tetrapeptide 3-VVVV-Den (limited solubility ∼10 mM),
we were unable to identify any long-range cross peaks in
their 2D NOESY and ROSEY spectra.
To determine the binding mode of the tripeptides, hydro-
gen/deuterium (H/D) exchange experiments were performed
according to the method described by Linton.13 It was found
that NÀH1 and NÀHC exchanged more slowly than the NH
of amide 8, indicating they acted as H-bond donors, while
NÀH2 served as a H-bond acceptor (see p S29 of SI for
details). For the carbamate NÀHN, it possessed a significant
shorter exchange half-life in comparison to that of car-
bamate 9 and, hence, served as a H-bond acceptor.
1
Figure 2. Dependence of H NMR (700 MHz, CDCl3, 25 °C)
amide NHc chemical shift values of 1-VV-Prg, 2-VVV-Prg,
2-VVV-Den, and 3-VVVV-Den on sample concentration. Note
that the x-axis scales are different in the two figures.
values of the various tripeptides 2 were higher (25À
280 MÀ1) but werestrongly dependent onthe C-endgroup.
Hence, for 2-VVV-Prg, the Kdimwas about 240( 30MÀ1 in
CDCl3, but it dropped to 25 ( 5 MÀ1 for 2-VVV-Den.
Vapor pressure osmometry (VPO) analysis of a CHCl3
solution of 2-VVV-Prg using concentrations at the higher
end range (50À133 mM) gave an apparent molecular
weight (MW) corresponding to that of the dimeric species,
while using concentrations at the lower end range (5À
16 mM) produced an apparent MW of the monomeric
species. These results were consistent with a mono-
merÀdimer equilibrium with a Kdim of 240 MÀ1. Inciden-
tally, the apparent MW determined by VPO at the 50À
133 mM concentration range in n-PrOH was about 1.8
times the theoretical value, suggesting it also exists pre-
dominately in dimeric form even in protic solvents. For the
soluble tetrapeptide 3-VVVV-Den, the Kdim was found to
be 680 ( 100 MÀ1. Interestingly, the triester analog 7 was
found to exist as a monomer according to VPO studies,
suggesting the importance of the amide units in the asso-
ciation binding process. Hence, both 1H NMR and VPO
studies confirmed that the larger the number of amide
units, the stronger the Kdim value. The enhanced self-
association with increasing number of amide units had
been attributed to a zip effect reported earlier by us.12
Similar findings had also been demonstrated by Hunter11
in studies of aromatic amide oligomers.
2D NOESY 1H NMR studies were then conducted on 2-
LLL-Prg and 2-VVV-Prg in CDCl3 to probe their solution
structure. At low concentrations (1À5 mM), no long-range
cross peaks could be identified. At 100 mM, NOE cross
peaks were noted between the protons of the Boc group
and NÀHC, protonsof theBoc group and the acetylenicH,
and R CÀHs of the first (HR1) and the third (HR3) amino
acids (see p S31 of SI for details). Hence, the N-end Boc
moiety of one molecule is in close proximity to the
C-terminal acetylene group of another molecule. Unfor-
tunately, for the dipeptide 1-VV-Prg (small Kdim) and
Figure 3. Stacked solution FT-IR spectra (30 mM in CHCl3) of
compounds 2-VVV-Prg, 7, and 9.
Data from the N;H and CdO stretching regions in FT-
IR spectra (at 2 cmÀ1 spectral resolution) provided addi-
tional information into the degree of H-bonding of these
click peptides in nonpolar solvents (Figure 3). For the ester
analog 7, three major absorption peaks, attributed to the
stretching frequencies of the carbamate N;H (3444cmÀ1),
the ester CdO (1743 cmÀ1), and the carbamate CdO
(1710 cmÀ1) bonds were found. These values were closely
related to the corresponding absorption frequencies of
non-H-bonded esters and carbamates. For tripeptide
2-VVV-Prg, four absorption bands, due to the stretching
frequencies of the carbamate N;H (3435 cmÀ1), the
H-bonded amide N;H (3309 and 3074 cmÀ1), and the
H-bonded CdO (1677 cmÀ1) were found. There was also a
small peakat 3162cmÀ1 in the IRspectra ofnon-H bonded
triester 7, which could be assigned to the stretching fre-
quencies of the triazole C;H. For the self-associating
2-VVV-Prg, this signal was red-shifted to 3156 cmÀ1. This
finding suggested that the triazole C;H may function as a
H-bond donor.14
(13) Steffel, L. R.; Cashman, T. J.; Reutershan, M. H.; Linton, B. R.
J. Am. Chem. Soc. 2007, 129, 12956.
(14) (a) Li, Y.; Flood, A. H. Angew. Chem., Int. Ed. 2008, 47, 2649.
(b) Juwarker, H.; Lenhardt, J. M.; Pham, D. M.; Craig, S. L. Angew.
Chem., Int. Ed. 2008, 47, 3740. (c) Juwarker, H.; Lenhardt, J. M.;
Castillo, J. C.; Zhao, E.; Krishnamurthy, S.; Jamiolkowski, R. M.;
Kim, K.-H.; Craig, S. L. J. Org. Chem. 2009, 74, 8924.
(12) (a) Lau, K.-N.; Chow, H.-F.; Chan, M.-C.; Wong, K.-W.
Angew. Chem., Int. Ed. 2008, 47, 6912. (b) Chow, H.-F.; Lau, K.-N.;
Chan, M.-C. Chem.;Eur. J. 2011, 17, 8395.
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Org. Lett., Vol. 14, No. 1, 2012