genic side chains, we selected and prepared a monomer with a
pyrrolidine ring, resembling proline, without prejudging the
conformational outcome. The compatibility of the 2.5-helix
geometry of oligoureas for noncanonical substitution patterns
such as N-alkylated residues has not been investigated
previously. In a peptides and proteins, proline is frequently
found at the ends of a-helical and b-sheet structures but rarely
at their center.[15,16] This location preference is due in part to
the lack of an amide proton at the Xaa-Pro bond that could
participate in hydrogen-bond stabilization of regular secon-
dary structures, and to steric constraints imposed by the
pyrrolidine ring. In oligoureas, one donor site still remains in
the trisubstituted urea formed by insertion of a proline-type
residue, and could eventually participate in helix stabilization
through intramolecular hydrogen bonding.
Activated oligomers 1 and 2 bearing a succinimidyl
(pyrrolidin-2-ylmethyl)carbamate terminus were readily pre-
pared in good overall yields. Segment condensation of the
tetramer 1 and heptamer 2 with short oligoureas (trimer and
hexamer) bearing the side chains of Val, Ala, and Leu in DMF
using 3 equivalents of diisopropylethylamine (DIEA) gave
the corresponding oligoureas 3–5 with a N-(pyrrolidin-2-
ylmethyl)ureido unit at the segment junctions (Scheme 1) in
yields ranging from 66–89%. Iterative segment coupling
starting from 3 and 5 readily afforded the 11-mer 6, 20-mer 7,
and 15-mer 8 with two to three pyrrolidine units in good to
high coupling yields (70–93%).
The secondary structure propensity of oligoureas contain-
ing N-pyrrolidin-2-ylmethyl)ureido units was first examined
by circular dichroism (CD) in 2,2,2-trifluoroethanol (TFE).
Oligoureas forming right-handed 2.5 helices (i.e. consisting of
units with an S configuration) have been shown previously to
exhibit a typical CD signature in TFE with an intense positive
band at l = 203 nm, zero crossing around l = 193 nm, and a
trough at l = 188 nm.[17] Remarkably, all spectra of oligoureas
3–7 having one and two pyrrolidine units were found to
display the hallmarks of the canonical 2.5-helical structure
(Figure 1). However, the difference in per residue molar
ellipticity (PRME) observed at l = 203 nm for the spectra of
oligoureas of equal length, with and without a pyrrolidine unit
(e.g. 7-mers 3 and 9 (see Scheme 2 for structures); Figure 1a),
suggests partial destabilization of the helical structure caused
by the proline-type residue.
Figure 1. Circular dichroism spectra of oligoureas with pyrrolidine
junction in TFE. a) Spectra for the 4-mer 10 and 7-mers 3 and 9. Inset:
temperature dependence of the CD signal of 3 and 9 at l=203 nm
between 08C and 608C in TFE. b) Spectra for the 7-mer 3, 11-mer 6,
15-mer 8, and 20-mer 7. Samples were studied at 0.1 or 0.2 mm.
thermal stability of helices formed by oligoureas, we con-
ducted comparative temperature experiments between 3 and
9 (Figure 1a, inset). A linear and gradual decrease in helicity
measured at l = 203 nm was observed in both cases between
08C and 608C. The slope is similar for both oligomers
irrespective of the presence of a pyrrolidine unit, thus
suggesting that the insertion of a proline-type residue does
not exacerbate thermal unfolding in an organic solvent such
as TFE.
1H NMR spectra of oligoureas 3–6 and 8 recorded in
[D3]methanol and [D5]pyridine show features characteristic
of 2.5-helical oligoureas, including dispersion of the urea NH
groups, large vicinal coupling constants between NH and
CH(R) protons (> 9 Hz), and strong differentiation between
vicinal coupling constants of main chain diasterotopic CH2
protons. Additional insight into the helical conformation of
oligoureas containing pyrrolidine units was gained by mon-
itoring 1H NMR diastereotopicity (Dd) of the main chain CH2
protons. We reported previously that Dd values are useful
descriptors of the conformational homogeneity of helical
N,N’-linked oligoureas.[17]
However, the finding that the 7-mer 3 exhibits a much
higher PRME than the 4-mer 10 (Scheme 2) bearing a
pyrrolidine unit at the terminus (Figure 1a) suggests that an
internal pyrrolidine unit is not acting as a helix breaker and
that the helix spans the entire sequence. This conclusion is
additionally supported by the finding that PRME is gradually
increasing upon elongation of 3 to 6 and 8 (Figure 1b). To
determine the impact of a pyrolidine residue insertion on the
Diastereotopicity measurements for the main chain CH2
protons in the sequence of the 7-mer 3 revealed Dd values in
the range 0.82–1.23 ppm, which are indicative of 2.5-helical
folding (Table 1). However, comparison with the Dd values
measured for 9 suggests that the insertion of a central
pyrrolidine unit has a local destabilizing effect at the
Scheme 2. Structures for 9 and 10.
Angew. Chem. Int. Ed. 2011, 50, 11382 –11385
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim