Bayer AG, Wuppertal, Germany; Tel.: / ; Fax.: / ; 2

Transcription

1 75 7 Polyaspartic Acids Dr. Winfried Joentgen, Dr. Nikolaus M ller 2, Dr. Alfred Mitschker 3, Dr. Holger Schmidt 4 Bayer AG, Wuppertal, Germany; Tel.: /36-266; Fax.: / ; winfried.joentgen.wj@bayer-ag.de 2 Bayer AG, 5368 Leverkusen, Germany; Tel.: 49 24/ ; Fax.: 49 24/ ; nikolaus.mueller.nm@bayer-ag.de 3 Bayer AG, 5368 Leverkusen, Germany; Tel.: 49 24/ ; Fax.: 49 24/ ; alfred.mitschker.am@bayer-ag.de 4 Bayer AG, 5368 Leverkusen, Germany; Tel.: 49 24/30-638; Fax.: 49 24/ ; holger.schmidt.hs2@bayer-ag.de Introduction Historical Outline Chemical Structure Chemical Analysis H-NMR Spectroscopy C-NMR Spectroscopy N-NMR Spectroscopy Gel-permeation Chromatography Isotachophoresis Detection in Technical Applications Fluorescence Spectroscopy Precipitation Titration Polyelectrolyte Titration Biodegradation Production of PASP... 85

2 76 7 Polyaspartic Acids 8 World Market and Applications Patents Outlook and Perspectives References bpp DOC GPC HEMA NMR OECD PASP SSNC bovine phosphophoryn dissolved organic carbon gel-permeation chromatography hydroxyethylmethacrylate nuclear magnetic resonance Organisation for Economic Cooperation and Development polyaspartic acid Swedish Society for Nature Conservation Introduction The presence of deposits of inorganic crystals in living organisms is a ubiquitous phenomenon, and the processes of formation of the crystalline phases are grouped together under the term biomineralization, where bio suggests the active involvement of the cells of the host organism in the process. Control of this inorganic crystallization is essential to living systems of virtually all types. For example, inorganic crystals that have been modified by association with organic components are used to produce skeletons, shells, and teeth, as well as a fascinating array of other structures and cellular inclusions. The study of such composites is inherent in the more general field of biomineralization, which has been the subject of numerous volumes and some excellent recent reviews (Addadi and Weiner, 992; Mann, 993; Allemand and Cuif, 994). A multitude of organic molecules from biological systems potentially interact with and modify inorganic crystallization. Principal among these is a class of polyanionic proteins, the main anionic residues of which are aspartic acid and serine, with up to 90% of the serines being phosphorylated. Aspartic acid and phosphoserine together account for approximately 80 mol.% of the total amino acid composition (Wheeler et al., 988; Rusenko et al., 99). One example of such a highly anionic protein is bovine phosphophoryn (bpp) which is found in mineralizing tissues such as dentine tissues. bpp is a large molecule (M w 50,000 Da) which contains about 30 amino acid residues of which aspartic acid accounts for some 450 residues and serine (90% phosphorylated) for another 550. Thus, about 950 of the total 30 residues (84%) are potentially anionic (Stetler-Stevenson and Veis, 983). When dissolved in solution, these proteins can inhibit crystal nucleation and growth as well as alter the morphology of the crystals that are formed. On the other hand, when the proteins are immobilized, perhaps by cross-linking to a structural framework such as collagen or chitin (as are found in tissues), they can promote nucleation and the growth

