Synthesis of Polypeptides by Ring-Opening Polymerization of a-amino Acid N-Carboxyanhydrides

Transcription

1 Top Curr Chem (2011) DI: /128_2011_173 # Spriger-Verlag Berli eidelberg 2011 Sythesis of Polypeptides by ig-peig Polymerizatio of a-amio Acid -Carboxyahydrides Jiaju Cheg ad Timothy J. Demig Abstract This chapter summarizes methods for the sythesis of polypeptides by rig-opeig polymerizatio. Traditioal ad recetly improved methods used to polymerize a-amio acid -carboxyahydrides (CAs) for the sythesis of homopolypeptides are described. Use of these methods ad strategies for the preparatio of block copolypeptides ad side-chai-fuctioalized polypeptides are also preseted, as well as a aalysis of the sythetic scope of differet approaches. Fially, issues relatig to obtaiig highly fuctioal polypeptides i pure form are detailed. Keywords Amio acid Block copolymer -Carboxyahydride Polymerizatio Polypeptide Cotets 1 Itroductio 2 Polypeptide Sythesis Usig CAs 2.1 Covetioal Methods 2.2 Trasitio Metal Iitiators 2.3 ecet Developmets 3 Copolypeptide ad Fuctioal Polypeptide Sythesis via CA Polymerizatio 3.1 Block Copolypeptides 3.2 de-chai-fuctioalized Polypeptides 4 Polypeptide Deprotectio ad Purificatio 5 Coclusios ad Future Prospects efereces J. Cheg Departmet of Materials Sciece ad Egieerig, Uiversity of Illiois at Urbaa-Champaig, Champaig, IL 61801, USA T.J. Demig (*) Departmet of Bioegieerig, Uiversity of Califoria, Los Ageles, CA 90095, USA [email protected]

2 J. Cheg ad T.J. Demig Abbreviatios AM B-Glu bpy CD CD DMF DMS EDTA EG FTI GPC GTP MDS MS ACE CA PBLG PDMS PEG TFA-Lys TF TMS TMSDC Z-Lys Activated moomer g-bezyl-l-glutamate 2,2 0 -Bipyridyl Circular dichroism 1,4-Cyclooctadiee Dimethylformamide Dimethyl sulfoxide Ethyleediamie tetraacetic acid Ethylee glycol Fourier trasform ifrared spectroscopy Gel permeatio chromatography Group trasfer polymerizatio examethyldisilazae Mass spectroscopy o-aqueous capillary electrophoresis a-amio acid--carboxyahydride Poly(g-bezyl L-glutamate) Poly(dimethylsiloxae) Poly(ethylee glycol) e-trifluoroacetyl-l-lysie Tetrahydrofura Trimethylsilyl Trimethylsilyl dimethylcarbamate e-carbobezyloxy-l-lysie 1 Itroductio Biological systems produce proteis that possess the ability to self-assemble ito complex, yet highly ordered structures [1]. These remarkable materials are polypeptide copolymers that derive their properties from precisely cotrolled sequeces ad compositios of their costituet amio acid moomers. There has bee recet iterest i developig sythetic routes for preparatio of these atural polymers as well as de ovo desiged polypeptide sequeces to make products for applicatios i biotechology (e.g., artificial tissues ad implats), biomieralizatio (e.g., resiliet, lightweight ad ordered iorgaic composites), ad aalysis (e.g., biosesors ad medical diagostics) [2, 3]. To be successful i these applicatios, it is importat that materials ca selfassemble ito precisely defied structures. Peptide-based polymers have may advatages over covetioal sythetic polymers sice they are able to hierarchically assemble ito stable, ordered coformatios [4]. Depedig o the substituets of the amio acid side chai, polypeptides are able to adopt a multitude of

3 Sythesis of Polypeptides by ig-peig Polymerizatio coformatioally stable, regular secodary structures (e.g., helices, sheets, ad turs), tertiary structures (e.g., the b-strad-helix b-strad uit foud i b-barrels), ad quaterary assemblies (e.g., collage microfibrils) [4]. The sythesis of polypeptides that ca assemble ito o-atural structures is a attractive challege for polymer chemists. Sythetic peptide-based polymers are ot ew materials: homopolymers of polypeptides have bee available for may decades but have oly see limited use as structural materials [5, 6]. owever, ew methods i chemical sythesis have made possible the preparatio of icreasigly complex polypeptide sequeces of cotrolled molecular weight that display properties far superior to ill-defied homopolypeptides [7]. These polymers are well suited for applicatios where polymer assembly ad fuctioal domais eed to be at legth scales ragig from aometers to micrometers. These block copolymers are homogeeous o a macroscopic scale, but dissimilarity betwee the block segmets typically results i microphase heterogeeity yieldig materials useful as surfactats, micelles, membraes, ad elastomers [8]. Sythesis of simple hydrophilic/hydrophobic hybrid diblock copolymers, whe dispersed i water, allows formatio of peptide-based micelles ad vesicles potetially useful i drug ad gee delivery applicatios [9, 10]. The regular secodary structures obtaiable with the polypeptide blocks provide opportuities for hierarchical self-assembly that are uobtaiable with typical block copolymers or small-molecule surfactats. Upo examiig the differet methods for polypeptide sythesis, the limitatios of these techiques for preparatio of well-defied copolymers readily become apparet. Covetioal solid-phase peptide sythesis is either useful or practical for direct preparatio of large polypeptides (> 100 residues) due to uavoidable deletios ad trucatios that result from icomplete deprotectio ad couplig steps. The most ecoomical ad expediet process for sythesis of log polypeptide chais is the polymerizatio of a-amio acid--carboxyahydrides (CAs) (Scheme 1) [11, 12]. This method ivolves the simplest reagets, ad high molecular weight polymers ca be prepared i both good yield ad large quatity with o detectable racemizatio at the chiral ceters. The cosiderable variety of CAs that have bee sythesized (> 200) allows exceptioal diversity i the types of polypeptides that ca be prepared [11, 12]. ce the late 1940s, CA polymerizatios have bee the most commo techique used for large scale preparatio of high molecular weight polypeptides [13]. owever, these materials have primarily bee homopolymers, radom copolymers, or graft copolymers that lack the sequece specificity ad moodispersity of atural CA ucleophile or base polypeptide + C 2 Scheme 1 Polymerizatio of a-amio acid--carboxyahydrides (CA)

4 J. Cheg ad T.J. Demig proteis. Util recetly, the level of cotrol i CA polymerizatios has ot bee able to rival that attaied i other sythetic polymerizatios (e.g., viyl additio polymerizatios) where sophisticated polymer architectures have bee prepared (e.g., stereospecific polymers ad block copolymers) [14]. Attempts to prepare block copolypeptides ad hybrid block copolymers usig covetioal CA polymerizatio has usually resulted i polymers whose compositios did ot match moomer feed compositios ad that cotaied sigificat homopolymer cotamiats [15 17]. Block copolymers could oly be obtaied i pure form by extesive fractioatio steps, which sigificatly lowered the yield ad efficiecy of this method. The limitatio of CA polymerizatios has bee the presece of side reactios (chai termiatio ad chai trasfer) that restrict cotrol over molecular weight, give broad molecular weight distributios, ad prohibit formatio of welldefied block copolymers [18]. ecet progress i elimiatio of these side reactios has bee a major breakthrough for the polypeptide materials field. A variety of metal- ad orgao-catalysts have bee developed ad utilized i recet years for the formatio of multiblock polypeptides or polypeptide-cotaiig hybrid materials with well-defied structures via cotrolled polymerizatio of CAs [19 22]. 2 Polypeptide Sythesis Usig CAs 2.1 Covetioal Methods CA polymerizatios are traditioally iitiated usig may differet ucleophiles ad bases, the most commo beig primary amies ad alkoxide aios [11, 12]. Primary amies, beig more ucleophilic tha basic, are good geeral iitiators for polymerizatio of CA moomers. Tertiary amies, alkoxides, ad other iitiators that are more basic tha ucleophilic have foud use sice they are i some cases able to prepare polymers of very high molecular weights where primary amie iitiators caot. ptimal polymerizatio coditios have ofte bee determied empirically for each CA ad thus there have bee o uiversal iitiators or coditios by which to prepare high molecular weight polymers from ay moomer. This is i part due to the differet properties of idividual CAs ad their polymers (e.g., solubility ad reactivity) but is also strogly related to the side reactios that occur durig polymerizatio. The most likely pathways of CA polymerizatio are the so-called amie ad the activated moomer (AM) mechaisms [11, 12]. The amie mechaism is a ucleophilic rig-opeig chai growth process where the polymer could grow liearly with moomer coversio if side reactios were abset (Scheme 2). the other had, the AM mechaism is iitiated by deprotoatio of a CA, which the becomes the ucleophile that iitiates chai growth (Scheme 3). It is importat to ote that a give system ca switch back ad forth betwee the amie ad AM mechaisms may times durig a polymerizatio: a propagatio step for oe

5 Sythesis of Polypeptides by ig-peig Polymerizatio ' 2 + ' ' 2 + C 2 C 2 + ' 2 CA Scheme 2 Proposed mechaism for CA polymerizatio iitiated by ucleophilic amies amioacyl CA 2 +CA + trasfer C reactio with CA or -amioacyl CA oligopeptides further codesatio 2 Scheme 3 Proposed mechaism for CA polymerizatio iitiated by activated moomers mechaism is a side reactio for the other, ad vice versa. It is because of these side reactios that block copolypeptides ad hybrid block copolymers prepared from CAs usig amie iitiators ofte have structures differet to those predicted by moomer feed compositios ad most likely have cosiderable homopolymer cotamiatio. These side reactios also prevet cotrol of chai-ed fuctioality, which is crucial for preparatio of hybrid copolymers. e iheret problem i covetioal CA polymerizatios is that there is o cotrol over the reactivity of the growig polymer chai-ed durig the course of the polymerizatio. ce a iitiator reacts with a CA moomer, it is o loger active i the polymerizatio ad the resultig primary amie, carbamate, or CA aio ed group is free to udergo a variety of udesired side reactios. Aother problem is associated with the purity of CA moomers. Although most CAs are crystallie compouds, they typically cotai miute traces of acid, acid chlorides, or isocyaates that ca quech propagatig chais. The presece of other advetitious impurities, such as water, ca cause problems by actig as chai-trasfer agets or eve as catalysts for side reactios. verall, the sheer abudace of potetial reactios preset i reactio media make it difficult to achieve a livig polymerizatio system where oly chai propagatio occurs.

6 J. Cheg ad T.J. Demig 2.2 Trasitio Metal Iitiators e strategy for elimiatig side reactios i CA polymerizatios is the use of trasitio metal complexes as active species to cotrol additio of CA moomers to polymer chai-eds. The use of trasitio metals to cotrol reactivity has bee prove i orgaic ad polymer sythesis as a meas to icrease both reactio selectivity ad efficiecy [23]. Usig this approach, substatial advaces i cotrolled CA polymerizatio have bee realized i recet years. ighly effective zerovalet ickel ad cobalt iitiators (i.e., (PMe 3 ) 4 Co [24], ad bpyi(cd), where bpy ¼ 2,2 0 -bipyridie ad CD ¼ 1,5-cyclooctadiee [19]) were developed by Demig that allow the livig polymerizatio of CAs ito high molecular weight polypeptides via a uprecedeted activatio of the CAs ito covalet propagatig species. The metal ios ca be coveietly removed from the polymers by simple precipitatio or dialysis of the samples after polymerizatio. Mechaistic studies o the iitiatio process showed that both these metals react idetically with CA moomers to form metallacyclic complexes by oxidative additio across the ahydride bods of CAs [19, 24, 25]. These oxidative-additio reactios were followed by additio of a secod CA moomer to yield complexes idetified as six-membered amido-alkyl metallacycles (Scheme 4). These itermediates were foud to further cotract to five-membered amido-amidate metallacycles upo reactio with additioal CA moomers. This rig cotractio is thought to occur via migratio of a amide proto to the metal-boud carbo, which liberates the chai-ed from the metal (Scheme 5)[26]. The resultig amidoamidate complexes were thus proposed as the active polymerizatio itermediates. Propagatio through the amido-amidate metallacycle was evisioed to occur by iitial attack of the ucleophilic amido group o the electrophilic C 5 carboyl of a CA moomer (Scheme 6). This reactio would result i a large metallacycle that (L) M + M = Co, i C 5 C " " (L)M CA 2 C 2 (L)M Scheme 4 xidative-additio of CAs to zerovalet cobalt ad ickel complexes (L)M CA C 2 (L)M proto migratio M(L) Scheme 5 Metallacycle rig cotractio mediated by CA additio

7 Sythesis of Polypeptides by ig-peig Polymerizatio (L)M + polymer (L)M C 2 polymer proto migratio M(L) polymer Scheme 6 Chai propagatio i trasitio-metal-mediated CA polymerizatio could cotract by elimiatio of C 2. Proto trasfer from the free amide to the tethered amidate group would further cotract the rig to give the amido-amidate propagatig species, while i tur liberatig the ed of the polymer chai ad becomig available for reactio with the ext icomig CA molecule. I this maer, the metal is able to migrate alog the growig polymer chai, while beig held by a robust chelate at the active ed. The formatio of these chelatig metallacyclic itermediates appears to be a geeral requiremet for obtaiig livig CA polymerizatios usig trasitio metal iitiators. These cobalt ad ickel complexes are able to produce polypeptides with arrow chai legth distributios (give by the polydispersivity idex, i.e., the weight-average molecular weight divided by the umber-average molecular weight, M w /M, which i this case is < 1.2) ad cotrolled molecular weights (500 < M < 500,000 g/mol) [26]. This polymerizatio system is very geeral, ad gives cotrolled polymerizatio of a wide rage of CA moomers as pure eatiomers (D- or L-cofiguratio) or as racemic mixtures. By additio of differet CA moomers, the preparatio of block copolypeptides of defied sequece ad compositio is feasible [7, 27]. The trasitio metal iitiators for CA polymerizatio described above should provide a meas for cotrolled sythesis of polypeptide hybrid block copolymers. owever, a limitatio of this methodology whe usig zerovalet metal complexes as iitiators is that the active propagatig species are geerated i situ, where the C-termial ed of the polypeptide is derived from the first CA moomer. Cosequetly, this method does ot allow attachmet of fuctioality (e.g., polymer or small molecule) to the carboxyl chai-ed of the polypeptides. To facilitate cotrol over the C-termial chai-ed, Demig ad coworkers pursued alterative methods for direct sythesis of the amido-amidate metallacycle propagatig species ad developed allyloxycarboylamioamides as uiversal precursors to amido-amidate ickelacycles. These simple amio acid derivatives udergo tadem oxidative additios to ickel(0) to give active CA polymerizatio iitiators (Scheme 7) [28]. These complexes were foud to iitiate polymerizatio of CAs yieldig polypeptides with defied molecular weights, arrow molecular weight distributios, ad with quatitative icorporatio of the iitiatig ligad as a C-termial ed-group. This chemistry provides a very facile way to icorporate diverse molecules such as polymers, peptides, oligosaccharides, or other ligads oto the carboxyl chai-eds of polypeptides via a robust amide likage (Scheme 8), ad was further elaborated by Mezel s group to grow polypeptides off polystyree particles [29].

8 J. Cheg ad T.J. Demig X = ligad, peptide, polymer X " " i(cd) 2 i L 2 L 2 X L 2 i X Scheme 7 Formatio of chai-ed fuctioalized ickelacycle iitiators (L) i x x C 2 x Scheme 8 Preparatio of PEG-polypeptide diblock copolymers by macroiitiatio depe i (PBLG) x 1) xs PEG C 2) 3 + (PBLG) x (PEG) Scheme 9 Preparatio of PEG-polypeptide diblock copolymers by isocyaate ed-cappig As a extesio of this work, Demig also developed a meas to ed-cap livig polypeptide chais with electrophilic reagets. Whe a macromolecular electrophile is used, the resultig product is a polypeptide hybrid block copolymer. It is well kow that i CA polymerizatios the electrophiles, such as isocyaates, act as chai-termiatig agets by reactio with the propagatig amie chai-eds [11, 12]. Demig ad coworkers reported that the reactive livig ickelacycle polypeptide chai-eds could be quatitatively capped by reactio with excess isocyaate, isothiocyaate, or acid chloride [30]. Usig this chemistry, they prepared isocyaate ed-capped poly(ethylee glycol) (PEG) ad reacted this, i excess, with livig poly(g-bezyl L-glutamate) (PBLG) to obtai PBLG-PEG diblock copolymers (Scheme 9). eactio with livig ABA triblock copolymers (vide ifra) gave the correspodig PEG-capped CABAC hybrid petablock copolymers, where A was PBLG; B was polyocteemer, PEG or PDMS; ad C was PEG. ce excess PEG was used to ed-cap the livig polypeptide chais, the petablock copolymers required purificatio, which was achieved by repeated precipitatio from TF ito methaol. verall, it ca be see that the use of cotrolled CA polymerizatio allows formatio of very complex hybrid block copolymer architectures that rival those prepared usig ay polymerizatio system. 2.3 ecet Developmets I recet years, a umber of ew approaches have bee reported for obtaiig cotrolled CA polymerizatios. These approaches share a commo theme i that

9 Sythesis of Polypeptides by ig-peig Polymerizatio they are all improvemets o the use of classical primary amie polymerizatio iitiators. This approach is attractive sice primary amies are readily available ad because the iitiator does ot eed to be removed from the reactio after polymerizatio. I fact, if the polymerizatio proceeds without ay chai-breakig reactios, the amie iitiator becomes the C-termial polypeptide ed-group. I this maer, there is potetial to form chai-ed-fuctioalized polypeptides or eve hybrid block copolymers if the amie is a macroiitiator. The challege i this approach is to overcome the umerous side reactios of these systems without the luxury of a large umber of experimetal parameters to adjust. I 2004, the group of adjichristidis reported the primary-amie-iitiated polymerizatio of CAs uder high vacuum coditios [21]. The strategy here was to determie if a reduced level of impurities i the reactio mixture would lead to fewer polymerizatio side reactios. Ulike the viyl moomers usually polymerized uder high vacuum coditios, CAs caot be purified by distillatio. Cosequetly, it is doubtful whether the CAs themselves ca be obtaied i higher purity uder high vacuum recrystallizatio tha by recrystallizatio uder a rigorous iert atmosphere. owever, the high vacuum method does allow for better purificatio of polymerizatio solvets ad the -hexylamie iitiator. It was foud that polymerizatios of g-bezyl-l-glutamate CA (B-Glu CA) ad e-carbobezyloxy-l-lysie CA (Z-Lys CA) uder high vacuum i DMF solvet displayed all the characteristics of a livig polymerizatio system [21]. Polypeptides could be prepared with cotrol over chai legth, chai legth distributios were arrow, ad block copolypeptides were prepared. Cotrolled polymerizatio of CAs uder high vacuum was later cofirmed by Messma ad coworkers [31]. The authors cocluded that the side reactios ormally observed i amieiitiated CA polymerizatios are simply a cosequece of impurities. ce the mai side reactios i these polymerizatios do ot ivolve reactio with advetitious impurities such as water, but istead reactios with moomer, solvet, or polymer (i.e., termiatio by reactio of the amie-ed with a ester side chai, attack of DMF by the amie-ed, or chai trasfer to moomer) [11, 12], this coclusio does ot seem to be well justified. It is likely that the role of impurities (e.g., water) i these polymerizatios is very complex. A possible explaatio for the polymerizatio cotrol observed uder high vacuum is that the impurities act to catalyze side reactios with moomer, polymer, or solvet. I this sceario, it is reasoable to speculate that polar species such as water ca bid to moomers or the propagatig chai-ed ad thus ifluece their reactivity. Further isights ito amie-iitiated CA polymerizatios were also reported i 2004 by the group of Giai ad coworkers [32]. This group studied the polymerizatio of e-trifluoroacetyl-l-lysie CA (TFA-Lys CA) i DMF usig -hexylamie iitiator as a fuctio of temperature. I cotrast to the high vacuum work, the solvet ad iitiator were purified usig covetioal methods ad the polymerizatios were coducted uder a itroge atmosphere o a Schlek lie. After complete cosumptio of CA moomer, the crude polymerizatio mixtures were aalyzed by gel permeatio chromatography (GPC) ad o-aqueous capillary electrophoresis (ACE). A uique feature of this work was the use of ACE to

10 J. Cheg ad T.J. Demig C + 2 Scheme 10 Polypeptide chai termiatio by reactio with CA aios Me Me + 2 Me Me + Scheme 11 Polypeptide chai termiatio by reactio with DMF solvet separate ad quatify the amout of polymers with differet chai-eds, which correspoded to livig chais (amie ed-groups) ad dead chais (carboxylate ad formyl ed-groups from reactio with CA aios ad DMF solvet, respectively; see Schemes 10 ad 11). ot surprisigly, at 20 C, the polymer products cosisted of 78% dead chais ad oly 22% livig chais, which illustrates the abudace of side reactios i these polymerizatios uder ormal coditios. A itriguig result was foud for polymerizatios coducted at 0 C where 99% of the chais had livig amie chai-eds, ad oly 1% were foud to be dead chais. To verify that these were truly livig polymerizatios, additioal CA moomer was added to these chais at 0 C, resultig i icreased molecular weight ad o icrease i the amout of dead chais. Although this was oly a prelimiary study ad further studies eed to be coducted to explore the scope of this method, this work clearly shows that the commo CA polymerizatio side reactios ca also be elimiated by lowerig the temperature. The effect of temperature is ot uusual, as similar treds ca be foud i catioic ad aioic viyl polymerizatios [33]. At elevated temperature, the side reactios have activatio barriers similar to chai propagatio. Whe the temperature is lowered, it appears that the activatio barrier for chai propagatio becomes lower tha that of the side reactios ad chai propagatio domiates kietically. A remarkable feature of this system is that the elevated levels of impurities, as compared to the high vacuum method, do ot seem to cause side reactios at low temperature. This result further substatiates the idea that the growig chais do ot react with the advetitious impurities, but that they maily affect these polymerizatios by alterig the rates of discrete reactio steps. The same group reported recetly that additio of urea i the polymerizatio solutio could also improve the polymerizatio ad miimize the tedecy of formatio of bimodal molecular weight distributio [34]. Aother iovative approach to cotrollig amie-iitiated CA polymerizatios was reported i 2003 by Schlaad ad coworkers [20]. Their strategy was to avoid formatio of CA aios, which cause sigificat chai termiatio after rearragig to isocyaocarboxylates [11, 12], through use of primary amie hydrochloride salts as iitiators. The reactivity of amie hydrochlorides with CAs was first explored by the group of Kobler, who foud that they could react

11 Sythesis of Polypeptides by ig-peig Polymerizatio Cl ' C Cl ' 3 + Cl 3 + Cl Scheme 12 Polypeptide sythesis usig amie-hydrochloride iitiators hydrochlorides with CAs to give sigle CA additio products [35, 36]. Use of the hydrochloride salt takes advatage of its dimiished reactivity as a ucleophile compared to the paret amie, which effectively halts the reactio after a sigle CA isertio by formatio of a iert amie hydrochloride i the product. The reactivity of the hydrochloride presumably arises from formatio of a small amout of free amie by reversible dissociatio of Cl (Scheme 12). This equilibrium, which lies heavily toward the dormat amie hydrochloride species, allows for oly a very short lifetime of reactive amie species. Cosequetly, as soo as a free amie reacts with a CA, the resultig amie ed-group o the product is immediately protoated ad is preveted from further reactio. The acidic coditios also assist elimiatio of C 2 from the reactive itermediate ad, more importatly, suppress formatio of uwated CA aios. To obtai cotrolled polymerizatio, ad ot just sigle CA additio reactios, Schlaad s group icreased the reactio temperature (from 40 to 80 C), which was kow from Kobler s work to icrease the equilibrium cocetratio of free amie, as well as icrease the exchage rate betwee amie ad amie hydrochloride [35, 36]. Usig primary amie hydrochloride ed-capped polystyree macroiitiators to polymerize Z-Lys CA i DMF, Schlaad s group obtaied polypeptide hybrid copolymers i 70 80% yield after 3 days at elevated temperature. Although these polymerizatios are slow compared to amie-iitiated polymerizatios, the resultig polypeptide segmets were well defied with very arrow chai legth distributios (M w /M < 1.03). These distributios were much arrower tha those obtaied usig the free amie macroiitiator, which argues for dimiished side reactios i the polypeptide sythesis. The molecular weights of the resultig polypeptide segmets were foud to be approximately 20 30% higher tha would be expected from the moomer-to-iitiator ratios. This result was attributed to termiatio of some fractio of iitiator species by traces of impurities i the CA moomers, although the presece of ureacted polystyree chais was ot reported. Although more studies eed to be performed to study the scope ad geerality of this system, the use of amie hydrochloride salts as iitiators for cotrolled CA polymerizatios shows tremedous promise. Fast, reversible deactivatio of a reactive species to obtai cotrolled polymerizatio is a prove cocept i polymer chemistry, ad this system ca be compared to the persistet radical effect employed i all cotrolled radical polymerizatio strategies [37]. Like those systems, success of this method requires a carefully cotrolled matchig of the

12 J. Cheg ad T.J. Demig Scheme 13 Polypeptide sythesis usig MDS iitiator MDS polymer chai propagatio rate costat, the amie/amie hydrochloride equilibrium costat, ad the forward ad reverse exchage rate costats betwee amie ad amie hydrochloride salt. This meas that it is likely that reactio coditios (e.g., temperature, halide couterio, solvet) will eed to be optimized to obtai cotrolled polymerizatio for each differet CA moomer, as is the case for most viyl moomers i cotrolled radical polymerizatios. Withi these costraits, it is possible that cotrolled CA polymerizatios utilizig simple amie hydrochloride iitiators ca be obtaied. A ew approach of cotrollig CA polymerizatio was reported by the Cheg group i 2007 [22]. I a scree of amie iitiators for the polymerizatio of B-Glu CA, they foud that hexamethyldisilazae (MDS) showed remarkable cotrol of polymerizatios ad led to formatio of PBLG with excellet chai legth cotrol, with less tha 22% deviatio from the expected molecular weights, ad arrow molecular weight distributios (M w /M < 1.2) (Scheme 13). The CA polymerizatios iitiated with MDS differed greatly from those iitiated with covetioal secodary amie iitiators, e.g., diethylamie, i which elevated PBLG molecular weights (three to four times higher tha the expected molecular weights) ad broad molecular weight distributios were observed. The cotrolled CA polymerizatios observed with MDS suggested that their iitiatio ad chai propagatio mechaisms differ from either the amie (Scheme 2) or the AM mechaism (Scheme 3), typically observed with amie iitiators. As a secodary amie, MDS ca either fuctio as ucleophile to ope the CA rig at C 5, or act as a base to deprotoate the group [11]. Previous studies showed that secodary amies with bulky alkyl groups (e.g., diisopropylamie) exclusively deprotoated CAs [38]. Therefore, it is ulikely that MDS, a secodary amie cotaiig two bulky TMS groups, attacks the C 5 of B-Glu CA. If the first step ivolves the deprotoatio of the CA group by MDS, a -TMS CA would form that should udergo rapid rearragemet to form a a-isocyaatocarboxylic acid [39]. owever, o isocyaate stretch (~2,230 2,270 cm 1 ) was observed whe a mixture of equimolar B-Glu CA ad MDS was aalyzed by FTI. Iterestigly, it was observed that the absorptio bad of MDS at 932 cm 1 i FTI disappeared, idicatig the cleavage of a bod durig iitiatio. It therefore seems likely that a TMS group is trasferred to C 2 from MDS i a coordiated maer (Scheme 14). Istead of formig a isocyaate, the CA-TMS itermediate istead udergoes rapid rig-opeig by the i-situ-geerated TMS-amie to form a TMS-carbamate. Cheg ad coworkers cofirmed the formatio of TMS-carbamates through a combiatio of 13 C M ad mass spectroscopy (MS) aalysis of a equimolar

13 Sythesis of Polypeptides by ig-peig Polymerizatio MDS CA 2 Me 3 C Polypeptide TMS " trasfer" to icomig CA air or Scheme 14 Proposed mechaism for polypeptide sythesis usig MDS iitiator mixture of B-Glu CA ad MDS i DMS-d 6. The MS aalysis of a mixture of B-Glu CA ad MDS (at a 5:1 molar ratio) further showed peaks correspodig to the molecular weights of the dimer through the petamer cotaiig the TMScarbamate ed-group. This suggests that polypeptide chais were ideed propagated through the trasfer of the TMS group from the termial TMS-carbamate to the icomig moomer to form a ew TMS-carbamate termial propagatig group (Scheme 14). To demostrate that cotrolled CA polymerizatios ca be mediated by the TMS-carbamate group, Cheg sythesized trimethylsilyl dimethylcarbamate (TMSDC), a TMS-carbamate compoud, ad used it as the iitiator for B-Glu CA polymerizatio. Polymerizatios usig this iitiator yielded PBLG chais with cotrollable molecular weights ad arrow molecular weight distributios, as i the MDS-mediated polymerizatios. These TMS-carbamate-mediated CA polymerizatios resemble to some extet the group-trasfer polymerizatio (GTP) of acrylic moomers iitiated by orgaosilico compouds [40]. Ulike GTPs that typically require Lewis acid activators or ucelophilic catalysts to facilitate the polymerizatio [41], TMS-carbamatemediated CA polymerizatios do ot appear to require ay additioal catalysts or activators. owever, it is still uclear whether the TMS trasfer proceeds through a aioic process as i GTP [41] or through a cocerted process as illustrated i Scheme 14. As the polypeptide chais are propagated oly at the amie-ed through the trasfer of the termial TMS-carbamate to the icomig moomer to form a ew TMS-carbamate termial propagatig group, Cheg ad his team reasoed that use of a -TMS amie as the iitiator will geerate a amie ad a TMS group

14 J. Cheg ad T.J. Demig 2 -TMS 1 CA C : Glu = (C 2 ) 2 CC 2 C 6 5 Lys = (C 2 ) 4 C()C 2 C 6 5 Scheme 15 Polypeptide sythesis usig TMS-amie iitiators (Scheme 15) that ca subsequetly form a correspodig amide at the C-termius ad a TMS-carbamate at the -termius after CA rig opeig. Thus, chai propagatio should proceed i the same maer as MDS-mediated polymerizatio. Because a large variety of -TMS amies are readily available, this method should allow facile fuctioalizatio at the C-termial eds of polypeptides. It has bee demostrated that a variety of primary amies, ragig from small molecules to polymers or cotaiig a variety of fuctioal groups (e.g., orboree, alkye, azide, PEG, etc.), could be readily itroduced to the C-termial eds of polypeptides via -TMS amie-mediated, cotrolled CA polymerizatio [42]. The polymerizatios iitiated by MDS ad -TMS amies usually complete withi 24 h at ambiet temperature with quatitative moomer cosumptio. These polymerizatios i geeral are slower tha those mediated by Demig s i(0) or Co (0) iitiators (about mi at ambiet temperature) [19, 24, 25], but are much faster tha those iitiated by amies at low temperature or usig amie hydrochloride iitiators [20]. These MDS ad -TMS amie-mediated CA polymerizatios ca also be applied to the preparatio of block copolypeptides of defied sequece ad compositio [22]. This orgaosilico-mediated CA polymerizatio, which was also show by Zhag ad coworkers to be useful for cotrolled polymerizatio of g-3-chloropropayl-l-glu CA [43], offers a advatage for the preparatio of polypeptides with defied C-termial ed-groups. 3 Copolypeptide ad Fuctioal Polypeptide Sythesis via CA Polymerizatio 3.1 Block Copolypeptides For the examiatio of model protei protei iteractios ad the assembly of ovel three-dimesioal structures, block copolypeptides are required that have

15 Sythesis of Polypeptides by ig-peig Polymerizatio structural domais (i.e., amio acid sequeces) whose size ad compositio ca be precisely adjusted. Such materials have prove elusive usig covetioal techiques. CA polymerizatios iitiated by a strog base are very fast. These polymerizatios are poorly uderstood ad block copolymers geerally caot be prepared. CA polymerizatios iitiated by primary amies are also ot free of side reactios. Eve after fractioatio of the crude preparatios, the resultig polypeptides are relatively ill-defied, which may complicate uequivocal evaluatio of their properties ad potetial applicatios. evertheless, there are may reports o the preparatio of block copolypeptides usig covetioal primary amie iitiators [44]. Examples iclude may hydrophilic hydrophobic ad hydrophilic hydrophobic hydrophilic di- ad triblock copolypeptides (i which hydrophilic residues were glutamate ad lysie, ad hydrophobic residues were leucie [28, 29], valie [45], isoleucie [46], pheylalaie [30], ad alaie [47]) prepared to study coformatios of the hydrophobic domai i aqueous solutio. These coformatioal prefereces of differet amio acid residues were used as the basis for early models for predictig protei coformatios from sequece. Cosequetly, these copolypeptides were studied uder coditios favorig isolated sigle chais (i.e., high dilutio), ad self-assembly of the polymers was ot ivestigated. These copolymers were ofte subjected to oly limited characterizatio (e.g., aalysis of amio acid compositio) ad, as such, their structures, ad the presece of homopolymer cotamiats, were ot coclusively determied. Some copolymers, which had bee subjected to chromatography, showed polymodal molecular weight distributios cotaiig substatial high ad low molecular weight fractios [30]. The compositios of these copolymers were foud to be very differet from the iitial moomer feed compositios ad varied widely for differet molecular weight fractios. It appears that most, if ot all, block copolypeptides prepared usig amie iitiators have structures differet to those predicted by moomer feed compositios ad probably have cosiderable homopolymer cotamiatio due to the side reactios described above. Polypeptide block copolymers prepared via trasitio-metal-mediated CA polymerizatio are well-defied, with the sequece ad compositio of block segmets cotrolled by the order ad quatity of moomer, respectively, added to iitiatig species. These block copolypeptides ca be prepared with the same level of cotrol foud i aioic ad cotrolled radical polymerizatios of viyl moomers, which greatly expads the potetial of polypeptide materials. The uique chemistry of these iitiators ad CA moomers also allows CA moomers to be polymerized i ay order, which is a challege i most viyl copolymerizatios, ad the robust chai-eds allow the preparatio of copolypeptides with may block domais (e.g., more tha four). The self-assembly of these block copolypeptides has also bee ivestigated (e.g., to direct the biomimetic sythesis of ordered silica structures [48]) for formatio of polymeric vesicular membraes [49 51], or for preparatio of self-assembled polypeptide hydrogels [52] ad aoscale emulsio droplets [53]. Furthermore, poly(l-lysie)-block-poly(l-cysteie) block copolypeptides have bee used to geerate hollow, orgaic iorgaic hybrid microspheres composed of a thi ier layer of gold aoparticles surrouded by a thick layer of

16 J. Cheg ad T.J. Demig silica aoparticles [54]. Usig the same procedure, hollow spheres could also be prepared, which cosisted of a thick ier layer of core shell CdSe/CdS aoparticles ad thicker silica aoparticle outer layer [55]. The latter spheres are of iterest because they allow for microcavity lasig without the use of additioal mirrors, substrate spheres, or gratigs. 3.2 de-chai-fuctioalized Polypeptides There have bee may examples i which polypeptides were chemically modified to improve their properties for delivery applicatios. Typically, this strategy ivolves the hydrophobic modificatio of poly(lysie) or poly(glutamate/aspartate) side chais by covalet attachmet of lipophilic groups [5, 6]. These modificatios are aki to polymer graftig reactios ad thus result i radom placemet of these hydrophobic substituets (typically log alkyl chais) alog the polypeptide chais. These modificatios were performed i order to icrease the polypeptide s ability to bid hydrophobic drugs, aggregate i aqueous solutio, ad/or peetrate the lipid bilayers of cell walls. The radom placemet of the hydrophobes alog the chai meas that they caot act as a distict domai i supramolecular assembly, as i a block copolymer, thus limitig their ability to orgaize. ther types of chemical polypeptide modificatio iclude additio of sugars, or sugar-bidig groups [27, 43, 56 58], to icrease fuctioality, ad the additio of oioic, polar groups to icrease solubility ad blood circulatio lifetime [8]. Icreasig bioavailability is a major cocer for drug delivery usig sythetic polypeptides. The amio acid fuctioalities that provide water solubility (e.g., the amio group of lysie, or the carboxylate groups of glutamate ad aspartate) are also detrimetal i that their polymers behave as polyelectrolytes. As such, they bid strogly to oppositely charged biomolecules (i.e., proteis, polyucleic acids, polysaccharides, lipids) resultig i aggregatio ad either rapid removal from the bloodstream or rapid digestio withi cells [59]. To circumvet this problem, oioic, water-solubilizig polypeptide residues have bee sought. Followig the discovery that optically pure polyserie is ot water-soluble at chai legths greater tha 20 residues [21], there have bee may attempts to prepare chemically modified residues that would impart these desired features. The simplest of these approaches is the graftig of PEG chais (1,000 < M < 5,000) oto side-chai fuctioalities, as described above, which results i highly heterogeeous materials that retai cosiderable charge [9]. A more sophisticated solutio was the developmet of hydroxyalkylglutamie polymers, prepared by the reactio of poly(alkylglutamate) esters with a,o-amio alcohols (Scheme 16). These polypeptides, particularly poly(hydroxypropylglutamie) ad poly(hydroxybutylglutamie), were foud to be oioic ad soluble i water over a wide p rage [32]. As such, these polymers should be useful as solubilizig domais i polypeptidebased drug delivery systems. The major detrimets of these materials, however, are that they are recogized as foreig etities ad rapidly degraded i vivo, they are

