Synthesis of lipidated eNOS peptides by combining enzymatic, noble metal- and acid-mediated protecting group techniques with solid phase peptide synthesis and fragment condensation in solution

Article abstract of DOI:10.1002/1521-3765(20010702)7:13<2940::AID-CHEM2940>3.0.CO;2-U

Lipid-modified proteins play decisive roles in important biological processes such as signal transduction, organization of the cytoskeleton, and vesicular transport. Lipidated peptides embodying the characteristic partial structures of their parent lipida

Full text of DOI:10.1002/1521-3765(20010702)7:13<2940::AID-CHEM2940>3.0.CO;2-U

FULL PAPER
Synthesis of Lipidated eNOS Peptides by Combining Enzymatic,
Noble Metal- and Acid-Mediated Protecting Group Techniques with
Solid Phase Peptide Synthesis and Fragment Condensation in Solution
Rainer Machauer[b] and Herbert Waldmann*[a]
Abstract: Lipid-modified proteins play
decisive roles in important biological
processes such as signal transduction,
organization of the cytoskeleton, and
vesicular transport. Lipidated peptides
embodying the characteristic partial
structures of their parent lipidated pro-
teins and semisynthetic proteins synthe-
sized from such peptides are valuable
tools for the study of these biological
phenomena. We have developed an
efficient synthesis strategy that allows
for the synthesis of long multiply lipi-
dated peptides embodying various side
chain functional groups. The strategy
was successfully applied in the synthesis
of the N-terminal undetrigintapeptide of
endothelial NO-synthase and related
lipopeptides. Key elements of the syn-
thesis strategy are the combined use of
the enzyme-labile para-phenylacetoxy-
benzyloxycarbonyl (PhAcOZ) urethane
as N-terminal blocking group, the Pd0-
sensitive allyl ester as C-terminal pro-
tecting function and acid-labile side
chain protecting groups for solution-
phase synthesis of labile S-palmitoylated
building blocks under the mildest con-
ditions with solid-phase techniques and
solution-phase fragment condensations.
The successful synthesis of the triply
lipidated 29-mer eNOS peptide convinc-
ingly demonstrates the full capacity of
the protecting group methods.
Keywords: enzymes
´ peptides ´
protecting groups ´ protein lipida-
tion ´ NO synthase
quantify the ability of a given lipidated peptide moiety to
direct a given protein to the plasma membrane or the
biological activity of a given protein depending on its plasma
membrane localization.[4] In addition, the use of such con-
jugates has shed light on the mechanism by which farnesylated
and palmitoylated Ras-proteins are selectively targeted to the
plasma membrane,[5] on the precise orientation of the lipid
groups in membranes,[6] the precise mode of action of
lipidating proteins such as Rab geranylgeranyltransferase[7]
and they yielded hints that biological signalling itself can be
amplified with small lipidated peptide conjugates.[8]
The scope of this approach critically depends on the
availability of efficient methods for the synthesis of multi-
functional lipidated peptides and strategies for the correct
orchestration of these methods in the context of the synthesis
of large lipidated peptides embodying for instance several
lipidated amino acid residues and amino acids with side chain
functional groups that require temporary protection during
the synthesis. In particular, these methods and strategies have
to take into account that lipidated peptides are acid- and base-
sensitive[3, 9] calling for sophisticated combinations of protect-
ing groups that can be removed selectively under the mildest
conditions yet are orthogonally stable to each other.
Introduction
Numerous lipid modified proteins are involved in biological
events of paramount importance, such as signal transduction,
organization of the cytoskeleton, and vesicular transport.[1, 2]
Lipidation of these proteins is a prerequisite to correct
biological function, and the lipid groups are believed to
participate in protein ± protein and protein ± membrane inter-
actions, which, for instance, may determine the selective
intracellular localization of lipid-modified proteins. Lipidated
peptides embodying the characteristic lipidated structures of
their parent proteins and semisynthetic lipoproteins obtained
from such peptides have proven to be invaluable tools in the
study of biological phenomena.[3] Thus, by means of such
peptide and protein conjugates we have developed a general
biological readout system that allows to determine and
[a] Prof. Dr. H. Waldmann
Max-Planck-Institut für molekulare Physiologie
Abteilung Chemische Biologie, Otto-Hahn-Strasse 11
44227 Dortmund (Germany) and
Universität Dortmund, Fachbereich 3, Organische Chemie
44227 Dortmund (Germany)
Fax : (‡49)231-133-2499
E-mail: herbert.waldmann@mpi-dortmund.mpg.de
Here we describe a synthesis strategy that meets these
demands. It relies on the combined use of enzyme-labile, acid-
sensitive and noble-metal-sensitive protecting groups for
solution-phase synthesis of labile S-palmitoylated building
[b] Dr. R. Machauer
Universität Karlsruhe, Institut für Organische Chemie
Richard-Willstätter-Allee 2
76128 Karlsruhe (Germany)
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Chem. Eur. J. 2001, 7, No. 13

2940±2956
blocks under very mild conditions with solid-phase and
fragment-condensation techniques.[10]
the blood vessel and activates soluble guanylate cyclase.
Thereby a signal transduction cascade is triggered which leads
to relaxation of the muscle cells. The correct orchestration of
this sequence of events is paramount to the regulation of
blood pressure.[11, 13]
Furthermore eNOS is involved in vascular remodelling and
angiogenesis,[13] and contributes to the pathogenesis of blood
vessel related disorders such as arteriosclerosis.[14] The local-
ization of eNOS to the plasma membrane and its concen-
tration in the caveolae, membrane microdomains highly
enriched in various signal trans-
As both biologically relevant and synthetically challenging
target compound we chose the triply lipidated N-terminal
undetrigintapeptide 1 of endothelial NO-synthase (eNOS;
Scheme 1). In response to exogenous signals transduced, for
example, by G-protein coupled receptors (GPCR), PKB or
change of the intracellular Ca2‡ this enzyme generates and
releases NO from arginine (Figure 1). NO then diffuses across
the endothelium to the surrounding smooth muscle cells of
ducing proteins is crucial for its
correct biological function-
ing.[15]
HN-Gly-Asn-Leu-Lys-Ser-Val-Gly-Gln-Glu-Pro-Gly-Pro-Pro-Cys-Gly-(Leu-Gly)4-Leu-Cys-Gly-Lys-Gln-Gly-OH
1
S
S
O
O
O
In contrast to the other iso-
forms of NO-synthase identi-
fied so far, the N-terminus of
eNOS is N-myristoylated at the
terminal glycine and twice S-
palmitoylated at cysteines 14
and 25 (see Scheme 1).[16] The
lipid groups are required for
plasma membrane localization
and biological activity.[15, 17]
However, the precise biological
roles fulfilled by the lipidated
part of the protein are subject
to various hypotheses. In par-
ticular, notions have been for-
warded that the lipid groups
might be responsible for selec-
tive targeting of eNOS at the
plasma membrane and caveo-
lae, for example, by mediation
of protein/protein or protein/
lipid interactions, and that pal-
mitoylation/depalmitoylation
might be involved in signalling
through eNOS.[1, 2]
C13H27C(O) = myristoyl (Myr)
C15H31C(O) = palmitoyl (Pal)
Gly-Asn(Trt)-Leu-Lys(Boc)-Ser(tBu)-Val-Gly-Gln(Trt)-Glu(OtBu)-Pro-OH
2
Myr
PhAcOZ-Gly-Pro-Pro-Cys-Gly-OAll
3
S Pal
Boc-Leu-Gly-Leu-Gly-Leu-Gly-Leu-Gly-OAll
4
PhAcOZ-Pro-Cys-Gly-OAll
S Pal
6
PhAcOZ-Leu-Cys-Gly-Lys(Boc)-Gln(Trt)-Gly-OtBu
PhAcOZ-Gly-Pro-OAll
5
S Pal
7
O
PhAcOZ:
O
O
PhAcOZ-Leu-Cys-Gly-OAll
Z-Lys(Boc)-Gln(Trt)-Gly-OtBu
O
S Pal
9
8
All:
Scheme 1. Retrosynthetic analysis of the N-myristoylated and twice S-palmitoylated N-terminal 29-mer peptide
of endothelial NO-synthase. Boc ˆ tert-butoxycarbonyl, Trt ˆ trityl ˆ triphenylmethyl, Z ˆ benzyloxycarbonyl.
Results and Discussion
The synthesis of lipidated pep-
tide 1 is complicated by several
parameters.
1) The palmitic acid thioesters
are sensitive to bases; that is,
they spontaneously hydro-
lyze at pH >7 and are sub-
ject to base-induced b-elim-
ination.[3] Therefore, base-
labile protecting groups can
not be employed in the syn-
thesis of 1.
2) For the synthesis of S-palmi-
toylated peptides solid-
phase methods are not avail-
able.[3]
Figure 1. Membrane association and enzymatic activity of endothelial NO-synthase.
Chem. Eur. J. 2001, 7, No. 13
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H. Waldmann and R. Machauer
FULL PAPER
3) Peptide 1 embodies several amino acids with side chain
functional groups that require protection during the
synthesis and final deprotection under conditions mild
enough to guarantee that the sensitive thioester bonds are
not attacked.
PhAcOZ-AA-OH
10: AA = Pro
11: AA = Leu
i
(PhAcOZ-AA-Cys-OAll)2
14: AA = Pro
15: AA = Leu
In developing a plan for the synthesis of 1 we utitlized our
experience in the synthesis of sensitive peptide conju-
gates.[3, 18] We chose a combination of enzyme-labile, acid-
sensitive, and noble-metal-sensitive protecting groups and
aimed to combine the use of solid-phase techniques with
solution-phase fragment condensations. Thus, in a retrosyn-
thetic sense, 1 was divided into N-myristoylated decapep-
tide 2, S-palmitoylated pentapeptide 3, octapeptide 4, and S-
palmitoylated hexapeptide 5 (Scheme 1). For the synthesis
and selective deprotection of S-palmitoylated building blocks
the enzyme-labile para-phenyl-acetoxybenzyloxycarbonyl
(PhAcOZ) urethane group[19] and the Pd0-labile allyl ester[20]
were chosen as temporary N- and C-terminal protecting
functions. The side chains of Asn, Lys, Ser, Gln, and Glu were
masked with acid-labile protecting groups to be cleaved off
simultaneously in the final step of the synthesis. We planned
to build up the N-terminal decapeptide on the solid phase by
means of an Fmoc/tert-butyl strategy and to assemble the
entire 29-mer in solution by appropriate fragment condensa-
tions. The retrosynthetic cuts were placed at the C-termini of
glycine and proline residues to exclude epimerization during
the fragment condensations.
ii, iii
PhAcOZ-AA-Cys-OAll
S Pal
16: AA = Pro
17: AA = Leu
iv, v
PhAcOZ-AA-Cys-Gly-OAll
S Pal
6: AA = Pro
8: AA = Leu
vi
O
O
NH-AA-Cys-Gly-OAll
O
S Pal
H2O
_ CO2
O
CH2
HO
OH
H-AA-Cys-Gly-OAll
S Pal
The base labile S-palmitoylated building blocks required
for the fragment condensations were synthesized as shown in
Scheme 2. PhAcOZ-protected amino acids 10 and 11 were
synthesized according to the method described earlier[19] and
then condensed with cystine bis(allyl ester) 13 to give
homodimeric peptides 14 and 15 in high yields. After cleavage
of the disulfide by treatment with dithiothreitol (DTT), the
liberated mercapto groups were acylated with palmitoyl
chloride to yield the fully masked dipeptides 16 and 17 in
high overall yields. To elongate the peptide chain in the
C-terminal direction, the allyl ester protecting group was
selectively cleaved by means of Pd0-catalyzed allyl transfer
with N,N'-dimethylbarbituric acid (DMB) as the accepting
C-nucleophile.[21] Coupling of the liberated carboxylic
acids 18 and 19 with glycine allyl ester 20 gave masked
intermediates 6 and 8 which had to be deblocked next at the
N-terminus. Upon treatment of these PhAcOZ-protected
compounds with immobilized penicillin G acylase in 0.2m
Na3PO4 buffer at pH 6.8, the phenylacetic acid ester incorpo-
rated into the urethane was saponified. Thereby a phenolate
was generated which spontaneously fragmented into a
quinone methide, CO2, and the desired selectively deprotect-
ed lipidated tripeptides 21 or 22 (Scheme 2). Notably, the
conditions of this enzyme-initiated blocking group fragmen-
tation are so mild that the base-labile palmitic acid thioester,
the C-terminal allyl ester and the peptide bonds remained
completely intact.
21: AA = Pro
22: AA = Leu
AA = Pro vii, viii
H-Gly-Pro-Pro-Cys-Gly-OAll
S Pal
24
Scheme 2. Synthesis of the sensitive S-palmitoylated building blocks with a
combination of the enzyme-labile PhAcOZ urethane and the Pd0-sensitive
allyl ester protecting groups. i) [H-Cys-OAll]2 ´ 2pTosOH (13), HOBt,
EDC, NEt3, CH2Cl2, 14: 88%, 15: 90%; ii) DTT, NEt3, CH2Cl2;
iii) H3C(CH2)14COCl, NEt3, 08C, CH2Cl2, 16: 72% (two steps), 17: 85%
(two steps); iv) [Pd(PPh3)4], DMB, THF, 18: 91%, 19: 86%; v) H-Gly-
OAll ´ pTosOH (20), HOBt, EDC, CH2Cl2, 6: 91%, 8: 94%; vi) penicillin G
acylase, phosphate buffer pH 6.8, 0.1m DMB or KI, dimethyl-b-cyclo-
dextrin, 20% (v/v) MeOH, 21: 53%, 22: 56%; vii) PhAcOZ-Gly-Pro-OH
23, HOAt, EDC, CH2Cl2, 3: 82%; viii) penicillin G acylase CLEC,
phosphate buffer pH 6.8, 0.1m KI, dimethyl-b-cyclodextrin, 20% (v/v)
ˆ
MeOH, 39%. HOBt ˆ 1-hydroxybenzotriazole, EDC N-ethyl-N'-(3-dime-
thylaminopropyl)carbodiimide hydrochloride, DTTˆ 1,4-dithio-d,l-threi-
tol, DMB ˆ N,N'-dimethylbarbituric acid, pTos ˆ para-toluenesulfonyl,
HOAt ˆ 1-hydroxy-7-azabenzotriazole.
liberated amino groups), this potent electrophile had to be
trapped by a good nucleophile.
Thus, reagents were sought that are nucleophilic enough to
trap the quinone methide efficiently but do not attack the
thioesters at the same time. For this purpose several reagents
were tested including NaI, KI, NaHSO3, Na2S, and DMB.
Surprisingly, in the case of leucine derivative 8 addition of
KI proved to be best, whereas in the case of proline peptide 6
the rather weak nucleophile DMB gave the best results. We
speculate that the reactivity of the additives is not only
determined by the aqueous medium but also by the micro-
environment in the active site of penicillin acylase which
In order to achieve reproducible, preparatively useful
results in the enzymatic transformations, several reaction
parameters had to be adjusted.
Firstly, to rule out undesired side reactions of the interme-
diary generated quinone methide (in particular with the
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Lipidated eNOS Peptides
2940±2956
makes predictions on the right choice of nucleophile rather
difficult.
PhAcOZ-Leu-Cys-Gly-OAll
S Pal
8
Secondly, peptides 6 and 8 are only sparingly soluble in
aqueous buffer so that solubilizing additives were required to
render the substrates accessible to the biocatalysts. Initially,
different cosolvents miscible with water were investigated.
However, even in the presence of up to 50 vol% of methanol,
ethanol, isopropanol, 1,4-dioxane, ethyleneglycol, DMF or
DMSO a satisfying conversion could not be achieved. There-
fore, cyclodextrins were additionally introduced, since these
had already earlier served well in related problematic cases.[9]
Finally, the combined use of 20 vol% methanol and 38 equiv-
alents dimethyl-b-cyclodextrin gave the best results. The
cyclodextrin most probably slips over the hydrophobic
palmitoyl groups, thereby solubilizing the peptides and
shielding the thioester from undesired hydrolysis.
Selectively deprotected lipotripeptide 21 was then con-
densed with PhAcOZ-masked dipeptide 23 and the resulting
lipidated pentapeptide 3 was N-terminally deprotected by
means of the enzyme-initiated fragmentation detailed above.
In this case the use of cross-linked enzyme crystals
(CLECs)[22] of penicillin G acylase proved to be superior to
the use of soluble or immobilized enzyme. This preparation of
the acylase is particularly stable in the presence of potentially
denaturing cosolvents and gave access to the desired building
block 24 in reasonable yield.
For the synthesis of lipid-modified fragment 5 tripeptide 8
was C-terminally deprotected by Pd0-catalyzed allyl transfer
to morpholine as accepting nucleophile (Scheme 3). Subse-
quent condensation of the resulting tripeptide carboxylic
acid 25 with N-terminally unmasked tripeptide 26 yielded
fully protected hexapeptide 5. Enzymatic deprotection of 5
was attempted under the conditions and including the
variations of the reaction conditions described above for
tripeptides 6, 8, and pentapeptide 3. However, due to the
significantly higher hydrophobicity of hexapeptide 5 all
attempts failed. In order to increase the hydrophilic character
of the palmitoylated hexapeptide an analogue without trityl-
masked glutamine side chain was synthesized by coupling of
lipotripeptide 25 with tripeptide ester 27 (Scheme 3). Tripep-
tides 26 and 27 were obtained using standard methods of
peptide chemistry (see the Experimental Section). Although
hexapeptide 28 clearly is more polar than trityl-masked
analogue 5 it could not be rendered accessible to the
biocatalyst under all conditions described above. Since this
variation of the protecting group pattern did not overcome
the synthetic problem the reaction medium was varied.
Since the use of deoxytaurocholic acid improves the results
in the lipase-catalyzed removal of the heptyl ester protecting
group from phosphopeptides[23] in an initial series of experi-
ments various detergents were investigated. However, neither
in the presence of anionic (cholic acid, deoxycholic acid,
taurocholic acid), cationic (cetyltrimethylammonium bro-
mide), non-ionic (Triton X-100, TritonX-114, TWEEN 20,
TWEEN 80, NonidetP 40, n-octylglycoside, digitonin, Pril)
detergents or other cyclodextrin derivatives (dimethyl a-
cyclodextrin, hydroxypropyl b-cyclodextrin, hydroxypropyl g-
cyclodextrin) in different concentrations was the solubility of
hexapeptides 5 and 28 significantly enhanced.
i
PhAcOZ-Leu-Cys-Gly-OH
S Pal
iii
25
ii
PhAcOZ-Leu-Cys-Gly-Lys(Boc)-Gln(X)-Gly-OtBu
S Pal
5: X = Trt
28: X = --
H-Leu-Cys-Gly-Lys(Boc)-Gln(X)-Gly-OtBu
S Pal
Scheme 3. Synthesis of the sensitive S-palmitoylated hexapeptides 5 and
28 by fragment condensation. i) [Pd(PPh3)4], morpholine, THF, 80%; ii) H-
Lys(Boc)-Gln(Trt)-Gly-OtBu (26), HOBt, EDC, CH2Cl2, 62%; iii) H-
Lys(Boc)-Gln-Gly-OtBu (27), HOBt, EDC, CHCl3/CF3CH2OH 3:1 (v/v),
32%.
Finally, upon use of the zwitterionic detergents N-dodecyl-
N,N-dimethyl-3-ammonium-1-propanesulfate (SB 3-12) and
lauryldimethylamine N-oxide (LDAO) at 25 ± 50 mm concen-
tration and in the presence of 10 ± 20 vol% DMSO as
cosolvent clear solutions were obtained. Subsequent attempts
to deprotect the lipopeptides with penicillin G acylase on
Eupergit or with penicillin G acylase CLECs were not
successful. Conversion of the starting materials could not be
detected, and inhibition of the biocatalyst preparations by the
detergents was ruled out by separate determination of its
activity in the presence of the additives.
Furthermore, attempts to unmask hexapeptide 5 with
penicillin G acylase CLEC in an organic solvent (ethyl
acetate, toluene) saturated with water or in a reversed micelle
system[24] were not successful. Finally, instead of hexapep-
tides 5 and 28 the corresponding octapeptides with the
peptide chain elongated in N-terminal direction by dipeptide
Gly-Leu were investigated. However, in these cases a
successful enzymatic deprotection could not be achieved
either.
These results shed light on the limitations of enzymatic
protecting group techniques in lipopeptide synthesis. In order
to guarantee that the substrates will be accessible to the
biocatalyst their amino acid/lipid residue/protecting group
composition must be chosen carefully. It appears that for the
synthesis of longer lipidated peptides building blocks are
preferable in which a combination of hydrophobic lipid
residues with hydrophobic protecting groups and/or amino
acids is kept minimal. We would like to stress that, in general,
the enzymatic techniques are fairly tolerant of hydrophobic
structures, allowing for instance for the successful removal of
the PhAcOZ and the related AcOZ urethane from palmitoy-
lated and farnesylated Ras peptides.[3, 4, 9] Obviously, however,
the combination of the PhAcOZ urethane with a palmitoyl
group, a Boc group, and a tert-butyl ester in 28 and even more
so with a trityl group in 5 renders the substrates too
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H. Waldmann and R. Machauer
FULL PAPER
hydrophobic and prevents an enzymatic transformation.
Reasons for this may be the low solubility of the lipidated
peptides in aqueous systems or a tendency to form micells
making the substrates inaccessible to the biocatalyst. In
addition, we consider it possible that the hydrophobic
peptides interact directly with hydrophobic patches on the
surface of the protein thereby blocking its activity.
Fmoc-Pro-O
Cl
36
i, ii
In the light of the difficulties encountered the strategy was
changed and two tripeptide units were employed instead for
the elongation of the peptide chain (see below), that is,
palmitoylated tripeptide 22 (Scheme 2) and N-terminally
deprotected non-lipidated tripeptide 26 (Scheme 3).
9 cycles
Fmoc-Gly-
8 AA
-Pro-O
Cl
Octapeptide building block 4 was synthesized as shown in
Scheme 4. Due to the fourfold repetition of the Leu-Gly
dipeptide unit a highly convergent solution-phase synthesis
37
iii, iv, v
98 % (5 steps)
Boc-Leu-Gly-OAll
29
Gly-Asn(Trt)-Leu-Lys(Boc)-Ser(tBu)-Val-Gly-Gln(Trt)-Glu(OtBu)-Pro-OH
i
ii
Myr
2
Boc-Leu-Gly-OH
HClïH-Leu-Gly-OAll
Scheme 5. Solid-phase synthesis of decapeptide 2. i) piperidine/NMP 1:4
(v/v); ii) 4 equiv Fmoc-AA-OBt; (9 cycles); iii) piperidine/NMP 1:4 (v/v);
iv) 4 equiv H3C(CH2)12COCl, 8 equiv NEt(iPr)2; v) AcOH/CF3CH2OH/
CH2Cl2 1:1:8 (v/v/v), 98% (all steps). NMP ˆ N-methyl-pyrrolidinone.
31
30
iii
Boc-Leu-Gly-Leu-Gly-OAll
32
iv
v
solid support was achieved by means of the very acid-sensitive
2-chlorotrityl linker (see 36).[25] After the entire peptide chain
had been assembled, the N-terminal glycine residue was
unmasked by removal of the Fmoc-urethane and then the
myristic acid amide was formed on the solid support by
treatment with myristoyl chloride (four equivalents) and
Hünigꢁs base (eight equivalents). The use of N-myristoylated
glycine directly was problematic due to the low solubility of
this building block. Finally, the desired lipidated and side-
chain protected peptide 2 was released from the polymeric
carrier 37 by treatment with acetic acid/2,2,2-trifluoroethanol/
methylene chloride 1:1:8 (v/v/v) without any attack on the
other side chain protecting groups. Peptide 2 was obtained in
98% overall yield.
N-Myristoylated decapeptide 2 turned out to be only
sparingly soluble in most regular solvents and solvent systems
employed usually for HPLC analysis such as CH3CN/H2O
mixtures. Appreciable solubility could only be reached by
using denaturing solvents such as DMSO or solvent mixtures
containing fluorinated solvents such as CHCl3/2,2,2-trifluoro-
ethanol 3:1 (v/v) and CH2Cl2/hexafluoroisopropanol 95:5
(v/v). In accordance with earlier findings we assume that this
is due to the formation of b-structures by intra- or intermo-
lecular hydrogen bonding.[26, 27, 28]
Boc-Leu-Gly-Leu-Gly-OH
HClïH-Leu-Gly-Leu-Gly-OAll
33
34
vi
Boc-Leu-Gly-Leu-Gly-Leu-Gly-Leu-Gly-OAll
4
vii
CF3CO2HïH-Leu-(Gly-Leu)3-Gly-OAll
35
Scheme 4. Synthesis of the octapeptide 35. i) [Pd(PPh3)4], morpholine,
THF, 99%; ii) HCl/Et2O, CH2Cl2, 98%; iii) HOBt, EDC, NEt3, CH2Cl2,
90%; iv) [Pd(PPh3)4], morpholine, THF, 99%; v) HCl/Et2O, CH2Cl2, 99%;
vi) HOOBt, EDC, CHCl3/CF3CH2OH 3:1 (v/v), 82%; vii) CF3COOH,
CHCl3, 98%. HOOBt ˆ 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine.
from dipeptide Boc-Leu-Gly-OAll (29) was developed. Thus,
selective removal of the N- and the C-terminal blocking
groups from 29 and condensation of the fragments gave
tetrapeptide Boc-Leu-Gly-Leu-Gly-OAll (32) (90%) which
was then subjected to the same sequence (82% coupling
yield). Selective removal of the Boc group was carried out
with HCl/ether and cleavage of the allyl ester was performed
with [Pd(PPh3)4]/morpholine (98 ± 99% yields in all cases).
Finally, the Boc group was removed from octapeptide 4 by
treatment with trifluoroacetic acid in CHCl3 to yield building
block CF3COOH ´ H-(Leu-Gly)4-OAll (35) in 98% yield.
N-Myristoylated decapeptide 2 was synthesized on the solid
phase (Scheme 5). For N-terminal protection the 9-fluorenyl-
methoxycarbonyl (Fmoc) group was employed and the side
chains of the trifunctional amino acids were masked with acid
labile trityl- or tert-butyl protecting groups. Attachment to the
With all required and selectively unmasked building blocks
in hand, the assembly of the eNOS 29-mer peptide 1 by
fragment condensation from the N- towards the C-terminus
was approached (Scheme 6). Due to the limited solubility of
myristoylated decapeptide 2 (see above) solvents that are
usually used in peptide chemistry such as CH2Cl2 or DMF
could not be employed in the coupling of 2 with palmitoylated
building block 24. However, by running the condensation
reaction in CHCl3/CF3CH2OH 3:1 (v/v) pentadecapeptide 38
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Lipidated eNOS Peptides
2940±2956
yield (92%). Therefore, all
further C-terminal deprotec-
tions were carried out in
DMSO.
Gly-Asn(Trt)-Leu-Lys(Boc)-Ser(tBu)-Val-Gly-Gln(Trt)-Glu(OtBu)-Pro-OH
2
Myr
i
Gly-Asn(Trt)-Leu-Lys(Boc)-Ser(tBu)-Val-Gly-Gln(Trt)-Glu(OtBu)-Pro-Gly-Pro-Pro-Cys-Gly-OAll
Coupling of pentadecapep-
tide 39 with octapeptide 35 in-
itially was attempted in
CHCl3/CF3CH2OH mixtures
as described above for 2, how-
ever, in this case conversion
was incomplete. By analogy to
the calcitonin synthesis by
Schäfer et al.[29] DMSO was
used as solvent and HOAt/
EDC as coupling reagents.
Under these conditions the
fragment condensation pro-
ceeded with appreciable rate
and resulted in a product mix-
ture from which the desired
23-mer peptide 40 could be
isolated readily by precipita-
tion and washing. From this
compound the allyl ester was
removed selectively under the
conditions described above to
give carboxylic acid 41 in high
yield. Elongation of the pep-
tide chain with the third lipi-
dated building block proceed-
ed smoothly in N-methyl-pyr-
rolidinone (NMP) as solvent
to yield 26-mer peptide 42.
38
S Pal
Myr
ii
Gly-Asn(Trt)-Leu-Lys(Boc)-Ser(tBu)-Val-Gly-Gln(Trt)-Glu(OtBu)-Pro-Gly-Pro-Pro-Cys-Gly-OH
39
S Pal
Myr
iii, iv
Gly-Asn(Trt)-Leu-Lys(Boc)-Ser(tBu)-Val-Gly-Gln(Trt)-Glu(OtBu)-Pro-Gly-Pro-Pro-Cys-(Gly-Leu)4-Gly-OH
41
S Pal
Myr
v, vi
Gly-Asn(Trt)-Leu-Lys(Boc)-Ser(tBu)-Val-Gly-Gln(Trt)-Glu(OtBu)-Pro-Gly-Pro-Pro-Cys-Gly-(Leu-Gly)4-Leu-Cys-Gly-OH
43
S Pal
S Pal
Myr
vii, viii
HN-Gly-Asn-Leu-Lys-Ser-Val-Gly-Gln-Glu-Pro-Gly-Pro-Pro-Cys-Gly-(Leu-Gly)4-Leu-Cys-Gly-Lys-Gln-Gly-OH
S
S
O
1
O
O
Scheme 6. Synthesis of the lipidated 29-mer target peptide 1 by fragment condensation. i) H-Gly-Pro-Pro-
Cys(Pal)-Gly-OAll (24), HOOBt, EDC, CHCl3/CF3CH2OH 3:1 (v/v), 91%; ii) [Pd(PPh3)4], DMB, DMSO, 92%;
iii: H-Leu-(Gly-Leu)3-Gly-OAll ´ CF3COOH (35), HOAt, EDC, NEt3, DMSO, 40: 72%; iv) [Pd(PPh3)4], DMB,
DMSO, 87%; v) H-Leu-Cys(Pal)-Gly-OAll (22), HOAt, EDC, NMP, 42: 86%; vi) [Pd(PPh3)4], DMB, DMSO,
69%; vii) H-Lys(Boc)-Gln(Trt)-Gly-OtBu (26), HOAt, EDC, NMP, 44: 86%; viii) CF3COOH/ethanedithiol/H2O
95:2.5:2.5 (v/v/v), 31%.
was obtained in 91% yield. Best results were obtained by
employing a slight excess (1.3 equiv) of pentapeptide 24 and
extraction of surplus palmitoylated peptide with methanol
following precipitation of the product after the reaction was
complete. This simple purification procedure was much more
efficient than all attempts to purify doubly lipidated pep-
tide 38 by HPLC on RP18 or RPC4 columns employing H2O/
CH3CN or H2O/iPrOH gradients or size-exclusion chroma-
tography on Sephadex LH-20 or LH-60 using DMSO,
CF3CH2OH/CHCl3/CH3OH or CHCl3/CH3OH/0.1n HCl mix-
tures as eluents.
In addition, analysis of the crude product mixture by
MALDI-MS indicated that the coupling had proceeded
efficiently since starting peptide 2 could not be detected.
Therefore, in all subsequent fragment condensation steps the
smaller peptide fragments were used in 1.3 ± 1.5-fold excess
and the products were purified by precipitation and washing
with methanol. Selective removal of the allyl ester from fully
masked peptide 38 with [Pd(PPh3)4]/N,N'-dimethylbarbituric
acid initially was attempted in CHCl3/CF3CH2OH 3:1 (v/v) as
well. However, mass spectrometrical analysis of the product
mixture indicated that under these conditions the pentadeca-
peptide trifluoroethyl ester was formed as by-product.
Screening for alternative solvents revealed that in DMSO
no undesired side reaction occurred. Under these conditions
the C-terminal allyl ester was removed selectively and in high
The selective removal of the allyl ester from the 26-mer
intermediate 42 was considered to be particularly challenging
since this peptide embodies two base-labile thioesters and
several acid-labile groups. In addition, this fully masked
compound is highly hydrophobic, so that formation of
aggregates and poor solubility, which would render the
compound poorly accessible to the Pd0 catalyst, had to be
anticipated. However, to our great pleasure this deprotection
could also be effected without any undesired side reaction,
and C-terminally deprotected 26-mer peptide 43 was obtained
in high yield. Finally, the peptide chain was elongated with
tripeptide 26 and, in the last step, all acid-labile side chain
protecting groups were cleaved from the resulting undetri-
gintapeptide 44 by treatment with trifluoroacetic acid in the
presence of ethanedithiol as cation scavenger.
All attempts to purify the completely masked lipopeptide
intermediates by HPLC failed. However, deprotected target
compound 1 could be purified by HPLC at elevated temper-
ature employing RPC4 column material as the stationary
phase and a water/acetonitrile gradient for product elution.
Although this unmasked peptide is significantly more polar
than its fully protected congener the influence of the three
lipid residues remains dominant. Notably, appropriate prod-
uct application to the column was crucial and best achieved
after dissolving the peptide in pure formic acid and subse-
quent dilution with water to a 1:1 (v/v) mixture.
Chem. Eur. J. 2001, 7, No. 13
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
0947-6539/01/0713-2945 $ 17.50+.50/0
2945

