Effect of proline analogues on the conformation of elastin peptides

Article abstract of DOI:10.1039/c3nj41001c

Three elastin model peptides containing the repetitive motif -VGVXGVG-, where X corresponds to (2S)-proline (Pro), (2S,4R)-4-hydroxy-proline (Hyp) and (2S,4R)-4-methoxy-proline (Mop), were synthesized in order to define the effect of the introduction of an electronegative group on the conformation and self-assembling properties of the peptides. Circular dichroism, NMR and FT-IR spectroscopies evidenced outstanding differences at molecular and supramolecular level. This work constitutes the basis for the proper design of elastin-inspired drugs with tailored conformational properties.

Full text of DOI:10.1039/c3nj41001c

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Effect of proline analogues on the conformation of elastin peptides
*a
a
*a
Antonietta Pepe, Maria Antonietta Crudele and Brigida Bochicchio
Received (in XXX, XXX) Xth XXXXXXXXX 200X, Accepted Xth XXXXXXXXX 200X
First published on the web Xth XXXXXXXXX 200X
DOI: 10.1039/b00000000x
Three elastin model peptides containing the repetitive motif ꢀVGVXGVGꢀ, where X corresponds
to (2S)ꢀproline (Pro), (2S,4R)ꢀ4ꢀhydroxyꢀproline (Hyp) and (2S,4R)ꢀ4ꢀmethoxyꢀproline (Mop)
were synthesized in order to define the effect of the introduction of an electronegative group on
the conformation and selfꢀassembling properties of the peptides. Circular Dicroism, NMR and
FTꢀIR spectroscopies evidenced oustanding differences at molecular and supramolecular level.
This work constitutes the basis for the proper design of elastinꢀinspired drugs with tailored
conformational properties.
case it is strictly related to the elastic function of the protein.
Introduction
Actually, it has the function of a flexible hinge structure, that
is able to easily convert itself into other secondary structures
such as ꢀstrands, ꢀturns and unordered conformations
The (2S,4R)ꢀ4ꢀhydroxyꢀproline (Hyp) is the most common
postꢀtranslational modification observed in the vertebrates . It
1
14
originating the high entropy of the relaxed state .
is usually associated with increased strength and thermal
stability of collagen, the main component of the extracellular
Elastin is characterized by a pseudoꢀperiodic, low complexity
sequence with several type of repeated motifs. For example,
the ꢀVPGVGꢀ motif is abundant in porcine and bovine elastin
where it is tandem repeated many times, while it is less
2
matrix . The primary sequence of collagen is dominated by
the presence of the triad XaaꢀYaaꢀGly (where a proline is
usually occupying the Xaa position while the Yaa is
preferentially occupied by Hyp). Glycine residues at the third
position are required in order to allow the twisting of three
polypeptide chains in polyproline II conformation (PPII) to
form the triple helix . It was initially proposed that the
stabilization induced by Hyp was due to water molecules
forming a hydrogen bonding network linking the prolyl
15
present in human tropoelastin . It is found in the polypeptide
sequences encoded by exon 7, exon 18, exon 20 and exon 30
of human tropoelastin gene (HTE) (see Figure S1).
Differentꢀsized (VPGVG)n oligomers and polymers were
3
synthesized as soluble models of elastin and
their
conformation was analyzed by CD, NMR and IR
16
spectroscopy . The conformational studies on many different
4
hydroxyl groups and the mainꢀchain carbonyl groups .
repeatꢀcontaining peptides gave a detailed resolution of the
elastin structure useful for theorizing a molecular mechanism
of elastin elasticity. Elastinꢀinspired polypeptides containing
the –VPGVGꢀ motif were firstly studied by Urry while those
Afterwards, it was shown that the increase in stability is
primarily due to stereoelectronic effects and that hydration of
the hydroxyproline residues provides little or no additional
5, 6
stability . The importance of the stereoelectronic effect is
essentially due to the presence of electronꢀwithdrawing
containing –XGGZGꢀ motif (X,
Z = V, L, A) were
investigated by Tamburro. For –VPGVGꢀ repeated sequences
substituents in 4R configuration which determines
a
17
the βꢀspiral structure was proposed . It was characterized by
favourable preorganization of the polypeptide chain thus
promoting folding into triple helix. The consequences of the
introduction of the electronegative group in 4R configuration
are a higher trans/cis ratio as well as a favoured c ꢀexo ring
puckering, fundamental for the correct packing of the three
a helical arrangement of consecutive type II βꢀturns having ꢀ
PGꢀ at the corners, considered to play a strategic role in the
elasticity mechanism of elastin. Actually, the βꢀspiral structure
is characterized by large amplitude, low frequency motions

(
librations) responsible for the high entropy of the relaxed
7
, 8
polypeptide chains into the triple helix
.
state. The decrease in amplitude of the librations on extension
causes a decrease in the entropy of the peptide, providing the
Hyp is a postꢀtranlational modification observed also in other
vertebrate proteins, such as in the hypoxia ꢀinducible
transcription factor (HIF) and is responsible for changes in
gene expression in response to hypoxic conditions .
