Synthesis and high-resolution NMR structure of a β3- octapeptide with and without a tether introduced by olefin metathesis

Article abstract of DOI:10.1002/hlca.200900311

Bridging between (i)- and (i+3)-positions in a β³-peptide with a tether of appropriate length is expected to prevent the corresponding 3₁₄-helix from unfolding (Fig. 1). The β³-peptide H-β³hVal-β³hLys

Full text of DOI:10.1002/hlca.200900311

Helvetica Chimica Acta – Vol. 92 (2009)
2643
Synthesis and High-Resolution NMR Structure of a b3-Octapeptide with and
without a Tether Introduced by Olefin Metathesis
by Marc-Olivier Ebert*a), James Gardinera)b)c), Steven Balletb)d), Andrew D. Abell*b), and
Dieter Seebacha)
a) Laboratory of Organic Chemistry, Department of Chemistry and Applied Biosciences, ETH Zꢀrich,
HCI Hçnggerberg, Wolfgang-Pauli-Strasse 10, CH-8093 Zꢀrich
(phone: þ 41-44-633-4726; e-mail: marc-olivier.ebert@org.chem.ethz.ch)
b) School of Chemistry and Physics, University of Adelaide, Adelaide 5000, Australia
(phone: þ 61-8-8303-5652; fax: þ 61-8-8303-4358; e-mail: andrew.abell@adelaide.edu.au)
c) Bio21 Institute, University of Melbourne, Melbourne 3010, Australia
d) Department of Organic Chemistry, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussel
Bridging between (i)- and (i þ 3)-positions in a b3-peptide with a tether of appropriate length is
expected to prevent the corresponding 314-helix from unfolding (Fig. 1). The b3-peptide H-b3hVal-
b3hLys-b3hSer(All)-b3hPhe-b3hGlu-b3hSer(All)-b3hTyr-b3hIle-OH (1; with allylated bhSer residues in 3-
and 6-position), and three tethered b-peptides 2 – 4 (related to 1 through ring-closing metathesis) have
been synthesized (solid-phase coupling, Fmoc strategy, on chlorotrityl resin; Scheme). A comparative
CD analysis of the tethered b-peptide 4 and its non-tethered analogue 1 suggests that helical propensity is
significantly enhanced (threefold CD intensity) by a (CH2)4 linker between the b3hSer side chains
(Fig. 2). This conclusion is based on the premise that the intensity of the negative Cotton effect near
215 nm in the CD spectra of b3-peptides represents a measure of ꢁhelical contentꢂ. An NMR analysis in
CD3OH of the two b3-octapeptide derivatives without (i.e., 1) and with tether (i.e., 4; Tables 1 – 6, and
Figs. 4 and 5) provided structures of a degree of precision (by including the complete set of side chain –
side chain and side chain – backbone NOEs) which is unrivaled in b-peptide NMR-solution-structure
determination. Comparison of the two structures (Fig. 5) reveals small differences in side-chain
arrangements (separate bundles of the ten lowest-energy structures of 1 and 4, Fig. 5, A and B) with little
deviation between the two backbones (superposition of all structures of 1 and 4, Fig. 5, C). Thus, the
incorporation of a CH2ꢀOꢀ(CH2)4ꢀOꢀCH2 linker between the backbone of the b3-amino acids in 3-
and 6-position (as in 4) does accurately constrain the peptide into a 314-helix. The NMR analysis,
however, does not suggest an increase in the population of a 314-helical backbone conformation by this
linkage. Possible reasons for the discrepancy between the conclusion from the CD spectra and from the
NMR analysis are discussed.
1. Introduction. – b-Peptides, oligomers composed of homologated proteinogenic
amino acids, have received considerable attention for their ability to adopt rather stable
and predictable conformations in solution [1a]1). One of the most common, and most
1
)
As compared to a-amino acids, the homologated proteinogenic amino acids have an additional
rotatable CꢀC bond. This is in contrast to the situation in trans-2-aminocyclopentane and
-cyclohexane carboxylic acids (originally studied by Gellman and co-workers [1b]); the cyclohexane
(and the 1,2-dithiane [1c]) amino-carboxylic-acid moiety is actually the strongest 314-helix-
stabilizing unit in b-peptides [1d] (an inversion of the six-membered-ring chair conformation with
equatorial CONH and NHCO moieties would put these substituents and the attached chains in axial
positions!).
ꢃ 2009 Verlag Helvetica Chimica Acta AG, Zꢀrich

2644
Helvetica Chimica Acta – Vol. 92 (2009)
studied, conformations is that of the 314-helix, where b-peptides composed of six or
more b3-amino-acid residues fold in solution to a regular helical structure stabilized by
14-membered H-bonded rings with approximately three residues per helix turn. Such
structures have found interest not only in their own right, but also for their ability to
interact with proteins and receptors and to display biological activity (for general
reviews on b-peptides, see [2]).
As such, the stabilization of b-peptidic 314-helices, particularly in aqueous solutions,
has offered a challenge in recent times. For a-peptides, helix stabilization has been
achieved using a number of different strategies (for recent reviews on a-helix
stabilization, see [3]). The most common of these involves the covalent linking of
adjacent turns of the helix by means of constraints between amino acid side chains (i.e.,
disulfides, lactams [4], triazoles [5], and C-tethers via ring-closing metathesis (RCM)
[6 – 8]), or the replacement of weak H-bonds with alkyl or hydrazone bridges [9].
Early work on b-peptidic 314-helices showed that helix stabilization could be
accomplished through the incorporation of cyclic b-amino acids [1b,c][2d][10] or
strategically placed salt bridges [11]2). Incorporation of a disulfide linkage, common in
a-peptides, between the b3-cysteine residues in i and i þ 3 positions of a 314-helix was
also investigated, and represented the first example of a covalent constraint used in the
stabilization of 314-helical b-peptides [12] (Fig. 1). Recently, Vaz and Brunsveld have
shown that the covalent linking of b-amino acid residues at i and i þ 3 positions via an
amide bond results in b-peptides with a high degree of 314-helicity in H2O, as concluded
from CD spectra [13] and from a number of typical [1a][2c] nuclear Overhauser effects
(NOEs) observed in the NMR spectra [1d].
Here, we describe the first detailed NMR solution-structure analysis of a
constrained 314-helical b3-peptide (prepared through RCM), including a comparison
with the NMR structure of its non-constrained analog, and we compare the results of
the NMR analysis with conclusions from CD spectra.
2. Design and Synthesis of the i-to-(i þ 3)-Bridged b-Peptides 2 – 4, and of the
Linear Analog 1. – b3-Octapeptides 1 – 4 (Scheme) are designed to adopt a 314-helix in
solution. The b-amino-acid sequence contains several features promoting helical
stability: i) it consists of b3-amino acids that are known to adopt particularly stable
helical structures [1][2][11]; ii) a bhLys residue at position 2 and a bhGlu residue at
position 5 provide potential for salt-bridge formation in a 314-helix; iii) a bhPhe residue
at position 4 and a bhTyr residue at position 7 provide potential for aromatic p/p
interaction; iv) finally, and most importantly, peptides 2, 3, and 4, contain a covalent
C-bridge between bhSer residues at positions 3 and 6 (giving a 21-membered
heterocycle) to prevent unfolding of the helix (cf. Fig. 1)3). The b3-octamers 1 – 4 allow
2
)
)
Multiple salt bridging, ꢁcappingꢂ of the macrodipole, and introducing amphipathic character of 314-b-
peptidic helices by Schepartz and co-workers led to helix stability in H2O and aggregation to
protein-type quaternary structures [11c – h].
The thus tethered b-peptide happens to contain a macrocycle of the same ring size (21 atoms) as
that in the b-peptide with an amide bond between (i)- and (i þ 3)-positioned bhLys and bhGlu
residues [1d][13], while in the b-peptide with a disulfide bridge, shown in Fig. 1 [12], the macrocycle
contains only 17 atoms.
3

