Schematic of polymerizations of N-carboxyanhydrides initiated by Ni(0) complexes. Adapted from Reference 285

The oxidative addition reactions are followed by the attachment of a second NCA molecule and yield six-membered amido-alkyl metallacyles. Further, these cyclic intermediates contract to a

35

five-membered ring upon addition of the NCA monomers. This process was explained to occur via proton migration from an amide group to the metal bound carbon, thus liberating the chain end from the metal. The cyclic intermediate was proposed as the active specie capable of initiating the nucleophilic attack of the amido group on the electrophilic C-5 of the NCA.

316-318,320

The large cycle formed as the result of the propagation could contract through CO2 release.

The amido amidate propagating species formed as a result of the proton transfer from the free amide to amidate group enables ring contraction. In fact, the end chain of the polymer becomes free and the metal chelate can migrate along the growing polymer.³²¹ Despite some ambiguity regarding the correct mechanistic route, Deming’s work spans a variety of preparative applications. Variation of the metal allowed good control over the molecular weight and PDI of poly(glutamate)s, and the use of the different NCA precursors resulted in a broad range of homopolypeptides. No less important is the multitude of the architectural copolypeptides designed by using metal chelate initiation.^322-345

The common theme of improving the classical amine-initiated polymerization initiators was also shared by other groups. Hadjichristidis conducted primary amine initiated polymerizations of NCAs under high vacuum conditions. ^346-353 Even though doubts were placed on the purification efficiency of NCA monomer under high vacuum when compared with the recrystallization under an inert atmosphere, the polymerization of the γ-benzyl-L-glutamate NCA (BLG-NCA) and N-carbobenzyloxy-L-lysine NCA, (CBL-NCA) in DMF under high vacuum was found to have a living character. The main conclusion drawn by the authors outlines that the side reactions in the amine-initiated mechanism are a result of the impurities contained in the monomer. Furthermore, the continuous removal of CO₂ generated during the reaction can mediate the polymerization.^346-353 Low-polydispersity products were obtained with predictable

36

molecular weight but the method needs more insight on the chain-end characterization. In a recent report of Pickel et al., the preparation of the poly(o-benzyl-L-tyrosine), PoTYR, in DMF was performed both using high vacuum techniques and glovebox environment.³⁵⁴ Polymers prepared by initiation with 1,6-diaminohexane in the high vacuum followed the normal amine pathway, the termination occurring with DMF. Products prepared in the glovebox showed initiation by both amine and activated monomer mechanisms and the termination yielded several species. The nano-assisted desorption/ionization time-of-flight mass spectrometry, NALDI-TOF MS, was used for the chain-end analysis and the conclusion pointed to the end-group fidelity obtained via high vacuum.³⁵⁴

N-trimethylsilylamines were used for controlled ring opening polymerization of NCAs by Cheng and coworkers.³⁵⁵ The unique feature of this technique is that the amine end-groups are obtained after cleaving the protecting trimethylsilyl end-group. A narrow distribution was achieved and the ¹³C NMR, the fast atom bombardment mass spectrometry, FAB-MS,³⁵⁶ the matrix assisted laser adsorption ionization time of flight, MALDI-TOF,³⁵⁵ and the electrospray ionization mass spectrometry, ESI-MS,³⁵⁷ determined the nature of terminal groups. In order to avoid the formation of NCA anions during the polymerization, Schlaad et al. have utilized primary amine hydrochloride salts as initiating species. Their goal was to inhibit the polymerization through the activated monomer mechanism, obtaining in this way low dispersity.

A drawback of this work points to the lack in the end-group determination. Higher values of molecular weight were obtained for products when compared with those theoretically computed.^358-360

The identification of the terminal groups is also important from another point of view.

For a long time in the NCA ring opening polymerization era it was believed that resulting

37

adducts were linear, unless branching was intentionally desired. One exception stated otherwise³⁶¹ until Kricheldorf confirmed the in situ formation of the cyclic polypeptides along with the linear ones.^297,362 Solvents with high nucleophilicity and donor ability such as pyridine, DMF, N-methylpyrrolidone, NMP, and dimethylsulfoxide, DMSO, were found to catalyze zwitterionic polymerizations.³⁶³ The Scheme 1.13 displays the interactions between the polymerization species when the solvent involved is pyridine.

