Solution phase synthesis and purification of the minigramicidin ion channels and a succinyl-linked gramicidin

Article abstract of DOI:10.1016/S0040-4020(02)00179-5

Peptides with alternating D- and L-configured residues as found in the natural ion channel active peptide gramicidin A (gA) are important building blocks for artificially engineered ion channels. We detail an optimised solution phase synthesis employing a

Full text of DOI:10.1016/S0040-4020(02)00179-5

TETRAHEDRON
Pergamon
Tetrahedron 58 ,2002) 2789±2801
Solution phase synthesis and puri®cation of the minigramicidin
ion channels and a succinyl-linked gramicidin
Hans-Dieter Arndt,a Andrea Vescovi,a Anna Schrey,a Jochen R. Pfeifera,b and Ulrich Koerta,b,p
a
È
Institut fur Chemie der Humboldt-Universitat zu Berlin, Brook-Taylor-Str. 2, D-12489 Berlin, Germany
Fachbereich Chemie, Philipps-Universitat Marburg, Hans-Meerwein-Str., D-35032 Marburg, Germany
È
b
È
Received 18 September 2001; revised 17 December 2001; accepted 18 February 2002
AbstractÐPeptides with alternating d- and l-con®gured residues as found in the natural ion channel active peptide gramicidin A ,gA) are
important building blocks for arti®cially engineered ion channels. We detail an optimised solution phase synthesis employing a convergent
assembly of peptide building blocks, giving access to the minigramicidines and a succinyl linked gA derivative on preparative scale.
Moreover, the minigramicidines were investigated for secondary structure formation by CD-spectroscopy, which was discovered to be
medium and capping-group dependent. A parallel, left-handed double-b-helix seems to be generally favoured in organic solvents for the
minigramicidines. q 2002 Elsevier Science Ltd. All rights reserved.
1. Introduction
A possible solution of this general problem is to focus on
unimolecular channels, i.e. to avoid intermolecular associa-
tion equilibria, and to use a lead conformation with a de®ned
folding motif, which will allow to study the action of
deliberately shaped molecular interactions.
Ion channels allow the passive transport of ions through a
phospholipid bilayer.1 The driving force for the channel
transport can be a concentration gradient or an electric
potential. In line with progress in the structural understand-
ing of biological ion channels2 stands the active area of
synthetic ion channels.3 Two different approaches to
synthesise ion channels were investigated in the past: a
non-peptidic one,4 using ether motifs, and a peptidic
approach.5 Recently hybrid channels have been reported,
which combine peptides with synthetic subunits.6
With these requirements in mind, we decided to explore
gramicidin A ,gA) as a structural lead ,Fig. 1). This lipo-
philic peptide with alternating d- and l-con®gured residues
is synthesised by the bacterium bacillus brevis in its
sporulation phase.8 The biological function of this anti-
bacterial peptide remains unclear, but its potency for ion
channel formation in cell membranes together with its
commercial availability has made this compound the most
prominent model substance for ion channel studies to date.9
In a membrane-like environment gA folds into a head-to-
head associated dimer of two right handed, single stranded
b-helices with a pitch of 6.3 residues per turn.10a This ion-
channel active conformation is populated in the
membrane,10b±d while in organic solvents gA forms a multi-
tude of generally dimeric b-helical species.10e
Our interest in ion channels results from the possibility to
understand a functional molecule on the basis of its
molecular structure. The functional analysis of ion channels
by conductivity measurements is well established.7 In
contrast, many questions remain unanswered concerning
the nature of the distinct ion channel properties observed
for the synthetic channels described so far. In our view, this
is most likely due to the delicate self-assembly nature of
many of the arti®cial systems reported. Many of them do
need a membrane environment to assemble properlyÐbut
the fraction of properly assembled molecules may be
minute. These facts severely complicate the characterisation
of the channel active conformation by direct methods such
as NMR or X-ray crystallography.
A covalent linker joining two N-termini of the d-l-peptide
strands should severely con®ne the conformational space of
the molecule. This will avoid the entropic cost for the head-
to-head dimerisation of gA and decrease the portion of
solvent-exposed backbone amide bonds. Considering the
spatial structure of the gA dimer,10a a C4-linker should be
the optimal substitute for two formamides. The NMR-
structure of succinyl-linked gA has been determined in
SDS-micelles and its conformation was found to be similar
to the gA dimer in this environment.11a Schreiber et al. have
employed more elaborate C4-linkers to connect two gA
moieties and studied their in¯uence on the channel
Keywords: biologically active compounds; peptides and polypeptides; ion
channels; gramicidin; b-helix; CD-spectroscopy.
Corresponding author. Address: Fachbereich Chemie, Philipps-
È
Universitat Marburg, Hans-Meerwein-Str., D-35032 Marburg, Germany.
Tel.: 149-6421-282-6970; fax: 149-6421-282-5677;
p
e-mail: koert@chemie.uni-marburg.de
0040±4020/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020,02)00179-5

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H.-D. Arndt et al. / Tetrahedron 58 42002) 2789±2801
Figure 1. Molecular structure of gA and the N-terminally linked target structures 1±4. The a-/d-heteropeptides 6 and 8 employ ether-d-amino acids and
deliberately chosen fragments from the gA sequence.6a,b
behaviour in some detail,11b but further information on
covalently linked d-l-peptide ion channels is scarce.
with the advent of solid-phase peptide synthesis. Optimised
protocols for the solid-phase assembly of gA are available.14
But after some experimentation, we decided to refocus on a
fragment coupling strategy for our d-l-peptide targets due
to three main reasons:
In this report we give a detailed account on the high-
yielding synthesis and puri®cation of N-terminally succinyl-
linked gramicidin 1 and the membrane-selective mini-
gramicidines 2±4,12 which also provides important building
blocks for the incorporation of the ether-d-amino acids 5
and 7 into the backbone-modi®ed a-/d-heteropeptides 6
and 8.6a,b
1. It is by no means trivial to ensure that an observed
speci®c ion channel property is not caused by an
impurity. Due to the single-molecule resolution of the
electrophysiological measurement techniques, highly
active minor components can be seriously misleading.
A coupling process employing pure fragments can be
scrutinised by all analytical techniques available and
separation problems, e.g. with co-eluting miscoupling
products are minimised. This will become especially
important for larger d-l-peptide fragments ,.10), as
these generally do not behave benign on both normal
and reversed phase columns.
2. Synthetic considerations
In a seminal contribution, Sarges and Wittkopcon®rmed the
molecular structure of gA by total synthesis using fragment
couplings in solution.13a This strategy has later been
explored to provide gA-analogues,13b but was abandoned
Scheme 1. Retrosynthetic disconnection of target 1 leading to the strategic peptide fragments.

H.-D. Arndt et al. / Tetrahedron 58 42002) 2789±2801
2791
Scheme 2. Synthesis of the undecamer 11. ,a) HOBt ,1.5 equiv.), NEt3 ,1.5 equiv.), EDC ,1.25 equiv.), CH2Cl2; ,b) methylester deprotection: 2.5 equiv.
LiOH, THF/H2O 3:1, 08C; Z-groupcleavage: 0.02 M, 15 wt% Pd/C ,5%), H ,1 bar); coupling: HOBt ,2.5 equiv.), DIEA ,2.5 equiv.), HBTU ,1.5 equiv.),
CH2Cl2/DMF 10:1, 0±208C.
2
2. Larger amounts of materials have to be provided for
structural investigations or studies with living cells.
Solution phase chemistry is competitive on scale,
especially if the amounts of resin, coupling reagents
,.4 equiv.), d-amino acids ,4 equiv., double couplings),
and solvents are taken into account, which are routinely
employed in solid phase synthesis.14
3. A convergent retrosynthetic strategy will provide a
library of fragments, which can be used in multiple, inter-
changed or substituted by new entities when they are
assembled in a block-wise fashion. This opens the door
for hybrid channels like 6 or 8.6a,b
Scheme 2. The dipeptides 21±23 and ethanolamine-tagged
24 were synthesised from the respective monomers15 15±20
in very good yield and purity using HOBt/EDC-mediated
couplings in CH2Cl2. Best results were obtained when
the initial reaction temperature was kept at 08C. The
Z-deprotection of all fragments was accomplished by
hydrogenolysis, but the polyaromatic compounds 14, 25
and 11 behaved somewhat capricious in line with early
®ndings.13a Generally the proper choice of catalyst ,Degussa
E101 NO/W), dilution ,0.02 M in MeOH) and purity of
starting material proved to be essential for a clean conver-
sion, in some cases slightly elevated temperatures ,30±
408C) and cosolvents ,THF, DMF or H2O) were bene®cial.
Our retrosynthetic strategy is shown in Scheme 1. The end-
capped succinyl-linked peptide dimer 1 is disconnected into
a desymmetrised `clamp' 9, the interconnecting dipeptide
10, and the undecamer 11. This key compound will be
assembled from three fragments of equal size 12, 13 and
14. All peptide fragments employ terminal Z/-OMe protec-
tion due to their availability and mild cleavage conditions,
and they allow leaving the Trpside chains unprotected. The
ethanolamine will carry a bulky, lipophilic TBDPS-
protecting group to aid solubility and ease chromatographic
puri®cation of the intermediates.
3. Results and discussion
3.1. Synthesis of the minigramicidines
Figure 2. HPLC-trace of heptamer 25: lˆ280 nm, method M5, but
50!100% B in 25 min.
The synthesis of the key undecapeptide 11 is comprised in

