Water-induced switching of β-structure to polyproline II conformation in the 4 S -aminoproline polypeptide via H-bond rearrangement

Article abstract of DOI:10.1021/ol1021993

4S-Aminoproline polypeptide 2 forms unusual β-structure in trifluoroethanol that switches to the polyproline II (PPII) form in aqueous medium, while 4R-aminoproline peptide 1 retains PPII form in both solvents. This first instance of a polyproline derivat

Full text of DOI:10.1021/ol1021993

ORGANIC
LETTERS
2010
Vol. 12, No. 23
5390-5393
Water-Induced Switching of ꢀ-Structure
to Polyproline II Conformation in the
4S-Aminoproline Polypeptide via H-Bond
Rearrangement
Mahesh V. Sonar† and Krishna N. Ganesh*,†,‡
DiVision of Organic Chemistry, National Chemical Laboratory, Dr Homi Bhabha
Road, Pune 411008, India, and Indian Institute of Science Education and Research,
900, NCL InnoVation Park, Dr Homi Bhabha Road, Pune 411008, India
kn.ganesh@iiserpune.ac.in
Received September 14, 2010
ABSTRACT
4S-Aminoproline polypeptide 2 forms unusual ꢀ-structure in trifluoroethanol that switches to the polyproline II (PPII) form in aqueous medium,
while 4R-aminoproline peptide 1 retains PPII form in both solvents. This first instance of a polyproline derivative showing a ꢀ-structure is
attributed to competitive pH-dependent (4-NH3+/NH2) stereoelectronic effect (4R vs 4S) and the overriding importance of stereospecific intra/
intermolecular H-bonding in (2,4)-cis-4S-aminoproline in contrast to (2,4)-trans-4R-aminoproline oligomers.
The polyproline type II (PPII) helix is a prevalent conforma-
tion in both folded and unfolded proteins1 and plays an
important role in a wide variety of biological processes, such
as signal transduction, transcription, immune response, and
cell motility.2 Each strand of collagen triplex with the Pro-
Hyp-Gly tripeptide repeat unit adopts a left-handed PPII-
like conformation.3 Oligoprolines and their derivatives have
found utility as cell penetrating agents4 and as molecular
spacers in biomimetic systems for energy/electron transport.5
The PPII helix is a fully extended left-handed structure with
all amide bonds in the trans conformation, while the right-
handed PPI helix is compact, with all amide bonds in the
cis conformation.1 It is well-known that polyprolines adopt
PPII conformation in water6 and PPI conformation in
hydrophobic solvents of short chain aliphatic alcohols.6 It
has been well demonstrated that the stereoelectronic effect
† National Chemical Laboratory.
‡ Indian Institute of Science Education and Research.
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10.1021/ol1021993  2010 American Chemical Society
Published on Web 11/04/2010

