High-throughput chemical and chemoenzymatic approaches to saccharide-coated magnetic nanoparticles for MRI

There is a need for biofunctionalised magnetic nanoparticles for many biomedical applications, including MRI contrast agents that have a range of surface properties and functional groups. A library of eleven adducts, each formed by condensing a reducing sugar with a catechol hydrazide, for nanoparticle functionalisation has been created using a high-throughput chemical synthesis methodology. The enzymatic transformation of an N-acetylglucosamine (GlcNAc) adduct into an N-acetyllactosamine adduct by β-1,4-galactosyltransferase illustrates how chemoenzymatic methods could provide adducts bearing complex and expensive glycans. Superparamagnetic iron oxide nanoparticles (8 nm diameter, characterised by TEM, DLS and SQUID) were coated with these adducts and the magnetic resonance imaging (MRI) properties of GlcNAc-labelled nanoparticles were determined. This straightforward approach can produce a range of MRI contrast agents with a variety of biofunctionalised surfaces.


General Methods and Equipment
Reagents were purchased from Sigma-Aldrich Co. Ltd., Dorset, UK. In the case of 3,4dihydroxybenzhydrazide 1, the supplier was Fluorochem, Derbyshire. Permanent magnets were bought from e-magnets UK, Hertfordshire, UK.
Reversed-phase HPLC purification was performed on an Agilent 1100 series system with an Agilent Eclipse XDB-C18 (9.4 mm × 250 mm) column. Sonication of nanoparticles during the coating process was performed with a Sonics VCX130PB Ultrasonic Processor with a stepped micro tip (3 mm × 136 mm) at 20 kHz.
NMR spectra were recorded in deuterated solvents using a Brüker 400 MHz Avance spectrometer with broadband probe or a Brüker 800 MHz Avance III. NMR chemical shift values are referenced to residual peaks from non-deuterated solvent and measured in ppm. Splitting patterns are reported as singlets (s), doublets (d), triplets (t), quartets (q), multiplets (m) or a combination of the above and coupling constants are measured in Hz. Electrospray mass spectrometry was performed on a Micromass LCT instrument using a Waters 2790 separations module with electrospray ionization and TOF fragment detection. High resolution mass spectrometry was performed on a Water Q-TOF micro with an ES+/-ion source. Elemental analysis was performed using a Thermo Scientific FLASH 2000 series CHNS/O Analyser. DLS and zeta potential measurements were carried out using a Malvern Zetasizer Nano.

General experimental procedure for adduct synthesis
The synthesis of compounds 2-12 was performed according to the following general procedure.
The saccharide (0.3 mmol) and 3,4-dihydroxybenzhydrazide 1 (0.3 mmol) were added to methanol (10 mL) containing aniline (5 mM from stock solution) and heated to reflux at 65 °C under nitrogen with stirring overnight. The solvent was removed in vacuo and the crude product was dissolved in a minimum of water, filtered if necessary to remove particulate material and purified by HPLC (see Section 2.3). The product containing fractions were collected and freeze dried to give the product as a white powder in all cases.

Comparison of different basic catalysts
NMR tube reactions were carried out in d 4 -methanol in order to assess the effectiveness of each catalyst. Each tube was filled with 3,4-dihydroxybenzhydrazide 1 (3 mg, 0.018 mmol) and glucose (4.8 mg, 0.018 mmol), as well a 5 mM stock solution of the catalyst in d 4 -methanol (0.6 mL). A control was also set up containing 1, glucose and d 4 -methanol with no catalyst present. 1 H NMR spectra were measured at 0 hours and the NMR tubes were then placed in an oil bath at 65 °C. Further 1 H NMR spectra were measured at 1, 2, 4, 6, 8 and 24 h. The yields were calculated by integrating the anomeric peaks of glucose and the glucose adduct 2. Taking the integrals of the α-and β-anomers of the product and dividing by the integrals of all glucose anomeric peaks (i.e. α and β for both product and starting material) gave a crude relative yield which could be converted into a percentage yield by multiplying by 100.
The aniline-catalysed reaction produced the product in better yields that the uncatalysed reaction. Both pphenylenediamine and anthranilic acid catalysed the reaction much better than aniline ( Figure  General procedure for adduct purification 1 H NMR spectra of the crude products (e.g. the glucose adduct 2, Figure S2.2) showed that the reaction did not produce many by-products, although the reaction does not go to completion after 24 h. 1 H NMR spectroscopy shows the crude product as a mixture of anomers as well as the anomeric peaks from the starting saccharide and the aromatic peaks from the starting hydrazide 1. The peaks from the catalyst can also be observed further downfield ( Figure S2.2 inset), although they are sometimes obscured by the aromatic peaks of the benzhydrazide 1. It was possible to remove some unreacted hydrazide 1 from the crude reaction mixture by first removing the methanol under reduced pressure before redissolving the crude reaction mixture in Milli-Q filtered water (5 mL), causing some of the hydrazide to precipitate out, and filtering through cotton wool. However, this did not remove all of the hydrazide and did not remove unreacted saccharide and catalyst from the filtrate. For this reason the reaction mixture (typically ~60 mg of crude material) was purified by high-performance liquid chromatography (HPLC) in the reversed-phase mode. The non-polar stationary phase was a C 18 column and the polar mobile phase was a mixture of water and tetrahydrofuran (THF) (v/v 95%/5%). A semi-preparative column (9.4 mm × 250 mm) was used and through purification of several aliquots of 0.5 mL, the entire crude reaction mixture of approximately 60 mg could usually be purified during one day. In order to elute less polar compounds, the proportion of THF (the less polar solvent) was increased from 5% to 50% over 40 mins. Typical retention times for monosaccharide adducts were from 14 to 18 mins at 1 mL/min. The column was then washed with a mixture of water/THF 5%/95% for a further 30 mins to remove all remaining material before the next crude sample was purified. The collected fractions were then concentrated under reduced pressure in order to remove the THF, before being lyophilised to give the products, as a mixture of α and β anomers, as white powders. In all, eleven small saccharides were used to form the library of saccharide adducts 2-12 ( Figure S2.3).

Characterisation data for adducts 2 -12
In each case the reported NMR spectroscopy data is for both anomers, but usually only the major β anomer is detected in the 13 C NMR spectra.

TEM measurements of uncoated MNP size distribution
The diameters of the synthesized nanoparticles was measured from TEM images using imageJ software (n = 100). The sizes were grouped in intervals of 2 nm, and the number of particles in each group was counted ( Figure S6.1). The mean diameter was calculated as 8.3 nm, with a standard deviation of 2.4 nm.