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H.-D. Arndt et al. / Tetrahedron 58 42002) 2789±2801
gramicidin 27 ,Scheme 3). This was desilylated with HF
in CHCl3/CH3CN to give minigramicidin 28 in 59% yield
after silica gel chromatography. For the linked minigrami-
cidin, amine 26 was derivatised with succinic anhydride, the
resulting acid puri®ed and homocoupled with a second
equivalent of 26 using HATU/HOAt17 in CH2Cl2/DMF to
provide dimer 2 in excellent yield. MeOH as solvent was
found to give the cleanest deprotection ,HPLC-monitoring),
and deprotected minigramicidin 3 could be obtained in a
gratifying 90% yield. Interestingly, the linked minigrami-
cidines 2±4 can be separated by FCC under optimised
conditions, so the asymmetrically protected ion channel 4
could be isolated if the reaction was quenched at 50±70%
conversion.
Figure 3. HPLC-trace of undecamer 11: lˆ280 nm, method M5, but
70!100% B in 25 min.
In the ®rst instance, reaction monitoring and preparative
puri®cation of 11, 27, 28, 2±4 were not straightforward.
Schreiber et al. have given a procedure to purify gA by
FCC on silica gel using CHCl3/MeOH doped with HOAc
and H2O.18 After a careful examination of solvent systems,
mixtures of CHCl3, MeOH ,upto 10 vol%) and anhydrous
HCOOH ,up to 10 vol%) proved to have superior resolving
power. They are easy to adjust and ensured good recovery
on preparative scale for all the compounds described here,
and gA itself. The HCOOH was removed by extraction with
aqueous buffer before evaporation. With this optimised
puri®cation protocol, linked minigramicidin 2 can be
provided on gram scale in .95% purity ,600 MHz 1H
NMR) without the need for large scale HPLC puri®cation.
Preparative RP-HPLC puri®cation of the ion channels 2±4
was frequently accompanied by signi®cant losses of
material and thus was only used to deliver high-purity
material for ion-channel studies on the 1±5 mg scale.
Neutral reaction conditions were preferred, as acidic hydro-
genation- or transfer-hydrogenation protocols delivered less
pure, coloured products. The Z-deprotection of heptamer 25
was best not driven to completion in order to minimise side
product formation. In contrast, the LiOH-mediated16
saponi®cation of the methylesters performed smoothly
with no detectable epimerisation even for the hindered
tetramer 12.
After some experimentation, a fragment coupling protocol
was established employing HBTU/HOBt/DIEA in CH2Cl2/
DMF-mixtures, which provided superior reactivity for these
peptides even at 08C. Epimerisation of residues was
generally weak ,,3%), only the dif®cult coupling leading
to 11 was hampered by a decreased reaction rate and con-
comitant side product formation ,majorly the 4Val-epimer).
All oligomers 12±14, 25 and 11 were obtained dia-
3.2. CD-spectroscopy
1
steromerically pure after careful FCC as evidenced by H
NMR, MS and HPLC ,Figs. 2 and 3). Following the pro-
cedure outlined in Scheme 2, multigram quantities of the
stable key intermediate 11 could be prepared.
To gain insight into the propensity of the minigramicidines
for secondary structure formation, CD-spectra of ion
channels 2±4 were recorded in several organic solvents
,1025 M). Each different conformer of gA including the
ion-channel active conformation gives rise to a distinct
CD-spectrum,10e so conformational preferences may be
concluded by comparison. The bis-TBDPS protected
To proceed towards the minigramicidines,12 undecamer 11
was deprotected and the resulting amine 26 formylated
,HCOOH, DIEA, EDC) to yield TBDPS-protected mini-
Scheme 3. Preparation of the minigramicidines 2±4. ,a) 25 wt% Pd/C ,5%), H2 ,1 bar), MeOH/DMF 10:1, 408C; ,b) HCOOH ,10 equiv.), DIEA ,10 equiv.),
cat. HOBt, EDC ,6 equiv.), CHCl3, 0!208C; ,c) ,i) succinic anhydride ,10 equiv.), pyridine, CH2Cl2/DMF 3:1, ,ii) 26 ,1 equiv.), HOAt ,4 equiv.), DIEA
,3 equiv.), HATU ,2.5 equiv.), CH2Cl2/DMF 3:1, 0!208C; ,d) 2.5 vol% HF in CH3CN/CHCl3 1:1; ,e) 2.5 vol% HF in CH3CN/MeOH 1:1.

H.-D. Arndt et al. / Tetrahedron 58 42002) 2789±2801
2793
Figure 4. CD-spectra of diprotected minigramicidin 2 ,1025 M) in several
organic solvents; Ð CH3CN, ´´´ MeOH, - - - TFE, ± ± ± n-hexane/i-PrOH
10:1, ±´±´± i-PrOH.
Figure 6. CD-spectra of minigramicidin 2 ,1025 M) in i-PrOH/H2O 99:1 in
the presence of different metal salts ,1 mM); Ð i-PrOH/H2O 99:1, ± ± ±
KPF6, ´´´ CsCl, ±´±´± ZnBr2, - - - Ca,ClO4)2.
compound 2 shows different types of secondary structure,
and the conformations may be addressed by the choice of
solvent ,Fig. 4). In MeOH, a mixture of conformations is
present, which can also be concluded from the extremely
complex 1H NMR spectra in this solvent. A presently
unknown right-handed species is favoured in i-PrOH,
which can be concluded from the positive ellipticity at
230 nm. This conformation is also signi®cantly populated
in n-hexane ,doped with i-PrOH for solubilisation). TFE is
known to deaggregate d-l-peptides, so the spectrum
obtained for 2 is somewhat similar to gA and is thought to
represent a mixture of random-coil and right-handed b4.4-
helical species. But in acetonitrile and in aqueous i-PrOH
,not shown), a left-handed parallel double helix is formed
exclusively,19 which indicates a symmetric pairing of two
subunits.
gates may be the reason for the losses associated with the
preparative RP-HPLC-puri®cation of this compound, where
strong denaturing conditions ,low pH, TFE or DMSO as
solvents) cannot be applied.
The in¯uence of metal ions on the conformation of 2 was
brie¯y examined ,Fig. 6). In i-PrOH ,containing 1% H2O to
solubilise the salts), the presence of monovalent cations
,1 mM) did not signi®cantly change the CD-spectrum, and
the LEWIS-acidic Zn21 also did not have any in¯uence. In
contrast to the former, the presence of Ca21 led to a
characteristic change in the spectrum. Its appearance
resembles the left-handed parallel double helix found for
2 in CH3CN, but the doubled negative ellipticity at
208 nm and decreased positive ellipticity at 195 nm indicate
moderate structural deviations from this type of secondary
structure.19 A lowered symmetry or differences in helical
pitch may account for the difference.
The mono-deprotected compound 4 has rather weak ellipti-
cities in organic solvents and probably exists mainly as a
mixture of conformers ,not shown). But the complete
desilylation in the ion-channel 3 reduces the conformational
freedom ,Fig. 5): in all the solvents examined for 3, a left-
handed parallel double helical species predominates
,n-hexane) or is the only one present ,CH3CN, i-PrOH
and MeOH). Only TFE leads as expected to a merely
random-coil conformation. The propensity to form aggre-
3.3. Synthesis of the succinyl-linked gA )1)
In our initial attempts to prepare the succinyl-linked grami-
cidin 1, we tried to assemble the two pentadecapeptide
fragments 29 and 30 as shown in Scheme 4. To our surprise,
we were hardly able to isolate any coupling product under
the various conditions we tried. The HATU/HOAt condi-
tions proven for the minigramicidines also did not lead to
appreciable product formation, as evidenced by a HPLC-
trace of the crude product mixture ,Fig. 7, dotted trace).
1
For whatever reason, the coupling of fragments to Val
seemed to be strongly retarded and we decided to shift the
Figure 5. CD-spectra of desilylated minigramicidin 3 ,1025 M) in several
organic solvents; Ð CH3CN, ´´´ MeOH, - - - TFE, ± ± ± n-hexane/i-PrOH
10:1, ±´±´± i-PrOH.
Scheme 4. Attempted synthesis of succinyl-linked 1 by the union of two
pentadecapeptide fragments 29 and 30.

