DOI: 10.1002/chem.201102223

Metallopolymer–Peptide Hybrid Materials: Synthesis and Self-Assembly ofFunctional, Polyferrocenylsilane–Tetrapeptide Conjugates

Siree Tangbunsuk,[a] George R. Whittell,[a] Maxim G. Ryadnov,[a, b]

Guido W. M. Vandermeulen,[c] Derek N. Woolfson,*[a, d] and Ian Manners*[a]

Introduction

The combination of peptide and synthetic polymers in bio-conjugates has afforded hybrid materials that benefit fromthe presence of each component.[1–6] For instance, the secon-dary structure of the former may pattern the latter into anotherwise inaccessible form, while the synthetic segmentserves to stabilise the hierarchical structure. Initial researchin this field utilised homopolypeptide sequences that wereknown to afford a-helices.[7,8] Advances in the syntheses ofsynthetic polymer–peptide conjugates,[9–12] however, have en-abled other helical structures and related coiled-coil motifs

to be realised.[13–16] Nonetheless, the majority of work hasbeen focused on the incorporation of b-sheet-forming aminoacid sequences,[17,18] as these are key components of manystructurally interesting natural products, such as amyloidalfibres, natural silks and elastin.

It was a desire to understand the mechanism for the ag-gregation of the amyloid precursor protein that led Lynnand co-workers to couple an oligopeptide with a polyethy-leneglycol (PEG).[19–21] They found that addition of PEG tothe C terminus allowed for the lamination of b-sheets thatwas common to the native protein, but inhibited further ag-gregation to afford soluble fibrils. By replacing the amor-phous segments of the proteins comprising Bombyx moriand Nephila clavipes silks with PEG, Sogah and co-workershave obtained materials that retained the anti-parallel b-sheet structure characteristic of the natural product, alongwith the associated desirable mechanical properties.[22–24]

Shao and co-workers have also prepared silk-inspired co-polymers by conjugating A5 (A=alanine) with isopreneoligomers.[25] The general ability of PEG to prevent the lat-eral aggregation of b-sheet-forming sequences has beendemonstrated by conjugation with another peptide, namely,Ac-QQKFQFQFEQQ-NH2 (Ac= acetyl, Q=glutamine,K= lysine, F =phenylalanine, E=glutamic acid) for whichthe conjugate formed soluble fibrils, whereas the parentpeptide afforded a hydrogel in solution.[26] Utilising proteinengineering and well-defined PEG oligomers, the group ofvan Hest prepared the monodisperse conjugates, PEG-[(AG)3EG]n-PEG (n=10 and 20; G =glycine), which alsocontained the silk-inspired alanylglycine repeating unit.[27]

Complementing the solid-state studies performed by Sogah

Abstract: Conjugates of poly(ferroce-nyldimethylsilane) (PFDMS) with Ac-(GA)2-OH, Ac-A4-OH, Ac-G4-OH andAc-V4-OH have been prepared by re-action of the tetrapeptide units withthe amino-terminated metallopolymer.The number average degree of poly-merisation (DPn) of the PFDMS wasapproximately 20 and comparable ma-terials with shorter (DPn�10) and/oramorphous chains have been preparedby the same procedure. Poly(ferroceny-lethylmethylsilane) (PFEMS) was em-

ployed for the latter purpose. All con-jugates were characterised by GPC,MALDI-TOF MS, NMR and IR spec-troscopy. With the exception of Ac-V4-PFDMS20, all materials exhibited someanti-parallel b-sheet structure in thesolid state. The self-assembly of theconjugates was studied in toluene by

DLS. The vast majority of the materi-als, irrespective of peptide sequence orchain crystallinity, afforded fibres con-sisting of a peptidic core surrounded bya PFS corona. These fibres were foundin the form of cross-linked networks byTEM and AFM. The accessibility ofthe chemically reducing PFS coronahas been demonstrated by the localisedformation of silver nanoparticles onthe surface of the fibres.

Keywords: nanoparticles · peptides ·polymers · self-assembly ·supramolecular chemistry

[a] Dr. S. Tangbunsuk, Dr. G. R. Whittell, Dr. M. G. Ryadnov,Prof. D. N. Woolfson, Prof. I. MannersSchool of Chemistry, University of BristolCantock�s Close, Bristol BS8 1TS (UK)Fax: (+44) 117-925-1295Fax: (+44) 117-929-0509E-mail : D.N.Woolfson@bristol.ac.uk

ian.manners@bristol.ac.uk

[b] Dr. M. G. RyadnovNational Physical Laboratory, Hampton RoadTeddington, Middlesex, TW11 0LW (UK)

[c] Dr. G. W. M. VandermeulenBASF, Carl-Bosch-Strasse 3867056 Ludwigshafen (Germany)

[d] Prof. D. N. WoolfsonDepartment of Biochemistry, University of BristolUniversity Walk, Bristol BS8 1TD (UK)

Supporting information for this article is available on the WWWunder http://dx.doi.org/10.1002/chem.201102223.

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and co-workers, the authors found that these materials self-assemble from solution to afford well-defined fibrils, thewidths of which are related to the length of the b-sheet-forming block. The same group also prepared a conjugatecontaining the oligopeptide sequence VPGVG (V=valine,P= proline), common to tropoelastin, located in the side-chain structure.[9] Although combined with a polymethacry-late instead of PEG, this material still displayed the lowercritical solution temperature (LCST) common to the pep-tide. Castelletto and Hamley have also obtained fibrillarstructures based on b-sheets by using F4 as the aggregatordomain.[28] This work has been contrasted with V4-contain-ing conjugates and the effect of PEG molecular weight hasalso been investigated.[29] Recently, Tam and co-workershave prepared conjugates of poly(acrylic acid) with poly ACHTUNGTRENNUNG(l-valine) and demonstrated that the b-sheet structure alsodominates in solution for these materials.[30–32]

In addition to having linear and side-chain conjugates,structurally intermediate materials have been realised thatcontain reverse-turn mimics. Sogah and co-workers em-ployed a phenoxathiin derivative in this role and demon-strated the ability of this rigid spacer to template intramo-lecular hydrogen bonding.[22,23] More recently, Bçrner andco-workers have employed a carbazole unit to pre-organisetwo (TV)2 (T= threonine) units for intermolecular interac-tions.[33] It is noteworthy that this material exhibited a supe-rior tendency to aggregate than the un-templated conjugatecontaining (TV)4. In further work with aggregator domainsbased on (TV)2, the same group have devised functional sys-tems in which the self-assembly of the conjugate can effec-tively be “switched-on” by chemical changes to a peptidicprecursor.[34,35]

The use of PEG as the synthetic component of the conju-gate has enabled the self-assembly of these materials to bestudied in aqueous solution. Utilising poly(n-butylacrylate),however, Bçrner and co-workers attempted similar process-es in organic media.[36] The use of non-polar solvents elimi-nates any contribution to self-assembly from the hydropho-bic effect, allowing hydrogen bonding alone to dictate themorphology. These studies afforded wound-tape structurescontaining anti-parallel b-sheet cores. Since this report,other groups have also achieved the self-assembly of pep-tide–polymer conjugates in organic solvents by utilising hy-drophobic chains.[25,37–41] The use of functional polymers forthe stabilisation of b-sheet structures, however, has beenlimited. Frauenrath and co-workers employed an elegant ap-proach in which diacetylenes were incorporated into theconjugate, which could be converted into functional polyace-tylenes post self-assembly.[37,42, 43] Preformed, semiconductingoligothiophenes have also been self-assembled as part of apeptide conjugate.[44] Herein, as an extension to our previouscommunication,[38a] we report the synthesis of a range of tet-rapeptide conjugates with polyferrocenylsilanes (PFSs), aclass of functional metallopolymer,[38b] constituting the func-tional synthetic component. The self-assembly of these ma-terials in organic media was investigated and the effect ofthis process on the functionality of the PFS was evaluated.

