Synthesis and membrane binding properties of a lipopeptide fragment from influenza virus A hemagglutinin

Article abstract of DOI:10.1002/1521-3765(20020802)8:15<3362::AID-CHEM3362>3.0.CO;2-0

Hemagglutinin from influenza virus A is a S-palmitoylated lipoglycoprotein in which the lipid groups are thought to influence the interaction between cell membrane and capsid during budding of viral offspring as well as fusion processes of the viral membr

Full text of DOI:10.1002/1521-3765(20020802)8:15<3362::AID-CHEM3362>3.0.CO;2-0

FULL PAPER
Synthesis and Membrane Binding Properties of a Lipopeptide Fragment from
Influenza Virus A Hemagglutinin
Frank Eisele,[b] J¸rgen Kuhlmann,*[a] and Herbert Waldmann*[c]
Abstract: Hemagglutinin from influen-
za virus A is a S-palmitoylated lipogly-
coprotein in which the lipid groups are
thought to influence the interaction
between cell membrane and capsid dur-
ing budding of viral offspring as well as
fusion processes of the viral membrane
with the endosome after entry of the
viral particle into the cell. The paper
describes the development of a method
for the synthesis of characteristic lipi-
dated hemagglutinin derived peptides
which additionally carry the fluorescent
lin G acylase; this results in the forma-
tion of a phenolate. This intermediate
spontaneously undergoes fragmentation
thereby releasing the desired carboxy-
lates. The combined use of this enzyme-
labile fragmenting ester with the acid-
labile Boc group, the Pd0-sensitive allyl
ester and the corresponding Aloc ure-
thane gave access to a mono-S-palmi-
toylated and a doubly S-palmitoylated
NBD-labelled hemagglutinin peptide.
The binding of these lipopeptides to
model membranes was analyzed in a
biophysical setup monitoring the trans-
fer of fluorescent-labelled lipopeptide
from vesicles containing the non-ex-
changeable
fluorescence
quencher
Rho-DHPE to quencher-free vesicles.
The experiments demonstrate that one
lipid group is not sufficient for quasi-
irreversible membrane insertion of lipi-
dated peptides. This is, however, ach-
ieved by introduction of the bis-palmi-
toyl anchor. The intervesicle transfer
always implies release of peptides local-
ized at the outer face of the vesicles into
solution followed by diffusion to and
insertion into acceptor vesicles. For
peptides bound at the inner face of the
vesicle membrane, however, an addi-
tional flip flop diffusion to the outer
face has to occur beforehand. The ki-
netics of these processes were estimated
by fast chemical quench of the outside
fluorophores by sodium dithionite.
7-nitrobenz-2oxa-1,3-diazole
(NBD)
group. To achieve this goal the en-
zyme-sensitive para-phenylacetoxyben-
zyloxycarbonyl (PAOB) ester was de-
veloped. It is cleaved from the peptides
and lipidated peptides under very mild
conditions and with complete selectivity
by treatment with the enzyme penicil-
Keywords: enzyme
catalysis
¥
hemagglutinin ¥ lipidated peptides ¥
peptides ¥ protecting groups
lipid-bilayer. For instance hemagglutinin from influenza
virus A is glycosylated in the extracellular domain (Fig-
ure 1)[1, 2] and the glycoprotein part is responsible for initiation
of viral infection through selective binding to sialic acid
receptors on the surface of the host cell. In addition, the
protein is S-palmitoylated next to the transmembrane region
(Figure 1);[3, 4] the lipid residues are required for the inter-
action between the cell membrane and the free capsid during
budding of viral offspring.[5, 6] They are also thought to
mediate protein protein and protein lipid interactions in
the viruses[7] and may play an important role in fusion
processes of the viral membrane with the endosome after
entry of the viral particle into the cell.[8, 9] However, this
proposal is controversial, since other investigations indicated
that the lipidated cytoplasmic tail of the complex viral
lipoglycoproteins is not essential for its membrane fusion
activity.[10]
Introduction
The infection of healthy cells by viruses is a complex multi-
step process which is decisively influenced and determined by
posttranslationally modified proteins embedded in the viral
[a] Dr. J. Kuhlmann
Max-Planck-Institut f¸r molekulare Physiologie
Abteilung Strukturelle Biologie
Otto-Hahn-Strasse 11, 44227 Dortmund (Germany)
[b] Dr. F. Eisele
Universit‰t Karlsruhe, Institut f¸r Organische Chemie
Richard-Willst‰tter-Allee 2
76128 Karlsruhe (Germany)
[c] Prof. Dr. H. Waldmann
Max-Planck-Institut f¸r molekulare Physiologie
Abteilung Chemische Biologie
Otto-Hahn-Strasse 11, 44227 Dortmund (Germany)
and
Universit‰t Dortmund, Fachbereich 3, Organische Chemie
44221Dortmund (Germany)
Also, the orchestration of fatty acid attachment to the viral
glycoproteins is not well-known. Thus, until today a consensus
sequence for acylation by a putative palmitoyl transferase
Fax : (‡49) 231/133-2499
E-mail: herbert.waldmann@mpi-dortmund.mpg.de
3362
¹ WILEY-VCH Verlag GmbH, 69451Weinheim, Germany, 2002
0947-6539/02/0815-3362 $ 20.00+.50/0
Chem. Eur. J. 2002, 8, No. 1 5

3362 3376
O
S
base
C15H31
O
H
NH
(ax)n
N
H
O
S
2
C15H31
Nu
O
O
NH
(ax)n
N
H
O
HS
3
Scheme 1. Lability of palmitoylated peptides in the presence of bases and
nucleophiles. The cysteines can be separated by one or more peptide
residues (ax)n, n ˆ 0, 1, 2, ...
Results and Discussion
In developing an enzyme-labile ester group which can be
removed selectively under conditions that are mild enough for
lipopeptide synthesis we resorted to the principle of fragmen-
tation after cleavage of a suitable enzyme-labile bond. To this
end, N-protected dipeptide PAOB esters 4 were synthesized
(Schemes 2 and 3). The PAOB group embodies a phenyl-
PAOB
O
O
Figure 1. Schematic drawing of glycosylated and lipidated influenza
virus A hemagglutinin and doubly palmitoylated target peptide 1.
penicillin G acylase
R
O
O
R
O
O
O
O
4
5
OH
from several palmitoylated viral proteins could not be
identified.[11] One of the few related facts known is that fatty
acylation obviously takes place after exit from the endoplas-
matic reticulum.[12]
spontaneous
fragmentation
O
+
O
R
O
6
7
H2O
For the study of these and related processes in molecular
detail, lipidated peptides which represent the characteristic
linkage region between the protein backbone and the lipid
groups and which additionally carry a marker by which they
can be traced in biological systems, that is a fluorescent group
or a biotin unit, may be employed as efficient molecular
probes.[13] However, their synthesis is complicated by the
pronounced base lability of the palmitic acid thioesters which
hydrolyse spontaneously at pH >7 or undergo base-mediated
b-elimination reactions (Scheme 1). For the synthesis of
sensitive lipidated peptides a combination of classical acid-
labile, noble-metal sensitive, and enzymatically cleavable
protecting groups may provide efficient solutions. These three
types of protecting functions can be combined in such a way
that they are orthogonally stable.[12, 14] However, an enzyme-
labile carboxy protecting group, which meets these demands
and is also compatible with the sensitivity of the palmitic acid
thioester, is lacking. In this paper we report on the develop-
ment of the p-phenylacetoxybenzyl (PAOB) ester, a new
enzyme-labile carboxy protecting group, and its application in
the construction of a fluorescent-labelled lipopeptide 1 from
influenza virus A hemagglutinin.[15]
OH
HO
8
Scheme 2. Principle of PAOB-ester-deprotection by enzyme-initiated
fragmentation.
acetate which is specifically recognized and cleaved by the
enzyme penicillin G acylase. Upon enzymatic hydrolysis a
phenolate 5 is formed that fragments spontaneously to a
quinone methide 7 and the desired carboxylic acids 6. The
quinone methide is trapped by water to give p-hydroxybenzyl
alcohol 8 or by added nucleophiles. This principle of
deprotection by enzyme-induced fragmentation has been
used for the development of enzyme-labile urethanes be-
fore.[16, 17] However, already in the case of the urethanes the
success of the enzyme-mediated fragmentation at pH 6
8
was rather surprising. Non-enzymatic induction of the frag-
mentation reaction requires the use of strong bases like
ammonia[18] whereas in the presence of the biocatalysts the
unmasking occurs already at neutral pH. In addition, in the
case of the urethane protecting groups the entire unmasking is
Chem. Eur. J. 2002, 8, No. 15
¹ WILEY-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002
0947-6539/02/0815-3363 $ 20.00+.50/0
3363

F. Eisele, J. Kuhlmann and H. Waldmann
FULL PAPER
driven by the liberation of CO2, thereby shifting the
equilibrium to the product side. This driving force is no
longer operative in the case of an ester blocking function.
Thus, it was highly questionable whether this principle could
be transferred successfully from an amino to a carboxy
protecting group.
The synthesis of PAOB esters 4 commenced with the
esterification of Boc-protected amino acids 9 with p-phenyl-
acetoxybenzyl alcohol (10), subsequent acid-mediated cleav-
age of the Boc group and coupling of the resulting amino acid
PAOB esters 12 with differently masked amino acids
(Scheme 3). In order to achieve high yields in the coupling
reactions it is necessary to preactivate the N-terminally
protected amino acid first followed by addition of the PAOB
amino acid esters.
Upon treatment of dipeptide PAOB esters 4 with penicil-
lin G acylase at pH 7 and room temperature the desired
selective C-terminal deprotection occurred smoothly. The
enzyme saponifies the phenylacetate, even at pH 7, and
without the additional driving force of CO2 liberation the
intermediary formed phenolates 5 undergo fragmentation to
the desired unmasked dipeptides 6a e which were obtained
in high yield (Scheme 3). Quinone methide 7 is efficiently
stable enzyme with a broad substrate tolerance which does
not attack peptide bonds or urethanes. In addition, in the
PAOB group the site of the enzymes× attack is remote from
the differing amino acids of the substrates. Consequently, the
efficiency of the enzyme-induced deprotection is nearly
independent of steric bulk and structure (i.e., d- or l-amino
acid, acyclic or cyclic amino acid). This is apparent from the
differing steric demand of the C-terminal amino acids
incorporated into 6. Thus, 6a d are obtained in similar yields
and the cyclic amino acid proline is tolerated at the
C-terminus without any problem. In this context it should
be noted that from peptides terminating in a proline amide[19]
or a proline heptyl ester[20] the C-terminal enzyme-labile
protecting group could not be removed by means of amidase-
or lipase-catalyzed hydrolysis. The low yield obtained for the
C-terminal deprotection of 4e is not a result of the steric
demand of the C-terminal phenylalanine (compare the results
for 4d and e which both terminate in this amino acid). Rather
it reflects the very limited solubility of the hydrophobic
dipeptide 4e in the buffer/cosolvent mixtures employed. This
observation was already indicative of the problems encoun-
tered later on in the enzymatic unmasking of unpolar
lipidated peptides (see below).
On the other hand the Boc- and the Aloc groups can be
removed selectively from dipeptide esters 4 without harm to
the PAOB esters, that is the protecting groups are orthogo-
nally stable to each other (data not shown, see also below).
The full capacity of the enzyme-labile PAOB ester became
evident in the synthesis of fluorescent labelled derivatives of
virus hemagglutinin peptide 1. This synthesis is complicated
by the base lability of the thioesters and the need to
additionally protect and deprotect the basic arginine side
chain functionality. Thus, a set of three orthogonally stable
protecting groups is required, whereby the use of base-labile
and hydrogenolytically removable blocking groups is not
permitted. This problem was overcome by using the acid-
labile Boc group for the N-terminus, the enzymatically
removable PAOB ester for the C-terminus and the Pd0-
sensitive Aloc group for the arginine guanidino side chain
function.
It was planned to generate the base-labile palmitic acid
thioesters at the dipeptide stage employing peptides with
C-terminal cysteine residues to avoid a possible S ! N acyl
migration which may occur after N-terminal unmasking of
S-acylated cysteine peptides.[21] Intermediary reversible pro-
tection of the thiol functions was avoided by synthesizing
cystine peptides as intermediates. Thus, Boc-cystine 9g was
esterified with alcohol 10 to yield ester 11e which was
selectively deprotected at the N-terminus (Scheme 4). The
resulting cystine ester 12e was then condensed with Boc-
isoleucine and Boc/Aloc-protected arginine to yield fully
masked dipeptides 13a and 13b, respectively. Reduction of
the disulfide bonds incorporated into these intermediates with
dithiothreitol (DTT) and subsequent S-palmitoylation gave
peptides 14a and 14b from which the Boc group could be
removed selectively in high yields. Finally, the peptide chain
was elongated by coupling with Boc-threonine and Boc-
methionine to yield lipidated tripeptide esters 16a and 16b,
respectively.
O
Boc-AA2-OH
+
HO
i
O
10
9
O
Boc-2AA-O
O
11
12
ii
O
H-2AA-O
O
iii
iv
O
PG-1AA-2AA-O
O
4
PG-AA1-AA2-OH
O
+
6
7
H2O
OH
HO
8
Scheme 3. Synthesis and selective enzymatic deprotection of N-protected
dipeptide PAOB esters 6. i) DIC/DMAP(cat.), CH2Cl2, 64 90%; ii) HCl/
Et2O or TFA/CH2Cl2, 73 98%; iii) PG-AA1-OH 9, EEDQ, NEt3, CH2Cl2
or DIC/HOBt, NEt3, CH2Cl2, 71 87%; iv) phosphate buffer (pH 7), 10%
methanol, penicillin G acylase; for 6e: phosphate buffer (pH 7), 30%
acetone, penicillin G acylase. DIC: N,N'-diisopropylcarbodiimide; DMAP:
4-(dimethylamino)-pyridine; EEDQ: 2-ethoxy-1-ethoxycarbonyl-1,2-dihy-
droquinoline; HOBt: 1-hydroxybenzotriazole; TFA: trifluoroacetic acid.
trapped by water, addition of stronger nucleophiles is not
necessary. Penicillin G acylase is a readily available (immo-
bilized, native, and as cross-linkes enzyme crystals) and very
3364
¹ WILEY-VCH Verlag GmbH, 69451Weinheim, Germany, 2002
0947-6539/02/0815-3364 $ 20.00+.50/0
Chem. Eur. J. 2002, 8, No. 1 5

Lipopeptides
3362 3376
solved well in phosphate buffer (pH 7) and treatment of this
solution with penicillin G acylase at 258C resulted in a smooth
and completely selective removal of the C-terminal enzyme-
labile protecting function (Scheme 5). Similarly, from S-pal-
Boc-Cys-OH
O
+
S
HO
O
2
9g
10
i
Boc-Cys-OPAOB
S
i
Boc-Thr-Ile-Cys-OPAOB
Boc-Thr-Ile-Cys-OH
2
SPal
16a
SPal
11e
17a
ii
Boc-Met-Arg-Cys-OPAOB
i
i
Boc-Met-Arg-Cys-OH
H-Cys-OPAOB . HCl
(Aloc)2
(Aloc)2
SPal
SPal
16b
17b
S
2
12e
Boc-Arg-Cys-OPAOB
Boc-Arg-Cys-OH
iii
iv
(Aloc)
(Aloc)2
2 SPal
SPal
14b
18
Boc-Arg-Cys-OPAOB
Boc-Ile-Cys-OPAOB
Scheme 5. Enzymatic deprotection of palmitoylated peptides. i) penicil-
lin G acylase, dimethyl-b-cyclodextrin, 0.05m phosphate buffer (pH 7),
258C, 16a: 81 %,16b: isolation not possible, 14b: 77%.
(Aloc)
2 S
S
13b
13a
2
2
v
v
Boc-Ile-Cys-OPAOB
Boc-Arg-Cys-OPAOB
(Aloc)2
mitoylated dipeptide PAOB ester 14b the enzyme labile
blocking group was cleaved without any undesired side
reaction. The mildness of the reaction conditions and the
substrate specificity of the biocatalyst guarantee that neither
the thioester nor the bis-acylated guanidino group are
attacked, and the selectively unmasked S-palmitoylated
peptides were isolated in high yields.
SPal
SPal
14a
14b
vi
vi
H-Ile-Cys-OPAOB . TFA
H-Arg-Cys-OPAOB . TFA
SPal
(Aloc)2
SPal
viii
15b
15a
vii
Also, tripeptide 16b was readily soluble in phosphate
buffer/dimethyl-b-cyclodextrin and deprotection with penicil-
lin G acylase proceeded smoothly and to completeness.
However, separation of lipotripeptide 17b from the cyclo-
dextrin could not be achieved. Thus, although the results
detailed above clearly demonstrated that the conditions
required for enzymatic removal of the PAOB ester group
are fully compatible with the demands of lipopeptide syn-
thesis, the strategy for the synthesis of hemagglutinin peptide
1 had to be modified.
Boc-Thr-Ile-Cys-OPAOB
Boc-Met-Arg-Cys-OPAOB
(Aloc)2
SPal
SPal
16a
16b
Scheme 4. Synthesis of palmitoylated tripeptide PAOB esters 16a and
16b. i) DIC/DMAP (cat.), CH2Cl2, 86%; ii) HCl/Et2O, 95%; iii) Boc-Ile-
OH, EEDQ, NEt3, CH2Cl2, 78%; iv) Boc-Arg(Aloc)2-OH, DIC/HOBt,
NEt3, CH2Cl2, 62%; v) a) DTT, NEt3, CH2Cl2, b) 2Pal-Cl, NEt3, CH2Cl2,
14a: 83%, 14b: 81%, two steps; vi) TFA/CH2Cl2, quant.; vii) Boc-Thr-
OH, EEDQ, NEt3, CH2Cl2, 74%; viii) Boc-Met-OH, DIC/HOBt, NEt3,
CH2Cl2, 65%; Pal: palmitoyl.
To this end, S-palmitoylated tripeptide carboxylic acid 17a
was condensed with isoleucine allyl ester and the Boc group
was removed to deliver lipotetrapeptide 20 in high overall
yield (Scheme 6). Compound 20 was blocked as allyl ester at
the C-terminus to allow for simultaneous Pd0-catalyzed
deprotection of both the arginine side chain function and
the C-terminal carboxylic acid towards the end of the
synthesis. C-terminally deprotected and S-palmitoylated di-
peptide building block 18 and N-terminally unmasked lip-
otetrapeptide 19 were then condensed to give double-
palmitoylated lipohexapeptide 23. The N-terminal Boc group
was removed from 23 and the fluorescent 7-nitrobenz-2-oxo-
1,3-diazole (NBD) label was introduced by coupling with the
NBD aminocaproyl amide of methionine (NBAca-Met) 26 to
obtain a high degree of convergency in the synthesis. Finally,
the three allyl-type blocking functions were cleaved by Pd0-
catalyzed allyl transfer to N,N'-dimethylbarbituric acid as the
accepting C-nucleophile.[24] Similarly, introduction of the
NBD label followed by selective removal of the allyl ester
yielded lipopeptide 22. The desired fluorescent and palmitoy-
lated influenza virus hemagglutinin lipopeptides 22 and 28
were obtained in high overall yield.
It was planned to enzymatically deprotect 16a and 16b in
order to obtain two key intermediates for the final assembly of
the desired heptapeptide target. C-terminal deprotection of
16a and subsequent elongation of the peptide chain by an
isoleucine ester would yield the (still N-masked) C-terminal
tetrapeptide unit of the target peptide. Enzymatic unmasking
of 16b would yield the N-terminal tripeptide unit of the final
target compound. Enzyme-catalyzed deprotection of lipo-
philic tripeptide PAOB ester 16a with immobilized penicil-
lin G acylase was attempted in various water/cosolvent
(methanol, acetone, DMF, THF, dioxane) mixtures but due
to the low solubility of the substrate in these solvents an
appreciable cleavage of the ester could not be observed. Also
the application of cross-linked enzyme crystals[22] or the use of
organic solvents saturated with water did not improve this
situation.
Finally, the use of dimethyl-b-cyclodextrin as solubilizing
agent was successful. This cyclic hexasaccharide most prob-
ably slips over the hydrophobic fatty acid group[16, 23] thereby
rendering the substrates soluble. After addition of the b-
cyclodextrin and ultrasonication lipidated peptide 16a dis-
Chem. Eur. J. 2002, 8, No. 15
¹ WILEY-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002
0947-6539/02/0815-3365 $ 20.00+.50/0
3365

