Helix-forming oligoureas: Temperature-dependent NMR, structure determination, and circular dichroism of a nonamer with functionalized side chains

Article abstract of DOI:10.1002/1522-2675(200211)85:11<3692::AID-HLCA3692>3.0.CO;2-W

To further investigate the degree of structural homology between γ-peptides A and N,N′-linked oligoureas B, we prepared oligourea nonamer 2 containing Ala, Val, Leu, Phe, Tyr and Lys side chains. Oligomer 2 was synthesized on solid support from activated monomers, i.e., from enantiomerically pure succinimidyl {2-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}ethyl}carbamates 3a-f that are further substituted at C(2) of the ethyl moiety. These precursors were conveniently prepared from N-Fmoc-protected β³-amino acids with corresponding side chains. Detailed NMR studies (DQF-COSY, TOCSY, and ROESY) in (D₅)pyridine revealed that 2 adopts a regular (P)-2.5 helical secondary structure very similar to that previously determined for oligourea heptamer 1 and closely related to the (P)-2.6₁₄ helix of γ-peptides. Temperature-dependent NMR further demonstrated the conformational homogeneity and remarkable stability of the structure of 2 in pyridine. The CD spectrum of 2 (0.2 mm) was recorded in MeOH with the aim to gain more information about the conformation of oligoureas. In contrast to 2.6-helical γ-peptides, which display only a weak or no Cotton effect, oligourea 2 exhibits an intense positive Cotton effect at ca. 203 nm ([O] = +373000 deg cm² dmol^-1) that decreases only slowly upon increasing the temperature.

Full text of DOI:10.1002/1522-2675(200211)85:11<3692::AID-HLCA3692>3.0.CO;2-W

3692
Helvetica Chimica Acta Vol. 85 (2002)
Helix-Forming Oligoureas: Temperature-Dependent NMR, Structure
Determination, and Circular Dichroism of a Nonamer with Functionalized
Side Chains
by Christine Hemmerlin and Michel Marraud
LCPM, UMR CNRS-INPL 7568, ENSIC-INPL, B.P. 451, F-54001 Nancy
and
Didier Rognan
Laboratoire de Pharmacochimie de la Communication Cellulaire, UMR CNRS, ULP 7081, 74 Route du Rhin,
B.P. 24, F-67401 Illkirch
and
Roland Graff
¬
Faculte de Chimie, 1 Rue Blaise Pascal, F-67008 Strasbourg
and
Vincent Semetey1), Jean-Paul Briand, and Gilles Guichard*
¬
¬
Immunologie et Chimie Therapeutiques, UPR CNRS 9021, Institut de Biologie Moleculaire et Cellulaire, 15
Rue Descartes, F-67084 Strasbourg
Dedicated to Professor Dieter Seebach on the occasion of his 65th birthday
To further investigate the degree of structural homology between g-peptides A and N,N'-linked oligoureas
B, we prepared oligourea nonamer 2 containing Ala, Val, Leu, Phe, Tyr and Lys side chains. Oligomer 2 was
synthesized on solid support from activated monomers, i.e., from enantiomerically pure succinimidyl {2-{[(9H-
fluoren-9-ylmethoxy)carbonyl]amino}ethyl}carbamates 3a f that are further substituted at C(2) of the ethyl
moiety. These precursors were conveniently prepared from N-Fmoc-protected b3-amino acids with correspond-
ing side chains. Detailed NMR studies (DQF-COSY, TOCSY, and ROESY) in (D5)pyridine revealed that 2
adopts a regular (P)-2.5 helical secondary structure very similar to that previously determined for oligourea
heptamer 1 and closely related to the (P)-2.614 helix of g-peptides. Temperature-dependent NMR further
demonstrated the conformational homogeneity and remarkable stability of the structure of 2 in pyridine. The
CD spectrum of 2 (0.2 mm) was recorded in MeOH with the aim to gain more information about the
conformation of oligoureas. In contrast to 2.6-helical g-peptides, which display only a weak or no Cotton effect,
oligourea 2 exhibits an intense positive Cotton effect at ca. 203 nm ([V] ˆ ‡373000 deg cm2 dmolÀ1) that
decreases only slowly upon increasing the temperature.
Introduction. Inspired by the unique three-dimensional structures of biomacro-
molecules (e.g., proteins, nucleic acids), chemists are now creating synthetic oligomeric
materials (−artificial bio-oligomers×) in which enough conformational control can lead
to the formation of well-defined secondary structures stabilized by noncovalent
interactions [1]. In the field of peptidomimetics, seminal work by Seebach and co-
1
)
New address: Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street,
Cambridge, MA 02138, USA.

Helvetica Chimica Acta Vol. 85 (2002)
3693
workers [2 4], Gellman and co-workers [5][6], and Hanessian and co-workers [7] has
demonstrated that properties such as folding and structural diversity are not restricted
to natural a-polypeptides but are shared by synthetic peptides consisting exclusively of
higher w-amino acids such as b- and g-amino-acids2). Indeed, short-chain b- and g-
peptides with defined substitution patterns can form stable helices [2][4a][5][7a],
sheets [3][6], and turns [3][4d][6][7b] in solution and in the solid state. Since this early
work, some reports have highlighted the potential of other w-amino acids including
conformationally restricted d-amino acids (e.g., sugar amino acids) for the construction
of folding oligomers [9]. Interestingly, within each w-peptide subclass, isosteric
backbone modifications can be introduced to generate peptidomimetics that, in turn,
might well form stable and novel secondary structures. Although the syntheses of a
number of such oligomeric peptidomimetics have been described in the literature,
detailed conformational analyses demonstrating unambiguously the presence of
regular secondary structures have been limited to a few3).
The substitution of a heteroatom for the C(a) or C(w) of the amino acid
constituents of w-peptides represent an interesting modification that has been used to
generate a-[13], b-[12][14], and g-peptide [15][16] mimetics. In these systems, the
degree of structural homology with the reference w-peptide is somewhat difficult to
predict, and the answer needs detailed case-by-case conformational investigation. For
example, oligomers (as short as trimers) of a-aminooxy acids obtained by replacement
of the C(b) of each residue of b-peptides with an O-atom adopt a twisted 28 helical
structure in solution [12], whereas the corresponding b2-peptides exhibit a 314 helical
secondary structure. Conversely, ab initio calculations conducted on hydrazinopeptides
(i.e., the C(b) of each residue within a b-peptide is substituted with an N-atom)
revealed a variety of secondary structures including a highly stable 3.3-helical structure
that topologically differs from the 314 helix [14a]4)
We have recently started a program aimed at studying the conformational
preference of urea-based peptidomimetics [17 19]. In this context, we postulated
that the replacement of the C(a) of g-amino acid residues by an N-atom in the
backbone of g-peptides of type A, which generates N,N'-linked oligoureas of type B5),
2
)
)
For reviews on b-peptides, see [8].
3
a-Peptide mimetics that have been shown to form helical secondary structure in solution include oligo(N-
substituted glycines) with a-chiral aromatic side chains [10], and conformationally restricted polyimides
(homo-oligomers of l-pyroglutamic acid) [11]. Oligomers of a-aminooxy acids that belong to the b-
peptide lineage have also been found to fold into helical structure [12].
4
5
)
However, although preliminary CD studies have been performed on hydrazinopeptides [14b],
experimental data to ascertain these predictions are still needed.
These oligomers were initially synthesized by Burgess and co-workers [15].
)

