Poly( r-alkyl γ-glutamate)s of Microbial Origin. 2. On theMicrostructure and Crystal Structure of Poly( r-ethyl

γ-glutamate)s

Antxon Martınez de Ilarduya,† Najim Ittobane,‡ Marta Bermudez,† Abdelilah Alla,†

Mustafa El Idrissi,‡ and Sebastian Munoz-Guerra*,†

Departament d’Enginyerıa Quımica, Universitat Politecnica de Catalunya E.T.S. d’Enginyers Industrials deBarcelona, Diagonal 647, 08028 Barcelona, Spain; and Laboratoire de Chimie Organique,

Faculte des Sciences, Universite Moulay Ismail, BP 4010 Beni m’hammed, Meknes, Morocco

Received May 14, 2002

The stereochemical microstructure and crystalline structure of nearly racemic poly(R-ethyl γ,DL-glutamate)obtained by esterification of biosynthetic poly(γ-glutamic acid) were examined by NMR, DSC, and powderX-ray diffraction. The two enantiomerically pure poly(R-ethyl γ-glutamate)s, as well as the racemicstereocopolymers with random and alternating microstructure, were prepared by chemical synthesis andstudied in parallel to help in the interpretation of the data. The13C NMR analysis revealed that biosyntheticpoly(R-ethylγ,DL-glutamate) consists of a block stereocopolymer accompanied by minor amounts of a mixtureof the two optically pure homopolymers. The polymer is crystalline, with a degree of crystallinity andcrystal structure essentially similar to those displayed by the optically pure polymers but clearly differentfrom the alternating copolymer. Conversely, the racemic stereocopolymer with a random microstructureprepared by chemical synthesis is amorphous. The crystal structure of the racemic mixture of theD- andL-homopolymers seems to be very close to that of the biosynthetic stereocopolymer, although some indicationssuggesting the existence of a stereocomplex were found.

Introduction

Poly(γ-glutamic acid) (PGGA) (I , R ) H) is poly(γ-aminobutyric acid) bearing a carboxylic group attached to

the γ-carbon atom of each repeating unit. This capsularbiopolymer is produced by a variety of bacterial species1

and has also been identified in some eucaryotes.2 It is alsopresent innatto, a traditional Japanese food made fromsoybeans fermented byBacillus strains.3 The bacteriallyproduced PGGA may have molecular weights of up tomillions, and may contain varying amounts ofD- andL-units,depending on the microorganism and fermentation conditions.

An industrial process usingBacillus subtilis F-201 hasrecently been developed for the production of nearly racemicPGGA on a large scale.4 Currently there is renewed interestin this biopolymer as a biocompatible and biodegradablematerial of potential use in biomedicine.

The biosynthesis of PGGA has been thoroughly investi-gated by a variety of authors, and various approaches havebeen described for its chemical modification to usable

products.1 Esterification of PGGA is generally considered areliable and adequate method to render more-resistantmaterials with improved thermal and mechanical properties.Thus, the preparation and characterization of poly(R-alkylγ-glutamate)s, denominated PAAG-nswith n standing forthe number of carbon atoms in the alkyl side chainshavebeen reported forn ranging from 1 to 22.5-8 Other poly(γ-glutamate)s bearing either benzyl9 or ethoxyalkyl10 sidechains have also been reported. The chemical synthesis ofpoly(γ-glutamate)s from glutamic acid was well-establishedlong ago by Hungarian researchers11 and has been recentlyrevisited by Sanda et al.,12 in a report describing thepreparation of several poly(R-methyl γ-glutamate)s withdifferent chain stereochemistry.

Despite the considerable volume of effort dedicated toinvestigating the biosynthesis, chemical synthesis and chemi-cal modification of PGGA, very little is still known aboutthe microstructure and crystal structure of this biopolymerand its derivatives. The stereochemical microstructure ofoptically heterogeneous PGGA produced by bacteria is along-standing problem since the earliest works dealing withits characterization.13 Although from the beginning there havebeen more-or-less sound interpretations based on sideobservations,13-15 the only study specifically devoted toelucidating the microstructure of PGGA is that of Tanaka etal.16 Those authors analyzed the peptide fractions resultingfrom the action ofγ,L-glutamyl hydrolase on PGGA pro-duced byB. subtilis, and they proposed the existence of anoptically heterogeneous polymer made of both large and

* Corresponding author: sebastian.munoz@upc.es.† Universitat Polite´cnica de Catalunya E.T.S. d’Enginyers Industrials de

Barcelona.‡ UniversiteMoulay Ismail.

