Synthesis and characterization of abietadiene, levopimaradiene, palustradiene, and neoabietadiene: Hydrocarbon precursors of the abietane diterpene resin acids

Article abstract of DOI:10.1016/S0040-4020(01)00605-6

The abietane diterpenes - abietadiene, levopimaradiene, palustradiene, and neoabietadiene (1b-4b) - were prepared from the corresponding resin acids by diazomethane esterifications, LiAlH₄ reductions, tosylations, and Zn/NaI reductions. Abietadiene was also obtained less efficiently by catechol borane reduction of abietadienal tosylhydrazone and Li/NH₃ reduction of its 18-phenylthio derivative. These conjugated dienes were characterized by chromatographic properties (HPLC, TLC, GC), MS fragmentation patterns, optical rotations, and UV, IR, ¹H NMR, and ¹³C NMR data. Assignments for the ¹H and ¹³C NMR spectra were made by COSY, DEPT, HMQC, HMBC, NOE data, H-H coupling analysis, and comparisons with literature data. The four diterpenes were identified as cyclization products of recombinant abietadiene synthase, supporting their likely role in the biosynthesis of the abietane resin acids in conifer oleoresin.

Full text of DOI:10.1016/S0040-4020(01)00605-6

TETRAHEDRON
Pergamon
Tetrahedron 57 (2001) 6155±6167
Synthesis and characterization of abietadiene, levopimaradiene,
palustradiene, and neoabietadiene: hydrocarbon precursors of the
abietane diterpene resin acids
Hyung-Jae Lee, Matthew M. Ravn and Robert M. Coatesp
Department of Chemistry, University of Illinois, 600 S. Mathews Avenue, Urbana, IL 61801, USA
Received 12 April 2001; accepted 30 May 2001
AbstractÐThe abietane diterpenesÐabietadiene, levopimaradiene, palustradiene, and neoabietadiene (1b±4b)Ðwere prepared from the
corresponding resin acids by diazomethane esteri®cations, LiAlH4 reductions, tosylations, and Zn/NaI reductions. Abietadiene was also
obtained less ef®ciently by catechol borane reduction of abietadienal tosylhydrazone and Li/NH3 reduction of its 18-phenylthio derivative.
These conjugated dienes were characterized by chromatographic properties (HPLC, TLC, GC), MS fragmentation patterns, optical rotations,
and UV, IR, 1H NMR, and 13C NMR data. Assignments for the 1H and 13C NMR spectra were made by COSY, DEPT, HMQC, HMBC, NOE
data, H±H coupling analysis, and comparisons with literature data. The four diterpenes were identi®ed as cyclization products of recombi-
nant abietadiene synthase, supporting their likely role in the biosynthesis of the abietane resin acids in conifer oleoresin. q 2001 Elsevier
Science Ltd. All rights reserved.
1. Introduction
were separated from the essential oil of Juniperus sabina
L. berries12 and from the oleoresin of Picea schrenkiana.13
Some characterization data are available for these more
frequently isolated abietane hydrocarbons,10±14 and partial
syntheses of abietadiene by Wolff±Kishner reduction of
abietadienal have been recorded.15 The detection of these
diterpenes, and occasionally neoabietadiene, by GC
analyses of oleoresin or essential oils from Cupressus
species,11 Pinus species,16 and Abies nord-munnia (Cauca-
sian ®r),17 various commercial gum and wood rosins,18 and
southern pine tree oil have been reported.1,19 Characteristic
MS peaks used for GC/MS identi®cation of abietadiene and
palustradiene have been presented.15 A plausible but tenta-
tive identi®cation of all four isomers in Cupressus arizonica
essential oil based on palmitic acid-catalyzed interconver-
sions at 2008C and GC comparisons, in accord with
characteristic thermal equilibrations of the corresponding
resin acids,1,20 was reported.11
Abietic, levopimaric, palustric, and neoabietic acids (Fig. 1,
1a±4a) are widely occurring tricyclic diterpene carboxylic
acids which together with the biogenetically related pimaric
acids comprise the majority of the C20 fraction of conifer
oleoresins.1,2 This viscous secretion serves an important
defensive function in ®lling and sealing wounds by solidi-
®cation and oxidative crosslinking.3,4 The pine rosins are
abundant natural chemicals which have many industrial
applications including paper sizings, polymerization
emulsi®ers, adhesive tacki®ers, printing ink resins, and
waterproo®ng material as well as other uses.5 The easily
isolated abietic6 and levopimaric7 acids are common starting
materials for synthesis of other natural products8,9 and
numerous diterpene derivatives.1
The abundance and widespread occurrence of the four
abietane-type resin acids stand in marked contrast to the
rarity and paucity of information in the literature concerning
the corresponding hydrocarbonsÐabietadiene, levopimara-
diene, palustradiene, and neoabietadiene (1b±4b)Ðdespite
their likely role as intermediates in resin acid biosynthesis.
Abietadiene has been isolated from Larix sibirica rosin,10a
Pinus sibirica resin,10b Cedrus libani cones,11a and Cupres-
sus arizonica oil,11b and both abietadiene and palustradiene
Signi®cant advances have been made recently in the
characterization of the enzymes and genes responsible for
resin acid biosynthesis in conifers. Abietadiene synthases
from Abies grandis (grand ®r),21 Pinus contorta (lodgepole
pine),21 and Pinus pinaster22 have been partially character-
ized. The gene encoding abietadiene synthase (AS) from
grand ®r has been cloned and the cDNA was functionally
expressed as a single polypeptide bearing a presumed
N-terminal plastidial targeting sequence.23,24 Cytochrome
P450 mixed function oxygenases and a dehydrogenase
which carry out the sequential oxidation of abietadiene to
abietadienol, abietadienal, and abietic acid have been
Keywords: abietane; terpenes and terpenoids; diterpenes; dienes; reduction;
zinc.
Corresponding author. Tel.: 1217-333-4280; fax: 1217-244-8024;
e-mail: r-coates@uiuc.edu
p
0040±4020/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(01)00605-6

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H.-J. Lee et al. / Tetrahedron 57 (2001) 6155±6167
Figure 1. Structures of the abietane-type resin acids and the related hydro-
carbons.
isolated from grand ®r and lodgepole pine.25 The regulation
of induced oleoresinosis in grand ®r has been studied by
monitoring changes in cyclase activity levels and steady-
state transcript abundances for monoterpene, sesquiterpene,
and diterpene synthases.4,26
The recent discovery that cyclization of (E,E,E)-geranylger-
anyl diphosphate catalyzed by native and recombinant AS
from grand ®r produces a mixture of abietadiene 1b and
three isomers presumed to be 2b±4b based on MS data,24
and the similarity of product ratios to those of 1a±4a in
fresh oleoresin,27a prompted the present investigation. Levo-
pimaradiene has recently been identi®ed as the cyclization
product of a diterpene synthase isolated from Gingko
Scheme 1.
the tosylates to be a general and ef®cient approach (Scheme
2), and this method provided dienes 1b±4b in high purity
and satisfactory yields.
Diazomethane esteri®cations (CH2N2, ether, 08C, 10 min) of
abietic and levopimaric acids and hydride reductions
(LiAlH4, ether, 08C, 1 h) of their esters provided abietadie-
nol 1d and levopimaradienol 2d in high yields (99%) and
purity. However, oxidations of 1d with PCC (1.5 equiv.,
CHCl2, rt, 6 h) or with (COCl)2/DMSO (Swern oxidation:
2788C, 15 min; Et3N) afforded abietadienal 515a in only fair
yields (44 and 53% at 70% purity, respectively) and accom-
panied by side products that were dif®cult to separate. A
plausible explanation for the low yields is competing oxida-
tion of the conjugated diene by PCC since allylic oxidation
1
biloba.27b In this paper we report the synthesis, H NMR,
13C NMR, and MS data and assignments, and chromato-
graphic behavior of these abietane diterpene precursors to
the resin acids.
2. Results and discussion
2.1. Conversion of resin acids to abietadiene isomers
of cycloalkenes by this reagent is well known.31b,33
similar PCC oxidation (1.0 equiv., CHCl2, rt, 1 h) of
levopimaradienol gave a 6:5:1 mixture (31%) of levopimar-
adienal/dehydroabietadienal/abietadienal (not shown) and
A
The conversion of the hindered carboxyl groups commonly
found in natural diterpenes to methyl groups has been
accomplished by Wolff±Kishner reductions of the corre-
sponding aldehydes,15,28,29 by catechol borane reduction of
the aldehyde tosylhydrazone,9 by Li/NH3 reduction of
phenylthiomethyl groups,30,31 and by Zn/NaI reductions of
tosylates and mesylates.29,32 However, application of these
or other methods to the resin acids 1a±4a might be jeopar-
dized by the sensitivity of the conjugated dienes to acids,
oxidative reagents, reducing conditions, and free radical
reactions. In the present work, three of these known deox-
ygenation methods were evaluated with abietic acid 1a
(Scheme 1), the most stable isomer, and the Li/NH3 and
Zn/NaI procedures were also applied to levopimaric acid
2a, known to be the least stable member of the abietane
resin acids.20 We have found the Zn/NaI reduction29,32 of
a
substantial recovered alcohol fraction of similar
Scheme 2.

H.-J. Lee et al. / Tetrahedron 57 (2001) 6155±6167
6157
(ca. 85% purity), and the presence of small amounts of
abietene contaminants from over-reduction could be seen
in the GC and H NMR spectra in the latter case. Li/NH3
1
reductions of levopimaradienyl sul®de afforded variable
mixtures of levopimaradiene, abietadiene, and three uniden-
ti®ed abietene isomers. In the best run the product was a
2.6:1:3.1 mixture (36%) of 2b/1b/abietenes. In another run
the less polar abietene isomers resulting from over-reduc-
tion were separated by chromatography as a 1:1.3:1.7
mixture and partially characterized by GC/MS and 1H
NMR spectra. Evidently base-catalyzed isomerization and
reduction of the cisoid diene were competitive with the
reductive cleavage of the sul®de under the various condi-
tions tried.
Reductive cleavages (Scheme 2) of the four tosylates 1e±4e
with Zn/NaI in HMPA at 1058C for 1±2 d according to the
procedure of Fujimoto and Tatsuno32 afforded all four diter-
pene hydrocarbons 1b±4b in satisfactory yields (74, 68, 48,
and 70%) and good purities (100, 88, ,95, and 100%)
according to GC and/or NMR analyses. Recently levopi-
maradiene (2b) was synthesized by reduction of 2e with
LiEt3BH in THF at re¯ux.27b Unsuccessful trials to effect
reduction of 1e with Zn/NaI in DMF, diglyme, and diglyme-
HMPA under similar conditions underscore the importance
of the HMPA solvent. The reaction of palustradienyl tosy-
late 3e with Zn/NaI HMPA (Scheme 3) under slightly
milder conditions (1008C, 1 d) gave the corresponding
iodide 8 (67%) which was converted to palustradiene 3b
(70%) at 1058C.
Scheme 3.
composition. Evidently double bond isomerization and
aromatization compete with the oxidation of the CH2OH
group. Although abietadienal tosylhydrazone 6 was readily
obtained (TsNHNH2, EtOH, rt, 1 d; 77%) and reduced ef®-
ciently by the catechol borane procedure34 (9 equiv., 4:1
CHCl3/THF, rt, 12 h; NaOAc, re¯ux, 1 h) to abietadiene
1b (81%), the low yields of the preceding oxidation step,
the dif®culty in puri®cation of abietadienal, and the evident
sensitivity of the cisoid diene to oxidation prompted the
investigation of other methods.
It therefore seems likely that these tosylate reductions
proceed by SN2 displacements forming the 18-iodo diter-
penes followed by electron-transfer from zinc. The lack of
deuterium incorporation into levopimaradiene 2b after a
reduction of 2e and addition of methanol-d4 implicates
a free radical intermediate which abstracts a hydrogen
atom from HMPA. (Eq. (1)) If an organozinc iodide is
formed, it must undergo thermal decomposition under
the high temperature conditions. The HMPA solvent
evidently facilitates these reductions through its dipolar
aprotic character which promotes the normally slow SN2
displacement by iodide anion, and by serving as an
ef®cient hydrogen atom donor for a free radical inter-
mediate.
Conversions of abietadienol to the tosylate 1e (5 equiv.
TsCl, pyridine, rt, 1 d) and mesylate 1f (1.5 equiv. MsCl,
Et3N, CH2Cl2, 08C, 30 min), and levopimaradienol 2d to its
tosylate 2e (5 equiv. TsCl, pyridine, rt, 1 d) all proceeded in
excellent yield (90±97%). However, the slow tosylation
reaction necessitated the use of excess tosyl chloride and
separation of the remaining reagent by either careful
chromatography or better by reaction with 3-(dimethyl-
amino)propylamine. The presence of small amounts
(5±10%) of tosyl chloride in chromatographically puri®ed
1
tosylates 2e and 4e was readily detected in the H NMR
spectra. In one run, incomplete separation of tosyl chloride
from palustradienyl tosylate 3e resulted in decomposition
during storage, presumably owing to hydrolysis and acid-
induced reactions. SN2 displacements of abietadienyl
mesylate 1f and levopimaradienyl tosylate 2e with
PhSNa (5 equiv., DMF, 1158C, 1 h) afforded 18-(phenyl-
thio)abietadiene 7 and 18-(phenylthio)-levopimaradiene
(structure not shown, 80% purity) in excellent yields
(96 and 90%) despite the steric hindrance at the neopentyl
positions.
ꢀ1†
Although the Li/NH3 reduction of hindered phenyl sul®des
has proven to be effective in carboxyl to methyl conversions
in the literature,30,31 the known capability of this reagent
to reduce conjugated dienes was reason for concern.
Reductions of the two phenylthio diterpenes with Li/NH3
(2±3 equiv. Li, ,1:1 NH3/THF, 2788C, ,5 min) were
carried out by adding the metal last and rapidly quenching
excess lithium with 3-hexyne and methanol. The yields of
abietadiene 1b from 7 varied from 59 (100% purity) to 86%
2.2. Characterization of abietadiene isomers
Synthesis of the diterpene hydrocarbons provided the oppor-
tunity to characterize the compounds fully by chemical,
chromatographic, MS, optical, and NMR data. A Diels±
Alder reaction of levopimaradiene with maleic anhydride
(toluene, 258C) (Eq. (2)) afforded the stable, crystalline
adduct 9 (66%), previously prepared from abietadiene at

