Synthesis of (R)- and (S)-10,16-dihydroxyhexadecanoic acid: Cutin stereochemistry and fungal activation

Article abstract of DOI:10.1016/S0031-9422(03)00003-7

The first asymmetric syntheses of the cutin monomers (R)- and (S)-10,16-dihydroxyhexadecanoic acid (10,16-DHPA) and confirmation of (S)(+)-absolute configuration for 10, 16-DHPA derived from tomato are reported. The individual DHPA stereoisomers display differences in their ability to activate the fungal pathogen Colletotrichum trifolii.

Full text of DOI:10.1016/S0031-9422(03)00003-7

Phytochemistry 63 (2003) 47–52
www.elsevier.com/locate/phytochem
Synthesis of (R)- and (S)-10,16-dihydroxyhexadecanoic acid:
cutin stereochemistry and fungal activation
Aqeel Ahmeda, Terry Crawforda, Stephanie Goulda, Y.S. Hab, Monica Hollraha,
Farhana Noor-E-Aina, Martin B. Dickmanb, Patrick H. Dussaulta,*
aDepartment of Chemistry, University of Nebraska-Lincoln, Lincoln, NE 68588, USA
bDepartment of Plant Pathology, University of Nebraska-Lincoln, Lincoln, NE 68588, USA
Received 2 August 2002; received in revised form 18 December 2002
Abstract
The first asymmetric syntheses of the cutin monomers (R)- and (S)-10,16-dihydroxyhexadecanoic acid (10,16-DHPA) and con-
firmation of (S)(+)-absolute configuration for 10,16-DHPA derived from tomato are reported. The individual DHPA stereo-
isomers display differences in their ability to activate the fungal pathogen Colletotrichum trifolii.
# 2003 Elsevier Science Ltd. All rights reserved.
Keywords: Tomato; Lycopersicon esculentum; Solonaceae; Cutin monomers; DHPA; Colletotrichum trifolii; Dihydroxyhexadecanoic acid; Asym-
metric synthesis
1. Introduction
assignment of 10,16-DHPA from tomato. The relative
ability of the individual enantiomers to elicit transcrip-
tion of fungal genes in Colletotrichum trifolii, the causal
agent of alfalfa anthracnose (Dickman, 2000) is also
reported.
Cutin, the outer coating on all aerial surfaces of
plants, is a polyester primarily composed of poly-
hydroxylated fatty acids (Kolattukudy, 2001). Upon
attack by some pathogenic fungi, the cutin layer is
cleaved by cutinolytic enzymes, and the presence of
cutin monomers released has been found to induce
expression of the fungal gene coding for these same
cutinases (Kolattukudy et al., 1995a,b). Consequently, it
is possible that the interaction of cutinase with cutin
and cutin derived monomers serves as a signal for
fungal gene expression associated with pathogenic
development.
One of the most abundant classes of cutin monomers is
the dihydroxypalmitic acids (DHPAs). Surprisingly, no
asymmetric synthesis of a DHPA has been reported
(Tulloch, 1980) and the single report assigning the
absolute stereochemistry of cutin-derived DHPA is only
based on ORD (Espelie and Kolattukudy, 1978; Blee
and Schuber, 1993). We now describe the enantio-
selective synthesis of (R)- and (S)-10,16-dihydroxyhex-
adecanoic acid (10,16-DHPA) and the stereochemical
2. Results and discussion
Our synthetic approach (Scheme 1) was based on the
use of a common precursor of both (R)- and (S)-10,16-
DHPA (1). The initial portion of the synthesis, illu-
strated in Scheme 2, began with addition of the lithiated
alkyne to 10-undecenal to furnish racemic enynol 2
(Frantz et al., 2000). The derived alkynone 3 underwent
reduction with (R)- or (S)-Alpine borane to furnish
enantiomerically enriched propargyl alcohols (R)-2 or
(S)-2, respectively, which were carried individually
through the remainder of the synthesis (Singh, 1992).