1078 Biomacromolecules 2002,3, 1078-1086

10.1021/bm025568+ CCC: $22.00 © 2002 American Chemical SocietyPublished on Web 06/27/2002

small stereoblocks unevenly distributed along the polymerchain.

The situation is not much better with regard to the structurein the solid state. The only information available on poly-(γ-glutamate)s of microbial origin is a preliminary com-munication in which the powder diffraction data of severalesters are compared.17 On the other hand, the crystal structureof poly(R-benzyl γ,L-glutamate) and poly(R-methyl γ,L-glutamate) prepared by chemical synthesis was examined byus 2 decades ago.18 It was then found that these enantio-merically pure PGGA esters are crystalline and able to adopthelical conformations similar to theR-helix characteristicof poly(R-peptide)s. Newly published research carried outon γ-oligopeptides by different workers has corroboratedthose pioneering results.19,20

In this paper, we report on the microstructure and crystalstructure of poly(R-ethyl γ-glutamate) (PAAG-2) obtainedby esterification of bacterial PGGA. This PGGA with anearly racemicD:L composition was produced by fermenta-tion with B. subtilisF-201. We use high-resolution13C NMRto prove the presence of microstructural heterogeneities inthe polymer chain and to characterize the sequence distribu-tion of enantiomeric units in the stereocopolymer. Calorim-etry and X-ray diffraction were also undertaken to supportthe NMR results. We carry out a parallel study on the twoenantiomerically pure polymers and on the racemic stereo-copolymers having random and alternating microstructure.These esters were prepared by chemical synthesis for thefirst time to help in the interpretation of the data. Theconclusions derived from this study are straightforwardlyapplicable to the parent PGGA, since no racemization wasobserved to happen upon esterification.

Results and Discussion

Synthesis of Poly(r-ethyl γ-glutamate)s.As illustratedin Scheme 1, PAAG-2 were prepared by two differentprocedures, i.e., by esterification of biosynthetic poly(γ-DL-glutamic acid) and fromD- andL-glutamic acids by applyingthe methodology well established for peptide synthesis.

Esterification of biosynthetic poly(DL,γ-glutamic acid) toPAA(D,L)G-2 was performed inN-methyl pyrrolidone withethyl bromide and sodium hydrogen carbonate according tothe methodology developed by Kubota et al.6 The reactionproceeded as expected, producing the ethyl ester in goodyields with a conversion higher than 99% and negligibleracemization. The molecular weight of the esterified polymerfell to near half the initial value as a consequence ofoccasional breakage of the polypeptide main chain occurringunder the basic conditions used for the alkylation reaction.These results are in agreement with those reported by Kubota,and reproduce satisfactorily those obtained by us in aprevious work.8

The chemical synthesis of PAAG-2 was undertaken in thiswork with the aim of preparing model polymers withpredetermined enantiomeric composition and sequence dis-tribution to be used as references in the investigation of themicrostructure of biosynthetic PAA(D,L)G-2. It is well-knownthat the synthesis of poly(R-alkyl γ-glutamate)s by poly-condensation requires the use of glutamylglutamate dimersin order to avoid undesired cyclization of the startingmonomers to pyroglutamate. The synthesis procedure wehave adopted for the preparation ofL-L, D-D, and L-D

dimers is depicted in Scheme 2, and follows (with someminor modifications) that reported by Sanda et al.12 for the

Scheme 1

Poly(R-alkyl γ-glutamate)s Biomacromolecules, Vol. 3, No. 5, 2002 1079

synthesis of the analogous methyl esters. The polymerizationof the dimers to PAAG-2 was carried out at room temper-ature in DMSO added with a molar excess of triethylamine.Either pure dimers or their equimolar mixtures were used toobtain the corresponding homopolymers or stereocopolymers,respectively (Table 1). For clarity and systematic nomen-clature, the acronyms used for the polymers keep theconfigurational notation used for the corresponding dimers;notice however that PAA(D-D)G-2 and PAA(L-L)G-2 couldbe simply denoted as PAA(D)G-2 and PAA(L)G-2. Theresulting polymers have weight-average molecular weightsbetween 10 000 and 15 000 and polydispersities close to 2.The use of other solvents, such as dimethylformamide orchloroform, afforded much poorer results regarding bothreaction yield and polymer size, and their use was thereforelimited to exploratory experiments.

