Identification and isolation of lantibiotics from culture: A bioorthogonal chemistry approach

Article abstract of DOI:10.1039/c2ob26050f

A distinguishing feature of the lantibiotic family of cyclic peptides is the presence of thioethers. Treatment of a lantibiotic with an alkaline solution at high pH gives rise to a β-elimination reaction yielding the corresponding ring opened precursor, c

Full text of DOI:10.1039/c2ob26050f

View Article Online / Journal Homepage / Table of Contents for this issue
Dynamic Article Links
Organic &
Biomolecular
Chemistry
Cite this: Org. Biomol. Chem., 2012, 10, 8677
www.rsc.org/obc
PAPER
Identification and isolation of lantibiotics from culture: a bioorthogonal
chemistry approach†
Jing Li,a Genevieve Girard,b Bogdan I. Florea,a Paul P. Geurink,a Nan Li,a Gijsbert A. van der Marel,a
Mark Overhand,a Herman S. Overkleeft*a and Gilles P. van Wezel*b
Received 31st May 2012, Accepted 10th September 2012
DOI: 10.1039/c2ob26050f
A distinguishing feature of the lantibiotic family of cyclic peptides is the presence of thioethers.
Treatment of a lantibiotic with an alkaline solution at high pH gives rise to a β-elimination reaction
yielding the corresponding ring opened precursor, containing a dehydro-amino acid residue. We here
reveal in a proof-of-concept study that a ring opened lantibiotic (mersacidin) can be captured for pull-
down from a culture broth, subsequently released and identified by mass spectrometry.
bacteria by pore formation or inhibition of peptidoglycan biosyn-
Introduction
thesis.6 Some lantibiotics combine more than one mode of
action, allowing them to escape resistance mechanisms.7 These
Due to the rapid spread of drug-resistant infectious diseases a
point of no return has been reached where novel antibiotics are
an absolute necessity.1 However, it has become increasingly
difficult to find novel classes of antibiotics with efficacy against
multi-drug resistant pathogens such as MDR-TB (multi-drug
resistant Mycobacterium tuberculosis), MRSA (methicillin-
resistant Staphylococcus aureus) and the rapidly emerging MDR
Gram-negative pathogens. Actinomycetes are prolific antibiotic
producers, and full genome sequencing efforts have established
that even the widely studied species are still relatively untapped
sources of natural products. Around 20 new gene clusters encod-
ing natural products were found on the genome of the actino-
mycete Streptomyces coelicolor alone.2 This suggests the
presence of silent antibiotics, and new approaches are required to
identify these.3 One promising class of antibiotics is that of the
lantibiotics, peptide antibiotics that are characterized by the pres-
ence of one or more cyclic thioether linkages.4 Nisin, the first
discovered lantibiotic and produced by the firmicute Lactococcus
lactis, is used as a food preservative and no significant bacterial
resistance has thus far been encountered.5 At present, more than
50 different lantibiotics produced by Gram-positive bacteria are
known with diverse structure and function, and due to the next-
generation sequencing technology many new lantibiotic gene
clusters are being discovered. Lantibiotics show substantial
specificity for bacterial cell membrane components, including
lipid II and phosphoethanolamine, and act on pathogenic
properties classify the lantibiotics as an interesting class of mole-
cules and a viable target for the discovery and development of
novel antimicrobial therapeutics.8
Lantibiotics are ribosomally synthesized as linear pro-peptides
and post-translationally modified to their biologically active thio-
ether containing forms. Following secretion, the leader peptide is
removed, creating the mature lantibiotic (typically around
2 kDa). Studies of the biosynthesis of lantibiotics suggest that
the post-translational modifications generally involve a two-step
procedure in which threonine and serine residues of the precur-
sor oligopeptides are enzymatically dehydrated to form dehydro-
butyrine and dehydroalanine residues, respectively.9 These
unsaturated amino acid residues serve as electrophiles on which
the thiolates of cysteine residues can attack via intra-molecular
1,4-addition followed by stereoselective protonation of the result-
ing enolate to create a β-methyllanthionine or lanthionine
moiety, respectively. Lantibiotics containing several thioether
rings may result from programmed post-translational modifi-
cation steps. Recently, it was found that even more complex ring
structures can be obtained when the resulting enolate is not pro-
tonated but serves as a nucleophile in an additional intramole-
cular 1,4-addition reaction.10 The thioether rings of certain
lantibiotics can be chemically reversibly opened following an
elimination reaction at high pH and elevated temperature
(Scheme 1). The resulting ring opened intermediates can be
trapped by the addition of an excess of a thiol reagent, such as
mercaptoethanol. Several ring opened derivatives can thus be
prepared and this strategy has been successfully applied for the
sequence analysis of the lantibiotics galidermin, Pep5 and acta-
gardine using NMR spectroscopy.11 It occurred to us that this
chemical feature of lantibiotics may be generally applied in a
two-step labeling strategy12 for the discovery of novel
aLeiden Institute of Chemistry and Netherlands Proteomics Centre,
Gorlaeus Laboratories, Einsteinweg 55, 2333 CC Leiden,
The Netherlands. E-mail: h.s.overkleeft@chem.leidenuniv.nl
bInstitute Biology Leiden, Sylviusweg 72, 2300 RA Leiden,
The Netherlands
†Electronic supplementary information (ESI) available. See DOI:
10.1039/c2ob26050f
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 8677–8683 | 8677

View Article Online
Scheme 1 The opening of a thioether linkage of a lantibiotic anti-
microbial peptide and the trapping of the ring opened intermediate using
excess thiol reagent, and the structures of the designed probes 2 and 1
(generated in situ from 3) used in the two-step labeling strategy.
lantibiotics produced by bacillus and streptomyces bacterial
strains. For this purpose we designed the probes 2 and 3
(Scheme 1). The free thiol 1, generated in situ from 3 under the
strong basic reaction conditions of the elimination reaction, is
equipped with an acetylene moiety for a projected click reac-
tion13 with the azide moiety of probe 2. Probe 2 contains a
biotin handle for recognition using streptavidin beads and a
hydrazine sensitive cleavable linker we previously described for
chemical proteomics purposes.14 We here reveal that probes 2
and 3 can be used in a two-step labeling protocol to identify the
known lantibiotic mersacidin15 from Bacillus subtilis fermenta-
tion broth.