Helvetica Chimica Acta – Vol. 92 (2009)
2645
Fig. 1. Juxtaposition of substituents in i- and (i þ 3)-position of a b-peptidic (M)-314-helix and disulfide
bridging between these positions in a b3-hexapeptide [12]
for two complete turns of a 314-helix (with up to seven intramolecular H-bonds
[1a][2c]).
The synthesis of the peptides 1 – 4 involved an on-bead strategy with incorporation
of two O-allyl-b3hSer residues [14] for subsequent RCM (Scheme). Removal of the on-
bead assembled peptide from the resin with simultaneous deprotection of the side-
chain protecting groups under standard conditions gave the linear b3-octapeptide
derivative 1.
Initial efforts to carry out RCM ꢁon-resinꢂ proved unsuccessful4). Instead, the fully
side-chain-protected linear peptides were cleaved from the resin under mild acidic
conditions (HFIP, CH2Cl2) affording compounds soluble in organic solvents. Ring
closing metathesis proceeded smoothly in the presence of Grubbs II ruthenium catalyst
5 in CH2Cl2, to give the tethered peptides 2 and 3 as predominantly the (E)-isomers
(ratio ca. 4 :1 by 1H-NMR). Removal of the side-chain protecting groups under acidic
conditions gave the CH2CH¼CHCH2-tethered peptide 2 (or its N-terminal Fmoc
derivative 3)5) in good yield. Subsequent hydrogenation of 2 provided the b3-
4
)
In this case, it was only possible to explore variations in thermal conditions. For examples, where
microwave conditions have been successfully employed in this regard, see [8].
The Fmoc derivative was initially prepared to allow for adequate detection of the peptide by UV
during HPLC analysis and purification.
5
)

2646
Helvetica Chimica Acta – Vol. 92 (2009)

Helvetica Chimica Acta – Vol. 92 (2009)
2647
octapeptide 4 with a C4 alkyl-chain tether between the b3hSer residues at positions 3
and 6. Preparative RP-HPLC purification produced samples of 1 – 4 with purities
> 98%.
3. CD Analysis of the b3-Octapeptides with and without Tether. – With purified
material in hand, we first used circular dichroism (CD) spectroscopy to analyze the
ꢁhelical contentꢂ of the linear b-peptide 1 (containing two O-allyl-b3hSer residues) and
the tethered b-peptide analogue 4 at 258, in MeOH, and also in H2O (Fig. 2). The CD
spectra of 1 and 4 in MeOH (Fig. 2,a) show curves characteristic of 314-helices [2c], i.e.,
minima at or near 215 nm, maxima between 195 and 205 nm, and a crossover from
positive to negative ellipticity between 205 and 210 nm. The mean ellipticity minimum
displayed by linear b-peptide 1 (ꢀ10000) in MeOH is consistent with that of similar
linear b-peptides analyzed in the past [1][2][11 – 13]. A significant increase in the
negative Cotton effect is seen for the tethered analogue 4 (ca. ꢀ 25000, almost
threefold that of 1), indicating enhanced helical propensity in MeOH as a result of the
covalently linked b3hSer residues. Significantly, this enhanced effect is also apparent in
H2O with maximum negative mean molar ellipticities of ꢀ 5000 and ꢀ 15000 observed
for 1 and 4, respectively. Based on the assumption that a correlation exists between
intensity of the negative Cotton effect near 215 nm and the 314-helical propensity of a b3-
peptide6), we conclude that 4 displays an enhanced helical propensity in MeOH, and
especially in H2O. The observed effect of (CH2)4-tethering on the CD spectra compares
favorably with that of the b-peptides tethered with an amide linker [1d][13].
Fig. 2. Normalized CD spectra of the linear b-peptide 1 (blue) and the tethered b-peptide 4 (red) in
MeOH (a), and in H2O (b; pH 7.1)
In summary, CD analysis of 4 suggests a significant increase in 314-ꢁhelical contentꢂ,
in both organic and aqueous solution, as a result of the incorporation of an alkane
tether. To further examine the influence of this tether on the conformation of the 314-
helix, a detailed NMR-solution analysis of peptides 1 and 4 was carried out. A
6
)
In our experience, CD-only analysis of b-peptides can be grossly misleading; we have observed
ꢁtypical helix-indicatingꢂ CD spectra of b-peptides, which cannot possibly fold to 314-helices [2c][15].
For a scholarly discussion of helical conformational manifolds and helical propensities of a- and b-
peptides, see [16].