3694
Helvetica Chimica Acta Vol. 85 (2002)
OH
O
O
O
O
H
N
H
N
H
N
H
N
H
N
H
N
H2N
N
N
H
N
H
N
H
N
N
H
N
H
NH2
OH
H
H
O
O
O
OH
1
NH2
NH2
OH
O
O
O
O
O
H
N
H
N
H
N
H
N
H
N
H
N
H
N
H
N
H2N
N
N
H
N
H
N
H
N
H
N
H
N
H
N
H
N
NH2
H
H
O
O
O
O
OH
2
was compatible with the 2.6 helical backbone of g-peptides. NMR Structure
determination in (D5)pyridine of heptamer 1 with Ala, Val, and Tyr side chains
confirmed that most of the structural information encoded in the reference g-peptide
was preserved upon heteroatom substitution (Fig. 1). Heptamer 1 was shown to adopt
a right-handed 2.5 helix of 5.1 ä pitch characterized by the presence of 12- and 14-
membered H-bonded pseudocycles [18].
To gain further insight into the conformational preferences of N,N'-linked
oligoureas and learn about the influence of the sequence on the folding, we prepared
nonamer 2 with Ala, Val, Leu, Phe, Tyr, and Lys side chains and performed a detailed
conformational analysis in solution.
2. Preparation of Oligourea 2. N,N'-Linked oligoureas are readily accessible by
solid-phase-synthesis methodology with a variety of appropriate monomers. Originally,
Burgess and co-workers utilized enantiomerically pure 2-phthalimido-1-substituted
ethyl isocyanates prepared by treatment of the corresponding 2-phthalimido-1-
substituted ethylamines with phosgene [15]. A number of alternative building blocks
including activated carbamates have since been proposed (e.g., 4-nitrophenyl (2-azido-
1-X-ethyl)carbamates [20], 4-nitrophenyl {2-{[(tert-butoxy)carbonyl]amino}-2-X-ethyl}-
carbamates [21], or succinimidyl {2-{[(tert-butoxy)carbonyl]amino}-2-X-ethyl}carba-
mates [22]). Because of the utility of the N-Fmoc protecting group in the solid-phase
synthesis of peptides, we have recently described the preparation of succinimidyl {2-
{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}-2-X-ethyl}carbamates as novel building
blocks for the synthesis of oligoureas on solid support [23]. Enantiomerically pure N-
Fmoc-protected succinimidyl carbamates 3a f with Ala, Val, Leu, Phe, Tyr, and Lys
side chains required for the synthesis of 2 were prepared as previously described
starting from N-Fmoc-b3-amino acids.

Helvetica Chimica Acta Vol. 85 (2002)
3695
Fig. 1. Comparison ofthe helical secondary structures and H-bonding patterns of a) g-peptides A and b) N,N'-
linked oligoureas B. a) Side view of the right-handed 2.614 helix with a pitch of 5.0 ä formed by g4-peptides in
solution as determined by NMR in (D5)pyridine (adapted from [4a]). The structure is stabilized by CˆO(i) ¥¥¥
HÀN(i ‡ 3) H-bonds that close 14-membered H-bonded rings. b) Side view of the right-handed 2.5 helical
secondary structure of oligoureas. The structure depicted is a section of the NMR structure of heptaurea 1
determined in (D5)pyridine [18]. The helix has a pitch of ca. 5.1 ä and is characterized by the simultaneous
presence of CˆO(i) ¥¥¥ HÀN'(i ‡ 2) and CˆO(i) ¥¥¥ HÀN(i ‡ 3) H-bonds that close 12- and 14-membered H-
bonded rings, respectively.
H
H
H
N
Fmoc
N
OSu
Fmoc
N
OSu
Fmoc
OSu
N
N
H
N
H
H
O
O
O
3b
3c
3a
NHBoc
OtBu
OSu
H
H
N
H
Fmoc
N
Fmoc
OSu
Fmoc
N
OSu
N
N
H
N
H
H
O
O
O
3d
3e
3f
Su = succininidyl

3696
Helvetica Chimica Acta Vol. 85 (2002)
Briefly, N-Fmoc-b3-Xaa-OH [24] were first converted to the corresponding acyl
azides, and, following Curtius rearrangement, the resulting isocyanates were treated
with N-hydroxysuccinimide to give carbamates 3a f in good yields (51 86%).
Carbamates 3a f are white solids that can be stored for months at 48 or at room
temperature without degradation. Nonamer 2 was assembled on Rink amide resin [25]
via successive coupling and deprotection cycles (see Scheme). The purity of the crude
oligomer, obtained after cleavage from the resin and lyophilization was 32% as checked
by reversed-phase HPLC. Oligomer 2 was purified by reversed-phase HPLC (C18) to a
final purity >95% and lyophilized.
Scheme. Synthesis ofOligourea 2 on Solid Support
Ri
1) 3a-f, NMM, DMF, 2 x 70 min
H
H
N
H
N
H2N
N
2) 20% piperidine in DMF
H
O
Rink amide resin
9
(repeat 9 times)
Ri
CF3COOH/H2O
H
H
N
NH2
N
H
O
9
3. Temperature-Dependent NMR and Structure Determination of 2 in (D5)Pyr-
idine. The NMR conformational analysis of 2 was conducted in a manner analogous
to that previously described for 1 [18]. The choice of (D5)pyridine as a solvent
was dictated by the larger dispersion and better resolution observed particularly
in the NH region compared to CD3OH6). The consequences were: i) an easier
identification of spin systems for all residues, ii) the access to 3J(NH,HÀC(b))
3
3
for residues 2 9 as well as to some J(N'HsiÀC(a)) and J(N'H,HReÀC(a)), iii)
the possibility to study temperature dependence over a large range of temperature
(80 K).
DQF-COSY and TOCSY Measurements allowed the assignment of proton
resonances for all residues. The chemical shifts are collected in Table 1. In the
fingerprint NH/HÀC(b) and N'HsiÀC(a) region (for stereospecific assignment of
diastereotopic C(a) protons, vide infra) of the DQF-COSY experiment recorded at
308 K, almost all cross-peaks were properly resolved (Fig. 2). Interestingly, N'H of
residues 4 9 appear systematically downfield with respect to the corresponding NH,
while the opposite is observed for residues 2 and 37). Unequivocal sequence-specific
assignment was accomplished by analysis of short range N'H(i)/NH(i ‡ 1) NOE
connectivities in the ROESY experiment recorded at 308 K (tm ˆ 300 ms).
6
)
The original conformational analyses of g-peptides reported independently by the groups of Seebach [4a]
and Hanessian [7a] were also carried out in (D5)pyridine.
Residues in 2 were numbered consecutively from 1 to 9 starting from the amino-terminal residue with a
Tyr side chain.
7
)

