Tetrahydropyran-amino acids: Novel building blocks for gramicidin-hybrid ion channels

Article abstract of DOI:10.1002/ejoc.200600068

The stereoselective synthesis of a cis-2,6-disubstituted tetrahydropyran bearing a δ-amino acid has been achieved starting from N-Boc-leucinal. The THP amino acid was incorporated into peptide sequences and the structural consequences were studied by X-ra

Full text of DOI:10.1002/ejoc.200600068

FULL PAPER
DOI: 10.1002/ejoc.200600068
Tetrahydropyran-Amino Acids: Novel Building Blocks for Gramicidin-Hybrid
Ion Channels
Sabine Schröder,[a] Anna K. Schrey,[b] Andrea Knoll,[a] Philipp Reiß,[c] Burkhard Ziemer,[a]
and Ulrich Koert*[c]
Keywords: THP amino acid / Stereoselective synthesis / Peptide / Gramicidin A / Ion channel
The stereoselective synthesis of a cis-2,6-disubstituted tetra- the THP amino acid is a suitable substitute for positions 11
hydropyran bearing a δ-amino acid has been achieved start-
ing from N-Boc-leucinal. The THP amino acid was incorpo-
rated into peptide sequences and the structural conse-
quences were studied by X-ray crystallography and NMR
analysis. Single-channel current measurements showed that
and 12 of the gramicidin ion channel. The resulting hybrid
ion channel revealed Eisenman I ion selectivity and an ion-
dependence of the channel dwell time.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2006)
Awhere Trp11--Leu12 of the gA sequence are replaced (2
Ǟ 3) by the THP amino acid 1. Positions 11 and 12 were
chosen because they are part of the channel entrance where
the partial desolvation of the conducted ion takes place.
The THP oxygen offers an additional binding site for the
cation and can assist the partial desolvation process.
Introduction
The channel-mediated transport of ions through the
phospholipid bilayer of cellular membranes is an important
biological process.[1] Parallel to the progress in the struc-
tural biology of ion channels,[2] synthetic chemists address
ion channels as target functions.[3] Gramicidin A (gA) is a
well suited lead structure for the design of synthetic ion
channels.[4] gA is a naturally occurring pentadecapeptide
with 15 amino acids in length (Scheme 1). Its alternating
sequence of - and -amino acids favours the formation of
a β-helical secondary structure. In the membrane gA forms
Results and Discussion
The synthesis of the N-Fmoc-protected THP amino acid
a head-to-head dimer of two right-handed single-stranded 10 started with N-Boc -leucinal 5 (Scheme 2).[11] A chela-
β-helices, which is the accepted ion-channel active struc- tion-controlled[12] reaction of 5 with ethereal 4-pentenyl-
ture.[5,6] When synthetic building blocks are coupled with magnesium bromide to yield alcohol 6 gave the best diastero-
the gA-motif, hybrid channels result which show new func- selectivity (Ͼ 98:2), when the aldehyde was dissolved in
tionality (photomodulation with hemithioindigo[3e] and azo- CH2Cl2 and precomplexed with magnesium bromide. Epox-
benzene,[7] ion selectivity with cyclohexyl ether amino ac- idation of the terminal double bond in 6 followed by an
ids[8] and aza-crown ethers[9]). Tetrahydrofuran amino acids acid-catalyzed intramolecular epoxide opening gave the
(THF amino acids) were extensively explored as building trans THP alcohol 7 and the cis THP alcohol 8 in nearly
blocks for gramicidin hybrid ion channels.[10] The exchange equal amounts.[10c] Both epimers could be separated by sil-
of the THF ring by a tetrahydropyran (THP) leads to THP- ica gel chromatography. Compound 8 was subjected to a
amino acids of type 1. Here we present a synthetic route to Boc Ǟ Fmoc exchange to produce 9, which was oxidized
THP-amino acids and gramicidin hybrid channels contain- to the N-Fmoc-protected THP amino acid 10 by a two step
ing this novel ion-channel building block motif. As one ex- procedure (Swern[13] followed by Pinnick[14]).
ample, the synthesis of the hybrid channel 4 is described,
The N-Boc-protected THP amino acid 11 was accessible
from the alcohol 8 (Scheme 3, A). From 11, the THP amide
12 was prepared. The relative configuration of 12 was un-
ambiguously proven by X-ray crystal-structural analysis
(Scheme 3, B), which secured the stereochemical assign-
ments of compounds 6, 7, and 8.
The hexapeptide 17 was chosen as a model compound
for the studies concerning the solution structure of peptides
containing the THP amino acid building block. A segment
coupling strategy[15] was applied for the synthesis of 17
[a] Institut für Chemie, Humboldt-Universität zu Berlin,
Brook-Taylor-Strasse 2, 12489 Berlin
[b] Forschungsinstitut für Molekulare Pharmakologie,
Robert-Rössle-Straße 10, 13125 Berlin
[c] Fachbereich Chemie, Philipps-Universität Marburg,
Hans-Meerwein-Strasse, 35032 Marburg
E-mail: koert@chemie.uni-marburg.de
Supporting information for this article is available on the
WWW under http://www.eurjoc.org or from the author.
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FULL PAPER
Scheme 1. Generation of the THP-amino-acid gramicidin A hybrid 4 by replacement of positions 11,12 in gA with the THP amino acid
1 (2 Ǟ 3).
Scheme 2. Synthesis of the N-Fmoc-protected THP amino acid 10.
a) H2C=CH(CH2)2CH2MgBr, MgBr2, Et2O/CH2Cl2, –50 °C; b) i:
meta-chloroperbenzoic acid, CH2Cl2, 20 °C; ii: camphor sulfonic
acid, CH2Cl2, 20 °C; c) TFA, CH2Cl2, 20 °C; ii: Fmoc-succinimide,
NaHCO3, CH3CN/H2O, 20 °C; d) i: (COCl)2, DMSO, Et3N,
CH2Cl2, –60 Ǟ 0 °C; ii: NaClO2, NaH2PO4, amylene, tBuOH/H2O,
20 °C.
(Scheme 4). Coupling of the Boc-protected THP acid 11
Scheme 3. A. Synthesis of THP-amino acid derivatives: a) i: (COCl)2,
DMSO, Et3N, CH2Cl2, –60 Ǟ 0 °C; ii: NaClO2, NaH2PO4, amyl-
with the dipeptide 13[15] provided the tripeptide 14. The lat-
ter was elaborated to the pentapeptide 15, whose coupling ene, tBuOH/H2O, 20 °C; b) benzylamine; HBtU, HOBt, DIEA,
with the tryptophan derivative 16[15] led to the desired hexa-
DMF/CH2Cl2, 20 °C. B: solid state conformation of 12 obtained
by X-ray crystal-structural analysis.
peptide 17.
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The 11,12-THP-gA hybrid (4) was synthesized by solid
phase synthesis on Wang resin using Fmoc-N-protection
and HBtU-HOBt coupling conditions (Scheme 5). Starting
from Wang resin PS-PHB-Trp-Fmoc 18, the Fmoc-THP
acid 10 was introduced in the third coupling step to yield
19. N-Formyl--valine was used in the final coupling step
(20 Ǟ 21). The cleavage of the peptide was carried out with
10% ethanolamine in DMF at 55 °C for 48 h. Chromato-
graphic purification gave the 11,12-THP-gA hybrid (4) in
43% yield.
The solution structures of the THP-amide 12 and the
model THP-peptide 17 were investigated by NMR spec-
troscopy combined with MD-calculation (details are given
in the Exp. Sect.).
THP-Benzylamide 12
The NMR-data were collected with 12, while the calcula-
tions were done with a replacement of Boc by Ac on the
corresponding benzylamide 22 (Figure 1, A). The structure
calculation afforded 100 structures, none of them violating
the upper boundary of the distance restraints by more than
0.13 Å. Figure 1 (B) shows an ensemble of the 20 energy-
lowest structures, fitted on the heavy atoms of the central
THP amino-acid building block. The closest-to-average co-
ordinates structure is highlighted. The mean rms deviation
of the heavy-atom fit excluding the benzyl group was
0.035 0.027 Å.
Scheme 4. Synthesis of the hexapeptide 17 by segment coupling in
solution. a) i: 13, H2, Pd/C, MeOH, 20 °C; ii: EDC, HOBt, Et3N,
CH2Cl2, 20 °C; b) i: 13, LiOH, THF/H2O, 20 °C; ii: 14, TFA,
CH2Cl2, 20 °C; iii: HOBt, EDC, DIEA, HBtU, CH2Cl2, 20 °C; c) i:
16, H2, Pd/C, MeOH, 20 °C; ii: 15, LiOH, THF/H2O, 20 °C; iii:
HOBt, DIEA, HBtU, CH2Cl2, 20 °C.