17 Sythesis of Polypeptides by ig-peig Polymerizatio m 2 m Scheme 16 Sythesis of water-soluble poly(hydroxyalkyl glutamies) z -Z- L -Lysie ac 3 TF/ 2 Z Cl 2 CC 3 (PMe 3 ) 4 Co TF x = 1 or 2 Scheme 17 Sythesis of water-soluble EG-grafted poly(l-lysie)s difficult to prepare without sigificatly degradig the polypeptide chais, ad they lack ordered secodary structure i solutio [32]. The last property is importat sice oe of the mai reasos for usig polypeptide scaffolds i biomedical applicatios is to take advatage of their ordered chai coformatios to mimic protei structures. Demig ad, more recetly, Klok have take a differet approach toward the developmet of oioic, water-soluble polypeptides. They icorporated the solubilizig ad protective properties of PEG ito polypeptides by cojugatio of short ethylee glycol (EG) repeats oto amio acid moomers, as opposed to the welldocumeted approach of graftig PEG to the eds or side chais of polypeptides [60]. Demig has also recetly exteded this work by attachig oioic moosaccharides to amio acid moomers to give oioic, sugar-fuctioalized polypeptides [57] (Scheme 17). The fuctioalized CA approach avoids the eed for expesive amio- or carboxylato-fuctioalized PEG molecules ecessary for couplig, ad avoids difficulties associated with derivatizatio of polymers that are usually associated with polypeptide chai cleavage ad broadeig of molecular weight distributios. I particular, the presece of short EG repeats or saccharides o every residue resulted i a high desity of hydrophilic moieties aroud the polymer chai. I effect, the polypeptides are surrouded by a EG or saccharide sheath that should mimic the physical properties of PEG or polysaccharides [33], respectively, yet ot deleteriously affect the secodary structure of the polypeptide core. For istace, circular dichroism (CD) aalysis revealed that EG-grafted poly (L-lysie) is essetially 100% a-helical i p 7 water at 25 C. This coformatio was uaffected by may evirometal factors. Its helical structure was stable i water over a examied p rage of EG-grafted poly(l-lysie) was also stable i solutios cotaiig various deaturig agets, such as up to 3 M acl, 1 M urea, or 1 M guaidiium Cl. The thermal stability of the helical

18 J. Cheg ad T.J. Demig coformatio of EG-grafted poly(l-lysie) was also very high, ad as much as 75% of its helicity is retaied at 85 C. This polymer is also soluble ad helical i may orgaic solvets (e.g., TF, Me, ad CCl 3 ), ad was ot digested by hydrolytic ezymes that readily digest poly(l-lysie) (e.g., papai, trypsi) [7], idicative of the PEG-like properties imparted by the EG sheath. The polymer has surface properties similar to ustructured PEG, but also possesses a rod-like backboe due to its a-helical coformatio. milar moomers ad polymers were prepared by Klok usig succiate likages betwee the EG segmets ad lysie [61]. I these polymers, the ester likages to the EG segmets are potetially degradable i water, ad the polymers were foud to prevet ospecific protei adsorptio whe used to coat surfaces. ecetly, Demig ad coworkers also reported glycopolypeptides via livig polymerizatio of glycosylated-l-lysie CAs [57], demostratig the feasibility of sythesizig water-soluble, highly helical (ca. 88% helicity) polypeptides via moosaccharide-fuctioalized CA moomers. Measuremet of CD spectra from 4to90 C revealed that the a-helical coformatio of poly(galactosyl-l-lysie) was gradually disrupted as temperature was icreased, with roughly 40% helicity beig retaied at 90 C. This behavior is probably due to disruptio of amide -bodig by iteractios with water molecules [6]. These disordered polypeptides remaied water-soluble, ad their a-helical coformatios were completely regaied upo coolig, showig that this process is reversible. The molecular weights of these rodlike PEG-mimic [60] or polysaccharide-mimic [57] polymers could also be easily adjusted by varyig the CA to-co(0) iitiator ratios. milar oligo-eg modificatios to the b-sheet preferrig amio acids L-serie ad L-cysteie were foud to allow facile aqueous processig of their correspodig b-formig polymers. The EG side chais were foud to provide good water solubility to the polymers, which could the form b-sheet structures upo solvet evaporatio or by cotrolled additio of a solvet that stabilizes the b-coformatio. The sythesis of EG-modified polyserie is show i Scheme 18, where the EG repeats were coupled oto the amio acid usig a ether likage [62]. The modified amio acids were the coverted to their correspodig CA moomers to allow subsequet polymerizatio. CD aalysis of the water-soluble polymer i deioized water at p 7 revealed that it was i a disordered chai coformatio [35, 36]. The CD spectra of this polymer were also ivariat with solutio p ad buffer stregth, cosistet with this result. owever, films cast from aqueous solutios of this polymer from a variety of buffers all gave CD spectra idicative of the b-sheet coformatio. The trasformatio from disordered coformatio to b-sheet was also achieved i water by additio of icreased percetages of methaol or 2 CCl 2 (PMe 3 ) 4 Co x Scheme 18 Sythesis of water-soluble EG-grafted poly(l -serie)s

19 Sythesis of Polypeptides by ig-peig Polymerizatio acetoitrile. Wide-agle X-ray scatterig data from films of oligo-eg-grafted poly (L-serie) revealed reflectios that were also commesurate with the atiparallel b-sheet structure. verall, these EG-modified polypeptides provide PEG-like a-helix ad b-sheet formig segmets that ca be icorporated ito block copolypeptide delivery agets. Such domais provide ot oly improved solubility ad bioavailability, but allow icorporatio of secodary structure to cotrol selfassembly of the drug complexes. 4 Polypeptide Deprotectio ad Purificatio Although quite complex copolypeptide architectures ca ow be sythesized, obtaiig these materials i a state of high purity typically requires additioal measures. As discussed above, may of the copolypeptides sythesized usig covetioal methods cotai homopolymer impurities, which must be removed by selective solvet extractios or fractioal precipitatio, whe possible. ce covetioal CA polymerizatios also usually give polypeptide segmets with large chai legth distributios, these samples are ideally also fractioated to give samples of well-defied compositio. A additioal purificatio issue arises from the amphiphilic ature of may of these copolymers, e.g., PEG-PBLG. Such polymers ted to associate i most solvets, leadig to trapped solvets or solutes i the copolymer sample, which ca complicate aalytical studies. I the case of polymerizatios iitiated by a trasitio metal, removal of the metal from the sample is also importat for most applicatios. For rigorous purificatio of these amphiphilic copolymers, Demig s group has foud that exhaustive dialysis of the samples agaist deioized water is very effective at removig small molecule cotamiats. I cases where a polymer segmet ca bid strogly to metals such as Co 2+ ad i 2+, the additio of a potet metal chelator, such as EDTA, i the early stages of dialysis was foud to be sufficiet to remove all traces of the metal ios. A highly useful feature of copolypeptide materials is their fuctioality. The commo aturally occurrig amio acids cotai umerous acidic ad basic fuctioal groups that provide iterestig p-resposive character to these materials. These fuctioal groups are masked by protectig groups before sythesis of the CA moomers, sice they will typically iterfere with polypeptide sythesis or CA stability [11, 12]. Cosequetly, these protectig groups must be removed after polymerizatio if the fuctioal group chemistry is to be used. The first cocer with polypeptide deprotectio is whether or ot all the protectig groups have bee removed. Small amouts of residual protectig groups ca sigificatly ifluece the resultig polypeptide properties, especially sice the protectig groups are typically hydrophobic ad the deprotected chai is typically hydrophilic. Fortuately, most of the commo protectig groups are removed without difficulty, ad deprotectio levels greater tha 97% are readily attaied. The secod, ad more serious, cosequece of deprotectio is cleavage of the peptide chai, or racemizatio of the optically pure amio acid residues.

