Linear and cyclic β3-oligopeptides with functionalised side-chains (-CH2OBn, -CO2Bn, -CH2CH2CO2Bn) derived from serine and from aspartic and glutamic acid

Article abstract of DOI:10.1039/a805478i

The natural β-amino acid derivative Boc-Asp(β-OH)-OBn, as well as Boc-β-HGlu(OBn)-OH and Boc-β-HSer(OBn)-OH (prepared from appropriately protected glutamic acid and serine, respectively, by Arndt-Eistert homologation), were employed as building blocks for the synthesis of linear (11-20) and cyclic (21-23) β-oligopeptides consisting of two to six β-amino acids [using trichloroethyl (TCE) ester groups for C-terminal protection and pentafluorophenyl-ester activation for macrocyclisation]. While the linear derivatives are soluble enough for reactions and structural investigations in solution, the cyclo-β-tri- and -hexapeptides are not (according to FT-IR measurements they form networks of hydrogen bonds, perhaps consisting of so-called nanotubes). The CD spectra of the Boc-OTCE-protected (19) and of the unprotected (20) β-hexapeptides [β-Asp(OBn)-β-HGlu(OBn)-β-HSer(OBn)]₂ differ drastically, and only the unprotected form shows the familiar pattern of a negative Cotton effect between 210 and 220 nm (indicative of a 3₁₄ helix). An NMR analysis in methanol of the β-hexapeptide 20 with free termini reveals the presence of a single, central, left-handed helix turn (14-membered hydrogen-bonded ring). The results are discussed and compared with those obtained previously for analogous β-peptides carrying non-functionalised side chains.

Full text of DOI:10.1039/a805478i

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Linear and cyclic â3-oligopeptides with functionalised side-chains
(-CH2OBn, -CO2Bn, -CH2CH2CO2Bn) derived from serine and
from aspartic and glutamic acid
Jennifer L. Matthews,† Karl Gademann,‡ Bernhard Jaun and Dieter Seebach*
Laboratorium für Organische Chemie der Eidgenössischen Technischen Hochschule,
ETH-Zentrum, Universitätstr. 16, CH-8092 Zürich, Switzerland
Received (in Cambridge) 14th July 1998, Accepted 18th August 1998
The natural β-amino acid derivative Boc-Asp(β-OH)-OBn, as well as Boc-β-HGlu(OBn)-OH and Boc-β-HSer(OBn)-
OH (prepared from appropriately protected glutamic acid and serine, respectively, by Arndt–Eistert homologation),
were employed as building blocks for the synthesis of linear (11–20) and cyclic (21–23) β-oligopeptides consisting of
two to six β-amino acids [using trichloroethyl (TCE) ester groups for C-terminal protection and pentafluorophenyl-
ester activation for macrocyclisation]. While the linear derivatives are soluble enough for reactions and structural
investigations in solution, the cyclo-β-tri- and -hexapeptides are not (according to FT-IR measurements they form
networks of hydrogen bonds, perhaps consisting of so-called nanotubes). The CD spectra of the Boc–OTCE-
protected (19) and of the unprotected (20) β-hexapeptides [β-Asp(OBn)-β-HGlu(OBn)-β-HSer(OBn)]2 differ
drastically, and only the unprotected form shows the familiar pattern of a negative Cotton effect between 210 and 220
nm (indicative of a 314 helix). An NMR analysis in methanol of the β-hexapeptide 20 with free termini reveals the
presence of a single, central, left-handed helix turn (14-membered hydrogen-bonded ring). The results are discussed
and compared with those obtained previously for analogous β-peptides carrying non-functionalised side chains.
of proteinogenic α-amino acids with functionalised side chains
Introduction
(e.g., Lys, Glu, Thr, Trp) is possible, that these β3-amino acids
The synthesis and characterisation of peptides consisting of β-
can be incorporated into β-peptides using standard techniques
amino acids, the so-called β-peptides, is a field of research that
and that these β3-peptides do not undergo rapid degradation by
has been receiving more and more interest in recent years.1
a variety of enzymes.15 It is also of interest to note that the
Whilst some groups have concentrated their efforts on building
biodegradability of poly(α,β-,-aspartates) has been studied
β-peptides with cycloalkyl substituents2,3 or with allyl substitu-
and the degree of degradation was found to change according
ents,4 we have devoted our attentions to β-amino acids which
to the extent of branching found within the polymer structure.16
bear proteinogenic side chains.1,5 We began initially with
We now report our findings on the stepwise synthesis of lin-
ear and cyclic oligo(β--aspartates) and β-peptides containing
other functionalised β-amino acids together with an investi-
gation into the structural characterisation of these compounds.
simple alkyl side chains and prepared β3-peptides containing
β3-HVal-β3-HAla-β3-HLeu subunits6 and also oligopeptides
based entirely on β3-HAla—these being peptidic analogues
of oligomers of 3-hydroxybutanoic acid.7 Our work and that of
other groups came to the spectacular conclusion that these
Results and discussion
Synthesis
short-chain oligomers (6 or 7 residues in length) form
remarkably stable 314 helical structures in solution. Further
work has shown that other types of structures (a 2.51 helix3 and
an irregular helix5 containing 10- and 12-membered hydrogen-
bonded rings) also exist. Early work on polymers based on
β-amino acids (the nylon-3 derivatives) has also demon-
strated8,9 the presence of well defined secondary structures.
More recently, calculations on poly-β-aspartates have suggested
that three different types of helix (174, 41 and 134) could be
adopted by these compounds.10,11 Oligo-β-aspartates have been
synthesised; however, no structural characterisation has been
performed.12 Powder X-ray measurements showed that certain
cyclic β-peptides form tube-like stacked structures in the solid
state,13 and, recently, cyclic β3-peptides have been shown to
induce Kϩ conductance through lipid bilayers.14
The first stage of the synthetic strategy design was to identify a
suitable set of orthogonal protecting groups. The amino acids
Boc-Glu(OBn)-OH 1, Boc-Ser(OBn)-OH 2 (Scheme 1) and
Boc-Asp(OH)-OBn 9 (Scheme 2) are all commercially available
and hence provided an attractive starting point. We opted to
protect the C-termini as 2,2,2-trichloroethyl (TCE) esters which
are easily prepared using standard techniques and deprotection
can be achieved by several methods which will leave the Boc
group and benzyl ester or ether groups unaffected.17
In order to prepare the β3-amino acids derived from glutamic
acid and serine, we followed the well established procedures18
using the Arndt–Eistert homologation protocols. Formation of
the diazoketones 3, 4 proceeded without any difficulties, and
homologation with silver trifluoroacetate and triethylamine in
aq. THF produced the free amino acids 5, 6 in excellent yields.
Subsequent protection of the C-terminus with trichloroethanol
and DCC–DMAP yielded the fully protected amino acids 7, 8
(Scheme 1). Boc-Asp(OH)-OBn 9 was also subjected to the
conditions of the final protection step to give the building block
Boc-Asp(OTCE)-OBn 10 in excellent yield, Scheme 2.
We have also recently demonstrated that the homologation
† Royal Society Postdoctoral Research Fellow 1995–1996; Schweizer-
ischer Nationalfonds zur Förderung der wissenschaftlichen Forschung
Fellowship Holder 1996–1997 (Grant No. 21-40659.94). Present
address: Department of Chemistry, University of Glasgow, Joseph
Black Building, Glasgow, UK G12 8QQ.
‡ Part of the Diplomarbeit (Master’s thesis, 1996) and of the projected
Ph.D. thesis of K. G., ETH Zürich.
The β-peptides derived entirely from aspartic acid or glu-
J. Chem. Soc., Perkin Trans. 1, 1998, 3331–3340
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malic acid. However, the previous calculations carried out on
R
R
H
poly(β-aspartates) considered10,11 compounds bearing ‘pro-
tected’ side chains and so we continued to investigate the struc-
tures of our protected oligopeptides.
Boc
OH
Boc
i
N
N
N2
H
H
O
O
We also felt that the lack of solubility, necessary for the
deprotection step, might be overcome by using peptides based
on more than one amino acid and therefore we developed a
synthesis, based on a strategy analogous to that shown above,
of β3-peptides derived from a sequence of β-Asp-β3-HGlu-β3-
HSer. These compounds were built up via the dimer Boc-
β3-HGlu(OBn)-β3-HSer(OBn)-OTCE 17, the trimer Boc-β-
Asp(OBn)-β3-HGlu(OBn)-β3-HSer(OBn)-OTCE 18 and the
1 R = [CH2]2CO2Bn
2 R = CH2OBn
3 R = [CH2]2CO2Bn (82%)
4 R = CH2OBn (83%)
ii
hexamer
Boc-[β-Asp(OBn)-β3-HGlu(OBn)-β3-HSer(OBn)]2-
R
O
R
O
iii
OTCE 19. Once again, deprotection of the terminal protect-
ing groups of compound 19 proceeded smoothly to yield the
hexamer 20 but all attempts to deprotect the side chains (both
before and after deprotection of the termini) failed. Undeterred
by this, we went on to investigate the structures adopted by
these molecules.
Boc
Boc
N
O
CCl3
N
OH
H
H
5 R = [CH2]2CO2Bn (71%)
6 R = CH2OBn (58%)
7 R = [CH2]2CO2Bn (92%)
8 R = CH2OBn (94%)
Scheme 1 Reagents and conditions: i, (1) ClCO2Et, Et3N, THF, Ϫ20 ЊC,
(2) CH2N2, Et2O; ii, CF3CO2Ag, aq. THF; iii, CCl3CH2OH, DCC,
DMAP, CH2Cl2.
CO2Bn
OBn
O
O
CO2Bn
CO2Bn
O
O
Boc
i
(92%)
N
N
H
O
CCl3
Boc
H
Boc
N
O
CCl3
N
OH
17 (64%)
H
H
CO2Bn
9
10
OBn
CO2Bn
Scheme 2 Reagents and conditions: i, CCl3CH2OH, DCC, DMAP,
O
O
O
O
CH2Cl2.
