Identification of the amino acid labionin and its desulfurised derivative in the type-III lantibiotic LabA2 by means of GC/MS

Article abstract of DOI:10.1039/c1cc11573a

A GC-MS method for the rapid and unambiguous identification of the amino acid labionin (Lab) occurring in type-III lantibiotics is presented. This method will constitute a valuable tool for the characterisation and structure elucidation of labyrinthopeptins and their differentiation from lanthionine-type lantibiotics.

Full text of DOI:10.1039/c1cc11573a

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Identification of the amino acid labionin and its desulfurised derivative in
the type-III lantibiotic LabA2 by means of GC/MSw
Alexander Pesic,z Maik Henkelz and Roderich D. Su
¨
ssmuth*
Received 18th March 2011, Accepted 19th April 2011
DOI: 10.1039/c1cc11573a
A GC-MS method for the rapid and unambiguous identification
of the amino acid labionin (Lab) occurring in type-III lantibiotics
is presented. This method will constitute a valuable tool for the
characterisation and structure elucidation of labyrinthopeptins
and their differentiation from lanthionine-type lantibiotics.
Lantibiotics are ribosomally synthesised peptide antibiotics
containing the thioether amino acids (2S,6R)-meso-lanthionine
(Lan, 1) and/or (2S,3S,6R)-3-methyl-lanthionine (MeLan, 2).1
As further structural modifications lantibiotics may contain
a,b-unsaturated amino acids such as 2,3-didehydroalanine
(Dha, 3) and 2,3-didehydroaminobutyric acid (Dhb, 4).
Lantibiotics are produced by a wide range of Gram-positive
bacteria, e.g. Staphylococci and Lactococci, and mostly have
antimicrobial activity against other Gram-positive bacteria.
Recently, we identified a new structural type of lantibiotics,
named labyrinthopeptins, which are produced by the Actino-
mycete Actinomadura namibiensis DSM6313.2 The structure
model of Labyrinthopeptin A2 (LabA2, 6a) showed the
presence of an unusual a,a-disubstituted amino acid, named
labionin (Lab, 5). From X-ray data it was deduced that Lab
(5) has (2S,4S,8R)-configuration. Lab (5) can be structurally
considered as a triamino triacid consisting of a basic lanthionine
motif, which is further extended at the quaternary C-a-atom
by a methylene bridge and an a-amino acyl residue, thus
enabling the formation of two ring systems within a peptide
chain. From the sequence of labyrinthopeptin prepropeptides
it was deduced that a Ser/Ser/Cys motif is the precursor of Lab
(5). Searches in databases containing genomic data showed
that gene clusters with similar architecture and high sequence
homology to the labyrinthopeptin gene cluster are widespread
among Actinomycetes, e.g. Streptomyces coelicolor, S. avermitilis,
and Saccharopolyspora erythraea. Based on those similarities
we assume that they are producers of labyrinthopeptin-type
lantibiotics. It is particularly interesting that their lantibiotic
precursor peptides also contain the Ser/Ser/Cys motif, which
could be a precursor of the Lab (5) motif (Fig. 1).
Fig. 1 (A) Structures of the amino acids Lan (1), MeLan (2), Dha (3)
and Dhb (4) characteristic of lantibiotics. Amino acid Lab (5) in
LabA2 is characteristic for recently discovered type-III lantibiotics of
the labyrinthopeptin-type. (B) Schemes of LabA2 (6a) and desulfo-
LabA2 (6b). (C) Scheme of hydrolysis and microderivatisation for
subsequent GC/MS analysis of Lab (7) and a-methyl-2,4-diamino-
glutaric acid (Dmg, 9): N-trifluoroacetyl/ethyl ester derivatisation to 8
and 10 by (i) 6N HCl; 24 h; 110 1C, (ii) reductive desulfurisation with
NiCl2/NaBH4; 6N HCl; 24 h; 110 1C, (iii, iv) AcCl; EtOH; TFAA.
However, structure elucidation of labyrinthopeptins as well
as of related type-III lantibiotics from abovementioned
bacterial producers is challenging. The high cyclisation degree
of LabA2 (6a) poses significant problems to mass spectro-
metric sequencing. This is aggravated by two possible
alternative isobaric posttranslational modifications Dha+Lan
(3+1) and Lab (5) in type-III lantibiotics. Furthermore,
structure elucidation of labyrinthopeptins by NMR spectro-
scopy is hampered by aggregation-prone properties.2 In this
context GC-MS appears as a very useful technique for the
detection, quantitation and structure elucidation of building
blocks or modifications of peptidic natural products, e.g.
amino acids, carbohydrates and lipids. Amino acids are
commonly derivatised as N-trifluoroacetyl or N-pentafluoro-
phenyl amino acid esters.3 The analytics of building blocks
with higher molecular mass by GC-MS however is aggravated
by a decrease in volatility of the derivatives with increasing
molecular mass. Polar functional groups require additional
Technische Universitat Berlin, Institut fur Chemie, Strasse des 17,
¨
¨
Juni 124, 10623 Berlin, Germany.
