Determination of the absolute configuration of peptide natural products by using stable isotope labeling and mass spectrometry

Article abstract of DOI:10.1002/chem.201103479

Structure elucidation of natural products including the absolute configuration is a complex task that involves different analytical methods like mass spectrometry, NMR spectroscopy, and chemical derivation, which are usually performed after the isolation of the compound of interest. Here, a combination of stable isotope labeling of Photorhabdus and Xenorhabdus strains and their transaminase mutants followed by detailed MS analysis enabled the structure elucidation of novel cyclopeptides named GameXPeptides including their absolute configuration in crude extracts without their actual isolation. Copyright

Full text of DOI:10.1002/chem.201103479

DOI: 10.1002/chem.201103479
Determination of the Absolute Configuration of Peptide Natural Products by
Using Stable Isotope Labeling and Mass Spectrometry
Helge B. Bode,* Daniela Reimer, Sebastian W. Fuchs, Ferdinand Kirchner,
Christina Dauth, Carsten Kegler, Wolfram Lorenzen, Alexander O. Brachmann, and
Peter Grꢀn[a]
Abstract: Structure elucidation of natural products including the absolute configu-
ration is a complex task that involves different analytical methods like mass spec-
trometry, NMR spectroscopy, and chemical derivation, which are usually per-
formed after the isolation of the compound of interest. Here, a combination of
stable isotope labeling of Photorhabdus and Xenorhabdus strains and their transa-
minase mutants followed by detailed MS analysis enabled the structure elucidation
of novel cyclopeptides named GameXPeptides including their absolute configura-
tion in crude extracts without their actual isolation.
Keywords: configuration determi-
nation · isotopic labeling · mass
spectrometry · peptides · structure
elucidation
Introduction
and has been used extensively for metabolomics in plants
and bacteria.[4–8] Here we describe the application and fur-
ther development of this simple and robust approach for the
structure elucidation of natural products from entomopatho-
genic bacteria, applied to four novel cyclopeptides. The ap-
proach allows for differentiation between isobar building
blocks such as leucine or isoleucine, as well as the determi-
nation of the absolute configuration in the case of amino
acids.
An important and time-consuming step in natural product
research is the structural elucidation of novel compounds.
Usually a crude extract from the producer of a natural prod-
uct is analyzed by HPLC/UV or HPLC/MS to clarify wheth-
er the respective peak in the chromatogram represents a
compound that is worth its isolation. In that case, the com-
pound of interest must be purified and its structure subse-
quently elucidated by means of NMR spectroscopic analy-
sis.[1] Today, high-resolution mass spectrometry allows the
determination of the mass of the respective compound to
such accuracy that a sum formula can be predicted, which
can be used for database searches for rapid identification of
novel compounds. Whereas the possible compositions for
protein-derived peptides or normal lipids would allow only
a certain number of possible sum formulae due to their
known building-block composition,[2,3] this is different for
natural products, which can be built from very unusual pre-
cursors and thus increase the number of possible sum for-
mulae. To address this problem and to speed up the identifi-
cation and structure elucidation of natural products especial-
ly from microorganisms, one can apply an isotope-labeling
strategy followed by mass spectrometry. This approach
allows the reliable determination of the correct sum formula
Results and Discussion
Sum formula determination: Entomopathogenic bacteria of
the genus Xenorhabdus and Photorhabdus were analyzed
because these bacteria are usually multiproducers of several
different natural products simultaneously.[9] Additionally,
highly similar compounds have been observed in different
strains, and thus a method was needed to differentiate these
compounds. When Photorhabdus luminescens strain TT01
was grown in standard growth medium, the sum formulas of
all peaks that appeared in an HPLC/MS analysis (Figure 1a)
could be predicted from high-resolution MS analysis, as ex-
emplified by compound 1 with m/z 586.39545 (Figure 1b).
