Synthesis of homopolypeptides with PPII structure

Article abstract of DOI:10.1002/pola.26705

Polyprolines are attractive polymers because of their folding property into polyproline II (PPII) structure, their significance in protein/protein interactions, and their potential as new therapeutic targets. Silaproline (Sip) is an analogue of proline, which exhibits similar conformational properties. The presence of dimethylsilyl group confers to Sip a higher lipophilicity as well as an improved resistance to biodegradation. Enantiomerically pure Sip was available in gram quantities from resolution of the enantiomers by chiral high performance liquid chromatography. This study describes the first synthesis of Sip N-carboxyanhydride (NCA) and shows preliminary results on comparison of polymerization of (l)Pro-NCA and (d)Sip-NCA to obtain homopolypeptides with PPII structure, polyproline, and polysilaproline polymers.

Full text of DOI:10.1002/pola.26705

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Synthesis of Homopolypeptides with PPII Structure
1
2
1
1
ꢀ
Charlotte Martin, Aurelien Lebrun, Jean Martinez, Florine Cavelier
1
ꢀ
IBMM, UMR-CNRS-5247, Universites Montpellier I and II, Place Euge`ne Bataillon, 34095 Montpellier, France
2
ꢀ
LMP, IBMM, Universite Montpellier II, Place Euge`ne Bataillon, 34095 Montpellier, France
Correspondence to: F. Cavelier (E-mail: florine@univ-montp2.fr)
Received 12 March 2013; accepted 18 March 2013; published online 22 April 2013
DOI: 10.1002/pola.26705
ABSTRACT: Polyprolines are attractive polymers because of
their folding property into polyproline II (PPII) structure, their
significance in protein/protein interactions, and their potential
as new therapeutic targets. Silaproline (Sip) is an analogue of
proline, which exhibits similar conformational properties. The
presence of dimethylsilyl group confers to Sip a higher lipo-
philicity as well as an improved resistance to biodegradation.
Enantiomerically pure Sip was available in gram quantities
from resolution of the enantiomers by chiral high perform-
ance liquid chromatography. This study describes the first
synthesis of Sip N-carboxyanhydride (NCA) and shows pre-
liminary results on comparison of polymerization of (L)Pro-
NCA and (D)Sip-NCA to obtain homopolypeptides with PPII
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structure, polyproline, and polysilaproline polymers.
2013
Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem.
2013, 51, 3103–3109
KEYWORDS: biopolymers; conformational analysis; N-carbox-
yanhydride; PPII helix; ring opening polymerization (ROP);
silaproline
In this context, we thought that it could be interesting to
demonstrate the possibility to synthesize both polyproline
and polysilaproline by polymerization of corresponding
amino acid NCA.
INTRODUCTION Polypeptides syntheses are widely investi-
gated, attempting to control the length and the architecture
of the resulting polymers, especially for homopolypeptides,
constituted by the same residue all along the polymeric
chain. Since the late 1940s,1 polymerization of N-carboxyan-
hydride (NCA) has been extensively used for the synthesis of
high-molecular weight polypeptides and this old method has
been reviewed recently.2,3 However, for proline-rich peptides
or polyprolines, one of the most interesting homopolypepti-
des first discovered in collagen,4 and attractive for their PPII
inducing structure, the synthesis was usually performed on
solid support following a step-by-step Fmoc-based strategy.
This was the case when the PPII helix was used as backbone
for cell penetrating peptides (CPP) for example.5 To modu-
late physicochemical properties of the resulting helix, which
is water soluble in the case of polyproline, hydrophobic resi-
dues such as silaproline (Sip) were introduced.6 Sip is a sili-
con-containing analogue of proline, which exhibits similar
conformational properties.7 The presence of the dimethylsilyl
group confers to Sip a higher lipophilicity as well as
an improved resistance to biodegradation.8 Sip was synthe-
To synthesize homopolypeptides, the most extensively used
method is based on polymerization of NCA, first reported by
Leuchs in the early nineteenth.12 The purity of NCAs has been
proved to be very important and it is necessary to obtain NCA
in satisfactory quality for controlled polymerization. Alterna-
tively to classical recrystallization, high vacuum techniques13
and flash chromatography14 have been proposed recently.
Although usual amino acids NCAs are quite well docu-
mented,15 proline is a special case in that it bears a second-
ary amine. Moreover, the cyclic structure of this imino acid
causes some constrains and conformational restrictions.
