GB2371052A - Poly(2-Acrylamido-2-Methyl-1-Propanoic Amide)-PAMPA: A Neutral Water-Soluble Synthetic Polymer with double-Stranded Helix Conformation - Google Patents

Abstract

Present invention involves a new synthetic polymer system: poly(2-acrylamido-2-methyl-1-propanoic amide) or PAMPA. From the chemical point of view, this homopolymer is homologous to a polypeptide and its molecular architecture can be viewed as inherited from poly(leucine) and poly(glutamine). An important difference between PAMPA and polypeptides or synthetic peptide-mimicking oligomers is that PAMPA has a nonpolar substituted poly(ethylene) backbone and includes peptide bonds in its side chain, while the latter have peptide groups on their backbone.

Description

Poly (2-Acrylamido-2-Methyl-t-Propanoic Amide) -PAMPA: A Neutral, Water-Soluble Synthetic Polymer with Double-Stranded Helix Conformation Piet Herdewijn and Jing Wang Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium Laboratory of Medicinal Chemistry, Rega Institute for Medical Research.

Molecular self-assembly plays a fundamental role in biological systems, as evidenced by proteins and polynucleotides. They exhibit intramolecular self-organization in aqueous solution by their spontaneous and reversible folding into well-defined conformations. * Understanding self-assembly and the associated non-covalent interactions is a central concern in structural biochemistry. The research on chemical systems with a tendency to spontaneous self-organization is also emerging as a new strategy in chemical synthesis. 2 Non-biological, simple synthetic polymers may provide alternative systems for investigating self-organization and allow researchers to understand particular interactions. Furthermore, the folding of linear polymers may provide synthetically simple tools to generate architectures that could potentially rival the biopolymers in their complexity and functionality.

Natural proteins carry out sophisticated chemical functions via folding into specific and compact conformations that are thermodynamically and kinetically stable. These tertiary folding structures provide'active sites'comprising of functional groups drawn from different regions of the linear polypeptides backbone. So far a synthetic polymer that folds in a specific tertiary structure has not been described. Protein tertiary structure arises from the assembly of regular secondary structures (helices, sheets and turns). The starting point for designing a synthetic polymer with the tendency to self-organization might therefore start with identification of a polymeric backbone with well-defined secondary structural preferences.

The folding of proteins involves complex interactions of a variety of non-covalent cohesive forces: hydrogen bonds, electrostatic interactions involving charged groups and electrical dipoles, van der Waals interactions, steric packing and hydrophobic effects. 3 One of the most important secondary structures is the helix, which forms the basis of more complicated structure. Duplex formation in nucleic acids and a helix formation in proteins are the most prominent examples of helical supramolecular structures of biological origin.

The a-helix as most abundant secondary structure in proteins was first postulated by Pauling et al. 4 It is generally believed that the primary reason for the self-assembly of polypeptides into a helix conformation in solution is intramolecular hydrogen bonding of the backbone. 5 This helix conformation is further stabilized by the hydrophobic interactions of the side chains. 6 During the past decade, several types of synthetic polymers with secondary helical structure have been reported and have involved the engineering of hydrogen bonds, whereas

7 the backbones used have been closely related to peptides. 7 The most well studied example is the 0-peptides consisting of P-amino acids. They have a rigid modified backbone and have a tendency to adopt a helical conformation. Other peptide-mimicking foldmers are vinylogous peptides and sulfonopeptides. Spontaneous self-assembly into double-or triple helices of single stranded oligo (2,2'-bipyridyl) has been observed and this process is driven by the formation of Cu (I) complex. 8 More recently, a set of meta-linked phenylacetylene oligomers have been described and they are claimed to collapse into helical conformations which are solvent-dependent. 9 They represent examples of non-hydrogen-bonded helices. However, all the reported synthetic systems consist of only a small number of residues and the longest oligomer so far has 18 residues. Natural proteins typically require more than 100 residues to display stable tertiary structure. Polymers consisting of preorganized monomers also require about 40 residues for a stable tertiary structure. The synthesis of large molecules being able to form a stable tertiary structure poses a great synthetic challenge.

Another main problem is that all synthetic systems using H-bonding disintegrate in aqueous or polar solvents that are able to compete for hydrogen bonds. 1o A central question in supramolecular chemistry is how hydrogen bond interaction and hydrophobic interaction can be built cooperatively into synthetic systems in order to generate the helix conformation via self-organization, which remains stable in aqueous medium. Furthermore, the solubility of a synthetic polymer in polar media is an important issue. For instance, the natural poly (alanine) peptides, which adopt a helix conformation, are scarcely soluble in water.