Helvetica Chimica Acta Vol. 85 (2002)
3697
Fig. 2. Fingerprint NH/HÀC(b) and N'H/HSiÀC(a) region ofthe DQF-COSY experiment of 2, recorded at
308 K in (D5)pyridine. Residues are numbered consecutively from 1 9 starting from the amino-terminal
residue with the Tyr side chain.
Table 1. 1H-NMR Chemical Shifts [ppm] ofNonamer 2 in (D5)Pyridine
Residue
1
NH
N'H
HÀC(b)
4.23
HSiÀC(a)
4.01
HReÀC(a)
3.59
HÀC(g)
3.39, 3.20
Others
8.01
7.33 (Ho)
7.09 (Hm)
2
3
7.48
6.97
7.25
6.81
4.20
4.26
3.85
3.91
2.79
2.60
1.98, 1.89
1.88, 1.32
1.53 (HÀC(d))
1.60 (HÀC(e))
3.31 (HÀC(x))
1.14 (HÀC(d))
0.95 (HÀC(e))
0.96 (HÀC(d))
7.19 (Ho)
4
5
6.66
6.76
7.13
7.20
3.86
4.55
3.95
4.22
2.59
2.86
1.59
2.86, 2.78
7.41 (Hm)
7.29 (Hp)
6
6.55
7.03
4.21
4.11
2.64
1.88, 1.72
1.34 (HÀC(d))
1.50 (HÀC(e))
3.24 (HÀC(x))
1.21, 0.90 (HÀC(d))
7.33 (Ho)
7
8
6.54
6.68
6.93
6.83
4.22
4.88
4.05
4.21
2.65
2.72
1.70
2.72
7.09 (Hm)
9
6.75
7.28
4.51
4.08
3.18
1.15
1
Qualitative comparison of H-NMR spectra of 2 and 1 recorded in (D5)pyridine
revealed a number of striking similarities. These include large 3J(NH,HÀC(b)) values,
large chemical-shift differences between diastereotopic HÀC(a) protons for central
residues, strong differentiation between vicinal coupling constants of each pair of

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Helvetica Chimica Acta Vol. 85 (2002)
diastereotopic HÀC(a) along the sequence, as well as very little temperature
dependence for these parameters. Taken together, these data represent indicators of
conformational homogeneity [26] as well as of conformational similarity with 1.
3
1
The J(NH,HÀC(b)) values extracted from the H-NMR spectra of 2 recorded
between 292 and 362 K with 10 K increments are collected in Table 2. For residues 3 8,
these values are not significantly affected by temperature and remain ꢀ10 Hz even at
362 K. This is consistent with a highly stable anti-periplanar arrangement of NH and
HÀC(b)8). The coupling constants of flanking residues 2 and 9 have slightly smaller
values (8 10.2 Hz) within the same temperature range.
Table 2. Variation of 3J(NH,HÀC(b)) Values ofResidues 2 9 ofNonamer 2 upon Temperature Increase from
292 to 362 K. n.d. ˆ not detected.
Temperature [K]
Residue number i
2
3
4
5
6
7
8
9
292
302
312
322
332
342
352
362
n.d.
n.d.
9.2
10.2
n.d.
n.d.
n.d.
9.5
n.d.
n.d.
10.5
10.4
10.2
10.2
10.5
10.0
10.2
10.4
10.7
10.4
10.4
10.4
10.4
10.5
10.5
10.4
10.8
10.4
10.4
10.4
10.5
10.2
10.2
10.2
10.8
10.4
10.4
10.2
10.5
10.5
10.2
10.5
10.7
10.8
10.4
10.4
10.7
10.5
10.2
10.2
10.7
10.7
10.5
10.2
10.4
10.5
8.5
8.5
9.7
8.9
8.4
8.0
8.4
n.d.
At first sight, the COSY and TOCSY experiments of 2 reveal that the two geminal
C(a) protons for residues 2 9 display markedly different spectroscopic behaviors. The
differences in chemical shifts (Dd) determined for each residue between 286 362 K is
reported in Table 3. In the case of residues 3 8, this difference is large (1.37 < Dd <
1.53 ppm at 286 K) and decreases only slowly upon increasing the temperature (1.29 <
Dd < 1.45 ppm at 362 K)9). Smaller Dd values were measured for residue 1 (Dd <
0.45 ppm), and, to a lesser extent, for residues 2 (Dd ꢁ 1.1 ppm) and 9 (Dd < 1 ppm).
Dd Values of flanking residues 1 and especially 9 are the most sensitive to variations of
temperature. Nevertheless, the nonequivalence of diastereotopic C(a) protons within
residues 2 8 at high temperature is consistent with their location in a distinct spatial
environment that is not affected by temperature changes and, thus, confirms the
existence of a well-defined and stable secondary structure. The conformation around
the C(a)ÀC(b) bond was then examined in more details.
Fig. 3 shows Newman projections along the C(a)ÀC(b) bond for the three possible
conformers I III with experimental requirements to be fulfilled so as to validate one
form or another. The exact measurement of 3J(HÀC(b),HÀC(a)) values was not
possible due to overlap of HÀC(b) resonances. However, careful examination of DQF-
COSY data obtained between 282 362 K qualitatively indicates that, for all residues,
the downfield C(a) proton exhibits a small coupling constant with HÀC(b) (the
corresponding cross-peaks were not detectable in the DQF-COSY plot), whereas the
8
)
)
Similarly, in the case of b- and g-peptides, the rotation is highly restricted around NÀC(b) and NÀC(g)
bonds, respectively, and the corresponding protons are situated in a nearly anti-periplanar arrangement.
In our previous study of heptamer 1, Dd values measured for central residues 3 6 were in the same range.
9

Helvetica Chimica Acta Vol. 85 (2002)
3699
Table 3. Chemical-Shift Differences (Dd) Between Geminal C(a) Protons for Residues 1 9 of Oligomer 2 as
Determined from DQF-COSY Experiments Recorded in (D5)Pyridine between 282 to 362 K
Temperature [K]
Residue number i
1
2
3
4
5
6
7
8
9
282
292
302
312
322
332
342
352
362
0.43
0.42
0.40
0.41
0.40
0.39
0.38
0.36
0.35
1.11
1.10
1.08
1.05
1.05
1.03
1.00
0.99
0.98
1.37
1.32
1.31
1.30
1.30
1.30
1.29
1.28
1.29
1.39
1.37
1.37
1.36
1.36
1.35
1.35
1.34
1.34
1.38
1.37
1.38
1.36
1.35
1.35
1.34
1.33
1.34
1.53
1.51
1.50
1.48
1.47
1.47
1.45
1.44
1.45
1.39
1.40
1.40
1.41
1.41
1.41
1.41
1.41
1.41
1.52
1.50
1.47
1.46
1.43
1.40
1.38
1.35
1.32
0.97
0.94
0.90
0.86
0.83
0.79
0.75
0.72
0.69
Fig. 3. Representation ofthe three possible conformers
I
III along the C(a)ÀC(b) bond in Newman projection
together with required NMR criteria to validate one particular conformation. Measurements of
3J(HÀC(b),HÀC(a) coupling constants and intensities of intra-residue NOE between NH and C(a) protons
in 2 indicate that exclusively conformer II is populated and that the downfield C(a) proton corresponds to
HSiÀC(a).
upfield C(a) proton has a large coupling constant with HÀC(b). Hence, conformer I
can be excluded. Comparison of intra-residue NOE intensities between NH and both
HÀC(a) protons for residues 2 9 reveals that the downfield C(a) proton consistently
displays a weaker NOE to NH than the corresponding upfield C(a) proton
(exemplified for residue 2 in Fig. 4). This result excludes the possibility to have
conformer III populated, and confirms that form II is the only one to be in full
agreement with experimental data (this is verified for all residues but 1). In addition,
this careful examination allowed the unambiguous stereospecific attribution of
diastereotopic C(a) protons, the downfield proton being assigned to HSiÀC(a).
3
Extraction of J(N'H,HÀC(a)) values to gain information on the conformational
restriction around the N'ÀC(a) bond was problematic because of severe overlap of the
aromatic protons of 2 with residual pyridine protons. Qualitative examination of DQF-

