Azobenzene-containing poly(l-glutamates). Photochromism and conformation in solution

Photochromic polypeptides having various contents of azobenzene chromophores attached to th side chains have been prepared by condensing poly(l-glutamic acid) with p-amino-azobenzene. The photochemical and thermal cis-trans isomerizations of the azo chromophores have been investigated by absorption and circular dichroism spectroscopy, and the photochromic behaviour has been related to the conformation in solution. The azopolypeptides exhibit the α-helix c.d. pattern, in trimethylphosphate. The α-helix content markedly depends on the azo content, but it is not affected by the cis or trans geometric forms of the azo side chains. Strong solvent effects by H₂O or trifluoroethanol on the extrinsic azo c.d. bands, suggest the existence, in trimethylphosphate solution, of a super-ordered secondary structure involving a regular arrangement of the side chains, on th periphery of the helical peptide backbone.     

Metal complexs of poly (α-amino acids): Cu(II) chelates of acetoacetylated poly(L-lysine), poly(L-ornithine), and poly(L-diaminobutyric acid)

The cupric complexes of poly(N^ε-acetoacetyl-L -lysine), [Lys(Acac)]_n′ poly(N^δ-acetoacetyl-L -ornithine), [Orn(Acac)]_n′ and poly(N^γ-acetoacetyl-L -diaminobutyric acid), [A₂bu-(Acac)]_n, as well as of the model compound n-hexyl acetoacetamide, have been investigated by means of absorption, potentiometric, equilibrium dialysis, and CD measurements. While in the complex of the model compound, one chelating group is bound to one cupric ion, in the polymeric complexes two β-ketoamide groups are bound to Cu(II) under the same experimental conditions. The binding constant of cupric ions to the three polymers and the formation constant of the Cu(II)-nhexylacetoacetamide complex have been evluated. Investigation on the chiroptical properties of the three polymeric complexes shows that the peptide backbone does not undergo conformational transitions, remaining α-helical when up to 20% of the side chains are bound to Cu(II). The optical activity of the β-ketoamide chromophores is substantially affected by complex formation and is discussed in terms of asymmetric induction from the chiral backbone.     

Synthesis and characterization of a cationic poly(beta-hydroxyalkanoate)

Poly(beta-hydroxyalkanoates) (PHAs) are biodegradable polyesters produced by a wide range of bacteria. The structures of these polymers may be tuned by controlling the carbon source composition in the feed stock, but the range of functional groups accessible in this manner is limited to those that the organism is able to metabolize. Much effort has been made to chemically modify the side chains of these polymers to achieve new materials. Here, we report the synthesis of the first cationic PHA, poly(beta-hydroxy-octanoate)- co-(beta-hydroxy-11-(bis(2-hydroxyethyl)-amino)-10-hydroxyundecanoate) (PHON). Pseudomonas putida Gpo1 was used to produce poly(beta-hydroxy-octanoate)- co-(beta-hydroxy-10-undecenoate) (PHOU), whose vinyl-terminated side chains were first converted to terminal epoxides and then modified with diethanolamine. The modification of PHOU was examined using (1)H, COSY, and HSQC NMR and GPC and resulted in a loss of molecular weight due to aminolysis and also in the introduction of side chains terminated with tertiary amine groups, which are protonated at physiological pH. The polycationic PHA is soluble in polar solvents such as DMSO, DMF, and water. The new biodegradable cationic polymers are envisioned as nucleic acid delivery systems.     

Stopped-flow kinetic analysis of the interaction of anthraquinone anticancer drugs with calf thymus DNA, poly[d(G-C)].poly[d(G-C)], and poly[d(A-T)].poly[d(A-T)]