Nature has provided us with excellent examples of water soluble and conformational stable supramolecular structures, which may be used as model for the design of synthetic alternatives. Thus, efforts have been made to increase the water solubility of poly (alanine) and other peptides via insertion of polar amino acids or via introduction of polar functional groups on the side chains of natural peptides. 11 Therefore, when designing pre-organized helical synthetic polymer candidates, their aqueous solubility should be a major concern.

SYNTHESIS We hereby report a new synthetic polymer system: poly (2-acrylamido-2-methyl-l- propanoic amide) or PAMPA (Figure 1). From the chemical point of view, this homopolymer is homologous to a polypeptide and its molecular architecture can be viewed as inherited from poly (leucine) and poly (glutamine). Leucine and glutamine are known to be helix forming residues. An important difference between PAMPA and polypeptides or synthetic peptidemimicking oligomers is that PAMPA has a nonpolar substituted poly (ethylene) backbone and includes peptide bonds in its side chain, while the latter have peptide groups on their backbone.

PAMPA was prepared as shown in Scheme 1. The known acid 2 12 was generated from a mixture of acrylic chloride and acetone in the presence of NaCN and NH4CI, followed by acid hydrolysis of the resulting cyanide 1. Aminolysis using the ammonium salt of N hydroxysuccinimidel in the presence of DCC then gave the monomer amide 3.

Polymerization was carried out in DMF at 70 C, initiated by a catalytic amount of AIBN. 14 Purification using Sephadex G-25 eluting with water, to remove monomer and DMF, followed by dialysis against water afforded PAMPA as a light white fiber. PAMPA is completely miscible with water, DMF and DMSO. Its molecular weight averages about 42000 D, as determined by Gel Permeation Chromatography (GPC) using proteins as standard.

FT-IR CONFORMATIONAL ANALYSIS Synthetic polyacrylamide polymers such as poly (N-isopropylacrylamide) (PNIPAM) undergo temperature-, salt concentration-or solvent induced transitions. As such they show a behavior similar to natural proteins and have therefore been used as simple models to study the physical behavior of the proteins. 15 Due to the similarity between the constitution of PAMPA and a polypeptide, the techniques for determining the secondary structure of proteins were applied to investigate the structural motif of the PAMPA system. Since the PAMPA polymer is a racemic system, Circular Dichroism (CD) is not suited to study its secondary structure. We therefore turned to Fourier Transform Infrared Spectroscopy (FT-IR), a technique that has been extensively used to study the different types of conformations of peptides, 16-19 to compare the spectral patterns of synthetic PAMPA with those of natural proteins and to investigate the secondary structure of PAMPA.

FT-IR measurements of PAMPA were taken in DzO as solvent. Apart from the polyethylene backbone of PAMPA, the main mid-infrared absorption of the PAMPA side chains is expected in the region between 1600 and 1700 cm-1. In this region the absorption is almost completely (80%) due to the C=O stretching vibration. There is a minor contribution of the N-H bending. Figure 2 shows the slightly resolution enhanced spectrum of the C=O stretching band of PAMPA (k=1.5) as well as the Gaussian bands that contribute to it. The three Gaussians have their maximum at 1623,1634 and 1651 cm', respectively. Their respective contribution to the overall structure is 18%, 36% and 46%. Normally the band

position at 1651 cm-l is typical of a classical a-helix, while the bands at 1623, 1634 cm' are usually assumed to indicate (3-sheets and random coil or disordered structure.

However, the absorption pattern analysis of the C=O stretching band of PAMPA shows striking similarity with the amide I band (1600-1700 cm-1) of double stranded coiled-coil helical proteins, such as desmin and tropomyosin. I ? Heimburg et al. showed that a coiled-coil helix structure is characterized by three bands at 1628 (28%), 1640 (40%) and 1653 (32%) cm respectively. All three bands are assigned to a helix structure. Their findings are confirmed by normal mode calculations of coiled coil models. 18 It is argued that the lower wavenumber Gaussians are due to the strong hydrogen bonding of the backbone C=O groups with the solvent in addition to the intra-helical hydrogen bonds. The similarity between PAMPA and coiled coil helix proteins might suggest that PAMPA is not just a globule, but that it might adopt a coiled-coil helix structure in solution. Considerations about the function of the methyl groups for structural organization of PAMPA support this conclusion. Indeed, the coiled-coil tertiary structure of natural proteins is formed via hydrophobic interactions of the side chains of the double stranded a-helices. As PAMPA has two hydrophobic methyl groups on the side chain, it is expected to exhibit a strong tendency to form a coiled-coil helical structure. The fact that the maxima of the three Gaussians for PAMPA do not exactly match those found for the proteins investigated, is explained by the dependence of their position on the relative alignment and the number of hydrogen bonds. 19 The temperature stability of PAMPA was investigated. It was found that PAMPA does not undergo major transition up to 95 C. Only a shift of the wavenumber of the C=O stretching band maximum can be observed (Figure 3). This can be explained by the temperature-induced distortion of hydrogen bonds. This finding is confirmed by differential scanning calorimetry (DSC, unpublished data). In contrast, other polyacrylamide compounds such as PNIPAM undergo transitions at low temperature (39 oC for PNIPAM). These transitions have been characterized as coil-globule transitions. 15, Also isolated natural ahelices are marginally stable in aqueous solution. They are forming rapidly but their rate of unraveling is just as high. The absence of such a temperature transition, as well as the fact that PAMPA shows significant temperature stability, might be interpreted as further evidence for its stable double helical structure.

