The synthesis of acid- and base-labile lipopeptides on solid support

Article abstract of DOI:10.1002/chem.200304822

Lipidated peptides, including characteristic partial structures of human Ras proteins, were synthesized by means of a new solid-phase technique in 22-68% yield. This technique gives access to farnesylated, palmitoylated, and doubly lipidated peptides as methyl esters or carboxylic acids carrying a fluorescent tag or a maleimide moiety for coupling to proteins. The peptide backbones were built up on the resin by using 9-fluorenylmethoxycarbonyl chemistry together with the oxidatively cleavable hydrazide linker. As a key step, the acid-labile farnesyl and basic-labile palmitoyl lipid groups were introduced onto the resin after the cleavage of appropriate acid- or reduction-sensitive protecting groups from the cysteine residues. Optional introduction of different fluorescent tags or a maleimide group into the peptide was followed by release of the resin-bound target peptide as the methyl ester or carboxylic acid by very mild copper(II)-mediated oxidation in slightly acidic or basic media. This new methodology should substantially facilitate the access to lipidated peptides for the study of important biological phenomena like biological signal transduction, localization, and vesicular transport.

Full text of DOI:10.1002/chem.200304822

FULL PAPER
The Synthesis of Acid- and Base-Labile Lipopeptides on Solid Support
Bjˆrn Ludolph and Herbert Waldmann*[a]
Abstract: Lipidated peptides, including
characteristic partial structures of hu-
man Ras proteins, were synthesized by
means of a new solid-phase technique in
22 68% yield.This technique gives
access to farnesylated, palmitoylated,
and doubly lipidated peptides as methyl
esters or carboxylic acids carrying a
fluorescent tag or a maleimide moiety
for coupling to proteins.The peptide
backbones were built up on the resin
by using 9-fluorenylmethoxycarbonyl
chemistry together with the oxidatively
cleavable hydrazide linker.As a key
group into the peptide was followed by
release of the resin-bound target peptide
as the methyl ester or carboxylic acid by
very mild copper(ii)-mediated oxidation
in slightly acidic or basic media.This
new methodology should substantially
facilitate the access to lipidated peptides
for the study of important biological
phenomena like biological signal trans-
duction, localization, and vesicular
transport.
step, the acid-labile farnesyl and basic-
labile palmitoyl lipid groups were intro-
duced onto the resin after the cleavage
of appropriate acid- or reduction-sensi-
tive protecting groups from the cysteine
residues.Optional introduction of differ-
ent fluorescent tags or a maleimide
Keywords: lipopeptides
teins protecting groups
proteins ¥ solid-phase synthesis
¥
lipopro-
¥
¥
Ras
Introduction
Lipidated proteins play important roles in the transduction of
extracellular signals across the cell membrane and in numer-
ous intracellular processes like organization of the cytoske-
leton, vesicle formation, and targeting.[1] Lipidation of these
proteins is a prerequisite for correct biological function.The
lipid groups are believed to be involved in protein protein
and protein lipid interactions and to serve as anchors of the
proteins to different membranes.Several different lipid
modifications are known.For instance, the Ras proteins
embody both farnesyl thioethers and palmitic acid thioesters
and terminate in a cysteine methyl ester, whereas the Rab
proteins are S-geranylgeranylated and carry either a carbox-
ylic acid or a methyl ester at the C terminus (Scheme 1).[1a, 2]
Tailormade lipidated peptides representing the characteristic
lipid-modified partial structures of their parent proteins are
efficient tools for the investigation of these biological
processes in molecular detail.[2c, 3]
Scheme 1.Structures of the lipidated C terminus of N-Ras and of the Rab
The synthesis of lipidated peptides is severely complicated
by the pronounced acid- and base-sensitivity of the isoprenyl
proteins.Far ˆ farnesyl, GerGerˆ geranylgeranyl, Pal ˆ palmitoyl.
thioethers and the palmitoyl thioesters, respectively
(Scheme 2), and requires the application of blocking groups
that are cleavable under the mildest conditions.[2a, 3c] Currently
differently functionalized and differently lipidated peptides
are only available through multistep solution-phase methods
[a] Prof.Dr.H.Waldmann, Dipl-.Chem.B.Ludolph
Max-Planck-Institut f¸r molekulare Physiologie
Abteilung Chemische Biologie
Otto-Hahn-Strasse 11, 44227 Dortmund (Germany)
and
Universit‰t Dortmund
employing, for instance, enzyme- or noble-metal-catalyzed
Fachbereich 3, Organische Chemie
Fax : (‡49)231-133-2499
c, 4]
transformations as key steps.[3a
The solid-phase synthesis
E-mail: herbert.waldmann@mpi-dortmund.mpg.de
of exclusively S-palmitoylated[5] or S-farnesylated[6] peptides
Chem. Eur. J. 2003, 9, 3683 3691
DOI:10.1002/chem.200304822
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H.Waldmann and B.Ludolph
FULL PAPER
Scheme 2.Acid-labile isoprenyl thioethers and base- and nucleophile-
labile palmitoyl thioesters in lipopeptides.AA ˆ amino acid, PG ˆ pro-
tecting group.
has been described in a few cases but no solid-phase method
that gives access to lipopeptides incorporating isoprenoid
groups together with thioesters is available.The major
challenge to be met by such a flexible solid-phase technique
lies in the development of a set of suitable orthogonally stable
protecting groups and a linker to the solid support that allows
for selective introduction of the different lipid groups as well
as additional functional groups for tracing such peptides in
biological systems (for example, a fluorescent group) or for
coupling them to expressed proteins (such as a maleimide
moiety).Also, the linker should allow for selective elongation
of the peptide chain and, finally, release of the desired
products into solution as the ester or acid under the mildest
conditions and without any harm to the acid- and base-
sensitive lipid groups.Herein we report on the development
of a solid-phase technique that fulfils the demands raised
above.The technique relies on the combined use of the base-
labile 9-fluorenylmethoxycarbonyl (Fmoc) group, the Pd0-
sensitive allyloxycarbonyl (Aloc) group, and the very acid-
sensitive trityl (Trt) group for protection of the amino groups,
the application of acid- and reduction-labile protecting groups
for the cysteine side chain, S-farnesylation and S-palmitoyla-
tion of the growing peptide chain on the solid support, and the
use of the oxidation-sensitive hydrazide linker.Part of this
work was published in a preliminary communication.[7]
Scheme 3.Cleavage of the hydrazide linker.Nu ˆ nucleophile.
imposed by the sensitivity of the lipids.To this end, the Fmoc
group of commercially available Fmoc-4-hydrazinobenzoyl
resin (NovaGel resin, Novabiochem) was cleaved and S-trityl-
protected Fmoc-cysteine was coupled to the hydrazide resin
to yield immobilized amino acid 3 with a loading of
0.35 mmolgÀ1.The S-trityl group was removed by treatment
with 50% TFA in dichloromethane and then S-farnesylation
was carried out by treatment of the liberated thiol with
farnesyl bromide in the presence of a tertiary amine, that is,
under basic conditions (Scheme 4).The farnesylated inter-
mediate was cleaved from the resin by treatment with
Scheme 4.Solid-phase synthesis of lipidated cysteine methyl esters 4 and
5.a) 50% TFA, TES, CH 2Cl2; b) Far-Br, DIEA, DMF; c) Pal-Cl, HOBt,
Et3N, CH2Cl2, DMF; d) Cu(OAc)2, pyridine, O2, CH2Cl2, MeOH, and in
the case of (c) additional acetic acid.TFA ˆ trifluoroacetic acid, TES ˆ
triethylsilane, Far-Brˆ trans,trans-farnesyl bromide, DIEA ˆ N,N'-diiso-
propylethylamine, DMF ˆ N,N-dimethylformamide, Pal-Cl ˆ palmitoyl
chloride, HOBt ˆ 1-hydroxybenzotriazole.
Cu(OAc)2 and oxygen in dichloromethane and in the
presence of methanol and pyridine to give ester 4 in 55%
yield.This is the first example of peptide farnesylation on a
solid support.
Results
To develop the principle set-up of the method, we first focused
on the oxidation-sensitive hydrazide linker 1 for attachment
to the solid support.It can be cleaved by oxidation with Cu
In a similar series of experiments, the on-resin palmitoyla-
tion and release of an S-palmitoylated model compound were
investigated.To this end, the trityl group was cleaved from
polymer-bound S-trityl-protected cysteine 3 and the liberated
thiol was converted into the corresponding palmitic acid
thioester by treatment with palmitoyl chloride in the presence
of HOBt and triethylamine (Scheme 4).The best results were
observed when 10 equivalents of both reagents were used for
15 h; application of only four equivalents for 2 h resulted in
lower yields and impure product. S-palmitoylated cysteine
methyl ester 5 was released in a yield of 56% from the solid
support by oxidation with Cu(OAc)2 in dichloromethane and
in the presence of pyridine and acetic acid.Under these
II
or N-bromosuccinimide to form an intermediate acyldiazene
2 that is then trapped by an added nucleophile to form an
ester or amide bond, thereby releasing the molecule from the
solid support (Scheme 3).This linker has been employed
before in the synthesis of small peptide esters and amides[8]
and cyclic peptides.[9]
Farnesylated and palmitoylated cysteine methyl esters: In an
initial series of experiments farnesylated and palmitoylated
cysteine methyl esters were synthesized to investigate the
compatibility of the hydrazide linker with the demands
¹ 2003 Wiley-VCH Verlag GmbH & Co.KGaA, Weinheim
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Acid- and Base-Labile Lipopeptides
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conditions undesired hydrolysis of the thioester was not
observed.Cleavage from the resin with an excess of Fe(NO 3)3
as the oxidant instead of Cu(OAc)2 resulted in lower yield
(34%).
out as described above to yield doubly lipidated polymer-
bound peptide 8.Cleavage with Cu II/O2 under acidic con-
ditions yielded doubly lipidated peptide methyl ester 9 in
25% overall yield.(Overall yields are given with respect to
the amount of the first amino acid bound to the resin.)