F. Eisele, J. Kuhlmann and H. Waldmann
FULL PAPER
cence quencher (N-(lissamine rhodamine sulfonyl)phospha-
tidylethanolamine, Rho-DHPE). Both, lipid and quencher
were dissolved in methanol. Buffer was added to generate
mixed vesicles corresponding to a POPC concentration of
1 mm. Vesicles are trimmed by freeze thaw cycles and
extruder treatment[26] to generate vesicles of a defined size
distribution (approximately 100 nm Æ). If the NBD fluoro-
phor of the lipopeptide incorporated in such a vesicle is
excited at 460 nm its fluorescence emission around 520
540 nm is directly absorbed by the rhodamine dye of the
quencher when Rho-DHPE is close to the lipopeptide. Mixing
those vesicles with an excess of pure POPC-vesicles allows
mobile lipopeptides to leave their original environment. They
enter the quencher-free vesicles where their NBD fluores-
cence is not quenched any longer resulting in an increase in
fluorescence emission at 525 nm. Figure 2b shows the fluo-
rescence emission spectra of vesicles loaded with Rho-DHPE
Boc-Thr-Ile-Cys-OH
SPal
17a
i
Boc-Thr-Ile-Cys-Ile-OAll
19
SPal
ii
H-Thr-Ile-Cys-Ile-OAll.TFA
20
SPal
Boc-Arg-Cys-OH
18
iii
v
(Aloc)
SPal
2
NBDac-Thr-Ile-Cys-Ile-OAll
Boc-Arg-Cys-Thr-Ile-Cys-Ile-OAll
(Aloc)2
21
SPal 23 SPal
SPal
iv
vi
NBDac-Thr-Ile-Cys-Ile-OH
22
H-Arg-Cys-Thr-Ile-Cys-Ile-OAll.TFA
SPal
~
(Aloc)2
SPal
SPal
24
and lipopeptide 22 before addition of empty vesicles ( ) and
after the end of the exchange reaction (Â). Note that the
quencher is a fluorophore itself with an emission maximum at
585 nm. Excitation wavelength was 465 nm.
vii
NBDAca-Met-OAll
NBDAca-Met-OH
viii
25
26
Mixed vesicles were generated containing 1mol% of
peptide 22 and 28, respectively and 2 mol% of Rho-DHPE.
Only tetrapeptide 22 with a single palmitoyl-thioester showed
an increase in fluorescence in two hours observation time
after mixing a solution of 5mm POPC (mixed vesicles) with a
40-fold excess of pure POPC-vesicles (Figure 3). This indi-
cates the transfer of the lipopeptide to the vesicles free of
Rho-DHPE and shows that one lipid modification is not
sufficient for irreversible membrane insertion. In contrast the
NBD emission of the heptapeptide 28 with two palmitoyl-
residues was not affected by the dilution with quencher-free
vesicles. This observation demonstrates that two C16 anchors
are sufficient to fix the lipopeptide in its original environment.
The intervesicle transfer of lipopeptide 22 is composed of
two separate processes. Peptides anchored at the outer face of
the vesicle can directly migrate to the acceptor vesicle by
diffusion. This step can be described as an irreversible first
order mechanism if the acceptor vesicles are present in high
excess. Depending on their distribution between inner and
outer face of the vesicles the intravesicular lipopeptides have
to perform a reversible flip flop diffusion to appear on the
outer face of the vesicles. In a first estimation we fitted the
overall change in fluorescence for the tetrapeptide by a
monoexponential function including a linear term for the
flip flop process. Best fits were obtained for five independent
experiments by a dissociation rate of 1.1Æ 0.3  10À3 sÀ1 (n ˆ
5) and a linear drift of 1.0Â 102 units of fluorescence sÀ1. The
drift corresponds to approximately 40% of the fitted ampli-
tude.
NBDAca-Met-Arg-Cys-Thr-Ile-Cys-Ile-OAll
(Aloc)2
SPal
ix
SPal
27
O
N
N
O
H
O2N
N
Met-Arg-Cys-Thr-Ile-Cys-Ile-OH
O
O
S
S
28
NBDAca
Scheme 6. Final steps of the synthesis of fluorescent labeled lipopeptides
22 and 28. i) H-Ile-OAll ¥ HOTos, EEDQ, NEt3, CH2Cl2, 71%; ii) TFA/
CH2Cl2, quant.; iii) NBDAca-OH, EDC/HOBt, NEt3, CH2Cl2, 86%;
iv) [Pd(PPh3)4], DMB, THF, 86%; v) EEDQ, NEt3, CH2Cl2, 46%;
vi) TFA/CH2Cl2, quant.; vii) [Pd(PPh3)4], DMB, THF, 94%; viii) EDC/
HOBt, NEt3, CH2Cl2, 72%; ix) [Pd(PPh3)4], DMB, THF, 96%. NBDAca:
N-(7-Nitrobenz-2-oxa-1,3-diazole-4-yl)-aminocaproyl; EDC: N-ethyl-N'-
(3-dimethylaminopropyl)-carbodiimide hydrochloride; DMB: dimethyl-
barbituric acid.
Fluorescent lipidated model peptides such as mono-palmi-
toylated tetrapeptide 22 and doubly palmitoylated heptapep-
tide 28 may serve as efficient tools to gain insight into the
membrane binding properties of the parent lipoproteins.
In order to demonstrate this potential the binding of the
lipidated peptides 22 and 28 to liposome model membranes
was analysed in a biophysical setup.[25] Here, the lipopeptides
were incorporated into lipid vesicles containing a suitable,
non-exchangeable fluorescence quencher molecule that ab-
sorbs the fluorescence signal of the excited lipopeptide
fluorophore as long as it is in sufficient vicinity. Addition of
an excess of pure vesicles without quencher leads to an
increase in fluorescence signal (see Figure 2).
To obtain more information about the slow phase of the
kinetics which putatively reflects the flip flop diffusion we
performed a long-term measurement over 16 h (Figure 4).
Again no fluorescence increase could be observed for the
vesicles containing the doubly palmitoylated lipopeptide 28.
The curve for lipopeptide 22 could be best fitted with a double
exponential function with a fast rate constant of 6.6 Â 10À4 sÀ1
and a slow rate constant of 8.7 Â 10À5 sÀ1. Here, the amplitude
of the fast reaction was about 40% of the total amplitude.
In our assay methanolic solutions of the fluorescent-labeled
lipopeptides 22 and 28 were mixed with a hundredfold molar
excess of lipid (here: palmitoyl oleoyl phosphatidylcholine,
POPC) and a twofold excess of a nonexchangeable fluores-
3366
¹ WILEY-VCH Verlag GmbH, 69451Weinheim, Germany, 2002
0947-6539/02/0815-3366 $ 20.00+.50/0
Chem. Eur. J. 2002, 8, No. 1 5

Lipopeptides
3362 3376
intervesicle transfer experiment
(Figure 4). In contrast to the
latter, the kinetics of the con-
secutive fluorescence decay
when fitted as a monoexponen-
tial function showed an appa-
rent rate constant of 2.1 Â
10À3 sÀ1 which was significantly
faster than the slow phase of the
intervesicle transfer. This accel-
erated decay therefore might
reflect a leakage of the vesicles
rather than the true flip flop
reaction.
a)
I
l
e
C
y
s
-
r
h
T
-Il
e-
OH
intervesicle
transfer
quenched vesicle
non exchangeable fluorescence quencher
quencher-free vesicle
=
A second finding of the di-
thionite experiment was the
different amplitude of the fast
fluorescence shift for lipopep-
tide 22 and 28. While dithionite
reduces the fluorescence signal
of the mono-palmitoylated lip-
opeptide 22 by 40% (see
above) the lipopeptide with
two palmitoylation sites shows
a decay of 80% (Figure 5b).
We interpret this finding as an
indication of an asymmetric
distribution of lipopeptide 28
between the inner and the outer
surface of the vesicles.
b)
emission spectra of NBDac-Thr-Ile-Cys(Pal)-Ile-OH (22 )
4.0E+06
3.0E+06
2.0E+06
rel. fluores-
cence
1.0E+06
0.0E+00
470
520
570
620
The apparent rate constant
for the intervesicle transfer of
tetrapeptide 22 and heptapep-
tide 28 between POPC vesicles
can be compared with data for
wavelength/ nm
~
)
Figure 2. a) General principle of intervesicle transfer detection; b) emission spectra of NBD in the presence (
and absence (Â) of the quencher Rho-DHPE.
4e+005
4.0E+05
lipopeptides with a single farnesyl modification or two
hydrophobic residues (farnesyl thioether and palmitoyl thio-
ester).[27] Here, a half-life of 21s for a peptide with the
sequence NBD-GCMGLPC(Far)-OMe and 155 h for NBD-
GC(Pal)MGLPC(Far)-OMe were calculated for experiments
at 378C, while tetrapeptide 20 has a half-life of about 11 min
at 208C. Thus, in comparison to a farnesyl thioether a single
3.5e+005
3.5E+05
3e+005
3.0E+05
2.5e+005
2.5E+05
rel.fluores-
cence
2e+005
2.0E+05
1.5e+005
1.5E+05
0
0
2000
2000
4000
4000
6000
6000
3.00E+06
2.50E+06
2.00E+06
1.50E+06
t / s
Figure 3. Intervesicle transfer of single palmitoylated tetrapeptide 22.
A separate analysis of the flip flop exchange is possible by
monitoring the fluorescence of vesicles loaded with lipopep-
tides after treatment with sodium dithionite. The addition of
sodium dithionite resulted in fast reduction of all accessible
NBD fluorophors at the outer face of the vesicles. This first
loss of emission signal was followed by a slower decay,
indicating the flip flop of the intravesicular lipopeptides to
the outside (Figure 5).
rel.fluores-
cence
--- NBD-Thr-Ile-Cys(Pal)-Ile-OH (22)
--- NBD-Met-Arg-Cys(Pal)-Thr-Ile-Cys(Pal)-Ile-OH (28)
1.00E+06
5.00E+05
0.00E+00
0
10000
20000
t / s
30000
40000
50000
60000
For lipopeptide 22 the fluorescence drops by 40% after
dithionite addition, which corresponds to the amplitude of the
fast phase double exponential fit of the long time range
Figure 4. Intervesicle transfer of single palmitoylated tetrapeptide 22
compared with doubly palmitoylated heptapeptide 28.
Chem. Eur. J. 2002, 8, No. 15
¹ WILEY-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002
0947-6539/02/0815-3367 $ 20.00+.50/0
3367

F. Eisele, J. Kuhlmann and H. Waldmann
FULL PAPER
sensitive lipidated peptides. It was successfully applied in the
synthesis of differently lipidated and fluorescent-labelled
peptides which represent the characteristic linkage region
between the protein backbone and the lipid groups of
influenza virus A hemagglutinin. The analysis of the mem-
brane binding properties of these model peptides in a
biophysical setup led to the conclusion that one palmitoyl
group is not sufficient for quasi-irreversible membrane local-
ization of the lipidated molecules. Rather a bis-palmitoylated
membrane anchor is required for this purpose. The avail-
ability of such hemagglutinin-derived lipopeptides and their
application in a more detailed biophysical analysis should
allow for an extrapolation to the membrane-binding proper-
ties of the parent lipoglycoprotein. This knowledge will
further our understanding of the biological processes influ-
enced by hemagglutinin.
a)
1.0E+06
8.0E+05
6.0E+05
4.0E+05
addition of dithionite
rel. fluores-
cence
2.0E+05
0.0E+00
0
500
1000
1500
2000
2500
3000
t / s
b)
4.0E+05
Experimental Section
3.0E+05
General procedures: 1H und 13C NMR spectra were recorded on Bruker
AC-250, Bruker AM-400 and Bruker DRX-500 spectrometers. EI and FAB
mass spectra were measured on a Finnigan MAT MS 70 Workstation.
Specific rotations were measured with a Perkin Elmer polarimeter 241.
Elementary analyses were performed on a Heraeus CHN-Rapid apparatus.
Melting points were determined in open capillaries using a B¸chi 530
apparatus and are uncorrected.
addition of dithionite
2.0E+05
rel.fluores-
cence
1.0E+05
0.0E+00
0
Materials: Analytical chromatography was performed on E. Merck silica
gel 60 F254 coated plates. Flash chromatography was performed on Baker
silica gel (40 64 mm). Size-exclusion chromatography was performed on
Pharmacia Sephadex LH 20. Penicillin G acylase (E.C. 3.5.1.11) was
obtained in immobilized form on Eupergit C from Roche (Boehringer
Mannheim). Penicillin G acylase CLEC were obtained from Altus Bio-
logics. All solvents were distilled using standard procedures. Commercial
reagents were used without further purification. Where indicated the
reactions were performed under argon. Several compounds were prepared
according to literature methods: 4-(phenylacetoxy)benzyl alcohol (10),[17]
Boc-Arg(Aloc)2-OH (9h).[29, 30]
500
1000
1500
2000
2500
3000
t / s
Figure 5. Time course of fluorescence of vesicles containing lipotetrapep-
tide 22 (a) or lipoheptapeptide 28 (b) after dithionite reduction.
palmitoyl group confers a significantly enhanced stability of
membrane insertion, however, desorption from the model
membrane still occurs at a relatively fast rate. Combination of
a farnesyl thioether with a palmitic acid thioester results in a
very slow but still detectable intervesicle transfer, but in the
presence of two palmitoyl groups desorption of membrane-
bound lipidated peptides cannot be detected at all. Thus, a bis-
palmitoyl membrane anchor will lead to quasi irreversible
membrane anchoring of doubly palmitoylated proteins which
can only be reversed by hydrolysis of the palmitic acid
thioester bonds.
Synthesis of Boc-protected amino acid PAOB-esters: DIC (0.570 g,
4.10 mmol) was added to
a solution of Boc-protected amino acid
(4.10 mmol) 9a d, 4-(phenylacetoxy)benzyl alcohol 10 (1g, 4.10 mmol)
and DMAP (0.010 g, 0.10 mmol) in CH2Cl2 (150 mL) and the mixture was
stirred at room temperature for 16 h. Then the solution was extracted with
acetic acid (5%, 2 Â 50 mL) and water (2 Â 50 mL), the organic layer was
dried with MgSO4 and the solvent was removed under reduced pressure.
The products were purified by flash chromatography on silica gel using
solvent mixtures as indicated.
These values correspond well to data recorded for model
peptides which are derived from other lipidated proteins
(such as Rho) and which have been used to predict the
membrane binding properties of their parent proteins.[27, 28]
Thus, determining a full set of data for several different
hemagglutinin-derived lipopeptides in a detailed biophysical
analysis should allow for an extrapolation to the membrane-
binding properties of this lipoglycoprotein as well.
Boc-Ala-OPAOB (11a): Purification by flash chromatography using n-
hexane/ethyl acetate 5:2 yielded the title compound as a colorless solid
(1.53 g, 90%). M.p. 768C; [a]2D0 ˆ À10.5 (c ˆ 1.0 in CHCl3); Rf ˆ 0.41( n-
1
hexane/ethyl acetate 5:2); H NMR (400 MHz, CDCl3): d ˆ 7.40 7.24 (m,
7H, C6H5, OCCHCH, aromatic), 7.06 7.03 (m, 2H, OCCH, aromatic),
5.16 (d, 2J ˆ 12.4 Hz, 1H, CH2a-O), 5.09 (d, 2J ˆ 12.4 Hz, 1H, CH2b-O), 5.10
(br, 1H, NH), 4.35 4.32 (m, 1H, a-CH, Ala), 3.84 (s, 2H, CH2-COO), 1.43
(s, 9H, tBu), 1.36 (d, 3J ˆ 7.2 Hz, 3H, b-CH3, Ala); 13C NMR (100.6 MHz,
ˆ
ˆ
CDCl3): d ˆ 173.10, 169.81 (2  C O, ester), 155.06 (C O, carbamate),
150.63 (Cq-O, aromatic), 133.28, 133.08 (2 Â Cq, aromatic), 129.31, 129.26,
128.71, 127.36, 121.61 (CH, aromatic), 79.78 (Cq, tBu), 66.21(CH 2-O), 49.32
(a-CH), 41.33 (CH2-COO), 28.29 (CH3, tBu), 18.46 (b-CH3); MS (EI): m/z:
calcd: 413.184; found: 413.185; elemental analysis calcd (%) for C23H27NO6
(413.5): C 66.81, H 6.58, N 3.39; found: C 66.73, H 6.56, N 3.40.
Conclusion
Boc-Ile-OPAOB (11b): Purification by flash chromatography using n-
hexane/ethyl acetate 4:1yielded a colorless oil (1.16 g, 64%). [ a]2D0 ˆ À1.2
(c ˆ 1.0 in CHCl3); Rf ˆ 0.50 (n-hexane/ethyl acetate 4:1); 1H NMR
(500 MHz, CDCl3): d ˆ 7.39 7.29 (m, 7H, C6H5, OCCHCH, aromatic),
7.07 7.03 (m, 2H, OCCH, aromatic), 5.18 (d, 2J ˆ 12.4 Hz, 1H, CH2a-O),
We have developed a new enzyme-labile carboxy protecting
group which can be removed by treatment with penicillin G
acylase under very mild conditions and with complete
selectivity. The PAOB ester meets all demands posed by
3368
¹ WILEY-VCH Verlag GmbH, 69451Weinheim, Germany, 2002
0947-6539/02/0815-3368 $ 20.00+.50/0
Chem. Eur. J. 2002, 8, No. 1 5

