Volatile methyl esters of medium chain length from the bacterium Chitinophaga Fx7914

Article abstract of DOI:10.1002/cbdv.201000190

The analysis of the volatiles released by the novel bacterial isolate Chitinophaga Fx7914 revealed the presence of ca. 200 compounds including different methyl esters. These esters comprise monomethyl- and dimethyl-branched, saturated, and unsaturated fatty acid methyl esters that have not been described as bacterial volatiles before. More than 30 esters of medium C-chain length were identified, which belong to five main classes, methyl (S)-2-methylalkanoates (class A), methyl (S)-2,(ω-1)-dimethylalkanoates (class B), methyl 2,(ω-2)-dimethylalkanoates (class C), methyl (E)-2-methylalk-2-enoates (class D), and methyl (E)-2,(ω-1)-dimethylalk-2- enoates (class E). The structures of the compounds were verified by GC/MS analysis and synthesis of the target compounds as methyl (S)-2-methyloctanoate (28), methyl (S)-2,7-dimethyloctanoate ((S)-43), methyl 2,6-dimethyloctanoate (49), methyl (E)-2-methylnon-2-enoate (20a), and methyl (E)-2,7-dimethyloct-2- enoate (41a). Furthermore, the natural saturated 2-methyl-branched methyl esters showed (S)-configuration as confirmed by GC/MS experiments using chiral phases. Additionally, the biosynthetic pathway leading to the methyl esters was investigated by feeding experiments with labeled precursors. The Me group at C(2) is introduced by propanoate incorporation, while the methyl ester is formed from the respective carboxylic acid by a methyltransferase using S-adenosylmethionine (SAM).

Full text of DOI:10.1002/cbdv.201000190

2228
CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
Volatile Methyl Esters of Medium Chain Length from the Bacterium
Chitinophaga Fx7914
by Thorben Nawratha), Klaus Gerthb), Rolf Mꢀllerb)c), and Stefan Schulz*a)
a) Institut fꢀr Organische Chemie, Technische Universitꢁt Braunschweig, Hagenring 30,
D-38106 Braunschweig (phone: þ49-531-3917353; e-mail: stefan.schulz@tu-bs.de)
b) Helmholtz-Zentrum fꢀr Infektionsforschung, Inhoffenstrasse 7, D-38124 Braunschweig
c) Institut fꢀr Pharmazeutische Biotechnologie, Universitꢁt des Saarlandes, Gebꢁude 2.3,
D-66123 Saarbrꢀcken
The analysis of the volatiles released by the novel bacterial isolate Chitinophaga Fx7914 revealed the
presence of ca. 200 compounds including different methyl esters. These esters comprise monomethyl- and
dimethyl-branched, saturated, and unsaturated fatty acid methyl esters that have not been described as
bacterial volatiles before. More than 30 esters of medium C-chain length were identified, which belong to
five main classes, methyl (S)-2-methylalkanoates (class A), methyl (S)-2,(wꢀ1)-dimethylalkanoates
(class B), methyl 2,(wꢀ2)-dimethylalkanoates (class C), methyl (E)-2-methylalk-2-enoates (class D),
and methyl (E)-2,(wꢀ1)-dimethylalk-2-enoates (class E). The structures of the compounds were
verified by GC/MS analysis and synthesis of the target compounds as methyl (S)-2-methyloctanoate (28),
methyl (S)-2,7-dimethyloctanoate ((S)-43), methyl 2,6-dimethyloctanoate (49), methyl (E)-2-methyl-
non-2-enoate (20a), and methyl (E)-2,7-dimethyloct-2-enoate (41a). Furthermore, the natural saturated
2-methyl-branched methyl esters showed (S)-configuration as confirmed by GC/MS experiments using
chiral phases. Additionally, the biosynthetic pathway leading to the methyl esters was investigated by
feeding experiments with labeled precursors. The Me group at C(2) is introduced by propanoate
incorporation, while the methyl ester is formed from the respective carboxylic acid by a methyltransfer-
ase using S-adenosylmethionine (SAM).
Introduction. – Fatty acids are one of the ubiquitous building blocks of biological
systems. The typical chain length varies in bacteria between 14 and 20, with the most
common fatty acids containing 16 or 18 C-atoms. In addition to these compounds, many
bacteria also produce methyl-branched fatty acids, carrying an additional Me group
often in the iso- or anteiso-position [1]. These acids are usually conjugated to other
compounds forming the various lipid classes of living organisms. In bacteria, a second
group of acids can be produced, short-chain, often amino-acid derived, volatile acids
with up to six C-atoms [2]. Fatty acids with a chain length between these two classes are
known, but are encountered relatively rarely in bacteria, often present only in low
amounts compared to the more common acids with a longer C-chain.
While short-chain fatty acids are volatile and have a distinct odor, the long-chain
fatty acids possess a much lower vapor pressure. Some bacteria increase the volatility
by transformation of the fatty acids into the methyl ester, making the compound also
unavailable for other biosynthetic transformations.
Methyl esters of intermediate chain length have only rarely been reported from
bacteria. Methyl 5-methylhexanoate and methyl 9-methyldecanoate have been
ꢂ 2010 Verlag Helvetica Chimica Acta AG, Zꢀrich

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
2229
detected in volatiles released by Stigmatella aurantiaca [3], while methyl 4-methyl-
pentanoate is produced by different actinomycetes [4]. Furthermore, methyl octanoate
was found in the odor of different Pecorino cheeses, most likely originating from
bacteria [5].
Chitinophaga belongs to the sphingobacteria and is a gliding bacterium not well
investigated for the production of secondary metabolites. The antibiotic elansolide is
the only natural compound known to be produced by these bacteria [6]. We became
interested in the volatiles released by Chitinophaga Fx7914 which proved to be very
complex. Beside several other components like sulfur compounds, amino acid
derivatives, and diterpenes, a complex set of saturated and unsaturated fatty acid
methyl esters of medium chain length was identified, all of them carrying a Me branch
at C(2). Many of these compounds have not been reported from bacteria or other
natural sources, beside a few exceptions [7–10]. The identification of these esters by
GC and GC/MS, the syntheses of representative examples as well as the determination
of the absolute configuration by stereoselective synthesis, followed by GC/MS on chiral
phases, will be presented here. Furthermore, some aspects of the biosynthesis of these
compounds were probed by feeding experiments using deuterium (2H1)-labeled
precursors. Finally, the bouquet of volatiles released by Chitinophaga Fx7914 is
described.
Results and Discussion. – Liquid cultures of Chitinophaga Fx7914 were analyzed
using the closed-loop-stripping methodology (CLSA) adopted to microbiological work
as described in [11][12]. The volatiles emitted by the bacteria were trapped on
activated charcoal. Elution with CH2Cl2 furnished an extract that was analyzed by GC/
MS. Chitinophaga cultures release a large variety of compounds which were identified
by comparison of mass spectra and GC retention indices with library data and with
synthetic standards (Fig. 1 and Table 1).
Fig. 1. Total ion chromatogram of a headspace extract of a liquid culture of Chitinophaga Fx7914
Characteristic compounds, repeatedly detected in large amounts in different
experiments, were 2-methyl- and 3-methylbutanoic acid (1 and 2, resp.), 2-methylte-
trahydrothiophenone (3), as well as the corresponding alcohol 4, 2-phenylethanol (5),
methyl phenylacetate (6), sesquiterpenes of the cadinane family such as g-cadinene (7)
and a-cadinol (8), several alcohols, ketones, and lactones, as well as diterpenoids of
unknown structure. The biosynthesis of 3 has recently been investigated by us [13].

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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
Table 1. Volatiles Released by Chitinophaga Fx7914
Ia)
Entry
Compound
Ident.b)
Conc.c)
1
2
3
4
5
6
7
8
9
Methyl 2-methylpropionate
4-Methylpentan-2-one
Methyl 3-methylbutanoate
3-Hydroxypentan-2-one
2-Hydroxypentan-3-one
5-Methylhexan-3-one
4-Hydroxy-4-methylpentan-2-one
2-Hydroxy-4-methylpentan-3-one
Ethylbenzene
MSd)
MS
MS
synf)
syn
MS
MS, RIg)
MS
xxxe)
xxx
xxx
xxx
xxx
xxx
xxx
xxx
xxx
xxx
xxx
xxx
xxx
xxx
xxx
xxx
xxx
xi)
x
809
816
839
846
853
860
862
868
870
877
892
898
903
908
910
913
914
916
923
931
948
953
957
958
962
964
968
976
983
990
999
1000
1011
1013
1013
1018
1020
1025
1027
1030
1032
1035
1039
1044
1050
1054
MS, RI
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
Unknown 55, 73, 98h)
m-Xylene
syn
Methyl 2-methylbut-2-enoate
2-Methoxy-1-methylethyl acetate
o-Xylene
2-Hydroxyhexan-3-one
3-Methylbutanoic acid (2)
2-Methylbutanoic acid (1)
2-Butoxyethanol
2,5-Dimethylpyrazine
Butano-4-lactone
But-2-eno-4-lactone
MS, RI
MS
syn
MS
syn
syn
MS
syn
syn
MS
syn
x
x
x
x
Dimethylsulfone
Hexane-2,5-dione
Unknown 41, 73, 100
Pentano-4-lactone
MS
xxx
x
syn
MS, RI
syn
6-Methylheptan-2-one
xxj)
xx
Benzaldehydek)
Unknown 82, 132
Dimethyl trisulfide
Unknown 45, 88
Aniline
2-Methyltetrahydrothiophen-3-one (3)
2,4,6-Trimethylpyridinek)
Methyl 4-methylhexanoate
3-Methylpenten-2-eno-4-lactone
2,4-Dimethylpent-2-eno-4-lactone
Unknown 43, 111, 126
Unknown 43, 69, 113
Mesitylene
xx
xx
x
x
xxx
xxx
xxx
xxx
x
x
x
x
x
x
xxx
x
MS, RI
syn
syn
MS
MS, RI
MS
MS
MS, RI
Unknown 43, 83, 98, 101
Unknown 68, 100
2-Methyltetrahydrothiophen-3-ol (4)
syn
MS
syn
syn
2-Ethylhexan-1-olk)
Benzyl alcohol
xx
2-Methyltetrahydrothiophen-3-ol (4)
Unknown 43, 109
xxx
xxx
x
x
x
Methyl 2-ethylhexanoatek)
7-Methyloctan-3-one
Unknown 66, 132
MS
MS, RI

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
2231
Table 1 (cont.)
Entry
Compound
Ia)
Ident.b)
Conc.c)
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
Unknown 84, 129
1-Phenylethanol
Acetophenone
N-Methylaniline
Methyl 2-methylheptanoate (A1)
2-Ethyl-4,6-dimethylpyridinek)
Methylthiobenzene
2-Phenylpropan-2-ol
o-Guaicol
Unknown 71, 95, 110, 128
Unknown 43, 57, 83
2-Phenylethanol (5)
Heptano-4-lactone
Methylundecene
8-Methylnonan-2-one
Methyl 2-methyloctanoate (A2)
Unknown 43, 77, 105
Unknown 91, 119, 134
Methyl 2-phenylacetate (6)
Decanal
1056
1059
1062
1062
1064
1067
1079
1082
1086
1103
1106
1109
1150
1154
1156
1161
1164
1168
1175
1204
1215
1224
1226
1248
1251
1255
1257
1260
1269
1271
1277
1279
1281
1286
1300
1311
1324
1328
1348
1353
1356
1358
1360
1373
1389
1394
1400
1408
1417
1423
xx
xx
xxx
xxx
xxx
x
x
xxx
x
x
x
xx
x
x
x
xx
x
x
xxx
x
xxx
xxx
x
x
x
syn
syn
syn
syn
MS
MS
MS
MS
syn
MS, RI
MS
MS, RI
syn
syn
syn
MS, RI
syn
syn
Benzothiazole
Methyl 2,7-dimethyloctanoate (B1)
Methyl 2,6-dimethyloctanoate (C1)
Methyl (E)-2-methyloct-2-enoate (D1)
9-Methyldecan-3-one
Octano-4-lactone
Benzyl propanoate
Methyl 2-methylnonanoate (A3)
Unknown 58, 87, 95
Undecan-6-one
Unknown 77, 106, 135, 159
4-(Methylsulfanyl)thiophenol
Nonano-5-lactone
Ethyl 4-methylbenzoate
Tridecane
Methyl (E)-2,7-dimethyloct-2-enoate (E1)
Methyl geranate
Methyl 3-methylalkanoate
Methyl (E)-2-methylnon-2-enoate (D2)
Unknown 111, 184
Methyl 2-methyldecenoate
Unknown 55, 70, 84, 124, 142
Methyl 2-methyldecanoate (A4)
2-Vinylnaphthalenek)
Dodecan-3-one
Dodecan-2-one
Tetradecane
Methyl (E)-2,8-dimethylnon-2-enoate (E2)
Methyl 2,9-dimethyldecanoate
Methyl 2,9-dimethyldecenoate (B2)
syn
MS, RI
MS, RI
MS
x
x
x
x
syn
MS, RI
x
xxx
xxx
x
x
x
xxx
x
x
xx
x
x
x
xx
x
x
x
x
x
x
xxx
MS
MS, RI
MS
MS, RI
syn
MS, RI
MS
syn
MS
syn
MS
MS, RI
MS, RI
MS, RI
MS, RI
MS
MS, RI