Purification of the final product was readily achieved by
simple column chromatography during which the copper was
completely removed.An undesired attack on the acid-
sensitive farnesyl thioether or the base-labile palmitoyl
thioester was not recorded.
However, a loss of peptide from resin could be observed
during peptide synthesis.Determination of the Fmoc groups
remaining on the resin by the established UV method[11]
showed a lower loading of the resin than expected, based on
the expected increase of weight of the resin-bound peptide
during the course of the synthesis.We assume that under basic
conditions oxidation of the hydrazide linker can take place
and subsequent cleavage reduces the amount of peptide on
resin.This view was supported by double linker experiments
similar to the one mentioned above.Also, in a related case it
has been shown that a hydrazide-based linker system was not
stable against oxygen in the presence of amines and this
resulted in hydrazide cleavage as well.[12] To avoid these side
reactions, in subsequent experiments all steps were carried
out under argon and piperidine was freshly distilled prior to
use.
These results clearly demonstrate that the hydrazide linker
is compatible with the demands posed by acid- and base-labile
lipid residues and is therefore, in principle, suitable for
lipopeptide synthesis on the solid support.It is, however,
intriguing that the overall yield for the reaction sequence is
only moderate, a fact that raises the question of whether
undesired side reactions occur in the lipidation or cleavage
procedures.To investigate the possibility of such processes, a
model peptide incorporating a double linker composed of the
hydrazide linker and the Wang linker was built up.After
releasing the product from the resin by oxidation of the
hydrazide, the Wang linker was cleaved under acidic con-
ditions.However a major side product could not be identified
by LC-MS.Rather, several minor side products were formed
which could not be identified unambiguously.
Lipopeptide synthesis: Based on these results the synthesis of
a variety of mono- and double-lipidated peptides was carried
out.Commercially available Fmoc-4-hydrazinobenzoyl No-
vaGel resin (Novabiochem) was employed for Fmoc-protect-
ed solid-phase peptide synthesis methods.Typically resins
with a loading of 0.35 0.43 mmolgÀ1 of the first amino acid
were used, as determined by quantification of Fmoc groups on
the resin by UV analysis.Peptide 9 was prepared as the first
example for the solid-phase synthesis of an acid-labile S-
isoprenylated and base-labile S-palmitoylated peptide
(Scheme 5).After assembling dipeptide 6 on the resin the
Farnesylated tripeptide 11 was prepared as shown in
Scheme 6.Either the acid-sensitive trityl or the orthogonally
stable reduction-sensitive tert-butyl disulfide (StBu) was
Scheme 6.Synthesis of farnesylated tripeptide 11.a) 50% TFA, TES,
CH2Cl2; b) Far-Br, DIEA, DMF; c) piperidine, DMF; d) HBTU, HOBt,
DIEA, Fmoc-Thr(Trt)-OH, DMF; e) 1% TFA, TES, CH2Cl2; f) Cu(OAc)2,
acetic acid, pyridine, O2, CH2Cl2, MeOH; g) PBu3, H2O, DMF, CH2Cl2.
HBTU ˆ N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-meth-
ylmethanaminium hexafluorophosphate N-oxide.
Scheme 5.Synthesis of the farnesylated and palmitoylated tripeptide 9.
a) 50% TFA, TES, CH2Cl2; b) Far-Br, DIEA, DMF; c) piperidine, DMF;
d) DIC, HOBt, Fmoc-Cys(Mmt)-OH, DMF; e) 1% TFA, TES, CH2Cl2;
f) Pal-Cl, HOBt, Et3N, CH2Cl2, DMF; g) Cu(OAc)2, acetic acid, pyridine,
O2, CH2Cl2, MeOH.DIC ˆ N,N'-diisopropylcarbodiimide.
employed as the protecting group for the cysteine residue.In
the case with the trityl-protected cysteine, 6, the trityl group
was removed at the stage of the dipeptide.The thiol function
was farnesylated and this was followed by subsequent
elongation with protected threonine.The trityl protecting
group of the threonine hydroxy function was cleaved from the
readily assembled tripeptide with 1% TFA and the N-termi-
nally Fmoc-protected peptide was liberated with copper
acetate in an acetic acid buffer to give target peptide 11 in
46% overall yield.Alternatively, farnesylation at the stage of
the tripeptide was achieved after cleaving the tert-butyl
disulfide used as a masking group for the cysteine thiol
function from 10 with PBu3 and H2O.Again, as the last step on
the resin, the trityl group of the threonine was removed with
1% TFA and the product was obtained after copper-mediated
trityl group was removed with 50% TFA and the cysteine
thiol group farnesylated under basic conditions.The Fmoc
group was removed and the peptide elongated to tripeptide 7.
The mercapto group of the second cysteine in the sequence
had been protected as a monomethoxytrityl (Mmt) thioether.
Cleavage of the methoxytrityl group was achieved with 1%
TFA in dichloromethane and in the presence of triethylsilane.
The farnesyl group remained unattacked under these mild,
weakly acidic conditions.[10] S-palmitoylation was then carried
Chem. Eur. J. 2003, 9, 3683 3691
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H.Waldmann and B.Ludolph
FULL PAPER
cleavage in 41% overall yield.Thus, both protecting group
strategies were successfully established for lipopeptide syn-
thesis on the solid support.
Having established all the necessary protecting-group and
lipidation procedures, we next turned to the synthesis of
doubly lipidated heptapeptides 16 and 17, which mimic the C
terminus of N-Ras (Scheme 7).Individual steps were per-
formed as described above for the synthesis of peptide 9.
peptide 17.As described for 16, peptide 17 was assembled on
the solid support, but the mercapto group of the second
cysteine residue in the sequence was protected with tert-butyl
disulfide as a masking group, which was removed with PBu3
and H2O to give the free thiol.After palmitoylation, cleavage
was carried out under very weakly acidic conditions with only
a slight excess of acetic acid to give 17 in an overall yield of
68%.However, under these conditions partial cleavage
(approximately 10%) of the palmitoyl thioester was observed
as well.During all steps the very acid-labile N-terminal trityl
protecting group present was not affected.We would like to
point out that the N-terminal Aloc urethane or trityl protect-
ing group can be removed selectively without any harm to the
S-palmitoyl thioester or the S-farnesyl thioether from lipi-
dated peptides including 16 and 17;[3d, 14] this method should
thereby give access to intermediates which can be equipped
with, for example, fluorescent groups or a biotin tag to be used
in further biological experiments.
Synthesis of fluorescently labeled and maleimidocaproyl
(MIC) labeled lipidated peptides: To investigate whether
such differently lipidated and additionally tagged peptides are
directly accessible by means of the solid-phase method
described above, the synthesis of fluorescently labeled lip-
opeptides and of a lipopeptide carrying a maleimido group
(for coupling to proteins through conjugate addition of
cysteine thiol groups[3a,b]) was investigated.
By employing the methodology delineated above S-farne-
sylated tetrapeptide 20 incorporating the fluorescent 4-nitro-
benz-2-oxa-1,3-diazole (NBD) group was synthesized
(Scheme 8).After assembly of the peptide chain to give
intermediate 18 and subsequent N-terminal deprotection, the
NBD fluorescent label was coupled to the N terminus.The
resulting labeled compound 20 was released from the solid
support and was obtained in 47% overall yield and with high
purity after simple flash column chromatography.In the
course of this synthesis neither the acid-labile trityl protecting
group nor the reduction-sensitive NBD label and tert-butyl
disulfide masking group were attacked.By using the same
methodology N-Ras peptide 21 incorporating the fluorescent
N-methylanthraniloyl (Mant) label was prepared in 49%
overall yield.In order to demonstrate that not only can
peptide esters be obtained by this solid-phase technique but
that lipidated peptides with an unmasked carboxylic acid at
the C terminus (as, for instance, are required for Rab-derived
peptides; see Scheme 1) are also available, the activated
intermediate formed in the oxidative release of the Mant-
labeled peptide from the solid support was alternatively
trapped with water instead of methanol to give lipopeptide
carboxylic acid 22.
Scheme 7.Synthesis of farnesylated and palmitoylated peptides 16 and 17
which resemble the C terminus of N-Ras.a) 1% TFA, TES, CH 2Cl2; b) Pal-
Cl, HOBt, Et3N, CH2Cl2, DMF; c) PBu3, H2O, DMF, CH2Cl2;
d) Cu(OAc)2, acetic acid, pyridine, O2, CH2Cl2, MeOH.