These features result in failure to obtain NCA with usual
methods, either by treatment with halogenating reagents
(Leuchs method12) [Scheme 1(a)], or with phosgene or phos-
gene substitutes like triphosgene16 (Fuchs–Farthings
method17,18) [Scheme 1(b)].
€
sized successfully via asymmetric synthesis by Schollkopf
method,9,10 but with this approach the scale up was difficult.
More recently, a racemic synthesis was carried out for the
gram scale preparation of enantiomerically pure Sip, requir-
ing resolution of the enantiomers by chiral high performance
liquid chromatography (HPLC).11
EXPERIMENTAL
Materials and Reagents
Hydrogene chloride solution in dioxane 4.0 M (Aldrich), tri-
phosgene reagent grade 98% (Aldrich), diethylaminomethyl-
Additional Supporting Information may be found in the online version of this article.
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SCHEME 1 Preparation of NCAs using (a) halogenating reagents and (b) phosgene.
polystyrene 200–4000 mesh crosslinked with 2% divinylbenzene Synthesis of Polypeptides
with a loading 3.2 mmol/g (Aldrich), and L-proline benzyl ester Polypeptides were prepared by ring-opening polymerization
hydrochloride 98% (Aldrich) were used as received. All reactions (ROP) of the NCA of L-proline, L-Sip, or D-Sip in the presence
involving air-sensitive reagents were performed under argon. Sol- of initiator such as water or L-proline benzyl ester. NCA was
vents and reagents were purchased from Aldrich and Fluka. Tetra- dissolved in dry THF under argon, and then the initiator was
hydrofurane (THF) was freshly distilled from benzophenone/ added (after neutralization of the hydrochloride with trie-
sodium prior to use. Ethyl acetate and hexane were anhydrous.
thylamine in ether in the case of HCl, H-(L)Pro-Bzl ester).
The reaction was stirred for 24 h at room temperature. The
reaction mixture was concentrated in vacuo and the polymer
was obtained as a white powder.
Synthesis
Synthesis of HCl, H-(D)Sip-OH
Boc-(D)Sip-Ot-Bu (1 g, 3.17 mmol) was dissolved with HCl 4 N in
dioxane (10 mL). The reaction was stirred for 1 h at room tem-
perature. The residue obtained after evaporation was dissolved
in a mixture of acetonitrile and water and lyophilized affording
HCl, H-(D)Sip-OH as a white solid in quantitative yield.
NMR Spectroscopy
Proton nuclear magnetic resonance (1H NMR) and carbon
nuclear magnetic resonance
(
13C NMR) spectra were
recorded on a Bruker spectrometer advance 300 at 300 and
75 MHz respectively for H-Sip-OH in D2O and for NCA in
CDCl3 at room temperature. The spectra of the polymers
were performed in CF3COOD on a Bruker spectrometer
advance 600 at 600 and 150 MHz, respectively.
1H NMR (300 MHz, D2O) d 4.19 (dd, J 5 11.4, 7.3, 1
H,CHa(Sip)), 2.77 (d, J 5 15.1, 1 H, SiCHHN), 2.52 (d, J 5 15.1,
1H, SiCHHN), 1.60 (dd, J 5 15.1, 7.3, 1 H, SiCH2Ca), 1.18 (dd,
J 5 15.1, 11.4, 1H, SiCH2Ca), 0.40–0.28 (m, 6H, Si(CH3)2.
Optical Rotation
13C NMR (75 MHz, D2O) d 175.13 (COOH), 62.49 (CHa(Sip)), 35.26
(SiCH2N), 16.75 (SiCH2Ca), 2 1.84 (Si(CH3)2), 2 2.00 (Si(CH3)2).
Optical rotation values were measured on a Perkin–Elmer
341 (20 ꢀC, sodium ray), with a concentration of 1 g/100
mL in HCl 1 N.
HCl, H-(D)Sip-OH: a2D0 5 1 14 (c 5 1, HCl 1 N).
Mass Spectroscopy
Synthesis of Sip-NCA
Analyses were conducted on the IBMM analytical platform
located in the Laboratoire de Mesures Physiques of University
Montpellier 2. Matrix assisted laser desorption/ionization-
time of light (MALDI-Tof) mass spectra were recorded on an
Ultraflex III Tof/Tof instrument (Bruker Daltonics, Wissem-
bourg, France) equipped with a pulsed Nd:YAG laser at a
wavelength of 355 nm. The source was operated in the posi-
tive mode. A solution of the matrix, a-cyano-4-hydroxycin-
namic acid [10 mg/mL in water/acetonitrile (HCCA) (vol/vol,
70/30)] or 2,5-dihydroxybenzoic acid (DHB) [10 mg/mL in
water/acetonitrile (vol/vol, 50/50)], was mixed with the sam-
ple in equal amount and 0.5 lL of this solution was deposited
onto the MALDI target according to the dried droplet proce-
dure. After evaporation of the solvent, the MALDI target was
introduced into the mass spectrometer ion source. Ions were
detected over a mass range from m/z 500 to 4000.