Scheme 2 (a) NaH, propargyl bromide in THF. (b) TsCl, NEt3 in
CH2Cl2. (c) KSAc in DMF. (d) Ethyl 5-azido-4-oxopentanoate, Pd/C,
H2 in methanol. (e) 2 M NaOH in methanol. (f) PPh3, THF/H2O 4/1
v/v. (g) 4-Hydroxy-3,5-diisopropylbenzaldehyde, NaOH, NaBH4 in
methanol. (h) Ac2O. (i) Compound 6, EDC, DMAP in DMF.
pure mersacidin (Scheme 3) and used the optimized conditions
to identify this lantibiotic from B. subtilis (Scheme 4). Complete
conversion of mersacidin was accomplished in aqueous metha-
nol at pH > 10 at 50 °C for 1 h using 30 equiv. of compound 3
(Scheme 2, step A). LC/MS analysis showed that mersacidin
was completely converted into its anticipated products with the
addition of one, two and three sulfide-alkyne moieties (HSA)
(Fig. S1, ESI†). This is indicated by the ions m/z 1037.00 Da
[M + HSA + Ac + 2H+]2+, 1139.00 Da [M + 2HSA + Ac +
2H+]2+ and 1240.67 Da [M + 3HSA + Ac + 2H+]2+. By per-
forming MS/MS sequencing it was found that the single mers-
acidin modified product is labeled at position 13 in the sequence
(Fig. S2, ESI†). The additional acetyl group in the products at
the N-terminus is probably obtained by a reaction involving the
deacetylation of 3. In addition, the in situ generated reactive
intermediate 1 was found to dimerize during the reaction. To
prevent labeling via S–S bond formation, a reduction step (using
DTT) and an alkylation step (using iodoacetamide) were intro-
duced (Scheme 3 step B, LC/MS analysis is shown in Fig. S3,
ESI†). Copper(I)-catalyzed azide–alkyne cycloaddition between
the alkyne modified mersacidin and biotin–azide 2 (Scheme 3,
step C) was complete within 17 h at room temperature according
to LC/MS analysis (Fig. S4, ESI†). The corresponding labeled
Results and discussion
The synthesis of compounds 2 and 3 is described in Scheme 2.
Triethylene glycol was alkylated using sodium hydride and pro-
pargyl bromide, subsequently converted into its tosylate and
treated with potassium thioacetate to obtain compound 3 in 25%
yield over 3-steps. Compound 417 was treated with 5-azido-4-
oxopentanoate in the presence of 10% Pd/C to give 5 in 73%
yield. Ester 5 was readily saponified to provide compound 6 in
87% yield. Carboxylic acid 6 was reacted with the phenol 7,
obtained in a three step one pot procedure from 1,2-bis(2-azido-
ethoxy)ethane, in the presence of EDC/DMAP to give product
2 in only 15% yield. The low yield of the former reaction is
likely due to severe steric hindrance of the aromatic hydroxyl.
Once synthesized, we proceeded to evaluate their reactivity
using the known lantibiotic mersacidin (C80H120N20O21S4, MW
1824.78 Da), containing three methyllanthionine residues, one
dehydroalanine residue and one S-aminovinyl-2-methylcysteine
moiety.16 We first investigated the efficiency of our probes using
8678 | Org. Biomol. Chem., 2012, 10, 8677–8683
This journal is © The Royal Society of Chemistry 2012

View Article Online
Scheme 3 Protocol for the two-step labeling of the lantibiotic
mersacidin.
products are indicated by ions m/z 1466.53 Da [M + HSA + Ac
+ Biotin + 2H+]2+ and 1494.60 Da [M + HSA + Ac + AA +
Biotin + 2H+]2+. With these products in hand the optimal con-
ditions for the cleavage of the levulinolate-linkage were exam-
ined (Fig. S5, ESI†) and subsequently applied by pull down of
the labeled products using streptavidin coated magnetic beads
followed by treatment of the beads with 0.1 M hydrazine at
37 °C for 4 h (Scheme 3, step D). The resulting solution was
characterized with LC/MS. The corresponding products were
found as a major (m/z 1240.00 Da [M + HSA + Ac + Biotin-C +
2H+]2+) and a minor signal (m/z 1268.47 Da [M + HSA + Ac +
IAA + biotin-C + 2H+]2+, Fig. S6, ESI,† HRMS, Fig. S7†).
In the key experiment, Bacillus sp. H1LY 85 54728 was incu-
bated, the supernatant collected and the crude products applied
on a size-exclusion chromatography column. As indicated by
LC/MS mersacidin is present in the fermented sample along
with numerous other components, and the size-exclusion chrom-
atography step thus mainly serves to concentrate the fermentation
sample (Scheme 4). The obtained sample was treated with the
above described protocol steps A–D involving reaction with
in situ generated probe 1 at high pH and elevated temperature,
followed by S–S bond reduction, alkylation, dialysis, click reac-
tion using probe 2, pull down with streptavidin-coated magnetic
beads and hydrazine cleavage. The resulting products were ana-
lyzed with LC/MS. Following this protocol mersacidin was iso-
lated from culture and identified by the mass of the resulting
derivative with mass m/z 1239.93 Da [M + HSA + Ac + biotin-
Scheme 4 Two-step labeling strategy for the identification of mers-
acidin from cultured Bacillus sp. H1LY85 54728.
