Concise preparation of tetra-orthogonally protected (2S,6R)-lanthionines

Article abstract of DOI:10.1021/jo802415c

Lantibiotics are antimicrobial peptides containing the unique bis-amino acids lanthionine and β-methyllanthionine. While previous syntheses of lanthionine have often involved the coupling of precursors derived from D-serine and L-cysteine, we here report

Full text of DOI:10.1021/jo802415c

a number of unique structural features including thioether
linkages and dehydrated amino acid side chains.6-8
Concise Preparation of Tetra-orthogonally
Protected (2S,6R)-Lanthionines
Nathaniel I. Martin
Department of Medicinal Chemistry and Chemical Biology,
UniVersity of Utrecht, Sorbonnelaan 16,
3584 CA Utrecht, The Netherlands
n.i.martin@uu.nl
FIGURE 1. Structure of the lantibiotic peptide nisin (1).
ReceiVed October 30, 2008
The biosynthetic origin of these structural features involves
the enzymatic dehydration of serine and threonine residues to
dehydroalanine(Dha)anddehydrobutyrine(Dhb),respectively.9-11
Subsequent enzyme-mediated Michael addition of cysteine
thiolates to Dha and Dhb residues generates the thioether cross-
links lanthionine and ꢀ-methyllanthionine respectively (these
moieties provide lantibiotic peptides their family name).12Given
the interest in lantibiotic peptides, various methods for their
synthetic preparation have been pursued. Critical to such
investigations is the ability to incorporate lanthionine residues
containing the (2S,6R) stereochemistry found in nature
(Figure 2).
Lantibiotics are antimicrobial peptides containing the unique
bis-amino acids lanthionine and ꢀ-methyllanthionine. While
previous syntheses of lanthionine have often involved the
coupling of precursors derived from D-serine and L-cysteine,
we here report an inverted strategy whereby D-cysteine and
L-serine are employed as building blocks. This approach
provides for a concise preparation of tetra-orthogonally
protected (2R,6S)-lanthionines while allowing convenient
introduction of orthogonal protecting groups not previously
incorporated into lanthionines.
FIGURE 2. Naturally occurring (2S,6R)-lanthionine and (2S,3S,6R)-
ꢀ-methyllanthionine.
In the only total synthesis of a lantibiotic to date, the Shiba
group utilized a desulfurizing ring-contraction approach to
convert cystines into lanthionines for the preparation of nisin.13
This methodology provides lanthionines in moderate yields and
is not amenable to solid-phase peptide synthesis (SPPS). As an
alterative, dehydro-residues have been incorporated into pep-
tides, after which Michael addition by a neighboring cysteine
thiolate can provide lanthionine-containing peptides.14-17 The
diastereoselectivity of lanthionine bridge formation in such
cases, however, has been shown to be highly dependent upon
preorganization of the peptide, and the desired stereochemical
outcome cannot be guaranteed in all cases. An alternate strategy
toward the synthesis of lantibiotic peptides involves the
The rapid emergence of drug-resistant bacteria poses a serious
threat to human health and underscores the importance of
developing new antibiotics. In this regard, the lantibiotic family
of antimicrobial peptides is rapidly gaining recognition.1-3
Lantibiotics are ribosomally synthesized and highly modified
bacterial defense peptides with potent antibacterial activity
against a wide range of infectious (and drug-resistant) bacteria.4,5
The most thoroughly studied lantibiotic nisin (Figure 1) contains
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der Donk, W. A. Science 2004, 303, 679.
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Leeuwenhoek 2002, 82, 165–185.
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R.; Jung, G. Nature 1988, 333, 276.
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Leiden, 1991.
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Jackson, D Biochem. J. 1992, 283 (Pt 2), 413–420.
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M.; Swali, V.; Bradley, M. Chem. Eur. J. 2000, 6, 1455–1466.
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946 J. Org. Chem. 2009, 74, 946–949
10.1021/jo802415c CCC: $40.75 2009 American Chemical Society
Published on Web 12/02/2008

