Lipolanthionine peptides act as inhibitors of TLR2-mediated IL-8 secretion. Synthesis and structure-activity relationships

Article abstract of DOI:10.1021/jm050585d

Lipoproteins from Gram-positive and -negative bacteria, mycoplasma, and shorter synthetic lipopeptide analogues activate cells of the innate immune system via the Toll-like receptor TLR2/TLR1 or TLR2/TLR6 heterodimers. For this reason, these compounds constitute highly active adjuvants for vaccines either admixed or covalently linked. The lanthionine scaffold has structural similarity with the 5-(2,3-dihydroxypropyl)-cysteine core structure of the lipopeptides. Therefore, lanthionine-based lipopeptide amides were synthesized and probed for activity as potential TLR2 agonists or antagonists. A collection of analytically defined lipolanthionine peptide amides exhibited an inhibitory effect of the TLR2-mediated IL-8 secretion when applied in high molar excess to the agonistic synthetic lipopeptide Pam₃Cys-Ser-(Lys)₄-OH. Structure-activity relationships revealed the influence of the chirality of the two α-carbon atoms, the chain lengths of the attached fatty acids and fatty amines, and the oxidation level of the sulfur atom on the inhibitory activity of the lipolanthionine peptide amides.

Full text of DOI:10.1021/jm050585d

1754
J. Med. Chem. 2006, 49, 1754-1765
Lipolanthionine Peptides Act as Inhibitors of TLR2-Mediated IL-8 Secretion. Synthesis and
Structure-Activity Relationships
Tobias Seyberth,† So¨hnke Voss,‡ Roland Brock,‡ Karl-Heinz Wiesmu¨ller,§ and Gu¨nther Jung*,†
Institute of Organic Chemistry and Institute of Cell Biology, UniVersity of Tu¨bingen, 72076 Tu¨bingen, Germany, and
EMC microcollections GmbH, Sindelfingerstr. 3, 72070 Tu¨bingen, Germany
ReceiVed June 20, 2005
Lipoproteins from Gram-positive and -negative bacteria, mycoplasma, and shorter synthetic lipopeptide
analogues activate cells of the innate immune system via the Toll-like receptor TLR2/TLR1 or TLR2/TLR6
heterodimers. For this reason, these compounds constitute highly active adjuvants for vaccines either admixed
or covalently linked. The lanthionine scaffold has structural similarity with the S-(2,3-dihydroxypropyl)-
cysteine core structure of the lipopeptides. Therefore, lanthionine-based lipopeptide amides were synthesized
and probed for activity as potential TLR2 agonists or antagonists. A collection of analytically defined
lipolanthionine peptide amides exhibited an inhibitory effect of the TLR2-mediated IL-8 secretion when
applied in high molar excess to the agonistic synthetic lipopeptide Pam3Cys-Ser-(Lys)4-OH. Structure-
activity relationships revealed the influence of the chirality of the two R-carbon atoms, the chain lengths of
the attached fatty acids and fatty amines, and the oxidation level of the sulfur atom on the inhibitory activity
of the lipolanthionine peptide amides.
Introduction
Toll-like receptors (TLRs) are prominent pattern recognition
receptors (PRRs) of the innate immune system recognizing
various invading microorganisms through conserved motifs
termed “pathogen-associated molecular patterns” (PAMPs),11
which are essential for and unique in microorganisms. In the
1980s, the Toll receptor was found in studies of embryonic
development in Drosophila melanogaster. To date 11 mam-
malian TLRs (TLR1-TLR11) have been identified12 and only
for TLR10 no ligands have been described so far. Interaction
of PAMPs with TLRs induces a variety of host defense
responses, including the production of proinflammatory cytok-
ines and the activation of adaptive immunity.12-15 Among the
TLRs, TLR2 mediates the response to the most diverse set of
molecular structures from a variety of microbial organisms,
including lipoteichoic acid, lipoarabinomannan, and bacterial
lipopeptides/proteins as well as some lipopolysaccharids (LPS).12
Among these PAMPs lipopeptides are the primary candidates
for analyzing the structure-activity relationships of immune
modulators. The role of the peptide moiety, the number and
structure of the fatty acids, and the stereochemistry of lipopep-
tides based on Dhc for TLR activation has been analyzed in
great detail.16,17 In contrast, variation of the Dhc core structure
has found little attention so far.18,19 The apparent similarity of
the scaffolds S-(2,3-dihydroxypropyl)cysteine (1) and lanthion-
ine (2; Figure 1) led us to the assumption that lanthionine-based
lipopeptide amides might show TLR2 agonistic activities similar
to those of Pam3Cys-peptides5-10 or, due to its structural
differences, inhibitory activities.
Lipoproteins are cell wall components of Gram-positive and
-negative bacteria1,2 and mycoplasma.3,4 The N-terminal cysteine
residue of these bacterial membrane proteins is posttranslation-
ally modified to S-(2,3-dihydroxypropyl)cysteine (Dhca), which
is acylated by three or two fatty acids. Lipoproteins and synthetic
analogues such as the water-soluble lipohexapeptide Pam3Cys-
Ser-(Lys)4-OH5 stimulate B cell proliferation and possess very
favorable adjuvant activities in the generation of T-cell responses
to vaccines.6-10
* Corresponding author. Tel.: +49 (7071) 29-76925. Fax: +49 (07071)
29-5560. E-mail: guenther.jung@uni-tuebingen.de.
† Institute of Organic Chemistry, University of Tu¨bingen.
‡ Institute of Cell Biology, University of Tu¨bingen.
§ EMC microcollections GmbH.
a Abbreviations: BEMT, 2-bromo-3-ethyl-4-methyl thiazolium tetrafluo-
roborate; BMTB, 2-bromo-N-methylthiazolium bromide; Boc, tert-butoxy-
carbonyl; CIP, 2-chloro-1,3-dimethylimidazolidium hexafluorophosphate;
DCM, dichloromethane; DIC, diisopropylcarbodiimide; DIEA, diisoprop-
ylamine; Dhc, S-(2,3-dihydroxypropyl)cysteine; DMF, dimethylformamide;
DMSO, dimethyl sulfoxide; ES-MS, electrospray ionization mass spec-
trometry; ELISA, enzyme linked immuno sorbent assay; Fmoc, 9-fluore-
nylmethoxycarbonyl; FT-ICR-MS, Fourier transform ion-cyclotron reso-
nance mass spectrometry; FCS, fetal calf serum; GC-MS, gas chromatog-
raphy-mass spectrometry; HATU, 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate; HOBt, 1-hydroxybenzotriazole;
HONB, N-hydroxybicyclo[2.2.1]hept-5-ene-2,3-dicarboximide; HPLC, high
performance liquid chromatography; IC50, 50% inhibitory concentration;
IL-8, interleukin-8; LPS, lipopolysaccharide; mAB, murine antibody;
MALDI, matrix-assisted laser desorption/ionization; mCPBA, 3-chloro-
perbenzoic acid; MSNT, 1-(mesitylene-2-sulfonyl)-3-nitro-1H-1,2,4-triazole;
MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide;
Myr, myristoyl; Myr2LanHda-, (2R,6R)-N2,N6-bis(myristoyl)-ꢀ-hexadecyl-
amide-lanthioninyl-; NMR, nuclear magnetic resonance; Pam, palmitoyl;
Pam3Cys, S-[2,3-bis(palmitoyloxy)-2(RS)-propyl]-N-palmitoyl-R-cysteine;
Pam2LanHda-OH, (2R,6R)-N2,N6-bis(palmitoyl)lanthionine-ꢀ-hexadecyl-
amide; Pam2LanHda-, (2R,6R)-N2,N6-bis(palmitoyl)-ꢀ-hexadecylamide-
lanthioninyl-; Pam-OH, palmitic acid; PAMP, pathogen-associated molecular
pattern; PRR, pattern recognizing receptor; PyBOP, O-benzotriazol-1-yloxy-
tris(pyrrolidino)phosphonium hexafluorophosphate; rt, room temperature;
SDS, sodium dodecyl sulfate; TBTU, O-(benzotriazol-1-yl)-N,N,N′,N′-
tetramethyluronium tetrafluoroborate; tBu, tert-butyl; TFA, trifluoroacetic
acid; TFA-OPfp, pentafluorophenyl trifluoroacetate; TFFH, fluoro-N,N,N′′,N′′-
tetramethylformamidinium hexafluorophosphate; TIPS, triisopropylsilane;
THF, tetrahydrofuran; TLC, thin-layer chromatography; TLR, Toll-like
receptor.
TLR agonists and antagonists are potential candidates for the
treatment of various immune-associated diseases.20 TLR2
antagonists have been suggested to have beneficial effects in
chronic inflammation, acute inflammation, skin diseases such
as acne, and treatment of sepsis. To explore these therapeutic
options, there is an imminent need for synthetically accessible
TLR agonistic and antagonistic compounds. Recently it was
demonstrated in a sepsis model that lethal shocklike syndromes
could be prevented by treatment of mice with the TLR2
antagonistic antibody mAB T2.5 prior to lipopeptide or Bacillus
10.1021/jm050585d CCC: $33.50 © 2006 American Chemical Society
Published on Web 02/09/2006