However, because usually several sum formulae are chemi-
cally and biologically possible, it is often difficult to find the
correct one. It was previously shown that the number of pos-
sible chemical formulae can be significantly reduced by
taking into account the isotope pattern of ultrahigh-resolu-
tion MS data.[10] Similarly, growing bacteria in culture media
fully labeled with 13C drastically reduces the number of pos-
sible sum formulae for lipids[11] and therefore we have ap-
plied this approach to Photorhabdus luminescens. When
comparing HPLC/MS analyses of strain TT01 grown in stan-
dard growth medium (natural abundance of all isotopes),
[a] Prof. Dr. H. B. Bode, D. Reimer, Dipl.-Biol. S. W. Fuchs, F. Kirchner,
Dipl.-Chem. C. Dauth, Dr. C. Kegler, Dipl.-Pharm. W. Lorenzen,
Dr. A. O. Brachmann, P. Grꢀn
Institut fꢀr Molekulare Biowissenschaften
Molekulare Biotechnologie, Goethe Universitꢁt Frankfurt
Max-von-Laue-Strasse 9, 60436 Frankfurt am Main (Germany)
Fax : (+49)69-798-29527
E-mail: h.bode@bio.uni-frankfurt.de
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201103479.
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Figure 1. a) HPLC/MS analysis (base peak chromatogram) of P. lumines-
cens TT01 highlighting compound 1 (arrow). b) Sum formula prediction
for 1 using HRESI-MS data. c) Determination of the number of carbon
and nitrogen atoms for 1 as determined from growth of strain TT01 in
standard growth medium, or medium fully labeled with 15N or 13C.
Figure 2. a) HPLC/MS analysis of X. nematophila HGB081 indicating the
positions of compounds 2–4. b) Identification of 2–4 in the Dictionary of
Natural Products (http://dnp.chemnetbase.com/dictionary-search.do?me-
thod=view&id=2265390&struct=start&props=&&si= last accessed
on November 3, 2011) based on the mass range and the number of
carbon and nitrogen atoms identified from labeling experiments. c) Struc-
tures of xenortide A (2), xenortide B (3), and xenematide (4).[7]
15N or 13C medium allowed the rapid identification of the
correct sum formula as the number of nitrogen and carbon
atoms can be easily determined from the mass shifts of 1 in
the different growth media (Figure 1c). Despite its simplici-
ty, this approach is also cheap as culture volumes as little as
1 mL can be used (media costs are around 1 EmLÀ1). More-
over, the sum formulae for all compounds produced under
the selected growth conditions (even produced in minute
amounts) can be determined in parallel (Table S3 in the
Supporting Information) as has also been described in me-
tabolome experiments in different organisms.[4–8] Indeed, the
approach is so powerful that it works even without HRMS
data. Analysis of Xenorhabdus nematophila HGB081 led to
the identification of compounds 2–4 with m/z 410.6, 449.6,
and 663.7. A database search in the Dictionary of Natural
Products with a total of 234378 entries using the number of
carbon and nitrogen atoms (C25N3, C27N4, and C37N6) as de-
termined by labeling experiments and the molecular-weight
range (409–410, 448–449, and 662–663) resulted in only six,
one, and one hit, respectively, and thus led to the identifica-
tion of xenortide A (2) and xenortide B (3), and xenematide
(4), which were already known from another Xenorhabdus
strain (Figure 2).[12]
Building-block determination: Similarly, this labeling ap-
proach can be used to identify building blocks of natural
products such as amino acids. Here, labeled precursors (13C,
2H, 15N) are usually added to a producing culture and their
incorporation would confirm the involvement of the precur-
sor in the natural product biosynthesis. Again, MS-based de-
tection allows for the analysis of compounds produced in
minute amounts. However, because not all possible precur-
sors are available for a reasonable price, one can also add a
[12C/14N] precursor with natural abundance to a culture
grown in a fully labeled 13C or 15N medium as described
above. Precursor incorporation in such an ꢃinverseꢄ labeling
experiment is easily visible by a shift to lower masses. In the
case of compound 1, its composition was determined to be
cycloACHTUNGTRNE(NUNG FLLLV) or a positional isomer thereof, as shown by
inverse labeling experiments with valine (Val), leucine
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H. B. Bode et al.
(Leu), and phenylalanine (Phe) in 13C medium (Figure 3).