E-mail: suessmuth@chem.tu-berlin.de; Fax: +49 30 314 79651;
Tel: +49 30 314 78774
w Electronic supplementary information (ESI) available: MS data and
synthesis of amino acid standard. See DOI: 10.1039/c1cc11573a
z Both authors contributed equally to this work.
c
This journal is The Royal Society of Chemistry 2011
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derivatisations exhausting the limits of GC-MS as an analytical
method.
show characteristic fragmentations, which strongly depend on
the structure of analysed N-trifluoroacetyl amino acid ester.5 The
XIC for [M + H]+ = 668 Da (Fig. 2B) revealed also a peak at
Rt = 35.9 min with identical mass spectrum (data not shown) as
for Rt = 36.3 min (Fig. 2C), which suggests epimerization of Lab
as a likely explanation for this observation.
The identification of the amino acid Lan (1) and MeLan (2)
in lantibiotics by means of GC/MS is known for several years,4
in contrast, no analytical protocols were available for the
detection of recently discovered triamino triacid Lab (7).
In this contribution for the first time we report on the
identification of the amino acid Lab (7) and its desulfurised
derivative acid a-methyl-2,4-diaminoglutaric acid (Dmg, 9)
from lantibiotics of the labyrinthopeptin-type by means of
GC-PCI-MS (PCI = Positive Chemical Ionisation). This
achievement provides rapid access to an unambiguous
identification of Lab (7) and analogues and will ultimately
assist in the assignment of unknown lantibiotics to distinct
lantibiotic classes.
The relative signal intensities in the GC-MS compared to
other amino acid constituents were significantly reduced and
several other compounds eluting from the column could not be
assigned (Fig. 2A).
In order to give additional proof on the occurrence and
the assignment of Lab (7), degradation of LabA2 (6a) by
desulfurisation of thioethers was applied as previously reported
for subtilosin A6 using NiCl2/NaBH4 treatment under retention
of the stereochemistry.7 Progress of desulfurisation was
monitored by LC-ESI-MS and the reaction was completed
within 24 hours. Fig. 3 shows the HR-ESI(+)-orbitrap mass
spectra of LabA2 (6a, in black) before [(M + 2H)2+ = 962 Da]
and after (6b, in red) desulfurisation [(M + 2H)2+ = 931 Da]
corresponding to a formal loss of two sulfur atoms and formal
addition of four hydrogen atoms. Under the reaction conditions
applied, the integrity of cystine remained untouched.
We sought to develop robust protocols for a differentiation
between Lan/MeLan (1/2) and Lab (5). Therefore, total
hydrolysis of the lantibiotic nisin as a model system was
performed and the hydrolysate was derivatised as N-trifluoro-
acetyl amino acid ethylesters. Detection of Lan/MeLan (1/2)
by means of GC-PCI-MS in Nisin (ESIw, 25, Fig. S7 and S10)
was established according to previous reports.4 In the same
manner we subsequently performed total hydrolysis of LabA2
(6a) and derivatisation of the hydrolysate. As expected, the
signals of the corresponding N-trifluoroacetyl/ethyl esters
could be identified and assigned to Ala, Gly, Thr, Leu, Asp,
Glu, Phe and Trp with the exception of (Cys)2 (Fig. 2A).
Additionally the GC-MS chromatogram revealed a signal at
Rt = 36.3 min (Fig. 2B) assigned to derivatised Lab (8). This
From the progress of the desulfurisation monitored by
LC-MS transformation of Lab (7) into a-methyl-2,4-diamino-
glutaric acid (Dmg, 9) and Ala was expected. Conversion
of the hydrolysate of desulfo-LabA2 (6b) into the
N-trifluoroacetyl/ethyl esters and subsequent GC-MS analytics
gave a new signal at Rt = 20.9 min (Fig. 4A and 5B) whereas
the previous signal from derivatised Lab (8) had disappeared.
Trp was not detected after desulfurisation (Fig. 4A) of
LabA2 (6a) whereas several other compounds eluted from
the column which could not be identified. Nevertheless,
characteristic molecular ions for Dmg derivative (10) (Rt =
20.9 min), [M + H]+ = 425 Da and [M + C2H5]+ = 453 Da
shown in Fig. 4B and characteristic mass fragments were
assigned (Fig. S12, ESIw).
was corroborated by indicative molecular ions [M + H]+
=
668 Da and [M + C2H5]+ = 696 Da of derivatised Lab (8)
under mild GC-PCI-MS conditions (Fig. 2C) and assignment
of characteristic ion fragments (Fig. S11, ESIw). These ions
Our intention was to independently verify the identity of
Dmg (9) by synthesis of the racemate according to Scheme 1.
Synthetic access was obtained by Claisen-type rearrangement
reaction of Cbz-L-alanine-allylester (13) to Cbz-a-allyl-alanine
as the central step.8 Subsequent to this step a Sharpless
dihydroxylation9 of the allyl moiety (15) and selective protection
of the primary alcohol with TBDMS (ꢀ17a/b) was performed.