Boc
Boc
N
H
N
H
N
H
O
CCl3
tamic acid were then readily prepared using a solution-phase
block-coupling procedure. This resulted in the formation of the
dimer 11, trimer 12 and hexamer 13 of aspartic acid (Scheme 3)
and the analogous oligomers 14, 15, 16 of β3-HGlu (Scheme 4).
18 (31%)
CO2Bn
OBn
CO2Bn
O
O
N
H
N
N
H
O
2
CCl3
Repetitive
Fragment
Coupling
CO2Bn
CO2Bn
O
O
H
Boc
Boc
OR
OR
N
N
i) or ii) then iii)
19 (55%)
H
H
n
m
11 m = 2 (51%)
12 m = 3 (84%)
13 m = 6 (54%)
CO2Bn
OBn
CO2Bn
O
O
O
Scheme 3 Reagents and conditions: i, Boc deprotection (CF3CO2H);
ii, Ester deprotection (Zn, AcOH); iii, Coupling of two half-protected
fragments (EDC, HOBt, NEt3); R = CH2CCl3; Bn = CH2Ph.
F3CCO2H • H
N
H
N
H
N
H
OH
2
20 (55%)
CO2Bn
O
CO2Bn
O
Repetitive
Fragment
Coupling
In analogy to our previous work,6,7,13 we also synthesised the
cyclo-β3-peptides based on these β-amino acids. Using the
established procedures, we converted the trimers 12 and 15 into
pentafluorophenyl esters by deprotection of the trichloroethyl
esters and coupling of the resulting acids with pentafluoro-
phenol with 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide
(EDC) in DMF. Subsequent deprotection of the N-terminus
and then addition of a dilute solution of the pentafluorophenyl
ester in acetonitrile to a solution of ethyldiisopropylamine
(DIPEA) in acetonitrile at 70 ЊC resulted in the formation
of the expected cyclo-β3-peptides 21 and 22 in good yields
(Scheme 5). The hexamer 13 of aspartic acid was also subjected
to the same procedures and this resulted in the formation of the
cyclo-β3-peptide 23 in good yield. The analogous compounds
based on β3-HAla had been found to be extremely insoluble
white powders. We had hoped that these new cyclopeptides
would prove to be more soluble given the nature of their
aromatic side chains. However, this proved not to be the case,
with white precipitates forming as before which proved to be the
pure cyclic peptides. These compounds turned out to have very
similar properties when compared with the β3-HAla analogues
Boc
Boc
OR
OR
N
N
i) or ii) then iii)
H
H
n
m
14 m = 2 (83%)
15 m = 3 (81%)
16 m = 6 (72%)
Scheme 4 Reagents and conditions: i, Boc deprotection (CF3CO2H);
ii, Ester deprotection (Zn, AcOH); iii, Coupling of two half-protected
fragments (EDC, HOBt, NEt3); R = CH2CCl3; Bn = CH2Ph.
The protecting groups at the N- and C-termini were readily
removed under the appropriate conditions. However, all
attempts to remove the benzyl protecting groups from the side
chains failed; we believe this can be accounted for by the poor
solubility of the oligopeptides in the reaction solvent. Given
our previous experience of a similar problem,7 we attempted the
hydrogenolysis using trifluoroethanol as the solvent but this
also proved to be fruitless. We have still been unable to remove
these protecting groups, which is unfortunate as we had hoped
to make a direct comparison of the structural characteristics of
these oligopeptides with the analogous esters19 derived from
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J. Chem. Soc., Perkin Trans. 1, 1998, 3331–3340

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CO2Bn
CO2Bn
CO2Bn
O
O
O
Boc
N
N
N
O
CCl3
H
H
H
12
i, ii, iii, iv
BnO2C
O
O
NH HN
CO2Bn
H
N
O
CO2Bn
21 (72%)
Fig. 1 CD Spectra of β-hexapeptides 19 (solid line) and 20 (dashed
line) (~0.2 m in MeOH; θ in deg cm2 dmolϪ1). The β-hexapeptide 19
exhibits a CD pattern that was never observed for β-peptides before; it
may be indicative of a new secondary structure in solution.23 Upon
deprotection, the CD spectrum of compound 20 is reminiscent of the
CD pattern indicative of a 314 helix.
BnO2C
O
O
NH HN
CO2Bn
H
N
IR Spectroscopy
O
It is widely accepted in the literature that Fourier transform
(FT)-IR spectroscopy may deliver valuable information about
the hydrogen-bonding environment of peptides and proteins,
both in solution and in the solid state.20 In our previous work,
we have used powder X-ray techniques13 to show that cyclic
β-peptides composed of β3-HAla adopt stacked tube-like
structures (so-called nanotubes) in the solid state. These com-
pounds also exhibit distinct bands in their FT-IR spectra, indi-
cating strong hydrogen bonding in a β-sheet-type manner.
FT-IR Spectroscopy on the cyclic β-peptides 21, 22 and 23
revealed very similar bands when compared with cyclo-β3-
HAla; i.e. for 22, the amide A band, indicative of the hydrogen-
bonding status, appears near 3294 cmϪ1, whereas the Amide I
(1648 cmϪ1) and Amide II (1559 cmϪ1, shoulders at 1554 and
1535 cmϪ1) bands are characteristic of β-sheet-type structure.7,20
These bands indicate the existence of a network of tight hydro-
gen bonds and, hence, it can be concluded that these cyclic
peptides also exist as nanotubes in the solid state.21
CO2Bn
22 (54%)
BnO2C
O
O
NH HN
CO2Bn
O
BnO2C
O
NH
HN
CO2Bn
H
N
H
N
O
O
CO2Bn
CO2Bn
23 (56%)
Scheme 5 Reagents and conditions: i, Ester deprotection (Zn, AcOH);
ii, C6F5OH, EDC, CH2Cl2; iii, Boc deprotection (CF3CO2H, CH2Cl2);
iv, Pri2NEt, CH3CN, syringe pump, several hours, 70 ЊC.
CD Spectroscopy
Due to their low solubility, it proved to be impossible to charac-
terise the cyclic β-peptides by CD-spectroscopy. However,
linear β-hexapeptides 19 and 20 were examined. The fully
protected β-hexapeptide gave rise to a CD spectrum of a type
that had not been previously observed (Fig. 1, solid line). It is
characterised by an intense maximum at 198 nm, with two
shoulders at 207 and 216 nm.22 It may be that a novel secondary
structure, as indicated by the new CD spectrum of β-hexa-
peptide 19, remains yet to be discovered by 2D-NMR analyses.
Upon deprotection of the N- and C-terminal protecting
groups, the CD spectrum of the deprotected β-peptide 20
changed dramatically (Fig. 1, dashed line). We observed a
minimum at 222 nm with a shoulder at 210 nm, a zero crossing
at 208 nm and a maximum at 198 nm. On the one hand, this
CD-pattern was somewhat reminiscent of that of a 314 helix
(maximum and zero crossing) and on the other hand, more
surprisingly to us, of that of an α-helix of α-peptides (line shape
and position of the minimum). However, CD cannot give
definitive proof of structure and we decided therefore to carry
out extensive 2D-NMR studies on β-hexapeptide 20.
and we therefore assume that they also adopt a similar stacked
tube-like structure13 and it is conceivable that the side chains
also stack in such a way as to increase the insoluble nature of
these compounds. There are ongoing attempts being made to
analyse the structures of these cyclo-β3-peptides using powder
diffraction techniques.
Spectroscopic characterisation of the â-peptides
So far, we had focused only on β-peptides with β-amino acids
corresponding to valine, alanine and leucine, i.e. peptides
bearing aliphatic and apolar side chains. It was therefore
of great importance to investigate the effects of amino acid
substitution on the remarkable ability of small β-peptides to
form distinct secondary structures in solution. The correlation
between the amino acid sequence and induced structure, well
known for α-peptides and α-proteins, is yet to be established for
β-peptides. We wondered whether small β-peptides composed
of protected β-amino acids (Asp, Glu, Ser) with polar groups,
hydrogen-bond acceptors and large benzyl-ester side chains
would also form stable and distinct secondary structures in
solution and in the solid state. Hence, we investigated their
structural properties by optical and NMR spectroscopic
techniques.