Sip (63 mg, 0.32 mmol) was suspended with magnetic stir-
ring in dry THF freshly distilled (5 mL) in a round bottom
flask (with CaCl2 tube) and under dry argon gas. Once this
suspension reached 50 ꢀC, triphosgene (83 mg, 0.28 mmol)
was added in one portion, and reaction mixture was stirred
for 1 h. The final solution was evaporated in vacuo and then
the oily residue was dissolved in dry THF (5 mL) and added
into the diethylamine polystyrene (3 equiv.), which had been
swollen previously in dry THF in a solid phase reactor with
a filter disk and kept under argon atmosphere for 3 h at
room temperature under mechanical stirring. Sip-NCA was
obtained by filtration and washing with additional dry THF.
The crude Sip-NCA was recrystallized from AcOEt/hexane at
ꢀ
2 20 C to afford to Sip-NCA crystals. Overall yield: 72%.
1H NMR (300 MHz, CDCl3) d 4.32 (dd, J 5 11.8, 7.0, 1 H,
CHa(Sip)), 3.18 (d, J 5 14.8, 1 H, SiCHHN), 2.52 (d, J 5 14.8,
1H, SiCHHN), 1.45 (dd, J 5 14.4, 7.0, 1 H, SiCHHCa), 1.01
(dd, J 5 14.4, 11.8, 1H, SiCHHCa), 0.34 (m, 6H, Si(CH3)2).
Circular Dichroism Spectroscopy
Samples for circular dichroism (CD) were prepared at a con-
centration of 0.1 mg/mL in trifluoroethanol (TFE). At first a
background spectrum was taken with 100% TFE. Then the
solution was pipetted into a quartz cell with a 0.1-mm path
length. Spectra were measured using a Jasco J-815 CD
13C NMR (75 MHz, CDCl3) d 170.20 (COO), 152.45 (NCO),
62.20 (CHa(Sip)), 32.60 (Si CH2N), 16.15 (SiCH2Ca), 2 2.41
(Si(CH3)2), 2 2.48 (Si(CH3)2).
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23
ꢀ
SCHEME 2 Synthesis of proline NCA according to Gulın et al.
SCHEME 3 Synthesis of (D)Sip-NCA.
Spectrometer. The instrument was operated with a data pitch
of 0.05 nm, scan speed of 100 nm/min, and response time
of 4 s. For thermal denaturation studies, we used the inter-
val spectra measurementꢀ mode of this CD instrument. The
as it occurs with other amino acids, and the addition of HCl
scavenger is required, by addition of silver oxide19,20 or or-
ganic bases.21,22 Recently, an efficient procedure using poly-
mer-supported bases has been reported.23 Using this method
no side-product formation like diketopiperazine was observed,
and (L)Pro-NCA was obtained in good yields with the assis-
tance of polymer-supported diethylamine to trigger the cycli-
zation (Scheme 2). The easy elimination of the supported
base hydrochloride led to good yields and purity. Recently,
pure (L)Pro-NCA has been obtained from Boc-protected pro-
line. However the purification method, including NCA sensi-
tive water extraction, led to rather low yields.24
ꢀ
interval ramp rate was 1 C/min starting from 0 to 70 C.
X-Ray Crystallography
CCDC 866402 contains the supplementary crystallographic
data for this paper.
These data can be obtained free of charge at www.ccdc.cam.
ac.uk/conts/retrieving.html [or from the Cambridge Crystallo-
graphic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK;
fax: 1 44 1223 336033; or e-mail: deposit@ccdc.cam.ac.uk].
We chose to reproduce the synthesis using a supported terti-
ary amine and we obtained the (L)Pro-NCA in 78% yield.
With this synthesis in hands, we attempted to prepare the
NCA of L-Sip and D-Sip.
Electron Microscopy Imaging
Analyses were performed at the ICG analytical platform located
at the University Montpellier 2. Scanning electron microscopy
(SEM) images were obtained using a Hitachi S2600N. Trans-
mission electron microscopy (TEM) images were obtained
using a Jeol 1200EX2 with operating parameters of 100 kV.
