1
Characterization of Polypeptides and Polypeptoides - Methods and Challenges

David Huesmann and Matthias Barz

Institute of Organic Chemistry, Johannes Gutenberg-Universität Mainz, Mainz, Germany

1.1 Introduction

Materials made from polypeptides, and recently also polypeptoids, have received considerable and growing attention in recent years. Since synthetic polypeptides, just like natural proteins, are made up of amino acids, they can be non-toxic, biocompatible, and degradable in the body while they remain stable in aqueous solution. The multitude of different side chains enables the design of peptidic superstructures like polyion complexes [1,2], polymer micelles [3,4], polymer vesicles [5,6], nanofibers or -tubes [7], and hydrogels [8].

Apart from exactly defined polypeptides (i.e., proteins) that show a defined sequence of amino acids, there are also natural polypeptides that resemble less defined classic synthetic polymers. One of these polypeptides is poly(&ip.gamma;-glutamic acid) [9,10], which is produced by bacteria and cnidaria [11]. It is the major constituent of natto (Japanese food from fermented soy beans) and approved by the FDA for cosmetic applications.

1.2 Synthesis of Poly(peptide)s

Synthetic polypeptides were first described by Leuchs in the beginning of the twentieth century, although their polymeric nature was not acknowledged at that time [12-14]. Many researchers have explored synthetic polypeptides through the twentieth century [15,16] partially with poor results regarding polymerization kinetics, end-group integrity, or dispersity, in particular with more complex systems such as block copolypeptides, star-like polypeptides, or bottle-brush polymers. The reasons for this are manifold, including monomer purity, monomer instability over prolonged periods of time and the fact that the polymerization does not necessarily follow a single mechanism (Figure 1.1). The most prominent competing pathways are the normal amine mechanism (NAM, which leads to a classical chain growth) and the activated amine mechanism (AMM, which leads to undefined polymers through condensation of polymer chains). Further, addition of an N-carboxyanhydride, NCA, monomer before decarboxylation can lead to carbamates, which can rearrange into urea units, while a deprotonated NCA can open to form an isocyanate. A more in-depth discussion of the reaction mechanism is outside of the focus of this chapter and can be found in excellent reviews and books [15-17].

Figure 1.1 Mechanisms of NCA polymerization: Normal amine mechanism (NAM) and activated monomer mechanism (AMM).

The complex reaction mechanism has led to the development of controlled NCA polymerization methods starting in the end of the last century. In the late 1990s, the group of Timothy Deming was the first to demonstrate that the NCA polymerization using transition metal catalysts proceeds in a living manner and yields well-defined polypeptides (Figure 1.2) [18]. While this approach has been very successful for the preparation of well-defined and complex polypeptide architectures [5,6,19], it has the need for a transition metal catalyst. Additionally, the synthesis of hybrid structures remains challenging since the transition metal catalyst needs to be modified [20].

Figure 1.2 Initiation and propagation of metal catalyzed NCA polymerization.

Source: Deming 2000 [21]. Reproduced with permission of American Chemical Society.

As a complementary approach, Cheng and coworkers have reported silylated amine initiators, which allow control over NCA polymerization [22,23]. The trimethylsilyl residue remains at the polymer terminus over the course of the polymerization, allowing the preparation of defined polypeptides (Figure 1.3). The rate of polymerization is not slowed down by this polymerization technique as the polymerization (M/I?=?300) was reported to be completed within 24?h or less. Amine initiated polymerization has been reported to be complete within the same time frame (17?h for Xn?=?438, poly(benzyl glutamic acid), (PGlu(OBn)) [24].

Figure 1.3 Mechanism of trimethylsilyl-mediated NCA polymerization.

Source: Lu 2007 [22]. Reproduced with permission of American Chemical Society.