H. Waldmann and R. Machauer
FULL PAPER
spectra on a Voyager BioSpectrometry Workstation. Specific rotations
were measured with a Perkin ± Elmer Polarimeter 241. UV spectra were
recorded on a Perkin ± Elmer Lambda 2 UV/VIS spectrometer. Elemen-
tary analyses were performed on a Hereaus CHN-Rapid apparatus.
Melting points were determined in open capillaries using a Büchi 530
apparatus and are uncorrected. Solid-phase peptide synthesis was done on
an Applied Biosystems 430A Peptide Synthesizer employing the Fmoc/
tBu-strategy, using the standard
By analogy to the acid-mediated removal of the side chain
protecting groups from 29-mer peptide 44, also N-myristoy-
lated decapeptide 2, doubly lipidated pentadecapeptide 39,
doubly lipidated 23-mer peptide 41 and triply lipidated 26-
mer peptide 43 were deprotected in appreciable yields
(Scheme 7). Thereby, altogether a set of five peptides
protocol. HPLC was performed on
a
Macherey&Nagel
Nucleosil
i
Gly-Asn-Leu-Lys-Ser-Val-Gly-Gln-Glu-Pro-OH
2
CC250/4.6 120-5 C4 analytical col-
umn, flow: 1 mLmin and Nucleo-
74 %
45
1
Myr
sil CC250/8 120-5 C4 semi prepara-
1
tive column, flow: 3 mLmin
.
i
Gly-Asn-Leu-Lys-Ser-Val-Gly-Gln-Glu-Pro-Gly-Pro-Pro-Cys-Gly-OH
Materials: Analytical chromatogra-
phy was performed on E. Merck
silica gel 60F254 aluminum plates.
Flash chromatography was per-
formed on Baker silica gel (40 ±
64 mm). Penicillin G acylase was
obtained in immobilized form on
Eupergit C from Roche (Diagnos-
tics). Penicillin G acylase CLECs
were obtained from Altus Biologics
Inc. All solvents were distilled using
standard procedures. Commercial
reagents were used without further
purification. Where indicated the
reactions were performed under
argon.
39
41
43
53 %
46
S Pal
Myr
i
Gly-Asn-Leu-Lys-Ser-Val-Gly-Gln-Glu-Pro-Gly-Pro-Pro-Cys-(Gly-Leu)4-Gly-OH
52 %
47
S Pal
Myr
i
Gly-Asn-Leu-Lys-Ser-Val-Gly-Gln-Glu-Pro-Gly-Pro-Pro-Cys-Gly-(Leu-Gly)4-Leu-Cys-Gly-OH
S Pal S Pal
42 %
48
Myr
Scheme 7. Unmasking of the lipidated fragments 2, 39, 41, and 43. i) CF3COOH/ethanedithiol/H2O 95:2.5:2.5
(v/v/v).
representing the N-terminus of eNOS but differing in size
and lipidation pattern were accessible. These compounds may
now be employed to determine the parameters (e.g. by surface
plasmon resonance[4, 9]) that are responsible for permanent
and/or reversible membrane binding of these model lipopep-
tides, and in extrapolation from the values to be obtained, of
eNOS itself.
Synthesis of PhAcOZ-protected amino acids: Trimethylsilyl chloride
(3.80 mL, 30 mmol) was added to stirred suspension of the amino acid
(15 mmol) in CH2Cl2 (70 mL) under argon in one portion. The mixture was
refluxed for 1 h and cooled to 08C and triethylamine (3.60 mL, 26 mmol)
was added. Then a solution of 4-(phenylacetoxy)benzyl chloroformate[19]
(3.04 g, 10 mmol) in CH2Cl2 (30 mL) was added dropwise (30 min) and the
reaction mixture was stirred for 30 min at 08C and at room temperature for
2 h. The solvent was removed under reduced pressure and the resulting
residue was dissolved in a mixture of NaHCO3 (2.5%, 125 mL) and diethyl
ether (100 mL). The organic layer was separated and the aqueous phase
was extracted with diethyl ether (2 Â 40 mL). The combined organic layers
were extracted with water (2 Â 25 mL) and the pH of the combined
aqueous layers was adjusted to pH 2 with HCl (2n). The aqueous solution
was extracted with ethyl acetate (3 Â 40 mL). The combined organic layers
were dried with MgSO4 and concentrated under reduced pressure to give
the desired amino acids.
Conclusion
We have developed a highly efficient synthesis of the N-
myristoylated and twice S-palmitoylated N-terminus of endo-
thelial NO-synthase and related peptides. The strategy relies
on the combined use of enzyme-labile, acid-sensitive and
noble-metal-sensitive protecting groups for solution-phase
synthesis of labile S-palmitoylated building blocks under the
mildest conditions with solid-phase and fragment-condensa-
tion techniques. The results demonstrate convincingly the full
capacity of the protecting group methods for the synthesis of
large and multiply lipidated peptides. Together with the
recently developed methods for the synthesis of entire
functional proteins by a combination of organic synthesis
with molecular biology[4] they should open up new opportu-
nities to study the chemical biology of endothelial NO-
synthase in precise molecular detail, particularly the param-
eters determining its localization to the plasma membrane
and caveolae of endothelial cells.
PhAcOZ-Pro-OH (10): Colorless oil (3.42 g, 89%); [a]D22
ˆ
40.1 (c ˆ 1.0
in CHCl3); 1H NMR (500 MHz, CDCl3): d ˆ 7.37 ± 7.26 (m, 7H; C6H5,
3
2CH), 7.04 (d, J ˆ 8.5 Hz, 2H; 2CH), 6.05 (brs, 1H; COOH), 5.18 ± 5.04
(m, 2H; CCH2O), 4.39 ± 4.30 (m, 1H; Pro a-CH), 3.85 (s, 2H; CCH2C(O)),
3.59 ± 3.39 (m, 2H; Pro d-CH2), 2.24 ± 2.04 (m, 2H; Pro b-CH2), 1.98 ± 1.85
(m, 2H; Pro g-CH2); 13C NMR (125.7 MHz, CDCl3): d ˆ 177.1 (COOH),
ˆ
169.9 (COO), 156.0 (OCON), 150.6 (arom. C O), 133.9 (arom. q), 133.4
(arom. q), 129.3, 128.7, 127.4, 121.6 (9arom. CH), 66.9 (CH2O), 59.5 (Pro a-
CH), 46.7 (Pro d-CH2), 41.4 (CH2) 29.1 (Pro b-CH2), 24.3 (Pro g-CH2); MS
‡
(FAB, 3-NBA/TFA 10:1): m/z (%): 384.2 [M‡H] ; C21H21NO6 (383.40).
PhAcOZ-Leu-OH (11): Colorless oil (3.30 g, 83%); [a]D2
ˆ
2.1 (c ˆ 1.0 in
CHCl3); 1H NMR (500 MHz, CDCl3): d ˆ 7.38 ± 7.29 (m, 7H; C6H5, 2CH),
7.03 (d, 3J ˆ 8.5 Hz, 2H; 2CH), 6.60 (brs, 1H; COOH), 5.16 (d, 3J ˆ 8.6 Hz,
1H; OCONH), 5.08 (s, 2H; CCH2O), 4.40 ± 4.35 (m, 1H; Leu a-CH), 3.84
(s, 2H; CCH2C(O)), 1.74 ± 1.65 (m, 2H; Leu g-CH, Leu b-CH2a), 1.57 ± 1.51
(m, 1H; Leu b-CH2b), 0.94 (m, 6H; 2Leu w-CH3); 13C NMR (125.7 MHz,
CDCl3): d ˆ 177.5 (COOH), 169.9 (COO), 156.1 (OCONH), 150.6 (arom.
ˆ
C O), 133.9 (arom. q), 133.4 (arom. q), 129.3, 128.8, 127.4, 121.6 (9 arom.
CH), 66.4 (CH2O), 52.4 (Leu a-CH), 41.4 (CH2, Leu b-CH2), 24.8 (Leu g-
‡
CH), 22.8, 21.7 (2Leu w-CH3); HRMS (EI, 70 eV): m/z (%): calcd for [M]
Experimental Section
399.1682; found: 399.1696; C18H17NO6 (399.44).
General procedures: 1H NMR and 13C NMR spectra were recorded on
Bruker AC-250 and Bruker DRX-500 spectrometers. EI and FAB mass
spectra were measured on a FinniganMATMS 70 and MALDI-TOF mass
PhAcOZ-Gly-OH (12): Colorless solid (4.17 g, 81%); m.p. 1628C;
1H NMR (500 MHz, CDCl3): d ˆ 7.38 ± 7.26 (m, 7H; C6H5, 2CH), 7.03 (d,
3J ˆ 8.5 Hz, 2H; 2OCCH), 5.31 (brs, 1H; OCONH), 5.08 ± 5.04 (s, 2H;
2946
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
0947-6539/01/0713-2946 $ 17.50+.50/0
Chem. Eur. J. 2001, 7, No. 13