Polymers were observed after a color negative preparation.
The deprotected (D)Sip starting material was obtained from
acid treatment of enantiopure Boc-(D)Sip-Ot-Bu.11 The N-car-
bamoyl intermediate synthesis was very easily monitored
since the reaction mixture became totally transparent when
all the starting amino acid was reacted. In the case of Sip,
we observed the presence of NCA in higher proportion
(50%) than for proline (33%), the spontaneous cyclization
being facilitated by the less constrained five-membered
ring7,8,25 (Scheme 3, Fig. 1).
RESULTS AND DISCUSSION
Monomer Synthesis
In the case of proline, the N-carbamoyl intermediate obtained
by treatment with phosgene does not cyclize spontaneously
FIGURE 1 1H NMR after triphosgene addition to (a) proline, (b) Sip, showing the proportion of N-carbamoyl chloride and NCA.
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FIGURE 2 1H NMR in CDCl3 and X-ray structure of (D)Sip-NCA.
However, various stoichiometry, reaction time, or temperature
did not allow us to optimize the cyclization, and only the
addition of polystyrene-supported diethylamine afforded the
(D)Sip-NCA in 72% yield and high purity. The (D)Sip-NCA was
fully characterized, and crystallized [X-ray structure (Fig. 2)].
from water initiation prompted us to use H-(L)Pro-OBzl as
initiator to take advantage of the benzyl group as a clear
NMR signal (Scheme 5).
In addition, the initiation reaction by H-(L)Pro-OBzl on
L-Proline, L-Sip, or D-Sip NCA, led to the NCA opening and
gave a new N-terminal “proline-like” amine, which acted as a
new initiator. The propagation of polymerization yielded
homopolypeptides with proline benzyl ester termination.
Homopolymerization
Homopolypeptides were achieved by ROP of NCAs.3 Several
species can initiate the NCA polymerization and afford poly-
peptides with a molecular weight essentially determined by
the NCA to initiator ratios. Two mechanisms are described
for this reaction, normal amine mechanism26 (NAM) and
activated monomer mechanism (AMM),3 depending on the
relative basicity and nucleophilicity of the initiator. When the
initiator is significantly basic, deprotonation of the NCA
occurs, and the resulting nucleophile initiates ring opening
via the AMM. The AMM requires the presence of a proton on
the nitrogen. When the initiator is more nucleophilic than
basic, it attacks the C-5 (C@O) position of the NCA, resulting
in the ROP via the NAM (Scheme 4). Decomposition of the
carbamic acid with liberation of CO2 releases a newly formed
amine that propagates the polymerization. In the particular
case of (L)Pro-NCA, and imino acids NCA in general, the
AMM cannot occur since no labile proton is available and
NAM is the only possible mechanism.
In this case, the 1H NMR allowed us to evaluate the DPn by rel-
ative integration of the aromatic protons of the C-terminal
benzyl ester and the protons borne by all Ca. Heteronuclear
single quantum coherence spectroscopy (HSQC) experiments
allowed us to determine the CHa range accurately. However
this range includes also the methylene of the benzyl group,
therefore two protons have to be excluded from the total (Fig.
3). As shown in Table 1 the DPn evaluated by NMR according
to this method was consistent with the theoretical DPn result-
ing from the stoichiometry of the reaction in all cases.
MALDI analyses, which are widely used for characterization of
homopolypeptides,28,29 displayed distributions of homopoly-
mers (see Supporting Information). Polymerization is attested
by the sequence of signals distant of the weight of each resi-
due, both for Pro and Sip. In all cases, as focused on P3 mass
spectra (Fig. 4), the main distribution was assigned to the
expected polymer with sodium cationization (represented by
square). Other major distributions (respectively represented
by a triangle and a circle) corresponded to the protonated and
potassium cationizated patterns of the same polymer.
Firstly, NCAs were subjected to polymerization reaction with
known quantities of water as initiator in anhydrous THF at
room temperature. NMR spectroscopy is often employed for
checking the polymer purity and for the determination of
the degree of polymerization (DPn) by integration of signals
assigned to monomer and initiator respectively.27 The diffi-
culty of verifying the DPn by NMR with a carboxylic acid
end-group added to the poor purity of polymers resulting
Besides these three distributions of the desired polymer, we
could sometimes observe low intensity signals corresponding
to secondary products like cyclic peptides and carboxylic
SCHEME 4 NAM mechanism for ring opening polymerization of NCA.
