Solid-supported synthesis and biological evaluation of the lantibiotic peptide bis(desmethyl) lacticin 3147 A2

Article abstract of DOI:10.1002/anie.200802919

(Chemical Equation Presented) Lan-tastic! A lanthionine analogue of lacticin 3147 A2 (Lan-A2, 2) containing multiple thioether bridges (see picture) has been successfully synthesized by a combination of solid- and solution-phase peptide synthesis. Chemically synthesized Lan-A2 (2) exhibits synergistic biological activity similar to natural lacticin A2 (1) in the presence of natural lacticin A1 against Gram-positive bacteria.

Full text of DOI:10.1002/anie.200802919

Communications
DOI: 10.1002/anie.200802919
Lantibiotic Synthesis
Solid-Supported Synthesis and Biological Evaluation of the Lantibiotic
Peptide Bis(desmethyl) Lacticin 3147 A2**
Vijaya R. Pattabiraman, Shaun M. K. McKinnie, and John C. Vederas*
Lantibiotics are a class of bacteriocins (antimicrobial peptides
from bacteria) that undergo extensive post-translational
[
1]
processing. Their biosynthesis involves enzymatic dehydra-
tion of serine and/or threonine residues with subsequent
intramolecular Michael addition of cysteine thiols to form
[
2]
lanthionine or b-methyllanthionine rings. Lantibiotics are
produced by Gram-positive bacteria either as single peptide
[3]
antibiotics (e.g., nisin A) or as two peptide systems. Many
lantibiotics bind lipid II, the precursor of peptidoglycan,
thereby hindering bacterial cell wall formation, and in some
cases, creating pores in the membrane at nanomolar concen-
[
4]
trations. They are generally nontoxic to mammals, and some
are very active against Gram-positive bacteria that are
resistant to methicillin (e.g., methicillin-resistant Staphylo-
coccus aureus (MRSA)) and vancomycin (e.g., vancomycin-
[
1]
resistant enterococcus (VRE)). They are already used in
food preservation and have considerable potential in human
medicine. The two-component lantibiotic, lacticin 3147, con-
[
5]
sists of A1 (1) and A2 (2) peptides that exhibit synergistic
Figure 1. Lacticin 3147 components A1 (1) and A2 (2). The bis(des-
methyl) lanthionine analogue (R=H) of lacticin A2 is Lan-A2 (3). It is
proposed that the a-carbon atoms of residues 26, 22, and 16 in 2, and
antimicrobial activity in nanomolar concentration (Figure 1).
[4b]
The mechanism involves initial binding of A1 (1) to lipid II.
[5]
This complex is then recognized by lacticin A2 (2) to give a
three-component assembly that promotes the formation of
pores in the cell membrane. Studies on structure–activity
relationships of these lantibiotics are being pursued to
alanine residues 9 and 12 have the d configuration.
[
6]
uncover the principles for designing new antibiotics. In
this respect, the development of chemical methods for the
synthesis of lantibiotics and their analogues has interested a
lanthionines. Biological evaluation of 3 shows that it unex-
pectedly retains potent synergistic activity with A1, but loses
its inherent independent antimicrobial activity, which indi-
cates two independent mechanisms for the natural A2 pep-
tide.
[
7,8]
number of research groups.
nisin A represents the only total chemical construction of a
A solution-phase synthesis of
[
9]
lantibiotic. Recently, we reported a solid-supported syn-
thesis of an inactive analogue of lacticin 3147 A2 wherein all
of the lanthionine bridges were replaced by larger carbocyclic
rings. We now describe a solid-phase synthesis of bis(des-
methyl) lacticin 3147A2 (Lan-A2, 3), an analogue of A2 (2)
that has the two b-methyllanthionine bridges replaced by
The synthetic approach to 3 utilizes solid-supported (9H-
fluoren-9-ylmethoxy)carbonyl (Fmoc) peptide synthesis with
an orthogonally protected lanthionine precursor, which is
coupled to the growing chain and eventually deprotected at
the distal sites for intramolecular ring formation (Figure 2).
The N-terminal residues (1–5) are synthesized in solution and
coupled as a unit onto the peptide. The lanthionine protection
was inspired by elegant studies by Tabor and co-workers for
[
10]
[
*] V. R. Pattabiraman, S. M. K. McKinnie, Prof. J. C. Vederas
Department of Chemistry, University of Alberta
Edmonton, Alberta T6G 2G2 (Canada)
Fax: (+1)780-492-2134
[
8]
the preparation of the monocyclic ring of nisin.
