Two prolines with a difference: contrasting stereoelectronic effects of 4R/S-aminoproline on triplex stability in collagen peptides [pro(X)-pro(Y)-Gly]n.

Article abstract of DOI:10.1039/b308581c

4R/S-Aminoprolines when present in the X-position of collagen peptide [pro(X)-pro(Y)-Gly]n exhibit pH- and stereochemistry-dependent effects on triplex stability.

Full text of DOI:10.1039/b308581c

Two prolines with a difference: contrasting stereoelectronic effects of
4R/S-aminoproline on triplex stability in collagen peptides
[Pro(X)-Pro(Y)-Gly]n†
M. Umashankara, I. Ramesh Babu and Krishna N. Ganesh*
Division of Organic Chemistry (Synthesis), National Chemical Laboratory, Pune 411008, India.
E-mail: kng@ems.ncl.res.in; Fax: 91 20 589 3153; Tel: 91 20 589 3153
Received (in Cambridge, UK) 24th July 2003, Accepted 28th August 2003
First published as an Advance Article on the web 17th September 2003
4R/S-Aminoprolines when present in the X-position of
collagen peptide [pro(X)-pro(Y)-Gly]n exhibit pH- and
stereochemistry-dependent effects on triplex stability.
followed by capping of the N-terminal residue. TFA cleavage of
the product directly yielded the N-acetylated C-terminal
amidated peptide 6. Similarly, the collagen peptide 5, having
4R-aminoproline at the X position, was synthesized from 4R-
aminoproline. The peptides 5 and 6 were purified on a semi-
preparative RP-C8 HPLC column and characterized by
MALDI-TOF mass spectrometry.8 The amino acid Phe in-
cluded at the N-terminus enabled accurate determination of the
peptide concentrations by UV absorbance at 259 nm (e = 200
M21 cm21). The N,C-end capped peptides 5 and 6 were used for
biophysical studies in order to eliminate the effects arising from
charge–charge repulsion in terminally ionized forms, a phenom-
enon well established in collagen peptides.
Collagen-like triple helical structures in solution exhibit
fingerprint CD spectra with a characteristically large negative
band around 200 nm, a crossover at around 213 nm, and a small
positive band around 215–227 nm.9 The ratio of the intensiy of
the positive to the negative band (Rpn), 0.07–0.18, is an
established criterion for triplex formation.10 All CD spectra
were recorded at 0.2 mM concentration which is higher than the
critical triple helical concentration seen in these systems. Fig. 2
shows the CD spectra of peptides 5 and 6 measured at different
pHs. Based on the Rpn criterion, 4S-Amp-peptide 6 forms a
triplex at pHs 3, 7 and 9, while 4R-Amp-peptide 5 forms a
triplex only at pHs 3 and 7 and at pH 12.0 neither form triplexes.
The formation or non-formation of triplexes was further
Collagen is the most abundant structural protein in mammals
and its mechanical properties are related to the high thermal
stability of its triple helical structure.1 The primary structure of
collagen is characterized by a repeating X–Y–Gly triplet motif.
Proline is the most abundant residue in the triple helix, with the
Y-position mostly occupied by trans-4R-hydroxyproline. While
this amino acid causes remarkable collagen stabilization when
present in the Y position, polypeptides with this moiety at the X-
position , Pro(OH)-Pro-Glycine, do not form a triplex in
aqueous solution.2 Differing conformational preferences for the
two prolines at the X and Y positions are thought to play a key
role in imparting unusual stability to collagen triple helices3 and
understanding the conformation–stability interplay is presently
an area of active research. It is now emerging that the
conformational preferences of the two prolines are strongly
dictated by the nature of the 4-substituent.4 Others5 and we6
+
have respectively demonstrated that 4R-F and 4R-NH2/NH3
(1) substituted prolines in the Y postion show improved triplex
properties of the derived collagen peptides, but the true
molecular origin of the stability is yet to be fully deciphered.
Fluorine at the 4-position has been shown to contribute to
stability by its strong electronegativity and the concomitant
influence on pyrrolidene ring conformation.5b The situation is
not that clear for the stability offered by 4-NH2/NH3+ functions,
where the conformational control may be exercised by a
combination of hydrogen bonding, electronegativity and long
range interstrand electrostatic contributions from the protonated
NH3+ groups.
To decipher the influence of these factors, we analyzed the
dependence of pyrrolidene ring conformation on the ster-
eochemistry of 4-NH2/NH3 by vicinal H–1H-coupling con-
stants in 1H-NMR of 4R-trans (1) and 4S-cis (2) aminoprolines.
Analysis, done according to previously established methods,7
indicated that 4R-trans-aminoproline 1 prefers a C4-exo pucker
(Fig. 1) for the pyrrolidene ring as in 4R-trans-hydroxyproline,
whereas the preferred conformation for 4S-cis-aminoproline 2
is C4-endo. Although for proline with no C4 substituent, these
two puckers are isoenergetic, theoretical calculations indicate
that C4-exo is not favourably accommodated in the X-
position.3,4 The preferred C4-endo conformation for 4S-cis-