Basic conditions were applied under argon, and according to
quantification of Fmoc groups by the UV method after the
addition of the second, third, and sixth amino acids no major
loss of peptide was observed.Part of the resin was subjected to
cleavage at the stage of the farnesylated tripeptide 12 to give
Fmoc-Leu-Pro-Cys(Far)-OMe (13) in an overall yield of
50%.After elongation to the heptapeptide 14 and depro-
tection of the second cysteine residue, palmitoylation in
dichloromethane, that is, under conditions which had worked
very well in case of the tripeptide 9, was not successful.This
might be due to the occurrence of peptide aggregation or
secondary structure formation on the solid support.To
overcome this problem, DMF was added to the palmitoyla-
tion mixture and the reaction proceeded smoothly to yield
intermediate 15.Finally, the desired N-Ras peptide methyl
ester 16 was released from the solid support in 42% overall
yield.By means of this procedure multimilligram amounts of
pure lipopeptide are readily obtained within days, whereas the
synthesis of the same compound by means of solution-phase
methods[4c, 13, 14] requires weeks.
In addition, doubly lipidated peptides carrying a tag for
subsequent biological experiments were prepared.To this
end, farnesylated tetrapeptide 24 was assembled on the solid
support (Scheme 9).It incorporates a reduction-sensitive tert-
butyl disulfide as a masking group for the cysteine thiol, an
acid-labile trityl blocking function, and a lysine with an N-
Aloc-protected side-chain amino group.This set of protecting
groups and the hydrazide linker are orthogonally stable.
The tert-butyl disulfide group was cleaved by treatment with
A different set of protection groups had to be used for the
synthesis of the N-terminally trityl-protected N-Ras hepta-
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PBu3/H2O and this was followed by palmitoylation of the
liberated mercapto group.The N-terminal trityl group was
removed from the formed intermediate under weakly acidic
conditions and then the Mant label was introduced by
coupling with Mant-aminocaproic acid, DIC, and HOBt in
the presence of triethylamine to yield immobilized fluores-
cently labeled lipopeptide 25.Finally, desired lipopeptide
methyl ester 26 was released from the solid support by
oxidation with CuII in methanol and in the presence of
pyridine and acetic acid.After flash column chromatography
it was obtained in pure form in 29% overall yield for 13 steps
on the polymeric carrier.
Finally, we investigated the possibility of introducing a
maleinimido group, which is an important linker group for the
coupling of chemically synthesized lipopeptides to expressed
proteins.For instance, it has been shown that lipopeptides
equipped with a maleimido group can react with the mercapto
groups of proteins to yield biologically fully functional Ras
proteins.[3a, b] With tetrapeptide 24 as the starting material,
removal of the tert-butyl disulfide group, subsequent palmi-
toylation of the free thiol, and N-terminal deprotection
(removal of the trityl group) were carried out as described
above.Commercially available maleimidocaproic acid was
coupled to the immobilized peptide with DIC, HOBt, and
triethylamine to yield the resin-bound target peptide.Cleav-
age from the resin by oxidation with CuII in methanol and in
the presence of pyridine and acetic acid furnished the desired
MIC-equipped lipopeptide 27 in 22% overall yield after
column chromatography (Scheme 10).Figure 1 displays the
Scheme 8.Solid-phase synthesis of fluorescently labeled and S-farnesy-
lated peptides 20 22.a) piperidine, DMF; b) HBTU, HOBt, DIEA,
NBDAca-OH (23), DMF; c) Cu(OAc)2, pyridine, O2, CH2Cl2, MeOH.
NBDAca ˆ N-(4-nitrobenz-2-oxa-1,3-diazol-7-yl)aminocaproyl.
Scheme 10.Solid-phase synthesis of MIC-tagged, doubly lipidated peptide
27.a) PBu 3, H2O, DMF, CH2Cl2; b) Pal-Cl, HOBt, Et3N, CH2Cl2, DMF;
c) 1% TFA, TES, CH2Cl2; d) HBTU, HOBt, DIEA, MIC-OH, DMF;
e) Cu(OAc)2, acetic acid, pyridine, O2, CH2Cl2, MeOH.MIC ˆ malein-
imidocaproyl.
HPLC trace of the product.For the peptides discussed above
similar traces were obtained after release from the solid
support.These findings demonstrate that the method detailed
in this paper yields very pure lipidated peptides that require
hardly any further purification.
Conclusion
Scheme 9.Solid-phase synthesis of Mant-labeled and doubly lipidated
peptide 26.a) PBu 3, H2O, DMF, CH2Cl2; b) Pal-Cl, HOBt, Et3N, CH2Cl2,
DMF; c) 1% TFA, TES, CH2Cl2; d) HBTU, HOBt, DIEA, MantAca-OH,
DMF; e) Cu(OAc)2, acetic acid, pyridine, O2, CH2Cl2, MeOH.MantAca ˆ
N-methylanthraniloylaminocaproyl.
We have developed the first solid-phase method for the
synthesis of differently lipidated and additionally labeled
peptides.It gives access to farnesylated, palmitoylated, and
Chem. Eur. J. 2003, 9, 3683 3691
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H.Waldmann and B.Ludolph
FULL PAPER
Peptide coupling: Unless stated otherwise all amino acids were coupled by
using HBTU/HOBt chemistry.Typically, amino acid (4 equiv) was treated
for 2 3 min with HBTU (3.6 equiv), HOBt (4 equiv) and DIEA (8 equiv)
in DMF.The solution was added to the resin and agitated for 2 h at room
temperature.Cysteine derivatives were coupled by using DIC/HOBt
chemistry in order to avoid extensive racemization.[15] Typically amino acid
(4 equiv) was treated with DIC (4 equiv) and HOBt (6 equiv) in DMF for
2
3 min and then added to the resin.Reaction times varied from 3 4 h at
room temperature.
Cleavage of the Trt group from cysteine: The Trt group was cleaved with
3% TES and 50% TFA in dichloromethane for 1 h.The resin was washed
several times with dichloromethane and then with DMF.
Removal of the Trt group from nitrogen atoms/removal of the Mmt group
from cysteine: Cleavage was achieved with 1% TFA and 2% TES in
dichloromethane.In the case of deprotection of a nitrogen atom the
procedure was repeated twice.The resin was washed six times with
dichloromethane and then with DMF.
Figure 1.HPLC trace of compound 27.An RP-C4 column with a water/
acetonitrile gradient and detection at 210 nm was employed.
Removal of the tert-butyl disulfide group from cysteine: The tert-butyl
disulfide group was cleaved with PBu3 (100 equiv) and H2O (400 equiv) in
doubly lipidated peptides as methyl esters or carboxylic acids
carrying an additional fluorescent group or a maleimido
moiety for further biological studies.This new methodology
should substantially facilitate the use of lipidated peptides,
and the lipidated proteins accessible from them by combina-
tion of organic synthesis and molecular biology technique-
s,[3a, b] in the study of important biological phenomena like
biological signal transduction, localization, and vesicular
transport.
DMF/dichloromethane (1:1) for 12 h.Typically 64. mL of PBu
together
3
with 2 mL of H2O were used in 40 mL of solvent.The resin was washed six
times with dichloromethane and then with DMF.
Farnesylation: The farnesyl group was introduced onto the freshly
deprotected thiol with farnesyl bromide (5 equiv) and DIEA (12 equiv)
in DMF for 4 h.The resin was washed six times with DMF.
Palmitoylation: The palmitoyl group was introduced with palmitoyl
chloride (20 equiv), HOBt (20 equiv), and Et3N (22 equiv) in DMF/
dichloromethane (1:3) for 15 h.The resin was washed six times with
dichloromethane and then with DMF.