NMR Analysis of the Microstructure. The 1H NMRspectra recorded in DMSO from different PAAG-2 at exactlythe same concentration of polymer are compared in Figure1. The spectra were taken at 353 K in order to optimizeresolution. These spectra show in the low-field region theNH proton signal at 7.90 ppm split into two peaks due to

coupling with theγ-CH. The remaining main chain protons,R-CH2, â-CH2, andγ-CH, are seen at 2.21, 1.90, and 4.25ppm respectively, the first one as a well-resolved triplet. Thesignal due to theâ-CH2 is particularly complex due to thediastereotopic nature of these two protons. The two signalsarising from the methylene and methyl protons of the ethylside group appear at 4.10 and 1.18 ppm. The only noteworthydifference detectable between these spectra is the broaderline width observed for PAA(D-D,L-L)G-2 and, to a lesserextent, for PAA(L-D,L-L)G-2. Although such broadeningshould be attributed to the presence of microstructureinhomogeneities, the resolution of these spectra is not highenough for conclusions to be drawn about the distributionof the enantiomeric units in the copolymers.

Scheme 2 Table 1. PAAG-2 Polymers Studied in This Work

name comonomersc D:Ld Mw/Mne

Chemical Synthesisa

PAA(L-L)G-2 9L-L n.d. 11800/6300PAA(D-D)G-2 9D-D n.d. 14000/7300PAA(D-D,L-L)G-2 9D-D + 9L-L n.d. 10100/5700PAA(L-D, L-L)G-2 9L-D + 9L-L n.d. 16000/9000PAA(L-D)G-2 9L-D n.d. 15900/8200

Biosynthesisb

PAA(D,L)G-2 59:41 210000/64000ns-PAA(D,L)G-2f 33:67 285000/80000s-PAA(D,L)G-2f 64:36 210000/58000

a Obtained by active ester polycondensation according to Scheme 1.b Obtained by esterification of biosynthetic PGGA according to Scheme1. c Obtained from D- and L-glutamic acid according to Scheme 2.d Determined by HPLC analysis of diastereomeric mixtures with Marfey’smethod. N.d. ) not determined. e Determined by GPC in HFIP againstpoly(ethylene oxide) standards and given in g mol-1 units. f Nonsoluble(ns) and soluble (s) fractions of PAA(D,L)G-2 in chloroform.

Figure 1. Comparison of 1H NMR spectra of PAAG-2. (*, #) Waterand solvent peaks.

1080 Biomacromolecules, Vol. 3, No. 5, 2002 Martınez de Ilarduya et al.

In contrast, the13C NMR spectra could successfully beused for this purpose. These spectra are compared in Figure2 for the whole series of PAAG-2 under investigation. Theyshow the peaks corresponding to every different carbonpresent in the repeating units of the polymers. The twocarbonyl groups (ester at 171.6 ppm and amide at 171.5 ppm)appear well resolved in the low-field region, as do CH2 andCH3 of the ethyl side group at 60.3 and 13.9 ppm.R-CH2,γ-CH, andâ-CH2 main signals appear at 31.4, 51.8, and 27.0

ppm, respectively, the latter two showing splitting in someof the spectra. This splitting is interpreted as being due tostereosequence effects, and its precise assignment could bemade using the model polymers prepared by chemicalsynthesis.

The information provided byγ-CH andâ-CH2 signals in13C NMR spectra will therefore constitute the basis of ouranalysis. These signals are shown in Figure 3 after beingenlarged for a detailed comparison. Theâ-CH2 signal ofsynthetic PAAG-2 made from mixtures ofL-L andD-D orL-D dimers splits into two peaks, whereas a single peak wasobserved for the PAA(D-D)G-2 and PAA(L-L)G-2 poly-mers, as well as for the alternating copolymer PAA(D-L)G-2. Theâ carbon is therefore sensitive to dyads, the higher-field peak arising fromDD andLL dyads and the lower onefrom DL andLD dyads. The dyad content was quantified bydeconvolution of the signal, followed by integration of thetwo deconvoluted peaks. (DD + LL) to (DL + LD) ratios of 1and 3 should be expected for PAA(L-D,L-L)G-2 and PAA-(D-D,L-L)G-2 copolymers, provided that they have arandom distribution of dimeric units. The experimental valuesobtained by analysis of theâ-CH2 signal were found to bein excellent correlation with those anticipated from theoreticalcalculations. The biosynthetic PAA(D,L)G-2 shows these twopeaks with an approximate area relation of 1/10. Thepresence of the aforementioned signal splitting proves thatthe biosynthetic polymer cannot be a mixture of the twoenantiomerically pure polymers, as has been suggestedpreviously in the reported literature.13 Furthermore, the lowintensity ratio observed for the heterotactic-to-isotactic dyadpeaks leads to the conclusion that the biosynthetic polymershould be a stereocopolymer in blockssand not at random,as was pointed out by other authors.21

Corroborating results were attained in the analysis of theγ-CH signal, which was found to be sensitive to triads. Inthe spectrum of PAA(L-D,L-L)G-2, this signal appearsclearly split into three peaks of similar intensities, attributable

Figure 2. Comparison of 13C NMR spectra of PAAG-2. (/) Solventpeak.