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 8677–8683 | 8679

View Article Online
C + 2H+]2+ (Scheme 4, the S-acetamide alkylated product could
not be clearly detected).
spectrometer was calibrated prior to measurements with a cali-
bration mixture (Thermo Finnigan). LC-MS analysis was per-
formed on a Jasco HPLC system with a Phenomenex Gemini
3 lm C18 50 × 4.60 mm column (detection simultaneously at
214 and 254 nm), coupled to a PE Sciex API 165 mass spec-
trometer with ESI (System A) or a Finnigan Surveyor HPLC
system with a Gemini C18 50 × 4.60 mm column (detection at
200–600 nm), coupled to a Finnigan LCQ Advantage Max mass
spectrometer with ESI (System B). The applied buffers were
H2O, ACN and 1.0% aq. TFA. The gradient used was 10% →
90% ACN/0.1% aq. TFA.
Conclusion
Two probes were designed and synthesized for a two-step label-
ing strategy to identify lantibiotic peptides following opening of
one (or more) of the characteristic thio-ether linkages. Using this
strategy, involving a first step labeling via an elimination/1,4-
addition reaction, a second step labeling with a click reaction,
and pull-down of the modified peptide products using streptavi-
din beads and hydrazine-induced release, we positively identified
the lantibiotic mersacidin in a fermentation sample of Bacillus
sp. H1L Y85 54728. The concentration of mersacidin in a fer-
mented sample is typically 1–10 mg L−1. Our procedure
involves the use of a size-exclusion chromatography step to
concentrate the sample. We are currently evaluating the here-
described approach to identify unknown lantibiotics from
cultures produced by actinomycetes.
S-2-(2-(2-(Prop-2-ynyloxy)ethoxy)ethoxy)ethyl ethanethioate
(3). To a stirred solution of triethylene glycol (5.30 g, 35 mmol)
and NaH (60% in mineral oil, 1.4 g, 35 mmol) in THF (100 mL)
at 0 °C after 30 min was added propargyl bromide (3.80 mL,
80% in toluene, 35 mmol) and stirring was continued for
30 min. The reaction mixture was diluted with ether (200 mL)
and washed with water (50 mL), brine (50 mL) and dried over
MgSO4. The organic solution was concentrated and the
residue was purified by silica gel column chromatography
(EtOAc : petroleum ether, elution with 1 : 2 → 3 : 1 v/v) to give
2.43 g of a colorless oil. 1.75 g (9.3 mmol) of this intermediate
was dissolved in CH2Cl2 (20 mL), treated with Et3N (3 mL) and
subsequently tosyl chloride (2.30 g, 12.1 mmol) was added
slowly at 0 °C. The reaction mixture was stirred at 0 °C for
30 min and at room temperature for 1 h, then poured onto water
(10 mL), and extracted with ether (3 × 20 mL). The organic
layer was washed with brine (10 mL), dried with MgSO4 and
concentrated under reduced pressure. The residue was purified
by silica gel column chromatography (EtOAc : petroleum ether,
elution with 1 : 10 → 1 : 1 v/v) to give 2.53 g of a colorless oil.
This intermediate was dissolved in DMF (20 mL) and treated
with KSAc (1.69 g, 14.8 mmol). The solution was stirred for 4 h
at 70 °C, and concentrated under diminished pressure. The
remaining reddish oil was purified by silica gel column chrom-
atography (EtOAc : petroleum ether, elution with 1 : 10 to 4 : 1
v/v) to give 3 (1.5 g, 25% yield in three steps) as a colorless oil;
Rf 0.55 (EtOAc : petroleum ether 1 : 2 v/v); 1H NMR (400 MHz,
CDCl3) δ 4.21 (d, J = 2.4 Hz, 2 H), 3.72–3.63 (m, 8 H), 3.60
(t, J = 6.4 Hz, 2 H), 3.10 (t, J = 6.4 Hz, 2 H), 2.44 (t, J =
2.4 Hz, 1 H), 2.34 (s, 3 H); 13C NMR (100 MHz, CDCl3)
δ 195.74, 79.83, 74.71, 70.73, 70.65, 70.48, 69.94, 69.28, 58.59,
30.76, 29.02; IR neat (cm−1) 3251.9, 2867.7, 1686.1, 1353.5,
1096.7, 1032.7, 626.2; HRMS m/z calculated for (M + H+)
C11H19O4S 2479.0926. Found: 2479.0999.
Experiment section
General methods
All reagents used were purchased from commercial sources and
were used without further purification. Solvents that were used
in reactions were stored over 4 Å molecular sieves, except
methanol and acetonitrile, which were stored over 3 Å molecular
sieves. Molecular sieves were flame dried before use. Tetra-
hydrofuran was distilled over LiAlH4 prior to use. The eluents
ethyl acetate (EtOAc) and petroleum ether (40–60 °C boiling
range) were distilled prior to use. Mersacidin was provided by
Novacta (UK). Bacillus was cultured in an Innova 4000 incu-
bator shaker. Column chromatography was performed on Screen-
ing Devices b.v. Silica Gel, with a particle size of 40–63 μm and
a pore diameter of 60 Å. Unless stated otherwise, all reactions
were monitored by TLC on Merck aluminum sheets (Silica gel
60 F254). Spots were detected under UV light (254 nm), and/or
by spraying with a solution of (NH4)6Mo7O24·4H2O (25 g L−1
)
and (NH4)4Ce(SO4)4·2H2O (10 g L−1) in 10% sulfuric acid, or a
solution of KMnO4 (20 g L−1) and K2CO3 (10 g L−1) in water,
followed by charring at ca. 150 °C. 1H NMR spectra were
recorded at 298 K on a Bruker AV-400 (400 MHz) spectrometer.