Structure determination by NMR spectroscopy
2D-NMR studies of β-hexapeptide 20 were carried out on a
J. Chem. Soc., Perkin Trans. 1, 1998, 3331–3340
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Table 1 Assignment of all 1H resonances (500 MHz; CD3OH) and NH, C(β)-H coupling constants for compound 20. Aromatic protons and NH3
of the terminal β-HAsp(OBn) have not been assigned (n/a)
Residue
β--Asp(OBn)1
β-HGlu(OBn)2
β-HSer(OBn)3
β--Asp(OBn)4
β-HGlu(OBn)5
β-HSer(OBn)6
NH, NH3
C(β)-H
C(α)-H
C(α)-HЈ
C(γ)-H
C(γ)-HЈ
C(δ)-H
n/a
4.34
3.1
7.87
4.39
2.61
2.21
2.4
2.39
1.86
1.75
9.3
8.23
4.43
2.67
2.53
3.41
3.41
8.07
4.98
2.72
2.49
7.82
4.39
2.43
2.27
2.35
2.34
1.82
1.61
9.5
7.87
4.46
2.53
2.49
3.28
3.28
2.13
C(δ)-HЈ
J[NH, C(β)-H]
n/a
8.9
8.7
9
Table 2 NOEs extracted from the 150 ms ROESY spectrum (500
MHz; CD3OH) for compound 20. The NOEs have been classified
in three distance categories according to their relative volume in the
contour plot: Strong, medium and weak corresponding to 3.0 Å, 3.5 Å
and 4.5 Å. SD stands for sequential difference, an SD of 0 indicating an
intraresidual and an SD of 1 a sequential NOE
Residue
Atom
Residue
Atom
Intensity
SD
1
2
2
2
3
3
3
3
4
4
4
4
5
5
5
5
6
6
6
C(α)-H
C(δ)-H2
C(α)-H
C(α)-H
C(γ)-H2
C(α)-H
C(α)-HЈ
C(α)-H
NH
C(α)-H
C(α)-H
C(α)-H
C(β)-H
C(β)-H
C(α)-H
C(δ)-H2
C(β)-H
C(β)-H
C(γ)-H2
4
2
3
5
3
3
3
4
3
4
4
5
3
5
5
5
3
4
6
C(β)-H
C(β)-H
NH
C(β)-H
C(β)-H
NH
NH
NH
NH
NH
C(β)-H
C(β)-H
NH
C(α)-H
NH
NH
NH
medium
medium
strong
medium
strong
medium
weak
strong
weak
medium
strong
medium
weak
medium
weak
weak
medium
weak
medium
3
0
1
3
0
0
0
1
1
0
0
1
2
0
0
0
3
2
0
NH
C(β)-H
500 MHz spectrometer for solutions in CD3OH. We used
DQF-COSY§ and TOCSY§ techniques to assign all 1H reson-
ances (except aromatic and benzylic protons) in their respective
spin-systems. HSQC¶ and HMBC¶ experiments resulted in
assignment of the sequence (long-range C,H correlations across
the peptide bond) and the 1H chemical shifts are given in Table
3
1. From the large J[NH, C(β)-H] coupling constants, ranging
from 8.7 to 9.5 Hz (see Table 1), it can be clearly concluded23
that the NH and C(β)-H protons must be fixed in an anti-
periplanar arrangement; this indicates an ordered secondary
structure in solution as random rotation around this bond
would result in much smaller coupling constants. We there-
fore decided to perform rotating-frame nuclear Overhauser
enhancement (ROESY) experiments to gather information
about the three-dimensional structure in solution, and the
extracted NOEs are summarised in Table 2. Due to resonance
overlap of the NH-resonance of β-HGlu2(OBn) and β-HSer6-
(OBn), NOEs for these protons could not be properly assigned.
From the 19 observed NOEs, 10 were intraresidual, 4 were
sequential, 2 were from residue i to (i ϩ 2) and 3 were from
residue i to (i ϩ 3). This i-to-(i ϩ 2) and -(i ϩ 3) pattern,
observed before by us,6 is charateristic for a 314 helical structure
in solution. These NOEs were then classified according to their
relative volume in the contour plot of the 150 ms ROESY spec-
trum in three distance categories with the following upper-
Fig. 2 Side views of the helical conformation of β-hexapeptide 20.
NMR solution structure of β-hexapeptide 20 in MeOH solution. This
small β-hexapeptide has a defined, clearly helical structure, with a
hydrogen bond from NH of residue 2 to the C᎐O of residue 4. All
᎐
carbon-bound hydrogens and all side chains have been omitted for
clarity, β-HAsp(OBn) residues are coloured in blue, β-HGlu(OBn)
residues coloured in green and β-HSer(OBn) residues are in yellow. This
figure was generated using VMD24 and Raster3D.25
bound distance limits: strong < 3.0 Å, medium < 3.5 Å and
weak < 4.5 Å. These distance restraints were then used together
with 5 dihedral angle restraints for the NH,C(β)-H dihedral
angles, derived from the coupling constants, in simulated
annealing. This calculation yielded 10 structures, which are
superimposed and displayed in Fig. 2. We selected this bundle
as representative for the structure in solution.
The structure is clearly helical, left-handed or (M), with a 14-
membered hydrogen-bonded ring from NH of residue 2 to C᎐O
of residue 4. The other possible hydrogen bonds, NH of res-
᎐
§ Double-quantum-filtered chemical shift-correlation spectroscopy;
phase-sensitive two-dimensional total correlation spectroscopy.
¶ Heteronuclear single-quantum coherence spectroscopy; two-
dimensional heteronuclear multiple-bond correlation spectroscopy.
idues 1 and 3 to C᎐O of residue 3 and 5, that would be closed in
᎐
the clear 314 helix are not present here. Therefore, the dominant
3334
J. Chem. Soc., Perkin Trans. 1, 1998, 3331–3340

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conformational type may be characterised as a transition struc-
ture between a random coil and a 314 helix. The additional
hydrogen-bond acceptors in or the bulkiness of the side chains
appear to weaken the helix. Furthermore the large aromatic
groups dominate the structure and may therefore have a destab-
ilising effect on the helix. There is no experimental evidence for
aromatic stacking (lack of interresidual side-chain NOEs), and
hence this effect appears not to be a stabilising contribution.
or Degussa. All other reagents were used as received from
Fluka. Reactions carried out with the exclusion of light were
performed in flasks completely wrapped in aluminium foil.
Flash column chromatography was carried out using silica
gel (Fluka 60, 0.04–0.053 mm). TLC was carried out on com-
mercially available pre-coated plates (Fluka Kieselgel 60)
and the compounds were detected with anisaldehyde (9.2 ml
anisaldehyde, 3.75 ml acetic acid, 12.6 ml conc. H2SO4, 340 ml
ethanol). Analytical HPLC was performed on a Waters HPLC
System (pump type 515, automated gradient controller 680,
data module type 746, tunable absorbance detector type 484),
Macherey-Nagel Nucleosil 5 C18 column. Preparative HPLC
was performed on a Knauer HPLC system [pump type 64, pro-
grammer 50, UV detector (variable-wavelength monitor)],
Macherey-Nagel 7 C18-column. Mps were measured on a
Büchi 510 apparatus and are uncorrected. IR spectra were
measured on a Perkin-Elmer 782 infrared spectrometer. 1H and
13C NMR spectra were recorded on Bruker AMX-400 (400
MHz), Varian Gemini 300 (300 MHz) or Gemini 200 (200
MHz) spectrometers: internal standard CHCl3 signal [δ = 7.26
(1H); δ = 77.0 (13C)]. Chemical shifts are quoted in parts per
million; J-values are given in Hz. Mass spectra were recorded
with VG-ZAB2-SEQ (FAB) or Bruker-Reflex-MALDI-TOF
(MALDI-TOF) spectrometers. 3-Nitrobenzyl alcohol (3-
NOBA) was used as matrix for FAB. Optical rotations were
measured with a Perkin-Elmer 241 polarimeter at rt (1 dm
cells, concentration c in g/100 cm3). Circular dichroism (CD)
spectra were recorded on a Jobin-Yvon Mark III system
between 190 and 300 nm (peptide concentration 0.2 mol
dmϪ3 in TFE); the molar ellipticity (θ) is reported in deg cm2
dmolϪ1. Microanalyses were carried out by the Mikro-
analytisches Laboratorium of the Laboratorium für Organ-
ische Chemie, ETH-Zürich.
Conclusions
A successful strategy for the synthesis of β-peptides with side
chains carrying OH and CO2H functional groups has been
developed, using an N-Boc/O-Bn/CO2-Bn/CO2-TCE combin-
ation of protecting groups, and the derived cyclic β-tri- and
-hexapeptides have been shown to be readily available. So far,
we have not found conditions for cleavage of the side chain
benzyl-ether and -ester groups (LiCl-solubilisation conditions26
have not yet been tested).
The dramatic influence of terminal protection upon the
secondary structure of β-peptides (CD spectra of compounds
19 and 20 in Fig. 1) has been observed before, and it was inter-
preted as a consequence of pole/charge interaction.5 The pres-
ence of only a single 314-helical turn in methanol solution of the
terminally unprotected β-peptide 20 (Fig. 2) is intriguing, espe-
cially since this turn appears to suffice to cause a CD pattern
(albeit of weak intensity), typical of the 314-helix of β-peptides:
the NMR structure reported herein may be considered as the
first secondary structure on the way from a random-coil to a
full-helix structure of a β-peptide, and this is in aggreement
with computational results (using the GROMOS96 force
field),27 as well as with our interpretation of thermochemical
measurements (temperature-dependent CD and NMR spectro-
scopy, microcalorimetry),28 indicating that helical folding/
unfolding of β-peptides might occur by a non-cooperative
mechanism. Whether the CD spectrum of the protected β-hexa-
peptide 19 arises from a hitherto unknown β-peptide secondary
structure, or from a mixture of different (known and/or
unknown) conformations, remains to be seen. Also, it will be
exciting to learn about the structure of a fully deprotected,
certainly very water-soluble, β-peptide of the series described
herein, should we ever obtain it!
General procedure A: preparation of diazoketones
In a similar fashion to the literature procedure,18 the N-Boc-
protected amino acid was dissolved under argon in THF (0.2 )
and then cooled to Ϫ25 ЊC. Triethylamine (1 equiv.) and ethyl
chloroformate were added to the solution. After 15 min the
suspension was allowed to warm to 0 ЊC. A solution of diazo-
methane in ‘ether’ was added until the intensive yellow colour
persisted over a long period. The mixture was allowed to warm
to rt and then stirred for 3 h. Excess of diazomethane was
destroyed by addition of a small amount of acetic acid. After
aqueous work-up by successive extraction with saturated aq.
sodium hydrogen carbonate, aq. ammonium chloride and
aq. sodium chloride, the organic layer was separated, dried
(MgSO4) and concentrated. Recrystallisation or FC afforded
the pure diazoketone.
The insolubility of the cyclic β-peptides [β-Asp(OBn)]3,6
(21, 23), [β-HGlu(OBn)]3 (22) as well as FT-IR data compares
well with that of the previously reported [β-HAla]4,7,13 [β-HVal-
6
β-HAla-β-HLeu]1,2 and [β-HLeu]4.14 For the cyclo-β-tetra-
peptides a stacking of the rings (with formation of tubes,
involving an infinite network of hydrogen bonds as in pleated
sheets) has been found in the solid state.7,13 This kind of
structure has not yet been demonstrated for cyclo-β-tri- or
-hexapeptides,29 and we have started collaborations with groups
specialising in structure determinations by new X-ray and
NMR methodologies amenable to substances of which no
single crystals can be obtained.