The presence of a significant amount of Hyp has been also
observed in elastin . Elastin, another structural protein of the
extracellular matrix responsible for the elasticity and
resilience of vertebrate tissues and organs, is constituted by
the crossꢀlinking of its soluble precursor tropoelastin . The
molecular structure of tropoelastin is highly flexible with lack
of stable secondary or tertiary structures. This behaviour,
that could let the protein contemplate among the natively
unstructured proteins, is fundamental for the entropically
driven elasticity mechanism . As a matter of facts, a multiꢀ
conformational dynamical equilibrium among PPII,
unordered, and ꢀturn conformation has been associated with
the relaxed state
The presence of PPII is fundamental also in the case of
tropoelastin, the soluble precursor of elastin, even if in this
18
driving force for returning to the relaxed state . While this
model provides a probable mechanistic description for local
regions of elastin, essentially those comprising PG segments,
it was not able to explain the elasticity of the whole protein.
In fact, several experimental and molecular dynamics
9
1
0
19
simulations show that short stretches of βꢀspiral are indeed
possible, but they are transient structures quickly interꢀ
converting into isolated βꢀturns, and extended structures such
as βꢀstrands as proposed by Tamburro. The presence of βꢀ
11
20
strands in elastin in the solid state has been shown . The
presence of nonꢀrecurring, isolated βꢀturns in elastin was
firstly proposed for the glycineꢀrich repeating sequences such
as XGG and XGGZG (X, Z =V, L) by Tamburro. They have
XG or GG segments at the corner with a 4→1 hydrogen bond
connecting the first and the fourth glycine or the X and Z
residue. The turns are labile and therefore can interconvert
each other giving rise to dynamic βꢀturns sliding along the
1
2
1
3
.
This journal is © The Royal Society of Chemistry [year]
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2
1
chain . The sliding increases the entropy of the chain and
contributes to the elasticity of the protein. MD simulations of
microsecond timeꢀscale have recently shown that also
Table 1 Sequence of E7X peptides (X=Pro, Hyp, Mop)
(
VPGVG)
7
polypeptide populates a highly heterogeneous
Peptide
E7P
E7H
Sequence
Mw (Da)
583.3
599.3
2
2
VGVProGVG
VGVHypGVG
VGVMopGVG
ensemble of conformations . The flexibility of the
polypeptide chain determines continuous exchanging of
conformations, determining only locally some fluctuating
ordered structures such as PPII and hydrogenꢀbonded βꢀturns
Therefore, a conformational equilibrium can be proposed for
elastin comprising PPII, βꢀturns and unordered conformations.
E7M
613.3
The heptapeptides E7P and E7H show very similar
spectroscopic features in aqueous solution (Fig. 2a and 2c). A
negative band at 198 nm, usually assigned to random coil is
visible in the CD spectra of the two peptides. The reduction of
the intensity of the negative band on heating is clearly
discernible and is not expected for peptides suggested to be
in random coil conformation. Actually, this temperatureꢀ
induced behaviour is commonly observed in elastin peptides
characterized by the presence of PPII conformation
Nevertheless, it is possible to assign these spectral features to
an inverse temperature transition from unordered to more
folded conformations, such as βꢀturns, typically observed for
elastin peptides of similar sequence . The CD spectra of E7M
in aqueous solution are shown in Fig. 2e. At 0°C the spectrum
shows a slight positive band at 211 nm that is blueꢀshifted at
5 and 70°C. Furthermore, they are characterized by a strong
negative band centred at 198 nm that is twoꢀfold higher than
the one observed for E7P and E7H. These spectral findings are
These
findings
support
the
general
equilibrium
extended→folded considered in the Tamburro model to be the
2
3
source of the high entropy of elastin in the relaxed state .
