Keywords:
Enzyme catalysis
Prenylation
Flavonoid
Polyketide
Kinetics
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
erated from malonyl-CoA by a type III polyketide synthase, and tai-
lored by installation of
a geranyl moiety that undergoes
To date, natural products with one or more prenyl groups have
been isolated predominantly from higher plants. These compounds,
originating from multiple natural product classes, often possess
anti-microbial, anti-oxidant, anti-inflammatory, anti-viral, and
anti-cancer activities.1 For example, several prenylated flavonoids
including 8-geranyl chrysin and 6-dimethylallyl galangin counter-
act multi-drug resistance.2 Recent structure–activity relationships
revealed that the activities of these prenylated compounds correlate
with their enhanced interaction with plasma membrane ABC (ATP-
binding cassette) transporters compared with their non-prenylated
parent compounds.3 Given their enhanced and distinct activities,
prenylated flavonoids show promise as lead compounds for the
development of non-toxic anti-microbials and nutraceuticals in
plants and new pharmacological agents for the treatment of animal
diseases.1 However, prenylated compounds often exist at trace lev-
els in natural sources, and are often not amenable to cost effective
synthesis. Given the recent identification of soluble prenyltransfe-
rases with relaxed substrate specificity displaying regiospecificity
in prenyl group transfer and prenyl chain selectivity,4 these biocata-
lysts can serve as an alternate production platform particularly
when combined with structure-based enzyme engineering.
subsequent cyclization.5–7 We identified a soluble prenyltransfer-
ase, Orf2, now renamed NphB, in the naphterpin biosynthetic gene
cluster from Streptomyces sp. strain CL190, that could catalyze the
transfer of a geranyl group to an unidentified THN-derived metab-
olite (Scheme 1).4 In addition, we solved the X-ray crystal structure
of NphB, an architecturally new prenyltransferase.4,8 Further, we
demonstrated that NphB accepts a diverse collection of hydroxyl-
containing aromatic substrates, including several flavonoids.
Although substrate specificity, regiospecificity, and a physiological
substrate of NphB have not been fully elucidated, it is clear that the
prenyltransferase may serve as a biocatalyst for the production of
prenylated compounds both in vivo and in vitro. Here, we report
multiple chemoenzymatic syntheses of prenylated aromatic com-
pounds by using two prenyltransferases NphB and SCO7190, a
NphB homolog from Streptomyces coelicolor A3(2). In addition, ki-
netic studies provide insight into the reaction mechanism of the
prenylation reaction.
2. Results and discussion
2.1. Chemoenzymatic syntheses of prenylated dihydroxy
naphthalene, prenylated flavonoids, and prenylated plant
polyketides
The anti-oxidant naphterpin is a natural product biosynthesized
from the polyketide 1,3,6,8-tetrahydroxynaphthalene (THN) gen-
Recombinant NphB and SCO7190 were overproduced in Esche-
richia coli and purified to homogeneity.4 As prenyl acceptors, we
* Corresponding author. Tel.: +81 3 5841 3073; fax: +81 3 5841 8030.
E-mail address: utkuz@mail.ecc.u-tokyo.ac.jp (T. Kuzuyama).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.07.052

8118
T. Kumano et al. / Bioorg. Med. Chem. 16 (2008) 8117–8126
OH
HO
O
HO
OH
P
P
HO
OH
O O
PPi
O
O
OH OH
THN
H
HO
NphB
Mg2+
H
O2
O
O
OH
HO
OH
O
HO
OH
O
P
P
HO
Naphterpin
O O
OH
O
GPP
Flaviolin
Scheme 1. Putative reaction catalyzed by NphB in naphterpin biosynthesis.
focused on 1,3-dihydroxynaphthalene (DHN), 1,6-DHN 1,
2,7-DHN 2, 4-hydroxyphenylpyruvate, 2,5,7- trihydroxynaphtho-
quinone (flaviolin), and the eight plant polyketides, olivetol 3, resve-
ratrol 4, piceatannol, pterostilbene and the (iso)flavonoid sub-class
including naringenin 5, apigenin 6, genistein 7, and daidzein 8. All
substrates, except for 1,3-DHN, 4-hydroxyphenylpyruvate, flaviolin,
piceatannol, and pterostilben underwent facile geranylation, yield-
ing between one and three products (Fig. 1 and Tables 1–4).
Most NphB reaction products contain a geranyl group in the ortho
position with respect to a neighboring hydroxy moiety. Only 1,6-
DHN underwent geranylation at the para position, albeit at a rela-
tively low catalytic efficiency (Table 1). Interestingly, NphB appended
two geranyl groups to 2,7-DHN to yield 1,6-digeranyl 2,7-DHN 13
(Table 1), which was formed from 1-geranyl 2,7-DHN 12 (Supple-
mental figure 1). With all flavonoids tested, NphB catalyzed the ger-
anylation of a hydroxy group leading to the formation of an O-prenyl
linkage (Table 2). In addition to this O-geranylation, NphB geranylat-
ed the C6 position of naringenin 5 and apigenin 6 and the C8 carbon
of daidzein 8 to yield 8-geranyl daidzein 20. NphB also geranylated
the plant polyketides, olivetol 3 and resveratrol 4 (Table 3).
2.2. Kinetic study of the NphB-catalyzed reaction
In the case of NphB incubation with 1,6-DHN and Mg2+, three ger-
anylated products, 2-geranyl, 4-geranyl, and 5-geranyl 1,6-DHNs 9–
11, formed (Fig. 1), indicating that 1,6-DHN must adopt at least three
productive binding orientations in the NphB’s active site. We previ-
ously reported the crystal structure of NphB complexed with a non-
hydrolysable geranyl diphosphate (GPP) analog, geranyl S-thiodi-
phosphate (GSPP) and 1,6-DHN (PDB ID, 1zb6).4 Recently, Merz
and co-workers reported simulation studies suggesting two alterna-
tive binding orientations for 1,6-DHN.9 In the light of these results,
we then estimated apparent Km value for each putative binding ori-
entation of 1,6-DHN. The kcat value for the corresponding product
was also estimated from the concentration dependence of the initial
velocity of the NphB reaction under conditions of saturating GPP.
Apparent steady-state kinetic constants were estimated for each
1,6-DHN product by quantitatively measuring the amount of each
geranylated 1,6-DHN using HPLC analysis (Table 1).
When 1,6-DHN was used as a substrate, the lowest Km values
were calculated for 2-geranyl 1,6-DHN 11 (0.51 0.08 mM) and
for 5-geranyl 1,6-DHN 10 (0.54 0.07 mM) biosynthesis. In our
crystal structure of NphB complexed with GSPP and 1,6-DHN
(Fig. 2),4 the 1,6-DHN molecule orients in the active site so that
its C5 position is closest to the C1 position of GSPP. This binding
orientation correlates with the Km value obtained for the orienta-
tion of 1,6-DHN leading to the formation of 5-geranyl 1,6-DHN.