SCHEME 1. Proposed Route to Tetra-orthogonally
Protected Lanthionines Employing IvDde Protection
Strategy
incorporation of orthogonally protected lanthionine building
blocks. Methods reported for the preparation of such building
18-20
blocks include the ring openings of serine ꢀ-lactones,
aziridines,21,22 and cyclic sulfamidates23,24 by protected cys-
teines. Apart from these ring-opening approaches, ꢀ-bromo-20,25,26
and ꢀ-iodoalanines27-29 derived from D-serine have also been
described as useful precursors in lanthionine preparation. In this
regard, the orthogonally protected lanthionine 4 was recently
employed by Tabor’s group in the solid-phase synthesis of the
nisin C-ring (Figure 3).29
bromide 6 (Scheme 1). To this end, D-serine was N-IvDde
protected by treatment with isovaleryl substituted dimedone 8
followed by conversion to allyl ester 10 (Scheme 2). Subsequent
conversion of 10 to the bromide 6, however, was problematic.
Under all reaction conditions explored, the undesired dehydro
elimination product was formed in significant quantities.
Complicating matters was the observation that bromide 6 was
found to further decompose to the dehydro side product upon
attempted chromatographic purification.
FIGURE 3. Tabor’s protected lanthionine building block for use in
SPPS.29
The development of additional methods for the preparation
of orthogonally protected lanthionines continues to be of
importance, and our recent progress in this area is here reported.
To date, compound 4 is the only protected lanthionine building
block described as suitable for use in SPPS. While the reported
preparation of 4 stereoselectively provides the requisite (2S,6R)
lanthionine stereochemistry, the route employed is a multistep
process providing 4 in a moderate overall yield.29 As well, given
that compound 4 contains both allyl ester and allyloxy carbamate
protection, its use in SPPS requires a relatively low resin loading
so as to avoid the interstrand cross-linking possible after
simultaneous allyl/alloc deprotection.29 Our objective was
therefore the development of a concise approach toward
lanthionine building blocks compatible with SPPS while em-
ploying an improved orthogonal protection strategy in place of
the allyl/alloc system. To this end the IvDde group developed
by Bycroft and co-workers30-32 was investigated as protection
for one of the two R-amines present in lanthionine 5 (Scheme
1). IvDde protection is stable to conditions used for removal of
tert-butyl and allyl esters as well as Fmoc deprotection, and no
racemization is observed when it is used to protect the R-amine
of amino acid building blocks used in SPPS.30 As removal of
the IvDde group is facilitated by treatment with dilute hydrazine,
compound 5 was designed such that Fmoc removal would
precede IvDde deprotection, thus avoiding protecting group
incompatibility issues.
SCHEME 2. Attempted Preparation of Bromide 6
Given that protected cysteine 7 would be expected to react
nonspecifically with the dehydro compound (yielding a diaster-
eomeric mixture of products), bromide 6 was not pursued
further. Somewhat surprisingly, treatment of 10 with MsCl/NEt3
led to near quantitative formation of the mesylate with no
detectable formation of the dehydro elimination product (Scheme
3). Subsequent treatment of the mesylate with protected L-
cysteine 7 under the mildly basic phase transfer conditions
previously described by Zhu and Schmidt25 yielded the lanthion-
ine product in high yield. Close inspection of the 13C NMR
spectrum, however, revealed a number of duplicate peaks
suggesting that the product was either formed as an inseparable
mixture of diastereomers or exists in a rotameric equilibrium.
To distinguish between these two possibilities the coupling of
Protected lanthionine 5 was envisioned to be assembled from
the previously reported cysteine 729 and the D-serine derived
(18) Shao, H.; Wang, S. H. H.; Lee, C. W.; Osapay, G.; Goodman, M. J.
Org. Chem. 1995, 60, 2956–2957.
(19) Smith, N. D.; Goodman, M. Org. Lett. 2003, 5, 1035–1037.
(20) Narayan, R. S.; VanNieuwenhze, M. S. Org. Lett. 2005, 7, 2655–2658.
(21) Nakajima, K.; Oda, H.; Okawa, K. Bull. Chem. Soc. Jpn. 1983, 56, 520–
522.
SCHEME 3. Preparation of Lanthionine 5 (Diastereomeric
Mixture) via Mesylate Approach
(22) Hata, Y.; Watanabe, M. Tetrahedron 1987, 43, 3881–3888.
(23) Cobb, S. L.; Vederas, J. C. Org. Biomol. Chem. 2007, 5, 1031–1038.
(24) Avenoza, a.; Busto, J. H.; Jimenez-Oses, G.; Peregrina, J. M. Org. Lett.
2006, 8, 2855–2858.
(25) Zhu, X. M.; Schmidt, R. R. Eur. J. Org. Chem. 2003, 4069–4072.
(26) Seyberth, T.; Voss, S.; Brock, R.; Wiesmuller, K. H.; Jung, G. J. Med.
Chem. 2006, 49, 1754–1765.
(27) Dugave, C.; Menez, A. Tetrahedron: Asymmetry 1997, 8, 1453–1465.
(28) Swali, V.; Matteucci, M.; Elliot, R.; Bradley, M. Tetrahedron 2002,
58, 9101–9109.
(29) Bregant, S.; Tabor, A. B. J. Org. Chem. 2005, 70, 2430–2438.
(30) Bycroft, B. W.; Chan, W. C.; Chhabra, S. R.; Teesdalespittle, P. H.;
Hardy, P. M. J. Chem. Soc., Chem. Commun. 1993, 776–777.
(31) Bycroft, B. W.; Chan, W. C.; Chhabra, S. R.; Hone, N. D. J. Chem.
Soc., Chem. Commun. 1993, 778–779.
(32) Chhabra, S. R.; Hothi, B.; Evans, D. J.; White, P. D.; Bycroft, B. W.;
Chan, W. C. Tetrahedron Lett. 1998, 39, 1603–1606.
J. Org. Chem. Vol. 74, No. 2, 2009 947