of the 4-substituent plays a major role in determining the
pucker of the pyrrolidine ring of proline and hence the
conformational stability of proteins.7
In this context, we reported earlier that 4-amino
substitution on proline in collagen peptide stabilizes the
triple helix8 at both acidic and basic pHs. In collagen,
the tripeptide repeat unit [Pro-Hyp-Gly] has glycine whose
amide linkage is involved in an interchain H-bond, leading
to the triple helix structure. In contrast, polyproline
peptides lack amide NH and hence are unable to form a
triplex via interchain H-bonds, ending up as a single helix
of PPI or PPII type. Recently, 4S(OH/NH3+) groups on
proline were shown to form intramolecular H-bonds with
the amide carbonyl, increasing the trans/cis amide ratio
and thereby promoting PPII conformation in the derived
polypeptides.9 Unlike other 4-substituents on proline
studied so far (OH, SH, CH3, F), the ionizable 4-NH2
group is a good probe to examine the pH effects on
polyproline conformation. Herein, we report the novel
behavior of 4S-aminoproline polypeptide to form a novel
ꢀ-structure in trifluoroethanol (TFE) that switches to PPII
form in aqueous medium. This property exclusive to 4S-
aminoproline polypeptide arises from a stereospecific
intramolecular H-bonding that stabilizes the PPII form,
while the unusual ꢀ-structure results from interchain
H-bonding. To our knowledge, this is perhaps the first
report of formation of ꢀ-structure in any polyproline
derivatives and its switch over to PPII form induced by
water.
Figure 1. CD profiles of polypeptides 1, 4R-Amp (b); 2, 4S-amp
(2); and 3, Pro9 (9), all at 100 µM (pH 7.2).
fashion. At acidic pH (4.0-5.0), the PPII helicity of 4S-
amp9 2 was low (20% of 4R-Amp9 1) but increased by 2-fold
at pH 10.0. The ellipticity of peptide Pro9 3 remained
Synthesis and Conformational Studies of 4-Aminopro-
line Oligomers. The oligopeptides 1-3 were synthesized
from appropriate N-Fmoc-protected monomers asssembled
in the solid phase, purified by HPLC, and characterized by
MALDI-TOF (for details, see Supporting Information). The
CD spectral analyses were carried out as a function of
temperature, pH, urea, and solvents (buffer and trifluoroet-
hanol, TFE). All three peptides (100 µM, pH 7.2) show CD
spectra (Figure 1) with a positive band between 220 and 230
nm and a negative band between 200 and 210 nm that are
the established patterns of the PPII conformation.10 The
intensity of the positive band at 225-227 nm is proportional
to the PPII helical content which is seen to decrease in the
order 4R-Amp9 1 > 4S-amp9 2 > Pro9 3.
The effect of protonation of the 4-amino group on PPII
helical content was examined by the CD spectra of peptides
1-3 (Figure 2A) recorded at different pH (4.0-10.0). The
positive ellipticity at 225 nm for 4R-Amp9 1 decreased by
10% with increasing pH up to 7.2 and did not change further
until pH 10.0. In the case of 4S-amp9 2, positive intensity
was enhanced in the pH range 4.0-10.0 in a sigmoidal
Figure 2. (A) Intensity of the positive band of CD spectra of
peptides 1-3 as a function of pH. (B) Thermal stability of PPII
helices in peptides 1-3 as a function of pH, followed at 225 nm.
constant with pH. This suggests a pivotal role for both
stereochemistry and the protonation status of the 4-amino
group in eliciting the PPII helicity of 4(R/S)-aminoproline
polypeptides 1 and 2.
The pH-dependent thermal stability (Tm) of PPII helices
in peptides 1-3 was measured from temperature-dependent
CD spectral data (Figure 2B and Supporting Information).
It is seen that (i) 4R-Amp9 1 has maximum Tm at all pHs;
almost invariant (ii) 4S-amp9 2 has the lowest Tm among the
peptides at pH 4.0 but increased gradually with raise in pH
to 10.0 to a value closer to the Tm of 4R-Amp9 1; and (iii)
Pro9 3 with intermediate Tm at pH 4.0 remained constant
(7) Bretscher, L. E.; Jenkins, C. L.; Taylor, K. L.; DeRider, M. L.;
Raines, R. T. J. Am. Chem. Soc. 2001, 123, 777–778.
+
over the pH range. The 4-NH3 group at pH 4.0 stabilized
(8) Babu, I. R.; Ganesh, K. N. J. Am. Chem. Soc. 2001, 1232079-2080.
(b) Umashankara, M.; Babu, I. R.; Ganesh, K. N. Chem. Commun. 2003,
2606–2607.
the PPII helix most in the 4R-form and least in the 4S-form,
while 4-NH2 at pH 10.0 stabilized both 4R- and 4S-peptides
to a similar extent. The 4S-amp9 2 thus exhibited significant
pH-dependent PPII stability that is maximum in the unionized
amino form.
(9) (a) Shoulders, M. D.; Kotch, F. K.; Choudhary, A.; Guzei, I. A.;
Raines, R. T. J. Am. Chem. Soc. 2010, 132, 10857–10865. (b) Kuemin,
M.; Nagel, Y. A.; Schweizer, S.; Monnard, F. W.; Ochsenfeld, C.;
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Org. Lett., Vol. 12, No. 23, 2010
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Figure 3
. (A) CD spectra of peptides 1-3 in trifluoroethanol (TFE). (B) CD spectra of 4S-amp9 2 in TFE with incremental addition of
phosphate buffer (pH 7.2) from (a) 0.1% to (k) 1.0% in 0.1% steps and (l) 2.0%. (C) Increasing concentration of 2 from 50 to 250 µM.
Urea is known to enhance the PPII helical content through
rigidification of the polypeptide backbone.11 The low PPII
helicity of 4S-amp9 2 was enhanced enormously (>300%)
by addition of 1 M urea (pH 7.2), while that of 4R-Amp9 1
and (Pro9) 3 increased by a mere 15-20% (Supporting
Information). The larger changes seen specifically for 4S-
amp9 2 in the presence of urea and upon increasing the pH
suggest the combined role of H-bonding and stereoelec-
tronic12 effects in dictating the PPII conformation.
Solvent plays a key role in modulating the H-bonding
effects, and hence the CD spectra of peptides 1-3 were
recorded in a flourinated solvent trifluoroethanol (TFE)
(Figure 3A). The 4R-Amp9 1 and Pro9 3 show CD spectra
typical of PPII form. Very interestingly, the CD spectra of
4S-amp9 2 in TFE are unlike the PPII pattern, showing a
negative maximum around 214 nm and a broad shoulder at
228 nm that is typical of ꢀ-structure.13
The formation of ꢀ-structure in polyproline peptides under
any conditions is unprecedented in the literature since they
lack H-bond donor sites. In 4S-amp9 2, the NH2 group can
form an intramolecular H-bond with the amide carbonyl of
the same proline moiety, promoting a PPII conformation.9b
The possibility of the 4S-NH2 group engaging the amide
carbonyl of another chain of 4S-amp9 through an intermo-
lecular H-bond would lead to ꢀ-structure. Such an interchain
H-bonded structure should be facilitated at higher peptide
concentration.
A plausible molecular picture for this conversion is
depicted in Scheme 1. The intramolecular H-bonding of 4S-
Scheme 1. Solvent-Derived Rearrangement of H-Bonds in
4S-amp6 Leading to Conformational Switch
When aqueous phosphate buffer (pH 7.2) was titrated into
TFE solution of 4S-peptide 2 in tiny incremental steps of
0.1%, the 214 nm negative band slowly shifted to 205 nm,
accompanied by a growing of the broad negative shoulder
at 228 nm into a positive band at about 224 nm (Figure 3B)
typical of PPII form. The isosbestic point seen at 215 nm is
indicative of the conversion of 4S-peptide 2 from ꢀ-structure
in 100% TFE to full PPII form with 0.8% buffer in TFE.
Upon increasing the concentration of 4S-amp9 2 from 50
to 250 µM in TFE, the CD spectra exhibited a nice growth
in the negative band intensity at 210 nm, accompanied by
its shift to 216 nm and a large increase of the positive band
at 200 nm (Figure 3C). As expected for intermolecular
hydrogen bonding increasing with concentration, this strongly
points to a consolidation of ꢀ-structure in the peptide 4S-
amp9 2. In the case of 4R-Amp9 1, increasing the peptide
concentration leads to enhancement of the PPII form without
any other changes. The overall results imply that 4S-amp9 2
assumes a ꢀ-structure in TFE that is transformed to the PPII
form in aqueous medium, unlike 4R-amp9 1 which retains
the PPII form in both conditions.
NH2 with amide carbonyl possible only in 4S-amp9 2 (I)
promotes the PPII form in buffer. Urea rigidifies the
backbone11 by complementary H-bonding (II) among the cis-
disposed 4S-NH2 group and the amide carbonyl to strengthen
the PPII form. In a fluorinated solvent TFE, the intramo-
lecular H-bonding between 4S-NH2 and the amide carbonyl
switches to interchain H-bonding (III) giving rise to anti-
parallel ꢀ-structure. The trans disposition of 4R-NH2 and
(11) (a) Robinson, D. R.; Jencks, W. P. J. Am. Chem. Soc. 1965, 87,
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Chim. Acta 1996, 79, 913–941.
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Org. Lett., Vol. 12, No. 23, 2010