Results and Discussion

To investigate the effect of amino acid sequence, as well assynthetic-polymer molecular weight and crystallinity on theobserved solution-phase self-assembly of the conjugates, weembarked on the synthesis of ten materials. Inspired by thecrystalline region of B. mori silk, we decided to couple Ac-(GA)2-OH to poly(ferrocenyldimethylsilane) (PFDMS). Inall cases the tetrapeptides were acylated at the N terminus(denoted by Ac-), and were coupled to the polymer at the Cterminus, which existed as the free acid (denoted by -OH).The role of the alanylglycine repeating units in self-assemblywas then judged by comparison with analogous materialscontaining Ac-G4-OH and Ac-A4-OH. Finally, Ac-V4-OHwas studied because of the strong propensity for this aminoacid to form b-structures in natural proteins.[45] PFDMS is asemicrystalline polymer and thus, to probe the effect of crys-tallinity on the self-assembly process, we also prepared ananalogue containing the amorphous poly(ferrocenylethylme-thylsilane) (PFEMS). The synthetic polymer is consideredto play a key role in preventing the lateral aggregation of b-sheets and we have, therefore, also chosen to vary thenumber of repeat units constituting this block (degree ofpolymerisation, DPn�10 and 20). Finally, we have examinedthe effect of the specific amino acid sequences upon self-as-sembly. In non-polar solvents, which are thermodynamicallygood for the PFS block, hydrogen bonding is expected to bethe dominant force governing self-assembly. Although thenumber of H-bond donors and acceptors in all native tetra-peptides will be the same, the hydropathy of the sidechain[46] relative to PFS may be expected to play a role indetermining the morphology.

Synthesis and characterisation of Ac-X4-PFS conjugates :PFDMS and PFEMS were both prepared by living anionicring-opening polymerisation of the appropriately substitutedsila[1]ferrocenophane by using n-butyllithium as the intiator(Scheme 1). The reactions were quenched with 1-(3-bromo-propyl)-2,2,5,5-tetramethyl-1-aza-2,5-disilacyclopentane and,after purification, amino-terminated PFSs were obtained aslight orange solids.[41]

All four materials were characterised by 1H NMR spec-troscopy, gel permeation chromatography (GPC) andmatrix-assisted laser desorption/ionisation time-of-flightmass spectrometry (MALDI-TOF MS). The results of thesevarious analyses (Table 1) were in close agreement with thetarget DPn values of 10 and 20.

The tetrapeptide sequences were prepared by solid-phasepeptide synthesis utilising standard 9-fluorenylmethoxycar-bonyl (Fmoc) chemistry.[47] These were characterised by1H NMR spectroscopy and electrospray ionisation massspectrometry (ESI MS) before being coupled in ten-foldexcess with the amino-terminated PFSs by using PyBOPand DIPEA (Scheme 1). The Ac-X4-PFS conjugates wereultimately obtained as orange powders in high yield (e.g.,72 % for Ac-A4-PFDMS20).

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The conjugates were soluble in good solvents for PFS,such as toluene, tetrahydrofuran (THF) and chlorocarbons,which allowed for solution-phase characterisation by GPCand 1H NMR spectroscopy (Table 2). The chromatogramsfrom the former were monomodal but indicated number-

averaged molecular weights that were consistently lowerthan those determined by MALDI-TOF MS. The 1H NMRspectra for the proposed conjugates exhibited resonances as-signable to the tetrapeptides, and thus afforded evidencethat the coupling reaction was indeed successful. Definitivesupport for conjugate formation was provided by MALDI-TOF MS, which exhibited peaks that coincided with thevalues calculated from the molecular formulae (Table 2). Itis noteworthy that no signals corresponding to the amino-terminated polymer could be detected by this technique.

The MALDI-TOF MS spectra of two representative Ac-X4-PFS conjugates are displayed in Figure 1. An inspectionof the peak-to-peak mass differences reveals a value of242 g mol�1 for Ac-A4-PFDMS9 and 256 g mol�1 for Ac-V4-PFEMS9. These masses are consistent with the compositionof the repeat units of the respective PFS chains.

Physical properties of the Ac-X4-PFS conjugates : The ther-mal properties of the PFS–tetrapeptide conjugates were in-vestigated by thermal gravimetric analysis (TGA) and dif-ferential scanning calorimetry (DSC). By way of compari-son, the amino-terminated precursor polymers were alsostudied and these results are presented, along with those ofrepresentative conjugates, in Table 3.

The amino-terminated polymers (PFDMS20-NH2 andPFEMS18-NH2) started to decompose under a nitrogen at-mosphere at approximately 429 and 403 8C, respectively. De-composition was essentially complete by 600 8C, affordingan involatile material with a yield of approximately 36 %.

Scheme 1. Synthesis of Ac-X4-PFS conjugates (n�10 and 20; R1 =R2 =

Me and R1 = Me, R2 = Et; X =unspecified amino acid; PyBOP = (benzo-triazole-1-yloxy)trispyrrolidinophosphonium hexafluorophosphate;DIPEA=N,N-diisopropylethylamine; Ac =MeCO- and HO- denotes thefree acid at the C terminus of the peptide).

Table 1. Molecular weight data for amino-terminated PFS homopoly-mers.

Mn [gmol�1][a] Mw/Mn[a] m/z [gmol�1][b] DPn

[c]

PFDMS9-NH2 2305 1.14 2295 9PFDMS20-NH2 5380 1.12 4956 20PFEMS9-NH2 2575 1.15 2421 9PFEMS18-NH2 5270 1.13 4724 18

[a] Determined by GPC. [b] Determined by positive ion MALDI-TOF MS with dithranol as the matrix. The value is quoted for the ion of100 % relative intensity. [c] Calculated from MALDI-TOF MS data.

Figure 1. Positive ion MALDI-TOF mass spectra of a) Ac-A4-PFDMS9

and b) Ac-V4-PFEMS9. Dithranol was used as the matrix.Table 2. Molecular weight data for Ac-X4-PFS conjugates.

Mn [gmol�1][a] Mw/Mn[a] m/z [g mol�1][b]

found calcd

Ac-(GA)2-PFDMS20 6490 1.22 5257 5257Ac-(GA)2-PFDMS9 3520 1.30 2835 2835Ac-A4-PFDMS20 8230 1.28 5258 5258Ac-A4-PFDMS9 3820 1.29 2621 2621Ac-A4-PFEMS18 9930 1.27 5566 5565Ac-A4-PFEMS9 5400 1.30 2747 2747Ac-G4-PFDMS20 6660 1.35 5227 5229Ac-V4-PFDMS20 11 370 1.21 5398 5397Ac-V4-PFEMS18 12 150 1.24 5676 5677Ac-V4-PFEMS9 5930 1.22 3115 3116

[a] Determined by GPC. [b] Determined by positive ion MALDI-TOF MS with dithranol as the matrix. The value is quoted for the ion of100 % relative intensity.

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This behaviour is similar to that observed for high molecularweight linear PFDMS,[48] with the exception that two distinctmass-loss processes were reported.

The TGA thermograms for the conjugates showed twoprocesses (Table 3), with that at lower temperature alwayscorresponding to the smaller percentage mass decrease. Themagnitude of this change is in close agreement with thatpredicted theoretically for complete loss of the tetrapeptidesegment (ca. 6 and 11 % mass reductions for the high andlow molecular-weight materials, respectively). The observeddiscrepancy most probably arises from limited decomposi-tion/depolymerisation of the metallopolymer chain. If thesecond mass-loss process is taken to correspond to forma-tion of the a-Fe-containing ceramic generally afforded uponpyrolysis of linear PFDMS under nitrogen, then the percent-age mass loss is larger than expected, especially for the highmolecular-weight materials. The presence of the tetrapeptidewould, therefore, appear to aid depolymerisation and thusreduce the ceramic yield from the PFS block.