Figure 3. Comparison of 13C NMR spectra of PAAG-2 in the γ-CH (left) and â-CH2 (right) region, showing the peaks used for estimation of triad(left) and dyad (right) contents in the stereocopolymers.

Poly(R-alkyl γ-glutamate)s Biomacromolecules, Vol. 3, No. 5, 2002 1081

- from higher to lower field- to (LLL ), (LLD + DLL) and(LDL + DLD) triads. This is in full agreement with astereocopolymer composed of an equimolar mixture ofL-D

andL-L dimeric units distributed at random. According toexpectations, only the two highest-field peaks appeared inthe spectrum of PAA(D-D,L-L)G-2, sinceLDL andDLD triadsare not feasible in this copolymer. Analogously, onlyhomotriads were observed for enantiomerically pure PAA-(D-D)G-2 and PAA(L-L)G-2 polymers, and only sym-metrical triads (LDL + DLD), for the alternating PAA(L-D)G-2copolymer. The same three peaks were observed for bio-synthetic PAA(D,L)G-2, the one corresponding to (DDD +LLL ) triads appearing with much greater intensity than theother two. These results are completely consistent with thoseobtained in the analysis of theâ-CH2 signal, and are alsodemonstrative of the block microstructure present in bio-synthetic PAA(D,L)G-2. The number-average sequence lengthsand randomness calculated on a theoretical basis and usingthe data obtained in the analysis of theγ-CH andâ-CH2

signals are compared in Table 2. Stereoblocks with a number-average sequence length of 7-11 enantiomeric units seemto be the most probable sequences existing in biosyntheticPAA(D,L)G-2.

To evaluate its homogeneity, the PAA(D,L)G-2 copolymerwas subjected to exhaustive extraction with boiling chloro-form. A soluble fraction,s-PAA(D,L)G-2, was obtained,which amounted to nearly 90% of the initial weight and hada D:L ratio of 64:36 and anMn of 58 000. The minornonsoluble fraction,ns-PAA(D,L)G-2, had aD:L ratio of34:67 and anMn of 80 000. These two fractions wereanalyzed by13C NMR in the same terms as previouslydescribed for the unfractionated polymer; their spectra in theγ-CH andâ-CH2 regions are compared in Figure 4. A singlepeak is seen for the two signals ofns-PAA(D,L)G-2, withno shred of evidence indicative of microheterogeneity. Onthe other hand, the profiles of the signals arising froms-PAA-(D,L)G-2 are essentially indistinguishable from those of theinitial polymer, indicating that the two must be similarlyconstituted.

Calorimetric Measurements. The comparative DSCanalysis of the PAAG-2 afforded unambiguous evidence ofthe similarities and divergences in crystallinity between thedifferent stereomorphs. Illustrative thermograms are depictedin Figure 5, and melting and crystallization parameters aregiven in Table 3. For a rigorous comparison, samples wereprepared as films by casting from chloroform containing a

Table 2. NMR Analysis of the Microstructure of PAAG-2a

â-CH2

dyadsγ-CHtriads

no. av seqlength

polymer D:L DD + LL LD + DL DDD + LLL DDL + LLD + LDD + DLL LDL + DLD nL nD

randomnessø

PAA(D-D,L-L)G-2a (50:50) 76.7 23.3 53.0 47.0 0 3.3 3.3 0.60(75.0) (25.0) (50.0) (50.0) (0) (3.5) (3.5) (0.57)

PAA(L-D,L-L)G-2b (25:75) 50.2 49.8 36.9 29.2 33.9 3.0 1.0 1.30(50.0) (50.0) (37.5) (25.0) (37.5) (2.6) (1.0) (1.38)

PAA(D,L)G-2c 59:41 89.1 10.8 79.7 15.7 4.6 10.7 6.8 0.24(51.6) (48.4) (27.3) (48.4) (24.2) (2.4) (1.7) (1.00)

s-PAA(D,L)G-2c 64:36 88.0 12.0 79.2 15.9 4.9 10.4 6.3 0.26(54.0) (46.0) (31.0) (46.0) (23.0) (2.8) (1.6) (1.00)

ns-PAA(D,L)G-2c 33:67 100 0 100 0 0 500 500 0(55.6) (44.4) (33.5) (44.3) (22.2) (1.49) (3.01) (1.00)

a In parentheses are given values of theoretical fractions of the different dyads and triads calculated for the copolymers with a random distribution of(a) DD and LL, (b) LD and LL, and (c) D and L units.24

Figure 4. Comparison of 13C NMR spectra of fractionated and unfractionated PAA(D,L)G-2 in the γ-CH (left) and â-CH2 (right) region.