Our kinetic data (Tables 1–3) indicate that all substrates tested
thus far with NphB possess Km values ranging from 0.29 to 2.1 mM.
These steady-state measurements bode well for the possible use of
NphB or mutants thereof for the biocatalytic production of regio-
specifically prenylated aromatic compounds of natural and syn-
thetic origin.
In contrast, SCO7190 only catalyzed carbon–carbon-based pre-
nylation by attaching a dimethylallyl moiety to 1,6-DHN 1, 2,7-
DHN 2, naringenin 5, olivetol 3, and resveratrol 4 (Table 4). Given
the structural diversity of the substrates employed and the large
number of possible C- and O-accepting atoms, the enzyme assays
revealed significantly relaxed substrate specificity while often pre-
serving regioselectivity for prenyl group transfer.
a
1,6-DHN
2.0
1.0
0
2.3. Reaction mechanism of NphB
0
5
10
15
20
To gain insight into the kinetic mechanism of NphB, double-re-
ciprocal plots (1/initial velocity vs 1/substrate concentration) of
GPP and naringenin turnover were calculated (Fig. 3). In both plots,
the lines intersect at a common point to the left of the 1/initial-
velocity axis, indicating that the NphB reaction follows a sequential
mechanism, where both GPP and naringenin must bind in succes-
sion at the enzyme’s active site before product formation can oc-
cur. However, these plots fail to elucidate the binding order of
the two substrates. We solved the 3D structures of NphB com-
plexed with GPP alone (PDB ID, 1zcw) and with GSPP and 1,6-
DHN.4 However, we repeatedly failed to obtain NphB structures
complexed with any aromatic acceptor alone (i.e., 1,6-DHN or res-
veratrol). This observation suggests that 1,6-DHN cannot bind to
NphB in the absence of bound GPP. Thus, GPP most likely binds
2
b
2.0
1.0
0
3
1,6-DHN
1
0
5
10
/min
15
20
t
Figure 1. HPLC analysis of geranylated products formed from 1,6-DHN by the
action of NphB. (a) No NphB added. (b) After a 36-h incubation with NphB. Peak 1,
4-geranyl 1,6-DHN 9; peak 2, 5-geranyl 1,6-DHN 10; peak 3, 2-geranyl 1,6-DHN 11.

T. Kumano et al. / Bioorg. Med. Chem. 16 (2008) 8117–8126
8119
Table 1
first in the sequential ordered mechanism of the NphB-catalyzed
reaction (Supplemental figure 2).
Geranylated DHNs synthesized by NphB
kcat ðsꢀ1Þ ꢁ 103
cat=Km ðMꢀ1 sꢀ1
We have proposed an electrophilic aromatic substitution reac-
tion in NphB, where ionic cleavage of the carbon–oxygen bond in
GPP and the resulting formation of a geranyl carbocation occur be-
fore prenylation of the electron rich aromatic acceptor.4 Together
with the results of the present kinetic study, we posit that the
NphB reaction proceeds as follows. (1) GPP binds to the NphB ac-
tive site and orients through diphosphate–cation interactions
(Mg2+ and/or basic side chains), (2) the aromatic acceptor then
binds triggering, (3) the formation of a geranyl carbocation through
pyrophosphate loss whereupon (4) the carbocation performs an
electrophilic attack on the aromatic acceptor, terminated by (5)
acceptor neutralization through proton loss.
Substrate
OH
Km (mM) Product
k
Þ
No products
OH
OH
1,3-DHN
OH
HO
4
9
1'
1.05 0.17
0.32 0.02
0.30
HO
2.4. Time course of the NphB-catalyzed reaction
9'
1
Finally, we determined yields of geranylated product formation
(Fig. 4). Naringenin 5, apigenin 6, and genistein 7 underwent nearly
complete geranylation during a 36-h incubation. On the other
hand, the yield of the geranylated products of daidzein 8 and olive-
tol 3 ranged from 20% to 80% over the same time period. Time
courses of the NphB reactions indicate that NphB maintains stabil-
ity for the entire 36-h incubation.
10'
8'
OH
OH
HO
5
10
0.54 0.07
4.2 0.2
7.7
HO
1
3. Conclusion
The present study describes multiple chemoenzymatic synthe-
ses of various prenylated compounds from aromatic substrates
including flavonoids using a newly described structural and func-
tional class of small molecule prenyltransferase isolated from bac-
teria. In particular, one such prenyltransferase, NphB possesses
synthetically useful relaxed substrate specificities while maintain-
ing in some cases regioselectivity for prenyl group transfer. As a
prenyltransferase belonging to the same class as that of NphB,
Fnq26 of furanonaphthoquinone biosynthesis has been identi-
fied.10 However, Fnq26 possesses substantially different substrate
specificities than that of NphB, although the two enzymes share
42% of sequence identity. For example, Fnq26 catalyzes the gerany-
lation of a hydroxy group of 1,3-DHN and 4-hydroxybenzoic acid,
both of which are not geranylated by NphB. In addition, interest-
ingly, Fnq26 is able to catalyze attachment of the C3 carbon of
the ionized geranyl group rather than the C1 carbon to flaviolin.
In contrast, Fnq26 does not accept 1,6-DHN and 2,7-DHN as a sub-
strate. Finally, the flavanone naringenin undergoes limited Fnq26
directed prenylation. Thus, in order to more easily predict and
engineer the substrate selectivity/promiscuity and regioselectivity
of newly identified prenyltransferases of the NphB/Fnq26 class, it
will be important to carry out a combination of structural studies
and additional biochemical experiments.
OH
0.51 0.08
1.1 0.1
2.2
OH
HO
2
1
HO
11
HO
OH
0.34 0.06
0.47 0.01
1.4
1
HO
OH
2
12
The potential of employing chemoenzymatic approaches as de-
scribed above provides a convenient starting point to explore novel
prenylation chemistry and the bioactivity of aromatic compounds
using structure-based enzyme engineering. In addition, such ratio-
nally based engineering of NphB or related prenyltransferases will
provide another powerful synthetic tool to expand the diversity
and bioactivities of many synthetic and natural compounds
through enzyme directed regiospecific prenylation.
HO
OH
HO
OH
N.D.a
N.D.a
2
6
13
O
O
4. Experimental
HO
OH
Negligible products
4.1. Enzyme assay
OH
flaviolin
Assays for the NphB-catalyzed reaction were carried out in
50 mM Tris–HCl (pH 9.0), containing 5 mM MgCl2, 5 mM aromatic
a
Not determined.

