J. M. Peregrina, F. Corzana et al.
Experimental Section
aMeSer was synthesized following our methodology.[20]
Compound 1: 1H NMR (400 MHz, D2O): d =0.84–0.93 (m, 6H, 2MeVal),
3
3
1.19 (d, JH,H =6.1 Hz, 3H, MeThr), 1.30 (d, JH,H =7.1 Hz, 3H, MeAla), 1.96
(s, 3H, MeGalNAc), 1.98 (s, 3H, MeN), 1.99–2.05 (m, 1H, HbVal), 2.65 (s,
3H, MeC), 3.64–3.70 (m, 2H, 2H6), 3.74–3.78 (m, 2H, HbSer), 3.78–3.83
(m, 1H, H3), 3.83–3.86 (m, 1H, H5), 3.87–3.91 (m, 2H, HaGly), 3.92–3.98
(m, 1H, H4), 4.02 (dd, 3JH,H =10.9, 3.0 Hz, 1H, H2), 4.11–4.16 (m, 1H,
HaAla), 4.20–4.28 (m, 2H, HaVal,
HbThr), 4.41 (t, 3JH,H =5.5 Hz, 1H,
HaSer), 4.54–4.57 (m, 1H, HaThr), 4.84 ppm (s, 1H, H1); 1H NMR
(400 MHz, H2O/D2O=9:1): d =7.62 (d, 3JH,H =9.5 Hz, 1H, NHGalNAc),
7.78–7.89 (m, 1H, NHMe), 8.05 (d, 3JH,H =7.6 Hz, 1H, NHVal), 8.16 (ꢅt’,
3JH,H =5.5 Hz, 1H, NHGly), 8.30 (d, 3JH,H =7.4 Hz, 1H, NHSer), 8.38 (d,
3JH,H =5.7 Hz, 1H, NHAla), 8.51 ppm (d, 3JH,H =8.7 Hz, 1H, NHThr);
13C NMR (100 MHz, D2O): d =16.6 (MeThr), 17.6 (MeVal), 18.1
18.5(MeAla), 21.6(MeCO), 22.2 (MeCO), 25.8(NHMe), 30.1(CbVal), 42.4
ACHTUNGTRENNUNG(MeVal),
A
E
N
ACHTUNGTRENNUNG
(CaGly), 49.7 (C2), 49.9 (CbAla), 54.8 (CaSer), 57.1 (CaThr), 59.4 (CaVal),
61.3 (C6), 61.5 (CbSer), 68.1 (C3), 68.6 (C5), 71.3 (C4), 76.3 (CbThr), 98.8
(C1), 171.0, 171.0, 171.5, 173.9, 174.8, 175.2 (CO) ppm; elemental analysis
calcd (%) for C28H49N7O13: C 48.62, H 7.14, N 14.17; found: C 48.56, H
7.12, N 14.13.
