Novel fatty acid methyl esters from the actinomycete Micromonospora aurantiaca

Article abstract of DOI:10.3762/bjoc.7.200

The volatiles released by Micromonospora aurantiaca were collected by means of a closed-loop stripping apparatus (CLSA) and analysed by GC-MS. The headspace extracts contained more than 90 compounds from different classes. Fatty acid methyl esters (FAMEs) comprised the major compound class including saturated unbranched, monomethyl and dimethyl branched FAMEs in diverse structural variants: Unbranched, α-branched, γ-branched, (ω-1)-branched, (ω-2)-branched, α-and (ω-1)-branched, γ-and (ω-1)-branched, γ-and (ω-2)-branched, and γ-and (ω-3)-branched FAMEs. FAMEs of the last three types have not been described from natural sources before. The structures for all FAMEs have been suggested based on their mass spectra and on a retention index increment system and verified by the synthesis of key reference compounds. In addition, the structures of two FAMEs, methyl 4,8-dimethyldodecanoate and the ethyl-branched compound methyl 8-ethyl-4-methyldodecanoate were deduced from their mass spectra. Feeding experiments with isotopically labelled [ ²H₁₀]leucine, [²H₁₀]isoleucine, [²H₈]valine, [²H₅]sodium propionate, and [methyl-²H₃]methionine demonstrated that the responsible fatty acid synthase (FAS) can use different branched and unbranched starter units and is able to incorporate methylmalonyl-CoA elongation units for internal methyl branches in various chain positions, while the methyl ester function is derived from S-adenosyl methionine (SAM).

Full text of DOI:10.3762/bjoc.7.200

Novel fatty acid methyl esters from the actinomycete
Micromonospora aurantiaca
Jeroen S. Dickschat*, Hilke Bruns and Ramona Riclea
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2011, 7, 1697–1712.
Institut für Organische Chemie, Technische Universität Braunschweig,
Hagenring 30, 38106 Braunschweig, Germany
doi:10.3762/bjoc.7.200
Received: 20 August 2011
Accepted: 28 November 2011
Published: 20 December 2011
Email:
Jeroen S. Dickschat* - j.dickschat@tu-braunschweig.de
* Corresponding author
This article is part of the Thematic Series "Biosynthesis and function of
secondary metabolites".
Keywords:
actinomycetes; FAMEs; fatty acid biosynthesis; GC–MS; volatiles
Associate Editor: S. Flitsch
© 2011 Dickschat et al; licensee Beilstein-Institut.
License and terms: see end of document.
Abstract
The volatiles released by Micromonospora aurantiaca were collected by means of a closed-loop stripping apparatus (CLSA) and
analysed by GC–MS. The headspace extracts contained more than 90 compounds from different classes. Fatty acid methyl esters
(FAMEs) comprised the major compound class including saturated unbranched, monomethyl and dimethyl branched FAMEs in
diverse structural variants: Unbranched, α-branched, γ-branched, (ω−1)-branched, (ω−2)-branched, α- and (ω−1)-branched, γ- and
(ω−1)-branched, γ- and (ω−2)-branched, and γ- and (ω−3)-branched FAMEs. FAMEs of the last three types have not been
described from natural sources before. The structures for all FAMEs have been suggested based on their mass spectra and on a
retention index increment system and verified by the synthesis of key reference compounds. In addition, the structures of two
FAMEs, methyl 4,8-dimethyldodecanoate and the ethyl-branched compound methyl 8-ethyl-4-methyldodecanoate were deduced
from their mass spectra. Feeding experiments with isotopically labelled [2H10]leucine, [2H10]isoleucine, [2H8]valine, [2H5]sodium
propionate, and [methyl-2H3]methionine demonstrated that the responsible fatty acid synthase (FAS) can use different branched and
unbranched starter units and is able to incorporate methylmalonyl-CoA elongation units for internal methyl branches in various
chain positions, while the methyl ester function is derived from S-adenosyl methionine (SAM).
Introduction
Lipids in general, and particularly fatty acids (FAs), belong to earth. In bacteria the cell membranes are mainly formed from
the most important building blocks of biological systems. They phospholipids such as phosphatidylcholines that contain a FA
fulfill various physiological functions, such as cell-membrane diglyceride, a phosphate, and a phophate-bound choline. The
assembly or, as highly reduced carbon compounds, energy simplest type of phospholipid is made up from unbranched satu-
storage, and are therefore found in every single living cell on rated FAs with typical chain lengths of 16 or 18 carbon atoms,
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but sometimes also shorter or longer FAs can be found. The with equal functions to the individual and respective megasyn-
fluidity of bacterial cell membranes can be tuned, e.g., by the thase domains. In both cases a starter unit, usually acetyl-CoA,
introduction of methyl branches or olefinic double bonds [1]. is selected by the acetyl transferase (AT) and loaded onto an
acyl-carrier-protein (ACP), or, more precisely, onto the thiol
The biosynthesis of FAs is a repetitive chain elongation process end of a phosphopantetheinyl linker that is attached to a highly
catalysed in animals and fungi by multifunctional megasyn- conserved serine residue of the ACP (Scheme 1A). The acetyl
thases, and in plants or bacteria by a set of discrete enzymes moiety is then taken over by a conserved cystein residue of the
Scheme 1: Fatty acid biosynthesis.