Lipopeptides
3362 3376
5.09 (d, 2J ˆ 12.4 Hz, 1H, CH2b-O), 5.02 (d, 3J ˆ 8.8 Hz, 1H, NH), 4.31
4.29 (m, 1H, a-CH, Ile), 3.86 (s, 2H, CH2-COO), 1.90 1.82 (m, 1 H, b-CH,
Ile), 1.44 (s, 9H, tBu), 1.38 1.31 (m, 1 H,g-CH2a, Ile), 1.16 1.10 (m, 1H, g-
CH2b, Ile), 0.90 0.85 (m, 6H, 2 Â CH3, Ile); 13C NMR (125.7 MHz,
45 min and at room temperature for 1h. Then the excess HCl and the
solvent were removed under reduced pressure. The residue was triturated
three times with diethyl ether (10 mL) to yield the desired compound as a
colorless solid (0.532 g, 98%). M.p. 1318C; [a]2D0 ˆ À28.2 (c ˆ 1.0 in
CH3OH); 1H NMR (500 MHz, CD3OD): d ˆ 7.44 (d, 3J ˆ 8.6 Hz, 2H,
OCCHCH, aromatic), 7.38 7.27 (m, 5H, C6H5, aromatic), 7.09 (d, 3J ˆ
8.6 Hz, 2H, OCCH, aromatic), 5.29 (d, 2J ˆ 12.1 Hz, 1H, CH2a-O), 5.26 (d,
2J ˆ 12.1 Hz, 1H, CH2b-O), 4.45 (dd, 3J1 ˆ 8.8 Hz, 3J2 ˆ 7.1Hz, 1H, a-CH,
Pro), 3.89 (s, 2H, CH2-COO), 3.41 3.32 (m, 2H, d-CH2, Pro), 2.45 2.38
(m, 1H, b-CH2a, Pro), 2.14 2.01 (m, 3H, b-CH2b, g-CH2, Pro); 13C NMR
ˆ
ˆ
CDCl3): d ˆ 172.19, 169.82 (2  C O, ester), 155.54 (C O, carbamate),
150.69 (Cq-O, aromatic), 133.31, 133.10 (2 Â Cq, aromatic), 129.50, 129.28,
128.74, 127.39, 121.61 (CH, aromatic), 79.77 (Cq, tBu), 66.12 (CH2-O), 57.93
(a-CH), 41.38 (CH2-COO), 38.02 (b-CH), 28.31(CH 3, tBu), 24.93 (g-CH2),
15.55, 11.38 (2 Â CH3); MS (EI): m/z: calcd: 455.231; found: 455.229;
elemental analysis calcd (%) for C26H33NO6 (455.5): C 68.55, H 7.30, N 3.07;
found: C 69.10, H 7.41, N 3.27.
ˆ
(125.7 MHz, CD3OD): d ˆ 171.77, 169.88 (2  C O, ester), 152.59 (Cq-O,
aromatic), 135.08, 134.03 (2 Â Cq, aromatic), 131.02, 130.43, 129.68, 128.32,
122.95 (CH, aromatic), 68.71 (CH2-O), 60.74 (a-CH, Pro), 47.14 (d-CH2,
Pro), 41.84 (CH2-COO), 29.27 (b-CH2, Pro), 24.45 (g-CH2, Pro); MS (FAB,
Boc-Pro-OPAOB (11c): Purification by flash chromatography using n-
hexane/ethyl acetate 3:1yielded a colorless oil (1.56 g, 89%). [ a]2D0 ˆ À21.4
(c ˆ 1.0 in CHCl3); Rf ˆ 0.41( n-hexane/ethyl acetate 3:1); 1H NMR
(250 MHz, CDCl3): d ˆ 7.40 7.31(m, 7H, C 6H5, OCCHCH, aromatic),
7.06 (d, 3J ˆ 7.9 Hz, 2H, OCCH, aromatic), 5.20 (d, 2J ˆ 12.2 Hz, 1H, CH2a-
‡
3-NBA): m/z: calcd for C20H22NO4: 340.1549; found: 340.1576 [M À Cl] .
Synthesis of N-terminally protected dipeptide-4-phenylacetoxybenzyl
esters: EEDQ (1.2 equiv) was added at 08C to a solution of N-terminally
protected amino acid (1equiv) 9 in CH2Cl2 (30 mL). Then l-amino acid
4-phenylacetoxybenzyl ester protected trifluoroacetate (1equiv) 12 and
triethylamine (1equiv) were added and the mixture was stirred at room
temperature for 16 h. The solvent was removed under reduced pressure and
the residue was purified by flash chromatography on silica gel using solvent
mixtures as indicated.
2
3
3
O), 5.08 (d, J ˆ 12.2 Hz, 1H, CH2b-O), 4.24 (dd, J1 ˆ 7.8 Hz, J2 ˆ 5.3 Hz,
1H, a-CH, Pro), 3.88 (s, 2H, CH2-COO), 3.61 3.38 (m, 2H, d-CH2, Pro),
2.30 2.13 (m, 1H, b-CH2a, Pro), 2.02 1.81 (m, 3H, b-CH2b, g-CH2, Pro),
1.43 (s, 9H, tBu); MS (EI): m/z: calcd for C25H29NO6: 439.1995; found:
439.2021.
Boc-Phe-OPAOB (11d): Purification by flash chromatography using n-
hexane/ethyl acetate 4:1yielded a colorless solid (1.63 g, 81%). M.p. 66 8C;
[a]2D0 ˆ À1.1 (c ˆ 1.0 in CHCl3); Rf ˆ 0.38 (n-hexane/ethyl acetate 4:1);
1H NMR (500 MHz, CDCl3): d ˆ 7.38 7.21(m, 10H, aromatic), 7.04 7.02
(m, 4H, aromatic), 5.11 (d, 2J ˆ 12.4 Hz, 1H, CH2a-O), 5.04 (d, 2J ˆ 12.4 Hz,
1H, CH2b-O), 5.00 (d, 3J ˆ 7.2 Hz, 1H, NH), 4.61 4.59 (m, 1H, a-CH, Phe),
3.85 (s, 2H, CH2-COO), 3.06 3.04 (m, 2H, b-CH2, Phe), 1.41 (s, 9H, tBu);
Aloc-Val-Ala-OPAOB (4a): Purification by flash chromatography using n-
hexane/ethyl acetate 2:1yielded a colorless solid (0.354 g (1equiv
ˆ
1.00 mmol), 71 %). M.p. 1388C; [a]2D0 ˆ À17.5 ( c ˆ 1.0 in CHCl3); Rf ˆ 0.26
(n-hexane/ethyl acetate 2:1); 1H NMR (400 MHz, CDCl3): d ˆ 7.37 7.31
(m, 7H, C6H5, OCCHCH, aromatic), 7.06 7.04 (m, 2H, OCCH, aromatic),
13
ˆ
ˆ
6.71(br, 1H, NH, amide), 5.94 5.84 (m, 1H, C H CH2), 5.50 (br, 1H, NH,
C NMR (125.7 MHz, CDCl3): d ˆ 171.76, 169.69 (2  C O, ester), 155.15
2
ˆ
ˆ
carbamate), 5.31 5.18 (m, 2H, CH CH2), 5.16 (d, J ˆ 12.4 Hz, 1H, CH2a-
(C O, carbamate), 150.81 (Cq-O, aromatic), 135.85, 133.28, 133.08 (3 Â Cq,
2
O, benzyl), 5.09 (d, J ˆ 12.4 Hz, 1H, CH2b-O, benzyl), 4.64 4.55 (m, 3H,
aromatic), 129.78, 129.35, 128.81, 128.61, 127.46, 121.68 (CH, aromatic),
80.04 (Cq, tBu), 66.42 (CH2-O), 54.53 (a-CH), 41.44 (CH2-COO), 38.32 (b-
CH2), 28.35 (CH3, tBu); MS (EI): m/z: calcd: 489.2151; found: 489.2171;
elemental analysis calcd (%) for C29H21NO6 (489.6): C 71.15, H 6.38, N 2.86;
found: C 71.02, H 6.44, N 2.55.
CH2-O, allyl, a-CH, Ala), 4.03 4.01(m, 1H, a-CH, Val), 3.85 (s, 2H, CH2-
COO), 2.07 2.04 (m, 1H, b-CH, Val), 1.38 (d, 3J ˆ 7.2 Hz, 3H, CH3, Ala),
0.94 (d, 3J ˆ 6.7 Hz, 3H, CH3, Val), 0.91(d, 3J ˆ 6.7 Hz, 3H, CH3, Val);
13
ˆ
C NMR (100.6 MHz, CDCl3): d ˆ 172.45, 171.00 (2  C O, ester), 169.87
ˆ
ˆ
(C O, amide), 156.28 (C O, carbamate), 150.74 (Cq-O, aromatic), 133.28,
Synthesis of N-terminally deprotected amino acid PAOB esters: TFA
(3.00 mL, 40.0 mmol) was added at 08C to a solution of N-tert-butyloxy-
carbonyl-l-amino acid-4-phenylacetoxybenzyl ester 11a d (2.0 mmol) in
CH2Cl2 (30 mL) and the mixture was stirred at room temperature for 2 h.
TFA and the solvent were coevaporated with toluene under reduced
pressure. The residue was triturated five times with diethyl ether to yield
the desired compound.
132.60 (2 Â Cq, aromatic), 132.91 (CH, allyl), 129.43, 129.28, 128.75, 127.41,
ˆ
121.69 (CH, aromatic), 117.78 (CH CH2), 66.47 (CH2-O, benzyl), 65.84
(CH2-O, allyl), 60.08 (a-CH, Ala), 48.07 (a-CH, Val), 41.38 (CH2-COO),
31.31 (b-CH, Val), 19.11 (CH3, Ala), 18.04, 17.79 (2 Â CH3, Val); MS (EI):
m/z: calcd: 496.221; found: 496.220; elemental analysis calcd (%) for
C27H32N2O7 (496.6): C 65.31, H 6.50, N 5.64; found: C 65.27, H 6.55, N 5.55.
Aloc-Ser-Ile-OPAOB (4b): Purification by flash chromatography using n-
H-Ala-OPAOB ¥ TFA (12a): Colorless solid (0.760 g, 89%); m.p. 698C;
[a]2D0 ˆ À2.0 (c ˆ 1.0 in CHCl3); Rf ˆ 0.22 (n-hexane/ethyl acetate 1:4 with
2 vol.% triethylamine); 1H NMR (250 MHz, CDCl3): d ˆ 8.23 (br, 3H,
NH3), 7.37 7.24 (m, 7H, C6H5, OCCHCH, aromatic), 7.04 6.99 (m, 2H,
OCCH, aromatic), 5.12 (d, 2J ˆ 15.0 Hz, 1H, CH2a-O), 5.07 (d, 2J ˆ 15.0 Hz,
1H, CH2b-O), 4.00 (q, 3J ˆ 7.1Hz, 1H, a-CH, Ala), 3.85 (s, 2H, CH2-COO),
1.51 (d, 3J ˆ 7.1Hz, 3H, b-CH3, Ala); MS (FAB, 3-NBA/TFA): m/z: calcd
hexane/ethyl acetate 3:2 yielded
a
colorless oil (0.270 g (1equiv ˆ
0.60 mmol), 81%). [a]2D0 ˆ À31.3 (c ˆ 1.0 in CHCl3); Rf ˆ 0.22 (n-hexane/
ethyl acetate 3:2); 1H NMR (400 MHz, CDCl3): d ˆ 7.41 7.29 (m, 7H,
C6H5, OCCHCH, aromatic), 7.21(d, 3J ˆ 7.7 Hz, 1H, NH, amide), 7.06
3
7.04 (m, 2H, OCCH, aromatic), 5.67 (d, J ˆ 7.2 Hz, 1H, NH, carbamate),
5.18 (d, 2J ˆ 12.3 Hz, 1H, CH2a-O), 5.09 (d, 2J ˆ 12.3 Hz, 1H, CH2b-O),
4.57 4.54 (m, 1H, a-CH), 4.18 (br, 1H, b-CH2a, Ser), 4.02 (d, 3J ˆ 10.0 Hz,
1H, b-CH2b, Ser), 3.85 (s, 2H, CH2-COO), 3.62 3.60 (m, 1H, a-CH), 3.49
(br, 1H, OH), 1.93 1.90 (m, 1H, b-CH, Ile), 1.44 (s, 9H, tBu), 1.37 1.29
(m, 1H, g-CH2a, Ile), 1.17 1.08 (m, 1 H, g-CH2b, Ile), 0.92 0.80 (m, 6H,
‡
for C20H20F3NO6: 314.139, found: 314.146 [M] .
H-Ile-OPAOB ¥ TFA (12b): Colorless solid (0.680 g, 73%); m.p. 658C;
[a]2D0 ˆ ‡2.3 (c ˆ 1.0 in CHCl3); Rf ˆ 0.31( n-hexane/ethyl acetate 1:2 with 2
vol.% triethylamine); 1H NMR (250 MHz, CDCl3): d ˆ 8.23 (br, 3H, NH3),
7.37 7.27 (m, 7H, C6H5, OCCHCH, aromatic), 7.06 6.99 (m, 2H, OCCH,
aromatic), 5.18 (d, 2J ˆ 10.0 Hz, 1H, CH2a-O), 5.07 (d, 2J ˆ 10.0 Hz, 1H,
CH2b-O), 3.95 3.92 (m, 1H, a-CH, Ile), 3.85 (s, 2H, CH2-COO), 2.06 1.92
(m, 1H, b-CH, Ile), 1.49 1.18 (m, 2H, g-CH2, Ile), 0.95 0.84 (m, 6H, 2 Â
CH3, Ile); MS (EI): m/z: calcd for C23H26F3NO6: 356.186; found: 356.184
2  CH3, Ile); 13C NMR (100.6 MHz, CDCl3): d ˆ 171.60, 171.47 (2  C O,
ˆ
ˆ
ˆ
ester), 169.86 (C O, amide), 156.14 (C O, carbamate), 150.71 (Cq-O,
aromatic), 133.26, 132.90 (2 Â Cq, aromatic), 129.56, 129.27, 128.73, 127.38
(CH, aromatic), 80.39 (Cq, tBu), 66.36 (CH2-O), 62.67 (b-CH2, Ser), 56.75
(a-CH, Ile), 54.52 (a-CH, Ser), 41.35 (CH2-COO), 37.41( b-CH, Ile), 28.25
(CH3, tBu), 24.83 (g-CH2, Ile), 15.57, 11.50 (2 Â CH3, Ile); MS (EI): m/z:
calcd for C29H38N2O8: 542.263; found: 542.264.
‡
[M] .
H-Phe-OPAOB ¥ TFA (12d): Colorless solid (0.917 g, 91%); m.p. 1178C;
[a]2D0 ˆ ‡5.5 (c ˆ 1.0 in CH3OH); Rf ˆ 0.67 (n-hexane/ethyl acetate 1:2 with
2 vol.% triethylamine); 1H NMR (250 MHz, CD3OD): d ˆ 7.40 7.26 (m,
10H, aromatic), 7.17 7.03 (m, 4H, aromatic), 5.25 (d, 2J ˆ 14.4 Hz, 1H,
CH2a-O), 5.18 (d, 2J ˆ 14.4 Hz, 1H, CH2b-O), 4.43 (t, 3J ˆ 6.8 Hz, 1H, a-CH,
Boc-Thr-Phe-OPAOB (4d): Purification by flash chromatography using n-
hexane/ethyl acetate 1:1 yielded a colorless solid (0.230 g (1 equiv ˆ
0.39 mmol), 85%). M.p. 1418C; [a]2D0 ˆ À61.3 (c ˆ 1.0 in CHCl3); Rf ˆ
0.43 (n-hexane/ethyl acetate 1:1); 1H NMR (500 MHz, CDCl3): d ˆ 7.40
7.30 (m, 7H, C6H5, PAOB, CCH, Phe, aromatic), 7.26 (d, 3J ˆ 7.6 Hz, 2H,
OCCHCH, aromatic), 7.06 7.03 (m, 5H, OCCH, PAOB, Phe, aromatic),
3
Phe), 3.92 (s, 2H, CH2-COO), 3.19 (d, J ˆ 6.8 Hz, 2H, b-CH2, Phe); MS
(EI): m/z: calcd: 390.171; found: 390.169; elemental analysis calcd (%) for
C26H24F3NO6 (504.6): C 62.03, H 4.80, N 2.78; found C 62.06, H 4.82, N 2.42.
3
5.42 (d, J ˆ 7.7 Hz, 1H, NH, carbamate), 5.12 (d, 2J ˆ 12.2 Hz, 1H, CH2a-
O), 5.07 (d, 2J ˆ 12.2 Hz, 1H, CH2b-O), 4.61 4.59 (m, 1H, a-CH, Phe),
4.28 4.26 (m, 1H, b-CH, Thr), 4.06 (d, 3J ˆ 7.2 Hz, 1H, a-CH, Thr), 3.86 (s,
2H, CH2-COO), 3.12 (dd, 2J ˆ 13.8 Hz, 3J ˆ 5.9 Hz, 1H, b-CH2a, Phe), 3.03
H-Pro-OPAOB ¥ HCl (12c): A saturated solution of HCl in diethyl ether
(10 mL) was added at 08C to a solution of Boc-Pro-OPAOB (11c; 0.530 g,
1.20 mmol) in CH2Cl2 (30 mL) and the mixture was stirred at 08C for
2
3
(dd, J ˆ 13.8 Hz, J ˆ 6.7 Hz, 1H, b-CH2b, Phe), 1.43 (s, 9H, tBu), 1.12 (d,
Chem. Eur. J. 2002, 8, No. 15
¹ WILEY-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002
0947-6539/02/0815-3369 $ 20.00+.50/0
3369