8120
T. Kumano et al. / Bioorg. Med. Chem. 16 (2008) 8117–8126
Table 2
Geranylated flavonoids synthesized by NphB
kcat ðsꢀ1Þ ꢁ 103
cat=Km ðMꢀ1 sꢀ1
Substrate
Km (mM)
Product
k
Þ
8''
10''
OH
9''
1''
HO
HO
HO
O
O
OH
1.6 0.2
2.3 0.1
1.4
1'
O
O
O
OH
OH
OH
7
5
5
6
OH
14
OH
HO
O
OH
OH
OH
6
O
O
2.1 0.5
0.67 0.08
0.31
OH
O
15
O
O
OH
0.58 0.06
0.33 0.01
0.57
O
O
O
OH
16
17
OH
HO
O
O
HO
O
O
0.53 0.06
0.60 0.03
1.1
OH
OH
6
HO
O
O
0.33 0.01
0.48 0.01
1.5
O
O
O
OH
OH
7
OH
OH
18
HO
O
O
0.29 0.05
0.17 0.01
0.59
O
O
O
OH
8
OH
19

T. Kumano et al. / Bioorg. Med. Chem. 16 (2008) 8117–8126
8121
Table 2 (continued)
kcat ðsꢀ1Þ ꢁ 103
Substrate
Km (mM)
Product
k
cat=Km ðMꢀ1 sꢀ1
Þ
HO
O
O
N.D.a
N.D.a
HO
O
O
OH
8
OH
20
a
Not determined.
Table 3
Geranylated polyketides synthesized by NphB
kcat ðsꢀ1Þ ꢁ 103
Substrate
Km (mM)
Product
k
cat=Km ðMꢀ1 sꢀ1
Þ
9'
10'
OH
OH
1'
2
0.64 0.16
0.043 0.003
0.067
8'
1
HO
C5H11
HO
C5H11
3
21
OH
OH
4
0.52 0.03
0.027 0.001
0.052
HO
HO
C5H11
HO
C5H11
22
3
OH
α
1'
OH
1
HO
1''
β
4
0.44 0.06
0.053 0.002
0.12
OH
23
OH
4
OH
OH
HO
2
4
HO
1'''
1''
N.D.a
N.D.a
OH
OH
24
4
OH
OH
HO
No products
OH
piceatannol
OH
H3CO
No products
OCH3
pterostilbene
a
Not determined.

8122
T. Kumano et al. / Bioorg. Med. Chem. 16 (2008) 8117–8126
Table 4 (continued)
Table 4
Prenylated products synthesized by SCO7190
Substrate
Product
Substrate
OH
Product
OH
OH
HO
No products
No products
OH
OH
OH
1,3-DHN
piceatannol
OH
OH
H3CO
HO
No products
HO
1
OCH3
25
26
pterostilbene
HO
HO
OH
OH
HO
OH
2
O
O
No products
OH
flaviolin
OH
OH
HO
O
HO
O
O
OH
O
OH
27
5
Figure 2. Crystal structure of NphB complexed with GSPP and 1,6-DHN (PDB ID,
1z6b).4 C5 of 1,6-DHN is the closest atom from C1 of GSPP. The distance is 4.2 Å. On
the other hand, the distance between C1 of GSPP and C2 of 1,6-DHN is 7.3 Å. The
distance between C1 of GSPP and C4 of 1,6-DHN is 5.5 Å.
OH
HO
HO
O
O
No products
No products
No products
OH
6
substrate, 5 mM geranyl diphosphate (GPP), 1 mg/ml NphB, in a to-
tal volume of 60
for overnight, and then extracted with 200
tracts were evaporated in a centrifugal vacuum concentrator, and
the residues were resolved in 20 l of methanol. The methanol
ll. The reaction mixtures were incubated at 25 °C
ll ethyl acetate. The ex-
O
O
l
solution was spotted on silica plates (Merck 20 cm ꢁ 20 cm silica
gel 60 F254, 0.2 mm). The plates were developed with chloroform:
methanol (15:1 v/v) and visualized by UV254. The reaction products
were also analyzed by high performance liquid chromatography
(JASCO, Tokyo) equipped with a MD-2010 Plus photodiode array
with PEGASIL ODS (4.6 ꢁ 250 mm, Senshu, Tokyo). Assays for
SCO7190-catalyzed reaction were carried out in 50 mM Tris–HCl
(pH 9.0), containing 5 mM aromatic substrate, 5 mM dimethylallyl
diphosphate (DMAPP), 1 mg/ml SCO7190, in a total volume of
OH
7
OH
OH
HO
O
O
8
60 ll. All the subsequent steps were identical to those of the NphB
assay.
OH
OH
OH
4.2. Product analysis
HO
C5H11 HO
C5H11
HO
HO
C5H11
3
28
29
Large-scale productions of prenyl products in a total volume of
10 ml were carried out in the same condition as that for the en-
zyme assay described above. The reaction mixtures were incubated
at 25 °C for overnight, and then extracted with 10 ml ethyl acetate
three times. The extracts were evaporated and the residues were
resolved in 1 ml of methanol. Prenylated products were purified
by high performance liquid chromatography with PEGASIL ODS
(20 ꢁ 250 mm, Senshu, Tokyo) employing an isocratic elution of
80% methanol, monitored at 254 nm. The chemical structures of
OH
OH
HO
OH
OH
4
30

T. Kumano et al. / Bioorg. Med. Chem. 16 (2008) 8117–8126
8123
an Enzyme Kinetic Module 1.3 (Systat Software, Point Richmond,
CA). The concentrations of GPP were 0.05–2.5 mM, while the con-
centration of naringenin was 5 mM. Under the condition, no sub-
strate inhibition of NphB was observed and Km value for GPP was
5
4
3
calculated to be 130 lM. For DHNs, the concentrations were 0.5,
1.0, 2.5, and 5 mM, while the concentration of GPP was 2.5 mM.
Incubation times were 3, 6, 9, and 15 min. For naringenin, apigenin,
and olivetol, the concentrations were 0.5, 1.25, 2.5, and 5.0 mM.
For genistein, the concentrations were 0.3, 0.5, 1.0, and 2.5 mM.
For resveratrol, the concentrations were 0.15, 0.3, 0.5, and
1.0 mM. For daidzein, the concentrations were 0.15, 0.3, 0.25, and
0.5 mM. The incubation times for these reactions were 10, 20, 30,
and 40 min. To gain insight into mechanism of the NphB-catalyzed
reaction, Lineweaver–Burk plots (Fig. 3) were created with 0.5,
1.25, 2.5, and 5 mM of naringenin and 0.05, 0.1, 0.2, 0.5, and
1 mM of GPP. The incubation times were 5, 17, 29, and 41 min.
We confirmed that all the reactions proceeded in a time-dependent
manner under each substrate concentration.
2
1
0
-1
0
-10
10
20
1/[GPP] (1/mM)
5
4
3
All the reactions were performed in 50 mM Tris–HCl (pH
9.0), containing 5 mM MgCl2, each aromatic substrate, 2.5 mM
GPP, 0.5 mg/ml NphB, in a total volume of 1 ml. The reaction
mixture was initiated by adding NphB and incubated at 25 °C.