Fmoc-Cys(Far)-OMe (4): Fmoc-4-hydrazinobenzoyl NovaGel resin was
deprotected with piperidine, and Fmoc-Cys(Trt)-OH was coupled to the
resin by using DIC/HOBt chemistry to give cysteine-bound resin (137 mg)
with a loading of 0.35 mmolgÀ1 (0.047 mmol). The Trt group was cleaved
and the thiol was farnesylated.The resin was treated with a solution of
Cu(OAc)2 (18 mg, 0.1 mmol), pyridine (280 mL, 3.46 mmol), and methanol
(1 mL) in dichloromethane (10 mL) for 2 h under oxygen.The resin was
filtered off, the solvent was evaporated under reduced pressure, and the
crude reaction mixture was dried in vacuo.Flash column chromatography
with ethyl acetate/cyclohexane (1:4) as the eluent furnished 4 (14.5 mg,
Experimental Section
General: Unless otherwise noted, reagents and chemicals were obtained
from Acros, Chimica, Advanced Chemtech, Aldrich, AppliChem, Avoca-
do, Biosolve, Fluka, Novabiochem, or Senn Chemicals and used without
further purification.Dichloromethane and piperidine were refluxed under
argon over CaH2 and freshly distilled prior to use. 1H and 13C NMR
spectroscopic data were recorded on a Varian Mercury 400 or a Bruker
DRX500 spectrometer at room temperature. 1H and 13C NMR spectra
were afterwards calibrated to the solvent signals of CDCl3 (d ˆ 7.26 ppm
and 77.16 ppm, respectively). ESI-MS was carried out by using a Agilent
1100 series binary pump together with a reversed-phase HPLC column
(Macherey-Nagel) and a Finnigan Thermoquest LCQ.FAB MS measure-
ments were taken with a Jeol SX102A apparatus by using a 3-nitrobenzyl
alcohol (3-NBA) matrix.Optical rotations were measured with a Perkin
Elmer Polarimeter 341.Flash chromatography was performed with Merck
0.026 mmol, 55%). Rf ˆ 0.35 (cyclohexane/ethyl acetate (4:1)); [a]D20
ˆ
À48.4 (c ˆ 0.5 in CHCl3); 1H NMR (400 MHz, CDCl3): d ˆ 7.76 (d, 3J ˆ
7.4 Hz, 2H; Fmoc), 7.62 7.60 (m, 2H; Fmoc), 7.40 (t, 3J ˆ 7.2 Hz, 2H;
3
3
Fmoc), 7.32 (t, J ˆ 7.4 Hz, 2H; Fmoc), 5.59 (d, J ˆ 8.2 Hz, 1H; NH), 5.21
3
ˆ
À
ˆ
(t, J ˆ 7.7 Hz, 1H; C CH CH2S), 5.10 5.07 (m, 2H; 2  C CH Far),
4.62 4.58 (m, 1H; aCH Cys), 4.35 4.45 (m, 2H; CH2 Fmoc), 4.22 4.26
(m, 1H; CH Fmoc), 3.78 (s, 3H; OCH3), 3.21 3.12 (m, 2H; CH2 Far),
2.85 3.00 (m, 2H; bCH2 Cys), 2.12 1.95 (m, 8H; CH2 Far), 1.68 (s, 3H;
CH3 Far), 1.66 (s, 3H; CH3 Far), 1.60 (s, 6H; CH3 Far) ppm; 13C NMR
(125 MHz, CDCl3): d ˆ 171.4, 155.8, 143.9, 141.3, 140.2, 135.4, 131.4, 127.8,
127.2, 125.2, 124.4, 123.7, 120.0, 119.6, 67.4, 53.8, 52.8, 47.3, 40.0, 38.9, 33.8,
30.3, 26.9, 26.7, 25.9, 17.9, 16.4, 16.3 ppm; MS (ESI ‡): m/z calcd for
silica gel 60.TLC was performed with aluminium-backed silica gel 60 F
254
plates (Merck).MALDI MS was carried out with a Voyager-DE Pro
BioSpectrometer from PerSeptive Biosystems by using a 2,5-dihydrox-
ybenzoic acid (DHB) matrix.The yield and scale of the solid-phase
reactions are given with respect to the amount of the first amino acid
coupled onto the resin and the resin loading was determined by measuring
the Fmoc groups remaining on the resin by the established UV method.
After cleavage from the resin no major byproducts could be detected by
HPLC.However, column chromatography was carried out to remove
copper salts.
‡
C34H44NO4S [M ‡ H] : 562.3; found: 562.2; MS (FAB, 3-NBA): calcd for
‡
[M ‡ H] : 562.2991; found: 562.3004.
Fmoc-Cys(Pal)-OMe (5): The Trt group was cleaved from resin-bound
Fmoc-Cys(Trt) (210 mg, with a loading of 0.38 mmolgÀ1, 0.080 mmol), and
the thiol function was palmitoylated.The resin was treated with a solution
of Cu(OAc)2 (6 mg, 0.033 mmol), pyridine (123 mL, 1.52 mmol), acetic acid
(175 mL, 3.06 mmol), and MeOH (400 mL, 9.88 mmol) in dry dichloro-
methane (20 mL) for 2 h under oxygen.The resin was filtered off, the
solvent was evaporated under reduced pressure, and the crude reaction
mixture was dried in vacuo.Flash column chromatography with ethyl
acetate/cyclohexane (1:2) as the eluent furnished 5 (26.7 mg, 0.045 mmol,
56%). Rf ˆ 0.56 (ethyl acetate/cyclohexane (1:2)); [a]2D0 ˆ ‡18.9 (c ˆ 1.04 in
General conditions for the synthesis of lipopeptides with the phenylhydra-
zide linker: For all reactions commercially available Fmoc-4-hydrazinoben-
zoyl NovaGel resin from Novabiochem was used.All reactions were carried
out under an argon atmosphere in a 50-mL solid-phase peptide synthesis
reactor.Agitation was achieved by bubbling argon gas through the glass
sinter or by using an orbital shaker.After liberation of the thiol group of
cysteine the subsequent reaction was carried out without any delay.Loading
of the resin was determined by the amount of Fmoc groups on the resin.To
this end, a small amount of dried resin (5 9 mg) was treated with freshly
prepared piperidine/DMF (1:4; 18 mL) for 10 min and then the UV
absorption of the solution at 301 nm (e ˆ 7800 mÀ1 cmÀ1) was determined.
1
3
CHCl3); H NMR (400 MHz, CDCl3): d ˆ 7.76 (d, J ˆ 7.3 Hz, 2H; Fmoc),
7.60 (d, 3J ˆ 7.2 Hz, 2H; Fmoc), 7.40 (t, 3J ˆ 7.6 Hz, 2H; Fmoc), 7.31 (t, 3J ˆ
7.5 Hz, 2H; Fmoc), 5.56 (s, 1H; NH), 4.61 (m, 1H; aCH Cys), 4.38 (d, 3J ˆ
7.0 Hz, 2H; CH2O Fmoc), 4.24 (t, 3J ˆ 7.0 Hz, 1H; CH Fmoc), 3.77 (s, 3H;
OMe), 3.39 (m, 2H; bCH2 Cys), 2.57 (t, 3J ˆ 7.4 Hz, 2H; aCH2 Pal), 1.65 (m,
2H; bCH2 Pal), 1.25 (m, 24H; Pal), 0.88 (t, 3J ˆ 6.8 Hz, 3H; wCH3
Pal) ppm; 13C NMR (100 MHz, CDCl3): d ˆ 199.0, 170.9, 155.9, 144.0,
Fmoc cleavage: Fmoc cleavage was achieved by using a solution of 50%
piperidine in DMF two times for 7 min.
¹ 2003 Wiley-VCH Verlag GmbH & Co.KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2003, 9, 3683 3691
3688

Acid- and Base-Labile Lipopeptides
3683 3691
141.6, 127.9, 127.3, 125.4, 120.2, 67.5, 53.9, 53.0, 47.3, 44.3, 32.2 29.1 (several
coupling of Fmoc-Leu-OH to give the resin-bound tripeptide 12.The resin
was treated with a solution of Cu(OAc)2 (6.7 mg, 0.037 mmol), pyridine
(119 mL, 1.47 mmol), acetic acid (170 mL, 2.97 mmol), and MeOH (374 mL,
9.2 mmol) in dichloromethane (15 mL) for 2 h under oxygen. The resin was
filtered off, the solvent was evaporated under reduced pressure, and the
crude reaction mixture was dried in vacuo.Flash column chromatography
with ethyl acetate/cyclohexane (1:1) as the eluent furnished 13 (15.8 mg,
signals), 25.8, 22.9, 14.2 ppm; MS (ESI ‡): m/z calcd for C35H50NO5S [M ‡
‡
‡
H] : 596.3; found: 596.1; MS (FAB, 3-NBA): calcd for [M ‡ H] : 596.3410;
found: 596.3419.
Fmoc-Cys(Pal)-Lys(Aloc)-Cys(Far)-OMe (9): Fmoc-Lys(Aloc)-OH was
coupled to resin-bound Fmoc-Cys(Trt) (488 mg, with
a loading of
0.38 mmolgÀ1, 0.185 mmol) by using HBTU/HOBt chemistry. The Trt
group was cleaved and the free thiol was farnesylated.The Fmoc group was
removed and Fmoc-Cys(Mmt)-OH was coupled onto the dipeptide by
using DIC/HOBt chemistry.The Mmt group was cleaved and the free thiol
0.021 mmol, 50%). Rf ˆ 0.4 (cyclohexane/ethyl acetate (1:1)); [a]D20
ˆ
À102.4 (c ˆ 0.41 in CHCl3); 1H NMR (400 MHz, CDCl3): d ˆ 7.75 (d,
3J ˆ 7.6 Hz, 2H; Fmoc), 7.59 (m, 2H; Fmoc), 7.39 (t, 3J ˆ 7.4 Hz, 2H; Fmoc),
7.31 (t, 3J ˆ 7.4 Hz, 2H; Fmoc), 5.48 (d, 3J ˆ 9.0 Hz, 1H; NH), 5.19, (t, 3J ˆ
was palmitoylated.The resin was treated with a solution of Cu(OAc)
2
ˆ
À
ˆ
7.6 Hz, 1H; C CH CH2S Far), 5.08 (m, 2H; 2 Â C CH Far), 4.70 4.65 (m,
1H; aCH), 4.60 4.56 (m, 1H; aCH), 4.37 4.34 (m, 2H; CH2 Fmoc),
4.22 4.19 (m, 1H; CH Fmoc), 3.50 3.80 (m, 5H; OMe, CH2 Pro), 3.18
3.08 (m, 2H; CH2 Far), 2.94 2.97 (m, 1H; bCH2a Cys), 2.80 2.75 (m, 1H;
bCH2b Cys), 2.20 1.10 (m, 27H; gCH Leu, bCH2 Leu, 4 Â CH2 Far, 2 Â
CH2 Pro, 4 Â CH3 Far), 1.01 0.94 (m, 6H; 2 Â dCH3 Leu) ppm; 13C NMR
(100 MHz, CDCl3): d ˆ 173.2, 171.3, 171.0, 156.5, 144.1, 141.5, 140.2, 135.6,
131.5, 129.7, 127.9, 127.3, 125.4, 124.5, 124.0, 120.2, 119.8, 67.3, 60.0, 52.7,
52.0, 51.1, 47.4, 42.7, 42.4, 39.9, 33.5, 30.0, 27.4, 27.0, 26.7, 25.9, 25.2, 24.9, 23.7,
21.8, 17.9, 16.4, 16.2 ppm; MS (ESI ‡): m/z calcd for C45H62N3O6S [M ‡
(7.9 mg, 0.043 mmol), pyridine (140 mL, 1.74 mmol), acetic acid (200 mL,
3.50 mmol), and MeOH (441 mL, 10.9 mmol) in dry dichloromethane
(20 mL) for 2 h under oxygen.The resin was filtered off, the solvent was
evaporated under reduced pressure, and the crude reaction mixture was
dried in vacuo.Flash column chromatography with ethyl acetate/cyclo-
hexane (1:1) as the eluent furnished 9 (51.8 mg, 0.047 mmol, 25%). Rf ˆ
0.45 (cyclohexane/ethyl acetate (1:1)); [a]2D0 ˆ ‡46.8 (c ˆ 2.06 in CHCl3);
1H NMR (400 MHz, CDCl3): d ˆ 7.68 (d, 3J ˆ 7.6 Hz, 2H; Fmoc), 7.52 (d,
3J ˆ 9.6 Hz, 2H; Fmoc), 7.32 (t, 3J ˆ 7.4 Hz, 2H; Fmoc), 7.23 (t, 3J ˆ 7.4 Hz,
2H; Fmoc), 6.94 (s, 1H; NH), 6.82 (s, 1H; NH), 5.83 5.75 (m, 2H; NH,
‡
‡
H] : 772.4; found: 772.3; MS (FAB, 3-NBA): calcd for [M ‡ H] : 772.4359;
CH CH2), 5.19 (d, 3J ˆ 18 Hz, 1H; CH CH2a), 5.15 4.97 (m, 5H;
ˆ
ˆ
found: 772.4387.