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Helvetica Chimica Acta Vol. 85 (2002)
Fig. 4. Parts ofthe a) TOCSYand b) ROESY plots showing cross-peaks between NH and C(a) protons ofresidue
2. Strong NOE cross-peaks are observed in the ROESY plot (tm ˆ 300 ms) between NH and HÀC(b) as well as
between NH and HReÀC(a). These data, together with the absence of a significant NH/HSiÀC(a) NOE cross-
peak, exclude the possibility to have conformer III (see Fig. 3) populated. Similar results were obtained for
other residues.
COSY data recorded between 292 362 K revealed that, for all residues except residue
1, HSiÀC(a) exhibits a large coupling constant with N'H, while the HReÀC(a) has a
small one (the N'H/HReÀC(a) cross-peaks were hardly detectable in the DQF-COSY
plot). Still, decoupling experiments performed by irradiation of HSiÀC(a) or
HReÀC(a) at chosen temperatures between 295 and 329 K allowed the determina-
3
tion of J(N'H,HÀC(a)) values for residues 1, 2, 4 7, and 9. With the exception of
residue 1 (3J(N'H,HSiÀC(a)) ˆ 3J(N'H,HReÀC(a)) ˆ 5 Hz), 3J(N'H,HSiÀC(a)) and
3J(N'H,HReÀC(a)) were between 7.4 10.5 Hz and 1 2 Hz, respectively, implying for
these residues that N'H is nearly anti-periplanar to HSiÀC(a) and syn-clinal to
HReÀC(a).
As for a- [27] and b-peptides [2e], information about the solvent accessibility of NH
protons in 2 and their possible engagement in H-bonds can be obtained from
temperature coefficients. It is generally assumed that, for peptides in a rigidly folded
state, amide protons with a temperature coefficient less negative than À 4 ppb/K are
involved in H-bonding. Table 4 shows the temperature coefficients of the urea proton
resonances determined for residues 1 9 over a range of 58.5 K. All NH signals shift
upfield upon warming and thus display negative temperature gradients. Absolute
values of temperature coefficients are ꢀ6.9 Hz in the case of the first two urea bonds,
thus indicating high solvent accessibility. The values are very similar for both NH and
N'H protons within these urea bonds (compare À Dd/DT of N'H(1) and NH(2)). In
contrast, the particularly low absolute values determined for NH of residues 4 9
(3.6 ꢀÀ Dd/DTꢀ 2) suggest solvent shielding and intramolecularly H-bonded protons.

Helvetica Chimica Acta Vol. 85 (2002)
3701
Table 4. Calculated Temperature Coefficients À Dd/DT [ppb/K] ofNH( i) and N'H(i) Protons for i ˆ 1 9, in
(D5)Pyridine
Residue number i
1
2
3
4
5
6
7
8
9
NH (i)
N'H(i)
9
7
6.9
3.9
3.5
5.6
2
5.6
2
4.4
3.1
5.2
2.3
4.4
3.6
5.6
9
For i ˆ 3 9, absolute values of temperature coefficients of N'H(i) are systematically
higher than those of NH(i ‡ 1). Although the same trend was observed for heptamer 1,
the difference contained between 0.4 and 2.6 ppb/K is probably not large enough
(particularly in the case of the third, sixth, and eighth urea bond) to distinguish an
increased solvent accessibility of N'H(i) compared to NH(i ‡ 1).
The ROESY data obtained at 308 K (tm ˆ 300 ms) was selected to determine the
three-dimensional structure of 2. A total of 134 NOEs (interresidue and intraresidue)
were extracted and classified according to their cross-peak volume in three categories:
strong, medium, and weak. The 21 inter-residue NOEs (7 sequential, 12 with (j À i) ˆ 2,
and 2 with (j À i) ˆ 3) are collected in Table 5.
Table 5. Interresidue NOEs Observed in the ROESY NMR Plot (tm ˆ 300 ms) ofNonamer 2, in (D5)Pyridine at
308 K
H-Atom
Residue i
H-atom
Residue j
NOEa)
k ˆ j À i
Hm
Ho
N'H
N'H
1
1
2
3
3
3
4
4
4
4
5
5
5
5
5
6
6
7
7
7
8
HÀC(d)
HÀC(d)
NH
NH
NH
N'H
NH
NH
N'H
N'H
NH
NH
N'H
HReÀC(a)
HReÀC(a)
NH
N'H
NH
4
4
3
4
5
5
5
6
6
6
6
7
7
7
7
7
8
8
9
9
9
s
m
s
s
s
s
s
s
s
s
s
s
s
m
m
s
s
s
3
3
1
1
2
2
1
2
2
2
1
2
2
2
2
1
2
1
2
2
1
HÀC(b)
HÀC(b)
N'H
HÀC(b)
HÀC(b)
HSiÀC(a)
N'H
HÀC(b)
HÀC(b)
HÀC(b)
Hm
N'H
HSiÀC(a)
N'H
HSiÀC(a)
HÀC(d2)
N'H
N'H
m
s
s
HReÀC(a)
NH
a) Strong (s), <2.8 ä; medium (m), <3.8 ä; weak (w), <5.5 ä.
Medium-range connectivities of the type HÀC(b)i/NH(i ‡ 2), HÀC(b)i/N'H(i ‡ 2),
and HSiÀC(a)(i)/N'H(i ‡ 2) are particularly informative. Two regions of the ROESY
plot containing the representative set of i/(i ‡ 2) NOE connectivities between residues

3702
Helvetica Chimica Acta Vol. 85 (2002)
4 and 6 are shown in Fig. 5. This NOE pattern repeated along the sequence and also
found in the ROESY plot of heptamer 1 is consistent with the presence of a helical
conformation. However, in contrast to what was previously observed with 1, a number
of i/(i ‡ 2) NOE connectivities (between residues 2 and 4, 6 and 8, and 7 and 9) could
not be assigned unequivocally because of resonance overlaps and, thus, were not used
in the calculations.
Fig. 5. Representative set of i/i ‡ 2 NOE connectivities between residues 4 and 6 extracted from the ROESY plot
(tm ˆ 300 ms) of 2. a) Part of the TOCSY plot showing N'H/HÀC(b) and N'H/HSiÀC(a) cross-peaks for
residues 4 and 6. b) ROESY plot with inter-residue N'H(6)/HÀC(b)4 and N'H(6)/HSiÀC(a)4 . c) Part of the
TOCSY plot showing NH/HÀC(b) and NH/HSiÀC(a) cross-peaks for residues 4 and 6. d) ROESY plot with
inter-residue NH(6)/HÀC(b)4 NOE cross-peaks. These i/i ‡ 2 NOE connectivities, which are found for i ˆ 3, 5,
6, and 7, are consistent with 2 adopting a regular helical structure.
The conformational analysis of nonamer 2 was further investigated by restrained
molecular-dynamics calculations. A 100-ps simulated annealing refinement of 30
randomly chosen starting structures, under experimental NMR restraints (distance and
dihedral-angle restraints), converged towards a regular right-handed 2.5 helix (well
defined from residues 1 9) of ca. 5.1 ä pitch (the overall length of the helix is ca.
18 ä). The resulting structures show no NOE-violations greater than 0.50 ä and