1082 Biomacromolecules, Vol. 3, No. 5, 2002 Martınez de Ilarduya et al.

few drops of trifluoroacetic acid, to help in the solubilizationof the polymer. As expected, almost exactly the same tracewas produced by the two optically pure enantiomorphs PAA-(D-D)G-2 and PAA(L-L)G-2. They showed an exothermalpeak due to cold crystallization at 146 and 133°C,respectively, and a melting peak at 247-248 °C. The DSCtrace recorded from biosynthetic PAA(D,L)G-2 displays themelting peak at 247°C and the cold crystallization exothermat 157 °C. The differences observed for crystallizationtemperatures can be attributed to differences in molecularweight, the larger polymer needing higher temperatures toachieve the chain mobility required for crystal growth.

Conversely, a nearly flat thermogram was obtained fromthe stereocopolymer PAA(D-D,L-L)G-2 prepared by chemi-cal synthesis, which did not change significantly aftersubjecting the initial sample to prolonged annealing treat-ment. This behavior is taken as demonstrating the amorphous

nature of the polymer, such as might be expected for arandom microstructure. On the other hand, the alternatingcopolymer PAA(L-D)G-2 shows a weak melting peak at 210°C, although no sign indicating cold crystallization wasperceivable in the thermogram. The much lower enthalpyof fusion observed in this case than that measured for theother crystallizable PAAG-2 is consistent with the absenceof cold crystallization and demonstrates the difficulty for thealternating microstructure to crystallize.

X-ray Diffraction

A comparative X-ray diffraction analysis was also madeon the PAAG-2 films obtained as described above and thensubjected to heating at 200°C to promote crystallization.The Debye-Sherrer patterns obtained are depicted in Figure6, and the Bragg spacings obtained from them are listed inTable 3.

Figure 5. Comparison of DSC heating traces of PAAG-2.

Table 3. DSC Data and X-ray Spacings of PAAG-2

synthesisa

PAA(D-D)G-2 PAA(L-L)G-2 PAA(D-D)G-2 + PAA(L-L)G-2 PAA(D-D,L-L)G-2 PAA(L-D)G-2biosynthesisb

PAA(D,L)G-2

Cast Filmc

Tc (°C)d 146 (30) 133 (18) 133 (12) n.o. n.o. 157 (11)dhkl (nm)e ∼1.0 s ∼1.0 s ∼1.0-1.5 df ∼1.0-1.5 df ∼1.5 m; 1.25 s ∼1.0-1.5 df

0.50 w 0.50 w 0.60 s0.42 s 0.42 s ∼0.4 df ∼0.4 df 0.46 s; 0.44 s ∼0.4 df

Crystallizedf

Tm (°C)d 247 (55) 248 (44) 259 (27) n.o. 210 (9) 247 (41)dhkl (nm)e ∼1.0-1.2 df ∼1.0-1.2 df ∼1.0-1.5 df ∼1.5 w; 1.25 s ∼1.0-1.5 df

0.92 s 0.90 w 0.92 sg

∼0.82 df ∼0.82 df 0.66 m 0.78 m ∼0.80 dfh

0.65 w 0.65 w 0.64 m 0.64 w0.50 w 0.55 m 0.60 s

0.50 w 0.47 s ∼0.40 df 0.52 w 0.51 w0.47 s 0.47 s 0.46 s 0.46 s

0.42 s 0.44 s0.42 s 0.42 s 0.38 m 0.41 m 0.42 s0.38 m 0.38 m 0.35 m 0.38 m 0.38 m0.35 m 0.35 m 0.35 m 0.35 m

a PAAG-2 obtained by chemical synthesis according to Scheme 1. b PAAG-2 obtained by esterification of biosynthetic PGGA according to Scheme 1.c Film cast from chloroform containing a few drops of TFA. d Cold crystallization and melting temperatures measured by DSC; in brackets, crystallizationor melting enthalpies in J g-1. e Visual intensities denoted as df: diffuse; s: strong; m: medium; w: weak. f Film heated to 200 °C at 20 °C min-1.g Reflection disappearing upon annealing. h Reflection strengthening upon annealing.