The connectivity of these building blocks can be determined
easily from MS-MS experiments as shown for 1 (Figure S1
in the Supporting Information), which was demonstrated to
be cycloACTHNGUETRNNU(G VLFLL) and named GameXPeptide A (1). This
approach additionally facilitates the differentiation of isobar
building blocks as leucine, isoleucine, and N-methyl valine,
which are often found in peptides from entomopathogenic
bacteria. To demonstrate this, the method was applied to en-
tolysin A (5) from Pseudomonas entomophila, for which
previous structure-elucidation efforts could not differentiate
between leucine and isoleucine.[13] From labeling experi-
ments with deuterated [2H9]leucine followed by tandem MS
analysis, the correct peptide sequence was determined
(Figure 4). This could have been done for the entolysin iso-
topologue that exhibited the incorporation of all four ex-
pected deuterated leucine residues. However, since the fully
labeled isotopologue is usually present in minute amounts,
the positional analysis was performed with the isotopologue
carrying only one labeled leucine. If the random incorpora-
tion of one [2H9]leucine into one of the four possible posi-
tions of each fragmented molecule is considered, the ratio
of individual sequence ions (in this case, b ions) with and
without the incorporated [2H9]leucine residue would change
in a predictable manner. As one isoleucine (Ile) and four
Leu residues are present in 5 as deduced from the building-
block analysis (data not shown), the following expectations
for ratios of labeled and unlabeled b ion isotopologues can
Figure 3. Determination of building blocks of 1 derived from growth of
strain TT01 in fully labeled 13C medium without (bottom) or with the ad-
dition of different l-12C amino acids (natural abundance).
Figure 4. Structure of entolysin A (5) and determination of the position of leucine and isoleucin residues in 5. Depicted is an MS2 resulting from 5 carry-
ing one [2H9]leucine; b ions are labeled with dotted lines highlighting deuterated (light gray) and nondeuterated (dark gray) isotopologues.
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Absolute Configuration Determination
FULL PAPER
be made for 5 labeled with one [2H9]leucine: Every b ion
that results from fragmentation at the N-terminal position
of Leu should exhibit an altered ratio of the b ion isotopo-
ing enolate anion and vice versa.[15] Thus, by using an amino
acid with a H label at the a position, the presence of an E
2
domain can be probed indirectly: Is the label found in the
final product? Then the amino acid at this position must be
l due to a missing or nonfunctional E domain. Is the label
ACHTUNGTRENNUNG
logues ([2H9]/[1H9]) in comparison to the b+1 ion isotopo-
logues, which still contain the Leu, in which the relative
amount of the unlabeled b ion ([1H9]) would increase.
Therefore b ions that harbor 3 of 4, 2 of 4, and 1 of 4 Leu
should exhibit ratios of unlabeled to labeled isotopologues
1
lost? Then it has been exchanged against H from the cul-
ture medium by means of E-domain-catalyzed racemization,
and the amino acid at this position must be d (Figure 5a) if
no other factor contributes to the peptide configuration.
Unfortunately, in wild-type strains, this approach cannot
be used because of the catalytic activity of transaminases in
the cells, which convert amino acids into the corresponding
2-keto carboxylic acids and vice versa and that are involved
in the last step in amino acid biosynthesis and the first step
ACHTUNGTRENNUNG
[1H9]/[2H9] of 1:3 (b11/b8), 1:1 (b7/b5), and 3:1 (b4), respective-
ly, as was indeed the case (Figure 2). The b13 ion could ex-
clusively be detected as its labeled isotopologue, thereby in-
dicating that the C-terminal amino acid residue of entolysin
is isoleucine (Figure 4).
Determination of amino acid configuration: The absolute
configuration of the amino acid building blocks still needs to
be determined. Peptides like 1–5 are usually derived from
nonribosomal peptide synthetases (NRPS).[14] These multi-
functional giant multienzyme complexes consist of single do-
mains for amino acid activation: adenylation (A domain),
condensation (C domain) of two amino acids as peptidyl
carrier protein, or thiotemplate (T domains) for covalent
binding of peptide intermediates. All domains necessary for
the incorporation of a single amino acid are grouped into
modules and thus the number of modules usually reflects
the number of amino acids in the final natural product and
vice versa. In most cases in which d-amino acids are present
in the natural product, epimerization (E) domains or mixed
C/E domains are present at the corresponding position in
the NRPS. These E domains racemize the enzyme-bound l-
amino acid or the bound l-peptide intermediate, and subse-
quently only the d derivative is further processed. During
this process, the amino acid is converted to the correspond-
Figure 6. Labeling of GameXPeptide A (1) with
[2H10]leucine, or [2H8]phenylalanine in wild type and
mutant of P. luminescens.