Helvetica Chimica Acta Vol. 85 (2002)
3703
dihedral-angle violations greater than 108. The superimposition of the 20 lowest-energy
structures is represented in Fig. 6.
Fig. 6. NMR Structure ofoligourea 2 in (D5)pyridine (stereo view along the helix axis). Bundle of the 20 best
structures of lowest energy. Root-mean-squares (rms) differences of bond and angle deviations were less than
0.02 ä and 38, respectively. Rms deviations of all heavy backbone atoms from a mean structure were 0.56 Æ 0.15
for residues 1 8 and 0.85 Æ 0.06 for residues 1 9.
The urea planes are approximately parallel to the helix axis. The helical structure is
stabilized by a number of CˆO(i) ¥¥¥ HÀN'(i ‡ 2) and CˆO(i) ¥¥¥ HÀN(i ‡ 3) H-bonds
that close 12- and 14-membered rings, respectively10). Urea protons located near the
amino terminus (N'H of residue 1 and 2 and NH of residues 2 and 3) can not be H-
bonded11). The CˆO of residue 1 is H-bonded to NH(4) and closes a 14-membered
ring. In contrast, for i ˆ 2 7, CˆO(i) is nearly equidistant from both urea protons
N'H(i ‡ 2) and NH(i ‡ 3). Hence, intramolecular H-bonds are regularly distributed
between the CˆO(i) and urea N'H(i ‡ 2) and NH(i ‡ 3), respectively, with a frequency
above 95% (Table 6). The CˆO(i) ¥¥¥ HÀN'(i ‡ 2) H-bond is the strongest for i ˆ 2, 4,
and 6, whereas the CˆO(i) ¥¥¥ HÀN(i ‡ 3) H-bond dominates for i ˆ 3, 4, and 7.
Whether this dichotomy in H-bonding strength throughout the backbone structure of 2
is a stabilizing property of the helix cannot, however, be inferred from the present study
and requires the structural study of longer oligomers. In the structure of heptaurea 1,
the H-bond pattern is slightly different, the 14-membered rings being systematically
more populated than the 12-membered rings.
The side chains are relatively well superimposed for all residues except aromatic
ones for which multiple orientations of the ring are populated. Examination of the top
view of the helix (Fig. 7) indicates that for i ˆ 1 4, side chains of residues i and i ‡ 5 are
10
)
)
Additionally, a less-frequent but still significant H-bond occurs between side chains of residues 1 and 6.
This observation is consistent with the high absolute values of temperature coefficients determined for
these protons (see Table 4).
11

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Helvetica Chimica Acta Vol. 85 (2002)
Table 6. Estimated CˆO(i) ¥¥¥ HÀN'(i ‡ 2) and CˆO(i) ¥¥¥ HÀN(i ‡ 3) H-Bonding Pattern for i ˆ 1 7 Based on
Statistical Analysis ofthe Final 20 Structures ofLowest Energy
CˆO(i) ¥¥¥ HÀN'(i ‡ 2)
CˆO(i) ¥¥¥ HÀN(i ‡ 3)
Frequencya)
Distanceb)
Anglec)
Frequencya)
Distanceb)
Anglec)
i ˆ 1
i ˆ 2
i ˆ 3
i ˆ 4
i ˆ 5
i ˆ 6
i ˆ 7
80
100
95
100
95
3.18 Æ 0.13
2.93 Æ 0.06
3.19 Æ 0.11
2.85 Æ 0.03
3.12 Æ 0.10
2.95 Æ 0.04
3.06 Æ 0.01
162 Æ 10
162 Æ 4
155 Æ 3
156 Æ 3
152 Æ 4
160 Æ 2
158 Æ 1
95
95
100
100
100
95
3.17 Æ 0.13
2.98 Æ 0.04
3.06 Æ 0.06
2.95 Æ 0.11
2.92 Æ 0.04
2.95 Æ 0.01
161 Æ 12
165 Æ 3
157 Æ 4
167 Æ 5
157 Æ 3
167 Æ 1
100
100d)
a) Frequency of occurrence of the H-bond over the 20 lowest-energy structures, in %. An H-bond was counted
when the donor-acceptor distance was less than 3.25 ä and the donor-H-acceptor angle was higher than 1208.
b) Average OÀN distance, in ä. c) Average OÀHÀN angle, in 8. d) The terminal urea N-atom is considered
here as i ‡ 3.
located nearly on top of each other. The distance between corresponding HÀC(b)i and
HÀC(b)i ‡ 5 determined from an average structure is between 9.8 and 11.2 ä. With an
internal diameter of ca. 3 ä, the helix is particularly compact and is devoid of empty
volume in the interior.
Fig. 7. NMR Structure ofoligourea 2 in (D5)pyridine (top view). Bundle of the 20 best structures of lowest
energy.
Overlay of the structures of 1 and 2 (Fig. 8), both determined in (D5)pyridine,
reveals a very similar backbone conformation for the heptamer and the nonamer. The
differences observed in the H-bond pattern of the two structures (vide supra) have little
or no influence on the overall topology of the helix.

Helvetica Chimica Acta Vol. 85 (2002)
3705
Fig. 8. Overlay ofthe backbone structures ofheptamer 1 [18] and ofnonamer 2, both in (D5)pyridine (stereo
view). C-Atoms of the 7-mer and of the 9-mer are displayed in yellow and white, respectively. Rms deviations of
all heavy backbone atoms are 0.60 ä for residues 1 7.
4. Circular-Dichroism Spectroscopy. CD Spectroscopy allows the study in solution
of the extrinsic asymmetry resulting from preferential conformations of optically active
molecules with chromophores. Applications of this powerful technique include
structural characterization of biomacromolecules (proteins, nucleic acids, polysacchar-
ides), polypeptides constituted of a-amino acids, and, more recently, nonnatural folding
oligomers [28] including b- [8] and g-peptides [4e]. Surprisingly, experiments with g4-
hexapeptides of type A (which are related to oligoureas B and which adopt a 2.614
helical structure in CD3OH and (D5)pyridine [4a]) did not reveal any characteristic CD
signature (no Cotton effect)12). With the aim to gain additional and complementary
information about the conformation of oligoureas in solution, we have performed CD
experiments with nonamer 2. The CD Spectrum of 2 (Fig. 9,a), measured in MeOH
between 194 and 250 nm at a concentration of 0.2 mm, exhibits a remarkably strong
positive band at ca. 203 nm ([F] ˆ ‡3.7 ¥ 105 molar-ellipticity units) suggesting the
presence of a defined secondary structure in MeOH. This CD pattern of 2 in MeOH is
neither sensitive to change in concentration (data not shown) nor to increase in
temperature. The intensity of the maximum at 203 nm recorded between 278.5 and
328 K decreased only slowly upon temperature increase (by less than 1% per 10 K)
(Fig. 9,b). The decrease is roughly linear and does not show any melting in the
temperature range studied.
Similarly, g2,4-peptides built from 2,4-disubstituted g-amino acids of like configuration and shown to adopt
a more stable 2.6 helical structure do not display typical CD curves [7a]. In contrast, CD spectra of 2.6
helical g2,3,4 peptides present a positive Cotton effect around 213 nm [4e].
12
)