Scheme 1.13 General route of the pyridine-mediated ring opening polymerization of N-protected N-carboxyanhydrides via zwitterionic ionic intermediates. Adapted from Reference 285.

Consequently, the synthesis of homo- and copolypeptides in these solvents should take into account that the polymerization mixture might be a blend of most linear chains with cyclic

“contaminations”. Soluble polypeptides that adopt a random coil conformation showed a high tendency toward cyclization.³⁶⁴ It has been known that traditional NCA ring opening polymerization leads to low-molecular weight polymers. Once removed (and usually discarded)

38

the remaining high-molecular weight polymer is very good (Mw/Mn ~ 1.2). Thus, it will be more efficient to fractionate and keep both high-molecular weight and low-molecular weight polymers.

From the perspective of the next section dedicated to core shell systems where the core consists of an inorganic hard sphere and a shell built from a fuzzy homopolypeptide, several words will be spent first on the enormous potential of polypeptides as shell materials. Depending on their side chain substituent, polypeptides can deliver a variety of versatile properties such as chirality, ability to form liquid crystals (LC), secondary structure structural changes as a response to external stimuli, etc. No less important than self assembly in spherical and cylindrical micelles or vesicles, the ability of polypeptides to form liquid crystalline phases has attracted a lot of interest. One of the most studied polypeptides is poly(γ-benzyl-L-glutamate), PBLG.³⁶⁵ Most of the early publications concentrated on PBLG lyotropic LCs are mentioned in the book by Block.³⁶⁶ The first reports of PBLG LC phases date in the 1950s365,367,368

and since then an immense volume of the work was dedicated to its LC behavior.366,369-388

Studies on glutamates with different side chain moieties were reviewed by Daly et al. ³⁸⁹ The Russo group focused on PBLG’s and other glutamate-based liquid crystals, aggregation and gelation behavior.274,390-407

The driving force behind such phenomenon is the change in the secondary structure and its effect on the phase behavior. Substituents attached to the side chains were found to induce thermotropic transition.

A very commonly used technique in the phase behavior for concentrated solutions of poly(glutamate ester)s was optical rotation dispersion, ORD. It has an advantage over circular dichroism, CD, because the interference from the solvent can be better discriminated. ORD can also deal very well with solutions prepared in solvents not suitable for CD. Most studies focused

39

on the characterization of the cholesteric phase.^407-409 It was found that the pitch of the cholesteric helix varies inversely proportional to the optical rotation. More or less recent investigations dealt with the specific features of helix formation and tried to simulate different models. Yet, below a critical point (temperature, molecular weight), the ORD signal suddenly became intense.⁴¹⁰ This jump in the intensity was attributed to the short range order of the long axes of the α helices. The orientational effect, as well the distorsions (disclinations) in the cholesteric phase were studied by varying temperature,^373,411 solvent,⁴¹² polydispersity of the polymer^413,414 , surfactants,^415,416 and concentration.^417,418 Small-angle X-ray scattering, SAXS, and dynamic/static³⁷³ light scattering, DLS/SLS, techniques added more information to the mechanism of helix formation and LC stabilization. Α helices possess a high dipole moment which can be influenced by electric^381,419 and magnetic fields.^420,421 The helix order vector aligns parallel to an external field. Strong fields can hamper the formation of the cholesteric phase.

Chiral and special solvents,^422-424 surfactants and fluorescent tags were additional tools used to influence the molecular orientation in the LC phases. The diffusion coefficients of the rod-like mesogens (PBLG) were measured by the means of the pulse field gradient nuclear magnetic resonance, PFG-NMR.^425-428 The diffusion coefficient for a solution of PBLG in chloroform at 30°C decreased from the isotropic to the cholesteric phase. The diffusion parallel, D^II, to the helix axis was higher than perpendicular, D┴. The diffusion behavior of the PBLG in concentrated solutions was found to follow Kirkwood theory. Very recently investigations with fluorescence photobleaching recovery, FPR, on the magnetically aligned fluorescein (FITC) labeled PBLG have found that the rate of the parallel diffusion in the direction of α-helix declined with the concentration while the rate perpendicular remained approximately constant.