2232
CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
Table 1 (cont.)
Entry
Compound
Ia)
Ident.b)
Conc.c)
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
Methyl 2,8-dimethyldecanoate (C2)
1429
1440
1448
1453
1460
1474
1493
1499
1510
1512
1517
1521
1523
1526
1547
1552
1559
1563
1567
1571
1573
1576
1597
1600
1600
1609
1613
1618
1621
1626
1629
1638
1640
1644
1647
1652
1659
1662
1664
1677
1699
1707
1710
1717
1733
1747
1750
1753
1759
MS, RI
MS
syn
MS
syn
MS, RI
MS, RI
MS, RI
MS, RI
MS, RI
x
x
xx
xx
x
x
x
x
xxx
xxx
x
x
x
x
x
xx
xx
x
1,2-Dihydro-2,2,4-trimethylquinolinek)
Methyl (E)-2-methyldec-2-enoate (D3)
Methyl 2-methylundecenoate
Methyl 2-methylundecanoate (A5)
Dodecan-1-ol
Decano-5-lactone
Tridecan-2-ol
Methyl (E)-2,9-dimethyldec-2-enoate (E3)
g-Cadinene (7)
Sesquiterpene
Sesquiterpene
Sesquiterpene
Methyl 3-methylalkanoate
(E)-Methyl 2-methylundec-2-enoate (D4)
Unknown 153, 237
Methyl 2-methyldodecanoate (A6)
Nerolidol
12-Methyltridecan-2-ol
Undecano-4-lactone
Sesquiterpene
MS
syn
syn
MS, RI
MS, RI
MS, RI
x
x
x
x
Tridecan-1-ol
Unknown 41, 73, 88, 243
Sesquiterpene
MS, RI
x
xx
xx
x
x
x
xxx
x
x
xx
xx
x
xxx
x
xx
x
xx
xxx
x
xxx
xxx
xx
x
xxx
xx
x
Hexadecane
MS, RI
MS, RI
MS
Methyl (E)-2,10-dimethylundec-2-enoate (E4)
Methyl 2,11-dimethyldodacenoate
Sesquiterpene
Methyl 2,11-dimethyldodecanoate (B3)
1-Epicubenol
Methyl 2,10-dimethyldodecanoate (C3)
t-Cadinol
Sesquiterpene
syn
MS, RI
MS, RI
MS, RI
d-Cadinol
MS, RI
syn
MS, RI
syn
Methyl (E)-2-methyldodec-2-enoate (D5)
a-Cadinol (8)
Methyl 2-methyltridecanoate (A7)
Unknown 88, 101, 256 (ester)
13-Methyltetradecan-3-ol
Tetradecan-1-ol
MS, RI
MS, RI
MS
MS
syn
MS
MS
syn
MS
Methyl alkanoate
2-Ethylhexyl benzoatek)
Methyl (E)-2,11-dimethyldodec-2-enoate (E5)
Methyl alkenoate
Methyl 2-methyltetradecenoate
Methyl (E)-2-methyltridec-2-enoate (D6)
Methyl 2-methyltetradecenoate
Unknown 105, 126
Methyl 2-methyltetradecanoate (A8)
syn
xx

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
2233
Table 1 (cont.)
Entry
Compound
Ia)
Ident.b)
MS
Conc.c)
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
188
189
190
191
192
193
194
195
196
Alcohol
Methyl farnesoate
1779
x
x
1784
1791
1795
1800
1805
1811
1814
1822
1829
1833
1845
1848
1855
1858
1859
1863
1869
1881
1889
1894
1900
1905
1906
1911
1914
1918
1922
1929
1935
1935
1941
1944
1949
1951
1957
1966
1971
1980
1995
2003
2058
2066
2071
2079
2115
2125
2133
Diterpene
Methyl alkenoate
Octadecane
Diterpene
Methyl 2,13-dimethyltetradecenoate
Diterpene
Methyl 2,13-dimethyltetradecanoate (B4)
Methyl 2,12-dimethyltetradecanoate (C4)
Methyl alkanoate
Hexahydrofarnesylacetone
Methyl (E)-2-methyltetradec-2-enoate (D7)
Diterpene
Diterpene
Methyl 2-methylpentadecanoate (A8)
Diterpene
Diterpene
Diterpene
Diterpene
Diterpene
Nonadecane
Diterpene
Diterpene
Methyl (E)-2,13-dimethyltetradec-2-enoate (E6)
Diterpene
Diterpene
Diterpene
Diterpene
Diterpene
Methyl ester
Diterpene
Diterpene
Methyl (E)-2-methylpentadec-2-enoate (D8)
Diterpene
Diterpene
Diterpene
Diterpene
Diterpene
Diterpene
Diterpene
Diterpene
Diterpene
Diterpene
Diterpene
Diterpene
Diterpene
Diterpene
xxx
xx
x
xxx
xxx
xxx
xxx
x
MS
MS, RI
MS
MS, RI
MS, RI
MS
syn
syn
x
xx
xxx
x
x
x
syn
x
xxx
xxx
xxx
x
x
xx
xx
xxx
xx
xxx
xxx
xx
x
MS, RI
xxx
xxx
x
x
x
xxx
xx
xx
xx
xx
xxx
xx
xx
xxx
xxx
xxx
xxx
syn

2234
CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
Table 1 (cont.)
Entry
Compound
Ia)
Ident.b)
Conc.c)
197
198
199
200
Diterpene
Diterpene
Diterpene
Diterpene
2141
2153
2173
2186
xxx
xx
xxx
xxx
a) I¼GC Retention index. b) Ident.¼Identification method. c) Conc.¼concentration. d) MS¼Mass
spectrum. e) xxx¼Main compound (> 0.1%). f) syn: Synthetic compound. g) RI¼Retention index.
h) Numbers indicate characteristic ions in the mass spectra of unknown compounds. i) x¼Trace
compound (< 0.04% of total area of the TIC). j) xx¼Minor compound (0.04–0.1%). k) Medium
constituent.
In addition to these compounds, a series of esters was detected featuring
characteristic ions at m/z 88 and 101 (Fig. 2). The ion corresponding to m/z 88 is
generated by a McLafferty rearrangement of the ester function, while the ion ascribed
to m/z 101 is caused by a primary H shift from C(6) to the CO group, followed by H-
transfer and cleavage of the C(3)ꢀC(4) bond [14]. Characteristic for ethyl esters,
usually associated with the ions corresponding to m/z 88 and 101, are those at m/z 73
and [Mꢀ45]þ that arise by cleavage next to the CO group. The latter ions were
missing; instead, m/z 59 and [Mꢀ31]þ were present, indicating the presence of a
methyl ester functional group. In this case, peaks at m/z 88 and 101 indicate the
presence of a Me branch at C(2), as shown in Scheme 1. These methyl 2-
methylalkanoates were accompanied by compounds which exhibit a similar mass
spectrum but a molecular-ion 2 amu lower than the parent compound, indicating the
presence of a C¼C bond equivalent. A derivatization of the extract with dimethyl
disulfide (MeSSMe) to locate the position of the C¼C bond in the chain furnished no
derivatized esters. This behavior strongly suggested the presence of a C¼C bond at
C(2), because a CO-conjugated C¼C bond does not react with MeSSMe due to its
lower reactivity [15–17]. The occurrence of ions with peaks at m/z 88 and 101 in the
unsaturated methyl esters, at first hand surprising when a C¼C bond is located at C(2),
might be explained by the migration of the C¼C bond out of conjugation before
fragmentation [18]. A diagnostic ion peak of considerable intensity in the spectra of
some unsaturated esters is at m/z 127. Its formation can be explained by the mechanism

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
2235
Fig. 2. Mass spectra of a) methyl 2-methylnonanoate (29; A3); b) methyl 2,7-dimethyloctanoate (43; B1);
c) methyl 2,7-dimethylnonanoate (49; C1); d) methyl 2-methylnon-2-enoate (20a; D2); and e) methyl 2,7-
dimethyloct-2-enoate (41a; E1)
Scheme 1. Characteristic Mass-Spectral Fragmentations of Saturated and Unsaturated Methyl Esters

2236
CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
shown in Scheme 1 [19]. Transfer of a H-atom of C(6) to the CO group is followed by
loss of the g-alkyl group leading to an extended conjugated C¼C bond system. This
reaction can only occur in a,b-unsaturated methyl esters, pointing again to a C¼C bond
at C(2).
More than 30 different methyl esters containing between 9 to 17 C-atoms were
detected in the natural samples (Tables 1 and 2). The determination of the GC
retention indices (RIs) revealed the presence of five different subtypes of these esters
[20]. The saturated methyl esters had RIs ending around 60, 22, and 28 (classes A, B,
and C), while the unsaturated ones exhibited values around 48 and 10 (class D and E).
The RIs of aliphatic compounds with a terminal functional group (FG) can be
calculated using an empirical RI model developed by us and used successfully in several
projects [3][21–24]. The functional group (FG) increment for methyl esters is 331,
calculated from the retention index of 1331 of methyl decanoate. The FG of a Me group
in a-position to a CO group was determined to be 30 [25]. The compound class A can
thus be identified to constitute methyl 2-methylalkanoates, exemplified by identifica-
tion of compound A3, the most prominent member of this class. The molecular-ion
peak at m/z 186 in the mass spectrum (Fig. 2,a) indicates a methyl ester with ten C-
atoms in the acid part. The fragmentation pattern establishes the position of a Me
group at C(2). The calculated retention index RIcalc for methyl 2-methylnonanoate
(A3) is therefore 900þ331þ30¼1261, in good agreement with the observed value of
1259 (Table 2) of the synthetic material (see below). The class-B compound B1 (mass
spectrum Fig. 2,b) has the same molecular mass as A3, but is eluted earlier in GC. The
RI of 1224 indicates an additional Me branch in the chain. By use of the known
increments for a Me branch at various positions along the chain, a wꢀ1 Me group
seems to be most plausible [21]. The value RIcalc ¼800þ331þ30þ60¼1221 is very
close to the observed value. The second best position would be wꢀ3 with RIcalc ¼800þ
331þ30þ56¼1217. The mass spectra of the class-B compounds were very similar to
those of the class A compounds, but showed a small intensity increase in the [Mꢀ43]þ
ion, typical for iso-esters [26]. Methyl esters branched at wꢀ3 also show a [Mꢀ43]þ
ion, but with higher intensity [26]. In addition to the classes A and B, the class C esters
proved to have a very similar mass spectrum (Fig. 2,c), also with a molecular-ion peak
at m/z 186 in case of C1. The class C esters are eluted directly after the corresponding
class B esters, pointing to a second Me branch in a different position than wꢀ1. Only
the anteiso-esters show a higher RI value compared to the iso-esters [21]. Therefore,
the second Me branch in the class-C compounds is located at the wꢀ2 position. The
corresponding calculated value for C1 is RIcalc ¼800þ331þ30þ73¼1234 which is
close to the observed value of 1226. The mass spectra of the class-C esters (Fig. 2,c)
also showed an enhanced formation of [Mꢀ29]þ which can be explained the loss of the
Et group at the end of the chain. The unsaturated esters of class D and E showed a RI

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
2237
Table 2. Different Methyl Esters from Chitinophaga Fx7914
b
c
d
Compound
Peaka)
RInat
)
RIcalc
)
RIexp
)
Methyl 2-methylheptanoate (27)
Methyl 2-methyloctanoate (28)
Methyl 2-methylnonanoate (29)
Methyl 2-methyldecanoate (30)
Methyl 2-methylundecanoate (31)
Methyl 2-methyldodecanoate (32)
Methyl 2-methyltridecanoate (33)
Methyl 2-methyltetradecanoate (34)
Methyl 2-methylpentadecanoate (35)
Methyl 2,7-dimethyloctanoate (43)
Methyl 2,9-dimethyldecanoate
Methyl 2,11-dimethyldodecanoate (44)
Methyl 2,13-dimethyltetradecanoate
Methyl 2,6-dimethyloctanoate (49)
Methyl 2,8-dimethyldecanoate
A1
A2
A3
A4
A5
A6
A7
A8
A9
B1
B2
B3
B4
C1
C2
C3
C4
D1
D2
D3
D4
D5
D6
D7
D8
E1
E2
E3
E4
E5
E6
1064
1161
1259
1360
1460
1559
1659
1759
1859
1224
1423
1621
1822
1226
1429
1629
1829
1248
1348
1448
1547
1647
1747
1848
1949
1311
1408
1510
1609
1710
1912
1061
1161
1261
1361
1461
1561
1661
1761
1861
1221
1421
1621
1821
1234
1434
1634
1834
1251
1351
1451
1551
1651
1751
1851
1951
1311
1411
1511
1611
1711
1911
1060
1161
1258
1359
1459
1559
1660
1759
1859
1223
1620
1229
Methyl 2,10-dimethyldodecanoate
Methyl 2,12-dimethyltetradecanoate
Methyl (E)-2-methyloct-2-enoate (19a)
Methyl (E)-2-methylnon-2-enoate (20a)
Methyl (E)-2-methyldec-2-enoate (21a)
Methyl (E)-2-methylundec-2-enoate (22a)
Methyl (E)-2-methyldodec-2-enoate (23a)
Methyl (E)-2-methyltridec-2-enoate (24a)
Methyl (E)-2-methyltetradec-2-enoate (25a)
Methyl (E)-2-methylpentadec-2-enoate (26a)
Methyl (E)-2,7-dimethyloct-2-enoate (41a)
Methyl (E)-2,8-dimethylnon-2-enoate
Methyl (E)-2,9-dimethyldec-2-enoate
Methyl (E)-2,10-dimethylundec-2-enoate
Methyl (E)-2,11-dimethyldodec-2-enoate (42a)
Methyl (E)-2,13-dimethyltetradec-2-enoate
1245
1346
1446
1547
1648
1746
1848
1949
1313
1710
a) Peak: see Fig. 1. b) RInat: Retention index of the naturally occurring ester. c) RIcalc: Calculated
retention index. d) RIexp: Retention index of the synthetic standard.
value 90 units higher than that of the respective saturated compounds of class A and B.
This retention behavior is typical for a,b-unsaturated CO compounds and point
together with the MS data to a C¼C bond at C(2) (Fig. 2,d and e). In conclusion, class-
D and -E compounds were suggested to be analogs of the class A and B compounds
with an additional C¼C bond at C(2). Besides these esters, several other methyl esters
of low abundance occurred in the headspace extract, but their concentration was to low
to confirm their structure (Table 1).
Several of the methyl esters of classes A–E were then synthesized to verify the
proposed structures and to clarify the configuration of the C¼C bond in the
unsaturated esters.
Synthesis of Methyl Esters. Different aldehydes 9–17, ranging from pentanal (9) to
tridecanal (17), were transformed by a Horner–Wadsworth–Emmons reaction with