The sodium dodecyl sulfate driven dissociation reactions of daunorubicin (1), mitoxantrone (2), ametantrone (3), and a related anthraquinone without hydroxyl groups on the ring or side chain (4) from calf thymus DNA, poly[d(G-C)]2, and poly[d(A-T)]2 have been investigated by stopped-flow kinetic methods. All four compounds exhibit biphasic dissociation reactions from their DNA complexes. Daunorubicin and mitoxantrone have similar dissociation rate constants that are lower than those for ametantrone and 4. The effect of temperature and ionic strength on both rate constants for each compound is similar. An analysis of the effects of salt on the two rate constants for daunorubicin and mitoxantrone suggests that both of these compounds bind to DNA through a mechanism that involves formation of an initial outside complex followed by intercalation. The daunorubicin dissociation results from both poly[d(G-C)]2 and poly[d(A-T)]2 can be fitted with a single exponential function, and the rate constants are quite close. The ametantrone and 4 polymer dissociation results can also be fitted with single exponential curves, but with these compounds the dissociation rate constants for the poly[d(G-C)]2 complexes are approximately 10 times lower than for the poly[d(A-T)]2 complexes. Mitoxantrone also has a much slower dissociation rate from poly[d(G-C)]2 than from poly[d(A-T)]2, but its dissociation from both polymers exhibits biphasic kinetics. Possible reasons for the biphasic behavior with the polymers, which is unique to mitoxantrone, are selective binding and dissociation from the alternating polymer intercalation sites and/or dual binding modes of the intercalator with both side chains in the same groove or with one side chain in each groove.     

Photoresponsive polypeptides: Photochromism and conformation of poly (L-glutamic acid) containing spiropyran units

Spirobenzopyran units were bound to the side chains of poly (L -glutamic acid) and partially methylated poly(L -glutamate)s. The modified polymers were found to exhibit “reverse photochromism” in hexafluoro-2-propanol (HFP), so the samples kept in the dark were characterized by an intense absorption band in the visible range of the spectrum, which was completely erased upon exposure to sunlight or irradiation at 500–550 nm. The CD spectra showed that the macromolecules adopted a random coil conformation in the dark, whereas the bleached solutions after exposure to light displayed the typical CD pattern of the α-helix. The back reaction in the dark was accompanied by the progressive decrease of the helix content and recovery of the original disordered conformation. The photoinduced conformational changes resulted in large and reversible viscosity variations. When spiropyran side chains were converted to “spiropyran salts” of trifluoroacetic acid, the system was still photochromic, but the macromolecules were disordered both in the dark and light conditions. However, when appropriate amounts of methanol were added as a cosolvent to the HFP solutions, the system responded to light, giving reversible variations of the α-helix content. Irradiation at appropriate solvent compositions allowed modulation of the extent of the photoresponse. © 1993 John Wiley & Sons, Inc.     

Conformation of poly(l-glutamic acid) at the air/water interface

The conformation of the monolayer of poly(l-glutamic acid) on subsolutions of different pH values was studied by the film-balance technique, obtaining surface pressure measurements, together with polarized infrared spectroscopy and Raman spectroscopy. The monolayers of poly(l-glutamic acid) gave different surface pressure-area curves on subsolutions of various pH values. It was found that the conformation of poly(l-glutamic acid) monolayer spread at the air/water interface differs from that in solution. It can be presumed that poly(l-glutamic acid) in a monolayer is in the form of an α-helix at pH 2.0, in the β-form at pH 3.5 and in an ‘intramolecular’ heterogeneous conformation (consisting of a random coil and an α-helix) at pH 4.0.     

An in-depth analysis of polymer-analogous conjugation using DMTMM

Combinatorial libraries have become increasingly popular in the field of functional biomaterials. One approach for creating diverse polymer libraries is polymer-analogous conjugation of functional groups to polymer scaffolds. In this study, we show that a water-soluble condensing agent, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM), can be employed to conjugate two disparate model ligands, d-(+)-galactosamine (Gal) and agmatine (Agm), to the side chains of either poly(methacrylic acid) (pMAA) or poly(acrylic acid) (pAA) at various substitution ratios. The degree of substitution was found to be directly influenced by media pH, polymer concentration, structure of ligands, and polymer precursor. A nearly 2-fold increase in conjugation efficiencies for both ligands to pAA was achieved as compared to pMAA under identical conditions reaching up to 56% and 78% of Gal and Agm of total content, respectively. These two structurally similar polymers showed remarkably different performances, which reveals that the selection of a polymer precursor is crucial for the optimal design of polymeric libraries, particularly when complex structural ligands are involved. The approach employed provides a basis from which larger and more diverse combinatorial libraries of functionalized polymers with multiple moieties can be generated.     

