Synthesis of homopolypeptides with PPII structure

Synthesis of Homopolypeptides with PPII Structure

Charlotte Martin,1 Aur�elien Lebrun,2 Jean Martinez,1 Florine Cavelier1

1IBMM, UMR-CNRS-5247, Universit�es Montpellier I and II, Place Eugene Bataillon, 34095 Montpellier, France2LMP, IBMM, Universit�e Montpellier II, Place Eugene Bataillon, 34095 Montpellier, France

Correspondence to: F. Cavelier (E-mail: [email protected])

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

structure, polyproline, and polysilaproline polymers. VC 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

INTRODUCTION Polypeptides syntheses are widely investi-gated, attempting to control the length and the architectureof the resulting polymers, especially for homopolypeptides,constituted by the same residue all along the polymericchain. Since the late 1940s,1 polymerization of N-carboxyan-hydride (NCA) has been extensively used for the synthesis ofhigh-molecular weight polypeptides and this old method hasbeen reviewed recently.2,3 However, for proline-rich peptidesor polyprolines, one of the most interesting homopolypepti-des first discovered in collagen,4 and attractive for their PPIIinducing structure, the synthesis was usually performed onsolid support following a step-by-step Fmoc-based strategy.This was the case when the PPII helix was used as backbonefor cell penetrating peptides (CPP) for example.5 To modu-late physicochemical properties of the resulting helix, whichis 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 similarconformational properties.7 The presence of the dimethylsilylgroup confers to Sip a higher lipophilicity as well asan improved resistance to biodegradation.8 Sip was synthe-sized successfully via asymmetric synthesis by Schollk€opfmethod,9,10 but with this approach the scale up was difficult.More recently, a racemic synthesis was carried out for thegram scale preparation of enantiomerically pure Sip, requir-ing resolution of the enantiomers by chiral high performanceliquid chromatography (HPLC).11

In this context, we thought that it could be interesting todemonstrate the possibility to synthesize both polyprolineand polysilaproline by polymerization of correspondingamino acid NCA.

To synthesize homopolypeptides, the most extensively usedmethod is based on polymerization of NCA, first reported byLeuchs in the early nineteenth.12 The purity of NCAs has beenproved to be very important and it is necessary to obtain NCAin 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 acidcauses some constrains and conformational restrictions.These features result in failure to obtain NCA with usualmethods, either by treatment with halogenating reagents(Leuchs method12) [Scheme 1(a)], or with phosgene or phos-gene substitutes like triphosgene16 (Fuchs–Farthingsmethod17,18) [Scheme 1(b)].

EXPERIMENTAL

Materials and ReagentsHydrogene 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.

VC 2013 Wiley Periodicals, Inc.

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polystyrene 200–4000mesh crosslinked with 2% divinylbenzenewith a loading 3.2 mmol/g (Aldrich), and L-proline benzyl esterhydrochloride 98% (Aldrich) were used as received. All reactionsinvolving air-sensitive reagents were performed under argon. Sol-vents and reagentswere purchased fromAldrich and Fluka. Tetra-hydrofurane (THF) was freshly distilled from benzophenone/sodiumprior to use. Ethyl acetate and hexanewere anhydrous.

SynthesisSynthesis of HCl, H-(D)Sip-OHBoc-(D)Sip-Ot-Bu (1 g, 3.17 mmol) was dissolved with HCl 4 N indioxane (10 mL). The reaction was stirred for 1 h at room tem-perature. The residue obtained after evaporation was dissolvedin a mixture of acetonitrile and water and lyophilized affordingHCl, H-(D)Sip-OH as a white solid in quantitative yield.

1H NMR (300 MHz, D2O) d 4.19 (dd, J 5 11.4, 7.3, 1H,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.

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).

HCl, H-(D)Sip-OH: a20D 5 1 14 (c 5 1, HCl 1 N).