Figure 1. A: Formula structure of 22. B Solution structure of 22:
20 lowest-energy conformations from NMR calculations. Bold:
Structure closest to the average coordinates. C Superposition be-
tween solution (blue) and X-ray structure (green).
The THP moiety including the isobutyl side chain as well
as the acetamide group converged very well. Deviations are
found in the benzylamide group because of the lack of
Scheme 5. Solid-phase synthesis of 11–12-THPgA (4). a) piperidine,
DMF, b) Fmoc-l, HOBt, DIEA, HBtU, DMF, c) Fmoc-W, HOBt,
DIEA, HBtU, DMF, d) 10, HOBt, DIEA, HBtU, DMF, e) N-for-
myl-V, HOBt, DIEA, HBtU, DMF, f) ethanolamine, DMF, 55 °C. structure-defining distance restraints. Nevertheless, in 18 of
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Novel Building Blocks for Gramicidin-Hybrid Ion Channels
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20 structures, a similar orientation of the aromatic moiety side chain, it does not. The C-terminal carboxamides in
can be found. The calculated solution structures are in good both the THP and the THF structures exhibit similar
agreement with the X-ray structure of 12. A superposition orientations.
of the solution and the X-ray structures is shown in Fig-
ure 1 (C). The rms deviation between both structures in-
cluding the orientation of the isobutyl group lies with
0.35 Å in the range of the experimental error of the X-ray
Model THP Peptide 17
structure (0.822 Å). The planes of the amide groups differ
The NMR spectroscopic data were collected with 17
slightly in their orientation towards the central THP ring, while for practical reasons the calculations were done with
which might be due to the different N-terminal side chains compound 23, where the CBz group was replaced by an
and the lack of structure-determining restraints for the acetyl group (Figure 2, A). The THP-containing peptide 23
benzamide moiety. None of the structures shows any pro- forms a complex, hydrogen-bonded structure. The tempera-
pensity to form a hydrogen-bonded β-turn structure. The ture dependence of the amide proton shifts is linear, sug-
amide protons are pointing towards each other. The dihe- gesting a stable secondary structure over the whole tem-
dral angle between the α and β protons is about 60 deg perature range. The corresponding temperature coefficients
(gauche+). In contrast, in a similar THF amide with a (see Supporting Information, Table S1a) imply hydrogen
methyl instead of a isobutyl side chain which is known to bonding of the amide protons at THP3, Trp4, and EAN7
form a hydrogen-bounded β-turn structure in solid state as (∆∆T Ͻ 6 -ppb/K) and partial hydrogen bonding for the
well as in solution,[10c] those protons form a dihedral angle amide protons at Trp1 ad Trp7 (6 -ppb/K Ͻ∆∆T Ͻ 9 -ppb/
in the range 180 deg (trans). In the case of the THF-amide, K), respectively. In the NMR structure calculations, 5 of
hydrogen-bond formation might override the steric 100 structures violated the restraint boundaries given in the
hindrance between the THF ring and the less bulky methyl methods section. Figure 2 (B) shows the ensemble of the 20
side chain. In case of the bulkier THP ring and isobutyl energy-lowest, unviolated structures. The structure closest
Figure 2. A: Formula structure of compound 23. B 20 lowest-energy conformations of 23 from NMR calculations. Bold: Structure closest
to the average coordinates. C Schematic representation of the multiturn hydrogen-bonding pattern.
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S. Schröder, A. K. Schrey, A. Knoll, P. Reiß, B. Ziemer, U. Koert
FULL PAPER
to the average coordinates is highlighted. In the other struc- man I selectivity for compound 4 (Cs+ ϾK+ Ͼ Na+) follows
tures, the side chains are omitted for clarity. The structures the order known for gA.[16] Long dwell times in the order
were fitted on N, Cα, Cβ, Cζ, C of the THP moiety and the of seconds were observed for NH4+, while burst-type events
backbone atoms of the adjacent Leu3 and Trp4 residues dominated for Cs+. This interesting dwell time behavior is
with a mean rms deviation of 0.510 0.117 Å. For the not known for gA and shows the ability of the THP to
whole backbone, a mean rmsd deviation of 1.153 0.215 Å modulate the ion channel functionality.
is found for this superposition.
The THP residue forms a sharp kink in the peptide
chain. The central ring is found in two different orienta-
tions, with torsion angles between Hα and Hβ of about 120
deg or –120 deg, respectively. This finding, however, has to
be taken with care, because the ring orientation is mainly
defined by the coupling constant between the two protons
which has been found to be about 3 Hz by J-sensitive 2D
NMR experiments and thus not unambiguous. Neverthe-
less, both orientations of the THP ring result in similar ori-
entations of the adjacent peptide chains which exhibit a hy-
drogen-bonded turn pattern. A schematic representation of
this pattern is shown in Figure 2 (C). The amide proton of
THP3 is involved in a hydrogen bond to the terminal acet-
amide oxygen, resulting in a type II β-turn formed by Trp1
and Leu2. This is in good agreement with the temperature
coefficients for the amide proton of THP3 (low) and Leu2
(high), the latter being accessible by the solvent. This hydro-
gen bond forces a reorientation of the amide plane between
Leu2 and THP3 with the carbonyl oxygen of Leu2 (instead
of the THP3 amide proton as in 12 and 22) pointing in the
direction of the THP ring. This reorientation enables an
interaction of this oxygen to the amide proton of Trp4.
The amide protons of residues 6 and 7 are involved in
fluctuating H-bonds to the carbonyl oxygens of THP3 and
Trp4, resulting of a suite of β- and γ-turns. The finding that
only the amide proton of the ethanolamine moiety and not
Figure 3. Single-channel current traces for 11,12-THP gA (4) in aso-
lectine at 100 mV. A: 1  NH4Cl, B: 1  CsCl, C: 1  KCl, D: 1 
NaCl.
that of Trp6 can be involved in a β-turn-forming hydrogen
bond which is geometrically more favoured than that of a
γ-turn is in good agreement with the lower temperature co-
efficient of the latter in contrast to the former proton shift.
In addition, the N-terminal and the C-terminal turn
structures exhibit a tendency for a crosslink formed by a
hydrogen bond between the amide proton of Trp1 and the
carbonyl oxygen of Leu5. This crosslink explains the me-
dium temperature coefficient for the proton shift which in-
dicates its participation in a weak H-bond.
Conclusions
The synthesis of the enantiomerically pure, novel THP
amino acid 1 was achieved using -leucine as a chiral-pool
source. The structural properties of 1 as a dipeptide isoster
were studied with compound 12 and the model peptide 17
by a combination of X-ray crystallography and solution-
structure-determination via NMR spectroscopy. The func-
tional analysis of the THP-gramicidin hybrid 4 shows, that
1 is a suitable substitute for positions 11 and 12 of gA. The
solid-phase-synthesis as described for 4 should allow the
broad study of substitutions at variable positions by syn-
thetic building blocks and their functional consequences
with respect to ion-channel activity.
Taken together, a structural ensemble with a decent con-
vergence could be calculated for 23. In spite of the uncer-
tainities in the THP region, the overall structure of the pep-
tide can be considered as valid.
Ion-Channel Activity of 11–12-THP-gA (4)
Single-channel current measurements in planar lipid bi-
layers were done in asolectine to characterize the ion-chan-
nel activity of 11–12-THP-gA (4).[10a]
Compound 4 displayed single-channel activity for mono-
valent cations (Figure 3). Compared to gA, compound 4
showed single-channel currents with about 50% reduced
Experimental Section
General: All reactions sensitive to air or moisture were conducted
in flame-dried glassware under an atmosphere of dry Argon. THF
conductivity. The observed ion selectivity exhibits Eisen- and Et2O were distilled from purple sodium benzophenone.
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CH2Cl2, toluene, hexanes, pyridine, and Et3N were distilled under
Ar from CaH2. All starting materials and reagents were used as
received unless noted otherwise. PE: light petroleum, boiling range
40–60 °C. MTBE: Methyl tert-butyl ether. Thin-layer chromatog-
raphy (TLC) was performed on glass-supported Merck silica gel 60
F254 plates. Spots were visualized by UV light and by heat staining
with 2% molybdophosphoric acid in ethanol. Flash column
chromatography (FCC) was performed on Merck silica gel 60 (63–
200 µm). Melting points were determined from pulverized material
(C-9), 160.7 (Boc-C=O) ppm. HR-MS calcd. 285.23039 found
285.2304.