1
Analysis of derived diastereomeric esters by H and 19F
NMR (Scheme 3) indicated that (R)-2 and (S)-2 were
each formed in at least 88% enantiomeric excess (Dale
and Mosher, 1973).
Completion of the synthesis is illustrated in Scheme 4.
Ozonolysis under standard conditions (CH2Cl2/MeOH,
followed by Me2S reduction) led mainly to the dimethyl
acetal. However, ozonolysis in methanol cleanly furnished
* Corresponding author. Tel.: +1-402-472-2732; fax: +1-402-472-
9402.
E-mail address: pdussault@unl.edu (P. H. Dussault).
0031-9422/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(03)00003-7

48
A. Ahmed et al. / Phytochemistry 63 (2003) 47–52
resulting diastereomeric carbamates 8 could be dis-
1
tinguished by the H NMR signals for the Me2Si, t-
BuSi, or the MeO groups; details of this analysis are
provided in the Experimental section. Although quanti-
tation was limited by partial peak overlap, the assay
largely confirmed the enantiomeric purity of the indivi-
dual synthetic samples of (R)- and (S)-DHPA. A com-
parison with the corresponding derivative of tomato-
derived 10,16-DHPA (vida infra) demonstrated that the
material from tomato possesses mainly the 10(S) abso-
lute configuration but also contains a small amount
(10–20%) of the 10(R)-stereoisomer.
Scheme 1. DHPA structure and retrosynthetic analysis.
Scheme 2. Introduction of the C10 stereocenter. (a) NaH, BnBr, DMF
(88%); (b) n-BuLi, 10-undecenal (86%); (c) PDC(95%); (d) ( R)-
Alpine borane; (e) (S)-Alpine borane (90%).
2.1. Isolation and analysis of 10,16-DHPA( 1) from cutin
Samples of 10,16-DHPA methyl ester were prepared
by digestion of tomato cutin using a variant of a repor-
ted procedure (Gerard et al., 1992). Silica gel chroma-
tography furnished material which was pure according
to GC/MS and which could be saponified to obtain
samples of DHPA (1). The low magnitude of the optical
rotations for individual 10,16-DHPA (1) enantiomers
rendered specific rotation of only moderate use in deter-
mination of stereochemical purity. However, the corre-
lation of the sign of specific rotations for tomato-derived
and synthetic samples of DHPA or DHPA methyl ester
does substantiate the derivative-based stereochemical
assignment described above.
Scheme 3. Determination of stereochemical purity.
Interestingly, both the synthetic and tomato-derived
samples of DHPA (1) are stable, while the dihydroxy
methyl esters undergo rapid oligomerization. We are
currently investigating the basis for this phenomenon,
which may be related to the biosynthesis of cutin.
2.2. Influence of cutin monomers on fungal gene expression
Scheme 4. Completion of the synthesis of DHPA. (a) (i) O3, MeOH;
(ii) Ac2O, pyridine (80%, two steps); (b) Pt/C, H2 then Pd/C, H2
(70%); (c) 10% aq. NaOH (96%).
Synthetic samples of DHPA (1), tomato-derived
10,16-DHPA (1) and the known fungal activator
aleuritic acid, were compared for their ability to activate
a lipid-activated protein kinase (LIPK) and a cutinase
both of which are known to be activated by the presence
of dihydroxypalmitic acids in the fungal pathogen C.
trifolii, causal agent of alfalfa anthracnose (Dickman et
al., 2003). To date, all major classes of cutins have been
shown to be essentially interchangeable with respect to
induction of biological activities (Kolattukudy et al.,
1995a). RNA was isolated from C. trifolii mycelia. Fig. 1
illustrates induction of LIPK after 30 min. (+)-10,16-
the 1-methoxy-1-hydroperoxyacetal, which was easily
fragmented to methyl ester 5 (Schreiber et al., 1982).
Sequential hydrogenation over Pt/Cand Pd/Csaturated
the alkyne and deprotected the benzyl ether to furnish 6.
Saponification of both acetate and methyl ester afforded
(R)-, (S)-, and (RS)-10,16-DHPA (1).