General procedure B: preparation of the homologated amino acid
derivatives
In a similar fashion to the literature procedure,18 the diazo-
ketone was dissolved in THF (0.1 ) with the addition of 10%
(v/v) water under the exclusion of light at Ϫ25 ЊC. Silver tri-
fluoroacetate (0.11 equiv.) dissolved in triethylamine (2.9 equiv.)
was added and the resulting mixture was stirred for 3 h as it
was allowed to warm to rt. The solvent was removed under
reduced pressure and the residue was dissolved in ethyl acetate.
The organic phase was extracted with saturated aq. NaHCO3,
and the resulting aqueous phase was adjusted to pH 2 with aq.
HCl and extracted with ethyl acetate. The ethyl acetate extracts
were washed with saturated aq. sodium chloride, dried (MgSO4)
and the solvent was removed under reduced pressure.
Experimental
General
FAB = fast-atom bombardment; FC = flash chromatography;
HV = high vacuum [0.1–0.01 bar (103–104 Pa)]; β-HXxx =
β-Homoamino acid; MALDI-TOF = matrix-assisted laser de-
sorption ionisation-time of flight; TFE = trifluoroethanol.
DMF and acetonitrile were distilled under reduced pressure
from CaH2 and stored over 4 Å molecular sieves. Solvents for
chromatography and work-up procedures were distilled from
Sikkon (anhydrous CaSO4; Fluka). ‘Ether’ refers to diethyl
ether; other ethers are named systematically. Triethylamine was
distilled from CaH2 and stored over potassium hydroxide. Ethyl
chloroformate was distilled, and stored at Ϫ25 ЊC under argon.
Amino acid derivatives were purchased from Bachem, Senn
General procedure C: Boc deprotection of amino acids
In a similar fashion to the literature procedure,5 the Boc-
protected amino ester was dissolved in CH2Cl2 (0.5 ). The
J. Chem. Soc., Perkin Trans. 1, 1998, 3331–3340
3335

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mixture was cooled in an ice-bath and stirred. An equal volume
of TFA was added and the solution was stirred for 1 h at 0 ЊC
and for 1 h at rt. The solvent was removed under reduced
pressure, the residue was taken up twice in toluene and again
the solvent was removed. The residue was then taken up in
CH2Cl2 and the solvent was removed. The trifluoroacetate salt
was dried under HV for 1 h, characterised by 1H NMR and was
used for peptide coupling without further purification.
was transformed according to general procedure A. Recrystal-
lisation from ethyl acetate–diisopropyl ether yielded compound
4 (4.5 g, 83%) as yellow needles, mp 87–88 ЊC; [α]Dr.t. Ϫ24.2 (c
1.13, CHCl3) (Found: C, 60.0; H, 6.4; N, 13.0. C16H21N3O4
requires C, 60.2; H, 6.6; N, 13.2%); νmax(CHCl3)/cmϪ1 2112,
1710 and 1639; δH(300 MHz; CDCl3) 1.45 (9H, s, t-Bu), 3.58–
3.63 (1H, m, CHH), 3.84–3.87 (1H, m, CHH), 4.32 (1H, br m,
HCNH), 4.52 (2H, s, H2CPh), 5.42 (1H, br d, NH), 5.56 (1H,
br s, N2CH) and 7.27–7.35 (5H, m, Ph); δC(75 MHz; CDCl3)
28.4, 54.3, 57.8, 69.9, 73.6, 80.4, 128.0, 128.2, 128.7, 137.7,
155.7 and 193.2; m/z (FAB) 958 [14%, (3M ϩ 1)ϩ], 639 [47,
(2M ϩ 1)ϩ], 320 [97, (M ϩ 1)ϩ], 264 (40.4) and 236.1 (100).
General procedure D: peptide coupling using EDC
The trifluoroacetate salt of the amino ester was dissolved in
CH2Cl2 (0.2 ) and cooled to 0 ЊC. This was treated successively
with Et3N (5 equiv.), HOBt (1.2 equiv.), the Boc-protected
amino acid (1 equiv.) and EDC (1.2 equiv.). The mixture was
stirred at 0 ЊC for 30 min, then allowed to warm to rt and stirred
for 16 h. The solvent was removed under reduced pressure, the
residue was taken up in ethyl acetate and washed successively
with saturated aq. NaHCO3, aq. NaHSO4 (1 ) and aq. NaCl,
and the organic phase was separated, dried (MgSO4) and con-
centrated. FC or recrystallisation yielded the pure peptide.
(3S)-3-(tert-Butoxycarbonylamino)hexanedioic acid 6-benzyl
ester 5. The diazoketone 3 (10 g, 27.7 mmol) was transformed
according to general procedure B. Recrystallisation from ethyl
acetate–pentane gave compound 5 (6.9 g, 71%) as an amorph-
ous solid, mp 97–98 ЊC; [α]Dr.t. Ϫ12.7 (c 1.0, CHCl3) (Found: C,
61.5; H, 7.2; N, 4.0. C18H25NO6 requires C, 61.3; H, 7.2; N,
4.0%); νmax(CHCl3)/cmϪ1 3436, 3032, 2980, 1712, 1501 and
1454; δH(300 MHz; CDCl3) 1.42 (9H, s, t-Bu), 1.86–1.94 (2H,
m), 2.43–2.48 (2H, m), 2.58 (2H, m), 3.95 (1H, br m, HCNH),
5.05 [1H, br d, J 9.0, C(O)NH], 5.12 (2H, s, H2CPh), 5.96 (1H,
br, CO2H) and 7.29–7.39 (5H, m, arom. H); δC(75 MHz;
CDCl3) 28.4, 29.4, 31.2, 39.2, 47.1, 66.6, 79.9, 128.5, 128.5,
128.8, 136.1, 155.8, 173.5 and 176.5; m/z (FAB) 1076.4 [4.4%,
(3M ϩ Na)ϩ], 725.2 [45.9, (2M ϩ Na)ϩ], 703.2 [33.9,
(2M ϩ 1)ϩ], 374.1 [60.2, (M ϩ Na)ϩ], 352.1 [48.7, (M ϩ 1)ϩ],
296.1 (81.4), 252.1 (100), 192.1 (7.0) and 137.0 (29.56).
General procedure E: cyclisation of oligopeptides
In a similar fashion to the literature procedure,7 a solution of
the carboxylic acid derivative in DMF (0.4 ) was treated at rt
with pentafluorophenol (1 equiv.) and EDC (1 equiv.). After 16
h, the mixture was evaporated and the residue was dissolved in
CHCl3. The resulting solution was washed successively with 1 
aq. HCl and brine. The org. phase was dried (MgSO4) and
evaporated. The residue was dissolved in CH2Cl2 (0.5 ) and an
equal volume of TFA was added at 0 ЊC. The reaction mixture
was stirred for 1 h at 0 ЊC and for 1 h at rt. The solvent was
evaporated, the residue was taken up twice in toluene and evap-
orated. The residue was dissolved in CH3CN (0.025 ) and was
slowly added to a solution of Hünig’s base (DIPEA) in CH3CN
(3.3 m) at 70 ЊC (bath temp.) using a syringe pump over
several hours. The resulting precipitate was filtered off and
dried under HV.
(3R)-4-Benzyloxy-3-(tert-butoxycarbonylamino)butanoic acid
6. The diazoketone 4 (4.25 g, 13.3 mmol) was transformed
according to general procedure B. Recrystallisation from ethyl
acetate–‘ether’–pentane yielded compound 6 (2.39 g, 58%) as a
solid, mp 73–74 ЊC; [α]Dr.t. ϩ15.1 (c 1.05, CHCl3) (Found: C, 61.9;
H, 7.4; N, 4.5. C16H23NO5 requires C, 61.9; H, 7.5; N, 4.5%);
νmax(CHCl3)/cmϪ1 3438, 2982, 2931, 2865, 1710, 1501 and 1454;
δH(300 MHz; CDCl3) 1.44 (9H, s, t-Bu), 2.68 (2H, d, J 9.2),
3.55–3.61 (2H, m), 4.16 (1H, br m, HCNH), 4.52 (2H, s,
H2CPh), 5.17 [1H, br d, C(O)NH] and 7.29–7.38 (5H, m, arom.
H); δC(75 MHz; CDCl3) 28.4, 36.2, 47.2, 71.1, 73.4, 80.0, 127.9,
128.0, 128.7, 138.1, 155.8 and 176.2; m/z (FAB) 619.3 [15.9%,
(2M ϩ 1)ϩ], 332.1 [12.8, (M ϩ Na)ϩ], 310.1 [78.9, (M ϩ 1)ϩ],
254.1 (100), 210.1 [96.0, (M Ϫ Boc)ϩ], 154.0 (43.9) and 137.0
(38.8).
General procedure F: removal of the trichloroethyl ester
protecting group
The peptide was dissolved in 90% (v/v) HOAc (0.03 ).
Zn-powder (80 equiv.) was activated using 6  aq. HCl, filtered
off and washed successively with water and ‘ether’. The result-
ing solid was added to the reaction mixture and stirring was
continued for 16 h at rt. The resulting suspension was filtered
and evaporated. The residue was taken up in ethyl acetate and
washed successively with 1  aq. NaHSO4 and brine. The
organic layer was dried (MgSO4) and the solvent was removed
under reduced pressure.