(2S,6S,1ЈS)-2-[1Ј-(tert-Butoxycarbonylamino)-3Ј-methylbutyl]-6-(hy-
droxymethyl)tetrahydropyran (7) and (2S,6R,1ЈS)-2-[1Ј-(tert-But-
oxycarbonylamino)-3Ј-methylbutyl]-6-(hydroxymethyl)tetrahydro-
pyran (8): The alcohol 6 (3.27 g, 0.011 mol) was dissolved in
CH2Cl2 (70 mL). mCPBA (3.9 g, 0.022 mol) dissolved in CH2Cl2
(20 mL) was added to the solution. The reaction mixture was
stirred for 12 h at 20 °C. A saturated Na2SO3 solution (50 mL) was
added to the mixture and the layers were separated. The organic
layer was washed with saturated NaHCO3 solution (100 mL), with
saturated NaCl solution (100 mL) and dried with MgSO4. The fil-
tered solution was concentrated to 50 mL and camphorsulfonic
acid (400 mg) was added. The reaction mixture was stirred for 2 h
at 20 °C and subsequently quenched with saturated NaHCO3 solu-
tion (50 mL). The layers were separated and the aqueous layer was
extracted with CH2Cl2 (100 mL). The combined organic layers
were washed with saturated NaCl solution (50 mL) and dried with
MgSO4. The solvent was removed in vacuo. The purification by
column chromatography yielded 1.1 g (32%) of the cis product 8
and 1.25 g (36%) of trans product 7 as colourless foams.
1
in glass capillaries and are uncorrected. H and 13C NMR spectra
were obtained on Bruker DPX 300, DRX 400, DRX 500, or AMX
600 spectrometers, respectively. All resonances are referenced to re-
sidual solvent signals. Optical rotations: Perkin–Elmer spectropho-
topolarimeter 241, cuvette path length 10 cm. CHCl3 for spec-
troscopy was filtered through basic aluminium oxide before use.
MS: Finigan MAT 95 (EI: 70 eV. FAB), MSI Concept 1H (ESI),
Finnigan LTQ FT (ESI). Elemental analyses: Leco CHNS 932 An-
alysator (microanalytical facility, HU Berlin).
Boc-L-leucinal (5): Oxalyl chloride (5.35 mL, 0.062 mol) was dis-
solved in CH2Cl2 (100 mL) at –60 °C. DMSO (8.5 mL, 0.13 mol),
dissolved in CH2Cl2 (50 mL), was added and stirred for 5 min.
Then Boc--leucinol (9 g, 0.041 mol), dissolved in CH2Cl2 (50 mL),
cis-THP Alcohol 8: Rf = 0.37 (ethyl acetate/hexane, 1:1). [α]D
=
was added and stirred for 15 min. DIEA (33.7 mL, 0.242 mol) was –29.4 (c = 4.9, acetone, T = 25 °C). 1H NMR (300 MHz, CD3OD):
added and the reaction mixture was warmed to 0 °C and stirred
for 30 min. Then, saturated NaHCO3 solution (200 mL) was added
and the layers were separated. The aqueous layer was extracted
δ = 1.05 (d, J = 6.6 Hz, 6 H, CH3), 1.36–1.41 (m, 2 H, 5-H2), 1.31–
1.35 (m, 1 H, 2Ј-HA), 1.53 (s, 9 H, Boc-H3), 1.45–1.57 (m, 2 H, 3-
H2), 1.59–1.63 (m, 1 H, 2Ј-HB), 1.67–1.73 (m, 1 H, 3Ј-H), 1.95–
with CH2Cl2 (100 mL) and the combined organic layers were 1.99 (m, 2 H, 4-H2), 3.38–3.41 (m, 1 H, 2-H), 3.41–3.49 (m, 1 H,
washed with 0.1  citric acid (100 mL) and with saturated NaCl
solution (100 mL). The solvent was removed and the crude product
used directly for the next reaction. To check enantiomeric purity a
small amount was purified by column chromatography and the op-
tical rotation was measured. [α]D = –48.2 (c = 0.76, MeOH, T =
24 °C); ref.[11b] [α]D = –48.8 (c = 1.07, MeOH).
6-H), 3.59 (d, J = 8.5 Hz, 2 H, CH2OH), 3.65–3.68 (m, 1 H, 1Ј-H)
ppm. OH and NH not detectable due to H/D exchange. 13C NMR
(75.5 MHz, CD3Cl): δ = 22.3, 23.6 (CH3), 22.6 (C-4), 24.7 (C-3Ј),
26.9 (C-5), 27.9 (C-3), 28.3 (Boc-CH3), 41.9 (C-2Ј), 52.4 (C-1Ј),
66.2 (CH2OH), 78.2 (C-6), 78.9 (Cq), 79.1 (C-2), 156.1 (Boc-C=O)
ppm. HR-MS: calcd. 300.217483 found 300.21749.
trans-THP Alcohol 7: Rf = 0.28 (ethyl acetate/hexane, 1:1). [α]D
=
(4S,5S)-4-(tert-Butoxycarbonylamino)-2-methyl-9-decen-5-ol
(6):
–14.5, [α]578 = –15.0, [α]546 = –16, [α]436 = –8.4, [α]365 = –44.0 (c =
Magnesium turnings (0.80 g, 32.9 mmol) were covered with Et2O
(5 mL). Dibromoethane (6.2 g, 33 mmol, 2.84 mL) in Et2O (5 mL)
was added dropwise. A two-layer system with MgBr2 was formed.
Magnesium turnings (0.73 g, 30.1 mmol) were combined with Et2O
(10 mL) and 5-bromopent-1-ene (4.47 g, 30 mmol, 3.55 mL) in
Et2O (5 mL) was added dropwise. The reaction mixture was re-
fluxed. Boc-leucinal (5) (3.08 g, 14.3 mmol) dissolved in CH2Cl2
(50 mL) was added dropwise to the MgBr2 system and cooled to
–60 °C. The Grignard solution was transferred via cannula in a
dropping funnel and added dropwise to the reaction mixture while
maintaining a maximum temperature of –50 °C. After 2.5 h satu-
rated NH4Cl solution (100 mL) was added dropwise and the reac-
tion mixture was warmed to room temperature. The aqueous layer
was extracted with MTBE (100 mL). The combined organic layers
were washed with saturated NaHCO3 solution (80 mL), with satu-
rated NaCl solution (80 mL), dried with MgSO4, and the solvent
was removed in vacuo. After purification by column chromatog-
raphy (MTBE/PE, 1:6 Ǟ 1:1) 2.2 g (54%) of alcohol 6 was ob-
1
4.1, acetone, T = 26 °C). H NMR (300 MHz, CD3OD): δ = 1.01
(d, J = 6.7 Hz, 6 H, CH3), 1.32–1.33 (m, 2 H, 5-H2), 1.35–1.44 (m,
2 H, 2Ј-H2), 1.46–1.56 (m, 2 H, 3-H2), 1.53 (s, 9 H, Boc-CH3),
1.58–1.63 (m, 2 H, 4-H2), 1.68–1.73 (m, 1 H, 3Ј-H), 3.50–3.69 (m,
1 H, 2-H), 3.78 (d, J = 7.1 Hz, 2 H, CH2OH), 3.81–3.83 (m, 1 H,
1Ј-H), 3.93–3.97 (m, 1 H, 6-H) ppm. OH and NH not detectable
due to H/D exchange. 13C NMR (75.5 MHz, CD3Cl): δ = 18.5 (C-
4), 23.4, 24.7 (CH3), 25.3 (C-5), 26.9 (C-3Ј), 27.2 (C-3), 28.4 (Boc),
41.8 (C-2Ј), 50.6 (C-1Ј), 62.03(CH2OH), 72.5 (C-6), 72.8 (C-2), 79.1
(Cq), 156.3 (Boc-C=O) ppm. HR-MS calcd. 300.217483 found
300.21748.
(2S,6R,1ЈS)-2-[1Ј-(tert-Fluorenylmethoxycarbonylamino)-3Ј-meth-
ylbutyl]-6-(hydroxymethyl)tetrahydropyran (9): The cis-Boc-THP
alcohol 8 (301 mg, 1.3 mmol) was dissolved in CH2Cl2 (7 mL).