6158
H.-J. Lee et al. / Tetrahedron 57 (2001) 6155±6167
Figure 2. Electron impact mass spectra for GC/MS analyses of abietadiene (1b, top), palustradiene (3b), levopimaradiene (2b), and neoabietadiene (4b,
bottom).
high temperature.15c
isomeric dienes (Fig. 2) are similar, they show distinctive
differences that may be useful for identi®cation by GC/MS.
The relative intensities of the m/z 272 (M1), 257 (M2CH3),
and 229 (M2C3H7) appear to be diagnostic for 1b
(272.229.257),
2b
(272.257.229),
and
3b
ꢀ2†
(257.272.229). The MS for 4b exhibits much less intense
peaks at m/z 272 (22) and 257 (4), no detectable peak at 229,
and a base peak at 135. The MS data for abietadiene
correspond reasonably well to the limited data in the
literature.15a However, MS intensity patterns attributed
to palustradiene16 match better with those of levopimar-
adiene, indicating that this pine resin constituent may be
mis-identi®ed.
HPLC analysis of a mixture of the four isomers on a silica
column showed only two peaks: one for 1b12b13b and
another for the slower eluting 4b. TLC analyses on AgNO3´-
silica gel (27±29%) showed signi®cant mobility differences
(Rf order 1b.4b.3b.2b) for the transoid and cisoid
dienes. TLC analysis of the resin acid esters on AgNO3±
alumina showed similar behavior except the order of 1c and
4c was reversed.35,36 Separation of abietadiene and palustra-
diene by argentation chromatography has been reported.12
Temperature-programmed capillary GC analyses of the
mixture on a fused silica column displayed well-separated
peaks in the Rt order, 3b,2b,1b,4b, which is similar to
the literature order for the tentatively identi®ed hydrocar-
bons11 with the exception of 2b/1b reversal (SE-30 packed
column). This GC retention order also corresponds to that
usually observed for the corresponding resin acid esters
3c#2c,1c,4c, on non-polar stationary phases (fused silica
and silicone gum rubber); however, the palustrate/levopi-
marate elution order is often opposite on polar
columns.27a,35,37
The optical rotations for abietadiene and levopimaradiene
are strongly negative ([a]20 ˆ2121 and 22658) whereas
D
those for palustradiene and neoabietadiene are positive
([a]20 ˆ159 and11458), and the signs and magnitudes
(^ ,D10%) match those of the esters (see Section 4).
The 245 and 2688 optical rotations reported for palus-
tradiene12,13 are inconsistent with our measurements.
The UV spectral data for the isomeric dienes are in
good accord with existing literature data for 1a11 and
1d12 and with values for the related esters and
alcohols.38
The 1H and 13C NMR spectral data for 1b±4b (Tables 1 and
2) were assigned by means of DEPT, COSY, HMQC,
HMBC, NOE data (Table 3 and Fig. 3), H±H coupling
pattern analysis, and comparisons with 13C NMR correla-
tions in the literature.14,39,40 The logic in deriving the assign-
ments is illustrated for palustradiene 3b in Section 4.2.20.
Although the EI mass spectral fragmentation patterns for the

H.-J. Lee et al. / Tetrahedron 57 (2001) 6155±6167
Table 1. H NMR (500 MHz, C6D6) Data and assignments for abietadiene (1b), levopimaradiene (2b), palustradiene (3b), and neoabietadiene (4b)
6159
1
Position
1b
2b
3b
4b
1a
1b
2a
2b
3a
3b
4
0.90 (td, 12.7; 3.8)
1.75 (dq, 12.9; 3.2)
1.37±1.42 (m)
1.53 (qt, 12.8; 3.7)
1.14 (td, 13.2; 3.4)
1.37±1.42 (m)
0.71 (td, 13.0; 3.8)
1.63 (dtd, 13.3; 3.2; 2.0)
1.37 (dquin, 12.5; 3.4)
1.46 (qt, 13.5; 3.3)
1.09 (td, 13.5; 4.0)
1.40±1.31 (m)
1.02 (td, 13.1; 3.7)
1.75 (dtd, 12.6; 3.5; 1.3)
1.44 (dquin, 13.5; 3.6)
1.59 (qt, 13.8; 3.4)
1.14 (td, 13.5; 4.1)
1.38 (dtd, 13.1; 3.3; 1.6)
±
0.93 (td, 13.0; 3.6)
1.62 (dtd, 12.9; 4.3; 2.5)
1.43±1.37 (m)
1.43±1.37 (m)
1.16 (td, 14.5; 4.6)
1.47 (dtd, 14.2; 3.2; 0.8)
±
±
±
5
1.23 (dd, 12.4; 4.9)
2.09 (dtd, 18.3; 5.0; 1.8)
1.94 (dd, 19.0; 12.3)
0.91 (dd, 9.2; 6.6)
1.60 (ddt, 12.8; 5.3; 2.5)
1.12±1.01 (m)
1.17 (dd, 12.4; 1.9)
1.65 (ddt, 12.3; 5.5; 3.0)
1.47±1.40 (m)
0.99 (dd, 12.3; 2.3)
1.55 (ddt, 12.8; 5.8; 3.0)
1.33 (qd, 12.9; 5.0)
2.19 (td, 13.6; 5.6)
6a
6b
7a
2.04 (td, 12.9; 4.9)
5.48 (dt, 5.1; 2.6)
2.12±2.08 (m)
7b
8
9
10
11a
2.41±2.29 (m)
±
1.94 (dd, 12.6; 7.8)
±
2.40 (ddd, 14.6; 4.9; 1.8)
±
1.81 (dd, 16.1; 7.1)
±
1.42 (qdd, 13.4; 3.2; 0.7)
±
1.81 (dquin, 13.2; 3.4)
±
1.27 (ddd, 14.1; 12.6; 8.1)
±
±
±
2.41±2.29 (m)
5.22 (m)
2.08±1.97 (m)
2.08±1.97 (m)
11b
12a
1.70 (dq, 11.9; 3.6)
1.99 (m)
1.75 (dt, 12.8; 4.2)
1.96 (br t, 13.7)
12b
13
14
15
16
17
18
19
20
2.61 (dt, 13.9; 4.3)
±
6.43 (s)
±
1.78 (s)
1.72 (s)
0.88 (s)
0.84 (s)
0.80 (s)
±
5.96 (s)
2.20 (septet, 6.8)
1.02 (d, 7.0)a
1.03 (d, 6.8)b
0.85 (s)
±
5.66 (s)
±
5.56 (s)
2.27 (septet, 6.8)
1.04 (d, 6.9)
1.04 (d, 6.9)
0.89 (s)
2.21 (septet, 6.8)
1.06 (d, 7.0)
1.06 (d, 7.0)
0.83 (3H, s)
0.80 (3H, s)
0.93 (3H, s)
0.89 (s)
0.84 (s)
0.86 (s)
1.06 (s)
Chemical shifts (multiplicity, observed coupling(s) in Herz). Assignments based on COSY, HMBC, HMQC, and NOE (see text).
Pro-S.
Pro-R.
a
b
The 13C NMR assignments for the A and B ring carbons of
3b agree with those reported for the related diterpenes,
pimara-8,15-diene39 and podocarp-8-ene,40 except for
reversal of C8 and C9 in the former case.
The positional assignments for abietadiene, levopimara-
diene, and neoabietadiene were deduced by similar means
with appropriate consistency tests and con®rmations. The
better separation of the 13C NMR signals for the ring A
quaternary methyls avoided the overlap of HMQC and
HMBC crosspeaks that occurred in the 2D plots for palus-
tradiene. Consequently more de®nite assignments of the
corresponding 1H NMR peaks for these methyl groups
could be made, despite their proximity in the proton spectra.
The identi®cation of the C16 (pro-S) and C17 (pro-R) meth-
yl doublets from the isopropyl group of abietadiene with dH
1.02 and 1.03 was established previously by stereo-
controlled deuterium labelling.41 The vinyl methyl peaks
Table 2. 13C NMR (125 MHz, C6D6) Data and assignments for abietadiene
(1b), levopimaradiene (2b), palustradiene (3b), and neoabietadiene (4b)
Position
1b
2b
3b
4b
1
2
3
4
5
6
7
8
39.5 (CH2)
19.2 (CH2)
42.7 (CH2)
33.1 (C)
50.5 (CH)
24.4 (CH2)
121.7 (CH)
135.7 (C)
51.3 (CH)
35.2 (C)
23.0 (CH2)
27.7 (CH2)
144.2 (C)
123.7 (CH)
35.3 (CH)
21.7 (CH3)
22.1 (CH3)
33.5 (CH3)
21.1 (CH3)
13.9 (CH3)
38.0 (CH2)
19.3 (CH2)
42.4 (CH2)
33.6 (C)b
55.3 (CH)
24.1 (CH2)
36.5 (CH2)
139.2 (C)d
49.9 (CH)
41.0 (C)
23.1 (CH2)
115.2 (CH)
139.4 (C)d
119.3 (CH)
33.8 (CH)
21.7 (CH3)f
21.6 (CH3)f
33.6 (CH3)b
21.9 (CH3)
14.3 (CH3)
36.1 (CH2)
19.28 (CH2)a
42.1 (CH2)
33.37 (C)c
51.9 (CH)
19.30 (CH2)a
31.0 (CH2)
138.2 (C)
125.4 (C)
37.9 (C)
23.0 (CH2)
26.6 (CH2)
142.5 (C)
121.3 (CH)
34.7 (CH)
21.2 (CH3)g
20.8 (CH3)g
33.39 (CH3)c
21.7 (CH3)g
21.5 (CH3)g
39.7 (CH2)
19.3 (CH2)
42.4 (CH2)
33.4 (C)
54.7 (CH)
22.9 (CH2)
36.4 (CH2)
139.0 (C)
51.7 (CH)
38.4 (C)
22.9 (CH2)
26.4 (CH2)
129.0 (C)e
122.6 (CH)
122.8 (C)e
19.8 (CH3)
20.4 (CH3)
33.8 (CH3)
22.2 (CH3)
15.2 (CH3)
1
(dH 1.72 and 1.78) in the H NMR spectrum of neoabieta-
diene were readily associated with C16 (Z) and C17 (E),
respectively, from the NOEs shown in Fig. 3. The 13C
NMR data and assignments shown for abietadiene agree
with those in the literature14 except for reversal of the
close-lying signals for C4 and C10, and they correlate
well with assignments for isopimaradiene.39 The 13C NMR
assignments for the A and B ring carbons of levopimara-
diene and neoabietadiene are consistent with those in the
literature for pimara-8(14),15-diene,13 all of which have an
8(14) double bond.
9
10
11
12
13
14
15
16
17
18
19
20
2.3. Enzymatic synthesis of 1b±4b mixture
Chemical shifts (hydrogen substitution). Assignments based on DEPT,
HMBC, and HMQC (see text).
These assignments may be reversed (see Section 4.2.20 for explanation).
Incubation of (E,E,E)-geranylgeranyl diphosphate with
recombinant abietadiene synthase from grand ®r afforded
a mixture of all four abietadiene isomers (1b±4b) in a
a,c±g
b
Superimposed signals (greater intensity relative to other signals).