2648
Helvetica Chimica Acta – Vol. 92 (2009)
qualitative NMR analysis (NOE effects) was also performed with the cis/trans-mixture
3, indicating that these stereoisomers also fold to a 314-helix in MeOH7).
4. NMR Solution Structures of 1 and 4. – Two NMR samples were prepared by
dissolving 5 mg of 1 or 4 in 700 ml of CD3OH to give 6 mm solutions of the b-
octapeptides. Sequence-specific assignments of the 1H- and 13C-resonances were
accomplished by COSY, TOCSY, HSQC, and HMBC. Assignments for 1 and 4 are
listed in Tables 1 and 2. The atom-designation scheme used is illustrated in Fig. 3.
Table 1. The 1H- and 13C-NMR Chemical Shifts of 1 in CD3OH Referenced to Internal TMS
a
a
HN HaSi;Re
Hb Hg
)
Hd
H
d1a)b)
Hd2 He
)
Hx
HuZ;E
Hz
b3hVal1
–
2.65, 2.82 3.42 2
–
1.08
–
1.06
–
–
–
–
–
–
b3hLys2 8.18 2.47, 2.87 4.49 1.59*c), 1.53 1.44
1.71, 1.65 2.91
n.o.d)
b3hSer3 8.55 2.42, 2.72 4.35 3.41, 3.33
b3hPhe4 8.54 2.43, 2.81 4.56 2.87, 2.76
b3hGlu5 8.23 2.44, 2.62 4.39 1.84*, 1.71
b3hSer6 7.8 2.61, 2.39 4.62 3.46, 3.35
b3hTyr7 7.75 2.56, 2.34 4.69 2.72
–
–
2.24
–
–
–
–
–
–
–
–
–
–
–
3.98
7.16
–
3.99
7.02
–
7.26
–
–
6.62
–
5.15, 5.25 5.86
–
–
7.21
–
5.15, 5.25 5.88
–
–
–
–
–
–
b3hIle8 7.71 2.57, 2.36 4.24 1.48
1.47, 1.10 0.89 0.88
Ca Cb
Cg Cd
C
d1b) Cd2 Ce Cx
Cu
Cz
C
b3hVal1 36.9 57.0
b3hLys2 42.1 47.5
b3hSer3 38.9 47.5
b3hPhe4 41.5 49.5
b3hGlu5 41.7 47.5
b3hSer6 38.9 47.8
b3hTyr7 41.2 49.2
b3hIle8 38.2 52.6
32.3
36.3 24.1
73.1
43.5 139.7
32.8 32.8
73.2
42.0 130.0
40.8
–
19.7 19.0
–
–
–
–
–
–
172.5
174.0
172.2
172.6
171.9
171.6
171.8
177.2
–
–
–
–
–
–
–
–
–
–
–
–
28.5 41.0
73.2
130.7 129.6
177.4
73.3
–
–
117.4 136.2
–
–
117.4 136.2
–
–
127.7
–
–
–
–
131.8 116.5
12.3
157.3
–
26.8 15.6
–
g
b
a) Tabulated from lower to higher field. ) Val: Cd1 ¼ CdSi. c) * refers to HSi. d) n.o. ¼ Not observed.
For both b-peptides, stereospecific assignments were possible for the H-atoms
attached to Ca (all residues), the Me groups of b3hVal1, and the H-atoms at g positions
in residues 2 and 5. With exception of the constitutionally different parts in residues 3
1
and 6, the two b-peptides show remarkably similar H- and 13C-NMR chemical shifts.
Distance restraints for the structure calculations were generated using ROESY
spectra with 300 ms mixing time resulting in 113 ROE restraints for b-peptide 1 and 102
ROE restraints for b-peptide 4. H-Bond restraints were not included in the calculation.
The 3J values between the amide H-atom and the H-atom at b-position were used as
additional restraints. The 3J(HN,Hb) value for residues 2 – 8 lies between 8.2 and 9.7 Hz
in both b-peptides (see Table 3). According to the Karplus relation published by Wang
and Bax [17] for the corresponding dihedral angle in proteins, these values match a
backbone dihedral angle f of ꢀ 120 ꢁ 308 (see Fig. 4 for the definition of the dihedral
7
)
We thank Professor B. Jaun for sharing these unpublished results with us.

Helvetica Chimica Acta – Vol. 92 (2009)
2649
Table 2. The 1H and 13C-NMR Chemical Shifts of 4 in CD3OH Referenced to Internal TMS
a
a
a
HN HaSi;Re
2.6, 2.90
b3hLys2 8.23 2.4, 3.00
Hb
Hg
)
Hd
H
d1a)b)
Hd2
He
)
Hx
Hz
)
b3hVal1
–
3.43 2.03
–
1.08
–
1.07
–
–
–
–
4.50 1.59*c), 1.49 1.44
1.70, 1.64 2.91 n.o.d)
b3hSer3 8.24 2.15, 2.94 4.30 3.61, 3.31
b3hPhe4 8.47 2.421, 2.83 4.61 2.87, 2.78
b3hGlu5 8.30 2.40, 2.61 4.34 1.83*, 1.69
b3hSer6 8.06 2.71, 2.32 4.63 3.40, 3.33
b3hTyr7 7.68 2.54, 2.33 4.67 2.74, 2.72
b3hIle8 7.61 2.52, 2.29 4.27 1.48
–
–
2.23
–
–
–
–
–
–
–
–
–
–
–
3.54, 3.36
7.18
–
3.63, 3.57
7.02
–
1.81, 1.54
7.26 7.20
–
–
6.62
–
–
1.82, 1.53
–
–
–
–
1.48, 1.11 0.89 0.90
Ca
Cb
Cg
Cd
Cd1b
)
Cd2 Ce Cx
Cz
C
b3hVal1 36.6 56.9
b3hLys2 41.8 47.5
b3hSer3 38.7 48.7
b3hPhe4 41.2 49.5
b3hGlu5 42.1 47.6
b3hSer6 38.9 47.0
b3hTyr7 41.5 49.5
b3hIle8 39.1 53.0
32.3
36.6 24.2
74.6
43.5 139.8
32.9 33.1
73.0
42.1 129.9
41.0
–
19.6 18.8
–
–
–
–
172.6
174.0
–
–
–
–
–
–
–
–
–
–
–
–
28.6 41.0
73.4
130.8 129.6
177.9
72.3
–
–
27.8 172.2
127.7 172.5
171.9
–
–
–
–
28.4 171.9
157.3 171.8
–
131.9 116.5
12.3
–
26.9 15.6
–
178.1
g
b
a) Tabulated from lower to higher field. ) Val: Cd1 ¼ CdSi. c) * refers to HSi. d) n.o. ¼ Not observed.
Fig. 3. b3h-Amino acid atom-designation scheme used for the assignment of chemical shifts and the
definition of dihedral angles (cf. Fig. 4)
angles). In the structure calculations of b-peptides 1 and 4, the dihedral angle f in
residues 2 – 8 was restrained to a more generous region of ꢀ 120 ꢁ 408.

2650
Helvetica Chimica Acta – Vol. 92 (2009)
Table 3. 3J(HN,Hb) Values [Hz] Determined for b-Octapeptides 1 and 4
1
4
b3hVal1
b3hLys2
b3hSer3
b3hPhe4
b3hGlu5
b3hSer6
b3hTyr7
b3hIle8
–
–
9.5
8.8
9.4
9.1
8.5
9.2
9.2
9.3
8.2
9.7
8.9
8.6
8.9
9.3
Fig. 4. Dihedral-angle distribution in the accepted structures. Top row: dihedral angles in the 45 accepted
structures of b3-octapeptide 1. Bottom row: dihedral angles in the 45 accepted structures of b3-
octapeptide 4. The innermost circle of each dial shows the dihedral angles of the first residue in the
sequence (if defined). The adjacent rings show the dihedral angles of residues 2 – 8 (if defined). Each
tick corresponds to one structure. The definitions of the dihedral angles are shown at the bottom of the
figure.
Using a simulated annealing protocol, 45 structures for each molecule were
calculated by restrained molecular dynamics. The statistics of the structure calculations
are listed in Table 4. The backbone RMSD of helix 1 (0.16 ꢄ) is not significantly higher