TFA (1.75 mL) was added and the reaction mixture was stirred for
1.5 h at room temperature. The solvent was removed by codistil-
lation with toluene in vacuo. The residue was dissolved in acetoni-
trile/water (10 mL, 1:1.6) and NaHCO3 (453.6 g, 5.4 mmol) was
added. Fmoc-succinimide (606 mg, 1.8 mmol) was added to the re-
action mixture which was stirred overnight. The acetonitrile was
tained as a colourless oil. Rf = 0.44 (acetone/CH2Cl2, 1:20). [α]D
=
1
–24.3 (c = 3.18, CHCl3, T = 24 °C). H NMR (300 MHz, CDCl3):
δ = 0.89 (d, J = 6.4 Hz, 6 H, 1/1i-H3), 1.19–1.30 (m, 1 H, 3-HA),
1.41 (s, 9 H, Boc-H3), 1.37–1.49 (m, 5 H, 8-H2, 6-HA, 3-HB, 7-HA), removed in vacuo. The residue was partitioned between MTBE
1.58–1.67 (m, 2 H, 2-H, 6-HB), 2.04–2.06 (m, 1 H, 7-HB), 3.44– (50 mL) and H2O (10 mL). The layers were separated and the aque-
3.52 (m, 1 H, 5-H), 3.55–3.61 (m, 1 H, 4-H), 4.53–4.61 (m, 1 H,
ous layer was extracted with MTBE (50 mL). The combined or-
NH), 4.90–5.00 (m, 2 H, 10-H2), 5.76 (ddt, J = 16.9/10.1/6.7 Hz, 1 ganic layers were washed with 0.1  HCl (10 mL), with saturated
H, 9-H) ppm. 13C NMR (75.5 MHz, CDCl3): δ = 22.1, 23.2 (C-1/
NaCl (50 mL) and dried with Na2SO4. The solvent was removed
1i), 24.8 (C-2), 24.9 (C-6), 28.3 (Boc-CH3), 32.2 (C-8), 33.7 (C-7), in vacuo. Purification by column chromatography yielded 311 mg
41.6 (C-3), 52.4 (C-4), 73.9 (C-5), 79.6 (Cq), 114.7 (C-10), 138.6 (76 %) of 9 as a colourless foam. Rf = 0.33 (MTBE/PE, 2:1).
Eur. J. Org. Chem. 2006, 2766–2776
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
2771

S. Schröder, A. K. Schrey, A. Knoll, P. Reiß, B. Ziemer, U. Koert
FULL PAPER
[α]D = –26.0, [α]578 = –26.7, [α]546 = –30.2, [α]436 = –50.2,
[α]365 = –76.0 (c = 0.824,MeOH, T = 24 °C). H NMR (300 MHz, wise. After 5 min, Boc-THP-alcohol 8 (3.1 g, 10.29 mmol) dis-
(2.6 mL, 41 mmol) dissolved in CH2Cl2 (10 mL) was added drop-
1
CDCl3): δ = 0.88 (d, J = 6.4 Hz, 3 H, CH3), 0.89 (d, J = 6.4 Hz, 3
H, CH3), 1.04–1.15 (m, 1 H, 5-HA), 1.18–1.31 (m, 3 H, 3-HA, 2Ј-
solved in CH2Cl2 (20 mL) was added dropwise and the mixture was
stirred for 15 min. Then NEt3 (14.25 mL, 103 mmol) was added
HA, 5-HB), 1.39–1.45 (m, 1 H, 3-HB), 1.45–1.63 (m, 2 H, 2Ј-HB, dropwise. After 30 min the reaction mixture was warmed to 0 °C
3Ј-H), 1.78–1.89 (m, 2 H, 4-H2), 1.97 (br. s, 1 H, OH), 3.32 (d, J and saturated NaHCO3 solution (30 mL) was added dropwise. The
= 10.9 Hz, 1 H, 2-H), 3.37–3.59 (m, 3 H, CH2OH, 6-H), 3.61–3.74 layers were separated and the aqueous layer was extracted with
(m, 1 H, 1Ј-H), 4.20 (t, J = 6.6 Hz, 1 H, Fmoc), 4.44 (d, J = 6.8 Hz, CH2Cl2 (50 mL). The combined organic layers were washed with
2 H, Fmoc), 4.80 (d, J = 9.8 Hz, 1 H, NH), 7.29 (t, J = 7.4 Hz, 2
H, Ar), 7.38 (t, J = 7.4 Hz, 2 H, Ar), 7.58 (dd, J = 7.2/3.0 Hz, 2
H, Ar), 7.74 (d, J = 7.5 Hz, 2 H, Ar) ppm. 13C NMR (75.5 MHz,
0.1  citric acid (50 mL), with saturated NaCl solution (50 mL)
and dried with MgSO4. The solvent was removed in vacuo.The
residue was dissolved in tert-butyl alcohol (50 mL) and in amylene
CDCl3): δ = 22.2, 23.3 (CH3), 22.6 (C-4), 24.6 (C-3Ј), 26.9 (C-5), (100 mL, 500 mmol). NaH2PO4·2H2O (12.5 g, 80 mmol) and
27.9 (C-3), 41.9 (C-2Ј), 47.5 (CHCH2O), 52.6 (C-1Ј), 66.1 (CH2O NaClO2 (9.04 g, 100 mmol), dissolved in a small amount of water,
Fmoc), 66.3 (CH2OH), 78.3 (C-6), 79.1 (C-2), 119.9, 124.9, 126.9, were added to the reaction mixture and stirred for 12 h at room
127.6, 141.4, 143.9 (Ar), 156.5 (Fmoc-C=O) ppm. HR-MS calcd. temperature. The tert-butyl alcohol was removed in vacuo, the resi-
423.2410 found 423.24093.
due was covered with MTBE (100 mL), 2  HCl was added to the
mixture and the aqueous layer was extracted with MTBE (100 mL).
The combined organic layers were concentrated, K2CO3 solution
(1.2 g/50 mL water) was added and the aqueous layer was extracted
with MTBE. The aqueous layer was covered with ethyl acetate, 2 
HCl was added and the layers were separated. The aqueous layer
was extracted with ethyl acetate (100 mL) three times. The com-
bined organic layers were washed with saturated NaCl solution
(100 mL), dried with MgSO4 and the solvent was removed to yield
3.04 g (96%) of acid 11 as a colourless foam. Rf = 0.20 (CHCl3/
MeOH/AcOH, 200:10:1) [α]D = –23.4, [α]578 = –24.1, [α]548 = –275,
[α]436 = –47.5, [α]365 = –75.6 (c = 1.5, MeOH, T = 25 °C). 1H NMR
(300 MHz, CD3OH): δ = 0.80 (d, J = 6.4 Hz, 6 H, CH3), 1.08–1.15
(m, 1 H, 2Ј-HA), 1.16–1.22 (m, 1 H, 3-HA), 1.25–1.30 (m, 2 H, 5-
H2), 1.38–1.39 (m, 1 H, 3-HB), 1.42–1.45 (m, 1 H, 2Ј-HB), 1.45–
1.57 (m, 1 H, 3Ј-H), 1.47 (s, 9 H, Boc-H3), 1.80–1.84 (m, 2 H, 4-
H2), 3.26–3.31 (m, 1 H, 6-H), 3.46–3.51 (m, 1 H, 1i-H), 3.86–3.91
(dd, 1 H, J = 2.3 Hz, 11.7 Hz, 2-H), 5.08 (d, J = 5.0 Hz, 1 H, NH)
ppm. 13C NMR (75.5 MHz, CD3OD): δ = 22.4, 23.7 (CH3), 24.1
(C-4), 28.6 (C-3), 28.8 (C-3Ј), 29.2 (Boc-CH3), 30.2 (C-5), 42.6 (C-
2Ј), 53.1 (C-1Ј), 77.2 (C-6), 79.2 (Cq), 80.9 (C-2), 157.0 (Boc-C=O),
176.2 (COOH) ppm. HR-MS calcd. 315.20457 found 316.212398
[MH+].