2
1
0
Aliquots (200 ll) were removed from the reaction mixture at
appropriate incubation times mentioned above. Each aliquot
was extracted with an equal volume of ethyl acetate twice.
The organic layer was collected and evaporated. The residual
material was dissolved in 60 ll of methanol, and the formation
of each product was quantified by HPLC using the standard
curve. A plot of product versus time was used to determine ini-
tial velocity.
-1
-1
0
1
2
1/[naringenin] (1/mM)
The reactions to determine yield of geranylated products forma-
tion (Fig. 4) were carried out in 50 mM Tris–HCl (pH 9.0), contain-
ing 5 mM MgCl2, 0.5 mM aromatic substrate, 5 mM GPP, 1 mg/ml
NphB, in a total volume of 60 ll, because 5 mM daidzein is not
soluble in the buffer, but 0.5 mM daidzein is soluble.
Figure 3. Double-reciprocal plots of GPP and naringenin turnover catalyzed by
NphB. (a) Lineweaver–Burk plots for the NphB reaction with 0.5 mM (d), 1.25 mM
(s), 2.5 mM (j), 5 mM (h) of naringenin and varied [GPP]. (b) Lineweaver–Burk
plots for the NphB reaction with 0.05 mM (d), 0.1 mM (s), 0.2 mM (j), 0.5 mM (h),
1 mM ( ) of GPP and varied [naringenin]. 1/initial velocity versus 1/substrate
N
concentration was fit to the Lineweaver–Burk plot using a software SigmaPlot 10
(Systat Software, Point Richmond, CA).
4.4. 1H, 13C NMR, and HRMS data of the prenylated products
The structures were analyzed by their mass spectral data
[HRMS (ESIꢀ); JEOL JMS-T100LC] and nuclear magnetic resonance
(NMR) spectral data (600 MHz, JEOL ECA-600).
100
80
60
40
20
0
4.4.1. 4-Geranyl 1,6-DHN (9; product was synthesized from 1,6-
DHN and GPP by recombinant NphB)
HRMS (ESIꢀ) calcd for C20H23O2 (Mꢀ), 295.16980; found
1
295.16781. H NMR (CD3OD) d: 1.50 (s, 3H, H-100), 1.54 (s, 3H,
H-80), 1.84 (s, 3H, H-90), 1.94 (m, 2H, H-40), 2.03 (m, 2H, H-50),
3.67 (d, J = 6.2 Hz, 2H, H-10), 5.00 (m, 1H, H-60), 5.14 (m, 1H,
H-20), 6.51 (d, J = 7.6 Hz, 1H, H-2), 6.97 (m, 2H, H-3, H-7), 7.17
(s, 1H, H-5), 8.05 (d, J = 8.9 Hz, 1H, H-8).
4.4.2. 5-Geranyl 1,6-DHN (10; product was synthesized from
1,6-DHN and GPP by recombinant NphB)4
0
10
20
/hr
30
t
HRMS (ESIꢀ) calcd for C20H23O2 (Mꢀ), 295.16980; found
1
295.17000. H NMR (CD3OD) d: 1.50 (s, 3H, H-100), 1.54 (s, 3H,
Figure 4. Time course of the NphB-catalyzed reaction. Reactions were carried out
in 50 mM Tris–HCl (pH 9.0), containing 5 mM MgCl2, 0.5 mM aromatic substrate,
5 mM GPP, 1 mg/ml NphB at 25 °C in a total volume of 60
apigenin (s), genistein (j), daidzein (h), and olivetol ( ).
H-80), 1.84 (s, 3H, H-90), 1.94 (m, 2H, H-40), 2.03 (m, 2H, H-50),
3.67 (d, J = 6.2 Hz, 2H, H-10), 5.00 (m, 1H, H-60), 5.14 (m, 1H, H-
20), 6.58 (d, J = 7.6 Hz, 1H, H-2), 6.97 (d, J = 8.9 Hz, 1H, H-7), 7.16
(dd, J = 8.3, 7.6 Hz, 1H, H-3), 7.27 (d, J = 8.3 Hz, 1H, H-4), 7.91
(d, J = 8.9 Hz, 1H, H-8).
ll. Naringenin (d),
N
the reaction products were elucidated by analyzing 1H NMR, 13C
NMR, HMBC spectra, and HRESI-MS data.
4.4.3. 2-Geranyl 1,6-DHN (11; product was synthesized from
1,6-DHN and GPP by recombinant NphB)4
4.3. Steady-state kinetic parameters
HRMS (ESIꢀ) calcd for C20H23O2 (Mꢀ), 295.16980; found
1
Steady-state kinetic parameters were determined by the
Michaelis–Menten Equation using a software SigmaPlot 10.0 and
295.16691. H NMR (CD3OD) d: 1.50 (s, 3H, H-100), 1.54 (s, 3H,
H-80), 1.84 (s, 3H, H-90), 1.94 (m, 2H, H-40), 2.03 (m, 2H, H-50),

8124
T. Kumano et al. / Bioorg. Med. Chem. 16 (2008) 8117–8126
3.67 (d, J = 6.2 Hz, 2H, H-10), 5.00 (m, 1H, H-60), 5.14 (m, 1H, H-20),
6.98 (m, 2H, H-3, H-7), 7.05 (m, 2H, H-4, H-5), 8.05 (m, 1H, H-8).
129.1 (C-60), 131.6 (C-700), 141.8 (C-300), 157.7 (C-40), 161.7 (C-8a),
163.3 (C-2), 164.8 (C-5), 164.8 (C-7), 182.3 (C-4).
4.4.4. 1-Geranyl 2,7-DHN (12; product was synthesized from
2,7-DHN and GPP by recombinant NphB)
4.4.9. 6-Geranyl apigenin (17; product was synthesized from
apigenin and GPP by recombinant NphB)
HRMS (ESIꢀ) calcd for C20H23O2 (Mꢀ), 295.16980; found
HRMS (ESIꢀ) calcd for C25H25O5 (Mꢀ), 405.17027; found
1
1
295.16599. H NMR (CD3OD) d: 1.50 (s, 3H, H-100), 1.53 (s, 3H, H-
405.17115. H NMR (DMSO-d6) d: 1.48 (s, 3H, H-1000), 1.54 (s, 3H,
80), 1.85 (s, 3H, H-90), 1.95 (m, 2H, H-40), 2.03 (m, 2H, H-50), 3.62
(d, J = 6.2 Hz, 2H, H-10), 5.01 (m, 1H, H-60), 5.15 (m, 1H, H-20), 6.81
(d, J = 8.9 Hz, 1H, H-6), 6.86 (d, J = 8.9 Hz, 1H, H-3), 7.10 (s, 1H, H-
8), 7.42 (d, J = 8.9 Hz, 1H, H-5), 7.55 (d, J = 8.9 Hz, 1H, H-4).