Helvetica Chimica Acta – Vol. 92 (2009)
2651
Table 4. Statistics of the Accepted Structures of b-Octapeptides 1 and 4 in the Simulated Annealing
Calculations. Of the 45 calculated structures for each peptide, all met the acceptance criteria described in
the Exper. Part.
4
1
Number of NOE restraints
Intraresidual
Sequential
102
60
14
28
7
113
70
11
32
7
Others
Number of dihedral angle restraints
RMSD from experimental restraints
Distance restraints [ꢄ]
Dihedral angle restraints [8]
RMSD from holonomic restraints (force field)
Bonds [ꢄ]
Bond angles [8]
Impropers [8]
Atomic RMSD (residues 2 – 7) [ꢄ]a)
0.011 ꢁ 0.006
0.01 ꢁ 0.001
0.005 ꢁ 0.01
0.0002 ꢁ 0.0009
0.001 ꢁ 0.0003
0.14 ꢁ 0.04
0.16 ꢁ 0.009
0.15
0.001 ꢁ 0.0001
0.3 ꢁ 0.007
0.14 ꢁ 0.004
0.16
a) Superposition was carried out over backbone atoms N, C, Ca, and Cb of residues 2 – 7.
than that of its ꢁstabilized analogueꢂ 4. Fig. 5 (A and B) depicts separate bundles of the
ten lowest-energy structures of 1 and 4, respectively, where both sequences form well-
defined 314-helices. The good definition of both backbones is reflected in the backbone
dihedral angle distributions shown in Fig. 4 and listed in Tables 5 and 6. The backbone
dihedral angles f, y, and q of residues 2 – 7 show a RMSD of < 108 for all (for 1) or
most of the residues (for 4).
The b3-octapeptides 1 and 4 are potentially able to form six backbone – backbone
H-bonds from HNi to COiþ2. If we define criteria for the existence of a NH ··· OC H-
bond to be a maximal H ··· O separation of 2.4 ꢄ and a deviation from NH ··· O
linearity of less than 358, then each of the inner four H-bonds is observed at least eight
times within the ten structures of both 1 and 4 shown in Fig. 5, A and B. Within the same
subset of the calculated structures, the terminal H-bond from HN6 to CO8 is observed
eight times for 1 and four times for 4. The H-bond from HN1 to CO3 is absent in the ten
lowest-energy structures of 4 and observed only twice within the ten structures of 1
shown in Fig. 5, A. Using the same criteria, the potential salt bridge between the side
chains of b3hLys2 and b3hGlu5 was identified twice within the bundle of 4 shown in
Fig. 5, B, but is absent in the structures of 1 in Fig. 5, A.
The position of the side chains is defined fairly well in both structures, with only the
side chains of residues 3 and 6 in the non-tethered b-peptide 1 showing increased
positional variability. This is also apparent in the RMSDs of the side-chain dihedral
angle c1 of b3hSer3 and b3hSer6 in 1 (Table 5), which show – with the only exception of
c1 of b3hIle8 – by far the highest values among the dihedral angles listed for both b-
peptides. This is, however, presumably due to a lack of unambiguous distance restraints
for the side-chain H-atoms of these residues and does not necessarily reflect a real
increase in conformational mobility within these regions.
In Fig. 5, C, a backbone superposition is shown of all calculated structures of the
two b-peptide derivatives 1 and 4. The RMSD for this superposition is 0.21 ꢄ. This

2652
Helvetica Chimica Acta – Vol. 92 (2009)

Helvetica Chimica Acta – Vol. 92 (2009)
2653
Table 5. Statistics of Selected Dihedral Angles in the Structural Ensemble Calculated for b-Octapeptide 1.
All 45 accepted structures were used. For definitions of dihedral angles, see Fig. 4.
f
y
q
c1
b3hVal1
b3hLys2
b3hSer3
b3hPhe4
b3hGlu5
b3hSer6
b3hTyr7
b3hIle8
–
ꢀ 152 ꢁ 9
ꢀ 120 ꢁ 3
ꢀ 132 ꢁ 8
ꢀ 145 ꢁ 7
ꢀ 140 ꢁ 8
ꢀ 158 ꢁ 8
ꢀ 141 ꢁ 5
–
56 ꢁ 4
ꢀ 63 ꢁ 2
ꢀ 139 ꢁ 7
ꢀ 153 ꢁ 4
ꢀ 139 ꢁ 9
ꢀ 120 ꢁ 8
ꢀ 131 ꢁ 7
ꢀ 110 ꢁ 7
ꢀ 115 ꢁ 8
52 ꢁ 3
66 ꢁ 5
48 ꢁ 4
49 ꢁ 3
74 ꢁ 5
35 ꢁ 6
102 ꢁ 31
ꢀ 85 ꢁ 7
ꢀ 107 ꢁ 52
ꢀ 86 ꢁ 5
ꢀ 58 ꢁ 3
119 ꢁ 57
ꢀ 122 ꢁ 8
ꢀ 41 ꢁ 54
Table 6. Statistics of Selected Dihedral Angles in the Structural Ensemble Calculated for b-Octapeptide 4.
All 45 accepted structures were used. For definitions of dihedral angles, see Fig. 4.
f
y
q
c1
b3hVal1
b3hLys2
b3hSer3
b3hPhe4
b3hGlu5
b3hSer6
b3hTyr7
b3hIle8
–
ꢀ 155 ꢁ 14
ꢀ 136 ꢁ 17
ꢀ 136 ꢁ 11
ꢀ 147 ꢁ 6
ꢀ 148 ꢁ 5
ꢀ 170 ꢁ 6
ꢀ 144 ꢁ 10
–
30 ꢁ 37
50 ꢁ 6
78 ꢁ 8
48 ꢁ 8
56 ꢁ 5
71 ꢁ 7
50 ꢁ 17
72 ꢁ 19
ꢀ 40 ꢁ 36
ꢀ 147 ꢁ 9
ꢀ 137 ꢁ 12
ꢀ 141 ꢁ 11
ꢀ 123 ꢁ 8
ꢀ 124 ꢁ 4
ꢀ 112 ꢁ 8
ꢀ 128 ꢁ 16
ꢀ 83 ꢁ 11
40 ꢁ 31
ꢀ 94 ꢁ 1
ꢀ 61 ꢁ 7
ꢀ 129 ꢁ 32
ꢀ 94 ꢁ 38
31 ꢁ 56
leads to the conclusion that the bridge in 4 does not distort the structure of this helix
compared to 1. The average Cbi ··· Cibþ 3 distance (ꢁpitchꢂ of the helix) is ca. 4.8 ꢄ in both
structures.
5. Conclusions and Discussion. – The (CH2)4-hydrocarbon-tethered b-octapeptide
derivative 4 and the non-restrained analog 1 have CD spectra (in MeOH and H2O
solution) which can be interpreted by assuming that there is ꢁmore helicityꢂ (i.e.,
reduced conformational flexibility) caused by the covalently attached bridge in 4. The
origin of the trough near 215 nm in the CD spectra of b-peptides, the intensity of which
is used for drawing this conclusion, is, however, controversial: the typical CD spectrum
hardly changes when a MeOH solution of a b3-peptide8) is heated to 608 and above
[18a], or when it is treated with a ꢁdenaturingꢂ additive such as urea [18b]. On the other
hand, addition of H2O to MeOH solutions (or CD measurements in pure H2O)9) leads
8
)
...consisting of homologated, noncyclic, a-amino acid moieties, and having a 314-helical secondary
structure by NMR analysis! The situation is different when Gellmanꢂs cyclic b-amino-acid residues
(especially ACHC) are involved [1b – d][2d]. Also, when the b-peptide consists of ꢁdisubstitutedꢂ
b2,3-amino-acid residues of like-configuration [2c], the helix is very stable: in CD3OD at r.t. the
central NH H-atoms of a b-hexapeptide of this type [18i] undergo H/D exchange with a half-life of
ca. 9 days!
The results of NMR measurements in H2O, and of NMR titrations of MeOH solutions of the b-
octapeptides 1 and 4 with H2O will be reported and discussed in a forthcoming separate paper by
our groups.
9
)