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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
methyl 2-(dimethoxyphosphoryl)propanoate to the unsaturated methyl esters 18a/
18b–26a/26b, respectively, as diastereoisomeric mixtures in varying ratios (Scheme 2)
[27]. NMR Analysis verified the C¼C bond configuration of the product diaster-
eoisomers, and GC/MS comparison with the natural esters established that the class-D
esters are constituted of methyl (E)-2-methylalk-2-enoates.
Scheme 2. Synthesis of Unsaturated and Saturated Methyl 2-Methylalkanoates
a) NaH, methyl 2-(dimethoxyphosphoryl)propanoate; 30–78%. b) H2, PtO2; 40–100%.
In a final reaction step, the diastereoisomer mixtures of the unsaturated esters were
hydrogenated in MeOH with PtO2 as catalyst, giving racemic mixtures of the
corresponding saturated mono-Me-branched methyl esters 27–35 (Scheme 2) [28].
Comparison of the synthetic material with the natural extract proved that the class-A
esters were indeed methyl 2-methylalkanoates.
The iso-esters of classes B and E were synthesized as shown in Scheme 3. Isobutyl
bromide (36) was transformed into 5-methylhexan-1-ol (37) or 9-methyldecan-1-ol
(38) by a Li2CuCl4-mediated chain elongation with 3-bromopropan-1-ol or 6-
bromohexan-1-ol, respectively [29]. The alcohols 37 and 38 were then oxidized with
PCC to furnish 5-methylhexanal (39) and 9-methyldecanal (40) [30]. The trans-
formation to the unsaturated esters as just described afforded methyl 2,7-dimethyloct-
2-enoate (41a and 41b) and 2,11-dimethyldodec-2-enoate (42a and 42b), respectively,
as mixtures of diastereoisomers. After separation of the isomers, the (E)-isomers
showed the same mass spectrum and RIs as the corresponding volatiles from
Chitinophaga Fx7914, verifying the suggested structures for the class-E compounds.
Scheme 3. Synthesis of Iso-Branched Unsaturated and Saturated Methyl 2-Methylalkanoates
a) Mg, bromoalcohol, Li2CuCl4; 85% (37), 68% (38). b) Pyridinium chlorochromate (PCC); 69% (39),
86% (40). c) NaH, methyl 2-(dimethoxyphosphoryl)propanoate; 32% (41), 44% (42). d) H2, PtO2; 66%
(43), 61% (44).

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
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Finally, hydrogenation of 41 or 42 provided racemic methyl 2,7-dimethyloctanoate
(43) and methyl 2,11-dimethyldodecanoate (44), respectively (Scheme 3), confirming
the proposed structure for the class-B compounds of Chitinophaga Fx7914.
The synthesis of the anteiso-esters of class C started from 4-methylhexanoic acid
(45) that was reduced with LiAlH4 to give 4-methylhexan-1-ol (46; Scheme 4) [22].
Following oxidation with PCC afforded the corresponding aldehyde 47 that was
transformed as described into methyl (E)- and (Z)-2,6-dimethyloct-2-enoate (48)
[27][30]. In a final reaction, 48 was hydrogenated in MeOH using PtO2 as catalyst [28]
to yield methyl 2,6-dimethyloctanoate (49) which proved identical to the natural
compound C1.
Scheme 4. Synthesis of Anteiso-Branched Saturated Methyl 2-Methylalkanoates
a) LiAlH4; 75% (46). b) PCC (47). c) NaH, methyl 2-(dimethoxyphosphoryl)propanoate; 38% (over
two steps) (48). d) H2, PtO2; quant. (49).
Although not all esters listed in Table 2, i.e., A1–E6, were synthesized, the
structures of B2, B4, C2, C3, C4, E2, E3, E4, and E6 were confirmed by their MS data
and RIs, showing the expected class values.
Determination of the Absolute Configuration. The mono- and dimethyl-branched
saturated methyl esters both contained a stereogenic center at C(2). Therefore, a
representative of class-A and -B esters was synthesized stereoselectively for the
determination of their configuration by GC/MS on chiral phases. Methyl (S)-2-
methyloctanoate ((S)-28) was synthesized as depicted in Scheme 5.
Octanoyl chloride (50) was transformed with the Evans chiral auxiliary (4S)-4-
benzyl-1,3-oxazolidin-2-one to the corresponding derivative 51 [31]. The following
stereoselective methylation with MeI under basic conditions furnished compound 52 in
a high diastereoisomeric ratio of 98 :2, which was purified by column chromatography
to give only one diastereoisomer [32]. The chiral auxiliary was cleaved off with H2O2
and LiOH, providing (S)-2-methyloctanoic acid (53) [32]. Finally, an acid-catalyzed
esterification with methanol afforded enantiomerically pure methyl (S)-2-methylocta-
noate ((S)-28) [33].
The methyl ester (S)-28 and rac-28 were used for GC analyses using a chiral b-Dex
225 GC phase (Fig. 3,a). The analyses confirmed the ee value of synthetic (S)-28
to be 100%, and proved that the natural compound also shows this configuration.

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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
Scheme 5. Stereoselective Synthesis of Methyl (S)-2-Methyloctanoate ((S)-28)
a) BuLi, (4S)-4-benzyl-1,3-oxazolidin-2-one; 84%. b) Sodium hexamethyldisilazane (NaHMDS), MeI;
66%. c) H2O2, LiOH; 100%. d) MeOH, HCl; 92%.
Furthermore, natural 28 is produced as single enantiomer. This results suggest that all
esters of class A possess an (S)-configuration.
Fig. 3. Determination of the absolute configuration of a) methyl (S)-2-methyloctanoate ((S)-28) and
b) methyl 2,7-dimethyloctanoate ((S)-43) on a chiral b-Dex 225 phase. a) 1) Synthetic rac-28. 2) Synthetic
(S)-28. 3) Coinjection of rac- and (S)-28. 4) Natural extract of Chitinophaga Fx7914. 5) Coinjection of
rac-28 and natural extract. 6) Coinjection of (S)-28 and natural sample. b) 1) Synthetic rac-43. 2)
Synthetic (S)-43. 3) Coinjection of rac- and (S)-43. 4) Natural extract of Chitinophaga Fx7914. 5)
Coinjection of rac-43 and natural extract. 6) Coinjection of (S)-43 and natural sample.
To extend these investigations to the dimethyl-branched saturated methyl esters of
class B, methyl (S)-2,7-dimethyloctanoate ((S)-43) was prepared (Scheme 6). Pre-
viously prepared 7-methyloctanoic acid (54) was converted to the corresponding acid
chloride 55 that was transformed to the Evans oxazolidinone 56 [17][31][34].
Methylation led to compound 57 with a diastereoisomeric ratio of 96 :4 that gave

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
2241
Scheme 6. Stereoselective Synthesis of Methyl (S)-2,7-Dimethyloctanoate ((S)-43)
a) Oxalyl chloride; 80%. b) BuLi, (4S)-4-benzyl-1,3-oxazolidin-2-one; 48%. c) NaHMDS, MeI; 46%. d)
H2O2, LiOH; 89%. e) MeOH, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC),
4-(dimethylamino)pyridine (DMAP); 66%.
enantiomerically pure 57 after purification [32]. The following cleavage of the
oxazolidinone produced acid 58 [32]. Finally, EDC-assisted esterification with MeOH
furnished the target compound (S)-43 that was used for GC/MS analyses on a chiral b-
Dex 225 column (Fig. 3,b) [35].
The natural methyl 2,7-dimethyloctanoate ((S)-43) is also produced exclusively as
pure (S)-enantiomer. Most likely, all class-B compounds possess (S)-configuration as
the class A esters do. The configuration of the two stereogenic centers in the class-C
compounds was not determined because of their low natural abundance.
Biosynthesis of the Methyl Esters. It is well known that a (wꢀ1)-Me group in fatty
acids containing an even number of C-atoms in the chain originates from 3-
methylbutyryl-CoA, derived from the amino acid leucine, while, in case of an odd
number of C-atoms, isobutyryl-CoA, derived from valine, serves as starter compound.
Similarly, the (wꢀ2)-Me group occurs only in fatty acids with an even number of C-
atoms in the chain and is formed from 2-methylbutyryl-CoA derived from isoleucine
[24][36][37]. The Me groups in the esters of Chitinophaga located at the end of the
chain are most likely biosynthetically formed via these pathways. In contrast, the
biosynthetic origin of the Me group at C(2) was unknown. Possibilities are either the
addition of the Me group to a C¼C bond via (S)-adenosylmethionine (SAM) or
incorporation of methylmalonate in the carboxylic acid chain extension by fatty acid
synthases [27][38][39].
Liquid cultures of Chitinophaga Fx7914 were supplemented with either
[1’-2H3]methionine as precursor of SAM or with sodium [1-13C]propanoate as precursor
of methylmalonate to investigate whether one of the proposed biosynthetic pathways is
operating. The emitted headspace volatiles were then analyzed by GC/MS [11][12].
The labeled precursors were incorporated in both experiments. Upon feeding of
[1’-2H3]methionine the molecular ion of methyl 2,7-dimethyloctanoate (43) shifted
from m/z 186 to 189, while the characteristic ions at m/z 88 and 101 were replaced by
the corresponding values at m/z 91 and 104 respectively (Fig. 4,a), indicating the
formation of [2H3]methyl 2,7-dimethyloctanoate ([2H3]-43) (incorporation rate

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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
>95%). Furthermore, the [Mꢀ31]þ ion was not present, but instead a [Mꢀ34]þ
2
fragment appeared. This shift indicated incorporation of the H label only into the
MeO group of the ester, but not into the Me group at C(2).
Fig. 4. Mass spectra of methyl 2,7-dimethyloctanoate (43) from Chitinophaga Fx7914 after feeding of
a) [1’,1’,1’-2H3]methionine and b) sodium [1-13C]propanoate
The feeding of sodium [1-13C]propanoate furnished labeled esters (incorporation
rate 20%). Contrary to 2H isotopomers that have shorter GC retention times compared
to their parent compounds [3][22][24], 13C-labelled compounds show no retention-
time shift. In the mass spectrum of labeled methyl 2,7-dimethyloctanoate (43) an
intensity increase of the ion peaks at m/z 89, 102, 144, 156, 172, and 187, compared to
the spectrum of unlabelled 43, was observed, indicating the incorporation of labeled
propanoate into 43 (Fig. 4,b). The mass spectrum showed the position of the label at
C(1), thus proving that the Me branch at C(2) originates from methylmalonate and not
from SAM.
On the basis of the known fatty acid biosynthesis and the labeling experiments, a
biosynthetic pathway for the formation of the different methyl-ester classes can be

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
2243
postulated (Scheme 7). Starting either with acetyl-CoA, propionyl-CoA (class A and
D), 3-methylbutyryl-CoA derived from leucine (class B and E), 2-methylbutyryl-CoA
derived from isoleucine (class C), or isobutyryl-CoA derived from valine (class E),
methyl-branched saturated and unsaturated methyl esters are synthesized.
Scheme 7. Biosynthetic Pathway for the Formation of the Different Methyl Ester Classes
The chain is extended from the starter molecules via malonyl-SCoA [2][22][40].
The odd or even chain length of the esters can be explained by use of the various
starters, but an a-oxidation process, leading to a loss of one C-atom during or after
chain extension, cannot be ruled out [24][37]. The feeding experiments with
[13C]propanoate did not clarify whether a propionyl starter is really used, because
the few ions which contain the alkyl end of the esters are of low abundance in the mass
spectra. A definite proof for a propionyl-CoA starter would be the enhanced intensity