aminoproline 4 prompted us to examine its compatibility at the
X-position in collagen peptide 6 and compare the stability of the
derived triplex with that of the analogous triplex from 4R-
aminoproline collagen peptide 5.
+
1
The N1-Fmoc-N4-Boc protected 4S-aminoproline 7 was
synthesized from 4R-hydroxyproline in eight steps by sequen-
tial protection/deprotection strategies (ESI).† This was then
incorporated by solid phase synthesis on Rink resin into the
collagen model peptide Phe(X-Pro-Gly)6 at the X-position,
† Electronic supplementary information (ESI) available: 1H NMR spectra
and coupling constant analysis of 3 and 4; synthetic schemes for 3, 4, 5 and
6; FABMS of 3 and 4; MALDI-TOF MS of 5 and 6; HPLC of peptides 5 and
6. See http://www.rsc.org/suppdata/cc/b3/b308581c/
Fig. 1 Position dependent preferred proline puckers in collagen.
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CHEM. COMMUN., 2003, 2606–2607
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pH 9.0. The pKa of the 4-NH2 group is 10.5 and these results
suggest that protonation of the 4-amino group seems to be
essential for triplex formation of X-peptides. In ethylene glycol,
which stabilizes triple helices through hydrogen bonding
interactions,11 both X-peptides exhibited significantly higher
stability (DTm 12–16 °C) than the Y-peptide 8.
The results presented here demonstrate that the 4R/S-
aminoprolines are one of the first proline derivatives that
stabilize the collagen triplex when present in the X-position and
the 4S-Amp is better than 4R-Amp. The cause of the
stabilization is the C4-endo pyrrolidene conformation adopted
by 4S-Amp that is inherently favoured at the X-position. The pH
dependent stabilities in both 4R and 4S aminoprolines also
suggest that protonation of NH2 is a prerequisite for triplex
formation in the X-position, while it is not so in the Y-position.
It is possible that the conformation of pyrrolidine ring may also
depend on the protonation status of the 4-amino group and
influence the cis–trans amide rotameric equilibrium.3a The
results reinforce the important role of stereoelectronic effects,
well elucidated in 4-fluoroprolines,12 also to be determinants of
stability of 4-aminoproline collagen. Future potential of this
work lies in rationally combining the stabilities offered by the
two diastereomers to design collagens (e.g. Amp-Hyp/Amp-
Gly)n or chimeric collagens of unusual stability for applications
in collagen based biomaterials.13 The results also have implica-
tions for peptidomimetic designs and the control of peptide
conformations through 4-substituent effects on proline.
M. U. and I. R. B. thank CSIR, New Delhi, for Research
fellowships.
Fig. 2 CD spectra of X-peptides at different pHs. A, 5; B, 6.
confirmed by CD spectra recorded as a function of temperature
at different pHs as shown in Fig. 3. The observed sigmoidal
transitions (Fig. 3 A and B) confirmed the presence of triplexes
while the non-sigmoidal transitions indicated non-formation of
triplexes (Fig. 3C for 5 and D). The CD-triplex melting
temperatures obtained from the corresponding derivative curves
are shown in Table 1.
The thermal stability of 4S-Amp X-peptide 6 at pH 3.0 and
7.0 was higher compared to that of 4R-Amp X-peptide 5 (DTm,
5–8 °C). At pH 9.0, only the 4S-amp X-peptide formed a triplex
and at pH 12.0 both X-peptides failed to show any triplex. This
is in contrast to the 4R-Amp Y-peptide 8 that formed a triplex at
all pH ranges. The triplex from X-peptides 5 and 6 were
however of lower stability than that of the 4R-Amp-Y-peptide 8
at pH 3 and 7, while 4S-Amp X-peptide 3 was better than 8 at
Notes and references
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Fig. 3 CD thermal denaturation profiles, with normalized ellipticity for
peptides 5 and 6 monitored at 225 nm. A, pH 3.0; B, pH 7.0; C, pH 9.0; D,
pH 12.0. For buffer conditions, see Table 1, footnote a.
2001, 40, 823; (c) For C4-endo, J1–2 > J1–3, J2–4 > J3–4 and J4–5 < J4–6
,
and for C4-exo, J1–2, J1–3 (Ca–Cb), J2–4 < J3–4 (Cb–Cg) and J4–5
>
J4–6 (Cg–Cd). See ESI.
8 Ac-Phe(Amp-Pro-Gly)6-NH2 5: C83H122N26O20, Mcalc = 1084, Mobs
1085 (M + H); Ac-Phe(amp-Pro-Gly)6-NH2 6: C83H122N26O20, Mcalc
1084, Mobs = 1086 (M + 2H) and 1089 (M + 5H+).
=
=
Table 1 CD–Tm data for collagen peptidesa
pH
3.0
7.0
9.0
5
6
8b
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Soc., 1996, 118, 10351.
1
2
3
4
5
36 (0.08)
33 (0.08)
ND
ND
35
44 (0.12)
37 (0.08)
34 (0.08)
ND
60 (0.17)
56.3 (0.15)
26 (0.09)
49 (0.17)
23
12.0
EG : W (3 : 1)
11 F. R. Brown III, A. Di Corato, G. P. Lorenzi and E. R. Blout, J. Mol.
Biol., 1972, 63, 85.
39
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Elsevier Science, Barking, Essex, 1992.
a pH 3.0, 20 mM acetate; pH 7.0, 20 mM phosphate; pH 9.0 and 12.0. 20
mM borate buffers, all with 0.1 M NaCl. Tm values are ± 0.5 °C; b Data from
ref. 6, values in parentheses indicate Rpn values.
CHEM. COMMUN., 2003, 2606–2607
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