MPEG-conjugated

Hou Z, Zhan C, Jiang Q et al (2011) Both FA- and mPEG-conjugated chitosan nanoparticles for targeted cellular uptake and enhanced tumor tissue distribution. Nanoscale Res Lett 6 563-573... 

Figure 4. Scheme of the HELP (High Efficiency Liquid Phase) synthesis of the MPEG-conjugated oligonucleotides. 

Figure 5. Anion-exchange chromatographic patterns of MPEG-conjugated anti-HIV 12mers. Main synthetic data are reported.

To verify the effect of the different shape of the PEG unit, the same oligonucleotide has been conjugated to a new branched polyethylene glycol. The synthetic data are quite similar to those of the linear MPEG-conjugate, save some reduced yield during the first synthetic steps, likely due to some steric hindrance of the reactive functionality of the branched polymer. 

A first biological characterisation of these high-molecular mass MPEG-conjugated oligonucleotides has been performed by their thermal... 

Figure 6. Thermal denaturation studies of the MPEG-conjugated HIV-12mers first derivative of UV absorption at 260 nm as function of temperature.

Figure 7. Nuclease stability studies of the MPEG-conjugated anti-HIV 12mers in presence of a 1 1 mixture of diesterase and 5 -nucleotidase percent of intact oligonucleotides as function of time.

Figure 8, RNase H activity studies of MPEG-conjugated anti-HIV 12mers percent of cleavage against increasing concentration of the oligonucletides. Bottom scheme of the points of attack of the Rnasc H on the duplexes and autoradiography of the gel-electrophoresis of products.

Figure 9. Antisense activity of the linear MPEG-conjugated anti-HIV 12mer in tandem with the 8mer sequence, and of the related compounds.

A future, more difficult task will address the synthesis, and the activity studies, of mixed MPEG-conjugated biopolymers, as peptides and oligonucleotides, in search for new more specific and effective drugs. 

Originally, for preparation of such conjugates the hydroxyl groups of monomethoxy-PEG (mPEG) were activated with cyanuric chloride, and the resulting compound then coupled with proteins (10). This approach suffers from disadvantages, such as the toxicity of cyanuric chloride and its limited applicability for modification of proteins having essential cysteine or tyrosine residues, as manifested by their loss of activity. 

Currently, a common form of activated mPEG used for preparation of therapeutic enzymes is mPEG-succinate-N-hydroxysuccinimide ester (SS-PEG) (11). It reacts with proteins in short periods of time under mild conditions, producing extensively modified conjugates with well preserved biological activity. However, the ester linkage between the polymer and the succinic acid residue has limited stability in aqueous media (5,12). 

Add CDI-mPEG to this solution with stirring to bring the final concentration of the activated polymer to 180 mM. Note Other ratios of polymer-to-protein may be used, depending on the modification level desired. Some optimization of the derivatization level may have to be done to obtain conjugates having the best amount of polymer substitution with retention of protein activity. 

It seems that a maximum of about 10 ST-PHPMA chains can be attached to 1 CT molecule. The conjugation degree was lower than that of the chy-motrypsin conjugates with PEG-SC (succinimidyl carbonate of mPEG up to 14 chains per one CT molecule) [20,33]. This might be attributed to the solution stmcture difference between the two polymers. PHPMA has a random coil stmcture in aqueous solution, whereas PEG possesses a more extended one. The coiled PHPMA may cover more of the enzyme surface, and the steric effect may prevent more PHPMA macromolecules from attaching to the enzyme. 

Usually, PEG-protein conjugates are prepared by the reaction between methoxypolyethylene glycol (mPEG) carrying a reactive electrophile group... 

The three reactive chlorines on TsT have dramatically different reactivities toward nucleophiles in aqueous solution. The first chlorine is reactive toward hydroxyls as well as primary and secondary amine groups at 4°C and a pH of 9 (Mumtaz and Bachhawat, 1991 Abuchowski et al., 1977a). Once the first chlorine is coupled, the second one requires at least room temperature conditions at the same pH to efficiently react. If two chlorines are conjugated to nucleophilic groups, the third is even more difficult to couple, requiring at least 80°C. After activation of mPEG with TsT, it is therefore, for all practical purposes, only possible to couple one additional component to the triazine ring. 

To make scVEGF-lipid conjugate, add mPEG-DSPE-maleimide directly to deprotection reaction to a final molar protein-to-lipidratio of 1 2. Residual DTT will not affect modification reaction. Incubate lipidation reaction mixture at room temperature for 1 h. 

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