ˆ
ˆ
À
ˆ
CH CH2b,C CH CH2S, 2 Â C CH Far, NH), 4.66 (m, 1H; aCH Cys),
À
4.50 4.24 (m, 6H; Fmoc, aCH Lys, O CH2 allyl), 4.14 (m, 1H; aCH Cys),
Aloc-Gly-Cys(Pal)-Met-Gly-Leu-Pro-Cys(Far)-OMe (16): Resin-bound
tripeptide Fmoc-Leu-Pro-Cys(Far) 12 was synthesized from resin-bound
Fmoc-Cys(Trt) (302 mg, with a loading of 0.35 mmolgÀ1, 0.106 mol) as
described above.The Fmoc group was cleaved and this was followed by
subsequent HBTU/HOBt-mediated coupling with Fmoc-Gly-OH, Fmoc-
Met-OH, Fmoc-Cys(Mmt)-OH, and Aloc-Gly-OH.The Mmt group was
removed and the thiol was palmitoylated.The resin was treated with a
solution of Cu(OAc)2 (20 mg, 0.11 mmol), pyridine (280 mL, 3.47 mmol),
acetic acid (400 mL, 6.99 mmol), and MeOH (882 mL, 21.7 mmol) in
dichloromethane (20 mL) for 2 h under oxygen.The resin was filtered
off, the solvent was evaporated under reduced pressure, and the crude
reaction mixture was dried in vacuo.Flash column chromatography with
dichloromethane/methanol (20:1) as the eluent furnished 16 (53.3 mg,
3.67 (s, 3H; OMe), 3.30 2.75 (m, 8H; 2 Â bCH2 Cys, bCH2 Lys, aCH2 Far),
3
2.51 (t, J ˆ 7.4 Hz, 2 H ; aCH2 Pal), 2.1 1.81 (m, 8H; CH2 Far), 1.68 1.59
(m, 2H; bCH2 Pal ), 1.60 (s, 3H; CH3 Far), 1.58 (s, 3H; CH3 Far), 1.52 (s,
6H; 2 Â CH3 Far), 1.46 1.43 (m, 2H; dCH2 Lys), 1.36 1.33 (m, 2H; gCH2
Lys), 1.18 (s, 24H; Pal), 0.81 (t, 3J ˆ 7.0 Hz, 3H; wCH3 Pal) ppm; 13C NMR
(100 MHz, CDCl3): d ˆ 200.6, 171.3, 171.2, 170.3, 156.7, 144.0, 141.5, 140.4,
135.6, 133.3, 131.5, 128.0, 127.3, 125.4, 124.5, 124.0, 120.2, 119.7, 117.7, 67.8,
65.7, 55.8, 53.3, 52.9, 52.0, 47.3, 44.3, 40.5, 39.9, 33.1 22.3 (several signals),
17.9, 16.4, 16.2, 14.3 ppm; MS (ESI ‡): m/z calcd for C63H94N4O9S2 [M ‡
‡
‡
H] : 1115.7; found: 1115.3; MS (FAB, 3-NBA): calcd for [M ‡ H] : 1115.7;
[M ‡ Na]: 1137.6; found: 1115.4, 1137.4.
Fmoc-Thr-Lys(Aloc)-Cys(Far)-OMe (11): Fmoc-Lys(Aloc)-OH was cou-
0.044 mmol, 42%). Rf ˆ 0.2 (dichloromethane/MeOH (20:1)); [a]D20
ˆ
pled to resin-bound Fmoc-Cys(Trt) (332 mg, with
a loading of
À52.8 (c ˆ 0.74 in CHCl3); 1H NMR (500 MHz, CDCl3): d ˆ 7.96 (s, 1H;
0.37 mmolgÀ1, 0.123 mmol) by using HBTU/HOBt chemistry. The Trt
group was cleaved and the free thiol was farnesylated.The Fmoc protection
group was removed and Fmoc-Thr(Trt)-OH was attached; this was
followed by cleavage of the Trt protecting group of the hydroxy moiety.
The resin was treated with a solution of Cu(OAc)2 (10 mg, 0.055 mmol),
pyridine (280 mL, 3.47 mmol), acetic acid (409 mL, 7.15 mmol), and MeOH
(1 mL, 24.7 mmol) in dry dichloromethane (20 mL) for 2 h under oxygen.
The resin was filtered off, the solvent was evaporated under reduced
pressure, and the crude reaction mixture was dried in vacuo.Flash column
chromatography with dichloromethane/methanol (20:1) furnished 11
(49.6 mg, 0.057 mmol, 46%). The route with the tert-butyl disulfide
protecting group was analogous except that farnesylation was carried out
at the stage of the tripeptide. Rf ˆ 0.4 (dichloromethane/methanol (20:1)).
[a]2D0 ˆ À20.7 (c ˆ 0.99 in CHCl3); 1H NMR (400 MHz, CDCl3): d ˆ 7.76 (d,
3J ˆ 7.3 Hz, 2H; Fmoc), 7.60 (d, 3J ˆ 7.2 Hz, 2H; Fmoc), 7.40 (t, 3J ˆ 7.6 Hz,
NH), 7.60 (s, 1H; NH), 7.51 (s, 1H; NH), 7.34 (s, 1H; NH), 7.05 (s, 1H; NH),
3
ˆ
ˆ
5.88 5.84 (s, 1H; CH CH2 allyl), 5.31 (d, J ˆ 17.4 Hz, 1H; CH CH2a
3
ˆ
allyl), 5.21 (d, J ˆ 10.4 Hz, 1H; CH CH2b allyl), 5.19 5.16 (m, 1H;
ˆ
À
C CH CH2S), 5.08 (m, 2H; 2  C ˆ CH Far), 4.8 4.36 (m, 11H; 5  aCH,
À
O CH2 allyl, 2 Â CH2 Gly), 3.5 4.0 (m, 7H; CH2 Pro, OMe, bCH2 Cys),
3.36 2.76 (m, 4H; CH2 Far, bCH2 Cys), 2.54 (m, 4H; gCH2 Met, aCH2
Pal), 2.3 1.8 (m, 17H; 4 Â CH2 Far, 2 Â CH2 Pro, gCH2 Met, SCH3), 1.70
1.30 (m, 17H; gCH Leu, bCH Leu, 4 Â CH3 Far, bCH2 Pal), 0.90 1.20 (m,
24H; Pal), 0.88 0.70 (m, 9H; wCH3 Pal, 2 Â dCH3 Leu) ppm; 13C NMR
(100 MHz, CDCl3): d ˆ 200.5, 173.2, 172.6, 171.6, 171.2, 170.5, 169.6, 196.0,
157.4, 140.3, 135.6, 132.8, 131.5, 124.5, 123.9, 119.8, 118.1, 66.3, 60.1, 53.9,
53.0, 52.7, 52.0, 49.3, 47.6, 45.0, 44.3, 43.4, 42.0, 41.2, 33.3 24.0 (several
signals), 17.9, 16.4, 16.2, 16.0, 14.3 ppm; MS (ESI ‡): m/z calcd for
‡
C62H106N7O11S3 [M ‡ H] : 1220.7; found: 1220.5; MS (MALDI, DHB):
‡
‡
3
calcd for [M ‡ Na] : 1242.7; [M ‡ K] : 1258.8; found: 1243.1, 1259.0.