(2R,6S,1ЈS)-6-[1Ј-(Fluorenylmethoxycarbonylamino)-3Ј-methylbut-
yl]tetrahydropyran-2-carboxylic Acid (10): Oxalyl chloride
(0.08 mL, 0.94 mmol) was dissolved in CH2Cl2 (5 mL) at –60 °C
and DMSO (0.12 mL, 1.88 mmol) dissolved in CH2Cl2 (1 mL) was
added dropwise. After 5 min Fmoc-THP alcohol 9 (200 mg,
0.47 mmol) dissolved in CH2Cl2 (3 mL) was added dropwise and
stirred for 15 min. Then NEt3 (0.65 mL, 4.7 mmol) was added
dropwise. After 30 min, the reaction mixture was warmed to 0 °C
and saturated NaHCO3 solution (10 mL) was added dropwise. The
layers were separated and the aqueous layer was extracted with
CH2Cl2 (20 mL). The combined organic layers were washed with
0.1  citric acid (20 mL), with saturated NaCl solution (20 mL)
and dried with MgSO4. The solvent was removed in vacuo to yield
199 mg of the corresponding aldehyde, which was dissolved in tert-
butyl alcohol (10 mL) and in amylene (2.5 mL, 23.5 mmol).
NaH2PO4·2H2O (592 mg, 3.8 mmol) and NaClO2 (425 mg,
4.7 mmol), dissolved in a small amount of water, were added to the
reaction mixture, which was stirred for 12 h at room temperature.
The tert-butyl alcohol was removed in vacuo, the residue was cov-
ered with MTBE (50 mL), 2  HCl was added to the mixture
(10 mL) and the aqueous layer was extracted with MTBE (50 mL).
The combined organic layers were concentrated, K2CO3 solution
(1.2 g/50 mL water) was added and the aqueous layer was extracted
with MTBE (50 mL). The aqueous layer was covered with ethyl
acetate, 2  HCl was added and the layers were separated. The
aqueous layer was extracted with ethyl acetate (50 mL) three times.
The combined organic layers were washed with saturated NaCl
solution (50 mL), dried with Na2SO4 and the solvent was removed
in vacuo to yield 202 mg (98%) of the acid 10 as a colourless foam.
Rf = 0.25 (CHCl3/CH3OH/AcOH, 200:10:1). [α]D = –12.4 (c =
0.432, MeOH, T = 24 °C). 1H NMR (300 MHz, CD3OH): δ = 0.87
(d, J = 6.2 Hz, 3 H, CH3), 0.88 (d, J = 6.2 Hz, 3 H, CH3), 1.14–
1.41 (m, 4 H, 2Ј-HA, 5-H2, 3-HA), 1.41–1.52 (m, 2 H, 2Ј-HB, 3Ј-H),
1.84–1.92 (m, 3 H, 4-H2, 3-HB), 3.30–3.31 (m, 1 H, 6-H), 3.59–3.62
(m, 1 H, 1Ј-H), 3.97 (d, J = 11.7 Hz, 1 H, 2-H), 4.20 (t, J = 6.2 Hz,
1 H, Fmoc), 4.39 (dd, J = 10.6/6.4 Hz, 1 H, Fmoc), 4.54 (dd, J =
10.6/6.4 Hz, 1 H, Fmoc), 4.59 (d, J = 11.3 Hz, 1 H, NH), 7.27–
7.40 (m, 4 H, Ar), 7.67 (d, J = 7.5 Hz, 2 H, Ar), 7.79 (d, J = 7.5 Hz,
2 H, Ar) ppm. 13C NMR (75.5 MHz, CD3OD): δ = 22.3, 23.5
(CH3), 24.1 (C-4), 25.9 (C-3Ј), 28.6 (C-3), 30.3 (C-5), 42.5 (C-2Ј),
48.9 (Fmoc), 53.7 (C-1Ј), 67.0 (Fmoc), 77.3 (C-2), 80.9 (C-6), 120.9,
126.1/126.3, 128.1, 128.5, 142.8, 145.4 (Ar), 157.6 (Fmoc-C=O),
175.0 (COOH) ppm. HR-MS calcd. 437.2202 found [M+ Na+]
460.20996.
(2R,6S,1ЈS)-N-Benzyl-6-[1Ј-(tert-butoxycarbonylamino)-3Ј-methyl-
butyl]tetrahydropyran-2-carboxamide (12): The Boc-protected THP
amino acid 11 (50 mg, 0.158 mmol) and benzylamine (51 mg,
0.474 mmol) were dissolved in CH2Cl2 (2 mL) and cooled to 0 °C.
HOBt (61 mg, 0.395 mmol), dissolved in DMF (0.2 mL), was
added to the mixture. Subsequently, DIEA (0.07 mL, 0.395 mmol)
was added and stirred for 5 min. HBtU (90 mg, 0.237 mmol) was
added and the mixture was stirred for 2 h. Ethyl acetate/toluene
(20 mL, 1:1) was added to reaction mixture. 0.1  HCl (5 mL) was
added and the layers were separated. The aqueous layer was ex-
tracted with acetic acid/toluene (10 mL, 1:1). The combined or-
ganic layers were washed with saturated NaHCO3 solution
(20 mL), with saturated NaCl solution (20 mL), dried with Na2SO4
and the solvent was removed. The purification by column
chromatography (MTBE/PE, 1:3 Ǟ 1:2) yielded 33 mg (51%) of
the carboxamide 12. Rf = 0.14 (MTBE/PE, 1:1). m.p. 112 °C. [α]D
=
–14.3, [α]578 = –15.1, [α]546 = –16.6, [α]436 = –27.1, [α]365 = –30.3 (c
= 1.026, MeOH, T = 23 °C). 1H NMR (600 MHz, CDCl3): δ =
0.87 (d, J = 6.4 Hz, 6 H, CH3), 1.26 (m, 1 H, 2Ј-HA), 1.32 (m, 1
H, 2Ј-HB), 1.33 (m, 1 H, 3-Hax), 1.34 (m, 1 H, 5-Hax), 1.37 (s, 9 H,
Boc-H3), 1.55 (m, 1 H, 5-Heq), 1.56 (m, 1 H, 4-Hax), 1.64 (m, 1 H,
3Ј-H), 1.95 (m, 1 H, 4-Heq), 2.14 (m, 1 H, 3-Heq), 3.34 (m, 1 H, 6-
(2R,6S,1ЈS)-6-[1Ј-(tert-Butoxycarbonylamino)-3Ј-methylbutyl]tetra- H), 3.67 (m, 1 H, 1Ј-H), 3.86 (dd, J = 11.6/ 2.1 Hz, 1 H, 2-H), 4.41
hydropyran-2-carboxylic Acid (11): Oxalyl chloride (1.8 mL,
20.5 mmol) was dissolved in CH2Cl2 (10 mL) at –60 °C and DMSO
(d, J = 9.5 Hz, 1 H, THP-NH), 4.47 (m, 2 H, CH2NH), 6.91 (br. s,
1 H, Bn-NH), 7.23–7.32 (m, 5 H, Ar) ppm. 13C NMR (75.47 MHz,
2772
www.eurjoc.org
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2006, 2766–2776

Novel Building Blocks for Gramicidin-Hybrid Ion Channels
FULL PAPER
DMSO): δ = 22.6, 24.0 (CH3), 24.34 (C-4), 25.8 (C-3Ј), 29.1 (Boc- to 0 °C. HOBt (49 mg, 0.31 mmol), EDC (43 mg, 0.225 mmol) and
CH3), 30.9 (C-3), 42.6 (C-2Ј), 43.6 (CH2NH), 53.5 (C-1Ј), 79.1 (C- DIEA (0.047 mL, 0.297 mmol) were added and the mixture was
2), 79.8 (Cq), 81.6 (C-6), 128.5, 128.8, 129.5, 140.1 (Ar), 157.0 (Boc-
C=O), 189.6 (THP-C=O) ppm. HR-MS calcd. 404.26750 found
404.26750.
stirred for 12 h at room temperature. HBtU (15 mg, 0.039 mmol)
was added and stirred for 2 h. 0.1  HCl (2 mL) was added to the
reaction mixture and the solvent was removed. The aqueous layer
was extracted with ethyl acetate/toluene (1:1, 20 mL). The com-
bined organic layers were washed with saturated NaHCO3 solution
(10 mL), with saturated NaCl solution (10 mL), and dried with
MgSO4. The solvent was removed. Purification by column
chromatography (CHCl3/CH3OH, 40:1) yielded 71 mg (50%) of
pentapeptide 15. Rf = 0.53 (CHCl3/MeOH, 10:1). 1H NMR
(600 MHz, DMSO): δ = 0.63 (d, J = 6.5 Hz, 3 H, 3THP-δi-H3),
X-ray Structure Determination of 12: Compound 12 was measured
on a four-circle diffractometer. The crystal-structure analysis was
performed with the program package SHELXL-97.2. Crystal data:
C23H36N2O4, M = 404.54, monoclinic, P21/c, a = 9.789(2), b =
25.448(6), c = 10.088(2) Å, β = 110.13 (2)°, V = 2359.4(9) Å3, Z =
4, Dcalcd. = 1.139 gcm–1, (0.48×0.40×0.16) mm3, 15453 measured
reflections, 4253 independent (Rint = 0.0766), R1 = 0.0968, wR2 =
0.0821 (all data).