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H.-D. Arndt et al. / Tetrahedron 58 42002) 2789±2801
puri®cation of compound 1 by preparative HPLC was dif®-
cult, because for an acceptable peak shape in the HPLC high
column temperatures had to be applied ,.508C), which
were not applicable for preparative purposes. After a combi-
nation of preparative TLC and size-exclusion chromato-
graphy, 1 was obtained in 40% yield with .95% purity
,1H NMR). 5 mg were puri®ed further by repetetive
preparative HPLC for ion channel analysis, until the ion
channel activity did not increase further.
Compound 1 forms ion channels, which do hardly close
again once they are open ,Fig. 8). This observation and
the minute amounts needed ,,10214 M added to the bath
solution) to induce ion channel formation indicate, that a
chemically stable, simple succinate linker is fully suf®cient
for joining two gA units to form a stable, long-lived ion
channel in the membrane.
Figure 7. Overlay of the HPLC-traces of the attempted ,dotted line) and
productive ,solid line) coupling experiments leading to 1 after gross
removal of the coupling reagents by aqueous extraction ,remainings at
3.6 min). lˆ280 nm, method M3, column temperature 508C.
4. Conclusions
2
3
®nal coupling position between Gly and Ala as shown in
Scheme 1.
For the synthesis of d-l-peptide ion channels a fragment
coupling strategy was optimised. Solution-based chemistry
ensured maximum purity of the products and gives access to
the minigramicidines on a gram scale. The ¯exibility of this
building block approach has already been demonstrated by
the combination of ether-d-amino acids with d-l-peptide
fragments.6a,b In the synthesis of the linked gramicidin 1,
the disconnection into two equally sized gA-subunits was
identi®ed as a dead end and successfully overcome by a
productive 1315113 clamping strategy. Using a simple,
chemically robust succinate linker, d-l-peptide ion channels
of membrane spanning dimension are obtained in excellent
yield and purity.
To this end, the undecamer amine 26 was elongated with the
dimer 1020 to give the tridecamer 32 ,Scheme 5). The clamp
9 was assembled from 3121 by deprotection, succination and
coupling with H-Ala-Gly-OMe22 under standard conditions.
Even this coupling was extremely slow, supporting our
1
notion of Val being especially unreactive. Compound 9 is
highly insoluble and had to be isolated by recrystallisation
from boiling i-PrOH.
The union of 32 with the clamp 9 proceeded uneventfully to
deliver the octadecamer 33 in 63% puri®ed yield, which was
coupled to the tridecamer 32 using the conditions previously
established for the minigramicidines. In the event, the
starting materials were consumed, furnishing the linked
gramicidin 1 as the main product ,Fig. 7, solid trace). The
CD-spectroscopy in organic solvents revealed, that the
conformational space for the linked minigramicidines 2±4
appears to be much more con®ned than for natural gA. If the
Scheme 5. Productive synthesis of succinyl-linked gramicidin 1. ,a) ,i) CH2Cl2/TFA 4:1, ,ii) succinic anhydride ,1.1 equiv.), CH2Cl2/DMF 5:1, 81%,
,iii) H-Val-Gly-OMe ,1.25 equiv.), HOBt ,1.5 equiv.), NEt3 ,1.5 equiv.), EDC ,1.25 equiv.), 208C, 18 h, 70%; ,b) 26, HOBt ,1.5 equiv.), NEt3
,1.5 equiv.), EDC ,1.25 equiv.), 0!208C; ,c) cleavage of Z- and benzyl-ester groups: 0.02 M in MeOH, 15 wt% Pd/C ,5%), H2 ,1 bar), 408C; coupling:
HOBt ,2.5 equiv.), DIEA ,2.5 equiv.), HBTU ,1.5 equiv.), CH2Cl2/DMF 10:1, 208C; ,d) methylester saponi®cation: LiOH ,2.5 equiv.), THF/H2O 3:1; Z-group
cleavage according to ,c); coupling: HOAt ,4 equiv.), DIEA ,3 equiv.), HATU ,2.5 equiv.), CH2Cl2/DMF 3:1, 208C.

H.-D. Arndt et al. / Tetrahedron 58 42002) 2789±2801
2795
Column Macherey and Nagel C8, 250£4 mm, A: H2O, B:
CH3CN, 308C. Method 5 ,M5): Column Macherey and
Nagel C8, 250£4 mm, A: H2O, solvent B: CH3CN/i-PrOH
2:1, 80% B!100% B in 25 min, 0.7 mL/min, 308C. Reten-
tion times are uncorrected.
CD-spectroscopy. JASCO-710 spectropolarimeter with a
N2-¯ow of 2 L/min. Spectrum acquisition at 258C, 2 mm
cuvette. Seven accumulations, background correction and
FFT-based smoothing were performed using the JASCO
software package.
Fig. 8. Trace of a single channel conductance experiment in a black lipid
bilayer ,soy bean lecithin ,symmetrical 1 M CsCl, 100 mV)). Each stepin
the diagram corresponds to a channel opening event. Open channels did not
close any more during the period of observation ,30 min).
MALDI-TOF mass spectrometry. ,1) Sample preparation:
sample ,10±100 mM in MeOH, 10 mL) and matrix solutions
,50 mM 2,5-dihydroxybenzoic acid or 1,8,9-trihydroxy-
anthracene in CHCl3, 10 mL) were thoroughly mixed.
Then 1 mL of the solution was applied on a stainless-steel
target plate and air dried. ,2) Mass determination: Bruker
Re¯exe with delayed extraction in the re¯ectron mode
using external calibration.
termini are capped, a medium dependency of the secondary
structure was evident. Generally, left-handed double-
stranded b-helical species are favoured in organic solvents,
especially for the uncapped compound 3.
In our hands the minigramicidines can be provided in a pure
state in preparative amounts and are potent, unimolecular
ion channel formers in lipid bilayers.12 If the constructs get
larger, reliable puri®cation becomes tedious and low
yielding. Thus the minigramicidines are deemed to be
most valuable subunits for the construction of more
elaborate, unimolecular ion channel constructs.
6.2. General procedures
The phrase `aqueous work-up' refers to an extraction of the
organic layer with sat. NaHCO3-, 1 M NaHSO4-, deionised
H2O and sat. NaCl ,`brine') solutions consecutively ,a third
of the volume of the organic layer each), followed by drying
with Na2SO4 and evaporation.
5. Abbreviations
Thin layer chromatography ,TLC) and ¯ash column
chromatography ,FCC) were performed on silica gel
,Merck Si60, 40±60 mm). Columns for FCC were slurry-
packed. For dif®cult separations, the silca-gel slurry was
sonicated ,1 min) before packing. TLC-spots were
visualised by UV-light and by staining with 2% moly-
bdophosphoric acid in EtOH, 1% ninhydrine in EtOH or
2% p-methoxy-benzaldehyde in EtOH followed by 20%
H2SO4, and heating.
Amino acid residues, for the schemes the common one letter
code was used, where capital letters denote l- and small
letters d-con®guration; V, l-Val; G, Gly; A, l-Ala; l,
d-Leu; v, d-Val; W, l-Trp; SDS, sodium-dodecyl sulfate;
Z, carboxy-benzyl; TBDPS, t-butyl-diphenyl-silyl; HOBt,
1-hydroxy-benzotriazole monohydrate; EDC, N-ethyl-N0-
,3-dimethylamino-propyl)-carbodiimide
hydrochloride;
HBTU, O-,benzotriazol-10-yl)-1,1,3,3-tetramethyl-uronium
hexa¯uorophosphate; DIEA, di-iso-propyl-ethylamin;
HATU, O-,70-aza-benzotriazol-10-yl)-1,1,3,3-tetramethyl-
uronium hexa¯uorophosphate; HOAt, 1-hydroxy-7-aza-
benzotriazole; FCC, ¯ash column chromatography; TFE,
tri¯uorethanol; DMSO, dimethylsulfoxide; DMF, dimethyl-
formamide; sat., saturated solution.
Methylester deprotection ,GP 1). The respective ester was
dissolved in THF ,0.05 M) and cooled to 08C. 2.5 equiv.
LiOH´H2O was added as an aqueous solution ,1:4 v/v)
and stirred until conversion was complete. The mixture
was then neutralised with 1 M NaHSO4-solution ,pH-
paper), the THF was evaporated and the residue partitioned
between EtOAc ,0.02 M) and 1 M NaHSO4 ,2:1 v/v). The
aqueous layer was extracted twice with EtOAc ,equal
volume), then all organic fractions were pooled and washed
with brine ,1:3 v/v), dried with Na2SO4, evaporated and
dried to constant weight ,0.01 Torr). The crude carboxylic
acid was found ,TLC, NMR, HPLC) to be suf®ciently pure
in all cases.
6. Experimental
6.1. Analytical techniques
NMR-spectroscopy. Bruker AMX300 and DPX600. All
resonances are given in ppm and referenced to residual
solvent signals ,CDCl3: 7.25 ppm, DMSO-d6: 2.49 ppm).
Z-Group cleavage ,GP 2). The Z-protected compound was
dissolved in the indicated solvent ,0.02 M), 15 wt% Pd/C
,5%, Degussa E101 NO/W) were added and the mixture was
degassed and purged with H2 ,3£). The ¯ask was warmed to
the temperature given and hydrogenated ,1 bar) with
vigorous stirring until conversion ceased. The catalyst was
®ltered off using a pad of Celitew 512 ,2 cm), the pad rinsed
with MeOH, and the combined fractions evaporated,
coevaporated with toluene/EtOH 10:1 and dried to constant
HPLC. Rainin Dynamax HPLC-System, DA-Detector.
Method 1 ,M1): Column Rainin Dynamax C8, 250£
4 mm; solvent A: H2O, solvent B: CH3CN/i-PrOH 2:1;
linear gradient 70!100% B in 20 min, ¯ow 0.7 mL/min,
column thermostat at 308C. Method 2 ,M2): Column Jupiter
C5, 250£4 mm; A: H2O, B: CH3CN/i-PrOH 2:1, 75!85%
B in 20 min, 0.7 mL/min, 308C. Method 3 ,M3): Column
Jupiter C5, 250£4 mm; A: H2O, B: CH3CN/i-PrOH 2:1,
90!100% B in 15 min, 1 mL/min, 508C. Method 4 ,M4):