6160
H.-J. Lee et al. / Tetrahedron 57 (2001) 6155±6167
Table 3. NOEs observed upon irradiation of quaternary methyl groups in abietadiene (1b), levopimaradiene (2b), and palustradiene (3b)
Compound no.
Methyl position
Signals irradiated dH (ppm)
NOE observed (%)
1b
18
19
20
18
19
20
18
19
20
0.85
0.89b
0.84b
0.83
0.80
0.93
0.89b
0.86b
1.06
NDa
H18 (2.4), H20 (3.3)
H19 (2.4)
2b
3b
NDa
H20 (9.0)
H19 (4.8)
H5a (13.3), H6a (6.8)
H20 (11.8)
H1b (3.2), H2b (2.1), H6b (4.9),
H11b (3.7), H19 (7.8)
a
Not determined.
Irradiation caused some excitation of the proximal methyl and some uncertainty in assigning the NOE origin.
b
33:42:9:16 ratio according to GC analysis, in agreement
with a recent report by Peters et al.24 The identity of the
individual components was established by comparison of
their GC retention times and their mass fragmentation
patterns in GC±MS recordings with those of mixture
of synthetic samples of 1b±4b. This ®nding con®rms
that abietadiene diterpene synthase is a multi-product
cyclase which gives rise to mixture of abietane diterpenes,
which in vivo is further oxidized to the corresponding resin
acid mixture.
4. Experimental
4.1. General
The following solvents and reference values (ppm) were
used for NMR spectroscopy: CDCl3 (1H: 7.27, 13C: 77.0),
C6D6 (1H: 7.15, 13C: 128.0). Coupling constants are given in
Hz. Mass spectra (MS) were obtained on a 70-VSE or
TofSpec mass spectrometer. Electron impact (EI) MS ion
analysis was performed with the attached HP 5970 quadra-
pole MS and related data station (Fig. 2). GC±MS spectra
were obtained on a Quattro instrument. HPLC was
conducted with a Waters Model M-6000A delivery system
using a Schoeffel SF 770 variable wavelength UV detector
at 250 nm and an analytical silica column (0.46£25 cm)
with a ¯ow rate of 1 mL min21. GC analyses were carried
out with a Shimadzu Model 14A-GC on Rtx-5 30 m fused
silica capillary column (5% diphenyl- and 95% dimethyl-
polysiloxane; ID 0.32 mm, 0.25 mm ®lm thickness) with a
split ratio of 100:1. AgNO3-impregnated silica gel (27±29%
w/w) TLC plates were freshly prepared by immersing silica
gel TLC plates (Merck, 0.25 mm 60 F-254 silica gel) in a
solution of 10% AgNO3 in acetonitrile and drying for 3 h at
rt.31b After development AgNO3±SiO2 TLCs were visua-
lized by spraying with KMnO4 (5% KMnO4 and 10%
NaHCO3). HMPA was distilled from CaH2 and stored
3. Conclusion
Abietadiene, levopimaradiene, palustradiene, and neoabie-
tadiene (1b±4b) are now accessible by partial synthesis
from the corresponding resin acids (1a±4a) in four steps
(40±68% overall yield). The key step is the Zn/NaI reduc-
tion32 of the C18 tosylates. These diterpene hydrocarbons
are likely intermediates in resin acid biosynthesis, and the
availability of reference samples and extensive charac-
terization data will facilitate their identi®cation as
cyclase products, oxidase substrates, and oleoresin
constituents. They may also be useful as enantiomeri-
cally pure starting materials for natural product synth-
eses and other purposes.
Ê
over 4 A sieves. Li metal surface was cleaned by rinsing
in hexane, submerging in EtOH, and rinsing in fresh hexane
before use. NaH (60% dispersion in mineral oil) was rinsed
under N2 with hexane three times before use. Anhydrous
NH3 was vapor transferred directly from the compressed
gas cylinder. CDCl3 was passed through a pipette ®lled
with basic alumina immediately before use. Flash
column chromatography was performed on 230±400
42
Ê
mesh silica gel (Merck 60 A, grade 9385). Solvents
(EAˆethyl acetate) were removed by rotary evapora-
tion. The purity of all products was judged to be
.95% by H NMR analysis unless speci®ed otherwise.
All products were characterized by UV, IR, H NMR and
1
1
13C NMR spectra. If satisfactory spectral data for known
compounds are available in the literature, only optical rota-
tions and mp are reported. (E,E,E)-Geranylgeraniol,
prepared from (E,E)-geranyllinalool (Takasago Fine
Chemicals, Japan) by PCC oxidation43 and NaBH4
reduction (MeOH, 08C), was converted to (E,E,E)-geranyl-
geranyl pyrophosphate (GGPP) by procedures similar to
those in the literature for geranyl pyrophosphate.44
Figure 3. NOE intensities (500 MHz, C6D6) observed from the indicated
irradiations of abietadiene (1b), levopimaradiene (2b), palustradiene (3b),
and neoabietadiene (4b).

H.-J. Lee et al. / Tetrahedron 57 (2001) 6155±6167
6161
Truncated recombinant abietadiene synthase was kindly
provided by Dr Reuben Peters.24
(B) By pyridinium chlorochromate (PCC) oxidation. Oxida-
tion15a of 1d (68 mg, 0.24 mmol) with a suspension of PCC
(77 mg, 0.36 mmol) in CH2Cl2 (2 mL) with TLC monitoring
was carried out for 6 h at 258C under N2. Isolation of the
product by ether extraction and puri®cation by ¯ash column
chromatography (20% diethyl ether/pentane) afforded
known15a aldehyde 5 (30.4 mg, 44%) as a colorless oil:
UV (MeOH) lmax (log 1) 234 (4.28), 242 (4.31), 250
(4.14) nm; IR (neat) nmax 2930, 2838, 2691 (CHvO),
1724 (CvO), 1626 (CvC), 1458, 1382, 1234, 1032,
4.2. Resin acid sources and properties
Abietic acid (Aldrich) was puri®ed according to a literature
procedure:6 mp 170±1718C [lit.38b mp 172±1748C],
[a]20 ˆ21018 (c 1.60, EtOH) [lit.38b [a]20 ˆ21068,
D
D
EtOH]. Levopimaric, palustric, and neoabietic acids are
commercially available from Helix BioTech. Levopimaric
acid was generously provided from USDA collection at
Forest Products Laboratory, Madison, WI, with the help
of Dr Duane Zinkel. These air- and acid-sensitive materials
were stored as solids at 2208C under nitrogen.
1
899 cm21; H NMR (500 MHz, CDCl3) d 9.20 (1H, s),
5.76 (1H, s), 5.33 (1H, m), 2.21 (1H, septet, Jˆ6.9 Hz),
2.10±1.89 (5H, m), 1.81 (1H, dq, Jˆ12.0, 3.2 Hz), 1.76
(1H, dd, Jˆ12.2, 4.5 Hz), 1.66±1.59 (3H, m), 1.47±1.40
(1H, m), 1.30 (1H, d, Jˆ13.1 Hz), 1.25±1.16 (1H, m),
1.12 (3 H, s), 1.11±1.05 (1H, m), 1.00 (3H, d, Jˆ6.9 Hz),
0.99 (3H, d, Jˆ6.9 Hz), 0.83 (3H, s); 13C NMR (125 MHz,
CDCl3) d 206.3, 145.6, 135.7, 122.3, 120.1, 50.5, 49.0, 42.4,
38.3, 34.8, 33.8, 32.9, 27.4, 25.4, 22.5, 21.4, 20.8, 17.2,
14.3, 14.0; EIMS m/z 286.2 [M]1 (41.5), 271.2 (9.5),
255.2 (23.0), 243.2 (15.9), 225.1 (3.6), 215.2 (7.9), 199.1
(5.9), 187.1 (100.0), 171.1 (5.8), 159.1 (9.4), 145.1 (23.2),
131.1 (59.1), 105.1 (24.6), 93.0 (17.5), 83.9 (23.8), 69.1
(20.6); HREIMS m/z 286.2300 (calcd for C20H30O,
4.2.1. Methyl abietadien-18-oate (methyl abietate, 1c). A
solution of abietic acid (2.00 g, 6.62 mmol) in ether (10 mL)
was stirred and cooled at 08C under N2 as excess diazo-
methane in ether (40 mL) generated from N-methyl-N-nitro-
sotoluenesulfonamide according to the Aldrich Bulletin and
kit 210,025-O was added. After 10 min, solvent and
unreacted diazomethane were removed by rotary evapora-
tion. Puri®cation of the residue by ¯ash column chromato-
graphy on silica gel (20% diethyl ether/pentane) afforded
methyl ester 1c (2.07 g, 99%) as a colorless oil:
[a]20 ˆ2968 (c 0.54, CHCl3) [Lit.38a,b [a]20 ˆ2968
1
286.2297). The H NMR (60 MHz) and EIMS data agree
with those reported for impure aldehyde 5.15a
D
D
1
(95% EtOH)]; H NMR (400 MHz, CDCl3) d 5.76 (1H,
s), 5.35 (1H, m), 3.61 (3H, s), 2.21 (1H, septet,
Jˆ6.6 Hz), 2.10±2.00 (4H, m), 1.93 (1H, d, Jˆ12.9 Hz),
1.87 (1H, d, Jˆ12.9 Hz), 1.82±1.68 (3H, m), 1.64±1.52
(3H, m), 1.24 (3H, s), 1.22±1.10 (2H, m), 1.00 (3H, d,
Jˆ6.8 Hz), 0.98 (3H, d, Jˆ6.8 Hz), 0.81 (3H, s). UV and
13C NMR (50 MHz) data are available in the literature.14,38
4.2.4. Abietadien-18-al p-toluenesulfonylhydrazone (6).
A solution of aldehyde 5 (60 mg, 0.21 mmol) and
TsNHNH2 (39 mg, 0.21 mmol) in absolute ethanol (3 mL)
was stirred for 1 d at 258C under N2.48 Evaporation of
solvent and puri®cation by ¯ash column chromatography
(20% EA/Hexane) afforded the tosylhydrazone
6
(67.3 mg, 71%) as a white solid. Recrystallization from pen-
tane/ether gave the analytical sample: mp 84±858C; UV
(MeOH) lmax (log 1) 234 (4.43), 244 (4.41), 250
(4.20) nm; IR (thin ®lm) nmax 2915, 2868, 1737, 1641,
4.2.2. Abietadien-18-ol (1d). A suspension of LiAlH4
(46.8 mg, 1.23 mmol) in ether (4 mL) under N2 was stirred
and cooled at 08C as methyl ester 1c (130 mg, 0.41 mmol) in
ether (1 mL) was added. After 1 h at 08C, water (47 mL),
aqueous 15% NaOH (47 mL), and water (141 mL) were
added.45 The white salts were ®ltered and washed thor-
oughly with ether (20 mL). Concentration of the ®ltrate
and puri®cation by ¯ash column chromatography on silica
gel (20% diethyl ether/pentane) afforded alcohol 1d
(117 mg, 99%) as a white solid. Recrystallization from pen-
tane/ether gave the analytical sample: mp 88±898C [Lit.15a
1
1598, 1468, 1383, 1166, 619 cm21; H NMR (500 MHz,
CDCl3) d 7.77 (2H, d, Jˆ8.2 Hz), 7.29 (2H, d, Jˆ8.6 Hz),
7.15 (1H, br s), 6.79 (1H, s), 5.72 (1H, s), 5.15 (1H, m), 2.43
(3H, s), 2.20 (1H, septet, Jˆ6.8 Hz), 2.10±2.01 (2H, m),
1.84 (1H, d, Jˆ13.0 Hz), 1.81±1.73 (3H, m), 1.59±1.48
(5H, m), 1.37 (1H, d, Jˆ14.3 Hz), 1.33±1.25 (2H, m),
1.23±1.12 (2H, m), 1.08 (3H, s), 1.00 (3H, d, Jˆ6.8 Hz),
0.99 (3H, d, Jˆ6.8 Hz), 0.87±0.81 (2H, m), 0.78 (3H, s);
13C NMR (125 MHz, CDCl3) d 161.7, 145.3, 143.9, 135.4,
134.9, 129.4, 128.0, 122.3, 120.5, 50.8, 45.9, 41.1, 38.3,
37.3, 34.8, 34.3, 27.3, 24.4, 22.4, 21.5, 21.3, 20.8, 17.7,
16.8, 14.1, 14.0; EIMS m/z 454.3 [M]1 (3.1), 299.3 (33.6),
284.2 (12.4), 270.2 (84.1), 255.2 (55.1), 241.2 (14.0), 227.2
(44.5), 199.1 (36.7), 185.1 (26.8), 171.1 (14.4), 156.0 (19.4),
135.1 (36.3), 105.1 (45.7), 91.1 (100.0), 65.0 (37.3);
HREIMS m/z 454.2662 (calcd for C27H38N2O2S, 454.2654).
mp 80±848C; Lit.46 mp 85.5±878C]; [a]20 ˆ21498 (c 0.54,
D
CHCl3); UV (hexane) lmax (log 1) 234 (4.32), 242 (4.34),
1
250 (4.17) nm. IR, H NMR (60 MHz), 13C NMR (15 and
50 MHz), and EIMS data are available in the literature.14,15a
4.2.3. Abietadien-18-al (5). (A) By Swern oxidation. Swern
oxidation of abietadienol 1d (116 mg, 0.4 mmol) in CH2Cl2
(1 mL) was carried out with DMSO (88 mL, 1.24 mmol)
and oxalyl chloride (52 mL, 0.6 mmol) in CH2Cl2 (5 mL)
under N2 for 15 min at 2788C after which Et3N (0.2 mL,
1.5 mmol) was added.47 Isolation of the product by extrac-
tion and puri®cation by ¯ash column chromatography (20%
EA/hexane) afforded aldehyde 5 (60 mg, 53% yield, 70%
4.2.5. Abietadien-18-yl p-toluenesulfonate (1e). A solu-
tion of alcohol 1d (90 mg, 0.31 mmol) and tosyl chloride
(296 mg, 1.56 mmol) in pyridine (0.5 mL) was stirred for
49
1 d at 258C under N2 with monitoring by TLC. CH2Cl2
(30 mL) was added and the resulting solution was washed
with 5% aqueous NaHCO3 (2£20 mL) and satd. NaCl
(2£20 mL). Concentration and puri®cation by ¯ash column
chromatography on silica gel (20% diethyl ether/pentane)
affordedthe known15a tosylate1e (129 mg, 94%)asa colorless
1
purity based upon H NMR analysis) and a more polar
unidenti®ed byproduct (24.9 mg, 22%) as colorless oils.
1
Characteristic H NMR (500 MHz, CDCl3) data for the
byproduct are 9.20 (1H, s), 5.83 (1H, s), 5.53 (1H, m).