Smallꢀsized elastin peptides are involved in important
biological and pathological processes, such as inflammation,
tumor invasion and proteinase production . These effects are
mediated through the interaction of elastin peptides with
2
4
14, 29
.
2
5
elastin receptors and elastin peptides were considered of
potential therapeutic interests.
In order to find out the effect of the introduction of electronꢀ
withdrawing substituents on the conformational structure of
elastin peptides, we synthesized three peptides containing the
elastin repeated sequences VGVXGVG, commonly found in
different elastins, where X corresponds to proline (Pro),
30
2
(
2S,4R)ꢀ4ꢀhydroxyꢀproline (Hyp) and (2S,4R)ꢀ4ꢀmethoxyꢀ
proline (Mop) (Fig.1). The peptides were studied by CD,
NMR and FTꢀIR spectroscopy in order to highlight potential
differences in the secondary structures. The selfꢀassembling
propensities were investigated by Atomic Force Microscopy
indicative of the presence of
a
significantly higher
contribution of PPII conformation as PPII typical CD spectra
show a strong negative band around 200 nm and a weaker
31
positive band at about 217 nm .
(
AFM).
On this basis, we suggest for E7M the dominance of PPII
conformation while for E7P and E7H peptides the coexistence
of PPII (at low temperature), random coil conformation and
folded conformations (at high temperatures) (Figure 3).
Thermodynamic analyses were performed from CD spectra of
E7P, E7H, and E7P recorded in water at different
temperatures. The mean residue ellipticity at 198 nm were
plotted as
a function of temperature and wellꢀdefined
conformational transition curves were observed. The data
were fitted to Van't Hoff plot and a linear correlation was
found (r > 0.98) (Figure SI 2ꢀ4). The fitting of the data
allowed the calculation of the enthalpy change ꢀH°, the
entropy change ꢀS°, and the transition temperature Tm of the
conformational transition from the slope, the intercept and the
Fig.1: Chemical structure of (2S)ꢀProline (Pro, R
hydroxyproline (Hyp, R =OH) and (2S,4R) 4ꢀmethoxyproline (Mop,
=OCH
1
=H), (2S,4R)ꢀ4ꢀ
1
R
1
3
)
Results and Discussion
Three elastin model peptides were synthesized as listed in
Table 1. The sequence of the 7ꢀresidue peptides E7X is
ꢀ
H°/ꢀS° values, respectively. . (Table 2).
Table 2 Thermodynamic values of the temperature induced
conformational transition of the elastin peptides
(
VGVXGVG) where X= Pro, Hyp, and Mop, (E7P, E7H, and
E7M, respectively) . We added 2 residue to the Nꢀterminus of
the pentapeptide repeat sequence in order to reduce the
possible termination effect, that could alter the conformational
studies, by biasing the βꢀturn structure due to electrostatic
interactions. These peptide sequences were commonly used as
a simplified model of the elastin molecule . E7M envisages
the presence of the amino acid (2S,4R)ꢀ4ꢀmethoxyꢀproline
(
-1
-1 -1
Peptide
E7P
E7H
ꢀ
H°/ Kcal mol
17.2±1.7
14.5±0.7
17.8±1.4
ꢀ
S°/ Kcal mol K
Tm/K
313
314
0.055± 0.005
0.047± 0.002
0.059± 0.005
E7M
303
2
6
All peptides exhibited positive ꢀΗ and ꢀS values, supporting
the model of an entropyꢀdriven folding transition. The
midpoint of the transition is very similar for E7Pand E7H,
while for E7M is 10 °C lower, indicating a higher propensity
for folding. The folding of the peptides in more ordered (βꢀ
turns) structures on increasing temperature, as a consequence
of hydrophobic interactions, could be driven by positive
Mop). Oꢀmethylation of the 4ꢀhydroxy group, as previously
6
shown for collagen peptides , preserves the stereoelectronic
effect, as the electronꢀwithdrawing and hyperconjugative
ability of OH and OCH are similar, while eliminating the
2
7
28
3
contribution of the hydrogenꢀbonding.
3
2..
Conformational studies in solution
entropies of dehydration
This model is considered at the
basis of the peculiar selfꢀassembling process of elastin
peptides. In particular in VPGVG containg sequences, the
hydrophobic interactions, involving the valine residues at the
CD spectroscopy is a very sensitive tool, able to define the
conformational differences among peptides. The CD spectra
of peptides are shown in Fig. 2.

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Fig.2: CD spectra in acqueous solution at 0°C (empty squares), 25 °C (empty triangles), 70°C (empty circles) of a) E7P; c) E7H; e) E7M.