2H; Fmoc), 7.31 (t, J ˆ 7.5 Hz, 2H; Fmoc), 7.05 (s, 1H; NH), 6.99 (s, 1H;
NH), 5.89 5.87 (m, NH, 2H; CH CH2 allyl), 5.27 (d, 3J ˆ 18 Hz, 1H;
ˆ
Trt-Gly-Cys(Pal)-Met-Gly-Leu-Pro-Cys(Far)-OMe (17): Fmoc-Pro-OH
and Fmoc-Leu-OH were coupled to resin-bound Fmoc-Cys(Trt) (127 mg,
with a loading of 0.34 mmolgÀ1, 0.043 mmol). The Trt group was cleaved
and the thiol farnesylated to give 12.The peptide was elongated with Fmoc-
Gly-OH, Fmoc-Met-OH, Fmoc-Cys(StBu)-OH, and Trt-Gly-OH by using
Fmoc chemistry.The tert-butyl disulfide group was cleaved and the thiol
ˆ
ˆ
ˆ
À
CH CH2a allyl), 5.20 5.17 (m, 2H; CH CH2b allyl, C CH CH2S), 5.10
ˆ
5.07 (m, 2H; C CH Far), 4.98 (s, 1H; NH), 4.73 4.72 (m, 1H; aCH Cys),
4.54 4.21 (m, 8H; Fmoc, 2 Â aCH, bCH Thr, O CH2 allyl), 3.75 (s, 3H;
À
OMe), 3.19 3.10 (m, 4H; aCH2 Far, CH2 eLys), 2.97 2.92 (m, 1H; bCH2
Cys), 2.85 2.80 (m, 1H; bCH2 Cys), 2.10 1.91 (m, 8H; 4 Â CH2 Far), 1.68
(s, 3H; CH3 Far), 1.66 (s, 3H; CH3 Far), 1.59 (s, 6H; 2 Â CH3 Far), 1.51 1.36
(m, 4H; dCH2, gCH2 Lys), 1.19 (d, 3J ˆ 6.3 Hz, 3H; CH3 Thr) ppm;
13C NMR (100 MHz, CDCl3): d ˆ 171.6, 171.4, 171.1, 156.9, 143.9, 141.5,
140.4, 135.6, 133.2, 131.6, 128.0, 127.3, 125.3, 124.5, 123.9, 120.2, 119.6, 117.9,
67.5, 65.8, 59.1, 53.6, 53.4, 52.9, 52.0, 47.3, 40.4, 40.0, 33.2, 31.4, 30.0, 29.5,
27.0, 26.7, 25.9, 22.4, 18.7, 17.9, 16.4, 16.2 ppm; MS (ESI ‡): m/z calcd for
was palmitoylated.The resin was treated with a solution of Cu(OAc)
2
(18 mg, 0.10 mmol), pyridine (280 mL, 3.47 mmol), acetic acid (228 mL,
3.99 mmol), and MeOH (1 mL, 24.7 mmol) in dry dichloromethane
(20 mL) for 2 h under oxygen.The resin was filtered off, the solvent was
evaporated under reduced pressure, and the crude reaction mixture was
dried in vacuo.Flash column chromatography with dichloromethane/
methanol (20:1) as the eluent furnished 17 (40.6 mg, 0.029 mmol, 68%).
Rf ˆ 0.35 (dichloromethane/MeOH (20:1)); [a]2D0 ˆ À36.1 (c ˆ 0.76 in
CHCl3); 1H NMR (400 MHz, CDCl3): d ˆ 7.4 7.1 (m, 15H; Trt), 5.10
‡
‡
C48H67N4O9S [M ‡ H] : 875.5; [M ‡ Na] : 897.5; found: 875.2, 897.4; MS
‡
(FAB, 3-NBA): calcd for [M ‡ H] : 875.4629; found: 875.4617.
Fmoc-Leu-Pro-Cys(Far)-OMe (13): The Fmoc group was cleaved from
resin-bound Fmoc-Cys(Trt) (119 mg, with a loading of 0.35 mmolgÀ1
,
4.95 (m, 3H; 3 Â C CH Far), 4.8 4.1 (m, 8H; 4 Â aCH, 2 Â aCH2 Gly),
ˆ
0.0042 mmol) and Fmoc-Pro-OH was coupled. The Trt group was removed
and the dipeptide was farnesylated; this was followed by deprotection and
3.6 3.3 (m, 7H; CH2 Pro, OMe, bCH2 Cys), 3.3 2.7 (m, 4H; CH2 Far,
bCH2 Cys), 2.4 2.6 (m, 4H; gCH2 Met, aCH2 Pal), 2.2 1.6 (m, 17H; 4 Â
Chem. Eur. J. 2003, 9, 3683 3691
www.chemeurj.org
¹ 2003 Wiley-VCH Verlag GmbH & Co.KGaA, Weinheim
3689

H.Waldmann and B.Ludolph
FULL PAPER
3
1H; aCH2b Far), 2.85 2.89 (m, 1H; bCH2a Cys), 2.79 (d, J ˆ 4.9 Hz, 3H;
CH2 Far, 2 Â CH2 Pro, gCH2 Met, SCH3), 1.6 1.3 (m, 17H; gCH Leu, bCH
Leu, 4 Â CH3 Far, bCH2 Pal), 1.2 0.95 (m, 24H; Pal), 0.9 0.6 (m, 9H;
wCH3 Pal, 2  dCH3 Leu) ppm; MS (ESI ‡): m/z calcd for C77H116N7O9S3
NCH3), 2.67 2.64 (m, 3H; bCH2b Cys, gCH2 Met), 2.48 2.45 (m, 2H;
eCH2 Aca), 2.20 2.16 (m, 3H; CH2 Pro, CH2a Pro), 2.10 1.90 (m, 15H;
bCH2 Met, CH3S Met, gCH, Leu, 4 Â CH2 Far, CH2b Pro), 1.63 1.30 (m,
20H; bCH2 Aca, gCH2 Aca, dCH2 Aca, 4 Â CH3 Far, bCH2 Leu), 0.90 0.87
(m, 6H; 2  CH3 Leu) ppm; 13C NMR (100 MHz, CDCl3): d ˆ 173.5, 172.4,
172.0, 171.5, 171.3, 170.1, 168.6, 150.6, 140.1, 135.5, 132.7, 131.4, 127.6, 124.4,
123.8, 119.7, 115.6, 114.5, 111.0, 60.0, 52.5, 52.3, 52.1, 49.1, 47.5, 43.1, 41.7,
39.8, 39.5, 36.1, 32.9, 31.8, 30.1, 29.8, 29.7, 29.2, 28.4, 26.8, 26.6, 26.5, 25.8,
25.2, 24.9, 24.7, 23.4, 22.0, 17.8, 16.2, 16.1, 15.4 ppm; MS (ESI ‡): m/z calcd
‡
‡
[M ‡ H] : 1378.8; [M ‡ Na] : 1400.8; found: 1378.8, 1400.7; MS (FAB,
‡
3-NBA): calcd for [M ‡ Na] : 1400.8; found: 1400.1.
N-(4-Nitrobenz-2-oxa-1,3-diazol-7-yl)aminocaproic acid (23): DIEA
(850 ml, 5.00 mmol) and slowly, over an hour, solid aminocaproic acid
(315 mg, 2.40 mmol) were added to a solution of NBD-Cl (401 mg,
2.01 mol) in methanol (20 mL) at 08C.The reaction mixture was stirred
overnight at room temperature.The solvent was evaporated and the
remaining material was purified by chromatography on silica with ethyl
acetate/MeOH (10:1) as the eluent.The substance was recrystallized from
methanol/water to give 23 (339 mg, 1.15 mmol, 57%). 1H NMR (400 MHz,
DMSO): d ˆ 9.4 (s, 1H; COOH), 8.41 (d, 3J ˆ 8.8 Hz, 1H; CH NBD), 6.32
(d, 3J ˆ 8.8 Hz, 1H; CH NBD), 3.41 (m, 2H; CH2 Aca), 2.18 (t, 3J ˆ 7.4 Hz,
2H; CH2 Aca), 1.65 (m, 2H; CH2 Aca), 1.53 (m, 2H; CH2 Aca), 1.35 (m,
2H; CH2 Aca) ppm; 13C NMR (100 MHz, DMSO): d ˆ 175.0, 145.7, 145.0,
144.7, 138.5, 121.1, 99.6, 49.2, 34.2 28.0, 26.6, 24.8; MS (ESI ‡): m/z calcd for
‡
for C51H82N7O8S2 [M ‡ H] : 984.6; found: 984.6; MS (FAB, 3-NBA): calcd
‡
for [M ‡ H] : 984.5666; found: 984.5728.