6162
H.-J. Lee et al. / Tetrahedron 57 (2001) 6155±6167
oil: [a]20 ˆ2618 (c 0.46, CHCl3); UV (MeOH) lmax
135.7, 129.4, 129.0, 125.8, 122.7, 121.0, 51.1, 49.2, 47.2,
39.0, 38.2, 37.8, 35.2, 35.1, 27.7, 24.0, 22.9, 21.7, 21.1,
21.0, 18.7, 14.3. The aqueous solution should be kept
basic during the extractive product isolations. The genera-
tion of several side products during isolation of abietadienyl
sul®de 7 is attributed to the presence of thiophenol (or other
thiol byproducts) and the likelihood of radical reactions of
the transoid diene.
D
(log 1) 228 (4.20), 242 (3.87), 250 (3.83) nm; IR (neat)
nmax 2929, 1599, 1464, 1358, 1187, 1099, 965, 844, 812,
1
666 cm21; H NMR (500 MHz, CDCl3) d 7.74 (2H, d,
Jˆ8.3 Hz), 7.31 (2H, d, Jˆ8.4 Hz), 5.74 (1H, s), 5.25
(1H, dt, Jˆ5.1, 2.4 Hz), 3.65 (1H, d, Jˆ9.4 Hz), 3.52 (1H,
d, Jˆ9.4 Hz), 2.44 (3H, s), 2.21 (1H, septet, Jˆ6.8 Hz),
2.22±2.07 (2H, m), 1.87±1.68 (5H, m), 1.55±1.43 (3H,
m), 1.38±1.26 (2H, m), 1.21±1.11 (1H, m), 1.00 (3H, d,
Jˆ6.8 Hz), 0.99 (3H, d, Jˆ6.8 Hz), 0.86 (3H, s), 0.86±
0.81 (1H, m); 13C NMR (125 MHz, CDCl3) d 145.3,
144.5, 136.4, 132.9, 129.8, 127.9, 122.3, 120.2, 78.0, 50.5,
43.5, 38.4, 36.8, 35.7, 34.8, 34.6, 27.4, 23.6, 22.5, 21.6,
21.4, 20.8, 17.8, 17.4, 14.1; EIMS m/z 442.3 [M]1 (21),
270.3 (95), 255.2 (44), 227.2 (42), 199.2 (18), 185.2 (28),
155.0 (33), 136.1 (21), 105.1 (32), 91.1 (100), 71.1 (19);
HREIMS m/z 442.2538 (calcd for C27H38O3S, 442.2542).
4.2.8. Abieta-7,13-diene (1b). (A) By Catechol borane
reduction.
A solution of tosylhydrazone 6 (34 mg,
0.08 mmol) in CHCl3 (3 mL) under N2 was stirred at 258C
as catechol borane in THF (0.75 mL, 1 M, 0.75 mmol) was
added.31b,34 After 12 h at 258C anhyd. NaOAc (204 mg,
1.5 mmol) was added. The resulting suspension was stirred
at re¯ux for 1 h and cooled to rt. Water (10 mL) was added,
the product was extracted with ether (3£10 mL), and the
combined extracts were concentrated. Puri®cation by ¯ash
column chromatography (100% pentane) afforded abieta-
diene 1b (16.6 mg, 81% yield, 93.4% purity by GC) as a
colorless oil.
1
The H NMR and EIMS data agree with the less complete
60 MHz 1H NMR and MS data listings in the literature.15a
4.2.6. Abietadien-18-yl methanesulfonate (1f). A solution
of alcohol 1d (1.30 g, 4.5 mmol) and triethylamine
(1.88 mL, 13.6 mmol) in CH2Cl2 (20 mL) under N2 was
stirred and cooled at 08C as CH3SO2Cl (0.52 mL,
6.8 mmol) was added.31b After 30 min at 08C, the solution
was diluted with CH2Cl2 (20 mL), washed with 10% NH4Cl
(3£50 mL), and concentrated. Puri®cation by ¯ash column
chromatography (25% EA/hexane) afforded the known15a
mesylate 1f (1.60 g, 97%) as a colorless oil: IR (neat) nmax
1356, 1176 cm21; 1H NMR (500 MHz, CDCl3) d 5.75 (1H,
s), 5.38 (1H, q, Jˆ3.5 Hz), 3.90 (1H, d, Jˆ9.3 Hz), 3.74
(1H, d, Jˆ9.3 Hz), 2.96 (3H, s), 2.20 (1H, septet,
Jˆ6.8 Hz), 2.1±2.0 (4H, m), 1.9±1.8 (2H, m), 1.78 (1H,
dq, Jˆ12.4, 3.7 Hz), 1.6±1.5 (3H, m), 1.5±1.4 (1H, m),
1.43 (1H, d, Jˆ3.8 Hz), 1.18 (1H, tdd, Jˆ12.4, 10.4,
6.6 Hz), 1.1±0.95 (1H, m), 0.99 (3H, d, Jˆ6.8 Hz), 0.98
(3H, d, Jˆ6.8 Hz), 0.96 (3H, s), 0.81 (3H, s); 13C NMR
(125 MHz, CDCl3) d 145.8, 135.8, 122.5, 120.5, 77.8,
50.9, 44.0, 38.7, 37.4, 37.1, 36.1, 35.1, 35.0, 27.7, 24.1,
(B) By Li/NH3 reduction. NH3 gas was condensed into a
stirred solution of sul®de 7 (30 mg, 0.08 mmol) in THF
(1 mL) at 2788C until the cloud point was reached (ca.
1:1 NH3/THF).31b A single piece of hexane-washed lithium
(2.9 mg, 0.40 mmol) was added, and the reaction mixture
was stirred and cooled at 2788C for 5 min. The dark blue
color developed was discharged by adding 3-hexyne
(0.5 mL) and methanol (0.5 mL). After evaporation of
excess NH3, the remaining THF solution (ca. 1 mL) was
diluted with 5% aqueous NaOH (20 mL), and the product
was extracted with hexane (3£20 mL). The combined
extracts were concentrated. Puri®cation by ¯ash column
chromatography (100% pentane) afforded abietadiene 1b
(13.2 mg, 59% yield, 99.9% purity by GC) as a colorless
oil. When too much NH3 was used causing precipitation of
starting material, reductions were incomplete. Longer reac-
tion times or inadequate mixing led to increased amounts of
the abietene over-reduction byproducts.
1
22.8, 21.6, 21.1, 18.1, 17.6, 14.4. The H NMR and EIMS
data agree with the less complete 60 MHz 1H NMR and MS
values given in the literature for impure 1f.15a
(C) By Zn/NaI reduction. A solution of tosylate 1e (23.7 mg,
0.053 mmol) in HMPA (1 mL) was purged with N2 and NaI
(40 mg, 0.27 mmol) and zinc dust (35 mg, 0.53 mmol) were
added.29,32 The suspension was stirred and heated at 1058C
for 1 d, cooled to rt, and ®ltered. Water (10 mL) was added,
the product was extracted with pentane (3£10 mL), and the
combined extracts were concentrated. Puri®cation by ¯ash
column chromatography (100% pentane) afforded abieta-
diene 1b (10.6 mg, 74% yield, 99.9% purity by GC) as a
4.2.7. 18-(Phenylthio)abietadiene (7). A suspension of oil
free NaH (524 mg, 21 mmol) in DMF (4 mL) under N2 was
stirred at rt as thiophenol (2.32 mL, 22 mmol) was
added.31b,50 After 1 h at 258C, mesylate 1f (1.60 g,
4.3 mmol) was added to the homogenous solution of
NaSPh.50 The solution was heated at 1158C for 1 h, cooled
to rt, and diluted with 5% NaOH (150 mL). The product was
extracted with hexane (3£75 mL). Solvent evaporation and
puri®cation by ¯ash column chromatography on silica gel
(5% EA/hexane) afforded sul®de 7 (1.58 g, 96%) as a color-
less oil: IR (neat) nmax 2956, 2923, 2866, 1583, 1479, 1439,
1382, 1089, 1025, 885, 736, 689 cm21; 1H NMR (500 MHz,
CDCl3) d 7.5±7.1 (5H, m), 5.79 (1H, s), 5.39 (1H, dt, Jˆ5.1,
2.6 Hz), 2.95 (1H, ABd, Jˆ12.1 Hz), 2.85 (1H, ABd,
Jˆ12.1 Hz), 2.23 (1H, septet, Jˆ6.8 Hz), 2.2±1.9 (5H,
m), 1.85 (1H, d, Jˆ12.8 Hz), 1.81 (1H, dq, Jˆ12.3,
3.5 Hz), 1.62 (1H, dd, Jˆ11.5, 4.9 Hz), 1.6±1.5 (4H, m),
1.21 (1H, tdd, Jˆ12.6, 9.7, 7.2 Hz), 1.1±1.0 (1H, m), 1.07
(3H, s), 1.02 (3H, d, Jˆ6.8 Hz), 1.01 (3H, d, Jˆ6.8 Hz),
0.83 (3H, s); 13C NMR (125 MHz, CDCl3) d 145.5, 138.6,
colorless oil: Rf (100% hexane) 0.78; [a]20 ˆ21218 (c
D
0.80, CHCl3) [Lit.51 [a]20 ˆ21378 (CHCl3); Lit.11b
D
2166.08 (c 0.4); Lit.13 2788 (c 4.0); Lit.10b 2758 (c 2.9,
CHCl3); Lit.10a 279.68]; UV (hexane) lmax (log 1) 234
(4.36), 242 (4.35), 250 (4.22) nm [Lit.11b lmax (log 1)
(EtOH) 233 (4.34), 241 (4.36), 249 (in¯.) nm]; IR (neat)
nmax 2925, 1625 (CvC), 1462, 1380, 1363, 884 cm21
;
EIMS m/z 272.2 [M]1 (100.0), 257.2 (48.6), 244.0 (3.3),
229.2 (74.8), 215.2 (2.6), 201.1 (11.0), 187.1 (22.9), 159.1
(15.3), 148.1 (56.7), 133.1 (5.7), 105.1 (82.6), 83.9 (62.6),
69.1 (40.4). See Tables 1 and 2 for NMR data.11b,14
Levopimaric, palustric, and neoabietic acids were converted