3
0.64 (m, 1 H, THP-δ-HA), 0.70 (d, J = 6.1 Hz, 3 H, Leu–δ–H3),
0.73 (d, J = 6.6 Hz, 3 H, Leu-δ-H3), 0.74 (m, 1 H, 3THP-δ-HB),
0.76 (d, J = 6.5 Hz, 3 H, 3THP-δi-H3), 0.77 (m, 1 H, 5Leu-γ-H),
CCDC-291471 contains the supplementary crystallographic data
for this paper. These data can be obtained free of charge from The
Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/
data_request/cif.
2
0.78 (d, J = 6.1 Hz, 3 H, Leu-δ-H3), 0.80 (m, 1 H, Leu-γ-H), 0.82
(d, J = 6.6 Hz, 3 H, Leu-δ-H3), 1.13 (m, 2 H, 3THP-γ-H2), 1.17
(m, 1 H, 3THP-βi-HA), 1.18 (m, 1 H, 3THP-γi-H), 1.19 (m, 1 H,
3THP-ε-Heq), 1.29 (m, 1 H, 2Leu-β-HA), 1.34 (m, 1 H, 3THP-βi-
HB), 1.43 (m, 1 H, 2Leu-β-HB), 1.47 (m, 1 H, 5Leu-β-HA), 1.54 (m,
Boc-THP-Trp-D-Leu-OMe (14): Cbz-W-l-OMe (13) (296 mg,
0.63 mmol) was dissolved in CH3OH (5 mL) and Pd/C (200 mg)
was added. The reaction mixture was degassed, flushed with argon
and hydrogen was bubbled through the solution/mixture. Pd/C was
separated by filtration through Celite and the filtrate was concen-
trated. The residue was dissolved together with the N-Boc-THP
amino acid 11 (100 mg, 0.31 mmol) in CH2Cl2 (2 mL). EDC
(89 mg, 0.465 mmol) and HOBt (71 mg, 0.465 mmol) were added.
After addition of NEt3 (0.2 mL, 1.6 mmol) the reaction mixture
was stirred for 12 h at room temperature; 0.1  citric acid (2 mL)
was added and the layers were separated. The aqueous layer was
extracted with ethyl acetate/toluene (1:1, 20 mL). The combined
organic layers were washed with saturated NaHCO3 solution
(10 mL), with saturated NaCl solution (10 mL), dried with Na2SO4
and the solvent was removed. The purification by column
chromatography (cyclohexane/ethyl acetate, 1:2) yielded 168 mg of
tripeptide 14 (84%). Rf = 0.54 (CHCl3/CH3OH/AcOH, 100:10:1).
5
3
1
1 H, Leu-β-HB), 1.66 (m, 1 H, THP-ε-Hax), 2.93 (m, 1 H, Trp-
β-HA), 3.05 (m, 1 H, 1Trp-β-HB), 3.09 (m, 2 H, 4Trp-β-H2), 3.27
(m, 1 H, THP-β-H), 3.58 (s, 3 H, O-CH3), 3.66 (m, 1 H, THP-ζ-
H), 3.86 (m, 1 H, THP-α-H), 4.15 (m, 1 H, Leu-α-H), 4.29 (m, 1
H, Leu-α-H), 4.38 (dd, J = 15.1/7.8 Hz, 1 H, Trp-α-H), 4.68 (dd,
J = 14.4/8.4 Hz, 1 H, 4Trp-α-H), 4.91 (d, J = 12.8 Hz, 1 H, Z-
CHA), 4.98 (d, J = 12.8 Hz, 1 H, Z-CHB), 7.02 (m, 6 H, Ar), 7.27
(m, 7 H, Ar), 7.41 (d, J = 9.5 Hz, 1 H, 3THP-NH), 7.47 (d, J =
8.3 Hz, 1 H, Trp-NH), 7.57 (d, J = 8.0 Hz, 1 H, Ar), 7.64 (d, J =
8.1 Hz, 1 H, Ar), 7.68 (d, J = 7.6 Hz, 1 H, Trp-NH), 8.28 (d, J =
3
3
3
2
5
1
4
1
2
5
7.9 Hz, 1 H, Leu-NH), 8.58 (d, J = 7.9 Hz, 1 H, Leu-NH), 10.79
(s, 1 H, indole-NH), 10.81 (s, 1 H, indole-NH) ppm. HR-MS calcd.
961.5313 found 962.5393 [M+H].
Cbz-Trp-D-Leu-cis-THP-Trp-D-Leu-Trp-NH-(CH2)2OTBDPS (17):
Cbz-Trp-NH-(CH2)2-OTBDPS 16 (50 mg, 0.8 mmol) was dis-
solved in CH3OH (3 mL) and Pd/C (49 mg) was added under ar-
gon. Hydrogen was bubbled through the reaction mixture. After
1 h Pd/C was filtered off via Celite and the filtrate concentrated in
vacuo. Methyl ester 15 (50 mg, 0.052 mmol) was dissolved in THF
(3 mL) and LiOH (26 mg/1 mL water, 0.624 mmol) was added. The
reaction mixture was stirred and monitored by TLC. Upon comple-
tion, 0.1  HCl (4 mL) was added to adjust pH = 2. Ethyl acetate
(20 mL) was added and the layers were separated. The aqueous
layer was extracted with ethyl acetate (10 mL). The combined or-
ganic layers were washed with saturated NaCl solution (20 mL),
dried with Na2SO4 and the solvent was removed in vacuo. The free
acid resulting from 15 (49 mg, 0.067 mmol) and the Cbz-depro-
tected amine resulting from 16 (32 mg, 0.067 mmol) were dissolved
in CH2Cl2 (5 mL) at 0 °C and HOBt (27 mg, 0.17 mmol) was
added. DIEA (0.029 mL, 0.17 mmol) was added and the mixture
was stirred for 5 min. After addition of HBtU (32 mg,
0.0832 mmol) the reaction mixture was stirred for 4 h. Ethyl acetate
(20 mL) and 0.1  HCl (10 mL) were added and the layers were
separated. The aqueous layer was extracted with ethyl acetate
(10 mL). The combined organic layers were washed with saturated
NaHCO3 solution (20 mL), with saturated NaCl solution (20 mL),
[α]D = –19.7, [α]578 = –20.8, [α]546 = –24.6, [α]436 = –47.8, [α]365
=
–89.0 (c = 0.456, CH2Cl2, T = 25 °C). 1H NMR (600 MHz,
CD3OH): δ = 0.70–0.86 (m, 6 H, 3Leu-δ-H3), 0.96–1.07 (m, 4 H,
1THP-γ-H2, 1THP-δ-H2), 1.09–1.51 (m, 10 H, 1THP-βi-H2, 3Leu-
γ-H, 1THP-ε-HA, 1THP-δi-H3), 1.41 (s, 9 H, Boc-H3), 1.63–1.98
1
1
2
(m, 2 H, THP-γi-H, THP-ε-HB), 3.10–3.32 (m, 2 H, Trp-β-H2),
3
1
3.40 (s, 3 H, O-CH3), 3.50–3.61 (m, 3 H, Leu-β-H2, THP-β-H),
1
1
3.68 (dd, J = 11.7/ 2.3 Hz, THP-ζ-H), 4.36–4.47 (m, 2 H, THP-
α-H, 3Leu-α-H), 4.64–4.73 (m, 1 H, 2Trp-α-H), 6.31 (d, J = 7.9 Hz,
2 H, THP-NH, 2Leu-NH), 6.92 (d, J = 7.5 Hz, 1 H, 3Trp-NH),
7.00–7.15 (m, 3 H, Ar), 7.28 (d, J = 7.9 Hz, 1 H, Ar), 7.60 (d, J =
7.5 Hz, 1 H, Ar), 8.15 (s, 1 H, indole-NH) ppm. HR-MS calcd.
628.3836 found 628.3836.