F. Eisele, J. Kuhlmann and H. Waldmann
FULL PAPER
3J ˆ 6.4 Hz, 3H, g-CH3, Thr); 13C NMR (125.7 MHz, CDCl3): d ˆ 171.00,
ˆ
ˆ
amide), 155.86 (C O, carbamate), 133.63 (CH, allyl), 116.87 (CH CH2),
64.36 (CH2-O, allyl), 59.65 (a-CH, Ala), 47.43 (a-CH, Val), 30.48 (b-CH,
Val), 19.11 (CH3, Ala), 18.04, 17.79 (2 Â CH3, Val); MS (EI): m/z: calcd:
272.137; found: 272.139; elemental analysis calcd (%) for C12H20N2O5
(272.3): C 52.96, H 7.41, N 10.29; found C 52.81, H 7.38, N 10.38.
ˆ
ˆ
169.83 (3 Â C O, ester, amide), 156.24 (C O, carbamate), 150.85 (Cq-O,
aromatic), 135.55, 133.31, 132.67 (3 Â Cq, aromatic), 129.79, 129.29, 129.23,
128.76, 128.61, 127.42, 127.17, 121.68 (CH, aromatic), 80.29 (Cq, tBu), 66.88
(b-CH, Thr), 66.60 (CH2-O), 58.09 (a-CH, Phe), 53.34 (a-CH, Thr), 41.40
(CH2-COO), 37.84 (b-CH2, Phe), 28.28 (CH3, tBu), 18.07 (g-CH3, Thr); MS
Boc-Ser-Ile-OH (6b): Aloc-Ser-Ile-OPAOB (4b; 0.150 g, 0.28 mmol) was
suspended in phosphate buffer (90 mL) and methanol (10 mL) and
immobilized penicillin G acylase (300 U) was added. The crude product
was purified by flash chromatography using n-hexane/ethyl acetate 1:1 with
2 vol.-% acetic acid to yield a colorless oil (0.070 g, 80%). [a]2D0 ˆ À6.6 (c ˆ
0.5 in CH3OH); Rf ˆ 0.15 (n-hexane/ethyl acetate 1:1 with 2 vol.-% acetic
‡
(FAB, 3-NBA): m/z: calcd: 591.2706; found: 591.2732 [M‡H] ; elemental
analysis calcd (%) for C33H39N2O8 (590.7): C 67.10, H 6.48, N 4.74; found: C
66.72, H 6.47, N 4.63.
Aloc-Val-Phe-OPAOB (4e): Purification by flash chromatography using n-
hexane/ethyl acetate 2:1yielded a colorless solid (0.770 g (1equiv
ˆ
2.00 mmol), 67%). M.p. 1448C; [a]2D0 ˆ À4.0 (c ˆ 1.0 in CHCl3); Rf ˆ 0.20
(n-hexane/ethyl acetate 2:1); 1H NMR (500 MHz, CDCl3): d ˆ 7.39 7.17
(m, 10H, aromatic), 7.05 6.98 (m, 4H, aromatic), 6.45 (d, 3J ˆ 7.6 Hz, 1H,
1
3
acid); H NMR (400 MHz, [D6]DMSO): d ˆ 7.69 (d, J ˆ 8.4 Hz, 1H, NH,
amide), 6.75 (d, 3J ˆ 8.1Hz, 1H, NH, carbamate), 4.22 4.18 (m, 1H, a-CH,
Ile), 4.03 3.98 (m, 1H, a-CH, Ser), 3.55 (dd, 2J ˆ 11.0 Hz, 3J ˆ 4.9 Hz, 1H,
b-CH2a, Ser), 3.49 (dd, 2J ˆ 11.0 Hz, 3J ˆ 6.7 Hz, 1H, b-CH2b, Ser), 1.79
1.72 (m, 1 H, b-CH, Ile), 1.42 1.40 (m, 1 H, g-CH2a, Ile), 1.37 (s, 9H, tBu),
1.18 1.11 (m, 1H, g-CH2b, Ile), 0.85 0.79 (m, 6H, 2 Â CH3, Ile); 13C NMR
ˆ
ˆ
NH, amide), 5.94 5.86 (m, 1H, CH CH2), 5.37 5.19 (m, 3H, CH CH2,
NH, carbamate), 5.11 (d, 2J ˆ 12.2 Hz, 1H, CH2a-O, benzyl), 5.03 (d, 2J ˆ
12.2 Hz, 1H, CH2b-O, benzyl), 4.90 (dt, 3J1 ˆ 7.6 Hz, 3J2 ˆ 6.1Hz, 1H, a-CH,
Phe), 4.58 4.49 (m, 2H, CH2-O, allyl), 4.02 3.99 (m, 1H, a-CH, Val), 3.86
(s, 2H, CH2-COO), 3.07 (d, 3J ˆ 6.1Hz, b-CH2, Phe), 2.07 2.01(m, 1H, b-
ˆ
ˆ
(100.6 MHz, [D6]DMSO): d ˆ 172.94 (C O, carboxylic acid), 170.54 (C O,
ˆ
amide), 155.44 (C O, carbamate), 78.46 (Cq, tBu), 61.79 (b-CH2, Ser), 56.27
CH, Val), 0.90 (d, 3J ˆ 6.7 Hz, 3H, CH3, Val), 0.85 (d, 3J ˆ 6.7 Hz, 3H, CH3,
(2 Â a-CH), 36.94 (b-CH, Ile), 28.24 (CH3, tBu), 24.73 (g-CH2, Ile), 15.48,
11.39 (2 Â CH3, Ile); MS (EI): m/z: calcd for C14H18N2O6: 318.179; found:
318.180.
13
ˆ
Val); C NMR (125.7 MHz, CDCl3): d ˆ 171.03, 170.91 (2  C O, ester),
ˆ
ˆ
169.81 (C O, amide), 156.15 (C O, carbamate), 150.81 (Cq-O, aromatic),
135.38, 133.28, 132.59 (3 Â Cq, aromatic), 132.63 (CH, allyl), 129.79, 129.28,
129.22, 128.75, 128.60, 127.40, 127.15, 121.65 (CH, aromatic), 117.77
Boc-Ala-Pro-OH (6c): Boc-Ala-Pro-OPAOB (4c; 0.135 g, 0.26 mmol) was
suspended in phosphate buffer (90 mL) and methanol (10 mL) and
immobilized penicillin G acylase (500 U) was added. The crude product
was purified by recrystallisation using n-hexane/ethyl acetate to yielld
colorless solid (0.061g, 81%). M.p. 154 8C; [a]2D0 ˆ À95.0 (c ˆ 1.0 in
CH3OH); Rf ˆ 0.11 (n-hexane/ethyl acetate 1:1); 1H NMR (500 MHz,
CD3OD): d ˆ 4.48 4.45 (m, 1H, a-CH, Pro), 4.37 (q, 3J ˆ 7.0 Hz, 1H, a-
ˆ
(CH CH2), 66.54 (CH2-O, benzyl), 65.83 (CH2-O, allyl), 60.10 (a-CH,
Phe), 53.12 (a-CH, Val), 41.37 (CH2-COO), 37.89 (b-CH2, Phe), 31.09 (b-
CH, Val), 19.09, 17.68 (2 Â CH3, Val); MS (EI): m/z: calcd: 572.252; found:
572.251; elemental analysis calcd (%) for C33H36N2O7 (572.7): C 69.21, H
6.34, N 4.89; found C 68.98, H 6.33, N 4.73.
Boc-Ala-Pro-OPAOB (4c): HOBt (63 mg, 0.40 mmol) and DIC (60 mL,
0.33 mmol) were added at 08C to a solution of Boc-Ala-OH (9a; 63 mg,
0.33 mmol) in CH2Cl2 (20 mL). Then H-Pro-OPAOB ¥ HCl (12c; 125 mg,
0.33 mmol) and triethylamine (50 mL, 0.33 mmol) were added and the
mixture was stirred at room temperature for 16 h. Then the solution was
extracted with hydrochloric acid (1m, 10 mL) and water (10 mL) and the
organic layer was dried with MgSO4. After filtration, the solvent was
removed under reduced pressure and the residue was purified by flash
chromatography on silica gel using n-hexane/ethyl acetate 1:1 to yield a
colorless oil (0.148 g, 87%). [a]2D0 ˆ À56.8 (c ˆ 1.0 in CHCl3); Rf ˆ 0.59 (n-
hexane/ethyl acetate 1:1); 1H NMR (250 MHz, CDCl3): d ˆ 7.39 7.30 (m,
7H, C6H5, OCCHCH, aromatic), 7.06 (d, 3J ˆ 8.6 Hz, 2H, OCCH,
CH, Ala), 3.80 3.76 (m, 1H, d-CH2a, Pro), 3.67 3.62 (m, 1H, d-CH2b
,
Pro), 2.29 2.23 (m, 1H, b-CH2a, Pro), 2.07 1.99 (m, 3H, b-CH2b, g-CH2,
Pro), 1.43 (s, 9H, tBu), 1.29 (d, 3J ˆ 7.0 Hz, 3H, b-CH3, Ala); 13C NMR
ˆ
ˆ
(125.7 MHz, CD3OD): d ˆ 175.31 (C O, carboxylic acid), 174.04 (C O,
ˆ
amide), 157.61 (C O, carbamate), 80.50 (Cq, tBu), 60.31( a-CH, Pro), 48.49
(a-CH, Ala), 48.01( d-CH2, Pro), 30.02 (b-CH2, Pro), 28.71(CH 3, tBu),
25.82 (g-CH2, Pro), 17.12 (b-CH3, Ala); MS (EI): m/z: calcd: 286.1529;
found: 286.1517; elemental analysis calcd (%) for C13H22N2O5.H2O (304.4):
C 51.31, H 7.95, N 9.20; found: C 51.51, H 7.84, N 9.05.
Boc-Thr-Phe-OH (6d): Boc-Thr-Phe-OPAOB (4d; 0.100 g, 0.17 mmol)
was suspended in phosphate buffer (90 mL) and methanol (10 mL) and
immobilized penicillin G acylase (500 U) was added. The crude product
was purified by flash chromatography using n-hexane/ethyl acetate 1:2 with
2 vol.-% acetic acid to yield a colorless oil (0.051g, 82%). [ a]2D0 ˆ ‡10.0
(c ˆ 1.0 in CH3OH); Rf ˆ 0.52 (n-hexane/ethyl acetate 1:2 with 2 vol.-%
acetic acid); 1H NMR (500 MHz, CD3OD): d ˆ 7.26 7.17 (m, 5H, Phe,
aromatic), 4.68 4.66 (m, 1H, a-CH, Phe), 4.08 4.06 (m, 1H, b-CH, Thr),
3
aromatic), 5.38 (d, J ˆ 7.2 Hz, 1H, NH, carbamate), 5.19 (d, 2J ˆ 13.8 Hz,
1H, CH2a-O), 5.07 (d, 2J ˆ 13.8 Hz, 1H, CH2b-O), 4.59 (dd, 3J1 ˆ 8.5 Hz,
3J2 ˆ 6.9 Hz, 1H, a-CH, Pro), 4.52 4.40 (m, 1H, a-CH, Ala), 3.84 (s, 2H,
CH2-COO), 3.75 3.68 (m, 1H, d-CH2a, Pro), 3.62 3.53 (m, 1H, d-CH2b
,
Pro), 2.27 2.16 (m, 1H, b-CH2a, Pro), 2.04 1.91 (m, 3H, b-CH2b, g-CH2,
Pro), 1.43 (s, 9H, tBu), 1.79 (d, 3J ˆ 7.3 Hz, 3H, b-CH3, Ala); C28H34N2O7
(510.4).
4.00 3.98 (m, 1H, a-CH, Thr), 3.18 (dd, 2J ˆ 13.8 Hz, J ˆ 5.3 Hz, 1H, b-
3
CH2a, Phe), 3.04 (dd, 2J ˆ 13.8 Hz, 3J ˆ 7.4 Hz, 1H, b-CH2b, Phe), 1.43 (s,
3
9H, tBu), 1.13 (d, J ˆ 6.3 Hz, 3H, g-CH3, Thr); 13C NMR (125.7 MHz,
Enzymatic removal of the PAOB-ester group from the model dipeptides:
Immobilized penicillin G acylase was added to a suspension of N-termi-
nally protected dipeptide-4-phenylacetoxybenzyl ester 4a e in a mixture
of phosphate buffer (0.05m, pH 7) and cosolvent and the reaction mixture
was shaken at room temperature for 24 h. After the enzyme was filtered off
and washed with the cosolvent and water, the organic solvent was removed
under reduced pressure. The resulting aqueous phase was washed with
CH2Cl2 (3 Â 50 mL), adjusted to pH 2 and extracted with ethyl acetate (5 Â
50 mL). The combined ethyl acetate layers were dried with MgSO4, filtered
and the solvent was removed under reduced pressure.
ˆ
ˆ
CD3OD): d ˆ 174.49 (C O, carboxylic acid), 172.88 (C O, amide), 157.83
ˆ
(C O, carbamate), 138.10 (Cq, aromatic), 129.89, 129.37, 127.73 (CH,
aromatic), 80.81(C q, tBu), 68.47 (b-CH, Thr), 61.21 (a-CH, Phe), 55.10 (a-
CH, Thr), 38.43 (b-CH2, Phe), 28.67 (CH3, tBu), 19.87 (g-CH3, Thr); MS
‡
(FAB, 3-NBA): m/z: calcd for [M‡H] : 367.1869; found: 367.1894;
elemental analysis calcd (%) for C18H26N2O6 ¥ H2O (385.4): C 56.25, H
7.34, N 7.29; found: C 56.77, H 7.35, N 7.07.
Aloc-Val-Phe-OH (6e): Aloc-Val-Phe-OPAOB (4e; 0.286 g, 0.50 mmol)
was suspended in phosphate buffer (70 mL) and acetone (30 mL) and
immobilized penicillin G acylase (600 U) was added. The crude product
was purified by recrystallisation using n-hexane/ethyl acetate to yield a
colorless solid (0.068 g, 39%). M.p. 1578C; [a]2D0 ˆ À15.4 (c ˆ 0.5 in
CH3OH); Rf ˆ 0.32 (n-hexane/ethyl acetate 1:1 with 2 vol.-% acetic acid);
1H NMR (500 MHz, [D6]DMSO): d ˆ 12.67 (br, 1H, COOH), 8.12 (d, 3J ˆ
7.8 Hz, 1H, NH, amide), 7.26 7.17 (m, 5H, aromatic), 7.08 (d, 3J ˆ 9.1Hz,
Aloc-Val-Ala-OH (6a): Aloc-Val-Ala-OPAOB (4a; 0.248 g, 0.50 mmol)
was suspended in phosphate buffer (90 mL) and methanol (10 mL) and
immobilized penicillin G acylase (300 U) was added. The crude product
was purified by recrystallisation using n-hexane/ethyl acetate to yield a
colorless solid (0.109 g, 80%). M.p. 1638C; [a]2D0 ˆ À39.2 (c ˆ 1.0 in
CH3OH); Rf ˆ 0.16 (n-hexane/ethyl acetate 2:1with 2 vol.-% acetic acid);
1H NMR (400 MHz, [D6]DMSO): d ˆ 12.43 (br, 1H, COOH), 8.19 (d, 3J ˆ
6.9 Hz, 1H, NH, amide), 7.16 (d, 3J ˆ 5.5 Hz, 1H, NH, carbamate), 5.95
ˆ
1H, NH, carbamate), 5.93 5.86 (m, 1H, C H CH2), 5.30 5.16 (m, 2H,
CH CH2), 4.50 4.41(m, 3H, CH 2-O, a-CH, Phe), 3.84 (dd, 3J1 ˆ 9.0 Hz,
ˆ
5.85 (m, 1H, CH CH2), 5.31 5.16 (m, 2H, CH CH2), 4.47 4.46 (m, 2H,
CH2-O), 4.22 4.15 (m, 1H, a-CH, Ala), 3.89 3.85 (m, 1H, a-CH, Val),
1.99 1.91 (m, 1 H,b-CH, Val), 1.27 (d, 3J ˆ 7.3 Hz, 3H, CH3, Ala), 0.89 (d,
3J2 ˆ 7.4 Hz, 1H, a-CH, Val), 3.04 (dd, 2J ˆ 13.9 Hz, 3J ˆ 5.1Hz, 1H, b-
CH2a, Phe), 2.89 (dd, 2J ˆ 13.9 Hz, 3J ˆ 9.0 Hz, 1H, b-CH2b, Phe), 1.93 1.87
(m, 1H, b-CH, Val), 0.80 0.78 (m, 6H, 2 Â CH3, Val); 13C NMR
ˆ
ˆ
3J ˆ 6.7 Hz, 3H, CH3, Val), 0.84 (d, J ˆ 6.7 Hz, 3H, CH3, Val); 13C NMR
(125.7 MHz, [D6]DMSO): d ˆ 172.75 (C O, carboxylic acid), 170.09
3
ˆ
ˆ
ˆ
ˆ
ˆ
(100.6 MHz, [D6]DMSO): d ˆ 173.98 (C O, carboxylic acid), 170.94 (C O,
(C O, amide), 155.72 (C O, carbamate), 137.44 (Cq, aromatic), 133.59
3370
¹ WILEY-VCH Verlag GmbH, 69451Weinheim, Germany, 2002 0947-6539/02/0815-3370 $ 20.00+.50/0 Chem. Eur. J. 2002, 8, No. 1 5

Lipopeptides
3362 3376
ˆ
(CH, allyl), 129.07, 128.09, 126.34 (CH, aromatic), 116.86 (CH CH2), 64.36
(CH2-O, allyl), 59.91( a-CH, Phe), 53.26 (a-CH, Val), 36.71( b-CH2, Phe),
30.99 (b-CH, Val), 19.10, 18.02 (2 Â CH3, Val); MS (EI): m/z: calcd:
348.169, found: 348.170; elemental analysis calcd (%) for C18H24N2O5
(348.4): C 62.05, H 6.94, N 8.04; found: C 61.76, H 6.84, N 7.32.
(Boc-Arg(Aloc)2-Cys-OPAOB)2 (13b): HOBt (0.196 g, 1.45 mmol) and
DIC (0.19 mL, 1.21 mmol) were added at 08C to a solution of Boc-
Arg(Aloc)2-OH (9h; 0.535 g, 1.21 mmol) in CH2Cl2 (50 mL). Then (H-Cys-
OPAOB ¥ HCl)2 (12e; 0.460 g, 0.61mmol) and triethylamine (0.17 mL,
1.21 mmol) were added and the mixture was stirred at room temperature
for 18 h. The mixture was extracted with hydrochloric acid (1m, 40 mL),
NaHCO3 (1m, 40 mL) and brine (40 mL) and the organic layer was dried
with MgSO4. Then the solvent was removed under reduced pressure and
the residue was purified by flash chromatography on silica gel using n-
hexane/ethyl acetate 1:1 to yield a colorless oil (0.581 g, 62%). [a]2D0 ˆ ‡7.7
(c ˆ 1.0 in CHCl3); Rf ˆ 0.44 (n-hexane/ethyl acetate 1:1); 1H NMR
(400 MHz, CDCl3): d ˆ 9.40 (br, 2H, NH, guanidino), 9.24 (br, 2H, NH,
guanidino), 7.42 (d, 3J ˆ 6.7 Hz, 2H, NH, amide), 7.36 7.28 (m, 10H, C6H5,
aromatic), 7.31(d, 3J ˆ 8.4 Hz, 4H, OCCHCH, aromatic), 7.03 (d, 3J ˆ
(Boc-Cys-OPAOB)2 (11e): DIC (2.17 mL, 13.98 mmol) was added to a
solution of bis(N-tert-butyloxycarbonyl)-l-cystine 9g (3.082 g, 6.99 mmol),
4-(phenylacetoxy)benzyl alcohol 10 (3.390 g, 13.98 mmol) and DMAP
(0.005 g, 0.04 mmol) in CH2Cl2 (100 mL) and the mixture was stirred at
room temperature for 18 h. Then the solution was extracted with hydro-
chloric acid (1m, 2 Â 50 mL) and water (2 Â 50 mL), the organic layer was
dried with MgSO4 and the solvent was removed under reduced pressure.
The product was purified by flash chromatography on silica gel using n-
hexane/ethyl acetate 2:to yield a colorless solid (5.299 g, 86%). M.p. 788C;
[a]2D0 ˆ ‡42.6 (c ˆ 1.0 in CHCl3); Rf ˆ 0.43 (n-hexane/ethyl acetate 2:1);
1H NMR (400 MHz, CDCl3): d ˆ 7.40 7.30 (m, 10H, C6H5, aromatic), 7.35
(d, 3J ˆ 8.4 Hz, 4H, OCCHCH, aromatic), 7.05 (d, 3J ˆ 8.4 Hz, 4H, OCCH,
ˆ
8.4 Hz, 4H, OCCH, aromatic), 6.00 5.87 (m, 4H, CH CH2), 5.80 (d,
3J ˆ 7.8 Hz, 2H, NH, carbamate), 5.35 5.16 (m, 8H, CH CH2), 5.12 (d,
ˆ
2J ˆ 12.4 Hz, 2H, CH2a-O, benzyl), 5.08 (d, 2J ˆ 12.4 Hz, 2H, CH2b-O,
benzyl), 4.81 4.76 (m, 2H, a-CH, Cys), 4.67 (d, 3J ˆ 5.7 Hz, 4H, CH2-O,
allyl), 4.64 4.52 (m, 4H, CH2-O, allyl), 4.32 4.30 (m, 2H, a-CH, Arg),
4.04 3.86 (m, 4H, d-CH2, Arg), 3.84 (s, 4H, CH2-COO), 3.05 3.00 (m,
4H, b-CH2, Cys), 1.81 1.79 (m, 2H, b-CH2a, Arg), 1.72 1.65 (m, 6H, b-
CH2b, g-CH2, Arg), 1.41 (s, 18H, tBu); 13C NMR (100.6 MHz, CDCl3): d ˆ
3
aromatic), 5.42 (d, J ˆ 7.8 Hz, 2H, NH, carbamate), 5.14 (s, 4H, CH2-O),
4.61 4.59 (m, 2H, a-CH), 3.86 (s, 4H, CH2-COO), 3.10 (d, 3J ˆ 5.0 Hz, 4H,
b-CH2), 1.44 (s, 18H, tBu); 13C NMR (100.6 MHz, CDCl3): d ˆ 170.44,
ˆ
ˆ
169.81 (2 Â C O, ester), 154.96 (C O, carbamate), 150.69 (Cq-O, aromatic),
133.21, 132.63 (2 Â Cq, aromatic), 129.63, 129.22, 128.68, 127.33, 121.61 (CH,
aromatic), 80.21(C q, tBu), 66.72 (CH2-O), 52.85 (a-CH), 41.28 (CH2-
COO), 40.91( b-CH2), 28.23 (CH3, tBu); MS (FAB, 3-NBA): m/z: calcd:
888.3; found: 887.8; elemental analysis calcd (%) for C46H52N2O12S2 (889.1):
C 62.15, H 5.90, N 3.15; found: C 62.09, H 5.99, N 3.21.
ˆ
172.21, 169.76 (3 Â C O, 2 Â ester, amide), 163.47 (Cq, guanidino), 160.56,
ˆ
155.68, 155.60 (3 Â C O, carbamate), 150.69 (Cq-O, aromatic), 133.23,
ˆ
132.99 (2 Â Cq, aromatic), 132.61, 131.01 (2 Â CH CH2), 129.51, 129.22,
ˆ
128.67, 128.46, 127.32, 121.61 (CH, aromatic), 119.43, 117.70 (2 Â CH CH2),
79.69 (Cq, tBu), 67.65, 66.73, 66.07 (CH2-O, 2 Â allyl, 1 Â benzyl), 53.70 (a-
CH, Cys), 51.89 (a-CH, Arg), 44.05 (CH2-COO), 41.28 (d-CH2, Arg), 39.66
(b-CH2, Cys), 28.93 (b-CH2, Arg), 28.28 (CH3, tBu), 24.60 (g-CH2, Arg);
(H-Cys-OPAOB ¥ HCl)2 (12e): A saturated solution of HCl in diethyl ether
(100 mL) was added at 08C to a solution of (Boc-Cys-OPAOB)2 (11e;
4.501g, 5.20 mmol) in diethyl ether (20 mL) and the mixture was stirred at
08C for 45 min and at room temperature for 1h. Then the excess HCl and
the solvent were removed under reduced pressure. The residue was
triturated three times with diethyl ether (20 mL) to yield the desired
compound as a colorless solid (3.759 g, 95%). M.p. 1408C; [a]2D0 ˆ À10.8
‡
MS (FAB, 3-NBA): m/z: calcd for [M‡H] : 1538.7; found: 1538.4;
elemental analysis calcd (%) for C74H92N10O22S2 (1537.7): C 57.80, H 6.03,
N 9.11; found: C 57.86, H 6.04, N 8.79.
Boc-Ile-Cys(Pal)-OPAOB (14a): DTT (0.474 g, 3.08 mmol) and triethyl-
amine (0.21mL, 1.54 mmol) were added under argon to a solution of (Boc-
Ile-Cys-OPAOB)2 (13a; 0.858 g, 0.77 mmol) in CH2Cl2 (50 mL) and the
mixture was stirred at room temperature for 90 min. Then the solution was
extracted with hydrochloric acid (1m, 3 Â 30 mL) and the organic layer was
dried with MgSO4 and filtered. At 08C triethylamine (0.21mL, 1.54 mmol)
and palmitoyl chloride (0.845 g, 3.08 mmol) were added to the above
solution and the mixture was stirred at room temperature for 2 h. Then the
solvent was removed under reduced pressure and the product was isolated
by flash chromatography on silica gel using n-hexane/ethyl acetate 4:1to
yield a colorless solid (1.017 g, 83%). M.p. 598C; [a]2D0 ˆ À0.7 (c ˆ 1.0 in
CHCl3); Rf ˆ 0.42 (n-hexane/ethyl acetate 4:1); 1H NMR (400 MHz,
CDCl3): d ˆ 7.38 7.30 (m, 7H, C6H5, OCCHCH, aromatic), 7.06 (d, 3J ˆ
1
3
(c ˆ 1.0 in CH3OH); H NMR (500 MHz, CDCl3): d ˆ 7.43 (d, J ˆ 8.6 Hz,
4H, OCCHCH, aromatic), 7.37 7.28 (m, 10H, C6H5, aromatic), 7.06 (d,
3J ˆ 8.6 Hz, 4H, OCCH, aromatic), 5.28 (d, 2J ˆ 12.1 Hz, 2H, CH2a-O), 5.21
(d, 2J ˆ 12.1 Hz, 2H, CH2b-O), 4.40 (dd, 3J1 ˆ 6.1Hz, 3J2 ˆ 5.5 Hz, 2H, a-
CH), 3.89 (s, 4H, CH2-COO), 3.31 3.19 (m, 4H, b-CH2); 13C NMR
ˆ
(125.7 MHz, CD3OD): d ˆ 171.99, 168.76 (2  C O, ester), 152.56 (Cq-O,
aromatic), 135.02, 133.80 (2 Â Cq, aromatic), 131.39, 130.44, 129.69, 128.32,
122.99 (CH, aromatic), 68.78 (CH2-O), 52.81( a-CH), 41.81 (CH2-COO),
‡
37.52 (b-CH2); MS (FAB, 3-NBA): m/z: calcd for [M À 2HCl‡H] :
689.1991; found: 689.2004; C36H36N2O8S2 ¥ 2HCl (761.7).
(Boc-Ile-Cys-OPAOB)2 (13a): EEDQ (1.194 g, 4.83 mmol) was added at
08C to a solution of Boc-Ile-OH (9b; 1.117 g, 4.83 mmol) in CH2Cl2
(75 mL). Then (H-Cys-OPAOB ¥ HCl)2 (12e; 1.840 g, 2.42 mmol) and
triethylamine (0.67 mL, 4.83 mmol) were added and the mixture was
stirred at room temperature for 18 h. The mixture was extracted with
hydrochloric acid (1m, 50 mL), NaHCO3 (1m, 50 mL) and water (50 mL)
and the organic layer was dried with MgSO4. Then the solvent was removed
under reduced pressure and the residue was purified by flash chromatog-
raphy on silica gel using n-hexane/ethyl acetate 2:1to yield a colorless solid
(2.111 g, 78%). M.p. 1398C; [a]2D0 ˆ ‡25.8 (c ˆ 1.0 in CHCl3); Rf ˆ 0.34 (n-
hexane/ethyl acetate 2:1); 1H NMR (400 MHz, CDCl3): d ˆ 7.51(d, 3J ˆ
7.2 Hz, 2H, NH, amide), 7.36 7.28 (m, 10H, C6H5, aromatic), 7.31(d, 3J ˆ
8.4 Hz, 4H, OCCHCH, aromatic), 7.03 (d, 3Jˆ8.4 Hz, 4H, OCCH, aromatic),
3
8.5 Hz, 2H, OCCH, aromatic), 6.72 (d, J ˆ 6.5 Hz, 1H, NH, amide), 5.11
(s, 2H, CH2-O), 5.05 (d, 3J ˆ 8.2 Hz, 1H, NH, carbamate), 4.79 4.74 (m,
1H, a-CH, Cys), 3.99 3.96 (m, 1H, a-CH, Ile), 3.86 (s, 2H, CH2-COO),
3.37 (dd, 2J ˆ 13.1 Hz, 3J ˆ 6.1Hz, 1H, b-CH2a, Cys), 3.32 (dd, 2J ˆ 13.1 Hz,
3
3J ˆ 4.6 Hz, 1H, b-CH2b, Cys), 2.52 (t, J ˆ 7.5 Hz, 2H, a-CH2, Pal), 1.93
1.81 (m, 1 H,b-CH, Ile), 1.68 1.57 (m, 3H, g-CH2a, Ile, b-CH2, Pal), 1.44 (s,
9H, tBu), 1.25 (s, 24H, CH2, Pal), 1.17 1.08 (m, 1 H, g-CH2b, Ile), 0.91
0.86 (m, 9H, CH3, Ile, CH3, Pal); 13C NMR (100.6 MHz, CDCl3): d ˆ 199.37
ˆ
ˆ
ˆ
(C O, thioester), 171.45 (C O, amide), 169.82, 169.59 (2 Â C O, ester),
ˆ
155.56 (C O, carbamate), 150.80 (Cq-O, aromatic), 133.28, 132.63 (2 Â Cq,
aromatic), 129.73, 129.28, 128.74, 127.40, 121.68 (CH, aromatic), 79.83 (Cq,
tBu), 66.97 (CH2-O), 59.06 (a-CH, Cys), 52.41( a-CH, Ile), 43.96 (CH2-
COO), 41.38 (b-CH2, Cys), 37.40 (b-CH, Ile), 31.93 (a-CH2, Pal), 30.33,
29.69, 29.43, 29.37, 29.22, 28.92, 25.52, 24.72 (CH2, Pal), 28.31(CH 3, tBu),
22.70 (g-CH2, Ile), 15.35, 14.15, 11.58 (3 Â CH3, Ile, Pal); MS (EI): m/z:
calcd: 796.470; found: 796.475; elemental analysis calcd (%) for
C45H67N2O8S (796.1): C 67.81, H 8.60, N 3.51; found: C 67.81, H 8.55, N 3.70.
3
2
5.40 (d, J ˆ 8.9 Hz, 2H, NH, carbamate), 5.14 (d, J ˆ 12.2 Hz, 2H, CH2a-
O), 5.08 (d, 2J ˆ 12.2 Hz, 2H, CH2b-O), 4.91 4.87 (m, 2H, a-CH, Cys),
4.15 4.11 (m, 2H, a-CH, Ile), 3.84 (s, 4H, CH2-COO), 3.06 2.96 (m, 4H,
b-CH2, Cys), 1.91 1.79 (m, 2H,b-CH, Ile), 1.59 1.52 (m, 2H, g-CH2a, Ile),
1.39 (s, 18H, tBu), 1.19 1.09 (m, 2H, g-CH2b, Ile), 0.92 (d, 3J ˆ 6.8 Hz, 6H,
g-CH3, Ile), 0.85 (t, 3J ˆ 7.2 Hz, 6H, d-CH3, Ile); 13C NMR (100.6 MHz,
Boc-Arg(Aloc)2-Cys(Pal)-OPAOB (14b): DTT (0.109 g, 0.71 mmol) and
triethylamine (48 mL, 0.36 mmol) were added under argon to a solution of
(Boc-Arg(Aloc)2-Cys-OPAOB)2 (13b; 0.273 g, 0.18 mmol) in CH2Cl2
(20 mL)and the mixture was stirred at room temperature for 90 min. Then
the solution was extracted with hydrochloric acid (1m, 3 Â 20 mL) and the
organic layer was dried with MgSO4, and filtered. At 08C to the solution
was added triethylamine (48 mL, 0.36 mmol) and palmitoyl chloride
(0.244 g, 0.89 mmol) and the mixture was stirred at room temperature for
2 h. Then the solvent was removed under reduced pressure and the product
ˆ
ˆ
ˆ
CDCl3): d ˆ 172.27 (C O, amide), 169.79 (2  C O, ester), 156.43 (C O,
carbamate), 150.72 (Cq-O, aromatic), 133.24, 132.64 (2 Â Cq, aromatic),
129.59, 129.23, 128.69, 127.34, 121.61 (CH, aromatic), 79.69 (Cq, tBu), 66.70
(CH2-O), 58.80 (a-CH, Cys), 52.42 (a-CH, Ile), 41.31 (CH2-COO), 39.27 (b-
CH2, Cys), 37.07 (b-CH, Ile), 28.27 (CH3, tBu), 24.74 (g-CH2, Ile), 15.26,
‡
10.97 (2  CH3, Ile); MS (FAB, 3-NBA): m/z: calcd for [M‡Na] : 1137.5;
found: 1136.9; elemental analysis calcd (%) for C58H74N4O14S2 (1115.4): C
62.46, H 6.69, N 5.02; found: C 62.77, H 6.50, N 4.68.
Chem. Eur. J. 2002, 8, No. 15
¹ WILEY-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002
0947-6539/02/0815-3371 $ 20.00+.50/0
3371