H.-J. Lee et al. / Tetrahedron 57 (2001) 6155±6167
6163
to the respective methyl esters, alcohols, tosylates, and
hydrocarbons by procedures similar to those described
above for abietic acid. In the case of identical procedures,
only yields, purity, and characterization data for the
products are provided below. Abbreviated or complete
procedures are provided if signi®cant changes were
made.
(1H, m), 2.27 (1H, d, Jˆ4.4 Hz), 2.20 (1H, dd, Jˆ14.3,
3.3 Hz), 2.12 (1H, septet, Jˆ6.8 Hz), 2.02 (2H, dd,
Jˆ12.7, 7.2 Hz), 1.96 (1H, m), 1.70 (1H, d, Jˆ3.0 Hz),
1.48 (1H, m), 1.41±1.23 (6H, m), 0.95 (6H, d, Jˆ6.8 Hz),
0.84 (3H, s), 0.75 (3H, s); 13C NMR (125 MHz, CDCl3) d
144.6, 138.8, 138.4, 132.9, 129.8, 127.9, 119.0, 114.8, 77.8,
49.3, 47.9, 40.5, 37.2, 36.9, 35.5, 35.4, 33.2, 23.4, 22.6,
21.6, 21.4, 21.3, 17.9, 17.2, 14.4; EIMS m/z 442.3 [M]1
(14), 427.3 (6), 270.3 (75), 255.3 (84), 241.2 (28), 227.2
(36), 213.2 (8), 199.2 (19), 172.1 (37), 149.2 (18), 135.1
(40), 107.1 (42), 91.1 (100), 77.1 (27); HREIMS m/z
442.2538 (calcd for C27H38O3S, 442.2542). Tosylation of
2d (439.3 mg, 1.52 mmol) with tosyl chloride (1.44 g,
7.6 mmol) in pyridine (2.5 mL) as described above without
adding 3-(dimethylamino)propylamine and with chromato-
graphic puri®cation afforded tosylate 2e (605 mg, 90%).
4.2.9. Methyl levopimaradien-18-oate (methyl Levopi-
marate, methyl abieta-12(13),8(14)-diene-18-oate 2c).
Yield, 2.07 g (99%); mp 628C [Lit.9 mp 60±628C];
[a]20 ˆ22388 (c 0.51, CHCl3) [Lit.38a,b [a]20 ˆ22698
D
D
(95% EtOH)]; UV (hexane) lmax (log 1) 270 (3.97) nm
[Lit.38a lmax (log 1) 272.8 (3.76); Lit.38b 272 (3.76) nm];
13C NMR (125 MHz, CDCl3) d 179.0, 145.3, 135.5,
122.3, 120.6, 51.8, 50.9, 46.6, 45.1, 38.3, 37.1, 34.9, 34.5,
1
1
27.4, 25.7, 22.4, 21.4, 20.8, 18.1, 17.0, 14.0. H NMR
(200 MHz) and HRMS data agree with the literature
values.9
The H NMR spectrum showed the presence of 5±10% of
residual tosyl chloride.
4.2.12. 18-(Phenylthio)levopimaradiene. Yield, 207 mg
(90%); purity, 80% (GC); IR (neat) nmax 3010, 2917,
2847, 1670, 1583, 1479, 1438, 1382, 1090, 1024, 736,
4.2.10. Levopimaradien-18-ol (2d). Yield, 542 mg
(100%); [a]20 ˆ21848 (c 0.47, CHCl3); UV (hexane)
D
1
lmax 268 nm; IR (neat) nmax 3380 (br OH), 2923, 1636
(CvC), 1489, 1403, 1039, 889 cm21; 1H NMR (500 MHz,
CDCl3) d 5.77 (1H, s), 5.39 (1H, m), 3.35 (1H, d,
Jˆ10.8 Hz), 3.13 (1H, d, Jˆ11.0 Hz), 2.21 (1H, septet,
Jˆ6.8 Hz), 2.08±1.95 (3H, m), 1.86 (2H, t, Jˆ13.2 Hz),
1.80 (1H, dq, Jˆ1.21, 3.5 Hz), 1.59 (1H, dt, Jˆ13.2,
3.7 Hz), 1.56±1.49 (3H, m), 1.45 (1H, broad s), 1.37 (1H,
dd, Jˆ12.7, 3.7 Hz), 1.36±1.28 (1H, m), 1.23±1.10 (1H, m),
1.00 (3H, d, Jˆ6.8 Hz), 0.99 (3H, d, Jˆ6.8 Hz), 0.87 (3H,
s), 0.87±0.80 (1H, m), 0.81 (3H, s); 13C NMR (125 MHz,
CDCl3) d 145.3, 135.5, 122.4, 120.9, 72.2, 50.8, 43.7, 38.9,
37.5, 35.7, 34.9, 34.6, 27.5, 23.8, 22.7, 21.4, 20.8, 18.2,
17.7, 14.2; EIMS m/z 288.3 [M]1 (74), 271.2 (26), 253.2
(32), 241.2 (9), 225.2 (2), 213.2 (10), 199.2 (13), 185.2 (23),
173.2 (28), 159.1 (25), 148.1 (39), 133.1 (99), 105.1 (56),
91.1 (100); HREIMS m/z 288.2463 (calcd for C20H32O,
288.2453).
690 cm21; H NMR (500 MHz, CDCl3) d 7.40 (2H, dd,
Jˆ8.6, 1.3 Hz), 7.30 (2H, tt, Jˆ7.7, 1.1 Hz), 7.19 (1H, tt,
Jˆ7.3, 1.5 Hz), 5.56 (1H, q, Jˆ1.7 Hz), 5.20 (1H, t,
Jˆ3.6 Hz), 3.08 (1H, ABd, Jˆ12.2 Hz), 2.80 (1H, ABd,
Jˆ12.2 Hz), 2.39 (1H, dd, Jˆ9.0, 3.6 Hz), 2.36 (1H, t,
Jˆ4.9 Hz), 2.30 (1H, ddd, Jˆ13.5, 4.3, 2.4 Hz), 2.20 (1H,
septet d, Jˆ6.9, 0.9 Hz), 2.12 (1H, dd, Jˆ12.2, 7.3 Hz), 2.03
(1H, td, Jˆ12.9, 5.1 Hz), 1.79 (1H, dt, Jˆ13.1, 3.4 Hz),
1.64±1.55 (4H, m), 1.51±1.45 (2H, m), 1.42 (1H, dd,
Jˆ12.4, 4.5 Hz), 1.03 (6H, d, Jˆ6.9 Hz), 1.02 (3H, s),
0.95 (3H, s); 13C NMR (125 MHz, CDCl3) d 138.9, 138.8,
138.3, 129.4, 128.8, 125.6, 118.9, 114.8, 50.9, 49.5, 48.9,
40.7, 38.0, 37.5, 37.2, 35.5, 33.2, 23.6, 22.6, 21.4, 21.3,
20.9, 18.5, 14.3; EIMS m/z 380.2 [M]1 (43.0), 271.2 (6.6),
257.2 (40.2), 218.0 (40.6), 187.1 (12.7), 173.1 (31.0), 147.1
(22.4), 133.1 (100.0), 109.1 (90.1), 91.1 (42.4); HREIMS m/
1
z 380.2537 (calcd for C26H36S, 380.2538). The H NMR
spectrum showed some extra peaks attributed to dehydro-
abietadienyl sul®de [dH 7.22 (2H, d, Jˆ8.4 Hz), 7.04 (2H,
dd, Jˆ7.9, 1.9 Hz), 6.92 (1H, br s)] and diphenyl disul®de
[dH 7.54 (2H, dd, Jˆ8.6, 1.3 Hz), 7.34 (2H, t, Jˆ7.7 Hz),
7.26 (2H, tt, Jˆ7.5, 1.3 Hz)].
4.2.11. Levopimaradien-18-yl tosylate (2e). A solution of
alcohol 2d (36 mg, 0.13 mmol) and TsCl (119 mg,
0.63 mmol) in pyridine (1 mL) under N2 was stirred for
1 d at 258C and cooled to 08C before adding 3-(dimethyl-
amino)propylamine (153 mg, 1.5 mmol). After 30 min at
08C, solvent and excess amine were removed under reduced
pressure (0.15 mm Hg), and the residue was dissolved in
CH2Cl2 (10 mL). The solution was washed with satd
NaHCO3 (2£10 mL) and satd NaCl (2£10 mL) and concen-
trated to dryness. The 1H NMR spectrum showed the
absence of alcohol, amine, pyridine, and TsCl. Puri®cation
by ¯ash column chromatography (15% diethyl ether/
pentane) afforded tosylate 2e (54 mg, 94%) as a white
solid. Further elution with 30% MeOH/CH2Cl2 gave N-(3-
dimethylaminopropyl)toluenesulfonamide (55 mg, 44%) as
a colorless oil. Recrystallization of the tosylate from pen-
tane/ether gave the analytical sample: mp 82±838C;
4.2.13. PCC oxidation of levopimaradienol. Oxidation of
alcohol 2d (53.2 mg, 0.18 mmol) with PCC (40 mg,
0.18 mmol) in CH2Cl2 (2 mL) for 1 h at 258, isolation of
the crude product, and puri®cation afforded two fractions
containing aldehydes (16.5 mg, 31%) and alcohols (21 mg,
39%). The 1H NMR spectra showed the fractions were 6:5:1
(levopimaradienal/dehydroabietadienal/5) and 4.7:1.7:1
(dehydroabietadienol/levopimaradienol/1d) mixtures.
4.2.14. Levopimaradiene (abieta-12(13),8(14)-diene, 2b).
(A) By Li/NH3 reduction. Reduction of levopimaradienyl
sul®de (36.5 mg, 0.1 mmol) with Li/NH3 in THF as
described above for 1f and puri®cation by chromatography
gave 9.8 mg (36%) of a 2.6:1:3.1 mixture of 2b, 1b, and 3
abietene isomers according to GC. In another run the
abietene mixture was obtained as a separate, less polar
[a]20 ˆ21298 (c 0.52, CHCl3); UV (hexane) lmax (log 1)
D
268 (3.97) nm; IR (thin ®lm) nmax 2954, 2881, 1671, 1616
(CvC), 1459, 1364, 1189, 1118, 965, 848, 814, 665 cm21
;
1H NMR (500 MHz, CDCl3) d 7.77 (2H, d, Jˆ8.3 Hz), 7.33
(2H, d, Jˆ8.1 Hz), 5.49 (1H, s), 5.11 (1H, m), 3.71 (1H, d,
Jˆ9.4 Hz), 3.47 (1H, d, Jˆ9.4 Hz), 2.44 (3H, s), 2.31±2.26
1
fraction: GC±EIMS: m/z 274.3 [M]1; H NMR (500 MHz,
CDCl3) d 5.47 (m), 5.31 (s), and 5.09 (s).