The low magnitude of the optical rotations of (R)-
and (S)-DHPA (1), or their methyl esters, led us to seek
an alternative method for discriminating the two enan-
tiomers. The near symmetry of 10,16-DHPA (1) (the
two alkyl chains branching from the C10 chiral center
are identical for the first six methylene units) appeared
to rule out chiral GCor HPLC. Based on a procedure
originally applied to racemic dihydroxyoctadecanoates
(Sonnet et al., 1994), the (R)-, (S), and racemic (RS)
samples of DHPA were individually reacted with an
enantiomerically pure chiral isocyanate (Scheme 5). The
Scheme 5. Stereochemical correlation. (a) CH2N2, MeOH; (b)
t-BuMe2SiCl, DMAP; (c) (R)-1-naphthylethyl isocyanate.

A. Ahmed et al. / Phytochemistry 63 (2003) 47–52
49
were recorded in CDCl3 at 500 (1H) and 125 (13C) MHz.
1H NMR peaks are reported as (multiplicity, number of
H, coupling constant in Hz).
3.1.1. (RS)-1-Benzyloxy-heptadec-16-en-5-yne-7-ol (2)
Into a 0 ꢀCsolution of 5-hexyn-1-ol (10.1 g, 103
mmol) in 100 ml DMF was added, over 45 min, a 60%
suspension of NaH in mineral oil (4.89 g, 204 mmol),
followed by benzyl bromide (18.7 g, 109 mmol). The
reaction was allowed to warm to room temperature and,
after 18 h, was quenched with water (50 ml). The EtOAc
extracts were washed with brine and then dried
(MgSO4). Following concentration in vacuo, the residue
was purified by flash chromatography (5% EtOAc/hex-
ane) to furnish 16.93 g (88%) of the benzyl ether of
Fig. 1. Relative induction of LIPK activity. Northern analysis of
activity of C. trifolii LIPK 30 min following treatment. Values are
relative to strongest activity. Lane 1: (+)-10,16-DHPA; Lane 2: (À)-
10,16-DHPA; Lane 3: (Æ)-10,16-DHPA; Lane 4: no treatment; Lane
5: cutin-derived 10,16-DHPA; Lane 6: aleuritic acid.
DHPA (1) produces slightly greater induction than the
(À)-DHPA (1), the (+)-DHPA (1) derived from hydro-
lysis of tomato cutin, or aleuritic acid. It is curious that
the (+)- and (À)-isomers of 10,16-DHPA (1), each
consisting largely (>90%) of a single stereoisomer, each
displayed higher activity relative to the racemic sample.
Fig. 2 displays the corresponding Northern analysis
for induction of cutinase 60 min following chemical
treatment. In this case, the synthetic and tomato-
derived samples of (+)-DHPA (1) display similar levels
of activity; the (À)-DHPA (1) and the racemic DHPA (1)
are slightly lower. For both the LIPK and cutinase
assays, dose-dependent responses were linear for all cases
and at all time points (data not shown). Details of the
analyses are provided in the Experimental section.
In conclusion, we have reported the first asymmetric
syntheses of the enantiomers of 10,16-dihydroxypalmitic
acid and have also confirmed the (S)(+) absolute stereo-
chemistry of DHPA (1) derived from tomato cutin (Espe-
lie and Kolattukudy, 1978). The two DHPA enantiomers
are not identical in activation of C. trifolii gene expression,
suggesting that some aspects of this plant/fungal interac-
tion may involve stereochemical discrimination.
1
5-hexyn-1-ol: Rf=0.51 (5%EtoAc/hexane); H NMR ꢀ
7.46–7.22 (5H), 4.46 (s, 2H), 3.45 (t, 2H, 6.3), 2.19 (td,
2H, 2.6, 7.1), 1.92 (t, 1H, 2.7) 1.76–1.57 (m, 4H); 13C
NMR ꢀ 138.46, 128.20, 127.60, 127.46, 127.42, 127.35,
84.15, 72.70 71.94, 69.46, 68.36, 28.63, 25.10, 18.06; IR
3295, 3088, 3064, 3030, 2940, 2860, 2793, 1496, 1494,
1362, 1112 cmÀ1; HREI calc. for C13H15O (MÀH)+
187.1126, found 187.1123.