the amide carbonyl group in 4R-Amp9 (IV) is not conducive
to formation of either intramolecular or strong interchain
H-bonding in the derived peptide.
hydrogen bonds involving 4S-NH2 and amide carbonyl,
which are broken in water and rearranged to intramolecular
H-bonding that favors the PPII form via enriching the trans-
amide geometry. This structural conversion illustrates a fine
balance between stereoelectronic and H-bonding effects in
novel tuning of the secondary structure of 4R/S-aminoproline
polypeptides. ꢀ-Structure in polyproline peptides is hitherto
unknown, and the present results will add a new design
principle to a growing repertoire of strategies for engineering
peptide secondary structural motifs for new biomaterials and
nanoassemblies.15
Stereoelectronic vs H-Bonding Effects in 4S-amp9 2. The
stereoelectronic effect of 4R-X substituents on proline is
known to strongly favor the PPII form, over that of 4S-X
substituents by enhancing the trans-amide content.12 Al-
though the intramolecular H-bonding in 4S-NH3+ favors the
trans-amide form,9b the unfavorable stereoelectronic effect
+
+
of 4S-NH3 (unlike 4R-NH3 ) strongly negates the benefit
of H-bonding (V), leading to a low PPII form for 2 at acidic
pH. At higher pH, the effect of intramolecular H-bonding
dominates a weaker stereoelectronic effect of 4S-NH2,
promoting higher PPII content in peptide 2. While the
conversion of PPII to PPI takes many hours/days due to a
slow conversion of amide from trans to cis form,14 the
switching of ꢀ-structure to PPII form is fast, within minutes,
suggesting that the amide bond of 2 is also in trans form in
the ꢀ-structure. No PPI form was seen for any of the peptides
1-3, under different conditions of pH, n-propanol and TFE.
In conclusion, it is demonstrated here that 4S-amp9 2
adapts an unusual ꢀ-structure in TFE unlike most polyproline
peptides which prefer the PPI form in hydrophobic/
fluorinated media. The ꢀ-structure arises from interchain
Acknowledgment. MVS acknowledges an award of
fellowship by Lady Tata Memorial Trust and CSIR (New
Delhi). KNG holds JC Bose Fellowship of DST (New Delhi).
Supporting Information Available: Experimental syn-
thesis procedures, HPLC, mass spectral data, and CD spectra
of peptides 1-3 under different conditions of pH, urea, salt,
and concentration. This material is available free of charge
via the Internet at http://pubs.acs.org.
OL1021993
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