6164
H.-J. Lee et al. / Tetrahedron 57 (2001) 6155±6167
(B) By Zn/NaI reduction. Yield, 52 mg (68%); purity, 88.2%
143.4, 137.6, 132.9, 129.8, 127.9, 124.8, 120.1, 78.0, 44.9,
37.5, 36.9, 35.1, 35.0, 34.2, 29.9, 26.2, 22.5, 21.6, 21.2,
20.9, 20.8, 18.6, 17.9, 16.9; EIMS m/z 442.3 [M]1 (25),
255.2 (100), 227.2 (9), 213.2 (6), 185.1 (17), 149.1 (26),
91.1 (52); HREIMS m/z 442.2542 (calcd for C27H38O3S,
442.2542).
(GC); [a]20 ˆ22658 (c 0.52, CHCl3); UV (hexane) lmax
D
(log 1) 268 (3.93) nm; IR (neat) nmax 2956, 2868, 1669,
1617 (CvC), 1458, 1388, 1364, 862 cm21; EIMS m/z
272.3 [M]1 (90), 257.3 (48), 229.2 (28), 215.2 (2), 201.2
(6), 187.2 (14), 175.2 (8), 159.1 (15), 148.1 (56), 137.2 (88),
117.1 (30), 105.1 (58), 91.1 (100), 69.1 (57); HREIMS m/z
272.2503 (calcd for C20H32, 272.2504). See Tables 1 and 2
for NMR data.
4.2.18. 18-Iodopalustradiene (8). Reaction of tosylate 3e
(63.8 mg, 0.14 mmol) with Zn (94 mg, 1.44 mmol) and NaI
(108 mg, 0.72 mmol) for 16 h at 1008C, as described above
for 1b and similar puri®cation by chromatography with pen-
tane as eluant afforded iodide 8 (37.6 mg, 67%) as a color-
less oil: UV (hexane) lmax (log 1) 268 (3.96) nm; IR (neat)
nmax 2955, 2930, 2868, 1658 (CvC), 1467, 1380, 1212,
4.2.15. Methyl palustradien-18-oate (methyl palustrate,
methyl abieta-8,13-dien-18-oate, 3c). Yield, 209 mg
(100%); Lit.38b mp 24±278C; [a]20 ˆ1658 (c 1.60,
D
CHCl3) [Lit.52 [a]20 ˆ169.98 (c 2.09, CHCl3); Lit.38a,b
D
1
1678]; UV (hexane) lmax (log 1) 268 (3.95) nm; IR (neat)
nmax 2929, 2867, 1726 (CvO), 1660 (CvC), 1458, 1386,
1177, 861 cm21; H NMR (500 MHz, CDCl3) d 5.40 (1H,
s), 3.30 (1H, d, Jˆ9.9 Hz), 3.19 (1H, d, Jˆ9.9 Hz), 2.28
(1H, septet, Jˆ6.9 Hz), 2.22±1.97 (6H, m), 1.80 (1H, dt,
Jˆ12.9, 3.2 Hz), 1.64±1.50 (4H, m), 1.48 (1H, d,
Jˆ12.2 Hz), 1.41 (1H, td, Jˆ11.8, 5.6 Hz), 1.32 (2H, dd,
Jˆ8.8, 3.9 Hz), 1.03 (3H, s), 1.02 (3H, s), 1.01 (3H, s), 1.00
(3H, s); 13C NMR (125 MHz, CDCl3) d 143.3, 137.7, 124.9,
120.2, 48.5, 38.6, 37.7, 35.4, 35.3, 34.2, 30.1, 28.5, 26.2,
22.6, 21.2, 20.9, 20.3, 18.6, 18.4, 18.3; EIMS m/z 398.2
[M]1 (71), 383.1 (100), 369.0 (5), 306.2 (3), 291.2 (12),
271.3 (16), 256.2 (30), 241.2 (7), 213.2 (5), 188.2 (34),
173.1 (17), 149.1 (56), 123.1 (18), 105.1 (26), 91.1 (30),
69.1 (15); HREIMS m/z 398.1465 (calcd for C20H31I,
398.1470).
1
1249, 1170, 1112, 1038, 862 cm21; H NMR (500 MHz,
CDCl3) d 5.37 (1H, s), 3.64 (3H, s), 2.27 (1H, septet,
Jˆ6.8 Hz), 2.12±1.98 (7H, m), 1.83 (1H, d, Jˆ13.4 Hz),
1.72 (1H, dd, Jˆ11.5, 4.6 Hz), 1.66±1.53 (4H, m), 1.24±
1.19 (1H, m), 1.18 (3H, s), 1.15 (1H, td, Jˆ12.5, 4.2 Hz),
1.04 (3H, s), 1.01 (3H, s), 1.00 (3H, s); 13C NMR (125 MHz,
CDCl3) d 179.2, 143.4, 137.6, 124.9, 120.2, 51.8, 47.7, 46.2,
37.1, 36.6, 35.2, 34.3, 30.1, 26.2, 22.4, 21.6, 21.2, 21.0,
20.7, 18.2, 16.3; EIMS m/z 316.3 [M]1 (87), 301.3 (100),
273.2 (4), 257.3 (21), 241.2 (90), 227.2 (4), 213.2 (19),
199.2 (9), 185.2 (26), 173.2 (8), 149.1 (34), 128.1 (12),
117.1 (16), 105.1 (34), 91.1 (29), 79.1 (13); HREIMS m/z
316.2397 (calcd for C21H32O2, 316.2394). The UV data
agree with the literature values.38,52
4.2.19. Palustradiene (abieta-8,13-diene, 3b). Reductions
of iodide 8 (37.6 mg, 0.09 mmol) and tosylate 3e (85.1 mg,
0.19 mmol) with Zn/NaI in HMPA at 1058C for 1 and 2 d,
respectively, were carried out as described in Section 4.2.8.
The yields and purities were 18 mg (70% from 8, 95%
purity by GC) and 25 mg (48% from 3e, ca. 95% purity
4.2.16. Palustradien-18-ol (3d). Yield, 164 mg (99%); mp
130±1318C [Lit.51 mp 131±1328C]; [a]20 ˆ1508 (c 0.69,
D
CHCl3) [Lit.51 [a]20 ˆ1538 (c 2.64, CHCl3)]; H NMR
1
D
(500 MHz, CDCl3)
d
5.39 (1H, s), 3.43 (1H, d,
Jˆ10.3 Hz), 3.17 (1H, dd, Jˆ10.3, 4.5 Hz), 2.27 (1H,
septet, Jˆ6.6 Hz), 2.12±1.94 (5H, m), 1.82 (1H, broad t,
Jˆ12.6 Hz), 1.65 (1H, dt, Jˆ13.5, 3.9 Hz), 1.63±1.59
(2H, m), 1.55 (1H, dt, Jˆ14.1, 3.9 Hz), 1.46±1.42 (2H,
m), 1.38±1.29 (4H, m), 1.05 (3H, s), 1.02 (3H, s), 1.00
(3H, s), 0.80 (3H, s); 13C NMR (125 MHz, CDCl3) d
143.2, 138.2, 124.8, 120.2, 72.3, 45.3, 37.6, 37.5, 35.5,
35.0, 34.2, 30.3, 26.2, 22.6, 21.2, 21.0, 20.9, 18.6, 18.3,
17.3; EIMS m/z 288.3 [M]1 (56), 273.2 (100), 255.2 (20),
227.2 (9), 213.2 (6), 199.2 (10), 188.2 (5), 173.1 (13), 161.2
(16), 149.1 (38), 133.1 (21), 105.1 (30), 91.1 (26), 67.1 (13);
HREIMS m/z 288.2454 (calcd for C20H32O, 288.2453). UV,
by 1H NMR estimate): [a]20 ˆ1598 (c 0.72, CHCl3)
D
[Lit.13 [a]20 ˆ2688 (c 5.00), Lit.12 245.58 (CHCl3)]; UV
D
(hexane) lmax (log 1) 268 (3.95) nm [Lit.13 lmax 267 (3.92,
heptane) nm]; IR (neat) nmax 2958, 2933, 2865, 1673
(CvC), 1457, 1370, 862 cm21; EIMS m/z 272.3 [M]1
(33), 257.3 (100), 243.1 (2), 229.2 (9), 214.2 (4), 188.2
(5), 173.2 (7), 161.2 (12), 149.2 (23), 133.1 (13), 117.1
(9), 107.1 (11), 91.1 (16), 81.1 (8), 69.1 (25); HREIMS
m/z 272.2510 (calcd for C20H32, 272.2504). See Tables 1
and 2 for NMR data.
1
4.2.20. Logic used to assign H and 13C NMR data for
palustradiene (3b). The H NMR singlets for the three
1
1
IR, and limited H NMR data are available in the litera-
ture.51
quaternary methyl groups on ring A (dH 0.89, 0.86, 1.06)
were identi®ed with C18, C19, and C20, respectively, by the
NOE results presented in Table 3. Although irradiations
intended for one of the gem dimethyl groups inevitably
excited both, the distinctive NOEs observed at dH 1.06±
2.08 (H11b, 3.2%) and 0.86 (H19, 7.8%) from selective
irradiation at 1.06 (H20) allowed unambiguous assignments
of C20 and C19, and by process of elimination C18. These
methyl peaks were correlated with 13C NMR signals at dC
33.4 and 21.5±21.7 by HMQC cross peaks, but overlap of
the higher ®eld pair left the assignments to C19 and C20
ambiguous. The low ®eld position of the equatorial methyl
(C18, 33.4) in palustradiene is consistent with its location in
the 13C NMR spectra of the other 3 dienes (dC 33.5±33.8).
However, its proximity (DdC 0.02) to the signal for C4
4.2.17. Palustradien-18-yl p-toluenesulfonate (3e). The
tosylate was prepared by the procedure described for 2e.
Data for 3e: yield, 189 mg (85%); mp 68±698C;
[a]20 ˆ1618 (c 0.33, CHCl3); UV (hexane) lmax (log 1)
D
266 (4.01) nm; IR (thin ®lm) nmax 2956, 2871, 1658
(CvC), 1599, 1459, 1358, 1189, 1099, 965, 844,
1
814 cm21; H NMR (500 MHz, CDCl3) d 7.76 (2H, d,
Jˆ8.4 Hz), 7.33 (2H, d, Jˆ8.6 Hz), 5.36 (1H, s), 3.73
(1H, d, Jˆ9.2 Hz), 3.55 (1H, d, Jˆ9.2 Hz), 2.45 (3H, s),
2.27 (1H, septet, Jˆ6.9 Hz), 2.07±1.95 (6H, m), 1.78 (1H,
dt, Jˆ12.6, 3.0 Hz), 1.59 (1H, dt, Jˆ13.5, 3.9 Hz), 1.51 (1H,
dt, Jˆ13.9, 3.6 Hz), 1.46±1.26 (6H, m), 1.02 (3H, s), 1.00
(6H, s), 0.79 (3H, s); 13C NMR (125 MHz, CDCl3) d 144.6,