Chemical shifts are referenced to CDCl3 (7.26 ppm, CDCl3) or
CD3OD (3.31 ppm, CD3OD) or D2O (4.78 ppm, D2O).
13C NMR APT spectra were recorded at 100 MHz, and chemical
shifts are referenced to CDCl3 (77.23 ppm, CDCl3) or CD3OD
1
(48.9 ppm, CD3OD). H NMR data are reported as though they
Ethyl 5-azido-4-oxopentanoate. To a solution of ethyl 5-bromo-
4-oxopentanoate16 (0.35 g, 1.57 mmol) in THF (5 mL) was
added a solution of NaN3 (0.20 g, 3.14 mmol) in H2O (3 mL) at
0 °C. Stirring was continued for 1 h at room temperature and
then the reaction mixture was diluted with H2O (10 mL) and
extracted with EtOAc (3 × 10 mL). The organic layer was
washed with brine (10 mL), dried over MgSO4 and concentrated
under reduced pressure. The residue was purified by silica gel
column chromatography (EtOAc : petroleum ether 1 : 20 v/v) to
give ethyl 5-azido-4-oxopentanoate (0.27 g, 95%) as a slightly
are first order, and the peak assignments were made by 2D-NMR
spectroscopy (1H–1H COSY and HMQC). IR spectra were
recorded on a Perkin Elmer Paragon 1000 FT-IR Spectrometer.
High resolution mass spectra were recorded by direct injection
(2 μL of a 2 μM solution in water/acetonitrile 50/50 (v/v) and
0.1% formic acid) on a mass spectrometer (Thermo Finnigan
LTQ Orbitrap) equipped with an electrospray ion source in posi-
tive mode (source voltage 3.5 kV, sheath gas flow 10, capillary
temperature 250 °C) with resolution R = 60 000 at m/z 400
(mass range m/z = 150–2000) and dioctylphthalate (m/z =
391.28428) as a “lock mass”. The high-resolution mass
1
yellow oil; Rf 0.5 (EtOAc : petroleum ether 1 : 8 v/v); H NMR
(400 MHz, CDCl3) δ 4.13 (q, J = 7.2 Hz, 2 H), 4.04 (s, 2 H),
8680 | Org. Biomol. Chem., 2012, 10, 8677–8683
This journal is © The Royal Society of Chemistry 2012

View Article Online
2.72 (ddd, J = 8.8, 6.8, 1.7 Hz, 2 H), 2.66 (ddd, J = 8.8, 6.8, 1.7
Hz, 2 H), 1.26 (t, J = 7.2, 3 H); 13C NMR (100 MHz, CDCl3)
δ 203.08, 172.31, 60.89, 57.49, 34.40, 27.85, 14.11; IR neat
(cm−1) 2984.1, 2103.4, 1727.5, 1203.0; HRMS m/z calculated
for (M + H+) C7H12N3O3 186.0800. Found: 186.2195.
the reaction mixture was cooled to −10 °C and NaBH4 (30 mg,
0.77 mmol) was added. The reaction mixture was stirred for
30 min, then allowed to warm to room temperature and stirring
was continued for another 30 min. The reaction mixture was
then adjusted to pH 6 by adding acetic acid. To the solution was
added acetic anhydride (5 mL) and stirring was continued for
2 h. The reaction mixture was diluted with ether (20 mL) and
washed with 1 M NaOH (5 mL), water (5 mL), brine (5 mL) and
dried over MgSO4. The organic layer was concentrated under
reduced pressure and the residue was purified by silica gel
column chromatography (EtOAc : petroleum ether, elution with
1 : 2 → 3 : 1 v/v) to give compound 7 (0.27 g, 87%) as a color-
Ethyl 4-oxo-5-(6-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno
̲
[3,4-d]imidazol-4-yl)pentanamido)hexanamido)pentanoate (5).
Biotin-AHA-NHS 417 (0.1 g, 0.22 mmol) and ethyl 5-azido-4-
oxopentanoate (49 mg, 0.26 mmol) were dissolved in methanol
(20 mL). To this solution was added 10% Pd/C (5 mg) and stir-
ring was continued for 4 h under a hydrogen atmosphere. The
solution was concentrated and the residue was purified by silica
gel column chromatography (CH2Cl2 : CH3OH, elution with
30 : 1 → 7 : 1, v/v) to give compound 5 (80 mg, 73%) as a white
amorphous solid; Rf 0.19 (CH2Cl2 : CH3OH 10 : 1 v/v);