2796
H.-D. Arndt et al. / Tetrahedron 58 42002) 2789±2801
weight ,0.01 Torr). In several cases the crude amines such
obtained had to be further puri®ed.
1H), 3.55 ,s, 3H), 4.33±4.57 ,m, 2H), 4.94±5.06 ,m, 2H),
5.41 ,d, Jˆ5.7 Hz, 1H, NH), 6.07 ,d, Jˆ8.3 Hz, 1H, NH),
6.93 ,d, Jˆ2.3 Hz, 1H, ar.), 7.02 ,t, Jˆ7.4 Hz, 1H, ar.), 6.93
,m, 1H, ar.), 7.18±7.30 ,m, 6H, ar.), 7.50±7.58 ,m, 1H, ar.),
8.17 ,s, 1H, NH). aDˆ20.5, a365ˆ211.3 ,cˆ1.00, CHCl3,
258C). HRMS ,FAB): for C26H32N3O5 ,M1H1) calcd
466.2342, found 466.2352. C26H31N3O5 ,465.55) calcd C
67.08, H 6.71, N 9.03; found C 67.10, H 6.70, N 9.09.
HOBt-EDC-mediated couplings ,GP 3). The materials were
dissolved in CH2Cl2 ,indicated concentration) and cooled to
08C. HOBt ,1.5 equiv.) and NEt3 were added ,1.5, 3 equiv.
if an amine-hydrochloride was used), followed by EDC
,1.25 equiv.) after 15 min. After 15 min at 08C the mixture
was stirred at the indicated temperature until conversion was
complete.
6.3.3. Z-Trp-d-Leu-Trp-2-)t-butyl-diphenylsilyloxy)-ethyl-
amide )14). 1.58 g ,3.4 mmol) of dipeptide 23 was C-depro-
tected according to GP 1 ,Rfˆ0.09 in CHCl3/MeOH 10:1) as
well as 1.42 g ,2.3 mmol) of amide 24 was N-deprotected
according to GP 2 ,MeOH, 208C, 2 h, Rfˆ0.40 in CHCl3/
MeOH/aq. NH3 10:1:0.1). The combined crude materials
were coupled following GP 3 ,0.1 M, 210!208C, 3 h).
After dilution with CHCl3 ,200 mL), aqueous work-up,
FCC ,250 g, CHCl3/acetone 5:1!4:1) gave 1.90 g
,2.0 mmol, 87%) of tripeptide 14 as a colourless foam.
Rfˆ0.16 ,CHCl3/acetone 4:1). 1H NMR ,300 MHz,
DMSO-d6): dˆ0.61 ,d, Jˆ6.4 Hz, 3H), 0.65 ,d, Jˆ6.4 Hz,
3H), 0.97 ,s, 9H), 1.14±1.19 ,m, 1H), 2.86±2.96 ,m, 2H),
3.02±3.08 ,m, 2H), 3.19±3.30 ,m, 2H), 3.59±3.63 ,m, 2H),
4.14±4.26 ,m, 1H), 4.27±4.38 ,m, 1H), 4.41±4.52 ,m, 1H),
4.88 ,d, Jˆ12.4 Hz, 1H), 4.90 ,d, Jˆ12.8 Hz, 1H), 6.89±
6.97 ,m, 2H, ar.), 6.90±7.22 ,m, 6H, ar.), 7.24±7.45 ,m,
12H, ar.), 7.54±7.65 ,m, 5H, ar.), 7.98±8.06 ,m, 1H, NH),
8.03±8.19 ,m, 2H, NH), 10.78 ,d, Jˆ1.8 Hz, 1H, NH),
10.82 ,d, Jˆ2.2 Hz, 1H, NH). aDˆ24.0, a365ˆ91.5
,cˆ1.05, MeOH, 308C). HPLC: 15.7 min ,M1). HRMS
,FAB): for C54H63N6O6Si ,M2OH2) calcd 919.4578;
found 919.4591. C54H64N6O7Si ,14´H2O, 937.23) calcd C
69.20, H 6.88, N 8.97; found C 69.33, H 6.80, N 8.73.
HBTU-mediated couplings ,GP 4). The materials were
dissolved in CH2Cl2 ,0.03 M) and cooled to 258C, HOBt
,2.5 equiv.) dissolved in DMF ,1:10 v/v) and DIEA
,2.5 equiv.) were added to give a clear solution, followed
after 5 min by HBTU ,1.5 equiv.). The mixture was stirred
at the indicated temperature.
Formamide formation ,GP 5). The amine was dissolved in
CHCl3 ,0.01 M), the solution cooled to 08C and treated with
HCOOH ,10 equiv.), DIEA ,10 equiv.), HOBt ,cat. crystal)
and EDC ,6 equiv.) successively and stirred for 1 h at 08C
and 2 h at 208C.
HATU-mediated couplings ,GP 6). The materials were
dissolved in CH2Cl2 ,0.03 M) and cooled to 08C, HOAt
,4 equiv.) dissolved in DMF ,1:3 v/v) and DIEA
,3 equiv.) were added to give a clear yellow solution,
followed after 5 min by HATU ,2.5 equiv.). The mixture
was stirred at the indicated temperature.
6.3. Preparations
6.3.1. Z-Trp-2-)t-butyl-diphenylsilyloxy)-ethylamide )24).
5.40 g ,16.0 mmol) Z-Trp-OH 19 and 4.79 g ,16.0 mmol)
2-,t-butyl-diphenylsilyloxy)-ethylamine 20 were coupled
according to GP 3 ,0.4 M, 208C, 3 h). The mixture was
then diluted with CHCl3 ,200 mL), and after aqueous
work-up, FCC ,300 g, PE/EtOAc 3:1!2:1!1:1) gave
8.50 g ,13.7 mmol, 86%) of amide 24 as a colourless
6.3.4. Z-)Trp-d-Leu)2-OMe )13). 1.17 g ,2.50 mmol)
dipeptide 23 were C-deprotected following GP 1 ,Rfˆ0.09
in CHCl3/MeOH 10:1) as well as 1.24 g ,2.65 mmol,
1.05 equiv.) of 23 were N-deprotected following GP 2
,MeOH/THF 5:1, 258C, 1 h; Rfˆ0.26 in CHCl3/MeOH
10:1). The crude materials were combined and coupled
following GP 4 ,58C, 2 h) and the mixture diluted with
EtOAc ,200 mL). After aqueous work-up, FCC ,200 g,
1
foam. Rfˆ0.14 ,n-Hex/EtOAc 2:1). H NMR ,300 MHz,
CDCl3): dˆ0.89 ,s, 9H), 3.04 ,dd, Jˆ14.5, 7.7 Hz, 1H),
3.12±3.33 ,m, 3H), 3.34±3.53 ,m, 2H), 4.28±4.45 ,m,
1H), 5.00 ,d, Jˆ12.2 Hz, 1H), 5.05 ,d, Jˆ12.2 Hz, 1H),
5.33±5.47 ,m, 1H, NH), 5.75±5.90 ,m, 1H, NH), 6.80±
6.90 ,m, 1H, ar.), 6.96±7.14 ,m, 2H, ar.), 7.15±7.41 ,m,
12H, ar.), 7.41±7.51 ,m, 4H, ar.), 7.53±7.62 ,m, 1H, ar.),
7.72 ,s, 1H, NH). aDˆ23.1, a365ˆ217.1 ,cˆ1.00, CHCl3,
208C). HRMS ,EI): for C37H41N3O4Si ,M1) calcd 619.2866,
found 619.2870. C37H41N3O4Si ,619.84) calcd C 71.70, H
6.76, N 6.78; found C 71.59, H 6.75, N 6.69.
CHCl3/acetone
,2.41 mmol, 96 %) of tetrapeptide 13 as a colourless
5:1!4:1!3:1)
provided
1.84 g
1
foam. Rfˆ0.12 ,CHCl3/acetone 4:1). H NMR ,300 MHz,
DMSO-d6): dˆ0.62 ,d, Jˆ5.7 Hz, 3H), 0.67 ,d, Jˆ5.7 Hz,
3H), 0.73 ,d, Jˆ5.3 Hz, 3H), 0.78 ,d, Jˆ5.7 Hz, 3H), 1.12±
1.26 ,m, 3H), 1.33±1.61 ,m, 3H), 2.90 ,dd, Jˆ14.3, 9.4 Hz,
2H), 3.09 ,ddd, Jˆ21.0, 14.7, 5.8 Hz, 2H), 3.60 ,s, 3H),
4.15±4.41 ,m, 3H), 4.49±4.63 ,m, 1H), 4.88 ,d, Jˆ
12.8 Hz, 1H), 4.93 ,d, Jˆ12.8 Hz, 1H), 6.94 ,t, Jˆ
7.35 Hz, 2H, ar.), 7.04 ,dt, Jˆ7.3, 3.5 Hz, 2H, ar.), 7.12
,d, Jˆ4.9 Hz, 2H, ar.), 7.20 ,m, 2H, ar.) 7.24±7.37 ,m,
5H, ar.), 7.59 ,m, Jˆ3.0 Hz, 1H), 7.61 ,d, Jˆ3.4 Hz, 1H),
8.09 ,d, Jˆ7.9 Hz, 1H, NH), 8.18 ,d, Jˆ8.2 Hz, 1H, NH),
8.26 ,d, Jˆ7.5 Hz, 1H, NH), 10.80 ,s, 2H, NH). aDˆ213.8,
a365ˆ260.0 ,cˆ1.07, CH2Cl2, 258C). HPLC: 8.8 min ,M1).
HRMS ,FAB): for C43H53N6O7 ,M1H1) calcd 765.3976,
found 765.3968. C43H52N6O7 ,764.93) calcd C 67.52, H
6.85, N 10.99; found C 67.15, H 6.77, N 10.67.
6.3.2. Z-Trp-d-Leu-OMe )23). 8.46 g ,25.0 mmol) Z-Trp-
OH 19 and 4.78 g ,26.3 mmol, 1.05 equiv.) H-d-Leu-
OMe´HCl 20 were coupled according to GP 3 ,0.3 M,
208C, 2 h). The mixture was diluted with CHCl3
,200 mL), and after aqueous work-up, recrystallisation
from boiling cyclohexane/EtOAc ,1:1) gave 10.1 g
,21.8 mmol, 87%) of dipeptide 23 as colourless needles,
1
mpˆ154.5±1558C. Rfˆ0.26 ,n-Hex/EtOAc 1:1). H NMR
,300 MHz, CDCl3): dˆ0.70 ,d, Jˆ1.5 Hz, 3H), 0.72 ,d,
Jˆ1.5 Hz, 3H), 1.07±1.25 ,m, 2H), 1.26±1.40 ,m, 1H),
3.11 ,dd, Jˆ14.5, 7.7 Hz, 1H), 3.24 ,dd, Jˆ14.5, 5.1 Hz,
6.3.5. Z-)Trp-d-Leu)3-Trp-2-)t-butyl-diphenylsilyloxy)-
ethylamide )25). 640 mg ,696 mmol) of tripeptide 14