Lipolanthionine Peptides Inhibit IL-8 Secretion
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5 1755
ines. In particular, we considered the synthesis via â-bromoala-
nine derivatives as a promising route leading to enantiopure
lanthionines, similar to the procedure recently published by Zhu
et al.35 At first we examined a substitution reaction of (R)-â-
bromo-N-Fmoc-alanine-allyl ester with (R)-N-Fmoc-cysteine-
tert-butyl ester in 5% DIEA in DMF, analogous to the protocol
of the synthesis of N-palmitoyl-S-[2,3-bis(palmitoyloxy)di-
hydroxypropyl]cysteine.36 (S)-N-Fmoc-serine-allyl ester was
obtained in high yields from (S)-N-Fmoc-serine and allyl
bromide. The subsequent conversion to the (R)-â-bromo-N-
Fmoc-alanine-allyl ester with PPh3 and CBr4 led to a mixture
of N-Fmoc-dehydroalanine-allyl ester and (R)-N-Fmoc-â-bro-
moalanine-allyl ester in a 4:1 ratio. Comparison of published
syntheses of different â-iodoalanine derivatives indicated that
bulky protecting groups for either the amino30 or the carboxy
group31,32 are necessary to hinder the R-hydrogen abstraction.
Therefore, we changed the protecting groups and prepared (R)-
N-Fmoc-cysteine-allyl ester (5a) and (R)-â-bromo-N-Fmoc-
alanine-tert-butyl ester (8a; Scheme 1). (S)-N-Fmoc-serine (6a)
was esterified with tert-butyl alcohol in the presence of DIC
and CuCl (7, 65% yield).37 Reaction times longer than 4 h led
to increasing amounts of Fmoc-dehydroalanine, consistent with
the fact that such conditions are also used for dehydroalanine
synthesis.38
Figure 1. Structures of the natural thioether amino acid scaffolds
S-(2,3-dihydroxypropyl)cysteine (Dhc) 1 and lanthionine (Lan) 2.
Because of the structural relationship lanthionine is an interesting
scaffold for lipopeptides with potential TLR2 agonistic or antagonistic
activity. R1, R2, and R3 are long chain alkyl residues.
subtilis challenge.21 Synthetic low molecular weight compounds
with inhibitory activity against TLR2 have not been described
so far.
Lanthionine was first detected in alkaline hydrolysates of
wool. Moreover, this molecule was found in human hair and in
keratin. In human lenses, the photooxidative degradation of
cystine residues may also result in the formation of lanthionine,
accompanied by protein cross-bridging that leads to increased
tissue rigidity and hardening of the lens. Another highly
interesting natural occurrence of lanthionine is the class of
bioactive lantibiotics.22-24
The conversion of 7a to the bromide 8a was achieved with
PPh3 and CBr4 in good yield (85%). The (R)-N-Fmoc-cysteine-
allyl ester (5a) was synthesized by esterification of (R)-N-Fmoc-
S-trityl-cysteine (3a) with allyl bromide (4a, 92% yield)
followed by acidic cleavage of the trityl group (5a, 95% yield).
Synthesis of the fully protected (2R,6R)-lanthionine derivative
was carried out by stirring 5a and 8a in 5% DIEA in DMF to
obtain compound 9a in 65% yield. Cleavage of the tert-butyl
ester of 9a with neat TFA followed by lyophilization from tert-
butyl alcohol/water led to the desired (2R,6R)-N2,N6-bis(Fmoc)-
lanthionine-ꢀ-allyl ester 10a.
NMR Analysis of Lanthionine. Mustapa et al.33 described
the formation of norlanthionine during the synthesis of lanthion-
ine from â-iodoalanine. The comparison of the observed NMR
data of the product 9a with the simulated spectra of lanthionine
and the isomeric norlanthionine (Supporting Information) and
with the NMR data published by Mustapa et al.33 and Zhu et
al.35 showed a high purity of the lanthionine. The identity of
the compound 9a was finally proven by H/H-COSY 2D-NMR
(Supporting Information). Since we did not observe further
byproducts apart from dehydroalanine, norlanthionine formation
is not a problem of our protocol for lanthionine synthesis.
The methods of lanthionine peptide syntheses have been
summarized recently.24 None of the published syntheses can
be classified as a routine procedure to yield lanthionines in high
enantiomeric purity. One of the main problems is the formation
of dehydroalanine during the S-alkylation of L-cysteine due to
the strongly basic conditions.25 The desulfurization of cystine
also forms large amounts of dehydroamino acid.26 The use of
serine â-lactone for the preparation of triprotected lanthionines
may lead to low yields, due to competing O-acyl fission.27,28
Bradley et al.26 applied a biomimetic synthesis for the prepara-
tion of a fully protected lanthionine via Michael addition of
Boc-Dha-OMe and Fmoc-Cys-OAll.29 The resulting mixture of
stereoisomers was separated by HPLC. Dugave and Menez30
followed the original idea of Brown and du Vigneaud25 using
N-triphenylmethyl (trityl) protected â-iodoalanine for the S-
alkylation of cysteine. The trityl group protected the â-iodo-
alanine derivative from â-elimination. Differently protected
â-iodoalanine derivatives have been described by Jobron et al.31
and Gair et al.32 However, the application of this method to
lanthionine synthesis by Mustapa et al.33 led to a mixture of
lanthionine and norlanthionine in a 1:2 ratio.34
This fact and the restricted scope with respect to the protecting
groups limited the applicability of the thioalkylation methods
for our purposes. In our hands, the synthesis of lanthionine via
the â-iodoalanine and the serine â-lactone route was not
successful. Therefore, we first elaborated a new preparation
method for N,N-bis(Fmoc)lanthionine with orthogonally pro-
tected carboxy groups. Using this building block a representative
collection of lipolanthionine peptides was synthesized and tested
for biological activity, revealing the ability of these compounds
to inhibit the TLR2-mediated IL-8 secretion.
Synthesis and GC-MS Analysis of the Four Stereoisomers
of Protected Lanthionine. The efficient synthesis protocol
(Scheme 1) was applied to the preparation of the four configu-
rational isomers (10a-d). Using the appropriate L- or D-amino
acid derivatives, the desired isomers of protected lanthionine
(9a-d) were obtained in high enantiomeric purities (>95%;
Table 1). The enantiomeric purities were analyzed by chiral
GC-MS (Chirasil-Val column) after derivatization.39 Since the
allyl group proved to be stable during the cleavage of the tert-
butyl ester, we used N2,N6-bis(trifluoroacetyl)lanthionine-R-
allyl-ꢀ-methyl diester as volatile derivative for GC-MS analysis,
leading to the fragment-ion m/z ) 228 u, which was used for
single-ion-mode MS-detection (Supporting Information).
Results
Synthesis of the Lanthionine Scaffold. After several less
satisfying test series for the preparation of the desired lanthionine
scaffold by reaction of (R)-N-Fmoc-cysteine-tert-butyl ester with
(S)-N-Fmoc-serine â-lactone or (R)-N-Fmoc-â-iodoalanine-allyl
ester, we searched for a new, more efficient access to lanthion-
(2R,6R)-N2,N6-bis(palmitoyl)lanthionine-E-hexadecyl-
amide. In the next step, we synthesized the (2R,6R)-N2,N6-bis-
(Fmoc)lanthionine-ꢀ-hexadecylamide (13; Scheme 2) from
compound 9a by allyl cleavage with Pd(PPh3)4 (11, 91%)
followed by coupling of 11 to hexadecylamine with HOBt/DIC

1756 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5
Seyberth et al.
Scheme 1. Synthesis of N2,N6-Bis(Fmoc)lanthionine-R-allyl
Ester (10a-d)a
Table 1. Summary of the Enantiomeric Purities of Lanthionines 9a-d
as Determined by Chiral Phase GC-MS Analysis
% purity
(2S,6R),
lanthionine
(2R,6R)
(2R,6S)
(2S,6S)
2R,6R (9a)
2R,6S (9b)
2S,6R (9c)
2S,6S (9d)
97.3
1.3
2.6
0.1
2.7
97.1
96.7
4.1
0.0
1.6
0.7
95.8
The enantiomeric purity of product 15 was determined after
total hydrolysis and derivatization with TFA/methanol by chiral
GC-MS. The product contained 91% of the 2R,6R enantiomer
according to the peak ratios.
Lipolanthionine Peptide Amide Syntheses. As test com-
pounds for biological assays (2R,6R)-Myr2LanHda- and (2R,6R)-
Pam2LanHda-Ser-(Lys)4-NH2 were synthesized. The pentapep-
tide Ser-(Lys)4 amide moiety5 was introduced in order to
improve water solubility. The peptide was synthesized on Rink-
amide resin (0.74 mmol/g loading) by HOBt/DIC coupling using
N-Fmoc-Lys(Boc)-OH and N-Fmoc-Ser(tBu)-OH, leading to
resin bound Fmoc-Ser(tBu)-[Lys(Boc)]4 (16). The synthesis was
monitored by Kaiser test,40 and the Fmoc-protected peptide
amides, after cleavage, were characterized by ES-MS and HPLC
(>95%). Following the last coupling step, the resin-loading was
determined by cleavage of the Fmoc protecting group with 20%
piperidine in DMF, and spectrophotometric analysis of the
cleaved fluorene confirmed a quantitative yield of 16 (resin-
loading 0.415 mmol/g). Coupling 15 (Scheme 2) to the Fmoc-
deprotected resin 16 proved to be difficult, since 15 is insoluble
at room temperature. Quantitative coupling was achieved by
double couplings with HOBt/DIC at 45 °C in CHCl3/DMF
(8:1). Since the lanthionine moiety of the product was strongly
racemized (61% 2R,6R), this lipolanthionine peptide was not
used for the biological tests. To circumvent the racemization,
the urethane-protected lanthionine monoamide 13 was coupled
to the resin-bound pentapeptide 16 with HOBt/DIC repeatedly
until a negative Kaiser test was observed. Again, quantitative
coupling of 13 was proven by spectrophotometric Fmoc
quantification (resin-loading: 0.329 mmol/g). Fmoc deprotection
and coupling of myristic acid and palmitic acid respectively
with HOBt/DIC yielded the lipolanthionine peptide amides
(2R,6R)-Myr2LanHda- (17) and (2R,6R)-Pam2LanHda-Ser-
(Lys)4-NH2 (18) after cleavage.
Coupling the Fatty Amine to Resin-Bound Carboxy
Functions. The first biological tests of lipolanthionine peptide
amides (17, 18) showed a significant inhibition of the TLR2-
mediated IL-8 secretion for both of the test compounds.
Therefore, we decided to investigate the influence of different
fatty acids and fatty amines as well as of different configurations
of the compounds and different oxidation levels of the sulfur
atom. First, a suitable protocol for coupling the carboxy group
of resin-bound lanthionine to the fatty amines had to be
developed. The urethane-protected lanthionine derivative 10a
was coupled to Fmoc-deprotected resin 16 (Scheme 3) and the
allyl group cleaved quantitatively from the resulting resin-bound
compound 19 with phenylsilane and Pd(PPh3)4 within 2 h (20).
For the attachment of the fatty amine, a variety of coupling
conditions were tested: activation of the polymer-bound acid
component 20 by TFFH, CIP, BEMT, or BMTB and activation
as active esters using HOBt/DIC, HONB/DIC, MSNT, TBTU/
HOBt, TFA-OPfp, HATU, or PyBOP. Preactivation as penta-
fluorophenyl ester with pentafluorophenyl trifluoroacetate (TFA-
OPfp) in DMF/pyridine followed by treatment with the fatty
amine and DIEA in THF proved to be the most effective
a (i) 1.1 equiv of DIEA, allyl bromide/acetonitrile (1:2), rt, 16 h; (ii) 3.3
equiv of TIPS, TFA/DCM (1:1), rt, 1 h; (iii) 4 equiv of tBuOH, 3 equiv of
DIC, CuCl, rt for 16 h; add 6a/b in DCM, rt, 4 h; (iv) 1.6 equiv of PPh3,
1.22 equiv of CBr4, 0 °Cfrt, 3 h; (v) 1 equiv of 5a/b, 1.1 equiv of 8a/b,
5% DIEA/DMF, rt, 4 h; (vi) TFA, rt, 1 h.
(12, 67%). One-half of compound 12 was treated with neat TFA
for 1 h to yield (2R,6R)-N2,N6-bis(Fmoc)lanthionine-ꢀ-hexa-
decylamide (13) after lyophilization. The remaining amount of
12 was treated with piperidine/DMF (1:1) and, after removal
of the solvents in vacuo, Pam-OH was coupled with DIC/HOBt,
resulting in (2R,6R)-N2,N6-bis(palmitoyl)lanthionine-ꢀ-hexa-
decylamide-R-tert-butyl ester (14) in 63% yield. Treatment with
neat TFA followed by lyophilization led to (2R,6R)-N2,N6-bis-
(palmitoyl)lanthionine-ꢀ-hexadecylamide (Pam2LanHda-OH, 15).