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ketosynthase (KS) making the ACP in turn available for the (bp 118 °C) and methyl acetate (bp 57 °C). Some bacteria can
uptake of an elongation unit, in most cases malonyl-CoA, which methylate FAs to yield the corresponding methyl esters,
is again selected and transferred by the AT. The reaction resulting not only in an increased volatility, but making them at
between the ACP-bound malonyl and the KS-bound acetyl the same time unavailable for other biosynthetic transforma-
group under decarboxylation conditions results in the formation tions. Such fatty acid methyl esters (FAMEs) have previously
of acetoacetyl-S~ACP by release of the KS. A three-step reduc- been reported as headspace constituents of diverse bacteria [10].
tive process involving the subsequent actions of a ketoreduc- The saturated compounds methyl butanoate (1), methyl isobu-
tase (KR), a dehydratase (DH), and an enoyl reductase (ER) tyrate (2), methyl 2-methylbutyrate (3), methyl isovalerate (4),
yields butyryl-S~ACP via (R)-3-hydroxybutyryl-S~ACP and methyl 2-methylpentanoate (5), methyl isocaproate (6), and
crotyl-S~ACP. In summary of these transformations, the starter methyl 3-methylpentanoate (7) were found in actinomycetes
unit is elongated by two fully reduced carbon atoms, and n [11,12]. Methyl 9-methyldecanoate (8) is released by the
iterations of this elongation procedure yield a fatty acyl-S~ACP myxobacterium Stigmatella aurantiaca DW4/3-1 [13]. A com-
product with a chain length of (2n + 2) carbon atoms, with the plex mixture of several methyl 2-methylalkanoates (9–26) was
final chain length being solely dependent on the size of the recently reported from the gliding bacterium Chitinophaga
acting FAS’s active site (although FA biosynthesis is catalysed Fx7914 [14]. Some α,β-unsaturated FAMEs have also been
by several discrete enzymes in bacteria, the term FAS, strictly described, such as methyl 4-methylpent-2-enoate (27), methyl
speaking short for fatty acid synthase and thereby implying the tiglate (28), and methyl 3-methylcrotonate (29) from actino-
action of only one single enzyme, will be used here for the mycetes [12], and various methyl 2-methylalk-2-enoates
complete bacterial FA biosynthetic machinery for reasons of (30–43) from Chitinophaga [14]. The proposed building blocks
brevity and simplicity). Product release from the ACP is for the biosynthesis of these methyl esters are highlighted in
achieved by action of a thioesterase (TE) to provide the bold and by use of a colour code in Figure 1. For the methyl
unbound FA. The combination of an acetyl-CoA starter and 2-methylalkanoates and -alk-2-enoates from Chitinophaga, the
malonyl-CoA elongators always leads to unbranched FAs with origin of the methyl group from S-adenosyl methionine (SAM,
an even number of carbon atoms (even FAs). Structural varia- purple) and of the 2-methyl groups from methylmalonyl-CoA
tions are possible through the use of alternative starters, such as was determined by feeding experiments [14].
propionyl-CoA, for the synthesis of odd FAs (Scheme 1B). The
branched amino acids valine and leucine provide, by transami- During our ongoing analysis of the volatiles released by
nation and oxidative decarboxylation, the iso-branched starters different bacteria and fungi with high potential for secondary
isobutyryl-CoA (red) for iso-even FAs and isovaleryl-CoA metabolism, the actinomycete Micromonospora aurantiaca
(blue) for iso-odd FAs, whereas the same reactions from ATCC 27029 came to our attention. This gram-positive, sporu-
isoleucine yield (S)-2-methylbutyryl-CoA (green) for anteiso- lating bacterial genus includes producers of important anti-
odd FA biosynthesis. Internal methyl branches can be intro- biotics such as the aminoglycoside gentamycin [15] and the
duced through the use of methylmalonyl-CoA elongation units, antitumor antibiotics lomaiviticins A and B (Micromonospora
and occur due to the logic of FA biosynthesis in even-numbered lomaivitiensis) [16]. Here the results of the headspace analyses
positions of the FA carbon chain. An alternative mechanism of M. aurantiaca are described. Besides compounds from other
leading to the same methyl branching pattern is well-known classes, several methyl esters were identified. The identifica-
from polyketide biosynthesis and involves the incorporation of tion of these esters from their mass spectra and retention
a malonyl-CoA unit followed by SAM-dependent methylation indices, as well as the verification of the proposed structures by
of the new α-carbon. Further alternative starters are known synthesis of representative reference compounds is presented.
[2-4], but these cases are rare. In contrast, the usage of alter-
native elongation units such as ethylmalonyl-CoA [5], propyl- Besides several other compounds, such as terpenes, pyrazines,
malonyl-CoA [6], isobutylmalonyl-CoA [7], or methoxy- aromatic compounds and methyl ketones, more than half of the
malonyl-ACP [8] remains almost limited to polyketide syn- components identified are unbranched and mono- and dimethyl-
thesis and is only found in very exceptional cases of fatty acid branched FAMEs, many of this last group having not been
biosynthesis [9].
reported before.
Results and Discussion
Due to their ability to participate in hydrogen bonds and to form
stabilised dimers, carboxylic acids have relatively low vapour Volatiles from Micromonospora aurantiaca
pressures and, therefore, high boiling points. The volatility of The volatiles released by agar plate cultures of the actino-
carboxylic acids can be increased by their transformation into mycete M. aurantiaca ATCC 27029 were collected by use of a
methyl esters, e.g., compare the boiling points of acetic acid closed-loop stripping apparatus (CLSA), as described previ-
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Figure 1: Volatile methyl esters from bacteria.
ously [17,18], and the obtained headspace extracts were isobutyric acid (49), isovaleric acid (50), and 2-methylbutyric
analysed by GC–MS. The structures of the identified com- acid (51) as the main compounds, with minor amounts of
pounds (apart from FAMEs, which will be discussed below) are 5-methylhexanoic acid (55) and 4-methylhexanoic acid (56), the
shown in Figure 2, the chromatogram of a representative sample α,β-unsaturated 3-methylbut-2-enoic acid (53) and 2-methylbut-
is presented in Figure 3A, and the full results from two extracts 2-enoic acid (54), a homologous series of unbranched saturated
are summarised in Table S1 of Supporting Information File 1. compounds from butyric acid to decanoic acid (57–63), and the
M. aurantiaca released more than 90 compounds from different unusual 2,2-dimethylpropanoic acid (52). Compound 51 was
compound classes including carboxylic acids, FAMEs, lactones, accompanied by minor amounts of its methyl ester 3. The acids
alcohols, aldehydes, acyloins, nitrogen and oxygen hetero- 49–51 can arise from the amino acids valine, leucine, and
cycles, aromatic compounds, and terpenoids. Carboxylic acids isoleucine, respectively, by transamination and oxidative decar-
were dominating, and this class was composed of the branched boxylation, whereas 52 is equally available from the unusual
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Figure 2: Compounds found in the headspace extracts of M. aurantiaca.