To obtain multigram quantities of orthogonally protected
[
7–9,11]
lanthionine
in a minimum number of steps, a combina-
E-mail: john.vederas@ualberta.ca
tion of the phase-transfer conditions to make lanthionines
[12]
[
**] This work was supported by the Natural Sciences and Engineering
Research Council of Canada (NSERC), the Alberta Heritage
Foundation for Medical Research (AHFMR), the Advanced Food &
Materials Network (AFMNet), and the Canada Research Chair in
Bioorganic and Medicinal Chemistry. We thank Randy Whittal and
Jing Zheng (University of Alberta) for help with mass spectrometry
studies.
reported by Zhu and Schmidt was used with the orthogonal
[8a]
protection scheme of Bregant and Tabor. Reaction of Aloc/
allyl-protected b-bromo-d-alanine (4) (Aloc = allyloxycar-
bonyl) as the electrophile with Fmoc/tBu-protected l-cysteine
[
13]
(5) as the nucleophile
in the presence of (Bu) NBr in
4
EtOAc and NaHCO3 (0.5m, pH 8.5), gave orthogonally
protected lanthionine, with the desired isomer predominating
in a 9:1 ratio based on the C NMR spectrum (Scheme 1).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200802919.
1
3
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472
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Chemie
Figure 2. Synthetic approach to 3, a lanthionine analogue of
lacticin A2.
Removal of the tert-butyl group under acidic conditions gave
6
in quantitative yield.
Scheme 1. Synthesis of orthogonally protected lanthionine.
[
14]
Acid 6 was attached to 2-chlorotrityl chloride resin with
ꢀ
1
a low loading (0.16 mmolg ) to avoid complications from
intermolecular coupling after deprotection of the distal
carboxyl and amino groups of the lanthionine residue. The
amino acids required for the linear sequence of ring C (8)
were introduced by Fmoc solid-phase peptide synthesis
(
SPPS) using (benzotriazol-1-yloxy)tripyrrolidinophospho-
nium hexafluorophosphate (PyBOP) as the coupling reagent.
Aloc and allyl protecting groups of the lanthionine residue in
[8a]
8
were removed on-resin
with [Pd(PPh ) ] and PhSiH ,
3 4 3
followed by removal of the Fmoc group with piperidine.
Cyclization to 9 was accomplished by on-resin reaction of the
peptide with PyBOP/HOBt (HOBt = 1-hydroxy-1H-benzo-
triazole) for 2 h (Scheme 2).
Scheme 2. Solid-phase synthesis of residues (6!29) of Lan-A2.
NMM=N-methylmorpholine, Boc=tert-butoxycarbonyl,
Pmc=2,2,5,7,8-pentamethylchroman-6-ylsulfonyl, Trt=triphenylmethyl.
A small sample of the peptide was cleaved from the solid
support using trifluoroacetic acid (TFA) for analysis by
electrospray mass spectrometry, which indicated the complete
formation of lanthionine ring C in 9. SPPS was continued for
the introduction of the protected amino acids required for the
linear portion of ring B. Removal of the Aloc/allyl and Fmoc
protecting groups as before, followed by cyclization with
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PyBOP/HOBt (2 ꢀ 2.5 h) on the
solid support, gave an only parti-
ally ring-closed product. The on-
resin peptide was reacted for a
further 2.5 h to force the reaction
to completion to give 10. After
liberation of a sample of the pep-
tide from the solid support with
TFA, analysis by MALDI-TOF
mass spectrometry showed
a
major peak corresponding to
ring B. In an approach similar to
the construction of rings B and C,
on-resin extension to the linear
precursor of ring A was accom-
plished by SPPS to introduce
amino
acid
residues 14–21.
Removal of the protecting groups
led to the unprotected amino and
carboxylic acid functionalities
required for cyclization. To
ensure the reaction was complete,
cyclization using PyBOP was done
over an extended time period (3 ꢀ
Scheme 3. Coupling of residues (1–5) to complete the synthesis of lanthionine analogue of
lacticin 3147 A2 (Lan-A2, (3)). DIPCDI=diisopropylcarbodiimide.