Lipolanthionine Peptides Inhibit IL-8 Secretion
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5 1757
Scheme 2. Synthesis of (2R,6R)-N2,N6-Bis(Fmoc)lanthionine-ꢀ-hexadecylamide (13) and
N2,N6-Bis(palmitoyl)lanthionine-ꢀ-hexadecylamide (15)a
a (i) 1.3 equiv of morpholine, cat. Pd(PPh3)4, rt, 1 h; (ii) 1 equiv of HOBt, 1 equiv of DIC, 1.1 equiv of hexadecylamine, rt, 6 h; (iii) TFA, rt, 1 h; (iv)
piperidine/DMF (1:1), rt, 2 h; (v) 1.8 equiv of Pam-OH, 1.8 equiv of HOBt, 1.8 equiv of DIC, rt, 16 h.
coupling protocol on the solid phase. The drawback of the
synthesis is a partial Fmoc cleavage by the primary amine;
therefore, the resin-bound lanthionine peptide was subsequently
reprotected with a surplus of fluoren-9-ylmethyl chloroformate
in pyridine/DMF prior to further usage for analytics and
following syntheses.
influences the biological activity, the sulfoxide and sulfone of
resin-bound Pam2LanHda-Ser(tBu)-[Lys(Boc)]4 amide (60;
Scheme 4) were synthesized and cleaved from the resin. The
protocol used by Matteucci et al.41 to oxidize resin-bound
S-methylcysteine to its sulfoxide with H2O2 and scandium
trifluoromethanesulfonate in DCM/methanol (9:1) proved suit-
able for lanthionine-sulfur oxidation in the lanthionine-contain-
ing lipopeptides. The sulfoxide of Pam2LanHda-Ser-(Lys)4-NH2
was obtained after cleavage from the resin (61; Scheme 4).
Oxidation to the sulfone was achieved by treatment of resin-
bound Pam2LanHda-Ser(tBu)-[Lys(Boc)]4 amide with mCPBA
(5 equiv) in DCM, a method used by Mata42 for the oxidation
of resin-bound penicillin derivatives. The desired sulfone 62
was obtained after cleavage in high yield and good purity. Mata
also described a protocol for sulfone generation (1.4 equiv of
mCPBA in DCM, 0 °C), which in our case led to a mixture of
non-, mono- and dioxidized lanthionines. The sulfoxide 61 and
sulfone 62 of Pam2LanHda-Ser-(Lys)4-NH2 (18) were character-
ized by MALDI-MS and used for testing TLR2 inhibition.
Synthesis of the Lipolanthionine Peptide Amide Collec-
tion. To determine structure-activity relationships, we synthe-
sized a variety of lipolanthionine peptide amides (Figure 2)
following Scheme 3. Several n-fatty amines R1-NH2 (CnH2n+1
-
NH2) were attached to the resin 20, and the resin-bound N2,N6-
bis(Fmoc)-ꢀ-(CnH2n+1) amide-lanthionine peptide amides 21-
27 (n ) 6, 8, 10, ..., 18) were obtained in good purities (>88%,
HPLC). Fmoc was cleaved from these resin-bound lanthionine
derivatives, and myristic acid was coupled to both of the amino
functions, yielding the lipolanthionine peptides 17 and 28-33
(Chart 1) after cleavage from the resin.
Variation of the fatty acid moiety was carried out by coupling
different n-fatty acids (CmH2m+1-COOH; m ) 5, 7, ..., 11 and
15, 17, 19) to Fmoc-deprotected resin 26 (carrying Fmoc2-
LanHda-Ser(tBu)-[Lys(Boc)]4) to yield compounds 18 and 34-
39 after cleavage. To determine the influence of different
configurations, the stereoisomeric lanthionines 10b-d were
coupled to Fmoc-deprotected resin 16 (leading to 40-42). The
subsequent steps were carried out as described for (2R,6R)-Myr2-
LanHda-Ser-(Lys)4-NH2 17 in Scheme 3, first cleaving the allyl
group (43-45) and subsequently coupling hexadecylamine (46-
48) and myristic acid, which results in (2R,6S)-, (2S,6R)-, and
(2S,6S)-Myr2LanHda-Ser-(Lys)4-NH2 (49, 50, and 51; Chart 1)
after cleavage.
We were also interested in the generation of the stereoisomers
of Myr2Lan(OH)-Ser-(Lys)4-NH2. These lipopeptide amides lack
the lipophilic fatty amine moiety in the 7-position of the
lanthionine. The stereoisomers 10a-d were coupled to resin
16 (leading to 19, 40-42) and myristic acid was coupled to
the resin-bound lanthionines after Fmoc-deprotection (52-55).
The allyl group was cleaved to yield the Myr2Lan(OH)-Ser-
(Lys)4-NH2 (56-59) after cleavage from the resin. The resulting
lipolanthionine peptide amides are summarized in Chart 1.
Structure-Activity Relationships. The representative col-
lection of analytically defined single lipolanthionine peptide
amides (Chart 1) was tested for agonistic activity and for their
ability to inhibit the TLR2-mediated IL-8 secretion. To test the
inhibitory activity, the well-studied agonist Pam3Cys-Ser-(Lys)4-
OH was used for stimulation of THP-1 cells. The synthesized
lipolanthionine compounds enabled the analysis of the structure-
activity relationships for the (i) fatty amine chain length, (ii)
fatty acid chain length, (iii) stereochemical properties, and (iv)
the oxidation level of the sulfur.
First we investigated the influence of the fatty amine chain
length using the derivatives based on the bis-myristoylated
(2R,6R)-lanthionine lipopeptide amides (17, 28-33). At con-
centrations of 25 µM only for the hexylamine derivative (28) a
weak agonistic activity was observed.
Interestingly, except for the hexylamine derivative (28), all
analogues of lipolanthionine peptide amides exhibited a pro-
nounced and comparable TLR2 inhibitory activity (Figure 3).
When applied in a 500-fold excess (25 µM) over the highly
potent Pam3Cys-Ser-(Lys)4-OH TLR2 agonist (50 nM), IL-8
expression was reduced by 48-65%. Therefore, given a
minimum length of eight carbon atoms, the length of the fatty
Oxidation of the Lanthionine to Sulfone and Sulfoxide.
To investigate whether the oxidation level of the sulfur