1H NMR (400 MHz, CD3OD) δ 4.52 (dd, J = 7.8, 5.0 Hz, 1 H),
4.33 (dd, J = 7.8, 4.5 Hz, 1 H), 4.13 (q, J = 7.1 Hz, 2 H), 4.09
(s, 2 H), 3.26–3.18 (m, 1 H), 2.96 (dd, J = 12.7, 5.0 Hz, 1 H),
2.79 (t, J = 6.4 Hz, 2 H), 2.73 (d, J = 12.7 Hz, 1 H), 2.61 (t, J =
6.4 Hz, 1 H), 2.30 (t, J = 7.4 Hz, 1 H), 2.22 (t, J = 7.3 Hz, 1 H),
1.78–1.39 (m, 14 H), 1.26 (t, J = 7.1 Hz, 3 H); 13C NMR
(100 MHz, CD3OD) δ 205.03, 175.04, 174.58, 172.95, 164.70,
61.99, 60.34, 60.23, 55.62, 48.33, 39.67, 38.80, 35.42, 35.18,
33.83, 28.69, 28.38, 28.10, 27.37, 26.08, 25.52, 25.11, 13.10; IR
neat (cm−1) 3314.7, 2936.0, 1710.0, 1640.8, 1538.0, 1423.8,
1197.5. HRMS m/z calculated for (M + H+) C23H39N4O6S
499.2512. Found: 499.2582.
1
less oil; Rf 0.29 (EtOAc : petroleum ether 1 : 1 v/v); H NMR
(400 MHz, CDCl3) δ 6.93, 6.83 (2 × s, 2 H), 5.52, 5.38 (2 × s,
1 H), 4.58 (s, 2 H), 3.76–3.72 (m, 1 H), 3.68–3.52 (m, 9 H),
3.41–3.36 (m, 2 H), 3.18 (sept, J = 7.0 Hz, 2 H), 2.20, 2.15 (2 ×
s, 2 H), 1.23 (d, J = 7.0 Hz, 12 H); 13C NMR (100 MHz, CDCl3)
δ171.27 (b), 171.12 (a), 149.45 (b), 149.39 (a), 134.53 (2 C, b),
134.07 (2 C, a), 129.17(a), 128.26 (b), 123.45 (2 C, a), 121.32
(2 C, b), 70.98, 70.87, 70.75 (b), 70.70 (a), 70.42 (b), 70.40(a),
69.39 (a), 69.22 (b), 53.63 (a), 51.00 (b), 47.81 (a), 45.99 (b),
27.02 (2 C), 22.68 (4 C), 21.66 (a), 21.63 (b); Rf 0.29 (EtOAc :
petroleum ether 1 : 1 v/v); IR neat (cm−1) 3342.1, 2960.3, 2111.9,
1624.3, 1471.9, 1287.7, 1202.7, 1123.5, 784.1. HRMS m/z calcu-
lated for (M + H+) C21H35N4O4 407.2580. Found: 407.2649.
4-((N-(2-(2-(2-Azidoethoxy)ethoxy)ethyl)acetamido)methyl)-2,6-
diisopropylphenyl 4-oxo-5-(6-(5-((3aS,4S,6aR)-2-oxohexahydro-
1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamido)pentano-
ate (2). Acid 6 (18 mg, 0.038 mmol), phenol 7 (31 mg,
0.078 mmol) and EDC (15 mg, 0.078 mmol) were taken up in
DMF (3 mL). To the suspension was added DMAP (5 mg,
0.04 mmol) and stirring was continued for 16 h at 70 °C. The
reaction mixture was concentrated under reduced pressure and
the residue was purified by silica gel column chromatography
(CH2Cl2 : CH3OH, elution with 30 : 1 → 7 : 1 v/v) to give
product 2 (4.8 mg, 15%) as a colorless oil; Rf 0.6 (CH2Cl2 :
4-Oxo-5-(6-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]-
imidazol-4-yl)pentanamido)hexanamido)pentanoic acid (6). To a
solution of ester 5 (80 mg, 0.16 mmol) in methanol (10 mL) was
added NaOH (2 M, 1.5 mL). The solution was stirred at room
temperature for 3 h, and then adjusted to pH 5 by adding HCl
(2 M). The solution was concentrated under reduced pressure
and after adding H2O (5 mL) sonicated for 5 min at room temp-
erature. The resulting white solid was filtered to obtain the
product 6 (65 mg, 87%) without the need for further purification;
1H NMR (400 MHz, DMSO) δ 12.15 (s, 1 H), 8.11 (s, 1 H),
7.74 (s, 1H), 6.44 (s, 1 H), 6.37 (s, 1 H), 4.31 (s, 1 H), 4.13 (s,
1 H), 3.92 (s, 2 H), 3.34 (s, 4 H), 3.10–2.04 (m, 13 H),
1.60–1.26 (m, 12 H); 13C NMR (100 MHz, DMSO) δ 206.19,
174.11, 172.91, 172.23, 163.17, 61.49, 59.64, 55.89, 48.77,
40.37, 38.74, 35.67, 35.45, 34.43, 29.44, 28.68, 28.49, 27.87,
26.55, 25.79, 25.43; IR neat (cm−1) 3315.7, 2939.9, 1699.8,
1638.9, 1537.8, 1264.0; HRMS m/z calculated for (M + H+)
C21H35N4O6S 471.2199. Found: 471.2272.
1
CH3OH 8 : 1 v/v); H NMR (400 MHz, CD3OD) δ 7.07, 6.99
(2 × s, 2 H), 4.72, 4.63 (2 × s, 2 H), 4.48 (dd, J = 7.9, 4.7 Hz,
1 H), 4.29 (dd, J = 7.9, 4.3 Hz, 1 H), 4.08 (s, 2 H), 3.66–3.52
(m, 8 H), 3.37–3.34 (m, 2 H), 3.22–3.16 (m, 3 H), 2.99–2.88
(m, 5 H), 2.70 (d, J = 12.7 Hz, 1 H), 2.28 (t, J = 7.4 Hz, 2 H),
2.24, 2.12 (2 × s, 2 H), 2.19 (t, J = 7.3 Hz, 2 H), 1.75–1.36 (m,
14 H), 1.60 (d, J = 6.5 Hz, 12 H); 13C NMR (100 MHz,
CD3OD) δ 206.04, 176.50, 176.01, 174.16, 173.40, 166.12,
146.17 (a), 146.13 (b), 134.54 (2 C, a), 142.07 (2 C, b), 137.23
(b), 126.53 (a), 124.66 (2 C, b), 122.98 (2 C, a), 71.79, 71.67,
71.53 (b), 71.51 (a), 71.23 (b), 71.20 (a), 70.32 (a), 70.12 (b),
63.42, 61.65, 57.06, 54.27 (a), 51.82, 49.80 (b), 49.71 (a), 47.16
(b), 41.10, 40.23, 36.85, 36.60, 35.16, 34.72, 30.12, 29.82,
29.53, 28.63 (4 C), 27.51, 26.96, 26.57, 21.85 (2 C), 21.75; IR
neat (cm−1) 3290.0, 2926.3, 2109.1, 1695.9, 1653.9, 1559.9,
1458.0, 1142.0, 668.0. HRMS m/z calculated for (M + H+)
C42H67N8O9S 859.4673. Found: 859.4751.