tert-leucine. The acids 53 and 54 can arise from their saturated Acyloins were recently identified as precursors for pyrazines
counterparts 50 and 51 by dehydrogenation. A two-carbon elon- from Corynebacterium glutamicum in our laboratory [19]. The
gation of 50 and 51 by means of the fatty acid biosynthetic M. aurantiaca headspace extracts contained methylpyrazine
pathway can generate 55 and 56, while their reduction to the (64), 2,5-dimethylpyrazine (65), trimethylpyrazine (66),
respective alcohols provides 47 and 48. Acetoin (44) was also 2-ethyl-3,6-dimethylpyrazine (67), and 2-ethyl-3,5-
found as a major compound, together with its derivatives dimethylpyrazine (68), which may also arise from acyloins.
3-hydroxypentan-2-one (45) and 2-hydroxypentan-3-one (46). Further identified compounds were 2-acetylpyrrole (69),
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Figure 3: Total ion chromatograms of the headspace extract from M. aurantiaca (A), and expansions of the total ion chromatogram (18–36 min)
showing the relevant peaks of the FAMEs (B–D, for clarity the same expansion is shown in three replicates for FAMEs from different series). The
colour code used for these series of FAMEs is given in the respective figures.
2-phenylethanol (70), phenylacetone (71), 1-phenylbutan-2-one Besides the compounds mentioned above, several saturated
(72), methyl phenylacetate (73), methyl salicylate (74), methyl FAMEs were present in the headspace extracts (Figure 4). All
furan-2-carboxylate (75), 2-acetyl-5-methylfuran (76), decanal the identified FAMEs were divided into groups according to
(77), 7-methyloctan-4-olide (78), nonan-4-olide (79), and the their pattern of methyl branchings. Unbranched FAMEs
terpenoids linalool (80) and geranyl acetone (81).
(Figure 3B, red) included all even and odd members of the
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Figure 4: FAMEs identified in the headspace extracts from M. aurantiaca.
homologous series from methyl nonanoate to methyl tetrade- OMe). Further fragment ions [M − CnH2n+1]+ arise from
canoate, in addition to methyl hexadecanoate. These com- cleavage of the saturated unbranched alkyl chain.
pounds were readily identified from their mass spectra by com-
parison to library spectra and subsequent GC–MS analysis of All other FAMEs were isomers of these unbranched com-
synthetic standards. Mass spectra of unbranched FAMEs (for pounds and were assumed to be methyl branched FAMEs due to
mass spectrum of methyl dodecanoate see Figure 5A) are char- biosynthetic considerations as outlined above. The structures of
acterised by fragment ions at m/z = 74 (McLafferty rearrange- these branched compounds have been suggested based on
ment, Scheme 2), m/z = 87 (β-cleavage), and [M − 31]+ (loss of careful analysis of their mass spectra and on a modified reten-
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Figure 5: Mass spectra of (A) methyl dodecanoate (83), (B) methyl 2-methyldodecanoate (10), (C) methyl 4-methyldodecanoate (90), (D) methyl
8-methyldecanoate (95), (E) methyl 9-methyldecanoate (8), (F) methyl 2,9-dimethyldecanoate (24), (G) methyl 4,11-dimethyldodecanoate (110), (H)
methyl 4,8-dimethyldecanoate (112), and (I) methyl 4,8-dimethylundecanoate (114).
tion-index increment system [20]. Following this system, the The increment N(n) depends on the number of carbon atoms n
retention index Icalc. of a methyl branched compound can be in the longest alkyl chain and is N(n) = 100 n, FG is an incre-
calculated (Equation 1) by
ment for the functional group, and the increments Mei have to
be considered for methyl branches depending on the positions i
of branching. The increments FG and Mei have to be deter-
mined for each type of GC column. In a first approximation,
(1)
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of the product were identical to those of the natural compound.
By using Equation 3 the increment for a methyl branching in a
α-position was determined as Meα = 35, resulting in the calcu-
lated retention indices for the α-methyl branched FAMEs as
listed in Table S3 of Supporting Information File 1, column 4.
The calculated retention indices fitted perfectly for compounds
with a chain length of around 10 carbon atoms, which is not
surprising since Meα was determined from 10, but the indices
deviated slightly from the measured values for shorter (n = 7:
Scheme 2: McLafferty fragmentation of FAMEs.
these increments can be assumed to be constants, but as will be Inat. − Icalc. = 4) or longer (n = 15: Inat. − Icalc. = −2) FAMEs. In
discussed below both FG(n) and Mei(n) are slightly dependent other words, Meα was dependent on the chain length. A linear
on the length of the alkyl chain, giving better results for the regression analysis gave
calculated retention indices if this dependency is considered.
(4)
For all of the following analyses the functional group incre-
ment for FAMEs on a HP-5 MS column (FG(n)FAME, HP-5 MS)
was determined from the homologous series of unbranched Recalculation of the retention indices of the α-methyl branched
FAMEs (Table S2 and Figure S1 of Supporting Information FAMEs, taking into account the dependency of Meα on the
File 1). By linear regression (Figure 6) the functional group chain length, resulted in the values listed in Table S3 of
increment (Equation 2) was
Supporting Information File 1, column 5, which perfectly fitted
the measured retention indices.