2
.5 h). A small sample was cleaved
from the resin and analyzed by
mass spectrometry; this showed
the successful formation of the desired tricyclic lanthionine
rings in 11. During the three cyclization steps, none of the
interchain dimerized peptide was observed, presumably
because of the low resin loading. The unmodified linear
region consisting of amino acids 6–15 (including d-Ala at 9
and 12) was introduced by regular Fmoc SPPS to obtain 12.
Residues 1–5 of Lan-A2 (3) and lacticin A2 (2) consist of
two dehydrobutyrine groups and an N-terminal a-ketoamide
arising from post-translational modification of l-threonine.
The pentapeptide 13 was assembled in solution by using a
using FT-ICR-MALDI mass spectrometry (ICR = ion cyclo-
tron resonance). The structure of 3 was further supported by
detailed tandem mass spectrometry studies to confirm the
correct sequence as well as the connectivity of the lanthionine
rings.
Preliminary biological evaluation of bis(desmethyl) lacti-
cin 3147 A2 (Lan-A2, 3) was done with the aid of natural
lacticin A1 (1), against the Gram-positive indicator organism
Lactococcus lactis subspecies cremoris HP. When applied next
to natural lacticin A1 (1), Lan-A2 (3) displayed synergistic
antimicrobial activity similar to the natural lacticin A2 (2,
Figure 3). However, in contrast to natural A2 (2), the
synthetic bis(desmethyl) analogue 3 did not exhibit any
[10]
strategy developed by us
that involves coupling of a
dehydrodipeptide with a dehydrotripeptide moiety, followed
by a transamination to introduce the a-ketoamide. To finish
the synthesis of Lan-A2 (3), the Fmoc group of residue 6
(
proline) in 12 was removed and the coupling of 13 to the
amino group of proline was done on-resin using N,Nꢁ-
diisopropylcarbodiimide/1-hydroxy-1H-benzotriazole
(
DIPCDI/HOBt; Scheme 3).
The peptide was cleaved from the polymer support with
trifluoroacetic
7.5:2.5)and purified by reverse-phase HPLC. Analysis by
MALDI-TOF mass spectrometry showed a parent signal at
819.6 Da, which indicates the successful synthesis of the
lanthionine analogue of lacticin 3147 A2 in an overall yield of
.3% (over 22 coupling and 3 cyclization steps, as well as
deprotection). This corresponds to an average yield of
approximately 84% for each of the 25 sequential coupling/
cyclization steps on the solid phase. However, the attachment
of the pentapeptide fragment 13 proceeds with lower yields,
and it is likely that most other amino acid coupling steps occur
in greater than 90–95% yield. To determine the exact mass of
acid/triisopropylsilane
(TFA/TIPS
9
2
Figure 3. Spot-on-lawn tests for antimicrobial activity showing zones of
growth inhibition: a) natural lacticin A1 (1) and A2 (2) and b) natural
lacticin A1 (1) and bis(desmethyl) analogue Lan-A2 (3).
1
independent antimicrobial activity at comparable concentra-
tions against the indicator organism. A serial dilution assay
performed by mixing Lan-A2 (3) with lacticin A1 (1) showed
that 3 was approximately 100 times less effective than natural
3, a high-resolution MALDI mass spectrum was obtained
9
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Angewandte
Chemie
lacticin A2 (2) in its ability to exert synergistic activity.
However, as the synergistic activity of natural 2 is in the low
nanomolar range for many organisms, the desmethyl deriv-
ative 3 is still very potent.
Interestingly, peptide 12, which has an Fmoc group
instead of residues 1–5, also exhibited synergistic activity
with lacticin A1, albeit at higher concentrations. The syner-
gistic activity of 12 was lost when the Fmoc group on residue 6
was removed. This observation indicates that the Fmoc group
can act as a mimic for residues (1!5), probably because of its
planarity and hydrophobicity. Further optimization could
potentially lead to structurally simpler analogues with fewer
amino acids than natural lacticin A2 (2).
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lacticin 3147 operates by at least two modes of action. The
inherent antimicrobial activity of natural 1 or 2 in the absence
of its partner may arise from a mechanism that involves direct
binding and sequestering of lipid II, with resulting inhibition
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activity of lacticin 3147. A lead compound 12 for generation
of structurally simpler lacticin A2 analogues has also been
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Published online: October 20, 2008
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9475