MantAca-Met-Gly-Leu-Pro-Cys(Far)-OH (22): Synthesis was carried out
as described above for compound 21 with resin-bound Fmoc-Cys(Trt)
(239 mg,with a loading of 0.39 mmolgÀ1, 0.093 mmol). MantAca-OH was
coupled by using DIC/HOBt chemistry.The resin was treated with a
solution of Cu(OAc)2 (10 mg, 0.055 mmol), pyridine (280 mL, 3.47 mmol),
and acetic acid (400 mL, 6.99 mmol) in THF (10 mL) containing H2O
(350 mL, 19.4 mmol) for 2 h under oxygen. The resin was filtered off, the
solvent was evaporated under reduced pressure, and the crude reaction
mixture was dried in vacuo.A solution of the crude product in dichloro-
methane was absorbed onto trisaminoethyl HL resin (200 mg, Novabio-
chem) and shaken for 2 h.The resin was washed with dichloromethane/
H2O then the product was carefully eluted with THF/H2O (20:1) followed
by THF/H2O/AcOH (20:1:0.5) and dried in vacuo to give 22 (28 mg,
0.029 mmol, 31%). [a]2D0 ˆ À31.5 (c ˆ 1.3 in CHCl3); 1H NMR (400 MHz,
CDCl3): d ˆ 7.38 (d, 3J ˆ 7.8 Hz, 1H; Mant), 7.29 7.26 (m, 1H; Mant), 7.06
‡
C12H15N4O5 [M ‡ H] : 295.10; found: 295.2; MS (ESI À): m/z calcd for
‡
[M À H]À: 293.10; found: 293.2; MS (FAB, 3-NBA): calcd for [M ‡ H] :
295.1042; found: 295.1042.
NBDAca-Ser(Trt)-Cys(StBu)-Lys(Aloc)-Cys(Far)-OMe (20): The Fmoc
group was cleaved from resin-bound Fmoc-Cys(Trt) (269 mg, with a
loading of 0.38 mmolgÀ1
, 0.102 mmol) and Fmoc-Lys(Aloc)-OH was
coupled.The Trt group was removed and the liberated thiol group was
farnesylated.Subsequently, Fmoc-Cys(S tBu)-OH, Fmoc-Ser(Trt)-OH, and
NBDAca-OH were coupled.The resin was treated with a solution of
Cu(OAc)2 (7.8 mg, 0.043 mmol), pyridine (140 mL, 1.74 mmol), and MeOH
(441 mL, 10.9 mmol) in dichloromethane (20 mL) for 80 min under oxygen.
The resin was filtered and the crude mixture was directly subjected to flash
column chromatography with ethyl acetate/cyclohexane (1:5) and 2% Et3N
as the eluent to give 20 (64.5 mg, 0.048 mmol, 47%). Rf ˆ 0.5 (dichloro-
methane/MeOH (20:1)); [a]2D0 ˆ À55.1 (c ˆ 0.118 in CHCl3); 1H NMR
3
(m, 1H; NH), 6.75 (m, 1H; NH), 6.62 (d, J ˆ 8.4 Hz, 1H; Mant), 6.54 (t,
3
ˆ
À
J ˆ 7.2 Hz, 1H; Mant), 5.15 (m, 1H; C CH CH2S), 5.07 (m, 2H; 2 Â
ˆ
CH C, Far), 4.76 4.31 (m, 5H; 5 Â aCH), 4.0 4.38 (m, 2H; 4 Â aCH
Gly), 3.72 (m, 1H; CH2a Pro), 3.56 (m, 1H; CH2b Pro), 3.33 (m, 2H; aCH2
Aca), 3.31 2.91 (m, 4H; aCH2 Far, bCH2 Cys), 2.86 2.66 (m, 5H; NCH3,
gCH2 Met), 2.50 (m, 2H; eCH2 Aca), 2.26 1.86 (m, 15H; 2 Â CH2 Pro,
CH3S Met, 4 Â CH2 Far), 1.66 1.57 (m, 21H; bCH2 Aca, gCH2 Aca, dCH2
Aca, 4 Â CH3 Far, bCH2 Leu, gCH Leu), 0.87 (m, 6H; 2 Â CH3 Leu) ppm;
3
(400 MHz, CDCl3): d ˆ 8.36 (d, J ˆ 8.6 Hz, 1H; CH NBD), 7.45 7.06 (m,
‡
19H; Trt, 4 Â NH), 6.28 (m, 1H; CH NBD), 6.07 (s, 1H; NH), 5.79 (m, 1H;
MS (ESI ‡): m/z calcd for C50H80N7O8S2 [M ‡ H] : 970.5; found: 970.5; MS
ˆ
ˆ
CH CH2 allyl), 5.34 (s, 1H; NH), 5.19 5.00 (m, 7H; CH CH2 allyl,
(ESI À): m/z calcd for [M À H]À: 968.5; found: 968.5; MS (FAB, 3-NBA):
‡
ˆ
À
ˆ
C CH CH2S, 2 Â C CH Far, 2 Â aCH), 4.72 4.59 (m, 2H; 2 Â aCH),
calcd for [M ‡ Na] : 992.5329; found: 992.5376.
À
4.45 4.35 (m, 4H; 2 Â aCH, O CH2 allyl), 3.80 3.55 (m, 4H; OMe, bCH2a
MantAca-Gly-Cys(Pal)-Lys(Aloc)-Cys(Far)-OMe (26): The Fmoc group
was cleaved from resin-bound Fmoc-Cys(Trt) (249 mg, with a loading of
0.43 mmolgÀ1, 0.107 mmol) and Fmoc-Lys(Aloc)-OH was coupled. The Trt
group was removed and the liberated thiol group was farnesylated.
Subsequently Fmoc-Cys(StBu)-OH and Trt-Gly were coupled by using
standard HBTU/HOBt chemistry.The tert-butyl disulfide group was
cleaved and the liberated thiol was palmitoylated.The Trt group was
cleaved from the glycine residue and MantAca-OH was coupled (111 mg,
0.42 mmol) by treatment with DIC (61.9 mL, 0.40 mmol), HOBt (97 mg,
0.64 mmol), and Et3N (15 mL, 0.11 mmol) for 4 h. The resin was treated
Ser), 3.4 3.6 (m, 2H; aCH2 Aca), 3.35 3.04 (m, 7H; bCH2b Ser, aCH2 Far,
bCH2 Cys, eCH2 Lys), 2.85 2.84 (m, 1H; bCH2 Cys), 2.76 2.71 (m, 1H;
bCH2 Cys), 2.18 2.16 (m, 2H; eCH2 Aca), 1.99 1.86 (m, 8H; 4 Â CH2 Far),
1.71 1.30 (m, 24H; bCH2 Lys, gCH2 Lys, dCH2 Lys, 4 Â CH3 Far, bCH2
Aca, gCH2 Aca, dCH2 Aca), 1.21 (s, 9H; StBu) ppm; 13C NMR (100 MHz,
CDCl3): d ˆ 174.0, 171.5, 171.4, 170.6, 170.0, 156.8, 144.5, 144.3, 143.6, 143.4,
140.2, 136.8, 135.6, 133.2, 131.5, 128.7, 128.4, 127.7, 124.5, 123.9, 119.8, 117.6,
98.7, 87.7, 65.7, 63.0, 54.9, 54.9, 53.9, 52.8, 52.1, 49.1, 41.6, 40.7, 39.9, 36.0, 33.1,
31.2, 30.0, 29.8, 29.6, 28.2, 27.0, 26.7, 26.6, 25.9, 24.9, 24.8, 23.0, 17.9, 16.4,
16.2 ppm; MS (ESI À): m/z calcd for C70H92N9O12S3 [M À H]À: 1346.6;
[M ‡ Cl]À: 1382.6; found: 1346.5, 1382.5.
with
a solution of Cu(OAc)2 (18 mg, 0.10 mmol), pyridine (280 mL,
3.47 mmol), acetic acid (400 mL, 6.99 mmol), and MeOH (882 mL,
21.7 mmol) in dichloromethane (20 mL) for 2 h under oxygen. The resin
was filtered off, the solvent was evaporated under reduced pressure, and
the crude reaction mixture was dried in vacuo.Flash column chromatog-
raphy with dichloromethane/methanol (30:1) furnished 26 (36.7 mg,
MantAca-Met-Gly-Leu-Pro-Cys(Far)-OMe (21): The Fmoc group was
cleaved from resin-bound Fmoc-Cys(Trt) (517 mg, with a loading of
0.38 mmolgÀ1, 0.196 mmol) and Fmoc-Pro-OH was coupled. The Trt group
was removed and the liberated thiol group was farnesylated.Subsequently
Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Met-OH, and MantAca-OH were
coupled by using standard HBTU/HOBt chemistry.The resin was treated
with a solution of Cu(OAc)2 (7.8 mg, 0.043 mmol), pyridine (140 mL,
1.74 mmol), and MeOH (441 mL, 10.9 mmol) in dichloromethane (20 mL)
for 2 h under oxygen.The resin was filtered off, the solvent was evaporated
under reduced pressure, and the crude reaction mixture was dried in vacuo.