Cbz-Trp-D-Leu-cis-THP-Trp-D-Leu-OMe (15): Dipeptide 13
(200 mg, 0.43 mmol) was dissolved in THF (3 mL) and LiOH
(45 mg, 1 mmol) dissolved in water (1 mL) was added. The reaction
mixture was stirred at 20 °C until TLC control showed complete
turnover. 0.1  HCl was added to adjust pH = 2 and the layers
were separated. The aqueous layer was extracted with ethyl acetate
(20 mL). The combined organic layers were washed with saturated
NaCl solution (10 mL), dried with Na2SO4 and the solvent re-
moved. Boc-protected THP-tripeptide 14 (100 mg, 0.16 mmol) was and dried with Na2SO4. The solvent was removed. Purification by
dissolved in CH2Cl2 (4 mL) and TFA (1.5 mL) was added. The
reaction mixture was stirred for 1.5 h at room temperature. The
solvent was removed in vacuo, residual TFA was removed by aceo-
trope distillation with toluene in vacuo. The Boc-deprotected THP-
tripeptide was dissolved together with the corresponding acid pre-
pared from 13 (108 mg, 0.24 mmol) in CH2Cl2 (2 mL) and cooled
column chromatography (CH3OH in CHCl3 0.5%–1.25%) gave
24 mg (33%) of hexapeptide 17 as a colourless solid. Rf = 0.53
(CH3OH/CHCl3, 1:10). [α]D = +12.6, [α]578 = +13.2, [α]546 = +15.2,
1
[α]436 = +27.9, [α]365 = +32.2 (c = 0.348, CH2Cl2, T = 25 °C). H
5
NMR (600 MHz, CDCl3/CD3OH, 3:1): δ = 0.21 (m, 1 H, Leu-γ-
H), 0.48 (m, 6 H, 5Leu-δ-H3), 0.64 (m, 1 H, 2Leu-γ-H), 0.84 (m,
Eur. J. Org. Chem. 2006, 2766–2776
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
2773

S. Schröder, A. K. Schrey, A. Knoll, P. Reiß, B. Ziemer, U. Koert
FULL PAPER
12 H, Leu-δ-H3, THP-δi-H3), 0.87 (m, 1 H, Leu-β-HA), 0.94 (s,
11–12THP-α-H), 4.09 (m, 1 H, 14Leu-α-H), 4.14 (m, 1 H, Val-α-H),
4.15 (m, 1 H, 1Val-α-H), 4.20 (m, 1 H, Val-α-H), 4.22 (m, 1 H, Val-
2
3
5
9 H, tert-butyl-H3), 1.06 (m, 1 H, 3THP-γ-HA), 1.09 (m, 1 H,
2
4
3THP-ε-HA), 1.21 (m, 1 H, Leu-β-HA), 1.30 (m, 1 H, 5Leu-β-HB),
α-H), 4.26 (m, 1 H, Leu-α-H), 4.27 (m, 1 H, 10Leu-α-H), 4.34 (m,
3
3
5
9
1.34 (m, 1 H, THP-δ-HA), 1.37 (m, 1 H, THP-βi-HA),1.46 (m, 1 1 H, 3Ala-α-H), 4.36 (m, 1 H, Ala-α-H), 4.55 (m, 1 H, Trp-α-H),
H, THP-ε-HB), 1.56 (m, 1 H, 2Leu-β-HB), 1.69 (m, 1 H, THP-γi- 4.56 (m, 1 H, 15Trp-α-H), 4.64 (m, 1 H, 13Trp-α-H), 7.00–7.15 (m,
H), 1.76 (m, 1 H, 3THP-δ-HB), 1.77 (m, 1 H, 3THP-γ-HB), 1.79
11 H, Ar), 7.32–7.39 (m, 4 H, Ar), 7.48 (m, 1 H, EAN-NH), 7.68
(m, 1 H, 3THP-βi-HB), 2.83 (m, 1 H, 4Trp-β-HA), 3.01 (m, 1 H, (m, 1 H, 11–12THP-NH), 7.73 (m, 1 H, 13Trp-NH), 7.76 (m, 1 H,
3
3
4Trp-β-HB), 3.08 (m, 1 H, Trp-β-HA), 3.12 (m, 1 H, Trp-β-HA), Val-NH), 7.77 (m, 1 H, Val-NH), 7.83 (m, 1 H, 9Trp-NH), 7.84 (m,
6
1
7
1
3.15 (m, 2 H, EAN-α-H2), 3.17 (m, 1 H, Trp-β-HB), 3.19 (m, 1
1 H, Val-NH), 7.85 (m, 1 H, 15Trp-NH), 7.90 (m, 1 H, 14Leu-NH),
H, 6Trp-β-HB), 3.26 (m, 1 H, 3THP-β-H), 3.31 (m, 1 H, EAN-β-
7.92 (m, 1 H, 10Leu-NH), 7.97 (m, 1 H, Ala-NH), 8.02 (m, 2 H,
5
HA), 3.44 (m, 1 H, EAN-β-HB), 3.80 (m, 1 H, 3THP-ζ-H), 3.96 (m, 3Ala-NH, 4Leu-NH), 8.14 (m, 1 H, CHO), 8.21 (m, 1 H, 1Val-NH),
3
5
2
2
1 H, THP-α-H), 4.21 (m, 1 H, Leu-α-H), 4.27 (m, 1 H, Leu-α-
8.35 (m, 1 H, Gly-NH), 10.02 (s, 1 H, indole-NH), 10.13 (s, 1 H,
4
1
H), 4.47 (m, 1 H, Trp-α-H), 4.51 (m, 1 H, Trp-α-H), 4.52 (m, 1 indole-NH), 10.19 (s, 1 H, indole-NH) ppm. HR-MS calcd.
6
H, Trp-α-H), 6.67 (m, 1 H, indole-δ-H), 6.85 (d, J = 5.4 Hz, 1 H,
1Trp-NH), 6.86 (d, J = 5.6 Hz, 1 H, EAN-NH), 6.86 (m, 1 H,
indole-H), 7.03 (m, 1 H, indole-H), 7.02–7.15 (m, 9 H, Ar), 7.19–
7.25 (m, 8 H, Ar), 7.36–7.39 (m, 3 H, Ar), 7.45–7.47 (m, 2 H, Ar),
7.52–7.54 (m, 5 H, Ar) 7.57 (m, 1 H, 3THP-NH), 7.67 (d, J =
1779.04 found 1779.2.
NMR Spectroscopy and Peak Assignment for Restraint Generation:
NMR experiments were performed with a solution of THP peptide
17 (4.6 m in CDCl3/[D3]MeOH, 1:3) and with a solution of the
THP amide 12 (1.4 m in CDCl3), respectively. H2O proton reso-
nance in the CDCl3/MeOH mixture was presaturated for signal
suppression. The spectra were processed using the UXNMR pro-
gram; all resonances were calibrated to the residual CHCl3 proton
signal (δ = 7.24 ppm). The peak assignments and NOE peak vol-
ume calculations were done with SPARKY (www.cgl.ucsf.edu/
home/sparky). For the intraresidue resonance assignments of 17,
DQF-COSY(500 MHz) and TOCSY (500 MHz) spectra were used.
Sequential assignment was taken from the NOESY spectrum
(500 MHz, 300 ms mixing time). The high mixing time was neces-
sary to ensure a sufficient peak intensity. For 12, DQF-COSY
(600 MHz) and NOESY (600 MHz, 400 ms mixing time) spectra
were used (resonances, coupling constants and temperature coeffi-
cients see Table S1a and S1b, NOE peak lists see Table S2a and
S2b in the Supporting Information). NOESY peaks were integrated
using the Gaussian fit method. Coupling constants were taken di-
rectly from the corresponding 1D proton spectra (500 MHz, presat.
H2O suppression). The temperature coefficients for the amide pro-
tons of THP were calculated from a suite of 8 proton spectra at
different temperatures between 278 K and 313 K.
5
6
7.9 Hz, 1 H, Leu-NH), 7.77 (d, J = 7.9 Hz, 1 H, Trp-NH), 7.81
(d, J = 5.6 Hz, 1 H, 4Trp-NH), 8.28 (d, J = 7.4 Hz, 1 H, 2Leu-
NH), 9.39 (s, 1 H, indole-NH), 9.79 (s, 1 H, indole-NH), 9.84 (s, 1
H, indole-NH) ppm– HR-MS calcd. [M+Na] 1437.73 found
[M+Na] 1437.36.
form-V-G-A-l-A-v-V-v-W-l-THP-W-l-W-NH-CH2-CH2-OH
(4):
The Wang resin PS-PHB-Trp-Fmoc (0.63 mmol/g) from RAPP Po-
lymere GmbH was swelled in DMF. The deprotection of the Fmoc-
group was performed with 25% piperidine in DMF. The Fmoc-
protected amino acids (4 equiv.) and the THP amino acid
(2×2 equiv.) were coupled with 2.5 equiv. HOBt, 1.5 equiv. HBtU
and 2.5 equiv. DIEA in 1 mL DMF. The coupling time was 2:15 h.