(3S)-3-(tert-Butoxycarbonylamino)hexanedioic acid 6-benzyl
1-(2,2,2-trichloroethyl) diester 7. A solution of compound 5 (5.9
g, 16.8 mmol) in CH2Cl2 (25 ml) was cooled to 0 ЊC and 2,2,2-
trichloroethanol (1.8 ml, 18.5 mmol), DCC (3.6 g, 18.5 mmol)
and DMAP (226 mg, 1.85 mmol) were added. The reaction
mixture was allowed to warm to rt and stirring was continued
for 16 h. The solvent was evaporated off and the residue was
dissolved in ethyl acetate and filtered through Celite. Evapor-
ation of the solvent and purification by FC (ethyl acetate–
pentane, 1:5) gave compound 7 (7.4 g, 92%) as an amorphous
solid, mp 65–66 ЊC; [α]Dr.t. Ϫ13.6 (c 1, CHCl3) (Found: C, 49.6; H,
5.3; N, 2.9. C20H26Cl3NO6 requires C, 49.8; H, 5.4; N, 2.9%);
νmax(CHCl3)/cmϪ1 1709, 1501 and 1455; δH(300 MHz; CDCl3)
1.42 (9H, s, t-Bu), 1.88–1.96 (2H, m), 2.47 (2H, t, J 7.2), 2.70
(2H, d, J 5.5), 4.00 (1H, br m, HCNH), νA = 4.77, νB = 4.73 (2H,
AB, JAB 12.1, OCH2CCl3), 4.93 [1H, br d, J 8.7, C(O)NH], 5.12
(2H, s, H2CPh) and 7.30–7.40 (5H, m, arom. H); δC(75 MHz;
CDCl3) 28.4, 29.4, 31.1, 39.2, 47.3, 66.6, 74.1, 79.8, 94.9, 128.5,
128.8 (2C), 136.1, 155.6, 170.0 and 173.2; m/z (FAB) 967.0
[43.8%, (2M ϩ 1)ϩ], 484.0 [35.6, (M ϩ 1)ϩ] and 381.9 [100.0,
(M Ϫ Boc)ϩ].
(4S)-4-tert-Butoxycarbonylamino-6-diazo-5-oxohexanoic acid
benzyl ester 3. Boc-Glu(δ-OBn)-OH (20 g, 59.3 mmol) 1 was
transformed according to general procedure A. Recrystallis-
ation from ‘ether’–pentane yielded compound 3 (17.5 g, 82%)
as yellow needles, mp 68–70 ЊC; [α]Dr.t. Ϫ17.1 (c 1.07, CHCl3)
(Found: C, 59.9; H, 6.2; N, 11.6. C18H23N3O5 requires C, 59.8;
H, 6.4; N, 11.6%); νmax(CHCl3)/cmϪ1 2112, 1712 and 1643;
δH(300 MHz; CDCl3) 1.41 (9H, s, t-Bu), 1.77–1.89 (1H, m,
CHH), 2.10–2.20 (1H, m, CHH), 2.37–2.57 (2H, m, H2C), 4.24
(1H, br m, HCNH), 5.09–5.12 (2H, m, H2CPh), 5.32 [1H, br d,
J = 7.5, C(O)NH], 5.47 (1H, br s, N2CH) and 7.26–7.36 (5H, m,
arom. H); δC(75 MHz; CDCl3) 27.8, 28.4, 30.2, 54.2, 56.7, 66.7,
80.2, 128.5, 128.5, 128.8, 136.0, 155.8, 173.1 and 193.63; m/z
(FAB) 745.1 [1.2%, (2M ϩ Na)ϩ], 723.3 [2.4, (2M ϩ 1)ϩ], 362.1
[19.6, (M ϩ 1)ϩ], 278.1 (100), 236.1 (18.4), 192.1 (76.4), 154.1
(75.6), 136.0 (86.0) and 107.0 (43.2).
(1S)-(1-Benzyloxymethyl-3-diazo-2-oxopropyl)carbamic
acid tert-butyl ester 4. Boc-Ser(OBn)-OH (5.01 g, 16.9 mmol) 2
2,2,2-Trichloroethyl
onylamino)butanoate 8. A solution of compound 6 (4.34 g, 14.0
(3R)-4-benzyloxy-3-(tert-butoxycarb-
3336
J. Chem. Soc., Perkin Trans. 1, 1998, 3331–3340

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mmol) in CH2Cl2 (25 ml) was cooled to 0 ЊC and 2,2,2-
trichloroethanol (1.56 ml, 16 mmol), DCC (3.3 g, 16 mmol) and
DMAP (171 mg, 1.4 mmol) were added. The reaction mixture
was allowed to warm to rt and stirring was continued for 16 h.
The solvent was evaporated off and the residue was dissolved in
ethyl acetate and filtered through Celite. Evaporation of the
solvent and purification by FC (ethyl acetate–pentane, 1:5)
gave compound 8 (5.8 g, 94%) as a solid, mp 40.5–42.5 ЊC; [α]Dr.t.
ϩ2.3 (c 0.84, CHCl3) (Found: C, 49.1; H, 5.6; N, 3.3. C18H24-
Cl3NO5 requires C, 49.1; H, 5.5; N, 3.2%); νmax(CHCl3)/cmϪ1
1708, 1499 and 1454; δH(300 MHz; CDCl3) 1.46 (9H, s, t-Bu),
2.81 (2H, d, J 6.2), 3.53–3.68 (2H, m), 4.16–4.24 (1H, br m,
HCNH), 4.53 (2H, s), νA = 4.71, νB = 4.68 (2H, AB, JAB 12.0,
OCH2CCl3), 5.11–5.18 [1H, br m, C(O)NH] and 7.29–7.47
(5H, m, arom. H); δC(75 MHz; CDCl3) 25.9, 33.7, 44.9, 68.5,
70.9, 71.6, 77.3, 125.4, 125.5, 126.1, 135.4, 152.8 and 167.5; m/z
(FAB) 879.1 [8.6%, (2M ϩ 1)ϩ], 440.0 [62.6, (M ϩ 1)ϩ] and
340 [100.0, (M Ϫ Boc)ϩ].
J1 15.2, J2 4.7), 2.65–2.95 (4H, m), 3.09 (1H, br m), 4.54–4.57
(2H, br m), 4.69 (1H, d, J 12.0), 4.82 (1H, br s), 4.89 (1H, br s),
5.11–5.21 (6H, m), 5.79 (1H, br m), 6.54 (1H, br m), 6.86 (1H,
br m) and 7.27–7.38 (15H, m); δC(100 MHz; CDCl3) 28.3, 35.7,
37.6, 48.6, 49.2, 50.5, 67.2, 67.5, 68.0, 74.2, 77.2, 80.0, 94.4,
128.1, 128.3, 128.4, 128.5, 128.5, 128.6, 128.7, 128.8, 134.8,
135.2, 135.5, 155.6, 169.0, 169.8, 170.0 and 170.3; m/z (FAB)
886.2 [3.4, (M ϩ Na)ϩ], 864.2 [16.5, (M ϩ 1)ϩ] and 764.2 [100,
(M Ϫ Boc)ϩ].
Boc-N-[â-D-Asp(á-OBn)]6-OCH2CCl3 13. Peptide 12 (483
mg, 0.53 mmol) was deprotected in 20 ml 90% (v/v) HOAc with
2 g Zn-powder according to general procedure F. A further
portion of peptide 12 (484 mg, 0.53 mmol) was deprotected
according to general procedure C. Both fragments were dis-
solved in CH2Cl2, Et3N (0.31 ml, 2.24 mmol), HOBt (91 mg,
0.67 mmol) and EDC (130 mg, 0.67 mmol) were added, and the
mixture was stirred overnight at rt. The resulting yellow mixture
was diluted with CHCl3 and washed successively with 1  aq.
NaHSO4 and saturated aq. NaHCO3. The organic phase was
washed with aq. NaCl, dried (MgSO4) and concentrated.
Recrystallisation (MeOH–hexane) gave compound 13 (443 mg,
54%) as a solid, mp 138–141 ЊC (decomp.); [α]Dr.t. ϩ12.9 (c 1,
CHCl3); νmax(CHCl3)/cmϪ1 3416, 1744, 1676 and 1498; δH(400
MHz; CDCl3) 1.38 (9H, s, t-Bu), 2.50–3.10 (12H, m), 4.49–4.69
(3H, m), 4.76–4.89 (3H, m), 5.06–5.18 (12H, m, H2CPh), 5.93
[1H, d, J 8.9, O(CO)NH], 5.79 [1H, d, J 7.6, C(CO)NH], 6.98
[1H, d, J 7.8, C(CO)NH], 7.05–7.11 [3H, m, C(CO)NH] and
7.26–7.37 (30H, m, arom. H); δC(100 MHz; CDCl3) 28.3, 35.7,
37.1, 37.2, 37.3, 48.5, 49.2, 49.4, 50.5, 67.1, 67.4, 67.5, 67.6,
67.7, 68.0, 74.1, 77.2, 79.8, 94.4, 126.96, 127.61, 128.02, 128.11,
128.24, 128.29, 128.47, 128.53, 128.58, 128.66, 128.71, 134.7,
135.1, 135.2, 135.3, 135.4, 135.7, 155.7, 169.1, 169.6, 169.7,
170.1, 170.6, 170.8 and 171.7; m/z (FAB) 1501.7 [3.1%,
(M ϩ Na)ϩ], 1479.8 [3.1, (M ϩ 1)ϩ] and 1381.6 [100,
(M Ϫ Boc)ϩ].
(2S)-2-(tert-Butoxycarbonylamino)butanedioic acid 1-benzyl
4-(2,2,2-trichloroethyl) diester 10. A solution of compound 9
(1.66 g, 5.0 mmol) in 5:1 CH3CN–DMF (36 ml) was cooled to
0 ЊC and 2,2,2-trichloroethanol (0.53 ml, 5.3 mmol), DCC (1.15
g, 5.3 mmol) and DMAP (70 mg, 0.57 mmol) were added. The
reaction mixture was allowed to warm to rt and stirring was
continued for 16 h. The solvent was evaporated off and the
residue was dissolved in ethyl acetate and filtered through
Celite. Evaporation of the solvent and purification by FC (ethyl
acetate–pentane, 1:5) gave compound 10 (2.09 g, 92%) as a
solid, mp 52–54 ЊC; [α]Dr.t. ϩ10.1 (c 1.05, CHCl3) (Found: C, 47.7;
H, 5.0; N, 3.1. C18H22Cl3NO6 requires C, 47.4; H, 4.9; N, 3.1%);
νmax(CHCl3)/cmϪ1 3437, 2932, 1754 and 1710; δH(400 MHz;
CDCl3) 1.43 (9H, s, t-Bu), 3.02 (1H, dd, J1 17.1, J2 4.9), 3.15
(1H, dd, J1 17.1, J2 4.6), 4.62–4.71 (3H, m), 5.15–5.23 (2H, m),
5.48 [1H, d, J 7.9, C(O)NH] and 7.30–7.38 (5H, m, arom. H);
δC(100 MHz; CDCl3) 28.3, 36.6, 50.0, 67.6, 74.1, 80.3, 94.5,
109.2, 128.4, 128.5, 128.6, 135.1, 155.3, 169.3 and 170.5; m/z
(FAB) 454.2 [13.4%, (M ϩ 1)ϩ], 400.1 (100) and 354.1 [63.7,
(M Ϫ Boc)ϩ].
