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b2,3-aminoxy peptides with cyclic aliphatic (non-polar) side
chains: strong NOEs exist between its amide protons NHb and
both a (H2) and b (H1) protons.[3d] This distinct NOE pattern in-
dicates that the amide proton NHb points directly toward the
adjacent amide carbonyl group to form intramolecular hydro-
gen bonds. Thus, the NOE data suggest that 2 adopts a rela-
tively stable secondary structure (i.e., b NÀO turn) in both
methanol and aqueous buffer. For compound 3, these charac-
teristic NOEs were observed between NHc and both H4 and H3
while the NOE signals involving NHb could not be detected be-
cause of its fast exchange with the solvent.
Table 1. 1H NMR spectroscopic chemical shifts of the amide protons of
1–3.[a,b,c]
Compound
in methanol
dNHa [ppm]
dNHb [ppm]
dNHc [ppm]
1
2
3
1
2
3
8.44 (s)
11.00 (s)
11.09 (s)
N.D.
N.D.
N.D.
8.03 (t)
8.70 (t)
12.15 (s)
8.14 (t)
8.34 (t)
N.D.
–
–
8.62 (t)
–
–
in buffer
8.26 (t)
[a] Abbreviations: s, singlet; t, triplet; N. D., not detectable. [b] Spectra
were recorded at room temperature; concentration: 10 mm; solvent:
CD3OH or 0.1m phosphate buffer (pH 7.00). [c] The signals of aminoxy
amide protons became undetectable in buffer solutions.
CD spectroscopy is a common tool—one that is comple-
mentary to NMR spectroscopy—for probing the secondary
structures of foldamers.[4] Figure 3 presents the CD spectra of
2–4. In Figure 3a–c, we observed that the CD absorption pat-
terns of 2–4, taken at room temperature in 2,2,2-trifluoroetha-
nol (TFE), methanol, and neutral phosphate buffer (pH 7.00),
are essentially alike, with a strong absorption at about 200 nm.
The similarity of the CD patterns between 3 and 4 proves that
the tetrapeptide also adopts a helical secondary structure in-
duced by several b NÀO turns. In addition, the highest abso-
lute intensity was obtained from the sample in TFE for 2 and
3, which is a helix-promoting solvent;[5] the lowest intensity
was obtained in methanol, which disfavors secondary structure
formation primarily through competition by intermolecular hy-
drogen bonding. Interestingly, the intensity in aqueous buffer
is noticeable and is even higher than that in TFE for tetramer
4. The intensity hierarchy suggests that 2–4 adopt very similar
secondary structures, that is, b NÀO turns, in TFE, methanol,
and neutral buffer.
that of the NHb proton of 1 in both methanol and neutral
buffer. Furthermore, the chemical shift of aminoxy amide NHb
of 3 is about 1 ppm more downfield than that of the NHa
proton, which was intermolecularly hydrogen-bonded with the
solvent methanol, though both became undetectable in buf-
fers because of a fast exchange with water as a result of their
high acidities. This result suggests that the NHb protons of 2
and 3 and the NHc proton of 3 form intramolecular hydrogen
bonds while the NHa proton does not, presumably with hydro-
gen bonding patterns that are very similar to those observed
in b2-aminoxy peptides,[3g] b2,2-aminoxy peptides,[3b] b3-aminoxy
peptides,[3c] b2,3-aminoxy peptides possessing acyclic side
chains,[3f] and b2,3-aminoxy peptides possessing cyclic aliphatic
(non-polar) side chains.[3d]
We sought to use 2D NMR spectroscopic techniques to fur-
ther investigate the conformational features of 1–3 in metha-
nol and in phosphate buffer. NOESY experiments were per-
formed at room temperature for compounds 1 and 2 and
ROESY for 3 at 10 mm concentration in CD3OH or 20 mm in
neutral buffer (0.01mK2HPO4/KH2PO4, pH 7.00). The NOE pat-
terns observed in methanol and buffer were similar (Figure 2).
Reference compound 1 exhibited a strong NOE between its
amide proton NHb and a-proton H2, but no NOE between NHb
and b-proton H1. This pattern reveals that proton NHb is orien-
tated away from the adjacent amide plane; such a conforma-
tion prevents any intramolecular hydrogen bond formation
and allows the NHb group to form hydrogen bonds with sol-
vent molecules. By contrast, compound 2 exhibited a NOE pat-
tern that is consistent with those of b NÀO turns displayed by
To determine the stability of b NÀO turns in solution, we
performed CD studies in buffers at various pH values and tem-
peratures. As shown in Figure 4, the absorption pattern of 2–4
remained constant at pH values below 9. Above pH 9, howev-
er, the absorption peak red-shifted gradually upon increasing
the pH of the buffer. An isochromic point exists, suggesting
that there are two states undergoing exchange, possibly as
a result of deprotonation of the aminoxy amide protons on
the backbone (see the Supporting Information). This change
did not appear to dramatically affect the secondary structures
of 2–4 in basic buffer; we observed similar NOE patterns in
basic (pH 11.14) and neutral buffers (see the Supporting Infor-
mation). These results suggest that 2–4 adopt relatively stable
and similar secondary structures under acidic, neutral, and
basic conditions. In addition, we
found that at neutral pH (7.00),
the CD absorption intensity at
200 nm
albeit
decreased
somewhat
linearly,
modestly,
upon increasing the tempera-
ture from 26 to 788C (Figure 5).
This finding reflects the thermal
stability of the secondary struc-
tures of 2–4 in neutral aqueous
solution.
Figure 2. Summary of NOEs observed at room temperature for compounds 1–3 at concentrations of 10 mm in
Based on the NMR spectro-
scopic data and the characteris-
CD3OH and 20 mm in neutral buffer (0.01mK2HPO4/KH2PO4, pH 7.00); s, strong NOE; blue dashed line, hydrogen
bond.
Chem. Asian J. 2015, 10, 2126 – 2129
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