DSC thermograms were acquired on all materials as threecyclic scans between �60 and 300 8C. The first heating rampwas used to anneal the sample and thereby erase all thermalhistory. The thermograms of the PFDMS-containing materi-als exhibited no first-order transitions that could be assignedto melting or crystallisation processes involving the semi-crystalline metallopolymer block. This observation presuma-bly arises due to the crystallisation kinetics being slow rela-tive to the time scale of the experiment. As a result, theonly feature observed was a second-order process (19–25 8C)that was assigned to the glass transition (Tg) of this block(33 8C).[49] In the absence of processes associated with crys-tallisation, these thermograms were essentially similar tothose obtained for the materials with the amorphousPFEMS block. The position of the Tg, however, was margin-ally lower in the latter cases (16–23 8C), in accordance withthe reported value for the PFEMS homopolymers (Tg

�15 8C).[50] The absence of a first-order transition, assigna-ble to the melting point (Tm) of the tetrapeptide fragment, isparticularly noteworthy in the case of both the PFDMS andPFEMS conjugates with Ac-A4-OH. Similar experiments,however, failed to detect the Tm for a multi-block copolymercontaining A5 segments, despite evidence for a crystalline,anti-parallel b-sheet domain by FT-IR spectroscopy.[25] In

contrast, the DSC thermogram of Ac-(GA)2-PFDMS18 didexhibit a transition at 67 8C that we tentatively assigned tothe Tm of the tetrapeptide. The reason for the observed dif-ferences in thermal behaviour between the two materialswith different aggregator domains, however, is not apparent.

FT-IR spectroscopy has proven a useful technique for es-tablishing the presence and nature of secondary interactionsin both peptides and proteins.[51] The FT-IR spectrum of Ac-(GA)2-PFDMS18 initially exhibited only a broad absorptionin the amide I region at 1650 cm�1, which is consistent withthe presence of amorphous peptide regions. Upon thermalannealing, however, absorptions at 1695 and 1620 cm�1 alsobecame apparent, suggesting, in part, the development of ananti-parallel b-sheet structure. It was, therefore, interestingthat a comparable material, Ac-(GA)2-PFDMS20, synthes-ised since our communication,[38a] exhibited an IR spectrumcharacteristic of an anti-parallel b-sheet prior to thermal an-nealing (Table 4). This behaviour has since proven to be the

norm with the majority of materials containing domainswith an anti-parallel b-sheet structure upon simple precipita-tion. In the cases of the (GA)2 and A4 peptide segments,metallopolymer crystallinity and block length seemed tohave no effect on the propensity of the material to exhibitthe anti-parallel b-sheet structure. In addition to absorptionscorresponding to an anti-parallel b-sheet (1685, 1632 and1556 cm�1), the FT-IR spectrum of Ac-G4-PFDMS20 dis-played a further absorption at 1640 cm�1, which we havetentatively assigned to regions of amorphous peptide seg-ments. The three conjugates containing the V4 block display

Table 3. Thermal properties of the amino-terminated PFS homopolymersand representative Ac-A4-PFS conjugates. Scans were performed at arate of 10 8C min�1 for both TGA and DSC.

Tg [8C] Td [8C] ([%])[a,b]

PFDMS20-NH2 19 429 (64.52)PFEMS20-NH2 16 403 (64.29)Ac-A4-PFDMS20 25 345 (6.56)/414 (79.29)Ac-A4-PFEMS18 23 324 (7.52)/424 (74.16)Ac-A4-PFDMS9 20 418 (16.21)/468 (66.18)Ac-A4-PFEMS9 18 304 (17.42)/403 (44.32)

[a] Decomposition temperature (Td) was defined by the point at which5% of the original mass was lost. [b] Percentage mass lost during eachtransition is reported in parentheses.

Table 4. FT-IR data for Ac-X4-PFS conjugates.

Amide I [cm�1][b] Amide II[cm�1][b]

Random-coil[a] 1656(s) 1535(s)Anti-parallel b-sheet[a]

1632(s), 1685(w) 1530(s)

Ac-(GA)2-PFDMS18

1650(s)[c] –1695(w)[d]

1655(s)[d]

1620(s)[d]

Ac-(GA)2-PFDMS20

1624(s), 1694(w) 1535

Ac-(GA)2-PFEMS18 1624(s), 1694(w) 1539Ac-(GA)2-PFDMS9 1625(s), 1694(w) 1537Ac-A4-PFDMS20 1626(s), 1694(w) 1540Ac-A4-PFEMS18 1626(s), 1693(w) 1540Ac-A4-PFDMS9 1627(s), 1692(w) 1541Ac-A4-PFEMS9 1625(s), 1693(w) 1539Ac-G4-PFDMS20 1632(s), 1640(s), 1685(w),

1746 (s)1556

Ac-G4-PFEMS18 1631(s), 1683(w) 1552Ac-V4-PFDMS20 1630(s) 1545Ac-V4-PFEMS18 1630(s), 1694(w) 1547Ac-V4-PFEMS9 1630(s), 1690(w) 1543

[a] Observed frequencies for amide absorptions of polypeptides in vari-ous formations.[51] [b] Letters in parentheses indicate observed intensities(s= strong, m =medium, w= weak). [c] Prior to thermal annealing.[d] After thermal annealing.

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interesting behaviour. Those formed with the amorphousmetallopolymer (PFEMS) exhibited IR spectra consistentwith anti-parallel b-sheet formation, however, that withPFDMS20 gave rise to a single absorption in both amide re-gions (1630 and 1545 cm�1), most likely arising from arandom-coil conformation of the peptide. These observa-tions are consistent with the relatively long, semi-crystallinemetallopolymer block in the latter serving to prevent thecrystallisation of the V4 segment. An anti-parallel b-sheetstructure would be expected to be thermodynamically fav-oured over the parallel arrangement because this minimisesthe steric interactions between the metallopolymer segmentsof adjacent conjugates. In the absence of the hydrophobiceffect, one would expect further stabilisation of the formeras this arrangement maximises the H-bonding interactions.The only materials that do not exhibit an anti-parallel b-sheet structure contain the longer metallopolymer chains.These larger segments would be expected to be more effec-tive at kinetically stabilising the random-coil structure.

Self-assembly of PFS–tetrapeptide conjugates in solution :

TEM studies : After thermal annealing of Ac-(GA)2-PFDMS18, through three heating/cooling cycles, the materialwas no longer soluble in toluene. Heating a suspension ofthis material to 70 8C in toluene, however, afforded a stableyellow solution, which increased in viscosity on cooling. Thisbehaviour is consistent with H-bond formation during theannealing process, and their subsequent disruption uponheating in toluene. Analysis of the colloidal solution atroom temperature by transmission electron microscopy(TEM; Figure 2) revealed the presence of a fibrous network.Formally exchanging the semi-crystalline metallopolymerfor amorphous PFEMS of comparable length (DPn =18)had no obvious effect, with a similar fibrous network beingobtained. Furthermore, repeating the procedure with Ac-A4-PFDMS20 and Ac-A4-PFEMS18 (Figure S2 in the Sup-porting Information), which contain the aggregator domaincommon to Nephila clavipes silk, afforded the same fibrous

networks. Shortening the length of the metallopolymerblock, as with Ac-A4-PFDMS9 and Ac-A4-PFEMS9 (Fig-ure S2 in the Supporting Information), again resulted in for-mation of the fibrous aggregates, but in this instance noheating was required. Similarly, Ac-V4-PFEMS9 also formedfibres without the need to heat above room temperature(Figure S3 in the Supporting Information). This observationcan presumably be ascribed to the reduced steric protectionprovided by the shorter metallopolymer block, leading to alower activation barrier to solution-phase aggregation. Incontrast, neither Ac-V4-PFDMS20, Ac-G4-PFDMS20 nor Ac-V4-PFEMS18 exhibited well-defined aggregates upon heatingin toluene and cooling to room temperature. This result sug-gests that Ac-A4 and Ac-(GA)2 display a greater tendencyto form fibrous structures in dilute toluene solution thanAc-V4 and Ac-G4. The propensities of the amino acids toform b-sheet secondary structures are of the order V>A>

G,[46] which may account for the observed differences in be-haviour between the Ac-A4 and Ac-G4 conjugates. Valine,however, is considerably more hydrophobic than both gly-cine and alanine,[45] and this is likely to disfavour self-assem-bly of the Ac-V4 conjugates in toluene, despite its high pro-pensity to form b-sheets in natural, water-soluble proteins.

AFM studies : To confirm that the fibrous structure of theaggregate was not influenced by solvent evaporation withinthe transmission electron microscope or by the substrate, wealso imaged all samples by atomic force microscopy (AFM).Colloidal solutions comparable to those studied by TEM(0.2 mgmL�1 in toluene) were heated appropriately, dip-coated onto silicon wafers and the topology of the materialstudied in tapping mode. The micrographs revealed thepresence of fibrous networks in all instances for which theyhad been observed by TEM. Representative phase andheight images, as well as a sectional profile for Ac-(GA)2-PFDMS20, are displayed in Figure 3. The AFM images forAc-A4-PFDMS9 and Ac-V4-PFEMS9 are shown in Figure S4in the Supporting Information.