Poly(R-alkyl γ-glutamate)s Biomacromolecules, Vol. 3, No. 5, 2002 1083

The only amorphous pattern was that recorded from PAA-(D-D,L-L)G-2, and its appearance did not change afterannealing at 200°C. This behavior is consistent with thatobserved by DSC and in accord with what should beexpected for the random microstructure determined for thispolymer by NMR. The patterns obtained for both PAA(D-D)G-2 and PAA(L-L)G-2 are identical in both spacings andintensities, and consist of more-or-less discrete reflectionsindicating the presence of a partially crystallized phase.Surprisingly, the pattern produced by an equimolar mixtureof PAA(D-D)G-2 and PAA(L-L)G-2 displays sharper defi-nition and significant spacing differences in the reflectionslocated in the medium angle region. It is also significant thatthe melting temperature of the mixture is higher than thoseof the individual components. This suggests the formationof a stereocomplex able to crystallize more efficiently thanthe single polymer components. Interestingly, the patternobtained from biosynthetic PAA(D,L)G-2 displays featuresintermediate between those observed for the optically purepolymers and for their racemic mixture. In fact, it containsthe relatively sharp 0.92 nm reflection characteristic of thestereocomplex, together with the diffuse 0.82 nm reflectionpresent in the pattern of PAA(L-L)G-2. Even more strikingis the fact that the pattern changed upon annealing at 200°C, losing the reflection at 0.92 nm and becoming almostindistinguishable from that of PAA(L-L)G-2. The conclusiondrawn from these observations is that the crystal structureof biosynthetic PAA(D,L)G-2 participates from the charac-teristics of the other two and that it becomes apparentlyidentical to the structure of the optically pure polymers byeffect of temperature. A crystalline structure comprising bothenantiomerically pure crystallites and racemic crystallites canbe speculated for PAA(D,L)G-2. The small size of thesecrystallites, determined by the size of the blocks in thecopolymer, can be claimed as the reason for the instabilitythat the stereocomplex phase displays in this case. Last butnot least, the alternating copolymer PAA(L-D)G-2 yieldeda well-defined pattern, characteristic of a well-crystallized

material with a crystal structure clearly different from thatadopted by the optically pure polymers. Obviously, adedicated study on the crystal structure of PAAG-2, includingthe analysis of oriented samples, is needed to unravel thestereochemical reasons for such interesting and complexbehavior. Such work is currently under way in our laboratory,and will be published in the near future.

Concluding Remarks

The observations from this work have provided the datanecessary for the unambiguous characterization of thestereochemistry of PGGA produced byB. subtilis and toestablish the relationship existing between the racemic andthe enantiomerically pure poly(R-ethyl γ,glutamate)s withregard to crystallinity. The outstanding conclusions from thisstudy can be summarized as follows: (a) PAA(D,L)G-2 ofmicrobial originsand hence the parent PGGAsconsists ofa block stereocopolymer ofD and L units together with aminor amount (∼10%) of a mixture of the two enantiomeri-cally pure homopolymers. The number-average sequencelength of the stereoblocks is estimated to be between 7 and11 enantiomeric units. (b) PAA(D,L)G-2 is a semicrystallinepolymer with a crystal structure fluctuating between thoseadopted by the optically pure enantiomorphs and theirstoichiometric mixture, probably as a result of the differentmodes of packing in which the enantiomerically homoge-neous blocks can be arranged in the crystal lattice. Noconformational changes seem to be involved in this structuraltransition.

Experimental Section

Materials and Measurements.All chemicals were ob-tained commercially from either Aldrich or Merck. Theywere analytical grade or higher, and used without further

Figure 6. Powder X-ray diffraction patterns of PAAG-2.

1084 Biomacromolecules, Vol. 3, No. 5, 2002 Martınez de Ilarduya et al.

purification. Solvents to be used under anhydrous conditionswere dried by standard methods. The poly(γ,DL-glutamicacid) with a D:L ratio of 55:45 and a weight-averagemolecular weight of about 4× 105 was kindly supplied byDr. Kubota of Meiji Co. The process used for the preparationof this PGGA is described elsewhere.4

Enantiomeric compositions of PGGA and PAAG-2 weredetermined according to the method of Cromwick and Gross.The hydrolyzed polymer was made to react with Marfey’sreagent22 (1-fluoro-2,4-dinitrophenyl-5-L-alanine amide, Pierce,Rockford, IL) to form the diastereoisomeric dipeptides, whichwere then analyzed by HPLC, as previously described indetail.23 Gel permeation chromatography measurements wereperformed on a Waters GPC system, fitted with a refractive-index detector, on divinylbenzene cross-linked polystyrenecolumns, using hexafluoro-2-propanol as mobile phase. Themolecular weights were calculated against monodispersepoly(ethylene oxide) standards. NMR spectra were recordedon a Bruker AMX-300 spectrometer. All spectra wereobtained from a 1-2 wt % solution of the samples in DMSO-d6 at 353 K. Spectra were referenced against tetramethylsi-lane (TMS). 1H (300.13 Mz) and13C (75.47 Mz) NMRspectra were recorded with 16K and 64K data points and 1and 2 s ofrecycle times between FIDs, respectively. Toquantify the number fraction of dyads or triads from13CNMR spectra, Lorentzian deconvolution of the peaks wasdone with the WIN-NMR software (Bruker). To perform thisquantification, it was assumed that the relaxation times andNOE effects are similar for the same carbon in the differentstereosequences.24