CD spectra in TFE (filled squares), 25 °C (filled triangles), 70°C (filled circles) of b) E7P; d) E7H; f) E7M.
corner of the type II βꢀturn, are shown to be favoured at
higher temperature.
pronounced in the weaker intermolecular hydrogen bonds and
NMR data confirm the presence of random coil and/or PPII
conformations, as evidenced by the absence of any mediumꢀ
range NOEs connectivities, thus ruling out the presence of
folded structures such as αꢀhelix and βꢀturn.
Variable temperature 1D spectra were recorded in the
temperature range 25°ꢀ45°C, in order to determine the
temperature coefficients of the amide protons. The strong
temperature dependence of amide proton chemical shifts is
related to the presence of two types of hydrogen bonds: intraꢀ
molecular and interꢀmolecular (mainly with water
therefore chemical shifts of amide proton not involved in
Fig. 3. Two molecular models of the heptapeptides VGVXGVG
representing on the left the random coil/PPII conformation (dominant at
low temperature) and on the right the folded/type II βꢀturn conformation
(
dominant at high temperature). The green circle represent the substituent
3
3
(R=H, OH, OMe) present at 4R position of the proline ring, respectively
for X=Pro, Hyp, and Mop residues.
molecules) . On increasing the temperature the lengthening
3
4
of the average hydrogen bond determines a reduction of the
deshielding effect induced by the acceptor with a consequent
highꢀfield shift of the amide protons. The effect is more
intramolecular HB are more temperature dependent. Stronger
intramolecular hydrogen bonds are less susceptible to

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deformation and such amides show smaller chemical shift
changes with temperature. In the case of turn structures,
temperature coefficient values higher than ꢀ4.0 ppb/K are
usually observed. The measured temperature coefficients of
E7P, E7H, and E7M in aqueous solution evidenced in all cases
low values (< ꢀ6.0 ppb/K), thus ruling out the presence of
intraꢀmolecular hydrogenꢀbonds in water (Table S1ꢀ S3).
interaction between the oxygen of the prolyl peptide bond
8
, 36
(Oi–1) and the prolyl carbonyl group (C′i=Oi)
This
interaction can only occur when the prolyl peptide bond is in
its trans conformation, and thus stabilizes that conformation.
In order to find out if the same effect is observed for elastin
peptides the Kt/c constant of the trans to cis conversion has
been determined by integration of isolated amide proton
signals. In the case of E7M, the data were confirmed by the
integration of the methoxyl signals belonging to the two
isomers. 1D spectra in water highlight the altered cis/trans
ratio. While for the E7P peptide a Kt/c of 9.0 is measured,
E7H and E7M have
respectively.
a higher values, 31.0, and 20.0,
The CD spectra of E7P and E7H peptides in TFE, a solvent
that promotes folded conformations, show again high
similarities pointing to a significant contribution of type II ꢀ
turn conformations for both peptides (Fig. 2b and 2e). The
higher intensity of the positive band at 200 nm in E7M CD
spectra suggests a significantly higher content of ꢀturn in
comparison with E7P and E7H (Fig. 2f).
NMR data are in accordance with the CD results evidencing
all the typical spectral features of type II βꢀturns. As a matter
of facts, NMR data of the peptides recorded in TFEꢀd
3 2
/H O
8
0/20, point to the presence of type II ꢀturn in the sequence
3
5
V ꢀ(Pro/Hyp/Mop)GꢀV in all peptides, as confirmed by the
reduced temperature coefficients for V5 amide protons (Table
S4ꢀS6).
However, the most important diagnostic features of a reverse
turn conformation is the close approach of Hα of residue i+1
with the NH of residue i+3 of the turn. The NOE effect arising
from this interaction is usually weak, because the distance
involved is ~3.4 Å. However, the existence of this typical
4
6
medium range NOE, as those observed between X and V of
all three peptides, is a good indication of a significant
3
7
population of turn conformation present in solution .
Furthermore, type II β−turns are characterized by strong
sequential dαΝ (i+1,i+2) and dNN(i+2,i+3) NOEs. In the case
of the E7X peptides, we can detect strong dαΝ (i+1,i+2)
4
5
NOEs between
d
X and G , together with strong sequential
5
6
NN NOEs between G and V , typical for type II βꢀturns.