3 2 Historical Outline 77 of crystals, again in specific orientations. These compounds influence processes not only in vivo but also in vitro, with the latter effects including the dispersion, nucleation inhibition or modification of the crystal growth of minerals or weakly soluble salts. Whilst the formation of insoluble mineral precipitates or incrustations is common in nature, it is also a widespread problem in many large-scale industrial processes. Nowadays, in aqueous systems in particular, this can lead to severe environmental problems because the organic polymers used in such processes as either scale inhibitors or dispersing agents (namely polyacrylic acid and copolymers with maleic acid, respectively) suffer from a lack of biodegradability (Schwamborn, 996). The latter problem becomes increasingly important if these substances are released directly as effluents, for example as one component of the so-called blow-down of cooling water circuits. In order to improve the environmental acceptability of these water-soluble scalecontrolling agents, sequestrants and dispersants that ultimately reach the environment (generally via the surface water), several attempts have been made to develop new biodegradable alternatives. It was clear that although the above-mentioned proteinogenic structures might represent an interesting approach for the control of such processes, these proteins themselves are not accessible for industrial use. Hence, an attempt was made to mimic the structural and functional properties of these surface active proteins (Sikes et al., 99), and this led to the development of polyaspartic acids (PASPs) ± a new class of synthetic polyamides which are structural and functional analogues of subdomains of biomineralization-controlling proteins. As yet, PASPs as homo polypeptides have not been found in nature; nor are they accessible via biotechnological procedures, unlike polyglutamic acid (Goto and Kunioka, 992) or polylysine (Kurshwaha, 980). Today, PASPs can be synthesized via two different chemical pathways. Whilst peptide chemical procedures mostly allow the synthesis of uniformly directed a- or b-polyaspartic acid of various molecular masses (Rao et al., 993), thermal polymerization techniques are preferred for production on an industrial scale. In this way, the sodium salts of PASP can be synthesized via thermal polymerization of aspartic acid (Figure ). Aspartic acid itself can be manufactured chemically starting from maleic acid anhydride, or by fermentation starting from fumaric acid or glucose (Eyal and Cami, 997). 2 Historical Outline The first experiments with PASP were carried out during the second half of the nineteenth century. Both French and German scientists heated ammonium salts of maleic acids (Dessaignes, 850; Wolff, 850) or asparagine (Schaal, 87), and separated an organic material which was insoluble in water. Hydrolysis of this unknown substance with hydrochloric acid or with an aqueous solution of ammonia yielded optically inactive aspartic acids. At this time, Schaal supposed this material to be a condensation product of several monomers. At the turn of that century, Schiff heated aspartic acid at temperatures of 2008C for 20 h, and separated two different types of condensation product from the mixture; these he called octa- and tetra-aspartide (Schiff, 897). The treatment of these compounds with dilute caustic or ammonia yielded the corresponding PASPs.

4 78 7 Polyaspartic Acids Fig. Main synthetic pathways to thermal polyaspartic acids About 50 years later, Kovacs et al. proposed that heating aspartic acid results in the formation of a reactive internal anhydride which condenses to a polyimide (Kovacs et al., 953). This polyimide, when exposed to an alkaline solution, readily undergoes a ring-opening reaction to PASPs. A transformation to the corresponding amides, followed by a Hofmann degradation, leads to acetaldehyde and,2-diamino-propanic acid. Based on this result, Kovacs and Kˆnyves (954) postulated a :.3 ratio of the a,b-amide bonds in these PASPs. Several investigations have dealt with the thermal polycondensation of amino acids or their precursors, such as maleamic acid to yield proteinoids. Only homopolymers of aspartic acid or glycine can be prepared using this method (Meggy, 956; Fox et al., 959; Harada, 959). Heating of the other natural amino acids alone results ordinarily in products such as ketopiperazines, tars, and other pyrolytic material. However, in the presence of large proportions of aspartic acid, glutamic acid or lysine, it is possible to synthesize copolymeric peptides by pyrocondensation of amino acid mixtures (Fox and Harada, 960b). The formation of such proteinoids can also be catalyzed by phosphorus compounds such as orthophosphoric acid (Fox and Harada, 960a) or polyphosphoric acid (Harada and Fox, 964). During the late 960s, Fox and coworkers discussed these pathways to proteinoids in detail with regard to questions about the origin of life (Fox et al., 970). During the 970s and 980s, Pivcova and colleagues were investigating the structure of the PASPs, and found both a- and b- linkages of the aspartic acid units in all thermally condensed derivatives (Pivcova et al., 98; Pivcova and Saudek, 985). Some of the first publications concerning the applications of PASPs involved the inhibition of calcium sulfate scale formation (Sarig and Shifrin, 977). Syntheses of

