Maleic reaction with protein

Parenthetically, it should be noted that the reaction of maleic anhydride with sulfhydryl-containing proteins also leads to the formation of S-succinylcysteine derivatives, by the reaction sequence... 

Maleic anhydride, 2-methylmaleic anhydride (citraconic anhydride), and 2,3-dimethyImaleic anhydride react with proteins in a manner similar to succinic anhydride, but the products are much more labile to hydrolysis [5,6]. The reaction products of maleic anhydride and amino groups are stable at neutral pH but rapidly hydrolyze when acidified to pH 3.5. This lability increases in the order maleyl < citraconyl < dimethylmaleyl derivatives. Citraconylamides are stable enough to withstand many hours at neutral or alkaline pH s but... 

In this type of protein immobilization, reactions involve the acylation of an NH2 group on a protein or an enzyme by pendent groups of the carrier such as azide, acid anhydride, carbodiimide, sulfonyl chloride, and hydroxysuccinimide esters. Copolymers of acrylamide and maleic anhydride have been useful for enzyme immobilization through the acid anhydride reaction with the enzyme. This system of... 

Scheme 6.12. Clearly, reaction of 47 with RNase was not specific of one particular lysine residue but rather involved several lysines. Selective introduction of the metallo-carbonyl group at the RNase active site residue Lys41 was achieved in two steps. First, reaction of RNase with maleic anhydride in the presence of a competitive enzyme inhibitor as a means of protection of the catalytic site led to an enzyme derivative with 3.7 blocked lysines. Removal of the competitive inhibitor by dialysis followed by reaction with compound 47 led to a new enzyme derivative exhibiting 1.1 g-atom of Fe/mol of RNase but exhibited only 29% of the catalytic activity of the unmodified protein [84].

Maleic anhydride has been long known for its reaction with amino groups of the proteins in basic aqueous solution, the reaction being reversible at acidic conditions [69]. Maleic anhydride copolymers could also be covalently bound with proteins in basic aqueous media, the hydrolysis of anhydride units being a competing reaction [70-75]. A water-soluble carbodiimide could also be used to activate the carboxylic acid groups [76]. 

The NCS was also modified with DIVEMA copolymer. The in-vivo toxicity of the resulting conjugate was reduced, but the EPR effect was not observed. This was explained by the hydrophilicity of DIVEMA that did not favor the binding with albumin which would increase the apparent size, as in the case of SMANCS [103]. Some efforts were also made in order to obtain a conjugate of tumor necrosis factor with DIVEMA copolymer [104]. In this case, several free amino groups of the protein need to be protected before the reaction with the maleic copolymer. In the in-vivo test, the conjugate had a much higher antitumor effect than the native tumor necrosis factor. 

N-Maleyl derivatives of proteins are obtained at alkaline pH by reaction with maleic acid anhydride. The acylated product is cleaved at pH < 5, regenerating the protein ... 

One can of course ask why only PEG is chosen for protein modification instead of other polymers. Actually, very few examples of conjugation with other polymers have been reported so far, the most successful being the poly(styrene-co-maleic acid/anhydride) derivative of neocardnostatin (SMANCS) developed by Maeda [118]. The main reason Hes in the mono-functionahty of mPEG that avoids a cross-linking reaction with the polyfunc-tionalized proteins. The usual natural or synthetic polymers present multiple points of attachment in the same molecule. This is the case of polysaccharide, for instance, but also for the extensively studied poly(N-(2-hydroxypropyl) methacrylamide) copolymers (HPMA) [119]. 

A copolymer of polyethylene glycol and maleic anhydride with a comb-shaped form, activated PM, was developed by NOF Corp. (Nippon Oil and Fats Co., Tokyo, Japan) [37]. Two activated PM copolymers have been prepared (Fig. 3i) activated PM13 (MW 13,000, m = 8, rt 33, R = H) and activated PMioo (MW 100,000, m 50, n 40, R = CH3). Amino groups in a protein molecule are coupled with maleic anhydride residues in the PM modifier to form acid-amide bonds (Scheme 7). These comb-shaped modifiers possess unique properties covering a protein molecule with the reaction between amino groups in a protein and multivalent acid anhydrides in the modifier. 

Maleic acid is a linear four-carbon molecule with carboxylate groups on both ends and a double bond between the central carbon atoms. The anhydride of maleic acid is a cyclic molecule containing five atoms. Although the reactivity of maleic anhydride is similar to that of other cyclic anhydrides, the products of maleylation are much more unstable toward hydrolysis, and the site of unsaturation lends itself to additional side reactions. Acylation products of amino groups with maleic anhydride are stable at neutral pH and above, but they readily hydrolyze at acid pH values around 3.5 (Buder et al., 1967). Maleylation of sulfhydryls and the phenolate of tyrosine are even more sensitive to hydrolysis. Thus, maleic anhydride is an excellent reversible blocker of amino groups to mask them temporarily from reactivity while another reaction is being done. For additional information and a protocol for the modification of proteins with this reagent, see Section 4.2. 

A simple procedure to prepare 5-aryl- and 5-pyridyl-2-furaldehydes from inexpensive, commercially available 2-furaldehyde diethyl acetal was reported. The reaction proceeded in a four-step, one-pot procedure and the yield of coupling step was usually between 58-91% <02OL375>. A facile route to 3,4-furandicarboxylic acids was developed. DDQ-oxidation of 2,5-dihydrofuran derivatives, which were produced from dimethyl maleic anhydride, furnished the desired esters of furan-3,4-dicarboxylic acid <02S1010>. The furan-fused tetracyclic core of halenaquinol and halenaquinone possessing antibiotic, cardiotonic, and protein tyrosine kinase inhibitory activities was synthesized. Intramolecular cycloaddition of an o-quinodimethane with furan gave the adduct as a single isomer via an enrfo-transition state, which was converted to trisubstituted furan by oxidation-elimination reactions <02T6097>. 

The half-life (x) of e-N-maleyl lysine is 11 h at pH 3.5 and 37 °C. More rapid cleavage is observed with the 2-methyl-maleyl derivative (x < 3 min at pH 3.5 and 20 °C) and the 2,2,3,3-tetrafluoro-succinyl derivative (x very low at pH 9.5 and 0°C). Cysteine binds maleic anhydride through an addition reaction. The S-succinyl derivative is quite stable. This side reaction is, however, avoided when protein derivatization is done with exo-cis-3,6-end-oxo-l,2,3,6- tetrahydrophthalic acid anhydride ... 

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