Boc-N-â-HGlu(OBn)-â-HGlu(OBn)-OCH2CCl3 14. Boc-N-
β-HGlu(OBn)-OCH2CCl3 (7.5 g, 15.6 mmol) 7 was deprotected
according to general procedure C. The resulting aminoester and
5 (5.5 g, 15.7 mmol), Et3N (10.9 ml, 78.5 mmol), HOBt (2.54 g,
18.8 mmol) and EDC (3.6 g, 18.8 mmol) was transformed
according to general procedure D. Recrystallisation (ethyl
acetate–pentane 7:3) gave compound 14 (9.29 g, 83%) as an
amorphous solid, mp 113–114 ЊC; [α]Dr.t. Ϫ16.4 (c 1, CHCl3)
(Found: C, 55.6; H, 5.8; N, 4.0. C33H41Cl3N2O9 requires C, 55.4;
H, 5.7; N, 3.9%); νmax(CHCl3)/cmϪ1 3428, 1731 and 1498;
δH(400 MHz; CDCl3) 1.40 (9H, s, t-Bu), 1.73–1.98 (5H, m),
2.25–2.55 (5H, m), 2.63–2.76 (2H, m), 3.80–3.89 (1H, m,
HCNH), 4.26–4.35 (1H, m, HCNH), νA = 4.78, νB = 4.72 (2H,
AB, JAB 12.0, OCH2CCl3), νA = 5.12, νB = 5.10 (4H, AB, JAB
10.9, H2CPh), 5.29 [1H, s, O(CO)NH], 6.35 [1H, d, J 7.8, C-
(CO)NH] and 7.31–7.38 (10H, m, arom. H); δC(100 MHz;
CDCl3) 28.4, 28.6, 29.6, 31.1, 38.7, 41.1, 45.9, 47.7, 66.4, 66.6,
74.0, 77.2, 79.4, 94.7, 128.2, 128.3, 128.4, 128.5, 128.6 (3C),
135.7, 135.9, 155.7, 169.6, 170.5, 173.0 and 173.1; m/z (FAB)
1455.2 [5.8%, (2M ϩ Na)ϩ], 1433.3 [11.8, (2M ϩ 1)ϩ], 739.1
[18.1, (M ϩ Na)ϩ], 717.1 [51.9, (M ϩ 1)ϩ] and 615.0 [100,
(M Ϫ Boc)ϩ].
Boc-N-â-D-Asp(á-OBn)-â-D-Asp(á-OBn)-OCH2CCl3
11.
Boc-N-β--Asp(α-OBn)-OCH2CCl3 10 (9.78 g, 21.5 mmol) was
deprotected according to general procedure C. The resulting
aminoester and 9 (7.11 g, 22 mmol), Et3N (11.7 ml, 85.9 mmol),
HOBt (3.51 g, 25.8 mmol) and EDC (5.00 g, 25.8 mmol) were
transformed according to general procedure D. FC (ethyl
acetate–hexane 1:2) gave compound 11 (7.37 g, 51%) as an
amorphous solid, mp 97–101 ЊC; [α]Dr.t. ϩ20.4 (c 1, CHCl3);
νmax(CHCl3)/cmϪ1 3426, 1752, 1708 and 1498; δH(400 MHz;
CDCl3) 1.42 (9H, s, t-Bu), 2.75 (1H, dd, J1 15.8, J2 4.5), 2.89–
2.95 (2H, m), 3.12 (1H, dd, J1 17.4, J2 4.4), 4.54–4.58 (1H, m,
HCNH), νA = 4.53, νB = 4.69 (2H, AB, JAB 11.9, OCH2CCl3),
4.85–4.89 (1H, m, HCNH), 5.13–5.23 (4H, m, H2CPh), 5.69
[1H, d, J 8.0, O(CO)NH], 6.53 [1H, d, J 7.8, C(CO)NH] and
7.28–7.36 (10H, m, arom. H); δC(100 MHz; CDCl3) 28.3,
35.91, 37.8, 48.5, 50.4, 67.3, 67.9, 74.1, 80.0, 94.4, 128.5, 128.6,
134.8, 135.4, 155.6, 169.1, 169.8 and 171.1; m/z (FAB) 1317.4
[1.6%, (2M ϩ 1)ϩ], 659.2 [34.6, (M ϩ 1)ϩ] and 561.2 [100,
(M Ϫ Boc)ϩ].
Boc-N-â-D-Asp(á-OBn)-â-D-Asp(á-OBn)-â-D-Asp(á-OBn)-
OCH2CCl3 12. Peptide 11 (2.03 g, 3.08 mmol) was deprotected
according to general procedure C. The resulting aminoester and
compound 9 (1.03 g, 3.20 mmol), Et3N (1.68 ml, 12.30 mmol),
HOBt (504 mg, 3.7 mmol) and EDC (712 mg, 3.7 mmol) were
transformed according to general procedure D. FC (ethyl
acetate–hexane 1:2) gave compound 12 (2.24 g, 84%) as an
amorphous solid, mp 116–119 ЊC; [α]Dr.t. ϩ26.2 (c 1, CHCl3)
(Found: C, 55.5; H, 5.2; N, 4.9. C40H41Cl3N3O12 requires C,
55.7; H, 4.8; N, 4.9%); νmax(CHCl3)/cmϪ1 3426, 1746, 1677, 1498
and 1456; δH(400 MHz; CDCl3) 1.42 (9H, s, t-Bu), 2.58 (1H, dd,
Boc-N-â-HGlu(OBn)-â-HGlu(OBn)-â-HGlu(OBn)-OCH2-
CCl3 15. Peptide 14 (7.77 g, 10.2 mmol) was deprotected accord-
ing to general procedure C. The resulting aminoester and
compound 5 (3.81 g, 10.9 mmol), Et3N (7.56 ml, 54.3 mmol),
HOBt (1.61 g, 11.9 mmol) and EDC (2.29 g, 11.9 mmol) were
transformed according to general procedure D. Recrystallis-
ation (ethyl acetate–pentane 4:1) gave compound 15 (8.38 g,
81%) as an amorphous solid, mp 129–131 ЊC; [α]Dr.t. Ϫ16.2 (c
1, CHCl3) (Found: C, 57.9; H, 5.9; N, 4.5. C46H56Cl3N3O12
requires C, 58.2; H, 6.0; N, 4.4%); νmax(CHCl3)/cmϪ1 3427, 1732,
J. Chem. Soc., Perkin Trans. 1, 1998, 3331–3340
3337

View Article Online
1665, 1495 and 1454; δH(400 MHz; CDCl3) 1.40 (9H, s, t-Bu),
1.80–1.95 (6H, m), 2.24–2.54 (10H, m), 2.70–2.78 (2H, m),
3.86–3.87 (1H, m, HCNH), 4.12–4.13 (1H, m, HCNH), 4.20–
4.30 (1H, m, HCNH), νA = 4.78, νB = 4.71 (2H, AB, JAB 12.0,
OCH2CCl3), 5.06–5.14 (6H, m, H2CPh), 5.35 [1H, d, J 8.2,
O(CO)NH], 6.54 [1H, d, J 7.5, C(CO)NH], 6.79 [1H, d, J 7.4,
C(CO)NH] and 7.31–7.38 (15H, m, arom. H); δC(100 MHz;
CDCl3) 28.3, 28.7, 28.9, 29.7, 31.0, 31.1, 38.6, 40.5, 41.0, 46.0,
46.6, 47.7, 66.3, 66.4, 66.6, 74.0, 77.2, 79.3, 94.7, 128.2 (2C),
128.3 (2C), 128.4, 128.5, 128.6 (2C), 135.7, 135.8, 135.9, 155.7,
169.7, 170.6, 173.0 and 173.1; m/z (FAB) 1921.4 [6.2%, (2M ϩ
Na)ϩ], 1899.4 [22.0, (2M ϩ 1)ϩ], 972.2 [13.5, (M ϩ Na)ϩ], 950.2
[33.3, (M ϩ 1)ϩ] and 850.1 [100, (M Ϫ Boc)ϩ].
m), 2.75 (3H, d, J 6.6), 3.56–3.63 (2H, m), 4.07–4.12 (1H, m,
HCNH), 4.45–4.54 (4H, m), 4.64–4.71 (2H, m, OCH2CCl3),
5.07–5.12 (2H, m), 5.13–5.19 (2H, m), 5.87 [1H, d, J 8.2,
O(CO)NH], 6.33 [1H, d, J 8.1, C(CO)NH], 6.65 [1H, d, J 7.6,
C(CO)NH] and 7.26–7.37 (15H, m, arom. H); δC(100 MHz;
CDCl3) 28.3, 28.5, 31.1, 35.8, 37.7, 40.1, 45.9, 46.5, 50.6, 66.5,
67.3, 70.6, 73.4, 74.1, 77.2, 79.9, 94.7, 128.0, 128.1, 128.2, 128.3,
128.5, 128.6, 135.5, 135.8, 137.4, 155.7, 169.6, 169.9, 170.2,
171.7 and 173.1; m/z (FAB) 1759.4 [3.2%, (2M ϩ 1)ϩ], 880.2
[43.3, (M ϩ 1)ϩ] and 780.2 [100, (M Ϫ Boc)ϩ].
Boc-N-[-â-D-Asp(á-OBn)-â-HGlu(OBn)-â-HSer(OBn)]2-
OCH2CCl3 19. Peptide 18 (500 mg, 0.57 mmol) was deprotected
in 90% (v/v) HOAc (24 ml) with zinc powder (4 g) according to
general procedure F. A further portion of peptide 18 (501 mg,
0.57 mmol) was deprotected according to general procedure C.