2654
Helvetica Chimica Acta – Vol. 92 (2009)
to a reduction [1c] or breakdown [18b,c] of the Cotton effect, or to a total change of the
patterns of the spectra [15b,c][18d]. Furthermore, as mentioned above6), a ꢁhelix-
typicalꢂ CD spectrum is observed with b-peptides, which cannot possibly fold to a 314-
helix. For these reasons we have always considered CD spectra of b-peptides as a mere
qualitative kind of fingerprint.
The NMR solution-structure analysis of the two b3-octapeptides 1 and 4, reported
herein, was carried out with a precision (by including the complete set of side chain –
side chain and side chain – backbone NOEs), which is unprecedented for b-peptides.
While there are differences in the side-chain conformations with respect to salt-
bridging between the Glu and Lys, or p-interaction between the Phe and Tyr side
chains, the backbones of the two peptides are superimposable (Fig. 5) with an RMSD
of 0.2 ꢄ, which leads to the statement made above: ꢁthe bridge in 4 does not distort the
structure of this helix compared to 1ꢂ, or in other words no helix-stabilizing effect of the
tether can be derived from the NMR analysis of the two peptides in MeOH. The
discrepancy between this conclusion and that drawn from the CD spectra suggests that
the negative Cotton effect near 215 nm is actually not caused by the global nature of a
single helical backbone conformation of the b-peptides but rather by the local extent of
helicity also present in partially unfolded conformers.
In looking for an interpretation [2c][18e,f], one has to keep in mind that chemists
very often tend to think in terms of static structures of molecules. Contrary to this view,
most spectroscopic experiments are carried out on statistical ensembles. The ease, with
which information from CD spectroscopy and from NOEs gained from an ensemble of
molecules can be condensed into a single representative picture, depends, among
others, on two factors, which are important for this discussion:
1. For an ensemble also consisting of partially unfolded macromolecules, a H ··· H
distance derived from a NOE is likely to be biased towards smaller values, i.e., towards
the more compact members of the ensemble exhibiting strong NOEs10).
2. While the size of the Cotton effect represents a linear average from all
contributing conformers, it is much more ꢁshortsightedꢂ than NMR cross-relaxation
experiments in the sense that it mainly reports on the local electronic structure and
local conformation of the backbone.
As soon as more than one backbone conformation is populated to a significant
extent, problems reconciling information derived from CD spectroscopy and NOEs in
one ꢁrepresentative moleculeꢂ are, therefore, likely to occur. Based on qualitative
arguments, this is especially to be expected if the folding process is not cooperative, as it
is presumably the case for the shorter b3-peptides [18a]11).
To complement the data presented here, investigations aiming at the dynamics of
the peptides 1 and 4 in aqueous media and in MeOH are in progress.
10
)
)
This is the result of the functional dependence of the NOE on the internuclear distance. The effect
of angular fluctuations of the internuclear vectors is assumed to be negligible here.
From investigations of F-substituted b3-hepta- and b3-tridecapeptides, we have recently concluded
that longer-chain b3-peptides might be subject to cooperative folding [18g,h]. The central NH H-
atom of a b3-pentadecapeptide has been found to undergo H/D exchange in CD3OD at room
temperature with a half-life of ca. 59 days, which is not compatible with dynamic opening and
closing of H-bonds in this region of the peptide [18i]. Finally, for Schepartzꢂs b-peptides (mostly
dodecapeptides; cf. Footnote 2) cooperative behavior has also been reported [11c – h].
11