To a À78 ꢀCTHF (50 ml) solution of the benzyl ether
of 5-hexynol (6.0 g, 33 mmol) was added dropwise a
2.62 m solution of n-BuLi in hexanes (13.4 ml, 35 mmol,
1.1 eq). The reaction mixture was stirred for 20 min
prior to dropwise addition of 10-undecenal (6.6 ml, 32
mmol, 1.0 eq). The reaction was allowed to warm to RT
and stirred for 24 h and then quenched by sequential
dropwise addition of methanol (5 ml) and deionised
(DI) water (20 ml). The combined hexane and EtOAc
extracts were dried over MgSO4. The concentrated resi-
due was purified by flash chromatography (10%
EtOAc/hexane) to yield 9.6 g (85%) of the propargyl
alcohol: Rf=0.38 (20% EtOAc/hexane); 1H NMR ꢀ
7.3–7.27 (5H), 5.8 (ddt, 1H, 17.1, 10.0, 6.8), 4.95 (br dd,
1H, 17.1, 1.8) 4.92 (br dd, 1H, 10.0, 1.9), 4.5 (s, 2H),
4.33(t, 1H, 4.6), 3.49 (t, 2H, 6.4), 2.22 (td, 2H, 1.9, 7.1),
2.03 (app q, 3H, 6.9–7.5), 1.87–1.59 (5H), 1.42–1.28
(12H); 13CNMR (75 MHz) ꢀ 139.88, 139.18, 129.04,
128.28, 128.22, 114.83, 85.61, 82.45, 73.54, 70.44, 63.31,
38.84, 34.49, 30.18, 30.08, 29.97, 29.79, 29.60, 29.53,
26.08, 25.91, 19.18; IR 3396, 3064, 3032, 2928, 2854,
2210, 1672, 1640, 1454, 1360, 1274, 1204, 1104, 1028,
996, 908, 732, 696 cmÀ1; Anal. calc. for C24H35O2: C ,
80.85; H, 10.18; found: C, 80.99; H, 10.32; HRFAB
calc. (M+Li)+ 363.2875; found 363.2870.
3. Experimental
3.1. General
Standard experimental conditions are described else-
where (Cho et al., 1999). Unless noted, NMR spectra
3.1.2. 1-Benzyloxy-heptadec-16-en-5-yne-7-one (3)
A C HCl2 (5 ml) solution of propargylic alcohol (0.28
2
g, 0.77 mmol) and pyridinium dichromate (1.5 eq, 0.43
g, 1.1 mmol) was refluxed for 24 h and then filtered
through Celite to remove reduced chromium salts. Fol-
lowing removal of solvent at reduced pressure, the crude
product was purified by flash chromatography (20%
Fig. 2. Relative induction of cutinase activity. Northern analysis of
cutinase from C. trifolii 60 min following treatment. Values are rela-
tive to strongest activity. Lane 1: (+)-10,16-DHPA; lane 2: (À)-10,16-
DHPA; lane 3: (Æ)-10,16-DHPA; lane 4: no treatment; lane 5: natural
10,16-DHPA (see text); lane 6: aleuritic acid.

50
A. Ahmed et al. / Phytochemistry 63 (2003) 47–52
EtOAc/hexane) to furnish 0.27 g (95%) of the ketone.
On a larger scale (11.0 g, 30.8 mmol) the isolated yield
was lower (75%). Rf=0.70 (20% EtOAc/hexane); H
amount of Sudan Red B was bubbled a gaseous solution
of O3/O2. Ozonolysis was stopped after the reddish
color faded to yellow and little starting material could
be observed by TLC. Residual ozone was removed by
sparging with N2 and solvent was removed in vacuo.