The fragments were coupled according to general procedure D
with Et3N (0.4 ml, 2.85 mmol), HOBt (85 mg, 0.63 mmol) and
EDC (120 mg, 0.63 mmol). FC [CHCl3–7% (v/v) MeOH] gave
compound 19 (472 mg, 55%) as an amorphous solid, mp 166–
176 ЊC (decomp.); [α]Dr.t. Ϫ12.2 (c 0.93, CHCl3); νmax(CHCl3)/cmϪ1
3426, 1733, 1667, 1498 and 1455; δH(400 MHz; CDCl3) 1.38
(9H, s, t-Bu), 1.75–1.88 (4H, m), 2.20–2.56 (12H, m), 2.64–2.82
(4H, m), 3.48–3.63 (4H, m), 4.10–4.15 (2H, m, HCNH), 4.40–
4.73 (10H, m), 5.06–5.17 (8H, m), 5.93 (1H, d, J 8.4, NH), 6.31
(1H, d, J 8.7, NH), 6.89 (1H, d, J 8.4, NH), 6.94 (1H, d, J 8.4,
NH) and 7.2–7.38 (30 H, m, arom. H); δC(100 MHz; CDCl3)
23.0, 28.3, 28.5, 28.8, 31.1, 35.8, 37.1, 37.3, 37.6, 40.0, 40.3,
45.9, 45.9, 46.3, 46.4, 46.5, 46.7, 49.0, 49.6, 50.6, 66.4, 66.5,
67.2, 67.4, 67.5, 70.6, 71.1, 73.2, 73.4, 74.1, 77.2, 79.8, 94.7,
127.66, 127.86, 127.95, 128.00, 128.02, 128.08, 128.10, 128.15,
128.21, 128.23, 128.30, 128.36, 128.39, 128.42, 128.48, 128.56,
128.58, 128.60, 128.62, 135.36, 135.44, 135.6, 135.8, 135.9,
137.37, 137.43, 137.9, 169.7, 169.8, 169.9, 170.0, 170.2, 170.3,
170.5, 171.25, 171.34, 171.8, 172.9, 173.0 and 173.3; m/z (FAB)
1531.5 [20.8%, (M ϩ Na)ϩ], 1509.5 [28.4, (M ϩ 1)ϩ] and 1409.4
[100, (M Ϫ Boc)ϩ].
Boc-N-[â-HGlu(OBn)]6-OCH2CCl3 16. Peptide 15 (1.5 g, 1.6
mmol) was deprotected in 90% (v/v) HOAc (66 ml) with zinc
powder (10 g) according to general procedure F. A further por-
tion of peptide 15 (1.5 g, 1.6 mmol) was deprotected according
to general procedure C. Both fragments were dissolved in
CH2Cl2 (30 ml), Et3N (1.1 ml, 7.9 mmol), HOBt (235 mg, 1.7
mmol) and EDC (333 mg, 1.7 mmol) were added, and the mix-
ture was stirred overnight at rt. The resulting yellow mixture
was diluted with CHCl3 and washed successively with 1  aq.
NaHSO4 and saturated aq. NaHCO3. The organic phase was
washed with aq. NaCl, dried (MgSO4) and concentrated.
Recrystallisation (toluene, 60 ЊC) gave compound 16 (1.86 g,
72%) as a glass, mp 199–201 ЊC (decomp.); [α]Dr.t. Ϫ14.7 (c 0.88,
CHCl3); νmax(CHCl3)/cmϪ1 3366, 1731, 1661 and 1498; δH(400
MHz; CDCl3) 1.40 (9H, s, t-Bu), 1.60–2.00 (12H, m), 2.17–2.41
(24H, m), 3.80–3.91 (1H, m, HCNH), 4.00–4.15 (3H, m,
HCNH), 4.20–4.40 (2H, m, HCNH), νA = 4.76, νB = 4.70 (2H,
AB, JAB 12.0, OCH2CCl3), 5.07–5.10 (12H, m, H2CPh), 5.48
[1H, m, O(CO)NH], 6.64–7.02 [5H, m, C(CO)NH] and 7.29–
7.35 (30H, m, arom. H); m/z (FAB) 1671.5 [13.6%, (M ϩ Na)ϩ],
1649.6 [21.1, (M ϩ 1)ϩ] and 1549.5 [100, (M Ϫ Boc)ϩ].
Boc-N-â-HGlu(OBn)-â-HSer(OBn)-OCH2CCl3 17. Com-
pound 8 (958 mg, 2.17 mmol) was deprotected according to
general procedure C. The resulting aminoester and compound 5
(763 mg, 2.17 mmol), Et3N (1.51 ml, 10.85 mmol), HOBt (323
mg, 2.38 mmol) and EDC (458 mg, 2.38 mmol) were trans-
formed according to general procedure D. FC (ethyl acetate–
pentane 2:3) yielded compound 17 (942 mg, 64%) as an
amorphous solid, mp 108–109 ЊC; [α]Dr.t. Ϫ5.4 (c 1.07, CHCl3)
(Found: C, 55.3; H, 5.9; N, 4.3. C31H39Cl3N2O8 requires C, 55.2;
H, 5.8; N, 4.16%); νmax(CHCl3)/cmϪ1 3648, 3430, 1704 and 1497;
δH(400 MHz; CDCl3) 1.41 (9H, s, t-Bu), 1.82–1.94 (2H, m),
2.31–2.43 (4H, m), 2.77 (2H, d, J 6.2), 3.52–3.63 (2H, m), 3.83–
3.91 (1H, m, HCNH), 4.45–4.54 (3H, m), νA = 4.67, νB = 4.64
(2H, AB, JAB 12.0, OCH2CCl3), 5.07–5.14 (2H, m), 5.32 [1H, d,
J 7.9, O(CO)NH], 6.33 [1H, d, J 8.1, C(CO)NH] and 7.31–7.38
(10H, m, arom. H); δC(100 MHz; CDCl3) 28.4, 29.5, 31.1, 35.8,
41.0, 45.9, 47.8, 49.2, 66.4, 70.5, 73.4, 74.1, 79.4, 94.8, 127.9,
128.0, 128.2, 128.3, 128.4, 128.5, 128.6, 135.9, 137.5, 155.6,
169.7, 170.2 and 173.1; m/z (FAB) 1345.5 [8.3%, (2M ϩ 1)ϩ],
673.2 [77.5, (M ϩ 1)ϩ] and 573.2 [100, (M Ϫ Boc)ϩ].
H2N-N-[-â-D-Asp(á-OBn)-â-HGlu(OBn)-â-HSer(OBn)]2-OH
20. Peptide 19 (144 mg, 0.095 mmol) was deprotected in 90%
(v/v) HOAc (3.3 ml) with zinc powder (500 mg) according to
general procedure F. The supension was filtered and the residue
was taken up in CHCl3 and washed successively with 1  aq.
HCl and brine. The organic layer was dried (MgSO4) and
evaporated. The resulting peptide was deprotected according to
general procedure C. Purification by preparative RP-HPLC
[A: water (0.1% TFA); B: CH3CN; 10 min 60% B, 10 to 15 min
80% B, 15 to 20 min 99% B] yielded compound 20 (66 mg, 55%)
as a glass, mp 148–150 ЊC; δH(500 MHz; CDCl3) see Tables 1
and 2.
cyclo[-â-D-Asp(á-OBn)-â-D-Asp(á-OBn)-â-D-Asp(á-OBn)-]
21. Peptide 12 (1.73 g, 2 mmol) was deprotected in 90% (v/v)
HOAc (60 ml) with zinc powder (5.5 g) according to general
procedure F. The resulting peptide was transformed according
to general procedure E with pentafluorophenol (387 mg, 2.1
mmol) and EDC (402 mg, 2.1 mmol) in CH2Cl2. The resulting
ester was deprotected according to general procedure C in
CH2Cl2 (80 ml) and TFA (20 ml). The resulting peptide was
transformed according to general procedure E and the precipi-
tate was collected by filtration to give compound 21 (886 mg,
72%) as a powder, mp >300 ЊC (Found: C, 64.4; H, 5.7; N, 6.8.
C33H33N3O9 requires C, 64.4; H, 5.7; N, 6.8%); νmax(KBr)/cmϪ1
3288, 1740, 1674, 1560 and 1445; δH(400 MHz; CDCl3–TFA)
2.89 (3H, dd, J1 10.0, J2 14.4), 3.14 (3H, dd, J1 5.1, J2 14.4),
4.77 (3H, br s, HCNH), νA = 5.24, νB = 5.19 (6H, AB, JAB 12.0,
H2CPh), 7.26–7.37 (15H, m, arom. H) and 7.58 (3H, d, J 7.6,
NH) and 7.37–7.26 (15H, m, arom. H); δC(100 MHz; CDCl3–
TFA) 37.3, 50.6, 69.1, 133.9, 170.1 and 171.5; m/z (FAB) 638.2
[96.3%, (M ϩ Na)ϩ] and 616.3 [100, (M ϩ 1)ϩ].
Boc-N-â-D-Asp(á-OBn)-â-HGlu(OBn)-â-HSer(OBn)-OCH2-
CCl3 18. Compound 17 (3.47 g, 5.15 mmol) was deprotected
according to general procedure C. The resulting aminoester and
Boc-N--Asp(α-OBn)-CO2H (1.66 g, 5.15 mmol), Et3N (3.6 ml,
25.7 mmol), HOBt (765 mg, 5.66 mmol) and EDC (1.09 g, 5.66
mmol) were transformed according to general procedure D.