[2H8]valine,
DilvEDtyrB
a
2
Figure 5. a) Epimerization-domain-catalyzed conversion of l- into d-amino acids based on H-labeled amino acids. Adenylation (A), peptidyl carrier pro-
2
tein (PCP), epimerization (E), and condensation (C) domains are indicated. b) Transaminase-catalyzed loss of label from H-labeled amino acids.
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H. B. Bode et al.
in amino acid degradation[16]
(Figure 5b). In Gram-negative
bacteria, these transaminases
are encoded by ilvE, tyrB, and
aspC, which show specificities
to different sets of amino
acids. These genes are highly
conserved and could be identi-
fied easily in the genome of P.
luminescens. When fully la-
beled [2H8]valine, [2H10]leucine,
or [2H8]phenylalanine were
added to P. luminescens wild-
type cultures, the percentage
of the fully labeled amino
acids decreased to 34, 20, and
11% in favor of [2H7]valine,
[2H9]leucine,
or
[2H7]phenylalanine, respective-
ly (Figures S2 and S3 in the
Supporting Information). As
expected, a deletion of ilvE re-
sulted in 95% preservation of
[2H10]leucine after 24 h and a
deletion of tyrB in full preser-
vation of [2H8]phenylalanine.
Unfortunately, the loss of label
from deuterated valine could
not be decreased further in
DilvE, DtyrB, or DilvEDtyrB
mutants, and we have not been
able to construct an ilvE/tyrB/
aspC mutant.
When we tested the incorpo-
ration of [2H8]valine and
[2H8]phenylalanine into com-
pound 1 in DilvE or DilvED
tyrB mutants, we could only
Figure 7. MS3 spectra of 1 isolated from P. luminescens DilvEDtyrB a–e) labeled with [2H10]leucine and
[2H9]leucine (m/z 605.5) and f–h) nonlabeled 1 as control. The parent MS2 ions are labeled with a diamond.
Amino acid labels in white boxes refer to unlabeled amino acids; [2H10]leucine is shown in gray boxes and
[2H9]leucine in black boxes. i) The assignment of the stereochemistry as concluded from the spectra in (a)–(e).
observe isotopes of
1 that
showed the loss of one deuteri-
um for valine and phenylala-
nine (m/z 593.4) as expected
for the incorporation of
[2H7]valine or [2H7]phenylalanine, respectively. In the case
of leucine, we could observe signals from isotopes at m/z
595.4 and 596.4 in a ratio of 1:2, thereby indicating the in-
corporation of [2H9]leucine and [2H10]leucine (Figure 6).
Thus, 1 is composed out of d-Val, d-Phe, d-Leu, and two l-
Leu, and therefore the configuration of all amino acids has
been identified. Subsequently, MS3 fragmentation experi-
ments of the isotope that was carrying one [2H9]leucine and
one [2H10]leucine (m/z 605.4) to determine the l- and d-leu-
cine positions were performed. Fragmentation of m/z 458.4
(loss of phenylalanine; Figure 7a) showed a [2H9]leucine-de-
rived fragment, whereas for m/z 506.5 (loss of valine; Fig-
ure 7b), m/z 492.4 (loss of leucine; Figure 7c), and m/z 483.4
(loss of [2H9]leucine; Figure 7d), only [2H10]leucine-derived
fragments were observed. Fragmentation of m/z 482.4 (loss
of [2H10]leucine; Figure 7e) showed the expected loss of
valine. For comparison, unlabeled 1 was also fragmented
similarly (Figure 7f–h). Thus, from the different connections
of the five different building blocks as determined in Fig-
ure 7a–e and summarized in Figure 7i, the absolute configu-
ration of 1 was determined to be cycloACHTNUTRGNEUNG(vLflL). We con-
firmed the structure of 1 by solid-phase synthesis and could
show that synthetic 1 has indeed the same retention time
(Figure S4 in the Supporting Information) and identical
NMR spectroscopic data (Table S4 in the Supporting Infor-
mation) as the natural compound. Furthermore, a small
amount of 1 was isolated and subjected to Marfey’s ad-
vanced analysis, which also indicated the correct configura-
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Absolute Configuration Determination
FULL PAPER
tion of the amino acids (Figure S5 in the Supporting Infor-
mation). In addition, we identified the gene encoding of the
GameXPeptide NRPS gxpS by plasmid integration and the
expected loss of production of 1 when compared to the wild
type (Figure S4 in the Supporting Information). A detailed
analysis of GxpS allowed the prediction of all required do-
mains for GameXPpetide biosynthesis including three C/E
domains at the positions determined by the labeling experi-
ments (Figure 8a, Figure S6 in the Supporting Information).