On the other hand, several approaches have been investigated to optimize the conditions of conventional amine initiated NCA polymerization. Vayaboury et al. used non-aqueous capillary electrophoresis to show a dramatic increase of living chain ends by lowering the polymerization temperature to 0?°C [25]. Unfortunately, no GPC plots and polymer dispersities of the obtained polymers were presented. Heise and coworkers investigated the influence of reduced temperature further [26] and used vacuum for the removal of CO2 from the reaction to increase its speed [27]. CO2 liberation is a step in NCA polymerization, which depends highly on the pressure in the reaction vessel. Wooley and coworkers reported the removal of CO2 by nitrogen flow through the reaction mixture, thereby increasing also the polymerization speed [28]. Both findings are surprising since theoretical studies and experiments have shown that CO2 liberation is not the rate determining step of the polymerization [15,29-31]. However, the performed control polymerizations (no nitrogen flow) yielded polypeptides with high dispersities of 1.38 and 2.19 for Xn of 50 and 100, respectively, while dispersities of PGlu(OBn) initiated by primary amines are usually well below 1.2 [32].

Scholz and Vayaboury tackled the issue of different secondary structures in the growing peptide by introducing thiourea to suppress hydrogen-bond formation [33]. It was found that the dispersity of polypeptides decreased markedly, independent of whether macroinitiators (PEG-NH2) or low molar mass initiators (hexylamine) were used.

In a different approach, Schlaad and coworkers introduced HCl salts of primary amines as initiators, lowering the reactivity of the growing chain end [34]. Elevated temperatures (40-80?°C) were used to counteract the slow polymerization. This method was complemented by other ammonium salts, namely different acetates [35] and recently the non-nucleophilic tetraflouroborates by Vicent and coworkers [36].

Finally, Hadjichristidis and coworkers reported on the use of highly purified monomers, solvents and reagents under high vacuum techniques [24]. Interestingly, these results suggest that all the previously mentioned potential side reactions are impurity related and that control can be achieved by working with highly pure solvents, monomers, and initiators.

1.3 Characterization of Poly(peptide)s

The aim for better and better control over the NCA polymerization over the last century was also accompanied by the development of analytical methods that allowed for a better characterization of polypeptidic materials. As with many classes of polymers, polypeptides are often characterized by the most widely used analytical techniques NMR and GPC to determine composition, size and dispersity of the polymers. However, due to the periodical peptide bond in the polypeptide backbone, these polymers are often not in a random coil conformation - as is usually the case for other polymers. This leads to two major challenges in the characterization of polypeptides: (1) The secondary structures must be characterized using for example NMR, IR, CD spectroscopy, or X-ray diffraction and (2) the different secondary structures lead to a change in the hydrodynamic radius of the polymers, limiting the usefulness of methods that rely on the hydrodynamic radius to deduce other physical parameters (e.g., GPC). It is worth noting, that these challenges do not apply to most polypeptoids, since they lack the free hydrogen at the amide bond and therefore do usually not form secondary structures.

The combination of a complex polymerization mechanism with many potential side reactions on one hand and challenging characterization on the other calls for extremely careful interpretation of obtained data. In the following sections, we will introduce different analytical methods for analyzing polypeptides highlighting their advantages and limitations.

1.4 Gel Permeation Chromatography (GPC)

Gel permeation chromatography is certainly one of the most important analysis methods in polymer chemistry yielding not only average molecular weights, but also a value for polymer dispersity, describing the width of the molecular weight distribution. However, these molecular weight distributions are often not obtained directly, but indirectly using a calibration by polymer standards.

The separation in the GPC column itself is enabled by polymer beads with different pore sizes. Large molecules cannot enter the pores and elute first from the columns, while smaller molecules can diffuse into the pores, thus remaining in the column for a longer time.

However, the separation does not occur by molecular weight, but by polymer size (i.e., hydrodynamic volume) and molecular weight is only inferred from calibration. To obtain correct molecular weights from this method, two conditions should be fulfilled: (1) The polypeptide has to be in one conformation and (2) the standards for the calibration have to be the same polymer (or at least very similar in structure) and have...