Synthesis of Sip-NCASip (63 mg, 0.32 mmol) was suspended with magnetic stir-ring in dry THF freshly distilled (5 mL) in a round bottomflask (with CaCl2 tube) and under dry argon gas. Once thissuspension reached 50 �C, triphosgene (83 mg, 0.28 mmol)was added in one portion, and reaction mixture was stirredfor 1 h. The final solution was evaporated in vacuo and thenthe oily residue was dissolved in dry THF (5 mL) and addedinto the diethylamine polystyrene (3 equiv.), which had beenswollen previously in dry THF in a solid phase reactor witha filter disk and kept under argon atmosphere for 3 h atroom temperature under mechanical stirring. Sip-NCA wasobtained by filtration and washing with additional dry THF.The crude Sip-NCA was recrystallized from AcOEt/hexane at2 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).

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).

Synthesis of PolypeptidesPolypeptides were prepared by ring-opening polymerization(ROP) of the NCA of L-proline, L-Sip, or D-Sip in the presenceof initiator such as water or L-proline benzyl ester. NCA wasdissolved in dry THF under argon, and then the initiator wasadded (after neutralization of the hydrochloride with trie-thylamine in ether in the case of HCl, H-(L)Pro-Bzl ester).The reaction was stirred for 24 h at room temperature. Thereaction mixture was concentrated in vacuo and the polymerwas obtained as a white powder.

NMR SpectroscopyProton nuclear magnetic resonance (1H NMR) and carbonnuclear magnetic resonance (13C NMR) spectra wererecorded on a Bruker spectrometer advance 300 at 300 and75 MHz respectively for H-Sip-OH in D2O and for NCA inCDCl3 at room temperature. The spectra of the polymerswere performed in CF3COOD on a Bruker spectrometeradvance 600 at 600 and 150 MHz, respectively.

Optical RotationOptical rotation values were measured on a Perkin–Elmer341 (20 �C, sodium ray), with a concentration of 1 g/100mL in HCl 1 N.

Mass SpectroscopyAnalyses were conducted on the IBMM analytical platformlocated in the Laboratoire de Mesures Physiques of UniversityMontpellier 2. Matrix assisted laser desorption/ionization-time of light (MALDI-Tof) mass spectra were recorded on anUltraflex III Tof/Tof instrument (Bruker Daltonics, Wissem-bourg, France) equipped with a pulsed Nd:YAG laser at awavelength 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 inwater/acetonitrile (vol/vol, 50/50)], was mixed with the sam-ple in equal amount and 0.5 lL of this solution was depositedonto the MALDI target according to the dried droplet proce-dure. After evaporation of the solvent, the MALDI target wasintroduced into the mass spectrometer ion source. Ions weredetected over a mass range from m/z 500 to 4000.

Circular Dichroism SpectroscopySamples for circular dichroism (CD) were prepared at a con-centration of 0.1 mg/mL in trifluoroethanol (TFE). At first abackground spectrum was taken with 100% TFE. Then thesolution was pipetted into a quartz cell with a 0.1-mm pathlength. Spectra were measured using a Jasco J-815 CD

SCHEME 1 Preparation of NCAs using (a) halogenating reagents and (b) phosgene.

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3104 JOURNAL OF POLYMER SCIENCE, PART A: POLYMER CHEMISTRY 2013, 51, 3103–3109

Spectrometer. The instrument was operated with a data pitchof 0.05 nm, scan speed of 100 nm/min, and response timeof 4 s. For thermal denaturation studies, we used the inter-val spectra measurement mode of this CD instrument. Theinterval ramp rate was 1 �C/min starting from 0 to 70 �C.

X-Ray CrystallographyCCDC 866402 contains the supplementary crystallographicdata 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: [email protected]].

Electron Microscopy ImagingAnalyses were performed at the ICG analytical platform locatedat the University Montpellier 2. Scanning electron microscopy(SEM) images were obtained using a Hitachi S2600N. Trans-mission electron microscopy (TEM) images were obtainedusing a Jeol 1200EX2 with operating parameters of 100 kV.Polymers were observed after a color negative preparation.