The residue was redissolved in CH2Cl2 (10 ml) and the
solution was chilled to À78 ꢀC, whereupon excess acetic
anhydride (5 ml) and pyridine (5 ml) were added. The
reaction mixture was allowed to warm to room tem-
perature and stirred for 24 h. The residue following
concentration was subjected to flash chromatography
(20% EtOAc/hexane) to furnish the methyl ester (1.01
g, 80% yield) accompanied by 10–15% of the corre-
sponding aldehyde: Rf=0.29 in 20% EtOAc/hexane; 1H
NMR ꢀ 7.34À7.25 (5H), 5.33 (t, 1H, 6.6), 4.49 (s, 2H),
3.66 (s, 3H), 3.48 (t, 2H, 6.2), 2.29 (t, 2H, 7.5), 2.23 (t,
2H, 6), 2.05 (s, 3H), 1.72À1.58 (8H), 1.58–1.28 (10H);
13CNMR ꢀ 174.70, 170.53, 138.39, 128.77, 128.0,
127.93, 100.09, 86.21, 77.69, 70.18, 64.97, 51.86, 35.50,
34.50, 29.67, 29.55, 29.50, 29.46, 29.25, 25.68, 25.44,
25.33, 22.42, 25.33, 22.42, 21.56, 18.94, 17.98; IR 2924,
2847, 2361, 2225, 1741, 1454, 1362, 1229, 1104, 997,
953, 735, 691 cmÀ1; Analysis calc. for C26H38O5: C ,
72.53, H, 8.90; found: C, 72.36, H, 8.63; HRMS calc.
for (M+Li) 437.2879, found 437.2862; (R)-5:
[a]D=+39.8 (c=2.7, CHCl3); (S)-5: [a]D=À37.7
(c=2.4, CHCl3).
1
NMR ꢀ 7.33–7.24 (5H), 5.94 (ddt, 17.0, 10.3, 6.7), 5.12
(br d, 1H, 17), 5.06 (br d, 1H, 10), 4.47 (s, 2H), 3.47 (t,
2H, 6.0), 2.48 (t, 2H, 7.4), 2.37 (t, 2H, 6.6), 2.02 (app
quarter, 2H, 7), 1.72–1.61 (6H), 1.36–1.26 (10H); 13C
NMR ꢀ 188.34, 139.05, 138.37, 128.56, 128.30, 128.11,
127.65, 127.50, 114.08, 99.59, 93.60, 81.01, 72.86, 69.41,
45.45, 33.70, 29.19, 29.10, 28.96, 28.87, 28.80, 24.56,
24.14, 24.02,18.68; IR 3057, 2934, 2842, 2217, 1669,
1444, 1362, 1106, 983, 922, 737 cmÀ1; HRFAB calc.
(M)+ 354.2559; found 354.2559.
3.1.3. (R) or (S)-1-Benzyloxy-heptadec-16-en-5-yne-7-
ol (2) illustrated for (R-2)
The alkynone (3.64 g, 10.2 mmol) was dissolved in a
0.5 m THF solution of (R)-Alpine-borane (41.0 ml, 20.5
mmol). The reaction was stirred for 7 days and then
concentrated in vacuo. The residue was redissolved in
ꢀ
100 ml of diethyl ether at 0 C, and ethanolamine (1.0
ml, 16 mmol) was added dropwise. The resulting sus-
pension was filtered through Celite and concentrated.
Gradient flash chromatography (5–20% EtOAc/hexane)
furnished 3.4 g (90%) of propargyl alcohol which was
identical (1H and 13C) to racemic 2. Rf=0.41 (20%
EtOAc/hexane); [a]D=+1.7 (c=3.05, CHCl3). The (À)-
enantiomer was prepared from (S)-Alpine borane
through the same procedure: [a]D=À2.0 (c=1.05,
CHCl3).