Lipidated eNOS Peptides
2940±2956
CCH2O), 4.45 (brs, 1H; COOH), 3.98 (d, 3J ˆ 4.9 Hz, 2H; Gly a-CH2), 3.85
(s, 2H; CCH2C(O)); 13C NMR (125.7 MHz, CDCl3): d ˆ 173.5 (COOH),
(ethyl acetate/n-hexane 2:3 v/v); 1H NMR (500 MHz, CDCl3): d ˆ 7.38 ±
7.28 (m, 7H; C6H5, 2CH), 7.03 (brs, 2H; 2OCCH), 6.76 (brs, 1H; CONH),
3
ˆ
ˆ
ˆ
169.9 (COO), 156.3 (OCONH), 150.6 (arom. C O), 133.8 (arom. q), 133.3
5.88 (br, 1H; allyl CH ), 5.34 (d, Jtrans ˆ 15.9 Hz, 1H; allyl CH2a), 5.24 (d,
3
ˆ
(arom. q), 129.3, 129.2, 128.7, 127.4, 121.6 (9 arom. CH), 66.5 (CH2O), 42.5
Jcis ˆ 9.9 Hz, 1H; allyl CH2b), 5.15 (brs, 2H; CCH2O), 4.74 (brs, 1H; Cys
(Gly a-CH2), 41.4 (CCH2CO); HRMS (EI, 70 eV, 1658C): m/z (%): calcd
a-CH), 4.62 ± 4.56 (m, 2H; allyl OCH2), 4.34 ± 4.29 (m, 1H; Pro a-CH), 3.84
(s, 2H; CCH2C(O)), 3.70 ± 3.26 (m, 4H; Pro d-CH2, Cys b-CH2), 2.49 (t,
3J ˆ 7.5 Hz, 2H; Pal a-CH2), 2.31 ± 2.15 (m, 1H; Pro b-CH2a), 1.95 ± 1.88 (m,
3H; Pro b-CH2b, Pro g-CH2), 1.59 (t, 3J ˆ 7.1 Hz, 2H; Pal b-CH2), 1.37 ± 1.26
(brs, 24H; Pal (CH2)12), 0.88 (t, 3J ˆ 6.9 Hz, 3H; Pal w-CH3); 13C NMR
‡
for [M] 343.1056; found: 343.1053; C18H17NO6 (343.33).
(PhAcOZ-Pro-Cys-OAll)2 (14): HOBt (505 mg, 3.3 mmol) was added to a
solution of PhAcOZ-Pro-OH (10, 1.15 g, 3 mmol) and (pTosOH ´ H-Cys-
OAll)2 (13, 997 mg, 1.5 mmol) in CH2Cl2 (30 mL) and at 08C NEt3
(0.43 mL, 3.1 mmol) and DIC (0.48 mL, 3.1 mmol). The mixture was
stirred at 208C for 16 h. Then the solution was extracted with acetic acid
(5%, 3 Â 10 mL), NaHCO3 (2.5%, 3 Â 10 mL), and water and the organic
layer was dried with MgSO4. The solvent was removed under reduced
pressure and the product 14 was purified by flash chromatography on silica
gel using ethyl acetate/n-hexane 3:1 (v/v) as eluent to yield the title
ˆ
ˆ
(125.7 MHz, CDCl3): d ˆ 198.5 (C OS), 171.6, 169.8, 169.6 (3C O), 155.7
ˆ
(OCON), 150.5 (arom. C O), 134.1 (arom. q), 133.4 (arom. q), 131.5 (allyl
CH), 130.0, 129.4, 129.3, 129.2, 128.8, 128.6, 127.4, 121.5 (9arom. CH), 119.0
(allyl CH2), 66.7, 66.4 (CH2O, allyl OCH2), 60.5 (Pro a-CH), 52.2 (Cys a-
CH), 47.0 (Pro d-CH2), 44.0 (Cys b-CH2), 41.4 (CH2), 32.0, 31.0, 30.4, 29.7,
29.6, 29.5, 29.4, 29.3 (10Pal CH2), 29.0 (Pro b-CH2), 28.5, 25.6 (2Pal CH2),
24.5 (Pro g-CH2), 23.6, 22.7 (2Pal CH2), 14.2 (Pal w-CH3); HRMS (EI,
compound as a colorless oil (1.39 g, 88%). [a]D22
ˆ
22.4 (c ˆ 0.95 in
‡
70 eV, 1958C): m/z (%): calcd for [M] 764.4070; found: 764.4092;
CHCl3); Rf ˆ 0.32 (ethyl acetate/n-hexane 3:1 v/v); 1H NMR (500 MHz,
elemental analysis calcd (%) for C43H60N2O8S (765.02): C 67.51, H 7.91, N
3.66; found: C 67.61, H 7.84, N 3.53.
CDCl3): d ˆ 8.02 (brs, 2H; 2CONH), 7.37 ± 7.32 (m, 14H; 2C6H5, 4CH),
3
ˆ
7.05 (d, J ˆ 8.5 Hz, 4H; 2OCCH), 5.90 ± 5.85 (m, 2H; 2allyl CH ), 5.35 ±
ˆ
ˆ
5.29 (m, 2H; 2allyl CH2a), 5.24 (m, 2allyl CH2b), 5.12 ± 5.03 (m, 4H;
2CCH2O), 4.79 ± 4.75 (m, 2H; 2Cys a-CH), 4.61 (s, 4H; 2allyl OCH2),
4.33 ± 4.31 (m, 2H; 2Pro a-CH), 3.88 (s, 4H; 2CCH2C(O)), 3.61 ± 3.59 (m,
2H; 2Pro d-CH2a), 3.51 ± 3.45 (m, 2H; 2Pro d-CH2b), 3.19 ± 3.10 (m, 2H;
2Cys b-CH2a), 3.01 ± 2.97 (m, 2H; 2Cys b-CH2b), 2.21 ± 1.88 (m, 8H; 2Pro
b-CH2, 2Pro g-CH2); 13C NMR (125.7 MHz, CDCl3): d ˆ 172.1, 169.9
PhAcOZ-Leu-Cys(Pal)-OAll (17): DTT (190 mg, 1.24 mmol) and NEt3
(68 mL, 0.495 mmol) was added under argon to a solution of (PhAcOZ-
Leu-Cys-OAll)2 (15, 268 mg, 0.247 mmol) in CH2Cl2 (20 mL)and the
mixture was stirred at 208C for 1 h. Then the solution was extracted with
a degased mixture of HCl (1n)/brine/water 1:1:1 (v/v/v) (3 Â 20 mL) and
the organic layer was dried with MgSO4. At 08C to the solution was added
NEt3 (68 mL, 0.495 mmol) and palmitoyl chloride (164 mL, 0.544 mmol) and
the mixture was stirred at 208C for 1 h. Then the solvent was removed
under reduced pressure and product 17 was purified by flash chromatog-
raphy on silica gel using ethyl acetate/n-hexane 1:3 (v/v) as eluent to yield a
ˆ
ˆ
(3C O), 155.6 (OCON), 150.4 (arom. C O), 134.2 (arom. q), 133.3 (arom.
q), 131.4 (allyl CH), 129.3, 129.1, 128.7, 127.4, 121.5 (9 arom. CH), 119.1
(allyl CH2), 66.6, 66.4 (CH2O, allyl OCH2), 60.3 (Pro a-CH), 52.3 (Cys a-
CH), 47.5 (Pro d-CH2), 47.0 (Cys b-CH2), 41.4 (CH2), 29.0 (Pro b-CH2), 24.4
‡
colorless wax (330 mg, 85%). M.p. 748C; [a]D22
ˆ
12.0 (c ˆ 0.5 in CHCl3);
(Pro g-CH2); MS (FAB, 3-NBA): m/z (%): 1051.0 [M‡H] ; elemental
Rf ˆ 0.19 (ethyl acetate/n-hexane 1:3 v/v); 1H NMR (500 MHz, CDCl3):
analysis calcd (%) for C54H58N4O14S2 (1051.20): C 61.70, H 5.56, N 5.33;
found: C 61.63, H 5.78, N 5.08.
d ˆ 7.38 ± 7.29 (m, 7H; C6H5, 2CH), 7.04 (d, 3J ˆ 8.5 Hz, 2H; 2OCCH), 6.78
3
3
3
(d, 3J ˆ 7.5 Hz, 1H; CONH), 5.85 (ddt, Jtrans ˆ 17.0, Jcis ˆ 10.8, Jvic
ˆ
(PhAcOZ-Leu-Cys-OAll)2 (15): HOBt (322 mg, 2.1 mmol) was added to a
solution of PhAcOZ-Leu-OH (11, 799 mg, 2 mmol) and (pTosOH ´ H-Cys-
OAll)2 (13, 665 mg, 1.0 mmol) in CH2Cl2 (30 mL) and at 08C NEt3
(0.29 mL, 2.1 mmol) and EDC (384 mg, 2.0 mmol). The mixture was
stirred at 208C for 16 h. Then the solution was extracted with acetic acid
(5%, 3 Â 10 mL), NaHCO3 (2.5%, 3 Â 10 mL), and water and the organic
layer was dried with MgSO4. The solvent was removed under reduced
pressure and the product 15 was purified by crystallization from ethyl
acetate/n-hexane to yield a colorless solid (991 mg, 91%). M.p. 1268C;
[a]2D2 ˆ ‡32.0 (c ˆ 1.0 in CHCl3); Rf ˆ 0.43 (ethyl acetate/n-hexane 1:1 v/v);
3
ˆ
ˆ
5.6 Hz, 1H; allyl CH ), 5.33 (d, Jtrans ˆ 17.1 Hz, 1H; allyl CH2a), 5.25 (d,
3
3
ˆ
Jcis ˆ 10.7 Hz, 1H; allyl CH2b), 5.19 (d, J ˆ 8.2 Hz, 1H; OCONH), 5.11 ±
5.07 (m, 2H; CCH2O), 4.77 ± 4.74 (m, 1H; Cys a-CH), 4.62 (d, 3J ˆ 5.5 Hz,
2H; allyl OCH2), 4.20 ± 4.18 (m, 1H; Leu a-CH), 3.84 (s, 2H; CCH2C(O)),
3
3.37 ± 3.34 (m, Cys b-CH2), 2.52 (t, J ˆ 7.5 Hz, 2H; Pal a-CH2), 1.69 ± 1.60
(m, 4H; Pal b-CH2, Leu g-CH, Leu b-CH2a), 1.51 ± 1.48 (m, 1H; Leu b-
CH2b), 1.24 (brs, 24H; Pal (CH2)12), 0.94 (d, 3J ˆ 6.1 Hz, 6H; 2Leu w-CH3),
0.88 (t, 3J ˆ 7.0 Hz, 3H; Pal w-CH3); 13C NMR (125.7 MHz, CDCl3): d ˆ
ˆ
ˆ
199.3 (C OS), 172.0, 169.8, 169.5 (3C O), 155.9 (OCONH), 150.6 (arom.
3
ˆ
1H NMR (500 MHz, CDCl3): d ˆ 7.64 (d, J ˆ 7.7 Hz, 2H; 2CONH), 7.38 ±
C O), 133.9 (arom. q), 133.3 (arom. q), 131.4 (allyl CH), 129.3, 129.2, 128.8,
7.26 (m, 14H; 2C6H5, 4CH), 7.00 (d, 3J ˆ 8.4 Hz, 4H; 2OCCH), 5.85 (ddt,
127.4, 121.6 (9 arom. CH), 119.2 (allyl CH2), 66.5, 66.3 (CH2O, allyl OCH2),
53.4 (Cys a-CH), 52.5 (Leu a-CH), 44.0 (Cys b-CH2), 41.6, 41.4 (CH2, Leu
b-CH2), 32.1, 31.9, 30.3, 29.7, 29.6, 29.4, 29.2, 29.0, 28.9, 25.7, 25.6 (13 Pal
CH2), 24.6 (Leu g-CH), 22.9 (Leu w-CH3), 22.7 (Pal CH2), 22.0 (Leu w-
3
3
Jtrans ˆ 17.0, 3Jcis ˆ 10.8, 3Jvic ˆ 5.6 Hz, 2H; 2allyl CH ), 5.65 (d, J ˆ 8.7 Hz,
ˆ
2H; 2OCONH), 5.29 (dd, 3Jtrans ˆ 17.2, J ˆ 0.9 Hz, 2H; 2allyl CH2a), 5.21
2
ˆ
3
ˆ
(d, Jcis ˆ 10.5 Hz, 2H; 2allyl CH2b), 5.06 (d, J ˆ 12.5 Hz, 2H; 2CCH2aO),
‡
CH3), 14.2 (Pal w-CH3); HRMS (EI, 70 eV, 2008C): m/z (%): calcd for [M]
5.00 (d, J ˆ 12.5 Hz, 2H; 2CCH2bO), 4.89 ± 4.86 (m, 2H; 2Cys a-CH), 4.58
(d, 3J ˆ 5.6 Hz, 4H; 2allyl CH2O), 4.49 ± 4.44 (m, 2H; 2Leu a-CH), 3.84 (s,
4H; 2CCH2C(O)), 3.02 (dd, 2J ˆ 4.1 Hz, 3J ˆ 14.0 Hz, 2H; 2Cys b-CH2a),
2.86 (dd, 2J ˆ 7.2, 3J ˆ 14.0 Hz, 2H; 2Cys b-CH2b), 1.72 ± 1.59 (m, 4H; 2Leu
g-CH, 2Leu b-CH2a), 1.56 ± 1.51 (m, 2H; 2Leu b-CH2b), 0.92 (d, 3J ˆ 6.5 Hz,
6H; 2Leu w-CH3), 0.88 (d, 3J ˆ 6.4 Hz, 6H; 2Leu w-CH3); 13C NMR
780.4383; found: 780.4407; elemental analysis calcd (%) for C44H64N2O8S
(781.07): C 67.66, H 8.26, N 3.59; found: C 67.85, H 8.06, N 3.32.
PhAcOZ-Pro-Cys(Pal)-OH (18): N,N'-Dimethylbarbituric acid (62 mg,
0.4 mmol) and a catalytic amount of [Pd(PPh3)4] were added under argon to
a
solution of PhAcOZ-Pro-Cys(Pal)-OAll (16, 153 mg, 0.2 mmol) in
ˆ
(125.7 MHz, CDCl3): d ˆ 172.9, 169.8, 169.7 (3C O), 156.4 (OCONH),
tetrahydrofuran (20 mL) and the mixture was stirred at 208C for 1 h. The
solvent was evaporated under reduced pressure, and the residue was
dissolved in ethyl acetate (20 mL). Then the solution was extracted with
phosphate buffer (0.1m, pH 6.8, 3 Â 10 mL) and the organic layer was dried
over MgSO4 and concentrated under reduced pressure. Recrystallization
from CH2Cl2/n-hexane gave a colorless solid (132 mg, 91%). M.p. 113 ±
ˆ
150.4 (arom. C O), 134.0 (arom. q), 133.3 (arom. q), 131.3 (allyl CH), 129.3,
128.7, 127.4, 121.5 (9 arom. CH), 119.0 (allyl CH2), 66.3 (2OCH2), 53.3 (Cys
a-CH), 52.6 (Leu a-CH), 41.4 (CH2, Leu b-CH2), 39.9 (Cys b-CH2), 24.6
(Leu g-CH), 22.9, 21.9 (2Leu w-CH3); MS (FAB, 3-NBA): m/z: 1082.9
‡
[M‡H] ; elemental analysis: calcd (%) for C56H66N4O14S2 (1083.29): C
62.09, H 6.14, N 5.17; found: C 62.12, H 6.15, N 5.03.
1148C; [a]2D2
ˆ
28.4 (c ˆ 0.5 in CHCl3); 1H NMR (500 MHz, CDCl3/
PhAcOZ-Pro-Cys(Pal)-OAll (16): DTT (304 mg, 2.0 mmol) was added
CD3OD 10:1 v/v): d ˆ 7.41 ± 7.29 (m, 7H; C6H5, 2CH), 7.04 (brs, 2H;
2OCCH), 5.14 (brs, 2H; CCH2O), 4.64 (brs, 1H; Cys a-CH), 4.31 (brm,
1H; Pro a-CH), 3.87 (s, 2H; CCH2C(O)), 3.61 ± 3.42 (m, 3H; Pro d-CH2,
Cys b-CH2a), 3.28 ± 3.24 (m, 1H; Cys b-CH2b), 2.51 (brs, 2H; Pal a-CH2),
2.19 (brs, 1H; Pro b-CH2a), 2.11 ± 2.08 (m, 1H; Pro b-CH2b), 1.90 (brs, 2H;
Pro g-CH2), 1.60 (t, 3J ˆ 6.9 Hz, 2H; Pal b-CH2), 1.32 ± 1.26 (brs, 24H; Pal
(CH2)12), 0.88 (t, 3J ˆ 6.9 Hz, 3H; Pal w-CH3); 13C NMR (125.7 MHz,
under argon to
a solution of (PhAcOZ-Pro-Cys-OAll)2 (14, 415 mg,
0.4 mmol) in CH2Cl2 (20 mL) and NEt3 (110 mL, 0.8 mmol) and the
mixture was stirred at 208C for 2 h. Then the solution was extracted with a
degassed mixture of HCl (1n)/brine/water 1:1:1 (v/v/v) (3 Â 10 mL) and the
organic layer was dried with MgSO4. At 08C to the solution was added
NEt3 (110 mL, 0.8 mmol) and palmitoyl chloride (596 mL, 2.0 mmol) and the
mixture was stirred at 208C for 2 h. Then the solvent was removed under
reduced pressure and product 16 was purified by flash chromatography on
silica gel using ethyl acetate/n-hexane 2:3 (v/v) as eluent to yield a colorless
ˆ
ˆ
CDCl3/CD3OD 10:1): d ˆ 199.9 (C OS), 173.2, 171.5, 170.7 (3C O), 155.3
ˆ
(OCON), 150.6 (arom. C O), 134.3 (arom. q), 133.5 (arom. q), 129.7, 129.5,
129.4, 129.1, 129.0, 128.9, 128.8, 127.6, 121.7 (9 arom. CH), 67.0 (CH2O), 60.9
wax (439 mg, 72%). M.p. 418C; [a]D22
ˆ
27.2 (c ˆ 0.5 in CHCl3); Rf ˆ 0.25
(Pro a-CH), 52.4 (Cys a-CH), 47.6 (Pro d-CH2), 44.2 (Cys b-CH2), 41.5
Chem. Eur. J. 2001, 7, No. 13
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
0947-6539/01/0713-2947 $ 17.50+.50/0
2947

H. Waldmann and R. Machauer
FULL PAPER
(CH2), 32.1, 31.3, 30.4, 29.9, 29.8, 29.6, 29.4, 29.3 (10 Pal CH2), 29.1 (Pro b-
CH2), 25.7 (Pal CH2), 24.5 (Pro g-CH2), 23.7, 22.9 (2Pal CH2), 14.2 (Pal w-
2:3 (v/v) as eluent to yield a colorless wax (92 mg, 94%). M.p. 588C; [a]D22
ˆ
15.3 (c ˆ 1.0 in CHCl3); Rf ˆ 0.23 (ethyl acetate/n-hexane 2:3 v/v);
1H NMR (500 MHz, CDCl3): d ˆ 7.39 ± 7.29 (m, 7H; C6H5, 2CH), 7.20 (brs,
1H; CONH), 7.08 (d, 3J ˆ 7.3 Hz, 1H; CONH), 7.05 (d, 3J ˆ 8.5 Hz, 2H;
‡
CH3); HRMS (FAB, 3-NBA/TFA 10:1): m/z (%): calcd for [M‡H]
725.3836; found: 725.3804; C40H56N2O8S (724.95).
2OCCH), 5.86 (ddt, 3Jtrans ˆ 16.4, 3Jcis ˆ 10.8, 3Jvic ˆ 5.7 Hz, 1H; allyl CH ),
ˆ
PhAcOZ-Leu-Cys(Pal)-OH (19): N,N'-Dimethylbarbituric acid (78 mg,
0.5 mmol) and a catalytic amount of [Pd(PPh3)4] were added under argon to
a solution of PhAcOZ-Leu-Cys(Pal)-OAll (17, 195 mg, 0.25 mmol) in
tetrahydrofuran (20 mL) and the mixture was stirred at 208C for 1 h. The
solvent was evaporated under reduced pressure, and the residue was
dissolved in ethyl acetate (40 mL). Then the solution was extracted with
phosphate buffer (0.1m, pH 6.8, 3 Â 10 mL) and the organic layer was dried
over MgSO4 and concentrated under reduced pressure. Recrystallization
from CH2Cl2/n-hexane gave a colorless solid (160 mg, 86%). M.p. 1158C;
[a]2D2 ˆ ‡8.3 (c ˆ 0.5 in CHCl3); 1H NMR (500 MHz, CDCl3/CD3OD 10:1):
d ˆ 7.40 ± 7.29 (m, 7H; C6H5, 2CH), 7.05 (d, 3J ˆ 8.5 Hz, 2H; 2OCCH), 5.08
(d, J ˆ 4.2 Hz, 2H; CCH2O), 4.64 (dd, 3J1 ˆ 6.9 Hz, 3J2 ˆ 4.7 Hz, 1H; Cys a-
CH), 4.18 (dd, 3J1 ˆ 9.3 Hz, 3J2 ˆ 5.2 Hz, 1H; Leu a-CH), 3.87 (s, 2H;
CCH2C(O)), 3.47 (dd, 2J ˆ 14.0, 3J ˆ 4.4 Hz, 1H; Cys b-CH2a), 3.25 (dd,
2J ˆ 14.1, 3J ˆ 7.3 Hz, 1H; Cys b-CH2b), 2.53 (t, 3J ˆ 7.5 Hz, 2H; Pal a-CH2),
1.69 ± 1.58 (m, 4H; Pal b-CH2, Leu g-CH, Leu b-CH2a), 1.52 ± 1.46 (m, 1H;
Leu b-CH2b), 1.32 ± 1.25 (brs, 24H; Pal (CH2)12), 0.94 ± 0.92 (m, 6H; 2Leu
w-CH3), 0.88 (t, 3J ˆ 6.9 Hz, 3H; Pal w-CH3); 13C NMR (125.7 MHz,
3
2
3
ˆ
5.31 (dd, Jtrans ˆ 17.2, J ˆ 1.4 Hz, 1H; allyl CH2a), 5.23 (dd, Jcis ˆ 10.3,
2
ˆ
J ˆ 1.1 Hz, 1H; allyl CH2b), 5.12 ± 5.04 (m, 3H; OCONH, CCH2O), 4.61
(d, 3J ˆ 5.6 Hz, 3H; allyl OCH2, Cys a-CH), 4.15 ± 4.09 (m, 2H; Leu a-CH,
2
3
Gly a-CH2a), 3.93 (dd, J ˆ 18.0 Hz, J ˆ 5.0 Hz, 1H; Gly a-CH2b), 3.85 (s,
2H; CCH2C(O)), 3.35 ± 3.25 (m, 2H; Cys b-CH2), 2.53 (t, 3J ˆ 6.6 Hz, 2H;
Pal a-CH2), 1.70 ± 1.58 (m, 4H; Pal b-CH2, Leu g-CH, Leu b-CH2a), 1.49 ±
1.43 (m, 1H; Leu b-CH2b), 1.33 ± 1.24 (brs, 24H; Pal (CH2)12), 0.93 (d, 3J ˆ
3
5.1 Hz, 6H; 2Leu w-CH3), 0.88 (t, J ˆ 7.0 Hz, 3H; Pal w-CH3); 13C NMR
ˆ
ˆ
(125.7 MHz, CDCl3): d ˆ 201.6 (C OS), 172.8, 169.9, 169.1 (4C O), 156.4
ˆ
(OCONH), 150.7 (arom. C O), 133.7 (arom. q), 133.3 (arom. q), 131.5
(allyl CH) 129.4, 129.3, 128.8, 127.4, 121.6 (9 arom. CH), 118.9 (allyl CH2),
66.6, 65.9 (CH2O, allyl OCH2), 54.2 (Cys a-CH), 53.9 (Leu a-CH), 44.0 (Cys
b-CH2), 41.4, 41.3, 41.1 (CH2, Leu b-CH2, Gly a-CH2), 31.9, 30.3, 29.7, 29.6,
29.4, 29.2, 29.0, 25.6 (13Pal CH2), 24.8 (Leu g-CH), 23.0 (Leu w-CH3), 22.7
(Pal CH2), 21.7 (Leu w-CH3), 14.1 (Pal w-CH3); HRMS (EI, 70 eV, 2008C):
m/z (%): calcd for [M] 837.4598; found: 837.4637; elemental analysis calcd
(%) for C46H67N3O9S (838.46): C 65.92, H 8.06, N 5.01; found: C 66.17, H
8.01, N 4.86.
‡
ˆ
ˆ
CDCl3/CD3OD 10:1): d ˆ 199.8 (C OS), 173.2, 171.7, 170.6 (3C O), 156.6
ˆ
(OCONH), 150.6 (arom. C O), 134.3 (arom. q), 133.4 (arom. q), 129.4,
Enzymatic removal of the PhAcOZ group from the lipopeptides 6 and 8: A
solution of the PhAcOZ-protected tripeptide (10 mmol) in methanol
(0.4 mL) was added to a solution of dimethyl-b-cyclodextrin (500 mg,
0.37 mmol) in phosphate buffer (3.6 mL, 0.2m, pH 6.8 and 0.2m KI) (two
Eppendorf vials, each 2 mL). The turbid solution was sonified until it
became clear. To this solution penicillin G acylase (100 U) was added and
the mixture was shaken at 258C for 16 h. After filtering off the enzyme and
washing with methanol and water the organic solvent was removed under
reduced pressure. The resulting aqueous phase was treated with benzyl-
triethylammonium bromide (1 g, 3.7 mmol) and extracted with diethyl
ether (10 Â 10 mL). The combined organic layers were dried with MgSO4
and concentrated under reduced pressure. The product was isolated from
the residue by flash chromatography on silica gel using CHCl3/methanol
50:1 (v/v) as eluent.
129.2, 128.9, 127.6, 121.7 (9 arom. CH), 66.3 (CH2O), 53.5 (Cys a-CH), 52.3
(Leu a-CH), 44.1 (Cys b-CH2), 41.5, 41.4 (CH2, Leu b-CH2), 32.1, 30.3, 29.8,
29.7, 29.6, 29.5, 29.4, 29.1, 25.7 (13 Pal CH2), 24.7 (Leu g-CH), 22.9 (Leu w-
CH3), 22.8 (Pal CH2), 21.8 (Leu w-CH3), 14.1 (Pal w-CH3); HRMS (FAB,
‡
3-NBA/TFA 10:1): m/z (%): calcd for [M‡H] 741.4149; found: 741.4179;
elemental analysis calcd (%) for C41H60N2O8S (740.99): C 66.46, H 8.16, N
3.78; found: C 66.07, H 7.99, N 3.73.
PhAcOZ-Pro-Cys(Pal)-Gly-OAll (6): HOBt (34 mg, 0.22 mmol) was
added to a solution of PhAcOZ-Pro-Cys(Pal)-OH (18; 132 mg, 0.18 mmol)
and pTosOH ´ H-Gly-OAll (20, 52 mg, 0.18 mmol) in CH2Cl2 (50 mL) and
at 08C NEt3 (25 mL, 0.12 mmol) and finally EDC (39 mg, 0.2 mmol). The
mixture was stirred at 208C for 16 h and the solvent was washed with HCl
(0.5n, 3 Â 10 mL) and water. The organic layer was dried over MgSO4 and
concentrated under reduced pressure. The product 6 was isolated from the
residue by flash chromatography on silica gel using ethyl acetate/n-hexane
1:1 (v/v) as eluent to yield a colorless wax (137 mg, 91%). M.p. 788C;
[a]2D2 ˆ ‡18.3 (c ˆ 1.0 in CHCl3); Rf ˆ 0.36 (ethyl acetate/n-hexane 3:2 v/v);
1H NMR (500 MHz, CDCl3): d ˆ 7.53 (brs, 1H; CONH), 7.40 ± 7.28 (m, 7H;
C6H5, 2CH), 7.18 (d, 3J ˆ 7.5 Hz, 1H; CONH), 7.06 (d, 3J ˆ 8.3 Hz, 2H;
H-Pro-Cys(Pal)-Gly-OAll (21): Colorless oil (3 mg, 53%); [a]D22
ˆ
33.6
(c ˆ 0.15 in MeOH); Rf ˆ 0.35 (CHCl3/MeOH 10:1 v/v); 1H NMR
3
3
3
(500 MHz, CD3OD): d ˆ 5.94 (ddt, Jtrans ˆ 16.1, Jcis ˆ 10.9, Jvic ˆ 5.5 Hz,
2
1H; allyl CH), 5.33 (dd, 3Jtrans ˆ 17.2, J ˆ 1.5 Hz, 1H; allyl CH2a), 5.23 (dd,
ˆ
3
2
Jcis ˆ 10.5, J ˆ 1.3 Hz, 1H; allyl CH2b), 4.63 (d, 3J ˆ 5.6 Hz, 2H; allyl
ˆ
OCH2), 4.62 ± 4.59 (m, 1H; Cys a-CH), 4.28 (dd, 3J1 ˆ 8.4, 3J2 ˆ 6.4 Hz, 1H;
Pro a-CH), 3.98 (s, 2H; Gly a-CH2), 3.45 ± 3.38 (m, 2H; Pro d-CH2), 3.36 ±
3.32 (m, 1H; Cys b-CH2a), 3.17 (dd, 2J ˆ 13.9, 3J ˆ 7.7 Hz, 1H; Cys b-CH2b),
2.58 (t, 3J ˆ 7.5 Hz, 2H; Pal a-CH2), 2.47 ± 2.40 (m, 1H; Pro b-CH2a), 2.14 ±
2.01 (m, 3H; Pro b-CH2b, Pro g-CH2), 1.64 (t, 3J ˆ 7.0 Hz, 2H; Pal b-CH2),
3
3
3
ˆ
2OCCH), 5.88 (ddt, Jtrans ˆ 16.4, Jcis ˆ 10.8, Jvic ˆ 5.6 Hz, 1H; allylCH ),
3
2
3
ˆ
5.30 (dd, Jtrans ˆ 17.2, J ˆ 1.3 Hz, 1H; allyl CH2a), 5.21 (d, Jcis ˆ 10.4 Hz,
1H; allyl CH2b), 5.13 (s, 2H; CCH2O), 4.61 (brd, 3J ˆ 4.8 Hz, 3H; allyl
ˆ
OCH2, Cys a-CH), 4.28 (dd, 3J1 ˆ 8.5, 3J2 ˆ 4.6 Hz, 1H; Pro a-CH), 4.20
(dd, 2J ˆ 17.8, 3J ˆ 6.5 Hz, 1H; Gly a-CH2a), 3.89 ± 3.85 (m, 3H; Gly a-
CH2b, CCH2C(O)), 3.61 ± 3.56 (m, 1H; Pro d-CH2a), 3.50 ± 3.45 (m, 1H; Pro
d-CH2b), 3.37 ± 3.27 (m, 2H; Cys b-CH2), 2.50 (t, 3J ˆ 7.4 Hz, 2H; Pal a-
CH2), 2.24 ± 2.16 (m, 1H; Pro b-CH2a), 2.12 ± 2.04 (m, 1H; Pro b-CH2b),
1.32 ± 1.24 (brs, 24H; Pal (CH2)12), 0.89 (t, 3J ˆ 6.9 Hz, 3H; Pal w-CH3);
13
ˆ
C NMR (125.7 MHz, CD3OD): d ˆ 200.1 (C OS), 171.8, 170.5, 169.7
ˆ
(3C O), 133.2 (allyl CH), 118.7 (allyl CH2), 66.8 (allyl OCH2), 61.0 (Pro a-
CH), 54.3 (Cys a-CH), 47.5 (Pro d-CH2), 44.7 (Cys b-CH2), 42.0 (Gly a-
CH2), 33.0, 31.0, 30.9, 30.7, 30.6, 30.5, 30.4, 30.3 (12 Pal CH2, Pro b-CH2),
26.6 (Pro g-CH2), 24.9, 23.7 (2Pal CH2), 14.5 (Pal w-CH3); HRMS (EI,
3
1.91 ± 1.87 (m, 2H; Pro g-CH2), 1.58 (t, J ˆ 6.6 Hz, 2H; Pal b-CH2), 1.31 ±
1.13 (brs, 24H; Pal (CH2)12), 0.88 (t, 3J ˆ 6.9 Hz, 3H; Pal w-CH3); 13C NMR
‡
ˆ
ˆ
(125.7 MHz, CDCl3): d ˆ 202.2 (C OS), 172.1, 170.0, 169.8, 169.1 (4C O),
70 eV, 1808C): m/z: calcd for [M] 553.3549; found: 553.3524; C45H63N3O9S
ˆ
156.1 (OCON), 150.6 (arom. C O), 133.8 (arom. q), 133.3 (arom. q), 131.6
(553.80).
(allyl CH), 129.4, 129.3, 129.0, 128.7, 127.4, 121.6 (9 arom. CH), 118.6 (allyl
CH2), 67.1, 65.8 (CH2O, allyl OCH2), 61.6 (Pro a-CH), 54.4 (Cys a-CH), 47.0
(Pro d-CH2), 43.9 (Cys b-CH2), 41.4, 41.2 (CH2, Gly a-CH2), 31.9, 30.2, 29.7,
29.6, 29.4, 29.3 (10Pal CH2), 29.2 (Pro b-CH2), 28.9, 25.6 (2Pal CH2), 24.6
(Pro g-CH2), 23.6, 22.7 (2 Pal CH2), 14.1 (Pal w-CH3); HRMS (FAB,
H-Leu-Cys(Pal)-Gly-OAll (22): Colorless oil (3 mg, 56%); [a]D22
ˆ
5.8
(c ˆ 0.15 in CHCl3); Rf ˆ 0.29 (CHCl3/MeOH 10:1 v/v); 1H NMR
3
3
3
(500 MHz, CD3OD): d ˆ 5.94 (ddt, Jtrans ˆ 16.2, Jcis ˆ 10.6, Jvic ˆ 5.6 Hz,
3
2
ˆ
2
ˆ
1H; allyl CH ), 5.33 (dd, Jtrans ˆ 17.2, J ˆ 1.3, 1H; allyl CH2a), 5.23 (dd,
3
Jcis ˆ 10.5, J ˆ 1.0 Hz, 1H; allyl CH2b), 4.63 (d, 3J ˆ 5.6 Hz, 2H; allyl
ˆ
‡
3-NBA/TFA 10:1): m/z (%): calcd for [M‡H] 822.4363; found: 822.4343;
OCH2), 4.57 (t, 3J ˆ 6.8 Hz, 1H; Cys a-CH), 3.97 (d, J ˆ 2.0 Hz, 2H; Gly a-
elemental analysis calcd (%) for C45H63N3O9S (822.07): C 65.75, H 7.72, N
5.11; found: C 65.34, H 7.66, N 5.02.
2
3
CH2), 3.94 ± 3.89 (m, 1H; Leu a-CH), 3.39 (dd, J ˆ 13.8, J ˆ 6.1 Hz, 1H;
2
3
3
Cys b-CH2a), 3.20 (dd, J ˆ 13.8, J ˆ 7.7 Hz, 1H; Cys b-CH2b), 2.59 (t, J ˆ
7.4 Hz, 2H; Pal a-CH2), 1.76 ± 1.62 (m, 5H; Pal b-CH2, Leu g-CH, Leu b-
CH2), 1.29 (brs, 24H; Pal (CH2)12), 1.00 (d, 3J ˆ 3.1 Hz, 3H; 1Leu w-CH3),
PhAcOZ-Leu-Cys(Pal)-Gly-OAll (8): HOBt (22 mg, 0.14 mmol) was
added to a solution of PhAcOZ-Leu-Cys(Pal)-OH (19, 87 mg, 0.12 mmol)
and pTosOH ´ H-Gly-OAll (20, 34 mg, 0.12 mmol) in CH2Cl2 (20 mL) and
at 08C NEt3 (16 mL, 0.12 mmol) and finally EDC (25 mg, 0.13 mmol). The
mixture was stirred at 208C for 16 h and the solvent was washed with HCl
(0.5n, 3 Â 10 mL) and water. The organic layer was dried over MgSO4 and
concentrated under reduced pressure. The product 8 was isolated from the
residue by flash chromatography on silica gel using ethyl acetate/n-hexane
0.99 (d, 3J ˆ 3.2 Hz, 3H; 1Leu w-CH3), 0.90 (t, 3J ˆ 6.8 Hz, 3H; Pal w-
13
ˆ
CH3); C NMR (125.7 MHz, CD3OD): d ˆ 200.2 (C OS), 171.8, 170.6,
ˆ
170.4 (3C O), 133.3 (allyl CH), 118.7 (allyl CH2), 66.7 (allyl OCH2), 54.2
(Cys a-CH), 52.9 (Leu a-CH), 44.8 (Cys b-CH2), 42.0, 41.6 (Leu b-CH2, Gly
a-CH2), 33.0, 31.0, 30.7, 30.6, 30.5, 30.4, 30.3, 30.0, 26.6 (13 Pal CH2), 25.3
(Leu g-CH), 23.7 (Pal CH2), 23.2 (Leu w-CH3), 22.0 (Leu w-CH3), 14.5 (Pal
2948
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
0947-6539/01/0713-2948 $ 17.50+.50/0
Chem. Eur. J. 2001, 7, No. 13