F. Eisele, J. Kuhlmann and H. Waldmann
FULL PAPER
was isolated by flash chromatography on silica gel using n-hexane/ethyl
acetate 2:1to yield a colorless solid (0.294 g, 81%). M.p. 44 8C; [a]2D0 ˆ ‡8.3
(c ˆ 1.0 in CHCl3); Rf ˆ 0.43 (n-hexane/ethyl acetate 2:1); 1H NMR
(500 MHz, CDCl3): d ˆ 9.44 (br, 1H, NH, guanidino), 9.26 (br, 1H, NH,
guanidino), 7.38 7.26 (m, 7H, C6H5, OCCHCH, aromatic), 7.10 (d, 3J ˆ
7.2 Hz, 1H, NH, amide), 7.05 (d, 3J ˆ 8.5 Hz, 2H, OCCH, aromatic), 6.02
Pal), 1.27 (s, 24H, CH2, Pal), 0.89 (t, 3J ˆ 6.9 Hz, 3H, CH3, Pal); 13C NMR
ˆ
ˆ
(125.7 MHz, CD3OD): d ˆ 199.58 (C O, thioester), 171.55 (C O, amide),
ˆ
170.62, 170.08 (2 Â C O, ester), 163.06 (Cq, guanidino), 161.28, 156.08, (2 Â
ˆ
C O, carbamate), 152.19 (Cq-O, aromatic), 134.93, 134.38 (2 Â Cq, aro-
ˆ
matic), 134.01, 132.53 (2 Â CH CH2), 130.83, 130.62, 130.33, 129.60, 128.22,
ˆ
122.73 (CH, aromatic), 119.75, 118.47 (2 Â CH CH2), 69.09, 67.77, 67.29
5.89 (m, 2H, CH CH2), 5.59 (d, 3J ˆ 8.3 Hz, 1H, NH, carbamate), 5.37
(CH2-O, 2 Â allyl, 1 Â benzyl), 53.70 (a-CH, Cys), 53.42 (a-CH, Arg), 45.03
(CH2-COO), 44.62 (d-CH2, Arg), 41.80 (b-CH2, Cys), 32.96 (a-CH2, Pal),
30.67, 30.66, 30.60, 30.44, 30.37, 30.26, 30.15, 29.85, 26.43, 25.95, 23.63 (CH2,
Pal), 28.92 (b-CH2, Arg), 24.47 (g-CH2, Arg), 14.44 (CH3, Pal); MS (FAB,
ˆ
2
ˆ
5.18 (m, 4H, CH CH2), 5.11 (d, J ˆ 12.7 Hz, 1H, CH2a-O, benzyl), 5.09 (d,
2J ˆ 12.7 Hz, 1H, CH2b-O, benzyl), 4.72 4.70 (m, 1H, a-CH, Cys), 4.63 (d,
3J ˆ 5.6 Hz, 2H, CH2-O, allyl), 4.64 4.55 (m, 2H, CH2-O, allyl), 4.24 4.21
(m, 1H, a-CH, Arg), 4.04 4.00 (m, 1H, d-CH2a, Arg), 3.90 3.87 (m, 1H,
‡
3-NBA): m/z: calcd for [M À CF3COO] : 909.4922; found: 909.4897;
d-CH2b, Arg), 3.85 (s, 2H, CH2-COO), 3.38 (dd, 2J ˆ 14.1 Hz, J ˆ 5.0 Hz,
C48H70N5O10S ¥ C2F3O2 (1022.2).
3
1H, b-CH2a, Cys), 3.30 (dd, 2J ˆ 14.1 Hz, 3J ˆ 6.4 Hz, 1H, b-CH2b, Cys),
2.49 (t, 3J ˆ 7.5 Hz, 2H, a-CH2, Pal), 1.80 1.78 (m, 1 H, b-CH2a, Arg),
1.71 1.67 (m, 3H, b-CH2b, g-CH2, Arg), 1.61 1.58 (m, 2H, b-CH2, Pal),
Boc-Thr-Ile-Cys(Pal)-OPAOB (16a): EEDQ (53 mg, 0.22 mmol) was
added at 08C to a solution of Boc-Thr-OH (9 f; 47 mg, 0.22 mmol) in
CH2Cl2 (20 mL). Then H-Ile-Cys(Pal)-OPAOB ¥ TFA (15a; 174 mg,
0.22 mmol) and triethylamine (31 mL, 0.22 mmol) were added and the
mixture was stirred at room temperature for 18 h. Then the solvent was
removed under reduced pressure and the residue was purified by flash
chromatography on silica gel using n-hexane/ethyl acetate 2:1to yield a
colorless solid (146 mg, 74%). M.p. 948C; [a]2D0 ˆ À27.4 (c ˆ 1.0 in CHCl3);
Rf ˆ 0.19 (n-hexane/ethyl acetate 2:1); 1H NMR (500 MHz, CDCl3): d ˆ
7.38 7.30 (m, 7H, C6H5, OCCHCH, aromatic), 7.06 (d, 3J ˆ 8.5 Hz, 2H,
OCCH, aromatic), 6.95 (d, 3J ˆ 8.3 Hz, 1H, NH, amide), 6.93 (d, 3J ˆ
7.2 Hz, 1H, NH, amide), 5.51 (d, 3J ˆ 7.9 Hz, 1H, NH, carbamate), 5.11
(s, 2H, CH2-O), 4.76 4.72 (m, 1H, a-CH, Cys), 4.31 4.28 (m, 2H, a-CH,
Thr, b-CH, Thr), 4.10 (d, 3J ˆ 6.6 Hz, 1H, a-CH, Ile), 3.85 (s, 2H, CH2-
COO), 3.55 (br, 1H, OH, Thr), 3.32 (d, 3J ˆ 5.6 Hz, 2H, b-CH2, Cys), 2.53
(t, 3J ˆ 7.6 Hz, 2H, a-CH2, Pal), 1.98 1.91 (m, 1 H, b-CH, Ile), 1.63 1.58
(m, 2H, b-CH2, Pal), 1.45 (s, 9H, tBu), 1.31 1.19 (m, 1 H,g-CH2a, Ile), 1.25
(s, 24H, CH2, Pal), 1.17 (d,3J ˆ 6.4 Hz, 3H, g-CH3, Thr), 1.14 1.08 (m, 1 H,
g-CH2b, Ile), 0.90 0.86 (m, 9H, CH3, Ile, CH3, Pal); 13C NMR (125.7 MHz,
1.44 (s, 9H, tBu), 1.25 (s, 24H, CH2, Pal), 0.88 (t, 3J ˆ 6.9 Hz, 3H, CH3, Pal);
13
ˆ
ˆ
C NMR (125.7 MHz, CDCl3): d ˆ 198.61 (C O, thioester), 171.92 (C O,
ˆ
amide), 169.78, 169.54 (2 Â C O, ester), 163.54 (Cq, guanidino), 160.70,
ˆ
155.68, 155.53 (3 Â C O, carbamate), 150.78 (Cq-O, aromatic), 133.31,
ˆ
133.06 (2 Â Cq, aromatic), 132.74, 131.02 (2 Â CH CH2), 129.60, 129.28,
ˆ
128.74, 127.39, 121.64 (CH, aromatic), 119.62, 117.83 (2 Â CH CH2), 79.81
(Cq, tBu), 67.77, 66.85, 66.18 (CH2-O, 2 Â allyl, 1 Â benzyl), 53.87 (a-CH,
Cys), 52.20 (a-CH, Arg), 44.10 (CH2-COO), 43.96 (d-CH2, Arg), 41.38 (b-
CH2, Cys), 31.92 (a-CH2, Pal), 30.31, 29.69, 29.65, 29.60, 29.44, 29.35, 29.23,
28.75, 25.48, 22.69 (CH2, Pal), 28.94 (b-CH2, Arg), 28.34 (CH3, tBu), 24.60
(g-CH2, Arg), 14.13 (CH3, Pal); MS (FAB, 3-NBA): m/z: calcd for
‡
[M‡H] : 1008.536; found: 1008.535; elemental analysis calcd (%) for
C53H77N5O12S (1008.3): C 63.13, H 7.69, N 6.94; found: C 63.05, H 7.92, N
6.58.
H-Ile-Cys(Pal)-OPAOB ¥ TFA (15a): TFA (0.72 mL, 9.41mmol) was
added at 08C to a solution of Boc-Ile-Cys(Pal)-OPAOB (14a; 0.250 g,
0.31mmol) in CH 2Cl2 (30 mL) and the mixture was stirred at 08C for
45 min and at room temperature for 60 min. TFA and the solvent were
coevaporated with toluene (2 Â 40 mL) under reduced pressure to yield a
colorless solid (0.255 g, 100%). M.p. 928C; [a]2D0 ˆ À2.7 (c ˆ 1.0 in
CH3OH); Rf ˆ 0.06 (n-hexane/ethyl acetate 4:1); 1H NMR (400 MHz,
CD3OD): d ˆ 7.41(d, 3J ˆ 8.6 Hz, 2H, OCCHCH, aromatic), 7.38 7.26 (m,
5H, C6H5, aromatic), 7.06 (d, 3J ˆ 8.6 Hz, 2H, OCCH, aromatic), 5.17 (d,
2J ˆ 12.2 Hz, 1H, CH2a-O), 5.13 (d, 2J ˆ 12.2 Hz, 1H, CH2b-O), 4.68 (dd,
3J1 ˆ 7.2 Hz, 3J2 ˆ 5.6 Hz, 1H, a-CH, Cys), 3.88 (s, 2H, CH2-COO), 3.72 (d,
ˆ
ˆ
CDCl3): d ˆ 199.75 (C O, thioester), 171.78, 170.83 (2  C O, amide),
ˆ
ˆ
169.81, 169.62 (2 Â C O, ester), 156.40 (C O, carbamate), 150.86 (Cq-O,
aromatic), 133.30, 132.62 (2 Â Cq, aromatic), 129.70, 129.29, 128.76, 127.41,
121.70 (CH, aromatic), 80.32 (Cq, tBu), 67.01(CH -O), 66.74 (b-CH, Thr),
2
57.98 (a-CH, Cys), 57.65 (a-CH, Thr), 52.70 (a-CH, Ile), 44.00 (CH2-COO),
41.40 (b-CH2, Cys), 36.71( b-CH, Ile), 31.93 (a-CH2, Pal), 30.18, 29.69,
29.65, 29.61, 29.44, 29.36, 29.24, 28.96, 25.56 24.52 (CH2, Pal), 28.31(CH
,
3
tBu), 22.70 (g-CH2, Ile), 18.23 (g-CH3, Thr), 15.42, 14.13, 11.43 (3 Â CH3,
‡
Ile, Pal); MS (FAB, 3-NBA): m/z: calcd for [M‡H] : 898.5251; found:
3J ˆ 5.2 Hz, 1H, a-CH, Ile), 3.44 (dd, 2J ˆ 14.0 Hz, 3J ˆ 5.6 Hz, 1H, b-CH2a
,
898.5217; elemental analysis calcd (%) for C49H75N3O10S (898.2): C 65.52, H
8.41, N 4.67; found: C 65.05, H 8.74, N 4.20.
Cys), 3.25 (dd, 2J ˆ 14.0 Hz, 3J ˆ 7.2 Hz, 1H, b-CH2b, Cys), 2.53 (t, 3J ˆ
7.1Hz, 2H, a-CH2, Pal), 1.91 1.83 (m, 1 H, b-CH, Ile), 1.62 1.58 (m, 2H,
b-CH2, Pal), 1.55 1.47 (m, 1 H, g-CH2a, Ile), 1.27 (s, 24H, CH2, Pal), 1.21
Boc-Met-Arg(Aloc)2-Cys(Pal)-OPAOB (16b): HOBt (35 mg, 0.27 mmol)
and DIC (33 mL, 0.22 mmol) were added at 08C to a solution of Boc-Met-
OH (9i; 55 mg, 0.22 mmol) in CH2Cl2 (25 mL). Then H-Arg(Aloc)2-
Cys(Pal)-OPAOB ¥ TFA (15b; 225 mg, 0.22 mmol) and triethylamine
(33 mL, 0.22 mmol) were added and the mixture was stirred at room
temperature for 18 h. Then the solvent was removed under reduced
pressure and the residue was purified by flash chromatography on silica gel
using n-hexane/ethyl acetate 2:1to yield a colorless solid (163 mg, 65%).
[a]2D0 ˆ À13.9 (c ˆ 1.0 in CHCl3); Rf ˆ 0.19 (n-hexane/ethyl acetate 2:1);
1H NMR (500 MHz, CDCl3): d ˆ 9.47 (br, 1H, NH, guanidino), 9.29 (br,
1H, NH, guanidino), 7.39 7.32 (m, 7H, C6H5, OCCHCH, aromatic), 7.31
(d, 3J ˆ 6.1Hz, 1H, NH, amide), 7.17 (d, 3J ˆ 7.8 Hz, 1H, NH, amide), 7.05
3
1.11 (m, 1 H,g-CH2b, Ile), 0.96 (d, J ˆ 6.9 Hz, 3H, g-CH3, Ile), 0.92 0.87
(m, 6H, d-CH3, Ile, CH3, Pal); 13C NMR (100.6 MHz, CD3OD): d ˆ 199.85
ˆ
ˆ
ˆ
(C O, thioester), 171.64 (C O, amide), 170.68, 169.44 (2 Â C O, ester),
152.32 (Cq-O, aromatic), 135.04, 134.45 (2 Â Cq, aromatic), 130.87, 130.40,
129.66, 128.28, 122.78 (CH, aromatic), 67.84 (CH2-O), 58.77 (a-CH, Cys),
53.78 (a-CH, Ile), 44.67 (CH2-COO), 41.85 (b-CH2, Cys), 38.07 (b-CH, Ile),
33.05 (a-CH2, Pal), 30.75, 30.52, 30.46, 30.34, 30.22, 29.92, 26.53, 26.06,
25.27 (CH2, Pal), 23.72 (g-CH2, Ile), 14.94, 14.50, 11.77 (3 Â CH3, Ile, Pal);
‡
MS (FAB, 3-NBA): m/z: calcd for [M À CF3COO] : 697.4250; found:
697.4270; C40H60N2O6S ¥ C2F3O2 (810.0).
(d, 3J ˆ 8.5 Hz, 2H, OCCH, aromatic), 6.02 5.89 (m, 2H, CH CH2), 5.37
ˆ
H-Arg(Aloc)2-Cys(Pal)-OPAOB ¥ TFA (15b): TFA (0.62 mL, 8.11 mmol)
was added at 08C to a solution of Boc-Arg(Aloc)2-Cys(Pal)-OPAOB (14b;
0.272 g, 0.27 mmol) in CH2Cl2 (3 mL) and the mixture was stirred at 08C for
45 min and at room temperature for 60 min. TFA and the solvent were
coevaporated with toluene (2 Â 10 mL) under reduced pressure to yield a
colorless solid (0.288 g, quant.). [a]2D0 ˆ À13.7 (c ˆ 1.0 in CH3OH); Rf ˆ 0.03
(n-hexane/ethyl acetate 2:1); 1H NMR (500 MHz, CD3OD): d ˆ 7.40 (d,
2
ˆ
5.19 (m, 4H, CH CH2), 5.11 (d, J ˆ 12.4 Hz, 1H, CH2a-O, benzyl), 5.08 (d,
3
2J ˆ 12.4 Hz, 1H, CH2b-O, benzyl), 4.69 (d, J ˆ 5.7 Hz, 2H, CH2-O, allyl),
4.68 4.64 (m, 1H, a-CH, Cys), 4.62 4.56 (m, 2H, CH2-O, allyl), 4.53 4.50
(m, 1H, a-CH, Arg), 4.27 4.25 (m, 1H, a-CH, Met), 3.99 3.95 (m, 2H, d-
CH2, Arg), 3.86 (s, 2H, CH2-COO), 3.37 (dd, 2J ˆ 14.0 Hz, 3J ˆ 4.9 Hz, 1H,
b-CH2a, Cys), 3.29 (dd, 2J ˆ 14.0 Hz, 3J ˆ 6.6 Hz, 1H, b-CH2b, Cys), 2.54 (t,
3J ˆ 7.2 Hz, 2H, g-CH2, Met), 2.51(t, 3J ˆ 7.7 Hz, 2H, a-CH2, Pal), 2.12
3J ˆ 8.6 Hz, 2H, OCCHCH, aromatic), 7.37 7.28 (m, 5H, C6H5, aromatic),
3
ˆ
7.06 (d, J ˆ 8.6 Hz, 2H, OCCH, aromatic), 5.98 5.94 (m, 2H, CH CH2),
2.05 (m, 1H, b-CH2a, Met), 2.09 (s, 3H, SCH3), 1.94 1.87 (m, 1 H, b-CH2b,
2
ˆ
5.37 5.20 (m, 4H, CH CH2), 5.16 (d, J ˆ 12.7 Hz, 1H, CH2a-O, benzyl),
Met), 1.84 1.81 (m, 1 H,b-CH2a, Arg), 1.73 1.66 (m, 3H, b-CH2b, g-CH2,
Arg), 1.61 1.55 (m, 2H, b-CH2, Pal), 1.42 (s, 9H, tBu), 1.25 (s, 24H, CH2,
Pal), 0.88 (t, 3J ˆ 6.9 Hz, 3H, CH3, Pal); 13C NMR (125.7 MHz, CDCl3):
5.11 (d, 2J ˆ 12.7 Hz, 1H, CH2b-O, benzyl), 4.71(dt, 3J ˆ 5.6 Hz, 4J ˆ 1.3 Hz,
2H, CH2-O, allyl), 4.67 (dd, 3J1 ˆ 7.3 Hz, 3J2 ˆ 5.4 Hz, 1H, a-CH, Cys),
4.60 4.58 (m, 2H, CH2-O, allyl), 3.98 3.95 (m, 1H, a-CH, Arg), 3.88 (s,
2H, CH2-COO), 3.87 3.84 (m, 2H, d-CH2, Arg), 3.45 (dd, 2J ˆ 14.0 Hz,
3J ˆ 5.4 Hz, 1H, b-CH2a, Cys), 3.24 (dd, 2J ˆ 14.0 Hz, 3J ˆ 7.4 Hz, 1H, b-
ˆ
ˆ
d ˆ 198.73 (C O, thioester), 171.58, 171.13 (2  C O, amide), 169.81, 169.45
ˆ
ˆ
(2 Â C O, ester), 163.46 (Cq, guanidino), 160.91, 155.67 (3 Â C O, carba-
mate), 150.79 (Cq-O, aromatic), 133.30, 132.72 (2 Â Cq, aromatic), 133.03,
CH2b, Cys), 2.53 (t, 3J ˆ 7.0 Hz, 2H, a-CH2, Pal), 1.90 1.82 (m, 1 H,b-CH2a
,
ˆ
131.00 (2 Â CH CH2), 129.65, 129.29, 128.75, 127.40, 121.65 (CH, aromatic),
ˆ
Arg), 1.81 1.73 (m, 3H, b-CH2b, g-CH2, Arg), 1.62 1.57 (m, 2H, b-CH2,
119.63, 118.03 (2 Â CH CH2), 80.05 (Cq, tBu), 67.82, 66.89, 66.22 (CH2-O,
3372
¹ WILEY-VCH Verlag GmbH, 69451Weinheim, Germany, 2002 0947-6539/02/0815-3372 $ 20.00+.50/0 Chem. Eur. J. 2002, 8, No. 1 5