3.1.6. Methyl-10-acetoxy-16-hydroxy hexadecanoate: (6)
A mixture of 5 (0.50 g, 1.1 mmol) and 10% Pt/C(30
mol%, 50 mg, 0.33 mmol) in MeOH (10 ml) was
hydrogenated in a shaker at 50 psi for 3 days, where-
upon 10% Pd/C(30 mol%, 0.33 mmol) was added and
the hydrogenation was resumed. After 4 days, the reac-
tion mixture was filtered and solvent removed under
reduced pressure. Flash chromatography (40% EtOAc/
hexane) furnished 0.35 g (70%) of 6 accompanied by
10–15% of methyl 16-hydroxyhexadecanoate: Rf=0.44
(50% EtOAc/hexane); 1H NMR ꢀ 4.84 (app quintet, 1H,
6.6, 5.9, 6.2), 3.6 (s, 5H), 2.29 (t, 2H, 7.5), 2.05 (s, 3H),
1.62–1.50 (8H), 1.33–1.26 (18 H); 13CNMR ꢀ 174.31,
170.94, 74.28, 82.94, 51.43, 34.08, 34.0, 32.65, 29.42,
29.28, 29.22, 29.13, 28.08, 25.59, 25.25, 25.23, 24.91,
23.23, 21.28; IR 3436, 2919, 2858, 2361, 1726, 1557,
3.1.4. Diastereomeric Mosher esters (4)
To a solution of propargyl alcohol (R)-2 (12.6 mg,
0.03 mmol) in CH2Cl2 (1.0 ml) was added (R)-methoxy
trifluoromethyl phenylacetic acid (MTPA, 12 mg, 0.05
mmol, 1.5 eq), DMAP (0.25 eq), and dicyclohex-
ylcarbodiimide (11 mg, 0.06 mmol, 1.5 eq), resulting in
the formation of a white precipitate. After 2 h, the
reaction mixture was diluted with several volumes of
ether and then filtered through a plug of cotton. Fol-
lowing removal of solvent, the crude product was fil-
tered through silica (10% EtOAc/hexane) to afford an
82% yield of the MTPA esters, which were determined
to be in a 94:6 ratio based upon the ratio of 19F NMR
signals at À72.12 and À72.36 (CDCl3). A similar ratio
was indicated by the ratio of signals at 3.583 and 3.547
1460, 1439, 1367, 1239, 1168, 1050, 1009, 881, 722 cmÀ1
;
Anal calc. for C19H36O5: C, 66.24, H, 10.53; Found: C,
66.17, H, 10.88; HRMS calc. for (M+Li)+ 351.2723,
found 351.2706. (R)À6: [a]D=À0.28 (c=9.5, CH2Cl2).
(S)-6: [a]D=+0.48 (c=7.7, CH2Cl2).
1
ppm in the H NMR spectrum. Analysis of the MTPA
esters of (S-4) prepared similarly, indicated a 6:94 ratio
of diastereomers. The corresponding analysis of the
racemic alcohol, employed as a control, showed a 51:49
ratio of diastereomers.
3.1.7. 10, 16-Dihydroxyhexadecanoic acid (10,16-DHPA)
(1)
3.1.5. Methyl 10-acetoxy-16-benzyloxy-11-hexadecynoate
(5)
A suspension of 6 (191 mg, 0.55 mmol) in MeOHꢀ(5
mL) and 10% NaOH (5 ml, excess) was held at 30 C
for 90 min and then acidified to pH 2 with 10% HCl .