(2)
(3)
The next class of compounds (Figure 3B, green) showed char-
acteristic fragment ions at m/z = 87 and m/z = 74 similar to the
unbranched FAMEs, but in contrast the β-cleavage was more
important than the McLafferty fragmentation (cf. Figure 5C for
mass spectrum of methyl 4-methyldodecanoate). This, together
with an almost completely missing fragment ion at m/z = 101,
accounting for a γ-cleavage in unbranched FAMEs, suggested
the presence of a methyl group in a γ-position, which leads to a
γ-fragmentation with m/z = 115. A synthesis of methyl
4-methyldodecanoate (90) was performed starting from 10
(Scheme 3). Reduction to the aldehyde 123d and subsequent
Horner–Wadsworth–Emmons reaction gave the α,β-unsatu-
rated ester 124d, which upon catalytic hydrogenation yielded
90. The synthetic compound was identical to the natural FAME
as judged by mass spectrum and retention index (I = 1572). The
retention index of this reference compound was used for to
determine that Meγ = 51, resulting in the calculated retention
indices for all γ-methyl branched FAMEs as summarised in
Table S4 of Supporting Information File 1. Correction of the
increment Meγ by linear regression gave
resulting in a modified Equation 1
Figure 6: The functional group increment FG(n)FAME, HP-5 MS.
(5)
A series of compounds (Figure 3A and Figure 3B, blue) exhib-
ited mass spectra with two significant fragment ions at m/z = 88
as the base peak and at m/z = 101, indicating a methyl
branching in a α-position (Scheme 2, cf. Figure 5B for mass
spectrum of methyl 2-methyldecanoate). For the determination
The corrected calculated retention indices, taking Equation 5
into consideration, were in good agreement with the measured
retention indices.
of the increment Meα the reference compound methyl Another group of FAMEs (Figure 3C, red) showed mass spectra
2-methyldecanoate (10) was synthesised by α-alkylation of 82 with significant fragment ions at m/z = 74 and m/z = 87, like the
(Scheme 3). The mass spectrum and retention index (I = 1357) unbranched compounds (for mass spectrum of methyl
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Scheme 3: Synthesis of FAMEs identified from M. aurantiaca.
8-methyldecanoate cf. Figure 5D), and were assumed to be ated 1,4-addition of the Grignard reagent to methyl acrylate
branched towards the Me terminus, i.e., these FAMEs are furnished the desired FAME 95. Its mass spectrum and reten-
suggested to originate from a branched amino acid starter. tion index (I = 1392) were in good agreement with those of the
Within this group only the FAMEs derived from the odd FAs natural product. The increment Meω−2 = 70 was determined
were found, pointing to an isoleucine-derived starter and a from the retention index of compound 95. In contrast to the
methyl branching in the (ω−2)-position. Furthermore, this increments Meα and Meγ the increment Meω−2 proved to be
suggestion was supported by the intensity of the [M − 57]+ ion, largely independent of the chain lengths of the FAMEs and
which is typical for anteiso-FAMEs. The synthesis of methyl gave good results for the calculated retention indices over a
8-methyldecanoate (95) as a reference compound was started wide range of n, including all other natural FAMEs of this type
from 1-bromo-2-methylbutane (117b, Scheme 3). Copper-cata- from M. aurantiaca and the compounds 118b and 121c, which
lysed 1,4-addition of the respective Grignard reagent to methyl were obtained in the syntheses of 95 and methyl 4,8-dimethyl-
acrylate in the presence of trimethylchlorosilane, dimethyl decanoate (112) as described below (Table S5 of Supporting
sulfide, and 4-dimethylaminopyridine gave methyl 5-methyl- Information File 1). Furthermore, the value of Meω−2 was in
heptanoate (118b). A sequence of LiAlH4 reduction to the good agreement with a previously published value determined
alcohol 119b, conversion into the bromide 120b, and Cu-medi- on a BPX-5 column (Meω−2 = 73) [20].
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The next series of FAMEs (Figure 3C, blue) also had very valine or leucine starters and therefore γ- and (ω−1)-methyl
similar mass spectra compared to the unbranched and (ω−2)- branched. Calculations of the retention indices for the suggested
methyl branched compounds dominated by the fragments at structures were in good agreement with the measured data
m/z = 74 and m/z = 87 (the mass spectrum of methyl (Table S8 of Supporting Information File 1), thus providing
9-methyldecanoate is shown in Figure 5E). Within this series further evidence for the suggested structures. Final verification
both even and odd FAMEs were detected, and therefore, these was obtained by synthesis of methyl 4,11-dimethyldodecanoate
compounds were suggested to be (ω−1)-methyl branched and (110) from 24 by its reduction to the aldehyde 123a,
derived from leucine or valine starters, respectively. As typical Horner–Wadsworth–Emmons olefination to 124a, and catalytic
for (ω−1)-methyl branched compounds, these FAMEs eluted hydrogenation (Scheme 3). The synthetic material was iden-
slightly earlier than their (ω−2)-methyl branched counterparts. tical to the natural compound 110.
Additional support for this suggestion was given by the occur-
rence of relatively intense fragments at [M − 43]+, as can be A related group of compounds (Figure 3D, blue) proved to have
expected for iso-FAMEs. In direct analogy to the synthesis of very similar mass spectra, thus indicating a methyl group in a
95, methyl 9-methyldecanoate (8) was synthesised from 1-bro- γ-position (Figure 5H shows the mass spectrum of methyl 4,8-
mo-3-methylbutane (117a, Scheme 3) and was identical to the dimethyldecanoate). The relatively high intensity of the
volatile from M. aurantiaca in terms of mass spectrum and [M − 57]+ fragment ion suggested the structures of anteiso-
retention index (I = 1385), according to an increment of FAMEs, and accordingly, only the isoleucine-derived methyl
Meω−1 = 63. The retention indices of all other volatiles from M. esters of even-numbered FAs were found. Retention-index
aurantiaca and of the intermediate 118a in the synthesis of 8 calculations (Table S9 of Supporting Information File 1)
were calculated from this constant increment and were in good showed full agreement of the calculated retention indices with
agreement with the measured values (Table S6 of Supporting the experimental data. A synthesis of methyl 4,8-dimethyl-
Information File 1).