Another criterion that could be used to define the presence of
turn structures is the difference in chemical shift observed
5
between the two enantiotopic H
α
protons of G , involved in
the C10ꢀring of a βꢀturn. In all three peptides the ꢀΗ
.40 ppm, pointing to a great difference in the structural
environment of the two protons, due to βꢀturn structure.
α
is >
0
1
Figure 4: Amide proton regions of monoꢀdimensional H spectra of a)
In accordance with CD spectra, E7M shows a higher content
of turn structure, as deduced from a higher coefficient
temperature (ꢀδ/ꢀT=ꢀ4.3 ppb/K) with respect to E7P and
E7H, ꢀ4.6 and ꢀ5.1 ppb/K, respectively. Furthermore, the
3 2
E7P, b) E7H and c) E7M, recorded at 25°C in TFEꢀd /H O 80/20.
In most peptide bonds, the trans (Z) isomer is greatly favored
over the cis (E) isomer. In contrast, the trans isomer of a
prolyl peptide bond is only slightly favored over the cis
isomer. The interconversion of cis and trans isomers about
prolyl peptide bonds has been identified as the rateꢀlimiting
step in protein folding pathways,including that of collagen .
Electronegative substituents in the 4R position, as in Hyp or
Mop, lead to a high trans:cis ratio because of a favorable
4
6
intensity of the dαΝ (i+1,i+3) NOE between X and V is
significantly higher for E7M. Because the Kt/c depends on the
3
8
solvents , we measured it also in TFEꢀd /H O solution. 1D
3 2
3
5
spectra highlight the altered cis/trans ratio. While for the E7P
peptide a Kt/c of 9.0 is measured, E7H and E7M have higher
values, 20.0, and 23.0, respectively (Figure 3).

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Conformational studies in the solid state
The Amide I and II decomposed FTIR spectrum of E7P
peptide is shown in Fig. 5a. It shows four bands at 1736,
ꢀ
1
1
686, 1659 and 1625 cm in the Amide I region. The higher
ꢀ
1
band at 1659 cm
could be attributed to unordered
conformations, the one at 1686 cm to ꢀturn and the third
one at 1625 cm at strong hydrogen bonded conformations
compatible with the presence of ꢀstrand. The band at 1736 is
assigned to the Cꢀterminal COOH group and is present in the
ꢀ
1
ꢀ
1
3
9
FTꢀIR spectra of all peptides . The presence of ꢀ
conformations is confirmed by the Amide II band component
ꢀ
1
40
at 1526 cm usually attributed to the NH hydrogen bending .
ꢀ
1
The band at 1553 cm is tentatively assigned to unordered
conformations. The Amide I and II bands of the decomposed
FTIR spectra of E7H peptide are analogues to E7P and are
shown in Fig. 5b Similar results were obtained for E7M
peptide but in the different ratio among the intensities of the
components (Fig. 5c). As a matter of fact, the component at
ꢀ
1
1
679 cm is the dominant one thus suggesting the prevalence
ꢀ
1
of ꢀturn compared to unordered (1655 cm ) and −strand
1623 cm )conformations.
ꢀ
1
(
Self-assembling propensities
AFM has become an important technique for direct imaging of
biomolecules with a subꢀnanometer resolution. Indeed, it is a
powerful tool for imaging proteins as well as polypeptides
especially in Tapping Mode providing nanometerꢀscale
information about surface structure even of soft and fragile
samples. As a matter of fact, it gives useful information on the
surface roughness of nanomaterials.
It has been widely used for determining the supramolecular
structures
of
selfꢀassembling
peptides,
detecting
nanostructures of different types, such as fibers, vescicles,
4
1
sheets or films
.
The main advantage of AFM over other microscopy
techniques is constituted by the sample preparation mode that
is very simple and fast. It consists in spotting few microliters
of the solution on a mica, glass, or silicon substrate without
any special treatment such as metal coating that would change
or damage the sample. Furthermore, as AFM analysis does
not need of high vacuum environment it let work at
atmospheric pressure and even in liquid medium by better
mimicking the physiological conditions. The selfꢀassembling
propensities of the peptides were investigated by Atomic
Force Microscopy (AFM). The results are shown in Fig. 6.
E7P peptide shows a tendency to adopt globular structures
that interact among them giving rise to a thin film covering
the silicon substrate with a height of about 2 nm (Fig. 6a). The
introduction of Hyp in E7H peptide strongly disfavours the
selfꢀaggregation propensity of the peptide giving rise to
amorphous deposits with leopardskinꢀlike macules (Fig. 5b).
