New shells for magnetic nanoparticles based on polypyrrole functionalized with α -amino acids

New functionalized magnetic core-shell nanostructures were developed based on magnetite as magnetic core covered with pyrrole copolymers substituted by α -amino acids. The 3-substituted pyrrole monomers were synthesized by amide formation between an α -amino acid and 4-(1- phenylsulfonyl-(1 H -pyrrol-3-yl)-4-oxo-butyric acid. Further, these substituted pyrrole monomers were copolymerized with unsubstituted pyrrole by oxidation with ammonium persulfate in the presence of water based magnetic nanofluids yielding functionalized magnetic core-shell nanostructures. The morphology of these magnetic functionalized polypyrrole core-shell nanoparticles was investigated by TEM and HRTEM, while the molecular structures and magnetic properties were investigated by FTIR spectroscopy and magnetization measurements, respectively.


Introduction
0][21][22] For many medical and biomedical applications it is essential to functionalize their surface.The functions can be directly attached to the magnetic nanoparticles by adsorption or by using polymers, such as polypyrrole (PPy).The latter polymer was found to be biocompatible in vitro and in vivo 23,24 and increasing interest has evolved for biochemical and medical applications.In this field, proteins [25][26][27][28][29][30] (glycosidase and other enzymes, collagen, fibrinogen, heparin), biotin 31,32 or polysaccharides were immobilized on polypyrrole films.4][35][36][37][38][39] Coating with biomolecule-functionalized PPy was recently used to improve the biocompatibility of metallic surfaces in implant material for biomedical application. 40Fe 3 O 4 nanoparticles coated by biofunctionalized PPy are rare.So far, folic acid 41 and the cancer antibody herceptine 42 were used to functionalize PPy-Fe 3 O 4 nanoparticles.These functions were fixed to the PPy layer of coated nanoparticles by the carbodiimide method.In a preliminary communication we reported PPy-Fe 3 O 4 nanoparticles functionalized with L-isoleucine without measurmement of magnetism. 43Here, we report full experimental details and measuring of magnetism as well as two new examples of 3-substituted PPy-Fe 3 O 4 nanoparticles functionalized with L-valine and Lphenylalanine.

Synthesis of 4-oxo-4-[1-(phenylsulfonyl)-1H-pyrrol-3-yl]butanoic acid
We tried to introduce the 3-carboxypropionyl group into position 3 of pyrrole by Friedel-Crafts acylation of 1-phenylsulfonylpyrrole with succinic anhydride in the presence of AlCl 3 (Scheme 1).The phenylsulfonyl group is the most commonly used protective group for the pyrrole nitrogen atom in Friedel-Crafts acylation of pyrrole (Py). 44However, the formation of mixtures of regioisomeric 2-and 3-substitution products, which are difficult to separate, is a major problem in this chemistry.We tried to overcome this drawback by modifying the reaction conditions performed by M. Kakushima et al., 45 i.e. by decreasing the reaction temperature to -30 °C and could achieve higher yield (68 %) of the product 3 and eliminate the formation of unwanted 2-acylpyrrole (Scheme 1).In addition, flash column chromatography was used to purify 3. Scheme 1. FC-Acylation of 1-phenylsulfonylpyrrole.

Pyrroles 5 by amino acid coupling to 4-oxo-4-[1-(phenylsulfonyl)-1H-pyrrol-3-yl]butanoic acid 3 and their hydrolytic cleavage to unprotected pyrroles 6
Delabouglise et al. reported the synthesis of amino acid-substitued pyrroles and transformed them into PPy polymers by electropolymerisation.They acylated methyl esters of L-serine and Lvaline with 3-pyrrolylacetic acid. 46As compared with the 3-pyrrolylacetic acid the pyrrole 3 used by us as the acid component has a longer linker.Thus the introduced amino acids are far enough from the pyrrole nucleus to avoid possible unwanted change in the conjugation and electronic properties in the resulting polymers by electronic or steric effects.We coupled the methyl esters of L-isoleucine 4a, L-valine 4b, L-phenylalanine 4c with 3 by using an adapted procedure from Abell et al. 47 and obtained 5a-c in excellent yields (Scheme 2).For the envisaged application of these new functionalized pyrroles 5a-c in copolymerisation reactions with pyrrole it was necessary to remove the 1-phenylsulfonyl protective group.We modified a literature-known method to implement this deprotection under basic conditions (Scheme 3) 47 and got the free functionalized pyrroles 6a-c (Table 2).All compounds were purified by flash column chromatography on silica (CH 2 Cl 2 /MeOH/HCOOH, 90:10:1) affording good yields.Racemization did not occur under these conditions as was checked by chiral HPLC of 6b and 6c.Scheme 3. Deprotection of 3-substituted pyrroles 5.