The coupling of the THP amino acid building block 10 took 2×2 h
for complete reaction. After the coupling of the THP amino acid
the free amino groups were capped with 0.1 mL Ac2O, 0.04 mL
NEt3 in 1 mL DMF for 20 min. Formylated valine was used as the
final amino acid. After the last coupling, the resin was washed with
DMF, CH2Cl2, 30% CH3COOH and MeOH. The cleavage of the
peptide was carried out with 10% ethanolamine in DMF at 55 °C
in 48 h. The resin was filtered and washed with MeOH, CH2Cl2
and TFE. The filtrate was concentrated in vacuo to an approximate
volume of 2 mL and the product was precipitated with H2O. The
precipitate was centrifuged (4 °C, 14000 rpm, 2 h). The supernatant
was decanted and the residue dissolved in MeOH, concentrated in
vacuo and precipitated by addition of H2O. The suspension was
frozen in liquid nitrogen and lyophilised. The purification was per-
formed by column chromatography (CHCl3/MeOH/HCOOH,
100:3:7–100:5:7) and gave 24 mg (43%) of 11–12-THPgA (4). Rf =
0.28 (CHCl3/CH3OH/HCOOH, 100:7:7). 1H NMR (600 MHz,
CDCl3/CD3OH, 1:1): δ = 0.67–0.89 (m, 43 H, 2× 10Leu-δ-H3, 2×
14Leu-δ-H3, 8× Val-γ-H3, 10Leu-γ-H, 2× 4Leu-δ-H3), 0.90 (m, 6 H,
11–12THP-δi-H3), 0.93 (m, 1 H, 14Leu-γ-H), 1.05 (m, 1 H, 14Leu-β-
Computational Procedure: All calculations were performed on a
SGI Origin server (4x R12000). For practical reasons, both struc-
tures were calculated with an N-terminal acetamide group instead
of a carbamate group (12 Ǟ 22, 17 Ǟ 23). For 17, the C-terminal
TBDPS group was omitted. Parameterisation of the THP, benzyl,
and ethanolamine building blocks: Energies and ESP charges were
calculated using GAMESS (6-313G* basis set). The RESP fit was
performed using a two-step procedure described by Bayley et al.[17]
For all structures, standard AMBER atom types and standard
AMBER parameters could be assigned.
Structure Calculations: Structures were calculated using AM-
BER6[18] (sander_classic, 2 processors) in a restrained 40 ps simu-
HA), 1.22 (m, 2 H, 11–12THP-ε-HA, 11–12THP-γ-HA), 1.24 (m, 1 H, lated annealing (SA) procedure (300 Ǟ 1500 Ǟ 500 Ǟ 0 K) in
10Leu-β-HA), 1.28 (m, 1 H, 14Leu-β-HB), 1.34 (m, 1 H, Leu-γ-H), vacuo with subsequent restrained minimisation (for the exact con-
4
5
3
1.35 (m, 3 H, Ala-β-H3), 1.39 (m, 3 H, Ala-β-H3), 1.46 (m, 3 H,
ditions see Supporting Information). A total of 100 structures was
11–12THP-βi-H2, 11–12THP-γ-HB), 1.47 (m, 1 H, 11–12THP-δ-HA), sampled using the same start structure and different random seeds
1.52 (m, 1 H, 10Leu-β-HB), 1.56 (m, 1 H, Leu-β-HA), 1.58 (m, 1 for every SA run. All stereogenic carbon atoms as well as amide
4
H, 11–12THP-γi-H), 1.63 (m, 1 H, 4Leu-β-HB), 1.78 (m, 1 H,
11–12THP-δ-HB), 1.82 (m, 1 H, 11–12THP-ε-HB), 2.07 (m, 1 H, Val-
β-H), 2.10 (m, 2 H, 1Val-β-H, Val-β-H), 2.11 (m, 1 H, Val-β-H),
bonds were constrained using torsional restraints (fc = 50 kcal/
mol). The numbers of the used distance and J coupling restraints
as well as the force constants (fc) are summarized in Table 1. The
distances were calculated from the NOE peak volumina applying
the r6 relationship and using the distance between two geminal
3.15 (m, 1 H, 13Trp-β-HA), 3.17 (m, 3 H, Trp-β-H2, 15Trp-β-HA),
9
3.20 (m, 1 H, 13Trp-β-HB), 3.25 (m, 2 H, EAN-α-H2), 3.37 (m, 1
H, 15Trp-β-HB), 3.41 (m, 1 H, 11–12THP-β-H), 3.45 (m, 1 H, EAN- methylene protons (1.8 Å) as a reference. For the THP amide 12,
2
β-HA), 3.54 (m, 1 H, EAN-β-HB), 3.76 (m, 1 H, Gly-α-HA), 3.82
the ring protons at THP-Cε were used, for the THP peptide 17 the
protons on Trp4-Cβ were evaluated. Because of the high NOESY
(m, 1 H, 11–12THP-ζ-H), 3.95 (m, 1 H, Gly-α-HB), 4.00 (m, 1 H,
2
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© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2006, 2766–2776

Novel Building Blocks for Gramicidin-Hybrid Ion Channels
FULL PAPER
mixing time, all calculated distances had to be checked for consist-
ence manually to avoid spin-diffusion artefacts. The lower bound-
ary was set to d–0.3 Å, the upper boundary to d+0.3 Å. The
boundaries for the J coupling constants were set to J–0.5 and
J+0.5, respectively.
+200 mV and –200 mV were applied; the trans electrode was vir-
tually grounded. Thereupon current measurents were smoothed by
a 1 kHz low-pass filter, amplified (Patch Clamp amplifier AXO
Patch 200B, Axon Instruments) and digitized (5 kHz, DigiData A/
D converter). Finaly, data was filtered by a Gauss filter with a low-
pass frequency of 50 Hz.
Table 1. Number of distance and J coupling restraints (N) and their
force constants (fc) for the THP amide 12 and the THP peptide
17.
Supporting Information (see also the footnote on the first page of
this article): Table S1a, proton chemical shifts, coupling constants,
and temperature coefficients for 17 in CDCl3/[D3]MeOH (3:1).
Table S1b, proton chemical shifts for 12 in CDCl3. Table S2, assign-
ments and volume of the NOESY peaks for 17 in CDCl3/[D3]-
MeOH (3:1). Graphic, C-atom numbering used for the NMR-
based structure calculation.
12
N
17
N
fc[kcal/mol]
fc[kcal/mol]
Distance restraints
Intraresidue
Sequential
24
50
23
16
6
100
Medium range
J coupling restraints
1
8
–
–
10
Acknowledgments
The structure calculation proceeded in an iterative process, in which
H-bond length restraints for amide protons which are known to be
H-bonded from the temperature-dependent NMR experiments
were added. The first run was performed without any H-bond re-
straints. After analysis of the resulting structures, several combina-
tions of H-bonds were tested. The final H-bond length restraints
are summarized in Table 2.
Generous support by the Deutsche Forschungsgemeinschaft (FG
495) and the Fonds der Chemischen Industrie is greatfully ac-
knowledged.
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C=O
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Trp6
Ace
Leu2
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THP3
Trp4
2.2
3.2
3.2
3.2
3.2
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averaged using the CARNAL module (AMBER7). The fitting pro-
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electrodes, coated with AgCl by electrolysis in 0.1  HCl) a poten-
tially existing offset was corrected. A membrane was generated over
the cuvette aperture (ø = 200 or 150 µm) by painting. For this pur-
pose, 1 µL of the lipid solution (25 mg/mL, in n-decane) was given
onto a teflon-encased metal loop and painted over the aperture,
until a membrane formed (detected by current flow between both
chambers falling to 0 pA, optical evaluation by the typical inter-
ference). After measuring the capacity of the membrane (specific
membrane capacity 0.4 µF/cm2) a solution of the ion channel fom-
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both compartments. On the cis electrode voltages between
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www.eurjoc.org
2775

S. Schröder, A. K. Schrey, A. Knoll, P. Reiß, B. Ziemer, U. Koert
FULL PAPER
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Received: January 27, 2006
Published Online: April 10, 2006
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© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2006, 2766–2776