5 4 Chemical Analysis 79 PASPs starting from aspartic and phosphonic acids, as well as their application in health care, have been described by P. Neri and coworkers (Neri et al., 973). From an economical viewpoint, one of the most important properties of PASP is its ability to inhibit calcium carbonate scale formation, and this was investigated fully by Sikes and coworkers (Sikes and Wheeler, 985, 988). As a result, a large number of patents were filed during the 990s which reflected the commercial interest of the chemical industry in PASPs or their derivatives starting at that time (see Section 9). 4 Chemical Analysis The molecular structure of PASP has mainly been investigated using spectroscopic methods (Matsubara et al., 997). In particular, NMR was the most often used technique to obtain information about the structural composition of PASP. Alternative methods such as infrared spectroscopy also proved valuable in the analysis of PASP and/or its copolymers. 4. H-NMR Spectroscopy 3 Chemical Structure Naturally occurring PASP consists of a- linked aspartic acid units up to a chain length of 50 subunits (Rusenko et al., 99), these occurring as part of polyanionic proteins rich in aspartic acid and phosphoserine (Sikes and Wierzbicki, 996). In contrast to the linkage of aspartic acid in native materials, PASPs obtained by a thermal polymerization process contain a- and b-linked moieties in a constant molar ratio of 30:70 (Low et al., 996) in a random distribution over the polymer chain according to determinations by nuclear magnetic resonance (NMR) spectroscopy (Pivcova et al., 98, 982) (Figure 2). PASP can be identified from its NMR spectrum (Figure 3); in alkaline solution the following signals are measured (ph 3; 400 MHz), as shown in the spectrum in Figure 3. Due to the polymer structure of PASP there is an absence of sharp peaks, and the signals appear in the form of broader multiplets. In a mixture, of which the resonance signals of PASP and of other compounds do not overlap, it is possible to analyze quantitatively PASP by using integrals. It is also possible to identify different copolymer units in the molecule. Matsuyama et al. (980) were able to determine the ratio of a- and b-amide units in the main chain by integrating the separated methine signals in the H-NMR spectrum. Fig. 2 Structures of poly-a-aspartic acid and poly-b-aspartic acid.

6 80 7 Polyaspartic Acids Fig. 3 H-NMR spectrum of thermal PASP from aspartic acid in D 2 O at ph 3 (Source: Bayer AG.) C-NMR Spectroscopy 3 C-NMR spectroscopy has also been used to provide information about the molecular structure of PASP. Pivcova et al. (98) evaluated the ratio of a- and b-amide units using the methylene signals in the 3 C-NMR spectrum, and then analyzed the amide bond sequence using the amide carbonyl signals. Their conclusion was that the distribution of the a- and b-bonds was random (Pivcova et al., 982) N-NMR Spectroscopy might be assigned to the different tertiary structures, and especially to branching sites within the polymer ( Wolk et al., 994). By using 5 N-NMR spectroscopy of 5 N- enriched model compounds, it was possible to visualize the N- terminal groups and N- branching sites (Figure 4). It could also be shown in this way that maleic imide terminal groups () and amide branching sites (2) have an unfavorable influence on biodegradation. A comparison of 5 N-NMR spectra of PASP prepared either by catalytic polycondensation or by thermal condensation is shown in Figure 5 (see also Figure ). A further spectroscopic technique used to obtain greater detail of the PASP structure, and especially the three-dimensional structure of PASPs of different origin, is that of 5 N-NMR spectroscopy, both solid state and in solution. According to previous publications (Freeman et al., 994; Morall et al., 997), the limited biodegradation of PASP is dependent upon its process of manufacture. It has been suggested that the different biodegradation behaviors of various PASPs Fig. 4 N-terminal group () and N-branching site (2) of polyaspartic acid produced by thermal condensation.

7 4 Chemical Analysis 8 Fig. 5 Comparison of catalytic polyaspartic acid and thermal polyaspartic acid. (a) Polyaspartic acid from catalytic polycondensation. (b) Polyaspartic acid from thermal polycondensation. SSB, spinning side band. (Source: Bayer AG.) 4.4 Gel-permeation Chromatography The molecular weight of PASP was determined in aqueous solution using gel-permeation chromatography (GPC). With this method it is possible to use polyacrylates or polystyrene sulfonic acids as a standard polymer, though other PASPs of defined and unique chain length are commercially available and can be used as standard materials. During these measurements it is important to maintain a defined ph value of the solution as the molecule extension is heavily dependent on charge density, which in turn is influenced by the degree of dissociation. For the GPC procedure a crosslinked polyhydroxyethylmethacrylate (poly-hema) can be used as the column material. 4.5 Isotachophoresis Isotachophoresis is a method which is both environmentally friendly and inexpensive to operate, mainly because it uses only very small amounts of material and only dilute aqueous buffer solutions are required. The method is well suited for complex mixtures, and also under conditions of extreme concentration. The process of isotachophoresis is based on the fact that charged particles may be characterized by their migration behavior in an electric field, with different dissolved ions being sharply separated into