13C NMR TACTICITY STUDY Although the tacticity assignment of polyacrylamide is rather controversial, 13 C NMR is considered to be the most useful technique for this purpose. As the polymerization of the monomer was carried out under radical conditions, PAMPA is expected to consist of a mixture of isomers of different tacticity. Its tacticity was studied by 13C NMR (125 MHz) in D2O as solvent at room temperature. The signals of the two carbonyl carbons (at 182 and 179 ppm) show small splittings and cannot be used for the interpretation of the structure of the backbone, while the methylene carbons that appear at 37 ppm give a complex pattern. However, the methine carbons appear as three distinct peaks at 44.9, 44.0 and 43.0 ppm, with relative areas of 35%, 48% and 17%, assigned to syndiotactic (rr), heterotactic (mr) and tactic (mm) polymer, respectively. The stereoregularity of PAMPA was assigned by comparison of the methine carbon pattern with the one of the known polyacrylamide21 and polyacrylic acid. 22 As a general rule, the low-field and high-field peaks are assigned to syndiotactic and isotactic sequences, respectively. The fact that the syndiotactic polymer is favored over the isotactic polymer in this case might be due to the bulkiness of the side-chain group and to preorganization into a helical structure driven by the internal hydrogen bonding (see below).

MOLECULAR MODELING FT-IR experiments (see above) have indicated that PAMPA can adopt a doublestranded helical structure. Given the structural complexity of the double-stranded helical structure, we have performed molecular modeling of the single-stranded helical structure of PAMPA using MacroModel. A 14-mer was used for the modeling and the resulting single helix structure of syndiotactic, heterotactic and isotactic PAMPA is presented in Figure 4. All three types of PAMPA can self-assemble into a helical conformation. Intramolecular hydrogen bonds between nearest-neighbor amide groups assure the coherence of the resulting supramolecular structure. In protein helices (a and 310), H-bonding between nearest-neighbor backbone amide groups is unfavorable. However, the presence of conformationally repeating unites caused by the eight-membered H-bonding ring drives PAMPA to helicalization.

PAMPA may then adopt a regular helical structure in which approximately 5 residues make up a full turn. In this conformation, the secondary structure is optimally preorganized for the formation of the intra-catenate H-bonding patterns. The globular structure of the three types of helices is very similar. The isotactic polymer is the most compact one and has only one type of hydrogen bond. The CO H distance is 1.75 A and the helix turn is 9.5 . In the case of the syndiotactic polymer there are two alternating types of hydrogen bonds, one is 1.8 A and the other 2.1 A., and the helical turn is longer (10.9 A) as compared to the isotactic isomer. The heterotactic polymer is in fact a mixture of syndiotactic and isotactic isomers. It has three types of hydrogen bonds: the most stable one is 1.8 A, similar to the one found in the isotactic isomer, and the other two are comparable to those of the syndiotactic isomer.

The heterotactic helix has a turn of 10 A. The three helical conformations are further stabilized by hydrophobic interactions between the two lateral methyl groups. This hydrophobic effect is believed to be essential for the helix structure to be stable in aqueous solution and also leads to the formation of a double-stranded coiled-coil helical structure. Furthermore, the water solubility of the polymer is increased by the presence of an additional amide group at the end of the side chains. This amide group doesn't interfere with the internal H-bonding upon helix formation. This general model of PAMPA corresponds with the specific patterns of polar and nopolar groups found in a polypeptide chain. Polar groups on the surface form hydrogen bonds with water and polar groups localized internally are involved in intramolecular hydrogen bonding. The nonpolar side chains are removed from water and assembled into hydrophobic cores.