3706
Helvetica Chimica Acta Vol. 85 (2002)
Fig. 9. CD Analysis ofoligourea 2: a) CD Spectrum of 2 recorded in MeOH at room temperature at a
concentration of 0.2 mm. b) CD Temperature scan of 2 in MeOH for l ˆ 203 nm. Molar ellipticity V in deg ¥
cmÀ1 ¥ dmolÀ1
.
5. Conclusions. This study, which led to the NMR structure determination of
nonamer 2 with Ala, Val, Leu, Phe, Tyr, and Lys side chains, further illustrates the
remarkable homogeneity and stability of the 2.5 helical secondary structure adopted by
enantiomerically pure N,N'-linked oligoureas A in pyridine. Temperature-dependent
NMR experiments indicate that the helical structure is probably still present at 362 K.
Comparison of the structure of nonamer 2 with the previously reported structure of
heptamer 1 [18] reveals that the overall backbone traces are very similar thus
suggesting that N,N'-linked oligoureas share a unique three-dimensional fold when a
minimal length (still to be determined) is reached. As expected, this helix is reminiscent
of the 2.614 helical structure of g-peptides. Thus, the similarity between g-peptides and
N,N'-linked oligoureas is no longer restricted to an isosteric relationship (based
exclusively on the chemical formulae of the backbone) but is extended to a unique
three-dimensional structural relationship.
Essentially two factors may play a role in inducing and stabilizing the 2.5 helix: i) a
rigid (‡)-syn-clinal arrangement around the C(a)ÀC(b) bond very similar to what was
previously observed for b- [2e] and g-peptides [4a][7a], and ii) intramolecular H-
bonds. In the case of oligoureas, the helix is held by two types of H-bonds involving a
given CˆO, namely CˆO(i) ¥¥¥ HÀN'(i ‡ 2) and CˆO(i) ¥¥¥ HÀN(i ‡ 3) that close 12-
and 14-membered H-bonded rings, respectively. In the structure of 1, CˆO(i) was not
equidistant from the two urea protons13) and CˆO(i) ¥¥¥ HÀN(i ‡ 3) was systemati-
cally more populated than CˆO(i) ¥¥¥ HÀN'(i ‡ 2). In contrast, the two H-bonds are
almost symmetrical in the modeled structure of 2. However, in the absence of more
13
)
In the crystal structure of a N,N'-linked cyclo-tetraurea forming nanotubes, the two NH of each urea
fragment are H-bonded to a CˆO of a neighboring macrocycle. Interestingly, the two N ¥¥¥ O distances are
not equal, and one (3.28 ä) is just above the limit generally considered for H-bonding [19].

Helvetica Chimica Acta Vol. 85 (2002)
3707
conclusive data, the precise position of CˆO(i) relative to N'H(i ‡ 2) and NH(i ‡ 3) is
still a matter of discussion.
Keeping in mind the absence of a characteristic CD signature for 2.6 helical g4-
peptides, results of the first CD experiments performed on oligoureas were particularly
surprising. The CD spectrum of 2 recorded in MeOH displays an intense maximum at
ca. 203 nm (per-residue molar ellipticity: [Vr] > 40000 deg cm2 dmolÀ1), which could
reflect the presence of one or more regular conformations. Although it is tempting to
attribute this signature to the 2.5 helical secondary structure, NMR experiments aimed
at probing the occurrence of the helix in MeOH as well as CD measurements on shorter
oligomers are a prerequisite, and our results along that line will be reported in due time.
¬
Access to both the Bruker ARX-500 facilities of the Service Commun de RMN (Faculte de Chimie,
Strasbourg) and the Bruker DRX-600-NMR facilities of the Service Commun de Biophysicochimie des
¬
¬
¬
Interactions Moleculaires (Universite HenriPoni care , Nancy I) were deeply appreciated.
Experimental Part
1. General. Abbreviations: GP (general procedure), b3-Xaa (b-homoamino acid), NMM (N-methylmor-
pholine), DMF (dimethylformamide). (D5)Pyridine (100%, IE ꢀ 96%) and tetramethylsilane were purchased
from Euriso-top (France). THF was freshly distilled from Na/benzophenone under Ar before use. Toluene and
pyridine were distilled from CaH2 and stored over 4-ä molecular sieves. Amino acid derivatives and Rink amide
resin were purchased from Neosystem or Senn. All other reagents and solvents were of anal. grade. N-Fmoc-b3-
amino acids bearing the Ala, Val, Leu, Phe, Tyr(OtBu), and Lys(Boc) side chains were synthesized as described
starting from the corresponding N-Fmoc-protected a-amino acids and gave spectroscopic data in good
agreement with those previously reported [24]. TLC: Merck silica-gel 60 F254 plates; detection with UV and
ninhydrin. Optical rotations: 258; Perkin-Elmer 241 polarimeter (Saint Quentin, Yvelines, France). Anal.
HPLC: Beckman instrument (Gagny, France); Nucleosil-C18 column 5 mm (4.6 Â 150 mm); linear gradient of A
(0.1% CF3COOH) and B (MeCN containing 0.08% of CF3COOH); flow rate 1.2 ml/min; UV detection at
214 nm; tR in min. Prep. reversed-phase HPLC: Perkin-Elmer apparatus; Aquapore ODS-20mm column (100 Â
10); linear gradient of A (aq. 0.06% CF3COOH) and B (80% MeCN/20% A); flow rate 6 ml/min; UV detection
at 214 nm. Circular dichroism (CD): Jobin-Yvon CD-max6 system; recording between 190 and 300 nm; 2-mm
rectangular cell; average of five scans, corrected for the baseline; oligomer concentration 0.2 mm in MeOH;
molar ellipticity [F] in deg ¥ cm2 ¥ dmolÀ1 (l in nm). NMR Spectra: Bruker 200 (1H 200 MHz, 13C 50 MHz),
Bruker-Avance-300 (1H 300 MHz, 13C 75 MHz), Bruker DRX-500 and -600 spectrometers; chemical shifts d in
ppm downfield from internal SiMe4 (ˆ0 ppm); J values in Hz. Matrix-assisted laser desorption/ionization
(MALDI) MS: Protein TOF apparatus (Bruker Spectrospin, Bremen, Germany); in m/z.
2. Succinimidyl Carbamates 3a f: General Procedure. The N-Fmoc-b3-amino acid (1 equiv.) was dissolved
in THF (30 ml) under Ar and cooled to À 208. After addition of EtOCOCl (1.1 equiv.) and NMM (1.1 equiv.),
the mixture was stirred at À 208 for 20 min. The resulting white suspension was allowed to warm to À 108 and
treated with aq. NaN3 soln. (5 ml; 2.5 equiv.). The mixture was stirred for 5 min, diluted with AcOEt, washed
with sat. NaCl soln., dried (MgSO4), and evaporated to give the expected acyl azide, which was used without
further purification. Toluene was added under Ar, and the resulting soln. was heated to 658 under stirring. After
the gas evolution had stopped (ca. 10 min), N-hydroxysuccinimide (1 equiv.) and pyridine (1 equiv.) were
added. The mixture was stirred for 5 min at 658 and then cooled to r.t. Compounds 3a f precipitated from the
toluene soln. and were collected by filtration. Recrystallization from toluene or AcOEt/iPr2O afforded anal.
pure monomers.
Succinimidyl {(2S)-2-{[(9H-Fluoren-9-ylmethoxy)carbonyl]amino}propyl}carbamate (3a). From Fmoc-b3-
Ala-OH (1.95 g, 6 mmol) according to the GP. Recrystallization from toluene yielded 3a (2.26 g, 86%). White
25
D
solid. M.p. 161 1638. ‰aŠ ˆ À3.6 (c ˆ 1.08, DMF). HPLC (linear gradient, 30 100% B, 20 mi n).tR 10.44.
1H-NMR (200 MHz, CD3CN) : 1.03 (d, J ˆ 7, Me); 2.76 (s, CH2CH2); 2.99 3.18 (m, CH2NH); 3.46 3.69
(m, CHMe); 4.18 4.34 (m, CHCH2O); 7.22 7.45 (m, 4 arom. H); 7.70 (d, J ˆ 7, 2 arom. H); 7.89 (d, J ˆ 7,
2 arom. H); 8.33 (t, J ˆ 6, CH2NH). 13C-NMR (50 MHz, CD3CN): 17.8, 25.2, 45.8, 46.2, 46.7, 65.2, 120.0, 125.1,
‡
‡
127.0, 127.5, 140.7, 143.8, 143.9, 152.1, 155.4, 170.7. MALDI-TOF-MS: 476 ([M ‡ K] ), 460 ([M ‡ Na] ).