N-(2-(2-(2-Azidoethoxy)ethoxy)ethyl)-N-(4-hydroxy-3,5-diiso-
propylbenzyl)acetamide
(7). 1,2-Bis(2-azidoethoxy)ethane
(0.24 g, 1.29 mmol) was dissolved in a mixture of THF and
H2O (5 mL, 4 : 1 v/v). To this solution was added PPh3 (0.34 g,
1.29 mmol) and stirring was continued for 17 h. The solution
was diluted with H2O (10 mL) and washed with ether (2 ×
10 mL). The water layer was concentrated under reduced
pressure and the crude product was dissolved in methanol
(10 mL). The reaction mixture was cooled to 0 °C and
4-hydroxy-3,5-diisopropylbenzaldehyde (0.16 g, 0.77 mmol)
was added. The solution was adjusted to pH 8 with the aid of a
5 M NaOH solution. After stirring for 2 h at room temperature,
Bacterial strain culture conditions
For the preparation of culture stocks, the Bacillus sp. H1L Y85
54728 was grown in a nutrient broth at 30 °C for 24 h. A colony
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 8677–8683 | 8681

View Article Online
was transferred from the solid medium to a 3 mL tryptone soy
broth and incubated for 24 h at 200 rpm and 30 °C. The culture
was diluted 1 : 1 with sterile 20% glycerol and stored at −80 °C.
The seed culture (0.1 mL) was inoculated into 50 mL mersacidin
production medium 2 × BPM ((NH4)2SO4 (50 mM), MgSO4
(2 mM), CaCl2 (1 mM), FeSO4 (0.35 mM), MnSO4 (1 mM),
Tris-maleate buffer pH 7 (0.1 M), glucose (0.4 M), potassium
phosphate buffer pH 7 (0.04 M)) in a 200 mL spring flask and
was shaken at 30 °C and 200 rpm for 4–5 days. The culture
(50 mL) was centrifuged at 5000 rpm for 3 × 10 min. The super-
natant was collected and lyophilized. The foamy residue was
treated with methanol (50 mL) and sonicated for 10 min. The
suspension was centrifuged and the supernatant was collected
and concentrated to a small volume and applied on a Sephadex
LH 20 column using methanol as an eluent. The high molecular
weight fractions were collected, concentrated under reduced
pressure to give a sample of 8 mg. LC/MS analysis (Scheme 3)
showed that mersacidin (C80H120N20O21S4 calculated mass
1824.78) was present in the sample in a small amount, indicated
by ions m/z 913.27 Da [M + 2H+]2+ and 1826.47 Da [M + H+]+,
retention time 7.18 min.
mixture. The residue was dissolved in methanol (20 μL) and
pull-down (PD) buffer (50 mM Tris·HCl pH 7.5, 150 mM NaCl)
(1 mL) to afford a clear solution. The solution was incubated
with 50 μL pre-washed MyOne T1 Streptavidin grafted magnetic
beads (Invitrogen) at 4 °C with vigorous shaking for 1 h. The
beads were stringently washed with 300 μL PD buffer with 0.1%
SDS, 2 × 300 μL PD buffer, 2 × 300 μL wash buffer I,
2 × 300 μL wash buffer II and 2 × 300 μL water.
Step D: release from the beads and analysis. To the biotin
captured magnetic beads was added hydrazine (300 μL, 100 μM)
and the sample was shaken at 37 °C for 2 h (or at room temp-
erature for 17 h). The supernatant was removed and lyophilized
to give a white powder, which was dissolved in a mixture of
t-butanol, acetonitrile and water (40 μL, 1 : 1 : 1, v/v) that was
analyzed with LC/MS. From this sample the modified mers-
acidin product was identified having a retention time of 9.05 min
and m/z 1239.93 Da [M + HSA + Ac + biotin-C + 2H+]2+
(Scheme 3).
Analysis of the protocol
Opening of the thioether linkage(s) and labeling. A mixture
of pure mersacidin (0.25 mg, 0.137 μmol) kindly provided by
the company Novacta and thio-alkyne 3 (1.01 mg, 4.11 μmol, 30
equiv.), H2O (10 μL), EtOH (10 μL) in a 0.5 mL Eppendorf vial
was carefully flushed with argon for 2 min and 1 M NaOH
(4.1 μL, 4.11 μmol, 30 equiv.) was added. The mixture was
stirred at 50 °C for 1 h. A solution of 1 M acetic acid (4.1 μL)
was added to the reaction mixture. The solution was vortexed
and centrifuged. The supernatant was discarded and the white
precipitate was dissolved in EtOH and H2O (40 μL, 1 : 1, v/v).
The reaction mixture was analyzed by LC/MS. From the spec-
trum shown in Fig. S1,† mersacidin was modified at one, two
Protocol for the two-step labeling, pull-down, release and
identification of the lantibiotic mersacidin from culture
Step A: opening of the thioether linkage(s) and labeling. To
the obtained cultured sample (3 mg) in a 0.5 mL Eppendorf vial
were added thio-alkyne 3 (2 mg, 8.1 μmol), H2O (10 μL) and
EtOH (20 μL). The reaction mixture was carefully flushed with
argon for 2 min and a 1 M NaOH solution (8.1 μL) was added.