H.-D. Arndt et al. / Tetrahedron 58 42002) 2789±2801
2797
were N-deprotected following GP 2 ,MeOH/THF 5:1, 308C,
12 h) and puri®ed by FCC ,30 g, CHCl3/MeOH
100:5!100:7) to give 432 mg ,550 mmol, 79%) of the
pure amine. 535 mg ,700 mmol, 1.3 equiv.) of tetrapeptide
13 were C-deprotected following GP 1. The combined
materials were coupled following GP 4 ,58C, 2 h) and the
mixture was diluted with CHCl3 ,50 mL). After aqueous
work-up, FCC ,120 g, CHCl3/acetone 3:1!2:1!3:2) gave
698 mg ,460 mmol, 84%) of heptapeptide 25 as a colourless
GP 4 ,CH2Cl2/DMF 3:1, 08C, 1 h). The mixture was acid-
i®ed to pH 4 with 2 M HCl, partitioned between EtOAc/
H2O/brine ,100:30:10 mL) and extracted with EtOAc
,3£50 mL) and CHCl3/i-PrOH ,3:1, 50 mL). The combined
organic layers were washed with brine ,50 mL), dried with
Na2SO4 and concentrated. FCC ,150 g, CHCl3/MeOH
100:1) gave 2.44 g ,4.57 mmol, 90%) of the tetrapeptide
12 as a colourless solid, mpˆ212±2148C. Rfˆ0.66
,CHCl3/MeOH 20:1). 1H NMR ,300 MHz, DMSO-d6):
dˆ0.82 ,m, 18H), 1.20 ,d, Jˆ6.8 Hz, 3H), 2.00 ,m, 3H),
3.60 ,s, 3H), 4.16 ,m, 2H), 4.34 ,m, 2H), 5.00 ,s, 2H), 7.31
,m, 5H), 7.38 ,d, Jˆ7.4 Hz, 1H), 7.78 ,d, Jˆ9.0 Hz, 1H),
7.94 ,d, Jˆ9.0 Hz, 1H), 8.16 ,d, Jˆ8.3 Hz, 1H). aDˆ13.0,
a365ˆ18.1 ,cˆ1.00, MeOH, 238C). HPLC: 6.87 min ,M1).
HRMS ,FAB): for C27H43N4O7 ,M1H1) calcd 535.3132;
found 535.3149. C27H42N4O7 ,534.66) calcd C 60.65, H
7.92, N 10.48; found C 60.60, H 7.90, N 10.41.
1
foam. Rfˆ0.07 ,CHCl3/acetone 3:1). H NMR ,300 MHz,
DMSO-d6): dˆ0.25±0.47 ,m, 18H), 0.71±0.85 ,m, 12H),
0.86±1.01 ,m, 6H), 2.60±2.78 ,m, 4H), 2.79±3.08 ,m, 6H),
3.42 ,t, Jˆ6.2 Hz, 2H), 3.89±4.06 ,m, 3H), 4.07±4.19 ,m,
1H), 4.20±4.40 ,m, 3H), 4.63 ,d, Jˆ12.4 Hz, 1H), 4.71 ,d,
Jˆ12.8 Hz, 1H), 6.66±6.76 ,m, 4H, ar.), 6.77±6.93 ,m, 8H,
ar.), 6.94±7.00 ,m, 1H, ar.), 7.02±7.14 ,m, 7H, ar.), 7.15±
7.26 ,m, 6H, ar.), 7.30±7.46 ,m, 9H, ar.), 7.69 ,d, Jˆ7.2 Hz,
1H, NH), 7.79 ,d, Jˆ7.5 Hz, 1H, NH), 7.85±7.96 ,m, 3H,
NH), 7.99 ,d, Jˆ7.5 Hz, 1H, NH), 8.05 ,d, Jˆ8.7 Hz, 1H,
NH), 10.55 ,s, 2H, NH), 10.58 ,s, 1H, NH), 10.60 ,s, 1H,
NH). aDˆ211.3, a365ˆ251.6 ,cˆ1.64, CHCl3, 258C).
HPLC: 17.0 min ,M1). MS ,ESI) calcd for
C88H104N12O10SiNa ,M1Na1) 1539.8; found 1539.9.
C88H106N12O11Si ,25´H2O, 1535.98) calcd C 68.81, H
6.96, N 10.94; found C 68.82, H 6.93, N 10.97.
6.3.9.
Z-Ala-d-Val-Val-d-Val-)Trp-d-Leu)3-Trp-2-)t-
butyl-diphenylsilyloxy)-ethylamide )11). 568 mg ,374
mmol) of heptapeptide 25 were N-deprotected following
GP 2 ,MeOH/THF 5:1, 408C, 8 h) and puri®ed by FCC
,30 g, CHCl3/MeOH 40:1!20:1!10:1) to give 80 mg
,53 mmol, 14%) of unchanged 25 and 445 mg ,321 mmol,
86%) of pure amine. 257 mg ,480 mmol, 1.5 equiv.) of tetra-
peptide 12 were C-deprotected following GP 1 ,THF/
MeOH/H2O 3:1:1, 208C, 8 h). The combined amine and
carboxylic acid were coupled following GP 4 ,30 mM,
0!108C, 8 h) and the mixture was diluted with CHCl3
,50 mL). After aqueous work-up, FCC ,50 g, toluene/
EtOH 100:5!100:6!100:7) provided 445 mg ,236 mmol,
74%) of HPLC-pure undecapeptide 11. 47 mg ,34 mmol,
11%) of unchanged heptamer-amine were eluted with
CH2Cl2/MeOH 10:1. Rfˆ0.20 ,toluene/EtOH/HCOOH
6.3.6. Z-Ala-d-Val-OMe )21). 2.33 g ,10.0 mmol) Z-Ala-
OH 15 and 1.67 g ,10.0 mmol) H-d-Val-OMe´HCl 16 were
coupled according to GP 3 ,0.2 M, 0!208C, 12 h). After
aqueous work-up, FCC ,200 g PE/MTBE 1:1!MTBE)
gave 3.14 g ,9.30 mmol, 93%) of dipeptide 21 as a colour-
less powder. Rfˆ0.18 ,PE/MTBE 1:1). 1H NMR ,300 MHz,
DMSO-d6): dˆ0.83 ,dd, Jˆ6.8, 1.9 Hz, 6H), 1.20 ,d,
Jˆ7.2 Hz, 3H), 2.02 ,dq, Jˆ20.0, 6.8 Hz, 1H), 3.63 ,s,
3H), 4.13±4.22 ,m, 2H), 5.00 ,s, 2H), 7.26±7.35 ,m, 5H,
ar.), 7.40 ,d, Jˆ7.9 Hz, 1H, NH), 8.12 ,d, Jˆ8.7 Hz, 1H,
NH). aDˆ226.9, a365ˆ2106 ,cˆ0.99, CHCl3, 258C).
HRMS ,EI): for C17H24N2O5 ,M1) calcd 336.1685 found
336.1688. C17H24N2O5 ,336.39) calcd C 60.70, H 7.19, N
8.33; found C 60.93, H 7.17, N 8.27.
1
100:7:1). H NMR ,300 MHz, DMSO-d6): dˆ0.43±0.59
,m, 21H), 0.63 ,d, Jˆ6.4 Hz, 3H), 0.71±0.82 ,m, 12H),
0.83±0.92 ,m, 3H), 0.96 ,s, 9H), 1.03±1.24 ,m, 9H),
1.69±1.84 ,m, 1H), 1.89±2.05 ,m, 2H), 2.78±2.95 ,m,
4H), 3.02±3.26 ,m, 6H), 3.62 ,t, Jˆ6.4 Hz, 2H), 4.04±
4.23 ,m, 5H), 4.24±4.36 ,m, 2H), 4.40±4.56 ,m, 4H), 4.96
,s, 2H), 6.85±6.95 ,m, 4H, ar.), 6.96±7.04 ,m, 4H, ar.),
7.05±7.08 ,s, 4H, ar.), 7.23±7.32 ,m, 9H, ar.), 7.34±7.45
,m, 7H, ar.), 7.48±7.56 ,m, 4H, ar.), 7.57±7.64 ,m, 4H, ar.),
7.69±7.80 ,m, 2H, NH), 7.86±7.97 ,m, 4H, NH), 8.02±8.33
,m, 6H, NH), 10.68 ,s, 1H, NH), 10.73 ,s, 3H, NH).
aDˆ36.4, a365ˆ136 ,cˆ0.42, MeOH, 308C). HPLC:
18.7 min. ,M1). MS ,ESI): for C106H136N16O14SiNa
,M1Na1) calcd 1908.0; found 1908.1.
6.3.7. Z-Val-d-Val-OMe )22). 2.51 g ,10.0 mmol) Z-Val-
OH 17 and 1.67 g ,10.0 mmol) H-d-Val-OMe´HCl 16 were
coupled following GP 3 ,0.1 M, 5 equiv. NEt3, 208C, 18 h).
After aqeous work-up, the crude product was crystallised
from CHCl3/Et2O to give 3.36 g ,8.99 mmol, 90%) of
dipeptide 22 as a colourless powder, mpˆ161±1638C.
Rfˆ0.25 ,PE/MTBE 1:1). aDˆ211.1, a365ˆ248.0
1
,cˆ0.96, CHCl3, 258C). H NMR ,300 MHz, DMSO-d6):
dˆ0.81±0.86 ,m, 12H), 1.89±2.07 ,m, 2H), 3.62 ,s, 3H),
4.04 ,dd, Jˆ9.0, 6.8 Hz, 1H), 4.17 ,dd, Jˆ7.9, 6.4 Hz, 1H),
5.02 ,s, 2H), 7.21 ,d, Jˆ9.4 Hz, 1H, NH), 7.23±7.35 ,m, 5H,
ar.), 8.17 ,d, Jˆ8.3 Hz, 1H, NH). HRMS ,EI): for
C19H28N2O5 ,M1) calcd 364.1998, found 364.2001.
C19H28N2O5 ,364.44) calcd C 62.62, H 7.74, N 7.69;
found C 62.63, H 7.69, N 7.66.
6.3.10. H-Ala-d-Val-Val-d-Val-)Trp-d-Leu)3-Trp-2-)t-
butyl-diphenylsilyloxy)-ethylamide )26). 400 mg ,212
mmol) of undecapeptide 11 were deprotected following
GP 2 ,MeOH/DMF 10:1, 25 wt% of Pd/C, 408C, 4 h),
dissolved in Et2O ,70 mL), washed with H2O ,5 mL), sat.
NaHCO3 ,20 mL) and brine ,20 mL), dried with Na2SO4
and evaporated to give 356 mg ,203 mmol, 96%) of amine
26 as a colourless gum, which was stored at 2308C.
Rfˆ0.06 ,CH2Cl2/MeOH 10:1). HPLC: 23.6 min ,M41
0.1% TFA, 65!75% B in 25 min, 0.5 mL/min). MS ,ESI):
for C98H131N16O12Si ,M1H1) calcd 1752.0, found 1752.1.
6.3.8. Z-Ala-d-Val-Val-d-Val-OMe )12). 1.87 g ,5.56
mmol) dipeptide 21 were C-deprotected following GP 1
,Rfˆ0.31 in CHCl3/MeOH/HOAc 100:10:1). N-deprotec-
tion of 1.85 g ,5.08 mmol) dipeptide 22 was performed
according to GP 2 ,Rfˆ0.54 in CHCl3/MeOH/aq. NH3
100:10:1). The combined materials were coupled following
6.3.11. TBDPS-protected minigramicidin )27). 27.2 mg
,15.5 mmol) of amine 26 were converted to the formamide