Lipopeptides
3362 3376
2 Â allyl, 1 Â benzyl), 53.52 (a-CH, Cys), 52.67 (a-CH, Arg), 52.36 (a-CH,
Met), 44.05, 44.00 (CH2-COO, d-CH2, Arg), 41.39 (b-CH2, Cys), 31.92,
31.73 (a-CH2, Pal, g-CH2, Met), 30.17 (b-CH2, Met), 29.69, 29.66, 29.62,
29.45, 29.36, 29.25, 28.50, 25.50, 22.69 (CH2, Pal), 28.96 (b-CH2, Arg), 28.28
(CH3, tBu), 24.55 (g-CH2, Arg), 15.32 (SCH3, Met), 14.13 (CH3, Pal); MS
Boc-Thr-Ile-Cys(Pal)-Ile-OAll (19): Triethylamine (20 mL, 0.14 mmol) and
EEDQ (35 mg, 0.14 mmol) were added at 08C to a solution of Boc-Thr-Ile-
Cys(Pal)-OH (17a; 80 mg, 0.12 mmol) and l-isoleucine-allylester tosylate
(12 f; 42 mg, 0.12 mmol) in CH2Cl2 (20 mL) and the mixture was stirred for
18 h at room temperature. The solvent was removed under reduced
pressure and the product was purified by flash chromatography on silica gel
‡
(FAB, 3-NBA): m/z: calcd for C58H86N6O13S2: 1139.6; found: 1139.5 [M‡H] .
using n-hexane/acetone 7:1to yield a colorless solid (70 mg, 71%). [ a]D20
ˆ
Boc-Thr-Ile-Cys(Pal)-OH (17a): Boc-Thr-Ile-Cys(Pal)-OPAOB (16a;
0.050 g, 0.06 mmol) was added to a solution of dimethyl-b-cyclodextrin
(2.226 g, 1.67 mmol) in phosphate buffer (100 mL, 0.05m, pH 7) and the
mixture was sonicated until a clear solution resulted. Then immobilized
penicillin G acylase (500 U) was added and the reaction mixture was
shaken at room temperature for 24 h. After filtering off the enzyme the pH
of the aqueous phase was adjusted to pH 2, the aqueous solution was
treated with benzyltriethylammonium bromide (0.909 g, 3.34 mmol) and
extracted with diethyl ether (5 Â 30 mL). The combined organic layers were
dried with MgSO4 and the solvent was removed under reduced pressure.
The resulting crude product was triturated with n-hexane (3 Â 5 mL) to
separate the desired compound from side products to yield a colorless solid
(31mg, 81%). M.p. 128 8C; [a]2D0 ˆ À13.4 (c ˆ 1.0 in CHCl3); Rf ˆ 0.03 (n-
hexane/ethyl acetate 1:1); 1H NMR (500 MHz, CDCl3): d ˆ 7.38 (d, 3J ˆ
6.8 Hz, 1H, NH, amide), 7.30 (d, 3J ˆ 8.7 Hz, 1H, NH, amide), 5.66 (d, 3J ˆ
8.2 Hz, 1H, NH, carbamate), 4.66 4.62 (m, 1H, a-CH, Cys), 4.35 (dd, 3J1 ˆ
À39.9 (c ˆ 1.0 in CH3OH); Rf ˆ 0.13 (n-hexane/acetone 7:1); 1H NMR
3
3
3
(500 MHz, CD3OD): d ˆ 5.96 (ddt, Jtrans ˆ 17.2 Hz, Jcis ˆ 10.4 Hz, Jvic
ˆ
3
ˆ
ˆ
5.7 Hz, 1H, CH CH2), 5.36 (d, Jtrans ˆ 17.2 Hz, 1H, CH CH2a), 5.24 (d,
Jcis ˆ 10.4 Hz, 1H, CH CH2b), 4.65 (dd, 2J ˆ 13.2 Hz, 3J ˆ 5.8 Hz, 1H,
3
ˆ
CH2a-O), 4.62 (dd, 2J ˆ 13.2 Hz, 3J ˆ 5.7 Hz, 1H, CH2b-O), 4.56 (dd, 3J1 ˆ
8.4 Hz, 3J2 ˆ 5.6 Hz, 1H, a-CH, Cys), 4.38 (d, 3J ˆ 6.0 Hz, 1H, a-CH, Ile1),
4.26 (d, 3J ˆ 6.7 Hz, 1H, a-CH, Ile2), 4.16 4.13 (m, 1H, b-CH, Thr), 4.08 (d,
3J ˆ 4.0 Hz, 1H, a-CH, Thr), 3.33 (dd, 2J ˆ 13.9 Hz, 3J ˆ 5.6 Hz, 1H, b-
CH2a, Cys), 3.13 (dd, 2J ˆ 13.9 Hz, 3J ˆ 8.4 Hz, 1H, b-CH2b, Cys), 2.56 (t,
3J ˆ 7.4 Hz, 2H, a-CH2, Pal), 1.95 1.88 (m, 1 H, b-CH, Ile1), 1.88 1.81 (m,
1H, b-CH, Ile2), 1.65 1.61 (m, 2H, b-CH2, Pal), 1.58 1.42 (m, 2H, 2Â g-
CH2a, Ile), 1.46 (s, 9H, tBu), 1.28 (s, 24H, CH2, Pal), 1.26 1.15 (m, 2H, 2Â
g-CH2b, Ile), 1.18 (d, 3J ˆ 6.2 Hz, 3H, g-CH3, Thr), 0.93 0.88 (m, 15H,
13
ˆ
CH3, Ile, CH3, Pal); C NMR (125.7 MHz, CD3OD): d ˆ 200.22 (C O,
ˆ
ˆ
thioester), 173.27, 172.12, 171.92 (3 Â C O, amide), 171.78 (C O, ester),
ˆ
ˆ
ˆ
157.96 (C O, carbamate), 133.25 (CH CH2), 118.97 (CH CH2), 80.79 (Cq,
tBu), 68.48 (b-CH, Thr), 66.63 (CH2-O), 61.05 (a-CH, Cys), 59.23 (a-CH,
Ile), 58.34 (a-CH, Ile), 53.97 (a-CH, Thr), 44.74 (b-CH2, Cys), 38.32, 38.20
(2 Â b-CH, Ile), 33.02 (a-CH2, Pal), 31.09, 30.71, 30.67, 30.50, 30.42, 30.35,
29.97, 26.53, 26.23 (CH2, Pal), 28.71(CH 3, tBu), 25.73, 23.69 (2 Â g-CH2,
Ile), 20.01( g-CH3, Thr), 15.95, 14.46, 11.75, 11.69 (5 Â CH3, Ile, Pal); MS
(EI): m/z: calcd for C43H78N4O9S: 826.5490; found: 826.5487.
3
8.7 Hz, J2 ˆ 7.6 Hz, 1H, a-CH, Thr), 4.31 4.27 (m, 1H, b-CH, Thr), 4.18
(d, 3J ˆ 6.2 Hz, 1H, a-CH, Ile), 3.48 (dd, 2J ˆ 14.1 Hz, 3J ˆ 4.4 Hz, 1H, b-
CH2a, Cys), 3.30 (dd, 2J ˆ 14.1 Hz, 3J ˆ 7.4 Hz, 1H, b-CH2b, Cys), 2.57 (t,
3J ˆ 7.5 Hz, 2H, a-CH2, Pal), 1.98 1.94 (m, 1 H, b-CH, Ile), 1.67 1.61 (m,
2H, b-CH2, Pal), 1.45 (s, 9H, tBu), 1.31 1.27 (m, 1 H,g-CH2a, Ile), 1.25 (s,
24H, CH2, Pal), 1.17 (d, 3J ˆ 6.3 Hz, 3H, g-CH3, Thr), 1.15 1.09 (m, 1 H,g-
CH2b, Ile), 0.92 (d, 3J ˆ 6.8 Hz, 3H, g-CH3, Ile), 0.90 0.87 (m, 6H, d-CH3,
Ile, CH3, Pal); 13C NMR (125.7 MHz, CDCl3): d ˆ 200.27 (C O, thioester),
H-Thr-Ile-Cys(Pal)-Ile-OAll ¥ TFA (20): TFA (0.12 mL, 1.60 mmol) was
added at 08C to a solution of Boc-Thr-Ile-Cys(Pal)-Ile-OAll (19; 44 mg,
0.05 mmol) in CH2Cl2 (0.50 mL) and the mixture was stirred at 08C for
45 min and at room temperature for 60 min. TFA and the solvent were
coevaporated with toluene (2 Â 10 mL) under reduced pressure to yield a
colorless solid (45 mg, quant.). [a]2D0 ˆ À32.7 (c ˆ 1.0 in CH3OH); Rf ˆ 0.06
(n-hexane/acetone 7:1); 1H NMR (500 MHz, CD3OD): d ˆ 5.96 (ddt,
ˆ
ˆ
ˆ
171.91, 171.76 (2 Â C O, amide), 156.45 (C O, carbamate), 80.55 (Cq, tBu),
66.14 (b-CH, Thr), 58.08 (a-CH, Cys), 57.96 (a-CH, Thr), 53.06 (a-CH, Ile),
44.05 (b-CH2, Cys), 36.67 (b-CH, Ile), 31.93 (a-CH2, Pal), 29.84, 29.70,
29.66, 29.63, 29.47, 29.36, 29.27, 29.02, 25.58, 24.52 (CH2, Pal), 28.31(CH
,
3
tBu), 22.69 (g-CH2, Ile), 18.29 (g-CH3, Thr), 15.40, 14.12, 11.30 (3 Â CH3,
‡
Ile, Pal); MS (FAB, 3-NBA): m/z: calcd for [M‡Na] : 696.4234; found:
3
3
3
3
ˆ
698.4227; elemental analysis calcd (%) for C34H63N3O8S (674.0): C 60.59, H
9.42, N 6.23; found: C 60.44, H 9.10, N 6.13.
Jtrans ˆ 17.1 Hz, Jcis ˆ 10.6 Hz, Jvic ˆ 5.9 Hz, 1H, CH CH2), 5.36 (d,
3
ˆ
ˆ
Jtrans ˆ 17.1 Hz, 1H, CH CH2a), 5.25 (d, Jcis ˆ 10.6 Hz, 1H, CH CH2b),
4.65 (dd, 2J ˆ 13.3 Hz, 3J ˆ 5.8 Hz, 1H, CH2a-O), 4.63 (dd, 2J ˆ 13.3 Hz, 3J ˆ
Boc-Arg(Aloc)2-Cys(Pal)-OH (18): Boc-Arg(Aloc)2-Cys(Pal)-OPAOB
(14b; 5 mg, 5 mmol) was added to a solution of dimethyl-b-cyclodextrin
(200 mg, 150 mmol) in phosphate buffer (2 mL, 0.05m, pH 7) and the
mixture was sonicated until a clear solution resulted. Then immobilized
penicillin G acylase (50 U) was added and the reaction mixture was shaken
at room temperature for 24 h. After the enzyme was filtered off, the pH of
the aqueous phase was adjusted to 2, and the aqueous solution was treated
with benzyltriethylammonium bromide (81mg, 300 mmol) and extracted
with diethyl ether (5 Â 10 mL). The combined organic layers were dried
with MgSO4 and the solvent was removed under reduced pressure. The
resulting crude product was triturated with n-hexane (3 Â 1mL) to separate
the desired compound from side products to yield a colorless solid (3 mg,
77%). M.p. 838C; [a]2D0 ˆ ‡11.7 (c ˆ 1.0 in CHCl3); Rf ˆ 0.10 (n-hexane/
3
3
5.8 Hz, 1H, CH2b-O), 4.57 (dd, J1 ˆ 7.9 Hz, J2 ˆ 6.2 Hz, 1H, a-CH, Cys),
3
3
4.39 (d, J ˆ 6.0 Hz, 1H, a-CH, Ile1), 4.29 (d, J ˆ 7.4 Hz, 1H, a-CH, Ile2),
3
4.07 4.04 (m, 1H, b-CH, Thr), 3.75 (d, J ˆ 6.6 Hz, 1H, a-CH, Thr), 3.30
(dd, 2J ˆ 13.8 Hz, 3J ˆ 6.2 Hz, 1H, b-CH2a, Cys), 3.14 (dd, 2J ˆ 13.8 Hz, 3J ˆ
3
7.9 Hz, 1H, b-CH2b, Cys), 2.57 (t, J ˆ 8.0 Hz, 2H, a-CH2, Pal), 1.93 1.87
(m, 1H, b-CH, Ile1), 1.87 1.80 (m, 1 H, b-CH, Ile2), 1.65 1.63 (m, 2H, b-
CH2, Pal), 1.59 1.42 (m, 2H, 2 g-CH2a, Ile), 1.31 (d, 3J ˆ 3.8 Hz, 3H, g-
CH3, Thr), 1.29 (s, 24H, CH2, Pal), 1.27 1.14 (m, 2H, 2Â g-CH2b, Ile),
0.95 0.89 (m, 15H, CH3, Ile, CH3, Pal); 13C NMR (125.7 MHz, CD3OD):
ˆ
ˆ
d ˆ 200.23 (C O, thioester), 172.77, 172.16, 171.68 (3  C O, amide), 168.44
ˆ
ˆ
ˆ
(C O, ester), 133.19 (CH CH2), 119.03 (CH CH2), 67.56 (b-CH, Thr),
66.66 (CH2-O), 59.88 (a-CH, Cys), 59.35 (a-CH, Ile), 58.28 (a-CH, Ile),
53.81( a-CH, Thr), 44.73 (b-CH2, Cys), 38.35, 38.12 (2 Â b-CH, Ile), 33.00
(a-CH2, Pal), 31.14, 30.70, 30.65, 30.49, 30.40, 30.34, 29.95, 26.53, 26.19
(CH2, Pal), 25.76, 23.67 (2 Â g-CH2, Ile), 20.19 (g-CH3, Thr), 15.96, 15.84,
14.44, 11.70, 11.52 (5 Â CH3, Ile, Pal); MS (FAB, 3-NBA): m/z: calcd for
1
ethyl acetate 1:1); H NMR (500 MHz, CDCl3/CD3OD 5:1): d ˆ 6.02 5.94
3
4
ˆ
ˆ
(m, 2H, CH CH2), 5.40 5.22 (m, 4H, CH CH2), 4.74 (dt, J ˆ 5.8 Hz, J ˆ
1.1 Hz, 2H, CH2-O, allyl), 4.64 (dd, 3J1 ˆ 7.2 Hz, 3J2 ˆ 4.6 Hz, 1H, a-CH,
Cys), 4.62 (dt, 3J ˆ 4.5 Hz, 4J ˆ 1.3 Hz, 2H, CH2-O, allyl), 4.12 4.08 (m,
1H, a-CH, Arg), 4.01 3.96 (m, 1H, d-CH2a, Arg), 3.93 3.89 (m, 1H, d-
CH2b, Arg), 3.49 (dd, 2J ˆ 13.9 Hz, 3J ˆ 4.7 Hz, 1H, b-CH2a, Cys), 3.24 (dd,
2J ˆ 13.9 Hz, 3J ˆ 7.2 Hz, 1H, b-CH2b, Cys), 2.55 (t, 3J ˆ 7.6 Hz, 2H, a-CH2,
Pal), 1.80 1.75 (m, 1 H, b-CH2a, Arg), 1.72 1.68 (m, 1 H, b-CH2b, Arg),
1.66 1.60 (m, 4H, b-CH2, Pal, g-CH2, Arg), 1.45 (s, 9H, tBu), 1.26 (s, 24H,
CH2, Pal), 0.88 (t, 3J ˆ 6.9 Hz, 3H, CH3, Pal); 13C NMR (125.7 MHz,
‡
[M À CF3COO] : 728.5122; found: 728.5154; C38H71N4O7S ¥ C2F3O2 (841.1).
NBDAca-Thr-Ile-Cys(Pal)-Ile-OAll (21): HOBt (7 mg, 0.04 mmol), tri-
ethylamine (5 mL, 0.04 mmol) and EDC (7 mg, 0.04 mmol) were added at
08C to
a solution of H-Thr-Ile-Cys(Pal)-Ile-OAll ¥ TFA (20; 30 mg,
0.04 mmol) in CH2Cl2 (20 mL) and the mixture was stirred for 18 h at
room temperature. The solvent was removed under reduced pressure and
the product was purified by flash chromatography on silica gel using
chloroform/methanol 50:1to yield an orange solid (31mg, 86%). M.p.
1448C; [a]2D0 ˆ À91.6 (c ˆ 1.0 in CHCl3); Rf ˆ 0.80 (chloroform/methanol
5:1); 1H NMR (400 MHz, CDCl3): d ˆ 8.48 (d, 3J ˆ 8.4 Hz, CH, NBD), 7.29
(br, 1H, NH, amide), 7.17 (d, 3J ˆ 7.1, 2H, NH, amide), 6.86 (br, 1 H, NH,
ˆ
ˆ
CDCl3/CD3OD 5:1): d ˆ 199.10 (C O, thioester), 172.72 (C O, carboxylic
ˆ
acid), 171.36 (C O, amide), 163.38 (Cq, guanidino), 160.53, 156.09, 155.60
ˆ
ˆ
(3 Â C O, carbamate), 132.80, 130.99 (2 Â CH CH2), 119.53, 117.74 (2 Â
ˆ
CH CH2), 79.91(C q, tBu), 67.85, 66.28 (2 Â CH2-O, allyl), 54.28 (a-CH,
Cys), 51.89 (a-CH, Arg), 44.42 (d-CH2, Arg), 43.90 (b-CH2, Cys), 31.84 (a-
CH2, Pal), 30.21, 30.13, 29.59, 29.56, 29.51, 29.34, 29.26, 29.15, 29.02, 28.13,
25.46, 23.76, 22.58 (CH2, Pal), 28.88 (b-CH2, Arg), 28.13 (CH3, tBu), 24.82
(g-CH2, Arg), 13.88 (CH3, Pal); MS (FAB, 3-NBA): m/z: calcd: 784.4530;
amide), 6.65 (br, 1H, NH, amide), 6.17 (d, 3J ˆ 8.4 Hz, CH, NBD), 5.94
3
ˆ
ˆ
5.85 (m, 1H, CH CH2), 5.33 (d, Jtrans ˆ 17.2 Hz, 1H, CH CH2a), 5.25 (d,
3
ˆ
Jcis ˆ 10.6 Hz, 1H, CH CH2b), 4.67 4.58 (m, 4H, CH2-O, a-CH, Cys, a-
‡
found: 784.4556 [M‡H] ; elemental analysis calcd (%) for C38H65N5O10S
CH, Ile1), 4.55 4.52 (m, 1H, a-CH, Thr), 4.36 4.30 (m, 2H, a-CH, Ile2, b-
CH, Thr), 3.52 3.49 (m, 2H, a-CH2, aminocaproyl), 3.31 3.24 (m, 2H, b-
(784.0): C 58.22, H 8.36, N 8.93; found C 58.09, H 8.43, N 8.72.
Chem. Eur. J. 2002, 8, No. 15
¹ WILEY-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002
0947-6539/02/0815-3373 $ 20.00+.50/0
3373