decanoate (112) as a reference compound was carried out
starting from 3-methylpentan-1-ol (119c), by similar methods as
In addition to these mono-methyl branched FAMEs, several described above (Scheme 3). The alcohol 119c was trans-
classes of multiply methyl branched compounds were identi- formed into the bromide 120c. The copper-catalysed Michael
fied. The first group of compounds (Figure 3C, green) exhib- addition of the respective Grignard reagent to methyl acrylate
ited mass spectra that were very similar to those of the yielded methyl 6-methyloctanoate (121c), which was α-methyl-
α-branched FAMEs described above, with dominating frag- ated to 122c. Reduction with DIBAH to the aldehyde 123c,
ment ions at m/z = 88 and m/z = 101 (the mass spectrum of Horner–Wadsworth–Emmons olefination to 124c, and final
methyl 2,9-dimethyldecanoate is depicted in Figure 5F), but catalytic hydrogenation afforded 112. The product exhibited the
compounds of this class eluted significantly earlier than their same mass spectrum and retention index (I = 1441) as the
mono-branched isomers, pointing to a second methyl branch natural FAME. The slight deviations between the calculated and
within the alkyl chain, likely towards the methyl terminus. measured retention indices for compounds 106 (ΔI = 2) and 112
Since both even and odd compounds of this series were present, (ΔI = 3) may be attributed to the fairly close proximity of the
the structures of α- and (ω−1)-methyl branched FAMEs derived two methyl branches, which are only three methylene units
from leucine and valine starters, respectively, were suggested apart.
(Table S7 of Supporting Information File 1). The increments
FG(n)FAME, HP-5 MS, Meα(n), and Meω−1 as determined above The FAME 114 (Figure 3D, green) was also suggested to be
were used for the calculation of retention indices. The calcu- γ-methyl branched based on its mass spectrum (the mass spec-
lated values were in perfect agreement with the measured trum of methyl 4,8-dimethylundecanoate is presented in
values, thus corroborating the suggested structures. For unam- Figure 5I). This compound was an isomer of methyl 4-methyl-
biguous proof, an exemplary reference compound of this series dodecanoate (90) and methyl 4,10-dimethylundecanoate (107),
was synthesised (Scheme 3). α-Methylation of 8 yielded methyl but eluted slightly earlier than 107, resulting in a suggested
2,9-dimethyldecanoate (24), which was identical to the respec- structure of methyl 4,8-dimethylundecanoate. A synthesis of
tive FAME from M. aurantiaca.
this compound was performed starting with bromination of
2-methylpentan-1-ol (125) and subsequent Cu-catalysed 1,4-
A second series of dimethyl branched FAMEs (Figure 3D, red) addition of the respective Grignard reagent to methyl acrylate,
exhibited very similar mass spectra to the γ-methyl branched resulting in methyl 5-methyloctanoate (127, Scheme 4). Reduc-
esters (for mass spectrum of methyl 4,11-dimethyldodecanoate tion to the aldehyde 129 via the alcohol 128, addition of methyl-
cf. Figure 5G). Within this class again both even and odd magnesium bromide, and bromination resulted in the secondary
FAMEs were found, suggesting that they were derived from bromide 131. Michael addition of its Grignard reagent to methyl
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Scheme 4: Synthesis of the γ- and (ω−3)-methyl branched FAME 114.
acrylate was less efficient than for primary bromides, but gave C6H13, respectively). This pattern suggested an ethyl branch in
the desired product 114 in low yield. The identity to the natural the 8-position corresponding to the structure of methyl 8-ethyl-
product was confirmed by comparison of GC–MS data.
4-methyldodecanoate (116). This compound is interesting in
terms of its biosynthesis, because it may be formed by incorpor-
The structures of two additional FAMEs were suggested based ation of an ethylmalonyl-CoA elongation unit. However, since
on their mass spectra, but these were not verified by synthesis no further compounds with ethyl branches were found, another
and therefore only tentatively identified (Figure 7). Both com- biosynthetic option seems more likely. The compound 2-ethyl-
pounds were suggested to be γ-methyl branched due to the rela- hexanol is a widespread pollutant originating from plasticisers,
tive intensities of the fragment ions at m/z = 87 and m/z = 74. and this compound could have been oxidised to 2-ethylhexa-
The first compound (Figure 7A) showed a molecular ion at noic acid, transformed into its CoA derivative, and used as an
m/z = 242 and further fragment ions at m/z = 157 and m/z = 185 unusual starter unit by the FAS to make 116.
resulting from the loss of C4H9 or C6H13, whereas no fragment
ion accounting for the loss of C5H11 (m/z = 171) was observed. Several of the FAMEs emitted by M. aurantiaca are chiral,
This pattern is in accordance with the structure of methyl 4,8- including, e.g., the α-, γ-, and (ω−2)-methyl branched FAMEs.
dimethyldodecanoate (115). The second compound (Figure 7B) In addition, compounds such as the γ- and (ω−2)-methyl
exhibited a molecular ion at m/z = 256 and fragment ions at branched FAMEs exist in two different diastereomers.
m/z = 157 and m/z = 199 according to the loss of C4H9 and However, these diastereomers, which were both contained in
C7H15, but not at m/z = 171 and m/z = 185 (loss of C5H11 and the synthetic material, e.g., of compounds 112 and 114, were
Figure 7: Mass spectra of tentatively identified methyl 4,8-dimethyldodecanoate (115) and methyl 8-ethyl-4-methyldodecanoate (116).