At the opposite end, AFM of E7M is characterized by beaded
filaments constituted of small diameter globules interacting
sideꢀbyꢀside (Fig. 6c) and originating short protofilaments.
We tentatively try to find a rationale for the different
aggregation pattern observed, according to our molecular and
thermodynamic studies. The great difference observed
between E7H and E7M could be
Fig. 5: Decomposed FTIR spectra of Amide I and II regions of : a) E7P;
b) E7H; c) E7M. Experimental spectrum (solid line), calculated spectrum
(
dashed line). On the lower panel the respective second derivative spectra
are shown.

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Figure 6: AFM micrographies of: a) E7P; b) E7H; c) E7M deposited after dissolution. AFM images of d) E7P; e) E7H; f) E7M deposited after seven days
of incubation at 50°C.
certainly assigned to a decreased hydration of E7M induced
by the Oꢀmethylation of Hyp . Therefore, compared with
about 5 nm height not present in E7P micrography at 25°C
(Fig.6d). Even, E7H peptide shows an increased selfꢀ
assembling propensity comparable in morphology as well as
in height with the AFM shown by E7P at room temperature
(Fig. 6e). E7M peptide is able to cover the entire silicon
substrate while only short protofilaments were visible at room
6
E7H,the less hydrated OMe in E7M would make Mop less
disposable to expose its ring chain to water, leading to a gain
of interꢀmolecular hydrophobic interactions responsible of the
observed increased selfꢀaggregation. The thermodynamic
analysis of the conformational transition observed for the
three heptapeptides suggested for E7M a higher entropic
contribution possibly assigned to reduced peptideꢀwater
interactions. In order to verify the hypothesis of the
dominance of hydrophobic interactions in the selfꢀassembling
process, expected to increase with the high temperature, the
peptide solutions were incubated for one week at 50°C and
then observed by AFM. The results are shown in Fig. 6dꢀf.
The E7P micrography shows the presence of a dense film
temperature (Fig. 6f).
On summarizing, the incubated
samples show an increased ability to selfꢀassemble on
increasing temperature thus confirming the hydrophobic
forces as the responsible of the prominent selfꢀaggregation
propensities of E7M in comparison with E7P and E7H.
However, in order to have more significant insights in the
aggregation propensities, a detailed supramolecular study with
longer peptides could be more effective and is currently
ongoing.
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Conclusions
then lyophilized in order to eliminate TFA excess to afford
1
MopꢀOH. HꢀNMR (400 MHz, DMSOꢀd
6
) : 9.95 (bs, 1H),
In this work we have synthesized three elastinꢀinspired
peptides differing in one amino acidic
8
.92 (bs 1H9, 2.05 (m, 1H), 2.4 (m, 1H), 3.23 (s, 3H), 3.2ꢀ3.4
residue. The
13
(
m, 2H), 4.10 (m, 1H), 4.32 (dd, J=11.6; 8.1 Hz, 1 H). Cꢀ
heptapeptides were studied at molecular and supramolecular
level in order to find out the effect of the single residue on the
conformation and selfꢀassembling propensities of the full
peptide. Spectroscopic studies in solution (CD, NMR) as
well as in solid state (FTIR) showed the presence of more
folded conformations such as type II ꢀturn for E7M when
compared with E7P and E7H. This conformation let E7M
peptide maintain at the minimum level the interaction with
water molecules by the intramolecular hydrogen bonds
NMR (400 MHz, CDCl ) : 34.27, 50.81, 56.58, 58.28,
3
+
7
8.90, 170.84.
HRMSꢀESI (m/z): [M+H ] calcd for
6 12 1 3
C H N O
146.0812; found 146.0812.
Synthesis of N-9-Fluorenylmethoxycarbonyl-(2S, 4R)-4-
methoxy-proline (Mop-OH)
MopꢀOH as white solid was dissolved in saturated NaHCO
3
(
aq) (70 ml). A solution of FmocꢀOSu (2.385 g, 0.00708 mol)
in dioxane (62 ml) was added, and the resulting white
suspension was stirred for 24 h. The reaction mixture was
concentrated under reduced pressure, and the residue was
diluted withwater (122 ml) and washed with ether (3 x 122
ml). The aqueous layer was acidified to pH 1.0 with 1N HCl,
extracted with ether (3 x 122 ml), dried over anhydrous
MgSO (s), and concentrated under reduced pressure to afford
4
a yellow oil. The oil was crystallized from DCM/petroleum
ether to afford FmocꢀMopꢀOH as a white powder (2.167g,
involving the peptide backbone.