The suspension was extracted into CH2Cl2 and the dried
Into a À78 ꢀCsolution of alkene 2 (1.05 g, 2.95 mmol)
in MeOH (20 ml) tinted slightly pink with a small

A. Ahmed et al. / Phytochemistry 63 (2003) 47–52
51
organic layer was concentrated to give a white solid
which was purified by flash chromatography (225:25:1
isopropanol:hexane:acetic acid) to give 10,16 DHPA as
a white solid (mp 77–79 ꢀC) which could be further
purified by washing with 5% EtOAc/hexane: Rf=0.15
(225:25:1 isopropanol/hexane/acetic acid) or 0.22 (9:1
CH2Cl2:MeOH); 1H NMR (3:1 CDCl3:MeOHÀd4) ꢀ
3.37 (t, 2H, 6.8), 3.36–3.33 (m, 1H) 2.08 (t, 2H, 7.5),
1.43–1.11 (26H); 13CNMR ꢀ 176.42, 71.19, 61.88, 48.90,
48.73, 48.56, 48.39, 48.22, 48.05, 47.88, 36.84, 36.73,
32.03, 29.27, 29.11, 29.03, 28.84, 28.74, 25.21, 25.18,
24.57. IR 3460, 3422, 3364, 3328, 3296, 3266, 2922,
2912, 2848, 1700, 1468, 1412, 1284, 1188, 1122, 1058,
1042 cm=1; Analysis calc. for C16H32O4: C, 66.63, H,
11.18; found: C, 62.25, H, 10.74; HRFAB calc.
1736, 1454, 1250, 1096, 847 cmÀ1; HRFAB calc.
(C23H48O4SiLi)+ 423.3487; Found 423.3483. (R)-7 and
(S)-7 were individually prepared from the (R) or (S)-enan-
tiomers of methyl 10,16-DHPA by the same procedure.
3.1.10. Formation of diastereomeric carbamates (8)
To a solution of (RS)-7 (2.6 mg, 6.2 mmol) in toluene
(0.5 ml), was added (R)-(À)-1-(naphthyl)ethyl isocya-
nate (3.8 mg, ꢀ0.02 mmol, 3.3 ml). The reaction was
heated to 100 Cfor 3 h and then stirred overnight at
room temp. Following concentration under high
vacuum, the crude product was filtered through flash
silica (30% EtOAc/hexane) and directly analyzed by
NMR. (RS)-8: Rf=0.81 (30% EtOAC/hex); 1H NMR d
7.86–7.43 (m, 7H), 3.667, 3.66 (s, 3H), 3.625–3.56 (m,
1H), 2.3–2.16 (7H), 1.67–1.25 (33H), 0.895/0.885 (s, 9H)
0.048/0.035 (s, 6H); 13CNMR d 174.32, 155.84, 128.79,
126.34, 125.70, 125.23, 122.06, 114.28, 74.95, 63.25,
51.43, 34.48, 34.10, 32.77, 29.69, 29.49, 29.39, 29.16,
29.10, 25.98, 25.71, 25.25, 24.93, À5.26; IR 2924, 2847,
2356, 2330, 1740, 1710, 1644, 1629, 1562, 1506, 1541,
1378, 1244, 1091,1050, 835, 768 cmÀ1; HRFAB calc. for
(C36H59NO5SiLi)+ 620.4323; found 620.4328.
The corresponding diastereomers derived from (R)-7
and (S)-7 could be distinguished by small differences in
the singlets at ꢀ 3.66 (OCH3), 0.89 (t-BuSi), or 0.18
(MeSi) ppm in the 1H NMR. A mixture of the naphthyl
carbamates derived from synthetic (R)-DHPA and
tomato-derived DHPA displayed two sets of signals; the
corresponding mixture involving (S)-DHPA gave one
major set of signals.
(M+Li)+
295.2461;
found
295.2453.
(R)-1:
[a]RD.T.=À3.1 (c=0.7, 1:1 MeOH/CHCl3); (S)-1:
[a]RD.T.=+3.0 (c=0.5, 1:1 MeOH/CHCl3).