H-800), 1.70 (s, 3H, H-900), 1.88 (m, 2H, H-400), 1.96 (m, 2H, H-500),
3.19 (d, J = 6.9 Hz, 2H, H-100), 4.99 (m, 1H, H-600), 5.17
(t, J = 6.9 Hz, 1H, H-200), 6.44 (s, 1H, H-3), 6.67 (s, 1H, H-8), 6.86
(d, J = 8.2 Hz, 2H, H-30, H-50), 7.84 (d, J = 8.2 Hz, 2H, H-20, H-60),
13
13.15 (br, 1H, C-5-OH). C NMR (DMSO-d6) d: 16.5 (C-900), 18.0
4.4.5. 1,6-Digeranyl 2,7-DHN (13; product was synthesized from
2,7-DHN and GPP by recombinant NphB)
(C-1000), 21.6 (C-100), 26.0 (C-800), 26.7 (C-500), 39.9 (C-400), 93.9 (C-
8), 103.0 (C-3), 103.2 (C-4a), 111.7 (C-6), 116.5 (C-30), 116.5
(C-50), 121.7 (C-10), 122.9 (C-200), 124.7 (C-600), 128.8 (C-20), 128.8
(C-60), 161.9 (C-2), 131.1 (C-700), 134.4 (C-300), 155.8 (C-40),
158.7 (C-8a), 161.9 (C-5), 163.7 (C-7), 181.9 (C-4).
HRMS (ESIꢀ) calcd for C30H39O2 (Mꢀ), 431.29500; found
1
431.29322. H NMR (CD3OD) d: 1.45 (s, 3H, H-1000), 1.47 (s, 3H,
H-800), 1.58 (s, 3H, H-100), 1.68 (s, 3H, H-80), 1.69 (s, 3H, H-900),
1.83 (s, 3H, H-90), 1.92 (m, 2H, H-40), 2.00 (m, 2H, H-50), 2.04 (m,
2H, H-400), 2.10 (m, 2H, H-500), 3.59 (m, 2H, H-100), 3.60 (m, 2H, H-
10), 5.01 (m, 2H, H-60), 5.38 (m, 2H, H-600), 6.86 (d, J = 8.3 Hz, 1H,
H-3), 7.11 (s, 1H, H-8), 7.35 (m, 1H, H-4), 7.35 (s, 1H, H-5).
4.4.10. 7-O-Geranyl genistein (18; product was synthesized
from genistein and GPP by recombinant NphB)
HRMS (ESIꢀ) calcd for C25H25O5 (Mꢀ), 405.17027; found
1
405.16781. H NMR (DMSO-d6) d: 1.52 (s, 3H, H-1000), 1.57 (s, 3H,
4.4.6 7-O-Geranyl naringenin (14; product was synthesized
from naringenin and GPP by recombinant NphB)4
H-800), 1.68 (s, 3H, H-900), 2.02 (m, 2H, H-400), 2.04 (m, 2H, H-500),
4.63 (d, J = 6.8 Hz, 2H, H-100), 5.02 (m, 1H, H-600), 5.39 (m, 1H, H-
200), 6.35 (s, 1H, H-8), 6.60 (s, 1H, H-6), 6.78 (d, J = 7.6 Hz, 2H, H-30,
H-50), 7.34 (d, J = 7.6 Hz, 2H, H-20, H-60), 8.35 (s, 1H, H-2). 13C NMR
(DMSO-d6) d: 16.9 (C-900), 18.1 (C-1000), 25.9 (C-800), 26.2 (C-500),
39.8 (C-400), 65.9 (C-100), 93.5 (C-8), 99.1 (C-6), 105.9 (C-4a), 115.7
(C-30), 115.7 (C-50), 119.2 (C-200), 121.5 (C-10), 123.0 (C-3), 124.2
(C-600), 130.7 (C-20), 130.7 (C-60), 131.7 (C-700), 142.0 (C-300), 154.8
(C-2), 158.0 (C-8a), 158.0 (C-40), 162.3 (C-5), 164.9 (C-7), 180.9 (C-4).
HRMS (ESIꢀ) calcd for C25H27O5 (Mꢀ), 407.18515; found
1
407.18732. H NMR (DMSO-d6) d: 1.50 (s, 3H, H-1000), 1.57 (s, 3H,
H-800), 1.63 (s, 3H, H-900), 1.97 (m, 2H, H-400), 2.01 (m, 2H, H-500),
2.66 (dd, J = 17.2, 2.8 Hz, 1H, H-3eq), 3.25 (dd, J = 17.2, 13.0 Hz, 1H,
H-3ax), 4.53 (d, J = 6.2 Hz, 2H, H-100), 5.00 (m, 1H, H-600), 5.32 (m,
1H, H-200), 5.42 (dd, J = 13.1, 2.8 Hz, 1H, H-2), 6.01 (s, 1H, H-8), 6.03
(s, 1H, H-6), 6.75 (d, J = 6.9 Hz, 2H, H-30, H-50), 7.27 (d, J = 6.9 Hz,
1H, H-20, H-60), 9.67 (br, s, C-40-OH), 12.05 (br, s, C-5-OH). 13C NMR
(DMSO-d6) d: 16.7 (C-900), 18.1 (C-1000), 26.0 (C-800), 26.2 (C-500), 39.5
(C-400), 42.5 (C-3), 65.7 (C-100), 79.1 (C-2), 94.8 (C-8), 95.7 (C-6),
103.0 (C-4a), 115.7 (C-30), 115.7 (C-50), 119.3 (C-200), 124.2 (C-600),
128.9 (C-20), 128.9 (C-60), 129.2 (C-10), 131.7 (C-700), 141.8 (C-300),
158.3 (C-40), 161.0 (C-8a), 163.6 (C-5), 167.2 (C-7), 197.4 (C-4).
4.4.11. 7-O-Geranyl daidzein (19; product was synthesized from
daidzein and GPP by recombinant NphB)
HRMS (ESIꢀ) calcd for C25H25O4 (Mꢀ), 389.17528; found
1
389.17653. H NMR (DMSO-d6) d: 1.58 (s, 3H, H-1000), 1.61 (s, 3H,
H-800), 1.78 (s, 3H, H-900), 2.09 (m, 2H, H-400), 2.13 (m, 2H, H-500),
4.70 (d, J = 6.2 Hz, 2H, H-100), 5.07 (m, 1H, H-600), 5.47 (m, 1H, H-
200), 6.83 (d, J = 4.8 Hz, 2H, H-30, H-50), 7.02 (s, 1H, H-8), 7.03 (d,
J = 13.0 Hz, 1H, H-6), 7.36 (d, J = 4.8 Hz, 2H, H-20, H-60), 8.09 (d,
J = 13.0 Hz, 1H, H-5), 8.17 (s, 1H, H-2). 13C NMR (DMSO-d6) d:
14.5 (C-900), 17.1 (C-1000), 24.6 (C-800), 26.0 (C-500), 39.3 (C-400), 64.7
(C-100), 93.5 (C-8), 99.1 (C-6), 104.9 (C-4a), 115.7 (C-30), 115.7 (C-
50), 117.7 (C-5), 118.9 (C-200), 122.9 (C-3), 123.7 (C-600), 125.0
(C-10), 130.2 (C-20), 130.2 (C-60), 131.5 (C-700), 141.9 (C-300), 153.6
(C-2), 157.5 (C-40), 158.5 (C-8a), 164 (C-7), 176.7 (C-4).