Herein, the ꢀturn
conformation acts as a steric constrain inducing the proper
alignment of molecules favouring the onset of hydrophobic
interactions among the sideꢀchain functional groups. These
hydrophobic interactions are responsible for the greater selfꢀ
assembling propensity of E7M observed by AFM technique.
The hypothesis concerning the hydrophobic forces as the main
responsible for the outstanding selfꢀassembly propensity of
E7M is corroborated by AFM measurements results carried
out on the samples incubated at 50°C. As a matter of fact, all
the three peptides show an increased capacity to aggregate on
increasing the temperature as expected when hydrophobic
forces are the driving forces of the aggregation process. This
result constitutes an important finding toward the design of
elastinꢀbased drugs with tunable conformation, able to interact
with elastin receptors and/or elastases.
0
.005904 mol, yield 83.43%).
Peptide synthesis and purification
TM
The peptides were synthesized by SPPS on Tribute
automatic peptide synthesizer (Protein Technologies Inc.) The
synthesis scale was 0.25 mmol with Fmoc/HBTU chemistry.
Fmoc αꢀaminoacids were purchased from Inbios (Pozzuoli,
Italy). NꢀtertꢀButoxycarbonylꢀ(2S, 4R)ꢀ4ꢀhydroxyproline
(
BocꢀHypꢀOH) was purchased by Sigma Aldrich, St Louis,
Experimental
USA). Nꢀ9ꢀFluorenylmethoxycarbonylꢀ(2S, 4R)ꢀ4ꢀmethoxyꢀ
proline (MopꢀOH) was synthesized as before described. The
cleavage of peptides from resin was achieved by using an
aqueous mixture of 95% trifluoroacetic acid. The peptides
were lyophilized and purified by semipreparative reversedꢀ
phase highꢀperformance liquid chromatography. Binary
Synthesis of N-tert-Butoxycarbonyl-(2S, 4R)-4-
methoxyproline (Boc-Mop-OH)
This preparation is similar to that reported by Krapcho et al.
4
2
for CbzꢀMopꢀOH . To a solution of NꢀtertꢀButoxycarbonylꢀ
2S, 4R)4ꢀhydroxyproline (BocꢀHypꢀOH, 5.03 g, 0.022 mol)
in anhydrous THF (80 ml) was added NaH (50% oil, 2.20 g,
.046 mol). After 45 min at room temperature, the reaction
(
2
gradient was used and the solvents were H O (0.1% TFA) and
CH CN(0.1% TFA). The purity of peptides was assessed by
3
0
ESI (Electrospray) Fourier Transformed Mass Spectroscopy
mixture was treated with MeI (2.86 ml, 0.046 mol), and the
resulting mixture was heated under reflux for 2 h and then
kept at room temperature for 18 h. The reaction mixture was
concentrated under reduced pressure and partitioned between
1
(
FTMS) and HꢀNMR spectroscopy.
CD spectroscopy
CD spectra of 0.1 mg/ ml solutions of peptides in water and in
TFE were recorded at 0°, 25°, 70°C on a JASCO J815 CD
spectropolarimeter using a HAAKE waterbath as temperature
controller and a quartz cell with a 1ꢀmm optical path length.
H O and CH Cl . The aqueous mixture was acidified to pH 1
2
2
2
by the addition of 3N HCl (aq) and extracted with CH
The combined organic layers were dried over MgSO (s) and
evaporated under reduced pressure. HꢀNMR (400 MHz,
CDCl ) : 1.42 and 1.48 (s, 9H), 2.16ꢀ2.05 (m, 0.5H), 2.34ꢀ
.28 (m, 1H), 2.50ꢀ2.36 (m, 0.5H), 3.33 (s, 3H), 3.70ꢀ3.50 (m,
H), 3.99 (m, 1H), 4.34 (t, J=7.6 Hz, 0.5H), 4.43 (t, J=7.9 Hz,
2
Cl2.
4
1
1
6 scans were acquired in the range 190ꢀ 250 nm by taking
ꢀ
1
3
points every 0.1 nm, with a 50 nm min scan rate, an
integration time of 2 s, and a 1 nm bandwidth. The CD spectra
were processed by the JASCO Spectral analysis software.
After background subtraction a FFT filtering algorithm was
applied for smoothing, and data expressed as molar elipticity
2
2
0
3
1
1
3
.5H).