FC (ethyl acetate–pentane 2:3) with subsequent recrystallis-
ation from ethyl acetate–pentane yielded compound 18 (1.39 g,
31%) as an amorphous solid, mp 120–122 ЊC; [α]Dr.t. Ϫ10.1 (c
0.75, CHCl3) (Found: C, 57.4; H, 5.7; N, 4.8. C42H50Cl3N3O11
requires C, 57.4; H, 5.7; N, 4.8%); νmax(CHCl3)/cmϪ1 3430, 1711,
1667, 1498, 1454 and 1368; δH(400 MHz; CDCl3) 1.40 (9H, s,
t-Bu), 1.83–1.86 (2H, m), 2.21–2.41 (4H, m), 2.43–2.54 (1H,
3338
J. Chem. Soc., Perkin Trans. 1, 1998, 3331–3340

View Article Online
cyclo[-â-HGlu(OBn)-â-HGlu(OBn)-â-HGlu(OBn)-] 22. Pep-
tide 15 (500 mg, 0.53 mmol) was deprotected in 90% (v/v)
HOAc (22 ml) with zinc powder (4 g) according to general pro-
cedure F. The resulting peptide was transformed according to
general procedure E with pentafluorophenol (97 mg, 0.53 mmol)
and EDC (102 mg, 0.53 mmol) in CH2Cl2 (29 ml). The resulting
ester was deprotected according to general procedure C in
CH2Cl2 (1 ml) and TFA (1 ml). The resulting peptide was trans-
formed according to general procedure E and the precipitate
was collected by filtration to give compound 22 (200 mg, 54%)
as a powder, mp >215 ЊC; νmax(KBr)/cmϪ1 3294, 1734, 1687,
1648, 1559 and 1498; δH(400 MHz; CDCl3–TFA) 1.97–1.89
(5H, br m), 2.51–2.35 (10H, br m), 2.59–2.57 (3H, br d), 4.21
(3H, br s, HCNH), νA = 5.18, νB = 5.09 (6H, AB, JAB 12.1,
H2CPh), 7.25 (3H, br s, NH) and 7.36–7.33 (15H, m, arom. H);
δC(100 MHz; CDCl3–TFA) 29.0, 30.9, 40.9, 48.7, 67.8, 128.5,
128.71, 128.74, 134.7, 173.4 and 175.1; m/z (FAB) 1421.7 [3.3%,
(2M ϩ Na)ϩ], 1399.8 [3.2, (2M ϩ 1)ϩ], 722.4 [79.4, (M ϩ Na)ϩ]
and 700.4 [100, (M ϩ 1)ϩ].
and FT to 1K × 1K real/real datapoints after multiplication by
sine2 filter shifted by π/3 in ω2 and cos2 filter in ω1. Baseline
correction with 3rd-degree polynomial in both dimensions.
NMR Structure determination
Calculations were performed using AMBER* with BatchMin
5.035 on a Silicon Graphics O2 (R 10000) workstation under
Irix 6.3. Visualisation and manipulation were carried out using
Visual Molecular Dynamics (VMD)24 and MacroModel 5.0.35
Structure of β-hexapeptide 20 in CD3OH: 17 NOEs (Table 2)
were ordered according to their cross-peak volume in the con-
tour plot of the 150 ms ROESY in three categories: strong,
medium and weak with 3.0 Å, 3.5 Å and 4.5 Å as upper bound
distance restraints and their van der Waals radii as lower bound
distance restraints together with 4 dihedral angle restraints
obtained via the Karplus equation.23 A model of the β-hexa-
peptide was generated and subjected to a 500-steps energy
minimisation. 20 ps Unrestrained molecular dynamics at 500 K
yielded 50 starting structures. All restraints were applied and
each of these structures was subjected to a simulated annealing
calculation from 700 K to 1 K in vacuo with subsequent 500-
steps energy minimisation. The 10 structures lowest in energy
with no violation of constraints converged well to the final
structural bundle and were selected as representative for the
structure in solution.
cyclo[-â-D-Asp(á-OBn)-]6 23. Peptide 13 (120 mg, 0.081
mmol) was deprotected in 90% (v/v) HOAc (5 ml) with zinc
powder (0.3 g) according to general procedure F. The resulting
peptide was transformed according to general procedure E with
pentafluorophenol (17 mg, 0.09 mmol) and EDC (18 mg, 0.09
mmol) in CH2Cl2 (5 ml). The resulting ester was deprotected
according to general procedure C in CH2Cl2 (5 ml) and TFA
(5 ml). The resulting peptide was transformed according to gen-
eral procedure E and the precipitate was collected by filtration
to give compound 23 (55 mg, 56%) as a powder, mp >300 ЊC;
νmax(KBr)/cmϪ1 3289, 1736, 1544 and 1388; δH(400 MHz;
CDCl3–TFA) 2.73 (6H, dd, J1 9.0, J2 15.4), 2.86 (6H, dd, J1 4.8,
J2 15.4), 4.75–4.72 (6H, m, HCNH), 5.21–5.13 (12H, m,
H2CPh) and 7.32–7.27 (36H, m); δC(100 MHz; CDCl3–TFA)
36.4, 50.5, 69.4, 134.0 and 171.4; m/z (FAB) 1254.0 [8.1%,
(M ϩ Na)ϩ] and 1232.0 [100, (M ϩ 1)ϩ].
Acknowledgements
We gratefully acknowledge the support of the Royal Society
and the Schweizerischer Nationalfonds zur Förderung der
wissenschaftlichen Forschung. Donations of trifluoroacetic
acid (Solvay Deutschland, Hanover) and tetrahydrofuran
(BASF, Ludwigshafen) are appreciated as is the continuing
support of Novartis Pharma AG, Basle.
References
1 D. Seebach and J. L. Matthews, Chem. Commun., 1997, 2015.
2 D. H. Apella, L. A. Christianson, I. L. Karle, D. R. Powell and
S. H. Gellman, J. Am. Chem. Soc., 1996, 118, 13071.
3 D. H. Apella, L. A. Christianson, D. A. Klein, D. R. Powell,
S. Huang, J. J. Barchi and S. H. Gellman, Nature (London), 1997,
387, 381.
4 S. Hanessian, H. Yang and R. Schaum, J. Am. Chem. Soc., 1996,
118, 2507; S. Hanessian, personal communication.
5 D. Seebach, K. Gademann, J. V. Schreiber, J. L. Matthews,
T. Hintermann, B. Jaun, L. Oberer, U. Hommel and H. Widmer,
Helv. Chim. Acta, 1997, 80, 2033; D. Seebach, S. Abele,
K. Gademann, G. Guichard, T. Hintermann, B. Jaun, J. L.
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Helv. Chim. Acta, 1998, 81, 932.
6 D. Seebach, M. Overhand, F. N. M. Kühnle, B. Martinoni,
L. Oberer, U. Hommel and H. Widmer, Helv. Chim. Acta, 1996, 79,
913; D. Seebach, P. E. Ciceri, M. Overhand, B. Jaun, D. Rigo,
L. Oberer, U. Hommel, R. Amstutz and H. Widmer, Helv. Chim.
Acta, 1996, 79, 2043.
7 J. L. Matthews, M. Overhand, F. N. M. Kühnle, P. E. Ciceri and
D. Seebach, Liebigs Ann. Chem., 1997, 1371.
8 J. M. Fernández-Santín, J. Aymamí, A. Rodríguez-Galán,
S. Muñoz-Guerra and J. A. Subirana, Nature (London), 1984, 311,
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9 F. Chen, G. Lepore and M. Goodman, Macromolecules, 1974, 7,
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10 C. Alemán, J. J. Navas and S. Muñoz-Guerra, Biopolymers, 1997, 41,
721.
11 M. García-Alvarez, A. Martínez de Ilarduya, S. León, C. Alemán
and S. Muñoz-Guerra, J. Phys. Chem. A, 1997, 101, 4215 and
references cited therein.
12 M. Kajtar, M. Hollosi and Zs. Riedl, Acta Chim. Acad. Sci. Hung.,
1976, 88, 301.
NMR Spectroscopy of â-hexapeptide 20
Sample: 16 mg of compound 20 dissolved in 0.6 ml of CD3OH.
1
1D-NMR (AMX500): H NMR (500 MHz): 90K data points,
128 scans, 5.6 s acquisition time. 2D-NMR: Solvent peak
removed by presaturation DQF.COSY (500 MHz; CD3OH)
with pulsed field gradients (PFG) for coherence pathway selec-
tion.30 Acquisition: 2K(t2) × 512 (t1) data points. 1 scan per t1
increment, 0.21 s acquisition time in t2; relaxation delay 2.0 s.
Time proportional phase incrementation (TPPI) quadrature
detection in ω1. Processing: Zero filling and FT to 1K × 1K
real/real datapoints after multiplication with sine2 filter shifted
by π/3 in ω2 and π/2 in ω1. TOCSY (DIPSI-2 SL; 10 kHz;
CD3OH)31 (500 MHz; CD3OH): Acquisition: 2K(t2) × 512 (t1)
data points. 32 scans per t1 increment, mixing time 125 ms,
TPPI quadrature detection. Processing: Zero filling and FT to
1K × 1K real/real datapoints after multiplication with sine2
filter shifted by π/3 in ω2 and π/2 in ω1. HSQC with PFG32 (500/
125 MHz; CD3OH): Acquisition: 2K(t2) × 512 (t1) datapoints,
2 scans per t1 increment. 13C-GARP decoupling during t2. 0.21 s
Acquisition time in t2. Processing: Zero filling and FT to
1K × 1K real/real datapoints after multiplication with sine2
filter shifted by π/3 in ω2 and cos filter in ω1. HMBC
with PFG33 (500/125 MHz; CD3OH): Acquisition: no 13C-
decoupling, 8 scans per t1 increment, otherwise identical with
parameters for HSQC. Processing: Zero filling and FT to
1K × 1K after multiplication with cos2 filter in ω2 and Gaussian
filter in ω1; power spectrum in both dimensions. ROESY34 (500
MHz; CD3OH). Acquisition: A series of 3 ROESY spectra with
mixing times of 50, 100 and 150 ms was acquired. CW-spin lock
(3.8 kHz) between trim pulses, 4K(t2) × 768 (t1) datapoints,
32 scans per t1-increment. 0.422 s acquisition time in t2, other
parameters identical with DQF.COSY. Processing: Zero filling
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