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SCHEME 5 Polymerization of (D)Sip-NCA with H-(L)Pro-OBzl as initiator.
FIGURE 3 Determination of DPn of polymer P7 by NMR as an example.
C-termination. However, the molecular weight determined by
MALDI-Tof MS was lower than the calculated molecular
weight by NMR, probably due to the sample preparation on
the matrix. We also suspected a limitation of MALDI-Tof due
to molecular discrimination during ionization and the abun-
dances might not be in agreement with the real amount of
each peptide present in the sample. To illustrate the impor-
tance of analysis conditions, we compared MALDI Tof spectra
on two different matrices. The spectra in DHB displayed
higher molecular weight than HCCA for the same sample,
thus proving that longer polymers are not desorbed in the
later case. Such difference proved that MALDI Tof does not
permit reliable distribution and DPn determinations.
alcohols, with characteristic CD signals that include a strong
negative band at 202–206 nm and a weak positive band at
225–229 nm. As mentioned earlier, the high lipophilic
TABLE 1 Synthesis of Homopolypeptides of Different Length
Polymer
Monomer
Initiator
Monomer/Initiator DPna
–
P1
P2
P3
P4
P5
P6
P7
(L)Pro-NCA H2O
(L)Pro-NCA (L)ProOBzl 21
(D)Sip-NCA H2O
24
–
(D)Sip-NCA (L)ProOBzl
5
5
(D)Sip-NCA (L)ProOBzl 20
(D)Sip-NCA (L)ProOBzl 50
(L)Sip-NCA (L)ProOBzl 10
14
44
11
As a preliminary structural study of polySip in solution, we
report here the CD results. Polyproline oligomers are known
to adopt a type II helical conformation (PPII) in polar sol-
vents such as water, trifluoroethanol, and other fluorinated
a
Determined by NMR integration.
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FIGURE 4 MALDI spectrum of polySip P3.
character of homopolysilaproline prohibited the use of water
as solvent. We investigated other polar solvents to solubilize
polysilaproline oligomers inducing PPII structure. The best
solvents were HFIP (hexafluoroisopropanol) and TFE, this
one being the most used for thermal studies. We recorded
CD spectra of all polymers in TFE at a concentration of 0.1
mg/mL. All spectra are similar, despite the inversion due to
the difference of absolute configuration of monomers (L)Sip
NCA and (D)SipNCA (see supporting information). Spectrum
of polymer P7 showed the typical CD curve of PPII helix
with a strong negative band (minimum) at 207 nm and a
weak positive band (maximum) at 228 nm.
the same minimum at 207 nm. However the positive band
was not affected. In comparison to the polyproline counter-
part [Fig. 5(a)], the PPII helix is clearly thermally more sta-
ble [Fig. 5(b)].
Finally, SEM and TEM pictures displayed surface and struc-
ture of polymer P6 in the solid state (see Supporting
Information).
CONCLUSIONS
NCA derivatives of proline and silaproline were successfully
synthesized by means of polystyrene supported tertiary
amine, allowing direct polymerization into homopeptides.
The Sip-NCA has been obtained for the first time and was
fully characterized. Polymerization of the lipophilic Sip
resulted in non water-soluble peptides, which did not
desorb properly for MALDI analysis. However, CD of homo-
polysilaproline compared to homopolyproline confirmed a
stable PPII structure.
After these first results that clearly proved that the homopo-
lysilaproline adopted a PPII conformation, we studied the
thermal denaturation of this PPII structure. The polymers
ꢀ
dissolved in TFE were heated from 0 to 70 C using a sealed
cell, all spectra were recorded and displayed in Figure 5. We
could observe a slight diminution of the negative band with
FIGURE 5 CD spectra of thermal denaturation of (a) polyproline P2 and (b) polysilaproline P7 in TFE.
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ACKNOWLEDGMENTS
13 T. Aliferis, H. Iatrou, N. Hadjichristidis, Biomacromolecules
2004, 5, 1653–1656.
The authors thank the “Ministe`re de l’Education Nationale” for
MENRT grant of Charlotte Martin. Authors are grateful to
Michel Giorgy for X-ray structure of (D)Sip-NCA and Mathieu
14 J. R. Kramer, T. J. Deming, Biomacromolecules 2010, 11,
3668–3672.
15 T. J. Deming, J. Polym. Sci. Part A: Polym. Chem. 2000, 38,
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Dupre for MALDI spectra.
ꢀ
16 W. H. Daly, D. Poche, Tetrahedron Lett. 1988, 29, 5859–5862.
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