Compound 2: 1H NMR (400 MHz, D2O): d=0.82–0.91(m, 6H, 2MeVal),
1.33 (d, 3JH,H =6.6 Hz, 3H, MeAla), 1.47 (s, 3H, MeMeSer), 1.98 (s, 6H,
2MeCO), 2.01–2.10 (m, 1H, HbVal), 2.65 (s, 3H, MeC), 3.62–3.70 (m, 3H,
2H6, HbMeSer), 3.73–3.78 (m, 2H, H3, H5), 3.78–3.83 (m, 2H, HbSer),
3.84–3.88 (m, 2H, HaGly), 3.88–3.92 (m, 1H, HbMeSer), 3.92–3.96 (m, 1H,
H4), 4.06 (d, 3JH,H =7.2 Hz, 1H, HaVal), 4.07–4.12 (m, 1H, H2), 4.19–4.23
(m, 1H, HaAla), 4.25–4.30 (m, 1H, HaSer), 4.83 ppm (s, 1H, H1);
1H NMR (400 MHz, H2O/D2O=9:1): d =7.61–7.71 (m, 1H, NHMe),
7.88 (d, 1H, 3JH,H =6.2 Hz, 1H, NHSer), 7.98 (d, 3JH,H =9.1 Hz, 1H,
NHGalNAc), 8.03 (d, 3JH,H =7.2 Hz, 1H, NHVal), 8.10 (d, 3JH,H =6.3 Hz, 1H,
NHAla), 8.17 (ꢅt’, 3JH,H =5.5 Hz, 1H, NHGly), 8.54 ppm(s, 1H, NHMeSer);
13C NMR (100 MHz, D2O): d =16.5 (MeAla), 17.4 (MeVal), 18.5 (MeVal),
20.0 (MeMeSer), 21.6 (MeCO), 22.0 (MeCO), 25.9 (NHMe), 29.8 (CbVal),
42.4 (CaGly), 49.9 (CaAla,C2), 56.0 (CaMeSer), 59.4 (CaSer), 59.5 (CaVal),
60.8 (CbSer), 61.2 (C6), 67.6 (C5), 68.4 (C4), 69.5 (C3), 71.4 (CbMeSer), 97.6
(C1), 171.6, 171.7, 173.3, 174.5, 174.7, 174.8, 175.2 ppm (CO); elemental
analysis calcd (%) for C28H49N7O13: C 48.62, H 7.14, N 14.17; found: C
48.51, H 7.10, N 14.13.
Figure 3. a) Relative STD effects for glycopeptides 1 (dark gray) and 2
(light gray) bound to EcorL lectin. b) The STD spectrum (top) of a mix-
ture of the glycopeptides and EcorL (12:12:1), showing the anomeric and
aliphatic regions; bottom spectrum is the reference spectrum. This plot
clearly indicates a key difference in the recognition of the peptide back-
bone in 1 and 2.
NMR experiments: NMR experiments were recorded on
a Bruker
Avance 400 spectrometer at 293 K. Magnitude-mode ge-2D COSY spec-
tra were recorded with gradients and using the cosygpqf pulse program
with 90 degree pulse width. Phase-sensitive ge-2D HSQC spectra were
recorded by using z-filter and selection before t1 removing the decou-
pling during acquisition by use of invigpndph pulse program with CNST2
(JHC)=145. 2D NOESY experiments were conducted by using phase-
sensitive ge-2D NOESY with WATERGATE for H2O/D2O (9:1) spectra.
Selective ge-1D NOESY experiments were carried out by using the 1D-
DPFGE NOE pulse sequence. NOEs intensities were normalized with
respect to the diagonal peak at zero mixing time. Distances involving NH
protons were semi-quantitatively determined by integrating the volume
of the corresponding cross-peaks. The calculated 3J values were obtained
from the simulations by applying the appropriate Karplus equation[21] to
the corresponding torsion angles.
Interaction studies with EcorL lectin: STD experiments[22] were recorded
on a Bruker Avance 500 spectrometer at 293 K. Commercial EcorL was
purchased from Sigma. The binding of the glycopeptides was evaluated
by STD experiments performed with a 12:1 molar ratio of the glycopep-
tide/EcorL mixture. The concentration of the lectin was approximately
40 mm. A series of Gaussian-shaped pulses of 50 ms each was employed
with a total saturation time for the protein envelope of 2 s and a maxi-
mum B1 field strength of 60 Hz. An off-resonance frequency of d=
40 ppm and on-resonance frequency of d= 7.0 ppm (protein aromatic
signals region) were applied. See the Supporting Information for more
details.
Figure 4. Structures of glycopeptides 1 (left) and 2 (right) in the bound
state.
modulate the proximity between the peptide backbone and
a given receptor, as validated with EcorL. As expected, con-
sidering the short glycopeptides and the model character of
the employed lectin, this effect is not translated into a signif-
icant variation in the stability of the resulting complex.
However, when applied to larger glycopeptides with appro-
priate receptors, this strategy could modulate the binding
properties. Efforts toward this end are currently underway
in our group.
3108
ꢄ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2011, 17, 3105 – 3110