RESULTS AND DISCUSSION

Monomer SynthesisIn the case of proline, the N-carbamoyl intermediate obtainedby treatment with phosgene does not cyclize spontaneously

as it occurs with other amino acids, and the addition of HClscavenger 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 methodno 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 supportedbase 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

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 theNCA of L-Sip and D-Sip.

The deprotected (D)Sip starting material was obtained fromacid treatment of enantiopure Boc-(D)Sip-Ot-Bu.11 The N-car-bamoyl intermediate synthesis was very easily monitoredsince the reaction mixture became totally transparent whenall 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 cyclizationbeing facilitated by the less constrained five-memberedring7,8,25 (Scheme 3, Fig. 1).

SCHEME 2 Synthesis of proline NCA according to Gul�ın et al.23

SCHEME 3 Synthesis of (D)Sip-NCA.

FIGURE 1 1H NMR after triphosgene addition to (a) proline, (b) Sip, showing the proportion of N-carbamoyl chloride and NCA.



www.ccdc.cam.ac.uk/conts/retrieving.html

www.ccdc.cam.ac.uk/conts/retrieving.html

deposit&hx0040;ccdc.cam.ac.uk

deposit&hx0040;ccdc.cam.ac.uk

However, various stoichiometry, reaction time, or temperaturedid not allow us to optimize the cyclization, and only theaddition of polystyrene-supported diethylamine afforded the(D)Sip-NCA in 72% yield and high purity. The (D)Sip-NCA wasfully characterized, and crystallized [X-ray structure (Fig. 2)].

HomopolymerizationHomopolypeptides were achieved by ROP of NCAs.3 Severalspecies can initiate the NCA polymerization and afford poly-peptides with a molecular weight essentially determined bythe NCA to initiator ratios. Two mechanisms are describedfor this reaction, normal amine mechanism26 (NAM) andactivated monomer mechanism (AMM),3 depending on therelative basicity and nucleophilicity of the initiator. When theinitiator is significantly basic, deprotonation of the NCAoccurs, and the resulting nucleophile initiates ring openingvia the AMM. The AMM requires the presence of a proton onthe nitrogen. When the initiator is more nucleophilic thanbasic, it attacks the C-5 (C@O) position of the NCA, resultingin the ROP via the NAM (Scheme 4). Decomposition of thecarbamic acid with liberation of CO2 releases a newly formedamine that propagates the polymerization. In the particularcase of (L)Pro-NCA, and imino acids NCA in general, theAMM cannot occur since no labile proton is available andNAM is the only possible mechanism.

Firstly, NCAs were subjected to polymerization reaction withknown quantities of water as initiator in anhydrous THF atroom temperature. NMR spectroscopy is often employed forchecking the polymer purity and for the determination ofthe degree of polymerization (DPn) by integration of signalsassigned to monomer and initiator respectively.27 The diffi-culty of verifying the DPn by NMR with a carboxylic acidend-group added to the poor purity of polymers resulting

from water initiation prompted us to use H-(L)Pro-OBzl asinitiator to take advantage of the benzyl group as a clearNMR signal (Scheme 5).

In addition, the initiation reaction by H-(L)Pro-OBzl onL-Proline, L-Sip, or D-Sip NCA, led to the NCA opening andgave a new N-terminal “proline-like” amine, which acted as anew initiator. The propagation of polymerization yieldedhomopolypeptides with proline benzyl ester termination.

In this case, the 1H NMR allowed us to evaluate the DPn by rel-ative integration of the aromatic protons of the C-terminalbenzyl ester and the protons borne by all Ca. Heteronuclearsingle quantum coherence spectroscopy (HSQC) experimentsallowed us to determine the CHa range accurately. Howeverthis 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 accordingto 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 ofhomopolypeptides,28,29 displayed distributions of homopoly-mers (see Supporting Information). Polymerization is attestedby the sequence of signals distant of the weight of each resi-due, both for Pro and Sip. In all cases, as focused on P3 massspectra (Fig. 4), the main distribution was assigned to theexpected polymer with sodium cationization (represented bysquare). Other major distributions (respectively representedby a triangle and a circle) corresponded to the protonated andpotassium cationizated patterns of the same polymer.