the mesylate with 7 was repeated using D2O in place of H2O.
This investigation revealed complete loss of the C2 R-proton
indicating that formation of the lanthionine proceeds via an
elimination/addition mechanism to give the product as the
undesired diastereomeric mixture.
Furthermore, during the course of the reaction in forming
lanthionine 5, periodic TLC analysis showed complete disap-
pearance of thiol 13 while a significant quantity of bromide 14
remained unreacted. This observation suggested that the low
yield of 5 was due to a competing degradation of thiol 13 and
prompted us to explore an alternate protecting group strategy
for the D-cysteine derived precursor. In place of allyl protection,
N-IvDde protected S-trityl D-cysteine 11 was converted to
trimethylsilylethyl ester 15 (Scheme 5). Trityl group removal
proceeded cleanly (20 equiv of TFA, 2 equiv of TIS) to yield
thiol 16, which was found to be much more stable than 13.
The use of thiol 16 for the synthesis of an orthogonally protected
lanthionine in turn required the preparation of a new, alternately
protected L-serine derived ꢀ-bromoalanine. Fmoc-L-Serine allyl
ester 17 was thus transformed into bromide 18 in good yield.
Coupling of thiol 16 and bromide 18 was performed as before
providing lanthionine 19 in a yield much improved over that
obtained for lanthionine 5. The optical purity of 19 was also
verified by 13C NMR showing selective formation of a single
diastereomer. Repeated synthesis of 19 following this approach
proved to be readily scalable, allowing for the preparation of
multigram quantities of lanthionine 19 in similar overall yield.
Compound 19 was readily deprotected using Pd(PPh3)4 and
N-methylaniline in THF34 to provide lanthionine building block
20 in a form suitable for SPPS.
Reasoning that the N-IvDde protecting group present in
compound 10 was responsible for the instability of the corre-
sponding bromide and mesylate, an alternate approach for the
incorporation of IvDde protection into lanthionine target 5 was
considered. This approach involved an inverted strategy whereby
IvDde and allyl protection could be introduced via a D-cysteine
derived building block. S-Trityl D-cysteine was thus N-IvDde
protected followed by conversion to allyl ester 12 (Scheme 4).
Treatment with TFA/TIS provided thiol 13, which despite
difficulty in handling (decomposing to give unidentified species
other than the disulfide) could be used directly in the subsequent
step. Employing the same phase transfer conditions described
above, thiol 13 was then reacted with known bromide 1433 to
yield lanthionine 5.
SCHEME 4. Stereoselective Preparation of
Tetra-orthogonally Protected Lanthionine 5
SCHEME 5. Stereoselective Preparation of
Tetra-orthogonally Protected lanthionine 19
Despite the modest yield of 5 obtained, we were encouraged
by the optical purity of the product. Examination of the 13C
NMR spectrum obtained for 5 indicated formation of a single
diastereomer (Figure 4).
In conclusion, we have developed a concise route for the
preparation of new tetra-orthogonally protected (2S,6R)-
lanthionines. Different from most previously reported lanthion-
ine syntheses, we here describe an “inverse strategy” utilizing
D-cysteine and L-serine derived building blocks. This approach
provides for a more concise route toward protected lanthionines
while allowing for the incorporation of orthogonal and SPPS-
compatible protecting groups (TSME, IvDde) not previously
utilized in the preparation of such compounds. Access to these
new orthogonally protected lanthionines serves to expand
the repertoire of available tools for future explorations into the
lantibiotic family of antimicrobial peptides. In this regard, we
are currently investigating the use of compounds 5 and 19 in
the construction of lanthionine containing peptides. Our progress
toward this end will be reported in due course.
FIGURE 4. Expansions of the CR region in the 13C APT spectra
acquired for (A) the diastereomeric mixture of lanthionine 5 obtained
via mesylate (Scheme 3) and (B) diastereomerically pure lanthionine
5 obtained via inverted strategy (Scheme 4).
(33) Zhu, X. M.; Schmidt, R. R Chem. Eur. J. 2004, 10, 875–887.
(34) Waldmann, H.; Kunz, H. Liebigs Ann. Chem. 1983, 1712–1725.
948 J. Org. Chem. Vol. 74, No. 2, 2009