3.1.8. Methyl 10,16-dihydroxyhexadecanoate (DHPA
methyl ester)
To a solution of 10,16 DHPA (20 mg, 0.066 mmol) in
MeOH (10 ml) was added ethereal diazomethane until a
yellow tint persisted. The solvent was removed under
reduced pressure, and the crude product was purified by
flash chromatography (40% EtOAc/hexane) to furnish
ꢀ
the methyl ester (20.7 mg, 99%): mp 47–49 C; Rf=0.3
1
(50% EtOAc/hexane); H NMR ꢀ 3.67–3.61 (6H), 2.29
(t, 2H, 7.5), 1.60–1.24 (26H); 13CNMR ꢀ 174.29, 71.94,
62.99, 51.39, 37.49, 37.38, 34.09, 32.72, 29.58, 29.44,
29.36, 29.15, 29.10, 25.71, 25.58, 24.93; IR 3308, 2924,
2842, 2361, 1731, 1465, 1219, 1127, 1065, 1004, 768, 717
cmÀ1; Anal calc. for C17H34O4: C, 67.5, H, 11.33; found:
C, 68.73, H, 11.37; HRMS calc. (M+Na)+ 325.2355;
found 325.2355. (R)-10 [a]D=À1.6 (c=1.3, CHCl3);
(S)-10: [a]D=+ 1.6 (c=1.0, CHCl3).
3.2. Isolation of cutin monomer
Cutin polymers were isolated using a streamlined
modification of a published procedure (Gerard et al.,
1992). Small sections of peel from mature tomatoes
(1.42 kg) were washed in DI water and then air dried.
The peels were boiled in 1 l of oxalate buffer (4 g oxalic
acid and 16 g ammonium oxalate) for 90 min and washed
with DI water. The dried peels were subjected to over-
night Soxhlet extraction with CHCl3 and the residue was
dried under vacuum. Washing with water and vacuum
drying resulted in 2 g of a yellow-brown powder.
The cutin polymer (0.5 g) was refluxed for 2 days in a
solution of 14% (v/v) BF3 /MeOH (66 ml), whereupon
the cooled reaction was carefully quenched with satu-
rated bicarbonate (150 ml). Following extraction with
CH2Cl2 (3Â50 ml) and drying with Na2SO4, concen-
tration in vacuo furnished 0.3 g of a viscous orange
liquid. The crude product was dissolved in 50% EtOAc/
hexane and subjected to silica gel flash chromatography
(50% EtOAc/hexane) to furnish the methyl ester of
10,16-DHPA as an off-white/yellow powder which dis-
played NMR and IR spectra identical to synthetic
material: mp 48–49 ꢀC ; a[]D=+0.6 (c=1, CHCl3).
3.1.9. Methyl-10-hydroxy-16-(dimethyl-1,1-dimethylethyl-
silyloxy)hexadecanoate (7)
To a solution of methyl 10,16-DHPA (18 mg, 0.06
mmol) in CH2Cl2 (0.5 ml) was added excess Et3N (0.1
ml), catalytic dimethylaminopyridine (7 mg, 0.06 mmol)
and after five min, TBSCl (9 mg, 0.6 mmol) in CH2Cl2
(0.3 ml). After 24 h at room temp, the reaction was
quenched with saturated ammonium chloride solution
and extracted with diethyl ether. The organic layer was
washed with brine, dried, and concentrated. The residue
was purified by flash chromatography to yield the
monosilyl ether in 22% overall yield (5.5 mg) accom-
panied by the bissilyl ether and recovered starting
material (55%): Rf=0.33 (20% EtOAc/hexane); 1H
NMR ꢀ 3.66–3.58 (6H), 2.29 (t, 2H, 7.52), 1.62–1.25
(25H) 0.89 (s, 9H) 0.043 (s, 6H); 13CNMR ꢀ 174.32,
71.97, 63.26, 51.43, 37.46, 37.43, 34.09, 32.80, 29.60,
29.49, 29.38, 29.17, 25.97, 25.79, 25.63, 25.60, 24.92,
18.37, 13.57,-5.26; IR 3738, 3390, 2934, 2847, 2361,

52
A. Ahmed et al. / Phytochemistry 63 (2003) 47–52
Solutions of methyl DHPA appeared to undergo rapid
polymerization or oligomerization. Optical rotations
were acquired on freshly purified material using alu-
mina-filtered CHCl3.
USDA (MBD). We thank Ingrid Jordan-Thaden, Leah
Sandvoss, and Jonathan Fritz for assistance with cutin
isolation and monomer synthesis.
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Acknowledgements
This work was supported by the NSF-REU program
(SG), the Nebraska Research Initiative (PHD), and the