CONCLUSION In summary, a synthetic polymer that can adopt a stable double-stranded helical conformation in water is reported. In contrast to the backbone of natural peptides, PAMPA has a poly (ethylene) backbone and it has two amide functional group in its side chain. Due to this constitutional organization, intramolecular eight-membered rings stabilized by one hydrogen bond between nearest-neighbor amide groups make up the conformational repeating units of PAMPA. This eight-membered H-bonding motif for helical formation has also been observed in a peptide-mimicking synthetic system. 23 In proteins the double-stranded or coiled-coil helical structure consists of two helices wrapped around each other with a left-handed super twist. Regularly repeated hydrophobic leucine groups are responsible for the double-stranded helix formation. Some synthetic polymers can also form a double-stranded helical structure. For example, the crystal structure analysis of poly (methyl methacrylates) showed that either isotactic, 24 syndiotactic25 or mixtures of isotactic and syndiotactic stereocomplexes26 adopt double-stranded helices. The two helical chains are combined together mainly by non-bonded van der Waals interactions between the methyl end groups. Because PAMPA combines the structural features of a poly (leucine) peptide and a poly (glutamine) peptide, it has the tendency to adopt a doublestranded helical conformation. The 2-methyl-l-propanoic amide substituent of the aforementioned eight-membered ring confers as well stabilization of the helical conformation as water solubility. The two methyl groups on the side chain create a hydrophobic face which is essential for the stability of the double helical structure. The coiled-coil helical structure stabilizes the helix conformation of the polymer. PAMPA is additionally a neutral polymer and the external amide groups on the side chain are exposed to the environment and guarantee the aqueous solubility of the polymer. PAMPA represents the first polymer system of nonnatural origin that is capable of adopting a stable tertiary structure in aqueous solution.

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Legends 1 figure 1: The structure of PAMPA.

Figure 2: The infrared C=O stretching band (1600-1700 cm-1) of PAMPA in D20 at 25 OC. The band is slightly resolution enhanced (k=1.5). The three Gaussians underneath are the contributing components with maxima at 1623,1634 and 1651 cm'\ PAMPA was dissolved in D20 (50 mg/ml) and the solution was allowed to stand overnight to ensure complete H/D exchange. The infrared spectrum was obtained with a Bruker IFS66 FT-IR spectrometer

equipped with a liquid nitrogen cooled broad band mercury-cadmium-telluride solid state detector using a CaF2 cell with a teflon spacer of 50 go (Graseby Specac). 250 Interferograms were co-added after registration at a resolution of 2 cm'\ Fourier selfdeconvolution and fitting were done with a program developed in our laboratory. The deconvolution parameters used are a Lorentzian of 20 cm-l half-bandwidth, a resolution enhancement factor (k value) of 1.5 and a triangular square apodization function.

Figure 3: Temperature dependence of the maximum of the IR C=O band of PAMPA.

Figure 4: Computed model (MacroModel V5.0, AMBER* force field) of the proposed helix structure of isotactic (top), heterotactic (middle) and syndiotactic PAMPA (bottom). The surrounding water was mimicked by use of the GB/SA solvation method. The solvent accessible surface area was calculated with the DMS program of MidasPlus. All figures were created using a modified version of Molscript.

Scheme 1: Synthesis of PAMPA

Claims (1)

1. CLAIMS 1) A process of producing neutral, water-soluble synthetic polymers with coiled-coil or double-stranded helix conformation, said polymer including at least one repeating

   structural unit of 2-acrylamido-2-methyl-l-propanoic amide or of analogues or derivatives of said 2-acrylamido-2-methyl-1-propanoic amide.

   2) The process of claim 1, comprising: a) generating acid from a mixture of acrylic chloride and acetone in the presence of NaCN and NH4Cl, b) acid hydrolysis of the resulting cyanide, c) aminolysis using the ammonium salt of N-hydroxysuccinimide in the presence of DCC to obtain a monomer amide, d) polymerisation in DMF, initiated by a catalytic amount of AIBN e) removing said polymers from the monomers and DMF.

   3) The process of claim 3, further comprising dialysis.

   4) The process of claim 2 or claim 3, wherein polymerisation occurs at a temperature between 50 to 90 C.

   5) The process of claim 2 or claim 3, wherein polymerisation occurs at a temperature of about 70 C.

   6) The process of any of the claims 2 to 5, wherein said polymers are purified on size by filtration in a chromatographic medium or by gel filtration.

   7) Poly (2-acrylamido-2-methyl-l-propanoic amide) obtainable by the process of any of the claims 1 to 6.

   8) Neutral, water-soluble synthetic polymers, characterised in that said polymers includes at

   least one repeating structural unit of 2-acrylamido-2-methyl-l-propanoic amide unit or of analogues or derivatives of said 2-acrylamido-2-methyl-l-propanoic amide. 9) The neutral, water-soluble synthetic polymers of claim 8, whereby said polymers have a coiled-coil or double-stranded helix conformation. jU) A synthetic water-soluble polymer, characterised in that said synthetic polymer is capable of adopting a stable tertiary structure in an aqueous solution.

   11) The synthetic polymer of claim 10, characterised in that said synthetic polymer comprises units of2-acrylamido-2-methyl-l-propanoic amide or of analogues or derivatives thereof.

   12) The synthetic polymer of claim 10, characterised in that said synthetic polymer is poly (2

   acrylamido-2-methyl-l-propanoic amide)