1758 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5
Seyberth et al.
Chart 1. The Synthesized Lipolanthionine Peptide Amide
Collection: Definition of the Structures of All Tested
Compounds
Scheme 3. Protocol for the Solid Phase Synthesis of 17, 18,
28-39, and 49-51 on Rink-Amide Resina
a (i) 20% piperidine/DMF, rt, 20 min; (ii) 3 equiv of 10a-d, 3 equiv of
HOBt, 3 equiv of DIC, rt, 8 h; (iii) 0.25 equiv of Pd(PPh3)4, 24 equiv of
phenylsilane, rt, 2 h, argon (20, 43-45); (iv) pyridine/DMF/TFA-OPfp
(3:2:1); (v) 10 equiv of R1-NH2, 30 equiv of DIEA, rt, 6 h; (vi) 3 equiv of
R2COOH, 3 equiv of HOBt, 3 equiv of DIC, rt, 8 h; (vii) TFA, rt, 3 h.
acid derivative (C-8, 35) was an agonistic activity observed.
The derivatives containing lauric (C-12, 37), myristic (C-14,
17), and palmitic acid (C-16, 18) had the highest inhibitory effect
on the lipopeptide-induced IL-8 secretion (Figure 4). The
analogues with short fatty acids (C-6 to C-10, 34-36) exhibited
less inhibitory activities.
For two of the most active inhibitors (17, 18), the concentra-
tion dependence of the inhibitory activity was determined. For
both compounds a concentration-dependent decrease in IL-8
expression was observed (Figure 5). For the myristic acid
derivative, IL-8 expression was reduced by up to 86% for the
palmitic acid derivative by up to 68%. Both compounds
exhibited IC50 values of about 5 µM. However, the inhibitory
activity of compounds 17 and 18 varied between several
experiments (38%, 18%, and 14% residual activity for com-
pound 17, respectively, and 30% and 42% for compound 18).
Since these activities were obtained from independent cellular
experiments, such variations represent slight alterations that
could not be excluded for cellular assays. Nevertheless, for all
experiments, using the compounds 17 and 18, the inhibitory
activity in the presented range was reproducible.
Figure 2. Core structure of the synthesized lipolanthionine peptide
amides. (Definition of the residues and abbreviations for the figure and
Chart 1: R1, R2, alkyl chains; SOy, y ) 0, sulfide; y ) 1, sulfoxide; y
2
6
) 2, sulfone; configurations CR ,CR , 2R,6R, 2R,6S, 2S,6R, 2S,6S; X
) NH, O).
amine chain seemed to be of minor significance. Except for
the octadecylamine derivative (33), no cell toxic properties were
observed at the tested concentration as determined by the MTT
test. The derivative containing the hexadecylamine residue was
selected for further tests.
The agonistic activity of Pam3Cys-Ser-(Lys)4-OH and even
of lipopeptide vaccines43 is dependent on the configuration of
the S-glyceryl-cysteine scaffold.43-45 Therefore, we tested the
four stereoisomers of the bis-myristoylated hexadecylamide
derivative, the most active inhibitor identified so far. The
agonistic activity negatively correlated with the inhibitory
Next we studied the influence of the variation of the amide-
bound fatty acid moieties. In this case, only for the caprylic

Lipolanthionine Peptides Inhibit IL-8 Secretion
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5 1759
Scheme 4. Synthesis of the Sulfoxide and Sulfone of
Pam2LanHda-Ser-(Lys)4 Amide from the Resin-Bound Sulfidea
Figure 4. Inhibitory activities of (R2)2Lan(Hda)-SK4-NH2 with varying
fatty acid residues R2. THP-1 cells were incubated with lipolanthionine
peptides (25 µM) in the absence or presence of Pam3Cys-SK4-OH (50
nM) for 5 h. The secretion of IL-8 was detected by ELISA in cell-free
supernatants.
a (i) 5 equiv of H2O2, 0.2 equiv of Sc(OTf)3, DCM/methanol (9:1), rt,
40 min; (ii) 5 equiv of mCPBA, rt, 24 h; (iii) TFA, rt, 3 h.
Figure 5. Concentration dependence of the inhibitory activity of Myr2-
Lan(Hda)-SK4-NH2 (17) and Pam2Lan(Hda)-SK4-NH2 (18). THP-1 cells
were incubated with lipolanthionine peptides (25, 12.5, 6.25, 3.13, 1.56,
0.78 µM) in the presence of Pam3Cys-SK4-OH (50 nM) for 5 h. The
secretion of IL-8 was detected by ELISA in cell-free supernatants. Error
bars represent the standard deviation of triplicates.
Figure 3. Inhibitory activities of Myr2Lan(R1)-SK4-NH2 with varying
fatty amine residue R1. THP-1 cells were incubated with lipolanthionine
peptides (25 µM) in the absence or presence of the agonistic
lipohexapeptide Pam3Cys-SK4-OH (50 nM) for 5 h. The secretion of
IL-8 was detected by ELISA in cell-free supernatants. The Pam3Cys-
SK4-OH-induced IL-8 secretion is shown in relation to a sample that
was incubated with Pam3Cys-SK4-OH in the presence of the same
amount of solvent as the lipolanthionine-containing samples. Error bars
represent the standard deviation of triplicates.
Figure 6. Dependence of the inhibitory activities of Myr2Lan(Hda)-
SK4-NH2 (17) on the configuration of the lanthionine scaffold. THP-1
cells were incubated with lipolanthionine peptides (25 µM) in the
absence or presence of Pam3Cys-SK4-OH (50 nM) for 5 h. The secretion
of IL-8 was detected by ELISA in cell-free supernatants. Error bars
represent the standard deviation of triplicates.
activity. The (2R,6R)-lanthionine peptide showed the highest
inhibitory activity, closely followed by the (2S,6R) derivative
(17 and 50; Figure 6). In contrast, a change of the configuration
in the C-6 stereocenter led to a loss of the inhibitory activity
and to an increase of the agonistic activity. The agonistic activity
of the (2R,6S) derivative was higher than that of the (2S,6S)
one. Therefore, only the configuration of C-6 strongly influences
the TLR2 inhibitory activity of the compounds, whereas the
configuration at C-2 seems to be of minor significance.
Lipolanthionine peptide amides containing free carboxy
functions (56-59) were unable to suppress the Pam3Cys-Ser-
(Lys)4-OH-induced IL-8 secretion (data not shown). Interest-
ingly, the methyl ester derivatives possessed cell toxic properties
(MTT test, data not shown). The Pam2LanHda-Ser-(Lys)4-NH2

1760 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5
Seyberth et al.
oxides (61 and 62) showed no significant alteration of the
observed inhibitory effect (data not shown).
mechanism of inhibition based on the interaction with a receptor
molecule. It is moreover remarkable that the agonistic activity
of the lanthionine peptides negatively correlated with their
inhibitory activity. Therefore, a nonspecific mode of action by
insertion of the lipolanthionine peptides into membrane domains
such as lipid rafts seems to be unlikely. Instead of directly
interacting with any TLR, the lipolanthionine peptides may bind
to CD14, a coreceptor that is shared by LPS and lipopeptides
for activation of their respective TLR.52 Still, the high concen-
trations necessary for the inhibitory activity of the lipolanthion-
ine peptides may suggest a direct interaction with the agonists.
For example, the agonist Pam3Cys-Ser-(Lys)4-OH might be
integrated into micelles composed of lipolanthionine peptides.
Future studies will have to elucidate whether the lipolanthionine
peptide-mediated IL-8 inhibition follows one of the above-
described mechanisms.
Discussion
So far, analyses of structure-activity relationships of the well-
characterized lipopeptide and highly active TLR2 agonist Pam3-
Cys-Ser-(Lys)4-OH have focused mainly on the periphery of
the natural core scaffold (Dhc), i.e., variations of the peptide
and/or of the fatty acids. To vary the scaffold, we substituted
the Dhc by the structurally related thioether amino acid
lanthionine. The lanthionine scaffold possesses the same number
of functional groups, suitable for the introduction of structural
diversity for lipopeptide synthesis, as the Dhc scaffold. A
stereoselective synthesis for a generally applicable protected
lanthionine building block was established, the four configu-
rational isomers were synthesized, and the enantiomeric purities
were determined by chiral GC-MS analysis. The high enan-
tiomeric purities suggested that the reaction of cysteine with
â-bromoalanine follows a substitution mechanism similar to the
lanthionine synthesis via â-iodoalanine. Two lipolanthionine
peptide amides, derived from the lipopeptide building-block
Fmoc2LanHda-OH 13 were tested for their capacity to influence
the IL-8 secretion induced by the TLR2 agonist Pam3Cys-Ser-
(Lys)4-OH. These in vitro tests revealed the concentration-
dependent and saturable TLR2 inhibitory rather than agonistic
activities of the lipolanthionine peptides.
Detailed analyses of the structure-activity relationship for
the fatty acid and fatty amine moieties were conducted for the
(2R,6R) configuration. The chain length of the amide-bound fatty
acid had a strong influence on the activity, whereby the myristic
acid derivative (17) was the most effective inhibitor on the
lipopeptide-induced IL-8 secretion. In contrast, the chain length
of the amide-bound fatty amine residue had no influence on
the inhibitory activity for chain lengths from C-8 to C-16 (17,
29-32). The C-6 derivative (34) exhibited some agonistic
activity.
Conclusion
Lipolanthionine peptide amides have been prepared and
evaluated for their ability to inhibit TLR2-mediated IL-8
secretion in myelomonocytic THP-1 cells. Based on a versatile
lanthionine building block, a representative collection of lipo-
lanthionine peptide amides with fatty acid and fatty amine
moieties of different length as well as with different lanthionine
configurations and sulfur oxidation levels were synthesized and
analytically characterized. In vitro tests revealed a TLR2
inhibitory activity that was strongly dependent on the chain
length of the fatty acid moiety with a maximal inhibitory activity
found for the myristic acid derivative 17. In contrast, the chain
length of the amide bound fatty amine residue had no significant
influence on the inhibitory activity concerning chain lengths
from C-8 to C-16 (17, 29-32). Derivatives with free lanthionine
carboxy functions were not able to suppress the IL-8 secretion.
It was further shown, that the configuration of the lanthionine
C-6 position was crucial for the inhibitory activity of the
compounds. A change of the configuration at the lanthionine
C-2 position had little influence. Compound 17 [(2R,6R)-Myr2-
LanHda-Ser-(Lys)4-NH2] was identified as the best inhibitor,
expressing an IC50 value of about 5 µM. First tests concerning
the ability of the lipolanthionine peptides to inhibit the TLR4-
mediated IL-8 secretion revealed a similar inhibitory activity
as detected for TLR2. Thus, the synthesized lipolanthionine
peptide amides do not selectively inhibit the TLR2-mediated
IL-8 secretion.
The configuration at the C-6 position of the lanthionine
proved to be crucial for inhibitory activity, whereas the C-2
configuration showed little influence. In conclusion, the lipo-
lanthionine peptide amide (2R,6R)-Myr2LanHda-Ser-(Lys)4-NH2
(17) exhibited the strongest TLR2 inhibitory activity.
Several possibilities exist in which way the lipolanthionine
peptide amides might influence the TLR2-mediated IL-8 secre-
tion. First, the inhibitor may interact with the agonist, preventing
receptor recognition and initiation of signal transduction.46
Second, the inhibitor may influence the TLR2 and/or a core-
ceptor directly, by interacting with the respective ligand binding
site. Third, the lipolanthionine peptides could inhibit the
assembly of the TLR2 receptor complex by integrating into cell
membrane components, such as lipid rafts. For TLR4 it was
demonstrated that LPS induces the coclustering of CD14 and
TLR4 in the lipid rafts.47,48 Recently, it was shown that oxidized
phospholipid products inhibited the LPS-induced translocation
of TLR4 to lipid rafts (caveolae).49 It was also reported, that
n-3 unsaturated fatty acids caused the alteration of fatty acid
composition in membrane lipid rafts, resulting in the inhibition
of T-cell activation.50,51
A direct inhibition by selective blocking of the TLR2 receptor
and/or either the TLR1 or TLR6 coreceptor seems to be rather
unlikely, since the lipolanthionine peptides also inhibited the
LPS-induced, TLR4-mediated IL-8 secretion (data not shown).
On the other hand, the inhibitory potential of the lipolanthion-
ine peptides was strongly dependent on the configuration of
the lanthionine C-6 position, an observation that favors a
To the best of our knowledge, the lipolanthionine peptides
are the first lipopeptidic compounds exhibiting inhibitory activity
for TLR2. They may provide a basis for the design of further
compounds with enhanced TLR2 inhibitory activity to coun-
teract the lethal effects of septic shock.
Experimental Section
Water-free solvents were from Fluka (Neu-Ulm, Germany).
Acetonitrile, methanol, trifluoroacetic acid (Uvasol), and chloroform-
d1 were from Merck (Darmstadt, Germany). Protected amino acids
were from Novabiochem (Bad Soden, Germany) or Bachem
(Heidelberg, Germany). Rink-amide resin was obtained from Rapp-
Polymere (Tu¨bingen, Germany). The reference TLR2 agonist Pam3-
Cys-Ser-(Lys)4-OH was from EMC microcollections GmbH (Tu¨b-
ingen, Germany). All further chemicals were purchased from
Aldrich (Taufkirchen, Germany) and Fluka (Neu-Ulm, Germany),
respectively.
For HPLC analysis we used a Waters LC System with Waters
712 WISP autosampler, a Waters 626 pump combined with a
Waters 600S controller, and a Waters 486 UV-detector (λ ) 214
nm). A Nucleosil 300 C18 RP-column and a linear gradient of

