On the Crystal Structure of Poly(R-benzyl γ,DL-glutamate) of Microbial Origin Jose ´ Melis, David Zanuy, Carlos Alema ´ n, Montserrat Garcı ´a-Alvarez, and Sebastia ´ n Mun ˜ oz-Guerra* Departament d’Enginyerı ´a Quı ´mica, ETSEIB, Universitat Polite ` cnica de Catalunya, Diagonal 647, Barcelona 08028, Spain Received May 13, 2002; Revised Manuscript Received July 22, 2002 ABSTRACT: The conformation and crystal structure of the benzyl ester of microbial poly(γ,DL-glutamic acid) with a nearly racemic composition was investigated by X-ray diffraction, polarized infrared spectroscopy and molecular modeling simulations. Three different molecular arrangements were characterized. Form I was found to be a structure made of hydrogen-bonded sheets of chains in a nearly extended conformation with features similar to the well-known γ-form of nylons. Form III consisted of a hexagonal packing of 37/10 helices stabilized by intramolecular hydrogen bonds set between i and i + 3 amide groups. Form II was observed as a poorly oriented phase and identified with the 5/2 helical form previously described for optically pure poly(R-benzyl γ,L-glutamate). Energy calculations indicated that the helical conformations adopted by PAB(DL)G are incompatible with a polymer having a statistical distribution of D and L units and that a polymer made of D- and L-stereoblocks is the most appropriate to describe the crystal structure present in form I. Introduction Poly(γ-glutamic acid) (PGGA) is a naturally occurring poly(γ-peptide) that is produced by several species of bacteria of the genus Bacillus. The biological occurrence of PGGA was accounted at the beginning of the past century and since then, the number of publications dealing with the biosynthesis, solution properties and chemical modifications of this fascinating biopolymer has been continuously increasing. 1 The biodegradable character and well-proven bioassimilation of PGGA and its derivatives justify the present interest for this substance as a potential material with applications in food packing and biomedicine. PGGA is made of glutamic acid units linked between the γ-carboxylic and the R-amino functional groups. The biosynthesis of PGGA in the laboratory usually affords PGGA in satisfactory yields with high molecular weight and containing variable amounts of D- and L-enantio- meric units. 2 The stereochemical microstructure of biosynthetic PGGA with enantiomerically heteroge- neous composition has remained uncertain for long time. Recently, strong evidences favoring the occurrence of a microstructure made of stereoblocks have been pro- vided. 3 On the other hand, the ability of un-ionized PGGA to adopt regularly folded conformations in water solution was early revealed by spectroscopy measure- ments 4 and later supported by computational methods. 5 Esterification appears to be the preferred procedure to modify the properties of microbial PGGA and to render non-water-soluble products. Thus, the prepara- tion, characterization, and properties of a good number of poly(γ, DL-glutamate)s have been reported in the last 10 years. 6 Surprisingly, the structure adopted in the solid state by the PGGA esters, or even by PGGA itself, has been scarcely investigated. In fact, a communication describing some X-ray diffraction features of several poly(γ-glutamate)s 7 and a preliminary account of the crystal polymorphism observed for poly(R-ethyl γ,DL- glutamate) 3b amount the only information available today on the crystal structure of biosynthetic poly(γ- glutamate)s. Some years ago we reported on optically pure poly(R-benzyl γ,L-glutamate) and poly(R-methyl γ,L-glutamate) obtained by chemical synthesis, and it was then discovered that these compounds can adopt helical conformations comparable to those characteristic of poly(R,L-glutamate)s. 8 This finding closely followed those obtained with poly(,L-aspartate)s, a family of nylon 3 derivatives which distinguishes by its ability to adopt regular folded conformations of R-helix type. 9 Recent investigations carried out on oligo(-amino acid)s and oligo(γ-amino acid)s 10 have shown that both types of peptides are able to adopt R-helix-like conformations confirming previous results reported for high molecular weight polymers. In the present paper, we wish to report on the conformation and crystal structure of racemic poly(R- benzyl γ,DL-glutamate) of microbial origin, hereafter abbreviated to PAB(DL)G. Previous work carried out on optically pure poly(R-benzyl γ,L-glutamate) prepared by chemical synthesis, hereafter abbreviated to PAB- (L)G, will be used as the reference. It is known that PAB(L)G adopts two crystal forms, one made of hydro- gen-bonded sheets stacked in a layered structure with chains in almost extended conformation (form I) and a second form proposed to consist of a hexagonal packing of intramolecularly hydrogen-bonded 5/2 helices (form II). It should be stressed that the polymer investigated in that case entirely consisted of L-enantiomeric units and had a molecular weight of about 30 000. In the present case, we are concerned with a polymer of much higher molecular weight and composed of approximately equal amounts of D and L units. * Corresponding author. E-mail: sebastian.munoz@upc.es 8774 Macromolecules 2002, 35, 8774-8780 10.1021/ma020728v CCC: $22.00 © 2002 American Chemical Society Published on Web 10/03/2002