3708
Helvetica Chimica Acta Vol. 85 (2002)
Succinimidyl {(2S)-2-{[(9H-Fluoren-9-ylmethoxy)carbonyl]amino}-3-methylbutyl}carbamate (3b). From
Fmoc-b3-Val-OH (3.53 g, 10 mmol) according to the GP. Recrystallization from toluene yielded 3b (3.21 g,
25
D
69%). White solid. M.p. 109 1118. ‰aŠ ˆ ‡5.9 (c ˆ 1.18, DMF). HPLC (linear gradient, 30 100% B, 20 mi n):
tR 11.84. 1H-NMR (300 MHz, (D6)DMSO): 0.90 (d, J ˆ 8, Me); 0.95 (d, J ˆ 8, Me); 1.72 1.89 (m, CHMe2); 2.71
(s, CH2CH2); 3.08 3.42 (m, CH2NH); 3.53 3.65 (m, NHCH); 4.17 4.22 (m, CHCH2O); 4.35 4.51
(m, CHCH2O); 6.20 (t, NH); 7.25 7.45 (m, 4 arom. H); 7.60 (d, J ˆ 7, 2 arom. H); 7.75 (d, J ˆ 7, 2 arom. H).
13C-NMR (75 MHz, (D6)DMSO): 18.2; 19.4; 25.4; 30.1; 44.4; 47.3; 56.7; 66.9; 120.0; 125.2; 127.1; 127.7; 141.3;
‡
‡
143.8; 152.2; 157.1; 169.9. MALDI-TOF-MS: 504 ([M ‡ K] ), 488 ([M ‡ Na] ).
Succinimidyl {(2S)-2-{[(9H-Fluoren-9-ylmethoxy)carbonyl]amino}-4-methylpentyl}carbamate (3c). From
Fmoc-b3-Leu-OH (2.94 g, 8 mmol) according to the GP. Recrystallization from toluene yielded 3c (1.95 g, 51%).
25
D
White solid. M.p. 134 1378. ‰aŠ ˆ À10.8 (c ˆ 1.01, DMF). HPLC (linear gradient, 30 100% B, 20mi n):tR
12.63. 1H-NMR (200 MHz, (D6)DMSO): 0.80 (d, J ˆ 7, Me); 0.83 (d, J ˆ 7, Me); 1.14 1.33 (m, CH2CHMe2);
1.50 1.54 (m, CHMe2); 2.57 (s, CH2CH2); 3.04 3.07 (m, CH2NH); 3.51 3.58 (m, NHCH); 4.45 4.44
(m, CHCH2O); 7.10 (d, J ˆ 8, NHCH); 7.25 7.45 (m, 4 arom. H); 7.67 (d, J ˆ 7, 2 arom. H); 7.86 (d, J ˆ 7,
2 arom. H); 8.27 (t, J ˆ 6, CH2NH). 13C-NMR (50 MHz, CD3CN): 21.6; 23.3; 24.1; 25.2; 45.4; 46.8; 48.5; 65.1;
‡
120.0; 125.1; 126.9; 127.5; 140.7; 143.7; 144.0; 152.0; 155.7; 170.7. MALDI-TOF-MS: 518 ([M ‡ K] ), 502 ([M ‡
‡
Na] ).
Succinimidyl {(2S)-2-{[(9H-Fluoren-9-ylmethoxy)carbonyl]amino}-3-phenylpropyl}carbamate (3d). From
Fmoc-b3-Phe-OH (4.42 g, 11 mmol) according to the GP. Recrystallization from toluene yielded 3d (3.73 g,
25
66%). White solid. M.p. 175 1778. ‰aŠ ˆ À26.1 (c ˆ 1.13, DMF). HPLC (linear gradient, 30 100% B, 20 mi n).
D
1
tR 12.48 . H-NMR (300 MHz, (D6)DMSO): 2.55 (s, CH2Ph); 2.72 (s, CH2CH2); 3.13 (t, J ˆ 6, CH2NH); 3.66
3.83 (m, NHCH); 4.04 4.22 (m, CHCH2O); 7.06 7.43 (m, 9 arom. H); 7.60 (d, J ˆ 7.3, 2 arom. H); 7.84 (d, J ˆ
7.7, 2 arom. H); 8.38 (t, J ˆ 5.8, CH2NH). 13C-NMR (75 MHz, (D6)DMSO): 25.7; 37.6; 45.2; 47.1; 52.7; 65.7;
120.5; 125.6; 126.5; 127.5; 128.0; 128.6; 128.8; 129.6; 139.0; 141.1; 144.3; 152.6; 156.0; 171.3. MALDI-TOF-MS:
‡
‡
552 ([M ‡ K] ), 536 ([M ‡ Na] ).
Succinimidyl {(2S)-2-{[(9H-Fluoren-9-ylmethoxy)carbonyl]amino}-3-[4-(tert-butoxy)phenyl]propyl}car-
bamate (3e). From Fmoc-b3-Tyr(tBu)-OH (2.37 g, 5 mmol) according to the GP. Recrystallization from toluene
25
D
yielded 3e (2.28 g, 78%). White solid. M.p. 138 1408. ‰aŠ ˆ À22.9 (c ˆ 1.12, DMF). HPLC (linear gradient,
30 100% B, 20 mi n):tR 13.87. 1H-NMR (300 MHz, (D6)DMSO): 1.13 (s, tBu); 2.48 2.69 (m, CH2Ar); 2.72
(s, CH2CH2); 3.13 (t, J ˆ 6, CH2NH); 3.63 3.82 (m, NHCH); 4.02 4.23 (m, CHCH2O); 6.74 (d, J ˆ 8, 2 arom.
H); 7.06 (d, J ˆ 8, 2 arom. H); 7.22 7.42 (m, 4 arom. H); 7.57 7.66 (m, 2 arom. H); 7.84 (d, J ˆ 7, 2 arom. H);
8.38 (t, J ˆ 6, CH2NH). 13C-NMR (75 MHz, (D6)DMSO): 24.5; 27.7; 35.9; 44.1; 45.9; 51.5; 64.6; 76.7; 119.3;
122.6; 122.7; 124.5; 126.3; 126.8; 128.8; 132.3; 140.0; 143.1; 151.4; 152.5; 154.8; 170.1. MALDI-TOF-MS: 624
‡
‡
([M ‡ K] ), 608 ([M ‡ Na] ).
Succinimidyl {(2S)-2-{[(9H-Fluoren-9-ylmethoxy)carbonyl]amino}-6-{[(tert-butoxy)carbonyl]amino}hex-
yl}carbamate (3f). From Fmoc-b3-Lys(Boc)-OH (7.16 g, 13 mmol) according to the GP. Recrystallization from
25
D
toluene yielded 3f (6.81 g, 79%). White solid. M.p. 122 1248. ‰aŠ ˆ À4.7 (c ˆ 1.16, DMF). HPLC (linear
gradient, 30 100% B, 20 mi n):tR 12.67. 1H-NMR (300 MHz, (D6)DMSO): 1.09 1.45 (m, OtBu, (CH2)3); 2.71
(s, CH2CH2); 2.79 2.95 (m, CH2NH); 3.30 3.32 (m, CH2NH); 3.46 3.50 (m, NHCH); 4.16 4.29
(m, CHCH2O); 6.71 (t, J ˆ 6, NH); 7.12 (d, J ˆ 7, NH); 7.27 7.42 (m, 4 arom. H); 7.64 7.69 (d, J ˆ 7, 2 arom.
H); 7.85 (d, J ˆ 7, 2 arom. H); 8.27 (t, J ˆ 6, CH2NH). 13C-NMR (75 MHz, (D6)DMSO): 22.7; 25.2; 28.2; 29.4;
31.0; 39.8; 45.0; 46.8; 50.5; 65.2; 77.3; 120.0; 125.2; 127.0; 127.5; 140.7; 143.8; 152.0; 155.5; 170.8. MALDI-TOF-
‡
‡
MS: 702 ([M ‡ K] ), 686 ([M ‡ Na] ).
3. Oligourea 2. Assembly of the protected oligourea chain was carried out with a homemade multichannel
synthesizer [29] in a semi-automatic mode on a 50-mmol scale starting from Rink amide resin (0.62 mmol/g). The
Fmoc group at the starting resin was removed with 20% piperidine in DMF (3 Â 5 min) under N2 bubbling. The
resin was then filtered and washed with DMF (5 Â 1 min). For each coupling step, a soln. of the carbamate 3a
f
(5 equiv.) in DMF and NMM (2 equiv.) were added successively to the resin, and the suspension was mixed
under N2 bubbling for 70 min. A double coupling was performed systematically. Monitoring of the coupling
reaction was generally performed with the Kaiser ninhydrin test [30] or with 2,4,6-trinitrobenzenesulfonic acid
[31]. After the removal of the last Fmoc protecting group, the resin was washed with CH2Cl2 and Et2O and dried
under N2. Side-chain deprotection and cleavage of the oligomer from the resin was performed by treatment with
CF3COOH/H2O for 150 min at 208. After precipitation in cold Et2O and centrifugation, the crude oligomer was
solubilized and lyophilized to afford crude 2 as CF3COOH salt (90 mg, quant.), purity 32% (reversed-phase
HPLC). The crude oligomer was finally purified by reversed-phase HPLC (linear gradient, 0 80% B, 40 mi n)