The Specificity of Antibodies Elicited by Poly(dG)-Poly(dC)

Abstract

Antibodies have been raised to the synthetic DNA polymer poly(dG)·poly(dC). These antibodies have the ability to distinguish this right-handed polymer from natural mixed sequence DNA, as well as from other right- and left-handed synthetic DNA polymers. They show reduced but measurable binding to synthetic polymers which contain various combinations of guanine and cytosine polynucleotides suggesting that both helical shape and sequence are recognized by this antiserum.     

Photoresponsive polymers: Azobenzene-containing poly(L-lysine)

Poly(L -lysine) was reacted with various azo-reagents, including p-phenylazobenzoic acid, p-phenylazobenzoyl chloride, and p-phenylazobenzoic N-hydroxy-succinimide ester, to give polypeptides containing 5–44 mol % azobenzene units in the side chains. The conformation of the azo-modified polypeptides was investigated in connection with their photochromic behavior caused by the trans ? cis photoisomerization of the azo groups present in the side chains. In methanol/water solvent mixture, the 20% azo-poly(L -lysine) adopts the α-helix conformation. The helix stability was found to be higher when the azo side chains are in cis than when they are in trans configuration. So irradiation at 340 nm (trans-to-cis isomerization), and alternately at 450 nm (cis-to-trans isomerization), produced reversible variations of the α-helix content. In hexafluoro-2-propanol/water/sodium dodecyl sulfate mixture, the 43% azo-poly(L -lysine) adopts a β-structure, as indicated by CD spectra. Irradiation at 340 nm caused the disruption of the β-structure and promoted the α-helix conformation. The effect was reversed upon irradiation at 450 nm. The photoinduced β ? helix change was explained on the basis of the different geometry and hydrophobic character of the trans and the cis azobenzene units.     

Synthesis and cytotoxicity of salicylate-based poly(anhydride esters)

This paper describes the synthesis and cytotoxicity of poly(anhydride esters) that are composed of several salicylate derivatives, including halogenated salicylates, aminosalicylates, salicylsalicylic acid, and thiolsalicylic acid. The incorporation of these nonsteroidal antiinflammatory drugs (NSAIDs) into a biodegradable polymer backbone yields drug-based polymers that have potential for a variety of applications. The poly(anhydride esters) were synthesized by melt condensation polymerization. The halogenated salicylate derivatives yielded the highest molecular polymers as well as the highest glass transition temperatures. All polymers displayed in vitro degradation lag times from 1 to 3 days, depending on the water solubility of the salicylate derivative. Cell viability and proliferation were determined with L929 fibroblast cells in serum-containing medium to assess the polymer cytotoxicities, which varied as a function of the saliyclate chemistry. Cell morphology was normal for most of the polymers evaluated.     

Addition of "charge-shifting" side chains to linear poly(ethyleneimine) enhances cell transfection efficiency

We reported recently that the addition of ester-functionalized, "charge-shifting" side chains to linear poly(ethyleneimine) (LPEI) can be used to design polyamines that promote both self-assembly and self-disassembly with DNA in aqueous environments. This investigation sought to characterize the influence of charge-shifting side chains on the ability of LPEI to mediate cell transfection and understand the extent to which increases (or decreases) in levels of transfection could be understood in terms of time-dependent changes in the net charges of these polymers. We report that the addition of "charge-shifting" side chains to LPEI leads to significant increases in levels of LPEI-mediated transfection. In particular, polymer 1e, functionalized with 20 mol % ester-functionalized side chains, mediates levels of transgene expression in vitro up to 8-fold higher than LPEI. Experiments using an amide-functionalized analog of polymer 1e demonstrated that the esters in polymer 1e play an important role in promoting increased levels of transfection. These results, in combination with the results of additional gel electrophoresis experiments, provide support for the view that increases in transfection result from time-dependent changes in the net charge of polymer 1e and the disruption of ionic interactions in polyplexes. Additional support for this view is provided by the results of confocal microscopy experiments and measurements of fluorescence resonance energy transfer, which suggest that polymer 1e promotes the disruption of polyplexes in intracellular environments effectively. The approach reported here provides a means of addressing one important "late-stage" obstacle to polyplex-mediated transfection (polyplex unpackaging). If integrated successfully with methods that have been developed to address other important barriers to transfection, this general approach could lead to the development of multifunctional polyplexes that mimic more effectively the range of functions of viruses as agents for the delivery of DNA.     