20 J. Cheg ad T.J. Demig Basic polypeptides, such as polylysie or polyargiie, are readily deprotected [11, 12, 48]. Acidic polypeptides, such as polyglutamic acid or polyaspartic acid, require more care i deprotectio reactios due to a abudace of potetial side reactios. PBLG, for example, ca be debezylated usig strog acid, aqueous base, or catalytic hydrogeatio. Use of strog acid (e.g., gaseous Br or Br i acetic acid) avoids ay racemizatio, but is kow to lead to sigificat chai cleavage arisig from protoatio of side-chai ester groups that react with the amide backboe [49]. Basic coditios avoid this reactio, but ca lead to sigificat racemizatio uless the amout of base is carefully cotrolled [50, 51]. ydrogeatio would appear to be the most attractive method, however, it is oly effective for chais less tha 10 kda i mass. Larger PBLG chais adopt stable helical coformatios that prevet access of the hydrogeatio catalyst to the ester groups [50, 51]. Ester cleavage usig trimethylsilyl iodide was foud to give clea coversio to the readily hydrolyzed trimethylsilyl ester, without ay racemizatio or chai cleavage [52]. The major drawbacks of this reaget are its expese as well as its reactivity with most other fuctioal groups, such as the ether likages i PEG. The deprotectio of poly(b-bezyl-l-aspartate) shows less side reactios uder acidic coditios compared to PBLG. owever, it has bee reported that the polymer backboe udergoes partial rearragemet to b-peptide likages uder basic coditios, presumably through a imide itermediate [54]. The degree of racemizatio i these samples was ot discussed. To avoid the issues of deprotectio chemistry ad to allow facile side-chai fuctioalizatio, a emergig field is focused o the developmet of ew CA moomers bearig side-chai fuctioal groups that stay itact durig polymerizatio ad ca be used for highly efficiet cojugatio of fuctioal moieties after polymerizatio. The sythesis of g-alkeyl-l-glutamate CAs was reported by Poché et al. i 1997, ad these were utilized for preparig poly(l-glutamate) s cotaiig pedat alkee fuctioal groups that were modified by a variety of reactios (Scheme 19)[63]. I the past few years, a umber of other CAs bearig cojugatio-ameable side-chai fuctioal groups, as well as their polymerizatios, have bee reported. These iclude g-propargyl-l-glutamate CA by ammod [64] ad Che [56] (Scheme 20), g-3-chloropropyl-l-glutamate CA by Zhag (Scheme 21) [43], propargyl-dl-glycie CA by eise (Scheme 22) [65], 3 + couplig + 3 CA formatio polymerizatio m post-reactio modificatio m Scheme 19 Sythesis ad derivatizatio of alkee-termiated poly(glutamates)

21 Sythesis of Polypeptides by ig-peig Polymerizatio 3 + TMS-Cl 3 + Cl 3 C C CCl 3 amie - 3 Scheme 20 Sythesis ad derivatizatio of alkye-termiated poly(glutamates) MDS a 3 Cl Cl 3 "click" chemistry Scheme 21 Sythesis ad derivatizatio of azido-termiated poly(glutamates) Scheme 22 Alkee ad alkye bearig CAs propargyl-dl-glycie CA allyl-dl-glycie CA allyl-dl-glycie CA by Schlaad (Scheme 22) [58] ad g-(p-viylbezyl)-lglutamate CA by Cheg (Scheme 23) [66]. Through a variety of azide-alkye, thiol-ee ad other chemistries, these fuctioalized polypeptides were modified with a variety of fuctioal moieties icludig moosaccharides ad PEG chais. These reactive polypeptides have the potetial for geeratio of a large library of fuctioal polymers from a sigle precursor. For istace, coversio of alkee groups i poly(g-(p-viylbezyl)-l-glutamate) to aldehydes, followed by reductive amiatio with primary amies was used to geerate poly(l-glutamate) aalogs with charged groups distally situated o their side chais (Scheme 23) [66]. These

22 J. Cheg ad T.J. Demig CC l2 1. MDS + 5% 2 C CBZ-Cl CBZ m γ-(p-viylbezyl)-l-glutamate CA / CCl 3 / -78 ºC - 2 / [] Cl m m poly(γ-(p-viylbezyl)-l-glutamate) Scheme 23 Sythesis ad derivatizatio of aldehyde-termiated poly(glutamates) side-chai charged poly(l-glutamate) aalogs were foud to be water-soluble ad able to adopt stable a-helical coformatios over a wide rage of p (p 1 12), i cotrast to poly(l-lysie) or poly(l-glutamic acid) that are kow for their p-depedet helix coil trasitios. 5 Coclusios ad Future Prospects The sythesis of polypeptides by rig-opeig polymerizatio of CAs is a area that has bee uder study for more tha six decades. Iitially, this field suffered from limitatios i the sythesis of the polypeptide compoets, which required excessive sample purificatio ad fractioatio to obtai well-defied copolymers. I recet years, vast improvemets i CA polymerizatios, either usig metal iitiators [19, 67] or improved covetioal iitiators [21, 22, 68] ow allow the sythesis of hybrid ad block copolymers of cotrolled dimesios (molecular weight, sequece, compositio, ad molecular weight distributio). Such materials will greatly assist i the developmet ad idetificatio of ew self-assembled structures made possible with ordered polypeptide segmets, as well as yield ew materials with a wide rage of tuable properties. There are still may challegig issues that remai to be addressed i the field of sythetic polypeptides. CA purificatio has bee oe of the bottleecks limitig the availability ad scale-up of CA moomers. ecrystallizatio has log bee the

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