Lipidated eNOS Peptides
2940±2956
‡
w-CH3); HRMS (EI, 70 eV, 2208C): m/z (%): calcd for [M] 569.3862;
found: 569.3854; C30H55N3O5S (569.84).
CH), 129.3, 129.2, 129.1, 128.7, 127.4, 121.5 (9 arom. CH), 118.7 (allyl CH2),
66.2, 65.7 (CH2O, allyl OCH2), 61.5, 58.5 (2Pro a-CH), 54.3 (Cys a-CH),
47.5, 46.3 (2Pro d-CH2), 44.1 (Cys b-CH2), 44.0, 41.4 (2Gly a-CH2), 41.2
(CH2), 31.9, 30.4, 29.7, 29.6, 29.4, 29.3, 29.2, 29.0, 28.7, 28.5 (10Pal CH2, 2Pro
b-CH2), 25.7, 25.5, 25.3, 25.1, 25.0, 22.7 (4Pal CH2, 2Pro g-CH2), 14.1 (Pal
PhAcOZ-Gly-Pro-OAll: HOBt (367 mg, 2.4 mmol) was added to
a
solution of PhAcOZ-Gly-OH (12, 687 mg, 2.0 mmol) and pTosOH
´ H-Pro-OAll (655 mg, 2.0 mmol) in CH2Cl2 (50 mL) and at 08C NEt3
(277 mL, 2.0 mmol) and finally EDC (422 mg, 2.2 mmol). The mixture
was stirred at 208C for 16 h and the solvent was washed with HCl (0.5n,
3 Â 20 mL), NaHCO3 (2.5%, 3 Â 20 mL), water (3 Â 20 mL) and brine
(20 mL). The organic layer was dried with MgSO4 and concentrated under
reduced pressure. The dipeptide was isolated from the residue by flash
chromatography on silica gel using ethyl acetate/n-hexane 3:2 (v/v) as
‡
w-CH3); HRMS (FAB, 3-NBA/TFA 10:1): m/z: calcd for [M‡H]
976.5106; found: 976.5134; elemental analysis calcd (%) for C52H73N5O11S
(976.25): C 63.98, H 7.54, N 7.17; found: C 63.66, H 7.49, N 7.21.
H-Gly-Pro-Pro-Cys(Pal)-Gly-OAll (24): A solution of PhAcOZ-Gly-Pro-
Pro-Cys(Pal)-Gly-OAll (3, 10 mg, 10 mmol) in methanol (0.4 mL) was
added to a solution of dimethyl-b-cyclodextrin (250 mg, 0.16 mmol) in
phosphate buffer (3.6 mL, 0.2m, pH 6.8; 0.1m KI) (two Eppendorf vials,
each 2 mL). The turbid solution was sonified until it became clear. To this
solution penicillin G acylase CLEC slurry (200 mL) was added and the
mixture was shaken at 258C for 16 h. After filtering off the enzyme and
washing with methanol and water the organic solvent was removed under
reduced pressure. The resulting aqueous phase was treated with benzyl-
triethylammonium bromide (1 g, 3.7 mmol) and extracted with diethyl
ether (10 Â 10 mL). The combined organic layers were dried with MgSO4
and concentrated under reduced pressure. The product 24 was isolated
from the residue by flash chromatography on silica gel using CHCl3/
eluent to yield a colorless oil (765 mg, 80%). [a]D22
ˆ
52.2 (c ˆ 1.05 in
CHCl3); Rf ˆ 0.25 (ethyl acetate/n-hexane 3:2 v/v); 1H NMR (500 MHz,
CDCl3): d ˆ 7.39 ± 7.28 (m, 7H; C6H5, 2CH), 7.04 (d, 3J ˆ 8.5 Hz, 2H;
2OCCH), 5.88 (ddt, 3Jtrans ˆ 16.2, 3Jcis ˆ 10.6, 3Jvic ˆ 5.6 Hz, 1H; allyl CH ),
ˆ
3
2
ˆ
5.70 (brs, 1H; OCONH), 5.32 (dd, Jtrans ˆ 15.7, J ˆ 1.5 Hz, 1H; allyl
3
2
ˆ
CH2a), 5.24 (dd, Jcis ˆ 10.5, J ˆ 1.3 Hz, 1H; allyl CH2b), 5.08 (s, 2H;
CCH2O), 4.61 (d, 3J ˆ 5.2 Hz, 2H; allyl OCH2), 4.53 (dd, 3J1 ˆ 8.9, 3J2 ˆ
3.3 Hz, 1H; Pro a-CH), 4.03 (dd, J ˆ 17.2, 3J ˆ 4.8 Hz, 1H; Gly a-CH2a),
2
3.96 (dd, 2J ˆ 17.2, 3J ˆ 4.0 Hz, 1H; Gly a-CH2b), 3.85 (s, 2H; CCH2C(O)),
3.60 ± 3.56 (m, 1H; Pro d-CH2a), 3.48 ± 3.43 (m, 1H; Pro d-CH2b), 2.25 ± 2.15
(m, 1H; Pro b-CH2a), 2.10 ± 1.99 (m, 3H; Pro b-CH2b, Pro g-CH2); 13C NMR
methanol 50:1 (v/v) as eluent to yield a colorless oil (2.8 mg, 39%). [a]D22
98.4 (c ˆ 0.125 in methanol); Rf ˆ 0.17 (CHCl3/methanol 10:1 v/v);
ˆ
ˆ
(125.7 MHz, CDCl3): d ˆ 171.4, 169.9, 166.9 (3C O), 156.1 (OCON), 150.4
ˆ
(arom. C O), 134.1 (arom. q), 133.4 (arom. q), 131.7 (allyl CH), 129.5,
3
3
1H NMR (500 MHz, CD3OD): d ˆ 5.94 (ddt, Jtrans ˆ 16.1, Jcis ˆ 10.7,
129.3, 129.1, 128.7, 127.3, 121.5 (9 arom. CH), 118.6 (allyl CH2), 66.1, 65.8
(CH2O, allyl OCH2), 58.9 (Pro a-CH), 45.9 (Pro d-CH2), 43.3 (Gly a-CH2),
41.4 (CH2), 30.0 (Pro b-CH2), 24.6 (Pro g-CH2); HRMS (EI, 70 eV, 1658C):
Jvic ˆ 5.6 Hz, 1H; allyl CH ), 5.32 (dd, Jtrans ˆ 17.2, 2J ˆ 1.4 Hz, 1H;
3
3
ˆ
3
2
ˆ
ˆ
allyl CH2a), 5.23 (dd, Jcis ˆ 10.5, J ˆ 1.2 Hz, 1H; allyl CH2b), 4.75 (dd,
3J1 ˆ 8.1, 3J2 ˆ 3.4 Hz, 1H; Cys a-CH), 4.63 (d, 2H; 3J ˆ 5.6 Hz, allyl
OCH2), 4.49 (dd, 3J1 ˆ 8.3, 3J2 ˆ 5.3 Hz, 1H; Pro a-CH), 4.42 ± 4.40 (m, 1H;
Pro a-CH), 4.00 ± 3.81 (m, 4H; 2Gly a-CH2), 3.70 ± 3.49 (m, 4H; 2Pro d-
‡
m/z: calcd for [M] 480.1897; found: 480.1875; C26H28N2O7 (480.51).
PhAcOZ-Gly-Pro-OH (23): Morpholine (44 mL, 0.5 mmol) was added
under argon to a solution of PhAcOZ-Gly-Pro-OAll (240 mg, 0.5 mmol) in
tetrahydrofuran (10 mL) and a catalytic amount of [Pd(PPh3)4], and the
mixture was stirred at 208C for 1 h. The solvent was evaporated under
reduced pressure, and the residue was dissolved in ethyl acetate (50 mL).
Then the solution was extracted with HCl (0.5n, 3 Â 10 mL), water (3 Â
10 mL) and brine and the organic layer was dried over MgSO4 and
concentrated under reduced pressure to yield a pale yellow oil (126 mg,
2
3
2
CH2), 3.41 (dd, J ˆ 14.0, J ˆ 5.3 Hz, 1H; Cys b-CH2a), 3.20 (dd, J ˆ 14.0,
3J ˆ 8.4 Hz, 1H; Cys b-CH2b), 2.58 (t, 3J ˆ 7.4 Hz, 2H; Pal a-CH2), 2.45 ±
1.95 (m, 8H; 2Pro b-CH2, 2Pro g-CH2), 1.64 (t, 3J ˆ 7.0 Hz, 2H; Pal b-
CH2), 1.28 ± 1.22 (brs, 24H; Pal (CH2)12), 0.90 (t, 3J ˆ 6.9 Hz, 3H; Pal w-
13
ˆ
CH3); C NMR (125.7 MHz, CD3OD): d ˆ 200.7 (C OS), 174.0, 172.9,
ˆ
172.2, 170.5, 165.7 (5C O), 133.3 (allyl CH), 118.7 (allyl CH2), 66.7 (allyl
OCH2), 62.0, 59.9 (2Pro a-CH), 54.2 (Cys a-CH), 48.0, 47.7 (2Pro d-CH2),
44.7 (Cys b-CH2), 42.0, 41.0 (2Gly a-CH2), 33.0, 31.2, 30.7, 30.5, 30.4, 30.2,
30.0, 29.9, 29.6 (10 Pal CH2, 2 Pro b-CH2), 26.6, 26.0, 25.7, 23.7 (4Pal CH2,
2Pro g-CH2), 14.5 (Pal w-CH3); HRMS (EI, 70 eV, 2108C): m/z: calcd for
98%). [a]2D2
ˆ
56.9 (c ˆ 1.0 in MeOH); 1H NMR (500 MHz, CDCl3/
3
CD3OD 1:1): d ˆ 7.38 ± 7.23 (m, 7H; C6H5, 2CH), 7.03 (d, J ˆ 8.5 Hz, 2H;
2OCCH), 5.08 (s, 2H; CCH2O), 4.45 (dd, 3J1 ˆ 9.0, 3J2 ˆ 2.9 Hz, 1H; Pro a-
CH), 4.02 (d, 2J ˆ 17.1 Hz, 1H; Gly a-CH2a), 3.90 (d, 2J ˆ 17.1 Hz, 1H; Gly
a-CH2b), 3.87 (s, 2H; CCH2C(O)), 3.59 ± 3.52 (m, 1H; Pro d-CH2), 2.22 ±
‡
[M] 707.4292; found: 707.4260; C36H61N5O7S (707.97).
2.19 (m, 1H; Pro b-CH2a), 2.03 ± 1.99 (m, 3H; Pro b-CH2b, Pro g-CH2);
PhAcOZ-Leu-Cys(Pal)-Gly-OH (25): Morpholine (11 mL, 0.13 mmol) and
a catalytic amount of [Pd(PPh3)4] were added to a solution of PhAcOZ-
Leu-Cys(Pal)-Gly-OAll (8, 109 mg, 0.13 mmol) in tetrahydrofuran (10 mL)
was added under argon and the mixture was stirred at 208C for 1 h. The
solvent was evaporated under reduced pressure, and the residue was
dissolved in ethyl acetate (20 mL). Then the solution was extracted with
HCl (0.5n, 3 Â 10 mL), water (3 Â 10 mL) and brine, and the organic layer
was dried with MgSO4 and concentrated under reduced pressure. The
product 25 was isolated from the residue by flash chromatography on silica
gel using ethyl acetate/n-hexane 2:1 (v/v) and then ethyl acetate/ethanol 1:1
(v/v) as eluent to yield a colorless wax (84 mg, 80%). M.p. 76 ± 788C;
13
ˆ
C NMR (125.7 MHz, CDCl3/CD3OD 1:1): d ˆ 175.0, 171.5, 169.2 (3C O),
ˆ
158.2 (OCON), 151.5 (arom. C O), 135.4 (arom. q), 134.5 (arom. q), 130.7,
130.1, 130.0, 129.9, 129.8, 129.4, 129.1, 128.0 (9 arom. CH), 66.8 (CH2O),
60.0 (Pro a-CH), 46.9 (Pro d-CH2), 43.7 (Gly a-CH2), 41.7 (CH2), 29.7 (Pro
‡
b-CH2), 25.3 (Pro g-CH2); HRMS (EI, 70 eV, 2108C): m/z: calcd for [M]
440.1584; found: 440.1590; C23H24N2O7 (440.45).
PhAcOZ-Gly-Pro-Pro-Cys(Pal)-Gly-OAll (3): HOAt (27 mg, 0.2 mmol)
was added to a solution of PhAcOZ-Gly-Pro-OH (23, 74 mg, 0.17 mmol)
and H-Pro-Cys(Pal)-Gly-OAll (21, 90 mg, 0.15 mmol) in CH2Cl2 (10 mL)
and at 08C EDC (35 mg, 0.18 mmol). The mixture was stirred at 208C for
16 h and the solvent was extracted with HCl (0.5n, 3 Â 15 mL), water (3 Â
15 mL), and brine (15 mL). The organic layer was dried with MgSO4 and
concentrated under reduced pressure. The product 3 was isolated from the
residue by size-exclusion chromatography on Sephadex LH-20 using
CHCl3/methanol 1:1 (v/v) as eluent to yield a colorless oil (122 mg,
[a]2D2
ˆ
10.8 (c ˆ 0.5 in CHCl3); Rf ˆ 0.29 (ethyl acetate/n-hexane 3:2 v/v);
1H NMR (500 MHz, CDCl3/CD3OD 10:1 v/v): d ˆ 7.42 ± 7.29 (m, 7H; C6H5,
2CH), 7.05 (d, 3J ˆ 8.5 Hz, 2H; 2OCCH), 5.11 (d, 2J ˆ 12.2 Hz, 1H;
CCH2aO), 5.04 (d, 2J ˆ 12.4 Hz, 1H; CCH2bO), 4.58 (dd, 3J1 ˆ 8.8, 3J2 ˆ
4.4 Hz, 1H; Cys a-CH), 4.15 ± 4.09 (m, 1H; Leu a-CH), 4.03 (d, 2J ˆ
18.0 Hz, 1H; Gly a-CH2a), 3.87 ± 3.84(m, 3H; CCH2C(O), Gly a-CH2b),
3.41 (dd, 2J ˆ 14.2, 3J ˆ 4.5 Hz, 1H; Cys b-CH2a), 3.22 (dd, 2J ˆ 14.2, 3J ˆ
8.9 Hz, 1H; Cys b-CH2b), 2.53 (t, 3J ˆ 7.6 Hz, 2H; Pal a-CH2), 1.72 ± 1.56
(m, 4H; Pal b-CH2, Leu g-CH, Leu b-CH2a), 1.52 ± 1.46 (m, 1H; Leu b-
CH2b), 1.32 ± 1.22 (brs, 24H; Pal (CH2)12), 0.94 (m, 6H; 2Leu w-CH3), 0.88
82%). [a]2D2
ˆ
56.2 (c ˆ 1.0 in CHCl3); Rf ˆ 0.31 (CHCl3/methanol 10:1
v/v); 1H NMR (500 MHz, CDCl3): d ˆ 8.10 (d, 3J ˆ 7.9 Hz, 1H; CONH),
3
7.49 (t, J ˆ 5.7 Hz, 1H; CONH), 7.39 ± 7.29 (m, 7H; C6H5, 2CH), 7.03 (d,
3J ˆ 8.4 Hz, 2H; 2OCCH), 5.88 (ddt, 3Jtrans ˆ 16.2, 3Jcis ˆ 11.0, 3Jvic ˆ 5.6 Hz,
3
3
ˆ
1H; allyl CH ), 5.64 (t, J ˆ 4.3 Hz, 1H; OCONH), 5.32 (dd, Jtrans ˆ 17.2,
2
3
J ˆ 1.1 Hz, 1H; allyl CH2a), 5.29 ± 5.20 (m, 1H; allyl CH2b), 5.08 (s, 2H;
CCH2O), 4.70 ± 4.24 (m, 5H; allyl OCH2, Cys a-CH, 2Pro a-CH), 4.10 ±
3.78 (m, 6H; 2Gly a-CH2, CCH2C(O)), 3.68 ± 3.39 (m, 4H; 2Pro d-CH2),
3.35 ± 3.25 (m, 2H; Cys b-CH2), 2.58 ± 2.51 (m, 2H; Pal a-CH2), 2.32 ± 2.15
(m, 4H; 2Pro b-CH2), 2.06 ± 1.98 (m, 2H; 2Pro g-CH2b), 1.94 ± 1.84 (m, 2H;
Pro g-CH2b), 1.66 ± 1.59 (m, 2H; Pal b-CH2), 1.30 ± 1.22 (brs, 24H; Pal
(CH2)12), 0.88 (t, 3J ˆ 6.9 Hz, 3H; Pal w-CH3); 13C NMR (125.7 MHz,
(t, J ˆ 6.9 Hz, 3H; Pal w-CH3); 13C NMR (125.7 MHz, CDCl3): d ˆ 200.7
ˆ
ˆ
ˆ
ˆ
ˆ
(C OS), 173.9, 171.8, 170.7 (4C O), 157.2 (OCONH), 150.7 (arom. C O),
133.5 (arom. q), 132.2 (arom. q), 129.5, 129.4, 129.3, 129.0, 128.9, 128.8,
128.3, 127.6, 121.8 (9 arom. CH), 66.6 (CH2O), 54.3 (Cys a-CH), 53.2 (Leu
a-CH), 44.2 (Cys b-CH2), 41.5, 41.3, 41.1 (CH2, Leu b-CH2, Gly a-CH2),
32.1, 30.3, 29.9, 29.8, 29.6, 29.5, 29.4, 29.2, 29.0, 25.8, 24.9 (13 Pal CH2), 23.1
(Leu g-CH), 22.9 (Leu w-CH3), 21.6 (Pal CH2), 21.1 (Leu w-CH3), 14.2 (Pal
‡
ˆ
ˆ
CDCl3): d ˆ 202.9 (C OS), 172.5, 171.5, 170.0, 169.2, 167.0 (5C O), 156.2
w-CH3); HRMS (FAB, 3-NBA/TFA 10:1): m/z: calcd for [M‡H]
ˆ
(OCON), 150.5 (arom. C O), 134.1 (arom. q), 133.4 (arom. q), 131.7 (allyl
798.4363; found: 798.4276; C43H63N3O9S (798.05).
Chem. Eur. J. 2001, 7, No. 13
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
0947-6539/01/0713-2949 $ 17.50+.50/0
2949