H.-J. Lee et al. / Tetrahedron 57 (2001) 6155±6167
6165
precluded unique assignments for these close lying reso-
nances. Similarly the clustering of the four peaks for the
C16, C17, C19, and C20 methyls and the resulting overlap
of HMBC correlations prevented their individual 13C NMR
assignment.
2.51 (1H, dt, Jˆ13.9, 4.3 Hz), 2.35 (1H, d, Jˆ15.9 Hz), 2.19
(1H, broad t, Jˆ12.2 Hz), 1.91 (1H, t, Jˆ8.1 Hz), 1.84 (1H,
t, Jˆ12.9 Hz), 1.73 (3H, s), 1.69 (3H, s), 1.59 (1H, s), 1.54±
1.49 (3H, m), 1.44±1.28 (7H, m), 1.03 (1H, td, Jˆ12.4,
4.9 Hz), 0.80 (3H, s), 0.78 (3H, s); 13C NMR (125 MHz,
CDCl3) d 139.0, 128.3, 123.2, 121.8, 72.2, 51.3, 47.6,
39.0, 38.1, 37.8, 35.7, 35.4, 25.8, 22.5, 22.3, 20.3, 19.6,
18.3, 17.9, 15.7; EIMS m/z 288.3 [M]1 (29), 273.3 (2),
257.3 (6), 173.2 (5), 161.2 (7), 148.1 (29), 135.1 (100),
119.1 (8), 91.1 (6), 79.1 (4); HREIMS m/z 288.2449
(calcd for C20H32O, 288.2453). UV and elemental analysis
data are available in the literature.53
The proton peaks arising from the isolated C1±C2±C3, C5±
C6±C7, and C11±C12 segments were assigned by COSY
plots, and also were correlated to the assigned carbon reso-
nances by HMQC crosspeaks. The equatorial proton H1b
(dH 1.75) was identi®ed, and clearly distinguished from the
H3b proton (dH 1.38), by the NOE arising from irradiation
at C20 and density at the C9$H1 intersection in the HMBC
plot. The assignments for all of the A ring methylene
protons are consistent with the multiplicities and the usual
magnitudes for axial±axial, axial±equatorial, and equator-
ial±equatorial coupling on a chair cyclohexane conforma-
tion. In addition, long-range W coupling between H1b and
H3b (J ,1.4 Hz) was evident.
4.2.23. Neoabietadien-18-yl p-toluenesulfonate (4e). The
tosylate 4e was prepared as described above for 2e. Data for
4e: yield, 45.5 mg (63%); mp 112±1148C; [a]20 ˆ11058 (c
D
0.73, CHCl3); UV (hexane) lmax (log 1) 254 (4.45) nm; IR
(thin ®lm) nmax 2933, 2867, 1614 (CvC), 1462, 1365, 1189,
1
1099, 965, 846, 814 cm21; H NMR (500 MHz, CDCl3) d
7.77 (2H, d, Jˆ8.6 Hz), 7.34 (2H, d, Jˆ7.9 Hz), 6.16 (1H,
s), 3.72 (1H, d, Jˆ9.2 Hz), 3.50 (1H, d, Jˆ9.4 Hz), 2.50
(1H, dt, Jˆ14.1, 4.1 Hz), 2.45 (3H, s), 2.23 (1H, ddd,
Jˆ14.6, 4.3, 1.9 Hz), 2.04 (1H, td, Jˆ14.2, 4.3 Hz), 1.83
(2H, q, Jˆ13.7 Hz), 1.76±1.71 (1H, m), 1.72 (3H, s), 1.68
(3H, s), 1.67 (1H, dq, Jˆ13.9, 1.3 Hz), 1.49±1.38 (3H, m),
1.34±1.24 (5H, m), 1.00 (1H, ddd, Jˆ12.9, 9.9, 7.1 Hz),
0.79 (3H, s), 0.73 (3H, s); 13C NMR (125 MHz, CDCl3) d
144.6, 138.3, 133.0, 129.8, 128.2, 127.9, 123.5, 122.0, 78.0,
51.1, 47.3, 38.6, 38.0, 37.1, 35.3, 25.7, 22.4, 22.2, 21.6,
20.3, 19.6, 17.9, 17.6, 15.6; EIMS m/z 442.3 [M]1 (20),
270.3 (21), 255.2 (7), 241.2 (4), 187.2 (7), 173.1 (6),
155.0 (25), 135.1 (100), 119.1 (16), 105.1 (12), 91.1 (58),
71.1 (21); HREIMS m/z 442.2533 (calcd for C27H38O3S,
442.2542). Tosylation of 4d (160 mg, 0.55 mmol) with
tosyl chloride (526 mg, 2.77 mmol) in pyridine (1 mL) as
described above without the addition of 3-(dimethylamino)-
Identi®cation of H5 (d 1.17) was apparent from the NOE by
3
C18 irradiation, its multiplicity and coupling (dd, Jˆ12.4,
1.9 Hz), and the HMQC crosspeak to the CH peak at dC
51.9. Although severe peak overlap precluded coupling
analysis for H6b, H7a, H7b, H11a, H11b, H12a, and
H12b, chemical shift range assignments could be made by
NOEs observed at H6b and H11b (irrad. H20) and at H7b
(irrad. H14), and by the HMQC crosspeaks which identify
both attached protons of the four CH2 groups. The assign-
ments for the C11 and C12 methylene carbons and for the
®ve quaternary carbons were deduced from the following
HMBC crosspeaks: C12$H14, H15; C4$H5, H18/H19;
C10$H5, H20; C8$H7, H11, H14; C9$H1, H7, H14;
C13$H12, H15.
4.2.21. Methyl neoabietadien-18-oate (methyl abieta-
8(14),13(15)-dien-18-oate, 4c). Yield, 205 mg (100%);
1
propylamine afforded tosylate 4e (197 mg, 81%). The H
mp 628C [Lit.53 mp 61.5±628C]; [a]20 ˆ11338 (c 1.56,
NMR spectrum showed the presence of 5±10% of residual
tosyl chloride. The standard Zn/NaI reduction of this
contaminated tosylate resulted in the decomposition.
D
CHCl3) [Lit.53 [a]20 ˆ1147.88 (2% EtOH); Lit.38a,b
D
[a]20 ˆ1148.0 (95% EtOH)]; IR (thin ®lm) nmax 2930,
D
2867, 1725 (CvO), 1623 (CvC), 1444, 1384, 1242,
1
1187, 1144, 1101, 866 cm21; H NMR (400 MHz, CDCl3)
4.2.24. Neoabietadiene (abieta-8(14),13(15)-diene, 4b).
Reaction of tosylate 4e (20.7 mg, 0.05 mmol) with Zn
(30.1 mg, 0.46 mmol) and NaI (35 mg, 0.23 mmol) in
HMPA (1 mL) for 1 d at 1058C, as described above for
1b, gave neoabietadiene 4b (9 mg, 70% yield, 99.9% purity
d 6.17 (1H, s), 3.65 (3H, s), 2.50 (1H, dt, Jˆ14.2, 4.2 Hz),
2.31 (1H, dd, Jˆ14.4, 3.2 Hz), 2.19 (1H, td, Jˆ13.4,
4.9 Hz), 1.95 (1H, br t, Jˆ7.2 Hz), 1.92 (1H, dd, Jˆ12.5,
2.4 Hz), 1.84 (1H, br t, Jˆ13.2 Hz), 1.78±1.68 (3H, m),
1.72 (3H, s), 1.68 (3H, s), 1.58±1.50 (3H, m), 1.46 (1H,
dd, Jˆ12.7, 3.7 Hz), 1.34 (1H, dt, Jˆ13.2, 3.7 Hz), 1.19
(3H, s), 1.17±1.10 (2H, m), 0.77 (3H, s); 13C NMR
(100 MHz, CDCl3) d 179.4, 138.5, 128.2, 123.5, 122.1,
51.9, 51.4, 48.9, 47.5, 38.5, 37.7, 36.9, 35.5, 25.7, 24.8,
22.3, 20.3, 19.7, 18.2, 17.0, 15.3; EIMS m/z 316.3 [M]1
(47), 257.2 (7), 181.1 (11), 148.1 (29), 135.1 (100), 121.1
(41), 105.1 (15), 91.1 (22), 79.1 (13); HREIMS m/z
316.2399 (calcd for C21H32O2, 316.2394). UV data are
available in the literature.38,53
by GC) as a white solid: mp 628C; [a]20 ˆ11458 (c 0.57,
D
CHCl3); UV (hexane) lmax (log 1) 254 (4.30) nm; IR (thin
®lm) nmax 2936, 2865, 2842, 1622 (CvC), 1455, 1386,
1364, 1114, 880 cm21; EIMS m/z 272.3 [M]1 (22), 257.2
(4), 187.2 (3), 161.1 (4), 148.1 (26), 135.1 (100), 117.1 (5),
105.1 (13), 91.1 (18), 69.1 (13); HREIMS m/z 272.2509
(calcd for C20H32, 272.2504). See Tables 1 and 2 for NMR
data.
4.2.25. Maleic anhydride adduct (9) of levopimaradiene.
A
The procedure was based on those in the literature.54
4.2.22. Neoabietadien-18-ol (4d). Yield, 159 mg (97%);
solution of levopimaradiene 2b (58.5 mg, 0.21 mmol) and
maleic anhydride (84 mg, 0.86 mmol) in toluene (0.2 mL)
was stirred for 1 d at rt under N2. The solution was diluted
with CHCl3 (20 mL), washed with water (3£20 mL),
dried (MgSO4), and concentrated to dryness. Puri®cation
by ¯ash column chromatography (20% ether/pentane) and
mp 99±1008C [Lit.53 mp 98±99.58C]; [a]20 ˆ11808 (c
D
0.19, CHCl3) [Lit.53 [a]20 ˆ11878 (1% CHCl3)]; IR (thin
D
®lm) nmax 3375 (br OH), 2931, 2863, 1628 (CvC), 1448,
1440, 1040, 870 cm21; 1H NMR (500 MHz, CDCl3) d 6.18
(1H, s), 3.41 (1H, d, Jˆ10.9 Hz), 3.12 (1H, d, Jˆ10.7 Hz),