The reaction mixture was shaken vigorously (vortex) and stirred
at 50 °C for 1 h and subsequently quenched with a 1 M acetic
acid solution (8.1 μL). The reaction mixture was shaken vigor-
ously (vortex) and centrifuged for 3 min at 5000 rpm using an
Eppendorf centrifuge. The supernatant was discarded to obtain a
white precipitate.
and three positions in the sequence. This is indicated by the ions
2+
m/z 1037.00 Da [M + HSA + Ac + 2H+]2+, C91H140N20O25S5
,
calculated mass 1036.44, 1139.00 Da [M + 2HSA + Ac +
Step B: reduction of S–S linkages and reaction of the free
thiols with iodoacetamide. The white precipitate was dissolved
in EtOH and H2O (40 μL, 1 : 1 v/v) and a solution of DTT
(60 μL) as a 0.2 M solution in a 1 : 1 mixture of EtOH and
50 mM aq. (NH4)2CO3 was added. The Eppendorf vial was incu-
bated for 30 min at 37 °C using a water bath. Subsequently,
iodoacetamide (60 μL) as a 0.6 M solution in a 1 : 1 mixture of
EtOH and 50 mM aq. (NH4)2CO3 was added and the reaction
mixture was shaken for 30 min at room temperature in the dark.
The reaction mixture was dialysed with MWCO 1200 cellulose
tubing in a mixture of EtOH and H2O (1 : 1, v/v) for 24 h. The
resulting solution was collected and concentrated under reduced
pressure in an Eppendorf centrifuge.
2H+]2+ C100H156N20O28S62+, calculated mass 1138.48 and
2+
1240.67 Da [M + 3HSA + Ac + 2H+]2+, C109H172N20O31S7
,
calculated mass 1240.52, within 7.33–9.38 min retention times.
The modified mersacidin product [M + HSA + Ac + 2H+]2+ was
sequenced to show that the modification site is at position 13 in
the sequence (Fig. S2†). From the LC/MS data it was also
observed that the reactive compound 1, which is formed in situ
via deacetylation of 3 in the strong basic medium, also formed a
dimer during the reaction conditions (ion m/z 406.60 Da,
+
C18H31O6S2 , calculated mass 407.15). To exclude labeling via
the formation of S–S linkages we included a reduction and alkyl-
ation step in our protocol.
Reduction of S–S linkages and reaction of the free thiols with
iodoacetamide. The above obtained reaction mixture was treated
with a solution of 0.2 M DTT (40 μL) in a 1 : 1 mixture of EtOH
and 50 mM aq. (NH4)2CO3 for 30 min at 37 °C, and was alkyl-
ated with iodoacetamide (40 μL, 0.6 M in a 1 : 1 mixture of
EtOH and 50 mM aq. (NH4)2CO3) for 30 min at room temp-
erature in the dark. The mixture was dialysed with MWCO 1200
cellulose tubing in a mixture of EtOH and H2O (1 : 1, v/v) for
24 h. The resulting solution was collected and analyzed with
Step C: click reaction and pull-down using magnetic strepta-
vidin beads. The residue was dissolved in a mixture of t-butanol
and ethanol (10 μL, 2 : 1 v/v). The solution was flushed carefully
with argon and a solution of the biotin probe 2 (0.5 mg,
0.58 μmol) in t-butanol and ethanol (5 μL, 2 : 1 v/v) was added.
To the stirred reaction mixture were added CuSO4 (0.09 mg,
0.58 μmol) and sodium ascorbate (0.12 mg, 0.58 μmol). Stirring
was continued for 16 h followed by concentration of the reaction
8682 | Org. Biomol. Chem., 2012, 10, 8677–8683
This journal is © The Royal Society of Chemistry 2012

View Article Online
LC/MS (Fig. S3†). The spectrum showed that the thiol groups of
the thioether opened and modified mersacidin reacted with the
iodoacetamide as indicated by the ions m/z 1065.80 Da
[M + HSA + Ac + IAA + 2H+]2+, C93H143N21O26S52+, calcu-
lated mass 1064.95, rt 7.96 min; 1167.73 Da [M + 2HSA + Ac
+ IAA + 2H+]2+, C102H159N21O29S62+, calculated mass 1166.99,
rt 8.54 min. The product with ion 1037.40 Da [M + HSA + Ac
+ 2H+]2+, rt 8.25, 8.48 min, did not change.
(STW-Genbiotics program GenExpand) and by the Netherlands
Genomics Initiative (NGI).
Notes and references
1 (a) D. J. Payne, M. N. Gwynn, D. J. Holmes and D. L. Pompliano, Nat.
Rev. Drug Discovery, 2007, 6, 39–40; (b) D. J. Cooper and D. Shlaes,
Nature, 2011, 472, 32–32.
2 G. L. Challis and D. A. Hopwood, Proc. Natl. Acad. Sci. U. S. A., 2003,
100, 14555–14561.
3 G. P. Van Wezel and K. J. McDowall, Nat. Prod. Rep., 2011, 28,
1311–1333.
Click reaction. Pure mersacidin (2.1 mg, 1.15 μmol) was
labeled as described using compound 3 (8.5 mg, 34.5 μmol)
under basic conditions 1 M NaOH (34.5 μmol), reduced with
0.2 M DTT (400 μL) in a 1 : 1 mixture of EtOH and 50 mM aq.
(NH4)2CO3, alkylated with iodoacetamide (400 μL) 0.6 M in a
1 : 1 mixture of EtOH and 50 mM aq. (NH4)2CO3 and dialysed.