Lipolanthionine Peptides Inhibit IL-8 Secretion
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5 1761
acetonitrile/0.1% TFA (A) and water/0.1% TFA (B) (10 vol % A
to 100 vol % A in 35 min; flow 0.3 mL/min) were used.
ES-MS spectra were recorded on a Bruker Esquire 3000plus
LC-ES-MSn (Bruker Daltonics, Bremen, Germany). High-resolu-
tion FT-ICR-MS spectra were recorded using a Bruker Daltonics
APEX II system with a 4.7 T magnet and ES ionization. As internal
standard for high-resolution analysis, PEG or Ultramark were used.
GC-MS analyses were performed on a Agilent (formerly HP)
system consisting of a 6890 GC unit and a 5973 MS detector using
EI ionization. Helium was used as carrier gas and a Chirasil-Val
column as chiral stationary phase. The following temperature
program was used: 55 °C for 3 min; 25 °C/min to 145 °C for 10
min; 2.5 °C/min to 180 °C. MALDI-MS analysis was carried out
on a Hewlett-Packard G2025A matrix-assisted-laser-desorption
time-of-flight system using a DHAP matrix (20 mg of DHAP, 5
mg of ammonium citrate in 1 mL of 80% isopropyl alcohol) on a
gold target. For signal generation 20-50 laser shots were added
up in the single shot mode.
NMR data were collected on Bruker Avance 400 MHz or Bruker
AMX 600 MHz spectrometers using solutions of the samples in
chloroform-d1 (30 mg/mL). The NMR data are given in the
Supporting Information.
For TLC, silica gel plates 60 F254, 5 × 10 cm (Merck, Darmstadt,
Germany) and the following solvent systems (v/v) were used in
solvent saturated glass chambers for determination of Rf values at
room temperature: I, hexane/ethyl acetate 3:1; II, hexane/ethyl
acetate 2:1; III, hexane/ethyl acetate 7:1; IV, hexane/ethyl acetate/
acetic acid 8:4:1; V, chloroform/methanol/acetic acid 90:10:1; VI,
ethyl acetate/chloroform/25% ammonia 130:8:1.
Allyl Protection of the Carboxy Group (4; General Proce-
dure). The N-Fmoc protected amino acid was dissolved in a mixture
of 1.1 equiv of DIEA and acetonitrile (0.66 mL/g). Allyl bromide
(0.33 mL/g) was added and the solution stirred for 16 h at room
temperature. The reaction mixture was diluted with a 5-fold excess
of ethyl acetate, washed three times with 10% Na2CO3 and brine,
and dried over MgSO4. The solvent was evaporated and the product
purified by flash column chromatography (silica gel, hexane/ethyl
acetate 3:1) to yield the allyl ester of the Fmoc-amino acid as a
colorless amorphous powder.
(R)-N-(Fluoren-9-ylmethoxycarbonyl)-S-tritylcysteine-allyl
ester (4a) was synthesized with (R)-N-(fluoren-9-ylmethoxycar-
bonyl)-S-tritylcysteine (7.50 g, 12.81 mmol), DIEA (2.41 mL),
acetonitrile (50 mL), and allyl bromide (25 mL). Yield: 7.37 g,
92.0%. TLC: Rf (I) ) 0.23. ES-MS: m/z ) 648.4 [M + Na]+.
(S)-N-(Fluoren-9-ylmethoxycarbonyl)-S-tritylcysteine-allyl
ester (4b). (S)-N-(Fluoren-9-ylmethoxycarbonyl)-S-tritylcysteine
(2,00 g, 3.42 mmol); DIEA (643.03 µL; 3.75 mmol); acetonitrile
(13.33 mL); allyl bromide (6.67 mL). Yield: 1.92 g, 89.6%. TLC:
Rf (I) ) 0.23. ES-MS: m/z ) 648.4 [M + Na]+.
Cleavage of the S-Trityl Protecting Group (5; General
Procedure). The S-tritylated cysteine derivative was dissolved in
TFA/DCM (1:1) and 3.3 equiv of TIPS was added. After stirring
for 1 h at room temperature, the solvent was removed in vacuo
and the residual product 5 recrystallized from DCM/diethyl ether
(1:20) at -20 °C.
(R)-N-(Fluoren-9-ylmethoxycarbonyl)cysteine-allyl ester (5a)
was synthesized with 4a (7.00 g, 11.19 mmol), TFA/DCM (1:1,
40 mL), TIPS (7.58 mL, 36.93 mmol), and DCM/diethyl ether
(1:20, 315 mL). Yield: 4.08 g, 95.1% of a colorless amorphous
powder. TLC: Rf (I) ) 0.34. ES-MS: m/z ) 406.1 [M + Na]+
422.0 [M + K]+. GC-MS: 99.5% ee.
dropwise to the N-Fmoc amino acid in DCM/DMF (14:1, 7.5 mL/
g). After 4 h of stirring at room temperature, the mixture was filtered
and the filtrate was washed three times each with saturated NaHCO3
and brine. The organic layer was dried over MgSO4 and evaporated
to dryness. Purification was by flash column chromatography (silica
gel, hexane/ethyl acetate 3:1f1:1).
(S)-N-(Fluoren-9-ylmethoxycarbonyl)serine-tert-butyl ester
(7a) was synthesized with DIC (14.19 mL, 91.66 mmol), tert-butyl
alcohol (11.61 mL, 122.21 mmol), CuCl (60.00 mg, 0.61 mmol),
DCM (70 mL), (S)-N-(fluoren-9-ylmethoxycarbonyl)serine (10.00
g, 30.55 mmol), and DCM/DMF (14:1, 75 mL). Yield: 7.62 g,
65.0% of a colorless product. TLC: Rf (II) ) 0.15. ES-MS: m/z
) 406.2 [M + Na]+, 422.1 [M + K]+. GC-MS: 99.7% ee.
(R)-N-(Fluoren-9-ylmethoxycarbonyl)serine-tert-butyl ester-
(7b) was synthesized with DIC (2.84 mL, 18.33 mmol), tert-butyl
alcohol (2.32 mL, 24.42 mmol), CuCl (20.00 mg, 0.20 mmol), DCM
(14 mL), (R)-N-(fluoren-9-ylmethoxycarbonyl)serine (2.00 g, 6.11
mmol), and DCM/DMF (14:1, 15 mL). Yield: 1.30 g, 55.5% of a
colorless product. TLC: Rf (II) ) 0.15. ES-MS: m/z ) 350.0 [M
- tBu + Na]+ 406.1 [M + Na]+, 422.0 [M + K]+. GC-MS:
98.7% ee.
Conversion of the Hydroxy Group to the Bromide (8; General
Procedure). The serine derivative 7a or 7b and CBr4 (1.22 equiv)
were dissolved under argon in dry DCM (10 mL/g) and cooled to
0 °C. To the stirred solution was added PPh3 (1.6 equiv) in several
portions within 1 h. The solution was allowed to warm to room
temperature and stirred for an additional 2 h. The solvent was
removed in vacuo, and the residue was dissolved in a small amount
of DCM and poured in ethyl acetate. The precipitate was removed
by filtration and the filtrate cooled to -20 °C and filtered again.
The solvent was removed and the residual product purified by flash
column chromatography (silica gel; hexane/ethyl acetate 7:1).
(R)-â-Bromo-N-(Fluoren-9-ylmethoxycarbonyl)alanine-tert-
butyl ester (8a) was synthesized with 7a (7.00 g, 18.26 mmol),
CBr4 (7.39 g, 22.27 mmol), DCM (70 mL), PPh3 (7.66 g, 29.21
mmol), and ethyl acetate (100 mL).Yield: 6.91 g, 84.8% of a
colorless compound. TLC: Rf (III) ) 0.15. ES-MS: m/z ) 412.2,
414.2 [M - tBu + Na]+, 468.0, 470.0 [M + Na]+.
(S)-â-Bromo-N-(Fluoren-9-ylmethoxycarbonyl)alanine-tert-
butyl ester (8b) was synthesized with 7b (500 mg, 1.30 mmol),
CBr4 (527 mg, 1.59 mmol), DCM (5 mL), PPh3 (547 mg, 2.09
mmol), and ethyl acetate (20 mL). Yield: 451.0 mg, 77.49% of a
colorless compound. TLC: Rf (III) ) 0.15. ES-MS: m/z ) 412.2,
414.2 [M - tBu + Na]+, 468.0, 470.0 [M + Na]+.
General Procedure for the Synthesis of the Lanthionines 9a-
d. The appropriate cysteine derivative was dissolved in 5% DIEA
in DMF under argon. A solution of the corresponding bromide (1.1
equiv) in DMF was added dropwise. After stirring for 4 h at room
temperature the mixture was diluted with a 5-fold excess of diethyl
ether and washed five times with 10% citric acid and three times
with water. The combined water phases were washed three times
with diethyl ether and the combined organic phases again three
times with 10% citric acid and brine. After removal of the solvent,
the residual product was purified by flash column chromatography
(hexane/ethyl acetate 4.5:1f2:1), yielding 9a-d as colorless
compounds.
(2R,6R)-N2,N6-Bis(fluoren-9-ylmethoxycarbonyl)lanthionine-
E-allyl-r-tert-butyl diester (9a) was synthesized with 5a (5.00 g,
13.04 mmol), 8a (6.40 g, 14.34 mmol), and 5% DIEA/DMF (60
mL). Yield: 6.30 g, 64.5%. TLC: Rf (I) ) 0.14. ES-MS: m/z )
771.2 [M + Na]+, 787.1 [M + K]+. FT-ICR-MS: m/z )
771.2710250 [M + Na]+ (C43H44N2O8SNa ) 771.2710582) ∆ )
0.04 ppm. GC-MS: 97.3% R,R; 2.7% R,S/S,R; 0.0% S,S.
(S)-N-(Fluoren-9-ylmethoxycarbonyl)cysteine-allyl ester (5b)
was synthesized with 4b (1.80 g, 2.88 mmol), TFA/DCM (1:1, 20
mL), TIPS (1.95 mL; 9.50 mmol), and DCM/diethyl ether (1:20,
105 mL). Yield: 1.05 g, 95.2% of a colorless amorphous powder.
TLC: Rf (I) ) 0.34. ES-MS: m/z ) 406.1 [M + Na]+, 422.0 [M
+ K]+. GC-MS: 93.4% ee.
Introduction of the tert-Butyl Carboxy Protecting Group (7;
General Procedure). A mixture of 3 equiv of DIC, 4 equiv of
tert-butyl alcohol, and 0.02 equiv of CuCl was stirred under argon
for 5 d. After dilution with DCM (7.0 mL/g), the mixture was added
(2R,6S)-N2,N6-Bis(fluoren-9-ylmethoxycarbonyl)lanthionine-
E-allyl-r-tert-butyl diester (9b) was synthesized with 5b (350 mg,
0.91 mmol), 8a (448 mg, 1.00 mmol), and 5% DIEA/DMF (8 mL).
Yield: 420 mg, 61.5%. TLC: Rf (I) ) 0.14. ES-MS: m/z ) 771.2
[M + Na]+, 787.1 [M + K]+. FT-ICR-MS: m/z ) 771.2707890
[M + Na]+ (C43H44N2O8SNa ) 771.2710582) ∆ ) 0.3 ppm. GC-
MS: 1.3% R,R; 97.0% R,S/S,R; 1.7% S,S.

