C O M M U N I C A T I O N S
are enthalpic in origin. The effects of temperature on the values of
Kt/c ) (kct/ktc) were measured directly by NMR spectroscopy, and
the resulting data were analyzed by van’t Hoff plots (Supporting
Information). Values for ∆H° and ∆S° were calculated from linear
least-squares fits of these plots (Table 3). In all cases studied, the
trans isomer of 1-4b is more stable than the cis isomer. Moreover,
the values of Kt/c for 1-4b are dependent on temperature such that
the trans isomer becomes increasingly favored as the temperature
decreases.
ring pucker favors the prolyl trans amide isomer. Our results
therefore suggest that glycosylation of Hyp may lead to additional
stabilization of the Cγ-exo ring pucker of Hyp. However, this
stabilization does not translate in a measurable increase on Kt/c for
peptide mimics 4a and 4b when compared to unglycosylated 2.
Most likely the stabilization of the trans isomer in compounds 4a
and 4b is too small to be differentiated and remains within the
experimental error. Any stabilization of the Cγ-exo ring pucker
may be more apparent in larger glycopeptides that contain two or
more glycosylated Hyp units, where the effect may be additive.
Perhaps more importantly, nOe experiments show that the glyco-
sylation of 2 results in distant contacts between the proline and
galactose rings, suggesting that galactosylation of Hyp induces
conformational constraint into glycopeptides. Very likely this is
the result of increased steric strain induced upon glycosylation.
Additionally, glycosylation of Hyp may also affect other properties
including solvation, solubility, and thermostability. In conclusion,
while there is no significant influence on prolyl N-terminal amide
isomerization, the presence of both an enhanced inductive effect
and Gal-Pro contacts between distant positions in 4a and 4b
suggests that glycosylation of Hyp will have important implications
on peptide backbone conformation in HRGPs and glycosylated Hyp-
containing peptides.
The pucker of Hyp (2S,4R) in model peptide 2 in solution has
been previously assigned to the Cγ-exo conformation on the basis
of observed J-coupling constants.10 The prolyl ring pucker for
compounds 3, 4a, and 4b were similarly established as the Cγ-exo
conformation on the basis of 1H NMR coupling constants by
comparison to literature values. For example, in compound 4a we
3
3
observed both JRâ1 and JRâ2 couplings constants to be 8.2 Hz.
The expected coupling constants for the Cγ-exo conformer are
7-10 Hz and 7-11 Hz, respectively, whereas those for Cγ-endo
are 6-10 Hz and 2-3 Hz, respectively. Similarly, other couplings
show characteristic patterns for the Cγ-exo pucker (Supporting
Information).
Previous reports have shown that inductive effects in the
γ-position of proline have significant structural consequences on
the thermodynamics and kinetics of prolyl amide bond isomeriza-
tion.11 To assess the inductive effect caused by glycosylation of
hydroxyproline we determined the 13C NMR chemical shifts of the
Cγ atom, which can indicate electron withdrawal by pendant
function groups,12 and have previously been used to correlate the
electron-withdrawing effects in various Cγ-substituted proline
Acknowledgment. The authors thank the National Science and
Engineering Council of Canada (NSERC) and the University of
Manitoba for financial support.
Supporting Information Available: Synthetic procedures, 1H
NMR, 13C NMR, 1D-GOESY spectra, Eyring plots, van’t Hoff plots,
plots of intensity versus mixing time for the magnetization transfer
experiments of 1-4b. This material is available free of charge via the
analogues.11c The observed 13C NMR chemical shifts (δCγ(trans)
)
indicate that electron withdrawal increases in the order hydroxyl
(δCγ ) 69.9) (2) < tert-butoxyl (δCγ ) 70.1) (3) < R-galactosyl
(δCγ ) 76.5) (4a) < â-galactosyl (δCγ ) 77.9) (4b) (see Table 1 in
the Supporting Information).
References
To determine the extent of interaction between the galactose and
prolyl rings, we performed nOe transfer experiments with com-
pounds 4a and 4b in D2O. Selective inversion of one of the H-â
protons in 4a by a selective GOESY6 experiment resulted in a 1.5%
resonance transfer to a peak at δ ) 3.83 ppm corresponding to the
overlapped signals of both H-4 and H-5 of galactose. By compari-
son, selective inversion of H-2 in 4b produced nOe transfer (3.0%)
to H-R of Hyp. These results suggest that galactosylation of Hyp
induces close contacts between distant positions in the carbohydrate
and pyrrolidine rings.
In summary, we have found that glycosylation of Hyp in
compounds 4a and 4b has no apparent effect on the isomer
equilibrium (Kt/c) nor on the rate of isomerization (ktc, ktc) in water
between the cis and trans isomers when compared to unglycosylated
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