H. Waldmann and R. Machauer
FULL PAPER
Z-Gln(Trt)-Gly-OtBu: HOBt (562 mg, 3.67 mmol) was added to a solution
of Z-Gln(Trt)-OH (1.60 mg, 3.06 mmol) and pTosOH ´ H-Gly-OtBu
(928 mg, 3.06 mmol) in CH2Cl2 (80 mL) and at 08C NEt3 (424 mL,
3.06 mmol) and finally EDC (645 mg, 3.36 mmol). The mixture was stirred
at 208C for 16 h and the solvent was washed with HCl (0.5n, 3 Â 20 mL),
NaHCO3 (saturated, 3 Â 20 mL), water (3 Â 20 mL), and brine (20 mL).
The organic layer was dried with MgSO4 and concentrated under reduced
pressure. The product was isolated from the residue by flash chromatog-
raphy on silica gel using ethyl acetate/n-hexane 1:1 (v/v) as eluent to yield a
colorless solid (1.43 g, 74%). M.p. 1658C; [a]2D2 ˆ ‡16.2 (c ˆ 1.0 in CHCl3);
Rf ˆ 0.34 (ethyl acetate/n-hexane 1:1 v/v); 1H NMR (500 MHz, CDCl3):
d ˆ 7.35 ± 7.17 (m, 21H; CONH, Z, Trt), 6.40 (brs, 1H; CONH), 5.83 (brs,
1H; OCONH), 5.04 (s, 2H; CCH2O), 4.13 ± 4.09 (m, 1H; Gln a-CH), 3.83 ±
3.79 (m, 1H; Gly a-CH2a), 3.72 ± 3.68 (m, 1H; Gly a-CH2b), 2.62 ± 2.52 (m,
acetate/n-hexane 2:1 (v/v) and then ethyl acetate/ethanol 1:1 (v/v) as eluent
to yield a colorless solid (88 mg, 87%). M.p. 968C; [a]D22
ˆ
8.4 (c ˆ 1.0 in
methanol); 1H NMR (500 MHz, CD3OD): d ˆ 7.30 ± 7.18 (m, 15H; Trt),
5.52 (d, 3J ˆ 6.8 Hz, OCONH), 5.05 (d, 2J ˆ 12.2 Hz, 1H; CCH2aO), 4.94 (d,
2J ˆ 12.2 Hz, 1H; CCH2bO), 4.75 (brs, 1H; OCONH), 4.42 ± 4.40 (m, 1H;
Lys a-CH), 3.92 (d, 2J ˆ 17.6 Hz, 1H; Gly a-CH2a), 3.85 ± 3.77 (m, 1H; Gln
2
a-CH), 3.73 (d, J ˆ 17.4 Hz, Gly a-CH2b), 3.03 ± 3.00 (m, 2H; Lys e-CH2),
2.57 ± 2.48 (m, 2H; Gln g-CH2), 2.10 ± 2.05 (m, 1H; Gln b-CH2a), 1.97 ± 1.91
(m, 1H; Gln b-CH2b), 1.86 ± 1.76 (m, 2H; Lys b-CH2), 1.44 (brs, 22H;
2C(CH3)3, Lys d-CH2, Lys g-CH2); 13C NMR (125.7 MHz, CD3OD): d ˆ
ˆ
174.3, 173.4, 170.7, 170.2 (4C O), 158.4 (OCONH), 145.8 (arom. q), 129.9,
128.7, 127.8 (15 arom. CH), 82.9, 79.8 (2 tBu q), 71.5 (Trt q), 54.4 (Gln a-
CH), 54.1 (Lys a-CH), 42.7 (Gly a-CH2), 40.8 (Lys e-CH2), 33.6 (Gln g-
CH2), 32.4, 30.3 (2 Lys CH2), 29.1 (Gln b-CH2), 28.8 (tBu), 28.3 (tBu), 22.9
‡
2H; Gln g-CH2), 2.08 ± 2.04 (m, 2H; Gln b-CH2), 1.38 (s, 9H; C(CH3)3);
(Lys CH2); HRMS (EI, 70 eV, 2208C): m/z: calcd for [M] 729.4101; found:
13
ˆ
C NMR (125.7 MHz, CDCl3): d ˆ 172.3, 171.5, 169.0 (3C O), 156.2
729.4093; C41H55N5O7 (729.91).
(OCONH), 144.5 (arom. q), 136.3 (arom. q), 128.5, 128.2, 128.0, 127.0 (20
arom. CH), 82.2 (tBu q), 70.4 (Trt q), 66.9 (CH2O), 54.3 (Gln a-CH), 41.8
(Gly a-CH2), 33.0 (Gln g-CH2), 28.4 (Gln b-CH2), 28.0 (tBu); HRMS (EI,
Z-Lys(Boc)-Gln-OH: N-Hydroxysuccinimide (231 mg, 2.0 mmol) and
DCC (496 mg, 2.4 mmol) were added at 08C to a solution of Z-Lys(Boc)-
OH (760 mg, 2.0 mmol) in tetrahydrofuran (30 mL) and the mixture was
stirred at 208C for 16 h. The precipitated urea was filtered off and the
solvent was removed under reduced pressure. The remaining N-hydroxy-
succinimide ester was dissolved in dioxane (5 mL) and at 08C added
dropwise to a suspension of H-Gln-OH (439 mg, 3.0 mmol) in NaOH (2n,
1.5 mL, 3.0 mmol)/water (10 mL)/dioxane (5 mL). The mixture was stirred
at 08C for 3 h and at 208C for 5 d. Then the dioxane was removed under
reduced pressure and the solution was extracted several times with diethyl
ether. At 08C the pH of the aquaeous layer was adjusted to pH 2 with HCl
(2n) and extracted with ethyl acetate. The combined ethyl acetate extracts
were dried with MgSO4 and the solvent was removed under reduced
‡
70 eV, 1758C): m/z: calcd for [M] 635.2995; found: 635.3015; elemental
analysis calcd (%) for C38H41N3O6 (635.76): C 71.79, H 6.50, N 6.61; found:
C 71.70, H 6.48, N 6.54.
H-Gln(Trt)-Gly-OtBu: Palladium on charcoal (10%, 33 mg) was added to
a solution of Z-Gln(Trt)-Gly-OtBu (671 mg, 1.06 mmol) in methanol
(30 mL) and the mixture was stirred under a hydrogen atmosphere at 208C
for 16 h. Then the catalyst was filtered off and the solvent was removed
under reduced pressure to yield a colorless solid (534 mg (quant.). [a]D22
ˆ
1
‡10.6 (c ˆ 0.5 in methanol); H NMR (500 MHz, CD3OD): d ˆ 7.27 ± 7.18
(m, 15H; Trt), 6.40 (brs, 1H; CONH), 3.88 (d, 2J ˆ 17.6 Hz, 1H; Gly a-
CH2a), 3.77 (d, 2J ˆ 17.6 Hz, 1H; Gly a-CH2b), 3.35 ± 3.32 (m, 1H; Gln a-
CH), 2.48 ± 2.44 (m, 2H; Gln g-CH2), 1.96 ± 1.90 (m, 1H; Gln b-CH2a),
1.84 ± 1.78 (m, 1H; Gln b-CH2b), 1.45 (s, 9H; C(CH3)3); 13C NMR
pressure to yield a colorless solid (945 mg, 93%). M.p. 1478C; [a]D22
ˆ
5.0
(c ˆ 1.0 in CHCl3/MeOH 1:1 v/v); 1H NMR (500 MHz, CD3OD): d ˆ 7.36 ±
2
3
7.20 (m, 5H; C6H5), 5.08 (d, J ˆ 3.0 Hz, 1H; CCH2O), 4.36 (t, J ˆ 4.1 Hz,
1H; Lys a-CH), 4.09 (dd, 3J1 ˆ 8.3, 3J2 ˆ 5.3 Hz, 1H; Gln a-CH), 3.02 (t,
3J ˆ 6.4 Hz, 2H; Lys e-CH2), 2.33 ± 2.22 (m, 3H; Gln g-CH2, Gln b-CH2a),
1.97 ± 1.90 (m, 1H; Gln b-CH2b), 1.80 ± 1.78 (m, 1H; Lys b-CH2a), 1.66 ± 1.64
(m, 1H; Lys b-CH2b), 1.53 ± 1.42 (m, 13H; Lys d-CH2, Lys g-CH2,
C(CH3)3); 13C NMR (125.7 MHz, CD3OD): d ˆ 177.9, 175.3, 174.7
ˆ
(125.7 MHz, CDCl3): d ˆ 177.1, 174.7, 170.5 (3C O), 146.0 (arom. q),
130.0, 128.7, 127.8 (15 arom. CH), 83.0 (tBu q), 71.5 (Trt q), 55.2 (Gln a-
CH), 42.7 (Gly a-CH2), 33.7 (Gln g-CH2), 31.9 (Gln b-CH2), 28.3 (tBu);
‡
HRMS (EI, 70 eV, 1558C): m/z: calcd for [M] 501.2628; found: 501.2609;
elemental analysis calcd (%) for C30H35N3O4 * 0.5 H2O: C 70.57, H 7.11, N
8.23; found: C 70.68, H 7.02, N 8.31; C30H35N3O4 (501.62).
ˆ
(3C O), 158.5, 158.4 (2 OCONH), 138.1 (arom. q), 129.4, 128.9, 128.7 (3
Z-Lys(Boc)-Gln(Trt)-Gly-OtBu: HOBt (196 mg, 1.28 mmol) was added to
a solution of Z-Lys(Boc)-OH (404 mg, 1.06 mmol) and H-Gln(Trt)-Gly-
OtBu (534 mg, 1.06 mmol) in CH2Cl2 (70 mL) and at 08C EDC (225 mg,
1.17 mmol). The mixture was stirred at 208C for 16 h and the solvent was
extracted with HCl (0.5n, 3 Â 20 mL), NaHCO3 (saturated, 3 Â 20 mL),
water (3 Â 20 mL) and brine (20 mL). The organic layer was dried with
MgSO4 and concentrated under reduced pressure. The product was isolated
from the residue by flash chromatography on silica gel using ethyl acetate/
n-hexane 3:2 (v/v) as eluent to yield a colorless solid (826 mg, 90%). M.p.
arom. CH), 79.8 (tBu q), 67.6 (CH2O), 56.4 (Lys a-CH), 53.6 (Gln a-CH),
41.3 (Gly a-CH2), 40.9 (Lys e-CH2), 33.1 (Gln g-CH2), 32.7, 30.5 (2 Lys
CH2), 29.0 (Gln b-CH2), 28.8 (tBu), 24.0 (Lys CH2); HRMS (FAB, 3-NBA/
‡
TFA 10:1): m/z: calcd for [M‡H] 509.2611; found: 509.2650; elemental
analysis calcd (%) for C24H36N4O8 ´ H2O: C 54.74, H 7.27, N 10.64; found: C
54.47, H 6.74, N 10.26; C24H36N4O8 (508.57).
Z-Lys(Boc)-Gln-Gly-OtBu: HOBt (144 mg, 0.94 mmol) and NEt3 (108 mL,
0.78 mmol) were added to a solution of Z-Lys(Boc)-Gln-OH (397 mg,
0.78 mmol) and HCl ´ H-Gly-OtBu (131 mg, 0.78 mmol) in dimethylform-
amide (2.5 mL) and CH2Cl2 (70 mL) was added and at 08C EDC (165 mg,
0.86 mmol). The mixture was stirred at 208C for 16 h. After addition of
CH2Cl2 (30 mL) the solvent was extracted with HCl (0.5n, 3 Â 15 mL),
NaHCO3 (1n, 3 Â 15 mL), water (3 Â 15 mL) and brine (20 mL). The
precipitated product was filtered off and washed with water, CH2Cl2 and
diethyl ether, the organic layer was dried with MgSO4 and concentrated
under reduced pressure to yield a colorless solid (406 mg, 84%). M.p.
898C; [a]2D2
ˆ
21.2 (c ˆ 1.0 in CHCl3); Rf ˆ 0.10 (ethyl acetate/n-hexane
2:1 v/v); 1H NMR (500 MHz, CDCl3): d ˆ 7.41 (d, 3J ˆ 6.7 Hz, 1H; CONH),
3
7.32 ± 7.20 (m, 20H; Z, Trt), 7.06 (brs, 2H; 2CONH), 5.52 (d, J ˆ 6.8 Hz,
2
2
OCONH), 5.05 (d, J ˆ 12.2 Hz, 1H; CCH2aO), 4.94 (d, J ˆ 12.2 Hz, 1H;
CCH2bO), 4.75 (brs, 1H; OCONH), 4.31 (m, 1H; Lys a-CH), 4.06 (d, 3J ˆ
2
3
4.2 Hz, 1H; Gln a-CH), 3.87 (dd, J ˆ 17.9, J ˆ 6.0 Hz, 1H; Gly a-CH2a),
3.70 (dd, 2J ˆ 18.0, 3J ˆ 5.0 Hz, 1H; Gly a-CH2b), 3.05 (brs, 2H; Lys e-CH2),
2.56 ± 2.44 (m, 2H; Gln g-CH2), 2.04 ± 1.98 (m, 2H; Gln b-CH2), 1.76 ± 1.74
(m, 1H; Lys b-CH2a), 1.57 ± 1.54 (m, 1H; Lys b-CH2b), 1.42 (brs, 20H; Lys d-
CH2, 2C(CH3)3), 1.33 ± 1.24 (m, 2H; Lys g-CH2); 13C NMR (125.7 MHz,
1638C; [a]2D2
ˆ
7.6 (c ˆ 1.0 in CHCl3/methanol 1:1 v/v); 1H NMR
(500 MHz, CDCl3/CD3OD 1:1 v/v): d ˆ 7.36 ± 7.29 (m, 5H; C6H5), 5.09 (d,
ˆ
CDCl3): d ˆ 172.3, 171.9, 171.2, 168.6 (4C O), 156.3, 156.1 (2 OCONH),
2J ˆ 4.8 Hz, 1H; CCH2O), 4.42 (dd, J1 ˆ 8.7, J2 ˆ 4.7 Hz, 1H; Lys a-CH),
4.07 (dd, 3J1 ˆ 8.0, 3J2 ˆ 5.2 Hz, Gln a-CH), 3.86 (d, 3J ˆ 3.5 Hz, 2H; Gly a-
CH2), 3.05 (t, 3J ˆ 5.9 Hz, 2H; Lys e-CH2), 2.38 ± 2.28 (m, 2H; Gln g-CH2),
2.19 ± 2.15 (m, 1H; Gln b-CH2a), 2.00 ± 1.96 (m, 1H; Gln b-CH2b), 1.82 ± 1.76
(m, 1H; Lys b-CH2a), 1.69 ± 1.66 (m, 1H; Lys b-CH2b), 1.53 ± 1.34 (m, 22H;
Lys d-CH2, Lys g-CH2, 2C(CH3)3); 13C NMR (125.7 MHz, CDCl3/CD3OD
3
3
144.4 (arom. q), 136.2 (arom. q), 128.6, 128.4, 128.1, 128.0, 127.0 (20 arom.
CH), 81.9, 79.0 (2 tBu q), 70.6 (Trt q), 66.9 (CH2O), 55.0 (Gln a-CH), 52.6
(Lys a-CH), 41.9 (Gly a-CH2), 39.8 (Lys e-CH2), 33.3 (Gln g-CH2), 32.0,
29.4 (2 Lys CH2), 28.4 (tBu), 28.3 (Gln b-CH2), 28.0 (tBu), 22.4 (Lys CH2);
‡
HRMS (FAB, 3-NBA/TFA 10:1): m/z: calcd for [M‡H] 864.4548; found:
864.4493; elemental analysis calcd (%) for C49H61N5O9 (864.06): C 68.11, H
7.12, N 8.11; found: C 67.85, H 7.01, N 8.02.
ˆ
1:1): d ˆ 177.1, 174.0, 172.7, 169.5 (4C O), 157.8, 157.5 (2OCONH), 136.8
(arom. q), 128.9, 128.5, 128.4, 128.2 (5arom. CH), 82.6, 79.5 (2 tBu q), 67.4
(CH2O), 56.0 (Lys a-CH), 53.1 (Gln a-CH), 42.2 (Gly a-CH2), 40.2 (Lys e-
CH2), 31.8 (Gln g-CH2), 29.7 (2 Lys CH2), 28.6 (tBu), 28.1 (tBu), 28.0 (Gln
b-CH2), 23.2 (Lys CH2); HRMS (FAB, 3-NBA/TFA 10:1): m/z: calcd for
H-Lys(Boc)-Gln(Trt)-Gly-OtBu (26): Palladium on charcoal (10%, 12 mg)
was added to
a solution of Z-Lys(Boc)-Gln(Trt)-Gly-OtBu (120 mg,
0.14 mmol) in methanol (15 mL) and the mixture was stirred under a
hydrogen atmosphere at 208C for 16 h. Then the catalyst was filtered off
and the solvent was removed under reduced pressure. The product 26 was
isolated from the residue by flash chromatography on silica gel using ethyl
‡
[M‡H] 622.3452; found: 622.3479; elemental analysis calcd (%) for
C30H47N5O9 ´ H2O: C 56.32, H 7.72, N 10.95; found: C 56.10, H 7.24, N 10.66;
C30H47N5O9 (621.73).
2950
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
0947-6539/01/0713-2950 $ 17.50+.50/0
Chem. Eur. J. 2001, 7, No. 13

Lipidated eNOS Peptides
2940±2956
H-Lys(Boc)-Gln-Gly-OtBu (27): Palladium on charcoal (10%, 6 mg) was
added to a solution of Z-Lys(Boc)-Gln-Gly-OtBu (63 mg, 0.10 mmol) in
2.40 ± 2.30 (m, 2H; Gln g-CH2), 2.19 ± 2.13 (m, 1H; Gln b-CH2a), 2.06 ± 2.00
(m, 1H; Gln b-CH2b), 1.87 ± 1.84 (m, 1H; Lys b-CH2a), 1.76 ± 1.67 (m, 2H;
Lys b-CH2b, Leu g-CH), 1.65 ± 1.62 (m, 2H; Pal b-CH2), 1.61 ± 1.55 (m, 2H;
Leu b-CH2), 1.49 ± 1.38 (2 brs, 22H; 2C(CH3)3, Lys d-CH2, Lys g-CH2),
1.36 ± 1.24 (brs, 24H; Pal (CH2)12), 0.96 (d, 3J ˆ 6.6 Hz, 3H; Leu w-CH3),
methanol (10 mL) and the mixture was stirred under
a hydrogen
atmosphere at 208C for 16 h. Then the catalyst was filtered off and the
solvent was removed under reduced pressure to yield a colorless solid
1
(49 mg, 100%). M.p. 818C; [a]D22
ˆ
12.5 (c ˆ 1.0 in methanol); H NMR
0.94 (d, 3J ˆ 6.5 Hz, 3H; Leu w-CH3), 0.89 (t, 3J ˆ 7.0 Hz, 3H; Pal w-CH3);
(500 MHz, CD3OD): d ˆ 4.43 (dd, 3J1 ˆ 8.4 Hz, 3J2 ˆ 5.6 Hz, 1H; Lys a-
CH), 3.92 (d, 2J ˆ 17.5 Hz, 1H; Gly a-CH2a), 3.77 (d, 2J ˆ 17.5 Hz, 1H; Gly
a-CH2b), 3.55 (t, 3J ˆ 6.4 Hz, 1H; Gln a-CH), 3.04 (t, 3J ˆ 6.9 Hz, 2H; Lys e-
CH2), 2.39 ± 2.32 (m, 2H; Gln g-CH2), 2.17 ± 2.10 (m, 1H; Gln b-CH2a),
2.01 ± 1.92 (m, 1H; Gln b-CH2b), 1.80 ± 1.74 (m, 1H; Lys b-CH2a), 1.70 ± 1.63
(m, 1H; Lys b-CH2b), 1.52 ± 1.33 (m, 22H; Lys d-CH2, Lys g-CH2,
2C(CH3)3); 13C NMR (125.7 MHz, CD3OD): d ˆ 177.8, 173.8, 170.2
C NMR (125.7 MHz, CDCl3/CD3OD): d ˆ 201.1 (C OS), 177.4, 175.3,
13
ˆ
ˆ
173.4, 173.0, 172.0, 171.5, 171.3, 169.6 (8C O), 158.1, 157.7 (2OCONH),
151.3 (arom. C O), 134.9 (arom. q), 134.1 (arom. q), 129.9, 129.7, 129.3,
ˆ
128.0, 122.2 (9 arom. CH), 82.6 (tBu q), 79.6 (tBu q), 67.0 (CH2O), 55.1, 54.9,
54.8 (Cys a-CH, Gln a-CH, Lys a-CH), 53.6 (Leu a-CH), 44.5 (Cys b-CH2),
43.5, 42.5, 41.7, 41.2 (CH2, Leu b-CH2, 2Gly a-CH2), 40.6 (Lys e-CH2), 32.5
(Gln g-CH2), 31.9, 31.3, 30.3, 30.2, 30.1, 30.0, 29.9, 29.6 (13 Pal CH2, 2Lys
CH2, Gln b-CH2), 28.7 (tBu), 28.3 (tBu), 27.9 (Pal CH2), 26.2 (Lys CH2),
25.3 (Leu g-CH), 23.7 (Pal CH2), 23.3 (Leu w-CH3), 23.2 (Pal CH2), 21.9
(Leu w-CH3), 14.3 (Pal w-CH3); MS (FAB, 3-NBA/TFA 10:1): m/z: 1111.6
ˆ
(3C O),158.5 (OCONH), 82.9, 79.9 (2 tBu q), 55.3 (Lys a-CH), 53.9
(Gln a-CH), 42.7 (Gly a-CH2), 41.0 (Lys e-CH2), 34.5 (Gln g-CH2), 32.4,
30.6 (2Lys CH2), 29.1 (Gln b-CH2), 28.8 (tBu), 28.3 (tBu), 23.5 (Lys CH2);
HRMS (FAB, 3-NBA/TFA 10:1): m/z: calcd for [M‡H] 488.3084; found:
‡
‡
‡
‡
[M Boc tBu‡3H] , 1167.6 [M Boc‡2H] , 1267.6 [M‡H] , 1289.6
‡
488.3052; C22H41N5O7 (487.59).
[M‡Na] ; C65H102N8O15S (1267.65).
Boc-Leu-Gly-OAll (29): HOBt (3.37 g, 22 mmol), NEt3 (2.78 mL,
20 mmol) were added to a solution of Boc-Leu-OH (4.63 g, 20 mmol)
and pTosOH ´ H-Gly-OAll (20, 5.75 g, 20 mmol) in CH2Cl2 (100 mL) was
added and at 08C DIC (3.12 mL, 20 mmol). The mixture was stirred at
208C for 16 h, then solvent was extracted with acetic acid (5%, 3 Â
100 mL), NaHCO3 (1n, 3 Â 100 mL) and water (3 Â 100 mL). The organic
layer was dried with MgSO4 and concentrated under reduced pressure. The
product 29 was isolated from the residue by flash chromatography on silica
gel using ethyl acetate/n-hexane 1:2 (v/v) as eluent to yield a colorless oil
PhAcOZ-Leu-Cys(Pal)-Gly-Lys(Boc)-Gln(Trt)-Gly-OtBu (5): HOBt
(20 mg, 126 mmol), EDC (22 mg, 116 mmol) and H-Lys(Boc)-Gln(Trt)-
Gly-OtBu (26, 77 mg, 105 mmol) were added to a solution of PhAcOZ-Leu-
Cys(Pal)-Gly-OH (25, 84 mg, 105 mmol) in CH2Cl2 (8 mL) were added at
08C. The mixture was stirred at 208C for 16 h. After addition of CH2Cl2
(30 mL) the solvent was extracted with HCl (0.5n, 3 Â 15 mL), water (3 Â
15 mL) and brine (15 mL). The organic layer was dried with MgSO4 and
concentrated under reduced pressure. The product 5 was isolated from the
residue by size-exclusion chromatography on Sephadex LH-20 using ethyl
CHCl3/methanol 1:1 (v/v) as eluent to yield a colorless solid (98 mg, 62%).
(5.75 g, 88%). [a]D22
ˆ
27.4 (c ˆ 1.0 in CH2Cl2); Rf ˆ 0.29 (ethyl acetate/
n-hexane 1:2 v/v); 1H NMR (500 MHz, CDCl3): d ˆ 6.80 (brs, 1H; CONH),
[a]2D2
ˆ
7.2 (c ˆ 1.0 in CHCl3); Rf ˆ 0.60 (CHCl3/methanol 10:1 v/v);
3
3
3
1H NMR (500 MHz, CDCl3): d ˆ 7.48 (brs, 1H; CONH), 7.41 (brs, 1H;
CONH), 7.36 ± 7.18 (m, 23H; 3CONH; Z, Trt, 2CH), 7.01 (d, 3J ˆ 8.4 Hz,
2H; 2OCCH), 6.92 (brs, 1H; CONH), 6.31 (brs, 1H; OCONH), 5.04 (s,
2H; CCH2O), 4.74 (brs, 1H; OCONH), 4.61 (d, 3J ˆ 6.5 Hz, 1H; Cys a-
CH), 4.24 (brs, 1H; Lys a-CH), 4.03 (brs, 1H; Leu a-CH), 3.92 ± 3.84 (m,
5H; Gln a-CH, Gly a-CH2, CCH2C(O)), 3.71 ± 3.57 (m, 2H; Gly a-CH2),
3.04 ± 2.93 (m, 4H; Lys e-CH2, Cys b-CH2), 2.57 ± 2.51 (m, 3H; Pal a-CH2,
Gln g-CH2a), 2.42 ± 2.40 (m, 2H; Gln g-CH2b), 2.11 ± 2.01 (m, 2H; Gln b-
CH2), 1.86 ± 1.54 (m, 7H; Lys b-CH2, Pal b-CH2, Leu g-CH, Leu b-CH2),
1.49 ± 1.40 (brs, 22H; 2C(CH3)3, Lys d-CH2, Lys g-CH2), 1.30 ± 1.25 (brs,
ˆ
5.86 (ddt, Jtrans ˆ 16.3, Jcis ˆ 10.5, Jvic ˆ 5.7 Hz, 1H; allyl CH ), 5.32 (dd,
Jtrans ˆ 17.1, J ˆ 1.5 Hz, 1H; allyl CH2a), 5.26 (dd, Jcis ˆ 10.3, 2J ˆ 1.2 Hz,
3
2
3
ˆ
1H; allyl CH2b), 5.00 (d, J ˆ 6.7 Hz, 1H; OCONH), 4.64 (d, 3J ˆ 5.8 Hz,
2H; allyl OCH2), 4.20 (brs, 1H; Leu a-CH), 4.06 (t, 3J ˆ 5.2 Hz, 2H; Gly a-
CH2), 1.70 ± 1.66 (m, 2H; Leu g-CH, Leu b-CH2a), 1.52 ± 1.47 (m, 1H; Leu
3
ˆ
b-CH2b), 1.44 (s, 9H; C(CH3)3), 0.94 (d, 3J ˆ 6.1 Hz, 6H; 2Leu w-CH3);
13
ˆ
C NMR (125.7 MHz, CDCl3): d ˆ 173.0, 169.4 (2C O), 155.8 (OCONH),
131.5 (allyl CH), 119.0 (allyl CH2), 80.1 (tBu q), 66.0 (allyl OCH2), 52.9
(Leu a-CH), 41.3 (Leu b-CH2, Gly a-CH2), 28.3 (tBu), 24.7 (Leu g-CH),
23.0 (Leu w-CH3), 21.9 (Leu w-CH3); HRMS (EI, 70 eV, 908C): m/z: calcd
‡
3
for [M] 328.1998; found: 328.1980; C16H28N2O5 (328.41).
24H; Pal (CH2)12), 0.95 ± 0.91 (m, 6H; 2 Leu w-CH3), 0.87 (t, J ˆ 6.9 Hz,
3H; Pal w-CH3); 13C NMR (125.7 MHz, CDCl3): d ˆ 202.2 (C OS), 173.9,
ˆ
Boc-Leu-Gly-OH (30): A catalytic amount of [Pd(PPh3)4] and morpholine
(800 mL, 9.2 mmol) were added to a solution of Boc-Leu-Gly-OAll (29,
2.71 g, 8.24 mmol) in CH2Cl2 (40 mL) under argon, and the mixture was
stirred at 208C for 16 h. The solvent was evaporated under reduced
pressure, and the residue was dissolved in NaHCO3 (half saturated, 30 mL)
and extracted with CH2Cl2 (5 Â 10 mL). At 08C the solution was acidified
to pH 2 with HCl (2n) and extracted with ethyl acetate (3 Â 10 mL). The
combined organic layers were dried with MgSO4 and concentrated under
reduced pressure to yield a colorless solid (2.35 g, 99%). M.p. 1068C;
ˆ
172.5, 172.0, 171.6, 170.7, 170.4, 170.1, 168.9 (8C O), 156.8, 156.2
ˆ
(2OCONH), 150.7 (arom. C O), 144.6 (arom. q), 133.7 (arom. q), 133.3
(arom. q), 129.4, 129.3, 128.8, 128.7, 128.0, 127.4, 127.1, 121.7 (24 arom. CH),
81.8, 79.1 (2tBu q), 70.5 (Trt q), 66.6 (CH2O), 56.0, 54.3, 54.1 (Cys a-CH,
Gln a-CH, Lys a-CH), 52.9 (Leu a-CH), 44.0 (Cys b-CH2), 43.6, 42.1, 41.4,
40.3 (CH2, Leu b-CH2, 2Gly a-CH2), 39.9 (Lys e-CH2), 33.7 (Gln g-CH2),
31.9, 30.4, 29.7, 29.6, 29.5, 29.4, 29.3, 29.0 (13Pal CH2, 2Lys CH2, Gln b-
CH2), 28.4 (tBu), 28.1 (tBu), 27.5 (Pal CH2), 25.7 (Lys CH2), 24.7 (Leu g-
CH), 23.1 (Leu w-CH3), 22.7 (Pal CH2), 21.5 (Leu w-CH3), 14.2 (Pal w-
[a]2D2
ˆ
18.6 (c ˆ 1.0 in CHCl3); 1H NMR (500 MHz, CD3OD): d ˆ 4.11
‡
3
3
2
3
CH3); FAB-MS (FAB, 3-NBA/TFA 10:1): m/z: 1509.9 [M‡H] , 1532.9
(dd, J ˆ 10.0, J ˆ 4.8 Hz, 1H; Leu a-CH), 3.95 (dd, J ˆ 17.8, J ˆ 4.3 Hz,
‡
1H; Gly a-CH2a), 3.86 (dd, 2J ˆ 17.8, J ˆ 4.7 Hz, 1H; Gly a-CH2b), 1.74 ±
3
[M‡Na] ; elemental analysis calcd (%) for C84H116N8O15S (1509.95): C
66.82, H 7.74, N 7.42; found: C 66.89, H 7.70, N 7.41.
1.68 (m, 1H; Leu g-CH), 1.61 ± 1.47 (m, 2H; Leu b-CH2), 1.44 (s, 9H;
C(CH3)3), 0.95 (d, 3J ˆ 6.6 Hz, 3H; Leu w-CH3), 0.93 (d, J ˆ 6.6 Hz, 3H;
3
PhAcOZ-Leu-Cys(Pal)-Gly-Lys(Boc)-Gln-Gly-OtBu (28): HOBt (18 mg,
120 mmol) and EDC (20 mg, 110 mmol) were added at 08C to a solution of
PhAcOZ-Leu-Cys(Pal)-Gly-OH (25, 80 mg, 100 mmol) and H-Lys(Boc)-
Gln-Gly-OtBu (27, 70 mg, 106 mmol) in CHCl3/2,2,2-trifluoroethanol 3:1
(v/v) (4 mL) and the mixture was stirred at 208C for 16 h. Then the solvent
was removed under reduced pressure and the product 28 was isolated from
the residue by size flash chromatography on silica gel using CHCl3/
methanol 50:1 to 1:1 (v/v) as eluent to yield a colorless solid (40 mg, 32%).
13
ˆ
Leu w-CH3); C NMR (125.7 MHz, CD3OD): d ˆ 176.1, 172.6 (2C O),
157.8 (OCONH), 80.6 (tBu q), 54.4 (Leu a-CH), 42.2, 41.7 (Leu b-CH2, Gly
a-CH2), 28.7 (tBu), 25.8 (Leu g-CH), 23.5 (Leu w-CH3), 21.9 (Leu w-CH3);
‡
HRMS (EI, 70 eV, 1358C): m/z: calcd for [M‡H] 288.1685; found:
288.1678; elemental analysis calcd (%) for C13H24N2O5 (288.35): C 54.15, H
8.39, N 9.72; found: C 54.08, H 8.21, N 9.52.
HCl ´ H-Leu-Gly-OAll (31): A saturated solution of HCl in diethyl ether
(120 mL) was added at 08C to a solution of Boc-Leu-Gly-OAll (29, 2.71 g,
8.24 mmol) in CH2Cl2 (40 mL) and the mixture was stirred at 08C for
30 min and at 208C for 2 h. Then the excess HCl and the solvent was
removed under reduced pressure to yield a colorless, hygroscopic solid
(2.15 g, 98%). [a]2D2 ˆ ‡33.1 (c ˆ 1.0 in CHCl3); 1H NMR (500 MHz,
M.p. 194 ± 1958C (decomp); [a]D22
ˆ
21.5 (c ˆ 0.4 in CHCl3/methanol 1:1
v/v); Rf ˆ 0.35 (CHCl3/methanol 10:1 v/v); 1H NMR (500 MHz, CDCl3/
CD3OD 1:1 v/v): d ˆ 7.40 ± 7.29 (m, 7H; C6H5, 2CH), 7.06 (d, 3J ˆ 8.5 Hz,
2H; 2OCCH), 6.06 (brs, 1H; OCONH), 5.04 (d, J ˆ 4.4 Hz, 2H; CCH2O),
3
3
4.39 ± 4.36 (m, 2H; Cys a-CH, Lys a-CH), 4.25 (dd, J1 ˆ 8.1, J2 ˆ 5.0 Hz,
1H; Leu a-CH), 4.11 (dd, 3J1 ˆ 8.7, 3J2 ˆ 6.3 Hz, 1H; Gln a-CH), 4.02 (d,
2J ˆ 16.9 Hz, 1H; Gly a-CH2a), 3.89 (s, 2H; CCH2C(O)), 3.85 (s, 2H; Gly a-
CH2), 3.79 (d, 2J ˆ 16.8 Hz, 1H; Gly a-CH2b), 3.42 (dd, 2J ˆ 14.3, 3J ˆ
4.4 Hz, 1H; Cys b-CH2a), 3.21 (dd, 2J ˆ 14.2, 3J ˆ 9.0 Hz, 1H; Cys b-
CH2b), 3.05 ± 3.02 (m, 2H; Lys e-CH2), 2.57 (t, 3J ˆ 7.5 Hz, 2H; Pal a-CH2),
3
3
3
CD3OD): d ˆ 5.93 (ddt, Jtrans ˆ 16.1, Jcis ˆ 10.5, Jvic ˆ 5.7 Hz, 1H; allyl
3
2
3
ˆ
ˆ
CH ), 5.34 (dd, Jtrans ˆ 17.2, J ˆ 1.5 Hz, 1H; allyl CH2a), 5.24 (dd, Jcis
ˆ
2
3
2
ˆ
10.5, J ˆ 1.3 Hz, 1H; allyl CH2b), 4.64 (dd, J ˆ 5.6, J ˆ 2.8 Hz, 2H; allyl
2
OCH2), 4.12 (d, J ˆ 17.6 Hz, 1H; Gly a-CH2a), 3.99 ± 3.91 (m, 2H; Gly a-
CH2b, Leu a-CH), 1.82 ± 1.77 (m, 2H; Leu g-CH, Leu b-CH2a), 1.75 ± 1.67
Chem. Eur. J. 2001, 7, No. 13
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
0947-6539/01/0713-2951 $ 17.50+.50/0
2951