4.4.7. 6-Geranyl naringenin (15; product was synthesized from
naringenin and GPP by recombinant NphB)4
HRMS (ESIꢀ) calcd for C25H27O5 (Mꢀ), 407.18515; found
1
407.18454. H NMR (DMSO-d6) d: 1.48 (s, 3H, H-1000), 1.55 (s, 3H,
H-800), 1.65 (s, 3H, H-900), 1.85 (m, 2H, H-400), 1.94 (m, 2H, H-500),
2.63 (dd, J = 17.2, 2.8 Hz, 1H, H-3eq), 3.07 (d, J = 6.9 Hz, 2H, H-100),
3.16 (dd, J = 17.2, 13.1 Hz, 1H, H-3ax), 4.99 (m, 1H, H-600), 5.08 (m,
1H, H-200), 5.34 (dd, J = 13.1, 2.8 Hz, 1H, H-2), 5.91 (s, 1H, H-8),
6.75 (d, J = 8.3 Hz, 2H, H-30, H-50), 7.26 (d, J = 8.3 Hz, 2H, H-20, H-
60), 9.64 (br, s, C-40-OH), 12.39 (s, 1H, C-5-OH). 13C NMR (DMSO-
d6) d: 16.4 (C-900), 18.0 (C-1000), 21.1 (C-100), 26.0 (C-800), 26.7 (C-500),
39.9 (C-400), 42.6 (C-3), 78.8 (C-2), 95.0 (C-8), 101.9 (C-4a), 108.1
(C-6), 115.7 (C-30), 115.7 (C-50), 123.0 (C-200), 124.6 (C-600), 128.8
(C-20), 128.8 (C-60), 129.6 (C-10), 131.2 (C-700), 134.3 (C-300), 158.2
(C-40), 161.0 (C-8a), 161.1 (C-5), 165.4 (C-7), 196.8 (C-4).
4.4.12. 8-Geranyl daidzein (20; product was synthesized from
daidzein and GPP by recombinant NphB)
HRMS (ESIꢀ) calcd for C25H25O4 (Mꢀ), 389.1753; found
1
389.1794. H NMR (CD3OD) d: 1.57 (s, 3H, H-1000), 1.60 (s, 3H, H-
800), 1.89 (s, 3H, H-900), 2.04 (m, 2H, H-400), 2.11 (m, 2H, H-500),
3.63 (d, J = 6.2 Hz, 2H, H-100), 5.06 (m, 1H, H-600), 5.31 (m, 1H, H-
200), 6.90 (d, J = 7.6 Hz, 2H, H-30, H-50), 7.02 (d, J = 8.9 Hz, 1H, H-6),
7.43 (d, J = 7.6 Hz, 2H, H-20, H-60), 7.98 (d, J = 8.9 Hz, 1H, H-5),
4.4.8. 7-O-Geranyl apigenin (16; product was synthesized from
apigenin and GPP by recombinant NphB)
13
8.27 (s, 1H, H-2). C NMR (CD3OD) d: 15.0 (C-900), 16.3 (C-1000),
HRMS (ESIꢀ) calcd for C25H25O5 (Mꢀ), 405.17027; found
21.4 (C-100), 24.5 (C-800), 26.2 (C-500), 39.4 (C-400), 114.2 (C-6),
114.9 (C-30), 114.9 (C-50), 115.6 (C-8), 116.9 (C-4a), 121.6 (C-200),
123.0 (C-10), 123.9 (C-600), 124.0 (C-5), 124.2 (C-3), 130.1 (C-20),
130.1 (C-60), 130.7 (C-700), 135.2 (C-300), 153.3 (C-2), 156.2 (C-40),
157.3 (C-8a), 160.5 (C-7), 177.3 (C-4).
1
405.16686. H NMR (DMSO-d6) d: 1.52 (s, 3H, H-1000), 1.57 (s, 3H,
H-800), 1.69 (s, 3H, H-900), 2.02 (m, 2H, H-400), 2.04 (m, 2H, H-500),
4.63 (d, J = 6.9 Hz, 2H, H-100), 4.99 (m, 1H, H-600), 5.17 (t,
J = 6.9 Hz, 1H, H-200), 6.30 (s, 1H, H-3), 6.72 (s, 1H, H-8), 6.76 (s,
1H, H-6), 6.85 (d, J = 8.9 Hz, 2H, H-30, H-50), 7.88 (d, J = 8.9 Hz, 2H,
H-20, H-60), 12.93 (br, 1H, C-5-OH). 13C NMR (DMSO-d6) d: 16.9
(C-900), 18.1 (C-1000), 26.0 (C-800), 26.3 (C-500), 39.9 (C-400), 65.9 (C-
100), 93.8 (C-8), 99.0 (C-4a), 103.0 (C-3), 105.1 (C-6), 116.8 (C-30),
116.8 (C-50), 119.4 (C-200), 120.5 (C-10), 124.3 (C-600), 129.1 (C-20),
4.4.13. 2-Geranyl olivetol (21; product was synthesized from
olivetol and GPP by recombinant NphB)
HRMS (ESIꢀ) calcd for C21H31O2 (Mꢀ), 389.17528; found
1
389.17653. H NMR (DMSO-d6) d: 0.88 (t, J = 6.9 Hz, 3H, H-50),

T. Kumano et al. / Bioorg. Med. Chem. 16 (2008) 8117–8126
8125
1.32 (m, 6H, H-20, H-30, H-40), 1.55 (s, 3H, H-1000), 1.60 (s, 3H, H-800),
4.4.18. 1-Dimethylallyl 2,7-DHN (26; product was synthesized
1.71 (s, 3H, H-900), 1.94 (t, J = 6.8, 7.6 Hz, 2H, H-400), 2.04 (c, J = 6.8,
6.8, 7.6 Hz, 2H, H-500), 2.43 (t, J = 7.6 Hz, 2H, H-10), 3.21 (d,
J = 6.2 Hz, 2H, H-100), 5.04 (m, 2H, H-200, H-600), 6.10 (s, 1H, H-6),
from 2,7-DHN and DMAPP by recombinant SCO7190)
HRMS (ESIꢀ) calcd for C15H15O2 (Mꢀ), 227.10720; found
1
227.10488. H NMR (CD3OD) d: 1.59 (s, 3H, H-50), 1.78 (s, 3H, H-
13
6.12 (s, 1H, H-4). C NMR (DMSO-d6) d: 13.1 (C-50), 15.0 (C-900),
40), 3.48 (d, J = 6.2 Hz, 2H, H-10), 5.06 (m, 1H, H-20), 6.86 (d,
J = 8.9 Hz, 1H, H-30), 6.87 (d, J = 8.9 Hz, 1H, H-6), 6.97 (s, 1H, H-8),
7.41 (d, J = 8.9 Hz, 1H, H-5), 7.53 (d, J = 8.9 Hz, 1H, H-4).