DSC thermograms were recorded in a Pyris I Perkin-Elmercalorimeter at a heating rate of 20°C min-1 under a nitrogenatmosphere. Annealing treatments were performed in thecalorimeter, and DSC traces of annealed samples wererecorded directly on heating. X-ray diffraction patterns wererecorded at room temperature on flat films in a modifiedStatton camera (W. Warhus, Wilmington, DE), using nickel-filtered copper radiation of wavelength 0.1542 nm. Thepatterns were internally calibrated with molybdenum sulfide(d002 ) 0.6147 nm). Films were positioned transversally tothe beam in order to obtain isotropic patterns free fromincidental orientation effects.

Chemical Synthesis.The synthesis of monomers andpolymers was carried out according to the route depicted inScheme 2.

γ-Benzyl-L-glutamic acid (1L). To a mixture of 500 mLof diethyl ether containing 50 mL of concentrated sulfuricacid (98%) was added 500 mL of benzyl alcohol. Ether wasevaporated, andL-glutamic acid (73.5 g, 500 mmol) wasadded in portions over a period of 15 min. The mixture wasstirred at room temperature for 20 h and diluted with 1000mL of ethanol (96%), and 250 mL of pyridine was addedunder vigorous stirring. The mixture was cooled to 0°C andleft overnight at this temperature. The resulting precipitatewas filtered and washed with anhydrous ether. Recrystalli-zation from boiling water containing 10% of pyridineafforded1L in 62% yield; mp) 167-168 °C. The enanti-omer1D was obtained by the same procedure in 64% yield;mp ) 164-165 °C.

N-tert-Butoxycarbonyl-γ-benzyl-L-glutamic acid (2L).To a solution of1L (20 g, 84.2 mmol) in 400 mL of DMF/H2O (1:1) were added di-tert-butyl dicarbonate (18.5 g, 84.7mmol) and triethylamine (8.6 g, 84.9 mmol) at 0°C, andthe resulting mixture was stirred at room temperature for 12h. Evaporation under vacuum afforded an oily residue, whichwas suspended in ethyl acetate, acidified to pH 2 withaqueous KHSO4 solution, and extracted with ethyl acetate(2 × 200 mL). The extract was washed with H2O (100 mL)and dried over Na2SO4. Evaporation of the solvent afforded2L as an oil in 82% yield. The enantiomer2D was preparedby identical procedure in 63% yield.

N-tert-Butoxycarbonyl-r-ethyl-γ-benzyl-L-glutamate (3L).To a solution of2L (15 g, 44.5 mmol) in DMF (50 mL) at0 °C was added potassium carbonate (6.5 g, 47 mmol). Afterthe suspension was stirred for 10 min in an ice-water bath,ethyl iodide (3.6 mL, 6.9 g, 44.5 mmol) was added andstirring continued for 30 min. The mixture was allowed toreach room temperature, then stirred overnight and filteredby suction. The filtrate was portioned between ethyl acetate(100 mL) and water (100 mL). The organic phase waswashed with brine (2× 100 mL), dried over Na2SO4, filtered,and concentrated to give3L. Yield: 77%. Mp) 65 °C. Thesame procedure was used for the preparation of the enanti-omer3D. Yield ) 79%. Mp ) 65 °C.

N-tert-Butoxycarbonyl-r-ethyl-L-glutamate (4L). 3L (15g, 41.1 mmol) and 0.3 g of 10% palladium-carbon in 150mL of 2-propanol were shaken under a hydrogen atmospherefor 12 h. The catalyst was filtered off and the filtrateevaporated to give4L as a syrup in 89% yield. The sameprocedure was used to obtain the enantiomer4D. Yield )95%.

r-Ethyl-γ-benzyl-L-glutamate‚CF3CO2H (5L). To a so-lution of 3L (15 g, 41.1 mmol) in CH2Cl2 (100 mL) wasaddedtrifluoroacetic acid (37.5 mL) at 0°C, and the reactionwas stirred at room temperature for 12 h. The mixture wasthen concentrated by rotary evaporator to give a solid whichwas washed with diethyl ether, affording5L. Yield ) 90%;mp ) 64-65 °C. 5D was synthesized similarly to5L in ayield of 94%; mp) 63-64 °C.