F. Eisele, J. Kuhlmann and H. Waldmann
FULL PAPER
CH2, Cys), 2.56 (t, 3J ˆ 6.9 Hz, 2H, a-CH2, Pal), 2.38 2.34 (m, 2H, e-CH2,
aminocaproyl), 1.99 1.95 (m, 1 H, b-CH, Ile1), 1.91 1.88 (m, 1 H, b-CH,
Ile2), 1.86 1.82 (m, 2H, b-CH2, aminocaproyl), 1.80 1.74 (m, 2H, d-CH2,
aminocaproyl), 1.63 1.60 (m, 2H, b-CH2, Pal), 1.55 1.51 (m, 2H, 2Â g-
CH2a, Ile), 1.43 1.41 (m, 2H, g-CH2, aminocaproyl), 1.24 (s, 24H, CH2,
170.98, 170.84, 170.39, 169.70 (C O, 5 Â amide, 1 Â ester), 156.24, 155.51
ˆ
ˆ
ˆ
(3 Â C O, carbamate), 132.90, 131.80, 130.78 (3 Â CH CH2), 118.61 (3 Â
ˆ
CH CH2), 80.81(C q, tBu), 68.02, 65.62 (3 Â CH2-O, allyl), 66.75 (b-CH,
Thr), 59.27, 58.91, 56.93, 55.64, 55.14, 53.51 (6Â a-CH), 50.81( d-CH2, Arg),
44.02 (2 Â b-CH2, Cys), 37.54, 36.07 (2 Â b-CH, Ile), 31.91 (2Â a-CH2, Pal),
30.32, 29.95, 29.69, 29.65, 29.46, 29.35, 29.29, 29.24, 29.03, 28.62, 25.64, 25.54,
25.10, 24.92, 24.68, 22.68 (CH2, Pal, b-CH2, g-CH2, Arg, g-CH2, Ile), 28.33
(CH3, tBu), 19.62 (g-CH3, Thr), 15.62, 15.46, 14.11, 11.67, 11.59 (6 Â CH3,
Ile, Pal); MS (FAB, 3-NBA): m/z: calcd for C76H134N9O16S2: 1492.9; found:
Pal), 1.16 (d, 3J ˆ 4.9 Hz, 3H, g-CH3, Thr), 1.12 1.07 (m, 2H, 2 g-CH2b
,
Ile), 0.91 0.86 (m, 15H, CH 3, Ile, CH3, Pal); 13C NMR (100.6 MHz,
ˆ
ˆ
CDCl3): d ˆ 201.39 (C O, thioester), 171.32, 171.06, 169.50 (5  C O,
ˆ
amide, ester), 144.33, 144.01 (4 Â Cq, NBD), 136.51 (CH CH2), 131.57 (2 Â
‡
ˆ
CH, NBD), 118.97 (CH CH2), 66.98 (b-CH, Thr), 65.90 (CH2-O, allyl),
1492.5 [M‡H] .
58.49 (a-CH, Cys), 56.86 (a-CH, Ile), 53.83 (a-CH, Ile), 53.43 (a-CH, Thr),
44.07 (a-CH2, aminocaproyl), 43.64 (b-CH2, Cys), 37.73, 36.71(2 Â b-CH,
Ile), 35.83 (a-CH2, Pal), 31.94 (e-CH2, aminocaproyl), 30.60, 29.71, 29.46,
29.37, 29.27, 29.03, 27.90, 26.32, 26.19, 25.60, 25.09, 24.81, 24.64, 22.70 (CH2,
Pal, b-CH2, d-CH2, g-CH2, aminocaproyl, g-CH2, Ile), 18.46 (g-CH3, Thr),
15.63, 15.50, 14.14, 11.58 (5 Â CH3, Ile, Pal); MS (FAB, 3-NBA): m/z: calcd
H-Arg(Aloc)2-Cys(Pal)-Thr-Ile-Cys(Pal)-Ile-OAll ¥ TFA (24): TFA (54 mL,
0.67 mmol) was added at 08C to a solution of Boc-Arg(Aloc)2-Cys(Pal)-
Thr-Ile-Cys(Pal)-Ile-OAll (2; 20 mg, 13 mmol) in CH2Cl2 (0.12 mL) and the
mixture was stirred at 08C for 45 min and at room temperature for 60 min.
TFA and the solvent were coevaporated with toluene (2 Â 10 mL) under
reduced pressure to yield a colorless solid (20 mg, quant.). [a]2D0 ˆ À14.3
(c ˆ 0.5 in CHCl3); Rf ˆ 0.12 (chloroform/methanol 10:1); 1H NMR
‡
for C50H83N8O11S: 1003.590; found: 1003.586 [M‡H] .
ˆ
(500 MHz, CDCl3/CD3OD 5:1): d ˆ 6.00 5.89 (m, 3H, CH CH2), 5.41
NBDAca-Thr-Ile-Cys(Pal)-Ile-OH (22): Dimethylbarbituric acid (2 mg,
0.02 mmol) and a catalytic amount of [Pd(PPh3)4] were added under argon
to a solution of NBDAca-Thr-Ile-Cys(Pal)-Ile-OAll (21; 31mg, 0.03 mmol)
in dry THF (10 mL). The mixture was stirred for 15 min at room
temperature and then the solvent was removed under reduced pressure.
The resulting residue was triturated with diethyl ether (3 Â 2 mL) to yield
the desired compound as an orange solid (25 mg, 86%). M.p. 1628C;
[a]2D0 ˆ À12.7 (c ˆ 1.0 in CHCl3/CH3OH 1:); Rf ˆ 0.44 (chloroform/meth-
anol 5:1); 1H NMR (500 MHz, CDCl3/CD3OD 1:1): d ˆ 8.52 (d, 3J ˆ 8.6 Hz,
CH, NBD), 6.30 (d, 3J ˆ 8.7 Hz, CH, NBD), 4.59 4.56 (m, 1H, a-CH, Cys),
4.45 (d, 3J ˆ 3.9 Hz, 1H, a-CH, Thr), 4.41 4.38 (m, 1H, a-CH, Ile1), 4.30
4.27 (m, 1H, a-CH, Ile2), 4.21(dq, 3J1 ˆ 3.9 Hz, 3J2 ˆ 6.4 Hz, 1H, b-CH,
Thr), 3.57 3.50 (m, 2H, a-CH2, aminocaproyl), 3.35 (dd, 2J ˆ 13.1 Hz, 3J ˆ
3
4
ˆ
5.23 (m, 6H, CH CH2), 4.75 (dt, J ˆ 5.9 Hz, J ˆ 1.2 Hz, 2H, CH2-O, allyl),
4.65 4.59 (m, 4H, 2 Â CH2-O, allyl), 4.56 4.54 (m, 1H, a-CH), 4.48 4.44
(m, 2H, b-CH, Thr, a-CH), 4.35 (d, 3J ˆ 4.0 Hz, 1H, a-CH), 4.26 (d, 3J ˆ
6.3 Hz, 1H, a-CH), 4.22 4.20 (m, 1H, a-CH), 4.16 4.12 (m, 1H, a-CH),
3.56 3.48 (m, 2H, d-CH2, Arg), 3.45 3.40 (m, 1H, b-CH2a, Cys1), 3.32 (dd,
2J ˆ 14.1 Hz, 3J ˆ 5.4 Hz, 1H, b-CH2b, Cys1), 3.21(dd, 2J ˆ 14.0 Hz, 3J ˆ
2
3
7.7 Hz, 1H, b-CH2a, Cys2), 3.14 (dd, J ˆ 14.0 Hz, J ˆ 8.4 Hz, 1H, b-CH2b
,
Cys2), 2.56 (t, 3J ˆ 7.6 Hz, 4H, 2  a-CH2, Pal), 2.05 1.98 (m, 1 H, b-CH,
Ile1), 1.93 1.75 (m, 5H, b-CH, Ile2, b-CH2, g-CH2, Arg), 1.64 1.62 (m, 4H,
2 Â b-CH2, Pal), 1.51 1.42 (m, 2H, 2Â g-CH2a, Ile), 1.32 1.19 (m, 2H, 2Â
g-CH2b, Ile), 1.26 (s, 48H, CH2, Pal), 1.15 (d, 3J ˆ 5.6 Hz, 3H, g-CH3, Thr),
0.93 0.87 (m, 18H, 4 Â CH3, Ile, 2 Â CH3, Pal); 13C NMR (125.7 MHz,
2
3
ˆ
CDCl3/CD3OD 5:1): d ˆ 200.34, 199.85 (2  C O, thioester), 172.06, 171.29,
5.4 Hz, 1H, b-CH2a, Cys), 3.16 (dd, J ˆ 13.1 Hz, J ˆ 8.6 Hz, 1H, b-CH2b
,
3
3
ˆ
170.60, 170.35, 170.21, 169.64 (6 Â C O, 5 Â amide, 1 Â ester), 162.89 (Cq,
Cys), 2.57 (t, J ˆ 7.5 Hz, 2H, a-CH2, Pal), 2.36 (t, J ˆ 7.3 Hz, 2H, e-CH2,
aminocaproyl), 1.92 1.88 (m, 2H, 2Â b-CH, Ile), 1.86 1.80 (m, 2H, b-
CH2, aminocaproyl), 1.77 1.71 (m, 2H, d-CH2, aminocaproyl), 1.65 1.61
(m, 2H, b-CH2, Pal), 1.55 1.48 (m, 4H, 2Â g-CH2a, Ile, g-CH2, amino-
caproyl), 1.27 (s, 24H, CH2, Pal), 1.22 1.14 (m, 2H, 2Â g-CH2b, Ile), 1.18
ˆ
guanidino), 160.62, 155.38 (2 Â C O, carbamate), 132.74, 131.76, 130.99
ˆ
ˆ
(3 Â CH CH2), 120.05, 119.00, 118.56 (3 Â CH CH2), 68.52, 66.57, 66.00
(3 Â CH2-O, allyl), 67.23 (b-CH, Thr), 58.62, 58.47, 57.17, 54.26, 53.13, 52.57
(6 Â a-CH), 44.16 (d-CH2, Arg), 43.64 (2 Â b-CH2, Cys), 37.61, 36.83 (2Â b-
CH, Ile), 32.08 (2 Â a-CH2, Pal), 30.36, 29.84, 29.80, 29.77, 29.60, 29.51,
29.43, 29.15, 27.10, 25.69, 25.63, 25.24, 24.79, 23.54 (CH2, Pal, b-CH2, g-CH2,
Arg, g-CH2, Ile), 22.83 (g-CH3, Thr), 18.85, 17.30, 17.11, 15.56, 15.51, 14.16,
11.54, 11.41 (6 Â CH3, Ile, Pal); MS (FAB, 3-NBA): m/z: calcd for
3
(d, J ˆ 6.4 Hz, 3H, g-CH3, Thr), 0.95 0.87 (m, 15H, CH3, Ile, CH3, Pal);
13
ˆ
C NMR (125.7 MHz, CDCl3/CD3OD 1:1): d ˆ 200.61(C O, thioester),
ˆ
ˆ
175.37 (C O, carboxylic acid), 173.69, 172.76, 171.90, 170.86 (4 Â C O,
amide), 146.06, 145.17, 144.89 (4 Â Cq, NBD), 132.53, 129.46 (2 Â CH,
NBD), 67.78 (b-CH, Thr), 58.85, 58.70, 57.61, 53.62 (a-CH), 44.46 (a-CH2,
aminocaproyl), 44.19 (b-CH2, Cys), 37.97, 37.55 (2 Â b-CH, Ile), 36.25 (a-
CH2, Pal), 32.50 (e-CH2, aminocaproyl), 30.74, 30.23, 30.20, 30.18, 30.00,
‡
C71H126N9O14S2: 1393.9; found: 1393.7 [M] .
NBDAca-Met-OAll (25): HOBt (14 mg, 0.09 mmol), triethylamine (10 mL,
0.08 mmol) and EDC (14 mg, 0.08 mmol) were added at 08C to a solution
of N-(7-nitrobenz-2-oxa-1,3-diazole-4-yl)-aminocaproic acid (20 mg,
0.08 mmol) and l-methionine-allyl ester tosylate (12g; 29 mg, 0.08 mmol)
in CH2Cl2 (20 mL) and the mixture was stirred at room temperature for
18 h. The solvent was removed under reduced pressure and the product was
purified by flash chromatography on silica gel using chloroform/methanol
50:1to yield an orange oil (26 mg, 69%). [ a]2D0 ˆ ‡16.3 (c ˆ 1.0 in CHCl3);
Rf ˆ 0.74 (chloroform/methanol 5:1); 1H NMR (500 MHz, CDCl3): d ˆ 8.48
29.91, 29.84, 29.54, 28.47, 27.03, 26.10, 25.80, 25.62, 25.20, 23.21 (CH , Pal, b-
2
CH2, d-CH2, g-CH2, aminocaproyl, g-CH2, Ile), 19.47 (g-CH3, Thr), 15.77,
15.74, 14.28, 11.78, 11.60 (5 Â CH3, Ile, Pal); MS (FAB, 3-NBA): m/z: calcd
‡
for C47H79N8O11S: 963.5589; found: 963.5562 [M‡H] .
Boc-Arg(Aloc)2-Cys(Pal)-Thr-Ile-Cys(Pal)-Ile-OAll (23): Triethylamine
(5 mL, 38 mmol) and EEDQ (12 mg, 46 mmol) were added at 08C to a
solution of Boc-Arg(Aloc)2-Cys(Pal)-OH (18; 30 mg, 38 mmol) and H-Thr-
Ile-Cys(Pal)-Ile-OAll ¥ TFA (20; 32 mg, 38 mmol) in CH2Cl2 (20 mL) and
the mixture was stirred at room temperature for 18 h. The solvent was
removed under reduced pressure and the product was purified by size-
exclusion chromatography on Sephadex LH 20 using chloroform/methanol
1:1 to yield a colorless solid (26 mg, 46%). [a]2D0 ˆ À33.4 (c ˆ 1.0 in CHCl3);
Rf ˆ 0.31 (chloroform/methanol 10:1); 1H NMR (500 MHz, CDCl3): d ˆ
9.46 (br, 2H, NH, guanidino), 7.56 (br, 1H, NH, amide), 7.37 (br, 1H, NH,
amide), 7.24 (br, 1H, NH, amide), 7.09 (d, 3J ˆ 8.3 Hz, 1H, NH, amide), 6.99
3
(d, J ˆ 8.6 Hz, 1H, NBD), 6.68 (br, 1H, NH, amine), 6.27 (d, 3J ˆ 7.7 Hz,
1H, NH, amide), 6.17 (d, 3J ˆ 8.6 Hz, 1H, NBD), 5.95 5.87 (m, 1H,
3
3
ˆ
ˆ
CH CH2), 5.35 (d, Jtrans ˆ 17.2 Hz, 1H, CH CH2a), 5.27 (d, Jcis ˆ 10.3 Hz,
3
ˆ
1H, CH CH2b), 4.79 4.75 (m, 1H, a-CH, Met), 4.66 (d, J ˆ 5.3 Hz, 2H,
CH2-O, allyl), 3.55 3.51(m, 2H, a-CH2, aminocaproyl), 2.61 2.55 (m,
2H, g-CH2, Met), 2.31(t, 3J ˆ 7.0 Hz, 2H, e-CH2, aminocaproyl), 2.23 2.17
(m, 1H, b-CH2a, Met), 2.09 1.99 (m, 1 H, b-CH2b, Met), 2.05 (s, 3H, SCH3,
Met), 1.87 1.82 (m, 2H, b-CH2, aminocaproyl), 1.80 1.73 (m, 2H, d-CH2,
aminocaproyl), 1.57 1.51 (m, 2H, g-CH2, aminocaproyl); 13C NMR
(d, 3J ˆ 6.2 Hz, 1H, NH, amide), 6.02 5.87 (m, 3H, CH CH2), 5.40 (br,
ˆ
ˆ
(125.7 MHz, CDCl3): d ˆ 172.56, 171.83 (2  C O, amide, ester), 144.28,
1H, NH, carbamate), 5.37 5.23 (m, 6H, CH CH2), 4.73 (d, 3J ˆ 5.8 Hz,
ˆ
144.07, 143.95, 123.68 (4 Â Cq, NBD), 136.58, 98.57 (2 Â CH, NBD), 131.33
2H, CH2-O, allyl), 4.66 4.57 (m, 4H, 2 Â CH2-O, allyl), 4.52 4.49 (m, 2H,
2 Â a-CH, Cys), 4.33 4.26 (m, 3H, b-CH, Thr, 2 Â a-CH), 4.08 4.02 (m,
ˆ
ˆ
(CH CH2), 119.16 (CH CH2), 66.27 (CH2-O, allyl), 51.64 (a-CH, Met),
43.68 (a-CH2, aminocaproyl), 35.87 (g-CH2, Met), 31.50 (e-CH2, amino-
caproyl), 29.68 (b-CH2, Met), 27.91( b-CH2, aminocaproyl), 26.22 (d-CH2,
aminocaproyl), 24.54 (g-CH2, aminocaproyl), 15.46 (SCH3, Met); MS (EI):
m/z: calcd for C20H27N5O6S: 465.2; found: 465.2.
2H, 2  a-CH), 3.97 3.93 (m, 2H, d-CH2, Arg), 3.40 (dd, 2J ˆ 14.3 Hz, 3J ˆ
3
4.9 Hz, 1H, b-CH2a, Cys1), 3.33 (dd, 2J ˆ 14.4 Hz, J ˆ 4.3 Hz, 1H, b-CH2b
,
Cys1), 3.30 3.22 (m, 2H, b-CH2, Cys2), 2.59 2.53 (m, 4H, 2 Â a-CH2, Pal),
2.06 2.02 (m, 1H, b-CH, Ile1), 1.92 1.89 (m, 1 H,b-CH2a, Arg), 1.88 1.85
(m, 1H, b-CH, Ile2), 1.75 1.70 (m, 3H, b-CH2b, g-CH2 Arg), 1.68 1.48 (m,
6H, 2  b-CH2, Pal, 2  g-CH2a, Ile), 1.46 (s, 9H, tBu), 1.44 (d, 3J ˆ 4.8 Hz,
3H, g-CH3, Thr), 1.31 1.14 (m, 2H, 2Â g-CH2b, Ile), 1.25 (s, 48H, CH2,
Pal), 0.94 0.75 (m, 18H, 4 Â CH3, Ile, 2 Â CH3, Pal); 13C NMR
NBDAca-Met-OH (26): Dimethylbarbituric acid (5 mg, 0.03 mmol) and a
catalytic amount of [Pd(PPh3)4] were added under argon to a solution of
NBDAca-Met-OAll (25, 26 mg, 0.06 mmol) in THF (10 mL). The mixture
was stirred for 15 min at room temperature and then the solvent was
removed under reduced pressure. The resulting residue was triturated with
ˆ
(125.7 MHz, CDCl3): d ˆ 200.07 (2  C O, thioester), 173.15, 171.55,
3374
¹ WILEY-VCH Verlag GmbH, 69451Weinheim, Germany, 2002
0947-6539/02/0815-3374 $ 20.00+.50/0
Chem. Eur. J. 2002, 8, No. 1 5