16.4 (C-1000), 22.4 (C-100), 23.8 (C-40), 24.5 (C-800), 26.4 (C-500), 30.8
(C-30), 31.9 (C-20), 33.0 (C-10), 39.5 (C-400), 99.8 (C-4), 107.1 (C-6),
117.5 (C-2), 124.1 (C-200), 124.8 (C-600), 130.7 (C-700), 132.7 (C-300),
142.8 (C-1), 155.1 (C-5), 155.7 (C-3).
4.4.19. 6-Dimethylallyl naringenin (27; product was
synthesized from naringenin and DMAPP by recombinant
SCO7190)
4.4.14. 4-Geranyl olivetol (22; product was synthesized from
olivetol and GPP by recombinant NphB)
HRMS (ESIꢀ) calcd for C20H19O5 (Mꢀ), 339.12325; found
HRMS (ESIꢀ) calcd for C21H31O2 (Mꢀ), 389.17528; found
339.12554. H NMR (CD3OD) d: 1.54 (s, 3H, H-500), 1.62 (s, 3H, H-
1
1
389.17323. H NMR (DMSO-d6) d: 0.88 (t, J = 6.9 Hz, 3H, H-50),
400), 2.58 (dd, J = 13.1, 2.8 Hz, 1H, H-3eq), 3.01 (d, J = 6.9 Hz, 2H,
H-100), 3.09 (dd, J = 17.2, 13.1 Hz, 1H, H-3ax), 5.04 (t, J = 6.2,
6.9 Hz, 1H, H-200), 5.29 (dd, J = 13.1, 2.8 Hz, 1H, H-2), 5.81 (s, 1H,
H-8), 6.73 (d, J = 8.3 Hz, 2H, H-30, H-50), 7.25 (d, J = 8.3 Hz, 2H, H-
1.29 (m, 6H, H-20, H-30, H-40), 1.54 (s, 3H, H-1000), 1.59 (s, 3H, H-
800), 1.73 (s, 3H, H-900), 1.93 (t, J = 6.8, 7.6 Hz, 2H, H-400), 2.04 (c,
J = 6.8, 7.6, 6.8 Hz, 2H, H-500), 2.37 (t, J = 7.6 Hz, 2H, H-10), 3.22 (d,
J = 6.9 Hz, 2H, H-100), 5.05 (m, 1H, H-600), 5.21 (t, J = 6.9 Hz, 1H, H-
13
20, H-60), 12.36 (s, 1H, C-5-OH). C NMR (CD3OD) d: 18.1 (C-400),
13
200), 6.11 (s, 2H, H-2, H-6). C NMR (DMSO-d6) d: 13.1 (C-50),
21.1 (C-500), 26.0 (C-100), 42.4 (C-3), 78.7 (C-2), 95.4 (C-8), 108.3
(C-4a), 108.4 (C-6), 115.7 (C-30), 115.7 (C-50), 123.3 (C-200), 128.8
(C-20), 128.8 (C-60), 129.7 (C-10), 130.7 (C-300), 157.9 (C-40), 160.9
(C-8a), 161.0 (C-5), 165.4 (C-7), 196.8 (C-4).
14.5 (C-900), 16.4 (C-1000), 21.7 (C-100), 22.3 (C-40), 24.5 (C-800), 26.4
(C-500), 30.9 (C-30), 31.3 (C-20), 35.4 (C-10), 39.6 (C-400), 106.5 (C-2),
106.5 (C-6), 112.2 (C-4), 123.7 (C-200), 124.3 (C-600), 130.6 (C-700),
133.2 (C-300), 141.0 (C-1), 155.6 (C-3), 155.6 (C-5).
4.4.20. 4-Dimethylallyl olivetol (28; product was synthesized
from olivetol and DMAPP by recombinant SCO7190)
4.4.15. 4-Geranyl resveratrol (23; product was synthesized from
resveratrol and GPP by recombinant NphB)
HRMS (ESIꢀ) calcd for C16H23O2 (Mꢀ), 247.16980; found
1
HRMS (ESIꢀ) calcd for C24H26O3 (Mꢀ), 363.19602; found
247.17005. H NMR (CD3OD) d: 0.88 (t, J = 6.9,7.5 Hz, 3H, H-50),
1
363.19137. H NMR (DMSO-d6) d: 1.53 (s, 3H, H-1000), 1.59 (s, 3H,
1.29 (m, 6H, H-20, H-30, H-40), 1.62 (s, 3H, H-500), 1.72 (s, 3H, H-
400), 2.37 (t, J = 6.9, 7.6 Hz, 2H, H-10), 3.21 (d, J = 6.8 Hz, 2H, H-100),
5.18 (m, 2H, H-200), 6.11 (s, 1H, H-2, H-6).
H-800), 1.73 (s, 3H, H-9), 1.92 (m, 2H, H-400), 2.02 (m, 2H, H-500),
3.25 (d, J = 7.6 Hz, 2H, H-100), 5.20 (m, 2H, H-200, H-600), 6.47 (s, 2H,
H-2, H-6), 6.73 (d, J = 15.8 Hz, 1H, H-b), 6.75 (d, J = 8.3 Hz, 2H, H-
30, H-50), 6.89 (d, J = 15.8 Hz, 1H, H-
a
), 7.31 (d, J = 8.3 Hz, 2H, H-
4.4.21. 2-Dimethylallyl olivetol (29; product was synthesized
from olivetol and DMAPP by recombinant SCO7190)
13
20, H-60). C NMR (DMSO-d6) d: 15.0 (C-900), 16.4 (C-1000), 21.9 (C-
100), 24.6 (C-800), 26.4 (C-500), 39.6 (C-400), 104.5 (C-2), 104.5 (C-6),
114.6 (C-4), 115.2 (C-30), 115.2 (C-50), 123.2 (C-200), 124.1 (C-600),
HRMS (ESIꢀ) calcd for C16H23O2 (Mꢀ), 247.16980; found
1
247.17123. H NMR (CD3OD) d: 0.89 (t, J = 6.9 Hz, 3H, H-50), 1.32
125.9 (C-
a
), 127.0 (C-20), 127.3 (C-b), 127.3 (C-60), 129.4 (C-10),
(m, 6H, H-20, H-30, H-40), 1.63 (s, 3H, H-500), 1.71 (s, 3H, H-400),
2.42 (m, 2H, H-10), 3.20 (d, J = 6.8 Hz, 2H, H-100), 5.03 (m, 2H, H-
200), 6.09 (s, 1H, H-6), 6.11 (s, 1H, H-4).