2798
H.-D. Arndt et al. / Tetrahedron 58 42002) 2789±2801
using GP 5, the mixture was then diluted with CHCl3
,50 mL) and puri®ed after aqueous work-up by FCC
,10 g, 5.5!6!6.5!7.5% EtOH in CHCl3) to provide
23.9 mg ,13.4 mmol, 87%) of N1-formyl undecapeptide 27
as a colourless powder. Rfˆ0.45 ,CHCl3/MeOH 10:1). H
stirred vigorously for 1 h at 208C. The biphasic mixture
was partitioned between CHCl3 ,20 mL) and 2 M NaHSO4
,10 mL, Caution!), and the aqueous layer was extracted with
CHCl3 ,2£5 mL). The combined organic layers were
washed with brine ,10 mL), dried with Na2SO4, and evapo-
rated. FCC ,10 g, CHCl3/MeOH/HCOOH 100:4:1!
100:6:1) gave 82.4 mg ,44.5 mmol, 88%) of the N1-succinyl
undecapeptide as a colourless solid. Rfˆ0.16 ,CHCl3/
MeOH/HCOOH 100:5:1). HPLC: 10.4 min ,M5,
70!100% B).
1
NMR ,600 MHz, DMSO-d6): dˆ0.48 ,app. d, Jˆ6.5 Hz,
6H), 0.524 ,d, Jˆ6.5 Hz, 3H), 0.532 ,d, Jˆ6.3 Hz, 3H),
0.55 ,app. d, Jˆ6.4 Hz, 9H), 0.62 ,d, Jˆ6.5 Hz, 3H), 0.77
,d, Jˆ6.8 Hz, 3H), 0.78 ,app. d, Jˆ7.1 Hz, 6H), 0.80 ,d,
Jˆ6.8 Hz, 3H), 0.81±0.93 ,m, 3H), 0.96 ,s, 9H), 1.05±
1.10 ,m, 2H), 1.10±1.17 ,m, 4H), 1.18 ,d, Jˆ7.0 Hz, 3H),
1.76 ,m, 1H), 1.98 ,m, 2H), 2.82±2.92 ,m, 4H), 3.06±3.19
,m, 4H), 3.24 ,m, 1H), 3.33 ,m, 1H), 3.62 ,t, Jˆ6.8 Hz, 2H),
4.09±4.15 ,m, 3H), 4.19 ,m, 1H), 4.28 ,dd, Jˆ7.9, 6.5 Hz,
1H), 4.32 ,dd, Jˆ8.7, 6.4 Hz, 1H), 4.44±4.57 ,m, 5H),
6.88±6.92 ,m, 4H), 6.97±7.02 ,m, 4H), 7.06 ,m, 4H), 7.26
,m, 4H), 7.36±7.44 ,m, 6H), 7.50±7.55 ,m, 4H), 7.58±7.61
,m, 4H), 7.73 ,d, Jˆ8.1 Hz, 1H), 7.91 ,s, 1H, ±CHO), 7.88±
8.00 ,m, 5H), 8.08 ,t, Jˆ5.8 Hz, 1H), 8.12 ,d, Jˆ7.3 Hz,
1H), 8.17±8.20 ,m, 3H), 8.25 ,d, Jˆ8.0 Hz, 1H), 10.69 ,s,
1H), 10.74 ,m, 3H). HPLC: 18.9 min ,M4, 65!75% B in
25 min, 0.7 mL/min). MS ,ESI): for C99H130N16O13SiNa
,M1Na1) calcd 1802.0, found 1801.8.
This carboxylic acid was combined with 79 mg ,45 mmol,
1.0 equiv.) of amine 26 and coupled according to GP 6 ,08C,
14 h). After dilution with CHCl3 ,50 mL) and aqueous
work-up, FCC ,15 g, CHCl3/MeOH/HCOOH 100:2.5:7!
100:4:7) provided 142 mg ,39.6 mmol, 89%) of TBDPS-
protected minigramicidin 2 as a colourless foam. The
pooled column fractions containing 2 have been washed
with sat. NaHCO3-solution ,equal volume, Caution!) and
dried with Na2SO4 to remove the HCOOH before evapora-
1
tion. Rfˆ0.25 ,CHCl3/MeOH/HCOOH 100:3:7). H NMR
,600 MHz, DMSO-d6): dˆ0.49 ,app. d, Jˆ6.0 Hz, 12H),
0.53±0.58 ,m, 30H), 0.63 ,d, Jˆ6.6 Hz, 6H), 0.76±0.81
,m, 24H), 0.82±0.91 ,m, 6H), 0.96 ,s, 18H), 1.07±1.20
,m, 12H), 1.16 ,d, Jˆ7.0 Hz, 6H), 1.79 ,m, 2H), 2.00 ,m,
4H), 2.24 ,m, 2H, succ.), 2.38 ,m, 2H, succ.) 2.85±2.95 ,m,
8H), 3.09±3.21 ,m, 8H), 3.26 ,m, 2H), 3.33 ,m, 2H), 3.63 ,t,
Jˆ6.7 Hz, 2H), 4.08±4.16 ,m, 6H), 4.19 ,m, 2H), 4.24±4.29
,m, 4H), 4.33 ,m, 2H), 4.44±4.58 ,m, 8H), 6.88±6.93 ,m,
8H), 6.98±7.02 ,m, 8H), 7.06±7.08 ,m, 8H), 7.25±7.29 ,m,
8H), 7.36±7.43 ,m, 12H), 7.51±7.56 ,m, 8H), 7.59±7.62
,m, 8H), 7.77 ,m, 4H), 7.90±7.98 ,m, 8H), 8.01 ,d,
Jˆ7.1 Hz, 2H), 8.08 ,t, Jˆ5.7 Hz, 2H), 8.13 ,d, Jˆ6.3 Hz,
2H), 8.21 ,m, 4H), 8.26 ,d, Jˆ8.0 Hz, 2H), 10.69 ,s, 2H),
10.74 ,m, 6H). HPLC: 21.3 min ,M5). MS ,MALDI-TOF):
for C200H262N32O26Si2Na ,M1Na1) calcd 3607.0; found
3607.0.
6.3.12. Minigramicidin )28). 20.0 mg ,11.2 mmol) of
TBDPS-protected 27 was dissolved in CHCl3 ,2 mL),
cooled to 08C and HF was added ,500 mL, 5 vol% in
CH3CN). The mixture was stirred for 2 h at 08C and for
1 h at 208C and became pink during that time. The mixture
was then partitioned between CHCl3 and sat. NaHCO3-
solution ,10 mL each) and extracted with CHCl3/i-PrOH
,5:1, 3£10 mL). The combined organic layers were dried
with Na2SO4 and evaporated. FCC ,3 g, CHCl3/MeOH/
HCOOH
100:8:1!100:10:1)
delivered
10.5 mg
,6.8 mmol, 59%) of Minigramicidin 28 as an off-white
powder. Rfˆ0.27 ,CHCl3/MeOH/HCOOH 100:10:1). 1H
NMR ,600 MHz, DMSO-d6): dˆ0.49 ,d, Jˆ6.4 Hz, 3H),
0.50 ,d, Jˆ6.5 Hz, 3H), 0.53 ,d, Jˆ6.4 Hz, 3H), 0.54 ,d,
Jˆ7 Hz, 3H), 0.55 ,d, Jˆ6.9 Hz, 3H), 0.56 ,d, Jˆ6.9 Hz,
3H), 0.57 ,d, Jˆ6.9 Hz, 3H), 0.63 ,d, Jˆ6.6 Hz, 3H), 0.77
,d, Jˆ6.7 Hz, 3H), 0.78 ,d, Jˆ6.9 Hz, 3H), 0.79 ,d,
Jˆ7.0 Hz, 3H), 0.80 ,d, Jˆ7.0 Hz, 3H), 0.82±0.90 ,m,
2H), 0.91±0.99 ,m, 1H), 1.04±1.19 ,m, 6H), 1.18 ,d,
Jˆ6.9 Hz, 3H), 1.77 ,m, 1H), 1.98 ,m, 2H), 2.83±2.93
,m, 4H), 3.06±3.21 ,m, 6H), 3.40 ,m, 2H), 4.09±4.17 ,m,
4H), 4.27 ,dd, Jˆ8.5, 5.9 Hz, 1H), 4.31 ,dd, Jˆ8.6, 6.4 Hz,
1H), 4.43±4.57 ,m, 5H), 4.70 ,t, Jˆ5.5 Hz, 1H, ±OH),
6.89±6.95 ,m, 4H), 6.98±7.03 ,m, 4H), 7.05±7.08 ,m,
4H), 7.25±7.29 ,m, 4H), 7.52 ,m, 2H), 7.55 ,app. d,
Jˆ8.0 Hz, 2H), 7.75 ,d, Jˆ8.7 Hz, 1H), 7.90±7.97 ,m,
3H), 7.91 ,s, 1H, ±CHO), 7.98±8.05 ,m, 3H), 8.15 ,d,
Jˆ7.4 Hz, 1H), 8.18±8.22 ,m, 3H), 8.30 ,d, Jˆ8.3 Hz,
1H), 10.69 ,m, 1H), 10.73±10.76 ,m, 3H). HPLC: 9.5 min
,M4, 65!75% B in 25 min, 0.5 mL/min). MS ,MALDI-
TOF): for C83H112N16O13Na ,M1Na1) calcd 1563.8,
found 1563.9.
6.3.14. Succinyl-linked minigramicidin )3) and its mono-
TBDPS-protected-form )4). 25.0 mg ,6.97 mmol) of bis-
TBDPS-ether 2 were dissolved in MeOH ,2 mL), HF
,5 vol% in CH3CN, 2 mL) was added and the solution
stirred for 24 h at 208C ,HPLC control). The mixture was
partitioned between CHCl3 ,20 mL) and sat. NaHCO3 solu-
tion ,20 mL, CAUTION!) and the aqueous layer extracted
with CHCl3/i-PrOH ,5:1, 5£10 mL). The combined organic
layers were washed with brine ,15 mL), dried with Na2SO4
and evaporated. FCC ,2.5 g, CHCl3/HCOOH/MeOH
100:7:4!100:7:7!i-PrOH/H2O 99:1) followed by GPC
,Sephadex LH20, CHCl3/MeOH 1:1) provided 19.6 mg
,6.30 mmol, 90%) of minigramicidin 3 as an off-white
1
solid. Rfˆ0.25 ,CHCl3/MeOH/HCOOH 100:7:7). H NMR
,600 MHz, DMSO-d6): dˆ0.49±0.54 ,m, 18H), 0.55±0.59
,m, 24H), 0.64 ,d, Jˆ6.4 Hz, 6H), 0.74±0.79 ,m, 24H),
0.88±0.94 ,m, 4H), 0.94±1.00 ,m, 2H), 1.12±1.22 ,m,
12H), 1.17 ,d, Jˆ6.9 Hz, 6H), 1.80 ,m, 2H), 1.97±2.02
,m, 4H), 2.22±2.28 ,m, 2H, succ.), 2.34±2.40 ,m, 2H,
succ.), 2.85±2.93 ,m, 8H), 3.06±3.21 ,m, 12H), 3.40 ,m,
4H), 4.04±4.16 ,m, 8H), 4.17±4.23 ,m, 4H), 4.30 ,m, 2H),
4.42±4.54 ,m, 8H), 4.78 ,bs, 2H, ±OH), 6.88±6.95 ,m, 8H),
6.97±7.02 ,m, 8H), 7.07 ,app. d, Jˆ8.6 Hz, 8H), 7.26±7.29
,m, 8H), 7.49 ,app. d, Jˆ7.8 Hz, 4H), 7.54 ,d, Jˆ6.9 Hz,
2H), 7.55 ,d, Jˆ7.0 Hz, 2H), 7.94 ,t, Jˆ5.2 Hz, 2H),
6.3.13. TBDPS-protected succinyl-linked minigrami-
cidin )2). 89 mg ,51 mmol) of amine 26 were dissolved in
CH2Cl2 ,3 mL) and treated with pyridine ,30 mL) and
succinic anhydride ,50 mg dissolved in 1 mL DMF,
0.5 mmol, 10 eq). After the complete conversion ,1 h),
sat. NaHCO3-solution ,1 mL) was added and the mixture