Effect of temperature : The observation that colloidal solu-tions of the conjugates containing the longer metallopoly-mer blocks required heating to acquire the fibrous structureprompted us to probe the evolution of these mixtures withtemperature. To this end, the apparent hydrodynamic radius(Rh) of a 0.2 mg mL�1 solution of Ac-(GA)2-PFDMS20 wasmeasured by dynamic light scattering (DLS) on heatingfrom 25–75 8C and similarly on cooling back to the originaltemperature. The results of this study are displayed inFigure 4. Upon initial dissolution the conjugate exhibited anRh of 36 nm, which was significantly larger than expectedfor individual unimers in solution (see below). Heating hadno observable effect until 60 8C, at which point Rh began toincrease rapidly, reaching a maximum value of about150 nm at 70 8C. Further heating to 75 8C resulted in a slightdecrease in size (ca. 130 nm) and this trend continued oncooling with Rh ultimately tending to a value of 92 nm at25 8C. The assumptions of the Stokes–Einstein model make

Figure 2. TEM image of Ac-(GA)2-PFDMS20 after heating a sample intoluene (0.2 mg mL�1) to 70 8C and allowing it to cool to room tempera-ture.

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a quantitative interpretation of the apparent hydrodynamicsizes difficult, however, there clearly appear to be threecharacteristic regions: one corresponding to the initial size;another during which the size changes and a third whichrepresents the final fibrous structure.

In an attempt to garner further information regarding themorphology of the aggregates in each region, we recordedTEM micrographs on samples removed from the colloidalsolution at 25, 75 and 25 8C, the last being taken after previ-ously heating to 75 8C (Figure 5). In keeping with the rela-tively large Rh observed upon initial dissolution of the con-jugate, the original micrograph at 25 8C is consistent withthe presence of large, irregular aggregates in solution. It isalso noteworthy that there appear to be a few fibres present,but these can no longer be observed in the micrograph re-corded on the sample removed at 75 8C. In this instance,only irregular interconnecting aggregates can be detected. Itcan be postulated that it is the crystallisation of the oligo-peptides in these structures that ultimately affords the ob-served fibrous network.

In keeping with our initial observations that Ac-A4-PFDMS9 afforded a fibrous structure upon simple dissolu-tion in toluene at room temperature, we observed a temper-ature invariant Rh of 68 nm by DLS for this conjugate (Fig-ure S5 in the Supporting Information) and confirmed thatthe fibrous structure was present over the temperaturerange by TEM (Figure S6 in the Supporting Information).By means of comparison, the conjugates Ac-G4-PFDMS20

and Ac-V4-PFDMS20, which did not form fibrous structuresfrom solution, exhibited Rh values at 25 8C of 84 and 36 nm,respectively. These results suggest that these materials alsoexist as aggregates in solution.

Structure of the fibres : A colloidal solution of the fibrousAc-(GA)2-PFDMS18 network in toluene was characterisedby 1H NMR spectroscopy. The spectrum only exhibited sig-nals that could be assigned to the PFDMS block, which sug-gested that this portion of the macromolecule was solvatedand thus formed the corona. This observation was in keep-ing with our expectation based on the solubility of the mate-rial in relatively non-polar solvents. Preliminary measure-ments by AFM demonstrated that the narrowest fibresvaried from 22–50 nm in width and 3–5 nm in height. Analy-sis of TEM micrographs obtained on fibres formed from thecomparable material Ac-(GA)2-PFDMS20, indicate similarthicknesses for the narrowest fibres (ca. 23 nm). This resultoffers further evidence for the coronal location of the PFS

Figure 3. AFM images of Ac-(GA)2-PFDMS20: a) phase; b) height andc) cross sectional profile. Red arrows indicate the height of an individualfibre (6 nm). Blue arrows indicate the width of the fibre at half height(ca. 100 nm).

Figure 4. Relationship between Rh [nm] and T [8C] for Ac-(GA)2-PFDMS20 at 0.2 mg mL�1 in toluene (^= 25!75 8C ; &=75!25 8C).

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block, because in the absence of staining, high electron den-sity is required at the periphery of the fibre to provide theobserved contrast. Furthermore, use of AFM to examine thefibres after dip-coating onto a silicon substrate suggestedthat they consist of bundles of laterally aggregated smallerfibres (Figure 6).

The dimensions of one such fibre when isolated on thesubstrate were determined to be approximately 7 nm inwidth and 5.5 nm in height. From the molecular formula ofAc-(GA)2-PFDMS20, one would predict the tetrapeptide to

extend 1.4 nm,[52] with the metallopolymer chain contribu-ting a value between that predicted for a Gaussian coil (ca.0.5 nm) and the contour length (ca. 3.3 nm).[53] A tape con-sisting of an anti-parallel b-sheet core of tetrapeptides and ametallopolymer corona would, therefore, be expected tohave a width of approximately 2.4–7.0 nm. Bçrner et al.have studied the self-assembly of poly(n-butylacrylate)18-b-(TV)5-nPhe-G-OH (nPhe =4-nitrophenylalanine) in organicmedia and observed the formation of a b-barrel.[36] Thisstructure, consisting of a b-sheet twisted around the longaxis of the fibre, was suggested to occur due to the chemicalasymmetry of the two faces of the tape. The hydroxylgroups of the threonine residues thus point towards thecentre of the cylinder with the isopropyl groups of valinegrouped on the outside. The face asymmetry of the tetra-peptides employed in this study is considerably less, al-though accumulation of residual dipoles on the oligopeptideand steric repulsion between synthetic-polymer chains maystill have been expected to cause the tape to twist. It hasbeen demonstrated, however, that conjugates of similar oli-gopeptides with polyethylene oxide (PEO) self-assemble inwater to afford flat fibres consisting of double b-sheets.[33]

This is despite the application of a considerably longer syn-

Figure 5. TEM micrographs of Ac-(GA)2-PFDMS20 (0.2 mg mL�1 in tolu-ene) recorded at a) 25, b) 75 and c) 25 8C (after previously heating to75 8C).

Figure 6. AFM study of Ac-(GA)2-PFDMS20 (0.2 mg mL�1 colloidal solu-tion in toluene after cooling from 70 to 25 8C). a) Phase image annotatedwith coloured lines and blue arrows to show points at which fibre heightand width data, respectively, have been obtained from the correspondingphase image. b) The height profiles along the coloured lines shown in a).

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I. Manners, D. N. Woolfson et al.

thetic component (DPn�73) and in keeping with the in-creased effective screening of the accumulated charge bythe more polar solvent. The van Hest group has also report-ed the formation of flat fibres from the lateral aggregationof an ABA-type block copolymer that contains [(AG)3EG]20

as the b-sheet element.[27] On the basis of AFM measure-ments they postulated that the hydrogen-bonding directionwas parallel to the substrate and perpendicular to the longaxis of the fibre. Although this is similar to what we havepreviously suggested for our materials,[38a] the dimensions ofthese fibres, gleaned from more recent experiments, are con-sistent with coincidence of both the hydrogen bonding andfibril directions (Figure 7). It would be expected that aggre-

gation of the fibres into the observed microscopic networkwould arise from lateral aggregation of these tapes, occur-ring only in places in which the kinetic barrier provided bythe corona had been overcome. From the work of Lynn andco-workers on the structure of the b-amyloid fibril,[21] suchlamination would be expected to have periodicity of approx-imately 10 nm and this is comparable to the narrowest fibrespacing found by AFM. Furthermore, the side-on orienta-tion of the tapes upon the substrate, which is suggested bythese data, is supported by good agreement between thewidth calculated above and the observed fibre height. Theabsence of any features in the selective-area electron dif-fractogram of the fibres is also consistent with the proposedorientation of the tetrapeptide core.