Synthesis of functionalized magnetic core-shell nanoparticles based on 3-substituted pyrrole copolymers
The water based magnetic nanofluids (MF) were prepared by a two-stage process, involving first the synthesis of magnetic nanoparticles, followed by their stabilization/dispersion by surfactants.The magnetite nanoparticles (MN) were synthesized through co-precipitation of Fe 3+ and Fe 2+ ions in solution with NH 4 OH in excess.The temperature was maintained at 80-82 o C, in order to obtain exclusively magnetite nanoparticles and to ensure optimal conditions for chemisorption of the surfactant.Combination of the surfactants lauric acid (LA), dodecylbenzenesulfonate (DBS) and oleic acid (OA), having different chain lengths were used, such as: LA+DBS, LA+LA, DBS+DBS and OA+OA (see also Table 1). 48,49 hese nanofluids provided the magnetic cores which were covered by copolymer shells obtained from functionalized pyrroles 6 and pyrrole later on.The copolymerisation was carried out in water using ammonium persulfate (APS) as oxidant.The type of substituted pyrrole 6 and the ratio of monomeric pyrroles were varied (Table 1) affording five new core-shell magnetic nanoparticles.These were characterized by FTIR, TEM, HRTEM and measuring of the magnetism.

Morphologic characterization
The functionalized magnetic nanoparticles based on PPy, obtained by the copolymerization of different pyrrole monomers 6 in the presence of Fe 3 O 4 nanofluids (Table 1) have a core-shell structure where Fe 3 O 4 is the magnetic core and PPy forms a conducting shell.The TEM image of Fe 3 O 4 magnetic nanoparticles from the ferrofluid Fe 3 O 4 (DBS+DBS) (Figure 1a) shows almost spherical shapes with an average diameter in the range of 5-8 nm.The magnetic nanoparticles MN-Py-phe 3 covered by copolymer (Figure 1b) have average diameters of 15-25 nm, some of them form aggregates.The HRTEM images given in Figure 2 show core-shell structures of functionalized magnetic copolymer nanostructures, with a crystalline magnetic core covered by a thin (1.5 -3 nm) copolymer shell, which is similar to those found in magnetic core-shell nanoparticles formed with unsubstituted pyrrole.

FTIR investigations
FTIR spectra of PPy and several functionalized core shell PPy MN were recorded (Figure 3).The strong absorption band located at around 570 cm -1 in all spectra of functionalized magnetic nanoparticles samples is attributed to Fe 3 O 4 .A weak band found at 1702 cm -1 in MN-Py-ile, 1707 cm -1 in MN-Py-val and 1711 cm -1 in MN-Py-phe 2 is ascribed to the amide C=O bond, demonstrating that the functionalized pyrroles 6 were incorporated.This analytic tool was also used by Lam et al. for the characterisation of copolymers composed of biofunctionalized pyrroles. 50However, the ratio of unsubstituted to substituted pyrrole in the copolymer can not be determined in this way.It can be assumed that the substituted pyrroles 6 are less reactive in the copolymerisation than unsubstituted pyrrole and thus the ratio in the copolymer will not be equivalent to the ratio of reactants used in the polymersation.
The spectra of magnetic nanoparticles MN-Py-ile, MN-Py-val and MN-Py-phe 2 containing functionalized polypyrroles show significant changes of the intensities and peak positions of the typical pyrrole ring vibration bands as compared with polypyrrole PPy.The peaks located at 914, 1190, 1465 cm -1 for PPy are shifted to higher wave numbers in the functionalized cases.Furthermore, the adsorption bands ascribed to the collective vibration mode of intra-ring and inter-ring C=C/C-C were shifted from 1548 cm -1 in PPy to 1553 cm -1 in magnetic nanoparticles MN-Py-phe 2, to 1557 cm -1 in magnetic nanoparticles MN-Py-val and to1561 cm -1 in magnetic nanoparticles MN-Py-ile.The position of this band is correlated with the conjugation length of the polymer chain, 51 the shift to higher frequencies indicates a decrease of the conjugation length of the copolymer in the functionalized magnetic nanoparticles as compared to PPy.