1762 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5
Seyberth et al.
(2S,6R)-N2,N6-Bis(fluoren-9-ylmethoxycarbonyl)lanthionine-
E-allyl-r-tert-butyl diester (9c) was synthesized with 5a (350 mg,
0.91 mmol), 8b (448 mg, 1.00 mmol), and 5% DIEA/DMF (8 mL).
Yield: 394 mg, 57.6%. TLC: Rf (I) ) 0.14. ES-MS: m/z ) 771.2
[M + Na]+, 787.1 [M + K]+. FT-ICR-MS: m/z ) 771.27107380
[M + Na]+ (C43H44N2O8SNa ) 771.2710582) ∆ ) 0.4 ppm. GC-
MS: 2.6% R,R; 96.7% R,S/S,R; 0.7% S,S.
removed in vacuo. The residual mixture containing 12 was dissolved
in DCM/DMF (5:2, 49 mL) and added dropwise to the solution of
activated Pam-OH. After stirring for 16 h at room temperature and
removal of the solvent in vacuo, the residual product was dissolved
in CHCl3 and washed three times with 10% Na2CO3 and brine.
The solution was dried over MgSO4 and the solvent removed in
vacuo. 14 was precipitated from CHCl3/methanol (1:6) at -20 °C
as a colorless crystalline compound (752 mg, 63.2%). TLC: Rf
(V) ) 0.83, Rf (VI) ) 0.72. ES-MS: m/z ) 986.7 [M + Na]+,
998.7 [M + Cl]-.
(2S,6S)-N2,N6-Bis(fluoren-9-ylmethoxycarbonyl)lanthionine-
E-allyl-r-tert-butyl diester (9d) was synthesized with 5b (350 mg,
0.91 mmol), 8b (448 mg, 1.00 mmol), and 5% DIEA/DMF (8 mL).
Yield: 383 mg, 56.0%. TLC: Rf (I) ) 0.14. ES-MS: m/z ) 771.3
[M + Na]+, 787.2 [M + K]+. FT-ICR-MS: m/z ) 771.2704620
[M + Na]+ (C43H44N2O8SNa ) 771.2710582) ∆ ) 0.8 ppm. GC-
MS: 0.12% R,R; 4.05% R,S/S,R; 95.83% S,S.
(2R,6R)-N2,N6-Bis(palmitoyl)lanthionine-E-hexadecylamide (15).
14 (752 mg, 0.78 mmol) was deprotected in neat TFA (10 mL) for
1 h at room temperature. The TFA was removed in vacuo and the
residual product 15 was lyophilized from tert-butyl alcohol/H2O
(4:1), giving 15 as a colorless amorphous powder (708 mg, 100%).
TLC: Rf (VI) ) 0.00. ES-MS: m/z ) 906.8 [M - H]-, 930.7 [M
+ Na]+. GC-MS: 90.68% R,R; 8.23% R,S/S,R; 0.89% S,S.
C54H105N3O5S (MW ) 908.52): calcd C 71.39, H 11.65, N 4.63,
S 3.53; found C 71.29, H 11.40, N 4.55, S 3.50.
Peptide Synthesis (General Procedure). The Fmoc-amino acids
were coupled by activation of 3 equiv of Fmoc-amino acid with 3
equiv of HOBt and 3 equiv of DIC in DCM/DMF (8:1) for 1 h.
After adding the solution to the dry, Fmoc-deprotected Rink-amide-
resin or the corresponding Fmoc-deprotected resin-bound amino
acids the mixture was agitated for 8 h at room temperature. The
resin was filtered off, washed three times each with DMF/DCM/
methanol, and dried in vacuo. If the Kaiser test showed incomplete
coupling, the above protocol was repeated. Fmoc was cleaved by
agitating the resin in a surplus of 20% piperidine in DMF for 20
min. The resin was filtered off and the procedure repeated. The
solvent was removed by filtration, and the resin was washed three
times each with DMF/DCM/methanol and dried in vacuo. For
cleavage of the final products, the resin-bound peptides were treated
with TFA/H2O (95:5) for 3 h at room temperature. The resin was
removed by filtration and the TFA removed in vacuo. The residue
was lyophilized from tert-butyl alcohol/H2O (4:1). After each
coupling step peptide samples were cleaved from the resin and
analyzed by HPLC and ES-MS. All products showed at least 95%
purity and the expected [M + H]+ signals.
tert-Butyl Deprotection of Lanthionines 9a-d (10a-d; Gen-
eral Procedure). The corresponding fully protected lanthionine was
dissolved in neat TFA and stirred for 1 h at room temperature.
The TFA was removed in vacuo and the residue lyophilized from
acetonitrile/H2O 4:1 to yield the pure lanthionines in quantitative
yield.
(2R,6R)-N2,N6-Bis(fluoren-9-ylmethoxycarbonyl)lanthionine-
r-allyl ester (10a) was synthesized with 9a (3.00 g, 4.01 mmol)
and TFA (15 mL). Yield: 2.78 g of a colorless amorphous powder.
ES-MS: m/z ) 715.1 [M + Na]+.
(2R,6S)-N2,N6-Bis(fluoren-9-ylmethoxycarbonyl)lanthionine-
r-allyl ester (10b) was synthesized with 9b (420 mg, 0.56 mmol)
and TFA (5 mL). Yield: 389 mg of a colorless amorphous powder.
ES-MS: m/z ) 715.1 [M + Na]+.
(2S,6R)-N2,N6-Bis(fluoren-9-ylmethoxycarbonyl)lanthionine-
r-allyl ester (10c) was synthesized with 9c (394 mg, 0.53 mmol)
and TFA (5 mL). Yield: 365 mg of a colorless amorphous powder.
ES-MS: m/z ) 715.1 [M + Na]+.
(2S,6S)-N2,N6-Bis(fluoren-9-ylmethoxycarbonyl)lanthionine-
r-allyl ester (10d) was synthesized with 9d (383 mg, 0.51 mmol)
and TFA (5 mL). Yield: 354 mg of a colorless amorphous powder.
ES-MS: m/z ) 715.1 [M + Na]+.
(2R,6R)-N2,N6-Bis(fluoren-9-ylmethoxycarbonyl)lanthionine-
r-tert-butyl Ester (11). 9a (3.00 g, 4.01 mmol) was dissolved in
DCM (50 mL) under argon. Morpholine (454 µL, 5.21 mmol) and
Pd(PPh3)4 (100 mg, 0.09 mmol) were added, and the mixture was
protected from light and stirred at room temperature. After 1 h the
reaction was complete (TLC) and the solvent removed in vacuo.
11 was purified by flash column chromatography (silica gel; hexane/
ethyl acetate/acetic acid 8:4:1) to give a colorless amorphous powder
(2.58 g, 90.8%). TLC: Rf (IV) ) 0.11. ES-MS: m/z ) 731.4 [M
+ Na]+, 1439.5 [2M + Na]+, 707.1 [M - H]-, 1415.2 [2M -
H]-.
Immobilization of (S)-N2-(fluoren-9-ylmethoxycarbonyl)-N6-
(tert-butoxycarbonyl)lysine (N-Fmoc-L-Lys(Boc)-OH) to Rink-
amide resin used Rink-amide resin (3.00 g, 2.22 mmol), N-Fmoc-
L-Lys(Boc)-OH (3120 mg, 6.66 mmol), HOBt (1020 mg, 6.66
mmol), and DIC (1031 µL, 6.66 mmol).
Coupling of N-Fmoc-L-Lys(Boc)-OH used N-Fmoc-L-Lys(Boc)-
OH (3120 mg, 6.66 mmol), HOBt (1020 mg, 6.66 mmol), and DIC
(1031 µL, 6.66 mmol).
(2R,6R)-N2,N6-Bis(fluoren-9-ylmethoxycarbonyl)lanthionine-
E-hexadecylamide-r-tert-butyl Ester (12). 11 (2.60 g, 3.67 mmol),
HOBt (562 mg, 3.67 mmol), and DIC (568 µL, 3.67 mmol) were
dissolved in DCM/DMF (5:2, 20 mL) and stirred for 1 h at room
temperature. A solution of hexadecylamine (974 mg, 4.03 mmol)
in DCM/DMF (5:2, 20 mL) was added dropwise and stirring was
continued for 6 h at room temperature. The solvent was removed
in vacuo and the residual product purified by flash column
chromatography (silica gel; hexane/ethyl acetate 2:1) to yield 12
as a colorless compound (2.35 g, 68.72%). TLC: Rf (II) ) 0.23.
ES-MS: m/z ) 467.2 [(M + 2H)/2]+, 954.6 [M + Na]+.
(2R,6R)-N2,N6-Bis(fluoren-9-ylmethoxycarbonyl)lanthionine-
E-hexadecylamide (13). 12 (1.15 g, 1.23 mmol) was dissolved in
neat TFA (11.5 mL) and stirred at room temperature for 1 h. The
TFA was removed in vacuo and the product lyophilized from tert-
butyl alcohol/H2O. 13 was obtained as an amorphous colorless
powder in quantitative yield (1.08 g, 100.0%). TLC: Rf (II) ) 0.00.
ES-MS: m/z ) 898.2 [M + Na]+.
Coupling of N-Fmoc-L-Ser(tBu)-OH (16) used N-Fmoc-L-Ser-
(tBu)-OH (2553 mg, 6.66 mmol), HOBt (1020 mg, 6.66 mmol),
and DIC (1031 µL, 6.66 mmol).
Coupling of 13 to 16 used resin 16 (100.00 mg, 41.60 µmol),
13 (109.35 mg, 124.80 µmol), HOBt (19.11 mg, 124.80 µmol),
and DIC (19.32 µL, 124.80 µmol). The coupling was repeated until
a negative Kaiser test was observed. HPLC: 98.0%. MALDI-MS:
m/z ) 1474.2 [M + H]+.
Coupling of the Lanthionines 10a-d to Resin 16. All four
stereoisomers of lanthionine (10a-d) were coupled to the Fmoc-
deprotected resin 16 using the DIC/HOBt protocol. Subsequently,
the reaction mixture was filtered off and 1 equiv each of the
appropriate lanthionine, HOBt, and DIC was added to the mixture
and the above protocol was repeated.
Coupling of 10a to 16 (19) used resin 16 (500.00 mg, 208.00
µmol), 10a (432.30 mg, 624.00 µmol), HOBt (95.55 mg, 624 µmol),
and DIC (96.62 µL, 624 µmol) and 10a (144.10 mg, 208.00 µmol),
HOBt (31.9 mg, 208 µmol), and DIC (32.2 µL, 208 µmol). HPLC:
96.9%. ES-MS: m/z ) 1313.3 [M + Na]+, MALDI-MS: m/z )
1291.8 [M + H]+.
(2R,6R)-N2,N6-Bis(palmitoyl)lanthionine-E-hexadecylamide-
r-tert-butyl Ester (14). A solution of palmitic acid (1.11 g, 4.34
mmol), HOBt (664 mg, 4.3 mmol), and DIC (672 µL, 4.3 mmol)
in DCM/DMF (5:2, 70 mL) was stirred for 2 h at room temperature.
Meanwhile, 12 (1.15 g, 1.23 mmol) was stirred for 2 h at room
temperature in piperidine/DMF (1:1, 30 mL), and the solvents were
Coupling of 10b to 16 (40) used resin 16 (40.00 mg, 16.64
µmol), 10b (34.59 mg, 49.92 µmol), HOBt (7.64 mg, 49.92 µmol),
and DIC (7.73 µL, 49.92 µmol) and 10b (11.53 mg, 16.64 µmol),