H. Waldmann and R. Machauer
FULL PAPER
(m, 1H; Leu b-CH2b), 1.03 ± 1.00 (m, 6H; 2Leu w-CH3); 13C NMR
Boc-Leu-Gly-Leu-Gly-Leu-Gly-Leu-Gly-OAll (4): NEt3 (14 mL, 0.1 mmol)
and EDC (21 mg, 0.11 mmol) were added at 08C to a solution of Boc-Leu-
Gly-Leu-Gly-OH (33, 46 mg, 0.1 mmol), HCl ´ H-Leu-Gly-Leu-Gly-OAll
(34, 44 mg, 0.1 mmol), and HOOBt (16 mg, 0.1 mmol) in CHCl3/2,2,2-
trifluoroethanol 3:1 (v/v) (4 mL). The mixture was stirred at 208C for 16 h,
then the solvent was removed under reduced pressure. The residue was
washed with methanol (5 Â 2 mL) and dried to yield a colorless solid
ˆ
(125.7 MHz, CD3OD): d ˆ 171.2, 170.5 (2C O), 133.2 (allyl CH), 118.8
(allyl CH2), 66.9 (allyl OCH2), 52.9 (Leu a-CH), 41.9, 41.7 (Leu b-CH2, Gly
a-CH2), 25.3 (Leu g-CH), 23.0 (Leu w-CH3), 22.3 (Leu w-CH3); HRMS
‡
(EI, 70 eV, 1058C): m/z: calcd for [M HCl] 228.1474; found: 228.1461;
C11H21ClN2O3 (264.75).
Boc-Leu-Gly-Leu-Gly-OAll (32): HOBt (1.38 g, 9.0 mmol), NEt3 (1.04 mL,
7.5 mmol) were added to a solution of Boc-Leu-Gly-OH (30, 2.16 g,
7.5 mmol) and HCl ´ H-Leu-Gly-OAll (31, 1.99 g, 7.5 mmol) in CH2Cl2
(80 mL) was added and at 08C EDC (1.58 g, 8.25 mmol). The mixture
was stirred at 208C for 16 h, then solvent was extracted with HCl (0.5n, 3 Â
20 mL), NaHCO3 (1n, 3 Â 20 mL) and water (3 Â 20 mL). The organic layer
was dried with MgSO4 and concentrated under reduced pressure. The
product 32 was isolated from the residue by flash chromatography on silica
gel using ethyl acetate/n-hexane 1:1 to 4:1 (v/v) as eluent to yield a colorless
(69 mg, 82%). [a]D22
ˆ
14.2; (c ˆ 1.0 in hexafluoroisopropanol/CHCl3 1:3);
1H NMR (500 MHz, [D6]DMSO): d ˆ 8.42 (t, 3J ˆ 5.4 Hz, 1H; CONH),
8.21 (t, 3J ˆ 6.3 Hz, 2H; CONH), 8.03 (brs, 1H; CONH), 7.91 ± 7.87 (m, 3H;
3
3
3
CONH), 6.97 (d, J ˆ 7.6 Hz, 1H; OCONH), 5.90 (ddt, Jtrans ˆ 15.9, Jcis
ˆ
3
3
2
ˆ
10.5, Jvic ˆ 5.3 Hz, 1H; allyl CH ), 5.31 (dd, Jtrans ˆ 17.1, J ˆ 1.3 Hz, 1H;
allyl CH2a), 5.26 (d, Jcis ˆ 10.6 Hz, 1H; allyl CH2b), 4.57 (d, 3J ˆ 5.3 Hz,
2H; allyl OCH2), 4.35 ± 4.24 (m, 3H; 3Leu a-CH), 3.94 (m, 1H; Leu a-
CH), 3.86 (d, 3J ˆ 5.9 Hz, 1H; Gly a-CH2a), 3.83 (d, 3J ˆ 5.8 Hz, 1H; Gly a-
CH2b), 3.75 ± 3.67 (m, 6H; 3Gly a-CH2), 1.61 ± 1.51 (m, 4H; 4Leu g-CH),
1.47 ± 1.42 (m, 8H; 4Leu b-CH2), 1.38 (s, 9H; C(CH3)3), 0.88 ± 0.83 (m,
24H; 8Leu w-CH3); 13C NMR (125.7 MHz, [D6]DMSO): d ˆ 172.9, 172.5,
3
ˆ
ˆ
foam (3.36 g, 90%). M.p. 568C; [a]D22
ˆ
11.8 (c ˆ 1.0 in CHCl3); Rf ˆ 0.26
(ethyl acetate/n-hexane 4:1 v/v); 1H NMR (500 MHz, CDCl3): d ˆ 7.22 (m,
3
3
2H; 2CONH), 7.12 (brs, 1H; CONH), 5.89 (ddt, Jtrans ˆ 16.3, Jcis ˆ 10.3,
ˆ
172.2, 169.2, 168.7, 168.6, 168.4 (7C O), 155.3 (OCONH), 132.2 (allyl CH),
3
3
Jvic ˆ 5.7 Hz, 1H; allyl CH ), 5.32 (dd, Jtrans ˆ 17.4, 2J ˆ 1.2 Hz, 1H;
ˆ
117.7 (allyl CH2), 78.0 (tBu q), 64.6 (allyl OCH2), 52.7, 51.0, 50.9, 50.6 (4Leu
a-CH), 41.9, 41.8, 40.9, 40.7, 40.5 (4Leu b-CH2, 4Gly a-CH2), 28.1 (tBu),
24.1, 23.9 (4Leu g-CH), 22.9, 21.5, 21.4, 21.3 (8Leu w-CH3); MS (FAB,
3
2
ˆ
ˆ
allyl CH2a), 5.26 (dd, Jcis ˆ 10.5, J ˆ 1.2 Hz, 1H; allyl CH2b), 5.19 (br, 1H;
OCONH), 4.62 (d, 3J ˆ 5.8 Hz, 2H; allyl OCH2), 4.55 (m, 1H; Leu a-CH),
4.18 (brs, 1H; Leu a-CH), 4.03 (m, 3H; Gly a-CH2, Gly a-CH2a), 3.89 (dd,
3J ˆ 16.6, 2J ˆ 4.9 Hz, 1H; Gly a-CH2b), 1.76 ± 1.62 (m, 4H; 2Leu g-CH,
2Leu b-CH2a), 1.59 ± 1.48 (m, 2H; 2Leu b-CH2b), 1.43 (s, 9H; C(CH3)3),
0.95 ± 0.90 (m, 12H; 4Leu w-CH3); 13C NMR (125.7 MHz, CDCl3): d ˆ
‡
‡
3-NBA/TFA 10:1): m/z: 739.8 [M Boc‡2H] , 840.0 [M‡H] , 862.1
‡
[M‡Na] ; elemental analysis calcd (%) for C40H70N8O11 (839.04): C 57.26,
H 8.41, N 13.36; found: C 57.07, H 8.62, N 13.31.
TFA ´ H-Leu-Gly-Leu-Gly-Leu-Gly-Leu-Gly-OAll (35): Trifluoroacetic
acid (450 mL, 5.9 mmol) was added at 08C to a suspension of Boc-Leu-
Gly-Leu-Gly-Leu-Gly-Leu-Gly-OAll (4, 55 mg, 66 mmol) in CHCl3 (1 mL)
and the mixture was stirred at 08C for 30 min and 208C for 1 h. The
trifluoroacetic acid and the solvent were coevaporated with benzene under
ˆ
173.7, 172.5, 169.6, 169.3 (4C O), 156.0 (OCONH), 131.6 (allyl CH), 118.8
(allyl CH2), 80.4 (tBu q), 65.9 (allyl OCH2), 53.5, 51.9 (2 Leu a-CH), 43.3,
41.2, 40.6 (2Leu b-CH2, 2Gly a-CH2), 28.3 (tBu), 24.8, 24.7 (2Leu g-CH),
23.0, 22.8 (2Leu w-CH3), 21.9, 21.8 (2Leu w-CH3); HRMS (EI, 70 eV,
‡
2008C): m/z: calcd for [M‡H] 498.3053; found: 498.3067; elemental
reduced pressure to yield a colorless solid (55 mg, 98%). [a]D22
ˆ
7.4 (c ˆ
analysis calcd (%) for C24H42N4O7 (498.62): C 57.81, H 8.49, N 11.24; found:
C 58.18, H 8.46, N 10.90.
1.0 in CHCl3/2,2,2-trifluoroethanol 3:1 v/v); 1H NMR (500 MHz,
[D6]DMSO): d ˆ 8.75 (t, 3J ˆ 5.6 Hz, 1H; CONH), 8.43 (t, 3J ˆ 5.9 Hz,
1H; CONH), 8.25 ± 8.20 (m, 3H; CONH), 8.15 (brs, 1H; H3N), 7.92 (t, 3J ˆ
Boc-Leu-Gly-Leu-Gly-OH (33): A catalytic amount of [Pd(PPh3)4] and
morpholine (44 mL, 0.5 mmol) were added to a solution of Boc-Leu-Gly-
Leu-Gly-OAll (32, 250 mg, 0.5 mmol) in tetrahydrofuran (20 mL) under
argon, and the mixture was stirred at 208C for 1 h. The solvent was
evaporated under reduced pressure, and the residue was dissolved in
NaHCO3 (half saturated, 30 mL) and extracted with CH2Cl2 (5 Â 10 mL).
At 08C the solution was acidified to pH 2 with HCl (2n) and extracted with
ethyl acetate (3 Â 10 mL). The combined organic layers were dried with
MgSO4 and concentrated under reduced pressure to yield a colorless solid
3
3
3
8.0 Hz, 2H; CONH), 5.89 (ddt, Jtrans ˆ 16.0, Jcis ˆ 10.6, Jvic ˆ 5.3 Hz, 1H;
3
2
ˆ
allyl CH), 5.31 (dd, Jtrans ˆ 17.3, J ˆ 1.5 Hz, 1H; allyl CH2a), 5.26 (dd,
Jcis ˆ 10.5, J ˆ 1.2 Hz, 1H; allyl CH2b), 4.56 (d, 3J ˆ 5.3 Hz, 2H; allyl
3
2
ˆ
OCH2), 4.35 ± 4.26 (m, 3H; 3Leu a-CH), 3.89 ± 3.77 (m, 5H; Leu a-CH,
2Gly a-CH2), 3.75 ± 3.66 (m, 4H; 2Gly a-CH2), 1.70 ± 1.40 (m, 12H; 4Leu
g-CH, 4Leu b-CH2), 0.90 ± 0.82 (m, 24H; 8Leu w-CH3); 13C NMR
(125.7 MHz, [D6]DMSO): d ˆ 172.6, 172.3, 172.2, 169.4, 169.3, 168.6,
ˆ
168.5, 168.0 (8C O), 132.4 (allyl CH), 117.9 (allyl CH2), 64.8 (allyl
(228 mg, 99%). M.p. 618C; [a]D22
ˆ
18.0 (c ˆ 1.0 in CHCl3); 1H NMR
OCH2), 51.0, 50.9, 50.7 (4Leu a-CH), 41.9, 41.1, 41.0, 40.6, 40.3 (4Leu b-
CH2, 4Gly a-CH2), 24.2, 24.1, 24.0 (4Leu g-CH), 23.5, 23.1, 23.0, 22.6, 22.0,
21.6, 21.5 (8Leu w-CH3); HRMS (FAB, 3-NBA/TFA 10:1): m/z: calcd for
(500 MHz, CD3OD): d ˆ 4.46 (m, 1H; Leu a-CH), 4.06 (m, 1H; Leu a-
CH), 3.94 ± 3.82 (m, 4H; 2Gly a-CH2), 1.72 ± 1.60 (m, 4H; 2Leu g-CH,
2Leu b-CH2a), 1.57 ± 1.54 (m, 2H; 2Leu b-CH2b), 1.45 (s, 9H; C(CH3)3),
0.96 ± 0.91 (m, 12H; 4 Leu w-CH3); 13C NMR (125.7 MHz, CD3OD): d ˆ
‡
[M CF3COO] : 739.4718; found: 739.4772; elemental analysis calcd (%)
for C37H63F3N8O11 ´ 1.5 H2O: C 50.50, H 7.56, N 12.73; found: C 50.62, H
7.56, N 12.57; C37H63F3N8O11 (852.94).
ˆ
176.3, 174.9, 172.5, 171.4 (4C O), 157.9 (OCONH), 80.6 (tBu q), 54.7, 52.9
(2 Leu a-CH), 43.6, 41.7, 41.6 (2Leu b-CH2, 2Gly a-CH2), 28.7 (tBu), 25.8,
25.6 (2Leu g-CH), 23.4 (2Leu w-CH3), 21.8 (2Leu w-CH3); HRMS (EI,
Myr-Gly-Asn(Trt)-Leu-Lys(Boc)-Ser(tBu)-Val-Gly-Gln(Trt)-Glu(OtBu)-
Pro-OH (2): The peptide chain was assembled on a polystyrene resin,
‡
70 eV, 1908C): m/z: calcd for [M‡H] 458.2740; found: 458.2750; elemental
1
starting with Fmoc-Pro-2-ClTrt-polystyrene 36 (479 mg, 0.53 mmolg
)
analysis calcd (%) for C21H38N4O7 (458.55): C 55.01, H 8.35, N 12.22; found:
C 54.78, H 8.27, N 12.03.
using an Applied Biosystems430A peptide synthesizer and the standard
Fmoc/tBu strategy in N-methyl-pyrrolidinone (deprotection with piper-
idine [20%, 5 and 20 min], couplings [4 equiv Fmoc-AA-OBt, 60 min].
Yield: 995 mg.
H-Leu-Gly-Leu-Gly-OAll (34): A saturated solution of HCl in diethyl
ether (35 mL) was added at 08C to a solution of Boc-Leu-Gly-Leu-Gly-
OAll (32, 435 mg, 0.87 mmol) in CH2Cl2 (30 mL) and the mixture was
stirred at 08C for 30 min and at 208C for 2 h. Then the excess HCl and the
solvent was removed under reduced pressure to yield a colorless foam
(381 g, 99%). M.p. 988C; [a]2D2 ˆ ‡9.5 (c ˆ 0.75 in CHCl3); 1H NMR
From a portion of the resin 37 (100 mg, 53 mmol), the Fmoc group was
removed (piperidine 20%, 20 and 30 min) and the resin was washed with
NMP and CH2Cl2 (3 Â 5 mL). Then to the resin was added diisopropyl-
ethylamine (86 mL, 65 mg, 0.5 mmol) and myristoyl chloride (68 mL,
0.25 mmol) in NMP (3 mL) and the mixture was shaken for 4 h. The resin
was filtered off, washed with NMP and CH2Cl2 (3 Â 5 mL) and dried. To
cleave off the product 2 a mixture of acetic acid/2,2,2-trifluoroethanol/
CH2Cl2 1:1:8 (v/v/v) (5 mL) was added and the mixture was shaken for 1 h.
The resin was filtered off, trifluroacetic acid (0.5%, 5 mL) was added and
the resin was shaken for 100 min. After filtration the solvents were
removed under reduced pressure and traces of acid were removed by
addition of water and lyophilisation to yield a colorless solid (47.7 mg,
3
3
3
(500 MHz, CD3OD): d ˆ 5.93 (ddt, Jtrans ˆ 16.2, Jcis ˆ 10.6, Jvic ˆ 5.7 Hz,
3
2
ˆ
ˆ
1H; allyl CH ), 5.33 (dd, Jtrans ˆ 17.2, J ˆ 1.5 Hz, 1H; allyl CH2a), 5.23
(dd, 3Jcis ˆ 10.5, J ˆ 1.3 Hz, 1H; allyl CH2b), 4.62 (d, 3J ˆ 5.6 Hz, 2H; allyl
2
ˆ
OCH2), 4.45 (q, 3J ˆ 5.0 Hz, 1H; Leu a-CH), 4.04 ± 3.89 (m, 5H; Leu a-CH,
2Gly a-CH2), 1.77 ± 1.58 (m, 6H; 2Leu g-CH, 2Leu b-CH2), 1.03 ± 0.92 (m,
12 H; 4Leu w-CH3); 13C NMR (125.7 MHz, CD3OD): d ˆ 175.1, 171.2,
ˆ
170.9, 170.6 (4C O), 133.2 (allyl CH), 118.6 (allyl CH2), 66.6 (allyl OCH2),
53.0, 52.9 (2Leu a-CH), 43.2, 41.9, 41.4 (2Leu b-CH2, 2Gly a-CH2), 25.7,
25.3 (2Leu g-CH), 23.4, 22.9 (2Leu w-CH3), 22.1, 21.8 (2Leu w-CH3);
HRMS (FAB, 3-NBA/TFA 10:1): m/z: calcd for [M Cl] 399.2607; found:
98%). M.p. 238 ± 2398C (decomp); [a]D22
ˆ
26.3 (c ˆ 1.0 in CHCl3/2,2,2-
‡
trifluoroethanol 3:1 v/v); 1H NMR (500 MHz, [D6]DMSO): d ˆ 8.57 (s, 1H;
CONH), 8.50 (s, 1H; CONH), 8.29 (d, 3J ˆ 7.6 Hz, 1H; CONH), 8.09 ± 8.04
(m, 4H; CONH), 7.88 (d, 3J ˆ 7.8 Hz, 1H; CONH), 7.80 (d, 3J ˆ 7.5 Hz, 1H;
399.2630; elemental analysis calcd (%) for C19H35ClN4O5 ´ 0.5H2O: C 51.40,
H 8.17, N 12.62; found: C 51.50, H 8.25, N 12.21; C19H35ClN4O5 (434.96).
2952
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
0947-6539/01/0713-2952 $ 17.50+.50/0
Chem. Eur. J. 2001, 7, No. 13