6.4, 51.13, 51.8, 56, 58, 78.2, 78.4, 80.9, 81.7, 154.04,
56.34, 178.9. HRMSꢀESI (m/z): calcd for C11 Na
268.11563; calcd for
[M+K ] 284.08448, found 284.04061.
3
CꢀNMR (400 MHz, CDCl ) : 28.4, 28.5, 34.3,
2
ꢀ1
H
19
N
1
O
5
1
[
θ] expressed as deg cm dmol .
+
[
M+Na ] 268.11554, found
+
11 19 1 5 1
C H N O K
Thermodynamic analysis
Synthesis of (2S, 4R)-4-methoxyproline (Mop-OH)
For the thermodynamic analysis, variable temperature CD
spectra of the peptides were recorded in aqueous solution by
cooling the samples to 0°C and then stepwise increasing
temperature from 0° up to 70°C by a 10°C step, allowing
samples to equilibrate at each temperature for five minutes.
BocꢀMopꢀOH (0.00708 mol) was dissolved in DCM (5 ml)
and the resulting solution was stirred at room temperature.
After the addition of TFA (5 ml) the solution was left under
stirring at 0°C and then at room temperature for 3 h. The
reaction mixture was concentrated under reduced pressure and
dried briefly under high vacuum affording MopꢀOH as a clear
oil. After the addition of water the suspension was freezed
3
0
The processing of the data was performed according to .
o
Briefly, the standard Gibbs freeꢀenergy change, ꢁG , of the
conformational transition as well as the corresponding

New Journal of Chemistry
Page 8 of 10
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enthalpy and entropy changes, the temperatureꢀdependent CD
using rectangular Si cantilevers (NCHR, Park Systems)
data (at 198 nm) were fitted to a twoꢀstate model. The van't
Hoff plot (plot of lnK versus 1/T, K being the equilibrium
constant) are obtained from the CD data . It was constructed
from K = ([θ]obs − [θ] )/([θ] − [θ]obs), where [θ]obs are the CD
having the radius of curvature less than 10 nm and with the
nominal resonance frequency and force constant of 330 kHz
ꢀ
1
and 42 N m , respectively, or diamond tips (Mikromasch)
with typical spike curvature radius less than 7 nm, nominal
resonance frequency of 325 kHz and typical force constant 46
U
F
data for the experimentally observed ellipticity, and [θ]
the values for the previously fitted endpoints of the transition
highꢀtemperature folded form, [θ] , and lowꢀtemperature
F U
, [θ]
ꢁ
1
Nm .
(
F
unfolded form, [θ]U)
.
Acknowledgements
Thanks are due to Dr. Neluta Ibris for AFM images (CIGASꢀ
University of Basilicata). The financial support of MIUR
NMR spectroscopy
(
PRIN2008) is gratefully acknowledged.
1
All H NMR experiments were performed on a Varian Unity
INOVA 500 MHz spectrometer equipped with a 5mm tripleꢀ
resonance probe and zꢀaxial gradients. The purified peptides
Notes and references
a
Department of Science, University of Basilicata, Potenza, Italy. Fax:
were dissolved in 700 ꢁl of H
/H O (80/20), containing 0.1 mM of 3ꢀ(trimethylꢀsily1)ꢀ1ꢀ
propane sulfonic acid (DSS) as internal reference standard at
ppm. 3 mM peptide solutions were used. Oneꢀdimensional
spectra were acquired in Fourier mode with quadrature
2 2
O/D O (90/10, v/v) and TFEꢀ
+39 0971205678; Tel: +39 0971205486-5481; E-mail:
d
3
2
antonietta.pepe@unibas.it; brigida.bochicchio@unibas.it
0
1
.
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4
3
44
detection . Twoꢀdimensional TOCSY and ROESY spectra
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transients and 256 increments. Relaxation delays were set to
1
3
.
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.
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doubleꢀpulsed fieldꢀgradient spinꢀecho . Amide proton
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1
(
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1
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Gaussian and Lorentzian peak shapes and accounts for the
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0 ꢀl of peptide solutions (1 mg/ml) were deposited on Si
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TOC
Effect of proline analogues on the conformation of elastin peptides
Antonietta Pepe, Maria Antonietta Crudele and Brigida Bochicchio
Three elastin model peptides containing the
repetitive motif
-VGVXGVG-, where X
corresponds to (2S)-proline (Pro), (2S,4R)-4-
hydroxy-proline (Hyp) and (2S,4R)-4-methoxy-
proline (Mop), were synthesized. The peptides
were
investigated
at
molecular
and
supramolecular level.