H.-D. Arndt et al. / Tetrahedron 58 42002) 2789±2801
2799
7.96±8.01 ,m, 4H), 8.01±8.06 ,m, 4H), 8.06±8.12 ,m, 4H),
8.25 ,d, Jˆ7.0 Hz, 2H), 8.27±8.34 ,m, 8H), 10.71 ,bs, 1H),
10.77 ,bs, 1H), 10.78 ,bs, 1H), 10.79 ,bs, 1H). HPLC
12.4 min ,M5). MS ,MALDI-TOF): for C168H226N32O26Na
,M1Na1) calcd 3130.7; found 3130.9.
6.3.16. Z-Ala-d-Leu-Ala-d-Val-Val-d-Val-)Trp-d-Leu)3-
Trp-2-)t-butyl-diphenylsilyloxy)-ethylamide )32). 200 mg
,0.110 mmol) amine 26 was coupled with 40 mg
,0.12 mmol, 1.1 equiv.) Z-Ala-d-Leu-OH 1020 according
to GP 4 ,5 mM, 3 h, 0!208C). After aqueous work-up,
FCC ,40 g, 2!4% MeOH in CHCl3) gave 200 mg
,0.096 mmol, 87%) of tridecapeptide 32 as a colourless
solid. Rfˆ0.55 ,CHCl3/MeOH/HCO2H 100:10:1). 1H
NMR ,300 MHz, DMSO-d6): dˆ0.42±0.65 ,m, 24H),
0.69±0.90 ,m, 21H), 0.96 ,s, 9H), 1.04±1.25 ,m, 12H),
1.37±1.61 ,m, 3H), 1.70±1.85 ,m, 1H), 1.87±2.06 ,m,
2H), 2.78±2.95 ,m, 4H), 3.04±3.30 ,m, 6H), 3.57±3.66
,m, 2H), 4.00±4.38 ,m, 9H), 4.40±4.60 ,m, 4H), 4.93±
5.06 ,m, 2H), 6.85±6.95 ,m, 4H, ar.), 6.96±7.10 ,m, 7H,
ar.), 7.23±7.46 ,m, 16H, ar.), 7.47±7.57 ,m, 4H, ar.), 7.58±
7.63 ,m, 4H, ar.), 7.66±7.82 ,m, 2H, NH), 7.85±8.00 ,m,
5H), 8.03±8.32 ,m, 6H), 10.69 ,s, 1H, NH), 10.74 ,s, 3H,
NH). HPLC: 12.2 min ,M2). MS ,MALDI-TOF) calcd for
C115H152N18O16SiNa ,M1Na1) calcd 2092.1, found 2092.2.
The mono-TBDPS-protected 4 was obtained on quenching
the reaction at an earlier stage of conversion ,HPLC
control). In a typical experiment employing 36 mg
,10.1 mmol) of 2, FCC ,5 g, CHCl3/HCOOH/i-PrOH/
MeOH 100:10:2.7:1.3!100:10:4:2!i-PrOH/H2O 99:1)
gave 8.1 mg ,2.3 mmol, 23%) 2, 7.8 mg ,2.3 mmol, 23%)
4 and 12.4 mg ,4.0 mmol, 39%) 3. Data for 4: Rfˆ0.23
,CHCl3/HCOOH/i-PrOH/MeOH 100:10:4:2). 1H NMR
,600 MHz, DMSO-d6): dˆ0.47±0.51 ,m, 12H), 0.52±0.58
,m, 24H), 0.61 ,d, Jˆ7.1 Hz, 3H), 0.63 ,d, Jˆ7.1 Hz, 3H),
0.75±0.80 ,m, 24H), 0.83 ,d, Jˆ7.1 Hz, 3H), 0.85 ,d,
Jˆ6.8 Hz, 3H), 0.81±0.90 ,m, 4H), 0.96 ,s, 9H), 0.91±
0.99 ,m, 2H), 1.05±1.19 ,m, 12H), 1.15 ,d, Jˆ6.9 Hz,
6H), 1.76±1.81 ,m, 2H), 1.95±2.02 ,m, 4H), 2.19±2.27
,m, 2H, succ.), 2.33±2.40 ,m, 2H, succ.), 2.85±2.92 ,m,
8H), 3.07±3.21 ,m, 10H), 3.23±3.30 ,m, 4H), 3.62 ,t,
Jˆ6.8 Hz, 2H), 4.04±4.20 ,m, 8H), 4.23±4.27 ,m, 4H),
4.32 ,m, 2H), 4.43±4.56 ,m, 8H), 4.70 ,t, Jˆ5.7 Hz, 1H,
±OH), 6.88±6.95 ,m, 8H), 6.97±7.03 ,m, 8H), 7.05±7.09
,m, 8H), 7.25±7.29 ,m, 8H), 7.36±7.43 ,m, 6H, TBDPS),
7.49±7.56 ,m, 8H), 7.58±7.61 ,m, 4H, TBDPS), 7.74±7.78
,m, 4H), 7.89±7.93 ,m, 7H), 7.94 ,d, Jˆ6.5 Hz, 1H), 8.00
,app. d, Jˆ7.3 Hz, 2H), 8.02 ,app. d, Jˆ8 Hz, 1H), 8.07 ,t,
Jˆ5.8 Hz, 1H), 8.12 ,m, 2H), 8.18±8.22 ,m, 4H), 8.25 ,d,
Jˆ7.7 Hz, 1H), 8.29 ,d, Jˆ9.0 Hz, 1H), 10.69 ,m, 2H),
10.74 ,m, 6H). HPLC 15.1 min ,M5). MS ,MALDI-TOF):
for C184H244N32O26SiNa ,M1Na1) calcd 3368.8; found
3368.9.
6.3.17. Succinic-1-)Val-Gly-OMe)-4-)Val-Gly-Ala-d-Leu-
Ala-d-Val-Val-d-Val-)Trp-d-Leu)3-Trp-2-)t-butyl-di-
phenylsilyloxy)-ethylamide)-diamide )33). 19 mg ,40
mmol) Benzylester 9 was debenzylated according to GP 2
,MeOH, Rfˆ0.30 in CHCl3/MeOH/HCO2H 100:10:1) as
well as 41 mg ,20 mmol) tridecapeptide 32 was N-depro-
tected following the same procedure ,Rfˆ0.40 in CHCl3/
MeOH/aq. NH3 100:10:1, HPLC: 19.6 min ,M2)). The
crude products were combined and coupled without further
puri®cation according to GP 4 ,1 mM, 208C, 4 h). After
aqueous work-up, FCC ,2 g, 2!4% MeOH in CHCl3)
furnished 30 mg ,12.7 mmol, 63%) of octadecamer 33 as
an off-white solid. ,Rfˆ0.45 in CHCl3/MeOH/HCO2H
1
100:10:1). H NMR ,300 MHz, DMSO-d6): dˆ0.43±0.66
,m, 24H), 0.72±0.91 ,m, 33H), 0.96 ,s, 9H), 1.02±1.25 ,m,
12H), 1.39±1.64 ,m, 3H), 1.69±1.86 ,m, 1H), 1.89±2.08
,m, 4H), 2.35±2.46 ,m, 4H), 2.79±2.97 ,m, 4H), 3.04±
3.29 ,m, 6H), 3.56±3.91 ,m, 10H), 4.03±4.36 ,m, 11H),
4.40±4.60 ,m, 3H), 6.84±6.95 ,m, 4H, ar.), 6.96±7.10 ,m,
8H, ar.), 7.22±7.31 ,m, 4H, ar.), 7.32±7.46 ,m, 6H, ar.),
7.47±7.65 ,m, 8H, ar.) 7.67±8.30 ,m, 16H, NH), 8.37 ,t,
Jˆ5.7 Hz, 1H, NH), 10.70 ,s, 1H, NH), 10.74 ,s, 3H, NH).
HPLC: 9.1 min ,M2). MS ,MALDI-TOF) for
C126H176N22O21SiNa ,M11Na1): calcd 2384.3, found
2384.5.
6.3.15.
Succinic-1-)Val-Gly-OMe)-4-)Val-Gly-OBn)-
diamide )9). 550 mg ,1.51 mmol) Boc-Val-Gly-OBn21
was dissolved in CH2Cl2 ,40 mL) and treated with TFA
,10 mL). After 15 min the mixture was diluted with toluene
,50 mL) and the crude ammonium salt was recovered by
evaporation ,Rfˆ0.30, CHCl3/MeOH/aq. NH3 200:10:1).
This material was dissolved in CH2Cl2/DMF/pyridine
5:1:1 ,7 mL) and succinic anhydride ,140 mg, 1.4 mmol)
was added. After stirring for 3 h the mixture was diluted
with EtOAc ,50 mL) and extracted with sat. Na2CO3
solution ,3£10 mL). The combined aqueous layers were
acidi®ed with 4 N HCl to pH 2 and extracted with EtOAc
,4£20 mL). Evaporation and FCC of the residue ,30 g,
CHCl3/MeOH/HCO2H 20:1:0.1) gave 474 mg ,1.37 mmol,
81%) of succinyl-Val-Gly-OBn as a colourless solid. This
was coupled with 502 mg ,1.56 mmol) H-Val-Gly-OMe22
according to GP 3 ,10 mM, 18 h, 208C). After dilution
with CH2Cl2 ,100 mL), the insoluble product was ®ltered
off and recrystallised from boiling i-PrOH to give 500 mg
,0.93 mmol, 70%) of diamide 9 as a colourless powder,
mpˆ2388C. Rfˆ0.35 ,CHCl3/MeOH/HCO2H 100:10:1).
1H NMR ,300 MHz, DMSO-d6): dˆ0.72±0.95 ,m, 12H),
1.87±2.05 ,m, 2H), 2.39 ,s, 4H), 3.59 ,s, 3H), 3.68±3.96
,m, 4H), 4.14 ,t, Jˆ7.5 Hz, 2H), 5.09 ,s, 2H), 7.29±7.42 ,m,
5H), 7.89 ,d, Jˆ8.3 Hz, 2H), 8.31±8.49 ,m, 2H). HRMS
,EI): for C26H38N4O8 ,M1) calcd 535.2768; found
535.2759. C52H78N8O17 ,,9)2´H2O, 1087.24) calcd C
57.45, H 7.23, N 10.31; found C 57.67, H 7.13, N 10.43.
6.3.18. TBDPS-protected, succinyl-linked gramicidin
)1). 20 mg ,8.5 mmol, 1.1 equiv.) of octadecamer 33 were
C-deprotected according to GP 1 ,Rfˆ0.30 in CHCl3/
MeOH/HCO2H 100:10:1), as well as 16 mg ,7.8 mmol) of
tridecapeptide 32 were N-deprotected following GP 2
,MeOH, Rfˆ0.33 in CHCl3/MeOH/aq. NH3 100:10:1;
HPLC 15.33 min ,M2)). The crude materials were
combined and coupled according to GP 6 ,1 mM, 208C),
until conversion of the amine was complete ,4 h, HPLC
monitoring, M2). The reaction mixture was diluted with
CHCl3 ,40 mL) and after aqueous work-up, preparative
TLC ,20 cm£20 cm£1 mm, CHCl3/MeOH 10:2, 20 mg
runs) followed by GPC ,Sephadex LH 20, 1.5£9.0 cm2
column, CHCl3/MeOH 1:1) provided 13 mg ,3.0 mmol,
40%) of compound 1 as a colourless solid. 1H NMR
,300 MHz, DMSO-d6): dˆ0.35±0.69 ,m, 48H), 0.70±0.90
,m, 60H), 0.96 ,s, 18H), 1.04±1.31 ,m, 30H), 1.40±1.60 ,m,