The obtained reaction mixture was dissolved in a mixture of
t-butanol and ethanol (30 μL, 2 : 1, v/v). The solution was
flushed carefully with argon for 2 min and a solution of the
biotin probe 2 (0.98 mg, 1.15 μmol) in t-butanol and ethanol
(10 μL, 2 : 1 v/v), CuSO4 (0.18 mg, 1.15 μmol) and sodium
ascorbate (0.23 mg, 1.15 μmol) were added. The reaction
mixture was stirred for 16 h at room temperature and analyzed
by LC/MS. The corresponding 2-step labeled products are indi-
cated by ions m/z 1466.53 Da [M + HSA + Ac + Biotin +
2H+]2+, C133H206N28O34S62+, calculated mass 1465.67, with rt
8.31 min, 1494.60 Da [M + HSA + Ac + IAA + Biotin +
2H+]2+, C135H209N29O35S62+, calculated mass 1494.18, with rt
7.94 min (Fig. S4†).
4 (a) A. C. Ross and J. C. Vederas, J. Antibiot, 2011, 64, 27–34;
(b) H. Abriouel, C. M. A. P. Franz, O. N. Ben and A. Galvez, FEMS
Microbiol. Rev., 2011, 35, 201–232; (c) J. M. Wiley and W. A. van der
Donk, Annu. Rev. Microbiol., 2007, 61, 477–501.
5 (a) L. A. Rogers, J. Bacteriol., 1928, 16, 321–325; (b) P. D. Cotter,
C. Hill and R. P. Ross, Nat. Rev. Microbiol., 2005, 3, 777–788.
6 (a) H. E. Hasper, N. E. Kramer, J. L. Smith, J. D. Hillman, C. Zachariah,
O. P. Kuipers, B. de Kruijff and E. Breuking, Science, 2006, 313,
1636–1637; (b) E. Breukink and B. de Kruijff, Nat. Rev. Drug Discovery,
2006, 5, 321–332.
7 J. M. Wiley and W. A. van der Donk, Annu. Rev. Microbiol., 2007, 61,
477–501.
8 C. Piper, P. D. Cotter, R. P. Ross and C. Hill, Curr. Drug Discovery
Technol., 2009, 6, 1–18.
9 T. J. Oman and W. A. van der Donk, Nat. Chem. Biol., 2010, 6, 9–18.
10 K. Meindl, T. Schmiederer, K. Schneider, A. Reicke, D. Butz, S. Keller,
H. Gühring, L. Vertesy, J. Wink, H. Hoffmann, M. Brönstrup,
G. M. Sheldrick and R. D. Süssmuth, Angew. Chem., Int. Ed., 2010, 49,
1151–1154.
11 (a) H. E. Meyer, M. Heber, B. Eisermann, H. Korte, J. W. Metzger and
G. Jung, Anal. Biochem., 1994, 223, 185–190; (b) N. Zimmermann,
J. W. Metzger and G. Jung, Eur. J. Biochem., 1995, 228, 786–797;
(c) J. Cortes, A. N. Appleyard and M. J. Dawson, Methods Enzymol.,
2009, 458, 559–574; (d) L. Smith, J. Novak, J. Rocca, S. McClung,
J. D. Hillman and A. S. Edison, Eur. J. Biochem., 2000, 267, 6810–6816.
12 (a) Y. Oda, T. Nagasu and B. T. Chait, Nat. Biotechnol., 2001, 19, 379–
382; (b) A. J. Poot, E. Ruijter, T. Nuijens, E. H. C. Dirksen,
A. J. R. Heck, M. Slijper, D. T. S. Rijkers and R. M. J. Liskamp, Proteo-
mics, 2006, 6, 6394–6399.
13 J. E. Moses and A. D. Moorhouse, Chem. Soc. Rev., 2007, 36,
1249–1262.
14 P. P. Geurink, B. I. Florea, N. Li, M. D. Witte, J. Verasdonck, C. L. Kuo,
G. A. van derMarel and H. S. Overkleeft, Angew. Chem., Int. Ed., 2010,
49, 6802–6805.
15 (a) W. W. Niu and H. C. Neu, Antimicrob. Agents Chemother., 1991, 35,
998–1000; (b) S. Chatterjee, D. K. Chatterjee, R. H. Jani, J. Blumbach,
B. N. Ganguli, N. Klesel, M. Limbert and G. Seibert, J. Antibiot., 1992,
45, 839–845; (c) G. Bierbaum, H. Brotz, K. P. Koller and H. G. Sahl,
FEMS Microbiol. Lett., 1995, 127, 121–126; (d) H. Brotz, G. Bierbaum,
A. Markus, E. Molitor and H. G. Sahl, Antimicrob. Agents Chemother.,
1995, 39, 714–719.
Reactivity of the cleavable linker. The biotin labeled mersaci-
din dissolved in methanol (21 μL) was divided over six 0.5 mL
Eppendorf vials and two different concentrations of hydrazine
(0.05 M, 0.1 M) were added and each of the two sets was stirred
at room temperature, 37 °C and 50 °C, respectively. The reac-
tions were monitored by LC/MS. It was found that the samples
using 0.1 M hydrazine, at room temperature for 17 h or at 37 °C
for 4 h, gave complete conversion, as shown in Fig. S5.† The
cleaved products were found to be ions m/z 1239.93 Da
[M + HSA + Ac + biotin-C + 2H+]2+ with rt 9.00 min, 1268.93
Da [M + HSA + Ac + IAA + biotin-C + 2H+]2+ with rt
8.53 min.
Acknowledgements
We are very grateful to Mike Dawson for providing mersacidin
and for discussion, and to Mervyn Bibb for discussion. This
research was funded by the Dutch Technical Science Foundation
16 R. Vallinayagam, H. Bertschy, Y. Berger, V. Wenger and R. Neier, Syn-
thesis, 2007, 3731–3735.
17 H. Shi, A. Xu, S. Yao and K. Liu, Chem. Commun., 2009, 5030–5032.
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 8677–8683 | 8683