Helvetica Chimica Acta Vol. 85 (2002)
3709
and lyophilized to give 2 as a CF3COOH salt (16 mg, 18%). White solid. HPLC (linear gradient, 5 65% B,
1
‡
20 min): tR 14.95. H-NMR (500 MHz, (D5)pyridine): Table 1. MALDI-TOF-MS: 1393.0 ([M ‡ 1] ).
4. NMR Spectroscopy ofOligourea 2. Spectra were recorded with Bruker DRX-500 and -600 NMR
spectrometers by means of 5 mm inverse 1 H/X with Z gradient capabilities probes. The temperature was
maintained at 308 K for the structure determination. 1) DQF-COSY [32] was performed by a gradient-selection
pathway. Acquisition: 2K(t2) ¥ 512(t1) data points; relaxation delay of 3 s; QF mode in t1 and 2 scans per
increment. Processing was done after a sine-bell P/3-shifted multiplication in both dimensions, and Fourier
transformed in 1K ¥ 1K real data points. 2) TOCSY [33] plots were recorded with 2K(t2) ¥ 512(t1) data points in
echo-antiecho mode with Z-gradients selection; relaxation delay of 3 s and 16 scans per increments; two
experiments were recorded with mixing times of 124 and 70 ms, resp. Processing: Zero-filling and FT to 2K ¥ 2K
after multiplication by cosine in both dimensions. 3) An adiabatic off-resonance ROESY [34] was recorded with
2K(t2) ¥ 512(t1) data points; acquisition in states-TPPI mode; 8 scans per increment and 3 s relaxation delay. The
CW-spin lock field of 5 KHz was alternatively shifted by ‡ / À 3750 Hz to achieve a tg(q) ˆ 54.78 allowing an
efficient mismatch of the Hartmann-Hahn condition. The adiabatic pulse was a trapezoidal shape. The mixing
time was 300 ms. Processing was done with 1K ¥ 1K real data points after multiplication by cosine filters in both
dimensions. 4) Several 2D-NOESYexperiments with mixing times from 200 to 500 ms were also performed with
2K(t2) ¥ 512(t1) data points in states-TPPI mode with Z-gradients selection; relaxation delay of 1.5 s and 96 scans
per increments. The build-up curve for different NOE correlations showed that spin diffusion was negligible for
tm ˆ 300 ms. The adiabatic off-resonance ROESY experiment appeared more sensitive and was used for the
structure determination.
The spectral width in F1 was 5000 Hz. Data processing was performed with XWIN-NMR software. The
intensity of peaks was extracted from the ROESY plot with Sparky software [35] and the interproton distances
calculated taking as reference the distance of 1.78 ä between the different sets of C(a) protons. Distance
restraints were assigned as strong, medium, and weak, and set at intervals of 1.8 2.8, 2.8 3.8, and 3.8 5.5 ä,
resp.
5. Structure ofOligourea 2. All calculations were performed with the AMBER6 suite of programs [36].
Parameters and topology files were first generated for each residue by a previously described procedure [37].
Starting from a fully extended structure generated by the SYBYL 6.8 package [38], geometries and atomic
charges were calculated with the GAUSSIAN98 program [39] by means of the HF/6-31G* basis set. Therein,
atom-centered charges were fitted to an ab initio electrostatic potential with the RESP method [40]. With
SYBYL, 30 starting structures were randomly generated; they were further energy-minimized (1000 steps
steepest descent followed by 1000 steps conjugate gradient relaxation) with AMBER6 by means of the Cornell
force-field [41]. The ab initio simulated annealing protocol started for each conformation with a 75-ps high-
temperature phase at 600 K followed by a 25-ps cooling phase where the temperature was linearly lowered from
600 to 1 K. During the first 75 ps, the NMR restraints (103 NOE-derived intraresidue restraints, 21 inter-residue
distance restraints, 38 torsional angle restraints) were slowly turned on, and fully effective during the cooling
phase. NMR Restraints were treated with the sander potential by means of 4 boundaries for each restraint. The
penalty function is flat (equal to zero) between an inner set of upper and lower bounds (2.3 2.8, 3.3 3.8, and
5.0 5.5 for strong, medium and weak NOEs, resp.), then rises parabolically when the internal coordinates
violate these bounds, and are finally linear outside even wider upper and lower bounds (1.8 3.3, 2.8 3.3, and
4.0 6.0 for strong, medium, and weak NOEs, resp.).Torsional-angle restraints were assigned similarly with 4
bounds (t À 40, t À 20, t ‡ 20, and t ‡ 40 for a torsion to which an average value of t was measured). For all
restraints, a force constant of 32 kcal ¥ molÀ1 ¥ äÀ2 and 50 kcal ¥ molÀ1 ¥ radÀ2 was assigned from distance and
torsional-angles restraints, resp. The resulting structures were analyzed in terms of deviations to experimental
restraints (no NOE violations >0.50 ä and no dihedral-angle violation >108). The final 20 structures with the
lowest energy were used for the structural statistics.
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Received June 7, 2002