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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
of the [Mþ2]þ ion, requiring incorporation of two propanoate units. The abundance of
such ions was too low for a definite conclusion.
In the final chain-extension step methylmalonyl-CoA derived from propanoate is
added to the chain, leading to the Me branch at C(2). Hydrolysis from the acyl carrier
protein (ACP) before reduction and final methylation with SAM leads to the
unsaturated esters, while reduction of the C¼C bond with NADPH, followed by
hydrolysis from ACP and SAM mediated methylation, affords the saturated esters. The
methylation of free fatty acids with SAM, established here by the labeling experiments,
has been described before by the action of a methyltransferase in Mycobacterium phlei
and the myxobacterium S. aurantiaca, and also in the rat hypothalamus [41–43].
As discussed in the introduction, methyl esters of medium chain length have only
occasionally been reported from bacteria. Most of the compounds described in Table 2
have not been reported before from nature. The related unsaturated fatty acids are also
rare. In mycobacteria, 2-methylalk-2-enoates of longer chain length have been found as
constituents of complex lipids [44][45]. In contrast, simple 2-methylalkanoic acids are
found in bacteria, plants, and animals. The parent acids of the 2,X-dimethyl-branched
esters reported here are also not known from nature, except for 2,7-dimethyloctanoic
acid which occurs bound to glycerol in epidermal glands of the reptile, Sphenodon
punctatus [46].
In summary, the identification, biosynthesis, and synthesis of novel Me-branched
aliphatic methyl alkanoates and methyl alkenoates of medium chain length is de-
scribed. Similar compounds have not been reported before as a major volatile class
released by bacteria. Why these esters are formed is unknown, but they might function
as signals or spacing compounds, inhibiting microbial growth next to Chitinophaga cells,
thus contributing to the success of these gliding bacteria.
We thank the Deutsche Forschungsgemeinschaft for financial support (DFG Schu 984-6).
Experimental Part
General. Chemicals were purchased from Sigma–Aldrich (Germany), Fluka (Switzerland), Acros
(Belgium), Merck (Germany), Cambridge Isotope Laboratories (USA), and Deutero (Germany). All
solvents were purified by distillation and dried according to standard methods. TLC: 0.2-mm pre-coated
plastic sheets (Polygram Sil G/UV254; Macherey-Nagel). Column chromatography (CC): Merck silica gel
60 (SiO2; 70–200 mesh); solvent mixtures as used for the determination of the Rf values in TLC;
compounds were detected using a molybdatophosphoric acid soln. (5% in EtOH) or a KMnO4 soln.,
followed by heat-gun treatment. NMR Spectra: Bruker AVII-300, DRX-400, or AVIII-400 spectrom-
eters; chemical shifts d in ppm relative to Me4Si as internal standard and coupling constants J in Hz.
Media and Growth Conditions. T2 Medium of the following composition was used to study the
production of metabolites and to perform feeding experiments under defined conditions: glutamine,
0.2% (w/w); MgSO4 ꢁ7 H2O, 0.02% (w/w); Fe-EDTA, 8 mg/l; MnSO4, 5 mg/l; ZnCl2, 0.25 mg/l; HEPES,
12 g/l; XAD 16 adsorber resin (Rohm and Haas), 2%; pH adjusted to 7.0. 5 ml of a 20% (w/w) autoclaved
sucrose soln. and 1 ml of a 7% (w/w) KH2PO4 autoclaved soln. were added to 100 ml of the above
medium. Liquid cultures were started in 106 ml T2 medium in 250-ml Erlenmeyer flasks inoculated with
2% of a preculture and incubated on a rotary shaker at 308 for 4 d. The feeding experiments were carried
out by the addition of [2H3]methionine (50 mg, 5.5 mm final concentration) or sodium [1-13C]propanoate
(200 mg, 31.2 mm final concentration) to well grown cultures of Chitinophaga Fx7914.
Sampling of Volatiles. Volatile org. compounds emitted by bacterial cultures were collected by the
closed-loop stripping analysis (CLSA) using the mentioned 250-ml Erlenmeyer flasks [11][12]. The

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
2245
volatiles were adsorbed on charcoal (Chromtech; Precision Charcoal Filter, 5 mg) for 24 h and extracted
with 30 ml of CH2Cl2. These extracts were immediately analyzed by GC/MS and then stored at ꢀ308.
GC/MS Analysis. GC/MS Analyses were carried out on a HP-6890 GC system connected to a HP-
5973 mass-selective detector fitted with a BPX5 fused-silica cap. column (25 m, 0.22 mm i.d., 0.25 mm
film; SGE, Australia) or on an Agilent 7890A GC system connected to an Agilent 5975C inert mass
detector fitted with a HP-5MS fused silica cap. column (30 m, 0.25 mm i.d., 0.25 mm film; J&W Scientific,
USA). Conditions for the HP-6890/HP-5973 system were: inlet pressure: 77.1 kPa, 23.3 ml He minꢀ1
;
injection volume: 1 ml; transfer line: 3008; electron energy: 70 eV. GC Program: 5 min at 508, increasing
with 108 minꢀ1 to 3208, operated in either split or splitless mode (60 s valve time); for the Agilent 7890A/
Agilent 5975C system, the following conditions were used: inlet pressure: 77.1 kPa, 23.3 ml He minꢀ1
;
injection volume: 2 ml; transfer line: 3008; electron energy: 70 eV. GC Programm: 5 min at 408, increasing
with 38 minꢀ1 to 3208, operated in either split or splitless mode (60 s valve time); He carrier gas at 1 ml
minꢀ1 (HP-6890) or 1.2 ml minꢀ1 (Agilent 7890A).
RI Values were determined from a homologous series of n-alkanes (C8 –C35) [20]. Identification of
compounds was performed by comparison of mass spectra to the Wiley-6 Library and the Essential Oils
Library (Massfinder) and by comparison of RI data from the literature or with synthetic standards.
GC/MS on Chiral Stationary Phase. Enantiomer separations were carried out on the Agilent 7890A/
5975C system equipped with a b-Dex 225 fused-silica cap. column (30 m, 0.32 mm i.d., 0.25 mm film;
Supelco/Sigma–Aldrich, Germany). The carrier gas was He at 1.6 ml minꢀ1 (valve time: 60 s, spitless
injection), injection volume: 1 ml. The mono-methyl esters were separated with the following temp.
program: 50 min at 608, then with 108 minꢀ1 to 2108. The dimethyl esters were separated with the
following temp. program: 120 min at 508, then with 208 minꢀ1 to 2108.
Derivatization with Dimethyl Disulfide (DMDS). According to the method of Leonhardt and
DeVilbiss [16], a soln. of the natural extract (10 ml), DMDS (50 ml), and an I2 soln. (5 ml, 5% in Et2O) was
heated to 608 for 8 h. The soln. was diluted with pentane, and excess I2 was removed by washing with sat.
aq. Na2S2O3 soln. The org. layer was separated, dried (MgSO4), concentrated, and analyzed by GC/MS.
Preparation of Methyl 2-(Dimethoxyphosphoryl)propanoate. A mixture of methyl 2-bromopropa-
noate (18.37 g, 0.11 mol) and (MeO)3P (12.40 g, 0.1 mol) was stirred and heated to 105–1108 for 2 h [47].
The MeBr formed during the reaction was removed continuously, finally furnishing the crude product.
This preparation was purified on SiO2 using Et2O as eluent. The purified product still contained small
amounts of irremovable side product. Rf (Et2O) 0.27. GC (BPX5): I 1293. 1H-NMR (400 MHz, CDCl3):
1.42–1.50 (m, 3 H); 3.02–3.14 (m, 1 H); 3.76–3.77 (m, 3 H); 3.78–3.80 (m, 3 H); 3.81–3.83 (m, 3 H).
13C-NMR (100 MHz, CDCl3): 11.6 (Me); 38.6 (d, J¼134.5, CH); 52.6 (Me); 53.3 (Me); 53.4 (Me); 170.0
(C). EI-MS (70 eV): 196 (1, Mþ ), 181 (21), 165 (40), 151 (4), 141 (15), 137 (31), 127 (15), 109 (100), 93
(12), 79 (27), 55 (53).
Preparation of Unsaturated Methyl Esters. According to Dickschat et al. [27], NaH (1.3 equiv., 60% in
mineral oil) was suspended in THF (1 ml/mmol) and cooled to 08. Methyl 2-(dimethoxyphosphoryl)-
propanoate (1.3 equiv.) dissolved in THF (0.5 ml/mmol) was added dropwise to this suspension. The
resulting mixture was stirred for 1 h at 408. After cooling to 08 the respective aldehyde (1–1.3 equiv.) in
THF (0.5 ml/mmol) was added dropwise. The mixture was stirred under reflux for 24 h, and the reaction
was quenched by the addition of 2m HCl. The aq. layer was extracted three times with Et2O, the
combined org. phases were dried with MgSO4 and finally concentrated under vacuum. Column
chromatography on SiO2 using a gradient of pentane/Et2O (200 :1 to 100 :1) gave the respective ester as a
diastereoisomeric mixture.
Methyl 2-Methylhept-2-enoate (18). Diastereoisomeric ratio (crude product): (E)/(Z) 2 :1. Yield:
42% (328 mg, 2.10 mmol). Rf (pentane/Et2O 10 :1): (Z): 0.61; (E): 0.49. GC (HP-5MS): (Z): RI 1088;
(E): RI 1145. 1H-NMR (400 MHz, CDCl3): (E): 0.85–0.93 (m, 3 H); 1.24–1.46 (m, 4 H); 1.83–1.84 (m,
3 H); 2.17 (q, J¼7.1, 2 H); 3.73 (s, 3 H); 6.77 (tq, J¼1.5, 7.5, 1 H). 13C-NMR (100 MHz, CDCl3): (E): 12.3
(Me); 13.8 (Me); 22.4 (CH2); 28.3 (CH2); 30.7 (CH2); 51.6 (Me); 127.4 (C); 142.7 (CH); 168.7 (C). EI-
MS (70 eV): (Z): 156 (46, Mþ ), 127 (100), 101 (33), 95 (47), 88 (27), 81 (10), 67 (27), 55 (55), 41 (29);
(E): 156 (35, Mþ ), 127 (63), 114 (7), 101 (92), 88 (65), 81 (17), 69 (29), 55 (100), 54 (11), 41 (40).
Methyl 2-Methyloct-2-enoate (19). Diastereoisomeric ratio (crude product): (E)/(Z) 3 :1. Yield:
30% (257 mg, 1.48 mmol). Rf (pentane/Et2O 10 :1): (Z): 0.64; (E): 0.46. GC (HP-5MS): (Z): RI 1189;