Flash column chromatography with dichloromethane/methanol (20:1) as
the eluent furnished 21 (95 mg, 0.097 mmol, 49%). Rf ˆ 0.3 (dichloro-
methane/MeOH (20:1)); [a]2D0 ˆ À37.6 (c ˆ 2.5 in CHCl3); 1H NMR
(400 MHz, CDCl3): d ˆ 7.81 (m, 1H; NH), 7.53 (m, 1H; NH), 7.43 7.39
(m, 2H; NH), 7.34 (d, 3J ˆ 7.8 Hz, 1H; Mant), 7.22 (t, 3J ˆ 7.8 Hz, 1H;
Mant), 6.87 6.85 (m, 1H; NH), 6.73 (m, 1H; NH), 6.59 (d, 3J ˆ 8.4 Hz, 1H;
Mant), 6.50 (t, 3J ˆ 7.0 Hz, 1H; Mant), 5.12 (t, 3J ˆ 7.4 Hz, 1H;
0.031 mmol, 29%). Rf ˆ 0.3 (dichloromethane/MeOH (20:1)); [a]D20
ˆ
À19.3 (c ˆ 1.6 in CHCl3); 1H NMR (400 MHz, CDCl3): d ˆ 7.35 (d, 3J ˆ
6.6 Hz, 1H; Mant), 7.28 (d, 3J ˆ 8.6 Hz, 1H; Mant), 6.62 (m, 1H; Mant),
ˆ
6.56 6.52 (m, 1H; Mant), 5.88 5.81 (m, 1H; CH CH2 allyl), 5.25 5.07
ˆ
ˆ
À
ˆ
(m, 5H; CH CH2 allyl, C CH CH2S, 2 Â C CH Far), 4.80 4.39 (m, 5H;
3 Â aCH, OCH2 allyl), 4.08 3.96 (m, 2H; aCH2 Gly), 3.72 (s, 3H; OMe),
3.37 3.09 (m, 8H; bCH2 Cys, eCH2 Lys, aCH2 Far, aCH2 Aca), 2.92 2.80
(m, 2H; bCH2 Cys), 2.82 (s, 3H; CH3 Mant), 2.54 (t, 3J ˆ 7.1 Hz, 2H; aCH2
Pal), 2.31 2.27 (m, 2H; eCH2 Aca), 2.07 1.95 (m, 8H; 4 Â CH2 Far), 1.90
1.37 (m, 24H; bCH2 Aca, gCH2 Aca, dCH2 Aca, 4 Â CH3 Far, bCH2
Lys, dCH2 Lys, gCH2 Lys), 1.23 (s, 24H; Pal), 0.87 (t, 3J ˆ 6.6 Hz, 3H;
wCH3 Pal) ppm; 13C NMR (100 MHz, CDCl3): d ˆ 200.4, 174.2, 171.8,
171.4, 170.2, 169.8, 169.6, 156.7, 150.7, 140.3, 135.6, 133.3, 132.8, 131.5,
127.6, 124.7, 123.9, 119.7, 117.6, 115.7, 114.6, 111.2, 65.6, 53.2, 52.7, 52.5, 44.3,
43.9, 40.8, 39.9, 39.6, 35.9, 32.8 22.5 (several signals), 17.9, 16.4, 16.2,
ˆ
À
ˆ
C CH CH2S), 5.06 5.03 (m, 2H; 2 Â CH C Far), 4.79 4.76 (m, 1H;
aCH), 4.65 4.58 (m, 4H; 4 Â aCH), 4.00 (m, 2H; aCH Gly), 3.76 3.73
(m, 1H; CH2a Pro), 3.67 (s, 3H; OMe), 3.61 3.57 (m, 1H; CH2b Pro), 3.33
3.29 (m, 2H; aCH2 Aca), 3.16 3.11 (m, 1H; aCH2a Far), 3.04 2.98 (m,
‡
14.3 ppm; MS (ESI ‡): m/z calcd for C64H106N7O10S2 [M ‡ H] : 1196.7;
found: 1196.7.
¹ 2003 Wiley-VCH Verlag GmbH & Co.KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2003, 9, 3683 3691
3690

Acid- and Base-Labile Lipopeptides
3683 3691
MIC-Gly-Cys(Pal)-Lys(Aloc)-Cys(Far)-OMe (27): The synthesis was car-
ried out as described above for compound 26 with resin-bound Fmoc-
Cys(Trt) (128 mg, 0.055 mmol). After cleavage of the Trt group MIC-OH
was coupled by using MIC-OH (73.9 mg, 0.35 mmol), DIC (31 mL,
0.20 mmol), HOBt (46 mg, 0.30 mmol), and Et3N (34 mL, 0.20 mmol) for
3 h.The resin was treated with a solution of Cu(OAc) 2 (19 mg, 0.10 mmol),
pyridine (140 mL, 1.74 mmol), acetic acid (200 mL, 3.50 mmol), and MeOH
(441 mL, 10.9 mmol) in dichloromethane (20 mL) for 2 h under oxygen. The
resin was filtered off, the solvent was evaporated under reduced pressure,
and the crude reaction mixture was dried in vacuo.Flash column
chromatography with dichloromethane/methanol (20:1) furnished 27
(13.9 mg, 0.012 mmol, 22%). Rf ˆ 0.3 (dichloromethane/MeOH (20:1));
[a]2D0 ˆ À12.2 (c ˆ 0.85 in CHCl3); 1H NMR (400 MHz, CDCl3): d ˆ 7.40 (s,
[3] a) K.Kuhn, D.J.Owen, H.Waldmann, A.Wittinghofer, J.Kuhlmann,
Nature 2000, 403, 223 226; b) K.Kuhn, D.J.Owen, B.Bader, A.
Wittinghofer, J.Kuhlmann, H.Waldmann, J. Am. Chem. Soc. 2001,
123, 1023 1035; c) D.Kadereit, J.Kuhlmann, H.Waldmann, Chem-
BioChem 2000, 1, 144 169; d) K.Alexandrov, I.Heinemann, T.
Durek, V.Sidorovitch, R.S.Goody, H.Waldmann, J. Am. Chem. Soc.
2002, 124, 5648 5649.
[4] a) H.Schroeder, R.Leventis, S.Rex, M.Schelhaas, E.N‰gele, H.
Waldmann, J.R.Silvius, Biochemistry 1997, 36, 13102 13109; b) M.
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tinghofer, H.Waldmann, Chem. Eur. J. 1999, 5, 1239 1252; c) E.
N‰gele, M.Schelhaas, N.Kuder, H.Waldmann,
J. Am. Chem. Soc.
1998, 120, 6889 6902; d) M.Vˆlkert, M.Wagner, C.Peters, H.
Waldmann, Biol. Chem. 2001, 382, 1133 1145.
ˆ
1H; NH), 7.18 (s, 1H; NH), 6.67 (s, 2H; CH CH MIC), 6.52 (s, 1H; NH),
ˆ
ˆ
ˆ
À
5.90 (m, 1H; CH CH2 allyl), 5.29 5.08 (m, 5H; CH CH2, C CH CH2S,
[5] a) P.Mayer-Fligge, J.Volz, U.Kr¸ger, E.Sturm, W.Gernandt, K.P.
Sch‰fer, M.Przybylski, J. Pept. Sci. 1998, 4, 355 363; b) D.Denis, E.
Trifilieff, J. Pept. Sci. 2000, 6, 372 377; c) S.P.Creaser, B.R.Peterson,
J. Am. Chem. Soc. 2002, 124, 2444 2445.
ˆ
2 Â C CH Far), 4.68 4.52 (m, 5H; 3 Â aCH, OCH2 allyl), 4.00 3.85 (m,
2H; aCH2 Gly), 3.73 (s, 3H; OMe), 3.52 3.48 (m, 2H; NCH2 MIC), 3.27
3.09 (m, 6H; bCH2 Cys, eCH2 Lys, aCH2 Far), 2.95 2.78 (m, 2H; bCH2
Cys), 2.57 (t, 3J ˆ 6.0 Hz, 2H; aCH2 Pal), 2.28 2.24 (m, 2H; CH2 MIC),
2.1 1.90 (m, 8H; 4 Â CH2 Far), 1.8 1.1 (m, 48H; bCH2 Aca, gCH2 Aca,
dCH2 Aca, bCH2 Lys, dCH2 Lys, gCH2 Lys, 4 Â CH3 Far, Pal), 0.87 0.85 (m,
[6] a) E.K.Dolence, J.M. Dolence, C.D. Poulter,
Bioconjugate Chem.
2001, 12, 35 43; b) B.Ludolph, F.Eisele, H.Waldmann, ChemBio-
Chem 2002, 3, 901 904.
‡
3H; wCH3 Pal) ppm; MS (ESI ‡): m/z calcd for C60H99N6O11S2 [M ‡ H] :
[7] B.Ludolph, F.Eisele, H.Waldmann,
5954 5955.
J. Am. Chem. Soc. 2002, 124,
‡
1143.7; found: 1143.6; MS (MALDI, DHB): calcd for [M ‡ Na] : 1165.7;
found: 1165.9.
[8] a) A.N.Semenov, K.Y.Gordeev, Int. J. Pept. Protein Res. 1995, 45,
303 304; b) C.R.Millington, R.Quarrel, G.Lowe, Tetrahedron Lett.
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Acknowledgement
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This research was supported by the Deutsche Forschungsgemeinschaft and
the Fonds der Chemischen Industrie.
H.C.Hui, E.M.Gordon, D.V.Patel,
15890.
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[1] a) F.L.Zhang, W.R.Tschantz, P.J.Casey,
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1451, 1 16.
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[15] Y.Han, F.Albericio, G.Barany, J. Org. Chem. 1997, 62, 4307 4312.
[2] a) O.Seitz, I.Heinemann, A.Mattes, H.Waldmann,
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Received: February 5, 2003 [F4822]
Chem. Eur. J. 2003, 9, 3683 3691
www.chemeurj.org
¹ 2003 Wiley-VCH Verlag GmbH & Co.KGaA, Weinheim
3691