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Beilstein J. Org. Chem. 2011, 7, 1697–1712.
not separated on the HP5-MS column used for GC–MS structure of [2H9]-3. For the respective free acid 51 no molec-
analyses, and therefore the elucidation of the relative configura- ular ion is visible, but the fragment ion at m/z = 101, formed by
tions was not possible, at least not by our GC approach. The loss of one hydrogen from the carboxylic acid function, was
only very small differences in the physical properties of these detected at m/z = 110 for [2H9]-51, indicating the incorporation
compounds were also reflected by the occurrence of only one of nine deuteriums (Figures S2C and S2D of Supporting Infor-
set of signals in the NMR spectra of these mixtures of dia- mation File 1). Further fragment ions were observed at m/z = 66
stereomers. The separation of enantiomers of the chiral com- (α-cleavage, + 9 amu), m/z = 79 (McLafferty rearrangement, + 5
pounds described here on chiral GC columns is also a very hard amu), and m/z = 101 (loss of methyl group, + 6 amu), clearly
task, especially for internally methyl branched FAMEs, and was establishing the identity of [2H9]-51. The uptake of isoleucine
beyond the scope of our work.
in the (ω−2)-methyl branched FAMEs was also observed with
high incorporation rates (>90%) for methyl 12-methyltetrade-
canoate (97, Figures S2E and S2F of Supporting Information
Feeding experiments
To investigate the biosynthetic origin of the FAMEs, several File 1), methyl 10-methyldodecanoate (96), and methyl
feeding experiments with deuterated precursors were 14-methylhexadecanoate (not shown). The mass spectrum of
performed. These were directly added to the agar plate cultures [2H9]-97 is characterised by a molecular ion at m/z = 265
and the headspace extracts were prepared by CLSA after ca. showing the incorporation of nine deuteriums, whereas the frag-
2–3 days of growth. The CLSA extracts were then analysed by ment ions at m/z = 74 and m/z = 87 indicative of the structure of
GC–MS. Incorporation of deuterated precursors was observ- a methyl ester are not shifted relative to the unlabelled material.
able through the increased molecular masses and m/z ratios of The compound methyl 14-methylhexadecanoate was not found
certain fragment ions that could be used to localise the under the original experimental conditions without feeding of
deuterium incorporation. One advantage of using deuterated [2H9]isoleucine, demonstrating that the production of (ω−2)-
precursors is that deuterium incorporation results in a decrease methyl branched FAMEs by M. aurantiaca can be activated by
in the retention time of the analyte with respect to its unlabelled isoleucine supply. Unfortunately, the γ- and (ω−2)-methyl
counterpart, i.e., the deuterated isotopomers are separated and branched compounds 112 and 113 were not produced under the
their mass spectra can easily be interpreted [21].
conditions of isoleucine feeding, and therefore, their biosyn-
thetic origin remained elusive.
Feeding of [2H10]isoleucine
One possible pathway to (ω−2)-methyl branched FAMEs is Feeding of [2H10]leucine
through the use of 2-methylbutyryl-CoA as a starting unit, Feeding of [2H10]leucine was performed to investigate the
which is available from isoleucine by transamination and oxida- biosynthetic origin of the (ω−1)-methyl branched FAMEs.
tive decarboxylation. The alternative would be to use an acetyl- Incorporation was observed for a series of (ω−1)-methyl
CoA starter followed by incorporation of a methylmalonyl-CoA branched FAs including isovaleric acid, 5-methylhexanoic acid,
elongation unit. The question as to which of these two alter- 7-methyloctanoic acid, 9-methyldecanoic acid (Figures S3A
natives is operative in M. aurantiaca was addressed by feeding and S3B of Supporting Information File 1), and 11-methyldode-
of [2H10]isoleucine. In this feeding experiment M. aurantiaca canoic acid, all with high incorporation rates (>70%). The
produced large amounts of [2H9]-2-methylbutyric acid ([2H9]- uptake of deuterated leucine for the last compound was observ-
51) and its respective methyl ester [2H9]methyl 2-methylbu- able by a shift of the molecular ion from m/z = 186 to
tyrate ([2H9]-3), both with incorporation rates >70%. The m/z = 195. The fragment ions of the McLafferty rearrangement
transamination of [2H10]isoleucine to 2-oxo-3-methylpentanoic (m/z = 60) and the β-cleavage (m/z = 73) remained unchanged,
acid proceeds with the loss of one deuterium, and accordingly, whereas fragment ions arising from cleavage of the terminal
nine deuterium atoms were incorporated into 3, as indicative by isopropyl group (m/z = 43 and m/z =143) shifted to m/z = 50 and
a shift of the molecular ion of 3 from m/z = 116 to m/z = 125 m/z = 145 in agreement with the deuterium labelling of this
(compare Figures S2A and S2B of Supporting Information portion of the molecule.
File 1). The fragment ion at m/z = 101, arising through the loss
of a methyl group, shifted to m/z = 107, whereas no signal was The labelling was also introduced into the iso-odd FAMEs 102
detected at m/z = 110 in the mass spectrum of [2H9]-3. There- and 103 (Figures S3C to S3F of Supporting Information File 1)
fore, the respective fragment ion only arises by methyl cleavage and the higher homologue methyl 15-methylhexadecanoate (not
from the acyl moiety, and not by loss of the methyl group from shown). Methyl 15-methylhexadecanoate was only found
the ester function. Further diagnostic fragment ions formed by during feeding of [2H10]leucine, similar to the formation of
α-cleavage (m/z = 57) and McLafferty rearrangement (m/z = 88) methyl 14-methylhexadecanoate found only during feeding of
shifted to m/z = 66 and m/z = 93, in full agreement with the [2H10]isoleucine.
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Beilstein J. Org. Chem. 2011, 7, 1697–1712.