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(E): RI 1245. 1H-NMR (400 MHz, CDCl3): (E): 0.89 (t, J¼6.9, 3 H); 1.27–1.37 (m, 4 H); 1.40–1.49 (m,
2 H); 1.83–1.84 (m, 3 H); 2.17 (dq, J¼0.8, 7.4, 2 H); 3.73 (s, 3 H); 6.77 (tq, J¼1.4, 7.5, 1 H). 13C-NMR
(100 MHz, CDCl3): (E): 12.3 (Me); 14.0 (Me); 22.5 (CH2); 28.2 (CH2); 28.6 (CH2); 31.5 (CH2); 51.7
(Me); 127.4 (C); 142.8 (CH); 168.8 (C). EI-MS (70 eV): (Z): 170 (42, Mþ ), 139 (19), 127 (100), 101 (58),
95 (42), 88 (45), 83 (21), 81(14), 67 (25), 55 (18), 53 (20), 41 (63); (E): 170 (19, Mþ ), 139 (27), 127 (45),
101 (100), 95 (21), 88 (69), 82 (21), 69 (53), 59 (20), 55 (56), 41 (38).
Methyl 2-Methylnon-2-enoate (20). Diastereoisomeric ratio (crude product): (E)/(Z) 1.3 :1. Yield:
78% (714 mg, 3.88 mmol). Rf (pentane/Et2O 10 :1): (Z): 0.65; (E): 0.50. GC (HP-5MS): (Z): RI 1282;
(E): RI 1346. 1H-NMR (400 MHz, CDCl3): (E): 0.89 (t, J¼6.9, 3 H); 1.21–1.36 (m, 6 H); 1.40–1.47 (m,
2 H); 1.82–1.83 (m, 3 H); 2.17 (dq, J¼0.9, 7.4, 2 H); 3.73 (s, 3 H); 6.74–6.79 (m, 1 H). 13C-NMR
(100 MHz, CDCl3): (E): 12.3 (Me); 14.1 (Me); 22.6 (CH2); 28.5 (CH2); 28.7 (CH2); 29.0 (CH2); 31.7
(CH2); 51.6 (Me); 127.4 (C); 142.8 (CH); 168.8 (C). EI-MS (70 eV): (Z): 184 (34, Mþ ), 153 (20), 127
(100), 101 (55), 95 (37), 88 (43), 83 (14), 69 (27), 55 (34), 41 (28); (E): 184 (10, Mþ ), 153 (23), 127 (37),
101 (100), 88 (74), 82 (17), 69 (39), 55 (45), 41 (32).
Methyl 2-Methyldec-2-enoate (21). Diastereoisomeric ratio (crude product): (E)/(Z) 2 :1. Yield:
52% (514 mg, 2.60 mmol). Rf (pentane/Et2O 10 :1): (Z): 0.64; (E): 0.51. GC (HP-5MS): (Z): RI 1381;
(E): RI 1446. 1H-NMR (400 MHz, CDCl3): (E): 0.88 (t, J¼6.9, 3 H); 1.23–1.48 (m, 9 H); 1.60–1.67 (m,
1 H); 1.82–1.83 (m, 3 H); 2.13–2.19 (m, 2 H); 3.73 (s, 3 H); 6.77 (tq, J¼1.4, 7.5, 1 H). 13C-NMR
(100 MHz, CDCl3): (E): 12.3 (Me); 14.1 (Me); 22.6 (CH2); 28.6 (CH2); 29.0 (CH2); 29.1 (CH2); 29.3
(CH2); 31.8 (CH2); 51.6 (Me); 127.4 (C); 142.9 (CH); 168.8 (C). EI-MS (70 eV): (Z): 198 (29, Mþ ), 167
(18), 127 (100), 114 (12), 101 (67), 95 (33), 88 (52), 83 (13), 69 (24), 55 (32), 41 (29); (E): 198 (7, Mþ ),
167 (19), 127 (29), 101 (100), 95 (17), 88 (71), 83 (17), 69 (29), 55 (36), 41 (29).
Methyl 2-Methylundec-2-enoate (22). Diastereoisomeric ratio (crude product): (E)/(Z) 2 :1. Yield:
74% (601 mg, 2.84 mmol). Rf (pentane/Et2O 10 :1): (Z): 0.64; (E): 0.50. GC (HP-5MS): (Z): RI 1480;
(E): RI 1547. 1H-NMR (400 MHz, CDCl3): (E): 0.88 (t, J¼6.8, 3 H); 1.20–1.52 (m, 11 H); 1.56–1.68 (m,
1 H); 1.82–1.83 (m, 3 H); 2.16 (dq, J¼0.8, J¼7.4, 2 H); 3.73 (s, 3 H); 6.77 (tq, J¼1.4, 7.5, 1 H). 13C-NMR
(100 MHz, CDCl3): (E): 12.3 (Me); 14.1 (Me); 22.6 (CH2); 28.7 (CH2); 29.2 (CH2); 29.3 (CH2); 29.4
(CH2); 29.6 (CH2); 31.8 (CH2); 51.6 (Me); 127.4 (C); 142.8 (CH); 168.8 (C). EI-MS (70 eV): (Z): 212
(22, Mþ ), 181 (17), 127 (100), 114 (13), 101 (77), 95 (32), 88 (57), 83 (13), 81 (13), 69 (25), 55 (31), 41
(30); (E): 212 (6, Mþ ), 181 (16), 127 (27), 101 (100), 95 (17), 88 (73), 83 (15), 69 (28), 55 (33), 41 (28).
Methyl 2-Methyldodec-2-enoate (23). Diastereoisomeric ratio (crude product): (E)/(Z) 2 :1. Yield:
76% (665 mg, 2.94 mmol). Rf (pentane/Et2O 10 :1): (Z): 0.61; (E): 0.48. GC (HP-5MS): (Z): RI 1580;
(E): RI 1648. 1H-NMR (400 MHz, CDCl3): (E): 0.88 (t, J¼6.8, 3 H); 1.20–1.52 (m, 13 H); 1.55–1.69 (m,
1 H); 1.82–1.83 (m, 3 H); 2.13–2.19 (m, 2 H); 3.73 (s, 3 H); 6.77 (tq, J¼1.6, 7.5, 1 H). 13C-NMR
(100 MHz, CD3OD): (E): 12.3 (Me), 14.1 (Me), 22.7 (CH2), 28.7 (CH2), 29.3 (CH2), 29.4 (2 CH2), 29.5
(CH2), 29.6 (CH2), 31.9 (CH2), 51.6 (Me), 128.6 (C), 142.8 (CH), 168.7 (C). EI-MS (70 eV): (Z): 226
(23, Mþ ), 195 (16), 127 (100), 115 (17), 101 (66), 95 (32), 88 (59), 69 (23), 55 (29), 41 (30); (E): 226 (5,
Mþ ), 195 (16), 127 (25), 101 (100), 95 (17), 88 (77), 83 (17), 69 (26), 55 (31), 41 (27).
Methyl 2-Methyltridec-2-enoate (24). Diastereoisomeric ratio (crude product): (E)/(Z) 1:1. Yield:
60% (554 mg, 2.31 mmol). Rf (pentane/Et2O 10 :1): (Z): 0.61; (E): 0.48. GC (HP-5MS): (Z): RI 1677;
(E): RI 1746. 1H-NMR (400 MHz, CDCl3): (E): 0.88 (t, J¼6.8, 3 H); 1.23–1.34 (m, 14 H); 1.36–1.47 (m,
2 H); 1.82–1.83 (m, 3 H); 2.13–2.20 (m, 2 H); 3.73 (s, 3 H); 6.77 (dq, J¼1.4, 7.5, 1 H). 13C-NMR
(100 MHz, CDCl3) (E): 12.3 (Me); 14.1 (Me); 22.6 (CH2); 28.6 (CH2); 29.3 (2 CH2); 29.4 (2 CH2); 29.6
(2 CH2); 31.9 (CH2); 51.6 (Me); 127.3 (C); 142.8 (CH); 168.7 (C). EI-MS (70 eV): (Z): 240 (22, Mþ ), 209
(15), 143 (9), 127 (100), 114 (14), 101 (69), 95 (31), 88 (63), 83 (15), 81 (15), 69 (23), 55 (29), 41 (31);
(E): 240 (5, Mþ ), 209 (15), 127 (25), 114 (9), 101 (100), 95 (17), 88 (82), 83 (16), 81 (14), 69 (24), 55
(30), 41 (28).
Methyl 2-Methyltetradec-2-enoate (25). Diastereoisomeric ratio (crude product): (E)/(Z) 1:1. Yield:
73% (715 mg, 2.81 mmol). Rf (pentane/Et2O 10 :1): (Z): 0.59; (E): 0.48. GC (HP-5MS): (Z): RI 1778;
(E): RI 1848. 1H-NMR (400 MHz, CDCl3): (E): 0.88 (t, J¼6.9, 3 H); 1.23–1.33 (m, 16 H); 1.36–1.45 (m,
2 H); 1.82–1.83 (m, 3 H); 2.13–2.20 (m, 2 H); 3.73 (s, 3 H); 6.77 (dq, J¼1.4, 7.5, 1 H). 13C-NMR
(100 MHz, CDCl3): (E): 12.3 (Me); 14.1 (Me); 22.7 (CH2); 28.6 (CH2); 29.3 (2 CH2); 29.4 (2 CH2); 29.6
(3 CH2); 31.9 (CH2); 51.6 (Me); 127.4 (C); 142.8 (CH); 168.8 (C). EI-MS (70 eV): (Z): 254 (22, Mþ ), 223

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(14), 157 (9), 143 (21), 127 (100), 114 (14), 101 (69), 95 (30), 88 (65), 83 (14), 81 (15), 69 (23), 55 (29), 41
(31); (E): 254 (4, Mþ ), 223 (12), 143 (5), 127 (24), 114 (8), 101 (100), 95 (17), 88 (91), 83 (16), 81 (14),
69 (26), 55 (31), 41 (28).
Methyl 2-Methylpentadec-2-enoate (26). Diastereoisomeric ratio (crude product): (E)/(Z) 1:1.
Yield: 65% (667 mg, 2.49 mmol). Rf (pentane/Et2O 10 :1): (Z): 0.60; (E): 0.48. GC (HP-5MS): (Z): RI
1877; (E): RI 1949. 1H-NMR (400 MHz, CDCl3): (E): 0.88 (t, J¼6.8, 3 H); 1.23–1.33 (m, 18 H); 1.35–
1.45 (m, 2 H); 1.82–1.83 (m, 3 H); 2.13–2.19 (m, 2 H); 3.73 (s, 3 H); 6.77 (dq, J¼1.4, 7.5, 1 H). 13C-NMR
(100 MHz, CDCl3): (E): 12.3 (Me); 14.1 (Me); 22.7 (CH2); 28.6 (CH2); 29.3 (2 CH2); 29.4 (2 CH2); 29.6
(4 CH2); 31.9 (CH2); 51.6 (Me); 127.3 (C); 142.8 (CH); 168.7 (C). EI-MS (70 eV): (Z): 268 (22, Mþ ), 237
(14), 171 (9), 157 (23), 127 (100), 114 (14), 101 (73), 95 (30), 88 (69), 83 (15), 81 (15), 69 (24), 55 (30),
41 (32); (E): 268 (5, Mþ ), 237 (12), 157 (5), 127 (23), 114 (8), 101 (100), 99 (6), 98 (12), 95 (16), 88 (83),
83 (15), 81 (14), 69 (23), 55 (29), 41 (25).
Methyl (2E)-2,7-Dimethyloct-2-enoate (41a). Yield: 13% (91 mg, 0.49 mmol). Rf (pentane/Et2O
1
10 :1) 0.40. GC (HP-5MS): RI 1313. H-NMR (400 MHz, CDCl3): 0.88 (d, J¼6.5, 6 H); 1.17–1.24 (m,
2 H); 1.44 (dt, J¼7.6, 15.6, 2 H); 1.49–1.58 (m, 1 H); 1.83–1.84 (m, 3 H); 2.12–2.18 (m, 2 H); 3.74 (s,
3 H); 6.74–6.79 (m, 1 H). 13C-NMR (100 MHz, CDCl3): 12.3 (Me); 22.5 (2 Me); 26.4 (CH2); 27.9 (CH);
28.9 (CH2); 38.3 (CH2); 51.6 (Me); 127.4 (C); 142.8 (CH); 168.8 (C). EI-MS (70 eV): 184 (1, Mþ ), 152
(68), 141 (4), 124 (25), 113 (10), 101 (75), 95 (23), 88 (100), 83 (21), 82 (65), 81 (26), 69 (71), 55 (42), 41
(45).
Methyl (2Z)-2,7-Dimethyloct-2-enoate (41b). Yield: 19% (138 mg, 0.75 mmol). Rf (pentane/Et2O
10 :1) 0.53. GC (HP-5MS): RI 1246. EI-MS (70 eV): 184 (9, Mþ ), 153 (14), 152 (32), 141 (6), 127 (67),
114 (11), 101 (55), 95 (48), 88 (67), 83 (22), 82 (59), 81 (33), 69 (66), 55 (60), 41 (100), 39 (50).
Methyl (2E)-2,11-Dimethyldodec-2-enoate (42a). Yield: 25% (228 mg, 0.95 mmol). Rf (pentane/
Et2O 10 :1) 0.58. GC (HP-5MS): RI 1710. 1H-NMR (400 MHz, CDCl3): 0.86 (d, J¼6.8, 6 H); 1.12–1.17
(m, 2 H); 1.25–1.35 (m, 8 H); 1.40–1.46 (m, 2 H); 1.48–1.55 (m, 1 H); 1.82–1.83 (m, 3 H); 2.13–2.19 (m,
2 H); 3.73 (s, 3 H); 6.77 (tq, J¼1.4, 7.5, 1 H). 13C-NMR (100 MHz, CDCl3): 12.3 (Me); 22.6 (2 Me); 27.3
(CH2); 27.9 (CH); 28.6 (CH2); 28.7 (CH2); 29.4 (CH2); 29.5 (CH2); 29.8 (CH2); 39.0 (CH2); 51.6 (Me);
127.4 (C); 142.8 (CH); 168.7 (C). EI-MS (70 eV): 240 (5, Mþ ), 209 (11), 208 (10), 127 (24), 114 (8), 101
(100), 95 (22), 88 (72), 83 (17), 81 (17), 69 (32), 55 (31), 41 (35).
Methyl (2Z)-2,11-Dimethyldodec-2-enoate (42b). Yield: 19% (174 mg, 0.73 mmol). Rf (pentane/
Et2O 10 :1) 0.69. GC (HP-5MS): RI 1643. EI-MS (70 eV): 240 (26, Mþ ), 209 (15), 127 (100), 114 (14),
101 (67), 95 (36), 88 (59), 83 (15), 81 (17), 69 (27), 55 (30), 43 (33), 41 (36).
Methyl 2,6-Dimethyloct-2-enoate (48). Diastereoisomeric ratio (crude product): (E)/(Z) 6 :1. Yield:
38% (203 mg, 1.10 mmol, over two steps). Rf (pentane/Et2O 10 :1): (Z): 0.74; (E): 0.62. GC (BPX5):
(Z): RI 1247; (E): RI 1317. 1H-NMR (400 MHz, CDCl3): (E): 0.75–0.84 (m, 6 H); 1.04–1.42 (m, 4 H);
1.50 (m, 1 H); 1.75–1.78 (m, 3 H); 2.04–2.14 (m, 2 H); 3.66 (s, 3 H); 7.19 (s, 1 H). 13C-NMR (100 MHz,
CDCl3): (E): 11.3 (Me); 12.3 (Me); 19.0 (Me); 26.3 (CH2); 29.3 (CH2); 34.1 (CH); 35.3 (CH2); 51.7
(Me); 127.2 (C); 143.0 (CH); 168.8 (C). EI-MS (70 eV): (Z): 184 (19, Mþ ), 127 (100), 101 (21), 95 (28),
88 (7), 67 (15), 55 (1541 (17); (E): 184 (4, Mþ ), 153 (14), 127 (23), 114 (10), 101 (100), 95 (20), 88 (20),
84 (33), 69 (37), 55 (30), 41 (31).
Preparation of Saturated Methyl Esters. According to Suzukamo et al. [28], the respective
unsaturated ester (1 mmol) was dissolved in 10 ml of dry MeOH, and 1 mol-% PtO2 was added. The
resulting suspension was stirred, while H2 was passed through it, and the progress of the reaction was
checked with GC. After the reaction was completed, column chromatography on SiO2 with pentane/Et2O
10 :1 as eluent afforded the pure product.
Methyl 2-Methylheptanoate (27). Yield: quant. (332 mg, 2.10 mmol). Rf (pentane/Et2O 10 :1) 0.59.
GC (HP-5MS): RI 1060. 1H-NMR (400 MHz, CDCl3): 0.88 (t, J¼6.9, 3 H); 1.14 (d, J¼7.0, 3 H); 1.25–
1.46 (m, 7 H); 1.59–1.70 (m, 1 H); 2.44 (sext., J¼7.0, 1 H); 3.67 (s, 3 H). 13C-NMR (100 MHz, CDCl3):
14.0 (Me); 17.0 (Me); 22.5 (CH2); 26.9 (CH2); 31.7 (CH2); 33.8 (CH2); 39.4 (CH); 51.4 (Me); 177.4 (C).
EI-MS (70 eV): 143 (2, [Mꢀ15]þ ), 127 (6), 115 (8), 101 (25), 88 (100), 69 (5), 57 (28), 41 (12).
Methyl 2-Methyloctanoate (28). Yield: 57% (138 mg, 0.80 mmol). Rf (pentane/Et2O 10 :1) 0.53. GC
(HP-5MS): RI 1161. 1H-NMR (400 MHz, CDCl3): 0.88 (t, J¼6.9, 3 H); 1.14 (d, J¼7.0, 3 H); 1.22–1.33
(m, 8 H); 1.36–1.47 (m, 1 H); 1.61–1.69 (m, 1 H); 2.43 (sext., J¼7.0, 1 H); 3.67 (s, 3 H). 13C-NMR