2800
H.-D. Arndt et al. / Tetrahedron 58 42002) 2789±2801
Nijenhuis, W. F.; Zhou, X. J. Org. Chem. 1996, 61, 8866±
8874. ,l) Seebach, D.; Brunner, A.; BuÈrger, H.-M.; Reusch,
R. N.; Bramble, L. L. Helv. Chim. Acta 1996, 79, 507±517.
,m) Wagner, H.; Harms, K.; Koert, U.; Meder, S.; Boheim, G.
Angew. Chem. 1996, 108, 2836±2839 Angew. Chem., Int. Ed.
Engl. 1996, 35, 2643±2645. ,n) Tanaka, Y.; Kobuke, Y.;
Sokabe, M. Angew. Chem. 1995, 107, 717±719 Angew.
Chem., Int. Ed. Engl. 1995, 34, 693±694. ,o) Pregel, M. J.;
Jullien, L.; Canceill, J.; Lacombe, L.; Lehn, J.-M. J. Chem.
Soc., Perkin Trans 2 1995, 417±426 for earlier work see Ref.
3.
6H), 1.73±1.84 ,m, 2H), 1.89±2.05 ,m, 6H), 2.33±2.47 ,m,
4H), 2.80±3.01 ,m, 8H), 3.04±3.28 ,m, 12H), 3.55±3.76
,m, 8H), 4.02±4.32 ,m, 20H), 4.41±4.60 ,m, 8H), 6.84±
6.93 ,m, 7H, ar.), 6.95±7.03 ,m, 8H, ar.) 7.04±7.13 ,m,
8H, ar.), 7.23±7.31 ,m, 8H, ar.), 7.33±7.45 ,m, 13H, ar.),
7.47±7.66 ,m, 16H, ar.), 7.68±8.32 ,m, 32H, NH), 10.68 ,s,
2H, NH), 10.74 ,m, 6H, NH). HPLC: 9.3 min ,M3). MS
,ESI) for C232H318N40O34Si2: ,M12Na1) calcd 2156.7,
found 2156.1.
5. Clark, T. D.; Buehler, L. K.; Ghadiri, M. R. J. Am. Chem. Soc.
1998, 120, 651±656.
Acknowledgements
6. ,a) Arndt, H.-D.; Knoll, A.; Koert, U. Angew. Chem. 2001,
113, 2137±2140 Angew. Chem., Int. Ed. Engl. 2001, 40,
2076±2078. ,b) Schrey, A.; Vescovi, A.; Knoll, A.; Rickert,
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Support by the Volkswagen-Stiftung, the Deutsche
Forschungsgemeinschaft, Schering AG, the Fonds der
Chemischen Industrie and the Pinguin Stiftung is gratefully
acknowledged. We thank Dr P. Franke ,MALDI-TOF, FU
È
Berlin) and Dr G. Hohne ,TU Berlin) for mass spectrometric
support, and Dr A. Knoll ,HU-Berlin) for the single channel
conductance measurement of compound 1.
7. Neher, E.; Sakmann, B. Nature 1976, 260, 799±802.
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