Helvetica Chimica Acta – Vol. 92 (2009)
2655
The authors wish to thank Dr. Denis Scanlon (Bio21 Institute, University of Melbourne) for access
to HPLC purification facilities and Prof. Bernhard Jaun (ETH Zꢀrich) for insightful discussions. J. G.
acknowledges financial assistance from an ARC International Linkage Fellowship LX0881950, and
A. D. A. support from ARC DP077190.
Experimental Part
1. General. Abbreviations: DIPEA: EtN(i-Pr)2, HATU: O-(7-azabenzotriazol-1-yl)-N,N,N’,N’-
tetraammonium hexafluorophosphate, HFIP: 1,1,1,3,3,3-hexafluoropropan-2-ol, TFA: CF3COOH, h.v.:
high vacuum (0.01 – 0.1 Torr), TNBS: 2,4,6-trinitrobenzenesulfonic acid. TIS: 2-Chlorotrityl resin was
purchased from GL Biochem (China). Fmoc-b3-amino acids were purchased from Fluka or prepared
according to established procedures. Anal. reversed-phase (RP) HPLC: on an Agilent 1100 using a
Supelco C18 column (150 ꢂ 4.6 mm, 5 mm), using a linear gradient of A (MeCN) and B (0.1% TFA in
H2O) at a flow rate of 1 ml/min. Prep. HPLC: on an Agilent 1200 using an Agilent C18 column (250 ꢂ
9.4 mm, 5 mm), using a linear gradient of A (MeCN) and B (0.1% TFA in H2O) at a flow rate of
5 ml/min. Retention time (tR) given in min. Lyophilization: Virtis benchtop SLC to obtain peptides as
TFA salts. MS: Agilent 6510 QTOF LC/MS.
2. Preparation of Peptides 1 – 4. H-b3hVal-b3hLys-b3hSer(All)-b3hPhe-b3hGlu-b3hSer(All)-b3hTyr-
b3hIle-OH (1). 2-Chlorotrityl resin (150 mg, 0.8 – 1.5 mmol/g, ca. 0.17 mmol) was swollen in CH2Cl2
(5 ml) for 1 h. The resin was filtered, and a soln. of Fmoc-b3hIle-OH (121 mg, 0.33 mmol, 2 equiv.) and
DIPEA (0.144 ml, 0.85 mmol, 5 equiv.) in CH2Cl2 (2 ml) was added and mixed under N2 bubbling for 2 h.
The resin was filtered, and the addition of Fmoc-b3hIle-OH was repeated once more for 2 h. The resin
was filtered and washed with CH2Cl2 (5 ml, 5 ꢂ 1 min). The N-terminal Fmoc group was removed using
20% piperidine/DMF (4 ml, 4 ꢂ 10 min), and the resin was washed with DMF (5 ml, 5 ꢂ 1 min). The
remaining Fmoc-protected b-amino acids were incorporated according to standard solid phase synthesis
protocols with Fmoc-bAA-OH (2 equiv.), HATU (1.95 equiv.), and DIPEA (4 equiv.) as a soln. in DMF
with a reaction time of 1 h: Fmoc-b3Tyr(tBu)-OH (174 mg), Fmoc-b3hSer(All)-OH (140 mg), Fmoc-
b3hGlu(tBu)-OH (161 mg), Fmoc-b3hPhe-OH (148 mg), Fmoc-b3hSer(All)-OH (140 mg), Fmoc-
b3hLys(Boc)-OH (177 mg), Fmoc-b3hVal-OH (130 mg); HATU (136 mg); DIPEA (0.128 ml). Cou-
plings were monitored using TNBS [19]. Once the final residue was incorporated, the N-terminal Fmoc
group was removed (20% piperidine/DMF, 4 ml, 4 ꢂ 10 min), and the resin was washed with DMF (5 ml,
5 ꢂ 1 min). A soln. of Boc2O (157 mg, 0.72 mmol, 4.2 equiv.) and Et3N (0.1 ml, 0.72 mmol, 4.2 equiv.) in
CH2Cl2 (1 ml) was then added and mixed under N2 bubbling for 1 h. The resin was filtered and washed
with CH2Cl2 (5 ml, 5 ꢂ 1 min). The peptide – resin was then dried in h.v. for 12 h. A portion of the
peptide – resin (50 mg) was treated with TFA/TIPS/H2O (95 :2.5 :2.5, 10 ml) for 3 h. The resin was
filtered, washed with TFA (2 ꢂ 2 ml), and the volatiles were removed under reduced pressure. The
resulting residue was treated with cold Et2O to precipitate the crude peptide (44 mg). Purification by
prep. RP-HPLC (0 – 60% A in 25 min) gave 1 as TFA salt (5.5 mg). White solid. Anal. RP-HPLC (0 –
60% A in 25 min): tR 19.7, > 98%. HR-MS: 1164.6904 ([M þ H]þ, C60H94N9O1þ4 ; calc. 1164.6920).
H-b3hVal-b3hLys-b3hSer(X)-b3hPhe-b3hGlu-b3hSer(X)-b3hTyr-b3hIle-OH (X ¼ CH2CH¼CHCH2
tether; 2). A further portion of the peptide – resin from the preparation of 1 (200 mg) was treated
with a soln. of HFIP/CH2Cl2 (3 :7, 10 ml) for 30 min, filtered, and washed with CH2Cl2 (2 ꢂ 2 ml). The
solns. were combined, and the volatiles were removed to give the fully protected linear peptide as a pale
white solid (148 mg, LR-MS: 1475.9 (C60H94N9O14; calc. 1475.9). Grubbsꢂ II ruthenium catalyst 5 (4 mg,
5 mol-%) was added to a soln. of the fully protected b-peptide (140 mg, 0.095 mmol) dissolved in
degassed CH2Cl2 (5 ml) under N2, and the mixture was stirred at 358 under N2 for 2 h. An additional
portion of catalyst was added, and stirring was continued for a further 2 h. The solvent was removed
under reduced pressure to give the fully protected peptide as a light brown solid (145 mg, LR-MS: 1468
([M þ Na]þ)). Treatment with TFA/TIS/H2O (95 :2.5 :2.5, 5 ml) gave 2. Light brown solid. HR-MS:
1136.6658 ([M þ H]þ, C58H90N9O1þ4 ; calc. 1136.6607).
Fmoc-b3hVal-b3hLys-b3hSer(X)-b3hPhe-b3hGlu-b3hSer(X)-b3hTyr-b3hIle-OH (X¼ CH2CH¼CHCH2
tether; 3). Prepared in a similar manner as described for 2 except that the N-terminal Fmoc group of 1

2656
Helvetica Chimica Acta – Vol. 92 (2009)
was not removed before cleavage of the peptide from the resin. Purification of a portion of the crude
peptide (40 mg) by RP-HPLC (0 – 60%, in 40 min) gave 3 (5.6 mg). Grey solid. LR-MS: 1358
(C73H99N9O16; calc. 1358.6).
H-b3hVal-b3hLys-b3hSer(X)-b3hPhe-b3hGlu-b3hSer(X)-b3hTyr-b3hIle-OH (X ¼ (CH2)4 tether; 4).
To a soln. of 2 (80 mg), dissolved in MeOH (2 ml) under N2, was added 10% Pd/C (8 mg, 10% (w/w)),
and the vessel was evacuated and flushed with H2 (repeated 2 ꢂ ). The suspension was then vigorously
stirred under H2 for 3 h. The suspension was filtered through Celite (2-cm plug), and the solvent was
removed to give a light brown solid (60 mg). Purification by prep. RP-HPLC (0 – 60% A in 25 min) gave
pure 4 as a TFA salt (5.7 mg). White solid. Anal. RP-HPLC (0 – 60% A in 25 min): tR 17.9, > 98%. LR-
MS: 1137.6 (C58H91N9O14; calc. 1137.7).
3. NMR Experiments. All spectra were measured on a Bruker AVANCE III 600-MHz spectrometer
at 26.48. For both peptides, an identical set of spectra was acquired. The solvent signal was suppressed by
presaturation. TOCSY and 13C-HSQC were run in a sensitivity-enhanced manner [20]. 80 ms of DIPSI2
isotropic mixing with a spin-lock field strength of 8.9 kHz was employed in the TOCSY experiments.
Offset-compensated [21] ROESY spectra with 300-ms mixing time and a CW spin-lock field strength of
1.85 kHz were recorded. The spectral width was 5400 Hz in both dimensions. Quadrature detection in the
indirect dimension was achieved by States-TPPI. 2k ꢂ 512 total data points were acquired. The time
domain in both dimensions was extended to twice its size by zero-filling and apodized with a cos2
function. The baseline of the resulting spectra was corrected with a polynomial of 5th order. All spectra
were processed with Topspin 2.1 [22]. Resonance assignment, as well as integration and calibration of the
ROESY cross-peaks and were performed with SPARKY 3.113 [23].
4. Generation of Distance Restraints. Two groups of cross-peaks for each peptide were used for
calibration. Peptide 1: HaSi/HaRe of residues 1 – 5 (peak group 1) and HN/HB of residues 2 – 8 (peak
group 2); peptide 4: HaSi/HaRe of all residues (peak group 1) and HN/HB of residues 2 – 8 (peak group
2). For both peptides, the average volume of peak group 1 and peak group 2 was assumed to correspond
to distances of 1.9 and 2.9 ꢄ, resp. These volumes and distances were used to solve the system of Eqn. 1
for coefficients a and b, which were then used to generate distances from the remaining cross-peak
volumes in the ROESY spectra.
r1,2 ¼ a þ b · Vꢀ1;12=6
(1)
The upper/lower limit of each restraint was generated by adding/subtracting 20% of the obtained
distance. If a cross-peak could be integrated on both sides of the diagonal, the longer of the two resulting
distances was used for structure calculation. Distances involving groups of equivalent protons (e.g., Me
groups) were multiplied by the corresponding correction factor C (Eqn. 2, where nI is the number of
equivalent H-atoms in group I).
C ¼ (nInS)1/6
(2)
In these cases, the restraint was implemented using an average distance d between the pairs of H-
atoms involved (Eqn. 3).
ꢀ3
d ¼ (hd i)ꢀ1/3
(3)
ij
For cross-peaks involving diastereotopic H-atoms without stereospecific assignment, a similar
procedure was employed. The longer of the two generated distances to a remote H-atom was introduced
as a restraint on the average separation as defined in Eqn. 3. In these cases, the lower limit for the
distance restraint was set to zero.
5. Structure Calculation. A simulated annealing protocol [24] implemented in XPLOR-NIH 2.21 [25]
was employed to generate 45 structures for each peptide. The protocol consisted of 60 ps of high-
temperature torsional angle molecular dynamics (TAD) at 20000 K, followed by 60 ps of slow cooling to
1000 K (TAD), 12 ps of slow cooling with Cartesian molecular dynamics to 300 K, and a final energy
minimization in Cartesian coordinates. The only nonbonded interaction used was a repel potential. For
each new structure, the starting velocities of all atoms were assigned randomly. A calculated structure