Feeding of [2H8]valine
m/z = 242 to m/z = 245, whereas the McLafferty rearrangement
Feeding of [2H8]valine resulted in the incorporation of the and β-cleavage fragment ions were detected at m/z = 74 and
isotopic labelling into its transamination-oxidative decarboxyla- m/z = 87, as for the unlabelled compound. The deuterium
tion product isobutyric acid (49) and the iso-even FAMEs labelling of the γ-methyl group was indicated by a shift of the
methyl 12-methyltridecanoate (100) and methyl 14-methyl- fragment ion at m/z = 115 (γ-cleavage) to m/z = 118. No
pentadecanoate (101) with high incorporation rates (>50%), as incorporation was observed into the (ω−2)-methyl branches of
indicated by the increase of the molecular ions by 7 amu in the respective FAMEs, ruling out their alternative formation
combination with the overall expected fragmentation pattern from an acetate starter in combination with an initial methyl-
(Figure S4 of Supporting Information File 1). The transamina- malonyl-CoA elongation unit, instead of from isoleucine.
tion product of valine, 2-oxoisovaleric acid, can be used by
most organisms for the biosynthesis of the leucine precursor Feeding of [methyl-2H3]methionine
2-oxoisocaproic acid. The enzymes for catalysis of this pathway Feeding of [methyl-2H3]methionine was performed, first as a
are encoded in the genome of M. aurantiaca, but the pathway control experiment with respect to the biosynthetic origin of the
seemed not to be active under the experimental conditions, α- and γ-methyl branches, and second, to investigate the biosyn-
judged by the fact that no incorporation of [2H8]valine into the thetic origin of the methyl ester moiety of the FAMEs. The
iso-odd FAMEs was observed. Furthermore, no uptake of feeding experiment resulted in the incorporation into 103
labelling from [2H10]leucine into the iso-even FAMEs was (Figure S6 of Supporting Information File 1) and 97 (not
found, which also rules out a similar formation of the higher shown), as indicated by a shift of the molecular ion from
homologue 2-oxo-5-methylcaproic acid from 2-oxoisocaproic m/z = 256 to m/z = 259, of the McLafferty ion from m/z = 74 to
acid. These results also demonstrate that the FAs in M. auran- m/z = 77, and of the β-cleavage fragment ion from m/z = 87 to
tiaca are not subject to α-oxidation, a process in which FAs are m/z = 90. Further FAMEs were not produced during the course
oxidatively degraded by one carbon.
of this experiment. The results clearly demonstrate the forma-
tion of FAMEs from methionine via SAM as the methyl donor
for the methylation of FAs.
Feeding of [2H5]sodium propionate
To investigate the biosynthetic origin of the α- and γ-methyl
branches, a feeding experiment with [2H5]sodium propionate Conclusion
was performed. This compound was expected to be carboxy- In summary, the headspace extracts from M. aurantiaca contain
lated to yield methylmalonyl-CoA, which would serve as an unbranched even and odd FAMEs that are made from an acetyl-
elongation unit for the introduction of methyl branches. Alter- CoA or propionyl-CoA starter. In particular the even FAMEs
native mechanisms could include the chain elongation with are very widespread in nature. Several α-methyl branched
malonyl-CoA, followed by methylation with SAM by catalysis FAMEs were also present that were previously described from
of a C-methyltransferase. Indeed the incorporation of isotopic Chitinophaga [14]. These compounds can be synthesised by
labelling into the α- and γ-methyl branched FAMEs was incorporation of a final methylmalonyl-CoA elongation unit.
observed with high incorporation rates (>90%, Figure S5 of When methylmalonyl-CoA is used as the penultimate building
Supporting Information File 1). Incorporation into methyl 2,11- block followed by malonyl-CoA, the synthesis results in the for-
dimethyldodecanoate (25) and its higher homologue 26 was mation of γ-methyl branched FAs that upon O-methylation
observable by an increase of the molecular ion by 3 amu, while yield the respective FAMEs. Only one such compound has
the fragment ions at m/z = 88 and m/z = 101, indicative of an previously been found in nature represented by methyl
α-methyl branch, shifted to m/z = 91 and m/z = 104. The 4-methyloctanoate, and this is a constituent of the pheromone
incorporation of only three deuterium atoms from [2H5]sodium blend of the date palm fruit stalk borer Oryctes elegans [22],
propionate is in agreement with the biosynthesis of FAs whereas the related FAMEs 89–94 emitted by M. aurantiaca
(Scheme 1): One deuterium is lost during carboxylation of are all new natural products. The use of alternative starter units
propionyl-CoA to yield methylmalonyl-CoA, the second is from branched amino acids for the biosynthesis of FAMEs was
possibly exchanged with water due to the C,H-acidity of demonstrated in feeding experiments. Although the respective
malonyl-CoA derivatives, but it is lost, at the latest, in the dehy- FAs are widespread in nature, only a few reports of these
dration of the 3-hydroxy-2-methylacyl-S~ACP to the respec- FAMEs exist, e.g., the isoleucine-derived compounds 95 and 96
tive 2-methyl-2-enoyl-S~ACP intermediate. The incorporation occur in the volatile fraction from Medicago sativa [23], the
of [2H5]sodium propionate was also observed for the γ-methyl valine-derived FAME 99 is known from Capsicum annuum
branched compounds represented by methyl 4,11-dimethyldo- [24], and the leucine-derived compound 8 was previously found
decanoate (110). The uptake of deuterium was in the first in Stigmatella aurantiaca [13], whereas the compounds 102 and
instance visible by an increase of the molecular ion from 103 (from leucine), and 98 (from valine) have not been
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Beilstein J. Org. Chem. 2011, 7, 1697–1712.
8. Carroll, B. J.; Moss, S. J.; Bai, L.; Kato, Y.; Tölzer, S.; Yu, T.-W.;
Floss, H. G. J. Am. Chem. Soc. 2002, 124, 4176–4177.
doi:10.1021/ja0124764
described before. The same is true for all FAMEs described
here that are derived from a branched amino acid and that are in
addition α- or γ-methyl-branched, and there is only one report
on such unusual FAs with a similar methyl branching pattern
from Bacillus [25]. The biosynthesis of these compounds was
established in feeding experiments with [2H5]sodium pro-
pionate and [methyl-2H3]methionine, which allowed us to
distinguish between two possible pathways, i.e., the incorpor-
ation of methylmalonyl-CoA elongation units, or the alter-
native incorporation of malonyl-CoA elongations units fol-
lowed by methylation with SAM. The experiments clearly
demonstrated the usage of methylmalonyl-CoA building blocks,
whereas the feeding experiment with [methyl-2H3]methionine
gave proof for the origin of the methyl ester functions by SAM-
dependent methylation of the respective FAs.