Patterning of silver nanoparticles on Ac-X4-PFS tetrapep-tide fibrous networks : Previous studies have demonstratedthat cylindrical micelles formed from the self-assembly ofPI-b-PFDMS (PI =polyisoprene) in a selective solvent forPI form cylindrical micelles, which may be made permanentby intra-coronal cross-linking.[54, 55] Subsequent dissolution ofthese objects in a common solvent caused the PFS cores toswell, thus creating space for nanoparticle encapsulation.Utilising both the void created by the swollen core and theredox activity of the PFS chains, we were then able to tem-plate the formation of 1D arrays of silver and silver iodidenanoparticles within the core of the micelle.[56] Similar pat-terns of silver nanoparticles have also been grown on thepeptidic cores of self-assembled nanotapes, which were de-rived from PEO–peptide conjugates.[57] In this case, howev-er, the absence of an in situ reductant enabled the use of

visible light to promote the formation of Ag0 from AgI. Theaccess to fibres with a PFS corona provided by this study(see below) prompted us to investigate the use of the ferro-cene/ferrocenium redox couple to localise silver nanoparti-cles in the exterior of the fibres without the need for cross-linking.

Initially, we attempted to synthesise silver nanoparticleson the fibres by adding dropwise a solution of AgACHTUNGTRENNUNG[PF6] intoluene to a colloidal solution of the self-assembled conju-gate in toluene. Our reaction was conducted by simply ti-trating a solution of Ac-(GA)2-PFDMS20 in toluene with asaturated solution of AgACHTUNGTRENNUNG[PF6], also in toluene. The colour ofthe reaction mixture changed from pale amber to deep

yellow and then to dark brown,before a precipitate was finallyformed. After centrifugation,we removed the precipitate andobtained a clear, green superna-tant liquid. TEM was per-formed on samples preparedfrom the supernatant by allow-ing the solvent to evaporatefrom a drop on a carbon-coatedcopper grid. In the micrographs,we observed nanoparticles withdiameters ranging from 35 to

110 nm, randomly dispersed on fibres and consistent withthe redox-induced preparation of Ag nanoparticles(Figure 8).

We infer that upon addition of AgACHTUNGTRENNUNG[PF6], the AgI ionswere reduced to Ag0 by the FeII centres in the PFS corona.This process was accompanied by formation of ferroceniumcentres [Eq. (1)].

FeIIPFS þAgI ! FeIII

PFS þAg0 ð1Þ

Previous studies have demonstrated in the case of the PI-b-PFDMS cylindrical micelles that pre-oxidation of the PFSwith a specific oxidant aids confinement of nanoparticles tothe core.[56] We have found, however, that the fibrous struc-ture of the Ac-X4-PFS conjugates when suspended in tolu-ene became unstable after oxidation. Thus, we decided toconduct the pre-oxidation step on a film of the fibrous net-work before adding Ag ACHTUNGTRENNUNG[PF6].

The synthesis of the silver nanoparticles on PFS-contain-ing fibres was conducted in two steps, as depicted inScheme 2. Firstly, thin films of the fibres were prepared byself-assembling Ac-A4-PFDMS10 as a 0.2 mgmL�1 colloidalsolution in toluene (see below) and then dip-coating onto acarbon-coated copper grid. The grids were sequentiallydipped into a pre-oxidant solution of either tris(4-bromo-phenyl)aminium hexachloroantimonate (Magic Blue) in di-chloromethane (0.4 mgmL�1) or iodine in toluene(0.5 mgmL�1) and then a solution of the oxidant, AgACHTUNGTRENNUNG[PF6] intoluene (0.5 mg mL�1).

The reaction products that formed on the copper gridwere subsequently analysed by TEM and EDX. The struc-

Figure 7. Schematic representation of the Ac-X4-PFS conjugates adopting a b-sheet secondary structure andthe lamination of these sheets into the observed fibres.

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FULL PAPERPolyferrocenylsilane–Tetrapeptide Conjugates

tures presented in Figure 9 are of Ac-A4-PFDMS10 pre-treat-ed with Magic Blue or iodine and then oxidised by Ag ACHTUNGTRENNUNG[PF6].

TEM demonstrated that pre-oxidation with either MagicBlue or iodine resulted in most silver nanoparticles being lo-cated on the fibrous Ac-A4-PFDMS10 networks. Further-more, it would appear that the nature of the pre-oxidant hasan effect on both the density of coverage and size of thenanoparticles formed. For instance, the composite materialformed after pre-treatment with Magic Blue (Figure 9 c) hada higher density (5 particles/104 nm2) of small particles

(25 nm), whereas when iodine was employed (Figure 9 e) thedensity was lower (3 particles/104 nm2) and the particleswere larger (33 nm). Figure 9 d and 9f show the energy-dis-persive X-ray spectroscopy (EDX) data of silver nanoparti-cles on the fibrous networks when pre-oxidised by MagicBlue and iodine, respectively. In addition to the expectedsilver signals, we also observed signals arising from the pres-ence of chlorine and antimony when the samples were pre-oxidised by the former and iodine when the latter was em-ployed. These data suggest that silver nanoparticle growthon the PFS corona of the fibres is seeded by the precipita-tion of silver halide and that this process is more efficient inthe case of the chloride. A similar mechanism has previouslybeen used to explain the localised formation of similarnanoparticles in the cores of cross-linked PI-b-PFDMS cy-lindrical micelles.[56]

Conclusion

We have synthesised a range of PFS-containing tetrapeptideconjugates and studied the affect of polymer molecularweight (DPn�10 or 20), crystallinity and specific-peptide se-quence on their self-assembly. With the exception of Ac-G4-PFDMS20 and Ac-V4-PFEMS18, all of the materials preparedexhibited only anti-parallel b-sheet structures in the solidstate. These two conjugates, however, contained some or allof the sequences in a random-coil conformation. Upon dis-solution in toluene, all materials with the shorter metallopo-lymer segments self-assembled to afford fibrous networkswith peptidic cores and PFS coronae. In the conjugates with

Ac-(GA)2 and Ac-A4, lengthen-ing the PFS chain resulted inno observable networks atroom temperature, but gavesimilar results (i.e., fibrous net-works). The observed differ-ence was attributed to kineticstabilisation by the longerchains of what would otherwisebe aggregating filaments. Theconjugates of either Ac-V4 orAc-G4 with these larger macro-molecules, however, did notafford comparable networksunder any of the conditions at-tempted. On the basis of de-

tailed studies of the fibres, we have postulated that they areformed by the lateral aggregation of b-sheets. This explana-tion accounts for the variation in widths and fibre cross-link-ing. The coronal location of the PFS enables ready access tothe reducing iron(II) centres and this was demonstrated inthe patterning of Ag0 nanoparticles.

Scheme 2. Synthesis of silver nanoparticles after pre-oxidation with either Magic Blue or iodine.

Figure 8. TEM image (top) and energy dispersive X-ray spectra (EDX;middle and bottom) showing the fibrous structures of the Ac-(GA)2-PFSconjugate with silver nanoparticles randomly distributed. * denotescounts arising from the Cu grid.

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I. Manners, D. N. Woolfson et al.

Experimental Section

Materials and methods : Alanine, glycine, valine and (benzotriazole-1-yloxy)trispyrrolidinophosphonium hexafluorophosphate (PyBOP) werepurchased from Novabiochem. All other commercial chemicals were ac-quired from Sigma–Aldrich. Amino-terminated poly(ferrocenyldimethyl-silane) (PFDMS) and poly(ferrocenylethylmethylsilane) (PFEMS) wereboth synthesised by using the method published for the former.[41]

All manipulations involved in the preparation of the amino-terminatedpolyferrocenylsilanes were performed either under dry argon or in vacuoby using standard Schlenk-line and glovebox techniques. Solvents (dieth-yl ether and hexanes) were purified by using a Grubbs-type solventsystem.[58] Tetrahydrofuran (THF) and 1-(3-bromopropyl)-2,2,5,5-tetra-methyl-1-aza-2,5-disilacyclopentane were purified by vacuum distillationfrom the appropriate drying agents (Na/benzophenone and CaH2, respec-tively) and stored under nitrogen. The tetrapeptides were synthesised bysolid-phase peptide synthesis (SPPS) by using Fmoc chemistry.[47]