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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
(100 MHz, CDCl3): 14.0 (Me); 17.1 (Me); 22.6 (CH2); 27.2 (CH2); 29.2 (CH2); 31.7 (CH2); 33.8 (CH2);
39.5 (CH); 51.4 (Me); 177.4 (C). EI-MS (70 eV): 172 (1, Mþ ), 143 (3), 141 (4), 129 (4), 115 (6), 101 (24),
88 (100), 71 (7), 57 (16), 41 (11).
Methyl 2-Methylnonanoate (29). Yield: 40% (176 mg, 0.95 mmol). Rf (pentane/Et2O 10 :1) 0.53. GC
(HP-5MS): RI 1258. 1H-NMR (400 MHz, CDCl3): 0.88 (t, J¼6.9, 3 H); 1.14 (d, J¼7.0, 3 H); 1.21–1.33
(m, 10 H); 1.36–1.45 (m, 1 H); 1.60–1.69 (m, 1 H); 2.43 (sext., J¼7.0, 1 H); 3.67 (s, 3 H). 13C-NMR
(100 MHz, CDCl3): 14.0 (Me); 17.0 (Me); 22.6 (CH2); 27.2 (CH2); 29.1 (CH2); 29.5 (CH2); 31.8 (CH2);
33.8 (CH2); 39.4 (CH); 51.4 (Me); 177.4 (C). EI-MS (70 eV): 186 (1, Mþ ), 171 (1), 157 (3), 143 (8), 129
(4), 115 (3), 101 (27), 88 (100), 69 (5), 55 (9), 41 (11).
Methyl 2-Methyldecanoate (30). Yield: 80% (285 mg, 1.43 mmol). Rf (pentane/Et2O 10 :1) 0.52. GC
(HP-5MS): RI 1359. 1H-NMR (400 MHz, CDCl3): 0.88 (t, J¼7.1, 3 H); 1.14 (d, J¼6.8, 3 H); 1.19–1.46
(m, 13 H); 1.60–1.69 (m, 1 H); 2.43 (sext., J¼7.0, 1 H); 3.67 (s, 3 H). 13C-NMR (100 MHz, CDCl3): 14.1
(Me); 17.1 (Me); 22.7 (CH2); 27.3 (CH2); 29.3 (CH2); 29.5 (2 CH2); 31.9 (CH2); 33.8 (CH2); 39.5 (CH);
51.4 (Me); 177.4 (C). EI-MS (70 eV): 200 (2, Mþ ), 169 (3), 157 (7), 143 (9), 115 (3), 101 (30), 88 (100),
69 (6), 57 (11), 41 (10).
Methyl 2-Methylundecanoate (31). Yield: 76% (237 mg, 1.11 mmol). Rf (pentane/Et2O 10 :1) 0.53.
GC (HP-5MS): RI 1459. 1H-NMR (400 MHz, CDCl3): 0.88 (t, J¼6.9, 3 H); 1.14 (d, J¼7.0, 3 H); 1.20–
1.34 (m, 14 H); 1.35–1.45 (m, 1 H); 1.60–1.69 (m, 1 H); 2.43 (sext., J¼7.0, 1 H); 3.67 (s, 3 H). 13C-NMR
(100 MHz, CDCl3): 14.1 (Me); 17.1 (Me); 22.7 (CH2); 27.2 (CH2); 29.3 (CH2); 29.5 (3 CH2); 31.9 (CH2);
33.8 (CH2); 39.5 (CH); 51.4 (Me); 177.4 (C). EI-MS (70 eV): 214 (2, Mþ ), 183 (3), 171 (5), 157 (9), 143
(7), 115 (4), 101 (32), 88 (100), 69 (6), 57 (11), 41 (10).
Methyl 2-Methyldodecanoate (32). Yield: 71% (332 mg, 1.46 mmol). Rf (pentane/Et2O 10 :1) 0.52.
GC (HP-5MS): RI 1559. 1H-NMR (400 MHz, CDCl3): 0.88 (t, J¼6.9, 3 H); 1.14 (d, J¼7.0, 3 H); 1.22–
1.33 (m, 16 H); 1.35–1.44 (m, 1 H); 1.60–1.70 (m, 1 H); 2.43 (sext., J¼7.0, 1 H); 3.67 (s, 3 H). 13C-NMR
(100 MHz, CDCl3): 14.1 (Me); 17.0 (Me); 22.7 (CH2); 27.2 (CH2); 29.3 (CH2); 29.5 (2 CH2); 29.6
(2 CH2); 31.9 (CH2); 33.8 (CH2); 39.5 (CH); 51.4 (Me); 177.4 (C). EI-MS (70 eV): 228 (3, Mþ ), 197 (2),
185 (5), 171 (6), 157 (6), 143 (7), 115 (3), 101 (34), 98 (2), 88 (100), 69 (6), 57 (10), 41 (10).
Methyl 2-Methyltridecanoate (33). Yield: 76% (226 mg, 0.93 mmol). Rf (pentane/Et2O 10 :1) 0.53.
GC (HP-5MS): RI 1660. 1H-NMR (400 MHz, CDCl3): 0.88 (t, J¼6.8, 3 H); 1.14 (d, J¼6.8, 3 H); 1.21–
1.47 (m, 19 H); 1.57–1.70 (m, 1 H); 2.43 (sext., J¼7.0, 1 H); 3.67 (s, 3 H). 13C-NMR (100 MHz, CDCl3):
14.1 (Me); 17.0 (Me); 22.7 (CH2); 27.2 (CH2); 29.3 (CH2); 29.5 (2 CH2); 29.6 (3 CH2); 31.9 (CH2); 33.8
(CH2); 39.5 (CH); 51.4 (Me); 177.4 (C). EI-MS (70 eV): 242 (4, Mþ ), 211 (2), 199 (6), 185 (6), 157 (7),
143 (9), 115 (3), 101 (36), 88 (100), 69 (7), 55 (10), 41 (10).
Methyl 2-Methyltetradecanoate (34). Yield: 73% (336 mg, 1.31 mmol). Rf (pentane/Et2O 10 :1) 0.57.
GC (HP-5MS): RI 1759. 1H-NMR (400 MHz, CDCl3): 0.88 (t, J¼6.9, 3 H); 1.14 (d, J¼7.0, 3 H); 1.21–
1.46 (m, 21 H); 1.59–1.70 (m, 2 H); 2.43 (sext., J¼7.0, 1 H); 3.67 (s, 3 H). 13C-NMR (100 MHz, CDCl3):
14.1 (Me); 17.1 (Me); 22.7 (CH2); 27.2 (CH2); 29.4 (CH2); 29.5 (2 CH2); 29.6 (3 CH2); 29.7 (CH2); 31.9
(CH2); 33.8 (CH2); 39.5 (CH); 51.4 (Me); 177.4 (C). EI-MS (70 eV): 256 (5, Mþ ), 225 (2), 213 (6), 199
(8), 157 (8), 143 (9), 115 (3), 101 (38), 88 (100), 69 (7), 55 (10), 41 (10).
Methyl 2-Methylpentadecanoate (35). Yield: 83% (250 mg, 0.93 mmol). Rf (pentane/Et2O 10 :1)
1
0.56. GC (HP-5MS): RI 1859. H-NMR (400 MHz, CDCl3): 0.88 (t, J¼6.9, 3 H); 1.14 (d, J¼7.0, 3 H);
1.21–1.46 (m, 23 H); 1.59–1.70 (m, 2 H); 2.43 (sext., J¼7.0, 1 H); 3.67 (s, 3 H). 13C-NMR (100 MHz,
CDCl3): 14.1 (Me); 17.1 (Me); 22.7 (CH2); 27.2 (CH2); 29.4 (CH2); 29.5 (2 CH2); 29.6 (3 CH2); 29.7
(2 CH2); 31.9 (CH2); 33.8 (CH2); 39.5 (CH); 51.4 (Me); 177.4 (C). EI-MS (70 eV): 270 (7, Mþ ), 239 (2),
227 (5), 213 (8), 199 (2), 171 (2), 157 (9), 143 (8), 101 (38), 88 (100), 69 (8), 55 (11), 41 (10).
Methyl 2,7-Dimethyloctanoate (43). Yield: 66% (153 mg, 0.82 mmol). Rf (pentane/Et2O 10 :1) 0.58.
GC (HP-5MS): RI 1223. 1H-NMR (400 MHz, CDCl3): 0.86 (d, J¼6.6, 6 H); 1.12–1.18 (m, 5 H); 1.24–
1.35 (m, 4 H); 1.36–1.56 (m, 2 H); 1.60–1.70 (m, 1 H); 2.43 (sext., J¼7.0, 1 H); 3.67 (s, 3 H). 13C-NMR
(100 MHz, CDCl3): 17.1 (Me); 22.6 (2 Me); 27.3 (CH2); 27.5 (CH2); 27.9 (CH); 33.9 (CH2); 38.8 (CH2);
39.5 (CH); 51.4 (Me); 177.4 (C). EI-MS (70 eV): 186 (1, Mþ ), 171 (2), 155 (3), 143 (13), 129 (3), 115 (4),
101 (40), 88 (100), 69 (13), 57 (14), 55 (13), 43 (15), 41 (18).
Methyl 2,11-Dimethyldodecanoate (44). Yield: 61% (156 mg, 0.64 mmol). Rf (pentane/Et2O 10 :1)
0.59. GC (HP-5MS): RI 1620. 1H-NMR (300 MHz, CDCl3): 0.86 (d, J¼6.6, 6 H); 1.12–1.46 (m, 10 H);