Lipolanthionine Peptides Inhibit IL-8 Secretion
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5 1763
Table 2. Summary of the Fatty Amine Couplings to the Resin Bound, Allyl Deprotected Lanthionines [P/D/T ) pyridine/DMF/TFA-OPfp (1:1:1)]
amount
resin
used
of resin
(mg, µmol)
P/D/T
(µL)
amount
of amine
DIEA
(µL)
HPLC
(%)
resulting
resin
fatty amine
20
20
20
20
20
20
43
44
45
20
20, 7.12
20, 7.12
20, 7.12
20, 7.12
20, 7.12
300, 106.80
20, 7.12
20, 7.12
20, 7.12
30, 10.68
300
300
300
300
300
1200
300
300
300
300
hexylamine
octylamine
decylamine
dodecylamine
tetradecylamine
hexadecylamine
hexadecylamine
hexadecylamine
hexadecylamine
octadecylamine
9.42 µL, 71.20 µmol
14.14 µL, 71.20 µmol
14.14 µL, 71.20 µmol
13.20 mg, 71.20 µmol
15.19 mg, 71.20 µmol
257.88 mg, 1.07 mmol
17.19 mg, 71.20 µmol
17.19 mg, 71.20 µmol
17.19 mg, 71.20 µmol
28.78 mg, 106.80 µmol
36.57
36.57
36.57
36.57
36.57
548.50
36.57
36.57
36.57
54.85
98.4
96.5
96.0
95.7
92.4
93.9
88.7
90.0
92.5
97.9
21
22
23
24
25
26
46
47
48
27
Table 3. Summary of the Fatty Acid Couplings to Different Fmoc-Deprotected, Resin-Bound Lanthionine-ꢀ-hexadecylamide Peptides
amount
resin
used
resin
(mg, µmol)
amount
fatty acid
DIC
(µL)
HOBt
(mg)
MALDI-MS
resulting
compd
fatty acid
[M + H]+
26
26
26
26
26
46
47
48
26
26
26
10.82, 3.56
10.82, 3.56
10.82, 3.56
10.82, 3.56
10.82, 3.56
10.82, 3.56
10.82, 3.56
10.82, 3.56
10.82, 3.56
10.82, 3.56
10.82, 3.56
caproic
caprylic
capric
2.68 µL, 21.36 µmol
3.39 µL, 21.36 µmol
3.68 mg, 21.36 µmol
4.28 mg, 21.36 µmol
4.88 mg, 21.36 µmol
4.88 mg, 21.36 µmol
4.88 mg, 21.36 µmol
4.88 mg, 21.36 µmol
5.48 mg, 21.36 µmol
6.08 mg, 21.36 µmol
6.68 mg, 21.36 µmol
3.31
3.31
3.31
3.31
3.31
3.31
3.31
3.31
3.31
3.31
3.31
3.27
3.27
3.27
3.27
3.27
3.27
3.27
3.27
3.27
3.27
3.27
1225.9
1282.0
1338.4
1394.0
a
1450.4
1450.5
1450.7
1506.2
1561.9
1617.9
34
35
36
37
17
49
50
51
18
38
39
lauric
myristic
myristic
myristic
myristic
palmitic
stearic
arachidic
a MALDI- and FT-ICR-MS data are given in the Experimental Section.
HOBt (2.54 mg, 16.64 µmol), and DIC (2.57 mg, 16.64 µmol).
HPLC: 91.6%. ES-MS: m/z ) 1313.2 [M + Na]+.
6 h at room temperature. The solvents were removed by filtration,
and the resin was washed thrice each with DMF/DCM/diethyl ether.
For analytical purposes 1 mg of the resin was treated with 10 equiv
of fluoren-9-ylmethyl chloroformate in pyridine/DMF (1:4) prior
to cleavage. Table 2 gives a summary of the amounts of reagents
and the HPLC purities. The MALDI-MS spectra of the products
showed the expected [M + H]+ and [M + Na]+ peaks.
General Procedure for the Fmoc-Protection of the Resin
Bound Lanthionine with Fluoren-9-ylmethyl Chloroformate.
The resin was agitated with a mixture of 10 equiv of fluoren-9-
ylmethyl chloroformate (Fmoc-Cl) in pyridine/DMF (1:4). The
solvents were removed by filtration, and the resin was washed three
times each with DMF/DCM/methanol and dried in vacuo. The resins
21-25, 46-48 (36.83 mg, 71.20 µmol each), 26 (552.5 mg, 1.07
mmol), and 27 (55.26 mg, 106.80 µmol) were Fmoc protected using
this protocol and the appropriate amounts of the reagents.
General Procedure for the Coupling of the Fatty Acids. A
mixture of 3 equiv of the fatty acid, 3 equiv of HOBt, and 3 equiv
of DIC was stirred in DCM/DMF (8:1) for 1 h. The mixture was
then added to the freshly Fmoc-deprotected resin. After 8 h of
stirring at room temperature, the solvents were removed by
filtration, and the resin was washed three times each with DMF/
DCM/methanol and dried in vacuo. Table 3 summarizes the reaction
procedures for the coupling of different fatty acids to the resin-
bound and Fmoc-deprotected lanthionine N7-hexadecylamides, and
Table 4 summarizes the couplings of myristic acid to the different
resin-bound lanthionine amides.
Analytical Data for Coupling of Myristic Acid to Fmoc-
Deprotected Resin 26 (17). MALDI-MS: m/z ) 1450.6 [M +
H]+. FT-ICR-MS: m/z ) 726.07824 [(M + 2H)/2]+ ([C77H151N13O10-
S+H]+ ) 1451.15004; [(C77H151N13O10S+2H)/2]+ ) 726.07844)
∆ ) 0.28 ppm.
Coupling of 10c to 16 (41) used resin 16 (40.00 mg, 16.64
µmol), 10c (34.59 mg, 49.92 µmol), HOBt (7.64 mg, 49.92 µmol),
and DIC (7.73 µL, 49.92 µmol) and 10c (11.53 mg, 16.64 µmol),
HOBt (2.54 mg, 16.64 µmol), and DIC (2.57 mg, 16.64 µmol).
HPLC: 93.9%. ES-MS: m/z ) 1291.3 [M + H]+, 1313.2 [M +
Na]+.
Coupling of 10d to 16 (42) used resin 16 (40.00 mg, 16.64
µmol), 10d (34.59 mg, 49.92 µmol), HOBt (7.64 mg, 49.92 µmol),
and DIC (7.73 µL, 49.92 µmol) and 10d (11.53 mg, 16.64 µmol),
HOBt (2.54 mg, 16.64 µmol), and DIC (2.57 mg, 16.64 µmol).
HPLC: 93.0%. ES-MS: m/z ) 1291.3 [M + H]+, 1313.2 [M +
Na]+.
General Procedure for the Allyl Deprotection. A solution of
24 equiv of phenylsilane in DCM and a solution of 0.25 equiv of
Pd(PPh3)4 in DCM was prepared under argon. The phenylsilane
solution was added to the dry resin and the mixture agitated for 5
min under argon. After addition of the Pd(PPh3)4 solution the
mixture was protected from light and agitated for 2 h under argon
at room temperature. The resin was washed three times with a 0.5%
sodium diethyl dithiocarbamate trihydrate solution in DMF for 15
min and afterward thoroughly with DMF/DCM/methanol. The resin
was then dried in vacuo.
The resin-bound lanthionine derivatives 19 (590.00 mg, 205.91
µmol f20), 40 (30.00 mg, 10.47 µmol f43), 41 (30.00 mg, 10.47
µmol f44), 42 (30.00 mg, 10.47 µmol f45), 52 (7.00 mg, 2.47
µmol f56), 53 (7.00 mg, 2.47 µmol f57), 54 (7.00 mg, 2.47 µmol
f58), and 55 (7.00 mg, 2.47 µmol f59) were deprotected
following this procedure in good purities (HPLC, >89%). In the
MALDI- or ES-MS spectra the [M + H]+, [M + Na]+, and/or [M
- H + 2Na]+ peaks were observed.
General Procedure for the Amine Coupling to Resin-Bound,
Allyl-Deprotected Lanthionine. The appropriate resin (20 and 43-
45) was agitated in a mixture of pyridine/DMF/pentafluorophenyl
trifluoroacetate (TFA-OPfp) (3:2:1) for 2 h at room temperature.
The solvents were removed by filtration and the resin washed twice
each with DCM/diethyl ether. A mixture of 10 equiv of amine and
30 equiv of DIEA in THF was added and the mixture agitated for
Oxidation of the Lanthionine Sulfur to Its Sulfoxide (61). A
solution of 0.2 equiv of Sc(OTf)3 (0.32 mg, 0.65 µmol) in DCM/
ethanol (9:1, 500 µL) was added to the dry resin 60 (10.00 mg,
3.26 µmol), 5 equiv of H2O2 (30%, 1.68 µL, 16.30 µmol) was
added, and the mixture was agitated for 40 min at room temperature.
The solvents were removed by filtration, the resin was washed three