r-r′-Diethyl-γ′-benzyl-N-tert-butoxycarbonyl-γ-L -glutamyl-L-glutamate (6L-L). To a solution of4L (2 g, 7.3mmol) in CH2Cl2 (60 mL) was added 1-(3-(dimethylamino)-propyl)-3-ethylcarbodiimide hydrochloride (EDC‚HCl, 1.68g, 8.8 mmol) at 0°C, and the mixture was stirred at roomtemperature for 1 h. Then, 2-hydroxybenzotriazole hydrate(HOBt, 1.0 g, 7.3 mmol) was added and the mixture stirredat 0 °C for 1 h. After that,5L (2.75 g, 7.3 mmol) andtriethylamine (1.2 mL, 0.9 g, 8.8 mmol) were added, andthe resulting solution was stirred at room temperature for20 h. The reaction mixture was washed with saturatedaqueous NaHCO3 solution, water, aqueous 10% citric acidsolution, and then water and finally dried over anhydrousNa2SO4. The organic layer was concentrated by rotaryevaporator and the residue was poured into ether/n-hexaneto precipitate6L-L as a white powder. Yield) 74%; mp)72-73 °C. The same procedure was used for the preparation

Poly(R-alkyl γ-glutamate)s Biomacromolecules, Vol. 3, No. 5, 2002 1085

of both the diastereoisomer6L-D (yield ) 72%; mp) 81-82 °C) and the enantiomer6D-D (yield ) 80%; mp) 75-76 °C).

r-r′-Diethyl-N-tert-butoxycarbonyl-γ,L -glutamyl-L-glutamate (7L-L). A mixture of 6L-L (10 g, 18.6 mmol),2-propanol (100 mL), and Pd/C (10%, 0.3 g) was stirred atroom temperature for 20 h under a hydrogen atmosphere.The reaction mixture was filtered, and the filtrate wasevaporated to give7L-L as an oil. Yield) 98%. The sameprocedure led to the diastereoisomer7L-D (yield ) 85%;mp ) 101-102 °C) and to the enantiomer7D-D (yield )96%).

r-r′-Diethyl-γ′-pentachlorophenyl-N-tert-butoxycarb-onyl-γ,L-glutamyl-L-glutamate (8L-L). To a solution of7L-L (10 g, 22 mmol) in CH2Cl2 (50 mL) was added 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride(EDC‚HCl, 5.5 g, 28.7 mmol) at 0°C, followed after 1 h ofstirring by addition of pentachlorophenol (5.85 g, 22 mmol).The reaction mixture was stirred at room temperature for 3h and then successively washed with aqueous NaHCO3

solution (2× 20 mL), water (2× 20 mL), aqueous 10%citric acid solution (2× 20 mL), water (20 mL), and brine(20 mL) and finally dried over Na2SO4. The solvent wasevaporated, affording8L-L as a white powder. Yield) 72%;mp ) 146-147 (°C). The diastereoisomer8L-D wassynthesized in 68% yield with mp) 106-107 °C, and theenantiomer8D-D, in 70% yield with mp) 149-150 °C.

r-r′-Diethyl-γ′-pentachlorophenyl-γ,L -glutamyl-L -glutamate‚HBr (9L-L). A solution of8L-L (5 g, 7 mmol)in HBr/AcOH (15%, 30 mL) was kept at room temperaturefor 1.5 h. Then, the solvent was removed and ether was addedto precipitate a white powder which was washed repeatedlywith ether. Yield) 78%; mp) 176-177 °C. The sameprocedure was used for the preparation of the diastereoisomer9L-D (yield ) 94%, mp) 164-165°C) and the enantiomer9D-D (yield ) 86%; mp) 179-180 °C).

Polymerization. General Procedure.The polymeriza-tions were performed in dimethyl sulfoxide (DMSO) in thepresence of an excess of triethylamine. Either the purecompound or the mixture of the stereoisomers selected forpolymerization was stirred at room temperature for 7 days,and the solid mass finally formed was washed successivelywith ether, ethanol, and water. The residual solid was driedovernight under vacuum and subjected to chemical charac-terization. Polymerization results are given in Table 1.

Acknowledgment. The authors are greatly indebted toDr. Kubota from Meiji Co. for the kind gift of PG(D,L)GAsamples. Thanks are given to Dr. J. J. Bou and M. Morillofor their valuable assistance with the GPC and HPLC

measurements. Financial support to this work was given bythe Ministry of Science and Technology with Grant Nos.PB-99-0490 and MAT99-0578-CO2-02.

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