6166
H.-J. Lee et al. / Tetrahedron 57 (2001) 6155±6167
recrystallization from 2% ether/pentane afforded the
known15c adduct 9 (51.5 mg, 66%) as a colorless crystals:
References
mp 105±1078C [Lit.15c mp 110±1118C]; [a]20 ˆ2178 (c
1. Soltes, J.; Zinkel, D. F. In Naval Stores Production, Chemis-
try, and Utilization; Zinkel, D. F., Russell, J., Eds.; Pulp
Chemicals Association: New York, 1989; Chapter 9.
2. Dev, S.; Misra, R. In CRC Handbook of Terpenoids: Diterpe-
noids; Dev, S., Ed.; CRC: Boca Raton, FL, 1986; Vol. III.
3. Phillips, M. A.; Croteau, R. B. Trends Plant Sci. 1999, 4, 184±
190.
D
1.48, CHCl3); IR (thin ®lm) nmax 2930, 2881, 1857
(CvO), 1775 (CvO), 1465, 1386, 1227, 1085, 939, 908,
852 cm21; 1H NMR (500 MHz, CDCl3) d 5.53 (1H, s), 3.08
(1H, m), 3.07 (1H, dd, Jˆ8.6, 3.2 Hz), 2.68 (1H, d,
Jˆ8.4 Hz), 2.54 (1H, dt, Jˆ13.7, 3.2 Hz), 2.23 (1H, septet,
Jˆ6.9 Hz), 1.69±1.61 (2H, m), 1.55 (1H, td, Jˆ13.7,
4.7 Hz), 1.47 (1H, qt, Jˆ13.3, 3.0 Hz), 1.39±1.29 (4H,
m), 1.26±1.20 (2H, m), 1.11 (1H, td, Jˆ13.5, 3.9 Hz),
0.98 (3H, s), 0.97 (3H, s), 0.88 (3H, s), 0.87±0.77 (2H,
m), 0.79 (3H, s), 0.54 (3H, s); 13C NMR (125 MHz,
CDCl3) d 172.9, 171.2, 147.9, 125.4, 55.1, 53.3, 53.2,
45.6, 41.8, 40.3, 38.9, 38.2, 35.7, 35.3, 33.5, 32.8, 32.7,
27.4, 21.8, 20.5, 19.9, 18.9, 17.8, 15.1; EIMS m/z 370.3.3
[M]1 (7.8), 342.3 (23.7), 327.3 (11.4), 272.3 (100), 255.2
(11.6), 229.2 (2.6), 187.2 (3.1), 173.2 (5.2), 161.1 (2.5),
148.1 (26.9), 134.1 (48.7), 105.1 (14.5), 91.1 (29.9), 67.1
(11.3); HREIMS m/z 370.2508 (calcd for C24H34O3,
370.2508). IR and elemental analysis data are available in
the literature.15c
4. Steele, C. L.; Katoh, S.; Bohlmann, J.; Croteau, R. Plant
Physiol. 1998, 116, 1497±1504.
5. (a) Ref. 1, Chapters 16±21. (b) McCoy, M. Chem. Eng. News
2000, March 27, pp 13±15.
6. Harris, G. C.; Sanderson, T. F. Organic Synthesis Collective
Vol. IV; 1963; pp 1±4.
7. Lloyd, W. D.; Hedrick, G. W. Organic Synthesis Collective
Vol. V; 1973; pp 699±702.
8. Arno, M.; Gonzalez, M. A.; Marin, M. L.; Zaragoza, R. J.
J. Org. Chem. 2000, 65, 840±846.
9. Ayer, W. A.; Talamas, F. X. Can. J. Chem. 1988, 66, 1675±
1685.
10. (a) Lisina, A. I.; Pentegova, V. A. Izv. Sibirsk. Otd. Akad.
Nauk. SSSR. Ser. Khim. Nauk 1965, 2, 96±100 (Chem.
Abstr. 1966, 64, 3831f). (b) Pentegova, V. A.; Kashtanova,
N. K. Khim. Prir. Soedin. 1965, 223±224; Chem. Nat.
Comp. (Engl. Transl.), 1965, 171 (Chem. Abstr. 1965, 63,
16389g).
4.3. Incubation of GGPP with rAS and GC±MS analysis
of enzymatic products
GGPP (2 mmol, 200 mL) was added to truncated recombi-
nant abietadiene synthase24 (rAS, 0.4 mL) in 3.6 mL of
buffer (30 mM HEPES, pH 7.2, 7.5 mM MgCl2, 20 mM
MnCl2, 5% (v/v) glycerol, 5 mM DTT) and then were gently
mixed. After the mixture was incubated at 338C for 3 h,
2 mL of hexane containing 1.6£1024 M BHT was added.
The phases were vigorously mixed on a vortex genie and
centrifuged to separate the emulsion. The hexane layer was
carefully concentrated and dissolved in 10 mL of hexane
containing 1.6£1024 M BHT under nitrogen. GC±MS
spectra were obtained on a Quattro instrument. GC±MS
analysis of a mixture of the synthetic samples 1b±4b was
compared with that of abietadiene (1b, 33%, Rtˆ16.4 min),
levopimaradiene (2b, 42%, Rtˆ14.1 min), palustradiene
(3b, 9%, Rtˆ13.8 min) and neoabietadiene (4b, 16%,
Rtˆ19.7 min) of the hexane extract obtained from incuba-
tion. GC conditions were as follows: Rtx-5 30-m fused silica
capillary column with a split ratio of 100:1; initial tempera-
ture 1908C for 20 min followed by an increase of
158C min21 up to 2508C. Although the GC peaks for 2b
and 3b are very close, it proved possible to obtain mass
spectra with very little cross contamination, thus con®rming
the identity of these two products. No attempt was made to
identify other minor components.
11. (a) Norin, T.; Winell, B. Phytochemistry 1971, 10, 2818±
2821. (b) Carmen, R. M.; Sutherland, M. D. Aust. J. Chem.
1979, 32, 1131±1142.
12. Teresa, J. D. P.; Barrero, A. F.; Caballero, M. C.; San
Feliciano, A. Ann. Quim. 1978, 74, 1093±1096.
13. Raldugan, V. A.; Demenkova, L. I.; Pentegova, V. A. Khim.
Prir. Soedin. 1991, 323±327; Chem. Nat. Comp. (Engl.
Transl.), 1991, 279±282.
14. San Feliciano, A.; Miguel del Corral, J. M.; Gordaliza, M.;
Salinero, M. A. Magn. Reson. Chem. 1993, 31, 841±844.
15. (a) Cambie, R. C.; Rutledge, P. S.; Ryan, G. R.; Strange, G. A.;
Woodgate, P. D. Aust. J. Chem. 1990, 43, 867±881. (b)
Anthonsen, T.; Bergland, G. Acta Chem. Scand. 1970, 24,
1860±1861. (c) Ayer, W. A.; McDonald, C. E.; Iverach,
G. G. Tetrahedron Lett. 1963, 1095±1101.
16. Lange, W.; Weissmann, G. Holzforschung 1989, 43, 359±362.
17. Ha®zoglu, H.; Reunan, M. Holzforschung 1994, 48, 7±11.
18. Joye Jr., N. M.; Proveaux, A. T.; Lawrence, R. V. J. Am. Oil
Chem. Soc. 1973, 50, 104±107.
19. Conner, A. H.; Rowe, J. W. J. Am. Oil Chem. Soc. 1975, 52,
334±338.
20. (a) Takeda, H.; Schuller, W. H.; Lawrence, R. V. J. Org.
Chem. 1983, 33, 1683±1684. (b) Ref. 1, pp 289±291.
21. LaFever, R. E.; Vogel, B. S.; Croteau, R. Arch. Biochem.
Biophys. 1994, 313, 139±149.
22. Walter, J.; Laprebande, B.; Leferriere, A.; Saint-Guily, H.;
Saint-Guily, A. Plant Lipid Metab. 1995, 356±358; 11th
Intern. Meeting on Plant Lipids, 1994 (Chem. Abstr. 1995,
123, 106048).
Acknowledgements
We thank Duane Zinkel and the USDA Forest Products
Laboratory, Madison, WI for providing levopimaric acid,
R. Peters and R. Croteau for recombinant abietadiene
synthase and assistance in this work, Patti Silver for typing
the manuscript, Chad E. Davis for helpful comments, S.
Matsuda for a preprint of Ref. 27b, and the National
Institutes of Health for ®nancial support (GM 13956).
23. Vogel, B. S.; Wildung, M. R.; Vogel, G.; Croteau, R. J. Biol.
Chem. 1996, 271 (23), 262±268.
24. Peters, R. J.; Flory, J. E.; Jetter, R.; Ravn, M. M.; Lee, H.-J.;
Coates, R. M.; Croteau, R. Biochemistry 2000, 39, 15592±
15602.
25. Funk, C.; Croteau, R. Arch. Biochem. Biophys. 1994, 308,
258±266.

H.-J. Lee et al. / Tetrahedron 57 (2001) 6155±6167
6167
26. Steele, C. L.; Lewinsohn, E.; Croteau, R. Proc. Acad. Sci. USA
1995, 92, 4164±4168.
39. Wenkert, E.; Buckwalter, B. L. J. Am. Chem. Soc. 1972, 94,
4367±4369.
27. (a) Lewinsohn, E.; Savage, T. J.; Gizen, M.; Croteau, R.
Phytochem. Anal. 1993, 4, 220±225. (b) Schepmann, H. G.;
Pang, J.; Matsuda, S. P. T. Arch. Biochem. Biophys. 2001 in
press.
40. Wahlberg, I.; Almqvist, S.-O.; Nishida, T.; Enzell, C. R. Acta
Chem. Scand. 1975, B29, 1047±1058.
41. Ravn, M. M.; Coates, R. M.; Jetter, R.; Croteau, R. B. Chem.
Commun. 1998, 21±22.
42. Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43,
2923±2925.
28. Coates, R. M.; Bertram, E. F. J. Org. Chem. 1971, 36, 2625±
2631.
43. Sundararman, P.; Herz, W. J. Org. Chem. 1977, 42, 813±819.
44. (a) Davisson, V. J.; Woodside, A. B.; Neal, T. R.; Stremler,
K. E.; Muehlbacher, M.; Poulter, C. D. J. Org. Chem. 1986,
51, 4768±4779. (b) Woodside, A. B.; Huang, Z.; Poulter, C. D.
Organic Synthesis Collective Vol. VIII; 1993; p 616.
45. Fieser, L. F.; Fieser, M. Reagents for Organic Synthesis,
Wiley: New York, 1967; pp 581±595.
29. Ahad, A.; Arno, M.; Domingo, L. R.; Zaragoza, R. J.
Tetrahedron 1985, 41, 4937±4940.
30. Crossley, N. S.; Dowell, R. J. Chem. Soc. C 1971, 2496±2498.
31. (a) Coates, R. M.; Kang, H.-Y. J. Org. Chem. 1987, 52, 2065±
2074. (b) Chu, M.; Coates, R. M. J. Org. Chem. 1992, 57,
4590±4597.
32. Fujimoto, Y.; Tatsuno, T. Tetrahedron Lett. 1976, 3325±
3326.
46. Erdtman, H.; Westfelt, L. Acta Chem. Scand. 1963, 17, 1826±
1827.
33. Piancatelli, G. In Encyclopedia of Reagents for Organic
Synthesis; Paquette, L. A., Ed.; Wiley: Chichester, 1995;
Vol. 6, pp. 4356±4363.
47. (a) Omura, K.; Swern, D. Tetrahedron 1978, 34, 1651±1660.
(b) Mancuso, A. J.; Brownfain, D. S.; Swern, D. J. Org. Chem.
1979, 44, 4148±4150. (c) Ireland, R. E.; Norbeck, D. W.
J. Org. Chem. 1985, 50, 2198±2200.
34. (a) Kabalka, G. W.; Baker, Jr., J. D. J. Org. Chem. 1975, 40,
1834±1835. (b) Kabalka, G. W.; Baker, Jr., J. D.; Neal, G. W.
J. Org. Chem. 1977, 42, 512±517. (c) Kabalka, G. W.; Chand-
ler, J. H. Synth. Commun. 1979, 9, 275±279. (d) Kabalka,
G. W.; Hutchins, R.; Natale, N. R.; Yang, D. T. C.; Broach,
V. Organic Synthesis Collective Vol. IV; Wiley: New York,
1988; pp 293±298.
48. Hutchins, R. O.; Milewski, C. A.; Maryanoff, B. E. J. Am.
Chem. Soc. 1973, 95, 3662±3668.
49. Tipson, R. S. J. Org. Chem. 1944, 9, 235±241.
50. Sheehan, J. C.; Daves, Jr., G. D. J. Org. Chem. 1964, 29,
2006±2008.
51. Raldugin, V. A.; Pentegova, V. A. Khim. Prir. Soedin. 1974,
674675; Chem. Nat. Comp. (Engl. Transl.), 1974, 696±697.
52. Dauben, W. G.; Coates, R. M. J. Org. Chem. 1964, 29, 2761±
2764.
35. Ref. 1, Chapter 24.
36. Zinkel, D. F.; Rowe, J. W. J. Chromatogr. 1964, 13, 74±77.
37. Nestler, F. H. M.; Zinkel, D. F. Anal. Chem. 1967, 39, 1118±
1124.
53. Loeblich, V. M.; Lawrence, R. V. J. Am. Chem. Soc. 1957, 79,
1497±1499.
38. (a) Zinkel, D. F.; Zank, L. C.; Wesolowski, M. F. Diterpene
Resin Acids: A Compilation of Infrared, Mass, Nuclear
Magnetic Resonance, Ultraviolet Spectra, and Gas Chromato-
graphic Retention Data (of the Methyl Esters), US Department
of Agriculture, Forest Products Laboratory: Madison, WI,
1971. (b) Joye, Jr., N. M.; Lawrence, R. V. J. Chem. Eng.
Data 1967, 12, 279±282.
54. (a) Wienhaus, H.; Sandermann, W. Chem. Ber. 1936, 69,
2202±2207. (b) Ayer, W. A.; McDonald, C. E.; Stothers,
J. B. Can. J. Chem. 1963, 41, 1113±1126.