Lipidated eNOS Peptides
2940±2956
CONH), 7.77 (d, 3J ˆ 7.7 Hz, 1H; CONH), 7.70 (d, 3J ˆ 7.7 Hz, 1H;
CONH), 7.58 (d, 3J ˆ 8.7 Hz, 1H; CONH), 7.27 ± 7.24 (m, 12H; C6H5),
7.19 ± 7.15 (m, 18H; C6H5), 6.68 (brs, 1H; OCONH), 4.53 ± 4.49 (m, 2H;
2a-CH), 4.34 (d, 3J ˆ 7.6 Hz, a-CH), 4.28 ± 4.16 (m, 5H; 5a-CH), 3.82 (dd,
2J ˆ 16.5, 3J ˆ 5.7 Hz, 1H; Gly a-CH2a), 3.75 (dd, 2J ˆ 16.5, 3J ˆ 5.1 Hz, 1H;
Gly a-CH2b), 3.68 ± 3.39 (m, 6H; Ser b-CH2, Gly a-CH2, Pro d-CH2), 2.85
(dd, 3J1 ˆ 3J2 ˆ 6.6 Hz, 2H; Lys e-CH2), 2.66 ± 2.63 (m, 2H; Asn b-CH2),
2.29 ± 2.26 (m, 4H; Gln g-CH2, Glu g-CH2), 2.19 ± 2.09 (m, 5H; Myr a-CH2,
Val b-CH, Glx b-CH2a, Glx b-CH2a), 1.98 ± 1.42 (m, 15H; Glx b-CH2b, Glx
b-CH2b, Pro b-CH2, Pro g-CH2, Lys b-CH2, Myr b-CH2, Myr g-CH2, Leu g-
CH, Leu b-CH2), 1.39 (s, 9H; C(CH3)3), 1.36 (s, 9H; C(CH3)3), 1.33 ± 1.27
(m, 4H; Lys d-CH2, Lys g-CH2), 1.23 (brs, 18H; Myr (CH2)9), 1.08 (s, 9H;
C(CH3)3), 0.86 ± 0.82 (m, 15H; Myr w-CH3, 2Leu w-CH3, 2Val w-CH3);
13C NMR (125.7 MHz, [D6]DMSO): d ˆ 173.6, 173.1, 172.8, 171.8, 171.7,
(CH2)12), 1.08 (s, 9H; C(CH3)3), 0.86 ± 0.82 (m, 18H; Myr w-CH3, Pal w-
CH3', 2Leu w-CH3, 2 Val w-CH3); 13C NMR (125.7 MHz, [D6]DMSO): d ˆ
ˆ
198.2 (C OS), 172.8, 171.8, 171.5, 171.3, 171.2, 171.0, 170.9, 170.7, 170.6,
ˆ
169.9, 169.4, 169.2, 168.9, 168.8, 168.2, 166.4 (19C O), 155.4 (OCONH),
144.8 (arom q), 144.6 (arom. q), 132.2 (allyl CH), 128.5, 128.4, 127.3, 126.2,
125.3 (30 arom. CH), 117.7 (allyl CH2), 79.6, 77.2, 72.7 (3tBu q), 69.3, 69.1
(Trt q), 64.8, 64.7 (allyl OCH2, Ser b-CH2), 61.4, 59.5, 59.1, 57.5, 53.0, 52.6,
51.8, 51.5, 51.2 (3 Pro a-CH, Cys a-CH, Ser a-CH, Asn a-CH, Gln a-CH,
Glu a-CH, Lys a-CH, Leu a-CH, Val a-CH), 50.2, 49.5 (3 Pro d-CH2), 46.7,
43.3, 42.2, 41.6 (Leu b-CH2, Cys b-CH2, 4Gly a-CH2), 40.7 (Lys e-CH2),
38.1 (Asn b-CH2), 34.9, 32.5 (Glu g-CH2, Gln g-CH2), 31.2 (Myr CH2), 30.6
(Val b-CH), 30.0, 29.2, 28.9, 28.8, 28.7, 28.6 (8Myr CH2, 10Pal CH2, Pro b-
CH2, 2Lys CH2, Gln b-CH2, Glu b-CH2), 28.2 (tBu), 27.9 (Pal CH2), 27.7
(tBu), 27.0 (tBu), 26.4 (Myr CH2), 25.0, 24.9, 24.4, 24.2 (Lys CH2, 3 Pro g-
CH2), 24.0 (Leu g-CH), 23.0 (Leu w-CH3), 22.6, 22.0 (2Myr CH2), 21.4 (Leu
w-CH3), 19.1 (Val w-CH3'), 17.9 (Val w-CH3'), 15.1 (Pal w-CH3'), 13.8 (Myr
ˆ
171.3, 171.0, 170.9, 170.7, 169.4, 169.3, 169.2, 168.8, 168.2 (14 O), 155.4
(OCONH), 144.8, 144.6 (arom. q), 128.5, 128.4, 127.3, 126.2 (30 arom. CH),
79.6, 77.2, 72.8 (3tBu q), 69.3, 69.1 (Trt q), 61.4 (Ser b-CH2), 58.3, 57.5, 53.0,
52.6, 51.8, 51.2, 50.2, 49.3 (Pro a-CH, Ser a-CH, Asn a-CH, Gln a-CH, Glu
a-CH, Lys a-CH, Leu a-CH, Val a-CH), 52.9 (Leu a-CH), 48.4 (Pro d-
CH2), 46.4, 42.2, 41.6 (Leu b-CH2, 2 Gly a-CH2), 40.3 (Lys e-CH2), 38.2
(Asn b-CH2), 35.0, 32.5 (Glu g-CH2, Gln g-CH2), 31.2 (Myr CH2), 30.6 (Val
b-CH), 30.3, 30.0, 29.2, 29.0, 28.9, 28.8, 28.7, 28.6, 28.5, 28.4 (8Myr CH2, Pro
b-CH2, 2 Lys CH2, Gln b-CH2, Glu b-CH2), 28.2 (tBu), 27.7 (tBu), 27.0
(tBu), 26.4 (Myr CH2), 25.0, 24.4 (Lys CH2, Pro g-CH2), 24.0 (Leu g-CH),
23.0 (Leu w-CH3), 22.6, 22.0 (2Myr CH2), 21.4 (Leu w-CH3), 19.1 (Val w-
CH3'), 17.9 (Val w-CH3'), 17.1 (Myr CH2), 13.9 (Myr w-CH3); MS (FAB,
‡
w-CH3); MS (MALDI-TOF, DHB/TFA): m/z: calcd for [M‡Na] 2646.54;
‡
found: 2647.54, calcd for [M‡K] 2662.51; found: 2662.94; C144H210N18O25S
(2625.44).
Myr-Gly-Asn(Trt)-Leu-Lys(Boc)-Ser(tBu)-Val-Gly-Gln(Trt)-Glu(OtBu)-
Pro-Gly-Pro-Pro-Cys(Pal)-Gly-OH (39): N,N'-Dimethylbarbituric acid
(2.0 mg, 12.8 mmol) and a catalytic amount of [Pd(PPh3)4] were added
under argon to a solution allyl protected peptide 38 (16.7 mg, 6.4 mmol) in
DMSO (400 mL), and the mixture was stirred at 208C for 2 h. Then citric
acid (5%, 5 mL) was added at 08C and the precipitated 15-mer 39 was
filtered off and washed several times with citric acid (5%, 1 mL), water,
methanol. After drying in vacuo the peptide was isolated as colorless solid
‡
‡
3-NBA/TFA 10:1): m/z: 1834.8 [M Boc‡2H] , 1935.6 [M‡H] , 1957.4
‡
[M‡Na] ; C108H151N13O19 (1935.48).
(15.1 mg, 92%). [a]D22
ˆ
76.0 (c ˆ 0.25 in CHCl3/2,2,2-trifluoroethanol 3:1
v/v); 1H NMR (500 MHz, [D6]DMSO): d ˆ 8.57 (s, 1H; CONH), 8.54 (s,
2H; CONH), 8.29 (d, 3J ˆ 7.4 Hz, 1H; CONH), 8.09 ± 8.03 (m, 4H;
CONH), 7.92 (d, 3J ˆ 7.8 Hz, 1H; CONH), 7.88 (d, 3J ˆ 7.7 Hz, 2H;
CONH), 7.81 (d, 3J ˆ 7.5 Hz, 1H; CONH), 7.77 (d, 3J ˆ 7.7 Hz, 1H;
CONH), 7.72 ± 7.68 (m, 2H; CONH), 7.59 (d, 3J ˆ 8.8 Hz, 2H; CONH),
7.27 ± 7.24 (m, 12H; C6H5), 7.20 ± 7.14 (m, 18H; C6H5), 6.68 (brs, 1H;
OCONH), 4.57 ± 4.43 (m, 3H; 3a-CH), 4.38 ± 4.32 (m, 3H; 3a-CH), 4.26 ±
4.01 (m, 5H; 5a-CH, 1Gly a-CH2a), 3.85 ± 3.39 (m, 15H; 1Gly a-CH2b, 3
Gly a-CH2, Ser b-CH2, 3Pro d-CH2), 3.04 (d, 2J ˆ 13.3 Hz, 2H; Cys b-CH2),
2.85 (dd, 2J ˆ 13.2, 3J ˆ 6.5 Hz, 2H; Lys e-CH2), 2.64 (brs, 2H; Asn b-CH2),
2.54 (t, 3J ˆ 7.3 Hz, 2H; Pal a-CH2), 2.29 ± 2.24 (m, 4H; Gln g-CH2, Glu g-
CH2), 2.13 ± 2.08 (m, 3H; Myr a-CH2, Val b-CH), 1.99 ± 1.43 (m, 27H; Gln
b-CH2, Glu b-CH2, 3Pro b-CH2, 3Pro g-CH2, Lys b-CH2, Pal b-CH2, Myr
b-CH2, Myr g-CH2, Leu g-CH, Leu b-CH2), 1.39 (s, 9H; C(CH3)3), 1.36 (s,
9H; C(CH3)3), 1.32 ± 1.27 (m, 4H; Lys d-CH2, Lys g-CH2), 1.22 (brs, 42H;
Myr (CH2)9, Pal (CH2)12), 1.08 (s, 9H; C(CH3)3), 0.89 ± 0.82 (m, 18H; Myr
w-CH3, Pal w-CH3', 2Leu w-CH3, 2Val w-CH3); MS (MALDI-TOF, DHB/
Myr-Gly-Asn-Leu-Lys-Ser-Val-Gly-Gln-Glu-Pro-OH (45): Myr-Gly-
Asn(Trt)-Leu-Lys(Boc)-Ser(tBu)-Val-Gly-Gln(Trt)-Glu(OtBu)-Pro-OH
(2, 3.9 mg, 0.9 mmol) was dissolved in a mixture of CF3CO2H/ethanedithiol/
water 95:2.5:2.5 (v/v/v) (500 mL) and shaken at 208C for 20 min. Then the
solvent was removed under reduced pressure and 45 was precipitated and
washed with diethyl ether. After purification by HPLC the deprotected
peptide was obtained as pale colorless solid (9.5 mg, 74%). M.p. 213 ±
2158C; [a]2D2 ˆ ‡122 (c ˆ 0.025 in CHCl3/2,2,2-trifluoroethanol 3:1 v/v);
HPLC: tr ˆ 11.9 min (508C, 25% to 100% B in 20 min, then 10 min B; A:
H2O‡1% CH3CN‡0.1% TFA; B: CH3CN‡1% H2O‡0.1% TFA);
1H NMR (500 MHz, CDCl3/CD3OD): d ˆ 4.56 ± 4.21 (m, 8H; 8a-CH),
3.97 ± 3.63 (m, 8H; Ser b-CH2, 2Gly a-CH2, Pro d-CH2), 2.94 ± 2.90 (m,
2H; Lys e-CH2), 2.79 ± 2.78 (m, 2H; Asn b-CH2), 2.46 ± 2.25 (m, 4H; Gln g-
CH2, Glu g-CH2), 2.06 ± 1.91 (m, 6H; Myr a-CH2, Val b-CH, Glx b-CH2,
Glx b-CH2a), 1.81 ± 1.38 (m, 14H; Glx b-CH2b, Pro b-CH2, Pro g-CH2, Lys
b-CH2, Myr b-CH2, Myr g-CH, Leu g-CH, Leu b-CH2), 1.32 ± 1.11 (brs,
22H; Lys d-CH2, Lys g-CH2, Myr (CH2)9), 1.02 ± 0.85 (m, 15H; Myr w-CH3,
2Leu w-CH3, 2Val w-CH3); MS (MALDI-TOF, DHB/TFA): m/z: calcd for
‡
‡
TFA): m/z: calcd for [M‡Na] 2606.50; found: 2605.94, calcd for [M‡K]
‡
‡
2622.48; found: 2621.25; C141H206N18O25S (2585.37).
[M‡H] 1238.74; found: 1238.05, calcd for [M‡Na] 1261.49; found:
1262.07; C57H99N13O17 (1238.47).
Myr-Gly-Asn-Leu-Lys-Ser-Val-Gly-Gln-Glu-Pro-Gly-Pro-Pro-Cys(Pal)-
Gly-OH (46): The protected peptide 39 (5.9 mg, 2.3 mmol) was dissolved in
a mixture of CF3CO2H/ethanedithiol/water 95:2.5:2.5 (v/v/v) (200 mL) and
shakenfor 20 min at 208C. Then the solvent was removed under reduced
pressure and 46 was precipitated and washed with diethyl ether. After
separation by HPLC the deprotected peptide was obtained as colorless
solid (2.3 mg, 53%). [a]2D2 ˆ ‡37.4 (c ˆ 0.02 in CHCl3/2,2,2-trifluoroethanol
3:1 v/v); HPLC: tr ˆ 16.5 min (508C, 25% to 100% B in 20 min, then 10 min
B; A: H2O‡1% CH3CN‡0.1% TFA; B: CH3CN‡1% H2O‡0.1% TFA);
1H NMR (500 MHz, CDCl3/CD3OD 1:1): d ˆ 4.64 ± 3.36 (m, 27H; 11 a-CH,
4Gly a-CH2, Ser b-CH2, 3Pro d-CH2), 3.20 ± 3.19 (m, 2H; Cys b-CH2), 2.93
(d, 3J ˆ 7.1 Hz, 2H; Lys e-CH2), 2.77 (d, 3J ˆ 6.2 Hz, 2H; Asn b-CH2), 2.54
(t, 3J ˆ 7.5 Hz, 2H; Pal a-CH2), 2.44 ± 1.39 (m, 34H; Gln g-CH2, Glu g-CH2,
Myr a-CH2, Gln b-CH2, Glu b-CH2, 3Pro b-CH2, 3Pro g-CH2, Lys b-CH2,
Myr b-CH2, Myr g-CH2, Pal b-CH2, Val b-CH, Leu g-CH, Leu b-CH2),
1.36 ± 1.20 (brs, 46H; Lys d-CH2, Lys g-CH2, Myr (CH2)9, Pal (CH2)12),
1.01 ± 0.88 (m, 18H; Myr w-CH3, Pal w-CH3', 2Leu w-CH3, 2Val w-CH3);
Myr-Gly-Asn(Trt)-Leu-Lys(Boc)-Ser(tBu)-Val-Gly-Gln(Trt)-Glu(OtBu)-
Pro-Gly-Pro-Pro-Cys(Pal)-Gly-OAll (38): EDC (2.9 mg, 15.1 mmol) was
added at 108C to a solution of peptide 2, 18.5 mg, 9.6 mmol), H-Gly-Pro-
Pro-Cys(Pal)-Gly-OAll (24, 7.1 mg, 10.1 mmol) and HOOBt (2.5 mg,
15.1 mmol) in CHCl3/2,2,2-trifluoroethanol 3:1 (v/v) (250 mL) and the
mixture was stirred at 208C for 16 h. The solvent was removed under a
stream of nitrogen and the residue was washed with methanol (5 Â 1 mL)
and dried to yield a colorless solid (23.0 mg, 91%). [a]D22
ˆ
73.2 (c ˆ 0.25,
CHCl3/2,2,2-trifluoroethanol 3:1 v/v); 1H NMR (500 MHz, [D6]DMSO):
d ˆ 8.57 (s, 1H; CONH), 8.54 (s, 2H; CONH), 8.30 ± 8.28(m, 2H; CONH),
8.08 ± 8.01 (m, 4H; CONH), 7.95 ± 7.87 (m, 4H; CONH), 7.80 (d, 3J ˆ 7.0 Hz,
1H; CONH), 7.77 (d, 3J ˆ 7.3 Hz, 1H; CONH), 7.69 (d, 3J ˆ 8.0 Hz, 1H;
CONH), 7.58 (d, 3J ˆ 8.3 Hz, 1H; CONH), 7.27 ± 7.24 (m, 12H; C6H5),
7.19 ± 7.15 (m, 18H; C6H5), 6.68 (brs, 1H; OCONH), 5.93 ± 5.87 (m, 1H;
3
3
ˆ
ˆ
allyl CH ), 5.31 (d, Jtrans ˆ 17.2 Hz, 1H; allyl CH2a), 5.21 (d, Jcis ˆ 10.6,
ˆ
1H; allyl CH2b), 4.58 ± 4.02 (m, 13H; 11a-CH, allyl OCH2), 3.85 ± 3.36 (m,
‡
MS (MALDI-TOF, DHB/TFA): m/z: calcd for [M‡H] 1888.12; found:
16H; 4Gly a-CH2, Ser b-CH2, 3Pro d-CH2), 3.17 (dd, 2J ˆ 13.7, 3J ˆ 8.5 Hz,
2H; Cys b-CH2), 2.85 (d, 3J ˆ 5.8 Hz, 2H; Lys e-CH2), 2.64 (brs, 2H; Asn b-
‡
‡
1888.14, calcd for [M‡Na] 1910.11; found: 1909.82; calcd for [M‡K]
1926.08; found: 1926.45; C90H154N18O23S (1888.39).
3
CH2), 2.54 (t, J ˆ 7.3 Hz, 2H; Pal a-CH2), 2.36 ± 2.25 (m, 4H; Gln g-CH2,
Glu g-CH2), 2.11 ± 1.47 (m, 30H; Myr a-CH2, Gln b-CH2, Glu b-CH2, 3Pro
b-CH2, 3 Pro g-CH2, Lys b-CH2, Myr b-CH2, Myr g-CH2, Pal b-CH2, Val b-
CH, Leu g-CH, Leu b-CH2), 1.39 (s, 9H; C(CH3)3), 1.36 (s, 9H; C(CH3)3),
1.33 ± 1.26 (m, 4H; Lys d-CH2, Lys g-CH2), 1.23 (brs, 42H; Myr (CH2)9, Pal
Myr-Gly-Asn(Trt)-Leu-Lys(Boc)-Ser(tBu)-Val-Gly-Gln(Trt)-Glu(OtBu)-
Pro-Gly-Pro-Pro-Cys(Pal)-Gly-Leu-Gly-Leu-Gly-Leu-Gly-Leu-Gly-OAll
(40): NEt3 (0.7 mL, 6.9 mmol) and EDC (1.5 mg, 7.8 mmol) were added to a
solution of peptide 39 (10.0 mg, 3.9 mmol), TFA ´ H-Leu-Gly-Leu-Gly-Leu-
Chem. Eur. J. 2001, 7, No. 13
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
0947-6539/01/0713-2953 $ 17.50+.50/0
2953