Magnetic properties
The typical behaviour of the magnetization vs. applied magnetic field of the functionalized magnetic nanocomposites MN-Py-val, MN-Py-phe 1 and MN-Py-phe2 at room temperature is shown in Figures 4a and 4b, respectively.The values of the magnetization are calculated related to the Fe 3 O 4 content of each nanocomposite.For all the reported functionalized magnetic nanocomposites, the magnetization does not show a hysteresis loop, being consistent with superparamagnetic behaviour.The saturation magnetization, M S for the nanocomposites is found at around 66 emu/g Fe 3 O 4 , which is in agreement with that usually reported for nanometric size magnetite. 52

Conclusions
This study reports a convenient and effective synthetic route to new functionalized pyrrole monomers which are substituted by amino acids via a linker in position 3. Novel functionalized magnetic nanostructures with a magnetite core and different pyrrole copolymer shells were obtained by chemical oxidative polymerization in aqueous solutions in the presence of Fe 3 O 4 nanofluid.Formation of the functionalized core-shell magnetic nanoparticles was shown by HRTEM.FTIR spectra exhibited characteristic adsorption bands of both component materials, namely the functionalized polypyrrole and Fe 3 O 4 .
The missing hysteresis loop in the magnetization vs. applied magnetic field proved the superparamagnetic behaviour for the functionalized magnetic nanostructures based on functionalized polypyrrole.
Because the synthesis is very flexible, it could also be applied to other amino acids or peptides, in particular to those exhibiting biological recognition functions.Such investigations as well as the application of the magnetic core-shell nanoparticles for the separation of enantiomers are currently underway in our laboratories.

Experimental Section
General Procedures.All reagents were purchased from Aldrich Chemical Company and were used as received without further purification.Solvents were dried by distillation under an inert atmosphere.All reactions were performed under ambient conditions. 1 H-NMR and 13 C-NMR solution spectra were recorded at 300 MHz and 75 MHz, respectively; with a Bruker AC 300 in CDCl 3 as the solvent in 5mm NMR tubes with TMS as internal standard.The morphology of functionalized magnetic nanoparticles based on PPy was determined by TEM using 1010 JEOL microscope and HRTEM using Hitachi H9000NAR transmission electron microscope.FT-IR spectra were obtained with a JASCO FTIR 610 spectrophotometer.The magnetic measurements were performed at room temperature using a Vibrating Sample Magnetometer Cryogenics.The purity of all products was checked by HPLC.

Unprotected pyrroles 6 by basic hydrolysis. General procedure B
To a solution of the pyrrole ester 5 (1.0 eq.) in methanol 2 M NaOH (2.5 eq.) was added at 0 °C resulting in a 1:1 mixture (v:v).The mixture was stirred at 40 °C for 1 h, diluted with fivefold amount of water, and the methanol was removed under reduced pressure.The aqueous solution was cooled to 0 °C and acidified with 3 M HCl to pH 2-3 and thoroughly extracted with ethyl acetate.The combined extracts were washed with water (2x), with brine (2x), dried over Na 2 SO 4 and evaporated under reduced pressure.The crude product was purified by flash column chromatography.

H-NMR
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