IR spectra were recorded on the solid materials by using a Perkin–ElmerFT-IR 1720x spectrometer. 1H NMR spectra were acquired on either aJEOL GX 270 or GX 400 spectrometer and referenced to tetramethylsi-lane (TMS) via the resonance of the residual protonated solvent or di-rectly to TMS salt for samples in D2O. MALDI-TOF and ESI MS were

performed in positive ion mode on anApplied Biosystems 4700 spectrometerand Bruker Daltonics FT-ICR-MS, re-spectively, with dithranol as the matrixfor the former. GPC was carried outon a Viscotek Gel Permeation Chro-matograph, equipped with an automat-ic sampler, a pump, an injector, aninline degasser and a column oven(30 8C). The columns consisted of sty-rene/divinyl benzene gels with poresizes ranging from 500 to 100 000 �.THF (Fisher) containing [nBu4]Br(0.1 % wt) was used as the eluent, witha flow rate of 1.0 mL min�1. The elut-ing sample was detected by means of aVE 3580 differential refractometer,and the column was calibrated withmonodisperse polystyrene standards(Aldrich). Thermal analyses were per-formed on a TA instrumentsDSC Q100 modulated differential-scanning calorimeter, equipped with acooling unit and a TGA Q500 ther-mogravimetric analyser. All analyseswere performed at a scan rate of10 8C min�1. TEM was performed on aJEOL JEM 1200 EX MKI microscope,fitted with an Oxford instruments linkISIS EDX detector, at an acceleratingvoltage of 120 kV. The TEM speci-mens were prepared on a carbon-coated copper grid (200 mesh). DLSwas conducted by using a Malvern In-struments Zetasizer Nano-S fitted witha 4 mW He–Ne laser (633 nm). AFMwas performed in tapping mode usinga Multimode atomic force microscopeequipped with a Nanoscope V control-ler (Veeco Instruments Ltd, Santa Bar-bara, USA) and Picoforce for high res-olution AFM. Tap300Al cantilevers(Budget Sensors, Sofia, Bulgaria) witha rotated monolithic silicon probe witha tip radius of less than 10 nm wereused in all experiments except for highresolution AFM. In this case SSS-NCL

(SuperSharpSilicon) cantilevers were used with a rotated monolithic sili-con probe with a tip radius of around 2 nm and were obtained fromNanosensors (Neuchatel, Switzerland). Samples were dip-coated onto sil-icon wafers. Images were analysed by using Gwyddion: an open-sourcesoftware program for scanning probe microscopy (SPM) images.

Syntheses of Ac-(GA)2-OH, Ac-A4-OH, Ac-G4-OH and Ac-V4-OH : Thepeptides Ac-(GA)4-OH, Ac-A4-OH, Ac-G4-OH and Ac-V4-OH weresynthesised by solid-phase peptide synthesis (SPPS) by using Fmocchemistry.[47] The amino acid residues were used as Fmoc-(amino acid)-OH and couplings were facilitated by use of O-benzotriazole-N,N,N’,N’-tetramethyluronium hexafluorophosphate (HBTU) and N,N-diisopropyl-ethylamine (DIPEA). After assembly of the oligopeptide on the Wangresin, the NH2-X4-(Wang resin) was stirred in an esterification mixture[acetic anhydride (5 % v/v), pyridine (5 % v/v), N,N-dimethylformamide(DMF; 90 % v/v)] to obtain Ac-X4-(Wang resin). The resin was thencleaved by stirring in a trifluoroacetic acid (TFA; 95 % v/v), H2O (2.5 %v/v) and triisopropylsilane (TIS; 2.5% v/v) mixture and the peptide wasprecipitated with diethyl ether. All materials were obtained as colourlesspowders.

Ac-(GA)2-OH : 68 mg, 86 % yield; 1H NMR (400 MHz, D2O, 19 8C, TMSsalt): 4.31 (m, 2 H; -CH [Ala]), 3.89 (s, 4H; CH2 [Gly]), 2.10 (s, 3 H;

Figure 9. TEM images of Ac-A4-PFDMS9 fibres pre-treated with a) Magic Blue or b) iodine. TEM images ofAc-A4-PFDMS9 fibres after pre-treatment with c) Magic Blue or e) iodine and followed by oxidation with Ag-ACHTUNGTRENNUNG[PF6]. EDX spectra of Ac-A4-PFDMS9 fibres after pre-treatment with d) Magic Blue or f) iodine and followedby oxidation with Ag ACHTUNGTRENNUNG[PF6]. * denotes counts arising from the Cu grid.

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FULL PAPERPolyferrocenylsilane–Tetrapeptide Conjugates

-COCH3), 1.36 ppm (d, J ACHTUNGTRENNUNG(H,H) =8 Hz 6H; CH3 [Ala]); MS (70 eV): m/z(%): 316 (100) [M+].

Ac-A4-OH : 106 mg, 75% yield; 1H NMR (270 MHz, D2O, 19 8C, TMSsalt): 4.29 (m, 4H; CH [Ala]), 2.00 (s, 3H; CH3CO-), 1.39 ppm (m, 12H;CH3 [Ala]); MS (70 eV): m/z (%): 344 (100) [M+].

Ac-G4-OH : 113 mg, 78% yield; 1H NMR (400 MHz, D2O, 19 8C, TMSsalt): 7.87 (m, 4H; NH), 3.93 (s, 8H; CH2 [Gly]), 2.00 ppm (s, 3H;-COCH3); MS (70 eV): m/z (%): 288 (100) [M+].

Ac-V4-OH : 208 mg, 91 % yield; 1H NMR (270 MHz, DMSO, 19 8C,TMS): 7.90 (m, 4 H; NH), 4.19 (s, 4H; -CH-NH), 1.98 (m, 4H; -CH-),1.85 (s, 3 H; CH3CO-), 0.84 ppm (m, 24H; CH3-[Val]); MS (70 eV): m/z(%): 456 (100) [M+].

Synthesis of amino-terminated poly(ferrocenyldimethylsilane)s : Theamino-terminated poly(ferrocenyldimethylsilane)s were prepared accord-ing to the literature procedures (typical yield: 450 mg, 45%).[38a,41]

PFDMS20-NH2 : 1H NMR (400 MHz, C6D6, 19 8C, TMS): 4.27 (br s; Cp),4.10 (br s; Cp), 2.50 (t, J ACHTUNGTRENNUNG(H,H) =7 Hz; -CH2NH2), 1.49 (m; -CH2-), 0.92(m; CH3-), 0.54 (br s; CH3-Si), 0.20 ppm (s; -CH2-Si); MALDI-TOF MS:m/z (%): 4956 (100); GPC: Mn =5380 gmol�1; PDI (Mw/Mn)=1.12.

PFDMS9-NH2 : 1H NMR spectrum was effectively the same as that forPFDMS20-NH2; MALDI-TOF MS: m/z (%): 2295 (100); GPC: Mn =

2305 gmol�1; PDI (Mw/Mn)=1.14.

Synthesis of amino-terminated poly(ferrocenylethylmethylsilane): Theamino-terminated poly(ferrocenylethylmethylsilane)s were prepared bymodifying the procedure used for the dimethyl analogues. The materialswere obtained as red-orange gums after precipitation into MeOH (typicalyield: 187 mg, 47 %).

PFEMS18-NH2 : 1H NMR (400 MHz, C6D6, 19 8C, TMS): 4.29 (br s; Cp),4.13 (br s; Cp), 2.50 (t, J ACHTUNGTRENNUNG(H,H) =7 Hz, 2 H; -CH2NH2), 1.40 (m; -CH2-),1.17 (br t, J ACHTUNGTRENNUNG(H,H) =7 Hz; CH3-), 1.01 (m; -CH2-Si), 0.43 (br s; CH3-Si);MALDI-TOF MS: m/z (%): 4724 (100); GPC: Mn =5270 gmol�1; PDI(Mw/Mn)=1.13.

PFEMS9-NH2 : 1H NMR spectrum was effectively the same as that forPFEMS18-NH2; MALDI-TOF MS: m/z (%): 2431 (100); GPC: Mn =

2575 gmol�1; PDI (Mw/Mn)=1.15.