H. Waldmann and R. Machauer
FULL PAPER
Gly-Leu-Gly-OAll (35, 5.0 mg, 5.9 mmol) and HOAt (0.6 mg, 4.4 mmol) in
dimethylsulfoxide (300 mL), and the mixture was stirred at 208C for 16 h.
At 08C citric acid (5%, 5 mL) was added and the precipitated product was
separated, washed with citric acid, water and methanol and dried to yield a
Leu b-CH2, Lys d-CH2, Lys g-CH2), 1.24 (brs, 42H; Myr (CH2)9, Pal
(CH2)12), 0.88 ± 0.81 (m, 42H; Myr w-CH3, Pal w-CH3', 10Leu w-CH3, 2Val
‡
w-CH3); MS (MALDI-TOF, DHB/TFA): m/z: calcd for [M‡H] 2568.55;
‡
found: 2569.06, calcd for [M‡Na] 2590.53; found: 2591.02, calcd for
‡
colorless solid (9.2 mg, 72%). [a]D22
ˆ
27.48 (c ˆ 0.45 in CHCl3/2,2,2-
[M‡K] 2606.50; found: 2608.24; C122H210N26O31S (2569.24).
trifluoroethanol 3:1 v/v); 1H NMR (500 MHz, [D6]DMSO): d ˆ 8.57 (s, 1H;
CONH), 8.53 (s, 1H; CONH), 8.40 (s, 2H; CONH), 8.29 (d, 3J ˆ 7.4 Hz,
1H; CONH), 8.17 (brs, 2H; CONH), 8.08 ± 8.04 (m, 5H; CONH), 7.87 ±
7.81 (m, 8H; CONH), 7.80 (d, 3J ˆ 5.8 Hz, 1H; CONH), 7.76 (d, 3J ˆ 8.0 Hz,
1H; CONH), 7.69 (d, 3J ˆ 7.4 Hz, 2H; CONH), 7.58 (d, 3J ˆ 6.5 Hz, 1H;
Myr-Gly-Asn(Trt)-Leu-Lys(Boc)-Ser(tBu)-Val-Gly-Gln(Trt)-Glu(OtBu)-
Pro-Gly-Pro-Pro-Cys(Pal)-Gly-Leu-Gly-Leu-Gly-Leu-Gly-Leu-Gly-Leu-
Cys(Pal)-Gly-OAll (42): EDC (0.8 mg, 4.2 mmol) was added at 08C to a
solution of peptide 41 (6.5 mg, 2.0 mmol), H-Leu-Cys(Pal)-Gly-OAll (22,
2.4 mg, 4.0 mmol) and HOAt (0.6 mg, 4.4 mmol) in N-methyl-pyrrolidinone
(300 mL), and the mixture was stirred at 208C for 16 h. At 08C citric acid
(5%, 5 mL) was added and the precipitated product was separated, washed
with citric acid, water and methanol and dried to yield a colorless solid
CONH), 7.27 ± 7.24 (m, 12H; C6H5), 7.19 ± 7.11 (m, 18H; C6H5), 6.68 (brs,
3
ˆ
1H; OCONH), 5.93 ± 5.86 (m, 1H; allyl CH ), 5.32 (d, Jtrans ˆ 16.9 Hz, 1H;
3
ˆ
ˆ
allyl CH2a), 5.21 (d, Jcis ˆ 10.5, 1H; allyl CH2b), 4.57 ± 4.18 (m, 17H; 15 a-
CH, allyl OCH2), 3.89 ± 3.38 (m, 24H; 8 Gly a-CH2, Ser b-CH2, 3Pro d-
(6.6 mg, 86%). [a]D22
ˆ
26.1 (c ˆ 0.45 in CHCl3/2,2,2-trifluoroethanol 3:1
3
CH2), 3.07 (m, 2H; Cys b-CH2), 2.85 (d, J ˆ 5.4 Hz, 2H; Lys e-CH2), 2.63
v/v); 1H NMR (500 MHz, [D6]DMSO): d ˆ 8.57 (s, 1H; CONH), 8.53 (s,
1H; CONH), 8.30 ± 8.28 (m, 2H; CONH), 8.18 (brs, 4H; CONH), 8.12 ±
8.04 (m, 6H; CONH), 7.91 ± 7.87 (m, 9H; CONH), 7.80 (d, 3J ˆ 7.1 Hz, 1H;
CONH), 7.76 (d, 3J ˆ 6.8 Hz, 1H; CONH), 7.69 (d, 3J ˆ 7.7 Hz, 2H;
(brs, 2H; Asn b-CH2), 2.54 (m, 2H; Pal a-CH2), 2.29 ± 2.25 (m, 4H; Gln g-
CH2, Glu g-CH2), 2.11 ± 2.09 (m, 3H; Myr a-CH2, Val b-CH), 1.98 ± 1.45
(m, 39H; Gln b-CH2, Glu b-CH2, 3Pro b-CH2, 3Pro g-CH2, Lys b-CH2,
Myr b-CH2, Myr g-CH2, Pal b-CH2, 5Leu g-CH, 5Leu b-CH2), 1.39 (s, 9H;
C(CH3)3), 1.35 (s, 9H; C(CH3)3), 1.33 ± 1.29 (m, 4H; Lys d-CH2, Lys g-
CH2), 1.23 (brs, 42H; Myr (CH2)9, Pal (CH2)12), 1.08 (s, 9H; C(CH3)3),
0.86 ± 0.83 (m, 42H; Myr w-CH3, Pal w-CH3', 10Leu w-CH3, 2Val w-CH3);
CONH), 7.58 (d, 3J ˆ 7.8 Hz, 1H; CONH), 7.27 ± 7.15 (m, 30H; Trt), 6.68
3
ˆ
(brs, 1H; OCONH), 5.90 ± 5.87 (m, 1H; allyl CH ), 5.31 (d, Jtrans
ˆ
3
ˆ
ˆ
16.8 Hz, 1H; allyl CH2a), 5.20 (d, Jcis ˆ 10.2 Hz, 1H; allyl CH2b), 4.58 ±
4.45 (m, 4H; 2 a-CH, allyl OCH2), 4.38 ± 4.18 (m, 15H; 15 a-CH), 3.89 ±
3.34 (m, 26H; 9 Gly a-CH2, Ser b-CH2, 3 Pro d-CH2), 3.04 (dd, 2J ˆ
13.2 Hz, 3J ˆ 8.9 Hz, 4H; 2Cys b-CH2), 2.85 (d, 3J ˆ 5.5 Hz, 2H; Lys e-
CH2), 2.63 (brs, 2H; Asn b-CH2), 2.55 (m, 4H; 2Pal a-CH2), 2.28 ± 2.25 (m,
‡
MS (MALDI-TOF, DHB/TFA): m/z: calcd for [M tBu‡H‡Na]
‡
3270.90; found: 3270.02, calcd for [M‡Na] 3326.96; found: 3326.15, calcd
‡
for [M‡K] 3342.93; found: 3341.91; C176H266N26O33S (3306.29).
3
4H; Gln g-CH2, Glu g-CH2), 2.17 (t, J ˆ 8.0 Hz, 2H; Myr a-CH2), 2.12 ±
Myr-Gly-Asn(Trt)-Leu-Lys(Boc)-Ser(tBu)-Val-Gly-Gln(Trt)-Glu(OtBu)-
Pro-Gly-Pro-Pro-Cys(Pal)-Gly-Leu-Gly-Leu-Gly-Leu-Gly-Leu-Gly-OH
(41): N,N'-Dimethylbarbituric acid (1.0 mg, 6.4 mmol) and a catalytic
amount of [Pd(PPh3)4] were added under argon to a solution of peptide
40 (9.0 mg, 2.7 mmol) in DMSO (400 mL) and the mixture was stirred at
208C for 2 h. Then at 08C citric acid (5%, 5 mL) was added and the
precipitated 23-mer 41 was separated and washed several times with citric
acid (5%, 1 mL), water, methanol. After drying in vacuo the peptide was
2.07 (m, 2H; Val b-CH, Glx b-CH2a), 1.99 ± 1.46 (m, 43H; Glx b-CH2b, Glx
b-CH2, 3Pro b-CH2, 3Pro g-CH2, Lys b-CH2, Myr b-CH2, Myr g-CH2,
2Pal b-CH2, 6Leu g-CH, 6Leu b-CH2), 1.39 (s, 9H; C(CH3)3), 1.36 (s, 9H;
C(CH3)3), 1.32 ± 1.31 (m, 4H; Lys d-CH2, Lys g-CH2), 1.23 (brs, 66H; Myr
(CH2)9, Pal (CH2)12), 1.08 (s, 9H; C(CH3)3), 0.87 ± 0.82 (m, 51H; Myr w-
CH3, 2Pal w-CH3', 11 Leu w-CH3, 2Val w-CH3); MS (MALDI-TOF, DHB/
‡
‡
TFA): m/z: calcd for [M‡Na] 3838.30; found: 3837.85, calcd for [M‡K]
isolated as pale yellow solid (7.7 mg, 87%). [a]D22
ˆ
28.6 (c ˆ 0.25 in
3854.28; found: 3853.54; C203H315N29O37S2 (3818.06).
CHCl3/2,2,2-trifluoroethanol 3:1 v/v); 1H NMR (500 MHz, [D6]DMSO):
d ˆ 8.57 (s, 1H; CONH), 8.54 (s, 1H; CONH), 8.51 (s, 1H; CONH), 8.28 (d,
3J ˆ 7.7 Hz, 1H; CONH), 8.18 (brs, 4H; CONH), 8.08 ± 8.01 (m, 5H;
CONH), 7.88 ± 7.85 (brs, 6H; CONH), 7.80 (d, 3J ˆ 7.3 Hz, 1H; CONH),
7.76 (d, 3J ˆ 7.9 Hz, 1H; CONH), 7.70 (d, 3J ˆ 7.6 Hz, 1H; CONH), 7.64 ±
7.55 (m, 3H; CONH), 7.27 ± 7.15 (m, 30H; Trt), 6.69 (brs, 1H; OCONH),
4.58 ± 4.47 (m, 3H; 3 a-CH), 4.35 ± 4.17 (m, 12H; 12 a-CH), 3.84 ± 3.38 (m,
24H; 8Gly a-CH2, Ser b-CH2, 3 Pro d-CH2), 3.06 (dd, 2J ˆ 13.4 Hz, 3J ˆ
8.4 Hz, 2H; Cys b-CH2), 2.85 (d, 3J ˆ 6.3 Hz, 2H; Lys e-CH2), 2.63 (brs,
Myr-Gly-Asn(Trt)-Leu-Lys(Boc)-Ser(tBu)-Val-Gly-Gln(Trt)-Glu(OtBu)-
Pro-Gly-Pro-Pro-Cys(Pal)-Gly-Leu-Gly-Leu-Gly-Leu-Gly-Leu-Gly-Leu-
Cys(Pal)-Gly-OH (43): N,N'-Dimethylbarbituric acid (0.5 mg, 3.2 mmol)
and a catalytic amount of [Pd(PPh3)4] were added under argon to a solution
of peptide 42 (4.7 mg, 1.2 mmol) in DMSO (300 mL), and the mixture was
stirred at 208C for 2 h. Then at 08C citric acid (5%, 5 mL) was added and
the precipitated 26-mer 43 was separated and washed several times with
citric acid (5%, 1 mL), water, methanol. After drying in vacuo the peptide
was isolated as pale yellow solid. (3.2 mg, 69%). [a]D22
ˆ
23.5 (c ˆ 0.15,
3
CHCl3/2,2,2-trifluoroethanol 3:1 v/v); 1H NMR (500 MHz, [D6]DMSO):
d ˆ 8.57 (s, 1H; CONH), 8.53 (s, 1H; CONH), 8.30 ± 8.27 (m, 2H; CONH),
8.18 (brs, 3H; CONH), 8.09 ± 8.04 (m, 4H; CONH), 7.91 ± 7.86 (m, 6H;
CONH), 7.81 (brs, 1H; CONH), 7.76 (d, 3J ˆ 7.8 Hz, 1H; CONH), 7.69 (brs,
1H; CONH), 7.58 ± 7.41 (m, 8H; CONH), 7.27 ± 7.15 (m, 30H; Trt), 6.69
(brs, 1H; OCONH), 4.53 ± 4.46 (m, 3H; 3 a-CH), 4.34 ± 4.18 (m, 15H; 15 a-
CH), 3.88 ± 3.37 (m, 26H; 9Gly a-CH2, Ser b-CH2, 3 Pro d-CH2), 3.06 (m,
4H; 2Cys b-CH2), 2.85 (d, 3J ˆ 7.0 Hz, 2H; Lys e-CH2), 2.63 (brs, 2H; Asn
b-CH2), 2.54 (m, 4H; 2 Pal a-CH2), 2.37 ± 2.25 (m, 4H; Gln g-CH2, Glu g-
CH2), 2.11 ± 2.09 (m, 3H; Myr a-CH2, Val b-CH), 1.98 ± 1.46 (m, 44H; Gln
b-CH2, Glu b-CH2, 3Pro b-CH2, 3Pro g-CH2, Lys b-CH2, Myr b-CH2, Myr
g-CH2, 2Pal b-CH2, 6Leu g-CH, 6Leu b-CH2), 1.39 (s, 9H; C(CH3)3), 1.35
(s, 9H; C(CH3)3), 1.34 ± 1.26 (m, 4H; Lys d-CH2, Lys g-CH2), 1.23 (brs,
66H; Myr (CH2)9, Pal (CH2)12), 1.08 (s, 9H; C(CH3)3), 0.86 ± 0.83 (m, 51H;
Myr w-CH3, 2 Pal w-CH3', 11Leu w-CH3, 2Val w-CH3); MS (MALDI-
2H; Asn b-CH2), 2.54 (t, J ˆ 7.4 Hz, 2H; Pal a-CH2), 2.36 ± 2.26 (m, 4H;
Gln g-CH2, Glu g-CH2), 2.12 ± 2.09 (m, 3H; Myr a-CH2, Val b-CH), 1.99 ±
1.46 (m, 39H; Gln b-CH2, Glu b-CH2, 3 Pro b-CH2, 3 Pro g-CH2, Lys b-
CH2, Myr b-CH2, Myr g-CH2, Pal b-CH2, 5 Leu g-CH, 5 Leu b-CH2), 1.39
(s, 9H; C(CH3)3), 1.36 (s, 9H; C(CH3)3), 1.32 ± 1.28 (m, 4H; Lys d-CH2, Lys
g-CH2), 1.23 (brs, 42H; Myr (CH2)9, Pal (CH2)12), 1.08 (s, 9H; C(CH3)3),
0.88 ± 0.82 (m, 42H; Myr w-CH3, Pal w-CH3', 10Leu w-CH3, 2Val w-CH3);
‡
MS (MALDI-TOF, DHB/TFA): m/z: calcd for [M‡Na] 3286.93; found:
‡
3284.57, calcd for [M‡K] 3302.90; found: 3302.22; C173H262N26O33S
(3266.22).
Myr-Gly-Asn-Leu-Lys-Ser-Val-Gly-Gln-Glu-Pro-Gly-Pro-Pro-Cys(Pal)-
Gly-Leu-Gly-Leu-Gly-Leu-Gly-Leu-Gly-OH (47): Peptide 41 (3.9 mg,
1.2 mmol) was dissolved in a mixture of CF3CO2H/ethanedithiol/water
95:2.5:2.5 (v/v/v) (200 mL) and shaken at 208C for 20 min. Then the solvent
was removed under reduced pressure and 47 was precipitated and washed
with diethyl ether. After separation by HPLC the deprotected peptide was
‡
TOF, DHB/TFA): m/z: calcd for [M‡H] 3776.29; found: 3777.65, calcd for
‡
‡
[M‡Na] 3798.27; found: 3797.32, calcd for [M‡K] 3814.25; found:
obtained as colorless solid (1.6 mg, 52%). [a]D22
ˆ
34.3 (c ˆ 0.15 in CHCl3/
3815.12; C200H311N29O37S2 (3777.99).
2,2,2-trifluoroethanol 3:1 v/v); HPLC: tr ˆ 18.4 min (508C, 25% to 100% B
in 20 min, then 10 min B; A: H2O‡1% CH3CN‡0.1% TFA; B:
CH3CN‡1% H2O‡0.1% TFA); 1H NMR (500 MHz, [D6]DMSO): d ˆ
8.39 (s, 2H; CONH), 8.21 ± 8.17 (m, 10H; CONH), 7.88 ± 7.78 (m, 10H;
CONH), 7.43 (s, 1H; CONH), 7.27 (brs, 2H; CONH), 6.95 (s, 1H; CONH),
6.76 (s, 1H; CONH), 4.57 ± 4.47 (m, 3H; 3a-CH), 4.34 ± 4.08 (m, 12H; 12 a-
CH), 3.82 ± 3.49 (m, 24H; 8Gly a-CH2, Ser b-CH2, 3 Pro d-CH2), 3.08 (m,
2H; Cys b-CH2), 2.75 (brs, 2H; Lys e-CH2), 2.47 (brs, 2H; Pal a-CH2), 2.27
(brs, Myr a-CH2), 2.14 ± 2.01 (m, 7H; Asn b-CH2, Val b-CH, Gln g-CH2,
Glu g-CH2), 1.91 ± 1.36 (m, 43H; Gln b-CH2, Glu b-CH2, 3 Pro b-CH2, 3
Pro g-CH2, Lys b-CH2, Myr b-CH2, Myr g-CH2, Pal b-CH2, 5 Leu g-CH, 5
Myr-Gly-Asn-Leu-Lys-Ser-Val-Gly-Gln-Glu-Pro-Gly-Pro-Pro-Cys(Pal)-
Gly-Leu-Gly-Leu-Gly-Leu-Gly-Leu-Gly-Leu-Cys(Pal)-Gly-OH (48): Pep-
tide 43 (3.2 mg, 0.8 mmol) was dissolved in a mixture of CF3CO2H/
ethanedithiol/water 95:2.5:2.5 (v/v/v) (200 mL) and shaken at 208C for
20 min. Then the solvent was removed under reduced pressure and 48 was
precipitated and washed with diethyl ether. After separation by HPLC the
deprotected peptide was obtained as colorless solid (1.1 mg, 42%). [a]D22
ˆ
17.8 (c ˆ 0.15 in CHCl3/2,2,2-trifluoroethanol 3:1 v/v); HPLC: tr ˆ
21.2 min (508C, 25% to 100% B in 20 min, then 10 min B; A: H2O‡ 1%
CH3CN‡0.1% TFA; B: CH3CN‡1% H2O‡0.1% TFA); 1H NMR
2954
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
0947-6539/01/0713-2954 $ 17.50+.50/0
Chem. Eur. J. 2001, 7, No. 13

Lipidated eNOS Peptides
2940±2956
(500 MHz, [D6]DMSO): d ˆ 8.18 ± 8.14 (m, 6H; CONH), 8.09 (d, 3J ˆ
6.1 Hz, 1H; CONH), 8.02 ± 7.86 (m, 9H; CONH), 7.70 ± 7.55 (m, 5H;
CONH), 7.42 (brs, 1H; CONH), 7.33 ± 7.20 (m, 6H; CONH), 6.94 (s, 1H;
CONH), 6.75 (s, 1H; CONH), 4.53 ± 4.47 (m, 2H; 2a-CH), 4.37 ± 4.06 (m,
16H; 16a-CH), 3.78 ± 3.38 (m, 26H; 9Gly a-CH2, Ser b-CH2, 3Pro d-CH2),
3.05 (m, 4H; 2Cys b-CH2), 2.75 (brs, 2H; Lys e-CH2), 2.56 (brs, 4H; 2Pal
a-CH2), 2.34 ± 2.30 (m, 4H; Gln g-CH2, Glu g-CH2), 2.17 ± 1.98 (m, 5H;
Myr a-CH2, Asn b-CH2, Val b-CH), 1.91 ± 1.33 (m, 48H; Gln b-CH2, Glu b-
CH2, 3Pro b-CH2, 3Pro g-CH2, Lys b-CH2, Myr b-CH2, Myr g-CH2, 2Pal
b-CH2, 6Leu g-CH, 6Leu b-CH2, Lys d-CH2, Lys g-CH2), 1.23 (brs, 66H;
Myr (CH2)9, 2Pal (CH2)12), 0.86 ± 0.82 (m, 51H; Myr w-CH3, 2Pal w-CH3',
12Leu w-CH3, 2Val w-CH3); MS (MALDI-TOF, DHB/TFA): m/z: calcd
Acknowledgement
This research was supported by the Deutsche Forschungsgemeinschaft and
the Fonds der Chemischen Industrie.
[1] J. T. Dunphy, M. E. Linder, Biochim. Biophys. Acta 1998, 1436, 245 ±
261.
[2] M. D. Resh, Biochim. Biophys. Acta 1999, 1451, 1 ± 16.
[3] a) D. Kadereit, J. Kuhlmann, H. Waldmann, ChemBioChem 2000, 1,
144 ± 169; b) D. Kadereit, J. Kuhlmann, H. Waldmann in Stimulating
Concepts in Chemistry (Eds.: M. Shibaski, J. F. Stoddart, F. Vögtle),
Wiley-VCH 2000, pp. 369 ± 382.
‡
‡
for [M‡H] 3079.89; found: 3079.15, calcd for [M‡Na] 3101.88; found:
‡
3103.02, calcd for [M‡K] 3117.85; found: 3115.49; C149H259N29O35S2
(3081.01).
[4] B. Bader, K. Kuhn, D. J. Owen, H. Waldmann, A. Wittinghofer, J.
Kuhlmann, Nature 2000, 403, 223 ± 226.
Myr-Gly-Asn(Trt)-Leu-Lys(Boc)-Ser(tBu)-Val-Gly-Gln(Trt)-Glu(OtBu)-
Pro-Gly-Pro-Pro-Cys(Pal)-Gly-Leu-Gly-Leu-Gly-Leu-Gly-Leu-Gly-Leu-
Cys(Pal)-Gly-Lys(Boc)-Gln(Trt)-Gly-OtBu (44): EDC (0.6 mg, 3.2 mmol)
[5] a) H. Waldmann, M. Schelhaas, E. Nägele, J. Kuhlmann, A. Wit-
tinghofer, H. Schroeder, J. R. Silvius, Angew. Chem. 1997, 109, 2334 ±
2337; Angew. Chem. Int. Ed. 1997, 36, 2238 ± 2241; b) H. Schroeder, R.
Leventis, S. Rex, M. Schelhaas, E. Nägele, H. Waldmann, J. R. Silvius,
Biochemistry 1997, 36, 13102 ± 13109.
[6] D. Huster, K. Kuhn, D. Kadereit, H. Waldmann, K. Arnold, Angew.
Chem. 2001, 113, 1083 ± 1085; Angew. Chem. Int. Ed. 2001, 40, 1056 ±
1058.
[7] a) D. J. Owen, K. Alexandrov, E. Rostkova, A. J. Scheidig, R. S.
Goody, H. Waldmann, Angew. Chem. 1999, 111, 570 ± 573; Angew.
Chem. Int. Ed. 1999, 38, 509 ± 512; b) N. H. Thomä, A. Iakovenko, D.
Owen, A. S. Scheidig, H. Waldmann, R. S. Goody, K. Alexandrov,
Biochemistry 2000, 39, 12043 ± 12052.
[8] M. Langer, C. Appelt, H. Waldmann, A. Beck-Sickinger, unpublished
results.
[9] a) E. Nägele, M. Schelhaas, N. Kuder, H. Waldmann, J. Am. Chem.
Soc. 1998, 120, 6889 ± 6902; b) M. Schelhaas, E. Nägele, N. Kuder,
H. B. Bader, J. Kuhlmann, A. Wittinghofer, H. Waldmann, Chem. Eur.
J. 1999, 5, 1239 ± 1252.
[10] Part of this work was published as a short communication: R.
Machauer, H. Waldmann, Angew. Chem. 2000, 112, 1503 ± 1507;
Angew. Chem. Int. Ed. 2000, 39, 1449 ± 1453.
[11] a) R. M. J. Palmer, D. S. Ashton, S. Moncada, Nature 1988, 333, 664 ±
666; b) P. W. Shaul, E. J. Smart, L. J. Robinson, Z. German, I. S.
Yuhanna, Y. Ying, R. G. W. Anderson, T. Michel, J. Biol. Chem. 1996,
271, 6518 ± 6522.
was added at 08C to
a solution of peptide 43 (5.3 mg, 1.6 mmol),
H-Lys(Boc)-Gln(Trt)-Gly-OtBu 26 (1.8 mg, 2.4 mmol) and HOAt (0.5 mg,
3.2 mmol) in N-methylpyrolidinone (300 mL), and the mixture was stirred at
208C for 16 h. At 08C citric acid (5%, 5 mL) was added and the
precipitated was separated, washed with citric acid, water and methanol
and dried to yield a colorless solid (6.2 mg, 86%). [a]D22
ˆ
25.6 (c ˆ 0.25 in
CHCl3/2,2,2-trifluoroethanol 3:1 v/v); 1H NMR (500 MHz, [D6]DMSO):
d ˆ 8.57 (s, 1H; CONH), 8.54 (s, 1H; CONH), 8.52 (s, 1H; CONH), 8.30 ±
8.28 (m, 2H; CONH), 8.18 (brs, 4H; CONH), 8.08 ± 8.06 (m, 8H; CONH),
7.91 ± 7.86 (m, 7H; CONH), 7.81 ± 7.76 (m, 4H; CONH), 7.69 (m, 2H;
CONH), 7.60 ± 7.58 (m, 2H; CONH), 7.27 ± 7.15 (m, 30H; Trt), 6.69 (brs,
2H; OCONH), 4.53 ± 4.46 (m, 3H; 3a-CH), 4.34 ± 4.19 (m, 16H; 16 a-CH),
3.85 ± 3.39 (m, 28H; 10Gly a-CH2, Ser b-CH2, 3Pro d-CH2), 3.06 (m, 4H;
2Cys b-CH2), 2.85 (d, 3J ˆ 6.5 Hz, 4H; 2Lys e-CH2), 2.63 (brs, 2H; Asn b-
CH2), 2.55 (m, 4H; 2Pal a-CH2), 2.36 ± 2.25 (m, 6H; 2Gln g-CH2, Glu g-
CH2), 2.18 ± 2.09 (m, 3H; Myr a-CH2, Val b-CH), 1.99 ± 1.46 (m, 48H; 2Gln
b-CH2, Glu b-CH2, 3Pro b-CH2, 3Pro g-CH2, Lys b-CH2, Myr b-CH2, Myr
g-CH2, 2Pal b-CH2, 6Leu g-CH, 6Leu b-CH2), 1.38 (s, 9H; C(CH3)3), 1.36
(s, 9H; C(CH3)3), 1.35 (s, 9H; C(CH3)3), 1.32 ± 1.29 (m, 8H; 2 Lys d-CH2, 2
Lys g-CH2), 1.23 (brs, 66H; Myr (CH2)9, Pal (CH2)12), 1.08 (s, 9H;
C(CH3)3), 0.86 ± 0.82 (m, 51H; Myr w-CH3, 2 Pal w-CH3', 11Leu w-CH3,
‡
2Val w-CH3); MS (MALDI-TOF, DHB/TFA): m/z: calcd for [M‡Na]
4509.67; found: 4510.12; C241H364N34O43S2 (4489.90).
Myr-Gly-Asn-Leu-Lys-Ser-Val-Gly-Gln-Glu-Pro-Gly-Pro-Pro-Cys(Pal)-
Gly-Leu-Gly-Leu-Gly-Leu-Gly-Leu-Gly-Leu-Cys(Pal)-Gly-Lys-Gln-Gly-
OH (1): Protected peptide 44 (3.9 mg, 0.9 mmol) was dissolved in a mixture
of CF3CO2H/ethanedithiol/water 95:2.5:2.5 (v/v/v) (200 mL) and shaken at
208C for 20 min. Then the solvent was removed under reduced pressure
and 1 was precipitated and washed with diethyl ether. After separation by
HPLC the deprotected peptide was obtained as colorless solid (1.0 mg,
[12] a) E. G. Shesely, N. Maeda, H. S. Kim, K. M. Desai, J. H. Krege, V. E.
Laubach, P. A. Sherman, W. C. Sessa, O. Smithies, Proc. Natl. Acad.
Sci. USA 1996, 93, 13176 ± 13181; b) P. L. Huang, Z. H. Huang, H.
Mashimo, K. D. Bloch, M. A. Moskowitz, J. A. Bevan, M. C. Fishman,
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Natl. Acad. Sci. USA 1996, 93, 6448 ± 6453; b) P. W. Shaul, E. J. Smart,
L. J. Robinson, Z. German, I. S. Yuhanna, Y. Ying, R. G. W. Ander-
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31%). [a]2D2
ˆ
19.0 (c ˆ 0.05 in CHCl3/2,2,2-trifluoroethanol 3:1 v/v);
HPLC: tr ˆ 22.2 min (508C, 25% to 100% B in 20 min, then 10 min B; A:
H2O‡1% CH3CN‡0.1% TFA; B: CH3CN‡1% H2O‡s0.1% TFA);
1H NMR (500 MHz, [D6]DMSO): d ˆ 8.20 ± 8.10 (m, 9H; CONH), 7.95 ±
7.84 (m, 9H; CONH), 7.62 (brs, 5H; CONH), 7.43 (s, 1H; CONH), 7.35 ±
7.20 (m, 9H; CONH), 6.95 (s, 1H; CONH), 6.76 (s, 1H; CONH), 4.79 ± 4.73
(m, 1H; 1 a-CH), 4.53 ± 4.50 (m, 4H; 4 a-CH), 4.37 ± 4.17 (m, 14H; 14 a-
CH), 3.80 ± 3.54 (m, 28H; 9Gly a-CH2, Ser b-CH2, 3Pro d-CH2), 3.07 (m,
4H; 2Cys b-CH2), 2.75 (brs, 4H; 2Lys e-CH2), 2.56 (m, 4H; 2Pal a-CH2),
2.37 ± 2.32 (m, 3H; 3 Glx g-CH2a), 2.14 ± 1.98 (m, 8H; Myr a-CH2, 3Glx g-
CH2b, Asn b-CH2, Val b-CH), 1.91 ± 1.27 (m, 52H; Gln b-CH2, Glu b-CH2,
3 Pro b-CH2, 3 Pro g-CH2, Lys b-CH2, Myr b-CH2, Myr g-CH2, 2Pal b-
CH2, 6Leu g-CH, 6Leu b-CH2, 2Lys d-CH2, 2Lys g-CH2), 1.23 (brs, 66H;
Myr (CH2)9, 2Pal (CH2)12), 0.88 ± 0.84 (m, 51H; Myr w-CH3, 2 Pal w-CH3',
12Leu w-CH3, 2Val w-CH3); MS (ESI): m/z: calcd for [M‡2H]2‡ 1697.04;
‡
found: 1697.9; MS (MALDI-TOF, DHB/TFA): m/z: calcd for [M‡H]
‡
3393.07; found: 3394.16, calcd for [M‡Na] 3415.05; found: 3415.62, calcd
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Liu, G. García-CardenÄa, W. C. Sessa, Biochemistry 1995, 34, 12333 ±
12340.
‡
for [M‡K] 3431.02; found: 3432.46; C126H282N34O39S2 (3394.37).
[17] L. J. Robinson, T. Michel, Proc. Natl. Acad. Sci. USA 1995, 92,
11776 ± 11780.
Â
[18] K. Hinterding, D. Alonso-Diaz, H. Waldmann, Angew. Chem. 1998,
110, 716 ± 780; Angew. Chem. Int. Ed. Engl. 1998, 37, 688 ± 749.
Chem. Eur. J. 2001, 7, No. 13
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
0947-6539/01/0713-2955 $ 17.50+.50/0
2955

H. Waldmann and R. Machauer
FULL PAPER
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Received: December 22, 2000 [F2959]
2956
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
0947-6539/01/0713-2956 $ 17.50+.50/0
Chem. Eur. J. 2001, 7, No. 13