Lipopeptides
3362 3376
diethyl ether (3 Â 2 mL) to yield the desired compound an an orange oil
(24 mg, 94%). [a]2D0 ˆ ‡10.3 (c ˆ 1.0 in DMF); Rf ˆ 0.39 (chloroform/
polycarbonate filters (20 Â ). The remaining organic solvents were removed
in a speedvac apparatus (Concentrator 5301, Eppendorf).
1
methanol 5:1); H NMR (500 MHz, CDCl3/CD3OD 1:1): d ˆ 8.51(d, 3J ˆ
Vesicles generated according to the general procedure:
8.2 Hz, 1H, NBD), 6.26 (d, 3J ˆ 8.0 Hz, 1H, NBD), 4.33 (dd, 3J1 ˆ 7.3 Hz,
3J2 ˆ 5.2 Hz, 1H, a-CH, Met), 3.55 3.49 (m, 2H, a-CH2, aminocaproyl),
2.51 2.49 (m, 2H, g-CH2, Met), 2.29 (t, 3J ˆ 7.2 Hz, 2H, e-CH2, amino-
caproyl), 2.15 2.09 (m, 1H, b-CH2a, Met), 2.07 (s, 3H, SCH3, Met), 2.06
1.92 (m, 1 H, b-CH2b, Met), 1.84 1.78 (m, 2H, b-CH2, aminocaproyl),
1.74 1.68 (m, 2H, d-CH2, aminocaproyl), 1.52 1.48 (m, 2H, g-CH2,
aminocaproyl); 13C NMR (125.7 MHz, CDCl3/CD3OD 1:1): d ˆ 180.34
Lipid vesicles loaded with fluorescence quencher and lipopeptides:
a) A solution of NBDac-Thr-Ile-Cys(Pal)-Ile-OH (22; 1 5mL, 100mm) in
chloroform/methanol 1:4 and
a solution of N-(lissamine-rhodamine-
sulfonyl)phosphatidylethanolamine (15 mL, 200mm) in methanol.
b) A solution of NBDac-Met-Arg-Cys(Pal)-Thr-Ile-Cys(Pal)-Ile-OH (28;
15 mL, 100mm) in chloroform/methanol 1:4 and a solution of N-(lissamine-
rhodamine-sulfonyl)phosphatidylethanolamine (15 mL, 200mm) in metha-
nol.
ˆ
ˆ
(C O, carboxylic acid), 174.12 (C O, amide), 145.23, 144.34, 127.80 (4 Â Cq,
NBD), 136.89 (2 Â CH, NBD), 54.62 (a-CH, Met), 48.34 (a-CH2, amino-
caproyl), 36.25 (g-CH2, Met), 32.67 (e-CH2, aminocaproyl), 30.59 (b-CH2,
Met), 26.73 (b-CH2, aminocaproyl), 25.44 (d-CH2, aminocaproyl), 23.63 (g-
CH2, aminocaproyl), 17.94 (SCH3, Met); MS (FAB, 3-NBA): m/z: calcd for
Lipid vesicles loaded with lipopeptides:
a) A solution of NBDac-Thr-Ile-Cys(Pal)-Ile-OH (22; 1 5mL, 100mm) in
chloroform/methanol 1:4.
‡
C17H24N5O6S: 426.1466, found 426.1437 [M‡H] .
b) A solution of NBDac-Met-Arg-Cys(Pal)-Thr-Ile-Cys(Pal)-Ile-OH (28;
15 mL, 100mm) in chloroform/methanol 1:4.
NBDAca-Met-Arg(Aloc)2-Cys(Pal)-Thr-Ile-Cys(Pal)-Ile-OAll (27): HOBt
(3 mg, 5.0 mmol), triethylamine (1 mL, 7 mmol) and EDC (3 mg, 4.6 mmol)
were added at 08C to a solution of NBDAca-Met-OH (26; 2 mg, 4.6 mmol)
and H-Arg(Aloc)2-Cys(Pal)-Thr-Ile-Cys(Pal)-Ile-OAll ¥ TFA (24; 7 mg,
4.6 mmol) in CH2Cl2 (20 mL) and the mixture was stirred at room
temperature for 18 h. The solvent was removed under reduced pressure
and the product was purified by size-exclusion chromatography on
Sephadex LH 20 using chloroform/methanol 1:1 to yield a yellow solid
(6 mg, 72%). [a]2D0 ˆ À8.0 (c ˆ 0.2 in CHCl3/CH3OH 1:1); Rf ˆ 0.80
(chloroform/methanol 5:1); 1H NMR (500 MHz, CDCl3/CD3OD 5:1):
d ˆ 8.52 (d, 3J ˆ 5.2 Hz, 1H, CH, NBD), 6.28 (d, 3J ˆ 5.3 Hz, 1H, CH,
General procedure for the preparation of acceptor vesicles: A solution of
POPC in methanol (30 mL, 20 mm) was mixed with 150 mL buffer (10 mm
(N-2-hydroxylethyl)piperazine-N'-2-ethansulfonic acid, 150 mm KCl, 8 mm
NaCl, 1mm MgCl2; pH 7). The mixtures were freeze-thawed three times
before extruding through 0.1 mm pore size polycarbonate filters (20 Â ). The
remaining organic solvents were removed under reduced pressure.
Monitoring of flip flop kinetics by dithionite reduction: A mixture of
loaded vesicles (with NBDac-Thr-Ile-Cys(Pal)-Ile-OH 22 or NBDac-Met-
Arg-Cys(Pal)-Thr-Ile-Cys(Pal)-Ile-OH (28; 6.1 mL) was added at 208C to a
buffer solution (1200 mL, pH 7). After 15 min a solution of sodium
dithionite in the buffer (12 mL, 1m) was added and the time-dependent
fluorescence decrease was detected at the emission wavelength of NBD
(535 nm).[27] Finally a solution of Triton X-100 (60 mL, 20%) was added
after 50 min.
ˆ
ˆ
NBD), 6.02 5.90 (m, 3H, CH CH2), 5.48 5.27 (m, 6H, CH CH2), 4.85
4.74 (m, 2H, CH2-O, allyl), 4.69 4.61(m, 4H, 2 Â CH2-O, allyl), 4.48 4.42
(m, 2H, 2 Â a-CH, Cys), 4.34 4.29 (m, 2H, 2 Â a-CH), 4.27 4.19 (m, 3H,
b-CH, Thr, 2 Â a-CH), 4.11 4.05 (m, 1H, a-CH), 3.98 3.90 (m, 2H, d-
CH2, Arg), 3.64 3.59 (m, 1H, b-CH2a, Cys1), 3.56 3.47 (m, 3H, a-CH2,
aminocaproyl, b-CH2b, Cys1), 3.33 3.28 (m, 1H, b-CH2a, Cys2), 3.22 3.16
(m, 1H, b-CH2b, Cys2), 2.93 2.84 (m, 2H, g-CH2, Met), 2.60 2.53 (m, 4H,
2 Â a-CH2, Pal), 2.34 2.29 (m, 2H, e-CH2, aminocaproyl), 2.09 (s, 3H,
SCH3, Met), 2.08 2.01(m, 2H, b-CH2, Met), 2.01 1.90 (m, 3H, 2 Â b-CH,
Ile, b-CH2a, Arg), 1.86 1.80 (m, 3H, b-CH2b, g-CH2 Arg), 1.72 1.60 (m,
10H, 2Â b-CH2, Pal, 2 Â g-CH2a, Ile, b-CH2, d-CH2, aminocaproyl), 1.54
1.48 (m, 2H, g-CH2, aminocaproyl), 1.44 (d, 3J ˆ 5.0 Hz, 3H, g-CH3, Thr),
1.26 (s, 48H, CH2, Pal), 1.19 1.10 (m, 2H, 2Â g-CH2b, Ile), 1.00 0.81 (m,
18H, 4Â CH3, Ile, 2 Â CH3, Pal); MS (FAB, 3-NBA): m/z: calcd for
Intervesicle transfer of the single palmitoylated tetrapeptide 22: A mixture
of loaded vesicles (with NBDac-Thr-Ile-Cys(Pal)-Ile-OH (22) and the
fluorescence quencher (6.1 mL) was added at 208C to a buffer solution
(1200 mL, pH 7). Then the desired amounts of acceptor vesicle mixture
were added and the time-dependent fluorescence increase was detected at
the emission wavelength of NBD (535 nm).
Comparison of intervesicle transfer of the double palmitoylated heptapep-
tide 28 with the single palmitoylated tetrapeptide 22: A mixture of vesicles
loaded with lipopeptides 22 or 28 and the fluorescence quencher was added
to 1200 mL buffer. After 30 min an access of acceptor vesicles was added
and the time-dependent fluorescence increase was detected at the emission
wavelength of NBD (535 nm). All experiments were performed at 208C.
‡
C91H152N14O19S3: 1842.1; found: 1842.0 [M‡H] .
NBDAca-Met-Arg-Cys(Pal)-Thr-Ile-Cys(Pal)-Ile-OH (28): Dimethylbar-
bituric acid (1.5 mg, 6.6 mmol) and a catalytic amount of [Pd(PPh3)4] were
added under argon to a solution of NBDAca-Met-Arg(Aloc)2-Cys(Pal)-
Thr-Ile-Cys(Pal)-Ile-OAll (27; 6 mg, 3.3 mmol) in THF (10 mL). The
mixture was stirred for 30 min at room temperature and then the solvent
was removed under reduced pressure. The resulting residue was purified by
size exclusion chromatography on Sephadex LH 20 using chloroform/
methanol 1:1 to yield yellow solid (5 mg, 96%). [a]2D0 ˆ À22.5 (c ˆ 0.2 in
CHCl3/CH3OH 1:1); Rf ˆ 0.10 (chloroform/methanol 5:1); 1H NMR
(500 MHz, CDCl3/CD3OD 1:1): d ˆ 8.55 8.51(m, 1H, CH, NBD), 6.40
6.37 (m, 1H, CH, NBD), 4.41 4.36 (m, 3H, 3 Â a-CH), 4.34 4.31(m, 1H,
a-CH), 4.28 4.21(m, 3H, 2 Â a-CH, b-CH, Thr), 4.18 4.12 (m, 1H, a-
CH), 3.70 3.63 (m, 2H, d-CH2, Arg), 3.47 3.41(m, 2H, a-CH2, amino-
caproyl), 3.31 3.11 (m, 4H, 2 Â b-CH2, Cys), 2.93 2.88 (m, 2H, g-CH2,
Met), 2.62 2.55 (m, 4H, 2 Â a-CH2, Pal), 2.38 2.30 (m, 3H, e-CH2,
aminocaproyl, b-CH2a, Met), 2.09 2.03 (m, 1H, b-CH2b, Met), 2.06 (s, 3H,
SCH3, Met), 1.95 1.88 (m, 3H, 2Â b-CH, Ile, b-CH2a, Arg), 1.75 1.62 (m,
11H, b-CH2, d-CH2, aminocaproyl, b-CH2b, g-CH2, Arg, 2 Â b-CH2, Pal),
1.55 1.47 (m, 4H, 2Â g-CH2a, Ile, g-CH2, aminocaproyl), 1.27 (s, 48H,
CH2, Pal), 1.21 (d, 3J ˆ 6.0 Hz, 3H, g-CH3, Thr), 1.17 1.12 (m, 2H, 2  g-
CH2b, Ile), 0.96 0.88 (m, 18H, CH3, Ile, CH3, Pal); MS (FAB, 3-NBA):
Acknowledgements
This research was supported by the Deutsche Forschungsgemeinschaft and
the Fonds der Chemischen Industrie.
[1] a) I. A. Wilson, J. J. Skehel, D. C. Wiley, Nature 1981, 289, 366 373;
b) D. C.Wiley, J. J. Skehel, Annu. Rev. Biochem. 1987, 56, 365 394;
c) P. A. Bullough, F. M. Hughson, J. J. Skehel, D. C. Wiley, Nature
1994, 371, 37 43.
[2] a) W. Keil, R. Geyer, J. Dabrowski, U. Dabrowski, H. Niemann, S.
Stirm, H.-D.Klenk, EMBO J. 1985, 4, 2711 2720; b) S. Y. Mir-Shekar,
D. A. Ashford, D. J. Harvey, R. A. Dwek, I. T. Schulze, J. Biol. Chem.
1997, 272, 4027 4036.
[3] M. F. G. Schmidt, Biochim. Biophys. Acta 1989, 988, 411 426.
[4] R. J. A. Grand, Biochem. J. 1989, 258, 625 638.
[5] E. Ponimaskin, M. F. G. Schmidt, Biochem. Soc. Trans. 1995, 23, 565
568.
‡
m/z: calcd for C77H133N14O15S3: 1589.9; found: 1589.7 [M‡H] .
General procedure for the preparation of loaded lipid vesicles:[40]
A
solution of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC; 7.5 mL,
20 mm) in methanol was mixed with the desired volumes of lipopeptide
and/or quencher stock solutions. Subsequently 150 mL of aqueous buffer
(10 mm (N-2-hydroxylethyl)piperazine-N'-2-ethansulfonic acid, 150 mm
KCl, 8 mm NaCl, 1m m MgCl2; pH 7) was added. The mixtures were
freeze-thawed three times before extruding through 0.1 mm pore size
[6] T. Zurcher, G. Luo, P. Palese, J. Virol. 1994, 68, 5748 5754.
[7] a) J. Lazarovits, S. P. Shia, N. Ktistakis, M. S. Lee, C. Bird, M. G. Roth,
J. Biol. Chem. 1990, 265, 4760 4767; b) H. Y. Naim, M. G. Roth, J.
Virol. 1993, 67, 4831 4841; c) E. Fire, O. Gutman, M. G. Roth, Y. I.
Henis, J. Biol. Chem. 1995, 270, 21075 21081.
[8] B. Lambrecht, M. F. G. Schmidt, FEBS Lett. 1986, 202, 127 132.
Chem. Eur. J. 2002, 8, No. 15
¹ WILEY-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002
0947-6539/02/0815-3375 $ 20.00+.50/0
3375

F. Eisele, J. Kuhlmann and H. Waldmann
FULL PAPER
[9] C. W. Naeve, D. Williams, EMBO J. 1990, 9, 3857 3866.
[10] a) D. A. Steinhauer, S. A. Wharton, D. C. Wiley, J. J. Skehel, Virology
1991, 184, 445 448; b) H. Y. Naim, B. Amarneh, N. T. Ktistakis, M. G.
Roth, J. Virol. 1992, 66, 7585 7588; c) H. Jin, K. Subbarao, S. Bagai,
G. P. Leser, B. R. Murphy, R. A. Lamb, J. Virol. 1996, 70, 1406 1414.
[11] M. Veit, B. N¸rnberg, K. Spicher, C. Harteneck, E. Ponimaskin, G.
Schulz, M. F. G. Schmidt, FEBS Lett. 1994, 339, 160 164.
[12] S. Bonatti, G. Migliaccio, K. Simons, J. Biol. Chem. 1989, 264, 12590
12595.
[13] D. Kadereit, J. Kuhlmann, H. Waldmann, ChemBioChem 2000, 1,
144 169.
[14] a) D. Kadereit, P. Deck, I. Heinemann, H. Waldmann, Chem. Eur. J.
2001, 7, 1184 1193; b) R. Machauer, H. Waldmann, Chem. Eur. J.
2001, 7, 2940 2956.
[21] R. G. Hiskey, T. Mizoguchi, T. Inui, J. Org. Chem. 1966, 31, 1192
1195.
[22] For a short review see: T. Zelinski, H. Waldmann, Angew. Chem. 1997,
109, 746 748; Angew. Chem. Int. Ed. 1997, 36, 722 724.
[23] M. Schelhaas, E. N‰gele, N. Kuder, H. B. Bader, J. Kuhlmann, A.
Wittinghofer, H. Waldmann, Chem. Eur. J. 1999, 5, 1239 1252.
[24] H. Kunz, J. Maerz, Angew. Chem. 1989, 110, 1424 1425; Angew.
Chem. Int. Ed. Engl. 1989, 37, 1375 1377.
[25] a) J. R. Silvius, F. l×Heureux, Biochemistry 1994, 33, 3014 3022; b) S.
Shahinian, J. R. Silvius, Biochemistry 1995, 34, 3813 3822; c) for a
recent application of this assay to non-peptidic compounds see: A.
Anikin, A. Buchynskyy, U. Kempin, K. Stembera, P. Welzel, G.
Lantzsch, Angew. Chem. 1999, 111, 3931 3935; Angew. Chem. Int.
Ed. 1999, 38, 3703 3707.
[15] Part of this work was published in preliminary form: F. Eisele, J.
Kuhlmann, H. Waldmann, Angew. Chem. 2001, 113, 382 386; Angew.
Chem. Int. Ed. 2001, 40, 369 373.
[16] E. N‰gele, M. Schelhaas, N. Kuder, H. Waldmann, J. Am. Chem. Soc.
1998, 120, 6889 6902.
[26] R. C. Macdonald, R. I. Macdonald, B. P. Menco, K. Takeshita, N. K.
Subbarao, L. R. Hu, Biochim. Biophys. Acta 1991, 1061, 297 303.
[27] H. Schroeder, R. Leventis, S. Rex, M. Schelhaas, E. N‰gele, H.
Waldmann, J. R. Silvius, Biochemistry 1997, 36, 13102 13109.
[28] For a recent review on biophysical experiments see: J. R. Silvius,
Liposome Res. 1999, 9, 1 19.
[17] T. Pohl, H. Waldmann, J. Am. Chem. Soc. 1997, 119, 6702 6710.
¬
[18] G. LeCorre, E. Guibe-Jampel, M. Wakselman, Tetrahedron 1978, 34,
[29] M. Jetten, A. M. Peters, J. W. F. M. v. Nispen, H. C. J. Ottenheijm,
Tetrahedron Lett. 1991, 6025 6028.
3105 3112.
[19] D. Steinke, M.-R. Kula, Angew. Chem. 1990, 102, 1204 1206; Angew.
Chem. Int. Ed. 1998, 29, 1139 1140.
[20] a) P. Braun, H. Waldmann, W. Vogt, H. Kunz, Liebigs Ann. Chem.
1991, 165 170; b) D. Sebastian, A. Heuser, S. Schulze, H. Waldmann,
Synthesis 1997, 1098 1108.
[30] A. Loffet, H. X. Zhang, Int. J. Peptide Protein Res. 1993, 42, 346 351.
[31] R. C. MacDonald, R. I. MacDonald, B. P. M. Menco, K. Takeshita,
N. K. Subbarao, L. Hu, Biochim. Biophys. Acta 1991, 1061, 297 303.
Received: December 13, 2001 [F3738]
3376
¹ WILEY-VCH Verlag GmbH, 69451Weinheim, Germany, 2002
0947-6539/02/0815-3376 $ 20.00+.50/0
Chem. Eur. J. 2002, 8, No. 1 5