130.9 (C-700), 133.7 (C-300), 136.3 (C-1), 155.8 (C-3), 155.8 (C-5),
156.6 (C-40).
4.4.16. 2,4-Digeranyl resveratrol (24; product was synthesized
from resveratrol and GPP by recombinant NphB)
4.4.22. 2-Dimethylallyl resveratrol (30; product was
synthesized from resveratrol and DMAPP by recombinant
SCO7190)
HRMS (ESIꢀ) calcd for C34H43O3 (Mꢀ), 499.3212; found
1
499.3189. H NMR (CD3OD) d: 1.49 (s, 3H, H-1000), 1.54 (s, 6H, H-
1H NMR (CD3OD) d: 1.64 (s, 3H, H-500), 1.78 (s, 3H, H-400), 3.34 (d,
J = 7.6 Hz, 2H, H-100), 5.08 (m, 1H, H-200), 6.20 (s, 1H, H-4), 6.53 (s, 1H,
H-6), 6.74 (d, J = 8.3 Hz, 2H, H-30, H-50), 6.80 (d, J = 16.5 Hz, 1H, H-b),
800, H-10000), 1.60 (s, 3H, H-8000), 1.76 (s, 3H, H-9000), 1.77 (s, 3H, H-
900), 1.96 (t, J = 6.9 Hz, 4H, H-400, H-4000), 2.03 (m, 2H, H-5000), 2.06
(m, 2H, H-500), 3.34 (d, J = 6.9 Hz, 2H, H-1000), 3.39 (d, J = 6.9 Hz,
2H, H-100), 5.01 (t, J = 6.9 Hz, 1H, H-600), 5.05 (t, J = 6.9 Hz, 1H, H-
6000), 5.09 (t, J = 5.5, 6.9 Hz, 1H, H-200), 5.21 (t, J = 6.2, 6.9 Hz, 1H,
H-2000), 6.62 (s, 1H, H-6), 6.72 (d, J = 8.2 Hz, 2H, H-30, H-50), 6.76
7.10 (d, J = 16.5 Hz, 1H, H-a
), 7.29 (d, J = 8.3 Hz, 2H, H-20, H-60).
Acknowledgments
(d, J = 16.5 Hz, 1H, H-b), 7.06 (d, J = 16.5 Hz, 1H, H-a), 7.27 (d,
This work was supported by METI and Grant-in-Aid for Scien-
tific Research on Priority Areas ‘Applied Genome’ (18018008) from
MEXT, Japan (T. Kuzuyama) and HHMI (J.P.N.).
13
J = 8.2 Hz, 2H, H-20, H-60). C NMR (CD3OD) d: 15.0 (C-9000), 15.2
(C-900), 16.4 (C-1000), 16.4 (C-10000), 22.4 (C-1000), 24.5 (C-100),24.5 (C-
800), 24.6 (C-8000), 26.4 (C-500), 26.4 (C-5000),39.5 (C-4000), 39.6 (C-400),
103.9 (C-6), 115.1 (C-30), 115.1 (C-50), 115.2 (C-4), 118.6 (C-2),
Supplementary data
123.0 (C-2000), 124.0 (C-600), 124.1 (C-6000), 124.2 (C- ), 124.6 (C-200),
a
127.3 (C-20, C-60), 128.4 (C-b), 129.7 (C-10), 130.8 (C-700), 130.8 (C-
7000), 133.4 (C-300), 134.5 (C-3000), 135.3 (C-1), 152.9 (C-3), 153.4 (C-
5), 156.8 (C-40).
Supplemental figure 1 and supplemental figure 2 can be found
in the Supporting Information. Supplementary data associated
with this article can be found, in the online version, at
doi:10.1016/j.bmc.2008.07.052.
4.4.17. 5-Dimethylallyl 1,6-DHN (25; product was synthesized
from 1,6-DHN and DMAPP by recombinant SCO7190)
HRMS (ESIꢀ) calcd for C15H15O2 (Mꢀ), 227.10720; found
References and notes
1
227.10785. H NMR (CD3OD) d: 1.57 (s, 3H, H-50), 1.77 (s, 3H, H-
1. Di Pietro, A.; Conseil, G.; Perez-Victoria, J. M.; Dayan, G.; Baubichon-Cortay, H.;
Trompier, D.; Steinfels, E.; Jault, J. M.; de Wet, H.; Maitrejean, M.; Comte, G.;
Boumendjel, A.; Mariotte, A. M.; Dumontet, C.; McIntosh, D. B.; Goffeau, A.;
Castanys, S.; Gamarro, F.; Barron, D. Cell. Mol. Life Sci. 2002, 59, 307.
40), 3.56 (d, J = 6.9 Hz, 2H, H-10), 5.07 (m, 1H, H-20), 6.58 (d,
J = 8.9 Hz, 1H, H-7), 6.59 (d, J = 6.8 Hz, 1H, H-2), 7.16 (dd, J = 8.2,
7.6 Hz, 1H, H-3), 7.18 (d, J = 8.2 Hz, 1H, H-4).

8126
T. Kumano et al. / Bioorg. Med. Chem. 16 (2008) 8117–8126
2. Ahmed-Belkacem, A.; Pozza, A.; Munoz-Martinez, F.; Bates, S. E.; Castanys, S.;
Gamarro, F.; Di Pietro, A.; Perez-Victoria, J. M. Cancer Res. 2005, 65, 4852.
3. Botta, B.; Vitali, A.; Menendez, P.; Misiti, D.; Delle Monache, G. Curr. Med. Chem.
2005, 12, 717.
6. Shin-ya, K.; Furihata, K.; Hayakawa, Y.; Seto, H. Tetrahedron Lett. 1990, 31, 6025.
7. Seto, H.; Watanabe, H.; Furihata, K. Tetrahedron Lett. 1996, 37, 7979.
8. Tello, M.; Kuzuyama, T.; Heide, L.; Noel, J. P.; Richard, S. B. Cell. Mol. Life Sci.
2008, 65, 1459.
4. Kuzuyama, T.; Noel, J. P.; Richard, S. B. Nature 2005, 435, 983.
5. Shin-ya, K.; Imai, S.; Furihata, K.; Hayakawa, Y.; Kato, Y.; Vanduyne, G. D.;
Clardy, J.; Seto, H. J. Antibiot. 1990, 43, 444.
9. Cui, G.; Li, X.; Merz, K. M., Jr. Biochemistry 2007, 46, 1303.
10. Haagen, Y.; Unsöld, I.; Westrich, L.; Gust, B.; Richard, S. B.; Noel, J. P.; Heide, L.
FEBS Lett. 2007, 581, 2889.