Syntheses of Ac-(GA)2-PFSn, Ac-A4-PFSn, Ac-G4-PFSn and Ac-V4-PFSn :A solution of an amino-terminated PFS was prepared in CH2Cl2 and a10-fold excess of Ac-X4-OH in DMF added. PyBOP (20 equiv) andDIPEA (30 equiv) were then added as a solution in DMF, and the reac-tion mixture was stirred at room temperature for 24 h. All solvents wereremoved in vacuo and THF was added to the residue. The excess Ac-X4-OH was removed by filtration and the Ac-X4-PFSn conjugate precipitatedby addition to MeOH. Drying in vacuo then afforded the product as anamber solid. For FT-IR data, see Table 4.

Ac-(GA)2-PFDMS20 : 90 % yield; 1H NMR (400 MHz, CDCl3, 19 8C,TMS): 4.43 (m, 2H; -CH- [Ala]), 4.22 (br s; Cp), 4.01 (br s; Cp), 3.93 (s,4H; -CH2- [Gly]), 3.24 (br s, 2 H; -CH2NHCO), 2.04 (s, 3H; CH3CO),1.36 (d, J ACHTUNGTRENNUNG(H,H) =8 Hz, 6 H; CH3- [Ala]), 0.47 ppm (br s; CH3Si);MALDI-TOF MS: m/z (%): 5257 (100); GPC: Mn =6490 gmol�1; PDI(Mw/Mn)=1.22.

Ac-(GA)2-PFDMS9 : 88 % yield; 1H NMR spectrum is essentially thesame as that for Ac-(GA)2-PFDMS20 after consideration of the differentPFDMS chain length; MALDI-TOF MS: m/z (%): 2853 (100); GPC:Mn =3520 g mol�1; PDI (Mw/Mn) =1.30.

Ac-A4-PFDMS20 : 72% yield; 1H NMR (400 MHz, CDCl3, 19 8C, TMS):4.19 (br s; Cp), 4.00 (br s; Cp), 3.22 (m, 2H; CH2NH-), 2.26 (s, 3 H;CH3CO-), 1.35 (m, 12 H; CH3- [Ala]), 0.87 (t, J ACHTUNGTRENNUNG(H,H) =7 Hz, 3H;CH3CH2-), 0.62 (m, 2 H; -CH2-), 0.44 ppm (br s; CH3Si); MALDI-TOF MS: m/z (%): 5258 (100); GPC: Mn = 8230 g mol�1; PDI (Mw/Mn)=

1.28.

Ac-A4-PFDMS9 : 66 % yield; 1H NMR spectrum is essentially the same asthat for Ac-A4-PFDMS20 after consideration of the different PFDMSchain length; MALDI-TOF MS: m/z (%): 2621 (100); GPC: Mn =

3820 gmol�1; PDI (Mw/Mn)=1.29.

Ac-A4-PFEMS18 : 85% yield; 1H NMR (400 MHz, CDCl3, 19 8C, TMS):4.19 (br s; Cp), 3.99 (br s; Cp), 3.24 (m, 2 H; CH2NH-), 2.26 (s, 3H;

CH3CO-), 1.70 (m, 12H; CH3- [Ala]), 1.04 (t, J ACHTUNGTRENNUNG(H,H) =7 Hz; CH3CH2Si),0.91 (q, J ACHTUNGTRENNUNG(H,H) =7 Hz; -CH2Si), 0.43 ppm (br s; CH3Si); MALDI-TOFMS: m/z (%): 5566 (100); GPC: Mn =9930 gmol�1; PDI (Mw/Mn)=1.27.

Ac-A4-PFEMS9 : 63% yield; 1H NMR spectrum is essentially the same asthat for Ac-A4-PFEMS18 after consideration of the different PFEMSchain length; MALDI-TOF MS: m/z (%): 2747 (100); GPC: Mn =

5400 gmol�1; PDI (Mw/Mn)=1.30.

Ac-G4-PFDMS20 : 55% yield; 1H NMR (400 MHz, CDCl3, 19 8C, TMS):4.20 (br s; Cp), 4.00 (br s; Cp), 3.90 (s, 8H; -CH2- [Gly]), 3.24 (m, 2 H;CH2NH-), 2.26 (s, 3H; CH3CO-), 0.90 (t, J ACHTUNGTRENNUNG(H,H) =7 Hz; CH3CH2-), 0.64(m, 2H; -CH2-), 0.44 ppm (br s; CH3Si); MALDI-TOF MS: m/z (%):5227 (100); GPC: Mn =6660 g mol�1; PDI (Mw/Mn) =1.35.

Ac-V4-PFDMS20 : 38 % yield; 1H NMR (270 MHz, CDCl3, 19 8C, TMS):4.20 (br s; Cp), 3.99 (br s; Cp), 3.20 (m, 2H; CH2NH-), 2.26 (s, 3 H;CH3CO-), 2.06 (m, 4 H; -CH- [Val]), 1.84 (m, 4H; (CH3)2CH-), 0.88 (t,J ACHTUNGTRENNUNG(H,H) =7 Hz; CH3CH2-), 0.78 (m, 24H; (CH3)2CH-), 0.62 (m, 2 H;-CH2-), 0.44 (br s; CH3Si); MALDI-TOF MS: m/z (%): 5398 (100); GPC:Mn =11370 gmol�1; PDI (Mw/Mn)=1.21.

Ac-V4-PFEMS18 : 33% yield; 1H NMR (270 MHz, CDCl3, 19 8C, TMS):4.19 (br s; Cp), 3.98 (br s; Cp), 3.24 (m, 2H; CH2NH-), 2.26 (s, 3 H;CH3CO-), 2.06 (m, 4 H; -CH- [Val]), 1.84 (m, 4H; (CH3)2CH-), 1.03 (t,J ACHTUNGTRENNUNG(H,H) =8 Hz; CH3CH2Si), 0.89 (q, J ACHTUNGTRENNUNG(H,H) =8 Hz; -CH2Si), 0.88 (t,J ACHTUNGTRENNUNG(H,H) =7 Hz; CH3-CH2-), 0.78 (m, 24H; (CH3)2CH-), 0.67 (m, 2H;-CH2-), 0.41 ppm (br s; CH3Si); MALDI-TOF MS: m/z (%): 5676 (100);GPC: Mn =12150 g mol�1; PDI (Mw/Mn) =1.24.

Ac-V4-PFEMS9 : (54 % yield): 1H NMR spectrum is essentially the sameas that for Ac-V4-PFEMS18 after consideration of the different PFEMSchain length; MALDI-TOF MS: m/z (%): 3115 (100); GPC: Mn =

5930 gmol�1; PDI (Mw/Mn)=1.22.

Acknowledgements

The authors would like to acknowledge the Royal Thai GovernmentCivil Service Commission, European Union, Royal Society (London) andBBSRC for financial support, Kyoung Taek Kim for a sample ofPFDMS-NH2 used in the preliminary work and members of the Mannersgroup for helpful suggestions.

[1] G. W. M. Vandermeulen, H.-A. Klok, Macromol. Biosci. 2004, 4,383 – 398.

[2] H. G. Bçrner, H. Schlaad, Soft Matter 2007, 3, 394 – 408.[3] J.-F. Lutz, H. G. Bçrner, Prog. Polym. Sci. 2008, 33, 1 –39.[4] H. G. Bçrner, Prog. Polym. Sci. 2009, 34, 811 – 851.[5] H. G. Bçrner, H. K�hnle, J. Hentschel J. Polym. Sci. Part A: Polym.

Chem. 2010, 48, 1– 14.[6] O. D. Krishna, K. L. Kiick, Biopolymers 2010, 94, 32 –48.[7] B. Gallot, Prog. Polym. Sci. 1996, 21, 1035 –1088.[8] H.-A. Klok, Angew. Chem. 2002, 114, 1579 –1583; Angew. Chem.

Int. Ed. 2002, 41, 1509 – 1513.[9] L. Ayres, M. R. J. Vos, P. J. H. M. Adams, I. O. Shklyarevskiy,

J. C. M. van Hest, Macromolecules 2003, 36, 5967 –5973.[10] L. Ayres, P. H. H. M. Adams, D. W. P. M. Lçwik, J. C. M. van Hest,

Biomacromolecules 2005, 6, 825 –831.[11] M. A. Gauthier, H.-A. Klok, Chem. Commun. 2008, 2591 –2611.[12] D. W. P. M. Lçwik, E. H. P. Leunissen, M. van den Heuvel, M. B.