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
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1.47–1.56 (m, 1 H); 1.58–1.71 (m, 1 H); 2.43 (sext., J¼7.0, 1 H); 3.67 (s, 3 H). 13C-NMR (75 MHz,
CDCl3): 17.1 (Me); 22.6 (2 Me); 27.2 (CH2); 27.4 (CH2); 28.0 (CH); 29.5 (2 CH2); 29.6 (CH2); 29.9 (CH2);
33.8 (CH2); 39.0 (CH2); 39.5 (CH); 51.4 (Me); 177.4 (C). EI-MS (70 eV): 242 (4, Mþ ), 211 (3), 199 (11),
185 (7), 157 (8), 143 (10), 101 (41), 88 (100), 69 (9), 55 (13), 43 (15), 41 (13).
Methyl 2,6-Dimethyloctanoate (49). Yield: quant. (183 mg, 0.99 mmol). Rf (pentane/Et2O 10 :1)
1
0.55. GC (HP-5MS): RI 1229. H-NMR (400 MHz, CDCl3): 0.83–0.94 (m, 6 H); 1.07–1.15 (m, 5 H);
1.20–1.44 (m, 6 H); 1.54–1.70 (m, 1 H); 2.39–2.49 (m, 1 H); 3.67 (s, 3 H). 13C-NMR (100 MHz, CDCl3):
11.4 (Me); 17.1 (Me); 19.1 (Me); 24.7 (CH2); 29.4 (CH2); 34.1 (CH2); 34.2 (CH); 36.4 (CH2); 39.5 (CH);
51.4 (Me); 177.4 (C). EI-MS (70 eV): 186 (2, Mþ ), 157 (14), 129 (9), 115 (3), 101 (46), 88 (100), 69 (15),
55 (28), 41 (24).
Preparation of (wꢀ1)-Me-Branched Alcohols. According to the procedure of Fꢀrstner et al. [29], a
soln. of i-BuMgBr (40 mmol, 2.5 equiv.) in THF (12.5 ml), freshly prepared from Mg (40 mmol,
2.5 equiv.) and i-BuBr (40 mmol, 2.5 equiv.), was added to an ice-cooled soln. of the respective 1-
bromoalcohol (16 mmol, 1 equiv.) and Li2CuCl4 (0.1m soln. in THF, 3 mmol, 0.19 mmol). The mixture
was stirred for 1.5 h at 08 and was then allowed to warm up to r.t. Then, conc. HCl (10–15 ml) was added.
The aq. layer was separated and extracted three times with Et2O. The combined org. extracts were
successively washed with sat. NaHCO3 and brine, dried (MgSO4), and then concentrated under vacuum.
The crude product was purified by CC on SiO2 with pentane/Et2O (5 :1) as eluent to yield the pure
alcohol.
5-Methylhexan-1-ol (37). Yield: 85% (1.571 g, 13.55 mmol). Rf (pentane/Et2O 5 :1) 0.07. GC
(BPX5): RI 942. 1H-NMR (400 MHz, CDCl3): 0.88 (d, J¼6.5, 6 H); 1.16–1.22 (m, 2 H); 1.31–1.38 (m,
2 H); 1.51–1.59 (m, 3 H); 3.61–3.69 (m, 2 H). 13C-NMR (100 MHz, CDCl3): 22.5 (2 Me); 23.5 (CH2);
27.9 (CH); 32.9 (CH2); 38.7 (CH2); 62.9 (CH2). EI-MS (70 eV): 98 (1, [Mꢀ18]þ ), 83 (21), 70 (40), 69
(42), 56 (90), 55 (100), 43 (73), 42 (30), 41 (85).
9-Methyldecan-1-ol (38). Yield: 68% (1.161 g, 6.75 mmol). Rf (pentane/Et2O 5 :1) 0.07. GC (BPX5):
1
RI 1338. H-NMR (400 MHz, CDCl3): 0.86 (d, J¼6.8, 6 H); 1.12–1.17 (m, 2 H); 1.24–1.38 (m, 9 H);
1.46–1.60 (m, 4 H); 3.63 (t, J¼6.7, 2 H). 13C-NMR (100 MHz, CDCl3): 22.6 (2 Me); 25.7 (CH2); 27.4
(CH2); 27.9 (CH); 29.4 (CH2); 29.6 (CH2); 29.8 (CH2); 32.8 (CH2); 39.0 (CH2); 63.0 (CH2). EI-MS
(70 eV): 139 (2, [Mꢀ43]þ ), 126 (5), 111 (12), 98 (13), 97 (12), 83 (34), 69 (78), 57 (63), 56 (100), 55
(84), 43 (86), 41 (91).
Preparation of 4-Methylhexan-1-ol (46). The alcohol was prepared according to known methods by
LiAlH4 reduction of 4-methylhexanoic acid (0.5 g. 3.85 mmol) [22]. Aqueous workup and removal of the
solvents furnished pure 46. Yield: 75% (0.335 g, 2.89 mmol). Rf (pentane/Et2O 5 :1) 0.09. GC (BPX5):
RI 954. 1H-NMR (300 MHz, CDCl3): 0.84–0.89 (m, 6 H); 1.08–1.23 (m, 2 H); 1.28–1.42 (m, 3 H); 1.45–
1.67 (m, 2 H); 2.37 (s, 1 H); 3.60 (t, J¼6.7, 2 H). 13C-NMR (75 MHz, CDCl3): 11.7 (Me); 19.5 (Me); 29.8
(CH2); 30.6 (CH2); 32.9 (CH2); 34.6 (CH); 63.6 (CH). EI-MS (70 eV): 98 (4, Mþ ), 83 (8), 70 (100), 69
(88), 57 (27), 56 (32), 55 (40), 43 (18), 42 (19), 41 (74), 39 (17).
Preparation of (wꢀ1)- and (wꢀ2)-Me-Branched Aldehydes. The aldehydes were synthesized from
the corresponding alcohols with PCC according to the standard procedure of Corey and Suggs [30].
Purification was performed by CC on SiO2 using pentane/Et2O in the ratio mentioned for the respective
aldehyde.
5-Methylhexanal (39). Yield: 69% (1.017 g, 8.92 mmol). Rf (pentane/Et2O 3 :1) 0.61. GC (BPX5):
RI 869. 1H-NMR (400 MHz, CDCl3): 0.89 (d, J¼6.6, 6 H); 1.18–1.24 (m, 2 H); 1.50–1.68 (m, 3 H); 2.31–
2.43 (m, 2 H); 9.77 (t, J¼1.9, 1 H). 13C-NMR (100 MHz, CDCl3): 19.9 (CH2); 22.4 (2 Me); 27.8 (CH);
38.3 (CH2); 44.1 (CH2); 203.0 (CH). EI-MS (70 eV): (33, [Mꢀ18]þ ), 86 (8), 81 (38), 71 (49), 55 (81), 43
(100), 41 (90), 39 (48).
9-Methyldecanal (40). Yield: 86% (0.958 g, 5.64 mmol). Rf (pentane/Et2O 2 :1) 0.75. GC (BPX5): RI
1267. 1H-NMR (400 MHz, CDCl3): 0.86 (d, J¼6.8, 6 H); 1.12–1.17 (m, 2 H); 1.24–1.35 (m, 8 H); 1.44–
1.57 (m, 1 H); 1.63 (quint., J¼7.3, 2 H); 2.42 (dt, J¼1.9, 7.3, 2 H); 9.77 (t, J¼1.9, 1 H). 13C-NMR
(100 MHz, CDCl3): 22.1 (CH2); 22.6 (2 Me); 27.3 (CH2); 27.9 (CH); 29.2 (CH2); 29.4 (CH2); 29.7 (CH2);
39.0 (CH2); 43.9 (CH2); 202.9 (CH). EI-MS (70 eV): 152 (1, [Mꢀ18]þ ), 142 (3), 137 (6), 124 (9), 109
(25), 96 (35), 95 (39), 82 (49), 81 (41), 69 (60), 57 (89), 43 (94), 41 (100).

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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
4-Methylhexanal (47). Rf (pentane/Et2O 3 :1) 0.60. 1H-NMR (200 MHz, CDCl3): 0.84–0.92 (m,
6 H); 1.09–1.55 (m, 4 H); 1.57–1.76 (m, 1 H); 2.30–2.49 (m, 2 H); 9.78 (t, J¼1.9, 1 H). 13C-NMR
(50 MHz, CDCl3): 11.9 (Me); 19.5 (Me); 29.2 (CH2); 29.8 (CH2); 34.7 (CH); 42.4 (CH2); 203.6 (CH). EI-
MS (70 eV): 96 (13, Mþ ), 81 (17), 71 (29), 70 (100), 57 (63), 55 (63), 43 (31), 42 (19), 41 (78), 39 (29).
Preparation of 7-Methyloctanoyl Chloride (55). According to the procedure of Spessard et al. [34], 7-
methyloctanoic acid (54) (140 mg, 0.89 mmol) was dissolved in 5 ml of dry Et2O and cooled to 08. Oxalyl
chloride (225 mg, 1.77 mmol) was added dropwise, and the mixture was stirred for 24 h at r.t. The solvent
and excess oxalyl chloride were removed under vacuum to produce pure 55 (125 mg, 0.71 mmol, 80%). Rf
(pentane/Et2O 2 :1) 0.93. GC (BPX5): RI 1202. 1H-NMR (400 MHz, CDCl3): 0.87 (d, J¼6.5, 6 H); 1.14–
1.19 (m, 2 H); 1.26–1.38 (m, 4 H); 1.52 (non., J¼6.6, 1 H); 1.63 (quint., J¼7.3, 2 H); 2.88 (t, J¼7.3, 2 H).
13C-NMR (100 MHz, CDCl3): 22.6 (2 Me); 25.1 (CH2); 26.8 (CH2); 27.9 (CH); 28.7 (CH2); 38.6 (CH2);
47.1 (CH2); 173.9 (C). EI-MS (70 eV): 141 (15, [Mꢀ35/37]þ ), 125 (18), 123 (17), 107 (16), 98 (33), 97
(42), 96 (30), 84 (55), 69 (48), 55 (100), 43 (93), 41 (73).
Preparation of Evans Chiral Auxiliary Acid Derivatives. As described by Gage and Evans [31], a
soln. of (4S)-4-benzyl-1,3-oxazolidin-2-one (1 equiv.) in THF (3 ml/mmol) was cooled to ꢀ788. Then,
BuLi (1.6m in hexane, 1 equiv.) was added, and the resulting mixture was stirred for 30 min under these
conditions. The respective octanoyl chloride (1.1 equiv.) was added dropwise, and the stirring was
continued for 4 h at ꢀ788. The mixture was allowed to warm up to r.t., and the reaction was quenched by
the addition of sat. NH4Cl soln. Then, the org. solvents were removed under vacuum, and the resulting aq.
layer was extracted three times with CH2Cl2. The combined org. layers were successively washed with 1m
NaOH soln. and brine, dried (MgSO4), and concentrated under vacuum. Purification of the crude
product by CC on SiO2 with pentane/Et2O (5 :1) furnished the pure product.
(4S)-4-Benzyl-3-octanoyl-1,3-oxazolidin-2-one (51). Yield: 84% (2.54 g, 8.38 mmol). Rf (pentane/
Et2O 5 :1) 0.14. GC (BPX5): RI 2459. 1H-NMR (400 MHz, CDCl3): 0.89 (t, J¼6.9, 3 H); 1.25–1.42 (m,
8 H); 1.63–1.77 (m, 2 H); 2.77 (dd, J¼9.7, J¼13.4, 1 H); 2.86–3.01 (m, 2 H); 3.30 (dd, J¼3.4, 13.4, 1 H);
4.14–4.22 (m, 2 H); 4.64–4.70 (m, 1 H); 7.20–7.35 (m, 5 H). 13C-NMR (100 MHz, CDCl3): 14.1 (Me);
22.6 (CH2); 24.2 (CH2); 29.0 (CH2); 29.1 (CH2); 31.7 (CH2); 35.5 (CH2); 37.9 (CH2); 55.1 (CH); 66.1
(CH2); 127.3 (CH); 128.9 (2 CH); 129.4 (2 CH); 135.3 (C); 153.4 (C); 173.4 (C). EI-MS (70 eV): 303 (6,
Mþ ), 232 (3), 219 (8), 212 (18), 178 (4), 127 (100), 117 (10), 109 (4), 91 (17), 57 (32), 41 (9).
(4S)-4-Benzyl-3-(7-methyloctanoyl)-1,3-oxazolidin-2-one (56). Yield: 48% (96 mg, 0.31 mmol). Rf
(pentane/Et2O 5 :1) 0.12. GC (BPX5): RI 2521. 1H-NMR (400 MHz, CDCl3): 0.87 (d, J¼6.8, 6 H); 1.15–
1.21 (m, 2 H); 1.29–1.41 (m, 4 H); 1.48–1.58 (m, 1 H); 1.66–1.74 (m, 2 H); 2.77 (dd, J¼9.6, 13.1, 1 H);
2.85–3.01 (m, 2 H); 3.30 (dd, J¼3.3, 13.4, 1 H); 4.15–4.22 (m, 2 H); 4.64–4.69 (m, 1 H); 7.20–7.36 (m,
5 H). 13C-NMR (100 MHz, CDCl3): 22.6 (2 Me); 24.3 (CH2); 27.2 (CH2); 27.9 (CH); 29.4 (CH2); 35.6
(CH2); 38.0 (CH2); 38.8 (CH2); 55.2 (CH); 66.1 (CH2); 127.3 (CH); 129.0 (2 CH); 129.4 (2 CH); 135.3
(C); 153.5 (C); 173.5 (C). EI-MS (70 eV): 317 (13, Mþ ), 302 (2), 226 (33), 219 (19), 178 (18), 141 (100),
123 (78), 117 (27), 91 (37), 81 (28), 71 (15), 67 (14), 57 (20), 55 (23), 43 (28), 41 (20).
Preparation of Methylated Evans Chiral Auxiliary Acid Derivatives. According to the procedure of
Siebum et al. [32], a soln. of the acylated Evans oxazolidinone (1 equiv.) in dry THF (3 ml/mmol) was
added dropwise to a cold (ꢀ 788) soln. of NaHMDS (1.0m soln. in THF, 2.5 equiv.) in THF (3 ml/mmol).
The resulting mixture was stirred for 1 h at ꢀ788, and then MeI (2 equiv.) was added. The mixture was
stirred for 4 h at ꢀ788 and then slowly allowed to warm up to r.t. Workup was carried out by the addition
of sat. NH4Cl soln., H2O, and 1m H2SO4 to adjust the pH to 1–2. The aq. layer was first separated and then
extracted three times with Et2O. The combined org. extracts were successively washed with sat. NaHCO3,
sat. Na2S2O3, and brine, dried (MgSO4), and then concentrated applying vacuum. The crude product
(diastereoisomeric ratio 98 :2 (52) and 96 :4 (57), resp.) was purified by CC on SiO2 with pentane/Et2O
(gradient from 10 :1 to 7:1) as eluent. The obtained products were diastereoisomerically almost pure.
(4S)-4-Benzyl-3-[(2S)-2-methyloctanoyl]-1,3-oxazolidin-2-one (52). Diastereoisomeric ratio: 99 :1.
Yield: 66% (859 mg, 8.38 mmol). Rf (pentane/Et2O 10 :1) 0.13. GC (BPX5): RI 2442. 1H-NMR
(400 MHz, CDCl3): 0.87 (t, J¼6.8, 3 H); 1.22 (d, J¼6.8, 3 H); 1.24–1.35 (m, 6 H); 1.36–1.46 (m, 2 H);
2.77 (dd, J¼9.6, 13.4, 1 H); 3.27 (dd, J¼3.3, 13.1, 1 H); 3.71 (sext., J¼6.8, 1 H); 4.14–4.22 (m, 2 H);
4.64–4.70 (m, 1 H); 7.20–7.35 (m, 5 H). 13C-NMR (100 MHz, CDCl3): 14.0 (Me); 17.3 (Me); 22.6 (CH2);
27.2 (CH2); 29.3 (CH2); 31.7 (CH2); 33.4 (CH2); 37.7 (CH); 37.9 (CH2); 55.3 (CH); 66.0 (CH2); 127.3