Chiroptical and photochromic properties of stereoregular copolymers of 4-methacryloxyethylenoxyazobenzene with (−)-menthyl methacrylate

Copolymers of the photochromic monomer 4-methacryloxyethylenoxyazobenzene with the optically active comonomer (-)-menthyl methacrylate, having different stereoregularity, were investigated in order to obtain a better understanding of the relationship between microstructure and photochromism in synthetic macromolecules. No appreciable effect was observed by copolymer composition, sequence distribution, and microtacticity on the photoinduced trans → cis isomerization of the azobenzene side chains. This last, however, is reflected in changes of the chiroptical properties, type and entity of the photoinduced variation being dependent on chain structure. The long spacer separating the azo chromophore from the main chain limits the extent of chiroptical properties dependence on irradiation.     

The alpha-glycosidic bonds of poly(ADP-ribose) are acid-labile.

The poly(ADP-ribosyl)ation system of higher eukaryotes produces multiple ADP-ribose polymers of distinct sizes which exhibit different binding affinities for histones. Although precipitation with trichloroacetic acid (TCA) is the standard procedure for isolation of poly(ADP-ribose) from biological material, we show here that poly(ADP-ribose) is not stable under acidic conditions. Storage of poly(ADP-ribose) as TCA pellets results in acid hydrolysis of polymers, the extent of which is dependent on storage time and temperature. The alpha-glycosidic, inter-residue bonds are the preferred sites of attack, thus reducing polymer sizes by integral numbers of ADP-ribose to yield artefactually more and smaller polymers than originally present. Therefore, poly(ADP-ribosyl)ation studies involving TCA precipitation, histone extraction with acids, or acidic incubations of ADP-ribose polymers must account for the impact of acids on resulting polymer populations.     

Poly(vinyl amine)-silica composite nanoparticles: models of the silicic acid cytoplasmic pool and as a silica precursor for composite materials formation

The role of polymer (poly(vinylamine)) size (238-11000 units) on silicic acid condensation to yield soluble nanoparticles or composite precipitates has been explored by a combination of light scattering (static and dynamic), laser ablation combined with aerosol spectrometry, IR spectroscopy, and electron microscopy. Soluble nanoparticles or composite precipitates are formed according to the degree of polymerization of the organic polymer and pH. Nanoparticles prepared in the presence of the highest molecular weight polymers have core-shell like structures with dense silica cores. Composite particles formed in the presence of polymers with extent of polymerization below 1000 consist of associates of several polymer-silica nanoparticles. The mechanism of stabilization of the "soluble" silica particles in the tens of nanometer size range involves cooperative interactions with the polymer chains which varies according to chain length and pH. An example of the use of such polymer-poly(silicic acid) nanoparticles in the generation of composite polymeric materials is presented. The results obtained have relevance to the biomimetic design of new composite materials based on silica and polymers and to increasing our understanding of how silica may be manipulated (stored) in the biological environment prior to the formation of stable mineralized structures. We suspect that a similar method of storing silicic acid in an active state is used in silicifying organisms, at least in diatom algae.     

Conformational energy calculations on alginic acid I. Helix parameters and flexibility of the homopolymers

Conformational energy maps have been calculated for the 1-4-linked dimers of β-D -mannuronic acid and α-L -guluronic acid. Helix parameters have been calculated for poly(mannuronic acid) and for poly(guluronic acid), which are in reasonable agreement with data from x-ray fiber diffraction studies of these polysaccharides. The flexibility of the homopolymers was investigated by calculating the characteristic ratios, i.e., the ratio of the mean-square end-to-end lengths of the unperturbed chains to the product of the number of residues in the chains and the virtual bond lengths. The general conclusions are that both polymers are very stiff and extended, but that poly(mannuronic acid) is less extended than poly(guluronic acid).     