9. Smith, A.; Calder, A. G. Biomed. Mass Spectrom. 1979, 6, 347–349.
doi:10.1002/bms.1200060808
10.Schulz, S.; Dickschat, J. S. Nat. Prod. Rep. 2007, 24, 814–842.
doi:10.1039/b507392h
11.Schöller, C. E. G.; Gürtler, H.; Pedersen, R.; Molin, S.; Wilkins, K.
J. Agric. Food Chem. 2002, 50, 2615–2621. doi:10.1021/jf0116754
12.Wilkins, K.; Schöller, C. Actinomycetologica 2009, 23, 27–33.
doi:10.3209/saj.SAJ230202
13.Dickschat, J. S.; Bode, H. B.; Wenzel, S. C.; Müller, R.; Schulz, S.
ChemBioChem 2005, 6, 2023–2033. doi:10.1002/cbic.200500174
14.Nawrath, T.; Gerth, K.; Müller, R.; Schulz, S. Chem. Biodiversity 2010,
7, 2228–2253. doi:10.1002/cbdv.201000190
15.Weinstein, M. J.; Luedemann, G. M.; Oden, E. M.; Wagman, G. H.;
Rosselet, J. P.; Marquez, J. A.; Coniglio, C. T.; Charney, W.;
Herzog, H. L.; Black, J. J. Med. Chem. 1963, 6, 463–464.
doi:10.1021/jm00340a034
16.He, H.; Ding, W. D.; Bernan, V. S.; Richardson, A. D.; Ireland, C. M.;
Greenstein, M.; Ellestad, G. A.; Carter, G. T. J. Am. Chem. Soc. 2001,
123, 5362–5363. doi:10.1021/ja010129o
Supporting Information
Supporting Information contains experimental details for
the syntheses and analytical data of all synthesized
compounds, the tabulated full results of the headspace
analyses, and mass spectra of labelled FAMEs obtained
during feeding experiments together with the mass spectra
of the unlabelled compounds for comparison.
17.Dickschat, J. S.; Wenzel, S. C.; Bode, H. B.; Müller, R.; Schulz, S.
ChemBioChem 2004, 5, 778–787. doi:10.1002/cbic.200300813
18.Dickschat, J. S.; Zell, C.; Brock, N. L. ChemBioChem 2010, 11,
417–425. doi:10.1002/cbic.200900668
19.Dickschat, J. S.; Wickel, S.; Bolten, C. J.; Nawrath, T.; Schulz, S.;
Wittmann, C. Eur. J. Org. Chem. 2010, 2687–2695.
doi:10.1002/ejoc.201000155
20.Schulz, S.; Toft, S. Tetrahedron 1993, 49, 6805–6820.
doi:10.1016/S0040-4020(01)80424-5
Supporting Information File 1
Experimental details and analytical data.
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-7-200-S1.pdf]
21.Dickschat, J. S.; Citron, C. A.; Brock, N. L.; Riclea, R.; Kuhz, H.
Eur. J. Org. Chem. 2011, 3339–3346. doi:10.1002/ejoc.201100188
22.Rochat, D.; Mohammadpoor, K.; Malosse, C.; Avand-Faghih, A.;
Lettere, M.; Beauhaire, J.; Morin, J.-P.; Pezier, A.; Renou, M.;
Abdollahi, G. A. J. Chem. Ecol. 2004, 30, 387–407.
doi:10.1023/B:JOEC.0000017984.26917.52
Acknowledgements
This work was supported by the Deutsche Forschungs-
gemeinschaft with an Emmy Noether fellowship (to J. S. D.,
DI1536/1-1).
23.Core, R. J.; Henning, J. A.; Gardea-Torresdey, J. J. Agric. Food Chem.
1994, 42, 2932–2936. doi:10.1021/jf00048a054
24.Guadayol, J. M.; Caixach, J.; Ribé, J.; Cabañas, J.; Rivera, J.
J. Agric. Food Chem. 1997, 45, 1868–1872. doi:10.1021/jf960266i
25.Carballeira, N. M.; Miranda, C.; Lozano, C. M.; Nechev, J. T.;
Ivanova, A.; Ilieva, M.; Tzvetkova, I.; Stefanov, K. J. Nat. Prod. 2001,
64, 256–259. doi:10.1021/np000494d
References
1. Kaneda, T. Microbiol. Rev. 1991, 55, 288–302.
2. Wang, J.-F.; Dai, H.-Q.; Wei, Y.-L.; Zhu, H.-J.; Yan, Y.-M.; Wang, Y.-H.;
Long, C.-L.; Zhong, H.-M.; Zhang, L.-X.; Cheng, Y.-X.
Chem. Biodiversity 2010, 7, 2046–2053. doi:10.1002/cbdv.201000072
3. Hamilton, J. T. G.; Harper, D. B. Phytochemistry 1997, 44, 1129–1132.
doi:10.1016/S0031-9422(96)00697-8
4. Cropp, T. A.; Wilson, D. J.; Reynolds, K. A. Nat. Biotechnol. 2000, 18,
980–983. doi:10.1038/79479
5. Hutchinson, C. R.; Sherman, M. M.; McInnes, A. G.; Walter, J. A.;
Vederas, J. C. J. Am. Chem. Soc. 1981, 103, 5956–5959.
doi:10.1021/ja00409a076
6. Liu, Y.; Hazzard, C.; Eustaquio, A. S.; Reynolds, K. A.; Moore, B. S.
J. Am. Chem. Soc. 2009, 131, 10376–10377. doi:10.1021/ja9042824
7. Xu, Z.; Ding, L.; Hertweck, C. Angew. Chem., Int. Ed. 2011, 50,
4667–4670. doi:10.1002/anie.201008265
1711

Beilstein J. Org. Chem. 2011, 7, 1697–1712.
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