Besides these three distributions of the desired polymer, wecould sometimes observe low intensity signals correspondingto secondary products like cyclic peptides and carboxylic

FIGURE 2 1H NMR in CDCl3 and X-ray structure of (D)Sip-NCA.

SCHEME 4 NAM mechanism for ring opening polymerization of NCA.


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C-termination. However, the molecular weight determined byMALDI-Tof MS was lower than the calculated molecularweight by NMR, probably due to the sample preparation onthe matrix. We also suspected a limitation of MALDI-Tof dueto molecular discrimination during ionization and the abun-dances might not be in agreement with the real amount ofeach peptide present in the sample. To illustrate the impor-tance of analysis conditions, we compared MALDI Tof spectraon two different matrices. The spectra in DHB displayedhigher molecular weight than HCCA for the same sample,thus proving that longer polymers are not desorbed in thelater case. Such difference proved that MALDI Tof does notpermit reliable distribution and DPn determinations.

As a preliminary structural study of polySip in solution, wereport here the CD results. Polyproline oligomers are knownto adopt a type II helical conformation (PPII) in polar sol-vents such as water, trifluoroethanol, and other fluorinated

alcohols, with characteristic CD signals that include a strongnegative band at 202–206 nm and a weak positive band at225–229 nm. As mentioned earlier, the high lipophilic

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.

TABLE 1 Synthesis of Homopolypeptides of Different Length

Polymer Monomer Initiator Monomer/Initiator DPna

P1 (L)Pro-NCA H2O –

P2 (L)Pro-NCA (L)ProOBzl 21 24

P3 (D)Sip-NCA H2O –

P4 (D)Sip-NCA (L)ProOBzl 5 5



P7 (L)Sip-NCA (L)ProOBzl 10 11

a Determined by NMR integration.



character of homopolysilaproline prohibited the use of wateras solvent. We investigated other polar solvents to solubilizepolysilaproline oligomers inducing PPII structure. The bestsolvents were HFIP (hexafluoroisopropanol) and TFE, thisone being the most used for thermal studies. We recordedCD spectra of all polymers in TFE at a concentration of 0.1mg/mL. All spectra are similar, despite the inversion due tothe difference of absolute configuration of monomers (L)SipNCA and (D)SipNCA (see supporting information). Spectrumof polymer P7 showed the typical CD curve of PPII helixwith a strong negative band (minimum) at 207 nm and aweak positive band (maximum) at 228 nm.

After these first results that clearly proved that the homopo-lysilaproline adopted a PPII conformation, we studied thethermal denaturation of this PPII structure. The polymersdissolved in TFE were heated from 0 to 70 �C using a sealedcell, all spectra were recorded and displayed in Figure 5. Wecould observe a slight diminution of the negative band with

the same minimum at 207 nm. However the positive bandwas 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 SupportingInformation).

CONCLUSIONS

NCA derivatives of proline and silaproline were successfullysynthesized by means of polystyrene supported tertiaryamine, allowing direct polymerization into homopeptides.The Sip-NCA has been obtained for the first time and wasfully characterized. Polymerization of the lipophilic Sipresulted in non water-soluble peptides, which did notdesorb properly for MALDI analysis. However, CD of homo-polysilaproline compared to homopolyproline confirmed astable PPII structure.

FIGURE 4 MALDI spectrum of polySip P3.

FIGURE 5 CD spectra of thermal denaturation of (a) polyproline P2 and (b) polysilaproline P7 in TFE.


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ACKNOWLEDGMENTS

The authors thank the “Ministere de l’Education Nationale” forMENRT grant of Charlotte Martin. Authors are grateful toMichel Giorgy for X-ray structure of (D)Sip-NCA and MathieuDupr�e for MALDI spectra.

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Synthesis of homopolypeptides with PPII structure