filtered, and evaporated. The residue was applied to a silica column
and eluted with a gradient of 3:1 f 2:1 hexane/EtOAc. Product
containing fractions were pooled and evaporated to provide
compound 19 as a colorless oil that solidified upon storage at 4 °C
Experimental Section
Details pertaining to the preparation of the diastereomerically
pure, orthogonally protected lanthionine 19 are provided below as
a representative procedure.
1
(4.42 g, 88%): mp 73-75 °C; Rf 0.30 (4:1 hexanes/EtOAc); H
(R)-Allyl 2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-3-((S)-
2-(1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methyl-butylamino)-
3-oxo-3-(2-(trimethylsilyl)-ethoxy)-propylthio)propanoate (19). Pro-
tected D-cysteine 15 (5.02 g, 7.5 mmol) was dissolved in CH2Cl2
(100 mL), cooled on ice, and treated with triisopropylsilane (3.07
mL, 15.0 mmol, 2 equiv) followed by addition of TFA (11.14 mL,
150 mmol, 20 equiv) and warming to room temperature. After 1 h,
TLC analysis (4:1 hexanes/EtOAc) indicated complete consumption
of starting material and formation of a more polar product (Rf 0.50).
The mixture was diluted with water (100 mL) followed by slow
addition of solid NaHCO3 (12.6 g, 150 mmol, 20.0 equiv) to
neutralize the excess TFA. The CH2Cl2 layer was separated, dried
over Na2SO4, filtered, and evaporated. The residue was applied to
a silica column eluting with a gradient of 8:1 f 4:1 hexane/EtOAc.
Product-containing fractions were pooled and evaporated to provide
thiol 16 as a colorless oil (2.80 g, 87%), which was used
immediately in the next step so as to avoid possible disulfide
formation. The intermediate thiol 16 (2.80 g, 6.5 mmol) and bromide
18 (3.10 g, 7.2 mmol, 1.1 equiv) were dissolved in EtOAc (75 mL)
and treated with 75 mL of a saturated NaHCO3 solution containing
tetrabutylammonium bromide (8.40 g, 26.0 mmol, 4.0 equiv). The
mixture was stirred vigorously under N2 (g) for 14 h after which
TLC analysis (4:1 hexane/EtOAc) indicated complete consumption
of thiol 16 and formation of a more polar product (Rf 0.30). The
mixture was diluted with EtOAc (75 mL) and water (75 mL), and
the layers were separated. The EtOAc layer was dried over Na2SO4,
(300 MHz, CDCl3) δ 7.75 (d, 2H, J ) 7.4 Hz), 7.59 (d, 2H, J )
7.2 Hz), 7.39 (t, 2H, J ) 7.2 Hz), 7.30 (t, 2H, J ) 7.4 Hz), 6.0-5.82
(m, 1H), 5.78 (br d, 1H, J ) 8.0 Hz); 5.40-5.20 (m, 2H), 4.71-4.54
(m, 4H), 4.45-4.33 (m, 2H), 4.33-4.19 (m, 3H), 3.18-2.83 (m,
6H), 2.43 (s, 2H), 2.33 (s, 2H), 1.93 (septet, 1H, J ) 6.6), 1.05-0.93
(m, 14H), 0.02 (s, 9H); 13C NMR (75 MHz, CDCl3) δ 200.5, 196.5,
176.2, 170.2, 169.1, 156.0, 144.0, 143.9, 141.5, 131.5, 128.0, 127.3,
125.4, 120.2, 119.6, 108.0, 67.6, 66.8, 65.4, 56.8, 54.2, 54.1, 52.6,
47.3, 37.7, 36.0, 35.7, 30.1, 29.5, 28.5, 28.4, 22.7, 17.6, -1.3; [R]D
)
+14.4° (c 1.2, CHCl3); HRMS (MALDI) calcd for
C42H56N2O8SSi [M + H]+ 777.3605, found 777.3610.
Acknowledgment. This work was supported by The Neth-
erlands Organization for Scientific Research (NWO-VENI
fellowship to N.I.M.). Rob Liskamp is thanked for insightful
comments and critical reading of this manuscript. Mirjam
Damen and Kees Versluis are kindly acknowledged for provid-
ing HRMS analysis.
Supporting Information Available: Experimental proce-
dures for preparation of new compounds including spectral
characterization. This material is available free of charge via
the Internet at http://pubs.acs.org.
JO802415C
J. Org. Chem. Vol. 74, No. 2, 2009 949