(1)H NMR study of the states of water in equilibrium poly(HEMA-co-THFMA) hydrogels

The spin-spin relaxation times, T(2), of hydrated samples of poly(hydroxymethyl methacrylate), PHEMA, poly(tetrahydrofurfuryl methacrylate), PTHFMA, and the corresponding HEMA-THFMA copolymers have been examined to probe the states of the imbibed water in these polymers. The decay in the transverse magnetization of water in fully hydrated samples of PHEMA, PTHFMA, and copolymers of HEMA and THFMA was described by a multiexponential function. The short component of T(2) was interpreted as water molecules that were strongly interacting with the polymer chains. The intermediate component of T(2) was assigned to water residing in the porous structure of the samples. The long component of T(2) was believed to arise from water residing in the remnants of cracks formed in the polymer network during water sorption.     

Poly(organo phosphazene) nanoparticles surface modified with poly(ethylene oxide)

The use of biodegradable derivatives of poly(organo phosphazenes) for the preparation of nanoparticles and their surface modification with the novel poly(ethylene oxide) derivative of poly(organo phosphazene) has been assessed using a range of in vitro characterization methods. The nanoparticles were produced by the precipitation solvent evaporation method from the derivative co-substituted with phenylalanine and glycine ethyl ester side groups. A reduction in particle size to less than 200 nm was achieved by an increase in pH of the preparation medium. The formation (and colloidal stability) of these nanoparticles seems to be controlled by two opposite effects: attractive hydrophobic interactions between phenylalanine ester groups and electrostatic repulsions arising from the carboxyl groups formed due to (partial) hydrolysis of the ester bond(s) at the high pH of the preparation medium. The poly[(glycine ethyl ester)phosphazene] derivative containing 5000-Da poly(ethylene oxide) as 5% of the side groups was used for the surface modification of nanoparticles. Adsorbed onto the particles, the polymer produced a thick coating layer of approximately 35 nm. The coated nanoparticles exhibited reduced surface negative potential and improved colloidal stability toward electrolyte-induced flocculation, relative to the uncoated system. However, the steric stabilization provided was less effective than that of a Poloxamine 908 coating. This difference in effectiveness of the steric stabilization might indicate that, although both the stabilizing polymers possess a 5000-Da poly(ethylene oxide) moiety, there is a difference in the arrangements of these poly(ethylene oxide) chains at the particle surface. (c) 1996 John Wiley & Sons, Inc.     

Nmr study of poly(aspartic acid). II. α- and β-Peptide bonds in poly(aspartic acid) prepared by common methods

The nature of peptide bonds in poly(aspartic acid) prepared by debenzylation of poly(β-benzyl-L -aspartate) under various conditions has been studied by means of nmr spectroscopy. It was established that the majority of the polymers prepared, as well as the commercially obtained polymer, contained aspartic acid linked in both α- and β-peptide bonds. The purest polymer, having practically undetectable amounts of β-bond, was prepared by debenzylation by HBr in trifluoroacetic acid. It was established that the β-bonds are formed via succinimides.     

Macromolecularization of a tripeptide analogue of the Cu(II) binding site of human serum albumin: 2. Conformational and binding properties towards Cu(II) and Ni(II)ions of Gly-Gly-His derivatives of poly(l-lysine)

The conformational and binding properties towards Cu(II) and Ni(II) ions of Gly-Gly-His derivatives of poly(l-lysine) have been investigated mainly using circular dichroism (c.d.) spectroscopy. These derivatized polymers can be considered macromolecular analogues of the Cu(II) and Ni(II) binding site of human serum albumin. It has been shown that modification up to 53% of the ε-amino groups of lysine side chains by covalent binding of the tripeptide unit Gly-Gly-His does not induce appreciable alteration of the α-helix forming tendency of the polylysine backbone. The derivatized polymers exhibit strong affinity towards Cu(II) and Ni(II) ions. At neutral pH, complexes are formed in which each tripeptide chelating unit is linked to one metal ion. The spectral characteristics in the visible absorption region are consistent with a square planar geometry of the complexes, with deprotonated peptide groups and one imidazole nitrogen in the coordination sphere of the ion. C.d. measurements in the far u.v. indicate that complex formation in the side chains causes an increase of ordered structure of the peptide backbone at neutral pH. This fact is interpreted in terms of a reduced electrostatic repulsion among side chains due to charge neutralization in the tripeptide units linked to metal ions.