Helvetica Chimica Acta – Vol. 92 (2009)
2657
was accepted, if it showed no violation of a NOE restraint > 0.2 ꢄ, no violation of a dihedral angle
restraint > 58, no RMS deviation from the equilibrium values for bond lengths and bond angles > 0.02 ꢄ
and > 28, resp., and no deviation from the equilibrium values of the impropers > 1.58. Topology and
parameter files were adapted to accommodate b3-amino acid residues. The force constant for the
stretching of a bond was 100 kcal molꢀ1 ꢄꢀ2, and the ones for angles and impropers were set to 500 kcal
molꢀ1 radꢀ2
.
Note added in proof: While the present paper was in print, an independent synthesis of hydrocarbon-
chain-tethered b3-peptides by P. Perlmutter and his group was published [26]. For structural assignment, a
couple of 314-helix-typical NOEs and CD-intensity comparisons were used.
REFERENCES
[1] a) D. Seebach, M. Overhand, F. N. M. Kꢀhnle, B. Martinoni, L. Oberer, U. Hommel, 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, H. Widmer, Helv. Chim. Acta 1996, 79, 2043; b) D. H. Appella, L. A.
Christianson, I. L. Karle, D. R. Powell, S. H. Gellman, J. Am. Chem. Soc. 1996, 118, 13071; c) D.
Seebach, A. Jacobi, M. Rueping, K. Gademann, M. Ernst, B. Jaun, Helv. Chim. Acta 2000, 83, 2115,
and in ꢁ Hominatio – An International Tribute to Albert Eschenmoserꢂ, Ed. M. V. Kisakꢀrek, Verlag
Helvetica Chimica Acta, Zꢀrich, Wiley-VCH, Weinheim, 2001; d) E. Vaz, W. C. Pomerantz, M.
Geyer, S. H. Gellman, L. Brunsveld, ChemBioChem 2008, 9, 2254.
[2] a) D. Seebach, J. Gardiner, Acc. Chem. Res. 2008, 41, 1366; b) D. Seebach, D. F. Hook, A. Glattli,
Biopolymers 2006, 84, 23; c) D. Seebach, A. K. Beck, D. J. Bierbaum, Chem. Biodiversity 2004, 1,
1111; d) R. P. Cheng, S. H. Gellman, W. F. DeGrado, Chem. Rev. 2001, 101, 3898; e) D. Seebach, J. L.
Matthews, Chem. Commun. 1997, 2015.
[3] L. K. Henchley, A. J. Jochim, P. S. Arora, Curr. Opin. Chem. Biol. 2008, 12, 692; J. Garner, M. M.
Harding, Org. Biomol. Chem. 2007, 5, 3577.
[4] N. E. Shepherd, G. Abbenante, D. P. Fairlie, Angew. Chem. 2004, 116, 2741; Angew. Chem., Int. Ed.
2004, 43, 2687; P. Grieco, P. M. Gitu, V. J. Hruby, J. Pept. Res. 2001, 57, 250; K. A. Carpenter, R.
Schmidt, S. Y. Yue, L. Hodzic, C. Pou, K. Payza, C. Godbout, W. Brown, E. Roberts, Biochemistry
1999, 38, 15295.
[5] S. Cantel, A. Le Chavalier Isaad, M. Scrima, J. J. Levy, R. D. DiMarchi, P. Rovero, J. A. Halperin,
A. M. DꢂUrsi, A. M. Papini, M. Chorev, J. Org. Chem. 2008, 73, 5663.
[6] A. K. Boal, I. Guryanov, A. Moretto, M. Crisma, E. L. Lanni, C. Toniolo, R. H. Grubbs, D. J.
OꢂLeary, J. Am. Chem. Soc. 2007, 129, 6986; H. E. Blackwell, R. H. Grubbs, Angew. Chem. 1998, 110,
3469; Angew. Chem., Int. Ed. 1998, 37, 3281.
[7] Y.-W. Kim, G. L. Verdine, Bioorg. Med. Chem. Lett. 2009, 19, 2533; F. Bernal, A. F. Tyler, S. J.
Korsmeyer, L. D. Walensky, G. L. Verdine, J. Am. Chem. Soc. 2007, 129, 2456; L. D. Wolensky, K.
Pitter, J. Morash, K. J. Oh, S. Barbuto, J. Fisher, E. Smith, G. L. Verdine, S. J. Korsmeyer, Mol. Cell
2006, 24, 199; L. D. Walensky, A. L. Kung, I. Escher, T. J. Malia, S. Barbuto, R. D. Wright, G.
Wagner, G. J. Verdine, S. J. Korsmeyer, Science 2004, 305, 1466; C. E. Schafmeister, J. Po, G. L.
Verdine, J. Am. Chem. Soc. 2000, 122, 5891.
[8] M. A. Hossain, K. J. Rosengren, S. Zhang, R. A. D. Bathgate, G. W. Tregear, B. J. van Lierop, A. J.
Robinson, J. D. Wade, Org. Biomol. Chem. 2009, 7, 1547; A. J. Robinson, J. Elaridi, B. J. van Lierop,
S. Mujcinovic, W. R. Jackson, J. Pept. Sci. 2007, 13, 280.
[9] A. J. Vernall, P. Cassidy, P. F. Alewood, Angew. Chem. 2009, 121, 5785; Angew. Chem., Int. Ed. 2009,
48, 5675; D. Wang, W. Liao, P. S. Arora, Angew. Chem. 2005, 117, 6683; Angew. Chem., Int. Ed. 2005,
44, 6525; E. Cabezas, A. C. Satterthwait, J. Am. Chem. Soc. 1999, 121, 3862.
[10] D. H. Appella, J. J. Barchi Jr., S. R. Durell, S. H. Gellman, J. Am. Chem. Soc. 1999, 121, 2309.
[11] a) P. I. Arvidsson, M. Rueping, D. Seebach, Chem. Commun. 2001, 649; b) R. P. Cheng, W. F.
DeGrado, J. Am. Chem. Soc. 2001, 123, 5162; c) S. A. Hart, A. B. F. Bahadoor, E. E. Matthews, X. J.
Qiu, A. Schepartz, J. Am. Chem. Soc. 2003, 125, 4022; d) J. A. Kritzer, J. Tirado-Rives, S. A. Hart,