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
2251
(CH); 128.9 (2 CH); 129.4 (2 CH); 135.3 (C); 153.0 (C); 177.3 (C). EI-MS (70 eV): 317 (5, Mþ ), 233
(18), 226 (20), 178 (14), 141 (100), 134 (7), 117 (21), 113 (22), 91 (39), 86 (21), 71 (65), 57 (57), 43 (27),
41 (32).
(4S)-4-Benzyl-3-[(2S)-2,7-dimethyloctanoyl]-1,3-oxazolidin-2-one (57). Diastereoisomeric ratio:
1
100 :0. Yield: 46% (70 mg, 0.22 mmol). Rf (pentane/Et2O 5 :1) 0.12. GC (BPX5): RI 2494. H-NMR
(400 MHz, CDCl3): 0.86 (d, J¼6.8, 6 H); 1.12–1.19 (m, 2 H); 1.22 (d, J¼6.8, 3 H); 1.25–1.33 (m, 4 H);
1.37–1.56 (m, 2 H); 1.70–1.79 (m, 1 H); 2.77 (dd, J¼9.7, 13.4, 1 H); 3.27 (dd, J¼3.1, 13.4, 1 H); 3.67–
3.75 (m, 1 H); 4.15–4.22 (m, 2 H); 4.65–4.70 (m, 1 H); 7.21–7.35 (m, 5 H). 13C-NMR (100 MHz,
CDCl3): 17.4 (Me); 22.6 (2 Me); 27.4 (CH2); 27.5 (CH2); 27.9 (CH); 33.5 (CH2); 37.7 (CH); 37.9 (CH2);
38.8 (CH2); 55.4 (CH); 66.0 (CH2); 127.3 (CH); 128.9 (2 CH); 129.4 (2 CH); 135.3 (C); 153.1 (C); 177.4
(C). EI-MS (70 eV): 331 (5, Mþ ), 316 (2), 240 (19), 233 (23), 178 (22), 155 (100), 134 (8), 133 (8), 127
(22), 117 (22), 91 (30), 85 (34), 71 (50), 57 (30), 43 (29), 41 (23).
Preparation of Carboxylic Acids from Acylated Evans Oxazolidinones. As reported by Siebum et al.
[32], a H2O2 soln. (30% in H2O, 5 equiv.) and LiOH (2.5 equiv.), dissolved in H2O (0.5 ml/mmol), were
added at 08 to a soln. of the respective oxazolidinone (1 equiv.) in THF (10 ml/mmol) and H2O (5 ml/
mmol). The mixture was stirred for 2 h under these conditions. Sodium sulfite in H2O (1.9 equiv., 4 ml/
mmol) was added, and the mixture was stirred for 15 min at 08. The soln. was adjusted to pH 9–10 by the
addition of sat. NaHCO3 soln. THF was evaporated, and the residual aq. layer was extracted two times
with CH2Cl2. The combined org. phases were dried (MgSO4) and then concentrated under vacuum to
give the recovered auxiliary (4S)-4-benzyl-1,3-oxazolidin-2-one. The aq. soln. was acidified to pH 1–2
with 1m H2SO4 soln. and extracted three times with Et2O. The combined org. extracts were dried
(MgSO4), and the solvents were evaporated to furnish the respective pure carboxylic acid.
(2S)-2-Methyloctanoic Acid (53). Yield: quant. (300 mg, 1.90 mmol). GC (BPX5): RI 1269.
1H-NMR (400 MHz, CDCl3): 0.88 (t, J¼6.9, 3 H); 1.18 (d, J¼6.8, 3 H); 1.24–1.37 (m, 8 H); 1.39–1.47
(m, 1 H); 1.64–1.73 (m, 1 H); 2.46 (sext., J¼6.9, 1 H); 10.52 (br. s, 1 H). 13C-NMR (100 MHz, CDCl3):
14.0 (Me); 16.8 (Me); 22.6 (CH2); 27.1 (CH2); 29.2 (CH2); 31.7 (CH2); 33.5 (CH2); 39.4 (CH); 183.4 (C).
EI-MS (70 eV): 158 (1, Mþ ), 129 (5), 115 (6), 101 (8), 87 (30), 74 (100), 69 (5), 55 (11), 43 (14), 41 (17).
(2S)-2,7-Dimethyloctanoic Acid (58). Yield: 89% (32 mg, 0.19 mmol). GC (BPX5): RI 1337.
1H-NMR (400 MHz, CDCl3): 0.86 (d, J¼6.6, 6 H); 1.13–1.23 (m, 5 H); 1.24–1.35 (m, 4 H); 1.39–1.56
(m, 2 H); 1.64–1.73 (m, 1 H); 2.46 (sext., J¼7.0, 1 H). 13C-NMR (100 MHz, CDCl3): 16.8 (Me); 22.6
(2 Me); 27.3 (CH2); 27.4 (CH2); 27.9 (CH); 33.6 (CH2); 38.8 (CH2); 39.3 (CH); 183.0 (C). EI-MS (70 eV):
172 (1, Mþ ), 157 (4), 129 (21), 115 (6), 101 (4), 87 (56), 74 (100), 69 (20), 57 (16), 55 (21), 43 (25), 41
(28).
Preparation of Methyl (2S)-2-Methyloctanoate ((S)-28). Similar to the procedure of Wipf et al. [33],
the acid 53 (258 mg, 1.63 mmol) was dissolved in an excess of dry MeOH (16 ml), and a few drops of
conc. HCl were added. The mixture was stirred under reflux for 4 h, concentrated, and then filtered
through SiO2 with Et2O as eluent. After drying (MgSO4), all solvents were removed under vacuum to
give the pure product (257 mg, 1.49 mmol, 92%). Rf (pentane/Et2O 10 :1) 0.53. GC (HP-5MS): RI 1161.
1H-NMR (400 MHz, CDCl3): 0.88 (t, J¼6.9, 3 H); 1.14 (d, J¼7.0, 3 H); 1.22–1.33 (m, 8 H); 1.36–1.45
(m, 1 H); 1.61–1.69 (m, 1 H); 2.43 (sext., J¼7.0, 1 H); 3.67 (s, 3 H). 13C-NMR (100 MHz, CDCl3): 14.0
(Me); 17.0 (Me); 22.6 (CH2); 27.2 (CH2); 29.1 (CH2); 31.7 (CH2); 33.8 (CH2); 39.4 (CH); 51.4 (Me);
177.4 (C). EI-MS (70 eV): EI-MS (70 eV): 172 (1, Mþ ), 143 (5), 141 (4), 129 (4), 115 (6), 101 (25), 88
(100), 71 (6), 57 (17), 41 (12).
Preparation of Methyl (2S)-2,7-Dimethyloctanoate ((S)-43). According to Patel et al. [35],
compound 58 (16 mg, 0.09 mmol) was dissolved in 3.5 ml of dry CH2Cl2. A few drops of MeOH were
added to the soln. before it was cooled to 08. DMAP (15 mg, 0.01 mmol) and EDC were added. The
mixture was stirred for 1 h under these conditions and for 1.5 h at r.t. Then, Et2O was added, and the org.
layer was successively washed with H2O and sat. NaHCO3 soln. The combined org. layers were dried
(MgSO4) and concentrated under vacuum. Purification on SiO2 with pentane/Et2O 2 :1 as eluent
afforded the pure product (11 mg, 0.57 mmol, 61%). Rf (pentane/Et2O 10 :1) 0.58. GC (HP-5MS): RI
1223. 1H-NMR (400 MHz, CDCl3): 0.86 (d, J¼6.6, 6 H); 1.12–1.18 (m, 5 H); 1.24–1.35 (m, 4 H); 1.36–
1.56 (m, 2 H); 1.60–1.70 (m, 1 H); 2.43 (sext., J¼7.0, 1 H); 3.67 (s, 3 H). 13C-NMR (100 MHz, CDCl3):
17.1 (Me); 22.6 (2 Me); 27.3 (CH2); 27.5 (CH2); 27.9 (CH); 33.9 (CH2); 38.8 (CH2); 39.5 (CH); 51.4 (Me);

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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
177.4 (C). EI-MS (70 eV): 186 (1, Mþ ), 171 (2), 155 (3), 143 (14), 129 (3), 115 (4), 101 (41), 88 (100), 69
(13), 57 (14), 55 (13), 43 (14), 41 (17).
REFERENCES
[1] T. Kaneda, Microbiol. Rev. 1991, 55, 288.
[2] S. Schulz, J. S. Dickschat, Nat. Prod. Rep. 2007, 24, 814.
[3] J. S. Dickschat, H. B. Bode, S. C. Wenzel, R. Mꢀller, S. Schulz, ChemBioChem 2005, 6, 2023.
[4] C. E. G. Schçller, H. Gꢀrtler, R. Pedersen, S. Molin, K. Wilkins, J. Agric. Food Chem. 2002, 50, 2615.
[5] R. Coda, E. Brechany, M. De Angelis, S. De Candia, R. Di Cagno, M. Gobbetti, J. Dairy Sci. 2006,
89, 4126.
[6] K. Gerth, H. Steinmetz, G. Hçfle, to Helmholtz-Zentrum fꢀr Infektionsforschung, European Patent
Application (09/103534), 2009.
[7] B. Maudinas, J. Villoutreix, Phytochemistry 1977, 16, 1299.
[8] A. Sawabe, Y. Matsuda, H. Toyoda, K. Maeda, T. Nonomura, N. Shimizu, N. Fujita, S. Ouchi, J. Oleo
Sci. 2003, 52, 171.
[9] M. D. Forero, C. E. Quijano, J. A. Pino, Flavour Fragr. J. 2009, 24, 25.
[10] H. Tsuji, A. Mizuno, J. Food Sci. 2010, 75, C79.
[11] W. Boland, P. Ney, L. Jaenicke, G. Gassmann, in ꢃAnalysis of Volatilesꢄ, Ed. P. Schreier, de Gruyter,
Berlin, 1984, p. 371.
[12] J. S. Dickschat, T. Martens, T. Brinkhoff, M. Simon, S. Schulz, Chem. Biodiversity 2005, 2, 837.
[13] T. Nawrath, K. Gerth, R. Mꢀller, S. Schulz, ChemBioChem 2010, in press, doi: 10.1002/
cbic.201000296.
[14] H. Budzikiewicz, C. Djerassi, D. H. Williams, ꢃMass spectrometry of organic compoundsꢄ, Holden-
Day, San Francisco, 1967.
[15] N. M. Carballeira, F. Shalabi, C. Cruz, Tetrahedron Lett. 1994, 35, 5575.
[16] B. A. Leonhardt, E. D. DeVilbiss, J. Chromatogr., A 1985, 322, 484.
[17] V. Thiel, B. Kunze, P. Verma, I. Wagner-Dçbler, S. Schulz, ChemBioChem 2009, 10, 1861.
ˇ
[18] F. W. McLaffery, F. Turecek, ꢃInterpretation of mass spectraꢄ, University Science Books, Sausalito,
1993.
[19] C. C. van de Sande, C. DeMeyer, A. Maquestiau, Bull. Soc. Chim. Belges 1976, 85, 79.
[20] H. van den Dool, P. D. Kraatz, J. Chromatogr., A 1963, 11, 463.
[21] S. Schulz, Lipids 2001, 36, 637.
[22] J. S. Dickschat, S. C. Wenzel, H. B. Bode, R. Mꢀller, S. Schulz, ChemBioChem 2004, 5, 778.
[23] J. S. Dickschat, E. Helmke, S. Schulz, Chem. Biodiversity 2005, 2, 318.
[24] J. S. Dickschat, H. B. Bode, R. M. Kroppenstedt, R. Mꢀller, S. Schulz, Org. Biomol. Chem. 2005, 3,
2824.
[25] S. Schulz, unpublished results.
[26] R. Ryhage, E. Stenhagen, Ark. Kemi 1960, 15, 291.
[27] J. S. Dickschat, T. Nawrath, V. Thiel, B. Kunze, R. Mꢀller, S. Schulz, Angew. Chem., Int. Ed. 2007, 46,
8287.
[28] G. Suzukamo, M. Fukao, M. Tamura, Tetrahedron Lett. 1984, 25, 1595.
[29] A. Fꢀrstner, J. Ruiz-Caro, H. Prinz, H. Waldmann, J. Org. Chem. 2004, 69, 459.
[30] E. J. Corey, J. W. Suggs, Tetrahedron Lett. 1975, 16, 2647.
[31] J. R. Gage, D. A. Evans, Org. Synth. 1990, 68, 83.
[32] A. H. G. Siebum, W. S. Woo, J. Lugtenburg, Eur. J. Org. Chem. 2003, 4664.
[33] P. Wipf, Y. Uto, S. Yoshimura, Chem.–Eur. J. 2002, 8, 1670.
[34] G. O. Spessard, W. K. Chan, S. Masamune, Org. Synth. 1983, 61, 134.
[35] J. Patel, J. C. Hoyt, R. J. Parry, Tetrahedron 1998, 54, 15927.
[36] T. Kaneda, Bacteriol. Rev. 1977, 41, 391.
[37] H. B. Bode, J. S. Dickschat, R. M. Kroppenstedt, S. Schulz, R. Mꢀller, J. Am. Chem. Soc. 2005, 127,
532.

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
2253
[38] T. Ramakrishnan, P. S. Murthy, K. P. Gopinathan, Bacteriol. Rev. 1972, 36, 65.
[39] D. L. Rainwater, P. E. Kolattukudy, J. Biol. Chem. 1985, 260, 616.
[40] P. M. Dewick, ꢃMedicinal Natural Products: A Biosynthetic Approachꢄ, 3rd edn., John Wiley &
Sons, Chichester, 2009.
[41] Y. Akamatsu, J. H. Law, J. Biol. Chem. 1970, 245, 709.
[42] I. Mꢀller, S. Weinig, H. Steinmetz, B. Kunze, S. Veluthoor, T. Mahmud, R. Mꢀller, ChemBioChem
2006, 7, 1197.
[43] L. E. Eiden, R. T. Borchart, C. O. Rutledge, J. Neurochem. 1982, 38, 631.
[44] J. Fourche, M. Capdepuy, J. Texier-Maugein, J. Chromatogr., B. 1989, 487, 142.
[45] V. S. Dubey, T. D. Sirakova, M. H. Cynamon, P. E. Kolattukudy, J. Bacteriol. 2003, 185, 4620.
[46] B. Flachsbarth, M. Fritzsche, P. J. Weldon, S. Schulz, Chem. Biodiversity 2009, 6, 1.
[47] A. Friese, K. Hell-Momeni, I. Zꢀndorf, T. Winckler, T. Dingermann, G. Dannhardt, J. Med. Chem.
2002, 45, 1535.
Received July 2, 2010