At low concentrations of the LC domain, the ratio between D^II and D┴ was 5 but at higher

40

concentrations it was 2. The molecular mobility and the flexure effects on the LC phase in m-cresol were studied by rheology.372,429-435

Mesophases occurring as a result of the transition from various LC states were investigated in terms of stability.^436,437 The nature of the side chain substituents and their influence on the molecular orientation was studied by varying the temperature.⁴²⁴ The work of Watanabe et al. on random copolymers made of different glutamates and on polyglutamates with mesogenic groups was dedicated to thermotropic phase behavior.

438-455 The pitch of the molecular helix was found to increase with the length of the n-alkyl chains and with the temperature.⁴⁵⁶ A columnar hexagonal mesophase formed at temperatures below the cholesteric phase. Phase behavior of PBLG in m-cresol was also studied in depth.^457,458 Research performed by other groups used differential scanning calorimetry, DSC,⁴⁵⁹ NMR,425,460-462

electron spin resonance, ESR⁴⁶³, X-ray photoelectron spectroscopy, XPS,²⁰ and CD to understand better the LC behavior. Polarized light microscopy, POM, was another technique used to visualize the morphologies of the LC phases and to calculate the order parameter.

384,464-466 Many other investigations were extended to poly(L-lysine),^467-470 poly(L-alanine),⁴⁷¹ poly(γ-benzyl-L-aspartate),^472,473 and other unusual polypeptides.⁴⁷⁴

The end of this section briefly summarizes the ability of polypeptides to undergo conformational transitions induced by the temperature, solvent, pH and surfactants. Doty⁴⁷⁵, Blout⁴⁷⁶ and Applequist⁴⁷⁷ conducted pioneering studies on PBLG helix-coil transition. A well-defined transition temperature for PBLG in dichloroacetic acid, DCA, (37% vol.)-chloroform (63% vol.) mixture was identified.^478,479 The heat capacity associated to the transition was determined by calorimetry. Watanabe et al. measured the dielectric constant and electric birefringence of the PBLG in DCA–EDC, 1,2-dichloroethane, mixture. Addition of DCA in small amount (~0.1%) caused a sharp change of these properties and was assigned to the

helix-41

coil transition.⁴⁸⁰ Ackermann et al. investigated the thermodynamics of PBLG in the same solvent mixture by polarimetric measurements.⁴⁸¹ The critical transition temperature increased with the PBLG concentration at constant solvent composition. This trend was also noted when the PBLG solutions had equal concentration but the amount of DCA increased. Both studies seem to agree that parameters such as transition enthalpy or cooperativity depend greatly on the concentration, the temperature and the solvent composition. The present trend in the literature concerns PBLG copolymers and the aggregation behavior based on the secondary structure changes.^482,483 Polytyrosine, PTYR, undergoes a helix-coil transition in mixtures of dimethylsulfoxide, DMSO, and DCA.^484,485 Upon addition of a strong base, NaOH, PTYR changed the helix conformation into the β- sheet.⁴⁸⁶Poly(N^ε-carbobenzyloxy-L-lysine), PCBL, has a reverse transition from helix to coil in m-cresol as a function of the temperature.^487-489 Poly(β-benzyl-L-aspartate) is another polypeptide known to change its conformation in m-cresol.⁴⁹⁰ Deprotection of side chains of the PBLG, PCBL, etc., enables the pH-dependent conformational transitions.^491-493 Helix-to-helix⁴⁹⁴ or helix-to-sheet^495,496 changes were also reported. A picture of the processes involved in the structural changes will be given in other chapters of this document.

1.8 Silica Polypeptide-based Hybrids

The past decades encountered a rapid development of micro- and nano-scale materials which found applications in wide areas such as medicine, industry and technology. Core-shell particles having a well-defined solid core made from silica and a shell comprised of a polymer can display a range of projected behaviors besides colloidal stability and robustness. From all core-shell particles, silica particles featuring a polypeptide shell have become lately an expanding field of research because of their potential applications, still in the incipient phase of

42

the exploration. In this section early reports on silica polypeptide hybrids will be summarized and, as well, trends in present research. Methods used in hybrid material synthesis such as grafting to (onto) and growing from will be compared and the shortcomings in designing the

desired properties of the final product will be also addressed. The main steps involved in these two techniques are presented in Scheme 1.14.