2658
Helvetica Chimica Acta – Vol. 92 (2009)
J. D. Lear, W. L. Jorgensen, A. Schepartz, J. Am. Chem. Soc. 2005, 127, 167; e) J. X. Qiu, E. J.
Petersson, E. E. Matthews, A. Schepartz, J. Am. Chem. Soc. 2006, 128, 11338; f) D. S. Daniels, E. J.
Petersson, J. X. Qiu, A. Schepartz, J. Am. Chem. Soc. 2007, 129, 1532; g) E. J. Petersson, A.
Schepartz, J. Am. Chem. Soc. 2008, 130, 821; h) J. L. Goodman, M. A. Molski, J. Qiu, A. Schepartz,
ChemBioChem 2008, 9, 1576.
[12] M. Rueping, B. Jaun, D. Seebach, Chem. Commun. 2000, 2267; A. Jacobi, D. Seebach, Helv. Chim.
Acta 1999, 82, 1150.
[13] E. Vaz, L. Brunsveld, Org. Lett. 2006, 8, 4199.
[14] Y. Bergman, M. Ciampini, S. Jalal, H. R. Lagiakos, M.-I. Aguilar, P. Perlmutter, Tetrahedron:
Asymmetry 2008, 19, 2861.
[15] a) D. Seebach, T. Sifferlen, P. A. Mathieu, A. M. Hꢅne, C. M. Krell, D. J. Bierbaum, S. Abele, Helv.
Chim. Acta 2000, 83, 2849; b) J. V. Schreiber, D. Seebach, Helv. Chim. Acta 2000, 83, 3139; c) D.
Seebach, J. V. Schreiber, P. I. Arvidsson, J. Frackenpohl, Helv. Chim. Acta 2001, 84, 271; d) A.
Glꢅttli, X. Daura, D. Seebach, W. F. van Gunsteren, J. Am. Chem. Soc. 2002, 124, 12972; e) X.
Daura, D. Bakowies, D. Seebach, J. Fleischhauer, W. F. van Gunsteren, P. Krꢀger, Eur. Biophys. J.
2003, 32, 661.
[16] D. S. Kemp, Helv. Chim. Acta 2002, 85, 4392.
[17] A. C. Wang, A. Bax, J. Am. Chem. Soc. 1996, 118, 2483.
[18] a) K. Gademann, B. Jaun, D. Seebach, R. Perozzo, L. Scapozza, G. Folkers, Helv. Chim. Acta 1999,
82, 1; b) T. Etezardy-Esfarjani, C. Hilty, K. Wꢀthrich, M. Rueping, J. Schreiber, D. Seebach, Helv.
Chim. Acta 2002, 85, 1197; c) S. Abele, G. Guichard, D. Seebach, Helv. Chim. Acta 1998, 81, 2141;
ˇ
d) D. Seebach, T. Kimmerlin, R. Sebesta, M. A. Campo, A. K. Beck, Tetrahedron 2004, 60, 7455;
´
e) J. Kapitan, F. Zhu, L. Hecht, J. Gardiner, D. Seebach, L. D. Barron, Angew. Chem. 2008, 120,
6492; Angew. Chem., Int. Ed. 2008, 47, 6392; f) D. Seebach, H. Widmer, S. Capone, R. R. Ernst, T.
Bremi, I. Kieltsch, A. Togni, D. Monna, D. Langenegger, D. Hoyer, Helv. Chim. Acta 2009, 92, in
print, doi: 10.1002/hlca.200900279; g) R. I. Mathad, B. Jaun, O. Flçgel, J. Gardiner, M. Lçweneck,
´
J. D. C. Codee, P. H. Seeberger, D. Seebach, M. K. Edmonds, F. H. M. Graichen, A. D. Abell, Helv.
Chim. Acta 2007, 90, 2251; h) R. I. Mathad, F. Gessier, D. Seebach, B. Jaun, Helv. Chim. Acta 2005,
88, 266; i) D. Seebach, S. Abele, K. Gademann, G. Guichard, T. Hintermann, B. Jaun, J.-L. Matthews,
J. V. Schreiber, L. Oberer, U. Hommel, H. Widmer, Helv. Chim. Acta 1998, 81, 932.
[19] W. S. Hancock, J. E. Battersby, Anal. Biochem. 1976, 71, 260.
[20] A. G. Palmer III, J. Cavanagh, P. E. Wright, M. Rance, J. Magn. Reson. 1991, 93, 151; J. Cavanagh, M.
Rance, J. Magn. Reson. 1990, 88, 72.
[21] C. Griesinger, R. R. Ernst, J. Magn. Reson. 1987, 75, 261.
[22] Bruker Biospin GmbH, Reinstetten, 2007.
[23] T. D. Goddard, D. G. Kneller, University of California, San Francisco, 2004.
[24] E. G. Stein, L. M. Rice, A. T. Brꢀnger, J. Magn. Reson. 1997, 124, 154.
[25] C. D. Schwieters, J. J. Kuszewski, N. Tjandra, G. M. Clore, J. Magn. Reson. 2003, 160, 66.
[26] Y. E. Bergman, M. P. Del Borgo, R. D. Gopalan, S. Jalal, S. E. Unabia, M. Ciampini, D. J. Clayton,
J. M. Fletcher, R. J. Mulder, J. A. Wilce, M.-I. Aguilar, P. Perlmutter, Org. Lett. 2009, 11, 4438.
Received August 31, 2009