Figure 9. a) Structure results from Marfey’s advanced analysis showing
the d,l-FDLA derivatives (continuous lines) and the l-FDLA derivatives
(dashed lines) of N-methylvaline (m/z 426.2, I), and valine (m/z 412.2, II)
in b) the positive mode, and results from labeling experiments with
c) [2H8]valine of mevalagmapeptide (9) produced by P. luminescence
TT01. Mevalagmapeptide 9 (m/z 334.7, C33H67O5N9) is detected as a
double-charged ion, thus incorporation of [2H7]valine and [2H8]valine led
to m/z 338.2 (+3.5) and 338.7 (+4.0), respectively.
Figure 8. a) Domain organization of GxpS responsible for the biosynthe-
sis of 1 (enzyme-bound intermediates are shown) and 6–8. b) Structures
of GameXPeptides A–D (1 and 6–8).
A detailed analysis of the P. luminescens extract revealed
the presence of three additional derivatives of 1 named
GameXPeptide B–D (6–8, Figure 8b), the structure of which
was elucidated similarly (Table S3 in the Supporting Infor-
mation) and which all were lost in the gxpS mutant. Their
structural variability results from the exchange of Val1 and/
or Phe3 against Leu.
in contrast to the exclusive [2H7]valine-derived isotopomer
observed for 1 (Figure 6), thus confirming the l configura-
tion and indicating that even a presence of 66% of fully
deuterated valine (see Figure S3 in the Supporting Informa-
tion) is sufficient for a stereochemical analysis. Similarly, the
configuration of leucine in 2, 3, and xenocoumacin-1,[17] and
phenylalanine in 3 was determined in transaminase mutants
of X. nematophila HGB081. Comparison of the labeling re-
sults from the ilvE or tyrB mutants with the wild type al-
lowed the differentiation between d- and l-amino acids and
confirmed that all analyzed amino acids are l in these com-
pounds (Figures S7 and S8 in the Supporting Information)
as described previously.[12,18]
As the presence of [2H7]valine in 1 could not fully differ-
entiate between the incorporation of [2H8]valine followed by
epimerization-domain-catalyzed loss of one deuterium and
the incorporation of [2H7]valine that resulted from residual
transamination activity in the bacterial culture, we analyzed
another novel peptide named mevalagmapeptide (9) from
strain TT01 (Figure 9a) that is composed of l-valine exclu-
sively as determined by Marfey’s advanced analysis (Fig-
ure 9b) after the isolation of 9. Here, the presence of
[2H7]valine but also of [2H8]valine was detected almost in a
1:1 ratio in the wild type (Figure 9c, bottom line), which is
As the three transaminases are highly conserved at the
protein but also at the DNA level in Xenorhabdus and Pho-
torhabdus and other bacteria (data not shown), their corre-
sponding genes can probably be disrupted or deleted even
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H. B. Bode et al.
by means of homologous recombination based on gene se-
quences of closely related strains, thus enabling the determi-
nation of absolute configuration in similar peptides also in
strains in which no genome sequence is available.
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Acknowledgements
This work was supported by the Deutsche Forschungsgemeinschaft
(DFG) and the European Communityꢄs Seventh Framework Programme
(FP7/2007–2013) under grant agreement no. 223328. Work in the author’s
laboratory is additionally supported by the Bundesministerium fꢀr Bil-
dung und Forschung (BMBF) and the Frankfurt Initiative for Microbial
Sciences (FIMS).
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Received: November 4, 2011
Published online: January 20, 2012
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