1764 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5
Seyberth et al.
Table 4. Summary of the Myristic Acid Couplings to Different Resin
derivatized enantiomers (2R,6R)-lanthionine 9a and (2S,6S)-
lanthionine 9d and of a mixture of D-, L-, and meso-lanthionine;
fragmentation scheme of the lanthionine derivatives used for chiral
GC-MS analysis. This material is available free of charge via the
Internet at http://pubs.acs.org.
Bound Lanthionine Peptide Amidesa
amount
resin
(mg, µmol)
MALDI-MS
resulting
compd
resin used
[M + H]+
21
22
23
24
25
26
27
19
40
41
42
10.32, 3.56
10.44, 3.56
10.53, 3.56
10.63, 3.56
10.72, 3.56
10.82, 3.56
10.92, 3.56
10.00, 3.49
10.00, 3.49
10.00, 3.49
10.00, 3.49
1311.1
1339.0
1366.6
1395.0
1423.1
1450.6
1479.4
1267.3
1267.5
1267.1
1267.2
28
29
30
31
32
17
33
52
53
54
55
References
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a Myristic acid (4.88 mg, 21.36 µmol), DIC (3.31 µL, 21.36 µmol), HOBt
(3.27 mg, 21.36 µmol).
times each with DMF/DCM/methanol and dried in vacuo. MALDI-
MS: m/z ) 1522.6 [M + H]+.
Oxidation of the Lanthionine Sulfur to Its Sulfone (62). A
solution of 5 equiv of mCPBA (2.83 mg, 16.30 µmol) in DCM
(500 µL) was added to resin 60 (10.00 mg, 3.26 µmol) and the
mixture was agitated for 24 h at room temperature. After removal
of the solvents, the resin was washed three times each with DMF/
DCM/methanol and dried in vacuo. MALDI-MS: m/z ) 1538.5
[M + H]+.
Cell Culture. The human myelomonocytic cell line THP-1
(ATCC number TIB-202) was obtained from the Deutsche
Sammlung von Mikroorganismen und Zellkulturen (DSMZ)
(Braunschweig, Germany). The cells were cultured in RPMI 1640
medium (PAN Biotech, Aidenbach, Germany) supplemented with
10% FCS (PAN Biotech) in a 5% CO2 humidified atmosphere at
37 °C. Cells were passaged every third or fourth day.
IL-8 Assay. THP-1 cells were dispensed into 96-well culture
plates at a cell density of 4 × 104 cells per 200 µL of medium per
well. Stock solutions of lipolanthionine peptides and Pam3Cys-SK4-
OH (EMC microcollections, Tu¨bingen, Germany) in DMSO were
prepared at a concentration of 10 mM. Cells were incubated with
lipolanthionine peptides at the indicated concentrations for 10 min
at 37 °C. Controls with lipolanthionine-free medium containing
equal amounts of DMSO were included in each assay. The agonist
Pam3Cys-SK4-OH was added to the samples at a concentration of
50 nM. After a further incubation for 5 h at 37 °C cell-free
supernatants were collected and stored at -80 °C prior to the
quantification of IL-8 secretion by matched-pair ELISA (clone
G265-5 and clone G265-8, BDPharMingen, San Diego, CA)
according to the protocol provided by the manufacturer.
Cell Viability Assay. Cell viability was measured using the
colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) dye reduction assay. THP-1 cells were treated with
the corresponding lipolanthionine peptides and Pam3Cys-SK4-OH
for 5 h (see above), and after removing 120 µL of the supernatant
for ELISA analysis, cells were incubated with MTT (Sigma) at a
concentration of 1 mg/mL for 4 h. The formazan product was
solubilized with SDS (10% (m/v) in 10 mM HCl). The fraction of
viable cells was determined by measuring the absorbance of each
sample at 570 nm relative to the absorbance of untreated control
cells using the microplate reader.
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Acknowledgment. We thank Graeme Nicholson for chiral
GC-MS analysis, and Paul Schuler for NMR (600 MHz)
measurements. Tobias Seyberth received a stipend from the DFG
Graduiertenkolleg “Analytical Chemistry”. This work was
supported by the Federal Ministry of Education and Research,
project BioChance no. 0312662.
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Supporting Information Available: 1D- and 2D-NMR spectra
of the synthesized (2R,6R)-lanthionine derivatives 9a and Pam2-
LanHda 15; exemplary chiral GC-MS chromatograms of the

Lipolanthionine Peptides Inhibit IL-8 Secretion
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5 1765
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