Hansen, J. C. M. van Hest, Chem. Soc. Rev. 2010, 39, 3394 –3412.[13] M. Pechar, P. Kopeckova, L. Joss, J. Kopecek, Macromol. Biosci.

2002, 2, 199 –206.[14] G. W. M. Vandermeulen, C. Tziatzios, R. Duncan, H.-A. Klok, Mac-

romolecules 2005, 38, 761 –769.[15] G. W. M. Vandermeulen, C. Tziatzios, H.-A. Klok, Macromolecules

2003, 36, 4107 –4114.[16] D. N. Woolfson, Biopolymers 2010, 94, 118 –127.[17] H. M. Kçnig, A. F. M. Kilbinger, Angew. Chem. 2007, 119, 8484 –

8490; Angew. Chem. Int. Ed. 2007, 46, 8334 – 8340.

www.chemeurj.org � 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Chem. Eur. J. 2012, 18, 2524 – 25352534

I. Manners, D. N. Woolfson et al.

[18] Y.-b. Lim, M. Lee, J. Mater. Chem. 2008, 18, 723 – 727.[19] T. S. Burkoth, T. L. S. Benzinger, D. N. M. Jones, K. Hallenga, S. C.

Meredith, D. G. Lynn, J. Am. Chem. Soc. 1998, 120, 7655 – 7656.[20] T. S. Burkoth, T. L. S. Benzinger, V. Urban, D. G. Lynn, S. C. Mere-

dith, P. Thiyagarajan, J. Am. Chem. Soc. 1999, 121, 7429 – 7430.[21] T. S. Burkoth, T. L. S. Benzinger, V. Urban, D. M. Morgan, D. M.

Gregory, P. Thiyagarajan, R. E. Botto, S. C. Meredith, D. G. Lynn, J.Am. Chem. Soc. 2000, 122, 7883 – 7889.

[22] O. Rathore, M. J. Winningham, D. Y. Sogah, J. Polym. Sci. Part A:Polym. Chem. 2000, 38, 352 –366.

[23] O. Rathore, D. Y. Sogah, Macromolecules 2001, 34, 1477 –1486.[24] O. Rathore, D. Y. Sogah, J. Am. Chem. Soc. 2001, 123, 5231 –5239.[25] C. Zhou, B. Leng, J. Yao, J. Qian, X. Chen, P. Zhou, D. P. Knight, Z.

Shao, Biomacromolecules 2006, 7, 2415 –2419.[26] J. H. Collier, P. B. Messersmith, Adv. Mater. 2004, 16, 907 –910.[27] J. M. Smeenk, M. B. J. Otten, J. Thies, D. A. Tirrell, H. G. Stunnen-

berg, J. C. M. van Hest, Angew. Chem. 2005, 117, 2004 –2007;Angew. Chem. Int. Ed. 2005, 44, 1968 –1971.

[28] V. Castelletto, I. W. Hamley, Biophys. Chem. 2009, 141, 169 –174.[29] N. Tzokova, C. M. Fernyhough, M. F. Butler, S. P. Armes, A. J.

Ryan, P. D. Topham, D. J. Adams, Langmuir 2009, 25, 11082 –11089.[30] A. Sinaga, T. A. Hatton, K. C. Tam, Biomacromolecules 2007, 8,

2801 – 2808.[31] A. Sinaga, T. A. Hatton, K. C. Tam, Macromolecules 2007, 40, 9064 –

9073.[32] A. Sinaga, T. A. Hatton, K. C. Tam, J. Phys. Chem. B 2008, 112,

11542 – 11550.[33] D. Eckhardt, M. Groenewolt, E. Krause, H. G. Bçrner, Chem.

Commun. 2005, 2814 – 2816.[34] J. Hentschel, E. Krause, H. G. Bçrner, J. Am. Chem. Soc. 2006, 128,

7722 – 7723.[35] H. K�hnle, H. G. Bçrner, Angew. Chem. 2009, 121, 6552 –6556;

Angew. Chem. Int. Ed. 2009, 48, 6431 –6434.[36] J. Hentschel, H. G. Bçrner, J. Am. Chem. Soc. 2006, 128, 14142 –

14149.[37] E. Jahnke, I. Lieberwirth, N. Severin, J. P. Rabe, H. Frauenrath,

Angew. Chem. 2006, 118, 5510 –5513; Angew. Chem. Int. Ed. 2006,45, 5383 –5386.

[38] a) G. W. M. Vandermeulen, K. T. Kim, Z. Wang, I. Manners, Bio-macromolecules 2006, 7, 1005 –1010; b) G. R. Whittell, M. D. Hager,U. S. Schubert, I. Manners, Nature Mater. 2011, 10, 167 –188.

[39] K. T. Kim, C. Park, G. W. M. Vandermeulen, D. A. Rider, C. Kim,M. A. Winnik, I. Manners, Angew. Chem. 2005, 117, 8178 –8182;Angew. Chem. Int. Ed. 2005, 44, 7964 –7968.

[40] Y. Wang, S. Zou, K. T. Kim, I. Manners, M. A. Winnik, Chem. Eur.J. 2008, 14, 8624 –8631.

[41] K. T. Kim, G. W. M. Vandermeulen, M. A. Winnik, I. Manners, Mac-romolecules 2005, 38, 4958 –4961.

[42] E. Jahnke, N. Severin, P. Kreutzkamp, J. P. Rabe, H. Frauenrath,Adv. Mater. 2008, 20, 409 –414.

[43] E. Jahnke, J. Weiss, S. Neuhaus, T. N. Hoheisel, H. Frauenrath,Chem. Eur. J. 2009, 15, 388 – 404.

[44] E.-K. Schillinger, E. Mena-Osteritz, J. Hentschel, H. G. Bçrner, P.B�uerle, Adv. Mater. 2009, 21, 1562 – 1567.

[45] P. Y. Chou, G. D. Fasman, Biochemistry 1974, 13, 211 – 222.[46] J. Kyte, R. F. Doolittle, J. Mol. Biol. 1982, 157, 105 –132.[47] W. C. Chan, P. D. White, Fmoc Solid Phase Peptide Synthesis: A

Practical Approach, Oxford University Press, Oxford, 2000.[48] R. Petersen, D. A. Foucher, B.-Z. Tang, A. Lough, N. P. Raju, J. E.

Greedan, I. Manners, Chem. Mater. 1995, 7, 2045 – 2053.[49] K. Kulbaba, I. Manners, Macromol. Rapid Commun. 2001, 22, 711 –

724.[50] D. A. Rider, K. A. Cavicchi, K. N. Power-Billard, T. P. Russell, I.

Manners, Macromolecules 2005, 38, 6931 – 6938.[51] T. Miyazawa, E. R. Blout, J. Am. Chem. Soc. 1961, 83, 712 – 719.[52] J. M. Berg, J. L. Tymoczko, L. Stryer, Biochemistry 5th ed., W. H.

Freeman & Co, New York, 2002.[53] P. C. Hiemenz, T. P. Lodge, Polymer Chemistry, 2nd ed., CRC Press,

Boca Raton, FL, 2007.[54] X. Wang, K. Liu, A. C. Arsenault, D. A. Rider, G. A. Ozin, M. A.

Winnik, I. Manners, J. Am. Chem. Soc. 2007, 129, 5630 – 5639.[55] X.-S. Wang, A. Arsenault, G. A. Ozin, M. A. Winnik, I. Manners, J.

Am. Chem. Soc. 2003, 125, 12686 – 12687.[56] H. Wang, X. Wang, M. A. Winnik, I. Manners, J. Am. Chem. Soc.

2008, 130, 12921 –12930.[57] I. D�ez, H. Hahn, O. Ikkala, H. G. Bçrner, R. H. A. Ras, Soft Matter

2010, 6, 3160 –3162.[58] A. B. Pangborn, M. A. Giardello, R. H. Grubbs, R. K. Rosen, F. J.

Timmers, Organometallics 1996, 15, 1518 –1520.

Received: July 20, 2011Published online: January 19, 2012

Chem. Eur. J. 2012, 18, 2524 – 2535 � 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.chemeurj.org 2535

FULL PAPERPolyferrocenylsilane–Tetrapeptide Conjugates