the C-terminal amide can serve as a hydrogen bond donor,
and the N-terminal carbonyl can serve as a hydrogen bond
acceptor, forming the first hydrogen bonds necessary to
propagate a â-sheet outward from a central tether. The
presence and orientation of hydrogen bonding was estab-
lished through a variety of techniques. In each case, the
hydrogen bonding patterns in â-sheet mimics 1 and 2 were
compared with similar monomeric cysteine derivatives 3 and
4 to verify the role of the adjacent peptide strand.
suggesting that greater aggregation occurs as concentration
increases, more so for the diamide 4. The disulfide dimer 1
is not soluble above 250 mM but shows little evidence of
aggregation at these concentrations (b, 9). The acetamide-
functionalized disulfide 2 is not soluble above 50 mM, but
the changes in chemical shift suggest that it is engaging in
intermolecular hydrogen bonds well below this concentration.
The acetamide N-H protons (0) show changes at all
concentrations above 1 mM, with larger shifts (0.3 ppm)
between 25 and 50 mM. The butylamide N-H signal (O) is
largely insensitive to concentration over the given range and
only shows a small effect at 50 mM, suggesting that
aggregation is mainly occurring through the acetamide N-H.
The greater sensitivity of the acetamide N-H (0) to
concentration as compared to controls suggests that 2 is
aggregating to a greater extent at lower concentrations.
Additionally, these curves point out the importance of
performing subsequent experiments at concentrations beneath
10 mM to minimize intermolecular hydrogen bonding.
Changes in chemical shift with the addition of a hydrogen
bonding solvent also indicate the presence of hydrogen
bonds.6 In this case, the chemical shifts of 1-4 were
determined in 100% C6D6 and with increasing percentages
of DMSO-d6.7 Significant downfield shifts (>1 ppm) with
increasing DMSO were observed for both N-H protons of
cysteine monomers 3 and 4, as well as the carbamate of 1
and the acetamide of 2. The butyl amide of both 1 and 2, on
the other hand, showed a very small shift (<0.3 ppm) and
ultimately an upfield shift at higher concentration. These
results suggest that the butylamides of 1 and 2 are already
participating in significant hydrogen bonds and are less
sensitive to the competitive hydrogen bonding contribution
of the added DMSO.
A complete picture of the hydrogen bonding in these
â-sheet mimics can only be determined by compiling the
results from different techniques and includes both intramo-
lecular hydrogen bonding as well as aggregation between
molecules. Changes in NMR chemical shift that occur at
different concentrations are indicative of intermolecular
hydrogen bonding,5 and the concentration dependence for
1-4 is shown in Figure 2. Both monomeric cysteine
While the data above are consistent with the formation of
interstrand hydrogen bonds, an even more detailed picture
is possible using hydrogen/deuterium (H/D) exchange. This
technique can be used to correlate a slower rate of H/D
exchange with a stronger hydrogen bond donor and the
increased rate of H/D exchange with a hydrogen bond
acceptor.8 The H/D exchange kinetics in 10% CD3OD/CDCl3
for 1 can be seen in Figure 3, along with comparisons with
the analogous cysteine monomer 3 and controls 5 and 6,
which cannot engage in intramolecular hydrogen bonding.
Exponential curve fits are included for all H/D exchange
figures to indicate the correlation with pseudo-first-order
kinetics.
The butyl amide of cysteine 3 (2) exchanged more slowly
than control 5 (+), indicating it was acting as a hydrogen
bond donor. The carbamate of 3 ([) exchanged more quickly
than control 6 (×), suggesting it is functioning as a hydrogen
bond acceptor. Since these kinetics were performed at a
concentration below which significant aggregation was
observed (Figure 2), this indicated that the observed in-
Figure 2. Dependence of NMR chemical shifts on concentration;
including cystine dimer 1 (Boc-NH ) 9; NHBu ) b), cystine
dimer 2 (Ac-NH ) 0; NHBu ) O), cysteine monomer 3 (Boc-
NH ) [; NHBu ) 2), and cysteine monomer 4 (Ac-NH ) ],
NHBu ) 4).
derivatives 3 and 4 show appreciable changes in chemical
shift of both N-H protons (2, [, 4, ]) above 50 mM,
(6) (a) Venkatachalapathi, Y. V.; Prasad, B. V. V.; Balaram, P.
Biochemistry 1982, 21, 5502. (b) Pitner, T. P.; Urry, D. W. J. Am. Chem.
Soc. 1972, 94, 1399.
(7) Complete data can be found in the Supporting Information.
(8) Steffel, L. R.; Cashman, T. J.; Reutershan, M. H.; Linton, B. R. J.
Am. Chem. Soc. 2007, 129, 12956.
(4) Raj, P. A.; Soni, S. D.; Ramasubbu, N.; Bhandary, K. K.; Levine,
M. J. Biopolymers 1990, 30, 73.
(5) (a) Nowick, J. S.; Chung, D. M.; Maitra, K.; Maitra, S.; Stigers, K.
D.; Sun, Y. J. Am. Chem. Soc. 2000, 122, 7654. (b) Dado, G. P.; Gellman,
S. H. J. Am. Chem. Soc. 1993, 115, 4228.
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Org. Lett., Vol. 9, No. 26, 2007