By a Mitsunobu reaction an azide group was introduced
and further steps comprised TBDMS deprotection, oxidation
of the resulting alcohol, removal of protecting groups, hydro-
genolysis and N-trifluoroacetyl/ethylester derivatisation which
Fig. 2 (A) GC-MS chromatogram of the hydrolysate of LabA2 (6a)
(6N HCl, 24 h) after N-trifluoroacetyl/ethylester derivatisation.
(B) Enlarged section of (A) Lab (8) (TIC in black and normalised
XIC [M + H]+ = 668 Da in red). (C) PCI mass spectrum of
N-trifluoroacetyl/ethylester of Lab (8) at Rt = 36.3 min. Peaks marked
with asterisk (*) are unidentified byproducts.
Fig. 3 HR-ESI(+)-orbitrap mass spectra of LabA2 before desulfuri-
sation (black, 6a) [(M + 2H)2+ = 962 Da] and after desulfurisation
(red, 6b) [(M + 2H)2+ = 931 Da].
c
7402 Chem. Commun., 2011, 47, 7401–7403
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Fig. 5(A) shows the GC-MS analysis of synthetic ꢀ23a/b.
The gradient allowed to separate the diastereomeric pairs
ꢀ23b at Rt = 20.6 min and ꢀ23a at Rt = 20.9 min,
respectively. As expected both pairs showed identical
fragmentations patterns under PCI-MS conditions (ꢀ23a/b,
Fig. S8, ESIw) compared to the natural product derived
compound Dmg (10). The identity in retention time (Fig. 5A
and B) and fragmentation patterns of synthetic reference
compounds ꢀ23a unequivocally proved the identity of this
amino acid as Dmg (10, Fig. 4B and 5B).
The derivatisation protocol combined with GC-MS analysis
developed for Lab (7)/Dmg (9) was applied to the analysis
of Labyrinthopeptin A1 (LabA1) and desulfurised LabA1
(ESIw, 24a/b, Fig. S5 and S13), respectively. LabA1 is
a member of the labyrinthopeptin family, for which the
structure has mainly been derived from genomic data of the
biosynthesis gene cluster.2 In accordance to the analytics of
LabA2 (6a/b) the GC-MS data of LabA1 (ESIw, 24a/b,
Fig. S5, S9, and S13) also could unambiguously prove the
presence of Lab (7) and Dmg (9) for the first time.
Fig.
4 (A) GC-MS chromatogram of the desulfo-LabA2 (6b)
In summary, we have established a protocol for the
detection of the unusual triamino triacid Lab (7) by GC/MS
from LabA2 hydrolysates. Furthermore, the above strategy
combined with NiCl2/NaBH4-mediated desulfurisation of
Lab-containing type-III lantibiotics can be independently
applied for GC-MS analysis of derivative Dmg (9). Sub-
sequently, this protocol was successfully applied to the
detection of Lab (7) and Dmg (9) from labyrinthopeptin
LabA1 and desulfurised LabA1 (ESIw, 24a/b, Fig. S13),
respectively. The detection of Lab (7) and Dmg (9) comple-
ments the presently available methods for the analytics of
lantibiotics. Our future attempts will extend on other type-III
lantibiotics to prove the occurrence of Lab (7) (ESIw, 24a,
Fig. S13) in these lantibiotics and we hypothesise that Lab (7)
could be considered as a common structural motif characteristic
for many type-III lantibiotics.
hydrolysate after N-trifluoroacetyl/ethylester derivatisation. (B) PCI
mass spectrum of the N-trifluoracetyl/ethylester of Dmg (10) at Rt =
20.9 min.
Fig.
5 GC-MS analytics. (A) Synthetic diastereomers of Dmg
(ꢀ23a/b) (TIC in black and normalised XIC [M + H]+ = 425 Da
in red). (B) Dmg (10) from desulfurised hydrolysate of LabA2 (6b)
(TIC in black and normalised XIC [M + H]+ = 425 Da in red). Peaks
marked with asterisk (*) are undefined byproducts.
The work was supported by a research grant from the
Deutsche Forschungsgemeinschaft (SU239/8-1).
Notes and references
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Scheme 1 Synthesis of Dmg (ꢀ23a/b) as a standard for assignment of
Lab derivatives after desulfurisation, hydrolysis and N-trifluoroacetyl/
ethylester derivatisation. (i) LDA; ZnCl2, (ii) BF3*OEt; TBTA,
(iii) AD-Mix-b, (iv) TBDMSCl; imidazol, (v) PPh3; DEAD; DPPA,
(vi) TBAF, (vii) TEMPO; KBr; NaOCl, (viii) TFA; TES, (ix) Pd/C;
H2, (x) AcCl; EtOH; TFAA.
finally led to synthetic Dmg (ꢀ23a/b). The stereochemistry of
synthetic Dmg diastereomers (ꢀ23a/b) was afterwards
deduced by comparison of the retention time of Dmg (10).
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 7401–7403 7403